Novel Chemoenzymatic Synthesis of Peptide C-Terminal Amides from Ester Precursors

Article abstract of DOI:10.1016/0040-4039(93)88038-K

A novel two step chemoenzymatic procedure for the preparation of peptide C-terminal amides has been developed.

Full text of DOI:10.1016/0040-4039(93)88038-K

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Vol. 33. No.
Britain
PP.
in
Novel Chemoenzymatic Synthesis of
Ester Precursors
C-Terminal Amides from
Jeremy Green end Aiexey
Dow
21 E.
Rd.,
OH 45215
A novel IWO
developed.
the preparation of
C-terminal
has been
Recent advances in multiple
very large
synthesist make
libraries. It is due to this progress, that in the nearest future we may see a dramatic in&ease in
such as neuropeptide hormones.2 fact that almost half of ail
the simultaneous preparation and analysis of
the number of
known neuropeptides are C-terminal amides demonstrates the importance of the carboxamido
biological activity of these
for full
The preparation of substantial amounts of these
can be better achieved by solution-, solid-phase
Each of these methods has its own advantages and limitations. in
synthesis. or enzymatic techniques.
phase
amide intermediates in
synthesis, the preparation of
C-terminal amides often suffers due to the poor
used organic solvents. This can be use of C-terminal esters and
at the conclusion of the synthesis. However, this reaction is often slow, low yielding,
selectivity when other esters (eg. benzyl) are present in the Solid phase
the preparation of amides but the high cost of the required resins restricts the adaptability
on a large scale.
of
ammonoiysis with
prone to
and
synthesis
of this technique
can also be achieved by enzyme-catalyzed
These reactions
of
C-terminal Gly residues, transpeptidation or
precursors, the
sequences of which are either one amino acid shorter7 or contain an alternative C-terminal amino acid to the desired
final product.*
Here we report a novel two step
of desired First, regioselective enzyme-mediated coupling of the corresponding
DmbNH2
method for the preparation of C-terminal amides
the
ester to
is carried out in aqueous-organic solvent mixtures or in organic solvents using papain or
subtiiisin. Second, the resulting product is treated with
terminal amide (see Figure).
acid to give a fully deprotected
The resulting peptida di- and tri-methoxylwnzylamides were isolated in good yield and high purity after a
simple work-up In addition, papain-catalyzed reactions were fast and complete in less than 2 h. It is
worth mentioning that different esters, such as methyl, and plienacyl were successfully
To the versatility of this technique, which, in addition to
the benzylamide moiety, contains three of the most widely used protecting groups was prepared on a large scale
a
(1.1 g). This compound was treated with
in quantitative
to give the desired fully deprotected C-terminal amide
7759

Pg, -AA, ‘AA, -OR +
H-AA, . ‘AA, -NH,
ester
Enzyme
T i e
20 min
65 min
60 min
2h
Yield
76
69
63
87
Papain
Papain
Papain
Subtilisin
24h
64
a. Reaction was carried out in
alcohol with DmbNH2 (3 eq.)
References sad Notes
Jung, G.; Beck-Sickinger, A.
Eur. Papt. Symp. (Eds.
Ed.
1992.31,
D. Andreu), ESCOM,
1.
2.
3.
4.
1991,
B. A.; Mains, R.E.
Rev.
L.
1988, SO, 333-344.
1992,
G;
Katopodis, A.G.; Ping, D.;
Trends Biochem. Sci. 1991,
S.W.
114.
D.G.
CRC Press,
FL, 1987. Schellenberg,
Jakubke,
Chem.,
Ed.
1991.30, 1437-1449.
Margolin,
D-F.; Klibanov, A.M. Am. Chem.
1987,
F.;
M.
Prof. Res., 1991.37, 153-160. Henriksen, D.B.;
Am. Chem. 1992, 114, 1876-1877.
J.; Buchard,
In a typical experiment protected
6-8 0.2 M carbonate buffer
followed by papain (S-10 mg) and the reaction stirred at RT
ester (80 mg) and
containing 40%
hydrochloride (3eq.) are suspended in
is added
min). Water is added to complete
precipitation of
Nonstandard abbreviations: Dmb.
which is filtered, washed with water, and dried.
TFA,
acid; Pg,
All compounds were characterized by TLC, HPLC,
(1 was stirred in TFA (21
overnight at RT. Solvent was evaporated and residue taken up in H20, washed with Et20 and
group;
amino acid,
NMR and MS.
thioanisole (5.9
11.
12.
and H20 (2.1
In addition to standard analyses (Ref. 10)
was characterized by FAB MS and high
resolution MS.
(Received in USA 27 August 1992; accepted 21 September 1992)