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Modification of nucleosides and nucleotides
BIOCHIMICA ET BIOPHYSICA ACTA
455
BBA 95576
MODIFICATION OF NUCLEOSIDES AND NUCLEOTIDES VII. SELECTIVE CYANOETHYLATION OF INOSINE AND P S E U D O U R I D I N E IN YEAST T R A N S F E R RIBONUCLEIC ACID* M I T S U A K I YOSHIDA AND T Y U N O S I N U K I T A
Faculty o[ Pharmaceutical Sciences, University o[ Tokyo, Tokyo (Japan) (Received J a n u a r y 3rd, 1968)
SUMMARY
I. The reaction of nucleosides with acrylonitrile was studied. It was found that, at pH 8.5 and 37 ° in aqueous medium containing dimethylformamide, inosine and pseudouridine (T) were completely Cyanoethylated within 4 h. Under the same conditions, only 4-6 % of uridine was cyanoethylated after 4 h reaction and no reaction was observed between the reagent and adenosine, guanosine and cytidine. 2. The products were identified as Nl-cyanoethylinosine, Nl-cyanoethyl-T and N3-cyanoet hyluridine. 3. rnosine and T residues in yeast tRNA were selectively modified b y this reaction. The sedimentation analysis, gel filtration and chromatographic analysis on DEAE-cellulose of the modified tRNA revealed that no cleavage of internucleotide linkages in the tRNA Occurred by this modification reaction.
INTRODUCTION
Recent progress in nucleic acid chemistry has led to the determination of the primary structures of several yeast transfer RNA (tRNA) molecules, and it has clarified the location of a number of minor nucleotides in their primary structure. However, the significance of these minor nucleotides in the biological function of tRNA remained obscure. A possible approach to the study of these minor nucleotides involves their selective modification by chemical reagents. Several communications on chemical modification of the minor nucleotides have hitherto been reported; thus, 12 oxidation of sulfur-containing nucleoside residue by CARBON, HUNG AND JONES 9 and by LIPSETT 3, modification of isopentenyladenosine residue with iodine by FITTLER AND HALL 4, cyanation of 4-thiouridine b y SANEYOSHI AND NISHIMURA5 and photoreduction of dihydrouridine by CERUTTI AND MILLERe have been reported. Selective modification of pseudouridine (~) residue with acrylonitrile has also been independently reported b y CHAMBERS ~, OFENGAND 8, YOSHIDA AND UKITA1 and recently by RAKE
AND
TENER 9.
Abbreviations: tRNA, transfer RNA; ~//pseudouridine; CE-, cyanoethyl. * A preliminary report of this work has been communicated x.
Biochim. Biophys. Acta, 157 (1968) 455-465
450
i . YOSHIDA, T. UKITA
We have found that inosine was cyanoethylated more rapidly than W with acrylonitrile, whereas uridine reacted much more slowly with the reagent and adenosine, guanosine and cytidine did not react. It was also found that this reagent could be used for the selective modification of these minor nucleotides in tRNA, because the reaction conditions were mild enough to avoid the cleavage of phosphodiester bonds. In this paper, the experiments and results will be reported in detail. MATERIALS AND METHODS
Materials Acrylonitrile was obtained from Daiichi Pure Chemicals Co. Ltd., Tokyo, and purified by distillation. Dimethylformamide was distilled over a small amount of phosphorus pentoxide (approx. 500 rag/l) to remove trace amounts of dimethylamine. Inosine was prepared by deamination of adenosine 1°. ~v was isolated from normal human urine according to the method of CHAMBERS, K U R K O V AND SHAPIRO 11, and ~P 2'(3')-phosphate was isolated from alkaline hydrolysate of yeast RNA by the method described by COHN1.. Escherichia coli phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1.) was prepared according to the procedure of GAREN AND LEVINTHAL1~. Yeast (Torulopsis utilis) t R N A was kindly supplied by Dr. K. MIURA of Nagoya University and further purified on a DEAE-cellulose column according to the method of MONIER, STEPHENSON AND ZAMECNIK 14.
Unless otherwise mentioned, paper chromatography was carried out on Toyo Roshi No. 51 or 53 paper using the following solvents: (A) isopropanol-conc. ammonia-water (7:1:2, v/v/v), (B) n-butanol-water (86:14, v/v), (C) isobutyric acid-o.5 M NH4OH (lO:6, v/v), (D) n-butanol-85 % formic acid-water (77:1o:13, v/v/v), and (E) n-butanol-water (86:14, v/v) with ammonia as vapor phase.
Reaction o/ nucleosides and nucleotides with acrylonitrile Nucleosides or nucleotides were dissolved in a concentration of approx. IO mg/ml in o.I M phosphate or carbonate buffer ~ containing 2. 7 M dimethylformamide and to the mixture acrylonitrile was added in 2.2-2.4 M concentration. The reaction mixture was incubated at 37 °. Aliquots of the reaction mixture were withdrawn at appropriate time intervals, I drop of dilute acetic acid was added to terminate the reaction and the mixture spotted on paper. The paper was developed in Solvent A and the spots separated were eluted with o.oi M HC1 and estimated at 260 m/~.
N1-Cyanoethylinosine (C E-inosine, Ia ) A reaction mixture (3o ml) of inosine (5o rag), o.oi M phosphate (pH 8.5) containing 2.7 M dimethylformamide (25 ml) and acrylonitrile (5 ml) was incubated at 37 °. After 3 h, the mixture was acidified by adding I drop of glacial acetic acid * A m m o n i a , p r i m a r y a n d s e c o n d a r y a m i n e could n o t be u s e d as b u f f e r c o n s t i t u e n t s , since aerylonitrile r e a c t s w i t h t h e s e c o m p o u n d s . T h e c o n t a m i n a t i o n of d i m e t h y l a m i n e in d i m e t h y l f o r m a m i d e m u s t also be a v o i d e d .
Biochim. Biophys. Acta, 157 (1968) 455-465
MODIFICATION OF INOSINE AND PSEUDOURIDINE
457
and evaporated under reduced pressure. The residue was dissolved in a small amount of water and 4-5 ml of ethanol were added to the solution. The materials precipitated were filtered, the filtrate was concentrated to approx. 3 ml, and the concentrated solution was kept in a refrigerator. The crystals which appeared were collected and recrystallized from 95 % ethanol to furnish colorless prisms, m.p. 2o8-211 °, in a yield of 37 mg (63 %). (Found: C, 48.90; H, 5.03; N, 21.64. C13H15OsN5 requires: C, 48.59; H, 4.71; N, 21.8o). The product gave an electrophoretic mobility of R i n o s i n e = 0.52 in o.oi M borate at p H 9.4.
Ni-C yanoethylinosine 5'-phosphate (CE-I M P, Ib ) A reaction mixture which contained inosine 5'-phosphate (disodium salt, IOO rag), o.oi 1~ phosphate (pH 8.5) containing 2.7 M dimethylformamide (20 ml) and acrylonitrile (4 ml) was kept at 37 ° for 3 h. After the reaction, barium acetate was added to the mixture until no further precipitation occurred and the precipitate was removed by centrifugation. The barium salt of the nucleotide was then precipitated by addition of 2 vol. of ethanol to the supernatant, collected by centrifugation and redissolved in water. After removal of the insoluble materials b y filtration, the precipitation with ethanol was repeated three more times. The final precipitate was washed with 66 % ethanol, 99 % ethanol and ether, successively, and the yield of the dried barium salt of the product was 75 mg. (Found: C, 27.58; H, 3.02; N, 11-89; P, 5.33. C13H14OsNsP'Ba'2 H20 requires: C, 27.26; H, 3.17; N, 12.23; P, 5.41). On treatment of this barium salt with E. coli phosphomonoesterase in o.05 M TrisHC1 (pH 8.o), the product gave only one ultraviolet-absorbing spot, the R u r i d i n e value (0.95 in Solvent B) of which coincided with that of Ia.
N1-Cyanoethylpseudouridine (CE-}P, II) }/-' (50 rag) was dissolved in o.oi M phosphate (pH 8.5) (25 ml) containing 2.7 M dimethylformamide, and acrylonitrile (5 ml) was added to the solution. After 4.5 h at 37 °, the reaction was terminated by the addition of I drop of glacial acetic acid and the mixture was evaporated. Remaining dimethylformamide was removed by codistillation with n-butanol under reduced pressure. Residual sirup was dissolved in 95 ~/o ethanol and insoluble material was filtered off. Ethyl acetate was added to the filtrate to give a very slight turbidity and the solution was kept in a refrigerator. C E - T (II) was crystallized by scratching and recrystallized from a mixture of ethanol and ethyl acetate to give colorless needles, the yield of which was 40 mg (60 %). The product melted at 163-165 °. (Found: C, 48.29; H, 5.44; N, 13.98. CI2H1506N3requires: C, 48.48; H, 5.09; N, 14.I4). Paper chromatographic analysis of the reaction mixture after 4.5 h reaction revealed that a minor product (III) besides C E - ~ (main product) was produced. The ultraviolet absorption spectra of this minor product did not show remarkable change in the pH range from 2 to 13.
N1-Cyanoethyluridine (CE-uridine, IV) A mixture of uridine (2oo rag), o.oi M phosphate (25 ml) (pH 8.5), containing 2.7 M dimethylformamide, and acrylonitrile (5 ml) was incubated at 37 °. Even after
Biochim. Biophys. Acta, 157 (1968) 455-465
458
M. Y O S H I D A , T. U K I T A
50 h reaction, only about 55 % of the uridine used was converted to a major product (R,ridine = 1.18 in Solvent B). This product was purified b y cellulose column chromatography using Solvent B to a glass, which gave an ultraviolet absorption m a x i m u m at 264 m#. The spectrum of this compound did not remarkably change in the p H from 2 to 13.
Reaction o/yeast t R N A with acrylonitrile The typical reaction mixture contained yeast t R N A (2-4 mg/ml), o.oi M phosphate-2.7 M dimethylformamide (pH 8.6) and 2.2-2. 4 M acrylonitrile. Before the addition of acrylonitrile, the p H of the mixture was adjusted to p H 8.6. The mixture was tightly stoppered and incubated at 4 o°. The aliquots were withdrawn after various reaction times and cooled in ice water. The t R N A was precipitated by adding 2 vol. of cold ethanol and few drops of 2 M NaC1. The precipitation procedure of the t R N A from water with ethanol was repeated twice, then the t R N A collected was washed with 99 % ethanol and ether, successively, and dried in vacuo. Base analysis o~ modified tRNA Analysis of cyanoethylated t R N A was performed as follows. (a) C E - ~ and CE-uridine. The modified t R N A (approx. 5 rag) was hydrolyzed with I M HC1 (o.I ml) at IOO° for I h*. The hydrolysate was diluted to i ml with water and passed through a Dowex 50 (H + form, 200-400 mesh) column (I.2 c m x 5 cm) equilibrated with 0.05 M HC1. The column was eluted with 0.05 M HC1 and the eluate was monitered continuously by UVICORD. The first ultraviolet-absorbing fractions were collected (about IO ml). The fraction was neutralized with I M K O H to p H 8.0 and 20/~1 of E. coli phosphomonoesterase solution and o.I ml of i M TrisHC1 (pH 8.0) were added. After 14-18 h incubation at 37 °, the mixture was deionized by passing through each column (1.2 cm x i cm) of Dowex 50 (H +) and IR-4 B (OH-), successively, and the deionized solution was concentrated. The solution was spotted on W h a t m a n No. I paper and the paper was developed in Solvent B. After drying, the paper was developed in the same solvent in the same direction. Uridine, ribosylthymine, 7 t, CE-uridine and C E - ~ w e r e separated from each other b y these repeated developments. A typical chromatogram is given in Fig. I. The spots separated were eluted with o.oi M HC1 and estimated spectrophotometrically at 260 m/~. Contents of each component in t R N A were calculated from the ratio to uridine, of which content was 19 mole% in t R N A used. Molar absorptivities at 260 m# used for calculation were as follows: uridine, 9900; 7 j, 8000; CE-uridine, 9900; CE-7 t, 81oo. (b) CE-inosine (as CE-hypoxanthine). The modified t R N A (4-5 rag) was hydrolyzed as described in (a) and the hydrolysate was spotted on W h a t m a n 3 MM paper. The paper was developed by two dimensional chromatography using Solvent D in the first and Solvent E in the second dimensions. The spots corresponding to hypoxanthine and CE-hypoxanthine** were eluted with o.oi M HC1 and estimated spectrophotometrically at 250 m/~. The molar absorption coefficients used for hypoxanthine and CE-hypoxanthine were IO 700 and IO 200, respectively. * U n d e r t h e s e c o n d i t i o n s ~v w a s c o n v e r t e d t o t h e m i x t u r e o f f o u r i s o m e r s 17, b u t n o c h a n g e o f t o t a l a b s o r b a n c e o f ~/1 a t 2 6 0 m / , w a s o b s e r v e d b e f o r e a n d a f t e r t h i s t r e a t m e n t . ** T h e s t a n d a r d s a m p l e o f C E - h y p o x a n t h i n e w a s o b t a i n e d b y h y d r o l y s i s o f C E - i n o s i n e w i t h i M H C 1 a t i o o ° f o r I h.
Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 4 5 5 - 4 6 5
MODIFICATION OF INOSINE AND PSEUDOURIDINE
459
Fig. I. Separation of uridine, r i b o s y l t h y m i n e , kU, CE-uridine and CE-~r / d e r i v e d from modified t R N A . The p r e p a r a t i o n of the m i x t u r e f r o m t h e modified t R N A are described in MATERIALS AND METHODS. a, native t R N A ; b, c y a n o e t h y l a t e d t R N A . The t w o s p o t s h a v i n g RF values lower t h a n t h a t of s t a n d a r d ~ are the isomers of ~ which were p r o d u c e d u n d e r the conditons used for h y d r o lysis of t R N A .
RESULTS AND DISCUSSION
Speci[icity o/ the reaction Inosine, ~, uridine, adenosine, guanosine and cytidine were separately incubated with an excess of acrylonitrile in o.I M phosphate buffer (pH 8.5 and pH 7.5) in the presence of 2.7 M dimethylformamide at 37 °. Paper chromatographic analysis (Solvent B) of the reaction mixture after 4 h reaction showed that inosine and completely disappeared and each new compound was produced, whereas in the case of uridine only 4-6 % of uridine reacted after 4 h. These reaction rates were plotted against reaction time in Fig. 2. In the case of reaction with ~rJ, a faint spot besides that of a major product was observed on the paper chromatogram (Solvent B) prepared after 4 h reaction. No detectable reaction was observed for adenosine, guanosine and cytidine under similar conditions within 4 h. Biochim. Biophys. Acta, 157 (1968) 455-465
460
M. YOSHIDA, T. UKITA REACTION TIME ( h ) 2 3 4 5
1 O0
I
I
i
*
60
-.q.
-
(9 4 0 z Z
im 2o W _.Q 10 8
4 2
I
I
i
I
Fig. 2. R a t e s of c y a n o e t h y l a t i o n of inosine, ~/1 a n d u r i d i n e at p H 8.5 a n d p H 7.5. E a c h nucleoside w a s i n c u b a t e d at 37 ° w i t h 2.2 M acrylonitrile in o.i M p h o s p h a t e c o n t a i n i n g 2. 7 M d i m e t h y l formamide. O - - 0 a n d O - - - O, inosine a t p H 8.5 a n d p H 7.5, respectively; 0 - - - - 0 and 0 - - - O , g l a t p H 8.5 a n d p H 7.5, respectively; [] V1, u r i d i n e a t p H 8. 5.
In the case of the reaction of acrylonitrile with nucleotides, the paper chromatographic analysis (Solvent C) of the reaction mixture indicated specificity of the reaction similar to that for nucleosides. Thus, inosine 5'-phosphate, and ~v 2'(3')-phosphate reacted rapidly with acrylonitrile and uridine 2'(3')-phosphate reacted only slowly. Adenosine, guanosine and cytidine 2' (3')-phosphates were not affected.
Characterization o/reaction products Inosine was reacted with acrylonitrile on a preparative scale. A crystalline product (Ia) which was isolated from the reaction mixture gave elemental analysis data coinciding with mono-CE-inosine. The ultraviolet absorption spectra of Ia
q~ 0
10
10
8
8
6
1/I
Q
%6 X \
b);?,
X UJ
4
'\\%~ %X%XX x
2
230
250 270 290 wavelength (rap)
2
240
260
wovzlength
280
300
(rap)
Fig. 3. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of CE-inosine in n e u t r a l a n d alkaline media. T h e s p e c t r a were t a k e n a t p H 2.o (a), p H 7.0 (b) a n d p H 13 (c). Fig. 4. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of CE-t//. T h e s p e c t r a were t a k e n at p H 7.o (a) a n d p H 13 (b).
Biochim. Biophys. Acta, 157 ([968) 455-465
401
MODIFICATION OF INOSINE AND PSEUDOURIDINE
given in Fig. 3 indicated that the product (Ia) had no dissociatable group in alkaline medium. This character was confirmed b y the electrophoretic mobility of Ia in borate buffer at p H 9.4, thus, the borate complex of Ia traveled as a monobasic acid, whereas that of inosine moved as a dibasic acid. Thus, the product (Ia) was identified as N1-CE-inosine. The corresponding reaction product (fb) for inosine 5'-phosphate was isolated as the barium salt. After dephosphorylation b y E. coli phosphomonoesterase, Ib gave a single spot corresponding to Ia in paper chromatography (Solvent B). This indicates that the product (Ib) is NLCE-inosine 5'-phosphate. 0
Hr~N' N~NN'>,
CH2~CHC N
0
NCCH2CH2~
RI
I
"r R
0
0
0
,
H~I'~IL"~HCI~CHCN NCCH2CH2N~L'NCFI H 2=CHCNNCCH2CH2~2CH2CN R
R ~
O
R m 0
0~1 CH2=CHCN NCCH2CH2c~ R a, R=rJbose
RI E b, R=ribose~-oP 2',(~)-phosphate
The cyanoethylated product (II) from }/' was also obtained as crystals. From elemental analysis and the ultraviolet absorption spectra (Fig. 4), this product (I1) was identified as N1-CE-~. Another minor product (nI) which was detected in the cyanoethylation of }/' must be N1,N3-diCE-~ as identified b y CHAMBERS7 and OFENG A N D 8.
Although the product (IV) of cyanoethylation of uridine could not be isolated as crystals, we concluded that the product was Na-CE-uridine as reported by O F E N G A N D 8, since the ultraviolet absorption spectra showed no change in alkaline medium as it has been observed for N1,N~-disubstituted uraciP.
Properties o/cyanoethylated products By treatment with o.3 M K O H at 37 ° for 18 h, CE-inosine and CE-uridine were converted to unknown compounds and CE-7 t was partially hydrolyzed. On treatment with I M HC1 at IOO° for i h CE-inosine was completely hydrolyzed to CE-hypoxanthine, but neither CE-uridine nor CE-}P was affected b y this treatment.
pH profiles o/ the reaction As seen in Fig. 2, the reaction between acrylonitrile and inosine or ~ follows pseudo-first-order kinetics. The apparent rate constants were plotted against p H Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 4 5 5 - 4 6 5
462
M. YOSHIDA, T. UKITA
1 O0
.
.
.
.
.
.
.
.
.
.
.
80
60 tD
o x_ a 0
7.0
8.0
9.0
I0~3
11.0
pH Fig. 5' The p H profile of the reaction. The reaction conditons were similar to Fig. 2. Over p H 9.o, p h o s p h a t e w a s r e p l a c e d w i t h c a r b o n a t e buffer. T h e r a t e c o n s t a n t s w e re c a l c u l a t e d u s i n g t h e e q u a t i o n for f i r s t - o r d e r k i n e t i c s . G - G , inosine; 0 - - - 0 , ~r/.
(Fig. 5). The p K values for inosine and ~ calculated from these curves were approx. 9.0 and 9.6, respectively. The dissociated species of the nucleosides are probably involved in this reaction, since the p K values obtained from Fig. 5 were in agreement with p K a values 9.I and 9.4 of these compounds which were measured spectrophotometrically in 3.0 M dimethylformamide solution.
Side reaction in prolonged reaction time rt was found that when the reaction of acrylonitrile with several nucleosides, under the conditions described above, was prolonged to 48 h, all of the nucleosides including adenosine, guanosine and cytidine, were partially converted to the respective new products. The paper chromatographic analysis revealed that the yields of such products ranged from 8 to IO %. These products gave ultraviolet absorption spectra similar to those of the corresponding nucleoside used. Their spots on the chromatogram did not consume periodate and moved as neutral compounds in electrophoresis run in borate buffer. This non-specific reaction was found to be strongly inhibited b y adding o.ooi M borate in the reaction mixture. These observations indicated t h a t the products must be 2'- or 3'-0-CE-nucleosides. The RF values of TABLE
I
R e l a t i v e R F v a l u e s of c y a n o e t h y l a t e d nucleosides. A s c e n d i n g p a p e r c h r o m a t o g r a p h y w a s c a r r i e d o u t a t r o o m t e m p e r a t u r e a n d R F v a l u e s r e l a t i v e to u r i d i n e are given.
Nucleoside
Uridine NZ-CE -urid ine O-CE-uridine N1-CE-~ r/ N 1, N3 -d iCE-tF Inosine
R~r~e~,,e
Nucleoside
Solvent A
Solvent B
I.OO 1.7 ° 1.28 0.77 0.95 1.27 0.83
i.oo 1.81 -
-
0.49 0.67 1.19 0.69
Biochim. Biophys. Acta, 157 (1968) 455-465
Nx-CE-inosine Adenosine O-CE-adenosine Guanosine O-CE-guanosine Cytidine O-CE-cytidine
R~.~a~n. Solvent A
Solvent B
1.43 1.2z 1.45 0.62 0.87 1.13 1.4o
o.95 i. 13 1.7o 0.48 o.81 o.61 I. 16
4~3
MODIFICATION OF INOSINE AND PSEUDOURIDINE
these products and the reaction rate of adenosine compared with t h a t of uridine are given in Table I and Fig. 6, respectively. Such a non-specific reaction was not observed in the case of reaction for nucleoside 2'(3')-phosphate under similar conditions. 5O
"~ 4 0
c=3o ,,,, U
*-20 o
1o
1 O0
~
80
uridln¢
~/
8
~ ~o
2O
O-CE-adenosine
16
24
reoction
32 time
40
4t5
Z .... i_ oI 0 CE-~'
0
10
56
20 reaction
(h)
30
40
50
t~me {h}
Fig. 6. R a t e s of c y a n o e t h y l a t i o n of a d e n o s i n e a n d u r i d i n e w i t h acrylonitrile. T h e r e a c t i o n c o n d i t i o n s as in Fig. 2. Q ) - G , N - C E - u r i d i n e ; 0 - 0 , O-CE-adenosine. Fig. 7. R a t e s of c y a n o e t h y l a t i o n of inosine, krt a n d u r i d i n e r e s i d u e s in t R N A . t R N A w a s t r e a t e d w i t h 2. 4 M acrylonitrile in o.oi M p h o s p h a t e c o n t a i n i n g 2. 7 M d i m e t h y l f o r m a m i d e a t 39-4 o°.
Modi/ication o~ yeast tRNA with acrylonitrile To a solution of yeast t R N A in o.oi M phosphate (pH 8.6) containing 2.7 M dimethylformamide was added acrylonitrile to 2.2-2.4 M concentration and the mixture was incubated at 39-4 o°. The rates of cyanoethylation of inosine, kv and uridine residues were analyzed as described in MATERIALS AND METHODS and the results are given in Fig. 7- As is seen in Fig. 7, over 9 ° % of ~t and inosine residues in t R N A were cyanoethylated after 48 h reaction, while the extent of the modification of uridine was less than 8 °/o. The figure indicates t h a t the specificity of the reaction was similar to that observed for nucleosides, but in the case of t R N A the reaction rates remarkably decreased in comparison with those for nucleosides. This decrease in reaction rate is probably due to the secondary structure of tRNA. No cyanoethylated product of adenosine, guanosine or cytidine residues was detected in the alkaline hydrolysate of the modified t R N A when analyzed according to the method
o f T A D A , T A D A AND YAG115 o r K A T Z AND COMB 16.
Properties o~ the modi/ied tRNA In order to check whether or not the nucleotide linkages of t R N A are split b y the modification, the properties of the modified t R N A in sedimentation analysis, gel filtration and DEAE-cellulose column chromatography were compared with those of native tRNA. The ultracentrifugal pattern of the t R N A which was modified for 48 h indicated its homogeneity (Fig. 8b). The sedimentation coefficient, 3.63 S, obtained from this pattern was similar to that of native tRNA, 3.56 S. The elution patterns of the modified t R N A from both Sephadex G-5o and DEAE-cellulose columns revealed a single peak at the same position as native t R N A as shown in Figs. 9 and IO. Biochim. Biophys. Acta, 157 (1968) 455-465
404
M. YOSHIDA, T. UKITA
Fig. 8. S e d i m e n t a t i o n p a t t e r n s of native (a) and modified t R N A ' s (b). The modified t R N A was p r e p a r e d by t r e a t m e n t of t R N A u n d e r conditions similar to Fig. 7 for 48 h. The p a t t e r n s were o b t a i n e d for 0.2 M NaC1 solutions of the t R N A using a Spinco Model E, at 48 rain after reaching a m a x i m u m speed of 59 800 rev./min.
1.4
oE
I t
L o
2.0
0.6
G
c o
.~o 1.o
1.0
-S-
0.2
3.5
I-.. t
4
8
12 fraction
16
20
24
28
NO.
32
0
50
100 vo[ urn¢
150
(ml}
Fig. 9. Gel filtration of native a n d modified t R N A ' s t h r o u g h S e p h a d e x G-5o. Native ( - - - ) or modified t R N A ( ), p r e p a r e d as in Fig. 8, w a s applied to a c o l u m n (o.8 cm x 60 cm) of S e p h a d e x G-5o. E l u t i o n was carried o u t using 0.05 M a m m o n i u m acetate and 1.5-ml fractions were collected. The area m a r k e d with the a r r o w is the position for uridine 5 ' - p h o s p h a t e to be eluted f r o m the same column. Fig. IO. E l u t i o n p a t t e r n of t R N A from DEAE-cellulose column. N a t i v e (- - - ) or modified t R N A ( - - - - - - ) , p r e p a r e d as in Fig. 8, w a s charged on the DEAE-cellulose c o l u m n (0.9 cm × i2.o cm). The c o l u m n was eluted w i t h a linear gradient f o r m e d from IOO ml each of o.o2 M Tris-HC1 (pH 7.5) and I M NaCl-o.o2 M Tris-HC1 (pH 7.5). F r a c t i o n s of approx. 3 ml were collected.
These results indicate that no gross cleavage of phosphodiester bonds of t R N A occurred during tile modification reaction. The possibility that the modified t R N A is partially cleaved b y the modification reaction and still behaved similarly to native t R N A could be excluded, since, as will be reported in a forthcoming paper 19, the secondary structure of the t R N A was almost disrupted in the reaction conditions used for the modification. If the cleavage of t R N A chain should occur, the fragments could not be held together b y hydrogen bonding. Biochim. Biophys. Acta, 157 (1968) 455-465
0
MODIFICATION OF INOSINE AND PSEUDOURIDINE
405
In conclusion, inosine and 7 t in tRNA can selectively be cyanoethylated and the reaction conditions are mild enough to avoid the cleavage of polynucleotide linkages in tRNA.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. KIN-ICHIRO MIURA of Nagoya University for his gift of Torula yeast tRNA. We are also indebted to Mr. YASUHIKO FUTAMI of this laboratory for his isolation of pseudouridylic acid from yeast RNA.
REFERENCES i 2 3 4 5 6 7 8 9 lO II 12 13 14 15 16 17 18 19
M. YOSHIDA AND T. UKITA,J. Biochem. Tokyo, 57 (1965) 818. J. A. CARBON, L. HUNG AND D. S. JONES, Proc. Natl. Acad. Sci. U.S., 53 (1965) 979. M. N. LIPSETT, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 449. F. FITTLER AND R. H. HALL, Biochem. Biophys. Res. Commun., 25 (1966) 441. M. SANEYOSHI AND S. NISHIMORA, Biochim. Biophys. Acta, 145 (1967) 208. P. CERUTTI AND N. MILLER, J. Mol. Biol., 26 (1967) 55R. W. CHAMBERS, Biochemistry, 4 (1965) 219. J. OFENGAND,Biochem. Biophys. Res. Commun., 18 (1965) 192. A. V. RAKE AND G. M. TENER, Biochemistry, 5 (1966) 3992. P. A. LEVENE AND W. A. JACOB, Bet., 43 (191o) 315 o. R. W. CHAMBERS, V. KURKOV AND R. SHAPIRO, Biochemistry, 2 (1963) 1192. W. E. COHN, Biochem. Prepn., 8 (1961) 116. A. GAREN AND C. LEVINTHAL, Biochim. Biophys. Acta, 38 (1959) 47 o. R. MONIER, M. L. STEPHENSON AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 43 (196o) I. M. TADA, M. TADA AND K. YAGI, J. Biochem. Tokyo, 55 (1964) 136. S. KATZ AND D. G. COMB, J. Biol. Chem., 238 (1963) 3065 . R. W. CHAMBERS AND V. KURKOV, Biochemistry, 3 (1964) 326. D. SHUGAR AND J. J. F o x , Biochim. Biophys. Acta, 9 (1952) 199. M. YOSHIDA AND T. UKITA,Biochim. Biophys. Acta, 157 (1968) 466.
Biochim. Biophys. Acta, 157 (1968) 455-465
455
BBA 95576
MODIFICATION OF NUCLEOSIDES AND NUCLEOTIDES VII. SELECTIVE CYANOETHYLATION OF INOSINE AND P S E U D O U R I D I N E IN YEAST T R A N S F E R RIBONUCLEIC ACID* M I T S U A K I YOSHIDA AND T Y U N O S I N U K I T A
Faculty o[ Pharmaceutical Sciences, University o[ Tokyo, Tokyo (Japan) (Received J a n u a r y 3rd, 1968)
SUMMARY
I. The reaction of nucleosides with acrylonitrile was studied. It was found that, at pH 8.5 and 37 ° in aqueous medium containing dimethylformamide, inosine and pseudouridine (T) were completely Cyanoethylated within 4 h. Under the same conditions, only 4-6 % of uridine was cyanoethylated after 4 h reaction and no reaction was observed between the reagent and adenosine, guanosine and cytidine. 2. The products were identified as Nl-cyanoethylinosine, Nl-cyanoethyl-T and N3-cyanoet hyluridine. 3. rnosine and T residues in yeast tRNA were selectively modified b y this reaction. The sedimentation analysis, gel filtration and chromatographic analysis on DEAE-cellulose of the modified tRNA revealed that no cleavage of internucleotide linkages in the tRNA Occurred by this modification reaction.
INTRODUCTION
Recent progress in nucleic acid chemistry has led to the determination of the primary structures of several yeast transfer RNA (tRNA) molecules, and it has clarified the location of a number of minor nucleotides in their primary structure. However, the significance of these minor nucleotides in the biological function of tRNA remained obscure. A possible approach to the study of these minor nucleotides involves their selective modification by chemical reagents. Several communications on chemical modification of the minor nucleotides have hitherto been reported; thus, 12 oxidation of sulfur-containing nucleoside residue by CARBON, HUNG AND JONES 9 and by LIPSETT 3, modification of isopentenyladenosine residue with iodine by FITTLER AND HALL 4, cyanation of 4-thiouridine b y SANEYOSHI AND NISHIMURA5 and photoreduction of dihydrouridine by CERUTTI AND MILLERe have been reported. Selective modification of pseudouridine (~) residue with acrylonitrile has also been independently reported b y CHAMBERS ~, OFENGAND 8, YOSHIDA AND UKITA1 and recently by RAKE
AND
TENER 9.
Abbreviations: tRNA, transfer RNA; ~//pseudouridine; CE-, cyanoethyl. * A preliminary report of this work has been communicated x.
Biochim. Biophys. Acta, 157 (1968) 455-465
450
i . YOSHIDA, T. UKITA
We have found that inosine was cyanoethylated more rapidly than W with acrylonitrile, whereas uridine reacted much more slowly with the reagent and adenosine, guanosine and cytidine did not react. It was also found that this reagent could be used for the selective modification of these minor nucleotides in tRNA, because the reaction conditions were mild enough to avoid the cleavage of phosphodiester bonds. In this paper, the experiments and results will be reported in detail. MATERIALS AND METHODS
Materials Acrylonitrile was obtained from Daiichi Pure Chemicals Co. Ltd., Tokyo, and purified by distillation. Dimethylformamide was distilled over a small amount of phosphorus pentoxide (approx. 500 rag/l) to remove trace amounts of dimethylamine. Inosine was prepared by deamination of adenosine 1°. ~v was isolated from normal human urine according to the method of CHAMBERS, K U R K O V AND SHAPIRO 11, and ~P 2'(3')-phosphate was isolated from alkaline hydrolysate of yeast RNA by the method described by COHN1.. Escherichia coli phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1.) was prepared according to the procedure of GAREN AND LEVINTHAL1~. Yeast (Torulopsis utilis) t R N A was kindly supplied by Dr. K. MIURA of Nagoya University and further purified on a DEAE-cellulose column according to the method of MONIER, STEPHENSON AND ZAMECNIK 14.
Unless otherwise mentioned, paper chromatography was carried out on Toyo Roshi No. 51 or 53 paper using the following solvents: (A) isopropanol-conc. ammonia-water (7:1:2, v/v/v), (B) n-butanol-water (86:14, v/v), (C) isobutyric acid-o.5 M NH4OH (lO:6, v/v), (D) n-butanol-85 % formic acid-water (77:1o:13, v/v/v), and (E) n-butanol-water (86:14, v/v) with ammonia as vapor phase.
Reaction o/ nucleosides and nucleotides with acrylonitrile Nucleosides or nucleotides were dissolved in a concentration of approx. IO mg/ml in o.I M phosphate or carbonate buffer ~ containing 2. 7 M dimethylformamide and to the mixture acrylonitrile was added in 2.2-2.4 M concentration. The reaction mixture was incubated at 37 °. Aliquots of the reaction mixture were withdrawn at appropriate time intervals, I drop of dilute acetic acid was added to terminate the reaction and the mixture spotted on paper. The paper was developed in Solvent A and the spots separated were eluted with o.oi M HC1 and estimated at 260 m/~.
N1-Cyanoethylinosine (C E-inosine, Ia ) A reaction mixture (3o ml) of inosine (5o rag), o.oi M phosphate (pH 8.5) containing 2.7 M dimethylformamide (25 ml) and acrylonitrile (5 ml) was incubated at 37 °. After 3 h, the mixture was acidified by adding I drop of glacial acetic acid * A m m o n i a , p r i m a r y a n d s e c o n d a r y a m i n e could n o t be u s e d as b u f f e r c o n s t i t u e n t s , since aerylonitrile r e a c t s w i t h t h e s e c o m p o u n d s . T h e c o n t a m i n a t i o n of d i m e t h y l a m i n e in d i m e t h y l f o r m a m i d e m u s t also be a v o i d e d .
Biochim. Biophys. Acta, 157 (1968) 455-465
MODIFICATION OF INOSINE AND PSEUDOURIDINE
457
and evaporated under reduced pressure. The residue was dissolved in a small amount of water and 4-5 ml of ethanol were added to the solution. The materials precipitated were filtered, the filtrate was concentrated to approx. 3 ml, and the concentrated solution was kept in a refrigerator. The crystals which appeared were collected and recrystallized from 95 % ethanol to furnish colorless prisms, m.p. 2o8-211 °, in a yield of 37 mg (63 %). (Found: C, 48.90; H, 5.03; N, 21.64. C13H15OsN5 requires: C, 48.59; H, 4.71; N, 21.8o). The product gave an electrophoretic mobility of R i n o s i n e = 0.52 in o.oi M borate at p H 9.4.
Ni-C yanoethylinosine 5'-phosphate (CE-I M P, Ib ) A reaction mixture which contained inosine 5'-phosphate (disodium salt, IOO rag), o.oi 1~ phosphate (pH 8.5) containing 2.7 M dimethylformamide (20 ml) and acrylonitrile (4 ml) was kept at 37 ° for 3 h. After the reaction, barium acetate was added to the mixture until no further precipitation occurred and the precipitate was removed by centrifugation. The barium salt of the nucleotide was then precipitated by addition of 2 vol. of ethanol to the supernatant, collected by centrifugation and redissolved in water. After removal of the insoluble materials b y filtration, the precipitation with ethanol was repeated three more times. The final precipitate was washed with 66 % ethanol, 99 % ethanol and ether, successively, and the yield of the dried barium salt of the product was 75 mg. (Found: C, 27.58; H, 3.02; N, 11-89; P, 5.33. C13H14OsNsP'Ba'2 H20 requires: C, 27.26; H, 3.17; N, 12.23; P, 5.41). On treatment of this barium salt with E. coli phosphomonoesterase in o.05 M TrisHC1 (pH 8.o), the product gave only one ultraviolet-absorbing spot, the R u r i d i n e value (0.95 in Solvent B) of which coincided with that of Ia.
N1-Cyanoethylpseudouridine (CE-}P, II) }/-' (50 rag) was dissolved in o.oi M phosphate (pH 8.5) (25 ml) containing 2.7 M dimethylformamide, and acrylonitrile (5 ml) was added to the solution. After 4.5 h at 37 °, the reaction was terminated by the addition of I drop of glacial acetic acid and the mixture was evaporated. Remaining dimethylformamide was removed by codistillation with n-butanol under reduced pressure. Residual sirup was dissolved in 95 ~/o ethanol and insoluble material was filtered off. Ethyl acetate was added to the filtrate to give a very slight turbidity and the solution was kept in a refrigerator. C E - T (II) was crystallized by scratching and recrystallized from a mixture of ethanol and ethyl acetate to give colorless needles, the yield of which was 40 mg (60 %). The product melted at 163-165 °. (Found: C, 48.29; H, 5.44; N, 13.98. CI2H1506N3requires: C, 48.48; H, 5.09; N, 14.I4). Paper chromatographic analysis of the reaction mixture after 4.5 h reaction revealed that a minor product (III) besides C E - ~ (main product) was produced. The ultraviolet absorption spectra of this minor product did not show remarkable change in the pH range from 2 to 13.
N1-Cyanoethyluridine (CE-uridine, IV) A mixture of uridine (2oo rag), o.oi M phosphate (25 ml) (pH 8.5), containing 2.7 M dimethylformamide, and acrylonitrile (5 ml) was incubated at 37 °. Even after
Biochim. Biophys. Acta, 157 (1968) 455-465
458
M. Y O S H I D A , T. U K I T A
50 h reaction, only about 55 % of the uridine used was converted to a major product (R,ridine = 1.18 in Solvent B). This product was purified b y cellulose column chromatography using Solvent B to a glass, which gave an ultraviolet absorption m a x i m u m at 264 m#. The spectrum of this compound did not remarkably change in the p H from 2 to 13.
Reaction o/yeast t R N A with acrylonitrile The typical reaction mixture contained yeast t R N A (2-4 mg/ml), o.oi M phosphate-2.7 M dimethylformamide (pH 8.6) and 2.2-2. 4 M acrylonitrile. Before the addition of acrylonitrile, the p H of the mixture was adjusted to p H 8.6. The mixture was tightly stoppered and incubated at 4 o°. The aliquots were withdrawn after various reaction times and cooled in ice water. The t R N A was precipitated by adding 2 vol. of cold ethanol and few drops of 2 M NaC1. The precipitation procedure of the t R N A from water with ethanol was repeated twice, then the t R N A collected was washed with 99 % ethanol and ether, successively, and dried in vacuo. Base analysis o~ modified tRNA Analysis of cyanoethylated t R N A was performed as follows. (a) C E - ~ and CE-uridine. The modified t R N A (approx. 5 rag) was hydrolyzed with I M HC1 (o.I ml) at IOO° for I h*. The hydrolysate was diluted to i ml with water and passed through a Dowex 50 (H + form, 200-400 mesh) column (I.2 c m x 5 cm) equilibrated with 0.05 M HC1. The column was eluted with 0.05 M HC1 and the eluate was monitered continuously by UVICORD. The first ultraviolet-absorbing fractions were collected (about IO ml). The fraction was neutralized with I M K O H to p H 8.0 and 20/~1 of E. coli phosphomonoesterase solution and o.I ml of i M TrisHC1 (pH 8.0) were added. After 14-18 h incubation at 37 °, the mixture was deionized by passing through each column (1.2 cm x i cm) of Dowex 50 (H +) and IR-4 B (OH-), successively, and the deionized solution was concentrated. The solution was spotted on W h a t m a n No. I paper and the paper was developed in Solvent B. After drying, the paper was developed in the same solvent in the same direction. Uridine, ribosylthymine, 7 t, CE-uridine and C E - ~ w e r e separated from each other b y these repeated developments. A typical chromatogram is given in Fig. I. The spots separated were eluted with o.oi M HC1 and estimated spectrophotometrically at 260 m/~. Contents of each component in t R N A were calculated from the ratio to uridine, of which content was 19 mole% in t R N A used. Molar absorptivities at 260 m# used for calculation were as follows: uridine, 9900; 7 j, 8000; CE-uridine, 9900; CE-7 t, 81oo. (b) CE-inosine (as CE-hypoxanthine). The modified t R N A (4-5 rag) was hydrolyzed as described in (a) and the hydrolysate was spotted on W h a t m a n 3 MM paper. The paper was developed by two dimensional chromatography using Solvent D in the first and Solvent E in the second dimensions. The spots corresponding to hypoxanthine and CE-hypoxanthine** were eluted with o.oi M HC1 and estimated spectrophotometrically at 250 m/~. The molar absorption coefficients used for hypoxanthine and CE-hypoxanthine were IO 700 and IO 200, respectively. * U n d e r t h e s e c o n d i t i o n s ~v w a s c o n v e r t e d t o t h e m i x t u r e o f f o u r i s o m e r s 17, b u t n o c h a n g e o f t o t a l a b s o r b a n c e o f ~/1 a t 2 6 0 m / , w a s o b s e r v e d b e f o r e a n d a f t e r t h i s t r e a t m e n t . ** T h e s t a n d a r d s a m p l e o f C E - h y p o x a n t h i n e w a s o b t a i n e d b y h y d r o l y s i s o f C E - i n o s i n e w i t h i M H C 1 a t i o o ° f o r I h.
Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 4 5 5 - 4 6 5
MODIFICATION OF INOSINE AND PSEUDOURIDINE
459
Fig. I. Separation of uridine, r i b o s y l t h y m i n e , kU, CE-uridine and CE-~r / d e r i v e d from modified t R N A . The p r e p a r a t i o n of the m i x t u r e f r o m t h e modified t R N A are described in MATERIALS AND METHODS. a, native t R N A ; b, c y a n o e t h y l a t e d t R N A . The t w o s p o t s h a v i n g RF values lower t h a n t h a t of s t a n d a r d ~ are the isomers of ~ which were p r o d u c e d u n d e r the conditons used for h y d r o lysis of t R N A .
RESULTS AND DISCUSSION
Speci[icity o/ the reaction Inosine, ~, uridine, adenosine, guanosine and cytidine were separately incubated with an excess of acrylonitrile in o.I M phosphate buffer (pH 8.5 and pH 7.5) in the presence of 2.7 M dimethylformamide at 37 °. Paper chromatographic analysis (Solvent B) of the reaction mixture after 4 h reaction showed that inosine and completely disappeared and each new compound was produced, whereas in the case of uridine only 4-6 % of uridine reacted after 4 h. These reaction rates were plotted against reaction time in Fig. 2. In the case of reaction with ~rJ, a faint spot besides that of a major product was observed on the paper chromatogram (Solvent B) prepared after 4 h reaction. No detectable reaction was observed for adenosine, guanosine and cytidine under similar conditions within 4 h. Biochim. Biophys. Acta, 157 (1968) 455-465
460
M. YOSHIDA, T. UKITA REACTION TIME ( h ) 2 3 4 5
1 O0
I
I
i
*
60
-.q.
-
(9 4 0 z Z
im 2o W _.Q 10 8
4 2
I
I
i
I
Fig. 2. R a t e s of c y a n o e t h y l a t i o n of inosine, ~/1 a n d u r i d i n e at p H 8.5 a n d p H 7.5. E a c h nucleoside w a s i n c u b a t e d at 37 ° w i t h 2.2 M acrylonitrile in o.i M p h o s p h a t e c o n t a i n i n g 2. 7 M d i m e t h y l formamide. O - - 0 a n d O - - - O, inosine a t p H 8.5 a n d p H 7.5, respectively; 0 - - - - 0 and 0 - - - O , g l a t p H 8.5 a n d p H 7.5, respectively; [] V1, u r i d i n e a t p H 8. 5.
In the case of the reaction of acrylonitrile with nucleotides, the paper chromatographic analysis (Solvent C) of the reaction mixture indicated specificity of the reaction similar to that for nucleosides. Thus, inosine 5'-phosphate, and ~v 2'(3')-phosphate reacted rapidly with acrylonitrile and uridine 2'(3')-phosphate reacted only slowly. Adenosine, guanosine and cytidine 2' (3')-phosphates were not affected.
Characterization o/reaction products Inosine was reacted with acrylonitrile on a preparative scale. A crystalline product (Ia) which was isolated from the reaction mixture gave elemental analysis data coinciding with mono-CE-inosine. The ultraviolet absorption spectra of Ia
q~ 0
10
10
8
8
6
1/I
Q
%6 X \
b);?,
X UJ
4
'\\%~ %X%XX x
2
230
250 270 290 wavelength (rap)
2
240
260
wovzlength
280
300
(rap)
Fig. 3. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of CE-inosine in n e u t r a l a n d alkaline media. T h e s p e c t r a were t a k e n a t p H 2.o (a), p H 7.0 (b) a n d p H 13 (c). Fig. 4. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of CE-t//. T h e s p e c t r a were t a k e n at p H 7.o (a) a n d p H 13 (b).
Biochim. Biophys. Acta, 157 ([968) 455-465
401
MODIFICATION OF INOSINE AND PSEUDOURIDINE
given in Fig. 3 indicated that the product (Ia) had no dissociatable group in alkaline medium. This character was confirmed b y the electrophoretic mobility of Ia in borate buffer at p H 9.4, thus, the borate complex of Ia traveled as a monobasic acid, whereas that of inosine moved as a dibasic acid. Thus, the product (Ia) was identified as N1-CE-inosine. The corresponding reaction product (fb) for inosine 5'-phosphate was isolated as the barium salt. After dephosphorylation b y E. coli phosphomonoesterase, Ib gave a single spot corresponding to Ia in paper chromatography (Solvent B). This indicates that the product (Ib) is NLCE-inosine 5'-phosphate. 0
Hr~N' N~NN'>,
CH2~CHC N
0
NCCH2CH2~
RI
I
"r R
0
0
0
,
H~I'~IL"~HCI~CHCN NCCH2CH2N~L'NCFI H 2=CHCNNCCH2CH2~2CH2CN R
R ~
O
R m 0
0~1 CH2=CHCN NCCH2CH2c~ R a, R=rJbose
RI E b, R=ribose~-oP 2',(~)-phosphate
The cyanoethylated product (II) from }/' was also obtained as crystals. From elemental analysis and the ultraviolet absorption spectra (Fig. 4), this product (I1) was identified as N1-CE-~. Another minor product (nI) which was detected in the cyanoethylation of }/' must be N1,N3-diCE-~ as identified b y CHAMBERS7 and OFENG A N D 8.
Although the product (IV) of cyanoethylation of uridine could not be isolated as crystals, we concluded that the product was Na-CE-uridine as reported by O F E N G A N D 8, since the ultraviolet absorption spectra showed no change in alkaline medium as it has been observed for N1,N~-disubstituted uraciP.
Properties o/cyanoethylated products By treatment with o.3 M K O H at 37 ° for 18 h, CE-inosine and CE-uridine were converted to unknown compounds and CE-7 t was partially hydrolyzed. On treatment with I M HC1 at IOO° for i h CE-inosine was completely hydrolyzed to CE-hypoxanthine, but neither CE-uridine nor CE-}P was affected b y this treatment.
pH profiles o/ the reaction As seen in Fig. 2, the reaction between acrylonitrile and inosine or ~ follows pseudo-first-order kinetics. The apparent rate constants were plotted against p H Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 4 5 5 - 4 6 5
462
M. YOSHIDA, T. UKITA
1 O0
.
.
.
.
.
.
.
.
.
.
.
80
60 tD
o x_ a 0
7.0
8.0
9.0
I0~3
11.0
pH Fig. 5' The p H profile of the reaction. The reaction conditons were similar to Fig. 2. Over p H 9.o, p h o s p h a t e w a s r e p l a c e d w i t h c a r b o n a t e buffer. T h e r a t e c o n s t a n t s w e re c a l c u l a t e d u s i n g t h e e q u a t i o n for f i r s t - o r d e r k i n e t i c s . G - G , inosine; 0 - - - 0 , ~r/.
(Fig. 5). The p K values for inosine and ~ calculated from these curves were approx. 9.0 and 9.6, respectively. The dissociated species of the nucleosides are probably involved in this reaction, since the p K values obtained from Fig. 5 were in agreement with p K a values 9.I and 9.4 of these compounds which were measured spectrophotometrically in 3.0 M dimethylformamide solution.
Side reaction in prolonged reaction time rt was found that when the reaction of acrylonitrile with several nucleosides, under the conditions described above, was prolonged to 48 h, all of the nucleosides including adenosine, guanosine and cytidine, were partially converted to the respective new products. The paper chromatographic analysis revealed that the yields of such products ranged from 8 to IO %. These products gave ultraviolet absorption spectra similar to those of the corresponding nucleoside used. Their spots on the chromatogram did not consume periodate and moved as neutral compounds in electrophoresis run in borate buffer. This non-specific reaction was found to be strongly inhibited b y adding o.ooi M borate in the reaction mixture. These observations indicated t h a t the products must be 2'- or 3'-0-CE-nucleosides. The RF values of TABLE
I
R e l a t i v e R F v a l u e s of c y a n o e t h y l a t e d nucleosides. A s c e n d i n g p a p e r c h r o m a t o g r a p h y w a s c a r r i e d o u t a t r o o m t e m p e r a t u r e a n d R F v a l u e s r e l a t i v e to u r i d i n e are given.
Nucleoside
Uridine NZ-CE -urid ine O-CE-uridine N1-CE-~ r/ N 1, N3 -d iCE-tF Inosine
R~r~e~,,e
Nucleoside
Solvent A
Solvent B
I.OO 1.7 ° 1.28 0.77 0.95 1.27 0.83
i.oo 1.81 -
-
0.49 0.67 1.19 0.69
Biochim. Biophys. Acta, 157 (1968) 455-465
Nx-CE-inosine Adenosine O-CE-adenosine Guanosine O-CE-guanosine Cytidine O-CE-cytidine
R~.~a~n. Solvent A
Solvent B
1.43 1.2z 1.45 0.62 0.87 1.13 1.4o
o.95 i. 13 1.7o 0.48 o.81 o.61 I. 16
4~3
MODIFICATION OF INOSINE AND PSEUDOURIDINE
these products and the reaction rate of adenosine compared with t h a t of uridine are given in Table I and Fig. 6, respectively. Such a non-specific reaction was not observed in the case of reaction for nucleoside 2'(3')-phosphate under similar conditions. 5O
"~ 4 0
c=3o ,,,, U
*-20 o
1o
1 O0
~
80
uridln¢
~/
8
~ ~o
2O
O-CE-adenosine
16
24
reoction
32 time
40
4t5
Z .... i_ oI 0 CE-~'
0
10
56
20 reaction
(h)
30
40
50
t~me {h}
Fig. 6. R a t e s of c y a n o e t h y l a t i o n of a d e n o s i n e a n d u r i d i n e w i t h acrylonitrile. T h e r e a c t i o n c o n d i t i o n s as in Fig. 2. Q ) - G , N - C E - u r i d i n e ; 0 - 0 , O-CE-adenosine. Fig. 7. R a t e s of c y a n o e t h y l a t i o n of inosine, krt a n d u r i d i n e r e s i d u e s in t R N A . t R N A w a s t r e a t e d w i t h 2. 4 M acrylonitrile in o.oi M p h o s p h a t e c o n t a i n i n g 2. 7 M d i m e t h y l f o r m a m i d e a t 39-4 o°.
Modi/ication o~ yeast tRNA with acrylonitrile To a solution of yeast t R N A in o.oi M phosphate (pH 8.6) containing 2.7 M dimethylformamide was added acrylonitrile to 2.2-2.4 M concentration and the mixture was incubated at 39-4 o°. The rates of cyanoethylation of inosine, kv and uridine residues were analyzed as described in MATERIALS AND METHODS and the results are given in Fig. 7- As is seen in Fig. 7, over 9 ° % of ~t and inosine residues in t R N A were cyanoethylated after 48 h reaction, while the extent of the modification of uridine was less than 8 °/o. The figure indicates t h a t the specificity of the reaction was similar to that observed for nucleosides, but in the case of t R N A the reaction rates remarkably decreased in comparison with those for nucleosides. This decrease in reaction rate is probably due to the secondary structure of tRNA. No cyanoethylated product of adenosine, guanosine or cytidine residues was detected in the alkaline hydrolysate of the modified t R N A when analyzed according to the method
o f T A D A , T A D A AND YAG115 o r K A T Z AND COMB 16.
Properties o~ the modi/ied tRNA In order to check whether or not the nucleotide linkages of t R N A are split b y the modification, the properties of the modified t R N A in sedimentation analysis, gel filtration and DEAE-cellulose column chromatography were compared with those of native tRNA. The ultracentrifugal pattern of the t R N A which was modified for 48 h indicated its homogeneity (Fig. 8b). The sedimentation coefficient, 3.63 S, obtained from this pattern was similar to that of native tRNA, 3.56 S. The elution patterns of the modified t R N A from both Sephadex G-5o and DEAE-cellulose columns revealed a single peak at the same position as native t R N A as shown in Figs. 9 and IO. Biochim. Biophys. Acta, 157 (1968) 455-465
404
M. YOSHIDA, T. UKITA
Fig. 8. S e d i m e n t a t i o n p a t t e r n s of native (a) and modified t R N A ' s (b). The modified t R N A was p r e p a r e d by t r e a t m e n t of t R N A u n d e r conditions similar to Fig. 7 for 48 h. The p a t t e r n s were o b t a i n e d for 0.2 M NaC1 solutions of the t R N A using a Spinco Model E, at 48 rain after reaching a m a x i m u m speed of 59 800 rev./min.
1.4
oE
I t
L o
2.0
0.6
G
c o
.~o 1.o
1.0
-S-
0.2
3.5
I-.. t
4
8
12 fraction
16
20
24
28
NO.
32
0
50
100 vo[ urn¢
150
(ml}
Fig. 9. Gel filtration of native a n d modified t R N A ' s t h r o u g h S e p h a d e x G-5o. Native ( - - - ) or modified t R N A ( ), p r e p a r e d as in Fig. 8, w a s applied to a c o l u m n (o.8 cm x 60 cm) of S e p h a d e x G-5o. E l u t i o n was carried o u t using 0.05 M a m m o n i u m acetate and 1.5-ml fractions were collected. The area m a r k e d with the a r r o w is the position for uridine 5 ' - p h o s p h a t e to be eluted f r o m the same column. Fig. IO. E l u t i o n p a t t e r n of t R N A from DEAE-cellulose column. N a t i v e (- - - ) or modified t R N A ( - - - - - - ) , p r e p a r e d as in Fig. 8, w a s charged on the DEAE-cellulose c o l u m n (0.9 cm × i2.o cm). The c o l u m n was eluted w i t h a linear gradient f o r m e d from IOO ml each of o.o2 M Tris-HC1 (pH 7.5) and I M NaCl-o.o2 M Tris-HC1 (pH 7.5). F r a c t i o n s of approx. 3 ml were collected.
These results indicate that no gross cleavage of phosphodiester bonds of t R N A occurred during tile modification reaction. The possibility that the modified t R N A is partially cleaved b y the modification reaction and still behaved similarly to native t R N A could be excluded, since, as will be reported in a forthcoming paper 19, the secondary structure of the t R N A was almost disrupted in the reaction conditions used for the modification. If the cleavage of t R N A chain should occur, the fragments could not be held together b y hydrogen bonding. Biochim. Biophys. Acta, 157 (1968) 455-465
0
MODIFICATION OF INOSINE AND PSEUDOURIDINE
405
In conclusion, inosine and 7 t in tRNA can selectively be cyanoethylated and the reaction conditions are mild enough to avoid the cleavage of polynucleotide linkages in tRNA.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. KIN-ICHIRO MIURA of Nagoya University for his gift of Torula yeast tRNA. We are also indebted to Mr. YASUHIKO FUTAMI of this laboratory for his isolation of pseudouridylic acid from yeast RNA.
REFERENCES i 2 3 4 5 6 7 8 9 lO II 12 13 14 15 16 17 18 19
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Biochim. Biophys. Acta, 157 (1968) 455-465