Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids

Article abstract of DOI:10.1039/c1ob06276j

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most p

Full text of DOI:10.1039/c1ob06276j

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Impact of sugar stereochemistry on natural killer T cell stimulation by
bacterial glycolipids†
Shenglou Deng,a Jochen Mattner,b,c Zhuo Zang,a Li Bai,d Luc Teyton,e Albert Bendelacd and Paul B. Savage*a
Received 28th July 2011, Accepted 30th August 2011
DOI: 10.1039/c1ob06276j
Natural killer T (NKT) cells recognize glycolipids produced
by Sphingomonas bacteria, and these glycolipids contain C6-
oxidized sugars, either glucuronic acid or galacturonic acid,
linked to ceramides. Glycolipids with gluco stereochemistry
are the most prevalent. Multiple studies have demonstrated
that galactosylceramides are more potent stimulators of
NKT cells than their glucose isomers. To determine if this
stereoselectivity is retained in the context of the C6-oxidized
sugars found in bacterial glycolipids, we prepared two sets of
gluco and galacto-glycolipids oxidized at their C6 positions
and compared their NKT stimulatory properties. In the
context of carboxylic acid groups at C6, gluco stereochemistry
gave the more potent responses. We also prepared bacterial
glycolipids containing more complex ceramide groups to
determine if these chains impact NKT cell responses.
The first identified NKT cell antigens were isolated from a ma-
rine sponge,12 and while potent stimulators of NKT cells, these gly-
colipid are not considered “natural” antigens. Structure–activity
studies of the sponge-derived glycolipids, in the context of anti-
tumor properties, led to the generation of a-galactosylceramide
KRN700013 (Fig. 1). This glycolipid has been the primary antigen
used in elucidating antigen presentation by CD1d and NKT cell
responses.
Natural killer T (NKT) cells are a subset of T cells and play a key
role in regulating immune responses.1–3 This regulation impacts
responses to infection, tumor growth, and autoimmune diseases.
In addition, NKT cell stimulation has been shown to improve the
efficacy of vaccines.4 Consequently, there has been considerable
interest in the means by which these cells can be stimulated to
produce the cytokines that in turn control responses of other
aspects of the immune systems in higher organisms. NKT cells are
stimulated via presentation of glycolipids by CD1d, and a limited
number of glycolipids and lipid polyols have been identified as
antigens for NKT cells.5 These include a-glycosylceramides,6 a-
glycuronosylceramides,7,8 a-galactosyldiacylglycerols,9 isoglobo-
side iGb3,10 and threitolceramides.11
Fig. 1 Structures of glycolipids from Sphingomonas (GSL-1A and
GSL-1A¢), NKT cell agonists a-glucosylceramide, KRN7000 and glycol-
ipids 1 and 2.
aDepartment of Chemistry and Biochemistry, Brigham Young University,
Provo, UT, 84602, USA
The first natural, exogenous antigens for NKT cells were
identified among the membrane components of the Sphingomon-
adaceae family of bacteria (including Sphingomonas spp.).7,8 The
bacteria in this family are Gram-negative (possess two lipid
bilayers); however, their outer membrane is not made up of the
lipopolysaccharide found in many Gram-negative bacteria (e.g.,
Escherichia coli). In its place, bacteria in this family produce a
group of glycuronosylceramides including GSL-1A and GSL-1A¢
(Fig. 1).14
bDepartment of Immunobiology, Cincinnati Children’s Hospital, Cincinnati,
OH, 45229, USA
cMikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hy-
giene, Universita¨tsklinikum Erlangen and Friedrich-Alexander Universita¨t
Erlangen-Nu¨rnberg, Wasserturmstr. 3/5, D-91054 Erlangen, Germany
dHoward Hughes Medical Institute, Committee on Immunology, Department
of Pathology, University of Chicago, Chicago, IL, 60637, USA
eDepartment of Immunology, Scripps Research Institute, La Jolla, CA,
92037, USA
† Electronic supplementary information (ESI) available: Experimental
procedures for preparation of GSL-1B, GSL-1C, 1 and 2. 1H and 13C NMR
spectra of 1, 2, GSL-1B and GSL-1C. Methods of measuring cytokine
production stimulated by CD1d presentation of glycolipids to NKT cells.
See DOI: 10.1039/c1ob06276j
Sphingomonas bacteria are ubiquitous. They are found in
water sources,15 on household items,16 and are one of the most
prevalent airborne forms of life.17 Consequently, humans are
routinely exposed to these bacteria. There is evidence that these
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bacteria modulate immune responses18 and that glycolipids from
Sphingomonas can trigger allergic asthma.19
1B and GSL-1C and NKT cell stimulatory activities of these two
compounds have not been reported.
To better understand the NKT cell stimulatory properties of
the GSLs produced by Sphingomonas, we20 and Kronenberg et
al.21 reported the synthesis and NKT cell stimulatory activity of
some of the glycolipids produced by this type of bacteria. Both
studies showed that sugars appended on GSL-1A decrease activity
significantly, and that GSL-1A and GSL-1A¢ were less stimulatory
than KRN7000. Kronenberg et al. also showed that GSL-1A is
somewhat less stimulatory than GSL-1A¢.
Multiple syntheses of GSL-1A and GSL-1A¢ have been reported
(for examples see ref. 19,20,22 and 23). a-Glycoside bond forma-
tion with glucuronic and galacturonic acid derivatives is typically
complicated by low anomeric selectivities. Pilgrim and Murphy23
reported an ingenious means of generating the a-anomer in high
yields in the synthesis of GSL-1A and GSL-1A¢. This group
formed the corresponding b anomer in good yield, then used
chelation-induced anomerization to give the a anomer in very
high yield. Alternatively, GSL-1A can be formed in reasonable
yield by oxidizing glucose to glucuronic acid after glycoside bond
formation.24 We used the latter approach for the glycolipids 1 and
2 (Scheme 1) and the former approach for synthesis of GSL-1B
and GSL-1C (Scheme 2).
a-Galactosylceramide is a much more potent stimulator of
NKT cells than a-glucosylceramide (Fig. 1).6 Among Sphin-
gomonas bacteria, GSL-1A, with gluco stereochemistry, is gen-
erally more predominant than GSL-1A¢, which has galacto
stereochemistry (Fig. 1).14,22 If one of the major roles of NKT cells
is surveillance for Sphingomonas and related bacteria, it would be
expected that the more predominant glycolipid (GSL-1A (gluco
stereochemistry)) would be a more potent stimulator than the less
common isomer (GSL-1A¢ (galacto stereochemistry)). Among the
Sphingomonas glycolipids, C6 in the sugars is oxidized, and we
questioned if oxidation at this position might alter recognition of
gluco vs. galacto selectivity in NKT cell stimulation. To answer this
question, we prepared GSL-1A, GSL-1A¢ and glycolipids 1 and 2
(Fig. 1). KRN7000 and the Sphingomonas glycolipids differ in the
ceramide portions of the molecules, and ceramide structure has
been shown to play a role in NKT cell responses to glycolipids.
Compounds 1 and 2 contain the glucoronic and galacturonic
acids found in GSL-1A and GSL-1A¢ and the ceramide found
in KRN7000. These compounds proved useful in dissecting the
impact of the acid groups in the sugars on NKT cell responses.
To determine the full impact of differences in the ceramide
portions of glycolipids from Sphingomonas on NKT cell stimu-
lation, we also prepared GSL-1B and GSL-1C (Fig. 2), in which
the sphinganine chain is elongated and modified as compared to
GSL-1A.14 These two glycolipids are produced by bacteria but in
lower amounts as compared to GSL-1A. The syntheses of GSL-
Scheme 1 Synthesis of glycolipids 1 and 2.
Oxidation at C6¢¢ after glycoside bond formation required
preparation of C6-orthogonally protected glycoside donor 4
(Scheme 1). To access this compound, we used thioglycoside 3
Fig. 2 Structures of GSL-1B and GSL-1C.
Scheme 2 Synthesis of GSL-1B and GSL-1C.
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Fig. 3 Cytokine production by splenocytes in response to the indicated glycolipids.
and treated it with TMS-triflate in acetic anhydride to give the
corresponding 1,6-diacetate. Though the yield of this reaction is
moderate, the starting material is simple to prepare in large scale,
and purification of the product is straightforward. Removal of the
acetates and selective protection at C6¢¢ gave 4 in good overall yield.
Surprisingly, coupling of 4 with 5 gave the corresponding glycoside
with little anomeric selectivity. The anomers were not separated
but were carried forward through deprotection at C6¢¢, oxidation
and ester formation giving 6. At this point, separation of the a and
b anomers was easily accomplished. Reductive deprotection was
followed by peracylation to provide a compound that was easily
chromatographable. The final deprotection gave 1. For the synthe-
sis of 2 (Scheme 1), compound 7 was prepared as described for 4.
The remaining steps paralleled those used in the preparation of 2.
Synthesis of the sphinganine for GSL-1B and GSL-1C began
with 1-octyne (Scheme 2). Deprotonation and reaction with 1,9-
dibromononane followed by reduction with Lindlar’s catalyst
gave 9. Grignard formation and reaction with Garner’s aldehyde
gave the corresponding alcohol as a mixture of diastereomers
that were difficult to separate. Consequently, the mixture was
carried on through benzylation (giving 10), liberation of the amine
and alcohol at C1 and azide formation yielding 11. Glycoside
formation with 12 gave 13 in reasonable yield with good anomeric
selectivity. Isomerization, giving the a anomer, azide reduction,
and amide formation gave 14, which was readily separated from
the minor diastereomer. Treatment with sodium methoxide in the
presence of a small amount of water gave GSL-1B. Preparation of
GSL-1C required protection of the alcohol at C3 in 14 followed
by cyclopropane formation using Simmons–Smith chemistry. In
the identification of GSL-1C, the relative stereochemistry of
the cyclopropyl group was not determined.14 The diastereomers
formed from cyclopropanation of 16 proved to be inseparable,
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and in the NMR spectra of 16, individual diastereomers were not
observed. Deprotection of 16 gave GSL-1C.
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Acknowledgements
Financial support from the National Institutes of Health (NIAID,
P01 AI053725) is gratefully acknowledged. We thank Michael E.
Fusakio for aiding in measuring cytokine release from splenocytes.
7662 | Org. Biomol. Chem., 2011, 9, 7659–7662
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