β-1,4-Galactosyltransferase-catalyzed synthesis of the branched tetrasaccharide repeating unit of Streptococcus pneumoniae Type 14

Article abstract of DOI:10.1016/S0968-0896(98)00095-9

A chemoenzymatic approach is described towards the branched tetrasaccharide repeating unit, β-d-Galp-(14)-β-d-Glcp-(16)-[β-d-Galp-(14)]-β-d-GlcpNAc, of Streptococcus pneumoniae type 14 in a form suitable for conjugation. The linear trisaccharide acceptor,

Full text of DOI:10.1016/S0968-0896(98)00095-9

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1605±1612
ꢀ-1,4-Galactosyltransferase-catalyzed Synthesis of the
Branched Tetrasaccharide Repeating Unit of Streptococcus
pneumoniae Type 14
Jutta Niggemann,* Johannis P. Kamerling and Johannes F. G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB Utrecht, The Netherlands
Received 17 February 1998; accepted 9 April 1998
AbstractÐA chemoenzymatic approach is described towards the branched tetrasaccharide repeating unit, b-d-Galp-
(1!4)-b-d-Glcp-(1!6)-[b-d-Galp-(1!4)]-b-d-GlcpNAc, of Streptococcus pneumoniae type 14 in a form suitable for
conjugation. The linear trisaccharide acceptor, b-d-Galp-(1!4)-b-d-Glcp-(1!6)-b-d-GlcpNAc-(1!O)CH2CH=CH2,
was synthesized by coupling of peracetylated lactosyl trichloroacetimidate to a suitably protected glucosamine building
block and subsequent deprotection steps. The obtained derivative was found to be a good acceptor for bovine milk b-
1,4-galactosyltransferase, and the resulting branched tetrasaccharide b-allyl glycoside was isolated and characterized by
NMR spectroscopy and FAB mass spectrometry. Reaction of the anomeric allyl function with cysteamine under UV-
irradiation gave the b-aminoethylthio-extended glycoside suitable for further coupling of the tetrasaccharide to protein
carriers. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
patients.6 Conjugation of carbohydrate antigens to
protein carriers represents an approach to convert T-
independent antigens to more immunogenic T-depen-
dent antigens through the addition of T-helper cell
epitopes.7,8
Pneumococcal infections like otitis media, pneumonia
and meningitis are still signi®cant causes of morbidity
and mortality throughout the world.1,2 Disease preven-
tion by vaccination is of great interest due to increasing
resistance of pneumococci to penicillin and other anti-
biotics.3 The currently used polyvalent vaccines4 Pneu-
movax 231 (Merck, Sharp & Dohme) and Pnu-Immune
23 (Lederle-Praxis) which contain capsular poly-
saccharides of 23 out of the 90 known serotypes,5 o€er
90% protection in immunocompetent adults but are due
to the poor immunogenicity of polysaccharide antigens
of limited use for people at highest risk. Polysaccharides
are thymus-independent antigens, stimulating mainly
IgM antibodies with weak memory, readily induced
tolerance and poor immune response in infants up to
the age of two, elderly people and immunode®cient
For immunological binding studies and the develop-
ment of more ecacious synthetic glycoconjugate
vaccines we have been investigating the synthesis of
well-de®ned oligosaccharide fragments corresponding
to the capsular polysaccharides of Streptococcus
pneumoniae type 2,9 6A,10±12 6B,10±15 8,16 7F,9 14,17
18C,18,19 22F,9 and 23F.9,20,21 Here, we report on a
chemoenzymatic approach towards the branched tetra-
saccharide repeating unit of S. pneumoniae type 14 con-
taining a spacer for subsequent coupling to protein
carriers.
The capsular polysaccharide of S. pneumoniae type 14,
which is identical with the asialo core antigen of type III
group B Streptococcus (GBS),22 consists of a linear
repeating trisaccharide backbone bearing a b-d-galacto-
pyranose side chain attached to the N-acetyl-b-d-glucos-
amine residue:23
Key words: Streptococcus pneumoniae type 14; chemoenzymatic
oligosaccharide synthesis; b-1,4-galactosyltransferase; tetra-
saccharide allyl glycoside.
*Corresponding author: Tel.: +31-30-2532618; Fax: +31-30-
2540980; E-mail: j.niggemann@chem.ruu.nl
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00095-9

1606
J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
presence of p-methylbenzoyl blocking groups, and sub-
sequent coupling of peracetylated lactosyl donor 4 to
the unprotected 6-position of 3 (Scheme 1).
Allyl glycoside 146 was deacetylated (!2a) and selec-
tively silylated at the primary hydroxyl group with tert-
butyldiphenylsilyl chloride in the presence of 4-di-
methylaminopyridine and triethylamine yielding 2b in
89% yield. The tert-butyldiphenylsilyl47 (TBDPS) pro-
tecting group is considerably more stable than the tert-
butyldimethylsilyl48 (TBDMS) group, and was found to
be more suitable for the subsequent introduction of p-
methylbenzoyl groups at the 3- and 4-position of 2b
a€ording the completely protected glucosamine deriva-
tive 2c (89%). Whereas selective cleavage of secondary
silyl ether groups with tetrabutylammonium ¯uoride
often gives complicated mixtures attributable to exten-
sive acyl migration,49 clean deprotection of the silyl
ether function in the presence of p-methylbenzoyl
groups could be achieved by reaction with hydrogen
chloride in methanol49 prepared in situ from acetyl
chloride and methanol (!3, 86%). The trisaccharide
derivative 5 was obtained by coupling of peracetylated
lactosyl trichloroacetimidate 417 to the glucosamine
derivative 3 in dichloromethane in the presence of tri-
methylsilyl tri¯uoromethanesulfonate (63%). Deprotec-
Although several oligosaccharide fragments related to
the capsular polysaccharide of S. pneumoniae type 14
have been synthesized,17,24±30 immunological studies on
oligosaccharide-protein conjugates have up to now
been limited to oligosaccharide fragments derived by
degradation of the isolated pneumococcal poly-
saccharide.31±33 For an easier access to well-de®ned
synthetic oligosaccharide±protein conjugates, a glyco-
syltransferase-mediated chemoenzymatic approach
towards spacer-linked branched-chain oligosaccharides
using commercially available b-1,4-galactosyltransferase
was investigated.
In biosynthetic pathways b-1,4-galactosyltransferase
(E.C. 2.4.1.22) catalyzes the transfer of b-d-galactopyr-
anosyl groups from UDP-galactose to the 4-position of
terminal N-acetyl-b-d-glucosamine residues.34 The
transferase has been extensively studied with regard to
synthetic applications and substrate speci®city,35±37 and
it has been shown that the enzyme allows various mod-
i®cations at the GlcNAc acceptor residues. Diverse
aglycons,38,39 a variety of N-acyl groups40,41 and sub-
stitutions at the 3- and 6-position are tolerated. With
respect to the 6-position, 6-O-acetyl,42,43 6-O-methyl38 and
6-O-sulfate44 groups are accepted. The enzyme, commonly
regarded as synthesizing terminal N-acetyllactosamine
sequences, can moreover use 6-O-glycosylated com-
pounds, such as a-l-Fucp-(1!6)-b-d-GlcpNAc and a-
Neu5Ac(OMe)-(2!6)-b-d-GlcpNAc, as substrates.38
tion of trisaccharide derivative
5 by deacylation/
dephthaloylation with ethylenediamine in butanol50 at
80 ꢀC followed by re-N,O-acetylation gave besides the
N-acetyl protected glycoside 6 (62%), the corresponding
N-p-methylbenzoylated derivative as by-product in 32%
yield. Therefore, trisaccharide 5 was ®rst de-p-methyl-
benzoylated and deacetylated with sodium methoxide in
methanol. Subsequent dephthaloylation with ethylene-
diamine in butanol at 80 ꢀC and re-N,O-acetylation with
acetic anhydride in pyridine gave 6 after chromatog-
raphy in 98% overall yield. After de-O-acetylation with
sodium methoxide in methanol and subsequent pur-
i®cation on Sephadex LH-20 with methanol as eluent,
the unprotected trisaccharide allyl glycoside 7 was
obtained in 94% yield.
In order to evaluate the feasibility of an enzymatic
approach to branched-chain oligosaccharide structures
of S. pneumoniae type 14 we have synthesized the 6-O-
lactose substituted b-d-GlcNAc allyl glycoside 7 and
investigated its conversion into the branched tetra-
saccharide allyl glycoside 10 by use of bovine milk b-
1,4-galactosyltransferase. Conversion of the anomeric
allyl group into an amino-derivatized spacer via light-
induced free-radical addition of cysteamine45 was inten-
ded for the subsequent coupling of the tetrasaccharide
to carrier proteins.
The rate of galactose transfer to the synthetic tri-
saccharide acceptor 7 using UDP-Gal and bovine milk
b-1,4-galactosyltransferase was tested with a coupled
enzyme assay for UDP (Scheme 2)51 by measuring
spectrophotometrically the decrease in absorbance at
340 nm.
In order to investigate both the in¯uence of the anome-
ric allyl group and the 6-O-lactosyl substitution, initial
reaction rates were determined under standard condi-
tions for N-acetylglucosamine 8 (assigned relative rate
of 100), the corresponding b-allyl glycoside 952,53 and
trisaccharide allyl glycoside 7 (Table 1). Both com-
pounds 7 and 9 were found to retain at a 10 mM
Results and Discussion
The synthetic strategy for the preparation of the pre-
cursor of compound 7, trisaccharide derivative 5,
involved the selective temporary protection of the pri-
mary HO-6 group of allyl glycoside 2a as tert-butyldi-
phenylsilyl ether, selective de-O-silylation in the

J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
1607
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH; (b) tert-butylchlorodiphenylsilane, Et3N, 4-dimethylaminopyridine, pyr-
idine; (c) MBzCl, pyridine; (d) acetyl chloride, MeOH, toluene; (e) TMSOTf, CH2Cl2; (f) NaOMe, MeOH; (g) H2NCH2CH2NH2, n-
butanol, 80 ꢀC; (h) Ac2O, pyridine; (i) NaOMe, MeOH. TBDPS=tert-butyldiphenylsilyl. MBz=p-methylbenzoyl.
acceptor concentration good acceptor activity with
relative rates of 35% and 29%, respectively. The sub-
stitution at the 6-position of b-allyl glycoside 7 by lac-
tose is thus tolerated by the enzyme and decreases the
relative rate of galactosyl transfer only slightly (83%
compared with the non-substituted glycoside). The
kinetic parameters Vmax and Km for trisaccharide 7
were determined to be 5.2 nmol/min and 5.4 mM,
respectively.
Then, the galactosyltransferase reaction with UDP-Gal
as donor and trisaccharide acceptor 7 was performed on
a preparative scale. To prevent feedback inhibition by
released UDP,54 alkaline phosphatase was added to the
incubation mixture55 permitting a near quantitative
conversion of the acceptor to tetrasaccharide 10
(Scheme 3).
The progress of the reaction could be followed by TLC
(butanol:acetic acid:water, 2:1:1) showing the dis-
appearance of 7 (Rf 0.35) and the appearance of a new
more polar product (Rf 0.25). HPLC analysis on
Lichrospher-NH2 with 70:30, acetonitrile:water as elu-
ent showed retention times of 8.9 min for trisaccharide 7
and 12.7 min for tetrasaccharide 10 at a ¯ow rate of
1.2 mL/min. The product could be isolated by removal
of excess UDP-Gal on Dowex 1X8 (Cl- form) and sub-
sequent size-exclusion chromatography on Toyopearl
Scheme 2. Reagents: (a) b-1,4-galactosyltransferase; (b) pyr-
uvate kinase; (c) l-lactate dehydogenase.

1608
J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
Table 1. Relative rates of galactopyranosyl transfer to
substituted GlcNAc residues at a 10 mM acceptor concentration
Compd
Rel. rate (%)
100
8
9
7
35
29
HW-40S in 70% yield. FABMS con®rmed the mole-
cular mass of the isolated product 10 ([M+H]+ at m/z
748), and the structure of the tetrasaccharide was con-
®rmed by 2-D 1H COSY, TOCSY and ROE spectro-
scopy (Table 2). The signal for the anomeric proton of
the new galactose residue appeared at d 4.54 as a doub-
let with a coupling constant of 8.5 Hz. The ROE con-
nectivity between Gal H-1 and GlcNAc H-4 con®rmed
the transfer of galactose to the 4-position of the non-
terminal N-acetyl-b-d-glucosamine residue.
Scheme 3. Reagents: (a) b-1,4-galactosyltransferase; (b) alka-
line phosphatase; (c) cysteamine hydrochloride.
The allyl glycoside 10 was converted into the 3-(2-amino-
ethylthio)propyl glycoside 11 by reaction with cystea-
mine45 under UV-irradiation. The radical addition of 2-
aminoethanethiol to the double bond could be followed
by TLC showing the formation of a new compound
(Rf 0.65 ! Rf 0.48). Excess of cysteamine and traces of
unreacted allyl glycoside were removed by size-exclu-
sion chromatography on Toyopearl HW-40S, and 11
was obtained as an amorphous white powder in 71%
yield.
NMR spectroscopy. The introduction of an amino-
derivatized spacer for the subsequent coupling of the
tetrasaccharide to protein carriers could be achieved by
irradiation of the anomeric allyl group in the presence of
cysteamine.
Experimental
General methods
Reactions were monitored by TLC on Silica Gel 60 F254
(Merck) with detection by UV light and/or charring
with aq 50% H2SO4 or 0.2% orcinol in 20% methanolic
H2SO4. Evaporations were conducted under reduced
pressure at <40 ꢀC. Column chromatography was per-
formed on Silica Gel 60 (0.063±0.200 mm, Merck).
Optical rotations were measured with a Perkin±Elmer
241 polarimeter. 1H NMR spectra (300 MHz) were
recorded with a Bruker AC 300 spectrometer. Two-
dimensional double-quantum ®ltered 1H-1H correlated
spectra (2-D DQF 1H-1H COSY), two-dimensional
TOCSY spectra with 100 ms and 150 ms mixing
sequences, and 2-D 1H ROE spectra (300 ms mixing
Conclusion
A combined chemoenzymatic synthesis of the tetra-
saccharide repeating unit of S. pneumoniae type 14 could
be established. The trisaccharide 7 was synthesized using
a convenient approach by combination of p-methylben-
zoyl and tert-butyldiphenylsilyl protecting groups. b-
1,4-Galactosyltransferase from bovine milk was found
to utilize the synthetic trisaccharide 7 as acceptor. The
branched-chain tetrasaccharide 10 was prepared on a
milligram scale and the transfer of galactose to the non-
terminal N-acetylglucosamine residue was con®rmed by

J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
Table 2. 1H NMR data (COSY, TOCSY, ROESY) of 10
1609
Proton (dH in ppm)
GlcNAc
4.59 (8.5)c
3.76
Glc
Gala
Galb
H-1
H-2
4.56 (8.5)
3.39 (8.5)
3.68
4.45 (7.3)
3.55 (9.8)
3.64
4.53 (8.5)
3.54 (9.8)
3.64
H-3
H-4
3.69
3.84
3.65
3.61
3.93
3.74
n.d.d
3.93
3.72
H-5
H-6a
H-6b
3.71
4.29 (11.0)
3.96 (3.7)
4.33, 4.16 (2m, each 1H)
5.9 (m, 1H)
5.32±5.25 (m, 2H)
2.03 (s, 3H)
3.98 (11.0)
3.81
n.d
n.d.
n.d.
OCH2CH=CH2
OCH2=CH=CH2
OCH2=CH=CH2
NHCOCH3
aGal-(b1-4)-Glc.
bGal-(b1-4)-GlcNAc.
c1H-1H coupling constants (Hz) were determined from a 500 MHz 1-D spectrum.
dn.d.=not determined.
sequence) were recorded at 300 K using a Bruker AMX
500 spectrometer. Chemical shifts (d) are given in ppm
relative to the signal for internal Me4Si (d 0, CDCl3) or
acetone (d 2.225, D2O). 13C NMR spectra (75.5 MHz)
were recorded with a Bruker AC 300 spectrometer; d
(ppm) values are given relative to the signal for CDCl3
(d 76.9) or internal acetone (d 31.08). Fast-atom bom-
bardment mass spectrometry (FABMS) was carried out
on a JEOL JMS SX/SX 102A four-sector mass spectro-
meter, equipped with a JEOL MS-FAB 10 D FAB gun.
Size-exclusion chromatography was performed on
Sephadex1 LH-20 (2.5Â35 cm) or Toyopearl1 HW-40S
(2.0Â60 cm), and ion-exchange chromatography on
Dowex 1X8 (200±400 mesh, Cl- form) or Dowex 1X8
(200±400 mesh, OH- form). HPLC analysis was carried
out on a Lichrospher1 NH2 (250 mm, I.D. 4.6 mm)
column, using 70:30, CH3CN:H2O as eluent at a ¯ow
rate of 1.2 mL/min. Elemental analyses were carried out
by H. Kolbe Mikroanalytisches Laboratorium (Mul-
heim an der Ruhr, Germany).
uvate, 0.3 mM NADH, 25 U pyruvate kinase, 25 U l-
lactate dehydrogenase, 0.25±10 mM acceptor and
20 mM b-1,4-galactosyltransferase. UDP-formation was
followed by monitoring the decrease in absorbance at
340 nm. Kinetic parameters Vmax and Km were deter-
mined from the initial rate data using a millimolar
1
extinction coecient of 6.22 mM 1 cm
for NADH
absorbance and rate data were ®t to the Michaelis±
Menten equation using SigmaPlot.
Allyl
methylbenzoyl-2-phthalimido-ꢀ-D-glucopyranoside (2c).
To solution of allyl 3,4,6-tri-O-acetyl-2-deoxy-2-
6-O-tert-butyldiphenylsilyl-2-deoxy-3,4-di-O-p-
a
phthalimido-b-d-glucopyranoside (1)46 (1.43 g, 3.0 mmol)
in dry MeOH (35 mL) was added 0.2 M NaOMe in dry
MeOH (3 mL). After stirring for 2 h at room tempera-
ture, the solution was neutralized with Dowex 50W-X8
(H+ form), ®ltered, and concentrated (!2a). The resi-
due was dissolved in dry CH2Cl2 (15 mL) and dry
pyridine (1 mL), and after the addition of 4-di-
methylaminopyridine (30 mg), Et3N (480 mL) and tert-
butylchlorodiphenylsilane (900 mL), the solution was
stirred overnight at room temperature. The mixture was
poured onto ice-water, extracted with CH2Cl2, and the
organic layer was washed with satd NaHCO3, then
concentrated. Column chromatography (toluene:
EtOAc, 3:2) of the residue gave amorphous 2b (1.58 g,
89%). To a solution of 2b (1.58 g, 2.69 mmol) in dry
pyridine (10 mL) was added at 0 ꢀC a solution of 4-
methylbenzoyl chloride (0.9 mL, 6.7 mmol) in dry
CH2Cl2 (15 mL). The mixture was stirred overnight at
room temperature, diluted with CH2Cl2, poured onto
ice-water, extracted with CH2Cl2, and the organic layer
was washed with satd NaHCO3, dried (MgSO4), and
concentrated. Column chromatography (30:1, tolue-
ne:EtOAc) of the residue gave 2c (1.98 g, 89%). TLC
Materials
Bovine milk b-1,4-galactosyltransferase (E.C. 2.4.1.22),
UDP-galactose, b-NADH, phospho(enol)pyruvate,
pyruvate kinase (E.C. 2.7.1.40, type III from rabbit
muscle), l-lactate dehydrogenase (E.C. 1.1.1.27, type XI
from rabbit muscle), and alkaline phosphatase (E.C.
3.1.3.1, type I from bovine intestine) were obtained from
Sigma. Toyopearl HW1-40S was supplied by Supelco.
Cysteamine hydrochloride was bought from Fluka.
Measurement of ꢀ-1,4-galactosyltransferase activity.
Initial reaction rates were determined under standard
conditions at 20 ꢀC in 500 mL 100 mM sodium cacody-
late bu€er (pH 7.5), containing 10 mM MnCl2, 50 mM
KCl, 0.2 mM UDP-galactose, 1 mM phospho(enol)pyr-
20
(10:1, toluene:EtOAc): Rf 0.49 (2b), 0.56 (2c). [a]d

1610
J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
+17ꢀ (c 1; CHCl3). H NMR (CDCl3) d 7.76±7.01 (m,
22H, Phth, 2 COC6H4CH3, and 2 Ph), 6.21 (dd, 1H,
J2,3=10.7 Hz, J3,4=9.2 Hz, H-3), 5.79 (m, 1H,
OCH2CH=CH2), 5.62 (t, 1H, J4,5=9.5 Hz, H-4), 5.58
(d, 1H, J1,2=8.4 Hz, H-1), 5.24±5.06 (m, 2H, OCH2
CH=CH2), 4.58 (dd, 1H, H-2), 4.33 and 4.12 (2m, each
1H, OCH2CH=CH2), 3.99±3.83 (m, 3H, H-5,6a,6b),
2.33 and 2.24 (2s, each 3H, 2 COC6H4CH3), 1.05 (s, 9H,
C(CH3)3); 13C NMR (CDCl3) d 165.7 and 164.9 (2
COC6H4CH3), 117.5 (OCH2CH=CH2), 97.0 (C-1),
75.0, 71.3, and 69.6 (C-3,4,5), 62.9 and 60.3 (C-6 and
OCH2CH=CH2), 54.9 (C-2), 26.5 (C(CH3)3), 21.5
(COC6H4CH3), 19.1 (C(CH3)3). Anal. calcd for
C49H49NO9Si: C, 71.42; H, 5.99 %. Found: C, 71.40; H,
6.08 %. FABMS calcd for C49H49NO9Si (M+Na):
846.3; found: 846.5.
®ltered through Celite, and concentrated. Column
chromatography (toluene:EtOAc, 3:2) of the residue
1
gave amorphous
5 (66 mg, 63%). TLC (toluene:
20
EtOAc, 2:3): Rf 0.76 (3), 0.52 (4), 0.64 (5). [a]d
5ꢀ
(c 1; CHCl3). 1H NMR (CDCl3) d 7.80±7.02 (m, 12H,
Phth and 2 COC6H4CH3), 6.19 (dd, 1H, J2,3=10.7 Hz,
J3,4=9.2 Hz, H-3), 5.76 (m, 1H, OCH2CH=CH2), 5.53
(d, 1H, J1,2=8.4 Hz, H-1), 5.37 (t, 1H, J4,5=9.5 Hz,
00
00 00
H-4), 5.34 (d, 1H, J3 ,4 =4.0 Hz, H-4 ), 5.12 and 5.08
(2m, each 1H, OCH2CH=CH2), 4.63 (d, 1H,
0
0
J1 ,2 =7.7 Hz, H-1 ), 4.51 (dd, 1H, H-2), 4.47 (d, 1H,
0
00
00 00
J1 ,2 =7.8 Hz, H-1 ), 2.34 and 2.27 (2s, each 3H, 2
COC6H4CH3), 2.14±1.94 (m, 21H, 7 Ac); 13C NMR
(CDCl3) d 170.2, 170.1, 170.0, 169.9, 169.6, 169.4, and
168.9 (7 COCH3), 165.5 and 165.2 (2 COC6H4CH3),
133.1 (OCH2CH=CH2), 117.7 (OCH2CH=CH2), 101.0
and 100.3 (C-10,100), 97.0 (C-1), 76.3, 73.9, 72.8, 72.4,
71.5, 70.9, 70.8, 70.6, 69.9, 69.0, and 66.5 (C-3, 4, 5, 20, 30,
40, 50, 200, 300, 400, 500), 69.9, 68.1, 62.1, and 60.7 (C-6, 60, 600
and OCH2CH=CH2), 54.7 (C-2), 21.5 and 21.4 (2
COC6H4CH3), 20.6±20.3 (COCH3). Anal. calcd for
C59H65NO26: C, 58.85; H, 5.44%. Found: C, 58.74; H,
5.50%. FABMS calcd for C59H65NO26 (M+Na):
1226.4; found: 1226.5.
Allyl 2-deoxy-3,4-di-O-p-methylbenzoyl-2-phthalimido-
ꢀ-D-glucopyranoside (3). To a solution of acetyl chlor-
ide (6.5 mL) in dry MeOH (100 mL) was added at room
temperature a solution of 2c (830 mg, 1.0 mmol) in dry
toluene (100 mL). The mixture was stirred overnight at
room temperature, then neutralized with Et3N, and
concentrated. The residue was dissolved in EtOAc, and
the solution washed twice with H2O, dried (MgSO4),
and concentrated. Column chromatography (5:1, then
3:1, toluene:EtOAc) of the residue gave amorphous 3
(504 mg, 86%). TLC (toluene:EtOAc, 5:1): Rf 0.72 (2c),
Allyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-(1!4)-
(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1!6)-2-acetamido-
3,4-di-O-acetyl-2-deoxy-ꢀ-D-glucopyranoside (6).
A
0.30 (3). [a]d +6ꢀ (c 1; CHCl3). H NMR (CDCl3) d
7.84±7.03 (m, 12H, Phth and 2 COC6H4CH3), 6.30 (dd,
1H, J2,3=10.8 Hz, J3,4=9.2 Hz, H-3), 5.76 (m, 1H,
OCH2CH=CH2), 5.58 (d, 1H, J1,2=8.4 Hz, H-1), 5.48
(t, 1H, J4,5=9.5 Hz, H-4), 5.16 and 5.08 (2m, each 1H,
OCH2CH=CH2), 4.52 (dd, 1H, H-2), 4.33 and 4.13
(2m, each 1H, OCH2CH=CH2), 3.89±3.71 (m, 3H, H-
5,6a,6b), 2.34 and 2.27 (2s, each 3H, 2 COC6H4CH3);
13C NMR (CDCl3) d 166.0 and 165.5 (2 COC6H4CH3),
117.5 (OCH2CH=CH2), 97.2 (C-1), 74.3, 70.4, and 69.8
(C-3,4,5), 70.1 and 61.2 (C-6 and OCH2CH=CH2), 54.7
(C-2), 21.4 (COC6H4CH3). Anal. calcd for C33H31NO9:
C, 67.68; H, 5.34%. Found: C, 67.95; H, 5.60%.
FABMS calcd for C33H31NO9 (M+Na): 608.2; found:
608.3.
solution of 5 (108 mg, 0.09 mmol) in MeOH (15 mL) was
stirred with NaOMe at pH 8±9 for 3 h at room tem-
perature. The solution was neutralized with Dowex
50W-X8 (H+ form), ®ltered, and concentrated. The
residue was dissolved in butanol (15 mL) and 1,2-di-
aminoethane (3 mL), and the mixture heated under Ar
for 24 h at 80 ꢀC, then co-concentrated with toluene.
TLC (butanol:HOAc:H2O, 2:1:1): Rf 0.35. A solution of
the residue in pyridine (30 mL) and Ac2O (15 mL) was
stirred overnight at room temperature, then co-con-
centrated with toluene. Column chromatography
(CH2Cl2:acetone, 2:1) of the residue gave amorphous 6
20
1
(86 mg, 98%). TLC (CH2Cl2:acetone, 2:1): Rf 0.44.
20
[a]d
14ꢀ (c 1; CHCl3). 1H NMR (CDCl3) d 5.86
(m, 1H, OCH2CH=CH2), 5.58 (d, J2,NH=8.7 Hz,
NHCOCH3), 4.68 and 4.58 (2d, each 1H, J=8.3 and
00
7.6 Hz, H-1,10), 4.50 (d, 1H, J1 ,2 =7.8 Hz, H-1 ), 4.37
00 00
Allyl (2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-(1!4)
-(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)-(1!6)-2-deoxy-
3,4-di-O-p-methylbenzoyl-2-phthalimido-ꢀ-D-glucopyrano-
side (5). To a solution of 3 (58 mg, 0.10 mmol) and
peracetylated lactosyl trichloroacetimidate 417 (62 mg,
0.079 mmol) in dry CH2Cl2 (0.5 mL) was added
powdered 4 A molecular sieves (150 mg), and the
suspension was stirred for 1 h at room temperature.
At 0 ꢀC trimethylsilyl tri¯uoromethanesulfonate (25 mL,
0.14 mmol) was added, and the reaction mixture
stirred at room temperature for 3 h. Then the solu-
tion was neutralized with Et3N, diluted with CH2Cl2,
and 4.32 (2m, each 1H, OCH2CH=CH2), 2.14, 2.12,
2.06, 2.05, 2.04, 2.03, 2.02, 2.01, 1.96, and 1.94 (10s,
each 3H, 10 Ac); 13C NMR (CDCl3) d 170.6±168.8
(COCH3), 133.3 (OCH2CH=CH2), 117.6 (OCH2CH
=CH2), 100.9, 100.0, and 99.3 (C-1,10,100), 76.0, 73.1,
72.7, 72.5, 72.2, 71.4, 70.8, 70.4, 69.1, 69.0, and 66.5
(C-3, 4, 5, 20, 30, 40, 50, 200, 300, 400, 500), 69.6, 67.9,
61.9, and 60.6 (C-6, 60, 600 and OCH2CH=CH2), 54.6
(C-2), 23.1 (NHCOCH3), 20.6±20.3 (COCH3).
FABMS calcd for C41H57NO25 (M+Na): 986.3;
found: 986.5.

J. Niggemann et al./Bioorg. Med. Chem. 6 (1998) 1605±1612
1611
Allyl (ꢀ-D-galactopyranosyl)-(1!4)-(ꢀ-D-glucopyranosyl)-
(1!6)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (7). To
a solution of 6 (54 mg, 0.056 mmol) in dry MeOH
(5 mL) was added NaOMe (33 mg), and the solution was
stirred overnight at room temperature. Then, H2O
(1 mL) was added, and after stirring overnight at room
temperature, the mixture was neutralized with Dowex
50W-X8 (H+ form), ®ltered, and concentrated. The
residue was puri®ed on Sephadex LH-20 with MeOH as
3-(2-Aminoethylthio)propyl (ꢀ-D-galactopyranosyl)-(1!4)-
(ꢀ-D-glucopyranosyl)-(1!6)-[(ꢀ-D-galactopyranosyl)-
(1!4)]-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside
(11).
The allyl glycoside 10 (6.2 mg, 8 mmol) was dissolved in
solution of cysteamine hydrochloride (4.5 mg,
a
0.04 mmol) in H2O (200 mL) and the mixture was irra-
diated in a quartz vial under UV-light for 3 days at
room temperature. The product was puri®ed twice by
size-exclusion chromatography on a Toyopearl HW-40S
column, eluted with 0.1 M NH4OAc at a ¯ow rate of
13 mL/h. Product-containing fractions were lyophilized
and deionized on a Dowex 1X8 (OH- form) column with
H2O as eluent to give 11 (4.7 mg, 71%). TLC (iso-
eluent to give
nol:HOAc:H2O, 2:1:1): Rf 0.35. [a]d
MeOH). 1H NMR (MeOD)
7
(30 mg, 94%). TLC (buta-
25ꢀ (c 1;
20
d
5.87 (m, 1H,
OCH2CH=CH2), 5.26 and 5.12 (2m, each 1H,
OCH2CH=CH2), 4.44, 4.42, and 4.36 (3d, each 1H,
J=8.3, 7.7 and 7.4 Hz, H-1, 10,100), 4.31 and 4.06 (2m,
each 1H, OCH2CH=CH2), 1.96 (s, 3H, NHCOCH3);
1
propanol:HOAc:H2O, 2:1:1): Rf 0.65 (10), 0.48 (11). H
NMR (D2O) d 4.60±4.5100 (m, 3H, H-1, 10, 1000), 4.46
00 00
(d, 1H, J1 ,2 =7.7 Hz, H-1 ), 4.29 (d, 1H, J5,6=11.2 Hz,
13C NMR (MeOD)
d
173.7 (NHCOCH3), 135.6
H-6), 3.01, 2.74, and 2.61 (3t, each 2H,
CH2CH2SCH2CH2NH2), 2.04 (s, 3H, NHCOCH3), 1.85
(m, 2H, CH2CH2CH2S); 13C NMR (D2O) d 180.0
(NHCOCH3), 103.8, 103.6, 103.3, and 102.2 (C-1, 10, 100,
1000), 79.3, 78.7, 76.2, 76.1, 75.6, 75.1, 74.7, 73.5, 73.4 (2
C), 73.1, 71.8 (2 C), and 69.4 (2 C) (C-3, 4, 5, 20, 30, 40, 50,
200, 300, 400, 500, 2000, 3000, 4000, 5000), 69.6, 68.3, 61.9 (2 C), and
60.9 (C-6, 60, 600, 6000 and OCH2CH=CH2), 55.9 (C-2),
39.8, 31.8, 29.4, and 28.0 (OCH2CH2CH2SCH2CH2),
23.0 (NHCOCH3).
(OCH2CH=CH2), 117.0 (OCH2CH=CH2), 105.1,
104.6, and 101.9 (C-1, 10, 100), 80.6, 77.1, 77.0, 76.5, 76.4,
75.9, 74.8, 74.7, 72.5, 72.0, and 70.3 (C-3, 4, 5, 20, 30, 40,
50, 200, 300, 400, 500), 70.9, 69.8, 62.5, and 61.9 (C-6, 60, 600
and OCH2CH=CH2), 57.3 (C-2), 22.9 (NHCOCH3).
FABMS calcd for C23H39NO16 (positive-ion mode)
(M+H): 586.6; found: 586.2. (M+Na): 608.6; found:
608.2. (negative-ion mode) (M-H): 584.6; found: 584.4.
Allyl (ꢀ-D-galactopyranosyl)-(1!4)-(ꢀ-D-glucopyranosyl)-
(1!6)-[(ꢀ-D-galactopyranosyl)-(1!4)]-2-acetamido-2-
deoxy-ꢀ-D-glucopyranoside (10). To a solution of tri-
saccharide 7 (6.3 mg, 11 mmol) in 50 mM sodium caco-
dylate bu€er pH 7.5 (600 mL) containing 5 mM MnCl2,
bovine serum albumin (0.5 mg) and NaN3 (0.02%), were
added alkaline phosphatase (4 U), UDP-galactose
(6.5 mg, 11 mmol) and b-1,4-galactosyltransferase (1 U).
The reaction mixture was incubated at 37 ꢀC. After 4 h,
another batch of UDP-galactose (4.6 mg, 8 mmol) was
added and the incubation was continued for 16 h at
37 ꢀC. Then, H2O (100 mL) was added and UDP-galac-
tose was removed using a Dowex 1X8 (Cl- form) column
with H2O as eluent. The eluate was concentrated, and
the residue applied on a Toyopearl HW-40S column,
eluted with 5 mM NH4HCO3 at a ¯ow rate of 13 mL/h.
The appropriate fractions were freeze-dried to give 10
(5.6 mg, 70%). TLC (butanol:HOAc:H2O, 2:1:1): Rf
Acknowledgements
This work was supported by the European Union pro-
grams CARENET-1 (grant ERB CHRX CT940442)
and VACNET (grant ERB BIO 4CT960158). The authors
would like to thank Dr. P. H. Kruiskamp for recording
the NMR spectra of compound 10, and Mrs A. van der
Kerk-van Hoof for recording FABMS spectra.
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