Nanomolar versus millimolar inhibition by xylobiose-derived azasugars: Significant differences between two structurally distinct xylanases

Article abstract of DOI:10.1021/ja993805j

The synthesis of xylobiose-derived nitrogen-containing inhibitors of xylanase is described starting with accessible precursors through efficient synthetic schemes. Four disaccharides were identified as powerful competitive inhibitors of the retaining fami

Full text of DOI:10.1021/ja993805j

J. Am. Chem. Soc. 2000, 122, 2223-2235
2223
Nanomolar versus Millimolar Inhibition by Xylobiose-Derived
Azasugars: Significant Differences between Two Structurally Distinct
Xylanases
Spencer J. Williams, Roland Hoos, and Stephen G. Withers*
Contribution from the Protein Engineering Network of Centres of Excellence, Department of Chemistry,
UniVersity of British Columbia, 2036 Main Mall, VancouVer, Canada V6T 1Z1
ReceiVed October 25, 1999
Abstract: The synthesis of xylobiose-derived nitrogen-containing inhibitors of xylanase is described starting
with accessible precursors through efficient synthetic schemes. Four disaccharides were identified as powerful
competitive inhibitors of the retaining family 10 xylanase, Cex, from Cellulomonas fimi, namely imidazole
(Ki ) 150 nM), lactam oxime (Ki ) 370 nM), isofagomine (Ki ) 130 nM), and deoxynojirimycin (Ki ) 5800
nM) derivatives of xylobiose. By contrast, none of the compounds inhibited the family 11 xylanase, Bcx, from
Bacillus circulans, to an appreciable extent. Two possible explanations are provided for the discrimination
exhibited by the imidazole and the lactam oxime. One explanation relates to the different active site locations
of the acid/base residue in the two enzymes: anti to the C1-O5 bond for the family 10 Cex and syn for the
family 11 Bcx. The other explanation concerns proposed differences in the transition state conformation for
the two enzymes: half-chair for Cex and boat for Bcx. The reasons for the difference in inhibition values
between Cex and Bcx for the isofagomine and deoxynojirimycin derivatives are less clear-cut but may be
ascribed to destabilizing steric and electrostatic interactions between the inhibitors and an essential tyrosine
residue in the active site of Bcx.
Xylanases (EC 3.2.1.8) are glycosidases that catalyze the
hydrolytic cleavage of â-1,4-linked polymers of D-xylose. Their
potential use in biomass conversion, fuel production, and bread
baking has attracted considerable interest.1,2 Further, they already
enjoy widespread use in the paper industry as agents to facilitate
lignin removal, thereby reducing the need for bleaching
chemicals.3,4 As a consequence, considerable effort has been
expended in the study of xylanases, both in their cloning and
expression and in studies of structure, stability, and catalytic
mechanism, as reviewed in refs 5-9. Within the sequence-based
classification of glycosidases, most xylanases are found in
families 10 and 11, with a few being classified into families 5
and 43.10 Despite the fact that members of families 10 and 11
catalyze the same reaction, cleavage of the glycosidic bond with
net retention of anomeric configuration, there are significant
differences between the two groups, both in three-dimensional
(1) Gilbert, H. J.; Hazlewood, G. P. J. Gen. Microbiol. 1993, 139, 187-
194.
(2) Woodward, J. Xylanases: functions, properties and applications in
Topics in enzyme and fermentation biotechnology; John Wiley & Sons:
New York, 1984; Vol. 8, pp 9-30.
(3) Beguin, P.; Aubert, J.-P. FEMS Microbiol. ReV. 1994, 13, 25-58.
(4) Viikari, L.; Suurnakki, A.; Buchert, J. Enzyme-aided bleaching of
kraft pulps: Fundamental mechanisms and practical applications. In Enzymes
for pulp and paper processing; American Chemical Society: Washington,
1996; Vol. 655, pp 15-24.
structure and in the substrate specificity. Family 10 xylanases
are members of the so-called clan GH-A grouping of glycosidase
families, the catalytic domains of which fold as a (â/R)8 barrel,
with similar locations of the key active site groups.10,11 Many
members of this family are capable of cleaving both xylan and
cellulose. Family 11 xylanases, smaller enzymes that only cleave
xylan, belong to clan GH-C and fold as a primarily â-sheet
jelly roll.
The catalytic mechanism of retaining glycosidases was
initially proposed by Koshland, who suggested that the active-
site contains two key catalytic groups, one with the role of acid/
base and the other functioning as a nucleophile (Figure 1).12 It
has since been shown in the vast majority of cases that these
two groups are carboxyl groups and that a covalent intermediate
is formed, which then undergoes hydrolysis to afford a hemi-
acetal with net retention of anomeric stereochemistry.7,8 The
transition states leading to and from the covalent intermediate
have substantial oxacarbenium ion character, as indicated by
kinetic isotope effects and by the effects of electron-withdrawing
substituents on the sugar ring upon reaction rate.7,8,13 Detailed
structural insights into stable species along the reaction coor-
dinate have now been obtained for several glycosidases by
solving structures of the free enzyme, the Michaelis complex,
the 2-deoxy-2-fluoroglycosyl-enzyme intermediate, and the
product complex. Such studies on the retaining clan GH-A
cellulase Cel5A from Bacillus agaradhaerens revealed sub-
stantial distortion of the sugar moiety in the -1 binding subsite
(5) Royer, J. C.; Nakas, J. P. Enzyme Microb. Technol. 1989, 11, 405-
410.
(6) Sunna, A.; Antranikian, G. Crit. ReV. Biotechnol. 1997, 17, 39-67.
(7) Zechel, D. L.; Withers, S. G. Glycosyl transferase mechanisms. In
ComprehensiVe Natural Products Chemistry; Poulter, C. D., Ed.; Perga-
mon: Amsterdam, 1999; Vol. 5, pp 279-314.
(8) Davies, G.; Sinnott, M. L.; Withers, S. G. Glycosyl transfer. In
ComprehensiVe Biological Catalysis; Sinnott, M. L., Ed.; Academic Press:
London, 1997; Vol. 1, pp 119-209.
(9) Ly, H. D.; Withers, S. G. Annu. ReV. Biochem. 1999, 68, 487-522.
(10) Henrissat, B.; Bairoch, A. Biochem. J. 1996, 316, 695-696.
1
in the Michaelis complex into a S3 skew-boat and relaxation
of this conformation into a 4C1 chair in the covalent intermedi-
(11) Henrissat, B.; Davies, G. Curr. Opin. Struct. Biol. 1997, 7, 637-
644.
(12) Koshland, D. E. Biol. ReV. 1953, 28, 416-436.
(13) Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202.
10.1021/ja993805j CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/23/2000

2224 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
and these have been found to be of great utility in the study of
the glycosidase mechanism.20,21 Examples of some of the more
successful and general inhibitors include molecules having an
sp2-hybridized nitrogen atom in place of the glycosidic oxygen,
such as the imidazole (1)22,23 and the lactam oxime (2),24,25 and
various amidines and amidrazones.26 Another class of inhibitors
includes those having an sp3-hybridized nitrogen atom in place
of the sugar endocyclic oxygen or carbon 1, such as deoxy-
nojirimycin (3)20,27 and isofagomine (4).28,29 Most of these
inhibitors have, at some stage, been proposed to act as mimics
of the transition states of the enzyme-catalyzed mechanism of
glycoside hydrolysis, although only a few studies have attempted
to determine the degree to which some of these compounds
actually resemble the transition state.30
The planar nature of the sugar ring and the build-up of
positive charge on C1 and the ring oxygen are key features of
the hypothetical transition state that are mimicked to varying
degrees by the aforementioned inhibitors. Additionally, it has
been suggested that the trajectory of protonation of the glyco-
sidic oxygen in the first step of the catalytic mechanism is an
important feature.31 This trajectory is of course dictated by the
positioning of the acid catalyst; thus, a classification of some
glycosidases into two classes based on a syn- or anti-protonation
trajectory, relative to the C1-O5 bond, has been proposed by
Heightman and Vasella (Figure 2).32 Inhibitors such as the
imidazole (1) and lactam oxime (2) should therefore function
as good inhibitors of anti-protonators. In this vein, the X-ray
crystal structure of a cellobiose-derived imidazole bound to
Cel5A from B. agaradhaerens, an anti-protonator, has been
determined, and this structure clearly shows the lateral pro-
tonation of this inhibitor.33
Figure 1. Hydrolytic mechanism of a retaining â-xylanase proceeding
through an intermediate in a chair conformation.
ate.14 This was accompanied by almost no change in the position
4
of the protein atoms. A similar C1 chair was observed for the
2-deoxy-2-fluoro-cellobiosyl- and 2-deoxy-2-fluoroxylobiosyl-
enzyme intermediates formed on the family 10 xylanase/
cellulase Cex from Cellulomonas fimi15,16 as well as the
cellobiosyl-enzyme intermediate formed on a double mutant
of Cex without need for the C-2 fluorine substituent.17 Interest-
ingly, the equivalent 2-deoxy-2-fluoroxylobiosyl-enzyme in-
termediate trapped on family 11 xylanases accommodates the
-1 sugar in a distorted 2,5B conformation.18,19 Such a conforma-
tion is attained more easily for xylosides compared to glucosides,
owing to the lack of a bulky hydroxymethyl substituent that is
forced into a pseudoaxial orientation in the latter case. The 2,5
B
conformation places O5, C5, C1, and C2 in a plane, just as is
required to accommodate the double bond character that
develops between C1 and O5 in the proposed transition state.
It has therefore been speculated that family 11 xylanases carry
out catalysis via a boat transition state, rather than a half-chair,
and that this feature dictates the absolute specificity for xylan
hydrolysis exhibited by enzymes of this family.18,19
To gain further insight into the details of the hydrolytic
mechanism of glycosidases, specific inhibitors, ideally transition
state analogues, are necessary which can act as mechanistic and
structural probes. A diverse array of extremely potent, basic,
nitrogen-containing inhibitors has been developed over the years,
Although there are many nitrogen-containing inhibitors
known for monosaccharidases, there are but few of the corre-
(20) Stu¨tz, A. E. Iminosugars as glycosidase inhibitors: nojirimycin and
beyond; Wiley-VCH: Weinheim, 1999.
(21) Legler, G. AdV. Carbohydr. Chem. Biochem. 1990, 48, 319-385.
(22) Granier, T.; Panday, N.; Vasella, A. HelV. Chim. Acta 1997, 80,
979-987.
(23) Tatsuta, K.; Miura, S. Tetrahedron Lett. 1995, 36, 6712-6724.
(24) Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem. Soc. 1993,
115, 11682-11690.
(25) Hoos, R.; Naughton, A. B.; Thiel, W.; Vasella, A.; Weber, W.;
Rupitz, K.; Withers, S. G. HelV. Chim. Acta 1993, 76, 2666-2686.
(26) Ganem, B. Acc. Chem. Res. 1996, 29, 340-347.
(27) Hughes, A. B.; Rudge, A. J. Nat. Prod. Rep. 1994, 11, 135-162.
(28) Bols, M. Acc. Chem. Res. 1998, 31, 1-8.
(29) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J. Am. Chem.
Soc. 1998, 120, 3007-3018.
(14) Davies, G. J.; Mackenzie, L.; Varrot, A.; Dauter, M.; Brzozowski,
A. M.; Schulein, M.; Withers, S. G. Biochemistry 1998, 37, 11707-11713.
(15) White, A.; Tull, D.; Johns, K.; Withers, S. G.; Rose, D. R. Nature
Struct. Biol. 1996, 3, 149-154.
(16) Notenboom, V.; Birsan, C.; Warren, R. A. J.; Withers, S. G.; Rose,
D. R. Biochemistry 1998, 37, 4751-4758.
(17) Notenboom, V.; Birsan, C.; Nitz, M.; Rose, D. R.; Warren, R. A.
J.; Withers, S. G. Nature Struct. Biol. 1998, 5, 812-818.
(18) Sidhu, G.; Withers, S. G.; Nguyen, N. T.; McIntosh, L. P.; Ziser,
L.; Brayer, G. D. Biochemistry 1999, 38, 5346-5354.
(19) Sabini, E.; Sulzenbacher, G.; Dauter, M.; Dauter, Z.; Jorgensen, P.
L.; Schulein, M.; Dupont, C.; Davies, G. J.; Wilson, K. S. Chem. Biol.
1999, 6, 483-492.
(30) Withers, S. G.; Namchuk, M.; Mosi, R. Potent glycosidase
inhibitors: transition state mimics or simply fortuitous binders? In Imino-
sugars as glycosidase inhibitors: nojirimycin and beyond; Stutz, A. E., Ed.;
Wiley-VCH: Weinheim, 1999; pp 188-206.
(31) Heightman, T. D.; Locatelli, M.; Vasella, A. HelV. Chim. Acta 1996,
79, 2190-2200.
(32) Heightman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed. Engl.
1999, 38, 750-770.
(33) Varrot, A.; Schulein, M.; Pipelier, M.; Vasella, A.; Davies, G. J. J.
Am. Chem. Soc. 1999, 121, 2621-2622.

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2225
sponding inhibitors known for glycan hydrolases. Glycan
hydrolases have a low affinity for monosaccharide substrates
and inhibitors and typically require at least disaccharide
glycosides as minimal substrates as a consequence of their
multiple binding subsites. The development of the corresponding
disaccharide inhibitors with residues that occupy the -1 and
-2 binding subsites is synthetically challenging and has been
a stumbling block for the preparation of effective inhibitors for
glycan hydrolases. The syntheses of several cellobiose- and
cellotriose-derived cellulase inhibitors have been described in
recent years.26,34-37 Further, several natural product inhibitors
which bind in the -1 and -2 subsites have been known for
some time; examples include molecules such as the adiposins
and amylostatins,38 which inhibit amylase and are closely related
to acarbose, and allosamidin and its derivatives, which are
inhibitors of some chitinases.39 However, aside from recent
studies on Cel5A with a cellobiose-derived imidazole,33 an
earlier complex of hevamine, a Chitinase from HeVea brasil-
iensis, with allosamidin,40 and the structure of bulgecin A with
a lytic transglycosylase from Escherichia coli,41 there have been
no structural studies of complexes of â-glycanases with tight-
binding inhibitors and relatively few kinetic studies with a single
enzyme and a series of inhibitors.
Figure 2. Protonation trajectories of the glucosidic bond in the catalytic
hydrolysis mechanism proposed by Heightman and Vasella:32 (a) anti-
protonation and (b) syn-protonation.
Scheme 1a
The xylanases Cex and Bcx of families 10 and 11, respec-
tively, provide an ideal system for such model studies. Not only
are high-resolution crystal structures of both enzymes and their
glycosyl-enzyme intermediates available but also the enzymatic
reactions likely proceed via different transition states. Further,
they differ in their protonation trajectory of the glycosidic
oxygen in the catalytic mechanism, the family 10 Cex being an
anti-protonator and the family 11 Bcx being a syn-protonator.
In addition, the synthetic chemistry necessary for the introduc-
tion of the nitrogen atom(s) into potential inhibitors is more
accessible for xylosides due to the absence of a hydroxymethyl
substituent and thus of a stereogenic center at C5.
To conduct detailed kinetic and structural studies of xylanases,
the synthesis of a number of disaccharide inhibitors was
undertaken. The compounds of interest were the xylobiose-
derived imidazole (5), lactam oxime (6), deoxynojirimycin (7),
and isofagomine (8) each of which was chosen as being
representative of known, potent, nitrogen-containing inhibitors.
a (a) Reference 42. (b) NaN3, DMPU, 70°, 45% from D-xylose. (c)
NaOMe, MeOH, 90%. (d) BnBr, NaH, THF. (e) H2SO4, H2O, AcOH,
43% over two steps. (f) CrO3, Pyr. (g) Bu3P, H2O, THF, 65% over
two steps.
(5), the lactam oxime (6), and the deoxynojirimycin derivative
(7) required the partially protected lactam (9) as a common
precursor. D-Xylose was converted to the known tosylate (10)42
and then treated with NaN3 in DMPU to afford the azide (11)
(Scheme 1). Deacetylation with NaOMe in MeOH gave the diol
(12), which was benzylated to furnish the dibenzyl ether (13).
Hydrolysis of the methyl glycosides with H2SO4 in AcOH/H2O
gave the hemiacetal (14), which was oxidized with CrO3/
pyridine, affording the lactone (15). Staudinger reduction of 15
occurred with spontaneous cyclization to afford the desired
lactam (9).43
To convert the lactam (9) to the imidazole (5) required
activation of the carbonyl group as a thionolactam. Thionation
of 9 with Lawesson’s reagent was troublesome and furnished
the thionolactam (16) in poor yield. A higher yield was achieved
when 9 was acetylated to give 17 prior to thionation, affording
16 in 82% over two steps (Scheme 2). Conversion of 16 to the
imidazole (18) following a protocol of Granier et al.,22 using
aminoacetaldehyde dimethyl acetal in the presence of mercuric
acetate, followed by treatment with aqueous acid, proceeded
smoothly (78% yield) and led also to the D-lyxo imidazole 19
(11% yield). D-Xylosylation of 18 with the trichloroacetimidate
(20)44 was sluggish, giving the disaccharide (21) in a yield of
15% (Scheme 3). In this case the reaction was most likely
complicated by the presence of the basic nitrogen of the
imidazole moiety. Deprotection of 21 by sequential catalytic
hydrogenolysis and hydrolysis of the acetate protecting groups
afforded the xylobiose-derived lactam (5) in 80% yield.
Synthesis of the Imidazole (5), the Lactam Oxime (6), and
Xylobiodeoxynojirimycin (7). The syntheses of the imidazole
(34) Vonhoff, S.; Piens, K.; Pipelier, M.; Braet, C.; Claeyssens, M.;
Vasella, A. HelV. Chim. Acta 1999, 82, 963-980.
(35) Sulzenbacher, G.; Driguez, H.; Henrissat, B.; Schulein, M.; Davies,
G. J. Biochemistry 1996, 35, 15280-15287.
(36) Vonhoff, S.; Vasella, A. Synth. Commun. 1999, 29, 551-560.
(37) Kawaguchi, T.; Sugimoto, K.; Hayashi, H.; Arai, M. Biosci. Biotech.
Biochem. 1996, 60, 344-346.
(38) Truscheit, E.; Frommer, W.; Junge, B.; Muller, L.; Schmidt, D.
D.; Wingender, W. Angew. Chem., Int. Ed. Engl. 1981, 20, 744-761.
(39) Berecibar, A.; Grandjean, C.; Siriwardena, A. Chem. ReV. 1999,
99, 779-844.
(42) Kobori, Y.; Myles, D. C.; Whitesides, G. M. J. Org. Chem. 1992,
57, 5899-5907.
(43) Hannessian, S. J. Org. Chem. 1969, 34, 675-681.
(44) Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1990, 195, 199-
224.
(40) Scheltinga, A. C. T.v.; Armand, S.; Kalk, K. H.; Isogai, A.;
Henrissat, B.; Dijkstra, B. W. Biochemistry 1995, 34, 15619-15623.
(41) Thunnissen, A.-M. W. H.; Rozeboom, H. J.; Kalk, K. H.; Dijkstra,
B. W. Biochemistry 1995, 34, 12729-12737.

2226 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
Scheme 2a
Scheme 5a
a Reagents: (a) (i) 15NH2OH.HCl, NaHCO3, MeOH; (ii) Ac2O, Pyr,
62%. (b) NaOMe, MeOH, 82%.
Scheme 6a
a Reagents: (a) (i) Ac2O, Pyr; (ii) Lawesson’s reagent, C6H6, 82%.
(b) Lawesson’s reagent, C6H6, 29%. (c) (i) aminoacetaldehyde dimethyl
acetal, Hg(OAc)2, THF; (ii) toluene, H2O, TsOH.
Scheme 3a
a Reagents: (a) BF3‚Et2O, CH2Cl2. (b) LiAlH4, Et2O.
Scheme 7a
a Reagents: (a) BF3.Et2O, (CH2Cl)2, 15%. (b) (i) Pd/C, H2, MeOH;
(ii) aqueous NH3, MeOH, 80%.
Scheme 4a
a Reagents: (a) (i) LiAlH4, THF; (ii) H2O then CbzCl, 83%. (b) 20,
BF3.Et2O, (CH2Cl)2, 68%. (c) (i) NaOMe, MeOH; (ii) Pd/C, H2, EtOH,
78%.
ester (27). Compound 27 was cleanly deacetylated with sodium
methoxide in methanol to afford the lactam oxime (6). In an
identical manner, the thionolactam (26) was treated with an
excess of 15NH2OH to afford, first, the 15N-labeled oxime ester
(28) and, subsequently, the 15N-labeled lactam oxime (29)
(Scheme 5). The oxime (6) was assigned the (Z)-configuration
on the basis of a small chemical shift difference (δ 3.25 ppm)
between the 13C NMR resonance for C1 in the lactam oxime
(6) and the oxime ester (27).25 In addition, the 15N NMR
spectrum of (27) showed a single resonance, δ 252.8, consistent
with the assignment of the tautomer with an exocyclic CdN
bond to 6.25
a Reagents: (a) BF3.Et2O, (CH2Cl)2, 68%. (b) Pd/C, MeOH. (c)
Ac2O, Pyr, 94% over two steps. (d) Lawesson’s reagent, C6H6, 89%.
(e) (i) NH2OH.HCl, NaHCO3, MeOH; (ii) Ac2O, Pyr, 66%. (f) NaOMe,
MeOH, 89%.
The conversion of the lactam (9) into the xylobiose-derived
deoxynojirimycin (7) was next addressed. While reduction of a
benzylated disaccharide such as 30 with LiAlH4 seemed
appealing, a preliminary, small-scale experiment with 30
(prepared from 9 and 3145) indicated that the reduction caused
elimination, affording the xylitol (32) (Scheme 6). Instead,
reduction of the lactam (9) with lithium aluminum hydride
followed by quenching of the reaction mixture with water and
addition of benzyl chloroformate allowed for the direct isolation
of the Cbz derivative (33) (Scheme 7). D-Xylosylation of 33
with the trichloroacetimidate (20) afforded the disaccharide (34).
Deprotection of 34 was achieved by sequential treatment with
catalytic sodium methoxide in methanol and, subsequently,
catalytic hydrogenolysis to afford 7 in 78% yield.
To achieve the synthesis of 6, the D-xylonolactam (9) was
D-xylosylated with 20 in the same manner as for the imidazole
(18), providing 22 in 68% yield (Scheme 4). Hydrogenolysis
of 22 furnished the intermediate diol (23), which was treated
briefly with pyridine and acetic anhydride to give 24 (94%
yield). In this case, a prolonged treatment of the diol (23) with
pyridine and acetic anhydride afforded imidate (25) as the major
Synthesis of the Isofagomine (8). Synthesis of the isofago-
mine (8) demanded an efficient synthesis of the piperidine (35).
product. The lactam (24) was treated with Lawesson’s reagent
to afford the thionolactam (26) as an unstable yellow foam.
Treatment of 26 with hydroxylamine in methanol and acetylation
of the residue after removal of the solvent afforded the oxime
(45) Rosenbrook, W.; Riley, D. A.; Lartey, P. A. Tetrahedron Lett. 1985,
26, 3-4.

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2227
Scheme 8a
Scheme 9a
a Reagents: (a) 1-benzoyloxybenzotriazole, Et3N, CH2Cl2.
Scheme 10a
a Reagents: (a) BF3‚Et2O, (CH2Cl)2, 44%. (b) NaOMe, MeOH, 81%.
a (a) References 46, 47. (b) BH3‚Me2S, THF, 55%. (c) TsCl, Pyr,
97%. (d) KCN, MeOH. (e) Ac2O, Pyr, 47% over two steps. (f) PtO2,
H2, MeOH, 64%. (g) NaOMe, MeOH, 90%. (h) (i) TMSCl, HMDS,
MeCN; (ii) BH3‚Me2S, dioxane; (iii) 1 M HCl, reflux; (iv) NaHCO3,
CbzCl, 55%. (i) Pd/C, H2, EtOH.
(c) Pd/C, EtOH, 83%.
Scheme 11a
The synthesis of 35 has been achieved by Ichikawa et al. from
D-lyxose, but their route was complicated by deoxygenation
steps and was somewhat lengthy.29 It was of interest to prepare
35 from a precursor that contained only the two necessary
stereogenic centers to simplify the synthesis sufficiently to
provide reasonable quantities of 35. D-Tartaric acid was
converted in two steps to the known methyl ester (36)46,47
(Scheme 8). Reduction of 36 with borane methyl sulfide gave
the alcohol (37)48 which was cleanly converted to the unstable
tosylate (38). Treatment of 38 with potassium cyanide in MeOH
provided the nitrile (39), presumably by way of an intermediate
epoxide. However, it proved more convenient to acetylate the
crude reaction mixture after treatment of 38 with potassium
cyanide and to isolate the diacetate (40). Hydrogenation of 40
over PtO2 proceeded in moderate yield with cyclization to afford
the lactam (41), which was converted to the diol (42) upon
treatment with NaOMe in MeOH. Reduction of 42 according
to a procedure modified from that of Godskesen et al.49 directly
gave the carbamate (43), which was treated with hydrogen over
Pd/C completing a synthesis of D-xylo-isofagomine (35).
To complete the synthesis of 8, the carbamate (43) was treated
with 1-benzoyloxybenzotriazole to afford the separable mono-
benzoates 44 and 45 (Scheme 9). D-Xylosylation of 44 with
the trichloroacetimidate (20) afforded the disaccharide (46),
which was transesterified to afford the carbamate (47) (Scheme
10). Hydrogenolysis of 47 furnished the xylobiose-derived
isofagomine (8) in good yield.
a Reagents: (a) BF3‚Et2O, (CH2Cl)2. (b) NaOMe, MeOH, 45% over
two steps. (c) Pd/C, EtOH, 79%.
Table 1. Inhibition Constants for Cex and Bcx with Various
Nitrogen-Containing Inhibitors
Ki (µM)
compound
Cex
4800
0.15
Bcx
80000
520
xylobiose
5
6
7
0.37
5.8
1400
1500
8
0.13
190
110
790
1100
no inhibition
3100
47
48
50
no inhibition
could be purified with some care away from contaminating
trichloroacetamide; on a larger scale it proved more convenient
to treat the crude reaction mixture with NaOMe in MeOH and
isolate the tetrol (50) instead. As before, hydrogenolysis of 50
cleanly furnished 48 in good yield.
Xylanase Inhibition. Each of the inhibitors was evaluated
for its ability to inhibit the family 10 xylanase Cex, from
Cellulomonas fimi and the family 11 xylanase Bcx, from
Bacillus circulans (Table 1). Compounds 47 (Ki ) 190 µM)
and 50 (Ki ) 790 µM) exhibited rather weak inhibition against
Cex, consistent with the nonbasic character of these compounds.
The xylobiodeoxynojirimycin (7) was a good inhibitor of Cex,
displaying a Ki of 5.8 µM, whereas the fagomine derivative (48),
the 2-deoxy version of 7, was a much poorer inhibitor (Ki )
110 µM). The imidazole (5) and isofagomine (8) (Figure 3)
both showed extremely strong competitive inhibition of Cex
With quantities of 45 in hand we decided to prepare the
xylobiose-derived fagomine (48). In a fashion analogous to that
above, 45 was treated with the trichloroacetimidate (20) to
provide the disaccharide (49) (Scheme 11). This compound
(46) Shriner, R. L.; Furrow, C. L. Org. Synth. Collect. Vol. 1963, 4,
242-243.
(47) Lucas, H. J.; Baumgarten, W. J. Am. Chem. Soc. 1941, 63, 1653-
1657.
(48) Umemura, E.; Tsuchiya, T.; Umezawa, S. J. Antibiot. 1988, 41,
530-537.
(49) Godskesen, M.; Lundt, I.; Madsen, R.; Winchester, B. Bioorg. Med.
Chem. 1996, 4, 1857-1865.

2228 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
Figure 4. Protonation modes of the lactam oxime suggested by
Vonhoff et al.:34 (a) anti-protonation and (b) syn-protonation.
While these relative affinities are indeed in the range observed
for B. agaradhaerens Cel5A and are in fact much better than
those seen for the T. reesei Cel7A, they are still very poor
compared to those found for Cex. Initial analysis would suggest
two likely causes of this difference in affinities: differences in
the protonation trajectory and differences in transition state
conformation.
Figure 3. Dixon plot of inhibition of C. fimi xylanase by the xylobiose-
derived isofagomine 8. The concentrations of the substrate, 2,4-
dinitrophenyl â-cellobioside, used were 0.011 (9), 0.028 (2), 0.071
(b), 0.142 (0), 0.285 (1), 0.712 (O), and 1.42 (4) mM.
Cex belongs to family 10 of the glycosyl hydrolase clas-
sification scheme, thus clan GH-A, and has therefore been
classified as an anti-protonator.32 The imidazole (5) and the
lactam oxime (6) therefore presumably experience favorable
interactions of their exocyclic nitrogen atoms with the acid/
base residue, and this is reflected in the tight binding of 5 and
6 to Cex (Figure 4a). By contrast, Bcx belongs to family 11
(and clan GH-C) and has been classified as a syn-protonator.32
In this case, the lone pair on nitrogen in 5 and 6 must be directed
away from the acid/base group and no hydrogen bonding is then
possible. This might well account for a good portion of the
difference in affinities noted here. Some caution in this analysis
is necessary, though since, as noted earlier, it may be possible
for the oxygen atom of the lactam oxime to engage in productive
binding with a syn-carboxyl group (Figure 4b),34 though the Ki
values measured for 5 and 6 give no support to this hypothesis.
Further mimicry of the transition state may be achieved by
protonation of the “glycosidic” nitrogens of 5 and 6 by the acid/
base/residue, thus imitating the partially protonated glycosidic
oxygen at the transition state. One important caveat here is that
at the transition state the substrate aglycon must be directed in
a pseudoaxial orientation. By contrast, however, the “glycosidic”
nitrogens of 5 and 6 are in the plane of the sugar ring; thus,
some difficulty in matching the transition state may be expected.
Nonetheless, in the X-ray structure of the complex of a
cellobiose-derived imidazole with Cel5A from B. agaradhaerens
it was observed that, despite the less than ideal positioning of
the “glycosidic” nitrogen atom, the acid/base residue was able
to form a short (2.58 Å) hydrogen bond with this atom.33 Thus,
the in-plane orientation of the “glycosidic” nitrogens of 5 and
6 seems unlikely to have a major impact on the formation of a
strong hydrogen bond with the acid/base residue of Cex.
Another possible reason for the relatively high affinities of 5
and 6 for Cex but not for Bcx is that the 4H3 conformation they
are assumed to adopt is nicely complementary to that of the
4H3 transition state stabilized by Cex (Figure 1), but not the
2,5B transition state of Bcx (Figure 5). It is of interest to note
here that while single-crystal X-ray structures of D-glucono-
lactam and the D-gluco-lactam oxime 2 show these molecules
to be in conformations close to that of a 4H3 chair,25,50 expected
for the transition state of the enzyme-catalyzed reaction, the
X-ray structure determined for the complex of the cellobiose-
derived imidazole with Cel5A showed the imidazopyridine ring
of the inhibitor to be in the 4E conformation,33 even though, by
analogy with D-gluconolactam and the D-gluco-lactam oxime
(Ki ) 150 and 130 nM, respectively), with the lactam oxime
(6) being an only slightly poorer inhibitor (Ki ) 370 nM).
Interestingly, while substitution of the basic nitrogen of 8 with
a benzyloxycarbonyl group, giving 47, caused a 1000-fold
reduction in inhibitory potency, the same substitution on 48
(namely 50) merely caused a 2-fold reduction in potency. This
is quite surprising given the supposed importance of protonation
of the nitrogen in inhibitors of the deoxynojirimycin type.
However, this different response to N-acylation of the isofago-
mine (8) (to give 47) compared to the fagomine (48) (to give
50) is reminiscent of the consequences of N-alkylation noted
by Ichikawa.29
Interestingly, compounds 5-8 and 48 were all rather poor
inhibitors of Bcx. The best of these compounds was the
imidazole (5), which inhibited Bcx with a Ki of 0.4 mM.
Compounds 47 and 50 showed no inhibition of Bcx at
concentrations up to 5 mM.
Discussion
The principal series of inhibitors tested, compounds 5-8, can
be separated into two groups. Compounds 5 and 6 have an sp2-
hybridized anomeric carbon with an exocyclic double bond off
C1; thus, planarity is attained on the “amidine” structure of the
ring nitrogen, anomeric carbon, and endocyclic nitrogen.
Compounds 7 and 8 contain an sp3-hybridized nitrogen at
positions equivalent to the ring oxygen or the anomeric carbon,
respectively. All of these compounds proved to be remarkably
effective inhibitors of C. fimi Cex, with Ki values in the nano-
to micromolar range. Unlike the well-studied cellobiohydrolase
Cel7A from Trichoderma reesei, which binds to its disaccharide
product, cellobiose, relatively tightly (Ki ) 20 µM),34 Cex binds
to its disaccharide product, xylobiose, quite poorly (Ki ) 4.8
mM). This product affinity is similar to that seen for cellotriose
with Cel5A from B. agaradhaerens (Ki ) 4.5 mM). As a
consequence, these inhibitors bind to Cex some 6.0-3.7 × 104-
fold more tightly (as expressed in terms of the Ki ratio) than
does the equivalent disaccharide. Thus, they are bound much
more tightly, relatively, than does the cellobiose-derived imid-
azole to Cel5A (Ki ratio ) 55), and indeed the same inhibitor
binds some 6.5-fold worse to the T. reesei Cel7A than does
cellobiose. The inhibition values measured for Cex therefore
represent the highest relative affinities yet seen for any xylanase
or cellulase with a small molecule.
By contrast, the same inhibitors bind relatively poorly to Bcx
with Ki ratios (relative to xylobiose) in the 26-150-fold range.
(50) Ogura, H.; Furuhata, K.; Takayanagi, H.; Tsuzuno, N.; Iitaka, Y.
Bull. Chem. Soc. Jpn. 1984, 57, 2687-2688.

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2229
Figure 5. Formation of the xylobiosyl-enzyme intermediate on Bcx with the sugar moiety in the -1 subsite in a 2,5B conformation.
4
2, this molecule is expected to be in the H3 conformation in
Summary
the solid state. On this basis it is quite possible that the imidazole
We have prepared a series of potent, nanomolar inhibitors
of a family 10 xylanase, Cex, which, surprisingly, are relatively
ineffective inhibitors of a family 11 xylanase, Bcx. All syntheses
were conducted from readily available precursors in a highly
stereoselective manner. These new inhibitors all exhibit com-
petitive inhibition consistent with the formation of a noncovalent
enzyme-inhibitor complex in the active site. The behavior of
two of these inhibitors, namely the imidazole (5) and the lactam
oxime (6), is consistent with the classification of the family 10
enzyme, Cex, as an anti-protonator and with the family 11
enzyme, Bcx, as a syn-protonator. However, the differences in
affinities for the two classes of xylanases may also have their
origins in the different conformations of the transitions states
of the two enzyme-catalyzed reactions, Bcx favoring a boat
conformation and Cex preferring a half-chair conformation. The
efficient syntheses of these new inhibitors lay open the way
for detailed structural evaluation of the mode of interaction of
these inhibitors with these two enzymes and kinetic evaluation
of the degree to which these compounds resemble the transition
state of glycoside hydrolysis.
4
5 and the lactam oxime 6 may actually bind to Cex in E
4
conformations rather than the H3 conformation and thus may
in fact not mimic the conformation of the transition state exactly
even in the case of Cex.
Also of particular note is the large difference in relative
affinities seen for Cex and Bcx with compounds 7 and 8. These
compounds presumably owe their high affinity for Cex to being
protonated in the active site of the enzyme and interacting with
the charged acid/base or nucleophile. As mentioned earlier, Bcx
has the acid/base syn to the C1-O5 bond of the substrate. Such
a location could place this residue in close proximity to the
nitrogen of 7; thus, strong interactions might be expected.
Likewise, the nucleophile of Bcx should be directed toward C1
of the substrate (in order to form a glycosyl enzyme) and thus
should be able to interact with the nitrogen of 8. A possible
reason why this might not be true is that the inhibitors might
not readily adopt the conformation preferred by the enzyme.
At first glance both 7 and, particularly, 8 seem conformationally
mobile; thus, it is difficult to see why these compounds should
not be able to adopt a suitable conformation in the active site
that provides for a good interaction between the basic amine
and either the acid/base or nucleophile residues. However, even
in cyclohexane, the difference in free energy51 of a boat versus
a chair conformation is approximately 6.4 kcal mol-1. Thus,
such differences in conformational free energies might well
explain the difference in affinities of 7 and 8 for the two classes
of enzymes. Quite possibly then, xylobiose analogues with the
reducing-end sugar ring constrained into a 2,5B conformation
would serve as much better inhibitors.
Another crucial difference between Cex and Bcx is the
presence of an essential tyrosine residue in the active site of
Bcx whose oxygen atom lies in close proximity to the sugar
endocyclic oxygen. Mutation of this residue, Tyr69 to Phe,
provides a mutant enzyme that is a severely disabled catalyst.52
A role for this residue in catalysis has been suggested wherein
a hydrogen bond forms between Tyr69 and the anomeric oxygen
of the glycosyl enzyme as the catalytic nucleophile departs from
this intermediate.18 A partial negative charge thus develops on
the phenolic oxygen that could serve to stabilize the partial
positive charge that develops on the sugar as it approaches the
transition state. Possibly, unfavorable interactions of Tyr69 with
each of these inhibitors may cause the high Ki values observed
with Bcx.
Experimental Section
General. All melting points are uncorrected. Organic extracts were
dried over MgSO4 and concentrated in vacuo. 1H and 13C NMR spectra
were recorded on Bruker instruments and were referenced using the
solvent peak. NMR spectra were in CDCl3 unless otherwise noted. 15
N
NMR spectra were recorded on a Varian instrument and were referenced
to 15NH3. Flash chromatography was performed on Merck silica gel
60.53 Thin-layer chromatography was performed on Merck silica gel
60 F254 plates. Microanalyses were performed by Mr. Peter Borda at
the University of British Columbia.
Methyl 2,3-Di-O-acetyl-5-azido-5-deoxy-D-xylofuranosides (11).
A solution of 1042 (70 g, prepared from 30 g of D-xylose, 130 mmol)
in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (500 g) was
treated with NaN3 (56 g, 860 mmol) at 70 °C for 18 h. The mixture
was diluted with H2O (2 L), stirred for 1 h, and extracted with EtOAc
(3 × 200 mL). The combined organic phases were washed with
saturated aqueous NaHCO3 and brine, dried, and evaporated. Flash
chromatography (1:3 EtOAc-C6H6) of the residue gave 11 as a clear
oil (24.8 g, 45% from D-xylose); Rf 0.35 (1:2 EtOAc-C6H6). 1H NMR
(400 MHz) of R-11: δ 2.07, 2.07 (2 s, 6 H, Ac), 3.26-3.33 (m, 2 H,
H5,5), 3.35 (s, 3 H, OMe), 4.38 (ddd, q, J3,4 ≈ J4,5 ≈ J4,5 6.0 Hz, H4),
4.98 (dd, 1 H, J1,2 4.7, J2,3 5.5 Hz, H2), 5.10 (d, 1 H, J1,2 4.5 Hz, H1),
5.44 (dd, t, 1 H, J2,3 ≈ J3,4 6.2 Hz, H3). 1H NMR (400 MHz) of â-11:
δ 2.06, 2.07 (2s, 3 H, Ac), 3.26-3.33 (m, 1 H, H5), 3.38 (s, 3 H,
OMe), 3.49 (dd, 1 H, J4,5 8.0, J5,5 12.8 Hz, H5), 4.17 (ddd, dt, 1 H, J3,4
5.8, J4,5 ≈ J4,5 7.8 Hz, H4), 4.83 (br s, 1 H, H2), 5.04 (br s, 1 H, H1),
5.26 (dd, 1 H, J2,3 1.7, J3,4 6.2 Hz, H3). 13C NMR (75.5 MHz): δ
20.43, 20.54 (CH3CO), 50.12, 51.33 (C5), 55.42, 55.47 (OMe), 74.23,
74.86, 75.05, 76.96, 79.28, 80.68 (C2,3,4), 99.81, 107.06 (C1), 169.34,
169.68, 170.05 (CO). DCI HRMS (NH3, CH4): calcd for m/z [M +
H]+ 274.1039, found 274.1042.
The inhibition by the fagomine derivative (48) gives some
idea of the importance of interactions with the 2-OH group in
7 as removal of this group causes a 20-fold reduction in affinity.
It is therefore interesting to speculate on the potential for
reintroducing a hydroxyl group into an isofagomine structure
to improve still further the extremely powerful inhibition by 8.
(51) Stoddart, J. F. Stereochemistry of carbohydrates; John Wiley &
Sons: New York, 1971; p 53.
(52) Joshi, M. Personal communication.
(53) Still, W. C.; Kahn, M.; Mitra, A. J. J. Org. Chem. 1978, 43, 2923-
2925.

2230 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
Methyl 5-Azido-5-deoxy-D-xylofuranosides (12). A solution of 11
(5.00 g, 18.3 mmol) in MeOH (100 mL) was treated with NaOMe (100
mg, 1.8 mmol) at 25 °C for 30 min. Evaporation and flash chroma-
tography (1:1 EtOAc-hexane then EtOAc) of the residue gave 12 (3.12
g, 90%); Rf 0.35 (R-12) and 0.40 (â-12, EtOAc). 1H NMR (400 MHz)
of R-12: δ 2.60 (d, 1 H, J 5.0 Hz), 2.77 (d, 1 H, J 7.2 Hz, 2OH,
3OH), 3.43 (dd, 1 H, J4,5 5.1, J5,5 12.9 Hz, H5), 3.47 (s, 3 H, OMe),
3.51 (dd, 1 H, J4,5 4.4, J5,5 12.9 Hz, H5), 4.07-4.10 (m, 1 H), 4.22-
(d, 0.5 H, J 11.8 Hz, CH2Ph), 4.61 (d, 0.5 H, J 11.9 Hz, CH2Ph), 5.28
(s, 0.5 H), 5.45 (d, 0.5 H, J1,2 4.0 Hz, H1), 7.25-7.40 (m, 10 H, Ph).
13C NMR (75.5 MHz): δ 50.07, 50.72 (C5), 71.80, 72.05, 72.38, 72.94
(CH2Ph), 76.66, 79.81, 80.46, 80.73, 81.11, 84.67 (C2,3,4), 95.94,
101.29 (C1), 127.63-128.52, 136.56, 137.05, 137.16 (Ph). DCI HRMS
(NH3, CH4): calcd for m/z [M + H]+ 356.1610, found 356.1611.
5-Azido-2,3-di-O-benzyl-5-deoxy-D-xylono-1,4-lactone (15). Pyr-
idine (150 mL) was added to CrO3 (14.5 g, 145 mmol) and the mixture
was stirred for 1 h, then treated with a solution of 14 (10.3 g, 29.0
mmol) in pyridine (150 mL) and stirred at 25 °C overnight. Et2O (1 L)
was added and the mixture was filtered through Celite. The filtrate
was washed with 1 M H2SO4, saturated aqueous NaHCO3, and brine,
dried and the solvent evaporated to give crude 15 as an oil (9.31 g) an
analytical sample of which was purified by flash chromatography; Rf
0.23 (1:3 EtOAc-hexane). 1H NMR (400 MHz): δ 3.58 (dd, 1 H, J4,5
4.3, J5,5 13.3 Hz, H5), 3.64 (dd, 1 H, J4,5 4.6, J5,5 13.3 Hz, H5), 4.31
(dd, t, 1 H, J2,3 ≈ J3,4 6.3 Hz, H3), 4.37 (d, 1 H, J2,3 5.8 Hz, H2), 4.52
(d, 1 H, J 11.8 Hz, CH2Ph), 4.64 (d, 1 H, J 11.9 Hz, CH2Ph), 4.62
(ddd, dt, 1 H, J3,4 ≈ J4,5 6.6, J4,5 4.5 Hz, H4), 4.71 (d, 1 H, J 11.4 Hz,
CH2Ph), 5.04 (d, 1 H, J 11.4 Hz, CH2Ph), 7.23-7.40 (m, 10 H, Ph).
13C NMR (75.5 MHz): δ 49.91 (C5), 72.51, 72.56 (CH2Ph), 76.64,
77.10, 78.49 (C2,3,4), 127.82-128.49, 136.56 (Ph), 172.20 (CO). DCI
HRMS (NH3, CH4): calcd for m/z [M + H]+ 353.1375, found 353.1364.
5-Amino-2,3-di-O-benzyl-5-deoxy-D-xylono-1,5-lactam (9). A so-
lution of 15 (8.84 g, 25.0 mmol) in 10:1 THF-H2O (550 mL) was
treated with Bu3P (7.2 mL, 29.2 mmol) and stirred at 25 °C for 24 h.
Evaporation of the solvent and flash chromatography (EtOAc) of the
residue gave a solid which was crystallized from EtOAc-petroleum
ether. Flash chromatography (EtOAc) of the mother liquor gave 9 (5.3
1
4.30 (m, 2 H, H2,3,4), 4.96 (d, 1 H, J1,2 4.5 Hz, H1). H NMR (400
MHz) of â-12: δ 2.46 (d, 1 H, J2,OH ≈ 3.1 Hz, 2OH), 2.87 (d, 1 H,
J3,OH 10.9 Hz, 3OH), 3.40 (s, 3 H, OMe), 3.47-3.53 (m, 2 H, H5,5),
4.04 (dd, 1 H, J3,OH ≈ 10.5, J3,4 ≈ 4.1 Hz, H3), 4.18 (d, 1 H, J2,OH
≈
2.4 Hz, H2), 4.41 (ddd, q, 1 H, J3,4 ≈ J4,5 ≈ J4,5 4.8 Hz, H4), 4.85 (s,
1 H, H1). 13C NMR (75.5 MHz): δ 50.53, 51.26 (C5), 55.19, 55.43
(OMe), 75.71, 75.97, 76.53, 77.92, 79.60, 81.05 (C2,3,4), 101.19,
108.46 (C1). DCI MS (NH3): m/z 190 (2, [M + H]+), 158 (7), 134
(6), 133 (100), 115 (13).
Methyl 5-Azido-2,3-di-O-benzyl-5-deoxy-D-xylofuranosides (13).
(a) With BnBr and Ag2O. A solution of 12 (0.50 g, 2.64 mmol) in
DMF was treated with BnBr (1.6 mL, 13.5 mmol) and Ag2O (1.55 g,
6.7 mmol) at 25 °C for 18 h. Pyridine (1 mL) was added and the mixture
was filtered through Celite. The filtrate was diluted with water and
extracted with Et2O. The combined organic phases were washed with
1 M H2SO4, saturated aqueous NaHCO3, and brine and dried, and the
solvent was evaporated. Flash chromatography of the residue gave 13
(0.68 g, 70%).
(b) With BnBr and NaH. A solution of BnBr (24 mL, 202 mmol)
in THF (150 mL) was treated with NaH (60% in oil, 11 g, ca. 275
mmol), cooled to 0 °C, and treated dropwise with a solution of 12
(12.8 g, 67.7 mmol) in THF (50 mL) at such a rate that the temperature
of the solution did not exceed 10 °C. When gas evolution ceased, the
mixture was heated to 60 °C for 5 min, cooled to 25 °C, treated with
MeOH (30 mL) and pyridine (30 mL), and stirred overnight. Water
was added and the mixture was extracted with toluene. The combined
organic phases were washed with 1 M H2SO4, saturated aqueous
NaHCO3, and brine and dried, and the solvent was evaporated to give
crude 13 as an oil (33.0 g) an analytical sample of which was purified
by flash chromatography; Rf 0.31 (â-13), 0.38 (R-13; 1:3 EtOAc-
1
g, 65% from 14); mp 107-108 °C. Rf 0.19 (EtOAc). H NMR (400
MHz): δ 2.80 (br s, 1 H, 4OH), 3.16 (ddd, 1 H, J4,5 2.1, J5,5 12.2, J5,NH
6.8 Hz, H5), 3.42 (ddd, 1 H, J4,5 4.5, J5,5 12.2, J5,NH 3.1 Hz, H5), 3.71
(dd, t, 1 H, J2,3 ≈ J3,4 6.8 Hz, H3), 3.89 (ddd, td, 1 H, J3,4 ≈ J4,5 6.8,
J4,5 4.8 Hz, H4), 3.94 (d, 1 H, J2,3 6.6 Hz, H2), 4.58 (d, 1 H, J 11.5 Hz,
CH2Ph), 5.12 (d, 1 H, J 11.4 Hz, CH2Ph), 4.75 (d, 1 H, J 11.3 Hz,
CH2Ph), 4.77 (d, 1 H, J 11.4 Hz, CH2Ph), 6.14 (br s, 1 H, NH), 7.21-
7.42 (m, 10 H, Ph). 13C NMR (75.5 MHz): δ 44.35 (C5), 66.83, 77.86,
80.71 (C2,3,4), 73.63, 74.35 (CH2Ph), 127.94-128.51, 137.33, 137.56
(Ph), 170.89 (CO). DCI MS (NH3): m/z 323 (6, [M + H]+), 221 (10),
130 (10), 116 (6), 115 (100), 114 (13), 98 (21), 91 (27). Anal. Calcd
for C19H21NO4: C, 69.71; H, 6.47; N, 4.28. Found: C, 69.84; H, 6.42;
N, 4.21.
5-Amino-2,3-di-O-benzyl-5-deoxy-D-xylonothio-1,5-lactam (16).
A solution of 9 (100 mg, 0.31 mmol) in C6H6 (4 mL) was treated with
Lawesson’s reagent (86 mg, 0.21 mmol) at 25 °C for 36 h. Evaporation
of the solvent and flash chromatography (1:1 EtOAc-hexane) of the
residue gave 16 (32 mg, 29%); Rf 0.22 (1:1 EtOAc-hexane). 1H NMR
(400 MHz): δ 2.73 (br s, 1 H, 4OH), 3.35 (br d, 1 H, J 13.6 Hz, H5),
3.55 (br d, 1 H, J 12.8, H5), 3.75 (dd, t, 1 H, J2,3 ≈ J3,4 4.5 Hz, H3),
3.99 (br d, 1 H, J3,4 ≈ 4.3 Hz, H4), 4.30 (d, 1 H, J2,3 4.3 Hz, H2), 4.46
(d, 1 H, J 11.7 Hz, CH2Ph), 4.61 (d, 1 H, J 11.7 Hz, CH2Ph), 4.83 (d,
1 H, J 11.4 Hz, CH2Ph), 5.14 (d, 1 H, J 11.4 Hz, CH2Ph), 7.20-7.44
(m, 20 H, Ph), 8.37 (br s, 1 H, NH). 13C NMR (75.5 MHz): δ 48.62
(C5), 66.10, 78.00, 80.71 (C2,3,4), 72.61, 74.05 (CH2Ph), 127.85-
128.51, 136.86, 137.16 (Ph), 199.51 (CS). DCI MS (NH3): m/z 344
(4, [M + H]+), 238 (4), 237 (28), 146 (5), 133 (5), 132 (8), 131 (100),
130 (12), 91 (82).
1
hexane). H NMR (400 MHz) of R-13: δ 3.38 (s, 3 H, OMe), 3.38-
3.41 (m, 2 H, H5,5), 4.00-4.02 (m, 1 H, H2), 4.28-4.30 (m, 2 H,
H3,4), 4.52 (d, 1 H, J 11.9 Hz, CH2Ph), 4.66 (d, 1 H, J 11.8 Hz, CH2Ph),
4.56 (d, 1 H, J 11.9 Hz, CH2Ph), 4.63 (d, 1 H, J 11.8 Hz, CH2Ph),
4.80 (d, 1 H, J1,2 4.2 Hz, H1), 7.25-7.37 (m, 10 H, Ph). 1H NMR (400
MHz) of â-13: δ 3.37 (dd, 1 H, J4,5 4.7, J5,5 13.0 Hz, H5), 3.41 (s, 3
H, OMe), 3.54 (dd, 1 H, J4,5 8.5, J5,5 13.0 Hz, H5), 4.01 (dd, 1 H, J1,2
1.7, J2,3 3.2 Hz, H2), 4.08 (dd, 1 H, J2,3 3.3, J3,4 6.4 Hz, H3), 4.35
(ddd, 1 H, J3,4 6.4, J4,5 8.4, J4,5 4.5 Hz, H4), 4.42 (d, 1 H, J 12.2 Hz,
CH2Ph), 4.58 (d, 1 H, J 12.0 Hz, CH2Ph), 4.49 (d, 1 H, J ≈ 12.8 Hz,
CH2Ph), 4.55 (d, 1 H, J 11.7 Hz, CH2Ph), 4.90 (d, 1 H, J1,2 1.7 Hz,
H1), 7.25-7.37 (m, 10 H, Ph). 13C NMR (75.5 MHz): δ 50.95, 51.75
(C5), 55.08, 55.24 (OMe), 71.94, 72.10, 72.27, 72.49 (CH2Ph), 75.38,
79.66, 80.94, 81.67, 83.77, 86.56 (C2,3,4), 100.35, 108.01 (C1),
127.54-128.30, 137.24, 137.34, 137.60 (Ph). DCI HRMS (NH3,
CH4): calcd for m/z [M - 1]+ 368.1610, found 368.1617.
5-Azido-2,3-di-O-benzyl-5-deoxy-D-xylofuranoses (14). A solution
of crude 13 (32.0 g) in AcOH (400 mL) was treated with 1 M H2SO4
(80 mL) and heated to 50-60 °C for 6 h. Water (1 L) was added and
the mixture was extracted with CH2Cl2 (3 × 200 mL). The combined
organic phases were washed with saturated aqueous K2CO3, saturated
aqueous NaHCO3, and brine and dried, and the solvent was evaporated.
Flash chromatography (1:5 EtOAc-hexane) of the residue gave 14 as
an oil (10.3 g, 43% from 12); Rf 0.15 (1:3 EtOAc-hexane). 1H NMR
(400 MHz): δ 3.42 (dd, 0.5 H, J4,5 6.1, J5,5 12.6 Hz, H5), 3.50 (dd, 0.5
H, J4,5 6.5, J5,5 12.5 Hz, H5), 3.47 (dd, 0.5 H, J4,5 ≈ 6.4, J5.5 ≈ 13.9
Hz, H5), 3.60 (dd, 0.5 H, J4,5 6.7, J5,5 12.5 Hz, H5), 3.94 (dd, 0.5 H,
J2,3 2.5, J3,4 4.1 Hz, H3), 4.00 (dd, 0.5 H, J2,3 2.2, J3,4 4.4 Hz, H3),
3.98-3.99 (m, 1 H, H2), 4.30 (ddd, td, 0.5 H, J3,4 4.8, J4,5 ≈ J4,5 6.2
Hz, H4), 4.33 (ddd, dt, 0.5 H, J3,4 4.4, J4,5 ≈ J4,5 6.5 Hz, H4), 4.44 (d,
0.5 H, J 11.9 Hz, CH2Ph), 4.51 (d, 0.5 H, J ≈ 10.8 Hz, CH2Ph), 4.48
(d, 0.5 H, J 11.3 Hz, CH2Ph), 4.61 (d, 0.5 H, J 12.0 Hz, CH2Ph), 4.52
(d, 0.5 H, J 12.0 Hz, CH2Ph), 4.58 (d, 0.5 H, J 11.8 Hz, CH2Ph), 4.56
4-O-Acetyl-5-amino-2,3-di-O-benzyl-5-deoxy-D-xylonothio-1,5-
lactam (17). A solution of 9 (1.00 g, 3.05 mmol) in EtOAc (30 mL)
was treated with pyridine (2.5 mL) and Ac2O (1.5 mL) at 25 °C for 24
h and evaporated. The residue was dissolved in C6H6 (15 mL) and
treated with Lawesson’s reagent (860 mg, 2.13 mmol) at 25 °C for 24
h. Evaporation of the solvent and flash chromatography (1:2 EtOAc-
hexane) of the residue gave 17 (970 mg, 82%); Rf 0.41 (1:1 EtOAc-
hexane). 1H NMR (400 MHz): δ 1.97 (s, 3 H, Ac), 3.39 (ddd, dt, 1 H,
J
J
J
5,5 13.7, JNH,5 ≈ J4,5 4.7 Hz, H5), 3.62 (ddd, dt, 1 H, J5,5 13.7, JNH,5 ≈
4,5 4.2 Hz, H5), 3.80 (dd, 1 H, J2,3 4.9, J3,4 3.4 Hz, H3), 4.23 (d, 1 H,
2,3 5.1 Hz, H2), 4.58 (d, 1 H, J 11.7 Hz, CH2Ph), 4.64 (d, 1 H, J 11.7
Hz, CH2Ph), 4.70 (d, 1 H, J 11.3 Hz, CH2Ph), 5.09 (d, 1 H, J 11.4 Hz,
CH2Ph), 5.06 (ddd, q, J4,5 ≈ J4,5 ≈ J3,4 4.2 Hz, H4), 7.23-7.43 (m, 10
H, Ph), 8.49 (br s, 1 H, NH). 13C NMR (75.5 MHz): δ 20.89 (Me),

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2231
44.47 (C5), 72.81, 73.71 (CH2Ph), 70.45, 79.44, 82.78 (C2,3,4),
127.88-127.92, 137.28, 137.37 (Ph), 170.07 (CO), 201.01 (CS). DCI
HRMS (NH3, CH4): calcd for m/z [M + H]+ 386.1426, found 386.1431.
(6R,7S,8S)-7,8-Dibenzyloxy-6-hydroxy-5,6,7,8-tetrahydroimidazo-
[1,2-a]pyridine (18) and (6R,7S,8R)-7,8-Dibenzyloxy-6-hydroxy-
5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (19). A solution of 14 (800
mg, 2.08 mmol) in THF (8 mL) was treated with aminoacetaldehyde
dimethyl acetal (1.12 mL, 10.4 mmol) and Hg(OAc)2 (856 mg, 2.69
mmol) at 5 °C for 45 min and evaporated. The residue was dissolved
in 11:1 toluene-H2O (48 mL) and treated with TsOH‚H2O (148 mg,
0.78 mmol) at 65 °C for 18 h. EtOAc was added, the mixture was
washed with saturated aqueous NaHCO3 and brine and dried, and the
solvent was evaporated. The residue was purified by flash chromatog-
raphy (3:1 EtOAc-hexane) to give 18 (570 mg, 78%) and 19 (80 mg,
mg) and H2 (ca. 1 bar) at 25 °C overnight. More AcOH (1 mL) and Pd
black (50 mg) were added, and the mixture was again treated with H2
(ca. 1 bar) at 25 °C overnight and then filtered and evaporated. The
residue [Rf 0.18 (18:2:1 EtOAc-MeOH-H2O)] was dissolved in
MeOH (2 mL) and treated with aqueous NH3 (25%) at 25 °C for 3 h.
The solvent was evaporated and the residue purified by flash chroma-
tography (4:2:1 EtOAc-MeOH-H2O) to give 5 (15 mg, 80%); Rf 0.17
(4:2:1 EtOAc-MeOH-H2O). 1H NMR (400 MHz, D2O): δ 3.28 (dd,
1 H, J1′,2′ 7.5, J2′,3′ 9.3 Hz, H2′), 3.31 (dd, 1 H, J5′,5′ 11.6, J4′,5′ 10.5 Hz,
H5′), 3.44 (dd, t, 1 H, J2′,3′ ≈ J3′,4′ 9.2 Hz, H3′), 3.62 (ddd, 1 H, J4′,5′
10.4, J3′,4′ 9.0, J4′,5′ 5.4 Hz, H4′), 3.97 (dd, 1 H, J5′,5′ 11.6, J4′,5′ 10.4 Hz,
H5′), 4.08 (dd, 1 H, J4,5 6.1, J5,5 12.7 Hz, H5), 4.08 (dd, 1 H, J6,7 7.4,
J7,8 ≈ 6.0 Hz, H7), 4.33 (ddd, 1 H, J5,6 ≈ 6.6, J5,6 4.8, J6,7 7.7 Hz, H6),
4.37 (dd, 1 H, J4,5 4.7, J5,5 12.6 Hz, H5), 4.53 (d, 1 H, J1′,2′ 7.5 Hz,
H1′), 4.67 (d, 1 H, J7,8 5.8 Hz, H8), 7.06, 7.07 (2s, H2,3). LSIMS HRMS
(thioglycerol): calcd for [M + H]+ 303.1192, found 303.1194.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-5-amino-2,3-di-O-benzyl-
5-deoxy-D-xylono-1,5-lactam (22). BF3‚Et2O (270 µL, 2.2 mmol) was
added to a solution of 9 (706 mg, 2.16 mmol) and 2,3,4-tri-O-acetyl-
D-xylopyranosyl trichloroacetimidate 2044 (1.36 g, 3.24 mmol) in 1,2-
dichloroethane (10 mL) at 0 °C and the solution was allowed to warm
to room temperature and left to stand for 6 h. The solution was washed
with saturated aqueous NaHCO3 and brine and then dried and the
solvent evaporated. The residue was purified by flash chromatography
(9:1 EtOAc-hexane then EtOAc) to afford 22 as a white crystalline
solid (861 mg, 68%); mp 150-158 °C, Rf 0.16 (3:1 EtOAc-hexane).
1H NMR (400 MHz): δ 2.00, 2.01, 2.03 (3s, 9 H, Ac), 3.22 (ddd, 1 H,
J5,5 12.9, J4,5 6.8, J5,NH 3.3 Hz, H5), 3.29 (dd, 1 H, J4′,5′ 8.1, J5′,5′ 12.0
Hz, H5′), 3.37 (ddd, dt, 1 H, J5,5 13.0, J4,5 ≈ J5,NH 4.0 Hz, H5), 3.83
(dd, t, 1 H, J2,3 ≈ J3,4 6.2 Hz, H3), 3.94 (d, 1 H, J2,3 6.8 Hz, H2), 4.03
(ddd, td, 1 H, J3,4 ≈ J4,5 6.5, J4,5 ≈ 4.4 Hz, H4), 4.10 (dd, 1 H, J4′,5′
4.8, J5′,5′ 12.0 Hz, H5′), 4.62 (d, 1 H, J1′,2′ 6.2 Hz, H1′), 4.67 (d, 1 H,
J 11.3 Hz, CH2Ph), 4.71 (d, 1 H, J 11.4 Hz, CH2Ph), 4.69 (d, 1 H, J
11.7 Hz, CH2Ph), 5.00 (d, 1 H, J, 11.5 Hz, CH2Ph), 4.84 (dd, 1 H, J2′,3′
8.1, J1′,2′ 6.3 Hz, H2′), 4.89 (ddd, td, 1 H, J3′,4′ ≈ J4′,5′ 8.0, J4′,5′ 4.8 Hz,
H4′), 5.10 (dd, t, 1 H, J2′,3′ ≈ J3′,4′ 8.0 Hz, H3′), 6.43 (br s, 1 H, NH),
7.23-7.33 (m, 8 aromatic H), 7.35-7.38 (m, 2 aromatic H). 13C NMR
(75.5 MHz): δ 20.63, 20.67, 20.71 (Me), 41.15 (C5), 61.70 (C5′), 68.37,
70.45, 70.68, 75.24, 78.52, 81.55 (C2,3,4,2′,3′,4′), 73.64, 73.95 (CH2Ph),
98.58 (C1), 127.69-128.31, 137.68, 137.76 (Ph), 169.32, 169.77,
169.94 (OCO), 171.45 (NCO). DCI MS (NH3): m/z 586 (2, [M + H]+),
259 (6), 204 (10), 202 (9), 201 (10), 157 (16), 140 (13), 139 (14), 98
(41), 97 (32), 92 (9), 91 (100). Anal. Calcd for C30H35NO11: C, 61.53;
H, 6.02; N, 2.39. Found: C, 61.37; H, 5.93; N, 2.28.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-5-amino-2,3-di-O-acetyl-5-
deoxy-D-xylono-1,5-lactam (24). A suspension of 22 (690 mg, 1.18
mmol) and Pd on carbon (10%, 200 mg) in MeOH (50 mL) was treated
with H2 (ca. 1 bar) at 25 °C overnight. The mixture was filtered through
Celite, the solvent was evaporated, and the residue was dissolved in
pyridine (6 mL) and treated with Ac2O (2 mL) at room temperature
for 1 h. Water (1 mL) was added and the mixture was stirred for 5
min. Workup (CH2Cl2; water, 1 M HCl, saturated aqueous NaHCO3)
followed by evaporation and flash chromatography (9:1 EtOAc-hexane
then EtOAc) gave 24 as a clear oil (545 mg, 94%); Rf 0.17 (EtOAc).
1H NMR (400 MHz): δ 2.00, 2.02, 2.03, 2.06, 2.10 (5s, 15 H, Ac),
3.28 (ddd, 1 H, J4,5 7.6, J5,5 12.5, J5,NH 2.3 Hz, H5), 3.42 (dd, 1 H, J5′,5′
12.2, J4′,5′ 7.2 Hz, H5′), 3.46 (ddd, dt, 1 H, J4,5 ≈ J5,NH 4.0, J5,5 12.7
Hz, H5), 4.05 (ddd, td, 1 H, J3,4 ≈ J4,5 ≈ 7.5, J4,5 4.5 Hz, H4), 4.12
(dd, 1 H, J4′,5′ 4.5, J5′,5′ 12.1 Hz, H5′), 4.64 (d, 1 H, J2,3 5.5 Hz, H2),
4.76 (dd, 1 H, J2,3 5.5, J3,4 7.3 Hz, H3), 4.85 (ddd, td, 1 H, J3′,4′ ≈ J4′,5′
7.2, 4.5 Hz, H4′), 5.09 (d, 1 H, J1′,2′ 8.2 Hz, H1′), 5.06 (dd, t, 1 H, J2′,3′
≈ J3′,4′ 7.3 Hz, H3′), 5.34 (dd, t, 1 H, J1′,2′ ≈ J2′,3′ 8.0 Hz, H2′), 6.65 (br
s, 1 H, NH). 13C NMR (75.5 MHz): δ 20.44, 20.48, 20.61, 20.69 (5
C, Me), 41.30 (C5), 61.11 (C5′), 67.94, 69.73, 69.82, 70.58, 71.98,
72.38 (C2,3,4,2′,3′,4′), 97.97 (C1′), 167.48 (C1), 169.17, 169.42, 169.71,
169.77, 169.97 (5 C, CO). DCI HRMS (NH3, CH4): calcd for [M +
H]+ 490.1561, found 490.1560.
1
11%); data of 18: Rf 0.27 (EtOAc), mp 100-101 °C. H NMR (400
MHz): δ 4.05-4.11 (m, 2 H), 4.17-4.23 (m, 2 H, H5,5, H6,7), 4.53
(d, 1 H, J 11.9 Hz, CH2Ph), 4.65 (d, 1 H, J 11.9 Hz, CH2Ph), 4.76 (d,
1 H, J 11.7 Hz, CH2Ph), 4.93 (d, 1 H, J 11.7 Hz, CH2Ph), 4.78 (d, 1
H, J7,8 2.9 Hz, H8), 6.88 (d, 1 H, J2,3 1.0 Hz), 7.14 (d, 1 H, J2,3 1.0 Hz,
H2,3), 7.18-7.33 (m, 10 H, Ph). 13C NMR (75.5 MHz): δ 48.75 (C5),
66.59, 70.87, 75.71 (C6,7,8), 71.63, 72.48 (CH2Ph), 119.48 (C3),
127.75, 127.88, 128.01, 128.08, 128.42, 128.49, 137.06, 137.10 (Ph),
129.13 (C2), 141.33 (C8a). DCI MS (NH3): m/z 352 (4), 351 (18, [M
+ H]+), 320 (6), 259 (8), 214 (25), 197 (13), 138 (66), 137 (46), 121
(42), 92 (8), 91 (100). Anal. Calcd for C21H22N2O3: C, 71.98; H, 6.33;
N, 7.99. Found: C, 71.70; H, 6.34; N, 7.98. Data of 19: Rf 0.13
1
(EtOAc), mp 142-144 °C. H NMR (400 MHz): δ 3.01 (br s, 1 H,
OH), 3.62 (dd, 1 H, J6,7 9.7, J7,8 3.5 Hz, H7), 3.69 (dd, 1 H, J5,5 12.1,
J4,5 9.3 Hz, H5), 4.35 (dd, 1 H, J5,5 12.1, J4,5 6.6 Hz, H5), 4.43 (d, 1 H,
J 11.7 Hz, CH2Ph), 4.63 (d, 1 H, J 11.7 Hz, CH2Ph), 4.66 (ddd, td, 1
H, J5,6 ≈ J6,7 9.4, J5,6 4.8 Hz, H6), 4.69 (d, 1 H, J 11.9 Hz, CH2Ph),
4.77 (d, 1 H, J 12.0 Hz, CH2Ph), 4.82 (d, 1 H, J7,8 3.5 Hz, H8), 6.84
(d, 1 H, J2,3 1.1 Hz), 7.09 (d, 1 H, J2,3 1.2 Hz, H2,3), 7.24-7.34, 7.40-
7.44 (2m, 10 H, Ph). 13C NMR (75.5 MHz): δ 48.37 (C5), 64.12, 67.37,
79.33 (C6,7,8), 70.72, 71.26 (CH2Ph), 119.17 (C3), 127.64, 127.97,
128.01, 128.27, 128.33, 128.46, 137.18, 137.80 (Ph), 129.54 (C2),
143.08 (C8a). DCI MS (NH3): m/z 351 (3, [M + H]+), 320 (5), 259
(6), 214 (29), 138 (50), 137 (36), 121 (33), 92 (8), 91 (100).
(6R,7S,8S)-6-(Tri-O-acetyl-â-D-xylopyranosyloxy)-7,8-dibenzyloxy-
5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (21). BF3‚Et2O (34 µL, 0.27
mmol) was added to a mixture of 18 (233 mg, 0.665 mmol), 2,3,4-
tri-O-acetyl-D-xylopyranosyl trichloroacetimidate 2044 (443 mg, 1.00
mmol), and molecular sieves (4 Å) in 1,2-dichloroethane (10 mL) and
the mixture stirred at -15 °C for 1 h. The mixture was filtered and the
filtrate was washed with saturated aqueous NaHCO3 and brine and
dried. The solvent was evaporated and the residue was purified by flash
chromatography (3:1 EtOAc-hexane) and crystallized from EtOAc/
Et2O/hexane to give 21 (60 mg, 15%); Rf 0.20 (3:1 EtOAc-hexane).
1H NMR (400 MHz, C6D6): δ 1.59 (s, 3 H, Ac), 1.65 (s, 3 H, Ac),
1.66 (s, 3 H, Ac), 3.06 (dd, 1 H, J5′,5′ 12.0, J4′,5′ 7.7 Hz, H5′), 3.36 (dd,
1 H, J5,5 12.5, J4,5 7.2 Hz, H5), 3.56 (dd, 1 H, J5,5 12.5, J4,5 4.6 Hz,
H5), 3.79 (ddd, td, 1 H, J5,6 ≈ J6,7 7.1, J5,6 4.6 Hz, H6), 4.01 (dd, 1 H,
J6,7 7.0, J7,8 4.2 Hz, H7), 4.02 (dd, 1 H, J5′,5′ 12.0, J4′,5′ 4.3 Hz, H5′),
4.39 (d, 1 H, J1′,2′ 6.0 Hz, H1′), 4.52 (d, 1 H, J 11.6 Hz, CH2Ph), 4.57
(d, 1 H, J 11.6 Hz, CH2Ph), 4.77 (d, 1 H, J7,8 4.0 Hz, H8), 4.94 (ddd,
td, 1 H, J4′,5′ ≈ J3′,4′ 7.7, J4′,5′ 4.8 Hz, H4′), 5.02 (d, 1 H, J 11.9 Hz,
CH2Ph), 5.25 (d, 1 H, J 11.9 Hz, CH2Ph), 5.09 (dd, J1′,2′ 6.0, J2′,3′ 7.9
Hz, H2), 5.31 (t, 1 H, J3′,4′ ≈ J2′,3′ 7.8 Hz, H3′), 6.30 (d, 1 H, J2,3 0.9
Hz, H2 or H3), 7.06-7.27 (m, 9 H, 8 aromatic H, H2 or H3), 7.50 (br
d, Jvic ≈ 7.2 Hz, 2 aromatic H). 13C NMR (75.5 MHz, C6D6): δ 20.59,
20.74 (Me), 44.97 (C5), 61.60 (C5′), 68.28, 70.14, 70.38, 72.96, 74.05,
80.29 (C6,7,8,2′,3′,4′), 72.14, 73.41 (CH2Ph), 98.86 (C1′), 118.33 (C3),
127.56, 127.87, 128.04, 128.25, 128.36, 137.55, 137.99 (Ph), 129.33
(C2), 143.55 (C8a), 169.27, 169.76, 169.92 (CO). DCI MS (NH3): m/z
610 (9), 609 (25, [M + H]+), 517 (21), 444 (21), 443 (79), 395 (49),
198 (27), 197 (73), 122 (27), 121 (100), 91 (82). Anal. Calcd for
C32H36N2O10: C, 63.15; H, 5.96; N, 4.60. Found: C, 62.98; H, 6.07;
N, 4.57.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-5-amino-2,3-di-O-acetyl-5-
deoxy-D-xylonothio-1,5-lactam (26). A suspension of Lawesson’s
reagent (407 mg, 1.01 mmol) and 24 (492 mg, 1.01 mmol) in dry
benzene (10 mL) was heated under reflux under nitrogen for 2 h. The
(6R,7S,8S)-7,8-Dihydroxy-6-(â-D-xylopyranosyloxy)-5,6,7,8-tet-
rahydroimidazo[1,2-a]pyridine (5). A solution of 21 (38 mg, 62 µmol)
in MeOH (1.5 mL) and AcOH (1 mL) was treated with Pd black (50

2232 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
mixture was cooled and applied directly to a column of silica gel. Flash
chromatography (70-90% EtOAc-petrol) afforded 26 as an unstable
yellow foam (376 mg, 89%). 1H NMR (400 MHz): δ 2.01, 2.03, 2.04,
2.06, 2.15 (5s, 15 H, Ac), 3.34-3.44 (m, 1 H, H5), 3.43 (dd, 1 H, J4′,5′
7.1, J5′,5′ 12.1 Hz, H5′), 3.59 (dt, 1 H, J4,5 ≈ J5,NH 3.6, J5,5 13.8 Hz,
H5), 4.08-4.18 (m, 1 H, H4), 4.13 (dd, 1 H, J4′,5′ 4.4 Hz, H5′), 4.67
(d, 1 H, J2,3 5.5 Hz, H2), 4.77 (dd, 1 H, J3,4 7.3 Hz, H3), 4.87 (dt, 1 H,
J3′,4′ ≈ J4′,5′ 4.5, J4′,5′ 7.1 Hz, H4′), 5.08 (t, 1 H, J2′,3′ 7.3 Hz, H3′), 5.22
(dd, 1 H, J1′,2′ 7.7 Hz, H2), 5.43 (d, 1 H, H1′), 8.45 (br s, 1 H, NH).
DCI HRMS (NH3, CH4): calcd for [M + H]+ 506.1332, found
506.1320.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-2,3-tri-O-acetyl-D-xylono-
(Z)-hydroximo-1,5-lactam (27). A mixture of the thionolactam 26 (80
mg, 158 µmol), NaHCO3 (200 mg, 2.38 mmol), and NH2OH‚HCl (164
mg, 2.38 mmol) in MeOH (3 mL) was stirred at room temperature for
1 h and then was heated under reflux for 1 h. The solvent was
evaporated and the residue was treated with Ac2O (1 mL) and pyridine
(3 mL) at room temperature for 1 h. Water (1 mL) was added and the
mixture was stirred for 10 min. The mixture was diluted with CH2Cl2,
and the organic phase was washed with water and saturated NaHCO3
and dried. The solvent was evaporated and the residue was purified by
flash chromatography (4:1 EtOAc-petrol then EtOAc) to give the
oxime acetate 27 as a clear oil (57 mg, 66%). Crystallization of a portion
gave analytically pure needles, mp 147-148 °C (EtOH). 1H NMR (400
MHz): δ 2.00, 2.01, 2.02, 2.05, 2.08, 2.13 (5s, 15 H, 5 Ac), 3.30 (ddd,
1 H, J4,5 7.8, J5,5 12.4, J5,NH 1.5, H5), 3.39 (dd, 1 H, J4′,5′ 7.8, J5′,5′ 12.0
101.95 (C1′), 154.92 (J1,N 5.6 Hz, C1). 15N NMR (50.7 MHz, D2O) δ
252.8. LSIMS HRMS (thioglycerol): calcd for [M + H]+ 296.1112,
found 296.1101.
2,3-Di-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-
xylitol (33). A mixture of the lactam (9) (264 mg, 807 µmol) and
LiAlH4 (240 mg, 6.3 mmol) in dry THF (20 mL) was heated under
reflux for 2 h. Water (10 mL) was cautiously added and the mixture
was stirred for 20 min. Benzyl chloroformate (160 µL, 1.13 mmol)
was added and the mixture was stirred overnight. The mixture was
diluted with aqueous HCl (1 M) and extracted with CHCl3. The organic
phase was washed with saturated aqueous NaHCO3 and dried. The
solvent was evaporated and the residue was purified by flash chroma-
tography (30-40% EtOAc-petrol) to give the carbamate (33) as a
clear oil (302 mg, 83%). Crystallization of a portion gave an analytically
pure microcrystalline powder; mp 81-82° (Et2O/petrol). 1H NMR (400
MHz): δ 3.15-3.93 (m, 7 H, C1,1,2,3,4,5,5), 4.45-4.83 (m, 6 H,
PhCH2OCH), 5.16, s, PhCH2OCO), 7.18-7.45 (m, 15 H, Ph). 13C NMR
(75.5 MHz): δ 42.78, 43.46, 46.74 (2 C, C1,5), 67.26 (CH2OCO),
67.69, 71.48, 73.12 (C2,3,4), 75.04, 75.24, 77.84, 78.17 (2 C, CH2-
OCH), 127.61-128.45, 136.53-137.87 (Ph), 155.99 (CO). Anal. Calcd
for C27H29NO5: C, 72.46; H, 6.53; N, 3.13. Found: C, 72.23; H, 6.66;
N, 3.24. DCI HRMS (NH3, CH4): calcd for [M + H]+ 448.2124, found
448.2120.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-2,3-di-O-benzyl-N-ben-
zyloxycarbonyl-1,5-dideoxy-1,5-imino-D-xylitol (34). BF3‚Et2O (100
µL, 0.79 mmol) was added to a solution of the trichloroacetimidate
2044 (1.02 g, 2.42 mmol) and the alcohol (33) (773 mg, 1.73 mmol) in
dry 1,2-dichloroethane (25 mL) at 0 °C. After 10 min saturated aqueous
NaHCO3 (5 mL) was added and the mixture was stirred for 5 min.
The organic phase was separated and dried and the solvent was
evaporated. The residue was purified by flash chromatography (30-
40% EtOAc-petrol) to give the disaccharide (34) as a foam (825 mg,
Hz, H5′), 3.46 (dt, 1 H, J4,5 ≈ J5,NH 4.8 Hz, H5), 3.97 (dt, 1 H, J3,4
≈
J
4,5 4.8 Hz, H4), 4.09 (dd, 1 H, J4′,5′ 4.7 Hz, H5′), 4.62 (d, 1 H, J1′,2′ 6.2
Hz, H1′), 4.80 (dd, 1 H, J2′,3′ 7.8 Hz, H2′), 4.87 (dt, 1 H, J3′,4′ ≈ J4′,5′
7.8 Hz, H4′), 5.08 (t, 1 H, H3′), 5.30 (t, 1 H, J2,3 ≈ J3,4 4.8 Hz, H3),
5.38 (d, 1 H, H2), 5.50 (br s, 1 H, NH). 13C NMR (75.5 MHz): δ
19.68, 20.62, 20.66, 20.69, 20.76 (6 C, Me), 41.38 (C5), 61.57 (C5′),
67.59, 68.17, 70.18, 70.41, 71.62, 74.53 (C2,3,4,2′,3′,4′), 99.07 (C1′),
151.67 (C1), 168.62, 168.93, 169.08, 169.24, 169.73, 169.93 (6 C, CO).
Anal. Calcd for C22H30N2O14: C, 48.35; H, 5.53; N, 5.13. Found: C,
48.37; H, 5.56; N, 5.01. DCI HRMS (NH3, CH4): calcd for [M + H]+
547.1776, found 547.1770.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-2,3-tri-O-acetyl-D-xylono-
(Z)-(15N)hydroximo-1,5-lactam (28). The thionolactam (85 mg) (26)
was treated with 15NH2OH‚HCl as for 27 above to afford the labeled
oxime acetate (28) as needles (57 mg, 62%); mp 146-147 °C (EtOH).
The 13C NMR (75.5 MHz) spectrum was identical to that recorded for
the material above. Anal. Calcd for C22H30N2O14: C, 48.27; H, 5.52;
N, 5.12. Found: C, 48.46; H, 5.47; N, 4.97. DCI HRMS (NH3, CH4):
calcd for [M + H]+ 548.1746, found 548.1750.
1
68%). H NMR (400 MHz): δ 1.90-2.20 (9 H, m, Ac), 2.59-2.80,
2.74 (m, 1 H, dd, 1 H, J 10.1, 13.2 Hz, H1,5), 3.15-5.20 (m, 16 H,
H1,2,3,4,5,1′,2′,3′,4′,5′,CH2Ph), 4.07 (dd, 1 H, J4′,5′ 5.0, J5′,5′ 11.9 Hz,
H5′), 7.22-7.43 (15 H, Ph). 13C NMR (75.5 MHz): δ 20.55 (3C,
CH3CO), 45.14, 45.82 (C1,5), 67.38 (NCOCH2), 68.49, 70.86, 71.46,
76.31, 76.83, 83.47 (C2,3,4,2′,3′,4′), 99.39 (C1′), 127.45-128.41,
136.11-138.27 (Ph), 154.78 (NCO), 169.18, 169.65, 169.79 (3C, CO).
Xylobiodeoxynojirimycin: 1,5-Dideoxy-1,5-imino-4-O-(â-D-xylo-
pyranosyl)-D-xylitol (7). A solution of the disaccharide (34) (98 mg,
139 µmol) in dry MeOH (5 mL) was treated with a small piece of
sodium metal and the solution left at room temperature for 30 min.
The solvent was evaporated and the residue was dissolved in EtOH
(10 mL) and aqueous HCl (1 mL of 0.5 M) and treated with Pd/C
(10%, 20 mg) under hydrogen overnight. The mixture was filtered and
the solvent was evaporated. The residue was purified by ion-exchange
chromatography (Dowex 1X-8, OH- form, eluted with water; BioRad
AG 50W-X2, H+ form, eluted with water then 2 M aqueous NH3) to
afford the amine (7) as a white solid (28 mg, 78%). Crystallization
afforded an analytically pure powder; mp >200° (dec; H2O/acetone).
1H NMR (400 MHz, D2O): δ 2.32-2.45 (2 H, H1,5), 2.98-3.12, 3.14-
3.65 (2m, 9 H, H1,2,3,4,5,2′,3′,4′,5′), 3.94 (dd, 1 H, J4′,5′ 5.4, J5′,5′ 11.6
Hz, H5′), 4.43 (d, 1 H, J1′,2′ 7.8 Hz, H1′). 13C NMR (75.5 MHz, D2O):
δ 47.04, 49.29 (C1,5), 65.41 (C5′), 69.44, 71.28, 73.02, 75.88, 76.54,
78.64 (C2,3,4,2′,3′,4′), 101.70 (C1′), Anal. Calcd for C10H19NO7‚1/
2H2O: C, 43.79; H, 7.35; N, 5.11. Found: C, 43.96; H, 7.46; N, 4.94.
LSIMS HRMS (thioglycerol): calcd for [M + H]+ 266.1240, found
266.1247.
D-Xylobiono-(Z)-hydroximo-1,5-lactam (6). A solution of the
oxime acetate (27) (25 mg) in MeOH (2 mL) was treated with NaOMe
(1 mL of 1 mg/mL NaOMe in MeOH) and the solution was allowed
to stand at room temperature for 2 h. The solution was treated with
cation-exchange resin (Amberlite IR-120, H+ form) until it became
neutral and the solvent was evaporated. The residue was purified by
flash chromatography (7:2:1 then 4:2:1 EtOAc-MeOH-H2O) to give
the lactam oxime (6) as a pale yellow oil (12 mg, 89%). 1H NMR (400
MHz, D2O): δ 3.18 (dd, 1 H, J4,5 7.6, J5,512.5 Hz, H5), 3.22-3.55 (m,
2 H, H2′,5′), 3.46 (t, J2′,3′ ≈ J3′,4′ 9.3 Hz, H3′), 3.51 (dd, 1 H, J4,5 4.9
Hz, H5), 3.60 (ddd, 1 H, J3′,4′ 9.3, J4′,5′ 5.5 Hz, 10.3, H4′), 3.79 (t, 1 H,
J2,3 ≈ J3,4 6.9 Hz, H3), 3.95 (dd, 1 H, J5′,5′ 11.6 Hz, H5′), 3.92-4.01
(m, 1 H, H4), 4.17 (br d, 1 H, H2), 4.47 (d, 1 H, J1′,2′ 7.8 Hz, H1′). 13
C
NMR (75.5 MHz, D2O): δ 41.91 (C5), 65.49, 69.10, 69.46, 72.99,
74.17, 75.81, 76.96 (C2,3,4,2′,3′,4′,5′), 101.98 (C1′), 154.79 (C1).
LSIMS HRMS (glycerol): calcd for [M + H]+ 295.1141, found
295.1140.
D-Xylobiono-(Z)-(15N)hydroximo-1,5-lactam (29). A solution of the
labeled oxime acetate (28) (45 mg) in dry MeOH (5 mL) was made
basic with NaOMe and the solution was left to stand at room
temperature for 2 h. The solvent was evaporated and the residue purified
by flash chromatography (7:2:1 then 4:2:1 EtOAc-MeOH-H2O) to
give the lactam oxime (29) as a pale yellow oil (20 mg, 82%). The 1H
NMR (400 MHz) spectrum was identical to that recorded for the
material above. 13C NMR (75.5 MHz, D2O): δ 41.90 (C5), 65.46 (C5′),
69.02 (J2,N 6.5 Hz, C2), 68.42, 72.96, 74.08, 75.78, 76.86 (C3,4,2′,3′,4′),
Methyl 2,3-Di-O-acetyl-D-threonate (37). A solution of methyl
hydrogen di-O-acetyl-D-tartrate (36)46,47 (18 g, 73 mmol) in dry THF
(150 mL) was treated cautiously with BH3‚Me2S (14 mL of 10 M, 140
mmol) and the solution was left at room temperature for 3 days. MeOH
(10 mL) was added dropwise over 1 h and the solvent was evaporated.
The residue was diluted with water and extracted with CH2Cl2. The
organic extract was dried and the solvent was evaporated. The residue
crystallized from Et2O/petrol to afford the alcohol (37) as colorless
needles (9.4 g, 55%); mp 75-76 °C (Et2O/petrol). 1H NMR (200 MHz,
CDCl3) δ 2.05, 2.16 (2s, 6 H, Ac), 2.48 (t, 1 H, J4,OH 6.6 Hz, OH),
3.65-3.75 (m, 2 H, H4,4), 3.72 (s, OMe), 5.28-5.36 (m, 2 H, H2,3).
13C NMR (50.1 MHz, CDCl3) δ 20.41, 20.64 (2 C, CH3CO), 52.68

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2233
(CH3O), 60.39 (C4), 70.42, 72.26 (C2,3), 168.00 (C1), 170.21, 170.27
(CH3CO). Anal. Calcd for C9H14O7: C, 46.16; H, 6.03. Found: C,
46.12; H, 6.12.
H2,3), 4.75 (br m, 1 H, NH). 13C NMR (75.5 MHz): δ 28.19 (C4),
30.04 (C5), 69.73, 73.16 (C2,3), 173.95 (C1). Anal. Calcd for C5H9-
NO3: C, 45.80; H, 6.92; N, 10.68. Found: C, 46.03; H, 7.05; N, 10.55.
DCI HRMS (NH3, CH4): calcd for m/z [M + H]+ 132.0661, found
132.0660.
Methyl 2,3-Di-O-acetyl-4-O-(p-tolylsulfonyl)-D-threonate (38). A
solution of the alcohol (38) (659 mg, 2.82 mmol) and TsCl (803 mg,
4.22 mmol) in dry pyridine (8 mL) was allowed to stand overnight
under N2. Water (1 mL) was added and the mixture was stirred for 10
min. The mixture was diluted with EtOAc, washed successively with
water, 1 M HCl, saturated NaHCO3, and brine, and dried. The solvent
was evaporated and the residue was purified by flash chromatography
(30-60% EtOAc-petrol) to afford the tosylate (38) as a clear oil (1.06
g, 97%) which crystallized. 1H NMR (200 MHz, CDCl3) δ 1.97, 2.06
(2s, 6 H, Ac), 2.41 (s, ArCH3), 3.66 (s, OMe), 4.05-4.15 (m, 2 H,
H4,4), 5.16 (d, 1 H, J2,3 3.1 Hz, H2), 5.43 (ddd, 1 H, J3,4 6.4, 6.4 Hz,
H3), 7.28-7.35, 7.69-7.76 (2m, 4 H, Ar). 13C NMR (50.1 MHz,
CDCl3) δ 20.15, 20.30 (2 C, CH3CO), 21.52 (ArCH3), 52.67 (OCH3),
65.44 (C4), 68.38, 69.68 (C2,3), 127.86, 129.91, 132.21, 145.26 (Ar),
166.95 (C1), 169.20, 169.49 (2 C, CO).
N-Benzyloxycarbonyl-1,4,5-trideoxy-1,5-imino-D-threo-pentitol (43).
A mixture of the lactam (42) (592 mg, 4.52 mmol), hexamethyldi-
silazane (2.09 mL, 9.94 mmol), and TMSCl (0.1 mL) in acetonitrile (5
mL) was heated under reflux for 2 h. The solvent was evaporated and
the residue was dissolved in dry dioxane (10 mL) and treated with
BH3‚Me2S (2.26 mL, 22.6 mmol) at reflux for 2 h. The solution was
cooled and excess aqueous HCl (1 M) was added cautiously and the
resultant solution was heated under reflux for 1 h. The bulk of the
solvent was evaporated and the residue was treated carefully with
saturated aqueous NaHCO3 (30 mL) and then benzyl chloroformate
(0.90 mL, 6.3 mmol) and the mixture was stirred vigorously overnight.
The mixture was extracted with CH2Cl2, and the combined extracts
were dried and the solvent was evaporated. The residue was purified
by flash chromatography (90-100% EtOAc-petrol then 10% MeOH-
EtOAc) to give the diol (43) as a white solid (628 mg, 55%); mp 81-
82° (EtOH/Pri2O). 1H NMR (400 MHz): δ 1.34-1.51, 1.82-1.95 (2m,
2 H, H5,5), 2.66, 2.70-2.92 (dd, 2 H, J 9.9, 13.1 Hz, m, H2,6), 3.30-
3.55 (m, 2 H, H2,6), 3.90-4.21 (m, 2 H, H3,4), 5.07 (s, CH2Ph), 7.25-
7.55 (m, 5 H, Ph). 13C NMR (75.5 MHz): δ 31.35 (C5), 42.04, 47.72
(C2,6), 67.36 (CH2Ph), 71.35, 72.87, 73.36 (C3,4), 127.70, 128.00,
128.39, 136.16 (Ph), 155.33 (CO). Anal. Calcd for C13H17NO4: C,
62.14; H, 6.82; N, 5.57. Found: C, 62.37; H, 6.91; N, 5.60. DCI HRMS
(NH3, CH4): calcd for m/z [M + H]+ 252.1236, found 252.1231.
D-xylo-Isofagomine: 1,4,5-trideoxy-1,5-imino-D-threo-pentitol (35).
A suspension of the carbamate (43) (84 mg, 335 µmol) and Pd/C (10%,
10 mg) in EtOH (5 mL) and aqueous HCl (2 mL of 0.5 M) was treated
with H2 and stirred overnight. The mixture was filtered and the solvent
evaporated. The residue was purified by ion-exchange chromatography
(Dowex 1X-8, OH- form, eluted with water; BioRad AG 50W-X2,
H+ form, eluted with water then 2 M aqueous NH3) to afford the amine
(35) as an extremely hygroscopic, white solid (32 mg, 82%). 1H NMR
(400 MHz, MeOH-d4) δ 1.37-1.48 (m, 1 H, H4), 1.92 (dddd, 1 H, J3,4
∼ J4,5 3.0, J4,5 4.1, J4,4 13.2 Hz, H4), 2.37 (dd, 1 H, J1,1 12.4, J1,2 9.1
Hz, H1), 2.54 (ddd, 1 H, J4,5 11.3, J5,5 12.9 Hz, H5), 2.94 (dddd, 1 H,
J3,5 1.1, J4,5 4.1 Hz, H5), 3.06 (ddd, 1 H, J1,2 1.3, 4.3 Hz, H1), 3.29-
3.45 (m, 2 H, H2,3). 13C NMR (75.5 MHz, MeOH-d4) δ 33.92 (C4),
44.71, 51.34 (C1,5), 73.42, 73.89 (C2,3). LSIMS HRMS (glycerol,
MeOH): calcd for m/z [M + H]+ 118.0868, found 118.0867.
Methyl 4-Cyano-4-deoxy-D-threonate (39). A solution of the
tosylate (38) (721 mg, 1.86 mmol) and potassium cyanide (603 mg,
9.29 mmol) in dry MeOH (20 mL) was heated at reflux under nitrogen
for 2 h. The solvent was evaporated and the residue was applied directly
to a column of silica gel and the product was eluted (70-100% EtOAc-
petrol) to give the nitrile (39) as a white solid (115 mg, 39%). A small
portion was recrystallized to afford flakes; mp 91-92 °C (EtOH/Pri2O/
1
petrol). H NMR (400 MHz, MeOH-d4) δ 2.65-2.79 (m, 2 H, H4,4),
3.77 (s, 3 H, Me), 4.18 (d, 1 H, J2,3 2.6 Hz, H2), 4.23 (ddd, 1 H, J3,4
6.1, 7.3 Hz, H3). 13C NMR (75.5 MHz, MeOH-d4) δ 22.60 (C4), 52.69
(CH3), 69.80, 73.85 (C2,3), 119.24 (CN), 173.81 (CO). Anal. Calcd
for C6H9NO4: C, 45.28; H, 5.70; N, 8.80. Found: C, 45.41; H, 5.75;
N, 8.66.
Methyl 2,3-Di-O-acetyl-4-cyano-4-deoxy-D-threonate (40). A solu-
tion of the tosylate (38) (5.84 g, 15.1 mmol) and potassium cyanide
(4.89 g, 75.3 mmol) in dry MeOH (100 mL) was heated under reflux
for 1 h. The solvent was evaporated and the residue was treated with
Ac2O/pyridine (1:3, 40 mL) at room temperature for 1 h. The dark
mixture was quenched with water (5 mL) and stirred at room
temperature for 5 min. The reaction mixture was diluted with EtOAc,
washed sequentially with water, 1 M HCl, saturated NaHCO3, and brine,
and then dried, and the solvent was evaporated. The residue was purified
by flash chromatography (30-50% EtOAc-petrol) to afford the
diacetate (40) as a light yellow crystalline solid (1.70 g, 47%). A small
1
portion was recrystallized to afford flakes; mp 75-76 °C (EtOH). H
1
The H NMR (400 MHz, D2O) spectrum of 35‚HCl (prepared by
NMR (200 MHz): δ 2.06, 2.17 (2s, 6 H, Ac), 2.66-2.87 (m, 2 H,
H4,4), 3.72 (s, 3 H, OMe), 5.20 (d, 1 H, J2,3 3.3 Hz, H2), 5.48 (dt, 1
H, J3,4 ≈ J3,4 6.6 Hz, H3). 13C NMR (50.1 MHz): δ 19.69, 20.32,
20.39 (3C, C4,CH3CO), 52.92 (OMe), 66.72, 71.29 (C2,3), 115.33
(CN), 166.68 (CO2CH3), 169.32, 169.66 (COCH3). Anal. Calcd for
C10H13NO6: C, 49.38; H, 5.39; N, 5.76. Found: C, 49.47; H, 5.40; N,
5.70.
addition of HCl in MeOH to 35) was identical to that reported.29
2-O-Benzoyl-N-benzyloxycarbonyl-1,4,5-trideoxy-1,5-imino-D-
threo-pentitol (45) and 3-O-Benzoyl-N-benzyloxycarbonyl-1,4,5-
trideoxy-1,5-imino-D-threo-pentitol (44). A solution of the diol (43)
(290 mg, 1.16 mmol), 1-benzoyloxybenzotriazole (290 mg, 1.21 mmol),
and Et3N (177 µmol) in dry CH2Cl2 (25 mL) was allowed to stand
overnight at room temperature under N2. The solution was diluted with
CH2Cl2, washed with saturated aqueous NaHCO3, and dried. The solvent
was evaporated and the residue was purified by flash chromatography
(20-30% EtOAc-toluene) to give, first, the 3-O-benzoate (44) (181
5-Amino-2,3-di-O-acetyl-4,5-dideoxy-D-threo-pentono-1,5-lac-
tam (41). A suspension of the nitrile (40) (473 mg, 1.95 mmol) and
PtO2 (55 mg) in MeOH (20 mL) was treated with H2 overnight. The
mixture was filtered, the solvent evaporated, and the residue purified
by flash chromatography (90-100% EtOAc-petrol then 5% MeOH-
EtOAc) to give the lactam (41) as a clear oil (268 mg, 64%). 1H NMR
(400 MHz): δ 1.81-2.23 (2 H, H4,4), 1.99, 2.06 (2s, 6 H, Ac), 3.23-
3.35 (2 H, m, H5,5), 5.09-5.19 (2 H, m, H2,3), 7.32 (br s, 1 H, NH).
13C NMR (75.5 MHz): δ 20.56, 20.81 (2 C, CH3CO), 26.77 (C4),
37.58 (C5), 69.53, 71.25 (C2,3), 167.88 (C1), 169.91, 169.95 (2 C,
CH3CO).
1
mg, 44%). H NMR (400 MHz): δ 1.65-1.77, 2.12-2.21 (2m, 2 H,
H4,4), 3.10-4.15 (m, 4 H, H1,1,2,5,5), 4.98-5.05 (m, 1 H, H3), 5.13
(s, 2 H, CH2Ph), 7.27-7.58, 8.00-8.06 (m, 10 H, Ph). 13C NMR (75.5
MHz): δ 28.05 (C4), 41.24, 47.57 (C1,5), 67.42 (CH2Ph), 68.19, 68.59,
74.32, 75.01 (2 C, C2,3), 127.90-136.36 (Ph), 155.43 (NCO), 166.35
(COPh). DCI HRMS (CH4): calcd for m/z [M + H]+ 356.1498, found
356.1499.
1
Next to elute was the 2-O-benzoate (45) (168 mg, 41%). H NMR
5-Amino-4,5-dideoxy-D-threo-pentono-1,5-lactam (42). A solution
of the lactam (41) (3.24 g, 15.1 mmol) in dry MeOH (50 mL) was
treated with a small piece of sodium metal at room temperature for 2
h. The solution was neutralized with cation-exchange resin (IR-120,
H+ form) and the solvent was evaporated. The residue was purified by
flash chromatography (90% EtOAc-MeOH then 17:2:1 then 7:2:1
EtOAc-MeOH-H2O) to give the diol (42) as a light orange crystalline
solid (1.77 g, 90%). A small portion was recrystallized to afford flakes;
(400 MHz): δ 1.55-1.80, 1.92-2.21 (2m, 2 H, H4,4), 2.50-2.82 (m,
1 H, OH), 3.50-4.20 (m, 4 H, H1,1,2,5,5), 4.85-5.20 (m, 3 H, H3,
CH2Ph), 7.20-7.60, 7.95-8.05 (2m, 5 H, Ph). 13C NMR (75.5 MHz):
δ 30.01, 30.72 (1 C, C4), 40.21, 40.53, 43.99, 44.27 (2 C, C1,5), 67.27
(CH2Ph), 68.03, 68.92, 72.15, 72.75 (2 C, C2,3), 127.75-136.38 (Ph),
155.38 (NCO), 165.96 (COPh). DCI HRMS (CH4): calcd for m/z [M
+ H]+ 356.1498, found 356.1496.
1
mp 111-112 °C (EtOH). H NMR (400 MHz): δ 1.82-1.95, 2.06-
3-O-(Tri-O-acetyl-â-D-xylopyranosyl)-2-O-benzoyl-N-benzyloxy-
carbonyl-1,5-imino-1,4,5-trideoxy-D-threo-pentitol (46). BF3‚Et2O (90
2.14 (2m, 2 H, H4,4), 3.25-3.32 (m, 2 H, H5,5), 3.86-3.95 (m, 2 H,

2234 J. Am. Chem. Soc., Vol. 122, No. 10, 2000
Williams et al.
1
µL, 0.70 mmol) was added to a suspension of powdered 4 Å molecular
sieves (2 g), the alcohol (44) (437 mg, 1.24 mmol), and the trichloro-
acetimidate 2044 (782 mg, 1.86 mmol) in 1,2-dichloroethane (15 mL)
at 0 °C under N2, and the solution was allowed to stand for 2 h. Et3N
(1 mL) was added, the mixture was filtered through Celite, and the
solvent was evaporated. The oil was dissolved in EtOAc, washed with
saturated aqueous NaHCO3 and then brine, and dried, and the solvent
was evaporated. The residue was purified by flash chromatography (20-
25% EtOAc-toluene) to give the disaccharide (46) as a clear oil (332
bulk of the material was used directly in the next step. H NMR (400
MHz): δ 1.60-1.75, 2.05-2.21 (2m, 2 H, H2,2), 1.97, 2.00 (2s, 9 H,
Ac), 2.90-4.15 (m, 7 H, H1,1,4,5,5,5′,5′), 4.60-5.25 (m, 5 H,
H3,1′,2′,3′,4′), 5.11 (s, 2 H, CH2Ph), 7.26-7.58, 7.98-8.04 (2m, 10
H, Ph). 13C NMR (75.5 MHz): δ 20.54, 20.63 (3C, CH3), 27.01, 27.46,
28.01 (1 C, C2), 40.25, 41.18, 43.44, 44.00, 47.53 (2 C, C1,5), 61.57
(C5′), 67.29, (CH2Ph), 68.33, 68.49, 70.26, 70.82, 72.40, 73.07, 74.35,
74.87 (C3,4,2′,3′,4′), 98.02, 98.91 (1 C, C1′), 127.80-136.34, (Ph),
155.14, (NCO), 165.20, (PhCO), 169.27, 169.69, 169.87 (3C, CH3CO).
DCI HRMS (CH4, NH3): calcd for m/z [M + H]+ 614.2238, found
614.2244.
N-Benzyloxycarbonyl-1,5-imino-1,2,5-trideoxy-4-O-(â-D-xylo-
pyranosyl)-D-threo-pentitol (50). The material above was treated as
for 47 to afford the carbamate (50) as a clear oil (185 mg, 45% over
two steps). 1H NMR (400 MHz, MeOH-d4) δ 1.32-1.45 (m, 1 H, H2),
1.95-2.00 (m, 1 H, H2), 3.02-4.01 (m, 10 H, H1,1,3,4,5,5,2′,3′,4′,5′),
3.81 (dd, 1 H, J4′,5′ 5.3, J5′,5′ 11.4 Hz, H5′), 4.25-4.31 (br m, 1 H,
H1′), 5.02-5.13 (m, 2 H, CH2Ph), 7.24-7.35 (m, H, Ph). 13C NMR
(75.5 MHz, MeOH-d4) δ 31.37 (C2), 42.04, 45.83 (C1,5), 67.01
(CH2Ph), 68.43 (C5′), 70.19, 71.00, 74.52, 77.62, 78.64, 79.20 (5 C,
C3,4,2′,3′,4′), 103.62, 104.32 (1 C, C1′), 128.87, 129.13, 129.56, 138.03
(Ph), 157.02, 157.46 (1 C, NCO). LSIMS HRMS (thioglycerol): calcd
for m/z [M + H]+ 384.1658, found 384.1648.
2-Deoxyxylobiodeoxynojirimycin: 1,5-Imino-1,2,5-trideoxy-4-O-
(â-D-xylopyranosyl)-D-threo-pentitol (48). The carbamate (50) (91 mg)
was treated as for 47 above to afford the amine (48) as a white
crystalline solid (47 mg, 79%). This material was recrystallized to afford
a white powder; mp >220° (dec; water/acetone). 1H NMR (400
MHz): δ 1.34-1.48, 1.92-2.02 (2m, 2 H, H2,2), 2.42 (dd, 1 H, J4,5
9.4, J5,5 12.6 Hz, H5), 2.46-2.57, 2.88-2.95 (2m, 2 H, H1,1), 3.17
(dd, 1 H, J4,5 3.9 Hz, H5), 3.23 (dd, 1 H, J1′,2′ 7.8, J2′,3′ 9.3 Hz, H2′),
3.28 (dd, 1 H, J4′,5′ 8.4, J5′,5′ 11.5 Hz, H5′), 3.52 (ddd, 1 H, J4′,5′ 5.5 Hz,
H4′), 3.55-3.66 (m, 2 H, H3,4), 3.93 (dd, 1 H, H5′), 4.44 (d, 1 H,
H1′). 13C NMR (75.5 MHz): δ 31.96 (C2), 42.62, 46.73 (C1,5), 65.39
(C5′), 69.43, 70.55, 73.03, 75.87, 79.51 (C3,4,2′,3′,4′), 101.68 (C1′).
Anal. Calcd for C10H19NO6: C, 48.19; H, 7.68; N, 5.62. Found: C,
48.11; H, 7.80; N, 5.70. LSIMS HRMS (thioglycerol): calcd for m/z
[M + H]+ 250.1291, found 250.1287.
Kinetic Analysis. (a) Inhibition of C. fimi xylanase, Cex. Cex was
purified as described previously.54 Inhibition constants were determined
at 37 °C using a 0.05 M NaH2PO4/Na2HPO4 buffer (pH 7.0) and 2,4-
dinitrophenyl â-cellobioside as a substrate. Measurements were started
by addition of Cex. Measurements of the increase of absorption at 400
nm per min in a continuous assay yielded reaction rates. This increase
was linear during all measurements (1-3 min). Michaelis parameters
(Vmax and Km) were extracted from these data by best fit to the
Michaelis-Menten equation using the program Grafit.55 Estimates of
Ki values were obtained by measuring rates in a series of cells at a
fixed substrate concentration in the presence of a range of inhibitor
concentrations (6-10 concentrations) which encompassed the Ki value
ultimately determined, generally from 0.33Ki to 3Ki. The observed rates
were plotted in the form of a Dixon plot, and the Ki value was
determined by an intersection of this line with a horizontal line drawn
through 1/Vmax. Full Ki determinations were performed for those
inhibitors whose estimated Ki was <10 µM by measurement of rates
at a series of seven substrate concentrations (generally from 0.3Ki to
3Ki) in the presence of a range of inhibitor concentrations (typically
five concentrations) which bracket the Ki value ultimately determined.
Full Ki determinations were usually within 25% of the estimated Ki
values. Ki values were calculated from these data by three-dimensional
nonlinear regression analysis using the program Grafit. The full Ki
determination for xylobiose was performed using 4-nitrophenyl â-D-
xylopyranoside as substrate, monitored at 400 nm.
1
mg, 44%). H NMR (400 MHz): δ 2.00, 2.01, 2.03 (3s, 9 H, Ac),
1.60-1.71, 2.00, 2.10 (2m, 2 H, H4,4), 3.25-4.05 (m, 5 H, H1,1,3,5,5),
3.34 (dd, 1 H, J4′,5′ 8.9, J5′,5′ 11.7 Hz, H5′), 4.08 (dd, 1 H, J4′,5′ 5.0 Hz,
H5′), 4.62 (d, 1 H, J1′,2′ 6.6 Hz, H1′), 4.79-5.17 (m, 5 H,
H2′,3′,4′,PhCH2), 7.08-7.58, 7.89-7.96 (2m, 10 H, Ph). 13C NMR
(75.5 MHz): δ 20.66, 20.70 (CH3CO), 26.35 (C4), 39.15, 43.53 (C1,5),
62.02 (C5′), 67.14 (CH2Ph), 68.54, 69.12, 70.78, 71.12, 73.12
(C2,3,2′,3′,4′), 99.58 (C1′), 127.62-136.22 (Ph), 155.65 (NCO), 165.17,
PhCO), 169.21, 169.74, 170.01 (MeCO). DCI HRMS (CH4, NH3):
calcd for m/z [M + H]+ 614.2238, found 614.2243.
N-Benzyloxycarbonyl-1,5-imino-1,4,5-trideoxy-3-O-(â-D-xylo-
pyranosyl)-D-threo-pentitol (47). A solution of the disaccharide (46)
(291 mg) in dry MeOH (15 mL) was treated with a small piece of
sodium metal, and the solution was allowed to stand overnight. The
solution was neutralized with cation-exchange resin (IR-120, H+ form)
and the solvent was evaporated. The residue was purified by flash
chromatography (EtOAc then 27:2:1 then 17:2:1 EtOAc-MeOH-H2O)
to give the carbamate (47) as a clear oil (148 mg, 81%). 1H NMR (400
MHz, MeOH-d4) δ 1.49-1.60, 1.95-2.06 (2m, 2 H, H4,4), 3.10-
3.90 (m, 11 H H1,1,2,3,5,5,2′,3′,4′,5′,5′), 4.31 (d, 1 H, J1′,2′ 7.6 Hz,
H1′), 5.09 (s, 2 H, CH2Ph), 7.26-7.35 (m, 5 H, Ph). 13C NMR (75.5
MHz, MeOH-d4) δ 28.30, 28.62 (C4), 41.84, 47.96 (C1,5), 66.95
(CH2Ph), 68.32 (C5′), 69.61, 71.07, 74.62, 77.72, 79.37 (C2,3,2′,3′,4′),
101.41 (C1′), 128.81, 129.05, 129.49, 138.08 (Ph), 157.21 (CO). LSIMS
HRMS (thioglycerol): calcd for m/z [M + H]+ 384.1658, found
384.1660.
Xylobioisofagomine: 1,5-Imino-1,4,5-trideoxy-3-O-(â-D-xylopyran-
osyl)-D-threo-pentitol (8). A suspension of the carbamate (47) (104
mg), Pd/C (10%, 10 mg), and aqueous HCl (2 mL of 0.5 M) in EtOH
(10 mL) was treated with H2 overnight. The mixture was filtered and
the solvent evaporated. The residue was applied to a column of anion-
exchange resin (BioRad AG-1 × 8, 200-400 mesh, OH- form) and
eluted with water. The eluant was evaporated, the residue was applied
to a column of cation-exchange resin (BioRad AG-50W ×2, 200-400
mesh, H+ form), and the column was washed with water and then eluted
with aqueous ammonia (2 M). The solvent was evaporated to afford
the amine (8) as a white crystalline solid (56 mg, 83%). This material
was recrystallized to afford a white powder; mp 215 °C (dec; H2O/
1
acetone). H NMR (400 MHz): δ 1.34-1.47, 2.03-2.12 (2m, 2 H,
H4,4), 2.37 (dd, 1 H, J1,1 12.5, J1,2 9.8 Hz, H1), 2.42-2.53 (m, 1 H,
H5), 2.90-3.00, 3.05-3.15 (2m, 2 H, H1,5), 3.24 (dd, 1 H, J1′,2′ 7.8,
J2′,3′ 9.3 Hz, H2′), 3.27 (dd, 1 H, J4′,5′ 4.8, J5′,5′ 11.5 Hz, H5′), 3.41 (t,
1 H, J3′,4′ 9.3 Hz, H3′), 3.51 (ddd, 1 H, J4′,5′ 5.4 Hz, H4′), 3.55-3.67
(m, 2 H, H2,3), 3.96 (dd, 1 H, H5′), 4.47 (d, 1 H, H1′). 13C NMR
(75.5 MHz): δ 29.65 (C4), 42.94, 49.38 (C1,5), 65.35 (C5′), 69.46,
70.16, 73.03, 75.95, 80.28 (C2,3,2′,3′,4′), 100.79 (C1′). Anal. Calcd
for C10H19NO6: C, 48.19; H, 7.68; N, 5.62. Found: C, 48.14; H, 7.77;
N, 5.60. LSIMS HRMS (thioglycerol): calcd for m/z [M + H]+
250.1291, found 250.1279.
4-O-(Tri-O-acetyl-â-D-xylopyranosyl)-3-O-benzoyl-N-benzyloxy-
carbonyl-1,5-imino-1,2,5-trideoxy-D-threo-pentitol (49). BF3‚Et2O (70
µL, 0.55 mmol) was added to a suspension of powdered 4 Å molecular
sieves (2 g), the alcohol (45) (382 mg, 1.08 mmol), and the trichloro-
acetimidate (20)44 (683 mg, 1.62 mmol) in 1,2-dichloroethane (15 mL)
at 0 °C under N2, and the solution was allowed to stand for 2 h. Et3N
(1 mL) was added, the mixture was filtered through Celite, and the
solvent was evaporated. The oil was dissolved in EtOAc, washed with
saturated aqueous NaHCO3 and then brine, and dried, and the solvent
was evaporated. The residue was purified by flash chromatography (20-
25% EtOAc-toluene) to give the disaccharide (49) contaminated with
trichloroacetamide, as a clear oil (474 mg). This material could be
purified by careful chromatography for characterization; however, the
(b) Inhibition of Bacillus circulans xylanase, Bcx. Bcx was purified
as described previously.56 Inhibition constants were determined at 40
°C using a 0.02 M 2-morpholinoethanesulfonic acid buffer (pH 6.0)
(54) Gilkes, N. R.; Langford, M. L.; Kilburn, D. G.; Miller, R. C.;
Warren, R. A. J. J. Biol. Chem. 1984, 259, 10455-10459.
(55) Leatherbarrow, R. J. Grafit; Erithacus Software: Staines.
(56) Sung, W. L.; Luk, C. K.; Zahab, D. M.; Wakarchuk, W. Prot. Expr.
Purif. 1993, 4, 200-206.

Inhibition by Xylobiose-DeriVed Azasugars
J. Am. Chem. Soc., Vol. 122, No. 10, 2000 2235
containing 0.05 M NaCl, using 2,5-dinitrophenyl â-xylobioside as a
substrate and following changes in absorbance at 440 nm. Kinetic
studies and data analysis were performed as described above. Full Ki
determinations were performed for inhibitors whose estimated Ki was
<1.4 mM.
Engineering Network of Centres of Excellence of Canada for
financial support. R.H. thanks Professor A. Vasella (Zurich) for
generous support. S.J.W. thanks the Killam Trust for a Post-
doctoral Fellowship. Karen Rupitz is thanked for technical
assistance.
Acknowledgment. We thank the National Sciences and
Engineering Research Council of Canada and the Protein
JA993805J