Parasite glycoconjugates. Part 16: Synthesis of a disaccharide and phosphorylated di- and tri-saccharides from Leishmania lipophosphoglycan

Article abstract of DOI:10.1016/j.carres.2006.03.026

A neutral disaccharide β-d-Galp-(1→4)-α-d-Manp and phosphorylated di- and tri-saccharides β-d-Galp-(1→3)-[H₂PO₃-6]-β-d-Galp-O[CH₂]₈CH{double bond, long}CH₂ and β-d-Galp-(1→3)-[H₂PO₃-6]-β-d-Galp-(1→4)-α-d-Manp, which are fragments of the phosphoglycan portion of the surface lipophosphoglycan from Leishmania donovani (the disaccharide) or Leishmania major (all three compounds), were prepared and used as TLC standards to help the identification and differentiation of the elongating and branching β-d-galactosyl transferase activities in Leishmania. The phosphosaccharides were synthesised using the H-phosphonate method for phosphorylation.

Full text of DOI:10.1016/j.carres.2006.03.026

Carbohydrate Research 341 (2006) 1954–1964
Note
Parasite glycoconjugates. Part 16: Synthesis of
a disaccharide and phosphorylated di- and tri-saccharides
from Leishmania lipophosphoglycanI
Andrew J. Ross, Olga V. Sizovaꢀ and Andrei V. Nikolaev*
Faculty of Life Sciences, Division of Biological Chemistry and Molecular Microbiology, University of Dundee, Dundee DD1 5EH, UK
Received 16 December 2005; received in revised form 5 March 2006; accepted 21 March 2006
Available online 15 May 2006
Abstract—A neutral disaccharide b-D-Galp-(1!4)-a-D-Manp and phosphorylated di- and tri-saccharides b-D-Galp-(1!3)-[H2PO3-
6]-b-D-Galp-O[CH2]8CH@CH2 and b-D-Galp-(1!3)-[H2PO3-6]-b-D-Galp-(1!4)-a-D-Manp, which are fragments of the phospho-
glycan portion of the surface lipophosphoglycan from Leishmania donovani (the disaccharide) or Leishmania major (all three
compounds), were prepared and used as TLC standards to help the identification and differentiation of the elongating and branching
b-D-galactosyl transferase activities in Leishmania. The phosphosaccharides were synthesised using the H-phosphonate method for
phosphorylation.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Carbohydrates; Phosphates; Glycosylation; Leishmania enzymes
1. Introduction
with the species. For example, in L. donovani2 R = H
(100%), whereas in L. major2,12 R is predominantly
b-D-Galp (52%) or b-D-Galp-(1!3)-b-D-Galp (25%).
In the LPG produced by L. mexicana,6,13 R is b-D-Glcp
(25%) or H (75%). The importance of the LPG for
parasite infectivity and survival14,15 makes the enzymes
responsible for its biosynthesis of great interest.
The biosynthesis of the b-D-Galp-(1!4)-a-D-Manp
phosphate backbone was shown16,17 to be performed
by sequential action of the Leishmania elongating a-D-
mannopyranosyl phosphate transferase (eMPT) and
the elongating b-D-galactopyranosyl transferase (eGT).
There is also expected to be a branching b-D-galactopyr-
anosyl transferase (or transferases, bGT), which intro-
duces side-chain b-D-Galp residues (i.e., R = b-D-Galp)
in L. major,18 but not in L. donovani. In Parts 4,19 9,20
11,21 1222 and 151 of this series, we disclosed our interest
in the design and synthesis of various substrates/sub-
strate analogues/inhibitors to study the fine acceptor–
substrate specificity of the eMPT23,24 and to gain more
information about enzyme–substrate recognition. We
now report the chemical synthesis of disaccharide 4,
phosphotrisaccharide 5 and phosphodisaccharide 6
The Leishmania are sandfly-transmitted protozoan para-
sites that cause a variety of debilitating and often fatal
diseases throughout the tropics and sub-tropics. The life
cycle of the parasite involves an insect vector (the sand-
fly) and mammalian hosts. The parasites’ survival and
infectivity are due to the glycocalyx and in particular
the lipophosphoglycan (LPG), which is one of its major
components.2 LPGs are common to Leishmania major,
Leishmania donovani and Leishmania mexicana pro-
mastigotes3–8 and L. major amastigotes,9 but they are
absent in L. donovani and L. mexicana amastigotes.10,11
The LPG of the above Leishmania species contains a
polymeric phosphoglycan region consisting of [-6)-
(R!3)-b-D-Galp-(1!4)-a-D-Manp-(1-PO3H-]n repeat-
ing units where the nature of side-chain groups R varies
q See Ref. 1.
*
Corresponding author. Fax: +44 01382 386373; e-mail: a.v.nikolaev
@dundee.ac.uk
ꢀ On leave from Zelinsky Institute of Organic Chemistry, Russian
Academy of Sciences, Moscow, Russia.
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.026

A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
1955
Figure 1.
(Fig. 1), which then were used for the identification and
differentiation of the elongating and branching galactos-
yl transferase activities in Leishmania.
The synthesis of trisaccharide phosphate 5 was per-
formed starting from the recently described trisaccharide
compound 9,1 which was converted to the monohydroxyl
derivative 10 (96%) by detritylation with 3% trifluoroace-
tic acid (TFA) in dichloromethane. The H-phosphonate
approach to phosphomonoesters26 was then applied for
the phosphate formation. As we showed previously,26
efficacious phosphorylation of primary, secondary
and anomeric HO-groups of carbohydrates could be
performed via the consecutive preparation of ‘sugar’
H-phosphonates and corresponding ‘sugar’ 9-fluorene-
methyl phosphodiesters. In that way, the 9-fluorene-
methyl ester was used as P-protecting group to
facilitate the oxidation step. The protection was then
easily removed (with piperidine) to give the desired phos-
phomonoesters. In the present paper, triethylammonium
9-fluorenemethyl H-phosphonate 8 was used for phos-
phorylating alcohol 10 in the presence of pivaloyl chlo-
ride followed by oxidation with iodine, thus forming
trisaccharide 9-fluorenemethyl phosphodiester 11.
Subsequent P-deprotection (20% piperidine in CH2Cl2)
produced the O-protected phosphotrisaccharide 12
(64%), which gave phosphate 5 upon debenzoylation
with 0.3 M MeONa in methanol. The required H-phos-
phonate 8 was made essentially as described in Ref. 27,
that is, by the reaction of 9-fluorenemethanol and PCl3
followed by hydrolysis (producing crystalline 9-fluore-
nemethyl H-phosphonic acid) and neutralisation with
Et3N.
First, a biochemical assay was developed for a galac-
tose transfer in crude membrane preparation from
either L. donovani or L. major (a cell free system24 as
enzyme source), using UDP-[3H]Gal (as a b-D-galacto-
pyranose donor substrate) and synthetic LPG frag-
ments 1, 2 or 325 (as exogenous acceptor substrates).
Conditions for the assay and for the isolation of galac-
tosylated products were similar to those used previously
for the eMPT.24 It appeared that no [3H]Gal transfer
was detected for disaccharide monophosphate 1, but
the longer phosphoglycans 2 and 3 containing two
and three phosphodiester bridges, respectively, each
produced [3H]-labelled products of b-D-galactosylation
in the presence of both L. donovani and L. major
membrane preparation.
In order to analyse the structures and to identify the
galactosylation sites, which were expected to be A (cor-
responding to a putative eGT activity), B or C (both
corresponding to putative bGTs), the products of the
biochemical transfer of [3H]Gal to 3 were isolated24
(C18 Sep-Pak) and then hydrolysed with mild acid
(40 mM trifluoroacetic acid, 100 ꢁC). Since the condi-
tions were selective enough to hydrolyse the a-D-mannos-
yl phosphate bonds only,4 we expected the products to
be [3H]-labelled compounds 4, 5 or 6 (or mixtures of
any two or all three of them) with [3H]Gal residue at
the non-reducing terminus of each of them. Thus, the
authentic saccharides 4–6 were needed as standards to
properly analyse the hydrolysis products by TLC.
The preparation of disaccharide phosphate 6 was
started from galactosyl trichloroacetimidate 1320 and
the glycosyl acceptor 15 (Scheme 2), which in turn,
was synthesised from known 3,6-diol 1425 by the reac-
tion with tert-butyldiphenylsilyl chloride (TBDPS chlo-
ride) in pyridine. Trimethylsilyl triflate (TMS triflate)
assisted glycosylation reaction gave b-linked disaccha-
ride 16 in 45% yield and the recovered acceptor 15
(24%). The b-configuration of the newly created D-galac-
toside bond was confirmed by the characteristic value
2. Results and discussion
The neutral disaccharide 4 was prepared by conven-
tional debenzoylation of the O-protected derivative 720
(Scheme 1) with 0.06 M MeONa in methanol.

1956
A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
Scheme 1. Reagents: (a) NaOMe, MeOH; (b) TFA–CH2Cl2 (3:97); (c) (i) 8, pivaloyl chloride, C5H5N; (ii) I2, C5H5N–water; (d) piperidine, CH2Cl2.
˚
Scheme 2. Reagents: (a) TBDPS chloride, imidazole, C5H5N; (b) TMSOSO2CF3, MS 4 A, CH2Cl2; (c) n-Bu4NF, AcOH, THF; (d) (i) 8, pivaloyl
chloride, C5H5N; (ii) I2, C5H5N–water; (e) piperidine, CH2Cl2; (f) NaOMe, MeOH.
(7.6 Hz) for J1 ;2 -coupling constant in the 1H NMR
spectrum of 16. It was then desilylated with tetra-n-butyl-
ammonium fluoride (TBAF)–acetic acid reagent (an
equimolar mixture in THF) to give the monohydroxyl
derivative 17 (77%). Condensation of 17 with H-phos-
phonate 8 followed by oxidation (in the same manner
as described for the phosphorylation of trisaccharide
alcohol 10) then produced phosphodiester 18. It then
afforded the targeted phosphate 6 (76% for four steps
starting from 17) upon deprotection by consecutive
treatment with piperidine in CH2Cl2 (!19) and MeONa
in methanol–THF.
Since disaccharide 16 was synthesised in moderate
yield, we also attempted its preparation via the forma-
tion of the galactobiose derivative 25, followed by
transformation to disaccharide trichloroacetimidate 28
and glycosylation of dec-9-en-1-ol (Scheme 3). In this
way, glycosylation of 2-(trimethylsilyl)ethyl galactoside
22 with the benzoylated glycosyl donor 13 in the pres-
ence of TMS triflate proceeded slowly and resulted in
galactobiose 25 (29%) together with isomeric disaccha-
ride orthoester 26 (33%) and the recovered acceptor 22
(38%). The structure of the orthoester was confirmed
by the characteristic28 chemical shifts and coupling
0
0

A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
1957
˚
Scheme 3. Reagents: (a) H2, Pd(OH)2/C, AcOH–ethanol; (b) TMSOSO2CF3, MS 4 A, CH2Cl2; (c) Mg(OMe)2, MeOH; (d) benzoyl chloride, C5H5N;
˚
˚
(e) 22, TMSOSO2CF3, MS 4 A, CH2Cl2; (f) TFA–CH2Cl2 (2:1); (g) CCl3CN, DBU, CH2Cl2; (h) dec-9-ene-1-ol, TMSOSO2CF2, MS 4 A, CH2Cl2.
constants for 10-H (dH 6.43, d, J1 ;2 4.6) and 20-H (dH
ation of dec-9-en-1-ol with the trichloroacetimidate 28
in the presence of TMS triflate proceeded smoothly
and afforded disaccharide 16 in 72% yield. Similarly,
the reaction of dec-9-en-1-ol with trichloroacetimidate
29 provided the de-O-silylated compound 17 (38%) as
the main product.
0
0
1
0
0
4.58–4.70, m, J2 ;3 5.0) signals in the H NMR spec-
trum, and by the presence of the diagnostic signal28
of the quaternary orthoester carbon (dC 121.16) in
the 13C NMR spectrum. Orthoester 26 was then con-
verted to additional disaccharide 25 (51% from 26)
by the reaction with the recovered compound 22 in
the presence of TMS triflate, thus making the total
yield of 25 as 46%.
The synthetic saccharides 4–6 were used later as TLC
standards to identify products of acid hydrolysis of
[3H]-galactosylated phosphosaccharide 3 ([3H]Gal–3),
which was biochemically prepared with the use of
crude membrane preparation from L. donovani or L.
major (see Introduction section). Only disaccharide 4
(which corresponds to the galactosylation site A,
Fig. 1) was detected in the hydrolysate of the
[3H]Gal–3 made with L. donovani membranes, but both
disaccharide 4 and phosphotrisaccharide 5 (corre-
sponding to the galactosylation sites A and B, respec-
tively) were found in the hydrolysate of products
formed in the L. major assay. This shows the presence
of the elongating b-D-galactosyl transferase in both
species and the presence of the branching b-D-galacto-
syl transferase in L. major only. Phosphodisaccharide 6
was not detected in the L. major products hydrolysate.
This result (indicating that galactosylation does not
take place at site C) reflects, probably, a substrate spec-
ificity of the bGT enzyme. A detailed and comprehen-
sive discussion of identification and characterisation of
the elongating and branching b-D-galactosyl transfer-
ases in Leishmania using a set of compounds prepared
in this laboratory will be published elsewhere in due
course.
Glycosylation of the same acceptor 22 with the acetyl-
ated glycosyl donor 20 appeared to be more efficient and
provided the expected b-linked disaccharide 23
0
0
ðJ1 ;2 ¼ 7:8 HzÞ in 80% yield. Glycosyl trichloroacetimi-
date 20, in turn, was effectively made (72% yield) from
penta-O-acetyl-b-D-galactopyranose by selective ano-
meric de-O-acetylation with Me2NH in acetonitrile fol-
lowed by the reaction with CCl3CN in the presence of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).29 Disaccha-
ride 23 was then converted to 25 by consecutive de-O-
acetylation30 with Mg(OMe)2 in MeOH (!24, 75%)
and conventional benzoylation (91%).
To prepare disaccharide trichloroacetimidate 28, com-
pound 25 was first anomerically deprotected31 with TFA
in CH2Cl2 (!27) followed by the reaction with CCl3CN
in the presence of DBU. Very much to our surprise,
some of the de-O-silylated trichloroacetimidate 29
(31%) was isolated in addition to the main product 28
(69%). Since the structure of the hemiacetal 27 was
clearly confirmed by the 1H NMR spectrum (see Exper-
imental section), we assume that partial de-O-silylation
was caused by the excessive DBU treatment. Glycosyl-

1958
A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
3. Experimental
give compound 4 (11.7 mg, 95%) as an amorphous solid;
20
½aꢁD +16.4 (c 1, MeOH); Rf 0.38 [CHCl3–MeOH–water,
(10:10:3)]; dH (D2O) (inter alia) 3.44 (dd, J2 ;3 10.0, 20-
0
0
3.1. General procedures
H), 3.56 (dd, J3 ;4 3.3, 30-H), 3.88 (m, 2- and 40-H),
0
0
4.34 (d, J1 ;2 7.7, 10-H), 5.08 (d, J1,2 1.5, 1-H); b-anomer
0
0
Melting points were determined on a Reichert hot-plate
apparatus and are uncorrected. Optical rotations were
measured with a Perkin–Elmer 141 polarimeter; [a]D-
components: 3.43 (dd, J2 ;3 9.9, 20-H), 4.33 (d, J1 ;2 7.7,
10-H); dC (D2O) 60.70 (C-6), 61.48 (C-60), 68.97 (C-40),
69.39 (C-3), 70.54 (C-2), 71.33 (C-20), 71.39 (C-5),
72.87 (C-30), 75.75 (C-50), 76.92 (C-4), 94.16 (C-1) and
103.42 (C-10); b-anomer components: 71.00 (C-2),
72.18 (C-3), 72.74 (C-30), 75.32 (C-5), 76.52 (C-4) and
94.00 (C-1); ES-MS (ꢀ): m/z 341.07 (5%) [MꢀH]ꢀ,
377.07 (45%) [M+35Cl]ꢀ, 379.10 (12%) [M+37Cl]ꢀ,
387.07 (100%) [M+HCOO]ꢀ; ES-MS (+): m/z 343.17
(100%) [M+H]+, 365.06 (70%) [M+Na]+, 381.06 (50%)
[M+K]+ (calcd for C12H22O11: M, 342.12).
0
0
0
0
values are given in units of 10ꢀ1 deg cm2 gꢀ1. H, 13C
1
and 31P NMR spectra (1H at 300 MHz, 13C at
75 MHz, 31P at 121 MHz) were recorded with a Bruker
AM-300 spectrometer for solutions in CDCl3, unless
otherwise indicated. Chemical shifts (d in ppm) are given
relative to those for Me4Si (for 1H and 13C) and external
aq 85 % H3PO4 (for 31P); J values are given in hertz. ES
mass spectra were recorded with a VG Quattro system
(VG Biotech, UK) and with a Mariner system (Applied
Biosystems). TLC was performed on Kieselgel 60 F254
(Merck) with detection under UV light or by charring
with sulfuric acid–water–ethanol (15:85:5). Flash col-
umn chromatography (FCC) was performed on Kiesel-
gel 60 (0.040–0.063 mm) (Merck). Dichloromethane,
acetonitrile, pyridine and toluene were freshly distilled
from CaH2. Solutions worked up were concentrated
under reduced pressure at <40 ꢁC.
3.4. 2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl-
(1!3)-2,4-di-O-benzoyl-b-D-galactopyranosyl-(1!4)-
1,2,3,6-tetra-O-benzoyl-a-D-mannopyranose (10)
To a solution of trisaccharide derivative 9 (41 mg,
0.022 mmol) in CH2Cl2 (5 mL) was added 6% TFA in
CH2Cl2 (5 mL). After 30 min, the mixture was neutra-
lised with Et3N–MeOH–CH2Cl2 (1:1:2, 4 mL), concen-
trated and toluene was evaporated off from the
residue. FCC [toluene–ethyl acetate, (9:1)!(8:2)] of
the residue produced the 60-hydroxy derivative 10
3.2. 9-Fluorenemethyl H-phosphonic acid
A solution of 9-fluorenemethanol (588 mg, 3 mmol) in
acetonitrile (5 mL) was added to a stirred solution of
phosphorus trichloride (1.9 mL, 21.8 mmol) in the same
solvent (5 mL). After stirring for 15 min, the mixture
was concentrated and acetonitrile was evaporated off
from the residue. The residue was dissolved in aceto-
nitrile (20 mL), water (5 mL) was added dropwise (over
5 min) and the mixture was stored at room temperature
for 3 h, whereafter it was concentrated and acetonitrile
was evaporated off from the residue to leave a white
solid. The residue was recrystallised from the same sol-
vent (5 mL) to form crystalline 9-fluorenemethyl H-
phosphonic acid (663 mg, 85%); mp 114–116 ꢁC {lit.,27
mp 118–120 ꢁC}; dH 4.25 (1H, m, 9-H), 4.35 (2H, m,
22
(33 mg, 96%) as an amorphous solid; ½aꢁD +63.1 (c
1.1, CHCl3); dH 2.87 (1H, dd, J5 ;6a0 8.5, 60-Ha), 3.06 (1H,
0
b
dd, J6a0;6b 11.8, 60-H ), 3.38 (1H, dd, J5 ;6b 5.7, 50-H),
0
0
0
3.97–4.08 (3H, m, 30-H, 5-H and 600-Ha), 4.11 (1H, t,
J5 ;6a00 ¼ J5 ;6b ¼ 6:1, 500-H), 4.28 (1H, dd, J5,6a 2.5,
00
00
00
6-Ha), 4.38 (1H, br d, J6a,6b 12.1, 6-Hb), 4.43–4.58 (2H,
b
m, 4-H and 600-H ), 4.72 (1H, d, J1 ;2 7.9, 10-H), 4.85
0
0
(1H, d, J1 ;2 7.7, 100-H), 5.28 (1H, dd, J3 ;4 3.3, 300-H),
00 00
00 00
5.45 (1H, dd, J2 ;3 10.3, 200-H), 5.53 (1H, d, 400-H), 5.55
00 00
(1H, dd, J2 ;3 10.1, 20-H), 5.69 (1H, dd, J2,3 3.2, 2-H),
0
0
5.72 (1H, d, J3 ;4 3.0, 40-H), 5.81 (1H, dd, J3,4 9.1, 3-H),
6.38 (1H, d, J1,2 1.9, 1-H) and 6.88–8.06 (50H, m,
10 · Ph); dC 59.42 (C-60), 61.49 (C-600), 61.91 (C-6), 67.44
(C-400), 69.42 (C-200), 69.60 (C-40), 69.91 (C-2), 70.37 (C-
3), 71.07 (C-300), 71.32 (2C, C-20 and C-500), 71.58 (C-5),
72.77 (C-4), 73.69 (C-50), 78.41 (C-30), 91.10 (C-1),
101.10 (C-100), 101.71 (C-10), 127.94–130.38 and 133.14–
133.67 (Ph) and 164.00–171.05 (C@O). Anal. Calcd for
C88H72O26: C, 68.39; H, 4.70. Found: C, 68.29; H, 4.94.
0
0
1
CH2), 5.60 and 7.98 (1H, d, JH,P 711.6, P-H), 7.27–
7.80 (8H, m, 2 · C6H4) and 9.65 (1H, br s, P-OH); dP
1
3
9.19 (dt, JP,H 711.6, JP,CH 7.0).
2
3.3. b-D-Galactopyranosyl-(1!4)-a-D-mannopyranose (4)
To a solution of benzoylated disaccharide 7 (42 mg,
0.036 mmol) in MeOH (10 mL) was added 3 M methan-
olic NaOMe (0.2 mL). The mixture was stored for 3 h,
whereafter it was deionised with Dowex 50W-X4 (H+)
resin, filtered and concentrated. Water (5 · 5 mL) was
then evaporated off from the residue (to remove methyl
benzoate). The residue was then dissolved in water
(20 mL), the solution was extracted with diethyl ether
(2 · 10 mL) and the aqueous layer was concentrated to
3.5. b-D-Galactopyranosyl-(1!3)-6-O-phosphonato-
b-D-galactopyranosyl-(1!4)-a-D-mannopyranose, bis-
triethylammonium salt (5)
9-Fluorenemethyl H-phosphonate 8 was prepared by
evaporation of pyridine–Et3N (8:2) (3 · 3 mL) from
9-fluorenemethyl H-phosphonic acid (5.6 mg, 0.022
mmol). Trisaccharide 10 (30 mg, 0.019 mmol) was then

A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
1959
added to the residue and the mixture was dried by
evaporation of pyridine (3 · 3 mL) therefrom. The resi-
due was dissolved in pyridine (1 mL), pivaloyl chloride
(0.01 mL, 0.081 mmol) was added and the mixture was
stirred at room temperature for 1 h, whereafter a second
portion of pivaloyl chloride (0.01 mL, 0.081 mmol) was
added. After a further 25 min, a freshly prepared solution
of iodine (11 mg, 0.043 mmol) in 95% aq pyridine (1 mL)
was added to the mixture. After 30 min, the mixture was
diluted with CH2Cl2 and the solution was washed
successively with cold 0.5 M Na2S2O3 and cold 0.5 M tri-
ethylammonium (TEA) hydrogen carbonate, dried by
filtration through cotton wool and concentrated to pro-
duce crude phosphodiester 11 [dH (inter alia) 3.92 (1H,
br d, J4,5 9.7, 5-H), 4.26–4.44 (5H, m, 10-H, 600-Hb
and CH2–CH of 9-fluorenemethyl), 4.50 (1H, t,
J3,4 = J4,5 = 9.7, 4-H), 4.60 (2H, br s, 6-H2), 4.65 (1H, d,
3.6. Dec-9-enyl 2,4-di-O-benzoyl-6-O-(tert-butyldiphenyl-
silyl)-b-D-galactopyranoside (15)
To a solution of galactoside 1425 (104 mg, 0.197 mmol)
and imidazole (31 mg, 0.455 mmol) in pyridine (1 mL)
was added TBDPS chloride (0.059 mL, 0.227 mmol)
and the mixture was stirred at room temperature. After
17 h, the mixture was diluted with CH2Cl2, washed suc-
cessively with NaHCO3 and water, dried (MgSO4) and
concentrated. FCC [toluene–ethyl acetate, (99:1)!(9:1)]
of the residue produced compound 15 (127 mg, 84%)
20
as an amorphous solid; ½aꢁD +11.9 (c 1, CHCl3); dH
1.05 (9H, s, Me3C), 1.08–1.37 (10H, m, 5 · CH2), 1.54
(2H, m, OCH2CH2), 2.02 (2H, q, J 6.7, CH2CH2CH@),
2
2.83 (1H, d, JH,OH = 3.9, 3-OH), 3.52 (1H, dt, JH,H
3
9.8, JH,H 6.9, OHCHCH2), 3.82–3.98 (4H, m,
OHCHCH2, 5-H and 6-H2), 4.19 (1H, ddd, J2,3 10.0,
3
J1 ;2 7.8, 100-H), 5.21 (1H, dd, J3 ;4 3.2, 300-H), 5.33 (1H,
3-H), 4.66 (1H, d, J1,2 7.9, 1-H), 4.96 (1H, br d, JH,H
00 00
00 00
dd, J2 ;3 10.2, 200-H), 5.44 (1H, dd, J1 ;2 8.0, J2 ;3 9.8, 20-
10.2, CH@HCH), 5.02 (1H, br d, 3JH,H 17.0, CH@HCH),
00 00
0
0
0
0
H), 5.60 (1H, d, 400-H), 5.62 (1H, d, J3 ;4 3.1, 40-H), 5.65
(1H, dd, 2-H), 5.81 (1H, dd, J2,3 3.4, 3-H) and 6.38 (1H,
d, J1,2 1.6, 1-H); dP ꢀ1.06]. The residue was dissolved in
CH2Cl2 (1 mL), piperidine (0.2 mL) was added and the
mixture was stirred at room temperature for 40 min, then
diluted with CH2Cl2, washed successively with cold 0.1 M
HCl and cold 1 M TEA hydrogen carbonate, dried by
filtration through cotton wool and concentrated. FCC
[dichloromethane–MeOH–Et3N, (99:0:1)!(64:35:1)] of
the residue gave O-protected trisaccharide phospho-
monoester12 (22 mg, 64%; dP 1.78) as an amorphous
solid. Compound 12 (22 mg) was dissolved in 0.3 M meth-
anolic NaOMe (3.3 mL) and the mixture was stirred at
room temperature. After 16 h, the solution was deionised
with Dowex 50W-X4 (H+) resin, filtered and neutralised
with Et3N. After concentration, water (5 · 5 mL) was
evaporated off from the residue to remove methyl benzo-
ate. 1H NMR of the prepared compound then revealed the
presence of residual aromatic signals. After additional
treatment with 0.3 M NaOMe in MeON (24 h, room tem-
perature) followed by a work-up as described above, tri-
saccharide phosphate 5 (9.8 mg, 64% from 10) was
5.35 (1H, dd, 2-H), 5.82 (1H, ddt, JH,CH 6.7, CH@CH2),
0
0
2
5.86 (1H, d, J3,4 3.5, 4-H) and 7.14–8.18 (20H, m, 4 · Ph);
dC 19.02 (Me3C), 26.65 (Me3C), 25.82, 28.82, 28.95,
29.21, 29.24, 29.42 and 33.71 (CCH2C), 61.58 (C-6),
70.10 (OCH2), 70.38 (C-4), 72.20 (C-3), 73.77 (2C, C-2
and -5), 101.23 (C-1), 114.07 (@CH2), 127.58–130.11
and 132.69–135.52 (Ph), 139.15 (–CH@) and 166.52 and
166.59 (C@O). Anal. Calcd for C46H56O8SiÆH2O: C,
70.56; H, 7.47. Found: C, 70.94; H, 7.70.
3.7. Dec-9-enyl 2,3,4,6-tetra-O-benzoyl-b-D-galacto-
pyranosyl-(1!3)-2,4-di-O-benzoyl-6-O-(tert-butyl-
diphenylsilyl)-b-D-galactopyranoside (16)
(A) To a stirred and cooled (ꢀ60 ꢁC) solution of galac-
tosyl trichloroacetimidate 1320 (119 mg, 0.161 mmol)
and acceptor 15 (104 mg, 0.136 mmol) in dry dichloro-
methane (1.5 mL) containing freshly activated mole-
˚
cular sieves 4 A (240 mg) under argon was added a 2%
(v/v) solution of TMS triflate in the same solvent
(0.073 mL, 0.0081 mmol). The temperature was allowed
to rise to room temperature for 3 h, and the mixture was
stirred overnight. N,N-Diisopropylethylamine (0.1 mL,
0.58 mmol) was then added, the solids were filtered off
through a Celite pad and the solution was concentrated.
Two consecutive FCCs of the residue [first, in toluene–
ethyl acetate, (99:1)!(90:10) and then in petroleum
ether (40–60 ꢁC)–ethyl acetate, (99:1)!(80:20)] provided
b-linked disaccharide 16 (83 mg, 45%) as an amorphous
20
produced as an amorphous solid; ½aꢁD +11 (c 0.3,
water); Rf 0.14 [CHCl3–MeOH–water, (10:10:3)]; dH
(D2O) (inter alia) 1.20 (18H, t, J = 7.3, 6 · MeCH2),
3.13 (12H, q, 6 · MeCH2), 4.19 (dd, J3 ;4 3.2, 40-H), 4.46
0
0
(d, J1 ;2 7.9, 10-H), 4.55 (d, J1 ;2 7.3, 100-H) and 5.11 (d,
J1,2 1.6, 1-H); b-anomer component: 4.83 (d, J1,2 0.9, 1-
H); dC (D2O) 8.60 (MeCH2), 47.05 (MeCH2), 60.88 (C-
0
0
00 00
20
2
6), 61.30 (C-600), 64.40 (d, JC,P 4.1, C-60), 68.47 (C-40),
solid; ½aꢁD +78 (c 1.04, CHCl3); dH 1.06 (9H, s, Me3C),
68.97 (C-3), 69.44 (C-400), 70.46 (C-2), 70.53 (C-20), 71.27
1.10–1.45 (10H, m, 5 · CH2), 1.65 (2H, m, OCH2CH2),
3
2
(C-5), 71.40 (C-200), 72.90 (C-300), 74.02 (d, JC,P 7.9, C-
1.95 (2H, q, J 6.7, CH2CH2CH@), 3.38 (1H, dt, JH,H
3
50), 75.44 (C-500), 77.84 (C-4), 82.11 (C-30), 94.06 (C-1),
103.24 (C-10) and 104.75 (C-100); b-anomer components:
70.99 (C-2), 72.20 (C-3), 75.22 (C-5) and 77.46 (C-4);
dP (D2O) 1.65; ES-MS (ꢀ): m/z 583.08 (100%)
[Mꢀ2Et3NꢀH]ꢀ (calcd for C30H63N2O19P: M, 786.38).
9.5, JH,H 6.5, OHCHCH2), 3.78–3.91 (4H, m,
OHCHCH2, 5-H and 6-H2), 4.24 (1H, m, 50-H), 4.27
0
0
(1H, dd, J3,4 3.5, 3-H), 4.35 (1H, dd, J5 ;6a0 7.3, J6a0;6b
11.1, 60-Ha), 4.54 (1H, d, J1,2 8.0, 1-H), 4.77 (1H, dd,
J5 ;6b 6.1, 60-H ), 4.92 (1H, br d, JH,H 10.2, CH@HCH),
b
3
0
0

1960
A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
4.97 (1H, br d, 3JH,H 16.9, CH@HCH), 4.99 (1H, d, J1 ;2
6.91–8.15 (30H, m, 6 · Ph); dC 25.66, 28.80, 28.92,
29.10, 29.30, 29.66 and 33.71 (CCH2C), 60.03 (C-6),
61.65 (C-60), 67.55 (C-40), 69.48 (C-20), 70.08 (OCH2),
70.91 (C-4), 71.95 (2C, C-2 and -50), 71.39 (C-30), 73.63
(C-5), 78.43 (C-3), 101.54 (C-1), 101.79 (C-10), 114.03
(@CH2), 127.95–130.43 and 132.59–133.56 (Ph), 139.18
(–CH@) and 164.45–168.33 (C@O). Anal. Calcd for
C64H64O17: C, 69.55; H, 5.84. Found: C, 69.68; H, 6.21.
(B) To a stirred and cooled (ꢀ40 ꢁC) solution of
trichloroacetimidate 29 (27 mg, 0.024 mmol) and dec-9-
en-1-ol (0.043 mL, 0.241 mmol) in CH2Cl2 (1 mL) con-
0
0
7.6, 10-H), 5.37 (1H, dd, J3 ;4 3.3, 30-H), 5.52 (1H, dd, J2,3
0
0
10.0, 2-H), 5.57 (1H, dd, J2 ;3 10.4, 20-H), 5.78 (1H, ddt,
0
0
JH,CH 6.7, CH@CH2), 5.88 (1H, d, 40-H), 6.00 (1H, d,
2
4-H) and 6.98–8.20 (40H, m, 8 · Ph); dC 19.10 (Me3C),
26.67 (Me3C), 25.72, 28.82, 28.94, 29.14, 29.17, 29.33
and 33.71 (CCH2C), 61.58 (C-60), 62.32 (C-6), 67.55
(C-40), 69.63 (C-20), 69.73 (OCH2), 70.21 (C-4), 70.99
(C-50), 71.54 (C-30), 71.65 (C-2), 74.67 (C-5), 77.04 (C-
3), 101.29 (C-10), 101.45 (C-1), 114.03 (@CH2), 127.58–
130.18 and 132.51–135.56 (Ph), 139.18 (–CH@) and
164.40–165.97 (C@O). Anal. Calcd for C80H82O17Si: C,
71.51; H, 6.15. Found: C, 71.52; H, 6.38. Also isolated
was the galactoside 15 (25 mg, 24% recovery).
˚
taining freshly activated molecular sieves 4 A (200 mg)
under argon was added 2% (v/v) solution of TMS triflate
in the same solvent (0.022 mL, 0.0024 mmol). The stirring
was continued while the temperature was allowed to rise
to 0 ꢁC for 2 h, whereafter cooling was discontinued and
the mixture was stirred at room temperature for a further
16 h. After standard work-up (as described for the prep-
aration of compound 16), FCC [toluene–ethyl acetate,
(90:10)] gave the disaccharide derivative 17 (10 mg, 38%).
(B) To a stirred and cooled (ꢀ70 ꢁC) solution of tri-
chloroacetimidate 28 (72 mg, 0.053 mmol) and dec-9-
en-1-ol (0.1 mL, 0.56 mmol) in CH2Cl2 (1 mL) contain-
˚
ing freshly activated molecular sieves 4 A (200 mg)
under argon was added a 2% (v/v) solution of TMS
triflate in the same solvent (0.027 mL, 0.003 mmol).
The stirring was continued, while the temperature was
allowed to rise to ꢀ30 ꢁC for 1.5 h. A second portion
of 2% (v/v) solution of TMS triflate in CH2Cl2
(0.027 mL, 0.003 mmol) was then added, the mixture
was slowly warmed up to 0 ꢁC, and the stirring was con-
tinued at this temperature for a further 19 h. After stan-
dard work-up (as above), FCC [toluene–ethyl acetate,
(95:5)] gave the disaccharide 16 (52 mg, 73%).
3.9. Dec-9-enyl b-D-galactopyranosyl-(1!3)-6-O-phos-
phonato-b-D-galactopyranoside, bis-triethylammonium
salt (6)
9-Fluorenemethyl H-phosphonate 8 was prepared by
evaporation of pyridine–Et3N (8:2) (3 · 3 mL) from 9-
fluorenemethyl H-phosphonic acid (6.5 mg, 0.025 mmol).
The disaccharide derivative 17 (23 mg, 0.021 mmol) was
then added to the residue and the mixture was dried by
evaporation of pyridine (3 · 3 mL) therefrom. The resi-
due was dissolved in pyridine (1 mL), pivaloyl chloride
(0.011 mL, 0.089 mmol) was added and the mixture was
stirred at room temperature for 40 min, whereafter a
freshly prepared solution of iodine (12 mg, 0.047 mmol)
in 95% aq pyridine (1 mL) was added. After 30 min, the
mixture was diluted with CH2Cl2 and the solution was
washed successively with cold 0.5 M Na2S2O3 and cold
0.5 M TEA hydrogen carbonate, dried by filtration
through cotton wool and concentrated. A solution of
the residue in toluene–ethyl acetate (7:3) was passed
through a silica gel pad, using, first, the same solvent
and then CH2Cl2–MeOH–Et3N (89:10:1) for elution.
The latter fraction was concentrated to produce disaccha-
3.8. Dec-9-enyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyr-
anosyl-(1!3)-2,4-di-O-benzoyl-b-D-galactopyranoside
(17)
(A) To a solution of derivative 16 (50 mg, 0.037 mmol) in
THF (1 mL) was added a 10% (v/v) solution of acetic
acid in THF (0.064 mL, 0.112 mmol) and then 1 M
TBAF in THF (0.112 mL, 0.112 mmol). The mixture
was stored at room temperature for 23 h, whereafter a
second portion of the AcOH/THF solution (0.064 mL,
0.112 mmol) followed by 1 M TBAF/THF (0.112 mL,
0.112 mmol) was added. After 6.5 h, the mixture was
applied for a silica column and FCC [toluene–ethyl
acetate, (100:0)!(70:30)], then gave the 6-hydroxy deriv-
ative 17 (32 mg, 78%) as an amorphous solid; ½aꢁ2D2 +71 (c
0.5, CHCl3); dH 0.75–1.40 (12H, m, 6 · CH2), 1.87 (2H,
0
ride phosphodiester 18 [dH (inter alia) 4.05 (1H, dd, J5 ;6a0
2
3
a
7.8, J6a0;6b 11.1, 60-H ), 4.15–4.30 (4H, m, 1-H and CH2–
0
q, J 6.7, CH2CH2CH@), 3.32 (1H, dt, JH,H 9.4, JH,H
6.5, OHCHCH2), 3.51 (1H, dd, J5,6a 8.2, J6a,6b 11.3, 6-
b
CH of 9-fluorenemethyl), 4.53 (1H, dd, J5 ;6b 5.7, 60-H ),
0
0
Ha), 3.63–3.80 (3H, m, OHCHCH2, 5-H and 6-Hb),
4.74 (1H, d, J1 ;2 7.7, 10-H), 4.85 (1H, br d, JH,H 10.2,
3
0
0
a
3
4.11 (1H, dd, J5 ;6a0 6.8, J6a0;6b 10.8, 60-H ), 4.17 (1H,
CH@HCH), 4.89 (1H, br d, JH,H 17.0, CH@HCH),
0
0
dd, J3,4 3.8, 3-H), 4.19 (1H, m, 50-H), 4.49 (1H, d, J1,2
8.0, 1-H), 4.57 (1H, dd, J5 ;6b 5.7, 60-H ), 4.83 (1H, br
5.30 (1H, dd, J3 ;4 3.3, 30-H), 5.43 (1H, dd, J2 ;3 10.5,
20-H), 5.45 (1H, dd, J1,2 7.1, J2,3 9.9, 2-H) and 5.63–
5.78 (3H, m, H-4, -40 and CH@CH2); dC (inter alia)
8.33 (MeCH2), 25.67, 28.82, 28.94, 29.14, 29.27, 29.66
and 33.71 (CCH2C), 45.26 (MeCH2), 48.32 (d, 3JC,P 8.9,
OCH2CH of 9-fluorenemethyl), 61.16 (C-60), 64.34 (d,
2JC,P 3.7, C-6), 67.20 (br, OCH2CH of 9-fluorenemethyl),
0
0
0
0
b
0
0
3
3
d, JH,H 10.2, CH@HCH), 4.89 (1H, br d, JH,H 16.9,
CH@HCH), 4.97 (1H, d, J1 ;2 7.8, 10-H), 5.33 (1H, dd,
0
0
J3 ;4 3.3, 30-H), 5.52 (1H, dd, J2 ;3 10.0, 20-H), 5.57
0
0
0
0
(1H, dd, J2,3 9.8, 2-H), 5.70 (1H, ddt, JH,CH 6.7,
2
CH@CH2), 5.72 (1H, d, 4-H), 5.77 (1H, d, 40-H) and

A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
1961
67.49 (C-40), 69.54 (C-20), 69.97 (OCH2 of dec-9-enyl),
70.24 (C-4), 70.76 (C-50), 71.40 (C-2), 71.50 (C-30),
centrated (thus forming 2,3,4,6-tetra-O-acetyl-D-galac-
topyranose, 4.5 g). The residue was dissolved in
CH2Cl2 (40 mL), the solution was cooled (0 ꢁC) and
Cl3CCN (45 mL, 449 mmol) was added under argon fol-
lowed by DBU (1.9 mL, 12.7 mmol). The mixture was
stirred for 2 h at 0 ꢁC and was then concentrated.
FCC [toluene–ethyl acetate, (100:0)!(85:15)] of the res-
idue gave trichloroacetimidate 20 (4.57 g, 72%); mp 126–
3
72.86 (d, JC,P 7.6, C-5), 77.87 (C-3), 101.22 (2C, C-1
and -10), 114.0 (@CH2) and 139.21 (–CH@); dP ꢀ0.83].
The residue was dissolved in CH2Cl2 (1 mL), piperidine
(0.2 mL) was added and the mixture was stirred at room
temperature for 1 h, prior to being diluted with CH2Cl2
and washed successively with cold 0.1 M HCl and cold
1 M TEA hydrogen carbonate. The organic layer was
dried by filtration through cotton wool and concentrated,
thus providing crude phosphomonoester19 [dC (inter alia)
61.20 (C-60), 63.42 (br, C-6), 67.53 (C-40), 69.59 (C-20),
69.87 (OCH2), 70.11 (C-4), 70.75 (C-50), 71.61 (2C, C-2
25
127 ꢁC (from ether–hexane); ½aꢁD +124 (c 1, CHCl3);
{lit.,32 mp 122–124 ꢁC (from benzene–hexane); [a]D
+115.5}; dH 2.02, 2.03, 2.04 and 2.18 (12H, 4 · s,
4 · Ac), 4.09 (1H, dd, J6a,6b 11.3, 6-Ha), 4.18 (1H, dd,
J5,6b 6.6, 6-Hb), 4.45 (1H, ddd, J5,6a 6.8, 5-H), 5.37
(1H, dd, J2,3 10.8, 2-H), 5.44 (1H, dd, J3,4 3.0, 3-H),
5.58 (1H, dd, J4,5 1.8, 4-H), 6.61 (1H, d, J1,2 3.3, 1-H)
and 8.69 (1H, s, NH); dC 20.50 and 20.58 (CH3), 61.20
(C-6), 66.85 (C-2), 67.32 (C-4), 67.45 (C-3), 68.94 (C-
5), 90.72 (CCl3), 93.48 (C-1), 160.88 (C@NH) and
169.91–170.23 (C@O); ES-MS (+): m/z 514.00 (100%)
[M+Na]+ (calcd for C16H20Cl3NO10: M, 491.02).
3
and -30), 73.22 (d, JC,P 7.6, C-5), 77.40 (C-3), 100.91
(C-1), 101.27 (C-10), 114.0 (@CH2) and 139.20 (–CH@);
dP 2.07]. The residue was dissolved in 0.5 M NaOMe in
MeOH–THF (1:1, 1 mL) and the mixture was stirred at
room temperature. After 22 h, the solution was deionised
with Dowex 50W-X4 (H+) resin, filtered and neutralised
with Et3N. After concentration, water (5 · 5 mL) was
evaporated off from the residue to remove methyl benzo-
ate. The residue was then dissolved in water (20 mL) and
the solution was washed successively with CH2Cl2
(2 · 10 mL) and diethyl ether (2 · 10 mL) to remove dib-
enzofulvene and its piperidine adduct (formed at the P-
deprotection step). The aqueous layer was concentrated
to give the disaccharide phosphate 6 (12 mg, 76% from
3.11. 2-(Trimethylsilyl)ethyl 2,4-di-O-benzoyl-6-O-(tert-
butyldiphenylsilyl)-b-D-galactopyranoside (22)
A solution of compound 2133 (2.17 g, 2.62 mmol) in a
mixture of glacial acetic acid and ethanol (1:1, 60 mL)
containing 20% Pd(OH)2/C (3 g) was stirred under a
mild overpressure of hydrogen at room temperature for
24 h. More catalyst (0.5 g) was added and stirring was
continued for another 4 h. The mixture was filtered
through a Celite pad and the filtrate was concentrated.
FCC (toluene–ethyl acetate, 95:5) of the residue gave
the monohydroxy derivative 22 (1.81 g, 95%); mp 146–
20
17) as an amorphous solid; ½aꢁD +23 (c 0.1, water–
MeOH, 9:1); Rf 0.65 [CHCl3–MeOH–water, (10:10:3)];
dH (D2O) (inter alia) 1.00–1.30 (28H, m, 5 · CH2 and
6 · MeCH2), 1.59 (2H, m, OCH2CH2), 2.01 (2H, m,
CH2CH2CH@), 3.16 (12H, q, J 7.2, 6 · MeCH2), 4.44
(1H, d, J1,2 7.5, 1-H), 4.58 (1H, d, J1 ;2 6.5, 10-H), 4.93
(1H, br d, 3JH,H 10.2, CH@HCH), 5.01 (1H, br d, 3JH,H
0
0
24
148 ꢁC (from petroleum ether, 60–80 ꢁC); ½aꢁD +11.5 (c
16.9, CH@HCH) and 5.88 (1H, ddt, JH,CH 6.7,
1.02, CHCl3); dH 0.00 (9H, s, Me3Si), 0.97 (2H, m,
2
2
CH@CH2); dC (D2O) 9.31 (MeCH2), 26.11, 29.25,
29.36, 29.61, 29.83, 30.91 and 34.23 (CCH2C), 47.74
CH2SiMe3), 1.09 (9H, s, Me3C), 3.65 (1H, dt, JH,H
3
10.0, JH,H 6.7, OHCHCH2), 3.85–4.00 (3H, m, 5-H
2
3
(MeCH2), 62.02 (C-60), 64.53 (d, JC,P 4.0, C-6), 68.99
and 6-H2), 4.08 (1H, dt, JH,H 5.7, OHCHCH2), 4.21
(C-4), 69.68 (C-40), 70.94 (C-2), 71.76 (OCH2), 72.13
(1H, dd, J3,4 3.5, 3-H), 4.74 (1H, d, J1,2 7.9, 1-H), 5.37
(1H, dd, J2,3 10.0, 2-H), 5.88 (1H, d, 4-H) and 7.15–
8.22 (20H, m, 4 · Ph); dC ꢀ1.50 (Me3Si), 18.05
(CH2SiMe3), 19.01 (Me3C), 26.65 (Me3C), 61.62 (C-6),
67.49 (OCH2), 70.44 (C-4), 72.36 (C-3), 73.85 (2C, C-2
and -5), 100.68 (C-1), 125.27–130.11 and 132.74–135.54
(Ph), and 166.56 and 166.59 (C@O). Anal. Calcd for
C41H50O8Si2: C, 67.74; H, 6.93. Found: C, 67.30; H, 7.04.
3
(C-20), 73.62 (C-30), 74.25 (d, JC,P 7.9, C-5), 76.15 (C-
50), 83.43 (C-3), 103.47 (C-1), 105.47 (C-10), 115.00
(@CH2) and 141.49 (–CH@); dP (D2O) 3.34; ES-MS
(ꢀ): m/z 559.21 (100%) [Mꢀ2Et3NꢀH]ꢀ (calcd for
C34H71N2O14P: M, 762.46).
3.10. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl tri-
chloroacetimidate (20)
3.12. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl-(1!3)-2,4-di-O-benzoyl-6-O-(tert-
butyldiphenylsilyl)-b-D-galactopyranoside (23)
Anhydrous dimethylamine (4 mL, 60 mmol) was added
to a solution of 1,2,3,4,6-penta-O-acetyl-b-D-galacto-
pyranose (5 g, 12.81 mmol) in acetonitrile (50 mL) at
ꢀ20 ꢁC and the mixture was kept at room temperature.
After 50–60 min, the mixture was concentrated and tol-
uene was evaporated off from the residue. The residue
was dissolved in CH2Cl2 (70 mL) and the solution was
washed with water (3 · 20 mL), dried (MgSO4) and con-
To a stirred and cooled (ꢀ40 ꢁC) solution of galactosyl tri-
chloroacetimidate 20 (763 mg, 1.55 mmol) and acceptor
22 (562 mg, 0.77 mmol) in dry dichloromethane (15 mL)
˚
containing freshly activated molecular sieves 4 A (1 g)
under argon was added TMS triflate (0.074 mL,

1962
A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
0.39 mmol), whereafter the stirring was continued at
ꢀ40 ꢁC for 2 h. The temperature was allowed to rise and
the mixture was stirred at room temperature for a further
16 h. N,N-Diisopropylethylamine (0.12 mL, 0.7 mmol)
was then added, the solids were filtered off through a Celite
pad and the solution was concentrated. FCC [toluene–
ethyl acetate, (100:0)!(80:20)] of the residue provided
the b-linked disaccharide 23 (650 mg, 80%) as an amor-
derivative 24 (38 mg, 0.043 mmol) in pyridine (1 mL)
and the mixture was allowed to warm to room temper-
ature, whereafter it was kept for 5 h. The mixture was
then diluted with CH2Cl2, and the solution was washed
successively with saturated aq NaHCO3 and water,
dried by filtration through cotton wool and concen-
trated. Toluene was evaporated off from the residue to
remove traces of pyridine. FCC [toluene–ethyl acetate,
(95:5)] of the residue gave benzoylated disaccharide 25
25
phous solid; ½aꢁD +24.6 (c 1.06, CHCl3); dH 0.00 (9H, s,
20
Me3Si), 1.00 (2H, m, CH2SiMe3), 1.14 (9H, s, Me3C),
1.60, 1.94, 2.13 and 2.14 (12H, 4 · s, 4 · Ac), 3.65 (1H,
dt, 2JH,H 10.0, 3JH,H 6.8, OHCHCH2), 3.83–3.98 (4H, m,
5-, 50-H and 6-H2), 4.03–4.18 (2H, m, 60-Ha and
(51 mg, 91%) as an amorphous solid; ½aꢁD +84.8 (c
1.03, CHCl3); dH 0.00 (9H, s, Me3Si), 0.97 (2H, m,
2
CH2SiMe3), 1.18 (9H, s, Me3C), 3.64 (1H, dt, JH,H
3
10.0, JH,H 7.0, OHCHCH2), 3.93–4.05 (3H, m, 5-H
3
OHCHCH2), 4.25 (1H, dd, J3,4 3.5, 3-H), 4.28 (1H, dd,
and 6-H2), 4.12 (1H, dt, JH,H 6.1, OHCHCH2), 4.39
b
J5 ;6b 5.9, J6a0;6b 11.2, 60-H ), 4.70 (1H, d, J1 ;2 7.8,
(1H, m, 50-H), 4.41 (1H, dd, J3,4 3.4, 3-H), 4.49 (1H,
0
0
0
0
0
a
10-H), 4.73 (1H, d, J1,2 8.0, 1-H), 4.80 (1H, dd, J3 ;4 3.3,
dd, J5 ;6a0 7.3, J6a0;6b 11.0, 60-H ), 4.73 (1H, d, J1,2 8.0,
0
0
0
0
b
30-H), 5.06 (1H, dd, J2 ;3 10.4, 20-H), 5.34 (1H, d, 40-H),
5.68 (1H, dd, J2,3 9.9, 2-H), 5.83 (1H, d, 4-H), and 7.25–
8.20 (20H, m, 4 · Ph). Anal. Calcd for C55H68O17Si2: C,
62.48; H, 6.48. Found: C, 62.20; H, 6.69.
1-H), 4.90 (1H, dd, J5 ;6b 6.1, 60-H ), 5.13 (1H, d, J1 ;2
0
0
0
0
0
0
8.0, 10-H), 5.50 (1H, dd, J3 ;4 3.3, 30-H), 5.67 (1H, dd,
0
0
J2,3 10.0, 2-H), 5.72 (1H, dd, J2 ;3 10.5, 20-H), 6.02
(1H, d, 40-H), 6.13 (1H, d, 4-H) and 7.15–8.29 (40H,
m, 8 · Ph); dC ꢀ1.50 (Me3Si), 18.05 (CH2SiMe3), 19.01
(Me3C), 26.65 (Me3C), 61.57 (C-60), 62.30 (C-6), 67.13
(OCH2), 67.53 (C-40), 69.62 (C-20), 70.27 (C-4), 70.97
(C-50), 71.56 (C-30), 71.67 (C-2), 74.68 (C-5), 77.15 (C-
3), 100.92 (C-1), 101.28 (C-10), 127.58–130.18 and
132.51–135.56 (Ph) and 164.40–165.97 (C@O). Anal.
Calcd for C75H76O17Si2: C, 69.00; H, 5.87. Found: C,
68.73; H, 6.08.
0
0
3.13. 2-(Trimethylsilyl)ethyl b-D-galactopyranosyl-
(1!3)-2,4-di-O-benzoyl-6-O-(tert-butyldiphenylsilyl)-
b-D-galactopyranoside (24)
To a stirred and cooled (0 ꢁC) solution of the disaccharide
derivative 23 (400 mg, 0.378 mmol) in MeOH (60 mL)
was added 8% methanolic solution of Mg(OMe)2
(3.75 mL). The mixture was stirred at 0 ꢁC for 1.5 h,
whereafter it was deionised with Dowex 50W-X4 (H+) re-
sin, filtered and concentrated. FCC [MeOH–dichloro-
methane, (100:0)!(90:10)] of the residue gave the
deacetylated derivative 24 (253 mg, 75%) as an amor-
(B) To a stirred and cooled (ꢀ40 ꢁC) solution of
galactosyl
trichloroacetimidate
1320
(76 mg,
0.103 mmol) and acceptor 22 (62 mg, 0.085 mmol) in
dry dichloromethane (1 mL) containing freshly acti-
˚
vated molecular sieves 4 A (200 mg) under argon was
25
phous solid; ½aꢁD +37.1 (c 1, CHCl3); dH 0.00 (9H, s,
added a 2% (v/v) solution of TMS triflate in the same
solvent (0.045 mL, 0.005 mmol). The temperature was
allowed to rise to ꢀ30 ꢁC and the stirring was continued
for 2 h, whereafter the mixture was warmed up to 0 ꢁC
and stirred for a further 3 h. N,N-Diisopropylethyl-
amine (0.1 mL, 0.58 mmol) was then added, the solids
were filtered off through a Celite pad and the solution
was concentrated. FCC [petroleum ether (40–60 ꢁC)–
ethyl acetate, (95:5)!(80:20)] of the residue provided
first galactoside 22 (24 mg, 38% recovery). Continued
elution gave the disaccharide orthoester 26 (37 mg,
33%) as an amorphous solid; dH 0.00 (9H, s, Me3Si),
1.00 (2H, m, CH2SiMe3), 1.20 (9H, s, Me3C), 3.60
Me3Si), 1.00 (2H, m, CH2SiMe3), 1.11 (9H, s, Me3C),
3.43 (1H, dd, J3 ;4 3.2, 30-H), 3.50 (1H, m, 50-H), 3.54
0
0
2
3
(1H, dd, J2 ;3 9.6, 20-H), 3.66 (1H, dt, JH,H 10.0, JH,H
6.8, OHCHCH2), 3.81–3.99 (6H, m, 40-, 5-H, 6-H2 and
60-H2), 4.10 (1H, m, OHCHCH2), 4.17 (1H, dd, J3,4 3.4,
0
0
3-H), 4.41 (1H, d, J1 ;2 7.4, 10-H), 4.80 (1H, d, J1,2 8.0,
1-H), 5.54 (1H, dd, J2,3 9.8, 2-H), 6.06 (1H, d, 4-H) and
7.25–8.20 (20H, m, 4 · Ph); dC ꢀ1.50 (Me3Si), 18.01
(CH2Si), 19.03 (Me3C), 26.69 (Me3C), 61.70 (C-60),
63.49 (C-6), 67.41 (OCH2), 70.33 (2C, C-4 and -40),
71.08 (C-20), 71.98 (C-2), 72.92 (C-30), 73.69 (C-50),
74.02 (C-5), 79.33 (C-3), 100.60 (C-1), 104.31 (C-10),
127.63–130.16 and 132.78–135.52 (Ph) and 166.01 and
166.41 (C@O). Anal. Calcd for C47H60O13Si2ÆH2O: C,
62.23; H, 6.89. Found: C, 62.22; H, 6.86.
0
0
2
3
(1H, dt, JH,H 10.1, JH,H 7.0, OHCHCH2), 3.77–3.90
3
(3H, m, 5-H and 6-H2), 4.05 (1H, dt, JH,H 6.5,
OHCHCH2), 4.11 (1H, dd, J2,3 10.0, 3-H), 4.41–4.51
(2H, m, 50-H and 60-Ha), 4.58–4.70 (3H, m, 1-, 20-H
b
and 60-H ), 5.54 (1H, dd, J2 ;3 5.0, 30-H), 5.60 (1H, dd,
0
0
3.14. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl-(1!3)-2,4-di-O-benzoyl-6-O-(tert-butyl-
diphenylsilyl)-b-D-galactopyranoside (25)
J1,2 8.1, 2-H), 5.82 (1H, dd, J3 ;4 4.4, J4 ;5 2.9, 40-H),
0
0
0
0
5.92 (1H, d, J3,4 3.0, 4-H), 6.43 (1H, d, J1 ;2 4.6, 10-H)
and 7.20–8.25 (40H, m, 8 · Ph); dC ꢀ1.59 (Me3Si),
18.00 (CH2SiMe3), 19.09 (Me3C), 26.71 (Me3C), 61.68
(C-6), 62.20 (C-60), 66.16 (C-40), 67.27 (OCH2), 68.75
0
0
(A) Benzoyl chloride (0.1 mL, 0.86 mmol) was added to
a stirred and cooled (0 ꢁC) solution of the disaccharide

A. J. Ross et al. / Carbohydrate Research 341 (2006) 1954–1964
1963
(C-50), 68.96 (C-4), 69.97 (C-30), 71.02 (C-2), 72.77 (C-
20), 73.95 (C-3), 74.03 (C-5), 98.32 (C-10), 100.88 (C-1),
121.16 (orthoester quaternary C), 126.47 and 135.84
(orthoester Ph), 127.65–130.05 and 132.85–135.54 (rest
of Ph), and 164.95–166.64 (C@O). Also isolated was
the required b-linked disaccharide derivative 25
(32 mg, 29%). The prepared orthoester 26 (37 mg,
0.028 mmol) was combined with compound 22 (24 mg,
0.033 mmol) in dry dichloromethane (1 mL), and freshly
2-H), 5.86 (1H, d, 40-H), 6.14 (1H, d, 4-H), 6.64 (1H,
d, J1,2 3.8, 1-H), 6.81–8.08 (40H, m, 8 · Ph) and 8.41
(1H, s, NH); dC 19.10 (Me3C), 26.63 (Me3C), 61.91
(C-6), 62.13 (C-60), 67.68 (C-40), 69.56 (C-2), 69.75
(C-20), 70.40 (C-4), 71.17 (C-50), 71.54 (C-30), 72.89
(C-5), 73.78 (C-3), 90.97 (CCl3), 93.79 (C-1), 101.54
(C-10), 127.58–130.21 and 132.85–135.59 (Ph), 160.42
(C@NH) and 164.46–166.02 (C@O). Further elution
gave the 6-OH trichloroacetimidate derivative 29
(27 mg, 31%) as an amorphous solid; dH 4.25–4.38
(3H, m, 50-H, 6-Ha and 60-Ha), 4.49–4.56 (2H, m, 3-H
and 6-Hb), 4.60–4.74 (2H, m, 5-H and 60-Hb), 5.07
˚
activated molecular sieves 4 A (100 mg) were added to
the solution. The mixture was cooled (ꢀ10 ꢁC) and a
2% (v/v) solution of TMS triflate in the same solvent
(0.027 mL, 0.003 mmol) was added under argon, where-
after the mixture was stirred at room temperature for
72 h. After standard work-up (as above), FCC [tolu-
ene–ethyl acetate, (99:1)!(90:10)] produced additional
portion of the disaccharide derivative 25 (19 mg; total
yield 51 mg, 46% from acceptor 22).
(1H, d, J1 ;2 7.7, 10-H), 5.39 (1H, dd, J3 ;4 3.3, 30-H),
0
0
0
0
5.52 (1H, dd, J2 ;3 10.5, 20-H), 5.66 (1H, dd, J2,3 10.3,
2-H), 5.84 (1H, d, 40-H), 6.06 (1H, d, J3,4 3.2, 4-H),
6.66 (1H, d, J1,2 3.8, 1-H), 6.85–8.15 (30H, m, 6 · Ph)
and 8.45 (1H, s, NH).
0
0
3.15. 2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl-
(1!3)-2,4-di-O-benzoyl-6-O-(tert-butyldiphenylsilyl)-a-
D-galactopyranosyl trichloroacetimidate (28) and 2,3,4,6-
tetra-O-benzoyl-b-D-galactopyranosyl-(1!3)-2,4-di-O-
benzoyl-a-D-galactopyranosyl trichloroacetimidate (29)
Acknowledgement
This work and A.J.R. were supported by a Wellcome
Trust Grant 048564/Z/96/Z.
To a stirred and cooled (0 ꢁC) solution of compound 25
(102 mg, 0.078 mmol) in CH2Cl2 (1 mL) was added TFA
(2 mL). The mixture was left for 55 min, whereafter a
mixture of toluene (16 mL) and ethyl acetate (8 mL)
was added and the solution was concentrated. Toluene
was evaporated off from the residue 3 times (to remove
traces of TFA) to give the hemiacetal derivative 27 [dH
(inter alia) 0.93 (9H, s, Me3C), 3.64 (dd, J5,6a 6.7,
J6a,6b 10.3, 6-Ha), 3.75 (dd, J5,6b 6.3, 6-Hb), 4.23–4.40
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(m, 5-, 50-H and 60-Ha), 4.59 (dd, J3,4 3.3, 3-H), 4.68
b
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0
0
20
acetimidate 28 (73 mg, 69%) as an amorphous solid; ½aꢁD
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J3,4 3.3, 3-H), 4.71 (1H, dd, J5 ;6b 5.6, J6a0;6b 10.3, 60-H ),
0
0
0
5.08 (1H, d, J1 ;2 7.7, 10-H), 5.38 (1H, dd, J3 ;4 3.2, 30-H),
0
0
0
0
5.53 (1H, dd, J2 ;3 10.5, 20-H), 5.56 (1H, dd, J2,3 10.3,
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