Indian Journal of ChemistryVol. 27 A, January, 1988, pp. 4-5

Adenosine-sensitised Photolysis of Alanine

T MUKHERJEE, A M WAN It & J P MITIAL*Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085

Received 9 April 1987 ; revised and accepted 8 May 1987

Photoexcitation of an aqueous solution of adenosine containing alanine with 253.7 om light, leads to sensitised photo-decomposition of the latter to afford NH3• The presence of O2 reduces the quantum yield of NH3 formation, while the pres-ence of N20,CO2 and other quenchers reduces the yield to zero, indicating that the sensitisation occurs via photoionisationof adenosine mediated via the triplet excited state.

The effect of UV radiation on nucleic acids has beenextensively investigated 1- 3. As DNA intercalates tothe proteins in the cells, the effect of UV radiation onthe amino acids can be studied by selectively excitingnucleic acids and following subsequent reactions ofthe excited states of the latter. Electronically excitedpurines have been shown to react with alcohols",amines-" and aliphatic amino acids 7, to give substitut-ed purines. In several cases the functional groups ofthe substrates do not appear in the final product, sug-gesting that the substrates are cleaved by interactionwith the excited purines. With amino acids as sub-strates, loss of amino groups has also been observed",The effect ofUV irradiation on aliphatic amino acids(e.g. alanine) in the presence of adenosine shouldserve as a useful model for photosensitisation in bio-logical systcms''", since light is almost exclusively ab-sorbed by adenosine, producing excited adenosine,which then interacts with the amino acid. In this pa-per, estimation of ammonia formed and luminesc-ence studies have been used to elucidate the mechan-ism of photosensitisation.

Materials and MethodsAlanine (SISCO Research Laboratories, India)

was rccrystallised seven times from water. Adenosine(Sigma) was used as received. Triply distilled waterwas dc-ioniscd by passing it in succession through ca-tion and anion exchanger columns. High purity oxy-gen and purified N 20 (Indian Oxygen Ltd) were bub-hied through the solution when necessary. Carbon di-oxide used was generated hy reacting CaCO, withHCIO~ in situ.

Ammonia was estimated with an Orion 95-10 gas-sensing electrode fitted with improved multilayer gasdiffusion membranes, coupled with an Orion 407 Aspecific ion meter (sensitivity better than 0.1 mY).

+ Present address: Nuclear Research Laboratory, Sri nagarI YO O{)() U&K}

4

Degassing was done by freeze-pump-thaw method at77K, using a vacuum line. Photolysis was done inquartz tubes (int. diam. 15 mm), using a Rayonet RPR-100 photochemical reactor (Southern New EnglandUltraviolet Co., USA), fitted with 16 low pressuremercury resonance lamps (253.7 om) in a cylindricalsymmetry and a merry-go-round rotating at 4 rpm foruniform irradiation.

Absorption spectra were recorded on a Hitachimodel 200-10 spectrophotometer. Fluorescence atroom temperature and phosphorescence at 77Kwere studied using an Aminco Bowman spectro-photofluorometer (model 4-8202 B) fitted with anAminco Keirs phosphoroscope). The photon fluxwas determined using a uranyl oxalate actinometer!".Typical photon flux used was 1.65 x 10lYdm -3S -I fora 90-min irradiation.

The quantum yield (<1» for the sensitised photode-composition was defined as:-

<I> = (Number of Nl-l , molecules produced due to sen-sitisation)/(Number of photons absorbed by sensitis-er molecules)Results and Discussion

In solutions containing 5 x 10 - 5mol dm - 3 adeno-sine and 1 mol dm - J alanine, 253.7 nm UV light wasabsorbed exclusively by adenosine (Ec5J7 = l. 7 X 104

dm'mol-1cm-1 for adenosine and -0.014dmmol " Iern - I for alanine). Hence (CE l..ocno'in/(CE lalanine~ 61. Ammonia was found to be a productformed by the photosensitisation of alanine by excit-ed adenosine. Typical quantum yield in degassed so-lution was 5.6 x 10" ~.lncrease in 0, concentration insolution decreased the quantum yield of ammonia to2.0 X 10- 4 and 2.4 x I() - 5 in air and oxygen saturatedsolutions respectively, indicating th.ut riplcts of aden-osine were probably involved.

At room temperature, an aqueous solution of aden-osine (pH 7) was devoid of any fluorescence. At 1'1-\ I,a very weak tluorescence was observed with A.max at -3lJ5 nm (A.exc - 285 nrn), Addition of alanine had no

MUKHERJEE et III.: PHOTOLYSIS OF ALANINE

effect on this fluorescence, indicating that adenosinesinglet states do not possibly interact with alanine.Steele and Szent-Gyorgyi II also did not observe anyfluorescence from adenosine at any pH at room tem-perature. However, they reported phosphorescencefrom a 10~3mol dm ~3 adenosine solution frozen at77K with Amax- 420 nm and life-time (1:) of - 2.5 s.Due to such a long life-time ofT Istate, further photonabsorption to T, state becomes very probable. Heleneet all? and Rosenthal et al.s have actually establishedthat even in steady state photolysis, biphotonic excita-tion from TI to higher triplet states occurs in adeno-sine, followed by auto-ionisation. The n-electron ion-isation energy of adenosine has been calculated quan-tummechanicallyas - 8 eyt3, but 253.7 nm corre-sponds only to - 5 eV, while E(TI) - 3.4 eV:Evident-ly, direct photoionisation from TI state is not possible,while biphotonic absorption via TI state to T, > 8 eVfollowed by ionisation becomes feasible. Helene andcoworkers 12,14also suggested that any subsequentchemical transformation in the adenosine-sensitisedreactions is due to interaction between the photo-ejected electrons and the substrate,

Presently it has been observed that an increase in0, concentration decreases the quantum yield ofNH3 production via sensitisation, indicating that trip-lets of adenosine are probably involved. However,the autoionisation process must be competing withthe reaction with 02' since, otherwise O2 should havereacted completely with such a long-lived triplet, giv-ing no NH3 as the product. In the presence of metalions (Cu 2 +), no NH 3 could be detected, indicatingcomplete inhibition due to triplet quenching I'i.

At lower pH ( < 4) no sensitisation was observed.This is possibly due to the lowering of triplet yield ofadenosine, since it is found that the phosphorescencefrom adenosine is quenched by protonation(pKa "'"4). Acid inhibition could also be due to sca-venging of photoejected electrons by acid protons(see Eq, 1)

" ,(1)

When N,O, an efficient electron scavenger Ill,wasbubbled through the mixture before photolysis, thequantum yield for sensitiscd photodecompositiondecreased to zero, indicating the intermediacy of hy-drated electrons in the sensitised photodecomposi-tion in the absence ofN 20. Since the NH.1yield actual-ly decreased, involvement of OH produced by thereaction between e;<jand N20 was also ruled out. Si-milar results were observed when N20 was replacedby CO2, another efficient electron scavenger.

The mechanism involving the generation of tripletadenosine, followed by ionisation from higher Tnstate, explains our observations (see Scheme I)

hV 1--------t •.~ 'A (51) (2 )

(3)

(4)

------- ••- A+ + .oq (5 )

------- •• PRODUCTS (7)

(ISC - INTER - SYSTEM CROSSING)

SCHEME 1

Reaction (6) is also supported by some earlier observ-ations 17~19 that reactions between e~ and amino ac-ids lead to deamination. However, in Scheme 1, thefate of the adenosine cation is still not fully known. Itprobably reacts with water (see Eq. 8)

A++H20-AOH+H+ ,,, (8)

In conclusion, triplet state of adenosine and its sub-sequent photoionisation are involved in the sensitisedphotodecomposition of alanine.

AcknowledgementThe authors thank Ms Indira Rau, a National Sci-

ence Talent Search Scholar from lIT, Delhi, for help insome of the preliminary experiments,

ReferencesI Wang S Y, Photochemistry and photobiology ojnucleic acids,

Vol I & II (Academic Press, New York) 1976.2 Steiner R F & Weinryh I, Excited states of proteins and nucleic

acids (Plenum Press, New York) 1971.3 Bensasson R V,Land E J & Truscot T G, Flash photolysis and

pulse radiolysis (Pergamon Press, London) 19S3.4 Steinmaus H, Rosenthal I & Elad D, J org Chern, 36 ( 1971 )

3594.5 Stankunas A, Rosenthal I & Pitts (Jr) J N, Tetrahedron u».

(1971)4779.6 Solomon J & Elad D, Photochem Photobiol, 19 (1974) 21.7 Elad D & Rosenthal I, Chem Commun, (1969) 905.S Rosenthal I, Pupko R, Muszkat K A & Sharefi-Ozeri S, J phys

Chem,80(1976)454.9 Mittal LJ, MittalJ P& Hayon E,JphysChem, 77( 1973) 14R2.

10 Murov S L, Handbook of photochemistry (Marcel Dekker,New York) 1973,

II Steele R H & Szent-Gyorgyi A, Proc natl Aca Sci US, 43(1957)477.

12 Helene C, Santus R & Douzon P, l'hotochem Photobiol, 5(1966) 127.

13 Pullman A & Rossi M, Biochim Biophys Acta, 88 ( 19(4) 211.14 Santus R, Helene C & Ptak M, C R Acad Sci Paris, Series n.

262 (1966) 2077.15 Helene c. Ptak M & Santos R, J chim l'hvs. 65 (196X II)().

16 Gordon S, Hart E J, Matheson M S. Rabani J & Thomas J K,Dise F-im/{/lII'SoC, 36 (1961) 19".

17 Braams R, Radiat Res, 27 (1966) 319.IX Neta p, Simic M & Hayon D, J phvs Chern, 74 ( 1970) 1214.19 Sevilla M D, J phvs Chern. 74 (1970) 2096.