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Nucleic Acids Research Pages 778-786

2'-Deoxynucleoside 5'-triphosphates modified at [alpha]-, [beta]- and [gamma]-phosphates as substrates for DNA polymerases
Introduction
Results
   Synthesis
   Enzymology
Discussion
Materials And Methods
   DNA and enzymes
   Synthesis of modified dNTP, method A
   Method B
   General procedure for the reduction of an azido-group in modified dNTP, method C
   Synthesis of the dNTP IIc-e
Acknowledgements
References


2'-Deoxynucleoside 5'-triphosphates modified at [alpha]-, [beta]- and [gamma]-phosphates as substrates for DNA polymerases

2 '-Deoxynucleoside 5 '-triphosphates modified at [alpha]-, [beta]- and [gamma]-phosphates as substrates for DNA polymerases Ludmila A. Alexandrova*, Alexander Yu. Skoblov, Maxim V. Jasko, Lyubov S. Victorova and Alexander A. Krayevsky

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow 117984, Russia

Received October 8, 1997; Revised and Accepted December 9, 1997

ABSTRACT

Replacement of [alpha]-, [beta]- and [gamma]-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at [alpha]-, [beta]- and [gamma]-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that [gamma]-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the [gamma]-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase [alpha]. For dUTP analogues substituted at both [gamma]-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at [gamma]-phosphonate or C5 position. Meanwhile, C5-substituted [beta],[gamma]-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[[beta],[gamma]-(methylphosphinyl)methylphosphonyl]-[alpha]-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.

INTRODUCTION

Modified dNTPs and rNTPs are used widely in molecular biology and biochemistry as model substrates for enzymatic systems. However, these compounds cannot be used in cell biology because of their rapid dephosphorylation both, in intercellular media and during penetration through the cell membrane. Therefore, application of dNTPs and rNTPs with increased stability toward dephosphorylating enzymes would seems to have the potential to produce a high rate of success, especially for stable dNTPs and rNTPs which carry additional reporter or ligand groups. The presence of reporter (fluoresceinyl, tetramethylrhodaminyl) or ligand (biotinyl, 2,4-dinitrophenyl) groups makes it possible to monitor the diffusion of these compounds into cells and to observe their incorporation into DNA.

Diffusion of modified dNTP into the cell has been poorly studied. This problem seems to be resolved by increasing the hydrophobicity or covalent attachment of ligands for transporting the modified dNTP molecule into the cell.

Recently it has been shown that the replacement of the [gamma]-phosphate by methylphosphonate or phenylphosphonate in natural and glycon-modified dNTPs has little effect on the substrate activity of the compounds toward HIV and AMV Reverse Transcriptases (RTs) (1,2). In addition, dNTPs substituted at the [alpha]-phosphate (3,4), [beta],[gamma]-diphosphate (5,6) and even at all three phosphates (7,8), were shown to be substrates for RTs; however, the limitations for such modifications are unknown.

In this work we synthesized several novel modified dNTPs (Scheme 1, I-III) and evaluated them as substrates towards AMV RT and some other DNA polymerases.

RESULTS

Synthesis

The key nucleoside (IV) was synthesized according to Scheme 2. The principal stage was the replacement of bromine of 3',5'-di-O-acetyl-(5-bromomethyl)-2'-deoxyuridine (Va) (9) by 1,6-hexandiol (10).

To obtain 5-[(6-azidohexyl)oxymethyl]-2'-deoxyuridine (IV), nucleoside Vb was methanesulfonylated and the methanesulfonyloxy group in Vc was replaced by an azide group by reaction with sodium azide in DMF. Compound Vd was deprotected with aqueous ammonia; the yield of IV was 39% starting from Va.

To synthesize dNTPs modified at both, the nucleobase and the triphosphate fragment (I and II, Scheme 3), nucleoside IV was phosphorylated with POCl3 in triethylphosphate. Nucleotide VI was converted to the corresponding imidazolide, which was coupled without purification with bis-(tri-n-butylammonium) salts of phenylphosphonylphosphoric, pyrophosphoric or dibromomethylenediphosphonic acids. Triphenylphosphine, DTT and mercaptoethanol were used for reducing the azido group in Ia, Ie and IIa. The best results were obtained with DTT.


Scheme 1.

The dNTP analogues containing ligand (IIc) or reporter (IId,e) groups were synthesized by coupling IIb with either N-succinimidyl N-biotinyl-6-aminohexanoate or fluorescein isothiocyanate or N-succinimidyl tetramethylrhodamine carboxylate according to (11).


Scheme 2.

Compounds Ia-c were synthesized according to Scheme 4.

Compound VIIb (12) was coupled either with 2,4-dinitrofluorobenzene for VIIc or with N-succinimidyl 6-N-(2,4-dinitrophenyl)aminohexanoate for VIId. Compounds VIIa,c,d were converted to VIIIa,c,d by reaction with trimethylbromosilane, then activated with CDI to yield IXa,c,d and coupled with 2'-deoxythymidine 5'-diphosphate to yield Ia,c,d. The yields were 21, 12 and 25%, respectively; Ia was reduced to Ib with DTT with 53% yield.

2'-Deoxythymidine 5'-{[beta],[gamma]-[(methylphosphinyl)methylphosphonyl]-[alpha]-phosphate} (IIIa, R = OH) and its 3'-azido-analog (IIIb, R = N3) were synthesized by coupling of either 2'-deoxythymidine 5'-phosphoimidazolide or 3'-azido-2',3'-dideoxythymidine 5'-bis-(1,2,4-triazolyl)phosphate with (methylphosphinyl)methylphosphonate (13).

The structure of all compounds was confirmed by UV, 1H- and 31P-NMR, and mass spectrometry.


Scheme 3.

Enzymology

Several groups of modified dNTPs were studied in this work. Group A included different dTTP [gamma]-phosphonates (Ia-Id) and dNTPs containing additional modifications at the thymine base (Ie-If); compound Ig was used as a control for Ie-If. Group B included compounds containing the dibromomethylenediphosphonate instead of the [beta],[gamma]-diphosphate and modified at the thymine base (IIa-IIe); IIf was used as a control. Compounds III contained two modifications at the [gamma]-phosphate: two of its P-O bonds were replaced by P-C bonds.
Scheme 1.

It can be seen in Figure 1 that Ia (lanes 6-11) and Ib (lanes 12-17) are substrates for AMV RT; they were incorporated into the DNA chain several times (lanes 10-11 and 16-17). Compound Ib was several fold less effective as a substrate than Ia (compare lanes 12-13 and 6-7). The presence of a minor octadecanucleotide band on lanes 3, 10 and 16is attributable to the low fidelity of RTs.

It is evident from Figure 2 that DNA polymerase [alpha] incorporates Ia very weakly into the DNA chain (lanes 6-10), while Ib is utilized by the enzyme at 600 µM (lanes 11-15). Thus, DNA polymerase [alpha] is 2-3 orders of magnitude more specific towards [gamma]-phosphate-substituted dNTPs than AMV RT; this observation is in agreement with our earlier data (2). Compound IIIa also elongated the primer (lanes 16-18); after incorporation of one residue of IIIa the primer was efficiently extended in the presence of both natural dNTPs and IIIa (lanes 19-20). However, comparison of lanes 18 and 2 shows that for IIIa the efficiency of primer extension was 2 orders of magnitude lower than that for dTTP.


Figure 1. Primer extention catalyzed by AMV RT in the presence of Ia and Ib. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane 4, as in lane 1, plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane 5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 6-9, as in lane 1 plus 2 µM (lane 6), 10 µM (lane 7), 20 µM (lane 8), 100 µM (lane 9) Ia; lane 10, as in lane 1 plus 100 µM Ia, 2 µM dGTP; lane 11, as in lane 1 plus 100 µM Ia, 2 µM dGTP, 2 µM dATP; lanes 12-15, as in lane 1 plus 2 µM (12), 10 µM (13), 20 µM (14), 100 µM (15) Ib; lane 16, as in lane 1 plus 100 µM Ib, 2 µM dGTP; lane 17, as in lane 1 plus 100 µM Ib, 2 µM dGTP, 2 µM dATP.

The substrate activity of Ic and Id containing large and hydrophobic groups at the [gamma]-phosphonate was close to that for Ia and Ib when primer extension was catalyzed by AMV RT (Fig. 3, lanes6-12 and 13-19, respectively).

Introduction of two modifications into the dNTP molecule (to the [gamma]-phosphate and the nucleobase, as in compounds Ie and If) decreased its substrate activity towards AMV RT by 1-2 orders of magnitude as compared with the compounds with only one modification Ig (Fig. 4, lanes 11-15 for Ie; Fig. 5, lanes 6-10 for If; Ig was used as control, Fig. 4, lanes 6-10). Additional minor bands on lanes 6-9 (Fig. 4) can be ascribed to insignificant contamination of the test compounds with Ig and Ie which are not modified at the nucleobase. It is illustrated by their electrophoretic mobility, similar to that for dTTP extended primer (lane 2), whenever DNA fragments containing at the 3'-terminus the modified residues of Ig or Ie have decreased mobility on account of its additional hydrophobicity. Compound IIa was a substrate for AMV RT (Fig. 4, lanes 16-20), but its affinity to the enzyme was lower. The primer being extended by IIa was elongated by only 1 nucleotide residue in the presence of dGTP, dATP and dCTP (lanes 19-20). Reduction of the azido-group in IIa to an amino-function completely inactivated IIb as a substrate (Fig. 5, lanes 11-15). Compounds IIc-e were not utilized by AMV RT (data not shown).

[beta],[gamma]-Dibromomethylene-substituted dTTP (IIf) extended the DNA chain by AMV RT (Fig. 6, lanes 6-9), but it was 100 times less active than dTTP (compare lanes 9 and 2). Its incorporation was observed up to two (lane 10) or more times (lane 11) with similar effectivity.


Figure 2. Primer extention catalyzed by DNA polymerase [alpha] in the presence of Ia, Ib and IIIa. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 10 µM dTTP; lane 3, as in lane 1 plus 10 µM dTTP, 10 µM dGTP; lane 4, as in lane 1 plus 10 µM dTTP, 10 µM dGTP, 10 µM dATP; lane 5, as in lane 1 plus 10 µM dTTP, 10 µM dGTP, 10 µM dATP, 10 µM dCTP; lanes 6-8, as in lane 1 plus 10 µM (lane 6), 100 µM (lane 7), 600 µM (lane 8) Ia; lane 9, as in lane 1 plus 600 µM Ia, 10 µM dGTP; lane 10, as in lane 1 plus 600 µM Ia, 10 µM dGTP, 10 µM dATP; lanes 11-13, as in lane 1 plus 10 µM (lane 11), 100 µM (lane 12), 600 µM (lane 13) Ib; lane 14, as in lane 1 plus 600 µM Ib, 10 µM dGTP; lane 15, as in lane 1 plus 600 µM Ib, 10 µM dGTP, 10 µM dATP; lanes 16-18, as in lane 1 plus 10 µM (16), 100 µM (17), 600 µM (18) IIIa; lane 19, as in lane 1 plus 600 µM IIIa, 10 µM dGTP; lane 20, as in lane 1 plus 600 µM IIIa, 10 µM dGTP, 10 µM dATP.

The [beta],[gamma]-phosphonate derivative of dTTP (IIIa) was utilized by AMV RT, but the efficiency of its incorporation into the DNA was 50-100 times lower compared to dTTP (Fig. 7, series B). Similar results were obtained for the azido-dTTP derivative IIIb (data not shown). Both IIIa and IIIb nucleotide residues were incorporated into the primer by catalysis of HIV RT, but the IIIa and IIIb efficiency was about 10 times lower than by AMV RT (data not shown). At the same time, IIIa was practically not recognized by TDT (Fig. 7, series A).

Thermus aquaticus DNA polymerase did not incorporate IIc-f into the DNA chain (data not shown).

DISCUSSION

In this work we found that a dNTP substituted with a bulky group at the [gamma]-phosphate position Ia-d extended the DNA chain by AMV RT (Figs 1 and 3), but are poorly utilized by DNA polymerase [alpha] (Fig. 2). It should be mentioned that the substrate activity of Ic,d towards AMV RT is decreased insignificantly in spite of the large size and high hydrophobicity of the substituents at the [gamma]-phosphonate. These data are consistent with the absence of sterical hindrances in the [gamma]-phosphonate binding site of the [primer-template + RT + dNTP] complex (2). Such sterical availability was demonstrated earlier for one of the stereoisomers of 2'-deoxythymidine ([alpha]-methylphosphonyl) or ([alpha]-phenylphosphonyl) [beta],[gamma]-diphosphate and [RT + template-primer] complexes (14,15).


Figure 3. Primer extention catalyzed by AMV RT in the presence of Ic and Id. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane 4, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane 5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 6-9, as in lane 1 plus 2 µM (lane 6), 20 µM (lane 7), 50 µM (lane 8), 100 µM (lane 9) Ic; lane 10, as in lane 1 plus 100 µM Ic, 2 µM dGTP; lane 11, as in lane 1 plus 100 µM Ic, 2 µM dGTP, 2 µM dATP; lane 12, as in lane 1 plus 100 µM Ic, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 13-16, as in lane 1 plus 2 µM (lane 13), 20 µM (lane 14), 50 µM (lane 15), 100 µM (lane 16) Ib; lane 17, as in lane 1 plus 100 µM Id, 2 µM dGTP; lane 18, as in lane 1 plus 100 µM Id, 2 µM dGTP, 2 µM dATP; lane 19, as lane 1 plus 100 µM Id, 2 µM dGTP, 2 µM dATP, 2 µM dCTP.


Figure 4. Primer extention catalyzed by AMV RT in the presence of Ig, Ie and IIa. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane 4, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane 5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes6-8, as in lane 1 plus 2 µM (lane 6), 20 µM (lane 7), 200 µM (lane 8) Ig; lane 9, as in lane 1 plus 200 µM Ig, 2 µM dGTP; lane 10, as in lane 1 plus 200 µM Ig, 2 µM dGTP, 2 µM dATP; lanes 11-13, as in lane 1 plus 10 µM (lane 11), 100 µM (lane 12), 600 µM (lane 13) Ie; lane 14, as in lane 1 plus 600 µM Ie, 2 µM dGTP; lane 15, as in lane 1 plus 600 µM Ie, 2 µM dGTP, 2 µM dATP; lanes 16-18, as in lane 1 plus 20 µM (lane 16), 400 µM (lane 17), 800 µM (lane 18) IIa; lane 19, as in lane 1 plus 800 µM IIa, 2 µM dGTP; lane 20, as lane 1 plus 800 µM IIa, 2 µM dGTP, 2 µM dATP.


Figure 5. Primer extention catalyzed by AMV RT in the presence of If and IIb. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane 4, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane 5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 6-8, as in lane 1 plus 2 µM (lane 6), 20 µM (lane 7), 200 µM (lane 8) If; lane 9, as in lane 1 plus 200 µM If, 2 µM dGTP; lane 10, as in lane 1 plus 200 µM If, 2 µM dGTP, 2 µM dATP; lanes 11-13, as in lane 1 plus 20 µM (lane 11), 200 µM (lane 12), 800 µM (lane 13) IIb; lane 14, as in lane 1 plus 800 µM IIb, 2 µM dGTP; lane 15, as in lane 1 plus 800 µM IIb, 2 µM dGTP, 2 µM dATP.

It is evident from Figures 4 and 5 that the compounds, substituted at both, [gamma]-phosphate and the uracil C5 atom (Ie,f) were efficiently utilized by AMV RT, but their substrate affinity was 1-2 orders of magnitude lower than that for the analogues with the only substituent (at the [gamma]- or C5 position). On the contrary, C5-subsituted [beta],[gamma]-dibromomethylenediphosphonates IIa-e displayed low or no substrate activity even toward AMV RT (Figs 4 and 5).

As it is seen from Figures 2 and 7, compounds IIIa,b showed substrate properties towards AMV RT, although they were ~50-100 times less effective than dTTP. HIV RT extended primer in the presence of Ic-e and IIIa,b ~10 times weaker as compared to that of AMV RT. This observation illustrates higher specificity of HIV RT to some modification of the triphosphate residue dNTP. At the same time IIIa practically had no substrate properties towards TDT.

Most striking is the change of the hydrophobicity in modified dNTP, expressed in an increse of their Rf at TLC (Table 1).

Table 1 demonstrates that all the compounds can be separated into two groups according to their Rf value: (i) Most hydrophilic dNTP (dUTP, dTTP, AZTTP) which have Rf in the range of 0.04-0.06 in system 1. It should be mentioned that the uracil modification at the C5 position mainly does not significantly increase the Rf value of the compound (usually 1.5-3-fold as compared with dUTP; examples are Ig and IIa-c); moreover, in the case of IIb, as well as for dUTP[5CH2O(CH2)6NH2] or dUTP(5CH = CHCH2NH2) the Rf is even decreased. (ii) Substitution of a [gamma]-phosphate by a [gamma]-phosphonate with a hydrophobic residue sharply increases the Rf, thus getting the latter rather close to those of nucleoside monophosphates (Ia, Ic, Id and Ie as examples). These results are in agreement with our previous observation (2).

So, substitution of [alpha]-, [beta]- and [gamma]-phosphate groups in dNTP by phosphonate ones significantly change their substrate properties towards different DNA polymerases and RTs although the majority of our compounds keep a definite level of their substrate activities with respect to any of enzymes.


Figure 6. Primer extention catalyzed by AMV RT in the presence of IIf. Lane 1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane4, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 6-9, as in lane 1 plus 2 µM (lane 6), 10 µM (lane 7), 50 µM (lane 8), 150 µM(lane 9) IIf; lane 10, as in lane 1 plus 150 µM IIf, 2 µM dGTP; lane 11, as in lane 1 plus 150 µM IIf, 2 µM dGTP, 2 µM dATP.

One can summarize such properties as follows: (i) [gamma]-Substituted dTTP (Ia-d) effectively elongate the DNA chain when catalyzed by AMV RT, but they are poorly recognized by DNA polymerase [alpha]. (ii) When substitution of the [gamma]-phosphate and uracil C5 of dUTP were done simultaneously (Ie,f), the substrate properties of such compounds were decreased by 1-2 orders of magnitude as compared with both of their prototypes containing the substitution only at the [gamma]-phosphate or at the uracil C5. (iii) C5-uracil-substituted [beta],[gamma]-dibromomethylenediphosphonates (IIa-e) are either poorly active or completely inactive even for the least specific enzyme, AMV RT. (iv) 2'-Deoxythymidine 5'-[[beta],[gamma]-(methylphosphinyl)methylphosphonyl]-[alpha]-phosphate (IIIa) and its 3'-azido-analog (IIIb) are shown to be AMR RT substrates, although they are ~50-100 times less effective than dTTP. Primer extension catalyzed by HIV RT in the presence of IIIa,b was by 1 order of magnitude weaker. At the same time they were practically not substrates for TDT and human DNA polymerases.


Figure 7. Primer extention catalyzed by TDT (Series A) and by AMV RT (Series B) in the presence of IIIa. (A). Lane 1, primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lanes 3-6, as in lane 1 plus 2 µM (lane 3), 10 µM (lane 4), 20 µM (lane 5), 100 µM (lane 6) IIIa. (B) lane1, template-primer plus enzyme; lane 2, as in lane 1 plus 2 µM dTTP; lane 3, as in lane 1 plus 2 µM dTTP, 2 µM dGTP; lane 4, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP; lane 5, as in lane 1 plus 2 µM dTTP, 2 µM dGTP, 2 µM dATP, 2 µM dCTP; lanes 6-9, as in lane 1 plus 2 µM (lane 6), 10 µM (lane 7), 20 µM (lane 8), 100 µM(lane 9) IIIa; lane 10, as in lane 1 plus 100 µM IIIa, 2 µM dGTP; lane 11, as in lane 1 plus 100 µM IIIa, 2 µM dGTP, 2 µM dATP; lane 12, as in lane 1 plus 100 µM IIIa, 2 µM dGTP, 2 µM dATP, 2 µM dCTP.

MATERIALS AND METHODS

Diethyl 2-azidoethylphosphonate (VIIa) and diethyl 2-aminoethylphosphonate (VIIb) were prepared according to (12), (methylphosphinyl)methylphosphonate as in (13). Dibromomethylenediphosphonic acid was prepared as in (16). Chemicals were used: DEAE-Toyopearl 650M (Toyo Soda); silica gel 60 (63-100 µm), DTT, LiChroprep RP-18 (25-40 µm) and LiChroprep RP-8 (40-63 µm) (Merck); trimethylbromosilane, CDI and Dowex 50W (Fluka).

Table 1. TLC of synthesized compounds, Rf in different systems (v/v) 0.00
Compound Dioxane-25%
NH4OH-water 4:1:2		 i-PrOH-25%
NH4OH-water 7:1:2
Ia 0.41 0.27
Ib, IIc, IIe 0.14 0.00
Ic 0.43 0.23
Id 0.47 0.21
Ie 0.38 0.32
If 0.29 0.11
Ig, IIa 0.08 0.00
IIb 0.04 0.00
IId 0.22 0.06
IIIa 0.22 0.14
IIIb 0.29 0.19
dUTP, dTTP, AZTTP 0.04 0.00
dUTP(5CH=CHCH2NH2) 0.02 0.00
dUTP[5CH2O(CH2)6NH2] 0.02 0.00
dUTP[5CH2O(CH2)6NHCO(CH2)5NHBio] 0.15
dUMP[5CH2O(CH2)6N3] 0.48 0.35
dTMP 0.40 0.21
AZTMP 0.56 0.35

1H-NMR spectra were recorded on a Bruker WP-200 SY spectrometer (USA), at 200.13 MHz, 31P-NMR spectra were recorded at 81 MHz with 1H-decoupling in D2O with 85% H3PO4 as external standard. Chemical shifts ([delta]) are reported in p.p.m. and coupling constants (J) in Hz. Mass spectra were obtained in FAB regime and were made on a Kratos MS 50TC spectrometer, samples were mixed with glycerol. Absorption spectra were measured with a Karl-Zeiss instrument Specord UV-VIS M40.

DNA and enzymes

M13mp10 DNA was isolated from the culture medium of the recipient Escherichia coli K12XL1 strain according to (17). The tetradecadeoxynucleotide primer (see Scheme 5) was labeled at the 5'-terminus using [[gamma]-32P]ATP (Radioisotop, Russia) and T4 polynucleotide kinase (Amersham) as described in (18). The DNA (0.5 µM) was hybridized with 0.75 µM 5'-32P-labeled primer in the following buffers: 10 mM Tris-HCl (pH 8.2), 5 mM MgCl2, 40 mM KCl and 1 mM DTT (for RTs); 10 mM Tris-HCl (pH 7.4), 6 mM MgCl2 and 0.4 mM DTT (for DNA polymerase [alpha]).1
Scheme 5.

HIV RT was isolated according to (19). AMV RT was from Omutninsk Chemicals (Russia). DNA polymerase [alpha] was isolated from human placenta as described in (20). The primer extension assays were performed as described in (14). The reaction products were separated by electrophoresis in 20% polyacrylamide gel, and the gels obtained were radioautographed.

5-[(6-Hydroxyhexyl)oxymethyl]-3',5'-di-O-acetyl-2'-deoxyuridine (Vb). 1,6-Hexanediol (7.43 g, 63 mmol) was added to 5-bromomethyl-3',5'-di-O-acetyl-2'-deoxyuridine (Va) (1.03 g, 20 mmol) in DMF (15 ml). The reaction mixture was kept for 48 h at 37°C under an argon atmosphere, then the solvent was evaporated, the residue was dissolved in ethyl acetate (30 ml), washed with water (5 × 10 ml) and dried with Na2SO4. The solvent was removedand the product was purified by flash chromatography on a Silica gel column (2 × 5 cm) in ethyl acetate to yield 3.64 g (65%) as a white solid. Analytical sample was purified on a Silica gel plate (10 × 8 × 0.4 cm) in system chloroform-MeOH, 20:1 (v/v).

1H-NMR (CDCl3 + CD3OD): 7.54 (1H, s, H-6), 6.38 (1H, dd, J1',2' 6 and 8, H-1'), 5.41 (1H, m, H-3'), 4.65 (2H, s, 5-CH2), 4.52 (1H, m, H-4'), 4.06 (2H, m, H-5'), 3.67 (2H, t, J 6, CH2OH), 3.35 (2H, t, J 6, OCH2C5H10), 2.65 (1H, m, H-2'a), 2.35 (1H, m, H-2'b), 2.17, 2.12 (6H, two s, 3'-OAc and 5'-OAc), 1.34 [8H, m, (CH2)4]. Mass: m/z: 442[M+ + 1], 465[M+ + 1 + Na].

5-{[6-(Methanesulfonyloxy)hexyl]oxymethyl}-3',5'-di-O-acetyl-2'-deoxyuridine (Vc). The nucleoside Vb (3.55 g, 8 mmol) in Py (10 ml) was cooled to -5°C and methanesulfonyl chloride (1.03 g, 9 mmol) was added, the reaction was carried out for 20 h at 20°C, and then quenched with ice water (1 ml). The solvents were evaporated and the residue was dissolved in water (10 ml) and chloroform (20 ml). The organic layer was washed with NaHCO3 (10 ml) and water (10 ml) and then dried with Na2SO4. The solvent was removedand the product was purified by flash chromatography on a silica gel column (2 × 3 cm) in ethyl acetate to yield 3.95 g (95%) as a white solid. An analytical sample was purified on a silica gel plate (10 × 8 × 0.4 cm) in chloroform-MeOH, 20:1 (v/v).

1H-NMR (CDCl3): 8.91 (1H, s, 3-NH), 7.54 (1H, s, H-6), 6.38 (1H, dd, J1',2' 6 and 8, H-1'), 5.41 (1H, m, H-3'), 4.65 (2H, s, 5-CH2), 4.52 (1H, s, H-4'), 4.25 (2H, s, J 6, CH2OMs), 4.06 (2H, m, H-5'), 3.35 (2H, t, J 6, OCH2C5H10), 3.24 (3H, s, OMs), 2.65 (1H, m, H-2'a), 2.35 (1H, m, H-2'b), 2.17, 2.12 (6H, two s, 3'-OAc and 5'-OAc), 1.34 [8H, m, (CH2)4]. Mass: m/z: 520 [M+].

5-[(6-Azidohexyl)oxymethyl]-3',5'-di-O-acetyl-2'-deoxyuridine (Vd). Sodium azide (2.6 g, 40 mmol) was added to Vc (3.9 g, 7.5 mmol) in DMF (10 ml) and acetonitrile (2 ml), and the mixture was refluxed for 5 h. The solvent was evaporated and the residue was dissolved in water (10 ml) and chloroform (20 ml). The organic layer was washed with water (2 × 5 ml) and dried with Na2SO4. The solvent was removed, and the product was purified by flash chromatography on a silica gel column (2 × 4 cm) in ethyl acetate to yield 2.63 g (75%) as a white solid.

1H-NMR (CDCl3): 8.67 (1H, s, 3-NH), 7.54 (1H, s, H-6), 6.35 (1H, dd, J1',2' 6 and 8, H-1'), 5.41 (1H, m, H-3'), 4.63 (2H, s, 5-CH2), 4.35 (1H, m, H-4'), 4.11 (2H, m, H-5'), 3.39 (2H, t, J 6, OCH2C5H10), 3.28 (2H, t, J 7, CH2N3), 2.65 (1H, m, H-2'a), 2.35 (1H, m, H-2'b), 2.17, 2.12 (6H, two s, 3'-OAc and 5'-OAc), 1.58, 1.36 [8H, two m, (CH2)4]. Mass: m/z: 466[M+], 489[M+ + Na]. IR: 2130 cm-1 (N3).

5-[(6-Azidohexyl)oxymethyl]-2'-deoxyuridine (IV). Aqueous ammonia (25%, 10 ml) was added to the solution of Vd (1.4 g, 3 mmol) in MeOH (2 ml); the reaction mixture was kept for 4 h at 20°C. The solvent was evaporated and the compound was purified on a silica gel column (2 × 26 cm) using a linear gradient of MeOH in chloroform (0-20%) to yield 0.98 g (85%).

UV: [lambda]max 265 nm ([epsilon] 9800). 1H-NMR (D2O): 7.82 (1H, s, H-6), 6.12 (1H, t, J1',2' 6, H-1'), 4.37 (1H, m, H-3'), 4.07 (2H, s, 5-CH2), 3.99 (1H, m, H-4'), 3.86 (2H, m, H-5'), 3.31 (2H, t, J 6, OCH2C5H10), 3.10 (2H, t, J 7, CH2N3), 2.17 (2H, m, H-2'), 1.42, 1.17 [8H, two m, (CH2)4]. Mass: m/z: 383[M+ + 1], IR: 2130 cm-1 (N3).

5-[(6-Azidohexyl)oxymethyl]-2'-deoxyuridine 5'-phosphate (VI). POCl3 (140 µl, 1.5 mmol) was dropped to the precooled (-10°C)solution of IV (381 mg, 1 mmol) in triethylphosphate (2 ml), the reaction was kept at +4°C for 20 h, then 0.2 M NaHCO3 (5 ml) was added. After 18 h at +4°C the mixture was washed with chloroform (2 ml), diluted with water (150 ml) and applied onto a DEAE-Toyopearl column (2.5 × 22 cm), elution was performed with a linear gradient of aqueous NH4HCO3 (0-0.2 M, 1.4 l). The fractions containing the product were evaporated and co-evaporated with water (3 × 10 ml). The residue was purified on a LiChroprep RP-18 column (1 × 15 cm), elution was performed with water to yield 268 mg (54%).

UV: [lambda]max = 265 nm ([epsilon] 9800). 1H-NMR (D2O): 7.82 (1H, s, H-6), 6.12 (1H, t, J1',2' 6, H-1'), 4.37 (1H, m, H-3'), 4.07 (2H, s, 5-CH2), 3.99 (1H, m, H-4'), 3.86 (2H, m, H-5'), 3.31 (2H, t, J 6, OCH2C5H10), 3.10 (2H, t, J 7, CH2N3), 2.17 (2H, m, H-2'), 1.42, 1.17 [8H, 2 m, (CH2)4]. 31P-NMR: 1.67 (s). Mass: m/z: 462 [M+]: 463 [M+ + 1], 486 [M+ + 1 + Na].

Synthesis of modified dNTP, method A

CDI (2-4 mmol) was added to the solution of tri-n-butylammonium phosphonate (0.5 mmol). The reaction mixture was stirred at 20°C for 18 h and MeOH (0.82 ml, 20 mmol) was added. After 40 min a solution of bis-(tri-n-butylammonium) 2'-deoxythymidine 5'-diphosphate (0.16 mmol) in DMF (2 ml) was added under stirring. After 48 h at 20°C water (40 ml) was added and the solution was applied onto a DEAE-Toyopearl column (3 × 10 cm). Elution was made with a linear gradient of NH4HCO3 (0-0.3 M, 1 l). The target fractions were evaporated, coevaporated with 50% water ethanol, dissolved in water and freeze-dried.

2'-Deoxythymidine 5'-[[gamma]-(2-azidoethylphosphonyl)-[alpha],[beta]-diphosphate] (Ia) was synthesized from VIIIa, to yield 18 mg (21%).1H-NMR (D2O): 7.63 (1H, q, J6,5-Me 1, H-6), 6.22 (1H, t, J1',2', 7, H-1'), 4.51 (1H, m, H-3'), 4.06 (3H, m, H-4', 5'), 3.42 (2H, m, CH2N3), 2.24 (2H, m, H-2'), 1.99 (2H, m, JCH2,P 19, CH2P), 1.80 (3H, d, 5-CH3). 31P-NMR: 14.7 (d, JP[gamma],P[beta] 24, P[gamma]), -10.6 (d, JP[alpha],P[beta] 20, P[alpha]), -22.2 (dd, P[beta]).

2'-Deoxythymidine 5'-{[gamma]-[2-N-(2,4-dinitrophenyl)aminoethylphosphonyl]-[alpha],[beta]-diphosphate} (Ic) was synthesized from VIIIc, to yield 13 mg (12%). 1H-NMR (D2O): 8.87 [1H,d, J3,5 3, H-3 (DNP)], 8.08 [1H, dd, J5,6 10, H-5 (DNP)], 7.44 [1H, s, H-6 (Thy)], 6.98 [1H, d, H-6 (DNP)], 5.93 (1H, t, J1',2' 7, H-1'), 4.37 (1H, m, H-3'), 4.04 (3H, m, H-4', 5'), 3.58 (2H, m, CH2N), 2.04 (4H, m, H-2',CH2P), 1.62 (3H, d, 5-CH3). 31P-NMR: 14.8 (m, P[gamma]), -10.7 (m, P[alpha]), -22.2 (m, P[beta]).

2'-Deoxythymidine 5'-[[gamma]-{2-N-[6-N-(2,4-dinitrophenyl)aminohexanoyl]aminoethylphosphonyl}-[alpha],[beta]-diphosphate] (Id) was synthesized from VIIId, to yield 20 mg (16%). 1H-NMR (D2O): 8.92 [1H, d, J3,5 3, H-3, (DNP)], 8.11 [1H, dd, J5,6 10, H-5 (DNP)], 7.55 [1H, s, H-6 (Thy)], 6.98 [1H, d, H-6 (DNP)], 6.11 (1H, t, J1',2' 7, H-1'), 4.47 (1H, m, H-3'), 4.03 (3H, m, H-4', 5'), 3.35 (2H, t, J 7, CH2NH-DNP), 3.31 (2H, m, CH2NHCO), 2.17 (2H, m, H-2'), 2.12 (2H, t, J 7, CH2CO), 1.88 (2H, m, CH2P), 1.73 (3H, s, 5-CH3), 1.59, 1.51, 1.29 [6H, three m, (CH2)3]. 31P-NMR: 16.1 (d, JP[gamma],P[beta] 24, P[gamma]), -10.5 (d, JP[alpha],P[beta] 20, P[alpha]), -22.1 (dd, P[beta]).

Method B

CDI (66 mg, 0.4 mmol) was added tothe solution of tri-n-butylammonium 5-(6-azidohexyloxymethyl)-2'-deoxyuridine 5'-phosphate (VI) (60 µmol) in DMF (2 ml). The mixture was stirred at 20°C under an argon for 2 h, and MeOH (50 µl) was added. Stirring was continued for 40 min and a solution of bis(tri-n-butylammonium) pyrophophate analog (0.24 mmol) in DMF (1.2 ml) was added.The reaction mixture was stirred for 18 h at 20°C, diluted with water (50 ml) and applied onto a DEAE-Toyopearl column (2.5 × 23 cm). The column was washed with 5% MeOH, then compounds were eluted by a linear gradient of NH4HCO3 (0-0.35 M, 1 l) in 5% MeOH. The appropriate fractions were evaporated, co-evaporated with water (5 × 10 ml), and the residue was purified on a LiChroprep RP-18 column (1 × 15 cm); elution was made with water. The resulted solution was freeze-dried.

5-[(6-Azidohexyl)oxymethyl]-2'-deoxyuridine 5'-[[gamma]-(phenylphosphonyl)-[alpha],[beta]-diphosphate] (Ie) was synthesized from tri-n-butylammonium phenylphosphonylphosphate according to the method B, to yield 16 mg (38%).UV: [lambda]max 265 nm ([epsilon] 9800). 1H-NMR (D2O): 7.81 (1H, s, H-6), 7.70 (2H, m, Ph), 7.27 (3H, m, Ph), 6.05 (1H, t, J1',2' 6, H-1'), 4.23 (1H, m, H-3'), 4.05 (2H, s, 5-CH2), 3.85 (3H, m, H-4', 5'), 3.31 (2H, t, J 6, OCH2C5H10), 3.10 (2H, t, J 7, CH2N3), 2.24 (1H, m, H-2'a), 2.08 (1H, m, H-2'b), 1.37, 1.15 [8H, two m, (CH2)4]. 31P-NMR: 6.60 (d, JP[gamma],P[beta] 19, P[gamma]), -10.5 (d, JP[alpha],P[beta] 26, P[alpha]), -22.2 (dd, P[beta]).

5-[(6-Azidohexyl)oxymethyl]-2'-deoxyuridine 5'-triphosphate (Ig) was synthesized from bis-(tri-n-butylammonium) pyrophosphate according to the method B, to yield 11 mg (53%).UV: [lambda]max 265 nm ([epsilon] 9800). 1H-NMR (D2O): 7.82 (1H, s, H-6), 6.12 (1H, t, J1',2' 6, H-1'), 4.37 (1H, m, H-3'), 4.07 (2H, s, 5-CH2), 3.99 (1H, m, H-4'), 3.86 (2H, m, H-5'), 3.31 (2H, t, J 6, OCH2C5H10), 3.10 (2H, t, J 7, CH2N3), 2.17 (2H, m, H-2'), 1.42, 1.17 [8H, two m, (CH2)4]. 31P-NMR: -9.6 (d, JP[alpha],P[beta] 19, P[alpha]), -10.6 (d, JP[gamma],P[beta] 20, P[gamma]), -22.2 (dd, P[beta]). Mass: m/z: 624 [M+ + 1], 641 [M+ + 1 + Na].

5-[(6-Azidohexyl)oxymethyl]-2'-deoxyuridine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IIa) was synthesized from bis-(tri-n-butylammonium) dibromomethylenediphosphonate according to the method B, to yield 40 mg (51%).UV: [lambda]max 265 nm ([epsilon] 9800). 1H-NMR (D2O): 7.84 (1H, s, H-6), 6.11 (1H, t, J1',2' 6, H-1'), 4.45 (1H, m, H-3'), 4.12 (2H, s, 5-CH2), 3.99 (3H, m, H-4', H-5'), 3.33 (2H, t, J 6, OCH2C5H10), 3.17 (2H, t, J 7, CH2N3), 2.17 (2H, m, H-2'), 1.41, 1.17 [8H, two m, (CH2)4]. 31P-NMR: 8.5 (d, JP[gamma],P[beta] 15, P[gamma]), 0.3 (dd, JP[beta],P[alpha] 28, P[beta]), -10,1 (d, P[alpha]).

General procedure for the reduction of an azido-group in modified dNTP, method C

DTT (25mg, 160 µmol) was added to a solution of the dNTP (Ia, Ie or IIa) (20 µmol) in water (0.5 ml), 25% aqueous ammonia (0.2 ml) was dropped under argon and the reaction mixture was kept for 18 h at +4°C. The solution was diluted with water (50 ml) and applied onto a DEAE-Toyopearl column (2 × 12.5 cm), washed with water (200 ml). Substances were eluted with a linear gradient of NH4HCO3 (0-0.3 M, 1 l). Fractions containing Ib, If and IIb were evaporated, coevaporated with water (5 × 5 ml), dissolved in water (0.2 ml) and applied onto a LiChroprep RP-18 column (1 × 15 cm). Elution was made by water and corresponding fractions were freeze-dried.

2'-Deoxythymidine 5'-[[gamma]-(2-aminoethylphosphonyl)-[alpha],[beta]-diphosphate] (Ib) was obtained using method C from Ia, to yield 5 mg (49%). 1H-NMR (D2O): 7.58 (1H, q, J6,5-Me 1, H-6), 6.19 (1H, t, J1',2' 7, H-1'), 4.48 (1H, m, H-3'), 4.04 (3H, m, H-4', 5'), 3.14 (2H, m, CH2N), 2.22 (2H, m, H-2'), 2.02 (2H, m, {italic J} sub {{{C H} sub 2} , P},P 19, CH2P), 1.78 (3H, d, 5-CH3). 31P-NMR: 12.9 (d, JP[gamma],P[beta] 23, P[beta]), -10.6 (d, JP[alpha],P[beta] 20, P[alpha]), -22.1 (dd, P[beta]).

5-[(6-Aminohexyl)oxymethyl]-2'-deoxythymidine 5'-[[gamma]-(phenylphosphonyl)-[alpha],[beta]-diphosphate] (If) was synthesized by method C from Ie, to yield 10 mg (72%). 1H-NMR (D2O): 7.87 (1H, s, H-6), 7.67 (2H, m, Ph), 7.24 (3H, m, Ph), 6.15 (1H, t, J1',2' 6, H-1'), 4.24 (1H, m, H-3'), 4.15 (1H, s, 5-CH2), 3.81 (3H, m, H-4', 5'), 3.31 (2H, t, J 6, OCH2C5H10), 2.77 (2H, t, J6, CH2N), 2.21 (2H, m, H-2'), 1.41, 1.15 [8H, two m, (CH2)4]. 31P-NMR: 6.7 (d, JP[gamma],P[beta] 19, P[gamma]), -10.8 (d, JP[alpha],P[beta] 25, P[alpha]), -22.1 (dd, P[beta]).

5-[(6-Aminohexyl)oxymethyl]-2'-deoxythymidine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IIb) was obtained according to method C from IIa, to yield 10 mg (67%).1H-NMR (D2O): 7.81 (1H, s, H-6), 6.14 (1H, t, J1',2' 6, H-1'), 4.49 (1H, m, H-3'), 4.12 (1H, s, 5-CH2), 4.04 (3H, m, H-4', 5'), 3.36 (2H, t, J 7, OCH2C5H10), 2.79 (2H, t, J 7, CH2N), 2.18 (2H, m, H-2'), 1.41, 1.17 [8H, two m, (CH2)4). 31P-NMR: 7.9 (d, JP[gamma],P[beta] 14, P[gamma]), 0.9 (dd, JP[beta],P[alpha] 28, P[beta]), -10,8 (d, P[alpha]).

2'-Deoxythymidine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IIf) was synthesized as in method B from bis-(tri-n-butylammonium) 2'-deoxythymidine 5'-phosphate and bis-(tri-n-butylammonium) dibromomethylenediphosphonate, to yield 18 mg (47%). 1H-NMR (D2O): 7.63 (1H, q, J6,5-Me 1, H-6), 6.23 (1H, t, J1',2' 7, H-1'), 4.56 (1H, m, H-3'), 4.11 (3H, m, H-4', 5'), 2.26 (2H, m, H-2'), 1.81 (3H, d, 5-CH3). 31P-NMR: 7.4 (d, JP[gamma],P[beta] 14, P[gamma]), -0.9 (dd, JP[beta],P[alpha] 24, P[beta]), -11.0 (d, P[alpha]).

Synthesis of the dNTP IIc-e

N-Methylimidazole (20 µl, 144 µmol) and Et3N (10 µl, 125 µmol) were added to a solution of IIb (5 mg, 6.5 µmol) in water (0.4 ml), and then the solution of N-succinimidyl N-biotinyl-6-amino-hexanoate (4.5 mg, 10 µmol) or N-succinimidyl tetramethylrhodamine carboxylate (5 mg, 8 µmol) or fluoreceinyl isothiocyanate (4 mg, 10 µmol) in DMF (0.4 ml) was added. The reaction mixture was stirred for 4 h at 20°C, then 4 M aqueous KCl (20 µl) and water (2 ml) were added. The mixture was extracted with n-butanol (2 × 1 ml), the aqueous solution was concentrated up to 0.2 ml and applied onto a LiChroprep RP-18 column (1 × 10 cm). Starting IIb was eluted with water, compounds IIc-e, by a linear gradient MeOH in water (0-20%). Fractions containing IIc-e were evaporated to dryness. The yields of IIc, IIe and IId were 6 mg (76%), 6 mg (89%) and 6 mg(77%), respectively.

5-[{6-N-[6-N-(Biotinyl)aminohexanoil]aminohexyl}oxymethyl]-2'-deoxythymidine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IIc). UV: [lambda]max 265 nm. 31P-NMR: 8.5 (d, JP[gamma],P[beta] 14, P[gamma]), 0.4 (dd, JP[beta],P[alpha] 28, P[beta]), -10.2 (d, P[alpha]).

5-{[6-N-(Fluoresceinylaminothiocarbonyl)aminohexyl]oxymethyl}-2'-deoxythymidine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IId). UV: [lambda]max 492 nm. 31P-NMR: 8.7 (d, JP[gamma],P[beta] 15, P[gamma]), 0.7 (dd, JP[beta],P[alpha] 29, P[beta]), -9.8 (d, P[alpha]).

5-{[6-N-[Tetramethylrhodaminylcarbonyl]aminohexyl]oxymethyl}-2'-deoxythymidine 5'-[[beta],[gamma]-(dibromomethylenediphosphonyl)-[alpha]-phosphate] (IIe). UV: [lambda]max 552 nm. 31P-NMR: 8.0 (d, JP[gamma],P[beta] 15, P[gamma]), 0.2 (dd, JP[beta],P[alpha] 29, P[beta]), -9.9 (d, P[alpha]).

2-Azidoethylphosphonic acid (VIIIa). Trimethylbromosilane (12.5 ml, 96.6 mmol) was added to the precooled (-5°C) solution of diethyl 2-azidoethylphosphonate (VIIa) (5 g, 24.2 mmol) in DMF (10 ml). The reaction mixture was stirred for 24 h at 20°C. The solvent was evaporated and co-evaporated with dry toluene (3 × 30 ml). The residue was dissolved in water (50 ml), stirred for 18 h at 20°C and then evaporated, coevaporated with water (3 × 30 ml), dissolved in water (5 ml) and purified on a LiChroprep RP-18 column (3.5 × 26 cm) with water elution, to yield 2.6 g (71%).

1H-NMR (D2O): 2.94 (2H, dt, {italic J} sub {{{C H} sub 2} , {{C H} sub 2}} 8, {italic J} sub {{{C H} sub 2} , P} 9, CH2N3), 1.31 (2H, dt, {italic J} sub {{{C H} sub 2} , P} 17, CH2P). 31P-NMR: 18.7 (s). Mass: m/z: 152 [M+ + 1].

2-Deoxythymidine 5'-{[beta],[gamma]-[(methylphosphinyl)methylphosphonyl]-[alpha]-phosphate} (IIIa). CDI (100 mg, 0.62 mmol) was added to a solution of bis-(tri-n-butylammonium) 2'-deoxythymidine 5'-phosphate (100 mg,0.31 mmol) in DMF (3 ml). The reaction mixture was stirred for 1 h at 20°C, then MeOH (150 µl, 3.7 mmol) was added. After 40 min of stirring at 20°C the solution of bis-(tri-n-butylammonium) (methylphosphinyl)methylphosphonate (54 mg, 0.31 mmol) in DMF (1.5 ml) was added. After 24 h of vigorous stirring the reaction mixture was diluted with water (70 ml) and applied onto a DEAE-Toyopearl column (3 × 10 cm). Elution was performed by a linear gradient of NH4HCO3 (0-0.2 M), fractions containing IIIa were evaporated, coevaporated with 50% water in ethanol, dissolved in water and freeze-dried to yield 29 mg (19%).

1H-NMR (D2O): 7.68 (1H, q, J6,5-ME 1, H-6), 6.29 (1H, t, J1',2' 7, H-1'), 4.56 (1H, m, H-3'), 4.10 (3H, m, H-4', 5'), 2.28 (2H, m, H-2'), 2.18 (2H, t, JCH2,P 19, PCH2P), 1.84 (3H, d, 5-CH3), 1.34 (2H, dd, {italic J} sub {{{C H} sub 3} , P beta} 2, {italic J} sub {{{C H} sub 3} , P gamma} 14, CH3P). 31P-NMR: 34.7 (s, JP[gamma],P[beta] 0, P[gamma]), 8.5 (d, JP[beta],P[alpha] 26, P[beta]), -11.3 (d, P[alpha]). Mass: m/z: 479 [M+ + 1].

3'-Azido-2',3'-dideoxythymidine 5'-{[beta],[gamma]-[(methylphosphinyl) methylphosphonyl]-[alpha]-phosphate} (IIIb). The solution of Et3N (140 µl, 1 mmol) and 1,2,4-triazole (70 mg, 1 mmol) in CH3CN (1 ml) was cooled to 0°C and POCl3 (32 µl, 0.34 mmol) was added. After stirring for 40 min at 20°C the precipitate was removed by centrifugation. The supernatant was dropped to AZT (60 mg, 0.23 mmol) in CH3CN (1 ml); after 40 min at 20°C the solution of bis-(tri-n-butylammonium) (methylphosphinyl)methylphosphonate (60 mg, 0.34 mmol) in DMF (2 ml) was slowly added. After 18 h of vigorous stirring the reaction mixture was diluted with water (70 ml) and applied onto a DEAE-Toyopearl column (3 × 10 cm). Elution was made by a linear gradient of NH4HCO3 (0-0.2 M), fractions containing IIIb were evaporated, coevaporated with 50% water ethanol, dissolved in water and freeze-dried, to yield 34 mg (30%).

1H-NMR (D2O): 7.54 (1H, q, J6,5-Me 1, H-6), 6.07 (1H, t, J1',2' 7, H-1'), 4.36 (1H, m, H-3'), 4.02 (3H, m, H-4', 5'), 2.28 (2H, m, H-2'), 2.12 (2H, dd, {italic J} sub {{{C H} sub 2} , P} 18 and 21, PCH2P), 1.72 (3H, d, 5-CH3), 1.28 (2H, d, {[iota][tau][alpha][lambda][iota][chi] [thetav]} [sigma][upsi][beta] {{{X H} [sigma][upsi][beta] 3} , [Pi] [gamma][alpha]µµ[alpha]} 15, CH3P). 31P-NMR: 38.0 (s, JP[gamma],P[beta] 0, P[gamma]), 7.4 (d, JP[beta]2,Pa 26, P[beta]), -11.5 (d, P[alpha]). Mass: m/z: 504 [M+ + 1].

2-N-(2,4-Dinitrophenyl)aminoethylphosphonic acid (VIIIc). To a solution of diethyl 2-aminoethylphosphonate (VIIb) (290 mg, 1.6 mmol) in CH3CN (3 ml) was added 2,4-dinitrofluorobenzene (221 µl, 1.76 mmol) and Et3N (223 µl, 1.6 mmol) at 37°C. After 3 h stirring the reaction mixture was cooled to 0°C and Me3SiBr (828 µl, 6.4 mmol) was added; after 18 h at 20°C the reaction mixture was evaporated, coevaporated with CH3CN (3 × 5 ml) and 10% aqueous ammonia (5 ml). The residue was dissolved in water (3 ml) and purified on a LiChroprep RP-8 column (2 × 25 cm) with water elution, to yield 360 mg (77%).

1H-NMR (D2O): 8.87 [1H, d, J3,5 3, H-3 (DNP)], 8.14 [1H, dd, J5,610, H-5 (DNP)], 7.01 [1H, d, H-6 (DNP)], 3.63 (2H, m, CH2N), 1.97 (2H, dt, {italic J} sub {{{C H} sub 2} , {{C H} sub 2}} 8, {italic J} sub {{{C H} sub 2} , P}, 17, CH2P). 31P-NMR: 19.5 (s).

2-N-[6-N-(2,4-Dinitrophenyl)aminohexanoyl]aminoethylphosphonic acid (VIIId). To a solution of diethyl 2-aminoethylphosphonate (VIIb) (90 mg, 0.5 mmol) and N-succinimidyl 6-N-(2,4-dinitrophenyl)aminohexanoate (160 mg, 0.41 mmol) in THF (3 ml) Et3N (56 µl, 0.41 mmol) was added. After 10 min the reaction mixture was concentrated up to 1 ml, cooled to 0°C and Me3SiBr (52 µl, 2.1 mmol) was added, the reaction mixture was stirred for 2 h at 20°C, evaporated, coevaporated with CH3CN (3 × 5 ml) and 10% aqueous ammonia (5 ml). The residue was dissolved in water (0.5 ml) and purified on a LiChroprep RP-8 column (1 × 8 cm) with a linear gradient of MeOH in water (0-5%) elution, to yield 143 mg (86%).

1H-NMR (D2O): 9.05 [1H, d, J3,5 3, H-3 (DNP)], 8.24 [1H, dd, J5,6 10, H-5 (DNP)], 7.11 [1H, d, H-6 (DNP)], 3.50 (1H, t, J 7, CH2NH-DNP), 3.35 (2H, m, CH2NHCO), 2.26 (2H, t, J 7, CH2CO), 1.90-1.42 [8H, m, CH2P, (CH2)3]. 31P-NMR: 18.6(s).

ACKNOWLEDGEMENTS

The study was supported by Russian Foundation of Basic Research, grants 95-03-08142a, 96-04-48277, 96-04-48278 and Outstanding Schools, grant 96-15-97646. The authors are grateful to Dr E.Shirokova for help in preparation of English version of the manuscript.

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*To whom correspondence should be addressed. Tel: +7 095 135 22 55; Fax: +7 095 135 14 05; Email: aak@imb.imb.ac.ru

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