A polarographic study of pyrimidines

A Polarographic Study of Pyrimidines 1 Liebe F. Cavalieri and Bertram A. Lowy From the Laboratories of the Sloan-Kettering Institute for Cancer Research, New York, New York Received May 11, 1951

INTRODUCTION I t has become a p p a r e n t in recent years t h a t the elucidation of t h e structure of the nucleic acids will require a thorough understanding of the physicochemical behavior of all of their components, We h a v e been studying the pyrimidines and purines of these substances in order to accumulate knowledge which m i g h t prove useful in the s t u d y of the macromolecular structure of nucleic acids (1,2). Recently, we h a v e been interested (3) in the problem of the t a u t o m e r i s m of h y d r o x y and amino derivatives (4), since a knowledge of these t a u t o m e r i c s y s t e m s is essential in considering the extent to which hydrogen bonding and other secondary valence forces exist (5) in nucleic acids. I t appeared to us t h a t the polarographic m e t h o d would offer a new p a t h w a y for such studies (6).2 We h a v e therefore u n d e r t a k e n a detailed s t u d y of a large n u m b e r of pyrimidines related to the n a t u r a l l y occurring ones with the hope of gaining both theoretical and practical knowledge useful for the determination of the structure of the high molecular weight substances known as nucleic acids. EXPERIMENTAL Apparatus All measurements were made with a Leeds and Northrop Electrochemograph. Two capillaries were used with the following characteristics: capillary 1: droptime, t -- 4.0 sec. ; m = 0.58 mg./sec. ; m2/St116 0.876; capillary 2: droptime, t = 3.1 sec.; m = 1.68 =

1The authors wish to thank the James Foundation of New York, Inc.; the joint support of the Office of Naval Research and the Atomic Energy Commission, contract N6-ori-99, Task Order 1; and the National Cancer Institute of the United States Public Health Service for their financial assistance. Heath discusses the polarography of several of the naturally occurring purines and pyrimldines. 83

84

LIEBE F. CAVALIERI AND BERTRAM A. LOWY

mg./sec.; m ~/3 t1/6 = 1.69 (open circuit). The height of the mercury column was 62 cm. unless otherwise noted. The Micromax recorder was standardized against a standard 100,000-ohm resistor (~-1%). The electrolysis cells consisted of 15-ml. vessels provided with inlet tubes for the introduction of nitrogen. Potentials are referred to the saturated calomel electrode (SCE). All current-voltage curves were corrected for residual current.

Measurements Measurements were carried out at 23 -4- 0.5 ~ Samples (10-20 mg.) were introduced into a 10-ml. volumetric flask and brought to volume with distilled water. One milliliter was diluted to ten ml. with a buffer of appropriate pH, and a 2-ml. sample of this solution was electrolyzed. Further concentrations were determined by diluting the sample in the electrolysis cell with buffer. The average experimental error was about 5%.

Materials Nearly all compounds included in this communication were those used for spectral measurements in previous communications (3,7). The remaining compounds were prepared according to existing procedures. All compounds were recrystallized to constancy with respect to ultraviolet absorption spectra, counter-current distribution constants (9), elementary analyses, and melting points (whenever possible).

Buffer Solutions Measurements at pH 2.3, 3.6, and 4.5 were made in 0.09 M citrate buffer; at pH 5.6 and 6.8 in 0.09 M phosphate buffer. For measurements at pH 1.3, 0.09 M hydrochloride acid-potassium chloride was used.

Nitrogen Tank nitrogen was purified by passing it through ammonium hydroxide (containing copper turnings) saturated with ammonium chloride, and then passing it through sulfuric acid. RESULTS

The compounds included in this study may be represented by the generalized formula shown. Typical polarograms are shown in Fig. 1 and the data are summarized in Tables I and II. Nearly all of the compounds in Table I show one wave at the pH values studied. Pyrimidine exhibits two waves at pH 3.6 and4.5, while 2-aminopyrimidine shows two waves at pH 3.6, 4.5, and 5.6. In the case of 6-aminopyrimidine two waves are observed at pH 1.2. 2,4,6-Trisubstituted pyrimidines and tetrasubstituted pyrimidines do not reduce under these conditions (Table II).

POLAROGRAPHIC

STUDY

85

OF P Y R I M I D I N E S

O'~

I

I!1

~ o ~

I l l l l l l

~

~

,,,1~

I

I

.

.

7777

I I I I I I I I I

A 0

O" ~'~,

,.~ 0

~,~ ~ O ~

~O

86

LIEBE

F.

CAVALIERI

AND

BERTRAM

I0

A.

H~N

LOWY

~

I

0

.o__

o

o.

~ - /

_,.o _,.,

-

0L,

,

_,.,

-,.,

J

-,.,

-,.,

-,.,

-,.,

,/___S~T, ~ @ J

),5 - 0 . 6 " 0 . 7 "0.8 ~

-I.O -I.I

OCH~

-,.,/-,.,

~

t

.

.

_

-,.,/,.,/.,

I

J .

-,.,

l

I

- , . , -,.,,

I

I

-I.2 -I,3 -I.4 -I.5 -I.6 -I.7 - I . 8 - I . 9 - 2 . 0 Ede VS. S.C~

FIG. l. Polarograms of pyrimidines. Reading from left to right; the pH values are: 1.2, 2.3, 4.5, and 6.8. R

R is H, OH, NH~, or 0CH~ I n a plot of half-wave potential v s . pH, 2-amino- and 6-aminopyrimidine show deviation from linearity. I n the case of pyrimidine, a straight line is obtained in the more acidic regions, while at about p H 6 i a significant deviation occurs. A plot of the values of l o g . --. v s . the Zd

--

potential yields a straight line at p H 2.3 in all cases. T h e values of the

i

i

POLAROGRAPHIC STUDY OF P Y R I M I D I N E S

87

TABLE II Pyrimidines Exhibiting No Waves at the Dropping Mercury Electrode~

Uracil 1,3-Dimethyluracil Thymine O,O-Dimethylthymine 3-Methylthymine Isocytosine Barbituric acid 2,4,6-Trimethoxypyrimidine Carried out at pH 2.3, 4.5, and 6.8.

2,4,6-Triamino4,6-Diamino-2-hydroxy2,4-Diamino-6-hydroxy4,5-Diamino-6-hydroxy2,4,5,6-Tetramino2,4,5-Triamino-6-hydroxy4,5,6-Triamino-2-hydroxy4,5-Diamino-2,6-dihydroxy-

reciprocal of the slopes are in satisfactory agreement for a one electron reversible reaction. Compound

Reciprocal slope

Pyrimidine (pH 1.25) 2-Amino6-Amino6-Hydroxy4-Amino-6-hydroxy2-Amino-6-methoxy-

0.061 0.074 0.066 0.064 0.055 0.064

The proportionality, between diffusion current and concentration was checked in all cases and id/C was found to be sensibly constant in the range studied (10-3 M). The effect of temperature and height of the mercury column were observed in several instances in order to determine the behavior of tautomeric systems. These effects will be discussed below. DISCUSSION Pyrimidine exhibits a well-defined wave at pH 1.2 and one at pH 6.8 whose limiting current is nearly twice that at pH 1.2. At intervening pH values (3.6, 4.5) two curves are apparent, each of which is about equal in height to that at pH 1.2. At pH 2.3 there is a definite indication of a second wave in the region of El - 1.25 v., although it lies too close to the hydrogen discharge curve to be determined accurately. At pH 1.2 the inflection in this region is absent, probably because it lies close to the discharge curve. I n the case in which two waves are evident, the one occurring at the more negative potential appears to be independent of the pH and it is suggestive, therefore, that the capture of an electron

88

LIEBE

F.

CAVALIERI

AND

BERTRAM

A.

LOWY

is the primary step. On the other hand, the wave whose potential is dependent upon the pH probably requires a hydrogen ion in the first i stage of the reduction. A plot of log . vs. Ed, yields a value of ~a - i 0.061 for the reciprocal slope at pH 1.2. This is in good agreement for a one electron reversible reaction; however a plot of p H vs. E~ indicates irreversibility. Calculations were made to ascertain the number of electrons involved. Using a value of 7.1 X 10-6 cm3 sec. for the diffusion coefficient of pyrimidine (calculated from the Stokes-Einstein equation) and substituting this in the Ilkovic equation yields a value of 1.1 electrons for the reduction at p H 1.2. At p H 3.6 and 4.5 the combined heights yield a value of 2.2 electrons, and at p H 5.6 and 6.8 the single waves give a vMue of 2.4 and 2.2 electrons, respectively. It would appear that the reduction of pyrimidine involves two stages. The following mechanism is consistent with the data for neutral solutions. In acid solution one of +H++e

---,

,

.

N

N

OH-

N

N

the nitrogen atoms would possess a positive charge but the mechanism for the reduction could be identical to that in neutral solution, if the uncharged nitrogen were involved. The alternative mechanism would be as follows: H

H2

+H++e N

e

--,

H~

~ N

--~ N

H2N /

N

The above reactions are essentially 1,4-additions; however a 1,2-addition (at the 1,6-double bond) would appear equally probable in this case.

It should be noted that the double bond at the 2,3-position differs from that at the 1,6-position in that the former is p a r t of an amidine system and reduction is therefore unlikely (10). In this connection it is noteworthy that imidazole, which contains an amidine system similar to that of pyrimidine, exhibits no reduction wave. On the other hand 4(5)-amino-5(4)-imidazolecarboxamidine, which does not contain the

POLAROGRAPHIC

89

STUDY OF P Y R I M I D I N E S

2,3-double bond but which possesses the 4,5,6,1-system, exhibits a halfwave potential of - 1 . 5 7 v. at p H 6.8. It is of further interest to note t h a t N-isopropyl crotonaldimine (8), which contains a system similar to the 4,5,6,1-system of pyrimidine, exhibits a wave. ~ NH~

NH~

L

I

c

H

C

N// \ell i-Pr

HN~

N

~C j

~

C

\

H2NJC~N /

CH3

The polarographic behavior of 2-aminopyrimidine is similar to t h a t of pyrimidine. The appearance of two waves corresponds to t h a t of pyrimidine. Similarly, the second of a pair of waves (i.e., t h a t occurring at the more negative potential) appears to be largely independent of the pH. For a value for D of 6.7 X 10-6 cm.2/sec., 2.2 electrons are taken up at p H 6.8. In the case of 6-aminopyrimidine two waves appear at p H 1.2. The wave occurring at the more negative potential is independent of the height of the mercury (Table III) and it would appear t h a t a kinetic TABLE III Effect of Height of Mercury and Temperature on Wave Heights of 6-Aminopyrimidine pH

1.2

Temp.

cm.

22

82 72 62 82 72 62 62 52 42 32

53 5.5

Height of Hg

~

22

il

illh89

7.6 7.0 6.4 8.8 8.4 7.8 4.6 4.3 3.8 3.2

0.84 9 0.83 0.82 0.97 0.99 0.99 0.58 0.59 0.58 0.57

3.1 3.0 3.4

current (11) is produced during electrolysis. This effect could be explained on the basis of a tautomeric shift which would allow the formation of a reducible form. 6-Aminopyrimidine m a y exist in the following 8The general appearance of this curve resembled that of pyrimidine, but it was not well-defined.

9O

LIEBE F. CAVALIERI AND BERTRAM A. LOWY

forms: NH~

NH

N

N

This equilibrium would fulfill the conditions, provided that the shift is labile enough to give rise to a kinetic current. Evidence supporting a tautomeric shift is found in the effect of temperature. At 53 ~ only one wave appears at pH 1.2. The half-wave potential and the height correspond to that of the first wave at room temperature (El -1.13 v.) after the temperature correction has been applied. The current is diffusion controlled. It was suggested above (in the case of pyrimidine) that the - N = C in the 1,6-position is probably involved in the reduction. More definitive evidence regarding the site of reduction is found in the behavior of isocytosine (I) and O-methylisocytosine (II). The essential difference between these compounds is that II possesses a 1,6-double bond while in the former (I) it is likely that the double bond is outside the ring (i.e., - C = O ) . 4 Since II exhibits a wave, it would appear that the 1,6-double bond of II is responsible for the reduction. 0

H~N

N I

OCH3

H~N

N II

The waves for IIare in general well-defined; however, at intermediate pH values,, the shape of the lower part of the curve suggests the presence of a superimposed wave. This may be due to the presence of both charged and uncharged molecules at these pH values. Further, thymine (III), 3-methylthymine (IV), uracil (V), and 1,3-dimethyluracil (VI) are not reduced while 3,6-0-dimethylthymine (VII) is reduced. There may be some doubt as to the tautomeric forms of compounds III-V, 4 Unpublished infrared data from this laboratory confirm this suggestion.

91

POLAROGRAPHIC STUDY OF PYRIMIDINES

O

O

JY

JY

H

CHa VI

V

OCHa

I1

b

H

CHa IV

O

z ' vN

=: ]

III

0

0

I

OCHa ! CHa

CHa

jv

HaCO

N

I

CH8 VII

VIII

but t h e y p r o b a b l y exist as shown. H o w e v e r c o m p o u n d s VI and V I I Cannot t a u t o m e r i z e and the fact t h a t V I I is reducible m a y be a t t r i b u t e d to the presence of a 1,6-N = C - unit which is the only essential difference. Since 0 , 0 - d i m e t h y l t h y m i n e ( V I I I ) exhibits no wave, it is a p p a r e n t t h a t in order for reduction to occur in these derivatives the oxygen a t o m in the 2-position m u s t be present in carbonyl form with the concomitant presence of an - N = C - in the 1,6-position. I t m a y also be inferred from a comparison of these compounds t h a t in t h y m i n e ( I I I ) and uracil (V) the oxygen-6-atoms are present as carbonyl groups. ACKNOWLEDGMENT The authors wish to thank Dr. George B. Brown for continued interest and helpful discussions. SUMMARY

Of 26 pyrimidines investigated, 10 have been found to reduce at the dropping m e r c u r y electrode. The n u m b e r of electrons taking p a r t in the reduction has been calculated for a n u m b e r of cases. I t is suggested t h a t

I

I

I

the reducible group in these compounds involves the - C = C - C = N system. On the basis of an observed kinetic current it appears t h a t 6-aminopyrimidine m a y tautomerize.

92

LIEBE F. CAVALIERI AND BERTRAM A. LOWY REFERENCES

1. CAVALIER1,L. F., AND ANGELOS, A., J. Am. Chem. Soc. 72, 4686 (1950). 2. CAVALIEr, L. F., KERR, S. E., AND ANGELOS,J. Am. Chem. Soc. 73, 2567 (1951); CAVALIERI,L. F., ANGELOS,A., AND BALIS, M. E., J. Am. Chem. Soc. 73, 4902 (1951). 3. CAVALIERI,L. F., AND BENDICH, A., J. Am. Chem. Soc. 72, 2587 (1950). 4. HEYROTH, F., AND LOOFBOUROW, J., J. Am. Chem. Soc. 56, 1728 (1934). 5. GULLAND,J., Cold Spring Harbor Symposia Quant. Biol. 12, 99 (1947). 6. HEATH, J. C., Nature 158, 23 (1946). 7. CAVALIERI, L. F., BENDICH, A., TINKER, J., AND BROWN, G. B., J. Am. Chem. Soc. 70, 3875 (1948). 8. TIOLLAIS, R., Compt. rend. 224, 1116 (1947). 9. TINKER, J., AND BROWN, G. B., J. Biol. Chem. 173, 585 (1948). 10. RUNNER, M., KILPATRICK, IV[., AND WAGNER, E., J. Am. Chem. Soc. 69, 1406 (1947). 11. BRDI~KA, R., AND WIESNER, Collection Czechoslov. Chem. Commun. 12, 138 (1947).