L-Cysteine as a water-soluble cation scavenger in the removal of the 2,4,6-trimethoxybenzyl group from thiols

Article abstract of DOI:10.1016/S0040-4039(02)00808-0

L-Cysteine was used as a water-soluble cation scavenger in the acid-catalyzed removal of the 2,4,6-trimethoxybenzyl (Tmob) group from thiols. After aqueous extraction, the product thiols were isolated in good yields. For most substrates, trifluoroacetic acid (TFA) is the reagent of choice. However, for the deprotection of acid-sensitive compounds, formic acid with an extended reaction time is appropriate.

Full text of DOI:10.1016/S0040-4039(02)00808-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4531–4533
L
-Cysteine as a water-soluble cation scavenger in the removal of
the 2,4,6-trimethoxybenzyl group from thiols
Chia-En Lin,* Stewart K. Richardson and David S. Garvey
NitroMed, Inc., 12 Oak Park Drive, Bedford, MA 01730, USA
Received 5 January 2002; revised 22 April 2002; accepted 25 April 2002
Abstract—L-Cysteine was used as a water-soluble cation scavenger in the acid-catalyzed removal of the 2,4,6-trimethoxybenzyl
(Tmob) group from thiols. After aqueous extraction, the product thiols were isolated in good yields. For most substrates,
trifluoroacetic acid (TFA) is the reagent of choice. However, for the deprotection of acid-sensitive compounds, formic acid with
an extended reaction time is appropriate. © 2002 Elsevier Science Ltd. All rights reserved.
During the last decade nitric oxide (NO) has been
identified as an important signaling molecule associated
with neurotransmission, smooth muscle relaxation,
platelet inhibition, and immune system regulation.1
NitroMed is committed to the development of nitric
oxide enhanced (NitRx) medicines in which NO com-
plements the biological activity of existing drugs by
increasing the safety and/or efficacy of the parent
molecules. The nitrosothiol moiety is one of the NO-
donor groups used by NitroMed for the preparation of
NitRx medicines and frequently, the preparation of
nitrosothiol containing compounds requires protection
of an intermediate thiol.
however the desired products were always contami-
nated with several trimethoxybenzyl-containing
byproducts as well as the excess reagents. Conse-
quently, isolation of the desired product thiols often
required
a
tedious silica gel chromatographic
separation.
Herein we wish to report the successful use of
L-cys-
teine as a water-soluble cation scavenger in the removal
of the Tmob group. The Tmob protected thiols were
treated with 10 equiv. of L-cysteine in 50% TFA for 10
min. Following a simple aqueous work up the thiols
were obtained in good yields. Eight examples (2–9) are
shown in Fig. 1 along with the isolated yields of the
thiols, except compound 3 whose yield was determined
by quantitative mass spectra analysis. In each instance,
the resulting thiol was further purified by recrystaliza-
tion, elution through a pad of silica gel or was of
sufficient purity to carry forward in the reaction
sequence.
The 2,4,6-trimethoxybenzyl (Tmob) group was origi-
nally developed as a side-chain protecting group for
cysteine in the Na-9-fluorenylmethoxycarbonyl (Fmoc)
strategy of solid-phase peptide synthesis (SPPS).2
Deprotection of the Tmob group in SPPS was done
with trifluoroacetic acid (TFA)-methylene chloride in
the presence of phenol, thioanisole and water or in the
presence of triethylsilane or triisopropylsilane. When
employing the synthesis on solid support strategy,
removal of the byproducts and excess cation scavenger
can be simply done by filtration.
For the preparation of Tmob protected thiols, 2,4,6-
trimethoxybenzyl alcohol 1 was first prepared from
2,4,6-trimethoxybenzaldehyde according to the litera-
ture in 97% yield.2 Protected thiols 2–5 were prepared
by reacting the corresponding thiols with 1 in methyl-
ene chloride with 5% TFA. For the preparation of
compounds 6–9, the thiol containing acid 10,3 was first
converted into its S-Tmob derivative 11 with 2,4,6-
trimethxybenzyl alcohol. This was then coupled (1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
We have utilized the Tmob protecting group in the
course of preparing tertiary nitrosothiols via conven-
tional solution phase methods. Deprotection of the
intermediate Tmob protected thiols was successful with
TFA in the presence of phenol, anisole and water,
hydrochloride) to the corresponding thiol or alcohol to
give 6–9.
* Corresponding author. Fax: 781-685-9727; e-mail: glin@
nitromed.com
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00808-0

4532
C.-E. Lin et al. / Tetrahedron Letters 43 (2002) 4531–4533
Figure 1.
For the deprotection reactions, good yields were
obtained for all compounds examined except com-
pound 5, which was isolated in moderate yield. This
lower yield was due to the formation of other sterol
related side products in TFA. We therefore explored
the use of a weaker acid as a catalyst for the deprotec-
tion process. Substituting formic acid for TFA in the
deprotection of compound 5 improved the isolated
yield of the product thiol to 75% (Eq. (1)).
constant for removing the Tmob group from the ter-
tiary thiol indicated that the tertiary thiol has a lower
affinity for the Tmob cation. Therefore, it is somewhat
more favorable to remove the Tmob group from a
tertiary thiol than from a primary thiol. When formic
acid was used, the reaction time for achieving the
steady state equilibrium under these conditions from a
tertiary thiol was 40 min; from a secondary thiol, 5 h;
from a primary thiol, 30 h.
(1)
To better define the reaction kinetics, the concentration
of compounds 2 and 3 and the corresponding thiols at
the equilibrium state using TFA and 10 equiv. of
In summary, we have found that formic acid can serve
as an effective substitute for TFA in the removal of
Tmob groups from acid sensitive compounds. In addi-
L
-cysteine were measured by quantitative mass spectra
tion, using L-cysteine as a water soluble cation scav-
analysis. The equilibrium constants were determined to
be 2.3 and 14 for the primary thiol 2 and the tertiary
thiol 3, respectively. The somewhat bigger equilibrium
enger for Tmob deprotection (1) facilitates the isolation
of the desired product; (2) improves the overall yield;
and (3) presents itself as a practical alternative when

C.-E. Lin et al. / Tetrahedron Letters 43 (2002) 4531–4533
4533
contemplating larger scale synthetic applications
employing conventional solution phase methodology.
mass spectra analysis showed a 95% yield. The crude
product was purified by column chromatography (silica
gel, ethyl acetate/hexane: 15%/85%, then 20%/80%, and
then 25%/75%) to give the free thiol (149.9 mg, 0.3780
mmol, 93.3%, mp=77–79°C) and unreacted compound
Experimental
1
2 (15.7 mg, 0.0272 mmol, 6.7%). H NMR (CDCl3) l
Preparation of Tmob protected thiols as illustrated by the
7.31 (s, 5H), 7.19 (d, J=7.2 Hz, 1H), 5.68 (d, J=7.2
Hz, 1H), 5.10 (s, 2H), 4.82 (m, 1H), 4.33 (m, 1H), 4.22
(m, 2H), 2.95 (m, 2H), 1.66 (m, 2H), 1.53 (m, 2H), 1.27
(t, J=7.1 Hz, 3H), 0.92 (m, 6H). 13C NMR (CDCl3) l
172.3, 169.7, 156.1, 136.1, 128.4, 128.0, 127.9, 66.9,
61.8, 53.5, 41.2, 26.5, 24.5, 22.7, 21.9, 14.0. LRMS
synthesis of dipeptide 2. The thiol dipeptide (281.2 mg,
0.7092 mmol, which was prepared from L-cysteine ethyl
ester and N-cbz-L-leucine with EDC coupling in 55%
yield) and 2,4,6-trimethoxybenzyl alcohol (182.9 mg,
0.9227 mmol) were dissolved in methylene chloride (2.0
mL). Trifluoroacetic acid (100 mL) was added dropwise
to give a pale yellow solution. This was stirred at room
temperature for 10 min, concentrated to dryness,
treated with ethyl acetate and concentrated to dryness.
The crude product was dissolved in hot ethyl acetate
(1.1 mL) and hexane (3.3 mL) was added to give a
crystalline product. After cooling with ice-water, the
crystals were collected, washed with hexane, and dried
in vacuum to give compound 2 (276.6 mg, 0.4796
mmol, 68%, mp=124–125°C). The mother liquid was
concentrated and purified by chromatography (silica
gel, ethyl acetate/hexane: 20%/80% then 25%/75%) to
(APIMS) m/z 397 (M+H+), 414 (M+NH4 ), 419 (M+
+
Na+), 793 (2M+H+), 810 (2M+NH4 ), 815 (2M+Na+).
+
Removal of the Tmob group with formic acid as illus-
trated by deprotection of compound 5. A solution of
compound 5 (83 mg, 0.142 mmol) in methylene chloride
(8 mL) was added dropwise rapidly to a solution of
cysteine (171 mg, 1.42 mmol) in formic acid (8 mL).
The resultant solution was stirred at room temperature
for 5 h and the volatile material removed in vacuo. The
residue was neutralized with sodium bicarbonate solu-
tion and extracted with methylene chloride. The
organic phase was washed with more sodium bicarbon-
ate solution, dried (sodium sulfate), filtered and concen-
trated to give the product (54 mg, 95%) which was
further purified by chromatography on silica gel (ethyl
acetate:hexane 5:95) to give the thiol (43 mg, 75%).
1
give more compound 2 (88.2 mg, 0.153 mmol, 22%). H
NMR (CDCl3) l 7.32 (s, 5H), 6.65 (d, J=7.2 Hz, 1H),
6.12 (s, 2H), 5.15 (m, 1H), 5.10 (s, 2H), 4.80 (m, 1H),
4.20 (m, 3H), 3.79 (m, 11H), 2.95 (m, 2H), 1.70 (m,
2H), 1.51 (m, 1H), 1.25 (t, J=7.0 Hz, 3H), 0.96 (m,
6H). 13C NMR (CDCl3) l 171.9, 170.7, 160.5, 158.8,
156.0, 136.3, 128.5, 128.1, 128.0, 107.7, 90.6, 67.0, 61.5,
55.7, 55.3, 53.4, 52.1, 41.9, 33.6, 24.6, 24.3, 22.9, 22.0,
14.1. LRMS (APIMS) m/z 577 (M+H+), 594 (M+
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Removal of the Tmob group with TFA as illustrated by
deprotection of dipeptide 2. L-Cysteine (491.1 mg, 4.052
mmol) was dissolved in trifluoroacetic acid (10 mL).
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