836
D. A. Jeyaraj, H. Waldmann / Tetrahedron Letters 42 (2001) 835–837
1) PhAcOZ - AA2 - OH
H - AA1 - Ser - Ala - OAll
H - AA2 - AA1 - Ser - Ala - OAll
HATU, DMF, 1h, rt
NHAloc
N
O
O P O
O
85 - 91 % (7 a, b)
NHAloc
N
O
O P O
O
penicillin G acylase
5 a-c
N
O
0.05M phosphate buffer,
MeOH (20 vol %), 0.1M
KI, pH 6.8, 24 h, rt
O
N
O
2) penicillin G acylase,
0.05M phosphate buffer,
MeOH (20 vol %), 0.1M
KI, pH 6.8, 24 h, rt
O
OAc
OAc
6 a: AA1 = Val 60 %
b: AA1 = Phe 69 %
c: AA1 = Pro 65 %
8 a: AA2 - AA1 = Pro - Val 42 %
b: AA2 - AA1 = Ala - Phe 40 %
Scheme 2. Selective penicillin G acylase catalyzed removal of the N-terminal PhAcOZ urethane from nucleotri- and tetrapeptides.
demands of nucleopeptide chemistry concerning
chemoselectivity and mildness of cleavage conditions.
The PhAcOZ group is an enzyme-labile urethane that is
cleaved under very mild conditions by penicillin G
acylase-mediated hydrolysis of the phenylacetic acid
phenyl ester and subsequent fragmentation of the gen-
erated phenolate to give a quinone methide (which is
trapped by nucleophiles) and the desired unmasked
amino acid ester. In order to determine if the conditions
for the selective removal of this enzyme-sensitive block-
ing group are mild enough to allow for an efficient
nucleopeptide synthesis model nucleotripeptides were
synthesized. To this end, PhAcOZ protected tripeptides
1 were synthesized according to the literature7 from
PhAcOZ-masked amino acids and H-Ser-Ala-OAll by
means of established peptide coupling procedures. The
central serine of tripeptides 1 was then coupled to
selectively masked deoxycytidine 48 via a phosphodi-
ester bond (Scheme 1). Upon treatment of 1 with
phosphordiamidite 2 in the presence of tetrazole,
hydrolysis-sensitive intermediates 3 were formed. They
were converted without isolation into the desired pep-
tide conjugates 59 by reaction with deoxycytidine build-
ing block 4 and subsequent oxidation of the formed
phosphites to the corresponding phosphates.
In all enzyme-catalyzed deprotections undesired side
reactions did not occur. Thus, the high selectivity of
penicillin G acylase for the phenylacetic acid group
guarantees that the peptide bonds, the allyl ester, the
allyl urethane, the acetate protecting group and the
phosphate remain fully intact. The conditions of the
enzymatic transformation are so mild that the glyco-
sidic bond is not affected and that a b-elimination of
the phosphate (which occurs at pH>7) is not observed
at all. These findings prove that the PhAcOZ group is
an efficient protecting function for the selective synthe-
sis of sensitive and multifunctional nucleopeptides.
Acknowledgements
This research was supported by the Fonds der Che-
mischen Industrie and Dr. D. A. Jeyaraj is grateful to
the Alexander von Humboldt foundation (AvH) for a
fellowship.
References
Completely masked nucleotripeptides 5 were then sub-
jected to selective enzymatic deprotection with peni-
cillin G acylase. To this end the PhAcOZ protected
compounds were treated with the enzyme in a mixture
of 0.05 M phosphate buffer and methanol (20 vol%) at
pH 6.8 and room temperature and in the presence of
0.1 M KI as a trapping reagent for the quinone methide
formed in the enzyme-initiated cleavage of the ure-
thanes. Under these conditions the enzyme smoothly
cleaved the phenylacetic acid and the selectively
unmasked nucleotripeptides 6 were formed in high yield
(Scheme 2). In order to assure that this method is
widely applicable, the peptide chain of compounds 6
was elongated with a further PhAcOZ-protected amino
acid to yield nucleotetrapeptides 7 in high yields. Once
more treatment of these peptide conjugates with peni-
cillin G acylase under the conditions described above
resulted in a smooth and clean deprotection of the
N-terminus to yield selectively deprotected nucleotetra-
peptides 8.9
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