Synthesis of C6′′-modified α-C-GalCer analogues as mouse and human iNKT cell agonists

Article abstract of DOI:10.1039/c7ob00081b

α-GalCer analogues that combine known Th1 polarizing C6′′-modifications with a C-glycosidic linkage were synthesized. We employed a protecting group strategy that allowed the preparation of both saturated and unsaturated derivatives with variable C6′′-substituents. Selected analogues demonstrate promising activity in mice. Interestingly, the introduction of a 6′′-O-pyridinylcarbamoyl substituent to α-C-GalCer restores its antigenicity in human iNKT cells.

Full text of DOI:10.1039/c7ob00081b

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2017, DOI: 10.1039/C7OB00081B.
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Synthesis of C6”-modified α-C-GalCer analogues as mouse and
human iNKT cell agonists
Joren Guillaume,a Toshiyuki Seki,b Tine Decruy,c,d Koen Venken,c,d Dirk Elewaut,c,d Moriya Tsujib
Received 00th January 20xx,
Accepted 00th January 20xx
and Serge Van Calenbergha,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
α-GalCer analogues were synthesized that combine known Th1 Glycolipid synthesis groups furnished a plethora of α-GalCer
polarizing C6”-modifications with a C-glycosidic linkage. We analogues1 and to date, the best known Th1-polarizing
employed a protecting group strategy that allowed the preparation glycolipid is the C-glycoside analogue of α-GalCer, α-C-GalCer
of both saturated and unsaturated derivatives with variable C6”-
(2
),4 which shows increased activity against malaria and B16-
substituents. Selected analogues demontrate promising activity in melanoma in mice.5 The prolonged secretion of Th1-cytokines
mice. Interestingly, introduction of a 6”-O-pyridinylcarbamaoyl like IFN-γ and IL-12 forms the basis of the observed Th1-profile6
substituent to α-C-GalCer restores its antigenicity in human iNKT and is likely due to the increased metabolic stability of α-C-
cells.
GalCer. Unfortunately, in contrast to α-GalCer, α-C-GalCer is a
weak antigen for human iNKT cells. However C-glycosides
having an E-alkene linker between the galactose sugar and the
ceramide do stimulate human iNKT cells.7
Introduction
We have previously shown that introducing aromatic urea or
carbamate substituents at the C6”-position of the
galactopyranose unit, can result in powerful Th1-biasing iNKT-
In the past two decades immunogenic glycolipids have gained
increasing interest due to their potential as vaccine adjuvants to
fight microbial infections and cancer, as well as for the
treatment for autoimmune disorders.1 In this context, the most
extensively studied glycolipid is α-galactosylceramide (α-
cell antigens. The Th1-bias observed for α-NU-GalCer (3) arises
from an increased stability of the CD1d-glycolipid complex as
the naphthyl moiety induces and occupies an additional
hydrophobic pocket in CD1d, thereby enhancing the affinity.8
The pyridine ring of PyrC-α-GalCer (4) on the other hand makes
extra contacts with the TCR, thereby enhancing the stability of
the ternary complex.9
GalCer, 1, Figure 1), which consist of a ceramide moiety that is
α-anomerically coupled to a galactopyranosyl moiety. α-GalCer
is known to bind with high specificity to CD1d, a protein present
on antigen presenting cells.2 Upon recognition of the CD1d-α-
GalCer complex by the T cell receptor (TCR) of invariant natural
killer T cells (iNKT cells) large amounts of both pro-inflammatory
T-helper 1 (Th1) and anti-inflammatory Th2 cytokines are
secreted. The concomitant release of both cytokine types,
which have opposing roles in vivo, is believed to limit the clinical
outcome of α-GalCer therapy.3 Consequently, potent analogues
able to skew the cytokine response towards either Th1 or Th2
are of great interest.
a.Laboratory for Medicinal Chemistry (FFW), Faculty of Pharmaceutical Sciences,
UGent, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
b.Aaron Diamond AIDS Research Center, Affiliate of The Rockefeller University, New
York, New York, USA
c. Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent
University, B-9000 Ghent, Belgium.
d.VIB Inflammation Research Center, Ghent University, Ghent, Belgium
e. * To whom correspondence should be addressed. E-mail:
serge.vancalenbergh@ugent.be; Fax: +32 9 264 81 46; Tel: +32 9 264 81 24.
Electronic Supplementary Information (ESI) available: 1H- and 13C-NMR spectra
of new compounds + additional figures. See DOI: 10.1039/x0xx00000x
Figure 1. Structures of glycolipids
1
to
6
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The idea of combining the above mentioned C6”-modifications Next, the acetate group was removed with ammonia in
with a C-glycosidic linkage seems obvious, yet no synthetic methanol and the alcohol was protected as PMB-ether.
strategy has been developed to date with the exception of a Ozonolysis of 9 and in situ reduction of the product with sodium
C6”-methoxy analogue featuring an E-alkene connection borohydride gave primary alcohol 10, which was reoxidized to
together with a modified phytosphingosine lacking the 4- the desired galactose formaldehyde enabling Julia-Kocienski
hydroxy.10 Here, we report a divergent synthetic route towards olefination with the known benzothiazole 11 11
.
Unfortunately
C6”-modified α-C-GalCer derivatives 5a
metabolically stable iNKT cell agonists.
,b and 6a,b as new the ozonolysis/reduction sequence was poorly reproducible as
only trace amounts of desired alcohol were obtained in most
attempts. Olefin 12 was obtained without epimerization at the
anomeric center and deprotection of the PMB-ether was
accomplished with ceric ammonium nitrate at low temperature
and short reaction time to avoid unwanted acetonide cleavage.
The primary alcohol was converted to an azide by means of a
Mitsunobu reaction with DPPA, followed by deprotection of the
acid labile protecting groups with TFA and acylation of the
resulting amine with the p-nitrophenyl ester of cerotic acid. A
troublesome side reaction occurring during TFA treatment is
the conversion of the released amine to the trifluoroacetamide,
which significantly lowered the yield. Addition of triethylsilane
to suppress this side reaction did not particularly improve the
reaction outcome.6 Staudinger reduction followed by addition
of 1-naphthyl isocyanate provided protected urea 16. Catalytic
hydrogenolysis and alkene saturation using Pd-black under
hydrogen atmosphere proceeded sluggishly resulting in an
inseparable mixture of products, including saturation of the
naphthyl to the tetraline ring and incomplete double bond
saturation. As Birch reduction is incompatible with the naphthyl
moiety we were forced to reconsider our strategy.
Results and discussion
Chemical synthesis
Initially we modified the procedure of Chen et al.11 utilizing the
Julia-Kocienski olefination method to join the ceramide part to
the sugar, as it allows the formation of both saturated and
unsaturated derivatives. To allow swift manipulation of the C6”-
position an orthogonally protected galactose formaldehyde was
required. Treatment of perbenzylated α-methyl galactoside
with propargyltrimethylsilane and trimethylsilyl triflate,
followed by the addition of acetic anhydride, furnished allene
(Scheme 1).
7
8
Scheme 2.
Scheme 1.
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To circumvent the unwanted saturation of the naphthyl ring, The use of DPPA as azide source gave no reaction, while
protecting group cleavage without hydrogenation was mesylation followed by sodium azide treatment gave the
desirable. Considering the protecting group strategy of the undesired elimination product. To our delight Mitsunobu
sulfone we opted for PMB-groups on the sugar aldehyde, as a reaction with phthalimide swiftly yielded 27 in good yield
single acid hydrolysis step could remove all protecting groups. (Scheme 3). Treatment with hydrazine monohydrate not only
Rather than employing the allene approach, which suffers from liberated the free amine but conveniently also reduced the
low yields due to the unreliable ozonolysis/reduction step and alkene via diimide reduction. Switching hydrazine for
the requirement of expensive reagents such as methylhydrazine allowed to remain the double bond intact.
propargyltrimethylsilane, we synthesized propene 18 by Next, treatment of the crude amines with 1-naphthyl isocyanate
standard C-glycosylation of galactosyl pentaacetate followed by furnished the corresponding urea derivatives. To avoid
palladium-catalyzed isomerization of the double bond (Scheme trifluoroacetamide formation the acid-labile protecting groups
2). Zemplén deacetylation and protection of the primary alcohol were removed by dissolving the intermediates 28 and 29 in a 4
as triisopropylsilyl ether gave propenylgalactoside 20, which M HCl in dioxane solution. An attempt to acylate the resulting
was treated with PMBCl to mask the remaining hydroxyl amines by treatment with cerotoyl chloride in a biphasic
functions. Propene 21 was subjected to an osmium-catalyzed mixture of THF and saturated sodium acetate solution12
dihydroxylation to furnish vicinal diol 22
.
resulted in a mixture of the desired compound and the
Oxidation of 22 with sodium periodate afforded C-formyl corresponding acetamide, probably due to mixed anhydride
galactoside 23 amenable for Julia-Kocienski olefination with the formation. Addition of the p-nitrophenyl ester of cerotic acid in
previously used sulfone. Using the same coupling protocol as for DCM gave 5a, while acylation with cerotoyl chloride in a
12, an inseparable mixture of α- and β-anomers was obtained biphasic mixture of THF and 1 M KOH solution gave 5b. The low
together with side product 25 due to β-elimination. The α/β- yields associated with the final acylation are likely due to the
ratio could be increased by slow addition of the aldehyde to the low solubility of the final compounds and the cerotic acid
deprotonated sulfone in dilute conditions, which also reduced derivative.
the formation of elimination product. After deprotection of the Since 4-pyridylisocyanate is not commercially available the
silyl ether with tetrabutylammonium fluoride both anomers primary alcohol of 26 was first treated with p-nitrophenyl
could be easily purified by column chromatography to furnish chloroformate and the mixed anhydride was subsequently
alcohol 26 as the single α-anomer.
turned into the 4-pyridylcarbamate by addition of 4-
Mitsunobu reaction of 26 with hydrazoic acid to introduce an aminopyridine (Scheme 4).
azide functionality at C6” did not furnish the desired product.
Scheme 4.
Scheme 3
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although this is most prominent for the pyridinylcarbamate
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Deprotection with HCl and acylation gave 6a. Obtaining the analogues 6a-b
. This indicates that DtOhIe: 10.a1p03p9e/nCd7OeBd00C0861”B-
saturated carbamate through catalytic hydrogenation proved substituents have the ability to restore the antigenic potency of
arduous since the pyridine nitrogen poisons the palladium α-C-GalCer. Furthermore, in line with previous findings, the E-
catalyst. Addition of acetic acid to protonate the pyridine alkene unsaturated derivatives display superior antigenic
improved the conversion rate, although full saturation of the properties as compared to the saturated C-glycosides. In
double bond could not be attained. Instead, we used the accordance with the results in Balb/c mice, the pyridine
diimide reduction with hydrazine to saturate intermediate 26
Further conversion following previously used reactions compounds
.
carbamates
6
5
surpass the antigenic potency of the naphthylurea
. Although pyridine carbamate 6a is less antigenic
furnished saturated derivative 6a
.
than its parent O-glycoside 4 (Figure S2), it shows more
powerful human iNKT cell activation than α-GalCer.
Immunological activity
The biological activity of the C-glycosides 5-6 was assessed by
measuring the IFN-γ and IL-4 levels after intraperitoneal injection of
1 µg of the corresponding glycolipids in Balb/c mice (Figure 2).
Figure 3: IFN-γ secretion after coculture of human iNKT cells and
Hela CD1d cells with the corresponding C-glycosides
Conclusions
In summary, we developed a versatile synthetic strategy
allowing the preparation of both saturated and unsaturated α-
C-GalCer derivatives with variable C6”-substituents. All
prepared C-glycosides were able to stimulate human iNKT cells,
denoting that the appended C6”-substituents have the ability to
restore the antigenic potency of α-C-GalCer, which lacks activity
on human iNKT cells.
Experimental section
Figure 2: IFN-γ and IL-4 secretion, measured over the course of
72h, after intraperitoneal injection of 1 µg of the final
compounds in Balb/c mice.
(3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-O-tri-
isopropylsilyl-α-C-D-galactopyranosyl)-3-tert-butyloxy-
carbonylamino-4,5-Di-O-isopropylidene-1-nonadecene-4,5-
diol (24)
With the exception of 5b, all glycolipids show superior antigenic
properties as compared to α-C-GalCer. Moreover, pyridine
carbamate 6b is, to the best of our knowledge, the first C-
glycoside that shows superior antigenic properties as compared
to α-GalCer. In general the pyridine carbamate modified
analogues are more potent than the naphthylurea derivatives.
A similar trend is observed when the cytokine profile was
investigated in C57BL/6 mice (Figure S1). However, while in
Balb/c mice the unsaturated derivatives are less potent than the
saturated C-glycosides, the opposite is true in C57BL/6 mice.
In addition, the C-glycosides were evaluated for their ability to
stimulate human iNKT cells (Figure 3). We were delighted to
observe that all final compounds activated human iNKT cells, ,
To a solution of diol 22 (930 mg, 1.23 mmol) in THF (16 mL) and
H2O (8 mL) was added NaIO4 (1.32 g, 6.15 mmol) portionwise
over a period of 1h. The reaction mixture was stirred at room
temperature for 2.5h. Upon disappearance of the starting
material (as judged by TLC), the mixture was diluted with Et2O
(30 mL) and the aqueous layer was extracted with Et2O (3 x 30
mL). The combined organic phases were washed with saturated
NaHCO3 solution (100 mL), dried over Na2SO4, filtered and
concentrated at 30 °C to afford crude aldehyde which was used
without further purification in the next step.
Lithium bis(trimethylsilyl)amide (1 M in THF, 2.46 mL, 2.46
mmol) was slowly added to a solution of sulfone 11 (786 mg,
1.23 mmol) in anhydrous THF (40 mL) at -78 °C. After 1.5h, the
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above prepared crude aldehyde in THF (40 mL) was added Exact mass (ESI-MS) for C57H86NO12 [M+H]+ found, 976.6146;
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DOI: 10.1039/C7OB00081B
dropwise over a period of 2h. The mixture was allowed to warm calcd, 976.6145.
to room temperature and stirred overnight. The reaction was
quenched with water (50 mL) and extracted with Et2O (3x 50 (3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-
mL). The combined organic phases were washed with H2O and phtalimidoyl-6’-deoxy-α-C-
brine, dried over Na2SO4, filtered and concentrated. The residue butyloxycarbonylamino-4,5-Di-O-isopropylidene-1-
was purified by flash chromatography (5 25% EtOAc in nonadecene-4,5-diol (27)
D-galactopyranosyl)-3-tert-

hexanes) to afford a mixture of α and β alkenes 24 (640 mg, To a solution of alcohol 26 (100 mg, 0.1 mmol) in anhydrous THF
1
46%) as a colorless oil. H NMR (300 MHz, CDCl3) (mixture of (2 mL) at -20 °C was added PPh3 (79 mg, 0.3 mmol), DEAD (40%
anomers): δ 0.89 (t, J = 6.3 Hz, 3 H, terminal CH3) 0.99 – 1.11 (m, DEAD in toluene, 0.09 mL, 0.3 mmol) and phtalimide (44 mg, 0.3
21 H, i-prop) 1.20 – 1.35 (m, 25 H, CH2 and CH3 ) 1.36 – 1.46 (m, mmol) in this order. The resulting mixture was allowed to reach
12 H, tBu and CH3 ) 1.49 – 1.70 (m, 4 H, CH2) 3.56 (dd, J = 9.2 and room temperature and was stirred for 5h. Upon complete
2.5 Hz, 1 H, H-3) 3.71 – 3.78 (m, 3 H, H-3’ and CH2-6) 3.78 – 3.83 consumption of the starting material the solvents were
(m, 10 H, 3 x OCH3 and H-5’) 3.84 – 4.02 (m, 2 H, H-4 and H-5) removed under reduced pressure and the resulting residue was
4.02 – 4.20 (m, 2 H, H-4’ and H-2) 4.25 – 4.75 (m, 7 H, purified by flash column chromatography (10
 30% EtOAc in
CH2PhOMe, H-1 and NH) 4.82 (d, J = 10.8 Hz, 1 H, CH2PhOMe) hexanes) giving phtalimide 27 (100 mg, 88%) as a white waxy
1
5.87 – 6.01 (m, 2 H, CH=CH) 6.77 – 6.90 (m, 6 H, arom. H) 7.15 – solid. H NMR (300 MHz, CDCl3): δ 0.89 (t, J = 6.6 Hz, 3 H,
7.30 (m, 6 H, arom. H); 13 C NMR (75 MHz, CDCl3): δ 11.91, 12.49, terminal CH3) 1.20 – 1.35 (m, 26 H, CH3 and CH2) 1.35 (s, 3 H,
14.11, 18.06, 18.12, 22.69, 25.53, 26.79, 27.53, 28.35, 29.02, CH3) 1.42 (s, 9 H, tBu) 1.46 – 1.66 (m, 3 H, CH2) 3.50 – 3.60 (m, 1
29.36, 29.60, 29.65, 29.69, 31.92, 52.21, 55.23, 61.81, 72.62, H, H-5’) 3.69 – 3.74 (m, 1 H, H-4’) 3.80 – 3.81 (m, 2 H, CH2-6’)
72.80, 72.95, 73.98, 74.82, 79.47, 80.56, 107.97, 113.54, 113.65, 3.82 (s, 4 H, H-5 and OCH3) 3.82 (s, 3 H, OCH3) 3.83 (s, 3 H, OCH3)
113.68, 125.76, 128.92, 129.02, 129.36, 129.66, 129.71, 130.85, 3.98 (dd, J = 5.0 and 2.9 Hz, 1H, H-4) 4.04 – 4.13 (m, 1 H, H-3’)
131.05, 131.18, 154.90, 159.00, 159.08; Exact mass (ESI-MS) for 4.19 – 4.77 (m, 10 H, H-1’, H-2’, H-3, CH2PhOMe and NH) 5.67
C66H106NO12Si [M+H]+ found, 1132.7462; calcd, 1132.7479.
(dd, J = 15.8 and 6.2 Hz, 1 H, CH-1=CH) 5.91 (dd, J = 15.7 and 4.0
Hz, 1 H, CH=CH-2) 6.82 – 6.93 (m, 6 H, arom. H) 7.10 – 7.17 (m,
(3S,4S,5R)-1-(6’-hydroxy-2’,3’,4’-tri-O-(4-methoxybenzyl)-α-C- 2 H, arom. H) 7.22 – 7.27 (m, 2 H, arom. H) 7.32 – 7.38 (m, 2 H,
D
-galactopyranosyl)-3-tert-butyloxycarbonylamino-4,5-Di-O-
arom. H) 7.66 – 7.70 (m, 2 H, Phtal) 7.79 – 7.83 (m, 2 H, Phtal);
13 C NMR (75 MHz, CDCl3): δ 14.12, 22.69, 25.47, 26.99, 27.22,
isopropylidene-1-nonadecene-4,5-diol (26)
A solution of 24 (2.3 g, 2.03 mmol) in THF (20 mL) was cooled to 28.40, 29.36, 29.59, 29.69, 31.92, 51.72, 55.28, 71.52, 72.63,
0 °C and tetrabutylammonium fluoride (1 M in THF, 4.06 mL, 72.85, 76.79, 77.21, 77.82, 79.30, 79.72, 107.85, 113.73, 113.76,
4.06 mmol) was added dropwise. The reaction mixture was 123.10, 123.25, 123.59, 127.01, 129.19, 122.22, 129.50, 129.59,
allowed to reach room temperature and was stirred overnight. 129.80, 130.21, 130.38, 130.50, 130.64, 131.91, 132.08, 132.34,
The reaction mixture was diluted with DCM (150 mL) and 133.60, 133.92, 134.30, 154.81, 159.13, 159.32, 168.30, 168.73;
washed with saturated aqueous NH4Cl solution (75 mL) and H2O Exact mass (ESI-MS) for C65H88N2NaO13 [M+Na]+ found,
(75 mL). The aqueous layer was back extracted with DCM (2 x 1127.6172; calcd, 1127.6179.
50 mL) and the combined organic layers were washed with
brine (100 mL). The organics were dried over Na2SO4, filtered (3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-naphtureido-
and concentrated under reduced pressure and the residue was 6’-deoxy-α-C-
D-galactopyranosyl)-3-tert-butyloxycarbonyl-
purified by flash column chromatography (0 30% EtOAc in amino-4,5-Di-O-isopropylidene-nonadecane-4,5-diol (28)

hexanes) to provide α-glycoside 26 (1.28 g, 65 %) as a colourless A solution of alkene 27 (100 mg, 0.09 mmol) in EtOH (4 mL) and
oil together with the corresponding β-glycoside (362 mg, 18%). hydrazine monohydrate (0.4 mL) was heated till reflux and
1H NMR (300 MHz, CDCl3): δ 0.89 (t, J = 6.6 Hz, 3 H, terminal CH3) stirred for 18h. Upon completion of the reaction, DCM (20 mL)
1.31 (s, 3 H, CH3) 1.22 – 1.34 (m, 22 H, CH2) 1.40 (s, 3 H, CH3) was added and the organic layer was washed with 1 M NaOH
1.44 (s, 9 H, tBu) 1.48 – 1.71 (m, 4 H, CH2) 2.02 (br. s, 1 H, OH) solution (20 mL). The aqueous layer was back extracted with
3.51 – 3.62 (m, 2 H, H-5 and Ha-6’) 3.81 (s, 3 H, OCH3) 3.82 (s, 3 DCM (2 x 20 mL) and the combined organic phase was washed
H, OCH3) 3.82 (s, 3 H, OCH3) 3.79 – 4.01 (m, 5 H, H-2, H-4, H-4’, with H2O (50 mL). The organics were dried over Na2SO4, filtered
H-5’ and Hb-6’) 4.10 – 4.19 (m, 1 H, H-3) 4.25 – 4.36 (m, 1 H, H- and concentrated under reduced pressure. The crude amine
3’) 4.49 – 4.70 (m, 7 H, CH2PhOMe, H-1 and NH) 4.74 (d, J = 11.6 was used in the next step without further purification. The
Hz, 1 H, CH2PhOMe) 5.84 (dd, J = 16.3 and 3.7 Hz, 1 H, CH-1=CH) amine was dissolved in DMF (2 mL) and cooled to 0 °C. Next 1-
5.93 (dd, J = 16.3 and 4.4 Hz, 1 H, CH=CH-2) 6.83 – 6.90 (m, 6 H, naphthyl isocyanate (34 µL, 0.23 mmol) was added and the
13
arom. H) 7.19 – 7.27 (m, 6 H, arom. H); C NMR (75 MHz, mixture was stirred overnight at room temperature. The
CDCl3): δ 14.11, 22.69, 25.40, 26.85, 27.21, 28.37, 28.81, 29.36, solvents were removed under reduced pressure and the residue
29.56, 29.62, 29.65, 29.69, 31.91, 52.15, 55.26, 61.86, 72.07, was purified by flash column chromatography (10
 30% EtOAc
72.88, 73.00, 73.03, 74.25, 77.21, 77.73, 78.01, 79.50, 79.61, in hexanes) to furnish urea 28 (80 mg, 77% over 2 steps) as a
108.02, 113.71, 113.77, 113.82, 126.38, 129.13, 129.53, 129.88, light brown solid. 1H NMR (300 MHz, CDCl3): δ 0.89 (t, J = 6.6 Hz,
130.26, 130.50, 130.61, 131.43, 154.90, 159.17, 159.23, 159.35; 3 H, terminal CH3) 1.15 – 1.32 (m, 27 H, CH3 and CH2) 1.38 (s, 3
H, CH3) 1.42 (s, 9 H, tBu) 1.46 – 1.78 (m, 6 H, CH2) 3.43 – 3.56
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(m, 1 H, H-4) 3.64 – 3.75 (m, 3 H, H-5, CH2-6’) 3.79 (s, 3 H, OCH3)
3.82 (s, 3 H, OCH3) 3.82 (s, 3 H, OCH3) 3.86 – 3.99 (m, 4 H, H-3, (3S,4S,5R)-1-(6’-hydroxy-2’,3’,4’-tri-O-(4D-mOeI:t1h0o.10x3y9b/eCn7OzyBl0)-0α0-8C1B-
H-2’, H-4’ and H-5’) 4.02 – 4.10 (m, 1 H, H-3’) 4.37 (d, J = 12.4 -galactopyranosyl)-3-tert-butyloxycarbonylamino-4,5-Di-O-
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D
Hz, 1 H, CH2PhOMe) 4.41 – 4.68 (m, 6 H, H-1’ and CH2PhOMe) isopropylidene-nonadecane-4,5-diol (30)
5.69 (br. s, 1 H, NH) 6.78 – 6.91 (m, 6 H, arom. H) 7.11 – 7.26 (m, A solution of alkene 26 (121 mg, 0.12 mmol) in EtOH (5 mL) and
6 H, arom. H) 7.41 – 7.53 (m, 4 H, naphthyl and NH) 7.60 – 7.73 hydrazine monohydrate (0.5 mL) was heated till reflux and
(m, 1 H, naphthyl) 7.82 – 7.88 (m, 2 H, naphthyl) 7.98 – 8.06 (m, stirred for 18h. Upon completion of the reaction, DCM (20 mL)
1 H, naphthyl); 13 C NMR (75 MHz, CDCl3): δ 14.12, 22.69, 25.39, was added and the organic layer was washed with 1 M NaOH
25.77, 26.76, 27.11, 28.43, 28.92, 29.37, 29.43, 29.57, 29.66, solution (20 mL). The aqueous layer was back extracted with
29.71, 30.92, 31.92, 50.21, 55.25, 72.02, 72.53, 72.83, 73.53, DCM (2 x 20 mL) and the combined organic phase was washed
73.76, 77.20, 77.75, 79.82, 107.85, 113.74, 120.37, 121.62, with H2O (50 mL). The organics were dried over Na2SO4, filtered
124.50, 125.80, 126.02, 128.46, 129.31, 129.47, 129.63, 129.73, and concentrated under reduced pressure. The residue was
130.20, 130.37, 130.64, 134.11, 134.24, 155.87, 156.44, 156.96, purified by flash column chromatography (0
 50% EtOAc in
159.19, 159.34; Exact mass (ESI-MS) for C68H96N3O12 [M+H]+ hexanes) to provide saturated glycoside 30 (88 mg, 73 %) as a
1
found, 1146.6990; calcd, 1146.6989.
yellowish oil. H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.6 Hz, 3
H, terminal CH3) 1.31 (s, 3 H, CH3) 1.20 – 1.33 (m, 24 H, CH2) 1.39
(3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-naphtureido- (s, 3 H, CH3) 1.42 (s, 9 H, tBu) 1.47 – 1.78 (m, 6 H, CH2) 2.56 (br.
6’-deoxy-α-C-
D
-galactopyranosyl)-3-tert-butyloxycarbonyl-
s, 1 H, OH) 3.53 – 3.73 (m, 4 H, H-2’, H-3’, H-4’ and Ha-6’) 3.81
(s, 3 H, OCH3) 3.81 (s, 3 H, OCH3) 3.81 (s, 3 H, OCH3) 3.86 – 4.00
amino-4,5-Di-O-isopropylidene-1-nonadecene-4,5-diol (29)
To a solution of alkene 27 (243 mg, 0.22 mmol) in degassed (m, 4 H, H-2, H-3, H-4 and H-5) 4.03 – 4.14 (m, 2 H, H-1 and Hb-
EtOH (3 mL) was added methylhydrazine (46 µL, 0.88 mmol). 6’) 4.41 (d, J = 11.7 Hz, 1H, CH2PhOMe) 4.45 – 4.67 (m, 6 H,
The resulting mixture was heated till reflux and stirred for 18h CH2PhOMe and NH) 6.82 – 6.91 (m, 6 H, arom. H) 7.13 – 7.28
13
under nitrogen atmosphere. Upon completion of the reaction, (m, 6 H, arom. H); C NMR (75 MHz, CDCl3): δ 14.08, 22.64,
DCM (20 mL) was added and the organic layer was washed with 23.88, 25.50, 26.70, 27.37, 28.32, 28.63, 28.90, 29.31, 29.51,
1 M NaOH solution (20 mL). The aqueous layer was back 29.57, 29.65, 31.88, 50.21, 55.20, 60.32, 72.12, 72.50, 72.83,
extracted with DCM (2 x 20 mL) and the combined organic 73.73, 73.93, 75.02, 76.24, 77.82, 79.47, 79.81, 107.74, 113.71,
phase was washed with H2O (50 mL). The organics were dried 113.74, 129.22, 129.31, 129.48, 129.60, 130.29, 130.52, 155.26,
over Na2SO4, filtered and concentrated under reduced pressure. 159.16, 159.25; Exact mass (ESI-MS) for C57H88NO12 [M+H]+
The crude amine was used in the next step without further found, 978.6314; calcd, 978.6301.
purification. The amine was dissolved in DMF (5 mL) and cooled
to 0 °C. Next 1-naphthyl isocyanate (82 µL, 0.57 mmol) was (3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-O-(4-
added and the mixture was stirred overnight at room pyridinylcarbamoyl)-α-C-D-galactopyranosyl)-3-tert-butyloxy-
temperature. The solvents were removed under reduced carbonylamino-4,5-Di-O-isopropylidene-nonadecane-4,5-diol
pressure and the residue was purified by flash column (31)
chromatography (10

30% EtOAc in hexanes) to furnish urea To a solution of alcohol 30 (324 mg, 0.33 mmol) in anhydrous
1
29 (152 mg, 60% over 2 steps) as a light brown solid. H NMR DCM (12 mL) was added p-nitrophenyl chloroformate (100 mg,
(300 MHz, CDCl3): δ 0.89 (t, J = 6.6 Hz, 3 H, terminal CH3) 1.19 – 0.5 mmol) and pyridine (53 µL, 0.66 mmol). The mixture was
1.32 (m, 27 H, CH3 and CH2) 1.39 (s, 3 H, CH3) 1.44 (s, 9 H, tBu) heated to 40 °C for 8h. After complete disappearance of the
1.48 – 1.71 (m, 2 H, CH2) 3.40 – 3.59 (m, 3 H, H-3’and CH2-6’) starting material the mixture was diluted with DCM (30 mL) and
3.77 (s, 3 H, OCH3) 3.78 – 3.81 (m, 1 H, H-5) 3.81 (s, 3 H, OCH3) washed successively with saturated NaHCO3 solution (20 mL),
3.82 (s, 3 H, OCH3) 3.84 – 3.98 (m, 3 H, H-4, H-4’ and H-5’) 4.09 H2O (20 mL) and brine (20 mL). Next the aqueous layer was
– 4.19 (m, 1 H, H-2’) 4.24 – 4.34 (m, 1 H, H-3) 4.44 – 4.67 (m, 7 extracted with DCM (2 x 50 mL) and the combined organic layer
H, H-1’, CH2PhOMe and NH) 4.70 (d, J = 10.9 Hz, 1 H, was dried over Na2SO4, filtered and concentrated under
CH2PhOMe)5.07 (br. s, 1 H, NH) 5.78 (dd, J = 16.4 and 3.9 Hz, 1 reduced pressure. The crude p-nitrophenyl carbonate was used
H, CH-1=CH) 5.88 (dd, J = 16.4 and 4.0 Hz, 1 H, CH=CH-2) 6.78 – without further purification in the next reaction.
6.91 (m, 6 H, arom. H) 7.15 – 7.27 (m, 6 H, arom. H) 7.41 – 7.53 The crude carbonate was dissolved in anhydrous DCM (8 mL)
(m, 3 H, naphthyl) 7.69 (d, J = 7.9 Hz, 2 H, naphthyl) 7.83 – 7.88 followed by addition of 4-aminopyridine (155 mg, 1.65 mmol)
(m, 1 H, naphthyl) 7.95 – 8.00 (m, 1 H, naphthyl); 13 C NMR (75 and Et3N (91 µL, 0.66 mmol). The reaction mixture was heated
MHz, CDCl3): δ 14.11, 22.67, 25.51, 25.69, 26.76, 28.37, 28.41, to reflux temperature and was stirred overnight. The mixture
28.67, 29.34, 29.53, 29.60, 29.65, 29.69, 30.90, 31.91, 40.87, was diluted with DCM (30 mL) and washed successively with
55.23, 72.91, 72.97, 73.43, 74.13, 74.88, 76.22, 77.20, 77.85, saturated NaHCO3 solution (20 mL), H2O (20 mL) and brine (20
79.50, 108.13, 113.68, 113.71, 113.76, 113.79, 121.80, 125.80, mL). Next the aqueous layer was extracted with DCM (2 x 50 mL)
125.91, 126.12, 126.50, 126.76, 126.96, 128.47, 128.55, 129.10, and the combined organic layer was dried over Na2SO4, filtered
129.15, 129.39, 129.50, 129.79, 130.06, 130.32, 130.43, 130.47, and concentrated under reduced pressure. The residue was
130.53, 130.64, 130.75, 133.56, 134.30, 134.43, 154.88, 155.43, purified by flash column chromatography (0
 50% EtOAc in
159.16, 159.23; Exact mass (ESI-MS) for C68H94N3O12 [M+H]+ hexanes) to provide carbamate 31 (200 mg, 55 %) as a white
1
found, 1144.6827; calcd, 1144.6832.
foam. H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.5 Hz, 3 H,
6 | J. Name., 2012, 00, 1-3
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terminal CH3) 1.14 – 1.36 (m, 24 H, CH3 and CH2) 1.36 – 1.51 (m, 66.19, 70.45, 72.74, 73.03, 73.18, 74.08, 76.28, 77.20, 77.98,
View Article Online
DOI: 10.1039/C7OB00081B
16 H, tBu, CH2 and CH3) 1.57 – 1.71 (m, 4 H, CH2) 1.74 – 1.87 (m, 79.76, 80.43, 108.61, 112.43, 113.74, 113.80, 115.74, 125.36,
1 H, CH2) 3.56 – 3.73 (m, 2 H, H-3’ and H-5’) 3.81 (s, 3 H, OCH3) 126.15, 129.10, 129.44, 129.88, 130.12, 130.47, 130.60, 147.98,
3.82 (s, 3 H, OCH3) 3.83 (s, 3 H, OCH3) 3.84 – 3.97 (m, 4 H, H-3, 152.61, 155.46, 159.20, 159.35, 163.58; Exact mass (ESI-MS) for
H-4, H-5 and H-4’) 4.04 – 4.15 (m, 1 H, Ha-6’) 4.15 (dd, J = 6.6 C63H90N3O13 [M+H]+ found, 1096.6450; calcd, 1096.6468.
and 3.3 Hz, 1H, H-2) 4.27 (q, J = 6.3 Hz, 1 H, H-1) 4.44 – 4.67 (m,
7 H, Hb-6’, CH2PhOMe and NH) 4.72 (d, J = 11.4 Hz, 1H, (3S,4S,5R)-1-(6”-naphtureido-6”-deoxy-α-C-D-galacto-
CH2PhOMe) 6.82 – 6.92 (m, 6 H, arom. H) 7.16 – 7.30 (m, 6 H, pyranosyl)-3-hexacosylamino-1-nonadecene-4,5-diol (5a)
arom. H) 7.55 (d, J = 6.2 Hz, 2 H, pyridine) 8.44 (d, J = 6.4 Hz, 2 A solution of 29 (125 mg, 0.11 mmol) in 4 M HCl in dioxane (5
H, pyridine) 8.66 (s, 1 H, NHC(O)O); 13 C NMR (75 MHz, CDCl3): δ mL) was stirred 3h at room temperature. The reaction was
14.12, 22.69, 25.54, 26.35, 27.18, 27.92, 28.43, 28.73, 29.36, monitored with mass spectrometry and upon completion the
29.43, 29.51, 29.66, 29.69, 31.92, 51.17, 55.29, 70.34, 72.88, solvents were evaporated under reduced pressure. The crude
73.11, 73.84, 77.95, 79.56, 79.87, 108.14, 112.46, 113.74, amine thus obtained was used without further purification in
113.80, 129.24, 129.48, 129.74, 130.11, 130.41, 130.53, 147.91, the next step. The amine was suspended in THF (2 mL) and a
152.67, 155.66, 159.25, 159.37; Exact mass (ESI-MS) for catalytic amount of 4-DMAP was added. Next p-nitrophenyl
C63H92N3O13 [M+H]+ found, 1098.6619; calcd, 1098.6625.
hexacosanoate (85 mg, 0.17 mmol) was added followed by
addition of Et3N (1 mL). The resulting mixture was stirred for 3
days at room temperature. Evaporation of the solvents and
(3S,4S,5R)-1-(2’,3’,4’-tri-O-(4-methoxybenzyl)-6’-O-(4-
pyridinylcarbamoyl)-α-C-D-galactopyranosyl)-3-tert-butyloxy- purification of the resulting residue by means of column
carbonylamino-4,5-Di-O-isopropylidene-1-nonadecene-4,5-
diol (32)
chromatography (2
 12% MeOH in DCM) yielded 5a (32 mg,
29%) as a white solid. 1H NMR (300 MHz, pyridine-d5): δ 0.89 (t,
To a solution of alcohol 26 (187 mg, 0.19 mmol) in anhydrous J = 6.6 Hz, 6 H, terminal CH3) 1.00 – 1.49 (m, 64 H, CH2) 1.49 –
DCM (8 mL) was added p-nitrophenyl chloroformate (57 mg, 1.96 (m, 5 H, CH2) 2.20 – 2.38 (m, 2 H, CH2) 2.47 (t, J = 7.4 Hz,
0.29 mmol) and pyridine (31 µL, 0.38 mmol). The mixture was 2H, CH2) 3.22 (app t, J = 5.6 Hz, 1 H, CH2) 4.16 (t, J = 5.5 Hz, 2 H,
heated to 40 °C for 8h. After complete disappearance of the CH2-6”) 4.19 – 4.34 (m, 3 H, H-4, H-5 and H-3”) 4.34 – 4.38 (m, 1
starting material the mixture was diluted with DCM (30 mL) and H, H-5”) 4.59 (t, J = 6.2 Hz, 1 H, H-4”) 4.79 (dd, J = 9.7 and 6.2 Hz,
washed successively with saturated NaHCO3 solution (20 mL), 1 H, H-2”) 5.03 – 5.09 (m, 1 H, H-1”) 5.85 – 5.93 (m, 1 H, H-3)
H2O (20 mL) and brine (20 mL). Next the aqueous layer was 6.08 (br. s, 5 H, OH) 6.84 (dd, J = 16.2 and 3.8 Hz, 1 H, CH-1=CH)
extracted with DCM (2 x 50 mL) and the combined organic layer 6.94 (dd, J = 16.2 and 6.0 Hz, 1 H, CH=CH-2) 7.43 – 7.54 (m, 2 H,
was dried over Na2SO4, filtered and concentrated under naphthyl) 7.61 – 7.72 (m, 2 H, naphthyl) 7.84 – 7.91 (m, 1 H,
reduced pressure. The crude p-nitrophenyl carbonate was used naphthyl) 8.52 (dd, J = 7.6 and 1.1 Hz, 1 H, naphthyl) 8.57 – 8.62
without further purification in the next reaction.
(m, 1 H, naphthyl) 8.68 (d, J = 9.0 Hz, 1 H, NH) 9.69 (s, 1 H,
13
The crude carbonate was dissolved in anhydrous DCM (5 mL) NHC(O)NH); C NMR (75 MHz, pyridine-d5): δ 14.69, 23.35,
followed by addition of 4-aminopyridine (89 mg, 0.95 mmol) 26.72, 26.87, 30.02, 30.20, 30.23, 30.29, 30.32, 30.41, 30.46,
and Et3N (53 µL, 0.38 mmol). The reaction mixture was heated 30.55, 30.80, 32.54, 35.05, 37.37, 42.35, 44.63, 46.24, 54.46,
to reflux temperature and was stirred overnight. The mixture 70.27, 71.54, 72.71, 73.00, 76.93, 78.91, 119.65, 123.26, 124.45,
was diluted with DCM (30 mL) and washed successively with 126.25, 126.43, 126.87, 127.73, 128.19, 129.13, 132.28, 135.24,
saturated NaHCO3 solution (20 mL), H2O (20 mL) and brine (20 136.70, 158.30, 173.08; Exact mass (ESI-MS) for C62H108N3O8
mL). Next the aqueous layer was extracted with DCM (2 x 50 mL) [M+H]+ found, 1022.8133; calcd, 1022.8131; m.p. 173 – 175 °C.
and the combined organic layer was dried over Na2SO4, filtered
and concentrated under reduced pressure. The residue was (3S,4S,5R)-1-(6”-naphtureido-6”-deoxy-α-C-
D-galacto-
purified by flash column chromatography (0 50% EtOAc in pyranosyl)-3-hexacosylamino-nonadecane-4,5-diol (5b)

hexanes) to provide carbamate 32 (129 mg, 62 %) as a white A solution of 28 (150 mg, 0.13 mmol) in 4 M HCl in dioxane (6
1
foam. H NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.6 Hz, 3 H, mL) was stirred 2.5h at room temperature. The reaction was
terminal CH3) 1.13 – 1.34 (m, 24 H, CH3 and CH2) 1.34 – 1.50 (m, monitored with mass spectrometry and upon completion the
14 H, tBu, CH2 and CH3) 1.59 – 1.75 (m, 2 H, CH2) 1.74 – 1.87 (m, solvents were evaporated under reduced pressure. The crude
1 H, CH2) 3.54 (dd, J = 9.0 and 2.2 Hz, 1 H, H-3”) 3.71 – 3.79 (m, amine was used without further purification in the next step
1 H, H-4”) 3.81 (s, 3 H, OCH3) 3.81 (s, 3 H, OCH3) 3.83 (s, 3 H, and was dissolved in THF (5 mL) and 10% KOH solution (2.5 mL)
OCH3) 3.90 (d, J = 11.2 Hz, 1 H, Ha-6”) 3.99 (d, J = 9.0 Hz, 1 H, H- and cooled to 0 °C. In a separate flask cerotic acid (199 mg, 0.5
5”) 4.04 – 4.21 (m, 2 H, H-5, H-2”) 4.31 – 4.42 (m, 2 H, H-3, H-4) mmol) was refluxed for 2h in oxalylchloride (5 mL) and the crude
4.48 – 4.73 (m, 7 H, H-1”, Hb-6, CH2PhOMe and NH) 4.77 – 4.88 acid chloride was obtained after evaporation of the solvent by
(m, 2 H, CH2PhOMe) 5.89 (dt, J = 16.5 and 2.0 Hz, 1 H, CH-2=CH) a stream of nitrogen and subsequent drying on high-vacuum.
6.03 (dt, J = 16.5 and 2.2 Hz, 1 H, CH=CH-1) 6.82 – 6.94 (m, 6 H, The crude acid chloride was dissolved in THF (5 mL) to obtain a
arom. H) 7.20 – 7.31 (m, 6 H, arom. H) 7.53 (d, J = 5.6 Hz, 2 H, 0.1 M solution of acid chloride in THF and dropwise added (2
pyridine) 8.44 (d, J = 6.2 Hz, 2 H, pyridine) 8.55 (s, 1 H, NHC(O)O); mL, 0.2 mmol) to the biphasic mixture. The reaction mixture
13 C NMR (75 MHz, CDCl3): δ 14.11, 22.67, 25.60, 26.32, 27.19, was stirred for 3h at 0 °C and TLC showed complete conversion
28.37, 28.88, 29.34, 29.48, 29.56, 29.63, 31.91, 51.60, 55.26, of the starting material. Next the aqueous layer was extracted
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with THF (3 x 15 mL) and the combined organic layer was dried solvents were evaporated under reduced pressure. The crude
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over Na2SO4, filtered and evaporated.
The resulting residue was purified by column chromatography the next step. The amine was suspended in THF (1 mL) and a
amine thus obtained was used without DfuOrIt: h10e.r10p39u/rCif7iOcaBt0i0o0n81inB
(2

12% MeOH in DCM) followed by trituration with EtOAc and catalytic amount of DMAP was added. Next p-nitrophenyl
1
yielded 5b (38 mg, 29%) as a white solid. H NMR (500 MHz, hexacosanoate (29 mg, 0.055 mmol) was added followed by
pyridine-d5): δ 0.92 (t, J = 6.1 Hz, 6 H, 2 x terminal CH3) 1.18 - addition of Et3N (0.1 mL). The resulting mixture was stirred for
1.51 (m, 67 H, CH2) 1.62 - 1.98 (m, 4 H, CH2) 2.21 – 2.32 (m, 3 H, 2 days at room temperature. Evaporation of the solvents and
CH2) 2.42 - 2.63 (m, 4 H, CH2) 4.20 - 4.39 (m, 6 H, H-4, H-5, H-3”, purification of the resulting residue by means of column
H-5” and CH2-6”) 4.48 (br. s, 1 H, H-4”) 4.60 – 4.66 (m, 2 H, H-1” chromatography (4
 14% MeOH in DCM) yielded 6b (20 mg,
and H-2”) 5.09 – 5.16 (m, 1 H, H-3) 7.48 - 7.57 (m, 4 H, naphthyl) 44%) as a white solid. 1H NMR (300 MHz, pyridine-d5): δ 0.89 (t,
7.65 (d, J = 7.9 Hz, 1 H, naphthyl) 7.92 (d, J = 7.3 Hz, 1 H, J = 6.6 Hz, 6 H, terminal CH3) 1.09 – 1.58 (m, 63 H, CH2) 1.58 –
naphthyl) 8.43 (d, J = 8.6 Hz, 1 H, NH) 8.59 (t, J = 7.3 Hz, 2 H, 2.38 (m, 11 H, CH2) 4.20 – 4.32 (m, 3 H, H-4, H-5 and H-4”) 4.41
13
naphthyl and NH) 9.38 (s, 1 H, NH);
C NMR (125 MHz, (br. s, 1 H, H-3”) 4.61 – 4.69 (m, 1 H, H-5”) 4.76 (dd, J = 8.1 and
pyridine-d5): δ 14.68, 23.38, 23.58, 26.15, 27.02, 27.10, 27.76, 5.8 Hz, 1H, H-2”) 4.84 – 5.05 (m, 1 H, Ha-6”) 5.06 – 5.23 (m, 2 H,
30.05, 30.06, 30.18, 30.23, 30.29, 30.36, 30.38, 30.44, 30.46, H-1” and Hb-6”) 5.82 – 5.93 (m, 1 H, H-3) 6.33 (br.s, 1 H, OH)
30.49, 30.56, 30.83, 32.59, 32.60, 35.11, 37.68, 41.67, 52.90, 6.63 (br. s, 1 H, OH) 6.83 (dd, J = 16.5 and 3.5 Hz, 1 H, CH-1=CH)
70.43, 71.31, 72.79, 73.75, 73.86, 75.86, 78.23, 119.32, 123.13, 6.98 (dd, J = 16.5 and 5.9 Hz, 1 H, CH=CH-2) 7.84 (d, J = 5.6 Hz, 2
123.64, 124.22, 126.17, 126.41, 127.00, 128.14, 129.30, 135.40, H, pyridine) 8.60 - 8.72 (m, 3 H, pyridine and NH) 10.98 (s, 1 H,
13
136.20, 137.04, 150.72, 157.90, 174.67; Exact mass (ESI-MS) for NHC(O)O);
C NMR (75 MHz, pyridine-d5): δ 14.68, 23.33,
C62H110N3O8 [M+H]+ found, 1024.8292; calcd, 1024.8287; m.p. 26.80, 26.84, 30.00, 30.17, 30.20, 30.26, 30.32, 30.40, 30.52,
187 – 189 °C.
30.72, 32.52, 35.02, 37.31, 54.17, 66.06, 70.50, 70.74, 72.79,
72.97, 73.16, 75.75, 78.54, 113.56, 127.83, 131.79, 147.62,
151.36, 154.67, 173.13; Exact mass (ESI-MS) for C57H104N3O9
[M+H]+ found, 974.7764; calcd, 974.7767; m.p. 173-175 °C.
(3S,4S,5R)-1-(6”-O-(4-pyridinylcarbamoyl)-α-C-D-galacto-
pyranosyl)-3-hexacosylamino-nonadecane-4,5-diol (6a)
A solution of 31 (180 mg, 0.16 mmol) in 4 M HCl in dioxane (6
mL) was stirred 3h at room temperature. The reaction was
monitored with mass spectrometry and upon completion the
solvents were evaporated under reduced pressure. The crude
amine thus obtained was used without further purification in
the next step. The amine was suspended in THF (3 mL) and a
catalytic amount of 4-DMAP was added. Next p-nitrophenyl
hexacosanoate (125 mg, 0.24 mmol) was added followed by
addition of Et3N (0.5 mL). The resulting mixture was stirred for
2 days at room temperature. Evaporation of the solvents and
purification of the resulting residue by means of column
Determination of Serum Cytokine Concentrations.
The serum concentrations of IFN-γ and IL-4 were assessed 2, 6, 12,
24, 48, and 72 h after i.v. administration of 1 µg of α-GalCer, α-C-
GalCer, or the indicated C-glycoside analogues in Balb/c-mice.
Cytokine levels were measured by way of a sandwich ELISA
(eBioscience).
Quantification of the level of cytokines produced by human iNKT
cell lines by ELISA
1x104 human iNKT cells were cocultured with 4x104 Hela cells
transfected with a CD1d gene, in the presence of an indicated
concentration of each glycolipid. After 24-h incubation at 37 °C, the
culture supernatants were collected and the concentrations of
human IFN-γ and IL-4 in the supernatants were determined by ELISA
(eBioscience).
chromatography (4
 18% MeOH in DCM) yielded 6a (27 mg,
17%) as a white solid. 1H NMR (300 MHz, pyridine-d5): δ 0.89 (t,
J = 5.6 Hz, 6 H, terminal CH3) 1.01 – 1.60 (m, 60 H, CH2) 1.60 –
2.05 (m, 8 H, CH2) 2.05 – 2.40 (m, 6 H, CH2) 2.40 – 2.84 (m, 4 H,
CH2) 4.20 – 4.43 (m, 4 H, H-4, H-5, H-2” and H-5”) 4.43 – 4.62
(m, 2 H, H-3”, H-4”) 4.63 – 4.76 (m, 1 H, H-1”) 4.85 (d, J = 10.2
Hz, 1H, Ha-6”) 5.00 – 5.17 (m, 1 H, H-3) 5.24 (dd, J = 10.2 and 8.9
Hz, 1 H, Hb-6”) 7.92 (d, J = 5.3 Hz, 2 H, pyridine) 8.53 (d, J = 8.5
Hz, 1 H, NH) 8.68 (d, J = 5.6 Hz, 2 H, pyridine) 10.89 (s, 1 H, Acknowledgements
13
NHC(O)O);
C NMR (75 MHz, pyridine-d5): δ 14.69, 23.35,
Joren Guillaume is a fellow of the Agency for Innovation by
Science and Technology (IWT) of Flanders. S.V.C. and D.E.
received support of the Belgian Stichting tegen Kanker and the
FWO Flanders.
26.77, 26.93, 27.06, 27.91, 30.02, 30.20, 30.23, 30.29, 30.32,
30.43, 30.58, 30.78, 32.54, 34.49, 37.41, 52.67, 66.26, 70.36,
70.82, 72.39, 72.61, 73.45, 75.93, 77.87, 113.61, 115.68, 131.30,
144.52, 147.73, 151.36, 154.84, 173.87; Exact mass (ESI-MS) for
C57H106N3O9 [M+H]+ found, 976.7925; calcd, 976.7924; m.p.
177-179 °C.
Notes and references
1
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(3S,4S,5R)-1-(6”-O-(4-pyridinylcarbamoyl)-α-C-D-galacto-
9
,
pyranosyl)-3-hexacosylamino-1-nonadecene-4,5-diol (6b)
A solution of 32 (53 mg, 0.05 mmol) in 4 M HCl in dioxane (1 mL)
was stirred 4h at room temperature. The reaction was
monitored with mass spectrometry and upon completion the
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