Nucleoside N-acylation with active derivatives of amino acids
Simple procedure for N-acylation ofadenosine guanosine and cytidine by active derivatives of amino acids is proposed. Procedure is based on transient silyt protection ofribose hydroxy groups and consists of 3 steps: (a) silytotion of nucleoside by trimethylchloro-silane, (b) reaction of amino group...
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Інститут молекулярної біології і генетики НАН України
1996
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| Cite this: | Nucleoside N-acylation with active derivatives of amino acids / S.M. Yarmoluk, A.M. Kostenko, D.V. Kryvorotenko, I.Y. Dubey // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 50-55. — Бібліогр.: 9 назв. — англ. |
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irk-123456789-1541682019-06-16T01:28:12Z Nucleoside N-acylation with active derivatives of amino acids Yarmoluk, S.M. Kostenko, A.M. Kryvorotenko, D.V. Dubey, I.Y. Simple procedure for N-acylation ofadenosine guanosine and cytidine by active derivatives of amino acids is proposed. Procedure is based on transient silyt protection ofribose hydroxy groups and consists of 3 steps: (a) silytotion of nucleoside by trimethylchloro-silane, (b) reaction of amino group ofsilylated nucleoside with chloroanhydride or active ester of N-protected amino acid, (c) desilylation of intermediate. The yields of N-amino-acylated nucleosides were about 40–90 %. Запропоновано просту методику для N-ацилювання аденозину, гуазину і цитидіну активними похідними амінокислот. У процедурі використано тимчасовий захист сильними залишками гідроксильних груп рибози, який здійснюється в три етапи: силюванням нуклеозидів триметил-хлорсиланом (а); ацилюванням аміногруп силільованих нуклеозидів хлорангідридами або активными ефірами N-захищених амінокислот (б) і десилюванням ацильованого нуклеозиду (в). Виходи N-ацильованих нуклеозидів складають 40–90 %. Предложена простая методика для N-ацилирования аденозина, гуанозина и цитидина активными производными аминокислот. В процедуре использована временная защита силильными остатками гидроксильных групп рибозы, которую осуществляли в три этапа: силилованием нуклеозидов триметилхлорсиланом (а); ацилированием аминогрупп силированных нуклеозидов хлорангидридами или активными эфирами N-защищенных аминокислот( б) и десилированием оцифрованного нуклеозида ( в). Выходы N-ацилированных нуклеозидов составляют 40–90 %. 1996 Article Nucleoside N-acylation with active derivatives of amino acids / S.M. Yarmoluk, A.M. Kostenko, D.V. Kryvorotenko, I.Y. Dubey // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 50-55. — Бібліогр.: 9 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000449 http://dspace.nbuv.gov.ua/handle/123456789/154168 542.95 en Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
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Simple procedure for N-acylation ofadenosine guanosine and cytidine by active derivatives of amino acids is proposed. Procedure is based on transient silyt protection ofribose hydroxy groups and consists of 3 steps: (a) silytotion of nucleoside by trimethylchloro-silane, (b) reaction of amino group ofsilylated nucleoside with chloroanhydride or active ester of N-protected amino acid, (c) desilylation of intermediate. The yields of N-amino-acylated nucleosides were about 40–90 %. |
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Yarmoluk, S.M. Kostenko, A.M. Kryvorotenko, D.V. Dubey, I.Y. |
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Yarmoluk, S.M. Kostenko, A.M. Kryvorotenko, D.V. Dubey, I.Y. Nucleoside N-acylation with active derivatives of amino acids Биополимеры и клетка |
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Yarmoluk, S.M. Kostenko, A.M. Kryvorotenko, D.V. Dubey, I.Y. |
| author_sort |
Yarmoluk, S.M. |
| title |
Nucleoside N-acylation with active derivatives of amino acids |
| title_short |
Nucleoside N-acylation with active derivatives of amino acids |
| title_full |
Nucleoside N-acylation with active derivatives of amino acids |
| title_fullStr |
Nucleoside N-acylation with active derivatives of amino acids |
| title_full_unstemmed |
Nucleoside N-acylation with active derivatives of amino acids |
| title_sort |
nucleoside n-acylation with active derivatives of amino acids |
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Інститут молекулярної біології і генетики НАН України |
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1996 |
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Nucleoside N-acylation with active derivatives of amino acids / S.M. Yarmoluk, A.M. Kostenko, D.V. Kryvorotenko, I.Y. Dubey // Биополимеры и клетка. — 1996. — Т. 12, № 5. — С. 50-55. — Бібліогр.: 9 назв. — англ. |
| series |
Биополимеры и клетка |
| work_keys_str_mv |
AT yarmoluksm nucleosidenacylationwithactivederivativesofaminoacids AT kostenkoam nucleosidenacylationwithactivederivativesofaminoacids AT kryvorotenkodv nucleosidenacylationwithactivederivativesofaminoacids AT dubeyiy nucleosidenacylationwithactivederivativesofaminoacids |
| first_indexed |
2025-07-14T05:48:12Z |
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2025-07-14T05:48:12Z |
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1837600177439899648 |
| fulltext |
Nucleoside N-acylation with active derivatives of amino acids
Sergiy M. Yarmoluk*, Alexander M. Kostenko, Dmytro V. Kryvorotenko,
Igor Ya. Dubey1
Institute of Molecular Biology and Genetics, National Academy of Sciences of the Ukraine
150 Zabolotnogo str., 252143, Kyiv, Ukraine
institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of the Ukraine
1 Murmanska str., 253660, Kyiv, Ukraine
Simple procedure for N-acylation of adenosine, guanosine and cytidine by active deriva-
tives of amino acids is proposed. Procedure is based on transient silyl protection ofribose
hydroxy groups and consists of 3 steps: (a) silylotion of nucleoside by trimethylchloro-
silane, (b) reaction of amino group ofsilylated nucleoside with chloroanhydride or active
ester of N-protected amino acid, (c) desily lotion of intermediate. The yields of N-amino-
acylated nucleosides were about 40—90 %.
Introduction. Problems concerning the design of drug delivery forms for
biologically active modified nucleosides are now under investigation in many
laboratories, and a number of approaches has been developed [1]. Conjugates
of nucleosides with amino acids and peptides are promising compounds from
this point of view. O-aminoacylated nucleosides were used previously. 5'-
Aminoacyl derivatives of AZT (3'-azido-2',3'-dideoxythymidine) with Phe, Lys,
Ser, lie, Glu were tested as the depot-forms for drug delivery. Their activity in
cell cultures was similar to that of AZT itself [2]. The preparation of 5'-amino
acid phosphoroamides of zidovudine, 3'-deoxy-2\3'-didehydrothymidine and
3'-fluoro-3'-deoxythymidine allowed to increase their efficiency of selective
inhibition of HIV viral replication [3 ].
We propose the use of exocyclic amino groups of nucleosides as possible
alternative for their conjugation with amino acids and peptides via amide bond.
Probably amino acid derivatives of nucleosides can be hydrolyzed after their
penetration into the cell by intracellular enzymes being converted into
corresponding free nucleosides. The reaction of unprotected nucleosides with
N-protected amino acids in the presence of DCC (dicyclohexylcarbodiimide)
was studied before. Selective acylation of exocyclic amino functions was
achieved only for cytidine. For N-acylation of adenosine and guanosine,
equimolar amounts of N-phtaloylamino acids chloroanhydrides were used, but
ribose hydroxy groups were acylated too, and the yields of N-
aminoacylnucleosides were low [4].
Here we describe a simple method for the selective N-acylation of
nucleosides with active derivatives of amino acids using a transient silyl
protection strategy. Chloroanhydrides and active esters of amino acids were
•Correspondence address.
© S. M. YARMOLUK, A. M KOSTENKO, D. V. KRYVOROTENKO. I. Y. DUBEY, 1996
50
NUCLEOSIDE N-ACYLATION WITH ACTIVE DERIVATIVES
used as acylating reagent to obtain N-aminoacylated nucleosides. With the use
of active esters it was also possible to prepare under mild conditions nuclesides
conjugates with peptides and some another compounds containing carboxy
function which could not be easily obtained via chloroanhydrides.
Materials and Methods. Adenosine, guanosine and cytidine were purchased
from «Biolar» (Latvia), amino acids and N(benzyloxycarbonyl)-amino acids
from «Reanal» (Hungary), DCC and PFF (pentafluorophenol) from «Sigma»
(USA), HOBt (1-hydroxybenzotriazol) was synthesized according to [5]. N-Pht
(phtaloyl) amino acids and chloroanhydrides were prepared as described in [6 ].
Solvents and reagents were prepared as follows: Diox (1,4-dioxane) was
refluxed with sodium for 12 hrs and distilled; Py (pyridine) was distilled over
P4O10, ninhydrine, and then refluxed over CaH2 for 2 hrs and distilled;
acetonitrile was distilled over P4OI0; TMSC1 (trimethylchlorosilane) was
distilled over A1C13. The TLC was performed on Kieseigel 60 F254 plates
(«Merck», Germany) using n-butanol:acetic acid:water (4:1:1) system. NMR
spectra were recorded on Varian VXR-300 (75.4 MHz) NMR-spectrometer in
DMSO-d6 using tetramethyl silane as internal standard.
Reverse phase HPLC was performed on Beckman «Gold System» using
Ultraprep C18 (21x1.5 cm, «Beckman») column. A 0—30 % gradient of
acetonitrile in 0.1 M TEAB (triethylammonium bicarbonate) buffer (pH 6.5)
with flow rate 4 ml/min was used throughout.
GENERAL PROCEDURE 1 (GP1). Nucleoside (5 mmol) was coevaporated
with anhydrous Py in vacuo, suspended in 50 ml of the same solvent and
35 mmol of TMSC1 was added dropwise diiring 30 min with stirring. After 2 hrs
a solution of N-phtaloylaminoacyl chloride (7 mmol) in Py-Diox (4:1) was
added dropwise, with stirring. After TLC showed the absence of starting
nucleoside (in other case additional 3—5 mmol of chloroanhydride were
added), water (10 ml) and 15 min later 5 ml of concentrated aqueous ammonia
were added to the reaction mixture. After 10 min solvents were removed in
vacuo, the residue was treated with water and extracted with ethylacetate.
Crystallization of desired product from water layer began immediately. If no,
water layer was separated and evaporated in vacuo until crystallisation began.
The crystals were filtered off, recrystallized from aqueous alcohol, and dried in
vacuo over P4O10. Yields were 70—80 %.
GENERAL PROCEDURE 2 (GP2). N-protected amino acid (7 mmol) and
HOBt (or PFF) (7 mmol) were coevaporated with dry Py, dissolved in 30 ml
of Py-acetonitrile (1:3) and 7,5 mmol of DCC were added with stirring. After
3 hrs the reaction mixture was filtered from dicyclohexyl urea into the flask
with silylated nucleoside (see GP1) (5 mmol) and the course of reaction was
monitored by TLC. Usually the reaction was completed in 1—3 days. Products
were isolated as described in GP1. Yields were 40—70 %.
N2-(Phtaloylphenylalanyl)-guanosine (Pht-Phe-G) GP1; Rf 0.67; *H NMR
a 12.1 (s, 1H, HI), 8.3 (s, 1H, H8), 7.8 (m, 4H, Pht), 7.1 (m, 5H, Phe), 5.8
(d, 1H, HI'), 5.3 (t, 1H, CH Phe), 4.3 (t, 1H, H2'), 4.1 (s, 1H, H3'), 3.8 (m,
1H, H4'); m. p. 217—219; yield 73 %.
N6-(N-Carbobenzoxyphenylalanyl)-adenosine (Z-Phe-A); GP2 with HOBt;
Rf 0.70; *H NMR д 8.8—8.7 (2s, 2H, H2+H8), 7.5—7.1 (m, 10H, 2Phe), 6.0
(d, 1H, HI'); m. p. 104—106; yield 41 %.
N2-(N-Phtcdoyl-E-aminohexyl)-guanosine (Pht-Ahe-G): GP1; Rf 0.65; *H
NMR <5 12.1 (s, 1H, HI), 11.7 (s, 1H, NHCO), 8.3 (s, 1H, H8), 7.9—7.7 (m,
4H, Pht), 5.8 (d, 1H, HI'), 4.4 (t, 1H, H2'), 4.1 (t, 1H, H3'), 3.9 (m, 1H,
H4'), 3.7—3.5 (m, 4H, 2H5'+ 2H Ahe), 2.5—2.3 (m, 2H, Ahe), 1.7—1.5 (m,
4H, Acp), 1.4-1.2 (t, 2H, Ahe); m. p. 196-198; yield 86 %.
N2-(N-Phtaloylvalyl)-guanosine (Pht-Val-G): GP1; Rf 0.65; *H NMR <5
12.1-11.9 (br. s, 2H, HI, NHCO), 8.3 (s, 1H, H8), 7.9-7.7 (m, 4H, Pht),
51
а — Me3SiCl/Py; b — RCOX; с — NH40H/H20
В — heterocyclic base (adenine, guanine or cytosine)
R — N-protected amino acid residue
X — -CI, -PFF, -ONSu, -OBt
After the silylation of starting ribonucleoside I with TMSC1 in Py it was
treated with active derivative of N-protected amino acid, namely
chloroanhydride or active ester. Selective N-acylation of nucleoside was
achieved followed by the hydrolysis of silyl O-protecting groups to give
N-aminoacyl derivative III.
Chloroanhydrides are widely used for the preparation of nucleosides
52
YARMOLUK S. M. ET AL.
5.7 (d, 1H, HI'), 4.7 (d, 1H, CH Val), 4.3 (m, 1H, H2'), 4.1 (m, 1H, H3'>,
3.8 (d, 1H, H4'>, 3.7—3.4 <m, 2H, H5'), 2.7 <m, IH, CH VaO, 1.0 (d, 3H,
CH3), 0.8 (d, 3H, CH3); m. p. 184-186; yield 90 %.
N2-(N-Carbobenzoxyglycyl)-guanosine (Z-Gly-G): GP2 with HOBt; Rf 0.51;
*H NMR д 11.8 (br. s, 2H, HI, NHCO), 8.3 (s, 1H, H8), 7.3 (m, 5H, Ph),
5.8 (d, 1H, HI'), 5.0 (s, 2H, CH2), 4.4 (t, 1H, H2'), 4.1 (t, 1H, H4'), 4.0—3.8
(m, 3H, H3\ CH2 (Gly)), 3.7—3.5 (m, 2H, H5'); m. p. 173—175; yield 67 %.
N4-(N-Carbobenzoxyglycylglycyl)-cytidine (Z-Gly-Gly-C): GP2 with OBt;
Rf 0.55; *H NMR д 8.5 (d, 1H, H6), 7.4 (s, 5H, Ph), 7.3 (d, 1H, H5), 5.8 (d,
1H, HI'), 5.0 (s, 2H, CH2), 4.0-3.8 (m, 7H, 2CH2 (Gly), H2', НЗ', H4'),
3.7 (m, 2H, H5'); m. p. 138—140; yield 47 %.
N2-(2,4-dinitrophenylacetyl)-guanosine (DNPAc-G): GP2 with HOBt; Rf
0.50; 'H NMR д 12.2 (s, 1H, HI), 11.8 (s, 1H, NHCO), 8.8, 8.6, 7.9 (ms, 3H,
Ar), 8.3 (s, 1H, H8), 5.8 (d, 1H, HI'), 4.5—4.3 (m, ЗН, CH2, H2'), 4.1 (t,
1H, H3'), 3.9 m, 1H, H4'), 3.7—3.5 (m, 2H, H5'); m. p. 183—185; yield
80 %;
Determination of N-amionoacylation yields in model experiments. Model
N-aminoacylation reaction with various active esters were carried out according
to GP2 with 0.1 mmol of nucleosides. The course of reaction was monitored by
TLC. After a certain reactions time (see the Table) the mixture was treated
with water and aqueous ammonia, and an aliquot of the resulting solution was
analyzed by HPLC to determine the reaction yield.
Results and discussion. Since N-amiuoacyl derivatives of nucleosides are
compounds of considerable biological interest, we have developed a simple and
efficient method for their preparation based on the transient protection strategy
described by Jones et al. [7 ]. The idea of this strategy is to make the acylation
reaction N-specific by prior protection of free hydroxyl functions of ribose
residue with labile silyl groups. Our approach is illustrated by the following
scheme:
NUCLEOSIDE N-ACYLATION WITH ACTIVE DERIVATIVES
N-protected with common acyl groups like benzoyl and isobutyryl by transient
protection method. We employed chloroanhydrides of N-protected amino acids
as aminoacylating reagents. The reaction was as efficient as nucleoside
N-acylation with usual chloroanhydrides. Typically, when acyl chlorides of
N-phtaioylamino acids were used, the one-flask reaction was completed in
2—4 hrs (GP1). Usually N-aminoacylated nucleosides could be easily
separated from the reaction mixture by crystallization, and after the
recrystallization from aqueous alcohol the desired products were obtained in a
high yield (70—90 %). Nevertheless, the use of aminoacyl chlorides is far from
being a general way for nucleoside N-aminoacylation, as it has some important
limitations: 1) with the exception of N-Pht protected, aminoacyl chlorides are
usually hardly available; 2) many amino acids cannot be converted into
chloroanhydrides even with phtaloyl N-protecting group; 3) formation of
chloroanhydrides of optically active amino acids leads to their racemization.
To overcome these problems, we decided to use the active esters of amino
acids as mild N-acylating reagents. The use of some active esters for nucleoside
N-protection was described previously. 4-Nitrophenylbenzoate and
pentachlorophenylbenzoate [9 ] were used to obtain N-benzoylcytidine. Hydro-
xybenzotriazolyl ester of phenoxyacetic acid prepared from phenoxyacetyl
chloride and HOBt was used to protect guanosine amino function [8 ]. Almost
any compound containing a carboxy group can be converted into corresponding
active ester to be used as acylating reagent. We have studied the reactivity of
PFF, ONSu and HOBt active esters of amino acids and peptides towards
exocyclic amino groups of adenosine, guanosine and cytidine. Active esters were
prepared using well-established procedures of peptide synthesis, e. g. by
treating the mixture of protected amino acid, and corresponding hydroxy
component with DCC. To evaluate the reactivity of active esters, a series of
model experiments were carried out without the isolation of N-aminoacylated
nucleosides. The course of reaction \yas monitored by TLC, and the yields were
determined by HPLC. The results are presented in the Table.
YARMOLUK S. M. ET AL.
N-acylation efficiency depended strongly on the nature of active ester and
nucleoside. The best yields were observed-for the reaction of cytidine with OBt
and PFF esters (67—92 %). Adenosine and guanosine amino groups as less
nucleophilic than cytidine NH2 need more, active acylating reagents, and they
were acylated with OBt esters with 61—87 % yields, whereas PFF esters were
not enough active to react with A and G efficiently. At the same time, ONSu
esters did not react with A and G at all and only slowly with C. The results
obtained are in full agreement with known facts that the basicity of nucleoside
amino groups increases from G to C, and the reactivity of active esters towards
NH2 groups increases in the order ONSu < PFF < OBt [8 ]. The experimental
data show that OBt esters are the most efficient reagents suitable for
N-aminoacylation of all nucleosides and could be recommended as reagents of
choice for the preparative use. Pentafluorophenyl esters are quite active for
cytidine N-acylation, and ONSu esters cannot be considered as useful for the
preparation of N-aminoacylated nucleosides by the proposed method.
So, only OBt and PFF active esters of amino acids were used further in
this work for the preparative synthesis of N-aminoacyl nucleoside derivatives.
The general procedure for their use (GP2) was quite similar to that utilising
chloroanhydrides. The solution of freshly prepared active ester was added to
the silylated nucleoside. N-aminoacylation with active esters was slower than
with chloroanhydrides and required 1—3 days, depending on the nucleoside
and the active ester used. After the acylation reaction was completed labile
trimethylsilyl protecting groups were removed by short treatment with diluted
ammonia. The desired products were crystallized from the reaction mixture
after its evaporation and extraction and then recrystallized from aqueous alcohol
to give corresponding N-aminoacyl ribonudeosides with the yields of 40—80 % .
The structure of nucleoside derivatives was confirmed by NMR. The average
yields seem to be somewhat higher for the procedure GP1, although the direct
comparison of two procedures was not carried out. But the obvious advantage
of active esters is that they are mild reagents of general use (see above). It
should be noted that the procedure GP2 can be used not only for any amino
acid, but also for the conjugation of nucleosides with peptides and other classes
of compounds which are labile or contain a carboxy group that cannot be easily
converted into haloanhydro function. We were able to prepare successfully the
conjugates of nucleosides with Gly-Gly using the OBt ester of N-protected
dipeptide; the yield of Z-Gly-Gly-C was 47 %.
In addition, the use of active^ esters can be also preferable when the
corresponding chloroanhydride is highly reactive. For example, DNPAc chloride
is too active electrophilic reagent to be used directly for nucleoside acylation.
At the same time, N-(DNPAc)-guanosine was prepared in a high yield (80 %)
by the mild procedure GP2. Finally, it would be also interesting to study the
possible use of active esters of amino acids for direct selective N-acylation of
nucleosides without any protection of ribose residue, as they are known to be
highly N-specific reagents in peptide chemistry. It would simplify the method
considerably.
Thus, we have reported a convenient procedure for the preparation of
ribonucleoside N-aminoacyl derivatives ( of course, this method can be used
also for deoxyribonucleosides). Conjugates of this class could be able to release
free nucleoside within the cell being the depot-forms for their delivery. The
attachment of amino acids and peptides to biologically active modified
nucleosides would be therefore especially interesting, and derivatization of
azapyrimidine nucleosides by the proposed approach is now studied in our
laboratory.
The project is supported by National Academy of Sciences of Ukraine.
54
NUCLEOSIDE N-ACYLATION WITH ACTIVE DERIVATIVES
С. M. Ярмолюк, О. M. Костенко, Д. Криворотенко, І. Я. Дубей
N-ацилювання нуклеозидів активними похідними амінокислот
Резюме
Запропоновано просту методику для N-ацйлювання аденозину, гуазину і цитидіну активними
похідними амінокислот. У процедурі використано тимчасовий захист сильними залишками гід-
роксильних груп рибози, який здійснюється в три етапи: силюванням нуклеозидів триметил-
хлорсиланом (а); ацилюванням аміногруп силільованих нуклеозидів хлорангідридами або актив-
ными ефірами N-захищених амінокислот (б) і десилюванням ацильованого нуклеозиду (в). Вихо-
ди N-ацильованих нуклеозидів складають 40—90 %.
С. Я Ярмолюк, А. Я Костенко, Д В. Криворотенко, И. Я. Дубей
N-ацилирование нуклеозидов активными производными аминокислот
Резюме
Предложена простая методика для N-ацилирования аденозина, гуанозина и цитидина активны-
ми производными аминокислот. В процедуре использована временная защита силильными ос-
татками гидроксильных групп рибозы, которую осуществляли в три этапа: силилованием нук-
леозидов триметилхлорсиланом (а); ацилированием аминогрупп силированных нуклеозидов хло-
рангидридами или активными эфирами N-защищенных аминокислот( б) и десилированием оци-
фрованного нуклеозида ( в). Выходы N-ацилированных нуклеозидов составляют 40—90 %.
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