Synthesis and purity assessment of tetra- and pentaacyl lipid A of Chlamydia containing (R)-3-hydroxyicosanoic acid

Article abstract of DOI:10.1016/j.tet.2004.10.017

Based on structural data of lipid A from Chlamydia trachomatis strains, chemically pure tetra- and pentaacyl 1,4′-bisphosphoryl as well as the related 4′-monophosphoryl derivatives of lipid A were synthesized. (R)-3-Hydroxyicosanoic acid as a chiral constituent was prepared via Noyori-reduction of methyl-3-oxoicosanoic acid. Synthetic intermediates were O-acylated with myristoic acid residues at positions 3 and 3′ and N-acylated with (R)-3-hydroxyicosanoic acid at both glucosamine units. Efficient purification methods for highly hydrophobic long-chain tri-, tetra- and pentaacyl progenitors of lipid A have been developed. Purity and homogeneity of the synthetic target compounds were confirmed by NMR and MS-data as well as a sensitive immunostaining approach. The tetra- and pentaacyl species serve as biomedical probes to investigate the endotoxic potential of chlamydial lipid A and to clarify its role in Chlamydia associated infections. Graphical Abstract

Full text of DOI:10.1016/j.tet.2004.10.017

Tetrahedron 60 (2004) 12113–12137
Synthesis and purity assessment of tetra- and pentaacyl lipid A
of Chlamydia containing (R)-3-hydroxyicosanoic acid
Alla Zamyatina,a Harald Sekljic,b,† Helmut Bradeb and Paul Kosmaa,
*
aDepartment of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
bResearch Centre Borstel, Leibniz Centre for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany
Received 18 August 2004; revised 7 October 2004; accepted 7 October 2004
Available online 27 October 2004
Abstract—Based on structural data of lipid A from Chlamydia trachomatis strains, chemically pure tetra- and pentaacyl 1,40-bisphosphoryl
as well as the related 40-monophosphoryl derivatives of lipid A were synthesized. (R)-3-Hydroxyicosanoic acid as a chiral constituent was
prepared via Noyori-reduction of methyl-3-oxoicosanoic acid. Synthetic intermediates were O-acylated with myristoic acid residues at
positions 3 and 30 and N-acylated with (R)-3-hydroxyicosanoic acid at both glucosamine units. Efficient purification methods for highly
hydrophobic long-chain tri-, tetra- and pentaacyl progenitors of lipid A have been developed. Purity and homogeneity of the synthetic target
compounds were confirmed by NMR and MS-data as well as a sensitive immunostaining approach. The tetra- and pentaacyl species serve as
biomedical probes to investigate the endotoxic potential of chlamydial lipid A and to clarify its role in Chlamydia associated infections.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
enterobacterial LPS, and the structural basis for this low
potency was attributed to the higher hydrophobicity of
chlamydial lipid A containing fatty acyl groups of longer
chain length (up to C-21) and only non-hydroxylated fatty
acids ester-linked to the sugar backbone.10,11 Chlamydial
LPS, due to the obligatory intracellular growth of the
bacteria, is only available in minor quantities. Moreover,
chlamydial lipid A comprises a complex mixture of
structural homologs having four or five long-chain acyl
groups of different chain lengths. Thus, isolation of
homogeneous chlamydial lipid A is not possible, and the
study of potential agonistic or antagonistic properties of
differently acylated lipid A variants is not viable. On the
basis of the reported structures for lipid A from
C. trachomatis we have chemically synthesized two
representative forms, the chlamydial tetra- and pentaacyl
li0pid A derivatives 1 and 2 as well as the corresponding
4 -monophosphoryl analogues 3 and 4 (Fig. 2). The
compounds serve as model compounds for authentic
chlamydial lipid A, for immunobiological studies and as
substrates for chlamydial CMP-Kdo-transferases.
Chlamydiae are obligatory intracellular Gram-negative
pathogens with a biphasic lifecycle that cause acute and
chronic diseases in animals and humans.1 The species
Chlamydia trachomatis is implicated in eye trachoma and
chronic urogenital infections leading to infertility in
women.2 The common opportunistic pathogen Chlamydo-
phila pneumoniae is suspected to contribute to the
pathogenesis of human atherosclerosis, myocardial
infarction and stroke.3,4 Macrophages infected by Chl.
pneumoniae are thought to be responsible for the mediation
of inflammatory and autoimmune processes leading to
atherosclerosis.5 The role of chlamydial lipopolysaccharide
(LPS) in these chronic infections and the underlying
pathophysiological mechanisms have not yet been
clarified.6 The structure of lipid A—as the endotoxically
active component of LPS—has recently been established for
the lipid A from C. trachomatis serotypes L2, E and F as
well as Chl. psittaci 6BC, showing unique tetra- and
pentaacylated species with only minor variations in the lipid
A acylation pattern (Fig. 1).7–9 Chlamydial LPS has been
shown to be at least 10-fold less stimulatory than
2. Results and discussion
Keywords: Lipid A; Chlamydia; Lipopolysaccharide; Sugar phosphates;
Glycolipid.
The basic synthetic approach to a large variety of lipid A
and analogues has been elaborated in the groups of Shiba
and Kusumoto.12 A crucial issue in the context of evaluating
the bioactivity of lipid A relates to the purity of lipid A
samples. Controls of synthetically prepared lipid A mostly
* Corresponding author. Tel.: C43 1 36006 6055; fax: C43 1 36006 6059;
e-mail: paul.kosma@boku.ac.at
† Present address: Intervet Innovation GmbH, D-55270 Schwabenheim,
Germany.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.10.017

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rely upon spectral analysis of the final products by MS- and
NMR data. Highly amphiphilic, poorly soluble lipid A
samples display weakly resoluted signals in the NMR
spectra making impossible the identification of potential
lipid-derived impurities. Yet minor contaminants may be
detected by highly sensitive immunostaining using a
monoclonal antibody recognizing the bisphosphorylated
backbone of lipid A.13 Therefore, the synthetic strategy
towards divergently acylated chlamydial lipid A 1 and 2 was
also focused on the purity assessment of hydrophobic
synthetic intermediates and the development of efficient
purification protocols. The target lipid A derivatives 1 and 2
and their monophosphoryl analogues (MLA) 3 and 4 were
prepared in a highly convergent manner from the following
major constituents: optically pure (R)-3-hydroxyalkanoic
acids 8 and 11 (Scheme 1), the trichloroacetimidate glycosyl
donor 20 (Scheme 2) and the bisacylated 4-O-benzyl
protected acceptor 36 (see Scheme 3). The synthesis of
b-(1/6)-linked disaccharides 28 and 37 followed by final
N-acylation and phosphorylation (Schemes 4 and 5,
respectively), and approaches towards isolation of fully
acylated intermediates will be successively presented.
2.1. Preparation of optically pure 3-hydroxyalkanoic
acids
Figure 1. Structure of major lipid A species from C. trachomatis serotypes
L2, E and F.
Whereas the preparation of a variety of (R)-3-hydroxy-
alkanoic acids and their derivatives has been previously
reported,14–16 3-hydroxyicosanoic acid was only prepared
as a racemic mixture.17 Herein we present the first
enantioselective synthesis of (R)-3-hydroxyicosanoic acid
8 and (R)-3-(octadecanoyloxy)-icosanoic acid 11—the long
chain acyl constituents of chlamydial lipid A. To this end,
methyl 3-oxoicosanoate 6 was prepared from octadecanoyl
chloride 5 via chain lengthening with 2,2-dimethyl-1,3-
dioxane-4,6-dione (Meldrum’s acid) and subsequent
decarboxylation in 46% yield.18 Enantioselective Noyori
hydrogenation16,19 of the prochiral 3-oxo-ester 6 in
methanol using RuCl2[(R)-Binap] at 60 8C and
85 kg cmK2 hydrogen pressure afforded methyl (R)-
hydroxyicosanoate 7 in 77% yield with excellent optical
purity (eeR99%). Comparison of its specific optical
rotation value with those reported for shorter methyl (R)-
3-hydroxyalkanoates14 allowed to assign the absolute
configuration of 7 as (R). The enantiomeric purity was
determined by 1H NMR analysis using a chiral shift
reagent europium tris[3-(heptafluoropropylhydroxy-methyl-
ene)-(C)-camphorate] Eu(hfc)3.14,20 The 3-hydroxy group
in 7 was protected by one-pot reductive benzylation
according to improved21 Nishizawa method22 using
benzaldehyde, hexamethyldisiloxane, trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf) and triethylsilane
(Et3SiH), which afforded methyl (R)-3-(benzyloxy)icosano-
ate in 90% yield. Subsequent hydrolysis of the methyl ester
group with LiOH$H2O gave (R)-3-(benzyloxy)icosanoic
acid 8 in 90% yield. (R)-3-(Octadecanoyloxy)icosanoic acid
11 was prepared from 3-hydroxyalkanoic ester 7 in four
steps (Scheme 1).
Alkaline cleavage of the methyl ester group of 7 afforded
9a, and subsequent treatment with phenacyl bromide
followed by 3-O-acylation with octadecanoyl chloride in
CH2Cl2/DMAP afforded ester 10. Cleavage of the phenacyl
Figure 2. Synthetic chlamydial tetraacyl lipid A 1, pentaacyl lipid A 2 and
monophosphoryl lipid A analogues 3 and 4.

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12115
Scheme 1. Synthesis of icosanoic acid derivatives: (a) 2,2-dimethyl-1,3-dioxane-4,6-dione, pyridine/CH2Cl2; (b) MeOH, reflux, 46% (two steps);
(c) RuCl2[(R)-Binap], 60 8C, 85 kg/cmK2 H2, 77%; (d) C6H5CHO, (TMS)2O, TMSOTf, Et3SiH, 90%; (e) LiOH$H2O, 90%; (f) NaOH; (g) Dowex HC
(h) phenacyl bromide, Et3N, EtOAc, 45 8C; (i) C17H35COCl, 4-DMAP, CH2Cl2, 88% for steps (h)C(i); (j) Zn–Cu couple, AcOH/toluene, then aq HCl, 85%.
;
group by Zn–Cu couple in AcOH–toluene gave free acid 11.
The sodium salt 9a was converted into the free acid 9b by
treatment with Dowexw AG 50 (HC-form).
prepared from the known intermediate 1223 (Scheme 2). The
3-OH group in 12 was acylated with myristoyl chloride/
pyridine in 95% yield to give fully protected 13, which was
further employed for the preparation of both donor and
acceptor moieties. Reductive cleavage of the benzylidene
acetal in 13 to yield the 6-O-benzyl-protected derivative 14
was achieved first by application of a frequently used
2.2. Glycosyl donor and acceptor synthesis
A common glycosyl donor for the synthesis of 1–4 was
Scheme 2. Synthesis of glycosyl donor and acceptor: (a) MyrCl, pyridine, THF, 95%; (b) method A: BH3$Me2NH, BF3$OEt2, CH2Cl2, 0 8C/ rt; 62%;
method B: Et3SiH, TfOH, CH2Cl2, mol. sieves 0.4 nm, K78 8C, 92%; (c) di-O-benzyloxy(N,N-diisopropylamino)phosphine, 1H-tetrazole, CH2Cl2, then tert-
BuOOH, (method A: 16 44% 15 6%; method B: 16 78%); (d) N,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine, 1H-tetrazole, CH2Cl2, then
3-chloroperbenzoic acid (18 47%, 17 8%); (e) {[bis(methyldiphenyl)phosphine](1,5-cyclooctadiene)iridium(I)} hexafluorophosphate, THF, then aq I2, 91%
for 19, 83% for 21; (f) trichloroacetonitrile, Cs2CO3, CH2Cl2.

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A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
Scheme 3. Synthesis of glycosyl acceptor 27: (a) CH3(CH2)12COOH, DCC, 4-DMAP, CH2Cl2, 86%; (b) 90% aq AcOH, 90 8C, 96%; (c) Zn–Cu couple, AcOH,
70%; (d) 8, DCC, CH2Cl2, 86%.
method in lipid A chemistry employing dimethylamine–
borane complex BH3$Me2NH as reagent with BF3$OEt2 as
a promoter in acetonitrile.21,24,25 This approach proved to be
disadvantageous in our case with respect to moderate yields
of 6-O-benzyl derivative 14 (60%) and persistent contami-
nation of the latter with dimethylamine–borane, which is
partially soluble in organic solvents. In spite of multiple
treatments of the reaction mixture with aqueous 1 M HCl
and thorough chromatographic purification of the product
14, the presence of minor amounts of BH3$Me2NH was
detrimental in the next phosphorylation step. Thus,
phosphitylation of contaminated 14 with di-O-benzyl-
oxy(N,N-diisopropylamino)phosphine in the presence of
1H-tetrazole as an acid catalyst and subsequent oxidation
Scheme 4. Phosphorylation of 4-unprotected precursor 31: (a) TMSOTf, CH2Cl2, molecular sieves 0.4 nm, K25 8C, 76%; (b) Zn–Cu couple, AcOH, 66%;
(c) 8, IIDQ, CH2Cl2/CHCl3, 76%; (d) {[bis(methyldiphenyl)phosphine](1,5-cyclooctadiene)iridium(I)}hexafluorophosphate, THF, then aq I2, 94%;
(e) [(BnO)2P(O)]2O, (TMS)2NLi, THF, K78 8C, 19%.

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
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Scheme 5. Synthesis of 4-O-protected glycosyl acceptor: (a) Et3SiH, PhBCl2, CH2Cl2, mol. sieves 0.4 nm, K78 8C, 96%; (b) method A: Zn–Cu couple, AcOH,
(34 60%, 35 5%), method B: Zn, AcOH, 89% for 34; (c) 8, WSCD$HCl, HOBt, CH2Cl2/CHCl3, 85%.
with tBuOOH gave rise to the desired phosphate 16 (44%)
along with the co-migrating 3-O-alkyl analogue 15 (15%
crude yield, 6% isolated yield). Partial reduction of the 3-O-
acyl group took place independently of the phosphitylating
reagent used. Employment of the Watanabe reagent26
(N,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-
3-amine) and subsequent oxidation furnished the
expected 3-O-acyl-4-O-phosphate 18, but also the undesired
3-O-alkyl derivative 17.27 Complete separation of 3-O-acyl-
4-O-phosphotriesters 16 and 18 from their co-migrating
3-O-alkyl counterparts 15 and 17, respectively, required
repeated preparative HPLC purifications which resulted in
low yields (44 and 47%, respectively) of chemically pure
phosphates 16 and 18.
effectively used in the synthetic preparation of different
lipid A derivatives.20,23 Formation of a 1/6-glycosidic
linkage with this type of acceptor is known to proceed
regioselectively, and the 4-OH group was regarded to
remain unaffected during final BuLi-assisted 1-O-phos-
phorylation of the fully acylated b(1/6)-linked disacchar-
ide 40-phosphate progenitor of lipid A.20,23,31 In a first
approach, the diol acceptor 27 was prepared according to a
literature procedure.32 The known acetonide 23 was
O-acylated with tetradecanoic acid in the presence of
dicyclohexylcarbodiimide (DCC) and a catalytic amount of
4-N,N-dimethylaminopyridine which furnished 24. Sub-
sequent acidic hydrolysis of the isopropylidene group
afforded diol 25. Removal of trichloroethoxycarbonyl
(Troc) protection with Zn–Cu couple and final N-acylation
of the free amine 26 with (R)-3-(benzyloxy)icosanoic acid 8
under assistance of DCC furnished 4,6-diol derivative 27
(Scheme 3) in 57% yield (for three steps).33
These shortcomings could be avoided by using Et3SiH as a
reductive reagent at K78 8C in CH2Cl2 in the presence of
activated molecular sieves and trifluoromethanesulfonic
acid (TfOH)28,29 for the regioselective opening of the
benzylidene acetal.28 In this way, benzylidene acetal 13 was
efficiently converted in 92% yield into the corresponding
6-O-benzyl derivative 14 (Scheme 2). Furthermore, phos-
phitylation and subsequent oxidation of the alcohol 14
proceeded without by-product formation and the dibenzyl-
phosphotriester 16 was readily isolated in 78% yield.
The allyl group in 16 was isomerized by treatment with
H2-activated iridium complex {[bis(methyldiphenyl)-
phosphine](1,5-cyclooctadiene) iridium(I)}hexafluoro-
phosphate.30 The propenyl group was cleaved with aq
iodine to furnish 19 which was transformed into glycosyl
donor 20 by treatment with trichloroacetonitrile/Cs2CO3.
The 4,6-O-benzylidene acetal derivative 13 was also
employed for the preparation of the non-phosphorylated
donor 22 by successive cleavage of the allyl group to
afford 21 and treatment of 21 with trichloroacetonitrile/
Cs2CO3.
To test the regioselectivity of 1-O-phosphorylation in the
last step of the synthesis, the diol-acceptor 27 was first
utilized for the synthesis of 40,60-O-benzylidene-protected
1-O-monophosphoryl lipid A progenitor 31. Glycosylation
of 27 with 4,6-O-benzylidene protected trichloroacetimidate
22 afforded disaccharide 28 (76%). Reductive cleavage of
N-Troc protecting group with Zn–Cu couple in AcOH–
toluene and subsequent N-acylation of the free amine 29
with (R)-3-(benzyloxy)icosanoic acid 8 in the presence of
1-isopropyloxycarbonyl-2-isopropyloxy-1,2-dihydroquino-
line (IIDQ) furnished the tetraacyl precursor 30. After the
removal of the anomeric allyl group, the reducing diol 31
was subjected to phosphorylation with tetrabenzyl diphos-
phate in the presence of lithium bis(trimethylsilyl)amide at
K78 8C.25 The high hydrophobicity and low reactivity of 31
required the use of four-times excess of the reagents to
complete the reaction, which led to an undesired phos-
phorylation at the unprotected 4-position. The major
product isolated in 19% yield by chromatography was
identified as 1,4-O-bisphosphate 32, while the more polar
2.3. Disaccharide synthesis
4,6-Diol glucosamine acceptors have previously been

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A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
Scheme 6. N-Acylation, 1-O-phosphorylation and final deprotection: (a) TMSOTf, CH2Cl2, mol. sieves 0.4 nm, K25 8C, 93%; (b) Zn, AcOH, 84%; (c) 8,
HATU, DMF, DIPEA, 35 8C, 92%; (d) 11, HBTU, DIPEA, DMF/THF, 58 8C, 86%; (e) {[bis(methyldiphenyl)phosphine](1,5-cyclooctadiene)iridium(I)}hexa-
fluorophosphate, THF, then aq I2, 85% for 40, 88% for 42; (f) Pd/C, H2, then DEAE-cellulose, 60% for 3; (g) Pd/C, H2, 5:1 toluene/MeOH, then silica gel and
DEAE-cellulose chromatography, 49%; (h) [(BnO)2OP]2O, (TMS)2NLi, THF, K78 8C, 85% for 43, 86% for 44; (i) Pd/C, H2, 5:1 toluene/MeOH, then DEAE-
cellulose, 51% for 1, 45% for 2.
1-O-monophosphoryl component of the mixture was
destroyed due to the prolonged contact with silica gel.
furnished amphiphilic acceptor 36, which was extensively
purified by partition-adsorption chromatography on silica
gel using toluene as stationary phase and aqueous MeOH as
mobile phase.
Taking into consideration the lability of reducing phospho-
triesters and the difficulty of their isolation in chemically
pure form, the importance of a full-protection strategy and,
therefore, the option of selective 1-O-monophosphorylation
using excess of reagents became evident.34 To this end the
4-O-benzyl glycosyl acceptor 33 was obtained in an
excellent yield (96%) by regioselective reductive opening
of benzylidene acetal 13 using Et3SiH as reductive reagent
and phenyldichloroborane PhBCl2 as Lewis-acid catalyst
(Scheme 5).28 Reductive cleavage of Troc protection with
Zn–Cu couple in aqueous AcOH23 led unexpectedly to
partial reduction of trichloroethoxycarbonyl group with the
formation of N-2,2-dichloroethoxycarbonyl derivative 35,
thereby reducing the yield of the amine 34 to 66%. The
employment of Zn in AcOH, however, afforded free amine
34 in 89% isolated yield without any detectable partial
reduction of the trichloroethoxycarbonyl group.35 Sub-
sequent N-acylation with (R)-3-(benzyloxy)icosanoic acid
8 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (water soluble carbodiimide,
WSCD$HCl) and 1-hydroxy-benzotriazole (HOBt)21
For the assembly of synthetic chlamydial lipid A the
protected 6-OH acceptor 36 and trichloroacetimidate donor
20 were employed (Scheme 6). Glycosylation of 36 with
N-alkoxycarbonyl-protected trichloroacetimidate 20 gave
rise exclusively to b-disaccharide 37.36 The b(1/6)-
linkage was supported by 1H NMR experiments which
3
0
0
revealed a coupling constant J1 ,2 of the disaccharide of
8.3 Hz. The next step, reductive cleavage of Troc-protection
with Zn in AcOH at 50 8C, and thorough purification of the
generated free amine by chromatography afforded 38 in
84% yield.37,38 The isolation of 38 in chemically pure form
was crucial, since its Troc-protected precursor 37, when not
fully deprotected or being partially reduced to a dichloro-
ethoxycarbonyl derivative, co-migrates with tetra- and
pentaacyl lipid A progenitors 39 and 41 prepared in the
subsequent acylation step. A series of conditions
towards divergent N-acylation of the disaccharide 38 with
either (R)-3-(benzyloxy)icosanoic acid 8 or (R)-3-(octade-
canoyloxy)-icosanoic acid 11 was explored. Acylation of 38

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12119
Table 1. Conditions for the N-acylation of lipid A precursor 38 with acids 8 or 11 to yield tetraacyl- (39) or pentaacyl (41) derivatives
Entry
Acid
Conditions
Coupling reagent, auxiliary nucleophile, base
Yield (%)
1
2
3
4
5
6
7
8
9
8
8
8
8
8
8
11
8
THF, 20 8C, 24 h
DMF, 40 8C, 40 h
THF, 20 8C, 40 h
THF, 20 8C, 30 h
DMF/THF, 20 8C, 3 h
DMF, 35 8C, 5 h
DMF, 50 8C, 8 h
DMF, 35 8C, 6 h
DMF, 58 8C, 5 h
WSCD.HCl, HOBt
WSCD.HCl, HOAt, DIPEA
EEDQ
20
20
40
50
50
92
75
80
86
IIDQ
HATU, DIPEA
HATU, DIPEA
HATU, DIPEA
HBTU, DIPEA
HBTU, DIPEA
11
with less bulky and less hydrophobic acid 8 in the presence
of water-soluble carbodiimide (WSCD$HCl)21 provided the
tetraacyl derivative 39 in only 20% yield independently of
the nature of the auxiliary nucleophile, base and solvent
used (Table 1, entries 1 and 2). Employment of 1-
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)35
or IIDQ as coupling reagents in THF required prolonged
reaction times and only slightly improved the isolated yields
(40–50%). The optimal reaction conditions were established
by employing the uronium salts O-(7-azabenzotriazol-1-yl)-
separation which eliminates an excess of reagents and
non-lipid impurities only.40 To facilitate the detection of co-
migrating by-products by TLC a four-component one-phase
mixture composed of toluene/dichloromethane/methanol/
water (150:100:15:1), where toluene/CH2Cl2 served as
unpolar stationary phase and MeOH/H2O as polar mobile
phase, was elaborated. Apart from perfect resolution on
TLC, an application of this solvent mixture for preparative
adsorption–partition chromatography on silica gel allowed
efficient isolation of protected tetra- (39, 40) and pentaacyl
(41, 42) intermediates in chemically pure form.
N,N,N0,N0-tetramethyluronium
hexafluorophosphate
(HATU) and O-(benzotriazol-1-yl)-N,N,N0,N0-bis-tetra-
methyluronium hexafluorophosphate (HBTU)39 in the
presence of diisopropylethylamine (DIPEA), which pro-
vided tetraacyl disaccharide 39 and sterically hindered
pentaacyl derivative 41 in 92 and 86% yield, respectively
(Table 1, entries 6 and 9). Subsequent isomerisation of the
allyl protection of the fully acylated disaccharides 38 and 41
with H2-activated iridium complex and hydrolysis of the
propenyl group by treatment with aq iodine furnished
reducing disaccharides 40 and 42, respectively.
2.5. Phosphorylation and deprotection
For the introduction of a-phosphate at the reducing end of
the tetraacyl lipid A progenitor 40, the phosphoramidite
procedure was first examined. Phosphitylation of 40 with
di-O-benzyloxy-(N,N-diisopropylamino)phosphine in the
presence of 1H-tetrazole and in situ oxidation with tBuOOH
afforded a 3:2 a/b mixture of phosphotriesters. Although
a-selectivity of 1-OH phosphitylation using the phosphor-
amidite approach has been reported in the literature,41,42 and
was rationalized on the basis of the extreme instability of the
anomeric b-phosphate,42 exclusively a-linked phosphates
could not be prepared using this approach. Applying the
very efficient a-selective phosphorylation of reducing
disaccharides 40 and 42 via 1-O-lithiation using lithium
bis(trimethylsilyl)amide followed by phosphorylation with
tetrabenzyl diphosphate at K78 8C25 afforded a-configured
anomeric bisphosphotriesters 43 and 44, respectively. The
propensity of these compounds to undergo fast acidic
2.4. Purification of disaccharide intermediates
While a variety of synthetic approaches towards divergently
acylated lipid A structures is adequately presented in the
literature, the issues of isolation of protected synthetic
intermediates of lipid A containing three or more long chain
fatty acids received little attention. In the commonly used
solvent mixtures (CH2Cl2–acetone or CHCl3–MeOH) for
chromatographic purification of such synthetic inter-
mediates of lipid A21,23,25,32 the potential by-products
stemming from cleavage, migration, or reduction of acyl
groups display similar chromatographic behaviour to those
of the products, thus making the detection of the former on
TLC and the isolation of the latter rather intricate.
Consequently, minor co-migrating by-products accumulate
throughout the multistep synthesis, leading to unseparable
and hardly detectable contaminations in the final amphi-
philic lipid A preparations. Therefore, development of an
efficient purification protocol for highly hydrophobic tri-,
tetra- and pentaacyl synthetic intermediates of lipid A has
been strongly demanded. Herein a highly efficient, mild and
simple isolation procedure for synthetic precursors of
chlamydial lipid A is presented. The method comprises
sequential precipitation of the desired reaction product with
successively polar (EtOH or MeOH or acetone) and unpolar
(n-hexane) solvents from CH2Cl2 or CH2Cl2/toluene, such
that the lipid-derived by-products and excess of the reagents
are withheld in the filtrates (Fig. 3). This purification
scheme provides a high-yield alternative to affinity-
Figure 3. Thin layer chromatography offinal precipitate and filtrates arising
from purification of protected pentaacyl lipid A precursor 41 by sequential
precipitation: TLC of filtrates from sequential precipitations from CH2Cl2
with EtOH (lanes 1–3), n-hexane (lanes 4–6), EtOH (lanes 7–9) and TLC of
final precipitate (lane 10).

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A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
hydrolysis on silica gel with the loss of reducing phosphate
stipulated the waste of more than 50% of synthesized
diphosphates during chromatography. As reported in the
literature, 1-O-phosphotriesters of fully protected lipid A are
either not isolated and used as crude mixtures in the final
deprotection step25 or just roughly purified by flash
chromatography.20,21,23,34 Again, the precipitation method
proved to be superior to chromatography in the purification
step of reducing phosphotriesters 43 and 44, which were
isolated in 90% yield by sequential precipitation (compared
to 30–40% yield by conventional chromatography for the
same degree of purity). Thus, the purification conditions set
forth herein show a broad scope, as they proved equally
useful for both stable and labile acylated hydrophobic
intermediates of lipid A. Hydrogenolytic cleavage of benzyl
protecting groups in monophosphates 40 and 42 with Pd/C
in THF or toluene/methanol, respectively, afforded a
complex mixture of monophosphoryl lipid A (MLA) and
their partially benzylated intermediates. Purification by
chromatography on silica gel in 50:20:20:3:2 CHCl3/
n-hexane/MeOH/H2O/AcOH and subsequent ion-exchange
chromatography on DEAE-cellulose (CH3COOK-form)
afforded monophosphoryl analogues 3 and 4 in 60 and
49% yield, respectively.
For the hydrogenolytic deprotection of the labile bisphos-
photriesters 43 and 44, the employment of Pd/C in 5:1
toluene/methanol was found to be the best option; in spite of
concomitant formation of methyl glycosides, partial
hydrolysis of reducing 1-phosphates and adsorption of the
amphiphilic products on charcoal, the yields were consider-
ably higher than those obtained by application of Pd-black
in THF.34 Tetraacyl lipid A 1 and pentaacyl lipid A 2 were
finally purified by anion-exchange chromatography on
DEAE-cellulose43 (CH3COOK-form) in 2:3:1 CHCl3/
MeOH/aq CH3COOKHNEt3C using a stepwise gradient of
triethylammonium acetate and by subsequent desalting with
Bligh–Dyer solvent system.44
Tetra- and pentaacyl lipid A 1 and 2 and their mono-
phosphoryl analogs 3 and 4 were characterized and their
structures and purity were confirmed by NMR spectroscopy
(Fig. 4), positive MALDI-TOF and ESI- mass spectrometry
and immunostaining.
Figure 4. 1H–13C HMQC spectrum of synthetic pentaacyl lipid A 2.

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12121
In the NMR spectra of compounds 3 and 4 the anomeric
protons were seen at 5.10–5.13 ppm with a coupling
constant 3J1,2Z3.3 Hz confirming a-anomeric configuration
of the reducing end, whereas the coupling constant (3J1 ,2
Z
0
0
8.5 Hz) displayed by the anomeric protons of the distal
glucosamine unit proved the presence of a b-glycosidic
li0nkage. The sites of esterification of fatty acids at the 3 and
3
positions were consistent with the observed proton
downfield shifts to 5.10–5.18 ppm, whereas the downfield
shifts of the proton at 40-position (4.28 and 4.24 ppm,
respectively), confirmed the presence of the phosphate
group (Table 2). NMR experiments of the monotriethylam-
monium salts of lipid A derivatives 1 and 2 were attempted
in different non-destructive solvent mixtures (2:1 CDCl3/
DMF-d7, 2:3:1 CDCl3/CD3OD/D2O, 4:1 CDCl3/2-PrOD-
d7, 4:1 CDCl3/CD3OD) in the temperature range 27–37 8C
and in concentrations of 5–8 mg/mL. Limited solubility and
time-dependent aggregation led generally to broad
unresolved lines. Only in 4:1 CDCl3/CD3OD45 the 1H
NMR signals were sharp and sufficiently resolved and
significant dephosphorylation at the anomeric position was
not detected upon storage over several days at room
temperature. The latter fact might be attributed to the actual
presence of large amount of aggregated water in the samples
(which is observed as broad intensive signal of HDO at
approx. 3.9 ppm) in connection with the reported stability of
purified intact lipid A in CDCl3/CD3OD/D2O.46 The well-
resolved double–doublet resonances at 5.44 and 5.48 ppm
were assigned to the anomeric protons, the doublets at 4.78
and 4.72 were assigned to the H-10 signal of the distal sugar
(for 1 and 2, respectively). The respective small and large
3
coupling constants (3J1,2Z3.2–3.5 Hz, J1 ,2 Z8.3–8.5 Hz)
proved a- and b-pyranosyl forms for the proximal and distal
glucosamine rings, respectively. Two phosphorous reson-
ances confirmed the bisphosphorylated structure. The
downfield positions of H-1 (doublet of doublets) and
H-40(pseudo triplet) at 5.44–5.48 and 4.26–4.23 ppm,
0
0
3
respectively, and phosphorus–proton coupling J1,PZ7.0–
7.4 Hz supported the sites of phosphorylation (the coupling
value 3J43,PZ10.0–10.5 Hz was very close to that of vicinal
0
3
0
0
0
0
protons J3 ,4 Z J4 ,5 Z9.0 Hz). Two anomeric carbon
resonances for 1 and 2, respectively, were observed at 93–
94 and 100.4–101.4 ppm and represented the phosphoryl-
ated anomeric position C-1 of the proximal sugar unit and
C-100 of the distal glucosamine residue, respectively. H-2 and
H-2 signals correlated with the upfield-shifted carbon
resonances at 52.3–52.4 and 53.8–54.3 ppm (C-2 and C-20,
respectively) reflecting the presence of acylamido groups
(Table 3). Downfield-shifted signals of H-3 and H-30
displayed overlapped cross-peaks to downfield-shifted C-3
and C-30 carbon resonances at 72.7–73.6 and at 73.4–
71.4 ppm, respectively, thus confirming the presence of acyl
groups at both positions. The broad CD3OH and HOD
signals at 30.95–3.80 ppm ob0scured the resonances from H-5,
H 6a, H-6a , b-2CH and b-2 CH; the chemical shifts of these
1
protons could be determined from two-dimensional H–1H
1
COSY and H–13C HMQC-experiments (Fig. 4).
Purity and molecular masses of the tetra- and pentacyl lipid
A derivatives 1 and 2 were confirmed by MALDI TOF-MS
and high-resolution ESI FT-MS. Furthermore, compounds 1
and 2 were subjected to separation by TLC and subsequent
immunostaining with a monoclonal antibody against lipid

12122
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
Table 3. 13C NMR dataa (ppm) of tetraacyl lipid A (1), pentaacyl lipid A (2) and the monophosphoryl analogs 3 and 4
GlcN
C-1
93.0
94.3
91.4
91.9
C-2
C-3
C-4
C-5
C-6
bCH
aCH2
1b
2b
3c
4d
52.3
52.4
52.4
52.8
72.7
73.6
73.6
71.9
68.5
68.7
68.7
69.6
73.1
73.3
70.5
71.6
67.3
68.4
68.9
68.9
68.9
69.2
68.7
69.0
43.9
44.0
43.4
43.5
GlcN0
1b
100.4
101.4
101.7
102.0
53.8
54.3
53.8
54.6
73.4
71.4
73.4
74.2
70.8
71.5
70.7
72.0
75.3
75.8
75.8
76.7
60.3
60.7
60.4
61.3
68.9
69.2
68.7
69.0
43.9
41.1
43.6
41.9
2b
3c
4d
a Recorded at 75.47 MHz.
b Chemical shifts at 300 K in CDCl3/CD3OD (4:1, v/v).
c Chemical shifts at 300 K in CDCl3/2-PrOD-d7 (4:1, v/v).
d Chemical shifts at 318 K in CDCl3/2-PrOD-d7 (4:1, v/v).
60 (10 mm). Anion-exchange chromatography was per-
formed on BioRad DEAE-Cellulose (Pharmacia). Reactions
were monitored by TLC on (A): Silica gel 60 F254 precoated
glass plates (Merck) or on (B): Silica gel 60 F254 HPTLC
precoated glass plates with 2.5 cm concentration zone
(Merck), unless stated otherwise; spots were visualized by
spraying with anisaldehyde–H2SO4; phosphorus-containing
compounds were additionally detected with a molybdate
solution [0.02 M solution of ammonium cerium(IV)sulfate
dihydrate and ammonium molybdate(VI)tetrahydrate in aq
H2SO4]. Concentration of solutions was performed at
reduced
pressure
at
temperatures
!25 8C.
Diisopropylethylamine, dry tetrahydrofuran, dry CHCl3
and dry MeOH were purchased from Merck. Triethylamine
and dichloromethane were dried by refluxing with CaH2
(5 g/L) for 16 h, then distilled and stored under argon.
Toluene was distilled from phosphorus pentaoxide and
redistilled from CaH2. The liquids were stored over
molecular sieves 0.4 nm. DMF was stirred with CaH2
(5 g/L) for 16 h at 20 8C, distilled under reduced pressure
and stored over activated molecular sieves 0.3 nm. Triethyl-
ammonium acetate (TEAA) (1 M) buffer was purchased
from Fluka. Optical rotations were measured with a Perkin–
Elmer 243 B polarimeter. [a]2D0-Values are given in units of
10K1 deg cm3 gK1. NMR-spectra were recorded at 297 K in
CDCl3 (unless stated otherwise) with a Bruker DPX 300
Figure 5. Immunostaining of compounds 1 and 2. Compounds 1 and 2 were
separated by TLC (500 ng) each, transferred to PVDF membrane and
stained with a monoclonal antibody against the 1,40-bisphosphorylated
glucosamine disaccharide of lipid A.
A.47 This method was shown to detect lipid A in amounts as
low as 10–50 ng. As depicted in Figure 5, the pentaacyl lipid
A derivative 2 (left lane) was free of any visible
contaminants, whereas the tetraacyl lipid A derivative 1
showed a very minor trace contaminant which migrated
slightly lower than the main compound (right lane).
spectrometer (1H at 300.13 MHz, 13C at 75.47 MHz and 31
P
1
at 121.50 MHz) using standard Bruker NMR software. H
NMR spectra were referenced to tetramethylsilane or 2,2-
dimethyl-2-silapentane-5-sulfonic acid. 13C NMR spectra
were referenced to chloroform (d 77.00). 31P NMR spectra
were referenced externally to 85% aq H3PO4 (d 0.0).
Elemental analyses were provided by Dr. J. Theiner,
Mikroanalytisches Laboratorium, Institut fu¨r Physikalische
In summary, the synthesis of pure tetra- and pentaacyl lipid
A species from Chlamydiae has been achieved starting from
the readily available precursor 12 in 11 steps in excellent
overall yields. Studies of the endotoxic activities of
synthetic chlamydial lipid A and as acceptors for
chlamydial Kdo-transferase are currently in progress and
will be reported elsewhere.
¨
Chemie, Universitat Wien. MALDI-TOF-MS spectra were
recorded on a Dynamo (Thermo BioAnalysis) instrument in
the positive ion mode with 2% 2,5-dihydroxybenzoic acid
as matrix. Laser-desorption-MS spectra were recorded on a
laser microprobe mass analyzer (LAMMA 500, Leybold
AG). ESI-MS spectra were recorded on a 7-Tesla Apex II,
Bruker Daltonics instrument. Melting points were deter-
mined with a Kofler hot stage microscope and are
uncorrected. Thin layer chromatography and immuno-
staining: compounds (500 ng) were separated by TLC on
precoated silica 60 aluminum plates (Merck) using
30:70:16:10 CHCl3/pyridine/88% aq HCOOH/H2O. Dried
plates were transferred to polyvinylidene difluoride (PVDF)
3. Experimental
3.1. General
Column chromatography was performed on silica gel 60
(230–400 mesh, Merck), HPLC was performed on silica gel

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12123
membranes as described13 and stained with monoclonal
antibody A6 recognizing the bisphosphorylated glucosa-
mine disaccharide of lipid A.47
LiOH$H2O (0.44 g, 10.9 mmol, 10 mL) under reflux for
3 h. The mixture was cooled to rt and the pH was adjusted to
7.0 by slow addition of 1.5 M aq HCl. The mixture was
diluted with CH2Cl2 (300 mL), washed with aq NaHCO3
(100 mL), dried (Na2SO4) and concentrated. Purification of
the residue on silica gel (CHCl3/10:1 CHCl3/MeOH)
afforded 8 as white solid (0.7 g, 90%), mpZ42–44 8C;
3.1.1. Methyl 3-oxoicosanoate (6). Octadecanoyl chloride
5 (106.0 g, 0.35 mol) was added dropwise during 20 min at
0 8C to a solution of 2,2-dimethyl-1,3-dioxane-4,6-dione
(50.4 g, 0.35 mol) and pyridine (55.4 g, 0.70 mol) in
CH2Cl2 (150 mL). The mixture was allowed to warm to rt
over a period of 20 min and stirred for 3 h, then
concentrated. The residue was repeatedly evaporated with
toluene (2!300 mL), dissolved in CHCl3 (500 mL) and
extracted in turn with 1 M aq HCl (2!500 mL) and brine
(500 mL). The organic layer was dried (Na2SO4) and
concentrated. The residue was dissolved in methanol
(500 mL) and the solution was stirred under reflux for
6.5 h. During this period methanol was added twice (after
3.5 and 5 h, 100 mL each). The solution was cooled to 0 8C
and kept at 4 8C for 12 h. The precipitated solid was
collected by filtration and crystallized from ethanol
(400 mL, 4 8C) to afford 6 as yellowish crystals (55.0 g,
[a]2D0ZK2.4 (c 0.25, CHCl3); H NMR: d 7.35–7.25 (m,
1
5H, CH2Ph), 4.56 (s, 2H, CH2Ph), 3.87 (m, 1H, bCH), 2.62
2
3
(dd, 1H, JaCHHZ15.5 Hz, JaCHH,bCHZ6.8 Hz, aCHH),
3
2.57 (dd, 1H, JaCHH,bCHZ5.0 Hz, aCHH), 1.69–1.53 (m,
3
2H, CH2), 1.39–1.25 (m, 30H, 15CH2), 0.87 (t, 3H, JZ
6.8 Hz, CH3); LAMMA m/z 417.6 [MKH]K. Anal. Calcd
for C27H46O3: C, 77.46; H, 11.07. Found: C, 77.23; H,
11.17.
3.1.4. Sodium (R)-3-hydroxyicosanoate (9a). To a solution
of 7 (1 g, 2.3 mmol) in THF (20 mL) a solution of NaOH
(0.6 g, 15 mmol) in H2O (3 mL) was added and the mixture
was vigorously stirred at reflux for 4 h, then cooled to 25 8C.
The pH was adjusted to 7.0 by addition of 1.5 M aq HCl.
The mixture was taken up in EtOAc (200 mL) and washed
with aq NaHCO3 (200 mL), which afforded three phases.
The middle phase was collected and its volume was reduced
to 10 mL. n-Hexane (100 mL) was added, the suspension
was heated to 60 8C until the solids dissolved, then cooled to
0 8C, and the precipitate was collected on a filter. Yield
916 mg (95%), mpZ176–180 8C; LAMMA m/z 327.5
[MKH]K; 1H NMR (DMSO, 65 8C): d 3.80 (m, 1H,
bCH), 2.24–2.20 (m, 2H, aCH2), 1.34–1.24 (m, 32H,
1
46%), mpZ53–55 8C. H NMR: d 3.71 (s, 3H, CO2CH3),
3.42 (s, 2H, COCH2CO), 2.50 (t, 2H, 3JZ7.4 Hz, CH2CH2-
CO), 1.56 (t, 2H, CH2CH2CO), 1.35–1.20 (m, 28H, 14CH2),
0.85 (t, 3H, 3JZ6.9 Hz, CH3). Anal. Calcd for C21H40O3: C,
74.07; H, 11.84. Found: C, 73.85; H, 11.55.
3.1.2. Methyl (R)-3-hydroxyicosanoate (7). A suspension
of 6 (30.0 g, 88.0 mmol) and (R)-(C)-2,20-bis(diphenyl-
phosphino)-1,10-binaphthyl-RuCl2 {prepared from (R)-
Binap (100 mg, 0.16 mmol) and [RuCl2(cyclooctadiene)]
(41 mg)}19 in MeOH (40 mL) was stirred under 85 kg cmK2
H2 at 60 8C for 44 h. The mixture was cooled to 0 8C, H2
was evacuated and the solids were collected by filtration.
The residue was crystallized from hexane (100 mL), light-
yellow crystals were collected on the filter and washed with
cold (0 8C) MeOH. Yield 23.3 g (77%, eeO99%), mpZ62–
63 8C. [a]2D0ZK10.4 (c 1.0, CHCl3). LAMMA m/z: 365.0
3
16CH2), 0.86 (t, 3H, JZ6.9 Hz, CH3). Anal. Calcd for
C20H39NaO3!H2O: C, 66.82; H, 11.21. Found: C, 66.60;
H, 11.31.
3.1.5. (R)-3-Hydroxyicosanoic acid (9b). To a stirred
suspension of 9a (1 g, 2.27 mmol) in MeOH (300 mL)
Dowexw AG 50 W-X8 resin (HC-form) was gradually
added until pH 5 was reached and a clear solution was
obtained. The resin was filtered off and the filtrate was
concentrated to give 0.93 g, (98%) of 9b as a white solid.
MpZ88–90 8C; [a]D23ZK11.5 (c 0.3, CHCl3); 1H NMR: d
1
[MCNa]C. H NMR: d 3.99 (m, 1H, bCH), 3.70 (s, 3H,
2
CO2CH3), 2.78 (br s, 1H, OH), 2.49 (dd, 1H, JaCHH
Z
3
16.5 Hz, JaCHH,bCHZ3.1 Hz, aCHH), 2.40 (dd, 1H,
3JaCHH,bCHZ9.0 Hz, aCHH), 1.53–1.40 (m, 2H, CH2),
1.35–1.20 (m, 30H, 15CH2), 0.86 (t, 3H, 3JZ6.9 Hz, CH3).
Anal. Calcd for C21H42O3: C, 73.63; H, 12.36. Found: C,
73.60; H, 12.33.
2
4.02 (m, 1H, bCH), 2.59 (dd, 1H, JaCHHZ16.6 Hz,
3
3JaCHH,bCHZ3.3 Hz, aCHH), 2.47 (dd, 1H, JaCHH,bCH
Z
8.8 Hz, aCHH), 1.60–1.44 (m, 2H, CH2), 1.40–1.20 (m,
3
30H, 15CH2), 0.88 (t, 3H, JZ6.5 Hz, CH3). Anal. Calcd
for C20H40O3!H2O: C, 71.17; H, 12.24. Found: C, 71.84;
H, 12.60.
3.1.3. (R)-3-(Benzyloxy)icosanoic acid (8). To a solution of
7
(3.0 g, 8.8 mmol) and benzaldehyde (2.67 mL,
26.3 mmol) in THF (30 mL) were added hexamethyl-
disiloxane (11.2 mL, 52.6 mmol) and trimethylsilyl
trifluoromethanesulfonate (1.3 mL, 7 mmol) at 0 8C. After
the mixture was stirred for 15 min at 0 8C, triethylsilane
(4.9 mL, 30.7 mmol) was added and the mixture was stirred
at 0 8C for 3 h, then allowed to warm to rt. The reaction was
quenched by addition of satd aq NaHCO3 (30 mL), diluted
with EtOAc (400 mL) and extracted in turn with satd aq
NaHCO3 (200 mL) and brine (200 mL). The organic phase
was dried (Na2SO4) and concentrated. Chromatography on
silica gel (toluene/100:3 toluene/EtOAc) afforded methyl
(R)-3-(benzyloxy)icosanoate (3.4 g, 90%), Rf 0.6 (9:1
toluene/EtOAc). A solution of methyl (R)-3-(benzyloxy)-
icosanoate (0.8 g, 1.86 mmol) in THF–H2O (5:1, 30 mL)
was vigorously stirred with an aqueous solution of
3.1.6. Phenacyl (R)-3-(octadecanoyloxy)icosanoate (10).
To a stirred suspension of 9a (886 mg, 2.12 mmol) and
phenacyl bromide (517 mg, 2.6 mmol) in EtOAc (100 mL)
triethylamine (0.6 mL, 4.3 mmol) was added at 0 8C and the
mixture was stirred at 45 8C for 20 h. The precipitate was
removed by filtration and washed with EtOAc (200 mL).
The combined filtrates were extracted with aq NaHCO3
(30 mL), brine (80 mL), dried (Na2SO4) and concentrated.
Purification on silica gel (100:1/100:25 CH2Cl2/MeOH)
afforded 1.1 g (1.97 mmol, 93%) of phenacyl (R)-3-hydro-
xyicosanoate as white solid. Rf 0.35 (9:1 toluene/EtOAc).
To a solution of phenacyl (R)-3-hydroxyicosanoate
(400 mg, 0.75 mmol) in CH2Cl2 (10 mL) a solution of
octadecanoyl chloride (300 mg, 0.99 mmol) in CH2Cl2

12124
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
(10 mL) and DMAP (234 mg, 1.9 mmol) were added and
the mixture was stirred for 3 h at rt. The reaction mixture
was diluted with EtOAc (300 mL), washed with aq NaHCO3
(50 mL), brine (50 mL), dried (Na2SO4) and concentrated.
Purification by chromatography on silica gel (EtOAc/
100:5 toluene/EtOAc) gave 10 as a white solid (475 mg,
88%). [a]2D0ZG0.0 (c 0.55, CHCl3); 1H NMR: d 7.84–7.81
(m, 2H, ortho-Ph), 7.56–7.51 (m, 1H, para-Ph), 7.43–7.38
(m, 2H, meta-Ph), 5.26 (s, 2H, CH2COPh), 5.23 (m, 1H,
3.1.9. Allyl 6-O-benzyl-2-deoxy-3-O-tetradecanoyl-2-
(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyrano-
side (14). Method A. Boron trifluoride etherate BF3$OEt2
(0.33 mL, 2.80 mmol) was added to a stirred solution of 13
(400 mg, 0.56 mmol) and borane–dimethylamine complex
BH3$Me2NH (166 mg, 2.82 mmol) in acetonitrile (10 mL)
at 0 8C under N2. The mixture was stirred for 30 min at 0 8C
and for 1 h at rt. The solution was cooled to 4 8C and
neutralized with satd aq NaHCO3 (60 mL). The mixture was
diluted with EtOAc (200 mL) and washed with satd aq
NaHCO3 (80 mL), 1 M aq HCl (2!80 mL), H2O (100 mL),
NaHCO3 (30 mL). The organic phase was dried (Na2SO4)
and concentrated. The residue was purified on silica gel
(toluene/100:15 toluene/EtOAc) which afforded 14 as a
3
bCH), 2.74–2.61 (m, 2H, aCH2), 2.38 (t, 2H, JZ7.5 Hz,
3
0
0
aCH2 ), 2.24 (t, 2H, JZ7.8 Hz, bCH2 ) 1.63–1.51 (m, 6H,
3CH2), 1.27–1.20 (m, 54H, 27CH2), 0.81 (t, 6H, 3JZ6.9 Hz,
2CH3); MALDI-TOF-MS: m/z: 735.9 [MCNa]C. Anal.
Calcd for C46H80O5: C, 77.48; H, 11.31. Found: C, 77.22; H,
11.44.
syrup (247 mg, 62%). [a]2D0ZC49.3 (c 0.15, CHCl3); H
1
NMR: d 7.34–7.29 (m, 5H, CH2Ph), 5.89 (m, 1H, CH]),
3
5.29 (dq, 1H, ]CH2trans), 5.40 (d, 1H, JNH,2Z9.7 Hz,
3.1.7. (R)-3-(Octadecanoyloxy)icosanoic acid (11). A
solution of 10 (200 mg, 0.27 mmol) in 1:2 AcOH–toluene
(20 mL) was stirred with Zn–Cu couple [1.3 g, made from
Zn (1.0 g) and 5% aq CuSO4 (7.0 mL)] at rt for 2 h. The
solids were removed by filtration through a pad of Celite,
the filtrate was concentrated, redissolved in toluene and
concentrated (3!60 mL). The residue was redissolved in
EtOAc (200 mL) and extracted with satd aq NaHCO3
(50 mL), brine (2!70 mL) and acidified water (2!50 mL,
pH adjusted to 4.5 with HCl). The organic phase was dried
(Na2SO4), concentrated and purified on silica gel (CHCl3/
100:5 CHCl3/MeOH) to afford 11 as a white solid (142 mg,
NH), 5.22 (dq, 1H, ]CH2cis), 5.14 (dd, 1H, 3J3,2Z10.3 Hz,
3J3,4Z8.7 Hz, H-3), 4.91 (d, 1H, J1,2Z3.4 Hz, H-1), 4.73
3
2
and 4.67 (AB, 2H, JZ12.1 Hz, CH2CCl3), 4.65 and 4.58
(AB, 2H, JZ12.1 Hz, CH2Ph), 4.22 (m, 1H, OCHH, All),
4.03 (m, 1H, OCHH, All), 4.0 (ddd, 1H, H-2), 3.88–3.77 (m,
2
2
3H, H-4, H-5, H-6a), 3.74 (dd, 1H, J6a,6bZ10.3 Hz,
3J6b,5Z3.9 Hz, H-6b), 2.68 (d, 1H, OH), 2.35 (m, 2H,
aCH2), 1.59–1.55 (m, 2H, CH2), 1.30–1.17 (m, 20H,
10CH2), 0.88 (t, 3H, CH3); MALDI-TOF-MS: m/z: 716.5,
718.7 (24.47% 37Cl) [MCNa]C. Anal. Calcd for
C33H50Cl3NO8: C, 57.02; H, 7.25; N, 2.02. Found: C,
56.75; H, 7.14; N, 1.94.
85%), mpZ49–50 8C; [a]2D3ZK1.3 (c 0.3, CHCl3); 1H
2
NMR: d 5.21 (m, 1H, bCH), 2.62 (dd, 1H, JaCHH
Z
3
Method B. A solution of 13 (1.3 g, 1.83 mmol) in CH2Cl2
(20 mL) and powdered activated molecular sieves 0.4 nm
were stirred for 3 h under N2 at ambient temperature. The
suspension was cooled to K78 8C and Et3SiH (320 mg,
440 mL, 2.75 mmol) and a solution of TfOH (275 mL,
3.11 mmol) in CH2Cl2 (2 mL) were added successively at
K78 8C under N2. The mixture was stirred for 40 min at
K78 8C. Et3N (2 mL) and MeOH (2 mL) were added
successively. The mixture was stirred for 15 min, then
warmed up to rt, diluted with EtOAc (30 mL) and filtered
over a pad of Celite. The filtrate was diluted with EtOAc
(300 mL) and washed successively with satd aq NaHCO3
(50 mL), H2O (100 mL), and brine (100 mL). The organic
phase was dried (cotton) and concentrated. The residue was
purified by flash chromatography on silica gel (5:1/4:1
toluene/EtOAc) which afforded 14 as a syrup (1.2 g, 92%).
15.4 Hz, JaCHH,bCHZ7.3 Hz, aCHH), 2.57 (dd, 1H,
3JaCHH,bCHZ5.1 Hz, aCHH), 2.26 (t, 2H, 3JZ7.5 Hz,
0
aCH2 ), 1.61–1.58 (m, 6H, 3CH2), 1.29–1.15 (m, 56H,
28CH2), 0.88 (t, 6H, 3JZ6.9 Hz, 2CH3); MALDI-TOF-MS:
m/z: 617.7 [MCNa]C. Anal. Calcd for C38H74O4!H2O: C,
75.56; H, 12.51. Found: C, 75.80; H, 12.6.
3.1.8. Allyl 4,6-O-benzylidene-2-deoxy-3-O-tetra-
decanoyl-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-
glucopyranoside (13). To a stirred solution of allyl 4,6-O-
benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonyl-
amino)-a-D-glucopyranoside 1223 (3 g, 6.21 mmol) in dry
pyridine (10 mL) a solution of myristoyl chloride (2.22 g,
2.45 mL, 9 mmol) in dry THF (5 mL) was added during
15 min under N2. The mixture was stirred for 1 h, diluted
with EtOAc (200 mL), washed with satd aq NaHCO3
(50 mL), brine (50 mL), dried (Na2SO4) and concentrated.
Purification of the residue on silica gel (toluene/50:1
toluene/EtOAc) afforded 13 as a white solid (4.2 g, 95%);
3.1.10. Allyl 6-O-benzyl-4-O-[bis(benzyloxy)phos-
phoryl]-2-deoxy-3-O-tetradecyl-2-(2,2,2-trichloro-
ethoxycarbonylamino)-a-D-glucopyranoside (15) and
allyl 6-O-benzyl-4-O-[bis(benzyloxy)phosphoryl]-2-
deoxy-3-O-tetradecanoyl-2-(2,2,2-trichloroethoxycar-
bonylamino)-a-D-glucopyranoside (16). Variant A. To a
solution of 14 (prepared according to method B) (330 mg,
0.47 mmol) and di-O-benzyloxy-(N,N-diisopropylamino)
phosphine (228 mg, 222 mL, 0.66 mmol) in CH2Cl2
(10 mL) a solution of 1H-tetrazole (46 mg, 0.66 mmol) in
CH3CN (3 mL) was added. The mixture was stirred for
30 min under N2, then cooled to 0 8C and a solution of tert-
butylhydroperoxide (300 mL) in CH3CN (3 mL) was slowly
added. The stirring was continued for 6 h at 0 8C, the
mixture was diluted with EtOAc (200 mL) and washed with
satd aq NaHCO3 (30 mL) and brine (30 mL). The organic
1
mpZ85–88 8C; [a]2D0ZC36.3 (c 0.3, CHCl3); H NMR: d
7.45–7.43 (m, 2H, CHPh), 7.36–7.34 (m, 3H, CHPh), 5.90
(m, 1H, CH]), 5.52 (s, 1H, CHPh), 5.40 (t, 1H,
3
3J3,2Z3J3,4Z10.0 Hz, H-3), 5.36 (d, 1H, JNH,2Z10.0 Hz,
NH), 5.31 (dq, 1H, ]CH2trans), 5.24 (dq, 1H, ]CH2cis),
3
4.93 (d, 1H, J1,2Z3.5 Hz, H-1), 4.71 (s, 2H, CH2CCl3),
2
3
4.29 (dd, 1H, J6a,6bZ10.3 Hz, J5,6aZ4.9 Hz, H-6a), 4.22
(dd, 1H, OCHH, All), 4.07–4.01 (m, 2H, H-2, OCHH, All),
3.94 (dd, 1H, H-5), 3.78 (t, 1H, 3J5,6bZ10.3 Hz, H-6b), 3.71
(t, 1H, J4,5Z10.0 Hz, H-4), 2.34–2.24 (m, 2H, aCH2), 1.59–
1.55 (m, 2H, CH2), 1.30–1.17 (m, 20H, 10CH2), 0.87 (t, 3H,
CH3); MALDI-TOF-MS: m/z: 714.0, 716.0 (24.47% 37Cl)
[MCNa]C. Anal. Calcd for C33H48Cl3NO8: C, 57.19; H,
6.98; N, 2.02. Found: C, 57.18; H, 7.46; N, 2.28.

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12125
phase was dried (Na2SO4), concentrated and the residue was
purified by chromatography on silica gel (100:1/100:15
toluene/EtOAc) which afforded 350 mg (0.37 mmol, 78%)
of 16, Rf 0.50 (A, 3:1 toluene/EtOAc). [a]2D0ZC39.5 (c 1,
CHCl3); 1H NMR: d 7.40–7.25 (m, 15H, Ph), 5.91 (m, 1H,
(1,5-dihydro-3-oxo-3l5-3H-2,4,3-benzodioxaphos-
phepin-3-yl)-3-O-tetradecanoyl-2-(2,2,2-trichloro-
ethoxycarbonylamino)-a-D-glucopyranoside (18). A
solution of 14 (prepared according to method A) (250 mg,
0.36 mmol), N,N-diethyl-1,5-dihydro-3H-2,4,3-benzo-
dioxaphosphepin-3-amine (260 mg, 1.08 mmol) and 1H-
tetrazole (126 mg, 1.8 mmol) in CH2Cl2 (10 mL) was stirred
for 20 min under N2. The mixture was brought to K20 8C
and the solution was stirred with 3-chloroperoxobenzoic
acid (70%, 222 mg, 0.9 mmol) for 10 min. The reaction was
quenched with satd aq NaHCO3 (30 mL) at 0 8C. The
mixture was diluted with CHCl3 (100 mL) and washed with
satd aq NaHCO3 (20 mL) and brine (20 mL). The organic
phase was dried (Na2SO4) and concentrated. The residue
was purified by chromatography on silica gel (3:1 hexane/
Et2O/Et2O, then 4:1/3:2 toluene/EtOAc) and, in three
portions, by HPLC (3:2 toluene/EtOAc) to afford 18 as a
main product (148 mg, 0.17 mmol, 47%); Rf 0.53 (B, 2:1
hexane/EtOAc), Rf 0.62 (B, 2:1 toluene/EtOAc); [a]2D0ZC
30.5 (c 0.2, CHCl3); 1H NMR: d 7.37–7.17 (m, 9H, Ph), 5.90
(m, 1H, CH]), 5.40 (dd, 1H, 3J3,2Z10.3 Hz, 3J3,4Z9.5 Hz,
H-3), 5.32 (dq, 1H, ]CH2trans), 5.30–5.20 (m, 2H, NH,
]CH2cis), 5.17–5.02 [m, 4H, C6H4(CH2O)2P], 4.96 (d, 1H,
3
3
CH]), 5.39 (dd, 1H, J3,2Z10.7 Hz, J3,34Z9.3 Hz, H-3),
5.34 (dq, 1H, ]CH2trans), 5.28 (d, 1H, J2,NHZ10.0 Hz,
NH), 5.27 (dq, 1H, ]CH2cis), 5.0–4.87 (m, 4H, CH2Ph),
3
4.92 (d, 1H, J1,2Z5.3 Hz, H-1), 4.70 (br s, 2H, CH2Ph),
3
4.60 (t, 1H, J4,5Z9.3 Hz, H-4), 4.57 and 4.48 (2AB, 2H,
2JZ12.0 Hz, CH2CCl3), 4.23 (m, 1H, OCHH, All), 4.07–
3.92 (m, 3H, H-2, H-5, OCHH, All), 3.77 (dd, 1H, 2J6a,6b
Z
Z
3
3
11.0 Hz, J6a,5Z2.1 Hz, H-6a), 3.71 (dd, 1H, J6b,5
3
4.6 Hz, H-6b), 2.17 (t, 2H, JZ7.4 Hz, aCH2), 1.35–1.15
(m, 2H, bCH2), 1.35–1.25 (m, 20H, 10CH2), 0.90 (t, 3H,
CH3); 13C NMR(CDCl3): d 174.45 (1C, CO), 154.52 (1C,
CONH), 138.45, 136.37 (2C, C6H5), 133.51 (]CH),
128.98, 128.86, 128.72, 128.53, 128.35, 128.0 (16C, Ph),
118.83 (]CH2), 96.53 (C-1), 95.75 (CCl3), 75.06 (CH2Ph),
2
74.10 (C-4, J4,PZ6.0 Hz), 73.83 (CH2, Troc), 71.43 (C-3,
3J3,PZ2.3 Hz), 70.36 (C-5, 3J5,PZ6.0 Hz), 69.98 and 69.91
2
(2C, CH2Ph, JC,PZ5.3 Hz), 69.49 (OCH2, All), 68.60
(C-6), 54.54 (C-2), 34.69 (aCH2), 32.56, 30.10, 30.06,
30.04, 29.86, 29.76, 29.71, 29.49 (10CH2), 24.98 (b CH2),
14.55 (CH3); 31P NMR (CDCl3): d K0.9; MALDI-TOF-
MS: m/z: 975.83, 977.92 (24.47% 37Cl) [MCNa]C. Anal.
Calcd for C47H63Cl3NO11P: C, 59.09; H, 6.65; N, 1.47.
Found: C, 59.25; H, 6.70; N, 1.42.
3
3J1,2Z3.7 Hz, H-1), 4.77 (t, 1H, J4,5Z9.5 Hz, H-4), 4.73
and 4.69 (2AB, 2H, 2JZ12.0 Hz, CH2CCl3), 4.67 and 4.59
(2AB, 2H, 2JZ12.1 Hz, CH2Ph), 4.24 (m, 1H, OCHH, All),
4.10–3.97 (m, 3H, H-2, H-5, OCHH, All), 3.82 (dd, 1H,
3
2J6a,6bZ11.1 Hz, J6a,5Z2.3 Hz, H-6a), 3.75 (dd, 1H,
3J5,6bZ4.9 Hz, H-6b), 2.38 (t, 2H, 3JZ7.4 Hz, aCH2),
1.65–1.55 (m, 2H, bCH2), 1.35–1.25 (m, 20H, 10CH2), 0.90
(t, 3H, CH3); 13C NMR(CDCl3): d 174.49 (1C, CO), 154.47
(1C, CONH), 138.33 (]CH), 135.12, 135.09 (2C, Ph),
129.37, 128.94, 128.84, 128.75, 128.25, 128.01 (10C, Ph),
118.92 (]CH2), 96.58 (C-1), 95.73 (CCl3), 75.06 (CH2,
Variant B. The reaction was carried out from 14 (prepared
according to method A) (330 mg, 0.47 mmol) in the same
manner as described above. Purification, first by column
chromatography on silica gel (100:1/100:15 toluene/
EtOAc), then in three portions by HPLC (linear gradient
toluene/100:15 toluene/EtOAc) afforded 16 as the main
product (195 mg, 0.21 mmol, 44%) and 15 (28 mg,
0.03 mmol, 6%) as a by-product; Rf 0.46 (A, 3:1 toluene/
EtOAc); 1H NMR: d 7.25–7.35 (m, 15H, Ph), 5.91 (m, 1H,
CH]), 5.30 (dq, 1H, ]CH2trans), 5.24 (dq, 1H, ]CH2cis),
5.20 (d, 1H, 3J2,NHZ9.7 Hz, NH), 5.0 (m, 4H, CH2Ph), 4.90
2
Troc), 74.76 (C-4, J4,PZ5.9 Hz), 74.05 (CH2Ph), 71.43
(C-3), 70.25 (C-5, 3J5,PZ6.0 Hz), 69.15 (OCH2, All), 68.97
(C-6, J6,PZ6.0 Hz), 68.70 [2C, (CH2O)2P], 54.45 (C-2),
34.55 (aCH2), 32.31, 30.08, 30.07, 30.04, 29.90, 29.74,
29.57, 23.08 (10CH2), 25.07 (bCH2), 14.51 (CH3); 31P
NMR (CDCl3): d 0.0; MALDI-TOF-MS: m/z: 898.6, 900.4
(24.47% 37Cl) [MCNa]C.
3
2
(d, 1H, J1,2Z3.5 Hz, H-1), 4.80 and 4.71 (2AB, 2H, JZ
2
12.0 Hz, CH2CCl3), 4.57 and 4.45 (2AB, 2H, JZ12.0 Hz,
3
CH2Ph), 4.48 (t, 1H, J4,5Z9.5 Hz, H-4), 4.21 (m, 1H,
OCHH, All), 4.05–3.97 (m, 2H, H-2, OCHH, All), 3.88 (m,
1H, H-5), 3.80–3.69 (m, 3H, H-6a, H-6b, aCHH), 3.63 (dd,
Further elution gave 17 (25 mg, 0.03 mmol, 8%) as a minor
product; Rf 0.50 (B, 2:1 hexane/EtOAc), 0.55 (B, 2:1
toluene/EtOAc). H NMR: d 7.30–7.10 [m, 9H, CH2Ph,
1
3
3
1H, J3,2Z10.5 Hz, J3,4Z9.2 Hz, H-3), 3.54 (m, 1H,
aCHH), 1.48 (m, 2H, bCH2), 1.30–1.18 (m, 20H, 10CH2),
0.90 (t, 3H, CH3); 13C NMR (CDCl3): d 154.43 (CONH),
138.45, 136.37 (2C, Ph), 138.60 (1C, Ph), 136.30, 136.21
C6H4(CH2O)2P], 5.83 (m, 1H, CH]), 5.27–5.10 [m, 3H,
]CH2trans, ]CH2cis, C6H4(CHHO)2P], 5.07 (d, 1H,
3J2,NHZ6.8 Hz, NH), 4.95–5.20 [m, 1H, C6H4(CHHO)2P],
3
4.83 (d, 1H, J1,2Z3.7 Hz, H-1), 4.72 and 4.60 (2AB, 2H,
3
3
2JZ12.0 Hz, CH2CCl3), 4.52 (t, 1H, J4,5Z9.8 Hz, H-4),
(2C, Ph, JC,PZ4.0 Hz), 133.72 (]CH), 128.94–127.86
(15C, C6H5, Bn), 118.62 (]CH2), 96.90 (C-1), 95.82
4.53 and 4.48 (2AB, 2H, 2JZ12.0 Hz, CH2Ph), 4.13 (m, 1H,
OCHH, All), 3.97–3.82 (m, 3H, H-2, H-5, OCHH, All), 3.75
(CCl3), 79.39 (C-3), 75.96 (C-4, 2J4,PZ6.8 Hz), 75.17 (CH2,
3
2
3
Troc), 73.71 (CH2Ph), 72.78 (aCH2), 70.68 (C-5, J5,P
Z
(dd, 1H, J6a,6bZ11.0 Hz, J6a,5Z2.0 Hz, H-6a), 3.70 (dd,
4.5 Hz), 69.87 and 69.79 (2C, CH2Ph, 2JC,PZ2.0 Hz), 68.96
and 68.86 (2C, OCH2, All, C-6), 54.73 (C-2), 32.32 (bCH2),
30.59, 30.09, 30.02, 29.76, 26.36, 23.09 (10CH2), 14.51
(CH3); 31P NMR (CDCl3): d 0.1; MALDI-TOF-MS: m/z:
962.34, 964.20 (24.47% 37Cl) [MCNa]C.
3
1H, J6b,5Z5.1 Hz, H-6b), 3.63 (ddd, 1H, aCHH), 3.58 (t,
1H, 3J3,2Z3J3,4Z9.8 Hz, H-3), 3.53 (ddd, 1H, aCHH), 1.47
(d, 2H, bCH2), 1.20–1.15 (m, 20H, 10CH2), 0.81 (t, 3H,
CH3); 13C NMR(CDCl3): d 154.37 (CONH), 138.57
(]CH), 135.53, 133.66 (2C, Ph), 129.34, 129.02, 128.68,
128.11, 127.88 (10C, Ph), 118.67 (]CH2), 96.91 (C-1),
95.83 (CCl3), 79.20 (C-3), 76.48 (C-4, 2J4,PZ4.0 Hz), 75.14
(CH2, Troc), 73.83 (CH2Ph), 72.62 (aCH2), 70.51 (C-5,
3J5,PZ2.0 Hz), 68.90, 68.82, 68.71, 68.68 [4C, OCH2, All,
(CH2O)2P, C-6], 54.69 (C-2), 32.32 (b CH2), 30.57, 30.10,
3.1.11. Allyl 6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-
oxo-3l5-3H-2,4,3-benzodioxaphosphepin-3-yl)-3-O-
tetradecyl-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-
glucopyranoside (17) and allyl 6-O-benzyl-2-deoxy-4-O-

12126
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
29.76, 26.45, 23.08 (10CH2), 14.51 (CH3); 31P NMR
(CDCl3): d 0.4; MALDI-TOF-MS: m/z: 884.02, 885.85
(24.47% 37Cl) [MCNa]C.
135.38 (2C, C6H5), 128.58, 128.46, 128.42, 127.86, 127.53,
127.32 (16C, Ph), 95.13 (C-1), 94.50 (CCl3), 90.69
[C(NH)CCl3], 74.59 (CH2Ph), 73.37 (CH2, Troc), 72.64
2
3
(C-4, J4,PZ6.0 Hz), 72.44 (C-5, J5,PZ6.0 Hz), 70.20
3
2
(C-3, J3,PZ2.3 Hz), 69.63 and 69.55 (2C, CH2Ph, JC,P
Z
3.1.12. 6-O-Benzyl-4-O-[bis(benzyloxy)phosphoryl]-2-
deoxy-3-O-tetradecanoyl-2-(2,2,2-trichloroethoxycar-
bonylamino)-D-glucopyranose (19). To a degassed
solution of 16 (600 mg, 0.63) in THF (20 mL) {[bis(methyl-
diphenyl)phosphine](1,5-cyclooctadiene) iridium(I)}hexa-
fluorophosphate (25 mg, 0.03 mmol) was added. The
mixture was degassed four times and filled with He, then
three times degassed and filled with H2 (which was kept for
10 s each time), and then four times degassed and filled with
He. The mixture was stirred under He for 30 min, cooled to
0 8C and a solution of I2 (304 mg, 1.2 mmol) in 2:1 THF/
H2O (3 mL) was added dropwise. The mixture was stirred at
0 8C for 6 h, then for 2 h at 25 8C, diluted with EtOAc
(200 mL), washed with 5% aq Na2S2O3 (20 mL), satd aq
NaHCO3 (30 mL) and brine (30 mL). The organic phase
was dried (cotton), concentrated and the residue was
purified by chromatography on silica gel (85:15/75:25
toluene/EtOAc) to afford 19 (527 mg, 91%) as a solid. Rf
5.3 Hz), 67.46 (C-6), 54.04 (C-2), 34.69 (aCH2), 32.56,
30.10, 30.06, 30.04, 29.86, 29.76, 29.71, 29.49 (10CH2),
24.98 (bCH2), 14.55 (CH3); 31P NMR (CDCl3): d 0.8.
3.1.14. Allyl 2-deoxy-4,6-O-isopropylidene-3-O-tetra-
decanoyl-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-
glucopyranoside (24). A solution of allyl 2-deoxy-4,6-O-
isopropylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-
D-glucopyranoside 2332 (2.2 g, 5.06 mmol), tetradecanoic
acid (1.44 g, 6.3 mmol), 1,3-dicyclohexylcarbodiimide
(1.41 g, 6.83 mmol) and 4-N,N-dimethylaminopyridine
(5 mg, 0.04 mmol) in CH2Cl2 (25 mL) was stirred for
30 min at rt. Methanol (1.5 mL) and acetic acid (0.6 mL)
were added and the mixture was stirred for further 30 min.
Insoluble materials were filtered off and the filtrate was
concentrated to 10 mL. The suspension was filtered, the
filtrate was diluted with EtOAc (150 mL) and extracted with
satd aq NaHCO3 (2!100 mL), H2O (100 mL), dried
(Na2SO4) and concentrated. The residue was purified by
silica gel chromatography (toluene/100:5 toluene/EtOAc)
to afford 24 (2.8 g, 86%). [a]2D0ZC56.0 (c 0.3, CHCl3); 1H
1
0.45 (2:1 toluene/EtOAc); [a]2D0ZC16.0 (c 1, CHCl3); H
NMR (for a-anomer): d 7.35–7.25 (m, 15H, Ph), 5.40 (dd,
3
3
1H, J3,2Z10.9 Hz, J3,4Z9.3 Hz, H-3), 5.32 (d, 1H,
3J2,NHZ9.8 Hz, NH), 5.0–4.87 (m, 4H, 2CH2Ph), 4.92 (t,
3
3
1H, J1,2Z3J1,OHZ4.0 Hz, H-1), 4.94, 4.93, 4.92, 4.91
NMR: d 5.88 (m, 1H, CH]), 5.36 (d, 1H, J2,NHZ9.8 Hz,
NH), 5.32 (dq, 1H, ]CH2trans), 5.29–5.18 (m, 2H,
(2AB, 4H, 2JZ11.5 Hz, 2CH2Ph), 4.72 and 4.66 (2AB, 2H,
2JZ11.8 Hz, CH2Ph), 4.54 and 4.45 (2AB, 2H, 2JZ
3
]CH2cis, H-3), 4.89 (d, 1H, J1,2Z3.7 Hz, H-1), 4.73 and
3
2
12.0 Hz, CH2CCl3), 4.44 (t, 1H, J4,5Z9.3 Hz, H-4), 4.19
(ddd, 1H, J6a,5Z1.8 Hz, J6b,5Z7.1 Hz, H-5), 3.95 (ddd,
1H, H-2), 3.77 (dd, 1H, J6a,6bZ10.7 Hz, H-6a), 3.66 (d,
1H, OH-1), 3.64 (dd, 1H, H-6b), 2.16 (t, 2H, JZ7.4 Hz,
4.68 (AB, 2H, JZ12.0 Hz, CH2CCl3), 4.44–3.96 (m, 2H,
3
3
H-2, OCHH, All), 4.20 (m, 1H, OCHH, All), 3.90 (m, 1H,
H-6a), 3.82–3.68 (m, 3H, H-4, H-5, H-6b), 2.30 (m, 2H,
aCH2), 1.63 (m, 2H, bCH2), 1.49 (s, 3H) and 1.40 (s, 3H,
CMe2), 1.34–1.24 (m, 20H, 8CH2), 0.88 (t, 3H, CH3);
MALDI-TOF-MS: m/z: 666.4, 668.4 (24.47% 37Cl) [MC
Na]C. Anal. Calcd for C29H48Cl3NO8: C, 54.00; H, 7.50; N,
2.17. Found: C, 53.91; H, 7.62; N, 2.15.
2
3
aCH2), 1.50–1.40 (m, 2H, bCH2), 1.35–1.25 (m, 20H,
10CH2), 0.90 (t, 3H, CH3); 31P NMR (CDCl3): d K1.5;
MALDI-TOF-MS: m/z: 936.35, 938.35 (24.47% 37Cl) [MC
Na]C. Anal. Calcd for C44H59Cl3NO11P: C, 57.74; H, 6.50;
N, 1.53. Found: C, 57.52; H, 6.24; N, 1.58.
3.1.15. Allyl 2-[(R)-3-(benzyloxy)icosanoylamino]-2-
deoxy-3-O-tetradecanoyl-a-D-glucopyranoside (27). A
suspension of 24 (307 mg, 0.48 mmol) in 90% aq AcOH
(3 mL) was stirred at 90 8C for 20 min. The solution was
concentrated and the residue was purified by chromato-
graphy (2:1/1:1 toluene/EtOAc) to give 270 mg
(0.45 mmol, 95%) of 25 [allyl 2-deoxy-3-O-tetradecanoyl-
2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyrano-
3.1.13. 6-O-Benzyl-4-O-[bis(benzyloxy)phosphoryl]-2-
deoxy-3-O-tetradecanoyl-2-(2,2,2-trichloroethoxycar-
bonylamino)-D-glucopyranose trichloroacetimidate (20).
A solution of 19 (526 mg, 0.575 mmol) in CH2Cl2 (10 mL)
was stirred with trichloroacetonitrile (0.5 mL, 5 mmol) and
Cs2CO3 (98 mg, 0.3 mmol) for 1 h. The reaction was
stopped by addition of satd aq NaHCO3 (5 mL). The
mixture was diluted with CH2Cl2 (200 mL) and washed
with satd aq NaHCO3 (30 mL) and brine (30 mL). The
organic phase was dried (cotton) and concentrated, the
residue was purified by flash chromatography on silica gel
(4:1:0.5 toluene/EtOAc/Et3N) to give 20 as a syrup
(360 mg, 0.34 mmol, 59%). Rf 0.58 (3:1 toluene/EtOAc);
1
side] as a syrup. Rf 0.55 (1:2 toluene/EtOAc); H NMR: d
5.90 (m, 1H, CH]), 5.34 (d, 1H, 3JNH,2Z9.7 Hz, NH), 5.32
(dq, 1H, ]CH2trans), 5.25 (dq, 1H, ]CH2cis), 5.15 (m, 1H,
3
H-3), 4.92 (d, 1H, J1,2Z3.6 Hz, H-1), 4.76 and 4.68 (AB,
2H, 2JZ12.0 Hz, CH2CCl3), 4.22 (m, 1H, OCHH, All), 4.03
(m, 1H, OCHH, All), 3.98 (m, 1H, H-2), 3.88 (AB, 2H,
H-6a, H-6b), 3.80–3.72 (ABX, 2H, H-4, H-5), 2.72 (br s,
1H, OH), 2.35 (t, 2H, aCH2), 2.03 (br s, 1H, OH), 1.65–1.55
(m, 4H, 2CH2), 1.20–1.35 (m, 50H, 25CH2), 0.88 (t, 6H,
CH3). A solution of 25 (270 mg, 0.45 mmol) in acetic acid
(5 mL) was stirred with Zn–Cu couple [1.0 g, made from Zn
(0.8 g) and 5% aq CuSO4 (4 mL)] at rt for 2 h. The solids
were removed by filtration, the filtrate was concentrated and
was repeatedly evaporated with toluene (3!30 mL). The
residue was dissolved in EtOAc (100 mL) and extracted
with satd aq NaHCO3 (50 mL) and brine (50 mL). The
organic phase was dried (Na2SO4) and concentrated. The
1
[a]2D0ZC37.0 (c 1, CHCl3); H NMR: d 8.68 (s, 1H, NH),
3
7.26–7.15 (m, 15H, Ph), 6.34 (d, 1H, J1,2Z3.7 Hz, H-1),
3
3
5.37 (dd, 1H, J3,2Z11.2 Hz, J3,4Z9.3 Hz, H-3), 5.16 (d,
3
1H, Troc, J2,NHZ9.4 Hz, NH), 4.93–4.80 (m, 4H,
3
2CH2Ph), 4.70 (dd, 1H, J4,5Z9.3 Hz, H-4), 4.63 and 4.56
2
(2AB, 2H, JZ11.8 Hz, CH2Ph), 4.45 and 4.38 (2AB, 2H,
2JZ12.0 Hz, CH2CCl3), 4.10 (ddd, 1H, H-2), 3.97 (m, 1H,
3
H-5), 3.68 (m, 2H, H-6a, H-6b), 2.15 (t, 2H, JZ7.4 Hz,
aCH2), 1.40–1.30 (m, 2H, bCH2), 1.25–1.15 (m, 20H,
10CH2), 0.90 (t, 3H, CH3); 13C NMR(CDCl3): d 174.44 (1C,
CO), 160.39 (1C, O–C]N), 154.0 (1C, CONH), 137.82,

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12127
residue was purified by flash chromatography on silica gel
(100:5 EtOAc/MeOH) to give 135 mg (0.31 mmol, 70%) of
26 (allyl 2-amino-2-deoxy-3-O-tetradecanoyl-a-D-gluco-
trichloroacetonitrile (0.5 mL, 5 mmol) and Cs2CO3 (98 mg,
0.3 mmol) for 1 h. The reaction was stopped by addition of
satd aq NaHCO3 (2 mL), the mixture was diluted with
CH2Cl2 (150 mL) and washed with satd aq NaHCO3
(20 mL) and brine (20 mL). The organic phase was dried
(cotton), concentrated and dried by repeated evaporations
with dry toluene (2!15 mL). The crude 22 [4,6-O-
benzylidene-2-deoxy-3-O-tetradecanoyl-2-(2,2,2-trichloro-
ethoxycarbonylamino)-a-D-glucopyranosyl trichloro-
acetimidate] (0.24 mmol, crude) and 27 (150 mg,
0.18 mmol) were dissolved in CH2Cl2 (5 mL) and stirred
with powdered activated molecular sieves (0.4 nm) under
N2 for 2 h. The suspension was cooled to K25 8C and a
solution of trimethylsilyl trifluoromethanesulfonate (3.6 mL,
0.02 mmol) in CH2Cl2 (1 mL) was added. The stirring was
continued for 20 min and the reaction was stopped by
addition of dry Na2CO3. The solids were removed by
filtration over a pad of Celite, the filtrate was diluted with
CH2Cl2 (150 mL) and washed with satd aq NaHCO3
(30 mL), H2O (30 mL) and brine (30 mL). The organic
phase was dried (cotton) and concentrated. The residue was
purified by chromatography on silica gel (30:1 CHCl3/
acetone). Appropriate fractions were collected, concen-
trated to dryness and purified by repeated precipitations.
The residue was dissolved in CH2Cl2 (3 mL), then acetone
(30 mL) was added. The volume was reduced to 15 mL by
evaporation, the suspension was cooled to 4 8C, the white
fluffy precipitate was separated on a glass-filter and washed
with acetone (5 mL). The precipitate was redisolved in
CH2Cl2 (10 mL) and the solution was concentrated to
dryness which afforded 28 (200 mg, 0.14 mmol, 76% based
on the acceptor 27; 57% based on 22) as a white solid. Rf 0.4
(B, 3:1 toluene/EtOAc), Rf 0.6 (B, 10:1 CH2Cl2/acetone);
1
pyranoside). Rf 0.3 (15:1 EtOAc/MeOH); H NMR: d 5.90
(m, 1H, CH]), 5.30 (dq, 1H, ]CH2trans), 5.20 (dq, 1H,
3
]CH2cis), 4.96 (t, 1H, J3,4Z9.8 Hz, H-3), 4.87 (d, 1H,
3J1,2Z3.5 Hz, H-1), 4.19 (m, 1H, OCHH, All), 4.0 (m, 1H,
OCHH, All), 3.81 (AB, 2H, H-6a, H-6b), 3.69 (ABX, 1H,
3
H-5), 3.55 (t, 1H, H-4), 2.83 (dd, 1H, J2,3Z9.8 Hz, H-2),
2.23 (br s, 3H, OH, NH2), 2.40 (t, 2H, aCH2), 1.68–1.59 (m,
4H, 2CH2), 1.35–1.20 (m, 50H, 25CH2), 0.88 (t, 6H, 2CH3);
13C NMR (CDCl3): d 175.93 (1C, CO), 134.06 (]CH),
118.04 (]CH2), 99.03 (C-1), 95.73 (CCl3), 78.41 (C-3),
72.46 (C-5), 70.31 (C-4), 68.91 (OCH2), 62.46 (C-6), 54.79
(C-2), 34.83 (aCH2), 32.28, 30.04–29.54 (10CH2), 25.41
(bCH2), 14.48 (CH3).
The free amine 26 (135 mg, 0.31 mmol) was dissolved in
CH2Cl2 (5 mL) and the solution was stirred with 8 (200 mg,
0.48 mmol) and 1,3-dicyclohexylcarbodiimide (110 mg,
0.54 mmol) at rt for 2 h. Methanol (0.5 mL) and acetic
acid (0.2 mL) were added and the mixture was stirred for
further 30 min. Insoluble materials were separated and the
filtrate was concentrated to 5 mL volume. The precipitate
was filtered off, the filtrate was diluted with EtOAc
(100 mL) and washed with satd aq NaHCO3 (20 mL) and
brine (20 mL). The organic phase was dried (Na2SO4) and
concentrated. The residue was purified on silica gel (1:1/
1:3 cyclohexane/Et2O) to give 221 mg (0.27 mmol, 86%) of
27 as a solid. Rf 0.4 (1:1 EtOAc/toluene); mpZ84–86 8C;
[a]2D3ZC42.3 (c 0.3, CHCl3); H NMR: d 7.34–7.26 (m,
1
3
5H, Ph), 6.31 (d, 1H, JNH,2Z9.5 Hz, NH), 5.73 (m, 1H,
CH]), 5.19 (dq, 1H, ]CH2t3rans), 5.12–5.07 (m, 2H,
]CH2cis, H-3), 4.77 (d, 1H, J1,2Z3.5 Hz, H-1), 4.52
1
2
3
[a]2D0ZC4.0 (c 1.0, CHCl3); H NMR: d 7.45–7.25 (m,
(AB, 2H, JA,BZ11.5 Hz, CH2Ph), 4.29 (ddd, 1H, J2,3
Z
3
10H, Ph), 6.34 (d, 1H, J2,NHZ9.4 Hz, NH), 5.87 (d, 1H,
9.8 Hz, H-2), 4.03 (dd, 1H, OCHH, All), 3.87–3.82 (m, 2H,
OCHH, All), 3.80–3.69 (m, 4H, H-4, H-5, H-6a, H-6b), 3.20
(d, 1H, 3J4,OHZ4.9 Hz, 4-OH), 2.60 (t, 1H, 3J6,OHZ6.0 Hz,
6-OH), 2.40–2.27 (m, 4H, 2aCH2), 1.59–1.44 (m, 4H,
2CH2), 1.30–1.25 (m, 50H, 25CH2), 0.88 (t, 6H, 2CH3);
MALDI-TOF-MS: m/z: 852.3 [MCNa]C. Anal. Calcd for
C50H87NO8: C, 72.33; H, 10.56; N, 1.69. Found: C, 72.31;
H, 10.54; N, 1.69.
3
0
0
0
J2 ,NH Z9.3 Hz, NH ), 5.75 (m, 1H, CH]), 5.50 (s, 1H,
3
3
0
0
0
0
CHPh), 5.35 (dd, 1H, J3 ,2 Z10.2 Hz, J3 ,4 Z9.4 Hz,
H-30), 5.20 (dq, 1H, ]CH2trans), 5.12 (dq, 1H, ]CH2cis),
3
3
5.10 (dd, 1H, J3,2Z10.2 Hz, J3,4Z9.7 Hz, H-3), 4.79 (d,
1H, J1,2Z3.6 Hz, H-1), 4.78 and 4.68 (2AB, 2H, 2JZ
3
11.8 Hz, CH2, Troc), 4.56 and 4.52 (2AB, 2H, 2JZ12.0 Hz,
3
0
0
0
CH2Ph), 4.46 (d, 1H, J1 ,2 Z8.3 Hz, H-1 ), 4.34 (dd, 1H,
2
3
0
0
0
0
0
J6a ,6b Z10.2 Hz, J5 ,6a Z4.9 Hz, H-6a ), 4.29 (ddd, 1H,
H-2), 4.04 (m, 1H, OCHH, All), 3.99 (m, 1H, H-5), 3.89–
3.1.16. Allyl 6-O-[4,6-O-benzylidene-2-deoxy-3-O-tetra-
decanoyl-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-
glucopyranosyl]-2-[(R)-3-(benzyloxy)icosanoylamino]-2-
deoxy-3-O-tetradecanoyl-a-D-glucopyranoside (28). To a
degassed solution of 13 (200 mg, 0.29 mmol) in THF
(20 mL) iridium catalyst (25 mg, 0.03 mmol) was added and
activated with H2 as described for 19. The mixture was
stirred under He for 30 min, then cooled to 0 8C and a
solution of I2 (100 mg, 0.4 mmol) in 2:1 THF/H2O (3 mL)
was added dropwise. The solution was stirred at 0 8C for 4 h,
then for 2 h at 25 8C. The mixture was diluted with EtOAc
(200 mL), washed with 5% aq Na2S2O3 (20 mL), satd aq
NaHCO3 (30 mL) and brine (30 mL). The organic phase
was dried (Na2SO4), concentrated and the residue was
purified by chromatography on silica gel (2:1 n-hexane/
EtOAc) to afford 21 [allyl 4,6-O-benzylidene-2-deoxy-3-O-
tetradecanoyl-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-
glucopyranose] (157 mg, 0.24 mmol, 83%) as a solid. The
residue was taken up in CH2Cl2 (10 mL) and stirred with
3.70 (m, 6H, H-4, H-6a, H-6b, H-6b0, bCHOBn, OCHH
All), 3.68 (t, 1H, 3J4 ,5 Z9.4 Hz), 3.50 (dt, 1H, Hz, J5 ,6b
Z
3
0
0
0
0
10.5 Hz, H-50), 3.20 (br s, 1H, OH-4), 2.38–2.32 (m, 6H,
IcoaCH2, 2MyraCH2), 1.60 (m, 6H, 2MyrbCH2, IcogCH2),
1.35–1.15 (m, 74H, 37CH2), 0.90 (t, 9H, 3CH3); 13C NMR
(CDCl3): d 175.25 (CO), 174.31 (CO), 171.44 (CONH),
154.91 (CONH, Troc), 138.77, 137.30, 137.26 (2C, Ph),
133.75 (]CH), 129.41, 128.70, 128.59, 127.95, 127.92,
126.30 (10C, Ph), 118.22 (]CH2), 102.62 (C-10), 101.51
(CHPh), 96.86 (C-1), 95.83 (CCl3), 79.19 (C-40), 76.62
(
IcoCHOBn), 75.0 (CH2, Troc), 74.14 (C-3), 71.76 (CH2Ph),
71.45 (C-30), 70.90 (C-4), 69.50 (C-5), 69.29 (C-6), 68.84
(C-60), 68.64 (OCH2, All), 66.60 (C-50), 56.84 (C-20), 51.78
(C-2), 42.0 (IcoaCH2), 34.73, 34.57, 32.26 (2MyraCH2,
IcogCH2), 30.05, 29.99, 29.91, 29.79, 29.70, 29.58, 29.56,
29.35, 25.69, 25.35, 25.28, 23.03 (37CH2), 14.44 (3CH3);
MALDI-TOF-MS: m/z: 1485.95 [MCNa]C, 1501.90 [MC
K]C.

12128
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
3.1.17. Allyl 6-O-[2-amino-4,6-O-benzylidene-2-deoxy-3-
O-tetradecanoyl-b-D-glucopyranosyl]-2-[(R)-3-(benzyl-
oxy)icosanoylamino]-2-deoxy-3-O-tetradecanoyl-a-D-
glucopyranoside (29). A solution of 28 (136 mg,
0.093 mmol) in 3:2 acetic acid/toluene (5 mL) was stirred
with Zn–Cu couple [1.0 g, made from Zn (0.8 g) and 5% aq
CuSO4 (4 mL)] at rt for 1 h and at 40 8C for 30 min. The
solids were removed by filtration over a pad of Celite and
washed with 1:1 toluene/acetic acid (15 mL). The filtrate
was concentrated, the residue was redissolved in toluene
and concentrated (3!20 mL). The residue was dissolved in
CH2Cl2 (150 mL) and extracted with satd aq NaHCO3
(20 mL), water (20 mL) and brine (20 mL). The organic
phase was dried (cotton), concentrated and purified by
chromatography on silica gel (2!20 cm, 100:0/100:2
CH2Cl2/MeOH) to afford 29 (80 mg, 0.06 mmol, 66%), Rf
the white fluffy precipitate was separated on the glass-filter
and washed with acetone (5 mL). The precipitate was
redissolved in CH2Cl2 (10 mL), the solution was concen-
trated to dryness which afforded 30 (90 mg, 0.07 mmol,
76%). Rf 10.65 (B, 10:1 CH2Cl2/acetone); [a]2D0ZG0 (c 0.5,
CHCl3); H NMR: d 7.35–7.20 (m, 15H, Ph), 6.40 (d, 1H,
3
3
0
0
0
J2 ,NH Z9.0 Hz, NH ), 6.29 (d, 1H, J2,NHZ9.5 Hz, NH),
5.65 (m, 1H, CH]), 5.40 (s, 1H, CHPh), 5.12 (dd, 1H,
3
3
0
0
0
0
0
J3 ,2 Z10.2 Hz, J3 ,4 Z9.5 Hz, H-3 ), 5.11 (dq, 1H,
3
3
]CH2trans), 5.02 (dd, 1H, J3,2Z10.5 Hz, J3,4Z8.9 Hz,
3
H-3), 5.01 (dq, 1H, ]CH2cis), 4.67 (d, 1H, J1,2Z3.8 Hz,
H-1), 4.56 and 4.41 (2AB, 2H, 2JZ12.1 Hz, CH2Ph),
2
4.46 and 4.42 (2AB, 2H, JZ12.0 Hz, CH2Ph), 4.25 (dd,
1H, J6a ,6b Z10.6 Hz, J5 ,6a Z5.0 Hz, H-6a ), 4.19 (ddd,
2
3
0
0
0
0
3
0
0
0
0
1H, H-2), 4.17 (d, 1H, J1 ,2 Z8.1 Hz, H-1 ), 3.93 (m,
1H, OCHH, All), 3.90 (ddd, 1H, H-20), 3.81 (dd, 1H,
3
0.2 (B, 10:1 CH2Cl2/acetone); [a]2D0ZC1.0 (c 1.0, CHCl3);
2J6a,6bZ10.0 Hz, J6a,5Z1.8 Hz, H-6a), 3.75–3.55 (m,
3
1H NMR: d 7.45–7.25 (m, 10H, Ph), 6.29 (d, 1H, J2,NH
Z
9.5 Hz, NH), 5.73 (m, 1H, CH]), 5.50 (s, 1H, CHPh), 5.20
7H, H-4, H-5, H-6b, H-6b0, 2bCHOBn, OCHH, All),
3
0
0
0
3.57 (t, 1H, J4 ,5 Z9.5 Hz, H-4 ), 3.33 (ddd, 1H,
3
3
3
J5 ,6b Z10.0 Hz, H-5 ), 3.20 (d, 1H, 2JZ4.0 Hz, OH-
4), 2.30–2.18 (m, 8H, 2IcoaCH2, 2MyraCH2), 1.55–1.45
(m, 8H, 2MyrbCH2, 2IcogCH2), 1.3–1.10 (m, 110H,
55CH2), 0.80 (t, 12H, 4CH3); 13C NMR(CDCl3): d
175.04 (CO), 173.92 (CO), 172.03 (CONH), 171.27
(CONH), 138.80, 138.47, 137.26 (3C, Ph), 133.78
(]CH), 129.38, 129.05, 128.68, 128.53, 128.28,
128.16, 127.95, 127.088, 126.39 (15C, Ph), 117.97
(]CH2), 102.31 (C-1 ), 101.63 (CHPh), 96.92 (C-1),
79.06 (C-40), 76.63 (IcoCHOBn), 76.45 (Ico0CHOBn),
73.82 (C-3), 71.77 (CH2Ph), 71.35 (C-3 ), 70.87
(CH2Ph), 70.58 (C-4), 69.48 (C-5), 69.21 (C-6), 68.90
(C-60), 68.60 (OCH2, All), 66.88 (C-50), 54.85 (C-20),
51.85 (C-2), 42.10 and 41.47 (2IcoaCH2), 34.70, 34.61,
34.40, 33.98 (2MyraCH2, 2IcogCH2), 32.27, 30.05, 29.91,
29.80, 29.79, 29.70, 29.62, 29.54, 29.36, 25.68, 25.45,
25.41, 25.28, 23.03 (55CH2), 14.45 (4CH3); MALDI-
TOF-MS: m/z: 1712.37 [MCNa]C, 1728.34 [MCK]C;
calcd 1712.28 [MCNa]C, 1728.37 [MCK]C.
0
0
0
(dq, 1H, ]CH2trans), 5.14 (dd, 1H, J3,2Z10.5 Hz, J3,4
Z
8.9 Hz, H-3), 5.13 (dd, 1H, 3J3 ,2 Z10.0 Hz, J3 ,4 Z9.3 Hz,
3
0
0
0
0
3
H-30), 5.12 (dq, 1H, ]CH2cis), 4.80 (d, 1H, J1,2Z3.7 Hz,
2
H-1), 4.56 and 4.52 (2AB, 2H, JZ11.7 Hz, CH2Ph), 4.40
3
2
0
0
0
0
0
(d, 1H, J1 ,2 Z8.0 Hz, H-1 ), 4.36 (dd, 1H, J6a ,6b
Z
10.7 Hz, 3J5 ,6a Z5.2 Hz, H-6a ), 4.33 (ddd, 1H, H-2), 4.16
0
0
0
2
3
(dd, 1H, J6a,6bZ9.9 Hz, J5,6aZ1.5 Hz, H-6a), 4.05 (m,
1H, OCHH, All), 3.90–3.76 (m, 6H, H-4, H-5, H-6b, H-6b0,
3
0
0
0
bCHOBn, OCHH, All), 3.64 (t, 1H, 0 J4 ,5 Z9.5 Hz, H-40),
3.53 (ddd, 1H, 3J5 ,6b Z10.0 Hz, H-5 ), 2.95 (dd, 1H, H-2 ),
2.35–2.15 (m, 7H, IcoCH2, 2MyraCH2, OH-4), 1.70–1.55 (m,
6H, 2MyrbCH2, IcogCH2), 1.35–1.15 (m, 74H, 37CH2), 0.90
(t, 9H, 3CH3); 13C NMR(CDCl3): d 175.19 (CO), 174.09
(CO), 171.42 (CONH), 138.81, 137.40 (2C, 2C6H5), 133.76
(]CH), 129.38, 128.77, 128.57, 128.0, 126.45 (10C, Ph),
118.26 (]CH2), 105.85 (C-10), 101.76 (CHPh), 97.14
(C-1), 79.38 (C-40), 76.71 (IcoCHOBn), 74.06 (2C, C-3,
C-30), 71.84 (CH2Ph), 71.25 (C-4), 70.13 (C-6), 69.30
(C-60), 69.05 (C-5), 68.81 (OCH2, All), 67.10 (C-50), 57.36
(C-20), 51.79 (C-2), 42.10 (IcoaCH2), 34.78, 34.76, 34.42
(2MyraCH2, IcogCH2), 32.32, 30.11, 30.07, 29.95, 29.80,
29.75, 29.66, 29.58, 29.42, 25.76, 25.53, 25.34, 23.08
(37CH2), 14.51 (3CH3).
0
0
3.1.19. 6-O-{4,6-O-Benzylidene-2-deoxy-2-[(R)-3-(octa-
decanoyloxy)icosanoylamino]-3-O-tetradecanoyl-b-D-
glucopyranosyl}-2-[(R)-3-(benzyloxy)icosanoylamino]-
2-deoxy-3-O-tetradecanoyl-D-glucopyranose (31). To a
degassed solution of 30 (46 mg, 0.27 mmol) in THF
(10 mL) iridium catalyst (15 mg, 0.02 mmol) was added
and activated with H2 as described for 19 and the mixture
was stirred under He for 30 min, then cooled to 0 8C. A
solution of I2 (20 mg, 0.08 mmol) in 2:1 THF/H2O (1 mL)
was added dropwise. The mixture was stirred at 0 8C for 4 h,
then for 2 h at 25 8C. The solution was diluted with EtOAc
(100 mL), washed with 5% aq Na2S2O3 (20 mL), satd aq
NaHCO3 (20 mL), water (20 mL) and brine (20 mL). The
organic phase was dried (cotton), concentrated and the
residue was purified by precipitation with acetone from
CHCl3 (2!) (as described for 30) and by chromatography
on silica gel (10:1 CHCl3/acetone) to afford 31 (42 mg,
0.025 mmol, 94%) as a solid. Rf 0.3 (B, 10:1 CH2Cl2/
acetone); [a]2D0ZK10.0 (c 1.0, CHCl3); 1H NMR (for
a-anomer): d 7.45–7.30 (m, 15H, Ph), 6.45 (d, 1H,
3.1.18. Allyl 6-O-{4,6-O-benzylidene-2-deoxy-2-[(R)-3-
(octadecanoyloxy)icosanoylamino]-3-O-tetradecanoyl-
b-D-glucopyranosyl}-2-[(R)-3-(benzyloxy)icosanoyl-
amino]-2-deoxy-3-O-tetradecanoyl-a-D-glucopyranoside
(30). To a stirred solution of 29 (90 mg, 0.07 mmol) and 8
(50 mg, 0.12 mmol) in CH2Cl2 (3 mL) under N2 a solution
of 1-isopropyloxycarbonyl-2-isopropyloxy-1,2-dihydro-
quinoline (36 mg, 35 mL, 0.12 mmol) in CH2Cl2 (1 mL)
was added. After stirring for 3 h, a white precipitate was
formed. The mixture was diluted with CHCl3 (2 mL) and
purged with N2 until the volume of the mixture was reduced
to 2 mL. Stirring was continued for 10 h, the mixture was
diluted with CH2Cl2 (150 mL) and extracted with satd aq
NaHCO3 (20 mL), water (20 mL) and brine (20 mL). The
organic phase was dried (cotton), concentrated and purified
by repeated precipitations with acetone from CH2Cl2 (!2)
(see purification of 28) and with acetone from CHCl3 (!3)
as follows. The residue was dissolved in CHCl3 (2 mL), then
acetone (30 mL) was added. The volume was reduced to
10 mL by evaporation, the suspension was cooled to 4 8C,
3
3
0
0
0
J2 ,NH Z8.7 Hz, NH ), 6.34 (d, 1H, J2,NHZ9.4 Hz, NH),
3
3
0
0
0
0
5.50 (s, 1H, CHPh), 5.23 (t, 1H, J2 ,3 Z J3 ,4 Z9.9 Hz,
H-30), 5.0 (dd, 1H, 3J2,3Z10.4 Hz, 3J3,4Z9.7 Hz, H-3), 4.95
(dd, 1H, 3J1,2Z3.7 Hz, 2J1,OHZ2.5 Hz, H-1), 4.62 and 4.48

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12129
2
(2AB, 2H, JZ11.5 Hz, CH2Ph), 4.56 and 4.53 (2AB, 2H,
1H–13C HMQC (CDCl3): 102.4 (CHPh), 101.5 (C-10),
96.4 (C-1), 79.3 (C-40), 77.5 (C-5), 76.5 (IcobCH), 75.7
2JZ12.0 Hz, CH2Ph), 4.52 (d, 1H, J1 ,2 Z8.5 Hz, H-1 ),
4.36 (d, 1H, OH-1), 4.35 (dd, 1H, J6a ,6b Z10.0 Hz,
3
0
0
0
2
(
IcobCH), 72.2 (C-4), 71.3 (CH2Ph), 70.2 (CH2, Bn), 68.9
0
0
3
0
J5 ,6a Z5.1 Hz, H-6a ), 4.16 (ddd, 1H, H-2), 4.06 (dd, 1H,
(C-60), 66.9 (C-50), 66.8 (C-6), 54.5 (C-20), 52.3 (C-2), 41.7
IcoaCH2), 41.40 (IcoaCH2), 34.7–34.3 (2MyraCH2,
2
IcogCH2), 32.0–23.0 (55CH2), 14.4 (4CH3); 31P NMR
0
0
2J6a,6bZ11.5 Hz, J5,6aZ2.2 Hz, H-6a), 3.98 (ddd, 1H,
(
3
3
H-20), 3.90–3.82 (m, 2H, 2bCHOBn), 3.80 (t, 1H, J5 ,6b
Z
Z
0
0
3
3
10.0 Hz, H-6b0), 3.72 (ddd, 1H, J4,5Z9.7 Hz, J5,6b
(CDCl3): d K1.63 (attached to C-4), K2.26 (attached to C-
1); MALDI-TOF-MS: m/z: 2192.50 [MCNa]C, 2208.47
[MCK]C; calcd 2192.36 [MCNa]C, 2208.46 [MCK]C.
3
0
0
0
7.2 Hz, H-5), 3.68 (t, 1H, J4 ,5 Z9.5 Hz, H-4 ), 3.55 (dd,
3
3
1H, J5,6bZ7.2 Hz, H-6b), 3.45 (ddd, 1H, H-50, J5 ,6b
Z
0
0
2
10.0 Hz), 3.37 (dt, 1H, J4,OHZ6.5 Hz, H-4), 2.65 (d, 1H,
OH-4), 2.45–2.20 (m, 8H, 2IcoaCH2, 2MyraCH2), 1.65–1.40
(m, 8H, 2MyrbCH2, 2IcogCH2), 1.35–1.20 (m, 110H,
55CH2), 0.90 (t, 12H, 4CH3); 13C NMR(CDCl3): d 175.53
(CO), 173.93 (CO), 172.24 (CONH), 171.57 (CONH),
138.86, 138.11, 137.34 (3C, 3C6H5), 129.46, 129.06,
128.78, 128.059, 128.47, 128.18, 128.07, 126.47 (15C, Ph),
102.81 (C-1 ), 101.72 (CHPh), 91.92 (C-1), 79.09 (C-40),
76.89 (2C, 0 2IcoCHOBn), 73.99 (C-3), 71.85 (CH2, Bn),
71.71 (C-3 ), 71.48 (CH2Ph), 71.30 (C-5), 70.59 (C-4),
70.03 (C-6), 68.93 (C-60), 66.93 (C-50), 55.10 (C-20), 52.01
(C-2), 42.11 and 41.85 (2IcoaCH2), 34.68, 34.61, 34.45,
33.03 (2MyraCH2, 2IcogCH2), 32.26, 30.06, 30.0, 29.88,
29.80, 29.70, 29.64, 29.49, 29.37, 25.61, 25.38, 25.27, 23.02
(55CH2), 14.45 (4CH3); MALDI-TOF-MS: m/z: 1672.34
[MCNa]C, 1688.31 [MCK]C; calcd 1672.23 [MCNa]C,
1688.34 [MCK]C.
3.1.21. Allyl 4-O-benzyl-2-deoxy-3-O-tetradecanoyl-2-
(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyrano-
side (33). A solution of 13 (820 mg, 1.16 mmol) in CH2Cl2
(20 mL) and powdered molecular sieves 0.4 nm (500 mg)
were stirred for 3 h under N2 at ambient temperature. The
suspension was cooled to K78 8C and Et3SiH (202 mg,
278 mL, 1.74 mmol) and a solution of PhBCl2 (313 mg,
256 mL, 1.97 mmol) in CH2Cl2 (2 mL) were added
successively under N2. The mixture was stirred for 1 h.
Et3N (1 mL) and MeOH (1 mL) were added and the mixture
was stirred for 15 min, then warmed up to rt, diluted with
EtOAc (30 mL) and filtered over a pad of Celite. The filtrate
was diluted with EtOAc (300 mL) and washed successively
with satd aq NaHCO3 (50 mL), H2O (50 mL) and brine
(50 mL). The organic phase was dried (cotton) and
concentrated. The residue was purified on silica gel
(4:1/3:1 toluene/EtOAc) which afforded 33 as a syrup
(790 mg, 96%). Rf 0.53 (A, 2:1 toluene/EtOAc); [a]2D0ZC
56.8 (c 1, CHCl3); 1H NMR: d 7.40–7.25 (m, 5H, Ph), 5.89
(m, 1H, CH]), 5.40 (dd, 1H, 3J2,3Z10.7 Hz, 3J3,4Z8.3 Hz,
3.1.20. 6-O-{4,6-O-Benzylidene-2-deoxy-2-[(R)-3-(octa-
decanoyloxy)icosanoylamino]-3-O-tetradecanoyl-b-D-
glucopyranosyl}-2-[(R)-3-(benzyloxy)icosanoylamino]-
1,4-O,O-bis[(dibenzyloxy)phosphoryl]-2-deoxy-3-O-
tetradecanoyl-1-a-D-glucopyranose (32). A 1.0 M
solution of lithium bis(trimethylsilyl)amide in n-hexane
(60 mL, 0.06 mmol) was added to a stirred solution of 31
(32 mg, 0.019 mmol) and tetrabenzyl diphosphate (43 mg,
0.08 mmol) in anhydrous THF (5 mL) at K78 8C under N2.
The mixture was stirred for 30 min, then allowed to warm
up to 0 8C within 5 min and the reaction was quenched with
satd aq NaHCO3 (0.5 mL). The mixture was diluted with
CHCl3 (100 mL) and washed with satd aq NaHCO3
(20 mL), H2O (20 mL) and brine (20 mL). The organic
phase was dried (cotton), concentrated and purified by
chromatography on silica gel (100:0.2:0/100:0.2:15
CH2Cl2/acetone). Appropriate fractions were collected and
purified by a second chromatography (1.5!30 cm,
150:100:15:1 toluene/CH2Cl2/MeOH/H2O) which afforded
32 (8 mg, 0.004 mmol, 19%) as a solid. Rf 0.7 (B, 10:1
CH2Cl2/acetone); [a]2D0ZC1.0 (c 0.4, CHCl3); 1H NMR: d
7.38–7.15 (m, 36H, 7Ph, NH0), 6.05 (d, 1H, 3J2,NHZ8.8 Hz,
NH), 5.49 (dd, 1H, 3J1,2Z3.3 Hz, 3J1,PZ5.3 Hz, H-1), 5.80
(s, 1H, CHPh), 5.19 (dd, 1H, 3J3,2Z10.9 Hz, 3J3,4Z9.2 Hz,
3
H-3), 5.35 (d, 1H, JNH,2Z9.7 Hz, NH), 5.30 (dq, 1H,
3
]CH2trans), 5.23 (dq, 1H, ]CH2cis), 4.92 (d, 1H, J1,2
Z
3.6 Hz, H-1), 4.73 and 4.69 (AB, 2H, 2JZ12.1 Hz,
2
CH2CCl3), 4.70 and 4.65 (AB, 2H, JZ12.0 Hz, CH2Ph),
4.19 (m, 1H, OCHH, All), 3.96 (m, 1H, OCHH, All), 3.94
(ddd, 1H, H-2), 3.88–3.72 (m, 4H, H-4, H-5, H-6a, H-6b),
2.23 (m, 2H, aCH2), 1.82 (t, 1H, OH), 1.58–1.52 (m, 2H,
bCH2), 1.33–1.22 (m, 20H, 10CH2), 0.89 (t, 3H, CH3); 13
C
NMR (HMQC, in CDCl3): d 138.5 (]CH), 135.0–128.0
(Ph), 119.0 (]CH2), 96.5 (C-1), 76.0 (C-4), 75.3 (CH2Ph),
74.8 (CH2, Troc), 72.7 (C-3), 71.5 (C-5), 69.1 (OCH2, All),
61.8 (C-4), 55.0 (C-2), 34.5 (aCH2), 32.0–28.0 (9CH2), 25.0
(b CH2), 23.0 (CH2), 14.51 (CH3); MALDI-TOF-MS: m/z:
716.32, 718.31 (24.47% 37Cl) [MCNa]C. Anal. Calcd for
C33H50Cl3NO8: C, 57.02; H, 7.25; N, 2.02. Found: C, 56.78;
H, 7.26; N, 1.97.
3.1.22. Allyl 2-amino-4-O-benzyl-2-deoxy-3-O-tetradeca-
noyl-a-D-glucopyranoside (34). Method A. A solution of
33 (723 mg, 1.04 mmol) in AcOH (20 mL) was stirred with
Zn–Cu couple [made from Zn (1.2 g) and 5% aq CuSO4
(5 mL)] at ambient temperature for 1 h. The solids were
removed by filtration over a pad of Celite, the filtrate was
concentrated and repeatedly evaporated with toluene (3!
30 mL). The residue was dissolved in EtOAc (250 mL) and
washed with satd aq NaHCO3 (50 mL) and brine (50 mL).
The organic phase was dried (cotton) and concentrated. The
residue was purified by chromatography on silica gel
(100:1/100:5 EtOAc/MeOH) to give 35 [allyl 4-O-
benzyl-2-deoxy-2-(2,2-dichloroethoxycarbonylamino)-3-
O-tetradecanoyl-a-D-glucopyranoside] as faster eluted
product (34 mg, 0.05 mmol, 5%); Rf 0.52 (A, 2:1 toluene/
3
3
0
0
0
0
0
H-3), 5.07 (t, 1H, J3 ,2 Z J3 ,4 Z9.9 Hz, H-3 ), 4.94–4.82
2
(m, 8H, 4CH2Ph), 4.56 and 4.45 (2AB, 2H, JZ11.9 Hz,
3
CH2Ph), 4.50 (t, 1H, J4,5Z9.2 Hz, H-4), 4.42 and 4.31
(2AB, 2H, 2JZ11.8 Hz, CH2Ph), 4.38 (d, 1H, J1 ,2
Z
Z
3
0
0
2
8.5 Hz, H-10), 4.24 (ddd, 1H, H-2), 4.17 (dd, 1H, J6a ,6b
0
0
10.7 Hz, 3J5 ,6a Z5.2 Hz, H-6a ), 4.09 (ddd, 1H, H-2 ), 3.85
0
0
0
0
(m, 1H, H-6a), 3.81 (m, 1H, H-5), 3.72–3.56 (m, 3H, H-6b0,
2bCHOBn), 3.54 (t, 1H, 3J4 ,5 Z9.5 Hz, H-4 ), 3.37 (dd, 1H,
0
0
0
2J6a,6bZ8.7 Hz, J5,6bZ3.7 Hz, H-6a), 3.12 (ddd, 1H,
3
J5 ,6b Z10.0 Hz, H-5 ), 2.25–2.19 (m, 8H, 2IcoaCH2,
3
0
0
0
2
MyraCH2), 1.50–1.40 (m, 8H, 2MyrbCH2, 2IcogCH2),
1.30–1.10 (m, 110H, 55CH2), 0.90 (t, 12H, 4CH3);
1
EtOAc); H NMR: d 7.38–7.26 (m, 5H, Ph), 5.88 (m, 1H,

12130
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
CH]), 5.82 (t, 1H, 3JZ6.1 Hz, CH2CHCl2), 3.37 (dd, 1H,
80:10:0.4 toluene/MeOH/H2O) to give 36 (703 mg,
0.76 mmol, 85%) as a solid Rf 0.3 (B, 2:1 toluene/EtOAc)
or Rf 0.4 (A, 80:10:0.4 toluene/MeOH/H2O). [a]2D0ZC42.0
3
3J2,3Z10.8 Hz, J3,4Z8.7 Hz, H-3), 5.29 (dq, 1H,
3
]CH2trans), 5.23 (d, 1H, JNH,2Z9.7 Hz, NH), 5.23 (dq,
3
1
1H, ]CH2cis), 4.89 (d, 1H, J1,2Z3.6 Hz, H-1), 4.70 and
(c 1, CHCl3); H NMR: d 7.38–7.25 (m, 5H, Ph), 6.25 (d,
4.64 (AB, 2H, 2JZ12.1 Hz, CH2Ph), 4.39 (dd, 2H, 3JZ6.1,
7.7 Hz, CH2CHCl2), 4.18 (m, 1H, OCHH, All), 3.96 (m, 1H,
OCHH, All), 3.91 (ddd, 1H, H-2), 3.86–3.71 (m, 4H, H-4,
H-5, H-6a, H-6b), 2.24 (m, 2H, aCH2), 1.77 (t, 1H, OH),
1.58–1.52 (m, 2H, bCH2), 1.33–1.22 (m, 20H, 10CH2), 0.89
(t, 3H, CH3); 13C NMR(CDCl3): d 174.29 (1C, CO), 155.23
(1C, CONH), 138.04 (]CH), 133.55, 128.94, 128.42,
128.25 (6C, Ph), 118.63 (]CH2), 96.86 (C-1), 75.78 (C-4),
75.26 (CH2Ph), 73.30 (C-3), 71.61 (C-5), 69.26 (1C,
CH2CHCl2), 69.10 (OCH2, All), 68.94 (1C, CH2CHCl2),
61.84 (C-6), 54.88 (C-2), 34.75 (aCH2), 32.36, 30.09, 30.07,
29.86, 29.72, 29.55, 23.11 (10CH2), 25.27 (b CH2), 14.56
(CH3). Further elution gave 34 (320 mg, 0.62 mmol, 60%)
as a syrup; Rf 0.5 (A, 15:1 EtOAc/MeOH); [a]2D0ZC92.0 (c
1H, J2,NHZ9.5 Hz, NH), 5.73 (m, 1H, CH]), 5.38 (ddd,
3
3
3
5
1H, J2,3Z10.7 Hz, J3,4Z9.2 Hz, J3,CHZ3.8 Hz, H-3),
5.18 (dq, 1H, ]CH2trans), 5.12 (dq, 1H, ]CH2cis), 4.78 (d,
3
1H, J1,2Z3.5 Hz, H-1), 4.70 and 4.64 (AB, 2H, 2JZ
11.5 Hz, CH2, 4-O-Bn), 4.56 and 4.52 (AB, 2H, 2JZ
12.0 Hz, CH2Ph), 4.27 (ddd, 1H, H-2), 4.02 (m, 1H, OCHH,
All), 3.88–3.70 (m, 6H, H-4, H-5, H-6a, H-6b, bCH, OCHH,
2
3
0
All), 2.35 (d, 2H, J!1 Hz, JZ7.0 Hz, aCH2 ), 2.23 (m,
2H, aCH2), 1.76 (m, 1H, OH), 1.65–1.45 (m, 2H, bCH2,
gCH2 ), 1.33–1.22 (m, 50H, 25CH2), 0.89 (t, 6H, CH3); 13C
0
NMR(CDCl3): d 174.24 (CO), 171.58 (CONH), 138.92 and
138.18 (2C, 2C6H5, Bn), 133.74 (]CH), 128.87, 128.74,
128.31, 128.19, 128.02, 127.94 (10C, 2C6H5, Bn), 118.24
(]CH2), 97.05 (C-1), 76.70 (b0CH), 76.08 (C-4), 75.21
(CH2, 4-O-Bn), 73.50 (C-3), 71.83 (CH2, Bn), 71.61 (C-5),
68.87 (OCH2, All), 61.88 (C-6), 52.58 (C-2), 42.23 (a0CH2),
34.79, 34.47 (2C, aCH2, g0CH2), 32.33, 30.11, 30.08, 30.06,
29.91, 29.76, 29.74, 29.62 (23CH2), 25.72, 25.26 (2CH2),
23.09 (CH2), 14.29 (CH3). Anal. Calcd for C57H93NO8: C,
74.39; H, 10.19; N, 1.52. Found: C, 74.16; H, 9.90; N, 1.79;
MALDI m/z 942.75 [MCNa]C.
1, CHCl3); 1H NMR: d 7.37–7.26 (m, 5H, Ph), 5.97 (m, 1H,
3
CH]), 5.33 (dq, 1H, ]CH2trans), 5.31 (dd, 1H, J2,3
Z
3
10.5 Hz, J3,4Z9.3 Hz, H-3), 5.22 (dq, 1H, ]CH2trans),
3
5.18 (d, 1H, J1,2Z3.5 Hz, H-1), 4.68 and 4.64 (AB, 2H,
2JZ11.5 Hz, CH2, 4-O-Bn), 4.20 (m, 1H, OCHH, All), 4.12
(m, 1H, OCHH, All), 3.85–3.75 (m, 3H, H-5, H-6a, H-6b),
3.68 (t, 1H, 3J4,5Z9.4 Hz, H-4), 3.11 (dd, 1H, H-2), 2.50 (br
s, 1H, OH), 2.31 (m, 2H, aCH2), 1.62–1.52 (m, 2H, bCH2),
1.33–1.22 (m, 20H, 10CH2), 0.89 (t, 3H, CH3); 13C
NMR(CDCl3): d 174.82 (CO), 138.15 (1C, Ph), 134.07
(]CH), 128.86, 128.29, 128.05 (5C, Ph), 118.16 (]CH2),
99.14 (C-1), 76.63 (C-4), 76.16 (C-3), 74.59 (CH2Ph), 71.49
(C-5), 68.70 (OCH2, All), 61.66 (C-6), 55.51 (C-2), 34.69
(aCH2), 32.09, 29.84, 29.81, 29.77, 29.61, 29.52, 29.46,
29.41 (9C, CH2), 25.12 (b CH2), 22.86 (CH2), 14.29 (CH3);
MALDI m/z 542.40 [MCNa]C. Anal. Calcd for
C30H49NO6: C, 69.33; H, 9.50; N, 2.70. Found: C, 69.10;
H, 9.26; N, 2.70.
3.1.24. Allyl 4-O-benzyl-6-O-{6-O-benzyl-4-O-[bis-
(benzyloxy)phosphoryl]-2-deoxy-3-O-tetradecanoyl-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyrano-
syl}-2-[(R)-3-(benzyloxy)icosanoylamino]-2-deoxy-3-O-
tetradecanoyl-a-D-glucopyranoside (37). A solution of 20
(445 mg, 0.42 mmol) and 36 (385 mg, 0.42 mmol) in
CH2Cl2 (5 mL) was stirred with powdered activated
molecular sieves (0.4 nm) under N2 for 2 h. The suspension
was cooled to K25 8C and a solution of trimethylsilyl
trifluoromethanesulfonate (7.5 mL, 0.04 mmol) in CH2Cl2
(1 mL) was added. The stirring was continued for 20 min
and the reaction was stopped by addition of dry Na2CO3.
The mixture was let to warm up to rt and the solids were
removed by filtration through a pad of Celite. The filtrate
was diluted with CH2Cl2 (200 mL) and washed with satd aq
NaHCO3 (50 mL), H2O (50 mL) and brine (50 mL). The
organic phase was dried (cotton) and concentrated. The
residue was purified in three equal portions by chromato-
graphy on silica gel (1.5!60 cm, 30:40:15:0.3 toluene/
CH2Cl2/acetone/H2O) which afforded 37 as a solid (710 mg,
0.39 mmol, 93%). Rf 0.5 (B, 150:100:15:1 toluene/CH2Cl2/
MeOH/H2O); Rf10.7 (B, 2:1 toluene/EtOAc). [a]2D0ZC20.7
(c 1.0, CHCl3); H NMR: d 7.38–7.24 (m, 25H, 5Ph), 6.21
(d, 1H, 3J2,NHZ9.4 Hz, NH), 5.73 (m, 1H, CH]), 5.36 (dd,
Method B. A solution of 33 (218 mg, 0.314 mmol) in AcOH
(5 mL) was stirred with Zn powder at rt for 4 h, then diluted
with AcOH (20 mL). The solids were removed by filtration
over a pad of Celite, the filtrate was concentrated,
redissolved in toluene and concentrated (3!30 mL). The
residue was dissolved in EtOAc (100 mL) and washed with
satd aq NaHCO3 (20 mL) and brine (20 mL). The organic
phase was dried (cotton) and concentrated. The residue was
purified by chromatography on silica gel (1.5!60 cm,
150:100:15:1 toluene/CH2Cl2/MeOH/H2O) to give 34
(145 mg, 0.28 mmol, 89%); Rf 0.2 (B, 150:100:15:1
toluene/CH2Cl2/MeOH/H2O).
3
3
3.1.23. Allyl 4-O-benzyl-2-[(R)-3-(benzyloxy)icosanoyl-
amino]-2-deoxy-3-O-tetradecanoyl-a-D-glucopyranoside
(36). To a stirred solution of 8 (437 mg, 1.04 mmol) in
CH2Cl2 (3 mL) HOBt (140 mg, 0.91 mmol) and
WSCD$HCl (175 mg, 0.91 mmol) were added successively
under N2. The suspension was sonicated to afford a clear
solution, which was stirred for 1 h. A solution of 34
(467 mg, 0.90 mmol) in CHCl3 (3 mL) was added to the
mixture and stirring was continued for 1.5 h under N2. The
mixture was taken up in EtOAc (200 mL), washed with satd
aq NaHCO3 (50 mL) and brine (50 mL). The organic phase
was dried (cotton) and concentrated. The residue was
purified by chromatography on silica gel (3!40 cm,
1H, J3,2Z10.8 Hz, J3,4Z9.1 Hz, H-3), 5.32 (dd, 1H,
3 3
0
]CH2trans), 5.05 (dq, 1H, ]CH2cis), 4.85 (d, 1H,
0
0
0
0
J3 ,2 Z10.7 Hz, J3 ,4 Z9.1 Hz, H-3 ), 5.14 (dq, 1H,
3J2,NHZ7.8 Hz, NH), 4.93–4.82 [m, 4H, CH2,
3
(BnO)2P(O)–], 4.70 (d, 1H, J1,2Z3.6 Hz, H-1), 4.54 and
4.48 (2AB, 2H, 2JZ12.2 Hz, CH2Ph), 4.56 and 4.52 (2AB,
2
2
2H, JZ11.8 Hz, CH2Ph), 4.66 and 4.57 (2AB, 2H, JZ
2
11.8 Hz, CH2CCl3), 4.67 and 4.61 (2AB, 2H, JZ11.6 Hz,
3
0
0
0
CH2Ph), 4.47 (d, 1H, J1 ,2 Z8.3 Hz, H-1 ), 4.44 (t, 1H,
3
0
0
0
J4 ,5 Z9.1 Hz, H-4 ), 4.29 (ddd, 1H, H-2), 4.11 (dd, 1H,
2J6a,6bZ10.8 Hz, J6a,5Z1.6 Hz, H-6a), 4.03 (m, 1H,
OCHH, All), 3.88–3.82 (m, 2H, H-5, bCHOBn), 3.84–
3
3.60 (m, 7H, OCHH, All, H-6b, H-60a, H-60b, H-20,

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12131
3
H-4, H-50), 2.35 (m, 2H, IcoaCH2), 2.28–2.17 (m, 4H,
MyraCH2), 1.65–1.40 (m, 6H, 2MyrbCH2, IcogCH2), 1.35–
(C-30, J3 ,PZ1.6 Hz), 74.78 (CH2Ph), 74.70 (C-50), 74.40
0
2
0
2
(C-40, J4 ,PZ6.3 Hz), 73.53 (CH2Ph), 73.47 (C-3), 71.57
1.20 (m, 74H, 37CH2), 0.90 (t, 9H, CH3); 13C
NMR(CDCl3): d 174.06 (CO), 173.96 (CO), 171.62
(CONH), 154.11 (CONH, Troc), 138.81, 138.39 (2C, Ph),
135.80, 135.70 (2C, Ph), 133.64 (]CH), 128.91, 128.88,
128.87, 128.63, 128.61, 128.24, 127.90, 127.86, 127.82
(25C, Ph), 118.17 (]CH2), 101.01 (C-10), 96.87 (C-1),
(
IcoCH2, Bn), 70.38 (C-5), 69.69 and 69.62 [2CH2, JC,P
Z
2
3.2 Hz, (BnO)2P(O)–], 69.06 (C-60), 68.52 (OCH2, All),
68.39 (C-6), 56.25 (C-20), 52.08 (C-2), 42.0 (IcoaCH2),
34.52, 34.31, 34.23 (2MyraCH2, IcogCH2), 32.06, 29.85,
29.79, 29.64, 29.63, 29.50, 29.36, 29.30 (37CH2), 14.25
(3CH3); 31P NMR (CDCl3): d K1.12. Anal. Calcd for
C98H149N2O16P: C, 71.67; H, 9.15; N, 1.71. Found: C,
71.44; H, 8.94; N, 1.71; MALDI-TOF-MS: m/z: 1642.14
[MCH]C, 1664.12 [MCNa]C, 1680.06 [MCK]C.
76.58 (IcoCHOBn), 76.04 (C-4), 74.91 (CH2Ph), 74.82
2
(C-50), 74.65 (CH2Ph), 74.31 (C-40, J4 ,PZ6.0 Hz), 73.78
0
3
(C-3), 73.73 (CH2, Troc), 72.19 (C-30, J3 ,PZ1.5 Hz),
0
71.73 (CH2, IcoBn), 70.27 (C-5), 69.93 and 69.86 [2CH2,
2JC,PZ3.0 Hz, (BnO)2P(O)–], 68.98 (C-60), 68.61 (OCH2,
All), 67.95 (C-6), 56.52 (C-20), 52.20 (C-2), 42.17
3.1.26. Allyl 4-O-benzyl-6-O-{6-O-benzyl-2-[(R)-3-(ben-
zyloxy)icosanoylamino]-2-deoxy-4-O-[bis(benzyloxy)-
phosphoryl]-3-O-tetradecanoyl-b-D-glucopyranosyl}-2-
[(R)-3-(benzyloxy)icosanoylamino]-2-deoxy-3-O-tetra-
decanoyl-a-D-glucopyranoside (39). To a solution of 8
(100 mg, 0.24 mmol) and DIPEA (42 mL, 0.24 mmol) in
DMF (2 mL) a solution of O-(7-azabenzotriazol-1-yl)-
(
IcoaCH2), 34.71, 34.41, 34.24 (2MyraCH2 IcogCH2),
32.22, 30.0, 29.95, 29.82, 29.75, 29.65, 29.59, 29.53,
29.38, 25.62, 25.17, 24.89, 22.98 (37CH2), 14.60 (3CH3);
31P NMR (CDCl3): d K1.52; MALDI-TOF-MS: m/z:
1838.0, 1853.97 (24.47% 37Cl) [MCNa]C. Anal. Calcd
for C101H150Cl3N2O18P: C, 66.74; H, 8.32; N, 1.54. Found:
C, 66.17; H, 8.20; N, 1.44.
N,N,N0,N0-tetramethyluronium
hexafluorophosphate
(HATU, 110 mg, 0.29 mmol) in DMF (1 mL) was added
and the mixture was stirred under N2 for 30 min. The
solution containing activated acid 8 was added to a stirred
solution of 38 (137 mg, 0.083 mmol) in DMF (5 mL) and
the mixture was stirred under N2 for 20 min at rt and for 5 h
at 35 8C. The mixture was diluted with CH2Cl2 (200 mL)
and washed with satd aq NaHCO3 (20 mL), H2O (50 mL)
and brine (40 mL). The organic phase was dried (cotton),
concentrated and purified by precipitation with EtOH
from CH2Cl2 (3!) and by precipitation with hexane from
CH2Cl2 (2!) as follows: precipitation with EtOH from
CH2Cl2. The residue was dissolved in CH2Cl2 (3 mL), then
acetone (0.5 mL) and EtOH (25 mL) were successively
added. The volume was reduced to 10 mL by concentration,
the suspension was diluted with EtOH (10 mL), the white
fluffy precipitate was separated on the glass-filter and
washed with EtOH (10 mL). The precipitate was
redissolved in CH2Cl2 (10 mL) and the solution was
concentrated to dryness. Precipitation with hexane from
CH2Cl2. The residue was dissolved in CH2Cl2 (2 mL), then
hexane (30 mL) was added and the volume was reduced to
10 mL by concentration under diminished pressure. The
suspension was diluted with hexane (10 mL) and cooled to
4 8C, the transparent gel-like precipitate was separated on
the glass-filter and washed with cold (4 8C) hexane (10 mL).
The precipitate was recovered from the filter by dissolution
in CH2Cl2 (10 mL), the solution was concentrated to
dryness. Yield 155 mg (0.076 mmol, 92%) of 39 as a
white solid; Rf 0.55 (B, 150:100:15:1 toluene/CH2Cl2/
MeOH/H2O); Rf10.7 (B, 2:1 toluene/EtOAc); [a]2D0ZC11.6
(c 1.0, CHCl3); H NMR: d 7.40–7.22 (m, 30H, 6Ph), 6.46
3.1.25. Allyl 6-O-{2-amino-6-O-benzyl-2-deoxy-4-O-
[bis(benzyloxy)phosphoryl]-3-O-tetradecanoyl-b-D-
glucopyranosyl}-4-O-benzyl-2-[(R)-3-(benzyloxy)icosa-
noylamino]-2-deoxy-3-O-tetradecanoyl-a-D-gluco-
pyranoside (38). A solution of 37 (176 mg, 0.097 mmol) in
acetic acid (10 mL) was stirred with Zn powder (300 mg) at
50 8C for 2 h under N2. The solids were removed by
filtration over a pad of Celite, the filtrate was concentrated
and repeatedly evaporated with toluene (3!30 mL). The
residue was dissolved in CH2Cl2 (200 mL) and extracted
with satd aq NaHCO3 (30 mL), H2O (50 mL) and brine
(50 mL). The organic phase was dried (cotton), concen-
trated and purified in two equal portions by chromatography
on silica gel (1.5!60 cm, 30:40:15:0.3 toluene/CH2Cl2/
acetone/H2O) to afford 38 (134 mg, 0.081 mmol, 84%) as a
transparent foam. Compound 38 was further purified by
chromatography on silica gel (100:1/100:10 CH2Cl2/
acetone) which afforded 105 mg of 38 (0.064 mmol, 66%).
Rf 0.5 (B, 30:40:15:0.3 toluene/CH2Cl2/acetone/H2O);
1
[a]2D0ZC20.6 (c 1.0, CHCl3); H NMR: d 7.33–7.22 (m,
25H, 5Ph), 6.20 (d, 1H, 3J2,NHZ9.5 Hz, NH), 5.70 (m, 1H,
30
3
0
0
0
0
CH]), 5.36 (dd, 1H,
J
Z10.7 Hz, J3 ,4 Z9.2 Hz,
3 ,2
H-30), 5.15 (dq, 1H, ]CH2trans), 5.07 (dq, 1H, ]CH2cis),
3
3
5.05 (dd, 1H, J3,2Z10.8 Hz, J3,4Z9.2 Hz, H-3), 4.93–
3
4.87 [m, 4H, CH2, (BnO)2P(O)–], 4.77 (d, 1H, J1,2
Z
3.5 Hz, H-1), 4.65 and 4.60 (2AB, 2H, 2JZ12.2 Hz,
2
CH2Ph), 4.54 and 4.49 (2AB, 2H, JZ11.8 Hz, CH2Ph),
4.51 and 4.44 (2AB, 2H, 2JZ11.6 Hz, CH2Ph), 4.37 (t, 1H,
3
0
0
0
J4 ,5 Z9.2 Hz, H-4 ), 4.30 (ddd, 1H, H-2), 4.18 (d, 1H,
3
2
3
J1 ,2 Z8.0 Hz, H-1 ), 4.11 (dd, 1H, J6a,6bZ10.9 Hz,
(d, 1H, 3J2 ,NH Z8.8 Hz, NH ), 6.22 (d, 1H, J2,NHZ9.4 Hz
0
0
0
0
0
0
3
3J6a,5Z1.5 Hz, H-6a), 4.01 (m, 1H, OCHH, All), 3.90–
3.55 (m, 8H, H-05, bCHOBn, OCHH All, H-6b, H-60a,
H-60b, H-4, H-5 ), 2.90 (dd, 1H, H-20), 2.33 (m, 2H,
IcoaCH2), 2.30–2.10 (m, 4H, 2MyraCH2), 1.60–1.40 (m, 6H,
NH), 5.70 (m, 1H, CH]), 5.34 (dd, 1H, J3 ,2 Z0.7 Hz,
0
0
J3 ,4 Z9.2 Hz, H-3 ), 5.33 (dd, 1H, J3,2Z10.8 Hz, 3J3,4
9.1 Hz, H-3), 5.18 (dq, 1H, ]CH2trans), 5.07 (dq, 1H,
]CH2cis), 4.97–4.86 [m, 4H, 2CH2, (BnO)2P(O)–], 4.75 (d,
Z
3
3
0
0
0
3
2
MyrbCH2, IcogCH2), 1.35–1.20 (m, 74H, 37CH2), 0.90 (t,
1H, J1,2Z3.4 Hz, H-1), 4.59–4.42 (m, 8H, 4CH2Ph), 4.47
3
3
9H, 3CH3); 13C NMR(CDCl3): d 174.0 (CO), 173.87 (CO),
171.20 (CONH), 138.63, 138.35, 137.95 (3C, 3C6H5),
135.79, 135.69 [2C, Ph, 3JC,PZ5 Hz, (BnO)2P(O)–], 133.45
(]CH), 128.68, 128.61, 128.48, 128.42, 128.07, 127.99,
127.74, 127.69, 127.63 (25C, Ph), 118.0 (]CH2), 104.53
(C-10), 96.69 (C-1), 76.41 (IcoCHOBn), 76.36 (C-4), 75.22
(d, 1H, J1 ,2 Z8.3 Hz, H-1 ), 4.42 (t, 1H, J4 ,5 Z9.2 Hz,
H-40), 4.28 (ddd, 1H, H-2), 4.01 (m, 1H, OCHH, All), 3.99
(dd, 1H, 2J6a,6bZ10.5 Hz, 3J6a,5Z1.9 Hz, H-6a), 3.88–3.57
(m, 10H, H-5, H-20, 2bCHOBn, OCHH All, H-6b,
H-60a, H-60b, H-4, H-50), 2.33 (m, 4H, 2IcoaCH2), 2.25–
2.0 (m, 4H, 2MyraCH2, Myr), 1.60–1.40 (m, 8H,
0
0
0
0
0

12132
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
2
MyrbCH2, 2IcogCH2), 1.35–1.10 (m, 110H, 55CH2),
3.79 (m, 2H, 2bCHOBn), 3.67–3.54 (m, 4H, H-20, H-6b,
0.90 (t, 12H, 4CH3); 13C NMR(CDCl3): d 174.10 (CO),
173.85 (CO), 171.55 (CONH), 171.34 (CONH), 138.85,
138.51, 138.24 (4C, 4!C6H5), 135.90, 135.81 [2C,
H-6b0, H-50), 3.28 (t, 1H, H-4), 2.40–2.30 (m, 4H,
2
IcoaCH2), 2.25–2.10 (m, 4H, 2MyraCH2), 1.62–1.40 (m,
8H, 2MyrbCH2, 2IcogCH2), 1.32–1.13 (m, 110H, 55CH2),
0.90 (t, 12H, 4CH3); 13C NMR(CDCl3): d 174.23 (CO),
173.66 (CO), 171.23 (CONH), 171.20 (CONH), 139.33,
3
2C6H5, JC,PZ5.0 Hz, (BnO)2P(O)–], 133.69 (]CH),
128.95, 128.89, 128.75, 128.66, 128.63, 128.26, 128.19,
128.11, 127.98, 127.94, 127.89 (30C, Ph), 118.09
(]CH2), 100.91 (C-10), 96.68 (C-1), 76.59 (C-4),
3
139.01, 138.74 (4C, Ph), 136.40, 136.11 [2C, Ph, JC,P
Z
5.0 Hz, (BnO)2P(O)–], 129.01, 128.82, 128.58, 128.33,
128.30, 128.25, 128.17, 128.13, 128.0, 127.96 (30C, Ph),
101.16 (C-10), 91.74 (C-1), 77.55 (C-4), 76.78 and 76.64
76.49 and 76.45 (2C, 2IcobCH), 74.83 (C-50), 74.73
2
(CH2Ph), 74.61 (C-40, J4 ,PZ6.0 Hz), 73.70 (CH2Ph),
0
3
73.55 (C-3), 72.66 (C-30, J3 ,PZ1.5 Hz), 71.73 and
(2C, 2IcobCH), 74.74 (CH2Ph), 74.63 (C-50), 74.55 (C-40,
0
71.09 (2CH2, 2IcoBn), 70.42 (C-5), 69.91 and 69.85
J4 ,PZ5.8 Hz), 73.84 (CH2Ph), 73.47 (C-3), 72.99 (C-30,
2
0
2
3
[2CH2, JC,PZ3.2 Hz, (BnO)2P(O)–], 69.10 (C-60),
J3 ,PZ1.4 Hz), 71.89 and 71.48 (2CH2, 2IcoBn), 71.05
0
68.47 (OCH2, All), 68.94 (C-6), 54.53 (C-20), 52.41
(C-2), 42.18 (IcoaCH2), 41.44 (IcoaCH2), 34.68, 34.46,
34.36, 33.98 (2MyraCH2, 2IcogCH2), 32.27, 30.06, 30.02,
29.90, 29.71, 29.57, 25.68, 25.36, 25.19, 24.93, 23.04
(55CH2), 14.46 (4CH3); 31P NMR (CDCl3): d K1.57;
MALDI-TOF-MS: m/z: 2064.41 [MCNa]C, 2080.40
[MCK]C. Alternatively, 39 [prepared from the same
amount of 38 (137 mg, 0.083 mmol)] was purified in
three equal portions by adsorption-partition chromato-
graphy on silica gel (1.5!60 cm, 150:100:15:1 toluene/
CH2Cl2/MeOH/H2O), to yield 118 mg (0.058 mmol,
76%) of 39.
(C-5), 69.96 and 69.89 [2CH2, 2JC,PZ3.4 Hz, (BnO)2P(O)–],
69.36 (C-60), 69.13 (C-6), 55.57 (C-20), 52.84 (C-2), 42.29
(
IcoaCH2), 41.81 (IcoaCH2), 34.79, 34.63, 34.37, 34.02
(2MyraCH2, 2IcogCH2), 32.33, 30.12, 30.07, 29.96, 29.91,
29.77, 29.62, 29.59, 25.68, 25.22, 24.96, 23.09 (55CH2),
14.51 (4CH3); 31P NMR (CDCl3): d K1.61; MALDI-TOF-
MS: m/z: 2024.46 [MCNa]C, 2040.40 [MCK]C.
3.1.28. Allyl 4-O-benzyl-6-O-{6-O-benzyl-4-O-[bis-
(benzyloxy)phosphoryl]-2-deoxy-2-[(R)-3-(octadecanoyl-
oxy)icosanoylamino]-3-O-tetradecanoyl-b-D-gluco-
pyranosyl}-2-[(R)-3-(benzyloxy)icosanoylamino]-2-
deoxy-3-O-tetradecanoyl-a-D-glucopyranoside (41). To a
solution of 11 (62 mg, 0.104 mmol) in 1:1 DMF/THF
(4 mL) a solution of O-(benzotriazole-1-yl)-N,N,N0,N0-
bistetramethyluronium hexafluorophosphate (HBTU)
(86 mg, 0.20 mmol) in DMF (1 mL) was added and the
mixture was stirred under N2 for 30 min. The solution
containing activated fatty acid 11 was added to a stirred
solution of 38 (98 mg, 0.06 mmol) and DIPEA (21 mL,
0.12 mmol) in DMF (5 mL) under N2. The mixture was
heated to 50 8C and the reaction vessel was purged with N2
for 10 min (to blow off the THF). The reaction mixture was
stirred for 5 h at 58 8C under N2, cooled to rt, diluted with
CH2Cl2 (150 mL) and washed with satd aq NaHCO3
(20 mL), H2O (50 mL) and brine (40 mL). The organic
phase was dried (cotton), concentrated and purified by
sequential precipitations with EtOH from CH2Cl2 (3!) (see
purification of 39), with hexane from CH2Cl2 (3!) and with
EtOH from CH2Cl2 (2 times) to give 41 (114 mg,
0.052 mmol, 86%) as a white solid; Rf 0.56 (B,
150:100:15:1 toluene/CH2Cl2/MeOH/H2O) or Rf 0.7 (B,
3.1.27. 4-O-Benzyl-6-O-{6-O-benzyl-2-[(R)-3-(benzyl-
oxy)icosanoylamino]-2-deoxy-4-O-[bis(benzyloxy)phos-
phoryl]-3-O-tetradecanoyl-b-D-glucopyranosyl}-2-[(R)-
3-(benzyloxy)icosanoylamino]-2-deoxy-3-O-tetradeca-
noyl-D-glucopyranose (40). To a degassed solution of 39
(138 mg, 0.068 mmol) in THF (20 mL) iridium catalyst
(25 mg, 0.03 mmol) was added and activated with H2 as
described for 19. The mixture was stirred under He for
30 min and cooled to 0 8C. A solution of I2 (50 mg,
0.2 mmol) in 2:1 THF/H2O (2 mL) was added dropwise.
The mixture was stirred at 0 8C for 6 h, then for 2 h at 25 8C,
diluted with CH2Cl2 (200 mL), washed with 5% aq Na2S2O3
(20 mL), satd aq NaHCO3 (30 mL) and brine (30 mL). The
organic phase was dried (cotton) and concentrated. The
residue was purified by repeated precipitations (see
purification of 39) with EtOH from CH2Cl2 (3!), then
with hexane from CH2Cl2 (3!) to afford 40 (120 mg,
0.06 mmol, 89%) as a solid. Alternatively, 40 (prepared
from 127 mg, 0.062 mmol of 39) was purified by sequential
precipitations with EtOH from CH2Cl2 (1!), with hexane
from CH2Cl2 (1!) and subsequent column chromatography
on silica gel (1.5!60 cm, 150:100:15:1 toluene/CH2Cl2/
MeOH/H2O) which afforded 40 (106 mg, 0.053 mmol,
85%) as a solid. Rf 0.47 (a-anomer) and 0.38 (b-anomer)
(B, 30:40:15:0.3 toluene/CH2Cl2/acetone/H2O); [a]2D3ZC
2:1 toluene/EtOAc); [a]2D0ZC15.7 (c 1.0, CHCl3); 1H
3
NMR: d 7.35–7.20 (m, 25H, 5Ph), 6.22 (d, 1H, J2,NH
Z
3
0
0
0
9.5 Hz NH), 5.94 (d, 1H, J2 ,NH Z8.4 Hz, NH ), 5.70 (m,
30
3
0
0
0
0
1H, CH]), 5.45 (dd, 1H,
J
Z10.6 Hz, J3 ,4 Z9.0 Hz,
3 ,2
3
3
H-30), 5.34 (dd, 1H, J3,2Z11.0 Hz, J3,4Z9.1 Hz, H-3),
5.18 (dq, 1H, ]CH2trans), 5.08 (dq, 1H, ]CH2cis), 4.96
[bCH, (octadecanoyloxy)icosanoyl], 4.96–4.87 [m, 4H,
1
2.5 (c 1.0, CHCl3); H NMR: d 7.35–7.18 (m, 30H, 6Ph),
0
3
3
3
0
0
0
0
0
6.43 (d, 1H, J2 ,NH Z8.1 Hz, NH ), 6.20 (d, 1H, J2,NH
Z
2CH2, (BnO)2P(O)–], 4.86 (d, 1H, J1 ,2 Z8.5 Hz, H-1 ),
9.4 Hz, NH), 5.34 (dd, 1H, 3J3 ,2 Z10.4 Hz, J3 ,4 Z9.0 Hz,
4.77 (d, 1H, J1,2Z3.5 Hz, H-1), 4.58–4.44 (m, 6H,
3
3
0
0
0
0
3
3
3
H-30), 5.28 (dd, 1H, J3,2Z10.5 Hz, J3,4Z9.2 Hz, H-3),
3CH2Ph), 4.43 (t, 1H, J4 ,5 Z9.0 Hz, H-4 ), 4.30 (ddd,
0
0
0
4.93–4.85 [m, 4H, CH2, (BnO)2P(O)–], 4.95 (d, 1H, 3J1,2
Z
1H, H-2), 4.07 (dd, 1H, J6a,6bZ11.0 Hz, J6a,5Z1.6 Hz,
H-6a), 4.01 (m, 1H, OCHH, All), 3.87–03.62 (m, 9H, H-5, H-
20, bCHOBn, OCHH All, H-6b, H-6a , H-6b0, H-4, H-50),
2.43–2.10 (m, 10H, 2IcoaCH2, 2MyraCH2, SteaCH2), 1.65–
1.40 (m, 10H, 2MyrbCH2, StebCH2, 2IcogCH2), 1.35–1.10
(m, 134H, 67CH2), 0.90 (t, 15H, 5CH3); 13C NMR(CDCl3):
d 174.16 (CO), 174.10 (CO), 173.92 (CO), 171.34 (CONH),
169.98 (CONH), 138.81, 138.46, 138.17 (3C, Ph), 135.94,
2
3
3.5 Hz, H-1), 4.86 (d, 1H, 3J1 ,2 Z8.3 Hz, H-1 ), 4.71 (br s,
0
0
0
1H, 1-OH), 4.55 and 4.51 (2AB, 2H, 2JZ11.8 Hz, CH2Ph),
2
4.53 and 4.48 (2AB, 2H, JZ12.0 Hz, CH2Ph), 4.52 and
4.51 (2AB, 2H, 2JZ11.7 Hz, CH2Ph), 4.44 and 4.39 (2AB,
2H,0 2JZ12.1 Hz, CH2Ph), 4.42 (t, 1H, J4 ,5 Z9.2 Hz,
3
0
0
H-4 ), 4.13 (ddd, 1H, H-2), 4.02–3.90 (m, 2H, H-5, H-6a),
2
3
0
0
0
0
0
3.80 (dd, 1H, J6a ,6b Z10.3 Hz, J6a ,5 Z2.0 Hz, H-6a ),

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12133
3
135.81 [2C, Ph, JC,PZ5.0 Hz, (BnO)2P(O)–], 133.68
(]CH), 128.92, 128.89, 128.81, 128.69, 128.43, 128.29,
128.19, 128.04, 127.97, 127.88 (25C, Ph), 118.18 (]CH2),
2
MyrbCH2, StebCH2, 2IcogCH2), 1.35–1.15 (m, 134H,
67CH2), 0.90 (t, 15H, 5CH3); 13C NMR(CDCl3): d 174.97
(CO), 173.94 (CO), 173.86 (CO), 171.47 (CONH), 170.59
(CONH), 138.84, 138.30, 138.89 (3C, 3C6H5), 135.90,
100.58 (C-010), 96.82 (C-1), 76.58 (IcobCH), 76.43 (C-4),
2
3
74.85 (C-5 ), 74.79 (CH2Ph), 74.60 (C-40, J4 ,PZ6.2 Hz),
135.81 [2C, Ph, JC,PZ5.0 Hz, (BnO)2P(O)–], 128.86,
128.83, 128.74, 128.62, 128.23, 128.21, 128.12, 128.03,
0
3
73.68 (CH2Ph), 73.60 (C-3), 72.46 (C-30, J3 ,PZ1.5 Hz),
71.75 (CH2Ph), 71.31 [bCH, (octadecanoyloxy)icosanoyl],
0
127.99, 127.86 (25C, Ph), 99.96 (C-10), 91.77 (C-1), 76.23
2
2
(C-4), 76.70 (IcobCH), 74.81 (CH2Ph), 74.65 (C-50, J5 ,P
Z
0
70.43 (C-5), 69.92 and 69.83 [2CH2, JC,PZ5.3 Hz,
2
(BnO)2P(O)–], 69.08 (C-60), 68.57 (OCH2, All), 67.94
(C-6), 55.29 (C-20), 52.32 (C-2), 42.19 (2IcoaCH2), 34.81,
34.66, 34.50, 34.46, 34.29 (2MyraCH2, SteaCH2, 2IcogCH2),
32.26, 30.05, 30.0, 29.92, 29.88, 29.78, 29.70, 29.58, 29.54,
25.67, 25.61, 25.32, 25.18, 24.92, 23.02 (67CH2), 14.44
(5CH3); 31P NMR (CDCl3): d K1.59; MALDI-TOF-MS:
m/z: 2240.70 [MCNa]C, 2256.69 [MCK]C.
4.0 Hz), 74.51 (C-40, J4 ,PZ6.9 Hz), 73.74 (CH2Ph), 73.48
0
3
(C-3), 72.34 (C-30, J3 ,PZ1.2 Hz), 71.86 (CH2Ph), 71.80
[bCH, (octadecanoyloxy)icosanoyl], 70.66 (C-5), 69.86 and
0
2
69.79 [2CH2, JC,PZ5.4 Hz, (BnO)2P(O)–], 68.96 (C-
60), 67.93 (C-6), 55.77 (C-20), 52.69 (C-2), 42.50 and 42.26
(2IcoaCH2), 34.81, 34.70, 34.59, 34.57, 34.26 (2MyraCH2,
SteaCH2, 2IcogCH2), 32.24, 30.04, 29.98, 29.92, 29.88,
29.82, 29.68, 29.54, 29.50, 25.58, 25.25, 25.14, 24.94,
24.78, 23.0 (67CH2), 14.45 (5CH3); 31P NMR (CDCl3): d K
1.65; MALDI-TOF-MS: m/z: 2200.67 [MCNa]C.
3.1.29. 4-O-Benzyl-6-O-{6-O-benzyl-4-O-[bis(benzyl-
oxy)phosphoryl]-2-deoxy-2-[(R)-3-(octadecanoyloxy)-
icosanoylamino]-3-O-tetradecanoyl-b-D-glucopyrano-
syl}-2-[(R)-3-(benzyloxy)icosanoylamino]-2-deoxy-3-O-
tetradecanoyl-D-glucopyranose (42). To a degassed
solution of 41 (95 mg, 0.043 mmol) in THF (20 mL)
iridium catalyst (25 mg, 0.03 mmol) was added and
activated with H2 as described for the preparation of 19.
The mixture was stirred under He for 30 min and cooled to
0 8C. A solution of I2 (20 mg, 0.08 mmol) in 2:1 THF/H2O
(2 mL) was added dropwise and the solution was stirred at
0 8C for 3 h, diluted with CH2Cl2 (200 mL), washed with
5% aq Na2S2O3 (20 mL), satd aq NaHCO3 (30 mL) and
brine (30 mL). The organic phase was dried (cotton) and
concentrated. The residue was purified by precipitation with
EtOH from CH2Cl2 (2!) (see preparation of 39), then with
MeOH from 1:1 toluene/hexane (2!) (see below) and,
finally, by chromatography on silica gel (1.5!60 cm,
150:100:15:1 toluene/CH2Cl2/MeOH/H2O) which afforded
42 (82 mg, 0.038 mmol, 88%) as amorphous solid. Precipi-
tation with MeOH from 1:1 toluene/hexane. The residue was
dissolved in CH2Cl2 (2 mL), then toluene (1 mL) and
hexane (3 mL) were added and the volume was reduced to
2 mL by concentration. MeOH (20 mL) was added and the
suspension was kept at 4 8C for 30 min. The white fluffy
precipitate was separated on a glass-filter and washed with
MeOH (10 mL). The precipitate was collected from the
filter by dissolution in CH2Cl2 (10 mL), the solution was
concentrated to dryness. Rf 0.45 (B, 150:100:15:1 toluene/
CH2Cl2/MeOH/H2O), Rf 0.36 (a-anomer) and Rf 0.32
(b-anomer) (B, 10:1 CH2Cl2/acetone); [a]2D0ZC4.5 (c 1.0,
3.1.30. 4-O-Benzyl-6-O-{6-O-benzyl-2-[(R)-3-(benzyl-
oxy)icosanoylamino]-4-O-[bis(benzyloxy)phosphoryl]-2-
deoxy-3-O-tetradecanoyl-b-D-glucopyranosyl}-2-[(R)-3-
(benzyloxy)icosanoylamino]-1-O-[bis(benzyloxy)phos-
phoryl]-2-deoxy-3-O-tetradecanoyl-a-D-glucopyranose
(43). To a stirred solution of 40 (58 mg, 0.029 mmol) and
tetrabenzyl diphosphate (76 mg, 0.14 mmol) in anhydrous
THF (10 mL) a 1.0 M solution of lithium bis(trimethyl-
silyl)amide in n-hexane (60 mL, 0.058 mmol) was added at
K78 8C under N2. The mixture was stirred for 30 min, then
allowed to warm up to 0 8C within 5 min and the reaction
was quenched with satd aq NaHCO3 (0.5 mL). The mixture
was diluted with CHCl3 (100 mL) and washed with satd aq
NaHCO3 (20 mL), H2O (20 mL) and brine (20 mL). The
organic phase was dried (cotton), concentrated and purified
by repeated precipitation with EtOH from CH2Cl2 (5!)
(see preparation of 39) to give 43 (56 mg, 0.025 mmol,
85%) as a solid. Rf 0.46 (B, 2:1 toluene/EtOAc), Rf 0.43 (B,
150:100:15:1 toluene/CH2Cl2/MeOH/H2O). [a]2D0ZC11.3
1
(c 1.0, CHCl3); H NMR: d 7.35–7.19 (m, 40H, 8Ph), 7.01
0
3
(d, 1H, 3J2 ,NH Z9.1 Hz, NH ), 6.20 (d, 1H, J2,NHZ8.7 Hz,
0
0
3
3
NH), 5.67 (d, 1H, J1,2Z3.6 Hz, J1,PZ5.3 Hz, H-1), 5.26
3
3
0
0
0
0
0
(dd, 1H, J3 ,2 Z10.8 Hz, J3 ,4 Z9.2 Hz, H-3 ), 5.22 (dd,
1H, 3J3,2Z10.8 Hz, 3J3,4Z9.3 Hz, H-3), 5.05–4.85 [m, 8H,
3
0
0
4CH2, 2!(BnO)2P(O)–], 4.72 (d, 1H, J1 ,2 Z8.5 Hz,
3
H-10), 4.56–4.38 (m, 4CH2, 4Bn), 4.44 (t, 1H, J4 ,5
Z
0
0
9.2 Hz, H-40), 4.26 (ddd, 1H, 4J2,PZ1.5 Hz, H-2), 4.03 (ddd,
3
3
1H, J6a,5Z2.0 Hz, J6b,5Z5.6 Hz, H-5), 3.93 (ddd, 1H,
1
2
CHCl3); H NMR (for a-anomer): d 7.38–7.22 (m, 25H,
5Ph), 6.26 (d, 1H, J2,NHZ9.5 Hz, NH), 6.02 (d, 1H,
J2 ,NH Z8.0 Hz, NH ), 5.42 (dd, 1H, J3 ,2 Z10.9 Hz,
J3 ,4 Z9.4 Hz, H-3 ), 5.39 (dd, 1H, J3,2Z10.7 Hz,
H-20), 3.90 (dd, 1H, J6a,6bZ11.5 Hz, H-6a), 3.83 (dd, 1H,
3
2
3
0
6b), 3.77 (m, 1H, bCHOBn), 3.70 (m, 1H, bCHOBn), 3.67
0
0
0
0
J6a ,6b Z11.1 Hz, J6a ,5 Z1.8 Hz, H-6a ), 3.78 (dd, 1H, H-
3
3
3
3
0
0
0
0
0
3
(dd, 1H, 3J6b ,5 Z6.2 Hz, H-6b ), 3.55 (ddd, 1H, H-5 ), 3.54
0
0
0
0
0
0
0
3
3
J3,4Z9.1 Hz, H-3), 5.17 (d, 1H, J1 ,2 Z8.2 Hz, H-1 ),
(t, 1H, J5,4Z9.3 Hz, H-4), 2.48–2.28 (m, 4H, 2IcoaCH2),
0
0
0
5.11 (d, 1H, 3J1,2Z3.2 Hz, H-1), 4.96 [bCH, (octadecanoyl-
oxy)icosanoyl], 4.95–4.85 [m, 4H, 2CH2, (BnO)2P(O)–],
4.62 and 4.54 (AB, 2H, 2JZ11.8 Hz, CH2Ph), 4.58 and 4.51
2.27–2.05 (m, 4H, 2MyraCH2), 1.55–1.40 (m, 8H,
2
MyrbCH2, 2IcogCH2), 1.34–1.15 (m, 110H, 55CH2), 0.90
(t, 12H, 4CH3); 13C NMR(CDCl3): d 174.04 (CO), 173.82
(CO), 171.85 (CONH), 171.53 (CONH), 138.96, 138.75,
138.60, 137.78 (4C, Ph), 136.01, 135.92, 135.78, 135.68
2
(AB, 2H, JZ11.5 Hz, CH2Ph), 4.53 and 4.46 (AB, 2H,
2JZ12.0 Hz, CH2Ph), 4.52 (t, 1H, J4 ,5 Z9.4 Hz, H-4 ),
3
0
0
0
4.22 (ddd, 1H, H-2), 4.12 (ddd, 1H, 3J6a,5Z1.6 Hz, 3J6b,5
Z
[4C, Ph, JC,PZ4.5 Hz, 2(BnO)2P(O)–], 129.08, 129.02,
3
2
8.0 Hz, H-5), 3.98 (dd, 1H, J6a,6bZ12.0 Hz, H-6a), 3.84
(m, 1H, bCHOBn), 3.82 (dd, 1H, J6a ,6b Z10.5 Hz,
J6a ,5 Z2.0 Hz, H-6a ), 3.74 (dd, 1H, H-6b), 3.70–3.50
(m, 3H, H-20, H-6b0, H-50), 3.36 (t, 1H, H-4), 2.40–2.15 (m,
10H, 2IcoaCH2, 2MyraCH2, SteaCH2), 1.65–1.40 (m, 10H,
128.87, 128.84, 128.72, 128.65, 128.63 (20C, Ph), 128.38,
128.33, 128.24, 128.14, 128.10, 128.0, 127.93, 127.88,
127.80 (20C, Ph), 101.39 (C-10), 96.39 (C-1, 2J1,PZ6.6 Hz),
2
0
0
3
0
0
0
76.27 (IcobCH), 75.94 (C-4), 75.88 (IcobCH), 75.18
3
(CH2Ph), 74.79 (C-50, J5 ,PZ3.2 Hz), 74.70 (C-40,
0

12134
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
2
J4 ,PZ6.0 Hz), 74.13 (C-5), 73.64 (CH2Ph), 73.38 (C-30,
174.12 (CO), 173.85 (CO), 173.82 (CO), 171.59 (CONH),
170.34 (CONH), 138.79, 138.70, 137.68 (3C, 3C6H5),
136.09, 136.01, 135.78, 135.69 [4C, Ph, JC,PZ4.0 Hz,
2(BnO)2P(O)–], 129.17, 128.92, 128.88, 128.72, 128.68,
0
3
0
J3 ,PZ2.0 Hz), 72.10 (C-3), 71.31 and 71.29 (2CH2,
2
3
2
IcoBn), 70.26 and 70.19 [2CH2, JC,PZ1.7 Hz,
2
(BnO)2P(O)–], 69.87 and 69.79 [2CH2, JC,PZ6.0 Hz,
(BnO)2P(O)–], 69.06 (C-60), 66.61 (C-6), 55.57 (C-20),
128.43, 128.36, 128.31, 128.29, 128.15, 128.08, 127.97,
0
52.52 (C-2, JC,PZ8.8 Hz), 41.73 (IcoaCH2), 41.40
127.56 (25C, Ph), 100.24 (C-10), 96.39 (C-1, J1 ,P
Z
3
2
(
IcoaCH2), 34.67, 34.57, 34.39, 34.32 (2MyraCH2,
6.6 Hz), 76.10 (C-4), 75.94 (IcobCH), 75.31 (CH2Ph),
2
2
IcogCH2), 32.27, 30.07, 30.02, 29.93, 29.86, 29.76,
74.86 (C-50), 74.74 (C-40, J4 ,PZ5.3 Hz), 73.69 (CH2Ph),
0
29.71, 29.58, 29.55, 25.69, 25.62, 25.46, 25.11, 24.91,
23.04 (55CH2), 14.46 (4CH3); 31P NMR (CDCl3): d K1.60
(attached to C-40), K2.55 (attached to C-1); MALDI-TOF-
MS: m/z: 2284.59 [MCNa]C, 2300.58 [MCK]C.
73.42 (C-5), 72.15 (C-3), 72.14 (C-30), 71.34 (CH2Ph),
71.31 [bCH, (octadecanoyl oxy)icosanoyl], 69.36 and
2
70.30 [2CH2, JC,PZ1.8 Hz, (BnO)2P(O)–], 70.04 and
2
69.85 [2CH2, JC,PZ7.5 Hz, (BnO)2P(O)–], 68.13 (C-
2
60), 66.69 (C-6), 54.50 (C-20), 52.61 (C-2, J2,P
Z
8.7 Hz), 41.58 and 41.47 (2IcoaCH2), 34.85, 34.64,
34.42, 34.35, 34.30 (2MyraCH2, SteaCH2, 2IcogCH2),
32.33, 30.13, 30.08, 29.99, 29.92, 29.87, 29.77, 29.63,
25.78, 25.75, 25.42, 25.17, 24.98, 23.10 (67CH2), 14.51
3.1.31. 4-O-Benzyl-6-O-{6-O-benzyl-4-O-[bis(benzyl-
oxy)phosphoryl]-2-deoxy-2-[(R)-3-(octadecanoyloxy)-
icosanoylamino]-3-O-tetradecanoyl-b-D-glucopyrano-
syl}-2-[(R)-3-(benzyloxy)icosanoylamino]-1-O-[bis(ben-
zyloxy)phosphoryl]-2-deoxy-3-O-tetradecanoyl-a-D-
glucopyranose (44). To a stirred solution of 42 (45 mg,
0.021 mmol) and tetrabenzyl diphosphate (54 mg,
0.1 mmol) in anhyd THF (10 mL) a 1.0 M solution of
lithium bis(trimethylsilyl)amide in n-hexane (40 mL,
0.04 mmol) was added at K78 8C under N2. The mixture
was stirred for 30 min, then allowed to warm up to 0 8C
within 5 min and the reaction was quenched with 10%
aqueous NaHCO3 (0.5 mL). The mixture was diluted with
CHCl3 (100 mL) and washed with satd aq NaHCO3
(20 mL), H2O (20 mL) and brine (20 mL). The organic
phase was dried (cotton) and concentrated. Purification by
repeated precipitations with EtOH from CH2Cl2 (3!) (see
preparation of 39) and with MeOH from toluene (3!) (see
below) gave 44 (43 mg, 0.018 mmol, 86%) as a solid.
Precipitation with MeOH from toluene. The residue was
dissolved in CH2Cl2 (2 mL), then toluene (1 mL) was added
and the volume was reduced to 1 mL by concentration.
MeOH (15 mL) was added and the suspension was kept at
4 8C for 30 min. The white fluffy precipitate was filtered off
and washed with MeOH (10 mL). The precipitate was
recovered from the filter by dissolution in CH2Cl2 (10 mL),
the solution was concentrated to dryness. Alternatively,
purification of the same amount of diphosphate 44 (starting
from 45 mg of 42) by subsequent precipitation with EtOH
from CH2Cl2 (2!) and chromatography on silica gel (1.5!
10 cm, 100:7/100:10 CH2Cl2/acetone) afforded 21 mg
(0.088 mmol, 41%) of 44. Rf 0.64 (B, 150:100:15:1 toluene/
CH2Cl2/MeOH/H2O), Rf 0.45 (B, 10:1 CH2Cl2/acetone);
31
(5CH3); P NMR (CDCl3): d K1.6 (attached to C-40),
K2.9 (attached to C-1); MALDI-TOF-MS: m/z: 2200.67
[MCNa]C.
3.1.32. Monotriethylammonium 2-deoxy-6-O-{2-deoxy-
2-[(R)-3-hydroxyicosanoylamino]-3-O-tetradecanoyl-b-
D-glucopyranosyl}-2-[(R)-3-hydroxyicosanoylamino]-3-
O-tetradecanoyl-a-D-glucopyranose 1,40-bisphosphate
(1). A solution of 43 (27 mg, 0.012 mmol) in 5:1 toluene/
MeOH (10 mL) was hydrogenated in the presence of Pd/C
(10%, 80 mg) at rt and atmospheric pressure for 20 h. The
reaction mixture was diluted with 5:1 toluene/MeOH
(10 mL), sonicated (10 min) and filtered through a mem-
brane filter (0.45 mm, regenerated cellulose). The catalyst
was removed from the filter, resuspended in THF (20 mL)
and the suspension was sonicated (10 min) and again
membrane-filtered. The procedure was repeated twice.
The combined filtrates were concentrated at diminished
pressure under N2, dissolved in 4:1 CHCl3/MeOH (5 mL)
and let slowly adsorb on a DEAE-cellulose column
(CH3COOK-form, 1!8 cm) equilibrated with 2:3:1
CHCl3/MeOH/H2O. The column was washed with 2:3:1
CHCl3/MeOH/H2O (50 mL) and then developed with
the stepwise gradient of 2:3:1 CHCl3/MeOH/aq CH3-
COOKHNEt3C (30 mL of CHCl3/MeOH/0.06 M aq CH3-
COOKHNEtC3 , 60 mL of CHCl3/MeOH/0.08 M aq
CH3COOKHNEt3C, 30 mL of CHCl3/MeOH/0.2 M aq
CH3COOKHNEtC3 and 100 mL of CHCl3/MeOH/1 M
aq CH3COOKHNEtC3 ). Appropriate fractions (1 was eluted
at 0.1 M aq CH3COOKHNEt3C) were collected, the total
volume was adjusted to 240 mL by addition of 2:3:1 CHCl3/
MeOH/H2O. The solution was transferred to an extraction
funnel and converted to a two-phase Bligh–Dyer system by
changing the solvent proportions to 2:2:1.8 by addition of
CHCl3 (40 mL) and water (68 mL). The phases were
resolved, the lower phase was concentrated, the residue
was redissolved in 2:3:1 CHCl3/MeOH/H2O (180 mL) and
rendered to a Bligh–Dyer mixture by addition of CHCl3
(40 mL), methanol (10 mL) and water (60 mL). The phases
were resolved in the extraction funnel, the lower phase was
1
[a]2D0ZC15.0 (c 1.0, CHCl3); H NMR: d 7.38–7.22 (m,
35H, 7PH), 6.92 (d, 1H, 3J2 ,NH Z9.2 Hz, NH ), 6.24 (d, 1H,
0
0
0
3J2,NHZ8.8 Hz, NH), 5.67 (dd, 1H, J1,2Z3.2 Hz, J1,P
Z
Z
3
3
30
3
0
0
0
0
5.3 Hz, H-1), 5.27 (dd, 1H,
J
Z10.6 Hz, J3 ,4
3 ,2
3
3
9.1 Hz, H-30), 5.26 (dd, 1H, J3,2Z11.0 Hz, J3,4Z9.5 Hz,
H-3), 5.10 [m, 1H, bCH, (octadecanoyloxy)icosanoyl],
5.09–4.88 [m, 8H, 4CH2, 2(BnO)2P(O)–], 4.90 (d, 1H,
3
2
0
0
0
J1 ,2 Z8.3 Hz, H-1 ), 4.50 and 4.39 (AB, 2H, JZ12.0 Hz,
CH2Ph), 4.61 and 4.55 (AB, 2H, 2JZ11.7 Hz, CH2Ph), 4.52
and 4.49 (AB, 2H, 2JZ12.1 Hz, CH2Ph), 4.43 (t, 1H,
3
0
0
0
J4 ,5 Z9.1 Hz, H-4 ), 4.28 (ddd, 1H, H-2), 4.09 (m, 1H,
H-5), 3.95–3.80 (m, 4H, H-20, H-6a, H-6b, H-6a0), 3.73 (m,
separated and concentrated to afford 1 (10 mg,
2
3
0
0
0
0
1H, bCHOBn), 3.68 (dd, 1H, J6a ,6b Z11.2 Hz, J6b ,5
Z
0.0061 mmol, 51%). Rf 0.3 (A, 100:75:15 CHCl3/MeOH/
H2O) or Rf 0.3 (A, 50:20:30:5:2 CHCl3/n-hexane/MeOH/
H2O/CH3COOH); [a]D20ZC12 (c 0.2, 4:1 CHCl3/MeOH);
NMR data see Table 2; MALDI-TOF-MS: m/z: 1466.07
[MKH3PO4CNa–H]C, 1482.44 [MKH3PO4CK–H]C,
5.6 Hz, H-6b0), 3.55 (m, 1H, H-50), 3.51 (t, 1H, H-4), 2.50–
2.15 (m, 10H, 2IcoaCH2, 2MyraCH2, SteaCH2), 1.65–1.40
(m, 10H, 2MyrbCH2, StebCH2, 2IcogCH2), 1.35–1.15 (m,
134H, 67CH2), 0.90 (t, 15H, 5CH3); 13C NMR(CDCl3): d

A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
12135
1564.07 [MCNa]C, 1586.04 [MKHC2Na]C; calcd
1466.07 [MKH3PO4CNa–H]C, 1482.17 [MKH3PO4C
K–H]C, 1564.04 [MCNa]C, 1586.02 [MKHC2Na]C;
ESI-MS-neg.: m/z: 1541.05 [M], calcd 1541.05 [M].
collected and the total volume was adjusted to 120 mL by
addition of 2:3:1 CHCl3/MeOH/H2O. The solution was
transferred to an extraction funnel and rendered to a Bligh–
Dyer mixture by addition of CHCl3 (20 mL) and water
(34 mL). The lower phase was concentrated and purified by
chromatography on silica gel (1.5!20 cm) 50:20:20:3:2
CHCl3/n-hexane/MeOH/H2O/CH3COOH. Appropriate
fractions were collected and concentrated. The residue
was suspended in 1% aq Et3N in water (10 mL) and freeze-
dried which afforded 3 as monotriethylammonium salt
(9 mg, 0.006 mmol, 60%). Rf 0.2 (A, 50:20:20:3:2 CHCl3/
n-hexane/MeOH/H2O/CH3COOH) or Rf 0.65 (A, 100:75:15
CHCl3/MeOH/H2O); [a]2D0ZK16.4 (c 0.6, CHCl3/MeOH);
MALDI-TOF-MS: m/z: 1484.14 [MCNa]C, 1506.14 [MK
HC2Na]C; calcd 1484.09 [MCNa]C, 1506.06 [MKHC
2Na]C; MALDI-TOF: m/z: [MKH]K 1460.07, calcd
1460.09 [MKH]K.
3.1.33. Monotriethylammonium 2-deoxy-6-O-{2-deoxy-
2-[(R)-3-(octadecanoyloxy)icosanoylamino]-3-O-tetra-
decanoyl-b-D-glucopyranosyl}-2-[(R)-3-hydroxy-
icosanoylamino]-3-O-tetradecanoyl-a-D-glucopyranose
1,40-bisphosphate (2). A solution of 44 (25 mg, 0.01 mmol)
in 5:1 toluene/MeOH (10 mL) was hydrogenated in the
presence of Pd/C (10%, 80 mg) at rt and atmospheric
pressure for 20 h. The reaction mixture was diluted with 5:1
toluene/MeOH (10 mL), sonicated (10 min) and filtered
over membrane filter as described for 1. The combined
filtrates were concentrated under N2, dissolved in 4:1
CHCl3/MeOH (5 mL) and purified on a DEAE-cellulose
column (CH3COOK-form, 1!8 cm) equilibrated with 2:3:1
CHCl3/MeOH/H2O. The column was washed with 2:3:1
CHCl3/MeOH/H2O (70 mL) and then developed with a
stepwise gradient as described for 1. Appropriate fractions
(2 was eluted at 0.1 M aq CH3COOKHNEt3C) were
collected and the total volume was adjusted to 240 mL by
addition of 2:3:1 CHCl3/MeOH/H2O, the solution was
transferred to an extraction funnel and subjected to the
Bligh–Dyer extraction as described for 1. The phases were
resolved in the extraction funnel and the lower phase was
separated and concentrated under reduced pressure to afford
2 (8.5 mg, 0.0045 mmol, 45%) as monotriethylammonium
salt. Rf 0.37 (A, 100:75:15 CHCl3/MeOH/H2O) or Rf 0.4 (A,
50:20:30:5:2 CHCl3/n-hexane/MeOH/H2O/CH3COOH);
[a]2D0ZC8 (c 0.7, 4:1 CHCl3/MeOH); MALDI-TOF-MS:
m/z: 1733.16 [MKH3PO4CNa–H]C, 1749.17 [MK
H3PO4CK–H]C, 1755.16 [MKH3PO4C2Na–2H]C,
1853.13 [MKHC2Na]C, 1875.09 [MK2HC3Na]C;
calcd 1733.33 [MKH3PO4CNa–H]C, 1749.44 [MK
H3PO4CK–H]C, 1755.32 [MKH3PO4C2Na–2H]C,
1852.28 [MKHC2Na]C, 1874.26 [MK2HC3Na]C; ESI-
MS-neg.: m/z: 1541.05 [MKC18H35O], 1564.04 [MKSteC
Na], 1807.32 [M], calcd 1541.05 [MKC18H35O], 1564.05
[MKSteCNa], 1807.31 [M].
3.1.35. Monotriethylammonium 2-deoxy-6-O-{2-deoxy-
2-[(R)-3-(octadecanoyloxy)icosanoylamino]-3-O-tetra-
decanoyl-b-D-glucopyranosyl}-2-[(R)-3-hydroxyicosa-
noylamino]-3-O-tetradecanoyl-D-glucopyranose 40-
phosphate (4). Compound 42 (40 mg, 0.018 mmol) was
dissolved in 5:1 toluene/MeOH (10 mL) under sonication
and the solution was hydrogenated in the presence of Pd/C
(10%, 80 mg) at rt and atmospheric pressure for 20 h (the
reaction mixture was sonicated every 30 min during first
2 h). The reaction mixture was diluted with 5:1 toluene/
MeOH (10 mL), sonicated (10 min) and filtered over
membrane filter (0.45 m, regenerated cellulose). The
catalyst was removed from the filter and suspended in
THF (20 mL). The suspension was sonicated (10 min), and
filtered over membrane filter (0.45 m, regenerated cellu-
lose); the procedure was repeated twice. The combined
filtrates were concentrated, the residue was dissolved under
sonication and heating (30 8C) in 50:20:20:3:2 CHCl3/
n-hexane/MeOH/H2O/CH3COOH (3 mL) and purified by
chromatography on silica gel (2!30 cm, 50:20:20:3:2
CHCl3/n-hexane/MeOH/H2O/AcOH).
Appropriate
fractions were collected, concentrated under diminished
pressure, redissolved in 4:1 CHCl3/MeOH (5 mL) and
slowly adsorbed on a resin bed of DEAE-cellulose column
(CH3COOK-form, 1!10 cm) equilibrated with 2:3:1
CHCl3/MeOH/H2O. The column was washed with 2:3:1
CHCl3/MeOH/H2O (50 mL) and then developed with
stepwise gradient of 2:3:1 CHCl3/MeOH/aq CH3-
COOKNHC4 (0.02 M, 0.04 M, 0.06 M, 0.08 M, 0.1 M aq
CH3COOKNHEtC3 , 50 mL each). Appropriate fractions
(4 was eluted at 0.06–0.08 M aq CH3COOKNHEt3C) were
collected and the total volume was adjusted to 120 mL by
addition of 2:3:1 CHCl3/MeOH/H2O. The solution was
transferred to an extraction funnel and converted to a Bligh–
Dyer system by addition of CHCl3 (20 mL) and water
(34 mL). The phases were resolved, the lower phase was
concentrated to dryness to give 4 (16 mg, 0.0087 mmol,
49%) (triethylammonium salt). Rf 0.3 (A, 50:20:20:3:2
CHCl3/n-hexane/MeOH/H2O/CH3COOH) or Rf 0.75 (A,
100:75:15 CHCl3/MeOH/H2O); [a]2D0ZK38 (c 0.4, 4:1
CHCl3/MeOH); MALDI-TOF-MS: m/z: 1750.43 [MC
Na]C, 1772.41 [MKHC2Na]C; calcd 1750.35 [MC
Na]C, 1772.35 [MKHC2Na]C.
3.1.34. Monotriethylammonium 2-deoxy-6-O-{2-deoxy-
2-[(R)-3-hydroxyicosanoylamino]-3-O-tetradecanoyl-b-
D-glucopyranosyl}-2-[(R)-3-hydroxyicosanoylamino]-3-
O-tetradecanoyl-D-glucopyranose 40-phosphate (3). A
solution of 40 (20 mg, 0.01 mmol) in THF (10 mL) was
hydrogenated in the presence of Pd/C (10%, 100 mg), at rt
and atmospheric pressure for 20 h. The reaction mixture was
diluted with THF (20 mL), sonicated (10 min) and filtered
through PTFE-membrane (0.45 mm) syringe filter. The
catalyst was removed from the filter, suspended in THF
(20 mL) and the suspension was sonicated (10 min) and
filtered [PTFE-membrane]. The combined filtrates were
concentrated, dissolved in 4:1 CHCl3/MeOH (3 mL) and
slowly adsorbed on a resin bed of DEAE-cellulose column
(CH3COOK-form, 1.5!10 cm) equilibrated with 2:3:1
CHCl3/MeOH/H2O. The column was washed with 2:3:1
CHCl3/MeOH/H2O (50 mL) and then developed with a
stepwise gradient of 2:3:1 CHCl3/MeOH/aq CH3-
COOKNHC4 (0.02 M, 0.03 M, 0.04 M, 0.06 M, 0.08 M aq
CH3COOKNHC4 , 30 mL each). Appropriate fractions (3
was eluted at 0.04–0.06 M aq CH3COOKNH4C) were

12136
A. Zamyatina et al. / Tetrahedron 60 (2004) 12113–12137
17. Gensler, W. J.; Alam, I.; Prasad, R. S.; Radhakrishna, A. I.;
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¨
helpful discussions with Ulrich Zahringer. The authors also
thank Buko Lindner for recording MALDI TOF and ESI
FT-MS spectra, Michael Puchberger and Andreas Hofinger
for measuring NMR-spectra, Fritz Altmann for MALDI-
TOF data, Maria Hobel and Irina von Cube for technical
assistance. Financial support of this work by FWF-grant P
13843 and a grant from the Japanese Society for the
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