synthesis and glycosylation of various ceramide ......borinic acid catalysis has been an important...

Synthesis and Glycosylation of Various Ceramide

Derivatives via Borinic Acid Catalysis

By

Robert Ward

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Chemistry

University of Toronto

© Copyright by Robert Ward 2017

ii

Synthesis and Glycosylation of Various Ceramide

Derivatives via Borinic Acid Catalysis

Robert Ward

Master of Science

Department of Chemistry

University of Toronto

2017

Abstract

Glycosphingolipids (GSLs) have become of increased interest due to recent realization that they

may place a role in neurological diseases and cancer. Chemical synthesis of GSLs from simple

building blocks requires efficient, yet selective methods to develop the major two components:

ceramide and the carbohydrate. Much effort has been done to develop synthesis of the individual

components; however the construction of the O-glycosidic bond that connects them has been

plagued with regio- and stereoselectivity issues. In the past, both the ceramide and carbohydrate

had to be chemically modified for the glycosylation step to occur with good selectivity and

yields, but recently unprotected ceramides have been used with protected donors. Borinic acid

catalysis has been an important advance in this field, allowing for higher yields and excellent β-

selectivity to develop GSLs. This thesis describes the synthesis of various ceramide derivatives

and their corresponding glycosylation by borinic acid catalysis.

iii

Acknowledgements

It has definitively been a learning curve throughout the course of my degree, and I may not have

got through it without the constant support of people in and out of the department. First, I would

like to thank my supervisor, Mark S. Taylor, whose consisting encouragement and reassurance,

allowed me to stay hopeful and positive throughout. As well as, giving me the opportunity to

work in his lab and explore new frontiers of organic chemistry. Second, I would like to thank the

Taylor group members for putting up with me for the past year. A special thanks to my

fumehood neighbors, Kashif and Ross, for tolerating my orderly work-style; long live the

stairwell of graduated cylinders. Third, I would also like to thank those you have got me to this

point. I would like to thank my undergraduate teaching assistants at the University of Alberta,

for inspiring me to go further into organic chemistry. Additionally, I would like to thank, my

undergraduate supervisor, Dennis G. Hall, for all his support throughout my young career.

Furthermore, I would like to thank my previous mentor Tristan Verdelet and the rest of the Hall

group for teaching me the tools of the trade, which has allowed me to perform high quality

research. Last, I would like to thank my family; David, Annette, Kelsey, and Jennifer Ward, for

there encouragement and support. Their love and assistance has allowed me to keep going,

especially when things got rough for me during my degree.

iv

Table of Contents

Acknowledgements………………………………………………………………………………ii

Table of Contents………………………………………………………………………………..iii

List of Tables…………………………………………………………………………………….vi

List of Figures……………………………………………………………………………………vii

List of Schemes…………………………………………………………………………………viii

Abbreviations……………………………………………………………………………………..ix

1 Introduction…………………………………………………………………………………...…1

1.1 Ceramide Structure and Function…………………………………………………...……1

1.2 Chemical Synthesis of Ceramides………………………………………………..………3

1.3 Chemical Synthesis of Glycosphingolipids………………………………………..……..6

1.3.1 General Strategies and Limitations……………………………………………..…...6

1.3.2 Gervay-Hague One Pot Synthesis……………………………………………….…..7

1.3.3 Boron Catalyzed α-Selective Glycosylation…………………………………….…..8

1.3.4 β-Selective Glycosylation of Ceramides……………………………………...……..9

1.3.4.1 Synthesis of β-Glycolipids via Helferich Glycosylation…………………….…..9

1.3.4.2 Synthesis of β-Glycosphingolipids via Organotin Mediated Approach………..11

1.3.4.3 Synthesis of β-Glycosphingolipids via Borinic Acid Activation of 1,3

Diols……………………………………………………………………………………11

2 Results and Discussion………………………………………………………………………...13

2.1 Research Goals………………………………………………………………………….13

2.2 Substrate Synthesis………………………………………………………………..…….13

2.2.1 Synthesis of Ceramide Derivatives…………………………………………..…….13

v

2.2.2 Synthesis of Glycosylated Donors…………………………………………………15

2.3 Glycosylation Optimization……………………………………………………………..17

2.4 Glycolipids made from Borinic Acid Catalyzed Glycosylation Reaction………………18

2.5 Kinetic Resolution Study………………………………………………………………..20

2.6 Deprotonation of PMB and Boc Groups………………………………………………21

2.7 Future Work……………………………………………………………………………..21

3 Conclusion……………………………………………………………………………………..22

4 Experimental Procedures and Characterization.…………………………………………....….23

vi

List of Tables

Table 2.1 Crude Ratios of Components of the Glycosylation Reaction at Different Catalyst

Loading…………………………………………………………………………….…………….18

Table 2.2 Results of the Kinetic Resolution Study………………………………………………20

vii

List of Figures

Figure 1.1 Naturally Occurring Precursors, Ceramides and Ceramide Variations …………….…1

Figure 1.2 Issues with Glycosylation of Ceramides………………………………………………7

Figure 2.1 Products made from the Borinic acid Catalyzed Glycosylation Reaction…………...19

viii

List of Schemes Scheme 1.1 Ceramide Synthesis via Garner’s Aldehyde………………………………………….3

Scheme 1.2 Ceramide Synthesis via Garner’s Methyl Ester……………….………………..……4

Scheme 1.3 Chelation Controlled Reduction of Secondary Alcohols…………………...………..4

Scheme 1.4 Ceramide Synthesis via Thioester Coupling……………………………………..…..5

Scheme 1.5 Ceramide Synthesis via Arabinose……………………………………………….......6

Scheme 1.6 Chemical Synthesis of α-Glycosphingolipids……………………………….……….7

Scheme 1.7 Synthesis of α-Cerebrosides through Borinic Acid Catalysis………………...……...8

Scheme 1.8 Synthesis of β-Mannosides by Toshima and Coworkers……………………....….…9

Scheme 1.9 Helferich Glycosylation of a Protected Ceramide …………………………….……10

Scheme 1.10 Glycosylation of a TCP Protected Ceramide…………………………………...…10

Scheme 1.11 Organostannyl Glycosylation of Ceramides………………………………………11

Scheme 1.12 Synthesis of β-Glycosphingolipids……………………………………………..…12

Scheme 1.13 Mechanism of Glycosylation using Borinic Acid 1,3 Diol Binding……….…...…12

Scheme 2.1 Synthesis of β-Ketophosphate from L-Serine ……………………………………...14

Scheme 2.2 Synthesis of N-Boc Ceramide Derivatives, of different Chain Lengths.…………...15

Scheme 2.3 Synthesis of PMB-Galactose……………………………………………………..…16

Scheme 2.4 Synthesis of PMB-Glucose ……………………………………………………...…16

Scheme 2.5 Synthesis of PMB-Lactose.…………………………………………………………17

Scheme 2.6 All-Around Deprotection of PMB and Boc Groups……………………………...…21

Scheme 2.7 Synthesis of 1-Tetracosanal………………………………………………………...21

ix

Abbreviations

AcOH

Boc

acetic acid

tert-butyloxycarbonyl

Cer ceramide

DBU

DCC

1,8-diazabicycloundec-7-ene

N,N'-dicyclohexylcarbodiimide

DCM dichloromethane

DIBAL

DMMP

2,2-DMP

Et2O

diisobutylaluminum hydride

dimethyl methylphosphonate

2,2-dimethoxypropane

ether

g

Gal

Glc

GSL(s)

gram

galactose

glucose

glycosphingolipid(s)

h hour(s)

Hex hexane

HRMS

iPrOH

high-resolution mass spectrometry

2-propanol

J

Lac

LiAl(tert-butoxy)3H

coupling constant

lactose

lithium tri-tert-butoxyaluminum hydride

M

Me

MeOH

molarity

methyl

methanol

NMR

o/n

nuclear magnetic resonance

overnight

OH

PCC

PMB

Ph

hydroxyl

pyridinium chlorochromate

4-methoxybenzyl chloride

phenyl

r.t.

TBS

TCP

TFA

room temperature

tert-butyldimethylsilyl ether

tetrachlorophthlimide

trifluoroacetic acid

THF tetrahydrofuran

TLC

TMS

thin layer chromotography

trimethylsilyl

1

1 Introduction

1.1 Ceramides: Structure and Function

Ceramides are found in all mammalian cells and make up the bulk of the cell membrane.

They can be modified by the cell to perform or assist in specific functions, such as in signal

transduction pathways, cell growth, differentiation, cell death, and cell proliferation.1 Ceramides

are synthesized in biological systems by three pathways; the de novo, salvage and

sphinogomyelin hydrolysis pathways.2 The de novo pathway uses L-serine as a starting substrate

and converts the amino acid via enzyme catalysis to the desired ceramide. The salvage and

sphingomyelin hydrolysis pathways serve as breakdown and recovery pathways. These

pathways are essential for the cell to maintain homeostasis, since diseases like Farber and

Gaucher can arise from an excess or depletion of ceramides, respectively.

Figure 1.1. Naturally Occurring Precursor, Ceramide and Ceramide Variations.

1. Morales-Serna, J. O.; Boutureira, O.; Diaz, Y.; Matheu I. M.; Castillon S. Carbohydrate

Research. 2007, 342, 1595–1612.

2. Mencarelli,j C.; Martinez–Martinez, P. Cell. Mol. Life Sci. 2013, 70, 181.

2

As previously mentioned, lipids can be modified via their primary hydroxyl group to

form more complex molecules (Figure 1.1). There are two typical classes that result from

modification: phosphosphingolipids and glycosphingolipids (GSLs).2 The latter can be further

broken down into four general classes: cerebrosides, neutral glycosphingolipids, sulfatides and

gangliosides. Cerebrosides are composed of one neutral sugar moiety bonded to the ceramide

and are found in mammalian nerve and muscle cells. They can also be precursors themselves to

more complex derivatives of the other three classes. Neutral glycosphingolipids, as the name

suggests, consist of two or more uncharged saccharide moieties bonded to a ceramide. In terms

of chemical synthesis, lactosylceramide, a neutral glycosphingolipid, is commonly further

modified by enzyme catalysis to add a sialic acid saccharide and form the ganglioside, GM3.

Sulfatides arise from the sulfation of cerebrosides, most commonly galactocerebrosides and are a

key component of myelin sheaths in the nervous system.2 Additionally, ceramides can be

modified through their tail length. In natural occurring derivatives, such as those found in nerve

cells, the non-acyl ceramide tail length ranges from 18—28 carbons.3

As previously mentioned, there are several diseases states that results due to errors in

ceramide biosynthesis, and accordingly there has been increased research in ceramides and their

derivatives. However, there are many limitations and hurdles, including poor solubility in

aqueous solution2 and lack of internal standards

4 available to perform and/or analyze

experiments. Despite their abundance in biological membranes, harvesting specific ceramides

from cells remains a challenge. Therefore chemical synthesis of ceramides is usually needed to

access ceramides and glycosphingolipids.5 Additionally, these synthesized lipids can be used as

standards, for example, for mass spectrometry analysis of biological samples. Chemical

synthesis can also be used to synthesize ceramide derivatives, such as shorter N-acyl chain

derivatives, which are water soluble and membrane permeable.

3. Lim, Y. X.; Pickford, R.; Don, A. S. Methods in Molecular Biology. 2016, 1376, 11–22.

4. Mirzaian, M.; Wisse P.; Ferraz, M. .J.; Marques, A. R. A.; Gaspar, P.; Oussoren, S.V.; Kytidou, K.;

Codée, .D.C.; van der Marel, G.; Overkleeft, H.S.; Aerts, J.M. Clinica Chimica Acta , 2017, 466, 178–184.

5. Kouzel, I. U.; Pirkl, A.; Pohlentz, G.; Soltwisch, J.; Dreisewerd, K.; Karch, H.; Muthing, J. Anal. Chem.

2014, 86, 1215−1222.

3

1.2 Chemical Synthesis of Ceramides

The chemical synthesis of ceramides has been accomplished using a variety of

approaches. The most common way follows the de novo pathway. Usually this involves several

intermediates, the most common being Garner’s aldehyde (Scheme 1.1). From Garner’s

aldehyde, many groups have used organometallics, such as, organolithium6, organosilanes

7,

organozirconium8 and Grignard

9 reagents to not only create a new carbon—carbon bond, but

also to achieve desired diastereoselectivity. However, there are limitations to these methods

including alkene geometry selectivity (E/Z) issues, moderate yields, protection/deprotection

steps, substrate generation and the use of toxic reagents.

Scheme 1.1 Ceramide Synthesis via Garner’s Aldehyde.

6. Durantie, E.; Bucher, C.; Gilmour, R. Chemistry - A European Journal, 2012, 18, 8208−8215.

7. Montogomery, J.; Kanicha, S. Tetrahedron, 2009, 65, 6707−6711.

8. Murakami, T.; Furusawa, K.; Tamai, T.; Yoshikai, K.; Nishikawa, M. Bioorganic & Medicinal Chemistry

Letters, 2005, 15, 1115−1119.

9. Sanllehi, P.; Abad, J. L.; Bujons, J.; Casas, J.; Delgado, A. European Journal of Medicinal Chemistry, 2016,

123, 905−915.

4

As an alternative to Garner’s aldehyde, Garner’s methyl ester has been used and provides

some unique advantages over the former. The methyl ester can be transformed into a β-

ketophosphate, from which a new carbon−carbon bond can easily be made via a mild,

epimerization free Horner−Wadsworth Emmons reaction. Simple long chain aldehydes, oxidized

from the corresponding alcohols, can be used to easily install a variety of hydrocarbon tails on

the ceramide backbone. However, this approach does not permit the installation of the long

chain tail and the reduced secondary alcohol in a single step, as in some cases with Garner’s

aldehyde. Various reducing agents have been investigated10

, and it has been found that either

Zn(BH4)211

in ether (Scheme 1.2), presumably through chelation control (Scheme 1.3)12

, or

LiAl(tert-butoxy)3H in ethanol13

, have provided the best diastereoselectivities and yields.

Scheme 1.2 Ceramide Synthesis via Garner’s Methyl Ester.

Scheme 1.3 Chelation Controlled Reduction of Secondary Alcohols.

Another approach, developed by Liebeskind and coworker, involves thioester coupling

with alkenyl borinic acids (Scheme 1.4).13

This method starts with L-serine, which undergoes

10. Koskinen, A. M. P.; Koskinen, P. M. Tetrahedron Letters, 1993, 34, 6765−6768.

11. Saito, S.; Murai, Y.; Usuki, S.; Yoshida, M.; Hammam, M. A. S.; Mitsutake, S.; Yuyama, K.; Igarashi, Y.;

Monde, K. Eur. J. Org. Chem. 2017, 1045.

12. Lee, J.; Lim, H.; Chung, S.. Tetrahedron: Asymmetry. 2002, 13, 343–347.

13. Yang, H.; Liebeskind, L. S. Organic Letters. 2007, 9, 2993.

5

DCC coupling with thiophenol and subsequently alcohol protection with TBS to arrive at the

thioester coupling substrate. The alkenylboronic acid coupling partner is a limitation of this

approach. Such reagents are synthesized from terminal alkynes, which are relatively expensive

and show little commercially availability, meaning that less derivatization can be done.

Additionally, the coupling reaction is dependent on the type of protecting groups used, the best

being a Boc group on the nitrogen and a TBS group on the primary hydroxyl. Despite these

limitations, this is a concise (6 steps from N-Boc-L-serine) and very efficient way (71 % from N-

Boc-L-serine) of constructing sphingosine, which with N-acylation would provide a ceramide.

Scheme 1.4 Ceramide Synthesis via Thioester Coupling.

Instead of using L-serine as a starting point, there are other alternatives that have been

used to synthesize ceramides. Carbohydrates, such as D-arabinose (Scheme 1.5)15

and 2-amino-

2-deoxy-D-glucose16

, have chosen for starting substrates since stereochemistry is already built in

and there are modifiable hydroxyl groups for functional group interconversions. Additionally,

another strategy involves forming an aziridine17, 18

or epoxide intermediate19

, and attacking with

hydroxide or an amine nucleophile.

14. Dangerfield E. M.; Cheng, J. M. H.; Knight, D. A.; Weinkove, R.; Dunbar, P. R.; Hermans, I. F.; Timmer; M.

S. M.; Stocker, B. L. ChemBioChem, 2012, 13, 1349−1356.

15. Tamio, S.; Masayuki, N. Carbohydrate Research, 1989, 194, 125.

16. Liew, S. K.; Kaldas, S. J.; Yudin, A. K.. Org. Lett., 2016, 18 , 6268.

17. Llaveria, J.; Beltran, A.; Sameera, W. M. C.; Locati, A.; Díaz-Requejo, M. M.; Matheu, M. I.; Castillon, S.;

Maseras, F.; Perez, P. J. J. Am. Chem. Soc. 2014, 136, 5342.

18. Morales-Serna, J. A.; Llaveria, J.; Dıaz, Y.; Matheu, M. I.; Castillon, S. Org. Biomol. Chem., 2008, 6, 4502.

6

Scheme 1.5 Ceramide Synthesis via D-arabinose.

1.3 Chemical Synthesis of Cerebrosides and Glycosphinogolipids

1.3.1 General strategies and limitations

Although there are several efficient methods for synthesizing ceramides, procedures to

glycosylate them are limited. The general reaction usually consists of a glycosyl donor with a

leaving group at the anomeric position and an acceptor (ceramide) as the nucleophile.

Limitations arise due to the complexity of the glycosylation, where many side products are

possible (Figure 1.2). Usually, extra steps are taken to adequately protect/deprotect the free

hydroxyl groups on the saccharide and the ceramide. The secondary amide group is usually

masked since ceramides are known to form an intramolecular hydrogen bond between NH and

primary hydroxyl free lone pair, rendering it less nucleophilic, thus decreasing the yield of the

reaction. The use of an azide in place of the amine, to form an azidosphingosine, is a frequently

employed approach, and has been shown to substantially increase the yield of the reaction.20

Additionally, reagents that interact with the 1,3-diol such as organotin and boron reagents have

been used to bind to the free hydroxyls and effective remove the hydrogen bonding interactions.

Furthermore, choice of protecting groups/deprotecting procedures must be considered, because

the molecule has several functional groups that could be affected, such as the C4/C5 double

bond. Lastly and most crucial is anomeric selectivity, which is dictated by a variety of factors

including leaving group, solvent, protecting groups and substrate.

19. Nicolaou, K. C.; Caulfield, T. J.; Katoaka, H.. Carbohydrate Research, 1990, 202, 177−91.

7

Figure 1.2. Issues with Glycosylation of Ceramides.

1.3.2 Gervay−Hague One Pot Synthesis.

A noteworthy synthesis that covers many issues with glycosylation is the Gervay−Hague

one pot synthesis (Scheme 1.6).21, 22

TMS-protected galactose is first converted to galactosyl

iodide and then mixed with an unprotected ceramide and tetrabutylammonium iodide as a

promoter. The promoter has two functions: to accelerate the reaction and to promote α-

stereoselectivity via in situ anomerization. During this process, the galactosyl iodide, initially in

the α-configuration, would be converted to the isomer and a SN2 reaction would take place to

give the desired stereoselectivity. Upon methanolysis in the presence of an acidic resin, the

desired cerebrosides were obtained in good to excellent yields. With the exception of the

requirement for a fully protected donor, which is difficult to avoid, this method allows a

straightforward and mild approach for construction of α-cerebrosides.

Scheme 1.6 Chemical Synthesis of α-Glycosphingolipids.

20. Du, W.; Kulkarni, S. S.; Gervay-Hague, J. Chem. Commun., 2007, 2336.

21. Schombs, M.; Park, F. E.; Du, W.; Kulkarni, S. S.; Gervay-Hague, J. J. Org. Chem. 2010, 75, 4891.

8

1.3.3 Boron-Catalyzed, α-Selective Glycosylation of Ceramides.

Another method that promotes α-selectivity involves a borinic acid catalyzed approach,

developed by Toshima and coworkers (Scheme 1.7). In this approach, the ceramide first binds to

the borinic acid via its C1 hydroxyl, then subsequent binding of the resulting species to a 1,2—

anhydroglucose donor results in epoxide ring opening to form the intermediate in Scheme 1.7.

Attack of the boron—bound ceramide (via C1 hydroxyl) to the oxonium cation results in the

corresponding α-selectivity observed. Despite the good yield (80 %) with no C2 nitrogen

alteration or protection, this method does still possess some limitations. Firstly, the method

requires two equivalents of the relatively costly donor, which must be synthesized from D-

glucal. Secondly, the C3 position is protected to prevent 1,3 diol binding, increasing the number

of steps and decreasing the reaction efficiency. An additional note about the reaction is that

Scheme 1.7 Synthesis of α-Cerebrosides through Borinic Acid Catalysis.

22. Tanaka, M.; Takahashi, D.; Toshima, D. Org. Lett., 2016, 18 , 5030

23. Tanaka, M.; Nashida, J.; Takahashi, D.; Toshima, K.. Org. Lett., 2016, 18, 2288.

9

when the upward facing epoxide is used, the coupled β-mannoside products are produced

(Scheme 1.8). Therefore, obtaining the most common types of natural β-glycolipids with this

method may not be feasible.

Scheme 1.8 Synthesis of β-mannosides by Toshima and Coworkers.

1.3.4 β-Selective Glycosylation of Ceramides.

1.3.4.1 Synthesizing β-Glycolipids via Helferich Glycosylation.

The Helferich glycosylation has allowed a route to obtaining β—glycolipids that is

tolerant of acid sensitive protecting groups (Scheme 1.9).25

Despite providing moderate to good

yields and high β-selectivity, the use of toxic mercury (II) cyanide is a drawback. Interestingly,

to increase nucleophilicity of the ceramide, Panza and coworkers chose to doubly protect the C2

nitrogen therefore creating a tertiary carbamate instead of forming an azidosphingosine

derivative. While a unique idea, the presence of the oxazolidine ring can be problematic, if harsh

glycosylation conditions are used, leading to undesired side products. Furthermore, two

different deprotection steps have to be undertaken to remove the benzoyl groups and the

oxazolidine ring with the Boc group.

24. Michielett, M.; Sillani, L.; Panza, L. Synlett, 2009, 16, 2609−2612.

10

Scheme 1.9 Helferich Glycosylation of a Protected Ceramide.

A few years after reporting the Helferich glycosylation of synthesizing ceramides, Panza

and coworkers developed a new approach (Scheme 1.10). This approach involves an

intramolecular H-bond, but between the C1 hydroxyl hydrogen and an oxygen lone pair of the

phthalimide moiety.26

The glycosylation reaction involves a tetrachlorophthalimide (TCP)

protected ceramide as the acceptor, a benzoyl protected galactose with a Sbox moiety at the

anomeric position and AgOTf as a promoter. When a slight excess of donor was used,

glycosylation occurred at the C1 position (62 %) with only minor bis-glycosylated product. With

two equivalents of acceptor, the reaction yield increased to 69 % with trace bis-glycosylation

product. Although this is an interesting approach, two separate deprotection steps are needed for

the TCP and benzoyl groups as well as the needed two equivalents of acceptor, which was made

over several steps.

Scheme 1.10 Glycosylation of a TCP protected Ceramide.

25. Di Benedetto, R.; Zanetti, L.; Varese, M.; Rajabi, M.; Di Brisco, R.; Panza, L. Org. Lett. 2014, 16, 952−955.

11

1.3.4.2 Synthesis of β-Glycosphingolipids via Organotin Mediated Approach.

Another way to form β-glycosphingolipids is to couple glycosyl halide donors with

stannylceramides (Scheme 1.11).27

The organotin serves to enhance the nucleophilicity of the

C1 oxygen, therefore rendering protection on the C3 hydroxyl on the ceramide needless. The

reaction proceeds through an attack of iodide anion on the β-configured galactosyl iodide to form

the α-configured galactosyl iodide. Then, the orthoester is formed by an intramolecular attack of

the C2-acetoxy group on the saccharide at the anomeric carbon, and subsequent attack by the

stannylether. The reaction proceeds with high regio- and stereoselectivity and excellent yields.

The major downfall of this approach is the use of harsh acidic conditions and the toxic organotin

reagent.

Scheme 1.11 Organostannyl Glycosylation of Ceramides.

1.3.4.3 Synthesis of β-Glycosphingolipids via Borinic Acid Activation of 1,3-diols

Recent work by Taylor and coworker provides an alternative to organotin for 1,3-diol

activation (Scheme 1.12). The borinic acid binds to the 1,3-diol rendering it more nucleophilic,

allowing for displacement of a methanesulfonate group from the donor molecule in a SN2 type

reaction. The reaction proceeds via high regio- and anomeric selectivity, but with moderate to

good yields. The selectivity of the reaction results from the attacked of a sterically hinder

nucleophile on the less crowded β face (Scheme 1.13). Additionally, the insolubility of the

ceramide acceptor could prevent the competing background reaction, since the ceramide would

need to be in solution for a reaction to occur and this would happen primarily through the borinic

acid assisted transfer.

26. J. A., Morales-Serna; O., Boutureira; Y., Diaz; M. I., Matheu; S., Castillon. Org. Biomol. Chem., 2008, 6, 443.

12

Scheme 1.12 Synthesis of β-glycosphingolipids.

Scheme 1.13 Mechanism of Glycosylation using Borinic Acid 1, 3-diol Binding.

27. D’Angelo, K.; Taylor M. S. Chem. Commun., 2017, 53, 5978.

13

2 Results and Discussion

2.1 Research Goals

Encouraged by previous results indicating that an unprotected ceramide could be

selectivity glycosylated via borinic acid catalysis, it was postulated that a more soluble ceramide

derivative could be a better substrate for borinic acid catalyzed glycosylation. By replacing the

N-acyl hydrocarbon chain of the ceramide with a common nitrogen protecting group, the

molecule would be less hydrophobic and possibly have increased solubility in dichloromethane.

Additionally, the nitrogen protecting group could be removed rendering a free primary amine,

which could be N-acylated to give the desired ceramide. Instead of N-acylating, it could also be

left as a free amine and then be further functionalized by enzymes under aqueous conditions. A

limitation to this approach could be that the increased solubility of the ceramide derivative could

decrease the anomeric selectivity, due to an increase in the rate of uncatalyzed background

reaction, which is known to be alpha-selective.27

2.2 Substrate synthesis

2.2.1 Synthesis of Ceramide Derivatives

For the synthesis of the desired N-protected ceramides, it was decided that the route from

L-serine through Garner’s methyl ester was most applicable for synthesis of many different

derivatives (Scheme 2.1). First, L-serine (1) was treacted with thionyl chloride and methanol to

provide the corresponding methyl ester salt (2), through an acyl chloride intermediate (not

shown). Next, the C2 nitrogen was protected with a Boc group, since Boc would be deprotected

with PMB groups on the glycosylated donor under acidic conditions and it would appear as a

singlet, which would assist in characterization. 2,2-Dimethoxypropane was used with a Lewis

acid to form an oxazolidine ring to protect both the C1 hydroxyl and the C2 nitrogen to arrive at

Garner’s methyl ester (4). Reaction of DMMP with 4, under basic conditions resulted in a β-

ketophosphate (5).

14

Scheme 2.1 Synthesis of β-ketophosphate from L-serine

Since cerebrosides, glycosphingolipids and gangliosides have received particular research

interest in the past decade, a variety of natural ceramide precursors were synthesized with the

goal of ultimately further functionalizing them to generate tools for glycobiology research

(Scheme 2.2). The β-ketophosphate was treated with a variety of long chain aldehydes (6—10),

which were synthesized by from corresponding alcohols, in a modified Horner—Wadsworth

Emmons reaction (11—15). Diastereoselective reduction using Zn(BH4)2 in ether, yielded the

corresponding allylic alcohols (16—20), and deprotection with aqueous acetic acid gave the

ceramide derivatives(21—25). Overall yields from L-serine ranged from 16-25 %, over 7 steps

with diastereoselectivities of >95:5 anti:syn as determined by 1H NMR spectroscopy.

15

Scheme 2.2 Synthesis of N-Boc Ceramide Derivatives, of Different by Chain Lengths.

2.2.2 Synthesis of Glycosylated Donors

Synthesis of three glycosylated donors, PMB-Gal (Scheme 2.3), -Glu (Scheme 2.4) and -

Lac (Scheme 2.5) was undertaken for the optimization. Synthesis of PMB-Gal, started from 26

and was protected using NaH and PMBCl. Bromination, followed by sequential hydrolysis,

resulted in PMB-Gal. Synthesis of PMB-Glu and -Lac started from their corresponding acetyl-

Glu and -Lac precursors. First, an S-Ar group was installed at the anomeric position and then

sodium methoxide deprotection resulted in 31 and 36. Following same protection and hydrolysis

steps as PMB-Gal, PMB-Glu and -Lac were obtained.

16

Scheme 2.3 Synthesis of PMB-Galactose.

Scheme 2.4 Synthesis of PMB-Glucose.

17

Scheme 2.5 Synthesis of PMB-Lactose.

2.3 Glycosylation Optimization

Conditions previously reported for the glycosylation of natural ceramides by Taylor and

coworker, were slightly reoptimized for the newly synthesized ceramides (Table 2.1).27

It was

decided that the catalyst loading would be investigated first. To begin it should be noted that

integration values in Table 2.1 may be slightly inaccurate due to overlap of signals. At 0 mol %

catalyst loading the reaction occurs with about 51% conversion. The α:β ratio for C1

glycosylation is about 2:1, which fits previously reported results.27

Increasing to 5 mol %

catalyst loading there was a substantial increase in conversion (84%), and C1 β:α ratio (3:1).

18

Increasing the catalyst loading to 10 mol % resulted in full conversion of starting material and an

improved β:α (6:1). Further increase in catalyst loading resulted in higher β:α ratio (11:1) with

no decrease in conversion. No further optimization was undertaken.

Table 2.1 Crude Ratios of Components of the Glycosylation Reaction at Different Catalyst

Loading.*

Entry Catalyst loading

(X mol %)

C1α

(1.44 ppm)

C1β

(1.42 ppm)

Starting material

(1.45 ppm)

1 0 1 0.45 1.92

2 5 1 2.8 0.77

3 10 1 6.0 trace

4 20 1 11.1 trace

* minor peaks in spectra, which could be C3 glycosylated products are not included in this table.

Ratios were determined by integrating the Boc peaks. All reagent amounts are based on acceptor

and not donor.

2.4 Glycolipids made from Borinic Acid Catalyzed Glycosylation

Reaction

To date, three glycolipids have been made using the reaction conditions outlined in Table

2.1 at 20 mol% loading of catalyst (Figure 2.1). PMB-Gal-anti-Cer18/Boc was synthesized with

a crude ratio of 11:1 (β:α), and purified by silica gel column chromatography to result in pure β,

in a 78% yield. PMB-Gal-syn-Cer18/Boc was synthesized with a crude ratio of 6:1 (β:α), and

19

purified by silica gel column chromatography to result in pure β, in a 66% yield. PMB-Lac-anti-

Cer18/Boc was synthesized with a crude conversion of 64% and an β:α of (5:1). Purification by

conventional silica gel column chromatography was difficult, but after two attempts the desired

product was isolated with a 56% yield. Further re-optimization on PMB-Lac-anti-Cer18/Boc

would be desirable since this is a key precursor to GM3 (Figure 1.1), and a higher yield with

better anomeric selectivity would make purification of the product simpler.

Figure 2.1 Products made from the Borinic Acid Catalyzed Glycosylation Reaction

20

2.5 Kinetic Resolution Study

Before it was found that Zn(BH4)2 in ether could provide the desired selectivity (>9:1.

anti:syn), Luche reduction conditions were used, which provided a lower selectivity of around

2.8:1 (anti:syn). The diastereomers, being having similar polarity and RF values, were difficult to

fully separate using conventional silica gel column chromatography. Therefore, a kinetic

resolution study was employed to investigate whether the catalyst could selectively activate one

diastereomer (Table 2.2). An anti:syn (50:50) mixture of ceramide was used (together one

equiv.) and a decreased amount of donor was employed. It was hoped that the anti diastereomer

would bind better than the syn diastereomer, which would be indicated by higher conversion of

anti—Cer and little to no conversion of the other diastereomer. Unfortunately, this did not occur

based on NMR analysis of the crude reaction mixture, which showed roughly equal conversions

of the two diastereomers, and similar amounts of the corresponding products.

Table 2.2 Results of the Kinetic Resolution Study.*

Anti: starting

Material

(1.454 ppm)

Syn: starting

material

(1.446 ppm)

Anti: β-GalCer

(1.424 ppm)

Syn: β-GalCer

(1.418 ppm)

Integration 1 1.04 0.39 0.32

* minor peaks in spectra, which could be C3 glycosylated products are not included in this table.

Ratios were determined by integrating the C(CH3)3 resonances from the Boc groups. Catalyst

loading and reagent amounts are based on acceptor

21

2.6 Deprotection of PMB and Boc groups

Scheme 2.6 Global Deprotection of PMB and Boc groups

Deprotection of the PMB groups and the Boc group were done according to the

conditions outlined by Taylor and coworker (Scheme 2.6).27

Purification with conventional

methods, proved difficult due to the high polarity of the TFA—ammonium salt. It was found

that addition of a non-polar aromatic solvent such as toluene could remove the aromatic and/or

non-polar impurities, by decanting, while leaving the product. This was confirmed by NMR

analysis of both the decanted toluene and the remaining residue (product).

2.7 Future work

Future work includes synthesis of tetracosanal, in order to access the last of the natural

occurring ceramide derivatives found in nerve cells. Bromination of 1-octadecanol has already

been done, and will be used to form a Gringard and then coupled to an alkyl bromide. PCC

oxidation, followed by procedures outline in Scheme 2.2 would provide the corresponding N-

Boc ceramide (Scheme 2.7). Other future work includes chemoenzymatic glycosylation of

derivatives, N-acylation, and applications in collaborations, in the field of glycobiology.

Scheme 2.7 Synthesis of 1-Tetracosanal

22

3 Conclusions

In conclusion, N-Boc ceramide derivatives varying by chain length have been

synthesized in respectable overall yields ranging from 16—25 % in 7 steps. The 1,3 diols of

some of these ceramide derivatives have been subjected to borinic acid binding and subsequently

glycosylation at a lower catalyst loading than described in previous work. It was also found that

simple addition and decanting of toluene could remove most impurities from the deprotection

step. This method of glycosylation allows for use of unprotected ceramides, decreasing the step

count and increasing the overall yield in the synthesis. As well it does not use toxic heavy

metals, which could problematic if biological testing is to be done. Further work includes further

optimization to increase yields and selectivity of the glycosylation reaction and elaboration to

more complex glycolipid derivatives.

23

4 Experimental Procedures and Characterization data

General

Unless stated otherwise, all reactions were stirred with Telfon coated stirred bars dried at 140 °C

prior to use. Moisture-sensitive liquids were transferred by stainless steel needles and gas tight

syringes. Flash silica gel chromatography was carried out using (60 Å, 230-400 mesh)

(Silicycle). Aluminium-backed silica gel 60 F254 plates (EMD Milipore) were used to perform

TLC analysis and visualized with a UV254 lamp and/or potassium permanganate, ninhydrin or I2

stains.

Materials and methods

Anhydrous solvents from solvent purification system were used where indicated. Distilled water

was used for stock solution and aqueous acid/base reaction mixtures and was obtained from an

in-house supply. Cambridge Isotope Laboratories supplied all NMR solvents. Methanesulfonic

anhydride was supplied from Alfa Aesar and store in a glove box freezer at −17 °C.

Carbohydrates were purchased from Carbosynth Ltd. and all other reagents/solvents were

purchased from Sigma Aldrich.

Instrumentation 1H,

13C, 2D NMR spectra were obtained using Varian Mercury 400 (400 MHz), Agilent DD2-

500 (13

C, 126 MHz) equipped with an XSens cryoprobe, and/or an Agilent DD2-600 (600 MHz)

instrument at the University of Toronto. 1H NMR data presented in the following order:

chemical shift in ppm (𝛿) downfield from tetramethylsilane (multiplicity, coupling constant,

integration). NMR data was report using the following abbreviations: s, singlet; d: doublet; m,

multiplet.

24

Methyl L-serinate hydrochloride salt (2)

Synthesized based on modified procedures.

28 To anhydrous MeOH (100 mL) in an oven dried

three neck flask was added thionyl chloride (3.5 mL, 48.0 mmol, 1 equiv.) dropwise via an

addition funnel at 0 °C under Ar(g). The resulting solution was stirred at 0 °C for 30 mins at

which time L-serine (1) (5.01 g, 47.6 mmmol, 1 equiv.) was added to the mixture. The resulting

yellow solution was heated to reflux under Ar(g) for 4 h, at which time TLC analysis showed

complete conversion to product. The flask was allowed to cool to room temperature and then the

solvent was removed via a rotary evaporator and co-evaporated using toluene. Product was

further dried under high vacuum to remove any traces of thionyl chloride, yielding in a white

solid weighing 7.36 g (99%).

Rf =0.53(MeOH :DCM, 8:2)

1H NMR (500 MHz, CD3OD): δ (ppm) = 4.18-4.10 (m, 1H), 4.04-3.89 (m, 2H), 3.85 (s, 3H).

13C NMR (500 MHz, CD3OD): δ (ppm) = 167.9, 59.2, 54.7, 52.3.

Methyl (tert-butoxycarbonyl)-L-serinate (3)


29 To an oven dried flask equipped with a stir bar,

containing anhydrous dichloromethane (60 mL) under Ar(g), was added methyl L-serinate

hydrochloride salt (3 g, 19.32 mmol, 1 equiv.). Anhydrous triethylamine (8.03 mL, 57.96 mmol,

3 equiv.) was then added dropwise at 0 °C to the resulting mixture of a white solid and clear

liquid. After ½ h of stirring at 0 °C, di-tert-butyl dicarbonate (5.16 g, 23.64 mmol, 1.2 equiv.)

was added as a solid and the reaction mixture was stirred for 15 h at room temperature. TLC

28. Koch. S.; Schollmeyer, D.; Löwe, H.; and Kunz, H. Chem. Eur. J. 2013, 19, 7020 –7041.

29. Brandstätter, M.; Roth, F.; and Luedtke, N. W. J. of Org. Chem., 2015, 80, 40–51.

25

analysis (Hex:EA, 2:3) of the resulting white cloudy reaction mixture showed complete

conversion of methyl L-serinate to product. The reaction was quenched using 1M HCl(aq) (37.5

mL) and then placed in a separatory funnel. The reaction mixture was further diluted using

dichloromethane (75 mL) and the layers were separated. The resulting organic layer was washed

with saturated NaHCO3(aq (25 mL)), washed with brine (40mL), dried over MgSO4, filtered, and

solvent was removed by a rotary evaporator to result in a yellow oil. Purification by silica gel

column chromatography, using an eluent system of Hex/EtOAc, 6:4 to 4:6) resulted in thick clear

colourless oil weighing 3.251 g (77 %).

Rf = 0.38 (Hex:EtOAc, 2:3)

1H NMR (500 MHz, CD3OD): δ (ppm) = 5.44 (s, 1H), 4.39 (s, 1H), 4.00-3.88 (m, 2H), 3.79 (s,

3H), 2.30 (s, 1H), 1.45 (s, 9H).

13C NMR (500 MHz, CD3OD): δ (ppm) = 171.2, 155.2, 80.3 63.6, 55.7, 52.6, 28.3.

3-(tert-butyl) 4-Methyl (S)-2,2-dimethyloxazolidine-3,4-dicarboxylate (4)


30 To an oven dried flask equipped with a stir bar,

and containing dichloromethane, was added a solution of methyl (tert-butoxycarbonyl)-L-

serinate (3 g, 13.7 mmol, 1 equiv.) in dichloromethane (20 mL total). The resulting mixture was

then stirred and 2,2-dimethoxypropane (12 mL) and BF3• Et2O (0.205 mL, 1.37 mmol, 0.1

equiv.) were then added. The addition of BF3•Et2O resulted in a colour change from clear

colourless to orange. After 18 h of stirring, the solution was black in colour and placed on a

rotovap to result in an red-brown oil. The crude was then diluted with dichloromethane (60 mL)

and quenched using a 1:1 mixture of distilled water and NaHCO3 (40 mL). The organic extract

was washed with brine (40 mL), dried over MgSO4, filtered through filter paper and solvent

removed on a rotovap evaporator to result in an orange oil. Purification by silica gel column

chromatography, using an eluent system of Hex/EtOAc (10:1), resulted in a clear yellow oil

weighing 3.111 g (83 %), as a mixture of rotamers.

30. Foss, F. W.; Snyder, A. H.; Davis, M. D.; Rouse, M.; Okusa, M. D.; Lynch, K. R.; and Macdonald, T. R.

Bioorganic & Medicinal Chemistry, 2007, 15, 663–677.

26

Rf = 0.68 (Hex:EtOAc, 1:1))

1H NMR (500 MHz, CDCl3): δ (ppm) 4.49-4.46 (m, 1H), 4.38-4.34 (m, 1H), 4.17-4.10 (m, 1H),

4.06-4.00 (m, 1H), 3.76 (s, 3H), 1.67 (s, 2H), 1.64 (s, 1H), 1.53 (s, 1H), 1.49 (s, 5H), 1.41 (s,

5H).

13C NMR (500 MHz, CDCl3): δ 171.1, 151.2, 95.0, 94.4, 80.9, 80.3, 66.2, 66.0, 59.2, 59.2, 52.4,

52.2, 28.3, 28.2, 26.0, 25.1, 24.9, 24.4.

tert-Butyl (S)-4-(2-(dimethoxyphosphoryl)acetyl)-2,2-dimethyloxazolidine-3-carboxylate (5)


31 To an oven dried round bottom flask equipped

with a stir bar under Ar(g) was added THF (30 mL) and dimethoxymethylphosphine oxide (0.87

mL, 8.104 mmol, 2.1 equiv.) at room temperature. The reaction flask was placed in a dry

ice/acetone bath and nBuLi (3.24 mL, 8.104 mmol, 2.1 equiv.) was added at −78 °C dropwise, to

result in a clear yellow solution. After 2 h of stirring at −78 °C, a solution of Garner’s methyl

ester (1.036 g, 3.859 mmol, 1 equiv.) in THF (5 mL) was slowly added dropwise via a Ar(g)

purged cannula at −78 °C. The reaction mixture was further stirred for 2 h, after which the

dry/ice acetone bath was removed and the reaction flask was allowed to warm in a water ice bath

and stirred for an additional 2 h. The yellow reaction solution was quenched using 20 mL of

aqueous 10% citric acid and transferred to a separatory funnel. Organic and aqueous layers were

separated and the aqueous phase was extracted using 3 x 20 mL of EtOAc. Organic layers were

collected and washed with brine (20 mL), dried over MgSO4, filtered through filter paper, and

solvent was removed on a rotovap to result in an clear yellow oil. Purification by silica gel

column chromotography, using a eluent gradient of Hex:EtOAc (80:20 to 40:60) resulted in a

clear colourless oil weighing 835 mg. (61 %).

31. Ordóñez, M.; Lagunas-Rivera, S.; Hernández-Núñez. E.; and Labastida-Galván, V. Molecules, 2010, 15,

1291–1301.

27

Rf = 0.24 (EtOAc)

1H NMR (500 MHz, CDCl3): δ (ppm) 4.64-4.57 (m, 1H), 4.52-4.45 (m, 1H), 4.20-4.12 (m, 2H),

4.08-4.01 (m, 2H), 3.84-3.75 (m, 12H), 3.46-3.12 (m, 4H), 1.68 (s, 3H), 1.62 (s, 3H), 1.52 (s,

3H), 1.50 (s, 3H), 1.47 (s, 9H), 1.41 (s, 9H).

General Procedure A - Oxidation of long chain alcohols


32 To an oven dried flask, equipped with a stir bar

and under Ar(g) was added PCC and long chain alcohol. Dichloromethane was then added and

the resulting orange solution was stirred for 5 h, at which point TLC analysis determined

complete conversion to product. The black mixture was then carefully filtered through a silica

plug using dichloromethane as eluent. Solvent was removed via a rotovap to provide the

corresponding aldehyde.

Tetradecanal (6)

Prepared from 1-tetradecanol (1 g, 4.664 mmol), according to General Procedure A (93 %)

Rf = 0.67 (Hex:EtOAc, 4:1)

1H NMR (400 MHz, CDCl3): δ (ppm) =9.76 (m, 1H), 2.44-2.39 (m, 2H), 1.67-1.58 (m, 2H),

1.35-1.23 (m, 20H), 0.88 (t, J = 6.8 Hz, 3H).

13C NMR (400 MHz, CDCl3): δ (ppm) = 202.9, 43.9, 31.9, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3,

29.3, 29.1, 22.7, 22.1, 14.1.

Palmitaldehyde (7)

Prepared from 1-hexadecanol (1 g, 4.128 mmol), according to General Procedure A (60%)

Rf = 0.70 (Hex:EtOAc, 4:1)

32. Chen, J.; Li, Y; Xiao-Ping, C. Tetrahedron: Asymmetry, 2006, 17, 933-941.

28

1H NMR (400 MHz, CDCl3): δ (ppm) = 9.76 (m, 1H), 2.44-2.37 (m, 2H), 1.67-1.58 (m, 2H),

1.35-1.23(m, 24H), 0.87 (t, J = 6.8 Hz, 3H).

13C NMR (400 MHz, CDCl3): δ (ppm) =202.9, 43.9, 31.9, 29.6, 29.6, 29.6, 29.6, 29.6, 29.6,

29.4, 29.3, 29.2, 29.2, 22.7, 22.1, 14.1.

Stearaldehyde (8)

Prepared from 1-octadecanol (1 g, 3.697 mmol), according to General Procedure A (97%)

Rf = 0.72 (Hex:EtOAc, 4:1)

1H NMR (400 MHz, CDCl3): δ (ppm) = = 9.76 (m, 1H), 2.44-2.37 (m, 2H), 1.67-1.58 (m, 2H),

1.35-1.23 (m, 28H), 0.87 (t, J = 6.8 Hz, 3H).


29.4, 29.4, 29.3, 29.1, 22.7, 22.1, 14.1.

Icosanal (9)

Prepared from 1-eicosonol (1 g, 3.349 mmol), according to General Procedure A (89%)

Rf = 0.74 (Hex:EtOAc, 4:1)

1H NMR (400 MHz, CDCl3): δ (ppm) = 9.76 (m, 1H), 2.44-2.37 (m, 2H), 1.67-1.58 (m, 2H),

1.35-1.23 (m, 32H), 0.88 (t, J = 6.8 Hz, 3H).


29.4, 29.3, 29.2, 22.7, 22.1, 14.1.

Docosanal (10)

Prepared from 1-docosanol (1 g, 3.062 mmol), according to General Procedure A (94%)

Rf = 0.77 (Hex:EtOAc, 4:1)

1H NMR (400 MHz, CDCl3): δ (ppm) =9.76 (m, 1H), 2.44-2.37 (m, 2H), 1.67-1.58 (m, 2H),

1.35-1.23 (m, 36H), 0.87 (t, J = 6.8 Hz, 3H).


29.3, 29.1, 22.7, 22.1, 14.1.

29

General procedure B - Horner Wadsworth Emmons reaction


11 To an oven dried vial equipped with a stir bar

under Ar(g) was added β-ketophosphate (1 equiv.) in THF and K2CO3 (3 equiv.). Aldehyde (1.2

equiv.) was then added and the resulting cloudy solution was stirred for 5 minutes at room

temperature, at which point water was added. The reaction was stirred for 72-96 h at room

temperature, then quenched with saturated NH4Cl(aq). The biphasic mixture was placed in a

separatory funnel and the layers were separated. The aqueous phase was extracted using ethyl

acetate and resulting organic layers were combined and washed with brine, dried over MgSO4,

gravity filtered and solvent removed on a rotovap to result in a yellow oil. Purification on silica

gel column chromatography using an eluent system of Hex/EtOAc (95:5 to 90:10) resulted in the

enone.

tert-Butyl (S,E)-4-(hexadec-2-enoyl)-2,2-dimethyloxazolidine-3-carboxylate (11)

Prepared from β-ketophosphate (350 mg, 0.997 mmol) and tetradecanal (233 mg, 1.096 mmol),

according to General Procedure B as a mixture of rotamers. Clear colourless oil (81 %).

Rf = 0.60 (Hex:EtOAc, 4:1)

1H NMR (500 MHz, CDCl3): δ (ppm) = 7.01-6.92 (m, 1H), 6.33-6.21 (m, 1H), 4.71-4.66 (m,

0.3H), 4.52-4.48 (m, 0.6H), 4.20-4.12 (m, 1H), 3.98-3.94 (m, 1H), 2.26-2.19 (m, 2H), 1.71 (s,

2H), 1.5 (s, 2.50H), 1.5 (s, 1.5H) , 1.49 (s, 3 H), 1.47-1.42 (m, 2H), 1.37 (s, 5.51H), 1.33-1.23

(m, 20H), 0.88 (t, J = 6.9 Hz, 3H).

13C NMR (500 MHz, CDCl3): δ (ppm) = 196.6, 195.7, 152.2, 151.4, 149.7, 125.8, 125.1, 95.1,

94.4, 80.7, 80.4, 65.9, 65.5, 64.1, 63.8, 32.7, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3,

29.2, 28.3, 28.2, 28.0, 27.9, 26.1, 25.2, 25.1, 24.1, 22.7, 14.1.

30

tert-Butyl (S,E)-2,2-dimethyl-4-(octadec-2-enoyl)oxazolidine-3-carboxylate (12)

Prepared from β-ketophosphate (150 mg, 0.4272 mmol) and hexadecanal (120 mg, 0.5126

mmol), according to General Procedure B as a mixture of rotamers. White solid (86 %)

Rf = 0.60 (Hex:EtOAc, 4:1)


0.3H), 4.52-4.48 (m, 0.6H), 4.20-4.12 (m, 1H), 3.98-3.94 (m, 1H), 2.26-2.19 (m, 2H), 1.70 (s,

2H), 1.65 (s, 1H), 1.57 (s, 1H), 1.51 (s, 1H), 1.49 (s, 3H) 1.47-1.42 (m, 2H), 1.33-1.23 (s, 6H),

1.33-1.23 (m, 26H), 0.87 (t, J = 6.9 Hz, 3H).


94.4, 80.7, 80.4, 65.9, 65.5, 64.1, 63.8, 32.7, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3,

29.2, 28.3, 28.2, 28.0, 27.9, 26.1, 25.2, 25.1, 24.1, 22.7, 14.1.

tert-Butyl (S,E)-4-(icos-2-enoyl)-2,2-dimethyloxazolidine-3-carboxylate (13)

Prepared from β-ketophosphate (137 mg, 0.390 mmol) and octadecanal (126 mg, 0.468 mmol),

according to General Procedure B as a mixture of rotamers. White solid (80 %)

Rf = 0.61 (Hex:EtOAc, 4:1)


0.3H), 4.52-4.48 (m, 0.6H), 4.20-4.12 (m, 1H), 3.98-3.94 (m, 1H), 2.26-2.19 (m, 2H), 1.79 (s,

2H), 1.55 (s, 2H), 1.52 (s, 1H), 1.49 (s, 3H), 1.47-1.42 (m, 2H), 1.37 (s, 6H), 1.33-1.23 (m, 30H),

0.88 (t, J = 6.9 Hz, 3H).


94.4, 80.7, 80.4, 65.9, 65.5, 64.1, 63.8, 32.7, 31.9,29.7, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2,

28.3, 28.2, 28.0, 27.9, 26.0, 25.2, 25.1, 24.1, 22.7, 14.1.

31

tert-Butyl (S,E)-4-(docos-2-enoyl)-2,2-dimethyloxazolidine-3-carboxylate (14)

Prepared from β-ketophosphate (149 mg, 0.427 mmol) and 1-eicosanal (151 mg, 0.512 mmol),


Rf = 0.62 (Hex:EtOAc, 4:1)


0.3H), 4.52-4.48 (m, 0.6H), 4.20-4.12 (m, 1H), 3.98-3.94 (m, 1H), 2.26-2.19 (m, 2H), 1.70 (s,

2H), 1.64 (s, 1H), 1.58 (s, 1H), 1.55 (s, 2H), 1.51 (s, 1H), 1.49 (s, 3H), 1.48-1.42 (m, 2H), 1.37

(s, 5H), 1.33-1.23 (m, 34H), 0.88 (t, J = 6.9 Hz, 3H).


94.4, 80.7, 80.4, 65.9, 65.5, 64.1, 63.8, 32.7, 31.9, 29.7, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2,

28.3, 28.2, 28.0, 27.9, 26.0, 25.2, 25.1, 24.1, 22.7, 14.1.

tert-Butyl (S,E)-2,2-dimethyl-4-(tetracos-2-enoyl)oxazolidine-3-carboxylate (15)

Prepared from β-ketophosphate (150 mg, 0.427 mmol) and 1-eicosanal (168 mg, 0.512 mmol),


Rf = 0.64 (Hex:EtOAc, 4:1)

1H NMR (500 MHz, CDCl3): δ (ppm) 7.01-6.92 (m, 1H), 6.33-6.21 (m, 1H), 4.71-4.66 (m,

0.3H), 4.52-4.48 (m, 0.6H), 4.20-4.12 (m, 1H), 3.98-3.94 (m, 1H), 2.26-2.19 (m, 2H), 1.70 (s,

1.85H), 1.65 (s, 1.15 H), 1.58 (s, 0.66), 1.55 (s, 1.62H), 1.51 (s. 1.25H), 1.49 (s, 3.49H) , 1.48-

1.42 (m, 2H), 1.37 (s, 5.84H), 1.33-1.23 (m, 38H), 0.88 (t, J = 6.9 Hz, 3H).


94.4, 80.7, 80.4, 65.9, 65.5, 64.1, 63.8, 32.7, 31.9, 29.7, 29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2,

28.3, 28.2, 28.0, 27.9, 26.0, 25.2, 25.1, 24.1, 22.7, 14.1.

32

Synthesis of Zn(BH4)2

Synthesized based on previous methods.33

To a 50 mL Schlenk tube in the glove box was added

ZnCl2 (500mg, 3.7 mmol). The tube was removed from the glove box and anhydrous ether

(18.75 mL) was then added. The resulting white solid-clear liquid mixture was then heated at 35

°C for 0.5 h and then allowed to cool to room temperature. The resulting clear solution was

decanted out of the flask and added to an 100 mL Schlenk tube containing NaBH4 (333 mg, 8.8

mmol) and ether (6.25 mL). The resulting mixture was stirred at room temperature for at least

12 h under Ar(g). The white cloudy solution was allowed to sit (unstirred) for at least 1 h, and

then the clear colourless solution was decanted (via needle and syringe) into a 100 mL Schlenk

tube under Ar(g), and then placed in a 4 °C fridge until use (approximately 0.15 M solution).

General procedure C – Selective reduction of enone using Zn(BH4)2


11 To an oven dried Schlenk tube equipped with a stir

bar was added enone, followed by anhydrous ether. The resulting solution was placed in a dry

ice acetone bath and cool to −78 °C. Zn(BH4)2 (2.5 equiv.) was added to the reaction, and the

reaction flask was moved to a EtOH:ethylene glycol (0.8/0.2) dry ice bath at −30 °C for 1 h.

The Schlenk tube was removed from the bath and placed in a water-ice bath for 1 h, after which

the clear solution as quenched using saturated NH4Cl(aq). The biphasic mixture was separated

and the aqueous phase was extracted using EtOAc. The organic layers were combined and

washed with brine, then the organic layer was collected and dried over MgSO4, filtered through

filter paper and solvent removed via a rotovap evaporator to result in a white solid (anti:syn

>90:10, determined by NMR analysis). Purification by silica gel column chromotography, using

an eluent system of Hex/EtOAc (4:1) resulted in a white solid.

33. W.J. Gensler, F.A. Johnson, and A.D.B. Sloan. J. Am. Chem.Soc. 82, 6074 (1960).

33

tert-Butyl (S)-4-((R,E)-1-hydroxyhexadec-2-en-1-yl)-2,2-dimethyloxazolidine-3-carboxylate

(16)

Prepared from 11 (156 mg, 0.356 mmol), according to General Procedure C as a mixture of

rotamers. Clear colourless oil (65 %).

Rf = 0.27 (Hex:EtOAc, 4:1)


1H), 4.16-4.08 (m, 1H), 4.07-3.97 (m, 1H), 3.86-3.79 (m, 1H), 2.06-2.00 (m, 2H), 1.53 (s, 2H)

1.48 (s, 9H), 1.40-1.32 (m, 2H), 1.32-1.21 (m, 22H), 0.87 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.3, 29.2, 29.1, 28.4, 26.2, 24.6, 22.7, 14.1.

tert-Butyl (S)-4-((R,E)-1-hydroxyoctadec-2-en-1-yl)-2,2-dimethyloxazolidine-3-carboxylate

(17)

Prepared from 12 (115.6 mg, 0.250 mmol), according to General Procedure C as a mixture of

rotamers. Clear colourless oil (89%).

Rf = 0.29 (Hex:EtOAc, 4:1)


1H), 4.16-4.08 (m, 1H), 4.07-3.97 (m, 1H), 3.86-3.79 (m, 1H), 2.06-2.00 (m, 2H), 1.53 (s, 2H)

1.48 (s, 12H), 1.40-1.32 (m, 2H), 1.32-1.21 (m, 26H), 0.87 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.3, 29.2, 29.1, 28.4, 26.2, 24.6, 22.7, 14.1.

tert-Butyl (S)-4-((R,E)-1-hydroxyicos-2-en-1-yl)-2,2-dimethyloxazolidine-3-carboxylate (18)

34


rotamers. White cloudy oil (75%).

Rf = 0.32 (Hex:EtOAc, 4:1)


1H), 4.16-4.08 (m, 1H), 4.07-3.97 (m, 1H), 3.86-3.79 (m, 1H), 2.06-2.00 (m, 2H), 1.53 (s, 2H)

1.48 (s, 12H), 1.40-1.32 (m, 2H), 1.32-1.21 (m, 26H), 0.87 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.3, 29.2, 29.1, 28.4, 26.2, 24.6, 22.7, 14.1.

tert-Butyl (S)-4-((R,E)-1-hydroxydocos-2-en-1-yl)-2,2-dimethyloxazolidine-3-carboxylate

(19)



Rf = 0.33 (Hex:EtOAc, 4:1)


1H), 4.16-4.08 (m, 1H), 4.07-3.97 (m, 1H), 3.86-3.79 (m, 1H), 2.06-2.00 (m, 2H), 1.48 (s, 14H),

1.40-1.32 (m, 2H), 1.32-1.21 (m, 34H), 0.88 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.3, 29.2, 29.1, 28.4, 26.2, 24.6, 22.7, 14.1.

tert-Butyl (S)-4-((R,E)-1-hydroxytetracos-2-en-1-yl)-2,2-dimethyloxazolidine-3-carboxylate

(20)



Rf = 0.34 (Hex:EtOAc, 4:1)

35


1H), 4.16-4.08 (m, 1H), 4.07-3.97 (m, 1H), 3.86-3.79 (m, 1H), 2.06-2.00 (m, 2H), 1.48 (s, 12H),

1.40-1.32 (m, 2H), 1.32-1.21 (m, 34H), 0.88 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.3, 29.2, 29.1, 28.4, 26.2, 24.6, 22.7, 14.1.

General Procedure D - Deprotection of oxazoline ring


34 To a 2 dram vial containing a stir bar and the

oxazolidine-protected ceramide was added distilled water (0.1 mL) and AcOH (0.9 mL). The

resulting clear solution was stirred at 50 °C for 5 h after which the resulting solution was

quenched using NaHCO3 and diluted with EtOAc. The biphasic mixture was placed in a

separatory funnel and organic layer was separated. The aqueous layer was extracted two more

times with EtOAc and the organic layers were collected and wash with brine. The organic layer

was then dried over MgSO4, gravity filtered through filter paper and solvent removed on a

rotovap evaporator to result in a white solid. Purification via silica gel column chromatography

resulted in the corresponding ceramide.

tert-Butyl ((2S,3R,E)-1,3-dihydroxyoctadec-4-en-2-yl)carbamate (21)

Prepared from 16 (97.2 mg, 0.243 mmol), according to General Procedure D. White solid (86

%).

Rf = 0.17 (Hex:EtOAc, 6:4)

34. Murakami, T.; and Furusawa, K. Tetrahedron, 2002, 58, 9257 – 9263.

36

Rf = 0.17 (Hex:EtOAc, 6:4)


1H), 4.34-4.29 (m, 1H,) 3.96-3.91 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.57 (m, 1H), 2.66-2.45 (m,

2H), 2.09-2.02 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 22H), 0.88 (t, J = 6.9 Hz, 3H) .


29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 22.7, 14.1.

tert-Butyl ((2S,3R,E)-1,3-dihydroxyicos-4-en-2-yl)carbamate (22)


%).

Rf = 0.19 (Hex:EtOAc, 6:4)


1H), 4.34-4.29 (m, 1H,) 3.96-3.91 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.57 (m, 1H), 2.66-2.45 (m,

2H), 2.09-2.02 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 26H), 0.88 (t, J = 6.9 Hz, 3H).


29.7, 29.7 29.6, 29.6, 29.5, 29.3, 29.2, 29.1, 28.4, 22.7, 14.1.

tert-Butyl ((2S,3R,E)-1,3-dihydroxydocos-4-en-2-yl)carbamate (23)


%).

Rf = 0.20 (Hex:EtOAc, 6:4)


1H), 4.34-4.29 (m, 1H,) 3.96-3.91 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.57 (m, 1H), 2.66-2.45 (m,

2H), 2.09-2.02 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 30H), 0.88 (t, J = 6.9 Hz, 3H).

37


29.7, 29.7 29.6, 29.6, 29.5, 29.3, 29.2, 29.1, 29.1, 28.4, 22.7, 14.1.

tert-Butyl ((2S,3R,E)-1,3-dihydroxytetracos-4-en-2-yl)carbamate (24)

Prepared from 19 (109.2 mg, 0.226 mmol), according to General Procedure D. White solid (81.5

mg, 81 %).

Rf = 0.21 (Hex:EtOAc, 6:4)


1H), 4.34-4.29 (m, 1H,) 3.96-3.91 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.57 (m, 1H), 2.66-2.45 (m,

2H), 2.09-2.02 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 34H), 0.88 (t, J = 6.9 Hz, 3H).


29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.3, 29.2, 29.1, 28.4, 22.7, 14.1.

tert-Butyl ((2S,3R,E)-1,3-dihydroxyhexacos-4-en-2-yl)carbamate (25)


%).

Rf = 0.22 (Hex:EtOAc, 6:4)


1H), 4.34-4.29 (m, 1H,) 3.96-3.91 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.57 (m, 1H), 2.66-2.45 (m,

2H), 2.09-2.02 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 38H), (t, J = 6.9 Hz, 3H).


29.7, 29.7 29.6, 29.6, 29.5, 29.3, 29.2, 29.1, 29.1, 28.4, 22.7, 14.1.

38

Reduction of enone using Luche’s conditions


35 To an oven dried Schlenk tube containing a stir bar

was added enone (272 mg, 0.6215 mmol, 1 equiv.) and CeCl3•7H2O (51.08 mg, 0.1371, 0.2

equiv.) under Ar(g). Anhydrous MeOH (8 mL) was added and the resulted white cloudy solution

was cooled in an dry ice/acetone bath. NaBH4 (37.9 mg, 0.9012 mmol, 1.3 equiv.) was added

at 78 °C and stirred for a further 6 h. The reaction mixture was quenched with 30 mL of

saturated NH4Cl(aq), and resulting bilayer solution was placed in a separatory funnel. The layers

were separated and aqueous layer was extracted using 3 x 30 mL of ethyl acetate. The organic

extracts were collected, combined and washed with brine (30 mL), dried over MgSO4, filtered,

and solvent removed on a rotovap to result in a clear colourless oil (anti:syn, 3:1 determined by

NMR analysis).

Syn-Cer18/BOC:

Rf = 0.26 (syn), 0.18 (anti); (Hex:i-PrOH:CH3COOH; 18.5:1.3:0.2)

1H NMR (400 MHz, CDCl3): δ 5.75 (m, 1H), 5.51(m, 1H), 5.15 (s, 1H), 4.34 (m, 1H), 3.79 (m,

2H), 3.61 (m, 1H), 2.58 (m, 2H), 1.45 (s, 9H), 1.40-1.23 (m, 24H), (t, J = 6.9 Hz, 3H).

Phenyl 1-thio-, 2,3,4,6-tetraacetate D-Glucopyranose.

Synthesized and characterized as previously reported.27

35. Haberkant, P.; Stein, F.; Hoglinger, D.; Gerl, M. J.; Brugger, B.; Van Veldhoven, P., P.; Krijgsveld, K.; Gavin,

A. G.; and Schultz, C. ACS Chem. Biol., 2016, 11 (1), pp 222–23.

39

Phenyl 1-thio-D-Glucopyranoside.

Synthesized and characterized as previously reported.

27

Phenyl 2,3,4,6-tetrakis-O-[(4-methoxyphenyl)methyl]-1-thio-β-D-Glucopyranoside.


27

4-methoxyphenyl 2,3,4,6-tetrakis-O-[(4-methoxyphenyl)methyl]- D-Glucopyranoside.

Synthesized and characterized as previously reported.27

2,3,4,6-Tetrakis-O-[(4-methoxyphenyl)methyl]-isopropylthio-D-Glucopyranoside.


27

2,3,4,6-Tetrakis-O-[(4-methoxyphenyl)methyl]-D-glucopyranoside.


27

40

4-O-(2,3,4,6-Tetraacetate-β-D-galactopyranosyl)-2,3,6-tetraacetate-4-methylphenyl 1-thio-

D-glucopyranose.

Synthesized as previously reported.

27

1H NMR (400 MHz, CDCl3): δ (ppm) = 7.36 (m, 2H), 7.11 (m, 2H), 5.33 (m, 1H), 5.20 (m, 1H),

5.12-5.06 (m, 1H), 4.94 (m, 1H), 4.89-4.82 (m, 1H), 4.59 (d, 1H), 4.55-4.50 (m, 1H), 4.46 (m,

1H), 4.15-4.02 (m, 4H), 3.89-3.81 (m, 1H,) 3.77-3.69 (m, 1H), 3.64-3.57 (m, 1H), 2.34 (s, 3H),

2.16-1.94 (m, 21H).

4-O-β-D-galactopyranosyl)-4-methylphenyl 1-thio-D-glucopyranose.


27

1H NMR (500 MHz, CH3OD): δ (ppm) = 7.46 (m, 2H), 7.13 (m 2H), 4.51 (m, 1H), 4.34 (m,

1H), 3.93-3.65 (m, 5H), 3.60-3.36 (m, 6H), 3.28-3.19 (m, 1H), 2.31 (s, 3H).

13C NMR (500 MHz, CH3OD): δ (ppm) = 137.0, 132.4, 129.4, 129.1, 103.5, 88.0, 79.1, 78.7,

76.5, 75.7, 73.4, 71.9, 71.1, 68.9, 61.1, 60.6, 19.7.

4-O-(2ʹ,3ʹ,4ʹ,6ʹ-Tetra-O-4-methoxybenzyl-β-D-galactopyranosyl)-2,3,6-tri-O-4-

methoxybenzyl- 4-methylphenyl 1-thio-D-glucopyranose.


27

41

1H NMR (400 MHz, CDCl3): δ (ppm) = 7.45 (m, 2H) 7.37-7.27 (m, 4H), 7.23-7.14 (m, 10H),

7.00 (m, 2H), 6.92-6.75 (m, 16H), 6.68-6.64 (m, 2H), 4.98 (m, 1H), 4.88 (m, 1H), 4.74-4.53 (m,

10H), 4.49-4.29 (m, 5H), 4.23-4.17 (m, 1H), 3.93-3.85 (m, 2H), 3.83-3.65 (m, 32H), 3.60-3.50

(m, 2H), 3.42-3.32 (m, 6H), 2.29 (s, 3H).


132.7, 131.3, 131.2, 131.0, 130.8, 130.7, 130.2, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3, 129.1,

129.0, 113.8, 113.7, 113.7, 113.6, 113.6, 113.5, 113.4, 102.8, 87.6, 84.8, 82.4, 79.9, 79.8, 79.5,

76.3, 75.2, 74.9, 74.6, 74.3, 73.4, 73.1, 73.0, 72.7, 72.4, 68.2, 68.0, 55.3, 55.2, 55.2, 55.2, 55.2,

55.1, 21.1.

4-O-(2ʹ,3ʹ,4ʹ,6ʹ-Tetra-O-4-methoxybenzyl-β-D-galactopyranosyl)-2,3,6-tri-O-4-

methoxybenzyl-D-glucopyranose.


27

1H NMR (400 MHz, CDCl3): δ (ppm) = 7.31-7.27 (m, 2H), 7.25-7.11 (m, 11H), 6.88-6.74 (m

14H), 6.71-6.61 (m, 2H), 5.12-5.08 (m, 0.44H), 5.00-4.85 (m, 2H), 4.80-4.54 (m, 7H), 4.49-4.41

(m, 2H), 4.37-4.25 (m, 3H), 4.24-4.18(m, 1H), 3.95-3.63 (m, 32H), 3.58-3.24 (m, 7H).

1-(chloromethyl)-4-methoxybenzene


36 To an oven dried flask, equipped with a stir bar,

was added 4-methoxybenzyl alcohol (12 mmol, 1.5 mL). The flask was evacuated and purged

three times, after which dry ether (24 mL) was added. To the resulting clear solution was added

thionyl chloride (24 mmol, 1.8 mL) dropwise at room temperature. The resulting yellow

36. Jaschinski, T.; and Hiersemann, T. Org. Lett., 2012, 14 (16), pp 4114–4117.

42

solution was stirred for 5 h at room temperature. Water (24 mL) was added and the biphasic

mixture was transferred to a separatory funnel. The layers were separated, and the aqueous layer

was extracted with ether (3 x 25 mL). The organic layers were combined, washed with brine,

dried over MgSO4, filtered, and solvent was removed via a rotator evaporator. The product was

furthered dried under high vacuum to remove any traces of thionyl chloride, yielding a yellow oil

weighing 1.88 g (99%).

1H NMR (500 MHz, CDCl3): δ (ppm) = 7.31 (m, 2H), 6.88 (m, 2H), 4.57 (s, 2H), 3.81 (s, 3H).

13C NMR (500 MHz, CDCl3): δ (ppm) = 159.7, 130.0, 129.7, 114.1, 55.3, 46.3.

1,1,3,3-tetraphenyldiboroxane

Synthesis as previously described.

37

1H NMR (500 MHz, CDCl3): δ (ppm) = 7.91-7.79 (m, 4H), 7.55-7.39 (m, 6H).

General Procedure E – Synthesis of PMB protected N-Boc Glycosylceramides.27

Stock solution of Ms2O donor (per 1 glycosylation): An oven dried 2 dram vial containing

glycosyl hemiacetal (0.10 mmol) was fitted with a septum and evacuated and purged 3 times

with Ar(g). To a 25 mL Schlenk tube, in a glove box was added methanesulfonic anhydride

Ms2O (41.8 mg). The tube was taken out of the glove box and anhydrous dichloromethane (2.6

mL) was added to the Schlenk tube (1.6 mL) and 1 mL to the 1 dram vial containing glycosyl

hemiacetal. PMP (58 μL) was then added to the glycosyl hemiacetal followed by 1 mL of the

0.15 M Ms2O solution. Solution was stirred for 15-20 minutes, which resulted in a colour

change from colourless to yellow.

37. D. Lee, M. S. Taylor, J. Am. Chem. Soc., 2011, 133, 3724-3727.

43

To a 1 dram was added N-Boc-D-sphingosine (0.04 mmol; >95:5, anti:syn) and diphenylborinic

anhydride (0.008 mmol). The vial was then fitted with a septum and evacuated/purged 3 times

with Ar(g). 0.6 mL of freshly prepared Ms2O stock solution (0.30 M) was then added, and the

septum was quickly removed and the vial was fitted with a plastic cap sealed with Telfon tape.

The reaction media was stirred for 24 h, after which the reaction media was placed under a gentle

stream of air to remove the solvent. Resulting residue was purified by flash column

chromatography (Hex:ether, 30:70), to result in the desire glycosylated sphingosine.27

tert-Butyl((2R,3S,E)-1-(((2R,3R,4S,5R,6R)-3,5-bis((4-methoxybenzyl)oxy)-6-(((4-

methoxybenzyl)oxy)methyl)-4-(((2S,3R,4S,5S,6R)-3,4,5-tris((4-methoxybenzyl)oxy)-6-(((4-

methoxybenzyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-

3-hydroxyoctadec-4-en-2-yl)carbamate

Prepared from Cer18/Boc and PMB-Lac according to General Procedure E. White cloudy oil

(29.1 mg, 56 %).

Rf = 0.62 (Ether)

1H NMR (500 MHz, CDCl3): δ (ppm) = 7.27-7.13(m, 14H), 6.87-6.78 (m, 14H), 6.70-6.66 (m,

2H), 5.68 (m, 1H), 5.51 (dd, 1H), 5.11-5.06 (m, 1H), 4.96-4.85 (m, 2H),4.75-4.58 (m, 8H), 4.49-

4.40 (m, 2H), 4.36-4.27 (m, 4H), 4.23-4.18 (m, 1H), 4.14-4.08 (m, 2H), 3.87-3.84 (m, 2H), 3.82-

3.72 (m, 21H), 3.57-3.47 (m, 2H), 3.40-3.28 (m, 6H), 3.20 (s, 1H), 2.00 (m, 2H), 1.43 (s, 9H),

1.37-1.20 (m, 22H), 0.88 (m, 3H).


133.7, 132.9, 132.8, 131.3, 131.0, 130.7, 130.5, 130.2, 129.7, 129.7, 129.4, 129.4, 129.3, 129.1,

129.0, 128.8, 113.8, 113.7,113.7, 113.6, 113.5, 113.5, 113.4, 103.7, 102.8, 82.6, 82.4, 81.0, 80.8,

79.7, 79.4, 75.0, 74.9, 74.9, 74.3, 73.3, 73.2, 73.0, 72.9, 72.3, 69.3, 68.0, 56.2, 56.2, 55.2, 55.2,

55.2, 54.8, 32.4, 31.9, 30.3, 29.8, 29.7, 29.6, 29.5, 29.3, 29.3, 29.2, 28.4, 28.1, 22.7, 14.1

44

tert-Butyl ((2R,3S,E)-3-hydroxy-1-(((2R,3R,4S,5S,6R)-3,4,5-tris((4-methoxybenzyl)oxy)-6-

(((4-methoxybenzyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)octadec-4-en-2-

yl)carbamate


8H), 5.69 (m, 1H), 5.49 (m, 1H), 5.16-5.10 (m, 1H), 4.82 (d, 1H), 4.78-4.68 (m, 2H), 4.67-4.58

(m, 2H), 4.52 (m, 1H), 4.41-4.28 (m, 2H), 4.27 (m, 1H), 4.13 (m, 1H), 4.06 (m, 1H), 3.80-3.70

(m, 16H), 3.53-3.40 (m, 4H), 3.24-3.21 (m, 1H), 2.00 (q, 2H), 1.42 (s, 9H), 1.37-1.21 (m, 22H),

0.88 (m, 3H).

13C NMR (500 MHz, CDCl3): δ (ppm) = 159.3, 159.2, 159.2, 159.2, 133.9, 130.6, 130.5,

129.9,129.8,129.7,129.6,129.1,129.1, 113.8, 113.8,113.8, 113.4, 104.4, 81.9, 79.4, 78.8, 74.9,

74.0, 73.6, 73.2, 72.9, 72.6, 69.6, 68.5, 55.2, 55.2, 54.8, 32.4, 31.9, 29.7, 29.7, 29.6, 29.6, 29.5,

29.3, 29.2, 28.4, 22.7, 14.1.

General Procedures F – Deprotection of PMB and Boc groups on protected

glycosphingolipids

The protected glycosphingolipid was dissolved in HPLC-grade dichloromethane, in a two dram

vial equipped with a stir bar. Trifluoroacetic acid (10 % v/v) and anisole (5 % v/v) were then

added and the resulting clear colourless solution was stirred under ambient conditions for 2 h.

After 2 h, the reaction mixture was diluted with toluene, and solvent evaporated on a rotovap

evaporator. The crude was then co-evacuated with toluene 3 times to produce a white oily solid.

Toluene was added and decanted from the vial, 3 times, to remove aromatic non-polar

impurities, resulting in the corresponding TFA salt glycosphingolipid.27

45

(2R,3S,E)-3-hydroxy-1-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-

2H-pyran-2-yl)oxy)octadec-4-en-2-aminium 2,2,2-trifluoroacetate salt

Synthesized based on modified procedures. Characterization is still in progress.

1H NMR (500 MHz, CH3OD): δ (ppm) = 5.87 (m, 1H), 5.48 (m, 1H), 4.58 (s, 3H), 4.32-4.28 (m,

1H), 4.27 (m, 1H), 3.96-3.91 (m, 1H), 383-3.70 (m, 3H), 3.58-3.46 (m, 3H), 3.40- 3.35 (m, 1H),

2.10 (m, 2H), 1.41 (m, 2H), 1.29 (m, 22H), 0.89 (m, 3H).

46

1H NMR, 500 MHz (CDCl3)

47

13C NMR, 500 MHz (CDCl3)

48


49


50


51


52


53


54


55


56


57


58


59

gHSQC, 600 MHz (CDCl3)

60

gCOSY, 600 MHz (CDCl3)

61


62


63


64


65

gHBMC, 600 MHz (CDCl3)

66


67


68


69


70

gHMBC, 600 MHz (CDCl3)

71

1H NMR, 500 MHz (CD3OD)

synthesis and glycosylation of various ceramide ......borinic acid catalysis has been an important...

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