/ . Biochem., 78, 409-420 (1975)
Masao AZEGAMI1 and Koichi IWAP Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, December 27, 1974
1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4, 6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNPguanosine, O-TNP-guanosine, -adenosine, -cytidine, and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases Tt and T2 [EC 3.1.4. 8 and 3.1. 4. 23]. 4. Trinitrophenylation reactions using 2,4, 6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.
DNFB3 (7) and TNBS (.2, 3) are used to introduce DNP or TNP groups into amino acids 1
Present address: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, 0 ai ° ' •Present address: Institute of Endocrinology, Gunma University, Maebashi, Gunma 371. 3 Abbreviations are used: DNFB, 2,4-dinitrofluorobenzene; TNBS, 2,4,6-trinitrobenzenesulfonate; TNCB, 2,4,6-trinitrochlorobenzene; TNFB, 2,4,6trinitrofluorobenzene; DNP, dinitrophenyl; TNP, trinitrophenyl. Vol. 78, No. 2, 1975
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Trinitrophenylation of Nucleic Acids and Their Constituents
or proteins _under relatively mild conditions, TNBS is more specific than DNFB and reacts only with primary amino and sulfhydryl groups (4) W e attempted the modification of nucleic a d d s with T N B S T h i s r e p o r t d e a l s with the trinitrophenylation of guanine residues of nu^ ^ Qf t h e j r c o n s t i t u e n t s a n d t h e for. . , . . . . -. c ,, . matlOn ° f Meisenhe.mer-type complexes (5, 6) with the 2 '" a n d 3'-hydroxyl groups of the ribose moiety. A brief communication on this work has been published (7).
409
M. AZEGAMI and K. IWAI
410
MATERIALS AND METHODS
of Kay et al.
{11).
Ribonucleases—Ti [EC 3.1.4.8] and T 2 [EC 3.1.4.23] were kindly supplied by Dr. K. Takahashi, Tokyo University. Bovine pancreatic ribonuclease I [EC 3.1. 4. 22] was ob4
The amount of guanine residues in the case of nucleic acids. 5 Suspended in the reaction mixtures. 6 The wavelength was chosen to minimize the contribution of spontaneous degradation products of the trinitrophenylating reagents.
Paper Electrophoresis—Samples containing TNP derivatives or starting materials were applied to Toyo Roshi No. 51 paper. Electrophoresis was carried out in ice-cooled hexane for 1 hr at 400 or 1,000 volts per cm in 0.25 M formic acid or 0.1—1% sodium bicarbonate. Spots on the paper were treated as in paper / . Biochem.
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TNBS (sodium salt) was prepared from TNCB and sodium bisulfite by the method of Willgerodt (8) or was obtained from Tokyo Kasei Co. The reagent was used after recrystallization from hydrochloric acid-ethanol; mp above 300°. TNCB was prepared from picric acid and phosphorus pentachloride by the method of Jackson and Gazzallo (9) and crystallized from benzene-ethanol; mp 83°. TNFB was prepared by nitration of DNFB according to the method of Shaw and Seaton (10) and crystallized from carbon tetrachloride ; mp 131-132°. Nucleic Acids and Relating Compounds— Adenine, guanine, cytosine, uracil, thymine, adenosine, guanosine, cytidine, uridine, thymidine, and yeast ribonucleic acid were obtained from Nutritional Biochemical Corporation. Ribose, deoxyribose, deoxyadenosine, deoxyguanosine, deoxycytidine, adenosine 3'(2')phosphate, cytidine 3'(2')-phosphate, and uridine 3'(2')-phosphate were from Sigma Chemical Co. Hypoxanthine, xanthine, uric acid, theobromine, theophylline, inosine, xanthosine, adenosine 3'-phosphate, adenosine 5'-phosphate, and glucose were from Tokyo Kasei Co. Glycerol and ethylene glycol were from Wako Junyaku Co. Caffeine, N, N-dimethylguanine, benzyl riboside, and yeast transfer RNA were kindly supplied by Dr. C. Hashimoto, Teikoku Zoki Co., Dr. T. Saito, Tanabe Seiyaku Co., the late Prof. T. Ukita, Tokyo University, and Dr. W. Kawade, Kyoto University, respectively. Calf thymus DNA was prepared by the method
tained from Nutritional Biochemical Corporation. Trinitrophenylation—Reactions of nucleic acids, their consituents, and related substances (usually 5-10 mM1) with TNBS (10-100 mM), TNCB5 or TNFB 5 (10-100 ^moles/ml) were performed at 37° either in buffers or in a pHstat (Radiometer, Copenhagen) at various pH values in the dark, with stirring. Buffers used were 0.2 M sodium phosphate-0.1 M citrate, pH 3 - 6 ; 0.5M sodium phosphate, pH 7.0; 0.5 M sodium carbonate, pH 8—11; 0.2 M sodium borate, pH 10.0. The progress of reactions was monitored spectrophotometrically in terms of the increase in absorbance at 500 nm6 after dilution of the reaction mixtures with excess 1% sodium bicarbonate. The readings were corrected for degradation of the trinitrophenylating agents. Paper Chromatography—Aliquots of reaction mixtures were spotted on filter paper (Toyo Roshi No. 51) and developed overnight in the dark with the following solvents : 1.5 M sodium phosphate buffer, pH 6, 86% aqueous w-butanol, 2-propanol-hydrochloric acid (2-propanol 170 ml, cone, hydrochloric acid 41 ml, total 250 ml with water) and others as cited in Table I. Spots of TNP derivatives on chromatograms developed with acid solvents were colored faint yellow and were detected under UV light. However, they could be easily identified by spraying dilute alkali, as they turned reddish-orange. Each spot of TNP derivative was cut out, eluted with 1% sodium bicarbonate for 15-20 min at 50° and estimated spectrophotometrically at 410 or 430 nm. Their molar extinction coefficients were determined with the purified TNP derivatives. Spots of starting bases, nucleosides, or nucleotides which remained intact were each eluted with 0.1 N hydrochloric acid for 24 hr at room temperature and estimated spectrophotometrically at 260 nm.
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
Methylation—Uridine and O-TNP-uridine were methylated with dimethyl sulfate essentially according to the method of Bredereck et al. (12). In the case of O-TNP-uridine, 0.4 ml of dimethyl sulfate was added to a solution of 20 mg of O-TNP-uridine in 4 ml of water and this -was incubated for 2 hr at 40° and pH 9.5 in a pH-stat. The reaction mixture was extracted with ethyl acetate and after evaporation of the extract to dryness, the residue was purified by precipitation from acetonebenzene mp 201-203°. Base Analyses—TNP-RNA and TNP-DNA were hydrolysed with 1N hydrochloric acid for 1 hr at 100°. Under these conditions about 5% of N2-TNP-guanine was hydrolysed to picric acid and guanine. The hydrolysates were evaporated to dryness and analysed by Vol. 78, No. 2, 1975
paper chromatography, using 2-propanol-hydrochloric acid solvent. In some cases, the hydrolysate was directly applied to a Dowex 50x8 (H form, 200-400 mesh) column (0.8x15 cm). Uridylic and cytidylic acids were eluted separately with 1 N hydrochloric acid and then guanine and adenine with 2 N hydrochloric acid. N2-TNP-guanine was not eluted until adenine had been completely eluted. The recoveries were almost quantitative. PREPARATIONS O-TNP-Adenosine, O-TNP-Cytidine, and O-TNP-Uridine—Two hundred milligrams of each nucleoside and 600 mg of TNBS were dissolved in 10 ml of 0.2 M sodium carbonate buffer, pH 10, and the solution was incubated at room temperature in the dark. After 48 hr the reaction mixture was adjusted to pH 10 with sodium hydroxide and incubated for a further 24 hr with an additional 100 mg of TNBS. The red precipitate which crystallized out only in the case of O-TNP-adenosine was purified by reprecipitation from acetone solution by adding 10 volumes of benzene. This reprecipitation was repeated several times until contaminating picric acid was completely removed. The supernatant of the reaction mixture of O-TNP-adenosine- and the whole reaction mixture of O-TNP-uridine or O-TNP-cytidine were neutralized to pH 7—8 with hydrochloric acid, applied to the top of a potato-starch column (3.5x22 cm), and developed with 1 . 5 M phosphate buffer, pH 6 (600-800 ml). A reddish-orange band of O-TNP-nucleoside was retained at the top of the column and separated from a yellow picric acid band and two other unidentified bands, violet and dark yellow, which moved more rapidly. These latter three bands corresponded to the degradation products of TNBS itself. The top band of O-TNPnucleoside was cut out and eluted with 600800 ml of 1 N hydrochloric acid-acetone ( 4 : 1 , v/v). The eluate was neutralized with sodium carbonate, concentrated to 150 ml under reduced pressure and extracted with 600 ml of ethyl acetat. The extract was evaporated to dryness and the residue was dissolved in about
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chromatography. The mobilities of various TNP derivatives are shown in Table I. UV, Visible, and Infrared Absorption Spectra—UV and visible spectra were measured with a Beckman spectrophotometer, model DU or a Hitachi automatic recording spectrophotometer, model EPS-3. Infrared spectra were determined in potassium bromide discs with a Nippon Bunko infrared spectrophotometer, model DS-301, in the Central Spectroscopical Laboratory, Faculty of Pharmaceutical Science, University of Tokyo. Elementary Analyses—Elementary analyses were kindly performed in the Central Microanalytical Laboratory, Faculty of Pharmaceutical Science, University of Tokyo. Deamination—Deamination of TNP derivatives and unsubstituted nucleosides (controls) was performed with nitrous acid as follows: To a solution of 3 mg of a TNP derivative in 2.5 ml of water was added 0.5 ml of glacial acetic acid and 1 ml of 30% sodium nitrite, and the mixture was incubated for 5 hr at 30°, except for O-TNP-cytidine and N2-TNP-guanosine, which were incubated for 18 hr. The mixture was then neutralized to pH 8 with sodium bicarbonate and extracted with ethyl acetate. The extract was evaporated to dryness and the residue, after purification by precipitation from acetone-benzene was identified by paper chromatography or paper electrophoresis.
411
412
and 37° in pH-stat. The reaction mixture was centrifuged to remove the dark-red supernatant, and the sediment, containing insoluble guanine, was further incubated with 100 mg of TNBS in 10 ml of water in the same way. The first and second supernatants were combined and adjusted to pH 2—3 to give a yellow precipitate of N2-TNP-guanine. After washing with 0.1 N hydrochloric acid, the precipitate was dissolved in carbonate buffer, pH 9. A small amount of insoluble material was removed by centrifugation, and N2-TNP-guanine was reprecipitated by adjusting the pH to 2—3. These treatments were repeated until no insoluble material was found in the alkaline solution. The yield of the last dried acid-precipitate was 70 mg. This crude N2-TNP-guanine was recrystallized from acetone. Anal. Found: C, 40.31; H, 3.10; N, 27.07%; Calcd. for CuH6N8O7-CH8COCH3: C, 40.00; H, 2.88; N, 26.66%. TNP-Ethylene Glycol—Ethylene glycol was trinitrophenylated with TNFB because the reaction rate with TNBS was very low. The products with TNFB and TNBS were identified in paper chromatography, paper electrophoresis, and UV absorption spectra. To a mixture of 2 g of ethylene glycol and 1 g of TNFB, 3 N sodium hydroxide was added dropwise with stirring to keep the pH at 8-9 for 1 hr at 37°. The resulting red precipitate was collected and purified by acetonebenzene reprecipitation. The contaminating picric acid was removed by paper electrophoresis. mp (decomp.) above 230°. TNP - Aniline (2, 4, 6 - Trinitrodiphenyl amine)—TNP-aniline was prepared from aniline and TNCB. The reaction was performed in benzene for 1 hr at 40° with stirring and the resulting precipitate was purified by recrystallization from benzene and ligroin. mp 179—180°
{13). TNP-RNA—One hundred and forty mg of yeast RNA and 700 mg of TNBS were dissolved in 7 ml of water and the solution was incubated for 25 hr at pH 9.5 and 37° in a pH-stat. The reaction mixture was neutralized to pH 6.5 with hydrochloric acid and dialyzed against water until picric acid and other byproducts had been removed. TNP-RN A was / . Biocheni.
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5 ml of acetone. The O-TNP-nucleoside was precipitated from this acetone solution by adding 10 volumes of benzene. This precipitation was repeated to remove any contaminating picric acid. O-TNP-adenosine: Yield, 303 mg (81% of the theoretical value); mp 225°. Anal. Found C, 38.41; H, 3.26; N, 22.31%; Calcd. for C16H13N8O10Na: C, 38.47; H, 2.62; N, 22.40%. O-TNP-cytidine: Yield, 137 mg (37%); mp 211°. O-TNP-uridine: Yield, 163 mg (42%); mp 224°. O-TNP-uridine crystallized from 25% acetic acid: Anal. Found; C, 39.65; H, 2.71; N, 15.65% ; Calcd. for C15H13N5O12; C, 39.57; H, 2.88; N, 15.38%. N2-TNP- and N2,O-bis-T NP-Guanosines— Two hundred mg of guanosine (70.7 mM) was treated with 600 mg of TNBS (171 mM) as described above. After 48 hr the whole reaction mixture, from which the starting guanosine had almost disappeared, was applied to a starch column and developed with 800 ml of 1.5 M phosphate buffer. The bands containing TNP-guanosines were cut out together, and treated as before. The precipitates from acetone-benzene were applied to Whatman 3MM paper and developed with the same phosphate buffer. N2-TNP-guanosine gave Rf 0.3, the same value as picric acid. N 2 ,0-bis-TNPguanosine gave Rf 0.01, but contained a small quantity of O-TNP-guanosine which also gave the same Rf value. TNP-guanosines were eluted with water from each of the bands cut from the paper, and were purified by acetonebenzene precipitation. N2-TNP-guanosine: Yield, 158 mg; mp (decomp.) above 220°. N2-TNP-guanosine precipitated from aqueous solution by acidification with hydrochloric acid: Anal. Found: C, 38.99; H, 3.12; N, 22.11%; Calcd. for C 16 H 14 N 8 0 1 i: C, 38.87; H, 2.85; N, 22.67%. N2, O-bis-TNP-guanosine: Yield, 31 mg; mp (decomp.) above 210° (contained a small quantity of O-TNP-guanosine which could be separated by paper electrophoresis). N2-TNP-Guanine— A suspension of 150 mg of guanine and 400 mg of TNBS in 10 ml of water was incubated for 3 hr at pH 10—11
M. AZEGAMI and K. IWAI
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
RESULTS AND DISCUSSION 1. Trinitrophenylation of the Constituents of Nucleic Acids—Specificity of the reaction with TNBS: In the reaction with TNBS at 37° in buffers of pH 9-10, the following compounds were found to give TNP derivatives: guanine, guanosine, guanosine 3'(2')-phosphate,
deoxyguanosine, adenosine, adenosine 5'-phosphate, inosine, xanthosine, cytidine, uridine, benzyl riboside, and ethylene glycol. They are compounds containing either a guanine residue (forming N2-TNP derivatives) or adjacent free hydroxyl groups in the cis position, such as 2'- and 3'-hydroxyl groups of ribosides as well as polyhydric alcohols (forming O-TNP derivatives). The reaction mixtures usually became colored reddish-orange as the reaction proceeded. The reaction mixture of ribose or glucose with TNBS became brown and side reactions seemed to occur. Three TNP derivatives, N2-TNP-, O-TNP-, and N 2 ,0-bis-TNPguanosine, were obtained from the reaction mixture with 171 mM TNBS and 70.0 mM guanosine, as described in "PREPARATIONS," but only N2,0-bis-TNP-guanosine was found with 100 mM TNBS and 10 mM guanosine. The products were separated by paper chromatography or paper electrophoresis. Table I shows the Rf values and mobilities of various TNP derivatives. TNP derivatives of the following sub-
TABLE I. Rf values in paper chromatography and mobilities in paper electrophoresis of TNP derivatives.
2
N -TNP-guanine N2-TNP-deoxyguanosine N2-TNP-guanosine N2, O-bis-TNP-guanosine O-TNP-guanosine O-TNP-adenosine O-TNP-cytidine O-TNP-uridine . . - . O-TNP-inosine O-TNP-xanthosine O-TNP-ethylene glycol Picric acid TNBS
Mobility (mm)
Rf value
Compound (1) 0.15 0.24 0.31 0.01 0.01 0.01 0.11 0.11 0.06 0.01 0.30 0.29 0.50
(2) 0.53
(3) 0.03
(4) - 7 . 2 (0.16)
0.38 0.41 0.56 0.66 0.54
0.09 0 0 0 0 - 0,
- 1 . 9 (0.10)0 - 1 . 9 (0.10) - 1 7 (0.33) -34 (0.49) 0 0
0.50--
0 0 + 52
(5)
+37 + 37 +37 + 37 + 15 + 15 + 30 +40 + 30 +61 + 61 + 83
(1) 1.5 M Sodium phosphate buffer, pH 6. (2) 86% (v/v) w-Butanol-15 N ammonia (100: 1, v/v). (3) Saturated ammonium sulfate-O.l M sodium phosphate buffer, pH 6-2-propanol ( 7 9 : 1 9 : 2 , v/v). (4) 0.25 M Formic acid, 400v/37cm, 1 hr. Symbols: + and — indicate movement toward the anode and the cathode, respectively. Values in parentheses show ratios of mobilities of TNP derivatives to those of the unsubstituted substances. (5) 0.1% Sodium bicarbonate, 1,000 v/37 cm, 1 hr.. . . Vol. 78, No. 2, 1975
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then precipitated by the addition of 3 volumes of ethanol. The precipitates were dissolved in 10 ml of 0.2 M sodium acetate buffer, pH 5, reprecipitated with ethanol, washed with ethanol and ether, and dried under reduced pressure. Yield, 100 mg. TNP-DNA — Twenty-four milligrams of calf thymus DNA and 150 mg of TNBS were dissolved in 10 ml of 1M saline and the solution was incubated at pH 9.5 and 37° in a pHstat. After 18 hr, an additional 50 mg of TNBS was added and incubation was continued for a further 6 hr. The reaction mixture was fully dialyzed against water. This TNP-DNA solution was used for further experiments.
413
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M. AZEGAMI and K. IWAI
TNPation. For instance, the reaction occurred to the extent of over 95% after 1.5 hr with deoxyguanosine, after 5 hr with adenosine, and after 23 hr with uridine, as shown in Fig. 1.
0
1
2
3
4
5
6
7
8
TIME OF REACTION (hr)
Fig. 1. Rates of trinitrophenylation of nucleosides. The reaction and assay were performed as follows: 5 ml of the reaction mixture containing 0.01 M nucleoside and 0.1 M TNBS was incubated at 37° and pH 9.5 in a pH-stat, except for guanosine, and 0.1 ml aliquots of the reaction mixture were removed at intervals. To these was added 10 or 20 ml of 1% sodium bicarbonate, and the optical densities of the solutions and similarly treated controls were read at 500 nm. A, N-TNPation of deoxyguanosine ; • , O-TNPation of adenosine; D, N, O-bis-TNPation of guanosine in 0.5 M carbonate buffer, pH 10.0-9.5 ; • , O-TNPation of cytidine ; O, TNPation of uridine.
TABLE II. Comparison of the rates of trinitrophenylation of nucleosides and nucleotides at several pH's. The reaction mixture, containing 10 mM nucleoside or nucleotide and 100 mti TNBS in each buffer, was incubated at 37° and aliquots were removed at intervals and treated as described in Fig. 1; the 50% reaction time was calculated from the change of absorption during the initial 2 hr. Molar extinction coefficients of O-TNP-benzyl riboside and TNP-ethylene glycol at 410 nm were assumed to be the same as that of OTNP-adenosine. 50% reaction time (min) Compound
0.5 M Na phosphate pH 7.0
Guanosine 3'(2')-phosphate Deoxyguanosine Adenosine Adenosine 5'-phosphate Cytidine Uridine Benzyl riboside Ethylene glycol
2,400
0.5 M Na carbonate pH 8.3 130 714
pH 9.0 51 30 119 — 447 595 —
pH 10.0 23 IS 21 98 97 140 893 70,000
0.2 M Na borate pH 10.0 23 2,400
/ . Biochem.
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stances were not produced, as determined chromatographically or spectrophotometrically: adenine, cytosine, uracil, thymine, hypoxanthine, xanthine, uric acid, N, N-dimethyl guanine, theobromine, theophylline, caffeine, deoxyadenosine, deoxycytidine, thymidine, adenosine 3'-phosphate, cytidine 3'(2')-phosphate, and uridine 3'(2')-phosphate. The following starting materials were recovered quantitatively by paper chromatography from the 20 hr-reaction mixtures at pH 9: adenine, cytosine, uracil, thymine, adenosine 3'-phosphate, and cytidine 3'(2')-phosphate. The degradation products of TNBS gave two unidentified dark red spots above Rf 0.6 (1.5 M sodium phosphate buffer, pH 6) in addition to those of picric acid and TNBS itself. Rate of Reaction with TNBS—The rate of trinitrophenylation at the guanine residue (NTNPation) and the ribose residue (O-TNPation) depended on pH, and both N- and O-TNPations were scarcely observed below pH 7. The reactivity increased with pH between pH 7 and 11. The time courses for some nucleosides and nucleotides are shown in Fig. 1 and the 50% reaction time, as a measure of reaction rate, is listed in Table II. N-TNPation was more rapid than 0-
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
A
as seen in Table II, but not N-TNPation as expected from the specificity of the reaction.
Trinitrophenylation with TNFB and TNCB —In preliminary and qualitative experiments, O-TNPation and N-TNPation were found to occur with both TNFB and TNCB; though both reagents were sparingly soluble in aqueous solution, TNFB reacted with nucleosides and
D
1.0 1.0
PH
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10.1
A
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o
CO CD < 10
o
to CO
\ " O. 300
350
400
450
500
\
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250 550
300
350
400
450
500
550
E
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1.0 1.0 7.1 10.1
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pyrimidine nucleosides >polyhydric alcohols. The difference in reaction rate between ribosides and polyhydric alcohols may depend on the readiness with which the hydroxyl groups form a stable cis configuration. Borate ions inhibited O-TNPation,
415
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M. AZEGAMI and K. IWAI
TABLE III. Optical properties of TNP derivatives. Absorption maxima (nm)a
O-TNP-adenosine O-TNP-cytidine O-TNP-uridine O-TNP-guanosine O-TNP-benzyl riboside TNP-ethylene glycol N2-TNP-guanine N2-TNP-guanosine N2, O-bis-TNP-guanosine a
pH 7.1 ^max 1
^max 2
408 408 412 408 410 415 422 425 408
470 470 470 470 470 470
pH 1.0
=410
^max
in 1% NaHCO3
£
Masao AZEGAMI1 and Koichi IWAP Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, December 27, 1974
1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4, 6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNPguanosine, O-TNP-guanosine, -adenosine, -cytidine, and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases Tt and T2 [EC 3.1.4. 8 and 3.1. 4. 23]. 4. Trinitrophenylation reactions using 2,4, 6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.
DNFB3 (7) and TNBS (.2, 3) are used to introduce DNP or TNP groups into amino acids 1
Present address: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, 0 ai ° ' •Present address: Institute of Endocrinology, Gunma University, Maebashi, Gunma 371. 3 Abbreviations are used: DNFB, 2,4-dinitrofluorobenzene; TNBS, 2,4,6-trinitrobenzenesulfonate; TNCB, 2,4,6-trinitrochlorobenzene; TNFB, 2,4,6trinitrofluorobenzene; DNP, dinitrophenyl; TNP, trinitrophenyl. Vol. 78, No. 2, 1975
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Trinitrophenylation of Nucleic Acids and Their Constituents
or proteins _under relatively mild conditions, TNBS is more specific than DNFB and reacts only with primary amino and sulfhydryl groups (4) W e attempted the modification of nucleic a d d s with T N B S T h i s r e p o r t d e a l s with the trinitrophenylation of guanine residues of nu^ ^ Qf t h e j r c o n s t i t u e n t s a n d t h e for. . , . . . . -. c ,, . matlOn ° f Meisenhe.mer-type complexes (5, 6) with the 2 '" a n d 3'-hydroxyl groups of the ribose moiety. A brief communication on this work has been published (7).
409
M. AZEGAMI and K. IWAI
410
MATERIALS AND METHODS
of Kay et al.
{11).
Ribonucleases—Ti [EC 3.1.4.8] and T 2 [EC 3.1.4.23] were kindly supplied by Dr. K. Takahashi, Tokyo University. Bovine pancreatic ribonuclease I [EC 3.1. 4. 22] was ob4
The amount of guanine residues in the case of nucleic acids. 5 Suspended in the reaction mixtures. 6 The wavelength was chosen to minimize the contribution of spontaneous degradation products of the trinitrophenylating reagents.
Paper Electrophoresis—Samples containing TNP derivatives or starting materials were applied to Toyo Roshi No. 51 paper. Electrophoresis was carried out in ice-cooled hexane for 1 hr at 400 or 1,000 volts per cm in 0.25 M formic acid or 0.1—1% sodium bicarbonate. Spots on the paper were treated as in paper / . Biochem.
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TNBS (sodium salt) was prepared from TNCB and sodium bisulfite by the method of Willgerodt (8) or was obtained from Tokyo Kasei Co. The reagent was used after recrystallization from hydrochloric acid-ethanol; mp above 300°. TNCB was prepared from picric acid and phosphorus pentachloride by the method of Jackson and Gazzallo (9) and crystallized from benzene-ethanol; mp 83°. TNFB was prepared by nitration of DNFB according to the method of Shaw and Seaton (10) and crystallized from carbon tetrachloride ; mp 131-132°. Nucleic Acids and Relating Compounds— Adenine, guanine, cytosine, uracil, thymine, adenosine, guanosine, cytidine, uridine, thymidine, and yeast ribonucleic acid were obtained from Nutritional Biochemical Corporation. Ribose, deoxyribose, deoxyadenosine, deoxyguanosine, deoxycytidine, adenosine 3'(2')phosphate, cytidine 3'(2')-phosphate, and uridine 3'(2')-phosphate were from Sigma Chemical Co. Hypoxanthine, xanthine, uric acid, theobromine, theophylline, inosine, xanthosine, adenosine 3'-phosphate, adenosine 5'-phosphate, and glucose were from Tokyo Kasei Co. Glycerol and ethylene glycol were from Wako Junyaku Co. Caffeine, N, N-dimethylguanine, benzyl riboside, and yeast transfer RNA were kindly supplied by Dr. C. Hashimoto, Teikoku Zoki Co., Dr. T. Saito, Tanabe Seiyaku Co., the late Prof. T. Ukita, Tokyo University, and Dr. W. Kawade, Kyoto University, respectively. Calf thymus DNA was prepared by the method
tained from Nutritional Biochemical Corporation. Trinitrophenylation—Reactions of nucleic acids, their consituents, and related substances (usually 5-10 mM1) with TNBS (10-100 mM), TNCB5 or TNFB 5 (10-100 ^moles/ml) were performed at 37° either in buffers or in a pHstat (Radiometer, Copenhagen) at various pH values in the dark, with stirring. Buffers used were 0.2 M sodium phosphate-0.1 M citrate, pH 3 - 6 ; 0.5M sodium phosphate, pH 7.0; 0.5 M sodium carbonate, pH 8—11; 0.2 M sodium borate, pH 10.0. The progress of reactions was monitored spectrophotometrically in terms of the increase in absorbance at 500 nm6 after dilution of the reaction mixtures with excess 1% sodium bicarbonate. The readings were corrected for degradation of the trinitrophenylating agents. Paper Chromatography—Aliquots of reaction mixtures were spotted on filter paper (Toyo Roshi No. 51) and developed overnight in the dark with the following solvents : 1.5 M sodium phosphate buffer, pH 6, 86% aqueous w-butanol, 2-propanol-hydrochloric acid (2-propanol 170 ml, cone, hydrochloric acid 41 ml, total 250 ml with water) and others as cited in Table I. Spots of TNP derivatives on chromatograms developed with acid solvents were colored faint yellow and were detected under UV light. However, they could be easily identified by spraying dilute alkali, as they turned reddish-orange. Each spot of TNP derivative was cut out, eluted with 1% sodium bicarbonate for 15-20 min at 50° and estimated spectrophotometrically at 410 or 430 nm. Their molar extinction coefficients were determined with the purified TNP derivatives. Spots of starting bases, nucleosides, or nucleotides which remained intact were each eluted with 0.1 N hydrochloric acid for 24 hr at room temperature and estimated spectrophotometrically at 260 nm.
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
Methylation—Uridine and O-TNP-uridine were methylated with dimethyl sulfate essentially according to the method of Bredereck et al. (12). In the case of O-TNP-uridine, 0.4 ml of dimethyl sulfate was added to a solution of 20 mg of O-TNP-uridine in 4 ml of water and this -was incubated for 2 hr at 40° and pH 9.5 in a pH-stat. The reaction mixture was extracted with ethyl acetate and after evaporation of the extract to dryness, the residue was purified by precipitation from acetonebenzene mp 201-203°. Base Analyses—TNP-RNA and TNP-DNA were hydrolysed with 1N hydrochloric acid for 1 hr at 100°. Under these conditions about 5% of N2-TNP-guanine was hydrolysed to picric acid and guanine. The hydrolysates were evaporated to dryness and analysed by Vol. 78, No. 2, 1975
paper chromatography, using 2-propanol-hydrochloric acid solvent. In some cases, the hydrolysate was directly applied to a Dowex 50x8 (H form, 200-400 mesh) column (0.8x15 cm). Uridylic and cytidylic acids were eluted separately with 1 N hydrochloric acid and then guanine and adenine with 2 N hydrochloric acid. N2-TNP-guanine was not eluted until adenine had been completely eluted. The recoveries were almost quantitative. PREPARATIONS O-TNP-Adenosine, O-TNP-Cytidine, and O-TNP-Uridine—Two hundred milligrams of each nucleoside and 600 mg of TNBS were dissolved in 10 ml of 0.2 M sodium carbonate buffer, pH 10, and the solution was incubated at room temperature in the dark. After 48 hr the reaction mixture was adjusted to pH 10 with sodium hydroxide and incubated for a further 24 hr with an additional 100 mg of TNBS. The red precipitate which crystallized out only in the case of O-TNP-adenosine was purified by reprecipitation from acetone solution by adding 10 volumes of benzene. This reprecipitation was repeated several times until contaminating picric acid was completely removed. The supernatant of the reaction mixture of O-TNP-adenosine- and the whole reaction mixture of O-TNP-uridine or O-TNP-cytidine were neutralized to pH 7—8 with hydrochloric acid, applied to the top of a potato-starch column (3.5x22 cm), and developed with 1 . 5 M phosphate buffer, pH 6 (600-800 ml). A reddish-orange band of O-TNP-nucleoside was retained at the top of the column and separated from a yellow picric acid band and two other unidentified bands, violet and dark yellow, which moved more rapidly. These latter three bands corresponded to the degradation products of TNBS itself. The top band of O-TNPnucleoside was cut out and eluted with 600800 ml of 1 N hydrochloric acid-acetone ( 4 : 1 , v/v). The eluate was neutralized with sodium carbonate, concentrated to 150 ml under reduced pressure and extracted with 600 ml of ethyl acetat. The extract was evaporated to dryness and the residue was dissolved in about
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chromatography. The mobilities of various TNP derivatives are shown in Table I. UV, Visible, and Infrared Absorption Spectra—UV and visible spectra were measured with a Beckman spectrophotometer, model DU or a Hitachi automatic recording spectrophotometer, model EPS-3. Infrared spectra were determined in potassium bromide discs with a Nippon Bunko infrared spectrophotometer, model DS-301, in the Central Spectroscopical Laboratory, Faculty of Pharmaceutical Science, University of Tokyo. Elementary Analyses—Elementary analyses were kindly performed in the Central Microanalytical Laboratory, Faculty of Pharmaceutical Science, University of Tokyo. Deamination—Deamination of TNP derivatives and unsubstituted nucleosides (controls) was performed with nitrous acid as follows: To a solution of 3 mg of a TNP derivative in 2.5 ml of water was added 0.5 ml of glacial acetic acid and 1 ml of 30% sodium nitrite, and the mixture was incubated for 5 hr at 30°, except for O-TNP-cytidine and N2-TNP-guanosine, which were incubated for 18 hr. The mixture was then neutralized to pH 8 with sodium bicarbonate and extracted with ethyl acetate. The extract was evaporated to dryness and the residue, after purification by precipitation from acetone-benzene was identified by paper chromatography or paper electrophoresis.
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and 37° in pH-stat. The reaction mixture was centrifuged to remove the dark-red supernatant, and the sediment, containing insoluble guanine, was further incubated with 100 mg of TNBS in 10 ml of water in the same way. The first and second supernatants were combined and adjusted to pH 2—3 to give a yellow precipitate of N2-TNP-guanine. After washing with 0.1 N hydrochloric acid, the precipitate was dissolved in carbonate buffer, pH 9. A small amount of insoluble material was removed by centrifugation, and N2-TNP-guanine was reprecipitated by adjusting the pH to 2—3. These treatments were repeated until no insoluble material was found in the alkaline solution. The yield of the last dried acid-precipitate was 70 mg. This crude N2-TNP-guanine was recrystallized from acetone. Anal. Found: C, 40.31; H, 3.10; N, 27.07%; Calcd. for CuH6N8O7-CH8COCH3: C, 40.00; H, 2.88; N, 26.66%. TNP-Ethylene Glycol—Ethylene glycol was trinitrophenylated with TNFB because the reaction rate with TNBS was very low. The products with TNFB and TNBS were identified in paper chromatography, paper electrophoresis, and UV absorption spectra. To a mixture of 2 g of ethylene glycol and 1 g of TNFB, 3 N sodium hydroxide was added dropwise with stirring to keep the pH at 8-9 for 1 hr at 37°. The resulting red precipitate was collected and purified by acetonebenzene reprecipitation. The contaminating picric acid was removed by paper electrophoresis. mp (decomp.) above 230°. TNP - Aniline (2, 4, 6 - Trinitrodiphenyl amine)—TNP-aniline was prepared from aniline and TNCB. The reaction was performed in benzene for 1 hr at 40° with stirring and the resulting precipitate was purified by recrystallization from benzene and ligroin. mp 179—180°
{13). TNP-RNA—One hundred and forty mg of yeast RNA and 700 mg of TNBS were dissolved in 7 ml of water and the solution was incubated for 25 hr at pH 9.5 and 37° in a pH-stat. The reaction mixture was neutralized to pH 6.5 with hydrochloric acid and dialyzed against water until picric acid and other byproducts had been removed. TNP-RN A was / . Biocheni.
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5 ml of acetone. The O-TNP-nucleoside was precipitated from this acetone solution by adding 10 volumes of benzene. This precipitation was repeated to remove any contaminating picric acid. O-TNP-adenosine: Yield, 303 mg (81% of the theoretical value); mp 225°. Anal. Found C, 38.41; H, 3.26; N, 22.31%; Calcd. for C16H13N8O10Na: C, 38.47; H, 2.62; N, 22.40%. O-TNP-cytidine: Yield, 137 mg (37%); mp 211°. O-TNP-uridine: Yield, 163 mg (42%); mp 224°. O-TNP-uridine crystallized from 25% acetic acid: Anal. Found; C, 39.65; H, 2.71; N, 15.65% ; Calcd. for C15H13N5O12; C, 39.57; H, 2.88; N, 15.38%. N2-TNP- and N2,O-bis-T NP-Guanosines— Two hundred mg of guanosine (70.7 mM) was treated with 600 mg of TNBS (171 mM) as described above. After 48 hr the whole reaction mixture, from which the starting guanosine had almost disappeared, was applied to a starch column and developed with 800 ml of 1.5 M phosphate buffer. The bands containing TNP-guanosines were cut out together, and treated as before. The precipitates from acetone-benzene were applied to Whatman 3MM paper and developed with the same phosphate buffer. N2-TNP-guanosine gave Rf 0.3, the same value as picric acid. N 2 ,0-bis-TNPguanosine gave Rf 0.01, but contained a small quantity of O-TNP-guanosine which also gave the same Rf value. TNP-guanosines were eluted with water from each of the bands cut from the paper, and were purified by acetonebenzene precipitation. N2-TNP-guanosine: Yield, 158 mg; mp (decomp.) above 220°. N2-TNP-guanosine precipitated from aqueous solution by acidification with hydrochloric acid: Anal. Found: C, 38.99; H, 3.12; N, 22.11%; Calcd. for C 16 H 14 N 8 0 1 i: C, 38.87; H, 2.85; N, 22.67%. N2, O-bis-TNP-guanosine: Yield, 31 mg; mp (decomp.) above 210° (contained a small quantity of O-TNP-guanosine which could be separated by paper electrophoresis). N2-TNP-Guanine— A suspension of 150 mg of guanine and 400 mg of TNBS in 10 ml of water was incubated for 3 hr at pH 10—11
M. AZEGAMI and K. IWAI
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
RESULTS AND DISCUSSION 1. Trinitrophenylation of the Constituents of Nucleic Acids—Specificity of the reaction with TNBS: In the reaction with TNBS at 37° in buffers of pH 9-10, the following compounds were found to give TNP derivatives: guanine, guanosine, guanosine 3'(2')-phosphate,
deoxyguanosine, adenosine, adenosine 5'-phosphate, inosine, xanthosine, cytidine, uridine, benzyl riboside, and ethylene glycol. They are compounds containing either a guanine residue (forming N2-TNP derivatives) or adjacent free hydroxyl groups in the cis position, such as 2'- and 3'-hydroxyl groups of ribosides as well as polyhydric alcohols (forming O-TNP derivatives). The reaction mixtures usually became colored reddish-orange as the reaction proceeded. The reaction mixture of ribose or glucose with TNBS became brown and side reactions seemed to occur. Three TNP derivatives, N2-TNP-, O-TNP-, and N 2 ,0-bis-TNPguanosine, were obtained from the reaction mixture with 171 mM TNBS and 70.0 mM guanosine, as described in "PREPARATIONS," but only N2,0-bis-TNP-guanosine was found with 100 mM TNBS and 10 mM guanosine. The products were separated by paper chromatography or paper electrophoresis. Table I shows the Rf values and mobilities of various TNP derivatives. TNP derivatives of the following sub-
TABLE I. Rf values in paper chromatography and mobilities in paper electrophoresis of TNP derivatives.
2
N -TNP-guanine N2-TNP-deoxyguanosine N2-TNP-guanosine N2, O-bis-TNP-guanosine O-TNP-guanosine O-TNP-adenosine O-TNP-cytidine O-TNP-uridine . . - . O-TNP-inosine O-TNP-xanthosine O-TNP-ethylene glycol Picric acid TNBS
Mobility (mm)
Rf value
Compound (1) 0.15 0.24 0.31 0.01 0.01 0.01 0.11 0.11 0.06 0.01 0.30 0.29 0.50
(2) 0.53
(3) 0.03
(4) - 7 . 2 (0.16)
0.38 0.41 0.56 0.66 0.54
0.09 0 0 0 0 - 0,
- 1 . 9 (0.10)0 - 1 . 9 (0.10) - 1 7 (0.33) -34 (0.49) 0 0
0.50--
0 0 + 52
(5)
+37 + 37 +37 + 37 + 15 + 15 + 30 +40 + 30 +61 + 61 + 83
(1) 1.5 M Sodium phosphate buffer, pH 6. (2) 86% (v/v) w-Butanol-15 N ammonia (100: 1, v/v). (3) Saturated ammonium sulfate-O.l M sodium phosphate buffer, pH 6-2-propanol ( 7 9 : 1 9 : 2 , v/v). (4) 0.25 M Formic acid, 400v/37cm, 1 hr. Symbols: + and — indicate movement toward the anode and the cathode, respectively. Values in parentheses show ratios of mobilities of TNP derivatives to those of the unsubstituted substances. (5) 0.1% Sodium bicarbonate, 1,000 v/37 cm, 1 hr.. . . Vol. 78, No. 2, 1975
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then precipitated by the addition of 3 volumes of ethanol. The precipitates were dissolved in 10 ml of 0.2 M sodium acetate buffer, pH 5, reprecipitated with ethanol, washed with ethanol and ether, and dried under reduced pressure. Yield, 100 mg. TNP-DNA — Twenty-four milligrams of calf thymus DNA and 150 mg of TNBS were dissolved in 10 ml of 1M saline and the solution was incubated at pH 9.5 and 37° in a pHstat. After 18 hr, an additional 50 mg of TNBS was added and incubation was continued for a further 6 hr. The reaction mixture was fully dialyzed against water. This TNP-DNA solution was used for further experiments.
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M. AZEGAMI and K. IWAI
TNPation. For instance, the reaction occurred to the extent of over 95% after 1.5 hr with deoxyguanosine, after 5 hr with adenosine, and after 23 hr with uridine, as shown in Fig. 1.
0
1
2
3
4
5
6
7
8
TIME OF REACTION (hr)
Fig. 1. Rates of trinitrophenylation of nucleosides. The reaction and assay were performed as follows: 5 ml of the reaction mixture containing 0.01 M nucleoside and 0.1 M TNBS was incubated at 37° and pH 9.5 in a pH-stat, except for guanosine, and 0.1 ml aliquots of the reaction mixture were removed at intervals. To these was added 10 or 20 ml of 1% sodium bicarbonate, and the optical densities of the solutions and similarly treated controls were read at 500 nm. A, N-TNPation of deoxyguanosine ; • , O-TNPation of adenosine; D, N, O-bis-TNPation of guanosine in 0.5 M carbonate buffer, pH 10.0-9.5 ; • , O-TNPation of cytidine ; O, TNPation of uridine.
TABLE II. Comparison of the rates of trinitrophenylation of nucleosides and nucleotides at several pH's. The reaction mixture, containing 10 mM nucleoside or nucleotide and 100 mti TNBS in each buffer, was incubated at 37° and aliquots were removed at intervals and treated as described in Fig. 1; the 50% reaction time was calculated from the change of absorption during the initial 2 hr. Molar extinction coefficients of O-TNP-benzyl riboside and TNP-ethylene glycol at 410 nm were assumed to be the same as that of OTNP-adenosine. 50% reaction time (min) Compound
0.5 M Na phosphate pH 7.0
Guanosine 3'(2')-phosphate Deoxyguanosine Adenosine Adenosine 5'-phosphate Cytidine Uridine Benzyl riboside Ethylene glycol
2,400
0.5 M Na carbonate pH 8.3 130 714
pH 9.0 51 30 119 — 447 595 —
pH 10.0 23 IS 21 98 97 140 893 70,000
0.2 M Na borate pH 10.0 23 2,400
/ . Biochem.
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stances were not produced, as determined chromatographically or spectrophotometrically: adenine, cytosine, uracil, thymine, hypoxanthine, xanthine, uric acid, N, N-dimethyl guanine, theobromine, theophylline, caffeine, deoxyadenosine, deoxycytidine, thymidine, adenosine 3'-phosphate, cytidine 3'(2')-phosphate, and uridine 3'(2')-phosphate. The following starting materials were recovered quantitatively by paper chromatography from the 20 hr-reaction mixtures at pH 9: adenine, cytosine, uracil, thymine, adenosine 3'-phosphate, and cytidine 3'(2')-phosphate. The degradation products of TNBS gave two unidentified dark red spots above Rf 0.6 (1.5 M sodium phosphate buffer, pH 6) in addition to those of picric acid and TNBS itself. Rate of Reaction with TNBS—The rate of trinitrophenylation at the guanine residue (NTNPation) and the ribose residue (O-TNPation) depended on pH, and both N- and O-TNPations were scarcely observed below pH 7. The reactivity increased with pH between pH 7 and 11. The time courses for some nucleosides and nucleotides are shown in Fig. 1 and the 50% reaction time, as a measure of reaction rate, is listed in Table II. N-TNPation was more rapid than 0-
TNPATION OF NUCLEIC ACIDS AND CONSTITUENTS
A
as seen in Table II, but not N-TNPation as expected from the specificity of the reaction.
Trinitrophenylation with TNFB and TNCB —In preliminary and qualitative experiments, O-TNPation and N-TNPation were found to occur with both TNFB and TNCB; though both reagents were sparingly soluble in aqueous solution, TNFB reacted with nucleosides and
D
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350
400
450
500
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250 550
300
350
400
450
500
550
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pyrimidine nucleosides >polyhydric alcohols. The difference in reaction rate between ribosides and polyhydric alcohols may depend on the readiness with which the hydroxyl groups form a stable cis configuration. Borate ions inhibited O-TNPation,
415
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M. AZEGAMI and K. IWAI
TABLE III. Optical properties of TNP derivatives. Absorption maxima (nm)a
O-TNP-adenosine O-TNP-cytidine O-TNP-uridine O-TNP-guanosine O-TNP-benzyl riboside TNP-ethylene glycol N2-TNP-guanine N2-TNP-guanosine N2, O-bis-TNP-guanosine a
pH 7.1 ^max 1
^max 2
408 408 412 408 410 415 422 425 408
470 470 470 470 470 470
pH 1.0
=410
^max
in 1% NaHCO3
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