Photochemistry and Phorob~ology,1976, Vol. 23. pp. 131--114
Pergamon Press
Printed in Great Britain
RESEARCH NOTE
PHOTOCHEMISTRY OF GUANINES AT LOW TEMPERATURE J. P. MORGAN* and P. R. CALLIS Department of Chemistry, Montana State University, Bozeman, M T 5971 5, U.S.A.
(Rewitred 11 August 1975; uccepted 24 October 1975) INTRODUCTION
The purine components of nucleic acids have received little attention with regard to photochemistry. The principal reason for the lack of study has been the relative photostability of the purines in aqueous media at room temperature (McLaren and Shugar, 1964; Burr, 1968). However, Steinmaus rt al. (1969) have reported that adenosine and guanosine form photoadducts with 2-propanol. No change in absorption properties was seen. More recently Porschke (1973) has reported a specific photoreaction in polydeoxyadenylic acid which provides changes in the UV and circular dichroistn spectra. We report here that guanine and many of its derivatives show considerable change in optical properties when irradiated with UV light of L Q 300 nm and dissolved in a cold, viscous, diol-containing medium. The distinctive optical properties of the photoproduct could be used to monitor excited state interactions in guanine-containing dinucleotides, and indicate the existence of paramagnetic metal base complexes. A small amount of another product, stable at room temperature, can be obtained via thermal conversion under certain conditions; or it can be produced in quantity by acetonephotosensitization. MATERIALS AND METHODS
Nucleic acid components were purchased from either CalBiochem o r Sigma and used without additional purfication. Organic solvents were of reagent grade and either doubly distilled o r treated with activated charcoal and checked for optical purity. Metal ions were introduced as the nitrate salts. Pi buffer 1 x 10-2A4 was used to maintain the alcohol-water mixtures to ncar neutral pH. For the fluorescence and absorption experiments, the sample in a 0.6 x 0.6 x 2 cm supracil fluorescence cell was placed within a stainless steel Dewar which had 3 sets of supracil windows. Temperature was maintained by bathing the sample in cooled N 2 . When the absorption was monitored, photochemistry was carried out using a 200 W Osram HBO high-pressure Hg arc which was focused on the sample with a beam slightly wider than the width of the cell. The absorption was monitored on a C'ary 14 spectrophotometer. Fluorescence expcriments *Present address: Radiation Center, Oregon State University, Corvallis, OR 97330, U.S.A. 131 ?.A P
2312
E
were carried out with a tluorometer consisting of an EM1 955XQ photomultiplier tube, two 500 mm Bausch & Lomb monochromators and either a 200 W high-pressure Hg arc or a 150 W Xe arc. Action spectra were correctcd for lamp and instrument dependence by the response of concentrated rhodamine B (Melhuish, 1962). Ferrioxalate actinometry (Hatchard and Parker, 1956) was used for photochemical quantum yields.
RESULTS
Figure 1 shows the fluorescence from a solution of guanosine monophosphate (CMP) in ethylene glycol-water (6:4, V/V) at -11o"C, while being irradiated with 248 nm light. It is seen that as the time of the irradiation increases, the GMP fluorescence decreases, while a new fluorescence, La,= 418 nm, increases. After irradiation at 248 nm, excitation at wavelengths between 305 and 350 nm yields the 418 nm fluorescence, whereas only the very weak fluorescence from the solvent was observed before irradiation. The quantum yield for the photoprocess, based on the reduction of parent GMP fluorescence, is 2y; when LCx = 248 nm and increases with wavelength to a value of about 4% at 290 nm. This wavelcngth dependence is also manifested in the luminescence yields of many of the nucleic acid components (Wilson et al., 1975). The Q F of the product is 0.35 as min
-
GMP Fluorescence
-0
--__-
...........
2
Aex=240nrn
4
12
0
300
350
A,
400
450
nm
Figure I . Changes in thc fluorescence spectrum of W . 1 x lo-" M . guanosine monophosphate in cthylcne glycolwater solution (6:4, V/V) at - I 10°C while exciting with 248 nm light. The 418 nm band is present by itself when exciting with i> 300 nm after photolysis with 248 nm light.
J. P. MORGANand P. R. CALLIS
3
Figure 2. Changes in the UV absorption spectrum of a 0.8 x M solution of guanosine in ethylene glycol-water (6:4, V/V) at -110°C under intense UV irradiation (I, = 3 x lo-' einstein cm-' min- I).
compared to a value of 0.029 for GMP under these conditions. In Fig. 2, we seen the UV absorbance of a solution of guanosine, under conditions very similar to those of Fig. 1. The presence of the isosbestic points indicates that if the guanosine is converted into more than one absorbing product, the product ratio is independent of the extent of reaction. Considering photoconversion to be complete after a photolysis period of 2 h and assuming each guanosine is converted into only one absorbing product, the molar extinction coefficients of the product are estimated to be 8.3 x lo3, 3.7 x lo3 and 19.4 x lo3 d. mol-' cm-' at 305, 265, and 225 nm, respectively. The known molar extinction coefficient values of guanosine were used for comparison. Only one fluorescent product was formed and its fluorescence excitation spectrum and
fluorescence excitation polarization spectrum qualitatively confirm the above assumptions and assignments. The photoprocess occurs in guanine, guanosine, deoxyguanosine, guanosine monophosphate, 7-methylguanosine, N2,N2-dimethyl guanosine, 1-methyl guanosine, solutions containing GMP and metal ions in equimolar amounts, and in a number of guaninecontaining dinucleotide monophosphdtes. The yield varies significantly in many cases, however. The photochemical quantum yields as determined by the absorption of product formed are listed in Table I. The photoprocess also occurs in glycerine-water (1: l), 1,2-propanediol-water (6:4), and 1,3-propanediol-water (6:4) but does not occur in a glass composed of methanol-water (12:3) or 2-propanol-water (6:4). In ethylene glycol-water the rate is very slow
Table 1. Photochemical quantum yields (QPc) of guanine containing molecules as determined by absorption monitored photolysis. Initial optical density at 252 nm = 0.60 for 6 mm. I, = 3 x lo-' einstein M . Ethylene cm-2 min-I. Metal ion concentrations are 1 x glycol-water (6:4,V/V) at - 110°C is the solvent. Dinucleotides are the 3' -+ 5' derivatives. Chromophore
'PCx102
Chrornophore oPCx102
Chrornophore
oPCx102
1 Me-Guanosine
.40
G M P t MgZt
1.8
APG
1.9
7 Me-Guanosine
.50
G M P t 2"''
2.0
GP*
2. 1
NZ-Guanosine
1.6
G M P t CuZt
.12
CPG
.60
Guanine
2.0
G M P t NiZt
.24
GPC
.76
Guanosine
2.0
GMP t Ca2'
.4R
dpGpT
2.1
GMP
2.0
G M P t CaZt
2.0
GPC
2.0
C M P t Mn2+
2.0
GPU
.98
Research Note
at -78‘C and reaches a maximum at about - 115°C. A t - 140°C the rate is approximately four times slower than the maximum. In glycerol-water the photoprocess was observable at temperatures as high as -25°C. The characteristic fluorescence of the photoproduct grows upon excitation of a solution of GMP in ethylcne glycol-water and 1M acetone with 31 3 nm light at -110°C. No product formation is seen without the acetone under these conditions. A triplet precursor is thus implicated. The product reverts in high yield back to the parent guanine when the solution is allowed to warm rapidly to room temperature. If the photolyzed solution is maintained at a temperature of near -80°C in the dark, the 418 nm fluorescent product partially converts to a 355 nm fluorescent product which is found to be stable at room temperature, the fluorescence maximum shifting to 365 nm. It is also possible to produce a 365 nm product at room temperature by photosensitization with acetone in a solution which contains either a diol or 2-propanol, in addition to a guanine-containing compound. DISCUSSlON
Because of its thermal instability, the identity of the low temperature product can only be speculated upon. Its absorption and fluorescence properties are quite similar to the tricyclic compounds such as 1, N6-ethenoadenosine (Secrist et al., 1972) and the “ Y base (Nakanishi et al., 1970). The eventual identification of the stable product at room temperature will aid in making a structural assignment to the low temperature species. Photochemistry is usually not a factor in luminescence measurements of purines. This has been true for guanine derivatives when luminescence was observed at room temperature (Borresen, 1965), at dry ice temperature (Callis et al., 1964), or at liquid nitrogen temperature (Eisinger and Shulman, 1968). Helenc et ul. (1966) have seen a decrease in phosphorescence without change in fluorescence or absorption at 77 K for purines in ethanolic solution. Drobnik and Augenstein (1966) have observed a fluorescence characteristic of the photoproduct from solutions of deoxyguanosine in ethylene glycol -water at - lWC, and have attributed it to aggregation, not to photochemistry. The process reported here does not occur
I33
in the dark, and changes occur in both the luminescence and absorption as the result of the photoalteration. Because the photochemistry appears to be specific for guanines, the product yield is a reflection of the extent of population of an excited state (triplet) of guanine and thus can be used as a quantitative probe into excited state pathways. This is especially useful when one seeks to evaluate changes in excited state populations due to energy transfer, or excited state dimer or complex formation. The variation in photochemical quantum yield for the various guanine-containing dinucleotides shown in Table 1 could have many causes: differing solvation spheres, energy transfer at the singlet and triplet levels, variable stacking conformations and exciplex formation, wavelength dependence of radiative, nonradiative and photochemical yields, or combinations thereof. An analysis is in progress based on triplet lifetimes and luminescence and photochemical quantum yield measurements as a function of excitation wavelength for guanine-containing dinucleotides and their monomeric components (Morgan and Callis, in preparation). The paramagnetic ions Cu”, c‘ozc, and Ni2+ are known to be triplet quenchers and they indeed effectively reduce the photochemical yield by factors of 4-16 as seen in Table 1. This is further evidence that the triplet state of the guanine is the precursor. That this quenching occurs with equimolar concentrations (10-4M) of guanine and ion indicates a large amount of complexation in the region of the base portion of GMP. Photochemical studies with guanosine and guanine in solution with the metal ions would indicate whether the metals are held in a bridge between the phosphate and base in GMP. While it is recognized that the special conditions required for the formation of the low temperature product, together with its thermal instability, make it seem an unlikely cause of photodamage in a physiological state, its unique optical properties cause it to be a convenient fluorescent probe. On the other hand, since the low temperature product can convert to a stable product under certain conditions, i.e. - 80”C, and since the stable product at room temperature can be produced via photosensitization at room temperature, it cannot be discounted that the low temperature product is the intermediate to photoadducts of guanines under biological conditions.
REFERENCES
Borresen, H. C. (1965) Acru Chem. Scand. 19, 21W2112. Burr, J. (1968) In A h a n c e s in Phorocheriristry (Edited by G. Hanimond and J. N. Pitts) Vol. 6, pp. 267 282, Wiley, New York. Callis, P.R., E. J. Rosa and W. T. Simpson (1964) J . AIM. Cherti. Soc. 86, 2292-2294. Drobnik, J., and L. Augenstein (1966) Photochetn. Photobid. 5. 83-97. Eisinger, J. and R. G. Shulman (1968) Science 161, 1311-1319. Hatchard, C. G . and C. A. Parker (1956) Proc. l7o.1,.Soc. A235, 518-536. Helene, C., R. Santus and P. Douzou (1966) Photochem. Photobid. 5, 127-133.
134
J. P. MORGANand P. R. CALLIS McLaren, A., and D. Shugar (1964) Pho/ockeiiii.str~Of’Protrins and Nucleic Acids, pp. 163-278, Pergamon Press, Oxford. Melhuish, W. H. (1962) J . Opt. SOC.Am. 52, 125G-1258. Nakanishi, K., J. Furutachi, M. Funamizu, D. Gunborger and I. B. Weinstein (1970) J . AJII. C ~ J I . SOC.92, 7617-7619. Porschke, D. (1973) Proc. Nut/. Acad. Sci. U S . 70, 2683-2686. Secrist, 1. A,, J. R. Barrio and G . Weber (1972) B i o c h e ~ i i w q11, 3499-3509. Steinmaus, H., I . Rosenthal and D. Elad (1969) ./. Am. Chew. Soc. 91, 4921-4923. Wilson, R. W., J. P. Morgan and P. R. Callis (1975) C h e m P h p . Lvttrrs, 36. 618--623.
Pergamon Press
Printed in Great Britain
RESEARCH NOTE
PHOTOCHEMISTRY OF GUANINES AT LOW TEMPERATURE J. P. MORGAN* and P. R. CALLIS Department of Chemistry, Montana State University, Bozeman, M T 5971 5, U.S.A.
(Rewitred 11 August 1975; uccepted 24 October 1975) INTRODUCTION
The purine components of nucleic acids have received little attention with regard to photochemistry. The principal reason for the lack of study has been the relative photostability of the purines in aqueous media at room temperature (McLaren and Shugar, 1964; Burr, 1968). However, Steinmaus rt al. (1969) have reported that adenosine and guanosine form photoadducts with 2-propanol. No change in absorption properties was seen. More recently Porschke (1973) has reported a specific photoreaction in polydeoxyadenylic acid which provides changes in the UV and circular dichroistn spectra. We report here that guanine and many of its derivatives show considerable change in optical properties when irradiated with UV light of L Q 300 nm and dissolved in a cold, viscous, diol-containing medium. The distinctive optical properties of the photoproduct could be used to monitor excited state interactions in guanine-containing dinucleotides, and indicate the existence of paramagnetic metal base complexes. A small amount of another product, stable at room temperature, can be obtained via thermal conversion under certain conditions; or it can be produced in quantity by acetonephotosensitization. MATERIALS AND METHODS
Nucleic acid components were purchased from either CalBiochem o r Sigma and used without additional purfication. Organic solvents were of reagent grade and either doubly distilled o r treated with activated charcoal and checked for optical purity. Metal ions were introduced as the nitrate salts. Pi buffer 1 x 10-2A4 was used to maintain the alcohol-water mixtures to ncar neutral pH. For the fluorescence and absorption experiments, the sample in a 0.6 x 0.6 x 2 cm supracil fluorescence cell was placed within a stainless steel Dewar which had 3 sets of supracil windows. Temperature was maintained by bathing the sample in cooled N 2 . When the absorption was monitored, photochemistry was carried out using a 200 W Osram HBO high-pressure Hg arc which was focused on the sample with a beam slightly wider than the width of the cell. The absorption was monitored on a C'ary 14 spectrophotometer. Fluorescence expcriments *Present address: Radiation Center, Oregon State University, Corvallis, OR 97330, U.S.A. 131 ?.A P
2312
E
were carried out with a tluorometer consisting of an EM1 955XQ photomultiplier tube, two 500 mm Bausch & Lomb monochromators and either a 200 W high-pressure Hg arc or a 150 W Xe arc. Action spectra were correctcd for lamp and instrument dependence by the response of concentrated rhodamine B (Melhuish, 1962). Ferrioxalate actinometry (Hatchard and Parker, 1956) was used for photochemical quantum yields.
RESULTS
Figure 1 shows the fluorescence from a solution of guanosine monophosphate (CMP) in ethylene glycol-water (6:4, V/V) at -11o"C, while being irradiated with 248 nm light. It is seen that as the time of the irradiation increases, the GMP fluorescence decreases, while a new fluorescence, La,= 418 nm, increases. After irradiation at 248 nm, excitation at wavelengths between 305 and 350 nm yields the 418 nm fluorescence, whereas only the very weak fluorescence from the solvent was observed before irradiation. The quantum yield for the photoprocess, based on the reduction of parent GMP fluorescence, is 2y; when LCx = 248 nm and increases with wavelength to a value of about 4% at 290 nm. This wavelcngth dependence is also manifested in the luminescence yields of many of the nucleic acid components (Wilson et al., 1975). The Q F of the product is 0.35 as min
-
GMP Fluorescence
-0
--__-
...........
2
Aex=240nrn
4
12
0
300
350
A,
400
450
nm
Figure I . Changes in thc fluorescence spectrum of W . 1 x lo-" M . guanosine monophosphate in cthylcne glycolwater solution (6:4, V/V) at - I 10°C while exciting with 248 nm light. The 418 nm band is present by itself when exciting with i> 300 nm after photolysis with 248 nm light.
J. P. MORGANand P. R. CALLIS
3
Figure 2. Changes in the UV absorption spectrum of a 0.8 x M solution of guanosine in ethylene glycol-water (6:4, V/V) at -110°C under intense UV irradiation (I, = 3 x lo-' einstein cm-' min- I).
compared to a value of 0.029 for GMP under these conditions. In Fig. 2, we seen the UV absorbance of a solution of guanosine, under conditions very similar to those of Fig. 1. The presence of the isosbestic points indicates that if the guanosine is converted into more than one absorbing product, the product ratio is independent of the extent of reaction. Considering photoconversion to be complete after a photolysis period of 2 h and assuming each guanosine is converted into only one absorbing product, the molar extinction coefficients of the product are estimated to be 8.3 x lo3, 3.7 x lo3 and 19.4 x lo3 d. mol-' cm-' at 305, 265, and 225 nm, respectively. The known molar extinction coefficient values of guanosine were used for comparison. Only one fluorescent product was formed and its fluorescence excitation spectrum and
fluorescence excitation polarization spectrum qualitatively confirm the above assumptions and assignments. The photoprocess occurs in guanine, guanosine, deoxyguanosine, guanosine monophosphate, 7-methylguanosine, N2,N2-dimethyl guanosine, 1-methyl guanosine, solutions containing GMP and metal ions in equimolar amounts, and in a number of guaninecontaining dinucleotide monophosphdtes. The yield varies significantly in many cases, however. The photochemical quantum yields as determined by the absorption of product formed are listed in Table I. The photoprocess also occurs in glycerine-water (1: l), 1,2-propanediol-water (6:4), and 1,3-propanediol-water (6:4) but does not occur in a glass composed of methanol-water (12:3) or 2-propanol-water (6:4). In ethylene glycol-water the rate is very slow
Table 1. Photochemical quantum yields (QPc) of guanine containing molecules as determined by absorption monitored photolysis. Initial optical density at 252 nm = 0.60 for 6 mm. I, = 3 x lo-' einstein M . Ethylene cm-2 min-I. Metal ion concentrations are 1 x glycol-water (6:4,V/V) at - 110°C is the solvent. Dinucleotides are the 3' -+ 5' derivatives. Chromophore
'PCx102
Chrornophore oPCx102
Chrornophore
oPCx102
1 Me-Guanosine
.40
G M P t MgZt
1.8
APG
1.9
7 Me-Guanosine
.50
G M P t 2"''
2.0
GP*
2. 1
NZ-Guanosine
1.6
G M P t CuZt
.12
CPG
.60
Guanine
2.0
G M P t NiZt
.24
GPC
.76
Guanosine
2.0
GMP t Ca2'
.4R
dpGpT
2.1
GMP
2.0
G M P t CaZt
2.0
GPC
2.0
C M P t Mn2+
2.0
GPU
.98
Research Note
at -78‘C and reaches a maximum at about - 115°C. A t - 140°C the rate is approximately four times slower than the maximum. In glycerol-water the photoprocess was observable at temperatures as high as -25°C. The characteristic fluorescence of the photoproduct grows upon excitation of a solution of GMP in ethylcne glycol-water and 1M acetone with 31 3 nm light at -110°C. No product formation is seen without the acetone under these conditions. A triplet precursor is thus implicated. The product reverts in high yield back to the parent guanine when the solution is allowed to warm rapidly to room temperature. If the photolyzed solution is maintained at a temperature of near -80°C in the dark, the 418 nm fluorescent product partially converts to a 355 nm fluorescent product which is found to be stable at room temperature, the fluorescence maximum shifting to 365 nm. It is also possible to produce a 365 nm product at room temperature by photosensitization with acetone in a solution which contains either a diol or 2-propanol, in addition to a guanine-containing compound. DISCUSSlON
Because of its thermal instability, the identity of the low temperature product can only be speculated upon. Its absorption and fluorescence properties are quite similar to the tricyclic compounds such as 1, N6-ethenoadenosine (Secrist et al., 1972) and the “ Y base (Nakanishi et al., 1970). The eventual identification of the stable product at room temperature will aid in making a structural assignment to the low temperature species. Photochemistry is usually not a factor in luminescence measurements of purines. This has been true for guanine derivatives when luminescence was observed at room temperature (Borresen, 1965), at dry ice temperature (Callis et al., 1964), or at liquid nitrogen temperature (Eisinger and Shulman, 1968). Helenc et ul. (1966) have seen a decrease in phosphorescence without change in fluorescence or absorption at 77 K for purines in ethanolic solution. Drobnik and Augenstein (1966) have observed a fluorescence characteristic of the photoproduct from solutions of deoxyguanosine in ethylene glycol -water at - lWC, and have attributed it to aggregation, not to photochemistry. The process reported here does not occur
I33
in the dark, and changes occur in both the luminescence and absorption as the result of the photoalteration. Because the photochemistry appears to be specific for guanines, the product yield is a reflection of the extent of population of an excited state (triplet) of guanine and thus can be used as a quantitative probe into excited state pathways. This is especially useful when one seeks to evaluate changes in excited state populations due to energy transfer, or excited state dimer or complex formation. The variation in photochemical quantum yield for the various guanine-containing dinucleotides shown in Table 1 could have many causes: differing solvation spheres, energy transfer at the singlet and triplet levels, variable stacking conformations and exciplex formation, wavelength dependence of radiative, nonradiative and photochemical yields, or combinations thereof. An analysis is in progress based on triplet lifetimes and luminescence and photochemical quantum yield measurements as a function of excitation wavelength for guanine-containing dinucleotides and their monomeric components (Morgan and Callis, in preparation). The paramagnetic ions Cu”, c‘ozc, and Ni2+ are known to be triplet quenchers and they indeed effectively reduce the photochemical yield by factors of 4-16 as seen in Table 1. This is further evidence that the triplet state of the guanine is the precursor. That this quenching occurs with equimolar concentrations (10-4M) of guanine and ion indicates a large amount of complexation in the region of the base portion of GMP. Photochemical studies with guanosine and guanine in solution with the metal ions would indicate whether the metals are held in a bridge between the phosphate and base in GMP. While it is recognized that the special conditions required for the formation of the low temperature product, together with its thermal instability, make it seem an unlikely cause of photodamage in a physiological state, its unique optical properties cause it to be a convenient fluorescent probe. On the other hand, since the low temperature product can convert to a stable product under certain conditions, i.e. - 80”C, and since the stable product at room temperature can be produced via photosensitization at room temperature, it cannot be discounted that the low temperature product is the intermediate to photoadducts of guanines under biological conditions.
REFERENCES
Borresen, H. C. (1965) Acru Chem. Scand. 19, 21W2112. Burr, J. (1968) In A h a n c e s in Phorocheriristry (Edited by G. Hanimond and J. N. Pitts) Vol. 6, pp. 267 282, Wiley, New York. Callis, P.R., E. J. Rosa and W. T. Simpson (1964) J . AIM. Cherti. Soc. 86, 2292-2294. Drobnik, J., and L. Augenstein (1966) Photochetn. Photobid. 5. 83-97. Eisinger, J. and R. G. Shulman (1968) Science 161, 1311-1319. Hatchard, C. G . and C. A. Parker (1956) Proc. l7o.1,.Soc. A235, 518-536. Helene, C., R. Santus and P. Douzou (1966) Photochem. Photobid. 5, 127-133.
134
J. P. MORGANand P. R. CALLIS McLaren, A., and D. Shugar (1964) Pho/ockeiiii.str~Of’Protrins and Nucleic Acids, pp. 163-278, Pergamon Press, Oxford. Melhuish, W. H. (1962) J . Opt. SOC.Am. 52, 125G-1258. Nakanishi, K., J. Furutachi, M. Funamizu, D. Gunborger and I. B. Weinstein (1970) J . AJII. C ~ J I . SOC.92, 7617-7619. Porschke, D. (1973) Proc. Nut/. Acad. Sci. U S . 70, 2683-2686. Secrist, 1. A,, J. R. Barrio and G . Weber (1972) B i o c h e ~ i i w q11, 3499-3509. Steinmaus, H., I . Rosenthal and D. Elad (1969) ./. Am. Chew. Soc. 91, 4921-4923. Wilson, R. W., J. P. Morgan and P. R. Callis (1975) C h e m P h p . Lvttrrs, 36. 618--623.
