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[4] The reaction of hydrazine with pyrimidine bases
[4]
HYDRAZINE REACTION
31
[4] T h e R e a c t i o n of H y d r a z i n e w i t h P y r i m i d i n e B a s e s
By D. M. BBOWN Hydrazine can be used as a reagent for the specific alteration of cytosine and uracil type bases. 1 The outcome of the reaction differs according to the conditions used. Purine derivatives are essentially unaffected by the reagent. Hydrazine hydrate 1,2 and anhydrous hydrazine3,4 have been commonly used. They have the same qualitative effect on pyrimidines and the corresponding nucleosides and nucleotides, leading to ring cleavage. Thus uracil (I; R--H) gives rise to pyrazolone (II; R----H), thymine (I; R--Me) to 4-methylpyrazolone (II; R--Me), and cytosine (III) to 3-aminopyrazole (IV). In each ease urea is the other product. The rate order is uracil > cytosine > thymine (1:5:15 in the anhydrous reagent3). Nucleosides give, in addition, the hydrazone of ribose (or 2-deoxyribose), from which the sugar can be released by treatment with benzaldehyde.~ Nucleotides give the corresponding phosphorylated derivatives. Hydrazinolysis is conveniently followed spectrophotometrically by the decrease in absorption at the ;tmaxof the pyrimidine. Qualitatively different results are obtained with hydrazine solutions at lower pH values2 Thus at pH 6, an exchange reaction occurs with cytosine leading to N4-aminocytosine (V) and the disubstituted hydrazine (VI). Uracil and thymine are apparently stable at this pH2 At pit 8 another reaction is observed 7 for which oxygen is required and copper is catalyf,ic, probably due to generation of diimide in the solution.8 Under these conditions thymine is converted to 5,6-dihydrothymine (VII) and, while reduction of cytosine can be observed, it is very slow. The reaction of hydrazine hydrate with pseudouridine9 and with 1R. Fosse, A. Hieulle, and L. W. Bass, Compt. Rend. Acad. Sci. 178, 811 (1924); P. A. Levene and L. W. Bass, J. Biol. Chem. 71, 167 (1927). 2F. Baron and D. M. Brown, J. Chem. Soc. p. 6263 (1955). 3V. Habermann, Collection Czech. Chem. Commun. 26, 3147 (1961). ~A. Temperli, H. Tiirler, P. Rfist, A. Danon, and E. Chargaff, Biochim. Biophy.~. Acta 91, 462 (1964). 5F. Lingens and H. Schneider-Bernl6hr, Ann. Chem. 686, 134 (1965). e E. I. Budovskii, J. A. Haines, and N. K. Kochetkov, Dokl. Akad. Nauk SSSR 158, 379 (1964). ' D. M. Brown, A. D. McNaught, and P. Schell, Biochem. Biophys. Res. Commun.
24, 967 (1966). 8F. Aylward and M. Sawistowska, Chem. Ind. (London) p. 484 (1962); S. Hiinig, H. R. Miiller, and W. Thier, Angew. Chem. Intern. Ed. ;Engl. 4, 271 (1965). F. F. Davis and F. W. Allen, J. Biol. Chem. 227, 907 (1957).
NUCLEIC ACID COMPONENTS
32
[4]
o
HN~.
o
HN/~, R
R
H (I)
(n)
NI~
NH~ -.
~
NI~- CO" NI~
+
H
H (III)
(IV)
NH •NI~
NH.,
NH
0
H
H
H
H
(V)
(VI)
(VII)
uridine diphosphate glucose 1° have been studied. Hydrazinolysis of nucleic acids is discussed ill this volume [26]. Methylhydrazine also reacts with pyrimidine bases. 5 Examples of the various procedures are given below. Procedure Anhydrous hydrazine m a y be purchased or be prepared from the sulfate or hydrochloride as follows: • bydrazine sulfate (100 g) and sodium hydroxide (150 g) are mixed in a flask (1 liter), which is then connected to a water condenser. T h e mixture is heated carefully. The reaction is very fast and all the hydrazine distills off as a colorless fuming liquid within a few minutes. The hydrazine is then distilled in v a c u o over solid sodium hydroxide) °a 1"R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini, d. Biol. Chem. 184, 333 (1950). io. 1%fluxing 100% hydrazine hydrate with an equal weight of sodium hydroxide pellets for 2 hours and then distillation in a slow stream of nitrogen introduced through a capillary leak is recommended. Distillation in air can lead to an explosion.
[4]
HYDRAZINE REACTION
33
Hydrazinolysis Reactions Hydrazine Hydrate. ~ Cytidine (0.5 g) and hydrazine hydrate (1.5 ml) are heated together at 65 ° for 90 minutes. Water (2 ml) is added and 3-aminopyrazole is extracted with ethyl acetate. It has Xmax 239 m~ (~ 6040) in 0.1 N HCI, forms a picrate (m.p. 217-218 °) (decomp.) and a dixanthhydryl derivative (m.p. 216 °) (decomp.) when heated with xanthhydrol in dilute acetic acid. The aqueous phase from the original reaction is evaporated in vac~o to give an oil, essentially ribose hydrazone, from which ribose is liberated by shaking a warm aqueous solution with benzaldehyde; excess reagent and benzalazine are removed by ether extraction. Under the same conditions the reaction with uridine is complete in approximately 10 minutes. After removal of hydrazine under vacuum and addition of methanolic xanthhydrol, the dixanthhydryl derivatives of urea and pyrazolone crystallize overnight. The former (m.p. 266 °) is insoluble in acetone, whereby it is separated from the latter (m.p. 215217°). Anhydrous Hydrazine. ~ Thymidine 5'-phosphate or its sodium salt (1 millimole) is suspended in freshly distilled hydrazine (1 ml) and heated in a sealed tube at 60 ° for 4 hours. The reaction mixture is taken to dryness under reduced pressure over P205 and H2S04 during 2~ A.8 hours and the residue is evaporated with water in vac~o. 4-Methylpyrazolone crystallizes from a strong aqueous solution on cooling and has m.p. 232 °, '~max 247 m/~ (e 7215) in water and 230.5 m# in 0.1 N HCh It is detected on ehromatograms by viewing in UV light (as is 3-aminopyrazole). The mother liquors contain urea, Rs 0.68 in isopropanol-l~ ammonium sulfate (2:1 v/v) detected by the p-dimethylaminobenzaldehyde spray ~1 and the hydrazone of 2-deoxyribose 5-phosphate, RI 0.45. 2-Deoxyribose 5-phosphate is liberated from its hydrazone by treating an aqueous solution (1 ml) with 10% ethanolic benzaldehyde (0.2 ml), shaking the sample at room temperature for 12 hours, and then extracting with ether. The sugar phosphate is detected with the diphenylamine spray reagent ~ (which gives an anomalous reaction with the hydrazoneS,4). Other Reactions with Hydrazine Formation o] N4-A mi~ooytosine (4-H ydrazino-~-pyrimidone ) 2 Cytosine (2 g) is stirred at 80 ° in 50 ml 4 M hydrazine solution adjusted to ~ R. E. Cline and R. M. Fink, Anal. Chem. 28, 47 (1956) ; K. Fink, R. E. Cline, and R. M. Fink, Anal. Chem. 35, 389 (1963). ~K. Burton, Biochem. J. 62, 315 (1956).
34
NUCLEIC ACID COMPONENTS
[S]
pH 6.0 with acetic acid. After 6 hours the solution is cooled and the yellow product (VI) (0.35 g) is collected. It has m.p. > 300 ° (decomp.) and X..... 327 m~ (e 21,380) in 0.1 N HC1. The mother liquor is evaporated to one-third its volume and set aside at 0% N4-aminocytosine (V) (1.2 g) separates. It has m.p. > 300 ° (decomp.) and X.... 268 m~ (c 7585) in water and 276 m~ (c 10,960) in 0.1 N-HC1. It forms a picrate m.p. 215 ° (decomp.). This product can also be isolated from the reaction with aqueous hydrazine at pH 8 ~ and with hydrazine hydrate. ~ Formation of 5,6-Dihydrothymine. ~ Thymine (60 mg) is dissolved in M hydrazine solution (30 ml), pH 8.0. Air is bubbled through the solution until the optical density at 267 m~ has fallen to 5% of the original value. Hydrazine is removed by percolating the solution through a column of Dowex 50 (H ÷ form). Evaporation of the eluate and crystallization of the residue from water gives dihydrothymine, m.p. 262 ° detected on chromatograms, after spraying with N-alkali to ring-open, by p-dimethylaminobenzaldehyde. 11 It has h ..... 230 m~ in alkaline solution, the optical density rapidly decreasing with time. In addition a more basic product of unknown structure is formed which is eluted by ammonia from the Dowex 50 column after hydrazine. Both it and dihydrothymine are converted by hot 6 N HC1 to ~-methyl-fl-alanine, detected by the ninhydrin reagent. Catalysis can be effected by dipping a clean copper wire into the hydrazine solution (1-5 M) for 2 minutes. Reaction times are very variable but, in small-scale runs (0.1-0.2 mg/5 ml), approximate half-lives for the reduction are: thymine 5 minutes, uracil 10 minutes, 5-methylcytosine 4.75 hours, cytosine 24 hours.
[5] T h e R e a c t i o n of H y d r o x y l a m i n e
with Pyrimidine Bases
By JOHN H. PHILLIPS Hydroxylamine may be used as a reagent for specifically modifying cytosine or uracil residues in nucleic acids; it does not react with purines. Cytosine residues react optimally at pH 6, uracil at pH 10, and the reaction is selective if carried out at these pH values. 1 An even more specific reaction is given by O-methylhydroxylamine, which reacts only with cytosine? 1D. W. Verwoerd, W. Zillig, and H. Kohlhage, Z. Physiol. Chem. 33~, 184 (1963). N. K. Kochetkov, E. I. Budowsky, and R. P. Shibaeva, Biochim. Biophys. Acta 68, 493 (1963).
HYDRAZINE REACTION
31
[4] T h e R e a c t i o n of H y d r a z i n e w i t h P y r i m i d i n e B a s e s
By D. M. BBOWN Hydrazine can be used as a reagent for the specific alteration of cytosine and uracil type bases. 1 The outcome of the reaction differs according to the conditions used. Purine derivatives are essentially unaffected by the reagent. Hydrazine hydrate 1,2 and anhydrous hydrazine3,4 have been commonly used. They have the same qualitative effect on pyrimidines and the corresponding nucleosides and nucleotides, leading to ring cleavage. Thus uracil (I; R--H) gives rise to pyrazolone (II; R----H), thymine (I; R--Me) to 4-methylpyrazolone (II; R--Me), and cytosine (III) to 3-aminopyrazole (IV). In each ease urea is the other product. The rate order is uracil > cytosine > thymine (1:5:15 in the anhydrous reagent3). Nucleosides give, in addition, the hydrazone of ribose (or 2-deoxyribose), from which the sugar can be released by treatment with benzaldehyde.~ Nucleotides give the corresponding phosphorylated derivatives. Hydrazinolysis is conveniently followed spectrophotometrically by the decrease in absorption at the ;tmaxof the pyrimidine. Qualitatively different results are obtained with hydrazine solutions at lower pH values2 Thus at pH 6, an exchange reaction occurs with cytosine leading to N4-aminocytosine (V) and the disubstituted hydrazine (VI). Uracil and thymine are apparently stable at this pH2 At pit 8 another reaction is observed 7 for which oxygen is required and copper is catalyf,ic, probably due to generation of diimide in the solution.8 Under these conditions thymine is converted to 5,6-dihydrothymine (VII) and, while reduction of cytosine can be observed, it is very slow. The reaction of hydrazine hydrate with pseudouridine9 and with 1R. Fosse, A. Hieulle, and L. W. Bass, Compt. Rend. Acad. Sci. 178, 811 (1924); P. A. Levene and L. W. Bass, J. Biol. Chem. 71, 167 (1927). 2F. Baron and D. M. Brown, J. Chem. Soc. p. 6263 (1955). 3V. Habermann, Collection Czech. Chem. Commun. 26, 3147 (1961). ~A. Temperli, H. Tiirler, P. Rfist, A. Danon, and E. Chargaff, Biochim. Biophy.~. Acta 91, 462 (1964). 5F. Lingens and H. Schneider-Bernl6hr, Ann. Chem. 686, 134 (1965). e E. I. Budovskii, J. A. Haines, and N. K. Kochetkov, Dokl. Akad. Nauk SSSR 158, 379 (1964). ' D. M. Brown, A. D. McNaught, and P. Schell, Biochem. Biophys. Res. Commun.
24, 967 (1966). 8F. Aylward and M. Sawistowska, Chem. Ind. (London) p. 484 (1962); S. Hiinig, H. R. Miiller, and W. Thier, Angew. Chem. Intern. Ed. ;Engl. 4, 271 (1965). F. F. Davis and F. W. Allen, J. Biol. Chem. 227, 907 (1957).
NUCLEIC ACID COMPONENTS
32
[4]
o
HN~.
o
HN/~, R
R
H (I)
(n)
NI~
NH~ -.
~
NI~- CO" NI~
+
H
H (III)
(IV)
NH •NI~
NH.,
NH
0
H
H
H
H
(V)
(VI)
(VII)
uridine diphosphate glucose 1° have been studied. Hydrazinolysis of nucleic acids is discussed ill this volume [26]. Methylhydrazine also reacts with pyrimidine bases. 5 Examples of the various procedures are given below. Procedure Anhydrous hydrazine m a y be purchased or be prepared from the sulfate or hydrochloride as follows: • bydrazine sulfate (100 g) and sodium hydroxide (150 g) are mixed in a flask (1 liter), which is then connected to a water condenser. T h e mixture is heated carefully. The reaction is very fast and all the hydrazine distills off as a colorless fuming liquid within a few minutes. The hydrazine is then distilled in v a c u o over solid sodium hydroxide) °a 1"R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini, d. Biol. Chem. 184, 333 (1950). io. 1%fluxing 100% hydrazine hydrate with an equal weight of sodium hydroxide pellets for 2 hours and then distillation in a slow stream of nitrogen introduced through a capillary leak is recommended. Distillation in air can lead to an explosion.
[4]
HYDRAZINE REACTION
33
Hydrazinolysis Reactions Hydrazine Hydrate. ~ Cytidine (0.5 g) and hydrazine hydrate (1.5 ml) are heated together at 65 ° for 90 minutes. Water (2 ml) is added and 3-aminopyrazole is extracted with ethyl acetate. It has Xmax 239 m~ (~ 6040) in 0.1 N HCI, forms a picrate (m.p. 217-218 °) (decomp.) and a dixanthhydryl derivative (m.p. 216 °) (decomp.) when heated with xanthhydrol in dilute acetic acid. The aqueous phase from the original reaction is evaporated in vac~o to give an oil, essentially ribose hydrazone, from which ribose is liberated by shaking a warm aqueous solution with benzaldehyde; excess reagent and benzalazine are removed by ether extraction. Under the same conditions the reaction with uridine is complete in approximately 10 minutes. After removal of hydrazine under vacuum and addition of methanolic xanthhydrol, the dixanthhydryl derivatives of urea and pyrazolone crystallize overnight. The former (m.p. 266 °) is insoluble in acetone, whereby it is separated from the latter (m.p. 215217°). Anhydrous Hydrazine. ~ Thymidine 5'-phosphate or its sodium salt (1 millimole) is suspended in freshly distilled hydrazine (1 ml) and heated in a sealed tube at 60 ° for 4 hours. The reaction mixture is taken to dryness under reduced pressure over P205 and H2S04 during 2~ A.8 hours and the residue is evaporated with water in vac~o. 4-Methylpyrazolone crystallizes from a strong aqueous solution on cooling and has m.p. 232 °, '~max 247 m/~ (e 7215) in water and 230.5 m# in 0.1 N HCh It is detected on ehromatograms by viewing in UV light (as is 3-aminopyrazole). The mother liquors contain urea, Rs 0.68 in isopropanol-l~ ammonium sulfate (2:1 v/v) detected by the p-dimethylaminobenzaldehyde spray ~1 and the hydrazone of 2-deoxyribose 5-phosphate, RI 0.45. 2-Deoxyribose 5-phosphate is liberated from its hydrazone by treating an aqueous solution (1 ml) with 10% ethanolic benzaldehyde (0.2 ml), shaking the sample at room temperature for 12 hours, and then extracting with ether. The sugar phosphate is detected with the diphenylamine spray reagent ~ (which gives an anomalous reaction with the hydrazoneS,4). Other Reactions with Hydrazine Formation o] N4-A mi~ooytosine (4-H ydrazino-~-pyrimidone ) 2 Cytosine (2 g) is stirred at 80 ° in 50 ml 4 M hydrazine solution adjusted to ~ R. E. Cline and R. M. Fink, Anal. Chem. 28, 47 (1956) ; K. Fink, R. E. Cline, and R. M. Fink, Anal. Chem. 35, 389 (1963). ~K. Burton, Biochem. J. 62, 315 (1956).
34
NUCLEIC ACID COMPONENTS
[S]
pH 6.0 with acetic acid. After 6 hours the solution is cooled and the yellow product (VI) (0.35 g) is collected. It has m.p. > 300 ° (decomp.) and X..... 327 m~ (e 21,380) in 0.1 N HC1. The mother liquor is evaporated to one-third its volume and set aside at 0% N4-aminocytosine (V) (1.2 g) separates. It has m.p. > 300 ° (decomp.) and X.... 268 m~ (c 7585) in water and 276 m~ (c 10,960) in 0.1 N-HC1. It forms a picrate m.p. 215 ° (decomp.). This product can also be isolated from the reaction with aqueous hydrazine at pH 8 ~ and with hydrazine hydrate. ~ Formation of 5,6-Dihydrothymine. ~ Thymine (60 mg) is dissolved in M hydrazine solution (30 ml), pH 8.0. Air is bubbled through the solution until the optical density at 267 m~ has fallen to 5% of the original value. Hydrazine is removed by percolating the solution through a column of Dowex 50 (H ÷ form). Evaporation of the eluate and crystallization of the residue from water gives dihydrothymine, m.p. 262 ° detected on chromatograms, after spraying with N-alkali to ring-open, by p-dimethylaminobenzaldehyde. 11 It has h ..... 230 m~ in alkaline solution, the optical density rapidly decreasing with time. In addition a more basic product of unknown structure is formed which is eluted by ammonia from the Dowex 50 column after hydrazine. Both it and dihydrothymine are converted by hot 6 N HC1 to ~-methyl-fl-alanine, detected by the ninhydrin reagent. Catalysis can be effected by dipping a clean copper wire into the hydrazine solution (1-5 M) for 2 minutes. Reaction times are very variable but, in small-scale runs (0.1-0.2 mg/5 ml), approximate half-lives for the reduction are: thymine 5 minutes, uracil 10 minutes, 5-methylcytosine 4.75 hours, cytosine 24 hours.
[5] T h e R e a c t i o n of H y d r o x y l a m i n e
with Pyrimidine Bases
By JOHN H. PHILLIPS Hydroxylamine may be used as a reagent for specifically modifying cytosine or uracil residues in nucleic acids; it does not react with purines. Cytosine residues react optimally at pH 6, uracil at pH 10, and the reaction is selective if carried out at these pH values. 1 An even more specific reaction is given by O-methylhydroxylamine, which reacts only with cytosine? 1D. W. Verwoerd, W. Zillig, and H. Kohlhage, Z. Physiol. Chem. 33~, 184 (1963). N. K. Kochetkov, E. I. Budowsky, and R. P. Shibaeva, Biochim. Biophys. Acta 68, 493 (1963).