The modified nucleosides of tRNAs. II. Synthesis of 2′-O-methylcytidylyl (3′-5′) cytidine

Voiumo 2 number 6 June 1975 The modified nucleosldes of tRNAs. (3'-5') cytidine Nucleic Acids Research II. Synthesis of 2'-O-methylcytldylyl W. T. Markiewicz and M. Wiewidrowski Institute of Organic Chemistry, Polish Academy of Sciences, ul.Noskowskiego 12/14, 61-704 Poznaft, Poland Received 28 April 1975 ABSTRACT The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2 -trichloroethyl for phosphate hydroxyl group, pchlorophenoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion. INTRODUCTION 2'-O-Methylnucleosides have been found in RNAs from different sources, as well as in rRNAsfl] and tRNAs[l, 2], The knowledge of the chemical behaviour, structural and biological significance of 2'-O-methylnucleoside residues is still fragmentary. 2'-O-Methylated oligonucleotides are more stable under alkaline conditions than other oligoribonucleotides [3]. The synthesis of an alkali-stable diribonucleoside monophosphate, 2'-O-methylcytidylyl (3'-5')cytidine (fig. 1, CmpC) is presented in this communication*. The synthesis of 2'-O-methylcytidylyl (3'-5')cytldine (CmpC) * Since we completed the synthesis described in this communication, the chemical synthesis of another oligoribonucleotide containing a 2'-O-methylnu;lcoslde has been published by T.Neilson et at. [4]. 951 Nucleic Acids Research CmpC (fig. 1) was syntheslsed by the triester method (fig. 2) using as protecting HO OH HO II HO i. OCH "^"3 •t III ' VIII HO IX (C pi 0CH 3 ClPhacO-i^-Ov^C111 R'O&CK CH 3 O. ^JicHa IV R=CIPhac, R'=H V R = H, R'=CIPh«c VIR = R'=CIPhac CIPhac= p-chk>roph«noxyac«tyl NH 2 O^ yOO OCH3 C= CI3CCH,O/NO- XI N 952 " CmpC r B t HC^OCH3 HOn ^ O ^ P § 1 Nucleic Acids Research groups, 2,2,2-trichloroethyl for phosphate hydroxyl group [5], p-chlorophenoxyacetyl for the 5'-hydroxyl group [6], methoxymethylidene for the 2',3'-cis-diol system [7], and benzoyl for the exo-amlno group of cytldlne. Cytidine (fig.2.1) waa methylated using diazomethane in the presence of SnCl2.2H2O [8] giving 2'-O-methylcytldine (II) in 67% yield. We should like to point out that in our preparation 70% of the yield was obtained by crystallisation directly from the reaction mixture without laborious chromatography*. 3'-O-Methylcytidlne (Ila) was obtained also after Dowex 1 x 2 (OH") chromatography of mother liquors In ll%yield. N4-Benzoyl-2'-O-methylcytidine (III) was obtained in as described for cytidine [10]. N4-Benzoyl-2'-O-methylcytidine was treated with p-chlorophenoxyacetyl chloride in CH3CN and 2,6-lutidine. N*-Benzoyl-5'-O-p-chlorophenoxyacetyl-2'-O-methylcytidine (IV) was obtained after column chromatography as a crystalline product In 40% yield 3',5'-diester (VI) were observed also. The 3'-ester (V) and 4 N -Benzoyl-3',5'-di-0-p-chlorophenoxyacetyl 2'-O-methylcytldlne (VI) was obtained as a crystalllneproduct in 14% yield. Studies on the improvement of yield of IV are in progress. N4-Benzoyl-2',3'-O- methoxymethylidenecytidine (VIII) was synthesised from cytidine by a two step method in 49% overall yield, whereas the synthesis described in the literature [7] follows a three-step procedure resulting Ina48%overallyield. distinctly simplifies and shortens the synthesis. Ourprocedure 4 N -Benzoylcytidine (VII), synthesised according to the literature [10J was treated with trimethylorthofor mate leading to two main products In approximately equal amounts, the monoorthoester (VIII) and thebls-orthoester(IX). The bls-orthoester was quantitatively transformed into N4-benzoyl-2',3t-O-methoxymethylidene cytidine under mildly acidic conditions, i.e. In chloroform solution in the presence of silicic acid, or methylene chloride-methanol solution in the presence of catalytic amounts of acid. A similar result was obtained for N4-2'-O-dimethylcytldine [9]. 953 75% yield by selective N-benzoylation of 2'-O-methylcytidine with benzoyl anhydride Nucleic Acids Research N*-Benzoyl-5'-0-p-chlorophenaxyacetyl-2'-0-methylcytidlne (IV) was phosphorylated wlththepyrldlnlumsaltof2,2,2-trlchloroethylphosphate and 2,4,6-trlisopropylbenzenesulphonyl chloride (TPS) In pyridlne [5]. The obtained nucleotide (X) was con- 4 densed with N -benzoyl-2',3'-O-methoxymethylldenecytidine (VIII) in pyridlne in the presence of TPS. The protected 2'-O-methylcytidylyl (S'-S'Jcytidine (XI) was obtained after short column chromatography in 72% yield. The 5'-protecting group of (XI) may be selectively eliminated [11] and the obtained partially blocked dtnucleosidemonophosphate was used for the synthesis of longer ollgonucleotldes. The dirlbonucleoslde monophosphate (XI) was then fully deblocked. The phosphotrlester protecting group was removed with a Zn-Cu couple in DMF [5], the alkali labile saturated methanolic ammonia [5, 6} and the methoxymethylidene group was removed using 0-01N hydrochloric acid followed by ammonia [7]. The product was obtained in 25% yield after a purification by paper-chromatography, and was characterised by UV, electrophoresis, paper- and thin-layer-chromatography in several systems. The chromatographic, electrophoretic and UV analysis of the enzymatic digestions of synthetic dlnucleoslde monophosphate with a snake venom phosphodiesterase showed the presence of 2'-O-methylcytidine and cytidine 5'phosphate in approximately equal amounts and proved it to be 2'-O-methylcytldylyl (3'-5')cytidine. On the basis of above results, it can be stated that the chosen protecting groups had accomplished their task well. EXPERIMENTAL Pyridlne was dried over CaH2 and freshly distilled. TPS (2,4,6-trliso - propylbenzenesulphonyl chloride) was obtained according to [12]. tion reactions with TPS were carried out in darkness. uncorrected. meter, The condensa- Melting points are UV spectra were recorded with a Unicam SP 700 A spectrophoto - IR spectra were measured wfth a Unicam SP 200 G specfcrophotometer. NMR spectra 80 MHz were determined on a Tesla 8S 487 A spec^rcmeter using HMDSO and t-butanol as internal references for non-aqueous arid deuteriurc. oxide solutions respectively. The results of UV data and an elemental analysis of obtained compounds are listed lr_ Tah!e 1. cud thin -layer -chromatography are shown in Table 2. 954 The rss,i!*s of paper- protecting groups benzoyl and p-chlorophenoxyacetyl were removed with half- Nucleic Acids Research Table 1 L'V analysis 3 max x l C ilemtmtal analysis (,H,N) ound calculated for N H, C, H, N Compound Solvent CmpC nHl 282 II pHl MeOH pH12 280 238. 274 271 11 .7 7.2, 8.2 87 242 254 252 2.2 6.4 6.4 46.36 5.73, 16.27 46.69.5.88,16.30 pHl MeOH pH12 280 240, 273 233, 272 12 5 6.8, 7.5 6.5, 7.3 242 228, 255 251 1.6 6.7, 6.1 5.3 45.76 5.78, 16.0 m MeOH 260, 304 20.5, 9 . 4 (...truncated)