Russian Journal of Bioorganic Chemistry, Vol. 29, No. 6, 2003, pp. 560–565. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 6, 2003, pp. 616–622. Original Russian Text Copyright © 2003 by Antonov, Esipov, Gurevich, Chuvikovsky, Mikulinskaya, Feofanov, Miroshnikov.
Chemical and Chemoenzymatic Synthesis of Nucleoside 5'-a-Thiotriphosphates K. V. Antonov,1 R. S. Esipov, A. I. Gurevich, D. V. Chuvikovsky, G. V. Mikulinskaya, S. A. Feofanov, and A. I. Miroshnikov Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia Received June 13, 2002; in final form, September 04, 2002
Abstract—New methods of chemical and chemoenzymatic synthesis of nucleoside 5'-thiophosphates and 5'-αthiotriphosphates are developed. The 5'-α-thiotriphosphates are used as substrates both in template-dependent enzymatic PCR synthesis and in a T7–RNA transcription polymerase system. Key words: phage T5 deoxyribonucleoside monophosphate kinase, immobilized preparation; nucleotides, phosphorothioate analogues, stannyl ethers 1
INTRODUCTION Phosphorothioate analogues of nucleic acids are widely used in molecular biology. The stability of these RNA and DNA analogues toward nucleases makes them attractive as potential effective therapeutic agents.2 The most simple method of the obtaining of nucleic acid phosphorothioate analogues of nucleic acids is the template-dependent synthesis by means of RNA and DNA polymerases, which turned out to be capable of using Sp diastereomers of nucleoside α-thiotriphosphates as substrates [1, 2]. Therefore, the development of an effective synthetic method for ribo- and 2'-deoxyribonucleoside α-thiotriphosphates (dNTPs) is an important prerequisite for the preparation of thioanalogues of nucleic acids in amounts sufficient for various purposes. Two approaches to the chemical synthesis of nucleoside α-thiotriphosphates are known: direct selective thiophosphorylation of nucleosides with phosphorus thiotrichloride [3, 4] and oxidation with sulfur of the corresponding derivatives of trivalent phosphorus [5]. The first approach is obviously more promising from the technological point of view. The major disadvantages of this method are low (25–50%) yields of the target products, which we believe to be connected with a decreased reactivity of phosphorus thiotrichloride in comparison with phosphorus oxychloride. This limitation can be overcome by increasing the activity of the second reaction component, hydroxyl group of nucleo1 Corresponding
author; phone: +7 (095) 330-7247; e-mail:
[email protected] 2 Abbreviations: Acp, acetyl phosphate; (d)NMP , (d)NDP , and S S (d)NTPS, (2'-deoxy)ribonucleoside-5'-O-thiomono-, α-thiodiand α-thiotriphosphates, respectively; IEP, immobilized enzymic preparation; and PCR, polymerase chain reaction.
side, for example, using the corresponding alcoholates as described in [6]. RESULTS AND DISCUSSION We carried out here thiophosphorylation of nucleosides using a well-developed procedure of activation of hydroxy group via the formation of stannyl ethers [7, 8]. Tributylstannyl ethers are usually obtained by refluxing carbohydrates in toluene in the presence of bis(tributyltin)oxide (I). These ethers are unstable and are not isolated, and researchers judge their structures from the products of further transformations. Etherification of primary hydroxyl groups usually proceeds in the reaction of polyols with one equivalent of (I) [7]. We chose adenosine and deoxyadenosine as substrates for thiophosphorylation. The reflux of deoxyadenosine in toluene or dioxane with one equivalent of oxide (I) leads to the dissolution of the nucleoside, and the mixture remains homogeneous after cooling. According to TLC, the addition of phosphorus thiotrichloride causes a rapid disappearance of the starting nucleoside (stannyl ether decomposes to deoxyadenosine under the TLC conditions) and the formation of the only product with a higher Rf value. An analysis of the reaction mixture by 31P NMR showed, in addition to the resonance of starting PSCl3 at 30.7 ppm, the presence of two additional singlets of equal intensity at 45.3 and 56.8 ppm; they seem to belong to nucleoside thiophosphorodichloridate (IIa) and tributyltin oxidothiophosphorodichloridate (III) (Scheme 1). 2'-Deoxyadenosine 5'-thiophosphate (IVa) was obtained in 80% yield after the hydrolysis of the reaction mixture with a Na2CO3 solution and ion-exchange chromatography on DEAE Toyopearl. The structure of (IVa) was confirmed by 13C and 31P NMR spectra [as
1068-1620/03/2906-0560$25.00 © 2003 MAIK “Nauka /Interperiodica”
CHEMICAL AND CHEMOENZYMATIC SYNTHESIS
561
NH2 N
N
N
N Bu3SnO
O
(d)Ado + (Bu3Sn)2O (I)
+ Bu3SnOH OH R
NH2
NH2 N
N
PSCl3
S Cl2PO
N
N
N
N
O
(Bu3N)4H4P2O7
+ Bu3SnOPCl2 (III)
OH R
S O P O P –O O O P –O O
N
N
O
S
O
OH R
(IIa): R = H (IIb): R = OH H2O
H2O
NH2 N
N S O P O O–
–
NH2
N
N O
N
N –O
E1, E2, E3
O O S P O P O P O O– O– O–
OH R
N
N O
OH R (Va): R = H (Vb): R = OH
(IVa): R = H (IVb): R = OH
Scheme 1. The synthesis of nucleoside and 2'-deoxynucleoside 5'-thiophosphate and 5'-α-thiotriphosphates.
well as by enzymatic phosphorylation to α-thiotriphosphate (Va)]. In particular, thiophosphorylation of the deoxyribose moiety in position 5' (reaction regioselectivity) was confirmed by the phosphorus-caused splitting of the C4' and C5' signals in the 13C NMR spectrum. The addition to the reaction mixture of excess tetra(tri-n-butylammonium) pyrophosphate results in the disappearance of intermediate (IIa) (TLC data) and RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
the formation of a mixture in which the major product had the chromatographic mobility close to that of nucleoside triphosphates. After hydrolysis, this product was isolated by ion-exchange chromatography and characterized by 31P NMR spectra as the target α-thiotriphosphate (Va) (an equimolar mixture of Rp and Sp diastereomers). Note that high yields of (Va) (60–70%) could be only achieved when a freshly prepared DMF solution of tetra(tri-n-butylammonium) pyrophosphate
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ANTONOV et al. (a) A (5') (5')
(3') (3')
B Z
(b)
(5') ... mRNA
(5') ..
Scheme 2. The template-dependent synthesis using nucleoside and 2'-deoxynucleoside 5'-α-thiotriphosphates. (a) PCR with íaq polymerase on a proinsulin gene template [10] with oligodeoxynucleotide primers A and B and dÄíPS + dNTP as substrates; (b) transcription of the proinsulin gene [10] cloned into the plasmid pET-20b(+) using T7 RNA polymerase with ÄíPS + NTP as substrates. The mRNA sequence fragment and the structure of the oligodeoxynucleotide probe Z used for hybridization are shown.
was used or when preparing it in situ from tetrapyridinium pyrophosphate and tri-n-butylamine. In the first case, the product is separated from the solution as emulsion and, in the second case, a heterophase reaction proceeds, the rate of which does not depend on the used solvent (toluene or dioxane). Adenosine 5'-O-(α-thiotriphosphate) (Vb) was similarly obtained from adenosine in 70% overall yield. Note that the interaction of adenosine with (I) only occurred if a considerable amount (10–20%) of a polar aprotic solvent (DMF or trimethyl phosphate) was added to the mixture, which may be due to a lower solubility of the ribonucleoside stannyl ether. We also studied the stannylation–thiophosphorylation reaction of some adenosine derivatives, namely, 2'and 3'-mono- and 2',3'-di-O-benzoyladenosines (data not given). The reaction with PSCl3 proceeded only with 2'-benzoyladenosine. After the addition of tetra(tri-n-butylammonium) pyrophosphate and the complete hydrolysis with ammonia, thiotriphosphate (Vb) was obtained, although in a considerably lower overall yield. With the use of this thiophosphorylation procedure, we also synthesized thymidine, deoxyguanosine, and uridine 5'-α-thiotriphosphates. These results will be published elsewhere. An alternative method of (d)NTPs preparation from (d)NMPs is based on enzymatic phosphorylation. Its major advantage is the stereospecificity, which allows the preparation of only one (Sp) isomer. For the phosphorylation of dÄMêS (IVa), we used IEPs obtained from the extracts of E. coli control cells (control IEP) and from the E. coli cells transformed with plasmid containing the gene of deoxynucleotide monophosphate kinase (dNMP kinase, EC 2.7.4.13) from T5 bacteriophage. It is known that the phage
enzyme, unlike bacterial dNMP kinases, displays wide substrate specificity [9]. We believe that the process proceeds according to the following scheme: dÄMêS + ATP
E1
dÄDPS + ADP
dÄDPS + ATP
E2
dÄTPS + ADP
2ADP + 2AcP
E3
dAMPS + 2AcP
2ATP + 2AcO
–
dATPS + 2 AcO–,
where E1 is either E. coli or phage T5 dNMP kinase and E2 and E3 are bacterial nucleoside diphosphate kinase (EC 2.7.4.6) and acetate kinase (EC 2.7.2.1), respectively. After the addition into the reaction mixture of acetyl phosphate, a donor of high-energy phosphate, acetate kinase regenerates ATP, which allows 50–100 times reduce the ATP consumption and simplify dATPS isolation from the reaction mixture, because made unnecessary the separation of the product from a large amount of ATP. It was shown that the IEP containing bacteriophage T5 dNMP kinase phosphorylates dÄMêS, although with a lower rate than dAMP: under the selected conditions, the transformation degree of the nucleotide thioanalogue into thiotriphosphate was about 5% after 2 h and about 50% after a day. In the case of control IEP containing the E. coli enzymes, thiotriphosphate was not formed. Thus, the chemoenzymatic synthesis in our system with IEP is not as productive as the chemical synthesis. The resulting nucleoside α-thiotriphosphates were used for the template-dependent enzymatic synthesis on a proinsulin gene as a template using PCR with Taq DNA polymerase and primers A and B [10]; dÄTPS +
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2
3
M
bp
1
563 2
3
700
260 170 100
Fig. 1. Separation in 2% agarose gel in the presence of ethidium bromide of the PCR products of the proinsulin gene amplification: 1, with all NTP (a control); 2, with three dNTP (in the absence of ATP); and 3, with three dNTP and dATPS; M, standards of DNA sizes.
Fig. 2. Transcription of the proinsulin gene cloned into the plasmid pET-20b(+) with T7 RNA polymerase using as substrates 1, all NTP; 2, three dNTP (in the absence of ATP); and 3, three dNTP and dATPS. Electrophoresis was carried out in 2% agarose gel with ethidium bromide.
dNTP were substrates (Scheme 2a). Since the rate of dNTPS incorporation into the DNA chain at the catalysis by Taq polymerase was 20 times lower than that of dNTP [11], we tried to enhance the efficiency by increasing the time of the elongation stage to 90 s and the number of amplification cycles to 35. The separation of amplification products in 2% agarose gel is shown in Fig. 1. A mixture of ÄTPS and NTP as substrates was used for the RNA enzymatic synthesis (Scheme 2b) in the T7 RNA polymerase transcription system on a proinsulin gene template cloned into plasmid pET-20b(+) (Novogen). The formation of transcripts was confirmed by electrophoresis in 2% agarose gel (Fig. 2). The amount of mRNA in the samples was estimated according to hybridization with 32P-labeled probe Z (Scheme 2b). This amount achieved 60% of the mRNA amount in the samples with ÄTPS + 3NTP if compared with those obtained in the presence of all four NTP. Thus, it was shown that the α-thiotriphosphates obtained by us can be used in enzymatic reactions.
(C) 43 : 54 : 3 dioxane–5 M ammonia–1 M ammonium acetate. HPLC was carried out on a Nucleosil 100 ë18 (5 µm) column (4.6 × 125 mm) at isocratic elution with a mixture of 100 mM ammonium acetate (pH 7.9) and 6% acetonitrile at a flow rate of 1 ml/min. NMR spectra were recorded on a Bruker AM-300 spectrometer (250 MHz) in D2O with 75% H3PO4 (31P) and 1,4-dioxane (13C) as external standards. Tris, acrylamide, N,N'-methylenebisacrylamide, and ammonium persulfate were from Merck; PSCl3 and bis(tributyltin) oxide were from Fluka; agarose, ATP, dNTP, and ethidium bromide were from Sigma; urea of a special purity grade was from Reakhim; [γ-32P]ATP and [α-32P]dATP (2000 Ci/mmol) were from Obninsk; T4 DNA ligase (EC 6.5.1.1), Taq DNA polymerase (EC 2.7.7.7), and T7 DNA polymerase (EC 2.7.7.6) were from Fermentas (Lithuania). The protein amount in the enzymatic preparations was determined according to [12]. The preparation of the producer strain biomass of bacteriophage T5 deoxyribonucleoside monophosphate kinase (dNMP kinase). The E. coli BL21(DE3) cells were transformed with the plasmid containing the dNMP kinase gene (G.V. Mikulinskaya, A.A. Zimin, and S.A. Feofanov, personal communica-
EXPERIMENTAL TLC was carried out on precoated Kieselgel 60 F254 plates (Merck) in (A) 4 : 1 chloroform–methanol; (B) 15 : 10 : 3 isopropanol–saturated ammonia–water; or RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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tion). When optical absorption Ä550 achieved 1.0, the synthesis of dNMP kinase in the cells was induced by the addition of isopropyl β-thiogalactoside up to the concentration of 1 mM. The cells were centrifuged at 5000 g for 10 min 2 h after the cell induction. To obtain the control biomass, the E. coli BL21(DE3) cells were grown for the same time and then centrifuged. The IEP preparation of bacteriophage T5 dNMP kinase [9]. All the procedures were carried out at 4°ë. A mixture of 50 mM K-Pi (3 ml, pH 7.4) and lysozyme (2 mg/ml) was added to the biomass (1 g) and resuspended. After the start of lysis, the cells were degraded by sonication using a 40-W sound disintegrator for 1 min. After interphase fractionating in the PEG–dextran system, 20% dextran 500T (125 µl) and 40% PEG 6000 (400 µl) per ml of the mixture were added to the resulting extract. After 20 min, the mixture was centrifuged at 10000 g for 10 min. The upper phase containing the total protein (5 mg/ml) was immobilized on aminopropyl-Silochrom C-80 treated with glutaraldehyde [13]. The resulting IEP contained at least 20 mg of protein per ml of sorbent. The IEP of bacteriophage T5 dNMP kinase and that of control E. coli BL21(DE3) cells were used for the thiotriphosphate synthesis. 2'-Deoxyadenosine 5'-thiophosphate (IVa). Bis(tributyltin)oxide (600 µl, 1.1 equiv) was added to a suspension of 2'-deoxyadenosine (252 mg, 1 mmol) in dioxane (25 ml), the mixture was refluxed for 1 h while distilling off 15 ml of the solvent. The homogeneous solution was cooled to room temperature, and PSCl3 (210 µl, 2 equiv) was added. After the reaction was over (TLC-monitoring, system A), the mixture was hydrolyzed with a cold saturated solution of Na2CO3 (3 ml); diluted with water (7 ml); and extracted with chloroform (2 × 30 ml). The aqueous layer was separated, filtered, and loaded onto a DEAE Toyopearl column (4 × 15 cm), eluted with water (200 ml) and 0.1 M LiCl (400 ml). The fractions containing the target product were evaporated; the residue was treated with acetone and filtered. The precipitate was dissolved in water (5 ml), filtered through Celite, and diluted with methanol (5 ml) and acetone (90 ml). The precipitate was separated by centrifugation and twice reprecipitated. The resulting lithium salt of (IVa) was dried in a vacuum over P2O5 to give 290 mg (80%) of (IVa); 31P NMR (δ, ppm): 40.81; 13C NMR (δ, ppm, J, Hz): 155.49 (C6), 152.86 (C2), 148.57 (C4), 142.5 (C8), 118.56 (C5), 86.98 (d, JC4',P 9.17, C4'), 84.31 (C1'), 72.37 (C3'), 64.9 (d, 1C, JC'5,P 2.91, C5'), and 40.2 (C2'). 2'-Deoxyadenosine 5'-O-(a-thiotriphosphate) (Va). Bis(tributyltin)oxide (300 µl, 1.1 equiv) was added to a suspension of 2'-deoxyadenosine (126 mg, 0.5 mmol) in dioxane (15 ml), and the mixture was refluxed for 1 h while distilling off 8 ml the solvent. The homogeneous solution was cooled to room temperature and PSCl3 (110 µl, 2 equiv) was added. After the reac-
tion was over (1 h, TLC, system A), solid pyridinium pyrophosphate (500 mg) and tributylamine (0.5 ml) were added. The mixture was stirred for 0.5 h, diluted with water (5 ml), and kept overnight at 4°ë. The completeness of hydrolysis was monitored by TLC (system B). The mixture was extracted with chloroform (2 × 30 ml), the aqueous solution was separated, filtered, and loaded onto a column eluted by a step gradient of LiCl (0.1, 0.2, and 0.3 M, 500 ml each). The corresponding fractions were evaporated, the residue was dissolved in water (5 ml), filtered through Celite, and diluted with methanol (5 ml) and acetone (90 ml). The precipitate was separated by centrifugation and twice reprecipitated. The resulting lithium salt of (Va) was dried in a vacuum over P2O5 to give 160 mg (62%) of the product. Analytical samples of Rp- and Sp diastereomers were isolated by reversed-phase chromatography [3] on a Si100 Polyol RP 18 (0.003 mm, Serva) column eluted with 3% (300 ml) and 4% (200 ml) acetonitrile in 100 mM triethylammonium acetate (pH 7.8); HPLC (RT, min): 7.3 (Sp) and 9.4 (Rp). 31P NMR (δ, ppm, J, Hz): (Va) (Sp): 46.3 (d, JPα,Pβ 27.12, Pα)), –7.9 (d, JPγ,Pβ 19.44, Pγ), –21.14 (dd, JPβ,Pα 27.12, JPβ,Pγ 19.44, Pβ); (Va) (Rp): 45.9 (d, JPα,Pβ 27.7, Pα), –8.01 (d, JPγ,Pβ 19.8, Pγ), –21.25 (dd, JPβ,Pα 27.7, JPβ,Pγ 19.8, Pβ). Enzymatic synthesis of 2'-deoxyadenosine 5'-O(a-thiotriphosphate (Va) using IEP of bacteriophage T5 dNMP kinase. The reaction was carried out at 37°ë. The reaction mixture (400 µl) contained 0.2 M Tris–HCl (pH 7.4), 40 mM ågCl2, IEP (100 µl), 5 mM thiophosphate (IVa), 50 mM acetylphosphate, and 0.5 mM ATP. The samples were semiquantitatively analyzed by TLC (system B). Adenosine 5'-O-(a-thiotriphosphate (Vb). Bis(tributyltin)oxide (160 µl, 1.1 equiv) was added to a suspension of adenosine (67 mg, 0.25 mmol) in toluene (9 ml) and trimethyl phosphate (1 ml), and the mixture was refluxed for 0.5 h while distilling off 8 ml of the solvent. The homogeneous solution was cooled to room temperature, and PSCl3 (60 µl, 2 equiv) was added. After the reaction was over (1 h, TLC, system A), solid pyridinium pyrophosphate (350 mg) and tributylamine (400 µl) were added. The mixture was vigorously stirred for 1.5 h, diluted with water (5 ml), and kept overnight at 4°ë. The completeness of hydrolysis was monitored by TLC (system B). The mixture was extracted with chloroform (2 × 30 ml), aqueous layer was separated, filtered, and loaded onto a column eluted in a step gradient of LiCl (0, 0.1, 0.15, 0.2, 0.25, and 0.3 M, 200 ml each). The corresponding fractions were evaporated, the residue was dissolved in water (5 ml), filtered through Celite, and diluted with methanol (5 ml) and acetone (90 ml). The precipitate was separated by centrifugation and twice reprecipitated. The resulting lithium salt of (Vb) was dried in a vacuum over P2O5 to give 95 mg (72%) of the product; HPLC (RT, min): 4.3 (Sp) and 6.7 (Rp). 31P NMR ((Rp,Sp):
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41.4–40.96 (m, Pα), –7.2 to –7.4 (m, Pγ), and –23.5 to – 23.9 (m, Pβ). 13C NMR (δ, ppm, J, Hz): 155.92 (C6), 153.31 (C2), 149.45 (C4), 143.2 (C8), 118.96 (C5), 87.65 and 87.54 (ë4'-Rp,Sp), 84.57 and 84.37 (ë1'Rp,Sp), 75.16 (C2'), 71.11 (C3'), 66.31 (d, JC'5,P 7.1, ë5'R(S)), and 65.9 (d, JC'5,P 5.68, ë5'S(R)). Oligonucleotides were synthesized by a standard phosphoramidite procedure on an ASM-102T synthesizer (BIOSSET, Novosibirsk), deprotected, and isolated as described in [14]. The PCR procedures were carried out on a GeneCycler amplificator (Bio-Rad) in the incubation mixture (50 µl) containing the proinsulin gene DNA template (about 20 pmol) [14], the corresponding primers (80 pmol each), a mixture of dNTP and dÄTPS (0.5 mM each), 67 mM Tris–HCl (pH 8.3), 6 mM MgCl2, 1 mM dithiothreitol, 17 mM (NH4)2SO4, gelatin (9 µg), and Taq DNA polymerase (5 U). The incubation in the DNA amplificator (35 cycles) was performed as follows: denaturation for 30 s at 94°ë; annealing for 30 s at 52°ë; and elongation for 90 s at 72°ë. Amplification products were identified after separation in 2% agarose gel (Fig. 1). Transcription with T7 RNA polymerase. The reaction mixture (20 µl) contained the buffer (Fermentas), ribonuclease inhibitor (15 U, Stratagene), a mixture of ÄTPS and NTP (without ATP, 0.5 mM each), T7 RNA polymerase (8–10 U), and the proinsulin gene DNA template (0.3–0.8 µg) cloned into plasmid pET-20b(+) (Novogen) [14]. The mixture was incubated for 2–2.5 h at 37°ë. Mixture of all NTP and three NTP (without ATP) were used in control experiments as substrates (Fig. 2). The preparation was extracted with phenol (pH 5.0) and a 25 : 24 : 1 phenol–chloroform–isoamyl alcohol mixture; 3M sodium acetate (pH 5.2, 1/10 volume) and ethanol (3 volumes) were added, the mixture was kept at –20°ë overnight, and mRNA was precipitated by centrifugation (10 min at 12000 rpm). The precipitate was washed with 70% ethanol, dried, and dissolved in water (4 µl); 20 × SSC buffer (2 µl) was added, and aliquots were loaded onto three identical nitrocellulose membranes for dot hybridization. The membranes were heated for 2 h at 80°ë between Whatman 3MM paper sheets, and the RNA amount in the samples was estimated according to
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the results of hybridization with (Scheme 2b).
565 32P
labeled probe Z
ACKNOWLEDGMENTS The authors are grateful to L.O. Kononov (Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences) for recording and interpretation of NMR spectra; to A.L. Kayushin for the synthesis of oligodeoxynucleotides; and to K.N. Verevkina for obtaining the immobilized enzymic preparations. The work was supported by the Russian Foundation for Basic Research, project nos. 00-15-97947 and 01-04-97004. REFERENCES 1. Eckstein, F. and Gish G., Trends Biochem. Sci., 1989, vol. 14, pp. 97–100. 2. Eckstein, F., Annu. Rev. Biochem., 1985, vol. 54, pp. 367–402. 3. Goody, R.S. and Isakov, M., Tetrahedron Lett., 1986, vol. 27, pp. 3599–3602. 4. Arabshahi, A. and Frey, P.A., Biochem. Biophys. Res. Commun., 1994, vol. 204, pp. 150–155. 5. Ludwig, J. and Eckstein, F., J. Org. Chem., 1989, vol. 54, pp. 631–635. 6. Szczepanik, M.B., Desaubry, L., and Johnson, R.A., Tetrahedron Lett., 1998, vol. 39, pp. 7455–7458. 7. David, S. and Hanessian, S., Tetrahedron, 1985, vol. 41, pp. 643–663. 8. Wagner, D., Verheyden, J.P.H., and Moffatt, J.G., J. Org. Chem., 1974, vol. 39, pp. 24–30. 9. Bessman, M.J., Herriott, S.T., and van Bibber Orr, M.J., J. Biol. Chem., 1964, vol. 240, pp. 439–445. 10. Esipov, R.S., Gurevich, A.I., and Miroshnikov, A.I., RF Patent no. 2181771, 2002. 11. Ciafre, S.A., Rinaldi, M., Gasparini, P., Seripa, D., Bisceglia, L., Zelante, L., Farace, M.G., and Fazio, V.M., Nucleic Acids Res., 1995, vol. 23, pp. 4134–4142. 12. Bradford, M.M., Anal. Biochem., 1976, vol. 72, pp. 248– 254. 13. Yamauchi, H., Machida, H., Midorikawa, Y., and Kuninaka, A., J. Ferment. Technol., 1986, vol. 64, pp. 445– 457. 14. Gurevich, A.I., Kachalina, T.A., Kayushin, A.L., Korosteleva, M.D., and Miroshnikov, A.I., Bioorg. Khim., 1993, vol. 19, pp. 629–632.
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