Preparative route to N-glycolylneuraminic acid phenyl 2-thioglycoside donor and synthesis of Neu5Gc-α-(2→3′)-lactosamine 3-aminopropyl glycoside

Article abstract of DOI:10.1016/S0008-6215(02)00003-4

The spacer-armed trisaccharide, Neu5Gc-α-(2→3′)-lactosamine 3-aminopropyl glycoside, was synthesized by regio- and stereoselective sialylation of the suitably protected triol acceptor, 3-trifluoroacetamidopropyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(6-O-benzyl-β-D- galactopyranosyl)-β-D-glucopyranoside, with the donor methyl [phenyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero- α,β-D-galacto-2-nonulopyranosid]onate. The donor was obtained, in turn, from methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-α, β-D-galacto-2-nonulopyranosid]onate by N-tert-butoxycarbonylation of the acetamido group followed by total N- and O-deacetylation, per-O-acetylation, subsequent Boc group removal, and N-acetoxyacetylation.

Full text of DOI:10.1016/S0008-6215(02)00003-4

Carbohydrate Research 337 (2002) 451–457
www.elsevier.com/locate/carres
Note
Preparative route to N-glycolylneuraminic acid phenyl
2-thioglycoside donor and synthesis of
Neu5Gc-a-(2“3%)-lactosamine 3-aminopropyl glycoside
Andrei A. Sherman, Olga N. Yudina, Alexander S. Shashkov, Vladimir M. Menshov,
Nikolay E. Nifantiev*
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow B-334, Russia
Received 28 August 2001; accepted 20 December 2001
Abstract
The spacer-armed trisaccharide, Neu5Gc-a-(2“3%)-lactosamine 3-aminopropyl glycoside, was synthesized by regio- and
stereoselective sialylation of the suitably protected triol acceptor, 3-trifluoroacetamidopropyl 2-acetamido-3,6-di-O-benzyl-2-de-
oxy-4-O-(6-O-benzyl-b-
tetra-O-acetyl-3,5-dideoxy-2-thio-
methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-
D
-galactopyranosyl)-b-
D
-glucopyranoside, with the donor methyl [phenyl 5-acetoxyacetamido-4,7,8,9-
-galacto-2-nonulopyranosid]onate. The donor was obtained, in turn, from
-glycero-a,b- -galacto-2-nonulopyranosid]onate by N-tert-
D
-glycero-a,b-
D
D
D
butoxycarbonylation of the acetamido group followed by total N- and O-deacetylation, per-O-acetylation, subsequent Boc group
removal, and N-acetoxyacetylation. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Sialylation; Sialyllactosamine; N-Deacetylation
Sialo-oligosaccharides play an important role in the
process of cell adhesion and act as receptors of various
lectins.1 In order to study the carbohydrate specificity
of the sialyloligosaccharides-binding lectins—selectins
and siglecs—we have synthesized various spacer-armed
oligosaccharides and neoglycoconjugates thereof, in-
cluding sialyl Lewis A and sialyl Lewis X,2 sialyl-lacto-
N-neotetraose and sialyl-lacto-N-tetraose,3 and 5-N-
glycolyl-neuraminyl-a-(2“6%)-lactosamine.4 Extending
this research work, we report herein the synthesis
of 5-N-glycolyl-neuraminyl-a-(2“3%)-lactosamine 3-
aminopropyl glycoside (1).
The known syntheses of the sialyl donors of N-
modified analogues of N-acetylneuraminic acid used
harsh conditions for the N-deacetylation of Neu5Ac
derivatives, such as heating with Me4NOH,5,6 hydrazine
hydrate,7,8 Ba(OH)2,9 or methanesulfonic acid.10,11
A
few syntheses12,13 of the Neu5Gc containing oligosac-
charides started from N-glycolylneuraminic acid, which
can be obtained by enzymatic methods14,15 but still is
poorly available.
We have shown4 that the sialyl-a-(2“6%)-lactosamine
trisaccharide derivative that had the neuraminic acid
acetamido group N-tert-butoxycarbonylated could be
efficiently transformed into the desired 5-N-glycolyl-sia-
lyl oligosaccharide by mild N,O-deacetylation, per-O-
acetylation, Boc group removal, and N-glycolylation.
In the present work, the same reaction sequence was
applied for the preparation of N-glycolyl sialyl donor 6,
which was then used for the construction of the trisac-
charide 1.
Previous applications of such a methodology include
amide-to-carbamate transformation of amino acid
* Corresponding author. Fax: +7-095-1358784.
E-mail address: nen@ioc.ac.ru (N.E. Nifantiev).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00003-4

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A.A. Sherman et al. / Carbohydrate Research 337 (2002) 451–457
Scheme 1. Reagents and conditions: (i) Boc2O, DMAP, THF, reflux, see Ref. 4 for details; (ii) MeONa, MeOH; (iii) Ac2O, Py;
,
(iv) 90% aq CF3CO2H, CH2Cl2; (v) AcOCH2C(O)Cl, Et3N, CH2Cl2, 0 °C; (vi) AgOTf, MS-4 A, CH2Cl2, rt; (vii) NIS, TfOH,
MS-3 A, MeCN, 18 h, −20 °C; (viii) H2, Pd–C, MeOH; (ix) KOH, H2O.
,
derivatives,16 cleavage of lactams,17 and N-deacetylation
of methyl 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-
ppm, and the methyl protons as a singlet at l 2.04 ppm.
In addition to the N-glycolyl derivative 6, the tri-
fluoroacetamide 7 byproduct was formed in 25% yield
during acylation of the salt 5 with acetoxyacetyl chloride.
The structure of 7 was determined by NMR spec-
troscopy. Firstly, the 1H NMR spectrum of 7 contained
four Ac groups, and the downfield location of the signals
of H-4, H-7, H-8, H-9a, and H-9b (see Table 1) indicated
that all these positions were acylated. Secondly, a single
peak at −75.9 ppm was observed in the 19F NMR
spectrum of 7, while its 13C NMR spectrum showed two
large-constant quartets at 157.7 and 115.4 ppm, and all
these signals are characteristic for the trifluoroacetic acid
amides and esters. Thirdly, the value of lNꢀH 6.92 ppm,
which is too downfield for the AcNꢀH proton† but fit for
CF3C(O)NꢀH one, excluded any 5-acetamido-tri-O-ace-
tyl-O-trifluoroacetyl structure. The formation of tri-
fluoroacetamide byproduct during acylation of the
ammonium salts that have the trifluoroacetate as a
counter-ion has been rationalized to proceed via the
highly reactive mixed anhydride intermediate.20 Com-
pounds 613 and 721 have already been prepared by
different routes, but not fully characterized.
a-D
-glucopyranoside18 and benzyl 5-acetamido-4,7,8,
9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-
D-glycero-D-
galacto-non-2-enosonate.19
The mixed imide 3 was obtained by N-tert-butoxycar-
bonylation of the thioglycoside 2 as described,4 but the
thorough purification of 3 by gel-permeation chromatog-
raphy was found unnecessary for further steps, and the
crude 3 could be used equally effective (Scheme 1).
As expected,4,17 treatment of 3 with methanolic sodium
methoxide resulted in rapid removal of the N-acetyl
group with concomitant O-deacetylation. Reacetylation
with acetic anhydride in pyridine afforded carbamate 4
in near quantitative overall yield.
Deprotection of the amino group in 4 with 90%
aqueous trifluoroacetic acid in dichloromethane gave the
salt 5, which was then immediately acylated with acetoxy-
acetyl chloride and triethylamine into the N-glycolyl
derivative 6 in 74% overall yield. Stability of the thiogly-
coside function in the former reaction is worth noting,
because it is known from peptide chemistry that Boc
group removal from sulfur-containing derivatives, e.g.,
those of methionine or cysteine, may be problematic
without a scavenger.20 In contrast, deprotection of 4 with
both 2 M HCl in diethyl ether and 4 M HCl in
1,4-dioxane gave a significantly less clean reaction. The
presence of acetoxyacetyl moiety in 6 was evident from
For the synthesis of the sialyl acceptor 12, the known
spacer-armed glucosamine derivative 104 was glycosy-
lated with galactosyl bromide 822 in the presence of
AgOTf to afford lactosamine 11 in 73% yield. In con-
trast, NIS-TfOH promoted the coupling of 10 and
1
the H NMR spectrum, where its methylene protons
appeared as two doublets (J 15.4 Hz) at l 4.62 and 4.31
† E.g., for compound 2 lNꢀH is normally about 5.2 ppm.

A.A. Sherman et al. / Carbohydrate Research 337 (2002) 451–457
453

454
A.A. Sherman et al. / Carbohydrate Research 337 (2002) 451–457
Table 2
13C NMR data (l, ppm) for carbohydrate ring carbons in compound 1, 4 (b anomer), 6 (b anomer), 7 (b anomer), 11, and 12
in the solvent specified
Compound (solvent)
Unit
C-1
C-2
56.2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
1 (D2O)
GN
Gal
Neu
102.4
103.8
175.0
168.3
167.7
167.7
101.0
99.9
73.4
76.7
40.9
37.7
37.6
37.5
78.4
71.5
79.8
73.1
78.7
79.7
68.7
69.2
69.0
68.2
68.4
75.4
68.2
76.2
68.5
77.3
75.9
76.4
52.6
50.8
49.4
50.2
75.0
72.6
74.6
73.3
61.2
62.2
73.8
72.9
73.1
72.1
68.3
67.3
68.2
68.7
70.6
101.1
88.9
89.0
89.1
54.9
70.5
54.5
71.5
55.6
69.2
69.4
68.8
68.6
73.0
73.2
73.1
73.1
63.8
62.8
62.7
62.5
4 (CDCl3)
6 (CDCl3)
7 (CDCl3)
11 (CDCl3)
GN
Gal
GN
Gal
GN
Gal
Neu
12 (1:1 CD3OD–CDCl3)
13 (CDCl3)
100.9
102.9
97.7
37.6
49.7
thiogalactoside 923 was less effective, and gave 11 in
40% yield only (details are not included in the Experi-
mental). The b-Gal linkage in 11 was confirmed (Table
1) by the value of J1,2 coupling constant 8 Hz, and no
a isomer was formed. Debenzoylation of 11 with
methanolic sodium methoxide gave 12 in high yield.
1. Experimental
General methods.—Molecular sieve Union Carbide
,
,
type 4 A (Fluka), or molecular sieve UOP type 3 A
(Fluka) were activated by heating (180 °C) under vac-
uum (0.1 mmHg) for 8 h. The catalyst used for hy-
drogenolysis was 10% Pd–C, oxide form (E.
Merck–Schuchardt). TLC was performed on Silica Gel
60 F254 (E. Merck, Darmstadt, Germany) and for the
elution of deblocked oligosaccharides solvent systems
1:2:1 n-butanol–n-propanol–0.1 M aq HCl (BPHCl),
1:1:1 MeCN–MeOH–water (AMW), and their combi-
nation were used. Optical rotation was measured with
Jasco DIP-360 digital polarimeter at 26–30 °C. NMR
spectra were recorded at 27 °C with Bruker DRX-500
Sialylation of 12 with 6 (1.5 equiv) in MeCN at
‡
,
−20 °C, promoted by NIS, TfOH, and MS-3 A pro-
ceeded slowly (18 h) but regio- and stereoselectively,
and gave the a-linked trisaccharide 13 in 40% yield.
Unreacted acceptor 12 (43%) and glycal 14 (61%) were
also isolated from the reaction mixture. Analysis of the
1H NMR spectrum of 13, in particular the values of
lH-3eqNeu (2.75 ppm) and lH-4Neu (5.06 ppm), supported
a configuration of the neuraminic acid residue.24
Deprotection of 13 by catalytic hydrogenolysis fol-
lowed by saponification with aqueous KOH afforded
the target trisaccharide 1, the structure of which was
completely confirmed by NMR spectroscopy. In partic-
ular, the presence of N-glycolyl moiety in 1 was evident
instrument (500 MHz for H and 125 MHz for 13C),
1
assignments were aided by APT, COSY, TOCSY, and
1H–13C correlation spectroscopy. tert-Butyl alcohol
was used as an internal standard for D2O solutions
(1.24 ppm (1H) and 30.29 ppm (13C)) and Me4Si for
other ones. 19F NMR spectrum was recorded using
CFCl3 (l 0.0 ppm) as an external standard. The follow-
ing notation was used to define the NMR signals: GN
for the glucosamine unit, Gal for the galactose unit,
Neu for the neuraminic acid unit, and Sp for the spacer
unit. Numeration of the atoms in spacer is Sug-1-2-3.
Mass spectrum was recorded using matrix-assisted laser
desorption ionization (MALDI)-time of flight (TOF)
on a Vision 2000 mass spectrometer.
1
from H and 13C NMR spectra, where it appeared as a
two-proton singlet (l 4.12 ppm) and a signal at 62.2
ppm (HOCH2C(O)N), respectively. The chemical shifts
(Tables 1 and 2) of H-3eqNeu (l 2.77 ppm), H-3axNeu
(l 1.80 ppm), H-4Neu (l 3.73 ppm) together with
C-1Neu (l 175 ppm) and C-6Neu (l 73.8 ppm) indi-
cated the neuraminic acid linkage to be a.24
In conclusion, the Neu5Gc donor 6 was efficiently
prepared from the readily available Neu5Ac phenyl-
thioglycoside (2) and employed for the synthesis of the
Methyl [phenyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-bu-
spacer-armed
trisaccharide
Neu5Gc-a-(2“3%)-lac-
toxycarbonylamino)-3,5-dideoxy-2-thio-
D-glycero-h,i-
tosamine 3-aminopropyl glycoside (1). The use of the
neoglycoconjugates obtained from 1 in biological stud-
ies will be published elsewhere.
D
-galacto-non-2-ulopyranosid]onate (4).—MeONa (1
M) in MeOH (0.1 mL, 0.1 mmol) was added to a
solution of 34 (211 mg, 0.31 mmol, a:b 1:2.3) in abs
MeOH (5 mL), and after stirring for 1 h at rt under Ar,
TLC (EtOAc) indicated the deacetylation to be com-
‡ Below this temperature, virtually no activation of the
donor took place (TLC data).

A.A. Sherman et al. / Carbohydrate Research 337 (2002) 451–457
455
plete. Acetic acid (0.05 mL) was added, and the reac-
tion mixture was concentrated and dried in vacuo. The
residue was dissolved in pyridine (3 mL) and Ac2O (2
mL) was added. After stirring overnight at rt MeOH (2
mL) was added, and the reaction mixture was concen-
trated with toluene. Chromatography (toluene“3:2
toluene–EtOAc) of the residue on a column of silica gel
(10 g) afforded 4 (196 mg, 98%) as white foam: Rf 0.65
(1:1 petroleum ether–EtOAc); NMR (CDCl3, data for
b anomer): 1H, see Table 1 for carbohydrate ring
protons; l 7.52–7.12 (m, 5 H, Ph), 4.69 (d, 1 H, JNꢀH,5
10.6 Hz, NꢀH), 3.55 (s, 3 H, OMe), 2.05, 2.03, 1.99,
1.91 (4 s, 3 H each, 4 Ac), 1.37 (s, 9 H, C(CH3)3); 13C,
see Table 2 for carbohydrate ring carbons; l 170.9,
170.5, 170.3, 169.7 (4 OC(O)CH3), 155.3 (NC(O)OC-
(CH3)3), 136.1 (Ph), 129.6 (Ph), 129.0 (Ph), 80.0
(OC(CH3)3), 52.5 (OCH3), 28.2 (OC(CH3)3), 21.1–20.7
(4 OC(O)CH3). Anal. Calcd for C29H39NO13S (641.68):
C, 54.28; H, 6.13; N, 2.18. Found: C, 53.91; H, 5.99; N,
2.04.
carbohydrate ring protons; l 7.51–7.29 (m, 5 H, Ph),
6.92 (d, 1 H, JNꢀH,5 9.6 Hz, NꢀH), 3.57 (s, 3 H, OMe),
2.12 (s, 6 H, 2 Ac), 2.07 (s, 3 H, Ac), 1.96 (s, 3 H, Ac);
13C, see Table 2 for carbohydrate ring carbons; l 171.7,
2
170.9, 170.2, 169.8 (4 OC(O)CH3), 157.7 (q, JC,F 38
Hz, F3CC(O)N), 136.4 (Ph), 129.8 (Ph), 128.8 (Ph),
1
115.4 (q, JC,F 275 Hz, F3CC(O)N), 52.5 (OCH3), 20.9
(OC(O)CH3), 20.6 (OC(O)CH3), 20.5 (2 OC(O)CH3);
19F, l −75.9 (CF3). Anal. Calcd for C26H30F3NO12S
(637.58): C, 48.98; H, 4.74; F, 8.94; N, 2.20; S, 5.03.
Found: C, 48.81; H, 4.86; F, 9.06; N, 2.01; S, 5.27.
3-Trifluoroacetamidopropyl O-(2,3,4-tri-O-benzoyl-6-
O-benzyl-i-
D
-galactopyranosyl)-(1“4)-2-acetamido-
3,6-di-O-benzyl-2-deoxy-i-
D
-glucopyranoside (11).—
Acceptor 104 (250 mg, 0.45 mmol) was refluxed in abs
CH2Cl2 (15 mL) under Ar until dissolution, then the
mixture was allowed to attain rt, and 823 (376 mg, 0.58
,
mmol) was added, followed by MS-4 A (1.5 g). After
stirring for 15 min, powdered AgOTf (231 mg, 0.9
mmol) was added, and stirring was continued for 18 h
at rt. Et3N (0.5 mL) was added, and the mixture was
filtered through a pad of Celite, diluted with CH2Cl2
(150 mL), washed with satd aq NaHCO3, 3 M aq
Na2S2O3, dried, and concentrated. Chromatography
(toluene“2:1 toluene–acetone) on a column of silica
gel (50 g) afforded recovered acceptor 10 (44 mg, 17%)
and lactosamine 11 (368 mg, 73%) as a white foam: Rf
0.51 (3:2 toluene–acetone); [h]D 22° (c 0.6, CH2Cl2);
Methyl [phenyl 5-acetoxyacetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-D-glycero-h,i-D-galacto-non-
2-ulopyranosid]onate (6) and methyl [phenyl 4,7,8,
9-tetra-O-acetyl-3,5-dideoxy-2-thio-5-trifluoroaceta-
mido-D-glycero-h,i-D-galacto-non-2-ulopyranosid]onate
(7).—To a solution of 4 (200 mg, 0.31 mmol) in
CH2Cl2 (3 mL), 90% aq CF3CO2H (1 mL) was added,
and after stirring for 1 h at rt, the reaction mixture was
coevaporated with toluene (6×5 mL) with the bath
temperature below 30 °C, and dried extensively in
vacuo to give the expected ammonium salt 5, Rf 0.24
(20:1:1 CH2Cl2–MeOH–AcOH). To a solution of 5 in
abs CH2Cl2 (5 mL), AcOCH2C(O)Cl (0.045 mL, 0.41
mmol) was added at 0 °C, followed by Et3N (0.11 mL,
0.81 mmol). After stirring for 20 min at 0 °C, the
reaction mixture was diluted with CH2Cl2 (50 mL),
washed with satd aq NaHCO3, dried, and coevaporated
with toluene. Chromatography (toluene“2:1 toluene–
acetone) of the residue on a column of silica gel (20 g)
afforded (in order of elution) trifluoroacetamide 7 (50
mg, 25%) and acetoxyacetamide 6 (149 mg, 74%).
Data for 6: white foam; Rf 0.37 (2:1 toluene–ace-
1
NMR (CDCl3): H, see Table 1 for carbohydrate ring
protons; l 7.96, 7.91, 7.81 (3 d, 2 H each, J 7.4 Hz,
ortho protons of 3 Bz), 7.59–7.14 (m, 24 H, Ph), 5.92
(d, 1 H, JNꢀH,2 8 Hz, NꢀH GN), 4.98 and 4.77 (2 d, 1
H each, J 11.7 Hz, PhCH2), 4.69 and 4.43 (2 d, 1 H
each, J 12 Hz, PhCH2), 4.50 and 4.36 (2 d, 1 H each, J
11.9 Hz, PhCH2), 3.78 (m, 1 H, H-1aSp), 3.47 (m, 1 H,
H-3aSp), 3.32 (m, 2 H, H-1bSp, H-3bSp) 1.96 (s, 3 H,
Ac), 1.75 (m, 2 H, H-2aSp, H-2bSp); 13C, see Table 2
for carbohydrate ring carbons; l 170.5 (NC(O)CH3),
165.4 (OC(O)Ph), 157.1 (F3CC(O)N), 138.5, 137.7,
137.5 (3 ipso Bn), 133.5–127.7 (Ph), 114.8 (F3CC(O)N),
73.6 (3 PhCH2), 67.1 (C-1Sp), 37.5 (C-3Sp), 28.1 (C-
2Sp), 23.3 (NC(O)CH3). Anal. Calcd for C61H61-
F3N2O15 (1119.2): C, 65.47; H, 5.49; N, 2.50. Found: C,
65.42; H, 5.30; N, 2.56.
1
tone); NMR (CDCl3, data for b anomer): H, see Table
1 for carbohydrate ring protons; l 7.55–7.12 (m, 5 H,
Ph), 6.16 (d, 1 H, JNꢀH,5 10.3 Hz, NꢀH), 4.62 and 4.31
(2 d, 1 H each, J 15.4 Hz, AcOCH2C(O)N), 3.59 (s, 3
H, OMe), 2.21, 2.11, 2.09, 2.04, 1.93 (5 s, 3 H each, 5
Ac); 13C, see Table 2 for carbohydrate ring carbons; l
171.1, 171.0 (2 OC(O)CH3), 170.2 (2 OC(O)CH3),
169.7 (OC(O)CH3), 168.1 (NC(O)CH2OAc), 136.2
(Ph), 129.8 (Ph), 129.1 (Ph), 52.6 (OCH3), 62.7
(NC(O)CH2OAc), 21.5-20.8 (5 OC(O)CH3). Anal.
Calcd for C28H35NO14S (641.64): C, 52.41; H, 5.50; N,
2.18. Found: C, 52.65; H, 5.61; N, 2.01.
3-Trifluoroacetamidopropyl O-(2,3,4-tri-O-benzoyl-6-
O-benzyl-i-
3,6-di-O-benzyl-2-deoxy-i-
D
-galactopyranosyl)-(1“4)-2-acetamido-
-glucopyranoside (12).—
D
MeONa (1 M) in MeOH (0.03 mL, 0.03 mmol) was
added to a solution of 11 (328 mg, 0.29 mmol) in abs
MeOH (1.5 mL). After stirring overnight at rt, the
reaction mixture was neutralized with KU-2 (H+)
cation-exchange resign, and the resign was filtered off,
and the filtrate was concentrated. Chromatography
(EtOAc“9:1 EtOAc–MeOH) of the residue on a
column of silica gel (10 g) gave 12 (213 mg, 90%) as
amorphous mass: Rf 0.48 (1:2 toluene–acetone); [h]D
Data for 7: white foam; Rf 0.6 (2:1 toluene–acetone);
1
NMR (CDCl3, data for b anomer): H, see Table 1 for

456
A.A. Sherman et al. / Carbohydrate Research 337 (2002) 451–457
1
−6° (c 0.5, MeOH); NMR (1:1 CD3OD–CDCl3): H,
see Table 1 for carbohydrate ring protons; l 7.15–6.92
(m, 15 H, Ph), 4.66 (d, 1 H, J 11.3 Hz, PhCH2),
4.40–4.07 (m, 5 H, PhCH2), 1.61 (s, 3 H, Ac), 1.56 (m,
2 H, H-2aSp, H-2bSp); 13C, see Table 2 for carbohy-
drate ring carbons; l 171.6 (NC(O)CH3), 138.5 (ipso
Bn), 137.6 (2 ipso Bn), 128.0–127.0 (Ph), 73.1 (2
PhCH2), 73.0 (PhCH2), 66.2 (C-1Sp), 36.5 (C-3Sp), 28.2
(C-2Sp), 22.2 (NC(O)CH3). Anal. Calcd for
C40H49F3N2O12 (806.8): C, 59.55; H, 6.12; N, 3.47.
Found: C, 59.41; H, 6.11; N, 3.15.
(531.5): C, 49.72; H, 5.50; N, 2.64. Found: C, 49.90; H,
5.48; N, 2.81.
3-Aminopropyl O-(3,5-dideoxy-5-hydroxyacetamido-
D
-glycero-h-
3)-O-(i- -galactopyranosyl)-(1“4)-2-acetamido-2 -
deoxy-i- -glucopyranoside (1).—To a solution of 13
D
-galacto-2-nonulopyranosylonic acid)-(2“
D
D
(56.5 mg, 0.042 mmol) in MeOH (5 mL), Pd–C (20 mg)
was added, and the mixture was degassed under vac-
uum with stirring, and then refilled with hydrogen, and
stirred under H2 overnight at rt. The reaction mixture
was filtered through Celite, the pad was washed thor-
oughly with MeOH, and the filtrate was concentrated.
The residue was treated with 0.3 M aq KOH (2 mL)
and after stirring for 1 h at rt, the pH of the solution
was carefully made neutral by the addition of AcOH,
and the reaction mixture was concentrated. Chro-
matography of the residue on a 1.5×100 cm column of
TSK HW-40S gel by elution with 0.1 M aq AcOH and
subsequent lipohilization afforded compound 1 (26 mg,
3-Trifluoroacetamidopropyl O-[methyl (5-acetoxyac-
etamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-h-
D
-galacto-2-nonulopyranosyl)onate]-(2“3)-(6-O-benzyl-
i-
D
-galactopyranosyl)-(1“4)-2-acetamido-3,6-di-O-
-glucopyranoside (13).—A mixture
benzyl-2-deoxy-i-
D
of donor 6 (78 mg, 0.12 mmol), acceptor 12 (62 mg,
0.077 mmol), and abs MeCN (8 mL) was stirred with
,
freshly activated MS-3 A (800 mg) for 5 h, then cooled
1
to −30 °C and NIS (54 mg, 0.24 mmol) was added,
followed by TfOH (0.005 mL, 0.06 mmol). After stir-
ring for 18 h at −20 °C, the reaction was terminated
by the addition of Et3N (0.1 mL), the mixture was
filtered through a pad of Celite, diluted with CH2Cl2,
washed with satd aq NaHCO3, 3 M aq Na2S2O3, dried,
and concentrated. Chromatography (toluene“2:3 tolu-
ene–acetone) of the residue on a column of silica gel
afforded, in order of elution, glycal 14 (39 mg, 61%),
trisaccharide 13 (41 mg, 40%), and recovered acceptor
12 (27 mg, 43%).
82%). According to H NMR, the material contained
\5% of contamination, which appeared as a doublet
of doublets at l 2.48 ppm (J 4.7 and J 12 Hz) and a
triplet (J 12 Hz) at l 1.71 ppm and perhaps might be
tentatively assigned to be the b-Neu linked isomer of
1.24 Purification by reverse-phase column chromatogra-
phy on a RP C18 (5 micron particle size) 1×25 cm
column (IBM Instruments, Inc.) by elution with water
gave (in order of elution) the mixed fraction (3.6 mg)
and the a anomer 1 (22 mg, 70%), HPLC analysis of
which indicated 99% purity: hygroscopic amorphous
mass; Rf 0.45 in 1:1 BPHCl–AMW; [h]D −12° (c 0.5,
Data for 13: white foam; Rf 0.25 (1:1 toluene–ace-
1
1
tone); [h]D −6° (c 1, CH2Cl2); NMR (CDCl3): H, see
water); NMR (D2O): H, see Table 1 for carbohydrate
Table 1 for carbohydrate ring protons; l 7.57 (NꢀH
Sp), 7.33–7.15 (m, 15 H, Ph), 5.62 (d, 1 H, J 7.1 Hz,
NꢀH GN), 4.90 (d, 1 H, J 11.7 Hz, PhCH2), 4.65–4.56
(m, 5 H, 3 PhCH2, AcOCH2C(O)O, H-1Gal), 4.46
(broad s, 2 H, PhCH2), 4.33 (d, 1 H, J 15.6 Hz,
AcOCH2C(O)O), 3.81 (s, 3 H, OMe), 3.47 and 3.36 (2
m, 1 H each, H-3aSp, H-3bSp), 3.01 (s, 1 H, HO-2Gal),
2.62 (s, 1 H, HO-4Gal), 2.20, 2.17, 2.15 2.04, 2.00, 1.82
(6s, 3 H each, 6 Ac), 1.80 (m, 2 H, H-2aSp, H-2bSp);
13C, see Table 2 for carbohydrate ring carbons; l 74.0,
73.4, 73.0 (3 PhCH2), 53.1 (OMe), 37.6 (C-3Sp), 28.2
(C-2Sp), 23.4 (NC(O)CH3). Anal. Calcd for
C62H78F3N2O26 (1338.3): C, 55.64; H, 5.87; N, 3.14.
Found: C, 55.24; H, 6.02; N, 2.85.
ring protons; l 4.12 (NC(O)ꢀCH2ꢀOH), 4.01 (m, 2 H,
H-6aGN, H-1aSp), 3.71 (m, H-1bSp), 3.04 (t, 2 H, J 7
Hz, H-3aSp, H-3bSp), 2.02 (s, 1 H, Ac), 1.92 (m, 2 H,
H-2aSp, H-2bSp); 13C, see Table 2 for carbohydrate
ring carbons; l 177.0 (NC(O)CH3), 175.8 (NC(O)ꢀ
CH2ꢀOH), 69.2 (C-1Sp), 62.2 (NC(O)ꢀCH2ꢀOH), 38.8
(C-3Sp), 28.2 (C-2Sp), 23.3 (NC(O)CH3). MALDI-
TOF–MS: Calcd for [C28H49N3O20 (747.29)+Na]+:
770.3. Found 770.1.
Acknowledgements
This work was supported by GlycoSense AG (Jena,
Germany).
Data for 14: white foam; Rf 0.61 (1:1 toluene–ace-
1
tone); [h]D 33° (c 1, EtOAc); NMR (CDCl3): H, see
Table 1 for carbohydrate ring protons; l 6.34 (d, 1 H,
JNꢀH,5 9 Hz, N-H), 4.56 and 4.37 (2 d, 1 H each, J 15.3
Hz, AcOCH2(O)O), 3.81 (s, 3 H, OMe), 2.15, 2.12 (2 s,
3 H each, 2 Ac), 2.06 (s, 6 H, 2 Ac), 2.03 (s, 3 H, Ac);
13C, l 170.8–169.6 (CH3C(O)O), 161.4 (C-1), 144.9
(C-2), 108.1 (C-3), 76.6, 70.8, 67.7, 67.5, 63.7
(AcOCH2(O)O), 61.8 (C-9), 52.4 (OMe), 46.3 (C-5),
20.6–20.4 (CH3C(O)O). Anal. Calcd for C22H29N2O14
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