Horseradish peroxidase-mediated aerobic and anaerobic oxidations of 3-alkylindoles

Article abstract of DOI:10.1016/j.bmc.2005.02.013

Little is known about the HRP-mediated oxidations of 3-alkylindoles. Whereas 3-methylindole and 3-ethylindole were found to be turned over smoothly by HRP, reactions of tryptophol and N-acetyltryptamine were inefficient. Oxidations of the former two indoles by HRP under aerobic conditions led to the corresponding ring-opened o-acylformanilides and oxindoles, whereas anaerobic oxidations resulted in oxidative dimerization. The major products of anaerobic oxidation of 3-methylindole were shown to be two hydrated dimers, while anhydrodimers were obtained in the 3-ethyl case. The proposed mechanism involves HRP-mediated one-electron oxidation to give an indole radical that either dimerizes (anaerobic conditions) or reacts with O₂ (aerobic conditions). Measured kinetics of indole oxidation by HRP compounds I and II mirrored their relative reactivities under turnover conditions. The observed comparable binding affinities for all four indole substrates investigated suggest that the low reactivity of tryptophol and N-acetyltryptamine reflect binding to HRP in an orientation that is disadvantageous to electron transfer oxidation of the indole ring.

Full text of DOI:10.1016/j.bmc.2005.02.013

Bioorganic & Medicinal Chemistry 13 (2005) 3543–3551
Horseradish peroxidase-mediated aerobic and anaerobic
oxidations of 3-alkylindoles
Ke-Qing Ling and Lawrence M. Sayre*
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA
Received 18 December 2004; revised 5 February 2005; accepted 9 February 2005
Available online 6 April 2005
Abstract—Little is known about the HRP-mediated oxidations of 3-alkylindoles. Whereas 3-methylindole and 3-ethylindole were
found to be turned over smoothly by HRP, reactions of tryptophol and N-acetyltryptamine were inefficient. Oxidations of the for-
mer two indoles by HRP under aerobic conditions led to the corresponding ring-opened o-acylformanilides and oxindoles, whereas
anaerobic oxidations resulted in oxidative dimerization. The major products of anaerobic oxidation of 3-methylindole were shown
to be two hydrated dimers, while anhydrodimers were obtained in the 3-ethyl case. The proposed mechanism involves HRP-med-
2
iated one-electron oxidation to give an indole radical that either dimerizes (anaerobic conditions) or reacts with O (aerobic condi-
tions). Measured kinetics of indole oxidation by HRP compounds I and II mirrored their relative reactivities under turnover
conditions. The observed comparable binding affinities for all four indole substrates investigated suggest that the low reactivity
of tryptophol and N-acetyltryptamine reflect binding to HRP in an orientation that is disadvantageous to electron transfer oxidation
of the indole ring.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
The indole structure can be found in many biologically
important compounds such as the plant hormone in-
dole-3-acetic acid (IAA), the pineal gland hormone mel-
atonin, the neurotransmitter serotonin, the amino acid
tryptophan and many other naturally occurring prod-
ucts. Research on HRP mediated oxidation of indole
derivatives has been mainly focused on IAA in connec-
tion with its oxidative degradation by plant peroxidase,
Horseradish peroxidase (HRP) catalyzes the oxidation
of phenols, anilines, and a variety of other electron-rich
1
compounds by H O and alkylhydroperoxides. Reac-
2
2
tion of native HRP, PFe(III), with H O first produces
2
HRP compound I, a two electron oxidized species
known to be ferryl porphyrin radical cation
2
a
+
Å
3
P Fe(IV)@O. One-electron reduction of compound I
by substrate gives rise to compound II, PFe(IV)@O,
which is then further reduced by one electron to the rest-
ing state of the enzyme. In some cases, compound I can
be reduced directly to PFe(III) via an overall two-elec-
tron oxidation process such as in the case of oxidation
of sulfides, although this reaction appears to involve di-
rect oxygen transfer from compound I to the sulfur
important for regulating plant growth. IAA is known
to react with HRP either in the presence or absence of
H O through a complicated mechanism involving
HRP compounds I, II, and III, and ultimately the dead
2
2
4
enzyme P-670. Under physiological conditions, IAA
exists as a carboxylate, and its degradation by HRP in-
volves decarboxylation of the initially generated IAA
radical cation, leading to the formation of CO and ska-
2
2
atom. In the presence of high concentrations of H O ,
2
tolyl radical, the latter then reacting with O to form in-
2
2
compound II can be converted to compound III, which
can be subsequently converted to an irreversibly inacti-
vated form of the enzyme absorbing at 670 nm.
dole hydroperoxide and other secondary degradation
4
products. However, there has been only limited investi-
gation of the chemistry of HRP-mediated oxidation of
1
3-alkylindoles other than IAA, where decarboxylation
would not be the key reaction feature. A recent study re-
ported the ability of tryptophan, tryptamine (and its N-
acetyl derivative), melatonin, and serotonin to reduce
HRP compounds I and II, and the rates paralleled
Keywords: 3-Alkylindoles; Peroxidase.
*
Corresponding author. Tel.: +1 216 368 3704; fax: +1 216 368
006; e-mail: lms3@case.edu
5
3
the ease of one-electron oxidation of these indoles.
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.013

3544
K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
However, no turnover products were reported in this
study, and turnover would be limited in most cases by
the rather slow rate-limiting reduction of compound
II. In fact, whereas HRP-mediated oxidations of melato-
nin and tryptophan were found in another study to gen-
erate the corresponding ring-opened 2-ketoformanilides
anol, which does not alter enzyme behavior, though the
7
rates are slower. Reactions of indoles 1 were thus car-
ried out in 50% pH 7 phosphate buffered aqueous meth-
anol under turnover reaction conditions with a molar
ratio of 1:10,000 (HRP/substrate). Starting concentra-
tions were 15–25 mM indole substrate, 1.5–2.5 lM
HRP, and the concentration of H O was maintained
(
these products required a high concentration of H O ,
presumably via dioxetane intermediates), formation of
2
2
at <30 lM by dropwise addition of a dilute stock solu-
tion as described in Section 4. We found that, under
such turnover conditions, only 1a and 1b were good sub-
strates for HRP. Reactions of 1c and 1d were slow, aero-
bically and anaerobically, and only a low conversion of
substrates (<10%) could be reached after 5 h. Attempts
to increase conversion by adding excess H O resulted
2
2
was sensitive to superoxide dismutase, and was pro-
posed to involve compound III rather than the normal
6
peroxidative cycle.
In this paper, we chose to investigate HRP mediated
oxidations of several 3-alkylindoles (1a–d) as representa-
tives of this biologically important class of compounds.
Reactions were conducted in the presence and absence
of O , since O is often integrally involved in the product
2
2
in loss of enzyme activity, presumably through interme-
8
diacy of compound III formation.
2
2
outcome of reactions involving radical intermediates.
The observed differences in chemistry as a function of
structural differences provide insight into the interaction
of biological indoles with peroxidases.
Thus, compounds 1a and 1b became the focus of de-
tailed investigation for HRP-mediated oxidations under
both aerobic and anaerobic conditions, and the results
are listed in Table 1. In addition to the identifiable prod-
H
N
R
N
N
H
R
R
R
O
H
O
R
R
O
R
R
R
H
N
N
N
H
N
H
H
N
N
O
H
1
2
3
4
dl-5
meso-5
1
1
1
1
a: R = CH
3
b: R = C
2
H
5
c: R = CH
2
CH
2
OH
2a-11a: R = CH3
d: R = CH
2
CH
2
NHCOCH
3
2b-11b: R = C H5
2
H
N
H
N
H
N
H
N
HO
R
N
HO
N
R
R
R
R
H
R
R
R
R
OH
R
R
R
O
OH
N
H
N
N
H
N
H
N
H
erythro-7
N
H
6
erythro-8
9
10
1
1
2
. Results and discussion
ucts listed, the reactions produced variable amounts of
insoluble, probably polymeric material, which ac-
counted for the bulk of the remaining starting indole
not recovered. For aerobic reactions, cleavage products
2 (major) and oxindoles 3 (minor) were obtained (Table
1). In the absence of oxygen, however, the oxidation of
2
.1. HRP-mediated oxidation of 3-alkylindoles
HRP oxidations of organic substrates only sparingly
soluble in water have been carried out in aqueous meth-
a
Table 1. Reaction conditions and product distribution of HRP-mediated oxidation of 1a and 1b
b
Reaction time (h)
c
consumed (mol equiv)
d
Products (%)
Substrate
Atmosphere
H
2
O
2
Conversion (%)
2
3
4 + 7
5
1
1
1
1
a
a
b
b
Air
3
3
5
5
0.33
0.45
0.32
0.43
85
77
43
37
38
0
21
0
0
33
0
0
0
0
Argon
Air
Argon
38
0
21
0
0
32
a
b
c
Reactions conducted in 1:1 0.1 M pH 7.0 sodium phosphate buffer/methanol at 25 ꢁC.
The time at which point no further H
2
O
2
could be consumed, as verified by the guaiacol test (see Section 4).
With respect to converted indole substrate.
1
Percent of converted indole substrate as indicated by H NMR of the CH
d
2
Cl
2
extract of the reaction mixture. This number may underestimate the
true yields due to incompletely extraction, but otherwise, the remaining material (maximally 41–68%) represents unidentified oligomeric or
polymeric products.

K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
3545
Al
N
N
R
R
LiAlH
4
R
R
R
N
H
H
N
N
Al
1
2
5
1
4
Scheme 1. LiAlH reduction of the dimer 5.
any products corresponding to 10b and/or 11b. The best
explanation is that the two anhydrodimers from 1b are
meso- and DL-5b, both of which underwent facile C–C
1
Figure 1. Crude H NMR (300 MHz, DMSO-d
6
) for the dichloro-
methane extract of the reaction mixture of the anaerobic oxidation of
1a, showing the tertiary CH doublets (d 4.79 ppm, J = 4.5 Hz, and d
cleavage during LiAlH reduction to give 1b via interme-
4
5.11 ppm, J = 2.4 Hz) of the major (4a) and minor (7a) dimers,
respectively.
diate 12b (Scheme 1). The aromatization accompanying
C–C cleavage in 12b apparently provides the driving
force for occurrence of this reaction to the exclusion
of reduction of 12b to 11b. Lewis acid Al(III)-mediated
1
0
11
1
a yielded two different products, both of which repre-
C–C cleavage and 1,2-alkyl shifts have been ob-
served previously in LiAlH reductions.
sent initial oxidative coupling at C3. The dimer mono-
hydrate 4a arising from hydration and reorganization
of the initially formed dimer DL-5a was isolated after
4
The latter observation allowed us to tentatively assign
the structure of the single slow-moving minor dimer hy-
drate (erythro-7a or erythro-8a) arising from anaerobic
9
column chromatography. The isomeric meso-5a dimer,
if formed, could not be converted to a monohydrate
analogous to 4a, but could form the monohydrate 6a
or the dihydrate forms, erythro-7a or erythro-8a. The
presence of one of these compounds (6a–8a) could be
oxidation of 1a. We found that LiAlH reduction of
4
the crude reaction mixture of anaerobic oxidation of
1a, showing the presence of both 4a and the minor dimer
hydrate, gave only DL-10a along with 1a. Since DL-10a
arises from 4a, the precursor to 1a is presumably ery-
thro-7a, which is proposed to equilibrate to its dehy-
drated form 5a during the reaction with LiAlH4.
Reduction of erythro-8a would have been expected to
give the trans isomer of 10a, which was not found.
The finding that the downfield CH in 7a appears further
1
deduced from the crude H NMR of the concentrated
organic extract (Fig. 1), with the major doublet at
4
DMSO-d . We were unable to isolate the minor product
.79 ppm being identical with that of isolated 4a in
6
showing the doublet at 5.11 ppm by chromatography,
probably due to its higher polarity (suggesting it is 7a
or 8a rather than 6a) and lower stability (see Section
4
). Although 4a is the major product as determined by
downfield (d 5.11 in DMSO-d ) than for 4a (d 4.79 in
6
TLC prior to extraction, the yield of the second product
may be greater than is apparent in Figure 1 due to
incomplete extraction. In sharp contrast, anaerobic
DMSO-d ) may reflect the fact that in the rigid structure
9
6
of 4a, one of the two benzene rings is positioned to
cause partial shielding of this proton.
1
reaction of 1b gave a mixture of what H NMR showed
to be two anhydrodimers, whose structures were either of
two possible diastereomers of 5b and/or 9b. No dimer
hydrate was found and also no hydration reaction oc-
curred when these anhydrodimers (5b/9b) were dissolved
in aqueous methanol in the absence or presence of either
2.2. Reaction mechanism
Control experiments showed that no reaction occurred
in the absence of HRP and/or H O , indicating that
all the products are both HRP dependent and H O
2
2
2
2
acid (HOAc) or base (Et N).
3
dependent. Furthermore, since catalytic amounts of
HRP were used, both compounds I and II are involved
in product formation (i.e., products are not being
formed through a stoichiometric compound I pathway).
The concentration of H O was never >30 lM (at most
Since neither the structures 5 and 9, nor the structures 6–
8
could be readily distinguished from each other by
NMR (HMQC, HMBC, and NOESY) due to the sym-
metric feature of these molecules, we sought chemical
evidence to resolve the structural ambiguities. LiAlH4
reduction was chosen because no equilibrium inter-
2
2
20:1 over HRP), and no evidence for the involvement of
compound III was obtained. In fact, at higher [H O ],
2
2
where compound III should form, the reactions were
very slow, suggesting loss of enzyme activity. TLC anal-
ysis of the reaction mixtures revealed that neither the di-
mers 4a (7a) and 5b were formed in aerobic reactions,
nor products 2 and 3 were produced in anaerobic reac-
tions (Table 1). This product distribution immediately
leads to the conclusions that (1) products 2 and 3 are
both O2 dependent and (2) the dimeric products 4a
(7a) and 5b formed under anaerobic condition are to-
9
change among 4–9 could occur under such conditions.
Also, the anticipated reduction products 10 (from 4, 8,
or 9) and 11 (from 5, 6, or 7) would no longer undergo
any tautomerization, thus permitting their full charac-
terization. Dimer 4a (DL) was found to be reduced quan-
titatively to the expected cis isomer (DL) of amine 10a by
9
LiAlH in absolute ether. Surprisingly, similar LiAlH
4
4
reduction of the suspected anhydrodimers (5b/9b) from
anaerobic oxidation of 1b afforded only 1b without
tally suppressed by O under aerobic conditions.
2

3546
K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
H
N
H
N
R
N
H
HO
H
2
O
CH
3
2
-H O
R
N
H
3
H C
R
CH
OH
3
O
threo-
3
H C
H
1
R = CH
3
N
H
N
N
H
5
7a
4
a
HRPI
HRPII
HRP
HRPII
argon
R
R
R O O
R
R
H
O
2
-
H
+
O
OH
20
N
H
N
N
N
H
N
15
1
3
14
3
1
4
"
NIH shift"
1
R
R
O
H
N
R
R
OH
O
R O OH
O
H
O
N
N
H
N
N
16
HRP HRPI
1
8
H
H
19
O
H
1
7
2
Scheme 2. Proposed mechanism of HRP-mediated oxidation of 3-alkylindoles.
A possible mechanism for HRP mediated oxidation of
dioxetane intermediate 17, which would undergo rapid
C–C cleavage, leading to the formation of product 2,
in analogy to what has been reported for peroxidase-
3-alkylindoles is provided in Scheme 2, with one electron
oxidation of 1 by either HRP compounds I or II as the
key step. The oxidation potentials of 3-alkylindoles
(reduction potentials of the radical cations) have been
determined in aqueous solution at pH 7 to be about
6
mediated oxidation of melatonin and related indoles.
Although hydroperoxide 16 can theoretically also be
1
6
converted to 2 by a Criegee-like rearrangement, evi-
dence for the intermediacy of the dioxetane is that the
1
2,13
1
.0–1.1 V versus NHE.
These can be compared to
the redox potentials of HRP compounds I and II, both
6
reported HRP oxidations are highly chemiluminescent.
1
4
of which have been estimated to be ꢀ0.9 V, though
Dioxetane 17 could form instead by cyclization of per-
oxy radical 15, and subsequent hydrogen atom abstrac-
tion. Alternatively, 16 could be reduced to alcohol 18,
possibly by way of converting native HRP to compound
I. Alcohol 18 should be in equilibrium with epoxide 19,
which has been shown to arise from cytochrome P450-
mediated oxidation of 1a and which is converted, by
the NIH shift mechanism (labeling study) to oxindole
compound II reactions are still typically slower than
those of compound I. This suggests that the reactions
1
are only slightly uphill, though it has been found that
HRP compound I oxidation of a series of ring-substi-
tuted IAAs is significantly slower than oxidation of phe-
4
f
nols of similar potential. Such observation could signal
lower binding affinities or less-productive binding modes
1
7
(
for electron transfer) of the indole compounds.
3. Although 19 could also form directly from com-
pound I-mediated oxygenation of 1a (at least some sub-
strates for HRP are thought to be accessible to direct
oxygenation by compound I), such is not the case here,
because no 3 was produced in the absence of O2.
Since electron-transfer oxidation of aryl substrates by
HRP typically follows the same initial course as do other
types of chemical single-electron transfer (SET) and
electrochemical oxidations, it can be presumed that the
first reaction of the indole substrates with HRP com-
pounds I and II is SET to give iminium cation 13, which
2.3. Kinetic studies and binding experiments
has a low pK and can readily dissociate at pH 7 to give
a
the neutral imine radical 14. Radical 14 is not readily
oxidized further, and thus undergoes dimerization, with
The observations that oxidation of 1a by HRP was pre-
paratively more efficient than that of 1b (Table 1) and
that 1c and 1d are not turned over smoothly by HRP,
suggested a unique structural dependence for the enzy-
matic as opposed to simple chemical oxidation, which
would not be expected to discriminate among these four
compounds. To gain information on the enzymologic
structure–activity relationships, we investigated the
kinetics and binding features of indoles 1a–d with
HRP. First, the apparent second-order rate constants
kapp of 1a–d with HRP compounds I and II were deter-
mined by monitoring the generation of the product spe-
cies in each case, compound II and native HRP,
respectively, under pseudo-first order reaction condi-
1
5
5
b being a final stable products, whereas dimers 5a are
further hydrated to form 4a and 7a. The different behav-
iors of 5a and 5b suggest that the C@N bond in 5b is less
susceptible to water addition than 5a, possibly due to
the steric hindrance.
For the aerobic reactions, dimerization of 14 apparently
cannot compete with its trapping by O to afford the
hydroperoxy radical 15, which generates hydroperoxide
2
16 via hydrogen atom abstraction from, for example, the
starting indole 1. The hydroperoxide 16 can cyclize to

K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
3547
a
Table 2. Apparent second-order rate constants of HRP compound I and II with indole substrates
ꢁ1
ꢁ1
ꢁ1 ꢁ1
app or HRP compound II (M s )
Substrate
k
app or HRP compound I (M
s
)
k
b
b
c
c
f
f
g
g
pH 7
pH 8
pH 9
pH 10
pH 7
pH 8
pH 9
pH 10
d
5e
5e
4
4 h
4 h
3
1
1
1
1
a
b
c
2.83 · 10
3.54 · 10
1.59 · 10
3.77 · 10
(2.8 · 10 )
(1.7 · 10 )
1.40 · 10
8.67 · 10
32.9
10.8
d
2
4
4
4
2
2
2
1.56 · 103
3.76 · 10
1.58 · 103
3.83 · 10
1.63 · 103
3.75 · 10
4.37 · 10
4.18 · 10
1.43 · 10
92.0
30.0
3
2
d
1.51 · 10
a
b
c
All the experiments were conducted at 25 ꢁC at least twice, and the average values are reported with standard deviations less than 10%.
Phosphate buffer (0.1 M); [HRP] = 1.4 lM; [H ] = 14 lM and [substrate] P 14 lM.
Borate buffer (0.1 M); [HRP] = 0.7 lM; [H ] = 7 lM and [substrate] P 7 lM unless otherwise stated.
Rates at low pH for compounds I and II were too fast to permit their dissection.
2 2
O
2
O
2
d
e
Because the reactions were fast, only a 1.75- and 2.5-fold excess of 1a and 1b, respectively, were used and the rate constant was obtained from the
initial stage of reaction where the pseudo first-order plots were linear.
f
Phosphate buffer (0.1 M); [HRP] = [H
2
O
2
] = 2.1 lM and [substrate] P 21 lM.
] = 2.1 lM and [substrate] P 21 lM.
value of 8.7 for the protonated form of HRP compound II.
g
h
Borate buffer (0.1 M); [HRP] = [H
Estimated value using a pK
2 2
O
19
E
tions (Table 2). We chose to study the kinetics at neutral
to basic pH (7–10) because we are interested in kinetic
constants near physiologic pH, and because whereas
compound I oxidation rates are constant in this pH
range, there is a group on the enzyme that titrates at
pH 5.4 to give a 10-fold more weakly oxidizing com-
0.08
25
20
15
0
0
0
0
.06
.04
.02
.00
1
5
0
0
r = 0.99995
1
8
pound I conjugate acid. This would result in a com-
pression between compound I and II rates at lower
pH, making their kinetic dissection difficult. In our cho-
sen pH range (7–10), there is an ionization of an enzyme
group around pH 8.7 that converts compound II to an
0.0 0.5 1.0 1.5 2.0 2.5
[
E]/∆A mM
1
9
unreactive conjugate base form. Thus, the already
slower compound II rates decrease with increasing pH.
The expected constancy of compound I rates and de-
crease in compound II rates are supported by the data
obtained for substrates 1c and 1d (Table 2).
-0.02
-0.04
3
20 340 360 380 400 420 440 460 480 500
λ (nm)
For the more rapidly oxidized 1a and 1b, compound I
and II rates could be dissected only at the highest pH
Figure 2. Difference spectra obtained by titration of HRP (10.0 lM)
with 3-methylindole 1a in 0.1 M sodium phosphate buffer (pH = 7.00)
at 25 ꢁC and the corresponding 1/[S] vs [E]/DA plots. Over the course
of the titration shown, the concentration of HRP decreased from 10.0
to 9.65 lM, and the concentration of 1a increased from 0 to 2.48 mM.
(
10) we used, where the compound II rates were now slo-
wed by two-orders of magnitude relative to compound I
rates. Based on their pH independence, one can estimate
compound I rates at pH 7–9 to be the same as deter-
mined at pH 10 (Table 2). Furthermore, the rate con-
stants for compound II at pH 7–9 can be estimated by
multiplying the rate constants at pH 10 by the Hender-
son–Hasselbach correction calculated using a value of
Second, the binding of 1a–d to native HRP was exam-
2
0
ined using optical difference spectroscopy, an example
of which (titration of HRP with 1a) is shown in Figure
2. From these spectra, the apparent dissociation con-
1
9
8
tive (protonated) form of HRP compound II (the esti-
.7 for the acid dissociation constant pK for the ac-
E
mated value for pH 7 is listed in Table 2).
stants K and the molar absorptivity differences De of
d
the free and bound enzyme were obtained by correlating
1/[S] with [E]/DA (Fig. 2, Table 3, and Section 4). All
four indole substrates bind to HRP and exhibit similar
difference spectra with the same maxima (380 and
425 nm), minima (405 nm), and isosbestic points (393
and 417 nm). Although there are likely to be some differ-
ences between the binding of donor molecules to the
activated forms of HRP (compound I and compound
II) and the iron(III) resting form, it is unlikely that these
differences are large, and measured binding affinities to
activated HRP may not necessarily reflect productive ki-
Table 2 reveals that for all four indole substrates,
reaction of HRP compound II is rate-limiting, and that
the two simple 3-alkyindoles 1a and 1b are much more
reactive than 1c and 1d toward both compounds I and
II. Thus, despite 1b having a higher rate than 1a with
compound I, its slower rate with compound II explains
its lower reactivity under turnover conditions. Further-
more, despite the fast reactions of 1c and 1d with
HRP compound I, their rate-limiting reactions with
compound II are two orders of magnitude slower
than are the reactions with 1a and 1b, explaining
their near inertness under turnover conditions at neutral
pH.
2
1
netic complexes anyway. Evidence that the structures
of different forms of peroxidases may differ only
slightly is that in the crystal structure of cytochrome

3548
K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
Table 3. Difference spectrum characterization and apparent dissocia-
a
tion constants of HRP–substrate complexes
3. Conclusions
3
10 K
ꢁ3
ꢁ1
ꢁ1
)
Substrate
d
(M)
10 De (M cm
This is one of the first studies of HRP-mediated oxida-
tion of 3-alkylindoles that delineates the different fates
1
1
1
1
a
b
c
1.24
0.943
1.19
1.95
11.4
10.7
6.23
5.75
(
aerobic vs anaerobic) of the reaction following initial
electron transfer oxidation of the indole ring by HRP
compounds I and II. The indole redox potentials are
apparently sufficiently accessible that smooth turnover
is possible, but this was observed only for the simple
d
a
Experimental conditions are given in Figure 2, 0.1 M sodium phos-
phate buffer (pH = 7.00) at 25 ꢁC . In all four cases, the difference
spectra were isosbestic and showed maxima at 380 and 425 nm and a
minimum at 405 nm. Experiments were conducted at least twice and
the average values are reported.
3
-methyl and 3-ethyl indoles 1a and 1b. In these cases,
whereas anaerobic oxidations result in oxidative cou-
pling products, aerobic oxidations result in oxygenated
monomers. The much slower rates for 1c and 1d, de-
spite similar binding affinities, suggest that the more ex-
tended and functionalized side chain of these indoles
controls the orientation of the indole ring during bind-
ing in a manner that retards electron transfer oxidation
by the heme edge (compound I) and the iron center
(compound II). Binding of typical phenol and aniline
reducing substrates for HRP is thought to reflect mainly
hydrophobic interactions of the aryl rings with the
heme edge of the protein. The electron-rich indole ring
should be capable of interacting in a similar manner,
and 1a and 1b fit this model. In contrast, we propose
that hydrogen bonding of the b hydroxyl and acetam-
ido groups of 1c and 1d with peptide residues around
the heme edge preserve binding affinity despite removal
of the indole ring from binding in a manner that is opti-
mal for electron transfer, either due to increased dis-
c peroxidase, compound I exhibits only tenths of ang-
strom atomic coordinate deviations from the native en-
2
2
zyme. Most workers tend to use binding data for
2
0b
native HRP, obtained by optical spectroscopy
2
or by
NMR, to discuss modes of binding that determine
reactivity.
3
Studies on the redox potentials of indoles have shown
little variation for substitution on the alkyl side chains
e.g., IAA vs tryptamine). Thus, the divergent kinetic
1
3
(
behavior between 1a,b and 1c,d is unlikely to arise from
an electronic effect of the b hydroxyl and acetamido
groups on the ease of one-electron oxidation of 1c,d.
Assuming a similarity in the redox potentials for 1a–d,
the slower rates for 1c,d could be explained if the latter
two compounds bind proportionately more weakly to
compound II, the rate-limiting enzyme step. However,
the data in Table 3 shows that there is surprisingly very
little difference in binding affinity among the four in-
doles. Admittedly, the nearly twofold weaker binding
for 1d compared to 1c could contribute to the slower
rates for the former compound, but there must be an
alternative explanation for the much more rapid oxida-
tion of 1a and 1b relative to 1c and 1d. A clue to this en-
igma is that the molar absorptivity differences (De
values) for 1c and 1d are lower than for 1a and 1b, which
is consistent with the possibility that despite similar
affinities, the orientation mode of binding of 1c and 1d
differs from that of 1a and 1b in a manner that affects
the kinetics of electron-transfer oxidation. Since the
rates for 1c and 1d are retarded for both compounds I
and II, it appears that the more extended and function-
alized 3-alkyl side-chain impairs optimal positioning of
the indole ring for SET to both oxidants, namely the
heme edge in the case of compound I and the iron center
tance and/or
a
sub-optimal spatial orientation.
Further structure–reactivity studies on the HRP-medi-
ated oxidations of substituted indoles should be a
worthwhile objective.
4. Experimental
4.1. General procedures
In reporting NMR spectra, chemical shifts were refer-
enced to TMS or the solvent peak. High resolution mass
spectra (HRMS, EI, or FAB) were obtained on a Kratos
MS-25A instrument. Solution pH was measured with a
Fisher Accumet model 910 pH meter equipped with a
Fisher combination glass electrode, calibrated to ±0.01
pH units using commercial standard buffers.
UV–vis spectra were obtained using a Perkin–Elmer
Lambda 20 spectrophotometer equipped with a temper-
ature-controlled multiple-cell compartment. Time-drive,
spectral scan, and kinetics data were obtained using UV
WINLAB software. 3-Methylindole (1a) and tryptophol
(1c) were obtained from Aldrich Chemical Co. 3-Ethyl-
1
in the case of compound II.
Once one recognizes the possibility that rates may be af-
fected by binding orientation independent of binding
affinity, there are subtle proportionality differences in
the kinetic data (Table 2) that indicate that such binding
orientation may differentially affect compound I and
compound II rates. For example, although the slightly
higher reactivity of 3-ethyl (1b) vs 3-methyl (1a) indole
toward compound I may reflect more favorable binding
or electronic factors, the much slower reactivity of 1b
than 1a with compound II probably reflects a greater
steric binding requirement of compound II.
2
4
indole (1b) was prepared by the reported method. N-
Acetyltryptamine (1d) was prepared by treatment of
tryptamine with acetic anhydride. Horseradish peroxi-
dase (type VI, 250–330 units/mg solid using pyrogallol)
was obtained from Sigma Chemical Co. All yields
reported are based on starting material converted. The
enzyme concentration was determined spectro-
photometrically at 403 nm using a molar absorptivity
5
ꢁ1
ꢁ1 25
. Sodium phosphate mono-
of 1.02 · 10 M cm
basic and boric acid were reagent grade. All solutions

K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
3549
for kinetics and binding studies were prepared using
water purified by a Milli-Q water purification system
from Millipore. Hydrogen peroxide solutions were pre-
pared by diluting commercially available 30% hydrogen
peroxide with distilled water immediately before addi-
tion to enzyme reactions. Concentrations of diluted
hydrogen peroxide solutions were determined by mea-
suring the UV absorption at 240 nm, with
to check for accumulation of H O in the reaction mix-
2 2
ture. The reaction stopped when 0.041 mmol of H O
2
2
was consumed. Routine workup and chromatographic
separation afforded 24.8 mg of unreacted 1b (43% con-
version), and products 2b (8.7 mg, 38%) and 3b
(4.4 mg, 21%). Compound 2b: light brown oil, which
2
9 1
solidified on standing;
H NMR (300 MHz, CDCl3):
dH 1.22 (t, 3H, J = 7.5 Hz), 3.08 (q, 2H, J = 7.5 Hz),
7.16 (t, 1H, J = 7.5 Hz), 7.55 (t, 1H, J = 7.5 Hz), 7.95
ꢁ
1
ꢁ1 26
.
e = 43.6 M cm
(
J = 7.5 Hz), 11.66 (br, 1H, NH); C NMR (50 MHz,
d, 1H, J = 7.5 Hz), 8.49 (s, 1H), 8.74 (d, 1H,
1
3
4
(
.2. HRP mediated aerobic oxidation of 3-methylindole
1a)
APT, CDCl ): dC 8.5 (ꢁ), 33.2 (+), 121.8 (ꢁ), 121.9
3
(
(
+), 123.1 (ꢁ), 130.7 (ꢁ), 134.9 (ꢁ), 139.8 (+), 159.9
ꢁ), 205.4 (+); HRMS (FAB) calcd for C H NO
1
0
12
2
To a vigorously stirred solution of 1a (65 mg, 0.5 mmol)
and HRP (3 mg, 0.05 lmol) in a mixture of methanol
and 0.1 M pH 7.0 sodium phosphate buffer (1:1 v/v, to-
tally 20 mL) was added H O (34 mM) with a syringe in
(
brown oil;
M+1) 178.0868, found 178.0861. Compound 3b: light
H NMR (300 MHz, CDCl ): dH 0.93 (t,
3
0
1
3
3
3
7
H, J = 7.4Hz), 2.04 (dq, 2H, J = 7.4 and 5.6 Hz),
.46 (t, 1H, J = 5.6 Hz), 6.89 (d, 1H, J = 7.5 Hz),
.03 (t, 1H, J = 7.5 Hz), 7.19–7.27 (m, 2H), 8.56
2
2
a manner that delivered ꢀ10 lL drops. At the beginning
of the reaction, the formation of HRP compound I
(
13
(
br, 1H, NH); C NMR (50 MHz, APT, CDCl ): dC
3
green) and its subsequent conversion to the correspond-
1
1
0.1 (ꢁ), 23.7 (+), 47.2 (ꢁ), 109.5 (ꢁ), 122.3 (ꢁ),
24.2 (ꢁ), 127.9 (ꢁ), 129.6 (+), 141.8 (+), 180.6 (+);
ing HRP compound II (red) as well as the reproduction
of native HRP (brown-yellow) from HRP compound II
could be observed after each drop of H O was added.
As the reaction proceeded, however, such phenomenon
was hard to visualize due to the formation of insoluble
polymeric materials (minor oxidation products). Thus,
the lack of buildup of H O prior to more being added
was verified by analyzing small aliquots of the reaction
mixture for their ability to oxidize guaiacol. At the same
time, TLC was conducted to monitor the consumption
of starting material as well as the formation of products.
Only 0.14 mmol of H O was consumed when the reac-
tion stopped at a conversion of 85%. The reaction pro-
ceeded smoothly only when a very low concentration
of H O in the reaction mixture was maintained
HRMS (EI) calcd for C H NO 161.0841, found
11
1
0
2
2
1
61.0847.
4.4. HRP mediated anaerobic oxidation of 3-methylindole
2
2
(1a)
A solution of 1a (65 mg, 0.5 mmol) and HRP (3 mg,
.05 lmol) in a mixture of methanol and phosphate buf-
0
fer (1:1 v/v, totally 20 mL) was placed in a well-sealed
flask. The solution was stirred vigorously and bubbled
continuously with argon for 30 min using inlet and out-
let syringe needles. To this solution was added dropwise
freshly prepared argon-purged aqueous H O (34 mM)
2
2
2
2
2
2
throughout (if H O was added too quickly, the reaction
2
2
while argon was still kept bubbling throughout the reac-
tion course. The reaction stopped when 0.17 mmol of
H O had been added, with a conversion of 77%. Similar
appeared to stop at a point reflecting low conversion of
a). Then methanol was removed in vacuo and the aque-
1
2
2
ous solution was extracted with chloroform (3 · 20 mL).
The combined organic layer was dried (Na SO ) and
concentrated, and the residue was subjected to a silica
gel flash chromatographic separation (a gradient of hex-
anes–ethyl acetate as eluent) to afford 10 mg of unre-
acted 1a (85% conversion), and products 2a (26.3 mg,
workup and chromatographic separation as described
above afforded 15.3 mg of unreacted 1a (77% conver-
2
4
9
sion) and dimer 4a (15.9 mg, 30%): mp 202–203 ꢁC;
1
H NMR (200 MHz, DMSO-d ): d 1.18 (s, 6H), 4.78
6
H
(
d, 2H, J = 4.5 Hz, becomes a singlet on adding D O),
2
2
7 1
6
7
7
.53 (dd, 2H, J = 0.9, 7.9 Hz), 6.67 (dt, 2H, J = 1.3,
.5 Hz), 6.90 (d, 2H, J = 4.5 Hz, exchangeable, NH),
.03 (dt, 2H, J = 1.4, 7.5 Hz), 7.21 (d, 2H, J = 7.9 Hz);
3
8%) and 3a (13 mg, 21%). Compound 2a: H NMR
(
200 MHz, CDCl ): dH 2.68 (s, 3H), 7.18 (t, 1H,
3
J = 7.5 Hz), 7.57 (t, 1H, J = 7.5 Hz), 7.93 (d, 1H,
J = 7.5 Hz), 8.50 (s, 1H), 8.75 (d, 1H, J = 7.5 Hz),
1
13
C NMR (APT, DMSO-d ): d 13.1 (ꢁ), 42.5 (+), 94.8
6
2
8
1
(
(
(
ꢁ), 114.0 (ꢁ), 117.8 (ꢁ), 126.2 (ꢁ), 127.5 (ꢁ), 128.0
1.61 (br, 1H, NH). Compound 3a:
H NMR
+), 142.6 (+); HRMS (FAB) calcd for C H N O
1
8
19
2
(
(
200 MHz, CDCl ): dH 1.51 (d, 3H, J = 7.5 Hz), 3.47
3
M+1) 279.1497, found 279.1504.
q, 1H, J = 7.5 Hz), 6.89 (d, 1H, J = 7.5 Hz), 7.03 (t,
H, J = 7.5 Hz), 7.17–7.27 (m, 2H), 9.05 (br, 1H, NH);
1
1
3
The above oxidation was repeated with 13 mg of 1a and
.6 mg of HRP. After the reaction had finished, the mix-
C NMR (75 MHz, APT, CDCl ): dC 15.4 (ꢁ), 41.2
3
0
(
(
ꢁ), 109.9 (ꢁ), 122.5 (ꢁ), 123.9 (ꢁ), 128.0 (ꢁ), 131.4
ture was poured into water and extracted with dichloro-
methane. The combined organic layer was dried
+), 141.3 (+), 181.6 (+).
(Na SO ) and evaporated to dryness, and the residue
was analyzed by H NMR in different solvents. In
2 4
1
4
.3. HRP mediated aerobic oxidation of 3-ethylindole (1b)
CDCl , the apparent minor dimer product (tentatively
3
The same procedure as for 1a was employed, though
the amounts of 1b (43.5 mg, 0.3 mmol) and HRP
assigned as erythro-7a) exhibited NH and CH signals
9
appearing at 5.27 (br) and 5.31 (s), whereas in
DMSO-d6 (see Fig. 1), the CH signal appeared at
5.11 ppm (d, J = 2.4 Hz).
(
(
2 mg, 0.03 lmol) were smaller. Aqueous H O
34 mM) was added dropwise, using the guaiacol test
2
2

3550
K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
4
.5. Reduction of 4a by lithium aluminum hydride
and formation of compound II was monitored at
19 nm (pH = 7 and 8) or 420 nm (pH = 9 and 10). Since
4
To a suspension of LiAlH (19 mg, 0.5 mmol) in 5 mL of
4
there is an excess of substrate, and compound II is also
reduced by substrate, albeit more slowly, the formation
of compound II over time underestimates that which is
generated by reduction of compound I. The excess of
H O is used to regenerate compound I instantaneously
when compound II is reduced. The cycling described is
equivalent to establishment of an equilibrium between
compounds I and II, where kII represents the rate of
compound II reduction. According to this equilibrium,
absolute ether under argon was added 14 mg of 4a
0.050 mmol) with vigorous stirring. The mixture was
(
stirred at room temperature until 4a had disappeared
as monitored by TLC. Then the mixture was carefully
added to ice, and the resultant mixture was extracted
with ether. Separation, drying, and concentration of
the organic layer, and subsequent chromatographic sep-
2
2
9
aration of the residue afforded DL-10a (13 mg, 98%) as
a colorless viscous oil that solidified on standing. Reduc-
tion of the crude anaerobic oxidation reaction mixture
of 1a containing 4a and erythro-7a with LiAlH was
if kII is less than one tenth that of k , the data at early
I
stages of the reaction, where k (compound II) is negli-
II
gible, can be analyzed by pseudo first-order kinetics.
4
examined in an attempt to observe the reduction prod-
uct of the minor dimer erythro-7a. H NMR of the mix-
ture after workup revealed the presence of only DL-10a
and 1a.
1
k ðfastÞ
I
compound I ꢀ compound II
kIIðslowÞ
2
1
4
(
.6. HRP mediated anaerobic oxidation of 3-ethylindole
1b)
A fresh solution of HRP compound II was prepared
by addition of 1 mol equiv of H to the native HRP
O
2 2
solution (3 mL, 2.1 lM in buffer) and then waiting
45 min before initiating reaction with substrate, at which
point less than 5% of residual compound I was present,
as determined spectrophotometrically. After addition of
at least 10-fold excess of substrate to the cuvette con-
taining the HRP compound II, the reaction was fol-
lowed by monitoring the appearance of native HRP at
403 nm. The initial portion of the reaction, correspond-
ing to reduction of residual compound I, was ignored in
the processing of the data. Control experiments showed
that the substrates were all stable in buffer in the absence
To a stirred solution of 1b (43.5 mg, 0.3 mmol) and
HRP (2 mg, 0.03 lmol) under argon as described for
1
reaction stopped when 0.048 mmol of H O was con-
a was added dropwise aqueous H O (34 mM). The
2 2
2
2
sumed. Routine workup and chromatographic separa-
tion afforded 27.4 mg of unreacted 1b (37%
conversion) and dimer 5b as a 1:1.4 mixture of two dia-
1
stereomers (5.1 mg, 32%): mp 45–49 ꢁC; H NMR
(
300 MHz, CDCl ): d 0.22 (t, 6H, J = 7.5 Hz), 0.29 (t,
3 H
6
1
H, J = 7.5 Hz), 1.93 (dq, 2H, J = 14.6 and 7.5 Hz),
.97 (dq, 2H, J = 14.6 and 7.5 Hz), 2.05 (dq, 2H,
or presence of the same concentrations of H O used
2 2
J = 13.4 and 7.5 Hz), 2.41 (dq, 2H, J = 13.4 and
.5 Hz), 7.06 (d, 2H, J = 7.5 Hz), 7.14 (t, 2H,
J = 7.5 Hz), 7.22–7.30 (m, 6H), 7.39 (t,
H, J = 7.5 Hz), 7.52 (d, 2H, J = 7.5 Hz), 7.63 (d, 2H,
above. Blank cuvettes in these experiments contained
only the appropriate buffer. In all cases, the recorded
time-dependent absorption (A) curves were ana-
7
2
lyzed according to the equation, ln[(A
1
(A )] = kobsdt, to yield pseudo-first order rate con-
ꢁA
)/
0
1
3
J = 7.5 Hz), 7.97 (s, 2H), 8.11 (s, 2H); C NMR
1
ꢁA
t
(
(
1
1
1
50 MHz, APT, CDCl ): dC 7.83 (ꢁ), 8.05 (ꢁ), 23.2
stants (kobsd), which were then converted to the apparent
second-order rate constants listed (kapp, Table 2) by
dividing by the indole substrate concentration ([S],
1.2–30 lM). The second-order dependence was verified
by observing a linear dependence of kobsd on substrate
concentration in selected cases (1c with compound I
and II at pH 8.0), which obeyed the simple equation
3
+), 23.7 (+), 65.8 (+), 65.9 (+), 121.4 (ꢁ), 121.6 (ꢁ),
22.4 (ꢁ), 122.9 (ꢁ), 126.1 (ꢁ), 126.4 (ꢁ), 128.4 (ꢁ),
28.7 (ꢁ), 138.6 (+), 138.8 (+), 155.7 (+), 156.2 (+),
75.1 (ꢁ), 175.5 (ꢁ); HRMS (FAB) calcd for
C H N (M+1) 289.1705, found 289.1707.
2
0
21
2
4
.7. Reduction of 5b by lithium aluminum hydride
kobsd = kapp[S]. Each experiment was repeated at least
two times, and the average values were reported.
To a suspension of LiAlH (4 mg, 0.1 mmol) in 0.5 mL
4
of absolute ether under argon was added 5b (3 mg,
0
4.9. HRP binding constants from difference spectrum
measurements
.01 mmol) with vigorous stirring. The mixture was stir-
red at room temperature until all 5b had disappeared as
monitored by TLC. Then the mixture was worked up as
above to afford 1b (2.5 mg, 0.017 mmol, 85%), whose
spectral data ( H and C NMR) were identical to those
of the authentic sample.
Difference optical spectra (enzyme–substrate complex vs
enzyme) were recorded. Initially, both the sample and
reference cuvettes were filled with 3 mL of the HRP
solution (10 lM in 0.1 M pH 7.0 sodium phosphate buf-
fer) and an initial baseline was recorded to compensate
for slight differences between the two cuvettes. Then
small volumes of the substrate solution were successively
added to the sample cuvette, with concomitant addition
of the same volume of buffer to the reference cuvette.
After each addition, both cuvettes were well mixed,
and the difference spectra were recorded. Absorption
spectra of the substrates at all concentrations achieved
1
13
4.8. Kinetics of substrate oxidations by HRP compounds I
and II
HRP compound I was freshly prepared by mixing 3 mL
of the HRP stock solution (in buffer) with a 10-fold ex-
cess of hydrogen peroxide (2.1 mM) in a 3 mL quartz
cuvette, to which was added an excess of substrate,

K.-Q. Ling, L. M. Sayre / Bioorg. Med. Chem. 13 (2005) 3543–3551
3551
in the successive additions were also recorded using the
same procedure but in the absence of HRP. All the dif-
ferent spectra were then corrected by deduction of both
the initial baseline and the corresponding substrate
absorption spectra using ORIGIN 6.0 software. The
apparent dissociation constants Kd and the molar
absorptivity differences De were obtained from equation
Biophys. Res. Commun. 2001, 287, 130–134; (c) Ximenes,
V. F.; Campa, A.; Catalani, L. H. Arch. Biochem. Biophys.
2001, 387, 173–179.
. Sato, K.; Hasumi, H.; Tsukidate, A.; Sakurada, J.;
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1
253, 94–102.
. (a) Adediran, S. A.; Lambeir, A. M. Eur. J. Biochem.
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1
2
0b
1
/[S] = [E]De/(K DA)ꢁ1/K .
d
d
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A.; Nazari, K.; Ghadermarzi, M. Ital. J. Biochem. 1999,
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Acknowledgements
1
9
. Ling, K. Q.; Ren, T.; Protasiewicz, J. D.; Sayre, L. M.
Tetrahedron Lett. 2002, 43, 6903–6905.
This work was supported by National Institutes of
Health grant GM 48812.
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Supplementary data
7
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0a. Supplementary data associated with this article can
1
1
be found, in the online version, at doi:10.1016/
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