A photoremovable ligation auxiliary for use in polypeptide synthesis

Article abstract of DOI:10.1016/S0040-4039(03)01463-1

A photoremovable auxiliary for use in peptide synthesis via the native chemical ligation method is described. A 2-mercapto-1-(2-nitrophenyl)ethylamine (Mnpe-amine) moiety was attached to the N-terminus of a peptide via the periodate oxidation of a seryl p

Full text of DOI:10.1016/S0040-4039(03)01463-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6059–6061
A photoremovable ligation auxiliary for use in polypeptide
synthesis
Toru Kawakami and Saburo Aimoto*
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 2 June 2003; revised 10 June 2003; accepted 12 June 2003
Abstract—A photoremovable auxiliary for use in peptide synthesis via the native chemical ligation method is described. A
2-mercapto-1-(2-nitrophenyl)ethylamine (Mnpe-amine) moiety was attached to the N-terminus of a peptide via the periodate
oxidation of a seryl peptide. The resulting peptide was then ligated to a peptide thioester, and UV (365 nm) irradiation resulted
in the removal of the auxiliary from the peptide.
© 2003 Elsevier Ltd. All rights reserved.
Peptide thioesters are versatile synthetic intermediates
that are useful in polypeptide synthesis.1 For example,
native chemical ligation using peptide thioesters as
building blocks is a very useful and convenient method
for polypeptide synthesis.2 This can be performed under
neutral conditions in aqueous solutions without the
need for protecting groups, although a cysteine residue
is required at the condensation site. Recently several
groups have introduced auxiliaries for the ligation of
peptides that permit the condensation without the need
for a cysteine residue at the condensation site. Three
scaffolds of removable auxiliaries have been reported to
date: 2-mercaptoethoxy group,3 methoxy groups-
attached 2-mercaptobenzyl4 and 1-phenyl-2-mercapto-
ethyl5 groups. Since the conditions required for their
removal frequently cause the adsorption of the peptides
to zinc powder or their degradation under the harsh
acidic conditions used, we initiated an investigation of a
new auxiliary, that can be removed under mild condi-
tions.
1-(2-Nitrophenyl)-2-(triphenylmethylthio)ethylamine (1)
was prepared as shown in Scheme 1. Methyl t-butoxy-
carbonyloxy-2%-nitrophenylglycinate (3) was prepared
from 2-nitrobenzaldehyde (2) via a Strecker type amino
acid synthesis,7 and a transformation similar to a
reported literature procedure8 gave the amine 1.9
Scheme 2 shows a model peptide synthesis using the
Na-2-mercapto-1-(2-nitrophenyl)ethyl (Mnpe) auxiliary.
The Na-glyoxyloyl peptide, CHOCO-Ser-Arg-Ala-His-
Ser-Ser-His-Leu-Lys-NH2 (5), was prepared by the perio-
date oxidation of a peptide, Ser-Ser-Arg-Ala-His-
Ser-Ser-His-Leu-Lys-NH2 (4).4a This peptide was
treated with amine 1 and sodium cyanoborohydride in
DMF containing acetic acid (Fig. 1A), followed by
treatment with trifluoroacetic acid (TFA) containing
triisopropylsilane to give the Na-Mnpe peptide 7.10
We previously reported the 4,5-dimethoxy-2-mercapto-
benzyl group, an auxiliary that can be removed by acid
treatment.4a An o-nitro group on benzyl group confers
photolability,6 but its introduction on thiophenol scaf-
folds decreases its nucleophilicity, thus reducing
thioester exchange in the first step of the chemical
ligation reaction. Herein, we describe the use of the
2-mercapto-1-phenylethyl group as a scaffold.
* Corresponding author. Tel: +81-6-6879-8601; fax: +81-6-6879-8603;
e-mail: aimoto@protein.osaka-u.ac.jp
Scheme 1. Synthesis of auxiliary molecule 1.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01463-1

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T. Kawakami, S. Aimoto / Tetrahedron Letters 44 (2003) 6059–6061
Scheme 2. 2-Mercapto-1-(2-nitrophenyl)ethylamine (Mnpe-
amine)-assisted ligation of
a peptide with the peptide
thioester.
Peptide 7 was then ligated to a peptide thioester, Lys-
Asp-Ala-Gln-Ala-Gly-Lys-Glu-Pro-Gly-SCH2CH2CO-
Ala (8), in sodium phosphate buffer (0.10 M, pH 7.4)
containing 50 mM thiophenol.11 After stirring for 24 h,
dithiothreitol (DTT) was added to the reaction mixture,
and the ligation product, Lys-Asp-Ala-Gln-Ala-Gly-
Lys-Glu-Pro-Gly-(Mnpe)Gly-Ser-Arg-Ala-His-Ser-Ser-
His-Leu-Lys-NH2 (9),12 was purified by RP-HPLC
(Fig. 1B). The Mnpe group was removed by UV irradi-
ation at 365 nm in sodium phosphate buffer (pH 7.4)
for 6 h to give Lys-Asp-Ala-Gln-Ala-Gly-Lys-Glu-Pro-
Gly-Gly-Ser-Arg-Ala-His-Ser-Ser-His-Leu-Lys-NH2
(10) (Fig. 1C).4a,13
Figure 1. RP-HPLC elution profiles of the reaction mixtures.
(A) Reductive amination product 6 (24 h). (B) Ligation
product 9 (24 h-reaction followed by the addition of DTT).
Since the Mnpe moiety has chirality, the diastereomers 9 were
separated as split peaks by HPLC. An arrow, 11, indicates a
hydrolysis product of peptide thioester 8 and oxidized DTT.
(C) Removal of Mnpe group (6 h). Column: YMC-Pack Pro
C18 (4.6×150 mm), eluent: 0.1% TFA in aq. acetonitrile, 1.0
mL/min.
serine residue, followed by reductive amination with the
Mnpe amine derivative 1. After ligation with a peptide
thioester the Mnpe group can be removed under mild
conditions by UV irradiation to give a native peptide
bond.
In conclusion, the Mnpe group can be successfully used
as a chemical ligation auxiliary for peptide synthesis.
The Na-Mnpe-attached peptide, for condensation with
the peptide thioesters, can be prepared from a non-pro-
tected peptide via periodate oxidation of the N-terminal

T. Kawakami, S. Aimoto / Tetrahedron Letters 44 (2003) 6059–6061
6061
Acknowledgements
6. Pillai, V. N. R. Synthesis 1980, 1–26.
7. Ma, D.; Wang, G.; Wang, S.; Kozikowski, A. P.; Lewin,
N. E.; Blumberg, P. M. Bioorg. Med. Chem. Lett. 1999, 9,
1371–1374.
8. Marinzi, C.; Bark, S. J.; Offer, J.; Dawson, P. E. Bioorg.
Med. Chem. 2001, 9, 2323–2328.
This research was supported, in part, by Grants-in-Aid
for Scientific Research (15750143 and 14380287) from
the Ministry of Education, Culture, Sports, Sciences
and Technology, Japan.
9. 1 (as a TFA salt): 1H NMR (400 MHz, CDCl3) l 3.01
(dd, J=8.0, 14.0 Hz, 1H), 3.15 (dd, J=7.3, 14.0 Hz, 1H),
3.62 (t, J=7.5 Hz, 1H), 7.18–7.28 (m, 9H), 7.34–7.39 (m,
6H), 7.46–7.50 (m, 2H), 7.56–7.61 (m, 1H), 7.84–7.88 (m,
1H); 13C NMR (100 MHz, CDCl3) l 32.8, 49.7, 67.9,
125.3, 127.2, 128.3, 128.9, 129.3, 129.6, 130.6, 134.2,
143.7, 149.3.
References
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10. 6 (50% yield in the 10 mmol-scale reaction, in which 5
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13. 10 (37% yield after RP-HPLC purification): MS
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