The 3-deoxy analogue of α-GalCer: Disclosing the role of the 4-hydroxyl group for CD1d-mediated NKT Cell activation

Article abstract of DOI:10.1021/ml2000802

KRN7000, or α-GalCer, is a potent agonist for natural killer T (NKT) cells. The 3-hydroxyl group of its phytosphingosine moiety is important for activating NKT cells, whereas its 4-hydroxyl group is perceived to be less crucial. To experimentally determine the role of the 4-hydroxyl group, we synthesized the 3-deoxy analogue of α-GalCer. It was found that 3-deoxy-α-GalCer induced potent cytokine responses from NKT cells, comparable to those of both α-GalCer and 4-deoxy-α-GalCer. This result and our docking studies suggest that the effects of an absence of the 3-hydroxyl group are compensated by the presence of a hydroxyl group at the C-4 position. Thus, we conclude that the 4-hydroxyl group of α-GalCer is as important to the mechanism of action as the 3-hydroxyl group and that the two hydroxyl groups could play individual and cooperative roles in orienting the glycolipid into the proper position in CD1d to be recognized by the T cell receptor of NKT cells.

Full text of DOI:10.1021/ml2000802

LETTER
pubs.acs.org/acsmedchemlett
The 3-Deoxy Analogue of r-GalCer: Disclosing the Role of the
4-Hydroxyl Group for CD1d-Mediated NKT Cell Activation
Dong Jae Baek,† Jeong-Hwan Seo,† Chaemin Lim,† Jae Hyun Kim,† Doo Hyun Chung,‡ Won-Jea Cho,§
Chang-Yuil Kang,† and Sanghee Kim*,†
†College of Pharmacy, Seoul National University, San 56-1, Shilim, Kwanak, Seoul 151-742, Korea
‡College of Medicine, Seoul National University, 28 Yongon, Chongno, Seoul 110-799, Korea
§College of Pharmacy, Chonnam National University, Yongbong, Buk, Kwangju 500-757, Korea
S Supporting Information
b
ABSTRACT: KRN7000, or R-GalCer, is a potent agonist for natural killer T (NKT)
cells. The 3-hydroxyl group of its phytosphingosine moiety is important for activating
NKT cells, whereas its 4-hydroxyl group is perceived to be less crucial. To experimen-
tally determine the role of the 4-hydroxyl group, we synthesized the 3-deoxy analogue
of R-GalCer. It was found that 3-deoxy-R-GalCer induced potent cytokine responses
from NKT cells, comparable to those of both R-GalCer and 4-deoxy-R-GalCer. This
result and our docking studies suggest that the effects of an absence of the 3-hydroxyl
group are compensated by the presence of a hydroxyl group at the C-4 position. Thus,
we conclude that the 4-hydroxyl group of R-GalCer is as important to the mechanism of
action as the 3-hydroxyl group and that the two hydroxyl groups could play individual
and cooperative roles in orienting the glycolipid into the proper position in CD1d to be recognized by the T cell receptor of
NKT cells.
KEYWORDS: CD1d, natural killer T cells, T cell receptor, glycolipid, R-GalCer, deoxy analogues
RN7000 (also commonly called R-GalCer, 1, Figure 1) is a
structurally modified analogue of the marine natural product
studies on the phytosphingosine moiety of R-GalCer have shown
that the analogue lacking the 4-hydroxyl group on the phyto-
sphingosine (4-deoxy-R-GalCer, 2, Figure 1) exhibits slightly
reduced activity as compared to R-GalCer, whereas the analogue
that lacks both 3- and 4-hydroxyl groups (3,4-dideoxy-R-GalCer,
3, Figure 1) was inactive.19ꢀ22 These SAR results, along with the
observed hydrogen-bonding interactions of the 3-hydroxyl group
of R-GalCer, with residues of both the CD1d (Asp-80) and the
TCR (Arg-95), led to the general belief that the 3-hydroxyl group
of phytosphingosine was crucial for activating NKT cells,
whereas the 4-hydroxyl group was not crucial for activity.16,20,23
However, the analogue lacking only the 3-hydroxyl group on the
phytosphingosine (3-deoxy-R-GalCer, 4, Figure 1) had never
been prepared and evaluated to ascertain the individual impacts
of the 3- and 4-hydroxyl groups on NKT cell activation. This lack
of potentially important SAR data prompted us to synthesize and
evaluate the 3-deoxy analogue 4. Herein, we report our studies on
this subject.
K
agelasphins.1 This glycolipid is the first-defined agonist for
natural killer T (NKT) cells, and it remains the most extensively
studied compound for the exploration of NKT cell biology and
pharmacology.2ꢀ6 R-GalCer activates NKT cells in a CD1d
restricted manner; it first binds to the CD1d molecule of
antigen-presenting cells, and the resulting R-GalCer/CD1d
complex is then recognized by the conserved Rβ T cell receptor
(TCR) to form the ternary complex that leads to activation of the
NKT cells. The activated NKT cells promptly secrete large
amounts of T helper 1 and 2 (Th1 and Th2) cytokines, such
as interferon-γ (IFN-γ) and interleukin-4 (IL-4), which play
critical roles in the regulation of innate and adaptive immune
responses.7ꢀ9
The recently achieved crystal structure of R-GalCer com-
plexed with CD1d reveals that the acyl and phytosphingosine
lipid chains of R-GalCer fit tightly into two hydrophobic pockets
of the CD1d binding groove, whereas the galactose group
protrudes from the CD1d cleft.10ꢀ13 There are several hydro-
gen-bonding interactions between the surface residues of CD1d
and the hydroxyl groups of the galactose and the phytosphingo-
sine base that appear to be crucial for maintaining R-GalCer in
the correct position for recognition by the TCR of NKT cells.
The hydrogen-bonding network in the crystal structure is well
matched with the previous results from the structureꢀactivity
relationships (SAR) studies of R-GalCer analogues.6,14ꢀ18 SAR
For the synthesis of the desired 3-deoxy analogue of R-GalCer 4,
the known D-ribo-phytosphingosine-derived compound 524 was
chosen as an ideal starting material given that its 2-amino and
4-hydroxyl groups were already suitably protected (Scheme1). The
primary hydroxyl group of 5 was selectively protected as its silyl
Received: March 30, 2011
Accepted: May 11, 2011
Published: May 16, 2011
r
2011 American Chemical Society
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Figure 1. Structures of glycolipids 1ꢀ4.
Scheme 1. Synthesis of 3-Deoxy-r-GalCer 4a
Figure 2. Biological evaluation of deoxy analogues. (a) IL-2 secretion
by DN32.D3 NKT hybridoma cells. IL-2 production was measured
from cocultured supernatants of NKT hybridoma DN32.D3 and
mouse CD1d transfected RBL cells after 16 h of culture. Representative
data of two individual experiments are expressed as the mean ( SD
of duplicates. (b) IFN-γ and IL-4 secretion by mouse splenocytes.
Cytokine production was measured after 72 h of culture. Results are
expressed as relative activity. Representative data of two individual
experiments are expressed as means ( SDs of triplicates. The statistical
significance of the difference in secretion levels was determined by
Student's t test. **p < 0.01.
a Reagents and conditions: (a) TBDPSCl, Et3N, CH2Cl2, room tem-
perature, 99%. (b) CBr4, PPh3, CH2Cl2, 40 °C, 72%. (c) NaBH4, NiCl2,
EtOH, room temperature, 85%. (d) Bu4NF, THF, room temperature,
80%. (e) Hexacosanoic acid, EDCI, DMAP, CH2Cl2, room temperature,
78%. (f) Compound 12, AgClO4, SnCl2, 4 Å molecular sieve, THF, ꢀ10
°C, ca. 10%. (g) TfN3, K2CO3, CuSO4, MeOH, CH2Cl2, H2O, room
temperature, 79%. (h) Compound 15, Bu4NBr, N,N-tetramethylurea,
CH2Cl2, room temperature, 83%. (i) PPh3, benzene/H2O (10:1), 60
°C, 12 h. (j) Hexacosanoic acid, EDCI, DMAP, CH2Cl2, room
temperature, 54% from 14. (k) H2, Pd(OH)2, EtOH/CH2Cl2 (3:1),
room temperature, 72%.
After successful removal of the C-3 hydroxyl group of ribo-
phytosphingosine, the silyl protecting group in 8 was removed
with Bu4NF to furnish amino alcohol 9. Acylation of 9 with
hexacosanoic acid and EDCI gave ceramide 10. For selective
R-glycosyl bond formation, we employed the Mukaiyama glyco-
sylation reaction25 involving the reactive galactosyl fluoride 12
as the glycosyl donor. This reaction provided the desired R-
galactoside 11, but the yield was very low (ca. 10%). Attempts
to increase the yield of 11 by varying the glycosyl donor and
reaction conditions were not successful.
The low yield of glycosylation product 11 could be attributed
to the fact that the amide group of 10 diminishes the nuc-
leophilicity of the primary hydroxyl group through hydrogen
bonding.26 An alternative approach was therefore devised in
which an azido group was used in place of the amide during the
glycosylation. The free amine group of 9 was first reconverted to
ether to afford compound 6. To remove the C-3 hydroxyl group,
alcohol 6 was first converted to bromide 7 with CBr4 and PPh3.
The obtained bromide 7 was then reduced using NaBH4 and NiCl2
in EtOH. Under these reaction conditions, the azido group of 7 was
concomitantly reduced to give amine 8 in good yield (85%).
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Figure 3. Comparison of the docking model of 3-deoxy-R-GalCer 4 (green backbone) with the crystal structure of R-GalCer 1 (pink backbone) within
the hCD1d/TCR (Protein Data Bank code 2PO6). The key amino acid residues are shown in blue (TCR residues) and orange (CD1d residues). (a)
R-GalCer 1 (shown in pink) and 3-deoxy-R-GalCer 4 (shown in green) are overlaid. The galactose headgroup of 3-deoxy-R-GalCer 4 is shifted down
(ca. 1.3 Å) as compared to that of R-GalCer 1. H-bonds between R-GalCer 1 and hCD1d/TCR are indicated by pink dashed lines. (b) Docking model of
3-deoxy-R-GalCer 4. H-bonds are shown as dark blue solid lines.
an azide 13 via a modified diazo transfer reaction.27 When the
in situ-generated galactosyl bromide 1528 was employed as a
galactosyl donor, the glycosylation of 13 was accomplished effi-
ciently to give R-galactoside 14 as the only identifiable anomer
in good yield (83%). Staudinger reduction of the azido group of
14 followed by condensation of the resulting amine with hexaco-
sanoic acid led to the formation of 11. This route required two
more steps than the original but proved significantly more effi-
cient and practical for the preparation of 11. Finally, the global
deprotection of the benzyl ethers by hydrogenolysis afforded the
desired 3-deoxy analogue 4.
For a preliminary biological evaluation of 3-deoxy analogue 4,
we used CD1d-specific NKT cell hybridoma cells (DN32.D3)
that are immortalized NKT cells and that tend to produce IL-2
upon stimulation. The parent R-GalCer 1 and its 4-deoxy
analogue 2 were also tested for comparison.29,30 As shown in
Figure 2a, 4-deoxy analogue 2 displayed comparable stimulatory
effects to R-GalCer 1, confirming previous reports that the
activity of 4-deoxy analogue 2 does not differ significantly from
that of R-GalCer.19 Contrary to the expectation, however, the
3-deoxy analogue 4 was slightly more effective than R-GalCer at
promoting IL-2 production. Because IL-2 secretion is dependent
on antigen-loaded CD1d,31 these results indicate that the
3-deoxy analogue 4 efficiently binds to CD1d and triggers
TCR signaling in a manner similar to R-GalCer.
stimulation activity of 4-deoxy analogue 2 did not significantly
differ from that of R-GalCer 1. Our evaluation showed that
3-deoxy analogue 4 could induce NKT cell cytokine responses
similarly to 1. However, compound 4 seemed to bias cytokine
secretion toward the Th2 response; 3-deoxy analogue 4 showed a
comparable stimulatory effect on IL-4 production as R-GalCer 1,
whereas it promoted smaller amounts of IFN-γ production as
compared to 1.
The above biological results suggest that the 3-deoxy analogue
4 can be favorably accommodated within the CD1d binding
groove. To understand the binding mode of 4, we performed
molecular modeling studies on the interaction of analogues 2 and
4 with CD1d and the NKT TCR.32,33 The X-ray crystallographic
structure of ternary complex R-GalCer/hCD1d/TCR (PDB
code 2PO6)12 was used for this docking analysis. The best
possible geometry of the analogue within the CD1d/TCR
complex was searched using Surflex-Dock. The docking scores
of compounds were in partial agreement with the biological data.
The 3-deoxy analogue 4 gave a better docking score than either
R-GalCer 1 or its 4-deoxy analogue 2 (see Figure S1 in the
Supporting Information), although analogue 4 forms fewer
hydrogen bonds with the CD1d/TCR complex than 1 or 2
(see Figure S2 in the Supporting Information). In our docking
model, the 4-deoxy analogue 2 occupies virtually the same
position and forms the same set of hydrogen bonds as R-GalCer
in its crystalline complex with hCD1d/TCR (see Figure S3 in the
Supporting Information). However, 3-deoxy analogue 4 sits
considerably deeper in the groove than R-GalCer 1 as shown
in Figure 3a. The galactose headgroup is laterally shifted approxi-
mately 1.3 Å toward the center of the binding groove.34 The
absence of a hydroxyl group in the 3-position seems to cause the
To determine whether the 3-deoxy analogue 4 can stimulate
cytokine release from intact NKT cells, we measured the levels of
IFN-γ and IL-4 in in vitro culture supernatants of mouse
splenocytes stimulated with 8 ng/mL of compounds 1, 2, and
4. Figure 2b shows the relative IFN-γ and IL-4 production levels
of 2 and 4 when compared with those of 1. As expected, the NKT
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sphingoid base to shift itself lower in the pocket to establish new
hydrogen bonds with CD1d/TCR complex (Figure 3b).
Although the computational modeling studies do not provide
conclusive proof, our model offers a view of how 3-deoxy ana-
logue 4 may bind CD1d and present its galactose epitope to the
TCR of NKT cells. Throughout these molecular modeling and
biological studies, the effect of the absence of the 3-hydroxyl
group appears to be compensated by the presence of a hydroxyl
group at the C-4 position. Thus, the 3- and 4-hydroxyl groups
of phytosphingosine are both individually effective at orienting
the glycolipid in CD1d and inducing NKT cells responses,
although the position of the hydroxyl group affects how the
glycolipid binds to CD1d.
In summary, we have prepared the R-GalCer analogue lacking
the 3-hydroxyl group on the phytosphingosine (3-deoxy-
R-GalCer, 4) to ascertain the individual impacts of the 3- and
4-hydroxyl groups on NKT cell activation. Contrary to the pre-
vious perception, 3-deoxy-R-GalCer was found to be similarly
effective at inducing NKT cells responses as both R-GalCer and
4-deoxy-R-GalCer. Together with our docking studies, this result
suggests that the 4-hydroxyl group of R-GalCer is as important as
the 3-hydroxyl group and that the two hydroxyl groups could
play both individual and cooperative roles in orienting the
glycolipid into the proper position in CD1d to be recognized
by the TCR of NKT cells. We believe that 3-deoxy-R-GalCer
represents an important new tool for improving the under-
standing of glycolipidꢀCD1d interactions and the NKT re-
sponse. Additionally, it could provide guidance regarding the
development of nonstereotypical immunostimulating agents that
are structurally distinct from the typical phytosphingosine-con-
taining galactosylceramide.
(3) Balato, A.; Unutmaz, D.; Gaspari, A. A. Natural killer T cells: an
unconventional T-cell subset with diverse effector and regulatory
functions. J. Invest. Dermatol. 2009, 129, 1628–1642.
(4) Cerundolo, V.; Silk, J. D.; Masri, S. H.; Salio, M. Harnessing
invariant NKT cells in vaccination strategies. Nat. Rev. Immunol. 2009,
9, 28–38.
(5) Banchet-Cadeddu, A.; Hꢀenon, E.; Dauchez, M.; Renault, J.-H.;
Monneaux, F.; Haudrechy, A. The stimulating adventure of KRN 7000.
Org. Biomol. Chem. 2011, 9, 3080–3104.
(6) Mori, K.; Tashiro, T. Sphingolipids and glycosphingolipids—
Their syntheses and bioactivities. Heterocycles 2011, 83, 951–1003.
(7) Kronenberg, M. Toward an understanding of NKT cell biology:
Progress and Paradoxes. Annu. Rev. Immunol. 2005, 23, 877–900.
(8) Kaer, L. V. R-Galactosylceramide therapy for autoimmune
diseases: Prospects and obstacles. Nat. Rev. Immunol. 2005, 5, 31–42.
(9) Murphy, N.; Zhu, X.; Schmidt, R. R. R-Galactosylceramides and
analogues—Important immunomodulators for use as vaccine adjuvants.
Carbohydr. Chem. 2010, 36, 64–100.
(10) Zajonc, D. M.; Cantu, C., III; Mattner, J.; Zhou, D.; Savage,
P. B.; Bendelac, A.; Wilson, I. A.; Teyton, L. Structure and function of a
potent agonist for the semi-invariant natural killer T cell receptor. Nat.
Immunol. 2005, 6, 810–818.
(11) Koch, M.; Stronge, V. S.; Shepherd, D.; Gadola, S. D.; Mathew,
B.; Ritter, G.; Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.;
Cerundolo, V. The crystal structure of human CD1d with and without
R-galactosylceramide. Nat. Immunol. 2005, 6, 819–826.
(12) Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.;
Pellicci, D. G.; Koh, R.; Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.;
McCluskey, J.; Rossjohn, J. CD1d-lipid-antigen recognition by the semi-
invariant NKT T-cell receptor. Nature 2007, 448, 44–49.
(13) Pellicci, D. G.; Patel, O.; Kjer-Nielsen, L.; Pang, S. S.; Sullivan,
L. C.; Kyparissoudis, K.; Brooks, A. G.; Reid, H. H.; Gras, S.; Lucet, I. S.;
Koh, R.; Smyth, M. J.; Mallevaey, T.; Matsuda, J. L.; Gapin, L.;
McCluskey, J.; Godfrey, D. I.; Rossjohn, J. Differential Recognition of
CD1d-R-GalactogylCeramide by the Vβ8.2 and Vβ7 semi-invariant
NKT T cell receptors. Immunity 2009, 31, 47–59.
(14) Goff, R. D.; Gao, Y.; Mattner, J.; Zhou, D.; Yin, N.; Cantu, C.,
III; Teyton, L.; Bendelac, A.; Savage, P. B. Effects of lipid chain lengths in
R-galactosylceramides on cytokine release by natural killer T cells. J. Am.
Chem. Soc. 2004, 126, 13602–13603.
(15) Tsuji, M. Glycolipids and phospholipids as natural CD1d-
binding NKT cell ligands. Cell. Mol. Life Sci. 2006, 63, 1889–1898.
(16) Savage, P. B.; Teyton, L.; Bendelac, A. Glycolipids for natural
killer T cells. Chem. Soc. Rev. 2006, 35, 771–779.
(17) Wu, D.; Fujio, M.; Wong, C.-H. Glycolipids as immunostimu-
lating agents. Bioorg. Med. Chem. 2008, 16, 1073–1083.
(18) Nadas, J.; Li, C.; Wang, P. G. Computaional structure activity
relationship studies on the CD1d/glycolipid/TCR complex using
AMBER and AUTODOCK. J. Chem. Inf. Model. 2009, 49, 410–423.
(19) Lac^one, V.; Hunault, J.; Pipelier, M.; Blot, V.; Lecourt, T.;
Rocher, J.; Turcot-Dubois, A.-L.; Marionneau, S.; Douillard, J.-Y.;
Clꢀement, M.; Le Pendu, J.; Bonneville, M.; Micouin, L.; Dubreuil, D.
Focus on the controversial activation of human iNKT cells by 4-deoxy
analogue of KRN7000. J. Med. Chem. 2009, 52, 4960–4963.
(20) Ndonye, R. M.; Izmirian, D. P.; Dunn, M. F.; Yu, K. O. A.;
Porcelli, S. A.; Khurana, A.; Kronenberg, M.; Richardson, S. K.; Howell,
A. R. Synthesis and evaluation of sphinganine analogues of KRN7000
and OCH. J. Org. Chem. 2005, 70, 10260–10270.
(21) Sidobre, S.; Hammond, K. J. L.; Bꢀenazet-Sidobre, L.; Maltsev,
S. D.; Richardson, S. K.; Ndonye, R. M.; Howell, A. R.; Sakai, T.; Besra,
G. S.; Porcelli, S. A.; Kronenberg, M. The T cell antigen receptor
expressed by VR14i NKT cells has a unique mode of glycosphingolipid
antigen recognition. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12254–12259.
(22) Miyamoto, K.; Miyake, S.; Yamamura, T. A synthetic glycolipid
prevents autoimmune encephalomyelitis by inducing TH2 bias of natural
killer T cells. Nature 2001, 413, 531–534.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures showing the docking
b
scores of compounds 1, 2, and 4 (Figure S1), the hydrogen-
bonding modes of compounds 2 and 4 (Figure S2), and the
docking conformation of compound 2 superimposed with R-
GalCer (1) (Figure S3), detailed information on the experi-
mental procedures, analytical data for all new compounds, and
copies of 1H and 13C NMR spectra for selected compounds. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 82-2-880-2487. Fax: 82-2-888-0649. E-mail: pennkim@
snu.ac.kr.
Funding Sources
This work was supported by a National Research Foundation of
Korea grant funded by the Korea government (MEST) (Grant
20100027763).
’ REFERENCES
(1) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa,
E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. Structure-
activity relationship of R-galactosylceramides against B16-bearing mice.
J. Med. Chem. 1995, 38, 2176–2187.
(2) Tupin, E.; Kinjo, Y.; Kronenberg, M. The unique role of natural
killer T cells in the response to microorganisms. Nat. Rev. Microbiol.
2007, 5, 405–417.
(23) Wu, D.; Zajonc, D. M.; Fujio, M.; Sullivan, B. A.; Kinjo, Y.;
Kronenberg, M.; Wilson, I. A.; Wong, C.-H. Design of natural killer T
547
dx.doi.org/10.1021/ml2000802 |ACS Med. Chem. Lett. 2011, 2, 544–548

ACS Medicinal Chemistry Letters
LETTER
cell activators: structure and function of a microbial glycosphingolipid
bound to mouse CD1d. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
3972–3977.
(24) Trappeniers, M.; Goormans, S.; Beneden, K. V.; Decruy, T.;
Linclau, B.; Al-Shamkhani, A.; Elliott, T.; Ottensmeier, C.; Werner, J. M.;
Elewaut, D.; Van Calenbergh, S. Synthesis and in vitro evaluation of
R-GalCer epimers. ChemMedChem 2008, 3, 1061–1070.
(25) Mukaiyama, T.; Murai, Y.; Shoda, S. An efficient method for
glucosylation of hydroxy compounds using glucopyranosyl fluoride.
Chem. Lett. 1981, 431–432.
(26) Du, W.; Kulkarni, S. S.; Gervay-Hague, J. Efficient, one-pot
syntheses of biologically active R-linked glycolipids. Chem. Commun.
2007, 2336–2338.
(27) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Metal catalyzed diazo
transfer for the synthesis of azides from amines. Tetrahedron Lett. 1996,
37, 6029–6032.
(28) Nishida, Y.; Shingu, Y.; Dohi, H.; Kobayashi, K. One-pot
R-glycosylation method using appel agents in N,N-dimethylformamide.
Org. Lett. 2003, 5, 2377–2380.
(29) Compounds 1 and 2 were prepared according to the procedure
in the literature; see refs 30 and 14.
(30) Kim, S.; Song, S.; Lee, T.; Jung, S.; Kim, D. Practical Synthesis
of KRN7000 from phytosphinghosine. Synthesis 2004, 847–850.
(31) Zhou, D.; Mattner, J.; Cantu, C., III; Schrantz, N.; Yin, N.; Gao,
Y.; Sagiv, Y.; Hudspeth, K.; Wu, Y.-P.; Yamashita, T.; Teneberg, S.;
Wang, D.; Proia, R. L.; Levery, S. B.; Savage, P. B.; Teyton, L.; Bendelac,
A. Lysosomal glycosphingolipid recognition by NKT cells. Science 2004,
306, 1786–1789.
(32) For selected examples for other computational studies in the
R-GalCer analogues, see Zhang, W.; Xia, C.; Nadas, J.; Chen, W.; Gu, L.;
Wang, P. G. Introduction of aromatic group on 40-OH of R-GalCer
manipulated NKT cell cytokine production. Bioorg. Med. Chem. 2011,
19, 2767–2776. Also see refs 33 and 18.
(33) Hꢀenon, E.; Dauchez, M.; Haudrechy, A.; Banchet, A. Molecular
dynamics simulation study on the interaction of KRN 7000 and three
analogues with human CD1d. Tetrahedron 2008, 64, 9480–9489.
(34) The lateral shift of the sugar headgroup at the CD1d surface has
also been observed in the crystallographic structure of an mCD1d/GalA-
GSL complex. For more information, see ref 23.
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