Peptide bond formation mediated by 4,5-dimethoxy-2-mercaptobenzylamine after periodate oxidation of the N-terminal serine residue.

Article abstract of DOI:10.1021/ol0157813

[reaction in text] A thiol linker-attached peptide was prepared from a nonprotected peptide via an N(alpha)()-alpha-oxoacyl peptide. Selective oxidation of the N-terminal serine with sodium periodate gave the N(alpha)-glyoxyloyl peptide, reductive amination of which with 4,5-dimethoxy-2-(triphenylmethylthio)benzylamine gave an N(alpha)-4,5-dimethoxy-2-mercaptobenzyl glycyl peptide after removal of the trityl group. The N(alpha)-4,5-dimethoxy-2-mercaptobenzyl peptide can be condensed with a peptide thioester, and the linker is removable. This strategy provides a useful method for the synthesis of peptides using recombinant proteins.

Full text of DOI:10.1021/ol0157813

ORGANIC
LETTERS
2001
Vol. 3, No. 9
1403-1405
Peptide Bond Formation Mediated by
4,5-Dimethoxy-2-mercaptobenzylamine
after Periodate Oxidation of the
N-Terminal Serine Residue
Toru Kawakami, Kenichi Akaji, and Saburo Aimoto*
Institute for Protein Research, Osaka UniVersity, 3-2 Yamadaoka,
Suita, Osaka 565-0871, Japan
aimoto@protein.osaka-u.ac.jp
Received March 2, 2001
ABSTRACT
r
A thiol linker-attached peptide was prepared from a nonprotected peptide via an N -r-oxoacyl peptide. Selective oxidation of the N-terminal
r
serine with sodium periodate gave the N -glyoxyloyl peptide, reductive amination of which with 4,5-dimethoxy-2-(triphenylmethylthio)benzylamine
r
r
gave an N -4,5-dimethoxy-2-mercaptobenzyl glycyl peptide after removal of the trityl group. The N -4,5-dimethoxy-2-mercaptobenzyl peptide
can be condensed with a peptide thioester, and the linker is removable. This strategy provides a useful method for the synthesis of peptides
using recombinant proteins.
We wish to report herein a new strategy for the synthesis of
polypeptides using recombinant proteins (nonprotected pep-
tides) in combination with S-alkyl peptide thioesters (peptide
thioesters)1,2 for use as building blocks which do not contain
protecting groups.
tion as the result of the reaction of peptide hydrazide and
NR-glyoxyloyl peptides, which were prepared from recom-
binant proteins.4 Although these reactions were observed to
proceed chemoselectively, they do not give rise to a native
peptide bond. On the other hand, Dawson et al. reported on
a chemoselective reaction of peptide thioesters and peptides,
which have a cysteine residue at the N-terminus, which
results in the formation of a native peptide bond.5 This
peptide bond formation via an S-N acyl shift during the
reaction of the thioester and cysteine had been previously
reported by Wieland et al.6 Thiol-containing linkers, such
as 2-mercaptoethoxy,7 2-mercaptobenzyl,8 and 1-phenyl-2-
Semisynthesis using biologically prepared peptides has
been investigated via chemical ligation methods.3 For
example, Gaertner et al. reported on hydrazone bond forma-
(1) Aimoto, S. Biopolymers (Pept. Sci.) 1999, 51, 247-265.
(2) (a) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117.
(b) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.; Tanaka, I.; Hikichi, K.;
Aimoto, S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-2706. (c) Kawakami,
T.; Kogure, S.; Aimoto, S. Bull. Chem. Soc. Jpn 1996, 69, 3331-3338. (d)
Kawakami, T.; Yoshimura, S.; Aimoto, S. Tetrahedron Lett. 1998, 39,
7901-7904. (e) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.;
Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc.
1999, 121, 284-290. (f) Kawakami, T.; Hasegawa, K.; Aimoto, S. Bull.
Chem. Soc. Jpn. 2000, 73, 197-203.
(4) Gaertner, H. F.; Rose, K.; Cotton, R.; Timms, D.; Camble, R.; Offerd,
R. E. Bioconjugate Chem. 1992, 3, 262-268.
(5) (a) Dawson, P. E.; Muir, T. M.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779. (b) Muir, T. M.; Dawson, P. E.; Kent, S. B. H.
Methods Enzymol. 1997, 289, 266-298.
(3) Wallace, C. J. A. Protein Engineering by Semisynthesis; Wallace,
C. J. A., Ed.; CRC Press: Boca Raton, 2000; pp 45-71.
(6) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs
Ann. Chem. 1953, 583, 129-149.
10.1021/ol0157813 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/06/2001

Scheme 1. Strategy for the Preparation of Thiol Linker-Attached Peptides as C-Terminal Building Blocks from Free Peptides
mercaptoethyl9 groups on the terminal amino group, can be
used to replace the cysteine residue. These methods are quite
convenient for polypeptide synthesis, because protecting
groups are not required and the reaction can be carried out
in neutral aqueous solutions. Furthermore, biologically
prepared peptides have been used in polypeptide synthesis
using the native chemical ligation method.10,11 An expressed
peptide was also used as a C-terminal building block for
polypeptide synthesis, in which a partially protected peptide
segment was condensed with a peptide thioester in the
presence of silver ions.12
producing an NR-glyoxyloyl moiety at the N-terminus
(Scheme 2).14,15 This peptide was treated with 4,5-dimethoxy-
2-(triphenylmethylthio)benzylamine (8)16 and sodium cy-
anoborohydride in DMF containing acetic acid (Figure 1A),
Scheme 2. 4,5-Dimethoxy-2-mercaptobenzylamine-Mediated
Condensation of Peptide with Peptide Thioester
Here we present an alternative method for the use of
expressed peptides in the peptide bond formation at Xxx-
Gly sequences. In our method, peptide building block 2 are
prepared from recombinant proteins (nonprotected peptide
1), which have a serine residue at the N-terminus, by the
following steps (Scheme 1): first, the N-terminal serine or
threonine residue of free peptide 1 is oxidized with periodate
to give NR-glyoxyloyl peptide 3, then thiol-containing amine
derivative 4 is introduced to give thiol-liker attached glycyl
peptide 2 via peptide 5. Peptide 2 would undergo condensa-
tion with a peptide thioester which contains no protecting
groups. In this study, we chose the 4,5-dimethoxy-2-
mercaptobenzyl (Dmmb) group as a novel linker, because
this group can be readily removed under acidic conditions.
A model sequence, Gly-Ser-Arg-Ala-His-Ser-Ser-His-
Leu-Lys, was examined as a C-terminal segment. The NR-
glyoxyloyl peptide, CHOCO-Ser-Arg-Ala-His-Ser-Ser-
His-Leu-Lys-NH2 (7),13 was obtained by the periodate
oxidation of a peptide, Ser-Ser-Arg-Ala-His-Ser-Ser-His-
Leu-Lys-NH2 (6), which is a convenient procedure for
(7) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
118, 5891-5896.
(8) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23-26.
(9) Botti, P.; Carrasco, M. R.; Kent, S. B. H. Tetrahedron Lett. 2001,
42, 1831-1833.
(10) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705-6710.
(11) Evans, T. C., Jr.; Benner, J.; Xu, M.-Q. Protein Sci. 1998, 7, 2256-
2264.
(12) Kawakami, T.; Hasegawa, K.; Teruya, K.; Akaji, K.; Horiuchi, M.;
Inagaki, F.; Kurihara, Y.; Uesugi, S.; Aimoto, S. Tetrahedron Lett. 2000,
41, 2625-2628.
(13) Sodium periodate (6.9 mg, 32 µmol) was added to a solution of
peptide 6 (20 mg, 11 µmol) in 50 mM sodium phosphate buffer (pH 7.2,
1.0 mL). After stirring for 10 min, Ser (34 mg) was added, and product 7
was then purified by RP-HPLC [Cosmosil 5C18AR-II (4.6 × 250 mm),
0.1% TFA in aqueous acetonitrile] (11 mg, 7.1 µmol, 65%): MS (MALDI-
TOF) found 1077.6 (MH+), calcd 1077.6. Amino acid analysis: Ser2.6
-
Ala0.91Leu1Lys0.94His1.8Arg0.92
.
1404
Org. Lett., Vol. 3, No. 9, 2001

(Figure 1B). After stirring for 24 h, dithiothreitol was added
to the reaction mixture, and the product, Lys-Asp-Ala-Gln-
Ala-Gly-Lys-Glu-Pro-Gly-(Dmmb)Gly-Ser-Arg-Ala-His-Ser-
Ser-His-Leu-Lys-NH2 (12),20 was purified by RP-HPLC. The
Dmmb group was removed by treatment with trifluo-
romethanesufonic acid (TFMSA) in TFA to give Lys-Asp-
Ala-Gln-Ala-Gly-Lys-Glu-Pro-Gly-Gly-Ser-Arg-Ala-His-
Ser-Ser-His-Leu-Lys-NH2 (13).21
The condensation of NR-2-mercaptobenzyl peptides with
peptide thioesters has been examined by Offer and Dawson.8
The N-2-mercaptobenzyl group on the backbone of peptide
is too stable at acidic conditions even under conditions of
hydrogen fluoride treatment.8 The introduction of two
methoxy groups on the benzene ring permitted the Dmmb
group to be removed by treatment with 1 M TFMSA in TFA.
In conclusion, we reported the successful preparation of
a thiol linker-attached peptide, for condensation with the
peptide thioesters, from a nonprotected peptide via periodate
oxidation of the N-terminal serine residue, and reductive
amination with the Dmmb amine derivative. Transamination
reaction of N-terminal amino groups would also be used
instead of the periodate oxidation of serine and threonine
residues, in principle, though the stereochemistry, formed
in the reductive amination, should be controlled. After the
condensation with a peptide thioester, the thiol linker can
be removed by treatment with an acid, such as TFMSA.
Figure 1. RP-HPLC elution profiles of the reaction mixtures. (A)
Reductive amination product 9. (B) Condensation product 12. An
arrow, 14, indicates thiophenyl ester derived from peptide 11.
Column: Cosmosil 5C18AR-II (4.6 × 150 mm), eluent 0.1% TFA
in aqueous acetonitrile, 1.0 mL/min.
Acknowledgment. This research was supported, in part,
by a Grant-in-Aid for Scientific Research (10179103 and
12780440) from the Ministry of Education, Science, Sports
and Culture, Japan.
OL0157813
followed by trifluoroacetic acid (TFA) containing triisopro-
pylsilane to give the NR-Dmmb peptide 10.17
(18) Starting from Boc-Gly-SCH2CH2CO-Leu-OCH2-Pam resin,2 the
peptide chain was elongated by means of a peptide synthesizer 430A
(Applied Biosystems) using Boc chemistry to give Boc-Lys(Cl-Z)-Asp-
(OcHex)-Ala-Gln-Ala-Gly-Lys(Cl-Z)-Glu(OBzl)-Pro-Gly-SCH2CH2CO-
Leu-OCH2-Pam resin. This resin was treated with hydrogen fluoride
containing anisole (10%) on an ice bath for 1.5 h. After evaporation of the
hydrogen fluoride, the mixture was washed with ether and freeze-dried.
The peptide, 11, was purified by RP-HPLC [Cosmosil 5C18AR-II (10 ×
250 mm), 0.1% TFA in aqueous acetonitrile]: MS (MALDI-TOF) found
Peptide 10 was condensed with a peptide thioester, Lys-
Asp-Ala-Gln-Ala-Gly-Lys-Glu-Pro-Gly-SCH2CH2CO-Leu-
OH (11),18 in neutral phosphate buffer (pH 7.2) containing
6 M guanidine hydrochloride and 0.20 M thiophenol19
(14) Dixon, H. B. F. J. Protein Chem. 1984, 3, 99-108.
1160.0 (MH+), calcd 1159.5. Amino acid analysis: Asp0.99Glu2.0Pro0.78
Gly2.1Ala3Lys2.0
-
(15) Rose, K.; Chen, J.; Dragovic, M.; Zeng, W.; Jeannerat, D.;
Kamalaprija, P.; Burger, U. Bioconjugate Chem. 1999, 10, 1038-1043.
(16) Amine 8 was prepared by the reaction of 4,5-dimethoxy-2-
mercaptobenzylamine and triphenylmethanol in TFA: 55% yield; 1H NMR
(400 MHz, CDCl3) δ 3.36 (s, 3H), 3.62 (s, 2H), 3.85 (s, 3H), 6.70 (s, 1H),
6.91 (s, 1H), 7.19-7.31 (m, 15H); 13C NMR (100 MHz, CDCl3) δ 41.18,
55.58, 55.98, 71.37, 113.22, 118.77, 124.13, 127.33, 127.96, 130.03, 131.29,
143.66, 149.09, 150.71; MS (FAB) 442 (MH+), calcd 442.
.
(19) Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. J.
Am. Chem. Soc. 1997, 119, 4325-4329.
(20) Peptides 10 (3.0 mg, 1.9 µmol) and 11 (4.2 mg, 2.8 µmol) were
dissolved in 50 mM sodium phosphate buffer (pH 7.2, 0.95 mL) containing
6 M guanidine hydrochloride in the presence of thiophenol (20 µL). After
stirring for 25 h, dithiothreitol (30 mg) was added, the mixture was stirred
for an additional 1 h, and product 12 was purified by RP-HPLC [Cosmosil
5C18AR-II (4.6 × 250 mm), 0.1% TFA in aqueous acetonitrile] (5.2 mg,
1.6 µmol, 85%): MS (MALDI-TOF) found 2243.7 (MH+), calcd 2243.5
(average). Amino acid analysis: Asp1.1Ser2.5Glu2.2Pro0.98Gly2.3Ala3.2Leu1-
(17) A solution of sodium cyanoborohydride (1.6 mg, 25 µmol) in DMF
(0.10 mL) was added to a solution of peptide 7 (7.2 mg, 5.0 µmol) and
amine 8 (22 mg, 50 µmol) in DMF (0.90 mL) containing acetic acid (8.0
µL). After stirring for 24 h, product 9 was purified by RP-HPLC [Cosmosil
5C18AR-II (4.6 × 250 mm), 0.1% TFA in aq acetonitrile] (3.7 mg, 1.9
µmol, 38%): MS (MALDI-TOF) found 1260.4 [(M - Trt + H)+], calcd
Lys3.3His1.8Arg0.94
.
(21) Peptide 12 (5.2 mg, 1.6 µmol) was treated with 1 M TFMSA and
1 M thioanisole in TFA (0.20 mL) on an ice bath for 1 h, and the mixture
was then washed with ether and freeze-dried. The product, 13, was purified
by RP-HPLC [Cosmosil 5C18AR-II (4.6 × 250 mm), 0.1% TFA in aqueous
acetonitrile] (1.6 mg, 0.73 µmol, 45%): MS (MALDI-TOF) found 2061.1
1260.6. Amino acid analysis: Ser2.5GlyndAla1.1Leu1Lys0.89His1.8Arg0.87
.
Peptide 9 was treated with TFA in the presence of triisopropylsilane (5%)
for 0.5 h and then washed with ether and dried in vacuo. The residue was
used for condensation with peptide 10: MS (MALDI-TOF) found 1260.9
(MH+), calcd 1260.6.
(MH+), calcd 2061.3 (average). Amino acid analysis: Asp0.99Ser2.7Glu2.1
-
Pro1.2Gly2.9Ala2.9Leu1Lys2.9His1.9Arg0.99
.
Org. Lett., Vol. 3, No. 9, 2001
1405