Sodium N-Chlorobenzenesulfonamide as a Selective Oxidant for Hexosamines in Alkaline Medium: A Kinetic and Mechanistic Study

Article abstract of DOI:10.1021/jo971398t

Oxidation of D-mannosamine (1), D-glucosamine (2), and D-galctosamine (3) by sodium N-chlorobenzenesulfonamide or chloramine-B (CAB) at 313 K is followed by a shortening of carbon chain and obeys the rate law, rate = k[CAB][sugar][HO^-]^x, where x is less than unity. The products are arabinonic acid, ribonic acid, and erythronic acid for 1 and 2 with smaller amounts of glyceric and hexonic acids, while lyxonic and threonic acids are predominant in the oxidation of 3 with smaller amounts of glyceric and hexonic acids. Proton inventory studies made in a H₂)O-D₂)O mixture point toward a single transition state. In the proposed mechanism the alkoxy anion (S^-) of the hexosamine formed in a base-catalyzed reaction at C-1 carbon is subjected to an electrophilic rate-limiting attack by Cl⁺ of the oxidant. The hexonic acid formed is decarboxylated with loss of ammonia to form the respective pentose, which is further converted into the corresponding pentonic acid. The breaking of the bond between C-1 and C-2 carbons in pentose yields tetronic acids. The thermodynamic parameters for sugar alkoxy anion formation and activation parameters for the rate-limiting step have been evaluated.

Full text of DOI:10.1021/jo971398t

J . Org. Chem. 1998, 63, 531-536
531
Sod iu m N-Ch lor oben zen esu lfon a m id e a s a Selective Oxid a n t for
Hexosa m in es in Alk a lin e Med iu m : A Kin etic a n d Mech a n istic
Stu d y
Kanchugarakoppal S. Rangappa,* Manikanahally P. Raghavendra,
Dandinasivara S. Mahadevappa, and Doddegowda Channegowda†
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore - 570 006, India
Received J uly 28, 1997X
Oxidation of D-mannosamine (1), D-glucosamine (2), and D-galctosamine (3) by sodium N-
chlorobenzenesulfonamide or chloramine-B (CAB) at 313 K is followed by a shortening of carbon
chain and obeys the rate law, rate ) k[CAB][sugar][HO-]x, where x is less than unity. The products
are arabinonic acid, ribonic acid, and erythronic acid for 1 and 2 with smaller amounts of glyceric
and hexonic acids, while lyxonic and threonic acids are predominant in the oxidation of 3 with
smaller amounts of glyceric and hexonic acids. Proton inventory studies made in a H2O-D2O
mixture point toward a single transition state. In the proposed mechanism the alkoxy anion (S-)
of the hexosamine formed in a base-catalyzed reaction at C-1 carbon is subjected to an electrophilic
rate-limiting attack by Cl+ of the oxidant. The hexonic acid formed is decarboxylated with loss of
ammonia to form the respective pentose, which is further converted into the corresponding pentonic
acid. The breaking of the bond between C-1 and C-2 carbons in pentose yields tetronic acids. The
thermodynamic parameters for sugar alkoxy anion formation and activation parameters for the
rate-limiting step have been evaluated.
In tr od u ction
through the cleavage of both C-C and C-H bonds to
form a mixture of aldonic acids from these hexosamines.
On the basis of these data, a novel pathway for the
oxidation of hexosamines by CAB is proposed. It was
interesting to note that the oxidation products analyzed
by HPLC and GC-MS gave products which indicated that
oxidation went beyond the pentose stage. The products
were found to be the identical under both stoichiometric
and kinetic conditions.
The sodium salts of N-arylhalosulfonamides generally
known as organic haloamines are strong electrolytes1-3
in aqueous solution and behave as sources of halonium
cations. They are capable of effecting an array of
molecular transformations, including limited oxidation
of specific groups. The benzene analogue, chloramine-B
(C6H5SO2NClNa‚1.5H2O or CAB) is easy to prepare, and
it has been employed for the oxidation of diverse sub-
strates. An important reaction that has been well
documented is the decarboxylation and deamination of
amino acids.4 A survey of literature indicates limited
information5 on the mechanistic aspects of oxidation of
hexosamines by halogens. The hexosamines are oxidized
by NaOCl to the respective pentoses, thus decreasing the
carbon chain.6 Herbst7 reports the oxidation of hex-
osamines by chloramine-T to the corresponding pentoses,
but no mechanistic details are available. As a part of
our broad program on the oxidtion of monosaccharides
by the N-haloamines,5 we herein report the kinetic and
mechanistic aspects of the oxidation of three hex-
osamines, D-mannosamine, D-glucosamine, and D-galac-
tosamine, by CAB in alkaline medium at 40 °C. The
results of this study suggest that the oxidation occurs
Resu lts a n d Discu ssion
The kinetics of oxidation of hexosamines by CAB were
investigated at several concentrations of the reactants.
With hexosamine (S) in excess, plots of log [CAB] vs time
were linear (r > 0.9992, s e 0.01) indicating a first-order
dependence on [CAB]o. The pseudo-first-order rate con-
stants kobs calculated from these plots are given in Table
1. The values decrease slightly with varying [CAB]o
possibly owing to a side reaction8 involving the formation
of NaClO3 (3NaOCl f NaClO3 + 2NaCl). The rate
increases with increase in [S]o (Table 1) and a plot of log
kobs vs log[S]o was found to be linear (r > 0.9990, s e 0.01)
with unit slope, indicating a first-order dependence on
hexosamine as well. Further, a plot of kobs vs [S]o was
linear (r > 0.9980, s e 0.03) and passed through the
origin, showing that the sugar-oxidant complex has only
a transient existence. The rate of reaction shows a
fractional order dependence on [HO-] (Table 2), as plots
of log kobs vs log[HO-]eff were linear (r > 0.9988, s e 0.02)
with slopes less than unity.
* Author for correspondence.
† Department of Biochemistry and Molecular Biology, Georgetown
University Medical Center, Washington, DC 20007-2197.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
(1) Campbell, M. M.; J ohnson, G. Chem. Rev. 1978, 78, 65.
(2) Bishop, E.; J ennings, V. J . Talanta 1958, 1, 197.
(3) Mahadevappa, D. S.; Ananda, S.; Murthy, A. S. A.; Rangappa,
K. S. Tetrahedron 1984, 10, 1673.
(4) Gowda, B. T.; Mahadevappa, D. S. J . Chem. Soc., Perkin Trans.
2 1983, 323.
(5) Iyengar, T. A.; Puttaswamy; Mahadevappa, D. S. Carbohydr. Res.
1990, 197, 119; 1990, 204, 197.
Addition of the reaction products, benzenesulfonamide
(BSA) and Cl- ion did not alter the rate of reaction,
(6) Matsushima, Y. Sci. Pap. Osaka Univ. 1951, No. 31, 1 through
Chem. Abstr. 1952, 46, 7052.
(7) Herbst, R. M. J . Biol. Chem. 1937, 119, 85.
(8) Agarwal, M. C.; Mushran, S. P. J . Chem. Soc., Perkin Trans. 2
1973, 762.
S0022-3263(97)01398-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/09/1998

532 J . Org. Chem., Vol. 63, No. 3, 1998
Rangappa et al.
Ta ble 1. Effect of Rea cta n t Con cen tr a tion s on th e Ra te
of Oxid a tion of Hexosa m in es by CAB a t 40 °Ca
104kobs (s-1
)
103[CAB]o,
(M)
102[S]o,
(M)
D-mannos-
amine
D-glucos-
amine
D-galactos-
amine
1.0
1.5
2.0
2.5
3.0
3.5
4.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.0
3.0
4.0
5.0
6.0
3.40
3.29
3.20
3.16
3.10
3.14
3.05
1.52
4.68
6.30
7.60
9.20
5.22
5.16
5.15
5.10
5.15
5.10
4.98
2.50
7.50
10.00
12.70
14.84
18.80
18.66
18.50
18.56
18.53
18.45
18.20
8.95
27.45
36.12
46.20
57.57
a
[HO-]eff ) 30.0 × 10-2 M, I ) 0.6 M.
Ta ble 2. Effect of Va r yin g [HO-] on th e Rea ction Ra te
a t 40 °Ca
102[HO-]eff
(M)
D-mannosamine, D-glucosamine, D-galactosamine,
c c c
b
104kobs (s-1
)
104kobs (s-1
)
104kobs (s-1
)
5.0
10.0
20.0
30.0
40.0
50.0
1.29 (1.17)
1.90 (1.88)
2.60 (2.69)
3.20 (3.15)
3.70 (3.64)
4.25 (4.04)
2.15 (1.92)
3.10 (3.13)
4.20 (4.55)
5.15 (5.36)
5.95 (5.89)
6.66 (6.26)
7.50 (7.40)
10.94 (11.60)
15.05 (15.10)
18.50 (18.58)
20.80 (20.70)
22.30 (22.13)
F igu r e 1. Plots of log kobs vs I1/2: (A) D-mannosamine, (B)
D-glucosamine, (C) D-galactosamine. [CAB]o ) 2.0 × 10-3 M;
[S]o ) 2.0 × 10-2 M; [HO-] ) 30.0 × 10-2 M; temperature )
40 °C.
[CAB]o ) 2.0 × 10-3 M, [S]o ) 2.0 × 10-2 M, I ) 0.6 M.
a
[HO-]eff indicates that its neutralization by the hydrochloride of
b
hexosamine has been taken into account. c Values in parentheses
refer to rate constants calculated from eq 9.
indicating the absence of these compounds in a preequi-
librium, to the rate-limiting step.
The effect of varying ionic strength (I) of the medium,
maintained by the addition of NaClO4, was checked. The
rate increased with increase in ionic strength and a plot
of log kobs vs (I)1/2 gave straight lines (r > 0.9991, s e
0.001) with slopes of 0.6-0.8 (Figure 1).
The solvent composition of the medium was varied by
adding methanol (0-40% v/v). The rate decreased with
increase in methanol content. Plots of log kobs vs 1/D,
where D is the dielectric constant of the medium, were
linear (r > 0.9983, s e 0.02) with negative slopes (Figure
2).
The reaction was studied over a range of temperatures
and the Arrhenius plot, log kobs vs 1/T was linear (r >
0.9978, s e 0.03). The activation energy, Ea, for the
composite reaction was calculated from this plot. The
other parameters ∆Hq, ∆Sq, ∆Gq and the Arrhenius
parameter A (in terms of log A) were computed from the
Ea values (Table 3).
The rate increased in D2O medium, and proton inven-
tory studies were made in H2O-D2O mixtures of varying
deuterium content. Values of the solvent isotope effect,
k(H2O)/k(D2O) ) 0.50-0.60 for the three hexosamines.
Addition of reaction mixture to acrylamide solutions
did not initiate polymerization of the latter, indicating
the absence of free radical species in the mixture.
A comparison of the HPLC and GC retention times of
the reaction products with those of the standards indi-
cated that arabinonic, ribonic, erythronic, and glyceric
acids are the oxidation products for both D-glucosamine
and D-mannosamine. However, with D-galactosamine,
lyxonic, threonic, and glyceric acids were found to be the
products of oxidation. Besides these, small quantities of
hexosaminic acids were observed (Figure 3 and Table 4)
F igu r e 2. Plots of log kobs vs 1/D: (A) D-mannosamine, (B)
D-glucosamine, (C) D-galactosamine. [CAB]o ) 2.0 × 10-3 M;
[S]o ) 2.0 × 10-2 M; [HO-] ) 30.0 × 10-2 M; I ) 0.6 M;
temperature ) 40 °C.
in the oxidation of the hexosamines. The oxidation
products of hexosamines were also analyzed at 0.5, 1, 2,
4, 8, 16, and 24 h. The relative proportions of various
aldonic acids formed (Figure 3 and Table 4) were similar
at all time points analyzed (but the formation of six-
carbon aldonic acids were observed only after 4 h),
revealing that the lower-carbon aldonic acids were not
derived from the initially formed six-carbon aldonic acids.
The identities of all the oxidation products were con-
firmed from their mass fragmentation patterns.
The sodium salts of N-aryl-N-halosulfonamides behave
like strong electrolytes in aqueous solution. Bishop and

Sodium N-Chlorobenzenesulfonamide as a Selective Oxidant
J . Org. Chem., Vol. 63, No. 3, 1998 533
Ta ble 3. Kin etic Da ta for th e Oxid a tion of Hexosa m in es by CAB in P r esen ce of Na OH
Ea
∆Hq
∆Gq
∆Sq
hexosamine
temp (K)
104kobs (s-1
)
(kJ mol-1
)
(kJ mol-1
)
(kJ mol-1
)
(J K-1 mol-1
)
log A
D-mannosamine
308
313
318
323
308
313
318
323
308
313
318
323
1.75
3.20
6.20
11.52
2.89
5.15
105.0
97.9
81.1
102.4
95.3
78.5
97.8
96.6
93.2
14.7
16.0
D-glucosamine
-3.9
15.0
12.8
9.33
16.50
11.30
18.50
30.00
48.50
D-galactosamine
-46.9
Ta ble 4. HP LC An a lysis of th e P r od u cts F or m ed by th e Oxid a tion of Hexosa m in es by CAB in Alk a lin e Med iu m
products (mol %)a
hexosamine
arabinonic acid
ribonic acid
erythronic acid
lyxonic acid
threonic acid
glyceric acid
hexonic acid
D-mannosamine
D-glucosamine
D-galactosamine
36
37
-
24
20
-
32
33
-
-
-
33
-
-
52
5
7
10
3
3
5
a
Based on the peak areas normalized using response factors obtained by analyzing standard aldonic acid solution. The mole proportions
of products formed at 0.5, 1, 2, 4, 8, and 16 h were similar to those observed at 24 h, except that the presence of six-carbon aldonic acids
was evident only after 4 h. Similar product profiles were observed even when the reactions were carried out under kinetic conditions.
R ) C6H5SO2-) ionizes in aqueous solution (eq 1) and
furnishes different oxidant species. The anion is proto-
nated in acid solutions (eq 2):
RNClNa h RNCl- + Na+
(1)
(2)
RNCl- + H+ y
\K z RNClH
a
Ka ) 1.5 × 10-5 at 25 °C
Although the free acid has not been isolated, there is
sufficient experimental evidence for its formation in
solution.12 It can further undergo disproportionation/
hydrolysis (eqs 3 and 4, respectively) giving the sulfona-
mide RNH2, the dichloramine RNCl2, and HClO:
2RNClH h RNH2 + RNCl2
(3)
(4)
RNClH + H2O h RNH2 + HClO
In alkaline solutions, the following equlibria are re-
ported.
RNCl- + H2O h RNH2 + ClO-
RNCl- + H2O h RNClH + HO-
RNClH + HO- h RNH2 + ClO-
(5)
(6)
(7)
F igu r e 3. HPLC analysis of the products formed by the
oxidation of hexosamines by CAB in presence of alkali at 40
°C. (1) Glyceric acid; (2) erythronic acid; (3) threonic acid; (4)
arabinonic acid; (5) ribonic acid; (6) lyxonic acid; (7, 8, and 9)
hexonic acids Glc N, Man N, and Gal N, respectively, represent
the oxidation products of D-glucosamine, D-mannosamine, and
D-galactosamine.
Jennings,2 Morris and co-workers,9 Hardy and Johnston,10
and Higuchi and Hussain11 have explained the several
types of equilibria present in acid and alkaline solutions
of these compounds. Thus chloramine-B (RNClNa, where
Equations 5 and 7 suggest a retardation of rate with
the addition of the reaction product RNH2, while eq 6
predicts a decrease in rate by hydroxyl ions. Since
neither of these have been observed in the present set of
experiments, the most likely oxidizing species for hex-
osamines is the anion RNCl- itself.
(9) Morris, J . C.; Salazar, J . R.; Winemann, M. A. J . Am. Chem.
Soc. 1948, 70, 2036.
(10) Hardy, F. F.; J ohnston, J . P. J . Chem. Soc., Perkin Trans. 2
1973, 642.
(11) Higuchi, T.; Hussain, A. J . Chem. Soc. 1967, 549.
(12) Mahadevappa, D. S.; Rangaswamy, Rev. Roum. Chim. 1977,
22, 1233.

534 J . Org. Chem., Vol. 63, No. 3, 1998
Rangappa et al.
Ta ble 5. Th er m od yn a m ic Qu a n tities for Alk oxy An ion F or m a tion a n d Activa tion P a r a m eter s for th e Ra te-Lim itin g
Step a n d for th e Oxid a tion of Hexosa m in es by CAB
D-mannosamine
D-glucosamine
D-galactosamine
K1′,
102k2,
K1′,
102k2,
K1′,
102k2,
temp (K)
dm3 mol-1
dm3 mol-1 s-1
dm3 mol-1
dm3 mol-1 s-1
dm3 mol-1
dm3 mol-1 s-1
308
313
318
323
5.78
6.50
8.31
9.55
1.43
2.38
4.20
7.40
4.80
6.00
8.00
2.27
4.17
7.10
5.14
7.72
9.50
7.69
13.33
22.22
38.20
10.29
12.50
13.13
∆H (kJ mol-1
)
27.9
40.9
72.0
88.3
85.7
-2.6
86.6
15.1
42.7
35.9
∆S (J K-1 mol-1
∆G (kJ mol-1
Ea (kJ mol-1
∆Hq (kJ mol-1
)
-94.0
-72.4
95.8
93.2
25.5
-113.9
)
71.9
)
88.9
86.3
13.2
82.1
)
∆Sq (J K-1 mol-1
)
∆Gq (kJ mol-1
log A
)
85.1
16.5
15.9
In alkaline solutions, sugars undergo enolization to
form the alkoxy anions.13 In the absence of other
reactants, these anions undergo epimerization and isomer-
ization (Lobry de Bruyn-Alberda van Ekenstein trans-
formation) to form a mixture of aldoses and ketoses.13
However, in the presence of CAB, the alkoxy anion (S-)
reacts with RNCl- to form an intermediate, which in turn
undergoes cleavage to form the products (Table 4).
In view of the observed first-order dependence of rate
on [CAB]o and [S]o and fractional order dependence on
[HO-], the following reaction sequence (Scheme 1) is
proposed for the oxidation of hexosamines by CAB in
alkaline solutions:
From eq 8,
k2K1′[S]t[HO-]
kobs
)
or
1 + K1′[HO-]
1/kobs ) 1/k2K1′[S]t[HO-] + 1/k2[S]t (9)
Values of formation constant K1′ of alkoxy anion and
its decomposition rate constant k2 were evaluated from
the double reciprocal plots of 1/kobs vs 1/[HO-]. Using
the values of K1′ and k2 obtained at 40 °C (Table 5), kobs
values (in Table 2) were checked using eq 9. It is seen
that there is good agreement between the calculated and
experimental sets of values (Table 2), thus supporting
the proposed mechanism.
The thermodynamic parameters for the equilibrium
step i and activation parameters for the rate-limiting step
ii in Scheme 1 could be evaluated as follows: Hydroxyl
ion concentration (as in Table 2) was varied at several
temperatures, and values of K1′ and k2 were determined
at each temperature. A van’t Hoff plot was made for the
variation of K1′ with temperature (i.e, log K1′ vs 1/T), and
values of the enthalpy of reaction ∆H, entropy of reaction
∆S, and free energy of reaction ∆G were calculated. An
Arrhenius plot of log k2 vs 1/T yielded the activation
parameters for the rate-limiting step in Scheme 1. These
are shown in Table 5. A comparison of the latter values
with those obtained for the composite reaction shows that
these values mainly refer to the rate-limiting step,
supporting the fact that reaction before the rate-
determining step is fairly rapid, involving little activation
energy.
Sch em e 1
S + HO- yK1z S- + H2O
(i)
(ii)
fast
k2
S
- + RNCl- slow8 X
k3
X + 2RNCl- fast8 products
(iii)
If [S]t represents the total hexosamine concentration,
then [S]t ) [S] + [S-], from which
[H2O] + K1[HO-]
[S]t ) [S-]
K1[HO-]
[
]
Therefore
K1[S]t[HO-]
[H2O] + K1[HO-]
[S-] )
Scheme 1 and rate law 8 can explain the observed
experimental facts. The increase of rate in D2O medium
supports a fast preequilibrium hydroxyl ion transfer.14-16
A comparison of the proton inventory plots with standard
curves17 implies the involvement of a single proton in the
transition state. A primary salt effect is observed as the
rate increases with increase in ionic strength of me-
dium,18 thus supporting the involvement of two negative
ions in the rate-limiting step. The Debye-Huckel plot
Since rate ) k2[S-][CAB], substituting for [S-], rate law
8 can be derived as
k2K1[CAB][S]t[HO-]
[H2O] + K1[HO-]
-d[CAB]/dt )
or
k2K1′[CAB][S]t[HO-]
1 + K1′[HO-]
-d[CAB]/dt )
where K1′ ) K1/[H2O].
(8)
(14) Collins, C. J .; Bowman, N. S. Isotope Effects in Chemical
Reactions; Van-Nostrand-Reinhold: New York, 1970; p 267.
(15) Albery, W. J .; Davies, M. H. J . Chem. Soc., Faraday Trans.
1972, 68, 167.
(16) Gopalakrishnan, G.; Hogg, J . L. J . Org. Chem. 1985, 50, 1206.
(17) Isaacs, N. S. Physical Organic Chemistry; Longman: New York,
1987; p 273.
(13) Pigman, W.; Annet, E. F. L. J . The Carbohydrates: Chemistry
and Biochemistry, 2nd ed.; Pigman, W., Horton, D., Eds.; Academic
Press: New York, 1972; Vol. 1A, p 165.
(18) Laidler, K. J . Chemical Kinetics, 3rd ed.; Harper and Row: New
York, 1987; p 200.

Sodium N-Chlorobenzenesulfonamide as a Selective Oxidant
J . Org. Chem., Vol. 63, No. 3, 1998 535
Sch em e 2
F igu r e 4. Isokinetic plot of (a) ∆Hq vs ∆Sq and (b) log kobs
(308 K) vs log kobs (323 K).
has a slope of 0.6-0.8 (Figure 1) and the expected slope
of unity has not been realized. This may be due to the
fact that the ionic strength employed is well above the
formal Debye-Huckel range. Alternatively there could
be Bjerrum ion pair formation.18
The rate decreased with decrease in the dielectric
constant (D) of the medium. The effect of composition
of the solvent on rate for a reaction involving two negative
ions of charges ZAe and ZBe is given19 by the Scatchard
equation 10,
log k ) log ko - ZAZBe2/DkTdAB
(10)
where ko is the rate constant in a medium of infinite
dielectric constant, dAB refers to the size of activated
complex, and k and T are the Boltzmann constant and
absolute temperature, respectively. Figure 2 shows that
the plot of log kobs vs 1/D is linear and from the slope,
dAB is computed as 3.90, 3.40, and 2.50 Å for D-man-
nosamine, D-glucosamine, and D-galactosamine, respec-
tively. The values are found to be reasonable in com-
parison with those of other reactions of similar nature.20
An isokinetic relation was noted when plots of enthalpy
and entropy of activation were found to be linear (Figure
4, r ) 0.9996, s ) 0.01). The value of isokinetic temper-
ature from this plot was 409 K, which is greater than
the experimental temperature (313 K), indicating that
the reaction is enthalpy controlled. The relationship was
found to be genuine through the Exner criterion by
plotting log kobs (308 K) vs log kobs (323 K) (r ) 0.9989, s
) 0.02, Figure 4). The value of â calculated from the
relation, â ) T1(1 - q)/(T1/T2) - q, where q is the slope
of the Exner plot,21 was found to be 407 K in agreement
with the isokinetic point (409 K) calculated from the
enthalpy-entropy relation. Since the â-value is higher
than the experimental temperature (313 K), it indicates
an enthalpy-controlled reaction, which is further sup-
ported by the fact that Ea values (Table 3) are the least
for the fastest reaction and highest for the slowest
reaction.
The rate-determining step (Scheme 1) involves an
interaction between similarly charged ions which would
require a high activation energy. It is found to be so, as
the composite activation energy is around 90 kJ mol-1
for the reactions. The near constancy of ∆Gq values for
(19) Laidler, K. J . Chemical Kinetics, 3rd ed.; Harper and Row: New
York, 1987; p 193.
(20) Laidler, K. J . Chemical Kinetics, 2nd ed.; Tata-Mcgraw Hill:
Bombay, 1965; p 214.
(21) Exner, O. Collect. Czech. Chem. Commun. 1964, 29, 1094.

536 J . Org. Chem., Vol. 63, No. 3, 1998
Rangappa et al.
Research Centre, Trombay, India. The dielectric constant of
the reaction medium was altered by the addition of methanol
in varying proportions (% v/v), and values of permittivity for
methanol-water mixtures reported24 in literature were em-
ployed. Blank experiments performed showed that methanol
was not oxidized by CAB under the experimental conditions.
Stoich iom etr y. The stoichiometry run of oxidation of
hexosamines with excess CAB solution over that of sugar
indicated that three moles of oxidant were consumed per mole
of hexosamine, to form the aldonic acids.
P r od u ct An a lysis. The oxidation products were analyzed
by Dionx HPLC with pulsed amperometric detection using a
CarboPac PA1 high pH anion exchange column (4 × 250
mm).25 An isocratic elution with 0.2 M NaOH was used. The
products were identified by comparison of their HPLC reten-
tion times with retention times of the standard aldonic acids
and by GC-MS (see below).
For GC-MS characterization, the reaction mixture was
extracted with diethyl ether to remove benzenesulfonamide
and then passed through Ag 50W-X12(H+) and Ag 4-x4(base)
resins. The Ag 4-x4 resins were eluted with 1 M pyridine/1
M acetic acid, pH 5.2, and lyophilized. The products were
converted into their trimethylsilyl drivatives and then ana-
lyzed by GC-MS.
Kin etic Mea su r em en ts. Pseudo-first-order conditions
were maintained for the kinetic runs ([S]o . [CAB]o). The
oxidant and the requisite amounts of hexosamines, alkali,
NaClO4 solutions, and H2O (for constant total volume) were
thermostated for 30 min at 40 °C. The reaction was initiated
by the rapid addition of CAB to the mixture, and its progress
was monitored by iodometric estimation of unconsumed CAB
in known aliquots of the reaction mixture at regular intervals
of time. The reaction was studied for more than two half-lives.
Pseudo-first-order rate constants calculated from log [CAB]
vs time plots were reproducible within (5%. Allowance was
made in adjusting the alkali concentration [NaOH]eff for
neutralizing the hydrochloride of substrate during the kinetic
runs.
all the three hexosamines indicates that a similar mech-
anism is operative in the oxidation of hexosamines. The
rate of oxidation of D-galactosamine is faster than both
D-glucosamine and D-mannosamine, possibly because of
the fact that the former has a higher percentage of the
reactive â-anomer.22 This is also clearly reflected in the
values of k2, the rate constant for the rate-limiting step.
A possible mode of oxidation of D-glucosamine (or
D-mannosamine) by CAB in alkaline medium is shown
in Scheme 2. The hexosamine reacts with the anion of
oxidant in presence of alkali to form 2-aminogluconic
acid, which reacts with the oxidant to form D-arabinose
through decarboxylation followed by deamination in the
form of NH3 via hydrolysis of the intermediate imine. The
pentose is further oxidized to arabinonic acid and its
epimer ribonic acid. The former predominates over its
epimer. This is possibly due to stabilization of transition
state, as the preceeding intermediate has hydrogen
bonding involving hydroxyl hydrogen on C-5 with oxygen
on C-2. The C-C bond scisson between C-1 and C-2 on
pentose yields the tetronic acids. The H and OH groups
on C-3 are not significantly isomerized. This explains
the formation of erythronic acid. Further bond breaking
between C-2 and C-3 results in the formation of glyceric
acid.
A similar mechanism can be drawn for the oxidation
of D-galactosamine by CAB into a mixture of D-lyxonic
acid, D-threonic acid, and D-glyceric acids in alkaline
medium.
Exp er im en ta l Section
Ma ter ia ls. D-Mannosamine hydrochloride, D-glucosamine
hydrochloride, D-galactosamine hydrochloride, and D-ribono-
1,4-lactone were from Sigma Chemicals. D-Arabino-1,4-lactone
from Pfansfiehl Labs (Waukegan, IL) and analytical reagent
grade chemicals were used. Fresh aqueous solutions of
hexosamines were prepared using triply distilled water.
Chloramine-B, was prepared23 by passing chlorine through
a solution of benzenesulfonamide in 4.0 M NaOH for 1 h at
343 K. The product was collected, dried, and recrystallized
Regression analysis of experimental data, to obtain regres-
sion coefficient r, and s, the standard deviation of points from
the regression line, was performed with an EC-72 statistical
calculator.
Ack n ow led gm en t. We thank Dr. Roberta Merkle,
Complex Carbohydrate Research Centre, University of
Georgia, and Dr. Lewis Pannell, NIDDK, NIH, for GC-
MS analysis, M.P.R. is grateful to the University of
Mysore for the award of University Post Graduate
J unior Research Fellowship. We thank the Council of
Scientific and Industrial Research (CSIR), New Delhi,
for financial support.
1
from water (mp 170 °C dec). Its purity was checked by its H
and 13C NMR spectra and also by iodometric assay of active
chlorine in its aqueous solutions. Solutions of CAB were
preserved in brown bottles to arrest its photochemical dete-
rioration. Sodium perchlorate was used for maintaining a
constant high ionic strength “to swamp” the reaction. It was
found that the sugars were unaffected by the presence of this
compound in high concentrations. Heavy water (99.4% D) for
solvent isotope studies was supplied by the Bhabha Atomic
J O971398T
(22) Angyal, S. J . Adv. Carbohydr. Chem. Biochem. 1984, 42, 15.
(23) Chrzaszewska, A. Bull. Soc. Sci. Lett. Lodz, Cl. 3 1952, 16, 5
through Chem. Abstr. 1955, 49, 212.
(24) Akerloff, G. J . Am. Chem. Soc. 1932, 54, 4125.
(25) Hardy, M. R.; Townsend, R. R. Methods Enzymol. 1994, 230,
208.