The chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white lysozyme mechanism

Article abstract of DOI:10.1016/j.carres.2004.12.015

2,4-Dinitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→4)-2- deoxy-2-fluoro-β-d-glucopyranoside (GN2FG-DNP) and 2-acetamido-2-deoxy- β-d-glucopyranosyl-(1→4)-2-deoxy-2-fluoro-β-d-glucopyranosyl fluoride (GN2FG-F) were prepared using a divergent synthetic approach involving 10 steps. The key steps involved the preparation of 1-O-acetyl-3,6-di-O-benzyl- 2-deoxy-2-fluoro-α/β-d-glucopyranose using Selectfluor in the presence of acetic acid and the subsequent glycosylation of this acceptor to generate the core 2-fluorodisaccharide. After further elaboration, the target molecules were obtained and tested as probes of the mechanism of hen egg white lysozyme (HEWL). Compound GN2FG-DNP is not a substrate for the enzyme while compound GN2FG-F is cleaved slowly with an apparent K_m greater than 5 mM and a second-order rate constant of k_cat/K_m = 9.6 s^-1 M^-1. Comparison of this value to that estimated for the hydrolysis of β-chitobiosyl fluoride by HEWL (1200 s^-1 M^-1) [Ballardie, F. W.; Capon, B.; Cuthbert, M. W.; Dearie, W. M. Bioorg. Chem. 1977, 6, 483-509] revealed a 126-fold rate decrease upon substitution of a fluorine group for the 2-acetamido group of β-chitobiosyl fluoride. This decrease resulted in the steady-state accumulation of an intermediate as visualized by mass spectrometry and the ultimate crystallographic determination of its structure [Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature 2001, 412, 835-838].

Full text of DOI:10.1016/j.carres.2004.12.015

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 379–388
The chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of
the hen egg white lysozyme mechanism
David J. Vocadloꢀ and Stephen G. Withers*
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1
Received 4 April 2004; received in revised form 11 December 2004; accepted 14 December 2004
Abstract—2,4-Dinitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-deoxy-2-fluoro-b-D-glucopyranoside (GN2FG-
DNP) and 2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-deoxy-2-fluoro-b-D-glucopyranosyl fluoride (GN2FG-F) were
prepared using a divergent synthetic approach involving 10 steps. The key steps involved the preparation of 1-O-acetyl-3,6-di-O-
benzyl-2-deoxy-2-fluoro-a/b-D-glucopyranose using SelectfluorTM in the presence of acetic acid and the subsequent glycosylation
of this acceptor to generate the core 2-fluorodisaccharide. After further elaboration, the target molecules were obtained and tested
as probes of the mechanism of hen egg white lysozyme (HEWL). Compound GN2FG-DNP is not a substrate for the enzyme while
compound GN2FG-F is cleaved slowly with an apparent Km greater than 5 mM and a second-order rate constant of kcat
Km = 9.6 sÀ1 MÀ1. Comparison of this value to that estimated for the hydrolysis of b-chitobiosyl fluoride by HEWL (1200 sÀ1 MÀ1
/
)
[Ballardie, F. W.; Capon, B.; Cuthbert, M. W.; Dearie, W. M. Bioorg. Chem. 1977, 6, 483–509] revealed a 126-fold rate decrease
upon substitution of a fluorine group for the 2-acetamido group of b-chitobiosyl fluoride. This decrease resulted in the steady-state
accumulation of an intermediate as visualized by mass spectrometry and the ultimate crystallographic determination of its structure
[Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature 2001, 412, 835–838].
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Fluorine; Hen egg white lysozyme (HEWL); Catalytic mechanism; Mechanism-based inactivator; Glycosyl fluoride, inhibitor
1. Introduction
metastable ion pair formed between the oxocarbenium
ion and the enzymic carboxylate Asp-52 (Chart 1).
The arguments against a covalent intermediate are
based on several three-dimensional models of HEWL-
product complexes.1,4 According to these interpreta-
tions, the saccharide is positioned too far away from
Asp-52, the orientation of Asp-52 is incorrect for it to
act in a nucleophilic capacity, and the active site is too
congested for a covalent intermediate to form.4 A mech-
anism involving a long-lived ion-pair intermediate, how-
ever, is at odds with physical organic studies showing
that in the presence of a negative ion there is essentially
no barrier to the collapse of the glycosyl cation.5–7 Addi-
tionally, a-deuterium kinetic isotope effects (KIE) mea-
sured using substrates for which the rate-determining
step is breakdown of the intermediate provide evidence
for a covalent intermediate. Such isotope effects have
been carried out with several b-retaining glycosidases
including, for example, the b-galactosidases from E. coli
The catalytic mechanism of HEWL has been the subject
of many studies. Despite having gained textbook famil-
iarity, however, certain aspects of the mechanism have
long remained contentious. Early studies confirmed the
majority of the features of the catalytic mechanism pro-
posed by Phillips and coworkers.1 Yet the most conten-
tious point, which has until recently2 remained unclear,
is the nature of the reaction intermediate. The original
catalytic mechanism proposed by Phillips on the basis
of model building studies, in conjunction with the
three-dimensional structure determined by X-ray dif-
fraction,3 suggests that the intermediate is a long-lived
*
Corresponding author. Tel.: +1 604 822 3402; fax: +1 604 822 8869;
e-mail: withers@chem.ubc.ca
ꢀ Present address: Department of Chemistry, Simon Fraser University,
Burnaby, BC, Canada V5A 1S6.
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.12.015

380
D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
Glu 35
O
O
H
O
OH
NAG
O
O
H
NAG
HO
O
O
Glu 35
O
AcHN
Glu 35
O
Asp 52
O
H
O
H
H2O
OH
OH
NAG
NAG
O
O
OH
NAG
O
HO
O
O
HO
Glu 35
O
AcHN
O
AcHN
O
O
NAG
O
O
Asp 52
Asp 52
H2O
H
O
OH
NAG
O
O
H
HO
AcHN
O
O
Asp 52
Chart 1. Possible mechanisms for hen egg white lysozyme. The upper path shows a mechanism involving a metastable oxocarbenium ion as proposed
by Phillips and the lower path shows a covalent glycosyl-enzyme intermediate as first proposed by Koshland.
(LacZ,8 ebga, ebgb, and ebgab),9 the b-glucosidases from
Agrobacterium sp.,10Stachyobotrys atra, Botrydiplodia
theobromae,11 a b-(1,4) glycanase from Cellulomonas
fimi,12 b-xylosidases from Thermoanaerobacterium
saccharolyticum,13 and Geobacillus stearothermophilus14
and a b-mannosidase from Cellulomonas fimi.15 In each
of these cases the intermediate has been revealed as a
covalent species and indeed, there is no retaining glyco-
sidase for which the intermediate has been clearly dem-
onstrated to be a non-covalent ion pair. Unfortunately,
a-deuterium kinetic isotope effects have been unable to
clarify this point with HEWL as no substrate for this en-
zyme has been found for which the rate-determining
step is deglycosylation. Thus, in the absence of a suitable
substrate for KIE experiments, we aimed to synthesize
the first mechanism-based inhibitors of HEWL that
function by accumulation of a stabilized intermediate.
The 2-deoxy-2-fluoro glycosyl fluorides and corre-
sponding dinitrophenyl glycosides have proven very
effective at labeling the catalytic nucleophiles of many
b-retaining glycosidases. These inhibitors function by
forming fairly stable covalent glycosyl-enzyme interme-
diates. The highly electron-withdrawing fluorine at the
2-position inductively destabilizes the oxocarbenium
ion-like transition states, through which both steps of
the double displacement mechanism proceed, thereby
slowing both steps of the reaction. Inclusion of a very
good leaving group at the anomeric center, however,
accelerates the first step making the intermediate kineti-
cally accessible. Both 2,4-dinitrophenolate and fluoride
have been shown to be excellent leaving groups for use
in this strategy.16,17 Early studies with synthetic sub-
strates revealed that HEWL was capable of cleaving
disaccharide substrates and that it also had a marked
preference for a fluoride-leaving group over a DNP leav-
ing group.18 Additionally, other studies have shown that
the 2-acetamido group of the sugar in the À1 subsite is
not essential for catalysis by HEWL and can be replaced
by a hydroxyl group or a hydrogen.19 Substitution of the
2-acetamido group with fluorine, which is only slightly
larger than hydrogen, should therefore be tolerated
by HEWL. Consequently, we opted to synthesize 2,4-
dinitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!4)-2-deoxy-2-fluoro-b-D-glucopyranoside (11) and
2-acetamido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-deoxy-
2-fluoro-b-D-glucopyranosyl fluoride (13) as possible
inactivators of HEWL. Here we report the synthesis of
these compounds and the detailed kinetic analysis of
their mode of action. Indeed, one of these probes, 2-acet-
amido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-deoxy-2-
fluoro-b-D-glucopyranosyl fluoride, has recently permit-
ted the observation of a covalent intermediate on HEWL
(Chart 1) and revealed the slight structural changes in the
enzyme that allow such a complex to form.2
2. Results and discussion
2.1. Synthesis
Preparation of the target 2-deoxy-2-fluorodisaccharides
(11 and 13) was accomplished using a linear synthetic

D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
381
OBn
O
OAc
O
iv
OBn
O
i, ii
(3a + 3b)
HO
BnO
HO
BnO
iii
AcO
AcO
OAc
F
1
2
3
OAc
3a:α-gluco
O
AcO
AcO
3b:β-gluco
3c:α-manno
3d:β-manno
NH
O
NH
CCl3
O
CCl3
4
OR1
OR
O
O2N
OR
OR2
O
R1O
R1O
ix
RO
RO
O
O
O
O
O
R2O
RO
NHAc
OR3
R
F
F
NO2
1
3
2
10 R = Ac
5 R = NHCOCCl , R = R = Ac, R = Bn
3
v
x
1
3
2
6 R = NHAc, R = R = Ac, R = Bn
11 R = H
vi
1
3
2
7 R = NHAc, R = R = Ac, R = H
vii
OR
1
2
3
OR
8 R = NHAc, R = R = R = Ac
O
RO
RO
O
xi
viii
O
1
2
F
RO
9 R = NHAc, R = R = Ac, R = H
3
NHAc
F
12 R = Ac
13 R = H
xii
Scheme 1. Synthetic route for the preparation of the target compounds NAG-2FGDNP (11) and NAG-2FGF (13). Reagents and conditions: (i) (a)
NaOMe, MeOH, (b) H+, MeOH; (ii) (a) (Bu3Sn)2O, C6H6, D, (b) TBAB, BnBr, C6H6, D; (iii) SelectfluorTM, AcOH, MeNO2, 25%; (iv) TMSOTf,
˚
(CH2Cl)2, 4 A sieves, Et3N, 0 ꢁC, 80%; (v) Bu3SnH, AIBN, C6H6, D, 81%; (vi) H2, Pd–C, MeOH/EtOAc/H2O; (vii) Ac2O, py, 84% over two steps ;
(viii) H2NNH3OAc, DMF, 94%; (ix) FDNB, DABCO, DMF, 55%; (x) HCl, MeOH, 26%; (xi) DAST, DCM, THF, À30 ꢁC, 67%; (xii) (a) NaOMe,
MeOH, (b) H+, MeOH, 90%.
strategy (Scheme 1). The known, selectively benzylated,
glycal 2 was prepared according to literature proce-
dures.20 Electrophilic fluorination of glycals has
emerged as a reliable method for the preparation of
2-fluorosaccharides.21–24 Studies have clearly demon-
strated that the addition of SelectfluorTM across the
glycal double bond occurs syn to yield 2-deoxy-2-flu-
oro-N-glycosyl intermediates.22–24 These intermediates
can be intercepted in the same pot by the addition of a
wide range of nucleophiles.21–24 We were pleased to find
that addition of acetic acid during the electrophilic fluo-
rination of glycal (2) using SelectfluorTM afforded the
2-deoxy-2-fluoro-D-glucopyranoses (3a,b) and -manno-
pyranoses (3c,d) in a ratio of 45 (3a+3b) to 55 (3c+3d)
as a mixture of anomers (gluco, 2:3, 3a/3b; manno 9:1,
3c/3d). Purification of a mixture of the glucopyranose
acceptors (3a and 3b) was readily accomplished by flash
column chromatography on silica. This strategy enabled
us to rapidly generate the selectively protected 2-deoxy-
2-fluoro-D-glucopyranoses (3a,b) bearing a free hydr-
oxyl at the 4-position that could be used as an acceptor
in the subsequent glycosylation without further manipu-
lation. The generation of these 1-O-acetyl derivatives
rather than interception of the N-glycosyl intermediate
to generate the 2,4-dinitrophenyl glycoside permitted
divergence of the synthetic strategy toward compounds
11 and 13 at the penultimate step. We opted to install
the fluorine atom early on during the synthetic plan
rather than fluorinate a disaccharide glycal in order to
avoid potential problems in the separation of diastero-
mers later on in the synthesis. The choice of donor for
the preparation of the target molecule required some
consideration as it would be necessary to effect the con-
version of the 2-amino protecting group into the desired
acetamido functionality in the presence of the anomeric
acetate of the acceptors (3a and 3b). Thus, the known
2-deoxy-2-trichloroacetamide donor, 4 which is accessi-
ble25 in four steps from commercial materials, was cho-
sen. This trichloroacetimidate has been demonstrated
to be an efficient donor for glycosylations and to also
undergo facile conversion of the 2-trichloroacetamido
group to an acetamide in the presence of other sensitive
protecting groups. Coupling of the donor 4 to a mixture
of acceptors 3a and 3b afforded the disaccharide 5 in
good yield. Only one anomeric product was obtained
from this glycosylation reaction and on the basis of

382
D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
the large coupling constant (ꢀ8.5 Hz) observed between
H-10 and H-20 in all subsequent products, this product
was deemed to be the b-anomer. This very high ano-
meric selectivity is consistent with previous results using
this donor.25 Subsequent reduction of the trichloroace-
tamido group proceeded smoothly to give the desired
20-acetamido disaccharide 6. Hydrogenolysis of the ben-
zyl protecting groups of the 3,6-di-O-benzyl protected
disaccharide 6 on a moderate scale was complicated by
the presence of small amounts of residual alkyl tin from
the preceding reaction, which poisoned the palladium
catalyst. Rigorous purification of the disaccharide 6
was required before the removal of the benzyl groups
could be effected using Pd–C in an atmosphere of hydro-
gen to provide diol 7. Acetylation of the crude diol 7 to
give the peracetylated disaccharide, 8 followed by selec-
tive cleavage of the anomeric acetate with hydrazine ace-
tate yielded the hemiacetal 9 as a 1:2 mixture of b to a
anomers as could be determined from both the 19F
observed in compounds 12 (5.6 Hz) and 13 (6.9 Hz).
The magnitude of the F1 to F2 coupling (ꢀ16 Hz) also
supports assignment of compounds 12 and 13 as 2-flu-
oro b-glycosyl fluorides. Furthermore, the use of these
compounds as substrates for HEWL in the studies de-
scribed below and in earlier studies2 published by us
provide compelling evidence for the anomeric configura-
tion of compounds 11 and 13.
2.2. Enzymology
On incubation of 11 with HEWL (0.1 mM) and moni-
toring of the solution at 400 nm using a UV/Vis spectro-
photometer, no reaction could be observed under any
conditions. Thus, disappointingly, compound 11 is nei-
ther a substrate nor an inactivator of the enzyme. Upon
incubation of 13 with HEWL, however, a steady-state
release of fluoride ion was observed using a fluoride-
selective electrode, indicating that this compound is
cleaved by the enzyme. It is interesting to note that
although the leaving group pKa values in each case are
quite similar and many glycosidases cleave equivalent
substrates with similar catalytic efficiency, HEWL dis-
criminates between them. Ballardie and coworkers had
earlier observed this behavior when studying the
HEWL-catalyzed hydrolysis of b-chitobiosyl fluoride
and 2,4-dinitrophenyl b-chitobioside.18 They found that
the second-order rate constant for the cleavage of the
fluoride was 30-fold greater than that for the 2,4-dinitro-
phenyl compound and attributed it to the greater sym-
metry of the fluoride ion leaving group. Regardless of
the physical basis of this discrimination for the aglycon
moiety, the energy barrier for hydrolysis of 11 by
HEWL may simply be too high for the reaction to be
observed under the conditions of this study.
Kinetic analysis of the cleavage of 13 revealed that
each molecule of HEWL was able to catalyze the turn-
over of more than one molecule of 13. This demonstra-
tion of catalytic competence, in conjunction with the
observation of linear progress curves, clearly shows that
13 is acted upon by HEWL as a substrate and not as an
inactivator. A plot of the rate observed versus concen-
tration of the substrate (Fig. 1) revealed that the appar-
ent dissociation constant of 13 was too high to allow an
accurate determination of a Km value, which must be
significantly greater than 5 mM. Unfortunately, as satu-
ration kinetics could not be observed, it is impossible to
estimate the importance of the 2-acetamido group for
the binding of chitobiose and 13 to HEWL. Previous
studies, however, have shown that the 2-acetamido sub-
stituent is not critical for HEWL catalyzed hydrolysis of
synthetic substrates.19 Indeed, incubation of HEWL
with aryl chitobioside substrate analogues, wherein the
2-acetamido group had been replaced with either a
hydroxyl or hydrogen, resulted in production of only
the disaccharide, along with the liberated unnatural
1
and H NMR spectra. The hydroxyl protons could be
1
observed in the H NMR spectrum of this anomeric
mixture, with the resonance from the b anomer observed
at 6.38 ppm and that of the a anomer observed at
6.22 ppm. The ratio of the intensity of these signals
was consistent with that observed for the ratio measured
in the 19F NMR. The position of these resonances far
downfield and with reasonably large coupling constants
(6.6 Hz and 4.7 Hz for the b and a anomers, respec-
tively) is consistent with the significant deshielding of
these protons as seen for the hemiacetal of a 2-fluorosac-
charide.26 Reaction of disaccharide 9 with 2,4-dinitroflu-
orobenzene in DMF27 gave the protected target
molecule 10. Global deprotection of glycoside 10 using
HCl in methanol afforded the target 2-deoxy-2-fluoro-
glycoside 11. Fluorination of the hemiacetal 9 using
DAST yielded predominantly the desired b-glycosyl
fluoride 12 with good stereoselectivity (b:a; 9:1). The
anomeric configuration of 10 was supported by the
observation of a relatively large H-1 to H-2 coupling
1
(6.3 Hz) in the H NMR spectrum of 10 as well as the
O-deacetylated product 11 (7.6 Hz). The observation
of a small H-1 to F coupling (3.7 Hz for compound 10
and 3.0 Hz for compound 11) are also consistent with
assignment of both 10 and 11 as b-configured glycosides.
Indeed, the chemical shifts and coupling constants mea-
sured are in reasonable agreement with those observed
by Dax and coworkers for 2,4-dinitrophenyl 3,4,6-tri-
O-acetyl-2-deoxy-2-fluoro-b-D-glucopyranoside.23 Re-
moval of the acetyl protecting groups from 12 using
sodium methoxide in dry methanol provided the target
compound 13. Assignment of the anomeric configura-
tion of compounds 12 and 13 as b-glycosyl fluorides is
supported by the small coupling constant between H-1
and F-2 observed in the 1H and 19F NMR spectra
(3.4 Hz for compound 12 and 3.3 Hz for compound
13) and the relatively large H-1 to H-2 coupling that is

D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
383
manifested as a more dramatic decrease in the second-
order rate constant.
2.3. Conclusion
This paper describes in detail the synthesis and testing of
two compounds designed as mechanism-based probes of
HEWL. Kinetic studies using these compounds revealed
that, under the conditions of this study, compound 11
was untouched by the enzyme while 13 was acted upon
as a slow substrate for which the rates of glycosyl en-
zyme intermediate formation and breakdown were
approximately equal. These results indicate that; (i)
any attempts to devise 2-fluoro mechanism-based inacti-
vators of HEWL should incorporate a fluoride-leaving
group and: (ii) another method of preferentially slowing
the second (deglycosylation) step over the first (glycosyl-
ation) step must be incorporated into the inhibitor
design to generate a useful inhibition. Indeed stoicheo-
metric accumulation of an intermediate with this reagent
has been seen with a mutant of HEWL in which the
acid/base catalyst is removed.2
Figure 1. Michaelis–Menten plot showing the rate of fluoride release
versus the concentration of NAG-2FGF in the presence of 101 lM
HEWL at 25 ꢁC in 150 mM NaOAc buffer, pH 5.00.
aglycone. Conversely, the HEWL catalyzed hydrolysis
of pNP-chitobioside itself was found to generate a mix-
ture of products arising from cleavage of the internal
glycosidic bond in addition to the liberation of the phe-
nol group. Replacement of the 2-acetamido group in
chitobiose-based substrates with a smaller moiety thus
eliminates the undesirable cleavage of the internal glyco-
sidic bond, facilitating interpretation of the kinetic
results.19
3. Experimental
3.1. General methods
The apparently high Km value and the steady-state ki-
netic studies showing more than one turnover of com-
pound 13 provide some evidence that the first step of
the mechanism (glycosylation) is at least partially rate
determining with this substrate and indeed, this expecta-
tion has been confirmed.2 Despite the absence of satura-
tion kinetics, a second-order rate constant could readily
be extracted from the data (kcat/Km = 9.6 minÀ1 MÀ1).
Comparison of this value with that estimated for the
All buffer chemicals and other reagents were obtained
from Sigma/Aldrich Chemical Co. unless otherwise
noted. Synthetic reactions were monitored by TLC
using E. Merck Kieselgel 60 F254 aluminum-backed
sheets. Compounds were detected by charring with
10% ammonium molybdate in 2 M H2SO4 and heating.
Flash chromatography at a positive pressure was per-
formed with E. Merck Kieselgel 60 (230–400 mesh)
1
using the specified eluants. H NMR spectra were re-
hydrolysis of b-chitobiosyl fluoride by HEWL (kcat
/
revealed a decrease in the sec-
corded on a Bruker WH-400 spectrometer at 400 MHz
(chemical shifts quoted relative to CDCl3, CD3OD or
(CD3)2CO where appropriate). 19F NMR spectra were
recorded on a Bruker AC-200 at 188 MHz or a Bruker
AV-300 at 282 MHz and are proton-coupled with
CF3CO2H as a reference. 13C NMR spectra were re-
corded on a Bruker WH-400 spectrometer at 100 MHz
or a Bruker AV-300 at 75 MHz (chemical shifts quoted
relative to CDCl3 or to CD3OD).
Km = 1200 minÀ1 MÀ1 18
)
ond-order rate constant of 126-fold. Such a decrease in
the glycosylation step upon substitution of a fluorine
group for the 2-acetamido group of b-chitobiosyl fluo-
ride is small, yet is consistent with decreases found in
previous reports with other glycosidases where the 2-hy-
droxyl of an activated substrate was replaced with fluo-
rine. Indeed, for Agrobacterium sp. b-glucosidase, which
is known to proceed through a covalent glycosyl enzyme
intermediate, a 335-fold rate reduction was observed on
substitution of the 2-hydroxyl group with fluorine.10,17
For some other retaining b-glycosidases, much more
dramatic rate reductions have been observed ranging
from 1.2 · 104 to 1.0 · 106-fold.12,28,29 That the rate
reduction is so small provides further credence to a
covalent glycosyl enzyme intermediate for HEWL. For
a discrete oxocarbenium ion intermediate, one would
reasonably expect a more significant destabilizing effect
3.2. 1-O-Acetyl-3,6-di-O-benzyl-2-deoxy-2-fluoro-a-D-
glucopyranose (3a) and 1-O-acetyl-3,6-di-O-benzyl-2-
deoxy-2-fluoro-b-D-glucopyranose (3b)
To a soln of glycal 2 (3.60 g, 8.1 mmol) in a mixture of
dry MeNO2 and AcOH (5:1, 300 mL) SelectfluorTM
(7.17 g, 20.3 mmol) was added portionwise over 2 h.
The reaction mixture was then stirred for 16 h at room

384
D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
temperature after which it was refluxed for 30 min. After
cooling the reaction mixture to room temperature, the
solvent was removed under diminished pressure to yield
a gum that was dissolved in CH2Cl2 (200 mL). This
CH2Cl2 soln was washed with water (100 mL), saturated
NaHCO3 (2 · 100 mL), water (100 mL), and brine
(100 mL). The organic layer was then dried over
MgSO4, filtered, and concentrated under diminished
pressure to a pale yellow oil. Gradient flash silica col-
umn chromatography of the residue (1:5 to 3:17,
EtOAc–hexanes) yielded the desired products 3a and
3b as a mixture in a 3:2 ratio (1.10 g, 2.7 mmol, 25%).
Further careful chromatography (3:17 EtOAc–petro-
leum ether (35–60 ꢁC) to 1:5 EtOAc–petroleum ether
(35–60 ꢁC)) yielded pure samples of each anomer as
gums. 1-O-Acetyl 3,6-di-O-benzyl-2-deoxy-2-fluoro-a-
D-glucopyranose (3a); 19F NMR (188 MHz, CDCl3,
CF3CO2H reference): d À124.30 (1F, dd, JF2,2 49.7 Hz,
F-2, JF2,3 12.0 Hz); 1H NMR (400 MHz, CDCl3) d:
7.38–7.27 (10H, m, Ar · 10), 6.34 (1H, d, J1,2 3.9 Hz,
H-1), 4.91 and 4.70 (AB quartet, 2H, Jgem 11.4 Hz,
PhCH2), 4.60 (1H, ddd, J2,3 9.2 Hz, H-2), 4.58 and
4.51 (AB quartet, 2H, Jgem 12.1 Hz, PhCH2), 3.60–3.95
(5H, m, H-3, H-4, H-5, H-6, and H-6a), 2.59 (1H, d,
JOH,4 1.9 Hz, OH), 2.14 (3H, s, OAc); 13C NMR
(100 MHz, CDCl3): d 168.97 (s, CO), 138.09, 137.70,
128.56, 128.44, 128.00, 127.96, and 127.72 (s, Ph),
89.72 (d, J2,F2 191.35 Hz, C-2), 89.26 (d, J1,F2
22.97 Hz, C-1), 79.68 (d, J3,F2 16.37 Hz, C-3), 74.80 (d,
of the a and b anomers of 1-O-acetyl-3,6-di-O-benzyl-2-
deoxy-2-fluoro-D-mannopyranose (3c/d) in a 9:1 ratio
(1.3 g, 3.2 mmol, 29%).
3.4. 3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-b-
D-glucopyranosyl-(1!4)-1-O-acetyl-3,6-di-O-benzyl-2-
deoxy-2-fluoro-a/b-D-glucopyranose (5)
To acceptor 3 (654 mg, 1.62 mmol) was added the donor
˚
4 (1.06 g, 1.78 mmol, 1.1 equiv) along with activated 4 A
sieves. After the addition of 1,2-dichloroethane (10 mL)
the reaction mixture was stirred for 1 h at room temper-
ature under an atmosphere of dry argon. The reaction
mixture was then cooled to 0 ꢁC and trimethylsilyltri-
flate (60 lL) was added. After 2 h, analysis of the mix-
ture by TLC revealed that the acceptor had been
completely consumed and Et3N was added (0.5 mL).
The reaction mixture was diluted with CH2Cl2 (5 mL)
and filtered. The filtrate was then concentrated to a pale
yellow residue under diminished pressure. Careful flash
chromatography of this residue on a silica column with
gradient elution (4:1 petroleum ether (35–60 ꢁC)–EtOAc
to 3:1 petroleum ether (35–60 ꢁC)–EtOAc) yielded the
desired product 5 as a mixture of anomers (1.009 g,
1.21 mmol, 75%). 19F NMR (188 MHz, CDCl3,
CF3CO2H reference): d À122.26 (1F, dd, JF2,H2 51.9
Hz, JF2,H3 15.4 Hz, JF2,H1 3.3 Hz F-2, b-anomer),
À124.30 (1F, dd, JF2,H2 47.8 Hz, JF2,H3 12.2 Hz, a-ano-
mer); Selected 1H NMR data (400 MHz, CDCl3): d
7.50–7.21 (10H, m), 6.33 (0.65H, d, J1,2 4.0 Hz, H-1, a
anomer), 6.16 (0.65H, d, JNH,2 9.2 Hz, NH, a-anomer),
6.15 (0.35H, d, JNH,2 9.0 Hz, NH, b anomer), 5.62
(0.35H, dd, J1,2 8.1 Hz, H-1, b-anomer), 5.02–4.67
(5H, m), 4.52 (0.65H, ddd, J2,3 9.2 Hz, H-2, a-anomer),
4.36–4.25 (2.35H, m), 4.17–4.11 (1H, m), 4.04–3.83 (4H,
JOCH2Ph(C-3),
2.26 Hz, OCH2Ph(C-3)), 73.77, 72.21,
F2
70.00 (d, J4,F2 8.70 Hz, C-4), 69.17, 20.90 (s, CH3); Anal.
Calcd for C22H25FO6: C, 65.34; H, 6.23. Found: C,
65.07; H, 6.23.
3.3. 1-O-Acetyl-3,6-di-O-benzyl-2-deoxy-2-fluoro-b-D-
glucopyranose (3b)
m), 3.73–3.60 (2H, m), 3.54–3.35 (2H, s); CIMS(NH ):
3
m/z
857
(M(35Cl37Cl2)+NH4)+
(10.5%),
855
(M(35Cl237Cl)+NH4)+
(38.0%),
853
(M+NH4)+
19F NMR (188 MHz, CDCl3, CF3CO2H reference): d
À122.70 (1F, dd, JF2,2 51.2 Hz, JF2,3 13.7 Hz, JF2,1
3.4 Hz); 1H NMR (400 MHz, CDCl3): d 7.37–7.27
(10H, m, Ar ·10), 5.70 (1H, dd, J1,2 8.0, H-1), 4.92
and 4.90 (AB quartet, 2H, Jgem 11.5 Hz, PhCH2), 4.58
and 4.51 (AB quartet, 2H, Jgem 12.1 Hz, PhCH2), 4.41
(1H, ddd, J2,3 8.4 Hz, H-2), 3.78–3.50 (5H, m, H-3, H-
4, H-5, H-6, and H-6a), 2.58 (1H, d, JOH,4 2.3 Hz,
OH), 2.13 (3H, s, OAc). 13C NMR (75 MHz, CDCl3):
d 169.19 (s, CO), 137.77, 137.54, 128.56, 128.44,
128.08, 127.84, and 127.77 (s, Ph), 91.60 (d, J2,F2
186.8 Hz, C-2), 91.52 (d, J1,F2 24.8 Hz, C-1), 82.17 (d,
J3,F2 16.4 Hz, C-3), 74.86, 74.69 (d, JOCH2Ph(C-3),F2
2.3 Hz, OCH2Ph(C-3)), 73.66, 70.27 (d, J4,F2 8.6 Hz,
C-4), 68.95, 20.91 (s, CH3); CIMS(NH 3): m/z 422
(M+NH4)+ (7.6%), 91 (CH2Ph)+ (100%); HRCIMS:
(M+NH4)+: Calculated, 242.19788; Found, 242.19784.
Also obtained from the reaction mixture was a mixture
(26.7%); HRCIMS: (M+NH4)+: Calculated, 853.19202;
Found, 853.19537, (M(35Cl237Cl)+NH4)+ Calculated,
855.18909; Found, 855.18989. Anal. Calcd for
C36H41Cl3FNO14Æ0.5H2O: C, 51.11; H, 5.00; N, 1.66.
Found: C, 51.00; H, 4.92; N 1.90.
3.5. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluco-
pyranosyl-(1!4)-1-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-
fluoro-a/b-D-glucopyranose (6)
To disaccharide 5 (642 mg, 0.76 mmol) was added a
spatula tip of AIBN followed by benzene (7.5 mL) and
tributyltin hydride (1.25 mL, 4.6 mmol, 6 equiv). This
reaction mixture was stirred for 1 h under dry argon at
room temperature and then refluxed for 2 h. After cool-
ing the reaction to room temperature, the solvent was
removed under diminished pressure to yield a white

D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
385
solid. This residue was dissolved in MeCN (50 mL) and
washed with hexanes (3 · 50 mL). The MeCN phase was
concentrated under diminished pressure to yield 6 as a
white crystalline solid. Flash chromatography of this
residue on silica with an initial step (1:1 EtOAc–petro-
leum ether (35–60 ꢁC)) followed by gradient elution
(4:1 EtOAc–petroleum ether (35–60 ꢁC) to 3:1 EtOAc–
petroleum ether (35–60 ꢁC)) yielded the desired product
6 as fine white crystals (451 mg, 0.61 mmol, 81%). An
anomerically pure analytical sample was obtained
(2 mL). After stirring this mixture for 16 h, the solvent
was evaporated under diminished pressure to give a
tan powder. The desired product was then purified by
flash chromatography on a column of silica with gradi-
ent elution (1:4 petroleum ether (35–60 ꢁC)–EtOAc to
1:6 petroleum ether (35–60 ꢁC)–EtOAc) yielding the
desired product 8 as an off white powder (240 mg,
0.38 mmol, 84%). A small amount of the pure a anomer
8a was obtained during the chromatography; 19F NMR
(188 MHz, CDCl3, CF3CO2H reference): d À124.22 (1F,
dd, JF2,H2 48.5 Hz, JF2,H3 15.4 Hz, F-2); 1H NMR
(400 MHz, CDCl3): d 6.33 (1H, d, J1,2 3.9 Hz, H-1),
during
the
column
chromatography:
2-acet-
amido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranosyl-
(1!4)-1-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-fluoro-a-D-
glucopyranose (6a). 19F NMR (188 MHz, CDCl3,
0
5.85 (1H, d, JNH,2 8.8 Hz, NH), 5.50 (1H, ddd, J3,F2
12.0 Hz, J3,2 9.5 Hz, J3,4 9.5 Hz, H-3), 5.24 (1H, dd,
0
J2 ,3 10.4 Hz, J3 ,4 9.3 Hz, H-30), 5.02 (1H, dd, J4 ,5
0
0
0
0
0
CF3CO2H reference):
d
À121.90 (1F, dd, JF2,H2
9.5 Hz, H-40), 4.63 (1H, d, J1 ,2 3 Hz, H-10), 4.52 (1H,
ddd, H-2), 4.40–4.22 (3H, m, H-6, H-6a, H-60), 4.04–
3.96 (2H, m, H-5, H-60a), 3.76 (1H, ddd, H-20), 3.68
0
0
51.0 Hz, JF2,H3 16.0 Hz, JF2,H1 4.0 Hz F-2, b-anomer),
À124.11 (1F, dd, JF2,H2 47.3 Hz, JF2,H1 11.9 Hz, a-ano-
1
mer); H NMR (400 MHz, CdCl3): d 6.31 (1H, d, J1,2
0
0
0
0
0
0
0
0
4.0 Hz, H-1), 4.97 (1H, dd, J4 ,3 9.6 Hz, J4 ,5 9.6 Hz,
H-40) 4.91 and 4.71 (AB quartet, 2H, Jgem 11.7 Hz,
(1H, dd, H-4), 3.63 (1H, ddd, J5 ,6 4.3 Hz, J5 ,6a
2.4 Hz, H-50), 2.17 (3H, s, NAc), 2.14, 2.10, 2.08, 2.06,
1.99, 1.98, and 1.91 (3H each, s each, 5 · COCH3); 13C
NMR (75 MHz, CDCl3): d 170.97, 170.64, 170.50,
170.40, 169.72, and 169.34, 168.84 (s, CO), 100.77 (s,
C-10), 88.17 (d, J1,F2 22.27 Hz, C-1), 86.35 (d, J2,F2
196.46 Hz, C-2), 75.33 (d, J4,F2 7.26 Hz, C-4), 72.14,
71.83, 70.54 (s, C-20, C-30, C-40), 70.03, (d, J3,F2
19.07 Hz, C-3), 68.05 (s), 61.71, 61.44 (s each, C-6, C-
60), 54.97 (s, C-20), 23.16 (s, NHCOCH3), 20.91, 20.71,
20.66, 20.59 (s, 5 · COCH3); Selected NMR data for
the b anomer; 19F NMR (188 MHz, CDCl3, CF3CO2H
reference): d À125.91 (1F, dd, JF2,H1 12.2 Hz, JF2,H2
49.0 Hz, F-2); CIMS(NH 3): m/z 655 (M+NH4)+
(2.0%); 638 (M+H)+ (63.0%); HRCIMS(M+H) +: Cal-
culated, 638.20978; Found, 638.20966; Anal. Calcd for
C26H36FNO16: C, 48.98; H, 5.69; N, 2.20. Found: C,
48.85; H, 5.69; N 2.14.
PhCH2), 4.83 (1H, dd, J3 ,2 10.0 Hz, H-30), 4.81 and
4.34 (AB quartet, 2H, Jgem 12.1 Hz, PhCH2), 4.59 (1H,
0
0
0
d, JNH,2 9.3 Hz, NH), 4.53 (1H, ddd, J2,3 8.8 Hz, H-
2), 4.41 (1H, d, J1 ,2 8.5 Hz, H-10), 4.09 (1H, dd, Jgem
0
0
12.3 Hz, J6 ,5 4.4 Hz, H-60), 3.96–3.88 (3H, m, H-20,
0
0
0
0
0
H-3, H-4), 3.85 (1H, dd, J6a ,5 2.4 Hz, H-6a ), 3.74–
3.70 (1H, m, H-5), 3.61 (1H, dd, Jgem 11.0, J6,5 2.4 Hz,
H-6), 3.49 (1H, dd, J6a,5 2.0 Hz, H-6a), 3.42 (1H, ddd,
H-50); 2.13, 1.99, 1.97, 1.96, 1.92, 1.69 (s each, 3H each,
5 · COCH3); 13C NMR (75 MHz, CDCl3): d 182.98,
170.75, 170.60, 169.83, 169.30, and 169.15 (s, CO),
138.67, 137.50, 129.09, 129.00, 128.13, 127.37, 127.33,
and 127.03 (s, Ph), 100.93 (s, C-10), 88.90 (d, J1,F2
22.8 Hz, C-1), 88.84 (d, J2,F2 191.2 Hz, C-2), 78.40 (d,
J3,F2 17.2 Hz, C-3), 76.10 (d, J4,F2 8.4 Hz, C-4), 74.67
(s), 73.88 (s), 72.75 (s), 71.85 (s), 71.42 (s), 68.22 (s),
66.68 (s), 61.74 (s, C-60), 54.14 (s, C-20), 23.12 (CH3),
20.98 (s, CH3), 20.61 (s, CH3); CIMS+ (NH3): m/z 751
(M+NH4)+ (0.46%); 734 (M+H)+ (14.1%); 674
(25.6%); HRCIMS: (M+NH4)+: Calculated, 751.30896;
Found, 751.30957; (M+H)+: Calculated, 734.27968;
Found, 734.28241.
3.7. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluco-
pyranosyl-(1!4)-3,6-di-O-acetyl-2-deoxy-2-fluoro-a/b-D-
glucopyranose (9)
To a soln of 8 (230 mg, 0.36 mmol) in DMF (3 mL) was
added hydrazine acetate (45 mg, 0.49 mmol, 1.4 equiv)
and the reaction mixture was stirred at room tempera-
ture for 2 h. After this time, water (20 mL) was added
and a white precipitate formed immediately. This mix-
ture was extracted with CH2Cl2 (2 · 25 mL) and the or-
ganic layer was then washed with water (25 mL), twice
with satd NaHCO3 (2 · 25 mL), and once with brine
(25 mL). The organic layer was then dried with MgSO4,
filtered, and the solvent removed under diminished pres-
sure to yield 9 as a mixture of anomers (1:2, b to a)
appearing as a faintly yellow solid (202 mg, 0.34 mmol,
94%). 19F NMR (188 MHz, (CD3)2CO, CF3CO2H refer-
ence): d À117.96 (1F, ddd, JF2,H2 51.2 Hz, JF2,H3
3.6. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluco-
pyranosyl-(1!4)-1,3,6-tri-O-acetyl-2-deoxy-2-fluoro-a/b-
D-glucopyranose (8)
The disaccharide 6 (335 mg, 0.46 mmol) was dissolved in
a mixture of 6:2:1 MeOH–EtOAc–water (9 mL). Pd–C
(340 mg) was added and the reaction mixture was stirred
under an atmosphere of hydrogen for 4 d at which time
the reaction was judged to be complete by 19F NMR.
The catalyst was removed by suction filtration through
Celite and glass fiber and the solvent removed under
diminished pressure to yield crude 7 as a clear residue.
To this gum was added pyridine (6 mL) and Ac2O

386
D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
14.5 Hz, JF2,H1 2.3, F-2 (b anomer)), À118.94 (1F, dd,
JF2,H2 50.5 Hz, JF2,H3 12.3 Hz, F-2 (a anomer)); 1H
0
NMR (400 MHz, (CD3)2CO): 7.14 (d, JNH,2 8.4, Hz,
C60), 55.37 (s, C-20), 23.19 (s, NHCOCH3), 20.71,
20.67, 20.63, 20.58 (s, 5 · COCH3); Anal. Calcd for
C28H34FN3O18; C, 46.74; H, 4.76; N, 5.84. Found: C,
47.00; H, 4.74; N 5.53.
0
NH), 7.13 (d, JNH,2 8.2, Hz, NH), 6.38 (d, JOH,1
6.6, Hz, OH b-anomer), 6.22 (d, JOH,1 4.7, Hz, OH
a-anomer), 5.54 (ddd, J 11.9 Hz, J 9.5 Hz, J 9.5 Hz),
5.40–5.25 (m), 5.0–4.92 (m), 4.52–4.32 (m), 4.15–4.00
(m), 4.88–3.63 (m), 2.104, 2.096 (s, NHAc), 2.062,
2.057, 2.051, 2.046, 2.033, 1.957, 1.920, 1.916, 1.830,
3.9. 2,4-Dinitrophenyl 2-acetamido-2-deoxy-b-D-gluco-
pyranosyl-(1!4)-2-deoxy-2-fluoro-b-D-glucopyranoside
(11)
1.824 (s, OAc); CIMS(NH ): m/z 596 (M+H)+ (7.4%),
To a cool (0 ꢁC) soln of the per-O-acetylated glycoside
10 (22 mg, 0.029 mmol) in dry MeOH (1 mL) was added
AcCl (0.2 mL) to yield a 2.3 M soln of methanolic HCl.
This mixture was then maintained at 0 ꢁC for 20 h at
which point the solvent was evaporated under dimin-
ished pressure to yield a pale yellow crystalline residue.
Recrystallization of this material from MeOH yielded
pale yellow needles of 11 (4.2 mg, 0.008 mmol, 26%).
19F NMR (376 MHz, CD3OD, CF3CO2H reference): d
À124.60 (1F, ddd, JF,H2 51.2 Hz, JF,H3 16.5 Hz, JF,H1
3
536 (14.5%), 330 (100%); HRCIMS: (M+H)+: Calcu-
lated, 596.19910; Found, 596.20166.
3.8. 2,4-Dinitrophenyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-D-glucopyranosyl-(1!4)-3,6-di-O-acetyl-2-
deoxy-2-fluoro-b-D-glucopyranoside (10)
To a soln of hemiacetal 9 (93 mg, 0.16 mmol) in DMF
(1.8 mL) under argon was added 2,4-dinitrofluorobenz-
ene (65 mg, 0.34 mmol, 2.2 equiv) and DABCO
(82 mg, 0.73 mmol, 4.7 equiv). The dark orange soln
was stirred at room temperature for 3.5 h after which
CH2Cl2 (60 mL) was added. This soln was washed with
water (1 · 50 mL), satd NaHCO3 (2 · 50 mL), and brine
(2 · 50 mL). The organic layer was dried over MgSO4,
filtered, and concentrated under diminished pressure to
a yellow powder. This residue was then partially purified
by step gradient flash column chromatography on silica
(1:3 petroleum ether–EtOAc to 1:4 MeOH–EtOAc) to
yield a pale yellow solid containing a mixture of ano-
mers. Crystallization of this solid from EtOAc–EtOH–
hexane yielded the pure b-anomer 10 as fine white crys-
tals (67 mg, 0.088 mmol, 55%); mp 258–259 ꢁC (dec);
19F NMR (188 MHz, CDCl3, CF3CO2H reference): d
À120.85 (1F, ddd, JF,H2 49.8 Hz, JF,H3 16.6 Hz, JF,H1
1
3.0 Hz); H NMR (400 MHz, CD3OD): d 8.72 (1H, d,
JAR3,AR5 2.8 Hz, HAR3), 8.46 (1H, dd, JAR5,AR6
9.3 Hz, HAR6), 7.61 (1H, d, HAR6), 5.60 (1H, d, JH1,
7.6 Hz, H-1), 4.51 (1H, d, JH1 ,H2 8.5 Hz, H-10),
4.35 (1H, ddd, JH2,H3 8.7 Hz, H-2), 3.95 (1H, ddd, JH3,
0
0
H2
7.9 Hz, H-3), 3.91 (1H, dd, JH6, H6a 11.6 Hz, JH6,
0
H4
2.0 Hz, H-6), 3.84 (1H, d, JH6 ,H6a 11.6 Hz, H-60),
0
H5
3.73–3.62 (5H, m, H-6a, H-30, H-40, H-50, H-6a0), 3.44
(1H, dd, JH4, H5 10.3 Hz, H-4), 3.35 (1H, ddd, JH5, H6a
6.5 Hz, H-5), 1.99 (3H, s, CH3); 13C NMR (100 MHz,
CD3OD): d 173.78 (s, CO), 154.90, 143.13, 141.15,
129.74, 122.16, 118.90, 103.16, 99.19 (d, J1,F1 23.78 Hz,
C-1), 92.37 (d, J2,F1 189.20 Hz, C-2), 80.11 (d, J4,F1
7.63 Hz, C-4), 78.15 (s, C-50), 77.06 (s, C-5), 75.77 (s,
C-30), 74.58, (d, J3,F1 17.89 Hz, C-3), 72.05 (s, C-40),
62.61, 61.18 (s, C6, C60), 57.33 (s, C-20), 23.04 (s,
NHCOCH3); HRESIMS: (M+NH3)+: Calculated,
574.1297; Found, 574.1308; (M+H+): Calculated,
552.1477; Found 552.1470.
1
3.7 Hz); H NMR (400 MHz, CDCl3): d 8.70 (1H, d,
JAR3,AR6 8.4 Hz, HAR3), 8.41 (1H, dd, JAR6,AR5
0
9.7 Hz, HAR6), 7.37 (1H, d, HAR5), 5.76 (1H, d, JNH,H2
8.5 Hz, NH), 5.43 (1H, d, J1,2 6.3 Hz, H-1), 5.38 (1H,
3.10. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluco-
pyranosyl-(1!4)-3,6-di-O-acetyl-2-deoxy-2-fluoro-b-D-
glucopyranosyl fluoride (12)
0
ddd, J3,2 7.9 Hz, J3,4 7.9 Hz, H-3), 5.28 (1H, dd, JH3 ,H2
0
10.3 Hz, JH3 ,H4 9.4 Hz, H-30), 5.03 (1H, dd, JH4 ,H5
0
0
0
0
9.6 Hz, H-40), 4.74 (1H, d, JH1 ,2 8.2 Hz, H-10), 4.63
(1H, ddd, H-2), 4.42 (1H, dd, JH6, H6a 12.2 Hz, JH6, H5
0
0
To a cool (À30 ꢁC) soln of the hemiacetal 9, under an
atmosphere of argon, (90 mg, 0.15 mmol) in a mixture
of CH2Cl2 (3 mL) and THF (10 mL) was added DAST
(0.5 mL, 0.38 mmol). The reaction mixture was warmed
to 20 ꢁC and stirred for 30 min. The reaction was judged
to be complete by TLC analysis of the mixture and was
then cooled to À30 ꢁC at which time 100 lL of MeOH
were added. This mixture was then warmed to 20 ꢁC
and stirred for another 3 h. The solvent was then evap-
orated under diminished pressure to yield a dark yellow
gum, which was dissolved in CH2Cl2 (30 mL), washed
with satd NaHCO3 (30 mL) and brine (30 mL), and
dried over MgSO4. After concentration under dimin-
0
0
1.7 Hz, H-6), 4.35 (1H, dd, JH6 ,H6a 12.4 Hz, JH6 ,H5
0
0
4.3 Hz, H-60), 4.19 (1H, dd, JH6a,
4.5 Hz, H-6a),
H5
4.06 (1H, dd, JH6a ,H5 2.2 Hz, H-60a), 3.95–3.86 (2H,
0
0
m, H-5, H-4), 3.74–3.64 (2H, m, H-20, H-50), 2.13,
2.08, 2.02, 2.00, 1.99, 1.91 (3H each,
s each,
5 · COCH3); 13C NMR (100 MHz, CDCl3): d 182.98,
170.65, 170.51, 170.45, 170.40, and 169.65, 169.36 (s,
CO), 153.28, 142.18, 140.23, 128.57, 121.55, 117.70 (s,
Ph) 100.50 (s, C-10), 97.87 (d, J1,F1 26.56 Hz, C-1),
88.65 (d, J2,F1 191.04 Hz, C-2), 74.86 (d, J4,F1 5.75 Hz,
C-4), 73.54, 72.14, 71.98 (s, C-20, C-30, C-40), 72.06, (d,
J3,F2 16.33 Hz, C-3), 68.23 (s), 61.80, 61.58 (s, C6,

D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
387
ished pressure, the resulting gum was purified by flash
column silica chromatography (1:4 petroleum ether
(35–60 ꢁC)–EtOAc) to provide a white crystalline solid.
Recrystallization of this material from a mixture of
EtOAc and EtOH provided fine white needles of 12
(60 mg, 0.1 mmol, 67%); mp 258–259 ꢁC; 19F NMR
(282 MHz, CDCl3, CF3CO2H reference): d À138.65
(ddd, JF1,H1 52.8 Hz, JF1,F2 17.0 Hz, F-1), À199.25
(dddd, JF2,H2 49.6 Hz, JF2,H3 16.0 Hz, JF2,H1 3.4 Hz,
NMR (100 MHz, CDCl3): d 173.76 (s, CO), (Note: C-
1 was not observed above noise but is expected to be ap-
pear as a dd at approximately 105 ppm), 103.11 (s, C-
10), 83.2 (dd, J2,F2 190.2 Hz, J2,F1 28.4 Hz, C-2), 79.98
(d, J5,F1 6.8 Hz, C-5), 78.12, 76.48 (d, J4,F2 9.0 Hz, C-
4), 75.76, 74.02 (dd, J3,F2 14.6 Hz, J3,F1 8.1 Hz, C-3),
72.06, 62.61, 61.15, 57.34 (s, C-20), 23.01 (s,
NHCOCH3); CIMS(NH ): m/z 388 (M+H)+ (100%);
3
368 (MÀF)+ (12.3%); HRCIMS(M+H) +: Calculated,
1
F-2); H NMR (400 MHz, CDCl3): d 5.72 (1H, d, JNH,
388.14191; Found, 388.14280.
H20
8.4 Hz, NH), 5.40 (1H, ddd, J1,2 5.6 Hz, Hz, H-1),
0
Enzyme kinetics: HEWL was obtained from Sigma
Chemical Co (Lot # 89F8276). All kinetic studies were
performed at 25 ꢁC in 150 mM NaOAc buffer,
pH 5.00. Attempts to observe the liberation of DNP
from 11 (5 mM) in the presence of HEWL (101 lM)
were made by monitoring the reaction at 400 nm by
means of a UNICAM 8700 UV–vis spectrophotometer
equipped with a circulating water bath. Reaction rates
for the hydrolysis of 13 by HEWL were determined by
monitoring the release of fluoride using a 9606BN Ion-
plus Orion fluoride ion electrode interfaced to a pH/
ion selective meter (Fischer Scientific). Reactions were
initiated by the addition of an aliquot (50 lL) of a stock
soln of enzyme (12.7 mg/mL) to give a final concentra-
tion of 101 lM HEWL in a final vol of 440 lL. Reaction
cells were pre-equilibrated in a water bath to 25 ꢁC. Cal-
culation of the second-order rate constant for substrate
13 was carried out by directly fitting the initial rate data
to a linear equation using GraFit version 3.0.30
5.29 (2H, m, H-3, H-30), 5.01 (1H, dd, JH4 ,H3 JH4 ,H5
0
0
0
9.6 Hz, H-40), 4.74 (1H, dd, JH1 ,H2 8.3 Hz, H-10), 4.46
(1H, m, H-2), 4.42 (1H, d, JH6, H6a 12.2 Hz, H-6), 4.34
0
0
(1H, dd, JH6 ,H6a 12.4 Hz, JH6 ,H5 4.6 Hz, H-60), 4.21
0
0
0
0
0
0
(1H, dd, JH6, H5 2.8 Hz, H-6a), 4.02 (1H, dd, JH6a ,H5
2.2 Hz, H-60a), 3.83 (2H, m, H-5, H-4), 3.71–3.63 (2H,
m, H-20, H-50), 2.12, 2.09, 2.07, 2.00, 1.99, 1.91 (3H
each, s each, 5 · COCH3); 13C NMR (100 MHz, CdCl3):
d 170.80, 170.59, 170.41, 170.32, 169.52, and 169.36 (s,
CO), 105.63 (dd, J1,F1 218.7 Hz, J1,F2 28.6 Hz, C-1),
100.40 (s, C-10), 88.96 (dd, J2,F2 189.6 Hz, J2,F1
30.2 Hz, C-2), 74.79 (d, J5,F1 6.0 Hz, C-5), 72.90 (s, C-
4), 71.95 (s, 2 · C), 71.66 (dd, J3,F2 13.8 Hz, J3,F1
8.1 Hz, C-3), 68.22, 61.80, 61.66, 55.36 (s), 23.19 (s,
NHCOCH3), 20.87, 20.64, 20.59, 20.57 (s, 5 · COCH3);
Anal. Calcd for C24H33F2NO14; C, 48.24; H, 5.57; N,
2.34. Found: C, 48.45; H, 5.59; N 2.39.
3.11. 2-Acetamido-2-deoxy-b-D-glucopyranosyl-(1!4)-2-
deoxy-2-fluoro-b-D-glucopyranosyl fluoride (13)
Acknowledgements
To a soln of 12 (24 mg, 0.04 mmol) in 2 mL of dry
MeOH was added 50 mL of a 1 M soln of NaOMe in
dry MeOH. After 30 min, the reaction was judged com-
plete and Amberlite IR-120 (H+) cation exchange resin
was added until the mixture was neutral. The soln was
then filtered and the solvent evaporated under dimin-
ished pressure to yield a pale yellow gum. Preparative
thin layer chromatography of this gum (7:2:1 EtOAc–
MeOH–water) yielded the desired product as a transpar-
ent gum (14 mg, 0.036 mmol, 90%). 19F NMR
(282 MHz, CD3OD, CF3CO2H reference): d À145.59
(1F, ddd, JF1,H1 53.5 Hz, JF1,F2 14.1 Hz, JF1,H2
13.3 Hz, F-1), À203.64 (1F, dddd, JF2,H2 51.4 Hz,
JF2,H3 = 17.0 Hz, JF2,H1 = 3.3 Hz, F-2); 1H NMR
(400 MHz, CD3OD) d: 5.35 (1H, d, J1,2 6.9 Hz, H-1),
We thank the Natural Sciences and Engineering Re-
search Council of Canada (NSERC) and the Protein
Engineering Network of Centres of Excellence (PENCE)
for financial support. D.J.V. thanks NSERC for a fel-
lowship and S. J. Williams for useful discussions.
References
1. Blake, C. C. F.; Johnson, L. N.; Mair, G. A.; North, A. C.
T.; Phillips, D. C.; Sarma, V. R. Proc. R. Soc. Lond. B:
Biol. Sci. 1967, 167, 378–388.
2. Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G.
Nature 2001, 412, 835–838.
3. Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C.
T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757–
761.
4. Strynadka, N. C. J.; James, M. N. G. J. Mol. Biol. 1991,
220, 401–424.
5. Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc. 1991, 113,
7958–7963.
6. Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc. 1991, 113,
7951–7958.
7. Amyes, T. L.; Jencks, W. P. J. Am. Chem. Soc. 1986, 111,
7888–7900.
4.49 (1H, d, J1 ,2 8.5 Hz, H-10), 4.15 (1H, dddd, J2,3
8.5 Hz, H-2), 3.90 (1H, dd, JH6, H6a 11.6 Hz, JH6, H5
0
0
2.2 Hz, H-6), 3.85 (1H, ddd, JH3,
17.0 Hz, J3,4
0
F2
0
0
0
8.5 Hz, H-3), 3.80 (1H, dd, JH6 ,H6a 12.2 Hz, JH6 ,H5
2.0 Hz, H-60), 3.67 (1H, dd, JH4 ,H5 10.3 Hz, JH4 ,H3
0
0
0
0
8.6 Hz, H-40), 3.64–3.60 (3H, m, H-6a, H-6a0, H-30),
3.50 (1H, ddd, JH5, H4 10.3 Hz, JH5, H6a 4.4 Hz, H-5),
0
3.43 (1H, dd, H-4), 3.33 (1H, ddd, JH5,H6a 6.5 Hz, H-
50), 3.30–3.26 (1H, m, H-20), 1.8 (3H, s, CH3); 13C

388
D. J. Vocadlo, S. G. Withers / Carbohydrate Research 340 (2005) 379–388
8. Sinnott, M. L.; Souchard, I. J. L. Biochem. J. 1973, 133,
89–98.
20. Gallant, M.; Link, J. T.; Danishefsky, S. J. J. Org. Chem.
1993, 58, 343–349.
9. Li, B. F.; Holdup, C. A.; Morton, C. A.; Sinnott, M. L.
Biochem. J. 1989, 260, 109–114.
10. Kempton, J. B.; Withers, S. G. Biochemistry 1992, 31,
9961–9969.
21. Burkart, M. D.; Zhang, Z.; Hung, S.-C.; Wong, C.-H. J.
Am. Chem. Soc. 1997, 119, 11743–11746.
22. Albert, M.; Dax, K.; Ortner, J. Tetrahedron 1998, 54,
4839–4848.
11. Umezurike, G. M. Biochem. J. 1988, 254, 73–76.
12. Tull, D.; Withers, S. G. Biochemistry 1994, 33, 6363–6370.
13. Vocadlo, D. J.; Wicki, J. W.; Rupitz, K.; Withers, S. G.
Biochemistry 2003, 41, 9727–9735.
14. Bravman, T.; Zolotnitsky, G.; Belakhov, V.; Shoham, G.;
Henrissat, B.; Baasov, T.; Shoham, Y. Biochemistry 2003,
42, 10528–10536.
15. Zechel, D. L.; Reid, S. P.; Stoll, D.; Nashiru, O.; Warren,
R. A.; Withers, S. G. Biochemistry 2003, 42, 7195–7204.
16. Withers, S. G.; Street, I. P. J. Am. Chem. Soc. 1988, 110,
8551–8553.
23. Ortner, J.; Weber, A. H.; Dax, K. J. Carbohydr. Chem.
1999, 18, 297–316.
24. Vincent, S. P.; Burkart, M. D.; Tsai, C. Y.; Zhang, Z. Y.;
Wong, C.-H. J. Org. Chem. 1999, 64, 5264–5279.
25. Blatter, G.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res.
1994, 260, 189–202.
26. Adamson, J.; Marcus, D. M. Carbohydr. Res. 1972, 22,
257–264.
27. van Boom, J. H.; Koeners, H. J.; de Koke, A. J.; Romers,
C. Recl. Trav. Chim. Pay.-Bas 1980, 99, 355–
362.
17. Street, I. P.; Kempton, J. B.; Withers, S. G. Biochemistry
1992, 31, 9970–9978.
28. Mackenzie, L. F.; Davies, G. D.; Schulein, M.; Withers, S.
G. Biochemistry 1997, 36, 5893–5901.
18. Ballardie, F. W.; Capon, B.; Cuthbert, M. W.; Dearie, W.
M. Bioorg. Chem. 1977, 6, 483–509.
29. McCarter, J. D.; Adam, M. J.; Withers, S. G. Biochem. J.
1992, 286, 721–727.
19. Rand-Meir, T.; Dahlquist, F. W.; Raftery, M. A. Bio-
chemistry 1969, 8, 4206–4214.
30. Leatherbarrow, R. 4.0 Ed., Erithacus Software Ltd,
Staines, UK, 1990.