A novel synthesis of oligonucleotide-peptide conjugates with a base- labile phosphate linker between the two components according to the allyl- protected phosphoramidite strategy

Article abstract of DOI:10.1016/S0040-4020(00)00376-8

An efficient synthesis of base-labile nucleotide-peptide conjugates has been accomplished, in which the two components are directly linked between the terminal hydroxyl of a nucleotide and the hydroxyl of a serine or threonine residue of a peptide by a phosphodiester bond. This synthesis utilizes the phosphoramidite method with allyl for the phosphate linkages and the C-terminal of the peptide and allyloxycarbonyl for the nucleoside bases and the N-terminal of the peptide. In this synthesis, the removal of the allylic protecting groups and the detachment of the products was achieved under non-basic or mild basic conditions to bring about no conspicuous decomposition of the labile phosphate linker, and thus the target conjugates were obtained at a high purity and in high yields. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00376-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 4427±4435
A Novel Synthesis of Oligonucleotide±Peptide Conjugates with a
Base-Labile Phosphate Linker between the Two Components
According to the Allyl-Protected Phosphoramidite Strategy
*
Akira Sakakura and Yoshihiro Hayakawa
Laboratory of Bioorganic Chemistry, Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received 28 March 2000; accepted 1 May 2000
AbstractÐAn ef®cient synthesis of base-labile nucleotide±peptide conjugates has been accomplished, in which the two components are
directly linked between the terminal hydroxyl of a nucleotide and the hydroxyl of a serine or threonine residue of a peptide by a phospho-
diester bond. This synthesis utilizes the phosphoramidite method with allyl for the phosphate linkages and the C-terminal of the peptide and
allyloxycarbonyl for the nucleoside bases and the N-terminal of the peptide. In this synthesis, the removal of the allylic protecting groups and
the detachment of the products was achieved under non-basic or mild basic conditions to bring about no conspicuous decomposition of the
labile phosphate linker, and thus the target conjugates were obtained at a high purity and in high yields. q 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
the peptide (type A in Scheme 1),12±14 while the other is that
in which the two components are connected via a stable
alkyl linker (type B in Scheme 1).15±20 This paper refers
to these conjugates as `nucleotide±peptide conjugates of
type A' and as `nucleotide±peptide conjugates of type B',
respectively.21 Of importance in the synthesis of these
conjugates, particularly of type-A conjugates with the
base-sensitive phosphate linkage at the b position of the
peptide carbonyl function, is the choice of protecting groups
of the nucleoside bases and the N-terminal of the peptide.
Traditional methods with acyl protectors for these amino
groups, which require heating with conc. ammonia for the
deprotection, are usable for the synthesis of type-B conju-
gates which are suf®ciently stable under such harsh basic
conditions, and several ef®cient syntheses via this strategy
having been developed so far.15±20 On the other hand, the
N-acyl-protection approach is not quite effective to the
synthesis of type-A conjugates. According to the experi-
ments using a nucleotide±serine conjugate on solid supports
as the model,22 this conjugate is fairly stable under rather
mild basic conditions such as conc. ammonia at ambient
temperature which are generally employed for the detach-
ment of the product from solid supports, but is cleaved to a
considerable extent under the above-mentioned harsh basic
conditions required for removal of the N-acyl protectors.
Actually, type-A conjugates have not been prepared in
satisfactory yields and at a high level of purity by the
N-acyl-protection approach.12±14 Thus, a crucial require-
ment for achieving the ef®cient synthesis of such a base-
labile conjugate is the use of a protecting group removable
under non-basic or mild basic conditions for the nucleo-
side base and the N-terminal of the peptide. The
The conjugation of a certain non-ionic substance to oligo-
nucleotides that allows great enhancement of the cellular
uptake of the nucleotide in the absence of liposomal carriers
is an important requirement for the therapeutic use of oligo-
nucleotides such as antisense molecules.1±3 Further, the
conjugation sometimes increases the stability of the
relationship of nucleotides to the enzymes.1,3 Thus, oligo-
nucleotides with such a conjugation are greatly expected to
have a novel, useful biofunctionality.3 Among the nucleo-
tide-related bioconjugates, covalently linked oligodeoxy-
ribonucleotide±peptide hybrid molecules have attracted
particular interest in their potential to broaden the spectrum
of oligonucleotide properties, representing a new method of
chemically increasing the activity of synthetic oligonucleo-
tides used as inhibitors of gene expression.4±8 These conju-
gates are also important as fundamental structures of site-
speci®c arti®cial nucleases9 and of the genetic materials of
some viruses such as polyovirus10 and adenovirus.11 Thus
the development of an ef®cient synthesis of oligodeoxy-
ribonucleotide±peptide conjugates is an important subject.
The covalently linked nucleotide±peptide conjugates are
broadly classi®ed into two types: one is that derivatives in
which the nucleotide directly conjugates to the peptide by a
phosphate linkage using a hydroxyl of serine or threonine of
Keywords: nucleic acid analogs; peptide analogs; phosphoramidites; solid-
phase synthesis.
* Corresponding author. Tel.: 181-52-789-4848; fax: 181-52-789-5646;
e-mail: yoshi@info.human.nagoya-u.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00376-8

4428
A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
Scheme 1.
Scheme 2. Bˆnucleoside base; Bpˆprotected nucleoside base; R0ˆH, CH3.

A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
4429
Scheme 3. (a) NH2CH2COOCH2CHvCH2´TsOH, DCC, HOBT, (C2H5)3N, THF. (b) CH2vCHCH2OP[N(i-C3H7)2]2, diisopropylammonium 1H-tetrazolide,
CH3CH.
allyloxycarbonyl (AOC) group removable by the organo-
palladium reaction under nearly neutral conditions23±25 is
such a protector. Thus, we expected that the allyl-protection
strategy may allow the high-yield, high-purity synthesis of
the base-labile nucleotide±peptide conjugates of type A.
This paper reports ef®cient ways to produce two kinds of
type-A derivatives, in which the peptide links at the 30-end
of the hydroxyl (30-end-nucleotide±peptide conjugate) and
at the 50-end of the oligonucleotide (50-end-nucleotide±
peptide conjugate), via the allyl-protection strategy, i.e.
by the use of the AOC protector for the nucleoside
bases and the N-terminal of the peptide and the allyl
group for the phosphate linkages and the C-terminal of the
peptide.22
Results and Discussion
Synthetic plan
We planned to synthesize the two types of nucleotide±
peptide conjugates using the convergent strategy as shown
in Scheme 2, where construction of the nucleotide chain and
The peptide phosphoramidites with allyl protection, 13, 14,
and 18, were prepared according to the known procedure
with some modi®cations. For example, the dipeptide phos-
phoramidite 13 was obtained via the pathway shown in
Scheme 3. Thus, the condensation of the p-toluenesulfonic
acid salt of allyl glycinate29 (1.0 equiv.) and N-(allyloxy-
carbonyl)-l-serine (9)30 (1.3 equiv.) assisted by dicyclo-
hexylcarbodiimide (DCC) (1.9 equiv.) in the presence of
1-hydroxybenzotriazole (HOBT) (1.3 equiv.) and triethyl-
amine (1.1 equiv.) in THF afforded the dipeptide 11 in a
98% yield. Subsequently, 11 (1.0 equiv.) was reacted with
(allyloxy)bis(diisopropylamino)phosphine (1.5 equiv.) by
the aid of diisopropylammonium 1H-tetrazolide
(1.0 equiv.) in acetonitrile gave the phosphoramidite 13 in
a 68% yield. In a similar way using N-(allyloxycarbonyl)-l-
threonine (10)30 in place of 9 as the starting material, the
dipeptide phosphoramidite 14 was prepared in a 60% over-
all yield (Scheme 3). The tetrapeptide phosphoramidite 18
was prepared via the following pathway (Scheme 4): esteri-
®cation of l-aspartic acid (1.0 equiv.) with allyl alcohol
(10 equiv.) and p-toluenesulfonic acid (1.1 equiv.), and the
subsequent condensation of the resulting O,O-diallyl
aspartate with N-(tert-butoxycarbonyl)glycine (Boc-Gly-
OH) (1.2 equiv.) promoted by DCC (1.8 equiv.) in the
presence of HOBT (1.8 equiv.) and triethylamine
(1.0 equiv.) in THF gave the dipeptide 15 in a 63% yield.
the phosphate bridge linking the two components is
performed via the phosphoramidite approach. We supposed
that the use of a highly-reactive reagent for the activation of
the phosphoramidites is also important in obtaining the
target products in high yields, and accordingly employed
benzimidazolium tri¯ate26 as the activator. Thus, the
synthetic reaction scheme is (i) preparation of a 30-O-free
or 50-O-free oligodeoxyribonucleotide anchored at the
50-end or 30-end hydroxyl, respectively, on the solid
supports via the phosphoramidite approach using benz-
imidazolium tri¯ate as the promoter, (ii) construction of
the phosphate bridge connecting the oligonucleotide and a
peptide using the benzimidazolium tri¯ate-assisted reaction
with a separately prepared peptide phosphoramidite,
followed by oxidation, (iii) the deprotection on the solid
supports using the organopalladium(0) reagent, and (iv)
detachment of the target hybrid compounds.
Preparation of nucleoside phosphoramidites and peptide
phosphoramidites
The allyl-protected nucleoside 30- and 50-phosphoramidites,
1±8, required for the construction of the nucleotide chains,
were obtained using the procedures reported in the
literature.22,25,27,28

4430
A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
Scheme 4. (a) 1ÐCH2vCHCH2OH, p-TsOH´H2O, toluene, re¯ux; 2ÐBoc-Gly-OH, DCC, HOBT, (C2H5)3N, CH2Cl2. (b) 1ÐCF3COOH, CH2Cl2; 2ÐBoc-
Ser(OTBDMS)-OH, BOP, (C2H5)3N, THF. (c) 1ÐCF3COOH, CH2Cl2; 2ÐAOC-Ala-OH, BOP, (C2H5)3N, THF; 3Ð(n-C4H9)NF, CH3COOH, THF. (d)
CH2vCHCH2OP[N(i-C3H7)2]2, diisopropylammonium 1H-tetrazolide, CH3CN.
Treatment of 15 with tri¯uoracetic acid in dichloromethane
at 08C, removing the N-tert-butoxycarbonyl protector, and
with N-(tert-butoxycarbonyl)-O-(tert-butyldimethylsilyl)-
l-serine [Boc-Ser(OTBDMS)-OH]31 (1.2 equiv.) in the
presence of (benzotriazolyl-1-oxy)tris(dimethylamino)-
phosphonium hexa¯uorophosphate (BOP)32 (1.8 equiv.)
and triethylamine (4.9 equiv.) provided the tripeptide 16
in a 91% yield. After removal of the N-tert-butoxycarbonyl
protector, 16 was converted in a 52% overall yield to the
tetrapeptide 17 by condensation with the N-(allyloxy-
carbonyl)-l-alanine (AOC-Ala-OH) (1.2 equiv.) followed
by desilylation with a mixture of tetrabutylammonium
¯uoride and acetic acid. Finally, 17 was phosphoramidityl-
ated by the use of (allyloxy)bis(diisopropylamino)phosphine
(1.5 equiv.) and diisopropylammonium 1H-tetrazolide
(1.0 equiv.) in acetonitrile to afford 18 in a 63% yield.
anchored 50-O-free oligonucleotides were initially prepared
starting from 19.
The chain elongation was achieved via the (30!50) direction
with 1, 2, 3, and 4 as monomer units and benzimidazolium
tri¯ate26 as the promoter. As a representative example, the
0
0
synthesis of Ala-Ser(p5 TGTCGACACCCAATT3 )-Gly-
Asp (20) is described below. First, the 50-end-free oligo-
0
0
nucleotide, 5 TGTCGACACCCAATT3 , anchored at the
30-end hydroxyl on the solid supports was prepared in a
99.2% average coupling yield (estimated by the trityl
assay) according to the synthetic procedure shown in
Table 1. Subsequently, the solid-anchored 50-O-free nucleo-
tide was reacted with the peptide phosphoramidite 18 by
the aid of benzimidazolium tri¯ate, and the resulting
phosphite product was oxidized by tert-butyl hydroperoxide
(TBHP).33 Finally, all allylic protecting groups were
removed by treatment with a mixture of the tris-
(dibenzylideneacetone)dipalladium±chloroform complex
[Pd2(dba)3´CHCl3 complex], triphenylphosphine, and
diethylammonium formate in THF (508C, 60 min), follow-
ing which the target conjugate 20 was released from the
supports by brief exposure to conc. aqueous ammonia
(258C, 60 min). The HPLC and the capillary gel electro-
phoresis (CGE) (Fig. 1) indicate that the product has good
purity in crude forms. No peak at tRˆ14 min due to
Solid-phase synthesis of nucleotide±peptide conjugates
The synthesis of the nucleotide±peptide conjugates was
conducted on a controlled pore glass (CPG) support
Ê
(500 A pore size) with a long-chain alkylamine spacer
arm on an ABI 392 DNA/RNA synthesizer. In the synthesis
of the 50-end-nucleotide±peptide conjugates, solid-
Table 1. Reaction sequence of the solid-phase synthesis of nucleotide±
peptide conjugates
Step
Operation
Reagent
Time (min)
1
2
3
4
Washing
CH3CN
3% Cl3CCOOH/CH2Cl2
CH3CN
0.1 M phosphoramidite/
CH3CN10.2 M
benzimidazolium tri¯ate/
CH3CN
0.4
1.3
0.8
1.0
Detritylation
Washing
Coupling
0
0
p5 TGTCGACACCCAATT3 , which is conceived as a
major fragment resulting from decomposition of 20,
was detected. The yield of 20 estimated in a crude
form by HPLC using the parent oligonucleotide,
5
6
Washing
Capping
CH3CN
0.2
0.3
Ac2O-2,6-lutidine±THF
(1:1:8)116% N-methyl-
imidazole/THF
0
0
5 TGTCGACACCCAATT3 , as a standard was 82%. The
electrospray ionization-mass (ESI-MS) spectrum of the
puri®ed product showed dominant molecular ion peaks at
m/z 2460.5 (M22H)22 and 1640.3 (M23H)32, supporting
the structure of 20 with a molecular weight of 4922.23.
7
8
9
Washing
Oxidation
Washing
CH3CN
1.0 M t-C4H9OOH/toluene
CH3CN
0.2
0.5
0.6

A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
4431
0
0
Figure 1. HPLC (a) and capillary gel electrophoresis (b) pro®les of the crude product of Ala-Ser(p5 TGTCGACACCCAATT3 )-Gly-Asp (20).
According to a similar process, the hybrids in which the
nucleotide links with the hydroxyl of threonine moiety
were also prepared by the use of 14 as the peptide phosphor-
amidite, though the yields of the products were generally
lower because of the poor reactivity of 14. For instance,
between the nucleotide and peptide, to con®rm that such an
undesired reaction does not take place in this synthesis. No
racemization of the amino acid components was observed in
the products. In the syntheses using 14, the incomplete
phosphitylation was generally observed. Many investiga-
tions for completing the phosphitylation were carried out,
but none of them could accomplish this purpose.
0
0
Thr(p5 TGTCGACACCCAATT3 )-Gly (21) was prepared
in a 69% yield (HPLC assay). As the HPLC illustrated in
Fig. 2 indicates, the crude product was contaminated by
some impurities. Comparison with the authentic sample
shows that the major impurity providing the peak at
tRˆ18 min in the HPLC is 30,50-diend-O-free
Meanwhile, the 30-end-nucleotide2peptide conjugates were
similarly prepared via the construction of the 30-O-free
oligonucleotides, starting from solid-anchored thymidine
22, through the (50!30)-directed chain elongation using
the nucleoside 50-phosphoramidites, 5±8, and benzimida-
zolium promoter, followed by the formation of the peptide
phosphate bridge with the peptide phosphoramidites, 13, 14,
and 18.
0
0
5 TGTCGACACCCAATT3 , which is the product under-
going no phosphitylation by 14. Further, the HPLC showed
0
0
no formation of p5 TGTCGACACCCAATT3 , which is the
product resulting from the cleavage of the phosphate linkage
Table 2 lists several 50-end- and 30-end-nucleotide2peptide
conjugates prepared by the present method, together with
yields estimated by HPLC.
The stability of the type-A conjugates prepared above was
investigated under basic conditions. As predicted, the conju-
gates were stable to brief treatment with conc. aqueous
ammonia at 258C, which are the reaction conditions for
the detachment of the products from solid supports, and
no changes including cleavage of the phosphate linker
between nucleotide and peptide moieties and recemization
of amino acid components were observed. By contrast, the
conjugates were labile under heating with conc. ammonia at
558C for 12 h, which are generally used for the removal of
N-acyl protectors, to decompose to some extent, giving the
corresponding oligonucleotide 30- or 50-monophosphates as
the degradation products.34
Figure
2.
HPLC
pro®les
of
the
crude
product
of
0
0
Thr(p5 TGTCGACACCCAATT3 )-Gly (21).
Table 2. Synthesis of nucleotide±peptide conjugates
Nucleotide±peptide conjugate
yielda (%)
0
Ser(p5 CATCAT)-Gly
90
79
89
82
78
69
82
76
81
0
Ser(p5 TGTCGACACCCAATT)-Gly
0
Ala-Ser(p5 CATCAT)-Gly-Asp
0
Ala-Ser(p5 TGTCGACACCCAATT)-Gly-Asp (20)
0
Thr(p5 CATCAT)-Gly
0
Thr(p5 TGTCGACACCCAATT)-Gly (21)
0
(CATCAT3 p)Ser-Gly
0
Conclusions
(TGTCGACACCCAATT3 p)Ser-Gly
0
Ala-(CATCAT3 p)Ser-Gly-Asp
a Estimated by HPLC analysis.
We developed an ef®cient method for the synthesis of base-
labile nucleotide±peptide conjugates with a phosphodiester

4432
A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
linkage between the 50- or 30-end hydroxyl group of an
oligodeoxyribonucleotide and the side-chain hydroxyl
group of a serine or threonine residue of a peptide via the
allyl-protected phosphoramidite strategy. This method
generally provides the target compounds in high yields
and in good purity without conspicuous degradation of the
base-labile phosphate linkage between the nucleotide and
the peptide. Examination of the stability of the products
under various basic conditions indicated that the use of
the allyl-protection strategy is a crucial point to allow
such an ef®cient synthesis.
sine 50-(allyl N,N-diiso-propylphosphoramidite) (7),22 30-O-
(p, p0-dimethoxytrityl)thymidine 50-(allyl N,N-diiso-propyl-
phosphoramidite) (8),22 p-toluenesulfonic acid salt of allyl
glycinate,29 N-(allyloxycarbonyl)-l-serine (9),30 N-(allyloxy-
carbonyl)-l-threonine (10),30 N-(tert-butoxy-carbonyl)-O-
(tert-butyldimethylsilyl)-l-serine,31 and benzimidazolium
tri¯ate (BIT)26 were prepared by the reported methods.
Acetonitrile and dichloromethane were distilled from CaH2.
Tetrahydrofuran (THF) was distilled from sodium and benzo-
phenone. Other organic solvents were used after simple
distillation of the commercially supplied ones. Unless
otherwise noted, commercially supplied reagents were used
without further puri®cation. A solution of a nucleoside
Experimental
phosphoramidite (0.1 M),
a
peptide phosphoramidite
(0.1 M), and benzimidazolium tri¯ate (0.2 M) in acetonitrile
was prepared immediately before use. A 1:1:8 mixture of
acetic anhydride, 2,6-lutidine, and THF, and a 16% solution
of N-methylimidazole in THF were also freshly prepared
before use.
General
IR spectra were measured in a described phase on a JASCO
FT/IR-5300 spectrometer. UV spectra were taken in metha-
nol on a JASCO V-550 spectrometer. NMR spectra were
obtained in CDCl3 unless otherwise noted on a JEOL a-400
instrument. The 1H NMR chemical shifts are described as d
values in ppm relative to (CH3)4Si. Chemical shifts quoted
in 31P NMR spectra are down®eld from 85% H3PO4. Angle
of optical rotation, [a]D, was obtained in chloroform, unless
otherwise stated, on a JASCO DIP-140. Measurement of
electrospray ionization-mass (ESI-MS) spectra was carried
out at the Chemical Instrument Center, Nagoya University.
High-performance liquid chromatography (HPLC) using an
ODS-5 mm column with a 0.1 M ammonium acetate buffer
containing 5±13% acetonitrile (v/v) (linear gradient in
30 min) was carried out on a JASCO PU-980 chromato-
graph with a JASCO UV-970-absorption detector. Capillary
gel electrophoresis using a capillary charged with 5%T
polyacrylamide gel was carried out on a Photal CAPI-
3200 instrument. Fuji silysia silica gel BW-300S was used
for column chromatography. Elemental analysis was
achieved at the Faculty of Agriculture, Nagoya University.
Unless otherwise stated, reactions were carried out at ambi-
ent temperature. Solid-phase syntheses were carried out on
an Applied Biosystems Model 392 DNA/RNA synthesizer.
The allyl protected (seryl)glycine N,N-diisopropyl-
phosphoramidite 13. To a solution of the p-toluenesulfonic
acid salt of the glycine allyl ester (3.70 g, 13.4 mmol),
N-(allyloxycarbonyl)-l-serine (9) (3.06 g, 16.2 mmol),
1-hydroxybenzotriazole (HOBT) (2.20 g, 16.3 mmol), and
triethylamine (1.38 g, 1.90 mL, 13.6 mmol) in THF (40 mL)
was added a solution of dicyclohexylcarbodiimide (DCC)
(5.01 g, 24.3 mmol) in THF (14 mL), and the mixture was
stirred overnight. The occurring insoluble materials were
removed by ®ltration and the ®ltrate was concentrated.
The residue was dissolved in ethyl acetate (800 mL) and
the resulting organic solution was successively washed
with an NaHCO3-saturated aqueous solution (100 mL), a
10% aqueous solution of citric acid (100 mL), the
NaHCO3-saturated solution (100 mL), and brine (100 mL).
The organic layer was dried and concentrated. The residue
was subjected to silica gel (50 g) column chromatography
with a 1:1 to 5:1 mixture of ethyl acetate and hexane as the
eluent to afford 11 (3.63 g, 98% yield) as a colorless oil:
[a]2D9ˆ219.28 (c 1.04); IR (neat) 3340, 1725, 1670, 1530,
1
1200 cm21; H NMR 3.65 (br s, 1H), 3.72 (dd, Jˆ4.9,
10.7 Hz, 1H), 4.04 (dd, Jˆ3.4, 11.2 Hz, 1H), 4.05±4.10
(m, 2H), 4.33 (br s, 1H), 4.58 (d, Jˆ5.4 Hz, 2H), 4.64 (d,
Jˆ5.9 Hz, 2H), 5.22 (d, Jˆ10.3 Hz, 1H), 5.27 (d,
Jˆ10.7 Hz, 1H), 5.31 (d, Jˆ19.0 Hz, 1H), 5.34 (d,
Jˆ17.1 Hz, 1H), 5.85±5.95 (m, 2H), 6.07 (br d, Jˆ6.3 Hz,
1H), 7.31 (s, 1H). Anal. Calcd for C12H18N2O6: C, 50.35; H,
6.34; N, 9.79. Found: C, 50.35; H, 6.51; N, 9.71. To a stirred
solution of 11 (978 mg, 3.42 mmol), 1H-tetrazole (238 mg,
3.40 mmol), and diisopropylamine (339 mg, 0.470 mL,
3.35 mmol) in acetonitrile (10 mL) was added (allyloxy)-
bis(diisopropylamino)phosphine (1.50 g, 5.20 mmol) and
stirring was continued for 2 h. The reaction mixture was
diluted with ethyl acetate (200 mL) and washed with an
aqueous solution saturated with NaHCO3 (50 mL) and
brine (50 mL). The organic layer was dried and concen-
trated to give a residual material. This crude product was
subjected to silica gel (50 g) column chromatography with a
1:4 mixture of ethyl acetate and hexane containing a trace of
triethylamine to afford 13 (1.10 g, 68% yield) as a ca. 1:1
mixture of diastereomers: IR (neat) 1730, 1680, 1530,
Material and solvents
20-Deoxyadenosine (Yamasa), 20-deoxycytidine (Yamasa),
20-deoxyguanosine (Yamasa), thymidine (Yamasa), l-
alanine (nacalai tesque), l-aspartic acid (nacalai tesque),
glycine (nacalai tesque), l-serine (nacalai tesque), l-threo-
nine (nacalai tesque), and N-(tert-butoxycarbonyl)glycine
(nacalai tesque) were commercially supplied. N 6-(Allyloxy-
carbonyl)-50-O-( p,p0-dimethoxytrityl)-20-deoxyadenosine
30-(allyl N,N-diisopropylphosphoramidite) (1),25,28 N4-
(allyloxycarbonyl)-50-O-( p,p0-dimethoxytrityl)-20-deoxy-
cytidine 30-(allyl N,N-diisopropylphosphoramidite) (2),25,28
O6-allyl-N2-(allyloxycarbonyl)-50-O-(p,p0-dimethoxytrityl)-
20-deoxyguanosine 30-(allyl N,N-diisopropylphosphorami-
dite) (3),25,27 50-O-(p,p0-dimethoxytrityl)thymidine 30-(allyl
N,N-diisopropylphosphoramidite) (4),25 N 6-(allyloxycar-
bonyl)-30-O-(p,p0-dimethoxytrityl)-20-deoxyadenosine 50-
(allyl N,N-diisopropylphosphoramidite) (5),22 N4-(allyloxy-
carbonyl)-30-O-(p,p0-dimethoxytrityl)-20-deoxycytidine 50-
(allyl N,N-diisopropylphosphoramidite) (6),22 O6-allyl-N 2-
(allyloxycarbonyl)-30-O-( p-methoxytrityl)-20-deoxyguano-
1
1185 cm21; H NMR 1.14±1.18 (m, 12H), 3.52±3.67 (m,
2H), 3.68±3.83 (m, 1H), 3.97±4.25 (m, 5H), 4.36±4.43 (m,

A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
4433
1H), 4.60 (d, Jˆ5.3 Hz, 2H), 4.65 (d, Jˆ5.8 Hz, 2H), 5.13±
5.36 (m, 6H), 5.70 (br s, 0.5H), 5.84±6.03 (m, 3.5H), 6.93
(br s, 0.5H), 7.01 (br s, 0.5H); 31P NMR 149.0, 149.9. Anal.
Calcd for C21H36N3O7P: C, 53.27; H, 7.66; N, 8.87. Found:
C, 53.31; H, 7.90; N, 9.02.
solution was added a solution of DCC (3.71 g, 18.0 mmol)
in dichloromethane (10 mL), and the mixture was stirred
overnight. The resulting insoluble material was removed
by ®ltration. Concentration of the ®ltrate provided a residual
oil, which was dissolved in ethyl acetate (300 mL). The
organic solution was washed successively with a 10% solu-
tion of citric acid (50 mL), an NaHCO3-saturated solution
(50 mL), water (50 mL), and brine (50 mL), and then, after
drying, concentrated. The resulting oily residue was chro-
matographed on a silica gel (50 g) column using a 1:2 to 1:1
mixture of ethyl acetate and hexane as eluent to afford
diallyl [(N-tert-butoxycarbonyl)glycyl]-l-aspartate (15)
(2.35 g, 63% yield) as a colorless viscous oil: [a]3D0ˆ
132.68 (c 1.01); IR (neat) 3340, 1740, 1685, 1520, 1370,
1170 cm21; 1H NMR 1.45 (s, 9H), 2.89 (dd, Jˆ4.6, 7.2 Hz,
1H), 3.07 (dd, Jˆ4.6, 7.2 Hz, 1H), 3.70±3.95 (m, 2H),
4.57±4.66 (m, 4H), 4.91 (td, Jˆ4.6, 8.3 Hz, 1H), 5.20±
5.40 (m, 5H), 5.81±5.91 (m, 2H), 7.20 (br d, Jˆ8.3 Hz,
1H). Anal. Calcd for C17H26N2O7: C, 55.13; H, 7.07; N,
7.56. Found: C, 55.12; H, 7.19; N, 7.66. The dipeptide 15
(380 mg, 1.03 mmol) was dissolved in dichloromethane
(3.0 mL) and tri¯uoroacetic acid (3.0 mL) was added at
08C. After stirring for 0.5 h at 08C, the reaction mixture
was concentrated to give a viscous oil, which was dissolved
in THF (5.0 mL). To this solution were added N-(tert-
butoxycarbonyl)-O-(tert-butyldimethylsilyl)-l-serine (383 mg,
1.20 mmol), triethylamine (508 mg, 0.70 mL, 5.02 mmol),
and BOP (810 mg, 1.83 mmol), and the resulting mixture
was stirred for 3 h. Concentration of the mixture afforded
residual oil, which was dissolved in ethyl acetate (80 mL).
The organic solution was washed with an aqueous solution
saturated with NaHCO3 (20 mL) and brine (20 mL), dried,
and concentrated. The resulting crude product was subjected
to silica gel (30 g) column chromatography with a 1:4 to 1:1
mixture of ethyl acetate and hexane, affording Boc-
Ser(OTBDMS)-Gly-Asp(OCH2CHvCH2)2 (16) (533 mg,
91% yield) as a colorless amorphous powder: [a]2D9ˆ
12.818 (c 1.01); IR (KBr) 1750, 1710, 1645, 1540, 1510,
1210, 1175 cm21; 1H NMR 0.08 (s, 6H), 0.88 (s, 9H), 1.46
(s, 9H), 2.89 (dd, Jˆ4.9, 17.1 Hz, 1 H), 3.05 (dd, Jˆ4.4,
17.1 Hz, 1H), 3.71 (dd, Jˆ6.3, 9.8 Hz, 1H), 3.99±4.03 (m,
1H), 4.00 (d, Jˆ5.4 Hz, 2H), 4.19 (m, 1H), 4.58 (d,
Jˆ5.4 Hz, 2 ), 4.64 (d, Jˆ5.4 Hz, 2H), 4.89 (ddd, Jˆ4.4,
4.9, 8.3 Hz, 1H), 5.25 (d, Jˆ10.3 Hz, 2H), 5.31 (dd, Jˆ1.0,
17.1 Hz, 2H), 5.39 (br s, 1H), 5.83±5.94 (m, 2H), 7.09 (br d,
Jˆ8.3 Hz, 1H), 7.14 (br t, Jˆ5.4 Hz, 1H). Anal. Calcd for
C26H45N3O9Si: C, 54.62; H, 7.93; N, 7.35. Found: C, 54.64;
H, 7.93; N, 7.29. To a solution of 16 (605 mg, 1.06 mmol) in
dichloromethane (3.0 mL) was added tri¯uoroacetic acid
(3.0 mL) at 08C and the mixture was stirred for 0.5 h at
08C. Concentration of the reaction mixture gave a viscous
oil, which was dissolved in THF (5.0 mL). To this solution
were added N-(allyloxycarbonyl)-l-alanine (225 mg,
1.30 mmol), triethylamine (537 mg, 0.74 mL, 5.31 mmol),
and BOP (853 mg, 1.93 mmol) and the mixture was stirred
for 3 h. The organic solvent was removed by evaporation
and the resulting material was dissolved in ethyl acetate
(80 mL). The solution was washed with an NaHCO3-satu-
rated solution (20 mL) and brine (20 mL), dried, and
concentrated to afford an oil. This crude product was
subjected to silica gel (30 g) column chromatography eluted
with a 1:40:40 to 1:10:10 methanol±ethyl acetate±hexane
mixture to afford a solid material (722 mg). This material
The allyl protected (threonyl)glycine N,N-diisopropyl-
phosphoramidite 14. The phosphoramidite 14 was
prepared according to a similar procedure to that mentioned
above for the preparation of 13. Firstly, allyl [N-(allyloxy-
carbonyl)-l-threonyl]glycinate 12 (3.02 g, 67% yield) was
obtained starting from the p-toluenesulfonic acid salt of
allyl glycinate (3.70 g, 13.4 mmol) and N-(allyloxycarbo-
nyl)-l-threonine (10) (3.65 g, 16.2 mmol). 12: [a]2D6ˆ
232.88 (c 1.01); IR (neat) 3340, 1726, 1665, 1530,
1
1200 cm21; H NMR 1.20 (d, Jˆ6.4 Hz, 3H), 3.82 (br s,
1H), 4.06 (d, Jˆ5.4 Hz, 2H), 4.25 (d, Jˆ6.3 Hz, 1H), 4.34
(m, 1H), 4.58 (d, Jˆ5.4 Hz, 2H), 4.63 (d, Jˆ5.9 Hz, 2H),
5.26 (dd, Jˆ10.2, 1.0 Hz, 1H), 5.31 (d, Jˆ17.1 Hz, 1H),
5.33 (dd, Jˆ17.6, 1.5 Hz, 1H), 5.85±5.96 (m, 2H), 6.06
(d, Jˆ8.3 Hz, 1H). Anal. Calcd for C13H20N2O6: C, 51.99;
H, 6.71; N, 9.33. Found: C, 52.05; H, 6.86; N, 9.08. Subse-
quently, 12 (248 mg, 826 mmol) was converted to 14
(360 mg, 89% yield) by the 1H-tetrazole-promoted phos-
phoramiditation with CH2vCHCH2OP[N(i-C3H7)2]2. 14:
IR (neat) 1760 (sh), 1730, 1680, 1510, 1185 cm21 1H
;
NMR 1.17±1.29 (m, 15H), 3.48±3.71 (m, 2H), 3.94±4.37
(m, 5H), 4.45±4.51 (m, 1H), 4.59 (d, Jˆ5.4 Hz, 2H), 4.65
(d, Jˆ5.7 Hz, 2H), 5.11±5.37 (m, 6H), 5.77±6.02 (m, 4H),
7.09 (br s, 0.5H), 7.21 (br s, 0.5H); 31P NMR 147.5, 150.0.
Anal. Calcd for C22H38N3O7P: C, 54.20; H, 7.86; N, 8.62.
Found: C, 54.25; H, 7.92; N, 8.77.
N-(Allyloxycarbonyl)-l-alanine. A mixture of l-alanine
(1.78 g, 20.0 mmol) and allyl chloroformate (2.5 mL,
23.6 mmol) in a 1.0 M NaOH solution (20 mL, 20 mmol)
was stirred for 3 h. During this period, a 2.0 M NaOH solu-
tion (10 mL, 20 mmol) was additionally poured in four
portions into the reaction mixture. To the reaction mixture
was added conc. aqueous HCl until the pH of the mixture
became about 1. The resulting solution was extracted with
diethyl ether (150 mL£5). The combined organic layers
were washed with brine (100 mL), dried, and concentrated
to give N-(allyloxycarbonyl)-l-alanine (3.45 g, 100% yield)
as a colorless oil: [a]2D7ˆ22.498 (c 1.09, CH3OH); IR (neat)
3325, 1715, 1535, 1455, 1410, 1240 cm21 1H NMR
;
(CD3OD) 1.38 (d, Jˆ7.3 Hz, 3H), 4.17 (q, Jˆ7.3 Hz, 1H),
4.53 (d, Jˆ5.4 Hz, 2H), 5.17 (dd, Jˆ1.5, 10.2 Hz, 1H), 5.30
(dd, Jˆ1.5, 17.1 Hz, 1H), 5.92 (ddd, Jˆ5.4, 10.2, 17.1 Hz,
1H). Anal. Calcd for C7H11NO4: C, 48.55; H, 6.40; N, 8.09.
Found: C, 48.61; H, 6.55; N, 8.14.
The allyl protected tetrapeptide N, N-diisopropyl-
phosphoramidite 18. A mixture of l-aspartic acid
(1.33 g, 10.0 mmol), allyl alcohol (5.81 g, 6.80 mL,
100 mmol), and p-toluenesulfonic acid monohydrate
(2.10 g, 11.0 mmol) in toluene (80 mL) was heated under
re¯ux for 3 h in a ¯ask equipped with a Dean±Stark water
separator. The reaction mixture was concentrated to give a
viscous oil (4.43 g). This material was dissolved in dichloro-
methane (10 mL) together with N-(tert-butoxycarbonyl)-
glycine (2.11 g, 12.0 mmol), HOBT (2.43 g, 18.0 mmol),
and triethylamine (1.02 g, 1.40 mL, 10.0 mmol). To this

4434
A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
was dissolved in THF (8.0 mL), and acetic acid (114 mL,
1.99 mmol) and a 1.0 M solution of tetrabutylammonium
¯uoride in THF (2.00 mL, 2.00 mmol) were added. The
mixture was stirred for 2 h and the reaction was quenched
by the addition of an aqueous solution saturated with NH4Cl
(50 mL). The whole mixture was extracted with ethyl
acetate (3£50 mL). The combined organic layers were
washed with brine (20 mL), dried and concentrated.
Chromatography of the resulting residual oil on silica
gel (30 g) column using a 1:2 to 2:1 mixture of acetone
A typical procedure for deprotection, detachment, and
puri®cation of nucleotide±peptide conjugates
Firstly, a solution of the Pd catalyst in THF was prepared
by heating a mixture of the Pd2(dba)3´CHCl3 complex
(20.7 mg, 20.0 mmol), triphenylphosphine (52.5 mg,
200 mmol), diethylamine (87.7 mg, 124 mL, 1.20 mmol),
and formic acid (55.3 mg, 45.3 mL, 1.20 mmol) in THF
(2.0 mL) until the mixture turned to be a clean orange
solution. A protected oligonucleotide±peptide conjugate
(0.2 mmol) anchored on the CPG supports was washed
with acetonitrile (1 mL£3) and dried under reduced
pressure. To the CPG-supported materials was added a
suitable volume of the Pd-catalyst solution (2.5 equiv. of
the Pd catalyst/allyl) freshly prepared as above. The mixture
was heated at 508C for 1 h with occasional shaking. After
the supernatant ¯uid was decanted, the resulting CPG
supports were washed by the reported procedure.25 The
washed CPG supports were treated with conc. aqueous
ammonia for 1 h to give the crude target conjugate. To a
portion of an aqueous solution of the crude product was
added an aqueous solution containing a suitable amount of
the parent oligonucleotide. The resulting solution was
subjected to HPLC analysis and the area of peak due to
the target conjugate was compared with that of the peak
due to the standard oligonucleotide to estimate the yield
of the desired product in a crude form.
and
Asp(OCH2CHvCH2)2 (17) (368 mg, 74% yield):
toluene
furnished
AOC-Ala-Ser-Gly-
[a]2D9ˆ22.368 (c 1.03, CH3OH); IR (KBr) 1740, 1710,
1
1675, 1635, 1525, 1220, 1185 cm21; H NMR (CD3OD)
1.36 (d, Jˆ7.3 Hz, 3H), 2.87 (dd, Jˆ6.3, 17.1 Hz, 1H),
2.93 (dd, Jˆ5.9, 17.1 Hz, 1H), 3.78 (dd, Jˆ4.9, 11.2 Hz,
1H), 3.87 (dd, Jˆ5.4, 11.2 Hz, 1H), 3.89 (d, Jˆ17.1 Hz,
1H), 3.94 (d, Jˆ17.1 Hz, 1H), 4.15 (q, Jˆ7.3 Hz, 1H),
4.36 (dd, Jˆ4.9, 5.4 Hz, 1H), 4.54±4.83 (m, 6H), 4.86
(dd, Jˆ5.8, 6.3 Hz, 1H), 5.16±5.23 (m, 3H), 5.28±5.34
(m, 3H), 5.87±5.98 (m, 3H). Anal. Calcd for
C22H32N4O10: C, 51.56; H, 6.29; N, 10.93. Found: C,
51.55; H, 6.30; N, 10.91. The protected tetrapeptide 17
(284 mg, 672 mmol) was dissolved together with 1H-tetra-
zole (23.6 mg, 337 mmol) and diisopropylamine (34.0 mg,
47.0 mL, 340 mmol) in acetonitrile (6.0 mL) and to this
solution was added CH2vCHCH2OP[N(i-C3H7)2]2
(254 mg, 881 mmol). After 2 h, the reaction mixture was
diluted with ethyl acetate (80 mL) and washed with an
aqueous NaHCO3 solution (10 mL) and brine (10 mL).
The organic layer was evaporated to give a residual mate-
rial, which was subjected to silica gel (20 g) column chro-
matography. Elution with a 1:4 mixture of ethyl acetate and
hexane containing a trace of triethylamine afforded 18
(266 mg, 63% yield) as a ca 1:1 mixture of diastereomers:
Conjugates prepared via the typical procedures and
con®rmation of their structures
The products prepared according to the typical procedure
are listed in Table 3. Their structures were con®rmed by
dominant molecular ion peaks measured by the ESI-MS
analyses shown in Table 3.
IR (KBr) 1750, 1710, 1640, 1510, 1240, 1225, 1185 cm21
;
1H NMR 1.14±1.18 (m, 12H), 1.41 (d, Jˆ6.8 Hz, 1.5H),
1.43 (d, Jˆ6.8 Hz, 1.5H), 2.90 (dd, Jˆ4.9, 17.1 Hz,
0.5H), 2.91 (dd, Jˆ4.9, 17.1 Hz, 0.5H), 3.00 (dd, Jˆ4.9,
17.1 Hz, 0.5H), 3.02 (dd, Jˆ4.9, 17.1 Hz, 0.5H), 3.51±
3.63 (m, 2H), 3.77±3.84 (m, 1.5H), 3.94±4.29 (m, 5.5H),
4.44±4.64 (m, 7H), 4.89±4.94 (m, 1H), 5.15±5.35 (m, 8H),
5.39 (br s, 1H), 5.83±6.02 (m, 4H), 7.13 (br s, 0.5H), 7.15
(br s, 0.5H), 7.22 (br s, 1H), 7.47 (br s, 0.5H), 7.48 (br s,
0.5H); 31P NMR 149.3, 149.6. Anal. Calcd for
C31H50N5O11P: C, 53.21; H, 7.20; N, 10.01. Found: C,
53.33; H, 7.25; N, 10.21.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scienti®c Research (No. 11101001) from the Ministry of
Education, Science, Sports and Culture, by a grant from
the `Research for the Future' Program of the Japan Society
for the Promotion of Science (JSPS-RFTF97I00301), and by
contributions from the Asahi Glass Foundation and the
Daiko Foundation.
Table 3. Molecular weight and dominant molecular ion of nucleotide±peptide conjugates
Nucleotide±peptide conjugate
Molecular weight
Molecular iona
0
Ser(p5 CATCAT)-Gly
1975.31
4736.07
2161.47
4922.23
1989.33
4750.09
1975.31
4736.07
2161.47
1974.4 (M2H)21
4734.3 (M2H)21
2160.8 (M2H)21
2460.7 (M22H)22
1988.2 (M2H)21
4748.8 (M2H)21
1974.5 (M2H)21
4734.3 (M2H)21
2161.7 (M2H)21
0
Ser(p5 TGTCGACACCCAATT)-Gly
0
Ala-Ser(p5 CATCAT)-Gly-Asp
0
Ala-Ser(p5 TGTCGACACCCAATT)-Gly-Asp (20)
0
Thr(p5 CATCAT)-Gly
0
Thr(p5 TGTCGACACCCAATT)-Gly (21)
0
(CATCAT3 p)Ser-Gly
0
(TGTCGACACCCAATT3 p)Ser-Gly
0
Ala-(CATCAT3 p)Ser-Gly-Asp
a Obtained by ESI-MS analysis.

A. Sakakura, Y. Hayakawa / Tetrahedron 56 (2000) 4427±4435
4435
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34. Structures of degradation products resulting from the peptide
components were not determined.