The 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc) amino-protecting group: Use in the solid-phase synthesis of pyrrole-imidazole polyamides

Article abstract of DOI:10.1016/S0040-4039(03)00010-8

The development of the 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc) function, a novel base-sensitive amino-protecting group, and its application to the preparation of DNA-binding polyamides are described. Pyrrole-imidazole polyamides were synthesized by an efficient solid-phase method under conditions compatible with Fmoc chemistry using two Tsc-protected amino acids, Tsc-Py-OH 1a and Tsc-Im-OH 1b.

Full text of DOI:10.1016/S0040-4039(03)00010-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1607–1610
The 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc)
amino-protecting group: use in the solid-phase synthesis of
pyrrole-imidazole polyamides
Jin Seok Choi,a,† Younjoo Lee,a,† Eunmyoung Kim,a Nakcheol Jeong,a Hosung Yub and
Hogyu Hana,*
aDepartment of Chemistry, Korea University, Seoul 136-701, South Korea
bHanChem. Co., Ltd., 461-6, Jonmin-dong, Yuseong-gu, Daejeon 305-390, South Korea
Received 6 November 2002; revised 21 December 2002; accepted 25 December 2002
Abstract—The development of the 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc) function, a novel base-sensitive
amino-protecting group, and its application to the preparation of DNA-binding polyamides are described. Pyrrole–imidazole
polyamides were synthesized by an efficient solid-phase method under conditions compatible with Fmoc chemistry using two
Tsc-protected amino acids, Tsc-Py-OH 1a and Tsc-Im-OH 1b. © 2003 Elsevier Science Ltd. All rights reserved.
Recently, we reported the use of the 2-(4-nitrophenyl-
sulfonyl)ethoxycarbonyl (Nsc) base-labile amino-pro-
tecting group for the preparation of DNA-binding
polyamides containing pyrrole (Py) amino acids.1 The
Nsc group differs from the 9-fluorenylmethoxycarbonyl
(Fmoc) group regarding its greater chemical and ther-
mal stability when installed on the exocyclic amino
group of the heteroaromatic pyrrole amino acid.1,2
However, the general utility of the Nsc group in the
solid-phase synthesis of polyamides was hampered by
the poor solubility of the Nsc-protected imidazole (Im)
amino acid, Nsc-Im-OH, in organic solvents.1 To over-
come this problem, we set out to develop a different
protecting group superior in solubility to Nsc without
compromising its stability. With this new protection
scheme, polyamides containing both pyrrole and imida-
zole amino acids could be produced.
tion reaction. Here, we report the development of Tsc,
a novel amino-protecting group, and its use in the
efficient solid-phase synthesis of pyrrole–imidazole
polyamides. The results reveal that Tsc is superior in
solubility to Nsc and in stability to Fmoc in the protec-
tion of exocyclic (hetero)aromatic amines. Tsc is
expected to envision its extensions to numerous amine
protections.
In order to implement the Tsc methodology for the
synthesis of pyrrole–imidazole polyamides, two Tsc-
protected monomers, Tsc-Py-OH 1a and Tsc-Im-OH
1b, were prepared by a modified route used for the
synthesis of Nsc-protected aliphatic and aromatic
amino acids (Scheme 1).1,3 In contrast to the Fmoc
group, we could readily introduce the Tsc protecting
group into Py and Im amino acids starting from methyl
and ethyl esters 5a and 5b via base-resistant 2-(4-tri-
fluoromethylphenylthio)ethoxycarbonyl (Ttc) protec-
tion. The Ttc group could be oxidatively converted to
the corresponding base-labile Tsc group. For the syn-
thesis of 1a and 1b, the starting nitropyrrole 5a and
nitroimidazole 5b were reduced by catalytic hydrogena-
tion to provide aminopyrrole 6a and aminoimidazole
6b, respectively.4 These amine derivatives were not sta-
ble as the free base and were used immediately without
further purification. The aromatic amino group of 6a
and 6b was readily protected by reaction with 4-nitro-
phenyl-2-(4-trifluoromethylphenylthio)ethyl carbonate
(4)5 to give the corresponding carbamates 7a and 7b.
As a first attempt, we have chosen the 2-(4-tri-
fluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc) group
as a more soluble analog of the Nsc group. Substitution
of the trifluoromethyl group for the nitro group would
make the protecting group more soluble. Both groups
have similar electron-withdrawing abilities, so the sub-
stitution should not effect the base-promoted b-elimina-
Keywords: Tsc group; amino protecting group; amino acid; dis-
tamycin; DNA.
* Corresponding author. Tel.: +82-2-3290-3134; fax: +82-2-3290-3121;
e-mail: hogyuhan@korea.ac.kr
† These authors contributed equally to this work.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(03)00010-8

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J. S. Choi et al. / Tetrahedron Letters 44 (2003) 1607–1610
Figure 1. The piperidine adduct 9 that forms presumably via
10 upon deprotection of 1a and 1b with a 20% (v/v) solution
of piperidine in DMF.
<50% intact after 2 h at 55°C). These results indicate
that Tsc is superior to Fmoc and similar to Nsc in its
chemical and thermal stability when used to protect the
exocyclic amino group of heteroaromatic amino
acids.1,9 Based on this result, the general application of
Tsc chemistry to the protection of exocyclic (het-
ero)aromatic amines was deemed feasible.
The deprotection of Tsc with piperidine and related
nitrogen bases was performed by essentially the same
procedure to that of Fmoc and Nsc. Periodic TLC
monitoring of the deprotection of 1a and 1b revealed
that the cleavage of the Tsc group by treatment with
20% (v/v) piperidine in DMF proceeded to completion
within 5 min at comparable rates.11 The primary
byproduct that liberated upon treatment of 1a and 1b
with excess piperidine was a stable compound whose 1H
NMR, 13C NMR and mass spectra were consistent with
the structure 9 (Fig. 1).12 According to HPLC and H
1
Scheme 1. Reagents and conditions: (a) mercaptoethanol,
K2CO3, DMF, reflux, 68%; (b) 4-nitrophenyl chloroformate,
pyridine, CH2Cl2, rt, 86%; (c) 10% Pd/C, 40 psi H2, EtOAc
(5a) or MeOH (5b), rt; (d) 4, DIEA, DMAP, HOBt, CH2Cl2,
rt, (7a, 86% from 5a; 7b, 77% from 5b); (e) LiOH, THF:H2O
(2:1), rt, (8a, 66%; 8b, 81%); (f) Na2MoO4, H2O2, acetone, rt,
(1a, 65%; 1b, 68%).
NMR analyses of the deprotection reaction, no b-elimi-
nation byproduct 10 was observed at any point in the
reaction. Presumably the initially formed vinyl sulfone
10 underwent rapid conversion to the adduct 9 by
Michael-like attack by piperidine. This piperidine
adduct was easily removed from a DMF solution of the
deprotected amines by wash several times with DMF,
CH2Cl2 and MeOH. Thus, Tsc is similar to Nsc in that
the deprotection and adduct-formation occur simulta-
neously, alleviating the byproduct removal problem
encountered in the Fmoc deprotecting reaction.1
Analogous to Fmoc and Nsc chemistry, the presence of
the chromophore in the piperidine adduct 9 allows
facile UV detection and quantitation. This provides a
simple method for estimation of stepwise yields in
coupling of Py and Im aromatic amino acids whose
amino group does not react in the quantitative ninhy-
drin test.
N-Ttc amino acid esters thus obtained were hydrolyzed
under alkaline conditions to afford the acids 8a and 8b
in high yields. Subsequent oxidation of the Ttc moiety
with H2O2/Na2MoO4 in acetone gave the desired prod-
ucts, Tsc-Py-OH 1a and Tsc-Im-OH 1b.6,7
As expected, Tsc-Im-OH exhibited much greater solu-
bility than Nsc-Im-OH in DMF, NMP and 1:1 DMF/
NMP (>0.15 M versus <0.01 M).1,8 Tsc-Py-OH was
also more soluble than its Nsc analog (>0.15 M versus
>0.05/<0.15 M). The solubility of Tsc-Im-OH and Tsc-
Py-OH was sufficient to allow their use for the solid-
phase synthesis of polyamides containing both Im and
Py amino acids.
Peptide coupling of the Tsc protected Py and Im amino
acids was investigated under various standard reaction
conditions in solution using coupling reagents such as
HOBt, HOAt, PyBOP, TFFH, TFFH/HOAt, PyBroP
and EDCI/DMAP.13 We found that EDCI/DMAP is
superior to other reagents by quantitatively affording
dipeptides 11a–d in the solution-phase coupling reac-
tion between 1 and 6 (Fig. 2). The similar results were
observed for Nsc-Py-OH.1 Based on these results,
EDCI/DMAP was used for the solid-phase synthesis of
polyamides.
Premature deprotection of Tsc protected amino acids
was investigated under various coupling reaction condi-
tions.9,10 Careful HPLC monitoring of the decomposi-
tion of Tsc-Py-OH and Tsc-Im-OH at room
temperature revealed that >98% of the Tsc group
remained intact even after 4 h under coupling reaction
conditions, whereas about 25–30% of the Fmoc groups
were eliminated from Fmoc-Py-OH and Fmoc-Im-
OH.1,9 Tsc-Py-OH and Tsc-Im-OH were also less prone
to decomposition than their Fmoc analogs at higher
temperature under the same condition (>95% versus
With Tsc-Py-OH and Tsc-Im-OH in hand, we set out to
demonstrate their use in polyamide synthesis. Our
approach involves standard Fmoc-based solid-phase
synthesis of polyamides using Fmoc-b-alanine-Wang

J. S. Choi et al. / Tetrahedron Letters 44 (2003) 1607–1610
1609
DMF. An additional DCM wash was employed right
before and after capping reactions. The resin was
cleaved by aminolysis with 3-(dimethylamino)propyl-
amine at 55°C for 24 h. Polyamides 12a and 12b were
synthesized in nine steps using the manual solid-phase
protocol (Fig. 2).14 The stepwise yield for both
polyamides was established as >99% by HPLC analysis.
A
single HPLC purification of these three-ring
polyamides afforded an overall recovery of 61% and
55% for 12a and 12b, respectively, and a final purity
greater than 98% as determined by analytical HPLC
and mass spectrometry. We also successfully synthe-
sized the eight-ring polyamides 13a and 13b containing
one fluorescent probe Pr1 in >98% yield of each cou-
pling step and an overall recovery of 48% and 36%,
respectively.14 We could not obtain comparable high
coupling and overall yields for 13b using Fmoc-Py-OH
and Fmoc-Im-OH under reaction conditions as
described (we obtained an overall recovery of 5% for
13b with Fmoc chemistry. Dervan’s group reported an
overall recovery of 9% for ImPyPyPy-g-ImPyPyPy-b-
Dp).2 These results validate our Tsc strategy for the
incorporation of imidazole amino acids into polyamides
in an efficient manner.
In conclusion, we have demonstrated the suitability of
the Tsc methodology for the preparation of polyamides
containing heteroaromatic pyrrole and imidazole amino
acids. The Tsc group alleviates a solubility problem
associated with the Nsc protecting group. In addition,
the Tsc method shows advantages over Fmoc chemistry
in that the Tsc group is less sensitive to premature
deprotection when installed on the exocyclic (het-
ero)aromatic amines and its deprotection byproduct is
easy to remove. The milder Tsc method will allow the
preparation of polyamides with a wide range of modifi-
cations that are incompatible with Boc chemistry. We
anticipate that Tsc will be widely used as an alternative
to Fmoc and Nsc amino-protecting groups.
Acknowledgements
This work was financially supported by the Basic
Research (Grant No. 981-0302-009-2) and CRM pro-
grams from KOSEF. Fellowship support from the
BK21 program (J.S.C., Y.L., E.K.) is gratefully
acknowledged.
Figure 2. Structures of polyamides 11–13. HPLC14 (a) and
MALDI-TOF mass spectral (b) analyses of the polyamide
13b.
References
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4299.
resin.1,2,4 Each coupling cycle consists of a solvent
wash, removal of the Tsc group with 20% piperidine in
DMF for 30 min, a solvent wash, addition of monomer
(2.5 equiv.) preactivated with EDCI and DMAP, cou-
pling for 4 h, a solvent wash, capping with acetic
anhydride/DIEA, and a final solvent wash. Washing
was performed with DMF, MeOH, DCM and then
2. Wurtz, N. R.; Turner, J. M.; Baird, E. E.; Dervan, P. B.
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J. S. Choi et al. / Tetrahedron Letters 44 (2003) 1607–1610
10. The amount of intact 1a and 1b (0.1 M) after treatment
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with NH2-Gly-OEt·HCl (0.2 M) and DIEA (0.3 M) in
DMF was determined by analytical HPLC on a C18
column using UV absorption at 254/268 nm. tBoc-Py-
OH4 (0.1 M) was added as an internal standard. Fmoc-
Py-OH and Fmoc-Im-OH2 were used for comparison
experiments and its cleavage was monitored by UV
absorption at 254/290 nm.
4. Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118,
6141–6146.
5. Ttc-ONp 4. TLC (EtOAc:hexane=1:3) Rf=0.37; 1H
NMR (300 MHz, CDCl3) l 8.27 (d, J=9.3 Hz, 2H), 7.56
(d, J=8.7, 2H), 7.47 (d, J=8.1, 2H), 7.36 (d, J=8.7, 2H),
4.47 (t, J=6.9, 2H), 3.35 (t, J=7.1, 2H); 13C NMR (75
MHz, CDCl3) l 155.05, 152.08, 145.24, 139.89, 129.29,
128.75, 128.32, 128.17, 127.89, 127.45, 125.87, 125.82,
125.77, 125.72, 125.69, 125.15, 122.09, 121.55, 118.48,
66.78, 30.95.
11. The deprotection rate was slightly faster by addition of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1% v/v) to a
20% piperidine–DMF solution.
12. Piperidine adduct 9. TLC (EtOAc:hexane=1:1) Rf=0.38;
1H NMR (300 MHz, CDCl3) l 8.07 (d, J=8.4, 2H), 7.83
(d, J=8.4, 2H), 3.33 (t, J=7.2, 2H), 2.74 (t, J=7.2, 2H),
2.27 (s, 4H), 1.35 (s, 6H); 13C NMR (75 MHz, CDCl3) l
143.37, 135.87, 135.44, 135.00, 134.57, 128.65, 126.12,
126.07, 126.02, 125.97, 124.93, 121.32, 117.72, 54.08,
53.62, 51.95, 25.66, 23.97; HRMS (FAB+) for
C14H19F3NO2S (MH+), calcd 322.1089, found 322.1082;
UV (DMF) umax=268 nm (m=1.6×103 M−1 cm−1).
13. (a) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc.
1995, 117, 5401–5402; (b) Coste, J.; Fre´rot, E.; Jouin, P.
Tetrahedron Lett. 1991, 32, 1967–1970; (c) Boger, D. L.;
Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122,
6382–6394; (d) Krutzik, P. O.; Chamberlin, A. R. Bioorg.
Med. Chem. Lett. 2002, 12, 2129–2132 and references
cited therein.
6. Tsc-Py-OH 1a. TLC (EtOAc:hexane=4:1) Rf=0.38; 1H
NMR (300 MHz, DMSO-d6) l 12.15 (brs, 1H), 9.21 (brs,
1H), 8.14 (d, J=8.1, 2H), 8.00 (d, J=8.1, 2H), 6.95 (s,
1H), 6.56 (s, 1H), 4.34 (t, J=5.6, 2H), 3.84 (t, J=5.4,
2H), 3.77 (s, 3H); 13C NMR (75 MHz, DMSO-d6) l
161.98, 152.61, 143.37, 134.28, 133.85, 133.42, 132.99,
128.87, 126.67, 126.63, 126.23, 122.14, 121.61, 120.04,
118.98, 117.99, 107.84, 57.61, 54.43, 36.15; HRMS (FAB+)
for C16H15F3N2O6S (M+), calcd 420.0603, found
420.0609.
Tsc-Im-OH 1b. TLC (EtOAc:MeOH:H2O=24:5:4) Rf=
1
0.17; H NMR (300 MHz, DMSO-d6) l 9.82 (brs, 1H),
14. The polyamides 12 and 13 were synthesized on Fmoc-b-
Ala-Wang resin (30 mmol) in a stepwise fashion by a
manual solid-phase method as described.1,15 Purity of the
polyamide was determined to be >98% by reverse-phase
HPLC on a C18 analytical column (4 mm, 0.39×15 cm,
Nova-Pak, Waters, MA) under gradient conditions: 12,
3.33% CH3CN/min, 1 mL min−1 flow rate, Rv=9.06 mL
(12a) and 9.14 mL (12b); 13, 0–10 min 100% H2O, 10–60
min 2% CH3CN/min, 1 mL min−1 flow rate, Rv=37.50
mL (13a) and 38.83 mL (13b). The observed molecular
mass agreed to within 0.1% of the calculated polyamide
mass. MALDI-TOF: 12a, C28H40N9O5 (MH+), calcd
582.3152, found 582.4471; 12b, C27H39N10O5 (MH+),
calcd 583.3105, found 583.7133; 13a, C70H83N20O10/
C70H82N20O10Na (MH+/MNa+), calcd 1363.6601/
8.14 (d, J=7.8, 2H), 8.00 (d, J=7.8, 2H), 7.94 (brs, 1H),
7.06 (s, 1H), 4.36 (brs, 2H), 3.87 (brs, 5H); 13C NMR (75
MHz, DMSO-d6)
l 160.60, 152.41, 143.24, 136.34,
134.64, 134.15, 133.72, 133.29, 132.86, 128.90, 126.62,
125.16, 121.55, 117.93, 111.83, 57.77, 54.22, 35.28;
HRMS (FAB+) for C15H14F3N3O6SNa (MNa+), calcd
444.0453, found 444.0464.
7. 1a and 1b could also be synthesized by treatment of
tert-butyl esters, H-Py-OtBu and H-Im-OtBu, with Tsc-
Cl using literature protocols.2 However, tert-butyl esters
are more expensive and require harsher conditions for
reduction (500 versus >40 psi) and ester deprotection (0.2
M TiCl4 versus 0.1N LiOH) than methyl and ethyl esters.
8. (a) Stigers, K. D.; Koutroulis, M. R.; Chung, D. M.;
Nowick, J. S. J. Org. Chem. 2000, 65, 3858–3860; (b)
Carpino, L. A.; Wu, A.-C. J. Org. Chem. 2000, 65,
9238–9240.
1385.6420,
found
1364.4369/1386.4111;
13b,
C69H82N21O10/ C69H81N21O10Na (MH+/MNa+), calcd
1364.6554/1386.6373, found 1365.1753/1387.1616.
9. Bodanszky, M.; Deshmane, S. S.; Martinez, J. J. Org.
Chem. 1979, 44, 1622–1625.
15. Kent, S. B. H. Annu. Rev. Biochem. 1988, 57, 957–989.