Chemoenzymatic synthesis of nucleopeptides

Article abstract of DOI:10.1002/(SICI)1521-3765(19990201)5:2<669::AID-CHEM669>3.0.CO;2-V

Nucleoproteins, in which the hydroxy group of a serine, a threonine, or a tyrosine, is linked through a phosphodiester group to the 3′- or 5′-end of DNA or RNA, play decisive roles in important biological processes. They may even have a major part in the process of viral replication by nucleoprotein-primed elongation of the oligonucleotide strand. For the study of the biological phenomena, in which nucleoproteins are involved, nucleopeptides with the characteristic linkage between the peptide chain and the oligonucleotide of their parent nucleoproteins may serve as powerful tools. However, the synthesis of these compounds is complicated by their pronounced acid- and base-lability, as well as their multifunctionality. As a result, protecting groups, which can be removed under the mildest conditions, are required. For the construction of such peptide conjugates using a flexible building block strategy, a combination of enzyme-labile and chemical protecting groups was developed. The C-terminal blocking function can be removed selectively from fully protected nucleoamino acid methyl, 2-methoxyethyl (ME), and methoxyethoxyethyl (MEE) esters by saponification of the esters. After elongation of the peptide chain with amino acid or peptide methyl, ME, MEE, and choline esters, the C-terminal ester blocking group can again be removed easily. The methyl, ME, and MEE esters are cleaved off with lipase, and the choline ester group is selectively attacked by butyrylcholine esterase. The nucleoamino acids and peptides formed may be fully deprotected. To this end, the enzyme-labile N-phenylacetyl (PhAc) group, which was employed to mask the amino functions of the nucleobases, was removed. The O-acetate in the deoxyribose was saponified, and the allyl protecting groups present were cleaved by Pd⁰-mediated allyl transfer. By combination of these techniques, a nucleopeptide was produced, which represents the characteristic linkage region of the nucleoprotein of adenovions 2. The conditions, under which the enzymatic deprotections proceed, are so mild that no undesired side reaction is observed, that is no depurination or β elimination of the nucleosides occurs. In addition, the specificity of the biocatalysts ensures that the peptide bonds and the other protecting groups present are not attacked either.

Full text of DOI:10.1002/(SICI)1521-3765(19990201)5:2<669::AID-CHEM669>3.0.CO;2-V

FULL PAPER
Chemoenzymatic Synthesis of Nucleopeptides
Stefanie Flohr, Volker Jungmann, and Herbert Waldmann*[a]
Abstract: Nucleoproteins, in which the
hydroxy group of a serine, a threonine,
or a tyrosine, is linked through a phos-
phodiester group to the 3'- or 5'-end of
DNA or RNA, play decisive roles in
important biological processes. They
may even have a major part in the
process of viral replication by nucleo-
protein-primed elongation of the oligo-
nucleotide strand. For the study of the
biological phenomena, in which nucleo-
proteins are involved, nucleopeptides
with the characteristic linkage between
the peptide chain and the oligonucleo-
tide of their parent nucleoproteins may
serve as powerful tools. However, the
synthesis of these compounds is compli-
cated by their pronounced acid- and
base-lability, as well as their multifunc-
tionality. As a result, protecting groups,
which can be removed under the mildest
conditions, are required. For the con-
struction of such peptide conjugates
using a flexible building block strategy,
a combination of enzyme-labile and
chemical protecting groups was devel-
oped. The C-terminal blocking function
can be removed selectively from fully
protected nucleoamino acid methyl,
2-methoxyethyl (ME), and methoxy-
ethoxyethyl (MEE) esters by saponifi-
cation of the esters. After elongation of
the peptide chain with amino acid or
peptide methyl, ME, MEE, and choline
esters, the C-terminal ester blocking
group can again be removed easily. The
methyl, ME, and MEE esters are
cleaved off with lipase, and the choline
ester group is selectively attacked by
butyrylcholine esterase. The nucleoami-
no acids and peptides formed may be
fully deprotected. To this end, the en-
zyme-labile N-phenylacetyl (PhAc)
group, which was employed to mask
the amino functions of the nucleobases,
was removed. The O-acetate in the
deoxyribose was saponified, and the
allyl protecting groups present were
cleaved by Pd0-mediated allyl transfer.
By combination of these techniques, a
nucleopeptide was produced, which rep-
resents the characteristic linkage region
of the nucleoprotein of adenovions 2.
The conditions, under which the enzy-
matic deprotections proceed, are so mild
that no undesired side reaction is ob-
served, that is no depurination or b
elimination of the nucleosides occurs. In
addition, the specificity of the biocata-
lysts ensures that the peptide bonds and
the other protecting groups present are
not attacked either.
Keywords: bioorganic chemistry
´
enzyme catalysis ´ nucleopeptides ´
nucleotides ´ protecting groups
organisms starts with the covalent attachment of a nucleotide
to a protein primer, the ꢀterminal proteinꢀ, and then proceeds
by elongation of the oligonucleotide strand (Scheme 1).[3]
For the study of the biological processes in which nucleo-
proteins are involved, nucleopeptides with the characteristic
linkage between the peptide chain and the oligonucleotide of
their parent nucleoproteins may serve as powerful tools. In
particular, the possibility of describing the mechanism of viral
replication in detail and the possible development of a new
class of antiviral agents based thereupon (for example, against
hepatitis B virus) call for the development of efficient
methods for the construction of these peptide conjugates.
However, the synthesis of nucleopeptides poses two major
challenges (Scheme 2):
1) The multifunctionality of the peptide ± nucleotide conju-
gates requires the application of a variety of orthogonally
stable amino, carboxy, phosphate, and hydroxy blocking
groups.
2) Fully protected serine/threonine nucleopeptides are acid-
labile (under acidic conditions the purine nucleotides may
Introduction
Nucleoproteins are naturally occurring biopolymers, in which
the hydroxy group of a serine, a threonine, or a tyrosine is
linked, through a phosphodiester group to the 3'- or 5'-end of
DNA or RNA.[1] They play decisive roles in important
biological processes, for example, DNA topoisomerases and
DNA gyrases employ nucleopeptide bonds while carrying out
their biological functions,[2] and the DNA is bound to the
nuclear matrix as a nucleoprotein.[1] Particularly important is
the finding that these protein conjugates may hold a central
position in the process of viral replication. For instance, the
single-stranded RNA of the polio virus and the double-
stranded DNA of the Bacillus subtilis phage F 29 and
hepatitis B virus are nucleoproteins.[3] The replication of these
[a] Prof. Dr. H. Waldmann, Dr. S. Flohr, Dr. V. Jungmann
Universität Karlsruhe, Institut für Organische Chemie
Richard Willstätter Allee 2, D-76128 Karlsruhe (Germany)
Fax : (‡ 49)721-608-4825
E-mail: waldmann@ochhades.chemie.uni-karlsruhe.de
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H. Waldmann et al.
FULL PAPER
oligopeptide is synthesized, and in a subsequent series of
reactions the oligonucleotide chain is constructed at the
peptide backbone. We reasoned that for the development of a
highly flexible and efficient strategy for nucleopeptide syn-
thesis, the use of nucleoamino acid building blocks, which can
be selectively deprotected at their amino acid or nucleotide
part, would be particularly advantageous. Such building
blocks would have to have several orthogonally stable block-
ing groups, which on the one hand must tolerate the
conditions of peptide and nucleotide synthesis, and on the
other hand must be removable selectively and without
attacking the peptide oligonucleotide linkage. Thus, the
reaction takes place under the mildest conditions.
Enzymatic protecting group techniques[7] offer viable
alternatives to classical chemical methods. As a result of the
mild conditions under which they can be split off (pH 6 ± 8,
room temperature), enzyme-labile blocking functions have
served as efficient tools in the synthesis of lipo-,[8] glyco-,[9] and
phosphopeptides.[10] This paper presents an investigation into
the use of enzyme-labile protecting groups and their use as the
key method for the construction of complex nucleopepti-
des.[11]
Results and Discussion
In order to develop a general building block strategy for
nucleopeptide synthesis, the nucleoamino acids 8 were built
up (Schemes 3 and 4). To this end, the exocyclic amino groups
of the nucleosides 1 were masked with the enzyme-labile
phenylacetamido (PhAc) protecting group[12] by silylation,
followed by treatment of the resulting persilylated intermedi-
ate with phenylacetyl chloride and HOBt. The N-protected
nucleosides (2) obtained were then converted into the 5'-
dimethoxytrityl (DMTr) ethers[13] 3, and the 3'-OH groups
were masked as acetates.
Scheme 1. Priming of viral replication by a nucleoprotein. TP: terminal
protein, dNTP: deoxyribonucleotide triphosphate.
be depurinated),[4] and base-labile (under basic condi-
tions, that is at pH ꢀ 8, the entire oligonucleotide part
may be split off by b elimination) (Scheme 2).
BPhac
1. TMSCl, pyridine
2. PhacCl, HOBt
3. NH3, H2O
B
Base Lability
Acid Lability
HO
HO
O
O
H
N
O
H+
87 - 92 %
1a,b
2a,b
OH
OH
NH2
N
N
O
H
N
O
base
O
O P O
OR
DMTrCl,
pyridine
N
O P O
RO
O
B
O
O
96 - 98 %
OR'
BPhac
BPhac
DMTrO
DMTrO
Scheme 2. Acid- and base-lability of nucleopeptides. B ˆ adenine, cyto-
Ac2O, pyridine
O
O
sine, guanine, thymine.
80-84 %
4a,b
3a,b
OAc
OH
Consequently, in nucleopeptide chemistry not only a
variety of orthogonally stable blocking functions is needed,
but also 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.[5, 6] In the most successful of these investigations,
either an oligonucleotide is built up first, which is then
coupled with an activated peptide, or alternatively, an
ZnBr2,
CH3NO2
O
PhacCl:
78 - 92 %
Cl
BPhac
N
N
HO
O
HOBt:
N
5a,b
OAc
OH
Scheme 3. Synthesis of selectively protected nucleosides 5. a: B ˆ adenine;
b: B ˆ cytosine.
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Chemoenzymatic Synthesis of Nucleopeptides
669±681
The DMTr group was selectively removed from the fully
deprotected nucleotides 4 by treatment with ZnBr2.[14] The
selectively unmasked nucleosides 5 were then coupled with
serine and threonine derived phosphoramidites 7 in the
presence of tetrazole, and this reaction yielded, after subse-
quent oxidation with tert-butylhydroperoxide, fully masked
nucleoamino acids 8 (Scheme 4). The amino acid derived
phosphoramidites 7 were built up from the N-protected amino
acid esters 6, according to the method developed by Bann-
warth et al.[15] By analogy, serine methyl ester 9 was converted
into the phosphorylated serine derivative 10, which is of
interest as an intermediate for the chemoenzymatic synthesis
of phosphopeptides.
O
O
H
N
O
H
R
O
CH3
O
N
O
O
O
O
BPhac
R'
O
O P O
AllO
8a-e
10
O P OAll
AllO
O
OAc
papain,
phosphate buffer pH 6.6,
10 vol% acetone
70 - 95 %
73 %
O
H
N
O
H
O
O
N
OH
OH
O
O
O
BPhac
R'
O
O P O
AllO
O P OAll
AllO
O
12
O
H
OAc
R'
H
B
A
C
A
R
O
N
11
a
O
b H
O
c
Me
OH
R'
R
R'
tetrazole, HNiPr2,
Scheme 5. Selective C-terminal deprotection of the nucleoamino acids 8
and the phosphoamino acids 10 by enzymatic ester hydrolysis.
6
Me
H
a
b
c
d
AllO NiPr2
MEE H
Me Me
MEE Me
P
NiPr2
,
CH3CN
Table 1. C-terminal deprotection of nucleoamino acids 8a ± e to the acids 11a ± c.
Deprotection with Deprotection with
O
O
H
H
R
O
O
N
O
N
Lipase from
Papain from
O
O
Aspergillus niger Carica papaya
O
O
OH
Entry No
R'
B
R
No.
yield [%]
yield [%]
R'
9
P NiPr2
OAll
1
2
3
4
5
8a
8b
8c
8d
8e
H
H
H
A
A
C
Me
MEE
Me
11a
11a
11b
11c
11c
35
42
39
0
96
92
91
70
51
R
R'
H
1. tetrazole,
7
Me
a
b
c
d
AllO OAll
P
MEE H
Me Me
MEE Me
NiPr2
,
Me A Me
Me A MEE
0
CH2Cl2
2. m -CPBA
1. 5a,b, tetrazole,
CH2Cl2, CH3CN
2. tert -BuOOH
62 %
attacked. This observation is in line with findings that lipases
can not or can only just accommodate substrates in their
active sites, which have a branching point in b position to the
82 - 88 %
O
H
O
N
O
O
[18]
ˆ
H
ester C O group. Papain does not have this disadvantage,
O
R
O
N
O
O
10
transformations proceed smoothly, yielding the desired selec-
tively unmasked nucleoamino acids 11 and the phosphoamino
acid 12 in much higher yield (Table 1). The conditions of the
enzymatic transformations are so mild that no undesired side
reaction is observed; the acid-labile purine nucleosides
remain intact, and a b elimination of the nucleotide, which
readily occurs under weakly basic conditions (vide supra),
does not occur. The protease tolerates the presence of both
purine and pyrimidine bases and does not attack the PhAc
group, the acetate, and the phosphate at all.
O P OAll
OAll
O
O
BPhac
R'
O P O
OAll
O
OAc
R
Me
MEE
Me
Me
R'
H
H
H
B
A
A
C
A
A
8 a
b
c
d
e
Me = -CH3
MEE = -CH2CH2OCH2CH2OCH3
Me
MEE Me
Scheme 4. Synthesis of the fully protected nucleoamino acids 8.
In order to demonstrate the orthogonal stability of the
enzyme-labile ester groups to the other blocking functions
present, the phenylacetamide, the O-acetate, and the allyl-
type protecting groups were removed selectively from the
nucleoamino acid 8c (Scheme 6). Thus, upon treatment of 8c
with penicillin G acylase from E. coli[12] under neutral
conditions, the corresponding amine was liberated smoothly
and in high yield. The partially deblocked nucleoamino acid
derivative 13 obtained may, for instance, serve as a versatile
intermediate for the construction of artificial nucleopeptide
analogues carrying, for example, a fluorescent label.[19] The
acetate blocking group in the 3'-position was cleaved off by
In order to determine if the nucleoamino acid derivatives 8
are viable building blocks for nucleopeptide synthesis, the
orthogonal stability of the blocking groups present was
investigated. Both the C-terminal methyl (Me)[16] and the
methoxyethoxylethyl (MEE)[17] ester protecting groups can
be selectively removed from the amino acid esters 8 and 10 by
treatment with lipase from Aspergillus niger at pH 7 and
378C. However, much better results were obtained if the
protease papain from Carica papaya was used at pH 6.6 and
378C (Scheme 5, Table 1). The lipase only accepts the serine
derivatives as substrates, and the threonine esters are not
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H. Waldmann et al.
FULL PAPER
each other, and a b elimination or hydrolysis of the N-
glycosidic bond could not be observed.
O
O
H
N
CH3
O
O
O
Further construction of nucleopeptides employing the
C-terminally deprotected building blocks 11 was then at-
tempted. The amino acid chain of the nucleoamino acids
could be elongated by condensing the carboxylic acids 11 with
N-terminally deblocked amino acid and dipeptide esters in
the presence of N-ethyl-N'-dimethylaminopropyl carbodii-
mide hydrochloride (EDC) and N-hydroxybenzotriazole
(HOBt). This reaction gave the fully protected nucleopep-
tides 16 in moderate to high yields (Scheme 7, Table 2). The
CPhac
8c
O P O
AllO
O
OAc
(PPh3)4Pd(0),
PhSiH3, THF
penicillin G acylase, rt
phosphate buffer pH 7
89 %
77 %
O
O
O
H
+
H3N
CH3
CH3
O
N
O
O
O
O
CPhac
C
O P O
- O
13 O P O
O
O
AllO
15
OAc
OAc
lipase from wheat germ,
37°C, phosphate buffer
pH 6.5
64 %
O
O
H
N
CH3
O
O
O
CPhac
O P O
AllO
14
O
OH
Scheme 6. Selective deprotection of the nucleoamino acid 8c by different
methods.
employing lipase from wheat germ as
a biocatalyst
(Scheme 6).[20] The best results were obtained at pH 6.5 and
378C in the presence of 10 ± 20 vol% of ethylene glycol as
solubilizing cosolvent. In this process, the deoxyribose deriv-
ative is obtained, without the C-terminal methyl ester being
attacked. Thus, by appropriate choice of the lipase, nucleo-
amino acid derivatives like 8c can be regioselectively
deprotected at the carbohydrate or the amino acid part of
the molecule. Finally, the N-terminal allyloxycarbonyl ure-
thane and the allyl phosphate present in 8c were cleaved
chemoselectively by Pd0-mediated allyl transfer to an accept-
ing nucleophile. The nucleophile of choice in this case was
[21]
Â
phenylsilane as recommended by Guibe and Loffet et al.
The use of formic acid/n-butylamine mixtures introduced by
Noyori et al. for the unmasking of oligonucleotides[22] was
found to be less efficient. In all these deprotection reactions,
the blocking groups were completely orthogonally stable to
Scheme 7. Synthesis and selective enzymatic deprotection of the nucleo-
peptide methyl, ME, and MEE esters 16.
Table 2. Synthesis of the nucleopeptides 16a ± i and selective C-terminal deprotection with lipase from Aspergillus niger to the acids 17a ± e.
Coupling
Deprotection
Entry
B
AA1
AA2
AA3
R
No.
yield [%]
No
yield [%]
1
2
3
4
5
6
7
8
9
C
C
C
C
C
C
C
C
A
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Glu(OAll)
Glu(OAll)
Glu(OAll)
Phe
Val
Gly
Gly
Gly
Phe
-
-
-
-
Me
ME
MEE
MEE
MEE
Me
ME
MEE
MEE
16a
16b
16c
16d
16e
16 f
16g
16h
16i
95
90
62
75
69
60
56
75
71
17a
17a
17a
17b
17c
17d
17d
17d
17e
78
89
58
70
59
81
91
70
63
Phe
Asp(OAll)
Asp(OAll)
Asp(OAll)
-
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Chemoenzymatic Synthesis of Nucleopeptides
669±681
methyl and MEE esters mentioned above, were again used as
C-terminal protecting groups. The use of methoxyethyl (ME)
esters was found to work best, as this ester group is more polar
than the methyl ester and therefore has already pronounced
solubility enhancing properties. However, this group is not
quite as hydrophilic as the MEE ester, so the nucleopeptide
ME esters are easier to isolate than the corresponding MEE
esters. Furthermore, through the series methyl to the ME, and
finally the MEE ester, the size and the polarity of the enzyme-
labile protecting group increase. By varying this part of the
substrates, their properties may be finely adjusted to the steric
and electronic requirements of the active site of the particular
biocatalyst used in the deprotection. The protease papain was
not used for the removal of the C-terminal protecting group
from the nucleopeptide esters 16, since in the presence of this
enzyme an undesired attack on the peptide bonds was feared.
However, if the peptide conjugates 16 are treated with lipase
from Aspergillus niger, the C-terminally deprotected nucleo-
peptides 17 are obtained without any undesired attack on the
peptide bonds (Scheme 7, Table 2). Once more the conditions
of the enzymatic reactions were so mild that no undesired side
reaction was observed, and, in addition, the substrate
specificity of the enzyme guaranteed that the allyl esters
present in 16a ± c and 16 f ± h remained intact, too. The nature
of the alcohol blocking function has a marked influence on the
efficiency of the enzyme-mediated hydrolysis. Thus for 16a,
16b, and 16c, which only differ in the nature of the C-terminal
protecting group, as well as for 16 f, 16g, and 16h, for which
the same is true, the highest yields were recorded for the
deblocking of the methoxyethyl (ME) esters (Table 2; com-
pare entries 1, 2, and 3 as well as entries 6, 7, and 8). All
selectively unmasked nucleopeptides 17a ± 17e were obtained
in high yields.
protected amino acids 18 were converted into their 2-bro-
moethyl esters 19.
These were then treated with trimethylamine to give the
choline esters 20, from which the Boc group was cleaved off
by treatment with a solution of HBr in acetic acid, forming the
desired esters 21 in high yield. The choline esters 21 were then
coupled with the nucleoamino acid 11a and the phosphoami-
no acid 12 in the presence of diisopropylcarbodiimide (DIC)
and N-hydroxy-azabenzotriazole (HOAt)[25] to give the nu-
cleopeptide choline esters 22 and the phosphopeptide 23 in
high yield (Scheme 9, Table 3). Butyrylcholine esterase
Aloc-Ser-OH
O
Aloc-Ser-AA-OCho
O
DIC, HOAt,
DMF/CH2Cl2
21a-d
O
P R
O
P R
65 - 99 %
AllO
AllO
R
AA
R = 3'Ac-dAPhac
OAll
3'Ac-dAPhac Ala
3'Ac-dAPhac Leu
3'Ac-dAPhac Phe
3'Ac-dAPhac Tyr
22a
22b
22c
22d
23
11a
12
OAll
Ala
butyrylcholine
esterase,
73 - 96 %
phosphate
buffer pH 6.5
Aloc-Ser-AA-OH
O
N
HOAt:
N
N
O
P R
N
OH
AllO
R
AA
3'Ac-dAPhac Ala
3'Ac-dAPhac Leu
3'Ac-dAPhac Phe
3'Ac-dAPhac Tyr
24a
24b
24c
24d
25
DIC:
N C N
As an alternative to the methyl, ME, and MEE esters, the
choline ester protecting group[24] was investigated. This group
has already proven to be an advantageous enzyme-labile
blocking function in lipopeptide chemistry.[8] The charged
choline esters have pronounced solubility enhancing proper-
ties and should be particularly advantageous for enzymatic
transformations of hydrophobic substrates, which otherwise
would be only sparingly soluble in the aqueous media needed
for optimal performance of the biocatalysts. The amino acid
choline esters employed were synthesized according to
published procedures[24], as shown in Scheme 8. Thus, Boc-
OAll
Ala
Scheme 9. Synthesis and selective enzymatic deprotection of the nucleo-
peptide choline esters 22 and the phosphopeptide choline ester 23.
Table 3. Synthesis of the nucleo- and phosphopeptide choline esters 22a ±
d, 23 and selective C-terminal deprotection with butyrylcholine esterase to
the acids 24a ± d, 25 (AA ˆ amino acid, A ˆ 3'-Ac-dAPhac, P ˆ PO(OAll)2).
Coupling
no. yield [%]
Deprotection
no. yield [%]
Entry
AA
R
1
2
3
4
5
Ala
Leu
Phe
Tyr
A
A
A
A
P
22a
22b
22c
22d
23
99
91
96
65
85
24a
24b
24c
24d
25
96
78
61
86
73
HOCH2CH2Br,
DIC, DMAP,
CH2Cl2
O
O
Ala
H
H
O
N
O
N
Br
OH
O
O
R
O
R
90 - 95 %
1
(9 unitsmg ) was utilized for the enzymatic removal of the
protecting group. This enzyme was isolated from horse serum
by a combination of anion-exchange chromatography and
affinity chromatography on a procainamide column, accord-
ing to a published procedure.[26] Upon treatment of an
aqueous solution of the choline esters 22 and 23 (due to
solubilizing properties of the choline ester group, addition of
an organic cosolvent was unnecessary) with this enzyme at
pH 6.5, the C-terminal ester was cleaved off smoothly and
without any undesired side reaction. The selectively un-
18a-d
19a-d
NMe3,
acetone
93 - 99 %
Br -
H3N+
O
O
O
H
+N
Br -
O
N
+N
Br -
HBr / HOAc
O
91 - 99 %
R
O
R
21a-d
20a-d
Scheme 8. Synthesis of the amino acid choline esters 21.
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673

H. Waldmann et al.
FULL PAPER
masked nucleodipeptides 24 and the phosphodipeptide 25
were obtained in high yields. These results demonstrate that
the choline ester can also be employed advantageously for the
construction of nucleo- and phosphopeptides.
Finally, to prove that the combination of the enzyme-labile
and the classical chemical blocking functions also allows for
the synthesis of completely deprotected nucleopeptides, all
protecting groups were removed from the nucleotripeptide
17d (Scheme 10). To this end, the phenylacetamido protecting
Experimental Section
General procedures: 1H NMR and 13C NMR spectra were recorded on a
Bruker AC 250, AM 400, and DRX 500. Mass spectra were measured on a
Finnigan MAT MS 70 spectrometer. Analytical chromatography was
performed on E. Merck silica gel 60 F254 plates. Flash chromatography was
performed on Baker silica gel (40 ± 64 mm). Solvents were dried according
standard procedures. All reactions except the enzymatic transformations
were carried out under nitrogen. Penicillin G acylase was immobilized on
Eupergit C, lipase was purchased from Amano, papain from Fluka.
N-Phenylacetyl-2'-deoxynucleosides (2a ± c): A solution of the nucleoside
1 (4 mmol) and TMSCl (3 mL, 25 mmol) in pyridine (20 mL) was stirred for
30 min at 08C, and a solution of HOBt (1.08 g, 8.0 mmol) and phenylacetyl
chloride (1.65 mL, 12 mmol) in a mixture of CH3CN (1.8 mL) and pyridine
(0.9 mL) was added. After 12 h, water (5 mL) and concentrated aqueous
NH3 (7.4 mL) were added, the solution was stirred for 10 min, concen-
trated, and the residue was taken up in hot acetone and filtered. The filtrate
was concentrated, and the crude oil was purified by chromatography on
silica gel (chloroform/ethanol 9:1).
6-N-Phenylacetyl-2'-deoxyadenosine (2a): Yield: 87%; white solid; m.p.
1698C; Rf ˆ 0.53 (ethyl acetate/methanol 2:1); 1H NMR (250 MHz,
[D6]DMSO): d ˆ 10.97 (s, 1H), 8.68 (s, 1H), 8.65 (s, 1H), 7.40 ± 7.25 (m,
5H), 6.45 (t, J ˆ 7 Hz, 1H), 5.35 (d, J ˆ 5.1 Hz, 1H), 4.98 (t, J ˆ 6.5 Hz, 1H),
4.44 (s, 1H), 3.71 (m, 3H, 4'H), 3.64 ± 3.43 (m, 2H), 2.82 ± 2.71 (m, 1H),
2.36 ± 2.32 (ddd, J2'a,3' ˆ 3.8 Hz, J2'a,1' ˆ 6.5 Hz, J2'a,2'b ˆ 13.5 Hz, 1H); [a]D20
ˆ
15.1 (c ˆ 1, CHCl3); elemental analysis for C18H19N5O4(%): calcd C 58.53,
H 5.18, N 18.96; found C 58.49, H 5.14, N 18.83.
4-N-Phenylacetyl-2'-deoxycytidine (2b): Yield: 92%; m.p. 758C; Rf ˆ 0.57
(ethyl acetate/methanol 2:1); 1H NMR (250 MHz, [D6]DMSO): d ˆ 8.34 (d,
J ˆ 7.0 Hz, 1H), 7.94 (s, 1H), 7.35 ± 7.24 (m, 5H), 7.19 (d, J ˆ 7.0 Hz, 1H),
6.10 (t, J ˆ 6.3 Hz, 1H), 5.27 (d, J ˆ 4.2 Hz, 1H), 5.05 (t, J ˆ 5.2 Hz, 1H),
4.22 ± 4.19 (m, 1H), 3.87 ± 3.84 (m, 1H), 3.73 (s, 2H), 3.65 ± 3.53 (m, 2H),
2.29 (m, 1H), 2.01 (m, 1H); [a]2D0 ˆ 64.1 (c ˆ 1, DMSO); elemental analysis
for C17H19N3O5(%): calcd C 59.12, H 5.55, N 12.17; found C 58.99, H 5.28, N
11.99.
Scheme 10. Complete deprotection of the nucleopeptide 17d.
2-N-Phenylacetyl-2'-deoxyguanosine (2c): Yield: 89%; m.p. 1658C (de-
comp); Rf ˆ 0.42 (ethyl acetate/methanol 2:1); 1H NMR (250 MHz,
[D6]DMSO): d ˆ 11.97 (s, 1H), 8.24 (s, 1H), 7.35 ± 7.25 (m, 5H), 6.21 (t,
J ˆ 7 Hz, 1H), 5.31 (d, J ˆ 3.9 Hz, 1H), 4.95 (t, J ˆ 5.4 Hz, 1H), 4.37 (m,
1H), 3.84 (m, 1H), 3.80 (s, 2H), 3.54 ± 3.43 (m, 2H), 2.56 (m, 1H), 2.27 (m,
function was removed from the nucleobase first, to provide
the modified peptide 26 in high yield. Next, the three allyl
blocking groups present in 26 were removed by Pd0-catalyzed
allyl transfer with phenylsilane as the accepting nucleophile.
The reaction yielded the nucleotripeptide 27. Finally, 27 was
deacetylated by treatment with hydrazine in methanol.[27] The
completely deprotected nucleopeptide 28 obtained by this
reaction sequence represents the characteristic linkage region
of the nucleoprotein of adenovirus 2.[28]
1H); [a]2D0
ˆ
5.1 (c ˆ 1, DMSO); elemental analysis for C18H19N5O5: calcd
C 56.10, H 4.97, N 18.17; found C 55.95, H 5.00, N 18.00.
5'-O-Dimethoxytrityl-N-phenylacetyl-2'-deoxynucleosides (3a ± c): A sol-
ution of 2 (1 mmol) and DMTrCl (407 mg, 1.2 mmol) in pyridine (10 mL)
was stirred for 3 h at room temperature. A saturated solution of aqueous
NaHCO3 (30 mL) was added, and the mixture was extracted with CH2Cl2
(30 mL). The organic phase was dried over MgSO4 and concentrated. The
residual yellow foam was purified by chromatography on silica gel
(chloroform/ethanol 30:1).
5'-O-Dimethoxytrityl-6-N-phenylacetyl-2'-deoxyadenosine (3a): Yield:
98%; white foam; m.p. 978C; Rf ˆ 0.66 (ethyl acetate/methanol 2:1);
1H NMR (500 MHz, CDCl3): d ˆ 8.88 (s, 1H), 8.61 (s, 1H), 8.10 (s, 1H),
7.37 ± 6.76 (m, 18H), 6.43 (t, J ˆ 6.4 Hz, 1H), 4.68 (m, 1H), 4.19 ± 4.14 (m,
Conclusion
We have demonstrated that sensitive and multifunctional
nucleopeptides can be built up efficiently by means of a
flexible building block strategy. This strategy makes use of a
set of orthogonally stable enzyme-labile and classical chem-
ical protecting groups. In particular, the conditions of the
enzyme-mediated selective deprotection of the nucleopep-
tides are so mild that no undesired side reaction (that is b
elimination of the nucleotide or depurination) occurs. Fur-
thermore, no unwanted attack on the peptide bonds or the
other blocking groups present is observed either. The ready
accessibility and application of nucleopeptides may serve to
develop new reagents and tools for research at the interface
between chemistry and biology, that is in the study of viral
propagation.
3H, 4'H), 3.74 (s, 6H), 3.39 (m, 2H), 2.81 (m, 1H), 2.52 (m, 1H) ); [a]D20
ˆ
4.0 (c ˆ 1, CHCl3); elemental analysis for C39H37N5O6: calcd C 69.73, H
5.55, N 10.43; found C 69.85, H 5.51, N 10.33.
5'-O-Dimethoxytrityl-4-N-phenylacetyl-2'-deoxycytidine (3b): Yield:
96%; white foam; m.p. 1028C; Rf ˆ 0.68 (ethyl acetate ± methanol 2:1);
1H NMR (250 MHz, CDCl3): d ˆ 8.86 (s, 1H), 8.22 (s, 1H), 7.40 ± 6.82 (m,
19H), 6.27 (t, J ˆ 5.9 Hz, 1H), 4.48 (m, 1H), 4.14 (m, 1H), 3.77 (s, 6H), 3.74
(s, 2H), 3.47 ± 3.37 (m, 2H), 2.77 (m, 1H), 2.19 (m, 1H); [a]D20
1, CHCl3); elemental analysis for C38H37N3O7: calcd C 70.46, H 5.76, N 6.49;
found C 70.73, H 5.81, N 6.40.
ˆ
33.4 (c ˆ
5'-O-Dimethoxytrityl-2-N-phenylacetyl-2'-deoxyguanosine (3c): Yield:
93%; white foam; m.p. 1188C; Rf ˆ 0.61 (ethyl acetate ± methanol 2:1);
1H NMR (400 MHz, CDCl3): d ˆ 12.20 (s, 1H), 10.69 (s, 1H), 7.79 (s, 1H),
7.34 ± 6.68 (m, 18H), 6.12 (t, J ˆ 6.4 Hz, 1H), 4.57 (s, 1H), 4.14 (d, J ˆ
2.7 Hz, 1H), 3.71 (s, 2H), 3.65 (s, 6H), 3.25 (m, 2H), 2.52 ± 2.43 (m, 1H),
674
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Chem. Eur. J. 1999, 5, No. 2

Chemoenzymatic Synthesis of Nucleopeptides
669±681
2.38 ± 2.36 (m, 1H) ); [a]D20
C39H37N5O7: calcd C 68.11, H 5.42, N 10.18; found C 68.13, H 5.40, N 10.10.
ˆ
3.3 (c ˆ 1, CHCl3); elemental analysis for
8.72 (s, 1H), 8.46 (m, 1H), 7.37 ± 7.22 (m, 5H), 6.57 ± 6.46 (m, 2H), 5.91 ±
5.81 (m, 2H), 5.48 (m, 1H), 5.33 ± 5.15 (m, 4H), 4.63 ± 4.10 (m, 12H), 3.72
(m), 2.98 (m, 1H), 2.65 (m, 1H), 2.13 (s, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 170.45, 169.43, 169.38, 155.83, 152.37, 151.16, 149.34, 141.77,
134.26, 132.46, 131.88, 129.51, 128.51, 126.99, 118.93, 117.76, 84.29, 83.22,
3'-O-Acetyl-5'-O-dimethoxytrityl-N-phenylacetyl-2'-deoxynucleosides
(4a,b):
A solution of 3 (0.5 mmol) and acetic anhydride (0.26 mL,
2.5 mmol) in pyridine (8 mL) was stirred for 4 h. A sat. solution of aqueous
NaHCO3 (12 mL) was added, and the mixture was extracted with CHCl3
(20 mL). The organic phase was dried over MgSO4 and concentrated. The
residual foam was purified by chromatography on silica gel (chloroform ±
triethylamine 100:1).
74.19, 68.74, 67.55, 66.88, 65.90, 54.26, 52.84, 44.27, 37.02, 20.85; [a]D20
ˆ
1.25 (c ˆ 0.4, CH2Cl2); elemental analysis for C31H37O12N6P: calcd C
51.96, H 5.20, N 11.73; found C 51.95, H 5.17, N 11.65.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-serine-methoxyethoxyethyl ester (8b): Yield: 78%;
white hygroscopic foam; Rf ˆ 0.23 (CHCl3 ± EtOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.45 (m, 1H), 8.73 (m, 1H), 8.41 (d, 1H), 7.38 ±
7.25 (m, 5H), 6.55 ± 6.44 (m, 2H), 5.89 ± 5.82 (m, 2H), 5.48 (m, 1H), 5.35 ±
5.09 (m, 4H), 4.65 ± 4.09 (m, 12H), 3.70 (m, 2H), 3.62 (m, 2H), 3.52 (m,
2H), 3.35 (s, 3H), 2.94 (m, 1H), 2.66 (m, 1H), 2.14 (s, 3H); 13C NMR
(100.6 MHz, CDCl3): d ˆ 170.47, 168.97, 168.92, 155.84, 152.48, 151.18,
149.32, 141.59, 134.22, 132.53, 131.93, 129.56, 128.64, 127.14, 119.04, 117.81,
84.32, 83.34, 74.31, 71.79, 70.43, 68.84, 68.68, 67.64, 66.94, 65.05, 58.95, 54.39,
3'-O-Acetyl-5'-O-dimethoxytrityl-6-N-phenylacetyl-2'-deoxyadenosine
(4a): Yield: 80%; white foam; m.p. 588C; Rf ˆ 0.22 (chloroform ± ethanol
1
20:1); H NMR (250 MHz, CDCl3): d ˆ 8.72 (s, 1H), 8.65 (s, 1H), 8.12 (s,
1H), 7.45 ± 7.15 (m, 14H), 6.85 ± 6.72 (m, 4H), 6.47 (dd, J1',2'a ˆ 5.2 Hz,
J1',2'b ˆ 8.0 Hz, 1H), 5.51 (d, J ˆ 6.2 Hz, 1H), 4.33 ± 4.25 (m, 1H), 4.20 (s,
2H), 3.75 (s, 6H), 3.50 ± 3.37 (m, 2H), 2.87 ± 3.03 (m, 1H), 2.56 ± 2.68 (m,
1H), 2.12 (s, 3H); [a]2D0 ˆ 5.4 (c ˆ 0.5, CHCl3); MS EI: m/z: 713.3 ([M])
(calcd for C41H39N5O7 713.3); elemental analysis for sesquihydrate: calcd C
66.47, H 5.71, N 9.45; found C 66.21, H 5.68, N 9.34.
44.39, 37.24, 20.89; [a]D20
C35H45O14N6P: calcd C 52.24, H 5.64, N 10.44; found C 52.18, H 5.57, N
10.30.
ˆ
2.2 (c ˆ 0.5, CH2Cl2); elemental analysis for
3'-O-Acetyl-5'-O-dimethoxytrityl-4-N-phenylacetyl-2'-deoxycytidine (4b):
Yield: 84%; white foam; m.p. 958C; Rf ˆ 0.75 (chloroform/ethanol 9:1);
1H NMR (500 MHz, CDCl3): d ˆ 8.13 (s, 1H), 8.07 (d, J ˆ 7.6 Hz, 1H),
7.38 ± 7.21 (m, 14H), 7.16 (d, J ˆ 7.6 Hz, 1H), 6.85 ± 6.81 (m, 4H), 6.25 (t, J ˆ
5.9 Hz, 1H), 5.35 (m, 1H), 4.22 (m, 1H), 3.78 (s, 6H), 3.74 (s, 2H), 3.46 ±
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-serine-methyl ester (8c): Yield: 87%; white wax; Rf ˆ 0.32
(CHCl3 ± EtOH 20:1); 1H NMR (500 MHz, CDCl3): d ˆ 10.07 (s, 1H), 7.99
(m, 1H), 7.38 (m, 1H), 7.21 ± 7.16 (m, 5H), 6.22 (m, 2H), 5.83 ± 5.79 (m, 2H),
5.30 ± 5.09 (m, 5H), 4.52 ± 4.19 (m, 10H), 3.76 (m, 2H), 3.67 (m, 3H), 2.89
(m, 1H), 2.00 (m, 3H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.44, 170.20,
169.14, 162.65, 155.53, 154.77, 143.70, 133.63, 132.27, 131.67, 129.08, 128.45,
127.02, 118.85, 117.50, 96.75, 86.66, 83.17, 73.87, 68.61, 67.28, 66.91, 65.65,
3.40 (m, 2H), 2.76 ± 2.72 (m, 1H), 2.28 ± 2.22 (m, 1H), 2.05 (s, 3H); [a]D20
ˆ
67.3 (c ˆ 1, CHCl3); elemental analysis for C40H39N3O8, hemihydrate: calcd
C 68.75, H 5.77, N 6.01; found C 68.58, H 5.68, N 6.39.
3'-O-Acetyl-N-phenylacetyl-2'-deoxynucleosides (5a,b): A suspension of 4
(3.33 mmol) and ZnBr2 (3 g, 13.33 mmol) in CH3NO2 (30 mL) was stirred
for 15 min at room temprature. A solution of NH4OAc (1m, 50 mL) was
added, and the mixture was extracted with chloroform. The organic phase
was dried over MgSO4 and concentrated. The residual oil was purified by
chromatography on silica gel (chloroform ± ethanol 19:1).
54.07, 52.59, 43.95, 38.28, 20.55; [a]D20
analysis for C30H37O13N4P): calcd C 52.02, H 5.38, N 8.09; found C 51.95, H
5.40, N 7.96.
ˆ
40.2 (c ˆ 0.4, CH2Cl2); elemental
3'-O-Acetyl-6-N-phenylacetyl-2'-deoxyadenosine (5a): Yield: 78%; white
solid; m.p. 778C; Rf ˆ 0.14 (ethyl acetate ± methanol 50:1); 1H NMR
(500 MHz, CDCl3): d ˆ 8.90 (s, 1H), 8.70 (s, 1H), 8.09 (s, 1H), 7.26 ± 7.40 (m,
5H), 6.31 (dd, J1',2'a ˆ 6.2 Hz, J1',2'b ˆ 9 Hz, 1H), 5.75 (br, 1H), 5.56 (d, J ˆ
6.2 Hz, 1H), 4.27 (m, 1H), 4.23 (s, 2H), 3.91 (m, 2H), 3.09 ± 3.18 (m, 1H),
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-threonine-methyl ester (8d): Yield: 68%; white wax;
Rf ˆ 0.32 (CHCl3/EtOH 20:1); 1H NMR (400 MHz, CDCl3): d ˆ 9.31 (s,
0.5H), 9.27 (s, 0.5H), 8.72 (s, 1H), 8.39 (m, 1H), 7.46 ± 7.21 (m, 5H), 6.68 ±
6.47 (m, 2H), 5.96 ± 5.80 (m, 2H), 5.50 (m, 1H), 5.47 ± 5.02 (m, 5H), 4.67 ±
4.07 (m, 10H), 3.74 (m, 3H), 2.92 (m, 1H), 2.68 (m, 1H), 2.14 (s, 3H), 1.41
(m, 3H); 13C NMR (100.6 MHz, CDCl3): d ˆ 170.45, 169.75, 169.48, 156.83,
152.39, 151.14, 149.28, 141.70, 134.26, 132.46, 131.88, 129.51, 128.59, 127.10,
118.94, 117.73, 84.21, 83.25, 76.10, 74.30, 68.72, 66.32, 65.51, 58.39, 52.92,
2.40 ± 2.46 (m, 1H), 2.13 (s, 3H); [a]D20
ˆ
21.3 (c ˆ 0.4, CH3OH);
elemental analysis for C20H21N5O5, hemihydrate: calcd C 57.14, H 5.27, N
16.66; found C 57.14, H 5.27, N 16.36.
3'-O-Acetyl-4-N-phenylacetyl-2'-deoxycytidine (5b): Yield: 92%; white
solid; m.p. 1878C; Rf ˆ 0.53 (chloroform ± ethanol 9:1); 1H NMR
(500 MHz, CDCl3): d ˆ 8.28 (d, J ˆ 7.5 Hz), 7.37 (d, J ˆ 7.5 Hz, 1H), 7.29 ±
7.20 (m, 5H), 6.18 (t, J ˆ 5.8 Hz, 1H), 5.23 (m, 1H), 4.10 (m, 1H), 3.81 ± 3.72
(m, 2H), 3.66 (s, 2H), 2.59 ± 2.55 (m, 1H), 2.20 ± 2.13 (m, 1H), 2.03 (s, 3H);
[a]2D0 ˆ 21.5 (c ˆ 0.4, CH3OH); elemental analysis for C19H21N3O6, hemi-
hydrate: calcd C 58.90, H 5.46, N 10.84; found C 58.52, H 5.46, N 10.70.
44.29, 37.68, 20.85, 18.95; [a]D20
ˆ
1.5 (c ˆ 0.4, CH2Cl2); elemental analysis
for C32H39O12N6P ´ H2O: calcd C 51.34, H 5.51, N 11.23; found C 51.70, H
5.46, N 11.04.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-threonine-methoxyethoxyethyl ester (8e): Yield: 75%;
white wax; Rf ˆ 0.25 (CHCl3/EtOH 20:1); 1H NMR (500 MHz, CDCl3):
d ˆ 9.08 (s, 0.5H), 9.03 (s, 0.5H), 8.77 (s, 0.5H), 8.71 (s, 0.5H), 8.37 (m, 1H),
7.46 ± 7.25 (m, 5H), 6.68 ± 6.52 (m, 2H), 5.94 ± 5.81 (m, 2H), 5.51 ± 5.45 (m,
1H), 5.32 ± 5.04 (m, 5H), 4.68 ± 4.07 (m, 12H), 3.70 (m, 2H), 3.62 (m, 2H),
3.53 (m, 2H), 3.36 (s, 1H), 2.92 (m, 1H), 2.68 (m, 2H), 2.14 (s, 3H), 1.41 (m,
3H); 13C NMR (100.6 MHz, CDCl3): d ˆ 170.44, 169.81, 169.57, 156.65,
152.42, 151.13, 149.20, 141.65, 134.14, 132.24, 131.92, 129.57, 128.69, 127.19,
118.95, 117.70, 84.17, 83.32, 76.06, 74.47, 71.81, 70.51, 70.42, 68.71, 66.29,
Synthesis of the phosphoramidites (7a ± d): The phosphoramidites 7a ± d
were synthesized according to a published procedure.[29] A solution of 6a ±
d (3.32 mmol, 1.5 h, room temperature) in methylene chloride (4 mL) was
added to a solution of allyl-N,N,N',N'-tetraisopropylphosphordiamidite
(1.3 mL, 4 mmol), diisopropylamine (0.23 mL, 1.65 mmol), and tetrazole
(115 mg, 1.65 mmol) in methylene chloride (9 mL). After 2 h, the reaction
mixture was taken up in a saturated aqueous solution of NaHCO3 (15 mL).
The mixture was extracted four times with methylene chloride. The organic
layer was dried with MgSO4 and concentrated under reduced pressure. The
residual oil was filtered with silica gel using hexane/acetic acid ethyl ester
mixtures as eluent. The crude products were concentrated and immediately
used for subsequent steps without further purification.
65.05, 65.03, 58.99, 58.40, 44.44, 37.71, 20.87, 18.53; [a]D20
CH2Cl2); elemental analysis for C36H47O14N6P: calcd C 52.81, H 5.79, N
10.26; found C 52.74, H 5.47, N 10.55.
ˆ
0.75 (c ˆ 0.4,
N-Allyloxycarbonyl-(O-diallylphosphato)-l-serine-methyl ester (10):
Compound 9 (1.01 g, 4.14 mmol) was added dropwise at 08C to a solution
of 8 (700 mg, 3.45 mmol) and tetrazole (290 mg, 4.14 mmol) in CH2Cl2
(10 mL). After completion of the reaction, mCPBA (80%, 1.53 g,
6.9 mmol) was added. After 30 min, Et2O (200 mL) was added, and the
solution was extracted with aqueous NaHSO3 (10%, 50 mL) and saturated
NaHCO3 (50 mL). The organic phase was dried over MgSO4, concentrated,
and the residual oil was purified by chromatography on silica gel (hexane/
ethyl acetate 2:1).
yield: 62%; colorless oil; Rf ˆ 0.21 (hexane/ethyl acetate 1:1); 1H NMR
(250 MHz, CDCl3): d ˆ 6.03 ± 5.87 (m, 3H), 5.81 (d, J ˆ 6.7 Hz, 1H), 5.41 (d,
Jtrans ˆ 17.1 Hz, 3H), 5.21 (d, Jcis ˆ 10.4 Hz, 3H), 4.56 ± 4.53 (m, 6H), 4.49
(br, 1H), 4.38 (br, 1H), 4.19 ± 4.13 (m, 1H), 3.72 (s, 3H); 13C NMR
Synthesis of the nucleoamino acids (8a ± e): A solution of tetrazole (58 mg,
0.82 mmol) in acetonitrile (3 mL) at room temperature was added dropwise
to a solution of 5a,b (0.49 mmol) and 6a ± d (0.8 mmol) in CH2Cl2 (5 mL).
After completion of the reaction (18 h), tBuOOH (80%, 3.4 mL) was
added at 08C. After 10 min, the reaction was taken up in H2O (100 mL),
and the solution was extracted with CH2Cl2. The organic phase was dried
with MgSO4 and concentrated, and the residual oil purified by chromatog-
raphy on silica gel (chloroform/ethanol 40:1).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-serine-methyl ester (8a): Yield: 72%; white foam; Rf ˆ
1
0.29 (CHCl3 ± EtOH 20:1); H NMR (400 MHz, CDCl3): d ˆ 9.75 (s, 1H),
Chem. Eur. J. 1999, 5, No. 2
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675

H. Waldmann et al.
FULL PAPER
(62.8 MHz, CDCl3): d ˆ 168.9, 155.9, 132.5, 132.2 (2 C), 118.6, 117.9 (2 C),
68.5 (2JC,P ˆ 5.6 Hz, 2 C), 66.7 (d, 2JC,P ˆ 5.3 Hz), 66.5, 54.8 (d, 3JC,P ˆ 7.1 Hz),
258C. After 18 h, the mixture was filtered and extracted six times with
chloroform (20 mL). The organic phase was dried with MgSO4, concen-
trated, and the residue was purified by chromatography on silica gel
(chloroform/ethanol 40:1 ± 10:1).
52.59; [a]2D0
ˆ
2.7 (c ˆ 1, CH3OH); HRMS (EI): m/z: 363.1094 ([M])
(calcd for C14H22NO8P: 363.1083); elemental analysis: calcd C 46.28, H
6.10, N 3.86; found C 46.34, H 6.08, N 3.86.
yield: 77%; white solid; m.p. 908C; Rf ˆ 0.53 (ethyl acetate/methanol 4:1);
1H NMR (500 MHz, CDCl3): d ˆ 7.73 (m, 1H), 6.35 ± 5.78 (m, 5H), 5.41 ±
5.17 (m, 5H), 4.71 ± 4.15 (m, 10H), 3.85 (m, 3H), 2.60 (m, 1H), 2.23 ± 2.04
(m, 3H); 13C NMR (125.75 MHz, CDCl3): d ˆ 170.72, 170.32, 169.38,
164.88, 155.79, 154.33, 140.89, 132.44, 131.90, 119.29, 118.12, 95.14, 86.09,
82.98, 74.21, 68.98, 67.57, 67.42, 66.12, 54.36, 53.01, 38.11, 20.91; [a]2D0 ˆ 4.4
(c ˆ 0.5, CH2Cl2); elemental analysis for C22H31O12N4P ´ 1H2O: calcd C
44.60, H 5.61, N 9.46; found C 44.47, H 5.53, N 9.70.
Cleavage of the C-terminal ester of 8a ± e with lipase from Apergillus niger
(Method A): A solution of 8a ± e (0.15 mmol) in acetone (0.2 mL) was
added dropwise to a solution of lipase from Apergillus niger (20 mg) and
deoxytaurocholic acid (0.2 mg) in phosphate buffer (1.8 mL, 0.2m, pH 7).
The mixture was shaken at 378C until completion of the reaction (18 ±
72 h), and then the pH was adjusted to 3. The resulting suspension was
extracted eight times with chloroform (3 mL), and the chloroform layers
were dried over MgSO4. The solution was concentrated, and the residual oil
was purified by chromatography on silica gel (chloroform/ethanol 40:1 ±
10:1).
Alcohol (14): A suspension of wheat germ lipase (WGL) (4 U) and 7b
(20 mg, 29 mmol) in a mixture of phosphate buffer (pH 6.5, 50mm, 8 mL)
and ethylene glycol (2 mL) was shaken for 24 h in a polyethylene flask at
378C. After completion of the reaction, the solution was lyophilized, the
residue was taken up in CH3OH, and the product was isolated by
chromatography on silica gel (choroform/ethanol 19:1).
yield: 64%; colorless oil; Rf ˆ 0.35 (chloroform/ethanol 9:1); 1H NMR
(500 MHz, CDCl3): d ˆ 7.99 (m, 1H), 7.38 (m, 1H), 7.21 ± 7.16 (m, 5H), 6.12
(m, 1H), 5.83 ± 5.79 (m, 3H), 5.30 ± 5.09 (m, 5H), 4.52 ± 4.40 (m, 6H), 4.40 ±
4.31 (m, 2H), 4.31 ± 4.19 (m, 2H), 4.04 (m, 1H), 3.83 (s, 3H), 3.76 (s, 2H),
2.55 (m, 1H), 2.14 (m, 1H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.44,
170.20, 162.65, 155.53, 154.77, 143.70, 133.63, 132.27, 131.67, 129.08, 128.45,
127.02, 118.85, 117.50, 96.75, 86.66, 83.17, 73.87, 68.61, 67.28, 66.91, 65.65,
54.07, 52.59, 43.95, 38.28; [a]2D0 ˆ 18.0 (c ˆ 1, CH3OH); HRMS (EI): m/z:
651.2189 ([M‡H]) (calcd for C28H36N4O12P 651.2067).
O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-allyl-phosphato)-l-ser-
ine-methyl ester (15): Tetrakis(triphenylphosphane)palladium(0) (10 mg,
8 mmol) and phenylsilane (37 mL, 0.3 mmol) at room temperature were
added to a solution of 8c (52 mg, 75 mmol) in CH2Cl2 (4 mL). After 10 min,
the solution was concentrated and extracted with acetone. The remaining
residue was purified by chromatography on silica gel (chloroform/ethanol
20:1 ± 3:1).
yield: 89%; white solid; m.p. 1408C; 1H NMR (400 MHz, CD3OD): d ˆ
8.34 (d, J ˆ 7.5 Hz, 1H), 7.47 (d, J ˆ 7.5 Hz, 1H), 7.31 ± 7.22 (m, 5H), 6.26
(dd, J1',2'a ˆ 5.7 Hz, J1',2'b ˆ 8.1 Hz, 1H), 5.33 (d, J ˆ 6 Hz, 1H), 4.30 ± 4.10 (m,
6H), 3.79 (s, 3H), 3.73 (m, 2H), 2.60 (m, 1H), 2.29 (m, 1H), 2.09 (s, 3H);
13C NMR (125.75 MHz, CD3OD): d ˆ 173.51, 172.09, 168.72, 164.35, 157.74,
146.14, 135.61, 130.45, 129.58, 128.14, 98.48, 86.66, 85.74, 76.35, 66.67, 64.23,
54.87, 54.00, 44.71, 39.53, 20.93; [a]2D0 ˆ 15.08 (c ˆ 0.4, MeOH); HRMS
(FAB) (glycerol): m/z: 569.1649 ([M‡H]) (calcd for C23H30O10N4P:
569.1745).
Cleavage of the C-terminal ester of 8a ± e and 10 with papain (Method B):
A solution of 8a ± e or 10 (0.22 mmol) in acetone (2.4 mL) was added
dropwise to a suspension of papain (120 mg) and l-cysteine (95 mg) in
phosphate buffer (pH 6.6, 0.07m, 21 mL). The mixture was shaken at 378C
until completion of the reaction (6 ± 18 h). After filtration, NaCl was added,
the solution was extracted eight times with chloroform (4 mL), and the
chloroform layer was dried over MgSO4. The solution was concentrated,
and the residual oil was purified by chromatography on silica gel
(chloroform/ethanol 10:1).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-serine (11a): Yield: 92 ± 95% (Method B), 35 ± 42%
(Method A); white foam; m.p. 618C; Rf ˆ 0.26 (ethyl acetate ± methanol
4:1); 1H NMR (400 MHz, CDCl3): d ˆ 8.67 (s, 1H), 8.44 (m, 1H), 7.38 ± 7.21
(m, 5H), 6.56 ± 6.27 (m, 2H), 5.93 ± 5.81 (m, 2H), 5.41 ± 5.16 (m, 5H), 4.58 ±
4.10 (m, 12H), 2.79 ± 2.60 (m, 2H), 2.12 (s, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 172.51, 170.59, 170.55, 156.00, 152.65, 151.03, 149.09, 141.65,
134.19, 132.58, 131.87, 129.57, 128.67, 127.20, 119.26, 117.94, 84.51, 83.55,
74.21, 69.07, 68.29, 67.16, 65.99, 54.80, 44.33, 37.99, 20.95; [a]2D0 ˆ 0.5 (c ˆ 0.4,
CH2Cl2); elemental analysis for C30H35O12N6P ´ 2H2O): calcd C 48.98, H
5.32, N 11.38; found C 48.90, H 5.31, N 11.42.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-serine (11b): Yield: 91% (Method B); white solid; m.p.
718C; Rf ˆ 0.28 (ethyl acetate/methanol 4:1); 1H NMR (500 MHz, CDCl3):
d ˆ 8.19 (d, J ˆ 7.6 Hz, 0.5H), 8.12 (d, J ˆ 7.6 Hz, 0.5H), 7.55 (d, J ˆ 7.6 Hz,
0.5H), 7.50 (d, J ˆ 7.6 Hz, 0.5H), 7.38 ± 7.23 (m, 5H), 6.31 (m, 0.5H), 6.23
(m, 0.5H), 6.00 ± 5.85 (m, 2.5H), 5.73 (d, J ˆ 7.1 Hz, 0.5H), 5.44 ± 5.09 (m,
5H), 4.65 ± 4.18 (m, 10H), 3.70 (m, 2H), 2.82 (m, 1H), 2.30 ± 2.00 (m, 3H);
13C NMR (125.75 MHz, CDCl3): d ˆ 172.20, 171.94, 170.85, 162.30, 155.76,
153.44, 145.82, 133.31, 132.39, 131.72, 129.55, 128.65, 127.40, 119.57, 117.92,
96.40, 86.74, 83.91, 74.93, 69.18, 67.99, 66.94, 66.04, 54.17, 44.12, 38.55, 20.94;
[a]2D0 ˆ 50.25 (c ˆ 0.4, CH2Cl2); elemental analysis for C29H35O13N4P: calcd
C 51.33, H 5.20, N 8.26; found C 51.33, H 5.31, N 8.00.
Synthesis of the nucleo-di- and tripeptides (16a ± i): A solution of 11a or c
(0.13 mmol), HOBt (35 mg, 0.26 mmol), and EDC (50 mg, 0.26 mmol) in
dry CH2Cl2 (3.5 mL) was stirred for 30 min at 08C. Then, a solution of an
amino acid ester or dipeptide ester hydrosalt (0.29 mmol), and diisopro-
pylethylamine (34 mmol, 0.2 mmol) in dry CH2Cl2 (1 mL) was added. After
stirring for 15 h at room temperature, the solution was extracted with 0.1n
HCl and saturated NaHCO3. The organic layer was dried over MgSO4 and
filtered. The filtrate was concentrated, and the residue was purified by
chromatography on silica gel (chloroform/ethanol 50:1 ± 20:1).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-threonine (11c): Yield: 51 ± 70% (Method B); white
solid, m.p. 808C; Rf ˆ 0.30 (ethyl acetate/methanol 4:1); 1H NMR
(400 MHz, CDCl3): d ˆ 8.68 (s, 0.5H), 8.64 (s, 0.5H), 8.45 (m, 0.5H), 8.42
(m, 0.5H), 7.41 ± 7.23 (m, 5H), 6.46 ± 6.04 (m, 2H), 5.93 ± 5.72 (m, 2H),
5.38 ± 5.07 (m, 6H), 4.59 ± 4.07 (m, 10H), 2.65 ± 2.57 (m, 2H), 2.10 (m, 3H),
1.47 (m, 3H); 13C NMR (100.6 MHz, CDCl3): d ˆ 172.50, 170.73, 170.47,
156.67, 152.62, 150.89, 149.20, 141.68, 134.18, 132.51, 131.83, 129.52, 128.64,
127.16, 118.98, 117.95, 84.29, 83.46, 74.23, 74.13, 68.67, 66.89, 66.13, 58.47,
44.34, 37.95, 20.90, 18.65; [a]D20 ˆ 3.4 (c ˆ 0.3, CH2Cl2); elemental analysis
for C37H49O12N6P ´ 2H2O: calcd C 49.40, H 5.61, N 11.15; found C 49.71, H
5.62, N 10.95.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glutamic acid-g-allyl ester-methyl ester (16a):
Yield: 95%; white wax; Rf ˆ 0.22 (CHCl3/EtOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.81 (s, 1H), 8.27 (m, 0.5H), 8.10 (m, 1H), 8.03
(d, J ˆ 7.1 Hz, 0.5H), 7.46 (m, 1H), 7.33 (m, 5H), 6.24 ± 6.13 (m, 2H), 5.94 ±
5.83 (m, 3H), 5.38 ± 5.34 (m, 1H), 5.30 ± 5.18 (m, 6H), 4.99 (s, 0.5H), 4.89 (s,
0.5H), 4.60 ± 4.24 (m, 13H), 3.88 (m, 2H), 3.72 (m, 2H), 2.63 (m, 1H), 2.44
(m, 2H), 2.23 ± 2.03 (m, 6H); 13C NMR (125.75 MHz, CDCl3): d ˆ 172.34,
171.89, 171.86, 170.50, 168.65, 162.54, 156.00, 155.32, 144.20, 133.62, 132.44,
132.35, 132.01, 129.51, 128.88, 127.49, 119.33, 118.41, 117.96, 97.21, 86.84,
83.45, 73.83, 68.97, 67.57, 66.12, 65.33, 58.92, 54.39, 51.96, 44.34, 38.72, 30.22,
26.81, 20.81; [a]2D0 ˆ 15.3 (c ˆ 0.4, CH2Cl2); elemental analysis for
C38H48O16N5P ´ 2.5H2O: calcd C 50.33, H 5.89, N 7.12; found C 50.56, H
5.95, N 7.23.
N-Allyloxycarbonyl-O-diallylphosphato-l-serine (12): Yield: 73% (Meth-
1
od B); yellow oil; H NMR (500 MHz, CD3OD): d ˆ 6.01 ± 5.90 (m, 3H),
5.41 ± 5.37 (m, 2H), 5.37 ± 5.34 (m, 1H), 5.28 ± 5.25 (m, 2H), 5.19 (d, 1H,
Jcis ˆ 10.5 Hz), 4.58 ± 4.48 (m, 6H), 4.42 ± 4.40 (m, 1H), 4.38 ± 4.33 (m, 2H);
13C NMR (62.8 MHz, CD3OD): d ˆ 171.9, 158.1, 134.1, 133.7 (2 C), 118.9 (2
C), 117.7, 69.8 (2 C), 68.5, 66.7, 55.6; [a]2D0 ˆ 27.2 (c ˆ 0.5, CH2Cl2); HRMS
(FAB) (glycerol): m/z: 349.0976 ([M]) (calcd for C13H20NO8P 349.0926).
N-Allyloxycarbonyl-O-(3'-O-acetyl-2'-deoxycytidine-allyl-phosphato)-l-
serine-methyl ester (13): A solution of 8c (50 mg, 72 mmol) in CH3OH
(4 mL) was added dropwise to a suspension of immobilized penicillin G
acylase (500 mg, 60 units) in phosphate buffer (15 mL, 0.07m, pH 7) at
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glutamic acid-g-allyl ester-methoxyethyl ester
(16b): Yield: 90%; slightly yellow wax; Rf ˆ 0.21 (CHCl3/EtOH 20:1);
1H NMR (400 MHz, CDCl3): d ˆ 9.71 (s, 1H), 8.56 (s, 0.5H), 8.38 (d, 0.5H),
676
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0947-6539/99/0502-0676 $ 17.50+.50/0
Chem. Eur. J. 1999, 5, No. 2

Chemoenzymatic Synthesis of Nucleopeptides
669±681
8.07 (d, J ˆ 7.4 Hz, 0.5H), 7.98 (d, J ˆ 7.4 Hz, 0.5H), 7.42 (d, J ˆ 7.5 Hz,
0.5H), 7.37 (d, J ˆ 7.5 Hz, 0.5H), 7.28 ± 7.26 (m, 5H), 6.39 ± 6.21 (m, 2H),
5.92 ± 5.78 (m, 3H), 5.37 ± 5.06 (m, 8H), 4.65 ± 4.24 (m, 14H), 3.81 (m, 2H),
3.54 (m, 2H), 3.32 (s, 3H), 2.57 (m, 1H), 2.41 (m, 2H), 2.17 ± 2.06 (m, 6H);
13C NMR (125.75 MHz, CDCl3): d ˆ 172.31, 171.64, 171.52, 170.43, 168.69,
162.65, 156.01, 155.48, 143.95, 133.87, 133.80, 132.33, 132.01, 129.43, 128.78,
127.37, 119.26, 118.29, 117.84, 97.18, 86.51, 83.28, 73.69, 70.11, 68.85, 67.73,
66.47, 66.01, 65.26, 64.25, 58.87, 54.25, 51.97, 44.12, 38.59, 30.12, 26.73, 20.85;
[a]2D0 ˆ 15.6 (c ˆ 0.45, CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z:
906.3174 ([M‡H]) (calcd for C40H53O17N5P 906.3062).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glutamic acid-g-allyl ester-methoxyethoxyethyl
ester (16c): Yield: 62%; yellow wax; Rf ˆ 0.30 (CHCl3 ± EtOH 20:1);
1H NMR (500 MHz, CDCl3): d ˆ 9.42 (s, 1H), 8.50 (d, J ˆ 5.3 Hz, 0.5H),
8.29 (d, J ˆ 6 Hz, 0.5H), 8.11 (d, J ˆ 7.5 Hz, 0.5H), 8.01 (d, J ˆ 7.5 Hz,
0.5H), 7.45 (d, J ˆ 7.5 Hz, 0.5H), 7.40 (d, J ˆ 7.5 Hz, 0.5H), 7.35 ± 7.27 (m,
5H), 6.33 ± 6.21 (m, 2H), 5.97 ± 5.84 (m, 3H), 5.40 ± 5.35 (m, 1H), 5.30 ± 5.17
(m, 6H), 5.04 (s, 1H), 4.65 ± 4.24 (m, 14H), 3.84 (m, 2H), 3.71 (m, 2H), 3.63
(m, 2H), 3.54 (m, 2H), 3.38 (s, 1H), 2.62 (m, 1H), 2.45 (m, 2H), 2.24 ± 2.06
(m, 6H); 13C NMR (125.75 MHz, CDCl3): d ˆ 172.29, 171.46, 171.37, 170.45,
168.69, 162.63, 156.10, 155.34, 144.02, 133.79, 132.35, 132.01, 131.96, 129.48,
128.86, 127.53, 119.03, 118.37, 117.92, 97.13, 86.88, 83.31, 73.68, 71.87, 70.49,
68.93, 68.89, 67.65, 66.10, 65.27, 65.25, 64.42, 59.03, 54.31, 51.99, 44.32, 38.72,
30.19, 26.84, 20.91; [a]2D0 ˆ 13.0 (c ˆ 0.4, CH2Cl2); HRMS (FAB) (glycerol/
TFA): m/z: 950.3436 ([M‡H]) (calcd for C42H67O17N5P: 950.3306);
elemental analysis for C42H56O18N5P ´ 0.5H2O (%): calcd C 52.61, H 5.99,
N 7.30; found C 52.61, H 5.97, N 6.86.
127.35, 119.14, 118.51, 117.90, 97.24, 87.01, 83.41, 73.87, 69.01, 68.97, 68.87,
66.11, 65.57, 54.58, 52.66, 48.66, 44.19, 43.29, 38.44, 36.03, 20.85; [a]2D0 ˆ 37.8
(c ˆ 0.4, CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z: 905.2970 ([M‡H])
(calcd for C39H50O17N6P: 905.2839).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glycyl-l-aspartic acid-g-allyl ester-methoxyethyl
ester (16g): Yield: 56%; slightly yellow wax; Rf ˆ 0.24 (CHCl3/EtOH
1
20:1); H NMR (500 MHz, CDCl3): d ˆ 9.87 (s, 0.5H), 9.81 (s, 0.5H), 8.23
(s, 0.5H), 8.13 (s, 0.5H), 8.05 (m, 1H), 7.75 (d, J ˆ 7.4 Hz, 0.5H), 7.62 (d, J ˆ
8.1 Hz, 0.5H), 7.46 (m, 1H), 7.33 ± 7.28 (m, 5H), 6.60 (d, J ˆ 7.7 Hz, 0.5H),
6.35 (d, J ˆ 7.8 Hz, 0.5H), 6.25 ± 6.18 (m, 1H), 5.94 ± 5.82 (m, 3H), 5.37 (m,
0.5H), 5.34 (m, 0.5H), 5.31 ± 5.17 (m, 6H), 4.89 ± 4.82 (m, 1.5H), 4.74 (s,
0.5H), 4.57 ± 4.24 (m, 13H), 4.20 ± 3.97 (m, 2H), 3.82 (m, 2H), 3.55 (m, 2H),
3.33 (s, 1H), 3.01 ± 2.87 (m, 2H), 2.66 (m, 1H), 2.19 ± 2.09 (m, 4H);
13C NMR (125.75 MHz, CDCl3): d ˆ 170.53, 170.39, 170.21, 170.15, 169.05,
168.98, 162.64, 156.16, 155.48, 144.20, 133.71, 132.34, 131.89, 131.71, 129.44,
128.77, 127.34, 119.21, 118.50, 117.94, 97.13, 87.06, 83.37, 73.87, 69.99, 68.98,
68.92, 68.87, 67.64, 66.95, 64.55, 58.78, 54.62, 48.75, 44.20, 43.22, 38.39, 36.06,
20.84; [a]2D0 ˆ 32.5 (c ˆ 0.4, CH2Cl2); elemental analysis for C41H53O18N6P ´
H2O (%): calcd C 50.93, H 5.73, N 8.69; found C 50.91, H 5.89, N 8.56.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glycyl-l-aspartic acid-g-allyl ester-methoxyethoxy-
ethyl ester (16h): Yield: 61%; white wax; Rf ˆ 0.09 (CHCl3/EtOH 20:1);
1H NMR (500 MHz, CDCl3): d ˆ 9.78 (s, 0.5H), 9.68 (s, 0.5H), 8.39 (s,
0.5H), 8.22 (s, 0.5H), 8.07 (d, J ˆ 7.5 Hz, 0.5H), 8.02 (d, J ˆ 7.5 Hz, 0.5H),
7.80 (d, J ˆ 6.9 Hz, 0.5H), 7.62 (d, J ˆ 8.0 Hz, 0.5H), 7.47 (d, J ˆ 7.7 Hz,
0.5H), 7.45 (d, J ˆ 7.8 Hz, 0.5H), 7.35 ± 7.26 (m, 5H), 6.64 (d, J ˆ 7.5 Hz,
0.5H), 6.37 (d, J ˆ 7.8 Hz, 0.5H), 6.26 ± 6.20 (m, 1H), 5.93 ± 5.82 (m, 3H),
5.37 (m, 0.5H), 5.34 (m, 0.5H), 5.30 ± 5.17 (m, 6H), 4.89 ± 4.78 (m, 2H),
4.55 ± 4.25 (m, 13H), 4.20 ± 3.97 (m, 2H), 3.82 (m, 2H), 3.66 (m, 2H), 3.59
(m, 2H), 3.52 (m, 2H), 3.36 (s, 1H), 2.99 ± 2.87 (m, 2H), 2.63 (m, 1H),
2.17 ± 2.10 (m, 4H); 13C NMR (125.75 MHz, CDCl3): d ˆ 170.56, 170.50,
170.40, 170.16, 169.18, 169.08, 162.69, 156.23, 155.60, 144.26, 133.78, 132.39,
131.80, 131.77, 129.46, 128.84, 127.42, 119.17, 118.55, 117.95, 97.13, 87.13,
83.39, 73.92, 71.83, 70.39, 69.03, 68.99, 68.91, 68.76, 67.10, 66.14, 65.62, 58.98,
54.59, 48.80, 44.25, 43.36, 38.41, 36.08, 20.90; [a]2D0 ˆ 20.0 (c ˆ 0.4 in
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-phenylalanine-methoxyethoxyethyl ester (16d):
Yield: 75%; slightly yellow wax; Rf ˆ 0.20 (CHCl3 ± EtOH 20:1);
1H NMR (500 MHz, CDCl3): d ˆ 9.32 (s, 1H), 8.06 (d, J ˆ 7.5 Hz, 0.5H),
8.01 (d, J ˆ 7.5 Hz, 0.5H), 7.72 (s, 0.5H), 7.65 (d, J ˆ 6.8 Hz, 0.5H), 7.43 (d,
J ˆ 7.6 Hz, 0.5H), 7.41 (d, J ˆ 7.6 Hz, 0.5H), 7.35 ± 7.12 (m, 10H), 6.26 ± 6.19
(m, 1.5H), 6.14 (d, J ˆ 7.1 Hz, 0.5H), 5.95 ± 5.82 (m, 2H), 5.38 ± 5.15 (m,
5H), 4.84 (m, 1H), 4.75 (m, 1H), 4.55 (m, 4H), 4.44 (m, 2H), 4.40 ± 4.16 (m,
5H), 3.78 (m, 2H), 3.64 (m, 2H), 3.60 (m, 2H), 3.54 (m, 2H), 3.37 (s, 1H),
3.13 (m, 2H), 2.64 (m, 1H), 2.12 ± 2.04 (m, 4H); 13C NMR (125.75 MHz,
CDCl3): d ˆ 171.36, 171.14, 170.51, 168.24, 162.49, 156.08, 155.22, 144.06,
136.02, 133.50, 132.37, 131.94, 129.47, 129.32, 129.01, 128.47, 127.61, 126.98,
119.34, 117.96, 96.89, 87.02, 83.42, 73.95, 71.86, 70.47, 68.89, 68.75, 67.24,
66.86, 66.12, 64.47, 59.04, 54.42, 53.72, 44.50, 38.67, 37.60, 20.88; [a]2D0 ˆ 25.0
(c ˆ 0.4, CH2Cl2); elemental analysis for C43H64O17N5P (%): calcd C 55.66,
H 5.87, N 7.55; found C 55.38, H 5.76, N 7.21.
CH2Cl2); elemental analysis for C43H57O19N6P ´ 0.5H2O (%): calcd
51.55, H 5.85, N 8.39, found C 51.76, H 6.25, N 8.12.
C
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-threonyl-l-phenylalanine-methoxyethoxyethyl ester
(16i): Yield: 71%; white wax; Rf ˆ 0.27 (CHCl3/EtOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 8.78 (s, 0.5H), 8.44 (s, 0.5H), 7.75 (m, 2H), 7.55 (m,
0.5H), 7.44 ± 7.00 (m, 10.5H), 6.54 (m, 1H), 6.17 (d, J ˆ 8 Hz, 0.5H), 6.10 (d,
J ˆ 8 Hz, 0.5H), 5.89 ± 5.81 (m, 2H), 5.46 (m, 1H), 5.32 ± 5.18 (m, 4H),
5.10 ± 4.83 (m, 2H), 4.66 ± 4.08 (m, 12H), 3.58 (m, 4H), 3.52 (m, 2H), 3.35
(s, 1H), 3.11 (m, 2H), 2.90 (m, 1H), 2.64 (m, 1H), 2.13 (m, 3H), 1.33 (m,
3H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.08, 170.42, 168.95, 168.04,
156.18, 152.56, 151.23, 149.00, 135.81, 133.93, 132.40, 131.96, 129.59, 129.31,
128.71, 128.54, 127.03, 126.45, 119.02, 117.08, 84.32, 83.53, 75.11, 74.39, 71.84,
70.41, 68.88, 68.70, 66.99, 66.15, 64.50, 59.05, 58.31, 53.71, 44.48, 37.77, 37.65,
20.90; [a]2D0 ˆ 0.14 (c ˆ 0.15, CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z:
966.3650 ([M‡H]) (calcd for C45H67O16N7P: 966.3483); elemental analysis
for C45H66O16N7P (%): calcd C 55.90, H 5.94, N 10.14; found C 55.79, H
5.79, N 8.59.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-valyl-l-phenylalanine-methoxyethoxyethyl ester
(16e): Yield: 69%; white wax; Rf ˆ 0.16 (CHCl3/EtOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.87 (s, 1H), 8.06 (d, J ˆ 7.5 Hz, 0.5H), 7.99 (d, J ˆ
7.5 Hz, 0.5H), 7.92 (s, 0.5H), 7.87 (s, 0.5H), 7.47 (d, J ˆ 7.6 Hz, 0.5H), 7.38 (d,
J ˆ 7.6 Hz, 0.5H), 7.35 ± 7.13 (m, 11H), 6.56 (m, 1H), 6.24 (m, 1H), 5.94 ±
5.80 (m, 2H), 5.36 ± 5.10 (m, 5H), 4.85 (m, 1H), 4.74 (m, 1H), 4.58 ± 4.11
(m, 12H), 3.89 (m, 2H), 3.77 (m, 2H), 3.73 (m, 2H), 3.61 (m, 2H), 3.36 (s,
1H), 3.11 (m, 2H), 2.62 (m, 1H), 2.17 ± 2.04 (m, 4H), 0.89 (m, 6H);
13C NMR (125.75 MHz, CDCl3): d ˆ 171.79, 171.16, 171.04, 170.54, 168.58,
162.70, 156.07, 155.40, 144.33, 135.89, 133.71, 132.45, 132.00, 129.43, 129.37,
128.94, 128.54, 127.52, 127.06, 119.21, 118.00, 97.30, 87.26, 83.30, 73.91, 71.86,
70.44, 68.90, 68.75, 67.66, 66.82, 66.08, 64.40, 59.05, 56.07, 54.87, 53.41, 44.36,
38.41, 37.66, 30.53, 20.87, 19.18; [a]2D0 ˆ 0.75 (c ˆ 0.4, CH2Cl2); elemental
analysis for C48H64O17N6P (%): calcd C 56.08, H 6.27, N 8.17; found C 55.93,
H 6.45, N 7.83.
C-Terminal deprotection of the nucleopeptides 16a ± i by lipase from
Apergillus niger: A solution of the nucleopeptide 16a ± i (0.15 mmol) in
acetone (0.2 mL) was added dropwise to
a solution of lipase from
Apergillus niger (20 mg) and deoxytaurocholic acid (0.2 mg) in phosphate
buffer (1.8 mL, 0.2m, pH 7). The mixture was shaken at 378C until
completion of the reaction (4 ± 72 h), and then the pH was adjusted to 3.
The resulting suspension was extracted eight times with chloroform (4 mL),
and the chloroform layers were dried with MgSO4. The solution was
concentrated, and the residual oil was purified by chromatography on silica
gel (chloroform/ethanol 40:1 ± 10:1).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glycyl-l-aspartic acid-g-allyl ester-methyl ester
(16 f): Yield: 60%; white wax; Rf ˆ 0.27 (CHCl3/EtOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.88 (s, 0.5H), 9.78 (s, 0.5H), 8.34 (s, 0.5H), 8.18 (s,
0.5H), 8.06 (d, J ˆ 7.5 Hz, 0.5H), 8.02 (d, J ˆ 7.5 Hz, 0.5H), 7.89 (s, 0.5H),
7.66 (d, J ˆ 7.3 Hz, 0.5H), 7.46 (m, 1H), 7.32 ± 7.27 (m, 5H), 6.69 (d, J ˆ
7.0 Hz, 0.5H), 6.37 (s, 0.5H), 6.26 ± 6.19 (m, 1H), 5.95 ± 5.82 (m, 3H), 5.37
(m, 0.5H), 5.34 (m, 0.5H), 5.30 ± 5.17 (m, 6H), 4.87 ± 4.84 (m, 1.5H), 4.74 (s,
0.5H), 4.55 ± 4.25 (m, 11H), 4.12 ± 3.96 (m, 2H), 3.83 (m, 2H), 3.70 (s, 1H),
2.97 ± 2.86 (m, 2H), 2.64 (m, 1H), 2.17 ± 2.04 (m, 4H); 13C NMR
(125.75 MHz, CDCl3): d ˆ 170.89, 170.52, 170.45, 170.16, 169.11, 169.01,
162.67, 156.19, 155.52, 144.25, 133.80, 132.33, 131.94, 131.70, 129.40, 128.73,
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glutamic acid-g-allyl ester (17a): Yield: 58 ± 89%;
slightly yellow wax; Rf ˆ 0.47 (ethyl acetate/methanol 4:1); 1H NMR
(500 MHz, CDCl3): d ˆ 9.94 (s, 1H), 8.04 (m, 1.5H), 8.99 (d, J ˆ 7.5 Hz,
0.5H), 7.45 (d, J ˆ 7.5 Hz, 0.5H), 7.42 (d, J ˆ 7.5 Hz, 0.5H), 7.34 ± 7.25 (m,
5H), 6.30 ± 6.09 (m, 2H), 5.93 ± 5.83 (m, 3H), 5.36 ± 5.17 (m, 7H), 4.91 (s,
Chem. Eur. J. 1999, 5, No. 2
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677

H. Waldmann et al.
FULL PAPER
0.5H), 4.82 (s, 0.5H), 4.63 ± 4.22 (m, 12H), 3.75 (m, 2H), 2.61 (m, 1H), 2.45
(m, 2H), 2.26 ± 2.05 (m, 6H); 13C NMR (125.75 MHz, CDCl3): d ˆ 172.60,
172.01, 171.57, 170.44, 168.52, 162.63, 156.03, 155.06, 144.73, 133.58, 132.39,
132.06, 131.84, 129.48, 128.85, 127.49, 119.28, 118.37, 117.91, 97.06, 87.27,
83.43, 73.78, 69.08, 67.73, 66.53, 66.07, 65.33, 54.18, 52.21, 44.35, 38.45, 30.35,
26.60, 20.88; [a]2D0 ˆ 21.0 (c ˆ 0.4, CH2Cl2); HRMS (FAB) (glycerol/TFA):
m/z: 848.2634 ([M‡H]) (calcd for C37H47O16N5P: 848.2755).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-phenylalanine (17b): Yield: 70%; white wax; Rf ˆ
0.58 (ethyl acetate/methanol 4:1); 1H NMR (500 MHz, CDCl3): d ˆ 8.05 (d,
J ˆ 7.5 Hz, 0.5H), 7.98 (d, J ˆ 7.5 Hz, 0.5H), 7.43 (d, J ˆ 7.6 Hz, 0.5H), 7.41
(d, J ˆ 7.6 Hz, 0.5H), 7.33 ± 7.14 (m, 10H), 6.15 (m, 1.5H), 5.95 ± 5.83 (m,
2.5H), 5.37 ± 5.19 (m, 5H), 4.84 (m, 1H), 4.60 (m, 1H), 4.56 ± 4.50 (m, 4H),
4.33 ± 4.21 (m, 5H), 3.72 (m, 2H), 3.20 ± 3.04 (m, 2H), 2.64 (m, 1H), 2.19 ±
2.08 (m, 4H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.60, 171.49, 170.64,
168.28, 162.42, 156.12, 155.85, 144.97, 136.15, 133.28, 132.35, 131.83, 129.51,
129.44, 128.95, 128.49, 127.62, 126.96, 119.46, 118.01, 96.91, 87.71, 83.59,
73.86, 69.12, 67.48, 66.79, 66.10, 54.25, 53.92, 44.42, 38.43, 37.37, 20.91;
[a]2D0 ˆ 16.4 (c ˆ 0.28, CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z:
826.2701 ([M‡H]) (calcd for C38H45O14N5P: 826.2593).
3H); 13C NMR (100.5 MHz, CDCl3): d ˆ 172.95, 155.08, 79.85, 64.30, 49.17,
28.36, 28.27 (3 C), 18.45; [a]D20
ˆ
12.6 (c ˆ 1, CHCl3); elemental analysis
for C10H18BrNO4 (%): calcd C 40.56, H 6.12, N 4.73; found C 40.41, H 6.04,
N 4.84.
Boc-l-leucine-2-bromoethyl ester (19b): Yield: 90%; colorless oil; Rf ˆ
1
0.56 (hexane/ethyl acetate 2:1); H NMR (400 MHz, CDCl3): d ˆ 4.99 (d,
Jvic ˆ 8.2 Hz, 1H), 4.43 (ddt, J1±1' ˆ 6.0 Hz, J2±1' ˆ 6.1 Hz, J1±2 ˆ 14.9 Hz, 2H),
4.33 (q, Jvic ˆ 5.3 Hz, 1H), 3.52 (t, Jvic ˆ 6.1 Hz, 2H), 1.73 (t, Jvic ˆ 6.9 Hz),
1.64 (m, 1H), 1.44 (s, 9H), 0.94 (d, Jvic ˆ 6.5 Hz, 6H); 13C NMR (100.5 MHz,
CDCl3): d ˆ 173.02, 155.36, 79.81, 64.24, 52.07, 41.54, 34.86, 28.26 (3 C),
24.25, 21.83, 22.78; [a]D20
337.0911 (calcd for C13H24BrNO4: 337.0889); elemental analysis (%): calcd
C 46.16, H 7.15, N 4.14; found C 46.31, H 7.00, N 4.12.
ˆ
27.2 (c ˆ 2, CH3OH); HRMS (EI): m/z:
Boc-l-phenylalanine-2-bromoethyl ester (19c): Yield: 90%; slightly yellow
oil; Rf ˆ 0.56 (hexane/ethyl acetate 2:1); 1H NMR (500 MHz, CDCl3): d ˆ
7.25 ± 7.32 (m, 3H), 7.17 (d, Jvic ˆ 7.0 Hz, 2H), 4.98 (d, Jvic ˆ 7.6 Hz, 1H), 4.61
(q, Jvic ˆ 6.7 Hz, 1H), 4.42 (t, Jvic ˆ 3.2 Hz, 2H), 3.45 (t, Jvic ˆ 6.2 Hz, 2H),
3.11 (t, Jvic ˆ 5.2 Hz, 2H), 1.42 (s, 9H); 13C NMR (125.7 MHz, CDCl3): d ˆ
171.49, 155.04, 135.80, 129.29 (2 C), 128.59 (2 C), 127.10, 80.02, 64.44, 54.39,
38.23, 28.27 (3 C), 28.06; [a]2D0 ˆ 21.2 (c ˆ 1, CHCl3); elemental analysis for
C16H22BrNO4(%): calcd C 51.62, H 5.96, N 3.76; found C 51.69, H 5.92, N
4.09.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-valyl-l-phenylalanine (17c): Yield: 59%; white
1
wax; Rf ˆ 0.60 (ethyl acetate/methanol 4:1); H NMR (500 MHz, CDCl3):
Boc-l-tyrosine-2-bromoethyl ester (19d): Yield: 91%; colorless oil; Rf ˆ
0.19 (hexane/ethyl acetate 3:1); 1H NMR (500 MHz, CDCl3): d ˆ 6.97 (m,
2H), 6.75 (d, Jvic ˆ 7.9 Hz, 2H), 6.23 (br, 1H), 4.55 (q, Jvic ˆ 6.7 Hz, 1H),
4.36 (m, 2H), 3.45 (m, 2H), 3.04 (m, 2H), 1.45 (s, 9H); 13C NMR
(125.7 MHz, CDCl3): d ˆ 171.49, 155.04, 130.43, 130.38 (2 C), 127.28 (2 C),
d ˆ 8.14 (d, J ˆ 7.5 Hz, 0.5H), 8.09 (d, J ˆ 7.5 Hz, 0.5H), 7.59 ± 7.44 (m, 2H),
7.34 ± 7.16 (m, 10H), 6.98 (m, 1H), 6.30 ± 6.07 (m, 2H), 5.94 ± 5.82 (m, 2H),
5.37 ± 5.17 (m, 5H), 4.74 ± 4.19 (m, 12H), 3.75 (m, 2H), 3.25 ± 3.07 (m, 2H),
2.62 (m, 1H), 2.37 ± 2.04 (m, 4H), 0.85 (m, 6H); 13C NMR (125.75 MHz,
CDCl3): d ˆ 171.54, 171.16, 171.00, 170.79, 168.69, 162.57, 155.95, 154.88,
144.33, 135.89, 133.71, 132.33, 131.85, 129.46, 129.27, 128.94, 128.55, 127.58,
126.98, 119.36, 118.06, 96.09, 87.26, 83.62, 73.99, 69.08, 67.38, 66.40, 66.16,
59.58, 54.02, 53.85, 44.42, 38.05, 37.17, 30.38, 20.91, 19.12; [a]2D0 ˆ 4.0 (c ˆ 0.2,
CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z: 925.3385 ([M‡H]) (calcd for
C43H54O15N6P: 925.3564).
115.60, 80.02, 64.44, 54.39, 38.23, 28.27 (3 C), 28.06; [a]D20
CH3OH); HRMS (EI): m/z: 387.0694 ([M]) (calcd for C16H22NO5Br:
ˆ
0.7 (c ˆ 1,
387.0682).
Boc-amino acid choline ester bromides (20a ± d): At
trimethylamine (5 mL, 109 mmol) was added to
78 8C, liquid
solution of the
a
bromoethyl ester 19a ± d (17.5 mmol) in acetone (50 mL). The solution
was stirred for three days at room temperature, and the solvent and the
excess trimethylamine were removed under reduced pressure.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxycytidine-all-
yl-phosphato)-l-seryl-l-glycyl-l-aspartic acid-g-allyl ester (17d): Yield:
70 ± 91%; slightly yellow wax; Rf ˆ 0.17 (ethyl acetate/methanol 4:1);
1H NMR (500 MHz, CDCl3): d ˆ 9.98 (s, 1H), 8.11 (m, 1H), 8.10 (m, 1H),
7.90 (m, 1H), 7.47 (m, 1H), 7.39 ± 7.14 (m, 5H), 6.64 (s, 1H), 6.31 (s, 1H),
6.18 (m, 1H), 5.93 ± 5.82 (m, 3H), 5.36 ± 5.17 (m, 7H), 4.84 ± 4.61 (m, 2H),
4.60 ± 4.25 (m, 11H), 3.89 (m, 2H), 3.78 (m, 2H), 2.99 ± 2.88 (m, 2H), 2.65
(m, 1H), 2.18 ± 2.08 (m, 4H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.92,
171.85, 170.80, 170.73, 169.35, 169.13, 162.74, 155.40, 154.69, 144.90, 133.61,
132.36, 131.74, 131.20, 129.49, 128.82, 127.58, 119.38, 118.55, 118.10, 97.08,
87.59, 83.59, 74.06, 69.12, 68.07, 67.16, 66.26, 65.67, 54.89, 48.84, 44.31, 43.136,
38.42, 35.94, 20.91; [a]2D0 ˆ 15.0 (c ˆ 0.4, CH2Cl2); HRMS (FAB) (glycerol/
TFA): m/z: 891.2814 ([M‡H]) (calcd for C38H48O17N6P: 891.3037).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-threonyl-l-phenylalanine (17e): Yield: 63%; white
wax; Rf ˆ 0.3 (ethyl acetate/methanol 4:1); 1H NMR (500 MHz, CDCl3):
d ˆ 8.77 ± 8.56 (m, 2H), 7.42 ± 7.06 (m, 10H), 6.57 (m, 1.5H), 6.05 (m, 0.5H),
5.94 ± 5.87 (m, 2H), 5.49 (m, 1H), 5.35 ± 5.18 (m, 4H), 4.95 ± 4.76 (m, 2H),
4.68 ± 4.10 (m, 10H), 3.21 ± 3.11 (m, 2H), 2.67 (m, 1H), 2.64 (m, 1H), 2.15
(m, 3H), 1.26 (m, 3H); 13C NMR (125.75 MHz, CDCl3): d ˆ 171.11, 170.49,
168.90, 168.24, 156.28, 152.50, 151.21, 149.14, 135.91, 133.97, 132.46, 131.95,
129.79, 129.38, 128.78, 128.54, 127.23, 126.55, 119.32, 117.07, 84.39, 83.57,
75.14, 74.29, 68.80, 66.87, 66.13, 58.34, 53.79, 44.48, 37.67, 37.62, 20.89;
[a]2D0 ˆ 8.9 (c ˆ 0.19, CH2Cl2); HRMS (FAB) (glycerol/TFA): m/z: 864.2969
([M‡H]) (calcd for C40H48O13N7P: 864.2833).
Boc-l-alanine choline ester bromide (20a): Yield: 98%; very hygroscopic
white solid; 1H NMR (400 MHz, CD3OD): d ˆ 4.76 (d, Jvic ˆ 13.93 Hz, 1H),
4.61 (d, Jvic ˆ 14.2 Hz, 1H), 4.25 (t, Jvic ˆ 7.3 Hz, 1H), 4.20 (s, 2H), 3.55 (s,
9H), 1.43 (d, Jvic ˆ 8.6 Hz, 3H), 1.42 (s, 9H); 13C NMR (100.5 MHz,
CD3OD): d ˆ 172.75, 155.45, 80.11, 64.84, 58.65, 54.40 (3 C), 49.31, 28.34 (3
C), 17.54; [a]2D0
ˆ
23.5 (c ˆ 1, CHCl3); HRMS (FAB) (glycerol): m/z:
‡
275.1935 ([M] ) (calcd for C13H27BrN2O4: 275.1971); elemental analysis for
the hydrate (%): calcd C 42.86, H 7.75, N 7.69; found C 43.05, H 7.70, N 7.73.
Boc-l-leucine choline ester bromide (20b): Yield: 99%; very hygroscopic
white solid; 1H NMR (400 MHz, CDCl3): d ˆ 5.12 (d, Jvic ˆ 7.5 Hz, 1H),
4.81 (d, Jvic ˆ 14.5 Hz, 1H), 4.52 (d, Jvic ˆ 14.7 Hz, 1H), 4.18 (m, 3H), 3.55 (s,
9H), 1.70 (m, Jvic ˆ 6.6 Hz, 1H), 1.59 (t, Jvic ˆ 7.5 Hz, 2H), 1.42 (s, 9H), 0.95
(dd, J1 ˆ 6.6 Hz, J2 ˆ 6.7 Hz, 6H); 13C NMR (100.5 MHz, CDCl3): d ˆ
172.96, 155.78, 80.31, 64.92, 58.51, 54.50 (3 C), 52.36, 40.32, 28.31 (3 C),
24.76, 21.63, 22.87; [a]D20
m/z: 317.2389 ([M Br]) (calcd for C16H33N2O4: 317.2440); elemental
analysis for C16H33N2O4Br, hemihydrate (%): calcd C 47.29, H 8.43, N 6.89;
found C 47.38, H 8.45, N 7.01.
ˆ
23.7 (c ˆ 1, CH3OH); HRMS (FAB) (glycerol):
Boc-l-phenylalanine choline ester bromide (20c): Yield: 94%; very
hygroscopic white solid; 1H NMR (500 MHz, CDCl3): d ˆ 7.29 ± 7.37 (m,
3H), 7.21 (d, Jvic ˆ 7.3 Hz, 2H), 5.20 (d, Jvic ˆ 7.1 Hz, 1H), 4.67 (dd, Jvic
6.6 Hz, Jgem ˆ 14.4 Hz, 1H), 4.43 (q, Jvic ˆ 7.3 Hz, 2H), 4.11 (dd, Jvic
ˆ
ˆ
6.7 Hz, Jgem ˆ 13.8 Hz, 1H), 3.96 (dd, Jvic ˆ 6.6 Hz, Jgem ˆ 13.9 Hz, 1H),
3.03 ± 3.14 (m, 2H), 3.38 (s, 9H), 1.41 (s, 9H); 13C NMR (125.7 MHz,
CDCl3): d ˆ 171.68, 155.33, 129.25 (2 C), 128.92 (2 C), 127.41 (2 C), 80.55,
Boc-amino acid-2-bromoethyl esters (19a ± d): A solution of DIC (5.34 g,
26 mmol) in CH2Cl2 (40 mL) was added slowly at 0 8C to a solution of BOC
amino acid 18a ± d (21.7 mmol), DMAP (269 mg, 2.2 mmol), and 2-bro-
moethanol (1.86 mL, 26 mmol) in CH2Cl2 (80 mL). After stirring for 12 h,
the solution was filtered, extracted with dilute aqueous HCl, conc. aqueous
NaHCO3, and water. The organic phase was dried with MgSO4, concen-
trated, and the crude product was purified by chromatography on silica gel
(hexane/ethyl acetate 2:1).
64.78, 58.47, 55.20, 54.36 (3 C), 38.14, 28.30 (3 C); [a]D20
ˆ
2.3 (c ˆ 1,
CH3OH); elemental analysis for C19H31BrN2O4, hemihydrate (%): calcd C
51.82, H 7.32, N 6.36; found C 51.39, H 7.25, N 6.31.
Boc-l-tyrosine choline ester bromide (20d): Yield: 93%; very hygroscopic
white solid; 1H NMR (500 MHz, CD3OD): d ˆ 7.05 (m, 2H), 6.73 (m, 2H,
Jvic ˆ 7.9 Hz), 4.54 (m, 1H), 4.42 (m, 1H), 4.27 (t, Jvic ˆ 7.7 Hz, 1H), 3.70 (dd,
Jvic ˆ 6.7 Hz, Jgem ˆ 13.8 Hz, 1H), 3.55 (dd, Jvic ˆ 6.6 Hz, Jgem ˆ 13.9 Hz, 1H),
3.38 (s, 9H), 2.94 (m, 2H), 1.38 (s, 9H); [a]2D0 ˆ 2.7 (c ˆ 2, CH3OH);
HRMS (FAB) (glycerol): m/z: 367.2293 ([M Br]) (calcd for C19H31N2O5:
Boc-l-alanine-2-bromoethyl ester (19a): Yield: 95%; colorless oil; Rf ˆ
0.80 (hexane/ethyl acetate 1:1); 1H NMR (400 MHz, CDCl3): d ˆ 5.17 (d,
Jvic ˆ 6.7 Hz, 1H), 4.48 (ddt, J1±1' ˆ 6.0 Hz, J2±1' ˆ 5.9 Hz, 2H), 4.34 (t, Jvic
ˆ
7.3 Hz, 1H), 3.53 (t, Jvic ˆ 6.1 Hz, 2H), 1.45 (s, 9H), 1.43 (d, Jvic ˆ 7.3 Hz,
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
678
0947-6539/99/0502-0678 $ 17.50+.50/0
Chem. Eur. J. 1999, 5, No. 2

Chemoenzymatic Synthesis of Nucleopeptides
669±681
367.2233); elemental analysis for C19H31N2O5Br (%): calcd C 51.01, H 6.98,
N 6.26; found C 50.92, H 7.18, N 6.04.
141.65, 134.19, 132.58, 131.87, 129.57, 128.67, 127.20, 119.26, 117.94, 84.51,
83.55, 74.21, 69.07, 68.29, 67.16, 65.99, 65.66, 60.87, 54.80, 54.60 (3 C), 52.59,
44.33, 40.26, 37.99, 25.57, 22.26, 22.70, 20.95; [a]D20
ˆ
11.5 (c ˆ 1, CH3OH);
Amino acid choline ester bromide hydrobromides (21a ± d): A solution of
the choline esters 20a ± d (8.2 mmol) in HBr in glacial acetic acid (45%,
25 mL) was stirred for 30 min at room temperature. After addition of Et2O
(150 mL), the precipitate was filtered, dissolved in CH3OH (3 mL), and the
product reprecipitated with Et2O.
HRMS (FAB) (glycerol): m/z: 901.4031 ([M Br]) (calcd for
C41H58N8O13P: 901.3861).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-seryl-l-phenylalanine choline ester bromide (22c):
1
l-Alanine choline ester bromide hydrobromide (21a): Yield: 96%; slightly
Yield: 96%; colorless oil; H NMR (500 MHz, CD3OD): d ˆ 8.64 (s, 1H),
1
yellow, hygroscopic solid; m.p. 2158C; H NMR (400 MHz, CD3OD): d ˆ
7.94 (m, 1H), 7.44 ± 7.21 (m, 10H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H),
5.41 ± 5.16 (m, 5H), 4.75 ± 4.10 (m, 15H), 3.83 (m, 1H), 3.69 (m, 1H), 3.29
(d, Jvic ˆ 7.3 Hz, 2H), 3.14 (s, 9H), 2.79 ± 2.60 (m, 2H), 2.11 (s, 3H);
13C NMR (125.7 MHz, CD3OD): d ˆ 172.31, 170.58, 170.52, 169.52, 156.30,
152.65, 151.03, 149.09, 141.65, 135.62, 134.19, 132.58, 131.87, 130.62, 130.31,
129.57, 129.06, 128.67, 127.20, 119.26, 117.94, 84.51, 83.55, 74.21, 69.07, 68.29,
67.16, 65.99, 65.60, 60.73, 55.28, 54.80, 54.51 (3 C), 44.33, 37.99, 37.38, 20.95;
4.75 (t, Jvic ˆ 2.5 Hz, 2H), 4.30 (q, Jvic ˆ 7.2 Hz, 1H), 3.93 (t, Jvic ˆ 4.7 Hz,
2H), 3.32 (s, 9H), 1.61 (d, Jvic ˆ 7.3 Hz, 2H); 13C NMR (100.5 MHz,
CD3OD): d ˆ 170.38, 65.72, 60.84, 54.59 (3 C), 49.93, 16.07; [a]2D0 ˆ 2.8 (c ˆ
1, CH3OH); elemental analysis for C8H20Br2N2O2 (%): calcd C 28.59, H
6.00, N 8.34; found C 28.53, H 5.94, N 8.30.
l-Leucine choline ester bromide hydrobromide (21b): Yield: 99%; slightly
1
[a]2D0
ˆ
10.5 (c ˆ 1, CH3OH); HRMS (FAB) (glycerol): m/z: 935.3771
yellow, hygroscopic solid; m.p. 2108C; H NMR (400 MHz, CD3OD): d ˆ
4.82 (d, Jvic ˆ 14.2 Hz, 1H), 4.73 (d, Jvic ˆ 14.4 Hz, 1H), 4.23 (q, Jvic ˆ 6.9 Hz,
1H), 3.97 (t, Jvic ˆ 4.7 Hz, 2H), 3.35 (s, 9H), 1.75 ± 1.92 (m, 3H), 1.02 (t,
Jvic ˆ 6.2 Hz, 6H); 13C NMR (100.5 MHz, CD3OD): d ˆ 170.35, 65.66, 60.87,
54.60 (3 C), 52.59, 40.26, 25.57, 22.70, 22.26; [a]2D0 ˆ 13.9 (c ˆ 2, CH3OH);
HRMS (FAB) (glycerol): m/z: 217.1892 ([M 2Br H]) (calcd for
C11H25N2O2: 217.1916); elemental analysis for C11H26Br2N2O2 (%): calcd
C 34.94, H 6.93, N 7.41; found C 35.10, H 6.77, N 7.45.
([M Br]) (calcd for C44H56N8O13P: 935.3704).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-seryl-l-tyrosine choline ester bromide (22d): Yield:
99%; colorless oil; 1H NMR (500 MHz, CD3OD): d ˆ 8.65 (s, 1H), 7.97 (m,
1H), 7.38 ± 7.21 (m, 5H), 7.18 (m, 2H), 6.83 (m, 2H), 6.56 ± 6.27 (m, 1H),
5.93 ± 5.81 (m, 2H), 5.41 ± 5.16 (m, 5H, 3'-H), 4.74 (m, 1H), 4.62 (m, 1H),
4.58 ± 4.10 (m, 13H), 3.85 ± 3.71 (m, 2H), 3.38 (s, 9H), 2.94 (m, 2H), 2.80 ±
2.61 (m, 2H), 2.13 (s, 3H); 13C NMR (125.7 MHz, CD3OD): d ˆ 173.48,
171.26, 170.54, 170.51, 156.06, 152.65, 151.03, 149.09, 141.65, 134.19, 132.58,
131.87, 130.43, 130.38, 129.57, 128.67, 127.28, 127.20, 119.26, 117.94, 115.60,
84.51, 83.55, 74.21, 69.07, 68.29, 67.16, 66.76, 65.99, 60.54, 54.80, 54.55 (3 C),
l-Phenylalanine choline ester bromide hydrobromide (21c): Yield: 99%;
slightly yellow, hygroscopic solid; m.p. 1308C; 1H NMR (400 MHz,
CD3OD): d ˆ 7.33 ± 7.44 (m, 5H), 4.75 (dddd, J2±1' ˆ 2.4 Hz, J2±2' ˆ 6.8 Hz,
J2±1 ˆ 14.1 Hz, 1H), 4.60 (dddd, J1±1' ˆ 2.5 Hz, J1±2' ˆ 6.8 Hz, J1±2 ˆ 14.2 Hz,
1H), 4.52 (t, Jvic ˆ 7.3 Hz, 1H), 3.83 (dddd, J2±1' ˆ 2.3 Hz, J2±2' ˆ 7.1 Hz, J2±1
ˆ
54.39, 44.33, 38.23, 37.99, 20.95; [a]D20
(FAB) (glycerol): m/z: 951.3318 ([M Br]) (calcd for C44H56N8O14P
ˆ
13.3 (c ˆ 1.5, CH3OH); HRMS
14.7 Hz, 1H), 3.69 (dddd, J1±1' ˆ 2.3 Hz, J1±2' ˆ 7.0 Hz, J1±2 ˆ 14.7 Hz, 1H),
3.29 (d, Jvic ˆ 7.3 Hz, 2H), 3.14 (s, 9H); 13C NMR (100.5 MHz, CD3OD):
d ˆ 169.62, 135.62, 130.62 (2 C), 130.31 (2 C), 129.06 (p-CH), 65.60, 60.73,
55.28, 54.51 (3 C), 37.38; [a]2D0 ˆ 16.6 (c ˆ 1, CH3OH); HRMS (FAB)
(glycerol): m/z: 251.1782 ([M 2Br H]) (calcd for C14H23N2O2:
251.1760); elemental analysis for C14H24Br2N2O2, hemihydrate (%): calcd
C 39.93, H 5.98, N 6.65; found C 40.14, H 5.92, N 6.43.
951.3654).
N-Allyloxycarbonyl-O-diallylphosphato-l-seryl-l-alanine choline ester
1
bromide (23): Yield: 85%; colorless oil; H NMR (500 MHz, CDH3OD):
d ˆ 6.01 ± 5.90 (m, 3H), 5.41 ± 5.37 (m, 2H), 5.37 ± 5.34 (m, 1H), 5.28 ± 5.25
(m, 2H), 5.19 (d, Jcis ˆ 10.5 Hz, 1H), 4.75 (t, Jvic ˆ 2.5 Hz, 2H), 4.58 ± 4.48
(m, 6H), 4.42 ± 4.30 (m, 4H), 3.93 (t, Jvic ˆ 4.7 Hz, 2H), 3.32 (s, 9H), 1.45 (d,
Jvic ˆ 7.3 Hz, 2H); 13C NMR (125.7 MHz, CD3OD): d ˆ 171.9, 170.4, 158.1,
134.1, 133.7 (2 C), 118.9 (2 C), 117.7, 69.8 (2 C), 68.5, 66.7, 65.7, 60.8, 55.6,
l-Tyrosine choline ester bromide hydrobromide (21d): Yield: 91%;
hygroscopic white solid; m.p. 1818C; 1H NMR (500 MHz, CD3OD): d ˆ
7.18 (m, 2H), 6.83 (m, 2H), 4.74 (m, 1H), 4.62 (m, 1H), 4.41 (t, Jvic ˆ 7.7 Hz,
1H), 3.85 (dd, Jvic ˆ 6.7 Hz, Jgem ˆ 13.8 Hz, 1H), 3.71 (dd, Jvic ˆ 6.6 Hz,
Jgem ˆ 13.9 Hz, 1H), 3.38 (s, 9H), 2.94 (m, 2H); [a]2D0 ˆ 12.1 (c ˆ 1,
CH3OH); HRMS (FAB) (glycerol): m/z: 267.1662 ([M 2Br H]) (calcd
for C14H23N2O3 267.1709); elemental analysis for C14H24Br2N2O3 (%): calcd
C 39.27, H 5.65, N 6.54; found C 38.99, H 5.79, N 5.96.
54.6 (3 C), 49.9, 16.1; [a]D20
(glycerol): m/z: 506.2206 ([M Br]) (calcd for C21H37N3O9P: 506.2267).
ˆ
10.9 (c ˆ 1, CH3OH); HRMS (FAB)
C-Terminal deprotection of nucleo- and phosphopeptide choline esters by
means of butyrylcholine esterase (24a ± d, 25): A solution of 22a ± d or 23
(0.03 mmol) and butyrylcholine esterase (30 U) in phosphate buffer
(pH 6.5, 50mm, 20 mL) was shaken for 64 h at 37 8C. The solution was
lyophilized, and the residue was treated sequentially with CH2Cl2, CHCl3,
and CH3OH. The combined organic solutions were concentrated, and the
product was isolated after chromatography on silica gel (chloroform/
methanol 4:1).
Nucleo- and phospho-dipeptide choline ester bromides (22a ± d, 23): A
solution of 11a or 12 (0.036 mmol), HOAt (5.3 mg, 0.039 mmol), and DIC
(6.03 mg, 0.039 mmol) in dry CH2Cl2 (3 mL) was stirred for 30 min and then
added to
a solution of 21a ± d (0.036 mmol) and NBu3 (8.47 mL,
0.036 mmol) in DMF (3 mL). After 14 h, the solution was concentrated
under reduced pressure, and the product was precipitated by addition of
dry Et2O (10 mL). The precipitate was filtered, dissolved in dry CH3OH
(1 mL), and reprecipitated with dry Et2O.
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-seryl-l-alanine (24a): Yield: 96%; colorless oil;
1H NMR (500 MHz, CD3OD): d ˆ 8.65 (s, 1H), 8.42 (m, 1H), 7.38 ± 7.21
(m, 5H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H), 5.41 ± 5.16 (m, 5H), 4.58 ±
4.10 (m, 13H), 2.79 ± 2.60 (m, 2H), 2.02 (s, 3H), 1.61 (d, Jvic ˆ 7.3 Hz, 2H);
13C NMR (125.7 MHz, CD3OD): d ˆ 173.21, 170.77, 170.57, 170.34, 155.88,
152.68, 151.00, 149.11, 141.65, 134.19, 132.58, 131.77, 129.57, 128.67, 127.23,
119.26, 117.94, 84.51, 83.55, 74.21, 69.07, 68.29, 67.16, 65.72, 54.86, 54.59,
49.93, 44.33, 38.11, 20.88, 16.12; [a]2D0 ˆ 1.4 (c ˆ 1, CH3OH); HRMS
(FAB) (glycerol): m/z: 796.2228 ([M‡Na]) (calcd for C33H40N7O13PNa:
796.2319).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-seryl-l-alanine choline ester bromide (22a): Yield:
99%; colorless oil; 1H NMR (500 MHz, CD3OD): d ˆ 8.77 (s, 1H), 8.44
(m, 1H), 7.38 ± 7.21 (m, 5H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H), 5.41 ±
5.16 (m, 5H), 4.75 (t, Jvic ˆ 2.5 Hz, 2H), 4.58 ± 4.10 (m, 13H), 3.93 (t, Jvic
ˆ
4.7 Hz, 2H), 3.32 (s, 9H), 2.79 ± 2.60 (m, 2H), 2.14 (s, 3H), 1.51 (d, Jvic
ˆ
7.3 Hz, 2H); 13C NMR (125.7 MHz, CD3OD): d ˆ 172.51, 170.59, 170.55,
170.38, 156.11, 152.65, 151.03, 149.09, 141.65, 134.19, 132.58, 131.87, 129.57,
128.67, 127.20, 119.26, 117.94, 84.51, 83.55, 74.21, 69.07, 68.29, 67.16, 65.99,
65.72, 60.84, 54.80, 54.59 (3 C), 49.93, 44.33, 37.99, 20.95, 16.07; [a]D20
ˆ
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosine-
allyl-phosphato)-l-seryl-l-leucine (24b): Yield: 78%; colorless oil;
1H NMR (500 MHz, CD3OD): d ˆ 8.78 (s, 0.5H), 8.66 (s, 0.5H), 8.64 (m,
0.5H), 8.52 (m, 0.5H, 7.38 ± 7.21 (m, 5H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m,
2H), 5.41 ± 5.16 (m, 5H), 4.58 ± 4.10 (m, 13H), 2.79 ± 2.60 (m, 2H), 2.13 (s,
3H), 1.75 ± 1.92 (m, 3H), 0.90 (m, 6H); 13C NMR (125.7 MHz, CD3OD):
d ˆ 171.99, 170.48, 170.41, 170.25, 156.23, 152.65, 151.03, 149.09, 141.65,
134.03, 132.58, 131.87, 129.57 128.67, 127.20, 119.26, 117.86, 84.61, 83.66,
74.25, 69.07, 68.29, 67.16, 66.21, 54.80, 52.59, 44.38, 40.26, 37.92, 25.56, 22.26,
11.5 (c ˆ 1, CH3OH); HRMS (FAB) (glycerol): m/z: 859.3330 ([M Br])
(calcd for C38H52N8O13P: 859.3391).
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosyl-
allyl-phosphato)-l-seryl-l-leucine choline ester bromide (22b): Yield:
91%; colorless oil; 1H NMR (500 MHz, CD3OD): d ˆ 8.67 (s, 1H), 8.54
(m, 1H), 7.38 ± 7.21 (m, 5H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H), 5.41 ±
5.16 (m, 5H), 4.82 (d, Jvic ˆ 14.2 Hz, 1H), 4.73 (d, Jvic ˆ 14.4 Hz, 1H), 4.58 ±
4.10 (m, 13H), 3.97 (t, Jvic ˆ 4.7 Hz, 2H), 3.35 (s, 9H), 2.79 ± 2.60 (m, 2H),
2.13 (s, 3H), 1.75 ± 1.92 (m, 3H), 0.92 (m, 6H); 13C NMR (125.7 MHz,
CD3OD): d ˆ 172.53, 170.79, 170.54, 170.35, 156.00, 152.65, 151.03, 149.09,
22.70, 20.99; [a]D20
838.2690 ([M‡Na]) (calcd for C36H46N7O13PNa: 838.2789).
ˆ
12.0 (c ˆ 1, CH3OH); HRMS (FAB) (glycerol): m/z:
Chem. Eur. J. 1999, 5, No. 2
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0502-0679 $ 17.50+.50/0
679

H. Waldmann et al.
FULL PAPER
N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosine-
allyl-phosphato)-l-seryl-l-phenylalanine (24c): Yield: 61%; colorless oil;
1H NMR (500 MHz, CD3OD): d ˆ 8.78 (s, 1H), 8.63 (m, 1H), 7.44 ± 7.21 (m,
10H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H), 5.41 ± 5.16 (m, 5H), 4.75 ±
4.10 (m, 13H), 3.29 (d, Jvic ˆ 7.3 Hz, 2H), 2.79 ± 2.60 (m, 2H), 2.11 (s, 3H);
13C NMR (125.7 MHz, CD3OD): d ˆ 172.71, 170.63, 170.48, 169.68, 155.85,
152.62, 151.31, 149.19, 141.86, 135.62, 134.19, 132.71, 131.76, 130.62, 130.31,
129.57, 129.06, 128.67, 127.20, 119.26, 117.94, 84.51, 83.55, 74.21, 69.07, 68.36,
67.19, 65.88, 55.28, 54.86, 44.38, 38.11, 37.38, 20.95; [a]2D0 ˆ 3.8 (c ˆ 1.5,
CH3OH); HRMS (FAB) (glycerol): m/z: 872.2795 ([M‡Na]) (calcd for
C39H44N7O13PNa: 872.2632); elemental analysis for C39H44N7O13P, tetrahy-
drate (%): calcd C 50.81, H 5.69, N 10.85; found C 50.99, H 5.56, N 11.14.
Acknowledgments
This research was supported by the Bundesministerium für Bildung und
Forschung, the Fonds der Chemischen Industrie, and Boehringer Mann-
heim GmbH.
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N-Allyloxycarbonyl-O-(3'-O-acetyl-6-N-phenylacetyl-2'-deoxyadenosine-
allyl-phosphato)-l-seryl-l-tyrosine (24d): Yield: 86%; colorless oil;
1H NMR (500 MHz, CDCl3): d ˆ 8.67 (s, 1H), 8.44 (m, 1H), 7.38 ± 7.21
(m, 5H), 6.56 ± 6.27 (m, 1H), 5.93 ± 5.81 (m, 2H), 5.41 ± 5.16 (m, 5H), 4.58 ±
4.10 (m, 12H), 2.79 ± 2.60 (m, 2H), 2.12 (s, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 173.32, 170.75, 170.67, 155.87, 152.72, 151.01, 149.06, 141.87,
134.19, 132.67, 131.92, 129.57, 128.77, 127.24, 119.22, 117.83, 84.62, 83.58,
74.16, 69.09, 68.33, 67.18, 66.12, 54.88, 44.25, 37.88, 21.03; [a]2D0 ˆ 0.8 (c ˆ 1,
CH3OH); HRMS (FAB) (glycerol): m/z: 888.2462 ([M‡Na]) (calcd for
C39H44N7O14PNa: 888.2581).
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view: M. Salas, Virus Strategies - Molecular Biology and Pathogenesis,
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2311.
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G. M. Visser, G. A. van der Marel, J. H. van Boom, Recl. Trav. Chim.
Pays-Bas 1986, 105, 69 ± 70.
N-Allyloxycarbonyl-O-diallylphosphato-l-seryl-l-alanine (25): Yield:
73%; colorless oil; 1H NMR (500 MHz, CD3OD): d ˆ 6.01 ± 5.90 (m,
3H), 5.41 ± 5.19 (m, 6H), 4.58 ± 4.48 (m, 6H), 4.42 ± 4.30 (m, 4H), 1.39 (d,
[6] a) E. Kuyl-Yeheskiely, C. M. Tromp, A. W. M. Lefeber, G. A. van -
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J. H. van Boom, Nucleic Acids Res. 1989, 17, 2897 ± 2905; c) C. M.
Dreef-Tromp, J. C. M. van der Marel, H. van den Elst, G. A. van -
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d) Y. Ueno, R. Saito, T. Hata, Nucleic Acids Res. 1993, 21, 4451 ± 4457;
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59, 2482 ± 2486.
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Jvic ˆ 7.3 Hz, 3H); [a]D20
ˆ
3.0 (c ˆ 0.5, CH3OH); HRMS (FAB) (glycerol):
m/z: 421.1439 ([M‡H]) (calcd for C16H25N2O9P: 421.1376).
N-Allyloxycarbonyl-O-(3'-O-acetyl-2'-deoxycytidine-allyl-phosphato)-l-
seryl-l-glycyl-l-aspartic acid-g-allyl ester (26): A solution of 17d (29 mg,
32 mmol) in CH3OH (4 mL) was added dropwise to a suspension of
immobilized penicillin G acylase (500 mg, 60 units) in phosphate buffer
(15 mL, 0.07m, pH 7) at 258C. After 18 h, the mixture was filtered and
extracted with chloroform (8 Â 20 mL). The organic phase was dried with
MgSO4, concentrated, and the residue was purified by chromatography on
silica gel (chloroform/ethanol 40:1 ± 3:1). Yield: 91%; white solid; m.p.
1138C; 1H NMR (500 MHz, CDCl3, MeOD): d ˆ 7.61 (m, 1H), 7.28 (m,
1H), 6.24 (m, 1H), 5.99 ± 5.86 (m, 3H), 5.37 (m, 0.5H), 5.33 (m, 0.5H),
5.29 ± 5.14 (m, 6H), 4.54 ± 3.71 (m, 15H), 2.87 (m, 2H), 2.48 ± 2.04 (m, 5H);
13C NMR (125.75 MHz, CDCl3, MeOD): d ˆ 207.43, 176.34, 172.30, 170.72,
169.34, 165.07, 156.36, 155.39, 140.82, 132.55, 132.53, 132.05, 119.06, 118.27,
117.98, 95.97, 86.28, 82.86, 74.43, 68.94, 67.57, 67.44, 66.91, 65.48, 54.84, 50.14,
42.83, 37.65, 36.79, 20.92; [a]2D0 ˆ 5.3 (c ˆ 0.15, CH2Cl2); HRMS
(FAB) (glycerol/TFA): m/z: 773.2395 ([M‡H) (calcd for C30H42O15N6P:
773.2274).
O-(3'-O-acetyl-2'-deoxycytidine-phosphato)-l-seryl-l-glycyl-l-aspartic
acid (27): Tetrakis(triphenylphosphane)palladium(0) (5 mg, 4 mmol) and
phenylsilane (15 mL, 0.1 mmol) at room temperature were added to a
solution of 26 (14 mg, 18 mmol) in CHCl3 (1.5 mL). After 5 h, the solution
was concentrated, extracted with acetone, methanol, and water. The water
layer was concentrated to give 27. Yield: 73%; yellow solid; m.p. 192 8C
(decomp); 1H NMR (500 MHz, D2O): d ˆ 7.76 (d, J ˆ 7.7 Hz, 1H), 6.17 (dd,
J1',2'a ˆ 6.2 Hz, J1',2'b ˆ 7.8 Hz, 1H), 5.97 (d, J ˆ 7.1 Hz, 1H), 5.20 (d, J ˆ
6.3 Hz, 1H), 4.29 ± 3.60 (m, 9H), 2.58 ± 2.39 (m, 3H), 2.22 (m, 1H), 1.97
(s, 3H); 13C NMR (125.75 MHz, D2O): d ˆ 179.21, 179.07, 174.79, 171.06,
168.52, 165.67, 156.53, 143.22, 97.53, 87.45, 84.53, 76.16, 67.59, 66.61, 54.71,
53.61, 43.83, 38.16, 37.53, 21.65; [a]2D0 ˆ 4.0 (c ˆ 0.05, H2O); HRMS (FAB)
(glycerol/TFA): m/z: 609.1558 (calcd for C20H30O14N6P: 609.1440).
O-(2'-deoxycytidine-phosphato)-l-seryl-l-glycyl-l-aspartic acid (28):
A
mixture of 27 (8 mg, 13 mmol) in hydrazine hydrate (300 mL) was stirred
at room temperature, and after 2 h, acetone (3 mL) was added at 08C. The
mixture was concentrated, and extracted with methanol. The remaining
residue was dissolved in water, filtered through a Sep-Pak Cartridge from
Waters, and concentrated to give 28. Yield: 85%; white solid; m.p. 2308C
(decomp); 1H NMR (500 MHz, D2O): d ˆ 7.69 (d, J ˆ 6.9 Hz, 1H), 6.14 (s,
1H), 5.91 (s, 1H), 4.35 ± 3.68 (m, 10H), 2.52 ± 1.64 (m, 4H); 13C NMR
(125.75 MHz, D2O): d ˆ 178.97, 178.43, 174.71, 170.24, 166.13, 157.57,
141.41, 96.40, 85.99, 85.23, 70.69, 67.09, 64.91, 54.66, 52.92, 42.34, 39.46,
39.45; [a]2D0 ˆ 9.0 (c ˆ 0.1, H2O); HRMS (FAB) (glycerol/TFA): m/z:
565.1124 ([M H]) (calcd for 565.1295).
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Â
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Received: June 25, 1998 [F1233]
Chem. Eur. J. 1999, 5, No. 2
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