Synthesis of oligonucleotide derivatives with aryl(trifluoromethyl)diazirine moiety for the photoaffinity modification of proteins and nucleic acids

Article abstract of DOI:10.1023/A:1019539707586

1-4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4, 4′-dimethoxytrityl)-2,3-pro-panediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbe

Full text of DOI:10.1023/A:1019539707586

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 4, 2002, pp. 293–299. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 4, 2002, pp. 324–331.
Original Russian Text Copyright © 2002 by Agapkina, Agapkin, Zagorodnikov, Alekseev, Korshunova, Gottikh.
Synthesis of Oligonucleotide Derivatives
with Aryl(trifluoromethyl)diazirine Moiety for the Photoaffinity
Modification of Proteins and Nucleic Acids
1
Yu. Yu. Agapkina*, D. V. Agapkin**, A. V. Zagorodnikov**, Ya. I. Alekseev***,
G. A. Korshunova**, and M. B. Gottikh**
Institute of Chemical Physics Problems, Russian Academy of Sciences,
Institutskii pr. 14, Chernogolovka, Moscow oblast, 142432 Russia
*
*
*Chemical Faculty and Belozerskii Institute of Physicochemical Biology, Moscow State University, Moscow, 119899 Russia
*
**Syntol ZAO, Timiryazevskaya ul. 42, Moscow, 127550 Russia
Received July 27, 2001; in final form, November 22, 2001
Abstract—1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4'-dimethoxytrityl)-2,3-pro-
panediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligode-
oxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with
aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)15
were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8–
9
°C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found
to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the for-
mation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide
sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing
duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA–protein
complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-pro-
panediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins
that recognize them.
Key words: aryl(trifluoromethyl)diazirine, integrase, oligonucleotides, photoaffinity modification
1
INTRODUCTION
Diazirine absorbs UV-light with wavelengths of 355–
65 nm, which is not harmful for biomolecules, and
3
Photoaffinity modification is one of the most fre-
generates carbene (Scheme 1), which can interact with
any neighboring grouping, including groups containing
aliphatic C–H bonds [4]. This high reactivity of the
ATFMD group is its important advantage over other
photoreactive groups, which considerably widens the
possibilities of the photoaffinity modification method.
quently used approaches to studying the structure and
function of nucleic acids, proteins, and their com-
plexes. When irradiated, the photoaffinity groups
within nucleic acids or proteins are converted into
highly reactive particles (radicals, carbenes, or
nitrenes) that attack the neighboring atomic groups to
form covalent bonds [1]. The identification of the pho-
toreaction products allows the elucidation of the struc-
tural and dynamic organization of the systems.
2
The carbene lifetime lies within the nanosecond
range [4], which helps carry out not only structural but
also kinetic studies. Therefore, diazirines are consid-
The thio- and halogen-containing analogues of het-
erocyclic bases [1] and aryl azide derivatives of nucleic
acids [2] are usually used as photoactivatable deriva-
tives. Note that, upon irradiation, all the photoactivat-
able groups can interact only with the nucleophilic and
aromatic compounds [1, 2]. Great attention is now
attracted to the ATFMD derivatives of nucleic acids [3].
N N
.
.
CF3
C CF
3
hν
1
Please address correspondence to phone: +7 (095) 939-5407; fax:
+
7 (095) 939-3181; e-mail: jagapkina@rambler.ru.)
2
Abbreviations: ATFMD, aryl(trifluoromethyl)diazirinyl; HEPES,
4
-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and HIV,
human immunodeficiency virus.
Scheme 1. A carben generation from ATFMD.
068-1620/02/2804-0293$27.00 © 2002 MAIK “Nauka /Interperiodica”
1

2
94
AGAPKINA et al.
ered to be the promising reagents for studying biopoly- that involved the introduction of the ATFMD group
mers. Numerous examples of using the ATFMD deriv- (where Ar = Bz) within a nonnucleoside insert. A com-
atives of nucleic acids for the photoaffinity modifica- mercially accessible reagent, 1-amino-2,3-propanediol,
tion of proteins can be found in literature [3]; however, was used as such an insert. It should be mentioned that
until now, we have not found any data on the photoaf-
finity modification of nucleic acids themselves by these
derivatives.
the introduction of the ATFMD group into oligonucle-
otides is theoretically possible by a postsynthetic way
[the deprotected amino group of aminodiol, already
The ATFMD group can be introduced into the het- inserted into the oligonucleotide chain, can be acylated
erocyclic base, sugar fragment, internucleotide phos- by the N-succinimide ester of 4-(3-(trifluoromethyl)-
phate groups, and terminal phosphate groups of oligo- 3H-diaziridin-3-yl)benzoic acid or by a direct incorpo-
nucleotides [3]. It is known from literature that the syn- ration of the synthon already containing the ATFMD
thesis of the ATFMD-containing nucleosides is a
laborious and multistage process that results in a low
yield of target product [5]. At the same time, there
exists a method of the introduction into oligonucle-
otides of various groupings within nonnucleoside link-
ers [6]. It is this method we chose for obtaining the
ATFMD-oligonucleotide derivatives.
group during the process of automatic oligonucleotide
synthesis. We chose the second approach due to higher
yields of the oligonucleotides containing the photoac-
tive group it ensures. In this connection, the first stage
of our work was the synthesis of anATFMD-containing
phosphoramidite.
The 2-(N,N-diisopropylamido)-β-cyanoethyl phos-
The goal of this work was the synthesis of oligonu-
cleotide derivatives that contain ATFMD group within
a nonnucleoside linker and the study of the possibility
phite
of
1-(4-(3-(trifluoromethyl)-3H-diazirin-3-
yl)benzamido)-3-O-(4,4'-dimethoxytrityl)-2,3-propane-
of their use for the photoaffinity modification of both diol (6) was synthesized using Scheme 2. Preliminary
proteins and nucleic acids.
experiments showed that the acylation of the amino
group of 1-amino-2,3-propanediol with N-succinimide
ester of 4-(3-(trifluoromethyl)-3H-diaziridin-3-yl)ben-
zoic acid (4) proceeds in a low yield. This seems to be
RESULTS AND DISCUSSION
As mentioned above, we chose the approach to the due to a low solubility of amine (1) in organic solvents.
synthesis of photoreactive oligonucleotide derivatives Therefore, we introduced the ATFMD group into the
O
NH2
OH
F3C
HO
NH2
OH
TFA-OEt
NH
OH
1. DMTr-Cl
. NaOH
2
HO
DMTrO
(1)
(2)
(3)
N
N
CF3
O
O
O
N
O
(
4)
NH-ATFMD
O
Cl
P N(iPr)2
CNEtO
NH-ATFMD
OH
DMTrO
CNEtO
6)
P N(iPr)2
DMTrO
(
(5)
Scheme 2. The scheme of the synthesis of 2-(N,N-diisopropylamido)-β-cyanoethyl phosphite of 1-(4-(3-(trifluoromethyl)-3H-diaz-
irin-3-yl)benzamido)-3-O-(4,4'-dimethoxytrityl)-2,3-propanediol (6).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 4 2002

SYNTHESIS OF OLIGONUCLEOTIDE DERIVATIVES
295
5
'
3'
described by Nassal [8]. The preliminarily obtained
succinimide ester (4) and amine (3) were coupled in
THF in the presence of triethylamine. The resulting
product (5) was isolated by column chromatography on
silica gel and characterized by mass and NMR spectra.
TTTTTTT-X-TTTTTTT
(I)
TTTT-X-TTTTT-X-TTTT
(II)
TTT-X-TTT-X-TTT-X-TTT
(III)
(IV)
Phosphoramidite derivative (6) was obtained from
(
5) by phosphitylation with 2-cyanoethyl diisopropyl-
AC-X-GCTAGAGATTTTCCACAC
chlorophosphoramidite in anhydrous methylene chlo-
ride in the presence of triethylamine and characterized
O
H
31
by P NMR spectrum. The overall yield of (6) from (1)
N
N
was 38%.
X =
O
N
The insertion of (6) into oligonucleotide chain was
achieved in the process of automatic synthesis. A con-
ventional synthetic scheme was used, but was modified
at the stage of the insertion of the modified nonnucle-
otide unit using a prolonged coupling time (10 min). A
series of oligonucleotides with the AFTMD group
within the nonnucleoside insert was synthesized
O
CF3
GTATAGCTCCACACG
(V)
GTGTGGAAAATCTCTAGCAGT
ACTGCTAGAGATTTTCCACAC
(VI)
(VII)
(
Scheme 3).
Scheme 3. The structures of synthesized oligodeoxynucle-
otides (symbol d is omitted).
The Photoaffinity Modification of Nucleic Acids
The photoaffinity modification of nucleic acids by
the oligonucleotide derivatives containing photoacti-
vatable groups can be used in a number of studies in the
field of molecular biology. For example, these may
involve the elucidation of the accessibility of certain
regions of a complex mRNA molecule, the determina-
tion of the NA–NA contacts within the protein–nucleic
acid complexes (e.g., in ribosome), the regulation of the
gene expression (as an instrument), etc.
dimethoxytrityl derivative of 1-amino-2,3-propanediol
(3).
The first stage of the synthesis was the protection of
the amino function in 1-amino-2,3-propanediol (1)
with trifluoroacetyl group. The reaction was carried out
in DMF in the presence of triethylamine, with ethyl tri-
fluoroacetate, which selectively reacts with amino
groups, being chosen as the acylating agent.
In this work, we studied the possibility of use of the
oligonucleotides with the ATFMD-containing nonnu-
cleoside inserts for the modification of nucleic acids.
Oligonucleotides (I)–(III), which were the derivatives
of (dT) with one, two, and three inserts, respectively,
The primary hydroxyl group of 1-(2,2,2-trifluoroac-
etamido)-2,3-propanediol (2) was protected with 4,4'-
dimethoxytrityl chloride in anhydrous pyridine (3 h).
For similar aminodiols, the dimethoxytritylation time is
known to vary from 4 to 18 h [6, 7]. Our preliminary
experiments demonstrated that no side product of the
secondary hydroxyl group alkylation formed for 3 h,
and, therefore, this time was chosen optimal for the
reaction. The trifluoroacetyl protection was removed in
alkaline medium, and the target product (3) was puri-
fied by a column chromatography on silica gel.
15
were synthesized.
We started with the determination of melting tem-
peratures, T , of the duplexes formed by the modified
m
oligonucleotides and the complementary template
(
dA) . One can see from the table that the introduction
15
of each nonnucleoside insert with the AFTMD group
decreases the duplex T by 8–9°C in comparison with
m
The N-succinimidyl 4-(3-(trifluoromethyl)-3H-
diazirin-3-yl)benzoate (4) was synthesized from p-bro-
motoluene according to the multistage scheme
the unmodified DNA duplex.
When assuring ourselves of the stability of the
resulting duplexes, we carried out the photoreaction in
them by the irradiation of the oligonucleotide–template
mixture by the light with the wavelength of 351 nm at
The effect of the number of nonnucleoside inserts with
AT-FMD group in oligonucleotides (I)–(III) on the thermo-
stability of their duplexes with (dA)15 template
4
°ë. The reaction mixtures were analyzed by PAGE in
20% denatured PAG (Fig. 1a). The covalent bond was
formed between the chains in the duplexes with differ-
ent efficiency, which depended on the number of the
AFTMD-containing inserts. The yields of the photore-
action products were 4, 10, and 15% in the case of (I),
Oligonucleotide
Number of inserts Tm, °C ± 0.5°C
within duplex
dT15
0
1
2
3
40
32
24
15
(II), and (III), respectively.
(
(
(
I)
The fact that the photoreaction proceeded within the
complementary complex was confirmed by the irradia-
tion of a mixture that included a 15-membered oligonu-
II)
III)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 4 2002

2
96
AGAPKINA et al.
(
‡)
(b)
(c)
Photoaffinity Modification of Proteins
The photoaffinity modification of proteins is a use-
ful instrument for studying NA–protein interactions.
We tested oligonucleotide (IV) with the AFTMD group
within the nonnucleoside insert as a reagent for the pho-
toaffinity modification of proteins by the example of the
HIV-1 integrase.
Covalent
conjugate
Starting
oligonucleotide
A sequence similar to the 5'-terminal site of one of
the chains of the U5 region of the long terminal repeat
of the HIV-1 cDNA was chosen for oligonucleotide
1
2 3 4
1 2 3
1 2
(IV). This cDNA site is recognized by the viral enzyme
integrase [12]. The integrase belongs to the class of
transponases. It inserts the viral DNA into the DNA of
the host cells by catalyzing two consecutive reactions:
Fig. 1. The radioautograph of SDS-PAGE in 20% PAG of
the products of photoreaction between the chains in DNA
3
2
duplexes: (a) Duplexes formed after irradiation of 5'- P-
labeled (dA)15 and 1, 2, and 3, oligonucleotides (I), (II),
3
'-processing, which represents the splitting off of
dinucleotide GT from the 3'-end of each chain of viral
DNA and a nucleophilic attack of internucleotide phos-
phate of the human DNA by 3'-hydroxyl of the pro-
cessed viral DNA. It is known that integrase recognizes
the sites of the viral DNA localized in the U5 and U3
fragments of long terminal repeats and binds them [12].
However, the complete information on the participation
of definite amino acids and nucleotides in the DNA–
and (III), respectively; 4, the same without the modified
3
2
chain. (b) Duplex formed by (I) and 5'- P-labeled (dA)
1
5
(
1) without irradiation, (2) by (V) after irradiation, and (3)
dA)15 after irradiation. (c) (1) Duplex (VI) + (IV) without
(
irradiation and (2) after irradiation; the 32P label was intro-
duced into the 5'-end of (IV).
cleotide of an arbitrary sequence (V) instead of the protein interaction is still unknown.
complementary chain (dA) . In this case, we observed
1
5
It has been previously shown by the method of pho-
toaffinity modification using the modified substrate
analogues containing 5-iodouridine [13, 14] or aryl
azide photolabels [15] that a contact between Lys159 of
the integrase and adenosine situated in the position 3
from the 3'-end of the viral DNA chain to be processed
exists in the enzyme–substrate complex. However, the
information on the possible contacts of integrase with
the unprocessed chain of substrate is absent from liter-
ature. Oligonucleotide (IV) has the sequence similar to
that of the unprocessed chain, and, therefore, we intro-
duced the ATFMD-containing grouping into the posi-
tion 3 from 5'-end of this oligonucleotide in order to test
possible contacts of integrase with the unprocessed
chain of viral DNA.
no formation of the photoreaction product (Fig. 1b).
Next, we studied the photomodification of nucleic
acids in the duplex that consisted of oligonucleotide
IV) and 21-membered oligonucleotide (VI) comple-
mentary to (IV) under the same conditions. The cova-
lent conjugate was obtained with an efficiency of 5%
(
(Fig. 1c).
These results allow us to recommend the oligonu-
cleotides containing the AFTMD group within the non-
nucleoside insert as the reagents for the photoaffinity
modification of nucleic acids. It is known that the oli-
gonucleotides that contain, e.g., thio-, aryl azide, or
psoralen groups can be used for this purpose [9–11].
Each of these reagents has advantages and drawbacks.
Therefore, the choice of photoreagent depends on the
problem the researcher should solve. For example, the
aryl azide group helps obtain sufficiently high photore-
action yields (up to 65%) [11]; however, this group can
be introduced into an oligonucleotide only postsynthet-
ically, which makes the synthesis more laborious. The
thio group and psoralen can be inserted into an oligonu-
cleotide during the automatic oligonucleotide synthesis
and provide for high crosslinking yields; however, upon
irradiation, they interact only with pyrimidine hetero-
cyclic bases [9, 10], which restricts the possibilities of
their use. The efficiency of the photoaffinity modifica-
tion of nucleic acids by our reagents is lower than those
The study of the interaction of HIV-1 integrase with
the viral DNA is a rather difficult task; therefore, sim-
pler systems are usually used to mimic this process in
vitro [16, 17]. We used a 21-membered DNA duplex
including the integrase recognition site from the U5
fragment of long terminal repeat of viral DNA [duplex
(VI + VII)] as a substrate, whereas the duplex (VI + IV)
played the role of the ATFMD-containing substrate
analogue.
Before the protein photomodification, we have sat-
isfied ourselves of the possibility of the specific 3'-pro-
cessing of the duplex (VI + IV). As mentioned above,
the processing of the starting 21-membered oligonucle-
otide results in the splitting off of the GT dinucleotide
from the 3'-end of the chain to be processed and the for-
by the reagents mentioned above, but due to the high mation of 19-membered oligonucleotide (Fig. 2a). The
reactivity of the ATFMD group, they can react with any processing efficiency was analyzed by the denaturing
nucleotide sequence and, therefore, are more prospec- electrophoresis (Fig. 2b). An appearance of the band
tive, e.g., for studying the RNA spatial structure.
corresponding to the oligonucleotide truncated by two
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 4 2002

SYNTHESIS OF OLIGONUCLEOTIDE DERIVATIVES
297
(‡)
Integrase
Integrase
3
2
32
3
'-processing
GT
b)
5
' [ p]
'
5' [ p]
CA
CAGT
GTCA
3
GTCA
3'
–
(
2
1
1 nt
9 nt
1
2
3
4
Fig. 2. The effect of the ATFMD group on the efficiency of 3'-terminal processing. (a) The scheme of 3'-terminal processing. (b) A
radioautograph of the electrophoretic separation in 20% PAG and 7 M urea of the 3'-terminal processing of (1) duplex (VI + VII)
and (3) duplex (VI + IV) and starting duplexes (2) (VI + IV) and (4) (VI + VII), respectively. Arrows indicate the positions of the
3
2
starting P-labeled 21-membered oligonucleotide (VI) and 19-membered product of its processing.
units indicated the successful reaction. The processing
EXPERIMENTAL
efficiency of the unmodified substrate (VI + VII) was
We used in our work chloro-2-(N,N-diisopropyl-
amido)-β-cyanoethoxyphosphine, dithiothreitol, Tris-
HCl, HEPES, LiClO , MnCl , and acrylamide from
4
0%, whereas that of the substrate (VI + IV) with
ATFMD group was 38%. In other words, the modifica-
tion did not affect the interaction specificity and the cat-
alytic activity of the enzyme. This allowed us to use the
modified duplex (VI + IV) for studying the integrase
interaction with the substrate.
4
2
M, kDa
4
3
2
5
We carried out the photoactivated reaction of the
substrate analogue containing the ATFMD group with
integrase. To this end, the modified duplex (VI + IV)
was incubated with integrase for 30 min at 37°ë and
then irradiated at 351 nm for 10 min under cooling with
ice. The formation of covalent conjugate was detected
by protein SDS-PAGE. The radioactive label was intro-
duced into 5'-end of (IV). The result of the photoreac-
tion is shown in Fig. 3.
DNA integrase
1
1.5
14.5
Note that the recombinant integrase we used had the
molecular mass of 32 kDa. Therefore, the radioactive
band in the gel situated at the mass range between 31
and 45 kDa should correspond to the adduct of the
radioactively labeled 21-membered oligonucleotide to
the integrase molecule. The yield of the photorection
product was 3.5%. Thus, the ATFMD group within a
nonnucleoside insert introduced into the duplex sub-
strate of the HIV-1 integrase helped us to reveal an inte-
grase contact with the unprocessed chain of the viral
DNA.
DNA
6
.5
1
2
Fig. 3. The radioautograph of SDS-PAGE in 15% PAG of
2) the products of photoreaction of duplex (VI + IV) with
integrase; (1) the integrase–duplex (VI + IV) without irra-
diation; M, protein markers of molecular masses.
(
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2
98
AGAPKINA et al.
32
Aldrich (United States); [γ- P]ATP for the introduc- (5). A solution of (3) (500 mg, 1.2 mmol) and (4)
tion of radioactive label in the DNA fragments from (431 mg, 1.32 mmol) in a mixture of dry THF (4 ml)
Izotop (Russia); and polynucleotide kinase from bacte- and triethylamine (0.5 ml) was stirred for 12 h at room
riophages T4 (Promega, United States). The HIV-1 temperature and evaporated. The residue (oil) was dis-
recombinant integrase was a kind gift of Dr. J.-F. Mous- solved in chloroform (50 ml), and the solution was
cadet (Gustave Roussy Institute, Villejuif). The reaction washed with water (2 × 50 ml), dried with anhydrous
course during the synthesis of (5) was monitored by sodium sulfate, and evaporated. The resulting crude (5)
TLC on Kieselgel 60 F precoated plates (Merck). The was purified by chromatography on a silica gel column
2
54
eluted with a step gradient of methanol (0–5%) in chlo-
substances with aromatic groups were detected on
chromatograms according to UV light absorption. The roform; yield 600 mg (80%); R
substances with a free amino group were detected using (CDCl , ppm): 3.25 (2 H, d, CH ), 3.50 (2 H, t,
1
0.5 (Ä); H NMR
f
3
2
the ninhydrin reagent (a solution of 0.4 g of ninhydrin CH NH), 3.78 (6 H, s, 2 OCH ), 4.02 (1 H, m, CHOH),
2
3
in 1 : 4 pyridine–isopropanol mixture). Aliphatic com-
pounds were detected by iodine vapor. For TLC, the
following solvent systems were used: (A) 90 : 10 chlo-
roform–methanol, (B) 96 : 4 chloroform–methanol, and
6
.9–7.6 [17 H, m, CC H (CH OC H )
+
6
5
3
6
4
2
+
C H CN CF ]; MS (m/z): 578 [M + 1 – N ] .
6
4
2
3
2
2
-(N,N-Diisopropylamido)-b-cyanoethyl phos-
phite of 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-
yl)benzamido)-3-O-(4,4'-dimethoxytrityl)-2,3-pro-
panediol (6). Chloro-2-(N,N-diisopropylamido)-β-
cyanoethoxyphosphine (50 µl, 0.18 µmol) was added
dropwise to a solution of (5) (100 mg, 0.16 mmol) in
dry methylene chloride (2 ml). The mixture was stirred
for 1 h at room temperature and evaporated. The resi-
due was dissolved in chloroform (50 ml), the solution
(C) chloroform. Column chromatography was carried
out on silica gel G60 (Merck, Germany) using a column
2
0 × 250 mm at an elution rate of 2.5 ml/min.
N-Succinimidyl 4-(3-(trifluoromethyl)-3H-diazi-
rin-3-yl)benzoate (4) was synthesized according to the
Nassal scheme [8]. The overall yield of (4) from the
starting p-bromotoluene was 11%, R 0.9 (C).
f
2
-(N,N-Diisopropylamido)-b-cyanoethyl phos- was washed with a saturated solution of soda (50 ml),
phite of 1-(4-(3-(trifluoromethyl)-3H-diazirin- dried by anhydrous sodium sulfate, and precipitated
3
-yl)benzamido)-3-O-(4,4'-dimethoxytrityl)-2,3-pro- with hexane to give (6); yield 95 mg (73%); R 0.7 (B);
panediol (6) (Scheme 2).
f
3
1
P NMR (CDCl ): 148.75 and 148.02 ppm.
3
1
-(2,2,2-Trifluoroacetamido)-2,3-propanediol (2).
Oligonucleotides (I)–(VII) were synthesized on an
Ethyl trifluoroacetate (4 ml) and triethylamine (2 ml)
were added dropwise to a stirred solution of 1-amino-
automatic DNA synthesizer ASM 102U (Novosibirsk,
Russia) using a standard phosphoramidite synthetic
scheme. At the stage of the introduction of (6), which
contained ATFMD group, the time of coupling
increased up to 10 min. The oligonucleotides were iso-
lated by SDS-PAGE in 20% PAG.
2
,3-propanediol (1) (1 g, 11 mmol) in DMF (10 ml).
The reaction mixture was stirred for 24 h at room tem-
perature and evaporated. The residue was dissolved in
9
: 1 chloroform–methanol mixture (50 ml) and washed
with water (50 ml). The organic layer was evaporated,
and the product was used at the next stage without puri-
fication.
3
2
The 5'- P-Phosphorylation of oligonucleotides
was carried out using T4-polynucleotide kinase for
4
7
[
0 min at 37°ë in 10 µl of 50 mM Tris-HCl buffer, pH
1-Amino-3-O-(4,4'-dimethoxytrityl)-2,3-propane-
.6, containing 10 mM MgCl , 5 mM dithiothreitol, and
2
diol (3). Oily product (2) from the previous stage was
trice coevaporated with 20 ml of anhydrous pyridine
and then treated dropwise with a solution of dimethoxy-
trityl chloride (3.3 g, 9.7 mmol) in pyridine (5 ml) at
stirring. The solution was stirred for 3 h at room tem-
perature and evaporated. The oily residue was dissolved
in 10 ml of 1 M NaOH in aqueous ethanol (1 : 1), stirred
for 24 h, and evaporated. The resulting oil was dis-
solved in chloroform (50 ml); washed with water (2 ×
3
2
32
γ- P]ÄíP. When 5'- P-phosphorylating oligonucle-
otides (I)–(III), we used 10 activity units of the enzyme
per 50 pmol of an oligonucleotide, whereas (VI)
10 pmol) was also phosphorylated with 10 activity
(
units of the enzyme. The reaction was stopped with the
addition of 0.25 mM EDTA (1 µl), and then an equiva-
lent quantity of the complementary oligonucleotide
(
IV) or (VII) and 5 M NaCl (0.8 µl) was added to the
mixture. The mixture volume was adjusted to 40 µl
with water, and it was heated to 90°ë and cooled
slowly. The radioactively labeled DNA duplexes were
isolated by centrifugation on the chromatographic col-
umns Micro Bio-Spin®, and the radioactivities of the
5
0 ml); dried with anhydrous sodium sulfate; evapo-
rated; and purified by a column chromatography on sil-
ica gel in a step gradient of methanol (0–7%) in chloro-
form to give (3); yield 2.9 g [66% from (1)]; R 0.1 (A);
H NMR (CDCl , ppm): 2.76 (2 H, t, NH ), 2.79 (2 H,
m, CH NH ), 3.14 (2 H, d, CH ), 3.27 (6 H, s, 2 OCH ),
.77 (1 H, m, CHOH), 6.81–7.46 [13 H, m,
f
1
3
2
32
P-labeled preparations were measured according to
Cherenkov using a Delta-300 counter (Tracor, the
Netherlands).
2
2
2
3
3
+
CC H (CH OC H ) ]; MS (m/z): 394 [M + 1] .
6
5
3
6
4 2
The temperature dependence of the UV light
1
-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)ben- absorption of duplexes was studied at 260 nm on a
zamido)-3-O-(4,4'-dimethoxytrityl)-2,3-propanediol spectrophotometer Hitachi 150-20 (Japan) equipped
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 4 2002

SYNTHESIS OF OLIGONUCLEOTIDE DERIVATIVES
299
with a thermostated cell holder at the steady increase of experiments and Dr. J.-F. Mouscadet, Institut Gustave
temperature from 10 to 80°ë at the rate of 0.5°C/min. Roussy, Villejuif) for his kind gift of HIV-1 integrase.
Thermostated quartz cells with an optical length of
This work was supported by the Russian Foundation
for Basic Research (project nos. 02-04-48797, 00-04-
1
cm (Pye Unicam, UK) were used. The concentration
of each chain was 5 µM, and the buffer was 50 mM
4
8312, and RFFI-NCNI 00-04-22003).
Tris-HCl with 20 mM MgCl , pH 7.0.
2
The reaction of 3'-terminal processing. A solution
of 10 pmol of integrase in 10 µl of buffer A (20 mM
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2
added to a solution of 0.2 pmol of duplex (VI + VII) or
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3
4
(
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(
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4
PAGE in 20% PAG containing 7 M urea followed by
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5
6
7
The ATFMD-containing oligonucleotides were
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–3
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2
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1
1
1
1
1
1
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(
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ACKNOWLEDGMENTS
We thank V.I. Beketov (Chemical Faculty, Moscow
State University) for his help with the photochemical
1
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 4 2002