N-bromosuccinimide oxidation of dipeptides and their amino acids: Synthesis, kinetics and mechanistic studies

Article abstract of DOI:10.1002/kin.20161

Dipeptides (DP), namely valyl-glycine (Val-Gly), alanyl-proline (Ala-Pro), and valyl-proline (Val-Pro) were synthesized by classical solution phase methods and characterized. The kinetics of oxidation of amino acids (AA) and DP by N-bromosuccinimide (NBS) was studied in the presence of perchlorate ions in acidic medium at 28°C. The reaction was followed spectrophotometrically at λ_max = 240 nm. The reactions follow identical kinetics, being first order each in [NBS], [AA], and [DP]. No effect on [H⁺], reduction product [succinimide], and ionic strength was observed. Effects of varying dielectric constant of the medium and addition of anions such as chloride and perchlorate were studied. Activation parameters have been computed. The oxidation products of the reaction were isolated and characterized. The proposed mechanism is consistent with the experimental results. An apparent correlation was noted between the rate of oxidation of AA and DP.

Full text of DOI:10.1002/kin.20161

N-Bromosuccinimide
Oxidation of Dipeptides
and Their Amino Acids:
Synthesis, Kinetics and
Mechanistic Studies
N. S. LINGE GOWDA, M. N. KUMARA, D. CHANNE GOWDA, K. S. RANGAPPA
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 11 September 2005; revised 10 November 2005; accepted 10 November 2005
DOI 10.1002/kin.20161
Published online in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: Dipeptides (DP), namely valyl–glycine (Val–Gly), alanyl–proline (Ala–Pro), and
valyl–proline (Val–Pro) were synthesized by classical solution phase methods and character-
ized. The kinetics of oxidation of amino acids (AA) and DP by N-bromosuccinimide (NBS) was
◦
studied in the presence of perchlorate ions in acidic medium at 28 C. The reaction was fol-
lowed spectrophotometrically at λmax = 240 nm. The reactions follow identical kinetics, being
+
first order each in [NBS], [AA], and [DP]. No effect on [H ], reduction product [succinimide],
and ionic strength was observed. Effects of varying dielectric constant of the medium and ad-
dition of anions such as chloride and perchlorate were studied. Activation parameters have
been computed. The oxidation products of the reaction were isolated and characterized. The
proposed mechanism is consistent with the experimental results. An apparent correlation was
noted between the rate of oxidation of AA and DP. ꢀC 2006 Wiley Periodicals, Inc. Int J Chem
Kinet 38: 376–385, 2006
INTRODUCTION
dation of ꢀ-amino acids is one of the well-documented
biochemical processes. Several studies have been re-
ported on the kinetics of oxidation of various substrates
by NBS in different media [3–8]. Although, the kine-
tics of oxidation of amino acids and peptides with metal
ions [9–11] reported, the studies on the oxidation of
both amino acids and peptides by NBS have not been
reported. The rate of oxidation of dipeptides and the
corresponding amino acids is compared.
Oxidative reactions play an important role in a variety
ofbiochemicaleventsrangingfromnormalmetabolism
to ageing and disease process [1,2]. Peptides and pro-
teins represent major targets for modification in these
reactions, and the identification of sites and structures
of modification may lead to a mechanistic understand-
ing and approaches for prevention. In this context, oxi-
We have synthesized three dipeptides viz. valyl–
glycine (Val–Gly), alanyl–proline (Ala–Pro), and
valyl–proline (Val–Pro), which are fragments of elas-
tic sequences [10] to elucidate the mechanism of these
redox reactions.
Correspondence to: K. S. Rangappa; e-mail: rangappaks@
yahoo.com.
c 2006 Wiley Periodicals, Inc.
ꢀ

N-BROMOSUCCINIMIDE OXIDATION OF DIPEPTIDES AND THEIR AMINO ACIDS
377
EXPERIMENTAL
Boc-Ala-Pro-OBzl. Boc-Ala-OH (4.1 g, 0.02 mol)
was coupled to Pro-OBzl. HCl (4.8 g, 0.02 mol), using
EDCI with HOBt and in the presence of NMM. The
reaction was worked up the same as Boc-Val-Gly-OBzl
All the amino acids used except glycine are of
L-configuration unless otherwise specified. All tert-
butyloxycarbonyl (Boc) amino acids, amino acid
derivatives, 1-ethyl-3 (3-dimethylaminopropyl) car-
bodiimide (EDCI), 1-hydrobenzotriazole (HOBt), tri-
fluroacetic acid (TFA), and N-methylmorpholine
1
to obtain 6.8 g (yield 88.2%) of Boc-Ala-Pro-OBzl. R
f
2
◦
◦
0.50 and R 0.63, mp 68 C (Lit [12] 67–69 C).
f
Ala-Pro. Boc-Ala-Pro-OBzl (5.8 g, 0.015 mol) was
saponified in methanol (50 mL) using 1 N NaOH for
2 h at room temperature and worked up the same as
Boc-Val-Gly-OH to obtain 4.2 g (yield 93.7%) of Boc-
(NMM) were purchased from Advanced Chem.
Tech (Louisville, KY, USA). Thin-layer chromatog-
raphy (TLC) was carried out on silica gel plates
obtained from Whatmann Inc. with the follow-
ing solvent systems: chloroform–methanol–acetic
2
f
3
f
Ala-Pro-OH. R 0.24 and R 0.29. This was deblocked
with TFA for 40 min to obtain TFA. Ala-Pro-OH (yield
100%).
1
acid (95:5:3), R , chloroform–methanol–acetic acid
Boc-Val-Pro-OBzl. Boc-Val-OH. (4.3 g 0.02 mol)
was coupled to Pro-OBzl. HCl (4.8 g, 0.02 mol) us-
ing EDCI with HOBt and in the presence of NMM.
The reaction was worked up the same as Boc-Val-Gly-
OBzl to obtain 6.6 (yield 81.2%) of Boc-Val-Pro-OBzl.
f
2
(
(
90:5:3), R , and chloroform–methanol–acetic acid
f
3
85:15:3), R .
f
The compounds on TLC plates were detected by
UV light after spraying with ninhydrin or by chlorine/
tolidine. The melting points were determined by using
Thomas–Hoover melting point apparatus.
1
f
2
f
◦
◦
R 0.75 and R 0.84, mp 81 C (Lit [12] 80–82 C).
Val-Pro. Boc-Val-Pro-OBzl (6.1 g, 0.015 mol) was
Boc-Val-Gly-OBzl. Boc-Val (3.5 g, 0.02 mol) and
HOBt (3.37 g, 0.022 mol) in DMF (40 mL) was
cooled to –15 C and EDCI (4.21 g, 0.22 mol) was
added. After stirring for 20 min, a precooled solu-
tion of Gly-OBzl.Tos (6.78 g, 0.02 mol) and NMM
saponified in methanol (50 mL) using 1 N NaOH for
2 h at room temperature and worked up the same as
Boc-Val-Gly-OH to obtain 4.4 g (yield 93.5%) of Boc-
◦
2
3
Val-Pro-OH. R 0.29 and R 0.33. This was deblocked
f f
with TFA for 40 min to obtain TFA. Val-Pro-OH (yield
100%).
(2.4 mL, 0.022 mol) in DMF (50 mL) was added and
stirred overnight at room temperature. After evaporat-
ing DMF under reduced pressure, the residue was taken
up by chloroform and extracted with 10% citric acid,
water, and 5% sodium bicarbonate solution. The sol-
vent was removed under reduced pressure and recrys-
tallized from ether/ethyl acetate to obtain 6.34 g (87 %)
Preparation of NBS Solution
An aqueous solution of NBS was prepared afresh each
day from a GRS Merck sample of reagent, and its
strength was checked by the iodometric method [13].
Solutions of AA and DP were prepared by dissolving
thesampleinH2Oofknownstrength. Allotherreagents
were of analytical grade. Double-distilled water was
used throughout the investigation.
1
2
3
of Boc-Val-Gly-OBzl. R 0.58, R 0.66, and R 0.72,
f
f
f
◦
◦
mp 80 C (Lit [12] 80–82 C).
Val-Gly. Boc-Val-Gly-OBzl (0.015 mol) was
saponified in methanol (50 mL) using 1 N NaOH (2.0
equivalent) for 2 h at room temperature. After evapo-
rating the solvent under reduced pressure, the residue
was taken up in water and washed with chloroform
Kinetic Procedure
(
3 × 25 mL). The aqueous layer was cooled and neu-
Solutions containing the requisite amount of sub-
strate, perchloric acid (to maintain a known acid con-
centration), succinimide, mercuric acetate, and wa-
ter (to keep the total volume constant) were placed
in stoppered boiling tubes. The mixture was ther-
tralized with cold 1 M HCl and extracted with chlo-
roform (40 mL). The organic phase was washed with
cold 0.1 M HCl, 50% saturated NaCl, and dried over
Na2SO4. The solvent was removed in vacuo and tritu-
rated with ether, filtered, washed with ether, and dried
◦
mally equilibrated in a water bath at 28 C. To the
2
to obtain 3.78 g (92%) of Boc-Val-Gly-OH. R 0.22
and R 0.34.
solution in this tube was added an aliquot of pre-
equilibrated NBS stock solution to give a known over-
all concentration. the progress of the reaction was
monitored for two half-lives by measuring the ab-
sorbance of unreacted NBS at 240 nm using a spec-
trochem Elico SL 150 UV–Vis spectrophotometer.
f
3
f
Boc-Val-Gly-OH (0.01 mol) was deblocked with
TFA (10 mL/g of peptide) by stirring for 40 min.
The solvent was removed under reduced pres-
sure; the residue was triturated with ether and fil-
tered, washed with ether to obtain TFA.Val-Gly-OH
−
−3
6
The reaction mixture containing [NBS] = 1.0 × 10
,
,
−
4
(100%).
[AA/DP] = 1.0 × 10 , [HClO ] = 0.01 mol dm
4

378
LINGE GOWDA ET AL.
−
3
[
0
succinimide] = 0.1 mol dm , [Hg(CH3COO)2] =
Stoichiometric Equations for Dipeptides (DP)
−3
.0 01 mol dm was quenched appropriately, plots of
Alanyl–Proline (AP) and Valyl–Proline (VP)
log(absorbance)vs. timewerelinear. Therateconstants
kobs calculated from these plots were reproducible to
within ± 3% error.
Stoichiometry and Product Analysis
MixturescontainingAA/DP(0.0001M), acid(0.01M),
and excess of N-bromosuccinimide (0.003 M) were
◦
kept for 24 h at 28 C. The unconsumed NBS was
then determined; 1 mol of oxidant was sufficient to
oxidize 1 mol of glycine/alanine/valine. Two moles
of oxidant was sufficient to oxidize 1 mole of pro-
line (AA) and 1 mole of Val–Gly (DP) and 3 mole
of oxidant were sufficient to oxidize 1 mole of Ala–
Pro and Val–Pro (DP) leading to aldehydes, carbon
dioxide, ammonia, and succinimide. Based on these
results, the following stoichiometric equations are
suggested.
where
R = CH3 for AP and R = CH(CH3)2 for VP
Valyl–Glycine (V G. )
Stoichiometric Equations for Glycine (Gly), Alanine
(Ala), and Valine (Val)
After the reaction was completed, the reaction products
were extracted with diethyl ether and subjected to col-
umn chromatography on silica gel (60–200 mesh) us-
ing a gradient elution (dichloromethane to chloroform).
Aldehydes were analyzed qualitatively by gas chro-
matography. The (Rf) retention values of formalde-
hyde, acetaldehyde, isobutaraldehyde, and succinalde-
hyde are 6.0, 5.14, 27.4, and 31.9, respectively, which
are identical with authentic samples. NH3 and CO2
were detected by the conventional method.
Stoichiometric Equations for Proline
RESULTS
Effect of Varying Reactant
Concentration on the Rate
All kinetic runs were performed under pseudo-
first-order conditions with [AA] ꢁ [NBS] and
[
DP] ꢁ [NBS]. Plots of log [NBS] vs. time were linear
even beyond 75% of the reaction, showing a first-order
dependence of the rate on [NBS] (Table I) at constant
[
succinimide]0, [HClO4]0,[NaCl]0, and temperature,
the rate increased with increase in [AA]o and in [DP]o
Table I). Plots of log kobs vs. log [AA] (Fig. 1) were
(

N-BROMOSUCCINIMIDE OXIDATION OF DIPEPTIDES AND THEIR AMINO ACIDS
379
Table I Effect of Varying Reactant Concentration on the Rate of Oxidation of Dipeptides and Their Amino Acids with
−3
[
HClO ] = 0.01 mol dm−3, [succinimide] = 0.1 mol dm−3, [Hg(CH COO) ] = 0.001 mol dm T = 301 K
4
3
2
5
−1
×
10 kobs (s
)
6
4
×
10 [NBS]
× 10 [S]
−
3
−3
(mol dm
)
(mol dm
)
Gly
Ala
Val
Pro
Val–Pro
Ala–Pro
Val–Gly
0
0
1
1
1
0
0
0
0
0
0
.6
.8
.0
.2
.4
.6
.6
.6
.6
.6
.6
1.0
1.0
1.0
1.0
1.0
0.6
0.8
1.0
1.2
1.4
1.6
10.81
10.66
10.61
10.24
10.10
07.73
08.18
10.61
12.07
14.20
16.24
11.64
11.62
11.61
11.58
11.48
08.05
09.23
11.46
13.72
16.39
17.85
12.40
12.37
12.79
12.89
12.85
08.24
10.33
12.79
15.86
17.25
19.15
15.88
15.70
15.88
15.20
15.45
09.59
12.47
15.88
18.82
20.86
22.36
08.57
08.76
08.68
08.53
08.71
05.02
06.87
08.68
09.77
10.55
11.48
08.50
08.45
08.56
08.66
08.70
05.18
06.89
08.56
10.82
12.62
14.29
06.20
06.33
06.47
06.55
06.73
03.89
05.98
06.47
08.85
10.20
12.72
linear with slopes of 1.00, 0.99, and 0.97 for Gly, Val,
and Pro, respectively. Plots of log kobs vs. log [DP]
the product is not involved in pre-equilibrium with the
oxidant.
(Fig. 1) were linear with slopes of 1.05, 0.97, and 1.10
for Gly–Val, Ala–Pro, and Val–Pro, respectively.
Effect of Varying Solvent Composition
The solvent composition of the medium was varied by
adding methanol (0.0–40%), to the reaction mixture.
The rate increased with increase in methanol content
(Table II). The plots of log kobsvs. 1/D (D = dielectric
constant of the medium) were linear with positive
slopes (Fig. 2). Measurements of rate constants were
carried out in both the presence and absence of AA/DP
with NBS. The rate constants were taken for the calcu-
lation of the effective kobs, although the rate of oxida-
tion of methanol in the absence of both AA and DP is
negligible under the conditions employed.
Effect of [HClO4]
Kinetic measurements were performed in HClO4–
+
+
NaClO4 solution of different [H ]. The effective [H ]
used was evaluated with the aid of a calibration curve
+
+
of [HClO4] vs. [H ]. An increase in [H ] (0.1–2.0 M)
had no effect on the rate.
Effect of Rate on Product and Added Salts
The effect on the rate of varying the concentration of
succinimide (which is the reduction product of the ox-
idant) was investigated. An increase in [succinimide]
Effect of Temperature
(from 0.001 to 0.01 M) had no effect on the rate. Sim-
−
ilarly, the effects of the anions [Cl ] (from 0.001 to
To determine the activation parameters, the reactions
−
◦
0
.05 M) and ClO4 from (from 0.001 to 0.1 M) on
were carried out at different temperatures (22–34 C,
the rate were insignificant. The reaction product suc-
cinimide had no effect on the reaction, indicating that
Table III). The Arrhenius plots of log kobs vs. 1/T
(Fig. 3) were linear. The activation energies (Ea) were
−
6
−3
−4
Table II Effect of Varying Dielectric Constant on the Rate with [NBS] = 1.0 × 10 mol dm , [S] = 1.0 × 10 mol
−3
−3
−3
−3
+
−2
−3
dm , [Hg(CH COO) = 1.0 × 10 mol dm , [succinimide] = 0.1 mol dm , [H ] = 1.0 × 10 mol dm , T = 301 K
3
2
5
−1
×
10 kobs(s
)
MeOH
%, v/v)
Dielectric
Constant (D)
(
Gly
Ala
Val
Pro
Val–Pro
Ala–Pro
Val–Gly
0
1
2
3
4
76.73
72.37
67.48
62.71
58.06
10.61
12.47
15.88
17.54
20.06
11.61
12.39
14.48
15.85
17.85
12.79
14.25
16.63
18.10
20.25
15.88
18.51
20.06
23.11
26.05
8.68
9.81
10.46
11.19
12.44
8.56
9.24
10.25
12.62
14.29
6.47
7.61
8.69
10.20
12.47
0
0
0
0

380
LINGE GOWDA ET AL.
Table III Temperature Dependence of the Oxidation of Dipeptides and Their Amino Acids with [NBS] = 1.0 ×
−
6
−3
−4
−3
−3
+
−2
−3
1
0
mol dm , [S] = 1.0 × 10 mol dm , [succinimide] = 0.1 mol dm , [H ] = 1.0 × 10 mol dm
5
−1
)
×
10 kobs(s
3
Temperature
K)
× 10 1/T
−
1
(
(K
)
Gly
Ala
Val
Pro
Val–Pro
Ala–Pro
Val–Gly
2
2
3
3
3
95
98
01
04
07
3.389
3.355
3.322
3.289
3.257
5.89
7.86
10.61
13.54
16.47
08.46
10.48
12.39
14.46
16.23
8.41
10.23
12.79
15.67
18.10
9.12
12.02
15.88
20.64
25.47
6.87
7.73
8.68
9.59
10.66
6.31
7.19
8.56
9.24
10.05
4.11
5.35
6.47
7.61
8.69
−
6
−3
mol dm
Figure 1 Effect of varying reactant concentration on the reaction rate with [NBS] = 1.0 × 10
[HClO4] =
−
3
−3
−3
◦
0
.01 mol dm , [succinimide] = 0.1 mol dm , [Hg(CH3COO)2] = 0.001 mol dm , T = 28 C.

N-BROMOSUCCINIMIDE OXIDATION OF DIPEPTIDES AND THEIR AMINO ACIDS
381
Table IV Kinetic and Activation Data for the Oxidation of Dipeptides and Their Amino Acids with [NBS] = 1.0 ×
−
6
−3
−4
−3
−3
+
−2
−3
1
0
mol dm , [S] = 1.0 × 10 mol dm , [succinimide] = 0.1 mol dm , [H ] = 1.0 × 10 mol dm
#
#
ꢀG#
Ea
ꢀH
(kJ mol
ꢀS
(kJ mol 1)
−
−1
)
(J mol
−1 −1
K
)
(kJ mol
−1
)
log A
Substrate
Gly
Val
Pro
Ala
Val–Pro
Ala–Pro
Val–Gly
70.25
50.38
48.25
64.78
25.87
29.38
32.85
67.75
49.24
45.75
62.27
23.36
26.87
30.34
−93.37
−122.85
−167.91
−114.75
−245.90
−233.78
−224.94
95.87
94.90
96.29
96.82
97.38
97.24
98.05
8.36
6.40
4.57
7.24
0.40
1.03
1.5
−
6
−3
Figure 2 Effect of varying dielectric constant on the reaction rate, with [NBS] = 1.0 × 10 mol dm , [AA/DP] = 1.0 ×
−4
−3
−3
−3
−3
+
−2
mol
1
0
mol dm , [Hg(CH3COO)2] = 1.0 × 10
mol dm , [succinimide] = 0.1 mol dm , [H ] = 1.0 × 10
−3
◦
dm , T = 28 C.

382
LINGE GOWDA ET AL.
−
5
−3
Figure 3 Temperature dependence of the oxidation of substrate by NBS, with [NBS] = 1.0 × 10 mol dm , [AA/DP] =
−
4
−3
−2
−3
−3
−3
1
.0 × 10
.1 mol dm
mol dm
,
[HClO4] = 1.0 × 10
mol dm
,
[Hg(CH3COO)2] = 1.0 × 10
mol dm ,
[succinimide] =
−
3
0
.
calculated from the slope of the plots, from these
dicating the absence of in situ formation of free radical
species in the reaction sequence.
#
#
values, the thermodynamic parameters ꢀH , ꢀS ,
#
ꢀ
G , and the frequency factor (log A) were evaluated
(Table IV).
DISCUSSION
Test for Free Radicals
The results of the oxidation of amino acids and dipep-
tides, recorded here, have revealed that the reac-
tions have identical kinetics and thus appear to have
Addition of reaction mixture to aqueous acrylamide
monomer solutions did not initiate polymerization, in-

N-BROMOSUCCINIMIDE OXIDATION OF DIPEPTIDES AND THEIR AMINO ACIDS
383
common mechanism. Insignificant effect of mercuric
acetate on reaction rate rules out its involvement in
NBS oxidation and acts only as a scavenger [14,15]
where [S] = AA or DP. Hence, rate = k1[NBS] [AA or
DP].
−
for any Br formed in the reaction. It suppresses
completely the oxidation by Br2, which would have
been formed by the interaction of HBr and NBS as
follows:
CONCLUSION
The rates of oxidation of amino acids and dipep-
tides by NBS were compared under identical exper-
imental conditions, and it was found that the rates
of oxidation of dipeptides were slower than those of
free amino acids. The change in each case is due to
the increased distance between the functional groups
and consequently weaker electrostatic effects. Hence,
the oxidation of dipeptides is expected to be slower
than that of free amino acids. Further, an apparent
correlation was noted between the rate of oxidation
and hydrophobicity [17] of those sequences where in-
creased hydrophobicity results in an increased rate of
oxidation.
Mercuric acetate thus ensures the oxidation purely
through NBS. NBS is known to exist in acidic media
in the following equilibria:
The most hydrophobic dipeptide, Val–Pro, oxidized
at a faster rate than the less hydrophobic dipeptides
Ala–Pro and Val–Gly. The probable reason for the
increased oxidation rate for the more hydrophobic
dipeptides is that the carboxylic groups are more desta-
bilized, which enhances the rate of formation of a
transition state complex with NBS, and the oxidation
rate may be higher. Further, it was observed that the
DP with Pro as C-terminus, Val–Pro, and Ala–Pro are
more susceptible to oxidation than the DP with Gly as
C-terminus (Val–Gly).
+
+
The NBS itself or protonated NBS, i.e., N BSH or Br
may be the possible oxidizing species in acidic media.
In the presence of mercuric acetate protonated form
of NBS, i.e., N BSH has been considered [16] as a
+
reactive species of NBS in acidic medium.
In the present investigation, it was found that the
added succinimide has a negligible effect on the rate
+
of the reaction. This categorically excludes Br as the
oxidizing species. Hence, the active species may be
APPENDIX: MECHANISM FOR
AMINO ACIDS
+
NBS or N BSH. The order of the reaction with respect
+
+
to [H ] is zero, and hence N BSH does not participate
in the rate-determining step. All these factors indicate
that NBS is the only possible oxidant species taking
part in the reaction. In the light of the experimental
results, a suitable mechanism has been proposed.
Scheme 1 accounts for the observed experimental
results for AA and DP:
k
1
NBS
+
[ S ]
X
(i) slow and rds
(ii) fast
k
2
2
nH O
X
Products
where [ S ] = AA or DP
where R = H for glycine; R = CH3 for alanine;
R = CH (CH3)2 for valine.
Scheme 1

384
LINGE GOWDA ET AL.
For Proline (Pro)
For Dipeptides (Val–Pro and Ala–Pro)
where R = CH (CH3)2 for valyl-proline and R = CH3 for alanyl-proline.

N-BROMOSUCCINIMIDE OXIDATION OF DIPEPTIDES AND THEIR AMINO ACIDS
385
For Val–Gly
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