Synthesis of nucleopeptides by employing an enzyme-labile urethane protecting group

Article abstract of DOI:10.1002/1521-3765(20020415)8:8<1879::AID-CHEM1879>3.0.CO;2-5

Nucleoproteins are naturally occurring biopolymers in which the hydroxy group of a serine, a threonine, or a tyrosine moiety is linked through a phosphodiester group to the 3′- or 5′-end of a nucleic acid. For the study of the biological phenomena in whic

Full text of DOI:10.1002/1521-3765(20020415)8:8<1879::AID-CHEM1879>3.0.CO;2-5

FULL PAPER
Synthesis of Nucleopeptides by Employing an Enzyme-Labile
Urethane Protecting Group
Duraiswamy A.Jeyaraj, [a, b] Heino Prinz,[a] and Herbert Waldmann*[a, b]
Abstract: Nucleoproteins are naturally
occurring biopolymers in which the
hydroxy group of a serine, a threonine,
or a tyrosine moiety is linked through a
phosphodiester group to the 3'- or 5'-end
of a nucleic acid. For the study of the
biological phenomena in which nucleo-
proteins are involved, for example, viral
replication, nucleopeptides embodying
the characteristic linkage between the
peptide chain and the oligonucleotide
may serve as powerful tools. However,
as a result of the multifunctionality and
the pronounced acid and base lability of
nucleopeptides, their synthesis requires
the application of a variety of orthogo-
nally stable blocking groups, which can
be removed under the mildest condi-
tions. We have developed a new mild
enzymatic deprotection method, that is,
the penicillin G acylase-catalyzed hy-
drolysis of the N-phenylacetoxybenzyl-
oxycarbony (PhAcOZ) group, for the
synthesis of nucleopeptides. We demon-
strate the wide applicability of this
method by coupling the N-terminally
deprotected nucleopeptides 31a c with
PhAcOZ-protected amino acids and
subsequent removal of the N-PhAcOZ
group from fully protected nucleotetra-
peptides 32a,b with penicillin G acylase.
The reaction conditions are very mild
(pH 6.8) so that no undesired side re-
action such as cleavage of the nucleotide
bond or b-elimination of the nucleotide
was observed.
Keywords:
peptides
protecting groups
enzymes
¥
nucleo-
¥
penicillin G acylase
¥
For the study of the biological
phenomena in which nucleo-
proteins are involved, nucleo-
peptides embodying the char-
acteristic linkage between the
peptide chain and the oligonu-
cleotide may serve as powerful
tools. However, as a result of
the multifunctionality of nucleo-
peptides their synthesis re-
quires the application of a vari-
ety of orthogonally stable
blocking groups. For example,
different functionalities such as
hydroxyl, amine, phosphate,
acid, and amine groups require
the application of different types of protecting groups that can
be selectively cleaved under very mild conditions. In addition,
fully protected serine/threonine-containing nucleopeptides
are very acid and base labile; this makes most of the
established blocking functions inapplicable to nucleopeptide
synthesis. Under acidic conditions the purine nucleotide may
be depurinated[6] and under basic conditions the entire
oligonucleotide part may be split off by b-elimination (Fig-
ure 2).
Introduction
Nucleoproteins are naturally occurring biopolymers in which
the hydroxy group of a serine, a threonine, or a tyrosine
moeity is linked through a phosphodiester group to the 3'- or
5'-end of a nucleic acid.[1] These protein conjugates play
decisive roles in important biological processes such as viral
replication.[2] The emerging biological importance of nucleo-
proteins was recently highlighted by the finding that the
p53 protein is a nucleoprotein.[3] Nucleoprotein p53 is a
tumour suppressor protein that is critically involved in cell-
cycle regulation.[4] It can potently suppress cellular trans-
formation, inhibit tumorigenesis, arrest cell growth, and
induce apoptotic death. In its biologically active form, p53 is
covalently linked at Ser-386 to a nucleic acid through a 5'-
phosphodiester group[5] and the serine is flanked by two
aspartic acid residues (Figure 1).
Figure 1. Structure of the nu-
cleopeptide representing the
linking region between peptide
and nucleotide in the p53 pro-
tein.
[a] Prof. Dr. H. Waldmann, Dr. D. A. Jeyaraj, Priv.-Doz. Dr. H. Prinz
Max-Planck-Institut f¸r molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Str. 11, 44227 Dortmund (Germany)
Fax : (‡49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[b] Prof. Dr. H. Waldmann, Dr. D. A. Jeyaraj
Universit‰t Dortmund, Fachbereich 3
Thus, it is not surprising that only a few reports on the
successful synthesis of nucleopeptides have appeared.[7 In
10]
Organische Chemie, 44221 Dortmund (Germany)
Chem. Eur. J. 2002, 8, No. 8
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H. Waldmann et al.
FULL PAPER
acids of the aspartic acid (Asp) residues and the pronounced
tendency of Asp to undergo undesired side reactions (see
below) would significantly complicate the matter.
In initial experiments aimed at the enzymatic deprotection
of nucleopeptides, N-PhAcOZ-protected serine nucleotide
esters like 2 were treated with penicillin G acylase (PGA). In
the ensuing enzymatic reaction the urethane group was
indeed cleaved (Scheme 1).
Figure 2. Acid and base lability of nucleopeptides. B ˆ adenine, cytosine,
guanine, or thymine.
particular, the development of protecting groups allowing the
assembly of sensitive serine/threonine-containing nucleopep-
tides through sequential selective N-terminal deprotection
and chain elongation has posed a major problem. However,
the application of enzyme protecting-group techniques may
offer an alternative to classical chemical methods.[11] In this
paper we report that the enzyme-labile phenylacetoxy ben-
zyloxycarbonyl group (PhAcOZ)[12] fulfils the extraordinary
demands of nucleopeptide chemistry concerning chemoselec-
tivity and mildness of cleavage conditions.[13] The PhAcOZ
group is an enzyme-labile urethane that is cleaved under very
mild conditions by penicillin G acylase (PGA). PGA hydro-
lyzes the phenylacetic acid phenyl ester, and subsequent
fragmentation of the resultant phenolate gives a quinone
methide (which is trapped by nucleophiles) and the desired
unmasked amino acid ester (Figure 3).
Scheme 1. Penicillin G acylase-mediated cleavage of N-PhAcOZ-protect-
ed serine nucleotide 2.
However, the N-terminally unmasked nucleoamino acid
ester 4 could not be isolated. Rather, the reaction product
decomposed under the reaction conditions, probably through
elimination of the nucleotide.
To overcome this problem in a second series of experi-
ments, dipeptide nucleotide esters were subjected to the
enzymatic reaction conditions. As hoped, the deprotection
occurred smoothly and rapidly, but the liberated dinucleo-
peptide esters underwent competing diketopiperazine forma-
tion, a well-known and undesired side reaction in peptide
chemistry.
Therefore, nucleotripeptides were chosen as an appropriate
starting point to investigate the applicability of the PhAcOZ
group in nucleopeptide chemistry.
Figure 3. Principle of the enzyme-initiated cleavage of the N-phenyl-
acetoxybenzyloxycarbony (PhAcOZ) urethane.
Results and Discussion
In the quest for an enzymatic technique, we focussed on the
establishment of a general method for the synthesis of various
nucleopeptides under appropriate reaction conditions. We
also aimed to synthesize the p53-related nucleopeptide 1 as a
particularly demanding example. Thus, we anticipated the
need to selectively mask and unmask the side chain carboxylic
Initial experiments were carried out by employing an
aspartyl nucleopeptide corresponding to 1 as the substrate in
the enzymatic deprotection reaction. To this end, tripeptide 15
was prepared as summarized in Scheme 2. The fully protected
1880
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Chem. Eur. J. 2002, 8, No. 8

Synthesis of Nucleopeptides
1879 1887
Scheme 3. Synthesis of 3'-acetyl-4-N-allyloxycarbonyl-2'-deoxycytidine
(20).
with tetrabutylammonium fluoride (TBAF) gave the desired
partially protected deoxycytidine 20 in very high yield.
The central serine of tripeptide 15 was then coupled to the
selectively masked deoxycytidine 20 by formation of a
phosphodiester bond. Upon treatment of 15 with phosphor-
diamidite 21[18] in the presence of tetrazole and diisopropyl-
amine at 08C, a hydrolysis-sensitive intermediate 22 was
formed. This was converted without isolation into the desired
peptide conjugate 23 by treatment with deoxycytidine build-
ing block 20 in the presence of tetrazole, and subsequent
oxidation of the formed phosphites to the corresponding
phosphates by means of tert-butyl hydroperoxide at 08C
(Scheme 4). The product was purified by gel permeation
Scheme 2. Synthesis of N-PhAcOZ-protected tripeptide 15.
aspartic acid 7 was obtained in high yield from the cesium salt
of Boc-Asp-OH by treatment with allyl bromide.[14] Then, the
Boc group was cleaved from 7 with trifluoroacetic acid (TFA)
to give the aspartic acid diallyl ester TFA salt 8 in quantitative
yield. Aspartic acid diallyl ester 8 was coupled with Boc-Ser-
OH (9) in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-
dihydroquinoline (EEDQ), to furnish dipeptide 10 in high
yield. The Boc group was cleaved with TFA in dichloro-
methane to give the dipeptide TFA salt 11. To synthesize the
PhAcOZ-protected aspartic acid derivative 14, the g-carbox-
ylic acid of aspartic acid 12 was selectively esterified according
to a known procedure.[15] Treatment of 13 with PhAcOZ-Cl
then gave the aspartic acid derivative 14 in high yield.
Coupling of compounds 14 and 11 in the presence of
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochlor-
ide (EDC)/1-hydroxybenzotriazole (HOBT) and triethyl-
amine proceeded smoothly to yield tripeptide 15.
The required partially protected deoxycytidine was pre-
pared as shown in Scheme 3. The free amino group present in
deoxycytidine 16 was protected with an allyloxycarbonyl
(Aloc) protecting group by reaction with allyloxycarbonyloxy-
benzotriazole (Aloc-OBT) following a literature proce-
dure.[16] Then, the 5'-OH group of 17 was selectively masked
as a tetrabutyldimethylsilyl (TBDMS) ether by reaction with
one equivalent of TBDMSCl.[17] Compound 18 was acylated at
the 3'-OH group by means of acetic anhydride in pyridine to
yield the fully protected deoxycytidine 19 in quantitative
yield. Selective removal of the TBDMS ether by treatment
Scheme 4. Enzymatic cleavage of the PhAcOZ group from nucleotripep-
tide 23 with penicillin G acylase.
chromatography after filtering through silica gel to yield the
nucleotripeptide 23 in 40% yield.
Enzymatic cleavage of the PhAcOZ group by penicillin G
acylase (PGA): Completely masked nucleotripeptide 23 was
subjected to selective enzymatic deprotection with PGA. The
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H. Waldmann et al.
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PhAcOZ protected nucleopep-
tide 23 was treated with the
enzyme in a mixture of 0.05m
phosphate buffer and methanol
(20 vol%) at pH 6.8 and room
temperature, in the presence of
0.1m KI as a trapping reagent
for the quinone methide
formed in the enzyme-initiated
cleavage of the urethane.[12]
NMR spectroscopic investiga-
tion of the product mixture
initially revealed that the aro-
matic signals characteristic for
the PhAcOZ group had disap-
peared, indicating that the con-
version had reached at least
80%. However, unexpectedly,
the isolation of the desired
nucleopeptide from the reac-
tion mixture proved to be very
difficult and yielded only 10
15% of the product. In addi-
Figure 4. Three-dimensional representation of an HPLC-MS experiment (time vs. m/z vs. relative abundance).
The gradient (percentage acetonitrile) is given in the background. The reaction products after the enzymatic
deprotection were loaded onto a C18-HD reverse-phase column and eluted with increasing amounts of
acetonitrile in 0.1% HCOOH. Masses ranging from m/z 400 1200 are given in the time range from 4 40 mi-
nutes.
tion,
a major product was
formed, which could not be
separated readily by means of conventional techniques. These
findings suggested either that the enzymatic reaction had not
proceeded in the expected way or that an undesired side
reaction had occurred once more.
The major product provides a
peak at m/z 871, that is 40 mass
units less than the expected
product of m/z 911. This was in
accordance with the NMR
spectrum of the crude reaction
mixture, which had a major
compound with only four allyl
groups instead of five allyl
groups expected for the desired
peptide. We speculated that this
peak in the mass spectrum was
due to the formation of an
intermediate aminosuccinyl de-
rivative 25 (Figure 5).
It has been suggested[19] that aminosuccinyl derivative
formation in aqueous solution is a common problem with
aspartic acid. Thus aspartyl peptides, in particular sequences
that contain aspartylglycine (Asp-Gly) and aspartylserine
(Asp-Ser),[20, 21] are prone to cyclize to five-ring imides under
acidic or basic conditions.
Fragmentation of the ion at m/z 911 in the mass spectrom-
eter (MS/MS experiments) yielded fragments of m/z 518 and
438, which corresponded to the peptide cleaved at the
phosphate with and without HPO3, respectively. The desired
product 24, was thus identified by its molecular mass (911.1)
and from its fragmentation pattern.
Since the compound in the salt form was partially soluble in
water, we encountered difficulties in isolating the product
from the buffer and the nucleophile used during the enzy-
matic deprotection. Conventional separation on silica gel and
extraction was not effective. Instead, the immobilized enzyme
was filtered and the solution was lyophilized. The phosphate
salts and the KI were separated from the required compound
Numerous attempts were made to overcome this severe
problem, for example, by varying different reaction param-
eters (temperature, pH, co-solvent) and increasing the
amount of biocatalyst. However, an increase in yield could
not be achieved. In particular, progress was hampered by the
fact that conventional analysis and separation techniques did
not give access to pure product, or allow clear conclusions to
be drawn about the product spectrum formed. After sub-
stantial experimentation, this problem was finally fully
analyzed by employing mass-spectrometric techniques. Since
these techniques subsequently proved to be of major impor-
tance in general, they are described below in detail for the
example discussed above.
Figure 5. Possible aminosuc-
cinyl intermediate 25 formed
during the reaction.
Identification of the reaction products: Identification of the
products from the deprotection reaction comprised the main
obstacle in the nucleopeptide synthesis. Matrix assisted laser
desorption ionization (MALDI) mass spectrometry gave a
variety of molecular masses, the relative abundance of which
varied with the matrix employed. The mass of the desired
product 24 was calculated to be 911 and could not be
identified above background level. FAB mass spectrometry
likewise allowed the detection of masses ranging from 800
1000, but not of the desired product. Nano-electrospray mass
spectrometry indicated that indeed a mass of 911 was present,
but its relative abundance was lower than a peak at m/z 909,
and thus it might have been an isotope of the latter. Only the
coupling of reverse phase chromatography to ESI mass
spectrometry (HPLC-MS) allowed the unambiguous identi-
fication of the reaction products (Figure 4).
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Synthesis of Nucleopeptides
1879 1887
by filtering through a C18 Sep-Pak cartridge and washing with
water (2 mL). The compound adsorbed on the C18 cartridge
was then eluted with methanol. The compound isolated by
this method was almost pure.
Enzymatic cleavage of the PhAcOZ-protecting group: Com-
pletely masked nucleotripeptides 30a c were then subjected
to selective enzymatic deprotection with penicillin G acylase
under the conditions described above. The enzyme smoothly
cleaved the phenylacetic acid ester and the selectively
unmasked nucleotripeptides 31a c were formed in high yield
(Scheme 7). The immobilized enzyme was filtered and the
These results clearly demonstrated that the enzymatic
protecting group technique had, in principle, worked well.
However, once more, an amino-acid-dependent side reaction
that was independent of the newly investigated blocking
group prevented a clear demonstration of the suitability of the
PhAcOZ group for nucleopeptide synthesis being achieved.
To determine if the conditions for the selective removal of
this enzyme-sensitive blocking group were mild enough to
allow an efficient nucleopeptide synthesis, different model
nucleotripeptides were synthesized. PhAcOZ-AA1-OH were
prepared from the corresponding amino acids and PhAcOZ-
Cl by a known procedure in high yields.[12] The PhAcOZ-
masked amino acids 26a c and H-Ser-AlaOAll (27), pre-
pared by literature procedures,[22] were coupled in the
presence of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HATU) and diisopropyl-
ethylamine in DMF at 08C for one hour to yield the PhAcOZ-
protected tripeptides 28a c in very high yield (Scheme 5). In
the presence of EDC/HOBT, the coupling was not efficient
and the tripeptides were isolated in only 30 40% yield.
Scheme 7. Selective PGA-catalyzed removal of the N-terminal PhAcOZ
urethane from nucleotripeptides 30a c and tetrapeptides 32a,b.
Scheme 5. Synthesis of PhAcOZ protected tripeptides 28a c.
solution was lyophilized. The phosphate salts and KI were
separated from the desired compound by using a C18 Sep-Pak
cartridge, as described above. Then the mixtures containing
31a c were separated by HPLC to yield the desired
compounds in high yield. To assure that this method is widely
applicable, the peptide chains of compounds 31a and 31b
were elongated with a further PhAcOZ-protected amino acid
in the presence of HATU and diisopropylethyl amine at 08C
to yield nucleotetrapeptides 32a and 32b in 40 and 42%,
respectively (Scheme 7). Once more, treatment of these
peptide conjugates with PGA under the conditions described
above resulted in a smooth and clean deprotection of the N-
terminus to yield selectively deprotected nucleotetrapeptides
The central serine of tripeptides 28a c were then coupled
to selectively masked deoxycytidine 20 through a phospho-
triester bond. Upon treatment of 28a c with phosphordi-
amidite 21 in the presence of tetrazole and diisopropylamine,
hydrolysis-sensitive intermediates 29a c were formed. These
were converted without isolation into the desired peptide
conjugates 30a
c by the method described above in
Scheme 4. The product was purified by gel permeation
chromatography after filtration through silica gel to yield
the nucleotripeptides 30a c in high yields in overall three
steps (Scheme 6).
Scheme 6. Synthesis of model N-PhAcOZ-protected nucleotripeptides 30a c.
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H. Waldmann et al.
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aspartic acid 7 (1.9 g, 76%). 1H NMR (400 MHz, CDCl3): d ˆ 5.85 5.75
(m, 2H), 5.53 5.51 (d, J ˆ 8.36 Hz, 1H), 5.24 5.13 (m, 4H), 4.54 4.49 (m,
5H), 2.96 2.75 (m, 2H), 1.35 (s, 9H); 13C NMR (100.6 MHz, CD3OD): d ˆ
170.59, 170.45, 155.26, 131.65, 131.49, 118.41, 79.84, 77.34, 66.05, 65.46,
49.94, 36.59, 28.16.
33a and 33b. These nucleotetrapeptides were purified by the
method described above.
The N-terminally deprotected nucleopeptides 31a, 31b,
33 a and 33b can be converted to the fully deprotected
nucleopeptides by Pd0-catalyzed removal of the allyl groups.
The removal of the allyl blocking groups from nucleopeptides
under nonbasic conditions and without undesired decompo-
sition reactions has been demonstrated by us[23a] and Hay-
akawa et al.[23b]
In all enzyme-catalyzed deprotections, undesired side
reactions did not occur. Thus, the high selectivity of PGA
for the phenylacetic acid group guarantees that the peptide
bonds, the allyl ester, the allyl urethane, the acetate protecting
group, and the phosphate remain fully intact. The conditions
of the enzymatic transformation are so mild (pH 6.8) that the
glycosidic bond is not affected and b-elimination of the
phosphate (which occurs at pH > 7) is not observed at all.
These findings prove that the PhAcOZ group is an efficient
protecting function for the selective synthesis of sensitive and
multifunctional nucleopeptides.
l-Aspartic diallyl ester, TFA salt (8): Trifluoroacetic acid (6 mL) was added
to a solution of 7 (1.2 g, 3.8 mmol) in dichloromethane (50 mL), and the
solution was stirred for 24 h at room temperature. Evaporation of the
solvent and excess TFA gave the desired product 8 in quantitative yield.
1H NMR (400 MHz, CD3OD): d ˆ 8.80 8.40 (brs, 1H), 5.91 5.80 (m,
2H), 5.34 5.24 (m, 4H), 4.72 4.59 (m, 5H), 4.43 4.40 (t, J ˆ 4.84,
9.84 Hz, 1H), 3.18 3.17 (d, J ˆ 4.44 Hz, 2H); 13C NMR (100.6 MHz,
CD3OD): d ˆ 170.20, 167.78, 130.98, 130.37, 120.06, 119.32, 67.68, 66.56,
49.59, 33.34.
N-tert-Butyloxycarbonyl-l-seryl-l-aspartic diallyl ester (10): EEDQ (4.0 g,
16 mmol) was added to a solution of Boc-Ser-OH (9) (1.6 g, 8 mmol) in
dichloromethane (30 mL) at 08C. A solution of compound 8 (2.6 g,
8 mmol) in dichloromethane (10 mL) and then triethylamine (1.2 mL,
8 mmol) were added at 08C. The reaction mixture was stirred at RT
overnight, washed three times with 0.1m HCl, once with saturated NaHCO3
solution and once with brine. The organic layer was dried over MgSO4 and
evaporated in vacuo. The crude material was purified by silica gel flash
1
chromatography to yield dipeptide 10 (2.75 g, 86%). H NMR (400 MHz,
CDCl3): d ˆ 7.62 (d, J ˆ 7.8 Hz, 1H), 5.93 5.83 (m, 2H), 5.74 (d, J ˆ 7.2 Hz,
1H), 5.34 5.23 (m, 4H), 4.94 4.90 (m, 1H), 4.65 4.57 (m, 4H), 4.25 (brs,
1H), 3.99 (brs, 1H), 3.69 (brs, 2H), 3.07 2.89 (m, 2H), 2.64 (s, 1H), 1.44 (s,
9H); 13C NMR (100.6 MHz, CD3OD): d ˆ 171.27, 170.45, 170.27, 131.61,
131.35, 118.99, 118.82, 80.26, 66.55, 65.83, 63.00, 55.50, 48.84, 36.09, 28.28;
MS (FAB): m/z calcd: 400.42; found: 401.2 (3-NBA matrix).
Conclusion
We have developed a new mild enzymatic method to
synthesize nucleopeptides through selective N-terminal de-
protection. We also demonstrated the applicability of this
method by performing N-terminal chain elongations followed
by deprotection. Since the enzymatic cleavage is very mild
(pH 6.8, room temperature)and highly selective, no side
reaction was encountered during the deprotection reaction.
This method will open up new opportunities to synthesize
nucleopeptides of biological interest.
l-Seryl-l-aspartic diallyl ester, TFA salt (11): The deprotection of the Boc-
group was carried out as described above for 8 to afford 11. 1H NMR
(400 MHz, CD3OD): d ˆ 5.98 5.88 (m, 2H), 5.36 5.29 (m, 2H), 5.25 5.21
(m, 2H), 4.64 4.59 (m, 5H), 3.99 3.95 (m, 2H), 3.83 3.78 (m, 1H), 2.96
(d, J ˆ 5.8 Hz, 2H); 13C NMR (100.6 MHz, CD3OD): d ˆ 171.38, 171.22,
168.21, 133.23, 132.95, 122.90, 118.99, 118.75, 67.35, 66.69, 61.65, 56.07, 50.41,
36.65; MS (FAB): m/z calcd: 300.31; found: 301.2 (3-NBA matrix).
N-Phenyacetoxybenzyloxycarbonyl-l-aspartic b-allyl ester (14): Triethyl-
amine (7.5 mmol) and trimethylsilyl chloride (TMSCl) (1.63 g, 15 mol)
were added in one portion to a vigorously stirred suspension of 13 (1.57 g,
7.5 mmol) in CH2Cl2 (25 mL). The mixture was heated at reflux for 1 h,
cooled to 08C, and diisopropylethylamine (2.2 mL, 13 mmol) and PhAc-
OZ-Cl (1.5 g, 5 mmol) were added. The reaction mixture was stirred at 08C
for 30 min and at RT for 2 h. The solvent was removed in vacuo, and the
residue was taken up in Et2O (25 mL) and 0.5m NaHCO3 solution (50 mL).
The layers were separated, and the aqueous layer was extracted with Et2O
(10 mL). The combined organic layers were washed with H2O (10 mL). The
pH of the combined aqueous layers was adjusted to pH 2 with HCl (0.5m),
and the aqueous solution was extracted with ethyl acetate. The combined
organic layers were then dried with MgSO4 and concentrated to give the
Experimental Section
General: 1H, 13C, and 31P NMR spectra were recorded on Bruker 250,
Am400, and DRX500 spectrometers. Mass spectra were measured on a
HPLC/ESI-MS system with the HP (Agilent, 1100 series) HPLC. FAB-MS
was measured on a Funnigan MAT MS70 spectrometer. Optical rotation
was measured on Perkin Elmer 341 polarimeter. Analytical chromatog-
raphy was performed on Merck silica gel 60 F254 plates. Flash chromatog-
raphy was performed on Baker silica gel (40 64 mm). HPLC purifications
were made by varian (Pro Star 215). C18-HD RP-analytical and semi-
preparative and preparative columns were purchased from Macherey
Nagel. Melting point was measured in a B¸chi 510 melting point apparatus.
Solvents were dried according standard procedures. All reactions except
the enzymatic transformations were carried out under argon. PGA
immobilized on Eupergit C was from Fluka. Sep-Pak C18 cartridges were
purchased from Waters, USA. Compound 21 was purchased from Aldrich
and distilled before the reaction.
1
desired product 14 (1.55 g, 70%). H NMR (250 MHz, CDCl3): d ˆ 10.6
10.4 (brs, 1H), 7.4 7.1 (m, 7H), 7.1 7.0 (m, 2H), 5.9 5.7 (m, 2H), 5.3 5.2
(m, 2H), 5.1 5.0 (m, 2H), 4.7 4.5 (m, 4H), 3.9 (s, 2H), 3.1 2.8 (m, 2H);
MS (FAB): m/z calcd: 441.43; found: 442.2 (3-NBA matrix).
N-Phenyacetoxybenzyloxycarbonyl-b-allyl-l-aspartyl-l-seryl-l-aspartic di-
allyl ester (15): A solution of 11 (0.22 g, 0.52 mmol) in CH2Cl2 (5 mL) was
added to a solution of PhAcOZ-Asp(OAll)-OH (14) (0.24 g, 0.52 mmol),
HOBT (0.08 g, 0.5 mmol) and EDC (0.2 g, 1 mmol) in CH2Cl2 (5 mL) at
08C. After addition of diisopropylethylamine (0.12 mL, 0.7 mmol), the
mixture was stirred for 18 h at RT and washed with 0.1m HCl (2 Â 20 mL)
and saturated NaCl (10 mL). Drying over MgSO4 and evaporation of the
solvent gave the desired product 15 (0.19 g, 50%). 1H NMR (400 MHz,
CDCl3): d ˆ 7.67 (d, J ˆ 8 Hz, 1H), 7.56 (d, J ˆ 7.1 Hz, 1H), 7.43 7.18 (m,
7H), 7.02 (d, J ˆ 8.5 Hz, 2H), 6.18 (d, J ˆ 8.7 Hz, 1H), 5.96 5.80 (m, 3H),
5.41 5.18 (m, 7H), 5.18 5.00 (m, 2H), 4.95 4.85 (m, 1H), 4.67 4.43 (m,
7H), 4.03 3.90 (m, 1H), 3.84 (s, 2H), 3.78 3.57 (m, 2H), 3.01 2.64 (m,
4H); 13C NMR (100.6 MHz, CDCl3): d ˆ 171.16, 170.88, 170.41, 170.20, 170,
155.98, 150.54, 133.73, 133.32, 131.65, 131.37, 129.41, 129.30, 128.92, 128.73,
128.49, 127.38, 126.54, 121.59, 118.89, 118.72, 118.67, 77.45, 77.13, 76.81,
66.55, 66.46, 65.79, 62.66, 54.73, 51.26, 48.86, 41.33, 36.55, 35.99; MS (FAB):
m/z calcd: 723.72; found: 724.3 (3-NBA matrix).
N-tert-Butyloxycarbonyl-l-aspartic diallyl ester (7): Boc-Asp-OH (1.88 g,
8 mmol) was dissolved in THF (9 mL) and water was added dropwise until
the solution became turbid. It was neutralized by the addition of Cs2CO3
(2.64 g, 16.2 mmol) in water (3 mL) at room temperature. After a few
minutes, the solution was evaporated to dryness and the salt obtained was
suspended in water (15 mL) and treated with allyl bromide (1.75 mL). The
mixture was stirred for 4 h at room temperature. Evaporation of the solvent
and excess reagent gave the crude material, which was dissolved in ethyl
acetate and washed with 5% NaHCO3 solution and water. The organic
layer was dried over MgSO4, filtered, and evaporated. The compound was
purified by silica gel column chromatography to yield the fully protected
1884
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0947-6539/02/0808-1884 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 8

Synthesis of Nucleopeptides
1879 1887
5'-O-(tert-Butyldimetylsilyl)-3'-O-acetyl-4-N-allyloxycarbonyl-2'-deoxycy-
tidine (19): A solution of 18 (0.1 g, 0.23 mmol) and acetic anhydride
(0.25 mL) in pyridine (5 mL) was stirred for 3 h at room temperature.
Evaporation of excess acetic anhydride and pyridine gave a foam, and
purification by chromatography on silica gel (chloroform/methanol 10:0.5)
afforded 19 (0.106 g, 99%). Rf ˆ 0.73 (CH2Cl2/MeOH 20:1); 1H NMR
(500 MHz, CDCl3): d ˆ 8.20 (d, J ˆ 7.55 Hz, 1H), 7.13 (brs, 1H), 6.28 (dt,
J ˆ 5.7 Hz, 1H), 5.92 5.84 (m, 1H), 5.31 (dd, J ˆ 17.1, 1.3 Hz, 1H), 5.23
5.18 (m, 2H), 4.63 (d, J ˆ 5.6 Hz, 2H), 4.14 (d, J ˆ 1.85 Hz, 1H), 3.85 (dq,
J ˆ 11.4, 16.1 Hz, 2H), 2.65 (dd, J ˆ 13 Hz, 1H), 2.08 2.01 (m, 1H), 2.03 (s,
3H), 0.83 (s, 9H), 0.04 (s, 6H); 13C NMR (125 MHz, CDCl3): d ˆ 176.96,
170.50, 162.53, 144.14, 131.47, 118.89, 87.10, 86.04, 75.19, 66.68, 63.33, 39.48,
25.82, 21.18, 20.95, 18.21, À5.52, À5.63.
once with saturated NaHCO3 solution and 1m HCl. The organic layer was
dried over MgSO4 and concentrated, and the residue was purified by
chromatography on silica gel to yield 28a (0.26 g, 88%). Rf ˆ 0.24
(ethylacetate/cyclohexane 3:1); [a]2D2 ˆ À17.6 (c ˆ 1.0 in CH2Cl2); m.p.
1508C; 1H NMR (400 MHz, CDCl3): d ˆ 7.38 7.20 (m, 9H), 7.02 (d, J ˆ
8.3 Hz, 2H), 5.93 5.83 (m, 1H), 5.75 (d, J ˆ 8 Hz, 1H), 5.35 5.20 (m, 2H),
5.08 5.00 (m, 2H), 4.66 4.51 (m, 4H), 4.17 4.10 (m, 1H), 3.97 3.95
(brd, J ˆ 10.8 Hz, 1H), 3.85 (s, 2H), 3.67 3.59 (m, 1H), 2.15 2.02 (m,
1H), 1.40 (d, J ˆ 7 Hz, 3H), 0.95 (d, J ˆ 6.8 Hz, 3H), 0.90 (d, J ˆ 6.8 Hz,
3H); 13C NMR (100.6 MHz, CDCl3): d ˆ 172.39, 172.01, 170.23, 169.99,
150.46, 133.88, 133.28, 131.40, 129.27, 128.72, 127.36, 121.54, 118.83, 66.33,
66.09, 62.65, 60.26, 53.98, 48.34, 41.34, 37.29, 31.22, 19.15, 17.78, 17.71, À0.03;
MS (ESI ‡ ): m/z (%) calcd: 583.63; found: 584.10 (100). Compounds 28b
and 28c were prepared by the same procedure.
3'-O-Acetyl-4-N-alloxycarbonyl-2'-deoxycytidine (20): 1m TBAF (0.4 mL)
in THF was added to a solution of 19 (0.1 g, 0.21 mmol) in THF (6 mL) at
room temperature, and the mixture was stirred for 3 h at room temper-
ature. After quenching with methanol, the solvent was evaporated in vacuo.
The residual foam was purified by chromatography on silica gel (chloro-
form/methanol/triethylamine 100:5:0.5) to give 20 (0.07 mg, 98%) as a
white solid. Rf ˆ 0.1 (CH2Cl2/MeOH 20:1); [a]2D2 ˆ ‡43.2 (c ˆ 1.0 in
MeOH); m.p. 1648C; 1H NMR (400 MHz, CDCl3): d ˆ 8.34 (d, J ˆ
7.5 Hz, 1H), 7.27 (s, 1H), 6.27 (t, J ˆ 6.6 Hz, 1H), 5.97 5.89 (m, 1H),
5.39 5.35 (m, 2H), 5.30 (d, J ˆ 10.3 Hz, 1H), 4.68 (d, J ˆ 5.4 Hz, 2H), 4.19
(s, 1H), 3.95 (q, J ˆ 12.1, 16.3 Hz, 2H), 3.70 3.40 (brs, 1H), 2.67 (dd, J ˆ
4.12 Hz, 1H), 2.38 2.31 (m, 1H), 2.1 (s, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 170.84, 162.37, 145.03, 131.34, 119.18, 95.50, 87.83, 86.08, 77.33,
77.01, 76.70, 74.87, 66.83, 62.20, 38.71, 21.00; MS (FAB): m/z calcd: 353.33;
found: 354.2 (3-NBA matrix).
N-Phenyacetoxybenzyloxycarbonyl-l-phenlyalanyl-l-seryl-l-alanine allyl
ester (28b): Yield ˆ 75%; solid; m.p. 1158C; Rf ˆ 0.15 (ethylacetate/
cyclohexane 3:1); [a]D22 ˆ À12.6 (c ˆ 1 in CH2Cl2); 1H NMR (400 MHz,
CDCl3): d ˆ 7.40 7.12 (m, 13H), 7.02 6.97 (m, 2H), 5.92 5.78 (m, 2H),
5.34 5.20 (m, 2H), 5.04 4.92 (m, 2H), 4.64 4.48 (m, 6H), 3.92 3.84 (m,
1H), 3.84 (s, 2H), 3.76 3.68 (m, 1H), 3.64 3.56 (m, 1H), 3.14 3.06 (m,
1H), 3.02 2.94 (m, 1H), 1.44 1.36 (m, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 172.86, 172.65, 172.09, 170.23, 170.20, 156.29, 150.72, 136.41,
134.05, 133.56, 131.70, 129.514, 129.47, 129.43, 128.96, 128.85, 127.60, 127.26,
121.76, 119.02, 66.54, 66.31, 63.00, 54.61, 48.64, 41.59, 38.74, 17.89; MS
(ESI ‡ ): m/z (%) calcd: 631.68; found: 632.10 (100).
N-Phenyacetoxybenzyloxycarbonyl-l-prolyl-l-seryl-l-alanine allyl ester
(28c): Yield ˆ 85%; white wax; Rf ˆ 0.04 (ethylacetate/cyclohexane 3:1);
[a]2D2 ˆ À50.2 (c ˆ 0.5 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d ˆ 7.40
7.20 (m, 9H), 7.03 (d, J ˆ 6.4 Hz, 2H), 5.92 5.84 (m, 1H), 5.31 (d, J ˆ
13.8 Hz, 1H), 5.23 (d, J ˆ 8.4 Hz, 1H), 5.20 4.95 (m, 2H), 4.64 4.46 (m,
4H), 4.34 4.28 (m, 1H), 4.25 4.20 (brs, 0.5H), 3.98 3.97 (m, 0.5H), 3.84
(s, 2H), 3.76 3.64 (m, 1H), 3.60 3.50(m, 1H), 3.48 3.42 (m, 1H), 3.17
3.02 (brs, 1H), 2.20 2.02 (m, 2H), 1.99 1.82 (m, 2H), 1.41 1.30 (m, 3H);
13C NMR (100.6 MHz, CDCl3): d ˆ 173.35, 172.88, 172.80, 175.58, 170.49,
156.06, 155.09, 150.85, 134.62, 134.42, 133.70, 132.00, 129.69, 129.60, 129.13,
127.78, 121.97, 119.11, 118.98, 67.12, 66.90, 66.33, 63.04, 61.37, 60.93, 55.08,
45.52, 48.80, 47.90, 47.54, 41.73, 31.68, 30.15, 24.91, 24.04, 17.91; MS (ESI ‡ ):
m/z (%) calcd: 381.61; found: 382.10 (100).
Synthesis of nucleotripeptide 23
Synthesis of the phosphoramidite (22): A solution of 15 (0.72 g, 1 mmol) in
CH2Cl2 (1 mL) was added to a solution of allyl-N,N,N',N'-tetraisopropyl-
phosphordiamidite (21) (1.3 mmol, 0.415 mL), diisopropylamine
(0.5 mmol, 0.071 mL), and tetrazole (35 mg, 0.5 mmol) in CH2Cl2 (2 mL).
After 1 h, the reaction mixture was taken up in a saturated aqueous
solution of NaHCO3 (5 mL). The mixture was extracted four times with
CH2Cl2. The combined organic layers were dried with MgSO4 and
concentrated under reduced pressure. The crude products were used
immediately for subsequent steps without further purification.
Preparation of comopunds 30a c: Compounds 30a c were prepared by
the procedure described above for compound 23.
N-Phenyacetoxybenzyloxycarbonyl-b-allyl-l-aspartyl-l-seryl-O-(3'-O-ace-
tyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-aspartic
di-
allyl ester (23): A solution of tetrazole (0.14 g, 2 mmol) in acetonitrile
(2 mL) at room temperature was added dropwise to a solution of 20
(1 mmol) and the crude 22 in CH2Cl2 (2 mL). After completion of the
reaction (24 h), tBuOOH (80%, 2 mL) was added at 08C. After 10 min, the
reaction mixture was taken up in water (100 mL), and the solution was
extracted with CH2Cl2. The organic layer was dried with MgSO4 and
concentrated, and the residual oil was purified by chromatography on silica
gel (chloroform/ethanol 20:1) and gel filtration chromatography to give 23
(0.475 g, 40%). 1H NMR (500 MHz, CDCl3): d ˆ 8.09, 8.04 (2d, J ˆ 7.2 Hz,
1H), 7.99, 7.96 (2d, J ˆ 7.6 Hz, 1H), 7.84, 7.79 (2d, J ˆ 8 Hz, 1H), 7.40 7.20
(m, 7H), 7.03, 7.025 (2d, J ˆ 8 Hz, 2H), 6.34 6.31 (m, 1H), 6.21 6.16 (m,
1H), 5.96 5.82 (m, 5H), 5.37 5.19 (m, 12H), 5.11 5.02 (m, 2H), 4.91
4.85 (m, 2H), 4.78 4.74 (m, 1H), 4.65 4.45 (m, 12H), 4.35 4.28 (m, 3H),
4.22 (d, J ˆ 2.3 Hz, 1H), 3.85 (s, 2H), 3.04 2.85 (m, 4H), 2.60 2.56 (m,
1H), 2.24 2.16 (m, 1H), 2.06 (s, 3H); 13C NMR (125.77 MHz, CDCl3): d ˆ
171.20, 171.13, 170.91, 170.51, 170.21, 170.13, 169.93, 169.86, 168.21, 168.11,
162.71, 156.00, 150.56, 144.41, 144.29, 133.83, 133.36, 132.11, 132.06, 131.77,
131.74, 131.53, 131.48, 129.30, 129.26, 128.93, 128.74, 127.39, 121.59, 119.03,
118.68, 118.57, 95.61, 87.87, 87.51, 83.44, 77.34, 77.09, 76.83, 74.21, 74.09,
68.95, 68.90, 67.17, 67.05, 66.90, 66.70, 66.47, 66.29, 65.72, 65.66, 53.34, 51.36,
49.08, 41.36, 38.14, 38.06, 36.55, 35.98, 20.88; 31P NMR (202.48 MHz,
CDCl3): d ˆ À0.44; MS (FAB): m/z calcd: 1179.08; found: 1179.2 (3-NBA
matrix)
N-Phenylacetoxybenzyloxycarbonyl-l-valinyl-l-seryl-O-(3'-O-acetyl-6-N-
allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-alanine-allyl
ester
(30a): Yield ˆ 43%; white solid; m.p. 688C; Rf ˆ 0.17 (CHCl3/EtOH
1
20:1); [a]2D2 ˆ ‡3.6 (c ˆ 0.5 in CH3OH); H NMR (400 MHz, CDCl3): d ˆ
8.10 7.94 (m, 2H), 7.73 (d, J ˆ 7.0 Hz, 0.5H), 7.67 (d, J ˆ 7.0 Hz, 0.5H),
7.38 7.24 (m, 7H), 7.04 (d, J ˆ 8.5 Hz, 2H), 6.24 6.20 (m, 1H), 5.96 5.80
(m, 3H), 5.41 5.20 (m, 8H), 5.10 4.97 (m, 5H), 4.68 4.52 (m, 9H), 4.40
4.26 (m, 2H), 4.24 4.19 (m, 1H), 3.86 (s, 2H), 2.61 2.51 (m, 1H), 2.25
2.10 (m, 3H), 2.07 (s, 3H), 1.42 1.40 (d, J ˆ 7.3 Hz, 3H), 0.97, 0.96 (2d, J ˆ
6.5 Hz, 3H), 0.91, 0.905 (2d, J ˆ 6.8 Hz, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 172.56, 172.49, 172.38, 170.96, 170.94, 170.34, 168.74, 168.54,
163.36, 156.86, 155.84, 152.86, 150.91, 144.18, 134.36, 133.73, 132.35, 132.04,
131.92, 129.60, 129.12, 127.77, 121.98, 119.52, 119.43, 119.25, 118.98, 118.94,
96.23, 87.34, 83.82, 83.74, 74.45, 74.36, 69.30, 69.24, 67.59, 67.22, 67.00, 66.94,
66.66, 66.25, 60.29, 53.21, 48.80, 41.74, 38.68, 38.59, 31.76, 31.70, 21.31, 21.29,
19.65, 18.23, 18.19, 17.85, 17.79; 31P NMR (202.46 MHz, CDCl3): d ˆ 0.0381,
0.0016; MS (ESI ‡ ): m/z (%) calcd: 1038.99; found: 1039.10 (100).
N-Phenylacetoxybenzyloxycarbonyl-l-phenylalanyl-l-seryl-O-(3'-O-ace-
tyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-alanine allyl
ester (30b): Yield ˆ 58%; solid; m.p. 558C; Rf ˆ 0.13 (CHCl3/EtOH
20:1); [a]2D2 ˆ ‡8.0 (c ˆ 0.35 in CH3OH); 1H NMR (400 MHz, CDCl3):
d ˆ 8.60 8.40 (brs, 1H), 7.81 7.66 (brs, 1H), 7.43 7.13 (m, 13H), 7.00 (d,
J ˆ 7.5 Hz, 2H), 6.26 6.12 (m, 1H), 5.99 5.72 (m, 4H), 5.45 5.17 (m,
7H), 5.06 4.20 (m, 16H), 3.85 (s, 2H), 3.70 3.55 (m, 1H), 3.50 3.35 (m,
1H), 3.25 3.15 (m, 1H), 3.02 2.90 (m, 1H), 2.78 2.64 (m, 1H), 2.50 2.30
(m, 1H), 2.06 (s, 3H), 1.44 (d, J ˆ 7.3 Hz, 3H); 13C NMR (100.6 MHz,
CDCl3): d ˆ 172.37, 172.23, 170.97, 170.33, 168.45, 168.29, 163.22, 156.36,
152.77, 150.87, 144.30, 136.80, 134.26, 133.73, 132.42, 132.35, 132.06, 131.85,
129.69, 129.51, 129.13, 128.91, 127.78, 127.28, 121.94, 119.48, 119.31, 118.98,
118.93, 96.14, 96.1, 87.82, 87.55, 83.79, 74.48, 69.33, 69.28, 67.60, 67.56, 67.35,
Synthesis of the nucleotripeptides (28a c)
N-Phenyacetoxybenzyloxycarbonyl-l-valinyl-l-seryl-l-alanine allyl ester
(28a): A solution of 26a (0.5 mmol, 0.19 g) and HATU (0.6 mmol, 0.23 g)
in dry DMF (5 mL) was stirred for 5 min at 08C. Then, a solution of the
dipeptide trifluoroacetic acid ester 27 (0.7 mmol, 0.235 g) in dry DMF
(5 mL), and diisopropylamine (1.2 mmol, 0.2 mL) were added. After
stirring for 1 h, the reaction mixture was diluted with CH2Cl2 and washed
Chem. Eur. J. 2002, 8, No. 8
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0808-1885 $ 20.00+.50/0
1885

H. Waldmann et al.
FULL PAPER
67.15, 67.05, 67.00, 66.61, 66.25, 56.41, 52.32, 48.82, 41.75, 38.95, 38.73, 21.31,
21.28, 18.16, 18.11; MS (ESI ‡ ): m/z (%) calcd: 1087.04; found: 1087.1
(100).
N-Phenylacetoxybenzyloxycarbonyl-l-alanyl-l-phenylalanyl-l-seryl-O-
(3'-O-acetyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-ala-
nine-allyl ester (32b): Yield ˆ 91%; H NMR (400 MHz, CDCl3): d ˆ 8.19
1
(d, J ˆ 7.5 Hz, 0.5H), 8.14 (d, J ˆ 7.8 Hz, 0.5H), 7.60 (d, J ˆ 7.3 Hz, 1H),
7.39 7.13 (m, 13H), 7.06 7.02 (m, 3H), 6.20 6.12 (m, 1H), 5.98 5.82 (m,
3H), 5.46 5.21 (m, 8H), 5.12 4.94 (m, 3H), 4.71 4.67 (m, 4H), 4.63 4.59
(m, 2H), 4.56 4.46 (m, 3H), 4.45 4.37 (m, 1H), 4.35 4.24 (m, 4H), 4.17
4.10 (m, 2H), 3.86 (s, 2H), 3.28 3.18 (m, 1H), 3.07 2.98 (m, 1H), 2.71
2.63 (m, 1H), 2.35 2.23 (m, 1H), 2.10, 2.09 (2s, 3H), 1.42, 1.41 (2d, J ˆ
7.3 Hz, 3H), 1.27 1.24 (m, 3H); MS (ESI ‡ ): m/z (%) calcd: 1158.11;
found: 1158.10 (100).
N-Phenylacetoxybenzyloxycarbonyl-l-prolyl-l-seryl-O-(3'-O-acetyl-6-N-
allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-alanine allyl ester
(30c): Yield ˆ 44%; white solid; m.p. 608C; Rf ˆ 0.15 (CHCl3/EtOH
20:1); [a]2D2 ˆ À11.8 (c ˆ 0.5 in CH3OH); 1H NMR (400 MHz, CDCl3):
d ˆ 8.07 7.88 (m, 2H), 7.76 7.63 (m, 1H), 7.40 7.20 (m, 7H), 7.04 (d, J ˆ
8 Hz, 2H), 6.30 6.24 (m, 1H), 5.97 5.83 (m, 3H), 5.40 5.00 (m, 9H),
4.93 4.85 (brs, 1H), 4.70 4.18 (m, 5H), 3.86 (s, 2H), 3.68 3.58 (m, 1H),
3.52 3.42 (m, 1H), 2.70 2.60 (m, 1H), 2.20 1.84 (m, 9H), 1.42 (d, J ˆ
6.8 Hz, 3H), 1.46 1.32 (m, 4H); 13C NMR (100.6 MHz, CDCl3): d ˆ
172.48, 172.38, 170.94, 170.34, 168.73, 168.53, 163.35, 156.85, 155.84,
152.85, 150.90, 144.18, 134.36, 133.73, 132.35, 132.04, 131.91, 129.69,
129.59, 129.27, 129.12, 127.77, 121.98, 119.52, 119.43, 119.25, 118.98,
118.94, 96.23, 87.73, 87.34, 83.82, 83.74, 74.45, 74.36, 69.30, 69.24, 67.59,
67.22, 67.00, 66.94, 66.66, 66.25, 60.28, 53.20, 48.80, 41.74, 38.67, 38.59, 31.76,
31.70, 26.16, 21.30, 19.65, 18.23, 18.19, 17.85, 17.79; MS (ESI ‡ ): m/z (%)
calcd: 1036.97; found: 1037.10 (100).
l-Alanyl-l-phenylalanyl-l-seryl-O-(3'-O-acetyl-6-N-allyloxycarbonyl-2'-
deoxycytidyl-allyl-phosphato)-l-alanine-allyl ester (33b): Yield ˆ 40%;
1H NMR (400 MHz, CD3OD): d ˆ 8.19, 8.18 (2d, J ˆ 7.5 Hz, 1H), 7.33,
7.32 (2d, J ˆ 7.5 Hz, 1H), 7.27 7.20 (m, 5H), 6.22 6.18 (m, 1H), 6.03 5.87
(m, 3H), 5.43 5.20 (m, 7H), 4.70 4.56 (m, 8H), 4.46 4.23 (m, 6H), 3.87
3.83 (m, 1H), 3.21, 3.17 (2d, J ˆ 5.5 Hz, 1H), 3.00 2.92 (m, 1H), 2.71 2.62
(m, 1H), 2.37 2.25 (m, 1H), 2.11 (s, 3H), 1.48 (d, J ˆ 7.0 Hz, 3H), 1.41 (d,
J ˆ 7.3 Hz, 3H); (ESI ‡ ): m/z (%) calcd: 889.84; found: 890.2 (100).
N-Terminal deprotection of the nucleopeptides 30 and 32 by PGA: A
solution of the respective PhAcOZ-protected nucleopeptide (20 30 mg,
0.02 mmol) in a mixture of MeOH (12 mL), Na2HPO4 buffer (0.05m,
40 mL) and KI (0.1m, 10 mL) was treated with PGA (800 mg) at pH 6.8 for
24 h at room temperature. The immobilized enzyme was filtered, and the
solution was lyophilized. The residue was dissolved in the minimum
amount of water required (ca. 1 mL) and filtered through a C18 cartridge.
The cartridge was washed with water (2 mL), and the absorbed organic
materials were eluted with MeOH (2 mL). After evaporation of the
solvent, the crude material was purified by HPLC (C18-HD RP).
Acknowledgement
This research was supported by the Fonds der Chemischen Industrie. D.A.J.
is grateful to the Alexander von Humboldt Foundation for a Fellowship.
[1] B. A. Juodka, Nucleosides Nucleotides 1984, 3, 445 483; b) J. C.
Wang, Ann. Rev. Biochem. 1985, 54, 665 697; c) A. B. Vartapetian,
A. A. Bogdanov, Prog. Nucleic Acid Res. Mol. Biol. 1987, 34, 209 251;
d) T. J. Kelly, M. S. Wold, J. Li, Adv. Virus Res. 1988, 34, 1 42; d) M.
Salas in The Viruses, Vol. 1, The Bacteriophages (Ed.: R. Calender),
Plenum, New York, 1988.
l-Valinyl-l-seryl-O-(3'-O-acetyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-al-
lyl-phosphato)-l-alanine allyl ester (31a): Yield ˆ 60%; 1H NMR
(400 MHz, CD3OD): d ˆ 8.15 (2d, J ˆ 7.8 Hz, 1H), 7.30, 7.29 (2d, J ˆ
7.8 Hz, 1H), 6.22 6.15 (m, 1H), 6.05 5.85 (m, 3H), 5.43 5.19 (m, 7H),
4.78 4.74 (m, 1H), 4.69 (d, J ˆ 5.8 Hz, 2H), 4.64 4.54 (m, 4H), 4.50 4.23
(m, 6H), 3.82, 3.79 (2d, J ˆ 5.8 Hz, 1H), 2.72 2.64 (m, 1H), 2.41 2.32 (m,
1H), 2.27 2.18 (m, 1H), 2.12, 2.11 (2s, 3H), 1.42, 1.415 (2d, J ˆ 7.3 Hz,
3H), 1.07 (d, J ˆ 6.8 Hz, 3H), 1.04 (d, J ˆ 6.8 Hz, 3H); 31P NMR
(202.46 MHz): d ˆ À0.41, À0.51; MS (ESI ‡ ): m/z (%) calcd: 770.72;
found: 771.10 (100).
[2] For two reviews, see: a) M. Salas, Ann. Rev. Biochem. 1991, 160, 39
71; b) M. Salas, Virus Strategies Molecular Biology and Pathogenesis,
¬
VCH, Weinheim, 1993, pp. 1 23; c) L. Blanco, J. M. Lazaro, M.
de Vega, A. Bonnin, M. Salas, Proc. Natl. Acad. Sci. 1994, 91, 12198.
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1982, 1, 497 508; b) E. Kuyl-Yeheskiely, P. A. M. van der Klein,
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Pays-Bas 1986, 105, 69 70; c) E. Kuyl-Yeheskiely, C. M. Tromp,
A. W. M. Lefeber, G. A. van der Marel, J. H. van Boom, Tetrahedron
1988, 44, 6515 6523; d) E. Kuyl-Yeheskiely, C. M. Dreef-Tromp, A.
Geluk, G. A. van der Marel, J. H. van Boom, Nucleic Acids Res. 1989,
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van den Elst, G. A. van der Marel, J. H. van Boom, Nucleic Acids Res.
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4449 4452; b) J. Robles, E. Pedroso, A. Grandas, J. Org. Chem. 1994,
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681; b) V. Jungmann, H. Waldmann, Tetrahedron Lett. 1998, 39, 1139
1142; c) H. Waldmann, S. Gabold, Chem. Commun. 1997, 1861 1862.
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Angew. Chem. Int. Ed. Engl. 1996, 35, 1720 1723; b) T. Pohl, H.
Waldmann, J. Am. Chem. Soc. 1997, 119, 6702 6710.
l-Phenylalanyl-l-seryl-O-(3'-O-acetyl-6-N-allyloxycarbonyl-2'-deoxycy-
tidyl-allyl-phosphato)-l-alanine allyl ester (31b): Yield ˆ 69%; 1H NMR
(400 MHz, CD3OD): d ˆ 8.20 8.10 (m, 1H), 7.38 7.20 (m, 6H), 6.22 6.13
(m, 1H), 6.04 5.85 (m, 3H), 5.43 5.16 (m, H), 4.74 4.20 (m, 15H), 3.09
3.00 (m, 1H), 2.71 2.61 (m, 1H), 2.41 2.30 (m, 1H), 2.11, 2.10 (2s, 3H),
1.44, 1.42 (2d, J ˆ 7.2 Hz, 3H). MS (ESI ‡ ): m/z (%) calcd: 818.76; found:
819.10 (100).
l-Prolyl-l-seryl-O-(3'-O-acetyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-al-
lyl-phosphato)-l-alanine allyl ester (31c): Yield ˆ 65%; 1H NMR
(400 MHz, CD3OD): d ˆ 8.17, 8.15 (2d, J ˆ 7.5 Hz, 1H), 7.26, 7.24 (2d,
J ˆ 7.5, 1H), 6.18 6.14 (m, 1H), 6.04 5.87 (m, 3H), 5.43 5.19 (m, 7H),
4.77 4.73 (m, 1H), 4.70 4.67 (m, 2H), 4.63 4.56 (m, 4H), 4.49 4.25 (m,
7H), 3.48 3.40 (m, 1H), 3.40 3.33 (m, 1H), 2.71 2.66 (m, 1H), 2.50 2.34
(m, 2H), 2.11, 2.12 (2s, 3H), 2.10 2.02 (m, 3H), 1.43, 1.41 (2d, J ˆ 2.8 Hz,
3H); MS (ESI ‡ ): m/z (%) calcd: 768.71; found: 769.3 (100).
Coupling of 31a,b with PhAcOZ-protected amino acid: A solution of the
PhAcOZ-protected amino acid (0.0078 mmol) and HATU (6 mg,
0.0156 mmol) in dry DMF (1 mL) was stirred for 5 min at 08C. Then, a
solution of the 31a,b (0.0052 mmol) in dry DMF (1 mL), and diisopropyl-
amine (2 drops) were added. After stirring for 1 h, the reaction mixture was
concentrated under reduced pressure and purified by HPLC.
N-Phenylacetoxybenzyloxycarbonyl-l-prolyl-l-valinyl-l-seryl-O-(3'-O-
acetyl-6-N-allyloxycarbonyl-2'-deoxycytidyl-allyl-phosphato)-l-alanine-
allyl ester (32a): Yield ˆ 85%; 1H NMR (400 MHz, CDCl3): d ˆ 8.02 7.96
(m, 1H), 7.70 7.60 (m, 1H), 7.43 7.22 (m, 8H), 7.07 7.02 (m, 2H), 6.22
6.15 (m, 1H), 5.98 5.83 (m, 3H), 5.40 5.00 (m, 11H), 4.80 4.20 (m, 14H),
3.87 (s, 2H), 3.88 3.84 (m, 1H), 3.60 3.40 (m, 4H), 2.70 2.60 (m, 1H),
2.36 2.20 (m, 2H), 2.09 (s, 3H), 2.04 1.88 (m, 2H), 1.42 (d, J ˆ 6.4 Hz,
3H), 0.94 0.80 (m, 6H); MS (ESI ‡ ): m/z (%) calcd: 1136.10; found:
[13] Part of this work was published in preliminary form: D. A. Jeyaraj, H.
Waldmann, Tetrahedron Lett. 2001, 42, 835 837.
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108, 2068 2070; Angew. Chem. Int. Ed. Engl. 1996, 35, 1939 1942.
[15] B. J. Belshaw, S. Mzengeza, G. A. Lajoie, Synth. Comm. 1990, 20,
3157 3160.
‡
1136.20, 1158 (100) [M‡Na] .
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¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0808-1886 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 8

Synthesis of Nucleopeptides
1879 1887
¬
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J. Martinez, Synthesis 1981, 333 356, and references therein.
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[23] a) S. Flohr, V. Jungmann, H. Waldmann, Chem. Eur. J. 1999, 5, 669
681; b) A. Sakakura, Y. Hayakawa, Tetrahedron 2000, 56, 4427 4435.
Received: October 1, 2001 [F3584]
Chem. Eur. J. 2002, 8, No. 8
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0808-1887 $ 20.00+.50/0
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