Synthesis of nucleopeptides by an enzyme labile urethane protecting group

Article abstract of DOI:10.1016/S0040-4039(00)02121-3

The synthesis of acid- and base-labile nucleopeptides is accomplished by employing the enzyme labile phenylacetoxy benzyloxycarbonyl (PhAcOZ) urethane protecting group as the key technique.

Full text of DOI:10.1016/S0040-4039(00)02121-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 835–837
Synthesis of nucleopeptides by an enzyme labile urethane
protecting group
Duraiswamy A. Jeyaraj and Herbert Waldmann*
Max-Planck-Institut fu¨r molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Str. 11,
D-44227 Dortmund, Germany and Universita¨t Dortmund, Fachbereich 3, Organische Chemie
Received 24 October 2000; accepted 16 November 2000
Abstract—The synthesis of acid- and base-labile nucleopeptides is accomplished by employing the enzyme labile phenylacetoxy
benzyloxycarbonyl (PhAcOZ) urethane protecting group as the key technique. © 2001 Elsevier Science Ltd. All rights reserved.
Nucleoproteins are naturally occurring biopolymers in
which the hydroxy group of a serine, a threonine or a
tyrosine is linked via a phosphodiester group to the 3%-
or 5%-end of a nucleic acid.1 These protein conjugates
play decisive roles in important biological processes like
viral replication.2 For the study of the biological phe-
nomena in which nucleoproteins are involved, nucleo-
peptides embodying the characteristic linkage between
the peptide chain and the oligonucleotide may serve as
powerful tools. However, due to the multifunctionality
of nucleopeptides their synthesis requires the applica-
tion of a variety of orthogonally stable blocking
groups. In addition, fully protected serine/threonine
nucleopeptides are very acid- and base-labile making
most of the established blocking function not applicable
to nucleopeptide synthesis. Thus, it is not surprising
that only a few reports on the successful synthesis of
nucleopeptides have appeared.3–6 In particular, protect-
ing groups allowing for the assembly of sensitive serine/
threonine nucleopeptides via sequential selective
N-terminal deprotection and chain elongation have not
been developed yet. In this paper we report that the
enzyme-labile
phenylacetoxy
benzyloxycarbonyl
(PhAcOZ) group7 (Scheme 1) fulfills the extraordinary
O
(i-Pr)2N
PhAcOZ - AA1 - Ser - Ala - OAll
O
P OAll
Ph
O
AA1 - Ser - Ala - OAll
(i-Pr)2N
O
N
H
O
P
2
N
O
tetrazole, i-Pr2NH
CH2Cl2, 0° C, 1h
1
PhAcOZ
3
O
1)
HN
N
O
O
PhAcOZ - AA1 - Ser - Ala - OAll
N
NHAloc
HO
O
O P O
O
O
tetrazole,
CH3CN, CH2Cl2
(1 : 1), rt, 24h
N
5 a: AA1 = Val 43 %
b: AA1 = Phe 58 %
c: AA1 = Pro 44 %
4
N
O
O
OAc
2) t-BuOOH, 10 min., 0° C
OAc
Scheme 1. Synthesis of model nucleopeptides 5.
Keywords: nucleopeptides; enzyme labile protecting group; PGA; enzymatic cleavage.
* Corresponding author. E-mail: herbert.waldmann@mpi-dortmund.mpg.de
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02121-3

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
1. (a) Juodka, B. A. Nucleosides & Nucleotides 1984, 3,
445–483; (b) Wang, J. C. Ann. Rev. Biochem. 1985, 54,
665–697; (c) Vartapetian, A. B.; Bogdanov, A. A. Prog.
Nucleic Acid Res. Mol. Biol. 1987, 34, 209–251; (d) Kelly,
T. J.; Wold, M. S.; Li, J. Adv. Virus Res. 1988, 34, 1–42;
(e) M. Salas In The Viruses, Vol. 1 The Bacteriophages;
R. Calender, Ed.
2. (a) Review: Salas, M. Ann. Rev. Biochem. 1991, 160,
39–71; (b) Review: Salas, M. Virus Strategies–Molecular
Biology and Pathogenesis, VCH, Weinheim, 1993, pp. 1–
23; (c) Blanco, L.; La´zaro, J. M.; de Vega, M.; Bonnin, A.;
Salas, M. Proc. Natl. Acad. Sci. 1994, 91, 12198.
3. (a) Juodka, B. A.; Kirveliene, V.; Povilionis, P. Nucleosides
& Nucleotides 1982, 1, 497–508; (b) Kuyl-Yeheskiely, E.;
van der Klein, P. A. M.; Visser, G. M.; van der Marel, G.
A.; van Boom, J. H. Recl. Trav. Chim. Pay-Bas 1986, 105,
69–70; (c) Kuyl-Yeheskiely, E.; Tromp, C. M.; Lefeber, A.
W. M.; van der Marel, G. A.; van Boom, J. H. Tetra-
hedron 1988, 44, 6515–6523; (d) Kuyl-Yeheskiely, E.;
Dreef-Tromp, C. M.; Geluk, A.; van der Marel, G. A.; van

D. A. Jeyaraj, H. Waldmann / Tetrahedron Letters 42 (2001) 835–837
837
Boom, J. H. Nucleic Acids Res. 1989, 17, 2897–2905; (e)
Dreef-Tromp, C. M.; van der Marel, J. C. M.; van den
Elst, H.; van der Marel, G. A.; van Boom, J. H. Nucleic
Acids Res. 1992, 20, 4015–4020.
ether by treatment with TBAF (S. Gabold, Dissertation,
Univ. Karlsruhe 1998).
9. Characteristic data of compound 5b: H NMR (400 MHz,
1
CD3OD): l (ppm) 8.40–8.60 (bs, 1H); 7.66–7.81 (bs, 1H);
7.13–7.43 (m, 13H); 6.99–7.01 (d, J=7.52 Hz, 2H); 6.12–
6.26 (m, 1H); 5.72–5.99 (m, 4H); 5.17–5.45 (m, 7H); 4.2–
5.06 (m, 16H); 3.85 (s, 2H); 3.55–3.70 (m, 1H); 3.35–3.50
(m, 1H); 3.15–3.25 (m, 1H); 2.90–3.02 (m, 1H); 2.64–2.78
(m, 1H); 2.30–2.50 (m, 1H); 2.06 (s, 3H); 1.44 (d, J=7.28
Hz, 3H). [a]2D2=+8.0 (c 0.35, MeOH). ESI-MS: Calc.
1087.04. Found 1087.1 (100%, positive mode). 8b: 1H
NMR (400 MHz, CD3OD): l (ppm) 8.18–8.20 and 8.17–
8.18 (two d, J=7.52 Hz, 1H); 7.32–7.34 and 7.31–7.33
(two d, J=7.52 Hz, 1H); 7.20–7.27 (m, 5H); 6.18–6.22 (m,
1H); 5.87–6.03 (m, 3H); 5.20–5.43 (m, 7H); 4.56–4.70 (m,
8H); 4.23–4.46 (m, 6H); 3.83–3.87 (m, 1H); 3.20–3.21 and
3.16–3.18 (two d, J=5.52 Hz, 1H); 2.92–3.00 (m, 1H);
2.62–2.71 (m, 1H); 2.25–2.37 (m, 1H); 2.11 (s, 3H); 1.47–
1.48 (d, J=7.04 Hz, 3H); 1.40–1.42 (d, J=7.28 Hz, 3H).
ESI-MS: Calc. 889.84. Found 890.2 (100%, positive mode).
4. (a) Robles, J.; Pedroso, E.; Grandas, A. Tetrahedron Lett.
1994, 35, 4449–4452; (b) Robles, J.; Pedroso, E.; Grandas,
A. J. Org. Chem. 1994, 59, 2482–2486; (c) Robles, J.;
Beltran, M.; Marchan, V.; Perez, Y.; Travesset, I.;
Pedroso, E.; Grandas, A. Tetrahedron 1999, 55, 13252–
13264 and references cited therein.
5. Flohr, S.; Jungmann, V.; Waldmann, H. Chem. Eur. J.
1999, 5, 669–681 and references cited therein.
6. Ueno, Y.; Saito, R.; Hata, T. Nucleic Acids Res. 1993, 21,
4451–4457.
7. Pohl, T.; Waldmann, H. J. Am. Chem. Soc. 1997, 119,
6702–6710.
8. Compound 4 was synthesized from 2-deoxycytidine via
introduction of the Aloc group into the nucleobase, mask-
ing of the 5%-OH group as TBDMS ether, acetylation of
the 3%-OH group and selective removal of the TBDMS
.
.