Nucleic Acids Research, 1993, Vol. 21, No. 23
5337-5344
Dipentafluorophenyl carbonate—a reagent for the
synthesis of oligonucleotides and their conjugates
V.A.Efimov, A.LKalinkina and O.G.Chakhmakhcheva
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul.
Miklukho-Maklaya 16/10, Moscow 117871, Russia
Received September 9, 1993; Revised and Accepted October 26,1993
ABSTRACT
Dipentafluorophenyl carbonate has been successfully
used as condensing agent for the internucleotide bond
formation in the synthesis of oligonucleotides via Hphosphonate approach. The mechanism of a nucleotide
component activation with this reagent has been
investigated with the help of 31P NMR spectroscopy.
It was shown that preactivation of deoxynucleoside Hphosphonate with dipentafluorophenyl carbonate has
no influence on the efficiency of the synthesis. This
reagent is highly reactive, nonhygroscopic and stable
on storage at room temperature. The effectiveness of
dipentafluorophenyl carbonate in the oligonucleotide
chemistry has been demonstrated in the solid-phase
synthesis of 10 - 50-mers on 0.2,1 and 10 /*mol scales.
The use of this reagent for the derivatisation of polymer
supports as well as for the synthesis of oligonucleotide
conjugates with polyethylene glycol and a lipid is
described.
material are observed during condensation, and it leads to
decreasing the yield of the desired compound [5,6]. In particular,
preactivation of a nucleoside 3'-H-phosphonate followed by the
addition to a OH-component, that usually takes place in the
synthesis on polymer supports, resulted in lower yields of the
H-phosphonate diesters. The other side reaction is a modification
of heterocyclic bases of nucleotides during condensation.
In this paper we describe a convenient and efficient Hphosphonate method based on the use of a new condensing
agent—dipentafluorophenyl carbonate, which provides high
coupling ability and considerable decrease of the side reactions
extent during preactivation of a nucleotide component and the
amount of by-products caused by modification of heterocyclic
bases. Along with this, the effectiveness of this reagent for the
other aspects of oligonucleotide synthesis, such as derivatization
of polymer support and synthesis of some oligonucleotide
conjugates has been also investigated.
RESULTS AND DISCUSSION
INTRODUCTION
The key step in the chemical synthesis of oligonucelotides is the
specific formation of an internucleotide phosphate linkage. At
present, there exist several well established approaches to
oligonucleotide synthesis. One of them is the hydrogenphosphonate approach, which was introduced for the first time
by A.Todd et al.[l]. Now it is widely used for chemical synthesis
of oligonucleotides [ 2 - 4 ] . By contrast with phophotriester and
phosphoramidite methods, H-phosphonate chemistry requires no
phosphate protecting group, since an assembled oligonucleotide
chain containing internucleotide H-phosphonate diester links is
relatively inert to the conditions of coupling. The H-phosphonate
units are more stable than the phosphoramidite units. Usually,
activation of a nucleotide component is achieved with hindered
acyl chloride (e.g. pivaloyl chloride [2,3], or adamantoyl chloride
[4]), which couples the H-phosphonate to a nucleoside 5'-hydroxyl group. The resultant H-phosphonate diester is relatively
inert to further phosphitylation , thus the oligonucleotide chain
may be extended without prior oxidation. Oxidation of all
phosphorus centres is performed simultaneously at the end of
the oligonucleotide synthesis. At the same time, this approach
gives a fast and efficient procedure that can be applied to the
rapid synthesis of oligonucleotide analogues. However, some
side-reactions between the condensing reagent and the starting
Earlier, we investigated the reaction of H-phosphonate monoester
preactivation by pivaloyl chloride (PivCl) with the use of 31P
NMR spectroscopy. On the basis of the results obtained a
modification of the method, which minimized the extent of side
reactions and included the use of acetonitril-quinoline mixture
as a solvent in coupling reaction, has been developed [6, 7].
In our continuous study to develop the coupling reagents for
the internucleotide bond formation by H-phosphonate approach,
we have examined various potential coupling reagents,
particularly activated carbonic acid derivatives. Thus, it was
found that pentafluorophenyl esters of pivalic acid, and
triisopropylbenzenesulfonic acid, as well as l,l'-carbonyldiimidazole, did not promote the internucleotide bond formation. Other
reagents, such as pentafluorophenyl chloroformate and fluorenylmethoxycarbonyl chloride are more reactive than necessary, and
the rate of internucleotide condensation which these reagents assist
is very close to the rates of side-reactions: modification of
heterocyclic bases and blocking the 5'-hydroxyl of a nucleoside
component. The best results were obtained by us with the use
of dipentafluorophenyl carbonate.
Recently, it was shown that dipentafluorophenyl carbonate
(PFPC) is an effective reagent for the synthesis of peptides via
pentafluorophenyl esters of amino acids [8]. The preparation of
this reagent is simple. PFPC is a highly reactive, nonhygroscopic
5338 Nucleic Acids Research, 1993, Vol. 21, No. 23
100
100
30
60
90
TIME
120
(sec)
150
100
Figure 2. The rate of 5',3'-0-diacetyl-N2-isobutyryldeoxyguanosine (0.05 M,
1 equiv.) modification under the action of condensing agent (5 equiv.): PivCl
(1,2) and PFPC (3). The reactions were carried out in acetonitrile containing
25% of pyridine (1,3), or 20% of quinoline (2) as a solvent. The amount of
unconsumed starting deoxynucleoside was estimated by the analysis of reaction
mixture aliquots by TLC and reversed phase chromatography.
80
70
10
TIME
(min)
Figure 1. A. Coupling rates in the synthesis of H-phosphonate diester from
[(MeO) 2 Tr]Tp(H) (0.05 M, 1.2 equiv.) and T(Bz) (1 equiv.) in
acetonitrile—pyridine (3:1) in the presence of 5 equiv. of condensing agent:
1. — regular coupling reaction with PivCl; 2. — after 10 min preactivation of
P-component with PivCl; 3. — reqular coupling reaction with PFPC; 4. — after
10 min preactivation of P-component with PFPC. B. The dependence of coupling
yields on the time of a P-component preactivation. The equal volumes of a 0.05
M solution (5 equiv.) of [(MeO)2Tr]Tp(H) and 0.25 M solution (25 equiv.) of
condensing agent in acetonitrile—pyridine (3:1) were added in time intervals to
the polymer-bound thymidine (1 equiv.). After 3 min the yield of the coupling
reaction was estimated by the detritylation of polymer.
and stable on storage crystalline compound, and it has good
solubility in most organic solvents.
In the first set of experiments we investigated the coupling
properties of PFPC. The condensation between 5'-Odimethoxytritylthymidine H-phosphonate (I) (0.05 M, 2 equiv.)
and 3'-O-benzoylthymidine (1 equiv.) in the presence of PFPC
(6 equiv.) was carried out in acetonitrile-pyridine (3:1) mixture
as a solvent at room temperature. The reaction was monitored
by TLC. It was revealed that the process went to a completion
in about 1 min (Fig. 1A). Under the same conditions, the reaction
time with PivCl was 30 sec. If the H-phosphonate (I) was
premixed with activating agent PFPC before coupling during 10
min, no decrease in the speed of condensation reaction and in
the yield of the desired product was observed in solution, or on
polymer support (Fig. IB). At the same time, after 10 min
preactivation the yield of dinucleoside phosphonate in the
presence of PivCl was only about 60% in 1 min.
One of the side-reactions in the H-phosphonate oligonucleotide
synthesis is the modification of heterocyclic bases, particularly
N^acylguanine and thymine residues which are susceptible to
acylation by condensing agents and to phosphitylation by activated
Table 1. Selected
31
P NMR chemical shift values
Compound
8(ppm)
'JP-H
la
Ib
Ob
2.10
2.90
2.00
0.50
-1.60
134.40
135.40
9.35
8.80
8.60
139.60
2.62
2.72
122.70
135.8
606
604
605
620
659
nia
mb
IVa
rvb
Va
Vb
VIb
RO-PO(H)-OPiv
RO-P(OPiv)2
EtO-P(OC6F5)2
(Hz)
720
726
710
735
10
nucleoside H-phosphonate. Our experiments revealed that in the
absence of a nucleotide component the rate of N^acylguanine
interaction with PFPC was much slower than with PivCl (Fig.
2). In the presence of a nucleoside H-phosphonate, after 20 min
the content of modified products in the reaction mixture consisted
of S'^'-O-diacetyl-NMsobutyryldeoxyguanosine (0.1 M),
10-fold excess of condensing agent and 5-fold excess of 5'-Odimethoxytritylthymidine H-phosphonate was about 35 % in the
case of PivCl with the use of acetonitrile-pyridine (3:1) as a
solvent. In acetonitrile -quinoline mixture (4:1), the modification
proceeds slower in the presence of PivCl about 15% during 20
min. PFPC gave only about 2% of O6-modified products in
acetonitrile—pyridine mixture (3:1) during the same time [see
also 7, 9].
The other side-reaction, which usually can be observed in the
oligonucleotide synthesis, is the interaction of 5'-hydroxyl of a
nucleoside component with the condensing agent. For a good
Nucleic Acids Research, 1993, Vol. 21, No. 23 5339
0
la
o
II
II
RO-P-O-P-OR
1
I
(III)
H H
O
(I)
O
II
RO-P-O"
I
H
II
,O-C-OC6F5
RO-P-O-C-OC r
I
II
H
V
O
(I)
O-C-OC.F.
II
O (IV)
excess
R'OH
O
II
RO-P-OR'
I
H
(V)
IVo
(VI)
R = d[(HeO) 2 Tr)TR'= a - dT(Bz); b - Et
Va
Scheme 1. Proposed mechanism of a H-phosphonate activation by PFPC.
IVa
Ilia
Va
Ilia
1
.140
130
120
110
100
20
10
0
-10
Figure 3. 3 I P NMR data of the activation of 5'-O-dimethoxytrityl-thymidine Hphosphonate in acetonitrile-pyridine (3:1, v/v). Spectra are recorded after 3 min.
1 . - [(MeO)2Tr]Tp(H); 2.-[(MeO) 2 Tr]Tp(H)(l equiv.) + PivCl (3 equiv.);
3. — [(MeO)2Tr]Tp(H)(l equiv.) + PFPC (3 equiv.); 4. — reaction mixture
from (3) + T(Bz) (2 equiv.); 5. - [(MeO)2Tr]Tp(H)(l equiv.) + PFPC (1
equiv.); 6. — reaction mixture from (5) + T(Bz) (2 equiv.).
coupling reagent, the rate of internucleotide condensation which
it assists has to be considerably higher than the rate of its
interaction with 5'-hydroxyl of a nucleoside component. We have
found that in the absence of a P-component the time needed for
5'-OH group blocking in 3'-O-benzoylthymidine (1 equiv.) under
the action of PFPC (10 equiv.) was 30 min, that was much slower
than the rate of phosphitylation reaction in the presence of PFPC.
As a result, in the regular internucleotide condensation conditions,
where the competition between phosphitylation and carbonylation
reactions takes place, the amount of 5'-carbonylated by-product
not exceeded 1 % during the time needed for completion of the
coupling reaction with PFPC.
To investigate the activation process in the presence of PFPC
31
P NMR spectroscopy has been used. The spectrum of the
reaction mixture containing 5'-O-dimethoxytritylthymidine Hphosphonate (la) (1 equiv.), 3'-O-Bz-thymidine (2 equiv.) and
PFPC (3 equiv.) in acetonitrile-pyridine (3:1) showed only
signals corresponding to H-phosphonate diester (Va) (Fig. 3,
Table 1). The treatment of (la) with 1 equiv. of PFPC resulted
in its conversion, apparently via the step of mixed anhydride (Ha)
to the intermediate (TVa) with the chemical shift at 134.4 ppm.
However, we did not succeed to registrate the signal of (Ha) in
this experiment. At the same time, a small amount of Hpyrophosphonate (Ula) can be detected. The addition of 3-fold
excess of PFPC fully converted (la) into (TVa). By analogy with
the activation with PivCl, it was supposed that (TVa) represents
5' -O-(MeO) 2 Tr-thymidine-3' -0-bis(pentafluorophenoxycarbonyl)phosphite (Scheme 1). The addition of 3'-O-Bzthymidine excess to the compound (TVa) resulted in its rapid
convertion to dinucleoside phosphonate (Va). The examination
of the compound (TVa) reactivity has shown that, in contrast to
bisacylphosphite, it is as active phosphitylating agent as mixed
anhydride (II). As a consequence, the coupling reaction in solution
is complete in the standard reaction time (about 1 min) even after
preactivation of a P-component with PFPC during several
minutes, and the increase of preactivation time up to 1 h does
not lead to noticeable reduction in coupling yield.
To clarify the activation process further, ethyl H-phosphonate
(Ib) (5 = 2.9 ppm, 'Jp.H = 604 Hz, 3JP.H = 8 Hz) was used
as a model compound. The addition of 1 equiv. of PFPC to (Ib)
in pyridine-acetonitrile led to mixed anhydride (Hb)(6 = 2.0,
'JP.H = 605 Hz) formation, and the excess of PFPC converted
(lib) into the intermediate (TVb) with chemical shift 135.4 ppm.
Then, (TVb) can be converted into the starting material (Ib) upon
addition of water, or into triethylphosphite (VIb) (6 139.6 ppm)
upon addition of ethanol excess. As a reference compound,
dipentafluorophenylethyl phosphite (multiplet at 135.8 ppm) was
used. However, in contrast to the compound (TVb) this phosphite
triester was much less reactive, and the formation of
triethylphosphite (VIb) was not complete even after 3 0 - 4 0 min
interaction of dipentafluorophenylethyl phosphite with excess of
ethanol. The dependence of the rate of the compound (TV)
formation upon the basicity of solvent used has been also
examined. It was revealed that, as well as in the case of PivCl,
the conversion of mixed anhydride (II) into intermediate (TV)
speeded up in more basic conditions.
The feasibility of PFPC application for the synthesis of
oligonucleotides on polymer supports was proved in the
5340 Nucleic Acids Research, 1993, Vol. 21, No. 23
Table 2. Reaction cycle for the solid-phase synthesis of oligonucleotides by H-phosphonate method with the use
of PFPC
Step
1
2
I
4
5
6
7
8
Reagents and solvents
Acetonitrile wash
2% Trichloroacetic acid in
dichloromethane
Acetonitrile wash
Acetonitrile-pyridine (3:1, v/v)wash
Coupling mixture*
Acetonitrile-pyridine (3:1, v/v) wash
Capping mixture**
Acetonitrile-pyridine (3:1, v/v) wash
Time (min)
0.2-1 /anol
scale
scale
1.0
2.5
1.5
1.0
0.5
2.5
1.0
1.5
1.0
6.0
4.0
6.0
10 nmol***
54
4.0
4.0
2.0
*P-component (0.05 M, 100 /il) in acetonitrile-pyridine (1:1,v/v) and PFPC (0.15 M, 100 /il) in acetonitrile are
injected into a reaction vessel containing 10—30 mg of a support (about 30 /unol of dimethoxytritylnucleoside per g).
**A mixture of 2-cyanoethyl H-phosphonate (0.05 M, 100 /d) and PFPC (0.15 M, 100 /J) in acetonitrile-pyridine
(3:1).
***For 10 janol scale the volume of the reaction mixtures was 1.2 ml, The amount of support in the reaction
vessel was 200-300 mg for CPG and 20-40 mg for TentaGel.
B
4
260
10
T I M E
20
30
fm i n)
Figure 4. A. Polyacrylamide gel electrophoresis of crude reaction products in the synthesis of the 33-mer d(GTGAGATCTGGGGATGCTGCCCTCTTTGAGCCC)(a)
with the use of PFPC and the 40-mer d(CGGCACCGGAGCTCCTGGGTGGCCCGTCTGTTTTCCTGTT) with the use of PivCl (b) and PFPC (b') as condensing
agents. B. The FPLC analysis of 33-mer (a) and 40-mer (b') obtained with the use of PFPC after isolation by gel electrophoresis.
automated synthesis of DNA fragments ranging from 10- to
50-mers. At each step, the 5 —10-fold excess of P-component
over the resin capacity was used. The coupling reactions were
performed in the presence of 3-fold excess of PFPC with respect
to a P-component. The optimal coupling time on polymer support
was 2—2.5 min, that is slower than with PivCl (1.5 min).
The manipulations of a standard elongation cycle are listed in
Table 2. The overall time needed for performance of one cycle
on 0.2—1.0 /xmol scale was 10 min. The average yield per step
was about 98—99%. The reactions were carried out on a polymer
support on the base of standard porous glass beads in automatic
conditions. The removal of the protecting groups from the final
oligonucleotides after completion of the synthesis was carried
out by the action of ethanolamine as described earlier [10]. The
results on the analysis of the oligonucleotides obtained by this
method are shown in Fig.4. One of them, the 33-mer
d(GTGAGATCTGGGGATGCTGCCCTCTTTGAGCCC)
represents a primer for the isolation of a gene for Thermus
aquaticus DNA polymerase using the PCR technique and 40-mer
d(CGGCACCGGAGCTCCTGGGTGGCCCGTCTGTTTTCCTGTT) represents a part of the artificial gene for Fc fragment
of human immunoglobulin Gl, the synthesis of which is now
in progress in our laboratory.
It should be noted that in oligonucleotide synthesis on a large
scale (10-100 /rniol) economic considerations become more
important. In this regard, the H-phosphonate method has some
Nucleic Acids Research, 1993, Vol. 21, No. 23 5341
IjTrO-i^cKj
o
I
I
F 5 C 6 O-C(CH,) 2 -C=O
"0-C(CH 2 ) 2 -00
0
10
20
TIME (mini
< -XC
A
260
(MeO)2Tr0t -BPB
I
H-P=O
I
10
20
TIME
fm I n)
B
0
o
1
II
s~^
O-C(CH2)jC-NH—( P )
B « protected heterocyclic
^^^ base
(p)= LCAA-CPG, or TentaGel
Figure 5. The analysis of reaction products in the synthesis of the 14-mer
d(CTTTCTTTTCTCTT) on a 10 jimol scale. A. Polyacrylamide gel
electrophoresis (20%) of crude 14-mer obtained with the use of PFPC as
condensing agent (a) and the same oligonucleotide after isolation by FPLC (b).
Visualization by UV-shadowing. B. FPLC of crude reaction products in the
synthesis of the 14-mer with the use of PivCl (1) and PFPC (2) as condensing
agents.
advantages over the phosphoramidite approach, since the Hphosphonate monomers are more stable and potentially
recoverable. However, with acyl chlorides as condensing agents,
the lost in the yields during the preactivation step becomes a
problem on a preparative level, and usually the application of
special constructed large-scale synthesizers is necessary for its
solvation. The application of PFPC as condensing agent
eliminates this problem and allows to obtain preparative amounts
of oligonucleotides with high yields using routine synthesizers.
Moreover, the combination of the PFPC application with the use
of supports allowing high-loading of first monomer (such as
polyethylene glycol/polystyrene copolimer TentaGel [11]) makes
it possible to raise the level of oligonucleotide synthesis on
standard machines up to 30—40 jtmol.
To illustrate this, the direct comparison of two variants of Hphosphonate approach: with PivCl and with PFPC was made in
the synthesis of 14-mer d(CTTTCTTTTCTCTT) on a 10 junol
scale. This antisense oligonucleotide represents one of the
sequences optimal for oligonucleotide binding and translation
arrest of RNAs corresponding to fragments of the tick-borne
encephalitis genome [12]. The synthesis has been carried out in
the standard 10 /unol vessel for Model 381a Applied Biosystems
synthesizer. In both experiments, the synthetic conditions (starting
monomers, support,capping procedure, solvents), oxidation,
deprotection and purification procedures were similar. The overall
coupling yields of the desired oligonucleotide based on the first
nucleoside attached to a support were estimated by the
spectroscopic analysis of the dimethoxytrityl function liberated
from the support. In the case of PivCl it was about 10%, and
Scheme 2. Attachment of a nucleoside to aminated polymer support.
in the case of PFPC about 80%. After deprotection and isolation,
the yields were 5% and 52% with PivCl and PFPC, respectively.
Fig.5 shows the results obtained in the isolation and analysis of
this 14-mer.
Along with the application in the internucleotide condensations,
PFPC can be successuUy used in the other aspects of nucleic acids
chemistry. The procedure on the activation of carboxy group by
its conversion into pentafluorophenyl ester is not new. Routingly
N,N'-dicyclohexylcarbodiimide was used for obtaining
pentafluorophenyl esters of carboxyl-containing compounds [13,
14]. The application of PFPC for this purposes makes possible
to eliminate carbodiimide from the scheme of synthesis and
simplifies the procedure. The separation of the pentafluorophenyl
ester obtained from the excess of PFPC can be achieved by its
precipitation into hexane.
One of the examples of PFPC application is attachment of the
first nucleoside to a solid support (Scheme 2). Thus, the
3'-terminal deoxynucleoside can be attached to the aminated
support by conversion of its N-protected 5'-0-dimethoxytrityl-3'-O-succinate into the corresponding pentafluorophenyl
ester, which is subsequently reacted with amino group on the
support. All the procedure takes 6 - 7 hours. As it was determined
by spectrophotometrical analysis of dimethoxytrityl cation
released [15], the amount of polymer-bound nucleoside was in
the range of 3 0 - 5 0 /tmol/g for CPG support and in the range
of 160-180 /tmol/g for TentaGel support.
We investigated the potential of PFPC in the synthesis of some
oligonucleotide conjugates. The application of antisense
oligonucleotides and their analogous as highly specific inhibitors
of gene expression and viral replication has attached great interest
in recent years. However, the use of unmodified oligonucleotides
5342 Nucleic Acids Research, 1993, Vol. 21, No. 23
^260
1.0
xcXC-
0.5
B PBB P B-
30
20
T I M E (MIN)
Figure 6. A. Polyacrylamide gel electrophoresis (8%) in denaturing conditions of the 5'-pegulated (PEG6000) (a) and non-pegulated (b) 22-mer
d(TCATGGTCATAGCTGTTTCCTG) after the removal of blocking groups. The gel was stained with ethidium bromide. Photo in UV-light. B. Reversed-phase
chromatography of 22-mer (b) and its 5'-PEG conjugate (a). C. Polyacrylamide gel electrophoresis (20%) in denaturing conditions of d(T)12(a), d(T)22(d) and the
5'-lipid conjugates of d(T),2(b) and d(T)22(c). Visualization by UV-shadowing.
in living cells faces some obstacles connected with difficulties
in passing through the cell membrane and nuclease degradation.
Chemically modified oligonucleotides should be able to surmount
these obstacles. One of the promising modifications can be the
introduction of phospholipid, or polyethylene glycol (PEG)
residues at the oligomer. Recently, the antiviral activity of lipidoligonucleotide conjugates obtained by the covalent attachment
of a phospholipid to oligonuclotides via solid-phase Hphosphonate synthesis has been demonstrated [16]. Conjugates
of oligonucleotides with PEG are very stable to the exonuclease
digestion in vitro and to serum mediated degradation, and they
have improved penetration ability through cell membranes [17,
18]. These types of conjugate molecules can be used as DNA
probes and antisense inhibitors of gene expression, with the
oligonucleotide segment acting as a targeting moiety. We
examined the application of PFPC for the attachment of
phospholipid and PEG residues to oligonucleotides. Earlier,
polyethylene glycol was used as a soluble support for the synthesis
of oligonucleotides in preparative amounts, but the PEG residue
was removed after the completion of chain elongation [19]. We
have developed a simple and fast solid-phase procedure for the
synthesis of 3'-, 5'-, or 3',5'-PEG-oligonucleotide conjugates
with the use of PEG 1000 -PEG6000.
To obtain a 3'-PEG-oligonucleotide conjugate, the long chain
alkylamine controlled pore glass support was fiinctionalized by
the addition of pentafluorophenyl ester of dimethoxytrityl-PEG
phthalate, which was obtained with the help of PFPC essentially
as it described above for nucleoside succinates (Scheme 3). It
should be noted that the use of phthalic bond between the support
and PEG was caused by its higher stability in comparison with
the standard succinic bond. Our attempts to use the succinic bond
between the support and PEG revealed considerable loss of DMT-
PEG residues from the succinated support during the storage and
oligonucleotide synthesis [17]. The derivatized CPG containing
dimethoxytrityl-PEG residues (5—25 /unole/g, depending on the
PEG size) was used for the automatic synthesis of the target
oligomer containing 3'-PEG residue. The synthesis of the 3',5'and 5'-PEG-oligonucleotide conjugates has been made by the
addition of dimethoxytrityl-PEG H-phosphonate to the polymer
bound oligonucleotide chain during the last synthetic cycle
(Scheme 3) [17].
Similarly , 1,2-di-O-hexadecyl-rac-glucerol-3-H-phosphonate
[16] was attached to the oligonucleotide on the support (Scheme
4). After the completion of chain elongation, oxydation and
removal of dimethoxytrityl group, oligonucleotide conjugates
were fully deprotected by the action of monoethanolamine-ethanol
(1:1) mixture [10] and then isolated by the polyacrylamide gel
electrophoresis, or by FPLC (Fig.6).
It should be noted that on the analogy with the introduction
of 5'-PEG and 5'-lipid residues, different other tails, reporter
groups and non-radioactive labels, such as biotin, can be attached
to synthetic oligonucleotides.
A study carried out in our laboratory on the use of PFPC in
oligonucleotide chemistry demonstrated that this reagent is very
effective for the solid-phase H-phosphonate synthesis. At the same
time, it is the convenient reagent for the attachment of first
nucleoside to polymer supports, as well as for the synthesis of
oligonucleotide conjugates and for the introduction of different
labels to oligonucleotides. Moreover, PFPC can be very useful
for the synthesis of modified oligonucleotides, such as polyamide
analogues and analogues with carbamate internucleoside linkages,
via pentafluorophenyl esters. Studies on the application of PFPC
for these purposes are in progress and will be published
elsewhere.
Nucleic Acids Research, 1993, Vol. 21, No. 23 5343
HO(CH2CH2O)nH
1.DMTrCl
• - DMTr-0(CH 2 CH,O) n C-C.H,-C-0"
2.Phthalic
||
||
anhydride
o
o
I
»FPC, NR,
Ii
3—P-0
I
p-o"
I
H
DHTr-O(CH 2 CH 2 O) n -C-C 6 H 4 -C-OC 6 F 5
o
B
O
o
H
NJ—o-iC(CH,),C-NH
II
II
o
o
PFPC, Py
0
II
-P-0
1
H
B
I
0
II
-O-P-0
1
H
1. Oxidation
2. Deprotection
1. oligonucleotide chain elongation;
2. H+;
I
Osj-0
II
•->.
-P-0 (CH2CH2O)n -C-C$H4 - C - H H - V A - ( P )
-P—0
I
H
-C(CH ,),C-NH—(P )
II
^S
II
0
nC
18H33°-|
nC
»6 H 33<H
Lo-p-o
I
o
II
I
s|-0H
O-P-O-
OH
OH
1. DMTrO(CHjCH2O)n-P-O
,PFPC
O~
2. Oxidation
3. Deprotection
HO ( C H J C H J 0 )
5 I
8I
-P-Osht
I
— P - ONJ-0
= LCAA-CPG
—P-O(CH,CH2O)nH
OH
'MJr = dimethoxytrityl
(?) - LCAA-CPG
Scheme 3. Solid-phase synthesis of a 3',5'-PEG-oligonucleotide conjugates.
MATERIALS AND METHODS
The following reagents were purchased from commercial sources:
protected deoxynucleoside 3'-H-phosphonates and deoxynucleoside 3'-O-succinates (MilliGen/Biosearch), pivaloyl chloride,
triethyl phosphite (Aldrich), pyridine, acetonitrile, dichloromethane, dimethylformamide, trichloroacetic acid, tris, PEG6000
(Merck), acrylamide, bisacrylamide (Bio-Rad), silica gel F-254
TLC plates (Eastman Kodak), LCAA CPG support (Pierce),
TentaGel support (Rapp Polymer), 1,2-di-O-hexadecyl-racglycerol (Sigma). Dipentafluorophenyl carbonate was manufactured in Perm (Russia). Ethyl hydrogenphosphonate (as
triethylammonium salt) was prepared as described [20].
Silica gel plates were developed in chloroform-methanol (9:1,
v/v).
Reversed-phase chromatography of oligonucleotides was
performed with Pharmacia FPLC system on HR 5/10 column
in a gradient of acetonitrile (5 -40%) in 0.1 M triethylammonium
acetate (pH 7.5) with flow rate 1 ml per min.
31
P NMR spectra were recorded on a Bruker WM500 (202
MHz) spectrometer without lH-heteronuclear decoupling. The
values of chemical shifts are reported relative to 85% H3PO4 in
D2O (external standard).
The determination of the internucleotide bond formation rate
was carried out according to the described procedure [7].The
estimation of the extent of heterocyclic base modification and
the rate of 5'-OH group carbonilation in the presence of PFPC
were carried out as described earlier [7, 21].
Scheme 4. Synthesis of a lipid-oligonucleotide conjugate.
Oligonucleotide synthesis
Automatic synthesis of oligonucleotides was performed on an
Applied Biosystems 381A synthesizer on 0.2, 1 and 10 /imol
scales. The various chemical operations performed for the
addition of one coupling unit to a support are listed in Table 2.
The amount of a nucleoside attached to the support and the
coupling yields of intermediates were estimated by spectroscopic
analysis of the dimethoxytrityl function liberated from the support
as described [15]. After completion of the chain elongation, the
oxidation by 2% I2 solution in pyridine-water (98:2) was carried
out during 10—15 min. The subsequent cleavage of oligomers
from the support, deprotection and isolation were carried out as
described [10, 21].
General procedure for the preparation of pentafluoropheny 1
esters of carbon acid derivatives
A solution of triethylammonium salt of a carboxy-containing
compound (1 mmol) in dry acetonitrile (5-10 ml) was treated
during 1 h at room temperature with PFPC (1.1 mmol) in the
presence of N-methylmorpholine (1.1 mmol). The course of the
process was monitored by TLC. After the completion of the
reaction (40-60 min), the precipitate of N-methylmorpholine
pentafluorophenolate was separated by filtration and the filtrate
was evaporated in vacuum. The residue was dissolved in small
amount of methylene chloride, and the pentafluorophenyl ester
was precipitated into pentane. The precipitate was used for further
work without additional purification. Yield 9 0 - 9 8 % .
Derivatisation of solid supports
A. Attachment of a protected nucleoside. The solution of
pentafluorophenyl ester of N-protected 5'-O-dimethoxytrityl
nucleoside succinate (0.2 mmol) obtained as described above in
5 ml of dry dimethylformamide was mixed with 1 g (0.1 mmol
amino groups per g) of LCAA CPG, or with 0.2 g (0.24 meq
amino groups per g) of TentaGel, and triethylamine (1 mmol)
was added. The reaction mixture was swirling for 5 hours at room
temperature, and the support was filtered and washed with
5344 Nucleic Acids Research, 1993, Vol. 21, No. 23
dimethylformamide (2x 10 ml), acetonitrile (2x 10 ml), diethyl
ether (3 x 10 ml) and dried. Then, the support was treated with
a mixture of acetic anhydride—1-methylimidazole—acetonitrile
(1:1:8, v/v) for 30 min. After filtration and washing with
acetonitrile, methanole and ether, it was dried under vacuum.
The product was analyzed for dimethoxytrityl content according
to the literature method [15]. The loading of nucleoside was about
3 0 - 5 0 /unol per g of support for CPG and about 160-180
jimol/g for TentaGel.
B. Attachment of PEG. Pentafluorophenyl ester of dimethoxytrityl-PEG-phthalate (0.3 mmol) obtained as described above and
LCAA-CPG (1 g) were allowed to react in the presence of
triediylamine (0.35 mmol) at room temperature with shaking for
4—5 hours. The support was separated by filtration, washed and
the unreacted amino groups were acylated as describe in section
(A). The loading was about 5—25 jimol per g depending on the
PEG size.
Synthesis of 5'-lipid- and 5-PEG-oligonucleotide cojugates
Dimethoxytrityl-PEG-H-phosphonate and lipid-H-phosphonate
were obtained from (MeO)2Tr-PEG-OH and 1,2-di-O-hexadecyl-rac-glycerol as described earlier [16, 17]. Oligonucleotides
prepared using automated synthesizer were subjected to a final
coupling cycle with 0.04 M solution of 1,2-di-O-hexadecyl-rac-glycero-3-hydrogenphosphonate, or dimethoxytritylPEG-H-phosphonate in standard conditions (Table 2). The
products were then oxidized, deprotected and purified by
reversed-phase column chromatography.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. T.Balashova for recording
the 31P NMR spectra.
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