Synthesis of Azidoaniline Derivatives of Oligonucleotides and

Synthesis of Azidoaniline Derivatives of Oligonucleotides and Investigation of Their Photochemical ... Attachment of Reporter and Conjugate Groups to ...
0 downloads 0 Views 271KB Size
Download PDF
Bioconjugate Chem. 1996, 7, 343−348

343

Synthesis of Azidoaniline Derivatives of Oligonucleotides and Investigation of Their Photochemical Behavior Tatyana S. Godovikova,*,† Vera D. Knorre,‡ Galiya A. Maksakova,† and Vladimir N. Sil’nikov† Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, 630090 Novosibirsk, Lavrentiev av. 8, Russia, and M. M. ShemyakinsYu. A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117871 Moscow, Mikluho-Maklaya av. 10/16, Russia. Received April 11, 1995X

A series of aryl azides, p-N3C6H4NH(CH2)nNH2 with n ) 2-6, have been synthesized and used to prepare oligonucleotide derivatives carrying photoreactive the p-azidoaniline residue. Reactive moieties have been coupled to the 5′-terminal phosphate of d(pGATACCAA) [compounds IV(b), IV(c), and IV(e) with n ) 3, 4, and 6, respectively] and of d(pGCC) [compound V(b) with n ) 3] via a phosphoamide bond. Irradiation at wavelengths over >300 nm of IV(b) and V(b) (n ) 3) resulted in cleavage of the P-N bond. However, under the same reaction conditions, the P-N bond remained intact for compounds containing longer spacers [IV(c) and IV(e)]. Intraduplex reaction of the latter derivatives with d(GGTATCp)NH(CH2)6NH2 resulted in cross-linking dependent on the presence of an aliphatic amino group. The results obtained have demonstrated that the azidoaniline derivatives of oligonucleotides capable of the affinity modification of a specific target can be prepared. However, the sufficiently long aliphatic spacer group is necessary to prevent P-N bond cleavage within the photoreactive oligonucleotide.

INTRODUCTION

phate which was subjected to irradiation within the complex:

Previously, derivatives of the nucleoside 5′-triphosphates (NTP)1 carrying a p-azidoanilide residue attached to the γ-phosphate were proposed and used for modification of a number of the NTP binding proteins (1-4). It has been shown that under irradiation p-azidoanilide derivatives can be converted to quinonediimine derivatives via isomerization of the intermediate nitrene (5, 6). The reagent formed turned out to be a strong electrophile capable of the efficient modification in aqueous solution of a number of compounds containing aliphatic amino groups, imidazole rings, and thiol residues (6, 7). Therefore, these derivatives may be considered as strong electrophiles capable of being switched on under irradiation. It seemed interesting to prepare similar derivatives of oligonucleotides for the photoaffinity modification of the nucleic acid binding proteins. However, with the [γ-32P]dTTP-p-azidoanilide, it was found that the modification of the Klenow fragment of DNA polymerase I was not accompanied by equivalent incorporation of 32P labeling of the protein (3). A similar result was recently obtained for the oligonucleotide derivative containing a p-azidoaniline residue directly bound to the 5′-terminal phos-

[5′-32P]d(pGpGpTpApTpCp)NHCH2CH2CH2NHCOCHRNH2 3′dApApCpCpApTpApG5′p-(p-NHC6H4N3)

where R is either the (CH2)4NH2 residue or CH2C6H5 (8). Although modification of the amino acid-carrying fragment of the target definitely took place under irradiation, the oligonucleotide moiety of the reagent was absent from the modification product. This indicated that the P-N bond cleavage took place. Therefore, prior to design of the oligonucleotide derivatives with the photoswitchable reactive group, on the basis of the properties of p-azidoaniline, it seemed reasonable to change the way of attachment of pazidoaniline to oligonucleotide. This could be achieved by introduction of the aliphatic spacer between p-azidoaniline and the 5′-terminal phosphate of the oligonucleotide. In the present paper, we describe the preparation of the derivatives containing the (CH2)nNH spacer separating the p-azidoaniline residue and the terminal phosphate of the oligonucleotide It has been found that the presence of the sufficiently long spacer (n > 3) permits retention of the P-N bond within the irradiated reagent. EXPERIMENTAL PROCEDURES

* To whom correspondence should be addressed at Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, 630090 Novosibirsk, Lavrentiev av. 8, Russia. Telephone: 007-3832-396274. Fax: 007-3832353459. E-mail: [email protected]. † Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences. ‡ M. M. ShemyakinsYu. A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. X Abstract published in Advance ACS Abstracts, April 15, 1996. 1 Abbreviations: NTP, nucleoside 5′-triphosphates; Ph P, 3 triphenylphosphine; (PyS)2, 2,2′-dipyridyl disulfide; DMAP, (N,N-dimethylamino)pyridine.

S1043-1802(96)00020-1 CCC: $12.00

General Methods. Melting points were recorded on an electrochemical capillary melting point apparatus and are reported uncorrected. 1H NMR spectra were recorded on a Bruker WP-200 SY (200 MHz) spectrometer. All 1 H chemical shifts are reported relative to tetramethylsilane (TMS) as an internal standard. 31P NMR spectra were recorded on a Bruker AC-200 (81.015 MHz) spectrometer, and the 31P chemical shifts are reported relative to H3PO4 as an external standard. Infrared spectra were recorded on a Specord M60 instrument (Carl Zeiss, Yena). Mass spectra and exact masses were obtained on a Finnigat-matt-2000. Absorption spectra were recorded © 1996 American Chemical Society

344 Bioconjugate Chem., Vol. 7, No. 3, 1996

on a Specord M40 instrument (Carl Zeiss, Yena) in 0.15 M NaCl and 0.02 M Na2HPO4 at pH 9.02. Molar extinction coefficients at 260 nm of unmodified oligonucleotides IV and V were estimated to be 89 900 and 30 600 M-1 cm-1 according to ref 9. The coefficients of modified oligonucleotides IV(b,c,e) and V(b) were estimated as a sum of the oligonucleotide and the aryl azide (λ ) 260 nm,  ) 11 000, pH 9) molar extinction coefficients. Oligonucleotides were prepared as described by ref 10. The aryl azide-oligonucleotide conjugates containing the amino spacer and an aliphatic amino group containing oligodeoxynucleotides were prepared as described in ref 11. To obtain 3′-phosphorylated oligonucleotides, a solid support containing [[2-[O-(4,4′-dimethoxytrityl)oxy]ethyl]sulfonyl]ethanol moiety was synthesized (12). Isolation and purification of the oligonucleotide derivatives were performed by reversed-phase high-performance liquid chromatography using a LiChrosorb RP18, 10 µm (Merck, FRG), 4.6 × 250 mm column, a Waters 600E chromatograph, and a Waters 484 tunable absorbance detector (U.S.A.). A linear gradient (2 mL/min) from 0 to 20% of solvent B (solvent A ) 0.05 M LiClO4, solvent B ) acetonitrile) was used. Analytical anion-exchange microcolumn chromatography was performed on the Milichrom chromatograph (Russia) using a Polisil-SA (Russia), 2.5 × 30 mm column. A linear gradient (flow rate ) 50 µL/min) from 0 to 1 M NaCl in 7 M urea and 0.01 M Na2HPO4 (pH 9) was employed. Materials. N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, and acetonitrile were purified and dried according to standard procedures. 4-(N,NDimethylamino)pyridine (DMAP) was purchased from Bergkamen (Berlin), and triphenylphosphine (Ph3P), 2,2′dipyridyl disulfide (PyS)2, and polymethylenediamines were obtained from Fluka AG (Switzerland). N-CetylN,N,N-trimethylammonium bromide and TLC aluminum sheets (silica gel 60 F254) were purchased from Merck (Germany). Synthesis of N-(p-Nitrophenyl) Derivatives of Diamines [I(a-e)]. A mixture of 0.01 mol of p-chloronitrobenzene and 0.1 mol respective diamine was heated in an oil bath at 135-140 °C for 1 h. The excess of diamine was evaporated under reduced pressure. The yellow liquid residue was dissolved in boiling 10% hydrochloric acid and filtered to remove any insoluble material (bis derivative). The solution was adjusted to pH 10-11 with concentrated sodium hydroxide, and the precipitate was filtered. The resulting solid was washed with 20 mL of water and crystallized from 35-50 mL of ethanol/water (1:2, v:v). The yield of N-(p-nitrophenyl)-1,2-diaminoethane [I(a)] was 74%, mp 140-141 °C [lit. 139-141 °C (13)]. The yield of N-(p-nitrophenyl)-1,3-diaminopropane [I(b)] was 81%, mp 112-113 °C [lit. 111 °C (14)]. The yield of N-(p-nitrophenyl)-1,4-diaminobutane [I(c)] was 72%, mp 103.2-103.7 °C [lit. 101 °C (15)]. The yield of N-(p-nitrophenyl)diaminopentane [I(d)] was 48%, mp 102.2-102.5 °C [lit. 97 °C (16)]. The yield of N-(p-nitrophenyl)-1,6-diaminohexane [I(e)] was 86%, mp 98.5-99.0 °C. Anal. Found: C, 60.8; H, 8.07; N, 17.7. Calcd for C12H19N3O2: C, 60.7; H, 8.15; N, 17.7. Synthesis of N-(p-Azidophenyl) Derivatives of the Protected Diamines [II(a-e)] (Scheme 1). A mixture of the N-(p-nitrophenyl) derivative of diamine (1 mmol), trifluoroacetic anhydride (3 mmol), and N-ethyldiisopropylamine (3 mmol) in 10 mL of dry chloroform was stirred for 30 min at room temperature. The solvent and the excess of N-ethyldiisopropylamine were evaporated under

Godovikova et al.

Figure 1. Reversed-phase chromatography of the reaction mixture for synthesis of p-azidoaniline derivatives of oligonucleotide IV(b).

reduced pressure. The resulting residue was washed with 1 N hydrochloric acid. The reaction progress was monitored by TLC [CHCl3/MeOH, 10:1, v:v]. The solid residue was dissolved in 10 mL of ethanol, and compounds were catalytically hydrogenated for 2 h at room temperature using 0.1 g of 10% Pd/C. The reaction mixture was filtered, the solvent was removed by evaporation, and the residue was dissolved in 20 mL of 2 N H2SO4. The reaction mixture was cooled to 0 °C, and 1 mmol of sodium nitrite in 1 mL of water was added. The mixture was stirred for 30 min at 0 °C, and 3 mmol of sodium azide was added in the dark. After 1 h, compounds II(a-e) were extracted with chloroform and the solvent was removed. Compounds II(a,c) were crystallized from 10-30 mL of benzene/hexane (1:5, v:v). The yield of N,N′-bis(trifluoroacetyl)-N-(p-azidophenyl)1,2-diaminoethane [II(a)] was 67%: mp 87-90 °C dec; Rf 0.48-0.52 (ethanol/CHCl3, 1:10, v:v; UV (ethanol) λmax 254 nm ( ) 14 200); IR (CHCl3) 2128-2104 d.b. (N3), 1728 and 1696 cm-1 (CdO); 1H NMR (CDCl3) δ 3.64 (dd, 2H, J ) 6 Hz), 3.95 (t, 2H, J ) 6 Hz), 7.08 (d, 2H, J ) 9 Hz), 7.23 (d, 2H, J ) 9 Hz); mass spectrum m/z (( 1 × 10-4) found 369.0660, calcd for C12H9F6N5O6 369.0660. The yield of N,N′-bis(trifluoroacetyl)-N-(p-azidophenyl)1,3-diaminopropane [II(b)] was 54%: Rf 0.52-0.56 (ethanol/CHCl3, 1:10, v:v; UV (ethanol) λmax 254 nm ( ) 14 000); IR (CHCl3) 2128-2104 d.b. (N3), 1724 and 1688 cm-1 (CdO); 1H NMR (CDCl3) δ 1.83 (m, 2H), 3.47 (dd, 2H, J ) 6, 5 Hz), 3.90 (t, 2H, J ) 6, 5 Hz), 7.06 (d, 2H, J ) 9 Hz), 7.21 (d, 2H, J ) 9 Hz); mass spectrum m/z (( 1 × 10-4) found 383.0817, calcd for C13H11F6N5O2 383.0817. The yield of N,N′-bis(trifluoroacetyl)-N-(p-azidophenyl)1,4-diaminobutane [II(c)] was 58%: mp 80-82 °C; Rf 0.58-0.62 (ethanol/CHCl3, 1:10, v:v; UV (ethanol) λmax 254 nm ( ) 14 000); IR (CHCl3) 2128-2104 d.b. (N3), 1728 and 1696 cm-1 (CdO); 1H NMR (CDCl3) δ 1.61 (m, 4H), 3.39 (dd, 2H, J ) 7 Hz), 3.72 (t, 2H, J ) 7 Hz), 7.04 (d, 2H, J ) 9 Hz), 7.17 (d, 2H, J ) 9 Hz); mass spectrum m/z (( 1 × 10-4) found 397.0972, calcd for C14H13F6N5O2 397.0973. The yield of N,N′-bis(trifluoroacetyl)-N-(p-azidophenyl)1,5-diaminopentane [II(d)] was 61%: Rf 0.61-0.63 (etha-

Synthesis of Azidoaniline Derivatives

Bioconjugate Chem., Vol. 7, No. 3, 1996 345

Scheme 1

Scheme 2

Figure 2. Profile of analytical anion-exchange microcolumn chromatography of compounds IV(b) (A) and d(pGATACCAA) (B) and of the reaction mixture for photolysis of p-azidoaniline derivatives of oligonucleotides IV(b) (C) and IV(e) (D).

nol/CHCl3, 1:10, v:v); UV (ethanol) λmax 254 nm ( ) 14 000); IR (CHCl3) 2128-2104 d.b. (N3), 1726 and 1696 cm-1 (CdO); 1H NMR (CDCl3) δ 1.45 (m, 4H), 1.63 (m, 2H), 3.33 (dd, 2H, J ) 7 Hz), 3.70 (t, 2H, J ) 7 Hz), 7.05 (d, 2H, J ) 9 Hz), 7.17 (d, 2H, J ) 9 Hz); mass spectrum m/z (( 1 × 10-4) found 411.1129, calcd for C15H15F6N5O2 411.1130. The yield of N,N′-bis(trifluoroacetyl)-N-(p-azidophenyl)1,6-diaminohexane [II(e)] was 68%: Rf 0.61-0.63 (ethanol/CHCl3, 1:10, v:v; UV (ethanol) λmax 254 nm ( ) 14 000); IR (CHCl3) 2128-2104 d.b. (N3), 1728 and 1698 cm-1 (CdO); 1H NMR (CDCl3) δ 1.34 (m, 4H), 1.56 (m, 4H), 3.33 (dd, 2H, J ) 7 Hz), 3.69 (t, 2H, J ) 7 Hz), 7.05 (d, 2H, J ) 9 Hz), 7.16 (d, 2H, J ) 9 Hz); mass spectrum m/z (( 1 × 10-4) found 425.1284, calcd for C16H17F6N5O2 425.1284. Synthesis of N-(p-Azidophenyl) Derivatives of Diamines [III(a-e)]. Each of the protected derivatives III(a-e) (0.1 mmol) were dissolved in 5 mL of ethanol in the dark. Five milliliters of 1 N sodium hydroxide was added, and the mixture was stirred for 15 min at room temperature. The excess of ethanol was evaporated under reduced pressure, and the products III(a-e) were crystallized from water or were extracted with ethyl acetate, followed by subsequent removal of the solvent: UV (ethanol/water, 1:2, v:v, pH 8) λmax 274 nm ( ) 16 000); IR (CHCl3) 2112 cm-1 (N3). Synthesis of Aryl Azide Oligonucleotide Derivatives [IV(b,c,e) and V(b)]. The reaction mixture consisting of 0.05 µmol of the N-cetyl-N,N,N-trimethylammonium salt of the oligonucleotide, 15 µmol of Ph3P,

15 µmol of (PyS)2, and 30 µmol of DMAP in 0.1 mL of DMF was incubated for 12 min at room temperature. The 4-(N,N-dimethylamino)pyridine derivative of the oligonucleotide was precipitated with 2% LiClO4 in acetone. Each aryl azide III(b,c,e) (300 µmol) in 50 µL of 50% aqueous DMF was added to the derivatized oligonucleotide precipitate, and the solution was incubated in the dark for 12 h. The aryl azide oligodeoxyribonucleotide derivatives formed were isolated by reversed-phase highperformance liquid chromatography (Figure 1). The retention times for compounds IV(b,c,e) were found to be 20-25 min. Products IV(b,c,e) were precipitated by 2% LiClO4 in acetone. The yield was 70-90%. Studies of the Photochemical Behavior of Aryl Azide Oligodeoxyribonucleotide Derivatives. A 5 × 10-5 M solution (20 µL) of photoreagent V(b), IV(b), IV(c), or IV(e) in 0.15 M NaCl and 0.02 M Na2HPO4 (pH 9) were pipetted into a 96-well polystyrene plate at 4 °C. Irradiation was carried out by exposing the plate for 5 min to filtered light (303-365 nm) emitted by a highpressure mercury lamp with an intensity of 1015 quanta cm-2 s-1. Photoreagents IV(c) and IV(e) were also combined with 5 × 10-5 M complementary aliphatic amino containing oligodeoxynucleotides. Following irradiation, the reaction mixtures were subjected to ionexchange chromatography. RESULTS AND DISCUSSION

(A) Synthesis of Aryl Azides. Syntheses of azides were accomplished as shown in Scheme 1. Compounds I(a-c) were obtained using the method described earlier for compound I(b) (15), with yields ranging from 48 to 86%. The reaction of I(a-e) with trifluoroacetic anhydride in dry CHCl3 and in the presence of N-ethyldiiso-

346 Bioconjugate Chem., Vol. 7, No. 3, 1996

Godovikova et al.

Figure 4. Profile of analytical anion-exchange microcolumn chromatography of the compound d(pGATACCAA) (A), of the duplex IV(e)‚d(GGTATCp)NH(CH2)6NH2 without irradiation (B), and of the reaction mixture of irradiated duplex IV(e)‚ d(GGTATCp)NH(CH2)6NH2 (C). Concentrations of the reagents were 5 × 10-5 M. Reaction mixture was irradiated and incubated at 4 °C for 12 h in the dark prior to analysis.

Figure 3. 31P NMR spectra in 0.15 M NaCl (pH 9) of 5 × 10-3 M d(pGCC) (A) and V(b) before (B) and after irradiation (C). Scheme 3

propylamine leads to the formation of the corresponding protected diamines. The reduction of the NO2 group and subsequent conversion of the amino group to the azido were carried out without isolation of intermediates (Scheme 1). The trifluoroacetylated azides II(a-e) were stored at -10 °C and deprotected just prior to the preparation of the oligonucleotide derivatives. (B) Synthesis of Aryl Azide Oligodeoxynucleotide Derivatives. The photoreactive oligonucleotide derivatives IV(b,c,e) and V(b) were obtained by phosphorylation of the amino groups of III(b,c,e) with the activated 4-(N,N-dimethylamino)pyridinium derivatives of the respective oligodeoxyribonucleotides (Scheme 2). The activated oligonucleotides were prepared by reaction of the phosphate group with a mixture of triphenylphosphine

and 2,2′-dipyridyl disulfide in the presence of 4-(N,Ndimethylamino)pyridine (11). These derivatives were isolated prior to coupling to compound III in order to avoid reaction with triphenylphosphine. The products obtained were isolated by reversed-phase chromatography (Figure 1). The derivatized oligonucleotides were observed to elute after the underivatized oligonucleotide. The isolated compounds were homogeneous by anion-exchange chromatography. In addition, the photoreactive oligonucleotides eluted as compounds having one charge less than the parent oligonucleotide. The yields of IV(b,c,e) and V(b) were at least 70%. IR absorption spectra of the obtained compounds revealed the presence of a band at 2100 cm-1, characteristic of the azido group. UV absorption spectra of the compounds had shown λmax to be 260 nm, characteristic of the oligonucleotide component. Anion-exchange chromatography and 31P NMR spectroscopy (the presence of the peak with δ ) 8.7 ppm, D2O) proved that the oligonucleotide and p-azidoaniline residues were connected by a phosphoamide bond. (C) Photochemical Behavior of the Aryl Azide Oligodeoxyribonucleotide Derivatives. The photolytic behavior of four representatives of oligodeoxyribonucleotide derivatives IV(b,c,e) and V(b) was investigated. The solution of photoreagents (5 × 10-5 M) in 0.15 M NaCl and 0.02 M Na2HPO4 (pH 9) was subjected to photolysis for 5 min and was analyzed by anionexchange chromatography. The irradiation time was chosen to be 10-fold greater than the half-life of photolysis. The kinetics of photolysis was studied by recording the IR spectra (2000-3000 cm-1) of the samples irradiated for definite time intervals. As shown in Figure 2C, reagent IV(b) undergoes a transformation leading to three products differing in their retention times with the anion-exchange column. The retention time of the main product is greater as compared with that of the parent azide derivative and coincides

Synthesis of Azidoaniline Derivatives

Bioconjugate Chem., Vol. 7, No. 3, 1996 347

oligonucleotide moiety. To check this possibility, we have subjected the below duplex to irradiation.

d(GpGpTpApTpC)pNH(CH2)6NH2 3′dApApCpCpApTpApG5′p-NH(CH2)6NHC6H4N3 In Figure 4C, the result of the anion-exchange chromatography of the irradiated solution of the duplex is presented. As shown by the chromatographic profile, the peaks appear with greater retention time as compared with the retention time of the parent oligonucleotides. This indicates that the formation of the cross-linking products has taken place. In the same time, no crosslinking was observed in the irradiated duplex containing the parent oligonucleotide lacking an aliphatic amino group (Figure 5C). The same data were obtained for the duplex formed by IV(c). The structure of the cross-linked products is now under investigation and will be discussed in a separate communication. Thus, the results of the present investigation have demonstrated that the azidoaniline derivatives of the oligonucleotides are capable of affinity modification of the specific target without the loss of the modifying moiety. However, the aliphatic spacer group is necessary to prevent splitting of the address, and this group should be sufficiently long. ACKNOWLEDGMENT

The authors are grateful to Dr. T. M. Ivanova for the preparation and purification of the monomers for the oligonucleotide synthesis and to Academician D. G. Knorre for many helpful discussions. This work was supported in part by a grant from Russian Fund of Fundamental Investigation.

Figure 5. Profile of analytical anion-exchange microcolumn chromatography of compound IV(e) (A), of the duplex IV(e)‚ d(GGTATCp) without irradiation (B), and of the reaction mixture obtained by photolysis of the duplex IV(e)‚d(GGTATCp) (C). Concentrations of the reagents were 5 × 10-5 M. Reaction mixture was irradiated and incubated at 4 °C for 12 h in the dark and then analyzed.

LITERATURE CITED

with that of the parent oligonucleotide. This suggested that the P-N bond is cleaved under irradiation with liberation of the 5′-terminal phosphate. To prove this statement, a similar experiment has been carried out with V(b) with fewer phosphate residues, and reaction mixture was subjected to 31P NMR investigation. As shown in Figure 3, the resonance typical of an aliphatic phosphoamide group (8.7 ppm) disappears after irradiation and a new resonance at 3.7 ppm arises. The new resonance corresponds to the phospho monoester group. To explain these results, we propose the tentative reaction as shown in Scheme 3. The scheme is based on the suggestion that the N atom forming the P-N bond attacks the strongly electrophilic C atom, the second C in the polyene structure of intermediate quinonediimine. This reaction seems to be facilitated by the formation of the geometrically favorable six-membered cycle. If this is the case, the use of longer spacers, (CH2)nNH, should hinder cyclization and prevent the P-N bond scission. Therefore, we have investigated the irradiation behavior of derivatives IV(c) and IV(e) (with n ) 4 and 6). In Figure 2D, the result of the ion-exchange chromatography of the irradiated solution of IV(e) is presented. As shown, the appearance of the chromatographic profile differs strongly from that for IV(b). No peak corresponding to the parent oligonucleotide was observed. This indicates that no P-N bond scission had taken place. The same results were obtained for IV(c). These results permitted one to expect that derivatives IV(c) and IV(e) should be capable of intraduplex reaction with the nucleophilic groups of the complementary oligonucleotide target without the loss of the addressing

(1) Knorre, D. G., and Vlassov, V. V. (1989) Photoaffinity Modification. Affinity Modification of Biopolymers pp 111113, CRC Press Inc., Boca Raton, FL. (2) Grachev, M. A., Knorre, D. G., and Lavrik, O. I. (1981) Interaction of E. coli Phe-RSase with photoreactive ATP γ-amidates. Photoaffinity modification of some E. coli ARSase by ATP γ-azidoanilidate. γ-Derivatives of nucleoside5′-triphosphates as probes for enzyme and protein factor active sites, pp 125-127, Harwood Academic, New York. (3) Kudryashova, N. V., Shamanina, M. Yu., Godovikova, T. S., Anan’ko, E. A., Akhmadieva, F. F., and Romashchenko, A. G. (1993) Specific effects of dTTP γ-p-azidoanilide on Escherichia coli DNA polymeraze 1 and its Klenow fragment. Biokhimiya 58, 224-223. (4) Rasenick, M. M., Talluri, M., and Dunn, W. J., III (1994) Photoaffinity Guanosine 5′-Triphosphate Analogs as a Tool for the Study of GTP-Binding Proteins. Methods Enzymol. 237, 100-110. (5) Gall, T. S., Griezan, N. P., Myzina, S. D., Bazhin, N. M., and Nemzeva, E. V. (1983) p-Benzenequinonediimine reactive intermediate of photolysis p-azidoaniline in aqueous solutions. Dokl. AS USSR 273, 883-887. (6) Badashkeyeva, A. G., Gall, T. S., Efimova, E. V., Knorre, D. G., Lebedev, A. V., and Myzina, S. D. (1983) Reactive derivative of adenozine-5′-triphosphate formed by irradiation of ATP-γ-p-azidoanilide. FEBS Lett. 155, 263-266. (7) Badashkeyeva, A. G., Gall, T. S., Knorre, D. G., Lebedev, A. V., Efimova, E. V., and Myzina, S. D. (1984) A study on photoconversion products of nucleoside 5′-triphosphate γ-pazidoanilides in aqueous solution. Bioorg. Khim. 10, 696701. (8) Godovikova, T. S., Berezovskye, M. V., and Knorre, D. G. (1995) Photoaffinity modification of aminoacid derivatives of oligonucleotides in the complimentary complex. Bioorg. Khim. 21, 858-867.

348 Bioconjugate Chem., Vol. 7, No. 3, 1996 (9) Cantor, C. R., and Tinoco, I. (1965) Absorption and optical rotatory dispersion of seven trinucleoside diphosphates. J. Mol. Biol. 13, 65-77. (10) Gryaznov, S. M., Gorn, V. V., Zarytova, V. F., Kumarev, V. P., Levina, A. S., Polistchuk, A. S., Potapov, V. K., Potemkin, G. A., Sredin, Ju. G., and Shabarova, Z. A. (1987) Automatic synthesis of oligodeoxyribonucleotides using phosphitamide method on “Victoria 4M”. Izv. Sib. Otd. Akad. Nauk SSSR 2, 119-123. (11) Godovikova, T. S., Zarytova, V. F., Mal’tzeva, T. S., and Khalimskaya, L. M. (1989) Reactive derivatives of oligonucleotides with a zwitterionic terminal phosphate group for affinity reagents and probe construction. Bioorg. Khim. 15, 1246-1252. (12) Horn, T., and Urdea, M. S. (1986) A chemical 5′-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release. Tetrahedron Lett. 27, 4705-4708.

Godovikova et al. (13) Chung, T. E., Wu, Y. M., and Chang, C. H. (1984) Reduction of aromatic nitro compounds by ethylenediamine. A new selective reagent for the synthesis of symmetric azo compounds. J. Org. Chem. 49, 1215-1217. (14) Berkner, K. L., and Folk, W. R. (1977) Polynucleotide kinase exchange reaction. J. Biol. Chem. 252, 31763184. (15) Perillo, I., and Lamdan, S. (1973) Synthesis of 1,2-disubstituted 1,4,5,6-tetrahydropyridines. J. Heterocycl. Chem. 10, 915-923. (16) Perillo, I., Fernandez, B., and Lamdan, S. J. (1977) Synthesis and properties of 1,2-diaryl-4,5,6,7-tetrahydro-1H1,3-diazepines and 1,2-diaryl-1,4,5,6,7,8-hexahydro-1,3-diazocines. Comparison with five- and six-membered homologues. J. Chem. Soc., Perkin Trans. 2, 2068-2072.

BC960020J