Synthesis of a 2 -Deoxy-β-GalCer

Article abstract of DOI:10.3390/molecules190710090

Structural studies of ternary complexes of CD1d/glycosyl ceramides/iNKT cells and CD1d/sulfatide/sulfatide reactive Type II NKT cells have shown how the polar moieties on the glycolipids interact with both the antigen presenting protein (CD1d) and the T cell receptors. However, these structures alone do not reveal the relative importance of these interactions. This study focuses on the synthesis of the previously unknown 2''-deoxy-β-galactosyl ceramide 2. This glycolipid is also evaluated for its ability to stimulate iNKT cells and sulfatide-reactive Type II NKT cells.

Full text of DOI:10.3390/molecules190710090

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Molecules 2014, 19, 10090-10102; doi:10.3390/molecules190710090
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of a 2ꞌꞌ-Deoxy-β-GalCer
Meena S. Thakur 1, Archana Khurana 2, Mitchell Kronenberg 2 and Amy R. Howell 1,*
1
Department of Chemistry, University of Connecticut, 55 N. Eagleville Rd., Storrs, CT 06269, USA
2
La Jolla Institute of Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA
* Author to whom correspondence should be addressed; E-Mail: amy.howell@uconn.edu;
Tel.: +1-860-486-3460; Fax: +1-860-486-2981.
Received: 29 May 2014; in revised form: 25 June 2014 / Accepted: 30 June 2014 /
Published: 10 July 2014
Abstract: Structural studies of ternary complexes of CD1d/glycosyl ceramides/iNKT cells
and CD1d/sulfatide/sulfatide reactive Type II NKT cells have shown how the polar
moieties on the glycolipids interact with both the antigen presenting protein (CD1d) and
the T cell receptors. However, these structures alone do not reveal the relative importance
of these interactions. This study focuses on the synthesis of the previously unknown
2ꞌꞌ-deoxy-β-galactosyl ceramide 2. This glycolipid is also evaluated for its ability to
stimulate iNKT cells and sulfatide-reactive Type II NKT cells.
Keywords: β-GalCer; antigens; NKT cells; CD1d
1. Introduction
α-Galactosyl ceramide (α-GalCer) and related compounds have garnered increasing attention in the
last decade and a half because of their ability to elicit immune responses via CD1d mediated activation
of natural killer T (NKT) cells [1–5]. NKT cells are a heterogeneous group of T lymphocytes that
express a T cell antigen receptor (TCR) and that perform functions related to both innate and adaptive
immunity, including defense against microbes, maintenance of immune tolerance and anti-tumor
responses. While most T lymphocytes respond to peptides bound to antigen presenting molecules
encoded in the Major Histocompatibility Complex (MHC), a defining characteristic of NKT cells is
their recognition of lipid/glycolipid antigens presented by CD1d, which is related to the MHC antigen
presenting proteins. CD1d-reactive NKT cells are split into two classes, Type I and Type II. Type I
NKT (or iNKT) cells are characterized by the expression of an invariant (i) TCRα chain paired with a

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limited number of TCRβ chains, and these cells have been extensively studied [6–11]. They recognize
various lipid containing antigens, particularly glycosphingolipids. On the other hand, the Type II NKT
cells have diverse TCRs and are not as well characterized, although recent attention has focused on the
role of sulfatide-reactive cells that belong to this group [12–17].
The understanding of the role of the sugar and the ceramide in influencing iNKT cell responses has
been greatly enhanced by both structure activity relationship (SAR) studies [7,9,10,18,19] and by the
availability of an ever increasing number of binary (glycolipid/CD1d) [20–22] and ternary (NKT cell
TCR/glycolipid/CD1d) crystal structures [23–28]. Again, SAR and structural data [29–31] for Type II
NKT cells lag those for Type I cells. Structural studies show that the lipid moiety of the glycolipids is
buried in two pockets of CD1d and that it is predominantly the sugar that interacts with the TCR,
which recognizes a composite of the sugar and the surface of the CD1d protein. Although the hydrogen
bonding interactions of both CD1d and the NKT cell TCR with the CD1d-exposed sugar moiety are
well-characterized, access to deoxysugar analogs has provided a nuanced understanding of the relative
influence of each carbohydrate OH and the possibility for fine specificity in interactions [26]. Ternary
Type I and Type II structures show that the OH on C2ꞌꞌ has hydrogen bonding interaction with both
CD1d and the NKT cell TCR for α-galactosyl ceramides (α-GalCers) and sulfatides (which have
β-linked sugars). Indeed, 2ꞌꞌ-deoxy-α-GalCer 1 (Figure 1) does not activate Type I NKT cells [32].
Interestingly, however, both β-glucosyl ceramide (β-GluCer) [33] and β-mannosyl ceramide
(β-ManCer) [34] have been recently reported to stimulate Type I NKT cells. To our knowledge, no
2ꞌꞌ-deoxy-β-glycosyl ceramide has been prepared or evaluated. Herein we report the preparation of
2ꞌꞌ-deoxy-β-GalCer 2 as a tool for further developing an understanding of NKT cell activation.
Figure 1. 2ꞌꞌ-Deoxy galactosyl ceramides 1 and 2 and β-ManCer.
The synthesis of 2ꞌꞌ-deoxy-β-glycosides is challenging. If glycosylations targeting such compounds
are conducted on already deoxygenated sugar donors, which lack the functionality needed to exploit
anchimeric assistance, the stereoselectivity can be low. Moreover, the glycosidic bond is more labile
due to the lack of an electron withdrawing C2ꞌꞌ substituent [35]. The most common approach involves
the use of a heteroatom substituent at C2, followed by its reductive removal subsequent to
glycosylation [36–45]. Other methods include the use of α-glycosyl phosphites [46,47], SN2 reactions
of α-glycosyl halides [48–50], Pd-catalyzed glycosylation reactions [51], the use of glycosyl imidates
under oxidative conditions [52], and anomeric O-alkylation/arylation [53]. Although many of these

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approaches provided good yields and stereoselectivities with some acceptors, our biggest concern
centered on the well-known decreased reactivity of either ceramide or sphingoid base acceptors in
comparison to coupling partners used in the majority of these studies. Danishefsky and coworkers had
used azidosphingosine 3 in a reaction with 1α,2α-oxirane 4 (Figure 2) derived from galactal, and the
glycosylation gave 60% isolated yield of the β-anomer [54]. Although there was not a subsequent
deoxygenation in this case, the group has used tin mediated deoxygenations in many other cases.
However, our previous experience with difficult purifications in tin-mediated deoxygenations of
glycolipids led us to consider an alternative approach.
Figure 2. β-Glycosylation partners.
We elected to employ Magnusson’s NIS promoted glycosylation [55] in combination with
Danishefsky’s observation that beta-selectivity in 1,2-anhydrosugars was sometimes enhanced with a
carbonate (as compared to benzyl groups or an isopropylidene) [56]. Most approaches to glycosyl
ceramides involve coupling of a sugar and ceramide or of a sugar and sphingoid base, followed by
acylation. The latter is the pathway we chose (Scheme 1).
Scheme 1. Strategy for the synthesis of 2ꞌꞌ-deoxy-β-GalCer 2.
Thus, the strategy was to glycosylate via iodoetherification of galactal 5 with phytosphingosine
alcohol 6. Our initial plan was to do a multi-stage deprotection/removal of the iodide of 7 to provide

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fully deprotected glycosylated phytosphingsine 8. Final acylation with activated cerotic acid ester 9
would achieve the synthesis of 2ꞌꞌ-deoxy-β-GalCer 2.
2. Results and Discussion
Glycosyl donor 5 was prepared in two steps from D-galactal as described by Danishefsky and
coworkers [54]. Glycosyl acceptor 6 was synthesized in three steps from phytosphingosine as
previously reported [57]. With donor 5 (1 equiv.) and acceptor 6 (2 equiv.) in hand, NIS-induced
glycosylation in CH2Cl2 at room temperature was attempted. The reaction was stirred overnight, and
TLC analysis showed consumption of 5. However, separation of the desired product 7 from the excess
of acceptor 6 using column chromatography was extremely difficult, resulting in a poor isolated yield of 7.
A reaction with acceptor 6 as the limiting reactant was sluggish, not going to completion even after 7 days.
In order to have an acceptor that was more easily separated from the product, NIS-induced
glycosylation reaction using acceptor 10 was tried. The reaction was complete in 48 h, and
β-glycosylated phytosphingosine 11 was isolated in 48% yield. Compound 11 was then treated with
catalytic NaOMe in MeOH, followed by deiodohydrogenation using palladium on carbon (10 wt %) in
one pot, to give compound 12 in 67% yield (Scheme 2). However due to the absence of a
functionalizable group at C2, cleavage of the anomeric bond was observed in the subsequent Boc
deprotection reaction using HCl (4 M in dioxane). Hence the strategy needed to be modified.
Scheme 2. Initial attempt to prepare 2.
Glycosylated product 11 was subjected to a one pot, two-stage deprotection (Scheme 3). First, the
cyclic carbonate group was removed using cat NaOMe in MeOH; removal of the TBPDS group using
TBAF (1.0 M solution in THF), then gave 13 in 56% yield over two steps. Boc cleavage, followed by
N-acylation using the para-nitrophenol ester 9 of cerotic acid in pyridine, afforded 14 in 42% yield.
Deiodohydrogenation using palladium on carbon (10 wt %) concluded the synthesis of 2. A substantial
quantity of ceramide 15 was recovered due to the cleavage of the anomeric bond. Nevertheless, there
was sufficient material to evaluate 2ꞌꞌ-deoxy-β-GalCer 2 for its ability to stimulate both Type I and
sulfatide-active Type II NKT cells.
2ꞌꞌ-Deoxy-β-GalCer 2 was assessed for its ability to stimulate iNKT cells using well-characterized
immortalized cells or hybridomas. Hybridoma 1.2 is a well characterized iNKT cell hybridoma [58]