Large-scale chemical and enzymatic synthesis of a Clostridium difficile toxin A binding trisaccharide

Article abstract of DOI:10.1055/s-2004-831188

Trisaccharide 1, 8-methoxycarbonyloctyl-O-(α-D-galactopyranosyl) -(1-→3)-(β-D-galactopyranosyl)-(1-→4)-β-D-glucopyranoside, linked to a solid support can be used to remove Cloxtridium difficile toxin A from the intestinal tract. Therefore, multi-gram chem

Full text of DOI:10.1055/s-2004-831188

PAPER
2293
Large-Scale Chemical and Enzymatic Synthesis of a Clostridium difficile Toxin
A Binding Trisaccharide1
L
arge-Scale Synt
.
hesis of
a
Cl
M
ostridium difficile Tox
u
in ABinding
r
Trisacc
r
haride ay Ratcliffe,*a Vivekanand P. Kamath,a Robert E. Yeske,a Jonathan M. Gregson,a Ying R. Fang,b
Monica M. Palcic*b
a
Department of Research & Process Development, SYNSORB Biotech Inc., 201, 1204 Kensington Road, Calgary, AB T2E 6J7, Canada
E-mail: murratcliffe@pathcom.ca
b
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
E-mail: monica.palcic@ualberta.ca
Received 18 September 2003; revised 22 April 2004
This manuscript is dedicated to Professor Heinz G. Floss on the occasion of his 70th birthday.
tions. It has been shown that a simple trisaccharide 1 can
Abstract: Trisaccharide 1, 8-methoxycarbonyloctyl-O-(a-D-galac-
effectively bind the toxin when bound to a solid support.3
This paper describes a practical large-scale chemical syn-
thesis of the toxin binding trisaccharide O-(a-D-galacto-
topyranosyl)-(1→3)-(b-D-galactopyranosyl)-(1→4)-b-D-glucopyr-
anoside, linked to a solid support can be used to remove Clostridium
difficile toxin A from the intestinal tract. Therefore, multi-gram
chemical and enzymatic synthesis of the toxin binding trisaccharide pyranosyl)-(1→3)-(b-D-galactopyranosyl)-(1→4)-b-D-
1 from 8-methoxycarbonyloctyl b-lactoside disaccharide 2 were ex-
plored. Chemical conversion in 30% overall yield could be
achieved in seven steps. Alternatively a single step enzymatic con-
version of the lactoside using recombinant a(1→3)-galactosyltrans-
ferase with UDP-galactose or UDP-glucose/UDP-Glc epimerase
gave 1 in 85–90% yield.
glucopyranoside (1), in seven steps starting from 8-meth-
oxycarbonyloctanol-b-D-lactoside (2)4 (Scheme 1). The
chemical synthesis involves only one column chromatog-
raphy step, while most of the intermediates were isolated
by precipitation or taken forward without any purification.
The chemical synthesis is compared to enzymatic routes
from the lactoside employing a(1→3)-galactosyltrans-
ferase and UDP-Gal or UDP-Glc as donors.
Key words: Clostridium difficile toxin A, galactosyltransferase,
iso-globo trisaccharide, Gal(a1,3)Gal epitope
The synthesis of the toxin binding trisaccharide starts with
1.7 kg of 8-methoxycarbonyloctyl-b-D-lactoside (2),
which was synthesized in four steps from commercially
available lactose as previously described.4 Compound 2
was treated with neat 2,2¢-dimethoxypropane with a cata-
lytic amount of camphorsulfonic acid at 65 °C. Upon
completion, the reaction mixture was cooled, poured into
methanol, stirred for 10 min and then neutralized. The
Clostridium difficile associated diarrhea (CDAD) occurs
as a complication of antibiotic therapy, generally as a re-
sult of an unbalancing of the normal microbial ecology of
the gut by the antibiotics. This allows C. difficile to prolif-
erate and generate two potent exotoxins, toxin A and toxin
B that can trigger intestinal inflammation.2 Patients who
have recurrent CDAD infection have few treatment op-
OBz
R'O
HO
OBz
O
OR'
OR'
OH
O
OH
BzO
R'O
O
HO
O
a
O
c
O
OR
O
O
OR
OR
HO
O
HO
O
O
OR'
(100%)
(68% 2 steps)
OH
OBz
OR'
OH
OBz
d
3 R' = OH
4 R' = Bz
5 R' = OH
6 R' = Ac
2 R = (CH2)8CO2Me
b
(86%)
h or i
OH
HO
OBn
O
BnO
(85–90%)
O
OH
HO
OH
BnO
(72% 2 steps)
OR"
OR'
OH
O
e
OR'
O
g
HO
O
BnO
HO
O
O
R'O
(72%)
O
OR
O
OR
O
f
OH
OH
OR'
OR'
1
7 R' = Bz, R" = Ac
8 R', R" = OH
Scheme 1 Reagents and conditions: (a) DMP, p-TsOH, 65 °C; (b) benzoyl chloride, pyridine; (c) 80% AcOH, 60 °C; (d) 1. triethyl ortho-
acetate, p-TsOH, 2. 5% HCl; (e) 2,3,4,6-tetra-O-benzyl-b-thiobenzyl galactoside, CuBr2, DMF, CH2Cl2; (f) NaOMe, MeOH; (g) H2/Pd/C,
MeOH; (h) UDP-Gal, a1.3GalT; (i) UDP-Glc, UDP-Gal epimerase, a1.3GalT.
SYNTHESIS 2004, No. 14, pp 2293–2296
x
x.
x
x
.
2
0
0
4
Advanced online publication: 19.08.2004
DOI: 10.1055/s-2004-831188; Art ID: M04003SS
© Georg Thieme Verlag Stuttgart · New York

2294
R. M. Ratcliffe et al.
PAPER
crude compound was then benzoylated under standard a purity of >99.5%. This material was suitable for linking
conditions to give compound 4 in 68% yield after crystal- to solid supports to produce a toxin binding adsorbant for
lization from methanol. Removal of the isopropylidene use in clinical trials. An enzymatic synthesis was also de-
group gave the diol 5, which was then selectively vised that provided a higher yield of product with fewer
protected5 to furnish 6 in 90% yield.
steps and greatly reduced organic waste production. The
ultimate selection of an enzymatic or chemo-enzymatic
route would be determined by the amount of trisaccharide
required on an annual basis. Quantities of up to 20 kg per
year are feasible by chemical routes and will likely be lim-
ited by duration of batch run times as related to the num-
ber of steps involved. Chemo-enzymatic routes are
feasible from gram to 100 kg scale but these require ac-
cess to stable recombinant enzymes with high levels of
expression; they also require chemical methods for agly-
cone installation and access to large amounts of donor, or
efficient donor recycling systems.
The acceptor, 6 was reacted with the donor thiobenzyl
galactoside6 using copper(II) bromide and dimethylform-
amide in dichloromethane to furnish the desired trisaccha-
ride in good yield. Activation of thioglycosides using
copper(II) bromide was first reported by Ogawa et al.6b It
constitutes a mild efficient approach towards glycosyla-
tion as compared to other well established promoters such
as silver triflate, mercuric bromide. In our case we ob-
served that the reaction proceeded smoothly in presence
of copper(II) bromide alone and molecular sieves to gen-
erate the desired a-glycoside in an efficient and economi-
cal manner. The crude mixture was debenzoylated with
sodium methoxide in methanol to furnish compound 8
that was purified by flash chromatography.
All solvents were commercial grade and used without purification
or drying. Melting points were measured with Fisher John instru-
ment and are uncorrected. NMR spectra were recorded on a Bruker
AMX-300 (300 MHz) or a Varian UNITY 500 (500 MHz) spec-
trometer with either internal (CH3)4Si (d 7.26, CDCl3) or DOH (d
4.80, D2O) and 13C NMR at 75 MHz. Mass Spectra were recorded
on a Micromass ZabSpec Hybrid Sector-TOF instrument in positive
electrospray ionization mode using a 1% solution of AcOH in
MeOH–H2O (1:1) as the liquid carrier. FTIR spectra were recorded
on a Perkin-Elmer Spectrum 1000 instrument and optical rotation
was measured with a Perkin-Elmer 241 polarimeter.
The final step consisted of removal of the benzyl groups
using hydrogen and 10% Pd/C to produce the desired tox-
in binding trisaccharide methyl ester 1, which was then
crystallized from ethanol and isopropanol. In this way
0.72 kg of product was obtained in 30% overall yield from
the lactoside 2, ca. 25% from commercial lactose, with a
purity greater than 99.5% by HPLC.
Alternate chemical synthesis of a-galactosyl 1,3-trisac-
charides have been reported6a,7 in relation to their biolog-
ical properties related to human anti-Gal antibodies and
xenotransplantation. Of these the largest scale has been
0.23 g of the aminoethyl glycoside.7d Although direct
comparisons of glycosylation yields obtained here with
those previously reported in the literature6a,7 (41–90% of
the desired anomer) are difficult due to different blocking
patterns and points of characterization, it may be worth
noting that the present method gave the desired deprotect-
ed glycoside 1 (three steps, free of b-anomer) in an overall
yield of 51% from the alcohol 6.
8-Methoxycarbonyloctyl-3¢,4¢-O-isopropylidene-b-D-lactoside
(3)
8-Methoxycarbonyloctyl lactoside 2 (1.7 kg, 3.32 mol) was added
to 2,2-dimethoxypropane (15.3 kg, 156.1 mol). The resulting mix-
ture was heated to 65 °C followed by the addition of camphorsul-
fonic acid (122 g, 0.54 mol). The pH of the reaction was maintained
at £3. The reaction mixture was stirred at 65 °C until TLC (EtOAc–
MeOH, 80:20) showed that the starting material was consumed. The
mixture was cooled to r.t. followed by addition into MeOH (28.4
kg). The reaction was stirred for exactly 10 min followed by neu-
tralization with Et3N (60 g, 0.56 mol) or until the pH was 6. The
solvent was evaporated to dryness to furnish the desired product,
which was taken to the next step without any purification.
Enzymatic syntheses of 1 was carried out from lactoside 2
using a(1→3)-galactosyltransferase with two different
nucleotide donors, UDP-Gal for one step synthesis or
UDP-Glc which requires conversion to UDP-Gal with an
epimerase enzyme. The one step UDP-Gal route gave 2 g
of product with 85% yield based on acceptor. For UDP-
Glc, 62 g of product was obtained in 90% yield based on
acceptor. While UDP-Glc is considerably less expensive
than UDP-Gal, this route requires an additional enzymatic
step. Enzymatic syntheses of CD trisaccharide, most fre-
quently as a reducing sugar, have also been reported.
These syntheses encompass cell free extracts of the
a(1→3)-galactosyltransferase,8 whole cell reactions with
engineered cells containing the transferase and nucleotide
donor synthesizing genes9 and transglycosylation.10 The
largest enzymatic scales are 9 g of CD trisaccharide as a
reducing sugar and 1 g for the benzyl glycoside.8e
8-Methoxycarbonyloctyl-2,3,6,2¢,6¢-penta-O-benzoyl-3¢,4¢-O-
isopropylidene-b-D-lactoside (4)
The crude product 3 was dissolved in pyridine (20.7 kg). Benzoyl
chloride (4.21 kg, 30.07 mol) was added over a period of 15 min and
the reaction was stirred at r.t. until TLC (toluene–EtOAc, 85:15,
product Rf 0.45) showed the reaction to be complete. The reaction
mixture was quenched with MeOH (1.28 kg) and evaporated. The
residue was diluted with CH2Cl2 (37 kg) and washed with H2O (25
kg), 5% aq HCl (27 kg), 6% aq NaHCO3 (28 kg) and H2O (26 kg).
The organic layer was evaporated to a solid that was triturated with
hexanes (2 × 13.5 kg). After decanting, the residual hexane was
evaporated and the residue dissolved in MeOH (28 kg) at 55 °C.
This solution was allowed to cool to r.t. to give, after filtration and
drying, solid 4 (2.4 kg) in 68% yield (2 steps); [a]D20 +39.0 (c = 0.9,
CHCl3); mp 138–140 °C.
IR (KBr): 3063, 2934, 1731, 1451, 1267, 1027, 709, 650 cm–1.
1H NMR (CDCl3): d = 7.24–8.16 (m, 25 H, C6H5), 4.63 (d, J = 8 Hz,
1 H, H-1¢), 4.59 (d, J = 8 Hz, 1 H, H-1), 3.67 (s, 3 H, CO2CH3), 2.21
(m, 2 H, CH2CO2), 1.52 (s, 3 H, CH3), 1.25 (s, 3 H, CH3), 0.92–1.60
(m, 12 H, CH2).
In summary, this chemical synthesis provided near kg
quantities of 8-methoxycarbonyloctyl trisaccharide 1 with
Synthesis 2004, No. 14, 2293–2296 © Thieme Stuttgart · New York

PAPER
Large-Scale Synthesis of a Clostridium difficile Toxin A Binding Trisaccharide
2295
13C NMR (CDCl3): d = 174.2, 165.9, 165.8, 165.5, 165.0, 164.8,
133.3, 133.1, 133.0, 133.0, 132.8, 129.9, 129.8, 129.7, 129.5, 129.4,
129.4, 129.3, 128.6, 128.4, 128.3, 128.2, 128.0, 110.8, 101.1, 100.1,
77.1, 75.4, 73.6, 73.1, 72.9, 72.6, 71.9, 71.3, 70.2, 62.8, 62.6, 51.3,
34.0, 29.2, 28.9, 28.8, 27.4, 26.1, 25.6, 24.8.
HRMS: m/z calcd for C60H64O18 [M + Na]+: 1095.3990; found:
1095.3998.
mol), and anhyd DMF (1.7 kg,). The mixture was stirred for 30 min
at r.t., followed by addition of thiobenzyl 2,3,4,6-tetra-O-benzyl-b-
D-galactopyranoside (2.26 kg, 3.50 mol). The reaction mixture was
stirred for 16–24 h and the progress monitored by TLC (toluene–
EtOAc, 85:15, product Rf 0.44). The mixture was filtered over
Celite (2.0 kg) and the filter pad washed with CH2Cl2 (10 kg). The
filtrate was washed with 5% aq H2SO4 (36.8 kg), and 6% aq
NaHCO3 (38 kg). The organic fraction was evaporated and the
crude product 7 was dried and taken directly to the next step.
8-Methoxycarbonyloctyl-2,3,6,2¢,6¢-penta-O-benzoyl-b-D-lacto-
side (5)
8-Methoxycarbonyloctyl-O-(2,3,4,6-O-benzyl-a-D-galactopyra-
nosyl)-(1→3)-(b-D-galactopyranosyl)-(1→4)-b-D-glucopyrano-
side (8)
Compound 4 (2.40 kg, 2.23 mol) was dissolved in AcOH (18.9 kg)
and H2O (4.5 kg). The resulting mixture was heated to 60 °C until
TLC (toluene–EtOAc–MeOH, 85:15:5, product Rf 0.25) showed
complete consumption of starting material. The mixture was cooled
and diluted with CH2Cl2 (27.0 kg) and H2O (22.5 kg) and the organ-
ic layer was separated. The aqueous layer was back-extracted with
CH2Cl2 (13.0 kg). The combined organic layers were washed with
H2O (36.0 kg), 6% aq NaHCO3 (38 kg) and 5% brine (38 kg). The
organic layer was evaporated to give the desired product 5 (2.3 kg)
in quantitative yield; [a]D20 +33.8 (c = 1.1, CHCl3).
The crude blocked trisaccharide 7 was dissolved in MeOH (20.0 kg)
and 1 N anhyd NaOMe (1.95 kg) was added. The reaction mixture
was stirred at 45 °C until TLC showed complete consumption of the
starting material (CH2Cl2–MeOH, 95:5, product Rf 0.22). Upon
completion, the solution was cooled and neutralized by the addition
of glacial AcOH (0.195 kg) or until a pH 5–7 was achieved. The so-
lution was evaporated and the residue dissolved in EtOAc (22 kg)
and washed with H2O (23 kg) and the organic layer evaporated. The
resulting syrup was triturated with hexanes (2 × 13 kg) and the re-
sidual hexane was removed by evaporation. Crude compound 8 was
purified by flash chromatography (Biotage Flash 400M) sequential-
ly eluted with CH2Cl2 (100 kg), 2 times CH2Cl2–MeOH (99 kg:1.2
kg) and 4 times CH2Cl2–MeOH (99.6:1.8 kg). The fractions con-
taining purified product were pooled together and the solvents re-
moved by evaporation under vacuum to give the product 8 (1.54 kg)
in 72% yield; [a]D20 +30.2 (c = 1.0, CHCl3).
IR (KBr): 3468, 2932, 1727, 1601, 1451, 1271, 1069, 709 cm–1.
1H NMR (CDCl3): d = 7.24–8.16 (m, 25 H, C6H5), 4.60 (2 d, J = 8
Hz each, 2 H, H-1, H-1¢), 3.67 (s, 3 H, CO2CH3), 2.21 (m, 2 H,
CH2CO2), 0.92–1.62 (m, 12 H, CH2).
13C NMR (CDCl3): d = 174.1, 166.2, 165.9, 165.8, 165.0, 133.3,
133.1, 129.8, 129.6, 129.5, 129.5, 129.3, 129.1, 128.5, 128.3, 128.2,
100.9 (C-1, C-1¢, overlap), 77.2, 76.2, 73.5, 73.0, 72.9, 72.6, 72.5,
71.7, 70.1, 68.6, 62.6, 62.0, 51.4, 34.0, 29.2, 28.9, 28.8, 25.6, 24.8.
IR (KBr): 3438, 3030, 1736, 1496, 1079, 735, 697 cm–1.
HRMS: m/z calcd for C57H60O18 [M + Na]+: 1055.3677; found:
1055.3668.
1H NMR (CDCl3): d = 7.20–7.40 (m, 20 H, C6H5), 4.74 (s, 2 H,
CH2C6H5), 4.87 (d, J = 3 H, 1 H, H-1¢¢), 4.45 (d, J = 8 Hz, 1 H, H-
1¢), 4.26 (d, J = 8 Hz, 1 H, H-1), 3.64 (s, 3 H, CO2CH3), 2.27 (t, 2
H, CH2), 1.20–1.64 (m, 12 H, CH2).
8-Methoxycarbonyloctyl-4¢-O-acetyl-2,3,6,2¢,6¢-penta-O-ben-
zoyl-b-D-lactoside (6)
13C NMR (CDCl3): d = 174.2, 138.5, 138.4, 137.8, 128.4, 128.3,
128.3, 128.2, 128.1, 128.1, 127.9, 127.8, 127.6, 127.5, 127.5, 127.3,
104.5 (C-1), 102.8 (C-1¢), 96.0 (C-1¢¢), 79.2, 77.2, 75.7, 75.5, 74.8,
74.5, 74.2, 73.2, 72.5, 70.1, 69.7, 68.5, 66.5, 51.4, 34.0, 29.5, 29.2,
29.2, 29.0, 25.8, 24.8.
HRMS: m/z calcd for C56H74O18 [M + Na]+: 1057.4772; found:
1057.4777.
Compound 5 (2.3 kg, 2.22 mol) was dissolved in THF (23.8 kg).
Triethyl orthoacetate (0.770 kg, 4.75 mol) and camphorsulfonic
acid (59 g, 0.0.22 mol) were added and the reaction mixture was
stirred at r.t. The progress of the reaction was monitored by TLC
(toluene–EtOAc–MeOH, 85:15:5, product Rf 0.71). Upon comple-
tion, 5% aq HCl (23 kg) was added to the mixture and stirred for 1
h. Once TLC confirmed complete conversion to the desired product
(toluene–EtOAc–MeOH, 85:15:5, product Rf 0.40), the mixture
was diluted with CH2Cl2 (45 kg) and extracted. The organic layer
was washed with H2O (36 kg), 6% aq NaHCO3 (36 kg) and H2O (36
kg). The organic layer was evaporated and the resulting residue was
dried under vacuum to furnish compound 6 (2.14 kg) in 86% yield;
[a]D20 +17.0 (c = 1.0, CHCl3).
8-Methoxycarbonyloctyl-O-(a-D-galactopyranosyl)-(1→3)-(b-
D-galactopyranosyl)-(1→4)-b-D-glucopyranoside (1)
To a solution of 8 (1.54 kg, 1.49 mol) in MeOH (22.5 kg) was added
10% Pd/C (0.40 kg). H2 was bubbled through the reaction mixture
until the complete consumption of the starting material was ob-
served by TLC anlysis (CHCl3–MeOH–H2O, 65:35:8, product
Rf 0.39). The solution was filtered through an Alsop cellulose filter
pad (ErtelAlsop, Kingston N.Y.) followed by 0.22 mm filtration.
The filtrate was evaporated and dried to give the crude product. The
residue was dissolved in EtOH (21 kg) at 60 °C and i-PrOH (10.4
kg) added and the resulting solution was stirred and allowed to cool
to r.t. Filtration of the solid and vacuum drying gave 1 (0.721 kg) in
72% yield. The purity of this material was shown to be >95.5% by
HPLC using a YMC polyamine II, 120A, S-5 mm, 4.6 × 250mm col-
umn eluted with MeCN (75%)–(NH4)H2PO4 (25% of 10 mM aq so-
lution) at a flow rate of 1.0 mL/min. The absence of the b-anomer
was shown using the same conditions with MeCN (85)–
(NH4)H2PO4 (15% of 10 mM aq solution) as the mobile phase;
[a]D20 +67.2 (c = 0.9, H2O); mp 187–189 °C.
IR (KBr): 3485, 2934, 1730, 1601, 1451, 1371, 1270, 1070, 710
cm–1.
1H NMR (CDCl3): d = 7.30–8.14 (m, 25 H, C6H5), 4.65 (2 d, J = 8
Hz each, 2 H, H-1, H-1¢), 3.67 (s, 3 H, CO2CH3), 2.22 (m, 2 H,
CH2CO2), 2.02 (s, 3 H, O=CCH3), 0.92–1.58 (m, 12 H, CH2).
13C NMR (CDCl3): d = 174.2, 170.5, 166.3, 165.9, 165.6, 165.3,
165.1, 133.4, 133.3, 133.3, 133.0, 129.9, 129.8, 129.7, 129.6, 129.5,
129.4, 129.4, 128.9, 128.6, 128.5, 128.4, 128.2, 128.1, 101.1, 100.3,
75.6, 73.4, 72.9, 72.7, 71.6, 71.4, 71.1, 70.2, 69.3, 62.6, 51.3, 33.9,
29.2, 28.9, 28.8, 25.6, 24.8, 20.5.
HRMS: m/z calcd for C59H62O19 [M + Na]+: 1097.3783; found:
1097.3777.
8-Methoxycarbonyloctyl-O-(2,3,4,6-O-benzyl-a-D-galactopyra-
nosyl)-(1→3)-(4-O-acetyl-2¢,6¢-di-O-benzoyl-b-D-galactopyra-
nosyl)-(1→4)-2,3,6-tri-O-benzoyl-b-D-glucopyranoside (7)
To a solution of compound 6 (2.24 kg, 2.08 mol) in CH2Cl2 (31.5
kg) were added 4 Å molecular sieves (2.35 kg), CuBr2 (2.6 kg, 11.6
IR (KBr): 3388, 2928, 1737, 1436, 1151, 1075, 803 cm–1.
1H NMR (D2O): d = 5.15 (d, J1¢¢, 2¢¢ = 3.9 Hz, 1 H, H-1¢¢), 4.53 (d,
J
1¢, 2¢ = 7.8 Hz, 1 H, H-1¢), 4.48 (d, J1, 2 = 8.0 Hz, 1 H, H-1), 4.20 (m,
1 H, H-5¢¢), 4.19 (d, J = <1 Hz, 1 H, H-4¢), 4.03 (d, J = 1 Hz, 1 H,
Synthesis 2004, No. 14, 2293–2296 © Thieme Stuttgart · New York

2296
R. M. Ratcliffe et al.
PAPER
H-4¢¢), 3.99 (d, J = 2 Hz, 1 H, H-6a), 3.96 (dd, J = 3 Hz, 1 H, H-3¢¢),
3.92 (t, 1 H, OCH2), 3.87 (dd, J = 10 Hz, 1 H, H-2¢¢), 3.80 (d, J = 6
Hz, 1 H, H-6b), 3.79 (dd, J = 3 Hz, 1 H, H-3¢), 3.78 (m, 1 H, H-6a¢),
3.74 (m, 3 H, H-6b¢, H-6a¢¢, H-6b¢¢), 3.73 (m, 1 H, H-5¢) 3.69 (s, 4
H, CO2CH3, OCH2), 3.68 (dd, J = 10 Hz, 1 H, H-2¢), 3.67 (d, J = 10
Hz, 1 H, H-4), 3.65 (dd, J = 10 Hz, 1 H, H-3), 3.60 (m, 1 H, H-5),
3.31 (dd, J = 9 Hz, 1 H, H-2), 2.39 (m, 2 H, CH2CO2), 1.3–1.8 (m,
12 H, CH2).
13C NMR (D2O): d = 178.2, 103.8 (1 C, J = 162.7 Hz, C-1), 103.0
(1 C, J = 161.5 Hz C-1¢), 96.4 (1 C, J = 170.9 Hz C-1¢¢), 79.6 (C-4),
78.1 (C-3¢), 76.0 (C-5¢), 75.6 (C-5), 75.5 (C-3), 73.7 (C-2), 71.8 (C-
5¢¢), 71.6 (C-1ab), 70.5 (C-2¢), 70.2 (C-3¢¢), 70.0 (C-4¢¢), 69.1 (C-2¢¢),
65.7 (C-4¢), 61.8 (C-6¢), 61.8 (C-6¢¢), 61.1 (C-6), 52.9 (CH3), 34.5
(C8), 29.5 (C2), 28.9 (C4, C5), 28.9 (C6), 25.7 (C3), 25.0 (C7).
References
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HRMS: m/z calcd for C28H50O18 [M + H]+: 675.3075; found:
675.3084.
Enzymatic Synthesis of 1 Using a1,3-Galactosyltransferase and
UDP-Gal Donor
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W.; Palcic, M. M. In Carbohydrate Bioengineering,
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A solution of 2 (1.93 g, 3.76 mmol), UDP-Gal (3.13 g, 4.87 mmol),
a(1→3)-galactosyltransferase (26 U),11 bovine serum albumin
(20.6 mg) and alkaline phosphatase (Boehringer Mannheim) (257
U) in MOPS buffer (100 mL of 62 mM, pH 6.4), containing MnCl2
(2 mmol) was stirred gently at r.t. for 120 h. The progress of the re-
action was monitored by TLC (4:1:0.2, EtOAc–MeOH–H2O, prod-
uct Rf 0.18) and by radiochemical assay,12 which indicated
complete conversion of 2 to product 1. The product was purified
upon completion using a reverse-phase C18 column (column size: 45
mL) that was pre-equilibrated with MeOH and then with H2O. The
C18 column was washed with deionized H2O (800 mL) and the prod-
uct was eluted with HPLC grade MeOH (800 mL). The solvent was
evaporated to give a residue that was re-dissolved in H2O and lyo-
philized to dryness to give 1 as a white powder (2.15 g, 85%). The
spectral data matched those of the chemically synthesized material.
Enzymatic Synthesis of 1 Using a1,3-Galactosyltransferase and
UDP-Glc Donor
A solution of 2 (45 g, 87.8 mmol), UDP-Glc (67.3 g, 110 mmol),
a(1→3)-galactosyltransferase (500 U), UDP-Gal epimerase (3000
U),13 MOPS buffer (pH 6.4, 900 mL, 62 mmol) bovine serum albu-
min (0.2 mg/mL), MnCl2 (2 mmol) and alkaline phosphatase (6300
U) was stirred in a 2 L plastic beaker at r.t. for 112 h. The progress
of the reaction was monitored by TLC (4:1:0.2, EtOAc–MeOH–
H2O, product Rf 0.18) and by radiochemical assay. The product was
purified upon completion using a reverse-phase C18 column (col-
umn size: 840 mL) that was pre-equilibrated with MeOH and then
with H2O. The C18 column was washed with deionized H2O (3.9 L)
and the product was eluted with 1.8 L of HPLC grade MeOH. The
solvent was evaporated to give a residue that was redissolved in
H2O and lyophilized to dryness to give 1 as a white powder (62 g,
90%).
Acknowledgment
The chemical synthesis was devised at Synsorb Biotech Inc., Calga-
ry, Alberta. Funding for the enzymatic synthesis from the Natural
Sciences and Engineering Research Council of Canada is gratefully
acknowledged. We thank Dr. Peng George Wang for supplying the
UDP-Gal epimerase (Gal E) clone and Mr. Andrew Cross for con-
sultation on analytical methods.
(12) Qian, X.; Sujino, K.; Palcic, M. M.; Ratcliffe, R. M. J.
Carbohydr. Chem. 2002, 21, 911.
(13) (a) Chen, X.; Liu, Z.; Wang, J.; Fang, J.; Fan, H.; Wang, P.
G. J. Biol. Chem. 2000, 275, 31594. (b) Chen, X.; Kowal,
P.; Hamad, S.; Fan, H.; Wang, P. G. Biotechnol. Lett. 1999,
21, 131.
Synthesis 2004, No. 14, 2293–2296 © Thieme Stuttgart · New York