Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution

Article abstract of DOI:10.1016/j.carres.2008.11.007

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong α-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acet

Full text of DOI:10.1016/j.carres.2008.11.007

Carbohydrate Research 344 (2009) 278–284
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Stability studies of hydrazide and hydroxylamine-based
glycoconjugates in aqueous solution
*
Anna V. Gudmundsdottir, Caroline E. Paul, Mark Nitz
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong
Received 24 September 2008
Received in revised form 4 November 2008
Accepted 10 November 2008
Available online 19 November 2008
a
-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of gly-
coconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydr-
azide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for
the formation of these useful conjugates, and their stability. Their apparent association constants (9–
74 Mꢀ1) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 °C), were deter-
mined. The half-lives of the conjugates varied between 3 h and 300 days. All the compounds were
increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found
for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed
more rapidly.
Keywords:
Glycoconjugate
Glycosylhydrazides
Oligosaccharide labelling
Hydrolysis rates
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
instability and potential mutarotation of the intermediary glycosyl
amine.7
The synthesis of glycoconjugates has facilitated a wide variety
of techniques for the detailed study of carbohydrates and their
interactions in biological systems.1,2 The types of glycoconjugates
that have been synthesized differ greatly depending on the in-
tended use, but the majority of the conjugates are similar in that
the reducing terminus of the oligosaccharide is functionalized for
conjugate formation. Until recently, most glycoconjugates have
been generated by the total synthesis of the oligosaccharide with
the desired reducing terminal conjugating functional group in-
stalled early in the synthetic procedure. This approach is robust
and can reliably generate the desired glycoconjugates but it is
labour intensive, requiring many steps.3,4
Alternative approaches to glycoconjugate formation that di-
rectly functionalize the reducing terminal hemiacetal have been
developed. These methods are especially useful when oligosaccha-
rides can be isolated from natural sources or generated enzymati-
cally. Classically, reductive amination has been used for the
synthesis of glycoconjugates.5 Unfortunately, reductive amination
results in an acyclic structure at the reducing terminus of the oli-
gosaccharide, and this can have consequences on the glycoconju-
gate, potentially causing the loss of its biological activity. It is
possible to generate glycosyl amines from the free hemiacetal at
the reducing terminus, and then directly acylate these derivatives.6
However, this reaction can be challenging to control due to the
Recently, condensation approaches that enable direct function-
alization of the terminal hemiacetal with preservation of the
reducing monosaccharide have been re-investigated. These
chemoselective conjugations involve condensation with a nucleo-
phile such as an N-alkylhydroxylamine,8 an acylhydrazide,9–11
a
sulfonylhydrazide,10 a semicarbazide12,13 or a semithiocarbazide.14
A thermodynamic mixture of glycosides is produced from these
condensation reactions, which for reducing terminal gluco-config-
ured monosaccharides is predominately the b-pyranoside.15,16
The conditions for the chemoselective conjugations are mild and
can often be carried out in aqueous solution, making these meth-
ods efficient and amenable to work with small quantities of oligo-
saccharide. The simplicity and efficiency of the conjugations have
made them excellent methods for the formation of glycoconjugates
for applications such as biotin labelling,17,18 labelling for mass
spectrometry,19,20 labelling for chromatography,21,22 formation of
glycoarrays,23–25 the generation of glycopeptide analogues26–30
or neoglycoproteins,31 to ‘sweeten’ the structures of
therapeutics,9,32–34 and recently in the protecting group-free
formation of glycosyl donors.35
Despite the wide use of these chemoselective methods, the
properties of the linkages have not been thoroughly investigated
with regard to their stability and rates of hydrolysis under aqueous
conditions.18,32 A more complete understanding of these properties
will allow for the condensation conditions for glycoconjugate
formation to be optimized and for the conditions under which
the glycoconjugates are useful to be elucidated.
* Corresponding author. Tel.: +1 416 946 0640.
E-mail address: mnitz@chem.utoronto.ca (M. Nitz).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.11.007

A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
279
O
O
O
O
O
a
N
N
OH
O
4
S
R
R
R
NH
NH
O
N
O
O
N
NH
H2
N
O
R
O
b
O
F3C
O-
H-R
1
2
3
2
O
Scheme 1. Reagents and conditions: (a) NaH, BnBr, DMF (85%); (b) TFA, CH2Cl2
(1:1) (90%).
HO
HO
9
10
13
11
R
R
R
OH
OH
OH
the O-benzyl-N-methylhydroxylamine 2 in an overall yield of
73% (Scheme 1).
O
12
14
17
HO
HO
N-Methyl-O-(N0-benzylacetamide)hydroxylamine (3) was also
prepared from t-butyl N-methyl-N-hydroxycarbamate, shown in
Scheme 2, following the method of Niikura et al.38 Compound 5
was obtained by alkylating the hydroxylamine at oxygen with
ethyl bromoacetate. The ester was hydrolyzed with sodium
hydroxide in THF to form 6. The acid 6 was then activated with
DCC and coupled with N-hydroxysuccinimide to afford the active
ester 7. This compound is a versatile intermediate for forming
other N-alkylhydroxylamine-based glycoconjugates. Benzylamine
was condensed with the succinimide ester to give 8, which was
then deprotected using TFA in CH2Cl2 (1:1) to give N-methyl-O-
(N0-benzylacetamide)hydroxylamine 3 as the TFA salt.
OH
O
15
16
HO
HO
NHAc
Figure 1. Nine glycoconjugates synthesized and subjected to hydrolysis
experiments.
In a previous communication, we reported the hydrolysis rates
for derivatized 1-glycosyl-1-benzoylhydrazides. We found them to
be unstable in weakly acidic solutions.36 In this report, we have
evaluated nine glycoconjugates (Fig. 1) based on p-toluene-
sulfonylhydrazide and two N-methylhydroxylamines. The equilib-
rium constants for formation of these conjugates in aqueous
solution were determined, and the rates of hydrolysis of the conju-
gates were evaluated at pH 4.0, 5.0 and 6.0. These results have been
compared to the established mechanisms for oxime or hydrazone
hydrolysis as well as for the hydrolysis of O-glycosides.
To determine the optimal conditions for glycoconjugate synthe-
sis, the equilibrium constant for formation of the conjugates was
determined using 1H NMR spectroscopy at equimolar concentra-
tions of hydrazide or hydroxylamine at four different concentra-
tions: 50, 75, 100 and 125 mM, in deuterated sodium acetate
buffer (pD 4.5). The amounts of conjugate and free monosaccharide
were determined by integration of the 1H NMR absorptions. After 4
days of equilibration at 37 °C, no further change was observed in
the relative amounts of species present, and the integrated values
were used to determine the apparent equilibrium constants under
these conditions (Table 1).
2. Results
As seen from the apparent association constants, the p-toluene-
sulfonohydrazide glycoconjugates (9, 12 and 15) are moderately
more stable than the N-methylhydroxylamine conjugates. Larger
2.1. Formation of glycoconjugates
The conjugates are derived from three different monosaccha-
rides: xylose, glucose and N-acetylglucosamine. These monosac-
charides were chosen for their biological relevance and for the
wealth of information that is available about O-glycoside hydroly-
sis for these monosaccharides. N-acetylglucosamine and xylose are
the reducing terminal sugars found in the N-linked glycoproteins
and the glycosaminoglycans, respectively, thus knowledge of the
properties of conjugates derived from these monosaccharides will
be directly applicable to conjugates formed from these oligosac-
charides. The mechanisms of hydrolysis of the corresponding
methyl glycosides of xylose, glucose and N-acetylglucosamine have
been studied in detail. This knowledge allows parallels to be drawn
to the rates of hydrolysis determined for the glycoconjugates stud-
ied here, potentially allowing the results obtained to be extrapo-
lated to other glycoconjugates.
Boc
N
Boc
N
a
OR
c
OH
O
+
O
O
Br
5: R = Et
b
O
6: R = H
Boc
O
Boc
N
O
d
N
O
O
H
N
N
O
O
O
7
8
These monosaccharides were condensed with p-toluene-
sulfonylhydrazide (1), O-benzyl-N-methylhydroxylamine (2) and
N-methyl-O-(N0-benzylacetamide)hydroxylamine (3) (Fig. 1). The
p-toluenesulfonylhydrazide 1 is commercially available while the
two N-methylhydroxylamines, 2 and 3, were synthesized.
O-Benzyl-N-methylhydroxylamine (2) was prepared from t-bu-
tyl N-methyl-N-hydroxycarbamate,37 which was alkylated at the
hydroxylamine oxygen using benzyl bromide to give 4. The Boc-
protecting group was removed with TFA in CH2Cl2 (1:1) to obtain
e
H
N
H2N
O
O
O
3
F3C
O-
Scheme 2. Reagents and conditions: (a) NaH, THF (95%); (b) NaOH, THF (86%); (c)
DCC, N-hydroxysuccinimide, EtOAc (87%); (d) benzylamine, CH3CN (73%); (e) TFA–
CH2Cl2 (1:1) (87%).

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A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
Table 1
Equilibrium constants for the glycoconjugate formation
a
Monosaccharide
Ka (Mꢀ1
) (compound)
Xyl
Glc
GlcNAc
74b (9)
21 (10)
18 (13)
16 (16)
20 (11)
19 (14)
9 (17)
65b (12)
18b (15)
a
Ka = [Glycoconjugate]/[free nucleophile][free hemiacetal]. Apparent association
constants determined in D2O (pD 4.5, 37 °C); values are the average of four deter-
minations at different compound concentrations. Standard deviation was between
6% and 9%.
b
3% DMSO-d6 was used to solubilize the p-toluenesulfonylhydrazide.
association constants were observed with more electron-rich
monosaccharides (xylose > glucose > N-acetylglucosamine). All of
the equilibrium constants are small. Thus, only under concentrated
conditions do the conjugation reactions proceed to near comple-
tion. It has been reported that the formation of some N-meth-
ylhydroxylamine conjugates does not proceed to completion, and
that the products of the condensation reaction with less reactive
monosaccharides (i.e., N-acetylglucosamine) are produced in low
yields.8,39 These observations may be the result of the small equi-
librium constant for the condensation in aqueous solution.
Given the small equilibrium constants for formation of the gly-
coconjugates, the condensation reactions were carried out under
concentrated conditions (0.75 M, equimolar) at pH 4.5 (2 M
NH4OAc). The samples were incubated at 37 °C for 72 h, and glyco-
conjugates 9, 12 and 15 were crystallized from isopropanol, while
the remaining compounds were purified using column chromato-
graphy. The isolated yields from the conjugation reactions were
between 80% and 90%, and only b-pyranosides were isolated in
all cases.
Figure 2. Hydrolysis of N-(b-D-xylopyranosyl)-p-toluenesulfonohydrazide in
20 mM NaOAc (0.5% DMSO) pH 4.0 at 37 °C. Peaks correspond to glycoconjugate
9 (6.8 min), benzyl alcohol (9.2 min, internal standard) and p-toluenesulfonylhydr-
azide (10.5 min). The corresponding salts of p-toluenesulfinic and p-toluenesulfonic
acids eluted at the solvent front.
peaks derived from both the isomers were combined. Benzyl alco-
hol was used as an internal standard in all cases. Control experi-
ments showed no glycoconjugate formation upon incubating the
nucleophile and hemiacetal at equivalent concentrations (2 mM)
under the hydrolysis conditions for time frames consistent with
those for the hydrolysis experiments.
The observed rate constants for hydrolysis (pseudo-first-order
conditions) were determined by directly fitting the integrated
areas indicative of the glycoconjugates remaining compared to
an internal standard. The observed half-lives for hydrolysis of con-
jugates 9–17 are presented in Table 2. The pH rate profile is pre-
sented in Figure 3. The hydrolysis of all the conjugates fit well to
a first-order process despite the possibility of two hydrolysis path-
ways for the p-toluenesulfonohydrazide conjugates (vide supra).
2.2. Rates of glycoconjugate hydrolysis
To confirm the formation of the hemiacetal and hydrazide or
hydroxylamine, hydrolysis products of the conjugates were ana-
lyzed by 1H NMR (3% DMSO-d6 in 10 mM Na2DPO4 buffer at pD
6.0 with t-butanol as the internal standard). The hydroxylamine
conjugates hydrolyzed to the expected products. However, the p-
toluenesulfonylhydrazide derivatives 9, 12 and 15 gave not only
the aldose and p-toluenesulfonylhydrazide but also p-toluenesulfi-
nic and p-toluenesulfonic acids. The degradation of p-toluene-
sulfonylhydrazide to p-toluenesulfinic and p-toluenesulfonic
acids had previously been observed.40,41 Attempts were made to
determine the rate of degradation of p-toluenesulfonylhydrazide
under the hydrolysis conditions, but the data do not fit to a simple
first-order process (Fig. 13S, see Supplementary data). Qualita-
tively, the rate of decomposition of p-toluenesulfonylhydrazide is
faster than hydrolysis of the conjugate at pH 6.0 but slower than
hydrolysis of the conjugate at pH 4.0. Thus, with the analysis car-
ried out here, it is not possible to determine if the p-toluene-
sulfonohydrazide conjugates are hydrolyzing exclusively by an
initial C–N bond cleavage, or by competitive pathways involving
initial C–N and N–S bond cleavage.
To determine the hydrolysis rates, the glycoconjugates 9–17
were incubated at the physiologically relevant pH values of 4.0,
5.0 and 6.0 at 37 °C, and the hydrolysis reaction was followed by
HPLC (Fig. 2). The p-toluenesulfonohydrazide derivatives 12 and
15 elute as two peaks. This may be due to cis–trans isomerisation
about the hydrazide bond. Cis–trans isomerism in glycosylhydraz-
ides has been observed for 1-glycosyl-2-acetylhydrazides11 and
1-glycosyl-2-benzoylhydrazides.11,36 When the two isomers were
separated, and re-analyzed separately, two peaks were again
observed corresponding to re-equilibration of the isomers. In the
calculation of the hydrolysis rates, the integrated areas under the
3. Discussion
Methyl
a-
D-xylopyranoside undergoes specific acid-catalyzed
hydrolysis approximately five times faster than methyl
a-D-gluco-
pyranoside.42 The difference in the rate of hydrolysis between
these structures correlates well with difference in electron affinity
of the substituent at C-5, with glucose being less electron rich than
xylose.43 The rate-limiting step in the hydrolysis reaction is forma-
tion of the oxocarbenium ion, and due to the lack of a C-5
Table 2
Half-lives in aqueous solution of glycoconjugates 9–17
Compound
pH 4.0
pH 5.0
pH 6.0
t1/2 (h)
t1/2 (h)
t1/2 (h)
9
2.9
7.8
13
19
11
100
72
76
13
26
140
48
210
990
230
660
3100
29
59
540
71
890
5100
1100
2600
7500
10
11
12
13
14
15
16
17
350
a
2 mM sample (20 mM NaOAc pH 4.0, 20 mM NaOAc pH 5.0, 20 mM Na2HPO4 pH
L samples were taken out at different time
intervals and quenched with the addition of 400 L of 4 °C 200 mM Na2HPO4 buffer
6.0) was incubated at 37 °C. 200
l
l
at pH 7.0 and immediately analyzed by HPLC. Standard deviation between replicate
runs was between 3% and 5%.

A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
281
From the analysis performed here, it cannot be determined
whether the pathway of hydrolysis for the glycosyl-p-toluene-
sulfonohydrazides proceeds exclusively via initial C–N bond cleav-
age or by two competing pathways involving initial C–N or S–N
bond cleavage. The observation that the hydrolysis reaction does
fit well to a first-order rate law makes the analysis useful for eval-
uating experimental conditions under which these conjugates can
be applied without completing hydrolysis.
The conjugates formed with N-methyl-O-benzylhydroxylamine
2 and N-methyl-O-(N0-benzylacetamide)hydroxylamine 3 display a
four to ninefold differences in hydrolysis rates across all the
individual monosaccharides assessed. Those differences may be
explained by the electronic properties of the N-methylhydroxyl-
amines, where 3 is more electron-deficient than 2 due to the
inductive effect of the amide in comparison to the phenyl ring.
As was observed with the glycosyl-p-toluenesulfonohydrazide
conjugates, the N-methylhydroxylamine conjugates formed from
the less electron-rich monosaccharides (i.e., N-acetylglucosamine)
hydrolyze more slowly.
Extensive studies have supported a mechanism of hydrolysis of
oximes, and hydrazones over a pH range of ꢁ4.0–6.0 which in-
volves a rate-limiting addition of water at the sp2 carbon.47 Over
this pH range general base catalysis is observed. It is proposed that
the base aids in deprotonation of the nucleophilic water molecule
upon attack. We hypothesize that the mechanism of hydrolysis of
the N-methylhydroxylamine glycoconjugates follows a similar
mechanism.
Figure 3. pH rate profile for the glycoconjugates 9–17. (h) 9, (s) 10, (4) 11, (l) 12,
(g) 13, ( ) 14, (j) 15, (d) 16, (N) 17. Each value is an average of two experiments;
standard deviation was between 3% and 5%. Buffers: 20 mM NaOAc pH 4.0, 20 mM
NaOAc pH 5.0, 20 mM Na2HPO4 pH 6.0. 0.5% DMSO was added to maintain
solubility for glycoconjugates 9, 12 and 15.
The expected steps in hydrolysis of the hydroxylamine-based
glycoconjugates are shown in Scheme 3. The effect of pH on the
rate of hydrolysis is reflected in the position of the equilibrium be-
tween the conjugate I and the ring open oxyiminium species II in
solution; at lower pH more of species II will be present. This is con-
sistent with the observation that more electron-rich conjugates,
formed from either electron-rich monosaccharides (e.g., xylose)
or more electron-rich hydroxylamines (e.g., 2), hydrolyze more
rapidly because they are more readily protonated. In the elec-
tron-poor glycoconjugates it is less favourable to form species II.
The rate-limiting step is then attack of water on the oxyiminium
ion (II). Buffer catalysis was observed at pH 4 and pH 6 on the
hydrolysis of 13, which would be expected for a rate-limiting at-
tack of water on the oxyiminium (II) (see Supplementary data).
After water attack, the carbanolamine (III) would rapidly eliminate
the N-methylhydroxylamine, as the rate of attack of hydroxyl-
amine on aldehydes is rapid and reversible over the pH range
investigated.48,49 Although beyond the scope of this initial investi-
gation, further studies to confirm the buffer ion responsible for the
catalysis and an expansion of the pH range investigated are
required to support this mechanistic proposal. This mechanism is
consistent with that proposed by Dumy and co-workers for the for-
mation of N-methyl-O-methylhydroxylamine glycoconjugates.8
substituent the xylosyl oxocarbenium ion is of lower energy, lead-
ing to faster hydrolysis.44 Similar effects have been measured for a
range of glycosides with deoxy and deoxyfluoro substituents of dif-
fering stereochemistry, and in general, the field effect of the substi-
tution on the electron-deficient transition state directly influences
the hydrolysis rate.45 In contrast, the rate of hydrolysis of methyl
2-acetamido-2-deoxy-b-
D-glucopyranoside is faster than that of
methyl b- -glucopyranoside, despite the greater electron affinity
D
of the N-acetamido group than that of the hydroxyl substituent
at C-2. This observation is most likely due to neighbouring group
participation of the N-acetamido group during hydrolysis.46 Thus,
by analogy, comparison of the rates of hydrolysis of the xylo-
and glucopyranoside conjugates 9–14 synthesized here provides
insight into the importance of the electronic properties of the
monosaccharides on the rate of hydrolysis. The comparison of glu-
cose glycoconjugates 12–14 with N-acetylglucosamine derived
conjugates 15–17 provides insight into the possible influence of
neighbouring group participation on the rate of hydrolysis.
From analysis of the kinetic data, it is apparent that the rates of
hydrolysis for all the conjugates investigated are strongly pH
dependent (Fig. 3, Table 2). The p-toluenesulfonohydrazide conju-
gates 9, 12 and 15 hydrolyze more quickly than the N-methylhydr-
oxylamine conjugates at pH 5 and 6 by a factor of 2–71.
The N-(b-D-glucopyranosyl)-p-toluenesulfonohydrazide 12 has
a half-life of 19 h, and hydrolyzes significantly more slowly than
the previously investigated 1-glucosyl-2-benzoylhydrazide.36 This
observation is in agreement with the previous observations that
conjugates formed from less basic hydrazides hydrolyze more
slowly.36 Comparison of the p-toluenesulfonohydrazide conjugates
formed from different monosaccharides 9, 12 and 15 indicates that
the rates of hydrolysis are slower when the monosaccharide has
more electron-withdrawing groups on the pyranose ring (xylose
(fastest) > glucose > N-acetylglucosamine (slowest)). The observa-
tion that N-acetylglucosamine derivative 15 hydrolyzes more
slowly than the corresponding glucose derivative 12 suggests that
the field effect of the N-acetamido group destabilizes the rate-lim-
iting transition state, and that neighbouring group participation is
not a major factor in the hydrolysis mechanism.
O
OH
HA
A-
R
N
R
N
O
O
HO
R
HO
I
II
OH
±H+,±H2O
N
O
HO
O
OH
III
+
R
HN
O
OH
HO
Scheme 3. Putative mechanism for the hydrolysis of N-alkylhydroxylamine
glycoconjugates.

282
A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
different solutions, each containing the corresponding sugars: xy-
4. Conclusions
lose, glucose or N-acetylglucosamine, and the corresponding
nucleophile: 1, 2 or 3, in equimolar amounts at 50, 75, 100 and
125 mM using 500 mM deuterated sodium acetate buffer, pD 4.5.
3% DMSO-d6 was added for solubility when using p-toluenesulf-
onyl hydrazide. The samples were incubated at 37 °C for 4 days,
and their 1H NMR spectra were measured. Product and remaining
reagent concentrations were then determined using integration
values. Degradation of the p-toluenesulfonylhydrazide into p-tolu-
enesulfinic and p-toluenesulfonic acids was corrected for in the
calculations. The represented Ka value is an average of the four
samples.
Efficient methods to synthesize glycoconjugates enable a wide
variety of biophysical tools to be used to study the properties of
carbohydrates. Here, we have investigated the equilibrium con-
stants for formation, as well as the rates of hydrolysis of N-meth-
ylhydroxylamine and p-toluenesulfonylhydrazide glycoconjugates
with xylose, glucose and N-acetylglucosamine. The equilibrium
constants for formation of all the conjugates are small, between
9 Mꢀ1 and 74 Mꢀ1, revealing the importance of carrying out the
conjugation reactions under concentrated conditions. In cases
where only small amounts of oligosaccharides are available, this
can be addressed by using excess nucleophile, provided that a fac-
ile separation method for the glycoconjugate product is available.25
In all cases, the hydrolysis rates of the conjugates accelerated un-
der increasingly acidic conditions and the half-lives of hydrolysis
of these conjugates suggest that caution should be employed when
using these conjugates below pH 6.0. The hydrolysis rates are
strongly affected by the electronic properties of the monosaccha-
ride involved, as electron-rich monosaccharides hydrolyze signifi-
cantly more quickly than electron-poor monosaccharides. Given
the trends in hydrolysis rates of these conjugates, which parallel
those observed with the well studied O-glycosides, it is possible
to use the results presented here as a practical guide to extrapolate
to the conditions under which a glycoconjugate formed from a
novel mono- or oligosaccharide may be useful.
5.2. O-Benzyl-N-methylhydroxylamine (2)
Compound 4 (5.0 g, 21 mmol) was dissolved in 40 mL CH2Cl2–
TFA (1:1) and stirred at rt for 22 h. The solvent was then removed
under reduced pressure, and the product was purified with column
chromatography (pentane) to afford 2.6 g of 2 as oil, yield 90%. 1H
NMR (400 MHz, CDCl3): d 8.78 (s, 2H, NH2), 7.37 (s, 5H, Ar), 5.04 (s,
2H, OCH2), 2.91 (s, 3H, NCH3); 13C NMR (100 MHz, CDCl3): d 133.0,
129.8, 129.5 (2), 129.1 (2), 76.4, 35.9; HRMS m/z calcd for C8H12NO
[M+H]+: 138.0913, found: 138.0915.
5.3. N-Methyl-O-(N0-benzylacetamide)hydroxylamine (3)
Compound 8 (0.9 g, 3.1 mmol) was dissolved in 10 mL CH2Cl2–
TFA (1:1) and stirred at rt for 1 h. The solvent was then removed
under reduced pressure, and the product was crystallized from
Et2O–pentane to give 0.81 g of the TFA salt 3 as white plates, yield
87%. 1H NMR (400 MHz, CDCl3): d 9.18 (s, 2H, NH2CH3), 7.36–7.26
(m, 5H, Ar), 7.14 (s, 1H, CONH), 4.54 (s, 2H, OCH2), 4.46 (s, 2H,
PhCH2), 2.88 (s, 3H, NCH3); 13C NMR (100 MHz, CDCl3): d 169.4,
137.3, 129.0 (2), 128.0, 128.0 (2), 71.6, 43.5, 37.4; HRMS m/z calcd
for C10H15N2O2 [M+H]+: 195.1128, found: 195.1123.
5. Experimental
5.1. General methods
HPLC analyses were performed on a Dionex BioLC (PDA-100
Photodiode Array Detector, GS50 Gradient pump and A550 Auto-
sampler) using a Waters SymmetryÒ C18 5
l
m (4.6 ꢂ 150 mm) re-
verse phase analytical column. All NMR spectra were recorded at
25 °C with either a Varian 400 MHz (AutoX8308-400 probe) or a
Mercury 400 MHz (ATB8123–400 probe) spectrometer. Chemical
shifts were reported in ppm (d scale) using the solvent residue sig-
nals as reference, and assignments were made by gCOSY spectro-
scopy. Data are represented as follows: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), inte-
gration and coupling constant. High resolution mass spectra were
obtained from an ABI/Sciex QStar mass spectrometer with an ESI
source.
5.4. t-Butyl N-benzyloxy-N-methylcarbamate (4)
A 60% oil dispersion of NaH (1.6 g, 40 mmol) was added to t-
butyl N-methyl-N-hydroxycarbamate (5.3 g, 36 mmol) in 20 mL
anhydrous DMF and stirred at 0 °C for 30 min under nitrogen.
Benzyl bromide (4.7 mL, 40 mmol) was added, and the reaction
mixture was stirred for 16 h. The reaction mixture was diluted
with pentane (150 mL), and the organic layer was then washed
with water (3 ꢂ 50 mL) and brine (40 mL), dried over MgSO4,
filtered and concentrated. The residue was purified by column
chromatography (CH2Cl2–pentane, 2:3) to afford 7.8 g of 4 as pale
yellow oil, yield 85%. 1H NMR (400 MHz, CDCl3): d 7.42–7.31 (m,
5H, Ar), 4.81 (s, 2H, OCH2), 3.05 (s, 3H, NCH3), 1.40 (s, 9H,
C(CH3)3); 13C NMR (100 MHz, CDCl3): d 157.2, 135.8, 129.6 (2),
128.6, 128.5 (2), 81.4, 76.6, 37.0, 28.4 (3); HRMS m/z calcd for
C13H19NO3 [M+Na]+: 260.1257, found: 260.1260.
5.1.1. Hydrolysis methods
Each glycoconjugate sample was prepared in duplicate at 2 mM
(20 mM NaOAc pH 4.0, 20 mM NaOAc pH 5.0 or 20 mM phosphate
pH 6.0) and incubated at 37 °C. Glycoconjugates 9, 12 and 15 re-
quired 0.5% DMSO for solubility. Benzyl alcohol (1 mM) was used
as an internal standard for all runs. 200
out at time intervals and quenched with the addition of 400
lL samples were taken
lL
of 4 °C 200 mM phosphate buffer at pH 7.0 and immediately ana-
lyzed by HPLC (15–25% CH3CN in H2O, 4 min–15 min gradient).
The observed pseudo-first-order rate constants for the hydrolysis
were determined by directly fitting the areas of each glycoconju-
gate remaining as a percentage to an exponential decay using
ORIGIN 7.0, defined by the equation: v = kobs ꢃ [% glycoconjugate
remaining].
5.5. Ethyl (t-butoxycarbonyl-N-methylaminooxy)acetate (5)
A 60% oil dispersion of NaH (136 mg, 3.4 mmol) was added to a
solution of t-butyl N-methyl-N-hydroxycarbamate (500 mg,
3.4 mmol) in THF (10 mL) and stirred at 0 °C for 30 min under
nitrogen. Ethyl bromoacetate (452 lL, 4.1 mmol) was added, and
the reaction mixture was stirred for 4 h at rt. The reaction mixture
was then diluted with EtOAc (150 mL), and the organic layer was
washed with water (3 ꢂ 50 mL) and brine (2 ꢂ 15 mL), dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (1:9 EtOAc–pentane) to afford 755 mg of 5
as pale yellow oil, yield 95%. 1H NMR (400 MHz, CDCl3): d 4.45
(s, 2H, OCH2CO), 4.24 (q, 2H, J = 7.1 Hz, OCH2CH3), 3.21 (s, 3H,
5.1.2. Determination of equilibrium constants, Ka
The Ka values were determined using 1H NMR spectroscopy by
integrating the absorption of the methyl peak corresponding to the
N-methylhydroxylamine or the methyl peak of the p-toluene-
sulfonylhydrazide. The reactions were followed using four

A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
283
NCH3), 1.49 (s, 9H, C(CH3)3), 1.30 (t, 3H, J = 7.1 Hz, OCH2CH3); 13C
NMR (100 MHz, CDCl3): d 169.5, 157.9, 82.1, 72.2, 61.2, 38.5, 28.4
(3), 14.3; HRMS m/z calcd for C10H19NO5Na [M+Na]+: 256.1155,
found: 256.1154.
CD3OD): d 145.1, 137.1, 130.6 (2), 129.1 (2), 92.4, 78.2, 71.4, 71.2,
68.3, 21.5; HRMS m/z calcd for C12H18N2O6NaS [M+Na]+:
341.0777, found: 341.0788.
5.10. N-Methyl-O-benzyl-N-(b-D-xylopyranosyl)hydroxylamine
(10)
5.6. (t-Butoxycarbonyl-N-methylaminooxy)acetic acid (6)
Compound 5 (689 mg, 3.0 mmol) was dissolved in THF (6 mL)
and solution of NaOH (240 mg, 6.0 mmol) in H2O (2 mL) was added
and the solution was stirred for 4 h at rt. The reaction mixture was
then diluted with EtOAc (50 mL), and the organic layer was
washed with 1 M HCl (2 ꢂ 50 mL), water (2 ꢂ 50 mL) and brine
(1 ꢂ 20 mL), dried over MgSO4, filtered and concentrated. The res-
idue was purified by column chromatography (CH2Cl2) to afford
519 mg of 6 as pale yellow oil, yield 86%. 1H NMR (400 MHz,
CDCl3): d 4.46 (s, 2H, OCH2), 3.14 (s, 3H, NCH3), 1.51 (s, 9H,
C(CH3)3); 13C NMR (100 MHz, CDCl3): d 170.7, 159.9, 85.2, 73.4,
37.9, 28.2 (3); HRMS m/z calcd for C8H15NO5Na [M+Na]+:
228.0842, found: 228.0850.
The title compound (152 mg) was prepared by making a 0.75 M
-xylose (100 mg, 0.67 mmol) and compound (110 mg,
D
2
0.8 mmol) solution in 2 M NH4OAc buffer pH 4.5 and incubating
at 37 °C for 72 h. The product was purified by column chromato-
graphy (5% MeOH in CH2Cl2), yield 84%. 1H NMR (400 MHz,
CD3OD): d 7.39–7.27 (m, 5H, Ar), 4.75 (s, 2H, PhCH2), 3.95 (d, 1H,
J1,2 = 9.0 Hz, H-1), 3.88 (dd, 1H, J5a,5b = 11.2, J4,5a = 5.4 Hz, H-5a),
3.49–3.42 (m, 2H, H-2, H-4), 3.31 (under CD3OD peak, 1H, H-3),
3.14 (dd, J5a,5b = 11.2, J4,5b = 10.9 Hz, H-5b), 2.69 (s, 3H, PhCH3);
13C NMR (100 MHz, CD3OD): d 138.5, 130.2 (2), 129.3 (2), 129.0,
96.4, 79.4, 76.4, 71.7, 71.1, 68.9, 39.3; HRMS m/z calcd for
C13H20NO5 [M+H]+: 270.1335, found: 270.1328.
5.7. (t-Butoxycarbonyl-N0-methylaminooxyacetyl)-
N-hydroxysuccinimide ester (7)
5.11. N-Methyl-O-(N0-benzylacetamide)-
N-(b-D-xylopyranosyl)hydroxylamine (11)
To a stirred solution of 6 (750 mg, 3.6 mmol) in EtOAc (25 mL)
were added N-hydroxysuccinimide (630 mg, 1.5 equiv) and DCC
(900 mg, 1.2 equiv). The reaction mixture was then stirred at rt
for 2 h. The solid was removed by filtration and washed with
EtOAc. The organics were then washed with a solution of sodium
bicarbonate (1 M, 2 ꢂ 30 mL) and dried over MgSO4. After filtration
the solvent was removed under vacuum, and the resulting white
solid was recrystallized from EtOAc–hexanes giving 960 mg of 7,
yield 87%. 1H NMR (400 MHz, CDCl3): d 4.80 (s, 2H, OCH2), 3.19
(s, 3H, NCH3), 2.86 (s, 4H, NCOCH2), 1.50 (s, 9H, C(CH3)3); 13C
NMR (100 MHz, CDCl3): d 168.8 (2), 165.2, 158.0, 82.6, 70.0, 38.9,
28.3 (3), 25.7 (2); HRMS m/z calcd for C12H18N2O7Na [M+Na]+:
325.1013, found: 325.1006.
Using the same procedure as described for compound 10,
D
-xy-
lose (41 mg, 0.27 mmol) and 3 (100 mg, 0.32 mmol) gave 76 mg of
11 as white amorphous solid (86%). 1H NMR (400 MHz, CD3OD): d
7.33–7.21 (m, 5H, Ar), 4.45 (d, 1H, OCH2a, J = 15.0 Hz), 4.41 (d, 1H,
OCH2b, J = 15.0 Hz), 4.34 (d, 1H, ArCH2a, J = 15.1 Hz), 4.28 (d, 1H,
ArCH2b, J = 15.1 Hz), 3.98 (d, 1H, J1,2 = 9.0 Hz, H-1), 3.88 (dd, 1H,
J5a,5b = 11.2, J4,5a = 5.4 Hz, H-5a), 3.48–3.41 (m, 2H, H-2, H-4), 3.31
(under CD3OD peak, 1H, H-3), 3.16 (dd, J5a,5b = 11.2,
J4.5b = 10.6 Hz, H-5b), 2.73 (s, 3H, Ac); 13C NMR (100 MHz, CD3OD):
d 172.1, 139.6, 129.6 (2), 128.7 (2), 128.3, 96.3, 79.1, 72.4, 71.6,
71.1, 68.9, 43.7, 38.8; HRMS m/z calcd for C15H23N2O6 [M+H]+:
327.1550, found: 327.1566.
5.12. N-(b-
D-Glucopyranosyl)-p-toluenesulfonohydrazide (12)
5.8. (t-Butoxycarbonyl-N-methyl-O-(N0-
benzylacetamide))hydroxylamine (8)
Using the same procedure as described for compound 9,
D-glu-
cose (2.0 g, 11.1 mmol) and p-toluenesulfonylhydrazide (2.13 g,
11.43 mmol) gave 3.43 g of compound 12 as white solid (89%).
1H NMR (400 MHz, D2O): d 7.81 (d, 2H, J = 8.3 Hz, Ar), 7.40 (d,
2H, J = 8.3 Hz, Ar), 3.86 (dd, 1H, J6a,6b = 11.7, J5,6a = 1.9 Hz, H-6a),
3.67 (d, 1H, J1,2 = 8.5 Hz, H-1), 3.58 (dd, 1H, J6a,6b = 11.7,
J5,6b = 6.2 Hz, H-6b), 3.34 (under CD3OD peak, 1H, H-3), 3.29 (under
CD3OD peak, 1H, H-4), 3.20–3.11 (m, 2H, H-2, H-5), 2.44 (s, 3H,
PhCH3); 13C NMR (100 MHz, CD3OD): d 145.1, 137.4, 130.6 (2),
129.1 (2), 91.5, 79.2, 78.2, 71.8 (2), 63.2, 21.5; HRMS m/z calcd
for C13H20N2O7NaS [M+Na]+: 371.0883, found: 371.0901.
To a solution of 7 (186 mg, 0.62 mmol) in CH3CN (15 mL) was
added benzylamine (107 lL, 0.74 mmol). The reaction mixture
was stirred under nitrogen for 30 min. The reaction mixture was
then diluted with EtOAc (100 mL), and the organic layer was
washed with 1 M NaHCO3 (2 ꢂ 50 mL), 1 M HCl (2 ꢂ 50 mL), H2O
(2 ꢂ 50 mL) and brine (50 mL), dried over MgSO4, filtered and con-
centrated. This afforded 8 as a colourless oil (169 mg), yield 93%. 1H
NMR (400 MHz, CDCl3): d 8.51 (s, 1H, CONH), 7.27–7.18 (m, 5H, Ar),
4.47 (d, 2H, J = 6.0 Hz, PhCH2), 4.32 (s, 2H, OCH2), 3.05 (s, 3H,
NCH3), 1.37 (s, 9H, C(CH3)3); 13C NMR (100 MHz, CDCl3): d 168.9,
158.0, 138.2, 128.6 (2), 127.8 (2), 127.3, 83.0, 73.5, 42.9, 37.4,
28.1 (3); HRMS m/z calcd for C15H22N2O4Na [M+Na]+: 317.1471,
found: 317.1465.
5.13. N-Methyl-O-benzyl-N-(b-D-
glucopyranosyl)hydroxylamine (13)
Using the same procedure as described for compound 10, D-glu-
5.9. N-(b-D-Xylopyranosyl)-p-toluenesulfonohydrazide (9)
cose (103 mg, 0.57 mmol) and 2 (94 mg, 0.68 mmol) gave 150 mg
of 13 as white solid (88%). 1H NMR (400 MHz, D2O): d 7.41–7.30
(m, 5H, Ar), 4.81 (d, 1H, J = 10.4 Hz, OCH2a), 4.78 (d, 1H,
J = 10.4 Hz, OCH2b), 4.04 (d, 1H, J1,2 = 8.9 Hz, H-1), 3.84 (dd, 1H,
J6a,6b = 12.1, J5,6a = 2.2 Hz, H-6a), 3.68 (dd, 1H, J6a,6b = 12.1,
J5,6b = 5.1 Hz, H-6b), 3.49 (t, 1H, J3,4 = 8.9 Hz, H-3), 3.39 (t, 1H,
J3,4 = 8.9 Hz, H-4), 3.28 (under CD3OD peak, 1H, H-2), 3.22 (ddd,
1H, J4,5 = 9.5, J5,6b = 5.1, J5,6a = 2.2 Hz, H-5), 2.76 (s, 3H, NCH3); 13C
NMR (100 MHz, CD3OD): d 138.1, 130.5 (2), 129.4 (2), 129.3,
95.4, 79.6, 79.3, 76.7, 71.8, 71.1, 62.7, 39.4; HRMS m/z calcd for
C14H22NO6 [M+H]+: 300.1441, found: 300.1454.
The title compound (3.5 g) was prepared by making a 0.75 M
D
-
xylose (2.0 g, 13.3 mmol) and p-toluenesulfonylhydrazide (2.6 g,
13.7 mmol) solution in 2 M NH4OAc buffer pH 4.5 and incubating
at 37 °C for 72 h. The solution was then lyophilized, and the prod-
uct was recrystallized from isopropanol, yield 83%. 1H NMR
(400 MHz, CD3OD): d 7.79 (d, 2H, J = 8.3 Hz, Ar), 7.39 (d, 2H,
J = 8.3 Hz, Ar), 3.79 (dd, 1H, J5a,5b = 11.3, J4,5a = 5.4 Hz, H-5a),
3.70 (d, 1H, J1,2 = 8.7 Hz, H-1), 3.47–3.40 (m, 2H, H-2, H-4), 3.28
(under CD3OD peak, 1H, H-3), 3.07 (dd, J5a,5b = 11.3,
J4,5b = 10.7 Hz, H-5b), 2.43 (s, 3H, PhCH3); 13C NMR (100 MHz,

284
A. V. Gudmundsdottir et al. / Carbohydrate Research 344 (2009) 278–284
5.14. N-Methyl-O-(N0-benzylacetamide)-
Acknowledgement
N-(b-
D-glucopyranosyl)hydroxylamine (14)
Funding from the Natural Science and Engineering Research
Council of Canada is gratefully acknowledged.
Using the same procedure as described for compound 10,
D
-glu-
cose (23 mg, 0.13 mmol) and 3 (50 mg, 0.16 mmol) gave 38 mg of
14 as white amorphous solid (82%). 1H NMR (400 MHz, CD3OD):
d 7.34–7.22 (m, 5H, Ar), 4.47 (d, 1H, J = 14.9 Hz, OCH2a), 4.43 (d,
1H, J = 14.9 Hz, OCH2b), 4.37 (d, 1H, J = 15.1 Hz, ArCH2a), 4.31 (d,
1H, J = 15.1 Hz, ArCH2b), 4.05 (d, 1H, J1,2 = 8.8 Hz, H-1), 3.87 (dd,
1H, J6a,6b = 11.9, J5,6a = 2.1 Hz, H-6a), 3.68 (dd, 1H, J6a,6b = 11.9,
J5,6b = 5.2 Hz, H-6b), 3.45 (t, 1H, J = 8.8 Hz, H-3), 3.37 (t, 1H,
J = 8.7 Hz, H-4), 3.30–3.21 (m, 2H, H-2, H-5), 2.78 (s, 3H, NCH3);
13C NMR (100 MHz, CD3OD): d 172.1, 139.7, 129.6 (2), 128.6 (2),
128.3, 95.4, 79.7, 79.2, 72.5, 71.7, 71.4, 62.7, 43.7, 38.8; HRMS
m/z calcd for C16H24N2O7Na [M+Na]+: 379.1475, found: 379.1462.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.11.007.
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7.38 (d, 2H, J = 8.3 Hz, Ar), 3.94 (d, 1H, J1,2 = 9.2 Hz, H-1), 3.89
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Using the same procedure as described for compound 10, N-
acetyl-D-glucosamine (147 mg, 0.67 mmol) and
2
(110 mg,
0.8 mmol) gave 196 mg of 16 as white amorphous solid (86%). 1H
NMR (400 MHz, CD3OD): d 7.42–7.40 (m, 2H, Ar), 7.36–7.30 (m,
3H, Ar), 4.68 (d, 1H, J = 10.0 Hz, ArCH2a), 4.62 (d, 1H, J = 10.0 Hz,
ArCH2b), 4.19 (d, 1H, J1,2 = 9.8 Hz, H-1), 3.95 (t, 1H, J1,2 = 9.8 Hz,
H-2), 3.85 (dd, 1H, J6a,6b = 12.1, J5,6a = 2.2 Hz, H-6a), 3.69 (dd, 1H,
J6a,6b = 12.1, J5,6b = 5.3 Hz, H-6b), 3.42 (t, 1H, J3,4 = 8.9, J2,3 = 9.8 Hz,
H-3), 3.33 (t, 1H, J4,5 = 9.5, J3,4 = 8.9 Hz, H-4), 3.23 (ddd, 1H, J4,5
=
9.5, J5,6b = 5.3, J5,6a = 2.2 Hz, H-5), 2.72 (s, 3H, NCH3), 2.01 (s, 3H,
Ac); 13C NMR (100 MHz, CD3OD): d 173.4, 138.1, 130.7 (2), 129.3
(2), 129.2, 93.7, 79.6, 77.6, 76.1, 71.6, 62.8, 54.1, 39.1, 23.1; HRMS
m/z calcd for C16H24N2O6Na [M+Na]+: 363.1526, found 363.1530.
5.17. N-Methyl-O-(N0-benzylacetamide)-N-(2-acetamido-2-
deoxy-b-D-glucopyranosyl) hydroxylamine (17)
Using the same procedure as described for compound 10, N-
acetyl- -glucosamine (29 mg, 0.13 mmol) and (31 mg,
D
3
0.16 mmol) gave 44 mg of 17 as white amorphous solid (85%). 1H
NMR (400 MHz, CD3OD): d 7.34–7.28 (m, 4H, Ar), 7.26–7.22 (m,
1H, Ar), 4.47 (d, 1H, J = 14.8 Hz, OCH2a), 4.42 (d, 1H, J = 14.8 Hz,
OCH2b), 4.27 (d, 1H, J = 14.7 Hz, ArCH2a), 4.25 (d, 1H, J1,2 = 9.7 Hz,
H-1), 4.18 (d, 1H, J = 14.7 Hz, ArCH2b), 3.89 (dd, 1H, J6a,6b = 11.9,
J5,6a = 2.1 Hz, H-6a), 3.82 (t, 1H, J = 9.7 Hz, H-2), 3.71 (dd, 1H,
J6a,6b = 11.9, J5,6b = 5.4 Hz, H-6b), 3.42 (dd, 1H, J4,5 = 9.9, J3,4
=
8.5 Hz, H-3), 3.30 (under CD3OD, H-4), 3.25 (ddd, 1H, J4,5 = 9.6,
J5,6b = 5.4, J5,6a = 2.1 Hz, H-5), 2.75 (s, 3H, NCH3), 1.95 (s, 3H, Ac);
13C NMR (100 MHz, CD3OD): d 173.5, 171.9, 139.8, 129.6 (2),
128.6 (2), 128.3, 93.6, 79.7, 77.3, 72.9, 71.9, 62.7, 54.1, 43.6, 38.6,
23.0; HRMS m/z calcd for C18H27N3O7Na [M+Na]+: 420.1741,
found: 420.1726.
49. Egberink, H.; Van Heerden, C. Anal. Chim. Acta 1980, 118, 359–368.