Synthesis and evaluation of potential inhibitors of chondroitin AC lyase from Flavobacterium heparinum

Article abstract of DOI:10.1021/jo020089m

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Δ4,5-unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→4)-α -L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the "leaving group" sugar moiety. Surprisingly, compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-β- D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK_a 8.8. The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid. The anion of compound 2 was found to be a competitive inhibitor with a K_i value of 5 mM, whereas the conjugate acid had a K_i value of 35 mM.

Full text of DOI:10.1021/jo020089m

J . Org. Chem. 2002, 67, 4505-4512
4505
Syn th esis a n d Eva lu a tion of P oten tia l In h ibitor s of Ch on d r oitin
AC Lya se fr om Fla voba cter iu m h epa r in u m
Carl S. Rye and Stephen G. Withers*
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver,
British Columbia, Canada, V6T 1Z1
withers@chem.ubc.ca
Received February 4, 2002
Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosamino-
glycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with ∆4,5-
unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two
potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-â-D-galactopyranosyl)-
(1f4)-R-L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety
expected at the transition state, yet retains the “leaving group” sugar moiety. Surprisingly,
compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-â-
D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a
carbon acid of pKa 8.8. The rate of reprotonation of the anion generated at this center is sufficiently
low that the anion of 2 can be used directly in initial steady-state velocity measurements without
significant interference from the conjugate carbon acid. The anion of compound 2 was found to be
a competitive inhibitor with a Ki value of 5 mM, whereas the conjugate acid had a Ki value of 35
mM.
In tr od u ction
Glycosaminoglycans (GAGs) are highly sulfated, un-
branched oligosaccharide chains built of a sequence of
repeating disaccharide units consisting of hexosamine
and uronic acid residues. These important constituents
of the extracellular matrix serve four main roles in the
body: lubrication and cushioning of joints, as barriers
to diffusion across basement membranes, as anticoagu-
lant coatings of blood vessels, and as reservoirs for
specific binding of proteins in order to regulate or
stabilize their activity.1 GAGs may be divided up into four
main classes: chondroitin sulfate and dermatan sulfate,
hyaluronic acid, heparin and heparan sulfate, and kera-
F igu r e 1. Proposed mechanism of polysaccharide lyases.
tan sulfate. With the exception of hyaluronic acid, all
GAGs are linked covalently to a protein core, forming a
proteoglycan. Of these proteoglycans, chondroitin sulfates
are the most common type of GAG chain, consisting of
three major classes: chondroitin sulfate A (chondroitin
4-sulfate), chondroitin sulfate B (dermatan sulfate), and
chondroitin sulfate C (chondroitin 6-sulfate). The chon-
droitin sulfates consist of an N-acetyl-D-galactosamine
residue (usually O-sulfated at C4 or C6) attached through
a â-(1,4) linkage to D-glucuronic acid or L-iduronic acid
(dermatan sulfate) that is in turn attached via a â-(1,3)
linkage (R-(1,3) for dermatan sulfate) to the next hex-
osamine residue.
in disaccharides or oligosaccharides with ∆4,5-unsatur-
ated uronic acid residues at their nonreducing end
(Figure 1). Although the substrate specificity and product
compositions have been characterized,1-5 little is known
about the lyase mechanism at the molecular level.
Inhibitors of these enzymes have potential as antibiotics,
since these enzymes are only known to be present in
bacterial sources, not in mammalian cells.
Chondroitin AC lyase from the Gram-negative soil
bacterium Flavobacterium heparinum (EC 4.2.2.5) cleaves
GAG chains in a random endolytic fashion. It cleaves a
variety of GAGs, including chondroitin sulfates A (chon-
droitin 4-sulfate) and C (chondroitin 6-sulfate), as well
as the unsulfated chondroitin and hyaluronic acid. The
proposed mechanism involves acid/base catalysis (Figure
Two classes of enzymes that act on GAGs are polysac-
charide hydrolases (eukaryotic) and lyases (prokaryotic).
The hydrolases cleave the anomeric carbon-oxygen bond
with either retention or inversion of configuraion. The
lyases act via an elimination mechanism, resulting
(2) Gu, K.; Linhardt, R. J .; Laliberte, M.; Zimmermann, J . Biochem.
J . 1995, 312, 569-577.
(3) J andik, K. A.; Gu, K.; Linhardt, R. J . Glycobiology 1994, 4, 289-
* To whom correspondence should be addressed. Telephone: (604)
822-3402. Fax: (604) 822-8869.
(1) Ernst, S.; Langer, R.; Cooney, C. L.; Sasisekharan, R. Crit. Rev.
Biochem. Mol. Biol. 1995, 30, 387-444.
296.
(4) Michelacci, Y. M.; Dietrich, C. P. Biochim. Biophys. Acta 1976,
451, 436-443.
(5) Baker, J . R.; Pritchard, D. G. Biochem. J . 2000, 348, 465-471.
10.1021/jo020089m CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/01/2002

4506 J . Org. Chem., Vol. 67, No. 13, 2002
Rye and Withers
Sch em e 1
F igu r e 2. Potential inhibitors 1 and 2.
1), in which the developing anion at C5 is resonance
stabilized into the carboxylate, which itself interacts with
a cationic center on the protein. The identity of the
catalytic residues remains elusive, even though the high-
resolution X-ray crystal structure of the free enzyme has
been solved6 as well as structures of various enzyme-
oligosaccharide complexes.7 The development of tight
binding inhibitors would be extremely helpful to these
structural studies, facilitating the identification of key
residues involved in binding and catalysis. At the present
time there are no known specific inhibitors of polysac-
charide lyase enzymes.
(a) CCl3COCl, NaHCO3(aq); (b) Ac2O, pyridine; (c) hydrazine
acetate, DMF; (d) CCl3CN, DBU, CH2Cl2.
ity of carbon acids and of the carbanionic carbon.13 This
interesting phenomenon allows the use of a preformed
carbanion in initial steady-state velocity measurements
without significant interference from the conjugate car-
bon acid.
Two separate concepts for inhibitor design were ex-
plored on the basis of mimics of reaction intermediates
or transition states. The formation of the proposed aci-
carboxylate intermediate yields sp2 hybridization at C5
of the uronic acid moiety. The potential inhibitor 1
(Figure 2) attempts to take advantage of this tetrahedral
sp3 to planar sp2 hybridization transformation that takes
place between substrate and intermediate and/or product.
Compound 1 cannot be cleaved by the enzyme due to the
lack of a proton at C5, satisfies the trigonal geometric
requirements of the carbanion intermediate and/or tran-
sition state, and contains the hexosamine “leaving group”
sugar moiety from which it can derive binding interac-
tions. The second approach to an inhibitor was the
synthesis of the novel 5-nitro sugar 2 (Figure 2). This
design was based on the remarkable stability of the anion
formed by the loss of a proton from the carbon acid (C5)
adjacent to a nitro group. It was hoped that the anion of
2 would mimic the anionic transition state and thus bind
tightly to the enzyme. The nitronate anion thus formed
is a mimic of the aci-carboxylate anion, due to the fact
that sp2 hybridization of C5 has already occurred as a
result of the delocalization of electrons from the carban-
ion to the oxygens of the nitro group (Figure 3). Nitro
analogues of the substrates of several non-carbohydrate
lyases8-11 have been shown to bind more tightly to these
enzymes than do the corresponding substrates, acting as
potent inhibitors. Invariably, it has been found that the
inhibitory species was the ionized form of these nitroal-
kanes (pKa values from 9 to 11). Proton transfers to and
from carbon acids are usually much slower than those
to and from oxygen, nitrogen, and sulfur.12 This results
from the need for structural reorganization accompanying
the delocalization of the negative charge, solvent reor-
ganization, and from the poor hydrogen-bonding capabil-
Resu lts a n d Discu ssion
I. Syn t h esis of Met h yl O-(2-Acet a m id o-2-d eoxy-
â-D-ga la ctop yr a n osyl)-(1f4)-r-L-th r eo-h ex-4-en op y-
r a n osid e, 1. Commercially available D-galactosamine
hydrochloride, 3, was converted first to 414 (32%), follow-
ing the literature procedure for the glucopyranoside
analogue,15 and then to the donor 5 (57%) according to
published procedures14 (Scheme 1).
The acceptor 9 was prepared in five steps as follows
(Scheme 2). Commercially available methyl â-D-gluco-
pyranoside was reacted with p-anisaldehyde dimethyl
acetal and catalytic p-toluenesulfonic acid in DMF at 50
°C under aspirator pressure,16 and the resulting crude
material was treated with pyridine and acetic anhydride
to give 6 as a white crystalline solid (80%). The ben-
zylidene protecting group was removed using a 1% (w/v)
solution of iodine in methanol at reflux17 to afford 718
(87%). Selective oxidation of the primary hydroxyl group
was accomplished using 2,2,6,6-tetramethyl-1-piperidi-
nyloxy (TEMPO) and NaOCl under phase-transfer condi-
tions with tetrabutylammonium bromide (TBAB) in
NaHCO3(aq) and EtOAc.19 Formation of the methyl ester
using acidic ion-exchange resin in methanol yielded the
acceptor 9 (25% from 7).
Glycosylation of 9 with 5 was accomplished15 using a
catalytic amount of trimethylsilyl triflate (TMSOTf) in
1,2-dichloroethane at 0 °C, affording the disaccharide 10
(6) Fethiere, J .; Eggimann, B.; Cygler, M. J . Mol. Biol. 1999, 288,
635-647.
(13) Bernasconi, C. F.; Terrier, F. J . Am. Chem. Soc. 1987, 109,
7115-7121.
(7) Huang, W.; Boju, L.; Tkalec, L.; Su, H.; Yang, H. O.; Gunay, N.
S.; Linhardt, R. J .; Kim, Y. S.; Matte, A.; Cygler, M. Biochemistry 2001,
40, 2359-2372.
(14) Bartek, J .; Muller, R.; Kosma, P. Carbohydr. Res. 1998, 308,
259-273.
(15) Blatter, G.; Beau, J . M.; J acquinet, J . C. Carbohydr. Res. 1994,
260, 189-202.
(8) Porter, D. J .; Bright, H. J . J . Biol. Chem. 1980, 255, 4772-4780.
(9) Schloss, J . V.; Porter, D. J .; Bright, H. J .; Cleland, W. W.
Biochemistry 1980, 19, 2358-2362.
(16) J ohansson, R.; Samuelsson, B. J . Chem. Soc., Perkin Trans. 1
1984, 2371-2374.
(10) Schloss, J . V.; Cleland, W. W. Biochemistry 1982, 21, 4420-
(17) Szarek, W. A.; Zamojski, A.; Tiwari, K. N.; Ison, E. R. Tetra-
hedron Lett. 1986, 27, 3827-3830.
4427.
(11) Anderson, V. E.; Weiss, P. M.; Cleland, W. W. Biochemistry
1984, 23, 2779-2786.
(12) Crooks, J . E. In Proton-Transfer Reactions; Caldin, E., Gold,
V., Eds.; Wiley: New York, 1975; p 153-177.
(18) Oldham, J . W. H.; Rutherford, J . K. J . Am. Chem. Soc. 1932,
54, 366.
(19) Davis, N. J .; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181-
1184.

Potential Inhibitors of Chondroitin AC Lyase
J . Org. Chem., Vol. 67, No. 13, 2002 4507
F igu r e 3. Illustration of how a nitro group mimics the normal carboxylate of the substrate.
Sch em e 2
side 1 (48%). Purification of 1 proved to be somewhat
problematic; successive separations by silica gel and size
exclusion chromatography were necessary to obtain a
pure product.
II. Syn th esis of P h en yl (5S)-5-Nitr o-â-D-xylop yr a -
n osid e, 2. Phenyl 2,3,4-tri-O-acetyl-â-D-xylopyranoside,
16,24 was prepared from 1,2,3,4-tetra-O-acetyl-â-D-xylo-
pyranose, 15, (Scheme 4) by first making the R-bromide
with HBr/AcOH, followed by glycosylation using phase-
transfer catalysis with phenol and tetrabutylammonium
sulfate (TBAHS) in a vigorously stirred mixture of 1 M
NaOH and CH2Cl2 (42%). Bromination25 at C5 using NBS
and light in refluxing CCl4 gave 17 as a pale yellow solid
(25%). A coupling constant of 4.0 Hz was measured for
the H4-H5 protons, indicating that the bromine was in
the axial orientation. Extended reaction times not only
resulted in bromination of one or more of the acetate
groups but also gave a p-bromophenyl derivative that was
extremely difficult to separate from the desired com-
pound. Displacement of the bromide with sodium azide
in DMF25 gave the product of inverted stereochemistry,
(a) p-anisaldehyde dimethyl acetal, p-TsOH, DMF, 50 °C; (b)
Ac2O, pyridine; (c) I2, MeOH, reflux; (d) TEMPO, NaOCl, NaBr,
TBAB, NaHCO3(aq), EtOAc, 0 °C; (e) H+ resin, MeOH.
1
18, as a crystalline solid (45%). The H NMR spectrum
as a white foam (78%) (Scheme 3). The 1H NMR spectrum
of 10 showed a doublet with a coupling constant of 8.4
Hz for the anomeric proton (H1′), confirming that the
newly formed linkage was of the â-configuration. Conver-
sion of the N-trichloroacetate to an N-acetate was effected
by treating 10 with tributyltin hydride (TBTH) and AIBN
in refluxing benzene15 to give 11 as a white crystalline
solid (78%). Since the next key step involved a radical
bromination, which is not compatible with an acetamide,
it was necessary to introduce a second acetyl group onto
the nitrogen. This second acetate group was easily
introduced onto the nitrogen center by treating 11 with
isopropenyl acetate and catalytic p-toluenesulfonic acid20
at 65 °C to afford the N,N-diacetate compound 12 (98%).
Directed by the adjacent carbonyl functionality, bromi-
nation21 at C5 of the glucopyranosiduronate moiety using
NBS and light in refluxing CCl4 proceeded smoothly to
give 13 as a colorless foam (64%). The alkene functional-
ity was then introduced by the elimination of HBr with
DBU in DMF22 to afford 14 as a white foam (75%). Global
deprotection of 14 using NaOH in 5:1 MeOH:H2O23
afforded the desired methyl O-(2-acetamido-2-deoxy-â-
D-galactopyranosyl)-(1f4)-R-L-threo-hex-4-enopyrano-
of 18 showed the expected upfield shift of H5 relative to
that in 17, and the J 5,4 coupling constant was measured
3
to be 7.6 Hz, as compared to 4.0 Hz for 17, indicating
the equatorial stereochemistry of the C5-azido group. The
transformation of the azide into the nitro functionality
was accomplished via the reduction of the azide with
hydrogen over Adam’s catalyst (PtO2) in ethanol/EtOAc,
followed by the immediate oxidation with freshly pre-
pared dimethyldioxirane (DMDO)26 in acetone to afford
the crystalline 5-nitro compound 19 (42%). The oxidation
with DMDO was performed without purification of the
intermediate amine to avoid epimerization and hydrolysis
of this newly formed glycosylamine. From the standard
1
1D H NMR data, the stereochemistry of the nitro group
at C5 and assignment of each signal was not obvious.
Selective NOE and COSY NMR experiments (CDCl3)
allowed the assignment and indicated that the sugar ring
was not in the normal 4C1 chair conformation. The X-ray
crystal structure of 19 was obtained (crystallized from
ethanol), confirming the structure of the sugar and
revealing a twist boat conformation (4S0), with C1, C2,
C3, and C5 defining a plane (Figure 4). The coupling
constants in the 1H NMR spectrum correlated nicely with
the X-ray structure, indicating that the conformation in
solution was nearly the same as that in the crystal.
(20) Demchenko, A. V.; Boons, G. J . Chem.-Eur. J . 1999, 5, 1278-
1283.
(21) Ferrier, R. J .; Furneaux, R. H. J . Chem. Soc., Perkin Trans. 1
1977, 1996-2000.
(24) Izumi, K. Agric. Biol. Chem. 1979, 43, 95-100.
(25) Ferrier, R. J .; Tyler, P. C. J . Chem. Soc., Perkin Trans. 1 1980,
(22) Blattner, R.; Ferrier, R. J .; Tyler, P. C. J . Chem. Soc., Perkin
Trans. 1 1980, 1535-1539.
2767-2773.
(26) Adam, W.; Hadjiarapoglou, L. Top. Curr. Chem. 1993, 164, 45-
(23) J acquinet, J . C. Carbohydr. Res. 1990, 199, 153-181.
62.

4508 J . Org. Chem., Vol. 67, No. 13, 2002
Rye and Withers
Sch em e 3
(a) TMSOTf, 1,2-dichloroethane, 0 °C; (b) TBTH, AIBN, benzene, reflux; (c) p-TsOH, isopropenyl acetate, 65 °C; (d) NBS, CCl4, light,
reflux; (e) DBU, DMF; (f) NaOH, MeOH/H2O.
Sch em e 4
F igu r e 4. X-ray crystal structure of 19 showing the 4S0
(a) HBr/AcOH; (b) PhOH, TBAHS, 1 M NaOH, CH2Cl2; (c) NBS,
CCl4, light, reflux; (d) NaN3, DMF; (e) PtO2, H2, EtOAc/EtOH; (f)
conformation.
DMDO, acetone; (g) AcCl, MeOH.
these nonequilibrium bond lengths with stable com-
pounds. Thus, transition state analogue designs, intended
as enzyme inhibitors, take advantage of the differences
in electronic and geometric characteristics of the ground
and transition states in order to capture a fraction of the
immense binding energy for the transition state species.
Deprotection of the acetates with HCl/MeOH provided
the desired phenyl (5S)-5-nitro-â-D-xylopyranoside, 2, as
a white solid (78%). Selective NOE and COSY NMR
experiments (methanol) facilitated the assignment of the
1H spectrum and showed that the conformation of 2 was
4
a normal C1 chair.
Despite possessing the leaving group hexosamine sugar
and having sp2 hybridization at C5 of the uronic acid
moiety, no inhibition was observed with 1 at concentra-
tions up to 25 mM. The lack of anionic character in this
compound may provide a rationale for the surprising lack
of inhibition observed. Alternatively, the preformed C4-
C5 alkene structure may be too product-like to be tightly
bound by the enzyme, or perhaps a â-1,3 linked hexos-
amine residue is required at the reducing end of the chain
to afford proper recognition and binding. This latter point
is emphasized by the fact that chondroitinases do not
III. In h ibition of Ch on d r oitin AC Lya se by 1 a n d
2. Transition state theory27 predicts that an enzyme
should bind to the transition state several orders of
magnitude more strongly than to the ground state via
protein-substrate interactions that are optimized only
at the transition state. The short-lived nature of the
transition state with partially formed and/or broken
bonds presents an insurmountable barrier to the syn-
thetic organic chemist who cannot recreate perfectly
(27) Mader, M. M.; Bartlett, P. A. Chem. Rev. 1997, 97, 1281-1301.

Potential Inhibitors of Chondroitin AC Lyase
J . Org. Chem., Vol. 67, No. 13, 2002 4509
F igu r e 7. Dixon plot showing inhibition by the anion of
compound 2. Kinetics were carried out in 200 mM Tris buffer
and 100 mM NaCl, pH 8.0, 30 °C, using a fluorescent
substrate28 at concentrations of 0.75Km and 1.5Km.
F igu r e 5. Spectrophotometric titration of 2 measuring the
pKa of the carbon acid (H5) to be 8.8. For each point, an aliquot
of a solution of 2 was equilibrated at the desired pH (buffered)
at 30 °C for 75 min. Corrections were made for buffer effects
caused by the different buffers used at each pH.
to 200 mM buffer and 100 mM NaCl at pH 8 reduced
the half-life of the anion to 7 min. These kinetic results
show that the anion of 2 is sufficiently stable at pH 8.0
to be used directly as a preformed carbanion in initial
steady-state velocity measurements (performed within a
few minutes) without significant interference from its
conjugate carbon acid.
Inhibition studies showed the anion of 2 to be a
competitive inhibitor with a Ki of 5 mM at pH 8, as shown
by the Dixon plot in Figure 7. The conjugate acid, 2, was
also found to inhibit the enzyme (at pH 6.8) but with a
Ki of approximately 35 mM. Disappointingly, the inhibi-
tion shown by the anion of 2 is not representative of a
transition state analogue, which would be expected to
bind much more tightly to the enzyme. Possibly this is a
consequence of the inhibitor not occupying other binding
sites on the enzyme. Inhibition might therefore be
increased dramatically by combining the inhibitory strat-
egy of compound 1 in which the hexosamine “leaving
group” is present, as well as more saccharide units at
the reducing end with the hope of increasing the binding
to an enzyme that normally prefers a polymeric sub-
strate.
F igu r e 6. Rates of protonation (descending curves) of the
preformed carbanion of 2 and deprotonation (ascending curves)
of 2 at pH 8.0 and 6.8. The reactions were carried out at 30 °C
in 50 mM sodium phosphate (pH 6.8) or Tris (pH 8.0) buffers
and 100 mM NaCl.
cleave heparin or heparan sulfate, which are entirely 1,4-
linked, suggesting that the 1,3-linkage may be a strong
determinant for enzymatic activity.1 However, synthetic
monosaccharide substrates have been developed that are
turned over by this enzyme;28 thus, some inhibition might
have been expected.
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise stated, all reagents
were obtained from commercial suppliers and were used
without further purification. Column chromatography was
performed with silica gel (230-400 mesh). TLC was performed
on precoated silica plates and visualized using UV light and/
or by applying a solution of 10% ammonium molybdate in 2
M H2SO4 followed by heating. CH2Cl2, CCl4, pyridine, and
benzene were distilled over CaH2. Methanol was distilled over
magnesium and iodine. DMF was dried successively over 4 Å
molecular sieves (3×). Compounds 414,15 and 514 were prepared
as described in the literature.
In h ibition Assa ys. I. In h ibition by Com p ou n d s 1 a n d
2. Assays were carried out in glass vials, total solution volume
300 µL. Mixtures containing buffer (50 mM sodium phosphate,
100 mM NaCl, pH 6.8) and the desired amounts of substrate
(benzyl 4-deoxy-4-fluoro-â-D-glucopyranosiduronic acid)28 and
inhibitor were incubated at 30 °C for at least 15 min to
thermally equilibrate them. Compound 2 was equilibrated in
buffer for at least 1.5 h prior to testing to ensure that a
consistent ionization state of the carbon acid had been reached.
Enzyme (30 µL of 1.0 × 10-4 M chondroitin AC lyase in buffer)
was added and the change in fluoride ion concentration was
monitored at 30 °C with a fluoride ion-selective electrode
interfaced with a computer. The rates were constant over a
period of at least 10 min. The pH of the solutions was
measured both at the beginning and at the end of the reaction.
The novel 5-nitro sugar 2 was synthesized with the
hope that the carbon acid would have a pKa sufficiently
low, and the anion be sufficiently stable, to be used in
inhibition studies. As a first step it was necessary to
measure the pKa of this molecule. Fortunately, this is
easily achieved, since the formation of the anion of the
carbon acid 2 can be monitored by the absorbance of this
species at 242 nm. In this way, the pKa of the carbon acid
2 (H5) was spectrophotometrically measured to be 8.8
(Figure 5), which is well within the expected range. A
second consideration in using 2 as an inhibitor is the
kinetic stability of the anion. Rates of deprotonation and
reprotonation were determined simply by monitoring
changes in absorbance at 242 nm after altering the
solution pH. Figure 6 shows such data for the deproto-
nation of 2 and the protonation of its conjugate base at
pH 6.8 and 8.0 in 50 mM buffer and 100 mM NaCl at 30
°C. At pH 6.8, kprot ) 3.7 × 10-3 s-1 (t1/2 ) 4 min) and at
pH 8.0, kprot ) 1.7 × 10-3 s-1 (t1/2 ) 15 min). The
deprotonation and reprotonation rates were found to be
dependent on the concentration of the buffer. A change
(28) Rye, C. S.; Withers, S. G. Submitted to Anal. Biochem.

4510 J . Org. Chem., Vol. 67, No. 13, 2002
Rye and Withers
II. In h ibition by th e An ion of Com p ou n d 2. Assays were
carried out in fluorescence cuvettes (1 cm path length), total
solution volume 500 µL. Mixtures containing buffer (200 mM
Tris, 100 mM NaCl, pH 8.0) and the desired amount of
substrate (phenyl 4-methylumbelliferyl-â-D-glucopyranosid-
uronic acid)28 were incubated at 30 °C for at least 15 min to
thermally equilibrate them. The anion of 2 was prepared by
adding 1 equiv of NaOH and equilibrating for at least 1.5 h.
Desired amounts of the anion of 2 and enzyme (25 µL of 3.5 ×
10-5 M chondroitin AC lyase in buffer) were then added, and
the change in fluorescence intensity was monitored at 30 °C
over 5 min (λexcitation 360 nm, λemission 450 nm) using a fluores-
cence spectrometer equipped with a temperature-controlled
cuvette holder. Rates were calculated using a standard curve
of 7-hydroxy-4-methylcoumarin (4-methylumbelliferone). The
pH of the solutions was measured both at the beginning and
at the end of the reaction.
was concentrated. The residue was dissolved in EtOAc, washed
with saturated NaHCO3 (2×) and H2O (1×), dried over MgSO4,
and concentrated. The residue was purified by column chro-
matography (PE:EtOAc 1:1), giving 9 (587 mg, 36%) as a
colorless oil which crystallized over time: 1H NMR (400 MHz,
CDCl3) δ 5.08 (1 H, ddd, J ) 9.5, 7.4, J 1.6 Hz), 4.89 (1 H, dd,
J ) 9.5, 7.7 Hz), 4.44 (1 H, d, J ) 7.7 Hz), 3.97-3.88 (2 H, m),
3.81 (3 H, s), 3.48 (3 H, s), 2.04, 2.01 (6 H, 2 s).
Meth yl [Meth yl O-(3,4,6-Tr i-O-a cetyl-2-d eoxy-2-tr ich lo-
r oacetam ido-â-D-galactopyr an osyl)-(1f4)-2,3-di-O-acetyl-
â-D-glu cop yr a n osid ]u r on a te (10). A solution of 9 (578 mg,
1.89 mmol) and 5 (1.46 g, 2.45 mmol, 1.3 equiv) in dry 1,2-
dichloroethane (30 mL) was stirred over powdered 4 Å molec-
ular sieves for 1.5 h before cooling to 0 °C and adding
trimethylsilyl trifluoromethanesulfonate (68 µL, 0.38 mmol,
0.2 equiv). After 3 h, an additional 100 mg of 5 was added,
and the reaction mixture was stirred at 0 °C for another 45
min before triethylamine (0.37 mL) was added to quench the
reaction. The reaction mixture was diluted with CH2Cl2,
filtered, and concentrated. The residue was purified by column
chromatography (PE:EtOAc 5:4 to 2:3) to give 10 (1.02 g, 73%)
as a white foam: 1H NMR (200 MHz, CDCl3) δ 6.74 (1 H, d, J
) 8.8 Hz), 5.32 (1 H, d, J 2.5 Hz), 5.22-5.10 (2 H, m), 4.93 (1
H, d, J ) 8.4 Hz), 4.88 (1 H, dd, J ) 9.6, 7.1 Hz), 4.44 (1 H, d,
J ) 7.1 Hz), 4.15-3.88 (6 H, m), 3.82 (3 H, s), 3.48 (3 H, s),
2.14, 2.05, 2.03, 2.01, 1.95 (15 H, 5 s); ESI-MS m/z 761.0 [M +
Na]+.
Meth yl [Meth yl O-(2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-
d eoxy-â-D-ga la ct op yr a n osyl)-(1f4)-2,3-d i-O-a cet yl-â-D-
glu cop yr a n osid ]u r on a te (11). A solution of 10 (1.01 g, 1.37
mmol), tributyltin hydride (1.73 mL, 6.42 mmol, 4.7 equiv),
and 2,2′-azobisisobutyronitrile (AIBN, 36 mg, 0.22 mmol, 0.17
equiv) in dry benzene (45 mL) was stirred at room temperature
for 15 min and then heated to reflux for 40 min. The reaction
mixture was then cooled and concentrated in vacuo. The
residue was dissolved in acetonitrile and washed with hexanes
(5×) and concentrated, leaving a white solid that was recrys-
tallized from EtOAc/hexanes to yield 11 (677 mg, 78%) as fine
colorless needles: mp ∼195 °C dec; 1H NMR (400 MHz, CDCl3)
δ 5.42 (1 H, d, J ) 8.6 Hz), 5.28 (1 H, d, J ) 3.4), 5.18 (1 H,
dd, J ) 11.8, 3.4 Hz), 5.17 (1 H, dd, J ) 9.2, 9.2 Hz), 4.89 (1
H, dd, J ) 7.5, 9.2 Hz), 4.69 (1 H, d, J ) 8.4 Hz), 4.43 (1 H, d,
J ) 7.5 Hz), 4.11-4.03 (3 H, m), 3.97 (1 H, d, J ) 9.6 Hz),
3.90-3.77 (2 H, m), 3.81 (3 H, s), 3.42 (3 H, s), 2.12, 2.04, 2.03,
2.02, 1.96, 1.90 (18 H, 6 s); 13C NMR (100 MHz, CDCl3) δ
180.34, 180.23, 180.10, 179.81, 179.48, 178.60, 102.04, 100.69,
76.14, 72.42, 71.39, 70.72, 70.05, 66.50, 61.17, 57.21, 53.03,
51.65, 23.26, 20.72, 20.59; ESI-MS m/z 635.8 [M]+. Anal. Calcd
for C26H37NO18: C, 49.13; H, 5.87; N, 2.20. Found: C, 48.87;
H, 5.98; N, 2.26.
Meth yl [Meth yl O-(3,4,6-Tr i-O-a cetyl-2-(N-a cetyla ceta -
m ido)-2-deoxy-â-D-galactopyr an osyl)-(1f4)-2,3-di-O-acetyl-
â-D-glu cop yr a n osid ]u r on a te (12). A suspension of 11 (595
mg, 0.94 mmol) and p-toluenesulfonic acid monohydrate (84
mg, 0.44 mmol, 0.47 equiv) in isopropenyl acetate (12 mL) was
heated to 65 °C under argon for 3.5 h (11 has dissolved after
∼1 h). Triethylamine (1.5 mL) was added to quench the
reaction, and the solvent was removed in vacuo. The residue
was purified by column chromatography (PE:EtOAc 2:3),
giving 12 (622 mg, 98%) as a white foam: 1H NMR (200 MHz,
CDCl3) δ 5.75 (1 H, dd, J ) 11.0, 3.7 Hz), 5.38 (1 H d, J ) 3.7
Hz), 5.27 (1 H, d, J ) 7.6 Hz), 5.12 (1 H, dd, J ) 9.5, 9.5 Hz),
4.94 (1 H, dd, J ) 9.5, 7.5 Hz), 4.40 (1 H, d, J ) 7.5 Hz), 4.18-
4.08 (3 H, m), 3.95 (1 H, dd, J ) 7.3, 7.3 Hz), 3.86 (1 H, d, J
) 9.8), 3.80 (3 H, s), 3.46 (3 H, s), 2.49, 2.29, 2.12 (9 H, 3s),
2.06 (6H, s), 2.04, 1.93 (6 H, 2 s); 13C NMR (75 MHz, CDCl3):
δ 184.38, 184.08, 180.34, 180.20, 179.63, 179.32, 179.10,
177.50, 102.08, 97.66, 74.77, 74.26, 72.20, 70.80, 70.50, 67.32,
67.04, 60.79, 58.93, 57.24, 53.14, 27.71, 24.93, 20.90, 20.68,
20.62, 20.46, 20.36. Anal. Calcd for C28H39NO18: C, 49.63; H,
5.80; N, 2.07. Found: C, 50.02; H, 5.81; N, 2.20.
Meth yl 2,3-Di-O-a cetyl-4,6-O-(4-m eth oxyben zylid en e)-
â-D-glu cop yr a n osid e (6). A solution of methyl â-D-glucopy-
ranoside (10.0 g, 51.5 mmol), 4-methoxybenzaldehyde dimethyl
acetal (14.0 g, 76.8 mmol, 1.5 equiv), and p-toluenesulfonic acid
monohydrate (200 mg, 1.0 mmol, 0.02 equiv) in dry DMF (50
mL) was rotated under aspirator pressure at 50 °C for 4.5 h.
The temperature was raised to 70 °C and the reaction mixture
concentrated until a solid began to form. The mixture was
poured into a stirred mixture of saturated NaHCO3(aq) (50
mL), diethyl ether (50 mL), and ice. The resulting solid was
filtered off, washed successively with petroleum ether (PE) (bp
35-60 °C), and water, and dried over P2O5 in vacuo. The solid
was dissolved in pyridine (50 mL) and acetic anhydride (45
mL) and stirred at room temperature for 1.5 h. The reaction
mixture was diluted with CH2Cl2, washed with H2O (1×), cold
1 M HCl (4×), saturated NaHCO3 (5×), H2O (1×), dried over
MgSO4, and concentrated under vacuum, leaving 6 (17.2 g,
1
80%) as a white solid: mp 190-191 °C; H NMR (200 MHz,
CDCl3) δ 7.33. (2 H, d, J ) 8.8 Hz), 6.85 (2 H, d, J ) 8.8 Hz),
5.44 (1 H, s), 5.29 (1 H, dd, J ) 9.3, 9.3 Hz), 4.96 (1 H, dd, J
) 9.3, 7.8 Hz), 4.49 (1 H, d, J ) 7.8 Hz), 4.35 (1 H, dd, J )
10.4, 4.8 Hz), 3.84-3.72 (4 H, m) 3.67 (1 H, dd, J ) 9.3, 9.3
Hz), 3.50 (3 H, s), 3.49 (1 H, ddd, J ) 9.3, 9.3, 4.8 Hz), 2.04,
2.01 (6 H, 2 s).
Meth yl 2,3-Di-O-a cetyl-â-D-glu cop yr a n osid e (7).18
A
solution of 6 in 1% (w/v) of iodine in methanol (200 mL) was
heated at reflux overnight. The solvent was removed in vacuo
and the residue dissolved in ethyl acetate (EtOAc) and washed
with saturated Na2S2O3 (1×). The aqueous phase was ex-
tracted with EtOAc (5×), and the combined organic phases
were dried over MgSO4 and concentrated. The residue was
purified by column chromatography (PE:EtOAc 1:4 to 1:5) to
yield 7 (3.40 g, 87%) as an oil which crystallized over time:
1H NMR (200 MHz, CD3OD) δ 5.04 (1 H, dd, J ) 9.5, 9.0 Hz),
4.74 (1 H, dd, J ) 9.5, 7.8 Hz), 4.47 (1 H, d, J ) 7.8 Hz), 3.88
(1 H, dd, J ) 12.0, 2.4 Hz), 3.71 (1 H, dd, J ) 12.0, 5.1 Hz),
3.56 (1 H, dd, J ) 9.0, 9.0 Hz), 3.48 (3 H, s), 3.37 (1 H, ddd, J
) 9.0, 5.1, 2.4 Hz), 2.02, 1.99 (6 H, 2 s).
Meth yl 2,3-Di-O-a cetyl-â-D-glu cop yr a n osid u r on ic Acid
(8). A solution of commercial bleach (30 mL, 6% w/v NaOCl,
23 mmol), saturated NaHCO3 (15 mL), and saturated NaCl
(30 mL) at 0 °C was added dropwise to a cooled solution of 7
(1.7 g, 6.1 mmol), sodium bromide (76 mg, 0.74 mmol, 0.12
equiv), tetrabutylammonium bromide (135 mg, 0.42 mmol,
0.068 equiv), and TEMPO (24 mg) in EtOAc (30 mL) and
saturated NaHCO3 (17 mL). The reaction was stirred for 2.5
h at 0 °C and diluted with EtOAc and water. The organic layer
was extracted with half-saturated NaHCO3 (3×). The com-
bined aqueous phases were acidified to pH 2 with 2 M HCl
and then extracted with EtOAc (5×) and CH2Cl2 (3×), dried
over MgSO4, and concentrated to give 8 (1.25 g, 70%) as a
white solid: 1H NMR (200 MHz, CDCl3) δ 5.14 (1 H, dd, J )
9.5, 9.5 Hz), 4.93 (1 H, dd, J ) 9.5, 7.6 Hz), 4.51 (1 H, d, J )
7.6 Hz), 4.06-3.85 (2 H, m), 3.50 (3 H, s), 2.07, 2.04 (6 H, 2 s).
Met h yl (Met h yl 2,3-Di-O-a cet yl-â-D-glu cop yr a n osid )-
u r on a te (9). A solution of 8 (1.48 g, 5.06 mmol) in methanol
(25 mL) was stirred with acid resin (200-400 mesh) for 3.5 h.
The resin was removed by filtration and the reaction mixture
Meth yl [Meth yl O-(3,4,6-Tr i-O-a cetyl-2-(N-a cetyla ceta -
m ido)-2-deoxy-â-D-galactopyr an osyl)-(1f4)-2,3-di-O-acetyl-
5-br om o-â-D-glu cop yr a n osid ]u r on a te (13). NBS (309 mg,
1.74 mmol, 2 equiv) was added to a solution of 12 (588 mg,

Potential Inhibitors of Chondroitin AC Lyase
J . Org. Chem., Vol. 67, No. 13, 2002 4511
0.87 mmol) in dry CCl4 (15 mL) and irradiated with two 200
W light bulbs (reflux). After 45 min, a further 140 mg of NBS
was added and the reaction mixture irradiated for another 25
min. The mixture was cooled, filtered, and concentrated and
the residue was dissolved in CH2Cl2, washed with saturated
NaHCO3 (1×) and H2O (1×), dried over MgSO4, and concen-
trated. The residue was purified by column chromatography
(PE:EtOAc 1:1), giving 13 (422 mg, 64%) as a white foam: 1H
NMR (200 MHz, CDCl3) δ 5.86 (1 H, dd, J ) 11.0, 3.9 Hz),
5.56 (1 H, d, J ) 7.9 Hz), 5.46-5.33 (2 H, m), 5.06 (1 H, dd, J
) 9.8, 8.1 Hz), 4.91 (1 H, d, J ) 8.3 Hz), 4.20-4.06 (3 H, m),
4.20-3.71 (2 H, m), 3.88 (3 H, s), 3.50 (3 H, s), 2.36, 2.33, 2.12,
2.06, 2.05, 1.92 (21 H, 6 s); ESI-MS m/z 756.2 [M]+.
7.9, 8.8 Hz), 7.03 (1 H, tt, J ) 7.9, 0.9 Hz), 6.97 (2 H, dd, J )
8.8, 0.9 Hz), 5.26-5.13 (3 H, m), 4.99 (1 H, ddd, J ) 7.9, 7.9,
5.1 Hz), 4.20 (1 H, dd, J ) 12.0, 5.1 Hz), 3.50 (1 H, dd, J )
12.0, 7.9 Hz), 2.05 (9 H, s); 13C NMR (100 MHz, CDCl3) δ
179.86, 179.73, 179.27, 156.58, 129.53, 123.04, 117.83, 98.56,
70.75, 70.19, 68.49, 61.84, 20.65. Anal. Calcd for C18H20O8: C,
57.95; H, 5.72. Found: C, 57.79; H, 5.74.
P h en yl (5S)-2,3,4-Tr i-O-a cetyl-5-br om o-â-D-xylop yr a -
n osid e (17). A mixture of 16 (4.82 g, 13.7 mmol) and NBS
(7.30 g, 41.0 mmol, 3 equiv) in dry CCl4 (250 mL) was
irradiated with one 250 W heat lamp and one 200 W light bulb
(reflux). After 2.5 h an additional 2.4 g of NBS was added,
and the reaction mixture irradiated for a further 2.5 h, cooled,
filtered, and concentrated. The residue was dissolved in CH2-
Cl2, washed with saturated NaHCO3 (1×) and H2O (1×), dried
over MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography (toluene:EtOAc 14:1),
giving 17 (1.47 g, 25%) as a pale yellow solid: 1H NMR (400
MHz, CDCl3) δ 7.30 (2 H, dd, J 8.5, J 7.6 Hz, Phmeta), 7.06 (1
H, tt, J 7.6, J 1.2 Hz, Phpara), 6.99 (2 H, dd, J 8.5, J 1.2 Hz,
Phortho), 6.61 (1 H, d, J 5,4 4.0 Hz, H5), 5.71 (1 H, dd, J 1,2 8.2,
J 1,3 0.6 Hz, H1), 5.64 (1 H, dd, J 3,4 ) J 3,2 9.7 Hz, H3), 5.33 (1
H, dd, J 2,3 9.7, J 2,1 8.2 Hz, H2), 4.91 (1 H, dd, J 4,3 9.7, J 4,5 4.0
Hz, H4), 2.10 (3 H, s, Ac), 2.05 (6 H, s, 2 Ac).
Meth yl [Meth yl O-(3,4,6-Tr i-O-a cetyl-2-(N-a cetyla ceta -
m ido)-2-deoxy-â-D-galactopyr an osyl)-(1f4)-2,3-di-O-acetyl-
r-L-th r eo-h ex-4-en op yr a n osid ]u r on a te (14). DBU (9 µL,
0.06 mmol, 1.5 equiv) was added to a solution of 13 (30 mg,
0.04 mmol) in dry DMF (0.8 mL) at 0 °C. The reaction was
then allowed to attain ambient temperature and stirred for
2.5 h. The reaction mixture was diluted with CH2Cl2, washed
with H2O (1×), 1 M HCl (2×), and H2O (1×), dried over MgSO4,
and concentrated. The residue was purified by column chro-
matography (PE:EtOAc 2:3), giving 14 (20 mg, 75%) as a white
foam: 1H NMR (400 MHz, CDCl3) δ 5.78 (1 H, dd, J ) 11.0,
3.5 Hz), 5.68 (1 H, d, J ) 7.6 Hz), 5.49 (1 H, d, J ) 1.8 Hz),
5.40 (1 H, d, J ) 3.5 Hz), 4.98 (1 H, dd, J ) 2.4, 2.4 Hz), 4.92
(1 H, d, J ) 2.4 Hz), 4.18-4.08 (2 H, m), 4.04 (1 H, dd, J )
11.3, 6.5), 3.93 (1 H, ddd, J ) 6.5, 6.5, 0.7 Hz), 3.78 (3 H, s),
3.46 (3 H, s), 2.42, 2.37, 2.13, 2.11, 2.06, 2.02, 1.94 (21 H, 7 s).
Meth yl O-(2-Acetam ido-2-deoxy-â-D-galactopyr an osyl)-
(1f4)-r-L-th r eo-h ex-4-en op yr a n osid e (1). NaOH (3 M, 3.4
mL) was added dropwise to stirred solution of 14 (250 mg, 0.37
mmol) in 5:1 MeOH:H2O (7 mL). The reaction mixture was
stirred at room temperature for 4 h, neutralized with dilute
acetic acid, and concentrated in vacuo. The residue was
purified by column chromatography (EtOAc:MeOH:H2O 10:
8:2 + 0.4% acetic acid to 5:4:2 + 0.4% acetic acid), concentrated
to remove EtOAc and MeOH, and then freeze-dried to yield
785 mg white solid (mostly salts). The solid was then dissolved
in water (2 mL) and passed down a size exclusion column
(Sephadex G-10, 1.6 × 60 cm). Fractions containing the desired
product were pooled, concentrated, and repurified by column
chromatography (EtOAc:MeOH:H2O 5:4:1 + 0.4% acetic acid)
to yield 1 (72 mg, 48%) as a solid: 1H NMR (400 MHz, D2O) δ
4.91 (1 H, d, J 3,2 ) 4.0 Hz, H3), 4.67 (1 H, d, J 1′,2′ ) 8.4 Hz,
P h en yl (5S)-2,3,4-Tr i-O-a cetyl-5-a zid o-â-D-xylop yr a n o-
sid e (18). Sodium azide (265 mg, 4.08 mmol, 2 equiv) was
added to a solution of 17 (880 mg, 2.04 mmol) in dry DMF
(6.5 mL) at 0 °C and stirred for 20 min, before the reaction
was allowed to warm to ambient temperature and stirred for
a further 1.5 h. The reaction was diluted with EtOAc and
washed with H2O (1×). The aqueous phase was extracted once
with EtOAc, and the combined organic phases were washed
with H2O (1×) and brine (3×), dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography (PE:EtOAc 3:1 to 2.75:1), giving 18 (359 mg,
45%) as a pale yellow syrup which crystallized over time: 1H
NMR (400 MHz, CDCl3) δ 7.31 (2 H, dd, J 7.6, J 8.5 Hz, Phmeta),
7.08 (1 H, tt, J 7.3, J 0.9 Hz, Phpara), 7.02 (2 H, dd, J 8.5, J 0.9
Hz, Phortho), 5.33 (1 H, dd, J 2,3 8.8, J 2,1 6.7 Hz, H2), 5.27 (1 H,
d, J 1,2 6.7 Hz, H1), 5.26 (1 H, dd, J 3,4 ) J 3,2 8.8 Hz, H3), 5.12
(1 H, dd, J 4,3 8.8, J 4,5 7.6 Hz, H4), 4.87 (1 H, d, J 5,4 7.6 Hz,
H5), 2.07, 2.06, 2.04 (9 H, 3 s, 3 Ac); 13C NMR (100 MHz,
CDCl3) δ 180.00, 179.21, 156.47, 129.70, 123.64, 118.14, 97.94,
85.46, 71.52, 70.91, 70.82, 20.57; IR (thin film) 2120 (N3), 1857
(CdO) cm-1
.
H1′), 4.09 (1 H, d, J 1,2 ) 3.6 Hz, H1), 3.98 (1 H, dd, J 2′,3′
)
P h en yl (5S)-2,3,4-Tr i-O-a cetyl-5-n itr o-â-D-xylop yr a n o-
sid e (19). A solution of 18 (428 mg, 1.09 mmol) in EtOAc (14
mL) and ethanol (35 mL) was stirred with PtO2 (Adam’s
catalyst, 175 mg) under 1 atm of H2 for 50 min. The catalyst
was removed by filtration through a bed of Celite, and the
solvent was removed in vacuo, leaving a white solid that was
dissolved in HPLC-grade acetone (30 mL) and added dropwise
to a freshly prepared solution of dimethyldioxirane26 in acetone
(250 mL, ∼0.06 M). The reaction mixture was stirred at room
temperature for 2.5 h and then concentrated under vacuum.
The residue was purified by column chromatography (PE:
EtOAc 3:1), giving 19 (182 mg, 42%) as a colorless solid. The
product was then recrystallized from EtOAc/hexanes to give
CR-439 as fine, colorless needles. Crystals were grown from
ethanol for X-ray analysis (Note: 19 decomposes on a TLC
plate to produce a spot with a larger Rf value; thus, samples
should be eluted immediately after spotting to avoid this
decomposition): 1H NMR (400 MHz, CDCl3): δ 7.30 (2 H, dd,
J 8.5, J 7.6 Hz, Phmeta), 7.08 (1 H, tt, J 7.6, J 0.9 Hz, Phpara),
7.03 (2 H, dd, J 8.5, J 0.9 Hz, Phortho), 6.04 (1 H, dd, J 4,3 9.4,
10.7, J 2′,1′ ) 8.4 Hz, H2′), 3.89 (2 H, m, H2, H4′), 3.79 (1 H, dd,
J 6a′,6b′ ) 11.8, J 6a′,5′ ) 8.2 Hz, H6a′), 3.74 (1 H, dd, J 3′,4′ ) 3.3,
J 3′,2′ ) 10.7 Hz, H3′), 3.69 (1 H, dd, J 6b′,5′ ) 4.0, J 6b′,6a′ ) 11.8
Hz, H6b′), 3.64 (1 H, dd, J 5′,6a′ ) 8.2, J 5′,6b′ ) 4.0 Hz, H5′), 3.46
(3 H, s, OMe), 2.01 (3 H, s, Ac) (assignments based on COSY
experiment); 13C NMR (100 MHz, D2O) δ 185.41, 179.60,
139.39, 132.73, 100.59, 100.21, 75.62, 71.29, 70.18, 68.08,
66.62, 61.21, 56.67, 52.68, 22.65; HRMS (LSIMS-, thioglycerol)
m/z 408.1142, calcd for C15H22NO12 [M - 1]- 408.1141; IR
(KBr) 3412, 2940, 1740, 1704, 1408, 1068 cm-1
P h en yl 2,3,4-Tr i-O-a cetyl-â-D-xylop yr a n osid e (16).24
.
A
solution of HBr (19 mL, 5.7 M in acetic acid) was added to a
solution of 1,2,3,4-tetra-O-acetyl-â-D-xylopyranose 15 (11.26
g, 35.4 mmol) in dry CH2Cl2 (30 mL) at 0 °C. The reaction
mixture was then allowed to warm to ambient temperature,
stirred for 3 h, poured into an ice/H2O mixture, and diluted
with CH2Cl2. The aqueous phase was extracted once with CH2-
Cl2, and the combined organic phases were washed with
saturated NaHCO3 (3×) and H2O (1×), dried over MgSO4, and
concentrated, yielding 2,3,4-tri-O-acetyl-â-D-xylopyranosyl bro-
mide as a white solid (11.39 g, 95%). The bromide was then
dissolved in CH2Cl2 (85 mL) to which was added phenol (6.32
g, 67.2 mmol, 2 equiv), tetrabutylammonium hydrogen sulfate
(11.4 g, 33.6 mmol, 1 equiv), and 1 M NaOH (85 mL). The
mixture was rapidly stirred for 4 h, diluted with EtOAc,
washed with 1 M NaOH (4×), H2O (1×), and brine (2×), dried
over MgSO4, and concentrated. The residue was recrystallized
from ethanol to yield 16 (5.18 g, 42% overall) as plate-like
crystals: 1H NMR (300 MHz, CDCl3): δ 7.27 (2 H, dd, J )
J
4,5 7.3 Hz, H4), 5.56 (1 H, d, J 1,2 2.1 Hz, H1), 5.51 (1 H, d, J 5,4
7.3 Hz, H5), 5.28 (1 H, dd, J 3,4 9.4, J 3,2 5,5 Hz, H3), 5.24 (1 H,
dd, J 2,3 5.5, J 2,1 2.1 Hz, H2), 2.11, 2.09, 2.08 (9 H, 3 s, 3 Ac)
(assignments based on NOE difference and COSY experi-
ments); 13C NMR (75 MHz, CDCl3) δ 179.83, 179.37, 178.62,
155.57, 129.65, 123.87, 117.49, 100.59, 99.13, 73.42, 70.30,
67.23, 20.64, 20.61, 20.44. Anal. Calcd for C18H19NO10
: C,
51.39; H, 4.82; N, 3.53. Found: C, 51.51; H, 4.91; N, 3.57.
P h en yl (5S)-5-Nitr o-â-D-xylop yr a n osid e (2). To a solu-
tion of 19 (234 mg, 0.59 mmol) in dry MeOH (10 mL) at 0 °C

4512 J . Org. Chem., Vol. 67, No. 13, 2002
Rye and Withers
was added acetyl chloride (1.0 mL). The reaction mixture was
then stirred at 4 °C for 23 h and concentrated in vacuo and
the residue purified by column chromatography (PE:EtOAc 1:2
to 1:3) to yield 2 (124 mg, 78%) as a white foam: 1H NMR
(400 MHz, CD3OD): δ 7.33 (2 H, dd, J ) 7.3, 8.8 Hz, Phmeta),
7.12 (2 H, dd, J ) 8.8, 1.2 Hz, Phortho), 7.08 (1 H, tt, J ) 7.3,
1.2 Hz, Phpara), 5.58 (1 H, d, J 5,4 ) 8.8 Hz, H5), 5.30 (1 H, d,
J 1.2 ) 6.7 Hz, H1), 3.93 (1 H, dd, J 4,3 ) J 4,5 ) 8.8 Hz, H4), 3.68
(1 H, dd, J 2,3 ) 8.8, J 2,1 ) 6.7 Hz, H2), 3.62 (1 H, dd, J 3,2 ) J 3,4
) 8.8 Hz, H3) (assignments based on NOE difference and
COSY experiments); 13C NMR (75 MHz, CD3OD): δ 158.39,
130.53, 124.10, 118.88, 105.65, 101.71, 75.23, 74.62, 73.27; ESI-
MS m/z 294.2 [M + Na]+. Anal. Calcd for C11H13NO7: C, 48.71;
H, 4.83; N, 5.17. Found: C, 48.48; H, 4.90; N, 5.05.
Ack n ow led gm en t. We thank the Canadian Insti-
tutes of Health Research, the Natural Sciences and
Engineering Research Council (NSERC) of Canada, Dr.
Paul Trussell, and the British Columbia Science Council
for financial support, and IBEX Technologies Inc. and
Dr. Mirek Cygler for their generous donation of chon-
droitin AC lyase.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 1, 6-10, 13-14, 17, and 18 and X-ray crystal
structure data for compound 19. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O020089M