Synthetic study towards Pseudaminic acid and Acinetaminic ...

Synthetic study towards Pseudaminic acid and

Acinetaminic acid analogues

Jona Merx

921210560010

Supervisors

Tjerk Sminia

Tom Wennekes

1 MSc Thesis Jona Merx


Abstract Sialic acids are nine carbon α-ketoacid sugars which are incorporated into oligosaccharide chains of

glycollipids and glycoproteins. In these oligosaccharides sialic acids often occupy the terminal position

and due to this position the sialic acids play a role in transport, cell-cell contact and recognition. Sialic

acids were first discovered in mammals and long thought to occur only in mammals, however in recent

years sialic acids have been isolated from microbes. Sialic acids are found in microbes which are in close

contact with humans. While sialic acids have been studied extensively in eukaryotes, little is known about

sialic acid in microbes. Due to the role of Pseudaminic acid and derivatives in virulence, the overall goal

of the project is to design and synthesize a Pseudaminic acid and Acinetaminic precursor which can be

functionalized with relevant bio-orthogonal handles, such as azides. These analogues can then be fed to

bacteria which will hopefully incorporate this precursor in the oligosaccharide chain after the precursor is

functionalized into a sialic acid. The synthesis of two sialic acids analogues are explored, one is an

analogue of Pseudaminic acid, an important microbial sialic acid as it is a precursors to many derivatives.

The other microbial sialic acid is a recently found epimer of Pseudaminic acid named Acinetaminic acid.

The synthesis towards both sialic acids start from the commercially available L-threonine, an amino acid.

The synthesis towards Pseudaminic acid and Acinetaminic acid diverges from the common building block,

an allyl alcohol Garner aldehyde derivative. For the synthesis of Pseudaminic acid a benzyloxycarbonyl

protected ally alcohol has been successfully synthesized. With this the stereoselectivity of the key step,

the tethered amino hydroxylation, is tested. This key step selectively introduces a carbon nitrogen bond

based on the stereochemistry of the allylic alcohol. The key step towards the Acinetaminic acid analogue

is the ozonolysis of the successfully synthesized tert-butyloxycarbonyl protected allyl alcohol. The

ozonolysis of the allyl alcohol needs further testing. The above mentioned steps almost complete the

total synthesis of microbial sialic acids. In the future analogues of microbial sialic acids will be prepared

and thereafter biological studies will be performed to unravel the biological relevance of microbial sialic

acids in human-gut related microbes.


Contents Abstract ................................................................................................................................... 2

List of abbreviations ................................................................................................................... 5

Introduction .............................................................................................................................. 6

Sialic acids ............................................................................................................................. 6

Sialic acids in microbes ............................................................................................................ 6

Metabolic labelling ................................................................................................................ 7

Existing pathways to Pseudaminic acid and Acinetaminic acid ........................................................ 7

Schoenhofen et al. ................................................................................................................ 7

Zunk et al. .......................................................................................................................... 8

Lee et al. ........................................................................................................................... 11

Tsvetkov et al .................................................................................................................... 13

Retrosynthesis ...................................................................................................................... 14

Pseudaminic Acid ................................................................................................................ 14

Acinetaminic Acid ............................................................................................................... 15

Key steps in the synthesis towards Pse and Acinetaminic acid ...................................................... 17

Tethered aminohydroxylation ............................................................................................... 17

Silyl chemistry and Fleming-Tamao oxidation.......................................................................... 20

Goal of the project ................................................................................................................ 21

Results and discussion .............................................................................................................. 22

Stereoinversion ..................................................................................................................... 22

Varying the protecting group ................................................................................................... 24

Boc protected amine ........................................................................................................... 24

Cbz protected amine ........................................................................................................... 27

Acetyl protected amine ........................................................................................................ 29

Pseudaminic acid precursor: the tethered aminohydroxylation ..................................................... 30

Acinetaminic acid precursor: Exploring the ozonolysis ................................................................. 32

Conclusion .............................................................................................................................. 35

Pseudaminic acid analogue ..................................................................................................... 35

Acinetaminic acid analogue ..................................................................................................... 35

Future prospects ...................................................................................................................... 36

Pseudaminic acid analogue ..................................................................................................... 36

Acinetaminic acid analogue ..................................................................................................... 37

Ozonolysis ............................................................................................................................ 39

Experimental ........................................................................................................................... 40

Acknowledgements .................................................................................................................. 50

References .............................................................................................................................. 52


List of abbreviations Ac acetyl aq. Aqueous Bn benzyl Bz benzoyl Cbz benzyloxycarbonyl d doublet (in NMR) DCM dichloromethane dd double of doublets (in NMR) DiBAL-H diisobutylaluminium hydride DMF dimethylformamide equiv. equivalents ESI electron spray ionisation Et ethyl EtOAc ethyl acetate EtOH ethanol g gram h hour HCl hydrochloric acid MS mass spectrometry Hz hertz (in NMR) J coupling constant (in NMR) m multiplet (in NMR) Me methyl MeOH methanol min. minutes mL millilitre MS mass spectrometry NMR nuclear magnetic resonance PG protecting group Ph phenyl Pse Pseudaminic acid ppm parts per million q quartet (in NMR) quint. quintet (In NMR) Rf retardation factor (in TLC) rt room temperature s singlet (in NMR) sat. saturated t triplet (in NMR) TBDMS tert-butyldimethylsilyl t-Bu tert-butyl THF tetrahydrofuran TLC thin layer chromatography


Introduction

Sialic acids Sialic acids are a group of nine carbon α-ketoacid sugars. This versatile class of molecules, with more

than 50 derivatives, was first discovered in mammalian tissue in 1942 and has since been isolated in

other classes of organism including microbes. The general structure of a sialic acid (1) is illustrated in

figure 1. The most common sialic acid in mammals is N-acetylneuraminic acid (Neu5Ac, 2).1 Sialic acids

are rarely observed as monosaccharides in nature, they are mostly incorporated in oligosaccharide chains

of glycollipids and glycoproteins. Sialic acids often occupy the terminal position of these oligosaccharides.

The negative charge created by the sialic acids aides cell-cell and protein repulsion and attraction

phenomena as well as binding and transport of positive charged molecules and ions, such as Ca 2+.2 The

size and charge of the sialic acid creates a shield for the sub terminal part. This can prevent for example

degradation of the glycoprotein by proteases. Their negative charge causes repulsion between sialic acid

containing oligosaccharide chains which can stabilize the correct folding of the associated protein.3

Figure 1. General structure off a sialic acid 1, Neu5Ac 2; R can be H, amide or alkyl.

Due to their terminal position sialic acids play an important role in recognition between cells and

molecules. The immune system for instance can distinguish the pattern of natural or unnatural sialic acid

glycans. Sialic acids are also necessary components of certain receptors for hormones and cytokines. It

was long thought that sialic acids were unique to mammals.

Sialic acids in microbes The last years sialic acids have been isolated from microbes. Although sialic acids have been studied

intensively in eukaryotes little is known about them in microbes, (microbial sialic acid, MSA).3, 4

The sialic acids found in microbes are often found on pilli, flagella and capsular carbohydrates, which are

important for bacterial virulence. Knock-out experiments of genes encoding for enzymes involved in the

biosynthesis of MSAs were found to be important for the glycosylation of flagella. Without glycosylation,

glycans are not placed on the outer surface resulting in a decrease in motility and virulence. The effect of

knocking out the genes encoding the enzymes responsible for the biosynthesis of MSA’s, the flagella are

not glycosylated and the subunits remain intracellular, hampering the motility and the virulence of the

bacteria. MSA’s are therefore essential for the virulence of certain bacteria.3, 5 For instance in L.

pneumophila it has been found to be important for virulence capabilities.6

Figure 2. Pseudaminic acid 3, Legionaminic acid 4 and Acinetaminic acid 5.

Two main subclasses of MSA’s have been identified in microbial organisms, which consist of Pseudaminic

acid, 5,7-di-N-acetyl-pseudaminic acid (3), and Legionaminic acid, 5,7-di-N-acetyllegionaminic acid (4),

see figure 2. Recently an epimer of Pseudaminic acid has been discovered and was named Acinetaminic

acid, 5,7-di-N-acetyl-acinetaminic acid (5).7

The biosynthesis of Pseudaminic acid and Legionaminic acid have been extensively researched and many

enzymes which are essential for the biosynthesis have been identified, although the enzymes responsible


for incorporation of them in oligosaccharides, glycosyl transferases and eventual removal, glycosyl

hydrolases, have not been identified yet.3

Metabolic labelling

Due to the role of Pseudaminic acid and derivatives in virulence, the overall goal of the project is to

design and synthesize a Pseudaminic acid and Acinetaminic precursor which can be functionalized with

relevant bio-orthogonal handles, such as azides. This functionalized Pseudaminic acid precursor can then

be fed to bacteria which will hopefully incorporate this precursor in the oligosaccharide chain after the

precursor is functionalized into Pseudaminic acid. The introduction of sialic acids with azide functionalities

in bacteria have been performed successfully in the past by Bertozzi et al. and yield valuable insight into

the role of microbial sialic acids in microbes.8

The proposed methodology is a chemical biology method to study MSA’s and the involved enzymes in

contrast with the molecular biology methods used in previous research.

The hereby introduced azides or other bio-orthogonal handles can for instance be used to detect the

localization of MSA’s by coupling a fluorophore, or used to couple other relevant molecules to the

carbohydrate chain. This coupling allows for the study of the lifecycle of Pseudaminic acid in the target

microbes of interest.

Existing pathways to Pseudaminic acid and Acinetaminic acid Three synthetic strategies are available for the synthesis of Pseudaminic acid, one enzymatically and the

others synthetic, and one for Acinetaminic acid. In this section these currently available synthetic

strategies towards Pseudaminic acid, and to a lesser extent Acinetaminic acid, will be discussed with

special focus on their advantages and shortcomings. Synthetic pathways towards Legionaminic acid are

also known,9, 10 however in this thesis the focus lies with Pseudaminic acid and Acinetaminic acid.

Schoenhofen et al. The first pathway towards Pseudaminic acid is an enzymatic synthesis starting from UDP-GlcNAc, 6 and

was published by Schoenhoven et al. in 2006.11 The final product of this pathway is CMP-pseudaminic

acid however the reaction can be stopped at Pseudaminic acid. The synthesis is carried out in one pot by

sequential addition of the enzymes and the co-factors needed for every step. The first step in the

pathway is the dehydration of C-4,6 and epimerization of 6 with PseB to yield 7. This is followed by

aminotransfer to C-4 by PseC of 7 to yield product 8, see scheme 1.

Scheme 1. Dehydration and epimerization of 6 to ketone 7, aminotransfer to 7 to yield amine 8.

The next step is the acetylation of C-4 amino of 8 by PseH to yield 9. The subsequent step is the removal

of the UDP of 9 by PseG to yield 10, see scheme 2.

Scheme 2. Acetylation of amine 8 to alcohol 9. Deprotection of alcohol 9 to yield hemiacetal 10.

The last step towards the synthesis of Pseudaminic acid (3) is the condensation of 10 with pyruvate, see

scheme 3. The conversion up until this point is 100%. The product 3 can be converted to 11 via

activation of 3 with CTP, however it is advantageous to perform a work up before the last step is


performed; namely the last enzymatic step is inhibiting earlier step which affects the production of

Pseudaminic acid.

Scheme 3. Condensation of hemiacetal 10 with pyruvate to Pseudaminic acid 3. Activation of Pseudaminic acid 3 to CTP - Pseudaminic acid 11.

Advantages and shortcomings

The main advantage of this route is the high conversion of the reaction (100%); the isolated yield of

Pseudaminic acid (3) however is not given. Another advantage of this pathway is the easy and fast

synthesis of Pseudaminic acid, it is a one-pot reaction and for full conversion to Pseudaminic acid a

reaction time of 20 hour is needed.

There are a few drawbacks to this method. Firstly for this biosynthetic enzymes are needed, which brings

with it the need to isolate and purify the enzymes which can be a time consuming process. Another

drawback is the starting material; although it is commercially available it is rather expensive. The

cofactors used in the reaction can also be expensive however cofactor regeneration is possible which

might make the synthesis economically feasible. Another disadvantage is that only Pseudaminic acid can

be made via this pathway. With this pathway analogues for use in metabolic labelling studies cannot be

made.

Zunk et al. The route towards an epimer of Pseudaminic acid, 8-epi-pseudaminic acid 12, published by Zunk et al in

2014,12 originally started from the sialic acid 2-keto-3-deoxy nonic acid (Kdn, 16) however due to

discontinued availability of KDN on preparative scale β-methyl glycoside methyl ester of KDN

(KDN1,β2Me2, 17) was prepared from per-acetylated Neu5Ac1,β2Me2 (18) and used instead. The

acetylation of Neu5Ac is described in literature and not discussed here.13 In scheme 4 the retrosynthesis

is shown.

Scheme 4. Retrosynthesis of 8-epi-pseudaminic acid starting from KDN (R=H, 16) or KDN1,β2Me2 (R=Me, 17). KDN1,β2Me2 was synthesized from acetylated Neu5Ac (18), SiR3 is a suitable silyl ether protecting group.

KDN1,β2Me2 can be synthesized from per-acetylated Neu5Ac1,β2Me2 in 42% yield. The first step

starting KDN1,β2Me2 is the selective protection of the C-8 and C-9 hydroxyl leading to 13 without

formation of bis-isopropylidene 19. The formation of bis-isopropylidene 19 was avoided by short reaction

times, see scheme 5.


Scheme 5. Selective protection of KDN1,β2Me2 17 to isopropylidene 15 without formation of bis-isopropylidene 19.

The next step is the selective silylation of the C-4 hydroxyl of 15 with TBDMSCl, see scheme 6. The

selectivity of TBDMSCl towards the C-4 hydroxyl group is caused by the steric hindrance of the C-5 and

C-7 hydroxyl groups in concordance with the mild conditions used for the protection. The reaction was

also stopped before complete conversion was observed preventing di-protected products.

Scheme 6. Selective protection of tri-ol 15 to di-ol 20 with TBDMSCl.

In the next step triflate groups are introduced on C-5 and C-7 hydroxyl groups of 20 to convert them

into good leaving groups for the subsequent step with NaN3, see scheme 7. The reaction proceeds via

Sn2 mechanism, the desired stereo inversion is observed.

Scheme 7. Functionalization of di-ol 20 with triflate to 14. Introduction of azide to 14 to yield 13.

The next step is the reduction of the azide moieties of 13 to amines, followed by functionalization of the

amines with acetyl groups to yield 21, see scheme 8. The first hydrogenation was performed without

TsOH.H2O which led to an intramolecular attack of the C-5 amine on the ester leading to product 22.

Upon addition of a 5 mol % of TsOH.H2O the reaction proceeded as desired.

Scheme 8. Reduction of di-azide 13 followed by acetylation to di-acetal 21. Product 22 is observed without addition of TsOH.H2O.

The deprotection of both the silly ether and the acetonide of 21 was performed with trifluoroacetic acid

(TFA) to yield tri-ol 23. With the Appel reaction an iodane was introduced on the least hindered hydroxyl

group of 23, leading to product 24.


Scheme 9. Deprotection of silyl ether 21 to tri-ol 23. Selective Appel reaction of tri-ol 23 to di-ol 24.

In the last step the reduction of the 9-iodo group furnished β-methyl glycoside methyl ester of 8-epi-

pseudaminic acid (12). The total yield over 9 steps, starting from KDN1,β2Me2 (17) is 23%.

Scheme 10. Reduction of di-ol 24 to 8-epi-Pseudaminic acid 12.

Attempts to invert the stereochemistry of the C-8 have been performed, however all attempts proved

unsuccessful.14

Very recently an attempt of the stereoinversion was successful, see scheme 11.14 From intermediate 13

the alcohol 25 was synthesized in two steps. The stereoinversion of the alcohol of 25 is accomplished

with an oxidation-reduction approach to yield 26.

Scheme 11. Deprotection of 13 and subsequent selective protection of primary alcohol to 25. Oxidation-reduction of 25 to yield 26.


The main difficulty of synthesis of Pseudaminic acid is the stereochemistry, the advantage of this route is

the starting material. The starting material has most chiral centres in the right orientation and both chiral

centres that are undesired from the starting material (C-5,7) were in one step inverted. The yield of the

reactions are also high, an average yield of 83% was obtained over all the steps starting from

KDN1,β2Me2. KDN1,β2Me2 itself can be synthesized from per-acetylated Neu5Ac1,β2Me2 in 2 steps with

43% yield. The isolated product 22 can also be used to selectively functionalize the C-7 acetyl amine

without affecting the C-5 acetyl, this can be a useful intermediate for the synthesis of other Pseudaminic

acid derivatives.

The main shortcoming of this particular route is that an epimer of Pseudaminic acid is synthesized. The

final product of this synthesis is also still protected, the ester and C-2 hydroxyl-group.

Furthermore Neu5Ac is rather expensive to acquire and functionalization of this epimer to analogues

used for metabolic labelling is rather difficult.


Lee et al. The starting material for the synthesis of Pseudaminic acid published by Lee et al in 2011.15 is N-Acetylglucosamine (D-GlcNAc, 28).15 This starting material is commercially available and not expensive. The retro synthetic approach is depicted in scheme 12.

Scheme 12. Retrosynthetic approach used by Lee et al. towards Pseudaminic acid (3) starting from D-GlcNAc (28).

The first step is the protection of the anomeric hydroxyl and C-3 hydroxyl group of GlcNAc (28), see

scheme 13. The added effect of protecting the anomeric hydroxyl group is that mutarotation is not

possible making life easier with regard to NMR and column chromatography. The protection is followed

by an Appel reaction to form product 30.

Scheme 13. Protection of D-GlcNAc (28) to di-ol 29 followed by a selective Appel reaction of di-ol 29 to alcohol 30.

The next step is the protection of the C-4 hydroxyl group of 30 with TIPS to allow the subsequent

elimination reaction towards 32. The hydroxyl moiety is thereafter deprotected with

tetrabutylammonium fluoride, TBAF, see scheme 14. Beta elimination was also tried, without protection

of the hydroxyl, although this resulted in poorer yields.

Scheme 14. Protection of alcohol 30 to 31 followed by elimination of 31 to olefin 32.

The olefin 32 was selectively hydrogenated in presence of Wilkinson’s catalyst, which led to the main

product 33 with a minor amount of 34, 96:4, see scheme 15.


Scheme 15. Reduction of olefin 32 to yield alcohol 33 as main product with 34 as minor by product.

The next step is the introduction of the C-4 acetyl amine, analogous to the manner employed by Zunk et

al., first functionalize the C-4 hydroxyl to a sulfonate ester followed by treatment with NaN3. This method

was unsuccessful, a variety of sulfonate esters were made, however the reaction with NaN3 did not

succeed. Another route for introduction of the amino functionality was performed. First the hydroxyl

group at C-4 was oxidized to yield 35. This was subsequently reacted with O-methylhydroxylamine which

yielded oxime 36 in good yield. The E/Z ratio was 2:1 for the oximes, however both can be used for the

next step.

Scheme 16. Oxidation of alcohol 33 to ketone 35 followed by derivatization to oxime 36.

The reductive amination of oxime 36 followed by immediate acetylation resulted in product 37, see

scheme 17. This was the only observed isomer, in the transition state the axial amino moieties is

expected to be less favoured due to 1,3-diaxial hindrance of the C-2 amino acetyl. The reductive

amination is followed by hydrogenation of the protecting benzyl groups on C-1 and 3, to yield product

27, (α/β = 2:1).

Scheme 17. Reductive amination of oxime 36 followed by acetylation to yield di-acetal 37. Reduction of the di-acetal 37 to hemiacetal 27.

The sugar 27 obtained in the last step is elongated with a Barbier alkylation see scheme 18. The

erythro/threo ratio slightly favours the erythro product.

Scheme 18. Barbier alkylation of hemiacetal 27 to yield 38.

The last steps consist of ozonolysis and final deprotection of the ethyl ester to yield Pseudaminic acid 3 in

15 step with 13% overall yield, see scheme 19.


Scheme 19. Ozonolysis of olefin 39 to the ethyl ester of Pseudaminic acid 39, followed by deprotection of

39 to yield Pseudaminic acid (3).


The route begins with an inexpensive sugar with most of the chirality in the correct place, a main

advantage. However halfway through the synthesis a lot of product was lost with the reductive amination

that proceeded in a moderate yield of 66%, scheme 17.

The method employed of introducing the amino group on C-4 (C-7 of Pseudaminic acid) by Zunk et al.

was higher yielding however it did not work on this particular substrate. The other step where a lot of

product was lost (44% of the wrong isomer) is the Barbier alkylation, scheme 18, due to undesired

isomer formation. Although the average yield of the reaction is high (88%), the pathway is lengthy and a

lot of product is lost with the Barbier alkylation the overall yield is on the low side.

Tsvetkov et al.

To elucidate the stereochemistry of the side chain of sialic acid Tsvetkov et al.9 synthesised a total of

nine sialic acid which include Pseudaminic acid and Acinetaminic acid.9 The synthesis however was

targeted on a variety of sialic acids for structure elucidation and not performed on a preparative scale,

furthermore both Pseudaminic acid and Acinetaminic acid were only by-products with a very low yield of

3% and 8% for the last step, respectively. This is the result from unexpected epimerization in case of

Pseudaminic acid and for the formation of Acinetaminic acid no clear reason is found. For these reasons

the synthetic approach used by Tsvetkov et al. will not be discussed in depth.


Retrosynthesis In this section our synthetic strategy towards Pseudaminic acid and Acinetaminic acid precursors are

discussed.

Pseudaminic Acid

In scheme 20 our retrosynthetic analysis of Pseudaminic precursor 40 is shown.

Scheme 20. Retrosynthetic approach towards Pseudaminic acid precursor 40 starting from L-threonine

(44).

The first retrosynthetic transformation of the target molecule 40 is depicted in scheme 20, which results

in the moiety 41 with appropriate protection groups. At this stage all the desired chiral moieties have

been installed correctly and an oxidation and deprotection of the protection groups are needed to form

the hemiacetal. The second transformation results in the terminal olefin 42. The amine and the hydroxyl

moiety are installed in one reaction with a tethered amino hydroxylation, which is discussed in the next

section. The third transformation leads to L-allo-threonine 43. The protection of both amine and alcohol

leads in a cyclic system assures the right chirality for both centres and is necessary later to introduce the

hydroxyl group in the desired stereo. At this stage variations of the amine protection can be tested for

the effect on the tethered aminohydroxylation. L-allo-threonine (43) is commercially available however it

is prohibitively expensive to start a total synthesis route, whereas a stereoisomer, L-threonine (44) is

economically viable. Therefore L-allo-threonine is synthesized from L-threonine by means of a four step

stereo inversion pathway.

With orthogonal protecting groups the C-4 amine of 40 can be selectively deprotected and functionalized

to a N azido acetyl group, which functions as a handle for metabolic labelling, see scheme 21.

Scheme 21. Selective functionalization of C-4 amine of 40 to azide 46.

With precursor 46 in hand, the metabolic labelling study can be started by feeding this compound to the

appropriate bacteria, which can then hopefully metabolize this further into Pseudaminic acid analogue

49. Alternatively with a Barbier alkylation, and few other well documented additional steps, the precursor

can be synthetically transformed into a Pseudaminic acid analogue in the lab, see scheme 22. The main

difficulty with this step is the selectivity of the Barbier alkylation. The same procedure was applied by Lee

et al. which led to poor stereoselectivity.


Scheme 22. Late stage synthetic strategy for synthesis of Pseudaminic acid analogue 49 from Pseudaminic acid hexose precursor 46.

Acinetaminic Acid

In scheme 23 the retrosynthetic analysis of Acinetaminic precursor 50 is shown. The first part of the

synthesis is analogous to Pseudaminic acid, the pathways diverge from 42 onwards and therefore 42 is

taken as a starting point.

Scheme 23. Retrosynthetic approach towards Acinetaminic acid precursor 50 starting from 42.

The first retrosynthetic transformation to the target molecule 50 is the deprotection of the isopropylidene

and oxidation of the primary alcohol 51. The primary alcohol 51 functionality is installed via a Fleming-

Tamao oxidation of the silylcompound 52, discussed in the next section. At this stage the molecule

exhibits the desired stereochemistry. The silyl group of 52 is introduced via a Grignard reaction of imine

53 with the suitable silyl Grignard reagent. The stereospecific Grignard reaction ensures the right

stereochemistry of the benzyl protected amine. The last retrosynthetic transformation of intermediate 42

is the imine formation to 53. First the alcohol protected before the ozonolysis of the alkene is performed.

The resulting aldehyde converted to imine 53 with benzyl amine.

With orthogonal protecting groups the C-4 amine of 50 can be selectively deprotected and functionalized

to a N azido acetyl group, which functions as a handle for metabolic labelling. The benzyl protected

amine however has to be converted into another protective group as the hydrogenation is not compatible

with the azide.

Scheme 24. Selective functionalization of C-4 amine of 50 to azide 55.

Analogous to Pseudaminic acid the Acinetaminic hexose can be chain elongated by the Barbier alkylation

followed by an ozonolysis and final deprotection to yield Acinetaminic acid analogue (58), see scheme

25. As noted before the stereoselectivity of the Barbier alkylation was poor on the Pseudaminic hexose in


the synthesis by Lee et al.15 The selectivity of the Barbier reaction might be affected by the protecting

group of the amine.

Scheme 25. Late stage synthetic strategy for synthesis of Acinetaminic acid analogue (58) from Acinetaminic acid precursor 55.


Key steps in the synthesis towards Pse and Acinetaminic acid In the synthesis towards Pseudaminic acid and Acinetaminic acid we have several key steps. In this

section these steps are discussed in detail. The key step in the synthesis towards Pse precursors is the

tethered amino hydroxylation. The key step in the synthesis of Acinetaminic acid is the silyl-Grignard

reduction and the Fleming-Tamao oxidation.

Tethered aminohydroxylation

For stereoselective and regioselective synthesis of chiral vicinal amino alcohols from alkenes the

asymmetric aminohydroxylation, developed by Sharpless, can be used.16 In this reaction both an amine

and a hydroxyl group are introduced to the same enantiotopic face of the prochiral alkene. This reaction

can be performed with a range of nitrogen sources and in presence of a catalytically amount of Os(VI).

The regioselectivity of this reaction is substrate depended and although much research has been done to

improve this, some substrates have unfavourable regioselectivity. To improve the regioselective

incorporation of the amino moiety the tethered amino hydroxylation (TA) was investigated by Donohoe

and co-workers in 2003.17 By tethering the nitrogen source to be installed onto the substrate

regioselectivity can be markedly improved. In scheme 26 the mechanism can be found. Initially a free

amine 59 was oxidized to species 60 and with addition of the osmium catalyst the catalytic cycle is

entered by species 61. Addition of another nitrone equivalent reoxidises the osmium catalyst yielding

species 63. Hydrolysis of this species leads to the product 64 and 61 which enters the cycle again.

Scheme 26. Mechanism of the tethered aminohydroxylation.

Improvements to the reaction were developed by Donohoe et al., namely the leaving group attached to

the nitrogen was varied. The first developed improvement was the functionalization of the amine to a

sulfonyloxy carbamate. This improved the amount of catalyst needed and the reaction is excluded from

chlorinating reagents as well as hydroxides. The milder conditions of this improvement improved the

scope of the reaction. Two years later more improvements were suggested; first the amine 65 was

functionalized to a hydroxycarbamate 66 in a high yielding on pot reaction, see scheme 27. The

hydroxycarbamates were reacted with suitable acid chlorides to yield 67. The best results obtained in the

initial study was with the C6F5 derivative of 67, although 2,4,6-Me3C6H2 is used more in synthesis.


Table 1. R4 Group

Scheme 27. General strategy to synthesize suitable carbamates, in table 1 the R groups can be found.

The TA reaction has been successfully used in literature towards a variety of products. Below a complete

overview of the TA reaction in literature is provided. The reaction itself has been tested on several test

substrates by Donohoe et al.17-19 However, in the scheme only products are depicted which are used as

an intermediate in a more elaborate synthesis project.

In the synthesis towards ent-dysiherbain the TA reaction is used on substrate 68 with the initial

procedure, see scheme 28. During the synthesis the sulfonyloxy carbamate improvement was published

and this has been used on substrate 69.20 The yield of the sulfonyloxy carbamate was higher than the 68

substrate. The relative amount of syn/anti products is not stated in the article. The initial procedure for

the TA reaction is also used for an intermediate in the synthesis of (L)-chloramphenicol and (D)-

thiamphenicol.21 The yield and stereo specificity of the TA reaction of 72 to 73 is good when taken into

account the procedure used. The TA reaction with the initial procedure is also used in the synthesis of L-

arabino-[2R,3S,4R] and L-xylo-[2R,3S,4S]-C18-phytosphingosines, namely from 74 to 75.22 The yield of

this reaction is good considering the procedure used and the stereoselectivity also proved acceptable.

Scheme 28. TA reaction of a range of substrates, using the initial procedure and the OSOMe, improved procedure; R = NO2, SO2Me.

In scheme 29 the TA reaction used in the synthesis of β‑Amino Acid of Microsclerodermins A and B is

shown. The optimized TA reaction procedure is used with the mesitylene carbamate functionalized amine.

The yield of this particular reaction was 76% and the only isomer observed was 77 without the syn

product, the catalyst load however had to be increased to 5 mol% which was attributed to the steric

hinderance.23 Another use of the TA reaction using the optimized procedure is that of 78, an

R4

Me

2,4,6-Cl3C6H2

2,4,6-Me3C6H2

C6F5

p-ClC6H4

t-Bu


intermediate in the synthesis of (-)-Hygromycin.24 The yield of this reaction is high and 79 is the only

product which was observed, the catalyst load was 1% for this reaction.

Scheme 29. Two TA reaction using the latest procedure with the mesitylene carbamate; R = 2,4,6-

Me3C6H2

In the synthesis towards (-)-galantinic acid the TA reaction is used, see scheme 30.25 This reaction

makes use of the optimized procedure although not the mesitylene carbamate but the chlorobenzene

carbamate is used. The yield of this reaction is good however besides the syn product 81 also the anti

product is observed. The ratio is favourable for the desired syn product.

Scheme 30. TA reaction using the latest procedure with chlorobenzene carbamate; R = 2,4,6 Cl3C6H2

The last example where the TA reaction is used in literature is in a tandem catalysation reaction to

produce multi-substituted tetrahydrofurans.26 In this reaction two catalytic reactions occur in one pot to

give tetrahydrofurans. First the TA reaction was used to give yield to 83. After the completion of the first

reaction the reaction mixture is acidified for the oxidative cyclisation reaction to form product 84. Below

the reaction with a z,z alkene is shown, three other alkenes (z,e ; e,z ; e,e) have also been tested with

favourable results. The z,e and e,e alkene substrate worked well, with yields of their corresponding

products of 87-90%. The e,z and the shown z,z alkene were lower yielding, the TA reaction however was

not the problematic step, product 83 could be isolated as a single isomer. The problematic step was the

oxidative cyclisation, the hypothesis is that this is caused due to strain in the transition state of the

cyclisation.

Scheme 31. Tandem TA and oxidative cyclisation osmium catalysed reaction of 82; R = 2,4,6-Me3C6H2

The Tethered aminohydroxylation is an useful reaction and has one main advantage over the asymmetric

aminohydroxylation which is perfect control over the regiospecificity, as shown in the examples above.

The procedure is markedly improved from the first proposed procedure. The first improvement,

sulfonyloxy carbamates, broadened the scope of the reaction due to the milder conditions; no

chlorinating agent and hydroxide were needed. Furthermore the yield could be increased, not on all

substrates, while using less catalyst. The yields and stability of the sulfonyloxy carbamates could be

problematic. The next improvement was from sulfonyloxy carbamates to O-derivatized

hydroxycarbamates. A variety of the latter carbamates were tested and initially C6F5 seemed the best


candidate although 2,4,6-Me3C6H2 was most employed in synthetic strategies, as discussed above. The

TA reaction however has a more limited substrate set than the asymmetric aminohydroxylation. Only

allylalcohols are suitable and furthermore the reaction is stereospecific.

Silyl chemistry and Fleming-Tamao oxidation

Stereoselective introduction, both syn and anti, of a silyl group can be achieved with a suitable Grignard

reagents and depending on the desired product CeCl3 or CuI and BF3.Et2O. This has been developed by

Boons et al. in 1989.27 By precomplexation of the silyl Grignard reagent with CeCl3 the exclusive syn

addition can be achieved. Conversely precomplexation of the same silyl Grignard reagent with CuI and

BF3.Et2O will give the anti-adduct. The resulting organocopper reagent from the precomplexation of the

Grignard reagent with CuI, attacks the least hindered face. The least hindered face depends on the

functionalization of the alcohol group in our synthesis. If the free hydroxyl group is used, the ring system

becomes more bulky, especially with the Boc protecting group, leading to product 86 via Felkin-Ahn

model transition state 85. If the hydroxyl group is suitably functionalized to make it sufficiently bulky it

can lead to product 52, via transition state 87, see scheme 32.

Scheme 32. Transaction state of addition of organocopper reagent and the resulting products.

The Fleming-Tamao oxidation is the oxidation of a carbon silicon bond to furnish an alcohol. A silicon

carbon bond is more stable compared to a carbon - hydroxyl group. Furthermore the stereochemistry of

the carbon silicon bond is retained after Fleming-Tamao oxidation. Therefore these functional groups are

also known as masked alcohols.

In scheme 33 the mechanism of the Fleming-Tamao oxidation using KBr and per acetate is depicted.

First, KBr is oxidized in situ with peracetate to 89 to make Br an electrophile; KOH is neutralized by the

NaOAc buffer present. Halogenation of the silyl atom and subsequent attack of the peracetic acid yields

95 followed by rearrangement akin to that observed in Baeyer-Villiger Oxidation yields after a number of

rearrangements species 97. By hydrolysis the alcohol 98 is generated; the original stereochemistry of

the silyl moiety is retained in product 98.


Scheme 33. Mechanism of the Fleming-Tamao oxidation. R groups to indicate retention of

stereochemistry with R1 ≠ R1 ≠ R1

The stereo selective introduction of the silyl group and subsequent oxidation is an elegant way to enforce

the right stereochemistry upon the amine.

Goal of the project The goal of the project is the synthesis of Pseudaminic hexose and Acinetaminic hexose. This thesis

focusses on two things:

- The effect of the protection group of the amine of 42 on the tethered aminohydroxylation

- Exploration of the Acinetaminic acid pathway from the common precursor 42 for both Pse and

Acinetaminic acid.

Figure 3. Common precursor 42.


Results and discussion

Stereoinversion The first step in the synthesis towards Pseudaminic acid and Acinetaminic acid precursor is the stereo

inversion of L-threonine to L-allo-threonine. 30 grams of the starting material, L- threonine, is used to

yield enough product later in the synthesis to work with.

The first step in the stereoinversion is the protection of L-threonine (44) with thionyl chloride and

methanol to yield ester 99, see scheme 34. After a simple workup, concentration in vacuo, the product

was obtained in quantitative yield which is in agreement with literature.

Scheme 34. Esterification of L-threonine 44 with thionyl chloride and methanol to yield 99. Reagents and conditions: 0 oC to reflux, MeOH, 24h; quantitative yield.

The next step is the functionalization of the amine with a benzoyl group. Benzoyl chloride was added

dropwise to a solution of amine 99 and Et3N, in methanol to yield 100. Et3N is added to both neutralize

the acid from the previous step as well as a non-nucleophilic base as depicted in scheme 35. The product

was obtained in 82% yield, which is lower than reported in literature (95%).28 This is due to problems

encountered during workup, on a previous reaction on a small scale the literature reported yield was

obtained.

Scheme 35. Benzoylation of amine 99 with BzCl and Et3N to yield 100 . Reagents and conditions: 0 oC to rt, MeOH, 24h; 82% yield.

Next is the formation of an oxazoline 101 from amine 100. This is performed with thionyl chloride and

the mechanism is depicted in scheme 36. The attack of the carbonyl oxygen occurs via a Sn2 reaction,

leading to the desired inversion of that chiral centre. The product was obtained in 85% yield which is

lower than reported in literature (96%).28 Although DCM was previously used as solvent in this reaction,

the reaction worked well in thionyl chloride.


Scheme 36. Oxazoline formation of 100 with thionyl chloride to yield 101. Reagents and conditions: 0 oC to rt, 24h; 85% yield.

The oxazoline product 101 obtained in the previous reaction is hydrolysed in acidic conditions to yield

the HCl salt of L-allo-threonine 102, see scheme 37. The methyl ester is also hydrolysed under these

conditions. The product of this reaction was a thick brown oil/paste which was difficult to handle. This

oil/paste was impure, the main impurity was benzoic acid resulting from the hydrolysis of the oxazoline.

Purification of this oil/paste has been performed in the past with moderate yields and it was therefore

decided to proceed with the crude product without further purification, besides an extraction.

Scheme 37. Hydrolysis of oxazoline 101 to yield L-allo-threonine HCl salt 102. Reagents and conditions: Reflux, H2O, 24h.

After the stereo inversion of L-threonine 44 to L-allo-threonine HCl salt 102, 102 was converted to the

methyl ester 103. This was performed analogous to the esterification of L-threonine, see scheme 38. The

reaction had to be repeated as the first time only 75% conversion, estimated with 1H NMR, was

achieved. After the second reaction the product was obtained as a brown paste/oil. The crude reaction

mixture was used in the next step without any further purification.


Scheme 38. Esterification of L-allo-threonine HCl salt 102 with thionyl chloride and methanol to yield 103. Reagents and conditions: 0 oC to reflux, MeOH, 24h.

Varying the protecting group To study the effect of the amine protecting group on the tethered amino hydroxylation three different

protected amines were synthesized. The TA reaction has been performed in the past by my supervisor

Tjerk Sminia with the Boc protected amine and poor selectivity and moderate yield was observed. The

amine in the ring of 42 is close to the allyl alcohol and might have an effect on the selectivity of the TA

reaction. By varying the protecting group present in the amine earlier in the synthesis of amine 104, the

effect of this group can be studied. To this end three different protected amines were synthesised, a Boc

protected amine, a Cbz protected amine and an acetylated amine. In this section the synthesis of these

protected amines up until the allyl alcohol 42 is discussed for every protecting group.

Scheme 39. Approach to synthesize three different protected allyl alcohols 42. PG = Boc, Cbz, Ac.

Boc protected amine

The methyl ester of L-allo-threonine 103 was Boc protected under basic conditions, see scheme 40.

NaCO3 was used as a base and more equivalents were added to neutralize the acid from the previous

reaction. The product was obtained as a transparent oil in 75% yield over three steps.

Scheme 40. Boc protection of amine 103 with di-tert-butyl dicarbonate to yield amine 106. Reagents and conditions: Rt, H2O, MeOH (1:1), 16h; Yield 75%.

The protected amine 106 was protected with 2,2-dimethoxypropane with BF3 etherate as Lewis acid, in

acetone to isopropylidene 107, see scheme 41. The isopropylidene protection is chosen as it leads to a

commonly used product for preparation of amino acid alcohols, the Garner aldehyde.29 Additionally both

the alcohol and the amine are protected and the intermediate is conformationally fixed.

A critical improvement was made to this procedure. Previously bottled dry acetone was used, however

with this procedure the reaction did not reach complete conversion after 12 h. The reaction had to be

quenched, concentrated in vacuo and subsequently the procedure had to be repeated to reach a final

yield of 78%. When technical acetone, dried over MgSO4, was used the reaction showed complete

conversion after 4 h, with an excellent yield of 97% after purification with flash chromatography. This


reaction equilibrium is clearly very sensitive to water and it is advantageous to dry the acetone over

MgSO4 or NaSO4 prior to performing the reaction to minimize the amount of water. The isopropylidene

product and further products gave rise to rotamers, also for the other protecting groups. By dissolving

the product in different solvents it was concluded that it was a rotamer and not a diastereoisomer, as the

ratio of peaks changed.

Scheme 41. Isopropylidene protection of amine 106 with 2,2 dimethoxypropane. Reagents and conditions: Rt, acetone, 4h; Yield 91%.

The ester of the isopropylidene 107 was reduced in the subsequent step, see scheme 42. The reduction

was performed with LiAlH4. After acidic workup and purification by flash chromatography alcohol 108

was obtained as an off-white solid in 89% yield.

Scheme 42. Reduction of ester 107 to alcohol 108 with LiAlH4. Reagents and conditions: Rt, THF, 1.5h; Yield 89%.

The alcohol 108 was subsequently oxidized via a Swern oxidation to aldehyde 109, see scheme 43. A

Swern oxidation was preferred over a Dess-Martin due to the scale. A Swern oxidation is more atom

efficient than a Dess-Martin oxidation and the reagents needed for a Swern oxidation are cheaper than

the Dess-Martin reagent; therefore the Swern oxidation was preferred. The Swern oxidation procedure

furnished aldehyde 109 as a yellow oil in 80% yield.


Scheme 43. Swern oxidation of alcohol 108 to aldehyde 109. Reagents and conditions: -78 oC, DCM, 1.5h; Yield 80%.

The ester 107 can be selectively reduced in one step to aldehyde 109 with DiBAL-H, however the

reduction to the alcohol 108 and oxidation to aldehyde 109 is preferred due to the ease of synthesis.

While the selective reduction to the aldehyde looks good on paper, overreduction to the alcohol can be a

problem. To ensure only the aldehyde is made the full reduction and subsequent oxidation is preferred

because it is more robust. The reduction with DiBAL-H also leads to small epimerization while the

epimerization is less with the reduction oxidation sequence, the base used in the Swern oxidation is

important; N,N-diisopropylethylamine gave better results than Et3N.29

With aldehyde 109 in hand a selective Grignard reaction is performed, to yield allylalcohol 110. At low

temperatures the reactions occurs mostly via the energetically favourable Felkin-Ahn transition state,

resulting in the desired stereochemistry, depicted in scheme 44. The product was obtained after

purification in 68% yield.

Scheme 44. Reaction of aldehyde 109 with a vinyl Grignard to yield allylalcohol 110. Reagents and conditions: -78 oC, THF, 5h; Yield 68%.


Cbz protected amine

The methyl ester of L-allo-threonine 103 was protected with a Cbz group with N,N-diisopropylethylamine

as base, see scheme 45. The product 111 was obtained in 69% yield over three steps.

Scheme 45. Cbz protection of amine b103 with Benzyl chloroformate to yield amine 111. Reagents and conditions: Rt, THF, 16h; Yield 69%.

The Cbz protected amine 111 was protected with 2,2-dimethoxypropane in acetone to isopropylidene

112 in 93% yield, see scheme 46. The improved procedure, using technical acetone dried over MgSO4,

was used in the synthesis of 112.

Scheme 46. Isopropylidene protection of Cbz protected amine 111 with 2,2 dimethoxypropane. Reagents and conditions: Rt, acetone, 4h; Yield 93%.

The next step is the reduction of the ester 112 to alcohol 113, see scheme 47. The reduction was

initially tried under the same conditions as the Boc protected isopropylidene on small scale, however

degradation was observed, probably the Cbz carbamate was cleaved. The Cbz protecting group is known

to be less stable to LiAlH4 reductions than the Boc protecting group. Following this result a selective

reduction to the aldehyde with DiBAL-H was performed. Although degradation was not observed some

overreduction of the ester had occurred. Besides the overreduction the conversion of this reaction was

slow even when more than 1 equivalent of reducing agent was added. In principle overreduction to the

alcohol is not a problem as the product can be oxidized to the aldehyde as performed before, however

due to the slow conversion other test reactions were performed. The reduction was also tried with NaBH4

with methanol, NaBH4 is not reactive enough to reduce esters however addition of methanol increases

the reactivity. The procedure was applied successfully in literature on Cbz protected amino esters.30 The

reaction proceeded selectively, one spot was observed by TLC with an Rf corresponding to the desired

product as determined by earlier test reactions. The reaction was however very slow, after 24 h and 8

equivalents of NaBH4 only minor conversion was observed. The last test reaction was performed with

LiAlH4 at -50 oC. As the ester is more reactive than the carbamate the lower reaction temperature might

lead to selective reduction of the ester. The test reaction gave a positive result, selective reduction of the

ester was observed with an acceptable amount of by-product, some starting material was also regained

after purification with flash column chromatography. The big scale reduction of the ester 112 however

did not proceed well. The same procedure was used as the small scale reaction and complete conversion

was observed with minor by product formation. Upon quenching the reaction the mistake was made to

immediately increase the temperature instead of waiting until the reaction was fully quenched. Upon

warming up the alcohol product 113 probably reacted which resulted in reduction of the carbamate the

product was obtained in a disappointing yield of 38%. Although the yield on larger scale was low it can


easily be improved by adequate quenching. Fortunately enough product was obtained to continue with

the synthesis.

Scheme 47. Reduction of ester 112 to alcohol 113. Reagents and conditions: -50 oC, THF 24h; Yield 38%.

With the alcohol 113 in hand the oxidation to aldehyde 114 was performed, see scheme 48. This was

performed with the Swern oxidation as outlined before. The product was obtained as a yellow oil in 85%

yield.

Scheme 48. Swern oxidation of alcohol 113 to aldehyde 114. Reagents and conditions: -78 oC, DCM

1.5h; Yield 85%.

A Grignard reaction of aldehyde 114 with vinylmagnesium bromide was performed and product 115 was obtained in 61% yield. The selectivity of this reaction is analogous to that of the Boc protected aldehyde.


Scheme 49. Reaction of aldehyde 114 with a vinyl Grignard to yield allylalcohol 115. Reagents and conditions: -78 oC, THF, 5h; Yield 61%.

Acetyl protected amine

The last protective group of the amine 103 to be evaluated for the tethered aminohydroxylation was an

acetyl that was installed in the presence of catalytic amount of DMAP, see scheme 50. The product 116

was obtained in 75% yield over three steps. By using 1.05 equivalents of Ac2O acetylation of the

hydroxyl group could be prevented.

Scheme 50. Acetyl protection of amine 103 with acetic anhydride to yield acetylated amine 116.

Reagents and conditions: Rt, pyridine, 16h; Yield 75%.

Following this reaction the acetylated amine 116 was protected to the isopropylidene 117 with 2,2-

dimethoxypropane and catalytic amount of BF3 etherate, see scheme 51. The old procedure was used for

this specific protected amine, without the acetone dried over MgSO4. The reaction had therefore been

repeated twice as the first time full conversion was not obtained after 16h. The old procedure resulted in

a poorer yield of 117, 66%, in comparison to the Boc and Cbz isopropylidene.

Scheme 51. Isopropylidene protection of amine b116 with 2,2 dimethoxypropane. Reagents and conditions: Rt, acetone, 16h; repeated, yield 66%.

The reduction of ester 117 with LiAlH4 at room temperature to alcohol 118 was unsuccessful, see

scheme 52. Degradation was observed; the acetyl protecting group was reduced. The improvement of

this procedure by lowering the reaction temperature as tested with the Cbz protected ester, might lead

to favourable results. However due to time constraints further testing of the reduction of ester 117 has


not been performed. Instead it has been chosen to focus on further modifying the Cbz allyl alcohol for

the tethered amino hydroxylation reaction.

Scheme 52. The reduction of Acetyl protected ester 117 to alcohol 118. Reagents and conditions: Rt, THF, 2h.

With both the Boc protected allyl alcohol 110 and Cbz protected allyl alcohol 115 in hand the synthesis

towards Pseudaminic and Acinetaminic acid precursor is continued. With the Cbz allyl alcohol 115 the

route towards Pseudaminic acid, with as key step the tethered amino hydroxylation, is pursued;

discussed in the next section. With the Boc protected allyl alcohol 110 the first steps towards

Acinetaminic acid were explored; discussed in corresponding section. The Acetyl protected ester 117 is

suitably stored and not continued in this thesis.

Scheme 53. Boc and Cbz protected allyl alcohol, 110 and 115. Acetyl protected ester 117.

Pseudaminic acid precursor: the tethered aminohydroxylation For the tethered amino hydroxylation reaction, the amine has to be tethered to the allylic alcohol group

followed by functionalization to a suitable O-derivatized hydroxycarbamate as discussed in the section

“Tethered aminohydroxylation”.

The first step is the functionalization of the allylic alcohol 115 to amide 119 in a two-step one pot

procedure that involved initial activation with CDI and subsequent reaction of this intermediate with

hydroxylamine, see scheme 54. The product 119 was obtained as a yellow oil in 70% yield with 20%

starting material 115 recovered.

Scheme 54. Functionalization of allylic alcohol 115 to amide 119. Reagents and conditions: 40 oC to 0 oC, pyridine, 24h; Yield 70%.


The amide 119 is further functionalized with 2,4,6-trimethylbenzoyl chloride to form O-derivatized

hydroxycarbamate 120, see scheme 55. The product 120 is obtained in 61% yield. 2,4,6-

Trimethylbenzoyl chloride is chosen to functionalize the amide because testing a variety of

hydroxycarbamates by Tjerk Sminia revealed that 2,4,6-trimethylbenzoyl carbamate gave the best

results with the Boc protected substrate.

Scheme 55. Functionalization of amide 119 with that 2,4,6-trimethylbenzoyl chloride to yield amide 120. Reagents and conditions: Rt, DCM, 1h; Yield 61%.

With the 2,4,6-trimethylbenzoyl carbamate 120 in hand the tethered amino hydroxylation was tested.

The mass of the desired product was found and the amine hydrogen peak was observed in 1H NMR however two spot were present on TLC, indicating the formation of both isomers. Purification of the crude reaction mixture by automated flash chromatography was unsuccessful.

With no indication of improvement of the stereoselectivity of the tethered amino hydroxylation of the Cbz

protected amide in comparison with the Boc protected amide the focus of our research shifted towards

Acinetaminic acid. The reaction with 2,4,6-trimethylbenzoyl carbamate 120 however has been performed

on a larger scale by Tjerk Sminia. The results of this larger scale reaction is inconclusive, however

significant lower yield was obtained in comparison with the Boc protected substrate. Due to the lower

yield and no improvement with regard to the stereochemistry the Boc substrate is chosen towards the

synthesis of Pseudaminic acid analogue.

Scheme 56. Tethered aminohydroxylation of 120 to amide 121. Reagents and conditions: Rt, t-BuOH,

MeCN, H2O (4:4:1), 16h.


Acinetaminic acid precursor: Exploring the ozonolysis With Boc protected allyl alcohol 115 in hand the first steps towards Acinetaminic acid precursor were

explored. The first step is the protection of the allylic alcohol 115 with a group which is compatible with

the ozonolysis, with that in mind the benzyl protective group is selected. The ozonolysis reaction is

compatible with an unprotected allylic alcohol although in further steps the protection of the alcohol is

needed. The benzylic protecting group is advantageous because it can be simultaneously deprotected

with the benzyl amine which is introduced after the oxidation. It was therefore decided to protect the

allylic alcohol with a benzyl protecting group.

The protection reaction of alcohol 115 was performed by deprotonating with NaH and BnBr mediated

alkylation, with a catalytic amount of tetrabutylammonium iodine (TBAI), to yield benzyl protected olefin

122. The protecting reaction with BnBr was slow, even after 68 hours the reaction was not complete

however the amount of starting material still present, judged by TLC, was small enough to warrant work-

up and 115 was obtained in 63% yield. The yield of the reaction was lower than obtained on a similar

substrate in literature while the same reaction conditions were used.31 A small test reaction in another

solvent DMF did not help, an even lower yield was obtained. Addition of more equivalents of base did not

give any noticeable difference. Although the used NaH used had its characteristic grey colour it might be

still be partially degraded. Although the yield was disappointingly low, enough product was obtained to

continue. The resulting product appeared to be a single product as judged by TLC under a variety of

conditions however the 1H NMR spectrum was not conclusive as it showed rotamers.

Scheme 57. Protection of allyl alcohol 115 with BnBr to yield olefin 122. Reagents and conditions: Rt, THF, 68h; Yield 63%.

Another protection for the allylic alcohol 115, a protection with TBDMS ether was tested next. The yield

of this reaction was very poor, 123 was obtained in only 21% yield.

Scheme 58. Protection of allyl alcohol 115 with TBDMS to yield 123. Reagents and conditions: Rt, DMF, 72h; Yield 63%.

The next step was the ozonolysis, however testing of the ozonolysis reaction was first performed on a

model olefin compound instead of our precious product. The ozonolysis was tested first to determine if

ozone could be generated in the first place as well as to gain experience with the procedure. The reaction


performed with 1-dodecene in ethanol and quenched with Me2S. Analysis of the 1H NMR spectra revealed

a characteristic aldehyde proton, the ozonolysis had worked.

The reaction with 1-dodecene was repeated, this time in ethanol stabilized DCM and quenched with

Me2S. In the 1H NMR spectrum only a small amount of aldehyde proton was observed. The last reaction

was repeated with addition of ethanol as the reaction performed in this solvent was successful.

Unfortunately this had no effect on the outcome of the reaction.

Although test reactions in DCM proved unsuccessful, successful ozonolysis reactions with these

conditions, DCM/MeOH 9:1 and quenching with Me2S, on similar substrates as our allylalcohol 122 have

been reported in literature. It was therefore decided to perform a test reaction on substrate 122 under

these conditions, see scheme 59. While the indicative blue coloration was observed, after quenching with

Me2S and work up degradation was observed. In the 1H NMR spectra multiple peaks in the aldehyde

region were observed, however complete degradation was observed with TLC. The reaction was repeated

with more quenching agent as in literature this varies between 2 equivalents and over a 100 equivalents

in the procedures used.32-34 However this addition had no influence on the reaction.

The last test reaction performed on the Benzyl protected ally alcohol 122 was with the addition of a

small amount of base, NaHCO3, to the reaction mixture. Another change was the quenching agent, solid

Ph3P was added which dissolved slowly over a period of 1 hour. TLC after quenching was promising, 2

spots with a KMNO4 stain of which one spot stained selectively with 2,4-dinitrophenylhydrazine (2,4-

DNP), which indicates the presence of an aldehyde. The reaction mixture was separated in two portions

and one portion was concentrated in vacuo while the other was first extracted with DCM and washed with

brine. This was done to check if degradation occurred in the extractive work up. TLC of both portions

where comparable and showed a major product, which stained selectively with 2,4-DNP however multiple

smaller spots were observed which were not there in the initial TLC. Analysis of both 1H NMR spectra,

which were very comparable, showed multiple aldehyde peaks and not characteristic olefin proton. The

mass of the desired product was confirmed by mass spectrometry. After purification of the pooled

portions 124 was obtained in 28% yield.

Scheme 59. Ozonolysis of olefin 122 to aldehyde 124. Reagents and conditions: -78 oC, DCM, MeOH 9:1, 1h; Yield 28%.

There was one more test reaction performed with 1-dodecene. The reaction was performed in ethanol

stabilized DCM with addition of 3 equivalents of pyridine. This procedure is mechanistically somewhat

different therefore it warranted testing, the proposed mechanism is depicted in scheme 60.35 A downside

of this procedure however is the chance of over-oxidation. The reaction has to be stopped when blue

coloration is observed otherwise a significant part will oxidize to the acid.


Scheme 60. Proposed mechanism of ozonolysis in presence of pyridine.

While testing 1-dodecene the blue coloration was hard to notice. The reaction had proceeded to long and

extensive over oxidation had occurred. However, a good amount of aldehyde was still present and this

was the first reaction with 1-dodecene in DCM which showed a favourable result. Due to time constrains

this procedure and other attempts at ozonolysis were not investigated further.


Conclusion

Pseudaminic acid analogue The 2,4,6-trimethylbenzoyl carbamate 120 was successfully synthesized. The tethered

aminohydroxylation with 120 was successful however no significant difference in stereoselectivity was

observed in comparison with the Boc protected moiety tested by Tjerk Sminia. The yield was lower than

the Boc protected moiety therefore the route towards Pseudaminic acid analogue will be continued with

the Boc protected moiety.

Acinetaminic acid analogue The Bn protected allyl alcohol 122 has been successfully synthesized. Ozonolysis of this substrate was

unsuccessful; the best yield obtained was 28%. Further testing is needed to successfully oxidize the allyl

alcohol 122.


Future prospects In this section firstly end game strategies for the synthesis of the hexose precursor of Pseudaminic acid

and Acinetaminic acid are discussed. Secondly suggestions regarding the ozonolysis will be discussed.

Pseudaminic acid analogue Starting from 125, successfully synthesized during this thesis, a few steps remain towards the

Pseudsaminic hexose precursor. The first step is the protection of the primary alcohol with a suitable

group, see scheme 61. Trimethylsilane might be a good choice for the protecting group as it can be

deprotected simultaneously with the isopropylidene group later. The TMS is acid labile however the next

steps are performed under basic condition so it is compatible. The amide is first acetylated and

afterwards cleaved to furnish amine 127. This protection cleavage step has been performed successfully

in literature with a Boc protecting group in one pot.23 As the reagents and conditions used for Boc

protection and Acetylation are quite similar, the same procedure might be used for the acetylation. Next

the secondary alcohol is protected, the benzyl group is chosen as it can be simultaneously deprotected

with the Cbz group of the amine.

Scheme 61. First steps towards Pseudaminic acid hexose.

The next step is the deprotection of the isopropylidene and TMS protective groups, this can be performed

with an acidic ion exchange resin, see scheme 62. Subsequent selective oxidation of the primary alcohol

of 129 leads to aldehyde 130, a hexose precursor of Pseudaminic acid.

Scheme 62. Deprotection of the isopropylidene followed by oxidation of primary alcohol to yield Pseudaminic precursor 130.

Both amines of hexose precursor 130 are orthogonally protected, therefore they can be selectively

modified to an azide. A strategy to functionalize Cbz protected amine can be found in scheme 63. Firstly

hydrogenation of hexose precursor 130 is performed to yield primary amine 131, the hydrogenation also

deprotects the C-3 hydroxyl group. The primary amine can be selectively converted to an N azido acetyl

group with 2-Azidoacetic acid.

Scheme 63. Converting C-4 amine to azide 132.

This Pseudaminic hexose precursor 132 can be converted to Pseudaminic acid analogue 134 in three

steps, see scheme 64. The same strategy is used by Lee et al. as discussed in the introduction.15 The

strategy consists of a Barbier alkylation followed by ozonolysis and final deprotection.


Scheme 64. Chain elongation with Barbier alkylation of 132 to yield 133, ozonolysis of olefin 133 followed by deprotection of the ester to yield Pseudaminic acid analogue 134.

Acinetaminic acid analogue Provided that the ozonolysis of 122 can be performed successfully, the pathway towards an Acinetaminic

acid hexose starts with reaction of aldehyde 124 with benzylamine to yield imine 135, see scheme 65.

As described in the introduction, a selective introduction of a silyl carbon bond can be achieved by

precomplexing the suitable silyl Grignard reagent with CeCl3. Subsequent Fleming-Tamao oxidation,

discussed in the same section, leads to alcohol 137.

Scheme 65. Introduction of a silyl carbon bond via imine 135, followed by subsequent oxidation to alcohol 137.

The isopropylidene 137 is subsequently deprotected to yield diol 138, see scheme 66, with the same

procedure as the Pseudaminic acid hexose. Selective oxidation of the primary alcohol of 138 to aldehyde

139 leads to hexose precursor of Acinetaminic acid.

Scheme 66. Deprotection of isopropylidene 137 followed by selective oxidation to yield hexose precursor 139.

The next step is the deprotection of the Bn and Cbz protective groups by means of hydrogenation, this

will lead to 140, see scheme 67. Selective acetylation of the primary amine with can be performed to

yield 141. Acetylation of the hydroxyl group might occur, it might be beneficial to protect the alcohol

first with a silane protecting group, which can be deprotected afterwards with tetrabutylammonium

fluoride.


Scheme 67. Deprotection of the benzyl groups of 139 to amine 140 which is subsequently acetylated to 141.

Selective deprotection of the Boc protective group of amine 141 can be performed to yield amine 142

with anhydrous acid, see scheme 68. The primary amine can be selectively converted to an N azido

acetyl group with 2-Azidoacetic acid. This procedure results in hexose precursor of Acinetaminic acid

analogue 143.

Scheme 68. Deprotection of the C-4 amine followed by conversion to the azide 143.

The hexose precursor of Acinetaminic acid can be converted to Acinetaminic acid analogue 145 in three

steps, see scheme 69. The same procedure is used, firstly a Barbier alkylation followed by ozonolysis and

a deprotection of the ester to yield Acinetaminic acid analogue 145.

Scheme 69. Barbier alkylation, ozonolysis and deprotection of hexose 143.

The selectivity of the Barbier reactions performed on the hexose precursors might be influenced by the

protective groups, especially the C-2 acetyl protective group could have an effect due to the proximity. It

is beneficial to test the effect on the protective group of this amine on the selectivity of the Barbier

alkylation as this very reaction had poor selectivity in literature.12


Ozonolysis With regard to the ozonolysis reactions a few procedures can be explored. The ozonolysis has been

performed on a benzyl protected allyl alcohol, the effect of other protected alcohols can be tested on the

reaction. For that purpose the TBDMS protected alcohol was synthesized however due to time constraints

the ozonolysis was not tested. Alternative the ozonolysis can be tested without protection of the allylic

alcohol.

An alternative to the ozonolysis reaction at this stage might be with the silyl Grignard reagent. The

stereoselective addition of this moiety can be controlled, as discussed in the introduction, and the silyl

group can be subjected to the Fleming-Tamao oxidation yielding a primary alcohol, see scheme 70.

Oxidation of the resulting alcohol to the aldehyde furnishes the same product 148 and can be used in the

originally planned synthesis strategy.

Scheme 70. Alternative if ozonolysis does not lead to favourable results. Stereoselective reaction of 109 with a silyl Grignard followed by Fleming-Tamao oxidation yields product 147. Selective oxidation of the primary alcohol leads to product 148.


Experimental Reagents were obtained from commercial sources and used without further purification unless stated

otherwise. Flash column chromatography was performed on silica gel 40-63 μm mesh with the indicated

solvents. Dry column chromatography was performed on silica gel 20-40 μm mesh with the indicated

solvents. Automated column chromatography was performed on Biotage Isolera system with prepacked

silica columns. TLC was performed with pre-coated silica gel on aluminium sheets (Merck KGaA 60 F254),

UV detection or stained with nihydrin for primary and secondary amines, 2,4-Dinitrophenylhydrazine for

aldehydes and KmNO4 as a general stain was used.

Mass spectra were recorded on LXQ (ESI-MS) and the mass is reported as a m/z ratio. The NMR spectra

were recorded on a Bruker Avanche 400. The 1H and 13C chemical shifts are reported relative to TMS, by

reference to the solvent residual 1H or13C resonances, in δ units (ppm) and J values (Hz).

Methyl- L –threoninate.HCl (99)

Thionyl chloride (20.12 mL, 277.04 mmol) was dropwise added to dry and

cooled (0 oC) MeOH (500 mL) and stirred for 1 hour. L-Threonine (30.0 g,

251.85 mmol) was portion wise added and the reaction was refluxed overnight.

After the reaction was complete the reaction mixture was concentrated in vacuo

to yield a yellow oil of (99) in quantitative yield.

Rf = 0.23 (3:1:2; DCM:MeOH:EtOAc). 1H NMR (400 MHz, Methanol-d4) δ 4.24 (dq, J = 6.5, 4.2 Hz, 1H,

H-2), 3.89 (d, J = 4.0 Hz, 1H, H-3), 3.81 (s, 3H, H-5), 1.29 (d, J = 6.8 Hz, 3H, H-1).

Methyl benzoyl- L –threoninate (100)

To a 1L round bottom flask charged with Methyl- L –threoninate.HCl (99)

(~47.0 g, 251 mmol) dry MeOH (550 mL) was added. To this solution

trimethylamine (76.81 mL, 554.07 mmol, 2.2 equiv.) was added and the

solution was stirred for 15 minutes. The reaction mixture was cooled to 0 oC and

benzoylchloride (27.80 mL, 239.26 mmol, 0.95 equiv.) was dropwise added to

this solution and left stirring overnight. After completion the reaction mixture

was concentrated in vacuo. DCM (500 mL) was added and subsequently washed

with sat. aq. NaHCO3 (2 x 200 mL) and brine (2 x 200 mL). The organic phase

was dried, (MgSO4) and concentrated in vacuo to yield Methyl benzoyl- L -

threoninate (100) as yellow oil; 82% yield* (48.92 g, 206.19 mmol).

Rf = 0.31 (95:5; DCM:MeOH). 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.2 Hz, 2H, H-10), 7.52 (t, J =

7.3 Hz, 1H, H-12), 7.44 (t, J = 7.3 Hz, 2H, H-11), 6.96 (d, J = 8.6 Hz, 1H, H-7), 4.83 (dd, J = 2.4, 8.7

Hz, 1H, H-3), 4.46 (dq, J = 2.5, 6.4 Hz, 1H, H-2), 3.79 (s, 3H, H-5), 2.43 (bs, 1H, H-6), 1.29 (d, J = 6.5

Hz, 3H, H-1). 13C NMR (100 MHz, CDCl3) δ 171.7 (C-4), 168.2 (C-8), 133.7 (C-9), 132.0 (C-12), 128.7

(C-11), 127.3 (C-10), 68.2 (C-2), 58.0 (C-3), 52.7 (C-5), 20.1 (C-1). MS: found 260.08 [M+Na]+,

calculated for [C12H15NO4 + Na]+ 260.09.

Methyl (4S,5S)-5-methyl-2-phenyl-4,5-dihydrooxazole-4-carboxylate.HCl (101)

Methyl benzoyl- L -threoninate (100) (48.92 g, 206.19 mmol) was added

portionwise to precooled, 0 oC, thionyl chloride (150 ml, 2.07 mol, 10 equiv.). The

solution was stirred overnight while allowed to warm up to rt. The reaction

mixture was concentrated in vacuo followed by the addition of ice-cold DCM (350

mL) and slow addition of precooled water (300 ml). Solid NaHCO3 (5~7g) was

portion wise added and the white suspension was transferred to a separating

funnel. The layers were separated; the aqueous layer was extracted with DCM (2

x 150 mL), and the combined organic phases were dried (MgSO4), and

concentrated in vacuo to yield Methyl (4S,5S)-5-methyl-2-phenyl-4,5-

dihydrooxazole-4-carboxylate (101); 85% (38.40 g, 175.15 mmol).


Rf = 0.75 (90:10; DCM:MeOH). 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 7.4 Hz, 2H, H-8), 7.48 (t, J =

7.3 Hz, 1H, H-10), 7.40 (t, J = 7.6 Hz, 2H, H-9), 4.83 (dq, J = 6.3, 10.2 Hz, 1H, H-2), 4.98 (d, J = 10.2

Hz, 1H, H-3), 3.76 (s, 3H, H-5), 1.38 (d, J = 6.3 Hz, 3H, H-1).

L -allo-threonine.HCl (102)

Methyl (4S,5S)-5-methyl-2-phenyl-4,5-dihydrooxazole-4-carboxylate.HCl (101)

(38.40 g, 175.15 mmol) was dissolved in 6M HCl (270 mL) and refluxed overnight.

The reaction mixture was washed with ether (1 x 50 ml). The aqueous layer was

concentrated in vacuo to give L-allo-threonine.HCl (102) as a dark brown oil/paste

(32.37 g). The resulting oil/paste is used in further steps without further

purification.

Methyl L -allo-threoninate.HCl (103)

To a round bottom flask containing L-allo-threonine.HCl (102) (30 g, 195.83

mmol) is added dry MeOH (300 mL) and the resulting solution was cooled to 0 oC. Thionyl chloride (15.9 mL, 218.93 mmol) was dropwise added and was

stirred overnight at room temperature. The reaction mixture was concentrated

in vacuo and the procedure was repeated. After the final concentration in vacuo

Methyl L-allo-threoninate.HCl (103) was obtained as a yellow oil/paste; it was

used in subsequent reactions without further purification.

Rf = 0.23 (3:1:2; DCM:MeOH:EtOAc). 1H NMR (400 MHz, Methanol-d4) δ 4.23 (m, 1H, H-2), 4.05 (d, J =

2.3 Hz, 1H, H-3), 3.81 (s, 3H, H-5), 1.23 (d, 6.6 Hz, 3H, H-1).

Methyl (tert-butoxycarbonyl)- L –allo-threoninate (106)

To a round bottom flask charged with methyl L -allo-threoninate.HCL (103)

(4.53 g, 26.71 mmol)* in water (50 mL) and MeOH (50 mL) NaHCO3 (6.73 g,

80,11 mmol, 3 equiv.) was added followed by di-tert-butyl dicarbonate (8.74

g, 40.05 mmol, 1.5 equiv.). The reaction mixture was stirred overnight and

upon complete conversion the solution is concentrated in vacuo. EtOAc (150

mL) and sat. aq. NaHCO3 (100 mL) were added. The layers were separated;

the water layer was extracted with EtOAc (2 x 150 ml) and the combined

organic layers were washed with sat. aq. NaHCO3 (1 x 100 mL) and with sat.

aq. NH4Cl (2 x 100 mL). The combined organic layers were dried (Na2SO4),

and concentrated in vacuo. Flash column chromatography (SiO2, 2:1

Hept.:EtOAc » 1:1 Hept.:EtOAc) gave Methyl (tert-butoxycarbonyl)- L -allothreoninate (106) as a yellow

oil; 75%** (4.27 g, 18.31 mmol).

Rf = 0.46 (3:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) δ 5.46 (s, 1H, H-7), 4.37 (s, 1H, H-2), 4.12 (m,

1H, H-3), 3.76 (s, 3H, H-5), 3.07 (d, 4.8 Hz, 1H, H-6), 1.43 (s, 9H, H-10), 1.18 (d, 6.4 Hz, 3H, H-1).

MS: found 256.11 [M+Na]+, calculated for [C10H19NO5 + Na]+ 256.12.

*Although the starting product is known to be impure, the amount of impurities is hard to determine. For

the calculations regarding equivalents the starting product is assumed to be pure.

**The yield is calculated from (101) as the intermediate products were used without purification.


Methyl ((benzyloxy)carbonyl)- L –allo-threoninate (111)

A round bottom flask was charged with methyl L -allo-threoninate.HCL (103) in

dry THF (60 mL) and cooled to 0 oC. Di-isopropyldiethyl amine (10.52 mL, 60.43

mmol, 2.2 equiv.) was added to the solution as well as CBzCl (4.32 mL, 30.25

mmol, 1.1 equiv.). The reaction mixture was stirred overnight while allowed to

warm to rt. After complete conversion, 1M HCl (50 mL) is added followed by

addition of EtOAc (50 mL). The layers were separated; the water layer was

extracted with EtOAc (3 x 50 mL) and the combined organic layers were washed

with brine (1 x 50 ml) and subsequently dried (Na2SO4) and concentrated in

vacuo. Flash column chromatography (SiO2, 4:1 Hept.:EtoAc » 2:1 Hept.:EtOAc)

gave Methyl ((benzyloxy)carbonyl)- L–allo-threoninate (111) as a yellow oil;

69%* yield (4.66g, 17.45 mmol).

Rf = 0.20 (1:2; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) δ 7.35 (m, 5H, H-11,12,13), 5.75 (d, 6.6 Hz,

1H, H-7), 5.11 (s, 2H, H-9), 4.44 (dd, 3.1, 4.1 Hz, 1H, H-2), 4.14 (m, 1H, H-3), 3.76 (s, 3H, H-5), 2.88

(s, 1H, H-6), 1.20 (d, 6.4 Hz, 3H, H-1). MS: found 290.33 [M+Na]+, calculated for [C13H17NO5 + Na]+

290.10.

*The yield is calculated from (101) as the intermediate products were used without purification.

Methyl acetyl- L –allo-threoninate (116)

To a round bottom flask charged with methyl L -allo-threoninate.HCL (103)

(4.82 g, 28.42 mmol) in pyridine (50 mL) Et3N (5.8 mL, 42.63 mmol, 1.5 equiv)

and DMAP (0.18 g, 1.48 mmol, 0.05 equiv.) were added. The reaction mixture

was cooled to ~-10 oC with ice and NaCl. After 15 minutes Ac2O (3.20 g, 31.34

mmol, 1.1 equiv.) was added. After 6 hours the reaction was complete and the

reaction mixture was concentrated in vacuo. Flash column chromatography

(SiO2, 2.5% » 5% MeOH in DCM) afforded Methyl acetyl- L –allo-threoninate

(116) (3.41 g, 19.47 mmol) in 75% yield.*

Rf = 0.13 (5% MeOH in DCM). 1H NMR (400 MHz, CDCl3) δ 6.59 (d, 4.8 Hz, 1H, H-7), 4.67 (dd, 3.4, 7.3

Hz, 1H, H-2), 4.16 (dq, 3.5, 6.5 Hz, 1H, H-3), 3.78 (s, 3H, H-5), 2.07 (s, 3H, H-9), 1.17 (d, 6.5 Hz, 3H,

H-1).

*The yield is calculated from (101) as the intermediate products were used without purification.

3-(tert-butyl) 4-methyl (4S,5S)-2,2,5-trimethyloxazolidine-3,4-dicarboxylate (107)

To a round bottom flask charged with methyl (tert-butoxycarbonyl)- L –allo-

threoninate (106) (3.05 g, 13.08 mmol) in acetone (technical grade dried

over MgSO4, 45 mL), 2,2-Dimethoxypropane (16 mL, 130 mmol, 10 equiv.)

and Boron trifluoride diethyl etherate (86 μl, 0.70 mmol, 0.05 equiv.) were

added. After complete conversion Et3N (5 mL) was added and the reaction

mixture was concentrated in vacuo. The resulting residue was pulled over a

plug of silica to afford 3-(tert-butyl) 4-methyl (4S,5S)-2,2,5-

trimethyloxazolidine-3,4-dicarboxylate (107) (3.46 g, 12.67 mmol) as a

yellow oil in 97% yield.

Rf = 0.45 (3:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) (mixture of rotamers, main / minor) δ 4.38/4.27

(d, 6.4 Hz, 1H, H-3), 4.36/4.40 (m, 1H, H-2), 3.74 (s, 3H, H-8), 1.73/1.69 (s, 3H, H-6), 1.52/1.49 (s,

3H, CH3-5), 1.39/1.48 (s, 9H, H-11), 1.24/1.23 (d, 2.0 Hz, 3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture

of rotamers, main / minor) δ 170.7/170.4 (C-7), 151.1/152.1 (C-9), 94.4/93.8 (C-4), 80.2/80.8 (C-10)

71.4/71.2 (C-3), 63.6/63.4 (C-2), 51.75/51.82 (C-8), 28.3/28.3 (C-11), 25.5/26.5 (C-6), 24.4/25.2 (C-

5), 15.5/15.4 (C-1). MS: found 296.17 [M+Na]+, calculated for [C13H23NO5 + Na]+ 296.15.


tert-butyl (4R,5S)-4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3-carboxylate (108)

LiAlH4 (1.0M in THF, 17 mL, 1.1 equiv.) was added to an ice cooled solution of

methyl (tert-butoxycarbonyl)- L –allo-threoninate (107) (4.21 g, 15.40mmol)

in dry THF (30 mL). After addition the solution was allowed to warm up to rt.

After complete conversion, 90 minutes, the reaction mixture was cooled to 0 oC and EtOAc (10 mL) was added followed by the addition of sat. aq.

Rochelle’s salt (5 mL). The reaction mixture was vigorously stirred for 30

minutes. The layers were separated; the aqueous layer was extracted with

EtOAc (2 x 70 mL) and the combined organic phases were washed with brine

(1 x 20 mL). The combined organic phases were dried (MgSO4), and concentrated in vacuo. Flash column

chromatography (SiO2, 2:1 Hept.:EtoAc » 1:1 Hept.:EtOAc) afforded tert-butyl (4R,5S)-4-

(hydroxymethyl)-2,2,5-trimethyloxazolidine-3-carboxylate (108) (3.365 g, 13.72 mmol) as an off-white

solid in 89% yield.

Rf = 0.50 (1:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) (mixture of rotamers, main / minor) δ 4.26

(quint, 6.2 Hz, 1H, H-2), 4.00 (m, 1H, H-3), 3.83 (m, 1H, H-7-a, H-3), 3.64 (m, 1H, H-7-b, H-3), 3.57

(m, 1H, H-8), 1.56/1.59 (s, 3H, H-6), 1.51/1.53 (s, 3H, H-5), 1.48 (s, 9H, H-11), 1.27/1.34 (d, J = 6.4

Hz, 3H, H-1).13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ 154.5 (C-9), 92.7 (C-4),

81.1 (C-10), 71.3 (C-2), 63.1 (C-7), 61.6 (C-3), 28.4 (C-11),27.8 (C-6), 24.5 (C-5), 14.4 (C-1). MS:

found 268.17 [M+Na]+, calculated for [C12H23NO4 + Na]+ 268.15.

tert-butyl (4S,5S)-4-formyl-2,2,5-trimethyloxazolidine-3-carboxylate (109)

A 250 ml three necked round bottom flask equipped with a stirring bar and

two dropping funnels was flame dried. One dropping funnel was charged with

DMSO (2.0 mL, 28.6 mmol, 3 equiv., in 2.5 mL DCM) the other one with tert-

butyl (4R,5S)-4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3-carboxylate

(108) (2.32 g, 9.54 mmol in 15 mL DCM), a flow of nitrogen is added to latter

dropping funnel and an outflow was provided by means of a needle trough a

septum on the remaining neck. The three necked round bottom flask was

charged with oxalyl chloride (1.20 mL, 14.30 mmol, 1.5 equiv., in 20 ml

DCM). The flask was cooled to -78 oC and the DMSO solution was dropwise added over a period of 15

minutes. Next the solution of 108 was dropwise added followed by rinsing of the dropping funnel with

DCM (2 x 5 mL) and the dropping funnel was subsequently charged with DIEA (9.0 mL, 51.6 mmol, 5.5

equiv.). The reaction mixture was allowed to warm up to -40 oC and after 30 minutes DIEA was dropwise

added. The reaction mixture was allowed to warm up to -0 oC and 1M HCl (35 mL) was added. The layers

were separated; the aqueous layer was extracted with DCM (3 x 10 mL) and the combined organic

phases were washed with phosphate buffer (pH 7, 4 x 20 mL). The organic phase was dried (Na2SO4)

and concentrated in vacuo to afford tert-butyl (4S,5S)-4-formyl-2,2,5-trimethyloxazolidine-3-carboxylate

(109) (1.83 g, 7.52 mmol) as a yellow oil in 80% yield.


(d, J = 3.7/2.9 Hz, 1H, H-7), 4.40 (quint, J = 6.5 Hz, 1H, H-2), 4.04/4.20 (dd, J = 6.7, 2.9 Hz, 1H, H-3),

1.73/1.67 (s, 3H, H-6), 1.57/1.53 (s, 3H, H-5), 1.40/1.49 (s, 9H, H-10), 1.29/1.31 (d, J =6.4 Hz, 3H, H-

1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ 200.8/200.7 (C-7), 151.6/152.6 (C-

8), 94.6/93.9 (C-4), 81.0/81.3 (C-9), 72.4/72.0 (C-2), 68.2/68.0 (C-3), 28.4/28.5 (C-10), 26.9/27.7 (C-

6), 24.0/24.9(C-5), 15.4 (C-1). MS: found 298.25 [M+CH3OH+Na]+, calculated for [C12H21NO4 + CH3OH

+ Na]+ 298.16; found 266.17 [M+Na]+, calculated for [C12H21NO4 + Na]+ 266.14.

tert-butyl (4R,5S)-4-((R)-1-hydroxyallyl)-2,2,5-trimethyloxazolidine-3-carboxylate (110)

A round bottom flask is charged with tert-butyl (4S,5S)-4-formyl-2,2,5-

trimethyloxazolidine-3-carboxylate (109) (1.83 g, 7.52 mmol) in dry THF (15

mL) was cooled to -78 oC. After 15 minutes, to ensure sufficient cooling,

Vinylmagnesium bromide solution (1.0 M, 8.27 mL, 1.1 equiv.) was dropwise

added. After complete conversion, 5 hours, sat. aq. NH4Cl (20 mL) was added.

The layers were separated; the aqueous layer was extracted with EtOAc (4 x

50 ml) and the combined organic phases were washed with brine (1 x 50 mL).

The organic phase was dried (Na2SO4) and concentrated in vacuo. Flash


column chromatography (SiO2, 1:1 Hept.:EtOAc » 1:3 Hept.:EtOAc) afforded tert-butyl (4R,5S)-4-((R)-

1-hydroxyallyl)-2,2,5-trimethyloxazolidine-3-carboxylate (110) (1.39 g, 5.12 mmol) as a yellow oil in

68% yield.

Rf = 0.43 (2:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) (mixture of rotamers, main / minor) δ 5.99-5.83

(m, 1H, H-8), 5.31/5.38 (dt, J = 17, 1.5 Hz, 1H, H-9), 5.22-5.14 (m, 1H, H-9), 4.33 (t, J= 6.5 Hz, H-2),

4.26 (m, 2H, H-3,7), 1.53 (s, 3H, H-6), 1.45 (m, 12 H, H-5,13), 1.34 (d, J= 6.5 Hz, H-1). 13C NMR (100

MHz, CDCl3) (mixture of rotamers, main / minor) δ 155.4 (C-11), 138.5/139.3 (C-8), 115.0/116.0 (C-9),

93.1 (C-4), 81.4 (C-12), 72.5 (C-2), 71.4 (C-7), 65.8 (C-3), 26.5 (C-6), 24.4 (C-5), 28.4 (C-13), 15.3

(C-1). MS: found 294.17 [M+Na]+, calculated for [C14H25NO4 + Na]+ 294.17.

Tert-butyl (4S,5S)-4-((R)-1-(benzyloxy)allyl)-2,2,5-trimethyloxazolidine-3-carboxylate (122)

A flame dried round bottom flask was charged with NaH (60% in mineral oil,

272 mg, 6.80 mmol, 2.0 equiv.) in dry THF (13 mL) and cooled to 0 oC. To

this suspension was added tert-butyl (4R,5S) -4- ((R)- 1- hydroxyallyl)- 2,2,5

-trimethyloxazolidine -3-carboxylate (110) (0.922 g, 3.40 mmol), co-

evaporated with dry toluene (3 x 5 mL), in dry THF (20 mL). The suspension

was allowed to warm up to rt and stirred for 60 minutes then cooled to 0 oC.

Benzyl bromide (600 μL, 5.10 mmol, 1.5 equiv.) and TBAI (130 mg, 0.34

mmol, 0.1 equiv.) were added. The suspension was allowed to warm up to rt

and stirred for 46 h. Additional benzyl bromide (200 μL, 1.70 mmol, 0.5

equiv.) was added and stirred overnight. Sat. aq. NH4Cl (33 mL) was added.

The layers were separated; the aqueous layer was extracted with EtOAc (4 x

50 ml) and the combined organic phases were washed with brine (1 x 50 mL).

The organic phase was dried (Na2SO4) and concentrated in vacuo. Flash

column chromatography (SiO2, 9:1 Hept.:EtOAc » 3:1 Hept.:EtOAc) afforded tert-butyl (4S,5S)-4-((R)-

1-(benzyloxy)allyl)-2,2,5-trimethyloxazolidine-3-carboxylate (122) (773 mg, 2.14 mmol) as a colourless

oil in 63% yield.

Rf = 0.50 (4:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) (mixture of rotamers, inseparable) δ 7.32-7.28

(m, 5H H-12,13,14), 6.07-5.88 (m, 1H, H-8), 5.37-5.20 (m, 2H, H-9), 4.63-4.53 (m, 1H, H-10), 4.29-

4.23 (m, 2H, H-2, 10), 4.08-3.91 (m, 2H, H-3,7), 1.58-1.47 (m, 15H, H-5,6,17), 1.29-1.35 (m, 3H, H-

1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ 151.8/152.5 (C-15), 138.6-138.0

(C-11), 136.5-136.8 (C-8), 128.3-127.2 (C-12,13,14), 119.5/118.9 (C-9), 93.0-92.1 (C-4), 80.3/81.3

(C-7), 79.8-79.5 (C-16), 72.9-72.5 (C-2), 70.8-70.0 (C-10), 61.8/61.7 (C-3), 28.4/28.5 (C-17),

27.0/27.7 (C-6), 23.5/25.0 (C-5), 14.9/15.3 (C-1). MS: found 384.28 [M+Na]+, calculated for

[C21H31NO4 + Na]+ 384.22.

tert-butyl (4S,5S)-4-((R)-1-((tert-butyldimethylsilyl)oxy)allyl)-2,2,5-trimethyloxazolidine-3-carboxylate

(123)

A round bottom flask was charged with tert-butyl (4R,5S) -4- ((R)- 1-

hydroxyallyl)- 2,2,5 -trimethyloxazolidine -3-carboxylate (110) (104.9 mg,

0.39 mmol), co-evaporated with dry toluene (3 x 0.5 mL), in dry DMF (5.0

mL). Imidazole (65 mg, 0.97 mmol, 2.5 equiv.), TBDMSCl (70 mg, 0.46

mmol, 1.2 equiv.) and DMAP (pipet point) were added and stirred for 72 h.

Sat. aq. NaHCO3 (1.0 mL) and EtOAc (2.0 mL) were added. The layers were

separated; the aqueous layer was extracted with EtOAc (3 x 3 ml) and the

combined organic phases were washed with sat. aq. NaHCO3 (1 x 3 mL). The

organic phase was dried (MgSO4) and concentrated in vacuo. Dry column

chromatography (SiO2, Hept. » 3:1 Hept.:EtOAc) afforded tert-butyl (4S, 5S)

4 - ((R) -1- ((tert-butyldimethylsilyl) oxy)allyl)-2,2,5-trimethyloxazolidine-3-

carboxylate (123) (32 mg, 0.083 mmol) as a colourless oil in 21% yield.


(m, 1H, H-8), 5.19/5.04 (m, 2H, H-9), 4.61-4.19 (m, 2H, H-2,7), 3.91-3.69 (m, 1-H, H-3), 1.63-1.45

(m, 16H, H-1,5,6,15), 1.37 (d, 2H, J= 6.5 Hz, H-1), 0.89-0.87 (m, 9H, H-12), 0.12-0.02 (m, 6H, H-10). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main/minor δ 152.9/152.1 (C-13),140.2/138.8/138.5

(C-8), 116.9/116.1 (C-9), 92.7/93.2, 92.1/92.5 (C-4), 80.0/79.7 (C-14) 74.9,74.1,72.5 (C-2,7),


64.0/63.1 (C-3), 28.9-28.6, 27.7, 26.9, 24.9, 24.7 23.5, 23.8 (C-5,6,15), 26.2-26.0 (C-12), 15.8-15.3

(C-1), -3.0 - -4.53 (C-10, 11). MS: found 408.42 [M+Na]+, calculated for [C20H39NO4Si + Na]+ 408.25

Methyl (4S,5S)-3-acetyl-2,2,5-trimethyloxazolidine-4-carboxylate (117)

To a round bottom flask was charged Methyl acetyl- L –allo-threoninate (116)

(3.20 g, 18.27 mmol) in acetone (80 mL), 2,2-Dimethoxypropane (22.4 mL,

182.5 mmol, 10 equiv.) and boron trifluoride diethyl etherate (140 μl, 1.13 mmol,

0.06 equiv.) were added. The reaction mixture was stirred overnight; Et3N (5 mL)

was added and the reaction mixture was concentrated in vacuo. The procedure

was repeated once more followed by flash column chromatography (SiO2, 1:1

Hept.:EtOAc » 1:3 Hept.:EtOAc) of the residue to afford methyl (4S,5S)-3-acetyl-

2,2,5-trimethyloxazolidine-4-carboxylate (117) (2,59 g, 12.03 mmol) as an off

white solid in 66% yield.

Rf = 0.13 (5% MeOH in DCM). 1H NMR (400 MHz, CDCl3) (mixture of rotamers, main / minor) δ

4.45/4.36 (quint, J= 6.3 Hz, 1H, H-2), 4.31/4.58 (d, J= 6.2 Hz, 1H, H-3), 3.79/3.71 (s, 3H, H-8),

1.95/2.16 (s, 3H, H-10), 1.75/1.78 (s, 3H, H-6), 1.57/1.59 (s, 3H, H-5), 1.27/1.25 (d, J= 6.4 Hz, 3H, H-

1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ 170.1/170.0 (C-7), 167.3/167.4 (C-

9), 96.2/93.6 (C-4), 72.3/70.7 (C-2), 64.8/64.0 (C-3), 52.5/52.1 (C-8), 25.4/31.0 (C-6), 24.1/26.3 (C-

5), 24.0/22.0 (C-10), 15.6/15.4 (C-1). MS: found 238.11 [M+Na]+, calculated for [C10H17NO4 + Na]+

238.11.

3-Benzyl 4-methyl (4S,5S)-2,2,5-trimethyloxazolidine-3,4-dicarboxylate (112)

To a round bottom flask charged with Methyl ((benzyloxy)carbonyl)- L –allo-

threoninate (111) (4.66 g, 17.43 mmol) in acetone (technical grade dried

over MgSO4, 80 mL), 2,2-dimethoxypropane (22 mL, 180 mmol, 10 equiv.)

and boron trifluoride diethyl etherate (104 μl, 0.85 mmol, 0.05 equiv.) were

added. After complete conversion Et3N (5 mL) was added and the reaction

mixture was concentrated in vacuo. Flash column chromatography (SiO2, 3:1

Hept.:EtOAc » 1:1 Hept.:EtOAc) afforded 3-benzyl 4-methyl (4S,5S)-2,2,5-

trimethyloxazolidine-3,4-dicarboxylate (112) (4.96 g, 16.14 mmol) as a

yellow oil in 93% yield.

Rf = 0.41 (2:1; Hept.:EtOAc). 1H NMR (400 MHz, CDCl3) (mixture of

rotamers, main / minor) δ 7.36-7.28 (m, 5H, H-12,13,14), 5.17 (q, J= 12.0 Hz, 1H, H-10a), 5.09 (q, J=

12.5, 40.1 Hz, 1H, H-10b), 4.44-4.37 (m, 2H, H-2,3), 3.65/3.76 (s, 3H, H-8), 1.76/1.69 (s, 3H, H-6),

1.56/1.50 (s, 3H, H-5), 1.25/1.26 (d, 5.8 Hz, 3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers,

main / minor) δ 170.4/170.2 (C-7), 151.7/153.0 (C-9), 136.5/136.2 (C-11), 95.0/94.4 (C-4), 128.7-

127-7 (C-12,13,14), 66.8/67.7 (C-10), 63.24/63.9/72.0/71.6 (C-2,3), 52.1 (C-8), 25.6/26.7 (C-6),

24.3/25.4 (C-5), 15.6 (C-1). MS: found 330.20 [M+Na]+, calculated for [C16H21NO5 + Na]+ 330.13.

Benzyl (4R,5S)-4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3-carboxylate (113)

A round bottom flask was charged with 3-benzyl 4-methyl (4S,5S)-2,2,5-

trimethyloxazolidine-3,4-dicarboxylate (112) (4.65 g, 15.13 mmol), co-

evaporated with dry toluene (3 x 5 mL), in dry THF (35 mL) was cooled to -

50 oC. LiAlH4 (1.0M in THF, 15.9 mL, 1.05 equiv.) was addeddropwise over a

period of 15 minutes. The reaction mixture was left stirring overnight. The

reaction mixture was allowed to warm up to -30 oC followed by the addition

of LiAlH4 (1.0M in THF, 15.1 mL, 1.0 equiv), the reaction mixture was left

stirring for 5 hours. EtOAc (50 mL) was added and it was allowed to warm up

to 0 oC. Sat. Aq. Potassium sodium tartrate (=Rochelle salt) (20 mL) was

added and the reaction mixture was vigorously stirred for 30 minutes. The

layers were separated; the aqueous layer was extracted with EtOAc (3 x 20

ml) an the combined organic phases were dried (MgSO4) and concentrated in vacuo. Flash column

chromatography (SiO2, 9:1 Hept.:EtOAc » 2:1 Hept.:EtOAc) afforded Benzyl (4R,5S)-4-(hydroxymethyl)-

2,2,5-trimethyloxazolidine-3-carboxylate (113) (1.59 g, 5.67 mmol) as a yellow oil in 38% yield.



(m, 5H, H-12,13,14), 5.17/5.13 (s, 2H, H-10), 4.19 (quint., 6.2 Hz, 1H, H-2), 4.06 (q, J= 5.5 Hz, 1H, H-

3a), 3.88-3.63 (m, 2H, H-7,10b), 3.21 (m, 1H, H-8), 1.56/1.63 (s, 3H, H-6), 1.50/1.57 (s, 3H, H-5),

1.30/1.36 (d, J= 6.3 Hz, 3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ

154.9/152.3 (C-9), 136.0/136.5 (C-4), 128.7-128.1 (C-12,13,14), 93.2/93.4 (C-11), 71.6/72.2 (C-2),

67.8/66.9 (C-10), 62.5,62.1/70.0,60.8 (C-3,7), 27.9/26.7 (C-6), 24.8/23.4 (C-5), 14.5 (C-1). MS: found

302.25 [M+Na]+, calculated for [C15H21NO4 + Na]+ 302.33.

benzyl (4S,5S)-4-formyl-2,2,5-trimethyloxazolidine-3-carboxylate (114)

250 ml three necked round bottom flask equipped with a stirring bar and two

dropping funnels was flame dried. One dropping funnel was charged with

DMSO (1.2 mL, 17 mmol, 3 equiv., in 1.5 mL DCM) the other one with

Benzyl (4R,5S)-4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3-carboxylate

(113) (1.59 g, 5.67 mmol in 10 mL DCM). The three necked round bottom

flask was charged with oxalyl chloride (0.73 mL, 8.5 mmol, 1.5 equiv., in 10

ml DCM). The flask was cooled to -78 oC and the DMSO solution was

dropwise added over a period of 15 minutes. Next the solution of 113 was

added dropwise followed by rinsing of the dropping funnel with DCM (2 x 5

mL) and the dropping funnel was subsequently charged with DIEA (5.4 mL,

31.2 mmol, 5.5 equiv.). The reaction mixture was allowed to warm up to -40 oC and after 30 minutes DIEA was added dropwise. The reaction mixture was allowed to warm up to -0 oC and 1M HCl (20 mL) was added. The Layers were separated; the aqueous layer was extracted with

DCM (3 x 10 mL) and the combined organic phases were washed with phosphate buffer (pH 7, 4 x 12.5

mL). The organic phase was dried (MgSO4) and concentrated in vacuo which benzyl (4S,5S)-4-formyl-

2,2,5-trimethyloxazolidine-3-carboxylate (114) (1.33 g, 4.80 mmol) as a yellow oil in 83% yield.


(d, 3.2/2.5 Hz, 1H, H-7), 7.36-7.25 (m, 5H, H-11,12,13), 5.09/5.19 (q, J= 5.7, 12.5/2.7, 12.4 Hz, 2H,

H-9), 4.43 (quint., J= 6.5 Hz, 1H, H-2), 4.19/4.31 (dd, J= 3.2, 6.6/ 2.5, 6.6 Hz, 1H, H-3), 1.76/1.67 (s,

3H, H-6), 1.60/1.53 (s, 3H, H-5), 1.33/1.34 (d, J= 6.5 Hz, 3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture

of rotamers, main / minor) δ 199.9/199.8 (C-7), 151.9 (C-8), 136.0/135.8 (C-10), 128.6-127.8 (C-

11,12,13), 94.9/94.2 (C-4), 67.6/68.2 (C-3), 67.0/67.8 (C-2), 26.5/27.4 (C-6), 23.8/24.9 (C-5),

15.1/15.2 (C-1). MS: found 300.25 [M+Na]+, calculated for [C15H19NO4 + Na]+ 300.12; found 332.25

[M+CH3OH+Na]+, calculated for [C15H19NO4 + CH3OH + Na]+ 332.16

Benzyl (4R,5S)-4-((R)-1-hydroxyallyl)-2,2,5-trimethyloxazolidine-3-carboxylate (115)

A round bottom flask was charged with benzyl (4S,5S)-4-formyl-2,2,5-

trimethyloxazolidine-3-carboxylate (114) (1.33 g, 4.80 mmol) in dry THF (10

mL) was cooled to -78 oC. After 15 minutes, to ensure sufficient cooling,

Vinylmagnesium bromide solution (1.0 M, 5.3 mL, 1.1 equiv.) was dropwise

added and left stirring overnight. After complete conversion sat. aq. NH4Cl

(10 mL) and EtOAc (20 mL) were added. The layers were separated; the

aqueous layer was extracted with EtOAc (3 x 20 ml) and the organic phases

were dried (MgSO4) and concentrated in vacuo. Flash column

chromatography (SiO2, 5:1 Hept.:EtOAc » 1:1 Hept.:EtOAc) followed by a

second purification by flash column chromatography (SiO2, 6:1 Hept.:EtOAc »

1:3 Hept.:EtOAc) afforded benzyl (4R,5S)-4-((R)-1-hydroxyallyl)-2,2,5-

trimethyloxazolidine-3-carboxylate (115) (890 mg, 2.91 mmol) as a yellow

oil in 61% yield.


(m, 5H, H-14,15,16), 5.98-5.80 (m, 1H, H-8), 5.41-5.26 (d, J= 17.1 Hz, 1H, H-9a), 5.17-4.97 (m, 3H,

H-9b,15), 4.33-3.85 (m, 3H, H-2,3,7), 1.65/1.44 (s, 6H, H-5,6), 1.37-1.31 m, 3H, H-1). 13C NMR (100

MHz, CDCl3) (mixture of rotamers, inseparable) δ 155.3 (C-11), 137,9 (C-8), 134.5 (C-16), 128.5-128

(C-14,15,16), 115.1 (C-9), 93.2 (C-4), 72.3 (C-7), 71.6 (C-2), 67.8 (C-12), 65.6 (C-3), 26.4, 24.4 (C-

5,6), 15.0 (C-1).


benzyl (4S,5S)-4-((R)-1-((hydroxycarbamoyl)oxy)allyl)-2,2,5-trimethyloxazolidine-3-carboxylate (119)

Benzyl (4R,5S)-4-((R)-1-hydroxyallyl)-2,2,5-trimethyloxazolidine - 3 -

carboxylate (115) (890 mg, 2.91 mmol), co-evaporated with dry toluene (3

x 5 mL), was dissolved in dry pyridine (20 ml). To this solution

carbonyldiimidazole (2.83 g, 2.83 mmol, 6.0 equiv.) was added and the

reaction mixture was heated to 40 oC. After 2.5 hours the reaction mixture

was cooled to 0 oC and NH2OH.HCl (2.05 g, 29.5 mmol, 10 equiv.) was

added. The reaction was stirred overnight at 0 oC. The reaction mixture was

allowed to warm up to rt. EtOAc (20 mL) and demi-water (10 mL) were

added. The layers were separated; the aqueous layer was extracted with

EtOAc (3 x 50 ml) and the organic phases were dried (MgSO4) and

concentrated in vacuo. Dry column chromatography (SiO2, Hept. » 1:1.5

Hept.:EtOAc) afforded benzyl (4S,5S)-4-((R)-1-((hydroxycarbamoyl)

oxy)allyl)-2,2,5-trimethyloxazolidine-3-carboxylate (119) (743 mg, 2.04

mmol) as a yellow oil in 70% yield. Benzyl (4R,5S)-4-((R)-1-hydroxyallyl)-

2,2,5-trimethyloxazolidine - 3 - carboxylate (115) (0.183 mg, 0.60 mmol) was recovered.


(m, 5H, H-17,18,19), 7.03 (s, 1H, H-12), 5.97-5.80/5.74-5.65 (m, 1H, H-8), 5.40 (m, 1H, H-7), 5.34-

5.02 (m, 4H, H-9,15), 4.25 (m, 1H, H-2), 4.17-4.01 (m, 1H, H-3), 1.58-1.47 (m, 6H, H-5,6), 1.31 (m,

3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor) δ 171.35 (C-11), 157.9 (C-14),

134.0/133.5 (C-8,16), 128.7-128.1 (C-17,18,19), 119.3 (C-9),93.9 (C-4), 75.5,73.7 (C-7), 67.4 (C-15),

61.1 (C-2), 60.7, 60.0 (C-3), 27.4, 27.1, 26.6, 26,1, 25.0, 23.5 (C-5,6), 14.7 (C-1). MS: found 387.21

[M+Na]+, calculated for [C18H24N2O6 + Na]+ 387.39.

benzyl (4S,5S)-2,2,5-trimethyl-4-((R)-1-((((2,4,6-trimethylbenzoyl)oxy)carbamoyl)oxy)allyl)oxazolidine-

3-carboxylate (120)

Benzyl (4S,5S)-4-((R)-1-( (hydroxycarbamoyl) oxy)allyl)-2,2,5-

trimethyloxazolidine-3-carboxylate (119) (743 mg, 2.04 mmol)

was dissolved in dry DCM (10 mL). To this solution Et3N (0.62 ml,

4.48 mmol, 2.2 equiv.) was added followed by dropwise addition of

2,4,6-Trimethylbenzoyl chloride (374 μl, 2.24 mmol, 1.1 equiv.).

Upon complete conversion, after 1 hour, sat. aq. NaHCO3 was

added. The layers were separated; the aqueous layer was extracted

with DCM (3 x 10 ml) and the organic phases were dried (MgSO4)

and concentrated in vacuo. Purified with automated flash column

chromatography (SiO2, 9:1 Hept.:EtOAc » 4:1 Hept.:EtOAc)

afforded benzyl (4S,5S)-2,2,5-trimethyl-4-((R)-1-((((2,4,6

trimethylbenzoyl) oxy) carbamoyl) oxy) allyl) oxazolidine – 3 -

carboxylate (120) (638 mg, 1.25 mmol) as an off white brittle

powder in 61% yield.


(m, 5H, H-23,24,25), 6.87 (s, 2H, H-17), 6.00-5.89/5.78-5.69 (m, 1H, H-8), 5.56-5.04 (m, 5H, H-

7,9,21), 4.28 (m, 1H, H-2), 4.25-4.07 (m, 1H, H-3), 2.35-2.29 (m, 9H, H-15,19), 1.61-1.49 (m, 6H, H-

5,6), 1.36 (m, 3H, H-1). 13C NMR (100 MHz, CDCl3) (mixture of rotamers, main / minor)* δ 133.5 (C-8),

128.8-128.3 (C-17,23,24,25), 120.2 (C-9), 74.8 (C-7), 72.4 (C-2), 67.1 (C-21), 61.0 (C-3), 26.2,23.5

(C-5,6), 21.4 (C-19), 20.1 (C-15), 14.3 (C-1). MS: found 533.25 [M+Na]+, calculated for [C28H34N2O7 +

Na]+ 533.33.

*Due to poor 13C spectra assignment of the quaternary carbon atoms was not possible.


benzyl (4S,4'S,5S,5'R)-4'-(hydroxymethyl)-2,2,5-trimethyl-2'-oxo-[4,5'-bioxazolidine]-3-carboxylate

(121)

Benzyl (4S,5S) - 2,2,5 –trimethyl -4 - ((R) -1- (((( 2 , 4, 6 –trimethyl

benzoyl)oxy)carbamoyl)oxy)allyl)oxazolidine-3-carboxylate (120) (52 mg,

0.10 mmol) was dissolved in t-BuOH:MeCN:H2O, (4:4:1, 2 mL), a catalytic

amount of potassium osmate (VI) dehydrate (pipet point) was added and the

reaction mixture was stirred overnight. The reaction was quenched with sat.

Aq. Na2SO3. The layers were separated; the aqueous layer was extracted

with EtOAc (3 x 10 ml) and the organic phases were dried (MgSO4) and

concentrated in vacuo.

Rf = 0.36 (5% MeOH in DCM). MS: found 387.15 [M+Na]+, calculated for

[C18H24N2O6 + Na]+ 387.15.


Acknowledgements First of all I would like to thank Tjerk Sminia for his daily supervision. Due to his enthusiasm and

guidance I have learned much from him. I would like to thank Tom Wennekes for the weekly meetings, I

have received some good advice in these meetings. I would like to thank Bauke Albada as the second

examinatior of this thesis. I would also like to thank the attendees of the synthesis group meeting for

their suggestions and remarks, it has been helpful. Further I would like to thank the people in the office,

Iris, Wouter, Marian, Ruijo and Pepijn for a pleasant time her in the office.


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