Synthesis of N4-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-L-asparagine analogues. L-2-chloro-, L-2-bromo-, and D,L-2-methylsuccinamic acid analogues

Article abstract of DOI:10.1081/CAR-100102542

L-Chlorosuccinic anhydride, L-bromosuccinic anhydride, and D,L-methylsuccinic anhydride react with 2-acetamido-2-deoxy-β-D-glucopyranosylamine to give varying mixtures of N⁴-(β-GlcNAc)-2-substituted- and N⁴-(β-GlcNAc)-3-substituted-succinamic acid isomers. The two regioisomers are separated by anion exchange chromatography. The N⁴-(β-GlcNAc)-2-substituted-succinamic acid isomers are characterized as analogues of N⁴-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-L-asparagine.

Full text of DOI:10.1081/CAR-100102542

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SYNTHESIS OF N 4-(2-ACETAMIDO-2-DEOXY-β-D-
GLUCOPYRANOSYL)-L-ASPARAGINE ANALOGUES.
L-2-CHLORO-, L-2-BROMO-, AND D,L-2-
METHYLSUCCINAMIC ACID ANALOGUES
Yuan-Qing Xia a & John M. Risley b
a Department of Chemistry, The University of North Carolina at Charlotte, Charlotte,
North Carolina, 28223, U.S.A.
b Department of Chemistry, The University of North Carolina at Charlotte, Charlotte,
North Carolina, 28223, U.S.A.
Version of record first published: 11 Dec 2006.
To cite this article: Yuan-Qing Xia & John M. Risley (2001): SYNTHESIS OF N 4-(2-ACETAMIDO-2-DEOXY-β-D-
GLUCOPYRANOSYL)-L-ASPARAGINE ANALOGUES. L-2-CHLORO-, L-2-BROMO-, AND D,L-2-METHYLSUCCINAMIC ACID
ANALOGUES, Journal of Carbohydrate Chemistry, 20:1, 45-55
To link to this article: http://dx.doi.org/10.1081/CAR-100102542
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J. CARBOHYDRATE CHEMISTRY, 20(1), 45–55 (2001)
SYNTHESIS OF N4-(2-ACETAMIDO-2-DEOXY-ꢀ-D-
GLUCOPYRANOSYL)-L-ASPARAGINE
ANALOGUES. L-2-CHLORO-, L-2-BROMO-, AND
D,L-2-METHYLSUCCINAMIC ACID ANALOGUES
Yuan-Qing Xia and John M. Risley*
Department of Chemistry, The University of North Carolina at
Charlotte, Charlotte, North Carolina 28223 U.S.A.
ABSTRACT
L-Chlorosuccinic anhydride, L-bromosuccinic anhydride, and D,L-
methylsuccinic anhydride react with 2-acetamido-2-deoxy-ꢀ-D-glucopyra-
nosylamine to give varying mixtures of N4-(ꢀ-GlcNAc)-2-substituted- and N4-
(ꢀ-GlcNAc)-3-substituted-succinamic acid isomers. The two regioisomers are
separated by anion exchange chromatography. The N4-(ꢀ-GlcNAc)-2-substi-
tuted-succinamic acid isomers are characterized as analogues of N4-(2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-asparagine.
INTRODUCTION
The synthesis of analogues of N4-(2-acetamido-2-deoxy-ꢀ-D-glucopyra-
nosyl)-L-asparagine ((GlcNAc-)Asn), has not been widely reported. Two types of
analogues containing the N-glycosyl bond are possible, analogues of the sugar and
analogues of the amino acid. Four analogues of the sugar have been reported: N4-
(ꢀ-D-glucopyranosyl)-L-asparagine ((Glc-)Asn),1-3 N4-(ꢀ-D-mannopyranosyl)-L-
asparagine ((Man-)Asn),2,3 N4-(ꢀ-D-galactopyranosyl)-L-asparagine ((Gal-)
Asn),2,3 and N4-(2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-L-asparagine
((GalNAc-)Asn).2 Nine analogues of the amino acid have been reported: N-(2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl)acetamide,4-8 N-(2-acetamido-2-deoxy-ꢀ-
D-glucopyranosyl)chloroacetamide,4,7 N-(2-acetamido-2-deoxy-ꢀ-D-glucopyra-
nosyl)bromoacetamide,4 N-(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)azidoac
45
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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XIA AND RISLEY
etamide,4 N1-(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)glycinamide,4,7 N1-(2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-N2,N2-dimethylglycinamide,4 N1-(2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-N2-benzyloxycarbonylglycinamide,4 N-
(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)propionamide,8 and N1-(2-acetamido-
2-deoxy-ꢀ-D-glucopyranosyl)-3-amino-4-hydroxybutyramide.6 (Paul et al.4 re-
ported the synthesis of five additional analogues of the amino acid where the sugar
hydroxyl groups were acetylated; however, these analogues were not O-deacety-
lated and characterized.) The amide bond between N-acetylglucosamine and as-
paragine is the principle linkage in the structure of N-linked glycoproteins,9 and the
hydrolysis of this bond by glycosylasparaginase (GA, aspartylglucosaminidase
(AGA), N4-(ꢀ-N-acetyl-D-glucosaminyl)-L-asparaginase; EC 3.5.1.26) is a key
step in the catabolism of N-linked glycoproteins.10 For a study of the active site fin-
gerprint of glycosylasparaginase, the synthesis of the N4-(ꢀ-GlcNAc)-L-2-chloro-
succinamic acid (2), L-2-bromosuccinamic acid (5), and D,L-2-methylsuccinamic
acid (8) analogues of the amino acid of (GlcNAc-)Asn were required, and are de-
scribed in this paper.
RESULTS AND DISCUSSION
The reaction of monosubstituted succinic anhydrides with amines can occur to
give regioselectively an N4-(R2)-2-(R1)-succinamic acid (I) or an N4-(R2)-3-(R1)-
succinamic acid (II), as shown in Scheme 1. While studies of this reaction have been
reported for various 2-aryl- and 2-alkylsuccinic anhydrides, the primary focus has
been on the reactions of L-aspartic anhydride (L-aminosuccinic anhydride) because
of its biochemical significance. Naps and Johns11 reported that, based on previous
work, the reaction of 2-arylsuccinic anhydrides with ammonia in anhydrous ether
resulted exclusively in the formation of 2-aryl-succinamic acids (I: R2 ꢁ H), the re-
action of 2-arylsuccinic anhydrides with methylamine in anhydrous ether resulted
exclusively in the formation of N4-methyl-2-aryl-succinamic acids (I: R2 ꢁ CH3),
and the reaction of 2-aryl- and 2-alkylsuccinic anhydrides with aniline in benzene
resulted exclusively in the formation of N4-phenyl-2-aryl- and N4-phenyl-2-alkyl-
succinamic acids (I: R2 ꢁ phenyl). The reactions of L-aspartic anhydride have been
studied for N-trifluoroacetyl-,12-14 N-benzoxycarbonyl-,3,15-18 N-9-fluorenyl-
methoxycarbonyl-,18 and N-tert-butoxycarbonyl-L-aspartic anhydride.18 The fol-
lowing three general conculsions may be drawn from these studies. Reaction of the
anhydride with amines results in a mixture of products I and II; in only one study12
were single isomers reportedly formed. There is little regioselectivity for the reac-
tion, as the mixture of products ranges from I:II ꢀ 1:1 to I:II or II:I up to 6:1; in
only one study16 was the regioselectivity reportedly significant (I:II ꢀ 25:1).
There is evidence to indicate in some reactions a regioselectivity that is solvent de-
pendent; in nonpolar aprotic solvents II is slightly favored while in polar aprotic
solvents I is slightly favored,16-18 but this is not true in all instances.3,18 (This con-
trasts with the significant solvent effect on regioselectivity observed in some reac-
tions for L-glutamic anhydride, where I:II or II:I up to 100:0 is reported for the

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47
Scheme 1.
comparable regioisomers.18) We studied the reaction of L-chloro-, L-bromo-, and
D,L-methylsuccinic anhydrides with 2-acetamido-2-deoxy-ꢀ-D-glucopyranosy-
lamine (1) to give the succinamic acid analogues of GlcNAc.
The syntheses of the sugar-amine, 1, and the monosubstituted succinic acids,
3 and 6, and anhydrides, 4 and 7, were straightforward. The properties of 1, 3, 4, 6,
and 7 were satisfactory. The NMR data for 6 agree with the data obtained in
methanol-d4.19 Following the well-established preference for the reaction of acti-
vated carboxylic acids with the amino group rather than the sugar hydroxyl groups
on carbohydrate molecules,3,20,21 when 1 and the anhydrides were mixed, only the
amide (N-glycosyl) bond was formed. The reaction of each anhydride with 1 was
1
done in D2O in order to acquire a H NMR spectrum of each reaction prior to
workup. These 1H NMR spectra are shown in Figure 1, and show that a mixture of

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XIA AND RISLEY
Figure 1. 1H NMR spectra of the (A) 2-bromosuccinamic acid analogue, (B) 2-chlorosuccinamic
acid analogue, and (C) 2-methylsuccinamic acid analogue in D2O. The signals of interest are the dou-
blets at ꢂ 5.05-5.10 for the anomeric proton on the sugar.
1
I and II (R2 ꢁ ꢀ-GlcNAc) formed in each reaction. This is evidenced by the H
NMR signal for the ꢀ-anomeric proton which characteristically appears as a dou-
blet at ꢂ 5.05-5.10 ppm with a J1,2 ꢁ 9.7 Hz.21,22 Following separation of the iso-
mers (see below) we were able to assign these NMR signals to specific isomers.
The 1H NMR signal for the ꢀ-anomeric proton in regioisomer II appears slightly
1
downfield from that in regioisomer I. The integration of these signals in the H
NMR spectra shows that isomer I was 80% (I:II ꢀ 4:1) for the L-chlorosuccinic
anhydride reaction, 65% (I:II ꢀ 2:1) for the L-bromosuccinic anhydride, and 50%
(I:II ꢀ 1:1) for the D,L-methylsuccinic anhydride. These results are consistent with
those reported previously where a mixture of I and II is formed and there is little
regioselectivity for the reaction. The 1H NMR spectra also indicate that the reac-
tions of the anhydrides with the sugar-amine was greater than 95% with little ev-
idence for unreacted sugar-amine or hydrolyzed sugar produced in the reaction,
and there is no evidence for formation of an ꢃ-anomeric N-glycosyl bond (ꢂ ꢁ 5.27

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49
with J1,2 ꢁ 1 Hz).21 The reaction of L-chlorosuccinic anhydride was complete in 10
min at room temperature, L-bromosuccinic anhydride in 20 min at room tempera-
ture, and D,L-methylsuccinic anhydride in 30 min at 40 °C.
Tamura et al.3 showed that the difference in acidities of the carboxylate
groups in isoasparagine and asparagine sugar analogues could be used to separate
the two regioisomers by ion exchange chromatography. We used this property in
the separation of isomer I from isomer II. The acidity of the carboxylate group in
I is greater than that of the carboxylate group in II due to substitution at the 2-po-
sition. Therefore I will bind more strongly to an anion exchange resin than will II.
An Amberlite® IRA-400(Cl) anion exchange column was activated with 1.0 M
sodium acetate (25 mL used for a 1 x 20 cm column) and then washed with water.
Activation of the resin with sodium hydroxide, ammonium acetate, and sodium
chloride were not as successful. The solution of succinamic acid analogue was ad-
justed to pH 4.0-4.5 and the solution was loaded onto the column. Any free sugar
was washed through the column with a water wash. Several eluent solvents were
tested: sodium hydroxide, hydrochloric acid, ammonium acetate, sodium acetate,
and sodium chloride.23 Two solvents were found to elute the isomers from the col-
umn: 0.1 M acetic acid eluted II from the column and subsequent addition of 0.5
M acetic acid eluted I from the column. Lyophilization of the sample removed all
1
of the acetic acid in the eluent. Figure 2 shows the H NMR spectrum of the 2-
chlorosuccinamic acid analogue (2) obtained in the second eluent following
lyophilization; all of the signals can clearly be identified, utilizing the nomencla-
ture used for (GlcNAc-)Asn.22
The N4-(ꢀ-GlcNAc)-2-substituted-succinamic acid isomers (I) as analogues
of ꢀ-N-acetylglucosaminyl-L-asparagine were characterized; the N4-(ꢀ-GlcNAc)-
1
Figure 2. The H NMR spectrum of 2 following separation from its isomer by anion exchange
chromatography. The NMR signals are clearly resolved and can be assigned.

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XIA AND RISLEY
3-substituted-succinamic acid isomers (II) were not further characterized. The
properties of the analogues clearly indicate the successful synthesis of each ana-
logue. Of particular importance is the 1H NMR signal of the anomeric proton that
shows a ꢀ-N-glycosyl bond is present in each analogue. In the 1H NMR spectrum
of the D,L-2-methylsuccinamic acid analogue, separate signals for the D and L enan-
tiomers were detected for the ꢃ-methyl group, acetamido group, the ꢃ, ꢀ and ꢀ’
protons, and the anomeric proton; in the 13C NMR spectrum separate signals were
detected for the ꢃ-methyl group, both carbons in the acetamido group, the ꢃ and ꢀ
carbons, the anomeric carbon, the carboxyl group, and the N-glycosyl carbon.
CONCLUSIONS
We have synthesized and characterized three new amino acid analogues of
N4-(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-asparagine. The reactions be-
tween the sugar-amine and substituted succinic anhydrides are consistent with pre-
viously reported reactions in terms of the formation of regioisomers and the gen-
erally poor regioselectivity of these reactions. Separation of the isomers are
effected by anion exchange chromatography. While our use of these analogues is
in a study of the active site fingerprint of glycosylasparaginase, these analogues
have potential for incorporation into new glycopeptides and complex glycoconju-
gates.
EXPERIMENTAL
Materials and Methods. Chemicals purchased from the following suppliers
were: N-acetyl-D-glucosamine and Amberlite® CG-120 cation exchange resin
from Janssen Chimica; ammonium bicarbonate from Mallinckrodt; L-aspartic acid
and Amberlite® IRA-400(Cl) strongly basic gel-type anion exchange resin 16-50
mesh from Spectrum Chemical; acetyl chloride and deuterium oxide (99.9 atom %
2H) from Sigma; thionyl chloride from Fisher Scientific; D,L-methylsuccinic anhy-
dride from Aldrich; acetone-d6 (99.9 atom % 2H) from Cambridge Isotopes. Acetyl
chloride and thionyl chloride were distilled in vacuo before use. All other chemi-
cals were at least analytical grade.
A GE 300 spectrometer was used to record NMR spectra. 1H NMR spectra
were recorded at 300.2 MHz in a 5 mm probe at ambient temperature with a 2000
Hz sweep width, 90° pulse angle, and an 8k data block; no line-broadening factor
was applied to the accumulated FID. Natural abundance 13C NMR spectra were
recorded at 75.5 MHz in a 5 mm probe at ambient temperature with a 10,000 Hz
sweep width, 90° pulse angle, and an 8k data block; protons were broad-band de-
coupled and a line-broadening factor of 2.0 Hz was applied to the accumulated
FID. The error in the measured chemical shifts is ꢄ 0.002 ppm for 1H NMR and
ꢄ 0.033 ppm for 13C NMR; the error in the measured coupling constants is ꢄ 0.50
Hz. Infrared spectra were recorded on a Midac Laser FTIR. Elemental analyses
were done at Atlanta Microlabs, Inc. (Norcross, Georgia).

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51
2-Acetamido-2-deoxy-ꢀ-D-glucopyranosylamine (1). The synthesis of 1 was
carried out as described with slight modification.20 N-Acetyl-D-glucosamine (0.11
moles) and ammonium bicarbonate (1.01 moles) in water (350 mL) were allowed
to equilibrate at 30 °C over five days. Glacial acetic acid (3 mL) was added, the so-
lution filtered, and concentrated on a rotary evaporator in vacuo. Excess ammo-
nium bicarbonate was removed by dissolving the product in water (50 mL) and
concentrating in vacuo for four times. A 1H NMR spectrum showed 80 % 1 and 20
% N-acetyl-D-glucosamine. The mixture was applied to an activated column of
Amberlite® CG-120 cation exchange resin (1 x 25 cm), N-acetyl-D-glucosamine
washed through the column with water, and 1 was eluted from the column with
0.05 M NaOH. 1 was recovered by lyophilization and stored at -20 °C. Properties
of 1 agree with those reported in the literature.6,7
N4-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-2-chlorosuccinamic Acid
(2)
L-2-Chlorosuccinic acid (3). The synthesis of 3 followed the reported proce-
dure.24 L-Aspartic acid (5 g; 38 mmoles) and urea (0.5 g) were dissolved in a mix-
ture of concentrated hydrochloric acid/concentrated nitric acid (16 mL; 1:1 v/v),
stirred at 65 °C for 2 h, and allowed to crystallize at 4 °C overnight. Crude 3 was
collected by suction filtration, and recrystallized by dissolving 3 in acetone (15
mL), adding benzene to turbidity, and placed at 4 °C overnight. The yield of 3
(white crystals) was 4.27 g (74.5 %). mp 176-177 °C (lit.24 176 °C) 1H NMR (ace-
tone-d6, internal reference 1,4-dioxane (3.53 ppm)): ꢂ 2.87 (dd, 1H, Jꢃ,ꢀ ꢁ 6.83 Hz,
Jꢀ,ꢀ′ ꢁ 17.09 Hz, Hꢀ (CH2)), 3.08 (dd, 1H, Jꢃ,ꢀ′ ꢁ 7.08 Hz, Jꢀ,ꢀ′ ꢁ 17.09 Hz,
Hꢀ′(CH2)), 4.62 (dd, 1H, Jꢃ,ꢀ ꢁ 6.83 Hz, Jꢃ,ꢀ′ ꢁ 7.08 Hz, Hꢃ (CHCl)). 13C NMR
(D2O, pD ꢁ 1.00, internal reference 1,4-dioxane (66.50 ppm)): ꢂ 39.14 (C3), 52.30
(C2), 172.16 (C4), 173.29 (C1).
L-Chlorosuccinic anhydride (4). The synthesis of 4 followed the reported pro-
cedure with slight modification.11 3 (2.1 g; 14 mmoles) was dissolved in a mixture
of acetyl chloride/thionyl chloride (18 mL; 2:1 v/v) and gently refluxed at 70 °C
for 3 h. Excess acetyl chloride and thionyl chloride were removed in vacuo. The
yellow oil was dissolved in chloroform/acetone (4 mL; 1:1 v/v), ligroine was added
to turbidity, and kept at 4 °C overnight to crystallize. The yield of 4 (white crys-
tals) was 1.5 g (79 %). mp 75-76 °C 1H NMR (acetone-d6, internal reference 1,4-
dioxane (3.53 ppm)): ꢂ 3.31 (dd, 1H, Jꢃ,ꢀ ꢁ 5.38 Hz, Jꢀ,ꢀ′ ꢁ 19.05 Hz, Hꢀ (CH2)),
3.75 (dd, 1H, Jꢃ,ꢀ′ ꢁ 9.28 Hz, Jꢀ,ꢀ´ ꢁ 19.05 Hz, Hꢀ′ (CH2)), 5.27 (dd, 1H, Jꢃ,ꢀ ꢁ
5.38 Hz, Jꢃ,ꢀ′ ꢁ 9.28 Hz, Hꢃ (CHCl)).
N4-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-2-chlorosuccinamic acid
(2). 1 (50 mg; 0.23 mmoles) was dissolved in D2O (1 mL), 4 (34 mg; 0.26 mmoles)
was added, and vortex-mixed for 10 min at room temperature. The homogeneous
solution was adjusted to pH 4.15 with 1.25 M NaOH, and was applied to an Am-
berlite® IRA-400(Cl) column (1 x 20 cm) activated with 1.0 M sodium acetate. The
column was washed with water (10 mL), and eluted with 0.1 M acetic acid (50 mL)
and 0.5 M acetic acid (50 mL) at 1 mL/min. 2 eluted in the second eluent and was
recovered by lyophilization. mp 168-169 °C 1H NMR (D2O, pD ꢁ 2.47, reference

ORDER
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XIA AND RISLEY
acetone (2.225 ppm)): ꢂ 1.97 (s, 3H, COCH3), 3.03 (dd, 1H, Jꢃ,ꢀ ꢁ 6.84 Hz, Jꢀ,ꢀ´
ꢁ 17.09 Hz, Hꢀ (CH2)), 3.13 (dd, 1H, Jꢃ,ꢀ′ ꢁ 6.35 Hz, Jꢀ,ꢀ′ ꢁ 17.09 Hz, Hꢀ′
(CH2)), 3.47 (dd, 1H, J3,4 ꢁ 9.04 Hz, J4,5 ꢁ 8.79 Hz, H4 (CHOH)), 3.53 (m, 1H,
J
4,5 ꢁ 8.79 Hz, J5,6a ꢁ 1.93 Hz, J5,6b ꢁ 4.88 Hz, H5 (CHO-)), 3.61 (dd, 1H, J2,3 ꢁ
10.01 Hz, J3,4 ꢁ 9.04 Hz, H3 (CHOH)), 3.74 (dd, 1H, J5,6b ꢁ 4.88 Hz, J6a,6b
ꢁ
12.45 Hz, H6b (CH2OH)), 3.85 (dd, 1H, J1,2 ꢁ 9.76 Hz, J2,3 ꢁ 10.01 Hz, H2
(CHNH)), 3.87 (dd, 1H, J5,6a ꢁ 1.93 Hz, J6a,6b ꢁ 12.45 Hz, H6a (CH2OH)), 4.68
(dd, 1H, Jꢃ,ꢀ ꢁ 6.84 Hz, Jꢃ,ꢀ′ ꢁ 6.35 Hz, Hꢃ (CHCl)), 5.07 (d, 1H, J1,2 ꢁ 9.76 Hz,
H1 (CHNH)). 13C NMR (D2O, pD ꢁ 2.47, internal reference 1,4-dioxane (66.50
ppm)): ꢂ 21.93 (COCH3), 52.40 (Cꢀ), 54.31 (CHCl), 60.42 (C2), 65.85 (C6), 69.48
(C4), 73.81 (C3), 77.75 (C5), 78.82 (C1), 171.15 (COOH), 173.09 (CONH), 174.61
(COCH3). IR (cm-1): 3315, 1736, 1665, 1556, 1416, 1368, 1301, 1220, 1168,
1085, 919, 622.
Anal. Calcd for C12H19ClN2O8: C 40.63, H 5.40, Cl 9.90, N 7.90. Found: C
40.36, H 5.47, Cl 9.89, N 7.83.
N4-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-2-bromosuccinamic Acid
Hydrate (5)
L-2-Bromosuccinic acid (6). The synthesis of 6 followed the reported proce-
dure.24 L-Aspartic acid (10 g; 75 mmoles) and urea (1.5 g) were dissolved in a mix-
ture of hydrobromic acid/concentrated nitric acid (50 mL; 1:1 v/v) in an icebath,
and diethyl ether (100 mL) was added. The reaction was completed in 20 min with
stirring. The solution was extracted three times with diethyl ether (100 mL each
time) and the aqueous phase was set at 4 °C overnight where crude 6 crystallized
(yield 4.0 g; 27 %). The crystals were dissolved in diethyl ether (10 mL) and
ligroine (8 mL), and recrystallized at 4 °C overnight. The yield of 6 (white crys-
tals) was 2.0 g (13.5 %). mp 179-180 °C (lit.19 171-172 °C, lit.24 175 °C, lit.25 178-
180 °C) 1H NMR (acetone-d6, internal reference 1,4-dioxane (3.53 ppm)): ꢂ 2.93
(dd, 1H, Jꢃ,ꢀ ꢁ 6.34 Hz, Jꢃ,ꢀ′ ꢁ 17.33 Hz, Hꢀ (CH2)), 3.17 (dd, 1H, Jꢃ,ꢀ′ ꢁ 8.79
Hz, Jꢀ,ꢀ′ ꢁ 17.33 Hz, Hꢀ′ (CH2)), 4.56 (dd, 1H, Jꢃ,ꢀ ꢁ 6.34 Hz, Jꢃ,ꢀ′ ꢁ 8.79 Hz,
Hꢃ (CHBr)). 13C NMR (acetone-d6, internal reference 1,4-dioxane (66.50 ppm)):
ꢂ 38.75 (C3), 39.08 (C2), 169.86 (C4), 170.93 (C1).
L-Bromosuccinic anhydride (7). The synthesis of 7 followed the reported pro-
cedure with slight modification.11 6 (2.1 g; 12 mmoles) was dissolved in a mixture
of acetyl chloride/thionyl chloride (24 mL; 1:1 v/v) and gently refluxed at 70 °C
for 4 h. Excess acetyl chloride and thionyl chloride were removed in vacuo. The
yellow oil was dissolved in chloroform (8 mL), ligroine was added to turbidity, and
kept at 4 °C overnight to crystallize. The yield of 7 was 1.5 g (71 %). mp 66-68 °C
1H NMR (acetone-d6, internal reference 1,4-dioxane (3.53 ppm)): ꢂ 3.35 (dd, 1H,
J
ꢃ,ꢀ ꢁ 4.40 Hz, Jꢀ,ꢀ′ ꢁ 19.54 Hz, Hꢀ (CH2)), 3.87 (dd, 1H, Jꢃ,ꢀ′ ꢁ 9.03 Hz, Jꢀ,ꢀ′
ꢁ 19.54 Hz, Hꢀ′ (CH2)), 5.24 (dd, 1H, Jꢃ,ꢀ ꢁ 4.40 Hz, Jꢃ,ꢀ′ ꢁ 9.03 Hz, Hꢃ
(CHBr)). 13C NMR (acetone-d6, internal reference 1,4-dioxane (66.50 ppm)):
ꢂ?35.00 (C3), 39.37 (C2), 168.31 (C4), 168.92 (C1).
N4-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-L-2-bromosuccinamic acid
hydrate (5). 1 (52 mg; 0.24 mmoles) was dissolved in D2O (1 mL), 7 (42 mg; 0.26

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53
mmoles) was added, and vortex-mixed for 20 min at room temperature. The ho-
mogeneous solution was adjusted to pH 4.15 with 1.25 M NaOH, and was applied
to an Amberlite® IRA-400(Cl) column (1 x 20 cm) activated with 1.0 M sodium
acetate. The column was washed with water (10 mL), and eluted with 0.1 M acetic
acid (50 mL) and 0.5 M acetic acid (50 mL) at 1 mL/min. 5 eluted in the second
eluent and was recovered by lyophilization. mp 160-161 °C 1H NMR (D2O, pD ꢁ
2.94, internal standard acetone (2.225 ppm)): ꢂ 2.00 (s, 3H, COCH3), 3.07 (dd, 1H,
J
ꢃ,ꢀ ꢁ 6.84 Hz, Jꢀ,ꢀ′ ꢁ 17.58 Hz, Hꢀ (CH2)), 3.24 (dd, 1H, Jꢃ,ꢀ′ ꢁ 8.79 Hz, Jꢀ,ꢀ′
ꢁ 17.58 Hz, Hꢀ′ (CH2)), 3.46 (dd, 1H, J3,4 ꢁ 8.79 Hz, J4,5 ꢁ 8.55 Hz, H4
(CHOH)), 3.53 (m, 1H, J4,5 ꢁ 8.55 Hz, J5,6a ꢁ 1.96 Hz, J5,6b ꢁ 4.88 Hz, H5 (CHO-
)), 3.62 (dd, 1H, J2,3 ꢁ 10.01 Hz, J3,4 ꢁ 8.79 Hz, H3 (CHOH)), 3.74 (dd, 1H, J5,6b
ꢁ 4.88 Hz, J6a,6b ꢁ 12.21 Hz, H6b (CH2OH)), 3.84 (dd, 1H, J1,2 ꢁ 9.77 Hz, J2,3 ꢁ
10.01 Hz, H2 (CHNH)), 3.89 (dd, 1H, J5,6a ꢁ 1.96 Hz, J6a,6b ꢁ 12.21 Hz, H6a
(CH2OH)), 4.63 (dd, 1H, Jꢃ,ꢀ ꢁ 6.84 Hz, Jꢃ,ꢀ′ ꢁ 8.79 Hz, Hꢃ (CHBr)), 5.09 (d,
1H, J1,2 ꢁ 9.77 Hz, H1 (CHNH)). 13C NMR (D2O, pD ꢁ 2.94, internal reference
1,4-dioxane (66.50 ppm)): ꢂ 22.16 (COCH3), 40.72 (Cꢀ), 54.34 (CHBr), 60.48
(C2), 65.50 (C6), 69.48 (C4), 74.13 (C3), 77.63 (C5), 78.14 (C1), 172.03 (COOH),
173.40 (CONH), 174.61 (COCH3). IR (cm-1): 3265, 2930, 2370, 2341, 1653, 1561,
1540.
Anal. Calcd for C12H19BrN2O80.5H2O: C 35.31, H 4.94, N 6.86. Found: C
35.58, H 5.53, N 6.67.
N4-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-D,L-2-methylsuccinamic
Acid Hydrate (8). 1 (47 mg; 0.21 mmoles) was dissolved in D2O (1 mL), D,L-2-
methylsuccinic anhydride (25 mg; 0.21 mmoles) was added, and stirred at 40 °C
for 30 min. The homogeneous solution was adjusted to pH 4.15 with 1.25 M
NaOH, and was applied to an Amberlite® IRA-400(Cl) column (1 x 20 cm) acti-
vated with 1.0 M sodium acetate. The column was washed with water (10 mL), and
eluted with 0.1 M acetic acid (50 mL) and 0.5 M acetic acid (50 mL) at 1 mL/min.
8 eluted in the second eluent and was recovered by lyophilization. mp 161-163 °C
1H NMR (D2O, pD ꢁ 2.98, reference acetone (2.225 ppm)): ꢂ 1.13 and 1.15 (d,
3H, Jꢃ,Me ꢁ 6.7 Hz, CHCH3), 2.02 and 2.03 (s, 3H, COCH3), 2.39 (dd, 1H, Jꢃ,ꢀ ꢁ
6.7 Hz, Jꢀ,ꢀ′ ꢁ 15.7 Hz, Hꢀ (CH2)), 2.45 (dd, 1H, Jꢃ,ꢀ ꢁ 6.6 Hz, Jꢀ,ꢀ′ ꢁ 15.7 Hz,
Hꢀ (CH2)), 2.60 (dd, 1H, Jꢃ,ꢀ′ ꢁ 3 Hz, Jꢀ,ꢀ′ ꢁ 15.7 Hz, Hꢀ′ (CH2)), 2.63 (dd, 1H,
Jꢃ,ꢀ′ ꢁ 2 Hz Jꢀ,ꢀ′ ꢁ 15.7 Hz, Hꢀ′ (CH2)), 2.81 (m, 1H, Jꢃ,ꢀ ꢁ 6.7 Hz, Jꢃ,ꢀ′ ꢁ 3
Hz, Jꢃ,ꢀ ꢁ 6.6 Hz, Jꢃ,ꢀ′ ꢁ 2 Hz, Jꢃ,Me ꢁ 6.7 Hz, Hꢃ (CHCH3)), 3.50 (dd, 1H, J3,4
ꢁ 9.28 Hz, J4,5 ꢁ 9.03 Hz, H4 (CHOH)), 3.55 (m, 1H, J4,5 ꢁ 9.03 Hz, J5,6a not re-
solved, J5,6b ꢁ 4.95 Hz, H5 (CHO-)), 3.64 (dd, 1H, J2,3 ꢁ 9.77 Hz, J3,4 ꢁ 9.28 Hz,
H3 (CHOH)), 3.77 (dd, 1H, J5,6b ꢁ 4.95 Hz, J6a,6b ꢁ 12.46 Hz, H6b (CH2OH)), 3.86
(dd, 1H, J5,6a not resolved, J6a,6b ꢁ 12.46 Hz, H6a (CH2OH)), 3.88 (dd, 1H, J1,2 ꢁ
9.77 Hz, J2,3 ꢁ 9.77 Hz, H2 (CHNH)), 5.07 and 5.09 (d, 1H, J1,2 ꢁ 9.77 Hz, H1
(CHNH)). 13C NMR (D2O, pD ꢁ 3.16, reference 1,4-dioxane (66.50 ppm)): ꢂ
15.92 and 15.98 (CHCH3), 21.93 and 22.00 (COCH3), 36.75 and 37.20 (Cꢀ), 38.85
and 38.72 (CHCH3), 54.24 (C2), 60.48 (C6), 69.48 (C4), 74.13 (C3), 77.56 (C5),
78.21 and 78.34 (C1), 174.45 and 174.68 (COOH), 175.58 and 175.80 (CONH),

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54
XIA AND RISLEY
179.01 and 179.50 (COCH3). IR (cm-1): 3270, 2977, 2938, 2372, 1728, 1663,
1592, 1097, 1042, 617, 432.
Anal. Calcd for C13H22N2O80.25H2O: C 46.08, H 6.69, N 8.27. Found: C
45.85, H 6.68, N 8.21.
ACKNOWLEDGMENTS
This research was supported by a Cottrell College Science Award of Re-
search Corporation. Acknowledgment is made to the Donors of the Petroleum Re-
search Fund, administered by the American Chemical Society, for partial support
of this research. This research was supported, in part, by funds provided by the
University of North Carolina at Charlotte.
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Received July 12, 2000
Accepted November 9, 2000

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