Production mechanism of active species on the oxidative bromination following perhydrolase activity

Article abstract of DOI:10.1002/poc.3490

Hypobromous acid and molecular bromine have been described as the active species involved in the oxidative bromination using perhydrolase, which catalyzes the reaction from acetic acid and hydrogen peroxide to peracetic acid (AcOOH). However, the brominating activity of them in a chemical model system was lower than that of the active species produced by the spontaneous reaction between AcOOH and Br^-. Consequently, acetyl hypobromite (AcOBr) was suggested as new active species on the bromination by detection of the decarboxylation in the reaction between AcOOH and Br^- and the strong brominating power with some tolerance against H₂O₂. Its production mechanism was explained as the ionic reaction involving the protonated intermediate of AcOOH by kinetic analysis.

Full text of DOI:10.1002/poc.3490

Research article
Received: 14 June 2015,
Revised: 10 August 2015,
Accepted: 16 August 2015,
Published online in Wiley Online Library: 22 September 2015
(wileyonlinelibrary.com) DOI: 10.1002/poc.3490
Production mechanism of active species
on the oxidative bromination following
perhydrolase activity
Hideyasu Chinaa,b*, Yutaka Okadaa* and Hiroyasu Oginob
Hypobromous acid and molecular bromine have been described as the active species involved in the oxidative bromination
using perhydrolase, which catalyzes the reaction from acetic acid and hydrogen peroxide to peracetic acid (AcOOH). However,
the brominating activity of them in a chemical model system was lower than that of the active species produced by the spon-
taneous reaction between AcOOH and Brꢀ. Consequently, acetyl hypobromite (AcOBr) was suggested as new active species
on the bromination by detection of the decarboxylation in the reaction between AcOOH and Brꢀ and the strong brominating
power with some tolerance against H2O2. Its production mechanism was explained as the ionic reaction involving the proton-
ated intermediate of AcOOH by kinetic analysis. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: active species; oxidative bromination; non-enzymatic mechanism; monochlorodimedone; peracetic acid; acetyl
hypobromite; perhydrolase; kinetic analysis; ionic reaction; decarboxylation
ester using H2O2 (Fig. 1(a)).[16b,15,17] The oxidative bromination
INTRODUCTION
following perhydrolase activity is easily detected by using two
[18]
synthetic substrates of monochlorodimedone (MCD, 1)
and
Brominations are especially important reactions for the prepara-
tion of synthetic intermediates that contribute to forming
carbon–carbon bonds and changing the functional group. Chemi-
cal brominations are generally performed using electrophilic
reagents such as N-bromosuccinimide and molecular bromine
(Br2) in organic solvent; however, they are poisonous sacrificial
reagent. Accordingly, many researchers recently have focused on
the oxidative halogenation, [1] which makes use of a brominating
speciesproducedformthereactionbetweenBrꢀ andoxidantsuch
as H2O2,[1] O2,[2] DMSO,[3] oxone,[4] (NH4)S2O8,[5] tBuOOH,[6]
phenol red affording monobromomonochlorodimedone (MBMCD,
2) and bromophenol blue, respectively, in the presence of Brꢀ (Fig. 1
(b)). A brominating species is produced at enzyme outside by the
non-enzymatic reaction between Brꢀ and RCOOOH liberated from
theactivesiteintheenzyme.Thenon-enzymaticbrominationinvolv-
ingtheactivityhasbeenexplainedbyusingtheactivespeciessuchas
HOBr (Eqns 1 and 2) [13b,d,15a] or Br2 (Eqns 3 and 4).[16b,17a] However,
evidence for the non-enzymatic step still remains unclear. This step
should be contemplated for the application of perhydrolase. The
study on the oxidative bromination following perhydrolase activity
is simplified using RCOOOH instead of the enzyme in the model
system of the bromination in principle. In this paper, the detailed
investigation as to the oxidative bromination is described.
peracid,[7] (NH4)2Ce(NO3)6,[8] NaIO4,[9] PPh3O,[10] selectfluor,
[11]
and so on. This method employs the safety salt such as NaBr, KBr,
NH4Br, Bu4NBr, or available HBr as a bromide source. Although
the oxidative bromination under mild condition is often required
as a catalyst such as metal ion and haloperoxidase, the enzyme
realizes the aqueous bromination using low concentration of
H2O2 at room temperature. Haloperoxidase is expected as an
environmental harmony-type catalyst in the oxidative bromina-
tion. Because various enzymatic brominations of terpenes[12] have
been confirmed using several haloperoxidases, many brominated
natural products have been suggested as the product induced by
the enzymes. [12,13]
AcOOH þ Hþþ Br →AcOH þ HOBr þ H2O
MCD þ HOBr→MBMCD þ H2O
(1)
(2)
(3)
(4)
-
AcOOH þ 2Hþþ Br →AcOH þ 1=2 Br2 þ H2O
-
MCD þ Br2→MBMCD þ Hþþ Br
-
Haloperoxidases are classified into four groups of the heme-
[13]
[13,14]
dependent,
vanadium-dependent,
flavin-dependent,
[13a–c]
and metal-free types (which was renamed perhydrolase)
[13,15] containing iron protorphyrin IV, vanadate (HVO42ꢀ), FADH2,
and the Ser–His–Asp catalytic triad in the active site, respectively.
It has been believed that the heme-dependent, vanadium-
dependent, and flavin-dependent-type haloperoxidases catalyze
directproductionofhypobromous acid(HOBr). Ontheotherhand,
perhydrolase catalyze to produce percarboxylic acid (RCOOOH)
from carboxylic acid (RCOOH) and H2O2. [16] The catalytic mecha-
nism of perhydrolase was explained by esterification of RCOOH
to the nucleophilic Ser residue and then perhydrolyzation of the
* Correspondence to: H. China and Y. Okada, Department of Applied Chemistry,
College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu,
Shiga 525–8577, Japan.
E-mail: hchina7a@fc.ritsumei.ac.jp; ygvictor@sk.ritsumei.ac.jp
a H. China, Y. Okada
Department of Applied Chemistry, College of Life Sciences, Ritsumeikan
University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577, Japan
b H. China, H. Ogino
Department of Chemical Engineering, Osaka Prefecture University, 1-1
Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan
J. Phys. Org. Chem. 2016, 29 84–91
Copyright © 2015 John Wiley & Sons, Ltd.

PRODUCTION MECHANISM OF ACTIVE SPECIES
(δ) values are expressed in parts per million relative
to trimethylsilane (TMS) as the internal standard,
and all of the coupling constants (J) values are
expressed in hertz. Signal multiplicities are repre-
sented as singlet (s), doublet (d), and double dou-
blet (dd). The infrared (IR) spectra of the inorganic
compounds were recorded by a Spectrum BX FT-IR
system (Perkin-Elmer, Inc., MA, USA) using the KBr
tablet method and were reported in wavenumbers
(cmꢀ1). The IR spectra of the organic compounds
were recorded by
a FTIR-8400 spectrometer
(Shimadzu Co., Kyoto, Japan) using a KBr fixed thick-
ness liquid cell (0.1 mm) with dehydrated solvents.
Synthesis
2-Bromo-2-chloro-5,5-dimethyl-cyclohexan1,3-edione
(2). (Method 1) To a solution of 1 (47.2 mg, 270 μmol)
in 6 mL of 1.0 M AcONa-H2SO4 buffer (pH 5.5) con-
taining 0.5 M NaBr was added AcOOH (771 μL,
594 μmol, 0.77 M solution containing AcOH). After
the mixture was stirred at 25 °C for 10 min, the reac-
tion was quenched with 9.4 mM H2O2. The product
was filtered under reduced pressure and then the
filtrate was washed using water. Drying the filtrate
gave 2 (53.1 mg, 209 μmol) as a white powdery in
Figure 1. (a) Perhydrolase activity and (b) oxidative bromination
77% yield. (Method 2) To a solution of 3 (43.8 mg,
200 μmol) in 5 mL of CH2Cl2 was added tBuOCl
(22.6 μL, 200 μmol) at room temperature. After the mixture was stirred at
the temperature for 1 h, the mixture was diluted in 15 mL of CH2Cl2 and
washed with 20 mL of water once. The extract was filtered with filter paper
for dehydration and then removal of the solvent by evaporation gave 2
(48.1 mg, 190 μmol) in 95% yield. 1H NMR (CDCl3): δ 0.85 (3H, s, ax.CH3),
1.20 (3H, s, eq.CH3), 2.65 (2H, d, J = 14.6 Hz, eq.CH), 3.39 (2H, dd, J = 14.6,
0.9 Hz, ax.CH) ppm. 13C NMR (CDCl3): δ 25.9 (ax.CH3), 29.7 (eq.CH3), 30.6
(C5), 48.7 (C4 and C6), 74.0 (C2), 192.6 (C1 and C6) ppm. IR (CHCl3): ν 577,
613, 1375, 1397, 1728, 1751, 2967 cmꢀ1. The 1H and 13C NMR values are
consistent with those reported in the literature.[21,18b]
EXPERIMENTAL
Concentration determination of materials
The concentration of ca. 9%(w/v) AcOOH in an AcOH solution (Sigma-
Aldrich Co., St. Louis, USA) was determined by iodometric titration using
NaI, Na2S2O3, and starch indicator and by cerimetric titration using 0.1 M
Ce(SO4)2 solution (Kanto Chemical Co., Inc., Tokyo, Japan) and 1.5%(w/v)
ferroin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) indicator.[19]
The concentrations of ca. 9%(w/w) NaOBr aqueous solution (Kanto) and
1.01 g/mL Br2 aqueous solution (Wako) were determined by the iodometric
titration. The concentration of ca. 30%(w/w) H2O2 (Santoku Chemical Indus-
tries Co., Ltd., Tokyo, Japan) was determined by cerimetric titration.
2,2-Dibromo-5,5-dimethyl-cyclohexane1,3-dione (4). To a solution of
dimedone (37.8 mg, 270 μmol) in 6 mL of 1.0 M AcONa-H2SO4 buffer
(pH 5.5) containing 0.5 M NaBr was added AcOOH (1.542 mL, 1.188 mmol,
0.77 M solution containing AcOH). After the mixture was stirred at 25 °C
for 10 min, the reaction was quenched with 9.4 mM H2O2. The product
was filtered under reduced pressure, and then the filtrate was washed
using water. Drying the filtrate gave 4 (50.1 mg, 168 μmol) as a white
ꢀ
Measurement on consumption of 1 and production of Br3
Consumption of 1 (ε = 1.36 × 104 Mꢀ1 cmꢀ1) and production of Br3
ꢀ
(ε = 4.09 × 104 Mꢀ1 cmꢀ1
)
using three of the 45 μM oxidant reagents
[20]
1
powdery in 62% yield. H NMR (CDCl3): δ 1.02 (6H, s, C(CH3)2), 3.01 (4H, s,
(AcOOH, NaOBr, and Br2) in 1.0 M AcONa-H2SO4 buffer (pH 5.5) with or
without additives (0.5 M NaBr and 10 mM H2O2) was assayed by measur-
ing disappearance at 278 nm and appearance at 266 nm, respectively,
using UV–Vis spectrophotometer at 25 °C. The three oxidants were used
in dark room. Production of Br3ꢀ was performed in the absence of 1. The
initial consumption rate of 1 was assayed under 1.0 M AcONa-H2SO4
(pH 4.0–5.5), 0.1 M Na2HPO4-H2SO4 (pH 6.0–8.1), and 0.1 M H3BO3-NaOH
(pH 8.3–10.0) buffers with 50 mM NaBr. The efficiencies and initial rates
on the consumption and production were calculated using the three
CO-CH2) ppm. 13C NMR (CDCl3): δ 27.7, 30.6, 48.2, 66.4, 192.8 ppm. IR
(CHCl3): ν 546, 1200, 1457, 1722, 2322, 2359, 2965, 3030 cmꢀ1. The 1H
and 13C NMR values are consistent with those reported in the literature.[22]
Titration of MCD for determination of pKa
A stirred 45μM MCD aqueous solution (400 mL) was carefully neutralized
by addition of a small amount of 1 M NaOH aqueous solution using an
molar extinction coefficients of 1, ε
4.5) = 1.35 × 104 Mꢀ1 cmꢀ1, and ε
4.0) = 1.34 × 104 Mꢀ1 cmꢀ1, ε
(pH
5.0–10.0) = 1.36 × 104 Mꢀ1 cmꢀ1
.
(pH
(pH
Structure determination analysis
The 1H and 13C NMR spectra were recorded by an ECS 400 NMR spec-
trometer (JEOL Ltd., Tokyo, Japan) at 400 and 100 MHz, respectively,
using deuterated solvents such as CDCl3 and D2O. All of chemical shifts
Figure 2. Tautomerism and ionization of 1
J. Phys. Org. Chem. 2016, 29 84–91
Copyright © 2015 John Wiley & Sons, Ltd.
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H. CHINA, Y. OKADA AND H. OGINO
appropriate micropipetor and then the
afforded solution was titrated using
0.5 M HCl aqueous solution at 25 °C.
After pH and the corresponding absor-
bance at 278 nm were measured for
each dropping, the
ɛ values were
calculated by using Lambert–Beer
equation with the resulting concentra-
tion of MCD. The minimum and
maximam values assigned to enol
(ɛenol = 1.18 × 104 Mꢀ1 cmꢀ1
)
and
enolate (ɛenolate = 1.36 × 104 Mꢀ1 cmꢀ1),
respectively. Curve fitting of the plots
was performed using SigmaPlot2001
software v7 (SPSS Inc., Chicago, IL, USA).
Detection of carbon dioxide pro-
duced from the reaction between
AcOOH and Brꢀ
To a stirred 2.0 M NaBr solution (100 mL)
in the absence and presence of 9.4 M
H2O2 was added 0.2 M AcOOH-NaOH
solution (pH 7.0) containing AcOH
(100 mL) with dropping funnel under
argon atmosphere at 25 °C. After stirring
for 10 min, the reaction was quenched
by the addition of 9.4 mM H2O2. Gener-
ated gas was passed through a trap
Figure 3. Relation between ionization and UV adsorption of 1. The experiments about (a) effect of
water on the ε value at 278 nm in CH3CN, (b) determination of isosbestic point in water, (c) effect of
pH on λmax in water, and (d) determination of pKa on 1 at 278 nm in water were performed in a
45 μM of 1 in solution at 25 °C
with
a saturated Ca(OH)2 solution
(50 mL) by pressing with balloon-filled
argon. A given precipitate of CaCO3
was filtered under reduced pressure
and then washed using water and acetone. IR: ν 3468, 2967, 2878,
2513, 2363, 1799, 1422, 875, 712 cmꢀ1
.
RESULTS AND DISCUSSION
MCD assay system
Figure 4. Synthesis of 2. (a) AcOOH (2.2 eq), 1.0 M AcONa-H2SO4 buffer,
0.5 M NaBr, 25 °C, 10 min, 77% and (b) tBuOCl, CH2Cl2, r.t., 1 h, 95%
An MCD assay for perhydrolase is usually performed in 1.0 M
AcONa-H2SO4 buffer (pH 5.5) with NaBr. It has been believed that
the enol form of 1 equilibrates with the keto form in acidic
aqueous solutions[13g,23] (Fig. 2). It may be that a conception
about keto–enol tautomerism of 1 was associated with its
tautomerism of dimedone.[24] However, the keto form of 1 was
not observed by a detailed NMR analysis in several deuterated
organic solvents. [22b] In fact, the keto form was not also observed
by 1H NMR analysis in 1.0 M CD3COOD-NaOD buffer (pD 5.5).
The hyperchromic effect of 1 was caused by the deprotonation
accompanied by an increase in water (Fig. 3(a)). The effect of pH on
the UV spectra of 1 showed an isosbestic point (276.5 nm), which
implied the existence of two compounds corresponding to the
enol and enolate of 1 in the aqueous solution (Fig. 3(b)). The λmax
values of the enol and enolate were 270 and 291 nm, respectively
(Fig. 3(c)). An acid dissociation constant of 1 (pKa = 3.18) was
determinated using Eqn 5 (Fig. 3(d)). Compound 2 produced by
the bromination of 1 using AcOOH was confirmed by the chlorina-
tion of monobromodimedone (MBD, 3)[25] using tBuOCl (Fig. 4).
The bromination of the enolate of 1 without a consideration of
the keto form in the assay was ensured by the results. Although
we recently clarified that the MCD assay contains minor systematic
error caused by decomposition of 2,[18b] the relative discussion
using apparent consumption efficiency of 1 enable to investigate
the active species in the oxidative bromination.
εenolate þ εenol
ε ¼ εenol
þ
(5)
1 þ eln10ðpKaꢀpHÞ
Active species
The oxidative bromination using perhydrolase is usually per-
formed at pH 5.5 in the presence of NaBr and H2O2. The chemical
model system of the bromination using AcOOH instead of
perhydrolase has simplified the study on the brominating activ-
ity in the presence of the enzyme. An active species produced
from the reaction between AcOOH and NaBr at pH 5.5 was inves-
tigated by a UV–Vis analysis. The investigation was mainly per-
formed in the absence of H2O2 because Br2 equilibrating HOBr
and Br3ꢀ in the presence of Brꢀ (Eqns 6 and 7)[20a,26] are rapidly
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J. Phys. Org. Chem. 2016, 29 84–91

PRODUCTION MECHANISM OF ACTIVE SPECIES
decomposed by H2O2 (Eqns 8 and 9).[27] The λmax values of the
Table 1. Chemical bromination in the model system
product in CCl4 and the acetate buffer were 415 and 267 nm,
ꢀ [26b,29]
respectively. These values corresponded to Br2[28] and Br3
,
Entry
Oxidants
NaBr
H2O2
a (%)
b (%)
respectively. This result was the same to the bromination under
a strong acidic pH.[30]
1
2
3
4
5
6
7
8
AcOOH
AcOOH
AcOOH
AcOOH
NaOBr
NaOBr
NaOBr
NaOBr
Br2
+
+
ꢀ
ꢀ
+
ꢀ
+
89
59
1>
1>
8
1>
5
1>
78
5
74
1>
0
0
5
1>
1>
1>
67
1>
1>
1>
ꢀ
Br2 þ H2O⇌HOBr þ Hþþ Brꢀ
(6)
(7)
+
ꢀ
+
Br2 þ Brꢀ ⇌ Br3
ꢀ
+
ꢀ
ꢀ
+
ꢀ
HOBr þ H2O2→O2 þ Hþþ Brꢀþ H2O
(8)
(9)
+
9
ꢀ
+
Br2 þ H2O2→O2 þ 2Hþþ 2Br
-
10
11
12
Br2
Br2
Br2
+
ꢀ
ꢀ
77
1
ꢀ
+
Theꢀconsumption efficiency of 1 and production efficiency
of Br3 using oxidant such as AcOOH, NaOBr, and Br2 are
shown in Table 1. It was reported that 1 is consumed by
oxygen of a strong oxidant.[31] AcOOH is also a strong oxidant;
however, 1 was not consumed by AcOOH (Entries 3a and 4a).
AcOOH and Br2 strongly contributed to the bromination of 1
in the presence of NaBr and absence of H2O2 (Entries 1a and
9a), whereas the contribution of NaOBr was weak (Entry 5a).
Although the bromination of 1 using NaOBr remained low
activity, the bromination using
The brominations using oxidants (AcOOH, NaOBr, and Br2)
were performed in the buffer solution with or without
additives (NaBr and H2O2) for 1 min. Final concentrations of
oxidants, NaBr, and H2O2, are 45 μM, 0.5 M, and 10 mM,
respectively. (a) Consumption efficiency of
production efficiency of Br3 were calculated using the
corresponding ɛ value.
1 and (b)
ꢀ
AcOOH was proportional to the
equivalent of the oxidants in stoi-
chiometry as well as the bromina-
tion using Br2 (Fig. 5(a)). A reason
for the weak brominating power
of NaOBr was supposedly due to
the decomposition of HOBr as
shown by Eqns 10 and 11.[20b,32]
The results indicated that HOBr
was not an active species on the
oxidative bromination following
perhydrolase activity.
2HOBr ⇌ HBrO2 þ Hþþ Brꢀ (10)
HOBr þ HBrO2 ⇌ BrO3ꢀþ 2Hþþ Brꢀ
(11)
It is believed that the production
of Br2 from the reaction between
AcOOH and Brꢀ undergoes the pro-
duction of HOBr (Eqns 1 and 12).[30]
In the case that appearance of HOBr
is assumed in production of ꢀBr2, the
production efficiency of Br3 using
NaOBr must be higher than that
using AcOOH. However, the produc-
tion efficiency using NaOBr (Entry
5b) was significantly lower than that
using AcOOH (Entry 1b) and Br2 (En-
try 9b). Comparison between AcOOH
and Br2 on the brominating activity
against phenol was also performed
Figure 5. Relation between consumption efficiency of 1 and various factors such as (a) amount of
oxidants, (b) concentration of H2O2, (c) pH, and (d) temperature. The brominations using AcOOH (circle
symbols), Br2 (diamond symbols), and NaOBr (triangle symbols) in the presence (open symbols) and
in
a 1.0 M AcONa-H2SO4 buffer
(pH 5.5) containing 0.5M NaBr and
30 %(v/v) MeOH. The consumption absence (closed symbols) of 10 mM H2O2 were observed without 5 min.
J. Phys. Org. Chem. 2016, 29 84–91
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H. CHINA, Y. OKADA AND H. OGINO
efficiency of phenol using AcOOH, 66%, was higher than that
using Br2, 34%. These facts let us expect that new active species
is involved with the oxidative bromination.
2MCD þ H2O2→2MCD· þ H2O
(14)
(15)
(16)
MCD· þ Br2→MBMCD þ Br·
Br· þ MCD→MCD· þ Hþþ Br
-
HOBr þ Hþþ Br → Br2 þ H2O
(12)
-
For the bromination using AcOOH in a solution with NaBr and
without H2O2, the initial consumption rate of 1, 0.248 mMsꢀ1
was faster than the net initial production rate of Br3
,
,
ꢀ
On the other hand, the increase in the consumption efficiency
of 1 using AcOOH in the presence of NaBr accompanied with the
decreasing pH (Fig. 5(c)) implied that the production of active
species was driven by the protonation of AcOOH (Eqn 17). The
reaction in the presence of 0–2.5 M NaBr showed that the pro-
duction efficiency of Br3ꢀ increased with the increasing concen-
tration of Brꢀ despite a decreasing consumption efficiency of 1
0.110 mMsꢀ1. The reaction between Br2 and Brꢀ (Eqn 7) will not
become a rate-determining step because the reaction is too fast.
Although the bromination of 1 using Br2 was strongly interfered
with H2O2 (Entry 10a), the bromination using AcOOH maintained
the high consumption efficiency (Entry 2a, Fig. 5(b)–(d)). The
results indicated that a brominating species produced by the reac-
tion between AcOOH and Brꢀ possesses some tolerance against
H2O2. On the other hand, CO2 pro-
ꢀ
(Fig. 6(a)). The brominating power of Br3 is significantly lower
duced in its reaction was detected as
CaCO3 using Ca(OH)2 solution. The re-
action between AcOOH and Brꢀ in the
absence and presence of H2O2 gave
CaCO3 in 1.27 0.02 and 0.20 0.04 %
yields, respectively. It can be inter-
preted that the cause of the suppres-
sion on the production of CaCO3 by
H2O2 is responsible for the decrease in
abundance of active species accompa-
nied with decarboxylation. Acetyl
hypobromite (AcOBr), which is well
known as an intermediate of the
Hunsdiecker reaction (Eqn 13), is
decomposed into an alkyl bromide
(RBr) and CO2 via halodecarboxylation
by heating.[33]
A decrease in the
consumption efficiency of 1 with the
increasing temperature (Fig. 5(d))
reflected in the instability of the active
species. Therefore, AcOBr was sug-
gested to be a significant active species
for the oxidative bromination.
Figure 6. Behavior of active species for bromination via an ionic reaction. (a) Relation between concen-
tration of NaBr and the efficiencies on consumption of 1 (closed circle symbols) and production of Br3
(open circle symbols). The experiments were performed in a 1.0 M AcONa-H2SO4 buffer (pH 5.5) with
ꢀ
50 mM NaBr at 25 °C for 1 min. (b) UV–Vis spectra of AcOBr and Br2. Change from AcOBr to Br2 was
observed by the addition of excess of NaBr to 5 M AcOBr (ε = 203 10 Mꢀ1 cmꢀ1 [28b]
λ = 320 nm) in
,
CCl4 prepared by mixing AcOAg and Br2 (ε = 198.8 Mꢀ1 cmꢀ1 [35] λ = 415 nm)
,
AcOBr→CH3Br þ CO2
Reaction species
(13)
It was suggested that the bromina-
tion of 1 using haloperoxidase un-
dergoes the radical chain reaction
depending on the MCD free radical
(MCD), which was produced by the
one-electron oxidation with H2O2
(Eqns 14–16).[31] This is the reason
that the α-position of 1 is activated
by the chlorine substituent. How-
ever, the initial rate for the bro-
mination of 1 in the presence of
450 μM hydroxyl-TEMPO as a radi-
cal scavenger (0.243 mMsꢀ1) hardly
decreased. The bromination of
dimedone without a substituent
using AcOOH readily proceeded
Figure 7. Determination about (a) k25 °C and (b) Ea on the production of the active species from the
reaction between AcOOH and Brꢀ. The reaction was performed in 1.0 M AcONa-H2SO4 buffer (pH 5.5) with
50 mM NaBr for 1 min
affording
2-dibromodimedone
(DBD, 4) in 62% yield.
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PRODUCTION MECHANISM OF ACTIVE SPECIES
than that of Br2.[34] The conversion from AcOBr to Br2 was
confirmed by mixing AcOBr and NaBr in CCl4 (Fig. 6(b)). These
facts indicated that the active species produced from the
reaction between AcOOH and Brꢀ was the brominium cation
(Br+) donor and implied that the non-enzymatic step for the
bromination using perhydrolase is an ionic reaction.
facts implied that AcOOH+2 intermediate is readily produced in
an aqueous weak acidic solution; that is to say, the proton-
ation for the first step in the production mechanism is not
the decisive rate-determining step. On the other hand, it was
proved that the second step on the three-reaction mecha-
nisms are the rate-determining steps under strong acidic condi-
tion (Fig. 9B(b)–(d)).[36] There is a possibility that the three reactions
occur under the mild conditions of the MCD assay for perhydrolase.
The activation energy (Ea = 49.5 kJmolꢀ1) and pre-exponential factor
(A= 4.14 × 106 Mꢀ2sꢀ1) for the production of the active species from
the reaction between AcOOH and Brꢀ were obtained by Arrhenius
plots(Fig.7(b)).ItisreasonablethattheEavaluesofthethreereactions
were higher than the Ea value of the production and that the k25°C
values of the three reactions were lower than the k25°C value of the
production (Table 2). The initial rate of the bromination was directly
AcOOH þ Hþþ Br → AcOBr þ H2O
(17)
-
Rate-determining step
It was presumed that the oxidative bromination step is divided
into the production of AcOBr (Eqn 17) and consumption of
AcOBr and 1 (Eqn 18). A linear relationship between ln
[MCD]/[MCD]0 and time showed a pseudo-first-order reaction
with a kinetic constant at 25 °C (k25°C = 8.707 × 10ꢀ3sꢀ1) for
the consumption of 1 (Fig. 7(a)).
The initial rate of the bromination
depended on pH (Fig. 8(a)), temper-
ature (Fig. 8(b)), and concentrations
of AcOOH (Fig. 8(c)) and NaBr (Fig. 8
(d)), however, was not dependent
on the concentration of 1 (Fig. 8
(e)). These results indicating that
the consumption step is faster than
the production step reflected in
high activity of the active species
on the bromination. Indeed, the
direct bromination of 1 using AcOBr
in CCl4 is the rapid reaction beyond
our measurement limit. Thus, the
production step is the rate-
determining step represented as
Eqn 19, which is agreed with the
steady-state approximation for the
bromination, whereas the bromi-
nation was inactivated by H2O2
(Fig. 8(f)).
MCD þ AcOBr→MBMCD þ AcOH
(18)
v ¼ ꢀd½MCDꢁ=dt
¼ d½MBMCDꢁ=dt
¼ d½AcOBrꢁ=dt
(19)
Production mechanism of AcOBr
The production mechanism of
AcOBr was discussed by referring
to the three-reaction mechanisms
of production, hydrolysis, and spon-
taneous decomposition for AcOOH. It
is known that the three reactions
undergo the protonation of a car-
bonyl group for the formation of an
active intermediate in the first step
(Fig. 9(A)).[36] The protonated AcOOH
(AcOOH+2) with low energy structure
is affirmed by a proton affinity value
Figure 8. Initial rate for the oxidative bromination of 1 using AcOOH in the presence of Brꢀ. Depen-
dences on (a) pH, (b) temperature, and concentrations of (c) AcOOH, (d) NaBr, (e) 1, and (f) H2O2 were
observed within 10 min. Standard condition for the experiments is 45 μM of 1, 45 μM AcOOH, and
ofAcOOH(ꢀ775.9 kJmolꢀ1).[37] These 50 mM NaBr in the absence of H2O2 at pH 5.5 and 25 °C
J. Phys. Org. Chem. 2016, 29 84–91
Copyright © 2015 John Wiley & Sons, Ltd.
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H. CHINA, Y. OKADA AND H. OGINO
model system using oxidant, because its oxidative bromination
undergoes non-enzymatic mechanism. In the chemical bromina-
tion in the presence of Brꢀ, the activity of HOBr and Br2 was
lower than that of AcOOH instead of the enzyme. HOBr and Br2
have been described as the active species in the oxidative bromi-
nation using the enzyme was excluded by the comparison of the
oxidants on the activity, then AcOBr was suggested as a new
active species on the bromination by detection of the decarbox-
ylation in the reaction between AcOOH and Brꢀ and the strong
brominating power with some tolerance against H2O2. Kinetic
analysis of the bromination with a pseudo-first-order reaction
in MCD assay clarified the ionic mechanism and rate-
determining step during the production of the active species.
The production of the active species, possessing a strong depen-
dent on the concentration of NaBr, was superior to the three
reactions as the production, hydrolysis, and spontaneous
decomposition of AcOOH in terms of k25°C and Ea. The rate-
determining step about the production mechanism of AcOBr
was interpreted as the reaction between AcOOH+2 and Brꢀ.
Therefore, the oxidative bromination following perhydrolase
activity was summarized with catalytic cycle (Fig. 10).
MCD assay that depends on the halogenation is necessary to
characterization of perhydrolase. However, the production
mechanism of the active species indicated that the evaluation
of perhydrolase activity is influenced on pH dependence of the
non-enzymatic bromination. In principle, net perhydrolase activ-
ity at basic condition is not detected by this method. Indeed,
perhydrolase with the activity at basic condition has not been
observed. Thus, the assay for perhydrolase should be improved
in the future. On the other hand, production of AcOBr possessing
strong brominating power in aqueous solution under mild
condition is very interesting. AcOBr, which is vulnerable to the
proton source, is generally synthesized in a toxic aprotic solvent
such as CCl4. The oxidative bromination using AcOOH in aque-
ous solution with halide salt is quick and safe. Employment of
perhydrolase with low concentration of H2O2 in the bromination
enhances its safety. The production mechanism of the active
species under mild condition will provide a significant key for
the application of perhydrolase.
Figure 9. The mechanism on (A) formation of carbocation intermediate
by protonation at first step, (B) intermolecular reaction between
carbocation intermediate and counterparts at second step, and intramo-
lecular reaction at third step. The counterparts about (a) production of
AcOBr, (b) production of AcOOH, (c) hydrolysis of AcOOH, and (d) decom-
position of AcOOH are Brꢀ, H2O, H2O2, and AcOOH, respectively
Table 2. The values of Ea and k25°C for the four reactions
Entry
1
Reaction
Ea (kJ molꢀ1 k25°C (sꢀ1
) )
Production of AcOBr
49.5
57.8
60.4
88.4
8.707 × 10ꢀ3
1.393 × 10ꢀ6
4.878 × 10ꢀ6
9.797 × 10ꢀ6
2[36c] Production of AcOOH
3[36c] Hydrolysis of AcOOH
4[36d] Decomposition of AcOOH
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