Chemoenzymatic synthesis of nucleopeptides

Article abstract of DOI:10.1039/a704215i

Acid- and base-labile multifunctional nucleopeptides have been selectively constructed under mild conditions by means of enzymatic protecting group techniques.

Full text of DOI:10.1039/a704215i

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Chemoenzymatic synthesis of nucleopeptides
Herbert Waldmann* and Stefanie Gabold
Universita¨t Karlsruhe, Institut fu¨r Organische Chemie, Richard-Willsta¨tter-Allee 2, D-76128 Karlsruhe, Germany
Acid- and base-labile multifunctional nucleopeptides have
been selectively constructed under mild conditions by means
of enzymatic protecting group techniques.
in 2 were cleaved off without undesired side reaction by Pd0-
mediated allyl transfer to PhSiH3 as accepting nucleophile9 at
room temp. in 89% isolated yield (Scheme 1).
Whereas the protease papain could advantageously be used to
unmask the amino acid esters 1–3 its application to the
deprotection of nucleopeptides might result in an undesired
attack of the enzyme on the formed peptide bonds.10 Therefore,
for the selective C-terminal deprotection of nucleopeptides the
lipase-mediated cleavage of the 2-(2-methoxyethoxy)ethyl
(MEE) esters11 was investigated, since lipases do not display
any protease activity. To this end, the selectively deprotected
nucleoamino acids 4–6 were coupled with different amino acid
or dipeptide MEE esters 9 to give the fully masked nucleopep-
tides 10–14 in high yields (Scheme 1, Table 1). Upon treatment
of the nucleopeptide acid MEE esters 10–14 with lipase from
Nucleoproteins are naturally occuring biopolymers in which the
hydroxy group of a serine, a threonine or a tyrosine is linked via
a phosphodiester group to the 3A- or 5A-end of DNA or RNA.1
These protein conjugates play decisive roles in important
biological processes; in particular, they may hold a central
position in the process of viral replication via nucleoprotein-
primed elongation of the oligonucleotide strand.2 For the study
of the biological phenomena in which nucleoproteins are
involved, nucleopeptides embodying the characteristic linkage
between the peptide chain and the oligonucleotide of their
parent nucleoproteins may serve as powerful tools. In particular,
the possibility of delineating the details of the above-mentioned
mechanism of viral replication and developing a new class of
antiviral agents based thereupon calls for the development of
efficient methods for the construction of these peptide con-
jugates. However, their synthesis poses two major challenges:
(i) the multifunctionality of the peptide-nucleotide conjugates
makes the application of a variety of orthogonally stable amino,
carboxy-, phosphate- and hydroxy-blocking groups necessary,
and (ii) fully protected serine/threonine nucleopeptides are acid-
labile (under acidic conditions the purine nucleotides may be
depurinated3) and base-labile (under basic conditions, i.e. pH !
8, the entire oligonucleotide part may be split off via
b-elimination4).
Ser
O
H
OMe
H
N
Phac
O
P
O
C
O
OH
OAc
8
ii (2)
Aloc AA1
O
Aloc AA1 OH
O
OMe
HN Phac
HN Phac
O
P
O
iii
O P O
B
B
Consequently, in nucleopeptide chemistry not only is a
variety of orthogonally stable blocking functions needed, they
all must be removable selectively under mild, preferably neutral
conditions. In the light of these seemingly contradictory
demands it is not surprising that only a few reports on the
successful construction of nucleopeptides have appeared.4,5
We have now found that enzymatic protecting group
techniques6 are efficient methods for the selective deprotection
of the carboxylic acids and nucleobases of nucleopeptides.
In order to develop an enzymatic protecting group strategy
for nucleopeptide synthesis, the protected nucleoamino acids
1–3 were constructed by means of established methods of
oligonucleotide chemistry.† In these compounds, the nucleo-
bases were masked with enzyme-labile phenylacetamide
groups7 and the carboxy groups of the amino acids were
protected as methyl esters.
O
O
AllylO
AllylO
OAc
OAc
4 AA1 = Ser, B = A, 96%
5 AA1 = Ser, B = C, 92%
6 AA1 = Thr, B = A, 70%
1 AA1 = Ser, B = A
2 AA1 = Ser, B = C
3 AA1 = Thr, B = A
iv
i (2)
Aloc AA1 AA2 AA3
MEE
O
Aloc
OMe
Ser
O
P
O
HN Phac
B
NH2
O
O
O
P O
C
O
O
AllylO
AllylO
OAc
OAc
10–14
7
From the nucleoamino acid esters 1–3 the C-terminal
protecting groups could be selectively removed by saponifying
the methyl esters with the protease papain from Carica papaya
at pH 6.6 and 37 °C (Scheme 1).8 The conditions of the
enzymatic transformations were so mild that the selectively
unmasked nucleoamino acids 4–6 were obtained in high yield
and without any undesired side reactions, i.e. the acid labile
purine nucleosides remained intact and a b-elimination of the
nucleotide, which readily takes place under weakly basic
conditions (vide supra), was not observed. The protease did not
attack the O-acetates. On the other hand, the nucleobase of e.g.
2 could be selectively N-deprotected by means of penicillin
acylase-catalysed removal of the phenylacetamido base protect-
ing group7 at pH 7 and room temp. in 77% isolated yield
(Scheme 1). In addition, the allyl-type blocking groups present
v
Aloc AA1 AA2 AA3 OH
O
HN Phac
B
O
O
P O
MEE =
Phac =
OMe
O
AllylO
O
OAc
15–19
Scheme 1 Reagents and conditions: i, Penicillin acylae, phosphate buffer
pH 7)–MeOH, 80:20; ii, Pd(PPh3)4, PhSiH3; iii, papain from Carica
papaya, cysteine buffer, pH 6.6; iv, H–AA2–AA3–OMEE 9,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydro-
xybenzotriazole; v, lipase from Aspergillus niger, phosphate buffer (pH
7)–acetone, 90:10
Chem. Commun., 1997
1861

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Table 1 Synthesis of the nucleopeptide esters 8–12 and their selective
deprotection with lipase from Aspergillus niger (AA = amino acid, B
= nucleobase)
71–74%; iv, N-allyloxycarbonyl amino acid methyl ester phosphor-
oamidate, tetrazole, room temp., then ButOOH, 0 °C, 68–82%.
1 B. A. Juodka, Nucleosides Nucleotides, 1984, 3, 445; J. C. Wang, Annu.
Rev. Biochem., 1985, 54, 665; A. B. Vartapetian and A. A. Bogdanov,
Progress Nucleic Acids Res. Mol. Biol., 1987, 34, 209; T. J. Kelly,
M. S. Wold and J. Li, Adv. Virus Res., 1988, 34, 1.
Coupling
Yield (%)
Deprotection
Yield (%)
Entry AA1 AA2
AA3
B
2 Review: M. Salas, Annu. Rev. Biochem., 1991, 160, 39; L. Blanco,
J. M. La´zaro, M. de Vega, A. Bonnin and M. Salas, Proc. Natl. Acad.
Sci. USA, 1994, 91, 12198.
3 For a recent review on the synthesis of nucleotides which also adresses
the problems encountered in the removal of the blocking functions, see
S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223.
4 B. Juodka, V. Kirveliene and P. Povilionis, Nucleosides Nucleotides,
1982, 1, 497; E. Kuyl-Yeheskiely, P. A. M. van der Klein, G. M. Visser,
G. A. van der Marel and J. H. van Boom, Recl. Trav. Chim. Pays-Bas,
1986, 105, 69.
1
2
3
4
5
Ser
Ser
Ser
Ser
Thr
Gly
Glu (All)
Phe
Val
Phe
Asp (All) C
10
11
12
13
14
61
62
75
69
71
15
16
17
18
19
70
58
70
59
63
—
C
C
C
A
—
Phe
—
Aspergillus niger at pH 7 and 37 °C the C-terminal carboxylic
acid was smoothly deprotected (Scheme 1, Table 1). In the case
of these multifunctional, complex peptide conjugates, too, the
enzymatic transformations occured without any undesired side
reaction, i.e. neither the acetate, N-terminal urethane, allyl
phosphate, phenylacetamide and peptide bonds, nor the acid-
labile purine nucleoside and the extremely base-labile serine
phosphates were attacked. The biocatalyst again tolerated the
presence of purine and pyrimidine bases and different amino
acids and amino acid sequences in the nucleopeptides. Its
substrate specificity guaranteed that the allyl esters which were
employed for masking of the amino acid side chain functions in
10 and 11 were not removed either. By means of this enzymatic
protecting group technique the desired selectively unmasked
nucleopeptides 15–19 were obtained in high yield. Nucleopep-
tide 15 represents the characteristic linkage region of the
nucleoprotein of adenovirus 2,12 and 16 is derived from the
nucleoprotein of bacteriophage Ø 29.13
Overall, the enzymatic protecting group strategy and the set
of orthogonally stable blocking groups described here open a
route to the construction of complex and sensitive nucleopep-
tides by means of a flexible building block strategy. The ready
accessibility of these multifunctional peptide conjugates may
serve to develop new tools for research at the interface between
chemistry and biology.
This work was supported by the Bundesministerium fu¨r
Bildung und Forschung, the Boehringer Mannheim GmbH and
the Fonds der Chemischen Industrie.
5 E. Kuyl-Yeheskiely, C. M. Tromp, A. W. M. Lefeber, G. A. van der
Marel and J. H. van Boom, Tetrahedron, 1988, 44, 6515; E. Kuyl-
Yeheskiely, C. M. Dreef-Tromp, A. Geluk, G. A. van der Marel and
J. H. van Boom, Nucleic Acids Res., 1989, 17, 2897; C. M. Dreef-
Tromp, J. C. M. van der Marel, H. van den Elst, G. A. van der Marel and
J. H. van Boom, Nucleic Acids Res., 1992, 20, 4015; Y. Ueno, R. Saito
and T. Hata, Nucleic Acids Res., 1993, 21, 4451; J. Robles, E. Pedroso
and A. Grandas, Tetrahedron Lett., 1994, 35, 4449; J. Robles,
E. Pedroso and A. Grandas, J. Org. Chem., 1994, 59, 2482; J. Robles,
E. Pedroso and A. Grandas, Nucleic Acids Res., 1995, 23, 4151.
6 Reviews: (a) H. Waldmann and M. Schelhaas, Angew. Chem., 1996,
108, 2192; Angew. Chem., Int. Ed. Engl., 1996, 35, 2056; (b)
H. Waldmann and D. Sebastian, Chem. Rev., 1994, 94, 911; (c)
T. Kappes and H. Waldmann, Liebigs Ann. Recl., 1997, 803.
7 H. Waldmann and A. Reidel, Angew. Chem., 1997, 109, 642; Angew.
Chem., Int. Ed. Engl., 1997, 36, 647; H. Waldmann, A. Heuser and
A. Reidel, Synlett 1994, 65-67.
8 For the enzymatic removal of methyl esters from glycosylated amino
acids, see D. Cantacuzene, S. Attal and S. Bay, Bioorg. Med. Chem.
Lett., 1991, 1, 197; S. Attal, S. Bay and D. Cantacuzene, Tetrahedron,
1992, 48, 9251; H. Ishii, K. Unabashi, Y. Mimura and Y. Inoue, Bull.
Chem. Soc. Jpn., 1990, 63, 3042.
9 M. Dessolin, M.-G. Guillerez, N. Thieriet, F. Guibe´ and A. Loffet,
Tetrahedron Lett., 1995, 36, 5741.
10 For the undesired attack of proteases on peptide bonds in the course of
enzymatic removals of blocking functions from peptides see the
references given in ref. 6(b), in particular: E. Walton, J. O. Rodin,
C. H. Stammer and F. W. Holly, J. Org. Chem., 1962, 27, 2255; G. Kloss
and E. Schro¨der, Hoppe-Seylers Z. Physiol. Chem., 1964, 336, 248.
11 For the enzymatic removal of MEE esters from glycosylated amino
acids and glycopeptides, see J. Eberling, P. Braun, D. Kowalczyk,
M. Schultz and H. Kunz, J. Org. Chem., 1996, 61, 2638.
12 J. E. Smart and B. W. Stillmann, J. Biol. Chem., 1982, 257, 13499.
13 J. M. Hermoso and M. Salas, Proc. Natl. Acad. Sci. USA, 1980, 77,
6425; J. M. Hermoso, E. Mendez, F. Soriano and M. Salas, Nucleic
Acids Res., 1985, 13, 7715.
Footnote and References
† Synthesis of 1–3: i, Me3SiCl (6 equiv.), pyridine, room temp., then
PhCH2COCl (3 equiv.), 1-hydroxybenzotriazole (2 equiv.), MeCN–
pyridine (2:1), room temp., then conc. NH3,
0 °C, 87–92%; ii,
4,4A-dimethoxytrityl chloride (1.2 equiv.), pyridine, room temp., then Ac2O
(6 equiv.), DMAP, 81–89%; iii, ZnBr2, MeNO2, room temp., 10 min,
Received in Glasgow, UK, 16th June 1997; 7/04215I
1862
Chem. Commun., 1997