Design and synthesis of dinucleotide 5′-triphosphates with expanded functionality

Article abstract of DOI:10.1016/j.bmc.2008.09.029

We propose the new approach to the synthesis of 5′-triphosphate derivatives of natural and modified dinucleotides with expanded functionality. Our strategy includes the combination of the solution phase synthesis of necessary dimers using the wide range of nucleic acids chemistry methods and the subsequent introduction of the triphosphate residue. A number of the new potential substrates for the template dependent synthesis of nucleic acids with expanded functionality are obtained, namely, 5′-triphosphates of dinucleotides containing the functionally active groups in heterocyclic bases, in carbohydrate-phosphate backbone, and the groups mimicking the residues of natural amino acids. The abilities of the proposed synthetic route are also demonstrated by the synthesis of 5′-triphosphates of dinucleotides with modified carbohydrate-phosphate backbone.

Full text of DOI:10.1016/j.bmc.2008.09.029

Bioorganic & Medicinal Chemistry 16 (2008) 9127–9132
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of dinucleotide 50-triphosphates
with expanded functionality
*
Tatiana V. Abramova , Svetlana V. Vasileva, Ludmila S. Koroleva, Nina S. Kasatkina, Vladimir N. Silnikov
Institute of Chemical Biology and Fundamental Medicine, Lavrent’ev Ave 8, Novosibirsk 630090, Russia
a r t i c l e i n f o
a b s t r a c t
We propose the new approach to the synthesis of 50-triphosphate derivatives of natural and modified
dinucleotides with expanded functionality. Our strategy includes the combination of the solution phase
synthesis of necessary dimers using the wide range of nucleic acids chemistry methods and the subse-
quent introduction of the triphosphate residue. A number of the new potential substrates for the
template dependent synthesis of nucleic acids with expanded functionality are obtained, namely, 50-tri-
phosphates of dinucleotides containing the functionally active groups in heterocyclic bases, in carbohy-
drate–phosphate backbone, and the groups mimicking the residues of natural amino acids. The abilities
of the proposed synthetic route are also demonstrated by the synthesis of 50-triphosphates of dinucleo-
tides with modified carbohydrate–phosphate backbone.
Article history:
Received 30 May 2008
Revised 8 September 2008
Accepted 10 September 2008
Available online 13 September 2008
Keywords:
50-Nucleoside triphosphates
Modified nucleosides
Nucleic acids with expanded functionality
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The profound interest in the molecular biology investigations
during the last decades gave the impetus for the development of
synthetic methods in nucleic acids chemistry. Achievements and
new approaches in this area are summarized in the reviews.1,2
Chemical synthesis of modified oligonucleotides is routine proce-
dure now due to the fast introduction of innovations appearing
in scientific literature in commercial practice; the catalogs of lead-
ing companies dealing with the synthesis of oligonucleotides and
their derivatives are regularly updated.3–6 At the same time very
little progress has been attained in the enzyme mediated modified
nucleic acids synthesis. This situation is due for the most part to
objective causes, namely, modified triphosphates ought to be effec-
tive non-terminating substrates of the corresponding enzymes. Re-
cently, a number of works devoted to the investigation of substrate
properties of a wide range of modified nucleoside 50-triphosphates
appeared.7–10 Functionally active nucleic acids obtained by the
chemical or enzymatic methods are the perspective tools of molec-
ular biology used in the various areas of fundamental and applied
research.11–14 So, further development of the functionalized nu-
cleic acid synthesis and of the available modification spectrum is
an actual task. We believe that 50-triphosphates of dinucleotides
used as substrates for NA polymerases might be the solution of
the problem (Fig. 1). One can suggest that such triphosphates
would have more modification points non critical for substrate
properties. Preliminary experiments showed that 50-triphosphate
Figure 1. 50-Triphospho-{20-d-[cytidinyl(30-phospho-50)cytidine]}. The possible
sites of ribose-phosphate backbone and heterocyclic base modifications are
indicated.
dinucleotides are the substrates for some DNA-polymerases.15
The substrate properties of the modified 50-triphosphodinucleo-
tides are currently under intensive investigation, the results were
partially presented in the plenary lecture in International confer-
ence in Novosibirsk, Russia.16
A few methods for the synthesis of 50-triphosphates of di- and
oligonucleotides in solution17,18 and on the solid support19 are
known, however the yields of the desired products did not exceed
50%. Furthermore, the quantity of the product is limited by the
oligonucleotide synthesis scale in the case of the solid phase
* Corresponding author. Tel.: +7 383 3333762; fax: +7 383 3329582.
E-mail address: abramova@niboch.nsc.ru (T.V. Abramova).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.029

9128
T. V. Abramova et al. / Bioorg. Med. Chem. 16 (2008) 9127–9132
synthesis. The main problem in the synthesis of 50-triphosphate
derivatives of di- and oligonucleotides is the presence of an addi-
tional highly reactive center in the molecule – internucleotide
phosphate group. The convenient method for the synthesis of 50-
triphosphates of di- and oligonucleotides in the solution with high
yields starting from the corresponding deprotected 50-monophos-
phates was recently proposed.20 However, in general it is necessary
to use protected oligonucleotides. Functional groups introduced in
oligonucleotide usually also require protection both during the oli-
gonucleotide synthesis and the triphosphate residue addition.
We propose the new approach to obtaining the 50-triphosphate
derivatives of natural and modified dinucleotides consisting of the
necessary dimer synthesis in solution using all methods of oligonu-
cleotide chemistry following the introduction of the triphosphate
residue in the suitably protected oligomers. Our approach provides
the availability of 50-triphosphodinucleotides containing the wide
range of modifications both in ribose–phosphate backbone and
heterocyclic bases with high yield and in large quantity.
convenient,24 but the coupling reaction was carried out in solution
instead of usually employed solid phase approach. Methylphos-
phonate derivatives (compounds 1 and 2 in Table 1) were synthe-
sized by the phosphorodiimidazolide method.26 Thus we
constructed the nucleotide part of the target derivatives depending
on its structure using a wide range of the oligonucleotide chemis-
try methods. Solution phase chemistry allows easily obtaining sub-
stances in large quantities (up to several grams). The careful
purification of the dinucleotides with only the 50-hydroxyl function
free by the chromatography provided high yield during the intro-
duction of the triphosphate residue.
The choice of the triphosphate residue introduction method
was determined by the nucleotide structure and the presence
and type of protective groups. Mainly we used modified Ludwig’s
method (Scheme 1).27,28 We carried out the additional optimiza-
tion of the reaction conditions and succeeded in increasing the
yield of the target product (compound IV, Scheme 1) up to 80%
after purification against 20% in the earlier article.28 Purification
of the 50-triphosphodinucleotide derivatives was performed by
two sequential reverse phase chromatographies, initially before
the deprotection of the product and then after removing of the pro-
tective groups. This allowed us to easily separate the excess of the
pyrophosphate anion from the target products.
2. Results and discussion
We began the synthesis of target compounds from the construc-
tion of the nucleotide part of the molecule containing necessary
modifications and functional groups. The choice of synthetic
strategy was determined by the structure and the location of the
modification. The synthesis of 20-d-[cytidinyl-(30-phospho-50)-5-
(aminoallyl)uridine] 50-triphosphate is depicted in Scheme 1 as
an example. Nucleotide part of the other triphosphates was formed
by the similar approach. Monomer blocks I and II were synthe-
sized by the various methods21–23 or were commercially available
(Table 1), then we carried out coupling reaction to produce suit-
ably protected dimers III presumably using the phosphotriester
chemistry (compounds 3, 4, 7–43, Table 1).24,25
In some cases the 50-triphosphodinucleotide modification can
be carried out post-synthetically. For example, we introduced the
amino alkyl residue in the N4-position of cytosine (compounds
29–33, the Table 1) by the transamination catalyzed by the bisul-
fite anion analogously the published procedure28; 50-triphospho-
thymidinyl-(30-phospho-50)-20-deoxycytidine was used as
a
parent compound in this reaction. Azide group was used as a pre-
cursor of an amine function in 20-amino-20-deoxynucleosides; the
reduction of the intermediate 9 by the H2 over Pd/C similar to
the published procedure30 afforded the compound 10 (Table 1).
In order to the widening of the functional group repertoire pres-
ent in the 50-triphosphodinucleotides we subjected aliphatic amine
groups in the modified 50-triphosphodinucleotides further modifi-
cation (compounds 3, 4, 7, 11, 17-39, 41, 43 in Table 1, Fig. 2). We
introduced reporter or reactive groups (biotin, fluorescein, photo
reactive p-azidotetrafluorobenzoyl residue), and the groups mim-
icking side radicals of natural amino acids. Natural amino acid
character notation is used in Figure 2 and Table 1 to indicate the
residues mimicking the corresponding amino acid side radicals.
Necessary organic acids were first activated usually as N-hydroxy-
succinimide esters in the presence of DCC analogously one of the
numerous published procedures; activated derivatives without
purification were used for the transformation of the triphosphate
derivatives (see Supplementary data).9,31 Sometimes we employed
corresponding anhydrides, p-nitrophenyl and pentafluorophenyl
esters if they were commercially available. In the lactic acid case
we brought into use its methyl ester in anhydrous conditions
though the reactivity of this substance was found to be rather low.
Some modifications of the target molecules are specified in Ta-
ble 2. We employed ‘locked’ nucleosides, 20-deoxy-20-amino, 20-
OMe groups as the examples of carbohydrate modifications; ade-
nine, cytosine and uracil containing aminoalkyl linkers as the
examples of heterocyclic base modifications; methylphosphono-
and phosphamide groups as the examples of phosphate
modifications.
In the case of 30-N-50-O-phosphamide internucleotide bond and
chimerical ribo-deoxyribodinucleotides (compounds 5 and 6 in
Table 1) the phosphosphoramidite chemistry was found to be more
3. Conclusion
We synthesized more than 40 modified and natural 50-trip-
hosphodinucleotides. Modifications introduced in the dinucleo-
tides involve heterocyclic bases, carbohydrates and phosphate
backbone. Other modifications may be available as they are com-
patible with the conditions of oligonucleotide synthesis and pro-
tective group removing. The general efficacy of our approach is
determined most of all by method of the internucleotide bond for-
mation. Phosphotriester chemistry is the method of choice in our
approach because of better yields of solution phase coupling. Some
physical–chemical characteristics, parent monomer blocks, type of
the coupling reaction used and post-synthetic modifications are
Scheme 1. Synthesis of the 50-triphosphates of modified dinucleotides as exem-
plified by obtaining the 20-d-[cytidinyl-(30-phospho-50)-5-(aminoallyl)uridine] 50-
triphosphate. B = N4-benzoylcytosine, B1 = 5-(trifluoroacetamidoallyl)uracil, B2
=
cytosine, B3 = 5-(aminoallyl)uracil, R1 = O-p-chlorophenyl, R2 = acetyl, R = R3 = H,
R
4 = Oꢀ. (i) 2,4,6-Triizopropylbenzenesulfonylchloride, N-Me-imidazole, Py; (ii)
HOAc/H2O; (iii) POCl3, Py, then [(C4H9)3N]2H2P2O7 in acetonitrile, N(C4H9)3; (iv)
NH3/H2O.

T. V. Abramova et al. / Bioorg. Med. Chem. 16 (2008) 9127–9132
9129
Table 1
Modified 50-triphosphodinucleotides — compounds IV (Scheme 1)
pppNN
Monomers I and II, method of
Method of coupling of
Post-synthetic modification
synthesis
monomers I and II
1
2
3
pppd[G(pCH3)A], 20-d-[guanosinyl(30-methylphosphono-
50)adenosine] 50-triphosphate (diastereomer mixture)
pppd[C(pCH3)A], 20-d-[cytidinyl(30-methylphosphono-50)-
adenosine] 50-triphosphate
50-O-(DMTr)dGiBu, dABz-30-O-(Ac)a
Phosphorodiimidazolide26
—
50-O-(DMTr)dCAc, dABz-30-(Ac)a
- « -
—
pppd[U(5-Flu)pT], 20-d-{5-[3-
50-O-(DMTr)-20-dU(5-MeOPrNHTfa)b 21
,
Phosphotriester25
Fluorescein isothiocyanate31
(fluoreceinthiocarbamoylamido)propyloxymethyl]uridinyl(30- 50-O-(p-ClPhP)dT-30-O-(Lev)c,25
phospho-50)thymidine} 50-triphosphate
4
5
pppd[U(5-Bio)pT], 20-d-{5-[3-
- « -
- « -
Biotin p-nitrophenyl ester31
(biotinamidopropyl)oxymethyl]uridinyl(30-phospho-
50)thymidine} 50-triphosphate
pppd[T(30NHp)C], 30-d-30-amido-20-d-thymidinyl(30-N-
phospho-50)-20-d-cytidine 50-triphosphate
50-O-(DMTr)-30-NH2-30-dT,
reduction of the corresponding
azide derivative,23 50-O-
[CNC2H4OPN(iPr)2]dCAc–O-(Ac)
similar to21
50-O-(DMTr)-20-O-(TBDMS)-U-30-
O-[CNC2H4OPN(iPr)2], dT-O-(Ac)a
50-O-(DMTr)dCBz-30-O-(p-ClPhP),
20-dU(5-aaTfa)-30-O-(Ac)d similar
to21,25
Phosphoramidite24
—
6
7
pppUpdT, uridinyl(30-phospho-50)-20-d-thymidine 50-
triphosphate
- « -
—
pppd[CpU(5-L-Bio)], 20-d-[cytidinyl(30-phospho-50)-5-(biotin-6-
amidocaproyl-6-amidocaproylamidoallyl)uridine] 50-
triphosphate
Phosphotriester25
N-Hydroxysuccinimidyl biotin-6-
amidocaproyl-6-amidocaproate31
8
9
pppd[U(20-NH2)pC], 20-d-20-aminouridinyl(30-phospho-50)-20- 50-O-(DMTr)-20-dU-20-NH(Tfa)22
,
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
—
—
d-cytidine 50-triphosphate
50-O-(p-ClPhP)dCBz30-O-(Lev)25
pppd[A(20-apaN3)pC], 20-d-20-azidoarabinoadenosinyl(30-
phospho-50)-20-d-cytidine 50-triphosphate
50-O-(DMTr)-20-d-20-araN3-ABz 23
,
50-O-(P-ClPhP)dCBz-30-O-(Lev)25
10 pppd[A(20-araNH2)pC], 20-d-20-aminoarabinoadenosinyl(30-
phospho-50)-20-d-cytidine 50-triphosphate
- « -
Reduction of the corresponding
azide derivative similar to30
Fluorescein isothiocyanate31
11 pppd[GpU(5-aa-Flu)], 20-d-[guanosinyl(30-phospho-50)-5-
50-O-(DMTr)dGiBu30-O(-p-ClPhP),
(fluoresceinthiocarbamoylamidoallyl)uridine] 50-triphosphate 20-dU(5-aaTfa)-30-O-(Ac)21,25
12 pppd[(LNA-C)pT], locked-cytidinyl(30-phospho-50)-20-d-
thymidine 50-triphosphate
50-O-(DMTr)(LockedC)Ac a
ClPhP)dCBz30-O-(Lev)25
,
50-O-(p-
—
—
—
—
—
13 pppd[TpA(6-ae)],e 20-d-[thymidinyl(30-phospho-50)-6-(2-
aminoethyl)adenosine] 50-triphosphate
50-O-(DMTr)dT-30-O-(p-ClPhP),
dA(6-aeTfa)-30-O-(Ac)21,25
50-O-(DMTr)dABz-30-O-(p-ClPhP),
dA(6-aeTfa)(Ac)21,25
14 pppd[ApA(6-ae)], 20-d-[adenosinyl(30-phospho-50)-6-(2-
aminoethyl)adenosine] 50-triphosphate
15 pppd[CpA(6-ae)], 20-[d-cytidinyl(30-phospho-50)-6-(2-
aminoethyl)adenosine] 50-triphosphate
50-O-(DMTr)dCBz-30-O-(p-ClPhP),
dA(6-aeTfa)(Ac)21,25
16 pppd[GpA(6-ae)], 20-d-[guanosinyl(30-phospho-50)-6-(2-
aminoethyl)adenosine] 50-triphosphate
50-O-(DMTr)dGiBu-30-O-(p-ClPhP),
dA(6-aeTfa)-30-O-(Ac)21,25
50-O-(DMTr)dT-30-O-(p-ClPhP),
dA(6-aeTfa)-30-O-(Ac)21,25
- « -
17 pppd[TpA(6-ae-H)], 20-d-{thymidinyl(30-phospho-50)-6-[2-
(urocanoylamido)ethyl]adenosine} 50-triphosphate
18 pppd[TpA(6-ae-D)], 20-d-{thymidinyl(30-phospho-50)-6-[2-
(succinylamido)ethyl]adenosine} 50-triphosphate
19 pppd[TpA(6-ae-K)], 20-d-[thymidinyl(30-phospho-50)-6-[2-(6-
aminocaproylamido)ethyl]adenosine} 50-triphosphate
N-Hydroxysuccinimidyl
urocanoate31
Succinic anhydride similar to31
- « -
N-Hydroxysuccinimidyl 6-
(trifluoroacetylamido)hexanoate,31
ammonia treatment
20 pppd[TpA(6-ae-V)], 20-d-{thymidinyl(30-phospho-50)-6-[2-
(isobutyrylamido)ethyl]adenosine} 50-triphosphate
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
- « -
Isobutyric anhydride similar to31
21 pppd[TpA(6-ae-R)], 20-d-{thymidinyl(30-phospho-50)-6-[2-(4-
guanidinebutyrylamido)ethyl]adenosine} 50-triphosphate
22 pppd[TpA(6-ae-W)], 20-d-{thymidinyl(30-phospho-50)-6-[2-
(indole-3-acetylamido)ethyl]adenosine} 50-triphosphate
23 pppd[TpA(6-ae-M)], 20-d-{thymidinyl(30-phospho-50)-6-[2-(2-
methylthioacetylamido)ethyl]adenosine} 50-triphosphate
24 pppd[TpA(6-ae-T)], 20-d-{thymidinyl(30-phospho-50)-6-[2-(2-
hydroxypropionylamido)ethyl]adenosine} 50-triphosphate
25 pppd[TpA(6-ae-C)], 20-d-{thymidinyl(30-phospho-50)-6-[2-(3-
mercaptopropionylamido)ethyl]adenosine} 50-triphosphate
N-Hydroxysuccinimidyl 4-
guanidinobutyrate31
Pentafluorophenyl indole-3-
acetate similar to31
N-Hydroxysuccinimidyl 2-
methylthioacetate31
Methyl lactate (racemic mixture),
pyridine
Bis-N-hydroxysuccinimidyl 3,30-
dithiodipropionate;31 precipitation
in the presence of 2-
mercaptoethanol
26 pppd[TpA(6-ae-N)], 20-d-{thymidinyl(30-phospho-50)-6-[2-
(amidosuccinylamido)ethyl]adenosine} 50-triphosphate
27 pppd[TpA(6-ae-Y)], 20-d-{thymidinyl(30-phospho-50)-6-[2-(3-
(p-hydroxyphenyl)propionylamido)ethyl]adenosine} 50-
triphosphate
- « -
- « -
- « -
- « -
N-Hydroxysuccinimidyl
monoamidosuccinate31
N-Hydroxysuccinimdyl 3-(p-
hydroxyphenyl)propionate31
28 pppd[TpA(6-ae-F)], 20-d-{thymidnyl(30-phospho-50)-6-[2-
(phenylacetylamido)ethyl]adenosine}-50-triphosphate
29 pppd[TpC(4-ae)], 20-d-[thymidinyl(30-phospho-50)-4-(2-
aminoethyl)cytidine] 50-triphosphate
- « -
- « -
- « -
- « -
N-Hydroxysuccinimidyl
phenylacetate31
50-O-(DMTr)dTa, 50-O-(p-PhP)dCBz
-
HSOꢀ3 , EDAꢁ2HCl29
O-(Lev)25
30 pppd[TpC(4-ae-V)], 20-d-{thymidinyl(30-phospho-50)-4-[2-
(isobutyrylamido)ethyl]cytidine} 50-triphosphate
- « -
HSOꢀ3 , EDAꢁ2HCl;29 isobutyric
anhydride similar to31
(continued on next page)

9130
T. V. Abramova et al. / Bioorg. Med. Chem. 16 (2008) 9127–9132
Table 1 (continued)
pppNN
Monomers I and II, method of
Method of coupling
Post-synthetic modification
synthesis
of monomers I and II
31
32
pppd[TpC(4-ae-R)], 20-d-{thymidinyl(30-phospho-50)-4-[2-(4-
guanidinobutyrylamido)ethyl]cytidine} 50-triphosphate
- « -
- « -
- « -
HSOꢀ3 , EDAꢁ2HCl;29 N-
hydroxysuccinimidyl 4-
guanidinobutyrate31
HSOꢀ3 , EDAꢁ2HCl;29 bis-N-
hydroxysuccinimidyl 3,30-
dithiodipropionate;31
precipitation in the
presence of 2-
pppd[TpC(4-ae-C)], 20-d-{thymidinyl(30-phospho-50)-4-[2-(3-
mercaptopropionylamido)ethyl]cytidine} 50-triphosphate
- « -
mercaptoethanol
33
pppd[TpC(4-ae-H)], 20-d-{thymidinyl(30-phospho-50)-4-[2-
(urocanoylamido)ethyl]cytidine} 50-triphosphate
- « -
- « -
HSOꢀ3 , EDAꢁ2HCl;29 N-
hydroxysuccinimidyl
urocanoate31
34
35
pppd[CpA(6-ae-W)], 20-d-{cytidinyl(30-phospho-50)-6-[2-(indole-3-
acetylamido)ethyl]adenosine} 50-triphosphate
50-O-(DMTr)dCBz-30-O(-p-ClPhP),
dA(6-aeTfa)-30-O-(Ac)21,25
- « -
- « -
- « -
Pentafluorophenyl indole-
3-acetate similar to31
N-Hydroxysuccinimidyl 3-
(p-hydroxyphenyl)
pppd[CpA(6-ae-Y)], 20-d-{cytidinyl(30-phospho-50)-6-[2-(3-(p-
hydroxyphenyl)propionylamido)ethyl]adenosine} 50-triphosphate
propionate31
36
37
pppd[U(5-aa-Y)pT], 20-d-{5-[(3-(p-
50-O-(DMTr)-20-dU(5-aa-Tfa) 21
,
50-O(-p-
- « -
- « -
N-Hydroxysuccinimdyl 3-
(p-hydroxyphenyl)
hydroxyphenyl)propionylamido)allyl]uridinyl(30-phospho-
50)thymidine} 50-triphosphate
ClPhP)dT-30-O-(Lev)25
propionate31
pppd[U(5-aa-M)pT], 20-d-{5-[(2-
- « -
N-Hydroxysuccinimdyl 2-
methylthioacetylamido)allyl]uridinyl(30-phospho-50)thymidine}
50-triphosphate
methylthioacetate31
38
39
pppd[U(5-aa-D)pT], 20-d-{5-[(succinylamido)allyl]uridinyl(30-
phospho-50)thymidine} 50-triphosphate
pppd[U(5-aa-T)pT], 20-d-{5-[(2-
- « -
- « -
- « -
- « -
Succinic anhydride similar
to31
Methyl lactate (racemic
mixture), pyridine
hydroxypropionylamido)allyl]uridinyl(30-phospho-50)thymidine}
50-triphosphate
40
41
pppd[U(5-aa)pT], 20-d-[5-(aminoallyl)uridinyl(30-phospho-
50)thymidine] 50-triphosphate
- « -
- « -
- « -
- « -
—
pppd[U(5-l-Bio)pT], 20-d-{5-[(biotin-6-
N-Hydroxysuccinimidyl
amidocaproyl)amidoallyl]uridinyl(30-phospho-50)thymidine} 50-
triphosphate
biotin-6-amidocaproate31
42
43
pppd[CpU(5-aa)], 20-d-[cytidinyl(30-phospho-50)-5-
(aminoallyl)uridine] 50-triphosphate
50-O-(DMTr)dCBz-30-O(-p-ClPhP), 20-
dU(5-aaTfa)-30-(Ac)d similar to21,25
- « -
- « -
- « -
—
pppd[CpU(5-l-Bio)], 20-d-{cytidinyl(30-phospho-50)-5-[(biotin-6-
amidocaproylamido)allyl]uridine} 50-triphosphate
N-Hydroxysuccinimidyl
biotin-6-amidocaproate31
a
Monomers are commercially available.
Tfa—trifluoroacetyl.
O-p-ClPhP—p-chlorophenylphosphate.
aa—aminoallyl.
b
c
d
e
ae—2-aminoethyl.
Table 2
were dried and purified by standard procedures. NMR spectra were
acquired on Bruker AM-400 and AV-300 instruments (Bruker, Ger-
many) in appropriate deuterated solvents at 30 °C. Chemical shifts
(d) are reported in ppm relative to TMS signals. In the case of 31P
an external standard of 85% H3PO4 was used. Coupling constants J
are reported in Hertz. MALDI-TOF mass spectra were run on Reflex
III (Bruker Daltonics, Germany) in positive detector mode with
dihydroxybenzoic acid as a matrix.
Modifications of 50-triphosphodinucleotides (Scheme 1, compounds IV)
Ribose–phosphate backbone
X = NH, O, S
Heterocyclic bases
4-N-(Aminoalkyl)cytosine (including
5-Me derivatives)
5-(Aminoallyl)uracil
R = H, OH, N3, NH2 (including arabino
configuration), OCH3
R4 = O, S, CH3
5-(Aminopropyl)-
hydroxymethyluracil
6-N-(Aminoalkyl)adenine
Preparative silica gel chromatography was performed on Kiesel-
‘Locked’ nucleosides
gel 55–100 lm (Merck, Germany). Preparative reverse phase chro-
matography was performed on Porasil C18, 55–100 mkm, (Waters,
USA). Semi-preparative reverse phase HPLC was performed using
the Bruker ChromStar Chromatography system (Bruker, Bremen,
Germany) and a 1 ꢂ 25 cm column packed with LiChroprep C18
summarized in Table 1 and 3 (Supplementary data). We find our
approach very efficient and capable to produce 50-triphosphophate
derivatives of dinucleotides with high yields and in any quantities
independently of the structure of the target compounds.
(15–25 lm) (Merck, Darmstadt, Germany). Thin layer chromatog-
raphy was performed using Alufolien Kieselgel 60 F254 plates
(Merck, Darmstadt, Germany) in the appropriate solvent mixtures
and visualised by UV irradiation, ninhydrin (amine groups) or cy-
stein/aqueous sulfuric acid (nucleoside). All evaporations were
performed under reduced pressure. The analytical anion-exchange
chromatography was performed on Milichrom-4 chromatograph
(Econova, Novosibirsk, Russia) using a 2.5 ꢂ 60 mm column packed
4. Experimental
4.1. General
Nucleosides, their protected derivatives and some modified
protected nucleosides were purchased from ChemGenes Corpora-
tion (USA) and NanoTex-C (Novosibirsk, Russia). The other re-
agents were from Sigma-Aldrich, Inc. (USA). Organic solvents
with Polisil SA, 15
ent (flow rate 100
l
l
m (Vector, Novosibirsk, Russia). A linear gradi-
l/min) from 0 to 0.4 M of K2HPO4/KH2PO4 (pH
7.0) in 30% aqueous acetonitrile was employed.

T. V. Abramova et al. / Bioorg. Med. Chem. 16 (2008) 9127–9132
9131
Figure 2. Post-synthetic modification of the 50-triphosphodinucleotides containing the aliphatic amine group joined to heterocyclic bases. Residues mimicking natural amino
acid side radicals are designated by the corresponding character notation.
4.2. A general procedure for the phosphotriester condensation
of the monomers I and II and the removal of the DMTr group
(Scheme 1)
0,12 mmol) and imidazole (41 mg, 0.6 mmol) in dry acetonitrile
with stirring. After 2 h, the solution of the 30-(O-Ac)-protected
nucleoside with the free 50-OH group (0.1 mmol) in pyridine
(0.1 mL) and tetrazole (21 mg, 0.3 mmol) were added to the reac-
tion mixture. After 1 h, the reaction was stopped by the addition
of several drops of 5% aqueous NaHCO3, and the reaction mixture
was subjected to the standard treatment as described above. The
yield was 50–60%.
Monomers I (nucleotide component, 0.12 mmol) and II (nucle-
oside component, 0.1 mmol) were dissolved in anhydrous pyridine
(1 mL). Triisopropylbenzenesulfonyl chloride (91 mg, 0.36 mmol)
and the 1-methylimidazole (60 lL, 0.72 mmol) were added to the
reaction mixture. After 1 h, the reaction was stopped by the addi-
tion of several drops of 5% aqueous NaHCO3, and the reaction mix-
ture was evaporated. The residue was distributed between 20 mL
of dichloromethane and 20 mL of 5% aqueous NaHCO3. The organic
layer was separated, dried with Na2SO4, filtered and evaporated
several times with water to remove traces of pyridine. The residue
was dissolved in 80% aqueous acetic acid (5 mL). After 2 h, the
reaction mixture was chilled in an ice bath; the solution was care-
fully neutralized with the concentrated aqueous ammonia till pH
7.0. The reaction mixture was evaporated; the dry residue was sus-
pended in acetonitrile and filtered. The precipitate was washed
with acetonitrile and methylene chloride. The filtrate was evapo-
rated; the residue was dissolved in methylene chloride (2 mL).
The target product was purified by silica gel chromatography. Elu-
tion was performed with a linear gradient of acetone in dichloro-
methane (the final concentration of acetone in the eluting
solution depended on a particular compound III, usually 50–
70%). Appropriate fractions were pooled and evaporated to give
the suitably protected dimer III as a dry powder. The yield was
75–80%.
4.4. A general procedure for the phosphoroamidite
condensation in solution
Thoroughly dried phosphoramidite component of the reaction
(0.05 mmol), nucleoside component (0.05 mmol) and tetrazole
(14 mg, 0.2 mmol) were dissolved in dry acetonitrile (0.5 mL).
The reaction mixture was stirred for 30 min, then a 1% iodine solu-
tion in pyridine/H2O = 98:2 (1 mL) was added. After 15 min, sev-
eral drops of 5% aqueous Na2S2O3 were added, and the reaction
mixture was subjected to the standard treatment as described
above. The yield was 50–60%.
4.5. A general procedure for the introduction of the 50-
triphosphate group into the dinucleotides
The appropriately protected dinucleoside with only the 50-hy-
droxyl function free (compound III, Scheme 1, 0.1 mmol) was dis-
solved in anhydrous pyridine (1 mL) and the solution was chilled in
an ice bath. Phosphorous oxychloride (0.018 mL, 0.2 mmol) was
then added to the solution. The reaction mixture was stirred for
20 min and then was transferred to the mixture of 0.5 M solution
of the bis(tetra-n-butylammonium) pyrophosphate in acetonitrile
(1 mL) and tributylamine (1 mmol, 0.23 mL) under vigorous stir-
ring. The cooling was removed and the reaction mixture was stir-
red 30 min at room temperature. 1 M TEAB buffer pH 7.5 (30 mL)
was then added. After 1 h, the reaction mixture was carefully evap-
4.3. A general procedure for the phosphorodiimidazolide
condensation
The solution of the 50-(O-DMTr)-protected nucleoside with the
free 30-OH group (0.1 mmol) in pyridine (0.1 ml) was added grad-
ually to the solution of methyphosphonodichloride (16 mg,

9132
T. V. Abramova et al. / Bioorg. Med. Chem. 16 (2008) 9127–9132
6. Summerton, J. Lett. Pept. Sci. 2003, 10, 215.
7. Ohbayashi, T.; Kuwahara, M.; Hasegawa, M.; Kasamatsu, T.; Tamura, T.; Sawai,
H. Org. Biomol. Chem. 2005, 3, 2463.
8. Kuwahara, M.; Nagashima, J-i.; Hasegawa, M.; Tamura, T.; Kitagata, R.; Kazuo,
H.; Hososhima, S-i.; Kasamatsu, T.; Ozaki, H.; Sawai, H. Nucleic Acids Res. 2006,
34, 5383.
9. Li, Z.; Bai, X.; Ruparel, H.; Kim, S.; Turro, N. J.; Ju, J. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 414.
10. Schoetzau, T.; Langner, J.; Moyroud, E.; Roehl, I.; Vonhoff, S.; Klussmann, S.
Bioconjugate Chem. 2003, 14, 919.
11. Schubert, J.; Kurreck, J.. In Handbook of Experimental Pharmacology; Springer-
Verlag: Berlin Heidelberg, 2006; Vol. 173. p 261.
12. Ordoukhanian, F.; Joyce, G. F. J. Am. Chem. Soc. 2002, 124, 12499.
13. Kwok, P.-Y.; Chen, X. Curr. Issues Mol. Biol. 2003, 5, 43.
14. Khodyreva, S. N.; Lavrik, O. I. Curr. Med. Chem. 2005, 12, 641.
15. Kless, H. WO 01/16366, 2001; Chem. Abstr. 2001, 134, 217979.
16. Abramova, T. V.; Vasileva, S. V.; Kasatkina, N. S.; Belousova, E. A.; Lebedeva, N.
A.; Lavrik, O. I.; Silnikov, V. N. Proceedings of the International Conference
‘Physical Chemical Biology’. July 30–August 3, 2006, Novosibirsk, Russia, 60–
61.
orated. The residue was dissolved in water and the product was
purified by the reverse phase chromatography in the gradient of
ethanol in water 0–50%. Appropriate fractions were pooled and
evaporated. The resulting protected 50-triphosphodinucleotide
was subjected to the standard deprotection protocols (ammonia
treatment for the acyl, 2-cyanoethyl and p-chlorophenyl protective
groups, Fꢀ treatment for the silyl protective group if necessary).
After the deprotection was complete, the target 50-triphosphodinu-
cleotides (compounds IV, Scheme 1, Table 1) were purified by the
reverse phase HPLC with the gradient of acetonitrile in water (the
final concentration of acetonitrile in the gradient mixture de-
pended on the hydrophobicity of the target product, usually 10–
30%) in the presence of 0.1 M TEA–HOAc, pH 7.0. Appropriate frac-
tions were pooled and evaporated. Compounds IV were precipi-
tated as lithium salts as described earlier,20 yield 0.05–0.08 mmol
(50–80%).
17. Tomasz, J.; Simoncsits, A.; Kajtar, M.; Krug, R. M.; Shatkin, A. J. Nucleic Acids Res.
1978, 5, 2945.
18. Seto, Y.; Abe, K.; Itoh, M.; Sawai, H. Bioconjugate Chem. 2002, 13, 303.
19. Lebedev, A. V.; Koukhareva, I. I.; Beck, T.; Vaghefi, M. M. Nucleosides Nucleotides
Nucleic Acids 2001, 20, 1403.
Acknowledgments
20. Abramova, T. V.; Vasileva, S. V.; Serpokrylova, I. Yu.; Kless, H.; Silnikov, V. N.
Bioorg. Med. Chem. 2007, 15, 6549.
21. Abramova, T. V.; Vasil’eva, S. V.; Ivanova, T. M.; Shishkin, G. V.; Silnikov, V. N.
Russ. J. Bioorg. Chem. 2004, 30, 224.
22. Vasil’eva, S. V.; Abramova, T. V.; Ivanova, T. M.; Shishkin, G. V.; Sil’nikov, V. N.
Russ. J. Bioorg. Chem. 2004, 30, 234.
23. Vasileva, S. V.; Krasnousova, E. E.; Donina, A. A.; Abramova, T. V.; Zhdanova, L.
G.; Kovalenko, S. P.; Silnikov, V. N. Russ. Chem. Bull. 2006, 55, 1677.
24. Brown, D. M.. In Methods in Molecular Biology; Agrawal, S., Ed.; Humana Press:
Totowa, NJ, 1993; Vol. 20, pp 1–80.
We thank Dr. Yulia Gerassimova for recording the MALDI-TOF
mass spectra. This work was supported by The Russian Fund for
Fundamental Research, Grant No. 07-04-00990a and The Founda-
tion for Assistance to Small Innovative Enterprises, Program ‘Start’
N4924r/734.
Supplementary data
25. Abramova, T. V.; Komarova, N. I.; Mundus, D. A.; Pereboeva, O. S. Izv. Sib. Otd.
Akad. Nauk, Ser. Khim. 1990, 5, 45.
26. Miller, P. S.; Reddy, M. R.; Murakami, A.; Blake, K. R.; Lin, S.-B.; Agris, C. H.
Biochemistry 1986, 25, 5092.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.029.
27. Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131.
28. Abramova, T. V.; Bakharev, P. A.; Vasilyeva, S. V.; Silnikov, V. N. Tetahedron Lett.
2004, 45, 4361.
References and notes
1. Lönnberg, H. Annu. Rep. Prog. Chem. Sect. B 2001, 97, 177.
2. Vaijayanthi, B.; Kumar, P.; Ghosh, P. K.; Gupta, K. C. Indian J. Biochem. Biophys.
2003, 40, 377.
29. Wlassoff, W. A.; Dobrikov, M. I.; Safronov, I. V.; Dudko, R. Y.; Bogachev, V. S.;
Kandaurova, V. V.; Shishkin, G. V.; Dymshits, G. M.; Lavrik, O. I. Bioconjugate
Chem. 1995, 6, 352.
3. Sproat, B. S. J. Biotechnol. 1995, 41, 221.
30. Aurup, H.; Williams, D. M.; Eckstein, F. Biochemistry 1992, 31, 9636.
4. Efimov, V. A.; Chakhmakhcheva, O. G. Collect. Czech. Chem. Commun. 2006, 71,
929.
31. Sinha, N. D.; Striepeke, S. In Oligonucleotides and Analogues: A Practical
Approach; Eckstein, F., Ed.; IRL Press at Oxford University Press: Oxford, New
5. Efimov, V. A.; Chakhmakcheva, O. G.; Wickstrom, E. Nucleosides Nucleotides
Nucleic Acids 2005, 24, 1853.
York, Tokyo, 1991; pp 185–210.