Nsc-mediated solid-phase synthesis of polyamides containing pyrrole amino acid

Article abstract of DOI:10.1016/S0040-4039(02)00809-2

The synthesis of the Nsc-protected amino acid, Nsc-Py-OH 1a, and its oligomerization are described.

Full text of DOI:10.1016/S0040-4039(02)00809-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4295–4299
Nsc-mediated solid-phase synthesis of polyamides containing
pyrrole amino acid
Jin Seok Choi,a Hwa-Sun Lee,a Younjoo Lee,a Nakcheol Jeong,a Hack-Joo Kim,b Young-Deug Kimb
and Hogyu Hana,*
aDepartment of Chemistry, Korea University, Seoul 136-701, South Korea
bResearch Institute, A&PEP Inc., 213, Sosabon 1-dong, Sosa-gu, Bucheon 422-231, South Korea
Accepted 24 April 2002
Abstract—The synthesis of the Nsc-protected amino acid, Nsc-Py-OH 1a, and its oligomerization are described. © 2002 Elsevier
Science Ltd. All rights reserved.
Polyamides for the specific recognition of DNA
sequences can be designed by the linear alignment of
N-methylpyrrole (Py), N-methylimidazole (Im), and N-
methyl-3-hydroxypyrrole (Hp) amino acids.1 The
sequence specificity of the polyamides depends on side-
by-side ring pairings of these aromatic amino acids in
the minor groove of DNA. The four amino acid pairs
complete the minor-groove recognition code for all four
Watson–Crick base pairs. The ability of polyamides to
bind DNA with specificities and affinities comparable
to those of naturally occurring DNA-binding proteins
has generated considerable interests owing to their
potential roles in regulating gene expressions.1,2
Recently, several groups tried to apply the Fmoc pro-
tection method in the preparation of polyamides.4
However, we have found that the synthesis of two
Fmoc-protected monomer building blocks, Fmoc-Py-
OH and Fmoc-Im-OH amino acids, required longer
synthetic steps and showed a substantially lower overall
chemical yield than that of Boc-protected Py and Im
amino acids. This might be attributed to facile decar-
boxylation of Py/Im heteroaromatic (amino) acids and
insufficient stability of the Fmoc group installed on the
exocyclic (hetero)aromatic amine.2b,5 We therefore
explored the alternative established for amine protec-
tion but still retaining compatibility with Fmoc chem-
istry. The use of the 2-(4-nitrophenylsulfonyl)ethoxy-
carbonyl (Nsc) group may be more suitable for
polyamide synthesis because of its increased stability
when compared to the Fmoc group.6,7 Here, we
describe the efficient synthesis of Py and Im monomers
bearing Nsc protection, followed by studies to optimize
conditions for coupling/deprotection cycles.
Previous reports on polyamide synthesis utilized tert-
butoxycarbonyl (Boc)-based protocols.1e This involves
acidic procedures such as repeated exposure of the
polyamide to TFA in the removal of the temporary Boc
amino protecting group. Such conditions preclude the
incorporation of various acid-sensitive modifications
into the standard polyamide structures. An alternative
strategy for polyamide synthesis may employ the 9-
fluorenylmethoxycarbonyl (Fmoc) protecting group.3
The Fmoc protection scheme would complement the
Boc strategy for polyamide synthesis because of its
stability to acid and lability to base. Interestingly, rela-
tively little information on Fmoc protections of the
exocyclic (hetero)aromatic amine is available in the
literature.
To test the applicability of the Nsc group for polyamide
synthesis, two monomers, Nsc-Py-OH 1a and Nsc-Im-
OH 1b were prepared by a modified route used for
synthesis of Nsc-protected aliphatic amino acids
(Scheme 1).6 In contrast to the Fmoc group, we could
readily introduce the Nsc protecting group into Py and
Im amino acids starting from methyl and ethyl esters 5a
and 5b via base-resistant 2-(4-nitrophenylthio)ethoxy-
carbonyl (Ntc) protection. The Ntc group could be
oxidatively converted to the corresponding base-labile
Nsc group. For the synthesis of 1a and 1b, the starting
nitropyrrole 5a and nitroimidazole 5b were reduced by
catalytic hydrogenation to provide aminopyrrole 6a
Keywords: Nsc 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
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00809-2

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J. S. Choi et al. / Tetrahedron Letters 43 (2002) 4295–4299
Figure 1. The piperidine adduct 9 that forms upon deprotec-
tion of 1a and 1b with a 20% (v/v) solution of piperidine in
DMF.
whereas about 25–30% of the Fmoc groups were elimi-
nated from Fmoc-Py-OH and Fmoc-Im-OH.11 Nsc-Py-
OH was also less prone to decomposition than
Fmoc-Py-OH at higher temperature under the same
solution conditions (>95% versus <50% intact after 2 h
at 55°C). These results indicate that the Nsc group
differs from the Fmoc group regarding its increased
chemical and thermal stability when used to protect the
exocyclic amino group of heteroaromatic amino acids.10
Based on this result, general application of Nsc chem-
istry to exocyclic (hetero)aromatic amine protection
was deemed feasible.
The deprotection of Nsc with piperidine and related
nitrogen bases was just like that of Fmoc. Periodic TLC
monitoring of the deprotection of 1a and 1b revealed
that the cleavage of the Nsc group by treatment with
20% (v/v) piperidine in DMF proceeded to completion
within 5 min at comparable rates.12 The primary
byproduct that liberated upon treatment of 1a and 1b
with excess piperidine was a stable compound whose
1H, 13C NMR and mass spectra were consistent with
Scheme 1. Reagents and conditions: (a) mercaptoethanol,
KOH, 70°C, quantitative; (b) COCl2, THF, −20°C, 95%; (c)
10% Pd/C, 40 psi H2, EtOAc, rt; (d) 4, TEA, EtOAc, rt, 78%
from 5a; (e) LiOH, THF:H2O (2:1), rt, 79%; (f) Na2MoO4,
H2O2, 1,4-dioxane, rt, 64%; (g) 10% Pd/C, 40 psi H2, EtOAc/
EtOH (1:2), rt; (h) 4, TEA, EtOAc, rt, 50% from 5b; (i)
NaOH, 1,4-dioxane, rt, 98%; (j) Na2MoO4, H2O2, 1,4-diox-
ane/AcOH (4:1), rt, 31%.
1
the structure 9 (Fig. 1).13 According to HPLC and H
NMR analyses of the deprotection reaction, no b-elimi-
nation byproduct 10 was observed at any point of 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 extraction or wash several times
with DMF, CH2Cl2 and MeOH. Thus, Nsc alleviates
the byproduct removal problem associated with the
Fmoc protecting group.7 Analogous to Fmoc chem-
istry, the presence of the chromophore in the piperidine
adduct 9 allows facile UV detection and quantitation.
This provides a simple method for estimation of step-
wise yields in coupling of Py aromatic amino acid
whose amino group does not react in the quantitative
ninhydrin test.
and aminoimidazole 6b, respectively.1e These amine
derivatives were not stable as the free base and thus
used as, formed immediately without further purifica-
tion. The aromatic amino group of 6a and 6b was
readily protected by reaction with chloro-2-(4-nitro-
phenylthio)ethyl carbonate (4) to give the correspond-
ing carbamates 7a and 7b, respectively. N-Ntc amino
acid esters thus obtained were hydrolyzed under alkal-
ine conditions to afford the acids 8a and 8b. Sub-
sequent oxidation of the Ntc moiety with H2O2/
Na2MoO4 gave the desired products, Nsc-Py-OH 1a
and Nsc-Im-OH 1b.8
Nsc-Py-OH exhibited greater solubility than Nsc-Im-
OH in DMF, NMP, and DMF/NMP (1:1). In fact, the
poor solubility (<0.01 M) of Nsc-Im-OH in these and
other common organic solvents such as CHCl3, MeOH
and THF proved to be the major obstacle to its utility
in the solid-phase synthesis of polyamides.9 Therefore,
only Nsc-Py-OH was used for solid-phase polyamide
synthesis. Premature deprotection of Nsc protected
amino acids was investigated under various coupling
reaction conditions.10,11 Careful HPLC monitoring of
the decomposition of Nsc-Py-OH at room temperature
revealed that >98% of the Nsc group remained intact
even after 4 h under coupling reaction conditions,
Peptide coupling of the Nsc protected Py amino acid
was investigated under various standard reaction condi-
tions in solution using coupling reagents such as HOBt,
HOAt, PyBOP, TFFH, TFFH/HOAt and PyBroP.14
The coupling reagents, TFFH/HOAt and PyBroP
showed comparable moderate efficiency (ca. 60% iso-
lated yield) in the synthesis of dipeptide 11 from 1a and
6a (Fig. 2).15 On the other hand, other reagents gave a
lower yield than that obtained with TFFH/HOAt and
PyBroP (ca. 30–40% isolated yield). Since all these
coupling efficiencies were somewhat unsatisfactory for

J. S. Choi et al. / Tetrahedron Letters 43 (2002) 4295–4299
4297
their use in solid-phase polyamide synthesis, we
explored more effective coupling reagents. We found
that EDCI in combination with DMAP demonstrated
the superiority over TFFH/HOAt and PyBroP by
affording the quantitative yield in the solution phase
coupling reaction between 1a and 6a.16 Based on
these results, the solid-phase coupling reaction was
performed using a combination of EDCI and DMAP.
The polyamides 12 and 13 were prepared by solid-
phase synthesis using Fmoc-b-alanine-Wang resin.
Each coupling cycle consists of a solvent wash,
removal of the Nsc group with 20% piperidine in
DMF for 30 min, a solvent wash, addition of
monomer (2.5 equiv.) preactivated with EDCI and
DMAP, coupling for 4 h, a solvent wash, capping
with acetic anhydride and DIEA and a final solvent
wash. Washing were performed with DMF, MeOH,
DCM and then DMF. An additional DCM wash was
employed right before and after capping reactions.
The resin was cleaved by aminolysis with 3-(dimethy-
lamino)propylamine at 55°C for 24 h. Ac-PyPyPy-b-
Dp 12 was synthesized in nine steps using the
protocols as described (Fig. 2).17 The stepwise yield
was established as >99% by HPLC analysis. A single
HPLC purification of the three-ring polyamide 12
afforded a 57% overall recovery and a final purity
greater than 98% as determined by analytical HPLC
and mass spectroscopy. We also successfully synthe-
sized the polyamide 13 containing five pyrroles and
one fluorescent label X 1418 in >98% yield of each
coupling step and an overall 48% recovery. We could
not obtain comparable high coupling and overall
yields using Fmoc-Py-OH under reaction conditions
as described4a (<80–90% coupling yield and <5–10%
recovery for Fmoc-Py-OH).
In conclusion, we have demonstrated the suitability of
the Nsc methodology for preparation of polyamides
containing heteroaromatic amino acids. The Nsc
group alleviates a stability problem associated with
the Fmoc protecting group due to the less sensitivity
of the Nsc group toward premature deprotection
when installed on the exocyclic (hetero)aromatic
amine. In addition, the Nsc method shows advantages
over the Fmoc chemistry in that it eliminates
difficulties in removing the deprotection byproduct.
The milder Nsc method will allow for preparation of
polyamides with a wide range of modifications which
are incompatible with Boc chemistry.
Acknowledgements
Figure 2. Structures of Nsc-PyPy-OMe 11, Ac-PyPyPy-b-Dp
12, and XPyPy-g-PyPyPy-b-Dp 13. HPLC analysis of 12 (a)
and MALDI-TOF mass spectral analyses of purified
polyamides 12 (b) and 13 (c).
This work was financially supported by the Korea
Research Foundation Grant (KRF-2000-015-DP0254).
Fellowship support from the BK21 Program (J.S.C.

4298
J. S. Choi et al. / Tetrahedron Letters 43 (2002) 4295–4299
and Y.L.) is gratefully acknowledged. We are grateful
for the instrumental support from the equipment facil-
ity of CRM-KOSEF, Korea University.
M. E.; Iguchi, S.; Ionescu, D.; El-Faham, A.; Reimer, C.;
Warrass, R. J. Org. Chem. 1999, 64, 4324–4338.
8. Nsc-Py-OH 1a. TLC (EtOAc:hexane=3:1) Rf=0.30; 1H
NMR (300 MHz, DMSO-d6) l 12.15 (brs, 1H), 9.03 (brs,
1H), 8.40 (d, J=8.7 Hz, 2H), 8.17 (d, J=8.7, 2H), 6.88
(s, 1H), 6.50 (s, 1H), 4.34 (t, J=5.4, 2H), 3.89 (t, J=5.6,
2H), 3.76 (s, 3H); 13C NMR (75 MHz, DMSO-d6) l
161.59, 152.13, 150.16, 144.59, 129.27, 124.48, 121.79,
119.78, 118.69, 107.46, 57.59, 54.28, 36.12; HRMS (FAB+)
for C15H15N3O8S (M+), calcd 397.0580, found 397.0582.
Nsc-Im-OH 1b. TLC (EtOAc:MeOH:H2O=25:5:4) Rf=
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1
0.40; H NMR (300 MHz, DMSO-d6) l 9.77 (brs, 1H),
8.37 (d, J=7.8, 2H), 8.16 (d, J=8.4, 2H), 7.15 (s, 1H),
4.37 (brs, 2H), 3.91 (brs, 2H), 3.86 (s, 3H); 13C NMR (75
MHz, DMSO-d6)
l 159.64, 152.16, 150.09, 144.55,
136.86, 131.74, 129.27, 124.45, 113.30, 57.91, 54.20, 35.48;
LRMS (FAB+) for C14H15N4O8S (MH+), calcd 399.1,
found 399.0.
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11. The amount of intact Nsc-Py-OH 1a (0.1 M) after treat-
ment with NH2-Gly-OEt·HCl (0.2 M) and DIEA (0.3 M)
in DMF was determined by analytical HPLC on a C18
column at 254 and 268 nm. tBoc-Py-OH (0.1 M) was
added as an internal standard. Fmoc-Py-OH was selected
as a substrate for comparision experiments.4a Cleavage of
the Fmoc group was performed as described above, with
the exception of monitoring the absorbance at 254 and
290 nm.
12. 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.
13. Piperidine adduct 9. TLC (EtOAc:hexane=3:1) Rf=0.39;
1H NMR (300 MHz, CDCl3) l 8.40 (d, J=8.7, 2H), 8.14
(d, J=9.0, 2H), 3.36 (t, J=6.9, 2H), 2.75 (t, J=6.8, 2H),
2.26 (brs, 4H), 1.33 (brs, 6H); 13C NMR (75 MHz,
CDCl3) l 150.60, 145.73, 129.62, 124.12, 54.01, 53.56,
51.95, 25.52, 23.79; HRMS (FAB+) for C13H19N2O4S
(MH+), calcd 299.1066, found 299.1076; UV (DMF)
umax=267 nm (m=4.7×103).
14. (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.
15. Nsc-PyPy-OMe 11. TLC (EtOAc:hexane=3:1) Rf=0.22;
1H NMR (300 MHz, DMSO-d6) l 9.87 (brs, 1H), 9.20
(brs, 1H), 8.43 (d, J=7.5, 2H), 8.19 (d, J=7.5, 2H), 7.46
(s, 1H), 6.89 (s, 1H), 6.81 (s, 1H), 6.77 (s, 1H), 4.37 (brs,
2H), 3.90 (brs, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.74 (s,
3H); 13C NMR (75 MHz, DMSO-d6) l 160.81, 158.28,
152.52, 150.43, 144.74, 129.47, 124.67, 122.90, 122.76,
121.56, 120.76, 118.48, 117.36, 108.32, 103.85, 57.49,
54.29, 50.98, 36.19, 36.12.
16. Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem.
Soc. 2000, 122, 6382–6394.
17. Ac-PyPyPy-b-Dp 12 and XPyPy-g-PyPyPy-b-Dp 13. The
polyamides 12 and 13 were synthesized on Fmoc-b-Ala-
Wang resin (60 mmol) in a stepwise fashion by a manual
solid-phase method as described.19 Nsc-Py-OH (150
7. (a) Carpino, L. A.; Philbin, M.; Ismail, M.; Truran, G.
A.; Mansour, E. M. E.; Iguchi, S.; Ionescu, D.; El-
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Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E.

J. S. Choi et al. / Tetrahedron Letters 43 (2002) 4295–4299
4299
mmol) was activated with 1-ethyl-3-(3-dimethylamino-
propyl)-carbodiimide hydrochloride (EDCI, 300 mmol)
and 4-dimethylaminopyridine (DMAP, 300 mmol) and
coupled for 4 h. Deprotection of the Nsc groups was
performed using 20% (v/v) piperidine in DMF for 30 min.
Polyamide 12 was acetylated at the N-terminus. Polyamide
resin cleavage was performed using 3-(dimethylamino)-
propylamine (3 mL) in a 4 mL glass vial at 55°C for
24 h. After evaporation of the solvent, the crude poly-
amide was dissolved in DMF (4 mL). Purification was
achieved by reverse-phase HPLC with a Vydac C18 column
and a linear water/acetonitrile gradient containing 0.1%
(v/v) trifluoroacetic acid. Polyamides were detected by
monitoring the absorbance at 254 nm. Polyamides were
recovered upon lyophilization of the appropriate fractions
as a solid (12, 20 mg, 57% recovery; 13, 32 mg, 48%
recovery). 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 gradi-
ent conditions (12, 3.33% CH3CN/min, 1 mL min−1 flow
rate, Rv=9 mL; 13, 0–10 min 100% H2O, 10–60 min 3.33%
CH3CN/min, 1 mL min−1 flow rate, Rv=39 mL). The
molecular mass of each polyamide was measured using
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) on a PerSeptive Biosystems Voyager-
DE™RP mass spectrometer. The observed molecular mass
agreed to within 0.1% of the calculated polyamide mass.
MALDI-TOF: 12, C28H40N9O5 (MH+), calcd 582.3152,
found 582.6091; 13, C58H71N16O8 (MH+), calcd 1119.5641,
found 1119.7124.
1
18. Compound X-OH 14. H NMR (300 MHz, DMSO-d6) l
8.56 (d, J=4.8, 1H), 8.13 (s, 1H), 7.68 (d, J=4.8, 1H), 7.53
(d, J=14.7, 1H), 7.52 (d, J=8.7, 2H), 7.05 (d, J=16.2, 1H),
6.74 (d, J=8.1, 2H), 2.96 (s, 6H); 13C NMR (75 MHz,
DMSO-d6) l 166.25, 150.76, 149.17, 146.91, 134.64, 128.61,
123.57, 122.71, 120.94, 119.83, 112.00; LRMS (FAB+) for
C16H17N2O2 (MH+), calcd 269.1, found 269.0.
19. Kent, S. B. H. Annu. Rev. Biochem. 1988, 57, 957–989.