Observations on chemical and enzymatic approaches to α-2,3-sialylated octyl β-lactoside

Article abstract of DOI:10.1016/S0040-4020(02)00265-X

A comparison of chemical and chemo-enzymatic syntheses of α-2,3-sialylated octyl lactoside is reported. The chemical approach, starting from lactose and sialic acid, required 14 steps and proceeded in 5% overall yield; poor α-selectivity in the sialylation step necessitated a difficult and low yielding separation of anomers. A chemoenzymatic approach, employing recombinant Trypanosoma cruzi trans-sialidase to effect the key sialylation reaction, required 10 steps and gave a similar overall yield. Whereas the chemo-enzymatic synthesis required only three chromatographic purification steps overall, the chemical synthesis required at least nine.

Full text of DOI:10.1016/S0040-4020(02)00265-X

TETRAHEDRON
Pergamon
Tetrahedron 58 :2002) 3207±3216
Observations on chemical and enzymatic approaches to
a-2,3-sialylated octyl b-lactoside
W. Bruce Turnbull,a Jennifer A. Harrison,a K. P. Ravindranathan Kartha,a,² Sergio Schenkmanb
and Robert A. Fielda,p,²
aSchool of Chemistry and Centre for Biomolecular Sciences, University of St Andrews, St Andrews KY16 9ST, UK
bDisciplina de Biologia Celular, Escola Paulista de Medicina, 04023-062 Sao Paulo, Brazil
Received 6 November 2001; revised 4 February 2002; accepted 28 February 2002
AbstractÐA comparison of chemical and chemo-enzymatic syntheses of a-2,3-sialylated octyl lactoside is reported. The chemical
approach, starting fromlactose and sialic acid, required 14 steps and proceeded in 5% overall yield; poor a-selectivity in the sialylation
step necessitated a dif®cult and low yielding separation of anomers. A chemoenzymatic approach, employing recombinant Trypanosoma
cruzi trans-sialidase to effect the key sialylation reaction, required 10 steps and gave a similar overall yield. Whereas the chemo-enzymatic
synthesis required only three chromatographic puri®cation steps overall, the chemical synthesis required at least nine. q 2002 Elsevier
Science Ltd. All rights reserved.
1. Background
dases,10 and so-called `glycosynthase' variants thereof,11,12
which do not rely on expensive sugar-nucleotide donor
substrates. Of late, this last point has also been addressed.13
The ef®cient production of sugar nucleotides by recombi-
nant proteins is becoming more routine, earlier problems
with heterologous expression of active glycosyltransferase
have seemingly been overcome, multi-enzyme and fusion
protein-based syntheses are effective in vitro, and metabolic
engineering has also proved effective in kilogramscale
oligosaccharide production.14±17
1.1. Introduction
Sialylated oligosaccharides are widespread in nature and
ful®ll a host of biological roles, particularly relating to
cellular adhesion and recognition events.1 There is increas-
ing interest in therapeutics based on glycobiology targets,2
and at a more fundamental level, chemical biologists have
begun to address the manipulation of cellular glycosyl-
ation.3 It is widely accepted that whilst there are a plethora
of literature procedures for the chemical synthesis of such
molecules,4 few methods are general, or routinely reliable in
inexperienced hands. The major drawback of a chemical
approach to oligosaccharide synthesis is the necessity for
extensive :and tedious) protecting group chemistry, which is
often essential to control both the regio- and stereo-
selectivity of glycosylation reactions. This invariably
leads to multi-step reaction sequences and low overall yields
often result. Recent advances in `programmable' one-pot
synthesis5 and automated solid-phase synthesis6 of oligo-
saccharides have begun to make synthesis of this complex
class of natural products tractable. The alternative is to use
the enzymatic procedures on which Nature relies for
constructing and remodelling complex glycans in vivo.7±9
Increasingly these highly precise bio-catalysts are becoming
available for use as synthetic tools, especially the glycosi-
1.2. Objective
In connection with studies on sialylated glycoconjugates we
had a need to prepare a-2,3-sialylated octyl b-lactoside, 1,
along with the de-N-acetyl derivative thereof. With only
limited prior experience of preparative sialylation pro-
cedures, we embarked on a comparison of chemical and
chemo-enzymatic approaches to our desired targets.
1.3. Previous chemical and enzymatic syntheses of a-2,3-
sialyl lactosides
Trisaccharide 1 is based on the ganglioside GM3 structure,
several syntheses of which have been reported previously.18
Both chemical and enzymatic approaches have been
employed. More generally, chemical and enzymatic
methods for O-sialylation have been extensively reviewed
by Boons and Demchenko.19
Keywords: trans-sialidase; synthesis; biotransformations.
Corresponding author. Tel.: 144-1603-593983; fax: 144-1603-592003;
p
e-mail: r.a.®eld@uea.ac.uk
Present address: Centre for Carbohydrate Chemistry, School of Chemical
Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
²
The hydrolytic neuraminidases have not proved overly
effective in reverse synthetic mode,20a although recent
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020:02)00265-X

3208
W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
OH OBn
MeO
O
AcO
OBn
O
AcO
O BnO
O
OAc
O-octyl
HO
O
SMe
AcHN
OBn
OBn
AcO
Chemical coupling
HO
O
OH
OH
OH
HO
OH
O
OH
O
HO
O
O-octyl
O
O
AcHN
OH
HO
OH
α-2,3-sialylated octyl β-lactoside (1)
Enzymatic coupling
HO
O
OH OH
OH
OH
HO
OH
O
O
HO
O
O-octyl
O
O
NO2
HO
AcHN
OH
HO
OH
Scheme 1. Key disconnections for chemical and chemo-enzymatic syntheses of a-2,3-sialylated octyl lactoside 1.
HO OH
O
AcO OAc
O
Br
AcO
OH
O
OH
HO
O
AcO
O
i
HO
AcO
O
OH
OAc
OH
OAc
(2)
lactose
ii
method 1
method 2
Me
O-octyl
AcO OAc
O
AcO OAc
O
AcO OAc
O
O
O-octyl
HO
O
OAc
AcO
O
AcO
O
AcO
O
O-octyl
AcO
AcO
AcO
O
O
O
OAc
OAc
OAc
OAc
OAc
OAc
(4)
(3)
(5)
iii
AcO OAc
AcO OAc
O
AcO
OAc
OH
separable by flash
chromatography
O AcO
O
AcO
O
O-octyl
AcO
AcO
O
O
OAc
OAc
OAc
OAc
(3)
hemi-acetal
iv
HO OH
O
OH
HO
O-octyl
HO
O
O
OH
OH
(6)
Scheme 2. Synthesis of octyl b-lactoside 6. Reagents: :i) Ac2O, HBr±AcOH :95%); :ii) AgClO4, Ag2CO3, octanol :see Section 4); :iii) TFA, H2O, CH2Cl2;
:iv) NaOMe, MeOH :99%).

W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
3209
Me
Ag
O-octyl
AcO OAc
O
AcO OAc
O
O
O-octyl
HO
O
AcO
O
AcO
O
AcO
AcO
O
O
OAc
OAc
OAc
(5) OAc
Me
O-octyl
AgO
H2O
O
HO-Oct
AcO OAc
O
AcO
O
AcO
O
OAc
OAc
Scheme 3. Mechanismfor the formation of a-glycoside 5.
Ê
work fromThiem, in particular, highlights their poten-
tial.20b,c The trans-sialidase fromthe South American
with 4 A molecular sieves prior to addition of Ag2CO3 and
glycosyl bromide 2 gave a 56% yield of the desired product,
3 [dH 4.40, 4.50 :2£1H, 2d, J1a,2aˆJ1b,2bˆ7.7, 8.0 Hz, 1a-H,
1b-H)], but also a considerable quantity of the orthoester 4
[dH 4.62 :1H, d, J1b,2bˆ8.0 Hz, 1b-H), 5.64 :1H, d,
J1a,2aˆ5.2 Hz, 1a-H)]. Although formation of the orthoester
creates a new stereogenic centre only one diastereomer was
observed, presumably the exo-isomer. The orthoester and
glycoside were not easily separated by ¯ash chromato-
graphy, but treatment of the mixture with 50% aqueous
TFA prior to chromatography, to hydrolyse the orthoester
to the hemiacetal, proved effective :Scheme 2). Standard
deacetylation of per-O-acetylated octyl lactoside 3 with
sodium methoxide in methanol gave the corresponding
unprotected glycoside 625 in near quantitative yield [dH
4.40, 4.41 :2£1H, 2d, J1a,2aˆJ1b,2bˆ7.4, 8.0 Hz, 1a-H,
1b-H)].
parasite Trypanosoma cruzi was the enzyme of choice for
our particular study. This a-2,3-speci®c enzyme is used by
these parasites to `steal' sialic acid frominfected host glyco-
conjugatesÐthe parasite does not produce sialic acid.
Acquisition of a negatively charged surface coat is thought
to be key to host cell adhesion and hence the cell invasion
process by this obligate intracellular parasite.21 Whilst
trans-sialidase usually employs sialylated oligosaccharides
as donor substrates, it can also transfer sialic acid from
simple glycosides such as the 4-methylumbelliferyl and
the readily available p-nitrophenyl sialosides.22c There
have been several reports of the use of this enzyme in
synthesis.22 A 70 kDa recombinant form of the catalytic
domain of trans-sialidase is under investigation in
our laboratory23 and was employed in studies reported
herein.
Attempts to improve on the above glycosylation chemistry
produced unexpected results. In the presence of octanol,
AgClO4 suffered some degradation during overnight drying,
giving a brown solution. Repeating the reaction without
adding octanol to the other reagents until directly prior to
the bromide addition gave no orthoester, but instead a
different by-product was identi®ed. This proved to be the
a-con®gured, 2-de-O-acetyl octyl glycoside, 5 :Scheme 2),
which was formed in 28% yield, along with 44% of the
expected b-glycoside, 3. a-Glycoside 5 presumably results
fromloss of octyl acetate fromthe orthoester and attack by
octanol to give the thermodynamically favoured a-lacto-
side, there being no participating groups present on C-2
:Scheme 3).§ Whilst the yield of a-glycoside 5 obtained in
this study is not exceptional, it is hard to conceive of a more
straightforward route to a-linked alkyl lactosides.
2. Results and discussion
2.1. Disconnections
In the current study, two routes were considered for the
synthesis of a-2,3-sialylated octyl b-d-lactoside 1, one
purely chemical, the other using a combination of chemical
and enzymatic steps :Scheme 1).
³
2.2. Octyl b-lactoside synthesis 'Scheme 2)
Lactose, being inexpensive and readily available, was
chosen as the starting material for the preparation of octyl
b-lactoside, a key intermediate for both chemical and
enzymatic elaboration. Lactose was converted in a one-pot
reaction24 to hepta-O-acetyllactosyl bromide 2 in 95% yield
with acetic anhydride and 30% HBr/AcOH. However,
subsequent glycosylation of octanol with this glycosyl
halide in the presence of silver perchlorate/silver carbonate
gave variable results. A 10% excess of AgClO4 mono-
hydrate and 2.5 equiv. of octanol, dried together overnight
2.3. Chemical synthesis approach
2.3.1. Acceptor synthesis for chemical coupling
'Scheme 4). Octyl lactoside 6 was protected with an isopro-
pylidene ketal across the 3b-,4b-hydroxyl groups to give 7
§
Attempts to effect orthoester rearrangement in the glycosylation reaction
by increasing the quantity of AgClO4 to 1.5 mol equiv. :with respect to
glycosyl bromide) had little impact on the ratio of products formed.
³
For di- and tri-saccharides, the monosaccharide residues are labelled a, b,
c fromthe reducing terminus.

3210
W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
O
OR
O
OH
O
HO
O
O-octyl
O
OH
HO OH
O
Me Me
OMe
OR'
OH
O
i
HO
O
(8),(9) R and/or R' =
O-octyl
O-octyl
HO
OH
i
OH
(6)
O
OH
OH
O
HO
O
O-octyl
O
O
OH
ii,iii
O
OR
OH
OR
O
(7)
O
RO
O
O
OR
iv,v
OR
(10) R = Ac
(11) R = Bn
HO OBn
OBn
O BnO
O
O-octyl
HO
O
OBn
OBn
(12)
Scheme 4. Syntheses of lactoside acceptor 12. Reagents: :i) :a) Pri:OMe)2, p-toluenesulfonic acid, :b) TFA±H2O, :66%); :ii) Ac2O, pyridine :quant); :iii)
NaH, BnBr, DMF :95%); :iv) AcOH:aq) :98%).
in 66% yield. This was achieved using an excess of
dimethoxypropane and catalytic p-toluenesulfonic acid,
with subsequent selective hydrolysis of any 6a- and/or
6b-based mixed methanol ketals, 8/9, with aqueous TFA.
The position of the protecting group was con®rmed by
acetylation of a small portion of the product, to give per-
O-acetate 10, which showed a clear down®eld shift of the
6a-H and 6b-H signals. Ketal 7 was then benzylated under
standard conditions, giving 11, followed by hydrolysis of
the acetonide to give the diol acceptor 12 in 90% yield
:Scheme 4). Although two hydroxyl groups are still free
in acceptor 12, glycosylation has shown to be selective for
the equatorial 3b position of galactosides over the less
reactive, axial 4b-OH.26
the glycosylation reaction is one possibility, but the partial
conversion of sialoside methyl esters to lactones under
29
Â
Zemplen deacetylation conditions has also been reported.
Hydrogenolysis of each of the deacetylated fractions using
palladium hydroxide in MeOH gave a mixture of methyl
ester:s) 18 and lactone 19. The remaining product mixture
was debenzylated in a similar fashion and then treated with
hot 1 M potassiumhydroxide solution to hydrolyse both the
ester/lactone and also the sialic acid acetamide, as trial
experiments had shown the a and b anomers to be best
resolved on TLC as the amino compounds 20a/b. The
deprotected product was obtained in 49% yield from
acceptor 12 as a 6:1, a:b mixture, whereas the published
procedure noted total anomeric stereocontrol to give the
a-sialoside in similar yield.26 Although the anomers could
be separated on a preparative TLC plate, or by repeated ¯ash
chromatography, this tedious ®nal puri®cation step gave the
desired product in only 21% yield. Reacetylation of the
amino group was achieved using 3 equiv. of acetic
anhydride in MeOH solution giving trisaccharide 1 in high
yield.
2.3.2. Donor synthesis for chemical coupling. The sialic
acid donor 13 was prepared in three steps, by a literature
procedure,27 in 74% overall yield fromsialic acid. Brie¯y,
the per-O-acetylated sialic acid methyl ester, prepared from
sialic acid by methyl esteri®cation and per-O-acetylation,
was treated with :methylthio)trimethylsilane and TMSOTf
to give the methyl thioglycoside 13 as an anomeric mixture
:a/b, 1:1).
2.4. Enzymatic approach
2.3.3. Chemical glycosylation 'Scheme 5). Glycosylation
of diol acceptor 12 was performed with 2.5 equiv. of donor
13 and N-iodosuccinimide and catalytic tri¯ic acid as
promoter, according to a literature procedure.26 It proved
convenient to deacetylate the product mixture prior to
column chromatography, which yielded a mixture of
trisaccharides 14 and 15 :Scheme 5).
2.4.1. Donor synthesis for enzymatic coupling. Whilst we
have been unable to determine reliable kinetic parameters
for the hydrolysis or transfer of sialic acid fromits p-nitro-
phenyl glycoside by trans-sialidase,23 this substrate, which
is straightforward to prepare on a gramscale, is very
effective for milligram-scale biotransformations, which
proceed with high ef®ciency when stoichiometric acceptor
is present.23 A more detailed account of this observation can
be seen in the recent work of Crout and co-workers.22c The
p-nitrophenyl sialoside glycosyl donor substrate 2130 was
prepared fromthe corresponding protected glycosyl
chloride under phase transfer conditions. Deacetylation
and saponi®cation of the resulting methyl ester gave the
required Neu5AcapNP donor 21 in 45% overall yield
fromsialic acid.
A small sample of the mixture was partially separated by
careful chromatography and the fractions reacetylated to
give an a/b mixture of the methyl ester 16 and the a-linked
3c!4b lactone 17, which was identi®ed by FAB-MS and by
comparison of NMR spectral data for analogous literature
compounds :Section 4).28,29 It is unclear at what point the
lactonisation occurred; acid-promoted lactonisation during

W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
3211
MeO
O
HO OBn
O BnO
AcO
AcO
OBn
O
OAc
O-octyl
O
SMe
HO
O
AcHN
OBn
(12)
AcO
OBn
(13)
i,ii
O
MeO
OR1
OR1
R1O OR2
R1O
(14) R1=H, R2=Bn
iii (16) R1=Ac, R2=Bn
(18) R1=R2=H
OR2
O
R2O
O
OR2
O
O
O
O-octyl
iv
iv
AcHN
R1O
OR2
+
OR2
O
OR1
OR1
O
R1O
OR2
O
(15) R1=H, R2=Bn
(17) R1=Ac, R2=Bn
R2O
O
O
iii
O
O-octyl
AcHN
O
(19) R1=R2=H
R1O
OR2
OR2
v
K
O
O
OH
HO OH
HO
OH
O
OH
O
O
HO
O
O
O-octyl
RHN
AcO
OH
OH
(20α/β) R=H
(20α) R=H
(1) R=Ac
separate
vi
Scheme 5. Chemical synthesis of a-2,3-sialylated octyl b-lactoside, 1, and its de-N-acetyl derivative, 18. Reagents: :i) NIS, TfOH, MeCN; :ii) NaOMe,
MeOH; :iii) Ac2O, pyridine; :iv) H2, Pd:OH)2, MeOH; :v) KOH:aq) :49% from 14. a/b, 6:1); :vi) Ac2O, MeOH :88%).
2.4.2. Enzymatic glycosylation 'Scheme 6). Biotrans-
formations were conducted with a 70 kDa recombinant
construct of the Trypanosoma cruzi trans-sialidase. This
construct is truncated to remove C-terminal repeats, retains
the catalytic N-terminal part of the enzyme, and is His-
tagged to aid puri®cation.31 In our hands, trans-sialidase
proved labile in both partially and fully puri®ed form, and
retained its activity far better, both in reactions and on
frozen storage, as a crude E. coli lysate. For routine use,
therefore, cleared cell lysates were used rather than puri®ed
enzyme for enzymatic syntheses.
sialidase is fully reversible. One would therefore expect
the equilibriumproduct mixture to re¯ect the relative
concentrations of acceptors present. After incubating octyl
lactoside, 6, and 1.5 equiv. of the donor 21 with the trans-
sialidase overnight at 308C, TLC indicated that the reaction
had gone about half way to completion. Although the
reaction can be pushed further towards completion by
increasing the concentration of the donor, excess unreacted
donor can complicate product isolation. Reactions were
therefore not generally pushed to complete consumption
of the acceptor, which could be recovered during puri®ca-
tion in a straightforward manner. It proved more convenient
to stop the reaction after a relatively short reaction time and
The trans-glycosylation reaction catalysed by the trans-
Na
O
O
OH
OH
OH OH
OH
HO
O
HO
O
O-octyl
O
O
NO2
HO
AcHN
O
OH
HO
OH
(21)
(6)
i
HO
OH
O
OH
OH
OH
HO
OH
O
O
HO
O
O-octyl
O
O
AcHN
OH
HO
OH
(1)
Scheme 6. Enzymatic synthesis of a-2,3-sialylated octyl b-lactoside, 1. Reagents: :i) trans-sialidase, phosphate buffer pH 7 :42%).

3212
W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
separate the product fromthe reactants. For these particular
compounds :i.e. octyl glycosides) preparative reverse phase
chromatography on C-18 silica proved to be highly ef®cient
in this regard, and far less time consuming than gel ®ltration
on Biogel resin, which has proved useful for reducing sugar
products.22c
immersion in a dilute ethanolic solution of sulfuric acid. An
orcinol dip, prepared by the careful addition of concentrated
sulfuric acid :20 cm3) to an ice cold solution of 3,5-
dihydroxytoluene :360 mg) in EtOH :150 cm3) and water
:10 cm3), was used to detect deprotected compounds by
charring. Flash chromatography was performed with silica
gel 60 :Fluka). Reverse phase chromatography was typi-
cally performed on 15 g C-18 silica gel 100 :Fluka) eluting
with H2O :80 cm3) and then MeOH±H2O, 1:3 :20 cm3), 1:1
:20 cm3), 3:1 :20 cm3) and ®nally with MeOH :20 cm3). For
smaller scale work C-18 Sep-pak cartridges :500 mg;
Waters) were used and eluted with 2 cm3 of each of the
above eluents.
Having removed most of the protein by precipitation with
ethanol, the supernatant was concentrated and redissolved in
water before applying to a C-18 column. The excess
Neu5AcapNP and virtually all of the yellow p-nitrophenol
were eluted from the column with approximately four
column volumes of water. The product and acceptor were
then eluted using a fairly crude stepwise methanol gradient;
the sialylated product eluted at approximately 25±50%
MeOH, the unreacted acceptor eluting with 75% MeOH.
Unreacted lactoside acceptor was recovered in suitably
pure formfor direct re-use. Using the method outlined
10 mg batches of the tri-saccharide 1 could be prepared
and puri®ed within 24 h fromthe easily accessible donor
and acceptor 21 and 6, respectively. Treatment of
During work-up, organic solutions were washed two or
three times with equal volumes of each of the aqueous
solutions listed. Standard work-up A involved washing
organic solutions successively with water, saturated
NaHCO3 solution and water; standard work-up B involved
washing organic solutions successively with 1 M HCl solu-
tion, saturated NaHCO3 solution and water. All such organic
solutions were then dried over anhydrous Na2SO4, ®ltered
and concentrated in vacuo.
acetamido-trisaccharide
1
with hot 1 M potassium
hydroxide solution gave the amino-trisaccharide 20a in
near quantitative yield, so potentially providing access to
a range of N-acyl trisaccharide derivatives.
Optical rotations were measured at the sodium D-line and at
ambient temperature, with an Optical Activity AA-1000
polarimeter. [a]D values are given in units of
1021 deg cm2 g21. Melting points were measured using a
Gallenkamp melting point apparatus and are uncorrected.
IR spectra were recorded as thin ®lms on NaCl plates
using a Perkin±Elmer 1710 IRFT spectrometer. Fast atom
bombardment :FAB) mass spectra were recorded on a
Fisons VG Autospec spectrometer using a 3-nitrobenzyl
alcohol matrix. Electrospray mass spectra :ES-MS) were
recorded on a Fisons VG Biotech electrospray mass spectro-
3. Conclusions
The chemo-enzymatic synthesis of a-2,3-sialylated octyl
lactoside 1 was completed in 10 steps from lactose and sialic
acid, in an overall yield of 10% The longest linear sequence
was seven steps fromsialic acid. This should be compared
with the purely chemical approach, which was four steps
longer and gave an overall yield of only 5%. These two
yields would have been almost identical had it not been
for the poor a-selectivity of the chemical sialylation,
which necessitated a dif®cult and low yielding chromato-
graphic separation of anomers. It is also worth noting that
the chemo-enzymatic synthesis required only three puri®ca-
tions by silica gel or reverse phase chromatography,
whereas the chemical synthesis required at least nine
chromatographic puri®cation steps. A crude extrapolation
fromthe preparations described herein suggests that a 10L
culture of E. coli expressing trans-sialidase would provide
suf®cient enzyme for synthesis on a tens of grams scale. We
conclude that trans-sialidase is a useful tool in oligo-
saccharide synthesis, allowing rapid and technically
undemanding stereo- and regio-selective synthesis of
a-2,3-linked sialosides in overall yields in excess of those
routinely attainable by chemical synthesis, and on scales
ranging from radiochemical to at least tens of grams.
1
meter. Unless stated otherwise, H NMR and 13C NMR
spectra were recorded on a Varian Gemini 2000 spectro-
meter at 300 and 75 MHz, respectively. H NMR spectra
1
were referenced to the following internal standards: CHCl3,
dH 7.26 in CDCl3; CD2HOD, dH 3.31 in CD3OD; CH3OH,
dH 3.43 in D2O. 13C NMR spectra were referenced to the
following internal standards: CDCl3, dC 76.9 in CDCl3;
CD3OD, dC 49.15 in CD3OD; CH3OH dC 49.9 in D2O.
J-values are given in Hz. Only partial NMR data are
given for some compounds; other spectral features were in
accord with the proposed structures but are not suf®ciently
resolved at 300 MHz to be useful. Signals for CF3 were not
recorded in 13C NMR experiments of compounds containing
the tri¯uoromethanesulfonyl group.
4.2. Synthetic procedures
The following compounds were prepared essentially as
described in the literature: 2,3,4,6-tetra-O-acetyl-b-d-galac-
topyranosyl-:1!4)-2,3,6-tri-O-acetyl-a-d-glucopyranosyl
bromide 2;24 methyl :methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-d-glycero-a/b-d-galacto-2-
nonulopyranosid)onate, 13;27 4-nitrophenyl O-:sodium5-
acetamido-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulo-
pyranosylonate), 21.30,33
4. Experimental
4.1. General
All reagents and solvents were dried prior to use according
to standard methods.32 Commercial reagents were used
without further puri®cation unless stated. Analytical TLC
was performed on silica gel 60-F254 :Merck or Whatman)
with detection by ¯uorescence and/or by charring following
4.2.1. Octyl 2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl-
'1!4)-2,3,6-tri-O-acetyl-b-d-glucopyranoside, 3. Method

W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
3213
one. A suspension of silver perchlorate :4.96 g, 22 mmol),
4 A molecular sieves :10 g), octanol :4.56 cm , 50 mmol)
4.2.2. Octyl b-d-galactopyranosyl-'1!4)-b-d-gluco-
pyranoside, 6.25 A solution of both compound 3 :8.35 g,
11.2 mmol) and sodium metal :100 mg, 4.3 mmol) in dry
MeOH :80 cm3) was stirred for 1 h at roomtemperature.
More MeOH :120 cm3) was added to dissolve the resulting
precipitate and the solution was neutralised with Amberlite
IRC-50 :H1) resin :1 g). Filtration and concentration gave
the fully deprotected compound 6 as an amorphous white
solid :5.02 g, 99%); [a]D ˆ29.2 :c 1 in MeOH);
dH[CD3OD±D2O :1:1)] 0.86 :3H, t, Jˆ6.9 Hz,
C7H14CH3), 1.22±1.40 :10H, m, :CH2)5 CH3), 1.62 :2H,
m, OCH2CH2), 4.40, 4.41 :2£1H, 2d, J1a,2aˆJ1b,2bˆ7.4,
8.0 Hz, 1a-H, 1b-H); dC[CD3OD±D2O :1:1)] 13.7, 23.0,
26.4, 29.7, 29.9, 30.1, 32.0, 61.3, 61.8, 69.7, 70.3, 71.9,
74.15, 74.2, 75.9, 75.9, 76.5, 80.1, 103.6, 104.5; ES-MS
:2ve): m/z 453 :M2H)2, 489.5 :M1Cl)2 :C20H38O11
requires m/z 454). NMR data consistent with the literature.25
3
Ê
and dry CH2Cl2 :110 cm3) was stirred overnight at room
temperature in a tin foil covered ¯ask in order to exclude
light. Silver carbonate :8.27 g, 30 mmol) was added,
followed by glycosyl bromide 2 :13.99 g, 20 mmol) and
the mixture was stirred at room temperature for 24 h. The
mixture was then ®ltered through Celite and aqueous TFA
:1 cm3; 50% v/v solution) was added to the ®ltrate. After
stirring for 2 h, the solution was subjected to standard work-
up A. Concentration gave a syrup. Flash chromatography
:silica gel; hexane±EtOAc, 3:1!1:1) gave the desired
acetylated octyl lactoside 3 as an amorphous mass :8.45 g,
56%), [a]Dˆ27.7 :c 1.0, CHCl3) :Found: C, 54.77; H, 7.26.
C34H52O18 requires C, 54.54; H, 7.00%); dH:CDCl3) 0.86
:3H, t, Jˆ6.9 Hz, C7H14CH3), 1.25 :10H, m, :CH2)5 CH3),
1.55 :2H, m, OCH2CH2), 1.94±2.14 :7£3H, 7s, 7£AcO),
3.45 :1H, m, OCH2), 4.40, 4.50 :2£1H, 2d,
J1a,2aˆJ1b,2bˆ7.7, 8.0 Hz, 1a-H, 1b-H), 4.86 :1H, dd,
J1a,2aˆJ2a,3aˆ9.3 Hz, 2a-H), 4.94 :1H, dd, J2b,3bˆ10.2 Hz,
J3b,4bˆ3.2 Hz, 3b-H), 5.09 :1H, dd, J1b,2bˆJ2b,3b, 2b-H),
5.17 :1H, t, J2a,3aˆJ3a,4aˆ9.3 Hz, 3a-H), 5.33 :1H, d,
J3b,4bˆ3.3 Hz, 4b-H); dC:CDCl3) 14.1, 20.5, 20.7 :4),
20.9, 22.7, 25.85, 29.3 :2), 29.45 :2), 31.8, 60.9, 62.2,
66.8, 69.3, 70.3, 70.8, 71.1, 71.9, 72.7, 73.0, 76.5, 100.8,
101.2, 169.35, 169.85, 170.1, 170.3, 170.4, 170.6, 170.7.
4.2.3. Octyl 3,4-O-isopropylidene-b-d-galactopyranosyl-
'1!4)-b-d-glucopyranoside, 7. A solution of octyl lacto-
side 6 :2.00 g, 4.40 mmol) and p-toluenesulfonic acid
:80 mg, 0.4 mmol) in 2,2-dimethoxypropane :40 cm3) was
stirred at roomtemperature for 60 h. The reaction was
quenched with triethylamine :0.5 cm3) and concentrated to
3
a solid foamwhich was suspended in EtOAc :30 cm ).
Aqueous TFA :200 mm3; 50% v/v) was added and after
stirring for 1 h, the reaction was again quenched with
triethylamine and evaporated onto silica :10 g). Flash chro-
matography :silica gel, 100 g; EtOAc then EtOAc±MeOH,
19:1) gave the title compound 7 as an amorphous white solid
:1.44 g, 66%), :Found: C, 55.75; H, 8.4. C23H42O11 requires
C, 55.86; H, 8.56%); [a]Dˆ19.9 :c 0.87 in MeOH);
dH:CD3OD) 0.90 :3H, t, Jˆ6.9 Hz, C7H14Me), 1.26±1.40
:13H, m, :CH2)5CH3, CMe2), 1.48 :3H, s, CMe2), 1.62
Method two. A suspension of silver perchlorate :4.96 g,
Ê
22 mmol), 4 A molecular sieves :10 g), and dry CH2Cl2
:125 cm3) was stirred overnight at roomtemperature in a
tin foil covered ¯ask in order to exclude light. Octanol was
added :4.56 cm3, 50 mmol) followed by silver carbonate
:8.27 g, 30 mmol), glycosyl bromide 2 :13.99 g, 20 mmol)
and the mixture was stirred at room temperature for 24 h.
The mixture was then ®ltered through Celite and gave a
syrup on concentration. Flash chromatography :silica gel;
hexane±EtOAc, 3:1!1:1) gave the desired acetylated octyl
lactoside 3 as an amorphous mass :6.51 g, 44%) which gave
identical analytical data to the above. Further elution
:hexane±EtOAc, 1:2) gave octyl 2,3,4,6-tetra-O-acetyl-b-
d-galactopyranosyl-31!4)-3,6-di-O-acetyl-a-d-glucopyra-
noside 5 as a glassy solid :3.90 g, 28%), :Found: C, 54.16;
H, 7.09. C32H50O17 requires C, 54.18; H, 7.13%);
[a]Dˆ164.4 :c 1 in CHCl3); dH:CDCl3) 0.86 :3H, m,
C7H14CH3), 1.26 :10H, m, :CH2)5 CH3), 1.55 :2H, m,
OCH2CH2), 1.94±2.14 :6£3H, 6s, 6£AcO), 4.49 :1H, d,
J1b,2bˆ8.0 Hz, 1b-H), 4.81 :1H, dd, J1a,2aˆ3.8 Hz, 1a-H),
4.93 :1H, dd, J2b,3bˆ10.4 Hz, J3b,4bˆ3.5 Hz, 3b-H), 5.09
:2H, m, OCH2CH2), 4.28, 4.37 :2£1H, 2d, J1a,2a
ˆ
J1b,2bˆ7.4, 7.7 Hz, 1a-H, 1b-H); dC:CD3OD) 14.5, 23.8,
26.6, 27.2, 28.5, 30.5, 30.7, 30.9, 33.1, 62.1, 62.6, 71.1,
74.6, 75.0, 75.2, 75.5, 76.5, 76.6, 81.0, 81.2, 104.4 :2), 111.3.
A small sample :80 mg) of the above was acetylated using
pyridine±acetic anhydride and puri®ed by ¯ash chromato-
graphy :silica gel; toluene±EtOAc, 2:1) to give octyl 2,6-di-
O-acetyl-3,4-O-isopropylidene-b-d-galactopyranosyl-31!4)-
2,3,6-tri-O-acetyl-b-d-glucopyranoside 10 as a colourless
syrup :105 mg, 95%), :Found: C, 56.48; H, 7.72. C33H52O16
requires C, 56.25; H, 7.44%); [a]Dˆ16.4 :c 1 in CHCl3);
nmax/cm21 2930 :CH2, CH3), 1750 :CvO), 1370 :Pri, C±
H), 1042 :C±O); dH:CDCl3) 0.84 :3H, t, Jˆ6.9 Hz,
C7H14CH3), 1.20±1.30 :13H, m, :CH2)5CH3, CMe2), 1.50
:5H, m, OCH2CH2, CMe2), 2.00, 2.01, 2.05, 2.08, 3.00
:5£3H, 5s, 5£AcO), 3.42 :1H, m, OCH2), 3.58 :1H, ddd,
:1H, dd, J1b,2bˆJ2b,3b
,
2b-H), 5.20 :1H, t, J2a,3a
ˆ
J3a,4aˆ9.5 Hz, 3a-H), 5.33 :1H, dd, J3b,4bˆJ4b,5bˆ1.0 Hz,
4b-H). A sample was acetylated using pyridine±acetic
anhydride and puri®ed by ¯ash chromatography :silica
gel; hexane±EtOAc, 1:1) to give octyl 2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-31!4)-2,3,6-tri-O-acetyl-a-
0
J4a,5aˆ9.9 Hz, J5a,6aˆ4.9 Hz, J5a,6a ˆ1.9 Hz, 5a-H), 3.72
:1H, m, 4a-H), 3.80 :1H, m, OCH2), 3.90 :1H, m, 5b-H),
4.09±4.17 :3H, m, 6a-H, 3b-H, 4b-H), 4.25 :1H, dd,
1
0
d-glucopyranoside. The following H NMR data are con-
J5b,6bˆ7.1 Hz, J6b,6b ˆ11.5 Hz, 6b-H), 4.30 :1H, dd,
0
0
0
sistent with the peracetylated a lactoside: dH:CDCl3) 0.85
:3H, m, C7H14CH3), 1.28 :10H, m, :CH2)5 CH3), 1.55 :2H,
m, OCH2CH2), 1.94±2.20 :7£3H, 7s, 7£AcO), 4.46 :1H, d,
J1b,2bˆ8.0 Hz, 1b-H), 4.74 :1H, dd, J1a,2aˆ3.6 Hz,
J2a,3aˆ10.1 Hz, 2a-H), 4.86 :1H, dd, J2b,3bˆ10.4 Hz,
J3b,4bˆ3.6 Hz, 3b-H), 4.94 :1H, d, J1a,2a,, 1a-H), 5.09 :1H,
dd, J1b,2bˆJ2b,3b, 2b-H), 5.32 :1H, br d, J3b,4b, 4b-H), 5.44
:1H, t, J2a,3aˆJ3a,4aˆ10.1 Hz, 3a-H).
J5b,6b ˆ5.2 Hz, J6b,6b , 6b -H), 4.31 :1H, d, J1b,2bˆ7.7 Hz,
1b-H), 4.42 :2H, m, 1a-H, 6a0-H), 4.82 :1H, m, 2b-H),
4.87 :1H, dd, J1a,2aˆ8.0 Hz, J2a,3aˆ9.6 Hz, 2a-H), 5.16
:1H, m, 3a-H).
4.2.4. Octyl 2,6-di-O-benzyl-3,4-O-isopropylidene-b-d-
galactopyranosyl-'1!4)-2,3,6-tri-O-benzyl-b-d-gluco-
pyranoside, 11. Sodiumhydride :60% dispersion in oil;

3214
W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
350 mg, 8.6 mmol) was added in portions to a cooled :08C),
stirred solution of ketal 7 :660 mg, 1.33 mmol) in DMF
:13 cm3) and the mixture was stirred at 08C for 30 min.
Benzyl bromide :0.87 cm3, 7.32 mmol) was added dropwise
and the mixture was allowed to warmto roomtemperature
and stirred for a further hour. After careful addition of
MeOH :1 cm3) and concentration, the residue was par-
titioned between diethyl ether and water, the aqueous
phase being extracted twice with diethyl ether before
washing the combined organic extracts with saturated
NaCl solution, drying and concentration to a colourless
oil. Flash chromatography :silica gel; hexane±EtOAc, 6:1)
gave compound 11 as a colourless syrup :1.20 g, 95%),
:Found: C, 73.89; H, 7.87. C58H72O11 requires C, 73.70;
H, 7.68%); [a]Dˆ115.8 :c 1.1 in CHCl3); nmax/cm21
3030 :Ar-H), 2930, 2860 :CH2, CH3), 1370 :Pri C±H),
735, 700 :Ar-H); dH:CDCl3) 0.90 :3H, t, Jˆ6.9 Hz,
C7H14CH3), 1.27±1.46 :16H, m, :CH2)5CH3, CMe2), 1.68
:2H, m, OCH2CH2), 4.32±4.99 :10£1H, 10 AB d,
5£OCH2Ph), 4.41, 4.46 :2£1H, 2d, J1a,2aˆJ1b,2bˆ8.0,
7.7 Hz, 1a-H, 1b-H), 7.20±7.45 :25H, m, Ar-H);
dC:CDCl3) 13.9, 22.4, 25.9, 26.2, 27.7, 29.0, 29.2, 29.5,
31.6, 68.2, 68.7, 69.9, 71.8, 73.0 :2), 73.2, 73.5, 74.8,
74.9, 75.2, 76.3, 79.2, 80.5, 81.7, 82.8, 101.7, 103.6,
109.6, 127.2±128.2 :Ar C), 138.3, 138.4, 138.5, 138.7,
139.0.
4.2.6. Octyl 'potassium 5-amino-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonate)-'2!3)-b-d-galac-
topyranosyl-'1!4)-b-d-glucopyranoside, 20. A suspen-
sion of acceptor 12 :200 mg, 220 mmol), donor 13
Ê
:288 mg, 550 mmol) and 3 A molecular sieves :600 mg) in
dry acetonitrile :2 cm3) was stirred for 2 h at roomtempera-
ture under nitrogen. The mixture was cooled to 2358C and
N-iodosuccinimide :247 mg, 1.10 mmol) was added,
followed by tri¯uoromethanesulfonic acid :10 mm3,
110 mmol) and the mixture was stirred at this temperature
for 2 h. The mixture was diluted with CH2Cl2 :50 cm3),
®ltered through Celite and the ®ltrate was washed succes-
sively with 1 M solutions of sodiumcarbonate, sodiumthio-
sulfate and sodiumchloride, before drying and
concentration to a syrup. In order to facilitate separation
of products from starting materials, the mixture was
deacetylated prior to chromatography: a solution of the
crude product mixture and sodium methoxide :6 mg,
110 mmol) in MeOH :10 cm3) was stirred for 2 h at room
temperature. The mixture was neutralised with acetic acid
and concentrated on to silica :2 g). Flash chromatography
:silica gel; CH2Cl2±MeOH, 99:1!97:3) gave a mixture of
tri-saccharides 14/15 :150 mg).
A small portion :20 mg) of this mixture was partially
separated by careful column chromatography :silica gel;
CH2Cl2±MeOH, 99:1!97:3) to give ®rst, the lactone,
octyl 35-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-2-
nonulopyranosyloyl-1c!4b-lactone)-32!3)-2,6-di-O-benzyl-
b-d-galactopyranosyl-31!4)-2,3,6-tri-O-benzyl-b-d-gluco-
pyranoside 15; dH:CD3OD; cf. compound 14 in Ref. 29)
1.69 :1H, dd, J3cax,3ceqˆ13.8 Hz, J3cax,4cˆ11.3 Hz, 3cax-H),
2.01 :3H, s, AcN), 2.26 :1H, dd, J3cax,3ceqˆJ3ceq,4cˆ5.5 Hz,
3ceq-H), 3.25 :1H, dd, J1a,2aˆ8.0 Hz, J2a,3aˆ9.3 Hz, 2a-H),
4.10 :1H, dd, J2b,3bˆ9.3 Hz, J3b,4bˆ3.9 Hz, 3b-H), 4.42 :1H,
d, J1b,2bˆ7.7 Hz, 1b-H), 4.45 :1H, d, J1a,2a, 1a-H), and then
octyl 3methyl 5-acetamido-3,5-dideoxy-d-glycero-a/b-d-
galacto-2-nonulopyranosylonate)-32!3)-2,6-di-O-benzyl-
b-d-galactopyranosyl-31!4)-2,3,6-tri-O-benzyl-b-d-
glucopyranoside 14 as an anomeric mixture :a/b, 7:3);
dH:CD3OD) 1.73 :m, J3cax,3ceqˆ12.9 Hz, J3cax,4cˆ11.8 Hz,
b3cax-H), 2.01 :m, a/bAcN, a3cax-H), 2.68 :m, a/b3ceq-
H), 3.58 :s, bCO2Me), 3.80 :s, aCO2Me). Both samples
were acetylated with pyridine and acetic anhydride to verify
their structures.
4.2.5. Octyl 2,6-di-O-benzyl-b-d-galactopyranosyl-
'1!4)-2,3,6-tri-O-benzyl-b-d-glucopyranoside, 12. Com-
pound 11 :1.02 g, 1.08 mmol) was dissolved in aqueous
acetic acid :50 cm3; 80% v/v) and stirred at 808C for 2 h.
After cooling, the solution was concentrated and co-evapo-
rated several times with toluene. Flash chromatography
:silica gel; hexane±EtOAc, 3:2) gave compound 12 as a
colourless syrup :0.96 g, 98%), :Found: C, 73.25; H, 7.61.
C55H68O11 requires C, 72.98; H, 7.57%); [a]Dˆ118.8 :c 1
in CHCl3); nmax/cm21 3450 :OH br), 3030 :Ar-H), 2925,
2860 :CH2, CH3), 735, 700 :Ar-H); dH:CDCl3) 0.90 :3H, t,
Jˆ6.9 Hz, C7H14CH3), 1.25±1.46 :10H, m, :CH2)5CH3),
1.67 :2H, m, OCH2CH2), 2.40 :2H, br s, OH), 4.38±5.02
:10£1H, 10 AB d, 5£OCH2Ph), 4.40, 4.60 :2£1H, 2d,
J1a,2aˆJ1b,2bˆ8.0, 8.0 Hz, 1a-H, 1b-H), 7.20±7.40
:25H, m, Ar-H); d:CDCl3) 13.9, 22.4, 25.95, 29.0,
29.2, 29.5, 31.6, 68.2, 68.5, 68.6, 69.9, 72.7, 73.0, 73.3,
73.4, 74.7 :2), 75.0, 75.05, 76.7, 79.9, 81.7, 82.7, 102.5,
103.6, 127.2±128.4 :Ar C), 137.95, 138.25, 138.3, 138.6,
139.2.
Lactone 17; dH:CDCl3) 1.88 :3H, s, AcN), 1.92, 2.00, 2.03,
2.15, :4£3H, 4s, 4£AcO), 4.60 :1H, dd, J2b,3bˆ9.3 Hz,
J3b,4bˆ3.9 Hz, 3b-H), 4.18 :1H, m, 5c-H), 4.37 :1H, d,
J1a,2aˆ7.7 Hz, 1a-H), 5.05 :1H, m, 8c-H), 5.25 :1H, dd,
J6c,7cˆ1.9 Hz, J7c,8cˆ6.3 Hz, 7c-H), 5.30 :1H, d,
J5c,NHˆ10.2 Hz, NH), 5.49 :1H, m, 4c-H); FAB-MS: m/z
1369 :M1Na)1 :C74H91NO22 requires m/z 1346).
A small sample of the diol :100 mg) was acetylated using
pyridine±acetic anhydride and puri®ed by ¯ash chromato-
graphy :silica gel; hexane±EtOAc, 4:1) to give octyl 3,4-di-
O-acetyl-2,6-di-O-benzyl-b-d-galactopyranosyl-31!4)-
2,3,6-tri-O-benzyl-b-d-glucopyranoside as a colourless
syrup :100 mg, 92%), dH:CDCl3) 0.89 :3H, t, Jˆ6.9 Hz,
C7H14CH3), 1.26±1.44 :10H, m, :CH2)5CH3), 1.67 :2H,
m, OCH2CH2), 1.95, 1.99 :2£3H, 2s, 2£AcO), 3.71
a/b methyl esters 16; dH:CDCl3, cf. compounds 25 and 26
in Ref. 28) 1.75, 1.96, 1.99, 2.00, 2.08 :5£3H, 5s, 5£bAcO),
1.85 :3H, s, a/bAcN), 2.60 :m, a/b3ceq-H), 3.43 :s,
bCO2Me), 3.83 :s, aCO2Me), 5.05 :d, J3b,4bˆ3.3 Hz, a4b-
H), 5.32 :dd, J6c,7cˆ2.2 Hz, J7c,8cˆ8.5 Hz, a7c-H), 5.39 :t,
J6c,7cˆJ7c,8cˆ2.0 Hz, a7c-H), 5.59 :1H, m, a8c-H).
0
:1H, dd, J5a,6aˆ1.6 Hz, J6a,6a ˆ11.0 Hz, 6a-H), 3.79 :1H,
0
0
0
dd, J5a,6a ˆ4.1 Hz, J6a,6a , 6a -H), 3.91±4.03 :2H, m,
OCH2, 4a-H), 4.19±4.98 :10£1H, 10 AB d, 5£OCH2Ph),
4.38 :1H, d, J1a,2aˆ7.7 Hz, 1a-H), 4.53 :1H, d,
J1b,2bˆ7.7 Hz, 1b-H), 4.87 :1H, dd, J2b,3bˆ10.2 Hz,
J3b,4bˆ3.3 Hz, 3b-H), 5.39 :1H, d, J3b,4b, 4b-H), 7.15±7.40
:25H, m, Ar-H).
A suspension of the remaining tri-saccharide mixture
:130 mg) and palladium hydroxide on charcoal :130 mg;

W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
3215
10% w/w) in MeOH :30 cm3) was stirred for 24 h under an
atmosphere of hydrogen. The mixture was ®ltered through
Celite and concentrated. A solution of the residue in
by addition of EtOH :0.5 cm3) to precipitate the protein and
the resulting mixture was centrifuged at 13,000g for 3 min.
The supernatants from®ve such incubations were combined
and concentrated. The residue was redissolved in water
:2 cm3) and subjected to reverse phase chromatography
:C-18 silica gel; gradient H2O!MeOH). Elution at approxi-
mately 50% aqueous methanol gave the title compound 1 as
a white powder following freeze-drying fromwater :1.4 mg,
42%). Analytical data were identical to those obtained for
the chemically synthesised material.
3
potassiumhydroxide solution :10 cm ;1 M) was heated for
20 h at 908C. After cooling to roomtemperature, the
solution was neutralised with acetic acid and desalted on a
reverse phase column :C-18 silica gel; H2O!MeOH) to
give octyl 3potassium 5-amino-3,5-dideoxy-d-glycero-a/b-
d-galacto-2-nonulopyranosylonate)-32!3)-b-d-galacto-
pyranosyl-31!4)-b-d-glucopyranoside 20a/b as a glassy
solid :80 mg, 49%; a/b, 6:1) dH:CD3OD) b anomer 2.34
:dd, J3cax,3ceqˆ12.6 Hz, J3ceq,4cˆ4.4 Hz, 3ceq-H), 2.90 :t,
4.2.9. Octyl 'potassium 5-amino-3,5-dideoxy-d-glycero-
a-d-galacto-2-nonulopyranosylonate)-'2!3)-b-d-galac-
topyranosyl-'1!4)-b-d-glucopyranoside, 20-de-N-
acetylation of 1. A solution of enzymatically synthesised
1 :5 mg) and potassium hydroxide :1 cm3; 1 M) was heated
for 20 h at 908C. After cooling to roomtemperature, the
solution was neutralised with acetic acid and desalted on a
reverse phase column :C-18 silica gel; H2O!MeOH) to
give the de-N-acetyl compound 20 as a glassy solid
:4.5 mg, 95%) Analytical data were identical to those for
the chemically synthesised material.
J4c,5cˆJ5c,6cˆ9.6 Hz, 5c-H), a anomer 2.73 :t, J4c,5c
J5c,6cˆ9.9 Hz, 5c-H), 2.79 :dd, J3cax,3ceqˆ12.4 Hz, J3ceq,4c
4.7 Hz, 3ceq-H).
ˆ
ˆ
Repeated ¯ash chromatography :silica gel; CH2Cl2±
MeOH±H2O, 6:5:1) gave 20a as a glassy solid :35 mg,
21%), [a]Dˆ224.8 :c 0.5 in MeOH); dH:CD3OD) 0.90
:3H, t, Jˆ6.9 Hz, C7H14CH3), 1.26±1.42 :10H, m,
:CH2)5CH3), 1.47±1.70 :3H, m, OCH2CH2, 3cax-H), 2.73
:1H, t, J4c,5cˆJ5c,6cˆ9.9 Hz, 5c-H), 2.79 :1H, dd,
J3cax,3ceqˆ12.4 Hz, J3ceq,4cˆ4.7 Hz, 3ceq-H), 4.28 :1H, d,
J1a,2aˆ8.0 Hz, 1a-H), 4.43 :1H, d, J1b,2bˆ8.0 Hz, 1b-H);
dC:CD3OD) 14.5, 23.8, 27.2, 30.5, 30.7, 30.9, 33.1, 42.1,
54.6, 62.3, 62.9, 64.9, 68.9, 70.1, 71.1 :2), 72.25, 73.7, 74.9,
76.6 :2), 77.2, 77.5, 81.3, 101.3, 104.5, 105.3, 175.75; ES-
MS :1ve): m/z 704 :M2K12H)1 :C29H52NO18K requires
m/z 741).
Acknowledgements
This project was supported by project grants fromthe
Wellcome Trust and the MRC; studentship support from
the AICR :W. B. T.) and BBSRC :J. A. H.) is also gratefully
acknowledged.
4.2.7. Octyl 'potassium 5-acetamido-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosylonate)-'2!3)-b-
d-galactopyranosyl-'1!4)-b-d-glucopyranoside, 1-Re-
N-acetylation of 20a. Acetic anhydride :64 mm3,
64 mmol; 1 M solution in MeOH) was added to a stirred
solution of 20a :15 mg, 21.3 mmol) in MeOH. After 18 h
the solution was concentrated and the residue was redis-
solved in water :2 cm3). Reverse phase chromatography
:C-18 silica gel; H2O!MeOH) gave octyl 3potassium
5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulo-
pyranosylonate)-32!3)-b-d-galactopyranosyl-31!4)-b-d-
glucopyranoside, 1 as a glassy solid :14 mg, 88%),
[a]Dˆ27.2 :c 1 in MeOH); dH:CD3OD) 0.90 :3H, t,
Jˆ6.9 , C7H14CH3), 1.26±1.42 :10H, m, :CH2)5CH3), 1.62
:2H, m, OCH2CH2), 1.73 :1H, m, 3cax-H), 2.00 :3H, s,
AcN), 2.86 :1H, dd, J3cax,3ceqˆ12.4 Hz, J3ceq,4cˆ4.4 Hz,
3ceq-H), 4.28 :1H, d, J1a,2aˆ7.7 Hz, 1a-H), 4.43 :1H, d,
J1b,2bˆ7.7 Hz, 1b-H); dC:CD3OD) 14.5, 22.7, 23.8, 27.2,
30.5, 30.7, 30.9, 33.1, 42.25, 54.1, 62.2, 62.9, 64.8, 69.2,
69.5, 70.3, 71.1, 73.2, 75.0, 75.1, 75.15, 76.6, 77.2, 77.9,
81.2, 101.3, 104.5, 105.3, 175.3, 175.8; ES-MS :2ve): m/z
744 :M2K)2 :C31H54NO19K requires m/z 783).
References
1. :a) Schauer, R. Glycoconjug. J. 2000, 17, 485±499. :b) Rosen,
A. Biology of the Sialic Acid; PlenumPress: New York, 1995.
2. Koeller, K. M.; Wong, C.-H. Nat. Biotechnol. 2000, 18, 835±
841.
3. Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357±
2364.
4. Recent developments in oligosachharide synthesis are
reviewed in: Davis, B. G. J. Chem. Soc., Perkin Trans. 1
2000, 2137±2160.
5. :a) Sears, P.; Wong, C.-H. Science 2001, 291, 2344±2350.
:b) Ye, X. S.; Wong, C.-H. J. Org. Chem. 2000, 65, 2410±
2431.
6. :a) Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science
2001, 291, 1523±1527. :b) Seeberger, P. H.; Haase, W. C.
Chem. Rev. 2000, 100, 4349±4395.
7. Essentials of glycobiology; Varki, A., Cummings, R., Esko, J.,
Freeze, H., Hart, G., Marth, J., Eds.; CSHL Press: Cold Spring
Harbor, NY, 1999.
8. Various authors Carbohydr. Res. 1997, 305, 307±568 special
issue devoted to enzymatic synthesis.
4.2.8. Octyl 'potassium 5-acetamido-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosylonate)-'2!3)-b-
d-galactopyranosyl-'1!4)-b-d-glucopyranoside, 1-enzy-
matic sialylation of 6. Typical biotransformation: pNP
sialoside 2130,33 :3 mg, 6.7 mmol) was added to a solution
of octyl lactoside 1 :2 mg, 4.4 mmol) and trans-sialidase
:,1 mg crude protein) in 50 mM phosphate buffer :pH 7,
1 cm3). The reaction mixture was incubated for 18 h at
308C. TLC :CHCl3±MeOH±H2O, 6:4:1) indicated ,50%
turnover of the octyl lactoside. The reaction was quenched
9. :a) Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232±240.
:b) Wymer, N.; Toone, E. J. Curr. Opin. Chem. Biol. 2000, 4,
110±119.
10. Scigelova, M.; Singh, S.; Crout, D. H. G. J. Mol. Catal. B:
Enzym. 1999, 6, 483±494.
11. :a) Mayer, C.; Jakeman, D. L.; Mah, M.; Karjala, G.; Gal, L.;
Warren, R. A. J.; Withers, S. G. Chem. Biol. 2001, 8, 437±
443. :b) Mackenzie, L. F.; Wang, Q. P.; Warren, R. A. J.;
Withers, S. G. J. Am. Chem. Soc. 1998, 120, 5583±5584.

3216
W. B. Turnbull et al. / Tetrahedron 58 32002) 3207±3216
12. Davies, G. J.; Charnock, S. J.; Henrissat, B. Trends Glycosci.
Glycotechnol. 2001, 13, 105±120.
949. :e) Lee, K. B.; Lee, Y. C. Anal. Biochem. 1994, 216,
358±364. :f) Scudder, P.; Doom, J. P.; Chuenkova, M.;
Manger, I. D.; Pereira, M. E. A. J. Biol. Chem. 1993, 268,
9886±9891. :g) Ito, Y.; Paulson, J. C. J. Am. Chem. Soc. 1993,
115, 7862±7863.
13. Jiang, J. Q.; Biggins, J. B.; Thorson, J. S. Angew. Chem. Int.
Ed. 2001, 40, 1502±1507.
14. Koizumi, S.; Endo, T.; Tabata, K.; Ozaki, A. Nat. Biotechnol.
1998, 16, 847±850.
23. Harrison, J. A.; Kartha, K. P. R.; Turnbull, W. B.; Scheuerl,
S. L.; Naismith, J. H.; Schenkman, S.; Field, R. A. Bioorg.
Med. Chem. Lett. 2001, 11, 141±144.
15. Gilbert, M.; Bayer, R.; Cunningham, A. M.; Defrees, S.; Gao,
Y. H.; Watson, D. C.; Young, N. M.; Wakarchuk, W. W. Nat.
Biotechnol. 1998, 16, 769±772.
24. Kartha, K. P. R.; Jennings, H. J. J. Carbohydr. Chem. 1990, 9,
777±781.
16. Blixt, O.; Brown, J.; Schur, M. J.; Wakarchuk, W.; Paulson,
J. C. J. Org. Chem. 2001, 66, 2442±2448.
25. Qian, X.; Sujino, K.; Otter, A.; Palcic, M. M.; Hindsgaul, O.
J. Am. Chem. Soc. 1999, 121, 12063±12072.
17. Chen, X.; Fang, J. W.; Zhang, J. B.; Liu, Z. Y.; Shao, J.;
Kowal, P.; Andreana, P.; Wang, P. J. Am. Chem. Soc. 2001,
123, 2081±2082.
26. Hasegawa, A.; Nagahama, T.; Ohki, H.; Kiso, M. J. Carbo-
hydr. Chem. 1992, 11, 699±714.
27. Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H.; Kiso, M.
Carbohydr. Res. 1991, 212, 277±281.
18. Duclos, R. I. Carbohydr. Res. 2000, 328, 489±507 and
references cited therein.
28. Fujita, S.; Numata, M.; Sugimoto, M.; Tomita, K.; Ogawa, T.
Carbohydr. Res. 1992, 228, 347±370.
19. Boons, G.-J.; Demchenko, A. V. Chem. Rev. 2000, 100, 4539±
4567.
29. Wilstermann, M.; Kononov, L. O.; Nilsson, U.; Ray, A. K.;
Magnusson, G. J. Am. Chem. Soc. 1995, 117, 4742±4754.
30. :a) Rothermel, J.; Weber, B.; Faillard, H. Liebigs Ann. Chem.
1992, 799±802. :b) Rothermel, J.; Faillard, H. Carbohydr.
Res. 1990, 196, 29±40.
20. :a) Ajiska, K.; Fujimoto, H.; Isomura, M. Carbohydr. Res.
1994, 259, 103±115. :b) Schmidt, D.; Sauerbrei, B.; Thiem,
J. J. Org. Chem. 2000, 65, 8518±8526. :c) Schmidt, D.;
Thiem, J. Chem. Commun. 2000, 1919±1920.
21. :a) Acosta-Serrano, A.; Almeida, I. C.; Freitas, L. H.; Yoshida,
N.; Schenkman, S. Mol. Biochem. Parasitol. 2001, 114, 143±
150. :b) Frasch, A. C. C. Parasitol. Today 2000, 16, 282±286.
22. For example: :a) Lee, S. G.; Kim, B. G. Enzyme Microb.
Technol. 2001, 28, 161±167. :b) Wilson, J. C.; Kiefel, M. J.;
Albouz-Abo, S.; von Itzstein, M. Bioorg. Med. Chem. Lett.
2000, 10, 2791±2794. :c) Singh, S.; Scigelova, M.; Hallberg,
M. L.; Howarth, O. W.; Schenkman, S.; Crout, D. H. G. Chem.
Commun. 2000, 1013±1014. :d) Vetere, A.; Miletich, M.;
Bosco, M.; Paoletti, S. Eur. J. Biochem. 2000, 267, 942±
31. :a) Ribeirao, M.; Perreira-Chioccola, V. L.; Eichinger, D.;
Rodrigues, M. M.; Schenkman, S. Glycobiology 1997, 7,
1237±1246. :b) Schenkman, S.; Chaves, L. B.; Pontes de
Carvalho, L. C.; Eichinger, D. J. Biol. Chem. 1994, 269,
7970±7975.
32. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Puri®cation of
Laboratory Chemicals; Pergamon: London, 1966.
33. Eschenfelder, V.; Brossmer, R. Carbohydr. Res. 1987, 162,
294±297.