Polynucleotides and their components in the processes of aromatic nucleophilic substitution: I. Chemistry and dynamics of nucleotide arylation with pentafluoropyridine; obtaining of synthons for molecular design of nucleic acid analogues

Article abstract of DOI:10.1023/B:RUBI.0000015773.37915.3b

Pentafluoropyridine reacts with thymidine, adenosine, and uridine hydroxy groups to give quantitative yields of the corresponding nucleoside di- and triaryl ethers. The nucleophilic substitution reactions proceed successively and in parallel, with the slo

Full text of DOI:10.1023/B:RUBI.0000015773.37915.3b

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 1, 2004, pp. 47–52. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 1, 2004, pp. 54–60.
Original Russian Text Copyright © 2004 by Litvak, Mainagashev, Bukhanets.
Polynucleotides and Their Components in the Processes
of Aromatic Nucleophilic Substitution:
I. Chemistry and Dynamics of Nucleotide Arylation
with Pentafluoropyridine; Obtaining of Synthons
for Molecular Design of Nucleic Acid Analogues
1
V. V. Litvak, I. Ya. Mainagashev, and O. G. Bukhanets
Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences,
pr. akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
Received August 26, 2002; in final form, May 14, 2003
Abstract—Pentafluoropyridine reacts with thymidine, adenosine, and uridine hydroxy groups to give quanti-
tative yields of the corresponding nucleoside di- and triaryl ethers. The nucleophilic substitution reactions pro-
ceed successively and in parallel, with the slowest step being the nucleophilic substitution of the nucleoside sec-
ondary hydroxyls. The resulting ethers contain tetrafluoropyridyl moieties, which could be smoothly modified
by nucleophilic substitution of fluorine atoms. The ethers are useful intermediate synthons (both isolated and
in situ) for molecular design of oligonucleotide analogues.
Key words: arylation of nucleosides, pentafluoropyridine, SNAr
1
INTRODUCTION
One of the topical practical aspects of this prob-
lem is the development of new approaches to the
selective modification of complex polyfunctional
compounds of medicinal use in order to increase their
efficiency.
The development of new approaches to modification
of nucleosides, nucleotides, heterocyclic bases, and oli-
gonucleotides is important for the solution of a number
of fundamental and applied problems [1–6]. In most
cases, the nucleosides are primarily functionalized by
their interaction with electrophiles, whereas the subse-
quent transformations of the resulting electrophilic
moieties are carried out by the reactions with nucleo-
philes. This strategy is used for the synthesis of com-
pounds bearing reporter groups, conjugates with
nucleic acids, and other hybrid molecules. However,
the number of methods for introduction of electrophilic
moieties into the molecules of, for example, oligonu-
cleotides is rather limited [4].
It is obvious that biopolymers interacting with elec-
trophiles can be regarded as specific polyfunctional
reagents containing ensembles of nucleophilic targets
(amino and hydroxy groups) that mutually affect each
other. Such structural organization implies specific
chemical properties of these compounds in comparison
with the properties of monofunctional nucleophilic
compounds. A comparative study of chemical proper-
ties of an isolated molecule and that in an ensemble
with other molecules is among “a new set of great
tasks”, whose solution may be of fundamental signifi-
cance [7].
We believe that the reaction of polyfunctional mol-
ecules with activated arenes may be a fruitful approach
to their modification; the arenes could bear several
nucleophilic atoms or groups, which could ensure fur-
ther transformations of the resulting products in SNAr
processes. This work opens a cycle of publications, the
goal of which is the study of general regularities of
interaction of polyfunctional molecules (components
of NAs) with activated aromatic compounds, clarifica-
tion of the specificity of these processes in comparison
with reactions of the corresponding mononucleophiles,
and use of the results for the molecular design of more
complicated molecules, e.g., constructs similar to syn-
thetic analogues of nucleic acids. Some specific
schemes of molecular design of such analogues will be
presented in the next communication.
RESULTS AND DISCUSSION
We carried out the reaction of thymidine (II), ade-
nosine (IX), and uridine (X) with pentafluoropyri-
dine (I), which led to (V), (XI), and (XII) after the
substitution of the first halogen atom of (I) (Schemes 1
and 2). Each of the products possesses at least three
1
Corresponding author; phone: +7 (3832) 39-6227; e-mail: lit-
vak@niboch.nsc.ru
1068-1620/04/3001-0047 © 2004 MAIK “Nauka /Interperiodica”

48
LITVAK et al.
O
F
F
F
F
F
A
F
F
N
N
(VII)
F
O
F
A'
F
Thy
Thy
O
HO
Thy
F
ii
O
N
F
F
O
OH
OH
(II)
Thy
F
F
F
F
O
(III)
+ Et3N · HF
(VI)
i
N
O
HO
(I)
O
F
F
iii
C'
F
N
F
O
(V)
F
F
F
O
C
F
F
F
F
N
F
(IV)
N
i) DMF/Et3N, 57°C
ii) EtOH/DMF/Et3N, 20°C
iii) PriOH/DMF/Et3N, 20°C
(VIII)
Scheme 1.
F
F
N
A''
F
F
HO
F
O
B
Base
F
F
F
F
O
O
DMF
Et3N, 20°C
+
F
F
N
OH
OH
F
O
O
F
F
(I)
(IX), (X)
C''
D''
F
N
N
F
F
(IX), (XI): B = Ade
(X), (XII): B = Ura
(XI), (XII)
Scheme 2.
fluorine atoms capable of subsequent nucleophilic
We studied the spectra of ethers (VII) and (VIII)
substitutions, which predetermines the possibilities formed in the reactions of pentafluoropyridine with pri-
19
of their further modifications. The dynamics of the
mary or secondary alcohols in order to assign the F
NMR resonances of (III), (IV), and (V) resulting from
processes shown in Scheme 1 was monitored by ana-
19
lyzing homogeneous reaction mixtures by F NMR the arylation of primary or secondary hydroxy groups
(Fig. 1).
of thymidine (Scheme 1 and Fig. 2).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

POLYNUCLEOTIDES AND THEIR COMPONENTS
49
Component contents, %
As follows from the fragments of spectra presented
in Fig. 2, the multiplets of β,β'-fluorine atoms of (VII)
are shifted upfield (3.86 ppm; here and further, chemi-
cal shifts are given for the centers of symmetrical mul-
tiplets) in comparison with those in isopropyl ether
(VIII) (4.89 ppm). Similar shifts were observed for the
centers of multiplet signals of the fluorine atoms in
pyridine positions 2 and 6. On the basis of these data,
one can assign with a high probability the low-field
multiplets at 5.59 and 5.18 ppm to fluorine atoms in
pyridine positions 3 and 5 of ë' and ë rings of (V) and
(IV), respectively. Then, the higher field multiplets at
4.60 and 4.51 ppm should be assigned to fluorine atoms
in positions 3 and 5 of rings Ä and Ä' attached to pri-
mary hydroxyls in (III) and (V). Another argument in
favor of the proposed assignment can be the fact that
the rates of formation of ether (III) [as well as the ethyl
ether (VII)] were much higher than those of arylation
of secondary hydroxy groups.
90
5
4
50
1
2
3
0
5
10
Reaction time, h
Therefore, the dynamics of the process can be mon-
itored by means of direct measurement of integral
intensities of the resonances of fluorine atoms in ë' and
ë rings. The content of ether (III), the multiplet of
which is partially overlaps with the resonances of frag-
ment Ä' (Fig. 2), was determined by subtraction of the
integral intensity of the fluorine atoms of the diether
(V) ring C'.
Fig. 1. The time dependences of relative contents (I /I ) ×
i
0
100 of components in the process of thymidine arylation
with pentafluoropyridine. I and I are integral intensities of
i
0
fluorine resonances of the reaction components and the ini-
tial pentafluoropyridine content, respectively. 1, Dynamics
of substrate (I) expenditure; 2 and 3, accumulation and
decrease of intermediates (III) and (IV), respectively; 4,
formation of diether (V); 5, accumulation of triethylamine
hydrofluoride (VI) and the total content of ethers (III)–(V).
The experimental data in Fig. 1 confirm the transfor-
mations presented in Scheme 1. As expected, the fluo-
rine atom in position 4 of pentafluoropyridine is substi-
tuted in the course of reaction, and an exponential
decrease in the intensity of resonance at 29.5 ppm
(curve 1) reflects this fact. A correlation between the
decrease in the substrate amount and the accumulation
of reaction products is observed at any moment of the
reaction. The concentrations of ethers (III) and (IV)
grow from zero to their maxima after three hours and
then again fall to zero. The rate of formation of (V) is
initially equal to zero. Then, it reaches its maximum at
the highest total concentrations of ethers (III) and (IV),
with the tangent of the angle of slope being 0.6, 1.2, 1.4,
2.8, 1.8, and 1.5 after 1.5, 2.15, 2.65, 3.1, 3.6, and 4.05
h, respectively. The S-like character of curve 4 also con-
firms the formation of ether (V) from its precursors
(III) and (IV) in successive processes. The initial rate
of formation of (III) is almost three times higher than
that of (IV) (Fig. 1, curves 2 and 3, respectively), which
could result from both a higher ionization degree of the
primary hydroxy group in the presence of triethylamine
(the reaction did not proceed in the absence of the base)
and lower sterical hindrances in comparison with sec-
ondary hydroxyl. On the basis of these data, one can
presume that compounds of type (III) and (IV) can be
obtained separately due to an increased difference
between their formation rates, which could be achieved
using substrates containing sterically hindered reaction
centers (e.g., 3,5-dichloro-2,4,6-trifluoropyridine).
Our data on the differences in arylaton rates of pri-
mary and secondary hydroxy groups of thymidine and
mononucleophiles (ethanol and isopropanol) deserve a
special discussion for elucidation of probable specific
properties of polynucleophilic reagents (Scheme 1).
The ratios of formation rates of ethers (VII) and (VIII)
measured at the initial parts of kinetic curves and under
the conditions identical to those used for the accumula-
tion of thymidine ethers (III) and (IV) are as follows:
(III)/(IV) = 3.1 (1), (VII)/(VIII) = 4.3 (2), (III)/(VII) =
2.2 (3), and (IV)/(VIII) = 2.5 (4). The first two ratios
are reasonable and can be explained by a higher ioniza-
tion degree of the primary hydroxy function in the pres-
ence of triethylamine and lesser steric hindrances for its
arylation in comparison with the secondary hydroxyl.
On the other hand, the next two ratios (3) and (4) con-
tradict not only to the first two ratios, but also to the
published data [8], according to which just a simple
increase in the number of methylene groups in alcoho-
lates should decrease the nucleophile reactivity (which
is reflected in the 1/2 ratios) rather than increase the 3/4
ratio. Thus, the hydroxy groups of thymidine attached
to a more bulky pentose backbone turn out to be more
reactive than similar nucleophilic centers in simple iso-
lated molecules. A possible explanation of the specific-
ity of a polynucleophilic target can be the involvement
of hydrogen atom of one of the hydroxy groups in the
intramolecular salvation, which results in a stabiliza-
tion of intermediate anionic Meisenheimer-like σ com-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

50
LITVAK et al.
A(III)
(a)
A'(V)
C(IV)
C'(V)
5.6
5.4
5.2
5.0
4.8
4.6
4.4 ppm
A(III)
A'(V)
(b)
C'(V)
C(IV)
5.6
5.4
5.2
5.0
4.8
4.6
4.4 ppm
C'(V)
A'(V)
(c)
5.8
(d)
5.6
5.4
5.2
5.0
4.8
4.6
4.4 ppm
5.0
4.8 ppm
4.0
3.8
ppm
Ether (VIII)
Ether (VII)
19
Fig. 2. Parts of F NMR spectra of the reaction mixtures presented in Scheme 1 that correspond to β,β'-fluorine atoms of pyridyl
rings Ä, C, Ä', and C' of (III), (IV), and (V). The spectra were registered after (a) 0.75, (b) 3, and (c, final) 16 h; (d), the parts of
19
F NMR spectra of the reaction mixtures corresponding to β,β''-fluorine atoms of pyridyl rings of ethers (VII) and (VIII)
plex. The formation of such an intermediate is probably result from greater steric hindrances along with the
a rate-limiting stage in the process under study, as it is
commonly the case in the majority of reactions of acti-
vated arenes with anionic reagents. Another factor
favoring the nucleoside transformation into more active
reagent can be the formation of a spirocomplex with a
successive participation of two hydroxyls, which is a
more stable than the classical Meisenheimer complex
[9].
growing number of hydroxy groups and aryl residues in
the pentose backbone. These hindrances can hamper
both the formation of intermediates and the probable
involvement of hydroxy groups in the intramolecular
stabilization. As in the case of thymidine, primary
hydroxyl reacts noticeably faster than the secondary
hydroxyls, which clearly follows from the dynamics of
ratios of integral intensities of multiplets (4.78 and
4.82 ppm) of fluorine atoms disposed in β-positions
relative the ring Ä'' nitrogen atom and the total intensity
Modification of the third hydroxy group in adenos-
ine (IX) and uridine (X) under the same conditions
(Scheme 2) requires a longer reaction time, which may of overlapped downfield low-field resonances (6.72 and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

POLYNUCLEOTIDES AND THEIR COMPONENTS
51
6.18 ppm) of the corresponding diether fragments (ë'' from thymidine) with the purity of 91% (here and here-
and D''). Like diether (V), triethers (XI) and (XII) were inafter, according to HPLC). The product with Rf 0.5
quantitatively accumulated in the reaction mixture.
(A) was purified by column chromatography on silica
gel (elution with CHCl3). A portion of the product (100
mg) isolated by chromatography was recrystallized
from 20% isopropanol in hexane to give (V) (36 mg)
According to 19F NMR, diether (V) formed in quan-
titative yield, displays no visible changes for the next
54 h at 57°ë (data not given), which predefines its pos-
sible use [as well the use of triethers (XI) and (XII)] as
in situ substrates for further modifications in SNAr-like
processes. These compounds contain several mobile
fluorine atoms in positions 2 and 6 of pyridine rings and
can be regarded as intermediates for the molecular
design of polyfunctional compounds, e.g., oligonucle-
otide analogues. We hope to describe such studies in
future.
1
with purity exceeding 99%; mp 88–90°ë; H NMR:
1.86 (3 H, s, CH3), 2.48–2.79 (2 H, m, H2'), 4.60 (1 H,
br. s, H4'), 4.80 (2 H, br. s, H5'), 5.52 (1 H, br. s, H3'),
6.30 (1 H, apparent t, J1',2' 7.1, H1'), 7.17 (1 H, s, H6),
and 9.59 (1 H, br. s, 3-NH); 19F NMR: 4.58 [2 F, m, 5'-
(β,β'-difluoropyrid-4-yl)], 5.55 [2 F, m, 3'-(β,β'-difluo-
ropyrid-4-yl)], 70.69 [2 F, m, 5'-(α,α'-difluoropyrid-4-
yl)], and 71.16 [2 F, m, 3'-(α,α'-difluoropyrid-4-yl)].
Found, %: C 44.12 and 45.31; H 2.29 and 2.42; N 10.43
and 10.25; F 28.36 and 28.59. C20H12F8N4O5. Calc., %:
C 44.46, H 2.24, N 10.37, F 28.13.
EXPERIMENTAL
1ç and 19F NMR spectra were registered on a
Bruker WP 200 SY spectrometer (Germany) at 295 K
and working frequencies of 200.13 and 188.28 MHz,
respectively. The values of chemical shifts (δ, ppm)
were measured relative to internal standards SiMe4 in
2',3',5'-O-Tris(2,3,5,6-tetrafluoropyrid-4-yl)ade-
nosine (XI). Pentafluoropyridine (506 mg, 2.99 mmol)
and triethylamine (331 mg, 3.28 mmol) were added to
a solution of adenosine (250 mg, 0.94 mmol) in DMF
(2 ml), and the reaction mixture was kept for 8 days at
room temperature. The reaction was monitored and the
product (XI) was isolated as described above for (V)
with quantitative yield (810 mg) and 97% purity. After
recrystallization from propanol the purity of (XI)
exceeded 99%; mp 81–83°ë; 1H NMR: 4.78–5.00 (3 H,
m, H5', H4'), 5.92 (2 H, br. s, 6-NH), 6.12 (1 H, appar-
ent t, J 4.8, H3'), 6.34–6.40 (2 H, m, H1' and H2'), 7.91
(1 H, s, H8), and 8.18 (1 H, s, H2). 19F NMR: 4.78 [2 F,
m, 5'-(β,β'-difluoropyrid-4-yl)], 6.72 [4 F, m, 3'-(β,β'-
difluoropyrid-4-yl)], 70.57 [2 F, m, 5'-(α,α'-difluoropy-
rid-4-yl)], and 71.51 [4 F, m, 3'-(α,α'-difluoropyrid-4-
yl)]. Found, %: C 42.51 and 42.23; H 1.96 and 1.73; N
15.41; F 32.26 and 32.56. C25H10F12N8O4. Calc., %: C
42.03, H 1.41, N 15.69, F 31.91.
CDCl3 for 1H and ë6F6 in DMF for 19F NMR. The cou-
pling constants J are given in Hz. Melting points were
determined on a Koefler S 30 A/G table (Germany).
Thymidine was from Yamasa Shoyu Co., Ltd
(Japan), adenosine from Reanal (Hungary), and uridine
(pure grade) from NPO Biokhimreaktiv (Russia). Pen-
tafluoropyridine had no admixtures according to 19F
NMR spectrum. Silica gel with a particle size of 40–
100 µm was used for column chromatography. Solvents
were purified according to the known procedures [10]
and stored over molecular sieves 4Å.
The completeness of reactions and homogeneity of
the resulting products were monitored by TLC and
HPLC. TLC was carried out on precoated TLC plates
Kieselgel 60F254 (Merck, Germany) in (A) 10 : 1 and
(B) 20 : 1 CHCl3–MeOH mixtures. Analytical HPLC
was carried out on a microcolumn liquid chromato-
graph Milikhrom-1 (Nauchpribor, Orel, Russia) using a
CHROM system for collection and analysis of chro-
matographic data [11], a Nucleosil 100-5 C-18, 5 µm
column (Macherey–Nagel, Germany) and elution for
20 min in a gradient of ëç3CN (70–90%) in 0.1% TFA
at the flow rate of 100 µl/min.
2',3',5'-O-Tris(2,3,5,6-tetrafluoropyrid-4-yl)uri-
dine (XII). Pentafluropyridine (554 mg, 3.28 mmol)
and triethylamine (363 mg, 3.58 mmol) were added to
a solution of uridine (250 mg, 1.02 mmol) in DMF
(2 ml), and the reaction mixture was kept for 6 days at
room temperature. The product was isolated in a yield
86% (709 mg, the purity of 86%) as described above.
Purification by column chromatography on silica gel
(elution with 49 : 1 CHCl3–MeOH) yielded the product
with Rf 0.5 (B), which was recrystallized from 7 : 3 pro-
panol–water to give (XII) (315 mg) with the purity of
3',5'-O-Bis(2,3,5,6-tetrafluoropyrid-4-yl)thymidine
(V). Pentafluropyridine (166 mg, 0.98 mmol) and tri-
ethylamine (111 mg, 1.10 mmol) were added to a solu-
tion of thymidine (116 mg, 0.48 mmol) in DMF
(0.5 ml), and the reaction mixture was kept for 16 h at
1
99%; mp 78–80°ë. H NMR: 4.76–5.00 (3 H, m, H5'
and H4'), 5.61 (2 H, br. s, H2' and H3'), 5.77 (1 H, d, J
8.1, H5), 5.96 (1 H, s, H1'), 7.42 (1 H, d, J 8.1, H6),
56–58°ë. The completion of reaction was monitored by 9.52 (1 H, br. s, 3-NH). 19F NMR: 4.82 [2 F, m, 5'-(β,β'-
19F NMR according to the absence of pentafluropyri-
dine. The reaction mixture was poured into ice water
and neutralized with concentrated HCl. The resulting
precipitate was filtered, washed with distilled water,
and dried in air and then in vacuum of a water-jet pump
difluoropyrid-4-yl)], 6.18 [4 F, m, 3'-(β,β'-difluoropy-
rid-4-yl)], 70.64 [2 F, m, 5'-(α,α'-difluoropyrid-4-yl)],
71.52 [4 F, m, 3'-(α,α'-difluoropyrid-4-yl)]. Found, %:
C 41.79 and 42.08; H 1.59 and 1.71, N 10.01, F 32.56
and 32.84. C24H9F12N5O6. Calc., %: C 41.70, H 1.31, N
over NaOH to give the product; yield 250 mg (96% 10.13, F 32.97.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

52
LITVAK et al.
The study of thymidine interaction with pen-
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