Analysis of racemic acid derivitives by chiral high performance liquid chromatography

Tara Kuknyo Orech,

Knox College

Overview

Chiral HPLC Amino and Phosphonic

Acids α Hydroxy-

phosphonates Results and Future

Research

What is HPLC?

High Performance Liquid Chromatography

P can also stand for “Pressure”

Fast! Large number of

plates Detected by UV-Vis

Nelson, David L., Albert L. Lehninger, and Michael M. Cox. "3.3 Working with Proteins."Lehninger Principles of Biochemistry. New York: W.H. Freeman, 2008. Print.

http://www.cnwtech.eu/HPLC-CNW.png

http://www.onu.edu/files/images/chemistry/HPLC.jpg

Chirality Chiral molecules differ in “handedness”,

or 3-D orientation Common in organic chemistry and biology Very difficult to separate because they have

identical physical properties, except for light rotation

http://www.answersingenesis.org/images/chirality-rgb.jpg

Chiral Chromatography Chromatography…with chirality? Affinity Chromatography Separated by three chiral binding site or

chiral pocket stationary phases

"Online Guide to Chiral HPLC." Mark Earll's Homepage. Web. 20 Aug. 2009. <http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm>.

Column Used Nucleosil Chiral-3, 4mm diameter,

pores 100Å, made for organic solvents Family of columns that uses Nucleosil silica Charge-transfer interactions, hydrogen

bonds, dipole-dipole intereactions and steric effects

NO2

O2N

O

HN

O

X SPACER (SiO2)n

Chiral Selector: N(3,5-dinitrobenzoyl)-L-phenylglycineNucleosil Silica

"NUCLEOSIL CHIRAL." MACHEREY-NAGEL Homepage. 2011. Web. 13 Apr. 2011. <http://www.mn-net.com/tabid/6150/default.aspx>.

Disadvantages to Chiral HPLC Expensive ($1,000-$10,000) Affinity chromatography=specific column for

different molecules Interactions between stationary phase and

chiral molecule are fairly weak, making it difficult to separate

"Online Guide to Chiral HPLC." Mark Earll's Homepage. Web. 20 Aug. 2009. <http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm>.

Why Chiral Chromatography? Analyzes enantiomeric purity Optical Rotation does not work for new

compounds Less sensitive to impurities than opt. rot. Allows separation of enantiomers

NH2

OH

O

*

α β

NH2 P

OH

O

*

OH

OHNH2

O

*

* P

OHNH2

O

OH

What is a β-amino phosphonic acid?

Abrams, Martha Leigh. “Progress towards the synthesis of phosphorus analogs of β-amino acids.” Knox College Honors Thesis, May 2008.

β-Amino Phosphonic Acids

Tyrosine and phenylalanine derivatives R and S-Valine first, translate techniques

S-Phenylalanine S-Tyrosine S-Valine

FMOC-Derivative

Eluent100:18:1 heptane, dioxane and trifluoroacetic acid, very non polar

Can detect all amino acids with FMOC at similar wavelengths (~280 nm)

NH2

H

OH

O

+

O

Cl

O

Acetone:AcetonitrileWaterBuffer

O

HN

O

HO

O

FMOC-ClS-Valine

Kortenaar, Paul B.W. et al.""Rapid and Efficient Method for the Preparation of F-moc-amino Acids Starting from 9-fluorenylmethanol."" International Journal of Peptide and Protein Research 26.4 (1996): 398-400. Web. 12 Jan. 2009

Method

S and R Valine-FMOC separately

Optimize the Peak Run a racemic mixture

First Factor: Wavelength

Optimized Factors

Wavelength: 280 nm Flow Rate: 1 mL/min Output Range: 1.0 Amplifier Setting: 0-1V Sample Concentration: 12 mmol Sample Size: 20μL Solvent: Acetone:Acetonitrile, Buffer Eluent: 100:18:1 heptane, dioxane and

trifluoroacetic acid

Initial Readings

Problem: both at 8 minutes! S-Valine R-Valine

Mixed Example

Separation of the Peaks

Dilute from 12 mmol to 1 mmol

Slow flow rate to .5 mL/min

Alter eluent polarity All methods diluted

peaks, even if put them further downfield

FMOC-Cl? 8 mins!

L-Valine with x5 TFA

Extractions of FMOC-Cl

Before extractions One extraction Two extractions

Re-Optimized! Changes: wavelength at 265 nm, sample size

5 µL S-Valine at ~16 mins R-Valine at ~13 mins

S-valine

Future Research

Tyrosine and Phenylalanine β-amino phosphonic acid derivatives α-hydroxy phosphonates (racemic)

Project 2: Racemic Dimethyl 1-hydroxybenzylphosphonate

H

O

1. NaOMe

2. H+ (quench)

P

O

OMeOMe

P

O

OMeOMe

H

OH

HO

H

+P(OMe)2

O

H

Pogatchnik, D. M., Weimer, David F. “Enantioselective Synthesis of α-Hydroxy Phosphonates via Oxidation with (Camphor Sulfonyl). University of Iowa Department of Chemistry . 1997.

Optimal Settings

Flow Rate 1.0 mL/min

Wavelength 285 nm

Output Range 1.0

Amplifier Setting 0-200mV

Sample Concentration 4 mmol

Sample Size 5 μL

Solvent Heptane Dioxane TFA 100:20:.1

Eluent Heptane Dioxane TFA 100:20:.1

Separation of enantiomers

Results

Total Average 69.14 : 30.86 +/- 1.39

Average of Batch 1 68.5 : 31.5 +/- 1.42

Average of Batch 2 69.95 : 30.05 +/- .94

Future Research Optimize further to obtain 50:50 results Nucleosil Chiral-2 Polarimeter to determine on S vs R Complex products from HPLC to form diasteromers, determine S

vs R by NMR Another factor…temperature?

NO2

O2N

O

HN

O

X SPACER (SiO2)n

NO2

O2N

O

HN

O

X SPACER (SiO2)n

N(3,5-dinitrobenzoyl)-L-phenylglycine

N(3,5-dinitrobenzoyl)-D-phenylglycine

Thank You!

Howard Hughes Medical Internship

Knox College Dr. Larry Welch Dr. Diana Cermak American Chemical

Society Illinois State

Academe of Science