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Photochemically reversible pyrimidine dimer product of electrochemical reduction of pyrimidone-2
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97382
PHOTOCHEMICALLY R E V E R S I B L E P Y R I M I D I N E DIMER PRODUCT OF ELECTROCHEMICAL REDUCTION OF PYRIMIDONE-2 B A R B A R A C Z O C H R A L S K A AND D. S H U G A R
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Warszawa 22, and Institute of Biochemistry a~d Biophysics, Academy of Sciences, Warszawa ~2 (Poland) (Received April 25th, 1972)
SUMMARY
Pyrimidone-2 gives a single one-electron polarographic wave in aqueous buffered medium, and two one-electron waves of equal height in aqueous unbuffered (CH3)aNBr. The reduction product in neutral aqueous medium, identical with that corresponding to wave I in (CH3)4NBr, was isolated by macroelectrolysis, and shown to consist of a dimer of 3,6-dihydropyrimidone-2, the structure of which was identified as 6,6'-bis(3,6-dihydropyrimidone-2). This dimer reduction product could be readily oxidized polarographically to regenerate the original pyrimidone-2. On irradiation in neutral aqueous medium at 254 nm, the dimer reduction product was quantitatively photodissociated to the parent pyrimidone-2 with a quantum yield of about o.I. The reduction product corresponding to wave II in (CH~)4NBr was also isolated and shown to be 3,6-dihydropyrimidone-2. The mechanism for electroreduction of pyrimidone-2 to give these two products has been formulated. Both of these products are also formed during electroreduction of cytosine in aqueous medium at pH approx. 4.5. The dimer reduction product has been identified as a component of some photoproducts of nucleic acid constituents and, possibly, of nucleic acids and its photobiological significance is discussed.
The polarographic behaviour of cytosine and some of its derivatives was investigated by Smith and Elving 1 and Janik and Palecek 2, who proposed that the electroreduction of cytosine proceeds initially at the 3,4 bond, leading to elimination of NH~ to give 2-pyrimidone. Consequently the final products of reduction of cytosine and 2-pyrimidone should be identical. Smith and Elving 1 further suggested that reduction of pyrimidone-2 is a one-electron radical process, whereas Janik and Palecek 2 claimed a two-electron mechanism, with formation of 3,4-dihydro-2-pyrimidone, which was unstable and subject to ring scission at the 3,4 bond. The similarity of the ultraviolet absorption spectra of the products of electroreduction of cytosine and pyrimidone-2 with those of the catalytic reduction products of these two substances 3,4 prompted us to examine once again the polarographic behaviour, and the mechanism for electroreduction, of pyrimidone-2, largely on the basis of isolation and identification of the products of electrolysis. The results obBiochim. Biophys. Acta, 281 (1972) i - i o
2
B. CZOCHRALSKA, 1~. SH(~;AR
tained are of general interest not only in relation to the properties of the biologically important dihydropyrimidines, but also with respect to pyrimidine photoproducts of biological significance.
MATERIALS AND METHODS
The hydrochloride salt of pyrinfidone-2 was a product of Sigma Chemical Co. It was chromatographically homogeneous in several solvent systems and, at pH 7.2, exhibited a ~max at 299 nm with an emax of 4"57 " 103 (C/. ref. 5}.
Polarography Polarographic curves were recorded with a Radiometer polarograph Polariter PO 4, using a dropping mercury electrode with the following characteristics: flow rate of mercury, m = 2.05 mg • s-l; drop time t -- 3.9 s. at a mercury column height of 6o cm. The pyrimidone-2 concentration was 5 " IO-* M, either in buffered solutions made up to 0.2 M with KC1 or in o.I M aqueous (CHshNBr. Britton-Robinson buffers were employed over the pH range 2-12. The solutions were rendered oxygen-free by bubbling through them nitrogen previously purified by the procedure of Meites 6. All curves were recorded at 25-t-1 °C. A Radiometer PHM-4d compensating instrument was used for pH measurements. All potentials are referred to the saturated calomel electrode.
Macroelectrolysis Preparative electrolyses were conducted with a mercury pool electrode (approx. 20 cm2), fitted with a magnetic stirrer for stirring, in 0.2 M acetate buffer (pH 4.5) and phosphate buffer (pH 7.5), as well as in aqueous (CHs)4NBr , under anaerobic conditions, with depolarizator concentrations of 1.25 • IO-~ M and 5 " IO-~ M. A silver coulometer was used for coulometric measurements. Macroelectrolysis was also carried out in ~H20, buffered with phosphate to p2H 7.2, and the reduction product isolated in the same way. Yields were similar to those in water. For NMR spectroscopy the product was dried several times from water to remove exchangeable deuterium atoms.
Chromatography Ascending chromatography was employed with Whatman papers No. I and 3, using the solvent systems indicated in Table I. Spots were revealed with the aid of a dark ultraviolet lamp and/or by spraying with I M alkali and then with p-dimethylaminobenzaldehyde 7.
Ultraviolet irradiation Solutions were irradiated in Io-mm path-length quartz spectrophotometer cuvettes, the irradiation source being a Phillips 4o-W germicidal lamp (254 nm), the radiation from which was first passed through a 5-mm layer of 35 % aqueous acetic acid to eliminate traces of radiation below 23 ° n m . The intensity incident oil the surface of the cuvette was estimated from the rate of photohydration of a lO -4 M aqueous solution of uridine in the same cuvette, using the known quantum yield for Biochim. Biophys. Mcta, 281 (1972) I - I O
PYRIMIDINE DIMER
3
this reaction of 2.1 • lO -3 (ref. 8). When the irradiated solution was to be subjected to chromatography, a Io-fold higher concentration was irradiated in a I-mm pathlength cuvette.
Spectroscopy Ultraviolet absorption spectra were run on a Zeiss (Jena) VSU-2 instrument.
In/rared absorption spectra were recorded by means of a Zeiss (Jena) U R - I o spectrophotometer, the samples being embedded in KBr matrices in the standard manner. Poor solubility made it impossible to record solution spectra. N M R spectra were obtained with the aid of a Zeiss (Jena) ZKR-6o instrument. The d,-acetic acid employed as solvent (a Fluorochem product) contained more than 99 % atom 2H. Chemical shifts are with reference to trimethylsilane. Mass spectroscopy made use of an Associated Electrical Industries MS- 9 doublefocussing instrument, and spectra were recorded at 25 and 7° eV and at temperatures of 50, 17o and 280 °C. Melting points, uncorrected, were measured on a Boeitius microscope hot stage.
RESULTS
Over the pH range 2-12, pyrimidone-2 was found to give a single polarographic, diffusion-controlled, reduction wave the height of which was independent of pH within the limits of experimental error. El/2 was directly dependent on the pH, the slope of the Ell 2 vs pH curve being 80 mV/pH. These results are in agreement with those of Smith and Elving 1 and Janik and Palecek ~. However, macrocoulometric studies demonstrated that reduction of the compound was a one-electron process, in accord only with Smith and Elving 1. The slope of the plot of log(i/io--I) vs E, where i is the current at potential E, and id the diffusion-controlled limiting current, exhibited a value of 75 mV, indicative of a nonreversible process. The value of p, the number of protons involved in the electrode reaction 6'~° was calculated as one per molecule. The values of pK~ and pK 2 for pyrimidone-2, e~timated from the dependence of Ell 2 on pH, were 2.8 and 9.5, as compared with those determined by direct spectrophotometric titration, 2.24 and 9.17 (ref. 9). In aqueous o.i M (CH3)4NBr, with depolarizator concentrations in the range 4 " lO-4 to 2 • lO -3 M, pyrimidone-2 exhibited two reduction waves of identical height with half-wave potentials, E1/2, of --0.75 and --1.53 V (Fig. I). At lower concentrations a third wave was visible. In o.I M ammonium carbonate, pH 8.2, with a pyrimidone-2 concentration of 5 " IO-4 M, two reduction waves of equal height and with El~ 2 values of --1.24 and --1.8 V were recorded.
Macrodeetrolysis and product isolation Macroelectrolysis was carried out as described under Materials and Methods at a constant potential corresponding to the initial limiting current, i.e. E ---- --0. 9 V at pH 4.5 and --1.15 V at pH 7.0. The difference in ultraviolet absorption spectra between pyrimidone-2 and its reduction product, at neutral pH (see below and Fig. 3) was utilized to follow the progress of the reaction. Following termination ol electrolBiochim. Biophys. A6ta, 28I (1972) i - i o
4
B. CZOCHRALSKA, D. SHUGAR
,1
,
t
-0.6
t
I
-tl$
,
,
I
r
,
'
4.0
I
,
-'t2
t
t
,
,
,
'
-1.4
,
1
-46
I
,
Ii
t
,
t
- 8
I
-2.0
I
I
i
,
-.2
I
I
lm..
V
Fig. I. Polarogram of 2 • lO -3 M pyrimidone-2 in aqueous unbuffered o.I M (CH~)4NBr. Potential vs standard calomel electrode, showing w a v e I ( E { = -0.75 V) and w a v e I I ( E ½ = -1.53 V).
ysis, the solution was concentrated under vacuum, leading to precipitation of a white, amorphous powder, insoluble in a wide variety of solvents. The product was washed with water on a sintered glass filter and brought to dryness, m.p. 240 °C (decompn). It proved possible to obtain the product in crystalline form, from water or ethanol, with m.p. 24 ° °C (decompn) as for the amorphous substance. The product was chromatographically homogeneous with 4 solvent systems (Table I). Electrolysis of 20 mg of the HCI salt of pyrimidone-2 in a single run yielded in this way 6 mg (42 %) of purified reduction product. In ethanolic solution the product exhibited a single broad absorption band in the quartz ultraviolet with ~max 248 nm, emax 4.7 " 1°3, strikingly similar to that for 3,6-dihydropyrimidone-2 (ref. 4). Elementary analysis of the reduction product gave C, 48.98 %; H, 5.20 %; N, 28.58 %. This corresponds to the addition of one hydrogen per pyrimidine ring, and not to two as would be anticipated if the product were dihydrop3~imidone-2. TABLE I
RF VALUI~S, W I T H A S C E N D I N G CHROMATOGRAPHY ON W H A T M A N P A P E R NO. I , OF P W R I M I D O N E - 2 , 3 , 6 - D I H Y D R O P Y R I M I D O N E - 2 AND D I M E R R E D U C T I O N P R O D U C T T h e following s o l v e n t s y s t e m s were used: (A) water-saturated n-butanol; (B) i s o p r o p a n o l water, 7 : 3 (v/v); (C) a m m o n i u m a c e t a t e - e t h a n o l , 2 : 5 (v/ v); (D) water-saturated n-butanol with N H 3 in gas phase. Note: on s p r a y i n g with I M N a O H , followed b y d i m e t h y l - a m i n o b e n z a l d e h y d e (see ref. 7), pyrimidone-2 and the dimer reduction product were unaffected, whereas 3,6d i h y d r o p y r i m i d o n e - 2 gave a bright red color.
RF with solvent system Compound Pyrimidone- 2 3,6-Dihydr°pyrimidone-2
D i m e r reduction product
A o. 35 o.62 0.23
Biochim. Biophys. Aeta, 281 (1972) i - i o
B
C
o. 7 ° o.8o o.62
o. 80 o.86 o.72
D o. 33 o. 8o 0,56
PYRIMIDINE DIMER
5
The mass spectrum of the dimer reduction product exhibited an intense peak at elm 194 corresponding to a dimer ion resulting from the combination of two reduced rings of pyrimidone-2, and a weaker peak at 196. No detailed analysis of the spectrum was carried out, but peaks at elm 97 and 98, of equal intensity, corresponded to the protonated forms of pyrimidone-2, while additional peaks at 167, 153, 136, 116, IOO, 81, etc. are readily accounted for as dimer fragments resulting from elimination of NH 3, CHNO, etc. as reported for other pyrimidine derivatives ~1. The N M R spectrum of the electroreduction product in [2H4]acetic acid did not exhibit the time dependent peaks previously observed in CF3COO2H (ref. I2), and now known to have been due to decomposition in the latter solvent. The spectrum revealed three multiplets with ~ values of 6.2, 4.8 and 4.15 ppm with relative integral intensities of I : i : I, and an additional peak at 12.7 ppm, ascribed to ring N hydrogen (s). For 3,6-dihydropyrimidone-2 the NMR spectrum reported by Skaric et al. 4 exhibits similar multiplets at 6.2, 4.92 and 4.15 ppm, with relative integral intensities of i : I : 2 and assigned, respectively, to the protons at C4, C5 and C6. The spectrum of the reduction product of pyrimidone-2 in 2H20, also recorded in C2HaOOZH, was identical to that of the product of reduction in water, with the exception of the peak at 12.7 ppm, the integral intensity of which was reduced to one-half that of the product of reduction in water. This points to the stronger binding of one of the ring N deuterons, in the product of reduction in 2H20, probably that at N °°. The NMR data, together with the results of polarography, elementary analysis and mass spectroscopy, are fully consistent with the formulation of the reduction product as a dimer, shown in Scheme ], formulated as 6,6'-bis-(3,6-dihydropyrimidone-2). This is further substantiated by the ultraviolet and infrared spectra (see below).
N
~
I-4-
N Scheme I
The in/rared spectrum of the reduction product in KBr (see Fig'. 2) exhibited an intense band in the C=O region at 168o cm -1, resolved into two bands at 167o and 169o cm -1 as in pyrimidone-2 (ref. 13), and assigned to carbonyl groups 14-17. The pyrimidone-2 reduction product possesses likewise bands at 3095 and 323 ° cm-1 characteristic for monosubstituted acid amides is, also present in the cis-trans cyclobutane uracil photodimer 13. The reduction product also exhibited a number of bands resembling, but slightly shifted with respect to, those of 3,6-dihydropyrimidone-2, and including frequencies at 3257-3230, 12o1-121o, 1226-126o and 1146Biochim. Biophys. Acta, 281 (1972) I-IO
6
B. CZOCHRALSKA, D. SHUGAli
._==
E
100
41/00
4200
4400
@00
't800
Frequency (cm-~)
I
.o_ = E
100
ZSO0
3O0O
320O
MOO
Frequencg (cm -t) Fig. 2. Infrared absorption spectrurn of dimer reduction product, 6,6'-his- (3,6-dihydropyrimidone-2) ill K B r (I rag/coo m g KBr).
1155 cm -], as well as a band at 133o cm -1 not present in the dihydro derivative. Furthermore, the latter product possesses only one C=O band at 1666 cm -1. The reduction product, following reduction in 2H20, now exhibited characteristic N - D frequencies at 2270 cm -J (w), 235 ° cm -1 (s) and 247 ° cm -1 (w) not present in the product reduced in water. These were accompanied by a marked reduction in intensities of the bands at 3095 cm -J and 3230 cm -1 due to the amide group NH-CO, thus confirming the NMR analysis according to which electroreduction involve.~ attachment of a hydrogen (or deuteron) to one of the ring nitrogens (see Scheme I). Electrolysis in o.z M (CHa)4NBr and o.I M ammonium carbonate Preparative electrolysis of pyrimidine-2 in unbuffered (CHa)4NBr at a controlled potential on wave I of E = --0. 9 V, as well as in o.I M ammonium carbonate pH 8.2 on wave I at --1. 4 V, resulted in both cases in formation of a reduction product which was identical with that obtained in aqueous buffered medium over the pH range 2-12. After electrolysis at potentials corresponding to wave I, wave II also disappears. By contrast, electrolysis on wave II led to formation of two products, readily separated by paper chromatography, both of which exhibited ultraviolet spectra with Amax 248 nm. One of these, difficultly soluble, was identical chromatographically with that formed on wave I. The second product, readily soluble in water, gave a red coloration with pdimethylaminobenzaldehydeL It was obtained on a preparative scale by chromatography on Whatman paper No. 3, eluted with water, the eluate brought to dryness, and the residue sublimed to give white crystals, m.p. 144-146 °C. Its infrared spectrum in KBr corresponded to that reported quantitatively by Skaric et al. 4 for 3,6dihydro-2-pyrimidone. Elementary analysis gave, C, 48.48 %; H, 6.35 %; N, 27.9 ° °.o, in agreement with that calculated for 3,6-dihydropyrimidone-2, C, 48.97 °:o; H, 6.17 °/o; N, 28.56 %. The RF values for this product are shown in Table I. Its ultraviolet spectrum in neutral aqueous medium exhibited a ;trnax at 248 nm. Biochim. Biophys. ,4cta, 281 (i97 2) i - i o
7
PYRIMIDINE DIMER
Oxidation o/dimer reduction product. As in tile case of NAD +, where the electroreduction product corresponding to the first polarographic wave may be readily oxidized electrochemically19, the pyrimidone-2 dimer reduction product in buffered medium at pH 7 was found to give a polarographic anodic wave with a half-wave potential El/2 = --0.27 V. Oxidation of the dimer reduction product at this potential led to regeneration of pyrimidone-2, as checked by chromatography, ultraviolet absorption spectrum and polarographic behaviour. Photochemical trans/ormation o/reduction product Irradiation of an aqueous solution of the reduction product at pH 7 led to the stepwise disappearance of the characteristic absorption maximum at 248 nm and the simultaneous appearance of a new band with ~m~x of 299 nm. The conversion from one to the other was such that all the intermediate curves passed through two isosbestic points at 262 and 225 nm (Fig. 3), indicating that a single ultraviolet absorbing photoproduct was formed during irradiation from the reduction product of pyfimidone-2. Pyrimidone-2 itself at pH 7 is relatively radiation resistant. Paper chromatography with solvent systems A, B and C, spectrophotometry at various pH values of the eluates of the photoproduct, and polarographic analyses of the latter, all demonstrated unequivocally that the photoproduct was the parent substance, pyrimidone-2. Furthermore, from Fig. 3, the ratio of the absorbance of the photochemically regenerated pyrimidone-2 to that of the initial reduction pro-
t.4
4.2
4.0
0.8
O.6
0.4
0.2
220
0
I 0
0
.120
3,10
nm
Fig. 3. Photochemical t r a n s f o r m a t i o n , on irradiation a t 254 n m in o.oi M p h o s p h a t e buffer (pH 7.2), of 1. 7 • I o - i M 6,6'-bis-(3,6-dihydropyrimidone-2 ), with q u a n t i t a t i v e regeneration of pyrimidone-2, as s h o w n b y isosbestic p o i n t at 262 n m (see t e x t for f u r t h e r details). The curve m a r k e d "'o'" is t h a t for t h e initial reduction product: figures beside the o t h e r curves r e p r e s e n t the time of irradiation in rain.
Biochim. Biophys. Acta, 281 (1972) I - i o
8
B. CZOCHRALSKA, D. S H ( ' ( ; A R
duct is 1.25/o.65 = 1.92. The emaxOf pyrimidone-2 at neutral pH is 4.57 ' lO3 (ref. 5), whereas Smax for the reduction product is 4.7 ° • IO3. But, since the photodissociation reaction leads to formation of monomers, we m a y express the extinction coefficient of the dimer in terms of its monomeric units (on the assmnption that the linkage ~t two monomers to form a dimer does not appreciably affect the absorption)" to obtain a value of 2.35 " lO 3. The ratio of extinction coefficients is therefore 4.57/2.35 = 1.94, as compared to 1.92 for the ratio of absorbances, an agreement which is quite satisfactory, and testifying to quantitative conversion of the reduction product to pyrimidone-2. The quantum yield for the photoconversion reaction was calculated to be o.o9 mole/Einstein, and was identical both in the presence and absence of oxygen. The rate of the photodissociation reaction was likewise unchanged when the irradiation was performed in 2H20, as for photodissociation of pyrimidine cyclobutane pbotodimers.
Electroreduction o/cytosine and cytidine In a study of the electrochemical reduction of cytosine (ref. 12; Czochralska and Shugar, in preparation), it was found that the reduction products at a potential of --1.6o V and pH 4.5 include two substances chromatographically identical with the two products of reduction of pyrimidone-2 in aqueous (CH3)4NBr. One of these, with RF values in solvents A and D of o.21 and 0.56, respectively, was converted to pyrimidone-2 b y irradiation at 254 nm, and is identical with the pyrimidone-2 dimer reduction product, 6,6'-bis-(3,6-dihydropyrimidone-2). The other, with R r values in solvent A and D of 0.62 and 0.80, gave a red coloration with p-dimethylaminobenzaldehyde, and was identified as 3,6-dihydropyrimidone-2. Preliminary observations have also demonstrated formation of a photodissoeiable dimer reduction product from cytidine. This will form the subject of a separate study.
DISCUSSION
It is clear from the overall results that electrochemical reduction of pyrimidone-2 in aqueous buffered medium gives rise to a single one-electron wave; whereas in aqueous (CH3)4NBr there are two one-electron waves of equal height. According to Zuman 2°, when the half-wave potential is p H dependent and its height is unchanged with pH, as is the case with pyrimidone-2, it is most likely that reduction proceeds via the protonated form and that rapid protonation precedes reduction. Taking the foregoing into consideration, the mechanism for electrochemical reduction of pyrimidone-2 to give 6,6'-bis-(3,6-dihydropyrimidone-2) in buffered aqueous medium and on the first wave in aqueous (CH3)aNBr, and 3,6-dihydropyrimidone-2 on the second wave in aqueous (CH3)4NBr, m a y be formulated as in Scheme I. It is clear that both products arise as a result of a 1, 4 reduction, and not 1,2 as previously suggested J'*. Furthermore, the NMR data show that the dimer linkage is 6,6' (and not 5,5'), since the product shows the presence of single hydrogens * T h a t t h i s a s s u m p t i o n is j u s t i f i e d is s h o w n b y t h e f a c t t h a t t h e m o l a r e x t i n c t i o n coeffic i e n t of t h e d i m e r is 4.7 ° . I o 8, w h e r e a s t h a t of 3 , 6 - d i h y d r o p y r i m i d o n e - 2 is 2.34 • IO s, i.e. o n e - h a l f t h a t for t h e d i m e r .
Biochim. Biophys. ,4cta, 281 (1972) i - i o
P Y R I M I D I N E DIMER
9
at C4, C 5 and C s, and a C4=Cs double bond, the presence of which agrees with the NMR data of Skaric et al. a, and the ultraviolet and infrared spectra. I t is not without relevance that a similar 1, 4 type of electrochemical reductio]L has been observed for NAD + by Burnett and Underwood 19. The resemblance between reduction mechanisms for NAD + and pyrimidone-2 is indeed rather striking; in aqueous (CHs)4NBr both exhibit a second polarographic wave. For NAD + wave I corresponds to formation of a 4,4' dimer, and wave I I to N A D H 19. The 4,4' dimer was also polarographically oxidizable to NAD +. Relationship o/ reduction product to nucleic acid photoproducts: The foregoing proposed structure for the reduction product of pyrimidone-2, viz. 6,6'-bis-(3,6dihydropyrimidone-2), is of some interest in relation to a recent report b y Wang and Rhoades 2~ on the isolation and identification of a tetrameric photoproduct resulting from the photodimerization of a photoadduct of thymine and pyrimidone-2, viz. 6-(4'-pyrimidone-2)thymine. The latter photoproduct was initially isolated from ultraviolet-irradiated DNA 22, as well as from an irradiated frozen solution of thymine and uracil 23. The structure of the tetrameric photoproduct is illustrated in Scheme II, in which the dashed line encloses that portion of the tetramer which is formally identical with our reduction product (Scheme I).
o II
H
o II
\ 2o C.
',, ,\ u
-.
N H
\ H
i H
NH
"(9 //
/
S c h e m e "IT
The tetramer was reported to be stable in strong acids, as compared to the acid lability of the dimeric reduction product. Treatment of the tetramer with dimethylsulphate in alkaline medium led to methylation of all the ring N hydrogens except N s and N n (using the nomenclature of Rhoades and Wang23), i.e. the ring N 1 hydrogens of the dimeric reduction product; under similar alkylation conditions, no methylation of ring nitrogen could be detected in the dimer electroreduction product, pointing to the low acidity of the ring N protons. The potential photobiological significance ot the tetramer photoproduct of Wang and Rhoades 21 lends added interest to further studies on the mechanism of formation, and properties, of the pyrimidone-2 dimer reduction product, further underlined b y a recent report on photoadducts of pyrimidone-2 (ref. 24) and their relationship to the photochemistry of t R N A species containing 4-thiouracil. Particularly striking is the quantitative photodissociation of 6,6'-bis-(3,6dihydropyrimidone-2) to the parent pyrimidone-2. Photodissociation of pyrimidine Biochim. Biophys. Acta, 281 (1972) i - I o
I0
B. CZOCHRALSKA, I). SHU(;AR
dimers to the parent pyrimidines has hitherto been considered an exclusive property of eyclobutane-type dimers, although there exist a variety of other pyrimidine photoproducts which photodissociate to regenerate the parent pyrimidines 2b''~. The quantum yield for photodissociation of the pyrimidone-2 dimer reduction product, approx. o.I, is lower than that for dissociation of cyclobutane type pyrimidine photodimers, approx, o.5, but is about the same as those for photodissociation of other photoaddition products of pyrimidines25'2L
ACKNOWLEDGMENTS
We are indebted to Dr Dieter Tresselt (Institute of Microbiology, German Academy of Sciences, jena, German Democratic Republic) for the NMR spectra; to Mrs Anna Psoda for tile infrared spectra; and to Mr Pawel Przybora for excellent technical assistance. This investigation was supported by the Polish Academy of Sciences (Project o9. 3.I) and profited also from the support of The Wellcome Trust, the World Health Organization, and the Agricultural Research Service, U.S. Dept. of Agriculture. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
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Biochim. Biophys. Acta, 281 (1972) i - 1 o
BBA 97382
PHOTOCHEMICALLY R E V E R S I B L E P Y R I M I D I N E DIMER PRODUCT OF ELECTROCHEMICAL REDUCTION OF PYRIMIDONE-2 B A R B A R A C Z O C H R A L S K A AND D. S H U G A R
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Warszawa 22, and Institute of Biochemistry a~d Biophysics, Academy of Sciences, Warszawa ~2 (Poland) (Received April 25th, 1972)
SUMMARY
Pyrimidone-2 gives a single one-electron polarographic wave in aqueous buffered medium, and two one-electron waves of equal height in aqueous unbuffered (CH3)aNBr. The reduction product in neutral aqueous medium, identical with that corresponding to wave I in (CH3)4NBr, was isolated by macroelectrolysis, and shown to consist of a dimer of 3,6-dihydropyrimidone-2, the structure of which was identified as 6,6'-bis(3,6-dihydropyrimidone-2). This dimer reduction product could be readily oxidized polarographically to regenerate the original pyrimidone-2. On irradiation in neutral aqueous medium at 254 nm, the dimer reduction product was quantitatively photodissociated to the parent pyrimidone-2 with a quantum yield of about o.I. The reduction product corresponding to wave II in (CH~)4NBr was also isolated and shown to be 3,6-dihydropyrimidone-2. The mechanism for electroreduction of pyrimidone-2 to give these two products has been formulated. Both of these products are also formed during electroreduction of cytosine in aqueous medium at pH approx. 4.5. The dimer reduction product has been identified as a component of some photoproducts of nucleic acid constituents and, possibly, of nucleic acids and its photobiological significance is discussed.
The polarographic behaviour of cytosine and some of its derivatives was investigated by Smith and Elving 1 and Janik and Palecek 2, who proposed that the electroreduction of cytosine proceeds initially at the 3,4 bond, leading to elimination of NH~ to give 2-pyrimidone. Consequently the final products of reduction of cytosine and 2-pyrimidone should be identical. Smith and Elving 1 further suggested that reduction of pyrimidone-2 is a one-electron radical process, whereas Janik and Palecek 2 claimed a two-electron mechanism, with formation of 3,4-dihydro-2-pyrimidone, which was unstable and subject to ring scission at the 3,4 bond. The similarity of the ultraviolet absorption spectra of the products of electroreduction of cytosine and pyrimidone-2 with those of the catalytic reduction products of these two substances 3,4 prompted us to examine once again the polarographic behaviour, and the mechanism for electroreduction, of pyrimidone-2, largely on the basis of isolation and identification of the products of electrolysis. The results obBiochim. Biophys. Acta, 281 (1972) i - i o
2
B. CZOCHRALSKA, 1~. SH(~;AR
tained are of general interest not only in relation to the properties of the biologically important dihydropyrimidines, but also with respect to pyrimidine photoproducts of biological significance.
MATERIALS AND METHODS
The hydrochloride salt of pyrinfidone-2 was a product of Sigma Chemical Co. It was chromatographically homogeneous in several solvent systems and, at pH 7.2, exhibited a ~max at 299 nm with an emax of 4"57 " 103 (C/. ref. 5}.
Polarography Polarographic curves were recorded with a Radiometer polarograph Polariter PO 4, using a dropping mercury electrode with the following characteristics: flow rate of mercury, m = 2.05 mg • s-l; drop time t -- 3.9 s. at a mercury column height of 6o cm. The pyrimidone-2 concentration was 5 " IO-* M, either in buffered solutions made up to 0.2 M with KC1 or in o.I M aqueous (CHshNBr. Britton-Robinson buffers were employed over the pH range 2-12. The solutions were rendered oxygen-free by bubbling through them nitrogen previously purified by the procedure of Meites 6. All curves were recorded at 25-t-1 °C. A Radiometer PHM-4d compensating instrument was used for pH measurements. All potentials are referred to the saturated calomel electrode.
Macroelectrolysis Preparative electrolyses were conducted with a mercury pool electrode (approx. 20 cm2), fitted with a magnetic stirrer for stirring, in 0.2 M acetate buffer (pH 4.5) and phosphate buffer (pH 7.5), as well as in aqueous (CHs)4NBr , under anaerobic conditions, with depolarizator concentrations of 1.25 • IO-~ M and 5 " IO-~ M. A silver coulometer was used for coulometric measurements. Macroelectrolysis was also carried out in ~H20, buffered with phosphate to p2H 7.2, and the reduction product isolated in the same way. Yields were similar to those in water. For NMR spectroscopy the product was dried several times from water to remove exchangeable deuterium atoms.
Chromatography Ascending chromatography was employed with Whatman papers No. I and 3, using the solvent systems indicated in Table I. Spots were revealed with the aid of a dark ultraviolet lamp and/or by spraying with I M alkali and then with p-dimethylaminobenzaldehyde 7.
Ultraviolet irradiation Solutions were irradiated in Io-mm path-length quartz spectrophotometer cuvettes, the irradiation source being a Phillips 4o-W germicidal lamp (254 nm), the radiation from which was first passed through a 5-mm layer of 35 % aqueous acetic acid to eliminate traces of radiation below 23 ° n m . The intensity incident oil the surface of the cuvette was estimated from the rate of photohydration of a lO -4 M aqueous solution of uridine in the same cuvette, using the known quantum yield for Biochim. Biophys. Mcta, 281 (1972) I - I O
PYRIMIDINE DIMER
3
this reaction of 2.1 • lO -3 (ref. 8). When the irradiated solution was to be subjected to chromatography, a Io-fold higher concentration was irradiated in a I-mm pathlength cuvette.
Spectroscopy Ultraviolet absorption spectra were run on a Zeiss (Jena) VSU-2 instrument.
In/rared absorption spectra were recorded by means of a Zeiss (Jena) U R - I o spectrophotometer, the samples being embedded in KBr matrices in the standard manner. Poor solubility made it impossible to record solution spectra. N M R spectra were obtained with the aid of a Zeiss (Jena) ZKR-6o instrument. The d,-acetic acid employed as solvent (a Fluorochem product) contained more than 99 % atom 2H. Chemical shifts are with reference to trimethylsilane. Mass spectroscopy made use of an Associated Electrical Industries MS- 9 doublefocussing instrument, and spectra were recorded at 25 and 7° eV and at temperatures of 50, 17o and 280 °C. Melting points, uncorrected, were measured on a Boeitius microscope hot stage.
RESULTS
Over the pH range 2-12, pyrimidone-2 was found to give a single polarographic, diffusion-controlled, reduction wave the height of which was independent of pH within the limits of experimental error. El/2 was directly dependent on the pH, the slope of the Ell 2 vs pH curve being 80 mV/pH. These results are in agreement with those of Smith and Elving 1 and Janik and Palecek ~. However, macrocoulometric studies demonstrated that reduction of the compound was a one-electron process, in accord only with Smith and Elving 1. The slope of the plot of log(i/io--I) vs E, where i is the current at potential E, and id the diffusion-controlled limiting current, exhibited a value of 75 mV, indicative of a nonreversible process. The value of p, the number of protons involved in the electrode reaction 6'~° was calculated as one per molecule. The values of pK~ and pK 2 for pyrimidone-2, e~timated from the dependence of Ell 2 on pH, were 2.8 and 9.5, as compared with those determined by direct spectrophotometric titration, 2.24 and 9.17 (ref. 9). In aqueous o.i M (CH3)4NBr, with depolarizator concentrations in the range 4 " lO-4 to 2 • lO -3 M, pyrimidone-2 exhibited two reduction waves of identical height with half-wave potentials, E1/2, of --0.75 and --1.53 V (Fig. I). At lower concentrations a third wave was visible. In o.I M ammonium carbonate, pH 8.2, with a pyrimidone-2 concentration of 5 " IO-4 M, two reduction waves of equal height and with El~ 2 values of --1.24 and --1.8 V were recorded.
Macrodeetrolysis and product isolation Macroelectrolysis was carried out as described under Materials and Methods at a constant potential corresponding to the initial limiting current, i.e. E ---- --0. 9 V at pH 4.5 and --1.15 V at pH 7.0. The difference in ultraviolet absorption spectra between pyrimidone-2 and its reduction product, at neutral pH (see below and Fig. 3) was utilized to follow the progress of the reaction. Following termination ol electrolBiochim. Biophys. A6ta, 28I (1972) i - i o
4
B. CZOCHRALSKA, D. SHUGAR
,1
,
t
-0.6
t
I
-tl$
,
,
I
r
,
'
4.0
I
,
-'t2
t
t
,
,
,
'
-1.4
,
1
-46
I
,
Ii
t
,
t
- 8
I
-2.0
I
I
i
,
-.2
I
I
lm..
V
Fig. I. Polarogram of 2 • lO -3 M pyrimidone-2 in aqueous unbuffered o.I M (CH~)4NBr. Potential vs standard calomel electrode, showing w a v e I ( E { = -0.75 V) and w a v e I I ( E ½ = -1.53 V).
ysis, the solution was concentrated under vacuum, leading to precipitation of a white, amorphous powder, insoluble in a wide variety of solvents. The product was washed with water on a sintered glass filter and brought to dryness, m.p. 240 °C (decompn). It proved possible to obtain the product in crystalline form, from water or ethanol, with m.p. 24 ° °C (decompn) as for the amorphous substance. The product was chromatographically homogeneous with 4 solvent systems (Table I). Electrolysis of 20 mg of the HCI salt of pyrimidone-2 in a single run yielded in this way 6 mg (42 %) of purified reduction product. In ethanolic solution the product exhibited a single broad absorption band in the quartz ultraviolet with ~max 248 nm, emax 4.7 " 1°3, strikingly similar to that for 3,6-dihydropyrimidone-2 (ref. 4). Elementary analysis of the reduction product gave C, 48.98 %; H, 5.20 %; N, 28.58 %. This corresponds to the addition of one hydrogen per pyrimidine ring, and not to two as would be anticipated if the product were dihydrop3~imidone-2. TABLE I
RF VALUI~S, W I T H A S C E N D I N G CHROMATOGRAPHY ON W H A T M A N P A P E R NO. I , OF P W R I M I D O N E - 2 , 3 , 6 - D I H Y D R O P Y R I M I D O N E - 2 AND D I M E R R E D U C T I O N P R O D U C T T h e following s o l v e n t s y s t e m s were used: (A) water-saturated n-butanol; (B) i s o p r o p a n o l water, 7 : 3 (v/v); (C) a m m o n i u m a c e t a t e - e t h a n o l , 2 : 5 (v/ v); (D) water-saturated n-butanol with N H 3 in gas phase. Note: on s p r a y i n g with I M N a O H , followed b y d i m e t h y l - a m i n o b e n z a l d e h y d e (see ref. 7), pyrimidone-2 and the dimer reduction product were unaffected, whereas 3,6d i h y d r o p y r i m i d o n e - 2 gave a bright red color.
RF with solvent system Compound Pyrimidone- 2 3,6-Dihydr°pyrimidone-2
D i m e r reduction product
A o. 35 o.62 0.23
Biochim. Biophys. Aeta, 281 (1972) i - i o
B
C
o. 7 ° o.8o o.62
o. 80 o.86 o.72
D o. 33 o. 8o 0,56
PYRIMIDINE DIMER
5
The mass spectrum of the dimer reduction product exhibited an intense peak at elm 194 corresponding to a dimer ion resulting from the combination of two reduced rings of pyrimidone-2, and a weaker peak at 196. No detailed analysis of the spectrum was carried out, but peaks at elm 97 and 98, of equal intensity, corresponded to the protonated forms of pyrimidone-2, while additional peaks at 167, 153, 136, 116, IOO, 81, etc. are readily accounted for as dimer fragments resulting from elimination of NH 3, CHNO, etc. as reported for other pyrimidine derivatives ~1. The N M R spectrum of the electroreduction product in [2H4]acetic acid did not exhibit the time dependent peaks previously observed in CF3COO2H (ref. I2), and now known to have been due to decomposition in the latter solvent. The spectrum revealed three multiplets with ~ values of 6.2, 4.8 and 4.15 ppm with relative integral intensities of I : i : I, and an additional peak at 12.7 ppm, ascribed to ring N hydrogen (s). For 3,6-dihydropyrimidone-2 the NMR spectrum reported by Skaric et al. 4 exhibits similar multiplets at 6.2, 4.92 and 4.15 ppm, with relative integral intensities of i : I : 2 and assigned, respectively, to the protons at C4, C5 and C6. The spectrum of the reduction product of pyrimidone-2 in 2H20, also recorded in C2HaOOZH, was identical to that of the product of reduction in water, with the exception of the peak at 12.7 ppm, the integral intensity of which was reduced to one-half that of the product of reduction in water. This points to the stronger binding of one of the ring N deuterons, in the product of reduction in 2H20, probably that at N °°. The NMR data, together with the results of polarography, elementary analysis and mass spectroscopy, are fully consistent with the formulation of the reduction product as a dimer, shown in Scheme ], formulated as 6,6'-bis-(3,6-dihydropyrimidone-2). This is further substantiated by the ultraviolet and infrared spectra (see below).
N
~
I-4-
N Scheme I
The in/rared spectrum of the reduction product in KBr (see Fig'. 2) exhibited an intense band in the C=O region at 168o cm -1, resolved into two bands at 167o and 169o cm -1 as in pyrimidone-2 (ref. 13), and assigned to carbonyl groups 14-17. The pyrimidone-2 reduction product possesses likewise bands at 3095 and 323 ° cm-1 characteristic for monosubstituted acid amides is, also present in the cis-trans cyclobutane uracil photodimer 13. The reduction product also exhibited a number of bands resembling, but slightly shifted with respect to, those of 3,6-dihydropyrimidone-2, and including frequencies at 3257-3230, 12o1-121o, 1226-126o and 1146Biochim. Biophys. Acta, 281 (1972) I-IO
6
B. CZOCHRALSKA, D. SHUGAli
._==
E
100
41/00
4200
4400
@00
't800
Frequency (cm-~)
I
.o_ = E
100
ZSO0
3O0O
320O
MOO
Frequencg (cm -t) Fig. 2. Infrared absorption spectrurn of dimer reduction product, 6,6'-his- (3,6-dihydropyrimidone-2) ill K B r (I rag/coo m g KBr).
1155 cm -], as well as a band at 133o cm -1 not present in the dihydro derivative. Furthermore, the latter product possesses only one C=O band at 1666 cm -1. The reduction product, following reduction in 2H20, now exhibited characteristic N - D frequencies at 2270 cm -J (w), 235 ° cm -1 (s) and 247 ° cm -1 (w) not present in the product reduced in water. These were accompanied by a marked reduction in intensities of the bands at 3095 cm -J and 3230 cm -1 due to the amide group NH-CO, thus confirming the NMR analysis according to which electroreduction involve.~ attachment of a hydrogen (or deuteron) to one of the ring nitrogens (see Scheme I). Electrolysis in o.z M (CHa)4NBr and o.I M ammonium carbonate Preparative electrolysis of pyrimidine-2 in unbuffered (CHa)4NBr at a controlled potential on wave I of E = --0. 9 V, as well as in o.I M ammonium carbonate pH 8.2 on wave I at --1. 4 V, resulted in both cases in formation of a reduction product which was identical with that obtained in aqueous buffered medium over the pH range 2-12. After electrolysis at potentials corresponding to wave I, wave II also disappears. By contrast, electrolysis on wave II led to formation of two products, readily separated by paper chromatography, both of which exhibited ultraviolet spectra with Amax 248 nm. One of these, difficultly soluble, was identical chromatographically with that formed on wave I. The second product, readily soluble in water, gave a red coloration with pdimethylaminobenzaldehydeL It was obtained on a preparative scale by chromatography on Whatman paper No. 3, eluted with water, the eluate brought to dryness, and the residue sublimed to give white crystals, m.p. 144-146 °C. Its infrared spectrum in KBr corresponded to that reported quantitatively by Skaric et al. 4 for 3,6dihydro-2-pyrimidone. Elementary analysis gave, C, 48.48 %; H, 6.35 %; N, 27.9 ° °.o, in agreement with that calculated for 3,6-dihydropyrimidone-2, C, 48.97 °:o; H, 6.17 °/o; N, 28.56 %. The RF values for this product are shown in Table I. Its ultraviolet spectrum in neutral aqueous medium exhibited a ;trnax at 248 nm. Biochim. Biophys. ,4cta, 281 (i97 2) i - i o
7
PYRIMIDINE DIMER
Oxidation o/dimer reduction product. As in tile case of NAD +, where the electroreduction product corresponding to the first polarographic wave may be readily oxidized electrochemically19, the pyrimidone-2 dimer reduction product in buffered medium at pH 7 was found to give a polarographic anodic wave with a half-wave potential El/2 = --0.27 V. Oxidation of the dimer reduction product at this potential led to regeneration of pyrimidone-2, as checked by chromatography, ultraviolet absorption spectrum and polarographic behaviour. Photochemical trans/ormation o/reduction product Irradiation of an aqueous solution of the reduction product at pH 7 led to the stepwise disappearance of the characteristic absorption maximum at 248 nm and the simultaneous appearance of a new band with ~m~x of 299 nm. The conversion from one to the other was such that all the intermediate curves passed through two isosbestic points at 262 and 225 nm (Fig. 3), indicating that a single ultraviolet absorbing photoproduct was formed during irradiation from the reduction product of pyfimidone-2. Pyrimidone-2 itself at pH 7 is relatively radiation resistant. Paper chromatography with solvent systems A, B and C, spectrophotometry at various pH values of the eluates of the photoproduct, and polarographic analyses of the latter, all demonstrated unequivocally that the photoproduct was the parent substance, pyrimidone-2. Furthermore, from Fig. 3, the ratio of the absorbance of the photochemically regenerated pyrimidone-2 to that of the initial reduction pro-
t.4
4.2
4.0
0.8
O.6
0.4
0.2
220
0
I 0
0
.120
3,10
nm
Fig. 3. Photochemical t r a n s f o r m a t i o n , on irradiation a t 254 n m in o.oi M p h o s p h a t e buffer (pH 7.2), of 1. 7 • I o - i M 6,6'-bis-(3,6-dihydropyrimidone-2 ), with q u a n t i t a t i v e regeneration of pyrimidone-2, as s h o w n b y isosbestic p o i n t at 262 n m (see t e x t for f u r t h e r details). The curve m a r k e d "'o'" is t h a t for t h e initial reduction product: figures beside the o t h e r curves r e p r e s e n t the time of irradiation in rain.
Biochim. Biophys. Acta, 281 (1972) I - i o
8
B. CZOCHRALSKA, D. S H ( ' ( ; A R
duct is 1.25/o.65 = 1.92. The emaxOf pyrimidone-2 at neutral pH is 4.57 ' lO3 (ref. 5), whereas Smax for the reduction product is 4.7 ° • IO3. But, since the photodissociation reaction leads to formation of monomers, we m a y express the extinction coefficient of the dimer in terms of its monomeric units (on the assmnption that the linkage ~t two monomers to form a dimer does not appreciably affect the absorption)" to obtain a value of 2.35 " lO 3. The ratio of extinction coefficients is therefore 4.57/2.35 = 1.94, as compared to 1.92 for the ratio of absorbances, an agreement which is quite satisfactory, and testifying to quantitative conversion of the reduction product to pyrimidone-2. The quantum yield for the photoconversion reaction was calculated to be o.o9 mole/Einstein, and was identical both in the presence and absence of oxygen. The rate of the photodissociation reaction was likewise unchanged when the irradiation was performed in 2H20, as for photodissociation of pyrimidine cyclobutane pbotodimers.
Electroreduction o/cytosine and cytidine In a study of the electrochemical reduction of cytosine (ref. 12; Czochralska and Shugar, in preparation), it was found that the reduction products at a potential of --1.6o V and pH 4.5 include two substances chromatographically identical with the two products of reduction of pyrimidone-2 in aqueous (CH3)4NBr. One of these, with RF values in solvents A and D of o.21 and 0.56, respectively, was converted to pyrimidone-2 b y irradiation at 254 nm, and is identical with the pyrimidone-2 dimer reduction product, 6,6'-bis-(3,6-dihydropyrimidone-2). The other, with R r values in solvent A and D of 0.62 and 0.80, gave a red coloration with p-dimethylaminobenzaldehyde, and was identified as 3,6-dihydropyrimidone-2. Preliminary observations have also demonstrated formation of a photodissoeiable dimer reduction product from cytidine. This will form the subject of a separate study.
DISCUSSION
It is clear from the overall results that electrochemical reduction of pyrimidone-2 in aqueous buffered medium gives rise to a single one-electron wave; whereas in aqueous (CH3)4NBr there are two one-electron waves of equal height. According to Zuman 2°, when the half-wave potential is p H dependent and its height is unchanged with pH, as is the case with pyrimidone-2, it is most likely that reduction proceeds via the protonated form and that rapid protonation precedes reduction. Taking the foregoing into consideration, the mechanism for electrochemical reduction of pyrimidone-2 to give 6,6'-bis-(3,6-dihydropyrimidone-2) in buffered aqueous medium and on the first wave in aqueous (CH3)aNBr, and 3,6-dihydropyrimidone-2 on the second wave in aqueous (CH3)4NBr, m a y be formulated as in Scheme I. It is clear that both products arise as a result of a 1, 4 reduction, and not 1,2 as previously suggested J'*. Furthermore, the NMR data show that the dimer linkage is 6,6' (and not 5,5'), since the product shows the presence of single hydrogens * T h a t t h i s a s s u m p t i o n is j u s t i f i e d is s h o w n b y t h e f a c t t h a t t h e m o l a r e x t i n c t i o n coeffic i e n t of t h e d i m e r is 4.7 ° . I o 8, w h e r e a s t h a t of 3 , 6 - d i h y d r o p y r i m i d o n e - 2 is 2.34 • IO s, i.e. o n e - h a l f t h a t for t h e d i m e r .
Biochim. Biophys. ,4cta, 281 (1972) i - i o
P Y R I M I D I N E DIMER
9
at C4, C 5 and C s, and a C4=Cs double bond, the presence of which agrees with the NMR data of Skaric et al. a, and the ultraviolet and infrared spectra. I t is not without relevance that a similar 1, 4 type of electrochemical reductio]L has been observed for NAD + by Burnett and Underwood 19. The resemblance between reduction mechanisms for NAD + and pyrimidone-2 is indeed rather striking; in aqueous (CHs)4NBr both exhibit a second polarographic wave. For NAD + wave I corresponds to formation of a 4,4' dimer, and wave I I to N A D H 19. The 4,4' dimer was also polarographically oxidizable to NAD +. Relationship o/ reduction product to nucleic acid photoproducts: The foregoing proposed structure for the reduction product of pyrimidone-2, viz. 6,6'-bis-(3,6dihydropyrimidone-2), is of some interest in relation to a recent report b y Wang and Rhoades 2~ on the isolation and identification of a tetrameric photoproduct resulting from the photodimerization of a photoadduct of thymine and pyrimidone-2, viz. 6-(4'-pyrimidone-2)thymine. The latter photoproduct was initially isolated from ultraviolet-irradiated DNA 22, as well as from an irradiated frozen solution of thymine and uracil 23. The structure of the tetrameric photoproduct is illustrated in Scheme II, in which the dashed line encloses that portion of the tetramer which is formally identical with our reduction product (Scheme I).
o II
H
o II
\ 2o C.
',, ,\ u
-.
N H
\ H
i H
NH
"(9 //
/
S c h e m e "IT
The tetramer was reported to be stable in strong acids, as compared to the acid lability of the dimeric reduction product. Treatment of the tetramer with dimethylsulphate in alkaline medium led to methylation of all the ring N hydrogens except N s and N n (using the nomenclature of Rhoades and Wang23), i.e. the ring N 1 hydrogens of the dimeric reduction product; under similar alkylation conditions, no methylation of ring nitrogen could be detected in the dimer electroreduction product, pointing to the low acidity of the ring N protons. The potential photobiological significance ot the tetramer photoproduct of Wang and Rhoades 21 lends added interest to further studies on the mechanism of formation, and properties, of the pyrimidone-2 dimer reduction product, further underlined b y a recent report on photoadducts of pyrimidone-2 (ref. 24) and their relationship to the photochemistry of t R N A species containing 4-thiouracil. Particularly striking is the quantitative photodissociation of 6,6'-bis-(3,6dihydropyrimidone-2) to the parent pyrimidone-2. Photodissociation of pyrimidine Biochim. Biophys. Acta, 281 (1972) i - I o
I0
B. CZOCHRALSKA, I). SHU(;AR
dimers to the parent pyrimidines has hitherto been considered an exclusive property of eyclobutane-type dimers, although there exist a variety of other pyrimidine photoproducts which photodissociate to regenerate the parent pyrimidines 2b''~. The quantum yield for photodissociation of the pyrimidone-2 dimer reduction product, approx. o.I, is lower than that for dissociation of cyclobutane type pyrimidine photodimers, approx, o.5, but is about the same as those for photodissociation of other photoaddition products of pyrimidines25'2L
ACKNOWLEDGMENTS
We are indebted to Dr Dieter Tresselt (Institute of Microbiology, German Academy of Sciences, jena, German Democratic Republic) for the NMR spectra; to Mrs Anna Psoda for tile infrared spectra; and to Mr Pawel Przybora for excellent technical assistance. This investigation was supported by the Polish Academy of Sciences (Project o9. 3.I) and profited also from the support of The Wellcome Trust, the World Health Organization, and the Agricultural Research Service, U.S. Dept. of Agriculture. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
D. S m i t h a n d P. J. Elving, J. Am. Chem. Soc., 84 (1962) 2741. B. J a n i k a n d E. Palecek, Arch. Biochem. Biophys., lO5 (1964) 225. C. J a n i o n a n d D. S h u g a r , Acta Biochim. Polon., 14 (1967) 293. V. Skaric, B. G a s p e r a n d D. Skaric, Croat. Chem. Acta, 36 (1964) 87. D. J. B r o w n , Nature, 165 (195 o) lOlO. L. Meites, Polavographic Techniques, I n t c r s c i e n c e , New York, 1965. R . M. Fink, R. E. Cline, C. Mc. G a u g h e y a n d K. F i n k , Anal. Chem., 28 (1956) 4. D. S h u g a r , in The Nucleic Acids, Vol. I I I , A c a d e m i c Press, N e w York, 196o. A. Albert, in A. R. K a t r i t z k y , Physical Methods in Heterocyclic Chemistry, Vol. I., A c a d e m i c Press, N e w York, 1963. P. J. E l v i n g a n d S. J. Pace, Biological Aspects of Electrochemistry, Exp.Suppl. 18 (1971) 35J. M. Rice, G. O. D u d e k a n d M. B a r b e r , J. Am. Chem. Soc., 87 (1965) 4569B. C z o c h r a l s k a a n d D. S h u g a r , Exp. Suppl., 18 (1971) 251. S. G. L a l a n d a n d G. S e r c k - H a u s s e n , Biochem. J., 90 (1964) 76. A. J. Varghese, Biochemistry, IO (1971) 2t94. M. N. K h a t t a k a n d S. Y. W a n g , Science, 163 (1968) 1341. J. V a r g h e s e a n d S. Y. W a n g , Science, 16o (1968) 186. A. J. Varghese, Photochem. Photobiol., 13 (1971) 357. N. B. Colthup, H. D a l y a n d S. E. W i b e r l e y , Introduction to Infrared and Raman Spectroscopy, A c a d e m i c Press, N e w York, 1964 . J. N. B a r n e t t a n d A. L. U n d e r w o o d , Biochemistry, 4 (1965) 2060. P. Z u m a n , The Elucidation of Organic Electrode Processes, A c a d e m i c Press, N e w York, 1969. S. Y. W a n g a n d D. F. R h o a d e s , J. Am. Chem. Soc., 93 (1971) 25.54. A. J. V a r g h e s e a n d S. Y. W a n g , Science, 156 (1971) 955. D. F. R h o a d e s a n d S. Y. W a n g , Biochemistry, 9 (197 o) 4416. D. E. B e r g s t r o m a n d N. J. L e o n a r d , Biochemistry, II (1972) i. M. F i k u s a n d D. S h u g a r , Photochem. Photobiol., 4 (1965) 521. J. P i e t r z y k o w s k a a n d D. S h u g a r , Acta Biochim. Polon., 17 (197 o) 361. E. K r a j e w s k a a n d D. Shugar, Acta Biochim. Polon., in t h e press.
Biochim. Biophys. Acta, 281 (1972) i - 1 o