Glycomethanethiosulfonates: Powerful reagents for protein glycosylation

Article abstract of DOI:10.1016/S0957-4166(99)00497-8

Twelve novel glycomethanethiosulfonate (glyco-MTS) protein glycosylation reagents have been prepared. Their use in a controlled site-selective glycosylation strategy that combines site-directed mutagenesis with chemical modification allows protein glycosylation with concomitant control of (i) site, (ii) carbohydrate, (iii) anomeric stereochemistry, (iv) sugar to protein spacer arm nature and (v) degree of glycan protection. The ability of these highly selective and yet reactive reagents has been illustrated by the introduction of D-glucosyl and N-Ac-D-glucosaminyl residues to both external and hindered internal sites in a model protein - the serine protease enzyme subtilisin Bacillus lentus (SBL) - using corresponding gluco-MTS 1 and N-Ac-glucosamine-MTS 2. Molecular modelling studies provide a rationale for the strikingly different effects of these reagents on the properties of the protein despite differing only in the nature of their C-2 substituents. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(99)00497-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 245–262
Glycomethanethiosulfonates: powerful reagents for protein
glycosylation
Benjamin G. Davis,a,∗ Michael A. T. Maughan,a Martin P. Green,a Astrid Ullman b and
J. Bryan Jones b
aDepartment of Chemistry, University of Durham, South Road, Durham DH1 3LE, UK
bLash-Miller Chemical Laboratory, University of Toronto, 80 St George Street, Toronto M5S 3H6, Canada
Received 2 November 1999; accepted 8 November 1999
Abstract
Twelve novel glycomethanethiosulfonate (glyco-MTS) protein glycosylation reagents have been prepared. Their
use in a controlled site-selective glycosylation strategy that combines site-directed mutagenesis with chemical
modification allows protein glycosylation with concomitant control of (i) site, (ii) carbohydrate, (iii) anomeric
stereochemistry, (iv) sugar to protein spacer arm nature and (v) degree of glycan protection. The ability of these
highly selective and yet reactive reagents has been illustrated by the introduction of D-glucosyl and N-Ac-D-
glucosaminyl residues to both external and hindered internal sites in a model protein — the serine protease enzyme
subtilisin Bacillus lentus (SBL) — using corresponding gluco-MTS 1 and N-Ac-glucosamine-MTS 2. Molecular
modelling studies provide a rationale for the strikingly different effects of these reagents on the properties of
the protein despite differing only in the nature of their C-2 substituents. © 2000 Elsevier Science Ltd. All rights
reserved.
1. Introduction
The glycosylation of proteins plays a key role in determining their expression, folding,1 thermal and
proteolytic stability2 and, in the case of enzymes, catalytic activity.3 Furthermore, the role of glycopro-
teins as cell surface markers in communication events such as microbial invasion,4 inflammation5 and
immune responses6 depends crucially on the correct glycosylation pattern. There is evidence that even
very slight alterations in the sugars that decorate the exterior of a protein can cause remarkable changes
in these properties. For example, a turn is induced in aqueous solution in a SYSPTSPSYS segment of the
C-terminal domain of RNA polymerase II when the threonine side chain is N-acetyl-D-glucosaminylated,
whereas the corresponding non-glycosylated peptide adopts a randomly-coiled structure.7 Moreover,
∗
Corresponding author. E-mail: ben.davies@durham.ac.uk
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00497-8
tetasy 3132

246
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
different forms of pancreatic ribonuclease B displayed a fourfold variation in hydrolytic activity although
differing only in the nature of the glycans attached to the enzyme.3
Despite this striking ability of carbohydrates to influence protein properties, systematic studies of
the effects of protein glycosylation have been limited and have required extensive chromatographic
separations from natural abundant glycoprotein sources.3 These difficulties are exacerbated by the natural
occurrence of microheterogeneous mixtures of glycoforms:8 glycoprotein forms that possess the same
peptide backbone, but differ in both the nature and site of glycosylation.
Chemical glycosylation provides an alternative method for the preparation of glycosylated proteins.9
In particular, such methods allow control of the glycan structure and the nature of the sugar–protein
bond. However, despite these advantages, existing methods typically generate mixtures through the
indiscriminate modification of the wide variety of functionality found in proteins. Advances in the site-
specific glycosylation of the inactive protein bovine serum albumin (BSA) have been made.10 Pioneering
work by Flitsch and co-workers established the validity of cysteine modification as a strategy for
chemoselective protein glycosylation. Thus, reaction of the α-iodoacetamide of N-acetyl-D-glucosamine
with oxidized bovine serum albumin (BSA) allowed modification of the single free cysteine that is
naturally present.10a Later this method was applied by Dwek and co-workers to introduce chitotriose and
a heptasaccharide from horseradish peroxidase to BSA.10b Boons and co-workers have also described
the use of a glycosyl dithiopyridine as another valuable addition to the cysteine-reactive reagents
available for protein glycosylation.10c However, these methods rely upon modification of an existing
cysteine in BSA and, as such, allow no flexibility in the choice of glycosylation site. For full control
of glycosylation, both choice of site (site-selectivity) and glycan are needed. This paper describes a
combined site-directed mutagenesis and chemical modification approach using well-defined and highly
specific glycosylating agents. This method is rapid, efficient and is virtually unlimited in the scope of
both the sites and the glycans that may be conjugated. The strategy involves the introduction of cysteine
as a chemoselective tag at preselected positions within a given protein and then reaction of its thiol
residue with glycomethanethiosulfonate reagents (Scheme 1). Methanethiosulfonate (MTS) reagents
react specifically and quantitatively with thiols11 and allow the controlled formation of neutral disulfide
linkages.
Scheme 1. A combined site-directed mutagenesis and chemical modification approach to site-selective glycosylation
This paper reports the detailed syntheses of these powerful reagents (Fig. 1) and their use in the
modification of the model protein: the serine protease subtilisin B. lentus (SBL). SBL is an ideal
protein for evaluating the validity of this strategy as it does not contain a natural cysteine and is not
naturally glycosylated. We chose four sites in SBL for glycosylation. S15612 is a surface-exposed
residue that permits the introduction of externally-disposed glycans resembling those found naturally
in glycoproteins.13 Furthermore, we were interested in probing the effect of glycosylation on enzyme
activity as part of an ongoing programme to tailor activity, specificity and stability for use in synthesis.

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
247
Promising results have indicated the potential of glycoenzymes14 but due to a lack of site-selectivity
in glycosylation no precise structure–activity relationship (SAR) determinations have until now been
possible. For this reason, we chose three further sites with internally oriented side chains, one in each
15
of0three key substrate binding pockets: N62 in the S2 pocket, S166 in the S1 pocket and L217 in the
S1 pocket. Moreover, position 62 is occupied by an asparagine residue, and positions 156 and 166 are
occupied by serine residues in the wild-type enzyme and, in this respect, are representative of natural
N-(Asn) and O-(Ser/Thr) glycosylation sites, respectively.16
Fig. 1.
The synthesis of a range of glycomethanethiosulfonates (glyco-MTS) 1–12 allows the preparation
of glycoproteins with control of (i) site, (ii) parent carbohydrate, (iii) anomeric stereochemistry, (iv)
length of the glycan–protein tether and (v) degree of glycan protection (thereby allowing the effects
of increased steric bulk and hydrophobicity to be assessed); some of this work has appeared as a
preliminary publication.14h The determination of valuable glycosylation–activity SARs is illustrated
by the glycosylation of SBL with reagents derived from D-glucose 1 and N-acetyl-D-glucosamine 2.
Molecular modelling studies are reported which provide a good rationale for the strikingly different
effects of these two reagents upon the catalytic activity of SBL.
2. Results and discussion
2.1. Preparation of directly linked glyco-MTS 1 and 2
Previous synthetic work has demonstrated17,18 that, of the wide variety of methods available,19 direct
nucleophilic displacement of a halide group by methanethiosulfonate ion provides an efficient and
high yielding method for the preparation of alkyl methanethiosulfonates. Furthermore, the ease with
which the anomeric leaving group of peracetylated glycosyl halides may be displaced by certain sulfur
nucleophiles20 prompted the synthesis of 1 and 2 by direct displacement using sodium methanethio-
sulfonate as a nucleophile (Scheme 2).21 Thus, brief treatment (20 min) of a solution of sodium me-
thanethiosulfonate in ethanol at 90°C with acetobromo-D-glucose 13 gave the fully protected anomeric
β-D-gluco-MTS 1 in 63% yield; prolonged treatment or the use of other solvents gave substantially lower
yields. Similarly, 2 was synthesized from acetochloro-N-acetyl-D-glucosamine 14, through displacement

248
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
of the anomeric chloride by methanethiosulfonate to give crystalline β-GlcNAc-MTS 2 in a modest
25% yield. In contrast to the synthesis of 1, highest yields of 2 were obtained without heating and by
the use of DMF as a solvent. Deprotection of 1 and 2 was attempted under a variety of conditions,22
but in all cases resulted either in the hydrolysis of the thioglycosidic bond, cleavage of the anomeric
methanethiosulfonate group or failed to cleave the acetate protecting groups. Gratifyingly, careful control
of pH and conditions during subsequent protein glycosylation (vide infra) allowed in situ deprotection of
1 and the introduction of partially and fully deprotected D-glucose to our model protein.
Scheme 2. (i) NaSSO2CH3, EtOH, 90°C for 1 or DMF, rt for 2; (ii) acetonitrile, aqueous buffer (various pH; see Section 2.3)
2.2. Preparation of ethyl tethered α-gluco-, β-gluco, β-galacto-, α-manno- and lacto-MTS 3–12
For the preparation of the ethyl glyco-MTS 3–12, we chose a strategy that allowed the introduction of
the methanethiosulfonate group as a final step and which would therefore avoid the problems encountered
in the deprotection of 1 and 2. The preparation of these reagents in fully protected 3,5,7,9,11 and
deprotected 4,6,8,10,12 forms allowed the effects of increased steric bulk and hydrophobicity to be
assessed. Furthermore, the choice of a range of commonly occurring parent carbohydrate structures
allowed us to explore the generality of the method. BF3·Et2O-catalyzed Fischer glycosidation of D-
glucose 15 in 2-bromoethanol afforded a bromoethyl α-D-glucopyanoside, which for ease of purification
was isolated as its tetraacetate 16. Treatment of 16 with NaSSO2CH3 in DMF at 50°C allowed the
preparation in 90% yield of the peracetylated ethyl α-D-gluco-MTS 3 (43% overall yield from 15).
Alternatively, Zemplén deprotection22a of bromide 16 using a catalytic amount of NaOMe gave the fully
deprotected bromide 17. Nucleophilic displacement of bromide by methanethiosulfonate ion proceeded
smoothly at 50°C to yield the deprotected ethyl α-gluco-MTS 4 in 73% yield (33% overall yield from
15).
The β-D-gluco-MTS reagents 5 and 6, epimeric at C-1 to 3 and 4, respectively, were prepared from
the corresponding peracetylated β-bromide 18.23 The preparation of 18 took advantage of well-defined
methodology utilizing Lewis acid-catalyzed displacement of anomeric acetate groups by alcohols.24 As
a consequence of anchimeric assistance by the neighbouring C-2 acetate group, the β-glucoside 18 is the
major product of the treatment of a solution of pentaacetyl glucose and 2-bromoethanol with BF3·Et2O.
Compound 18 was then elaborated to the corresponding peracetylated, 5, and deprotected, 6, ethyl
β-gluco-MTS reagents in an essentially identical manner to that used for the epimeric α-gluco-MTS
reagents. Thus, using NaSSO2CH3, 18 gave 5 in 80% yield (48% overall yield from 15) and, following
deprotection, 1925 afforded 6 in 74% yield (42% overall yield from 15).

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
249
A parallel route starting from D-galactose 20 allowed the efficient preparation of the β-D-galacto-MTS
7 (55% overall yield from 20) and 8 (43% overall yield from 20), epimeric at C-4 to 5 and 6, respectively,
via bromide 2123 (Scheme 3). Similarly, ethyl α-D-manno-MTS 9 and 10, epimeric at C-2 to 3 and
4, respectively, were prepared using the same acetate displacement methodology. Thus, treatment of
peracetyl D-mannose with 2-bromoethanol and BF3·etherate again proceeded with anchimeric assistance
by the C-2 acetate and yielded bromoethyl α-mannoside 24 in 64% yield. This key intermediate was
elaborated to fully protected ethyl D-manno-MTS 9 (in 57% overall yield from D-mannose 23) and after
deprotection ethyl D-manno-MTS 10 (49% overall yield from 23).
Scheme 3. (i) Br(CH2)2OH, BF3·Et2O; (ii) Ac2O, py.; (iii) Br(CH2)2OH, BF3·Et2O, DCM; (iv) NaSSO2CH3, DMF, 50°C; (v)
NaOMe, MeOH
The adaptability of this method to disaccharides was illustrated by the preparation of the peracetylated
11 and fully deprotected 12 disaccharide ethyl lacto-MTS, without cleavage of the interresidue glycosidic
bond, in overall yields from lactose 27 of 47% and 32%, respectively.
2.3. Site-selective protein glycosylation
To illustrate the ability of glyco-MTS as protein glycosylation reagents we glycosylated the model
protein SBL using β-D-gluco-MTS 1 and N-acetyl-β-D-glucosamine MTS 2. This allowed us to explore
the effects of two configurationally identical reagents bearing different substituents at C-2. Thus, 1 and 2
were reacted in aqueous buffer with four cysteine mutants prepared by site-directed mutagenesis:26 SBL-

250
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
N62C, -S156C, -S166C and -L217C. These reactions were rapid (complete within 1 h) and quantitative,
as judged by monitoring changes in specific activity and by titration of residual free thiols with Ellman’s
reagent.27 The glycosylated proteins were purified by size-exclusion chromatography and dialysis, and
their structures were confirmed by rigorous ES-MS analysis (m/z ꢀ 5). The high level of purity indicated
by ES-MS was confirmed by non-denaturing gradient polyacrylamide gel electrophoresis (PAGE) and
fast protein liquid chromatography (FPLC). In all cases, complete integrity of the site selectivity was
retained. The active enzyme concentration of the resulting glycosylated SBL solutions was determined
by active site titration with α-toluenesulfonyl fluoride (PMSF) using a fluoride ion-sensitive electrode.28
The extent of deacetylation during modification is highly reagent-dependent (Table 1). Modification of
L217C with reagent 1 at pH 9.5 was accompanied by complete in situ deacetylation, and the sole product
was the fully deprotected glucosylated-SBL, L217C-S-β-Glc. Furthermore, through the variation of pH
the level of deacetylation was controlled. Thus, the reaction of L217C with 1 at pH 7.5 and 5.5 yielded
products in which the glucosyl residue introduced retained two and three acetate groups, forming L217C-
S-β-Glc(Ac)2 and L217C-S-β-Glc(Ac)3, respectively. In contrast, treatment of cysteine mutant L217C
with 2 under identical conditions resulted in no concomitant deacetylation during glycosylation and
the formation of the fully protected glucosylated-SBL, L217C-S-β-GlcNAc(Ac)3. We have previously
attributed this valuable site-dependent deacetylation to a novel intramolecular SBL-catalyzed process.14h
Due to the similarity in structure of 1 and 2 we may attribute their different reactivities solely to the
presence of an NHAc at C-2 in 2 but an OAc group at C-2 in 1 and we speculate that deacylation of 2 is
inhibited by the presence of a less labile acetamide rather than acetate group. Glycosylation at sites 62,
156 and 166 with 1 and 2 at pH 9.5 introduced only fully acetylated glucosyl residues.
Table 1
Kinetic parameters of glycosylated enzymes in amide hydrolysisa
The effects of glycosylation upon the enzyme SBL were assessed by the determination of kinetic
parameters kcat and KM for the hydrolysis of the amide substrate succinyl-AAPF-p-nitroanilide (Suc-
AAPF-pNA) and the ester substrate succinyl-AAPF-S-benzyl (Suc-AAPF-SBn) at pH 8.6 (Tables 1 and
2, respectively).
In all cases and at all positions, glycosylation lowers SBL’s amide hydrolysis activity. The two
0
most dramatic reductions are caused by glycosylations in the S1 and S1 pockets. Introduction of

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
251
Table 2
Kinetic parameters of glycosylated enzymes in ester hydrolysisb
0
fully deprotected untethered glucose to site 217 in the S1 pocket, forming L217C-S-β-Glc, lowered
kcat/KM to sixfold lower than unglycosylated SBL-WT. Interestingly, the results of glycosylation with
both acetylated D-glucose or N-Ac-D-glucosamine to form L217C-S-β-Glc(Ac)2, L217C-S-β-Glc(Ac)3
or L217C-S-GlcNAc(Ac)3 are far less marked, leading to reductions of no more than threefold in
activity, and suggest that capping of the hydrophilic hydroxyl groups reduces this deleterious effect of
glycosylation.
The introduction of peracetylated N-Ac-D-glucosamine to site 166 in the S1 pocket to form S166C-
S-β-GlcNAc(Ac)3 reduces amide hydrolysis activity 15.5-fold relative to WT. This striking decrease
is in contrast to the effect of introducing peracetylated D-glucose to form S166C-S-β-Glc(Ac)4 which
causes only a fourfold reduction in activity. Differences between these two glycosylated enzymes can
be attributed to changing a single group at C-2 in the sugar introduced. The effect of changing a C-2
-OAc group in S166C-S-β-Glc(Ac)4 to a -NHAc in S166C-S-β-GlcNAc(Ac)3 halves catalytic turnover
(kcat) and binding interactions (as judged by KM), perhaps due to unfavourable interactions of the N–H
hydrogen bond donor found only in S166C-S-β-GlcNAc(Ac)3 with the phenyl side-chain of the substrate
suc-AAPF-pNA.
The two glyco-MTS reagents 1 and 2 had generally opposite effects upon the ester hydrolysis activity
of SBL. Whereas 2 gave glycosylated enzymes with lower activities than the unglycosylated enzyme
in all cases, excitingly 1 gave three enzymes with better than WT activities. These three enhanced
0
enzymes all resulted from glycosylations at position 217, in the S1 pocket. Glycosylation with fully
deprotected D-glucose to form L217C-S-β-Glc increased kcat/KM by 1.8-fold. Furthermore, increasing
the number of acetate groups present in the introduced glycosyl residue further improved this beneficial
effect to 2.4-fold greater than WT for L217C-S-β-Glc(Ac)2 and to a dramatic 8.4-fold greater than WT
for L217C-S-β-Glc(Ac)3.14j Interestingly, the corresponding effect of introducing a peracetylated N-Ac-
D-glucosaminyl residue at the same site (217) is almost negligible. The striking differences in behaviour
caused by glycosylation at site 217 between 1 and 2 were further investigated through molecular
modelling.

252
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
2.4. Molecular modelling
The results of molecular mechanics analysis of the structures of L217C-S-β-Glc(Ac)3 and L217C-S-
β-GlcNAc(Ac)3 are shown in Fig. 2. Similar overall minimum energy structures emerge as a result of the
extensive solvation of the C-3, 4 and 6 substituents of the carbohydrate residues (bulk solvent omitted for
clarity). This in turn forces the C-2 substituent (OAc or NHAc) towards the key nucleophilic serine of the
catalytic triad of SBL. We have previously suggested14j that the carbonyl oxygen of the C-2 acetate of
L217C-S-β-Glc(Ac)3 might stabilize a crucial nucleophilic water molecule by acting as a hydrogen bond
(1.89 Å) acceptor. This may create a channel of solvent to the active site of SBL, leading to enhanced
esterase activity. This is supported by the minimum energy structure shown in Fig. 2, left.
Fig. 2. Molecular mechanics analysis of L217C-S-β-Glc(Ac)3 (left) and L217C-S-β-GlcNAc(Ac)3 (right). The substrate mimic
0
AAPF (purple) is shown binding in the S1 subsite, whilst glycan occupies the S1 subsite. These carbohydrate moieties allow a
channel of water molecules (yellow) to enter the active site from bulk through hydrogen bonding interactions
In L217C-S-β-GlcNAc(Ac)3 (Fig. 2, right) the mode of bonding and hence the role of the C-2
substituent (here NHAc) is reversed. The N–H of NHAc now acts as a hydrogen bond (2.10 Å) donor
to the oxygen atom of an equivalent nucleophilic water. This serves to reduce the nucleophilicity by
lowering electron density. There is consequently no enhancement of the esterase activity of L217C-S-β-
GlcNAc(Ac)3.

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
253
3. Conclusions
With the aim of creating precise structure–activity relationships (SARs) for protein glycosylation
which may be of use in the preparation of, for example, novel therapeutics or enhanced catalysts,30 we
have developed a novel chemical glycosylation strategy that uses powerful glyco-MTS reagents. These
reagents are readily synthesized and allow a high level of flexibility in the nature of SARs that can be
surveyed. For example, experiments on heterogeneous glycoconjugate samples have demonstrated that
the nature, and in particular the length, of the spacer arm that is used to conjugate carbohydrates to
proteins influences both the efficiency of conjugation31 and the strength of binding interactions of the
resulting conjugate.32 In order to probe the effect of distance between sugar and protein in a precise
manner, we also intend to construct glyco-MTS in which the tether length may be systematically varied.
The results of these further syntheses and full details of SARs created using reagents 1–12 will be
published elsewhere.14i,j
4. Experimental
4.1. General experimental
Mutants of subtilisin B. lentus (SBL) were generated, and WT and mutant enzymes purified as descri-
bed previously.26 These mutants were glycosylated with 1 and 2 and purified as described previously.14i
The preparation of NaSSO2CH3 has been described previously.18 Acetobromo-D-glucose 1733 in 73%
yield, pentaacetylglucose34 in 99% yield and pentaacetylmannose34 in 92% yield, pentaacetylgalactose34
in 99% yield, octaacetyllactose35 in 82% yield were prepared according to literature methods. DMF was
distilled under N2 from CaH2 and stored over molecular sieve under N2 before use. Dichloromethane,
methanol and ethanol were distilled under N2 from CaH2, Mg/I2 and Mg/I2, respectively, immediately
prior to use. Br(CH2)2OH was stood over and distilled from CaO under reduced pressure and stored under
N2 prior to use. All other chemicals were used as received from Sigma-Aldrich, Fluorochem or Baker.
Thin layer chromatography (TLC) was carried out on aluminium sheets coated with silica gel 60F254
.
Plates were developed using an ammonium molybdate dip. Flash chromatography was performed using
silica gel (Whatman, 60 Å, 230–400 Mesh). Melting points were determined using an Electrothermal
IA9000 series digital or Gallenkamp melting point apparatus and are uncorrected. Optical rotations were
measured on an Optical Activity Ltd. AA-10 or Perkin–Elmer 241 polarimeter with a path length of 1 dm.
Concentrations are given in g/100 ml. IR spectra were recorded on Bomem MB, Perkin–Elmer Spectrum
1
1000 or Perkin–Elmer Series 1600 Fourier Transform spectrophotometers. H NMR and 13C NMR
spectra were recorded on Varian Gemini 200, Unity 300, Unity 400 or Unity 500 NMR spectrometers at
the frequencies indicated. Where indicated, NMR peak assignments were made using COSY or DEPT
experiments; all others are subjective. All chemical shifts were referenced to residual solvent as an
internal standard; for 13C NMR in D2O 1,4-dioxan (67.6 ppm) was used. Protein ES-MS data were
acquired using a PE SCIEX API III biomolecular mass spectrometer. Mass spectra were acquired using
Micromass 70–250S, Micromass Autospec, Micromass ZAB-SE or VG Platform mass spectrometers
according to the ionization methods indicated. High Resolution ESMS were performed by the EPSRC
Mass Spectrometry Service at Swansea, UK. Microanalyses were performed by the microanalysis service
at Durham University, Chemistry Department. Solvents were removed in vacuo.

254
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
4.2. 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl methanethiosulfonate 1
Acetobromoglucose 13 (1 g, 2.43 mmol) was added to a solution of NaSSO2CH3 (380 mg, 2.84 mmol)
in ethanol (4 ml) at 90°C under N2. After 20 min the resulting suspension was cooled and the solvent
removed. The residue was purified by flash chromatography (EtOAc:hexane, 9:11) and the resulting solid
recrystallized from ether to give 1 (674 mg, 63%) as a white solid; mp 151–152°C, melts then decomp.
1
(ether); [α]D27=−19.0 (c 1.24, CHCl3); IR (KBr) 1749 (C_O), 1333, 1140 cm−1 (S–SO2); H NMR
(400 MHz, CDCl3) δ 2.00, 2.04, 2.06, 2.07 (s×4, 3H×4, Ac×4), 3.44 (s, 3H, CH3SO2-), 3.82 (ddd, J4,5
0
0
0
10.1 Hz, J5,6 5.9 Hz, J5,6 2.2 Hz, 1H, H-5), 4.08 (dd, J5,6 5.9 Hz, J6,6 12.5 Hz, 1H, H-6), 4.31 (dd, J5,6
2.2 Hz, J6,6 12.5 Hz, 1H, H-60), 5.05 (t, J 9.8 Hz, 1H, H-4), 5.07 (dd, J1,2 10.5 Hz, J2,3 9.4 Hz, 1H,
H-2), 5.25 (d, J1,2 10.5 Hz, 1H, H-1), 5.29 (t, J 9.3 Hz, 1H, H-3); 13C NMR (50 MHz, CDCl3) δ 20.5,
20.7 (CH3COO-×4), 52.8 (CH3SO2-), 61.8, 68.0, 68.7, 73.3, 76.6 (C-2, C-3, C-4, C-5, C-6), 86.4 (C-1),
169.3, 169.3, 169.7, 170.1 (CH3COO-×4); HRMS m/z (EI+): found 443.0636 (M+H+); C15H23O11S2
requires 443.0682.
0
4.3. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl methanethiosulfonate 2
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride 3,4,6-triacetate 14 (2 g, 5.722
mmol) was added to a solution of NaSSO2CH3 (900 mg, 6.72 mmol) in DMF (35 ml) under N2 and
stirred for 72 h. The reaction course was followed by TLC (EtOAc:hexane, 70:30) which showed the
consumption of starting material (Rf 0.2) and the formation a major product (Rf 0.4). The solvent was
removed and the residue purified by flash chromatography (EtOAc:hexane, 70:30) to give an off-white
powder. The powder was recrystallized (EtOAc/hexane) to give 2 as a white crystalline powder (0.584
g, 25%); mp 139–140°C; [α]D20=−33.0 (c 0.06, CHCl3); IR (KBr) 3400 (br, N–H), 1748 (C_O), 1657
(amide I), 1542 (amide II), 1311, 1139 cm−1 (S–SO2); H NMR (300 MHz, CD3CN) δ 1.84, 1.96, 1.99,
1
0
0
2.01 (s×4, 3H×4, Ac×4), 3.46 (s, 3H, SSO2CH3), 3.9–3.96 (ddd, J5,6 2.4 Hz, J5,6 6.2 Hz, J6,6 10.0 Hz,
0
1H, H-5), 4.05–4.15 (m, 2H), 4.20 (dd, J5,6 2.4 Hz, J6,6 12.4, 1H, H-6), 5.00 (pt, J 9.8 Hz, 1H, H-2), 5.28
(pt, J 9.8 Hz, 1H, H-3), 5.40 (d, J1,2 10.8 Hz, 1H, H-1), 6.6 (d, J 10 Hz, 1H, NHAc); 13C NMR (50.3 MHz,
CD3CN) δ 20.1, 20.2 (CH3COO-×3), 22.2 (CH3CONH-), 51.8 (SSO2CH3), 52.2, 62.4, 68.6, 72.8, 76.2
(C-2,3,4,5,6), 87.62 (C-1), 169.8, 170.4, 170.4, 170.6 (CH3COO×3, CH3CONH); m/z (ES): 464 (14,
M+Na+), 352 (100%, oxazoline+Na+). Found: C, 41.22; H, 5.43; N, 2.90%; C15H23O10NS2 requires: C,
40.81; H, 5.25; N, 3.17%.
4.4. 2-(2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl)ethyl methanethiosulfonate 3
BF3·Et2O (145 µL, 1.1 mmol) was added dropwise to a suspension of D-glucose 15 (1.45 g, 8.1 mmol)
in Br(CH2)2OH (19 ml) under N2 and the resulting mixture heated to 105°C. After 8 h the resulting
solution was cooled and the solvent removed. The residue was dissolved in Ac2O:pyridine (2:3 v/v, 16
ml) under N2. After a further 24 h the reaction solvent was removed and the residue purified by repeated
flash chromatography (EtOAc then EtOAc:hexane, 3:7) to give 2-bromoethyl 2,3,4,6-tetra-O-acetyl-α-
D-glucopyranoside 16 (1.76 g, 48%) as a colourless oil that crystallized on standing to give a white solid;
mp 86–88°C; [α]25D=+130.6 (c 0.21, CHCl3); IR (film) 1749 cm−1 (C_O); 1H NMR (400 MHz, CDCl3)
δ 2.01, 2.03, 2.07, 2.09 (s×4, 3H×4, Ac×4), 3.51 (t, J 5.9 Hz, 2H, -CH2Br), 3.83 (dt, Jd 11.6 Hz, Jt 5.8
Hz, 1H, -OCHH0-), 3.96 (dt, Jd 11.6 Hz, Jt 5.8 Hz, 1H, -OCHH0-), 4.10 (dd, J5,6 2.2 Hz, J6,6 12.0 Hz,
0
0
0
0
1H, H-6), 4.14 (ddd, J4,5 10.2 Hz, J5,6 2.2 Hz, J5,6 4.4 Hz, 1H, H-5), 4.24 (dd, J5,6 4.4 Hz, J6,6 12.0
Hz, 1H, H-60), 4.84 (dd, J1,2 3.8 Hz, J2,3 10.3 Hz, 1H, H-2), 5.05 (t, J 9.7 Hz, 1H, H-4), 5.14 (d, J1,2 3.8

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
255
Hz, 1H, H-1), 5.49 (dd, J2,3 10.3 Hz, J3,4 9.5 Hz, 1H, H-3); 13C NMR (100 MHz, CDCl3) δ 20.6, 20.7
(CH3COO-×4), 29.9 (-CH2Br), 61.9, 67.8, 68.5, 68.8, 70.0, 70.8 (-OCH2-, C-2, C-3, C-4, C-5, C-6),
96.0 (C-1), 169.6, 170.0, 170.2, 170.6 (CH3COO-×4); HRMS m/z (FAB+): found 477.0381 (M+Na+);
C16H23O10BrNa requires 477.0372. NaSSO2CH3 (75 mg, 0.56 mmol) was added to a solution of 16 (190
mg, 0.42 mmol) in DMF (6 ml) under N2 and warmed to 50°C. After 21 h the solution was cooled and
the solvent removed. The residue was purified by flash chromatography (EtOAc:hexane, 1:1) to give 3
(183 mg, 90%) as a colourless oil; [α]D27=+92.1 (c 0.39, CHCl3); IR (film) 1748 (C_O), 1322, 1134
1
cm−1 (S–SO2); H NMR (400 MHz, CDCl3) δ 2.01, 2.03, 2.07, 2.09 (s×4, 3H×4, Ac×4), 3.41 (t, J
5.7 Hz, 2H, -CH2S-), 3.41 (s, 3H, CH3SO2-), 3.75 (dt, Jd 10.8 Hz, Jt 5.7 Hz, 1H, -OCHH0-), 3.99–4.06
(m, 2H, H-5, -OCHH0-), 4.09 (dd, J5,6 2.4 Hz, J6,6 12.6 Hz, 1H, H-6), 4.25 (dd, J5,6 4.6 Hz, J6,6 12.6
Hz, 1H, H-60), 4.87 (dd, J1,2 3.9 Hz, J2,3 10.3 Hz, 1H, H-2), 5.06 (t, J 9.8 Hz, 1H, H-4), 5.12 (d, J1,2
3.9 Hz, 1H, H-1), 5.43 (t, J 9.8 Hz, 1H, H-3); 13C NMR (100 MHz, CDCl3) δ 20.6, 20.6, 20.7, 20.7
(CH3COO×4), 36.0, (-CH2S-), 50.8 (CH3SO2-), 61.8 (-OCH2-), 67.0, 67.8, 68.3, 69.8, 70.7 (C-2, C-3,
C-4, C-5, C-6), 96.0 (C-1), 169.5, 170.0, 170.6 (CH3COO-×4); HRMS m/z (FAB+): found 487.0946
(M+H+); C17H27O12S2 requires 487.0944.
0
0
0
4.5. 2-(α-D-Glucopyranosyl)ethyl methanethiosulfonate 4
A solution of NaOMe (0.1 M, 0.3 ml) was added to a suspension of 16 (300 mg, 0.66 mmol) in
MeOH (3 ml) under N2 and stirred vigorously. After 6 h the resulting solution was passed through a
Dowex 50W(H+) plug (2×1 cm, eluant MeOH) and the solvent removed to give 2-bromoethyl α-D-
glucopyranoside bromide36 17 (178 mg, 94%) as a white solid. NaSSO2CH3 (100 mg, 0.75 mmol) was
added to a solution of 4b (178 mg, 0.62 mmol) in DMF (7 ml) under N2 and warmed to 50°C. After 25
h the solution was cooled and the solvent removed. The residue was purified by flash chromatography
(MeOH:EtOAc, 1:9) to give 4 (144 mg, 73%) as a hygroscopic foam; [α]D27=+109.9 (c 1.11, H2O); IR
(film) 3423 (OH), 1309, 1128 cm−1 (S–SO2); 1H NMR (500 MHz, D2O, COSY) δ 3.16 (t, J 9.5 Hz, 1H,
H-4), 3.28 (t, J 5.9 Hz, 2H, -CH2S-), 3.30 (s, 3H, CH3SO2-), 3.31 (dd, J1,2 3.8 Hz, J2,3 9.9 Hz, 1H, H-2),
3.44 (t, J 9.5 Hz, 1H, H-3), 3.47–3.53 (m, 2H, H-6, H-60), 3.58–3.61 (m, 1H, H-5), 3.62 (dt, Jt 5.4 Hz,
Jd 10.8 Hz, 1H, -OCHH0-), 3.79 (dt, Jt 6.3 Hz, Jd 10.8 Hz, 1H, -OCHH0), 4.72 (d, J1,2 3.8 Hz, 1H, H-1);
13C NMR (100 MHz, D2O) δ 36.7 (-CH2S-), 50.7 (CH3SO2-), 61.5 (-OCH2-), 67.2, 70.5, 72.3, 73.2,
74.0 (C-2, C-3, C-4, C-5, C-6), 99.4 (C-1); HRMS m/z (FAB+): found 319.0517 (M+H+); C9H19O8S2
requires 319.0521.
4.6. 2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)ethyl methanethiosulfonate 5
BF3·Et2O (3.3 ml, 26.0 mmol) was added dropwise over the course of 15 min to a solution of 1,2,3,4,6-
penta-O-acetyl-α,β-D-glucose (2 g, 5.1 mmol) and Br(CH2)2OH (0.45 ml, 6.3 mmol) in CH2Cl2 (9 ml)
at 0°C under N2. After 1.5 h the solution was warmed to room temperature. After 20 h the reaction
solution was added to ice water (15 ml) and extracted with CH2Cl2 (15 ml×3). These extracts were
combined, washed with water (15 ml), satd NaHCO3 (aq. 15 ml), water (15 ml), dried (MgSO4),
filtered and the solvent removed. The residue was purified by flash chromatography (EtOAc:hexane,
1:3) to give 2-bromoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 18 (1.42 g, 61%) as a white solid;
mp 118–120°C (EtOAc/iso-octane) [lit.,23 mp 117.3°C (EtOH)]; [α]D27=−11.9 (c 1.65, CHCl3) [lit.,37
[α]D20=−12.3 (c 0.2, CHCl3)]; H NMR (200 MHz, CDCl3) δ 2.00, 2.02, 2.07, 2.09 (s×4, 3H×4,
1
Ac×4), 3.42–3.51 (m, 2H), 3.67–3.87 (m, 2H), 4.10–4.31 (m, 3H), 4.57 (d, J1,2 8 Hz, 1H, H-1), 4.97–5.27
(m, 3H). NaSSO2CH3 (260 mg, 1.94 mmol) was added to a solution of 18 (640 mg, 1.41 mmol) in

256
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
DMF (18 ml) under N2 and warmed to 50°C. After 25 h the solution was cooled and the solvent
removed. The residue was purified by flash chromatography (EtOAc:hexane, 1:1) and the resulting
solid recrystallized from EtOAc/hexane to give 5 (544 mg, 80%) as a white solid; mp 115–116°C
(EtOAc/hexane); [α]D27=+5.4 (c 1.06, CHCl3); IR (KBr) 1758, 1741 (C_O), 1314, 1133 cm−1 (S–SO2);
1H NMR (500 MHz, CDCl3, COSY) δ 1.99, 2.02, 2.06, 2.08 (s×4, 3H×4, Ac×4), 3.30–3.38 (m, 2H,
0
-CH2S-), 3.34 (s, 3H, CH3SO2-), 3.70 (ddd, J4,5 9.9 Hz, J5,6 2.2 Hz, J5,6 4.6 Hz, 1H, H-5), 3.83 (ddd, J
5.6 Hz, J 7.4 Hz, J 10.5 Hz, 1H, -OCHH0-), 4.13–4.18 (m, 2H, H-6, -OCHH0-), 4.24 (dd, J5,6 4.6 Hz,
0
J6,6 12.4 Hz, 1H, H-60), 4.55 (d, J1,2 8.1 Hz, 1H, H-1), 4.98 (dd, J1,2 8.1 Hz, J2,3 9.7 Hz, 1H, H-2), 5.07
0
(t, J 9.9 Hz, 1H, H-4), 5.19 (t, J 9.6 Hz, 1H, H-3); 13C NMR (125 MHz, CDCl3, DEPT) δ 20.5, 20.7
(q×2, CH3COO-×4), 36.0 (t, -CH2S-), 50.6 (q, CH3SO2-), 61.6 (t, -OCH2-), 68.1, 70.8, 71.9, 72.5 (d×4,
C-2, C-3, C-4, C-5), 68.4 (t, C-6), 100.8 (d, C-1), 169.3, 170.0, 170.5 (s×3, CH3COO-×4); HRMS m/z
(FAB+): found 487.0940 (M+H+); C17H27O12S2 requires 487.0944.
4.7. 2-(β-D-Glucopyranosyl)ethyl methanethiosulfonate 6
A solution of NaOMe (0.1 M, 0.3 ml) was added to a suspension of 18 (300 mg, 0.66 mmol) in
MeOH (3 ml) under N2 and stirred vigorously. After 4 h the resulting solution was passed through a
Dowex 50W(H+) plug (2×1 cm, eluant MeOH) and the solvent removed to give 2-bromoethyl β-D-
glucopyranoside 19 (176 mg, 93%) as a white solid that was used directly in the next step. A sample
was recrystallized from EtOH/EtOAc to give a colourless, crystalline solid; mp 74–78°C (EtOH/EtOAc)
[lit.,37 mp 74–75°C (EtOH/EtOAc)]; [α]D26=−22.4 (c 1.63, H2O) [lit.,37 [α]D19=−26.1 (c 3.0, H2O)];
1H NMR (400 MHz, CD3OD) δ 3.30 (t, J 8.4 Hz, 1H, H-2), 3.39–3.49 (m, 3H), 3.64–3.80 (m, 3H), 3.97
(br d, J6,6’ 11.7 Hz, 1H, H-60), 4.02 (dt, Jt 6.5 Hz, Jd 11.3 Hz, 1H, -OCHH0-), 4.23 (dt, Jt 6.5 Hz, Jd 11.3
Hz, 1H, -OCHH0-), 4.44 (d, J1,2 7.9 Hz, 1H, H-1). NaSSO2CH3 (100 mg, 0.75 mmol) was added to a
solution of 19 (176 mg, 0.61 mmol) in DMF (7 ml) under N2 and warmed to 50°C. After 15 h the solution
was cooled and the solvent removed. The residue was purified by flash chromatography (MeOH:EtOAc,
1:9) to give 6 (144 mg, 74%) as a hygroscopic foam; [α]D27=−15.8 (c 0.88, H2O); IR (KBr) 3400 (OH),
1310, 1131 cm−1 (S–SO2); 1H NMR (500 MHz, D2O, COSY) δ 3.07 (dd, J1,2 8.1 Hz, J2,3 9.4 Hz, 1H,
H-2), 3.16 (dd, J3,4 9.0 Hz, J4,5 9.8 Hz, 1H, H-4), 3.24 (ddd, J4,5 9.8 Hz, J5,6 6.0 Hz, J5,6’ 2.3 Hz, 1H,
H-5), 3.27 (t, J 9.0 Hz, 1H, H-3), 3.30–3.33 (m, 2H, -CH2S-), 3.34 (s, 3H, CH3SO2-), 3.50 (dd, J5,6 6.0
Hz, J6,6 12.4 Hz, 1H, H-6), 3.69 (dd, J5,6 2.3 Hz, J6,6 12.4 Hz, 1H, H-60), 3.81 (dt, Jt 5.8 Hz, Jd 11.5
Hz, 1H, -OCHH0-), 4.00 (dt, Jt 5.7 Hz, Jd 11.4 Hz, 1H, -OCHH0-), 4.30 (d, J1,2 8.1 Hz, 1H, H-1); 13
0
0
0
C
NMR (50 MHz, D2O) δ 36.9 (-CH2S-), 51.0 (CH3SO2-), 62.0 (-OCH2-), 69.5, 70.9, 74.3, 76.7, 77.3
(C-2, C-3, C-4, C-5, C-6), 103.7 (C-1); HRMS m/z (FAB+): found 341.0351 (M+Na+); C9H18O8S2Na
requires 341.0341.
4.8. 2-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)ethyl methanethiosulfonate 7
BF3·Et2O (8.5 ml, 67.0 mmol) was added dropwise to a solution of 1,2,3,4,6-penta-O-acetyl-α,β-D-
galactose (5.1 g, 13.1 mmol) and Br(CH2)2OH (1.15 ml, 16.2 mmol) in CH2Cl2 (24 ml) at 0°C under N2.
After 1 h the solution was warmed to room temperature. After 24 h the reaction solution was added to ice
water (20 ml) and extracted with CH2Cl2 (30 ml×3). These extracts were combined, washed with water
(20 ml), satd NaHCO3 (aq., 20 ml), water (20 ml), dried (MgSO4), filtered and the solvent removed. The
residue was purified by flash chromatography (EtOAc:hexane, 1:3) to give 2-bromoethyl 2,3,4,6-tetra-O-
acetyl-β-D-galactopyranoside 21 (4.01 g, 67%) as a white solid; mp 116–117°C (EtOAc/hexane) [lit.,23
111°C; lit.,39 114–116°C (EtOAc/light pet. ether)]; [α]D27=−3.8 (c 0.81, CHCl3) [lit.,39 [α]D23=−5 (c

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
257
1.4, CDCl3)]; 1H NMR (200 MHz, CDCl3) δ 1.98, 2.05, 2.08, 2.15 (s×4, 3H×4, Ac×4), 3.43–3.50 (m,
2H), 3.75–3.95 (m, 2H), 4.12–4.24 (m, 3H), 4.53 (d, J1,2 8 Hz, 1H, H-1), 5.02 (dd, J2,3 11 Hz, J3,4
3
Hz, 1H, H-3), 5.23 (dd, J1,2 8 Hz, J2,3 11 Hz, 1H, H-2), 5.40 (br d, J3,4 3 Hz, 1H, H-4). NaSSO2CH3
(85 mg, 0.63 mmol) was added to a solution of 21 (223 mg, 0.49 mmol) in DMF (6 ml) under N2 and
warmed to 55°C. After 30 h the solution was cooled and the solvent removed. The residue was purified
by flash chromatography (EtOAc:hexane, 1:1) to give 7 (198 mg, 83%) as a white foam; [α]D27=+9.1 (c
1.41, CHCl3); IR (film) 1747 (C_O), 1320, 1133 cm−1 (S–SO2); 1H NMR (500 MHz, CDCl3) δ 1.98,
2.05, 2.09, 2.15 (s×4, 3H×4, Ac×4), 3.35 (s, 3H, CH3SO2-), 3.35–3.38 (m, 2H, -CH2S-), 3.84 (ddd, J
6.1 Hz, J 7.1 Hz, J 10.5 Hz, 1H, -OCHH0-), 3.92 (td, J4,5 1.1 Hz, Jt 6.6 Hz, 1H, H-5), 4.10–4.21 (m,
3H, H-6, H-60, -OCHH0-), 4.52 (d, J1,2 8.0 Hz, 1H, H-1), 5.01 (dd, J2,3 10.3 Hz, J3,4 3.5 Hz, 1H, H-3),
5.20 (dd, J1,2 8.0 Hz, J2,3 10.3 Hz, 1H, H-2), 5.40 (dd, J3,4 3.5 Hz, J4,5 1.1 Hz, 1H, H-4); 13C NMR (100
MHz, CDCl3) δ 20.6, 20.7, 20.8 (CH3COO-×4), 36.1, (-CH2S-), 50.6 (CH3SO2-), 61.2 (-OCH2-), 67.0,
68.3, 68.5, 70.8, 71.0 (C-2, C-3, C-4, C-5, C-6), 101.3 (C-1), 169.5, 170.0, 170.1, 170.4 (CH3COO-×4);
HRMS m/z (FAB+): found 487.0936 (M+H+); C17H27O12S2 requires 487.0944.
4.9. 2-(β-D-Galactopyranosyl)ethyl methanethiosulfonate 8
A solution of NaOMe (0.104 M, 0.8 ml) was added to a solution of 21 (778 mg, 1.71 mmol) in MeOH
(10 ml) under N2. After 4 h the reaction solution was passed through a Dowex 50W(H+) plug (3×1
cm, eluant MeOH) and the solvent removed to give 2-bromoethyl β-D-galactopyranoside38 22 (450 mg,
92%) as a white solid which was used directly in the next step. NaSSO2CH3 (180 mg, 1.34 mmol) was
added to a solution of 22 (290 mg, 1.01 mmol) in DMF (12 ml) under N2 and warmed to 50°C. After 15
h the solution was cooled and the solvent removed. The residue was purified by flash chromatography
(MeOH:EtOAc, 1:9) to give 8 (229 mg, 71%) as a white foam; [α]D27=+2.9 (c 0.58, H2O); IR (film)
3358 cm−1 (br, O–H), 1306, 1120 cm−1 (S–SO2); 1H NMR (500 MHz, D2O, COSY) δ 3.29–3.33 (m,
2H, -CH2S-), 3.30 (dd, J1,2 7.7 Hz, J2,3 10.0 Hz, 1H, H-2), 3.35 (s, 3H, CH3SO2-), 3.43 (dd, J2,3 10.0
0
Hz, J3,4 3.6 Hz, 1H, H-3), 3.48 (ddd, J4,5 0.9 Hz, J5,6 4.3 Hz, J5,6 7.9 Hz, 1H, H-5), 3.52 (dd, J5,6 4.3
Hz, J6,6 11.7 Hz, 1H, H-6), 3.57 (dd, J5,6 7.9 Hz, J6,6 11.7 Hz, 1H, H-60), 3.70 (dd, J3,4 3.6 Hz, J4,5
0.9 Hz, 1H, H-4), 3.80 (dt, Jd 11.2 Hz, Jt 6.1 Hz, 1H, -OCHH0-), 4.01 (dt, Jd 11.4 Hz, Jt 5.8 Hz, 1H,
-OCHH0-), 4.24 (d, J1,2 7.7 Hz, 1H, H-1); 13C NMR (100 MHz, D2O) δ 36.7 (-CH2S-), 50.8 (CH3SO2-),
61.9 (-OCH2-), 69.2, 69.6, 71.7, 73.7, 76.2 (C-2, C-3, C-4, C-5, C-6), 104.0 (C-1); HRMS m/z (FAB+):
found 319.0523 (M+H+); C9H19O8S2 requires 319.0521.
0
0
0
4.10. 2-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl)ethyl methanethiosulfonate 9
BF3·Et2O (7.7 ml, 60.7 mmol) was added dropwise over the course of 15 min to a solution of 1,2,3,4,6-
penta-O-acetyl-α,β-D-mannose (4.7 g, 12.1 mmol) and Br(CH2)2OH (1.05 ml, 14.8 mmol) in CH2Cl2
(22 ml) at 0°C under N2. After 1 h the solution was warmed to room temperature. After 25 h the reaction
solution was added to ice water (20 ml) and extracted with CH2Cl2 (20 ml×2). These extracts were
combined, washed with water (20 ml), satd NaHCO3 (aq. 20 ml), water (20 ml), dried (MgSO4), filtered
and the solvent removed. The residue was crystallized from EtOAc/iso-octane to give 2-bromoethyl
2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside 24 (3.52 g, 64%). Purification of the resulting mother
liquor by flash chromatography (EtOAc:hexane, 1:3) gave further 24 (320 mg, 6%; 70% in total) as a
white highly crystalline solid; mp 121–123°C [lit.,39 118–119°C (EtOAc/iso-octane)]; [α]D28=+48.3 (c
1.31, CHCl3) [lit.,39 [α]D23=+45 (c 0.6, CDCl3)]; 1H NMR (200 MHz, CDCl3) δ 1.99, 2.05, 2.10, 2.16
(s×4, 3H×4, Ac×4), 3.52 (t, J 6 Hz, 2H, -CH2Br), 3.82–4.04 (m, 2H, -OCH2-), 4.09–4.16 (m, 1H, H-5),

258
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
4.13 (dd, J5,6 2 Hz, J6,6 12 Hz, 1H, H-6) 4.28 (dd, J5,6 6 Hz, J6,6 12 Hz, 1H, H-60), 4.87 (br s, 1H,
H-1), 5.22–5.40 (m, 3H, H-2, H-3, H-4). NaSSO2CH3 (230 mg, 1.72 mmol) was added to a solution
of 24 (600 mg, 1.32 mmol) in DMF (17 ml) under N2 and warmed to 55°C. After 20 h the solution
was cooled and the solvent removed. The residue was purified by flash chromatography (EtOAc:hexane,
9:11) and the resulting solid recrystallized from Et2O/hexane to give 9 (566 mg, 88%) as a white solid;
mp 128–129°C (Et2O/hexane); [α]D27=+53.2 (c 0.92, CHCl3); IR (KBr) 1739 (C_O), 1325, 1129 cm−1
(S–SO2); 1H NMR (500 MHz, CDCl3, COSY) δ 1.97, 2.04, 2.09, 2.14 (s×4, 3H×4, Ac×4), 3.37–3.40
(m, 2H, -CH2S-), 3.38 (s, 3H, CH3SO2-), 3.79 (dt, Jd 10.5 Hz, Jt 5.8 Hz, 1H, -OCHH0-), 3.98–4.03
0
0
0
(m, 2H, -OCHH0-, H-5), 4.09 (dd, J5,6 2.5 Hz, J6,6 12.5 Hz, 1H, H-6), 4.26 (dd, J5,6 5.6 Hz, J6,6 12.5
Hz, 1H, H-60), 4.85 (d, J1,2 0.7 Hz, 1H, H-1), 5.23–5.29 (m, 3H, H-2, H-3, H-4); 13C NMR (50 MHz,
CDCl3) δ 20.6, 20.7, 20.8 (CH3COO-×4), 35.7, (-CH2S-), 50.8 (CH3SO2-), 62.5 (-OCH2-), 66.0, 66.8,
69.0, 69.2, 69.3 (C-2, C-3, C-4, C-5, C-6), 97.7 (C-1), 169.7, 169.9, 170.0, 170.6 (CH3COO-×4); HRMS
m/z (FAB+): found 487.0954 (M+H+); C17H27O12S2 requires 487.0944.
0
0
0
4.11. 2-(α-D-Mannopyranosyl)ethyl methanethiosulfonate 10
A solution of NaOMe (0.143 M, 0.7 ml) was added to a suspension of 24 (1 g, 2.2 mmol) in
MeOH (10 ml) under N2. After 3 h the resulting solution was passed through a Dowex 50W(H+) plug
(2×1 cm, eluant MeOH) and the solvent removed. The residue was purified by flash chromatography
(MeOH:EtOAc, 2:25) to give 2-bromoethyl α-D-mannopyranoside40 25 (606 mg, 96%) as a white foam;
[α]D +50.7 (c 0.91, H2O); IR (KBr) 3417 cm−1 (OH); 1H NMR (500 MHz, D2O, COSY) δ 3.38–3.44
26=
(m, 3H, H-4, -CH2Br), 3.50–3.55 (m, 2H, H-5, H-6), 3.60 (dd, J2,3 3.5 Hz, J3,4 9.7 Hz, 1H, H-3), 3.66
(dd, J5,6 4.6 Hz, J6,6 11.2 Hz, 1H, H-60), 3.68 (ddd, J 4.6 Hz, J 5.4 Hz, J 11.7 Hz, 1H, -OCHH0-),
3.76 (dd, J1,2 1.8 Hz, J2,3 3.5 Hz, 1H, H-2), 3.81 (ddd, J 5.1 Hz, J 6.5 Hz, J 11.7, 1H, -OCHH0-), 4.71
(d, J1,2 1.8 Hz, 1H, H-1); 13C NMR (100 MHz, D2O) δ 32.1 (-CH2Br), 61.7 (-OCH2-), 67.5, 68.4,
70.7, 71.3, 73.8 (C-2, C-3, C-4, C-5, C-6), 100.5 (C-1); HRMS m/z (FAB+): found 308.9985 (M+Na+);
C8H15O679BrNa requires 308.9950. NaSSO2CH3 (150 mg, 1.12 mmol) was added to a solution of 25
(245 mg, 0.85 mmol) in DMF (10 ml) under N2 and warmed to 50°C. After 16 h the solution was cooled
and the solvent removed. The residue was purified by flash chromatography (MeOH:EtOAc, 1:9) to give
10 (217 mg, 80%) as a hygroscopic foam; [α]D29=+58.0 (c 1.34, H2O); IR (KBr) 3441 (OH), 1314, 1132
cm−1 (S–SO2); 1H NMR (500 MHz, D2O) δ 3.31 (t, J 5.8 Hz, 2H, -CH2S-), 3.35 (s, 3H, CH3SO2-), 3.45
0
0
0
(t, J 9.6 Hz, 1H, H-4), 3.49 (ddd, J4,5 9.8 Hz, J5,6 5.8 Hz, J5,6 1.9 Hz, 1H, H-5), 3.55 (dd, J5,6 5.8 Hz,
0
J6,6 12.1 Hz, 1H, H-6), 3.60 (dd, J2,3 3.4 Hz, J3,4 9.0 Hz, 1H, H-3), 3.66 (dt, Jd 10.7 Hz, Jt 5.7 Hz, 1H,
-OCHH0-), 3.69 (dd, J5,6 1.9, J6,6 12.1 Hz, 1H, H-60), 3.77 (dd, J1,2 1.6 Hz, J2,3 3.4 Hz, 1H, H-2), 3.83
(dt, Jd 11.0 Hz, Jt 5.9 Hz, 1H, -OCHH0-), 4.72 (d, J1,2 1.6 Hz, 1H, H-1); 13C NMR (125 MHz, D2O) δ
36.7 (-CH2S-), 50.7 (CH3SO2-), 61.9 (-OCH2-), 66.7, 67.7, 70.9, 71.5, 74.0 (C-2, C-3, C-4, C-5, C-6),
100.8 (C-1); HRMS m/z (FAB+): found 319.0528 (M+H+); C9H19O8S2 requires 319.0521.
0
0
4.12. 2-(2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)-
ethyl methanethiosulfonate 11
BF3·Et2O (4.0 ml, 31.5 mmol) was added dropwise to a solution of 1,2,3,6-tetra-O-acetyl-4-O-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside (5 g, 7.4 mmol) and Br(CH2)2OH
(0.65 ml, 9.2 mmol) in CH2Cl2 (15 ml) at 0°C under N2. After 1 h the solution was warmed to
room temperature. After 20 h the reaction solution was added to ice water (15 ml) and extracted with
CH2Cl2 (20 ml×2). These extracts were combined, washed with water (20 ml), satd NaHCO3 (aq.

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
259
20 ml), water (20 ml), dried (MgSO4), filtered and the solvent removed. The residue was purified by
flash chromatography (EtOAc:hexane, 1:1) to give 2-bromoethyl 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-
O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside 27 (2.94 g, 53%) as a white foam; [α]D27=−7.8
(c 1.28, CHCl3) [lit.,39 [α]D23=−11 (c 1.3, CHCl3)]; 1H NMR (500 MHz, CDCl3, COSY) δ 1.94, 2.02,
2.02 (s×3, 3H×3, Ac×3), 2.04 (s, 6H, Ac×2), 2.10, 2.13 (s×2, 3H×2, Ac×2), 3.38–3.46 (m, 2H, -
CH2Br), 3.59 (ddd, J5 ,6 2.2 Hz, J 4.9 Hz, J 9.9 Hz, 1H, H-50), 3.75–3.80 (m, 2H, H-40, -OCHH0-),
3.85 (td, J4,5 1.1 Hz, Jt 6.9 Hz, 1H, H-5), 4.03–4.12 (m, 4H, H-6, H-60, H-600, -OCHH0-), 4.45 (d, J1,2
0
000
12.1 Hz, 1H, H-6000), 4.50 (d, J1 ,2 7.9 Hz, 1H, H-10),
0 0
0
000
00,6000
7.8 Hz, 1H, H-1), 4.48 (dd, J5 ,6 2.2 Hz, J6
4.89 (dd, J1 ,2 7.9 Hz, J2 ,3 9.6 Hz, 1H, H-20), 4.92 (dd, J2,3 10.5 Hz, J3,4 3.4 Hz, 1H, H-3), 5.08 (dd,
J1,2 7.8 Hz, J2,3 10.5 Hz, 1H, H-2), 5.18 (t, J 9.6 Hz, 1H, H-30), 5.32 (dd, J3,4 3.4 Hz, J4,5 1.1 Hz, 1H,
H-4). NaSSO2CH3 (87 mg, 0.65 mmol) was added to a solution of 27 (357 mg, 0.48 mmol) in DMF (6
ml) under N2 and warmed to 50°C. After 22 h the solution was cooled and the solvent removed. The
residue was purified by flash chromatography (EtOAc:hexane, 11:9) to give 11 (327 mg, 88%) as a white
0
0
0
0
1
foam; [α]D27=−3.7 (c 1.0, CHCl3); IR (KBr) 1751 (C_O), 1323, 1134 cm−1 (S–SO2); H NMR (500
MHz, CDCl3, COSY) δ 1.94, 2.02, 2.02, 2.04, 2.04, 2.11, 2.13 (s×7, 3H×7, Ac×7), 3.29–3.40 (m, 2H,
-CH2S-), 3.32 (s, 3H, CH3SO2-), 3.59 (ddd, J4 ,5 9.9 Hz, J5 ,6 4.9 Hz, J5 ,6 2.2 Hz, 1H, H-50), 3.77 (t,
J 9.5 Hz, 1H, H-40), 3.79–3.86 (m, 2H, H-5, -OCHH0-), 4.03–4.13 (m, 4H, H-6, H-60, H-600, -OCHH0-),
0
0
0
00
0
000
4.46 (d, J1,2 7.8 Hz, 1H, H-1), 4.50 (d, J1 ,2 8.0 Hz, 1H, H-10), 4.52 (dd, J5 ,6 2.2 Hz, J6
11.9 Hz,
0
0
0
000
00,6000
1H, H-6000), 4.87 (dd, J1 ,2 8.0 Hz, J2 ,3 9.6 Hz, 1H, H-20), 4.93 (dd, J2,3 10.5 Hz, J3,4 3.5 Hz, 1H, H-3),
5.08 (dd, J1,2 7.8 Hz, J2,3 10.5 Hz, 1H, H-2), 5.17 (t, J 9.3Hz, 1H, H-30), 5.32 (dd, J3,4 3.5 Hz, J4,5 1.0 Hz,
1H, H-4); 13C NMR (125 MHz, CDCl3, DEPT) δ 20.5, 20.7, 20.8, 20.9 (q×4, CH3COO-×7), 36.0 (t,
-CH2S-), 50.6 (q, CH3SO2-), 60.7, 61.6, 68.5 (t×3, -OCH2-, C-6, C-60), 66.5, 69.0, 70.6, 70.9, 71.3, 72.6,
72.8, 76.0 (d×8, C-2, C-3, C-4, C-5, C-20, C-30, C-40, C-50), 100.7, 101.1 (d×2, C-1, C-10), 169.1, 169.7,
169.7, 170.1, 170.2, 170.3, 170.4 (s×7, CH3COO-×7); HRMS m/z (FAB+): found 775.1793 (M+H+);
C29H43O20S2 requires 775.1789.
0
0
0
0
4.13. 2-(4-O-β-D-Galactopyranosyl-β-D-glucopyranosyl)ethyl methanethiosulfonate 12
A solution of NaOMe (0.1 M, 0.6 ml) was added to a solution of 27 (877 mg, 1.18 mmol) in MeOH (6
ml) under N2. After 3 h the reaction solution was passed through a Dowex 50W(H+) plug (4×1 cm, eluant
MeOH) and the solvent removed to give 2-bromoethyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside
28 (476 mg, 90%) as a white foam which was used directly in the next step. NaSSO2CH3 (185 mg, 1.38
mmol) was added to a solution of 28 (476 mg, 1.06 mmol) in DMF (24 ml) under N2 and warmed to
50°C. After 21 h the solution was cooled and the solvent removed. The residue was purified by flash
chromatography (CHCl3:MeOH:AcOH:H2O, 60:30:3:5) to give 12 (346 mg, 68%) as a hygroscopic
foam; [α]D28=+1.5 (c 1.66, H2O); IR (KBr) 3416 (br, OH), 1311, 1131 cm−1 (S–SO2); 1H NMR (400
0
0
MHz, D2O, COSY) δ 3.10–3.13 (m, 1H, H-2), 3.30 (t, J 6.0 Hz, 2H -CH2S-), 3.31 (dd, J1 ,2 7.8 Hz,
J2 ,3 10.3 Hz, 1H, H-20), 3.34 (s, 3H, CH3SO2-), 3.38–3.54 (m, 5H), 3.44 (dd, J2 ,3 10.3 Hz, J3 ,4 3.3
0
0
0
0
0
0
Hz, 1H, H-30), 3.57 (dd, J 8.4 Hz, J 11.4 Hz, 1H), 3.59 (dd, J 4.9 Hz, J 7.3 Hz, 1H), 3.70 (br d, J3 ,4
0
0
3.3 Hz, 1H, H-40), 3.77 (dd, J 1.0 Hz, J 11.5 Hz, 1H), 3.79–3.83 (m, 1H, -OCHH0-), 3.97–4.02 (m, 1H,
-OCHH0-), 4.22 (d, J1 ,2 7.8 Hz, 1H, H-10), 4.33 (d, J1,2 7.8 Hz, 1H, H-1); 13C NMR (125 MHz, D2O) δ
36.7 (-CH2S-), 50.8 (CH3SO2-), 61.0, 62.1, 69.4, 69.6, 71.9, 73.5, 73.7, 75.3, 75.9, 76.4, 79.3 (-OCH2-,
C-2, C-3, C-4, C-5, C-6, C-20, C-30, C-40, C-50, C-60), 103.3, 103.9 (C-1, C-10); HRMS m/z (FAB+):
found 503.0886 (M+Na+); C15H28O13S2Na requires 503.0869.
0
0

260
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
4.14. Protein glycosylation
Cysteine mutants of SBL were glycosylated according to previously published methods14i in the
appropriate buffer: 70 mM CHES, 2 mM CaCl2 (pH 9.5); 70 mM HEPES, 2 mM CaCl2 (pH 7.5); or 70
mM MES, 2 mM CaCl2 (pH 5.5) before quenching in 5 mM MES, 1 mM CaCl2 (pH 6.5 or 5.5). The free
thiol content of all CMMs was determined spectrophotometrically by titration with Ellman’s reagent27
in phosphate buffer (0.25 M, pH 8.0). In all cases no free thiol was detected. Modified enzymes were
analyzed by nondenaturing gradient (8–25%) gels at pH 4.2, run towards the cathode, on the Pharmacia
Phast-system and appeared as a single band. Prior to ES-MS glycosylated enzymes were analyzed by
FPLC (BioRad, Biologic System) on a Source 15 RPC matrix (17-0727-20 from Pharmacia) with 5%
acetonitrile, 0.01% TFA as the running buffer and eluted with 80% acetonitrile, 0.01% TFA in a one
step gradient. MS m/z (ES-MS): N62C-S-β-GlcNAc(Ac)3 calculated 27048, found 27050; L217C-S-β-
GlcNAc(Ac)3 calculated 27049, found 27045; S156C-S-β-GlcNAc(Ac)3 calculated 27075, found 27080;
S166C-S-β-GlcNAc(Ac)3 calculated 27075, found 27077.
4.15. Enzyme kinetics
Kinetic constants were determined in duplicate by the method of initial rates in 0.1 M Tris buffer,
pH 8.6, 0.005% Tween 80, 1% DMSO. [S]=0.125 mM to 3 mM, [E]=1.5×10−8 M to 9.0×10−8 M for
amidase kinetics. [S]=0.015 mM to 3 mM, [E]=3.5×10−10 M to 2.3×10−9 M for esterase kinetics. The
kinetic parameters of esterase activity were determined indirectly following the release of thiobenzyl
alcohol from the substrate succinyl-Ala-Ala-Pro-Phe-SBn (suc-AAPF-SBn) with Ellman’s reagent.27
4.16. Molecular modelling
The X-ray structure of subtilisin B. lentus with the peptide inhibitor AAPF bound (Brookhaven
database entry 1JEA) was used as the starting point for calculations on wild type and CMMs. The enzyme
set-up was performed with Insight II, version 2.3.0.41 To create initial coordinates for the minimization,
hydrogens were added at the pH (8.6) used for kinetic measurements. This protonated all Lys and Arg
residues and the N-terminus and deprotonated all Glu and Asp residues and the C-terminal carboxyl
group. The protonated form of His 64 was used in all calculations. The model system was solvated with
a 5 Å layer of water molecules. The total number of water molecules in the system was 1143. The overall
charge of the enzyme–inhibitor complex resulting from this set-up was +4 for the WT enzyme. Energy
simulations were performed with the Discover program, version 2.9.5 on a Silicon Graphics Indigo
computer, using the consistent valence force field (CVFF) function. A non-bonded cut-off distance of
18 Å with a switching distance of 2 Å was employed. The non-bonded pair list was updated every 20
cycles and a dielectric constant of 1 was used in all calculations. The WT enzyme was minimized in
stages, with initially only the water molecules being allowed to move, followed by water molecules and
the amino acid side chains, and then finally the entire enzyme. The mutated and chemically modified
enzymes were generated by modifying the relevant amino acid using the Builder module of Insight.
These structures were then minimized in a similar manner. Initially the side-chain of the mutated residue
and the water molecules were minimized. Then all side-chains and the water molecules were minimized
while the backbones of the residues were constrained, then all of the atoms were minimized. The AAPF
inhibitor was free to move throughout all stages of the minimization. Each stage of energy minimization
was conducted by means of the method of steepest descents without Morse or cross terms until the
derivative of energy with respect to structural perturbation was less than 5.0 kcal/Å; then the method of

B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
261
conjugate gradients, without Morse or cross terms until the derivative of energy with respect to structural
perturbation was less than 1.0 kcal/Å; and finally the method of conjugate gradients with Morse and cross
terms until the final derivative of energy with respect to structural perturbation was less than 0.1 kcal/Å.
Acknowledgements
We acknowledge the generous financial support of the University of Durham, the Natural Sciences and
Engineering Research Council of Canada and Genencor International, Inc, and we thank Dr. Rick Bott for
very helpful discussions. We would also like to acknowledge the use of the EPSRC’s Mass Spectrometry
Service at Swansea, UK and of the EPSRC’s Chemical Database Service at Daresbury.
References
1. Helenius, A. Mol. Biol. Cell 1994, 5, 253–265.
2. Opdenakker, G.; Rudd, P. M.; Ponting, C. P.; Dwek, R. A. FASEB J. 1993, 7, 1330–1337.
3. Rudd, P. M.; Joao, H. C.; Coghill, E.; Fiten, P.; Saunders, M. R.; Opdenakker, G.; Dwek, R. A. Biochemistry 1994, 33,
17–22.
4. Sharon, N.; Lis, H. Essays in Biochem. 1995, 30, 59–75.
5. (a) Lasky, L. A. Annu. Rev. Biochem. 1995, 64, 113–139. (b) Weis, W. I.; Drickamer, K. Annu. Rev. Biochem. 1996, 65,
441–473.
6. (a) Varki, A. Glycobiology 1993, 3, 97–130. (b) Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
7. Simanek, E. E.; Huang, D.-H.; Pasternack, L.; Machajewski, T. D.; Seitz, O.; Millar, D. S.; Dyson, H. J.; Wong, C.-H. J. Am.
Chem. Soc. 1998, 120, 11567–11575.
8. Rademacher, T. W.; Parekh, R. B.; Dwek, R. A. Annu. Rev. Biochem. 1988, 57, 785–838.
9. For reviews, see: (a) Stowell, C. P.; Lee, Y. C. Adv. Carbohydr. Chem. Biochem. 1980, 37, 225–281. (b) Neoglycoconjugates:
Preparation and Applications; Lee, Y. C.; Lee, R. T., Eds.; Academic: London, 1994. (c) Abelson, J. N.; Simon., M. I., Eds.
Methods Enzymol. 1994, 242. (d) Lee, Y. C.; Lee, R. T., Eds. ibid. 1994, 247. (e) Bill, R. M.; Flitsch, S. F. Chem. Biol. 1996,
3, 145–149. (f) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384–1390. (g) Davis, B. G. J. Chem. Soc., Perkin
Trans. 1 1999, 3215–3237.
10. (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1991, 32, 6793–6796. (b) Wong, S. Y. C.; Guile, G. R.; Dwek, R. A.;
Arsequell, G. Biochem. J. 1994, 300, 843–850. (c) Macindoe, W. M.; van Oijen, A. H.; Boons, G.-J. J. Chem. Soc., Chem.
Commun. 1998, 847–848.
11. Wynn, R.; Richards, F. M. Methods Enzymol. 1995, 201, 351–356.
12. Standard one-letter amino acid codes are used in this paper, e.g. S=Ser=Serine and L=Leu=Leucine.
13. Molecular Glycobiology; Fukuda, M.; Hindsgaul, O., Eds.; Oxford University Press: Oxford, 1994.
14. (a) Maekawa, K.; Liener, I. E. Arch. Biochem. Biophys. 1960, 91, 108. (b) Longo, M. A.; Combes, D. FEBS Lett. 1995,
375, 63–66. (c) Longo, M. A.; Combes, D. J. Mol. Catal. B 1997, 2, 281–289. (d) Hill, T. G.; Wang, P.; Huston, M. E.;
Wartchow, C. A.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.; Callstrom, M. R. Tetrahedron Lett. 1991, 32, 6823–6826.
(e) Wang, P.; Hill, T. G.; Wartchow, C. A.; Huston, M. E.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.; Callstrom, M. R.
J. Am. Chem. Soc. 1992, 114, 378–380. (f) Wartchow, C. A.; Wang, P.; Bednarski, M. D.; Callstrom, M. R. J. Org. Chem.
1992, 60, 2216–2226. (g) Wang, P.; Hill, T. G.; Bednarski, M. D.; Callstrom, M. R. Tetrahedron Lett. 1991, 32, 6827–6830.
(h) Davis, B. G.; Lloyd, R. C.; Jones, J. B. J. Org. Chem. 1998, 63, 9614–9615. (i) Davis, B. G.; Lloyd, R. C.; Jones, J. B.,
submitted for publication, 1999. (j) Lloyd, R. C.; Davis, B. G.; Jones, J. B., submitted for publication, 1999. (k) Baek, W.
O.; Vijayalakshmi, M. A. Biochim. Biophys. Acta 1997, 1336, 394–402.
15. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157–162.
16. Molecular Glycobiology; Fukuda, M.; Hindsgaul, O., Eds.; Oxford University Press: Oxford, 1994.
17. Berglund, P.; DeSantis, G., Stabile, M. R.; Shang, X.; Gold, M.; Bott, R. R.; Graycar, T. P.; Lau, T. H.; Mitchinson, C.;
Jones, J. B. J. Am. Chem. Soc. 1997, 119, 5265–5266.
18. Kenyon, G. L.; Bruice, T. W. Methods Enzymol. 1977, 47, 407–430.
19. (a) Douglass, I. B.; Farah, B. S. J. Org. Chem. 1959, 24, 973–975. (b) Levitt, L. S.; Levitt, B. W. J. Org. Chem. 1972, 37,
332–334. (c) Weidner, J. P.; Block, S. S. J. Med. Chem. 1972, 15, 564–567. (d) Palumbo, G.; Caputo, R. Synthesis 1981,

262
B. G. Davis et al. / Tetrahedron: Asymmetry 11 (2000) 245–262
888–890. (e) Block, E.; Zhao, S.-H. J. Org. Chem. 1992, 57, 5815–5817. (f) Billard, T.; Langlois, B. R.; Large, S.; Anker,
D.; Roidot, N.; Roure, P. J. Org. Chem. 1996, 61, 7545–7550.
20. (a) Fischer, E.; Delbrück, K. Chem. Ber. 1909, 42, 1476–1482. (b) Schneider, W.; Gille, R.; Eisfeld, K. Chem. Ber. 1928,
61, 1244–1259. (c) Horton, D. Methods Carbohydr. Chem. 1963, 2, 433–437. (d) Cerny, M.; Stanek, J.; Pacak, J. Monatsch.
Chem. 1963, 94, 290–294. (e) Apparu, M.; Blanc-Muesser, M.; Defaye, J.; Driguéz, H. Can. J. Chem. 1981, 59, 314–320.
21. Initial approaches to untethered glyco-MTS reagents similar in type to 1,2 were based upon Danishefsky’s glycal
methodology: Halcomb, R. L.; Danishefsky, S. J. J. Am. Chem. Soc. 1989, 111, 6661–6666. Tris-TBS glucal was prepared
according to the method of Lesimple, P.; Beau, J.-M.; Jaurand, G.; Sinäy, P. Tetrahedron Lett. 1986, 27, 6201–6204, oxidized
to tris-TBS protected 1,2-anhydroglucose using dimethyldioxirane and then reacted with a solution of butylammonium
methanethiosulfonate in dichloromethane that had been prepared by ion pair exchange extraction. However, under a variety
of conditions and in contrast to the behaviour of other sulfur nucleophiles [see: (a) Gordon, D. M.; Danishefsky, S. J.
Carbohydr. Res. 1990, 206, 361–366. (b) Berkowitz, D. B.; Danishefsky, S. J.; Schultz, G. K. J. Am. Chem. Soc. 1992, 114,
4518–4529], methanethiosulfonate ion failed to open the epoxide moiety of tris-TBS protected 1,2-anhydro-D-glucose.
22. (a) Zemplén, G.; Kunz, A. Ber. Dtsch. Chem. Ges. 1923, 56, 1705–1710. (b) Plattner, J. J.; Gless, R. D.; Rappoport, H.
J. Am. Chem. Soc. 1972, 94, 8613–8615. (c) Mori, K.; Sasaki, M. Tetrahedron Lett. 1979, 20, 1329–1332. (d) Herzig, J.;
Nudelman, A. Carbohydr. Res. 1986, 153, 162–167. (e) Herzig, J.; Nudelman, A.; Gottlieb, H. E.; Fischer, B. J. Org. Chem.
1986, 51, 727–730. (f) Vekemans, J. A. J. M.; Franken, G. A. M.; Chittenden, G. J. F.; Godefroi, E. F. Tetrahedron Lett.
1987, 28, 2299–2300. (g) Cinget, F.; Schmidt, R. R. Synlett 1993, 168–170.
23. Coles, H. W.; Dodds, M. L.; Bergeim, F. H. J. Am. Chem. Soc. 1938, 60, 1020–1022.
24. Dahmén, J.; Frejd, T.; Grönberg, G.; Lave, T.; Magnusson, G.; Noori, G. Carbohydr. Res. 1983, 116, 303–307.
25. Helferich, B.; Lutzmann, H. Justus Liebigs Ann. Chem. 1939, 541, 1–16.
26. Stabile, M. R.; Lai, W. G.; DeSantis, G.; Gold, M.; Jones, J. B.; Mitchinson, C.; Bott, R. R.; Graycar, T. P.; Liu, C.-C.
Bioorg. Med. Chem. Lett. 1996, 6, 2501–2512.
27. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88–95.
28. Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal. Biochem. 1996, 242, 221–227.
29. Plettner, E.; DeSantis, G.; Stabile, M. R.; Jones, J. B. J. Am. Chem. Soc. 1999, 121, 4977–4981.
30. Davis, B. G.; Jones, J. B. Synlett 1999, 1495–1507.
31. Ragupathi, G.; Koganty, R. R.; Qiu, D. X.; Lloyd, K. O.; Livingston, P. O. Glycoconjugate J. 1998, 15, 217–221.
32. (a) Leteux, C.; Stoll, M. S.; Childs, R. A.; Chai, W.; Vorozhaikina, M.; Feizi, T. J. Immunol. Methods 1999, 227, 109–119.
(b) Leteux, C.; Childs, R. A.; Chai, W. G.; Stoll, M. S.; Kogelberg, H.; Feizi, T. Glycobiology 1998, 8, 227–236. (c) Kichler,
A.; Schuber, F. Glycoconjugate J. 1995, 12, 275–281. (d) Kretzschmar, G.; Sprengard, U.; Kunz, H.; Bartnik, E.; Schmidt,
W. Tetrahedron 1995, 51, 13015–13030.
33. Prepared from D-glucose according to Scheurer, P. G.; Smith, F. J. Am. Chem. Soc. 1954, 76, 3224.
34. Prepared from the corresponding parent carbohydrates according to the method of Verley, A.; Bölsing, F. Ber. Dtsch. Chem.
Ges. 1901, 34, 3354–3358, and purified by flash chromatography.
35. Prepared from lactose according to the method of Hudson, C. S.; Johnson, J. M. J. Am. Chem. Soc. 1915, 37, 1270–1275,
and purified by flash chromatography.
36. The synthesis of 4b as an intermediate has been described previously. In our hands this method gave only a poor yield of
product. Nagai, K.; Honda, A.; Kiho, T.; Ukai, S.; Tsuchiya, T. Carbohydr. Res. 1989, 190, 165–180.
37. Helferich, B.; Lutzmann, H. Justus Liebigs Ann. Chem. 1939, 541, 1–16.
38. The synthesis of unstable 4e has been described previously. Dahmén, J.; Frejd, T.; Magnusson, G.; Noori, G.; Carlstroem,
A. S. Carbohydr. Res. 1984, 125, 237–245.
39. Dahmén, J.; Frejd, T.; Grönberg, G.; Lave, T.; Magnusson, G.; Noori, G. Carbohydr. Res. 1983, 116, 303–307.
40. The use of 4d as a reactant has been described previously, although no details of preparation or characterization were given.
Nilsson, K. G. I. U.S. Patent 4 918 009, 1986.
41. Biosym Technologies, Inc. San Diego, CA, USA.