A convenient preparation of bis(4-methoxyphenyl)methanethiol and its application in the synthesis of biotin thioacid

Article abstract of DOI:10.1002/jccs.201300664

We have established a facile protocol for the preparation of bis(4-methoxyphenyl)methanethiol (4). Thiol 4 was used to prepare biotin thioester 7 which was later subjected to acidolysis to afford biotin thioacid (1b) in high yield. Compound 1b represents

Full text of DOI:10.1002/jccs.201300664

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Note
A Convenient Preparation of Bis(4-methoxyphenyl)methanethiol and Its Application in
the Synthesis of Biotin Thioacid
Tzyy-Chao Chou, Yu-Ling Hsu and Lee-Chiang Lo*
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
(Received: Dec. 23, 2013; Accepted: Feb. 15, 2014; Published Online: Mar. 11, 2014; DOI: 10.1002/jccs.201300664)
We have established a facile protocol for the preparation of bis(4-methoxyphenyl)methanethiol (4). Thiol
4 was used to prepare biotin thioester 7 which was later subjected to acidolysis to afford biotin thioacid
(1b) in high yield. Compound 1b represents a new biotinylating reagent worth exploring and its spectral
data was reported for the first time.
Keywords: Thiol; Thioester; Thioacid; Biotin; Bis(4-methoxyphenyl)methyl.
INTRODUCTION
Biotin (1a), a coenzyme with extraordinarily high af-
pound 1b. We have also performed a detailed spectral char-
acterization of the compound.
finity for avidin/streptavidin, has been extensively utilized
as a tag in biochemical research.1 In our efforts to develop
activity-based probes for various classes of enzymes, we
have also frequently utilized a biotin group to serve as the
key reporter group.2 The unique binding interaction of bio-
tin makes possible the detection as well as the enrichment
of the biotinylated biomolecules from a complicated sys-
tem.3 The biontinylation step in a probe construction was
mainly achieved by reacting an active ester of biotin, such
as biotin-OSu, with a free amino group. We envisioned that
increasing the repertoire of the biotinylating agents by ex-
ploiting a novel chemistry would offer great advantage and
flexibility in the synthetic planning of the probes. A prom-
ising strategy involves the thioacid chemistry which has re-
ceived much attention in recent years.4 Due to their soft and
powerful nucleophilic behavior, thioacids could selectively
react with a broad range of functional groups, such as
azides,5 sulfonylazides,6 and isonitriles.7 These character-
istic reaction features thus make biotin thioacid (1b) a po-
tentially useful and attractive biotinylating agent. This
viewpoint was also substantiated by a recent application of
compound 1b in “sulfo-click” ligation.8 Despite its inter-
esting application, compound 1b has never been purified
nor characterized previously. We herein describe a conve-
nient procedure for the preparation and isolation of com-
Amongst the available thioacid acquiring methods,9-13
the most commonly used approach in laboratory operation
utilizes the thioester precursors because it does not require
the use of toxic H2S reagent and offers the advantage that
these precursors could be easily obtained in a pure form in
large quantity. In this approach, thiols were first coupled to
the carboxylic acids and the resultant thioesters were sub-
jected to deprotection to release the corresponding thio-
acids. Although thiols such as HSFmoc (2)12 and HSTmob
(3)13 were commonly used in the preparation of thioesters,
these thiols were not readily accessible (Figure 2). In our
search for a convenient alternative, we found that 4,4¢-di-
methoxybenzhydryl group was previously reported as a
protecting group for the thiol functionality of cysteine
through a thioether linkage.14 In the present study, we fur-
ther explored this protecting group and established a facile
protocol for the preparation of bis(4-methoxyphenyl)meth-
anethiol (4), which will serve as a reliable thiol source for
thioester formation.
RESULTS AND DISCUSSION
As shown in Scheme 1, commercially available bis-
(4-methoxyphenyl)methanol (5) was easily converted to
thioacetate 6 by reacting it with thioacetic acid in the pres-
Fig. 1. The structures of biotin (1a) and biotin thioacid
(1b).
Fig. 2. The structures of thiols 2 (HSFmoc), 3
(HSTmob), and 4.
* Corresponding author. Tel: +886-2-3366-1669; E-mail: lclo@ntu.edu.tw
Supporting information for this article is available on the www under http://dx.doi.org/10.1002/jccs.201300664
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Note
Chou et al.
Scheme 2 Synthesis of biotin thioacid (1b)
Synthesis of bis(4-methoxyphenyl)meth-
Scheme 1
anethiol (4)
served, it was easily separated and removed by silica gel
column chromatography. Finally, the thioester 7 was sub-
jected to acidolysis under conditions that were previously
optimized (20-40% TFA) to release the biotin thioacid
(1b). The nonpolar byproduct 8 formed in the acidolysis
was conveniently removed by repeatedly washing the mix-
ture with ether. Compound 1b was thus obtained in high
yield (94%) without any hydrolyzed contaminant. More
importantly, compound 1b could be stored as a suspension
in n-hexane for a long period of time. We observed that the
purity of compound 1b remained greater than 95% even af-
ter 3 months of storage. This application demonstrates that
thiol 4 could be regarded as a useful synthetic equivalent of
ence of ZnI2 in CH2Cl2.15 Asimple aqueous workup and ex-
traction procedure gave compound 6 in high yield (93%).
This procedure could be readily applied to a multigram-
scale preparation of compound 6 without the need for te-
dious purification steps. Thioacetate 6 served two pur-
poses: firstly, it was used to generate thiol 4 by treatment
with K2CO3 in MeOH under argon atmosphere. Although it
was known that a free thiol group might undergo slow oxi-
dation to a disulfide or a sulfoxide under ambient condi-
tions,16 we found that the quality of thiol 4 could be main-
tained over a long period of time when it was frozen in
benzene solution.
Secondly, thioacetate 6 was used as a model to inves-
tigate the optimal conditions for thioacid formation under
acidolysis with TFA (Figure 3). We performed time course
experiments by incubating 6 (0.1 M) with varying concen-
trations of TFA (5%, 10%, 20%, 40%) and 8 equivalents of
Et3SiH in CDCl3. The progress of the acidolysis was moni-
tored by 1H-NMR spectroscopy and the extent of the reac-
tion was calculated from the integration of the signals in the
aromatic region of the residual 6 and the bis(4-methoxy-
phenyl)methane byproduct (8). The preliminary data re-
vealed that TFA concentrations higher than 20% were ef-
fective for the release of thioacid.
1
hydrosulfide. It is interesting to note that the H-NMR
spectrum of compound 1b resembles that of biotin, except
the a-methylene group of compound 1b displayed a dra-
matic downfield shift, from d 2.20 ppm to 2.65 ppm (Figure
4). A similar downfield shift phenomenon was also ob-
served for the thiocarboxylic group, from d 175.0 ppm to
193.7 ppm, in the 13C-NMR spectra.17 In addition, com-
pound 1b showed an absorption band at 2531 cm-1 in the IR
spectrum, which was assigned to the characteristic S-H
stretching.17,18
CONCLUSIONS
Thiol 4 was then coupled with biotin-OSu in DMF
under an atmosphere of Ar at room temperature overnight
to afford the biotin thioester 7 in 82% yield (Scheme 2). Al-
though formation of a trace amount of disulfide was ob-
In summary, we have established a facile protocol for
the preparation of bis(4-methoxyphenyl)methanethiol (4),
Fig. 4. 1H-NMR spectra of biotin (1a) and biotin thio-
acid (1b) in DMSO-d6. The asterisk denotes the
signals of the a-methylene group.
Fig. 3. Time-course acidolysis of thioacetate 6 with
TFA for thioacid formation.
708
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Synthesis and Characterization of Biotin Thioacid
which is a useful synthetic equivalent of hydrosulfide.
Thiol 4 was used to prepare biotin thioester 7 which was
later subjected to acidolysis to afford biotin thioacid (1b) in
high yield. Compound 1b represents a new biotinylating
reagent worth exploring. The results of its applications will
be described in due course.
135.8 (C), 128.7 (CH), 113.8 (CH), 55.2 (CH3), 46.6 (CH). IR
(neat): 3442, 2999, 2953, 2930, 2834, 2552 (SH), 2055, 1886,
1606, 1506, 1460, 1300, 1243, 1168, 1108, 1029, 810, 774, 626,
580 cm-1. ESI-MS m/z (%): 259 ([M-H]-, 100), 227 ([M-SH]-, 17);
HR-ESI-MS calcd for C15H15O2S (M-H)- 259.0793, found
259.0797.
Biotin Thioester (7): To a solution of compound 4 (258
mg, 0.99 mmol) and biotin-OSu (647 mg, 0.99 mmol) in DMF (10
mL) was added DIEA (246 mL, 1.49 mmol). The reaction mixture
was purged and filled with Ar, and was stirred for 18 h at room
temperature. The volatiles were removed in vacuo, and the resi-
due obtained was dissolved in EtOAc (100 mL). The organic solu-
tion was washed with 5% NaHCO3(aq) (30 mL, ´3), H2O (30 mL,
´3), and brine (30 mL, ´2). The organic phase was then dried over
anhydrous Na2SO4, filtered and concentrated. Purification by sil-
ica gel column chromatography (eluent: 90% CHCl3/MeOH) pro-
vided the desired product 7 as a pale yellow oil in 82% yield. Rf =
0.22 (CHCl3/MeOH = 9/1). 1H-NMR (400 MHz, CDCl3): d 7.21
(m, 4 H), 6.80 (m, 4 H), 5.83 (s, 1 H), 5.74 (s, 1 H, NH), 5.21 (s, 1
H, NH), 4.43 (m, 1 H), 4.20 (m, 1 H), 3.75 (s, 6 H), 3.06 (m, 1 H),
2.83 (dd, J = 12.8, 5.0 Hz, 1 H), 2.64 (d, J = 12.8 Hz, 1 H), 2.55 (t,
J = 7.9 Hz, 2 H), 1.75-1.53 (m, 4 H), 1.46-1.28 (m, 2 H); 13C-NMR
(100 MHz, CDCl3): d 197.8 (C), 158.6 (C), 133.3 (C), 133.3 (C),
129.3 (CH), 113.9 (CH), 61.9 (CH), 60.1 (CH), 55.2 (CH), 55.2
(CH), 50.5 (CH3), 43.3 (CH2), 40.5 (CH2), 29.7 (CH2), 28.1
(CH2), 25.3 (CH2). IR (neat): 3248, 2931, 1702, 1608, 1510,
1461, 1302, 1250, 1176, 1111, 817, 763, 587, 552 cm-1. ESI-MS
m/z (%): 509 ([M+Na]+, 100), 261 (8), 229 (17); HR-ESI-MS
calcd for C25H30NaN2O4S2 (M+Na)+ 509.1545, found 509.1545.
Biotin Thioacid (1b): To a solution of thioester 7 (100 mg,
0.20 mmole) in 2 mL of 40% TFA/CH2Cl2 was added Et3SiH (266
mL, 1.60 mmol). The reaction mixture was stirred for 1 h at room
temperature under an Ar atmosphere. The volatiles were then re-
moved in vacuo, and the residue was dissolved in a minimum
amount of CHCl3. The solution was diluted with ether until pre-
cipitation occurred. The ether layer was removed with a dropper
and the process was repeated for several times to afford the ana-
lytically pure 1b as a white solid in 94% yield. Rf = 0.36 (CHCl3/
MeOH = 9/1), mp: 170-172oC. 1H-NMR (400 MHz, DMSO-d6): d
6.42 (s, 1 H, NH), 6.35 (s, 1 H, NH), 4.30 (m, 1 H), 4.13 (m, 1 H),
3.10 (m, 1 H), 2.82 (dd, J = 12.6, 5.1 Hz, 1 H), 2.65 (t, J = 7.5 Hz, 2
H), 2.58 (d, J = 12.6 Hz, 1 H), 1.70-1.27 (m, 6 H); 13C-NMR (100
MHz, DMSO-d6): d 193.8 (C), 163.2 (C), 61.5 (CH), 59.7 (CH),
55.7 (CH), 42.4 (CH2), 40.2 (CH2), 28.4 (CH2), 28.1 (CH2), 25.4
(CH2). IR (KBr): 3250, 2916, 2531 (SH), 1703, 1671, 1476, 1397,
1166, 1082, 1022, 745, 663, 615 cm-1. ESI-MS m/z (%): 259
([M-H]-, 100); HR-ESI-MS calcd for C10H15N2O2S2 (M-H)-
EXPERIMENTAL
General Methods: All reagents and starting materials
were obtained from commercial suppliers and were used without
1
further purification. H and 13C NMR spectra were recorded at
400 MHz and 100 MHz, respectively. IR spectra were recorded at
room temperature. The reactions were monitored by analytical
TLC and spots were visualized under UV light and/or phospho-
molybdic acid/ethanol stain. Column chromatography was per-
formed with silica gel (230-400 mesh). Melting points are uncor-
rected.
S-4,4¢-Dimethoxybenzhydryl Thioacetate (6): To a solu-
tion of bis(4-methoxyphenyl)methanol (5, 3.00 g, 12.3 mmol)
and thioacetic acid (961 mL, 13.5 mmol) in 150 mL of anhydrous
CH2Cl2 was added ZnI2 (2.00 g, 6.14 mmol) and the reaction mix-
ture was refluxed for 18 h. Thereafter, the solution was allowed to
cool to room temperature and then evaporated to dryness. The res-
idue obtained was dissolved in EtOAc (200 mL) and the solution
was washed with 5% NaHCO3(aq) (30 mL ´3), H2O (30 mL ´3)
and brine (30 mL ´2). The organic phase was dried over anhy-
drous Na2SO4, filtered and concentrated. Without further purifi-
cation, the desired product 619 was obtained as a yellow oil in 93%
1
yield. Rf = 0.52 (hexane/EtOAc = 1/1). H-NMR (400 MHz,
CDCl3): d 7.24 (d, J = 8.7 Hz, 4 H), 6.82 (d, J = 8.7 Hz, 4 H), 5.87
(s, 1 H), 3.76 (s, 6 H), 2.32 (s, 3 H); 13C-NMR (100 MHz, CDCl3):
d 194.1 (C), 158.6 (C), 133.3 (C), 129.3 (CH), 113.8 (CH), 55.2
(CH), 50.7 (CH3), 30.3 (CH3). IR (neat): 3000, 2955, 2835, 1688,
1608, 1509, 1461, 1353, 1301, 1246, 1176, 1133, 1108, 1033,
955, 817, 778, 629, 585, 551 cm-1. EI-MS m/z (%): 259 ([M-Ac]+,
3), 227 ([M-SAc]+, 100); HR-EI-MS calcd for C17H18O3S (M+)
302.0977, found 302.0980.
Bis(4-methoxyphenyl)methanethiol (4): To a solution of
compound 6 (116 mg, 0.38 mmol) in 4 mL of MeOH was added
K2CO3 (159 mg, 1.15 mmol). The reaction mixture was purged
and filled with Ar, stirred for 1 h at room temperature. It was then
filtered and concentrated under reduced pressure. Purification by
silica gel column chromatography (eluent: 90% hexane/EtOAc)
provided thiol 420 as a colorless oil in 91% yield. Rf = 0.64 (hex-
ane/EtOAc = 65/35). 1H-NMR (400 MHz, CDCl3): d 7.35 (m, 4
H), 6.87 (m, 4 H), 5.38 (d, J = 4.7 Hz, 1 H), 3.78 (s, 6 H), 2.22 (d, J
= 4.7 Hz, 1 H, SH; 13C-NMR (100 MHz, CDCl3): d 158.5 (C),
J. Chin. Chem. Soc. 2014, 61, 707-710
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709

Note
Chou et al.
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259.0575, found 259.0570.
1H-NMR Time Course Acidolysis of Thioacetate 6: To
solutions of compound 6 (30.2 mg, 0.10 mmol) in an array of
TFA/CDCl3 (5%, 10%, 20%, 40% TFA, and the total volume was
1 mL) were added Et3SiH (129 mL, 0.80 mmol). The reaction mix-
tures were monitored with 1H-NMR at designated intervals. The
percent conversion for the reaction was calculated from the inte-
gration of the signals in the aromatic region of the residual 6 and
the bis-(4-methoxyphenyl)methane byproduct (8).
8. Rijkers, D. T. S.; Merkx, R.; Yim, C.-B.; Brouwer, A. J.;
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9. (a) Crich, D.; Sasaki, K.; Rahaman, M. Y.; Bowers, A. A. J.
Org. Chem. 2009, 74, 3886; (b) Mapp, A. K.; Dervan, P. B.
Tetrahedron Lett. 2000, 41, 9451.
ACKNOWLEDGEMENT
This research was supported by National Science
Council (NSC 101-2113-M-002-007).
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SUPPORTING INFORMATION AVAILABLE:
1H/13C NMR spectra for compounds 6, 4, 7, 1a, 1b,
and 8 are included. IR and MS spectra for compound 1b are
also included.
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