Synthesis of Ribonucleoside 5'-Triphosphates Derived from 2-Thiocytidine, 4-Thiouridine, and 5-Bromocytidine

B. NAWROT, P. HOFFMÜLLER, and M. SPRINZL*

Laboratorium für Biochemie, Universität Bayreuth, D-9544О Bayreuth, Germany

Received 11 October 1995

Dedicated to Professor K. Antoš, in honour of his 70th birthday

The synthesis of 5'-triphosphates of 4-thiouridine, 2-thiocytidine, 5-bromocytidine, and [6- 15N]adenosine is described. All compounds are characterized by chromatographic and spectral data. Phosphorylation of corresponding nucleotides was performed using phosphoryl chloride and con­ densation with pyrophosphate. Intermediates were not isolated. The final products were purified by chromatography and characterized by 31P NMR spectroscopy. The nucleoside triphosphates are substrates for RNA polymerase from bacteriophage T7 and were used for enzymatic synthesis of modified and isotopically labelled RNA.

5;-Triphosphates of modified nucleosides are of in­ lation and purification procedure as well as spectral terest to us for three main reasons. First, they serve and chromatographic data for the isolated products. as substrates for enzymatic preparation of modified RNA transcripts suitable for crosslink reactions with EXPERIMENTAL different protein molecules [1]. Second, according to the previously described procedure, enzymatic incor­ Solvents were purchased from Merck and then poration of s2C into position 75 in the CCA terminus additionally dried as follows: pyridine and DMF of tRNA allows modification of spectroscopic labels were stored for 24 h over dry molecular sieves [2—8]. Third, introduction of [6-15N]-labelled adeno­ (0.3 nm); toluene was distilled over sodium. Diethyl sine into position 76 of the CCA end of tRNAs pro­ ether and HPLC grade methanol were used as pur­ vides the possibility for 15N NMR investigations. chased. Triethylamině for triethylammonium bicar­ Early methods of chemical synthesis of nucleoside bonate buffer (ТЕAB) preparation was distilled before 5'-triphosphates involved condensation of an activated use. Reagents were purchased from Aldrich. Tributyl- nucleotide derivative, mostly 5'-phosphoromorpholi- amine and trimethyl phosphate were used directly, and date [9] or 5'-phosphoroimidazolidate [10—12] with phosphoryl chloride was freshly distilled before use. inorganic pyrophosphate [9]. The procedure of choice (Bu3NH)2H2P207 (0.5 M-DMF solution) was pre­ is presently the "one-pot" phosphorylation reaction pared according to the described procedure [9]. of nonprotected nucleosides with phosphoryl chloride 2-Thiocytidine (s2C) was purchased from Aldrich in trialkyl phosphates. This procedure provides pre­ (Steinheim, Germany). 4-Thiouridine (s4U) was syn­ dominantly nucleoside 5'-phosphorodichloridates [13] thesized by thionation of 2',3/,5'-tri-0-benzoyluridine which are transformed in a good yield to nucleoside and its subsequent deprotection [17]. 5-Bromocytidine 5 5'-triphosphates using an excess of (Bu3NH)2H2P207 (br C) was prepared by direct bromination of cyti- in Ar,A^-dimethylformamide (DMF) under anhydrous dine [18]. [6-15N] Adenosine was a kind gift of Profes­ conditions, followed by neutral hydrolysis [14—16]. sor Ishido of Tokyo University (Japan). Nucleosides The reaction may proceed via a highly reactive imi- were dried prior to use by coevaporation 3 times with doyl phosphate intermediate formed by condensation anhydrous pyridine and coevaporation 5 times with of phosphorodichloridate with DMF [16]. We have ap­ anhydrous toluene. plied this "one-pot" procedure for preparation of 5'- Uridine derivatives br5UTP, i5UTP, and s4UDP triphosphates of photolabile and relatively unstable and the cytidine derivative i5CTP were purchased modified nucleosides. Here we provide a detailed iso­ from Sigma (Deisenhofen, Germany). For purifica-

*The author to whom the correspondence should be addressed.

Chem. Papers 50 (3) 151—155(1996) 151 В. NAWROT, Р. HOFFMÜLLER, M. SPRINZL tion of triphosphates a DEAE-Sephadex A-25 ion- tion from concentrated methanol solutions with 1 % exchange column (40 mmxlO mm) (Pharmacia, Hei­ sodium Perchlorate in acetone solution (0.5 cm3), cen- delberg, Germany) was used. Elution was done at 4 trifugation and washing with acetone (4 x 0.2 cm3 °C in a TEAB buffer (pH 7.5) gradient with the flow portions) and dried over phosphorus pentoxide under rate 2 cm3 min"1. Evaporation of pooled fractions was vacuum. Yields were 79 % for s4UTP (Vb), 15 % for done under water pump pressure. s2CTP {Vb), 30 % for br5CTP (7c), and 70 % for Thin-layer chromatography was performed on pre­ [6-15N]ATP (Vd). dated TLC plates Cel 300-10 UV254 (0.1 mm) UV spectrum, Amax/nm (log{e}): for Va (in H20,

(Macherey—Nagel, Düren, Germany) in a 1-propanol— pH 6) 331 (4.31), Vb (in H20, pH 6) 250 (4.35), Vc ammonium hydroxide—water (phosphoric acid mixture with (q = 0.004 mol dm3) br5CTP, i5CTP, was used as an external reference. s2CTP or with br5UTP, i5UTP, s4UTP, respectively. Ultraviolet spectra were recorded on a UV VIS After incubation of the reaction mixtures for 2 h at recording spectrophotometer Shimadzu UV-160A 37 °C, EDTA was added to a final concentration of -3 (Shimadzu, Kyoto, Japan) under the conditions in­ 0.050 mol dm , and the reaction mixtures were pre­ dicated. cipitated with ethanol. The products of transcription were separated to single nucleotide resolution by ver­ General Phosphorylation Procedure tical gel electrophoresis on a denaturing 12 % Poly­ acrylamide gel (40 cm x 20 cm x 0.2 cm) containing A nucleoside (0.1 mmol) was dissolved in trimethyl 8 M-urea. The bands were located and their radioac­ phosphate (0.25 cm3) and cooled to 0°C, then phos- tivity counted in a Canberra Packard Instant Imager phoryl chloride (0.13 mmol, 12.1 mm3) was added 2024 (Packard, Frankfurt, Germany) to determine the dropwise under stirring and the reaction mixture was yield. left for 3 h at 0°C. A cold mixture of DMF solution 3 of (Bu3NH)2H2P207 (0.5 mmol, 1 cm ) and tributyl- RESULTS AND DISCUSSION amine (0.1 cm3) was added with shaking to the re­ action mixture. After 3 min of shaking (except br5C, Application of a "one-pot" synthesis of nucleoside for which 5 min shaking was necessary) at 0°C, cold 1 5'-triphosphates enabled us to obtain 5'-triphosphates M-TEAB buffer of pH 7.5 (5 cm3) was added to stop of the following modified nucleosides: 4-thiouridine the reaction. The reaction mixture was then extracted (s4U) /a, 2-thiocytidine (s2C) lb, 5-bromocytidine once with diethyl ether (5 cm3) and the aqueous layer (br5C) Ic, and [6-15N]adenosine [6-15N]A Id. The syn­ was applied onto DEAE-Sephadex A-25 column equi­ thesis (Scheme 1) was done according to the previously librated in TEAB buffer. The product was eluted with described method of Ludwig [16] with a slight modifi­ a gradient of 50—500 mM-TEAB buffer of pH 7.5 cation in the reaction time and isolation of products. (1 dm3 each), with the exception of s2CTP, where a Generally, the first step of the synthesis is based gradient of 50—800 mM-TEAB buffer of pH 7.5 was on the selective phosphorylation of the 5'-OH group applied. Fractions containing the triphosphate were of a nonprotected nucleoside with phosphoryl chlo­ combined, evaporated under reduced pressure and co- ride in trimethyl phosphate under anhydrous condi­ evaporated several times with methanol for remov­ tions [13]. The reaction was done for 3 h at 0°C ing traces of triethylammonium bicarbonate. Triphos­ to achieve sufficient yield. The progress of the re­ phates were obtained as sodium salts by precipita­ action was monitored by TLC of reaction aliquots

152 Chem. Papers 50(3)151—155 (1996) RIBONUCLEOSIDE 5'-TRIPHOSPHATES

ff -\& ~ a-nff ^ шь тч^ сн сн CH CH CH OH DMR, // III

(B«NH)ifcft07 ff ff ft ff DMF ;N=OÍ-^-po-T^-CS^? ^• , ro-'i-o-L-o-"гттт ^ CH ОН CH CH

IV V

Base Ш ,0NH2

ON N Y

Scheme 1

Table 1. Chromatographic Data of Nucleosides and Their 5'- ysis with TEAB buffer of pH 7.5, yielded nucleoside Mono- and 5-Triphosphates 5'-triphosphates. The reaction is catalyzed by forma­ tion of highly reactive imidoyl phosphate intermedi­ Compound TLC HPLCa Purity/% ate IVa—IVd, synthesized by condensation of phos- Ät/min phorodichloridate with DMF, which is the reaction s4U la 0.60 solvent [16]. The reaction time, which is crucial for s2C lb 0.65 this step, usually is not longer than 3 min (twice 5 br C Ic 0.77 as long as the reaction time used for the synthe­ [6-15N]A Id 0.61 4 sis of ATP or 2'-dATP [16], except for the case of s UMP Ilia 0.35 5 s2CMP im 0.27 br C, for which a longer reaction time was neces­ br5CMP IIIc 0.26 sary). In the most extreme cases, for hypermodified 5- [6-15N]AMP Hid 0.31 substituted uridine and cytidine derivatives, the reac­ 4 ь s UTP Va 0.13 8.90 80.5 tion time was prolonged up to 2—3 h [15]. The excess s2CTP Vb 0.10 29.98 89.0 5 of (Bu NH) H P207 and the prolonged reaction time br CTP Vc 0.11 17.31 98.0 3 2 2 [6-15N]ATP Vd 0.12 25.58 99.0 can lead, however, to appreciable formation of 1,4- UTP 0.10 18.95 (6.15ь) dinucleotide tetrapyrophosphates, which decompose ATP 0.12 25.13 in neutral conditions to 5'-nucleoside diphosphates, GTP 0.12 25.13 contaminating the desired product [9]. CTP 0.11 14.63 Isolation of the desired 5'-triphosphates, as well as a) 50 mM potassium phosphate (pH 6.7), 10 mM-TBHAS— their purification, was achieved by DEAE-Sephadex acetonitrile (y?r = 9 : 1); b) 50 mM potassium phosphate (pH A-25 column chromatography in triethylammonium

6.7), 10 mM-TBHAS—acetonitrile (y>r = 3 : 1). form (pH 7.5) at 4°C. Elution of products was con­ ducted by a gradient solvent system from 50 mmol dm"3 to 500 mmol dm"3 (800 mmol dm"3 for s2CTP) (2 mm3) decomposed with an excess of 1 M-TEAB. triethylammonium buffer, pH 7.5. Fractions eluted The relative mobilities (Rf) of the nucleoside 5'- with the buffer concentration in the range 250— monophosphates are listed in Table 1. The conden­ 380 mmol dm"3 were collected. Minor contaminants sation of 5'-phosphorodichloridates Ha—lid with an in the crude reaction product, nucleoside 5'-топо- excess (5 mol) of l/2(Bu3NH)2H2P207 in DMF un­ and diphosphates, were removed during this purifica­ der anhydrous conditions, followed by neutral hydrol­ tion step. Final purification of 5'-triphosphates was

Chem. Papers 50 (3) 151 —155 (1996) 153 В. NAWROT, Р. HOFFMÜLLER, M. SPRINZL

31 Table 2. P NMR Data for Nucleoside 5'-Triphosphates Va—Vd in D20—Methanol-^ (y>r = 9 : 1), T 277 К, с = 0.02 mol dm"3

Compound P-Q c5(J/Hz) P-/3 6( J/Hz) P-7 6( J/Hz)

-8.245 (19.8) -20.003 (19.89) -7.501 (19.4) -8.402 (18.48) -20.099 (br s) -7.769 (18.87) Vc -8.373 (15.46) -20.000 (br s) -7.528 (17.29) -8.212 (19.55) -20.006 (br s) -7.0464 (19.59)

achieved by their precipitation with sodium salt from Table 3. The Yields (n) of Synthesized tRNAphe Transcripts 1 % acetone solution of sodium Perchlorate. The yield per mol of Plasmid of the nucleoside 5'-triphosphates Va— Vd varied from tRNAPhe transcripts containing 15 % to 79 %, depending on the nature of the nucleo- 4 2 5 5 5 5 natural s U s C i C i U br C br U base. The phosphorylation of 2-thiocytidine provided NTPs the lowest yield. Here the reaction of POCl3 with the n(tRNAphe) relative 2-thio function led probably to decomposition 583 435 0 112 136 429 432 of the nucleoside [21]. Chromatographic characteriza­ n(template) tion of the products was performed by TLC and HPLC (Table 1). Reverse-phase ion-paired (RP—IP) analysis of nucleoside 5'-triphosphates on a C-18 column was br5C-, br5U-, and s4U-substituted tRNA transcripts achieved by application of the retaining agent tetra- were slightly lower. For 5-iodonucleotide-substituted butylammonium hydrogen sulfate in an aqueous ace- tRNA transcripts we observed a significant decrease tonitrile solvent system [22]. Chromatographic analy­ of the yield probably due to the slightly larger van der sis of the cytidine derivatives br5CTP and s2CTP, as Waals radius of iodine as compared to bromine. The well as the uridine derivative s4UTP showed a prolon­ lack of incorporation of 2-thiocytidine is surprising. gation of the retention times in comparison with the This polymerase must therefore be especially sensitive retention times of parent nucleoside 5'-triphosphates to modification of cytidine in position 2. Other poly­ due to their increased hydrophobicity. In the case of merizing enzymes, such as polynucleotide Phosphory­ the most lipophilic s4UTP, a less polar solvent sys­ lase [23] or ATP (CTP) tRNA nucleotidyltransferase tem had to be used for elution. All compounds were [24], accept 2-thiocytidine. Possibly, the highly spe­ homogeneous as shown by RP HPLC analysis. cific template-dependent RNA polymerase, which re­ 31P NMR analysis showed spectra typical for nu­ quires correct base-pairing between elongating rNTP cleoside 5'-triphosphates: three signals corresponding and template, cannot read a Watson—Crick s2C—G to a-, /3-, and 7-phosphorus atoms. The most up- base pair. In this base pair the hydrogen bonds are field shifted 6 signal (triplet or broad singlet) at widened by about 0.06 nm in comparison to С—G -20 belongs to the resonance of the /3-phosphorus base pair. This results in weaker hydrogen bonds or in atom, which is shielded by two adjacent electroneg­ a distortion of the base pair geometry [25]. ative phosphorus atoms. Both remaining phosphorus Previously s4UTP was randomly incorporated into atoms, a and 7 appear at lower magnetic field giving RNA by T7 RNA polymerase and the resulting RNA rise to doublets of 6 in the ranges —8.2 to —8.4 and was used for crosslinking studies [26]. In this case the -7.0 to -7.7, respectively (Table 2). photoreactive nucleotide was obtained enzymatically The synthesized nucleoside 5'-triphosphates were using nucleoside 5'-diphosphate kinase, which converts tested as substrates for DNA-dependent RNA poly­ s4UDP into s4UTP [27]. Our attempts to apply the merase from bacteriophage T7. The s2CTP, s4UTP, enzymatically synthesized s4UTP which was prepared and br5CTP as well as the commercially available according to [27] without chromatographic purifica­ br5UTP, i5UTP, and i5CTP were incorporated into tion were also successful, but complete replacement the corresponding position of the T. thermophilus of U by s4U could not be achieved. The latter tran­ tRNAPhe by in vitro transcription. In each transcrip­ scription assay was successful when a chemically syn­ tion experiment one nucleotide derivative was intro­ thesized s4UTP derivative was employed. The use of duced in place of the parent nucleotide. The nucleotide chemically synthesized s4UTP provided RNA in which analogues were accepted as substrates for T7 RNA uridines were completely replaced by 4-thiouridines. polymerase at distinctly different degrees (Table 3). The low activity of enzymatically prepared substrates In the case of the unmodified tRNA transcript, 500 is probably due to contamination. We found the chem­ to 600 tRNA molecules per mole of DNA template ical synthesis of nucleoside 5-triphosphates more con­ were synthesized, thus milligram quantities of tran­ venient for preparative purposes than the alternative script were prepared. The yields of the syntheses of enzymatic method.

154 Chem. Papers 50 (3) 151 —155 (1996) RIBONUCLEOSIDE ^-TRIPHOSPHATES

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