Regenerative labeling of saccharides

Article abstract of DOI:10.1039/c3ra41426d

A simple method for regenerative labeling of saccharides is devised by using (naphth-2-ylmethoxy)amine or N-methyl-(naphth-2-ylmethoxy)amine. The naphthyl group is optically responsive to facilitate the separation of labeled saccharides, which are subsequently reverted to the parental saccharides by one-pot hydrogenolysis-hydrolysis using co-catalysts Pd(OH)₂ and HCl under an atmosphere of H₂. The mechanistic study indicates that the naphthylmethyl group plays an essential role in the initial hydrogenolysis, followed by in situ hydrolysis, to regenerate the saccharides.

Full text of DOI:10.1039/c3ra41426d

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Regenerative labeling of saccharides3
Hao-Yu Wen,a Peng-Hao Hsu,a Guei-San Chenb and Jim-Min Fang*ab
Cite this: RSC Advances, 2013, 3, 9530
A simple method for regenerative labeling of saccharides is devised by using (naphth-2-ylmethoxy)amine
or N-methyl-(naphth-2-ylmethoxy)amine. The naphthyl group is optically responsive to facilitate the
separation of labeled saccharides, which are subsequently reverted to the parental saccharides by one-pot
hydrogenolysis–hydrolysis using co-catalysts Pd(OH)2 and HCl under an atmosphere of H2. The mechanistic
study indicates that the naphthylmethyl group plays an essential role in the initial hydrogenolysis, followed
by in situ hydrolysis, to regenerate the saccharides.
Received 6th November 2012,
Accepted 9th April 2013
DOI: 10.1039/c3ra41426d
www.rsc.org/advances
method is to regenerate the native oligosaccharides with
retention of the internal glycosidic bonds by a simple chemical
manipulation, i.e. tandem hydrogenolysis and hydrolysis in
Introduction
Carbohydrates play essential roles in living organisms, such as
in regulating cell–cell recognition and cellular developments.
To understand the exact mechanisms of complex carbohy-
drates in biological system, proper methods for analysis and
isolation are needed to gain fundamental information on their
structures and activities. However, carbohydrate molecules are
not easily tracked due to lack of a responsive chromophore.
The current techniques for separation of unmodified oligo-
saccharides rely on using gel permeation column chromato-
graphy and high pH anion exchange chromatography–pulsed
amperometric detection (HPAEC–PAD) in strong alkaline
conditions with special instrumentation. Alternatively, analy-
sis of carbohydrate composition can be facilitated by labeling
methods such as reductive amination,1–3 oxidative amidation,4
as well as oxime,5–8 hydrazide,9,10 thiazoline,11 and imidazole
formation.12,13
However, regeneration of parental carbohydrates from the
labeled derivatives remains a challenging task. There are some
methods for the labeling and reversion of saccharides,14–21
though most reports are demonstrated for analytic purposes
without details of preparation procedures, product yields or
rigorous structural determination. In this study, we wish to
address the issue of the regenerative labeling of saccharides
using (naphth-2-ylmethoxy)amine (reagent a) and N-methyl-
(naphth-2-ylmethoxy)amine (reagent b) (Fig. 1). The naphtha-
lene-containing tag, as the example in our initial study, not
only provides sensitive chromogenic/fluorescence detection of
the labeled saccharides but also exhibits hydrophobic proper-
ties to balance the hydrophilic nature of saccharides for better
separation with HPLC. The most attractive feature of this
the presence of catalytic amounts of Pd(OH)2 and HCl(aq)
.
Results and discussion
In our first approach, the condensation reaction of D-glucose
with reagent a was conducted in pyridine at 60 uC (method A)
to afford the oxime ether Glc-a (Table 1, entry 1). For the
purpose of isolation and structural elucidation, Glc-a was
subsequently treated with Ac2O to give peracetyl-D-glucose O-
(naphth-2-ylmethyl)oxime
(Glc-a)Ac5
(see
Electronic
Supplementary Information, ESI ). After a considerable study,
3
we found that the oxime ether Glc-a was smoothly reverted to
glucose under the hydrogenolysis–hydrolysis conditions using
Pd(OH)2 and HCl(aq) as co-catalysts (1 atm H2, 25 uC, 1 h, in
H2O). The reaction conditions for regeneration of saccharides
were very mild without interfering with the OAc or CO2H
groups (entry 7). Furthermore, no epimerization at the C-2
position of mannose occurred during the labeling–reversion
1
process (entry 3) according to the H NMR analysis (Fig. S1 in
aDepartment of Chemistry, National Taiwan University, Taipei 10617, Taiwan
E-mail: jmfang@ntu.edu.tw; Fax: (8862)23637812; Tel: (8862)33661663
bThe Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan
Fig. 1 Labeling and reversion of saccharides, using D-glucose as an example,
with (naphth-2-ylmethoxy)amine (reagent a) and N-methyl-(naphth-2-
ylmethoxy)amine (reagent b).
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
c3ra41426d
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Table 1 Labeling and reversion of saccharides using (naphth-2-ylmethoxy)amine (reagent a) and N-methyl-(naphth-2-ylmethoxy)amine (reagent b)
Entry
Saccharide
Methodabcd
Labeling product (yield, %)
Reversion yield (%)e
1
2
3
4
5
6
7
8
Glc
Glc
Man
Man
Xyl
Fru
GlcA
Lac
A
C
A
C
C
A
B
A
C
B
B
D
B
D
Glc-a (—)f
Glc-b (83)
Man-a (—)f
Man-b (—)f
Xyl-b (91)
Glc (83)g
Glc (82)
Man (83)g
Man (56)g
Xyl (81)
Fru-a (—)f
GlcA-a (67)
Lac-a (—)f
Lac-b (—)f
Man2-a (75)
Man5-a (64)
Glc6-b (76)
Glc7-a (73)
Glc7-b (68)
Fru (88)g
GlcA (77)
Lac (84)g
Lac (82)g
Man2 (70)
Man5 (72)
Glc6 (70)
Glc7 (64)
Glc7 (78)
9
Lac
10
11
12
13
14
Man2
Man5
Glc6
Glc7
Glc7
a
b
Method A: reagent a, pyridine, 60 uC, 3–12 h. Method B: reagent a, ammonium acetate buffer containing 20% MeOH, 60 uC, 3–12 h.
e
c
d
Method C: reagent b, pyridine, 60 uC, 3–24 h. Method D: reagent b, ammonium acetate buffer containing 20% MeOH, 60 uC, 3–12 h. The
f
labeling product in H2O was stirred with catalytic amounts of Pd(OH)2 and HCl under H2 (1 atm) at 25 uC for 1 h. The labeling product was
not isolated, but characterized by its peracetyl derivative. The overall yield of the two steps, for labeling and reversion.
g
ESI ). The oxime ether Lac-a was similarly prepared from
derivatives Glc6-b and Glc7-b, which were found to be well
separated on a reversed-phase HC-C18 column by elution with
gradients of CH3CN and H2O (Fig. 2).
3
lactose (entry 8). The condensation reactions of mannobiose,
mannopentaose and maltoheptaose with reagent a were
performed in ammonium acetate buffer containing 20%
MeOH (method B) to circumvent the problem of poor
solubility in pyridine, giving oxime ethers Man2-a, Man5-a
and Glc7-a. The oxime ethers were readily reverted to their
parental oligosaccharides with retention of the internal
glycosidic bonds by hydrogenolysis and in situ hydrolysis
(Fig. 1).
When a saccharide reacts with reagent a, the oxime product
might exist as a mixture of E- and Z-isomers, which might also
undergo cyclization to give N-alkoxy-glycosylamine.22–25
Indeed, the condensation product of maltoheptaose with
reagent a showed three isomers according to the HPLC
The one-pot hydrogenolysis–hydrolysis reaction for regen-
eration of saccharides was conducted in very mild conditions
to retain the internal glycosidic bonds and other functional
groups, such as acetyl and carboxyl groups. The reaction
mechanism via direct acid-catalyzed hydrolysis is less likely.
To distinguish whether the hydrogenolysis occurred at the N–
O bond or at the ArCH2–O bond, we treated (Glc-a)Ac5, the
oxime ether of peracetylglucose, with catalytic amounts of
Pd(OH)2 and HCl(aq) in MeOH under an atmosphere of H2. The
corresponding peracetylglucose oxime (Glc-oxime)Ac5 was
analysis (Fig. S2 in ESI ). To alleviate the problem of separation
3
of the isomers, we further explored another protocol using
reagent b as the labeling agent. For example, D-glucose was
treated with reagent b in pyridine at 60 uC (method C) to give
exclusively
N-methyl-N-(naphth-2-ylmethoxy)glucosylamine
(Glc-b) as a single isomer with the b-configuration (entry 2).
The condensation reactions of xylose, lactose, maltohexaose
and maltoheptaose with reagent b also proceeded smoothly to
give exclusively the corresponding glycopyranosylamines Xyl-b,
Lac-b, Glc6-b and Glc7-b in b-configuration (entries 5, 9, 12 and
14). The glycosylamines were readily reverted into their
parental saccharides by in situ hydrogenolysis–hydrolysis
using co-catalysts Pd(OH)2 and HCl(aq). However, the con-
densation reaction of mannose with reagent b proceeded
sluggishly to give Man-b as a mixture of a/b-anomers (entry 4),
presumably due to the steric effect between the C-2 axial
hydroxyl group and the incoming reagent b.
Fig. 2 HPLC analysis of N-alkoxy-N-methyl-glycosylamines prepared with
reagent b: (a) maltoheptaose derivative Glc7-b; (b) a 1 : 1 mixture of
maltohexaose derivative Glc6-b (tR 17.8 min) and maltoheptaose derivative Glc7-
b (tR 17.5 min). Loading: 1.5 6 1025 mol of each component in 20 mL solution
on a reversed-phase HC-C18 column (5 mm porosity, 4.6 mm inner diameter,
250 mm length). Flow rate: 1 mL min21; elution gradients: 0–20% CH3CN–H2O
for 10 min, 20–70% CH3CN–H2O for 20 min, and 100% CH3CN for 15 min. UV
detection at wavelength of 260 nm .
To demonstrate a potential application of our present
method, a mixture of maltohexaose and maltoheptaose (1 : 1)
was treated with reagent b in ammonium acetate buffer
containing 20% MeOH (method D) to give the glycosylamine
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obtained in 80% isolated yield, indicating that the hydro-
genolysis occurred at the ArCH2–O bond, instead of the N–O
bond. In contrast, N-methoxy-N-methyl peracetylglucosyla-
mine, an analog of (Glc-b)Ac4 having an N-methoxy group in
lieu of the naphthylmethoxy group, was inert in the conditions
containing Pd(OH)2 and HCl(aq). Taken together, the experi-
mental evidence supports a mechanism (Fig. 3) in which the
labeled saccharide derivatives undergo an initial hydrogeno-
lysis of the ArCH2–O bond, followed by an acid-catalyzed
hydrolysis in situ to regenerate the parental saccharides.
Reversion of the tagged compounds to the native carbohy-
drates can be realized by using co-catalysts Pd(OH)2 and
HCl(aq). Our current method thus provides a new approach to
label and regenerate oligosaccharides.
Experimental
General
Reactions were monitored by TLC on silica gel plates using
p-anisaldehyde or H2SO4 as the visualizing agent. The melting
points were not corrected. The chemical shifts in the NMR
spectra are given in ppm relative to dH 7.24/dC 77.0 for CDCl3,
dH 3.31/dC 49.15 for CD3OD and dH 4.80 for D2O.
Conclusions
We have developed a simple labeling method to introduce an
optically responsive naphthyl tag to the (oligo)saccharides; the
subsequent hydrogenolysis–hydrolysis of the labeled com-
pounds with co-catalysts Pd(OH)2 and HCl(aq) smoothly
regenerates parental (oligo)saccharides. This method can be
applied to facilitate the separation of bioactive oligosacchar-
ides from natural sources, followed by recovery of the parental
oligosaccharides for evaluation of their bioactivities. The
naphthyl moiety in reagents a and b may be modified by
substituents or replaced by other appropriate (hetero)aromatic
fluorophores26 to cope with the need of detection and
separation. Nevertheless, our present protocol still requires
elaboration to minimize the reaction scale and to improve the
yield in order to deal with complex carbohydrates in trace
amounts. One possible approach is to conjugate reagent b to
biotin or magnetic nanoparticles, via a cleavable linker, for
efficient extraction/enrichment of target carbohydrates from a
sample of mixture.5,27–29 After cleaving the linker, the released
carbohydrates tagged by reagent b can be further separated by
HPLC with the assistance of the optically responsive tag.
O-(Naphth-2-ylmethoxy)amine (reagent a)
A solution of 2-(bromomethyl)naphthalene (3.32 g, 15 mmol)
in DMF (10 mL) was added dropwise to a solution of
N-hydroxylphthalimide (2.7 g, 16.5 mmol) and DBU (2.53 g,
16.6 mmol) in DMF (20 mL) at room temperature. The mixture
was stirred for 2 h, poured into 1 M aqueous HCl (300 mL).
The precipitate was collected by filtration, rinsed with water,
and dissolved in CH2Cl2 (100 mL). Hydrazine (64% in water,
1.55 mL, 30 mmol) was added at room temperature. The
mixture was stirred for 1 h, Et2O (100 mL) was added, and
stirred for another 1 h. The precipitate was filtered off. The
filtrate was concentrated and acidified by concentrated HCl (5
mL). The solids were collected and dried in vacuo to afford
reagent a (as the HCl salt, 1.83 g, 59%). Mp 239–243 uC; 1H
NMR (400 MHz, CD3OD) d 7.96–7.89 (4 H, m), 7.56–7.54 (3 H,
m), 5.22 (2 H, s); 13C NMR (100 MHz, CD3OD) d 135.4, 134.7,
131.8, 130.4, 130.0, 129.4, 129.0, 128.2, 127.9, 127.5, 78.5; ESI-
TOF-HRMS calcd for C11H12NO: 174.0919, found: m/z 174.0914
[M + H]+.
N-Methyl-(naphtha-2-ylmethoxy)amine (reagent b)
Under an atmosphere of argon, NaH (780 mg, 19.5 mmol, 60%
in mineral oil) was washed with anhydrous hexane, and a
solution of O-(tert-butyl)-N-hydroxy-N-methyl carbamate30 (1.73
g, 11.8 mmol) in DMF (10 mL) was added dropwise at 0 uC.
After 30 min, a solution of 2-(bromomethyl)naphthalene (2.98
g, 13.5 mmol) in DMF (10 mL) was added. The mixture was
stirred at room temperature for 20 h, and quenched by careful
addition of 1 M HCl (2 mL). The mixture was diluted with
hexane (90 mL), and washed with water and brine. The organic
phase was dried (MgSO4), filtered, and concentrated under
reduced pressure to give O-(tert-butyl)-N-methyl-N-(naphtha-2-
ylmethoxy) carbamate (1.56 g), which was treated with TFA (5
mL) at room temperature for 1 h. The mixture was neutralized
with Amberlyst A-21 resin (2OH), filtered, and concentrated
under reduced pressure to give reagent b (as the TFA salt, 1.61
1
g. 45%). Mp 105–108 uC; H NMR (400 MHz, CDCl3) d 9.05 (1
H, br s), 7.83–7.77 (4 H, m), 7.50–7.44 (2 H, m), 7.40 (1 H, dd, J
= 8.4, 1.6 Hz), 5.17 (2 H, s), 2.86 (3 H, s); 13C NMR (100 MHz,
CDCl3) d 133.7, 132.9, 129.4 (26), 129.0, 128.1, 127.7, 127.1,
126.7, 126.0, 76.6, 35.6; ESI-TOF-HRMS calcd for C12H14NO:
188.1075, found: m/z 188.1075 [M + H]+.
Fig. 3 Reaction mechanism for reversion of the labeled (oligo)saccharides via
tandem hydrogenolysis and hydrolysis.
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