A divergent approach to the synthesis of iGb3 sugar and lipid analogues via a lactosyl 2-azido-sphingosine intermediate

Article abstract of DOI:10.1039/c4ob00241e

Isoglobotrihexosylceramide (iGb3, 1) is an immunomodulatory glycolipid that binds to CD1d and is presented to the T-cell receptor (TCR) of invariant natural killer T (iNKT) cells. To investigate how modifications to the lipid tail or terminal sugar residu

Full text of DOI:10.1039/c4ob00241e

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Biomolecular Chemistry
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A divergent approach to the synthesis of iGb3
sugar and lipid analogues via a lactosyl
2-azido-sphingosine intermediate†
Cite this: Org. Biomol. Chem., 2014,
12, 2729
Janice M. H. Cheng,a,b Emma M. Dangerfield,a,b Mattie S. M. Timmer*a and
Bridget L. Stocker*a,b
Isoglobotrihexosylceramide (iGb3, 1) is an immunomodulatory glycolipid that binds to CD1d and is pre-
sented to the T-cell receptor (TCR) of invariant natural killer T (iNKT) cells. To investigate how modifi-
cations to the lipid tail or terminal sugar residue of iGb3 influence iNKT cell activity, we developed an
efficient and divergent synthetic route that provided access to both sugar and lipid iGb3 analogues which
utilised a lactosyl 2-azido-sphingosine derivative as a common intermediate. In this way, iGb3 (1) and the
unprecedented analogues 6’’’-deoxy-iGb3-sphingosine 2, 6’’’-deoxy-iGb3-sphinganine 3, C12 N-acyl
iGb3 4 and C20:2 N-acyl iGb3 5 were prepared so that key structure–activity relationships can be
explored.
Received 31st January 2014,
Accepted 8th March 2014
DOI: 10.1039/c4ob00241e
www.rsc.org/obc
1. Introduction
The glycolipid isoglobotrihexosylceramide (iGb3, 1, Fig. 1) is a
β-linked triglycosyl ceramide that activates a distinct popu-
lation of T lymphocytes called invariant natural killer T (iNKT)
cells when presented by CD1d on dendritic cells (DCs).1–5
Here, the lipid tails of the glycolipid lodge themselves into the
deep hydrophobic pockets of the CD1d protein6,7 which leads
to the presentation of the sugar headgroup for recognition by
the T cell receptor (TCR) of the iNKT cell.8 β-Linked sphingo-
lipids were initially studied for their potential to be the
endogenous ligands responsible for the positive selection of
iNKT cells,9 however, more recently, such glycolipids have been
found to play a role in infection,10 as illustrated by β-glucosyl-
ceramide which accumulated in the lymphoid tissue of mice
and humans following the stimulation of DCs with Toll-like
receptor (TLR) agonists or live bacteria.11 Moreover, the
manipulation of the immune response, by altering the struc-
ture of iGb3 and hence the activity of iNKT cells, has potential
application in the treatment of a variety of diseases, such as
cancer and autoimmune disorders.12,13 Historically, studies of
iNKT cells focussed on the potent CD1d ligand α-galactosylcer-
amide (α-GalCer), however, iNKT cell activation using α-GalCer
aSchool of Chemical and Physical Sciences, Victoria University of Wellington,
PO Box 600, 6140 Wellington, New Zealand. E-mail: mattie.timmer@vuw.ac.nz,
bridget.stocker@vuw.ac.nz
bMalaghan Institute of Medical Research, PO Box 7060, 6242 Wellington,
New Zealand
†Electronic supplementary information (ESI) available: Experimental procedures
and 1H- and 13C-NMR spectra for all compounds. See DOI: 10.1039/c4ob00241e
Fig. 1 iGb3 and analogues.
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can lead to anergy and accordingly, there has been growing the amine, followed by selective orthoester opening to give the
interest in alternative CD1d-binding glycolipids.14–17 4″-O-acetyl derivative34 and glycosylation with galactosyl donor
Given their significant structural differences, it is remark- 722 or D-fucosyl donor 8. The distinction between the two
able that both α-GalCer and iGb3 can activate iNKT cells. To 6′′′-deoxy-iGb3 derivatives would be achieved at the final depro-
shed light on this phenomenon, Rossjohn and co-workers and tection step, whereby global deprotection using Birch con-
Yu et al. concurrently solved the crystal structures of ternary ditions would afford sphingosine 2, while catalytic
TCR-iGb3-CD1d complexes and determined that the TCR of hydrogenation followed by ester hydrolysis would give sphinga-
the iNKT cell pushes the protruding sugar headgroup of iGb3 nine 3. For the synthesis of the N-acyl derivatives 4 and 5,
flat against the α2-helix of CD1d, causing the internal glyco- however, lactosyl 2-azido-sphingosine intermediate 6 would
sidic bond to resemble an α-configuration.3,18 Moreover, the first be transformed into its 4″-O-acetyl derivative, followed by
6′′′-hydroxyl of iGb3 forms a hydrogen bond with Thr159 of glycosylation of the 3″-OH with donor 7 and installation of the
CD1d, and both the 4′′′- and 6′′′-hydroxyls partake in van der C12 or C20:2 N-acyl chain after reduction of the azide to an
Waals interactions with Thr159 and Met162, which has been amine. Lactosyl 2-azido-sphingosine 6 would be obtained by
suggested to be crucial for activity.3 Changes to the glycolipid the coupling of lactosyl donor 9 and sphingosine acceptor 11,
lipid chains, either at the N-acyl position or at the sphingoid with donor 9 being prepared from D-lactose (10)35 and sphin-
backbone, also affect CD1d presentation and iNKT cell gosine acceptor 11 from D-arabinose (12).22
activation, resulting in the induction of altered cytokine profiles,
a phenomenon mainly studied using α-GalCer analogues.8,19–23
To better understand the effects of structural modifications
to iGb3 on CD1d binding and iNKT cell activation, we became
2. Results and discussion
interested in developing an efficient and divergent synthetic The synthesis of the iGb3 derivatives began with the prepa-
route that would allow for the synthesis of both sugar and ration of key lactosyl 2-azido-sphingosine intermediate 6. To
lipid modified iGb3 analogues. In particular, we were inter- this end, a strategy similar to that developed by Xing et al.35
ested in the synthesis of 6′′′-deoxy derivatives of iGb3 contain- was adopted in order to prepare an orthogonally protected lac-
ing either the more typical sphingosine ceramide (2), or the tosyl donor (Scheme 2). D-Lactose (10) was thus peracetylated,
saturated sphinganine backbone (3) (Fig. 1). Biological evalu- the anomeric acetate converted into a thiophenyl glycoside,
ation of these compounds would allow for clarification about and the remaining acetate groups removed under Zemplén
the importance of the iGb3 6′′′-hydroxyl for CD1d binding and conditions to give thiolactoside 13 as a crystalline solid.36
iNKT cell activation and whether lessening the rigidity in the Thiolactoside 13 was then subjected to 2,2-dimethoxypropane
lipid backbone (i.e. 3) can compensate for the loss of the and a catalytic amount of p-toluenesulfonic acid (pTsOH) to
6′′′-hydroxyl H-bond. Of the N-acyl derivatives, we were inter- afford the crystalline 3″,4″-ketal in 56% yield,35 which was sub-
ested in the synthesis of the iGb3–C12 4, which will provide a sequently benzoylated to give the fully protected thioglycoside
platform to further explore how truncated lipids can affect the 14 in 94% yield, again as crystalline material. At this stage
orientation of the sugar headgroup and hence activity, and several glycosidation strategies were explored. The double
C20:2 iGb3 5, as the C20:2 lipid has been found to have bond in the sphingosine acceptor precluded the use of a thio-
superior activity compared to the saturated C20 analogue in philic promoter, however, the anomeric thiophenol could be
the case of α-GalCer and skews the immune system towards an readily hydrolysed with NBS in aqueous acetone to give the
anti-inflammatory (Th2) response.8,19,24 To date, both sphinga- lactol, which could be further functionalised. Conversion of
nine iGb3 and α-iGb3 have been synthesised and found to the lactol into the corresponding lactosyl bromide using
activate iNKT cells more efficiently than the parent bromine and triethyl phosphite proceeded smoothly, however
compound,25–27 and terminal sugar deoxy iGb3 derivatives the lactosyl bromide was not a suitable donor for the glycosyla-
containing a phytosphingosine have been synthesised28 and tion of sphingosine acceptor 11 as coupling yields did not
used to study iNKT cell TCR recognition of structurally diverse exceed 40%. Consequently, the lactol was equipped with a tri-
CD1d-restricted ligands.29 A selection of N-acyl chain homo- chloroacetimidate leaving group to give lactosyl imidate donor
logues of iGb3 have also been prepared, such as C8,2,30 C16,31 9 in 73% yield over the two steps. The coupling reaction
C18,32 C2433 and C26,1 however, no systematic structure between lactosyl imidate 9 and sphingosine acceptor 1122 was
activity relationship studies have been performed on this then performed and this reaction proceeded quantitatively
series.
when using 1.5 equivalents of donor to give β-lactosyl 2-azido
To prepare iGb3 (1) and analogues 2–5, we envisioned sphingosine 6 as a single anomer, as confirmed by the 7.8 Hz
using a lactosyl 2-azido-sphingosine intermediate 6 that could 1H-NMR coupling constant between H-1′ and H-2′. Interest-
be orthogonally functionalised (Scheme 1). In this way, both ingly, when Xing et al. coupled the identical lactosyl donor to
the terminal sugar residue and lipid chain could be varied the full ceramide sphingosine acceptor bearing a C18 acyl
with a minimal number of synthetic steps. The synthesis of lipid, the reported yield was 60%,35 which was presumably due
iGb3 (1) and the 6′′′-deoxy-iGb3 derivatives 2 and 3 would to poorer solubility and reactivity of the di-lipid acceptor.
involve the installation of the C26 N-acyl group after reduction
Having successfully prepared the lactosyl 2-azido-sphingo-
of the azide on lactosyl 2-azido-sphingosine intermediate 6 to sine intermediate 6, the next synthetic targets were the donors
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Scheme 1 Retrosynthesis for iGb3 analogues.
Scheme 2 Synthesis of key lactosyl 2-azido-sphingosine intermediate 6.
7 and 8 to install the terminal sugars. While D-galactosyl donor envisioned – the first requiring 6-steps and starting from
7 could be readily prepared from D-galactose in 7 steps and D-fucose (15), and the second commencing with the signifi-
45% overall yield,22 a new strategy was required for the syn- cantly less expensive D-galactose (18) though requiring an
thesis of the D-fucosyl donor 8. To this end, two strategies were additional four steps to reach the same common intermediate
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Scheme 3 D-Fucose building block synthesis.
16 (Scheme 3). Accordingly, D-fucose (15) was peracetylated removed via hydrolysis under acidic conditions to afford the
(→16) and converted into the ethyl thioglycoside via treatment corresponding diol and, using a versatile approach to acetylate
of 16 with ethanethiol and BF3·Et2O to give thioether 17 in the axial hydroxyl in a cis-diol system first reported by Lemieux
72% yield over the two steps. To reach the same intermediate et al.,34 the 4″-OH was regioselectively protected using tri-
16, D-galactose 18 was converted to the primary iodide 19 via methyl orthoacetate and camphorsulfonic acid to first install a
selective isopropylidene protection and iodination,37 followed methyl orthoacetate protecting group across the 3″-OH and
by reduction of the iodide with tributyltin hydride and protect- 4″-OH followed by ring opening during an acidic work-up to
ing group manipulations.38 The acetate groups in 17 were then afford 4″-OAc LacCer 23. The position of the acetate ester was
removed under Zemplén conditions and the non-participatory confirmed via an HMBC between the carbonyl carbon of the
benzyl protecting groups installed to give benzylated donor 20 acetate and the proton at the 4″-position. In addition, the
in 83% over two steps.39 Here, it should also be noted that small coupling constant (J3″,4″ = 3.5 Hz) characteristic of H-4″
thioethers (e.g. 20) are useful glycosyl donors which can be observed at a downfield shift (δ 5.22 ppm) relative to the
activated under mild conditions using CuBr2 and Pr4NBr,40 doublet of doublets (J2″,3″ = 9.8 Hz, J3″,4″ = 3.5 Hz) for H-3″
thus donor 20 was also used en route to the attempted syn- (δ 3.82 ppm) also corroborated our assignment. A TMSOTf
thesis of 6′′′-deoxy-iGb3 derivatives 2 and 3 (vide infra). To mediated glycosylation reaction between 4″-OAc LacCer 23 and
make the imidate donor, however, the anomeric thioethyl D-galactosyl imidate donor 7 was then undertaken to give the
group of 20 was removed via the agency of NBS and resultant fully protected iGb3 24, which was deprotected under Birch
lactol 21 was reacted with trichloroacetonitrile and DBU to conditions to give iGb3 (1).
afford fucose donor 8, with 1H and 13C NMR spectral data
matching that of the enantiomer and the optical rotation the 6′′′-deoxy-iGb3 derivatives 2 and 3 from 4″-OAc LacCer 23
being equal in magnitude but with opposite sign.41
and the D-fucosyl thioethyl donor 20 using the CuBr2 and
With iGb3 (1) in hand, attempts were then made to prepare
With the terminal building block prepared, we next Pr4NBr promoter system.40 Unfortunately, despite several
focussed on the synthesis of iGb3 (1) to explore the validity of attempts at this reaction, none of the desired product was
our route and to also provide iGb3 as a control for subsequent observed and unreacted lipid acceptor and hydrolysed donor
biological testing. Accordingly, the azide in lactosyl 2-azido- were isolated upon work-up (Scheme 4). Accordingly, the glyco-
sphingosine 6 was reduced with PPh3 to produce the corres- sylation reaction was repeated using the α-anomer of imidate
ponding amine, which was used without further purification donor 8, so as to follow Schmidt’s original coupling con-
in the EDCI/DMAP-mediated condensation with hexacosanoic ditions.41 Gratifyingly, this reaction proceeded smoothly to
(C26) acid (Scheme 4). Under these conditions the fully pro- give only the α-anomer (J1′′′,2′′′ = 3.5 Hz) of the fully protected
tected LacCer 22 was produced in 72% yield over two steps. 6′′′-deoxy-iGb3 25. An HMBC between C-3″ and H-1′′′ confirmed
Here, it should be noted that the C26 acyl lipid is notorious the attachment of the terminal fucose moiety to the 3″-posi-
for being poorly soluble and unreactive and therefore in the tion of LacCer. The 69% yield for this reaction was encoura-
syntheses of glycosphingolipids, the original C26 acyl chain, as ging as glycosylation reactions incorporating D- or L-fucose
is found in α-GalCer and iGb3, is often substituted by shorter donors are often poor yielding due to the increased acid labi-
variants. This presumably has the advantage of increasing lity of the α-fucosyl linkage.43,44 To provide the 6′′′-deoxy-iGb3
reaction yields, however, it can lead to differences in the solu- derivatives 2 and 3, the fully protected trisaccharide 25 was
bility of the glycolipids and variations in in vivo distribution either treated under Birch conditions or hydrogenated with
and pharmacology.42 From LacCer 22, the acetonide was then Pearlman’s catalyst followed by the hydrolysis of the esters to
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Scheme 4 Synthesis of iGb3 (1) and 6’’’-deoxy derivatives 2 and 3.
afford 6′′′-deoxy-iGb3-sphingosine 2 or the 6′′′-deoxy-iGb3- that 1,4-dienes (skipped-dienes) are particularly prone to auto-
sphinganine 3, respectively.
xidation to form conjugated diene hydroperoxides,45,46 and
Next, we turned our attention towards the synthesis of the indeed, we observed autoxidation of 11Z,14Z-eicosadienoic
N-acyl iGb3 analogues 4 and 5 (Scheme 5). Here, the isopropyl- acid by HRMS with molecular ion peaks corresponding to the
−
idene group in lactosyl 2-azido-sphingosine 6 was removed, 1,3-conjugated diene alcohol (m/z calcd for C20H35O3
:
and the corresponding orthoester selectively opened in excel- 323.2592, obsd: 323.2587) and the 1,3-conjugated diene ketone
lent yield to give the 3″-OH lactoside 26. TMSOTf-mediated (m/z calcd for C20H33O3−: 321.2435, obsd: 321.2435) if the acid
glycosylation of 26 with D-galactosyl imidate donor 7 yielded was exposed to oxygen for too long. To minimise this, the com-
triglycosyl 2-azido-sphingosine 27 in 56% yield and as the pounds were kept under an inert, oxygen-free atmosphere as
α-anomer only. The sphingosine backbone was then functiona- often as was practically feasible and fortunately, none of the
lised with fatty acids bearing the C12 or the C20:2 lipid, first by-products could be observed by HRMS or NMR spectroscopy
via reduction of the azide to the amine using triphenyl- in the purified fractions of the fully protected iGb3–C20:2 29,
phosphine and water, followed by an EDCI/DMAP-mediated or in the final product. Global deprotection of the fully pro-
coupling of the crude amine intermediate with either dodeca- tected iGb3-homologues 28 and 29 containing the sphingosine
noic (C12) or 11Z,14Z-eicosadienoic (C20:2) acid to give the backbone was then performed using a Birch reaction to give
fully protected iGb3–C12 28 or iGb3–C20:2 29, respectively. the target compounds, iGb3–C12 4 and iGb3–C20:2 5. At this
Here, the difference in the esterification yield for the different point, we were somewhat disappointed with the modest yield
acids (75% yield for C12 vs. 28% yield for C20:2) was attributed of the Birch reaction, although we were aware that the yields of
to the different solubilities and reactivities of the two deriva- Birch reactions of complex carbohydrates vary in the literature.
tives, with the coupling of the longer lipid to the trisaccharide Thus, we chose to investigate further and upon careful HRMS
being more problematic. In addition, it has also been reported analyses of aliquots taken from the crude reaction mixtures,
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(or similar) backbone followed by N-acylation.25,28,49,50 While
these reported strategies may be more convergent for the syn-
thesis of one specific target, the route designed in our studies
is robust and versatile and allows for the synthesis of multiple
targets of either the terminal sugar or the acyl chain series.
The yield and number of steps for the syntheses of all
iGb3 homologues reported to date are very similar and where
one gain is made, another may be lost – as illustrated by the
coupling of the sphingosine backbone which requires an
additional N-acylation step in our strategy, but which occurs in
better yield than the coupling of the full lipid.35 Thus far, iGb3
plus four unprecedented analogues have been prepared to
probe key aspects of CD1d-glycolipid-iNKT cell interactions,
however, it is envisioned this strategy will prove valuable in the
synthesis of further derivatives in the near future.
3. Conclusions
In summary, iGb3 (1), 6′′′-deoxy derivative 2, 6′′′-deoxy-iGb3-
sphinganine 3 and the two the N-acyl homologues C12 iGb3 4
and C20:2 iGb3 5 were prepared via a single synthetic strategy
involving a common lactosyl 2-azido-sphingosine intermedi-
ate. This intermediate was prepared by coupling a sphingosine
acceptor with a lactosyl imidate donor, which proceeded in
excellent yield and β-selectivity. Functionalisation of this key
lactosyl 2-azido-sphingosine intermediate at either the 3″-posi-
tion or the sphingoid nitrogen provided facile entry into a
range of iGb3 analogues, which will allow for the future assess-
ment of structure activity relationships in terms of iNKT cell
activation. These findings will be reported in due course.
Acknowledgements
The authors would like to thank Cancer Society of New
Zealand for financial support. B. L. S. also thanks the Health
Research Council of New Zealand (Hercus Fellowship, 13/049).
Scheme 5 Synthesis of iGb3–C12 4 and iGb3–C20:2 5.
observed that glycolipid fragmentation had occurred during
this final deprotection with the hydrolysed by-products
LacCer-C12, GlcCer-C12, and Cer-C12, and the analogous
C20:2 homologues, being observed. As glycosidic linkages are
generally considered to be stable under basic conditions, this
result was somewhat unexpected and while others have
noticed similar glycosidic cleavages,47 this is an area that
needs to be more thoroughly explored. The target iGb3 deriva-
tives, however, could be readily purified by silica gel and
reverse phase column chromatography to give N-acyl
iGb3 homologues 4 and 5.
Taken as a whole, the strategy presented herein differs from
all other iGb3 and analogue syntheses insomuch as lactosyl
2-azido-sphingosine glycolipid 6, itself readily accessible from
D-lactose, is used as the key intermediate. In other reported
routes, a trisaccharide donor is coupled directly to either the
complete ceramide27,32,33,48 or in two steps to the sphingosine
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