A model compound of the novel organic cofactor CTQ (cysteine tryptophylquinone) of quinohemoprotein amine dehydrogenase

Article abstract of DOI:10.1002/ejoc.200400191

A model compound of the novel organic cofactor CTQ (cysteine tryptophylquinone) of quinohemoprotein amine dehydrogenase (QH-AmDH) has been synthesized for the first time by the Michael addition of phenylmethanethiol to the 3-methyl-6,7-indolequinone derivative as a key step. The model compound replicates the characteristic absorption spectrum of native CTQ as well as its redox potential in the subunit of QH-AmDH. Detailed structural and spectroscopic characterizations of the model compound have provided important insights into the substituent effects of the thioether group of CTQ. Wiley-VCH Verlag GmbH & Co, KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400191

FULL PAPER
A Model Compound of the Novel Organic Cofactor CTQ (Cysteine
Tryptophylquinone) of Quinohemoprotein Amine Dehydrogenase
Yoko Murakami,[a] Yoshimitsu Tachi,[a] and Shinobu Itoh*[a]
Keywords: Amine dehydrogenases / Cofactors / Post-translational modification / Quinones / Quinoprotein
A model compound of the novel organic cofactor CTQ (cys-
subunit of QH-AmDH. Detailed structural and spectroscopic
teine tryptophylquinone) of quinohemoprotein amine dehy- characterizations of the model compound have provided im-
drogenase (QH-AmDH) has been synthesized for the first
time by the Michael addition of phenylmethanethiol to the
3-methyl-6,7-indolequinone derivative as a key step. The
model compound replicates the characteristic absorption
portant insights into the substituent effects of the thioether
group of CTQ.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
spectrum of native CTQ as well as its redox potential in the γ Germany, 2004)
Introduction
of lysyl oxidase,[6] Tyr-Cys (tyrosine covalently attached to
the thiol group of cysteine) of galactose oxidase,[7] and TTQ
(tryptophan tryptophylquinone) of methylamine and aro-
matic amine dehydrogenases.[8] Among such post-trans-
lationally derived organic cofactors, CTQ is closely related
to TTQ, since both cofactors possess the same indole-6,7-
quinone structure. Therefore, the oxidative deamination re-
action of QH-AmDH is considered to occur at the quinone
moiety of CTQ, as is the case for the TTQ-dependent amine
dehydrogenases.[9] However, detailed spectroscopic charac-
terization of CTQ as well as kinetic studies of the enzymatic
reaction have been hampered by the strong absorption
bands arising from the heme prosthetic groups in the α su-
bunit. Recently, Kano and co-workers succeeded in isolat-
ing the CTQ-containing γ subunit and characterized the
native CTQ cofactor by spectroscopic and electrochemical
techniques.[10] Nevertheless, a limited quantity of the
sample as well as the complexity of its structure due to the
peptide backbone have prevented a more detailed exami-
nation of the chemistry of the CTQ cofactor.
Quinohemoprotein amine dehydrogenase (QH-AmDH)
is a bacterial enzyme, isolated from Pseudomonas putida
and Paracoccus denitrificants, that catalyses the oxidative
deamination of primary amines to the corresponding alde-
hydes and transfers the substrate-derived electrons to
physiological electron acceptors such as cytochrome c-550
and azurin.[1,2] The enzyme is an αβγ heterotrimer, in which
there are two heme c prosthetic groups in the largest α su-
bunit and a carbonyl cofactor in the smallest γ subunit.[1,2]
Two independent research groups have recently analysed
the enzyme by X-ray crystallography and identified the or-
ganic cofactor as a tryptophan-6,7-quinone (Trq 43) cova-
lently attached to the thiol group of cysteine (Cys 37) at its
C-4 position, and is thus called CTQ (cysteine trypto-
phylquinone).[3,4]
In this paper, we report the synthesis and characteriza-
tion of the first model compound of CTQ having exactly
the same indole-6,7-quinone skeleton with a thioether
group at the C-4 position. Comparison of the spectroscopic
and redox properties of the CTQ model compound 1 and
the TTQ model compound 2[11,12] has provided important
insights into the electronic effects of C-4 substituents of in-
dole-6,7-quinone cofactors.
CTQ is a new member of the redox-active amino acid
cofactors that include TPQ (topa quinone) of the copper-
containing amine oxidases,[5] LTQ (lysine tyrosylquinone)
[a]
Department of Chemistry, Graduate School of Science, Osaka
City University,
3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
E-mail: shinobu@sci.osaka-cu.ac.jp
3074
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200400191
Eur. J. Org. Chem. 2004, 3074Ϫ3079

Model Compound of the Novel Organic Cofactor Cysteine Tryptophylquinone
FULL PAPER
oxy groups of 5 by trimethylsilyl iodide gave quinol 6,[15]
which was converted into indole-6,7-quinone derivative 7
by oxidation with ceric ammonium nitrate (CAN). Treat-
ment of 7 with 1 equiv. of phenylmethanethiol in DMF pro-
duced the Michael adduct of the mercaptan (8H2: the qui-
nol derivative of 8) together with quinol 6.[16] The quinol
mixture was subjected to column chromatography on SiO2
which induced air oxidation of the quinols to provide quin-
Results and Discussion
The CTQ model compound 1 has been synthesized as
outlined in Scheme 1. Reduction of the formyl group of
known compound 3[13,14] by LiAlH4 gave compound 4. The
indole nitrogen atom (N-1) of 4 was then protected by a p-
tosyl group to give compound 5. Deprotection of the meth-
Scheme 1
Figure 1. ORTEP drawings of compound 7 (A) and compound 8 (B) showing 50% probability thermal ellipsoids
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Eur. J. Org. Chem. 2004, 3074Ϫ3079
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3075

Y. Murakami, Y. Tachi, S. Itoh
FULL PAPER
˚
one 8 together with the recovery of some 7. Deprotection
of the tosyl group had to be carried out on quinol 8H2,
since quinone 8 was not stable enough under the basic con-
ditions (NaOH in ethanol) of the deprotection reaction.
Thus, compound 8 was first converted into 8H2 by re-
duction with NH2NH2, and then the resulting quinol 8H2
was treated with NaOH in ethanol to give the deprotected
product. Aerobic work up of the deprotected product gave
the CTQ model compound 1 as a dark red solid.[17]
Table 1. Selected bond lengths [A] and angles [°] of compounds 7
and 8
Compound 7
C(1)ϪC(2)
C(2)ϪC(9)
C(3)ϪC(8)
C(5)ϪC(6)
C(7)ϪC(8)
O(1)ϪC(6)
N(1)ϪC(1)
C(1)ϪC(2)ϪC(3)
1.373(4)
1.504(4)
1.391(3)
1.468(4)
1.440(4)
1.215(3)
1.379(3)
C(2)ϪC(3)
C(3)ϪC(4)
C(4)ϪC(5)
C(6)ϪC(7)
S(1)ϪN(1)
O(2)ϪC(7)
N(1)ϪC(8)
C(1)ϪC(2)ϪC(9)
C(2)ϪC(3)ϪC(4)
C(4)ϪC(3)ϪC(8)
C(4)ϪC(5)ϪC(6)
O(1)ϪC(6)ϪC(7)
O(2)ϪC(7)ϪC(6)
C(6)ϪC(7)ϪC(8)
N(1)ϪC(8)ϪC(7)
S(1)ϪN(1)ϪC(1)
C(1)ϪN(1)ϪC(8)
1.416(4)
1.457(4)
1.337(4)
1.560(4)
1.709(2)
1.219(3)
1.398(3)
The crystal structures of the o-quinone derivatives 7 and
8 (synthetic intermediates) have been determined by X-ray
crystallographic analysis, as shown in Figure 1. Selected C(3)ϪC(2)ϪC(9)
106.2(2)
126.1(3)
127.7(3)
108.7(2)
121.2(2)
122.9(3)
119.6(3)
127.4(3)
107.1(2)
122.7(3)
129.4(2)
110.1(3)
129.9(2)
121.4(3)
120.7(3)
117.5(3)
118.5(3)
114.0(2)
130.2(2)
122.3(2)
107.9(2)
C(2)ϪC(3)ϪC(8)
bond lengths and angles are summarized in Table 1. Al-
C(3)ϪC(4)ϪC(5)
though the indole nitrogen atoms (N-1) of these com-
O(1)ϪC(6)ϪC(5)
pounds are protected by the tosyl group, comparison of the
C(5)ϪC(6)ϪC(7)
detailed structures of 7 and 8 has provided an insight into
the substituent effects of the thioether group. Namely,
the deviation from planarity of the quinone moiety
O(1)ϪC(6)ϪC(7)ϪO(2) of 8 (dihedral angle ϭ 14.4°)
O(2)ϪC(7)ϪC(8)
N(1)ϪC(8)ϪC(3)
C(3)ϪC(8)ϪC(7)
S(1)ϪN(1)ϪC(8)
N(1)ϪC(1)ϪC(2)
is larger than that of
7
(9.6°), while the
C(10)ϪS(2)ϪC(4)ϪC(5)ϪC(6)ϪO(1) framework of 8 is
nearly flat [the dihedral angles of C(10)ϪS(2)ϪC(4)ϪC(5),
S(2)ϪC(4)ϪC(5)ϪC(6), and C(4)ϪC(5)ϪC(6)ϪO(1) are
2.5°, 3.6°, and 2.1°, respectively]. The C(5)ϪC(6) bond of 8
Compound 8
C(1)ϪC(2)
C(2)ϪC(9)
C(3)ϪC(8)
C(5)ϪC(6)
C(7)ϪC(8)
O(2)ϪC(7)
N(1)ϪC(1)
S(2)ϪC(4)
C(1)ϪC(2)ϪC(3)
C(3)ϪC(2)ϪC(9)
1.351(7)
C(2)ϪC(3)
C(3)ϪC(4)
C(4)ϪC(5)
C(6)ϪC(7)
O(1)ϪC(6)
S(1)ϪN(1)
N(1)ϪC(8)
S(2)ϪC(10)
C(1)ϪC(2)ϪC(9)
C(2)ϪC(3)ϪC(4)
C(4)ϪC(3)ϪC(8)
S(2)ϪC(4)ϪC(3)
C(4)ϪC(5)ϪC(6)
O(1)ϪC(6)ϪC(7)
O(2)ϪC(7)ϪC(6)
C(6)ϪC(7)ϪC(8)
N(1)ϪC(8)ϪC(7)
S(1)ϪN(1)ϪC(1)
C(1)ϪN(1)ϪC(8)
C(4)ϪS(2)ϪC(10)
1.416(7)
1.497(7)
1.387(7)
1.437(7)
1.438(7)
1.207(6)
1.380(7)
1.720(5)
1.476(7)
1.357(7)
1.550(7)
1.221(6)
1.707(4)
1.403(6)
1.815(5)
˚
˚
(1.437 A) is shorter than that of 7 (1.468 A), while the
˚
˚
˚
C(4)ϪC(5) and C(6)ϪO(1) bonds of 8 (1.357 and 1.221 A,
respectively) are longer than those of 7 (1.337 and 1.215 A,
respectively). Moreover, the S(2)ϪC(4) bond of 8 (1.720 A)
is slightly shorter than the typical SϪC bond in simple
106.7(5)
123.8(5)
[18,19]
˚
thioanisole derivatives (1.77 A).
These structural fea-
129.5(5)
108.0(4)
119.7(4)
124.1(4)
123.5(5)
119.0(4)
127.0(5)
107.5(4)
123.9(4)
128.3(3)
110.8(4)
132.2(5)
119.8(4)
116.2(4)
122.5(5)
117.4(5)
119.1(4)
113.8(4)
128.2(4)
121.8(3)
106.8(4)
103.2(2)
tures clearly suggest that there is a conjugative interaction C(2)ϪC(3)ϪC(8)
C(3)ϪC(4)ϪC(5)
between S(2) and the α,β-unsaturated carbonyl moiety
C(5)ϪC(4)ϪS(2)
[C(4)ϭC(5)ϪC(6)ϭO(1)] of compound 8. This conclusion
O(1)ϪC(6)ϪC(5)
is supported by the IR data; the CϭO stretching vibration
C(5)ϪC(6)ϪC(7)
of quinone 8 appears at 1636 cmϪ1, which is lower than
O(2)ϪC(7)ϪC(8)
that of compound 7 by about 30 cmϪ1 (see Exp. Sect.).
N(1)ϪC(8)ϪC(3)
C(3)ϪC(8)ϪC(7)
Figure 2 shows the UV/Vis spectrum of compound 1
S(1)ϪN(1)ϪC(8)
(solid line) which exhibits two distinct πϪπ* transition
N(1)ϪC(1)ϪC(2)
bands at 343 and 381 nm (ε ϭ 14800 and 12000 Ϫ1·cmϪ1
,
respectively) together with a weak nϪπ* transition band at
450Ϫ600 nm. These absorption bands are very similar to
those of native CTQ [350Ϫ380 nm (πϪπ*) and
450Ϫ600 nm (nϪπ*)], although the absorption band at ca.
350 nm of native CTQ is not well separated from the band
at 380 nm.[10]
The absorption bands in the visible region (λ Ͼ 350 nm)
of 1 are blue-shifted relative to those of the TTQ model
compound 2 (πϪπ*: 407 nm, ε ϭ 10700 Ϫ1·cmϪ1; nϪπ*:
500Ϫ650 nm), and the CϭO stretching vibration in the IR
spectrum of quinone 1 (1630 cmϪ1) is slightly higher than
that of quinone 2 (1628 cmϪ1).[11,12] These differences in
λmax. and νCϭO may reflect the different levels of conjug-
ative interaction between the quinone ring and the C-4 sub-
stituent (thioether group in 1 vs. indole ring in 2) of 1 and
2. In addition, the lower value of νCϭO (1630 cmϪ1) of 1
compared with that of 8 (1636 cmϪ1) suggests that there
is also a conjugative interaction between N-1 and the C-7
carbonyl group of compound 1.
Figure 2. UV/Vis spectra of 1 (5.0 ϫ 10Ϫ5 ) and its quinol deriva-
tive 1H2, generated by the reduction of 1 with MeNHNH2 (10
equiv.) in CH3CN
3076
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Eur. J. Org. Chem. 2004, 3074Ϫ3079

Model Compound of the Novel Organic Cofactor Cysteine Tryptophylquinone
FULL PAPER
software package.[22] The crystal structures were solved by direct
methods and refined by the full-matrix least-squares method using
SIR-92. All non-hydrogen atoms and hydrogen atoms were refined
anisotropically and isotropically, respectively. X-ray crystallo-
graphic data are presented in Table 2. Crystallographic data for the
structures reported in this paper have been deposited with the Cam-
bridge Crystallographic Data Centre as supplementary publication
no. CCDC-232604 (7) and -232605 (8). Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK [Fax: (internat.) ϩ 44-1223/336-033; E-
Compound 1 exhibits a quasi-reversible redox couple at
E°1/2 ϭ Ϫ0.187 V vs. Ag/AgCl in an aqueous solution con-
taining 30% CH3CN (pH ϭ 7.0), which is similar to that of
native CTQ (Ϫ0.132 V vs. Ag/AgCl at pH ϭ 7.0)[10] but
somewhat negative compared with that of the TTQ model
compound 2 (Ϫ0.090 V vs. Ag/AgCl at pH ϭ 7.0).[12] The
negative shift of E°1/2 reflects the electron-donating nature
of the thioether group of compound 1.
Reduction of 1 to its quinol form 1H2 with methylhydra-
zine caused a decrease in the visible absorption bands to- mail: deposit@ccdc.cam.ac.uk].
gether with an increase of the absorption bands at around
300 nm [λmax. ϭ 291 nm (ε ϭ 11600 Ϫ1·cmϪ1) and 312
(11900)] (dotted line in Figure 2). The spectral change due
Table 2. Summary of the X-ray crystallographic data for com-
pounds 7 and 8
to the two-electron reduction of 1 to 1H2 is also very similar
to that observed for native CTQ.[10]
7
8
Conclusion
Empirical formula
Formula mass
Crystal system
Space group
C16H13NO4S
315.34
C23H19O4NS2
437.53
We have succeeded in synthesizing the first model com-
pound of the novel organic cofactor CTQ of QH-AmDH,
which has exactly the same indole-6,7-quinone skeleton
with a thioether group at the C-4 position. The compound
replicates the characteristic absorption spectrum of CTQ
as well as its redox potential of the γ subunit. The model
compound has been synthesized by the nucleophilic attack
(Michael addition) of a thiol at the C-4 position of indole-
6,7-quinone 7. This kind of reaction may be involved in the
biogenetic pathway of CTQ in the QH-AmDH enzymatic
system.[20] Amine oxidation by the model compound is cur-
rently being investigated in order to shed light on the cata-
lytic mechanism of QH-AmDH.
monoclinic
P21/c (No. 14)
6.970(1)
8.451(1)
24.229(3)
95.464(5)
1420.6(4)
4
orthorhombic
Pbca (No. 61)
7.356(2)
17.053(6)
31.49(1)
90
3950(2)
8
1824.00
1.471
˚
a [A]
˚
b [A]
˚
c [A]
β [°]
3
˚
V [A ]
Z
F(000)
656.00
1.474
159
Dcalcd. [g·cmϪ3
T [K]
]
164
Crystal size [mm]
0.12 ϫ 0.13 ϫ 0.10 0.10 ϫ 0.17 ϫ 0.08
2.46
55.0
µ (Mo-Kα) [cmϪ1
2θmax. [°]
]
3.02
55.0
No. of reflns. measd. 13275
50266
3124 [I Ͼ 0.50σ(I)]
No. of reflns. obsd.
2133 [I Ͼ 1.00σ(I)]
No. of variables
213
291
R[a]
0.035
0.050
1.00
0.050
0.088
1.00
Rw[b]
GOF
Experimental Section
General: The reagents and solvents used in this study were commer-
cial products of the highest available purity and were further puri-
fied by standard methods, if necessary.[21] Melting points were de-
termined with a Yanaco Micro Melting Point Apparatus. FT-IR
spectra were recorded with a Shimadzu FTIR-8200PC and UV/Vis
spectra were taken with a HewlettϪPackard 8453 photodiode array
[a]
[b]
2 1/2
R ϭ Σ||Fo| Ϫ |Fc||/Σ|Fo|. Rw ϭ [Σw(|Fo| Ϫ |Fc|)2/ΣwFo ]
.
6,7-Dimethoxy-3-methylindole (4): 6,7-Dimethoxyindole-3-carbal-
dehyde (3) was prepared from vanillin in 7 steps according to re-
ported procedures.[13,14] Compound 3 (465 mg, 2.3 mmol) in dry
THF (23 mL) was added dropwise to LiAlH4 (175 mg, 4.6 mmol),
suspended in dry THF (12 mL) at 0 °C under N2, and the mixture
was stirred at reflux temperature for 6 h. The reaction was
quenched by adding saturated aq. Na2SO4, and insoluble materials
were removed by filtration. The filtrate was then extracted with
diethyl ether (30 mL ϫ 4), and the combined organic layers were
dried with MgSO4. After removal of MgSO4 by filtration, evapora-
tion of the solvent gave a crude product, from which 4 was isolated
as a yellow-brown oily material in 99% yield by column chromatog-
raphy (SiO2; CHCl3). IR (neat): ν˜ ϭ 3415 (NH), 1301 and 1207
1
spectrophotometer. H NMR spectra were recorded with a JEOL
FT NMR Lambda 300WB or a JEOL FT NMR GX-400 spec-
trometer. Mass spectra were recorded with a JEOL JMS-700T Tan-
dem MS-station mass spectrometer. Elemental analyses were per-
formed with a Fisons Instrument EA1108 Elemental Analyzer. The
cyclic voltammetry measurement was performed with an ALS 630
electrochemical analyzer in a deaerated 0.10  phosphate buffer
solution containing 30% CH3CN. The working electrode was a
glassy carbon electrode (BAS) which was polished with BAS pol-
ishing alumina suspension and rinsed with H2O and dried before
use. The counter electrode was a platinum wire and the reference
electrode was Ag/AgCl.
1
X-ray Structure Determination: The single crystals of compounds 7
and 8 were mounted on a glass fiber. X-ray diffraction data were
collected with a Rigaku RAXIS-RAPID imaging plate two-dimen-
(CϪO) cmϪ1. H NMR (CDCl3): δ ϭ 2.29 (s, 3 H, CH3), 3.92 (s,
3 H, OCH3), 3.98 (s, 3 H, OCH3), 6.85 (d, J ϭ 8.6 Hz, 1 H, 5-H),
6.88 (br. s, 1 H, 2-H), 7.21 (d, J ϭ 8.6 Hz, 1 H, 4-H), 7.97 (br. s, 1
H, NH) ppm. 13C NMR (CDCl3): δ ϭ 9.7 (CH3), 57.5, 60.8
sional area detector using graphite-monochromated Mo-Kα radi-
˚
ation (λ ϭ 0.71069 A) and 2θmax. ϭ 55.0°. All the crystallographic (OCH3), 107.9, 111.9, 113.7, 121.2, 125.2, 130.7, 134.4, 147.0 (8
calculations were performed by using the Crystal Structure
aromatic C) ppm. HRMS (FAB): m/z ϭ 191.0948 [Mϩ]; calcd. for
3077
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Y. Murakami, Y. Tachi, S. Itoh
C11H13NO2 191.0947. C11H13NO2 (191.2): calcd. C 69.09, H 6.85, 4-(Benzylsulfanyl)-3-methyl-1-(p-tosyl)indole-6,7-dione (8): Phenyl-
FULL PAPER
N 7.32; found C 69.07, H 6.96, N 7.17.
methanethiol (7.44 µL, 0.063 mmol) was added to a dry DMF solu-
tion (4.0 mL) of 7 (20.0 mg, 0.063 mmol) at room temperature un-
der N2, and the resulting mixture was stirred for 2.5 h. The reaction
mixture was then diluted with water (50 mL), extracted with EtOAc
(20 mL ϫ 3) and dried with MgSO4. After removal of the MgSO4
by filtration, evaporation of the solvent gave a reddish brown solid,
from which compound 8 was isolated as a red solid in 45% yield
by column chromatography (SiO2; CHCl3) under air. M.p.
242Ϫ244 °C (dec.). IR (KBr): ν˜ ϭ 1636 (CϭO), 1385 and 1180
6,7-Dimethoxy-3-methyl-1-(p-tosyl)indole (5): Compound 4 (1.00 g,
5.2 mmol) in dry DMF (4.0 mL) was added dropwise to NaH
(318 mg, 11 mmol, 65% content in a mineral oil suspension that
was washed with dry n-hexane three times before use) suspended
in dry DMF (10 mL) at room temperature under N2, and the mix-
ture was stirred for 10 min. A dry DMF (3.0 mL) solution of p-
toluenesulfonyl chloride (1.95 g, 11 mmol) was then added to the
mixture, and the resulting solution was stirred at 0 °C for 2 h. The
reaction was quenched by adding 20% aq. Na2CO3 (100 mL), and
the mixture was extracted with EtOAc (30 mL ϫ 5). After drying
with MgSO4, removal of the solvent gave a crude product, from
which compound 5 was isolated as a pale green solid in 68% yield
by column chromatography (SiO2; n-hexane/EtOAc, 9:1). M.p.
77Ϫ79 °C. IR (neat): ν˜ ϭ 1354 and 1173 (SO2), 1258 and 1038
(CϪO) cmϪ1. 1H NMR (CDCl3): δ ϭ 2.23 (d, J ϭ 1.3 Hz, 3 H, 3-
CH3), 2.35 (s, 3 H, C6H4CH3), 3.83 (s, 3 H, OCH3), 3.86 (s, 3 H,
OCH3), 6.89 (d, J ϭ 8.5 Hz, 1 H, 5-H), 7.11 (d, J ϭ 8.5 Hz, 2 H,
4-H), 7.22 (d, J ϭ 8.3 Hz, 2 H, C6H4CH3), 7.48 (d, J ϭ 1.3 Hz, 1
H, 2-H), 7.76 (d, J ϭ 8.3 Hz, 2 H, C6H4CH3) ppm. 13C NMR
(CDCl3): δ ϭ 9.5 (CH3), 21.5 (C6H4CH3), 56.7 (OCH3), 60.6
(OCH3), 109.6, 113.9, 116.0, 121.1, 125.0, 127.3, 128.7, 129.5,
136.7, 137.0, 143.9, 150.6 (12 aromatic C) ppm. HRMS (FAB):
1
(SO2) cmϪ1. H NMR (CDCl3): δ ϭ 2.34 (s, 3 H, 3-CH3), 2.42 (s,
3 H, C6H4CH3), 4.17 (s, 2 H, SCH2C6H5), 6.05 (s, 1 H, 5-H),
7.33Ϫ7.39 (m, 7 H, C6H4CH3, SCH2C6H5), 7.63 (s, 1 H, 2-H), 8.05
(d, J ϭ 8.6 Hz, 2 H, C6H4CH3) ppm. HRMS (FAB): m/z ϭ
439.0917 [M
ϩ 2
H]ϩ; calcd. for C23H21NO4S2 439.0913.
C23H19NO4S2 ϩ 1/6H2O: calcd. C 62.71, H 4.42, N 3.18; found C
62.63, H 4.37, N 3.08.
4-(Benzylsulfanyl)-3-methylindole-6,7-dione (1): Hydrazine mono-
hydrate (5.5 µL, 0.11 mmol, 5 equiv.) was added to a suspension of
8 (10.0 mg, 0.023 mmol) in dry CH3CN (3 mL, deoxygenated by
bubbling N2 for 10 min), and the reaction mixture was stirred under
anaerobic conditions (N2) for 20 min. Removal of the solvent under
reduced pressure gave a yellow solid material (8H2), to which 3 
NaOH in H2O/EtOH (9:5, v/v) (278 µL) was added. The mixture
was then stirred at room temperature for 30 min, and was extracted
with EtOAc (15 mL ϫ 6). After the extract was dried with MgSO4,
evaporation of the solvent gave a dark red residue, from which com-
pound 1 was isolated in 82% yield by column chromatography
(SiO2; CHCl3/EtOAc, 3:2). M.p. 239Ϫ240 °C. IR (KBr): ν˜ ϭ ca.
m/z
ϭ
345.1049 [Mϩ]; calcd. for C18H19NO4S 345.1036.
C18H19NO4S (345.4): calcd. C 62.59, H 5.54, N 4.06; found C
62.35, H 5.60, N 4.26.
3-Methyl-1-(p-tosyl)indole-6,7-diol (6): Compound
5 (253 mg,
0.73 mmol) was treated with trimethylsilyl iodide (2.08 mL,
15 mmol) in dry CH3CN (10 mL) at reflux temperature under N2
for 30 h. The reaction was quenched by adding water (30 mL) and
the mixture was extracted with EtOAc (30 mL ϫ 5). The combined
organic layers were washed with aqueous Na2S2O3 and dried with
MgSO4. After removal of the MgSO4 by filtration, evaporation of
the solvent gave a crude material, from which compound 6 was
isolated in 55% yield by column chromatography (SiO2; n-hexane/
EtOAc, 7:1). M.p. 162Ϫ163 °C. IR (KBr): ν˜ ϭ 3449 (OH), 1261
and 1088 (SO2) cmϪ1. 1H NMR (CDCl3): δ ϭ 2.13 (d, J ϭ 1.2 Hz,
3 H, 3-CH3), 2.35 (s, 3 H, C6H5CH3), 5.71 (s, 1 H, OH), 6.83 (d,
J ϭ 8.3 Hz, 1 H, 5-H), 6.94 (d, J ϭ 8.3 Hz, 1 H, 4-H), 7.03 (d, J ϭ
1.2 Hz, 1 H, 2-H), 7.22 (d, J ϭ 8.1 Hz, 2 H, C6H4CH3), 7.63 (d,
J ϭ 8.1 Hz, 2 H, C6H4CH3), 8.96 (s, 1 H, OH) ppm. HRMS (FAB):
m/z ϭ 317.0723 [Mϩ]; calcd. for C16H15NO4S 317.0723.
3500 (br. NϪH), 1630 (CϭO), 700 (SϪC) cmϪ1 1H NMR
.
([D6]DMSO): δ ϭ 2.19 (s, 3 H, 3-CH3), 4.39 (s, 2 H, SCH2C6H5),
5.94 (s, 1 H, 5-H), 7.13 (s, 1 H, 2-H), 7.29Ϫ7.48 (m, 5 H, C6H5),
12.7 (br. s, 1 H, NH) ppm. 13C NMR ([D6]DMSO): δ ϭ 13.0
(CH3), 35.4 (SCH2Ph), 114.1, 119.7, 125.0, 127.7, 127.9, 128.7,
129.2, 129.7, 134.8, 156.8 (10 aromatic C), 167.9, 178.7 (CϭO)
ppm. HRMS (FAB): m/z ϭ 284.0743 [M ϩ H]ϩ; calcd. for
C16H14NO2S 284.0746. C16H13NO2S ϩ 1/4CHCl3: calcd. C 62.32,
H 4.26, N 4.47; found C 62.54, H 4.46, N 4.33.
4-(Benzylsulfanyl)-3-methylindole-6,7-diol (1H2): Methylhydrazine
(3.4 µL, 6.4 ϫ 10Ϫ2 mmol), was added to a deaerated CH3CN
solution (2.0 mL) of 1 (1.81 mg, 6.39 ϫ 10Ϫ3 mmol) and the reac-
tion mixture was stirred under anaerobic conditions (N2) for
30 min. Removal of the solvent under reduced pressure gave a dark
yellow material of 1H2 (1.5 mg, 83% yield). 1H NMR ([D6]DMSO):
δ ϭ 2.20 (s, 3 H, 3-CH3), 3.97 (s, 2 H, SCH2Ph), 6.59 (s, 1 H, 5-
H), 6.82 (s, 1 H, 2-H), 7.18Ϫ7.28 (m, 5 H, SCH2C6H5), 10.3 (s, 1
H, NH) ppm. MS (EI): m/z ϭ 285 [Mϩ].
3-Methyl-1-(p-tosyl)indole-6,7-dione (7): CAN (86.3 mg, 0.16
mmol) was added to an acetonitrile solution (4.0 mL) of 6
(50.1 mg, 0.16 mmol), containing 1.0 mL of water at 0 °C. The mix-
ture was stirred at 0 °C for 10 min. The reaction was then quenched
by adding aqueous K2CO3, and the mixture was extracted with
CH2Cl2 (20 mL ϫ 4) and dried with MgSO4. After removal of the
MgSO4 by filtration, evaporation of the solvent gave a red solid,
from which compound 7 was isolated in 89% yield by column chro-
matography (SiO2; CHCl3). M.p. 197Ϫ198 °C. IR (KBr): ν˜ ϭ 1663
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, and Cul-
ture of Japan (13480189 and 15350105). The authors also acknowl-
edge Professor Kenji Kano of Kyoto University and Professor Ken
Hirotsu of Osaka City University for their helpful discussions.
1
(CϭO), 1377 and 1092 (SO2) cmϪ1. H NMR (CDCl3): δ ϭ 2.16
(s, 3 H, 3-CH3), 2.42 (s, 3 H, C6H5CH3), 6.11 (d, J ϭ 10.0 Hz, 1
H, 5-H), 7.22 (d, J ϭ 10.0 Hz, 1 H, 4-H), 7.33 (d, J ϭ 8.6 Hz, 2
H, C6H4CH3), 7.60 (s, 1 H, 2-H), 8.08 (d, J ϭ 8.6 Hz, 2 H,
C6H4CH3) ppm. 13C NMR (CDCl3): δ ϭ 8.67 (CH3), 21.14
(C6H4CH3), 120.6, 125.9, 126.2, 128.4, 129.9, 130.9, 133.4, 134.9,
135.4, 146.1 (10 aromatic C), 165.3, 181.0 (CϭO) ppm. HRMS
(FAB): m/z ϭ 316.0649 [M ϩ H]ϩ; calcd. for C16H14NO4S
316.0644. C16H13NO4S (315.3): calcd. C 60.94, H 4.16, N 4.44;
found C 61.15, H 3.96, N 4.49.
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Received March 23, 2004
Eur. J. Org. Chem. 2004, 3074Ϫ3079
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3079