Synthesis of N4-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-L-asparagine analogues: Succinamide, L-2-hydroxysuccinamide, and L-2-hydroxysuccinamic acid hydrazide analogues

Article abstract of DOI:10.1016/S0008-6215(00)00224-X

The syntheses of three analogues of N⁴-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-L-asparagine are described. N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-

Full text of DOI:10.1016/S0008-6215(00)00224-X

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Carbohydrate Research 329 (2000) 487–493
Synthesis of
N4-(2-acetamido-2-deoxy-b-
D-glucopyranosyl)-
L
-asparagine analogues: Succinamide,
L
-2-hydroxysuccinamide, and -2-hydroxysuccinamic acid
L
hydrazide analogues
De Hua Huang, John M. Risley *
Department of Chemistry, Uni6ersity of North Carolina at Charlotte, 9201 Uni6ersity City Bl6d., Charlotte,
NC 28223, USA
Received 20 June 2000; accepted 21 July 2000
Abstract
The syntheses of three analogues of N4-(2-acetamido-2-deoxy-b-
D
-glucopyranosyl)-L-asparagine are described.
N-(2-Acetamido-2-deoxy-b- -glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl
succinamate with 2-acetamido-2-deoxy-b- -glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b- -glu-
copyranosylamine was synthesized, and the complete assignment of the H NMR spectrum is given. Reaction of the
protected b- -glycosylamine with -malic acid chloralid in the presence of a coupling agent (EEDQ) gave
N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
-glucopyranosyl)- -malamic acid chloralid that was deprotected two
-glucopyranosyl)- -2-hydroxysuccinamide, and
-glucopyranosyl)- -2-hydroxysuccinamic acid hydra-
D
D
D
1
D
L
D
L
ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-b-
D
L
(2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-b-
D
L
zide. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: b-N-Acetylglucosaminyl-
L
-asparagine analogues; N4-(2-Acetamido-2-deoxy-b-
D-glucopyranosyl)succinamides; Penta-
fluorophenyl succinamate; L-Malic acid chloralid; Succinamic acid hydrazide
N4-(b-
Asn) [1–3], N4-(b-
paragine ((Man-)Asn) [2,3], N4-(b-
topyranosyl)- -asparagine ((Gal-)Asn) [2,3],
and N4-(2-acetamido-2-deoxy-b-
-galactopy-
ranosyl)- -asparagine ((GalNAc-)Asn) [2].
Nine analogues of the amino acid have
been reported: N-(2-acetamido-2-deoxy-b-
glucopyranosyl)acetamide [4–8], N-(2-acetam-
ido-2-deoxy-b- -glucopyranosyl)chloroaceta-
mide [4,7], N-(2-acetam-ido-2-deoxy-b- -glu-
copyranosyl)bromoacetamide [4], N-(2-acet-
D
-glucopyranosyl)-
-mannopyranosyl)-
-galac-
L
-asparagine ((Glc-)-
1. Introduction
D
L
-as-
The synthesis of analogues of N4-(2-acetam-
ido-2-deoxy-b- -glucopyranosyl)- -asparagine
D
D
L
L
((GlcNAc-)Asn) has not been widely reported.
Two types of analogues containing the N-gly-
cosylic bond are possible: analogues of the
sugar and analogues of the amino acid. Four
analogues of the sugar have been reported:
D
L
D
-
D
* Corresponding author. Tel.: +1-704-6874844; fax: +1-
704-6873151.
E-mail address: jmrisley@email.uncc.edu (J.M. Risley).
D
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00224-X

488
D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
amido - 2 - deoxy - b -
D
- glucopyranosyl)azido-
acetamide [4], N1-(2-acetamido-2-deoxy-b-
-
D
glucopyranosyl)glycinamide [4,7], N1-(2-acet-
amido-2-deoxy-b-
-glucopyranosyl)-N2,N2-
dimethylglycinamide [4], N1-(2-acetamido-2-
deoxy-b-
-glucopyranosyl)-N2-benzyloxycar-
bonylglycinamide [4], N-(2-acetamido-2-de-
oxy-b-
-glucopyranosyl)propionamide [8], N1-
D
D
D
(2-acetamido-2-deoxy-b- -glucopyranosyl)-3-
D
2. Results and discussion
amino-4-hydroxybutyramide [6], and three
that we recently reported [9], N4-(2-acetam-
The synthesis of the succinamide analogue 1
of GlcNAc-Asn (Scheme 1) required activa-
tion of the carboxylic acid group of succin-
amic acid. While there are many methods to
activate a carboxylic acid, we chose the pen-
tafluorophenyl ester [12], which was straight-
forward and gave crystals of 5 of high purity.
The reaction of 5 with the b amino group of 4
in N,N-dimethylformamide (DMF) occurred
exclusively at the amino group to form the
N-glycosylic bond, which followed the well-es-
tablished preference of activated carboxylic
acids to react with the amino group to form
the N-glycosylic (amide) bond rather than the
sugar hydroxyl groups on carbohydrate
molecules [3,9,12,13]. Thus, although 4 used in
the reaction contained approximately 24%
GlcNAc, reaction of 5 did not occur with a
sugar hydroxyl group to form a carboxylate
ester between GlcNAc and succinamic acid.
We found that the use of aqueous DMF re-
duced the yield of 1 by approximately 75%. In
order to purify 1, pentafluorophenol released
during the reaction was first removed on an
anion-exchange column. We thought that it
might be possible to oxidize the anomeric
carbon of unreacted GlcNAc under very mild
conditions using Tollen’s reagent [14] for re-
moval of the aldonic acid by anion-exchange
chromatography; this was only partially suc-
cessful (data not shown). Reversed-phase
HPLC provided a direct route to the purifica-
tion of 1. The properties of 1 indicated the
successful synthesis of the analogue. The
nomenclature used to assign the NMR signals
follows that used for GlcNAc-Asn [15]. The
NMR spectra agreed with data reported for
GlcNAc-Asn [15] and other analogues [9]. The
characteristic doublet for the anomeric proton
ido-2-deoxy-b-
succinamic acid, N4-(2-acetamido-2-deoxy-b-
-glucopyranosyl)- -2-bromosuccinamic acid,
and N4-(2-acetamido-2-deoxy-b-
-glucopyra-
nosyl)- -2-methylsuccinamic acid. (Paul et
D
-glucopyranosyl)-
L
-2-chloro-
D
L
D
D,L
al. [4] reported the synthesis of five additional
analogues of the amino acid where the sugar
hydroxyl groups were acetylated; however,
these analogues were not O-deacetylated and
characterized.) The amide bond between N-
acetylglucosamine and asparagine is the princi-
pal linkage in the structure of N-linked
glycoproteins [10], and the hydrolysis of this
bond by glycosylasparaginase (GA, aspartyl-
glucosaminidase (AGA), N4-(b-N-acetyl-
-
D
glucosaminyl)- -asparaginase; EC 3.5.1.26) is
L
a key step in the catabolism of N-linked glyco-
proteins [11]. The synthesis of the GlcNAc(b1-
N)-succinamide 1, -2-hydroxysuccinamide 2,
L
and
L
-2-hydroxysuccinamic acid hydrazide
((3S) - 3 - hydrazinocarbonyl - 3 - hydroxypro-
pionamide) 3 analogues of the amino acid of
GlcNAc-Asn are described in this paper. In
1
addition, the complete assignment of the H
NMR spectrum of 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b- -glucopyranosylamine is
D
given.
1
in the H NMR spectrum appears at l 5.068
Scheme 1.
with J1,2 9.77 Hz [13,15].

D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
489
amido-2-deoxy-3,4,6-tri-O-acetyl-b-
D
-gluco-
pyranosyl)acetate [20], 2-C-(2-acetamido-3,4,
6-tri-O-acetyl-2-deoxy-b-
D
-glucopyranosyl)-
nitroethene [21], and 4-C-(2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-b- -glucopyranosyl)-1-
D
methoxybenzene [22]. Upon hydrogenation of
the intermediate glycosyl azide to give 6, sig-
1
nificant changes occur in the H NMR spec-
trum for H-1, H-2, H-3, H-4 and H-5.
Specifically, H-3 and H-4 shift upfield, but the
shift of H-3 is much larger than the shift of
H-4 such that the positions of the protons in
the spectrum have changed, and importantly
the coupling changes from a first-order spec-
trum in the glycosyl azide to an ABMX (or
MABX) second-order spectrum in 6; the high
symmetry arises because J2,3 and J4,5 are
nearly identical. The assignments of the pro-
tons in 6 were verified in decoupling experi-
ments. In particular, decoupling of H-2 or H-5
in 6 breaks the symmetry of the second-order
spectrum for H-3 and H-4, respectively, in an
identical, but opposite, manner to give half of
a typical AB spectrum, as expected. To
confirm the second-order spectrum for H-3
and H-4, a theoretical computation was done
that was identical to the observed spectrum
(data not shown). Our assignments differ from
the broad, general chemical shifts reported by
Paul and Korytnyk [18] only for H-1 and H-4,
which are reversed. The properties of 6 were
satisfactory.
Scheme 2.
The
L
-2-hydroxysuccinamide analogue 2
and the -2-hydroxysuccinamic acid hydrazide
analogue 3 of GlcNAc-Asn were synthesized
(Scheme 2) from a common intermediate, the
L
protected sugar
The protected b-
L
-malamic acid chloralid 8.
-glycosylamine 6 is an im-
D
portant intermediate in the synthesis of N-gly-
cosylic carbohydrates; the procedure most
often cited to synthesize 6 is that of Bolton
et al. [16,17]. The protected sugar 6 was syn-
thesized in three steps from GlcNAc using
well-established procedures: acetylation–
chlorination with acetyl chloride to give the
glycosyl chloride, azide substitution to give
the glycosyl azide and hydrogenation to give
the glycosyl amine 6. Bolton et al. reported
1
that the 60 MHz H NMR spectrum of the
intermediate glycosyl azide in CDCl3 confir-
med the b anomeric configuration because the
H-1 proton was a doublet with J1,2 9 Hz, but
no additional NMR data were given. In the
characterization of the glycosyl amine 6, no
1H NMR data were reported by Bolton et al.
Paul and Korytnyk [18] reported limited, se-
lected H NMR data for both the glycosyl
azide and 6 in CDCl3 at 100 MHz. The com-
plete H NMR assignments for the glycosyl
azide were reported by Tropper et al. [19] in
1992, but we were not able to find a report of
the complete H NMR assignments for 6 de-
spite the many citations to its synthesis. All of
the signals for the glycosyl azide are first
order, and our results agree with Tropper et
al. [19]. The data also generally agree with
data reported for similar compounds, such as
the C-glycosyl compounds ethyl 2-C-(2-acet-
The a-carboxyl and a-hydroxyl groups of
L
-malic acid were protected by reaction with
chloral hydrate to give -malic acid chloralid
L
7. The chloralid is sufficiently unreactive that
the b-carboxyl group may be activated; we
activated the b-carboxyl group as the pen-
tafluorophenyl ester and found that reaction
with the b amino group of 4 occurred exclu-
sively at the b-carboxyl group, and not with
the chloralid (data not shown). However, it
was more convenient to couple 7 directly with
6 using the coupling agent, EEDQ, without
the intermediate synthesis of an activated es-
ter. Again, we found that the chloralid was
unreactive toward the b amino group of 6
under these conditions as the protected sugar
1
1
1
L
-malamic acid chloralid 8 was synthesized in
high purity. Formation of the amide (N-glyco-
sylic) bond at the anomeric carbon results in a

490
D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
1
shift of the H-3 and H-4 protons in the H
tafluorophenol,
2-ethoxy-1-ethoxycarbonyl-
NMR spectrum to give a first-order spectrum.
1,2-dihydroquinoline and hydrazine monohy-
drate from Aldrich Chemical Co.; D2O (99.9
The properties of the protected sugar
L
-
2
2
malamic acid chloralid 8 were satisfactory.
The -2-hydroxysuccinamide analogue 2 was
atom% H), Me2SO-d6 (99.9 atom% H), and
2
L
CHCl3-d (99.8 atom% H) from Cambridge
synthesized by deprotecting 8 in ethanol in the
presence of ammonia from concentrated am-
monium hydroxide. Analogue 2 was insoluble
in ethanol and precipitated from solution dur-
ing the 4 days of the deprotecting reaction.
Compound 2 was purified by HPLC, and the
eluent with a retention time of approximately
16 min was lyophilized. The properties of 2
were satisfactory and indicate successful syn-
thesis of the analogue.
Isotopes Laboratories; chloral hydrate from
Fischer Scientific Co.; N,N%-dicyclohexylcar-
bodiimide,
Amberlite®
IRA-400
(Cl−)
strongly basic gel-type anion-exchange resin,
acetyl chloride, -malic acid from Sigma
Chemical Co.; ammonium bicarbonate from
Spectrum. All other chemicals were at least
analytical grade.
General methods.—A GE 300 spectrometer
was used to record NMR spectra. H NMR
L
1
The
L
-2-hydroxysuccinamic acid hydrazide
spectra were recorded at 300.2 MHz in a
5-mm probe at ambient temperature with a
2000 Hz sweep width, 30° pulse angle, and an
8K data block; no line-broadening factor was
applied to the accumulated FID. Natural
analogue 3 was synthesized by deprotecting 8
in ethanol in the presence of hydrazine by
addition of hydrazine monohydrate. Analogue
3 was insoluble in ethanol and precipitated
from solution during the 2 days of the depro-
tecting reaction. Compound 3 was purified by
HPLC, and the eluent with a retention time of
approximately 18 min was lyophilized. The
properties of 3 were satisfactory and indicate
successful synthesis of the compound. Depro-
tection of 8 with sodium methoxide gave a
mixture of products that included the methyl
ester analogue, and dehydration to give the
fumaramic and maleamic acid analogues (data
not shown)1.
13
abundance C NMR spectra were recorded at
75.5 MHz in a 5-mm probe at ambient tem-
perature with a 10,000 Hz sweep width, 30°
pulse angle, and an 8K data block; protons
were broad-band decoupled and a line-broad-
ening factor of 2.0 Hz was applied to the
accumulated FID. The error in the measured
1
chemical shifts is 90.002 ppm for H NMR
and 90.033 ppm for 13C NMR; the error in
the measured coupling constants is 90.50 Hz.
Reversed-phase HPLC was done on a Perkin–
Elmer Series
2
Liquid Chromatograph
equipped with an LC-75 spectrophotometric
UV detector and an ODS-3 preparative
column. Water was used as the mobile phase
at a flow rate of 1.0 mL/min, and the column
was monitored at 220 or 240 nm. Infrared
spectra were recorded on a Bio-Rad FTS
175C FTIR. Evaporation of solvents was con-
ducted on a rotary evaporator at ꢀ60–70 °C
at water aspirator vacuum. Elemental analyses
were done at Atlantic Microlabs, Inc. (Nor-
cross, GA).
3. Experimental
Materials.—Chemicals purchased from the
following suppliers were: 2-acetamido-2-de-
oxy- -glucopyranose, succinamic acid, pen-
D
1 W.M. York, J.M. Risley, unpublished data. The fu-
maramic acid analogue and the maleamic acid analogue were
synthesized independently by reaction of 4 with maleic anhy-
dride and allowed to equilibrate overnight. The two ana-
logues were separated by chromatofocusing on an Amberlite
IRA-400 (Cl−) anion-exchange column: the fumaramic acid
analogue eluted at pH 3.9 and the maleamic acid analogue
Preparation of 2-acetamido-2-deoxy-i-
glucopyranosylamine [6,7,9,12] (4).—2-Acet-
amido-2-deoxy- -glucopyranose (GlcNAc)
D
-
D
1
eluted at pH 3.0. The H NMR spectrum of the fumaramic
was dissolved in satd aq ammonium bicarbon-
ate (ꢀ1.5 M) to give a molar ratio of 5:1
ammonia–sugar. After 6 days at 37 °C,
with the intermittent addition of solid am-
monium bicarbonate to maintain saturation,
acid analogue has three characteristic signals (D2O): l 5.12 (d,
1 H, J1,2 9.77 Hz, H-1), 6.00 and 6.50 (d, 1 H each, Ja,b 15.9
1
Hz, H-a and H-b (trans-CHꢀCHꢁ)). The H NMR spectrum
of the maleamic acid analogue has three characteristic signals
(D2O): l 5.15 (d, 1 H, J1,2 9.77 Hz, H-1), 6.35 and 6.45 (d, 1
H each, Ja,b 12.5 Hz, H-a and H-b (cis-CHꢀCHꢁ)).

D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
491
lyophilized to give 1 as a white solid: mp
248.5–249.0 °C; IR (KBr): 3300, 2960, 2927,
1656.9, 1635.5, 1539.9, 1426.8 cm−1; 1H NMR
(D2O, reference acetone (2.225 ppm)): l 2.050
(s, 3 H, COCH3), 2.556 (center of A2B2 second
solid ammonium bicarbonate was removed by
filtration, and excess ammonium bicarbonate
was removed by repeated rotary evaporation
of aq solns. The white solid contained 76% 4
and 24% a and b anomers of GlcNAc as
1
order spectrum not resolved,
4
H,
determined by integration of the H NMR
NHCOCH2CH2CONH2), 3.474 (dd, 1 H, J3,4
10.25, J4,5 8.79 Hz, H-4 (CHOH)), 3.518 (m, 1
H, J5,6a 1.46, J5,6b 4.39 Hz, H-5 (CHOꢁ)),
3.611 (dd, 1 H, J2,3 9.77 Hz, H-3 (CHOH)),
3.746 (dd, 1 H, J6a,6b −12.21 Hz, H-6b
(CH2OH)), 3.819 (dd, 1 H, J1,2 9.77 Hz, H-2
(CHNHAc)), 3.882 (dd, 1 H, H-6a (CH2OH)),
5.068 (d, 1 H, H-1 (CHNH)); 13C NMR (D2O,
reference 1,4-dioxane (66.66 ppm)): l 22.06
signals in D2O for the anomeric protons at l
4.14 (J1,2 9.28 Hz), l 5.19 (J1,2 3.42 Hz), and l
4.34 (J1,2 9.77 Hz), respectively. The crude
compound was used without further purifica-
tion.
Preparation of pentafluorophenyl succinam-
ate (5).—Reaction of succinamic acid (1.43 g,
12.2 mmol), pentafluorophenol (2.25 g, 12.2
mmol), and N,N%-dicyclohexylcarbodiimide
(2.52 g, 12.2 mmol) in DMF (15 mL) followed
the general procedure of Urge et al. [12]. The
mixture was stirred in an ice bath for 5 h, the
precipitated dicyclohexylurea was removed by
filtration, and the solvent was evaporated at
70 °C. The slightly yellow solid was recrystal-
lized from 1,4-dioxane overnight at −20 °C.
The white crystals were collected by filtration,
washed with CH2Cl2, and air-dried for 24 h;
yield 2.55 g (74%): mp 138–139.5 °C; 1H
NMR (Me2SO-d6, reference Me4Si): l 2.51 (t,
2 H, Ja,b 6.59 Hz, H-a (CH2)), 2.93 (t, 2 H, Ja,b
6.59 Hz, H-b (CH2)), 6.9 and 7.5 (s, 1 H,
(COCH3),
29.85
(CH2CONH2),
30.48
(CH2CONH), 54.37 (C-2), 60.58 (C-6), 69.57
(C-4), 74.29 (C-3), 77.69 (C-5), 78.43 (C-1),
174.82 (CONH2), 175.62 (CONH), 177.76
(COCH3). Anal. Calcd for C12H21N3O7·
0.25H2O: C, 44.51; H, 6.69; N, 12.98. Found:
C, 44.52; H, 6.66; N, 13.26.
Preparation of 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-h- -glucopyranosyl chloride
D
[23].—The 1H NMR spectrum in CDCl3
showed a doublet for the anomeric proton at
l 6.197, J1,2 3.42 Hz.
Preparation of 2-acetamido-3,4,6-tri-O-ace-
tyl-2-deoxy-i- -glucopyranosyl azide [16].—
D
13
NH2); C NMR (Me2SO-d6, reference Me4Si):
This compound was synthesized from
l 28.92 (C-b), 29.63 (C-a), 134–140 (pen-
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a- -
D
tafluorophenyl,
JCFꢀ250
Hz),
173.07
glucopyranosyl chloride (above) and gave
(CONH2), 173.94 (COO).
crystals: mp 165–166 °C, lit. 166–168 °C [16],
N-(2-Acetamido-2-deoxy-i-
D
-glucopyran-
1
lit. 166–167 °C (dec) [19]; H NMR (CDCl3,
osyl)succinamide hydrate (1).—Compounds 4
(0.60 g, 2.2 mmol) and 5 (2.52 g, 8.9 mmol)
were dissolved in DMF (24 mL) and stirred in
an ice bath for 72 h [12]. After the solvent was
evaporated at 60 °C, the light yellow solid was
dissolved in distilled water (4 mL), and the
insoluble material was removed by filtration.
To remove pentafluorophenol, an Amberlite
IRA-400 (Cl−) anion-exchange column (1×
25 cm) was activated with 0.1 M NaOH and
washed with water. The filtrate was loaded
onto the column and washed with distilled
water (25 mL) at a rate of 1 mL/min. The
eluent was collected and lyophilized to give
150 mg of a white solid, containing approxi-
mately 50% of 1. Portions of the solid were
purified by HPLC. The eluent with a retention
time of 19.1–20.4 min was collected and
reference Me4Si): l 1.993, 2.046, 2.053, 2.115
(s, 3 H each, 4 COCH3), 3.811 (m, 1 H, J4,5
9.77, J5,6a 2.44, J5,6b 4.88 Hz, H-5 (CHOꢁ)),
3.930 (m, 1 H, J1,2 9.28, J2,3 9.77, J2,NH 8.79
Hz, H-2 (CHNHAc)), 4.177 (dd, 1 H, J6a,6b
−12.21 Hz, H-6a (CH2OAc)), 4.284 (dd, 1 H,
H-6b (CH2OAc)), 4.782 (d, 1 H, H-1 (CHN3)),
5.111 (dd, 1 H, J3,4 9.28 Hz, H-4 (CHOAc)),
5.265 (dd, 1 H, H-3 (CHOAc)), 5.779 (d, 1 H,
NH (NHAc)).
Preparation of 2-acetamido-3,4,6-tri-O-ace-
tyl-2-deoxy-i- -glucopyranosylamine [16,17]
D
(6).—Hydrogenation of 2-acetamido-3,4,6-tri-
-glucopyranosyl azide
O-acetyl-2-deoxy-b-
D
(above) over Adams’ platinum oxide catalyst
in anhyd EtOH at ambient temperature gave
crystals: mp 150–151 °C (dec), lit. 150 °C
1
(dec) [17]; H NMR (CDCl3, reference Me4Si):

492
D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
l 1.983, 2.036, 2.051, 2.105 (s, 3 H each, 4
COCH3), 3.650 (m, 1 H, J4,5 9.28, J5,6a 2.44,
J5,6b 4.88 Hz, H-5 (CHOꢁ)), 4.018 (m, 1 H, J1,2
9.28, J2,3 9.77, J2,NH 8.79 Hz, H-2 (CHN-
HAc)), 4.114 (dd, 1 H, J6a,6b −12.21 Hz, H-6a
(CH2OAc)), 4.133 (d, 1 H, H-1 (CHNH2)),
4.226 (dd, 1 H, H-6b (CH2OAc)), 5.042 (MA
part of ABMX second-order spectrum, 1 H,
J3,4 9.28 Hz, H-3 (CHOAc)), 5.089 (BX part
of ABMX second-order spectrum, 1 H, H-4
(CHOAc)), 5.716 (d, 1 H, NH (NHAc)).
9.77, J2,NH 7.81 Hz, H-2 (CHNHAc)), 4.095
(m, 1 H, J1,NH not resolved, H-1 (CHN-
HCOR)), 4.104 (dd, 1 H, J6a,6b −12.70 Hz,
H-6a (CH2OAc)), 4.323 (dd, 1 H, H-6b
(CH2OAc)), 4.813 (dd, 1 H, H-a (CH2CH)),
5.033 (dd, 1 H, J3,4 9.28 Hz, H-3 (CHOAc)),
5.138 (dd, 1 H, H-4 (CHOAc)), 5.917 (s, 1 H,
chloralid (CHCCl3)), 6.126 (d, 1 H, NH
(NHAc)). Anal. Calcd for C20H25Cl3N2O12: C,
40.67; H, 4.27; Cl, 17.78; N, 4.75. Found: C,
40.73; H, 4.32; Cl, 17.82; N, 4.73.
Preparation of
L
-malic acid chloralid [24]
N4-(2-Acetamido-2-deoxy-i-
osyl)- -2-hydroxysuccinamide
D
-glucopyran-
(2).—Com-
(7).— -Malic acid (40.2 g, 0.30 mol) and
L
L
chloral hydrate (59.5 g, 0.36 mol) were mixed
with concd H2SO4 (30 mL) in an ice bath,
stirred for 2 h, and allowed to warm to rt
overnight. The mixture was poured onto ice
(200 g) and extracted with EtOAc (4×90
mL). The extracts were combined, washed
with water (3×180 mL), and dried over
sodium sulfate. The solvent was evaporated to
give a light yellow solid that was recrystallized
from toluene (300 mL) at 4 °C overnight to
give a white solid that was dried to give 13.70
g (52%) of 7: mp 135–136.5 °C, lit. 140 °C
pound 8 (0.30 g, 0.51 mmol) was dissolved in
EtOH (25 mL), and concd ammonium hy-
droxide (1.25 mL) was added [26]. The soln
was stirred at rt for 4 days. The white precip-
itate was collected, washed with EtOH and
diethyl ether, dissolved in water (2 mL), and
purified by HPLC. The eluent with a retention
time of 15.1–16.8 min was collected and
lyophilized to give 65 mg (38%) of 2 as a white
powder: mp 218–218.5 °C; IR (KBr): 3300,
2960, 2940, 2927, 2910, 1671, 1659, 1556
cm−1
;
1H NMR (D2O, reference acetone
1
[24]; H NMR (CDCl3, reference Me4Si): l
(2.225 ppm)): l 2.004 (s, 3 H, COCH3), 2.594
(dd, 1 H, Ja,b 8.79, Jb,b% −15.14 Hz, H-b
(CH2CH)), 2.773 (dd, 1 H, Ja,b% 3.91 Hz, H-b%
(CH2CH)), 3.478 (dd, 1 H, J3,4 10.25, J4,5 8.30
Hz, H-4 (CHOH)), 3.536 (m, 1 H, J5,6a 1.46,
J5,6b 4.39 Hz, H-5 (CHOꢁ)), 3.612 (dd, 1 H,
J2,3 9.77 Hz, H-3 (CHOH)), 3.749 (dd, 1 H,
J6a,6b −12.21 Hz, H-6b (CH2OH)), 3.830 (dd,
1 H, J1,2 9.77 Hz, H-2 (CHNHAc)), 3.887 (dd,
1 H, H-6a (CH2OH)), 4.473 (dd, 1 H, H-a
(CH2CH)), 5.097 (d, 1 H, H-1 (CHNHCOR));
13C NMR (D2O, reference 1,4-dioxane (66.66
ppm)): l 22.060 (COCH3), 40.074 (C-b),
54.240 (C-2), 60.515 (C-6), 67.986 (C-a),
69.506 (C-4), 74.228 (C-3), 77.657 (C-5),
78.271 (C-1), 173.363 (NHCOR), 174.848
(CONH2), 178.471 (COCH3). Anal. Calcd for
C12H21N3O8: C, 42.97; H, 6.32; N, 12.54.
Found: C, 42.71; H, 6.28; N, 12.81.
3.055 (dd, 1 H, Ja,b 3.42, Jb,b% −18.07 Hz, H-b
(CH2)), 3.149 (dd, 1 H, Ja,b% 3.91 Hz, H-b%
(CH2)), 4.904 (dd, 1 H, H-a (CH2CH)), 5.903
(s, 1 H, chloralid (CHCCl3)).
N4-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
i-D
-glucopyranosyl)- -malamic acid chloralid
L
(8).—Compounds 6 (1.74 g, 5 mmol) and 7
(1.32 g, 5 mmol) were dissolved in CHCl3 (100
mL), to which was added 2-ethoxy-1-ethoxy-
carbonyl-1,2-dihydroquinoline (EEDQ) (1.24
g, 5 mmol) [25]. The reaction was stirred at
ambient temperature for 22 h, and the solvent
was evaporated at a temperature of less than
40 °C. The white solid was dissolved in EtOAc
(40 mL), petroleum ether was added to turbid-
ity, and the mixture was allowed to crystallize
at rt for 8 h. The crystals were collected and
washed with diethyl ether to give 2.13 g (72%)
N4-(2-Acetamido-2-deoxy-i -
nosyl)- -2-hydroxysuccinamic acid hydrazide
dihydrate (3)2.—Compound 8 (0.25 g, 0.42
D
-glucopyra-
1
of 8: mp 199–200 °C; H NMR (CDCl3, refer-
L
ence Me4Si): l 1.991, 2.054, 2.082, 2.100 (s, 3
H each, 4 COCH3), 2.806 (dd, 1 H, Ja,b 3.42,
Jb,b% −17.09 Hz, H-b (CH2CH)), 2.968 (dd, 1
H, Ja,b% 3.91 Hz, H-b% (CH2CH)), 3.746 (m, 1
H, J4,5 9.77, J5,6a 1.95, J5,6b 4.39 Hz, H-5
(CHOꢁ)), 4.089 (m, 1 H, J1,2 not resolved, J2,3
2 IUPAC name: (3S)-N-(2-acetamido-2-deoxy-b-
ranosyl)-(3-hydrazinocarbonyl-3-hydroxypropionamide).
D-glucopy-

D.H. Huang, J.M. Risley / Carbohydrate Research 329 (2000) 487–493
493
mmol) was dissolved in EtOH (20 mL), and
hydrazine monohydrate (1.25 mL) was added
[27]. The soln was stirred at rt for 2 days.
The white precipitate was collected, washed
with EtOH and diethyl ether, dissolved in
water (3 mL), and purified by HPLC. The
eluent with a retention time of 16.6–19.3 min
was collected and lyophilized to give 106 mg
(65%) of 3 as a white powder: mp 207–
208 °C; IR (KBr): 3315, 2929, 2902, 1652,
the University of North Carolina at Char-
lotte.
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1
1555, 1423 cm−1; H NMR (D2O, reference
acetone (2.225 ppm)): l 2.004 (s, 3 H,
COCH3), 2.591 (dd, 1 H, Ja,b 8.30, Jb,b%
−15.14 Hz, H-b (CH2CH)), 2.762 (dd, 1 H,
Ja,b% 3.91 Hz, H-b% (CH2CH)), 3.479 (dd, 1 H,
J3,4 10.25, J4,5 8.30 Hz, H-4 (CHOH)), 3.533
(m, 1 H, J5,6a 1.95, J5,6b 4.88 Hz, H-5
(CHOꢁ)), 3.616 (dd, 1 H, J2,3 9.77 Hz, H-3
(CHOH)), 3.747 (dd, 1 H, J6a,6b −12.21 Hz,
H-6b (CH2OH)), 3.827 (dd, 1 H, J1,2 9.77 Hz,
H-2 (CHNHAc)), 3.887 (dd, 1 H, H-6a
(CH2OH)), 4.518 (dd, 1 H, H-a (CH2CH)),
13
5.089 (d, 1 H, H-1 (CHNHCOR)); C NMR
(D2O, reference 1,4-dioxane (66.66 ppm)): l
22.12 (COCH3), 40.24 (C-b), 54.31 (C-2),
60.58 (C-6), 67.76 (C-a), 69.57 (C-4), 74.29
(C-3), 77.72 (C-5), 78.37 (C-1), 173.23
(CONHNH2), 173.62 (NHCOR), 174.95
(COCH3). Anal. Calcd for C12H22N4O8·2H2O:
C, 37.30; H, 6.78; N, 14.50. Found: C, 37.10;
H, 6.70; N, 14.27.
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Acknowledgements
The authors thank Yuan-Qing Xia for his
preliminary work on the syntheses of the suc-
cinamide analogue 1 and
L
-malic acid chlo-
ralid 7, and Dr Clifford M. Carlin for the
computation of the second-order spectrum.
This research was supported by a Cottrell
College Science Award of Research Corpora-
tion. Acknowledgment is made to the Donors
of the Petroleum Research Fund, adminis-
tered by the American Chemical Society, for
partial support of this research. This research
was supported, in part, by funds provided by
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