Synthesis and testing of 2-deoxy-2,2-dihaloglycosides as mechanism-based inhibitors of α-glycosidases

Article abstract of DOI:10.1021/jo702565q

(Chemical Equation Presented) The synthesis of a series of 2-deoxy-2,2-dihaloglycosyl halides as potential α-glycosidase inactivators has been achieved via the halogenation of protected 2-fluoroglycal precursors. Direct chlorination of per-O-acetylated 2-

Full text of DOI:10.1021/jo702565q

Synthesis and Testing of 2-Deoxy-2,2-Dihaloglycosides as
Mechanism-Based Inhibitors of r-Glycosidases
Ran Zhang,† John D. McCarter,† Curtis Braun,† Wai Yeung,† Gary D. Brayer,‡ and
Stephen G. Withers*,†,‡
Department of Chemistry, UniVersity of British Columbia, 2036 Main Mall, VancouVer, British Columbia,
Canada V6T 1Z1, and Department of Biochemistry and Molecular Biology, UniVersity of British
Columbia, VancouVer, British Columbia, Canada, V6T 1Z3
withers@chem.ubc.ca
ReceiVed NoVember 29, 2007
The synthesis of a series of 2-deoxy-2,2-dihaloglycosyl halides as potential R-glycosidase inactivators
has been achieved Via the halogenation of protected 2-fluoroglycal precursors. Direct chlorination of
per-O-acetylated 2-fluoro-D-glucal and 2-fluoromaltal followed by basic deprotection yielded the
corresponding 2-chloro-2-deoxy-2-fluoroglycosyl chlorides. Reaction of the per-O-acetylated 2-fluoro-
glycals with acetyl hypofluorite or Selectfluor yielded the 2-deoxy-2,2-difluoroglycosyl derivatives, which
were converted to their R-chlorides using thionyl chloride and deprotected under basic conditions.
Trinitrophenyl glycosides of the 2-deoxy-2,2-difluoro mono- and disaccharides were synthesized by
arylation of the hemiacetals with picryl fluoride, then deprotected with HCl in methanol. All three
monosaccharide derivatives caused active site-directed, time-dependent inactivation of yeast R-glucosidase
Via the trapping of covalent glycosyl-enzyme intermediates, and kinetic parameters for inactivation by
each compound were determined. Surprisingly neither of the 2-deoxy-2,2-dihalomaltosyl chlorides caused
time-dependent inactivation of human pancreatic R-amylase, despite the fact that the trinitrophenyl 2-deoxy-
2,2-difluoromaltoside functioned in that mode. The trinitrophenyl glycosides appear to be approximately
1000-fold more reactive than the corresponding chlorides in the enzyme active sites.
Introduction
but also provide guidance for the rational design and optimiza-
tion of reversible inhibitors. Indeed, given the importance of
such irreversible-inhibitor drugs as penicillin and aspirin,
mechanism-based inhibitors of glycosidases and other enzymes
may also prove valuable, despite industry prejudices against such
approaches.
The vast majority of glycosidases employ one of two
fundamental mechanisms, first described by Koshland,6 that are
based on the stereochemical outcomes of the hydrolysis reactions
they catalyze.7 Inverting enzymes use a single displacement
mechanism with acid/base catalysis and oxocarbenium ion-like
transition states. Retaining glycosidases generally use a double
displacement mechanism (Figure 1) in which the first step
involves an acid-catalyzed nucleophilic displacement of the
aglycone by a carboxylate residue at the active site (glycosy-
lation step). In a second step the covalent glycosyl-enzyme
Glycosidases are involved in a range of important biological
processes, and potent inhibitors of these enzymes have shown
promise as therapeutics for diseases such as diabetes, obesity,
lysosomal storage diseases and influenza.1 Indeed several
glycosidase inhibitors are already used clinically for treating
some of these diseases.2-3 While interest has generally focused
on reversible inhibitors of glycosidases, irreversible inhibitors
have proved to be extremely important in providing structural
and mechanistic insights4-5 that not only are of academic interest
† Department of Chemistry, University of British Columbia.
‡ Department of Biochemistry and Molecular Biology, University of British
Columbia.
(1) Asano, N. Glycobiology 2003, 13, 93R-104R.
(2) Laube, H. Clin. Drug InVest. 2002, 22, 141-156.
(3) Moscona, A. New Engl. J. Med. 2005, 353, 1363-1373.
(4) Mosi, R. M.; Withers, S. G. Methods Enzymol. 2002, 354, 64-84.
(5) Wicki, J.; Rose, D. R.; Withers, S. G. Methods Enzymol. 2002, 354,
84-105.
(6) Koshland, D. E. Biol. ReV. 1953, 28, 416.
(7) Zechel, D. L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11-18.
10.1021/jo702565q CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/18/2008
3070
J. Org. Chem. 2008, 73, 3070-3077

2-Deoxy-2,2-Dihaloglycosides
FIGURE 1. Double displacement mechanism of retaining R-glycosidases.
intermediate is then hydrolyzed, with general base catalysis
provided by the same group that earlier served as the acid
catalyst (deglycosylation step). Both steps involve oxocarbenium
ion-like transition states.
effects of fluorine substitution at the C-2 position tend to be
proportionally greater on the glycosylation step than the
deglycosylation step. Consequently the 2-deoxy-2-fluoro-R-
glycosides tend to function as slow substrates, for which the
glycosylation step is rate-limiting. Two solutions to this problem
have been devised. In one approach a fluorine introduced at
C-5 seems to inductively destabilize the deglycosylation step
for R-glycosidases more than the glycosylation step, thus when
C-5 fluorination is coupled with the incorporation of a good
leaving group (fluoride) at C-1, inactivation may be achieved.17
Unfortunately, synthetic approaches to the disaccharide deriva-
tives that would be needed for amylases, for which tight-binding
inhibitors might serve as novel anti-diabetes drugs, have not
been developed since selective fluorination at C-5 and not C-5′
has not proved possible, and reaction yields are insufficient to
effect successful fluorination at both sites. The other approach
has been to introduce two fluorines at C-2, thereby slowing
down both steps enormously.18-19 This requires the incorpora-
tion of a yet better leaving group at the anomeric center in order
to allow the glycosylation step to proceed. To date this has been
achieved by the use of a 2,4,6-trinitrophenyl (picryl) group
(TNP). The presence of the two fluorines at C-2 ensures that
the compound has the necessary inherent solvolytic stability to
be deprotected and survive in aqueous solution. Three such
compounds, all with two fluorines at C-2 and a TNP group at
the anomeric center, have been reported previously20-21 and all
act as excellent, mechanism-based inactivators for their corre-
2-Deoxy-2-fluoroglycosides with activated leaving groups
have proved to be a very useful class of mechanism-based
inhibitors for retaining â-glycosidases that function Via the
formation of a relatively stable R-linked 2-deoxy-2-fluorogly-
cosyl-enzyme intermediate.8-9 The presence of a fluorine at
C-2 destabilizes the oxocarbenium ion-like transition states both
inductively and via the removal of key transition state-stabilizing
interactions that are normally present at C-2. As a consequence
both the formation and the hydrolysis of the glycosyl-enzyme
intermediate are slowed down. However, the incorporation of
a very good leaving group ensures that the intermediate is
kinetically accessible, thus that it accumulates. This approach
has been applied to many retaining â-glycosidases and the
resultant trapped intermediates have been characterized by NMR
spectroscopy, MS, and X-ray crystallography, thereby providing
great insights into each enzyme’s mechanism.10-15
While this strategy has been highly successful with a range
of retaining â-glycosidases, it has not been generally successful
with R-glycosidases.9,16 In these latter cases the deglycosylation
step tends to be fast relative to the glycosylation step and the
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(12) McIntosh, L. P.; Hand, G.; Johnson, P. E.; Joshi, M. D.; Korner,
M.; Plesniak, L. A.; Ziser, L.; Wakarchuk, W. W.; Withers, S. G.
Biochemistry 1996, 35, 9958-9966.
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Struct. Biol. 1996, 3, 149-154.
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A. M.; Schulein, M.; Withers, S. G. Biochemistry 1998, 37, 11707-11713.
J. Org. Chem, Vol. 73, No. 8, 2008 3071

Zhang et al.
sponding enzymes. Indeed the 2,2-difluorogalactosyl derivative
has been used to identify the catalytic nucleophile in an
R-galactosidase from family 27.21 However, details of the
synthesis of this class of compounds have not been reported.
While the use of gem-difluoro substitution has a certain
esthetic appeal, and minimizes the chances of steric conflicts,
it is not clear that both halogens need to be fluorine. A chlorine
substituent would provide a similar inductive effect at C-2, and
while larger than fluorine, it is still of a comparable size to the
hydroxyl group originally at that position. Replacement by
chlorine would also provide certain advantages in terms of
synthesis since the introduction of chlorine is generally simpler
than introduction of fluorine. Yet more importantly, through
the use of Cl2 as chlorinating agent it may be possible to also,
simultaneously, introduce an excellent leaving group at C-1 in
the form of a chloride. We have found that sugars bearing
anomeric chlorides have generally proved to be too labile to be
deprotected, even in the presence of a fluorine substituent at
C-2, vividly illustrating the relatively high reactivity achievable
with such an aglycone. However, it seems probable that, when
accompanied by dihalo substitution at C-2, such deprotected
sugars may prove sufficiently stable to survive aqueous condi-
tions, but still reactive enough to undergo the first step of a
retaining glycosidase mechanism and thereby trap the intermedi-
ate. An additional potential advantage is that, by introducing
halides of different sizes at C-2 it may be possible to generate
inhibitors that are selective for R-glucosidases over R-mannosi-
dases, by incorporating an equatorial chlorine and an axial
fluorine, or Vice Versa to produce selective R-mannosidase
inhibitors.
especially challenging for the synthesis of disaccharides. The
third approach, which was the one we followed, involves the
initial synthesis of a 2-fluoroglycal, which is then reacted with
an electrophilic fluorine source such as Selectfluor, F2, acetyl-
hypofluorite or trifluoroxymethane.25,30-31 The gem-difluoro
sugars can be obtained in respectable yields in this manner. This
route is also highly compatible with the synthesis of 2-deoxy-
2-chloro-2-fluoro sugars, since the same fluoroglycal intermedi-
ate can be reacted with electrophilic chlorine sources. Indeed,
by use of Cl2 as the chlorinating agent it should be possible to
introduce chlorines at both the C-2 and C-1, with the latter
potentially serving as a suitably reactive leaving group. The
initial choice of a trinitrophenyl leaving group at C-1 of these
gem-dihalo sugars was based upon its substantially better leaving
group ability (pKa of picric acid ) 0.4) than that of the 2,4-
dinitrophenyl leaving group (pKa ) 4.0) used with the mono-
fluorosugar inhibitors, as needed to offset the rate retardation
in the glycosylation step effected by the two adjacent halogens.6
It was also advantageous synthetically since it could be installed
by reaction of the sugar hemi-acetal with picryl fluoride, thereby
avoiding potentially challenging displacement reactions at a
highly deactivated anomeric center using a very weak phenol
nucleophile.
This manuscript describes the synthesis of several 2-deoxy-
2,2-dihaloglycosyl derivatives in both a monosaccharide and a
disaccharide series, with anomeric leaving groups of either
chloride or 2,4,6-trinitrophenolate. It further describes the kinetic
evaluation of these reagents as mechanism-based inhibitors of
both the yeast R-glucosidase and the human pancreatic R-amy-
lase.
There are three general strategies by which sugars containing
geminal fluorine substituents have been synthesized, though in
most of the cases published the products have not additionally
included a highly reactive leaving group. The most common
approach is to selectively oxidize the hydroxyl group at the site
in question and then react the ketone with DAST to introduce
the gem-difluoro functionality.22-25 However, the success of this
reaction is highly dependent on the structure of the substrate,
particularly with respect to the identities and stereochemistry
of neighboring groups.26-27 In some cases yields of over 80%
were obtained while in other cases, moderate yields or even no
detectable yields were observed due to competing side reactions
such as migrations and eliminations. A second approach involves
a Reformatsky reaction in which acyclic gem-difluoro precursors
such as ethyl bromotrifluoroacetate or bromodifluoromethyl
phenyl acetylene are coupled with a correctly configured
aldehyde, then the product cyclized to complete the synthesis
of the gem-difluoro-substituted sugar ring.28-29 While this
approach was successful for the assembly of 2-deoxy-2,2-
difluororibose, it requires maintenance of exquisite control over
the stereochemistry of the ring hydroxyls and would be
Results and Discussions
I. Synthesis of 2-Chloro-2-deoxy-2-fluoro-r-D-glucosyl
chloride (1) and 2-chloro-2-deoxy-2-fluoro-r-maltosyl chlo-
ride (2) (Scheme 1). 3,4,6-Tri-O-acetyl-2-fluoro-D-glucal, 3,
prepared essentially as described previously from commercially
available D-glucal,31 was dissolved in cooled carbon tetrachloride
(-23 °C) and subjected to chlorination with chlorine gas.32-34
Early studies on the addition of chlorine to the double bond of
tri-O-acetyl-D-glucal had shown that the stereochemical outcome
for this addition depends on the solvent polarity, with nonpolar
solvents favoring syn addition while polar solvents favor anti
addition.34 Indeed, the major product isolated in this case was
that of syn addition from the R-face, namely 3,4,6-tri-O-acetyl-
2-chloro-2-deoxy-2-fluoro-R-D-glucopyranosyl chloride, 4. This
compound was readily deprotected using ammonia in methanol
to yield the desired target 1, which was purified by silica gel
SCHEME 1
(22) Middleton, W. J. J. Org. Chem. 1975, 40, 574-578.
(23) Sharma, R. A.; Kavai, I.; Fu, Y. L.; Bobek, M. Tetrahedron Lett.
1977, 18, 3433-3436.
(24) An, S. H.; Bobek, M. Tetrahedron Lett. 1986, 27, 3219-3222.
(25) Dessinges, A.; Escribano, F. C.; Lukacs, G.; Olesker, A.; Thang,
T. T. J. Org. Chem. 1987, 52, 1633-1634.
(26) El-Laghdagh, A.; Echarri, R.; Matheu, M. I.; Barrena, M. I.;
Castillon, S.; Garcia, J. J. Org. Chem. 1991, 56, 4556-4559.
(27) Nicolaou, K. C.; Ladduwahetty, T.; Randall, J. L.; Chucholowski,
A. J. Am. Chem. Soc. 1986, 108, 2466-2467.
(28) Fried, J.; Halliman, E. A.; Szwedo, M. J. J. Am. Chem. Soc. 1984,
106, 3871-3872.
(29) Hanzawa, Y.; Inazawa, K.; Kon, A.; Aoki, H.; Kobayashi, Y.
Tetrahedron Lett. 1987, 28, 659-662.
(30) Adamson, J.; Foster, A. B.; Westwood, J. H. Carbohyd. Res. 1971,
18, 345-347.
(31) McCarter, J. D.; Adam, M. J.; Braun, C.; Namchuk, M.; Tull, D.;
Withers, S. G. Carbohydr. Res. 1993, 249, 77-90.
(32) Bradley, P. R.; Buncel, E. Can. J. Chem. 1968, 46, 3001-3006.
(33) Lemieux, R. U.; Fraser-Reid, B. Can. J. Chem. 1965, 43, 1460-
1475.
(34) Igarashi, K.; Honma, T.; Imagawa, T. J. Org. Chem. 1970, 35, 610-
616.
3072 J. Org. Chem., Vol. 73, No. 8, 2008

2-Deoxy-2,2-Dihaloglycosides
SCHEME 2
chromatography. Likewise, hexa-O-acetyl-2-fluoromaltal (5)
(synthesis available in the Supporting Information) was similarly
reacted with chlorine gas dissolved in cold carbon tetrachloride
then warmed to room temperature, yielding 3,6-di-O-acetyl-4-
O-[2′,3′,4′,6′-tetra-O-acetyl-R-(1,4)-D-glucosyl]-2-chloro-2-deoxy-
2-fluoro-R-D-glucopyranosyl chloride (6) as the major product
in 49% yield. After purification by flash chromatography and
characterization, the disaccharide derivative 6 was de-O-
acetylated in 82% yield using sodium methoxide in methanol.
The syn addition stereochemistry in each case was clearly shown
mixed solvent.35 This route employs a much more stable and
safer fluorinating agent, but in this case suffered from a lower
yield (around 20%) in the fluorination step. However, when
applied to the fluorination of the 2-fluoromaltal, an excellent
yield (>80%) was obtained. Deprotection of 9 was simply
achieved using ammonia in methanol.
Similar chemistry was employed in the conversion of hexa-
O-acetyl-2-fluoromaltal (5) to the 2-deoxy-2,2-difluoro-4-O-[R-
(1,4)-D-glucopyranosyl]-R-D-arabinohexopyranosyl chloride (14)
except that thionyl chloride could not be employed for the
chlorination step since the acidic conditions resulted in cleavage
of the interglycosidic linkage. Simple lowering of the reaction
temperature was not useful since under these conditions no
chlorination occurred. After unsuccessfully experimenting with
a range of chlorination conditions, including the use of triph-
enylphosphine with N-chlorosuccinimide,36 we were delighted
to discover that the addition of bismuth(III) oxychloride (BiOCl)
to thionyl chloride allows the chlorination reaction to proceed
at room temperature, albeit in low yield.37 BiOCl is thought to
act as a pro-catalyst for BiCl3, which is difficult to handle, but
which itself acts as an efficient Lewis acid catalyst for a range
of reactions.38-39
III. Synthesis of 2,4,6-Trinitrophenyl 2-deoxy-2,2-difluoro-
r-D-arabinohexopyranoside (16) and 2,4,6-trinitrophenyl-
2-deoxy-2,2-difluoro-4-O-[r-(1,4)-D-glucopyranosyl]-r-D-ar-
abinohexopyranoside 18. (Scheme 3). Reaction of 3,4,6-tri-
O-acetyl-2-deoxy-2,2-difluoro-R-D-arabinopyranose (8) with
1-fluoro-2,4,6-trinitrobenzene in dichloromethane in the presence
of the hindered base 2,6-di-tert-butylpyridine for 10 days at
room temperature in the dark under nitrogen provided a
satisfactory yield (51%) of the desired product 15, which was
deprotected using acetyl chloride in methanol. Conversion of
the maltose derivative 12 was achieved in an excellent yield
(91%) under identical conditions, and the protected disaccharide
was also deprotected using acetyl chloride in methanol to yield
the final compound 18 in 71% yield after purification by flash
1
by analysis of the H and 19F NMR spectra of the addition
products. Large J3,F coupling constants of 22.8 and 23.3 Hz
clearly indicated an axial fluorine at C-2 with a trans-diaxial
coupling to H-3. In addition, the presence of relatively small
J1,F coupling constants of 6.0 and 6.1 Hz clearly indicated the
absence of a trans-diaxial coupling in that case, thus the presence
of an equatorial proton at C-1, and therefore of an axial anomeric
chlorine.
II. Synthesis of 2-Deoxy-2,2-difluoro-R-D-arabinohexopy-
ranosyl chloride 10 and 2-deoxy-2,2-difluoro-4-O-[r-(1,4)-
D-glucopyranosyl]-r-D-arabinohexopyranosyl chloride 14
(Scheme 2). 3,4,6-Tri-O-acetyl-2-fluoro-D-glucal 3 was sub-
jected to electrophilic fluorination with acetyl hypofluorite, as
described previously31 to afford 1,3,4,6-tetra-O-acetyl-2-deoxy-
2,2-difluoro-R-D-arabinohexopyranose (7) in a good yield (76%).
All attempts to convert this directly to the R-chloride by
treatment with HCl proved unsuccessful, lending support to the
notion that the presence of geminal fluorine substituents at C-2
would severely slow down the displacement chemistry at the
anomeric center. A two-step chlorination process was therefore
used in which hydrazine acetate was first used to selectively
deprotect the anomeric acetate, yielding the R-hemiacetal 8.
Reaction of this product with thionyl chloride at 60 °C for 3
days provided a reasonable yield (62%) of 3,4,6-tri-O-acetyl-
2-deoxy-2,2-difluoro-R-D-arabinohexopyranosyl chloride (9).
The formation of the R-chloride is most likely a consequence
of an initial â-chloride formation from displacement of the
R-sulfinyl chloride, followed by equilibration to the thermody-
namically favored R-chloride driven by the excess chloride in
solution. This anomeric stereochemistry is clearly evidenced by
the two small JF,1 coupling constants of 4.8 and 2.0 Hz
measured. An axial anomeric proton would exhibit one large
trans-diaxial coupling and one small. Alternatively, synthesis
of 8 can be achieved by directly reacting 3,4,6-tri-O-acetyl-2-
fluoro-D-glucal (3) with Selectfluor in a nitromethane/water
(35) Francisco, C. G.; Gonzalez, C. C.; Paz, N. R.; Suarez, E. Org. Lett.
2003, 5, 4171-4173.
(36) Sugimoto, O.; Mori, M.; Tanji, K. Tetrahedron Lett. 1999, 40,
7477-7478.
(37) Ghosh, R.; Chakraborty, A.; Maiti, S. Tetrahedron Lett. 2004, 45,
9631-9634.
(38) Repichet, S.; Le Roux, C.; Roques, N.; Dubac, J. Tetrahedron Lett.
2003, 44, 2037-2040.
(39) Sun, H. B.; Li, B.; Hua, R. M.; Yin, Y. W. Eur. J. Org. Chem.
2006, 18, 4231-4236.
J. Org. Chem, Vol. 73, No. 8, 2008 3073

Zhang et al.
SCHEME 3
chromatography. In both cases, the R-anomeric configurations
of the products are clearly indicated by the small (3.6 and 5.3
Hz) J1, F2(ax) coupling constants. A â-configured product would
have exhibited a large, trans-diaxial coupling constant J1, F2(ax)
with the axial anomeric proton.
Kinetic Studies. Yeast R-glucosidase is a well behaved
R-glycosidase whose inhibition and inactivation has been studied
extensively previously, thus was chosen as the model enzyme
for the testing of the monosaccharide-based inactivators.20,40-41
Indeed, the time-dependent inactivation of yeast R-glucosidase
by compound 16, 2,4,6-trinitrophenyl-2-deoxy-2,2-difluoro-R-
arabinohexopyranoside, had been studied previously20 and
yielded a second-order rate constant for inactivation of ki/Ki )
0.25 min-1 mM-1. Similarly, both the 2-chloro-2-fluoro-R-
glycosyl chloride, 1, and the 2,2-difluoro-R-glycosyl chloride,
10, caused time-dependent inactivation of yeast R-glucosidase,
as discussed below.
Pseudo-first-order kinetics of inactivation were seen at each
concentration of 1, allowing the determination of a series of
rate constants for inactivation (kobs) at each inactivator concen-
tration (Figure 2). A replot of these inactivation rate constants
versus inactivator concentration allowed the determination of
inactivation parameters of ki ) 0.25 ( 0.06 min-1 and Ki ) 47
( 19 mM, thus a second-order rate constant for inactivation of
ki/Ki ) (5.3 ( 3.4) × 10-3 min-1 mM-1. Active site-directed
inactivation is demonstrated by the protection against inactiva-
tion afforded by the known competitive inhibitor 1-deoxynojiri-
mycin (63 µM; Ki ) 25 µM42), which reduced the kobs at 11.3
mM of compound 1 from 0.056 min-1 to 0.020 min-1. No
reactivation of inactivated enzyme that had been dialyzed to
remove excess inactivator was seen upon incubation at 37 °C
in buffer, even after incubation for 10 days.
Similarly, time-dependent inactivation of yeast R-glucosidase
was observed upon incubation with the 2-deoxy-2,2-difluoro-
R-D-arabinohexopyranosyl chloride 10 (data not shown). Indi-
vidual inactivation parameters of Ki ) 9.7 ( 2.7 mM and ki )
(8.8 ( 1.0) × 10-4 min-1 were determined, yielding a second-
order rate constant for inactivation of (9.1 ( 3.3) × 10-5 min-1
mM-1. Protection against inactivation by 200 µM 1-deox-
ynojirimycin reduced kobs from 3.7 × 10-4 min-1 to 9.9 × 10-5
min-1 at 11 mM of compound 10, showing that inactivation is
active-site directed. Once again no reactivation was observed
after incubation of the inactivated enzyme, which had been freed
of excess inactivator in buffer over 3 days at 37 °C. This is
consistent with formation of an extremely stable glycosyl-
enzyme intermediate.
FIGURE 2. Inactivation of yeast R-glucosidase by 2-chloro-2-deoxy-
2-fluoro-R-D-glucosyl chloride (1). A) Plot of residual activity versus
time at the indicated inhibitor concentrations: 3.78 mM (×), 5.67 mM
(1), 11.34 mM (0), 22.7 mM (9), 34 mM (O), and 60 mM (2). (B)
Replot of kobs values from above. (C) Protection against inactivation
by 1. Inactivation at a single concentration of 1 (11.3 mM) in the
presence of the indicated concentrations of 1-deoxynojirimycin: 0 µM
(B); 63 µM (0).
Surprisingly, no time-dependent inactivation of human pan-
creatic R-amylase16,43 was observed upon incubation with either
of the 2,2-dihalomaltosyl chlorides 2 or 14. This was initially
surprising since the trinitrophenyl-2-deoxy-2,2-difluoromaltoside
18 had previously proven to be a slow, but effective time-
dependent inactivator of the human amylase,20 and since
inherently, chloride should be a better leaving group than
trinitrophenolate. Presumably binding interactions with the aryl
moiety in the enzyme active site44-45 accelerate, relatively, the
reaction of amylase with the trinitrophenyl glycoside. Indeed,
a similar phenomenon is seen with yeast R-glucosidase upon
comparison of the inactivation parameters for the different
inactivators. (Table 1) The trinitrophenyl-2,2-difluoroglucoside
16 (ki/Ki ) 0.25 min-1 mM-1) inactivates yeast R-glucosidase
2000-fold faster than does the corresponding R-chloride 10. On
that basis the lack of inactivation of human pancreatic R-amylase
by the 2,2-difluoromaltosyl chlorides is not so surprising given
that even the trinitrophenyl 2,2-difluoromaltoside is a slow (ki/
Ki ) 7.3 × 10-3 min-1 mM-1) inactivator. Interestingly the
replacement of a chlorine at the C-2 position by a fluorine in
the 2,2-dihaloglucosyl chloride results in a 50-fold slower
inactivation of yeast R-glucosidase. This rate difference is
undoubtedly due, at least in part, to the greater electronegativity
of fluorine than of chlorine thus a bigger inductive effect on
the transition state, but may also be partly attributed to better
(43) Rydberg, E. H.; Li, C. M.; Maurus, R.; Overall, C. M.; Brayer, G.
D.; Withers, S. G. Biochemistry 2002, 41, 4492-4502.
(44) Nerinckx, W.; Desmet, T.; Claeyssens, M. FEBS Lett. 2003, 538,
1-7.
(45) Lairson, L. L.; Watts, A. G.; Wakarchuk, W. W.; Withers, S. G.
Nat. Chem. Biol. 2006, 2, 724-728.
(40) McCarter, J. D.; Withers, S. G. J. Biol. Chem. 1996, 271, 6889-
6894.
(41) Howard, S.; Withers, S. G. Biochemistry 1998, 37, 3858-3864.
(42) Dong, W.; Jespersen, T.; Bols, M.; Skrydstrup, T.; Sierks, M. R.
Biochemistry 1996, 35, 2788-2795.
3074 J. Org. Chem., Vol. 73, No. 8, 2008

2-Deoxy-2,2-Dihaloglycosides
TABLE 1. Summary of Inactivation Parameters for 2-Deoxy-2,2-dihaloglycosides with Two Model Enzymes
potential inactivator
enzyme
ki/Ki (min-1 mM-1
)
TNP22DFGlc (16)
2Cl2FGlcCl (1)
22DFGlcCl (10)
TNP22DFMal (18)
2Cl2FMalCl (2)
22DFMalCl (14)
yeast R-glucosidase
0.25a
yeast R-glucosidase
(5.3 ( 3.4) × 10-3 b
(9.1 ( 3.3) × 10-5 b
0.0073a
yeast R-glucosidase
human pancreatic R-amylase
human pancreatic R-amylase
human pancreatic R-amylase
no inactivationb
no inactivationb
a Reference 20. b This work.
3,6-Di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-r-(1,4)-D-glu-
copyranosyl]-2-chloro-2-deoxy-2-fluoro-r-D-glucopyranosyl chlo-
ride (6). Protected 2-fluoro maltal (5) (0.80 g, 1.38 mmol) was
dissolved in dry carbon tetrachloride (80 mL) over 4 Å molecular
sieves and cooled in a CCl4/dry ice bath (-23 °C). Chlorine was
bubbled through the solution until it turned yellowish green (5 min),
then the flask was wrapped in aluminum foil to exclude light,
allowed to slowly warm up to room temperature and stirred
overnight. Upon completion of the reaction, excess chlorine was
purged with a stream of dry nitrogen for several minutes until the
solution was colorless, and the solvent was evaporated in Vacuo.
The resulting material was chromatographed twice (petroleum
ether: ethyl acetate ) 3:2; then petroleum ether:diethyl ether )
2:3), giving the disaccharide chloride as a white foam (0.44 g, 0.68
complementarity of the equatorial 2-chloro substituent with the
active site than is the case with an equatorial fluorine.
Conclusion
Convenient synthetic routes have been developed to a series
of 2-deoxy-2,2-dihaloglycosyl derivatives of glucose and mal-
tose involving common 2-fluoroglycal precursors. Useful mech-
anism-based inhibitors of R-glucosidases that function Via the
formation of relatively stable glycosyl-enzyme intermediates
were thereby developed, though chloride leaving groups were
not found to be very effective in trapping of intermediates, based
on our kinetic experiments. Such reagents may be useful in the
structural analysis of glycosyl-enzyme intermediates on R-gly-
cosidases46 and could also prove to have clinical importance as
therapeutics that function by ablating R-glycosidase activities.
1
mmol, 49%) H NMR data (CDCl3, 300 MHz): δ 6.03 (d, 1 H, J
) 6.1 Hz), 5.83 (dd, 1 H, J ) 23.3 Hz, J ) 9.2 Hz), 5.37 (d, 1 H,
J ) 4.0 Hz), 5.36 (t, 1 H, J ) 10.6 Hz), 5.08 (t, 1 H, J ) 9.7 Hz),
4.88 (dd, 1 H, J ) 10.6 Hz, J ) 4.0 Hz), 4.59 (dd, 1 H, J ) 12.5
Hz, J ) 2.4 Hz) 4.50-4.20 (m, 4 H), 4.06 (dd, 1 H, J ) 13.4 Hz,
J ) 2.3 Hz), 3.98 (dt, 1 H, J ) 9.7 Hz, J ) 2.3 Hz), 2.18 (s, 3 H),
2.15 (s, 3 H), 2.10 (s, 3 H), 2.09 (s, 3 H), 2.03 (s, 3 H), 2.01 (s, 3
H). 13C NMR (CDCl3, 75 MHz): δ 170.8, 170.6, 170.4, 170.0,
169.6, 169.5, 108.6 (d, J ) 248.0 Hz), 96.1, 91.5 (d, J ) 30.7 Hz),
73.1 (d, J ) 17.9 Hz), 71.7, 71.4, 70.1, 69.3, 68.9, 68.0, 61.8, 61.5,
20.9, 20.8 (3C), 20.7 (2C). 19F NMR data (CDCl3, 282 MHz): δ
-119.9 (dd, J ) 23.0 Hz, J ) 5.0 Hz) HRMS data: calcd for
C24H31O15F35Cl2 + Na+, 671.0922; found, 671.0923.
Experimental Section
3,4,6-Tri-O-acetyl-2-chloro-2-deoxy-2-fluoro-r-D-glucopyra-
nosyl chloride (4). 3,4,6-tri-O-acetyl-2-fluoro-D-glucal (3) (0.80
g, 2.76 mmol)31 was dissolved in dry carbon tetrachloride (150 mL)
over 4 Å molecular sieves and cooled in a CCl4/dry ice bath (-23
°C). Chlorine was bubbled through the solution until it turned a
yellowish green (5 min). The flask was wrapped in aluminum foil
to exclude light, allowed to slowly warm up to room temperature
and stirred for 18 h. Upon completion of the reaction, excess
chlorine was purged with a stream of dry nitrogen for several
minutes until the solution was colorless, and the solvent was
evaporated in Vacuo. The resulting material was chromatographed
twice (petroleum ether:diethyl ether ) 2:1; then petroleum ether:
diethyl ether ) 3:1) giving the dihaloglycopyranosyl chloride (4)
2-Chloro-2-deoxy-2-fluoro-4-O-[r-(1,4)-D-glucopyranosyl]-r-
D-glucopyranosyl chloride (2). Protected disaccharide chloride (6)
(80 mg, 0.12 mmol) was dissolved in 10 mL of dry methanol, a
small piece of sodium metal was added then the mixture was stirred
at room temperature for 2 h. Acidic ion-exchange resin was added,
stirred for 10 min until the solution became weakly acidic, then
the resin was filtered off and the solvent was evaporated in Vacuo.
The product was purified by flash column chromatography (ethyl
acetate:methanol:water ) 7:2:1) yielding (2) (41 mg, 0.10 mmol,
82%) as a white foam. 1H NMR data (D2O, 300 MHz): δ 6.26 (d,
1 H, J ) 6.3 Hz), 5.35 (d, 1 H, J ) 4.0 Hz), 4.40 (dd, 1 H, J )
24.3 Hz, J ) 9.8 Hz), 4.13 (dt, 1 H, J ) 9.8 Hz, J ) 2.3 Hz), 3.90
(t, 1 H, J ) 9.8 Hz), 3.77-3.53 (m, 6 H), 3.45 (dd, 1 H, J ) 10.0
Hz, J ) 4.0 Hz), 3.32 (t, 1 H, J ) 9.7 Hz). 13C NMR data (D2O,
100 MHz): δ 110.52 (d, J ) 242.0 Hz), 99.71, 91.79 (d, J ) 31.0
Hz), 73.86, 73.83, 73.68, 72.99, 72.86, 71.64, 69.40, 60.53, 59.98.
19F NMR data (D2O, 282 MHz): δ -123.87 (dd, J ) 24.3 Hz, J
) 6.3 Hz) HRMS data: calcd for C12H19O9FCl2 + Na+, 419.0288;
found, 419.0289.
1,3,4,6-Tetra-O-acetyl-2-deoxy-2,2-difluoro-r-D-arabinohexo-
pyranose (7) (Acetyl Hypofluorite Method). Sodium acetate (280
mg), glacial acetic acid (3.5 mL) and CFCl3 (35 mL) were mixed
in a 3-necked round-bottom flask and cooled in a dry ice/acetone
bath (-78 °C). A gas reservoir (∼1 L) was filled with 20% fluorine
in neon (10 psi) which was diluted 4-fold with helium to 40 psi.
This mixture was then bubbled through the slurry. The flask was
thus charged twice before compound (3) (0.800 g, 2.76 mmol)
dissolved in CFCl3 (10 mL) was added to the mechanically stirred
mixture. The loosely stoppered flask was allowed to warm to room
temperature. After general workup, column chromatography (pe-
1
as a yellow oil (0.17 g, 0.47 mmol, 17%). H NMR data (CDCl3,
400 MHz): δ 6.06 (d, 1 H, J ) 6.0 Hz), 5.71 (dd, 1 H, J ) 22.8
Hz, J ) 9.7 Hz), 5.28 (dt, 1 H, J ) 9.7 Hz, J ) 1.5 Hz), 4.4-4.05
(m, 3 H), 2.14 (s, 3 H), 2.07 (s, 3 H), 2.03 (s, 3 H). 19F NMR data
(CDCl3, 188 MHz): δ -120.4 (dd, J ) 22.8 Hz, J ) 6.0 Hz).
Anal. Calcd for C12H15O7FCl2: C, 39.91; H, 4.19; Found: C, 39.73;
H, 4.08.
2-Chloro-2-deoxy-2-fluoro-r-D-glucopyranosyl Chloride (1).
The acetylated glycopyranosyl chloride (4) (0.12 g, 0.33 mmol)
was dissolved in HPLC grade methanol (10 mL) and cooled to
0 °C. Dry ammonia was bubbled in for 10 min. Then the ice bath
was removed and the reaction was allowed to warm up to room
temperature and further reacted for 4.5 h. Column chromatography
(ethyl acetate:methanol:water ) 27:2:1) gave a yellow solid which
was further purified using ethyl acetate:methanol ) 24:1 affording
a yellow oil (0.039 g, 0.17 mmol, 50%). 1H NMR data (D2O, 200
MHz): δ 6.36 (d, 1 H, J ) 6.6 Hz), 4.21 (dd, 1 H, J ) 24.0 Hz,
J ) 9.0 Hz), 4.10-3.69 (m, 4 H). 19F NMR data (D2O, 188 MHz):
δ -123.25 (dd, J ) 24.0 Hz, J ) 6.6 Hz). Anal. Calcd for C6H9O4-
FCl2: C, 30.66; H, 3.86; Found: C, 30.91; H, 3.93.
(46) Uitdehaag, J. C. M.; Mosi, R.; Kalk, K. H.; Van der Veen, B. A.;
Dijkhuizen, L.; Withers, S. G.; Dijkstra, B. W. Nat. Struct. Biol. 1999, 6,
432-436.
J. Org. Chem, Vol. 73, No. 8, 2008 3075

Zhang et al.
troleum ether: ethyl acetate ) 4:1) afforded the product 7 (0.77 g,
2.1 mmol, 76%) as an anomeric mixture, only the spectrum of the
R-anomer is given here. 1H NMR data (CDCl3, 200 MHz): δ 6.19
(t, 1 H, J ) 3.0 Hz), 5.50 (dt, 1 H, J ) 20.0 Hz, J ) 10.0 Hz),
5.20 (t, 1 H, J ) 10.0 Hz), 4.3-4.0 (m, 3 H), 3.88 (s, 3 H), 3.85
(s, 3 H), 3.81 (s, 3 H), 3.74 (s, 3 H). 19F NMR data (CDCl3, 188
MHz): δ -121.0 (F-2/eq., F-2/ax. co-incident). MS: calcd for
C14H18F2O9 + Na+, 391.1; found, 391.0.
nose (11) (Acetyl Hypofluorite Method). Per-O-acetyl-2-fluoro-
maltal 5 (0.99 g, 1.7 mmol) was fluorinated according to the general
fluorination procedure mentioned in the Supporting Information.
The reaction mixture was partially purified by flash chromatography
(hexanes: ethyl acetate ) 1: 1) to afford 0.67 g (1.0 mmol, 59%)
of a syrup containing both R- and â anomers of 11. Only the spectra
1
of the R-anomer are given here. H NMR (CDCl3, 300 MHz): δ
6.14 (d, 1 H, J ) 4.1 Hz), 5.57 (dt, 1 H, J ) 16.7 Hz, J ) 8.4 Hz),
5.43 (d, 1 H, J ) 3.9 Hz), 5.36 (t, 1 H, J ) 9.8 Hz), 5.07 (t, 1 H,
J ) 9.8 Hz), 4.86 (dd, 1 H, J ) 10.5 Hz, J ) 3.9 Hz), 4.50-3.90
(m, 7 H), 2.24 (s, 3 H), 2.14 (s, 3 H), 2.13 (s, 3 H), 2.09 (s, 3 H),
2.06 (s, 3 H), 2.02 (s, 3 H), 2.00 (s, 3 H). 13C NMR (CDCl3, 150
MHz): δ 170.8, 170.7, 170.6, 170.2, 169.8, 169.6, 168.0, 114.8
(dd, J ) 258 Hz, J ) 246 Hz), 96.0, 88.8 (dd, J ) 37.7 Hz, J )
31.7 Hz), 71.5 (d, J ) 4.5 Hz), 71.1 (t, J ) 19.6 Hz), 70.7, 70.3,
69.3, 68.9, 68.0, 62.2, 61.5, 21.0, 20.94, 20.91, 20.88, 20.80 (3C).
HRMS: calcd for C26H34F2O17 + Na+, 679.1662; found, 679.1650.
This anomeric mixture was directly used in the next step without
further purification.
3,4,6-Tri-O-acetyl-2-deoxy-2,2-difluoro-r-D-arabinohexopy-
ranose (8). Fully acetylated difluorosugar (7) (0.5 g, 1.36 mmol)
was dissolved in DMF (10 mL), hydrazine acetate (280 mg, 3.0
mmol) was added and allowed to react 3 days at 50 °C. Evaporation
of the solvent in Vacuo, followed by flash chromatography (hexanes:
ethyl acetate ) 1:1) afforded the hemiacetal (280 mg, 66%) as a
1
colorless gum. H NMR data (CDCl3, 300 MHz): δ 5.56 (ddd, 1
H, J ) 19.0 Hz, J ) 10.0 Hz, J ) 6.0 Hz), 5.24-5.10 (m, 2 H),
4.32-4.00 (m, 3 H), 3.48 (broad, 1 H), 2.09 (s, 3 H), 2.06 (s, 3 H),
2.00 (s, 3 H). 13C NMR data (CDCl3, 75 MHz): δ 171.1, 170.0,
169.5, 115.6 (dd, J ) 257.0 Hz, J ) 246.0 Hz), 91.4 (dd, J ) 36.0
Hz, J ) 28.7 Hz), 68.7 (t, J ) 18.5 Hz), 68.2, 67.7 (d, J ) 6.3
Hz), 61.9, 20.8, 20.7, 20.6 19F NMR data (CDCl3, 188 MHz): δ
-120.7 (dd, J ) 251.0 Hz, J ) 6.0 Hz), -122.7 (ddd, J ) 251.0
Hz, J )19 Hz, J ) 5.0 Hz). HRMS: calcd for C12H16F2O8 + Na+,
349.0711; found, 349.0710.
3,6-Di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-r-(1,4)-D-glu-
copyranosyl]-2-deoxy-2,2-difluoro-r-D-arabinohexopyranose (12).
To 0.67 g (1.0 mmol) of syrup 11 was added 0.13 g (1.4 mmol,
1.4 equiv) of hydrazine acetate in DMF (20 mL), and the reaction
was continued for 3 days at 50 °C. The reaction was stopped by
redissolving the mixture in ethyl acetate then the organic layer was
washed successively with water and brine. Evaporation of the
solvent in Vacuo, followed by flash chromatography (hexanes:ethyl
acetate ) 1:1) gave the R-hemiacetal 12 (0.42 g, 0.68 mmol, 68%)
3,4,6-Tri-O-acetyl-2-deoxy-2,2-difluoro-R-D-arabinohexopy-
ranose (8) (Selectfluor Method). Protected 2-fluoroglucal 3 (0.64
g, 2.2 mmol) was dissolved in 25 mL of CH3NO2/water (v/v )
4:1) mixed solvent, and Selectfluor (1.17 g, 3.3 mmol) was added.
The reaction mixture was stirred at room temperature for overnight
and then was heated to 95 °C for 4 h. After the mixture had cooled
down, most of the solvent was evaporated under diminished
pressure. The residue was redissolved in EtOAc and washed
successively with water, saturated NaHCO3(aq), and brine. After
drying over MgSO4 and evaporation of the solvent, flash chroma-
tography (petroleum ether: ethyl acetate ) 1:1) yielded a colorless
syrup of 8 (130 mg, 20%).
1
as a white foam. H NMR (CDCl3, 300 MHz): δ 5.63 (ddd, 1 H,
J ) 18.5 Hz, J ) 9.2 Hz, J ) 6.5 Hz), 5.42 (d, 1 H, J ) 4.0 Hz),
5.35 (dd, 1 H, J ) 10.4 Hz, J ) 9.6 Hz), 5.18 (d, 1 H, J ) 4.7
Hz), 5.05 (t, 1 H, J ) 9.8 Hz), 4.84 (dd, 1 H, J ) 10.4 Hz, J ) 4.0
Hz), 4.6-4.0 (m, 7 H), and 2.2-1.9 (5 s, 6 OAc). 13C NMR data
(CDCl3, 75 MHz): δ 170.9, 170.8 (2C), 170.2, 169.8, 169.6, 115.8
(dd, J ) 258.0 Hz, J ) 245.0 Hz), 95.7, 91.0 (dd, J ) 35.0 Hz, J
) 28.8 Hz), 71.9 (d, J ) 5.4 Hz), 70.9 (t, J ) 19.0 Hz), 70.2, 69.5,
68.6, 68.5, 68.1, 62.6, 61.5, 20.93, 20.85, 20.79, 20.7 (3C) 19F NMR
(CDCl3, 282 MHz): δ -120.9 (dd, J ) 253.0 Hz, J ) 6.3 Hz),
-122.7 (ddd, J ) 253.0 Hz, J ) 18.4 Hz, J ) 4.5 Hz). HRMS
data: calcd for C24H32F2O16 + Na+, 637.1556; found, 637.1562.
3,6-Di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-r-(1,4)-D-glu-
copyranosyl]-2-deoxy-2,2-difluoro-r-D-arabinohexopyranose (12)
(Selectfluor Method). Protected 2-fluoromaltal 5 (20 mg, 0.035
mmol) and 18 mg (0.05 mmol) of Selectfluor were dissolved in 5
mL of CH3NO2/water (v/v ) 4:1) mixed solvent. The reaction
mixture was stirred at room temperature overnight and then heated
to 95 °C for 4 h. After cooling down the mixture, most of the solvent
was evaporated under diminished pressure. The residue was
redissolved in EtOAc and washed successively with water, saturated
NaHCO3(aq) and brine. After drying over MgSO4 and evaporation
of the solvent, flash chromatography (petroleum ether: ethyl acetate
) 1: 1) yielded colorless syrup 12 (18 mg, 84%).
3,4,6-Tri-O-acetyl-2-deoxy-2,2-difluoro-r-D-arabinohexopy-
ranosyl chloride (9). The hemiacetal (8) (0.079 g, 0.23 mmol) was
dissolved in freshly distilled thionyl chloride (1 mL, 11 mmol) and
stirred at refluxing (∼65 °C) for 80 h. Column chromatography
(petroleum ether:ethyl acetate ) 5:1) yielded the acetylated glycosyl
1
chloride (0.049 g, 0.14 mmol, 62%). H NMR data (CDCl3, 300
MHz): δ 5.92 (dd, 1 H, J ) 4.8 Hz, J ) 2.0 Hz), 5.68 (dt, 1 H, J
) 15.0 Hz, J ) 10.0 Hz), 5.19 (t, 1 H, J ) 10.0 Hz), 4.2-4.0 (m,
3 H), 2.13 (s, 3 H), 2.09 (s, 3 H), 2.05 (s, 3 H). 13C NMR data
(CDCl3, 75 MHz): δ 170.6, 169.6, 169.2, 115.3 (dd, J ) 257.4
Hz, J ) 252.6 Hz), 88.2 (t, J ) 34.0 Hz), 71.1, 68.1 (t, J ) 19.4
Hz), 66.7 (d, J ) 4.2 Hz), 61.0, 20.7, 20.6, 20.5. 19F NMR data
(CDCl3, 188 MHz): δ -115.5 (ddd, J ) 249.8 Hz, J ) 15.0 Hz,
J ) 4.8 Hz), -116.9 (ddd, J ) 249.8 Hz, J ) 10.0 Hz). Anal.
CalcdforC12H15O7F2Cl: C,41.81;H,4.39.Found: C,41.76;H,4.50.
2-Deoxy-2,2-difluoro-r-D-arabinohexopyranosyl Chloride (10).
The acetylated glycosyl chloride (9) (0.049 g, 0.14 mmol) was
dissolved in methanol (10 mL) and cooled to 0 °C, and dry ammonia
gas was bubbled in for 5 min. The reaction was then left stirring at
room temperature for overnight. The resulting light orange oil was
purified by column chromatography (ethyl acetate: methanol: water
) 147: 2: 1) to afford compound 10 as a colorless oil (0.016 g,
3,6-Di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-r-(1,4)-D-glu-
copyranosyl]-2-deoxy-2,2-difluoro-r-D-arabinohexopyranosyl
Chloride (13). The hemiacetal 12 (250 mg, 0.41 mmol) was
dissolved in 10 mL of dry CH2Cl2 along with 3.5 mL of SOCl2
(large excess to prevent the evaporation of SOCl2) and then BiOCl
(0.35 g) was added. The reaction flask was wrapped with aluminum
foil to exclude light, and then the mixture was stirred vigorously
at room temperature under a nitrogen atmosphere for 3 days when
TLC showed that most of the starting material had been consumed.
The reaction mixture was poured into ice-cold water, stirred, then
transferred to a separatory funnel. The organic phase was washed
successively with water, saturated NaHCO3(aq), water, and brine.
The organic layer was dried with MgSO4, filtered and the solvent
was evaporated in Vacuo. Two rounds of flash column chroma-
tography (hexanes: ethyl acetate ) 1:1 then CHCl3: acetone )
20:1) yielded the pure product (34 mg, 0.054 mmol, 13%) as a
colorless syrup. 1H NMR data (CDCl3, 300 MHz): δ 5.93 (d, 1 H,
1
0.073 mmol, 52%). H NMR data (D2O, 300 MHz): δ 6.15 (d, 1
H, J ) 7.6 Hz), 4.25 (ddd, 1 H, J ) 21.0 Hz, J ) 9.5 Hz, J ) 5.0
Hz), 3.92-3.62 (m, 4 H). 13C NMR data (CDCl3, 75 MHz): δ
170.6, 169.6, 169.2, 115.3 (dd, J ) 258.0 Hz, J ) 253.0 Hz), 88.2
(t, J ) 34.0 Hz), 71.1, 68.1 (t, J ) 19.4 Hz), 66.7 (d, J ) 4.2 Hz),
61.0, 20.7, 20.6, 20.5. 19F NMR data (D2O, 188 MHz): δ -116.1
(dd, J ) 248.7 Hz, J ) 5.0 Hz), -119.45 (ddd, J ) 248.7 Hz, J )
21.0 Hz, J ) 6.0, Hz). Anal. Calcd for C6H9O4F2Cl: C, 32.97; H,
4.15; Found: C, 33.15; H, 4.16.
1,3,6-Tri-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-r-(1,4)-D-glu-
copyranosyl]-2-deoxy-2,2-difluoro-r/â-D-arabinohexopyra-
3076 J. Org. Chem., Vol. 73, No. 8, 2008

2-Deoxy-2,2-Dihaloglycosides
J ) 6.4 Hz), 5.78 (ddd, 1 H, J ) 18.8 Hz, J ) 9.6 Hz, J ) 5.2
Hz), 5.45 (d, 1 H, J ) 4.0 Hz), 5.37 (t, 1 H, J ) 10.4 Hz), 5.09 (t,
1 H, J ) 10.0 Hz), 4.87 (dd, 1 H, J ) 10.4 Hz, J ) 4.0 Hz), 4.59
(dd, 1 H, J ) 12.6 Hz, J ) 2.4 Hz), 4.34 (dt, 1 H, J ) 9.6 Hz, J
) 2.4 Hz), 4.29-4.23 (m, 2 H), 4.18 (t, 1H, J ) 9.6 Hz), 4.08 (dd,
1 H, J ) 12.6 Hz, J ) 2.4 Hz), 3.95 (dt, 1 H, J ) 10.0 Hz, J ) 2.4
Hz), 2.17 (s, 3 H), 2.16 (s, 3 H), 2.11 (s, 3 H), 2.08 (s, 3 H), 2.04
(s, 3 H), 2.02 (s, 3 H). 13C NMR data (CDCl3, 100 MHz): δ 171.6,
171.5, 171.2, 170.8, 170.4, 170.3, 116.2 (dd, J ) 258.6 Hz, J )
249.5 Hz), 96.9, 88.8 (t, J ) 35.0 Hz), 72.3, 72.0 (d, J ) 5.4 Hz),
71.1, 71.0 (t, J ) 19.3 Hz), 70.2, 69.8, 68.9, 62.6, 62.3, 21.7, 21.63,
21.61, 21.56 (3C). 19F NMR data (CDCl3, 282 MHz): δ -115.8
(ddd, J ) 247.5 Hz J ) 18.8 Hz, J ) 7.0 Hz) 116.9 (dd, J ) 247.5
Hz, J ) 5.2 Hz) HRMS data: calcd for C24H31O1535ClF2 + Na+,
655.1217; found, 655.1215.
acetyl chloride (80 µL), and the mixture was allowed to stir at
4 °C for 2 days. After workup, flash chromatography (hexanes:
ethyl acetate: ) 1:1) afforded pure 2,4,6-trinitrophenyl-2-deoxy-
2,2-difluoro-R-D-arabinohexopyranoside (14 mg, 19%) as a yel-
lowish gum, which was freeze-dried to give a yellowish white solid.
Its characterization is consistent with an earlier description.20
2,4,6-Trinitrophenyl-3,6-di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-
acetyl-r-(1,4)-D-glucopyranosyl]-2-deoxy-2,2-difluoro-r-D-ara-
binohexopyranoside (17). To a solution of protected difluorohe-
miacetal 12 (535 mg, 0.87 mmol) dissolved in dry CH2Cl2 (50 mL)
were added 2,6-di-tert-butyl pyridine (0.6 mL, 2.7 mmol) and
fluoro-2,4,6-trinitrobenzene (460 mg, 2.0 mmol), and the mixture
was allowed to stir for 10 days at room temperature in the dark
under N2. Evaporation of the solvent in Vacuo, followed by flash
chromatography (hexanes: ethyl acetate ) 1: 1) afforded 2,4,6-
trinitrophenyl-3,6-di-O-acetyl-4-O-[2′,3′,4′,6′-tetra-O-acetyl-R-(1,4)-
D-glucopyranosyl]-2-deoxy-2,2-difluoro-R-D-arabinohexopyrano-
2-Deoxy-2,2-difluoro-4-O-[R-(1,4)-D-glucopyranosyl]-R-D-ara-
binohexopyranosyl Chloride (14). Protected disaccharide 13 (20
mg, 0.03 mmol) was dissolved in 5 mL of methanol (HPLC grade),
cooled to 0 °C and dry ammonia was bubbled in for 1 min. After
removal of the ice bath the reaction was stirred at room temperature
overnight. The solvent was then evaporated to dryness and the
residue purified by flash column chromatography (ethyl acetate:
methanol:water ) 7:2:1) to afford 14 (10 mg 0.026 mmol, 83%)
1
side (668 mg, 91%) as a yellowish gum. H NMR (CDCl3, 400
MHz): δ 8.85 (s, 2 H), 5.71 (ddd, 1 H, J ) 20.3 Hz, J ) 8.8 Hz,
J ) 3.6 Hz), 5.61 (d, 1 H, J ) 5.3 Hz), 5.44 (d, 1 H, J ) 4.0 Hz),
5.33 (t, 1 H, J ) 10.0 Hz), 5.02 (t, 1 H, J ) 9.9 Hz), 4.83 (dd, 1
H, J ) 10.5 Hz, J ) 4.0 Hz), 4.4-3.9 (m, 7 H), and 2.2-1.9 (6 s).
13C NMR data (CDCl3, 100 MHz): δ 171.7, 171.5, 171.1, 170.9,
170.4, 170.0, 145.75, 145.73, 143.78, 124.7 (3 × C), 114.9 (dd, J
) 262.0 Hz, J ) 246.1 Hz), 100.9 (dd, J ) 40.2 Hz, J ) 28.9 Hz),
96.7, 73.5, 74.3 (d, J ) 5.6 Hz), 71.0, 70.8 (t, J ) 20.3 Hz), 70.2,
69.8, 68.9, 62.8, 62.4, 21.64 (2 × CH3CO), 21.59, 21.55 (3 × CH3-
CO). 19F NMR (CDCl3): δ -126.0 (ddd, J ) 264.0 Hz, J ) 19.3
Hz, J ) 5.1 Hz), -128.5 (dd, J ) 264.0 Hz, J ) 4.7 Hz). HRMS
data: calcd for C30H33F2N3O22 + Na+, 848.1421; found, 848.1416.
2,4,6-Trinitrophenyl-2-deoxy-2,2-difluoro-4-O-[r-(1,4)-D-glu-
copyranosyl]-r-D-arabinohexopyranoside (18). To acetylated
difluoroglycoside 17 (96 mg, 0.12 mmol) in dry MeOH (2 mL) at
0 °C was added freshly distilled acetyl chloride (80 µL), and the
mixture was allowed to stir at 4 °C for 2 days. After workup, flash
chromatography (ethyl acetate: methanol: water ) 20: 2: 1)
afforded 18 (47 mg, 71%) as a yellowish gum, which was freeze-
dried to give a yellowish white solid. Its characterization is
consistent with an earlier description.20
1
as a white foam. H NMR data (D2O, 400 MHz): δ 6.13 (d, 1 H,
J ) 7.3 Hz), 5.36 (d, 1 H, J ) 3.8 Hz), 4.46 (ddd, 1 H, J ) 21.2
Hz, J ) 9.2 Hz, J ) 5.5 Hz), 4.07 (dt, 1 H, J ) 9.2 Hz, J ) 3.0
Hz), 3.85 (t, 1 H, J ) 9.2 Hz), 3.77-3.52 (m, 6 H), 3.44 (dd, 1 H,
J ) 10.0 Hz, J ) 3.8 Hz), 3.31 (t, 1 H, J ) 9.1 Hz). 13C NMR
data (D2O, 100 MHz): δ 119.42 (t, J ) 244.0 Hz), 102.24, 91.05
(t, J ) 36.0 Hz), 76.32, 76.15, 75.71, 75.59, 74.39, 73.14 (t, J )
19.0 Hz), 72.14, 63.28, 62.65. 19F NMR data (D2O, 282 MHz): δ
-117.7 (dd, J ) 249.0 Hz, J ) 5.5 Hz) 119.7 (ddd, J ) 249.0 Hz,
J ) 21.2 Hz, J ) 7.3 Hz). HRMS data: calcd for C12H1935ClF2O9
+ Na+, 403.0583; found, 403.0589.
2,4,6-Trinitrophenyl-3,4,6-tri-O-acetyl-2-deoxy-2,2-difluoro-
r-D-arabinohexopyranoside (15). To a solution of the difluoro-
hemiacetal 8 (93 mg, 0.28 mmol) dissolved in dry CH2Cl2 (1 mL)
were added 2,6-di-tert-butyl pyridine (0.1 mL, 0.44 mmol) and
fluoro-2,4,6-trinitrobenzene (90 mg, 0.39 mmol), and the mixture
was allowed to stir for 10 days at room temperature in the dark
under N2. Evaporation of the solvent in Vacuo, followed by flash
chromatography (hexanes:ethyl acetate ) 1:2) afforded 2,4,6-
trinitrophenyl 3,4,6-tri-O-acetyl-2-deoxy-2,2-difluoro-R-D-arabino-
Acknowledgment. We thank Prof. Michael J. Adam and
Dr. Nathaniel C. Lim at TRIUMF located on the campus of the
University of British Columbia for help with fluorination
reactions. The Canadian Institutes for Health Research is thanked
for financial support of this work.
1
hexopyranoside 15 (76 mg, 51%) as a yellowish gum. H NMR
(CDCl3): δ 8.84 (s, 2 H), 5.68 (ddd, 1 H, J ) 19.2 Hz, J ) 9.9
Hz, J ) 6.0 Hz), 5.62 (d, 1 H, J ) 5.0 Hz), 5.23 (dt, 1 H, J ) 10.0
Hz, J ) 1.4 Hz), 4.3-4.0 (m, 3 H), 2.2-2.0 (2 s, 3 OAc). 19F
NMR (CDCl3): δ -118.2 (ddd, J ) 263 Hz, J ) 19.0 Hz, J ) 6.0
Hz), -121.3 (dd, J ) 263.0 Hz, J ) 6.0 Hz).
2,4,6-Trinitrophenyl-2-deoxy-2,2-difluoro-r-D-arabinohexo-
pyranoside (16) To the protected difluoroglycoside 15 (84 mg,
0.18 mmol) in dry MeOH (2 mL) at 0 °C was added freshly distilled
Supporting Information Available: Text giving general ex-
perimental methods, including the synthesis of precursor 5 and
enzyme kinetic studies and figures showing 1H and 13C NMR
spectra of compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO702565Q
J. Org. Chem, Vol. 73, No. 8, 2008 3077