Synthesis of a novel α-galactosyl ceramide haptenated-lipid antigen, a useful tool in demonstrating the involvement of i NKT cells in the production of antilipid antibodies

Article abstract of DOI:10.1021/bc9005255

A new haptenated derivative of α-galactosyl ceramide (α-GalCer) has been synthesized to assist in the study of the mechanism of T cell help for the production of B cell antibodies. Our synthetic route provides access to an amine intermediate which can eas

Full text of DOI:10.1021/bc9005255

Bioconjugate Chem. 2010, 21, 741–747
741
Synthesis of a Novel r-Galactosyl Ceramide Haptenated-Lipid Antigen, a
Useful Tool in Demonstrating the Involvement of iNKT Cells in the
Production of Antilipid Antibodies
Natacha Veerapen,† Elizabeth A. Leadbetter,‡ Michael B. Brenner,‡ Liam R. Cox,§ and Gurdyal S. Besra*,†
School of Biosciences and School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United
Kingdom, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Harvard Medical School, 1
Jimmy Fund Way, Boston, Massachusetts 02115. Received November 30, 2009; Revised Manuscript Received March 4, 2010
A new haptenated derivative of R-galactosyl ceramide (R-GalCer) has been synthesized to assist in the study of
the mechanism of T cell help for the production of B cell antibodies. Our synthetic route provides access to an
amine intermediate which can easily be extended to generate an array of compounds, useful in various ongoing
studies. Herein, we also describe the biological evaluation of the nitrophenyl (NP) haptenated R-GalCer and
demonstrate its importance in such mechanistic studies. For instance, in Vitro studies showed that NP-R-GalCer
stimulates both T and B cell proliferation while in ViVo studies in immunized mice showed the production of IgG
anti-NP antibodies after exposure to NP-R-GalCer. The interpretation of these results helps toward a better
understanding of T cell help for the production of antibodies.
INTRODUCTION
1
Antigen-presenting cells (APC) and lymphocytes are two
major cell types that work together to ensure an effective
immune response through the recognition and elimination or
neutralization of pathogenic organisms and other foreign invad-
ers in mammals (1-4). APC include macrophages, B lympho-
cytes, and dendritic cells, while T cells, invariant natural killer
(iNKT), and natural killer (NK) cells are three major types of
lymphocytes important for immune defense. Specifically, B cells
recognize their epitopes as a part of either an intact protein,
lipid molecule, or polysaccharide, whereas T cells only recog-
nize epitopes which are subcomponents of an intact molecule
and are found in association with antigen-presenting molecules,
such as MHC class II or CD1.
Figure 1. iNKT cells provide cognate help for lipid-specific B cells.
A distinctive class of T cells, known as invariant Natural
Killer T (iNKT) cells, display characteristics of both T cells
and NK cells and play a crucial role in diverse immune
responses and other pathologic conditions (5). The synthetic
glycolipid R-galactosyl ceramide (R-GalCer) (6), known as
KRN7000 (1), is a powerful agonist for these cells. When it is
presented by CD1d, it activates iNKT cells to release diverse
cytokines, including both Th1 and Th2 cytokines (7-9). It is
believed that the release of Th1 cytokines may contribute to
antitumor and antimicrobial functions, while Th2 cytokines may
help alleviate autoimmune diseases (10-12), such as multiple
sclerosis (13) and arthritis (14) or helminth infections. Once
stimulated, iNKT cells also activate other cells, such as dendritic
cells, NK cells, peptide-reactive T cells, and B cells (2).
The detection of IgG antilipid antibodies, generated by lipid-
specific B cells during infection and autoimmune disease, such
as malaria (15) and systemic lupus erythematosus (16), respec-
tively, suggests the existence of cooperation between iNKT cells
and B cells (17). To better understand the mechanism of iNKT
cell help for the production of antilipid antibodies, haptenated
lipid antigens, containing both a B cell recognition component
and an iNKT cell agonist are useful tools (Figure 1) (17). As
part of ongoing studies on the emerging role for CD1d and iNKT
cells/B cell responses that are crucial in defense against
infection, autoimmunity, and tumor immunity, easy access to
hybrid haptenated lipids is therefore desired. Here, we propose
that the modified R-GalCer derivative 2 (Figure 2) represents
an ideal template which is easily amenable to such molecules.
We believe that the amine functionality in compound 2 allows
conjugation to different haptens, e.g., biotin or 4-hydroxy-3-
nitrophenyl (NP) to yield substrates such as compound 3, thereby
making it an ideal scaffold. In this paper, we describe the
syntheses of compounds 2 and 3 and describe the biological
activities of compounds 3 and 4 (Figure 2). The hapten,
4-hydroxy-3-nitrophenyl (NP), was chosen as the B cell
recognition component because the anti-NP B cell response has
been extensively studied in mice and there are a number of
B-cell specific immunologic tools available to investigate this
response (18-20). Conversely, R-GalCer was selected as the
iNKT cell component of this conjugated molecule because it is
also a well-studied antigen which stimulates robust activation
* To whom correspondence should be addressed. Phone: (44)1214158123;
E-mail: g.besra@bham.ac.uk.
† School of Biosciences, University of Birmingham.
‡ Harvard Medical School.
§ School of Chemistry, University of Birmingham.
1Abbreviations: APC, antigen-presenting cell; IgG, immunoglobin
G; IgM, immunoglobin M; DMF, dimethylformamide; CFSE, carboxy-
fluorescein succinimidyl ester.
10.1021/bc9005255  2010 American Chemical Society
Published on Web 03/26/2010

742 Bioconjugate Chem., Vol. 21, No. 4, 2010
Veerapen et al.
and evaporated. The residue was then purified by flash chro-
matography using hexanes/EtOAc (4:1) to give the desired
1
product as a colorless oil (1.80 g, 2.72 mmol, 75%). H NMR
(CDCl3): δ 7.65-7.75 (3H, m, Ar-H), 7.49-7.55 (2H, m, Ar-
H), 7.32-7.44 (8H, m, Ar-H), 7.19-7.27 (2H, m, Ar-H), 4.55
(1H, d, J1,2 ) 9.72 Hz, H-1), 4.28 (1H, dd, J4,3 ) 5.6, J4,5 ) 1.9
Hz, H-4), 4.15 (1H, dd, J3,2 ) 6.5 Hz, H-3), 3.96-3.83 (3H,
m, H-5, H-6a, H-6b), 3.72 (1H, ddd, J1a′,1b′ ) 10.7, J1a′,2a′
J1a′,2b′ ) 6.1 Hz, OCH2C3H6CH2N3), 3.59 (1H, ddd, J1b′,2a′
)
)
8.1, J1b′,2b′) 5.2 Hz, OCH2C3H6CH2N3), 3.34-3.39 (1H, m,
H-2), 3.26-3.31 (2H, m, OCH2C3H6CH2N3), 1.56-1.70 (4H,
m, CH2), 1.34, 1.47 (6H, 2s, C(CH3)2), 1.09 (2H, s, CH2), 1.06
(9H, s, t-Bu). 13C NMR (75 MHz, CDCl3): δ 135.6, 134.8,
133.4, 131.4, 131.5, 129.7, 128.8, 127.7, 127.6, 127.2 (18 CAr),
109.8 (O2C(CH3)2), 86.6 (C-1), 79.7, 79.2, 76.7, 73.4 (C-2, C-3,
C-4, C-5), 71.7 (OCH2C3H6CH2N3), 63.0 (C-6), 51.4
(OCH2C3H6CH2N3), 29.5, 28.6 (2 × CH2), 28.0, 26.8, 26.6, 26.3
(C(CH3)3, C(CH3)2), 23.3 (CH2), 19.2 (C(CH3)3). HRMS calcd
for C36H47N3O5SiS [M+Na]+: 684.2904, found 684.2908.
Phenyl 2-O-(5-Azapentyl)-1-thio-ꢀ-D-galactopyranoside 10.
To a solution of 13 (1.50 g, 2.27 mmol) in THF (30 mL) was
added a 1 M solution of tetrabutylammonium fluoride (4.50 mL,
2 equiv). The reaction mixture was stirred at room temperature
for 4 h. Upon completion of the reaction, the reaction mixture
was diluted with EtOAc (20 mL) and washed successively with
water (20 mL) and brine (20 mL). The organic layer was dried
over anhydrous Na2SO4 and evaporated. The residue was then
dissolved in CH2Cl2, and TFA (5 mL) was added. The mixture
was stirred at room temperature for 2 h, after which time TLC
analysis indicated that the reaction was complete. The solvents
were removed in vacuo, and the residue was purified by flash
chromatography using EtOAc to give the desired product as a
Figure 2. CD1d agonist KRN7000 and analogues.
of iNKT cells and for which there are a number of important
immunologic tools available.
EXPERIMENTAL PROCEDURES
1
General Experimental Procedures. H NMR spectra were
recorded at 400 or 300 MHz, using Bruker AMX 400, Bruker
AV 400, Bruker AV 300, and Bruker AC 300 spectrometers.
13C NMR spectra were recorded at 100 or 75 MHz, respectively,
using Bruker AMX 400, Bruker AV 400, Bruker AV 300, and
Bruker AC 300 spectrometers. Chemical shifts are reported as
δ values (ppm) referenced to the following solvent signals:
CHCl3, δ H 7.26; CDCl3, δ C 77.0; CH3OH, δ H 3.34; CD3OD,
δ C 49.9. Quaternary carbons were reported only when
observed. HRMS were recorded on a Micromass LCT spec-
trometer using a lock mass incorporated into the mobile phase.
All reagents were obtained from commercial sources and were
used without further purification unless stated otherwise.
Anhydrous solvents were purchased from Sigma-Aldrich, U.K.,
stored over 4 Å molecular sieves and under an argon atmo-
sphere. Analytical thin layer chromatography (TLC) was
performed on aluminum plates precoated with Merck silica gel
60A F-254 as adsorbent. The developed plates were air-dried,
visualized by UV detection (at 254 nm) and/or stained with 5%
phosphomolybdic acid in EtOH (MPA spray). Compounds were
purified by flash column chromatography on Merck silica gel
(particle size 40-63 lm mesh) or Fluka 60 (40-60 lm mesh)
silica gel.
Phenyl 3,4-O-Isopropylidene-6-O-tert-butyldiphenylsilyl-2-
O-(5-azapentyl)-1-thio-ꢀ-D-galactopyranoside 13. To a cold
solution of phenyl 3,4-O-isopropylidene-6-O-tert-butyldiphe-
nylsilyl-1-thio-ꢀ-D-galactopyranoside 12 (2.00 g, 3.64 mmol)
in dry DMF (30 mL) was added sodium hydride (0.44 g, 18.2
mmol, 5 equiv) in small amounts at 0 °C. The solution was
allowed to stir for 20 min before the addition of 1,5-dibro-
mopentane (4 mL, 18.2 mmol, 5 equiv). The reaction mixture
was stirred at room temperature for two hours, and then taken
up in water (50 mL). The resulting mixture was then extracted
with CH2Cl2 (3 × 20 mL). The combined organic layers were
washed with brine (30 mL), dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude bromide was further dissolved
in DMSO (30 mL) and sodium azide (1.20 g, 18.2 mmol, 5
equiv) was added. The reaction was stirred overnight at 60 °C,
after which it was taken up in water (50 mL) and extracted
with CH2Cl2 (3 × 20 mL). The combined organic layers were
then washed with brine (30 mL), dried over anhydrous Na2SO4,
1
colorless oil (0.68 g, 1.78 mmol, 78%). H NMR (CDCl3): δ
7.48-7.54 (2H, m, Ar-H), 7.26-7.35 (3H, m, Ar-H), 4.59 (1H,
d, H-1, J1,2 ) 9.7 Hz), 4.06 (1H, d, J4,3 ) 3.1 Hz, H-4), 3.95
(1H, dd, J6a,6b ) 11.9, J6a,5 ) 5.9 Hz, H-6a), 3.90 (1H, dd, H-6b),
3.80-3.69 (2H, m, OCH2C3H6CH2N3), 3.63 (1H, dd, J3,2 ) 8.9
Hz, H-3), 3.58 (1H, m, H-5), 3.45 (1H, t, H-2), 3.28 (2H, t,
OCH2C3H6CH2N3), 1.56-1.71 (4H, m, CH2), 1.07 (2H, s, CH2).
13C NMR (75 MHz, CDCl3): δ 134.0, 131.1, 129.0, 127.4 (6
CAr), 87.7 (C-1), 78.5, 77.9, 75.1, 69.8 (C-2, C-3, C-4, C-5),
73.2 (OCH2C3H6CH2N3), 62.6 (C-6), 51.4 (OCH2C3H6CH2N3),
29.7, 28.6 (2 × CH2), 23.2 (CH2), 19.2 (C(CH3)3). HRMS calcd
for C17H25N3O5S [M+Na]+: 406.1413, found 406.1426.
Phenyl 3-O-Benzyloxy-4,6-O-di-tert-butylsilylene-2-O-(5-aza-
pentyl)-1-thio-ꢀ-D-galactopyranoside 8. To a solution of 10 (0.52
g, 1.36 mmol) in dry DMF (15 mL) was added di-tert-butylsilyl
bis(trifluoromethanesulfonate) (0.90 g, 2.04 mmol, 1.5 equiv)
at 0 °C, and the mixture was stirred for 30 min, then neutralized
with Et3N. The solution was taken up in water and extracted
with EtOAc (3 × 20 mL). The organic layers were combined,
washed with brine (20 mL), dried over anhydrous Na2SO4, and
evaporated. The residue was then dissolved in pyridine (20 mL)
and benzoyl chloride (0.27 g, 1.92 mmol, 1.4 equiv) was added.
The mixture was stirred at room temperature for 2 h, after which
time TLC analysis indicated that the reaction was complete.
The reaction was quenched with methanol, diluted with CH2Cl2
(30 mL), washed successively with 1 M HCl (3 × 20 mL),
water (2 × 20 mL), brine (20 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was finally
purified by flash chromatography using hexanes/EtOAc (4:1)
to give the desired product as a colorless oil (0.82 g, 1.31 mmol,
96%). 1H NMR (CDCl3): δ 8.06-8.13 (2H, m, Ar-H), 7.41-7.65
(6H, m, Ar-H), 7.27-7.34 (2H, m, Ar-H), 5.03 (1H, dd, J3,2
)
9.4, J3,2 ) 3.1 Hz, H-3), 4.69-4.75 (2H, m, H-1, H-4),
4.22-4.28 (2H, m, H-6a, H-6b), 3.82-3.94 (2H, m, H-2,
OCH2C3H6CH2N3), 3.66 (1H, ddd, J1a′,1b′ ) 9.0, J1a′,2a′ ) J1a′,2b′

R-Galactosyl Ceramide Haptenated-Lipid Antigen
Bioconjugate Chem., Vol. 21, No. 4, 2010 743
) 6.2 Hz, OCH2C3H6CH2N3), 3.50 (1H, s, H-5), 3.05-3.18 (2H,
m, OCH2C3H6CH2N3) 1.26-1.57 (4H, m, CH2), 1.09 (2H, s,
CH2), 1.16, 0.97 (18H, 2s, 2 × t-Bu). 13C NMR (75 MHz,
CDCl3): δ 133.2, 132.0, 131.9, 129.6, 128.8, 128.5, 127.4 (12
CAr), 88.5 (C-1), 77.3 (C-3), 76.7, 74.6, 70.6 (C-2, C-4, C-5),
73.3 (OCH2C3H6CH2N3), 67.2 (C-6), 51.2 (OCH2C3H6CH2N3),
29.7, 28.5 (2 × CH2), 27.7, 27.4 (2 × C(CH3)3), 23.3, 20.7 (2
× C(CH3)3). HRMS calcd for C32H45N3O6SiS [M+Na]+:
650.2696, found 650.2693.
J4,3 ) 3.1 Hz, H-4), 3.70 (1H, dd, J1a′,1b′ ) 10.9, J1a′,2a′ ) 4.4
Hz, H-1aCer), 3.65 (1H, dd J3,2 ) 9.8 Hz, H-3), 3.54-3.62 (5H,
m, H-1bCer, H-2Cer, H-3Cer, H-4Cer, H-5), 3.43 (1H, dd, H-2),
3.31-3.37 (2H, m, H-6a, H-6b), 3.20 (2H, ddd, J1a′,1b′ ) 8.9,
J1a′,2a′ ) J1a′,2b′ ) 6.8 Hz, OCH2C3H6CH2N3), 2.02-2.11 (2H,
m, OCH2C3H6CH2N3), 1.50 (9H, s, NHCO2C(CH3)3) 1.35-1.60
(m, 8H, CH2), 1.22-1.32 (2H, m, CH2), 1.20-1.23 (68H, m,
CH2), 0.71 (6H, t, J ) 6.8 Hz, CH3). 13C NMR (75 MHz,
CDCl3): δ 98.1 (C-1), 78.2, 76.1, 75.2, 71.4, 70.2, 69.2 (C-2,
C-3, C-4, C-5, C-4Cer, C-3Cer), 71.2 (OCH2C3H6CH2N3), 68.2
(C-1Cer), 61.4 (C-6), 51.3 (OCH2C3H6CH2N3), 50.1 (C-2Cer),
36.5-22.0 (17 × CH2), 27.5, 27.3 (2 × C(CH3)3), 14.0 (CH3Cer).
HRMS calcd for C34H66N4O10[M+Na]+: 713.4677, found
713.4676.
(2S,3S,4R)-2-N-tert-Butoxycarbonylamino-3,4-di(benzyloxy)1-
[3-O-benzyloxy-4,6-O-di-tert-butylsilylene-2-O-(5-azapentyl)-r-
D-galactopyranosyloxy)]-octadecane 14. A suspension of 10
(0.75 g, 1.20 mmol), 9 (0.90 g, 1.44 mmol, 1.2 equiv), silver
triflate (0.18 g, 2.4 mmol), and 4 Å molecular sieves in
anhydrous CH2Cl2 (10 mL) was stirred, with the exclusion of
light for 10 min at room temperature under Ar before it was
cooled to -78 °C. A solution of p-nitrobenzenesulfenyl chloride
(0.24 g, 1.20 mmol, 95% purity) in anhydrous CH2Cl2 (2 mL)
was dropped into the above suspension at -78 °C. The reaction
mixture was maintained at -78 °C for 30 min and allowed to
warm up to 0 °C over the following 30 min, after which TLC
analysis showed that the reaction was complete. The reaction
was quenched with saturated NaHCO3 (2 mL) and diluted with
CH2Cl2 (5 mL) to give a suspension, which was filtered through
Celite, and the Celite pad was washed with CH2Cl2 (10 mL).
The filtrate was separated, and the organic phase was dried over
anhydrous Na2SO4 and evaporated. The residue was purified
by flash chromatography using hexanes/EtOAc (5:1) to give the
glycosylated compound 14 as a light yellow oil (1.02 g, 0.89
(2S,3S,4R)-2-Amino-N-hexacosanoyl-1-[2-O-(5-azapentyl)-r-
D-galactopyranosyloxy)]-3,4-octadecanediol 16. Compound 15
(0.38 g, 0.550 mmol) was dissolved in CH2Cl2/MeOH (2:1) (10
mL) and TFA (2 mL) was added. The reaction was stirred for
2 h and concentrated in vacuo to give the crude amine which
was used in the next step without purification. The crude amine
was then dissolved in a 1:1 mixture of THF/NaOAC (saturated
solution) (5 mL) and the freshly prepared acid chloride of
hexacosanoic acid (0.25 g, 0.603 mmol, 1.1 equiv) was added.
The reaction was allowed to stir overnight at room temperature.
The organic phase was then concentrated, and the residue was
purified by flash chromatography (gradient from CHCl3 to 15%
MeOH in CHCl3) to give the acylated compound 16 as a white
solid (0.38 g, 0.39 mmol, 71%). 1H NMR (CDCl3/CD3OD 2:1):
δ 4.86 (1H, d, J1,2 ) 3.6 Hz, H-1), 3.79 (1H, d, J4,3 ) 3.2 Hz,
H-4), 3.72 (1H, dd, J1a′,1b′ ) 10.9, J1a′,2a′ ) 4.6 Hz, H-1aCer),
1
mmol, 75%). H NMR (CDCl3): δ 7.89-8.13 (6H, m, Ar-H),
7.32-7.64 (12H, m, Ar-H), 5.64 (1H, dd, J3,2 ) 8.5, J3,4 ) 3.4
Hz, H-3Cer), 5.39-5.52 (2H, m, NHBOC, H-4Cer), 5.24 (1H,
dd, J3,2 ) 10.3, J3,2 ) 3.1 Hz, H-3), 5.00 (1H, d, J1,2 ) 3.5 Hz,
H-1), 4.27 (1H, d, H-4), 4.06-4.32 (3H, m, H-6a, H-6b, H-2Cer),
4.00 (1H, dd, H-2), 3.73-3.84 (2H, m, H-5, H-1aCer), 3.60-3.71
3.64 (1H, dd J3,2 ) 9.8 Hz, H-3), 3.52-3.60 (5H, m, H-1bCer
,
H-2Cer, H-3Cer, H-4Cer, H-5), 3.43 (1H, dd, H-2), 3.31-3.37 (2H,
m, H-6a, H-6b), 3.20 (2H, ddd, J1a′,1b′ ) 8.9, J1a′,2a′ ) J1a′,2b′
6.8 Hz, OCH2C3H6CH2N3), 2.00-2.10 (2H,
)
m,
OCH2C3H6CH2N3), 1.36-1.59 (m, 8H, CH2), 1.25-1.35 (2H,
m, CH2), 1.20-1.24 (68H, m, CH2), 0.73 (6H, t, J ) 6.8 Hz,
CH3). 13C NMR (75 MHz, CDCl3): δ 97.7 (C-1), 77.2, 75.2,
72.1, 70.4, 69.6, 69.3 (C-2, C-3, C-4, C-5, C-4Cer, C-3Cer), 71.2
(OCH2C3H6CH2N3), 67.4 (C-1Cer), 61.5 (C-6), 51.1
(OCH2C3H6CH2N3), 49.8 (C-2Cer), 36.2, 32.9, 31.6, 29.4, 29.1,
28.3, 25.6, 22.8, 22.4 (CH2), 13.7(CH3Cer). HRMS calcd for
C55H108N4O9 [M+Na]+: 991.8014, found 991.8018.
(2H, m, OCH2C3H6CH2N3, H-1bCer), 3.44 (1H, ddd, J1a′,1b′
)
8.9, J1a′,2a′ ) J1a′,2b′ ) 6.6 Hz, OCH2C3H6CH2N3), 2.84-3.06
(2H, m, OCH2C3H6CH2N3), 1.80-2.02 (2H, m, H-5aCer, H-5bC-
er), 1.49 (9H, s, NHCO2C(CH3)3), 1.15-1.13 (24H, m, CH2),
1.06-1.14 (2H, m, CH2), 0.94-098 (2H, m, CH2), 1.03, 0.92
(18H, 2s, 2 × t-Bu), 0.87 (3H, t, CH3). 13C NMR (75 MHz,
CDCl3): δ 133.3, 132.9, 133.2, 132.0, 131.9, 129.9, 129.7, 129.6,
129.4, 128.8, 128.5, 127.4 (18 CAr), 97.4 (C-1), 80.3 (NH-
COOC(CH3)3), 77.1 (C-3), 76.5, 74.8, 70.2 (C-2, C-4, C-5), 73.8
(C-3Cer), 73.2 (OCH2C3H6CH2N3), 71.8 (C-4Cer), 67.0 (C-6), 61.7
(C-1Cer), 51.2 (OCH2C3H6CH2N3), 49.9 (C-2Cer), 32.1-22.8 (13
× CH2Cer), 29.8, 28.4 (2 × CH2), 27.6, 27.3 (2 × C(CH3)3),
23.1, 20.5 (2 × C(CH3)3), 14.8 (CH3Cer). HRMS calcd for
C63H94N4O13Si[M+Na]+: 1165.6484, found 1165.6488.
(2S,3S,4R)-2-Amino-N-hexacosanoyl-1-[2-O-(5-aminopentyl)-
r-D-galactopyranosyloxy)]-3,4-octadecanediol 3. Compound 16
(0.32 g, 0.33 mmol) was dissolved in MeOH (10 mL) and stirred
with Pd-C (5 mg) under H2 overnight, after which time it was
filtered through a pad of Celite, which was subsequently washed
with MeOH. The combined filtrates were concentrated and the
target compound 3 was obtained as a white solid (0.28 g, 0.297
mmol, 90%). The crude amine was used without any further
purification in the following acylation reactions. Acylation to
give compound 3: (3-hydroxy-4-nitrophenyl) acetic acid (17
mg, 0.09 mmol) was added to neat oxalyl chloride (1 mL) and
stirred at 70 °C for 2 h, after which time the solution was cooled
to rt and the unreacted oxalyl chloride was removed under a
stream of argon. The residual volatiles were removed under
reduced pressure. The resulting crude acyl chloride was dis-
solved in THF (1 mL) and added to a solution of amine 3 (50
mg, 0.05 mmol) in THF/NaOAc(sat) (1:1, 1 mL). The reaction
was stirred vigorously overnight after which the organic phase
was removed. The aqueous phase was further extracted with
THF (2 × 1 mL), and the combined organic phases were
concentrated. The residue was finally purified by flash chro-
matography (gradient from CHCl3 to 15% MeOH in CHCl3) to
give the acylated compound 17 as a yellow solid (42 mg, 0.39
(2S,3S,4R)-2-N-tert-Butoxycarbonylamino-1-[2-O-(5-azapen-
tyl)-r-D-galactopyranosyloxy)]-octadecanediol 15. To a solution
of 14 (0.93 g, 0.814 mmol) in THF was added a 1 M solution
of tetrabutylammonium fluoride (2 mL, 2 equiv). The reaction
mixture was stirred at room temperature for 6 h. Upon
completion of the reaction, the reaction mixture was diluted with
EtOAc (20 mL) and washed successively with water (20 mL)
and brine (20 mL). The organic layer was dried over anhydrous
Na2SO4 and evaporated. The residue was then dissolved in
MeOH and a 1 M solution of NaOMe (5 mL) was added. The
mixture was stirred at room temperature for 2 h, after which
time TLC analysis indicated that the reaction was complete.
The reaction was neutralized by the addition of Dowex
50WX8-200 resin. The latter was then filtered and the filtrate
concentrated to give a residue, which was purified by flash
chromatography using EtOAc/MeOH (7:1) to give the title
compound as a colorless syrup (0.49 g, 0.710 mmol, 88%). 1H
NMR (CDCl3): δ 4.90 (1H, d, J1,2 ) 3.8 Hz, H-1), 3.81 (1H, d,
1
mmol, 71%). H NMR (CDCl3/CD3OD 2:1): δ 7.85 (1H, d, J
) 2.4 Hz), 7.36 (1H, dd, J ) 8.6, J ) 2.4 Hz), 6.90 (1H, d, J

744 Bioconjugate Chem., Vol. 21, No. 4, 2010
Veerapen et al.
) 8.6 Hz), 4.81 (1H, d, J1,2 ) 3.6 Hz, H-1), 3.74 (1H, d, J4,3
)
C-4Cer, C-3Cer), 71.1 (OCH2C3H6CH2NHCO), 63.1 (C-1Cer), 60.4
3.1 Hz, H-4), 3.25-3.69 (10H, m, H-1aCer, H-1bCer, H-2Cer
,
(C-6),
49.9
(C-2Cer),
42.0
(COCH2Ar),
39.5
H-3Cer, H-4Cer, H-2, H-3, H-5, H-6a, H-6b), 3.00 (2H, t, J1a′,2a′
) J1a′,2b′ ) 6.4 Hz, OCH2C3H6CH2NHCO), 2.00-2.06 (2H, m,
OCH2C3H6CH2NHCO), 1.32-1.50 (m, 8H, CH2), 1.20-1.24
(68H, m, CH2), 0.68 (6H, t, J ) 6.5 Hz, CH3). 13C NMR (75
MHz, CDCl3): δ 173.2 (CdO), 138.8, 125.1, 120.4 (CAr), 98.1
(C-1), 77.3, 75.2, 72.9, 70.5, 69.8, 69.2 (C-2, C-3, C-4, C-5,
(OCH2C3H6CH2NHCO), 31.9-21.1 (CH2), 14.1 (CH3Cer). HRMS
calcd for C63H115N3O13 [M+Na]+: 1144.8328, found 1144.8326.
Mice. All mice were maintained in Dana Farber Animal
Facility according to IACUC guidelines. C57BL/6 WT were
obtained from Jackson Laboratories. C57BL/6 VR14 Tg mice
(iNK T TcR Tg) created by A. Bendelac (University of Chicago,
Chicago) were provided by Mark Exley (Beth Israel Hospital,
Boston). C57BL/6.SJL congenic B1-8hi B cell receptor knock-
in mice, previously created by insertion of a high-affinity NP-
specific BcR transgene into the BCR coding region to maintain
class switch components, were provided by M. Nussenzweig
(Rockefeller University, New York). Mice were immunized as
noted with 200 µL intraperitoneally (IP) in PBS/0.1%BSA.
Serum was collected by retro-orbital bleed and stored at -20
°C until evaluation.
Scheme 1. Synthesis of Compound 4
Murine Serum Antibody ELISA. NP-conjugated antigen
challenge induces a heteroclitic response, where the resulting
antibodies have higher affinity for NIP, so antibody is detected
with an NIP-specific ELISA. Plates (Immulon 2 HB) were
coated with 1 mg NIP (5)-OVAL in PBS and blocked with 10%
soy milk/0.05% Tween/PBS. Serum was serially diluted in 0.1%
soy milk/PBS and antibody detected with HRP-labeled goat
antimouse IgG, IgM (Southern Biotech) developed with ABTS
(Sigma A-1888). Concentration was extrapolated from IgG anti-
NP (clone Pevchγ1) or IgM anti-NP (clone J558) (provided by
A. Ferguson, Boston University School of Medicine, Boston)
standard curves.
Scheme 2. Retrosynthetic Ananlysis of Target Compound 3
In Vitro Proliferation Assay. B and T cells were purified
by pan-B or pan-T MACS bead separation (Milteny-Biotec)
according to the manufacturer’s instructions. iNK T TcR Tg
total splenic T cells were approx 40% iNK T cells (data not
shown). Purified B and T cells were mixed at 1:1 ratio (1 ×
105 cells per well each) and labeled with 0.5 µM CFSE (Sigma
21888) for 9 min in PBS, then quenched with FCS and washed
extensively before culture. Proliferation was assessed by FACs
as CFSE dilution on day 3. Murine-specific antibodies were anti-
CD19 PerCP-Cy5.5 (1D3), anti-Thy1.2 APC(53-2.1), NA/LE
anti-CD3 (145-2C11), and isotype controls (all BD Biosciences
PharMingen). Cells were preblocked with unlabeled anti-
FcγRIII, II (clone 2.4G2).
RESULTS AND DISCUSSION
Compound 4 was synthesized as reported via a pseudogly-
cosylation reaction of compound 6 with a suitably protected
R-GalCer 5 (Scheme 1) (17). In this route, the six-carbon linker
was first attached to the hapten (p-nitrophenol) before being
added onto the sugar residue. While this remains an effective
strategy for synthesizing derivatives of R-GalCer, we have
observed that glycosylation reactions at this slightly hindered
C-2 position can sometimes be tricky and dependent upon
various factors, such as the reactivity of the donor, thus offering
Scheme 3. a
a (a) C4H9Si(C6H5)2Cl, Pyridine, quant; (b) (BOC)2O, CH3CN, Et3N, 72%; (c) BzCl, Pyridine, 84%; (d) TBAF, THF, 82%.

R-Galactosyl Ceramide Haptenated-Lipid Antigen
Bioconjugate Chem., Vol. 21, No. 4, 2010 745
Scheme 4. a
Scheme 6. a
a (a) THF/NaOAc (1:1), 71%.
a (a) DMF, NaH, (CH2)5Br2, quant; (b) NaN3, DMSO, 60 °C, 75%;
(c) TBAF, THF, quant; (d) TFA, CH2Cl2, 78%; (e) Si(OTf)2(C4H9)2,
DMF, Et3N, quant; (f) BzCl, Pyridine, 96%.
The phytosphingosine acceptor 9 (22) was synthesized from
(2S,3S,4R)-2-amino-1,3,4-octadecanetriol using standard pro-
cedures summarized in Scheme 3. The spectroscopic data of
compound 9 matched those previously reported (22). Regarding
the synthesis of 10, an appropriately protected galactosyl
derivative had to be carefully chosen at the outset. Importantly,
the protecting groups should withstand the basic reaction
conditions for the formation of an alkoxide at C-2. In galactose,
the cis-orientation of the hydroxyl groups at C-3 and C-4
facilitate their simultaneous protection as the acetonide, while
the primary hydroxyl group at C-6 can be selectively blocked
using diphenyltertiarybutylsilyl chloride, thereby leaving C-2
free for modification.
Scheme 5. a
The crucial intermediate 12 was thus synthesized from ꢀ-D-
galactopyranose pentaacetate according to published report (23).
The alkoxide of 12, which was generated in situ by treatment
with NaH in DMF, was then reacted with commercially
available 1,5-dibromopentane to afford the bromide. The amine
functional group was then introduced into the molecule by an
SN2 displacement of the bromide with sodium azide in DMSO.
With the linker in place at C-2, we then proceeded to the
preparation of the glycosyl donor 8. The protecting groups were
sequentially removed to give 10 in quantitative yields. Finally,
introduction of the R-directing bulky DTBS group at C-4 and
C-6, followed by benzoylation at C-3, gave compound 8 as
colorless syrup in 96% yield after purification.
With both the donor 8 and the acceptor 9 in hand, we next
turned our attention to the glycosylation reaction. Because of
the possible cleavage of the acid-sensitive BOC group on the
acceptor, we preferred avoiding the usual method of activation
of the thioglycoside with NIS/TfOH. When using Crich’s (24)
relatively new method of activation with the commercially
available p-nitrobenzenesulfenyl chloride (p-NO2PhSCl) in
conjunction with silver triflate in anhydrous CH2Cl2 at -78 °C
over 2 h, we were pleased to observe that the desired glycoslated
product 14 was obtained as the R-anomer exclusively in 75%
yield after purification by flash chromatography. The formation
of the R-linkage was confirmed by the H-1 and C-1 signals in
1H and 13C NMR, respectively.
Stepwise deprotection of compound 14 was then carried out.
To avoid any probable base-induced migration of the benzoate
to the free amine, we chose to remove the BOC protecting group
on the aglycon last. Hence, treatment with TBAF and Zemplen’s
deprotection of the benzoate protecting groups produced the
intermediate 15 as a thick syrup in quantitative yields. The BOC
group was subsequently cleaved with TFA and the resulting
free amine was acylated with the fully saturated fatty acid,
hexacosanoic acid. This was achieved by the reaction of the
corresponding acid chloride with the free amine in a 1:1 mixture
of THF and saturated sodium acetate solution. The azide
intermediate 16 was obtained as a white solid after concentration
of the organic phase and purification of the residue by flash
a (a) p-NO2PhSCl, AgOTf, CH2Cl2, -78 °C, 75%; (b) TBAF, THF,
quant; (c) NaOMe/MeOH, 88%; (d) TFA, CH2Cl2, quant; (e)
C25H51COCl, THF/NaOAc (1:1), 71%; (f) H2, Pd, MeOH, 90%.
limited scope for hapten diversity. Therefore, we reason that
the introduction of a linker into the sugar residue via a reaction
other than glycosylation is worth exploring. Also, in the interest
of adaptability, the linker should facilitate the introduction of
different haptens or other molecules via simple and diverse
reactions.
Retrosynthetic analysis (Scheme 2) indicated that target
compound 3 can be obtained by acylation of compound 7, which
in turn can be accessed from the coupling of sugar donor 8
with the sphingosine derivative 9. This route is quite efficient
as it entails the introduction of the linker at C-2 prior to
glycosylation via an alkylation reaction, thereby eliminating the
complications discussed above. It is noteworthy that compound
7 bears amine functionalities on both the sugar and the
sphingosine base moieties. Judicious orthogonal protection of
the two amino groups is therefore required, as they are to be
acylated with different carboxylic acids at distinct stages in the
course of the synthetic route. Since the sugar moiety is to be
subjected to a wider range of chemical reactions, we chose to
protect the amino group as the corresponding azide. The latter
is known to be very stable and compliant to diverse reaction
conditions. As for the amino group on the sphingosine moiety,
we opted for the acid-sensitive BOC protecting group, which
is also compatible with the benzoate protecting group. To ensure
R-selectivity in the crucial glycosylation step, we relied on the
directing effect of the bulky 4,6-O-di-tert-butylsilylene (DTBS)
group on the sugar moiety (21).

746 Bioconjugate Chem., Vol. 21, No. 4, 2010
Veerapen et al.
Figure 3. NP-haptenated derivative (4) of RGalCer, but not RGalCer, stimulates in vivo production of NP-specific IgM (left panel) and IgG (right
panel) by B1-8 BcR Tg mice. (n ) 2-4 mice/group, representative example from more than 2 experiments).
chromatography. Ultimately, hydrogenation of the azide in
methanol provided the desired scaffold 2. Conjugation of the
amine with the NP hapten was then accomplished by acylation
with its corresponding activated carboxylic acid group. Com-
pound 3 was obtained after stirring freshly prepared acid chloride
of the (3-hydroxy-4-nitrophenyl) acetic acid with 2 in a 1:1
mixture of THF and saturated sodium acetate solution for 12 h
(Scheme 6).
Following successful synthesis, the NP-derivatives (3 and 4)
were tested in ViVo and in Vitro for recognition by iNKT and B
cells. Mice expressing an increased frequency of B cells with
an NP-specific B cell receptor (B1-8 BcR Tg) were found to
develop high titers of both IgM and IgG anti-NP antibodies in
their serum (Figure 3) when immunized with 4, but not RGalCer
or PBS/0.1%BSA. Because T cell help is required for class
switch to IgG, the presence of NP-specific IgG antibodies
following immunization with 4 suggests T cell help is being
provided, most likely by iNKT cells recognizing the R-GalCer
component of the compound (Figure 1). In depth investigations
of this immunologic cooperation have been published elsewhere
(17). Furthermore, the requirement of iNKT activation and
Figure 4. Alternatively synthesized NP-haptenated compounds 3 and
response for both the specific antibody production and class-
switch was demonstrated by Batista et al. (25) in their in ViVo
studies using R-GalCer conjugated with the antigen CGG
(chicken gamma globulin). Therefore, it can be inferred that
iNKT cells help anti-lipid antibody production.
4 stimulate similar levels of iNKT and B cell proliferation. Mixtures
of T cells from VR14 Tg mouse and B cells from B1-8 BcR Tg mouse
were stimulated with 10-0.01 ng/mL NP-RGalCer (top panel), or
media, 1 µg/mLLPS, 10 µg/mL anti-CD3, 10 ng/mL RGalCer as
controls (bottom panel). Proliferation is measured as % B and T cells
diluting CFSE. Statistics performed by Prism software ANOVA analysis
(* ) p < 0.05 for 2 vs 17). Each bar represents the mean of pools of
triplicate wells from two experiments.
The biological functions of 3 and 4 haptenated with NP (3-
hydroxy-4-nitrophenyl) using the two different approaches
described above were then compared in vitro. To compare
functional recognition of 3 and 4 by iNKT cells and B cells,
isolated cell populations were mixed, labeled with CFSE,
incubated in vitro, and then assessed for proliferation after three
days of culture. Proliferation in response to control stimuli
(Figure 4, lower panel) confirms the specificity of the assay,
since only B cells respond to the B cell stimulus, LPS
(Salmonella enterica, Sigma); iNKT cells respond more strongly
to the T cell stimulus, anti-CD3; and neither cell type responds
to the media alone. iNKT cells respond to RGalCer, and while
there is no selective advantage for NP-specific B cells, there
are likely some B cells specific for R-GalCer in any population
which contribute to the low level of proliferation observed in
the RGalCer stimulated group. Our data show that proliferative
responses by iNKT cells and B cells were predominantly
comparable following stimulation with 3 and 4 despite their
slight structural differences. (Figure 4, upper panel). Importantly,
previous in vitro experiments (17) showed that NP-R-GalCer 4
stimulated 3.2 µg/mL anti-NP IgG by day 7, whereas R-GalCer
stimulated <0.05 µg/mL IgG anti NP, clearly indicating that
both 3 and 4 induce substantial proliferation of both B and T
cells.
CONCLUSION
We have described the synthesis of a highly biologically
attractive glycolipid template 2, which can be conjugated to
multiple molecules, exemplified by the synthesis of compound
3. Furthermore, the biological evaluation of the haptenated
derivatives of R-GalCer showed that the biologic recognition
of 3 and 4 is virtually the same and that they are both important
tools in studying the mechanism of iNKT cell help for the
production of anti-lipid antibodies.
ACKNOWLEDGMENT
We thank Michel Nussenzweig and Mark Exley for gener-
ously providing transgenic mice. This work was supported by
the Irvington Institute for Immunological Research (E.A.L.) and
National Institutes of Health Grant T32 AR007530-21(to

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