Large-scale chemical and chemo-enzymatic synthesis of a spacer-containing Pk-trisaccharide

Article abstract of DOI:10.1016/j.carres.2003.12.027

The Pk-trisaccharide, linked to a solid carrier, is a potential agent for neutralization of shiga-like toxin in the gastrointestinal tract. Two approaches to the multigram-scale synthesis of a linkable Pk-trisaccharide derivative were therefore investigat

Full text of DOI:10.1016/j.carres.2003.12.027

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1141–1146
Large-scale chemical and chemo-enzymatic synthesis of a
spacer-containing Pk-trisaccharideq
Vivekanand P. Kamath,a Robert E. Yeske,a Jonathan M. Gregson,a R. Murray Ratcliffe,a,*
Ying R. Fangb and Monica M. Palcicb,*
aDepartment of Research and Process Development, SYNSORB Biotech Inc., 201, 1204 Kensington Road Calgary,
Canada AB T2E 6J7
bDepartment of Chemistry, University of Alberta, Edmonton, Canada AB T6G 2G2
Received 16 September 2003; accepted 26 December 2003
Abstract—The Pk-trisaccharide, linked to a solid carrier, is a potential agent for neutralization of shiga-like toxin in the gastro-
intestinal tract. Two approaches to the multigram-scale synthesis of a linkable Pk-trisaccharide derivative were therefore investi-
gated. A four-step chemical synthesis yielded 8-methoxycarbonyloctyl b-lactoside in 75% yield from lactose. Further conversion of
this derivative through either multistep organic synthesis or one-step enzymatic galactosylation with UDP-galactose and
recombinant a-1,4-galactosyltransferase gave the Pk-trisaccharide derivative 8-methoxycarbonyloctyl a-
D-galactopyranosyl-
(1 fi 4)-b- -galactopyranosyl-(1 fi 4)-b- -glucopyranoside in 25% and 68% overall yields from commercial lactose, respectively.
D
D
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Pk-trisaccharide; Chemoenzymatic
1. Introduction
transferases and glycosidases8 to produce the intergly-
cosidic linkages. In this communication, we address the
preparative problems raised by the recent increased
interest in the development of carbohydrate drugs for
gastrointestinal diseases such as verotoxigenic E. coli
(VTEC) infections caused by food-borne pathogens.
These pathogens release a toxin whose receptor on
mammalian cell surfaces has been identified9 as the
carbohydrate corresponding to the Pk-antigen, which
Cell-surface carbohydrates mediate cell–cell interactions
through specific binding to mammalian lectins present
on cell surfaces.1–3 Whether this recognition involves
migration of metastatic cells,4 bacterial adherence,5 or
adhesion of neutrophils to the endothelium during
inflammatory responses,6 competitive carbohydrate
ligands have therapeutic potential. However, there are
barriers that remain in the practical synthesis7 and use
of oligosaccharides for these purposes. Cost and man-
agement of multistep synthesis is cumbersome and is
often seen as a limitation for entrance of this class of
molecule into the therapeutic arena. These challenges
can often be met by the use of chemical synthesis in
combination with the use of such enzymes as glycosyl-
has the a-
D
-Gal-(1 fi 4)-b-
D
-Gal(1 fi 4)-b-
D-Glc struc-
ture present in the glycoside 1 (Scheme 1). The compli-
cations arising from E. coli infections, such as acute
kidney failure, have been correlated with the expression
of Pk-antigen in the renal glomerulus.10;11 Currently,
there is no viable anti-toxin therapy available. The Pk-
trisaccharide, when covalently linked to silica gel parti-
cles, has been shown to adsorb and neutralize the toxin
from the gut. Several chemical or enzymatic syntheses of
Pk-trisaccharide as a reducing sugar and with a variety
of aglycones have been published in the literature.12–16 In
the chemical approaches, most of the intermediates
involved were purified after each step by column
qPresented, in part, at the Fourth Carbohydrate Bioengineering
Meeting Stockholm, Sweden, 10–13 June 2001.
* Corresponding authors. E-mail addresses: murratcliffe@pathcom.ca;
monica.palcic@ualberta.ca
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.12.027

1142
V. P. Kamath et al. / Carbohydrate Research 339 (2004) 1141–1146
OBz
OH
BzO
R'
OBz
OBz
O
a
HO
HO
BzO
d
OH
BzO
O
BzO
BzO
b
O
HO
O
OBz
BzO
O
O
OH
O
BzO
R
O
O
OBz
OBz
OH
OBz
OBz
OH
2
4 R' = Br, R = H
3
c
5 R' = H, R = O(CH2)8CO2CH3
Ph
O
OH
e
HO
HO
OH
f
OBn
O
OR'
HO
O
g
O
HO
BzO
O
OBz
BzO
R'O
O
O
R
R
O
O
R
O
OH
R'O
O
OR'
OH
OR'
OBz
OBz
7 R' = H, R = O(CH2)8CO2CH3
8 R' = Bz, R = O(CH2)8CO2CH3
6
R = O(CH2)8CO2CH3
9
R = O(CH2)8CO2CH3
a
i
HO
HO
OH
BnO
BnO
OBn
O
O
h
OH
OH
O
OBn
O
HO
O
OR'
HO
O
(CH2)8CO2CH3
O
O
R'O
O
BnO
R
O
OH
O
HO
OH
R'O
OR'
OR'
10 R' = Bz, R = O(CH2)8CO2CH3
11 R' = H, R = O(CH2)8CO2CH3
1
d
Scheme 1. Reagents and conditions: (a) benzoyl chloride, pyridine; (b) HBr in acetic acid; (c) AgOTf, HO(CH2)8CO2CH3, tetramethylurea; (d)
NaOCH3; (e) PhCH(OCH3)2, TsOH; (f) AlCl3, BH3ÆEt3N, trifluoroacetic acid; (g) 2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl chloride, AgOTf,
tetramethylurea; (h) H2, Pd–C; (i) UDP-Galactose, a-1,4-Gal-T.
chromatography. In this paper, we report a practical,
large-scale chemical synthesis of the Pk-trisaccharide
glycoside 1 in 10 steps starting from lactose. The syn-
thesis involves only one column chromatographic puri-
fication while all other intermediates were purified by
either crystallization or trituration, or were taken for-
ward without any purification. For comparison, a large-
scale enzymatic synthesis of 1 was also carried out,
starting from the synthesized lactose derivative 6.
a significant improvement from earlier reported syn-
theses in literature.13 For the chemical synthesis of 1, a
benzylidene group was introduced at the 40,60 position of
compound 6, using benzaldehyde dimethyl acetal and a
catalytic amount of camphorsulfonic acid. The resulting
product 7 was directly benzoylated with benzoyl chlo-
ride in pyridine to give 8, which could be crystallized
from methanol in 85% yield. Subsequent reductive
opening of the benzylidene ring using anhydrous alu-
minum chloride, borane–triethylamine complex, and
trifluoroacetic acid gave the desired glycosyl acceptor
9,17 which was crystallized readily from methanol in 86%
2. Results and discussion
1
yield. The structure of 9 was confirmed by H NMR
Both the chemical and enzymatic synthesis of 1 utilized
the 8-methoxycarbonyloctyl b-lactoside 6, which was
synthesized in four steps from commercially available
lactose (Scheme 1). The strategy involved converting
lactose into its octabenzoate 3, using benzoyl chloride in
pyridine. Treatment of the crude octabenzoate with
hydrogen bromide in acetic acid gave the lactosyl bro-
mide 4, which was in turn used in a silver triflate-pro-
moted glycosylation of 8-methoxycarbonyloctanol to
give 5. Debenzoylation of 5 with sodium methoxide and
precipitation gave 6 in a 75% yield from lactose, which is
spectroscopy using in situ derivatization with trichlo-
roacetyl isocyanate, which resulted in a downfield shift
of the H-400 proton signal to 6.65 ppm.
Several conditions were attempted for the glycosyl-
ation of 9. The best conditions found were as follows:
Compound 9 was reacted with 2,3,4,6-tetra-O-benzyl-a-
D
-galactopyranosyl chloride (obtained by treating
commercially available ethyl 2,3,4,6-tetra-O-benzyl-1-
thio-b- -galactopyranoside with N-chlorosuccinimide
D
and tetraethylammonium chloride) using silver triflate
promotion at ambient temperature18 and tetramethyl-

V. P. Kamath et al. / Carbohydrate Research 339 (2004) 1141–1146
1143
urea as acid scavenger. The crude reaction mixture,
containing 10, was directly debenzoylated (sodium
methoxide), and the resulting mixture was then sub-
jected to column chromatography using a dichloro-
methane–methanol gradient as eluant. The desired
fractions were pooled and evaporated to give pure 11 in
75% yield from the alcohol 9. Finally, the benzyl groups
were removed by catalytic hydrogenation using 10%
Pd/C. The desired Pk-trisaccharide glycoside 1 was
obtained in a crystalline form in 80% yield (from 11).
(2, 400 g, 1.10 mol) was stirred in pyridine (4.5 kg)
at ambient temperature. Benzoyl chloride (1.7 kg,
12.3 mol) was added to the reaction vessel and the
mixture was stirred at 65 ꢁC. The progress of the reac-
tion was monitored by TLC (85:15, toluene–EtOAc;
product a anomer Rf 0.58, b anomer Rf 0.51). Excess
benzoyl chloride was decomposed by the addition of
MeOH (250 g) and the mixture was concentrated to a
syrup. The crude syrup was redissolved in CH2Cl2 and
washed with water, 5% aq HCl, and 6% aq NaHCO3.
The organic layer was dried (Na2SO4) and this solution
was carried forward to the next step.
1
The structure of 1 was verified by high field H and 13C
NMR spectroscopy. The data agreed closely with those
previously published14 for the Pk-trisaccharide bromo-
ethyl glycoside.
For comparison, we also carried out enzymatic
galactosylation of 6 on a relatively large scale (Scheme 1),
utilizing UDP-galactose and recombinant a-1,4-galac-
tosyltransferase from N. meningitidis. This gave 27 g of
3.2.2.
2,3,4,6-Tetra-O-benzoyl-b-
D-galactopyranosyl-
-glucopyranosyl bromide
(1 fi 4)-2,3,6-tri-O-benzoyl-a-
D
(benzobromolactose, 4). To the solution of 3 in CH2Cl2
was added 30% w/w HBr–acetic acid (1110 g, 4.12 mol)
and the mixture was stirred at ambient temperature.
Progress of the reaction was monitored by TLC (85:15,
toluene–EtOAc; product Rf 0.53). The organic layer was
washed with water and 6% aq NaHCO3, then evapo-
rated to a syrup. The syrup was triturated with hexane
and the supernatant liquid was decanted. The resulting
solid was dried and taken forward to the next step
without any further purification.
compound 1 in 90% yield, similar to what has been
16a
reported before
for small-scale (210 mg) synthesis of
the corresponding methyl glycoside. The largest scale
reported for Pk-trisaccharide is 188 g/L of the reducing
sugar using metabolically engineered bacteria.15a
In summary, the enzymatic route to 1 gave a 68%
overall yield from lactose, whereas the corresponding
yield of 1 by the chemical route was 25%. Both routes
were shown to be suitable for pilot-plant size batches
of material.
3.2.3. 8-Methoxycarbonyloctyl b-
D-galactopyranosyl-
(1 fi 4)-b-
lactoside, 6). Compound 4 (1300 g, 1.17 mol) dissolved
D
-glucopyranoside (8-methoxycarbonyloctyl b-
in CH2Cl2 (2 kg) was added to a reaction vessel con-
ꢀ
taining CH2Cl2 (10 kg), 4 A molecular sieves (1.5 kg),
3. Experimental
8-methoxycarbonyloctanol (263 g, 1.40 mol), tetrame-
thylurea (147 g, 1.26 mol) and silver triflate (389 g,
1.51 mol). The mixture was stirred at ambient tempera-
ture until TLC (85:15 (v/v), toluene–EtOAc, product Rf
0.48) showed complete consumption of the starting
material. The mixture was neutralized with Et3N and
then filtered through Celite. The filtrate was washed
with 5% aq NH4OH and then neutralized with AcOH.
The organic layer was evaporated to a syrup, which was
triturated with hexane. The supernatant was decanted
and the resulting solid dried, and then dissolved in
MeOH (5.5 kg), followed by the addition of 1 M anhy-
drous NaOMe (300 g, to pH > 11). The mixture was kept
at 45 ꢁC until TLC (65:35:8, CHCl3–MeOH–H2O,
product Rf 0.48) showed complete conversion. The
mixture was neutralized by the addition of AcOH to pH
5–7. The solvent was evaporated and the crude product
triturated with hexane to give a crude solid. The com-
pound was finally crystallized from MeOH–EtOAc to
furnish the desired product 6 (75% yield, 400 g). Mp
3.1. General methods
All solvents were of commercial grade and used without
purification or drying. Melting points were measured
with a Fisher–Johns instrument and are uncorrected.
NMR spectra were recorded on a Bruker AMX-300
instrument using the signals from either internal Me4Si
(d 0.00, CDCl3) or DOH (d 4.80, D2O) as references.
FTIR spectra were recorded on a Perkin–Elmer Spec-
trum-1000 instrument. Mass spectra were recorded on a
Micromass ZabSpec Hybrid Sector-TOF instrument in
positive electrospray ionization mode using a 1% solu-
tion of AcOH in 1:1 methanol–water as the liquid car-
rier. Optical rotation was measured with a Perkin–Elmer
241 polarimeter. Abbreviations used: CH2Cl2; EtOAc,
ethyl acetate; MeCN, acetonitrile; MeOH, methanol;
Et3N, triethylamine; TFA, trifluoroacetic acid; EDTA,
ethylenediaminetetraacetic acid.
1
3.2. Synthesis
158–160 ꢁC; ½aŠ )1.41ꢁ (c 0.5, H2O); H NMR (D2–O/
D
CD3OD) d 4.51 (d, 1H, J 8 Hz, H-10), 4.47 (d, 1H, J
8 Hz, H-1), 3.71 (s, 3H, –OCH3), 2.40 (t, 2H, –CH2CO),
1.20–1.78 (m, 12H, –CH2–); 13C NMR (D2O/CD3OD) d
177.7, 103.8, 103.0, 79.3, 76.1, 75.5, 75.3, 73.7, 73.4,
3.2.1.
(1 fi 4)-1,2,3,6-tetra-O-benzoyl-a,b-
(lactose octabenzoate, 3). Commercially available lactose
2,3,4,6-Tetra-O-benzoyl-b-
D
-galactopyranosyl-
D
-glucopyranose

1144
V. P. Kamath et al. / Carbohydrate Research 339 (2004) 1141–1146
71.7, 71.2, 69.4, 61.7, 60.9, 52.5, 34.4, 29.6, 29.2, 29.1,
29.0, 25.8, 25.1; IR (KBr) 3395, 2920, 2851, 1729,
was recrystallized from MeOH to give the desired
product 9 (86% yield, 292 g), ½aŠ +55.30ꢁ (c 1.6,
D
1
1438, 1086, 1064, 1037 cmÀ1
;
HRMS calcd for
CHCl3); mp 80–82 ꢁC; H NMR (CDCl3) d 7.17–8.03
C22H40O13[M+H]þ 513.2547, found 513.2536.
(m, 30H, C6H5), 4.76 (d, 1H, J 8 Hz, H-10), 4.65 (d, 1H,
J 8 Hz, H-1), 3.64 (s, 3H, O@CAOACH3), 2.21 (m, 2H,
–CH2CO), 0.92–1.54 (m, 12H, –CH2–); 13C NMR
(CDCl3) d 174.2, 165.7, 165.6, 165.3, 165.2, 165.0, 137.6,
133.2, 133.1, 129.9, 129.8, 129.77, 129.7, 129.67, 129.65,
129.4, 129.0, 128.8, 128.4, 128.38, 128.3, 128.29, 127.7,
127.4, 101.2, 100.8, 76.3, 73.5, 73.2, 72.9, 71.9, 70.0,
69.9, 67.3, 51.4, 34.0, 29.2, 28.95, 28.9, 25.6, 24.8; IR
(KBr) 3448, 3066, 2930, 1729, 1602, 1452, 1274, 1107,
3.2.4. 8-Methoxycarbonyloctyl 2,3-di-O-benzoyl-4,6-O-
benzylidene-b-
D
-galactopyranosyl-(1 fi 4)-2,3,6-tri-O-
benzoyl-b- -glucopyranoside (8). Compound 6 (340 g,
D
0.66 mol) and benzaldehyde dimethyl acetal (322 g,
2.10 mol) was stirred in MeCN (5.7 kg). The pH of the
suspension was adjusted to 3 by addition of camphor-
sulfonic acid (14 g, 60 mmol). The mixture was kept at
40 ꢁC for 16–24 h until the reaction was complete as
indicated by TLC (65:35:8, CHCl3–MeOH–H2O; prod-
uct Rf 0.77). The mixture was then neutralized by
addition of Et3N, and the solvents were evaporated to
give crude 7, which was directly taken up in pyridine
(4.7 kg) containing 4-dimethylaminopyridine (84 g,
0.68 mol). Benzoyl chloride (742 g, 5.28 mol) was added
dropwise at room temperature and the reaction was then
stirred overnight. The progress of the reaction was
monitored by TLC (85:15, toluene–EtOAc; product Rf
0.35). Excess benzoyl chloride was decomposed by
addition of MeOH (250 g). The solvent was evaporated
and the residue was dissolved in CH2Cl2 and washed
with water, 5% aq HCl, 6% aq NaHCO3, and water. The
solvents were removed by evaporation and the residue
was crystallized from MeOH to give pure 8 (85% yield,
708 cmÀ1
;
HRMS calcd for C64H66O18 [M+Na]þ
1145.4146, found 1145.4152.
3.2.6. 8-Methoxycarbonyloctyl 2,3,4,6-tetra-O-benzyl-a-
D
-galactopyranosyl-(1 fi 4)-6-O-benzyl-b-
D-galactopyr-
anosyl-(1 fi 4)-b- -glucopyranoside (11). To a solution
D
ꢀ
of 9 (312 g, 0.28 mol) in CH2Cl2 (5.9 kg) was added 4 A
molecular sieves (312 g), tetramethylurea (84 g, 0.72 mol)
and silver triflate (173 g, 0.67 mol). The mixture was
stirred for 1 h and then 2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl chloride (470 g, 0.84 mol) was added.
The reaction was allowed to proceed at room tempera-
ture for 16–24 h and the progress of the reaction was
monitored by TLC (85:15, toluene–EtOAc; product Rf
0.5). The mixture was filtered and the filtrate extracted
with aq EDTA and water. The organic layer was dried,
filtered and the solvents evaporated. The residue, con-
taining crude 10, was taken directly to the next step
without further purification. Thus, 1 M anhydrous
NaOMe (164 g) was added to a solution of 10 (234 g) in
MeOH (3.0 kg) to give a pH > 11. The mixture was
stirred at 45 ꢁC and the progress of the reaction moni-
tored by TLC (95:5, CH2Cl2–MeOH; product Rf 0.25).
The mixture was neutralized by the addition of AcOH to
pH 5–7. The solvent was evaporated and the residue was
redissolved into EtOAc and washed with water, and 6%
brine. The organic layer was evaporated and the crude
product purified by silica gel column chromatography
using a Biotage 150 flash chromatography system. The
fractions containing product were pooled together and
the solvents evaporated to furnish pure 11 in 75% yield
1
632 g), mp 164–166 ꢁC; ½aŠ +98.18ꢁ (c 1.7, CHCl3); H
D
NMR (CDCl3) d 7.10–8.07 (m, 30H, C6H5), 5.29 (s, 1H,
benzylidene), 4.85 (d, 1H, J 8 Hz, H-10), 4.66 (d, 1H, J
8 Hz, H-1), 3.65 (s, 3H, O@CAOACH3), 2.21 (m, 2H,
–CH2CO), 0.92–1.7 (m, 12H, –CH2–); 13C NMR
(CDCl3) d 174.2, 166.0, 165.6, 165.3, 165.0, 164.8, 137.4,
133.1, 133.0, 132.9, 129.9, 129.8, 129.7, 129.65, 129.6,
129.5, 128.9, 128.8, 128.7, 128.4, 128.3, 128.28, 128.26,
128.2, 127.9, 126.3, 101.4, 100.7, 100.6, 76.8, 76.5, 74.0,
73.0, 72.7, 72.6, 72.3, 69.4 66.4, 51.4, 50.8, 34.0, 29.2,
29.0, 28.9, 25.6, 24.8; IR (KBr) 3433, 3064, 2936, 1724,
1602, 1452, 1274, 1070, 708 cmÀ1; HRMS calcd for:
C64H64O18 [M+Na]þ 1143.3990, found 1143.4004.
3.2.5. 8-Methoxycarbonyloctyl (2,3-di-O-benzoyl-6-O-
benzyl-b-
D
-galactopyranosyl)-(1 fi 4)-2,3,6-tri-O-ben-
1
zoyl-b- -glucopyranoside (9). Anhydrous aluminum
D
(234 g) as foam. ½aŠ +8.75ꢁ (c 2.7, CHCl3); H NMR
D
chloride (204 g, 1.5 mol) was slowly added to THF
(3.3 kg) at 0 ꢁC. After the reaction had subsided,
BH3ÆEt3N (112 g, 1.5 mol) and compound 8 (340 g,
0.3 mol) were added. Once the mixture had stabilized at
0 ꢁC, TFA (175 g, 1.5 mol) was added dropwise. The
reaction was monitored by TLC (85:15:2, toluene–
EtOAc–MeOH; product Rf 0.63). Upon completion of
the reaction as detected by TLC, the mixture was
washed twice with 5% aq H2SO4, and then with 6% aq
NaHCO3 until the pH was above 7. The solvents were
removed by evaporation to give a solid residue, which
(CDCl3) d 7.10–8.15 (m, 25H, C6H5), 3.65 (s, 3H,
O@CAOACH3), 2.29 (t, 2H, J 8 Hz, –CH2CO), 1.20–
1.70 (m, 12H, –CH2–); 13C NMR (CDCl3) d 174.2,
138.3, 138.0, 137.7, 137.3, 128.6, 128.5, 128.4, 128.38,
128.34, 128.3, 128.2, 127.9, 127.87, 127.84, 127.8, 127.7,
127.5, 104.2, 102.5, 100.5, 84.0, 80.9, 78.7, 76.5, 76.3,
74.9, 74.5, 74.4, 74.2, 74.1, 73.9, 73.8, 73.5, 73.3, 73.1,
71.7, 71.3, 70.1, 69.2, 63.2, 51.4, 34.0, 29.5, 29.1, 29.08,
29.0, 25.8, 24.8; IR (KBr) 3448, 3054, 2930, 2305, 1732,
1454, 1265, 1097, 896, 739 cmÀ1; HRMS calcd for
C63H80O18 [M+Na]þ 1147.5242, found 1147.5241.

V. P. Kamath et al. / Carbohydrate Research 339 (2004) 1141–1146
1145
3.2.7. 8-Methoxycarbonyloctyl a-
D
-galactopyranosyl-
-glucopyrano-
Swedish Agricultural University for his critical review of
this manuscript and Dr. Warren Wakarchuk of the
National Research Council of Canada for the a-1,4-
galactosyltransferase clone.
(1 fi 4)-b- -galactopyranosyl-(1 fi 4)-b-
D
D
side (1). To a nitrogen-purged solution of 11 (120 g,
0.1 mol) in MeOH (4.9 kg) was added 10% Pd/C (70 g).
The mixture was stirred while hydrogen gas was bubbled
through the solution for 5–8 h. The progress of the
reaction was monitored by TLC (65:35:8, CHCl3–
MeOH–H2O; product Rf 0.67). Upon completion of the
reaction, the catalyst was removed by filtration and the
filtrate was concentrated. The residue was crystallized
from MeOH–1-propanol to give 1 in 80% yield (57 g),
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1
mp 156–158 ꢁC; ½aŠ +46.98ꢁ (c 1.9, H2O); H NMR
D
00 ;200
(D2O) d 4.95 (d, 1H, J1
4.0 Hz, H-100 ), 4.51 (d, 1H,
J1 ;2 7.8 Hz, H-10), 4.48 (d, 1H, J1;2 8.1 Hz, H-1), 4.35 (wt,
1H, H-500 ), 4.04 (d, 1H, H-40), 4.03 (d, 1H, H-400), 3.99
(1H, H-6a), 3.93 (1H, H-6a0), 3.91 (1H, H-300 ), 3.84 (2H,
J 10.5 Hz, H-200 , H-6b0), 3.83 (1H, H-6b), 3.79 (1H, H-
50), 3.74 (2H, H-6a00 , H-3), 3.70 (m, 1H, H-6b00 ), 3.69 (s,
3H, –OCH3), 3.65 (1H, H-4), 3.64 (dd, J 9.8 Hz, H-3),
3.58 (2H, H-5, H-20), 3.30 (dd, 1H, J 9.2 Hz, H-2), 2.39
(m, 2H, –CH2CO), 1.30–1.80 (m, 12H, –(CH2)–); 13C
NMR (D2O) d 178.1, 103.5 (1C, C10), 102.3 (1C, C-1),
100.6 (1C, C-100 ), 79.0, 77.7, 75.7, 75.1, 74.8, 73.2, 72.5,
71.2, 71.1, 71.0, 69.4, 69.2, 68.9, 60.8, 60.7, 60.4, 52.4,
34.0, 29.0, 28.7, 28.6, 28.4, 25.3, 24.6; IR (KBr) 3401,
2930, 1736, 1438, 1073, 810, 700, 545 cmÀ1; HRMS calcd
for C28H50O18 [M+Na]þ 697.2894, found 697.2903.
0
0
12. (a) Shapiro, D.; Acher, A. J. Chem. Phys. Lipids 1978, 22,
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3.2.8. Enzymatic synthesis of 1. A 1.13 L solution of 6
(22.5 g, 43.9 mmol), UDP-Gal.2Na (35.7 g, 58.5 mmol),
recombinant N. meningitidis a-1,4-galctosyltransfer-
ase15;16 (283 U) in 68 mM MOPS-NaOH buffer, pH 7.5,
containing 0.2 mg/mL bovine serum albumin, 2 mM
MnCl2 and alkaline phosphatase (3660 U), was gently
stirred in a 2 L plastic beaker at room temperature for
38 h. The progress of the reaction was monitored by
TLC (4:1:0.2, EtOAc–MeOH–H2O) and by a radio-
chemical assay19 until conversion of 6 to product 1 was
complete (product 1 Rf 0.29, substrate 6 Rf 0.47). The
mixture was filtered and the filtrate was applied to a
reverse-phase C18 column (column size: 370 mL). The
column was washed with Milli-Q water (1.5 L) and then
the product was eluted with 0.5 L HPLC-grade MeOH.
The solvent was evaporated to dryness to give a residue,
which was re-dissolved in H2O and lyophilized to dry-
ness to furnish the desired product 1 as a white powder
(26.9 g, yield 90%). The spectral data matched those of
a chemically prepared batch of 1. HRMS calcd for
C28H50O18 [M+Na]þ 697.2894, found 697.2900.
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Acknowledgements
This work was funded in part by a grant from the
Natural Sciences and Engineering Research Council of
Canada. We thank Professor Thomas Norberg of the

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