The chemistry of L-Ascorbic acid derivatives in the asymmetric ...

The Chemistry of L-Ascorbic Acid Derivatives in the Asymmetric Synthesis of C2- and

C3- Substituted Aldono-γ-lactones

A Dissertation by

Ayodele O. Olabisi

M. S., Wichita State University, 2004

B. S., Wichita State University, 1999

Submitted to the College of Liberal Arts and Sciences and the Faculty of the Graduate School of

Wichita State University in partial fulfillment of the requirements for the Degree of

Doctor of Philosophy

August 2005

ii

The Chemistry of L-Ascorbic Acid Derivatives in the Asymmetric Synthesis of C2- and

C3- Substituted Aldono-γ-lactones

I have examined the final copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Chemistry.

______________________________________ Professor Kandatege Wimalasena, Committee Chair We have read this dissertation and recommend its acceptance: __________________________________________ Professor William C. Groutas, Committee Member __________________________________________ Professor Ram P. Singhal, Committee Member __________________________________________ Professor Francis D’Souza, Committee Member __________________________________________ Professor George R. Bousfield, Committee Member

Accepted for the College of Liberal Arts and Sciences

__________________________________________ Dr. William Bischoff, Dean

Accepted for the Graduate School

__________________________________________ Dr. Susan K. Kovar, Dean

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DEDICATION

To My Parents

iv

ACKNOWLEDMENTS

I wish to express my deepest and sincerest appreciation to my advisor, Dr

Kandatege Wimalasena for his positive guidance, enlightened mentoring and

encouragement. His passion for the subject matter has greatly improved my knowledge

and interest. My sincere appreciation extends to Dr. Shyamali Wimalasena and Dr.

Mathew Mahindaratne, who helped me with my initial research training and their

assistance in the preparation of my manuscripts. I would also like to give my appreciation

to my other committee members; Dr. William C. Groutas, Dr. Ram Singhal, Dr. Francis

D’Souza and Dr. George Bousfield for their significant recommendations.

I wish to express my heartfelt gratitude to my colleagues, Dr. Srimevan

Wanduragala, Dr. Mehul Bhakta, Dr. Rohan Perera and Samantha Ranaweera, for their

invaluable friendship. It was a joy to work with them.

Finally, I acknowledge some of the many people without whom I could not have

completed my education; my wife, Monica, and children, Angela, Dominique and Folade

for their love, support and encouragement even at times of difficulty. My special

gratitude deeply extends to my parents and sisters for their incomparable love, support

and prayers.

This work was supported by a grant from the National Institutes of Health (NS 39423).

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ABSTRACT

The antioxidant and redox properties of L-ascorbic acid are closely associated

with the electron rich 2, 3-enediol moiety of the molecule and therefore selective

functionalization of the 2- and 3-OH groups is essential for the detailed structure-activity

studies. Reactions of 5- and 6-OH protected ascorbic acid with electrophilic reagents

exclusively produce the corresponding 3-O-alkylated products under mild basic

conditions due to the high nucleophilicity of the C-3-OH. Based on the density functional

theory (B3LYP) electron density calculations, a novel and general method was devised

for the direct alkylation of the 2-OH group of ascorbic acid with complete regio- and

chemo-selectivity. A complete spectroscopic analysis of two complementary series of 2-

O-acetyl-3-O-alkyl and 2-O-alkyl-3-O-acetyl ascorbic acid derivatives was carried out to

define their spectroscopic characteristics and to resolve common inconsistencies in the

literature.

The asymmetric approach to the synthesis of natural products or other

biologically active compounds is impeded by low abundance of natural sources as well as

a limited number of efficient synthetic methods. Nevertheless, carbohydrate-based

systems such as the aldono-1,4-lactones (also known as aldono-γ-lactones) which

generate a host of chiral compounds have been particularly rewarding in this respect. This

study shows a practical approach using 5,6-O-isopropylidene-L-ascorbic acid (ketal of L-

ascorbic acid) as a single common starting material for facile asymmetric synthesis of

protected, optically pure and functionalized aldono-1,4-lactones derivatives, valuable in

the synthesis of derivatives of various pharmacologically active agents for structure-

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activity studies. The practicality of this new approach is demonstrated by the convenient

synthesis of a series of thermal Claisen-rearranged products of 5,6-O-isopropylidene-3-

O-allyl-L-ascorbic acid and 5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid as the

corresponding 5,6-O-isopropylidene-2-allyl-3-keto-L-galactono-γ-lactone and 5,6-O-

isopropylidene-3-allyl-2-keto-L-galactono-γ-lactone respectively. The synthetic routes

are economical, efficient, diastereospecific, and proceed in high yields.

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TABLE OF CONTENTS

CHAPTER 1 ....................................................................................................................... 1

INTRODUCTION .......................................................................................................... 1

CHAPTER 2 ....................................................................................................................... 4

BACKGROUND AND SIGNIFICANCE...................................................................... 4

2.1 Discovery and History of L-Ascorbic Acid .......................................................... 4

2.2 Sources of L-Ascorbic Acid................................................................................ 10

2.3 Tissue Distribution of L-Ascorbic acid............................................................... 12

2.4 Biosynthesis of L-Ascorbic Acid in Animals ..................................................... 14

2.5 Biosynthesis of L-Ascorbic Acid in Plants......................................................... 18

2.6 Commercial Scale Synthesis of L-Ascorbic acid................................................ 22

2.7 Biological Functions of L-Ascorbic Acid........................................................... 26

2.7.1 L-Ascorbic Acid as an Enzyme Cofactor .................................................... 26

2.7.2 L-Ascorbic Acid in Electron Transport ....................................................... 31

2.7.3 L-Ascorbic Acid as an Antioxidant in Biological Systems ......................... 33

2.8 L-Ascorbic Acid Metabolic Enzymes................................................................. 36

2.9 Degradation and Oxidation of L-Ascorbic Acid................................................. 37

2.10 Cellular Transport and Intestinal Absorption of L-Ascorbic Acid ................... 40

2.11 Molecular Structure of L-Ascorbic Acid .......................................................... 41

2.12 Chemical and Physical Properties of L-Ascorbic Acid .................................... 45

2.13 Synthetic Derivatives and Analogues of L-Ascorbic Acid............................... 46

CHAPTER 3 ..................................................................................................................... 53

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RESEARCH OBJECTIVE ........................................................................................... 53

CHAPTER 4 ..................................................................................................................... 55

RESULTS AND DISCUSSION................................................................................... 55

4.1 Chemo- and Regio-Selective Alkylation of L-Ascorbic Acid............................ 55

4.1.1 3-O-Alkylation of 5,6-O-Isopropylidene-L-Ascorbic Acid......................... 56

4.1.2 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid........................ 59

4.1.3 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid ................ 62

4.2 Acylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid........................................ 65

4.2.1 C3-O- to C2-O Rearrangements of 3-O-Acyl-L-Ascorbic Acid Derivatives

..................................................................................................................... 67

4.3 NMR Spectroscopic Analyses of L-Ascorbic Acid and its Derivatives ............. 70

4.3.1 NMR Spectroscopic Properties of 2-O- and 3-O-Substituted 5,6-O-

Isopropylidene-L-Ascorbic Acid ................................................................ 71

4.4 The Sigmatropic Claisen Rearrangement of L-Ascorbic Acid Derivatives........ 75

4.4.1 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-

Isopropylidene-3-O-Allylic Derivatives of L-Ascorbic Acid..................... 76

4.4.1.1 NMR Spectroscopic Analyses of Products from C3-O to C2

Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-3-O-

Allyl-L-Ascorbic Acid Derivatives ...................................................... 77


Isopropylidene-3-O-Cinnamyl-L-Ascorbic Acid Derivatives .................... 85

ix



Acetyl-3-O-Cinnamyl-L-Ascorbic Acid Derivative (10A) .................. 87


Isopropylidene-2-O-Allyl-L-Ascorbic Acid Derivatives............................ 90



Allyl-L-Ascorbic Acid Derivatives ...................................................... 91

4.5 Comparative Analysis and Identificaton of Products of C3-O to C2 and C2-O to

C3 Claisen Rearrangement of L-Ascorbic Acid Derivatives .......................... 102

4.6 Stereochemistry of Products of C3-O to C2 and C2-O to C3 Claisen

Rearrangement of L-Ascorbic Acid Derivatives............................................. 105

4.7 Chemo- and Diastereo-Selective Reduction of Claisen Rearranged Products (E

& F Series) of L-Ascorbic Acid Derivatives................................................... 110

CHAPTER 5 ................................................................................................................... 116

EXPERIMENTAL SECTION.................................................................................... 116

LIST OF REFERENCES............................................................................................ 144

APPENDIX................................................................................................................. 164

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LIST OF TABLES

Table 1 Approximate Levels of L-Ascorbic Acid in Tissues ........................................... 13

Table 2 List of Enzymes Requiring L-Ascorbic Acid as a Cofactor or as a Modulator of

Activity (adapted from Ref. 118) ......................................................................... 28

Table 3 Physical Properties of L-Ascorbic Acid (adapted from Ref. 211 & 212)............ 47

Table 4 Products from 3-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid........ 58

Table 5 Products of 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)....... 61

Table 6 Products of 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic

Acid ...................................................................................................................... 63

Table 7 Products of 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic

Acid ...................................................................................................................... 64

Table 8 Products of 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid..... 65

Table 9 1H NMR (C-4-H) and 13C NMR (C-2 & C-3) Chemical Shifts (δ) of 2-O-Alkyl

and 3-O-Alkyl Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1) ......... 72

Table 10 1H NMR (C4-H) and 13C NMR (C2 & C3) Chemical Shifts (δ) of 2,3-O-

Disubstituted Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1) ........ 74

Table 11 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-

(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone............................................... 79

Table 12 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-


Table 13 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-

prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone ................................................... 81

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Table 14 13C-NMR Chemical shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-


Table 15 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-

(1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone ............................... 83



Table 17 1H-NMR Chemical Shifts (δ) Claisen Rearranged 5,6-O-Isopropylidene-2-O-

Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone................ 88

Table 18 13C-NMR Chemical Shifts (δ) Claisen Rearranged 5,6-O-Isopropylidene-2-O-

Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone................ 89










methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone.................................... 98



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methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone.................................. 100


(1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone ............................. 101

Table 27 Comparison of Diastereoisomers of Allyl-L-Galactono-γ-Lactone ................ 103

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LIST OF FIGURES

Figure 1 Depiction of L-Ascorbic Acid Biosynthesizing Abilities of Various Species of

Animals in Relation to their Phylogeny ............................................................ 11

Figure 2 Cytochrome b561 in Trans-Membrane Electron Transport. ................................ 32

Figure 3 Chemical Illustration of Radical Reactions in the Cell and Antioxidant Activities

(adapted from Ref. 129) .................................................................................... 35

Figure 4 L-Ascorbic Acid Redox System......................................................................... 36

Figure 5 Degradation of L-Ascorbic Acid (adapted from Ref. 35) .................................. 39

Figure 6 L-Ascorbic Acid and its Diastereomers. ............................................................ 44

Figure 7 Structural Forms of Dehydro-L-Ascorbic Acid ................................................. 45

Figure 8 Regioselective O-Alkylation of Ascorbic Acid.................................................. 49

Figure 9 Potentials of L-Ascorbic Acid as a Chiral Synthon............................................ 52

Figure 10 Calculated Electrostatic Density Potential Diagrams of Monoanion Species of

1. Order of Electron Density: Blue < Green < Yellow < Red........................ 57

Figure 11 Calculated Electrostatic Density Potential Diagrams of Dianion Species of 1.

Order of Electron Density: Blue < Green < Yellow < Red............................ 60

Figure 12 Calculated Electrostatic Density Potential Diagrams of Neutral Species of 1.

Order of Electron Density: Blue < Green < Yellow < Red............................ 70

Figure 13 Diastereoselective Reduction of C3-keto of E Series via Metal Chelation.... 112

Figure 14 Diastereoselective Reductive Amination Products of 1E, 5E and 1F (X, Y and

Z Respectively) ............................................................................................ 115

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LIST OF SCHEMES

Scheme 1 Proposed Biosynthetic Pathway for L-Ascorbic Acid in Animals (adapted from

Ref. 118, 119 & 121)...................................................................................... 15

Scheme 2 The Smirnoff-Wheeler Biosynthetic Pathway for L-Ascorbic Acid in Plants

(adapted from Ref. 118, 119 & 121) .............................................................. 19

Scheme 3 The Reichstein Process for L-Ascorbic Acid Manufacture (adapted from Ref.

127) ................................................................................................................ 23

Scheme 4 Microbial-Engineered Pathway for L-Ascorbic Acid Manufacture (adapted

from Ref. 127) ................................................................................................ 25

Scheme 5 L-Ascorbic Acid (Exogenous Electron Donor) in DβM Enzymatic Reaction 30

Scheme 6 Four-Electron Reduction Process of Oxygen to Water.................................... 33

Scheme 7 3-O-Alkylation of 5,6-O-Isoprpylidene-L-Ascorbic Acid ............................... 58

Scheme 8 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1) ...................... 61

Scheme 9 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid. 63

Scheme 10 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid. 64

Scheme 11 2,3-O-Disubstituted 5,6-O-Isopropylidene-L-Ascorbic Acid ........................ 65

Scheme 12 Acylation of 5,6-O-Isopropylidene-L-Ascorbic Acid.................................... 67

Scheme 13 Irreversible Isomerization of 5,6-O-Isopropylidene-3-O-Acetyl-L-Ascorbic

Acid under Basic Conditions. ........................................................................ 68

Scheme 14 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3-

Keto-L-Galactono-γ-Lactone ......................................................................... 77

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Scheme 15 Direct Synthesis of 5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3-Keto-

L-Galactono-γ-Lactone from 1....................................................................... 86

Scheme 16 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1-

phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone................................. 86

Scheme 17 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-prop-2-enyl)-2-

Keto-L-Galactono-γ-Lactone ......................................................................... 92

Scheme 18 C3-O (A) to C2 (E) Claisen Rearrangement Transition-State Geometry .... 107

Scheme 19 C2-O (C) to C3 (F) Claisen Rearrangement Transition-State Geometry .... 107

Scheme 20 The Reduction Products of 5, 6-O-Isopropylidene-2-Allyl-3-Keto-L-

Galactono-γ-Lactones (E) ............................................................................ 111

Scheme 21 The Reduction Product of 5,6-O-Isopropylidene-3-Allyl-2-Keto-L-Galactono-

γ-Lactone 2F ................................................................................................ 112

1

CHAPTER 1

INTRODUCTION

Ascorbic acid is a versatile water soluble radical scavenger widely distributed in

aerobic organisms that plays a central role in the protection of cellular components

against oxidative damage by free radicals and oxidants that are involved in the

development and exacerbation of a multitude of chronic diseases such as cancer, heart

disease, brain dysfunction, aging, rheumatism, inflammation, stroke, emphysema, and

AIDS.1-17 & 217 It also plays a critical role as a physiological reductant for key enzymatic

transformations in catecholamine neurotransmitter, amidated-peptide hormone, and

collagen biosynthetic pathways. In addition, simple derivatives of L-ascorbic acid have

been shown to possess important pharmacological properties. For example, (a) 5,6-O-

modified ascorbic acid derivatives have been found to be effective anti-tumor agents for

various human cancers, and induce apoptosis in tumor cells;18-25 (b) C2 alkylated

derivatives have been shown to have immuno-stimulant activity;26-31 (c) C2-O and C3-O

alkylated derivatives are known to protect against peroxidation of lipids of the bio-

membrane.32-33 Recently, the chemistry of ascorbic acid has also been exploited to

develop strategies for central nervous system drug delivery.34 These antioxidant as well

as redox and pharmacological benefits of L-ascorbic acid and its derivatives are closely

associated with the electron rich C2,C3-enediol moiety of its five-membered lactone

ring.35 Therefore, the selective modification of its C2- and C3-OH groups is essential for

detailed structure-activity studies of L-ascorbic acid. Consequently, our research group

2

was interested in studies13-17 involving various ascorbate derivatives as probes for

Dopamine-β-mono-oxygenase and Cytochrome b561, both of which use ascorbate as a

source of physiological reductant in catecholamine neurotransmitter biosynthesis.

In addition to well known physiological and pharmacological properties of L-

ascorbic acid and its derivatives,35 L-ascorbic acid has also been commonly used as an

inexpensive chiral synthon for the synthesis of a variety of natural products and

pharmacologically active agents.36-53 The common usage of the oxidatively cleaved C6-

C3 fragment of L-ascorbic acid as a chiral synthon36-48 and the selective alteration or

modification of its C2- and/or C3-OH functional groups provides a unique route to

different classes of aldono-1, 4-lactone derivatives which are important precursors for the

synthesis of modified sugars and non-carbohydrate natural compounds.54 One of the

widely used aldonolactones in the synthesis of natural products is D-gulono-γ-lactone

also known as D-gulono-1, 4-lactone and is easily obtained from L-gulono-γ-lactone by

intramolecular Walden inversion.55 Gulono-1,4-lactone is a very versatile precursor for a

large number of pharmacologically active agents and natural products. For example, it is

used as a precursor in the synthesis of (a) rare sugars such as L-ribofuranose, which are

common starting materials for the synthesis of new nucleoside antibiotics such as

novobiocin and anti-bacterial agents against Gram-positive bacteria;56-61 (b)

pharmacological agents for the suppression of abnormal T-cell responses;62 (c) α-

hydroxy-β-amino acid natural products that are known to display a broad range of

biological activities which include antibiotic, antifungal, antitumor and potent

aminopeptidase protease inhibitors;63-72 and (d) non-carbohydrate natural alkaloids

known for their antitumor activity.73 Furthermore, gulono-1,4-lactone also has

3

applications in polymer chemistry for the synthesis of potentially renewable, biomedical

polymeric materials which are biodegradable.74 Besides the usefulness of aldono-1, 4-

lactones as synthetic chemical precursors,75-76 both L-galactono-γ-lactone and L-gulono-

γ-lactone are also the key intermediate precursors of vitamin C biosynthesis in plants and

animals, respectively.77-81

4

CHAPTER 2

BACKGROUND AND SIGNIFICANCE

2.1 Discovery and History of L-Ascorbic Acid

L-ascorbic acid is commonly referred to as vitamin C. The term “Vitamin C” is

applied to substances that have anti-scorbutic activity and includes two compounds and

their salts: L-ascorbic acid and its two-electron oxidized form, L-dehydroascorbic acid.

The major deficiency syndrome of vitamin C in animals is scurvy. Symptoms of scurvy

include anorexia, anemia, arthralgia, bleeding gums, coiled hair, depression, dry eyes and

mouth (Sjogren’s syndrome), ecchymosis, follicular hyperkeratosis, fatigue, frequent

infections, impaired wound healing, inflamed gums, joint effusions, myalgia, muscle

weakness, perifollicular hemorrhages, and petechiae. The disease’s later-stage conditions

include patients exhibiting extreme exhaustion, kidney and pulmonary problems, as well

as diarrhea, eventually leading to death. The necessity to take in raw animal flesh or fresh

plant food in the diet to prevent scurvy disease was known from ancient times. Eber’s

Papyrus, an ancient Egyptian medical treatise in 1,500 BC, described scurvy as a disease

characterized by spongy and bleeding gums and bleeding under the skin. Around 400 BC,

Hippocrates, a Greek physician known as the founder of medicine developed an Oath of

Medical Ethics for physicians to follow. This Oath known as the Hippocratic Oath is

taken by physicians today as they begin their medical practice. He preached against one-

sided nutrition and described how good a daily and healthy diet rich in foods that are

5

known today to contain great amounts of vitamin C could help prevent diseases such as

scurvy. In 1200 AD, the Crusaders were plagued with scurvy. From 1492 to 1600, world

exploration was threatened by scurvy. Ferdinand Magellan, a Portuguese sea captain

around 1520, lost 80% of his crew to scurvy (after he and his crew reached Cape

Virgennes on the southern tip of South America). Also, Vasco de Gama, a Portuguese

conquistador famously known as Henry the navigator, was the first to sail across the

African coast on his way to India in 1492 and lost 100 of his 160 crew to scurvy. Scurvy

was a severe threat to thousands of soldiers and sailors alike and many died of the disease

during military campaigns and lengthy ocean voyages, respectively, until in 1720, when

the physician J. G. H. Kramer found that fresh herbs and lemon cured the disease.82-111

In 1746, James Lind, a British naval surgeon on H.M.S. Salisbury, conducted a controlled

test on 12 of his seamen suffering from the debilitating effects of scurvy and became the

first person to give a scientific basis for the cause of scurvy. In 1753, James Lind

published the results of his famous findings in a 400-page book, Treatise of the Scurvy,

where for the first time, he established the benefit of citrus fruits in combating scurvy,

and by 1795, the royal navy had mandated the use of lime juice or other citrus fruits as a

scurvy preventative. In 1840, George Budd, a Londoner physicist, wrote that scurvy was

due to the absence of an essential food factor that will be discovered in the near future by

organic chemists. Up until 1907 scurvy was considered as a human-only disease as no

other animal was known to be susceptible to it. However, in 1907, Alex Holst and

Theodore Frohlich, two Norwegian biochemists confirmed that guinea pigs were also

susceptible to scurvy and later showed that laboratory monkeys were susceptible to

scurvy as well. They also described the prevention of the illness by feeding fruits and

6

vegetables to patients. In 1908, the classic lectures of Sir Archibald Garrod on the

“Inborn Errors of Metabolism”, in which he showed that missing enzymes could cause

diseases such as scurvy, were ignored and neglected at a time when modern, widely

accepted biochemical and genetic concepts were unknown or unrecognized.82-111

In 1912, for the first time, the vitamin hypothesis was suggested by Polish-

American chemist Funk, part of which stated that scurvy was a deficiency disease caused

by the lack of an unknown water-soluble substance called the anti-scorbutic factor. In

1920, Sir Jack Cecil Drummond, a Londoner, and the first Professor of Biochemistry in

the University of London, suggested calling this substance as Vitamin C because man,

guinea pigs, and certain monkeys unlike other mammals, cannot make their own ascorbic

acid. This unknown water-soluble antiscorbutic substance was isolated from Ox adrenal

cortex (and various plants) in 1928 by the Hungarian biochemist research team of Joseph

L. Svirbely and Albert Von Szent-Györgyi. In autumn of 1931, this reducing substance

with the molecular formula C6H8O6, which he named hexuronic acid, was unequivocally

proven in experimentation as the powerful anti-scorbutic substance, and that the anti-

scorbutic activity of plant juices corresponded to their hexuronic acid content. About the

same time, the Americans Charles Glen King and William A.Waugh also reported

crystals obtained from lemon juice, which were actively anti-scorbutic and resembled

hexuronic acid. In 1932, Albert Von Szent-Györgyi and British chemist Sir Walter

Norman Haworth subsequently renamed hexuronic acid as Ascorbic acid. In 1933, the

main features of the constitution of ascorbic acid and its formula as a lactone of 2-keto-L-

gulonic acid, capable of reacting in various tautomeric forms, was first announced from

the University of Birmingham. At about the same time, the Polish Tadeus Reichstein, in

7

Switzerland, as well as Haworth’s group independently achieved the organic synthesis of

vitamin C. The synthetic form of the vitamin was identical to the natural form and this

made possible the cheap mass production of vitamin C. Three patent applications were

filed in 1935 and the patents were granted in 1939 and 1940. Thus, the American

biochemist and chemical engineer Dr. Irwin Stone obtained the first patents on an

industrial application of ascorbic acid. Sir Walter Norman Haworth was awarded the

Noble Prize for chemistry largely for this contribution in 1937. Also, in 1937, Albert Von

Szent-Györgyi was awarded the Nobel Prize for the first isolation of vitamin C.82-111

In 1959, an American, J. J., Burns showed that the basic biochemical lesion in the

few mammals susceptible to scurvy was primarily due to their inability to produce the

active enzyme, L-gulonolactone oxidase which is the last of the four enzymes involved in

the mammalian conversion of blood glucose to ascorbic acid, in the liver. According to

Dr. Hickey of Manchester University, humans carry a mutated and ineffective form of the

enzyme. This was 51 years after Sir Archibald Garrod’s famous lecture pointed to the

lack of an enzyme as the reason for scurvy. Up until 1965, it was assumed that all

primates were unable to produce their own ascorbic acid and were as a result susceptible

to scurvy. Then it was suggested by Dr. Irwin Stone that the whole order of primates

should be examined for the presence of L-gulonolactone oxidase in their livers to

determine in which primate ancestor of man this important enzyme system was lost. This

challenge was picked up, tested and reported from Harvard University in 1966 by O.,

Eliott and 3 years later by the Yerkes Primate Research Center, wherein it was indicated

that members of the suborder Anthropoidea showed an inactive form of the enzyme, L-

gulonolactone oxidase, in their liver. As a result of these evolutionary studies, Dr. Irwin

8

Stone’s research on the genetics of scurvy had progressed to a point where it could then

be said that scurvy was not a dietary disorder, but rather was a potentially lethal problem

in medical genetics that was due to an ineffective gene, which produces an inactive

enzyme. Therefore, present day humans still suffer from a mammalian inborn error of

carbohydrate metabolism indigenous to the liver. He produced four papers by 1966

describing a human birth defect existing in 100% of the population due to a defective

gene. The potentially fatal genetic liver enzyme disease, which makes it necessary for

man to obtain ascorbic acid from exogenous sources was named “Hypoascorbemia”, and

stated as the cause of scurvy.82-111 Stone profoundly believed in the distinctive healing

qualities of vitamin C and became convinced of its effectiveness when he and his wife

had an accident involving a head on collision with a drunk driver and used large doses of

vitamin C in their speedy and remarkable recovery. In 1968, the American and two-time

Nobel laureate Linus Pauling, who was introduced to vitamin C by Dr. Irwin Stone,

indicated that this evolutionary mutation may have had survival values at the time simply

because it freed the biochemical machinery (glucose-consumption) for other purposes

and conserved energy. Vitamin C is a hexose derivative, similar in structure to the six-

carbon sugar glucose. Pauling was initially skeptical of Dr. Irwin Stone’s ideas but was

intrigued by Stone’s theory regarding genetic deficiencies and genetic mutation. Pauling

later decided to follow Dr. Stone’s advice by taking 3 grams of vitamin C daily for 3

years. Pauling soon noted that his sense of wellness improved and he was not

experiencing the dreaded cold that plagued him for 40 years. He later described vitamin

C as an essential nutrient in the maintenance of a healthy immune system for humans

(optimum intake of about 2.3 to 9.5 grams per day) in early 1970. Linus Pauling

9

concluded that the intake of vitamin C could improve, as well as extend life expectancy,

and therefore went forward to advocate its uses for various therapeutic uses for the

remaining 30 years of his life.82-111

In the present day civilization, ascorbic acid is less known as the anti-scorbutic

factor used for many centuries to cure the variety of clinical symptoms known as scurvy,

as this pathological state in no longer very common. L-ascorbic acid is largely known as

an antioxidant which efficiently scavenges toxic free radicals and other reactive oxygen

species (ROS) formed in cell metabolism. ROS are associated with several forms of

tissue oxidative damage by free radicals and oxidants that are involved in the

development and exacerbation of a multitude of chronic diseases. A complete list of

vitamin C uses can be found in the Clinical Guide to the Use of Vitamin C, edited by

Lendon H. Smith, M.D., Life Sciences Press, Tacoma, WA (1988). Some of these uses

includes its benefit in combating (a) Allergic Rhinitis, (b) Alzheimer’s disease, (c)

Asthma, (d) Atherosclerosis, (e) Breast Cancer, (f) Burns, (g) Cataracts, (h) Cervical

Dysplasia, (i) Common-Cold, (j) Diabetes-Mellitus, (k) Eczema (l) Gallbladder disease

(m) Glaucoma, (n) Hypertension, (o) Hypercholesterolemia, (p) Macular-Degeneration,

(q) Myocardial-Infarction, (r) Obesity, (s) Osteoarthritis, (t) Pancreatitis, (u) Parkinson’s

disease, (v) Photodermatitis, (w) Skin-Cancer, (x), Stroke, (y) Uveitis, and (z) Wounds.

Thus, this makes ascorbic acid ever more important than when scurvy was a major

menace confronting human health. Furthermore, ascorbic acid is widely used in the food

industry as a common additive to foods in order to improve the taste and as well as to

restore the vitamin C loss due to processing and storage. It is used as a preservative to

prevent oxidation or serve as a stabilizer in various food products and beverages. It is also

10

used in bread baking, brewing, wine making, and freezing of fruits. In addition, ascorbic

acid and some of its derivatives have important usage in industrial processes such as

polymerization reactions, photographic development and printing, and metal technology.

Most of these modern applications of ascorbic acid make use of the reducing properties

of the molecule.82-111

2.2 Sources of L-Ascorbic Acid

The main sources of L-ascorbic acid for humans are from plants and animals with

indigenous biosynthetic capabilities of producing L-ascorbic acid. The ubiquitousness

of L-ascorbic acid throughout the human body emphasizes its daily requirement and

vitality as a nutrient for healthy maintenance.112-114 Its biological half-life in humans

is 14-40 days after normal intake and a vitamin-C-free diet in a human develops

scurvy in about 3-4 months.115 It is required in the diet by only a few species of

animals (Figure 1): man, guinea pig, red-vented barbul, an Indian fruit-eating bat and

some related species of passeriform birds, and most but not all primates. Many

invertebrates and teleost fish are incapable of synthesizing vitamin C. L-Ascorbic

acid is also an essential nutrient for rainbow trout, carp, Coho salmon, and some

insects.

The vast majority of species of plants and animals are known to synthesize their own

vitamin C. A majority of vertebrates such as amphibians, reptiles, birds, and

mammals are able to synthesize L-ascorbic acid. Molecules similar to ascorbic acid

are made by some fungi but not by bacteria.

11

Figure 1 Depiction of L-Ascorbic Acid Biosynthesizing Abilities of Various Species of Animals in Relation to their Phylogeny

Adapted from Seib & Tolbert Am. Chem. Soc. Adv. Chem. Ser. 200; Washington, D. C., 1982 (Ref. 35)

All algal classes can synthesize vitamin C from glucose or other sugars. All higher

plant species can synthesize vitamin C and thus make it prevalent in the surrounding

food sources. For example, large concentrations of vitamin C are found in fruits such

as oranges, grapefruits, tangerines, lemons, limes, papaya, strawberries, and

12

cantaloupe. It is also found in the white linings of these fruits and other plants. Also,

many vegetables are known to pack in vitamin C and these include tomatoes,

broccoli, green and red bell peppers, raw lettuce and other leafy greens. A complete

listing of every food containing vitamin C according to the USDA food database is

available through The Vitamin C Foundation. 86

2.3 Tissue Distribution of L-Ascorbic acid

Tissue distribution of L-ascorbic acid (Table 1) offers a clue to its metabolic role

since its concentration in various tissues is found to be tightly controlled.114, 116-117 Its bio-

availability status in the body is found to influence many metabolic systems such as iron

and copper balance, fatty acid transport, hemostasis, endocrine function, control of blood

pressure, collagen synthesis, peptide metabolism, the immune system, endothelial

function, steroid metabolism and lipid metabolism.115 The biochemical mechanism of L-

ascorbic acid in each of the different systems appears to be related to its antioxidant

properties.35, 115-116 The plasma L-ascorbic acid concentration of a healthy person is 8-14

mg/L and it contributes around 10-15% of the total antioxidant strength of fasting

plasma.115 Some monocytes and adrenal cells such as the adrenal glands, corpus luteum,

pituitary, thymus and retina have L-ascorbic acid concentrations up to 100-fold that of the

plasma.115 The salivary gland, pancreas, leukocytes, kidney, thyroid, liver, small

intestinal mucosa, lymph glands, testicle, lung, spleen, ovary and the brain have in excess

of about 10-50 times that of the plasma L-ascorbic concentration.115

13

Table 1 Approximate Levels of L-Ascorbic Acid in Tissues

Tissues Human (mg / 100mg tissue)

Rat (mg / 100mg tissue)

Adrenal glands 30-40 280-400

Brain 13-15 35 Eye lens 25-31 8-10

Heart muscle 5-15 5-10 Kidney 5-15 15-20 Liver 10-16 25-40 Lungs 7 20-40

Pancreas 10-16 - Pituitary gland 40-50 100-130

Plasma 0.4-1.0 1.6 Saliva 0.07-0.09 -

Skeletal muscle 3-5 5 Spleen 10-15 40-50 Testes 3 25-30

Thymus 10-15 40 Thyroid 2 22

Adapted from Levine, M.; Mortia, K. In Vitamins and Hormones; Aurbach, G. D.; McCormick, D. B., Eds.; Vol. 42, academic Press Inc.: New York, NY., 1985, pp. 1-64.

The cardiac and smooth muscles, erythrocytes, and the skeletal muscle have

concentrations about 10 times that of plasma. The high level of L-ascorbic acid found in

vital organs suggests that these regions have elevated anti-oxidation requirements and

thus serve to protect them against dietary deficiencies as well as the maintenance of their

structural integrity through collagen synthesis. Thus, they are enabled in performing their

specialized functions.

14

2.4 Biosynthesis of L-Ascorbic Acid in Animals

The biosynthesis of L-ascorbic acid in animals (Scheme 1) is integrated with the

glucuronic acid metabolic pathway. This metabolic pathway is involved in the

metabolism of sugars under both normal and disease states and is regulated by the body’s

physiological functions.35, 115,118 It is an important pathway for major detoxification

processes in the body and the activities of the synthesizing enzymes vary from species to

species.35, 115,118 The well-known evolutionary distribution of L-ascorbic acid

biosynthesis suggests that it started in the kidney of lower vertebrates such as amphibians

and reptiles, then transferred to the liver of mammals, and eventually lost in primates,

fruit bats and guinea pigs.35, 115,118 Even in vertebrates capable of synthesizing L-ascorbic

acid, this biosynthesis only takes place in a few cell types. For mammals, these cells are

the hepatocytes, whereas in reptiles, amphibians and egg-laying mammals, the

biosynthesis takes place in the kidney cells. However, in birds with the exception of the

passeriforms, which are incapable of L-ascorbic acid biosynthesis, this biosynthesis is

known to take place in the kidney, liver or both.35, 115,118 Most of the research on ascorbic

acid synthesis in animals have been carried out using rats.35, 115,118

15

Scheme 1 Proposed Biosynthetic Pathway for L-Ascorbic Acid in Animals (adapted from Ref. 118, 119 & 121)

ATP ADP

Cyt Cox Cyt Cred

HHO

O

OHHO H

D-Glc D-Glc-1-P UDP-D-Glc UDP-D-GlcUA

D-GlcUA-1-PD-GlcUAL-GulA

L-GLL-Ascorbic acid

O H

OH

HO

HO

O

OHHO

1 3 4

5

678

9HO

D-Glc-6-P2

Catalytic Step Enzyme Substrate

1 Hexokinase D-Glucose

2 Phosphoglucomutase D-Glucose-6-phosphate

3 UDP-D-Glucose pyrophosphorylase D-Glucose-1-phosphate

4 UDP-D-Glucose dehydrogenase (EC 1.1.1.22)

UDP-D-Glucose

5 D-Glucuronate-1-phosphate uridylytransferase (EC 2.7.7.44)

UDP-D-Glucuronic acid

6 D-Glucurono kinase (Hydrolase) UDP-D-Glucuronic acid-1-phosphate

7 D-Glucuronate reductase (EC 1.1.1.19)

D-Glucuronic acid

8 Aldonolactonase (EC 3.1.1.17) L-Galacturonic acid

9 L-Gulono-1,4-lactone dehydrogenase (EC 1.1.3.8)

L-Gulono-1,4-lactone

16

In 1960, the de novo biosynthesis of L-ascorbic acid in animals was established and

known for utilizing intermediates of the D-glucuronic acid (hexuronic acid) pathway.115,

118 In vivo, the hexose skeleton of L-ascorbic acid originated from D-glucose that is

mainly derived from the breakdown of glycogen.115, 118 This in vivo biosynthesis takes

place either in the liver or kidney, which are both glycogen-storing organs.115, 118

The deficiency in the biosynthesis of L-ascorbic acid found in some animals and

humans has been localized to a lack of the terminal flavor-enzyme, L-gulono-1,4-lactone

oxidase (GuLO, EC 1.1.3.8), which completely blocks the liver production of L-ascorbic

acid in humans.35,115,118 This oxidizing enzyme is required in the last step of the

conversion of L-gulono-γ-lactone to 2-oxo-L-gulono-γ-lactone, which is a tautomer of L-

ascorbic acid that is spontaneously transformed into vitamin C. Although cloning and

chromosomal mapping studies have indicated that the gene encoding L-gulono-1,4-

lactone oxidase was found to be present in the human genome, nonetheless it is not

expressed due to the accumulation of a number of promoter defective mutations which

are without any selective pressure since it presumably ceased to function during

evolution.35,115,118 This terminal enzyme, L-gulono-1,4-lactone oxidase, is found not to

be 100% specific for L-gulono-γ-lactone as substrate, but also known to catalyze the

oxidation of related aldono-lactones such as D-altrono-γ-lactone(16%), D-manono-γ-

lactone (64%) and L-galactono-γ-lactone (70-90%), which is the direct precursor of L-

ascorbic aid biosynthesis in plants.115,118 Studies with radioactive labeling techniques

have indicated that D-glucose is converted into L-ascorbic acid sequentially via D-

glucuronic acid, L-gulonic acid, L-gulono-γ-lactone and 2-keto-L-gulono-γ-lacone (2-

oxo-L-gulono-γ-lactone) as intermediates.115, 118 Radiotracer studies with D-[6-14C]-

17

glucose, D-[2-14C]-glucose and D-[1-14C]-glucose indicated that the C1 carbonyl group

of L-ascorbic acid is derived from the oxidation of the C6 carbon rather than the C1 of D-

glucose115, 118and that this important reduction and oxidation conversion of the C1 and C6

respectively, takes place between D-glucuronic acid and L-gulonic acid, while the D-

glucose chain remains intact.115, 118 Consequently, L-ascorbic acid biosynthesis in animals

is known to follow a non-inversion type conversion of derivatives of D-glucose. Some

prokaryotic organisms that contain the enzyme, L-gulono-γ-lactone dehydrogenase,

which is able to synthesize L-ascorbic acid or one of its isomers have been isolated and

characterized.115, 118 However, the chemical and physical properties of this enzyme are

entirely different from those of eukaryotic organisms. Both in vivo and in vitro studies

have established that L-ascorbic acid biosynthesis in animals is controlled by a direct

feedback mechanism and that the concentration of L-ascorbic acid in the cell culture

medium or in the blood helps to regulate the amount of L-ascorbic acid synthesized in the

liver or in hepatocytes of rat or mice.115, 118 For example, in hepatocytes, L-ascorbic acid

synthesis is stimulated by glucagon, dibutyryl, cyclic adenosine monophosphate (cAMP),

phenylephrine, vasopressin and okadaic acid.115, 118 The hepatic L-ascorbic acid

biosynthesis in mice has also been shown to be stimulated by enhanced

glycogenolysis.115, 118 In rats, uridine diphosphate (UDP) glucuronosyltransferase gene

expression is shown to be involved in the stimulation of L-ascorbic acid biosynthesis by

exposure to xenobiotic compounds such as 3,4-benzpyrene, 3-methylcholanthrene and

sodium Phenobarbital.115, 118 Xenobiotic compounds are known to induce biosynthesis of

enzymes involved in the glucuronic acid pathway which is a part of the drug

detoxification process in the body.115, 118 The rate of in vitro L-ascorbic acid biosynthesis

18

shows close correlation with the glucose release by hepatocytes.115, 118 In mice, the

injection of glucagon increases L-ascorbic acid concentrations in the liver and plasma

membrane.115, 118 On the other hand, the biosynthesis of L-ascorbic acid is impaired by

the deficiency of vitamin A, vitamin E and biotin.

2.5 Biosynthesis of L-Ascorbic Acid in Plants

The biosynthesis of L-ascorbic acid in plants has not been clearly and easily

established when compared to its biosynthesis in animals. However, recent advances in

the understanding of L-ascorbic acid biosynthesis in plants have helped to resolve many

of the contradictions of the past decades. There is now a general consensus that the

biosynthetic pathway, which proceeds via GDP-D-mannose and GDP-L-galactose35, 118-

121 as proposed by the Smirnoff group,120 represents the major L-ascorbic biosynthetic

pathway in plants (Scheme 2). This pathway is known today as the Smirnoff-Wheeler L-

ascorbic acid biosynthetic pathway. The first part of the pathway is also utilized for the

synthesis of cell wall polysaccharide precursors, while the later steps following GDP-L-

galactose are solely dedicated to plant biosynthesis of L-ascorbic acid. The earlier

observation on the conversion of L-galactono-γ-lactone to L-ascorbic acid also applied in

this case since, interestingly, this pathway also utilizes the same terminal enzyme L-

galactono-γ-lactone dehyrogenase, just as in the route originally proposed by Isherwood

et al.122

19

Scheme 2 The Smirnoff-Wheeler Biosynthetic Pathway for L-Ascorbic Acid in Plants (adapted from Ref. 118, 119 & 121)

ATP ADP

GTPPPi

NAD NADH

Cyt Cox

Cyt Cred

HHO

HOO

OHHO H

D-Glc D-Glc-6-P D-Fru-6-P D-Man-6-P

D-Man-1-PGDP-ManGDP-L-Gal

L-Gal

L-GL

L-Asc

L-Gal-1-P O

H

OH

HO

HO

O

OHHO

GMP

Pi

1 2 3

4

567

89

10

Catalytic Step Enzyme Substrate

1 Hexokinase (E.C. 2.7.1.1) D-Glucose

2 Phosphoglucose isomerase (E.C. 5.3.1.9)

D-Glucose-6-phosphate

3 Phosphomannose isomerase (E.C. 5.3.1.8)

D-Fructose-6-phosphate

4 Phosphomannose mutase (E.C. 5.4.2.8)

D-Mannose-6-phosphate

5 GDP-Mannose pyrophoshorylase (E.C. 2.7.7.22)

D-Mannose-1-phosphate

6 GDP-Mannose-3,5-epimerase (E.C. 5.1.3.18)

GDP-D-Mannose

7 GDP-L-Galactose pyrophosphatase

GDP-L-Galactose

8 L-Galactose-1-phosphate phosphatase

L-Galactose-1-phosphate

9 L-Galactose dehydrogenase L-Galactose

10 L-Galactono-1,4-lactone dehydrogenase (E.C. 1.3.2.3)

L-Galactono-1,4-lactone

20

The conversion of D-glucose to L-ascorbic acid in this pathway occurs without the earlier

proposed inversion of the hexose carbon skeleton, which suggested a site-specific

epimerization of the D-glucose carbon skeleton that causes the conversion from D to L

configuration. Therefore, this Smirnoff-Wheeler biosynthetic pathway for L-Ascorbic

Acid in Plants reconciles the crucial radio-labeling evidence from earlier works by

Loewus group.123-124 They partially purified a new L-galactose-dehydrogenase enzyme

from pea and Arabidopsis thaliana and established L-galactose as an effective precursor

of L-ascorbic acid in vivo. This enzyme is known to catalyze the NAD-dependent

oxidation of the C1 of L-galactose to give L-galactono-γ-lactone with a Km of 0.3 mM for

L-galactose. This same enzyme was also able to slowly oxidize L-sorbosone to L-

ascorbic acid at a very low Km value, which may perhaps explain earlier literature

reports.118 GDP-L-galactose, which is synthesized from the double epimerisation of

GDP-D-mannose, is incorporated as a minor component of certain cell wall

polysaccharides.118 The reaction is catalyzed by a poorly characterized enzyme isolated

from Chlorella pyrenoidosa and flax, which is known as GDP-D-mannose-3,5-

epimerase.118 Nonetheless, the enzyme responsible for converting GDP-L-galactose to L-

galactose remains unidentified in plants. On the other hand, it has been reported that

incubations with radio-labeled GDP-D-mannose in vitro resulted in the incorporation of

radio-labels into L-galactono-γ-lactone.118 Furthermore, additional genetic data in support

of this pathway is beginning to emerge from the characterization of the L-ascorbic-acid-

deficient Arabidopsis mutants.118 The locus of one of these mutants has recently been

shown to be D-mannose pyro-phosphorylase. Also, independent work on the anti-sense

inhibition of this enzyme in potato was reported to have produced plants with foliar L-

21

ascorbic acid levels of about 44% to 72% of wild type, and with a 30% to 50% reduction

in their leaf cell wall mannose content.118 And upon transfer to soil, these plants

expressed developmental changes leading to early senescence.118 Therefore, the

importance of this new Smirnoff-Wheeler L-Ascorbic Acid biosynthetic pathway in

plants is that it integrates L-ascorbic acid biosynthesis into the pathways for central

carbohydrate metabolism and provides connections to protein glycosylation and

polysaccharide biosynthesis.118 Nevertheless, some important questions remained

unanswered such as the earlier reports on the in vivo conversion of uronic acid derivatives

for example, D-glucuronic acid, its lactone, D-glucuronolactone, and as D-galacturonic

acid methyl ester, which are found to be converted directly to L-ascorbic acid. These

conversions are found to occur without disruption of the carbon skeleton and with slight

redistribution of the radio-labels.118 The draw-back is that there are few available data on

the enzymes catalyzing these reactions and thus researchers are still uncertain of its

significance in L-ascorbic acid biosynthesis. It is possible that L-ascorbic biosynthesis

from these compounds may only be significant under certain cellular circumstances or in

specific tissue types. However, what is known is that D-glucuronic acid and D-

galacturonic acid are major components of plant non-cellulose type cell wall

polysaccharides and their conversion to L-ascorbic acid might in part represent a

mechanism to salvage carbon fragments arising from the breakdown of the cell walls,

such as those that take place during growth, cell expansion, abscission, pollen grain

maturation, fruit ripening and softening.118

22

2.6 Commercial Scale Synthesis of L-Ascorbic acid

At present, the bulk of commercially manufactured L-ascorbic acid is synthesized

via the seven-step Reichstein process (Scheme 3), which was developed soon after the

discovery of vitamin C by Albert Von Szent-Györgyi in 1928. The current world

production of L-ascorbic acid is estimated at 80,000 tons per year with a global market in

excess of US $600 million and with an annual growth rate of 3-4%.125-126 This enormous

demand for L-ascorbic acid is driven by its various uses in manufacturing, agricultural,

health and pharmaceutical industries. For example, (1) approximately 50% of the

synthetic L-ascorbic acid is used in vitamin supplements and in pharmaceutical

preparations such as in the making of ointments for the treatment of burns; (2) There is a

rapidly growing market in cosmetic products which use L-ascorbic acid as an additive,

due to its anti-oxidant properties and its potential to stimulate collagen production;35 (3)

This antioxidant properties are also exploited in food processing and beverage

manufacturing, to protect against pigment discoloration and enzymatic browning. This

helps to preserve flavor, aroma, and enhance or protect the nutrient content;35 (4) Farmers

frequently use L-ascorbic-acid-supplemented feeds to augment its bio-availability in

livestock for optimum growth and health.35

23

Scheme 3 The Reichstein Process for L-Ascorbic Acid Manufacture (adapted from Ref. 127)

CH3COCH3

HCl CH3OH

C2H5OH

Ni2+

Pd2+

D-Glucose

D-Sorbitol

L-Sorbose

Diacetone-L-Sorbose

2-Keto-L-Gulonic Acid

2-Keto-L-Gulonic Acid Methylester

L-Ascorbic Acid

80-125 atm

140-150 oC

Fermentation

100oC

Conc. H2SO4

The Reichstein process uses D-glucose as the starting material and involves six chemical

steps and one fermentation step for the oxidation of D-sorbitol to L-sorbose with an

overall yield of 50%.125 The synthetic process is based on chemical methods and bears no

relationship to the biochemical pathway used by L-ascorbic-acid-biosynthesizing

organisms. In spite of its many years in development, the Reichstein process is still

highly energy consuming and requires high temperatures and/or pressure for many of the

steps. These and other economic factors have generated a substantial interest in the

manufacturing of the Reichstein intermediates towards the synthesis of L-ascorbic acid in

24

a more economical and efficient manner. The more recent revelation of the plant

biosynthesis pathway and genomic advancement have broadened new opportunities to

explore recent innovations in technologies such as fermentation processes, cell-free bio-

catalytic systems, biochemistry and recombinant DNA technology for a more efficient

commercial synthesis of L-ascorbic acid via the Reichstein intermediates.127 Such

methods involve the use of genetically engineered prokaryotes for the large scale

synthesis of L-ascorbic acid. The two most commercially advanced methods are the

oxidation of D-glucose to 2-keto-L-gulonate (2-KLG) via D-gluconate, 2-keto-D-

gluconate and 2,5-diketo-D-gluconate [2,5-DKG pathway, (Scheme 4)] and the oxidation

of D-sorbitol or L-sorbose to 2-keto-L-gulonate via the intermediate L-sorbosone

[sorbitol pathway, (Scheme 4)]. The first synthesis of L-ascorbic acid from a non-

carbohydrate source was successfully attempted using enantiopure cis-1,2-

dihydrocatechol. This precursor was obtained from microbial oxidation of chlorobenzene

and converted via 3,5-O-benzylidene-L-gulonolactone into L-ascorbic acid.128 While this

synthetic method may not yet be economically suitable for commercial scale synthesis of

L-ascorbic acid, however it offers a reaction sequence open to the preparation of labeled

L-ascorbic and its derivatives that could then be used in probing the in vivo functions of

L-ascorbic acid.

25

Scheme 4 Microbial-Engineered Pathway for L-Ascorbic Acid Manufacture (adapted from Ref. 127)

COOHOHHHHOOHHOHH

CH2OH

CHOOHHHHOOHHOHH

CH2OH

CH2OHOHHHHOOHHOHH

CH2OH

COOHOHHOOHHOHH

CH2OH

CH2OHOHHOOHHHHO

CH2OH

COOHOHHOOHHO

CH2OH

COOHOHHOOHHHHO

CH2OH

CHOOHHOOHHHHO

CH2OH

C=OOHOHHOHHO

CH2OH

Glucose dehydrogenase Hydrogenation

D-Gluconic acid D-Glucose D-Sorbitol

Gluconate dehydrogenase D-Sorbitol dehydrogenase

2-Keto-D-Gluconic acid L-Sorbose

2-Keto-D-gluconate dehydrogenase L-Sorbose dehydrogenase

2,5-Diketo-D-Gluconic acid 2-Keto-L-Gulonic acid L-Sorbosone

2,5-DiKeto-D-Gluconic Acid Pathway

Esterification Lactonisation

D-Sorbitol Pathway

L-Ascorbic Acid

2,5-DiKeto-D-gluconate- dehydrogenase

L-Sorbose-dehydrogenase

26

2.7 Biological Functions of L-Ascorbic Acid

There are three main types of biological activity distinctive to L-ascorbic acid in

plants and animals. These are (1) its function as an enzyme co-factor; (2) as a direct

physiological radical scavenger and finally; (3) as a donor/acceptor in electron transport

in both plasma membrane and chloroplasts.35, 116, 118

2.7.1 L-Ascorbic Acid as an Enzyme Cofactor

L-ascorbic acid is involved in the modulation of a number of important enzymatic

reactions such as in the metabolism of several amino acids which lead to the formation of

hydroxyproline, hydroxylysine, norepinephrine, serotonin, carnitine and homogenistic

acid. It has also been found to be essential for the normal functioning of the osteoblasts,

fibroblasts, adrenal hormones and carnitine biosynthesis.35, 115, 118 Carnitine is a molecule

present in the liver, heart and skeletal muscles, which is responsible for the transport of

energy-rich activated long-chain fatty acids from the cytoplasm across the inner

mitochondrial membrane to the matrix side, where they are catabolized to acetates.115, 118

Carnitine is synthesized from methionine and lysine by two hydroxylases through a series

of reactions that require ferrous iron and L-ascorbic acid for optimum activity. Therefore,

the deficiency of L-ascorbic acid is found to cause a decrease in both the rate of carnitine

biosynthesis and the efficiency of carnitine renal re-absorption, and increase in the

urinary carnitine excretion; these effects are linked to the buildup of triglycerides in

blood, physical fatigue and lassitude in scurvy patients.115, 118 A list of enzymes that

27

requires L-ascorbic acid for optimal function is shown in Table 2. These enzymes are

typically mono or di-oxygenases that contain transition metals such as iron or copper at

their active sites and require L-ascorbic acid for optimum activity.35, 115, 118 The role of L-

ascorbic acid in these enzymes is to maintain the transition metal ion centers in the

reduced form, which is required for the optimum activity of the systems.35, 115, 118 For

example, many of the symptoms of scurvy in animals, particularly those having to do

with the connective tissues defects are traced back to the biochemical role of L-ascorbic

acid as a cofactor for the two mixed-function oxidases, which are prolyl and lysyl

hydroxylase enzymes involved in the formation of both hydroxyproline and

hydroxylysine, which are two important components of collagens and the fibrous

connective tissues in animals. Collagen is the principal components of tendons,

ligaments, skin, bone, teeth, cartilage, heart valves, intervertebral disks, cornea, eye lens

and the ground substances between cells. When collagen is synthesized, proline and

lysine are post-translationally hydroxylated on the growing peptide chain.

Hydroxyproline and hydroxylysine are required for the formation of a stable extracellular

matrix and cross-links in the fiber.

28

Table 2 List of Enzymes Requiring L-Ascorbic Acid as a Cofactor or as a Modulator of Activity (adapted from Ref. 118)

Enzyme Physiological Role Enzymatic Activity Metal Ion Centre

1-Aminocyclopropane-1-

carboxylate Oxidase Ethylene (plant hormone)

biosynthesis Oxidation of 1-Aminocyclopropane to

ethylene and cyanoformic acid Iron

Cholesterol 7-alpha monooxygenase E.C. 1.14.13.17

Cholesterol catabolism; bile acid synthesis (animals) Hydroxylation of Cholesterol -

Catechol-O-methyl transferase E.C. 2.1.1.6

Adrenaline (epinephrine) inactivation (animals)

Increased levels of adrenaline (epinephrine) -

Dopamine-β- monooxygenase E.C. 1.14.17.1

Noradrenaline (norepinephrine) synthesis β-hydroxylation of dopamine Copper

Deacetoxycephalosporin C synthetase Antiobiotic metabolism (fungi) Penicillin N to deacetylcephalosporin Iron

γ-Butyrobetaine-2-oxoglutarate-4-dioxygenase E.C. 1.14.11.1 Carnitine biosynthesis Hydroxylation of butyrobetaine to

carnitine Iron

Gibberellin-3-β-dioxygenase E.C. 1.14.11.15

Gibberin (plant hormone) biosynthesis

C20 oxidation decarboxylation and activation of gibberellins Iron

4-Hydroxylphenylpyruvate dioxygenase E.C. 1.13.11.27 Tyrosine metabolism

Decarboxylation and hydroxylation of 4-hydroxyphenyl pyruvic acid to

homogenistic acid Iron

Lysine hydroxylase E.C. 1.14.11.4

Collagen biosynthesis (animals) & Extensin biosynthesis (plants) Hydroxylation of Lysine Iron

Mitochondrial glycerol-3-phosphate dehydrogenase

E.C. 1.1.99.5

NAD(P) H and ATP production; aid in insulin release Dehydrogenation of triose phosphate Iron

Peptidyl glycine α-amidating monooxygenase E.C. 1.14.17.3

Peptide amidation in peptide hormone metabolism C-terminal glycine amidation Copper

Procollagen proline 2-oxoglutarate-3-dioxygenase E.C. 1.14.11.7

Procollagen biosynthesis (animals) Extensin biosynthesis (plants)

Hydroxylation of proline (3-hydroxylating) Iron

Proline hydroxylase E.C. 1.14.11.2 Procollagen synthesis (animals) Hydroxylation of proline (4-

hydroxylating) Iron

Pyrimidine deoxynucleoside 2’-dioxygenase E.C. 1.14.11.3 Pyrimidine metabolism (fungi) Deoxyuridine to uridine Iron

Thioglucoside glucohydrolase E.C. 3.2.3.1 Catabolism of glucosinolates (plants) Hydrolysis of S-glucosides -

Thymine dioxygenase E.C. 1.14.11.6 Pyrimidine metabolism (fungi) 7-Hydroxylation of thymine Iron

Trimethyllysine 2-oxoglutarate dioxygenase E.C. 1.14.11.8 Carnitine biosynthesis Hydroxylation of trimethyl lysine Iron

Violaxanthin de-epoxidase Zeaxanthin biosynthesis and the xanthophylls cycle (plants)

De-epoxidation of violaxanthin and antheroxanthin -

29

L-ascorbic acid acts a physiological electron donor to the ferric and cupric ions at the

metal centers of these enzymes, thus reducing them to their activated reduced states,

which is essential for the reactions to proceed. Some collagens that are biosynthesized in

the absence of L-ascorbic acid, such as what occurs in scurvy, are known to form

abnormal fibers, resulting in skin lesions, blood-vessel fragility, etc.35, 115,118 In plants, the

direct involvement of L-ascorbic acid in the biosynthesis of plant hydroxyproline-rich

proteins118 has implications for cell expansion and cell division.118 High levels of

hydroxyproline-rich glycoproteins such as the extensins found in the cell wall118 are

developmentally regulated and are involved in the cross-linking of the cell wall in

response to injury. Also, extensin genes are induced in response to wounding and

pathogenic attacks.118 Furthermore, in the biosynthesis of a variety of neurotransmitters

and hormones35, 115, 118 in animals, L-ascorbic acid is an important factor in many of the

hydroxylation and decarboxylation processes involved in these metabolic pathways. For

example, L-ascorbic acid is important for the initial hydroxylation step in the synthesis of

serotonin, a neurotransmitter and vasoconstrictor, which is catalyzed by tryptophan

hydroxylase. This step involves the hydroxylation and decarboxylation of tryptophan and

L-ascorbic acid is able to convert dihydrobiopterin (oxidized form) to tetrahydrobiopterin

(reduced form), which is the co-substrate for this hydroxylase enzyme.115, 118 Animals

with deficiency in L-ascorbic acid are unable to catabolize tyrosine to fumaric and

acetoacetic acid via homogenistic acid.115, 118 Also, tyrosine is metabolized in the

presence of L-ascorbic acid to catecholamines by hydroxylation and decarboxylation to

produce dopamine, norepinephrine, epinephrine, and adrenocrome. L-ascorbic acid is

30

directly involved as an electron donor to dopamine-β-monooxygenase (DβM) reaction for

the conversion of dopamine to norepinephrine (Scheme 5).

Scheme 5 L-Ascorbic Acid (Exogenous Electron Donor) in DβM Enzymatic Reaction

OHHO

NH2

OHHO

NH2

HO H2H + 2e-

DßM-E CUII2 + 2ASC DßM-E CUI

2 + 2Semidehydro-ASC

2Semidehydro-ASC ASC + Dehydro-ASC

Dopamine R-NorepinephrineH2OO2

Catecholamine biosynthesis occurs in the adrenal glands and brain, both with relatively

large amounts of L-ascorbic acid. L-ascorbic acid also protects catecholamines by direct

chemical interactions and elimination of adrenochrome, a toxic product of catecholamine

oxidation, which has been linked to certain mental diseases.115 There are complex

interactions among catecholamines and their receptors with L-ascorbic acid to protect

them from oxidative damage. Other enzymatic systems responsible for neurotransmitter

and hormone synthesis, and dependent on the presence of oxygen and L-ascorbic acid,

are the copper-containing peptidyl glycine amidating monooxygenases, which are found

in the skin, atrium, adrenal and pituitary glands.35, 115-116, 118 The microsomal enzymatic

system containing cytochrome P450-hydroxylases requires L-ascorbic acid for the

hydroxylation reaction involved in the stepwise conversion of cholesterol to bile acid via

31

7α-hydroxycholesterol.35, 115, 118 In L-ascorbic acid deficient animals including humans,

impaired cholesterol transformation to bile acids leads to cholesterol accumulation in the

blood and liver, atherosclerotic changes in coronary arteries, and formation of cholesterol

gallstones. Therefore, administration of L-ascorbic acid helps to lower the plasma

chlolesterol concentration. L-ascorbic acid is also essential for the oxidation and

decarboxylation of fatty acids in lipid metabolism. Animals with L-ascorbic acid-

deficiency exhibit high levels of plasma triglycerides with a decrease in post-heparin

plasma lipolytic activity and the half-life of plasma triglycerides increases, thereby

causing triglyceride accumulation in the liver and arteries.35, 115, 118

2.7.2 L-Ascorbic Acid in Electron Transport

The biochemical and physiological functions of L-ascorbic acid primarily depend

on its reducing properties and its role as an electron carrier.35, 115 L-ascorbic acid and its

single-electron oxidized product, semidehydro-L-ascorbate functions as a cycling redox

couple in various electron transport reactions and changes the activities of cytochromes,

the electron membrane-protein carriers. Several ascorbate oxidoreductases have been

identified and are involved in the electron transport reactions with a cytochrome b

protein.35, 115 For instance, L-ascorbic acid is known as a major electron donor for a trans-

membrane oxidoreductase of human erythrocytes.35, 115 Cytochrome b561, an electron

channel membrane-protein found in secretory and synaptic vesicles, catalyzes the trans-

membrane electron transport.

32

Figure 2 Cytochrome b561 in Trans-Membrane Electron Transport

Adapted from Stewart & Klinman, Annu. Rev. Biochem., 1988, Vol. 57, 551-592

The transported electrons mediate equilibration of the L-ascorbate/semidehydro-L-

ascorbate redox couple inside the secretory vesicles with those present in the cytoplasm.

The role of cytochrome b561 (Figure 2) is to regenerate L-ascorbic acid inside the vesicle

for use by intravesicular monooxygenases such as dopamine β-monooxygenase and

peptidylglycine α-amidating mooxygenase.35, 115, 118 The cytochrome is reduced by a

single reducing equivalent donated by L-ascorbic acid in the cytosol and is oxidized by

semidehydro-L-ascorbate in the granule matrix, thereby maintaining a redox equilibrium

33

between cytoplasmic and intravesicular pools of ascorbate and semidehydro-L-

ascorbate.35,115-116,118

2.7.3 L-Ascorbic Acid as an Antioxidant in Biological Systems

Since oxygen is required for cell viability in both plant and animal systems, it is

essential that a mechanism be available to control the reactive oxygen species (ROS)

generated during cellular metabolism and from exogenous sources and environmental

chemicals. L-ascorbic acid interacts enzymatically and non-enzymatically with ROS and

their derivatives to neutralize their cellular damaging effects. Radical reactions are

initiated by ROS mainly produced as side products from the mitochondria in animals and

choloroplast in plants, where cellular energy is produced by the reaction of an oxygen

molecule with 4 electrons and 4 protons resulting in the formation of water (Scheme 6).

These ROS such as superoxide and especially hydrogen peroxide undergo the so called

Fenton reaction in the presence of transition metal ions, especially Fe (II) to produce the

hydroxyl radical, an extremely reactive radical.

Scheme 6 Four-Electron Reduction Process of Oxygen to Water

O O O OO O

Oe

O2 O2 H2O2 2H2O

+2H+

eH H

+2H+

2e HH

O HH

-

34

The hydroxyl radicals (HO.) undergo facile radical reactions with susceptible cellular

components such as proteins, DNA, lipids and membrane lipids.129 For example,

membrane lipids possess allylic hydrocarbon chains that can undergo facile reactions

with hydroxyl radicals.129 The resulting carbon-centered radicals react with oxygen

rapidly at a diffusion-controlled rate to form alkyl peroxy radicals (LOO.). This alkyl

peroxy radicals abstract a hydrogen atom from lipids to generate LOOH.129 LOOH has a

sufficient life-time to migrate and finally generate reactive radicals by reacting with metal

ions to damage other cellular components in addition to the membrane. Therefore LOOH

is capable of causing extensive tissue damage that may lead to cell death due to its radical

effect called oxidative stress. L-ascorbic acid and glutathione, another water-soluble

reducing agent, function together as antioxidants against oxidative stress and free radical

damage in the body (Figure 3).129 Although, L-ascorbic acid cannot scavenge lipophilic

radicals directly within the lipid compartment, it acts as a synergist with tocopherol for

the reduction of lipid peroxide radicals. At the lipid-aqueous interphase, L-ascorbic acid

interacts with the membrane-bound oxidized tocopherol radical to regenerate active

reduced tocopherol for continued antioxidant functions.129 The biological importance of

the antioxidant behavior of L-ascorbic acid is unlike other low-molecular-weight

antioxidants (uric acid, carotenoids, flavonoids, α-tocopherol, etc.), in that it terminates

the radical chain reactions and itself is transformed into non-toxic oxidized products, i.e.,

semidehydro-L-ascorbic acid radical and dehydro-L-ascorbic acid. Semidehydro-L-

ascorbic acid radical, disproportinates back to L-ascorbic acid and dehydro-L-ascorbic

acid (Figure 4).

35

Figure 3 Chemical Illustration of Radical Reactions in the Cell and Antioxidant Activities (adapted from Ref. 129)

NADP+ NADPH

ASC

TOC

SOD

Glucose Oxidation

GSSG Reductase

GSSG GSH

LOH LOOH

ASC.

or DHASCGPX

Membrane

TOC.

LOOH LOO. L

. + O2

Initiation

LH + X.

Metal Ions

Nucleic Acids Proteins

Aldehydes Lipid Peroxidation

H2O+ O2Catalase, GSH PX

H2O2 O2-

Fe2+

HO.

Radical species in the cell (designated as X.) initiate radical chain reactions leading to oxidative stress. Thus, cell

antioxidants (e.g. L-ascorbic acid, Asc.), antioxidant enzymes and glucose supplies reducing power to fight oxidative stress (adapted from Ref. 129).

36

Figure 4 L-Ascorbic Acid Redox System

O O

OHO

OH

H

HO

O O

OO

OH

H

HO

O O

OO

OH

H

HO

L-ascorbate (Asc) Semidehydro-L-ascorbic acid(SDA) or L-ascorbyl free radical

Dehyro-L-ascorbic acid (DA)

- H+, - e-

- e-

+ H+, + 2e-

+H+, + e-

+e-

-H+, -2e-

The non-enzymatic antioxidant activity of L-ascorbic acid provides reducing equivalents

to a wide range of biological substrates to maintain their reduced and active forms. For

example, L-ascorbic acid maintain the reduced form of folic acid which is needed in the

many one-carbon transfer reactions, which are involved in the formation of a wide variety

of biologically important bio-molecules.129

2.8 L-Ascorbic Acid Metabolic Enzymes

L-ascorbic acid is directly oxidized by two enzymes, ascorbate peroxidase and

ascorbate oxidase. Ascorbate peroxidase is a hydrogen-peroxide-scavenging enzyme that

functions to protect cells from hydrogen peroxide accumulation under normal and

stressful conditions present in plants.35, 115-116,118 This enzyme is found both as membrane-

37

bound and soluble forms. In chloroplasts, it catalyzes the reduction of hydrogen peroxide,

as an electron donor, to yield water and semidehydro-L-ascorbate radical as the primary

product. Ascorbate oxidase is a member of the class of blue multicopper oxidases and

catalyzes the oxidation of L-ascorbic acid to dehydro-L-ascorbic acid with the conversion

of O2 to H2O2. This enzyme is associated with the rapidly growing regions in plants and

has been found as protein bound to the cell wall and as soluble protein in the cytosol.35,

115-116,118 Semidehydro-L-ascorbate radical and dehydro-L-ascorbate, which are the two

oxidized forms of L-ascorbic acid are respectively reduced by semidehydro-L-ascorbate

reductase and dehydro-L-ascorbate reductase. Semidehydro-L-ascorbate reductase

catalyzes the regeneration of L-ascorbic acid from semidehydro-L-ascorbic acid radical

using nicotinamide–adenine dinucleotide phosphate (NADPH) as the electron donor. This

enzyme scavenges toxic reactive oxygen species in plant tissues.35, 115-116,118 Dehydro-L-

ascorbate reductase functions as a reducing agent for the regeneration of L-ascorbic acid

from dehydro-L-ascorbic acid. It has been isolated from various plant and animal tissues.

Its ability to recycle L-ascorbic acid depends on the relative activity level of the enzymes

and concentration of glutathione.35, 115-116,118

2.9 Degradation and Oxidation of L-Ascorbic Acid

L-ascorbic acid is metabolized in the liver, and to some extent in the kidneys. The

principal pathway of L-ascorbic acid metabolism involves the direct loss of two electrons

which produces dehydro-L-ascorbic acid.35, 115 The loss of one electron produces

semidehydro-L-ascorbic acid radical, which can also undergo oxidation to reversibly

38

produce dehydro-L-ascorbic acid. Dehydro-L-ascorbic acid can irreversibly react with

water to produce physiologically inactive 2,3-diketogulonic acid product. 2,3-

diketogulonic acid is either cleaved to oxalic acid and threonic acid, or undergoes

decarboxylation to produce carbon dioxide, xylose, and xylulose and eventually leads to

the formation of L-xylonic acid and L-lyxonic acid. All these metabolites and L-ascorbic

acid are excreted in the urine. The amount of each metabolite varies from species to

species according to the amounts of L-ascorbic acid ingested. Some other metabolites

besides those already mentioned above, such as 2-O-sulfate L-ascorbic acid and 2-O-

methyl L-ascorbic acid have also been found in humans and rats.35, 115 A new metabolite,

2-O-β-glucuronide-L-ascorbic acid was recently identified in human urine and plasma.35,

115 The rate of chemical degradation of L-ascorbic acid depends on several factors among

which are temperature, oxygen level, light, transition metals (e.g. copper & iron), and pH

(most stable at pH 4-6).35, 115 L-ascorbic acid undergoes slow two-electron autooxidation

to dehydro-L-ascorbic acid depending on these factors mentioned above. In acidic

solutions, degradation of L-ascorbic acid metabolites proceeds to form L-(+)-tartaric acid,

2-furfuraldehyde, 3-hydroxy-2-pyrone, and other furan derivatives, as well as some

condensation products.35, 115 Dehydro-L-ascorbic acid nonenzymatically reacts with

several amino acids to form brown-colored products, a reaction known to contribute to

food spoilage.35, 115 L-Ascorbic acid content in foods significantly decreases during

storage and the degradation process is greatly enhanced during cooking.

39

Figure 5 Degradation of L-Ascorbic Acid (adapted from Ref. 35)

OHO OH

H

OHHO

O OHO OH

H

OO

O

OHHO OH

H

OO

O

HO

H2O

H2O CO2CO2

Anaerobic Aerobic

Delactonization

L-Ascorbic Acid Dehydro-L-Ascorbic Acid

XylosoneDeoxypentose

Furfural

Reductones± Amino Acid

Brown Pigments

2,3-Diketogulonic Acid

-2H+

+2H+

Thermal decomposition of L-ascorbic acid is extensively studied because of its

importance in food and beverage industries. Kurata and Sakurai131, 132 both studied the

degradation process in acidic medium, which was found to progess through

decarboxylation after the lactone ring opening and subsequent cyclization to give furfural

(Figure 5). Thermal degradation of L-ascorbic acid and dehydro-L-ascorbic acid also

resulted in the formation of some furan derivatives.130, 133 A group of volatile furan-type

compounds and reductones are detected by gas chromatography.115 Some of the browning

40

metabolite products have antioxidant activity, 35, 115 while others have destructive

prooxidant effects, which include cytotoxicity, lipid peroxidation, mutagenesis, the

adduct formation with proteins and nucleic acids.35, 115

2.10 Cellular Transport and Intestinal Absorption of L-Ascorbic Acid

L-ascorbic acid accumulates in human tissues as much as 50-fold compared to the

plasma.134 L-ascorbic acid and its oxidized metabolite, dehydro-L-ascorbic acid are both

transported and accumulated distinctly and neither competes with the other. L-ascorbic

acid is transported by sodium-dependent carrier-mediated active transport. Dehydro-L-

ascorbic acid transport and accumulation is at least 10-fold faster than L-ascorbic acid

transport and is sodium-independent and biologically separable from its reduction to L-

ascorbic acid. Studies have shown that dehydro-L-ascorbic acid, and not L-ascorbic acid,

is preferentially transported intracellularly.35, 115 Once transported, dehydro-L-ascorbic

acid is immediately reduced in the intracellular compartments to L-ascorbic acid. A

number of detailed experimental criteria have been used to distinguish the two systems,

which established that L-ascorbic acid and dehydro-L-ascorbic acid are transported into

various cells such as the human neutrophils and fibroblasts by two distinct mechanisms.

This has also established L-ascorbic acid as preferentially available for intracellular

utilization. Glucose Transporters I-V have been well characterized and are neither

sodium-dependent nor have sites indicative of sodium dependency. Such sodium

dependency is required for L-ascorbic acid but not for dehydro-L-ascorbic acid transport

and accumulation. The activity of sodium-dependent uptake of radio-labeled L-ascorbic

41

acid is reported to be inhibited by excess unlabeled L-ascorbic acid and not glucose, thus

implying that the putative L-ascorbic acid transporter is mediated by a different protein

than that responsible for Glucose.115, 135 Intestinal absorption of L-ascorbic acid is

achieved by this sodium-dependent transport system.115, 135 Its transport into the ileum is

a carrier-mediated process at low mucosal concentrations of L-ascorbic acid. However, at

high mucosal concentrations, the influx of L-ascorbic acid into the ileum is linearly

dependent on its concentration and absorption occurs predominantly by simple

diffusion.115, 135 Its gastro-intestinal absorption is inversely dependent on its dosage.115, 135

The amount of L-ascorbic acid absorbed decrease with the age of a person and L-ascorbic

acid in large doses can cause intestinal discomfort and osmosis diarrhea.115, 135

2.11 Molecular Structure of L-Ascorbic Acid

One of the most difficult tasks facing earliest organic chemists was to work out a

detailed structure of a compound from studying its reaction with a variety of known

materials and then sequentially fitting different possible models to account for the

chemistry. Therefore, it came as no surprise that L-ascorbic acid synthesis was first

accomplished long before the correct structure was determined. Micheel and Kraft

suggested that 2-(4,5-dihydo-3,4-dihydroxy-5-hydroxymethyl) furanyl-carboxylic acid as

the constitutional formula of ascorbic acid, in 1933, after analyzing the chemical

properties of the compound. This structure was later rejected after failing to account for

the characteristic chemical and physical properties of ascorbic acid. One such property

42

was the mild oxidations of ascorbic acid which resulted in loss of its acidic properties.136,

137

On the contrary, the structure constituting the C2,C3-enediol lactone moiety

suggested by Hirst received a worldwide recognition. In the same year, a detailed

synthetic report was published, considering D-xylosone has a furanose structure, with the

initial reaction involving the substitution of its hydroxyl group at the anomeric carbon by

cyanide to give 2-oxo-1-cyano-1-deoxy-xylofuranose as the primary product. And

successively upon its acid hydrolysis, the author suggested that the compound would lead

to give the final product as D-ascorbic acid, having the structure similar to that earlier

proposed by Micheel and Kraft. The physical and chemical properties of this final

product were found to be very similar to the natural L-ascorbic acid, except for the

differences in their specific optical rotations. Consequently, these findings were

considered as an excellent model for the structure of L-ascorbic acid, and it helped to

guide Haworth and coworkers using the same reaction sequence starting with L-xylosone

to obtain L-ascorbic acid.106, 110 As a result of this brilliant work, they were able to isolate

and characterize 1-imino-L-ascorbic acid as the final product, which upon hydrolysis

eventually produced L-ascorbic acid as the final product. As a result of this outstanding

work, they were able to give the precise structure of L-ascorbic acid that is used today.

The designation of the compound was changed from 2-oxo-L-threo-hexano-1,4-lactone-

2,3-enediol or vitamin C to L-ascorbic acid in 1965 by the IUPAC-IUB Commission on

Biochemical Nomenclature. The stereo-chemical assignments of ascorbic acid to the L-

configuration was first established by the synthesis from L-xylose and later confirmed by

X-ray crystallography and neutron diffraction analysis.138-140

43

The L-ascorbic acid molecule has a molecular formula of C6H6O6 and includes two

asymmetric carbon atoms, C4 and C5. Therefore, in addition to L-ascorbic acid itself,

there are three other stereoisomers: L-isoascorbic acid, D-isoascorbic acid, and D-

ascorbic acid. Of the four possible stereoisomeric forms of ascorbic acid, only the form

identical to the natural vitamin C, that is the (+)-ascorbic acid (L-ascorbic acid), has the

same anti-ascorbutic activity.141 However, all of the diastereomers show the same strong

antioxidant properties. There is often the misconception that (+)-Ascorbic acid and (-)-

isoascorbic acid, often labeled as L-ascorbic acid and D-erythrorbic acid, respectively,

are enantiomers. These are not enantiomers, but rather are diastereomers as the structures

are not mirror images. While L-ascorbic acid is utilized as a bioactive vitamin C nutrient,

both L-ascorbic acid and D-erythorbic acid are commercially important as antioxidant

preservatives, for instance in protecting the flavor profile of citrus soft drinks such as

orange soda. D-erythorbic acid exhibits only 5% of the anti-ascorbutic activity compared

to L-ascorbic acid.141 The structure of L-ascorbic acid and its three stereosiomers are

shown in Figure 6. L-ascorbic acid is a dibasic acid with a C2,C3-enediol moiety built

into a five-membered heterocyclic lactone ring. The ring is almost planar with a slight

distortion of the lactone oxygen atoms out of the enediol plane. The molecule is

stabilized by delocalization of the π electrons of the conjugated carbonyl with the enediol

system. The chemical and physical properties of L-ascorbic acid are directly related to its

structure. Dehydro-L-ascorbic acid, the first stable oxidation product of L-ascorbic acid is

also present in biological tissues and retains physiological or vitamin C activity.

44

Figure 6 L-Ascorbic Acid and its Diastereomers.

O O

OHHO

HO

H

HOH

O O

OHHO

HO

H

HOHO O

OHHO

HO

H

HOH

O O

OHHO

HO

H

HOH(S) (R)

(S)

(R) (S)

(R)(S)

(R)

L-Ascorbic acid D-Ascorbic acid

D-Arabo-ascorbic acid L-Arabo-ascorbic acid

= D-Erythorbic acid = L-Erythorbic acid

The structure of dehyro-L-ascorbic acid was postulated as C2,C3-diketo-lactone, which

has a side chain that forms a hydrated hemiketal (Figure 7, a). X-ray crystallography

analysis was used to determine dehydro-L-ascorbic acid as a dimer (Figure 7, b). Nuclear

magnetic resonance (NMR) studies have also indicated that dehyro-L-ascorbic acid in

aqueous solution exists as a bicyclic hydrated monomer. Electrochemical studies have

indicated that L-ascorbic acid and dehydro-L-ascorbic acid form a reversible redox

couple. 35,115

45

Figure 7 Structural Forms of Dehydro-L-Ascorbic Acid

O

O

O

OHOH

H

OH

HO

O

O

O

OH

H

O

HO

O

O OH

O

H

HO

O

(a) Dehydro-L-ascorbic acid hydrated Monomer

(b) Dehydro-L-ascorbic acid hydrated Dimer (crystal from)

2.12 Chemical and Physical Properties of L-Ascorbic Acid

The role of L-ascorbic acid in biological systems stems from its basic functional

structure. It is a five-membered lactone sugar acid and its C3 and C2 enolic hydroxyl

groups can dissociate to form a dibasic acid. The 2,3-enediol moiety of L-ascorbic acid

conjugated with its C1 carbonyl group, makes the proton on the C3 hydroxyl group

significantly acidic (pK1 = 4.25: comparable to acetic acid with pKa = 4.8) in comparison

to the proton on C2 hydroxyl group (pK2 = 11.79). These two acidic protons are the

reason for the acidic properties of the molecule. The 2,3-enediol moiety enables L-

ascorbic acid to donate one or two electrons (reducing equivalents) and form somewhat

stable oxidized intermediate (semidehydro-L-ascorbic acid) and the final oxidized

product (dehydro-L-ascorbic acid), a property from which, most if not all the chemical

and biological functions of L-ascorbic acid are derived. The two hydroxyl groups at C5

and C6 are normal alcohol groups and thus react with aldehydes and ketones to give

cyclic acetals and ketals, respectively. L-Ascorbic acid registers a positive value for

optical rotation due to the two asymmetric centers at C4 and C5. The optical rotation is

46

not significantly affected by the acidity of the solution, but in contrast, it varies greatly

with alkalinity, increasing over +160o in 2N NaOH solution.142 A number of physical

properties of L-ascorbic acid are listed in Table 3.

2.13 Synthetic Derivatives and Analogues of L-Ascorbic Acid

It is more convenient to correlate the literature with respect to the reactivity of L-

ascorbic acid with acylating and alkylating reagents under basic and acidic conditions, its

ketal and acetal derivatization, and lastly with respect to the asymmetric chemistry of its

oxidative cleavage and/or reduction towards producing chiral synthons. L-ascorbic acid

has several reactive positions that are open to derivatization towards producing a number

of compounds with interesting chemical and physical properties. There are many

substituted derivatives at the C2, C3, C5 and C6 positions of L-ascorbic acid reported in

the literature.35, 115 Electrophilic attack on L-ascorbic acid by acylating and alkylating

reagents depends on the acidity (pKa) and steric constraints of the four hydroxyl groups

at C2, C3, C5, and C6 of the molecule. The first ionization takes place at the most acidic

proton which is the enolic C3-hydrogen (pK1 = 4.25). However, the delocalization of the

negative charge in the monoascorbate anion causes susceptibility to alkylation at both the

C3-O and the C2 positions.

47

Table 3 Physical Properties of L-Ascorbic Acid (adapted from Ref. 211 & 212)

Property Comments

Appearance White, odorless, crystalline solid with sharp acidic state

Formula / Molar mass C6H8O6 / 176.13 g/mol

Melting point 190-192oC

Density 1.65 g/cm3

pH ~3 (5 mg/ml); ~2 (50mg/ml)

pK1 4.17

pK2 11.57

Redox potential First stage: E1O + 0.166 V (pH 4)

Spectral properties

UV pH 2:

Emax (1%, 1cm), 695 at 245nm (undissociated form)

Spectral properties

UV pH 6.4: Emax (1%, 1cm), 940 at 265nm

(monodissociated form)

Optical rotation [α]D at 25oC = +20.5o to +21.5o (C = 1 in

water)

[α]D at 23oC = +48o (C = 1 in methanol)

Solubility (g/ml)

Water 0.33

95% Ethanol 0.033

Propylene glycol 0.05

Glycerol (USP) 0.01

Fats and oil solvents: ether, chloroform, benzene, petroleum

ether, etc.

insoluble

48

The ambident (a chemical compound with two alternative and strongly interacting

distinguishable reactive centers, to either of which a bond may be made in a reaction with

the centers: the centers must be connected in such a way that reaction at either site stops

or greatly retards subsequent attack at the second site) characteristic of the C3-O-

monoanion to display nucleophilicity at both the C3-O and C2 was first reported by

Jackson and Jones,164 who were the first to report alkylation of sodium ascorbate with

benzyl chloride to afford a mixture of C3-O and C2 benzylated products. Likewise, Poss

and Belter obtained C2 allylated derivatives by treating potassium ascorbate with various

allylic bromides in acetone as solvent.165 Also, dealkylation of 2-O-(E)-cinnamoyl-5,6-O-

isopropylidene-3-O-methyl-L-ascorbic acid with lithium iodide and iodomethane in DME

afforded the C2-alkylated isomer, where the C2 acted as a sink for the equilibrating

mixture of dealkylated C3-O- and the C2.166 Furthermore, reaction of ascorbic acid with

various Michael acceptors with α, β-unsaturated carbonyl compounds undergoing

conjugated addition and 1,4-dialdehydes and 4-keto aldehydes giving aldol-derived

products have all been extensively studied as means of generating C2-alkylated

analogues.145-148 Therefore, reactions of C5- and C6-OH protected ascorbic acid with

electrophilic reagents under mild basic conditions exclusively takes place at the C3-OH

due to its high nucleophilicity.35 However, the C2-O-alkylated products could only be

obtained after the protection of the C3-OH group with protecting groups (Figure 8) such

as acetyl, MOM, benzyl, etc. On the other hand, in a strong basic conditions (pKa ≥ 12),

alkylation of the di-anion of L-ascorbic acid occurs preferentially at the less stable C2-O

position (pK2 11.79), allowing the direct and selective functionalization of this position

among the other three hydroxyl groups. Under highly basic conditions, ionization of the

49

C4-hydrogen of L-ascorbic acid occurs to produce tri-anionic form of L-ascorbic acid. If

a leaving group resides at the C5 position of L-ascorbic acid, elimination can occur via

the ionization of the C4-hydrogen to produce a 4,5-dehydro-L-ascorbic acid derivative.

This has been observed in the case of 2,3,5,6-tetra-O-methyl-L-ascorbic acid, which on

treatment with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) or potassium hydride afforded

the 5-deoxy-4,5-dehydro-2,3-di-O-methyl-L-ascorbic acid as a mixture of olefinic

isomers.143

Figure 8 Regioselective O-Alkylation of Ascorbic Acid

R1O

OOO O

OHH

H3C CH3

BnO

OHHO

O O

OH

H

HO

OHHOO O

OHH

OOO O

OH

H

H3C CH3

OR1

BnO

OHHOO O

OR2

H

R1O

OHHOO O

OHH

HO

OHHOO O

OR2

H

1. Acetone,AcCl, r.t. 4 h

2. R1X, K2CO3, DMSO/THF, 6 h

minor major

+

conc. HCl,THF, r.t., 5 h

BnBr, KHCO3,DMSO, 60 oC, 12 h

R2X, K2CO3,DMSO/THF

H2-Pd/CAcOEt, r.t. 6 h

1. DEAD, Ph3P,THF/DMF, -78 oC 15 min. 2. BnOH, r.t., 2 h

or

Adapted from Ref. 35,144,154-156,164-165, 217

In general, the selective C2-O and C3-O alkylation is difficult to achieve and requires

protection of the C5-O and C6-O positions prior to alkylation in order to minimize their

interference during reaction.217 However, Beifuss et al.144 has reported an efficient and

regioselective approach to C2-O and /or C3-O alkylation without the C5- and C6-OH

protection. Preferential and direct alkylation of C3-OH of the C5- and C6-OH

50

unprotected ascorbic acid under the Mitsunobu conditions has been recently reported.154-

156 Acid catalyzed esterification of L-ascorbic acid with an acylating reagent initially

produces the C6-O-acylated derivative, and under a more vigorous conditions, eventually

gives the 5,6-diester derivatives.35 Also, some C2-O-esters and C2 inorganic esters such

as C2-O-phosphate and C2-O-sulfate have been synthesized.35 A detailed characterization

of C2-O and C3-O-acetyl esters of 5,6-O-isopropylidine-L-ascorbic acid has been

previously reported.163 Activated (α, β-unsaturated) aldehydes and ketone reagents have

been used in a Michael addition reaction with L-ascorbic to protect the C2 and C3

positions.145-148 This process permits the selective modification of the primary and

secondary alcohol groups on the products to produce a number of side-chain-oxidized

derivatives.

The syntheses of 5,6-O-ketal or 5,6-O-acetal derivatives of L-ascorbic acid

under acidic catalyzed conditions helps to maximize its solubility in organic solvent and

also limit the interference of the 5,6-O-protected group in reactions involving the C2- and

C3-OH. For example, 5,6-O-isopropylidene and 5,6-O-benzylidene, which are ketal and

acetal derivatives respectively are well-known and extensively used in various organic

syntheses of L-ascorbic derivatives and analogues.217 In basic conditions, if both the C2-

and C3-OH groups are protected, base-promoted alkylation or acylation takes place at the

more sterically accessible primary hydroxyl group on C6 rather than C5 position.

Therefore, reactions at the C5 position occur only after derivatization of C2, C3, and C6

are completed. Since L-ascorbic acid possesses several chiral and pro-chiral centers,

significant attention has been focused on the chemistry of this important molecules and

its application in various asymmetric synthesis to obtain commercially unavailable and

51

highly functionalized chiral synthons (Figure 9). Some examples include a group of

bicyclic alklidene-dimethyloxy butenolides, glycerol acetonides, threitols, erythritol and a

series of hydroxyl-lactones, all synthesized using L-ascorbic acid as a starting material.36-

53 Compounds with quite similar structural features to L-ascorbic acid have been

synthesized. For example, an analogue such as cyclopentenone has been synthesized. In

this molecule, the C1-oxygen of the lactone ring is replaced with a carbon atom.149 Also,

the total synthesis of 2-deoxy-L-ascorbic acid from methyl-3,4-O-isopropylidene-L-

threonate has been reported.150, 151 Nitrogen analogues of L-ascorbic acid such as 2-

amino-2-deoxy- and 2,3-diamino-2,3-deoxy-L-ascorbic acid are also known. Also, 6-

halo-6-deoxy such as 6-fluoro-, 6-bromo-, 6-chloro- and 6-iodo-L-ascorbic analogues

have been synthesized.152 The crystal structure of erythroascorbic acid has been reported.

This molecule contains a side chain with a methoxy group instead of an ethoxy group.153

52

Figure 9 Potential of L-Ascorbic Acid as a Chiral Synthon

HO

OHHOO O

OHH

HO

OOO O

OH

CH3H3C

H

HO

OOO O

OH

CH3H3C

H

O

OOOH

CH3H3C

H H

O

OOOH

CH3H3C

H OH

OOOH

CH3H3C

H OH

HO

OOO O

OH

CH3H3C

H

O

OOO O

OH

CH3H3C

H

HO

OOO O

O

CH3H3C

H

53

CHAPTER 3

RESEARCH OBJECTIVE

The acetyl functional group has been commonly used as a C3-O protecting

group during the alkylation of the C2-OH of L-ascorbic acid. However, the high

instability of the C3-O-acetyl derivatives and facile migration of acyl groups from C3-O

to C2-O even under mild reaction conditions157-160 have led us161 and others162-163 to the

misidentification and characterization of C2-O and C3-O substituted L-ascorbic acid

derivatives. Although, the products of these reactions were once characterized as C3-O-

acetyl-C2-O-alkyl derivatives, in most cases they were C2-O-acetyl-C3-O-alkyl

derivatives which were predominantly formed due to the fast acetyl migration under

typical reaction conditions.161 In order to resolve the discrepancies of the structure

assignments of C2-O and C3-O substituted ascorbate derivatives, we sought to develop a

specific and a direct method to alkylate the C2-O position of 5,6-O-isopropylidine-L-

ascorbic acid, which to our knowledge had not been reported in the literatures. We used

density functional theory (B3LYP) calculations to determine the electron density

distributions and reactivities of the neutral, monoanion and dianion of L-ascorbic acid

and found that electrophilic reactions with the monoanion and dianion of L-ascorbic acid

should preferentially occur at the C3-O and C2-O positions, respectively. Based on these

findings, we have devised a novel and general method for the direct alkylation of C2-O of

5,6-O-isopropylidene-L-ascorbic acid in good yields with complete regio- and chemo-

selectivity with both activated and inactivated electrophiles. We have also carried out a

complete spectroscopic analysis of two complementary series of C2-O-acetyl-C3-O-alkyl

54

and C2-O-alkyl-C3-O-acetyl ascorbic acid derivatives in order to clearly define the

spectroscopic characteristics of these derivatives for future studies.

Our previous study161 showed that C2-O- and C3-O-allyl derivatives of 5,6-O-

isopropylidene-L-ascorbic acid, which are cyclic enol ethers, undergo facile thermal

Claisen rearrangement providing an excellent, convenient and stereo controlled access to

non-accessible C2- and C3-substituted L-galactono-γ-lactones. As a result, a direct and

practical route to the synthesis of unknown C2- and C3- substituted gulono-1,4-lactone

derivatives will be very valuable in the structure-activity studies of the various galactono-

γ-lactone-derived pharmacological agents for the improvement of their

pharamacokinetics and thus their therapeutic values. Consequently, we extended our

previous findings161 that thermal Claisen rearrangement of C2-O- and C3-O-allyl L-

ascorbic acid derivatives easily provides a convenient entry to non-accessible C2- and

C3-substituted L-galactono-γ-lactones. More importantly, we show that the Claisen

rearranged products could be stereoselectively reduced to produce a new series of C2-

substituted L-gulono-γ-lactone derivatives, which are synthetically more demanding and

could be used as chiral intermediates in the synthesis of a range of important natural

products and pharmacologically active materials.54-81

55

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Chemo- and Regio-Selective Alkylation of L-Ascorbic Acid

The alkylation and acylation of L-ascorbic acid under basic conditions is a

function of the acidity (pKa) and steric environments of the four hydroxyl groups at the

C2, C3, C5 and C6 positions. These four hydroxyl groups show different reactivity

toward electrophiles under basic reaction conditions. The presence of four hydroxyl

functional groups makes L-ascorbic acid very hydrophilic and insoluble in organic

solvents. Therefore, it is difficult to use L-ascorbic acid as a starting material for organic

synthesis. The most synthetically useful and well-studied class of modified L-ascorbic

acid is the 5, 6-O-isoproylidene-L-ascorbic acid derivatives (ketal of L-ascorbic acid).

These derivatives (5,6-O-ketal & 5,6-O-acetal) are significant in organic synthesis for

protection of the 5,6-hydroxyl functions, which makes them more soluble in organic

solvents and also limits the interference of the protected hydroxyl group from reactions

involving the C2- and C3-enol hydroxyls. Consequently, all our syntheses began with

5,6-O-isopropylidene-L-ascorbic acid (1) as the starting material, which is cheaply and

easily made from L-ascorbic and low-grade acetone (contaning small H2O%) under acid-

catalyzed conditions with excellent yield.

56

4.1.1 3-O-Alkylation of 5,6-O-Isopropylidene-L-Ascorbic Acid

The C3-OH group of L-ascorbic acid is more reactive towards electrophiles under

mild basic conditions in comparison to the C2-OH (Scheme 7). This is primarily due to

the preferential deprotonation of C3-OH over C2-OH under mild basic conditions to

produce the monoanion.35 The electron density distribution diagram of the monoanion of

1 (Figure 10) clearly shows that the negative charge of the monoanion is distributed

between the C3-O─ and C1-carbonyl of the lactone ring with little electron density on C2-

OH. Therefore, the reactions of C5-OH- and C6-OH-protected ascorbic acid with various

electrophilic reagents under mild basic conditions should predominantly occur at the C3-

OH position as experimentally observed with the synthesized compounds in Table 4. In

addition, the electron density distribution diagram of the monoanion of 1 noticeably

confirm that the electron density at the C2 of the monoanion is significantly higher than

that of the C2-OH suggesting that the C2 position of the monoanion may also be

susceptible to electrophilic reactions.

57

Figure 10 Calculated Electrostatic Density Potential Diagrams of Monoanion Species of 1. Order of Electron Density: Blue < Green < Yellow < Red

In agreement with the literature findings and the electron density distribution diagram

(Figure 10), C2-alkylated products were also observed as minor products in the alkylation

of 1 under mild alkaline conditions. The electrophile isoprenyl bromide (4-bromo-2-

methyl-2-butene) gave exclusively C2-alkylated product when reacted with 5,6-O-

isopropylidene-L-ascorbate.191 The observed steady increment in the production of the

C2-alkylated product over the 3-O-alkylated product, when the electrophilic reagents

changed from allyl, crotyl, benzyl, to cinnamyl bromide, clearly indicates the dependence

of the transition state stability on the electrophile, which apparently plays a significant

role in the product distribution (Table 4). Therefore, these empirical results show that the

reactions of 1 with simple electrophiles under mild basic conditions can produce both C3-

O and C2 alkylated products.

58

Scheme 7 3-O-Alkylation of 5,6-O-Isoprpylidene-L-Ascorbic Acid

O

OOO O

O

CH3H3C

H

R HHO

OO O O

OH

CH3H3C

OOO O

O

CH3H3C

H

H

RO

CH3I or RBr / K2CO3

DMSO:THF / r.t. / 4-6 h

1

+

A B

H

Table 4 Products from 3-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid

Products R Yielda (A:B ratio)b

1A CH3 91 (100:0)

2A CH2C6H5 86 (62:38)

3A CH2CH=CHCH3 72 (70:30)

4A CH2CH=CH2 80 (80:20)

5A CH2CH=CHC6H5 72 (35:65)

aAll the yields (%) are given for the chromatographically purified products. bCalculated based on the weights of the purified compound

or by 1H-NMR signals for the nonresolvable mixtures.

59

4.1.2 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid

The C2-O-alkylated products of 1 could only be obtained after the protection of

the C3-OH group with protecting groups such as acetyl, MOM, etc.144, 161, 167 because of

the high nucleophilicity of C3-OH under mild basic condtions as previously mentioned.

In the literature, the acetyl group has been commonly used as a C3-O-protecting group in

the alkylation of the C2-OH of ascorbic acid. However, the high instability of the C3-O-

acetyl derivatives and facile migration of acyl groups from C3-O to C2-O position even

under mild reaction conditions led to many misidentification157-163 and

mischaracterization157-163 of C2,C3-O-disubstituted ascorbic acid derivatives. In all these

cases, the products characterized as C3-O-acetyl-C2-O-alkyl derivatives of 1, were later

confirmed as the C2-O-acetyl-C3-O-alkyl derivatives of 1. These thermodynamically

favored C2-O-acetyl-C3-O-alkyl derivatives are predominantly formed due to the fast

acetyl migration under the reaction conditions as previously mentioned.

Therefore, we sought to develop a specific and a direct method to alkylate the C2-O

position of 5,6-O-isopropylidine-L-ascorbic acid (1), which has not been reported in the

literature, to our knowledge. We used the results of density functional theory (B3LYP)

calculations to determine the electron density distributions and thus the nucleophilicity of

the dianion species of 1, towards electrophiles. Based on the molecular calculation

results, we have devised a novel and general method for the direct alkylation of C2-OH

of 1 in good yields with complete regio- and chemo-selectivity with both activated and

unactivated electrophiles.

60

Figure 11 Calculated Electrostatic Density Potential Diagrams of Dianion Species of 1 Order of Electron Density: Blue < Green < Yellow < Red

Inspection of the electron density distribution of the dianion of 1 (Figure 11) shows that

the negative charge of the C3-O is highly delocalized to the lactone carbonyl similar to

that of the monoanion as previously discussed. However, the electron density of the C2-

O is highly localized in the dianion suggesting that electrophilic reagents should

preferentially react with the C2-O rather than C3-O of the dianion of 1.

In excellent agreement, the dianion of 1 (Scheme 8), generated by reacting two

equivalents of potassium tert-butoxide (t-BuOK) in DMSO/THF (3:2) at -10 oC, reacts

with an equivalent amount of activated or unactivated electrophilic alkylating agents to

exclusively produce the corresponding C2-O-alkylated products (Table 5) in good yields

(80-90%). Regardless of the nature of the electrophile used according to Scheme 8, no

61

detectable amounts of C3-O- and/or C2-substituted products were produced, under these

experimental conditions. However, the addition of two equivalents of the electrophile

cleanly produces the corresponding C2,C3-O-disubstituted derivatives.

Scheme 8 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)

HO

OO

OO

O

CH3H3C

H

RHO

OO O O

OH

CH3H3C

DMSO:THF / -10 oC / 3 h1 C

H

O

OOO O

O-

CH3H3C

H -2 equivs t-BuOK

-2H+RBr

Table 5 Products of 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)

R Products Yield (%)

CH2CH=CH2 1C 80

CH3 2C 91

CH2CH=CHCH3 3C 72

CH2C6H5 4C 83

CH2CH=CHC6H5 5C 87

CH2(CH2)5CH3 6C 96

62

4.1.3 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid

2,3-O-disubstituted products of 5,6-O-isopropylidene-L-ascorbic acid (1) are

known for their vast usefulness in synthetic chemistry and various biological and

pharmaceutical application. The synthesis of most disubstituted products of 1, are simply

achieved either by (a) the use of excess electrophilic reagents under the appropriate

reaction conditions to directly produce 2,3-O-disubstituted products of 1; or (b)

selectively alkylating C2-OH (Scheme 8) or C3-OH (Scheme 7) of 1 prior to the

subsequent modification of these mono-substituted proudcts to generate the desired

disubstituted products (Scheme 9-11) . Tables 6-8 show a summary of 2,3-O-

disubstituted derivatives synthesized in the present study. The C2-O-alkylation of 5,6-O-

isopropylidene-3-O-acetyl-L-ascorbic acid derivatives of 1 to produce 5,6-O-

isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid products (Table 7) could only be

achieved by this latter procedure. This is because the intra-molecular C3-O to C2-O

acetyl migration is much faster than the alkylation of the C2-OH group of 5,6-O-

isopropylidene-3-O-acetyl-L-ascorbic acid (1D), under the C2-O-alkylation conditions.

Therefore, C2-O-alkylation of 5,6-O-isopropylidene-3-O-acetyl-L-ascorbic acid produces

the 5,6-O-isopropylidene-2-O-acetyl-3-O-alkyl-L-ascorbic acid products instead of the

desired 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid derivatives.

Consequently, the synthesis of 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic

acid derivatives must proceed by first synthesizing the 5,6-O-isopropylidene-2-O-alkyl-

L-ascorbic acid derivatives (Scheme 8), followed by the subsequent acylation (Scheme

10) to give the 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid derivatives.

63

Scheme 9 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid

RO

OO

O O

O

CH3H3C

H O

CH3

CH3COCl / PyCH2Cl2 / RT / 2 h

RO

OO O O

OH

CH3H3C

1A-5A 6A-10A

H

Table 6 Products of 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid

Starting material R Product Yield (%)

1A CH3 6A 90

2A CH2C6H5 7A 90

3A CH2CH=CHCH3 8A 78

4A CH2CH=CH2 9A 70

5A CH2CH=CHC6H5 10A 76

64

Scheme 10 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid

O

OO O O

OR

CH3H3C

OH3C

CH3COCl / Py

CH2Cl2 / RT / 2 h

7C-11C

HO

OO O O

OR

CH3H3C

1C-5C

H

Table 7 Products of 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid

Starting material R Product Yield (%)

1C CH2CH=CH2 7C 70

2C CH3 8C 80

3C CH2CH=CHCH3 9C 84

4C CH2C6H5 10C 82

5C CH2CH=CHC6H5 11C 76

65

Scheme 11 2,3-O-Disubstituted 5,6-O-Isopropylidene-L-Ascorbic Acid

R1O

OO

OO

OR2

CH3H3C

HR1O

OO O O

OH

CH3H3C

DMSO:THF / RT / 3 h

RBr / K2CO3H

Table 8 Products of 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid

Starting material R1 R2 Product Yield (%)

4A CH2CH=CH2 CH3 11A 59

4A CH2CH=CH2 CH2CH=CH2 12A 61

2A CH2C6H5 CH2C6H5 13A 88

5C CH2CH=CHC6H5 CH2CH=CHC6H5 14A 75

1A CH3 CH2CH=CH2 12C 66

1A CH3 CH2CH=CHCH3 13C 65

1A CH3 CH2CH=CHC6H5 14C 71

2A CH2C6H5 CH2CH=CH2 15C 81

4.2 Acylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid

Ascorbic acid has four hydroxyl groups that are susceptible to acylation.

However, by working with 1, the 5,6-O-isopropylidene derivative of L-ascorbic acid, the

situation is simplified in that only the C2- and C3-hydroxyls remain accessible to

acylation. The huge gap in the pKa of C2- and C3-OH groups synthetically favors a C3-

66

O-acyl product, which is predominantly formed over the pH range of 4-7 under acylation

reaction condition.163 Therefore, many publications reported to have synthesized C3-O-

acyl derivatives of L-ascorbic acid, which were supported by non-crystallographic

structural evidence. In our acylation reactions, methylene chloride was used as the

solvent, which also served as an indicator to determine the completion of reaction. Since

the starting material (1) is only slightly soluble in methylene chloride, the reaction

mixture, which begins as a heterogeneous mixture slowly changes from a cloudy mixture

into a homogenous solution as the reaction progresses to completion. In order to avoid

the formation of 2-O-acyl, and 2,3-O-di-acyl derivatives of 1, pyridine, a weak organic

base with pKa close to 9168 was used to exclusively generate 3-O-acyl derivatives. When,

1 in methylene chloride was reacted with one equivalent of pyridine and one equivalent

of acetyl chloride (acylating agent), a mixture of products was obtained in about 85%

yield. TLC analysis of the crude mixuture shows three different products. 1H NMR

analysis of the crude product shows three different signals for the C4 methine carbon

proton doublet at 4.78, 4.84 and 5.15 ppm with calculated ratios of 21:49:30 respectively

(Scheme 12). Further purification and spectroscopic analysis of the crude mixture

revealed the products as 2D, 1D and 3D respectively.

67

Scheme 12 Acylation of 5,6-O-Isopropylidene-L-Ascorbic Acid

O

OOO O

O

CH3H3C

HH

HO

OO O O

OH

CH3H3C

O

H3CO

OOO O

O

CH3H3C

H

H

O

CH3 O

OOO O

O

CH3H3C

HO

H3C

O

CH3

1D 2D

CH3COCl / Py

1

+ +

3DCH2Cl2 / RT / 1 h

H

4.2.1 C3-O- to C2-O Rearrangements of 3-O-Acyl-L-Ascorbic Acid Derivatives

The favored formation of C2-O-acyl over C3-O-acyl has long been a serious

problem confronting the acetylation of L-ascorbic acid over the years. The possible acyl

migration (Scheme 13) was long associated with several inorganic esters of L-ascorbic

acid such as the phosphate and sulfate derivatives of L-ascorbic acid. For example, some

earlier reported C3-O-phosphate and C3-O-sulfate derivatives of L-ascorbic acid were

later confirmed by crystallography to be the corresponding C2-O-esters derivatives.163

68

Scheme 13 Irreversible Isomerization of 5,6-O-Isopropylidene-3-O-Acetyl-L-Ascorbic Acid under Basic Conditions.

O

OOO O

O

CH3H3C

HO

H3C

O

OOO O

O

CH3H3C

H O

CH3

1D

O

OOO O

O

CH3H3C

HO

H3C

O

OOO O

O

CH3H3C

H

CH3O

- + BH B

H

- + BH

- + BH

2D

It has not been possible to obtain a pure C3-O-acetyl ester of 1 under various reaction

conditions without the contamination with some C2-O-ester and C2,C3-O-di-esters

isomers. We observed that the ratio of the C3-O-acetyl over the C2-O-acetyl ester

derivatives, even during work-up procedures with increasing ratio of the C2-O-acetyl

ester over the C3-O-acetyl ester derivatives. The presence of proton donor contaminants

such as methanol or water led to a reduced amount of the C3-O-acetyl derivative and an

69

increase in the C2-O-acetyl ester derivative. Also, the use of neutral form of 1, prolonged

reaction time, and the use of inorganic bases such as K2CO3 and NaHCO3 in the acylation

reaction led to an increased formation of C2-O-acetyl ester derivatives. The change in

reaction condition from 25 oC to 0 oC did not substantially change the amount of C3-O-

acetyl ester formation; however, this accelerated the overall formation of the C2-O-acetyl

ester derivative. It is clearly shown from the molecular calculations result (Figure 12) that

the C2-O atom of the neutral species of 1 has a slightly higher electron density than the

C3-O atom. These results are consistent with the literature findings that the C2-O is more

nucleophilic than the C3-O atom in the neutral species of L-ascorbic acid and may have

added to the increased formation of the C2-O-acetyl ester derivative.35, 158, 163 It is well

known in the literature that the neighboring hydroxyl groups participate in the

monophosphate isomerization observed in ribonucleoside-2’-phosphate, glycerol-2-

phosphate, glycerol-1-phosphate and in some other β-hydroxy phosphate esters.169-186

Another possible factor influencing isomerization of C3-O-acyl esters of 1 is that the C2-

O and C3-O atoms are restricted to an eclipsed conformation as a result of the planar

lactone ring and such restriction could enhance the intramolecular rearrangement by

nucleophilic participation of the vicinal hydroxyl group.139, 187-190 There was no evidence

for the formation of C2,C3-O-diesters from C3-O-acetyl-esters of 1, when stirred with

1.2 equivalents of base under nitrogen gas for over 12 h, which resulted in complete

acetyl group migaration of the C3-O- to C2-O- position. Therefore, the findings that the

increase in susceptibility of the C3-O-acetyl esters to rearrangement in the presence of

protic impurities, prolonged reaction time and increase in temperature strongly suggests

70

an intra-molecular and not inter-molecular isomerization happening in the acetyl

migration of L-ascorbic acid derivatives.163

Figure 12 Calculated Electrostatic Density Potential Diagrams of Neutral Species of 1 Order of Electron Density: Blue < Green < Yellow < Red

4.3 NMR Spectroscopic Analyses of L-Ascorbic Acid and its Derivatives

The detailed structural analysis of L-ascorbic acid and its derivatives by NMR

spectroscopy is important in understanding their structural characteristics in relation to

their biological and chemical properties. The diagnostic 1H- and 13C-NMR spectral

signals of L-ascorbic acid has its most upfield resonance belonging to the C6, which is

followed by the C5, C4, C2, C3 and the C1 respectively. Consequently, derivatives of L-

ascorbic acid display distinctive characteristics in their 1H- and 13C-NMR chemical shifts

71

for the six carbon centers of L-ascorbic acid, which could be used to unequivocally

identify the derivatives.

4.3.1 NMR Spectroscopic Properties of 2-O- and 3-O-Substituted 5,6-O-Isopropylidene-L-Ascorbic Acid

The C2- and C3-OH group substitution of the 5,6-O-isopropylidene-L-ascorbic

acid (1) causes a diagnostic chemical shift of its indigenous carbon and hydrogen that are

useful in the characteristic identification of the various derivatives. The data presented in

Table 9 demonstrate that C2-O- and C3-O-monosubstituted derivatives of 1 display

characteristic 13C-NMR chemical shifts for their C2 and C3 carbon signals and the 1H-

NMR chemical shifts for their C4-H that could be used to unequivocally identify the C2-

O- and C3-O-monosubstituted derivatives. The standard 13C chemical shifts of C2 and C3

of ascorbic acid and 1 are 118.8 ppm, 120.5 ppm, 156.3 ppm, and 158.4 ppm

respectively.35, 161 The 13C-NMR signals of C2 and C3 are in the ranges of 119-120 ppm

and 148-150 ppm for C3-O-alkylated derivatives and 121-123 ppm and 156-158 ppm for

C2-O-alkylated derivatives, respectively (Table 9), and are in good agreement with the

previously reported literature values.144, 154-156, 167 Substitution at the C3-OH (3-O-

alkylation) causes an upfield shift (8.5 to 10.2 ppm) of 13C signals of C3 (1A-4A) with

respect to 1 depending on the nature of the alkyl substituent. On the other hand, the

effects of the C2-O-substitution (1C-6C) on the 13C signals of C-2 are considerably

smaller and in the range of 0.3 to 2.6 ppm (upfield). The large chemical shift difference

in the 13C signals of C3 in the C3-O-substitued derivatives (1A-4A) in comparison to the

C2 in the C2-O-substititued derivatives (1C-6C) must be due to the lack of efficient

delocalization of the C3-O electron density into the C1-carbonyl in C3-O-substituted

72

derivatives (1A-4A) in comparison to C2-O-substituted derivatives (1C-6C). This effect

is also clearly visible in 1H–NMR chemical shifts of C4-H (Table 9), where C3-O-

substitution (1A-4A) caused an upfield shift of the C4-H in comparison to the C2-O-

substituted derivatives (1C-6C).

Table 9 1H NMR (C-4-H) and 13C NMR (C-2 & C-3) Chemical Shifts (δ) of 2-O-Alkyl and 3-O-Alkyl Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1)

R1O

OO O O

OR2

CH3H3C

43 2

1

Derivatives R1 R2 13C

δ (2-C) 13C

∆(2-C)a 13C

δ(3-C) 13C

∆(3-C)a 1H

δ(4-C-H)

1 H H 120.5 - 158.4 - 4.91

1A CH3 H 119.5 -1.0 149.9 -8.5 4.53

2A CH2C6H5 H 119.5 -1.0 148.6 -9.8 4.57

3A CH2CH=CHCH3 H 119.1 -1.4 148.6 -9.8 4.55

4A CH2CH=CH2 H 119.2 -1.3 148.2 -10.2 4.58

1C H CH2CH=CH2 121.4 +0.9 156.4 -2.0 4.72

2C H CH3 123.1 +2.6 155.8 -2.6 4.71

3C H CH2CH=CHCH3 120.8 +0.3 157.9 -0.5 4.69

4C H CH2C6H5 121.1 +0.6 157.5 -0.9 4.60

5C H CH2CH=CHC6H5 121.2 +0.7 157.1 -1.3 4.64

6C H CH2(CH2)5CH3 121.8 +1.3 156.6 -1.8 4.71

aThe difference in 13C chemical shifts of C2 and C3 (∆(C-2) and ∆(C3)) were calculated by subtracting the chemical shifts of various derivatives from the corresponding values of the compound

73

In the C2,C3-O-disubstituted series (2,3-O-dialkylation), the C2 and C3 showed

characteristic shifts of 13C signals (Table 10) with respect to 1 that could be used to

distinguish the 2-O-alkyl-3-O-acetyl derivatives (7C-11C) from the 2-O-acetyl-3-O-alkyl

derivatives (6A-10A). The 13C signals of C3 of 2-O-alkyl-3-O-acetyl derivatives (7C-

11C) showed a large upfield shift in the range of 16.1 to 13.8 ppm with respect to 1. On

the other hand, 13C signals of C2 of these derivatives showed large downfield shifts (in

the range of 9.6 to 14.3 ppm) with respect to 1. As discussed above for C3-O-alkylated

derivatives (1A-4A), these large 13C shifts of the C3 signals of C3-O-acetylated

derivatives (7C-11C) must be due to the significant perturbation of the native electronic

structure of 1 by the electron withdrawing C3-O-acetate group. The inhibition of the

delocalization of C3-O electron to the C1-carbonyl group by the C3-O-acetate group

leads to an increase of the electron density at C3 causing 13C signal to shift upfield, and a

decrease of electron density at C2 causing 13C signal to shift significantly downfield. A

significant downfield shift of the 1H-NMR signal (Table 10) of C4-H of 7C-11C, in

comparison to 1, further confirms the significant perturbation of the native electronic

structure of 1 by the electron withdrawing nature of the C3-O-acetyl group. In 2-O-

acetyl-3-O-alkyl derivatives (6A-10A), the effects were more localized to the C2 as

expected. The 13C signals of the C2 of these derivatives shifted upfield in comparison to

1, which is in sharp contrast to the effects of C3-O-acetate substitution, suggesting that

the direct electron withdrawing effect of the C2-O-acetate group primarily determines the

13C chemical shift of the C2 of these derivatives.

74

Table 10 1H NMR (C4-H) and 13C NMR (C2 & C3) Chemical Shifts (δ) of 2,3-O-Disubstituted Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1)

R1O

OO O O

OR2

CH3H3C

43 2

1

Derivatives R1 R2 13C

δ (2-C) 13C

∆(2C)a 13C

δ(3-C) 13C

∆(3-C)a

1H δ(4-C)H

1 H H 120.5 - 158.4 - 4.91

6A CH2-CH=CH2 O=C-CH3 114.6 -5.9 159.5 +1.1 4.69

7A CH3 O=C-CH3 114.5 -6.0 160.7 +2.3 4.67

8A CH2-CH=CHCH3 O=C-CH3 114.4 -6.1 159.7 +1.3 4.66

9A CH2-C6H5 O=C-CH3 114.8 -5.7 159.8 +1.4 4.71

10A CH2-CH=CH-C6H5 O=C-CH3 114.7 -5.8 159.7 +1.3 4.70

11A* CH2CH=CH2 CH3 123.0 +2.5 155.1 -3.3 4.53

12A CH2CH=CH2 CH2CH=CH2 121.5 +1.0 155.7 -2.7 4.55

13A CH2C6H5 CH2C6H5 121.2 +0.7 156.4 -2.0 4.53

14A* CH2CH=CHC6H5 CH2CH=CHC6H5 121.3 +0.8 156.1 -2.3 4.55

7C O=C-CH3 CH2-CH=CH2 130.1 +9.6 143.8 -14.6 5.18

8C O=C-CH3 CH3 131.2 +10.7 142.3 -16.1 5.15

9C O=C-CH3 CH2-CH=CHCH3 132.2 +11.7 143.8 -14.6 5.18

10C O=C-CH3 CH2-C6H5 130.1 +9.6 144.6 -13.8 5.15

11C O=C-CH3 CH2-CH=CH-C6H5 134.8 +14.3 144.6 -13.8 5.17

12C* CH3 CH2CH=CH2 121.5 +1.0 157.1 -1.3 4.52

13C* CH3 CH2CH=CHCH3 121.4 +0.9 157.3 -1.1 4.51

14C* CH3 CH2CH=CHC6H5 121.4 +0.9 157.4 -1.1 4.51

15C* CH2C6H5 CH2CH=CH2 121.4 +0.9 155.9 -2.5 4.55

aThe difference in 13C chemical shifts of C2 and C3 (∆(C2) and ∆(C3)) were calculated by subtracting the chemical shifts of various derivatives from the corresponding values of the compound

*Data obtained from Mahindaratne, M. P. D. Thesis Ref. 191

75

The C4-H of C2-O-acetyl derivatives (6A-10A), showed a small but significant upfield

shift, again demonstrating the electron withdrawing inductive effect of the C2-O-acetate

group. Furthermore, in C2,C3-O-dialkyl derivatives (11A-14A & 12C-15C), the 13C

signals of C3 showed moderate upfield shifts in the range of 1.1 to 2.7 ppm with respect

to 1. In contrast, 13C signals of C2 of these derivatives (11A-14A & 13C-15C) showed

modest downfield shifts in the range of 0.7 to 2.5 ppm with respect to 1. As discussed

above for C3-O-alkylated derivatives (1A-4A), these upfield 13C shifts of C3 of C2,C3-

O-dialkyl derivatives (11A-14A & 13C-15C) must be due to the significant perturbation

of the native electronic structure of 1 by the C3-O-alkyl group. The inhibition of the

delocalization of C3-O electron to the C1-carbonyl by the C3-O-alkyl group leads to the

increase of the electron density at C3 causing 13C signal to shift upfield, and a decrease of

electron density at C2 causing 13C signal to shift downfield moderately. This effect is

also clearly visible in 1H–NMR chemical shifts of C4-H, where C3-O-substitution of the

C2,C3-O-dialkyl derivatives (11A-14A & 13C-15C) caused an upfield shift of the C4-H

in comparison to the free non-substituted ascorbate 1 and thus further confirms the

considerable perturbation of the native electronic structure of 1 by the inherent nature of

the substitution group.

4.4 The Sigmatropic Claisen Rearrangement of L-Ascorbic Acid Derivatives

Selective modification of the C2- and C3-OH groups of L-ascorbic acid provides

a unique route to different ascorbate derivatives with great potentials as chiral synthons.

Therefore, in order to further explore the versatility of L-ascorbic acid, our group had

76

previously exploited the possibility of using L-ascorbic acid derivatives as Claisen

substrates for the synthesis of new C2- and C3-substituted aldonolactone (L-galactono-γ-

lactone) derivatives. Therefore, the sigmatropic Claisen rearrangement of the allyl vinyl

ether’s moiety of a series of 5,6-O-isopropylidene-3-O-allyl-L-ascorbic acid (A, Scheme

14-16) and 5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid derivatives (C, Scheme 17) to

their corresponding 5,6-O-isopropylidene-2-allyl-3-keto-L-galactono-γ-lactone (E, Table

11-18) and 5,6-O-isopropylidene-3-allyl-2-keto-L-galactono-γ-lactone (F, Table 19-26)

respectively were synthesized and fully characterized for comparative purposes.


Isopropylidene-3-O-Allylic Derivatives of L-Ascorbic Acid

5,6-O-isopropylidene-3-O-allyl-L-ascorbic acid derivatives progressed through a facile

Claisen rearrangement (100% conversion) according to Scheme 14 to give products with

a β-keto-ester functional group known as 5,6-O-isopropylidene-2-allyl-3-keto-L-

galactono-γ-lactone. This aldono-γ-lactone products are less polar and less-UV-active

than their corresponding starting materials. 1H- and 13C-NMR analysis of the crude

products revealed a C2-allylated diastereomeric excess with a minor diastereomeric

product. These products were very visible in an iodine bath and were easily separated by

careful silica gel chromatography with ethyl acetate and hexane in high purity for

characterization.

77

Scheme 14 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

O

OOO O

OR2

CH3H3C

H O

OO O

O

OR2

CH3H3C

H1

23

456

7

89

10

11

12

12

345

6

7

8

9

10

11

1213

13

reflux / toluene(a)

6 h

A ER1

14

R1

14

Reactant R1 R2 Product(b)

4A H H 1E

9A H COCH3 2E

11A H CH3 3E

12A H CH2CH=CH2 4E

3A CH3 H 5E

8A CH3 COCH3 6E

(a)100% conversion of A to E was obtained in 6 h. (b) 1H-NMR analysis of crude reaction mixtures indicated that the product is a mixture of two diastereomers with > 90% of the major.

4.4.1.1 NMR Spectroscopic Analyses of Products from C3-O to C2 Sigmatropic

Claisen Rearrangement of 5,6-O-Isopropylidene-3-O-Allyl-L-Ascorbic Acid

Derivatives

The comparative 1H-NMR signals (E: Table 11, 13 & 15) of the Claisen rearranged

products of a series of 5,6-O-isopropylidene-3-O-allylic derivatives of L-ascorbic acid

and their corresponding starting materials (A: Scheme 14) showed characteristic chemical

78

shift patterns that could be used to identify the products. For instance, there is a

downfield shift of the C10 proton signals of the starting materials (A) by a range of 0.25

to 0.58 ppm in forming the corresponding products (E). The newly formed C2-C12 bond

on the products resulted in an upfield chemical shift of the C11-H, C12-H and C13-H

proton signals of the starting materials by about 0.05 to 0.32 ppm, 2.55 to 2.93 ppm and

0.54 to 2.77 ppm respectively. The C3-β-ketone-lactone moiety on the products showed

in general the deshielding of the product’s C4-H and C5-H proton signals when

compared to their corresponding starting materials.

The comparative 13C NMR signals of the starting material and their corresponding C2-

allylated products (E: Table 12, 14, & 16) revealed distinctive chemical shift patterns that

could be used in the identification of the compounds. For example, C10 carbon signals of

the products showed a downfield chemical shift ranging from 45.5 to 50.6 ppm when

compared to the corresponding starting materials (A). The newly created C2-C12 bond

resulted in a large upfield chemical shift of the C12 carbon signals of the products by

about 77.8 to 85.1 ppm when compared to their starting materials. Furthermore, C3 and

C2 carbon signals of the products appeared in the ranges of 200.9-206.1 ppm (~ 41.2 to

57.3 ppm downfield) and 73.8-80.4 ppm (~ 37.1 to 44.7 ppm upfield) respectively, when

compared to the corresponding starting materials. These significant chemical shift

patterns of the C2 and C3 carbon signals reflect the disappearance of the conjugated

enone moiety in the products.

79

Table 11 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H1

23

456

7

89

10

11

12

12

345

6

7

8

9

10

11

12

13

13

A E

Proton # R = H R = CH3

A (4A)

E (1E) ∆ (δ H)a A

(11A) E

(3E) ∆ (δ H)a

δ (4-H) 4.58 4.66 -0.08 4.53 4.54 -0.01

δ (5-H) 4.28 4.54 -0.26 4.30 4.65 -0.35

δ (6-H’) 4.04 4.08 -0.04 4.04 4.06 -0.02

δ (6-H”) 4.15 4.19 -0.04 4.14 4.17 -0.03

δ (8-CH3) 1.37 1.35 +0.02 1.36 1.32 +0.04

δ (9-CH3) 1.40 1.41 -0.01 1.40 1.38 +0.02

δ (10-H’) 4.97 5.26 -0.29 4.93 5.20 -0.27

δ (10-H”) 4.97 5.28 -0.31 4.93 5.25 -0.32

δ (11-H) 6.01 5.69 +0.32 5.98 5.73 +0.25

δ (12-H’) 5.31 2.66 +2.65 5.33 2.63 +2.70

δ (12-H”) 5.41 2.66 +2.75 5.40 2.63 +2.77

δ (13-H) - - - 3.85 3.34 +0.51

aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E

80

Table 12 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H1

23

456

7

89

10

11

12

12

345

6

7

8

9

10

11

12

13

13

A E

Carbon # R = H R = CH3

A (4A)

E (1E) ∆ (δ C)a A

(11A) E

(3E) ∆ (δ C)a

δ (1-C) 171.0 172.6 -1.60 168.7 171.2 -2.50

δ (2-C) 119.2 74.5 +44.7 123.0 80.4 +42.6

δ (3-C) 148.2 205.5 -57.3 155.1 206.1 -51.0

δ (4-C) 75.6 81.5 -5.90 74.6 81.5 -6.90

δ (5-C) 74.3 72.0 +2.30 74.0 74.3 -0.30

δ (6-C) 65.3 64.8 +0.50 65.3 64.9 +0.40

δ (7-C) 110.3 111.3 -1.00 110.3 110.9 -0.60

δ (8-C) 25.5 25.3 +0.20 25.5 25.5 0.00

δ (9-C) 25.9 25.5 +0.40 25.8 25.6 +0.20

δ (10-C) 72.3 122.9 -50.6 72.2 122.1 -49.9

δ (11-C) 132.2 127.6 +4.60 131.8 127.8 +4.00

δ (12-C) 119.1 39.8 +79.0 118.8 41.0 +77.8

δ (13-C) - - - 59.9 55.8 +4.10

aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen

compound E

81


O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H1

23

456

7

89

10

11

12

12

345

6

7

8

9

10

11

12

13

13

A E

Proton # R = COCH3 R = CH2CH=CH2

A (9A)

E (2E) ∆ (δ H)a A

(12A) E

(4E) ∆ (δ H)a

δ (4-H) 4.69 4.87 -0.18 4.55 4.54 +0.01

δ (5-H) 4.38 4.53 -0.15 4.30 4.64 -0.34

δ (6-H’) 4.08 4.10 -0.02 4.04 4.06 -0.02

δ (6-H”) 4.16 4.20 -0.04 4.14 4.17 -0.03

δ (8-CH3) 1.37 1.37 0.00 1.36 1.33 +0.03

δ (9-CH3) 1.41 1.42 -0.01 1.39 1.39 0.00

δ (10-H’) 4.81 5.19 -0.38 4.94 5.19 -0.25

δ (10-H”) 4.81 5.26 -0.45 4.94 5.20 -0.26

δ (11-H) 5.95 5.90 +0.05 5.99 5.74 +0.25

δ (12-H’) 5.35 2.80 +2.55 5.35 2.67 +2.68

δ (12-H”) 5.40 2.80 +2.60 5.39 2.67 +2.72

δ (13-H) 2.27 2.16 +0.11 4.62, 5.27, 5.31, 5.98

3.98, 5.25, 5.30, 5.89

+0.64, +0.02, +0.01, +0.09


82


O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H1

23

456

7

89

10

11

12

12

345

6

7

8

9

10

11

12

13

13

A E

Carbon # R = COCH3

R = CH2CH=CH2

A (9A)

E (2E) ∆ (δ C)a A

(12A) E

(4E) ∆ (δ C)a

δ (1-C) 167.6 170.9 -3.30 168.9 171.4 -2.50

δ (2-C) 114.6 73.8 +40.8 121.5 79.9 +41.6

δ (3-C) 159.5 201.0 -41.5 155.7 206.1 -50.4

δ (4-C) 75.3 82.1 -6.80 74.7 81.5 -6.80

δ (5-C) 73.7 73.7 0.00 74.0 74.2 -0.20

δ (6-C) 65.2 65.2 0.00 65.3 64.9 +0.40

δ (7-C) 110.6 110.8 -0.20 110.3 110.9 -0.60

δ (8-C) 25.5 25.5 0.00 25.6 25.4 +0.20

δ (9-C) 25.8 25.8 0.00 25.9 25.7 +0.20

δ (10-C) 72.5 120.8 -48.3 72.5 118.0 -45.5

δ (11-C) 131.0 128.3 +2.70 132.9 127.8 +5.10

δ (12-C) 119.6 34.5 +85.1 119.2 41.2 +78.0

δ (13-C) 20.3, 166.8

19.2, 169.7

+1.10, -2.90

72.3, 118.9, 131.9

69.2, 122.2, 133.2

+3.10, -3.30, -1.30

aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E

83

Table 15 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H

H3C

CH3

A E

1

23

456

7

89

10

11

12

12

345

6

7

89

10

11

1213

13

14

14

Proton # R = H R = COCH3

A (3A)

E (5E) ∆ (δ H)a A

(8A) E

(6E) ∆ (δ H)a

δ (4-H) 4.55 4.60 -0.05 4.66 4.51 +0.15

δ (5-H) 4.26 4.53 -0.27 4.36 4.51 -0.15

δ (6-H’) 4.02 4.07 -0.05 4.07 4.13 -0.06

δ (6-H’’) 4.13 4.18 -0.05 4.15 4.13 +0.02

δ (8-CH3) 1.37 1.34 +0.03 1.36 1.38 -0.02

δ (9-CH3) 1.40 1.40 0.00 1.40 1.48 -0.08

δ (10-H’) 4.89 5.26 -0.37 4.73 5.28 -0.55

δ (10-H’’) 4.89 5.28 -0.39 4.73 5.31 -0.58

δ (11-H) 5.90 5.75 +0.15 5.87 5.71 +0.16

δ (12-H’) 5.68 2.75 +2.93 5.62 2.90 +2.72

δ (13-H) 1.75 1.18 +0.57 1.76 1.22 +0.54

δ (14-H) - - - 2.27 2.16 +0.11


84

Table 16 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

O

OOO O

OR

CH3H3C

HO

OO O

O

OR

CH3H3C

H

H3C

CH3

A E

1

23

456

7

89

10

11

12

12

345

6

7

89

10

11

1213

13

14

14

Carbon # R = H R = COCH3

A (3A)

B (5E) ∆ (δ C)a A

(8A) B

(6E) ∆ (δ C)a

δ (1-C) 171.8 172.8 -1.00 167.6 170.3 -2.70

δ (2-C) 119.1 74.4 +44.7 114.4 77.3 +37.1

δ (3-C) 148.6 205.7 -57.1 159.7 200.9 -41.2

δ (4-C) 75.7 81.8 -6.10 75.3 84.8 -9.50

δ (5-C) 74.4 74.3 +0.10 73.7 75.1 -1.40

δ (6-C) 65.3 64.8 +0.50 65.2 65.2 0.00

δ (7-C) 110.3 111.0 -0.70 110.5 110.0 +0.50

δ (8-C) 25.6 25.3 +0.30 25.5 25.3 +0.20

δ (9-C) 25.9 25.4 +0.50 25.7 26.7 -1.00

δ (10-C) 72.4 120.0 -47.6 72.6 120.6 -48.0

δ (11-C) 132.6 134.3 -1.70 133.2 132.9 +0.30

δ (12-C) 125.2 44.3 +80.9 124.0 41.4 +82.6

δ (13-C) 17.8 12.5 +5.30 17.7 12.3 +5.40

δ (14-C) - - - 167.0, 20.2

169.3, 19.2

-2.30, +1.00

aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E

85

4.4.2 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-

3-O-Cinnamyl-L-Ascorbic Acid Derivatives

5,6-O-isopropylidene-2-O-acetyl-3-O-cinnamyl-L-ascorbic acid derivative (10A)

was rearranged according to Scheme 16 to give 5,6-O-isopropylidene-2-O-acetyl-2-(1-

phenyl-1-prop-2-enyl)-3-keto-L-galactono-γ-lactone, which is a C2-allylated aldono-γ-

lactone derivative. Interestingly, when 5,6-O-isopropylidene-L-ascorbic acid (1) was

treated with 2 molar equivalents of potassium tert-butoxide in DMSO/THF solvent

system for 3 hours (Scheme 15), the reaction exclusively produced a C2-allylated aldono-

γ-lactone derivative, 5,6-O-isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3-keto-L-

galactono-γ-lactone (7E). This reaction is unique to using cinnamyl chloride as the

electrophilic reagent. For instance, when cinnamyl bromide was used under the same

reaction condition, the product of this reaction was 5,6-O-isopropylidene-2-O-cinnamyl-

L-ascorbic acid (5A), a C3-O-allylated compound in place of the C2-allylated compound

(7E). The crude product of 10A was easily separated by careful silical gel

chromatography followed by recrystallization to give a diastereomeric excess (9E) and a

minor diastereomeric product (10E).

86

Scheme 15 Direct Synthesis of 5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone from 1

Scheme 16 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1-

phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

OOO O

O

CH3H3C

H1

23

456

7

89

10

1112

reflux / toluene(a)

6h

13

O

OO

OO

CH3H3C

H1

2

4

56

7

8

9

3

11

1213

10

OOC6H5

C6H5

10A E

O

CH3

O

CH3

Reactant E: Product (Major) E: Product (Minor)

10A 9E(b) 10E(b) (a)100% conversion of A to E was obtained in 6 h. (b)1H-NMR analysis of crude

reaction mixtures indicated that the product is a mixtures of two diastereomers with > 75% of the major.

OOO O

HO

CH3H3C

H1

23

456

7

89

2Eq t-Buok, DMSO/THF

Cinnamyl Chloride, 3 hOH

OO

OO

CH3H3C

H1

2

4

56

7

8

9

3

11

1213

10

OORC6H5

1 7E

CH2Cl2, -79 oC (quant.)

(Ac)2O /DMAP / Et 3N

OO

OO

CH3H3C

H1

2

4

56

7

8

9

3

11

12

13

10

O

OC6H5

8E

O

CH3

87


Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-Acetyl-3-O-Cinnamyl-L-

Ascorbic Acid Derivative (10A)

The 1H-NMR (Table 17) identifiable features of 5,6-O-isopropylidene-2-O-acetyl-3-O-

cinnamyl-L-ascorbic acid derivatives (10A) after sigmatropic Claisen rearrangement

showed distinctive chemical shift patterns that could be used in the identification of the

products. For example, there is a downfield shift of the C10 proton signal by a range of

0.33 to 0.41 ppm in the rearranged products (9E & 10E) when compared to the starting

material (10A). Also, the C12-H, C13-H and C14-H proton signals of the products shifted

upfield by about 2.24 to 2.31 ppm, 0.04 to 0.08 ppm and 0.14 to 0.15 ppm respectively

when compared to the starting material. The newly formed C2-C12 bond on the products

resulted in an upfield chemical shift of the C11-H proton signals by about 0.28 to 0.37

ppm than the starting material. In general, the C3-β-ketone-lactone moiety on the

products (E) resulted in the deshielding of the C4-H and C5-H proton signals when

compared to the starting materials (A). However, the C4-H of 10E and C5-H of 9E

showed a shielding of their proton signals by 1.00 ppm and 1.43 ppm respectively than

the starting compound (10A).

88

Table 17 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

OOO O

O

CH3H3C

H1

23

456

7

89

10

1112

13

OAC

OO

OO

CH3H3C

H1

2

4

56

7

8

9

3

11

1213

10

OOACC6H5

C6H5

A E Proton # A

(10A) E

(9E) ∆ (δ H)a E

(10E) ∆ (δ H)a

δ (4-H) 4.70 4.75 -0.05 3.70 +1.00

δ (5-H) 4.40 2.97 +1.43 4.42 -0.02

δ (6-H’) 4.09 3.82 +0.27 4.05 +0.04

δ (6-H”) 4.17 3.97 +0.20 4.05 +0.12

δ (8-CH3) 1.36 1.27 +0.09 1.32 +0.04

δ (9-CH3) 1.41 1.45 -0.04 1.34 +0.07

δ (10-H’) 4.97 5.17 -0.20 5.31 -0.34

δ (10-H”) 4.97 5.30 -0.33 5.38 -0.41

δ (11-H) 6.71 6.43 +0.28 6.34 +0.37

δ (12-H) 6.29 4.05 +2.24 3.98 +2.31

δ (13-H) 7.30 7.40

7.24 7.36

+0.06 +0.04

7.22 7.35

+0.08 +0.05

aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by

subtracting the chemical shifts of A from the corresponding values of the Claisen compound E

89

Table 18 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone

OOO O

O

CH3H3C

H1

23

456

7

89

10

1112

13

OAC

OO

OO

CH3H3C

H1

2

4

56

7

8

9

3

11

1213

10

OOACC6H5

C6H5

A E Carbon # A

(10A) E

(9E) ∆ (δ C)a E

(10E) ∆ (δ C)a

δ (1-C) 167.6 169.3 -1.70 169.1 -1.50

δ (2-C) 114.7 76.0 +38.7 76.7 +38.0

δ (3-C) 159.7 201.5 -41.8 201.8 -42.1

δ (4-C) 75.3 83.9 -8.60 85.1 -9.80

δ (5-C) 73.6 72.7 +0.90 75.0 -1.40

δ (6-C) 65.2 64.9 +0.30 65.1 +0.10

δ (7-C) 110.6 110.2 +0.40 109.8 +0.80

δ (8-C) 25.5 25.4 +0.10 25.3 +0.20

δ (9-C) 25.7 26.4 -0.70 26.6 -0.90

δ (10-C) 72.6 119.9 -47.0 121.0 -48.4

δ (11-C) 135.5 134.9 +0.60 133.8 +1.70

δ (12-C) 121.6 51.3 +70.0 52.9 +68.7

δ (13-C) 126.7, 128.6, 128.7, 129.3

128.5 129.3 129.8 132.5

-1.80 -0.70 -1.10 -3.20

128.8 129.1 129.3 131.1

-2.10 -0.50 -0.60 -1.80

aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of

the Claisen compound E

90

The differences in the C4-H and C5-H chemical shifts of 9E and 10E (Table 17) are

possibly due to the nature of the combined steric influence of the bulky substituents at

their C2, C4 and C12 positions. The comparative 13C NMR signals of C3-O (A: 10A) to

C2 (E: 9E & 10E) sigmatropic Claisen rearrangement (Table 18) revealed distinctive

chemical shift patterns that are useful in the identification of products. Notable examples

are the C10 (~ 47.3 to 48.4 ppm downfield), C12 (~ 68.7 to 70.0 ppm upfied), C2 (~ 38.0

to 38.7 ppm upfield) and C3 (~ 41.8 to 42.1 ppm downfield) carbon signals of the

products when they were compared to the starting material. These distinctive chemical

shift patterns are diagnostic of the disappearance of the conjugated enone moiety of the

starting material (A) and the formation of products (E) without the enone moiety and thus

the reason for their poor UV-activity. The C3-carbonyl signals of the β-keto-ester group

(E, 200.9 to 205.7 ppm) and their lactone C1-carbonyl (E, 170.3-172.8 ppm) are in the

same range of typical β-keto-γ-lactone carbonyl NMR signals, which usually appear

around 200 ppm and 170 ppm respectively. 208-210,215-216


Isopropylidene-2-O-Allyl-L-Ascorbic Acid Derivatives

5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid derivatives were subjected to

sigmatropic Claisen rearrangement according to Scheme 17 to give products with an α-

keto-ester functional group known as 5,6-O-isopropylidene-3-allyl-2-keto-L-galactono-γ-

lactone. This compound is a C3-substituted aldono-γ-lactone derivative and the 1H- and

13C-NMR analysis of the crude products revealed a diastereomeric excess (>95%). The

91

products are very visible in iodine and are very sensitive to hydrolysis in silica gel

chromatography even under very careful conditions.


Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-Allyl-L-Ascorbic Acid

Derivatives

The comparative 1H-NMR signals (Table 19, 21, 23 & 25) of the series of 5,6-O-

isopropylidene-2-O-allylic derivatives of L-ascorbic acid (C) after sigmatropic Claisen

rearrangement to produce their corresponding products (F), showed disntintive chemical

shift patterns that could be used to identify this series of compounds. For example, the

C10 methylene proton signals of the products showed a downfield chemical shift of about

0.45 to 0.77 ppm when compared to the corresponding starting materials (C). Also, as a

result of the newly created C3-C12 bond on the products (F), their C11-H, C12-H and

C13-H proton signals showed an upfield chemical shift in the ranges of 0.01 to 0.29

ppm, 2.40 to 2.96 ppm, and 0.07 to 0.64 ppm respectively, when compared to their

corresponding starting materials. The C2-α-ketone-ester moiety on the products showed

in general the deshielding of the C4-H and C5-H proton signals when compared to their

corresponding starting materials. However, 3F and 6F both showed a moderate shielding

of their C4-H and C5-H proton signals, perhaps due to the combined steric and electronic

influence of the bulky substituents at C4 and the acetate group at C3 position.

92

Scheme 17 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone

Reactant R1 R2 Product

1C H H 1F

7C H COCH3 2F

12C H CH3 3F

15C H CH2C6H5 4F

3C CH3 H 5F

9C CH3 COCH3 6F

13C CH3 CH3 7F

(a)100% conversion of C to F was obtained in 24 h. 1H-NMR analysis of the crude reaction mixtures indicated that only a single diastereomer was detectable

The comparative 13C NMR signals of C2-O (C) to C3 (F) sigmatropic Claisen

rearrangement (Table 20, 22, 24 & 26) showed distinguishable chemical shift patterns

that are useful for the identification of the series of compounds. For instances, the C10

carobn signals of the products revealed a downfield chemical shift ranging from 46.3 to

R2O

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

10

11

12

13

123

456

7

89

10

11

12 13

C F

reflux / toluene(a) OR2O

R1

14

14 R1

93

50.6 ppm when compared to the corresponding starting materials. Also, the newly formed

C3-C12 bond on the products resulted in a large shielding (80.0 to 84.0 ppm) of the non-

oxygen bearing C12 carbon signals when compared to the starting materials.

Furthermore, the C2 and C3 carbon signals of the products appear in the ranges of 186.3

to 195.3 ppm (~ 55.0 to 74.5 ppm downfield) and 77.2 to 79.3 ppm (~ 65.0 to 79.2 ppm

upfield) respectively, when compared to the corresponding starting materials. These

significant chemical shift patterns reflect the disappearance of the conjugated enone

moiety in the products and these carbon signals are within the same range of typical α-

keto-γ-lactone NMR signals.208-210,215-216

94


Proton # R = H R = CH3

C (1C)

F (1F) ∆ (δ H)a C

(12C) F

(3F) ∆ (δ H)a

δ (4-H) 4.72 4.69 +0.03 4.52 4.58 -0.06

δ (5-H) 4.43 4.53 -0.10 4.28 4.49 -0.21

δ (6-H’) 4.02 4.09 -0.07 4.03 3.99 +0.04

δ (6-H”) 4.16 4.16 0.00 4.13 4.14 -0.01

δ (8-CH3) 1.38 1.29 +0.09 1.36 1.33 +0.03

δ (9-CH3) 1.43 1.32 +0.11 1.39 1.33 +0.03

δ (10-H’) 4.62 5.24 -0.62 4.61 5.26 0.65

δ (10-H”) 4.62 5.30 -0.68 4.61 5.31 -0.70

δ (11-H) 5.98 5.81 +0.17 6.00 5.75 +0.25

δ (12-H’) 5.28 2.53 +2.75 5.28 2.51 +2.77

δ (12-H”) 5.36 2.53 +2.83 5.36 2.66 +2.70

δ (13-H) - - - 4.15 3.70 +0.45

aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F

RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

10

11

12

13

123

456

7

89

10

11

1213

C F

ORO

95


Carbon # R = H R = CH3

C (1C)

F (1F) ∆ (δ C)a C

(12C) F

(3F) ∆ (δ C)a

δ (1-C) 168.8 159.4 +9.40 169.9 159.9 +10.0

δ (2-C) 121.4 193.9 -72.5 121.5 192.2 -70.7

δ (3-C) 156.4 77.2 +79.2 157.1 79.3 +77.8

δ (4-C) 73.9 80.8 -6.90 74.5 79.5 -5.00

δ (5-C) 73.8 73.1 +0.70 73.8 72.5 +1.30

δ (6-C) 64.9 64.4 +0.50 64.2 65.0 -0.80

δ (7-C) 110.6 111.3 -0.70 110.3 111.3 -1.00

δ (8-C) 25.2 24.7 +0.50 25.5 24.7 +0.80

δ (9-C) 25.7 24.8 +0.90 25.8 25.2 +0.60

δ (10-C) 72.1 121.9 -49.80 72.7 121.7 -49.0

δ (11-C) 133.0 129.0 +4.00 132.8 129.1 +3.70

δ (12-C) 119.6 39.6 +80.0 119.3 35.0 +84.0

δ (13-C) - - - 59.6 52.8 +6.80

aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen

compound F

RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

10

11

12

13

123

456

7

89

10

11

1213

C F

ORO

96


Proton # R = COCH3 R = CH2C6H5

C (7C)

F (2F) ∆ (δ H)a C

(15C) F

(4F) ∆ (δ H)a

δ (4-H) 5.18 4.94 +0.24 4.55 4.63 -0.08

δ (5-H) 4.29 4.28 +0.01 4.30 4.55 -0.25

δ (6-H’) 4.02 4.04 -0.02 4.03 4.00 +0.03

δ (6-H”) 4.15 4.14 +0.01 4.11 4.11 0.00

δ (8-CH3) 1.36 1.32 +0.04 1.36 1.34 +0.02

δ (9-CH3) 1.38 1.34 +0.04 1.38 1.35 +0.03

δ (10-H’) 4.78 5.23 -0.45 4.54 5.30 -0.76

δ (10-H”) 4.78 5.23 -0.45 4.54 5.31 -0.77

δ (11-H) 5.97 5.68 +0.29 5.94 5.82 +0.12

δ (12-H’) 5.28 2.76 +2.52 5.26 2.57 +2.69

δ (12-H”) 5.37 2.97 +2.40 5.34 2.75 +2.59

δ (13-H) - - - 5.48 5.48 7.37

4.90 5.25 7.30

+0.58 +0.23 +0.07


RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

10

11

12

13

123

456

7

89

10

11

1213

C F

ORO

97


Carbon # R = COCH3 R = CH2C6H5

C (7C)

F (2F) ∆ (δ C)a C

(15C) F

(4F) ∆ (δ C)a

δ (1-C) 166.4 158.2 +8.20 168.8 159.8 +9.00

δ (2-C) 130.1 186.3 -56.0 121.4 192.2 -70.8

δ (3-C) 143.8 78.4 +65.0 155.9 79.3 +76.6

δ (4-C) 74.5 81.1 -6.60 74.7 79.4 -4.70

δ (5-C) 72.8 72.6 +0.20 73.9 72.7 +1.20

δ (6-C) 65.1 65.1 0.00 65.2 64.9 +0.30

δ (7-C) 110.5 111.1 -0.60 110.3 111.2 -0.90

δ (8-C) 25.3 25.0 +0.30 25.6 24.9 +0.70

δ (9-C) 25.6 25.2 +0.40 25.8 25.4 +0.40

δ (10-C) 71.3 121.9 -50.6 72.5 121.9 -49.4

δ (11-C) 132.4 128.1 +4.30 132.8 137.5 -4.70

δ (12-C) 118.9 36.9 +82.0 119.3 36.3 +83.0

δ (13-C) 20.6 20.0 +0.60 73.5, 127.7, 128.5, 128.7, 129.1

67.1, 127.5, 127.9, 128.4, 129.2

+6.40, +0.20, +0.60, +0.30, -0.10

aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen

compound F

RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

10

11

12

13

123

456

7

89

10

11

1213

C F

ORO

98


RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

1011

12

14

123

456

7

89

10

11

12 13

CF

ORO

CH3

CH3

13

14

Proton # R = H

C (3C)

F (5F) ∆ (δ H)a

δ (4-H) 4.69 4.75 -0.06

δ (5-H) 4.39 4.52 -0.13

δ (6-H’) 4.05 4.08 -0.03

δ (6-H’’) 4.17 4.18 -0.01

δ (8-CH3) 1.37 1.29 +0.08

δ (9-CH3) 1.41 1.32 +0.10

δ (10-H’) 4.49 5.18 -0.69

δ (10-H’’) 4.49 5.18 -0.69

δ (11-H) 5.80 5.79 +0.01

δ (12-H’) 5.64 2.71 +2.93

δ (13-H) 1.71 1.15 +0.56

δ (14-H) - - -

aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from

the corresponding values of the Claisen compound F

99


RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

1011

12

14

123

456

7

89

10

11

12 13

CF

ORO

CH3

CH3

13

14

Carbon # R = H

C (3C)

F (5F) ∆ (δ C)a

δ (1-C) 170.1 159.5 +10.6

δ (2-C) 120.8 195.3 -74.5

δ (3-C) 157.9 78.9 +79.0

δ (4-C) 74.6 80.0 -5.40

δ (5-C) 73.9 73.4 +0.50

δ (6-C) 65.0 64.4 +0.60

δ (7-C) 110.4 111.2 -0.80

δ (8-C) 25.3 24.8 +0.50

δ (9-C) 25.7 24.9 +0.80

δ (10-C) 72.3 118.6 -46.3

δ (11-C) 132.6 135.3 -2.70

δ (12-C) 125.8 43.8 +82.0

δ (13-C) 17.8 13.2 +4.60

δ (14-C) - - -

aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from

the corresponding values of the Claisen compound F

100


RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

1011

12

14

123

456

7

89

10

11

12 13

CF

ORO

CH3

CH3

13

14

Proton # R = COCH3 R = CH3

C (9C)

F (6F) ∆ (δ H)a C

(13C) F

(7F) ∆ (δ H)a

δ (4-H) 5.18 4.83 +0.35 4.51 4.63 -0.12

δ (5-H) 4.28 4.30 -0.02 4.27 4.45 -0.18

δ (6-H’) 4.01 4.04 -0.03 4.03 3.98 +0.05

δ (6-H’’) 4.14 4.13 +0.01 4.13 4.14 -0.01

δ (8-CH3) 1.36 1.33 +0.03 1.36 1.33 +0.03

δ (9-CH3) 1.38 1.35 +0.03 1.39 1.33 +0.06

δ (10-H’) 4.70 5.22 -0.50 4.52 5.14 -0.62

δ (10-H’’) 4.70 5.22 -0.52 4.52 5.17 -0.65

δ (11-H) 5.83 5.73 +0.10 5.82 5.69 +0.13

δ (12-H’) 5.64 3.17 +2.47 5.66 2.70 +2.96

δ (13-H) 1.73 1.10 +0.63 1.74 1.10 +0.64

δ (14-H) 2.31 2.16 +0.15 4.14 3.73 +0.41


101


RO

OOO O

O

CH3H3C

H

OO O

O

CH3H3C

H123

456

7

89

1011

12

14

123

456

7

89

10

11

12 13

CF

ORO

CH3

CH3

13

14

Carbon # R = COCH3 R = CH3

C (9C)

F (6F) ∆ (δ C)a C

(13C) F

(7F) ∆ (δ C)a

δ (1-C) 166.5 158.0 +8.50 169.1 159.8 +9.30

δ (2-C) 132.2 187.5 -55.0 121.4 192.6 -71.2

δ (3-C) 143.8 77.3 +66.0 157.3 78.5 +78.8

δ (4-C) 74.4 79.7 -5.30 74.5 81.1 -6.60

δ (5-C) 72.9 73.4 -0.50 73.9 72.9 +1.00

δ (6-C) 65.0 65.5 -0.50 65.2 65.2 0.00

δ (7-C) 110.5 111.0 -0.50 110.3 111.2 -0.90

δ (8-C) 25.3 25.2 +0.10 25.6 24.9 +0.70

δ (9-C) 25.6 25.5 +0.10 25.8 25.4 +0.40

δ (10-C) 71.4 119.7 -48.3 72.2 119.3 -47.1

δ (11-C) 130.1 134.7 -4.60 132.4 135.9 -3.50

δ (12-C) 125.4 42.1 +83.3 125.8 42.3 +83.5

δ (13-C) 17.7 12.8 +4.90 17.8 13.6 +4.20

δ (14-C) 20.5 166.3

19.9 170.9

+0.60 -4.60

59.6 54.6 +5.00

aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F

102

4.5 Comparative Analysis and Identificaton of Products of C3-O to C2 and C2-O

to C3 Claisen Rearrangement of L-Ascorbic Acid Derivatives

The Claisen rearrangement of C3-O-allyl ascorbate derivatives listed in Table 11-

18 quantitatively produced the C2-allylated products in about 6 h in boiling toluene. The

1H-NMR analysis of the crude reaction mixtures revealed that both possible

diastereomers were produced with more than 75% diastereomeric excess. In contrast to

the smooth rearrangement of C3-O-allyl ascorbate derivatives under relatively mild

conditions (Scheme 14-16), the rearrangement of their C2-O-allyl counterparts (Scheme

17) was found to be much slower and required much more drastic reaction conditions.

For example, C2-O-allyl ascorbate derivatives typically require about 12 h to 24 h

refluxing in toluene for the complete conversion in comparison to 6 h for corresponding

C3-O-allyl ascorbate derivatives. In addition, only a single diastereomer was detected by

1H-NMR analysis of the crude rearranged reaction mixtures of C2-O-allyl derivatives

(Table 19-26). Sterically more hindered C2-O-allyl ascorbate derivatives such as 11C &

14C do not undergo any significant rearrangement, even in boiling xylene or styrene for

72 h.

103

Table 27 Comparison of Diastereoisomers of Allyl-L-Galactono-γ-Lactone

O

OO

O O

OR2

CH3H3C

H

R1

O

OO O

O

OR2

CH3H3C

H

Reflux / toluene 11 2

2 33

4 45

5

6

6

R1

7

89

10

1112

13

14

14

12

10

9

11

13

7

8

A E

R2O

OO

O O

O

CH3H3C

H

R1

OO

O O

CH3H3C

H

OOR2

R1

Reflux / toluene1

23

456

89

10

11 12

13

14

7

FC

9 8

7

6 5123

4

12

13

10 1114

Carbon # R1 = H & R2 = H R1 = H & R2 = COCH3

A (4A)

E (1E)

∆a (δ C)

C (1C)

F (1F)

∆a (δ C) A

(9A) E

(2E) ∆a

(δ C) C

(7C) F

(2F) ∆a

(δ C)

δ (1-C) 171.0 172.6 -1.60 168.8 159.4 +9.40 167.6 170.9 -3.30 166.4 158.2 +8.20 δ (2-C) 119.2 74.5 +44.7 121.4 193.9 -72.5 114.6 73.8 +40.8 130.1 186.3 -56.0 δ (3-C) 148.2 205.5 -57.3 156.4 77.2 +79.2 159.5 201.0 -41.5 143.8 78.4 +65.0 δ (4-C) 75.6 81.5 -5.90 73.9 80.8 -6.90 75.3 82.1 -6.80 74.5 81.1 -6.60 δ (5-C) 74.3 72.0 +2.30 73.8 73.1 +0.70 73.7 73.7 0.00 72.8 72.6 +0.20 δ (6-C) 65.3 64.8 +0.50 64.9 64.4 +0.50 65.2 65.2 0.00 65.1 65.1 0.00 δ (10-C) 72.3 122.9 -50.6 72.1 121.9 -49.8 72.5 120.8 -48.3 71.3 121.9 -50.6 δ (11-C) 132.2 127.6 +4.60 133.0 129.0 +4.00 131.0 128.3 +2.70 132.4 128.1 +4.30 δ (12-C) 119.1 39.8 +79.0 119.6 39.6 +80.0 119.6 34.5 +85.1 118.9 36.9 +82.0 δ (13-C) - - - - - - - - - 20.6 20.0 +0.60

δ (14-C) - -

- -

- -

- -

- -

- - 20.3

166.8 19.2 169.7

+1.10 -2.90

- -

- -

- -

Carbon # R1 = H & R2 = CH3 R1 = CH3 & R2 = COCH3

A (11A)

E (3E)

∆a (δ C)

C (12C)

F (3F)

∆a (δ C) A

(8A) E

(6E) ∆a

(δ C) C

(9C) F

(6F) ∆a

(δ C)

δ (1-C) 171.8 172.8 -1.00 170.1 159.5 +10.6 167.6 170.3 -2.70 166.5 158.0 +8.50 δ (2-C) 119.1 74.4 +44.7 120.8 195.3 -74.5 114.4 77.3 +37.1 132.2 187.5 -55.0 δ (3-C) 148.6 205.7 -57.1 157.9 78.9 +79.0 159.7 200.9 -41.2 143.8 77.3 +66.0 δ (4-C) 75.7 81.8 -6.10 74.6 80.0 -5.40 75.3 84.8 -9.50 74.4 79.7 -5.30 δ (5-C) 74.4 74.3 +0.10 73.9 73.4 +0.50 73.7 75.1 -1.40 72.9 73.4 -0.50 δ (6-C) 65.3 64.8 +0.50 65.0 64.4 +0.60 65.2 65.2 0.00 65.0 65.5 -0.50 δ (10-C) 72.4 120.0 -47.6 72.3 118.6 -46.3 72.6 120.6 -48.0 71.4 119.7 -48.3 δ (11-C) 132.6 134.3 -1.70 132.6 135.3 -2.70 133.2 132.9 +0.30 130.1 134.7 -4.60 δ (12-C) 125.2 44.3 +80.9 125.8 43.8 +82.0 124.0 41.4 +82.6 125.4 42.1 +83.3 δ (13-C) 17.8 12.5 +5.30 17.8 13.2 +4.60 17.7 12.3 +5.40 17.7 12.8 +4.90

δ (14-C) - -

- -

- -

- -

- -

- - 20.2

167.0 19.2 169.3

+1.00 -2.30

20.5 166.3

19.9 170.9

+0.60 -4.60

aThe difference in 13C-NMR chemical shifts (∆) was calculated by subtracting the chemical shifts of various derivatives (E & F) from their corresponding starting materials (A & C).

104

The relative unfavorability for the rearrangement of C2-O-allyl in comparison to C3-O-

allyl derivatives could be due to a combination of steric and electronic effects. First, the

steric constraints on the transition state for the C2-O to C3 allyl migration (Scheme 14-

16) is more pronounced relative to that of the C3-O to C2 migration (Scheme 17), due to

the presence of a bulky 1,2-O-isopropylidene-1,2-ethanodiol moiety at the C4 of the

molecule. Second, the relatively high lability of C3-O-allylic ether linkage compared to

that of the C2-O-allylic ether linkage due to the direct interaction of the C3-O with the

conjugated enone moiety could also facilitate the rearrangement to produce the

thermodynamically more stable C2-allylated products. Therefore, the facile

rearrangement of the C3-O-allyl derivatives in comparison to the non-catalyzed

conventional Claisen rearrangement which requires high temperatures (150-240 oC)

suggests that the thermodynamic lability of C3-O-allyic ether bond facilitates an efficient

rearrangement to the C2 allylated products. This notion is further supported by the

observation that the diallyl ascorbate derivative such as 12A exclusively rearranged to

produce the C2-allylated product, 4E, and not the potential, corresponding C3-allylated

product.

A comparative 13C-NMR spectra analysis of C3-O to C2 and C2-O to C3 (Table

27) rearranged products show some interesting and characteristic features which could be

used in their unequivocal identification. For example, the C3 and C2 carbonyl signals of

two series of rearranged products, E and F, appear in the ranges of 205.7–200.9 and

186.3–195.3 respectively. The significant upfield shift of the 13C-NMR signals of C2

carbonyls of F series must be due to the electronic effects of the adjacent C1 lactone-

carbonyl in comparison to that of the isolated C3 carbonyl group of E series. Similarly,

105

the C1 13C-NMR carbonyl signals of the two series were also quite distinguishable and

appear in the ranges of 170.3–172.8 for E and 158–159.4 for F, again due to the

electronic effects of the C2 carbonyl of F in comparison to that of the C3 carbonyl of

E.215-216 Interestingly, C4 13C-NMR signals of the two series of rearranged products were

not significantly different. However, C4 13C-NMR signals of both E and F series show a

significant downfield shift (∆δ in the range of 5.3–9.5) in comparison to the

corresponding starting materials (A and C). This most likely reflects the deshielding

effects of the disappearance of the conjugated enone moiety of the starting materials due

to the rearrangement. Therefore, the comparative analysis of the 13C-NMR characteristics

of the starting materials and their products could conveniently be used to distinguish

between the two series of Claisen rearranged products (E and F).

4.6 Stereochemistry of Products of C3-O to C2 and C2-O to C3 Claisen

Rearrangement of L-Ascorbic Acid Derivatives

The intrinsic stereochemistry at C4 and C5 positions of all the starting materials

(A & C series), rearranged products (E & F series) and their derivatives are fixed, since

L-ascorbic acid is used in all cases. The stereochemistry of the C2 of the Claisen-

rearranged products (E) was assigned by their NMR spectroscopic characteristics and

confirmed by X-ray crystallographic studies. 191 For example, we unequivocally

established that the least-bulky C3-O-allyl Claisen substrate, 4A (A series), yielded a

major diastereomer (~90%) which is C2 allylated from the bottom face of the lactone ring

(A to E rearrangement). Therefore, we believe the preferential migration of the allylic

106

functionality from the bottom face of the lactone ring must be primarily due to the steric

constraints imposed by the bulky C4 side chain (1,2-O-isopropylidene-1,2-ethanediol

moiety) on the top face of the molecule. Based on these arguments, we also concluded

that C2-O-allyl (C series) Claisen rearrangements must exclusively occur from the

bottom face of the molecule, since the steric constraints of the C4 bulky substituent must

even be more pronounced for the C2-O to C3 migrations (C to F rearrangement) in

comparison to C3-O to C2 migration (A to E rearrangement). The importance of the C4

bulky side chain group (1,2-O-isopropylidene-1,2-ethanediol moiety) in the control of the

new stereogenic center was more profound in the C2-O to C3 Claisen rearrangement

(Scheme 17). For example, C2-O-crotyl (3C, 9C, & 13C) Claisen substrates rearranges to

give two additional chiral centers at their C3 and C12 positions (5F, 6F & 7F) and

therefore could have four isomers. However, since we detected only one major product

(~98%) from these Claisen substrates, it is safe to conclude that the Claisen

rearrangement went through the usual chairlike transition state conformation to mainly

produce products having the migrated C2-O-allylic group (crotyl) in opposite position

(trans) to the C4 bulky side chain group, especially given that they are brought much

more closer to each other than in the C3-O to C2 migrated products (Scheme 14-16).

However, 5,6-O-isopropylidene-2-O-trans-cinnamyl-3-O-acetyl-L-asorbic acid (11C) and

5,6-O-isopropylidene-2-O-trans-cinnamyl-3-O-methyl-L-asorbic acid (14C) were not

susceptible to sigmatropic rearrangement even under vigorous conditions (refluxing in

xylene and styrene for over 72 h).

107

Scheme 18 C3-O (A) to C2 (E) Claisen Rearrangement Transition-State Geometry

OC1

OR 2

H

H

H

HC4

R1

C1C4

OR 2OH2C

CC

R1

Boat

O

OO O

OOR 2

CH 3H3C

CH

CH 2

12

34

10

11

12

14

R113

O

OO O

OOR 2

CH 3H3C

CH

CH 2

12

34

10

11

12

14

13R1

=

10 14

1112

13

E,Z

H

H

OOO

H3C CH 3

O

H

1

23

4O

R1R2Othreo

OR1

C1

OR 2

HH

HH

C4

Chair

=OOO

H3C CH 3

OH

O

OR 2R1

123

4

erythro

(E)

(A)

Major Pathway

Minor Pathway

Schemes 15-17

(7E & 8E)

(S)

(R) (S)

(S)

H

(S)(S)(R)

(R)H

Scheme 19 C2-O (C) to C3 (F) Claisen Rearrangement Transition-State Geometry

O

OO

H3CCH 3

OH

OR2O

R12

4

31

56

7

89

C4 C1

R2O O CH 2

C C

R1

OC4

OR 2

H

H

H

HC1

R1

=

=

R2O

OO

O O

O

CH 3H3C

H 12

4

56

7

89

C

3

10

111213

CH 2

R1

14

R2O

OO

O O

O

CH 3H3C

H 12

4

C

3

10

111213

CH 2

R1

14

erythro

Boat

10

11 12

13

14

Z,EH

H

OR2O

C4

R1

H H

HH

C1

Chair

O

OO

H3CCH 3

OH

2

4

3

1

56

7

89

OR1 R2O threo

(F)

(C)

MinorPathway

MajorPathway

Schemes 19-20

(S)

(R)

(R)

(S)

(R)

(S)

(R)

(R)

H

H

108

This is in sharp contrast to 5,6-O-isopropylidene-2-O-acetyl-3-O-trans-cinnamyl-L-

asorbic acid (10A), which was susceptible to facile Claisen rearrangement even under

very mild conditions (at room temperature, 100% conversion was obtained within 3

weeks) to yield two diastereomeric products with a ratio similar to that obtained under

reflux in toluene for 6 h (Scheme 16). Since the rearrangement of C2-O-trans-crotyl (3C,

9C & 13C), C3-O-trans-crotyl (3A & 8A) and C3-O-trans-cinnamyl (10A) derivatives

generate an additional chiral center at their C12 positions, we have thoroughly

investigated these reactions in order to establish the stereochemistry of the C12 site on

these derivatives. The factors affecting the stereochemical outcome of the products of

thermal Claisen rearrangement have been extensively studied and established that the

stereochemistry of the newly generated stereogenic centers is controlled by the steric and

electronic features of the system in the transition state.192-204 The restriction imposed by

the orbital symmetry rules on the highly ordered cyclic transition state allows excellent

prediction of the stereochemical result.203 One of the well established strategies that has

been developed to control the stereoselectivity of Claisen rearrangement, is the

intraannular stereo-selection (i.e., the stereogenic elements accounting for selectivity are

incorporated into the cyclic structure of the transition state) by achiral auxiliary (i.e., e.g.

CH3 of crotyl derivatives and C6H5 of cinnamyl derivatives) intrinsic to the

stereochemistry of the vinylic double bond. Therefore, this makes it possible to determine

the stereoselectivty of the rearrangement process regardless of the optical purity of the

product’s newly created chiral centers, in which case, the E/Z selectivity rule of the new

double bond is considered. The Claisen rearrangement progresses preferentially through a

chair-like transition state in order to minimize the steric interactions of various

109

substituents as illustrated in Schemes 18 & 19. Thus, the relative stereochemistry

(erythro/threo) at the newly generated allylic stereo-centers is controlled by the relative

geometry of the allylic double bond of the Claisen substrate.203 Therefore, as shown in

Schemes 18 & 19, (Z,Z) and (E,E) Claisen substrates produce threo products, while (E,Z)

and (Z,E) Claisen substrates produce erythro as the major products. Based on these

arguments, and since the allylic functionality migrates from the bottom face of the

lactone ring during the C3-O to C2 and C2-O to C3 rearrangements as argued above, we

conclude that the major isomer of the Claisen-rearranged products of trans-crotyl (3A,

8A, 3C, 9C & 13C) and trans-cinnamyl (5A) derivatives must have R stereochemistry at

their C12 as shown in Schemes 18 & 19. In cyclic systems such as the five-membered

lactone ring, conformational constraints can take priority over the intrinsic preference for

the chair-like transition state in both Cope and Claisen rearrangements and consequently

lead to a partial involvement if not the dominance of a boat-like transition state structure,

204 which might explain why 10A produced 9E (S stereochemistry at C12) and 10E (R

stereochemistry at C12) as major and minor diastereomeric isomers respectively (Scheme

18). 1H- and 13C-NMR of the C2-O acetylation product of 7E revealed this product, 8E as

the same minor diastereomeric compound 10E, that was obtained as a minor product

from 10A Clasien rearrangement. The NMR signal patterns, the selective 2D-NMR

spectra and X-ray structures helped to unequivocally identify all the Claisen rearranged

products (E & F Series).

110

4.7 Chemo- and Diastereo-Selective Reduction of Claisen Rearranged Products (E &

F Series) of L-Ascorbic Acid Derivatives

In order to further explore the chemistry and synthetic utility of the Claisen-

rearranged products E and F, we carried out the selective reduction of their C3- and C2-

keto groups by conventional methods. The reduction of the major isomers of the

rearranged products of the E series (Scheme 20) with sodium borohydride in ethanol

proceeds with high chemo- and diastereo-selectivity in producing a ~7:1 diastereomeric

mixtures of alcohol products, G Series (diastereomeric excess), in almost quantitative

yield. The diastereomeric mixture was easily separated by normal phase silica gel column

chromatography using ethyl acetate/hexane solvent system. Subsequent acetylation of G

under the reaction conditions of Scheme 20 gave the corresponding acetates (H Series) in

quantitative yield. In contrast to the E series, the attempts made to reduce the rearranged

C2-keto products (F series) gave uncharacterizable complex reaction mixtures under the

same conditions. However, under carefully controlled conditions, we were able to obtain

a clean reduced product I from 2F in 60% yield (Scheme 21) after careful silica gel

chromatography with ethyl acetate/hexane solvent system.

The structures of the diastereomeric excess of sodium borohydride reduced

products (G) and their acetates (H) were unequivocally identified by their 1H- and 13C-

NMR characteristics. The stereochemistry of reduction by NaBH4, which is a

nucleophile, although with relatively small steric demand, delivered the hydride from the

less hindered direction perhaps due to the bulkier C4 substituent (1,2-O-isopropylidene-

111

1,2-ethanediol moiety on the top face of the molecule) generating cis alcohol

diastereomers (G), which were identified by NOESY-NMR studies.

Scheme 20 The Reduction Products of 5, 6-O-Isopropylidene-2-Allyl-3-Keto-L-

Galactono-γ-Lactones (E)

OO O

O

OH*

CH3H3C

HHO H R

NaBH4, EtOH12

345

6

OO O

O

OAc*

CH3H3C

HAcO H RCH2Cl2, -79 oC

(quant.)

(Ac)2O /DMAP / Et3N12

345

6E

G H

(E) (G) (H) R

1E 1G 1H H

4E 4G

*H = CH2CH=CH2

4H

*Ac = CH2CH=CH2

H

5E 5G 5H CH3

7E 7G 7H C6H5

It is understood that the involvement of sodium ions from NaBH4 is not required for

reduction to take place, 205-207 however; due to the inherent steric constraints of E, sodium

metal ions may well have played a crucial role in the stereochemical course of the

reduction to produce G. An elegant but ponderable explanation for the stereochemical

results (Scheme 20) is that reduction of starting materials (E) may possibly proceed via a

112

well-known chelation205-207 in which the sodium ion is coordinated by the β-carbonyl

oxygen atom (C3-O) and the oxygen atom of the α-hydroxy group of the E series (OR or

OHI) as depicted in Figure 13, thus, favoring and strengthening the diastereoselective

formation of products (G). Subsequent acetylation of these compounds (G) according to

Scheme 20 gave the corresponding acetate products (H) in quantitative yield.

Scheme 21 The Reduction Product of 5,6-O-Isopropylidene-3-Allyl-2-Keto-L-Galactono-γ-Lactone 2F

NaBH4, EtOHO

O

O

OH3C

H3C

H O

O CH3

H

OH

12

345

62F

I

Figure 13 Diastereoselective Reduction of C3-keto of E Series via Metal Chelation

H-

O

C4

C10

H'O

O

Na

C4

C10

C4

OHH

C10OH'

OH'

2

3

The nucleophile, H-, approaches C3-keto of the much more conformationally rigid chelated intermediate along the allylic side group trajectory, which is much smaller than the C4 (1,2-O-isopropylidene-1,2-ethanediol moiety on the top

face of the molecule) side group.

113

An unambiguous structural identification of reduced products G or I, and their

corresponding acetylated product H was established by their NMR spectroscopic

characteristics such as 1H-1H, 13C-1H and NOESY correlation studies. For example, upon

reduction, the C4-H doublets at 4.54-4.66 ppm in the starting materials (E, Scheme 20)

were shifted to doublet of doublets at 4.44-4.48 ppm for resultant cis-hydroxyl

derivatives (G, Scheme 20) or at 4.08-4.43 ppm for their corresponding acetates (H,

Scheme 20) as expected. In addition, a new 1H-NMR signals appeared in the range of

4.15-4.25 ppm for all G derivatives, and at 5.51-6.11 ppm for their corresponding

acetates H, due to the newly-generated C3-H of G and H upon reduction (Scheme 20).

Furthermore, the characteristic 13C-NMR β-carbonyl carbon singlets of starting materials

E in the range of 200.9-206.1 ppm had disappeared upon reduction and new C3 13C-

methine carbon signal (doublet) appeared in the range of 71.9-74.7 ppm for alcohols (G,

Scheme 20) and 72.2-73.0 ppm for corresponding acetates (H, Scheme 20). In contrast,

the reduction of the C2-carbonyl of C2-O to C3 rearranged product, 2F (I, Scheme 21),

resulted in one significant change in 1H-NMR and two visible changes in 13C-NMR of the

product. First, the appearance of a new 1H-NMR singlet at 4.09 ppm corresponding to the

newly-generated C2-H. Second, the C2-carbonyl carbon 13C-NMR singlet of the starting

material at 186.3 ppm is converted to a doublet at 71.8 ppm and there is a significant 11

ppm downfield shift of C1 carbon signal (from 158.2 ppm to 169.4 ppm) of the product

(I). These changes in the 1 H- and 13C-NMR characteristic, further confirms the

reduction of the C2-carbonyl of 2F to produce I (Scheme 21).

The stereochemistries of these compounds were confirmed by NOESY correlation

studies. For instance, an intense qualitative NOESY correlation between the newly

114

generated C3-H and the C4-H of all the products listed in Scheme 20 (G & H Series)

strongly suggests that the configuration of these protons are cis to each other and

identical to the overall configuration of L-gluono-γ-lactones. However, since the C2-O-

to C3-allylated rearranged product 2F and its reduced counterpart, I, showed no

significant qualitative NOESY correlation among the C4-H, C2-H, or C3-allylic-

substituent hydrogens (C12-H2), the C2-stereochemistry of the product I could not be

assigned with certainty. All the 1H- and 13C-NMR spectra of the sodium borohydride

reduced products of G or I, and corresponding acetylated products H series were in good

agreement with reported literature spectra for similar compounds.208-210

These studies have shown the importance of C2- and C3-allylated derivatives of

L-ascorbic acid and the potential for their applications in synthetic organic and

pharmaceutical chemistry. Also, it shows a new synthetic route to other interesting

compounds by possible derivatization of these L-ascorbic-acid-derived L-galactono-γ-

lactones (E and F series) through the reductive-amination of their keto groups. This could

potentially produce a series of biologically-active amines. We are currently working on

the chemo- and diastereo-selective reductive-amination of E and F series of compounds

and are currently working on developing a general method for synthesis and also the

complete structural characterization of all derivatives. A list of some compounds already

synthesized but yet to be fully characterized is listed in Figure 14.

115

Figure 14 Diastereoselective Reductive Amination Products of 1E, 5E and 1F (X, Y and Z Respectively)

O

O O

H

H3C CH3

O

H2NH OH

O

OO

H

H3CCH3

OH2N

HHO

CH3

H

O

OO

H

H3CCH3

O

HO NHX Y Z

116

CHAPTER 5

EXPERIMENTAL SECTION

General: All reagents and chemicals were obtained from various commercial sources

at the highest purity available and used without further purification. Chromatographic

separations were carried out using Davisil grade 1740 type 60A (200-424mesh, Fisher)

silica gel and the reaction products were eluted with a mixture of ethyl acetate and n-

hexane with varying ratios depending on the nature of the compound. The TLC analyses

were performed on pre-coated silica gel GF plates (250µm, Analtech) and the products

were observed under UV light and/or by exposure to iodine vapor. All solvents used were

dried with appropriate drying agents and freshly distilled. NMR Spectra were recorded on

a Varian XL-300-NMR spectrometer operating at 300 MHz for 1H and at 75.4 MHz for

13C or on a Varian UNITY INOVA 400-NMR spectrometer operating at 400 MHz for 1H

and at 100.6 MHz for 13C. All chemical shifts are reported on the δ (ppm) scale relative

to TMS (0.00 ppm) for 1H-NMR and to CDCl3 (77.0 ppm) for 13C-NMR. The elemental

analyses were carried out at Desert Analytical, Tucson, Arizona. The exact mass FAB

experiments were performed at the University of Kansas, Mass Spectrometry laboratory,

Lawrence, Kansas.

Computational Modeling: The initial calculations were carried with AM1 semi-

empirical method using Winmopac v2 to obtain a reasonable initial model to serve as a

foundation for the subsequent ab initio calculations. All the ab initio calculations were

carried out by the density function B3LYP method and basis set 6-31G* using Gaussian

117

98 programs.213 The electrostatic potential diagrams of the electronic density were

obtained from these calculations. The electron density distributions of the molecules are

plotted as the electrostatic potential with the standard color-coding using the standard

built in features of the Gaussian 98 programs213 (order of electron density: blue < green <

yellow < red).

5,6-O-Isopropylidene-L-ascorbic Acid (1). This was synthesized in 82% yield

according to the procedure of Jung et al., 36 mp 204-206 oC [Lit.33 201-203 oC]: 1H-NMR

(400 MHz, D2O) δ 1.37 (6H, s), 4.17 (1H, dd, J = 9.1, 5.0 Hz), 4.31 (1H, dd, J = 9.1, 7.3

Hz), 4.59 (1H, ddd, J = 7.3, 5.0, 2.4 Hz), 4.91 (1H, d, J = 2.4 Hz); 13C-NMR (100 MHz,

D2O): δ 26.70, 27.51, 67.75, 75.65, 78.54, 113.46, 120.54, 158.37, 176.08.

5,6-O-Isopropylidene-3-O-methyl-L-ascorbic Acid (1A). This compound was

synthesized according to the procedure of Wimalasena and Mahindaratne.161 A mixture

of 1 (1 g, 4.63 mmol) and 1.2 equiv of K2CO3 (0.77 g, 5.56 mmol) in DMSO/THF (9:8)

were stirred for 20 min at room temperature. Then 1.2 equiv of methyl iodide (0.79 g,

5.56 mmol) in the same solvent was added dropwise and the mixture was vigorously

stirred for 4-6 h at room temperature. The reaction mixture was diluted (4-fold) with

water and extracted with ethyl acetate. The organic layer was thoroughly washed with

water and dried over anhydrous Na2SO4 and the solvents were removed under reduced

pressure. The product was purified by conventional silica gel column chromatography

using 4:1 n-hexane:ethyl acetate to give 91% yield as a viscous oil: 1H-NMR (300 MHz,

CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 4.02 (1H, dd, J = 8.5, 6.6 Hz), 4.13 (1H, dd, J = 8.5,

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6.7 Hz), 4.18 (3H, s), 4.23 (1H, dt, J = 6.7, 3.8 Hz), 4.53 (1H, d, J = 3.8 Hz); 13C-NMR

(75 MHz, CDCl3): δ 25.2, 25.6, 59.4, 65.0, 73.9, 75.3, 110.0, 119.5, 149.9, 171.2.

5,6-O-Isopropylidene-3-O-benzyl-L-ascorbic Acid (2A). This was synthesized from

1 and benzyl bromide in 86% yield as a semisolid using the same procedure as for 1A:

1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.02 (1H, dd, J = 8.6, 6.7 Hz),

4.10 (1H, dd, J = 8.6, 6.7 Hz), 4.26 (1H, dt, J = 6.7, 3.8 Hz), 4.57 (1H, d, J = 3.8 Hz),

5.52 (2H, m), 7.35-7.42 (5H, m); 13C-NMR (75 MHz, CDCl3): 25.5, 25.9, 65.3, 73.5,

74.2, 75.7, 110.3, 119.5, 128.4, 128.6, 128.7, 135.7, 148.6, 171.1.

5,6-O-Isopropylidene-3-O-trans-crotyl-L-ascorbic Acid (3A). This was synthesized

from 1 and trans-crotyl bromide in 72% yield as a light yellow oil using the same

procedure as for 1A: 1H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 1.75 (3H,

dq, J = 6.5, 1.5 Hz), 4.02 (1H, dd, J = 8.6, 6.7 Hz), 4.13 (1H, dd, J = 8.6, 6.7 Hz), 4.26

(1H, dt, J = 6.7, 3.9 Hz), 4.55 (1H, d, J = 3.9 Hz), 4.89 (2H, m), 5.68 (1H, dtq, J = 15.3,

6.6, 1.5 Hz), 5.90 (1H, dtq, J = 15.3, 6.5, 1.5 Hz); 13C-NMR (75 MHz, CDCl3): δ 17.8,

25.6, 25.9, 65.3, 72.4, 74.4, 75.7, 110.3, 119.1, 125.2, 132.6, 148.6, 171.8.

5,6-O-Isopropylidene-3-O-allyl-L-ascorbic Acid (4A). This was synthesized from 1

and allyl bromide in 80% yield as a light transparent oil using the same procedure as for

1A: 1H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 4.04 (1H, dd, J = 8.6, 6.7

Hz), 4.15 (1H, dd, J = 8.6, 6.7 Hz), 4.28 (1H, dt, J = 6.6, 3.8 Hz), 4.58 (1H, d, J = 3.8

Hz), 4.97 (2H, d, J = 5.7 Hz), 5.31 (1H, dq, J = 10.4, 1.4 Hz), 5.41 (1H, dq, J = 17.2, 1.4

Hz), 6.01 (1H, ddt, J = 17.2, 10.4, 5.7 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.5, 25.9,

65.3, 72.3, 74.3, 75.6, 110.3, 119.1, 119.2, 132.2, 148.2, 171.0.

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5,6-O-Isopropylidene-3-O-trans-cinnamyl-L-ascorbic Acid (5A). This was

synthesized from 1 and cinnamyl chloride in 72% yield as light yellowish oil using the

same procedure as for 1A. However, this compound undergoes very fast and facile

Claisen rearrangement to its C2-allylated rearranged product (7E). Therefore, 7E was

characterized by NMR spectroscopy.

5,6-O-Isopropylidene-2-O-acetyl-3-O-methyl-L-ascorbic Acid (6A). This compound

was synthesized according to the procedure of Wimalasena and Mahindaratne.161 1A (1 g,

4.34 mmol) and 1.4 equiv of pyridine (0.481 g, 6.1 mmol) in CH2Cl2 was stirred for 20

min at room temperature and 1.2 equiv of acetyl chloride (0.409 g, 5.21 mmol) was

added drop-wise. The mixture was vigorously stirred until the solution became

homogenous and was further stirred for 2 h at room temperature. The reaction mixture

was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was

thoroughly washed with water and dried over anhydrous Na2SO4 and the solvents were

removed under reduced pressure. The product was isolated and purified with

conventional silica gel column chromatography using 7:1 n-hexane:ethyl acetate to give

90% yield as a transparent oil: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s),

2.31 (3H, s), 4.00 (3H, s), 4.02 (1H, dd, J = 8.5, 6.6 Hz), 4.15 (1H, dd, J = 8.5, 6.6 Hz),

4.29 (1H, dt, J = 6.6, 2.4 Hz), 5.14 (1H, d, J = 2.4 Hz); 13C-NMR (75 MHz, CDCl3): δ

20.5, 25.3, 25.6, 58.4, 65.1, 72.9, 74.4, 110.6, 131.3, 142.4, 166.3, 166.4.

5,6-O-Isopropylidene-2-O-acetyl-3-O-benzyl-L-ascorbic Acid (7A). This was

synthesized from 2A and acetyl chloride in 90% yield as a light yellow oil using the same

procedure as for 6A: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 2.22 (3H,

s), 4.07 (1H, dd, J = 8.6, 6.7 Hz), 4.14 (1H, dd, J = 8.6, 6.7 Hz), 4.38 (1H, dt, J = 6.7, 2.9

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Hz), 4.71 (1H, d, J = 2.9 Hz), 5.31 (1H, d, J = 11.5 Hz), 5.37 (1H, d, J = 11.5 Hz), 7.30-

7.50 (5H, m); 13C-NMR (75 MHz, CDCl3): δ 20.1, 25.5, 25.7, 65.1, 73.6, 73.9, 75.2,

110.5, 114.8, 127.5, 128.8, 129.0, 134.5, 159.8, 166.8, 167.5.

5,6-O-Isopropylidene-2-O-acetyl-3-O-trans-crotyl-L-ascorbic Acid (8A). This was

synthesized from 3A and acetyl chloride in 78% as a light yellow semisolid using the

same procedure as for 6A: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.40 (3H, s), 1.76

(3H, dq, J = 6.5, 1.6 Hz), 2.27 (3H, s), 4.07 (1H, dd, J = 8.6, 6.6 Hz), 4.15 (1H, dd, J =

8.6, 6.6 Hz), 4.36 (1H, dt, J = 6.6, 3.1 Hz), 4.66 (1H, d, J = 3.1 Hz), 4.73 (2H, m), 5.62

(1H, dtq, J = 15.4, 6.5, 1.6 Hz), 5.87 (1H, dtq, J = 15.4, 6.5, 1.6 Hz); 13C-NMR (75 MHz,

CDCl3): δ 17.7, 20.2, 25.5, 25.7, 65.2, 72.6, 73.7, 75.3, 110.5, 114.4, 124.0, 133.2, 159.7,

167.0, 167.6.

5,6-O-Isopropylidene-2-O-acetyl-3-O-allyl-L-ascorbic Acid (9A). This was

synthesized from 4A and acetyl chloride in 70% yield as a colorless semisolid using the

same procedure as for 6A: 1H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.41 (3H, s), 2.27

(3H, s), 4.08 (1H, dd, J = 8.6, 6.6 Hz), 4.16 (1H, dd, J = 8.6 , 6.6 Hz), 4.38 (1H, dt, J =

6.6, 3.0 Hz), 4.69 (1H, d, J = 3.0 Hz), 4.81 (2H, m), 5.35 (1H, dq, J =10.6, 1.6 Hz), 5.40

(1H, dq, J = 17.7, 1.6 Hz), 5.95 (1H, ddt, J = 17.7, 10.6, 5.5 Hz); 13C-NMR (75 MHz,

CDCl3): δ 20.3, 25.5, 25.8, 65.2, 72.5, 73.7, 75.3, 110.6, 114.6, 119.6, 131.0, 159.5,

166.8, 167.6.

5,6-O-Isopropylidene-2-O-acetyl-3-O-trans-cinnamyl-L-ascorbic Acid (10A). A

crude mixture of 5,6-O-isopropylidene-2-O-acetyl-L-ascorbic acid, 2D (1 g, 3.87 mmol)

and 1.2 equiv of K2CO3 (0.64 g, 4.65 mmol) in DMSO/THF (9:8) were stirred for 20 min

at room temperature. Then 1.2 equiv of cinnamyl bromide (0.92 g, 4.65 mmol) in the

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same solvent was added dropwise and the mixture was vigorously stirred for 4-6 h at

room temperature. The reaction mixture was diluted (4-fold) with water and extracted

with ethyl acetate. The organic layer was thoroughly washed with water and dried over

anhydrous Na2SO4 and the solvents were removed under reduced pressure. The product

was purified by conventional silica gel column chromatography using 7:1 n-hexane:ethyl

acetate to give 76% yield as colorless crystals: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H,

s), 1.41 (3H, s), 2.28 (3H, s), 4.09 (1H, dd., J = 8.6, 6.6 Hz), 4.17 (1H, dd., J = 8.6, 6.6

Hz), 4.40 (1H, d t, J = 6.6, 2.9 Hz), 4.70 (1H, d, J = 2.9 Hz), 4.90-5.03 (2H, m), 6.29 (1H,

dt, J = 15.9, 6.3 Hz), 6.71 (1H, d, J = 15.9 Hz), 7.23-7.36 (3H, m), 7.37-7.42 (2H, m);

13C-NMR (75 MHz, CDCl3): δ 20.2, 25.5, 25.7, 65.2, 72.6, 73.6, 75.3, 110.6, 114.7,

121.6, 126.7, 128.6, 128.7, 129.3, 135.5, 159.7, 166.9, 167.6.

5,6-O-Isopropylidene-3-O-allyl-2-O-methyl-L-ascorbic Acid (11A). This was

prepared from 4A and methyl iodide (Scheme 11) in 59% yield as a semi-solid: 1H-NMR

(300 MHz, CDCl3) δ 1.36 (3H, s), 1.40 (3H, s), 3.85 (3H, s), 4.04 (1H, dd, J = 8.5, 6.6


Hz), 4.93 (2H, dt, J = 5.6, 1.4 Hz), 5.33 (1H, dq, J = 10.5, 1.3 Hz), 5.40 (1H, dq, J = 17.2,

1.5 Hz), 5.98 (1H, ddt, J = 17.2, 10.5, 5.6 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.5, 25.8,

59.9, 65.3, 72.2, 74.0, 74.6, 110.3, 118.8, 123.0, 131.8, 155.1, 168.7.

5,6-O-Isopropylidene-2,3-O-diallyl-L-ascorbic Acid (12A). This was prepared from

4A and allyl bromide (Scheme 11) in 61% yield as a light yellow oil: 1H-NMR (300

MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.04 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (1H, dd, J

= 8.5, 6.7 Hz), 4.30 (1H, dt, J = 6.7, 3.2 Hz), 4.55 (1H, d, J = 3.2 Hz), 4.62 (2H, m), 4.94

(2H, dt, J = 5.6, 1.4 Hz), 5.27 (1H, dq, J = 10.8, 1.5 Hz), 5.31 (1H, dq, J = 10.5, 1.3 Hz),

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5.35 (1H, dq, J = 17.2, 1.5 Hz), 5.39 (1H, dq, J = 17.3, 1.5 Hz), 5.98 (1H, ddt, J = 17.3,

10.5, 5.6 Hz), 5.99 (1H, ddt, J = 17.2, 10.3, 6.1 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.6,

25.9, 65.3, 72.3, 72.5, 74.0, 74.7, 110.3, 118.9, 119.2, 121.5, 131.9, 132.9, 155.7, 168.9.

5,6-O-Isopropylidene-2,3-O-dibenzyl-L-ascorbic Acid (13A). This was prepared

from 2A and benzyl bromide (Scheme 11) in 88% yield as a light transparent semi-solid:



5.07 (1H, d, J = 11.3 Hz), 5.11 (1H, d, J = 11.7 Hz), 5.14 (1H, d, J = 11.3 Hz), 5.20 (1H,

d, J = 11.7 Hz), 7.15-7.25 (2H, m), 7.30-7.50 (8H, m); 13C-NMR (75 MHz, CDCl3): δ

25.7, 25.9, 65.3, 73.5, 73.7, 74.0, 74.6, 110.2, 121.2, 127.7, 128.6, 129.1, 135.4, 136.0,

156.4, 168.9.

5,6-O-Isopropylidene-2,3-O-dicinnamyl-L-ascorbic Acid (14A). This was prepared

from 5C and cinnamyl bromide (Scheme 11) in 75 % yield as a light transparent semi-

solid: 1H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.36 (3H, s), 4.05 (1H, dd, J = 8.5, 6.8


Hz), 4.82 (2H, m), 5.13 (2H, m), 6.32 (1H, dt, J = 15.9, 6.2 Hz), 6.36 (1H, dt, J = 15.9,

6.6 Hz), 6.68 (1H, d, J = 15.9 Hz), 6.69 (1H, d, J = 15.9 Hz), 6.36 (1H, dt, J = 15.8, 6.6

Hz), 6.68 (1H, d, J = 15.9 Hz), 6.69 (1H, d, J = 15.9 Hz), 7.27-7.38 (10H, m); 13C-NMR

(75 MHz, CDCl3): δ 25.6, 25.8, 65.3, 72.1, 72.3, 73.9, 74.7, 110.4, 121.3, 122.8, 123.8,

126.7, 128.1, 128.3, 128.6, 128.7, 134.7, 135.0, 135.8, 136.2, 156.1, 169.0.

5,6-O-Isopropylidene-3-Keto-2-benzyl-L-galactono-γ-lactone (2B). This was

isolated as a minor product from a reaction mixture of 1 and benzyl bromide (Scheme 7)

as a colorless semi-solid: 1H-NMR (300 MHz, CDCl3) δ 1.31 (3H, s), 1.37 (3H, s), 1.65-

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1.95 (1H, br s), 3.25 (1H, d, J = 12.6 Hz), 3.27 (1H, d, J = 12.6 Hz), 3.91 (1H, dd, J =

8.7, 7.0 Hz), 3.92 (1H, d, J = 2.0 Hz), 4.06 (1H, dd, J = 8.7, 7.0 Hz), 4.42 (1H, dt, J = 7.0,

2.0 Hz), 6.02 (1H, dt, J = 15.9, 7.8 Hz), 7.12-7.16 (2H, m), 7.26-7.35 (3H, m).

5,6-O-Isopropylidene-3-Keto-2-(trans-1-methyl-1-prop-2-enyl)-L-galactono-γ-

lactone (3B). This was isolated as a minor product from a reaction mixture of 1 and

trans-crotyl bromide (Scheme 7): 1H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.41 (3H,

s), 1.61-1.79 (3H, m), 2.60 (2H, dt, J = 7.5, 1.1 Hz), 3.00-3.40 (1H, br s), 4.08 (1H, dd, J

= 8.7, 7.0 Hz), 4.19 (1H, dd, J = 8.7, 7.0 Hz), 4.53 (1H, dt, J = 7.0, 2.0 Hz), 4.62 (1H, d, J

= 2.0 Hz), 5.29 (1H, dtq, J = 15.2, 7.6, 1.7 Hz), 5.69 (1H, dtq, J = 15.2, 6.5, 1.2 Hz); 13C-

NMR (75 MHz, CDCl3): δ 18.1, 25.3, 25.5, 38.9, 64.9, 72.2, 74.5, 81.4, 111.2, 119.7,

134.4, 172.8, 205.8.

5,6-O-Isopropylidene-3-Keto-2-(trans-3-phenyl-1-prop-2-enyl)-L-galactono-γ-

lactone (5B). This was isolated as a major product from a reaction mixture of 1 and

trans-cinnamyl bromide (Scheme 7) as a yellowish viscous oil: 1H-NMR (300 MHz,

CDCl3) δ 1.32 (3H, s), 1.41 (3H, s), 2.83 (2H, d, J = 7.8 Hz), 4.06 (1H, dd, J = 8.7, 7.0


Hz), 6.02 (1H, dt, J = 15.7, 7.8 Hz), 6.54 (1H, d, J = 15.8 Hz), 7.20-7.40 (5H, m); 13C-

NMR (75 MHz, CDCl3): δ 25.3, 25.5, 39.2, 64.8, 72.2, 74.5, 81.5, 111.2, 118.1, 126.5,

128.2, 128.6, 136.0, 137.5, 172.8, 205.8.

5,6-O-Isopropylidene-2-O-allyl-L-ascorbic Acid (1C). A solution of 2 equivalents of

potassium tert-butoxide (t-BuOK) (1.04 g, 9.26 mmol) in dry DMSO/THF (3:2) was

added dropwise to a solution of 1 (1 g, 4.63 mmol) in the same solvent at -10 oC under

nitrogen to produce a bright yellow solution with an orange tint. The stirring of the

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mixture was continued for about 2 min after which, 1.1 equivalents of allyl bromide (0.62

g, 5.09 mmol) in the same solvent was added dropwise over a period of 3 min with

stirring continued for an additional 5 min at -10 oC. The cooling bath was removed, and

the reddish orange solution was stirred for 3 h at room temperature. The reaction mixture

was quenched with a cold solution of 0.25 M HCl (20 mL) and extracted with ethyl

acetate (3 X 100 mL). The organic layer was dried over Na2SO4 and the solvents were

removed under reduced pressure. The product was purified by conventional silica gel

column chromatography using 3:1 n-hexane:ethyl acetate to give 80% yield as a white

solid:214 1H-NMR (300 MHz, CDCl3) δ 1.38 (3H, s), 1.43 (3H, s), 4.02 (1H, dd, J = 9.0,

6.8 Hz), 4.16 (1H, dd, J = 9.0, 6.8 Hz), 4.43 (1H, dt, J = 6.8, 3.6 Hz), 4.62 (2H, dt, J =

6.3, 1.2 Hz), 4.72 (1H, d, J = 3.6 Hz), 5.28 (1H, dq, J = 10.2, 2.0 Hz), 5.36 (1H, dq, J =

17.3, 2.0 Hz), 5.98 (1H, ddt, J = 17.3, 10.2, 6.3 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.2,

25.7, 64.9, 72.1, 73.8, 73.9, 110.6, 119.6, 121.4, 133.0, 156.4, 168.8. Anal. Calcd for

C12H16O6: C, 56.24; H, 6.29; O, 37.47. Found: C, 56.31; H, 6.21; O, 37.48.

5,6-O-Isopropylidene-2-O-methyl-L-ascorbic Acid (2C). This was synthesized from

1 and methyl iodide in 91% yield as a semisolid using the same procedure as for 1C: 1H-

NMR (300 MHz, CDCl3) δ 1.40 (3H, s), 1.45 (3H, s), 3.87 (3H, s), 4.07 (1H, dd, J = 9.0,

6.8 Hz), 4.19 (1H, dd, J = 9.0, 6.8 Hz), 4.46 (1H, dt, J = 6.8, 3.3 Hz), 4.71 (1H, d, J = 3.3

Hz); 13C-NMR (75 MHz, CDCl3); δ 25.4, 25.8, 59.6, 65.1, 73.96, 74.2, 111.0, 123.1,

155.8, 169.3. Anal. Calcd for C10H14O6: C, 52.17; H, 6.13; O, 41.70. Found: C, 52.43; H,

6.34; O, 41.23.

5,6-O-Isopropylidene-2-O-trans-crotyl-L-ascorbic Acid (3C). This was synthesized

from 1 and trans-crotyl bromide in 72% yield as a light yellow oil using the same

125

procedure as for 1C: 1H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.41 (3H, s), 1.71 (3H,

dq, J = 6.9, 1.7 Hz), 4.05 (1H, dd, J = 8.5, 6.8 Hz), 4.17 (1H, dd, J = 8.5, 6.8 Hz), 4.39

(1H, dt, J = 6.8, 3.6 Hz), 4.49 (2H, dt, J = 6.6, 1.0 Hz), 4.69 (1H, d, J = 3.6 Hz), 5.64

(1H, dtq, J = 15.7, 6.9, 1.6 Hz), 5.797 (1H, dtq, J = 15.7, 6.9, 1.6 Hz); 13C-NMR (75

MHz, CDCl3): δ 17.8, 25.3, 25.7, 64.99, 72.3, 73.9, 74.6, 110.4, 120.8, 125.8, 132.6,

157.9, 170.1. HRMS (FAB +) m/z exact mass calcd for C13H19O6 (M + 1) 271.1180,

found m/z 271.1182.

5,6-O-Isopropylidene-2-O-benzyl-L-ascorbic Acid (4C). This was synthesized from

1 and benzyl bromide in 83% yield as a semisolid using the same procedure as for 1C:

1H-NMR (400 MHz, CDCl3) δ 1.34 (3H, s), 1.37 (3H, s), 3.86 (1H, dd, J = 8.2 , 6.7 Hz),


5.11 (2H, two d), 7.31-7.41 (5H, m); 13C-NMR (100 MHz, CDCl3): δ 25.3, 25.8, 64.9,

73.3, 73.9, 74.2, 110.4, 121.1, 128.4, 128.5, 128.7, 136.4, 157.5, 169.3. Anal. Calcd for

C16H18O6: C, 62.74; H, 5.92; O, 31.34. Found: C, 62.95; H, 5.80; O, 31.25.

5,6-O-Isopropylidne-2-O-trans-cinnamyl-L-ascorbic Acid (5C). This was

synthesized from 1 and trans-cinnamyl bromide in 87% yield as a semisolid using the

same procedure as for 1C: 1H-NMR (300 MHz, CDCl3) δ 1.30 (3H, s), 1.35 (3H, s), 3.99

(1H, dd, J = 8.8, 6.7 Hz), 4.09 (1H, dd, J = 8.8, 6.7 Hz), 4.36 (1H, dt, J = 6.7, 3.6 Hz),

4.64 (1H, d, J = 3.6 Hz), 4.74 (2H, d, J = 6.6 Hz), 6.32 (1H, dt, J = 13.8, 6.6 Hz), 6.36

(1H, d, J =13.8 Hz), 7.213-7.375 (5H, m), 8.667 (1H, s); 13C-NMR (75 MHz, CDCl3): δ

25.13, 25.63, 64.85, 72.00, 73.73, 74.13, 110.59, 121.25, 123.77, 126.69, 128.15, 128.59,

135.09, 136.06, 157.11, 169.50. Anal. Calcd for C18H20O6: C, 65.05; H, 6.07; O, 28.88.

Found: C, 65.34; H, 5.75; O, 28.91.

126

5,6-O-Isopropylidene-2-O-heptyl-L-ascorbic Acid (6C). This was synthesized from 1

and 1-bromoheptane in 96% yield as a semisolid using the same procedure as for 1C: 1H-

NMR (400 MHz, CDCl3) δ 0.88 (3H, t), 1.28-1.35 (8H, m), 1.38 (3H, s), 1.42 (3H, s),

1.66 (2H, quin, J = 7.0 Hz), 4.03-4.10 (2H, m), 4.18 (2H, dd, J = 8.8, 7.0 Hz), 4.43 (1H,

dt, J = 6.6, 3.4 Hz), 4.71 (1H, d, J = 3.4 Hz), 8.79 (1H, s); 13C-NMR (100 MHz, CDCl3):

δ 13.7, 22.51, 25.18, 25.46, 25.72, 29.00, 29.57, 31.69, 64.99, 72.04, 73.82, 74.47,

110.50, 121.78, 156.58, 170.01. HRMS (FAB +) m/z exact mass calcd for C16H27O6 (M +

1) 315.1810, found m/z 315.1808.

5,6-O-Isopropylidene-3-O-acetyl-2-O-allyl-L-ascorbic Acid (7C). A mixture of 5,6-

O-isopropylidene-2-O-allyl-L-ascorbic acid, 1C (1 g, 3.90 mmol) and 1.4 equivalents of

pyridine (0.43 g, 5.46 mmol) in CH2Cl2 stirred for 20 min at room temperature. Then, 1.2

equivalents of acetyl chloride (0.37 g, 4.68 mmol) was added dropwise under nitrogen.

The mixture was vigorously stirred until the solution became homogenous and was

further stirred for 2 h at room temperature. The reaction mixture was diluted with water

(4-fold) and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and

the solvents were removed under reduced pressure. The product was purified by

conventional silica gel column chromatography using 7:1 n-hexane:ethyl acetate to give

in 70% yield a light yellow oil:214 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.38 (3H,

s), 2.31 (3H, s), 4.02 (1H, dd, J = 8.3, 6.6 Hz), 4.15 (1H, dd, J = 8.3, 6.6 Hz), 4.29 (1H,

dt, J = 6.6, 2.5 Hz), 4.78 (2H, tt, J = 5.1, 1.5 Hz), 5.18 (1H, d, J = 2.5 Hz), 5.28 (1H, dq, J

= 10.5, 2.1 Hz), 5.37 (1H, dq, J = 17.3, 2.1 Hz), 5.97 (1H, ddt, J = 17.3, 10.5, 5.1 Hz);

13C-NMR (75 MHz, CDCl3): δ 20.6, 25.3, 25.6, 65.1, 71.3, 72.8, 74.5, 110.5, 118.9,

127

130.1, 132.4, 143.8, 166.3, 166.4. HRMS (FAB +) m/z exact mass calcd for C14H19O7 (M

+ 1) 299.1130, found m/z 299.1131.

5,6-O-Isopropylidene-3-O-acetyl-2-O-methyl-L-ascorbic Acid (8C). This was

synthesized from 2C and acetyl chloride in 80% yield as a transparent viscous oil using

the same procedure as for 7C: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s),

2.32 (3H, s), 4.00 (3H, s), 4.04 (1H, dd, J = 9.0, 6.9 Hz), 4.16 (1H, dd, J = 9.0, 6.9 Hz),

4.30 (1H, dt, J = 6.9, 2.3 Hz), 5.15 (1H, d, J = 2.3 Hz); 13C-NMR (75 MHz, CDCl3): δ

20.5, 25.3, 25.6, 58.4, 65.1, 72.8, 74.4, 110.5, 131.2, 142.3, 166.2, 166.4. HRMS (FAB

+) m/z exact mass calcd for C12H17O7 (M + 1) 273.0970, found m/z 273.0974.

5,6-O-Isopropylidene-3-O-acetyl-2-O-trans-crotyl-L-ascorbic Acid (9C). This was

synthesized from 3C and acetyl chloride in 84% yield as a light yellow oil using the same


dq, J = 6.4, 1.8 Hz), 2.31 (3H, s), 4.01 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (1H, dd, J = 8.5, 6.6

Hz), 4.28 (1H, dt, J = 6.6, 2.4 Hz), 4.70 (2H, dt, J = 6.4, 1.7 Hz), 5.18 (1H, d, J = 2.4 Hz),

5.64 (1H, dtq, J = 16.8, 6.4, 1.7 Hz), 5.83 (1H, dtq, J = 16.8, 6.4, 1.7 Hz); 13C-NMR (100

MHz, CDCl3); δ 17.7, 20.5, 25.3, 25.6, 65.0, 71.4, 72.9, 74.4, 110.5, 125.4, 130.1, 132.2,

143.8, 166.3, 166.5. Anal. Calcd for C15H20O7: C, 57.69; H, 6.45; O, 35.86. Found: C,

57.74; H, 6.54; O, 35.72.

5,6-O-Isopropylidene-3-O-acetyl-2-O-benzyl-L-ascorbic Acid (10C). This was

synthesized from 4C and acetyl chloride in 82% yield as a light yellow oil using the same



Hz), 5.15 (1H, d, J = 2.4 Hz), 5.28 (1H, d, J = 11.6 Hz), 5.33 (1H, d, J = 11.6 Hz) 7.31-

128

7.39 (5H, m); 13C-NMR (100 MHz, CDCl3): δ 20.6, 25.3, 25.6, 65.0, 72.5, 72.8, 74.40,

110.5, 127.8, 128.4, 128.5, 130.1, 135.9, 144.6, 166.1, 166.5. HRMS (FAB +) m/z exact

mass calcd for C18H21O7 (M + 1) 349.1290, found m/z 349.1298.

5,6-O-Isopropylidene-3-O-acetyl-2-O-cinnamyl-L-ascorbic Acid (11C). This was

synthesized from 5C and acetyl chloride in 76% yield as viscous oil using the same



Hz), 4.89-5.00 (2H, m), 5.17 (1H, d, J = 2.4 Hz), 6.33 (1H, dt, J = 13.6, 6.5 Hz), 6.08

(1H, d, J = 13.6 Hz), 7.19- 7.41 (5H, m); 13C-NMR (75 MHz, CDCl3): δ 25.3, 25.4, 25.6,

65.1, 71.3, 72.9, 74.5, 110.6, 123.4, 126.7, 128.3, 128.6, 128.8, 134.8, 136.0, 144.6,

166.3, 166.6. HRMS (FAB +) m/z exact mass calcd for C20H23O7 (M + 1) 375.1440,

found m/z 375.1444.

5,6-O-Isopropylidene-2-O-allyl-3-O-methyl-L-ascorbic Acid (12C). This was

prepared from 1A and allyl bromide (Scheme 11) in 66 % yield as a yellowish viscous

oil: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.03 (1H, dd, J = 8.6, 6.6

Hz), 4.13 (1H, dd, J = 8.6, 6.6 Hz), 4.15 (3H, s), 4.28 (1H, dt, J = 6.6, 3.1 Hz), 4.52 (1H,

d, J = 3.1 Hz), 4.60 (1H, ddt, J = 12.3, 6.2, 1.2 Hz), 4.62 (1H, ddt, J = 12.3, 6.3, 1.1 Hz),

5.28 (1H, dq, J = 10.3, 1.5 Hz), 5.36 (1H, dq, J = 17.2, 1.5 Hz), 6.00 (1H, ddt, J = 17.2,

10.3, 6.2 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.5, 25.8, 59.6, 64.2, 72.7, 73.8, 74.5,

110.3, 119.3, 121.5, 132.8, 157.1, 169.9.

5,6-O-Isopropylidene-2-O-trans-crotyl-3-O-methyl-L-ascorbic Acid (13C). This

was prepared from 1A and trans-crotyl bromide (Scheme 11) in 65 % yield as yellowish

viscous oil: 1H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 1.74 (3H, dq, J =

129

6.4, 1.3 Hz), 4.03 (1H, dd, J = 8.5, 6.6 Hz), 4.13 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (3H, s),

4.27 (1H, dt, J = 6.6, 3.1 Hz), 4.51 (1H, d, J = 3.1 Hz), 4.52 (2H, m), 5.66 (1H, dtq, J =

15.3, 6.7, 1.5 Hz), 5.82 (1H, dtq, J = 15.3, 6.4, 1.5 Hz); 13C-NMR (75 MHz, CDCl3): δ

17.8, 25.6, 25.8, 59.6, 65.2, 72.6, 73.9, 74.5, 110.3, 121.4, 125.8, 132.4, 157.3, 169.1.

5,6-O-Isopropylidene-2-O-trans-cinnamyl-3-O-methyl-L-ascorbic Acid (14C). This

was prepared from 1A and trans-cinnamyl bromide (Scheme 11) in 71 % yield as

colorless solid: 1H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.36 (3H, s), 4.03 (1H, dd, J

= 8.5, 6.7 Hz), 4.12 (1H, dd, J = 8.5, 6.7 Hz), 4.16 (3H, s), 4.27 (1H, dt, J = 6.7, 3.0 Hz),

4.51 (1H, d, J = 3.0 Hz), 4.74 (1H, ddd, J = 12.1, 6.8, 1.2 Hz), 4.81 (1H, ddd, J = 12.1,

6.6, 1.2 Hz), 6.36 (1H, dt, J = 15.9, 6.6 Hz), 6.68 (1H, d, J = 15.9 Hz), 7.23-7.36 (3H, m),

7.37-7.42 (2H, m); 13C-NMR (75 MHz, CDCl3): δ 25.5, 25.8, 59.6, 65.2, 72.4, 73.8, 74.5,

110.3, 121.1, 123.8, 126.7, 128.1, 128.6, 135.1, 136.2, 157.4, 169.0.

5,6-O-Isopropylidene-2-O-allyl-3-O-benzyl-L-ascorbic Acid (15C). This was

prepared from 2A and allyl bromide (Scheme 11) in 80 % yield as a colorless semi-solid:


4.11 (1H, dd, J = 8.6, 6.7 Hz), 4.30 (1H, dt, J = 6.7, 3.2 Hz), 4.54 (2H, m), 4.55 (1H, d, J

= 3.2 Hz), 5.26 (1H, dq, J = 10.3, 1.6 Hz), 5.34 (1H, dq, J = 17.2, 1.5 Hz), 5.48 (2H, br

s), 5.94 (1H, ddt, J = 17.2, 10.3, 6.1 Hz), 7.37 (5H, br s); 13C-NMR (75 MHz, CDCl3): δ

25.6, 25.8, 65.2, 72.5, 73.5, 73.9, 74.7, 110.3, 119.3, 121.4, 127.7, 128.5, 128.7, 129.1,

132.8, 155.9, 168.8.

5,6-O-Isopropylidene-3-O-acetyl-L-ascorbic Acid (1D). A mixture of 5,6-O-

isopropylidene-L-ascorbic acid, 1 (1 g, 4.63 mmol) and 1.0 equivalents of pyridine (0.37

g, 4.63 mmol) in CH2Cl2 stirred at room temperature for 2 min. Then 1.0 equivalents of

130

acetyl chloride (0.36 g, 4.63 mmol) was added dropwise. The mixture was vigorously

stirred until the solution became homogenous and was further stirred for 15 min at room

temperature. The reaction was diluted (4-fold) with water and extracted with ethyl

acetate. The organic layer was thoroughly washed with water and dried over anhydrous

Na2SO4 and the solvents were removed under reduced pressure. The product could not be

obtained in its pure form with conventional silica gel column chromatography without a

significant amount of 2D and 3D contaminations due to its isomerization.

5,6-O-Isopropylidene-2-O-acetyl-L-ascorbic Acid (2D). A mixture of 5,6-O-

isopropylidene-L-ascorbic acid, 1 (1 g, 4.63 mmol) and 1.4 equivalents of pyridine (0.51

g, 6.48 mmol) in CH2Cl2 stirred at room temperature for 10 min. Then 1.2 equivalents of

acetyl chloride (0.44 g, 5.55 mmol) was added dropwise. The mixture was vigorously

stirred until the solution became homogenous and was further stirred for 2 h at room

temperature. The reaction was diluted (4-fold) with water and extracted with ethyl

acetate. The organic layer was thoroughly washed with water and dried over anhydrous

Na2SO4 and the solvents were removed under reduced pressure. The product was

purified with conventional silica gel column chromatography using 5:2 n-hexane:ethyl

acetate to give 90% yield as a white solid: 1H-NMR (300 MHz, CDCl3) δ 1.38 (3H, s),

1.41 (3H, s), 2.36 (3H, s), 4.10 (1H, dd, J = 8.71, 6.74 Hz), 4.20 (1H, dd, J = 8.71, 6.74

Hz), 4.44 (1H, dt, J = 6.74, 2.81 Hz), 4.70 (1H, d, J = 2.81 Hz), 9.70-9.80 (1H, s): 13C-

NMR (75 MHz, CDCl3): δ 20.68, 25.54, 25.72, 65.28, 73.40, 74.57, 110.72, 115.39,

155.13, 166.23, 171.29.

5,6-O-Isopropylidene-2,3-O-diacetyl-L-ascorbic Acid (3D). This was isolated as a

solid in the least polar fractions of 3D silica gel column separation: 1H-NMR (400 MHz,

131

CDCl3) δ 1.35 (3H, s), 1.39 (3H, s), 2.27 (3H, s), 2.30 (3H, s), 4.09 (1H, dd, J = 8.8, 6.6


Hz); 13C-NMR (100 MHz, CDCl3): δ 20.04, 20.42, 25.30, 25.62, 65.18, 72.86, 75.26,

110.81, 122.24, 150.82, 164.74, 165.41, 166.11.

5,6-O-Isopropylidene-3-keto-2-(1-prop-2-enyl)-L-galactono-γ-lactone (1E). This

was obtained as a major diastereomer with a small amount of a minor diastereomer161

from the Claisen rearrangement of pure 4A in refluxing toluene for 6 h (Scheme 14): 1H-

NMR (300 MHz, CDCl3) δ 1.35 (3H, s), 1.40 (3H, s), 2.65 (2H, d, J = 7.4 Hz), 4.09 (1H,

dd, J = 8.7, 6.8 Hz), 4.20 (1H, dd, J = 8.7, 6.8 Hz), 4.55 (1H, dt, J = 6.8, 2.0 Hz), 4.67

(1H, d, J = 2.0 Hz), 5.26 (1H, dq, J = 16.7, 1.4 Hz), 5.29 (1H, dq, J = 10.5, 1.0 Hz), 5.70

(1H, ddt, J = 16.7, 10.5, 7.5 Hz); 13C-NMR (75 MHz, CDCl3): δ 25.3, 25.5, 39.7, 64.8,

72.0, 74.5, 81.5, 111.4, 122.9, 127.6, 172.6, 205.5.

5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-prop-2-enyl)-L-galactono-γ-lactone

(3E). This was obtained as a major diastereomer with a small amount of a minor

diastereomer161 from the Claisen rearrangement of pure 9A in refluxing toluene for 6 h

(Scheme 14): 1H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.42 (3H, s), 2.16 (3H, s), 2.80

(2H, m), 4.10 (1H, dd, J = 8.5, 7.1 Hz), 4.20 (1H, dd, J = 8.5, 7.1 Hz), 4.53 (1H, dt, J =

7.1, 1.6 Hz), 4.87 (1H, d, J = 1.7 Hz), 5.19 (1H, dq, J = 17.0, 1.5 Hz), 5.26 (1H, dq, J =

10.2, 1.5 Hz), 5.90 (1H, ddt, J = 17.2, 10.1, 7.0 Hz); 13C-NMR (75MHz CDCl3): δ 19.2,

25.5, 25.8, 34.5, 65.2, 73.7, 73.8, 82.1, 110.8, 120.8, 128.3, 169.7, 170.9, 201.0.

5,6-O-Isopropylidene-3-keto-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-galactono-γ-

lactone (4E). This was obtained as a major diastereomer with a small amount of a minor


132

(Scheme 14): 1H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.40 (3H, s), 2.67 (2H, m),

4.00 (2H, m), 4.07 (1H, dd, J = 8.7, 6.9 Hz), 4.16 (1H, dd, J = 8.7, 6.9 Hz), 4.55 (1H, d, J

= 1.9 Hz), 4.65 (1H, dt, J = 6.9, 1.9 Hz), 5.20 (1H, dq, J = 10.5, 1.2 Hz), 5.22 (1H, dq, J

= 17.0, 1.2 Hz), 5.26 (1H, dq, J = 10.4, 1.2 Hz), 5.31 (1H, dq, J = 17.3, 1.6 Hz), 5.75 (1H,

dddd, J = 17.0, 10.4, 7.9, 6.9 Hz), 5.90 (1H, ddt, J = 17.3, 10.4, 5.7 Hz); 13C-NMR (75

MHz, CDCl3): δ 25.4, 25.7, 41.2, 64.9, 69.2, 74.2, 79.9, 81.5, 110.9, 118.0, 122.2, 127.8,

133.2, 171.4, 206.1. Anal. Calcd for C15H20O6: C, 60.80; H, 6.80. Found: C, 60.66; H,

6.70.

5,6-O-Isopropylidene-3-keto-2-(1-prop-2-enyl)-2-O-methyl-L-galactono-γ-lactone



(Scheme 14): 1H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.38 (3H, s), 2.63 (2H, m),

3.34 (3H, s), 4.06 (1H, dd, J = 8.5, 7.0 Hz), 4.17 (1H, dd, J = 8.5, 7.0 Hz), 4.54 (1H, d, J

= 1.7 Hz), 4.65 (1H, dt, J = 7.0, 1.7 Hz), 5.20 (1H, dt, J = 18.2, 1.0 Hz), 5.25 (1H, dt, J =

10.1, 1.0 Hz), 5.73 (1H, dddd, J = 18.1, 10.2, 8.0 Hz); 13C-NMR (75 MHz, CDCl3): δ

25.5, 25.6, 41.0, 55.8, 64.9, 74.3, 80.4, 81.5, 110.9, 122.1, 127.8, 171.2, 206.1.

5,6-O-Isopropylidene-3-keto-2-(1-methyl-1-prop-2-enyl)-L-galactono-γ-lactone



(Scheme 14): 1H-NMR (300 MHz, CDCl3) δ 1.19 (3H, d, J = 7.0 Hz), 1.35 (3H, s), 1.41

(3H, s), 2.69-2.81 (1H, m), 4.08 (1H, dd, J = 8.7, 6.8 Hz), 4.17 (1H, dd, J = 8.7, 6.8 Hz),

4.52 (1H, dt, J = 6.8, 2.1 Hz), 4.61 (1H, d, J = 2.2 Hz), 5.25 (1H, dt, J = 17.9, 1.0 Hz),

133

5.29 (1H, dt, J = 9.8, 1.0 Hz), 5.76 (1H, ddd, J = 17.9, 9.8, 8.2 Hz); 13C-NMR (75 MHz,

CDCl3): δ 12.5, 25.4, 25.5, 44.3, 64.8, 74.3, 74.4, 81.8, 111.1, 120.0, 134.3, 172.8, 205.7.

5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-methyl-1-prop-2-enyl)-L-galactono-γ-

lactone (6E). This was obtained as a major diastereomer with a small amount of a minor


(Scheme 14): 1H-NMR (300 MHz, CDCl3) δ 1.23 (3H, d, J = 6.8 Hz), 1.39 (3H, s), 1.48

(3H, s), 2.16 (3H, s), 2.95 (1H, dq, J = 7.8, 6.8 Hz), 4.08-4.18 (2H, m), 4.46-4.55 (2H,

m), 5.28 (1H, dt, J = 17.0, 1.1 Hz), 5.31 (1H, dt, J = 10.4, 1.1 Hz), 5.71 (1H, ddd, J =

17.0, 10.4, 7.8 Hz); 13C-NMR (75 MHz, CDCl3): δ 12.4, 19.3, 25.3, 26.7, 41.4, 65.2,

75.1, 77.3, 84.8, 110.0, 120.6, 132.9, 169.3, 170.3, 200.9.

5,6-O-Isopropylidene-3-keto-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ-lactone

(7E). This was synthesized as the major product of 1 and trans-cinnamyl chloride

reaction using the standard procedure for 1C (Scheme 15), and isolated as a yellowish

viscous oil: 1H NMR (400 MHz, CDCl3) δ 1.29 (3H, s), 1.30 (3H, s), 3.53 (1H, d, J = 2.0

Hz), 3.86 (1H, dd, J = 8.6, 7.0 Hz), 3.89 (1H, dd, J = 8.6, 7.0 Hz), 3.92 ( 1H, d, J = 2.0

Hz), 4.43 (1H, dt, J = 7.0, 2.0 Hz), 5.39-5.49 (2H, m), 6.35-6.50 (1H, m), 7.19-7.36 (5H,

m); 13C NMR (100 MHz, CDCl3): δ 25.2, 25.4, 56.7, 64.7, 74.0, 74.2, 81.9, 110.9, 122.3,

128.5, 128.7, 129.1, 131.1, 134.2, 172.0, 206.1.

5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ-

lactone (8E). This was synthesized from 7E (Scheme 15) as a white semisolid in

quantitative yield using the standard procedure for 1F: 1H NMR (400 MHz, CDCl3) δ

1.32 (3H, s), 1.34 (3H,s), 2.14 (3H, s), 3.70 (1H, d, J = 8.4 Hz), 3.98 (1H, d, J = 9.3 Hz),

4.05 (2H, dd, J = 6.0, 1.2 Hz), 4.42 (1H, dt, J = 8.4, 6.0 Hz), 5.31 (1H, dq, J = 16.8, 1.6

134

Hz), 5.38 (1H, dq, J = 10.4, 1.2 Hz), 6.34 (1H, dddd, J = 16.8, 10.4, 9.2, 1.2 Hz), 7.20-

7.24 (3H, m), 7.33-7.38 (2H, m); 13C NMR (100 MHz, CDCl3): δ 19.2, 25.3, 26.6, 52.9,

65.1, 75.0, 76.7, 85.1, 109.8, 121.0, 128.8, 129.1, 129.3, 131.1, 133.8, 169.1, 170.3,

201.8.


lactone (9E). This was obtained as a major product from refluxing clean 10A in toluene

for 6 h (Scheme 16), resulting in 100% conversion of the UV-active starting material to a

major less polar non-UV-active product with minute traces of minor diastereomers:161,218

1H NMR (300 MHz, CDCl3) δ 1.27 (3H, s), 1.45 (3H, s), 2.13 (3H, s), 2.97 (1H, dt, J =

6.8, 6.1 Hz), 3.82 (1H, dd, J = 8.8, 6.8 Hz), 3.97 (1H, dd, J = 8.8, 6.8 Hz), 4.05 (1H, d, J

= 8.6 Hz), 4.75 (1H, d, J = 6.8 Hz), 5.17 (1H, dt, J = 17.0, 1.1 Hz), 5.30 (1H, dt, J = 10.3,

1.1 Hz), 6.43 (1H, ddd, J = 17.0, 10.2, 8.6 Hz), 7.21-7.27 (2H, m), 7.32-7.40 (3H, m); 13C

NMR (75 MHz, CDCl3): δ 19.3, 25.4, 26.4, 51.3, 64.9, 72.7, 76.0, 83.9, 110.2, 119.9,

128.5, 129.3, 129.8, 132.5, 134.9, 169.3, 170.6, 201.5.


lactone (10E). This was obtained as a minor product from silica gel column separation of

9E (10E & 8E are the same compound with identical sets of 1H- & 13C-NMR spectra).

5,6-O-Isopropylidene-2-keto-3-(1-prop-2-enyl)-L-galactono-γ-lactone (1F). This

was obtained as a major diastereomer with an insignificant amount of a minor

diastereomer from the Claisen rearrangement of pure 1C in refluxing toluene for 12 h

(Scheme 17):218 1H-NMR (400 MHz, CDCl3) δ 1.30 (3H, s), 1.32 (3H, s), 2.54 (2H, dt, J

= 7.2, 1.2 Hz), 3.13 (1H, br), 4.10 (1H, dd, J = 8.8, 6.8 Hz), 4.17 (1H, dd, J = 15.2, 8.8

Hz), 4.52 (1H, dt, J = 13.2, 6.8 Hz), 4.70 (1H, d, J = 1.2 Hz), 5.25 (1H, dq, J = 17.2, 3.2

135

Hz), 5.35 (1H, dq, J = 10.4, 1.6 Hz), 5.81 (1H, ddd, J = 10.4, 7.2, 3.2 Hz); 13C-NMR (100

MHz, CDCl3): δ 24.6, 24.8, 39.6, 64.4, 73.1, 77.2, 80.8, 111.3, 121.9, 129.0, 159.4,

193.9.

5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone

(2F). This was obtained as a major diastereomer with an insignificant amount of a minor


(Scheme 17): 218 1H-NMR (400 MHz, CDCl3) δ 1.32 (3H, s), 1.34 (3H, s), 2.16 (3H, s),

2.76 (1H, m), 2.97 (1H, m), 4.04 (1H, dd, J = 8.6, 7.2 Hz), 4.14 (1H, dd, J = 8.6, 7.2 Hz),

4.28 (1H, dt, J = 7.2, 1.6 Hz), 4.94 (1H, d, J = 1.6 Hz), 5.23 (2H, m), 5.62-5.73 (1H, m);

13C-NMR (100 MHz, CDCl3): δ 20.0, 25.0, 25.2, 36.9, 65.1, 72.6, 78.4, 81.1, 111.1,

121.9, 128.1, 158.2, 170.6, 186.3.

5,6-O-Isopropylidene-2-keto-3-O-methyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone



(Scheme 17): 218 1H-NMR (300 MHz, CDCl3) δ 1.33 (6H, s), 2.51 (1H, dd, J = 15.0, 8.3

Hz), 2.66 (1H, ddt, J = 14.9, 5.6, 1.3 Hz), 3.70 (3H, s), 3.99 (1H, dd, J = 8.4, 7.0 Hz),


5.26 (1H, dq, J = 17.1, 1.3 Hz), 5.31 (1H, dq, J = 10.5, 1.3 Hz), 5.75 (1H, dddd, J = 17.0,

10.3, 8.3, 5.6 Hz); 13C-NMR (75 MHz, CDCl3): δ 24.7, 25.2, 35.0, 52.8, 65.0, 72.5, 79.3,

79.5, 111.3, 121.7, 129.1, 159.9, 192.2.

5,6-O-Isopropylidene-2-keto-3-O-benzyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone



136

(Scheme 17): 218 1H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.35 (3H, s), 2.57 (1H, dd,

J = 14.8, 8.5 Hz), 2.75 (1H, ddt, J = 14.8, 5.5, 1.4 Hz), 4.00 (1H, dd, J = 8.3, 7.3 Hz),


4.90 (1H, d, J = 10.9 Hz), 5.25 (1H, m), 5.30 (1H, d, J = 10.8 Hz), 5.31 (1H, d, J = 18.6

Hz), 5.82 (1H, dddd, J = 18.8, 10.3, 8.5, 5.5 Hz), 7.30 (5H, br s); 13C-NMR (75 MHz,

CDCl3): δ 24.9, 25.4, 36.3, 64.9, 67.1, 72.7, 79.3, 79.4, 111.2, 121.9, 127.5, 127.9, 128.4,

129.2, 137.5, 159.8, 192.2.

5,6-O-Isopropylidene-2-keto-3-(1-methyl-1-prop-2-enyl)-L-galactono-γ-lactone



(Scheme 17): 218 1H-NMR (400 MHz, CDCl3) δ 1.15 (3H, d, J = 6.9 Hz), 1.29 (3H, s),

1.32 (3H, s), 2.71 (1H, m), 3.23 (1H, br), 4.08 (1H, dd, J = 8.7, 7.1 Hz), 4.18 (1H, dd, J =

8.4, 7.1 Hz), 4.52 (1H, dt, J = 7.1, 1.5 Hz), 4.75 (1H, d, J = 1.5 Hz), 5.18 (2H, m), 5.72-

5.85 (1H, m); 13C-NMR (100 MHz, CDCl3): δ 13.2, 24.8, 24.9, 43.8, 64.4, 73.4, 78.9,

80.0, 111.2, 118.6, 135.3, 159.5, 195.3.

5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1-methyl-1-prop-2-enyl)-L-galactono-γ-

lactone (6F). This was obtained as a major diastereomer with an insignificant amount of

a minor diastereomer from the Claisen rearrangement of pure 9C in refluxing toluene for

24 h (Scheme 17): 218 1H-NMR (400 MHz, CDCl3) δ 1.10 (3H, d, J = 7.0 Hz), 1.33 (3H,

s), 1.35 (3H, s), 2.16 (3H, s), 3.17 (1H, m), 4.04 (1H, dd, J = 8.8, 7.1 Hz), 4.13 (1H, dd, J

= 8.8, 7.1 Hz), 4.30 (1H, dt, J = 7.2, 2.4 Hz), 4.83 (1H, d, J = 2.4 Hz), 5.15-5.28 (2H, m),

5.64-5.81 (1H, m); 13C-NMR (100 MHz, CDCl3): δ 12.8, 19.9, 25.2, 25.5, 42.1, 65.5,

73.4, 77.3, 79.7, 111.0, 119.7, 134.7, 158.0, 170.9, 187.5.

137

5,6-O-Isopropylidene-2-keto-3-O-methyl-3-(1-methyl-1-prop-2-enyl)-L-galactono-

γ-lactone (7F). This was obtained as a major diastereomer with an insignificant amount

of a minor diastereomer from the Claisen rearrangement of pure 13C in refluxing toluene

for 24 h (Scheme 17): 218 1H-NMR (300 MHz, CDCl3) δ 1.10 (3H, d, J = 6.8 Hz), 1.33

(6H, s), 2.70 (1H, dq, J = 9.0, 6.8 Hz), 3.73 (3H, s), 3.98 (1H, dd, J = 8.3, 7.3 Hz), 4.14

(1H, dd, J = 8.3, 7.1 Hz), 4.45 (1H, dt, J = 7.2, 1.3 Hz), 4.63 (1H, d, J = 1.3 Hz), 5.14

(1H, dt, J = 17.0, 1.3 Hz), 5.17 (1H, dt, J = 10.3, 1.3 Hz), 5.69 (1H, ddd, J = 17.0, 10.2,

9.1 Hz); 13C-NMR (75 MHz, CDCl3): δ 13.6, 24.9, 25.4, 42.3, 54.6, 65.2, 72.9, 78.5,

81.1, 111.2, 119.3, 135.9, 159.8, 192.6.

5,6-O-Isopropylidene-2-(1-prop-2-enyl)-L-gulono-γ-lactone (1G). To a stirred

solution of diastereomerically pure 1E (1.50 g, 5.85mmol) in absolute EtOH (80 mL) at -

79 oC was added NaBH4 (244 mg, 6.44 mmol). The reaction mixture was kept at -79 oC

for 5 min and then allowed to rise to room temperature for a period of no more than 30

min. The reaction mixture was concentrated under reduced pressure to about 25 mL and

diluted with a mixture of 1:1 cold water and ethyl acetate (250 mL) and stirred for 30

min. The ethyl acetate layer was separated and the aqueous layer was extracted two times

with ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous

Na2SO4, and the solvents were removed under reduced pressure. The residue was

chromatographed on silica gel using 3:1 n-hexane/ethyl acetate to give 1.39 g (92%) of

pure 1G as a white semisolid and ~200 mg of its diastereomer (Scheme 20): 218 1H-NMR

(400 MHz, CDCl3) δ 1.40 (3H, s), 1.43 (3H, s), 2.56 (1H, dd, J = 14.5, 8.8 Hz), 2.62 (1H,

dd, J = 14.5, 6.2 Hz), 2.77 (1H, d, J = 4.5 Hz), 3.65 (1H, br), 4.03 (1H, dd, J = 8.4, 7.0

Hz), 4.15 (1H, t, J = 6.4 Hz), 4.17 (1H, m), 4.37 (1H, dt, J = 7.0, 4.5 Hz), 4.44 (1H, dd, J

138

= 6.4, 4.5 Hz), 5.30 (1H, d, J = 12.0 Hz), 5.31 (1H, d, J = 16.4 Hz), 5.90-6.00 (1H, m);

13C-NMR (100 MHz, CDCl3) δ 25.5, 25.9, 36.4, 65.2, 74.3, 75.9, 77.2, 79.8, 110.7,

121.1, 130.9, 175.4. Anal. Calcd for C12H18O6: C, 55.81; H, 7.02. Found: C, 55.73; H,

7.04.

5,6-O-Isopropylidene-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-gulono-γ-lactone

(4G). This was synthesized from diastereomerically pure 4E in 90% yield as a semisolid

using the same procedure as for 1G: 1H-NMR (400 MHz, CDCl3) δ 1.38 (3H, s), 1.46

(3H, s), 2.47 (1H, m), 2.77 (1H, m), 3.01 (1H, d, J = 4.6 Hz), 3.83 (1H, dd, J = 8.4, 6.8

Hz), 4.21 (1H, dd, J = 8.8, 6.8 Hz), 4.25 (1H, t, J = 7.0 Hz), 4.28-4.31 (1H, m), 4.39 (1H,

dt, J = 8.0, 4.6 Hz), 4.48 (1H, dd, J = 7.0, 4.6 Hz), 4.55-4.60 (1H, m), 5.19-5.24 (2H, m),

5.25-5.26 (2H, m), 5.72-5.82 (1H, m), 5.86-5.96 (1H, m); 13C-NMR (100 MHz, CDCl3) δ

25.2, 26.6, 35.7, 65.5, 65.7, 71.9, 74.9, 78.2, 81.5, 110.1, 117.5, 120.8, 129.9, 133.7,

173.2. Anal. Calcd for C15H22O6: C, 60.39; H, 7.43. Found: C, 60.52; H, 7.63.

5,6-O-Isopropylidene-2-(1-methyl-1-prop-2-enyl)-L-gulono-γ-lactone (5G). This

was synthesized from diastereomerically pure 5E in 91% yield as a semisolid using the

same procedure as for 1G: 1H-NMR (400 MHz, CDCl3): δ 1.27 (3H, d, J = 7.2 Hz), 1.40

(3H, s), 1.43 (3H, s), 2.64 (1H, d, J = 4.5), 2.75 (1H, m), 4.03 (1H, dd, J = 8.8, 7.0 Hz),

4.15 (1H, dd, J = 8.8, 7.0 Hz), 4.23 (1H, t, J = 6.2 Hz), 4.35 (1H, dt, J = 7.0, 4.5 Hz), 4.45

(1H, dd, J = 6.2, 4.5 Hz), 5.26 (1H, d, J = 10.4 Hz), 5.29 (1H, d, J = 9.8 Hz), 5.99 (1H,

ddd, J = 17.6, 10.4, 8.0 Hz); 13C-NMR (100 MHz, CDCl3) δ 14.5, 25.6, 25.9, 41.9, 65.3,

74.7, 76.9, 77.7, 80.5, 110.7, 118.5, 138.4, 175.2. Anal. Calcd for C13H20O6: C, 57.34; H,

7.40. Found: C, 57.40; H, 7.22.

139

5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-L-gulono-γ-lactone (7G). This

was synthesized from diastereomerically pure 7E in 90% yield (crude) as a semisolid

using the same procedure as for 1G: Since analytically pure 7G could not be obtained by

traditional chromatographic techniques, it was characterized as acetate (7H) which was

easily obtained in its pure form (see below).

The Product of NaBH4 Reduction of 5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1-

prop-2-enyl)-L-galactono-γ-lactone (I). This was synthesized from diastereomerically

pure 2F in 60% yield as a white semisolid using the same procedure as for 1G (Scheme

21): 218 1H-NMR (400 MHz, CDCl3) δ 1.42 (3H, s), 1.46 (3H, s), 2.25 (3H, s), 2.48 (1H,

dd, J = 14.2, 8.0 Hz), 2.54 (1H, dd, J = 14.0, 7.5 Hz), 4.05 (1H, dd, J = 8.4, 6.8 Hz), 4.09

(1H, s), 4.20 (1H, dd, J = 8.4, 6.8 Hz), 4.30 (1H, d, J = 3.0 Hz), 4.53 (1H, dt, J = 6.8, 3.0

Hz), 5.24 (2H, dd, J = 15.2, 7.2 Hz), 5.52 (1H, br), 5.82-5.93 (1H, m); 13C-NMR (100

MHz, CDCl3) δ 20.5, 25.7, 40.9, 65.7, 71.8, 73.5, 77.7, 79.9, 111.1, 120.8, 130.8, 169.4,

170.1.

5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-prop-2-enyl)-L-gulono-γ-lactone (1H).

To a stirred solution of a mixture of diastereomerically pure 1G (1.20 g, 4.65 mmol), 4-

DMAP (284 mg, 2.32 mmol) and triethylamine (3.3 mL, 23 mmol) in dichloromethane

(30 mL) at -79 oC was added acetic anhydride (1.76 mL, 18.6 mmol). The reaction

mixture was stirred for 3 h at -79 oC and then quenched with saturated aqueous sodium

bicarbonate. The mixture was extracted with ethyl acetate, and the organic layer was

washed with water and brine, and then dried over anhydrous Na2SO4. The solvents were

removed under reduced pressure and the residue was chromatographed on silica gel using

10:1 n-hexane/ethyl acetate to give pure 1H (1.59 g, quant.) as a white semisolid

140

(Scheme 20): 218 1H-NMR (400 MHz, CDCl3) δ 1.37 (3H, s), 1.43 (3H, s), 2.15 (3H, s),

2.16 (3H, s), 2.58 (1H, m), 2.60 (1H, m), 3.98 (1H, dd, J = dd, J = 9.0, 6.0 Hz), 4.06 (1H,

dd, J = 9.0, 7.0 Hz), 4.30 (1H, dd, J = 8.0, 6.0 Hz), 4.43 (1H, dt, J = 11.6, 6.0 Hz), 5.27

(1H, m), 5.83 (1H, m), 5.94 (1H, J = 8.0 Hz); 13C-NMR (100 MHz, CDCl3) δ 20.4, 20.5,

25.0, 26.1, 36.2, 64.6, 72.5, 74.6, 78.1, 81.3, 110.5, 121.4, 128.8, 169.3, 169.8, 169.9.

Anal. Calcd for C16H22O8: C, 56.13; H, 6.48. Found: C, 56.35; H, 6.38.

5,6-O-Isopropylidene-3-O-acetyl-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-gulono-

γ-lactone (4H). This was synthesized from diastereomerically pure 4G in quantitative

yield as a white semisolid using the same procedure as for 1H: 1H-NMR (400 MHz,

CDCl3) δ 1.37 (3H, s), 1.46 (3H, s), 2.11 (3H, s), 2.58 (1H, dt, J = 14.8, 7.6 Hz), 2.71

(1H, dt, J = 14.8, 7.6 Hz), 3.69 (1H, dd, J = 8.8, 6.8 Hz), 4.01 (1H, dd, J = 8.8, 6.8 Hz),

4.19 (1H, m), 4.22 (1H, m), 4.33 (1H, m), 4.43 (1H, dd, J = 8.0, 4.2 Hz), 5.12-5.16 (2H,

m), 5.26-5.30 (2H, m), 5.51 (1H, d, J = 4.2 Hz), 5.78-5.90 (2H, m); 13C-NMR (75 MHz,

CDCl3) δ 20.6, 25.1, 26.5, 37.4, 65.2, 66.7, 72.4, 74.5, 79.2, 79.4, 110.3, 116.2, 120.8,

129.8, 134.0, 169.2, 172.4. Anal. Calcd for C17H24O7: C, 59.99; H, 7.11. Found: C, 60.23;

H, 7.39.

5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-methyl-1-prop-2-enyl)-L-gulono-γ-

lactone (5H). This was synthesized from diastereomerically pure 5G in quantitative yield

as a white semisolid using the same procedure as for 1H: 1H-NMR (400 MHz, CDCl3) δ

1.21 (3H, d, J = 7.2 Hz), 1.37 (3H, s), 1.43 (3H, s), 2.14 (3H, s), 2.15 (3H, s), 2.75-2.84

(1H, m), 3.90 (1H, dd, J = 8.8, 6.4 Hz), 4.04 (1H, dd, J = 8.8, 6.4 Hz), 4.31 (1H, dd, J =

8.0, 6.0 Hz), 4.42 (1H, dt, J = 12.8, 6.4 Hz), 5.16 (1H, d, J = 14.8 Hz), 5.17 (1H, d, J =

12.8 Hz), 5.89-5.94 (1H, m), 5.96 (1H, d, J = 8.0 Hz); 13C-NMR (100 MHz, CDCl3) δ

141

14.9, 20.5, 20.6, 25.0, 26.1, 41.6, 64.5, 73.0, 74.9, 78.7, 82.9, 110.4, 118.2, 135.8, 169.4,

169.8, 170. Anal. Calcd for C17H24O8: C, 57.30; H, 6.79. Found: C, 57.48; H, 6.50.

5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-phenyl-1-prop-2-enyl)-L-gulono-γ-

lactone (7H). This was synthesized from diastereomerically pure 7G in quantitative yield

as a white semisolid using the same procedure as for 1H: 1H-NMR (400 MHz, CDCl3) δ

1.34 (3H, s), 1.39 (3H, s), 1.94 (3H, s), 2.11 (3H, s), 3.91 (1H, dd, J = 9.0, 6.4 Hz), 4.00

(1H, dd, J = 9.0, 6.4 Hz), 4.08 (1H, dd, J = 8.0, 6.2 Hz), 4.31 (1H, dt, J = 11.9, 6.4 Hz),

5.20 (1H, d, J = 16.9 Hz), 5.26 (1H, d, J = 10.4 Hz), 6.11 (1H, d, J = 8.0 Hz), 6.22 (1H,

ddd, J = 16.9, 10.4, 9.0 Hz), 7.26-7.35 (2H, m), 7.39-7.41 (3H, m); 13C-NMR (75 MHz,

CDCl3) δ 20.3, 20.6, 24.9, 26.0, 52.4, 64.5, 72.2, 74.4, 77.6, 83.1, 110.4, 119.3, 127.7,

128.5, 130.2, 134.2, 136.4, 169.1, 169.4, 169.7. Anal. Calcd for C22H26O8: C, 63.15; H,

6.26. Found: C, 63.46; H, 6.04.

The Product of NaBH3CN Reduction of 5,6-O-Isopropylidene-3-keto-2-(1-prop-2-

enyl)-L-galactono-γ-lactone (X). To a stirred solution of diastereomerically pure 1E

(1.50 g, 5.85 mmol) and ammonium acetate (4.51 g, 58.5 mmol) in dry methanol (80 mL)

at 25 oC was added NaBH3CN (257 mg, 4.10 mmol) and 2.5 g of 4Å molecular sieve

beads. The reaction mixture was vigorously stirred for 24 h at room temperature and

remained cloudy throughout the reaction. The reaction mixture was concentrated under

reduced pressure to about 20 mL and diluted with a mixture of cold brine-NaHCO3

solution and ethyl acetate, 1:5 respectively (250 mL) and stirred for 3 min. The ethyl

acetate layer was separated and the aqueous layer was extracted two times with ethyl

acetate. The combined ethyl acetate extracts were dried with anhydrous Na2SO4, and the

solvents were removed under reduced pressure. The residue was chromatographed on

142

silica gel using 3:1 n-hexane/ethyl acetate to give 70% of clean X as a white semisolid

and with spectroscopic traces of its diastereomer: 1H-NMR (400 MHz, CDCl3) δ 1.40

(3H, s), 1.43 (3H, s), 2.53-2.64 (2H, m), 4.04 (1H, dd, J = 8.8, 7.2 Hz), 4.16 (1H, dd, J =

8.8, 7.2 Hz), 4.19 (1H, t, J = 3.2 Hz), 4.37 (1H, dt, J = 6.6, 3.2 Hz), 4.44 (1H, dd, J = 6.6,

4.4 Hz), 5.23-5.33 (2H, m), 5.79-6.00 (1H, m); 13C-NMR (100 MHz, CDCl3): δ 25.54,

25.86, 36.34, 65.22, 74.30, 75.92, 76.76, 79.83, 110.64, 121.12, 130.88, 175.47.

The Product of NaBH3CN Reduction of 5,6-O-Isopropylidene-3-keto-2-(1-methy-l-

prop-2-enyl)-L-galactono-γ-lactone (Y). This was synthesized from 5E in

diastereomeric excess to give Y in 70% yield as a white semisolid using the same

procedure as for X. Y was isolated as racemic mixtures of diastereomers: 13C-NMR (100

MHz, CDCl3) δ 13.87, 14.46, 25.50, 25.54, 25.84, 25.93, 41.19, 41.86, 65.16, 65.25,

74.44, 74.66, 76.83, 77.19, 77.69, 77.72, 78.89, 80.19, 80.46, 110.48, 110.68, 118.00,

118.52, 137.68, 138.38, 175.22, 175.35.

The Product of NaBH(OAc)3 Reduction of 5,6-O-Isopropylidene-2-keto-2-(1-prop-

2-enyl)-L-galactono-γ-lactone (Z). To a stirred solution of diastereomerically pure 1F

(1.50 g, 5.85 mmol) and 2.5 g of 4Å molecular sieve beads in methylene chloride (60

mL) at 0 oC was added phenethylamine (1.1 g, 8.78 mmol). The reaction mixture was

vigorously stirred for 12-24 h at room temperature, until the imine formation was

completed (determined by TLC analysis). The imine in methylene chloride was carefully

treated with NaBH(OAc)3 (1.62 g, 7.61 mmol) for 12 h. The reaction mixtures remained

cloudy throughout the reaction. The reaction mixture was concentrated under reduced

pressure to about 20 mL and diluted with a mixture of cold brine-NaHCO3 and ethyl

acetate, 1:5 respectively (250 mL) and stirred for 3 min. The ethyl acetate layer was

143

separated and the aqueous layer was extracted two times with ethyl acetate. The

combined ethyl acetate extracts were dried with anhydrous Na2SO4, and the solvents

were removed under reduced pressure. The residue was chromatographed on silica gel

using 3:1 n-hexane/ethyl acetate to give 90% of clean Z as a white semisolid and with

spectroscopic traces of its diastereomer: 1H-NMR (400 MHz, CDCl3) δ 1.37 (3H, s), 1.39

(3H, s), 2.24 (1H, dd, J = 14.0, 7.2 Hz), 2.48 (1H, dd, J = 14.0, 7.2 Hz), 2.77-2.89 (2H,

m), 2.99-3.06 (1H, m), 3.12-3.19 (1H, m), 3.82 (1H, s), 3.99 (1H, dd, J = 12.0, 8.0 Hz),

4.12 (1H, dd, J = 12.0, 8.0 Hz), 4.23 (1H, d, J = 2.0 Hz), 4.41 (1H, dt, J = 7.3, 2.0 Hz),

5.10-5.21 (2H, m), 5.76-5.87 (1H, m), 7.20-7.32 (5H, m); 13C-NMR (100 MHz, CDCl3):

δ 25.72, 25.73, 36.77, 39.14, 50.77, 65.34, 65.74, 74.11, 79.46, 79.65, 110.48, 120.59,

126.28, 128.48, 128.78, 131.81, 139.63, 174.60.

144

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APPENDIX

229

The X-ray Crystal Structure of 9A

230

The X-ray Crystal Structure of 2E

The chemistry of L-Ascorbic acid derivatives in the asymmetric ...

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