Analogues of glycosphingolipids and glycerolipids suitable for conjugation to gold- and amino-functionalised surfaces

Article abstract of DOI:10.1016/S0040-4020(00)00946-7

A general synthesis of analogues of natural lipids, (i.e. lactosylceramide, globotriasylceramide, and phosphatidylcholine) where one of the alkyl chains carries a terminal thiol- or carboxyl functionality, is described. The lipids were prepared by N- or O-acylation of sphingosine or monoacylglycerol derivatives. These lipids are suitable for anchoring to gold- or amino-functionalised surfaces, thus creating mimics of a cell membrane for use in the study of protein-carbohydrate interaction. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00946-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9975±9984
Analogues of Glycosphingolipids and Glycerolipids Suitable for
Conjugation to Gold- and Amino-Functionalised Surfaces
²
*
Jorgen Ohlsson and Goran Magnusson
È
È
Organic Chemistry 2, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
È
Dedicated to the memory of Professor Goran Magnusson
Received 29 June 2000; revised 22 September 2000; accepted 5 October 2000
AbstractÐA general synthesis of analogues of natural lipids, (i.e. lactosylceramide, globotriasylceramide, and phosphatidylcholine) where
one of the alkyl chains carries a terminal thiol- or carboxyl functionality, is described. The lipids were prepared by N- or O-acylation of
sphingosine or monoacylglycerol derivatives. These lipids are suitable for anchoring to gold- or amino-functionalised surfaces, thus creating
mimics of a cell membrane for use in the study of protein±carbohydrate interaction. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
glycol chains (PEG) and thioacetyl GM1 glycolipid for
biosensor applications. The PEG chain was reported to
cause some problem with non-speci®c protein adsorption
and phase separation in forming the SAM:s.7
Carbohydrates presented as glycolipids in cell membranes
serve as receptors for proteins, antibodies and other bio-
molecules, for example, for pathogens such as bacteria
and viruses during the initial phase of infection.1 The
study of these phenomena has traditionally been performed
with natural or synthetic glycolipids presented on TLC
plates, microtiter plates, glass or plastic particles, or in
liposomes. However, these procedures are not always
reliable; the mode of presentation could greatly in¯uence
the binding2 and many of the methods used only give an
answer to whether there is a binding or not.
We herein introduce a general synthesis of two series of
lipid analogues, where one of the alkyl chains carries a
terminal thiol or carboxyl group (Fig. 1). The ®rst series is
based on v-mercapto terminated lipids8 prepared from
acylation of glycosylsphingosines (compounds 1 and 3)
and acylation of monoacylated sn-glycerol (compound 5)
with modi®ed fatty acids. The second series of lipid
analogues are v-carboxyterminated analogues of lactosyl-
ceramide and globotriasylceramide (2 and 4). The corre-
sponding phosphatidylcholine analogue has been reported
earlier.9 Anchoring mixtures of these lipids to gold respec-
tively amino-functionalised surfaces is a possible route
towards mimics of cell membranes.
One improvement could be to synthesise analogues of the
naturally occurring glycosphingolipids and phoshoglycero-
lipids. If these analogues are equipped with a functionality
that allows covalent conjugation to surfaces, mimics of cell
membranes could be generated. Anchoring mixtures of
functionalised lipids to surfaces should result in a surface
where the carbohydrate will be presented in a way that
closely resemble natural cell membranes, thereby mini-
mising undesirable non-speci®c protein binding. The
anchoring of long-chain alkyl-thiols to gold is a well-
known phenomena,2,3 and in the adsorption process they
form self-assembled monolayers (SAM:s). Glycosides
carrying various single-chain alkyl-thiol linker arms have
been used in amperometric biosensors4 and surface plasmon
resonance measurements.5,6 A recent report7 describes an
investigation of SAM:s of thiolterminated polyethylene-
The problems with non-speci®c protein adsorption and
phase separation in forming the SAM:s using PEG chains7
might be circumvented by substituting the PEG chain for
natural lipids described in this paper. Additional advantages
of the present work are that analogues of the carbohydrate
part could be introduced and that the concentration of the
glycolipid could be varied.
Results and Discussion
Synthesis of modi®ed fatty acid
Keywords: glycolipid; phospholipids; cell membrane mimic.
* Corresponding author. Tel.: 146-222-8211; fax: 146-222-8209; e-mail:
jorgen.ohlsson@orgk2.lth.se
The hydroxyl acid 6 was transformed into the corresponding
bromide 7 by re¯uxing in aqueous HBr and acetic acid
for three days (Scheme 1). The product crystallised upon
²
Deceased June 2000
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00946-7

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J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
Figure 1.
Scheme 1. (a) HBr, AcOH, re¯ux, 3 days. (b) AcSK, DMF, 508C, 8 h. (c) N-hydroxysuccinimide, EDC, CH2Cl2, 12 h. (d) HSCH2CH2COOMe, Cs2CO3, DMF,
15 h.
cooling and could be ®ltered off yielding the crude v-bromo
palmitic acid 7 as a white solid. This could be converted into
either the thioacetate 8 in 90% yield by treatment with
KSAc in DMF at 508C or the methyl ester 10 in 84%
yield by treatment with methyl 3-mercaptopropionate and
Cs2CO3 in DMF. Treatment of 8 with N-hydroxysuccini-
mide(NHS) and ethyl-3-(3-dimethylaminopropyl)carbodi-
imide (EDC) furnished the activated acid derivative 9.
Synthesis of modi®ed glycolipids
The known lactosyl azidosphingosine10 derivative 11 was
converted into 12 in 81% yield via deacetylation in
methanolic NaOMe, azide reduction with H2S, and acyl-
ation overnight with the NHS-ester 9 (Scheme 2). Removal
of the protection group by treatment with methanolic
NaOMe furnished the thiol and a small amount of disul®de.
Scheme 2. (a) (i) NaOMe, MeOH. (ii) H2S, pyridine, Et3N, MeOH, (iii) 9, DMF, Et3N. (b) (i) NaOMe, MeOH, (ii) DTE, iPrNEt, DMF. (c) (i) H2S, pyridine/
H2O, 6:1, (ii) 10, EDC, CH2Cl2. (d) (i) NaOMe, MeOH, (ii) NaOH, H2O, MeOH.

J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
9977
Scheme 3. (a) NIS, TMSOTf, CH2Cl2/Et2O, 1:2, 2508C. (b) (i) H2, Pd/C, AcOH, (ii) Ac2O, pyridine. (c) (i) CF3COOH, CH2Cl2, (ii) Cl3CCN, DBU, CH2Cl2.
(d) (2S,3R,4E)-2-azido-3-benzoyloxy-octadec-4-ene-1-ol, BF3´Et2O, CH2Cl2, MS 300 AW. (e) (i) NaOMe, MeOH, (ii) H2S, pyridine, Et3N, MeOH, (iii) 9,
DMF, Et3N. (f) (i) NaOMe, MeOH, (ii) DTE, iPr2NEt, DMF. (g) (i) H2S, pyridine/H2O, 6:1, (ii) 10, EDC, CH2Cl2. (h) (i) NaOMe, MeOH, (ii) NaOH, CH2Cl2,
MeOH, H2O.
with the fatty acid derivative 10 and EDC in CH2Cl2 gave
the protected lactosyl ceramide mimic 13 in 79% yield from
11. Compound 13 was deprotected in two steps to give the
mimic 2 in 66% yield.
Figure 2.
The synthesis of globotriasylceramide analogues 3 and 4
(Scheme 3) started with the preparation of globotriose 16.
The galactose donor 1411,12 was glycosylated with lactose
acceptor 1513 using N-iodosuccinimide and TMS-tri¯ate14,15
In order to reverse the disul®de formation the mixture was
routinely treated with dithioerythritol and HuÈnig's base in
DMF6 to give the lactosyl ceramide mimic 1 in 88% yield.
The carboxyl-terminated lactosyl ceramide mimic 2 was
prepared from 11 by reduction with H2S in a mixture of
pyridine and water to give the amine 13. Under these less
basic conditions base promoted acylmigration is prevented,
although the reduction is somewhat slower than the condi-
tions used in the preparation of 12. Subsequent treatment
in CH2Cl2/Et2O at 2508C. By using electron withdrawing
benzoyl protecting groups on the acceptor, less than 3%
b-anomer is obtained still giving a 94% yield of the
globotriose derivative 16. This selectivity may be
explained by the low nucleophilicity of the 40-OH
induced by the surrounding electron withdrawing benzo-
ates.13,16 Debenzylation of 16 with catalytic hydrogenation,
followed by acetylation gave 17 in 91% yield.
Scheme 4. (a) (i) CH2Cl2, CH3CH2SS(CH2)15COOH, DMAP, DIC, 08C, 0.5 h, (ii) 228C, 4.5 h. (b) BCl3/hexane, EtS±SEt, CH2Cl2, 2788C, 1 h. (c) (i) PCl3,
immidazole, Et3N, toluene, (ii) cholin tosylate, NPCl, pyridine, (iii) I2, pyridine/H2O (49:1). (d) n-Bu3P, EtOH/H2O.

9978
J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
Conversion of the TMSEt-group of 17 into the corre-
sponding trichloroacetimidate 18 was accomplished by
treating 17 with CF3COOH in CH2Cl2,17 followed by
Cl3CCN and DBU in CH2Cl2.18 Glycosylation of 3-O-
benzoyl azidosphigosine19±21 with 18 using BF3´Et2O as
promoter in CH2Cl2 furnished 19 in 83% yield.
Conclusion
We have described the synthesis of thiol- and carboxy
analogues of naturally occurring lipids suitable for conjuga-
tion to gold- or amino-functionalised surfaces. Such
surfaces are expected to present the glycolipid in a more
natural way. The synthesis (Schemes 1±4) is general, i.e.
it allows the introduction of different carbohydrates,
including chemically modi®ed analogues.
The thiol-terminated globotriasylceramide mimic 3 was
prepared from 19, in a manner similar to that described
for the corresponding lactosylceramide mimic 1, via the
protected analogue 20 and ®nally 81% yield of 3 along
with 5% of the corresponding disul®de after deprotection
of the thioacetate followed by reduction. The carboxyl-
terminated globotriasylceramide mimic 4 was prepared
from 19 as described in the preparation of lactosylceramide
analogue 2, giving 82% of the protected intermediate 21 and
then 83% yield of 4 after deprotection.
Experimental
General
NMR spectra were recorded with a Bruker DRX-400 instru-
ment using residual CHCl3 as reference. H NMR spectral
1
assignments were made by COSY experiments. Concentra-
tions were made using rotary evaporation with a bath
temperature at or below 408C. Flash chromatography was
performed on Grace Amicon Silica gel 60 (0.035±
0.070 mm) and TLC was performed on Kieselgel 60 F254
plates (Merck). All non-aqueous reactions were run in
septum-capped, oven-dried ¯asks under Ar (1 atm).
CH2Cl2, toluene, and pyridine was distilled from CaH2.
Et2O was distilled from Na.
Synthesis of modi®ed phosphatidylcholine
Initial attempts using the S-acetyl protected fatty acid 8
turned out to give disappointingly low yield in the ®nal
de-S-acetylation of the corresponding phospholipid
analogue (shown in Fig. 2, prepared from 22). Selective
de-S-acylation was not accomplished in the presence of
the fatty acid esters under all conditions tried (for example,
methanolic NaOMe, methanolic NaSMe,22 and pyrrolidine
in CH2Cl2).23
S-Acetyl-16-mercaptohexadecanoic acid (8). To a solu-
tion of 16-hydroxyhexadecanoic acid (0.55 g, 2.0 mmol)
in AcOH (9.4 mL) was added aqueous HBr (48%, 9.4 mL)
and the solution was re¯uxed for 3 days. The product crys-
tallised upon cooling of the reaction mixture to room
temperature. The crystals were ®ltered off and washed
with ice-cold water. Drying gave the desired 16-bromo-
hexadecanoic acid 7 which was used in the next step without
further puri®cation.
As an alternative approach 16-(S-dithioethyl)hexadecanoic
acid24 was thus investigated (Scheme 4). The palmitoyl
derivative 22 was prepared as described8 from sn-3-O-
benzylglycerol and palmitic acid. Acylation of 22 with
16-(S-dithioethyl)hexadecanoic acid promoted by diiso-
propylcarbodiimide (DIC) in CH2Cl2/DMAP-solution,
gave the benzylprotected bis-acylglycerolderivative 23.
Debenzylation of 23 was performed with BCl3 in CH2Cl2
at 2788C25 yielding 24 in only 30%. One major side
reaction was disproportionation of the disul®de which
gave dimers via disul®de formation between the lipids.
This large molecule turned out to be dif®cult to handle
due to its poor solubility. In a ®rst attempt to circumvent
disul®de disproportion, changing the benzyl protection
group to p-methoxybenzyl, was expected to allow the
use of a milder Lewis' acid in the deprotection of 23.
Unfortunately the use of milder acids, such as BF3´Et2O,
SnCl4, or 1±5% CF3COOH, resulted in acyl migrations
leading to inseparable mixtures of regioisomers. The dis-
proportionation was ®nally solved by addition of diethyl
disul®de to the reaction mixture in the debenzylation of
23 under the original conditions with BCl3, which gave 24
in 74% yield.
To a solution of 16-bromohexadecanoic acid in freshly
destilled DMF (13 mL) was added AcSK (0.34 g,
3.0 mmol) and the mixture was stirred at 508C for 15 h,
then poured into Et2O/H2O. 1 M aqueous HCl was added
to the aqueous phase until pH 5±6 (moist pH-paper), which
was then extracted with Et2O (3£20 mL). The organic
phases were combined, dried (MgSO4), concentrated, and
¯ash chromatographed (SiO2, 10:1!2:1 heptane/EtOAc
gradient) to give 8 (0.60 g, 91%) as a pale yellow solid; n
(®lm) 2920, 2870, 1700 cm21; dH (400 MHz, CDCl3) 2.87
(t, 2H, Jˆ7.3 Hz, CH2SAc), 2.36 (t, 2H, Jˆ7.4 Hz,
CH2COOH), 2.33 (s, 3H, SAc), 1.63 (m, 4H, CH2CH2SAc,
CH2CH2COOH), 1.44±1.25 (m, 22H, CH2); dC
(100.6 MHz, CDCl3) 196.7, 179.9, 34.4, 31.1, 30.06,
30.04, 30.00, 29.92, 29.90, 29.85, 29.7, 29.60, 29.55, 29.5,
29.3, 25.1; HRMS (FAB): (M1H)1, found 331.2310.
C18H35O3S requires 331.2307.
Phosphorylation under dry conditions with PCl3/Et3N/
imidazole in freshly distilled toluene,26 coupling with
choline tosylate27 using 5,5-dimethyl-2-oxo-2-chloro-
1,3,2-dioxaphosphorinan (NPCl)28 as coupling agent, and
mild oxidation of the phosphatide with iodine, furnished
the phosphatidyl choline analogue 25. Compound 25
was deprotected under mild reducing conditions using
n-Bu3P in EtOH/H2O24 yielding the phosphatidyl choline
analogue 5.
N-(S-Acetyl-16-mercaptohexadecanoyloxy)-succinimide
(9). To a solution of 8 (300 mg, 0.91 mmol) in CH2Cl2
(8 mL) was added N-hydroxysuccinimide (418 mg,
3.63 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (725 mg, 3.63 mmol) and the mixture was stirred
overnight. The reaction mixture was diluted with CH2Cl2
(30 mL), washed with H2O (2£25 mL), dried (Na2SO4),
concentrated, and ¯ash chromatographed (SiO2, 4:1!2:1

J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
9979
heptane/EtOAc gradient) to give 9 (370 mg, 95%) as a white
solid; n (®lm) 2920, 2870, 1740, 1700 cm21; dH (400 MHz,
CDCl3) 2.87 (m, 6H, CH2SAc, COCH2CH2CO), 2.61 (t, 2H,
Jˆ7.4 Hz, CH2COON), 2.34 (s, 3H, SAc), 1.75 (dt, 2H,
Jˆ7.2, 7.9 Hz, CH2CH2COON), 1.59 (dt, 2H, Jˆ5.9,
7.5 Hz, CH2CH2SAc), 1.30±1.25 (m, 22H, CH2); dC
(100.6 MHz, CDCl3) 196.6, 169.6, 169.1, 31.4, 31.1,
30.04, 30.03, 29.99, 29.97, 29.92, 29.91, 29.8, 29.59,
29.55, 29.5, 29.3, 29.2, 26.0, 25.0; HRMS (FAB):
(M1H)1, found 428.2468. C22H38O3NS requires 428.2471.
3.98±3.30 (m, 11H), 3.20 (m, 1H), 3.10 (m, 1H, H-2), 2.65
(t, 2H, Jˆ7.3 Hz, CH2SAc), 2.08 (s, 3H, SAc), 1.95 (t, 2H,
Jˆ7.7 Hz, NCOCH2), 1.80 (m, 2H, vCH±CH2), 1.35 (m,
4H, NCOCH2CH2, CH2CH2SAc), 1.14±1.04 (m, 44H,
CH2), 0.68 (t, 3H, Jˆ6.6 Hz, CH3); dC (100.6 MHz,
CDCl3/CD3OD/D2O 65:35:10) 197.2, 174.5, 134.3, 129.2,
103.5, 102.7, 79.2, 75.4, 74.5, 73.2, 73.0, 71.7, 71.0, 68.8,
61.2, 60.4, 36.3, 32.2, 31.7, 30.2, 29.6, 29.53, 29.51, 29.49,
29.42, 29.35, 29.31, 29.25, 29.22, 29.19, 29.1, 28.99, 28.95,
28.6, 25.8, 22.5, 13.8; HRMS (FAB): (M1Na)1, found
958.5925. C48H89O14NSNa requires 958.5901.
16-(2-Methoxycarbonylethylthio)-hexadecanoic acid (10).
To a solution of crude 16-bromohexadecanoic acid 7
(200 mg), prepared as described for 8 in dry and
deoxygenated DMF (10 mL) was added Cs2CO3 (290 mg,
0.90 mmol) and methyl 3-mercaptopropionate (0.13 mL,
1.20 mmol) and the mixture was stirred at room temperature
for 15 h, then poured into Et2O/H2O. Aqueous HCl (1 M)
was added to the aqueous phase until pH 5±6 (moist pH-
paper). The aqueous phase was extracted with Et2O
(3£10 mL), the organic phases were combined, dried
(MgSO4), concentrated, and ¯ash chromatographed (SiO2,
4:1!3:1 heptane/EtOAc gradient) to give 10 (190 mg,
85%) as a white solid; n (®lm) 2920, 2870, 1750,
1700 cm21; dH (400 MHz, CDCl3) 3.71 (s, 3H, CH3), 2.79
(t, 2H, Jˆ7.4 Hz, CH2COOMe), 2.62 (t, 2H, Jˆ7.3 Hz,
SCH2CH2COOMe), 2.54 (t, 2H, Jˆ7.3 Hz, CH2S), 2.36 (t,
2H Jˆ7.5 Hz, CH2COOH), 1.66±1.55 (m, 4H,
CH2CH2COOH, CH2CH2S), 1.35±1.25 (m, 22H, CH2); dC
(100.6 MHz, CDCl3) 180.2, 172.9, 52.1, 35.1, 34.4, 32.5,
30.0, 29.91, 29.85, 29.8, 29.6, 29.4, 29.2, 27.3, 25.0;
HRMS (FAB): M1, found 374.2498. C20H38O4S requires
374.2491.
(2S,3R,4E)-3-Hydroxy-2-(16-mercaptohexadecanamido)-
octadec-4-enyl (b-d-galactopyranosyl)-(1!4)-b-d-gluco-
pyranoside (1). To a solution of 12 (40 mg, 0.044 mmol)
in MeOH (1.0 mL) and CH2Cl2 (1.0 mL) was added NaOMe
(0.150 mL, 1 M in MeOH). After 2 h, methanolic acetic
acid (20%) was added until neutral reaction on pH-paper
and the solution was evaporated. The residue was dissolved
in DMF (9.0 mL) followed by addition of dithioerythritol
(41 mg, 0.26 mmol) and diisopropylethylamine (0.023 mL,
0.13 mmol). After 5 h, the mixture was ®ltered through a
short SiO2-column (CH2Cl2/MeOH/H2O, 66:33:4), concen-
trated, and ¯ash chromatographed (SiO2, 7:1:0.2!7:3:0.2,
CH2Cl2/MeOH/H2O gradient) to give 1 (34 mg, 88%) as a
white solid; [a]D23ˆ12 (c 1.0, CHCl3/MeOH/H2O,
65:35:10); dH (400 MHz, CDCl3/CD3OD/D2O, 65:35:10)
7.33 (d, 1H, Jˆ9.2 Hz, NH), 5.46 (m, 1H, vCH±CH2),
5.22 (dd, 1H, Jˆ7.6 Hz, 15.4, vCH±CH(OH), 4.13 (d,
1H, Jˆ7.5 Hz, H-10), 4.08 (d, 1H, Jˆ7.8 Hz, H-1), 3.96
(dd, 1H, Jˆ4.3, 7.6 Hz, OCHaHb), 3.84 (t, 1H, Jˆ8.2 Hz,
CH(OH)), 3.74 (m, 1H, CH(NH)), 3.98±3.30 (m, 11H), 3.20
(m, 1H), 3.10 (m, 1H, H-2), 2.31 (t, 2H, Jˆ7.3 Hz, CH2SH),
1.96 (t, 2H, Jˆ7.4 Hz, NCOCH2), 1.82 (m, 2H, vCH±
CH2), 1.45±1.36 (m, 4H, NCOCH2CH2, CH2CH2SH),
1.14±1.06 (m, 44H, CH2), 0.68 (t, 3H, Jˆ6.6 Hz, CH3);
dC (100.6 MHz, CDCl3/CD3OD/D2O 65:35:10) 174.6,
134.6, 129.1, 103.3, 102.7, 78.8, 75.4, 74.8, 74.5, 73.03,
72.97, 71.6, 71.0, 68.7, 61.1, 60.2, 36.3, 33.9, 32.3, 31.8,
29.59, 29.56, 29.51, 29.47, 29.46, 29.42, 29.38, 29.32,
29.30. 29.21, 29.16, 28.9, 28.2, 25.9, 24.2, 22.5, 13.8;
(2S,3R,4E)-3-Hydroxy-2-(S-acetyl-16-mercaptohexadecan-
amido)octadec-4-enyl (b-d-galactopyranosyl)-(1!4)-b-d-
glucopyranoside (12). Compound 1110 (210 mg,
0.197 mmol) was dissolved in MeOH (15 mL), NaOMe
(0.080 mL, 1 M) was added, and the mixture was stirred
at room temperature overnight, then neutralised with
Amberlite IR-120 (H1) resin, ®ltered, concentrated, ¯ash
chromatographed (SiO2, 20:10, CH2Cl2/MeOH), and
concentrated. The residue (118 mg, 0.186 mmol) was
dissolved in pyridine (5.5 mL), Et3N (2.8 mL), and MeOH
(2.8 mL). The solution was saturated with H2S by bubbling
for 1 h at 08C. The mixture was kept under H2S at ambient
temperature for 15 h, N2 was bubbled through the mixture
for 1 h, and the mixture was concentrated and co-concen-
trated with toluene. The residue was dissolved in DMF
(10 mL) and compound 9 (119 mg, 0.28 mmol) and Et3N
(0.053 mL, 0.37 mmol) were added. The mixture was stirred
overnight, concentrated, and ¯ash chromatographed (SiO2,
30:10:1 CH2Cl2/MeOH/H2O). In order to remove residual
N-hydroxysuccinimide, the residue was further puri®ed on a
Bond±Elut C-18 column (1:9!1:0 MeOH/H2O gradient),
which gave pure 12 (145 mg, 85%) as a white solid;
[a]2D3ˆ11 (c 1.0, CHCl3/CH3OH/H2O, 65:35:10); n (®lm)
3850, 2920, 2870 cm21; dH (400 MHz, CDCl3/CD3OD/
D2O, 65:35:10) 7.33 (d, 1H, Jˆ9.2 Hz, NH), 5.46 (m, 1H,
vCH±CH2), 5.22 (dd, 1H, Jˆ7.6, 15.4 Hz, vCH±
CH(OH), 5.13 (d, 1H, Jˆ7.5 Hz, H-10), 5.08 (d, 1H,
Jˆ7.8 Hz, H-1), 3.96 (dd, 1H, Jˆ4.3, 7.6 Hz, OCHaHb),
3.84 (t, 1H, Jˆ8.2 Hz, CH(OH)), 3.74 (m, 1H, CH(NH)),
HRMS (FAB):
C46H87O13NSNa requires 916.5796.
(M1Na)1,
found
916.5784.
(2S,3R,4E)-3-O-Benzoyl-2-(16-(2-methoxycarbonylethyl-
thio)hexadecanamido)octadec-4-enyl (2,3,6-tri-O-acetyl-
b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl-b-d-
glucopyranoside (13). H2S was bubbled through a mixture
of 1110 (50 mg, 0.047 mmol) in pyridine/H2O (14 mL, 6:1)
at 08C for 1 h. The mixture was kept under H2S at ambient
temperature for 72 h, N2 was bubbled through the mixture
for 1 h, and the mixture was concentrated and co-concen-
trated with toluene. The residue was dissolved in CH2Cl2
(5 mL) and compound 10 (53 mg, 0.14 mmol) and EDC
(27 mg, 0.14 mmol) were added. The mixture was stirred
for 3 h, concentrated, and ¯ash chromatographed (SiO2,
3:1!1:1, heptane/EtOAc gradient) to give 13 (54 mg,
82%) as a white solid; [a]2D3ˆ21 (c 1.0, CHCl3); dH
(400 MHz, CDCl3) 8.01 (m, 2H, Ar-H), 7.55 (m, 1H,
Ar-H), 7.43 (m, 2H, Ar-H), 5.88 (m, 1H, vCH±CH2),
5.53±5.45 (m, 2H, vCH±CHOBz), 5.33 (d, 1H,
Jˆ2.6 Hz, H-40), 5.17 (t, 1H, Jˆ9.5 Hz, H-3), 5.08 (dd,
1H, Jˆ7.9, 10.4 Hz, H-20), 4.94 (dd, 1H, Jˆ3.4, 10.4 Hz,

9980
J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
H-30), 4.87 (dd, 1H, Jˆ7.8, 9.6 Hz, H-2), 4.49 (m, 3H, H-1,
H-10, CHNH), 4.35 (d, 1H, Jˆ9.6 Hz, H-6), 4.15±4.01 (m,
2H, H-60), 4.02±3.93 (m, 2H, H-6, CH2O), 3.87 (t, 1H,
Jˆ9.6 Hz, H-4), 3.69 (s, 3H, Me), 3.62 (dd, 1H, Jˆ4.6,
10.3 Hz, CH2O), 3.55 (m, 1H, H-5), 2.78 (t, 2H, Jˆ7.5 Hz,
CH2COOMe), 2.60 (t, 2H, Jˆ7.4 Hz, CH2CH2COOMe), 2.51
(t, 2H, Jˆ7.7 Hz, CH2SCH2CH2COOMe), 2.14, 2.07, 2.05,
2.02, 2.01, 1.96, 1.95 (7 s, 3H each, OAc), 2.03 (m, 2H,
vCH±CH2), 1.57 (m, 4H, NCOCH2, CH2CH2SCH2CH2-
COOMe), 1.35±1.20 (m, 44H, CH2), 0.87 (t, 3H, Jˆ6.6 Hz,
CH3); dC (100.6 MHz, CDCl3) 173.1, 172.9, 170.8, 170.7,
170.6, 170.5, 170.2, 170.1, 169.5, 165.6, 138.0, 133.5,
130.6, 130.0, 128.8, 125.0, 101.5, 100.7, 76.5, 74.5, 73.1,
73.0, 72.2, 71.4, 71.1, 69.4, 67.9, 67.0, 62.3, 61.2, 52.2,
51.0, 37.3, 35.1, 32.8, 32.6, 32.3, 30.1, 30.04, 29.97, 29.91,
29.87, 29.8, 29.7, 29.4, 29.3, 27.4, 26.2, 23.1, 21.2, 21.09,
21.07, 21.0, 20.9, 14.6; HRMS (FAB): (M1Na)1, found
1400.7142. C71H111O25NSNa requires 1400.7165.
added CH2Cl2 (12 mL) and Et2O (24 mL) and the solution
was cooled down to 2508C. Trimethylsilyl tri¯uoro-
methanesulfonate (0.020 mL, 0.110 mmol) was added and
the mixture was stirred for 2.5 h. Triethylamine (2 mL) was
added and the mixture was stirred for another 1 h at 2508C.
The mixture was allowed to obtain room temperature,
diluted with CH2Cl2 (20 mL), washed with 10% aqueous
Na2S2O3-solution (5 mL) and saturated aqueous NaHCO3-
solution (10 mL), dried (MgSO4), and concentrated. The
residue was ¯ash chromatographed (SiO2, 5:1!3:1,
heptane/EtOAc gradient) to give 16 (560 mg, 94%) as a
colourless oil; [a]2D3ˆ155 (c 1.0, CHCl3); n (®lm) 1740,
1260 cm21; dH (400 MHz, CDCl3) 8.06±7.09 (m, 50H,
Ar-H), 5.85±5.75 (m, 2H, H-300, H-20), 5.39 (dd, 1H,
Jˆ7.9, 9.5 Hz, H-200), 5.06 (dd, 1H, Jˆ2.7, 10.7 Hz,
H-30), 4.91 (d, 1H, Jˆ7.8 Hz, H-10), 4.84 (d, 1H,
Jˆ11.1 Hz, H-1), 4.79±4.50 (m, 10H, H-100, H-60), 4.48
(d, 1H, Jˆ2.3 Hz, H-40), 4.33 (d, 1H, Jˆ2.3 Hz, H-40),
4.31±4.18 (m, 5H, H-4, H-60), 4.11 (m, 1H), 4.00±3.88
(m, 4H), 3.71 (t, 1H, Jˆ6.4 Hz, H-50), 3.53 (dt, 1H,
Jˆ6.6, 10.1 Hz, OCH2CH2Si), 3.38 (t, 1H, Jˆ8.8 Hz, H-
6), 3.02 (dd, 1H, Jˆ4.9, 8.5 Hz, H-6), 0.85 (m, 2H,
CH2Si), 20.10 (s, 9H, SiMe3); dC (100.6 MHz, CDCl3)
166.9, 166.3, 166.1, 165.8, 165.5, 139.4, 139.3, 138.7,
133.7, 133.54, 133.45, 130.31, 130.29, 130.25, 130.21,
130.16, 130.1, 130.0, 129.0, 128.84, 128.76, 128.73,
128.70, 128.6, 128.48, 128.45, 128.1, 127.9, 127.8,
127.74, 127.71, 101.7, 101.6, 100.7, 79.6, 77.0, 76.3, 75.4,
75.2, 74.9, 73.8, 73.7, 73.6, 73.5, 73.4, 73.1, 72.9, 70.3,
70.2, 67.9, 67.8, 63.1, 62.5, 18.3, 21.1; HRMS (FAB):
(M1Na)1, found 1611.5756. C93H92O22SiNa requires
1611.5747.
(2S,3R,4E)-3-Hydroxy-2-(16-(1-thio)propionylhexadeca-
namido)octadec-4-enyl (b-d-galactopyranosyl)-(1!4)-
b-d-glucopyranoside (2). To a solution of 13 (54 mg,
0.039 mmol) in MeOH (5.0 mL) and CH2Cl2 (4.0 mL)
was added NaOMe (0.05 mL, 1 M in MeOH). After 16 h,
methanolic acetic acid (20%) was added until neutral
reaction (moist pH-paper) and the solution was ®ltered,
concentrated and ¯ash chromatographed (SiO2, 5:1!2:1,
CH2Cl2/EtOH gradient) to give the corresponding ester.
To a solution of the ester in CH2Cl2 (1.5 mL), MeOH
(2.5 mL) and H2O (1.0 mL) was added 1 M aqueous
NaOH (0.10 mL) and the resulting mixture was stirred for
24 h. Methanolic acetic acid (20%) was added until neutral
reaction on pH-paper and the solution was concentrated.
Flash chromatography of the residue (SiO2, 70:20:2!
66:33:4, CHCl3/MeOH/H2O gradient) gave 2 (24 mg,
64%) as a white solid; [a]2D3ˆ11 (c 1.0, CHCl3/MeOH/
H2O, 65:35:10); dH (400 MHz, CDCl3CD3OD/D2O,
65:35:10) 7.33 (d, 1H, Jˆ9.2 Hz, NH), 5.42 (m, 1H,
vCH±CH2), 5.22 (dd, 1H, Jˆ7.6, 15.4 Hz, vCH±
CH(OH)), 4.08 (d, 1H, Jˆ7.5 Hz, H-10), 4.03 (d, 1H,
Jˆ7.8 Hz, H-1), 3.96 (dd, 1H, Jˆ4.3, 7.6 Hz, OCHaHb),
3.84 (t, 1H, Jˆ8.2 Hz, CH(OH)), 3.74 (m, 1H, CH(NH)),
3.98±3.30 (m, 11H), 3.20 (m, 1H), 3.10 (m, 1H, H-2), 2.49
(t, 2H, Jˆ7.5 Hz, CH2COOH), 2.29 (m, 4H, CH2SCH2),
1.88 (t, 2H, Jˆ7.4 Hz, NCOCH2), 1.74 (m, 2H, vCH±
CH2), 1.45±1.36 (m, 4H, NCOCH2CH2, CH2CH2SCH2-
CH2COOH), 1.14±0.94 (m, 44H, CH2), 0.61 (t, 3H,
Jˆ6.6 Hz, CH3); dC (100.6 MHz, CDCl3/CD3OD/D2O
65:35:10) 174.5, 134.2, 129.1, 103.4, 102.7, 79.1, 77.4,
75.4, 74.8, 74.5, 73.1, 73.0, 71.6, 70.9, 68.7, 68.4, 67.6,
61.1, 60.2, 53.0, 36.2, 35.1, 33.9, 32.1, 31.7, 31.6, 29.4,
29.3, 29.2, 29.10, 29.06, 29.0, 28.9, 28.6, 26.9, 25.7, 25.2,
22.4, 13.6; HRMS (FAB): (M1Na)1, found 958.5925.
C48H89O14NSNa requires 958.5901.
2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-acetyl-a-d-galacto-
pyranosyl)-(1!4)-(2,3,6-tri-O-benzoyl-b-d-galactopyrano-
syl)-(1!4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside (17).
Compound 16 (0.50 g, 0.31 mmol) was dissolved in
AcOH (13 mL) and hydrogenated (H2, 1 atm, 10% Pd/C,
0.20 g) for 5 h. The mixture was ®ltered through Celite
and concentrated. The residue was dissolved in pyridine
(15 mL), Ac2O (12 mL) was added, and the mixture was
stirred overnight, then concentrated and ¯ash chromato-
graphed (SiO2, 2:1!1:1, heptane/EtOAc gradient) to give
17 (0.40 g, 91%) as a colourless oil; [a]2D3ˆ182 (c 1.0,
CHCl3); n (®lm) 2920, 1730, 1250 cm21; dH (400 MHz,
CDCl3) 8.06±7.21 (m, 30H, Ar-H), 5.80 (t, 1H, Jˆ9.2 Hz,
H-3), 5.69 (dd, 1H, Jˆ7.8, 10.8 Hz, H-20), 5.48 (d, 1H,
Jˆ2.2 Hz, H-400), 5.40 (dd, 1H, Jˆ7.9, 9.5 Hz, H-2), 5.34
(dd, 1H, Jˆ3.3, 11.0 Hz, H-300), 5.14 (m, 2H, H-30, H-200),
5.06 (d, 1H, Jˆ3.7 Hz, H-100), 4.81 (d, 1H, Jˆ7.8 Hz, H-10),
4.70 (d, 1H, Jˆ7.9 Hz, H-1), 4.59 (dd, 1H, Jˆ1.9, 11.9 Hz,
H-6), 4.48 (m, 2H, H-6, H-500), 4.26 (d, 1H, Jˆ2.5 Hz,
H-40), 4.21 (t, 1H, Jˆ9.5 Hz, H-4), 4.03±3.86 (m, 4H,
H-5, H-60, OCHaHbCH2Si), 3.79 (d, 1H, Jˆ8.0 Hz, H-600),
3.77 (m, 2H, H-50, H-600), 3.53 (dt, 1H, Jˆ6.6, 10.1 Hz,
OCHaHbCH2Si), 2.06, 2.02, 1.96, 1.93 (4 s, 3H each,
OAc), 0.85 (m, 2H, CH2Si), 20.10 (s, 9H, SiMe3); dC
(100.6 MHz, CDCl3) 170.9, 170.8, 170.5, 170.0, 166.6,
166.2, 165.9, 165.7, 165.5, 165.4, 134.1, 134.0, 133.7,
133.54, 133.50, 130.2, 130.10, 130.08, 130.05, 130.0,
129.9, 129.7, 129.09, 129.06, 129.0, 128.9, 128.8, 128.7,
101.7, 100.6, 100.7, 79.6, 74.0, 73.8, 73.3, 73.0, 72.6,
70.0, 68.7, 68.2, 67.9, 67.6, 61.2, 21.12, 21.07, 21.0, 18.3,
2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzyl-a-d-galacto-
pyranosyl)-(1!4)-(2,3,6-tri-O-benzoyl-b-d-galactopyrano-
syl)-(1!4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside (16).
To a mixture of 2-(trimethylsilyl)ethyl (2,3,6-tri-O-benzoyl
b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzoyl-b-d-glu-
copyranoside13 (400 mg, 0.368 mmol), phenyl 2,3,4,6-tetra-
O-benzyl-1-thio-b-d-galactopyranoside11,12 (330 mg, 0.515
mmol), and N-iodosuccinimide (210 mg, 0.92 mmol) were

J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
9981
21.1; HRMS (FAB): (M1Na)1, found 1419.4272.
C73H76O26SiNa requires 1419.4291.
10.0 Hz, H-60), 2.06, 2.02, 1.96, 1.92 (4 s, 3H each, OAc),
1.88 (m, 2H, vCH±CH2), 1.30±1.15 (m, 22H, CH2), 0.88
(t, 3H, Jˆ6.7 Hz, CH3); dC (100.6 MHz, CDCl3) 170.9,
170.8, 170.5, 170.0, 166.6, 166.2, 165.9, 165.5, 165.40,
165.36, 139.4, 134.1, 134.0, 133.7, 133.64, 133.55, 133.5,
130.31, 130.26, 130.2, 130.14, 130.07, 130.0, 129.94,
129.92, 129.7, 129.0, 128.9, 128.81, 128.79, 128.78,
122.8, 102.0, 100.9, 98.8, 75.7, 74.0, 73.7, 73.4, 73.1,
72.4, 70.0, 68.62, 68.55, 68.2, 67.9, 67.6, 63.8, 62.7, 61.7,
61.2, 32.7, 32.4, 30.12, 30.09, 30.06, 30.0, 29.79, 29.78,
29.5, 29.0, 23.1, 21.10, 21.07, 21.0, 14.6; HRMS (FAB):
(M1Na)1, found 1730.6440. C93H101O28N3Na requires
1730.6469.
(2,3,4,6-Tetra-O-acetyl-a-d-galactopyranosyl)-(1!4)-
(2,3,6-tri-O-benzoyl-b-d-galactopyranosyl)-(1!4)-2,3,6-
tri-O-benzoyl-a-d-glucopyranosyl trichloroacetimidate
(18). To a solution of 17 (281 mg, 0.198 mmol) in dry
CH2Cl2 (1.33 mL) was added tri¯uoroacetic acid
(2.70 mL) and the mixture was stirred at ambient tempera-
ture. After 1.5 h n-propylacetat (11 mL) and toluene
(11 mL) were added and the mixture was concentrated
and co-concentrated with toluene to give the corresponding
hemiacetal. The crude hemiacetal was dissolved in dry
CH2Cl2 (2.5 mL), and trichloroacetonitrile (0.75 mL) was
added. The mixture was cooled down to 08C, 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU, 0.045 mL, 0.30 mmol)
was added, the reaction mixture was stirred for 1.5 h, diluted
with CH2Cl2 (10 mL), washed with ice-cold saturated
aqueous NaHCO3 (5 mL), dried (Na2SO4), and concen-
trated. The residue was ¯ash chromatographed (SiO2,
2:1:0.01!1:1:0.01, heptane/EtOAc/Et3N gradient) to give
18 (220 mg, 76%) as a white solid; [a]2D3ˆ182 (c 1.0,
CHCl3); n (®lm) 3090, 2950, 1740 cm21; dH (400 MHz,
CDCl3) 8.55 (s, 1H, NH), 8.10±7.20 (m, 30H, Ar-H), 6.69
(d, 1H, Jˆ3.7 Hz, H-1), 6.18 (dd, 1H, Jˆ8.6, 10.0 Hz, H-3),
5.74 (dd, 1H, Jˆ7.9, 10.8 Hz, H-20), 5.47 (m, 2H, H-2, H-
100), 5.35 (dd, 1H, Jˆ3.3, 11.0 Hz, H-200), 5.15 (m, 2H, H-30,
H-300), 5.08 (d, 1H, Jˆ3.6 Hz, H-400), 4.93 (d, 1H, Jˆ7.8 Hz,
H-10), 4.56 (m, 2H, H-6), 4.46 (t, 1H, Jˆ7.2 Hz, H-50), 4.31
(m, 3H, H-4, H-5, H-40), 3.96 (m, 2H, H-60), 3.75 (dd, 1H,
Jˆ8.1, 11.0 Hz, H-600), 3.65 (m, 2H, H-50, H-600), 2.06, 2.02,
1.95, 1.91 (4 s, 3H each, OAc); dC (100.6 MHz, CDCl3)
170.9, 170.8, 170.6, 170.0, 166.6, 166.1, 166.0, 165.8,
165.5, 165.3, 161.1, 134.1, 134.0, 133.8, 133.7, 133.6,
130.4, 130.2, 130.07, 130.05, 130.03, 130.00, 129.95,
129.6, 129.1, 129.02, 129.01, 128.97, 128.95, 128.91,
128.88, 128.86, 128.8, 102.4, 98.7, 93.4, 91.1, 75.4, 74.1,
72.9, 71.6, 71.2, 71.0, 70.1, 68.6, 68.2, 67.9, 67.5, 62.3,
61.3, 61.1, 21.12, 21.09, 21.0; HRMS (FAB): (M1Na)1,
found 1462.2655. C70H64O25NCl3Na requires 1462.2680.
(2S,3R,4E)-3-Hydroxy-2-(S-acetyl-16-mercaptohexade-
canamido)octadec-4-enyl (a-d-galactopyranosyl)-(1!4)-
(b-d-galactopyranosyl)-(1!4)-b-d-glucopyranoside (20).
To a solution of 19 (204 mg, 0.12 mmol) in MeOH
(15 mL) was added NaOMe (0.050 mL, 1 M in MeOH)
and the resulting solution was stirred overnight, then
quenched by addition of methanolic acetic acid (10%)
until neutral reaction to pH-paper. Concentration and ¯ash
chromatography (SiO2, CH2Cl2/MeOH/H2O, 66:33:4) gave
the deprotected intermediate globotriasyl azidosphingosine
(90 mg, 91%). H2S was bubbled through a solution of the
residue (50 mg, 0.059 mmol) in pyridine (3 mL), MeOH
(1.5 mL), and Et3N (1.5 mL) at 08C for 1 h. The solution
was allowed to reach room temperature and after 24 h, N2
was bubbled through the solution for 1 h. The mixture was
concentrated and co-concentrated with toluene. To the resi-
due in DMF (3 mL) was added 9 (38 mg, 0.089 mmol) and
Et3N (0.017 mL, 0.12 mmol) and the mixture was stirred at
room temperature overnight. The mixture was evaporated
and the residue was ¯ash chromatographed (SiO2, CH2Cl2/
MeOH/H2O, 70:20:3) In order to remove residual N-hydro-
xysuccinimide the product was further puri®ed on a Bond±
Elut C-18 column (1:9!1:0 MeOH/H2O gradient), which
gave 20 (53 mg, 82%) as a white solid; [a]2D3ˆ128 (c 1.0,
CHCl3/MeOH/H2O, 66:33:10); dH (400 MHz, CDCl3/
CD3OD/D2O, 65:35:10) 7.33 (d, 1H, Jˆ9.1 Hz, NH), 5.47
(m, 1H, vCH±CH2), 5.21 (dd, 1H, Jˆ7.6, 15.4 Hz, vCH±
CH(OH)), 4.71 (d, 1H, Jˆ3.2 Hz, H-100), 4.21 (d, 1H,
Jˆ7.4 Hz, H-10), 4.10 (d, 1H, Jˆ7.7 Hz, H-1), 4.01 (t, 1H,
Jˆ6.1 Hz, H-500), 3.95 (dd, 1H, Jˆ4.3, 10.2 Hz, OCHaHb),
3.83 (t, 1H, Jˆ8.0 Hz, CH(OH)), 3.76±3.30 (m, 17H), 3.20
(m, 1H), 3.12 (m, 1H, H-2), 2.65 (t, 2H, Jˆ7.3 Hz,
CH2SAc), 1.95 (t, 2H, Jˆ7.7 Hz, NCOCH2), 1.80 (m, 2H,
vCH±CH2), 1.35 (m, 4H, NCOCH2CH2, CH2CH2SAc),
1.14±1.04 (m, 44H, CH2), 0.68 (t, 3H, Jˆ6.6 Hz, CH3);
dC (100.6 MHz, CDCl3/CD3OD/D2O, 65:35:10) 197.3,
174.6, 134.6, 129.1, 103.6, 102.6, 100.9, 79.1, 78.4, 77.4,
75.0, 74.8, 73.0, 72.8, 72.6, 72.3, 71.0, 69.5, 69.3, 68.8,
61.1, 60.3, 36.3, 32.3, 31.8, 30.2, 29.60, 29.55, 29.50,
29.47, 29.45, 29.4, 29.3, 29.24, 29.19, 29.1, 29.01, 28.95,
28.6, 25.9, 22.5, 13.8; HRMS (FAB): (M1Na)1, found
1120.6418. C54H99O19NSNa requires 1120.6430.
(2S,3R,4E)-2-Azido-3-benzoyloxyoctadec-4-enyl (2,3,4,
6-tetra-O-acetyl-a-d-galactopyranosyl)-(1!4)-(2,3,6-
tri-O-benzoyl-b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-
benzoyl-b-d-glucopyranoside (19). To a solution of 18
(220 mg, 0.15 mmol) and (2S,3R,4E)-2-azido-3-benzoyl-
oxy-octadec-4-ene-1-ol19±21 (97 mg, 0.23 mmol) in
CH2Cl2 (8.0 mL) was added MS 300 AW (780 mg) and
the resulting suspension was stirred for 1 h whereafter
BF3´Et2O (0.028 mL, 0.23 mmol) was added. After 2.5 h,
Et3N (0.30 mL) was added, the mixture was ®ltered through
Celite, concentrated, and ¯ash chromatographed (SiO2,
heptane/EtOAc gradient, 2:1!1:1) to give 19 (219 mg,
83%) as a white solid; [a]2D3ˆ154 (c 1.0, CHCl3) n (®lm)
2950, 2380, 2100, 1750 cm21; dH (400 MHz, CDCl3)
8.07±7.20 (m, 30H, Ar-H), 5.81 (t, 1H, Jˆ8.9 Hz, H-3),
5.72±5.64 (m, 2H, H-20, vCH±CH2), 5.54±5.34 (m, 5H,
H-2, H-300, H-400, vCH±CHOBz), 5.15 (m, 2H, H-30,
H-200), 5.06 (d, 1H, Jˆ3.6 Hz, H-100), 4.89 (d, 1H,
Jˆ7.9 Hz, H-10), 4.73 (d, 1H, Jˆ7.4 Hz, H-1), 4.62 (m,
1H, H-6), 4.48 (m, 2H, H-6, H-500), 4.27 (m, 2H, H-4,
H-40), 4.04±3.78 (m, 6H, H-5, H-50, H-60, H-600, CHN3,
CH2O), 3.68 (m, 2H, H-600, CH2O), 3.52 (dd, 1H, Jˆ6.0,
(2S,3R,4E)-3-Hydroxy-2-(16-mercaptohexadecanamido)-
octadec-4-enyl (a-d-galactopyranosyl)-(1!4)-(b-d-galacto-
pyranosyl)-(1!4)-b-d-glucopyranoside (3). To a solution
of 20 (15 mg, 0.014 mmol) in MeOH (0.4 mL) and CH2Cl2
(0.2 mL) was added NaOMe (0.034 mL, 1 M in MeOH).
After 2 h methanolic acetic acid (10%) was added until

9982
J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
neutral reaction on pH-paper and the solution was evapo-
rated. The residue was dissolved in DMF (2.0 mL) followed
by addition of dithioerythritol (13 mg, 0.082 mmol) and
diisopropylethylamine (0.007 mL, 0.041 mmol). After
2.5 h, the mixture was ®ltered through a short column
(SiO2, CH2Cl2/MeOH/H2O, 66:33:4), concentrated, and
¯ash chromatographed (SiO2, CH2Cl2/MeOH/H2O,
70:20:3 to give 3 (12 mg, 81%) as a white solid;
[a]2D3ˆ127 (c 1.0, CHCl3/MeOH/H2O, 66:33:10); dH
(400 MHz, CDCl3/CD3OD/D2O, 65:35:10) 7.33 (d, 1H,
Jˆ9.1 Hz, NH), 5.47 (m, 1H, vCH±CH2), 5.21 (dd, 1H,
Jˆ7.6, 15.4 Hz, vCH±CH(OH)), 4.71 (d, 1H, Jˆ3.2 Hz,
H-100), 4.21 (d, 1H, Jˆ7.4 Hz, H-10), 4.10 (d, 1H, Jˆ7.7 Hz,
H-1), 4.02 (t, 1H, Jˆ6.2 Hz, H-500), 3.95 (dd, 1H, Jˆ4.3,
10.2 Hz, OCHaHb), 3.83 (t, 1H, Jˆ8.0 Hz, CH(OH)), 3.76±
3.30 (m, 17H), 3.20 (m, 1H), 3.12 (m, 1H, H-2), 2.31 (t, 2H,
Jˆ7.3 Hz, CH2SH), 1.95 (t, 2H, Jˆ7.6 Hz, NC(O)OCH2),
1.79 (m, 2H, vCH±CH2), 1.43±1.31 (m, 4H,
NC(O)OCH2CH2, CH2CH2SH), 1.10±1.03 (m, 44H, CH2),
0.67 (t, 3H, Jˆ6.6 Hz, CH3); dC (100.6 MHz, CDCl3/
CD3OD/D2O, 65:35:10) 174.6, 134.5, 129.0, 103.6, 102.6,
101.0, 79.1, 78.5, 77.4, 75.3, 74.9, 74.8, 72.8, 71.6, 71.3,
71.0, 69.4, 69.3, 68.7, 61.1, 55.3, 36.3, 33.8, 32.2, 31.7,
29.7, 29.52, 29.50, 29.46, 29.45, 29.4, 29.3, 29.22, 29.19,
29.1, 29.01, 28.97, 28.1, 25.8, 22.4, 13.7; HRMS (FAB):
(M1Na)1, found 1078.6337. C52H97O18NSNa requires
1078.6324.
129.95, 129.9, 129.7, 129.5, 129.1, 129.03, 128.99,
128.95, 128.9, 128.8, 128.7, 125.2, 101.8, 101.1, 98.7,
74.5, 74.0, 73.5, 73.3, 73.0, 72.8, 69.9, 68.7, 68.2, 67.9,
67.6, 61.2, 52.2, 50.8, 36.9, 35.2, 32.7, 32.6, 32.4, 30.13,
30.10, 30.07, 30.05, 29.99, 29.97, 29.9, 29.8, 29.68, 29.67,
29.6, 29.34, 29.32, 27.4, 25.9, 23.1, 21.09, 21.06, 21.0, 14.5;
HRMS
C113H139O31NSNa requires 2060.8949.
(FAB):
(M1Na)1,
found
2060.8931.
(2S,3R,4E)-3-Hydroxy-2-(16-(1-thio)propionylhexadeca-
namido)octadec-4-enyl (a-d-galactopyranosyl)-(1!4)-
(b-d-galactopyranosyl)-(1!4)-b-d-glucopyranoside (4).
To a solution of 21 (50 mg, 0.024 mmol) in MeOH
(4.0 mL) and CH2Cl2 (1.0 mL) was added NaOMe
(0.050 mL, 1 M in MeOH). After 16 h, methanolic acetic
acid (10%) was added until neutral reaction on pH-paper
and the solution was ®ltered, concentrated and ¯ash chro-
matographed (SiO2, CHCl3/MeOH/H2O, 66:33:4) to give
the corresponding ester. To a solution of the ester in
CH2Cl2 (2.0 mL), MeOH (3.0 mL), and H2O (1.0 mL) was
added 1 M aqueous NaOH (0.10 mL) and the resulting
mixture was stirred overnight. Methanolic acetic acid
(10%) was added until neutral reaction on pH-paper and
the solution was concentrated. Flash chromatography of
the residue (SiO2, CHCl3/MeOH/H2O, 66:33:4!66:33:10)
gave 4 (22 mg, 83%) as a white solid; [a]2D3ˆ127 (c 1.0,
CHCl3/MeOH/H2O, 66:33:10); dH (400 MHz, CDCl3/
CD3OD/D2O, 65:35:10) 7.33 (d, 1H, Jˆ9.1 Hz, NH), 5.47
(m, 1H, vCH±CH2), 5.21 (dd, 1H, Jˆ7.6, 15.4 Hz, vCH±
CH(OH)), 4.71 (d, 1H, Jˆ3.2 Hz, H-100), 4.21 (d, 1H,
Jˆ7.4 Hz, H-10), 4.10 (d, 1H, Jˆ7.7 Hz, H-1), 4.02 (t, 1H,
Jˆ6.2 Hz, H-500), 3.95 (dd, 1H, Jˆ4.3, 10.2 Hz, OCHaHb),
3.83 (t, 1H, Jˆ8.0 Hz, CH(OH)), 3.76±3.30 (m, 17H), 3.20
(m, 1H), 3.12 (m, 1H, H-2), 2.54 (t, 2H, Jˆ7.5 Hz,
CH2COOH), 2.34 (m, 4H, CH2SCH2), 1.94 (t, 2H,
Jˆ7.6 Hz, NC(O)OCH2), 1.79 (m, 2H, vCH±CH2),
1.40±1.32 (m, 4H, NC(O)OCH2CH2, CH2CH2S), 1.15±
1.03 (m, 44H, CH2), 0.66 (t, 3H, Jˆ6.6 Hz, CH3); dC
(100.6 MHz, CDCl3/CD3OD/D2O, 65:35:10) 174.6, 134.6,
129.1, 103.6, 102.6, 100.9, 77.5, 75.0, 74.8, 74.4, 73.0, 72.7,
71.6, 71.2, 71.0, 69.4, 69.3, 68.73, 68.69, 61.0, 60.2, 60.1,
53.0, 31.9, 31.7, 29.6, 29.53, 29.48, 29.44, 29.43, 29.39,
29.37, 29.33, 29.30, 29.25, 29.2, 29.1, 29.0, 28.7, 26.9,
22.5, 13.8; HRMS (FAB): (M1Na)1, found 1150.6500.
C55H101O20NSNa requires 1150.6535.
(2S,3R,4E)-3-Hydroxy-2-(16-(2-methoxycarbonylethyl-
thio)hexadecanamido)octadec-4-enyl (2,3,4,6-tetra-O-
acetyl-a-d-galactopyranosyl)-(1!4)-(2,3,6-tri-O-benzoyl-
b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzoyl-b-d-
glucopyranoside (21). H2S was bubbled through a solution
of 19 (58 mg, 0.033 mmol) in pyridine/H2O (14 mL, 6:1) at
08C for 1 h. The solution was allowed to reach room
temperature and after 72 h, N2 was bubbled through the
solution for 1 h. The mixture was concentrated and co-
concentrated with toluene. To the residue in CH2Cl2
(3 mL) was added 10 (37 mg, 0.10 mmol) and EDC
(19 mg, 0.10 mmol) and the mixture was stirred at room
temperature for 2 h. The mixture was concentrated and the
residue was ¯ash chromatographed (SiO2, heptane/EtOAc
gradient, 3:1!1:1) to give 21 (56 mg, 82%) as a white solid;
[a]2D3ˆ160 (c 1.0, CHCl3); dH (400 MHz, CDCl3) 8.08±
7.21 (m, 35H, Ar-H), 5.82±5.72 (m, 2H, H-3, vCH±
CH2), 5.70±5.60 (m, 2H, H-20, NH), 5.47 (m, 2H, H-400,
CHOBz), 5.42±5.30 (m, 3H, H-2, H-300, vCH±CHOBz),
5.15±5.10 (m, 2H, H-30, H-200), 5.05 (d, 1H, Jˆ3.7 Hz, H-
100), 4.82 (d, 1H, Jˆ7.9 Hz, H-10), 4.63 (d, 1H, Jˆ7.7 Hz, H-
1), 4.47±4.37 (m, 4H, H-6, H-500, CHNH), 4.25 (d, 1H,
Jˆ2.5 Hz, H-40), 4.17 (t, 1H, Jˆ9.5 Hz, H-4), 4.07 (dd,
1H, Jˆ3.1, 10.1 Hz, CH2O), 3.96 (m, 2H, H-60), 3.85±
3.75 (m, 2H, H-5, H-600), 3.70±3.65 (m, 5H, H-50, H-600,
Me), 3.55 (dd, 1H, Jˆ3.8, 9.9 Hz, CH2O), 2.79 (t, 2H,
Jˆ7.5 Hz, CH2COOMe), 2.62 (t, 2H, Jˆ7.5 Hz,
CH2CH2COOMe), 2.53 (t, 2H, Jˆ7.3 Hz, CH2SCH2CH2-
COOMe), 2.06, 2.01, 1.95, 1.93 (4 s, 3H each, OAc), 2.03
(m, 2H, vCH±CH2), 1.78 (t, 2H Jˆ7.0 Hz, NHCHOCH2),
1.58 (m, 2H, CH2CH2SCH2CH2COOMe), 1.38±1.10 (m,
44H, CH2), 0.88 (t, 3H, Jˆ6.6 Hz, CH3); dC (100.6 MHz,
CDCl3) 173.0, 170.9, 170.8, 170.5, 170.0, 166.6, 166.1,
165.9, 165.8, 165.6, 165.4, 165.3, 137.7, 134.0, 133.7,
133.5, 133.3, 130.6, 130.2, 130.08, 130.05, 130.03,
2-O-(16-(Ethyldithio)hexadecanoyl)-1-O-hexadecanoyl-
3-O-benzyl-sn-glycerol (23). To a solution of 1-O-hexa-
decanoyl-3-O-benzyl-sn-glycerol (22)8 (118 mg, 0.28
mmol) and 16-(ethyldithio)hexadecanoic acid24 (108 mg,
0.31 mmol) in CH2Cl2 (3.0 mL) at 08C was added N,N-
dimethylamino-4-pyridine (DMAP, 25 mg). A solution of
diisopropylcarbodiimide (DIC, 0.052 mL, 0.34 mmol) in
CH2Cl2 (1.0 mL) was added dropwise over 10 min, and
the mixture was stirred at room temperature for 5 h. The
reaction mixture was ®ltered through Celite, the ®ltrate
was concentrated, and ¯ash chromatographed (SiO2,
heptane/EtOAc 5:1) to give 23 (200 mg, 96%) as a white
solid; n (®lm) 2950, 2870, 1750 cm21; [a]D23ˆ15 (c 1.0,
CHCl3); dH (400 MHz, CDCl3) 7.37±7.29 (m, 5H, Ar-H),
5.25 (m, 1H, H-sn2), 4.57/4.53 (ABq, 2H, Jˆ12.1, 18.0 Hz,
OCH2Ph), 4.20 (dd, 1H, Jˆ3.9, 12.0 Hz, H-sn1), 4.35 (dd,
1H, Jˆ6.4, 11.9 Hz, H-sn1), 3.60 (dd, 2H, Jˆ4.3, 5.2 Hz,

J. Ohlsson, G. Magnusson / Tetrahedron 56 (2000) 9975±9984
9983
H-sn3), 2.68 (m, 4H, CH2SSCH2), 2.35±2.24 (m, 4H,
OCOCH2), 1.70±1.55 (m, 6H, OCOCH2CH2, CH2CH2S),
1.35±1.23 (m, 49H, CH2, SCH2CH3), 0.89 (t, 3H,
Jˆ6.6 Hz, CH3); dC (100.6 MHz, CDCl3) 173.6, 173.3,
137.9, 128.6, 128.0, 127.8, 73.5, 70.2, 68.5, 62.9, 39.5,
34.6, 33.0, 32.2, 29.9, 29.7, 29.6, 29.51, 29.48, 29.34,
29.31, 28.8, 25.2, 25.1, 24.8, 22.9, 14.6, 14.4; HRMS
(FAB): (M1H)1, found 751.5377. C44H79O5S2 requires
751.5369.
chromatography of the residue (SiO2, CHCl3/MeOH/H2O
66:33:4) gave a crude product that was dissolved in THF/
H2O (10:1) and eluted through an ion-exchange column
(TMD-8, conditioned in the same solvents) to give 25
(16 mg, 41%) as a white solid; [a]2D3ˆ14 (c 1.6, CHCl3/
CH3OH/H2O, 65:35:10); dH (400 MHz, CDCl3/CD3OD/
D2O, 65:35:10) 4.99 (m, 1H, H-sn2), 4.18 (dd, 1H, Jˆ3.1,
12.1 Hz, H-sn1), 4.01 (m, 2H, b-CH2), 3.92 (dd, 1H, Jˆ7.1,
12.0 Hz, H-sn1), 3.75 (dt, 2H, Jˆ5.8, 6.4 Hz, H-sn3), 3.38
(m, 2H, a-CH2), 2.98 (s, 9H, N(Me)3), 2.40 (m, 4H,
CH2SSCH2), 2.12±2.04 (m, 4H, OCOCH2), 1.40±1.25 (m,
6H, OCOCH2CH2, SCH2CH2), 1.10±1.00 (m, 49H, CH2,
SCH2CH3), 0.64 (t, 3H, Jˆ6.6 Hz, CH3); dC (100.6 MHz,
CDCl3/CD3OD/D2O, 65:35:10) 174.1, 173.7, 70.3
(Jˆ7.9 Hz), 66.2, 63.5 (Jˆ5.4 Hz), 62.7, 59.0, 53.9,
(Jˆ3.6 Hz), 39.1, 34.1, 34.0, 32.6, 31.8, 29.6, 29.51,
29.45, 29.42, 29.39, 29.36, 29.21, 29.17, 29.1, 29.01,
28.98, 28.4, 24.8, 24.7, 22.5, 14.2, 13.8; HRMS (FAB):
(M1H)1, found 826.5466. C42H85O8NPS2 requires
826.5454.
2-O-(16-(Etyldithio)hexadecanoyl)-1-O-hexadecanoyl-
sn-glycerol (24). To a solution of 23 (100 mg, 0.127 mmol)
and diethyl disul®d (0.078 mL, 0.064 mmol) in CH2Cl2
(5.0 mL) at 2788C was added dropwise over 10 min a
1 M solution of BCl3 in hexane (0.57 mL, 0.57 mmol).
The mixture was stirred at 2788C for 30 min. More BCl3
(0.57 mL) was added and the resulting mixture was stirred
for another 30 min. The reaction mixture was poured into
ice-water (5 mL) and the aqueous phase was extracted with
CH2Cl2 (2£5 mL). The organic phases were combined,
dried (Na2SO4), concentrated, and ¯ash chromatographed
(SiO2, heptane/EtOAc 3:1) to give 24 (66 mg, 74%) as a
white solid; [a]2D3ˆ12 (c 1.0, CHCl3); n (®lm) 3500,
2920, 2870, 1750, 1410 cm21; dH (400 MHz, CDCl3) 5.09
(m, 1H, H-sn2), 4.33 (dd, 1H, Jˆ4.4, 11.9 Hz, H-sn1), 4.24
(dd, 1H, Jˆ6.3, 11.8 Hz, H-sn1), 3.74 (s, 2H, H-sn3), 2.69
(m, 4H, CH2SSCH2), 2.35±2.24 (m, 4H, OCOCH2), 2.05 (t,
1H, Jˆ6.2 Hz, OH), 1.70±1.60 (m, 6H, OCOCH2CH2,
CH2CH2S), 1.35±1.23 (m, 49H, CH2, SCH2CH3), 0.89 (t,
3H, Jˆ6.6 Hz, CH3); dC (100.6 MHz, CDCl3) 174.0, 173.7,
72.3, 62.2, 61.7, 39.5, 34.5, 34.3, 33.0, 32.1, 29.90, 29.87,
29.85, 29.83, 29.80, 29.72, 29.68, 29.6, 29.48, 29.45, 29.32,
29.29, 28.7, 25.13, 25.08, 22.9, 14.7, 14.3; HRMS (FAB):
M1, found 660.4815. C37H72O5S2 requires 660.4821.
2-O-(16-Mercaptohexadecanoyl)-1-O-hexadecanoyl-sn-
glyceryl-3-phosphocholine (5). To a solution of 25 (15 mg,
0.019 mmol) in ethanol (1.0 mL) and water (0.5 mL) was
added tri-n-butylphosphine (0.008 mL, 0.039 mmol) and
the resulting mixture was stirred at room temperature in
the dark for 7 h, after which the solvent was evaporated
under reduced pressure. Flash chromatography of the resi-
due (SiO2, CHCl3/MeOH/H2O 66:33:4) gave 5 (10 mg,
75%) as a white solid; [a]2D3ˆ14 (c 1.0, CHCl3/MeOH/
H2O, 65:35:10); dH (400 MHz, CDCl3/CD3OD/D2O,
65:35:10) 5.03 (m, 1H, H-sn2), 4.21 (dd, 1H, Jˆ3.1,
12.1 Hz, H-sn1), 3.96 (m, 2H, b-CH2), 3.88 (dd, 1H,
Jˆ7.1, 12.0 Hz, H-sn1), 3.75 (dt, 2H, Jˆ5.8, 6.4 Hz,
H-sn3), 3.38 (m, 2H, a-CH2), 2.98 (s, 9H, N(Me)3), 2.31
(dt, 2H, Jˆ6.2, 7.3 Hz, CH2SH), 2.15±2.09 (m, 4H,
OCOCH2), 1.45±1.30 (m, 6H, OCOCH2CH2, SCH2CH2),
1.10±1.00 (m, 46H, CH2), 0.68 (t, 3H, Jˆ6.6 Hz, CH3);
dC (100.6 MHz, CDCl3/CD3OD/D2O, 65:35:10) 174.1,
173.7, 70.3 (Jˆ7.9 Hz), 66.2, 63.4 (Jˆ5.4 Hz), 62.6, 59.0,
53.9, 34.1, 33.9, 33.8, 32.6, 31.7, 29.6, 29.51, 29.45, 29.42,
29.39, 29.36, 29.21, 29.17, 29.1, 29.01, 28.9 24.8, 24.7,
24.2, 22.5, 22.3, 13.8; HRMS (FAB): (M1H)1, found
766.5407. C40H81O8SNP requires 766.5421.
2-O-(16-(Etyldithio)hexadecanoyl)-1-O-hexadecanoyl-
sn-glyceryl-3-phosphocholine (25). To a stirred solution of
imidazole (45 mg, 0.67 mmol, co-concentrated once with
freshly distilled toluene) in toluene (0.6 mL) at 08C was
added dropwise freshly distilled PCl3 (0.013 mL,
0.146 mmol) in toluene (0.14 mL) followed by freshly
distilled triethylamine (0.054 mL, 0.38 mmol) in toluene
(0.14 mL). Stirring was continued for 10 min, the tempera-
ture was lowered to 2108C and 24 (34 mg, 0.049 mmol,
dried over P2O5 overnight) in toluene (0.7 mL), was added
dropwise over 1 h. After stirring for another 20 min at
2108C, the reaction was quenched by addition of water/
pyridine (3 mL, 1:4) and the mixture was allowed to reach
room temperature. CH2Cl2 (20 mL) was added and the
organic layer was washed with water (1£7 mL), dried
(Na2SO4), concentrated and dried (over P2O5 over night).
The residue was dissolved in freshly distilled pyridine
(1.0 mL) and cholintosylate27 (27 mg, 0.098 mmol, dried
over P2O5 over night) and 5,5-dimethyl-2-oxo-2-chloro-
1,3,2-dioxaphosphorinan28 (NPCl, 27 mg, 0.147 mmol)
was added. The resulting mixture was stirred for 15 min.
I2 (25 mg, 0.098 mmol) dissolved in pyridine/water
(1.0 mL, 98:2) was added and the mixture was stirred for
10 min. CH2Cl2 (35 mL) was added and the organic phase
was washed with 5% (w/v) aqueous solution of sodium
bisul®te (7 mL). The aqueous phase was extracted with
CH2Cl2 (2£10 mL). The combined organic phases were
concentrated and co-concentrated with toluene. Flash
Acknowledgements
This work was supported by the Swedish Natural Science
Research Council and by a grant from the programme
`Glycoconjugates in Biological Systems' sponsored by the
Swedish Foundation for Strategic Research. We are grateful
to Dr Ulf J. Nilsson for proof reading this manuscript.
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