`
`Nucleic Acids Research
`
`Chemical synthesis of 5-azacytidine nucleotides and preparation of tRNAs containing 5-azacytidine
`in its 3'-terminus
`
`
`
`Wojciech S.Zielinski* and Mathias Sprinzl
`
`
`Department of Biochemistry, University of Bayreuth, PO Box 3008, D-8580 Bayreuth, FRG
`
`
`Received 2 May 1984; Revised and Accepted 1 June 1984
`
`ABSTRACT
`
`5-azacytidine-5'-triphosphate prepared from 5-azacytidine by
`chemical phosphorylation is a substrate for AMP(CMP)tRNA nucleo-
`tidyl transferase from yeast.
`tRNAsPhe from yeast containing 5-
`azac tidine in their 3'-fermini were prepared enzymatically.
`tRNA he_cpn CpA and tRNA
`-n Cpn°cpa can be aminoacylated by
`phenylalanyl-tRNA synthetase from yeast and they are active in the
`poly(U)-dependent synthesis of poly(Phe) on E. coli ribosomes.
`The decomposition of 5-azacytidine via hydrolysis of the tri-
`azine ring is significantly accelerated by a phosphate group on
`the 5'-position of the nucleotide. After the incorporation of 5-
`azacytidine-5'-phosphate into a polynucleotide chain the rate of
`hydrolysis of the triazine ring decreases considerably.
`
`INTRODUCTION
`
`5-Azacytidine (1)
`
`is an analogue of cytidine containing an
`
`additional nitrogen heteroatom in the position 5 of the pyrimi-
`
`dine ring (2). As a potent inhibitor of the cell growth this drug
`
`exhibits pronounced antibacterial activity (3) and is used clini-
`
`cally in the treatment of leukemia (4). Recently it was reported
`
`that 5 -azacytidine is able to deblock inactive genes (5-8) pro-
`
`bably by the inhibition of the methylation of cytosine at speci-
`fic sites of the DNA.
`
`Inhibition of protein synthesis is probably the main target
`
`accounting for the strong cytotoxic activity of 5-azacytidine.
`
`Since this effect can be suppressed by actinomycin it was sugge-
`
`sted that the incorporation of the analogue into RNA is crucial
`
`for inhibition (9,10). It was later demonstrated that 5-azacyti-
`
`dine incorporation into mammalian tRNAs causes an inactivation of
`
`tRNA cytosine-5-methyltransferase leading to a marked reduction
`
`of S-methylcytidine content in tRNAs
`
`(11).
`
`5-Azacytidine is unstable and it undergoes a rapid hydro-
`
`
`© IRL Press Limited, Oxford, England.
`
`(cid:38)(cid:40)(cid:47)(cid:42)(cid:40)(cid:49)(cid:40)(cid:3)(cid:21)(cid:20)(cid:22)(cid:20)
`CE LG FE N FE 2 4 31
`5025
`(cid:36)(cid:51)(cid:50)(cid:55)(cid:40)(cid:59)(cid:3)(cid:89)(cid:17)(cid:3)(cid:38)(cid:40)(cid:47)(cid:42)(cid:40)(cid:49)(cid:40)
`APOTEX v. CELGENE
`(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:21)(cid:22)(cid:16)(cid:19)(cid:19)(cid:24)(cid:20)(cid:21)
`IPR2023-00512
`
`
`
`Nucleic Acids Research
`
`
`lytic degradation of its triazine ring at neutral pH (12). Once
`
`the analogue is incorporated into the polynucleotide chain such
`
`degradation could cause changes in RNA structure and account for
`
`inhibitory effects during the protein biosynthesis.
`
`In order to investigate the stability of 5-azacytidine
`
`-containing RNA we chemically synthesized 5-azacytidine-5'-tri-
`
`phosphate, and incorporated this nucleotide analogue into the 3'-
`
`end of tRNAby aATP(CTP)
`
`tRNA nucleotidyl transferase-catalysed
`
`reaction.
`
`In extension of our previous investigations with tRNA
`
`molecules having a modified 3'-terminus (13) we studied the acti-
`
`vity of 5-azacytidine-containing tRNAs
`
`in in vitro protein bio-
`
`synthesis.
`
`MATERIALS AND METHODS
`
`Ph
`
`?*®_apcpcpa anda tRNA’? ©_apcpc were isolated from yeast
`tRNA’
`bulk tRNA (Boehringer, Mannheim Germany)
`(14). The shortened
`tRNAs,
`tRNA??? _anc as well as erwa’??_g were prepared as descri-
`Phe_
`bed previously (15). The aminoacylation capacity of tRNA
`A
`tRNA nucleotidyl transferase, ATP
`in the presence of ATP(CTP)
`and CTP was 1250 pmol
`th4o] phenylalanine / A,,. unit tRNA. ATP
`
`(CTP)tRNA nucleotidyl transferase (E.C. 2.7.7.25)
`
`from baker's
`
`yeast of a specific acitvity of 44 000 units/mg protein (16) was
`
`a gift of Dr. Hans Sternbach, Géttingen. Phenylalanyl-tRNA syn-
`
`thetase (E.C.6.1.1.20.)
`
`from baker's yeast had a specific acti-
`
`vity of 1820 units/mg protein (17). 5-azacytidine was a product
`of Fluka AG (Buchs, Switzerland).
`(+4clphenylalanine 57 Ci/mol
`and [(“*elpnenylalanine
`521 Ci/mol were obtained from Amersham
`Buchler
`(Braunschweig, Germany). Trimethylphosphate was purified
`
`by distillation from sodium under reduced pressure. Dimethylform-
`amide was distilled from calcium hydride and stored over ah
`molecular sieves.
`315, nmr proton-decoupled spectra were recorded
`on a Bruker-Spectra-Spin spectrometer at 81 MHz,
`in aqueous so-
`
`lutions containing 0.1 M EDTA, pH 7.0, and 75 % D,0. Negative
`chemical shift values are assigned for compound absorbing at
`higher field than 85 $% H,P0, external standard. UV-spectra and
`the kinetics of the hydrolysis of 5-azacytidine and its deriva-
`
`tives were recorded with a Beckman DU-8 spectrophotometer.
`
`
`
`5026
`
`
`
`Nucleic Acids Research
`
`Ion exchange thin-layer chromatography (TLC) and thin-layer
`electrophoresis were performed on Polygram Cel 300 PEI/UV 5c,
`precoated sheets and precoated TLC plates Cel 300-10/UV
`(both
`from Macherey-Nagel, Dttren, Germany). For
`ion-exchange TLC the
`
`following systems were used: solvent A,
`
`1 M LiCl; solvent B, 1.5
`
`M LiCl; buffer C,
`
`1 M NaOAc pH 6.0. Thin layer electrophoresis
`
`was run in 0.1 M citrate buffer pH 5.6 at 23 V/cm.
`
`The poly(U)-dependent translation assay on E.coli ribosomes
`
`(18) was described previously (19). The hydrolysis of tRNA with
`ribonuclease Ty and the conditions for HPLC separation of the
`digestion products were described in (20).
`All evaporations were done below 20°C in vacuum using a
`rotary evaporator equipped with a dry-ice condenser. The molar
`
`extinction coefficient for phosphorylated 5-azacytidine deriva-
`
`tives was assumed to be the same as determined for 5-azacytidine,
`
`foes = 2,56 x 103 at pH 1
`
`(0.1 M HCl),
`
`fogs = 6.56 x 10? at pH
`
`7.6 (50 mM TRIS°*HC1).
`
`Aminoacylation of tRNA species was performed in a reaction
`
`mixture (O.1 ml) containing 150 mM TRIS*HCl,
`
`pH 7.5, 100 mM KCl,
`
`50 mM MgCl., 2.5 mM ATP, 2.5 mM 2-mercaptoethanol, 0.02 mM tt4e]-
`phenylalanine, specific acitivity 57 Ci/mol, 0.2 A
`units tRNA
`
`260
`
`and 5
`
`ig phenylanlanyl-tRNA synthetase (17).
`Ph
`Incorporation of 5-azacytidine into tRNA ©_a and tRNA
`
`Phe
`
`-
`
`ApC was accomplished as described previously (15). The reaction
`
`mixture (0.5 ml) contained
`
`150 mM TRIS*HC1, pH 9.0, 150 mM KCl,
`
`15 mM MgCl,
`
`1 mM 2-mercaptoethanol,
`
`2 mM n°CTP, 10-20 A560
`
`units of shortened tRNA and 0.2 mg/ml ATP(CTP)tRNA nucleotidyl
`
`tranferase. After incubation for 4 h at room temperature,
`
`the pH
`
`was adjusted to 5.0 by addition of 2 M sodium acetate pH 4.0.
`
`The reaction mixture was diluted with 0.6 ml water and the solu-
`
`tion was applied onto a column of Sephadex A 25 (0.8 x 5 cm)
`
`equilibrated with 20 mM sodium acetate,
`
`pH 5.2. The column was
`
`washed with 400 mM NaCl
`
`in 20 mM sodium acetate, pH 5.2 (50 ml)
`
`and finally with 1.0 M NaCl
`
`in the same buffer. The high salt
`
`portion of the eluant contained the modified tRNA. It was iso-
`
`lated by desalting on a Biogel P 2 column (15) and concentrated
`
`by evaporation.
`
`
`
`5027
`
`
`
`Nucleic Acids Research
`
`Preparation of 5-azacytidine-5'-monophosphate
`To a stirred suspension of 5-azacytidine (112 mg, 0.5 mmol)
`
`in trimethylphosphate (2 ml), phosphorus oxychloride (70 ul, 0.75
`mmol) was added at o°c. The mixture was stirred with exclusion of
`atmospheric moisture at o°c for 4 h. The resulting clear solution
`was added dropwise to 25 ml stirred ice-cold 0.1 M triethylammo-
`
`nium bicarbonate buffer pH 7.6. After 45 min the pH of the mix-
`
`ture was adjusted to about 5 by addition of the same buffer and
`
`solvents were removed by evaporation. The residue was dissolved -
`
`in water
`Germany)
`
`(2 ml) and applied on the AG 50 Wx2
`(BioRad, Miinchen,
`ion exchanger
`(100-200 mesh) column (1.8 x 27 cm)
`in H*
`
`form. The column was washed with water and the UV-absorbing frac-
`
`tions of the eluate were collected. The appropriate fractions
`
`were pooled. After addition of an equimolar amount of triethyl-
`
`the solution was evaporated. The residue was dissolved in
`amine,
`(1 ml) and freeze-dried.
`water
`The homogeneous product had R,-values
`of 0.63 (solvent A), 0.80 (solvent B), and 0.47 (buffer C) on TLC.
`
`=
`
`Electrophoretic mobility was equal
`to CMP. UV-spectrum:
`i
`max
`255 and 241 nn, Anin = 231 and 224 nm in 0.1 M HCl, and 50 mM
`TRIS - HCl
`(pH 7.6) respectively.
`Je
`ratio = 1.22 in 0.1 M
`©255/
`"244
`31
`(pH 7.6), respectively.
`
`HCl, and 0.78 in 50 mM TRIS-HCl
`
`Ponmr
`
`:
`
`6
`
`= + 2.312 ppm.
`
`H,P0,
`Preparation of 5-azacytidine-5'-triphosphate
`Triethylammonium salt of n°CMP (0.215 mmol) was dissolved in
`85 % aqueous dimethylformamide and n-tributylamine (0.43 mmol)
`
`was added. The resulting clear solution was evaporated, and the
`
`residue dried by repeated coevaporation with dry dimethylform-
`amide (6 x 4 ml). The final residue was dissolved in dimethylform-
`amide (3 ml) and carbonyldiimidazole (0.32 mmol, 52 mg) was added
`with stirring. After 40 min at room temperature the solution was
`
`cooled in ice and tetrakistributylammonium pyrophosphate (0.6
`mmol)
`in dimethylformamide was added dropwise with vigorous stir-
`ring. After 2 h at room temperature,
`the reaction mixture was
`
`cooled in ice,
`
`treated with 30 wl methanol and after 15 min eva-
`
`(5 ml) and applied
`porated. The residue was dissolved in water
`ion-exchange column
`Onto a Dowex 1 x 4
`(200-400 mesh,
`formate)
`(1.x 12 cm). The column was eluted with a linear gradient of am-
`
`5028
`
`
`
`Nucleic Acids Research
`
`
`monium formate (50 mM - 1.0 M, pH 4.2, 600 ml) and fractions con-
`
`taining the product were pooled, concentrated and desalted ona
`
`Sephadex G-10 column (2 x 54 cm). UV-spectrum in 100 mM HCl:
`cae 254, Amine 238;
`€539/£254
`ratio = 0.71; E554/"270 ratio =
`2.74. Electrophoretic mobility was equal
`to CTP. Rr values: 0.16
`(solvent A), 0.25 (solvent B); ai, mr (o in ppm):
`-10.04 (Po),
`-21.36 (P
`d -8.67 (P_);
`I
`= I
`= 19.7 Hz.
`(Pg) an Tae=Te,y(Py?
`
`
`
`RESULTS
`
`Synthesis and stability of 5-azacytidine nucleotides
`In order to avoid decomposition of the unstable triazine
`ring during the synthesis of S-azacytidine-5'-monophosphate and
`
`5-azacytidine-5'-triphosphate the common phosphorylation proce-
`
`dures (21,22) had to be modified. The reactions and purification
`
`steps were performed in the shortest possible time, and extreme
`
`PH conditions were avoided. The average yield for the phosphory-
`
`lation of S-azacytidine to 5-azacytidine-5'-monophosphate in
`
`several experiments was about 70 %. The yield of the triphosphate
`
`synthesis was only about 20 %, and could not be improved by va-
`
`riation of the reaction time or of the stoichiometry of the reac-
`
`ting components. Since no further UV-absorbing by-products were
`
`detected in the reaction mixture, we assume that the low yield of
`
`triphosphate reflects a high reactivity of the triazine ring of
`
`the S-azacytidine with nucleophiles leading to decomposition of
`
`the base and loss of UV-absorption.
`
`The 5-azacytidine-5'~-triphosphate was characterized by its
`34, nmr spectrum, UV-absorbance and chromatographic properties.
`It contained about
`5
`% unidentified impurities and could be
`stored for several weeks in lyophilized form at -20°C without de-
`
`composition. As compared to the enzymatic phosphorylation of 5-
`
`azacytidine, which was utilized previously (23)
`
`the chemical syn-
`
`thesis of 5-azacytidine-nucleotides is convenient especially for
`
`a large scale preparation of the compound.
`
`In aqueous solutions 5-azacytidine decomposes rapidly. The
`
`kinetics of the decomposition suggests that a nucleophilic attack
`
`of a hydroxyl
`
`ion on the position 6 of the triazine ring followed
`
`by an opening of the ring and the formation of a UV-absorbing
`
`aldehyde intermediate takes place during this reaction (12). This
`
`
`5029
`
`
`
`Nucleic Acids Research
`
`Absorbanceat245nm
`
`time [h]
`
`Figure i: UV-monitored
`decomposition of 5-azacytidine
`and its derivatives at 24°C. The
`compounds were transferred from
`a cooled stock solutions to
`quartz cells containing 50 mM
`TRIS-HCl, pH 7.56. The absorbance
`was measured at 245 nm.
`(—) 5-
`azacytidine,
`(---) 5-azacytidine-
`5'-monophosphate,
`(---) 5-
`azacytidine-5'-triphosphate.
`
`is manifested by a transient increase of the absorbance at 245 nm
`
`(fig. 1). The aldehyde is then decomposed to non UV-absorbing pro-
`
`ducts, guanidine and ribose. Measuring the decay of UV-absorbance
`
`at 245 nm curves similar to those obtained for the free nucleoside
`(12) were observed in the case of n° CMP and n°-CTP. By increasing
`the pH from 7.6 to 8.5 the rate of decomposition is accelerated
`
`about three-fold. From the part of the curve reflecting the se-
`
`cond, slow reaction, first order rate constants were calculated.
`
`The introduction of the phosphate group into 5'-position of the
`
`5-azacytidine increases the rate of decomposition. This effect is
`
`Table i First order rate constants and half ee of the decompo-
`sition of 5-azacytidine and its derivatives a),
`
`rate constant
`
`[h_
`
`half time [h]
`
`compound
`
`without Mg?
`
`15 mM Mg?
`
`15 mM Mg?
`
`
`
`
`
`
`
`a) Determined in 50 mM TRIS-HCL, pH 7.6 at 24°c. by monitoring the
`decay of UV-absorbance at 245 nm. The values were calculated for
`the second, slow reaction.
`
`
`5030
`
`
`
`Nucleic Acids Research
`
`
`more pronounced in the case of the 5-azacytidine-5'-triphosphate,
`
`which is more labile than the 5-azacytidine-5'-monophosphate.
`Whereas the Mg-* has no effect on the stability of 5-azacytidine
`or 5-azacytidine-5'-monophosphate,
`the rate of decomposition of
`
`the 5-azacytidine-5'-triphosphate decreases considerably upon
`
`addition of 15 mM magnesium ions.
`
`Synthesis and activity of 5-azacytidine-cortaining tRNAs
`
`ATP(CTP)tRNA nucleotidyl transferase recognizes 5-azacyti-
`
`dine-5'-triphosphate as a substrate and incorporates this modi-
`fied nucleotide into the 3'-end of tRNA. Starting from trnarhe_
`APCl, 6x tRNATPCa in the presence of ATP and n-CTP, respec-
`
`tive tRNAs containing one or two S-azacytidine residues in the
`
`3'-terminus can be prepared by this reaction. No differences in
`the rate or extent of incorporation of n°CMP as compared to the
`natural cytidine nucleotide were observed.
`In order to identify the n°c residues incorporated into the
`polynucleotide chain,
`the modified trnarhe species,
`trnaP?®_apn?
`Cpn°cpa and trna’"*-apepn°cpa were digested by RNase T, and the
`
`formed oligonucleotides were analyzed by HPLC (20). This analy-
`
`tical approach, however, did not provide clear results. Due to
`
`the lability of 5-azacytidine-containing oligonucleotides, se-
`
`veral peaks which are not present in the chromatograms of the
`
`T, digest of native trnaPhe appeared in the chromatograms Geri.
`ved from the modified species. Most probably the expected CpApn
`Cpn-cpa and CpApcpn° cpa Oligonucleotides decomposed during the
`incubation with the nuclease. The oligonucleotides CPAGH or
`CPAPOH originating from the 3'-end of T, ribonuclease digested
`trna? “HAs or tRNA’"°-apc.,, respectively, could not be found
`
`in the chromatograms. This demonstrates that the expected
`
`incorporation of 5-azacytidine into the shortened tRNAs took
`
`place.
`
`5
`5
`‘
`‘
`5
`:
`More direct evidence showing the incorporation of n CMP
`:
`Ph
`into the 3'-terminus of tRNA ° was obtained by enzymatic amino-
`
`acylation of shortened tRNAs in the presence of ATP(CTP)tRNA nu-
`Phe
`
`cleotidyl transferase. The aminoacylation of tRNA
`
`-A
`
`73
`
`and
`
`tRNAP"®-apc.
`
`takes place only in the presence of the CCA end-
`
`regenerating enzyme and is dependent on the presence of CTP.
`5
`<
`n CTP can substitute the natural substrate in these reactions.
`
`
`5031
`
`
`
`Nucleic Acids Research
`
` ;
`
`.
`.
`.
`tes
`Table 2 Enzymatic aminoacylation of native and modified tRNAs
`in the presence of ATP(CTP)tRNA nucleotidyl transferase 3).
`
`Ph
`
`©
`
`without
`
`
`
`
`
`
`Aminoacylation [%]>)
`
`CTP
`
`CTP or n CTP with
`
`~ ab)
`
`-Apc
`
`-ApCpn Cpa
`
`~Apn°Cpn°Cpa
`
`a) 0.2 mg/ml ATP(CTP)tRNA nucleotidyl transferase from yeast and
`2.5 mM ATP were always present in the aminoacylation mixture, 2.5
`mM CTP or n°CTP were added to the aminoacylation assay as indica-
`ted. b) 100 % corresponds to 1250 pmol Phe/Ay¢5 unit tRNA.
`
`Phe
`
`and trnaPP®_apc
`Almost complete aminoacylation of tRNA
`74
`“hr3
`with ATP(CTP)tRNA nucleotidyl transferase and n CTP present in
`Ph
`the aminoacylation mixture, demonstrates the formation of tRNA =.
`ApCpn Cpa and trna??°_apn°cpn cpa. These modified tRNA species
`could also be prepared in preparative scale in a reaction mixture
`Phe acp, n-CTP and ATP(CTP) tRNA
`nucleotidyl transferase. They were aminoacylated to the same ex-
`tent as the native trna?®®_cpcpa in a phenylalanyl-tRNA synthe-
`tase-catalyzed reaction. The yield of aminoacylation could not be
`
`containing the shortened tRNAs
`
`improved by the presence of ATP(CTP)tRNA nucleotidyl transferase
`
`and CTP in the aminoacylation mixture (table 2),
`
`indicating that
`
`the particular positions of the 3'-terminus are completely occu-
`pied by n° CMP residues.
`
`The rate of hydrolytic decomposition of 5-azacytidine, which
`
`P
`Table 3 Effect of incubation of native and modified tRNAs he in
`50 mM TRIS-HCL pH 7.6 on their aminoacylation capacity.
`
`INCUBATION
`
`°
`20°C;
`
`4 days
`
`o
`20 C;
`
`9 days
`
`sd
`38 C;
`
`4 days
`
`% of remaining activity
`
`-Apn°Cpn°Cpa
`
`-ApCpCpA
`5
`-ApCpn CpA
`
`5032
`
`
`
`Nucleic Acids Research
`
`>
`g
`oe
`3
`5
`Ba
`é
`z 2
`
`Figure 2: Poly(U) dependent synthesis
`of poly(Phe) on E. coli ribosomes.
`Experimental conditions are given
`under Materials and Methods.
`(-o-)
`tRNAPhe-cpycpa,
`(-G-)
`tRNAPhE_cpndcpa.
`(-A~)
`tRNAPhe-n5cpn°cpa,
`(-m-)
`tRNAPHE_cocpa but without poly(U).
`
`x
`time [min ]
`
`takes place with a half-time of about 8 h at 24°C (s.fig. 1) was
`compared with the rate of deactivation of 5-azacytidine-contai-
`
`ning tRNAs under identical conditions. For this purpose,
`
`the ef-
`
`fect of prolonged incubation at pH 7.6 on the aminoacylation ca-
`
`pacity of the modified tRNAs was measured. The results summarized
`in the table 3 demonstrate that a prolonged incubation of suena5
`ApCpn°Cpa at room temperature and pH 7.6 does not
`impair its ami-
`noacylation activity. This tRNA is as stable as the native trnaP he
`-~CpCpA. If both cytidines, 75 as well as 74, are replaced by 5-aza-
`
`cytidine a significantly higher loss of aminoacylation activity
`
`was observed upon incubation. Similar results were obtained when
`the incubation was performed at pH 7.6 and 38°C. Thus the presence
`of two neighbouring 5-azacytidines in the polynucleotides facili-
`
`tates the deactivation process. However, even in the unfavourable
`case of the double-modified trna’"*_apn°cpn°-cpa the rate of de-
`activation is about 25 times lower than the rate of hydrolytic
`decomposition of n°CMP.
`Poly(U)-dependent synthesis of poly(Phe) with 5-azacytidine-
`containing Phe-tRNas’@¢ was tested in an in vitro system derived
`from E. coli. The results shown in fig.
`2 demonstrate an inhibi-
`
`tion of the poly(Phe) synthesis, which is dependent on the pre-
`sence of the S-azacytidine in the 3'-end of trnal he, The inhibi-
`tion is higher when both Co, and Co. are replaced by S-azacyti-
`dine as compared to Phe-tRNaA’ Pe in which only the position 75 is
`occupied by the analogue. Since the amount of the aminoacylated
`
`tRNAs as determined by their precipitation in cold aqueous tri-
`
`the observed
`chloroacetic acid was the same in all three assays,
`differences reflect the inhibition of the interaction of Phe-
`
`a 5
`
`033
`
`
`
`Nucleic Acids Research
`
` 5
`
`trna’?®_apcpn°cpa and Phe-tRNa?
`BS aon cpa CpA with the components
`of the ribosomal elongation system, and are not related to the
`
`extent of aminoacylation of the tRNAs.
`
`In control experiments the
`
`tRNAT?®_a_, and tRNA’"®-apc. were regenerated in the presence of
`
`the native substrates CTP and ATP and subsequently aminoacylated.
`These Phe-tRNa’7e species showed identical kinetics of poly(Phe)
`synthesis as native trna’©_cpcpa. Thus the inhibition of poly(Phe)
`synthesis in the case of 5-azacytidine-containing tRNAs is due
`only to the presence of this cytidine analogue in the CCA-end.
`
`DISCUSSION
`
`In the present work S5-azacytidine-5'-monophosphate and 5-
`
`azacytidine-5'-triphosphate were synthesized and the rates of
`
`their decomposition in aqueous solution were measured. An increase
`
`in the rate of decomposition of both nucleotides as compared to
`
`the unphosphorylated 5-azacytidine was observed. The introduction
`
`of the phosphate group into the 5'-position of 5-azacytidine pro-
`
`bably facilitates the nucleophilic attack of water on the carbon
`
`6 atom of the heterocyclic ring presumably by the intramolecular
`
`involvement of the phosphate as a general base catalyst. This
`
`suggestion is compatible with the fact,
`
`that the enhancement of
`
`the rate of hydrolysis is more pronounced in the case of 5-azacy-
`
`tidine-5'-triphosphate as compared to 5-azacytidine-5'-monophos-
`phate. Moreover,
`the binding of Mg”* to triphosphate and a par-
`tial neutralization of the phosphate ion via a bidentate uy"
`complex reverses the hydrolysis-stimulation effect of the 6,yY-
`phosphate groups. No such Mg?*-effect is observed in the case of
`the monophosphate.
`
`Based on several investigations of the chemical modification
`
`of the CCA-end of tRNA (13,24,25) it can be assumed that a hydro-
`
`lytic decomposition of the 5-azacytidine in the 3'-end of tRNA
`
`would lead to complete loss of its aminoacylation activity. Sur-
`prisingly, under conditions under which the n° CMP monomer rapidly
`decomposes,
`the tRNA containing S-azacytidine in the position 75
`is as stable as the native trna?™©_apcpcpa. Most probably the
`structural elements discussed above, which are facilitating the
`
`hydrolytic decomposition of the triazine ring in the free nucleo-
`
`if S5-azacytidine is
`tide, are not existing, or less prominent,
`
`
`5034
`
`
`
`Nucleic Acids Research
`
`incorporated into the polynucleotide chain. Moreover, it was re-
`
`ported that stacking interactions inhibit the addition of
`
`bisulphite on the 5,6 double bond-of the cytidine ring (26,27).
`Since the residues Coy and Coe of trnaPhe from yeast reacted
`
`with 240- and 180-times, respectively, more slowly with bisul-
`
`phite as cytidine-5'-monophosphate (13) it was suggested,
`
`that
`
`the 3'-end of tRNA possess an ordered, stacked structure. It is
`likely,
`that 5-azacytidine in positions 74 and 75 of the trnaPhe
`is also protected by stacking interactions. Such stabilization,
`
`however, does not
`take place in shorter oligonucleotides. This
`can be concluded from our observation that the CpApn°Cpn cpa and
`CpApCpn°Cpa oligonucleotides which should be formed by T,-ribonu-
`
`clease degradation of 5-azacytidine-containing tRNAs are decon-
`
`posed rapidly at neutral pH and cannot be identified by HPLC.
`
`As one possibility to explain the cytotoxicity of 5-azacyti-
`
`dine, it was suggested,
`
`that the incorporation of 5-azacytidine
`
`into the 3'-end of tRNA may lead to deactivation of the tRNAS and
`
`to a general inhibition of the protein biosynthesis (3).
`
`In the
`
`present work we could demonstrate,
`
`that incorporation of 5-azacy-
`
`tidine into tRNA can indeed take place by the reaction catalyzed
`
`by ATP(CTP)tRNA nucleotidyl transferase. We could, however, also
`
`show that the incorporation of this modified nucleotide does not
`
`lead to inhibition of the aminoacylation of tRNA or to a strong
`
`inhibition of protein synthesis. Synthesis of polyphenylalanine
`
`on poly(U)-programmed E. coli 70 S ribosomes is only slightly
`
`slower with modified tRNAs. The consequences of this inhibitory
`
`effect of 5-azacytidine incorporation into tRNA under in vivo con-
`
`ditions may be, however, signifficant. Recent experimental data
`
`indicate,
`
`that the rate of protein biosynthesis and the accuracy
`
`of the translation are related (28). A drug leading even to
`
`slight inhibition of the rate of protein biosynthesis may cause
`
`in vivo a formation of abnormal proteins.
`
`ACKNOWLEDGEMENTS
`
`This work was supported by a short-term FEBS fellowship to
`
`W.Z. We
`
`thank the Deutsche Forschungsgemeinschaft and Fonds der
`
`Chemischen Industrie for financial support.
`
`*Present address: The Salk Institute, PO Box 85 800, San Diego, CA 92138, USA
`
`
`5035
`
`
`
`Nucleic Acids Research
`
`
`(1981) Proc. Natl. Acad. Sci. U.S.A.
`
`REFERENCES
`1. Abbreviations: n°C = 5-azacytidine = 1-(8,D-ribofuranosyl1)-
`4-amino-5-triazine-2(1H)-one; n°CMP and n°CTP = 5-azacytidine-
`5'-monophosphate, and 5-azacytidine-5'-triphosphate, respec-
`tively;
`tRNAPhe-apcpcpa = tRNAPhE = native phenylalanine tRNA
`from yeast;
`tRNAPhe-apCpn°cCpA and tRNAPhe-apn5cCpn5cpaA are
`tRnaPhe analogues,
`in which the C75 or both the C74 and C75,
`respectively, are replaced by S-azacytidine.
`Piskala, A. and Sorm, F.
`(1964) Coll. Czech. Chem. Commun.
`29, 2060-2076.
`(1978) Pharmac. Ther. A. 2, 813-840.
`Vesely,
`J. and Cihak, A.
`Saiki, J.H., Mc Cready, K.B., Veitti, T.J., Whitekar, J.
`and Hoogstraten, B.
`(1978) Cancer 42, 2111-2114.
`Jones, P.A. and Taylor, S.M.
`(1980) Cell 20, 85-93.
`Venolia, L., Gartler, S.M., Wassman, E.R., Yen, P., Mohandas,
`T. and Shapiro, L.J.
`(1982) Proc. Natl. Acad. Sci. U.S.A. 79,
`2352-2354.
`Niwa, O. and Sugahara, T.
`78, 6290-6294.
`Ley, T.J., De Simone, J., Anagnon, N.P., Keller, G.H., Hum-
`phries, R.K., Turner, P.H., Young, N.S., Heller, P. and Nien-~
`huis, A.W.
`(1982) N. Engl. J. Med. 307, 1469-1475.
`Reichmann, M. and Penman, S.
`(1973) Biochem. Biophys. Acta
`324, 282-289.
`(1976) Pharmacolocy 25, 1737-1742.
`Lee, T.T. and Karon, M.R.
`Randerath, K., Tseng, W.-C., Harris, J.S. and Lu L.-J.W.
`(1983)
`in Recent Results in Cancer Research, Vol. 84, Modified
`Nucleosides and Cancer, Nass, G. Ed. Springer-Verlag, Berlin,
`Heidelberg, pp. 283-297.
`Notari, R.E. and DeYoung, Y.L. (1975) J. Pharm. Sci. 64, 1148-1157.
`Sprinzl, M. and Cramer, F.
`(1979) Progr. Nucl. Acids Res. Mol.
`Biol. 22, 1-69.
`Schneider, D., Solfert, R. and von der Haar, F.
`Seyler'z Z. Physiol. Chem. 353, 1330-1336.
`Sprinzl, M. Sternbach, H., von der Haar, F. and Cramer, F.
`(1977) Eur. J. Biochem. 81, 579-589.
`Sternbach, H., von der Haar, F., Schlimme, E., Gaertner, E.
`and Cramer, F.
`(1971) Eur. J. Biochem. 22, 166-172.
`von der Haar, F.
`(1973) Eur. J. Biochem. 34, 84-90.
`Jelenc, P.C.
`(1980) Anal. Biochem. 105, 369-374.
`Wagner, T. and Sprinzl, M. (1980) Eur. J. Biochem. 108, 213-221.
`McLaughlin, L.W., Cramer, F. and Sprinzl, M.
`(1981) Anal.
`Biochem. 112, 60-69.
`Yoshikawa, M., Kato, T. and Takenishi, T.
`Lett. 5065-5068.
`Hoard, D.E. and Ott, D.G. (1965) J. Am.Chem. Soc. 87, 1785-1788.
`Lee, T.T. and Momparler, R.L.
`(1976) Anal. Biochem. 71, 60-67.
`Schulman, L.H. and Goddard, J.P.
`(1973) J. Biol. Chem. 248,
`1341-1345.
`
`(1972) Hoppe-
`
`(1967) Tetrahedron
`
`bt
`
`BmWw
`
`aw
`
`10.
`11.
`
`12.
`1,3 «
`
`14,
`
`LS.
`
`16.
`
`17.
`18.
`19.
`20.
`
`21.
`
`22 «
`2.3.6
`24.
`
`Riehl, N., Remy, P., Ebel, J.P. and Ehresmann, B.
`J. Biochem. 128, 427-433.
`Bhanot, 0O.S. and Chambers, R.W.
`2551-2559.
`Bhanot, 0O.S., Aoyagi, S. and Chambers, R.W.
`Chem. 252, 2566-2574.
`Thompson, R.C. and Karim, A.M.
`U.S.A. 79, 4922-4926.
`
`
`(1982) Eur.
`
`(1977) J. Biol. Chem. 252,
`
`(1977) J. Biol.
`
`(1982) Proc. Natl. Acad. Sci.
`
`25.
`
`26.
`
`27.
`
`28.
`
`5036
`
`