Nucleophilic catalysis of carbohydrate oxime formation by anilines

Article abstract of DOI:10.1021/jo902425v

("Figure Presented") Chemoselective formation of glycoconjugates from unprotected glycans is needed to further develop chemical biology involving glycans. Carbohydrate oxime formation is often slow, and organocatalysis by anilines would be highly promisin

Full text of DOI:10.1021/jo902425v

pubs.acs.org/joc
termed oximes. The obtained oximes are in equilibrium with
Nucleophilic Catalysis of Carbohydrate
Oxime Formation by Anilines
closed-ring N-glycosyloxyamines.2-4 Carbohydrate oxime
chemistry has been applied to, e.g., neo-glycopeptides,5
solution-phase tagging,6 microarrays,3,7,8 nanoparticles,4,9
and solid supports10,11 and generally displays high chemo-
selectivity and high yields; however, reaction rates are signi-
ficantly decreased in certain cases, especially for carbo-
hydrate electrophiles containing a 2-acetamido group (e.g.,
GlcNAc).11 Rate enhancements of oxime formation involving
exclusively open-chain reactants and products may be
obtained by nucleophilic catalysis, as reported initially by
Cordes and Jencks12 and recently revitalized by Dawson and
co-workers,13 for benzaldehyde and N-glyoxal electrophiles,
respectively. However, reactions of reducing glycans 1
and aniline under aqueous conditions are known to yield
N-phenylglycosylamines 4 (Keq∼1 at pH 4-5),14 indicating
that catalytic efficiency could be rather different from oxime
formation with strictly open-chain aldehydes (Scheme 1).
Specifically, the application of reducing glycans as electro-
philes in catalytic oxime formation includes additional equi-
libria due to (i) the dynamic masking of the reactive aldehydo
tautomer 2 as the cyclic pyranose (and furanose) hemiacetals
1 (>99.9%) and hydrate form (omitted in Scheme 1 for
clarity) and (ii) the trapping of iminium ions 3 as
(protonated) glycosyl amines 4, both of which involve po-
tentially rate-limiting intermediates.12,15 The high reactivity
of the imine intermediates is thought to be largely a result of
the ease of protonation of these species relative to the
carbonyl species, where subsequent elimination of aniline is
fast.12,16
Mikkel B. Thygesen,†, Henrik Munch,†, Jørgen Sauer,†,‡
Emiliano Clo, Malene R. Jørgensen, Ole Hindsgaul,
and Knud J. Jensen*,†,‡
§
§
ꢀ †
†IGM -Bioorganic Chemistry, Faculty of Life Sciences,
University of Copenhagen, Thorvaldsensvej 40, DK-1871
Frederiksberg, Denmark, ‡Centre for Carbohydrate
Recognition and Signalling, and §Carlsberg Laboratory,
Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
These authors contributed equally to this work.
kjj@life.ku.dk
Received November 12, 2009
Chemoselective formation of glycoconjugates from un-
protected glycans is needed to further develop chemical
biology involving glycans. Carbohydrate oxime forma-
tion is often slow, and organocatalysis by anilines would
be highly promising. Here, we present that carbohydrate
oxime formation can be catalyzed with up to 20-fold
increases in overall reaction rate at 100 mM aniline.
Application of this methodology provided access to
complex glycoconjugates.
(4) Thygesen, M. B.; Sauer, J.; Jensen, K. J. Chem.;Eur. J. 2009, 15,
1649–1660.
(5) Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem., Int. Ed. 1996, 35,
1230–1232. Zhao, Y. M.; Kent, S. B. H.; Chait, B. T. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 1629–1633. Spetzler, J. C.; Høeg-Jensen, T. J. Peptide Sci.
1999, 5, 582–592. Vila-Perello, M.; Gallego, R. G.; Andreu, D. ChemBio-
Chem 2005, 6, 1831–1838. Jimenez-Castells, C.; de la Torre, B. G.; Andreu,
D.; Gutierrez-Gallego, R. Glycoconjugate J. 2008, 25, 879–887.
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An important basis for the functional study of carbohy-
drate interactions and (automated) analysis of carbohydrate
mixtures is the access to well-defined glycoconjugates, in
solution or bound to solid phases.1 Preferably, these glyco-
conjugates should be constructed in highly chemoselective
reactions and in high yields. One methodology entails cova-
lent coupling of reducing glycans with aminooxy nucleo-
philes to form carbohydrate oxime O-ethers, commonly
ꢀ
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1752 J. Org. Chem. 2010, 75, 1752–1755
Published on Web 02/04/2010
DOI: 10.1021/jo902425v
r
2010 American Chemical Society

Thygesen et al.
JOCNote
SCHEME 1. Proposed Main Reaction Pathways of Nucleo-
philic Catalysis of Carbohydrate Oxime Formation at pH 4-5
SCHEME 2. Oxime Coupling with O-Benzylhydroxylamine
Herein we report for the first time that carbohydrate
oxime formation is catalyzed by nucleophiles such as aniline
in analogy with oxime formation from open-chain alde-
hydes. Kinetic studies showed a dramatic increase in reaction
rate in the presence of excess aniline, although the kinetic
efficiency (i.e., the kcat) was lower for carbohydrate oximes
than in reported cases of strictly open-chain reactants.
Catalytic effects were demonstrated in all investigated cases,
both in solution and on solid phase.
We based our kinetic experiments on previous studies of
aldose oxime formation with hydroxylamine (Scheme 1, R =
H) showing that these reactions typically proceed with a rate
optimum at pH 4-5 (coinciding with the pKa of the anili-
nium ion of 4.6), and with increasing equilibrium constants
with increasing pH in the pH range 2-5.15 The pH rate
optimum partly reflects a change in rate-determining step
from the dehydration of the carbinol oxyamine intermediate
(5) at higher pH to the nucleophilic attack on the aldehydo
tautomer (2) at lower pH. In the pH range 4-5, however, the
kinetics are also influenced by the ring-chain tautomerism
(mutarotation) of reducing glycans, which is catalyzed by
acid or base on either side of pH 4.6.17 Accordingly, we
first investigated oxime coupling between D-glucose and
O-benzylhydroxylamine in acetate buffer pH 4.6 at 25 °C
to allow facile monitoring of formation of oxime 918 by
HPLC (Scheme 2).
Initially, reactions were performed under equimolar con-
ditions at 1 mM reactants; however, the observed reaction
rates and product conversions (i.e., Keq) were generally too
low for straightforward kinetic analyses. In order to increase
the reaction rates we performed reactions with 10 equiv of
carbohydrate (10 mM) and the catalytic effect was assessed
under increasing concentrations of aniline (0, 5, 10, 20, and
100 mM). These conditions allowed the determination of
comparative rate constants from simple pseudo-first-order
kinetic treatments of the data (R2 > 0.99). The reaction is
first-order in electrophile (i.e., 2 or 3) even though glucose is
in excess. Measured kobs values displayed an approximately
linear increase with aniline concentration from 0.13 h-1 in
the absence of aniline to 2.6 h-1 in the presence of 100 mM
FIGURE 1. Kinetic studies of oxime coupling between D-glucose
(10 equiv.) and O-benzylhydroxylamine (1 mM) under nucleophilic
catalysis by different concentrations of aniline: 100 mM ((), 20 mM
(1), 10 mM (2), 5 mM (b), and no aniline (9) in 100 mM acetate
buffer pH 4.6. Solid lines represent fitting to pseudo-first-order
kinetics.
aniline corresponding to an overall 20-fold rate increase
(Figure 1). Thus, catalytic efficieny is substantial albeit
approximately 1 order of magnitude lower than for strictly
open-chain aldehydes.13
In analogy with reported reaction mechanisms, the expec-
ted rate-limiting step is the formation of an N-phenylgluco-
syliminium species.12 However, this species is a common
intermediate for formation of oxime product 9 and N-
phenylglucosylamine 10. Indeed, we observed concurring
formation of 10 during the oxime coupling reaction, reaching
levels of 10-15% conversion based on glucose in the pre-
sence of 100 mM aniline. Plots of initial rates of formation19
of 9 versus 10 revealed that glycosylamine formation became
increasingly dominant at higher aniline concentrations
(Supporting Information). Mutarotation20 and hydro-
lysis14,21 studies of N-phenylglucosylamines have shown that
the equilibrium between 10 and the imine intermediate is fast
at pH 4-5 relative to the mutarotation of glucose, and
preformed N-phenylglucosylamines have been reported as
imine precursors.22 Our results indicate, however, that glyco-
sylamine (4) formation at higher aniline concentrations
causes a lowering of the observed catalytic efficiency in
carbohydrate oxime formation due to the deprivation of
the reactive iminium intermediate (3). In agreement here-
with, the catalytic efficiency was lowered further to a ∼10-
fold increase at 100 mM aniline at high nucleophile condi-
tions (10 equivalents, 10 mM) (Supporting Information).
Next, we applied nucleophilic catalysis to neo-glycopep-
tide synthesis with galacturonic acid on Leu-Enkephalin
derived peptide 11 containing an N-terminal aminooxyacetyl
functionality (Table 1). After 1 h at 25 °C, a 3-fold increase in
conversion to 12 was observed in the presence of 100 mM
(19) Jencks, W. P. In Catalysis in Chemistry and Enzymology, 1st ed.;
McGraw-Hill: New York, 1969; pp 555-599.
(20) Pigman, W.; Cleveland, E. A.; Couch, D. H.; Cleveland, J. H. J. Am.
Chem. Soc. 1951, 73, 1976–1979.
(17) Isbell, H. S.; Pigman, W. W. J. Res. Nat. Bur. Stand. 1938, 20, 773–
798.
(18) Plenkiew, J; Szarek, W. A.; Sipos, P. A.; Phibbs, M. K. Synthesis
1974, 56–58.
(21) Capon, B.; Connett, B. E. J. Chem. Soc. 1965, 4497–4502.
(22) Bognar, R.; Nanasi, P. Nature 1953, 171, 475–476. Bognar, R.;
Nanasi, P. J. Chem. Soc. 1955, 189–193.
J. Org. Chem. Vol. 75, No. 5, 2010 1753

Thygesen et al.
JOCNote
TABLE 1. Neoglycopeptide Synthesis with GalAa
TABLE 2. Capture of Glycans on Heterobifunctional Linkersa
entry
catalystb
temp (°C)
time (h)
conversionc (%)
temp
entry glycan catalystb (°C)
time
(h)
conditions,
product
conver-
sionc (%)
1
2
3
4
5
6
7
25
25
25
25
60
60
60
1
70
1
6
1
30
96
87
90
79
91
95
aniline
aniline
1
2
3
4
5
6
7
8
9
Glc
Glc
Glc
Glc
Lac
Lac
Glc
Glc
Glc
25
25
25
25
50
50
25
25
60
25
25
25
25
25
60
25
25
25
25
25
25
25
25
25
25
25
3
3
18
18
3
3
1
1
1
6
6
16
1
1
1
6
6
48
1
a, 14a
a, 14a
a, 14a
a, 14a
b, 14b
b, 14b
c, 16a
c, 16a
c, 16a
c, 16a
c, 16a
c, 16a
c, 16b
c, 16b
c, 16b
c, 16b
c, 16b
c, 16b
c, 16c
c, 16c
c, 16c
c, 16d
c, 16d
c, 16d
c, 16e
c, 16e
14
43
52
84
62
100
35
88
97
90
100
74d
7
20
72
aniline
aniline
aniline
aniline
2
aniline
0.17d
aConditions: 11 (2.5 mM), GalA (6.5 mM), 100 mM NH4OAc buffer
pH 4.5. b100 mM. cBy HPLC. d10 min.
aniline. Gentle heating (60 °C) promoted a similar increase in
reaction rate, and aniline catalysis at 60 °C provided a
shortening of reaction time from 70 h to 10 min.
10 Glc
11 Glc
12 Glc
13 GlcNAc
14 GlcNAc aniline
15 GlcNAc
16 GlcNAc
17 GlcNAc aniline
18 GlcNAc
19 Gal
aniline
A series of experiments were conducted to investigate the
potential of nucleophilic catalysis of carbohydrate oxime
formation with heterobifunctional linkers containing amino
(138) or thiol (154) groups for anchoring to various surfaces.
The catalytic effect was assessed by comparison of reactant
conversion in the presence or absence of 100 mM aniline. The
observed increase in conversion levels of reaction of 13 was
3-fold with glucose (14a) and 2-fold with lactose (14b8) after
3 h. Reactions with 15 were performed in H2O-MeCN 1:2
due to the apolar nature of the nucleophile and with 300 mM
acetic acid (pH ca. 4.3). For glucose (16a4), the conversion
level was almost 3-fold higher after 1 h in the presence of
aniline, and the conversion for the less reactive N-acetyl-
glucosamine (16b) was 2-3-fold higher after 1 and 6 h. In
cases of galactose (16c), mannose (16d), and galacturonic
acid (16e), the uncatalyzed reactions proceeded too rapidly
for an evaluation of the catalytic effect, although in all cases
the conversion levels in the presence of aniline were higher.
The reaction rates for the various monosaccharides in
Table 2 follow the trends Man ≈ Gal > Glc and GlcA >
Glc > GlcNAc in accordance with previous data,15 confirming
that ring-chain tautomerism23 (to produce open-chain
aldehydo form) influences the overall rate of oxime formation.
It is worthy to note that conditions involving a raise of the
temperature to 50 or 60 °C in the absence of aniline in some
cases provided increases in reaction rate superior to aniline
catalysis conditions; however, in some of these cases addi-
tional byproducts were apparent after longer reaction times.
We subsequently applied carbohydrate oxime coupling
with linker 15 for chemoselective labeling of a complex
lipochitin oligosaccharide24 (nodulation factor) isolated
from Mesorhizobium loti. Oxime coupling with the isolate,
which contained a mixture of three main species (two
monoacetylated and a nonacetylated), could be accom-
plished under very mild conditions in 25 mM acetate buffer
37
80
69d
90
20 Gal
21 Gal
aniline
aniline
1
16
1
1
16
1
100
73d
88
100
75d
100
84d
22 Man
23 Man
24 Man
25 GalA
26 GalA
16
aConditions: (a) 13 (50 mM), glycan (150 mM), ammonium acetate
buffer pH 4.5; (b) 13 (5 mM), glycan (5 mM), ammonium acetate buffer
pH 4.5; (c) 15 (20 mM), glycan (40 mM), HOAc (300 mM), H2O-MeCN
1:2. b100 mM. cBy HPLC. dIsolated yield.
pH 4.6 at 25 °C in the presence of 100 mM aniline. Conver-
sion to oxime 17 (Scheme 3) was estimated by HPLC at 73%
(55% isolated yield) after 19 h as compared to only 8% in the
absence of aniline.
Finally, we examined the effect of added aniline in the
protocol for solid-phase oligosaccharide tagging (SPOT)
reported by Lohse et al.11 Specifically, amino-functionalized
controlled-pore glass (CPG) was derivatized through an
ester linkage to the p-carboxyl group of benzylhydroxyl-
amine (18, Scheme 4) at a density of 68 μmol/g. A solution of
Gal, GlcNAc, and maltoheptose (G7) (100 μM each) was
incubated overnight with beads under two different sets of
conditions expected to result in the trapping of Gal (19a),
GlcNAc (19b), and G7 (19c) as the oximes. The efficiency of
capture was evaluated following reduction, tagging with
tetramethylrhodamine isothiocyanate (TMR-NCS), and
cleavage using aqueous LiOH, and the products 20a-c were
quantitated by fluorescence detection in capillary electro-
phoresis (Table 3) as previously described.11 Under ideal
conditions for oxime formation (pH 4.5), aniline improved
the capture efficiency for the more difficult sugars but less
than 3-fold. At pH 7.0, conditions reported to be favorable
for 4-anisidine catalysis, the improvement was much
more dramatic for both aniline and 4-anisidine though not
(23) Zhu, Y. P.; Zajicek, J.; Serianni, A. S. J. Org. Chem. 2001, 66, 6244–
6251.
(24) Bek, A. S.; Sauer, J.; Thygesen, M. B.; Duus, J. Ø.; Petersen, B. O.;
Thirup, S.; James, E.; Jensen, K. J.; Stougaard, J.; Radutoiu, S. Mol. Plant-
Microbe Interact. 2010, 23, 58–66.
1754 J. Org. Chem. Vol. 75, No. 5, 2010

Thygesen et al.
JOCNote
SCHEME 3. Labeling of Isolated Lipochitin Oligosaccharide As Glycan Oxime
SCHEME 4. Solid-Phase Oligosaccharide Tagginga
aA carbohydrate mixture of Gal (a), GlcNAc (b) and maltoheptaose (G7) (c) at 100 μM each was used. The corresponding oximes (19a-c) were reduced
(8 M borane-pyridine), tagged (TMR-NCS), and cleaved by saponification to give 20a-c as described.11 Conditions: (a) 100 mM acetate buffer pH 4.5;
(b) 100 mM phosphate buffer pH 7.0.
TABLE 3. Aniline Catalysis of Solid-Phase Oligosaccharide Tagginga
minimum 10 min) were prepared by dissolution of the solids in
100 mM acetate buffer pH 4.6. Aniline solutions (0, 10, 20, 40, or
200 mM) were prepared by dissolution in 100 mM acetate buffer
followed by readjustment to pH 4.6 with acetic acid. Reactions
were initiated by mixing the above three solutions in the ratio
1:1:2 (v/v/v) to provide final concentrations of 1 mM oxyamine,
10 mM carbohydrate, and 0, 5, 10, 20, or 100 mM aniline.
Formation of 9 and 10 was monitored by HPLC.
Neoglycopeptide 12. To peptide 11 (1 μmol) was added
D-galacturonic acid (3 μmol) in 100 mM acetate buffer pH 4.5.
The conversion to the product was followed by HPLC.
Oxime Coupling with Heterobifunctional Linker 13. All coup-
ling reactions were conducted in 100 mM acetate buffer pH 4.5.
Conditions a: solutions of 13 (50 mM) and glucose (150 mM)
were shaken at room temperature either in the absence or
presence of aniline (100 mM). Product (14a) conversion was
monitored by HPLC-MS. Conditions b: solutions of linker 13
(5 mM) and lactose (5 mM) were shaken at 50 °C either in the
absence or presence of aniline (100 mM). Product (14b) conver-
sion was monitored by HPLC-MS.
Oxime Coupling with Heterobifunctional OEG Linker 15.
Reactions were conducted in water-acetonitrile 1:2 with 15
(20 mM), glycan (40 mM), and acetic acid (300 mM). Reactions
were performed either at (i) room temperature in the absence of
aniline, (ii) 60 °C in the absence of aniline, or (iii) room tempe-
rature in the presence of aniline (100 mM). Product (16a-e)
formation was monitored by HPLC-MS over periods of 48 h
Products were isolated by vacuum liquid chromatography
(methanol-dichloromethane 1:20 f 1:8).
Lipochitin Oxime 17. Coupling of lipochitin oligosaccharide
(3.3 mM) isolated from M. loti24 with 15 (6.6 mM) through
oxime formation was accomplished in water-acetonitrile 1:1,
100 mM aniline, 25 mM acetate buffer pH 4.6. Compound 17
was purified by preparative HPLC.
conversion (%)c
entry
catalystb
conditions
20a
20b
20c
1
2
3
4
5
6
a
a
a
b
b
b
100
100
83
19
74
18
51
38
0
9
29
38
53
42
0
20
40
aniline
4-anisidine
aniline
4-anisidine
80
aConditions: (a) 100 μM glycan mixture, 100 mM acetate buffer
pH 4.5, 50 °C; (b) 100 μM glycan mixture, 100 mM phosphate buffer
pH 7.0, 50 °C. b100 mM. cBy capilliary electrophoresis.
reaching the coupling efficiency observed at pH 4.5, in line
with previous reports.13
In conclusion, we have shown that carbohydrate oxime
formation can be catalyzed by anilines. Overall reaction rates
were increased up to 20-fold in the presence of 100 mM
catalyst. Concurring formation of the N-phenylglycosyl-
amines accounted for up to 15% (based on carbohydrate)
under the investigated conditions and likely contributes to the
lowering of the catalytic efficiency compared to catalysis of
oxime formation with strictly open-chain aldehydes. Never-
theless, significant rate enhancements were demonstrated in a
range of practical application of glycan capture. Reactions
were also well promoted by moderate heating (50-60 °C);
however, additional byproducts were apparent after longer
reaction times even for monosaccharides, and further degra-
dation may be expected with increasingly complex glycans.
The findings described herein are particularly useful for coup-
ling reactions involving complex glycans available in small
quantities and glycans containing labile decorations. As expec-
ted and in support of the mechanism in Scheme 1, preliminary
experiments confirm that these results are readily extendable
to carbohydrate hydrazones, N-glycosylhydrazides, as well as
N-glycosyl-N-(alkyl)oxyamines (data not shown).
Acknowledgment. Financial support to K.J.J. from The
Danish Council for Strategic Research, the Danish National
Research Foundation, and the Danish Council for Indepen-
dent Research-Technology and Production Sciences is
acknowledged.
Experimental Section
Kinetic Measurements on Formation of D-Glucose O-Benzyl-
oxime (9). Separate solutions of O-benzylhydroxylamine hy-
drochloride (4 mM) and R-D-glucose (40 mM, equilibrated15
Supporting Information Available: General experimental
methods, compound characterization, and kinetic data. This mate-
rial is available free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 5, 2010 1755