Preparation of Tetrapyrrole-Amino Acid Covalent Complexes

I'lunt I'ht.siol.Ritx ltt'nt. 1996. -14 (3). 393-39lt

Preparation of tetrapyrrole-amino acid covalent complexes

Leszek Fiedorl'2*, Varda Rosenbach-Belkinl, Maruthi Sail and Avigdor Scherzl

I BiochernistryDepartment. The Weizn-rannInstitute of Science.76100 Rehovot.Israel. I Prcscntaddress: Institute of Molecular BiologSr.Ja-ciellonian University. Al. Mickiewicza 3. 3 l- 120 Cracow. Poland. ':'Author to whom correspondenceshould bc addrcsscd(fax +48-12-336907:E-mail fiedor@)mol.uj.edu.pl)

Abstract The presentedsynthetic approach towards chcn'rical modifications of chlorophylls(Chls) provides a perspectivcto construct model systems. where tetrapyrrole-aminoacid and tetrapyrrole-peptideinteractions coulcl be studied in covalent rnodel compor,rncls. The approach relies on thc lact that in Chls the | 7r propionic rcid sidc chain docs not participatc in the tetrapl'rroleii--electron system. It makes use of a plant enzvmechlolophyllase (EC 3.1.1.1,+).which lrr lilo and in yitrc catalysesreactions at this sidc function. The transesterilicationand hyclrolysisenzymatic rerctions are useful on a preparativescale. ln the transesterificationreaction. a desiredamino acid rcsiduc posscssirrgprimary hydloxyl group can be directly attachedto the propiorric acid side chain o1' Chl. This mcthod allows to replace the phytyl moiety in Chls n'ith seline. The r:rtherreaction. enzyrratic hydrolysis of Chls, yields chlorophyllides and opens a convenientroutc fbr furthcr rnodifications.If sufliciently mild synthetic mcthodsarc uscd. such as catalysisw,ith ,l-dimethyl arnino pyridine or activationwith N-hvdroxvsuccinimide.an arrino acid or peptide residuecan be covalentlybound to chlorophyllides' carboxylic group. lear,'ingthe essentialclectlonic structure of Chl intact. The activation w'ith N-hydroxvsuccininridcallows fbr the coupling cvrn in aqueous rncdia. Followinc these two metl.rods.the chlorophyllideswere linked e.g. to tyrosine or melanocytestirnulating horrnone (rr-'l,7-MSH;. The spectralf'eatures of thesenrodel cornpounds indicate a lbrmation of a ground statecharge transl'er complex betweenthe tetrapl,rrolcand amino acid moieties.Thirnks to the high stereospecilicityof chlorophyllase.the describedurodel compounds are the non-primediastereisomers. They have chemicnlfeatures of both Chl and amino acid and thus can be uscd as modulesto build more cornplicatednrodel svstems.

Kel rvords Chlorophi,ll. chloropht'llase.chlorophyllides. nlrdel compoundssynthesis. interaction with arnino acids.char-rrc transfcr complex.

Abbreviations Bchla. bactcriochlorophvlla; Chla. chlorophl'll a: Chlase, chlorophyllase;Chlide, chlorophyllidcl DCC. dicl'clohexylcarbodiimide:DMAP. .l-dimethyl amino pyridine: HPLC. hi-ch pressure licluid chromatography:IR. infra-red; LHC, light harvesting complexl MSH. melanocytestimLrlating hormone: NHS. N-hyclroxysuccinimide;PDT, photodynarnictherapy: Phco. pheophytinl Phide. pheophorbidc:RC. reaction centlel THF. tetrahyclrofuran:Tl-C. thin layer chrontatoglaphy.

INTRODUCTION undergo certain modificationsto suit their functions in yit,o.It becameevident that this tunin-uof pigment In natural systems interacting with li-rrht. the properties is achieved by the interactions of the tetrapyrrolesplav a specialrole by virtue ol'their light pigmentswith their environment,which in most cases absorbin-eproperties. However. these properties ur consistsof proteins.The plant and bacterialphotosyn- lrlo difl'ermarkedly fion thoseof isolatedpigments in thetic systemsare well known exampleswhere such organicmedia. This is becausethe pigmentproperties interactionsoccLrr. The chloroohvlls.embedded in the

Plant Phr,siol.Biochern.. 09flI 9.1ltl/96/03/:1i-1.(Xyaal Ciauthicr-Villals L. F'iedor el a/. proteinmatrix closelyinteract with variousamino acid ln this respect,the enzymatic catalysisappears to residues(Deisenhofer and Michel. lc)89;McDermott be a promising approach towards the synthesisof et al.,1995).These interactions ale involvednot only covalent tetrapyrrole-aminoacid/peptide complexes. in maintainingthe structureof the photosyntheticrc'rLc- Thanks to the unique f'eaturesof enzymes. l.c. tion centresand light harvestingcomplexes (Zuber and high selectivity. specificity and good yields of the Brr-rnisholz.199 l: McDermott d/ ul.. 1995)but also catalysedreactions. the use of enzymaticmethods in in the tuning of pignent propertics.This was shown organicsynthesis receives much attention(Zaks. 1990; elegantly by site-directedr-nutagenesis of particular Hal-ea5,1992). ln application to Chl modifications, arninoacid residuesin severalbacterial RCs (Coleman the plant enzyme chlorophyllasehas been fbund to and Youvan. I990; Bylina and Youvan. 199I), be a convenientsynthetic tool (Michalski.1988). The fbllowed by spectroscopicor redox mcasurements. enzyne is known to catalyse in v'ivo and irt t'ittrt The presenceol'some amino acid residuesprofor-rndly the reactions at the propionic acid side chain of influences the ultrafhst electron transf-er process various Chls and their derivatives(Willstaedter and (DiMagno and Norris. 1993). AlthoLrghin such Stoll, l9l3; Fiedoret al.. 1992).As we have shown experimentsthe involvement of certain amino acid previously (Fiedor et al., 1992), in the hydrolysis. into the electrontransf'er or other processesis clearly transesterificationand reesterificationof this side shown. the exact mechanismsol' that involvement tunction. Chlase shows high stereospecificityand are not understood yet. This is due to the high catalyses the production of only the non-prime complexity of the natural systemsand the dilliculty Chl diastereoisomers.Here we describe the use of in spotting these weak single pigment-an'rinoacid Chlaseand other syntheticmethods in constructionof interactions.superimposed on the backgroundwhich chlorophyll-aminoacid/peptide covalent cornpounds. is a sum of many suchinteractions. Therefbre. it seems Thesemethods enabled us to synthesizethe covalent reasonableto investi-{atethe specific tetrapyrrole- complexesof Chls c.g. with the amino acids serine arninoacid and tetrapyrrole-peptideinteractions in less and tyrosine and a 14 amino acid peptide o-4,7- complicatedmodel systems.provided an appropriute MSH (melanocytestirnulating hormone). These model systemcan be constructed.ln tirct, a grcat eflbrt has compounds were characterizedby various spectral been put into the developrnentof artificial covalent techniques,such as absorptionand emissionspectro- model compoundsand other model systems.as they scopies.The model compoundscontaining aromatic proved to be very helpfLrlin studiesof rnany aspects amino acids,show occurrenceof char-oetransf'er com- of photosyntheticsystems (Wasielewski, 1992). In plexes betweenChla and these amino acid residues. order to achievedesired mirrricking effects in model Serine-(B)Chlidewas recently characterizedas effi- systems.each of their componentsmust preserveall cient photcrsensitizerfbr PDT (Fiedor et al., 1993lr. their structuraland chemicalcharacteristics. crucial to allow the specific interacticlnsto occur. Attempting to model the interacfionsbetween the photosynthetic RESULTS AND DISCUSSION pigmentsand arclmaticamino acids which are tound in the RC, in particular those that may all-ect Transesterification reaction the electron transf'er process. requires that thesc moleculesshould be brought into close contact and The Chlase catalysed transesterificationreaction their (stereo)electronicstructures need to be intact. was used to synthesizeSer-Chlide and Ser-Bchlide However. from the synthetic point of view it ntay esters. Almost pure non-prine epimeric forms of appear to be difficult. Due to the prcsence of a the pigmentswere producedwith yields up to JOC/a. labile centralmagnesium ion, the caseol'oxidation. The purificationof the transesterifiedcompounds was epimerizationand isocyclic ring opening, etc., Chls achievedon a CM-Sepharosecolumn or by reversed are known to be chemicallyunstable. Therefore. or.rly phaseHPLC. Thesesimple model compoundsposses very mild syntheticprocedures can be applied in the chernical f-eaturesof both Chl and the arnino acid constructionof model systemswhich contain Chls. and therefbrecan be used as modulesin cclnstructicln This seemsto be a sevefeobstacle since out of many of rrore advanced model systems. They are water- syntheticmodel systemsconstructed up to now only soluble and spectrclscopicallyresemble parent Chls f'ewaddress the aspectsof Chl-aminoacid interactions (see below). Recently, they were characterizedas (seee.g. Boxer, 1983;Verchere-Beaur et ttl., 1990). very efTectivephotosensitizers for PDT (Fiedor er a/..

Plunt Phvsiol. BioL'lterrt. Preparation of tetrapyrrole 395

1993).lmportantly, in comparisonto hematoporphyrin derivative, these compounds administeredinto the living organismsare expectedto undergomuch faster it biodegradation(Spikes and Bommer. l99l). I tt I I it Hydrolysis of chlorophylls , rl The chlorophyllideswere obtainedon a preparative lr scaleby enzymatichydrolysis of variouschlorophylls, ,l using Chlase.The productswere purified by column I chromatography on CM-Sepharose. The enzyme hydrolysesand produceswith very high yields (up 300 430 560 690 820 950 Io 90c/c)only the non-prinrediastereoisomers ol' the Wavelength Inrn I pigrnents(Fiedor et ul.. 1992).

Figure l. Ab.utrption .\pe(tftt ol Scr-Chlide und Ser-lJchlide in Modifications at the 172 carboxylic group of Chlides Ser-Chlideand Ser-Bchlidein methanol.are shown Thc l7- earboxylic group in chlorophyllides is in figure l. The spectra are almost identical to assumed not to participate in the tetrapyrrole those of the correspondingChlides (see e.g. Chlidea r-electron system. Therefore, a covalent linkage to spectrumin.ftg.2') or Chls (HofTand Amesz. l99l), this side function permits fbr the modificationsclf indicating that the Chl electronic structurein these Chl without irff-ectingthe r-electron systern. Two compoundsis preserved.Moreover. due to the enzyme mild methodswere used to link various amino acids stereospecificity,the Chl moiety of these model or peptidesto the carboxylic group of Chlides. The compounds is the non-prime diastereoisomer.as DMAP methodallows tor esterillcationof'the Chlides confirmedby rH-NMR spectroscopy(data not shown (e.9. with amino acids soluble in organic media (Fiedor. 1994)). dichloromethane).such as tyrosine,with yield of 30- The absorptionspectra of Tyr-O-Chlidein methanol 10c/r,. The other rnethod, the activation with NHS. (.fig 2l and MSH-Phide in 80% aqueousacetonitrile is more general as it f'acilitatesthe linkage to the (t'tg.3), diff'er somewhat from that of the corre- Chlide carboxylic gloup via an amide bond even rn spondin-qparent pigrnents.However. in both cases. aqueous milieu. Thus. through the activation with the alterations in the absorption protiles are of a NHS. the Chlides can be covalently bound to water similar character.The most prominent changesare soluble peptidesand proteins.This method was used to synthesizeChlide-MSH conjugate(yield 20c/o1. The modilied Chlides were puritied by reversed phaseHPLC. The Chlide esterifiedwith tyrosine(Tyr- O-Chlide) was elutecl with aqueous methanol. The conditionsfbr purificationof the MSH conjugatedto Chlide(MSH-Chlide/Phide) were similar to thoseused i ll tii tbr isolationof peptides.l.e. the elution with aqueous ! ri i, ilcetonitrilecontaining TFA at low concentration.The :t! presenceof the acid causedthe loss of magnesiumin the product.MSH-Chlide. resulting in thc separrtion i+,+ ti i of MSH-Phide. rii

230 360 490 620 754 Spectral characterization of the model compounds WavelengthInml The synthesizedmodel compoundswere character- yritlt ized by several spectroscopictechniques. Sorne of Figure 2. Ablorption sp?(trurn rtf Trr O-Cltlitlc t ontpuretl tltL' sltcctru ol ('ltlidea artd tyntsine irt rnttlrututl. The tyrosine currc is the resultsof these studiesare presentedbelow. The scaledto nratchthe absorptionvalucs of the pignrentsin the UV electronic spectraof the transesterificationproducts. rcgion.

vol. 3.1.n" 3 1996 L. Fiedor et al.

0L 0 360 490 620 750 3oo 4oo ttt 80( WavelengthInm I **1tt**,"1i-,

Figure 3. Ab.;orytlirtrt sp((trutr1 rI MSH-Pltilc in E0(,t utlueous l-igure 4. Entis.;irnt.\l)e(trum ol 7\'r-O-Chlide(e.rcitation ut 275 nnt) atekntitrile, cornpuretl witlt the.spt'ctnt rtl MSH untl Phideu in the t ontlxtred tritlt the erni.s.siortsp('(lrurn ol' l: I ntilure o.f ttrosinc urtd .sttntesolt ent. Tlre MSH curve is scaled lbr convenient Dresentrtiolr. ()ltlidet itt methrutol.The spectnture rtormalizcdto the sameO.D. Valuert the excitationu'avelcngth. observedin the r"elativeintensities of the Soretand Q\ on the tetrapyrrolemoiety in the model compounds. transitions.Thus. in the model compoundsthe raticr Moreover,the fluorescencespectra show an unusually Soret/Q,reaches 3:1. while in Chla (and Pheoa)it shaped bands in the UV region. which by the is closeto l:1 (Hofl'and Amesz. 199l). Compared resemblanceto the tyrosinate signal may reveal a to the parentcompounds, the Soret transitionsin the partial electronexcess on the tyrosine moiety (Szabo model compoundsbecome narrower (as measuredat et al.. l9l8). the half'-width)and their maxima are red-shilied by Altogether. the above observationsindicate the 7-8 nm whereasthe Q, transitionsbroaden at the half occlrrrenceol' specific interaction between the Chl hei-ghtwith no shift in the maximum position. ?r-electron systen.tand the aromatic amino acid ln the emission spectraof the model compounds. residues.Covalently linked Chls and tyrosine (and in comparisonto the Chla/Pheoaemission profiles, perhapsother aromaticamino acids)fbrrn an electron some significantditfbrences can be obscrvedtoo. The donor-acceptor(charge transf-er)complex. althor"rgh excitationar 275 nm o1'Tyr-O-Chlide(in methanol) they are not conjugated via thc r-electrt)n system. and MSH-Phide(in 80% aqueousacetonitrile) results This conclusion may have fr.rrtherbeirrings on the in the emission spectracomposed of two prominent photosyntheticRC functioning. where the tyrttsine bands (fg. 4 and.fg. 5. respectively).which are not just a sum of the emission profiles of the parent 1 pigmentsand the irmino acid (or the peptide). band is positioned in thc UV region One broad i (maximum at 345 nn-rfbr Tyr-O-Chlidc and 360 nm for MSH-Phide), which has characteristicshoulders at the longer wavelengths.The secondband, with its maximum in the near-IRis lower in the interrsityand MSH also red-shiftedby severalnanometers. relative to the correspondingband of parentcompounds. Interestingly.the ground state absorption spectra o1' the model compounds Tyr-O-Chlide and MSH- Phide resemblethc spectraof correspondingcation 0 700 800 radicals of fiee Chla or Pheoa (Borg ct ol., 19701. 300 400 500 600 Such absorption spectra as well as the quenched Wavelength[nm ] emission in the near-lR range are typical lbr charge Figure 5. Enti.;.sirntsp&tru ol MSH untl MSH Philt (..\(ildtit,n Ltt transf'ercomplexes of Chls (Droupadi and Krishnan. 275 nn) irt 80'tr ut1Lreousutt'tottitrile. Thc spcctraare norrnirlizedtcr 1984). These indicate a partial electron deficiency the sameO.D. r'aluc at the excitiitionuavelength.

P lunt Pl:l.viol.IJirrlrcnt. Preparation of tetrapyrrole residueis presentnear the electrontransf'er cof'actors containing0.87c Triton X-100 (r,'/v).The reactionmixture and would be able to interactwith thesecofactors. was then stirred lbr 6 h at 37"C in thc dark undcr argon atmosphere. The reaction progrcss was Inonitured by TLC on silica _seland afier the reaction completion CONCLUSION the productswere extractedftom the leactionmixture with dicthyl ether.The transesterifiedproducts werc purilicd by column chronatographyon a CM-Sepharose(Phalmacia) or The present studies delilonstratethat the chloro- alternatively.bl, rcvcrscd phase HPLC on a C-ltl Vydac phyllase catalysed reactions. transesterificationand column(218TP1022). A detaileddescription of thc mcthod hydrolysis. can be used on a preparativescale in is describedby Ficdor (199;l) and Scherzer al. (1994). synthesisof covalent chlorophyll-aminoacid/peptide complexes.The enzyme is highly stereospecificand therefbre only the non-prime epirneric firrms are Chlorophr,)l produced.The modifred chlorophylls with intact n- electron system can be synthesized in one step I synthesisvia the transesterificationreaction or with St'r--Chlidc the chlorophyllides as the interrnediates.via the I hydrolysis. The covalently linked chlorophylls and amino acids/peptidesare useful as model compounds C-hlorophvJlidt in the studiesof specificinteractions of tetrapyrroles D\,I.\P'DCC/,/ / \ .,,,."" \ with amino acids. Some of thesc interactionsmay " cata\* / \"" involve the formation of charge transf-ercomplexes / \'st'' pigment betweenthe and thc arornaticamino acids. Anrino acid Amino lcid ancl peptide clcrivatives (esters) cleriyatir es (arniues.l I'vr-O-Clhlide \{SII-Chlidc METHODS Figure 6. Outline ol tht' nill t'n:vntutit ultprrnch tottdrd.\ tlt(' .r.rltlrt,.ri,rol ontino utid/ptlttide -chloroplnll utvulenl t rtnple.rt.s. Pigment isolation. The pigments useclin the svnthesisof rrciclelcompounds were isolated according to the rnethods Thc hyclrolysisof Chls was carried out accordin_cto ir previously described.Chla was cxtractedtiom lyophilised proceduredescribccl by Ficdol et ul. (1992) with sorne cells crf Spirullinu g,ultieriand purified chromatogralicalll' nrodifications(Fiedor. 1994).Bchla or Chla was incubated on DEAE-Scphrnrsecoluntn. filllowing Omata and Murata with thc Clhlaseextl'act undel similar conclitionsls fbr (lt)83) with somc rnodilications(Roscnbach Belkin. 1988). the transesterification.Aftcr thc reaction completion. the Bchla was analogouslyobtained liom the lyophilisedcells products Chlides. were extracted into diethyl ether iind of photosynthetichactelir-rm Rhotlospirillurtt rtrbrurn. purified on a CM-Sepharosecolurnn.

Chlorophyllase catalyzed syntheses. For the svnthetic Chemical modifications at the 172 carboxylic group. applications.Chlase was preparedb1' Triton X- 100extrae tion The nrodifications of Chlides at thc sidc propionic of the so called acetone powder obtained fl'onr the acid carboxylic group were done by two methods. The chloroplastsof fiesh leaveso1'Chine Tree (Meliu u:.etleruch1. esterilication of Chlide with tyrosine was perfbrmed as describedbl, Ficdor t't ul. (1992) and Ficclor( 199;1). by a DMAP nucleophilic catalysis irr the presenceo1' The syntheticmethods described here relv on the reaetions DCC in anhydror-rsdichloromethane, accolding to the catalysedby Chlase and are outlined in ligure 6. On the plocedure previously reported tbr esterificationof Chlide preparativescalc. two enzynraticreactions can be applied. deriyatiyes (Wasiclcwski and Sycc. 1980). In aqueous that is the transesterificationancl hl,dlolysis. In the tbrrrer ntedia.a Chlide-NHS active esterwas usedto link Chlides reaction.the phytol moietvol'(ts)Chla is directlvreplaced by to MSH (Ac-Ser-T1'r-Ser-Nle-Gln-His-D-Phe-Arg-Trp-Gly- a primary'alcohol. The latter one vields (B)Chlidcs.which Lys-Prn-Val-NH:). fbllowing thc protocol described by 'Ihe furthercan be modifiedat the freepropionic acid sidc group. Schcrzt:/ ul. (1994)and Fiedor( 199,1). reactionprogress The synthesisof Ser-Bchlide/Chlideesters is basedon the wrs nionitoredby TLC on silica gel. The products were transcstclillcationreaction of Chla or Bchla in prcscnccof purified b1' rcvcrscd phasc HPLC on an analytical C-18 excessserine. catalysed by Chlase.The substrates.l0 rng of Vydaccolurnn (2l8TP) usingfirr clutiona methanol/wateror Chla (Bchla) and 200 mg ol' l-methyl serineh1'drochloridc acek)nitrilc/waterl0.l%TFA gradicnt.Thc opticalabsorption (Si-t:nra).were homogenizedwith 100 mg of' the acetone spcctra were recordedon a Milton Roy Spectronic l20l powdcl in 5 ml of 0.1 M sodiumphosphatc buflcr (pH 7.6) spectra/photomctcr.Thc fluorcsccncemcasurcmcnts were

rol. .lJ. n' I - 1996 398 1,. Fiedor et al. perfbrmed using a SLM Aminco 8000 spectrofluorimeter. McDermott G., Prince S. M., Freer A. A., All solvents(local supplier) were of analytical or HPLC Hawthornthwaite-Lawless A. M., Papiz M. 2., grade and if needed,werc dried by passingthrough active Cogdell R. J. and Isaacs N. W.' 1995. Crystal structure from alurninium oxide. of an integral membrane light-harvesting complex photosyntheticbacteria. Nuture, 374. 511-521. Michalski T. J., Hunt J. E., Bradshaw C', Wagner (ReceivedJuly 5, 1995;accepted November -5. 199-5) A. M.. Noris J. R. and Katz J. J., 1988. Enzyme- catalyzed synthescs: transesterilicationreactions of and deriratives REFERENCES Chlorophyll a. Bacteriochlorophylla, with Chlorophyllase."/. Am. Chem.Soc..ll0,,5888--589 l. Omata T. and Murata N., 1983. Preparation o1' BorgD. C.,Fajer J., FeltonR. H. andDolphin D.' 1970. Chlorophyll a, Chlorophyll D and Bacteriochlorophylla The r-cation radicalof Chlorophyll u. Prot. Nutl. Acatl. by colurnn chromatographywith DEAE-SepharoseCL- .Sci.USA, 67, 813-820. 68 and SepharoseCL-6B. Plant Cell. Pht'siol., 24. Boxer S. G., 1983. Model reactions in photosynthesis. r093-rr00. Biochim. Biopht's. AL'ru, 726. 265-292. Rosenbach-Belkin V., 1988. The primary reactants Bylina E. J. and Youvan D. C., 1991.Protein-chromophore in bacterial photosynthesis modelling by irt t'itnt inleractions in thc reactit'rn centcr ttf Rfuilobacter preparation.Ph. D. The.si.s,The Weizmann Institute of tttpsulatLts.lnChloroplulls. ScheerH.. ed.. CRC Pless, Science. Boca Raton. 105-719 Scherz A., Salomon Y. and Fiedor L.,1994. Preparationol' Coleman W. J. and Youvan D. C., 1990. Spectroscopie Chlorophylland Bacteriochlorophi,'11coniugates and their analysisof geneticallymodified photosyntheticreaction applicationas photosensitizersin photodynarnictherapy centers.Annu. Rev.Biopht.t. Biopht's.Chem.. 19. 333- and generaldiagnostics. Eur. Pat. Appl.. EP 584552. 361. Spikes J. D. and Bommer J. C., 1991. Chlorophyll Deisenhof'er J. and Michel H.' 1989. The photosynthetic and relatedpigments as photosensitizelsin biology and reaction centre from the purple bacieriunt Rhodopseu- mcdicir.re.ln Chlontphtll.i, Schccr H.. ed.. CRC Press, rlornontrsviridis. EMIIO J., 8. 2149-2170. Boca Raton.Il8l-1204. DiMagno T. J. and Norris .J. R., 1993. Initial Electron Szabo A. G.. Lynn K. R., Krajcarski D. T. and Rayner transf-erin photosyntheticbacteria. ln The photttsvnthetit' D. Nl., 1978. T1'rosinatefluoresccnce maxima at 345 nnl reactiotlcenter.,II, Deisenhol-erJ. and Norris J. R.. ed.. in proteinslacking tryptophanat pH 7. FEBS Letr..94. AcademicPress, San Diego. 105-132. 249-252. Droupadi P. R. and Krishnan V., 1984. Charge transf'er Verchere-Beaur C., Mikros E., Perree-Fauvet NI. complexesof pheophytinrr with nitroarontatics.Electron and Gaudemer A., 1990. Structural studics of transf-er fronr excited singlet of pheophytin rr tL) nrctalloporphyrins.Part XI. Cornplexesof water-soluble nitroaromatics.Phrtot:hem. Photobiol., 39. l6l-167. zinc(Il) porphyrinswith amino acicls:lnlluencc of ligand- Fiedor L., 1994. Preparation and characterizationof' nrod- ligand interactionson the stability of the complexes. ified chlorophyllsfor modelling prirnaly photosynthesis J. Inorq. Biochent..40, 127-139. and applicationin photodynamictherapy of cancer.P/r. Wasielewski M. R., 1992. Photoindrrcedelectron transt'er D. Thesis.The WeizrnannInstitute of Science. in supramolecularsystems firr artificial photosynthesis. Fiedor L.. Rosenbach-Belkin V. and Scherz A'., 1992. Clpn. Rev'.,92, 435-161. The stereospecificinteraction between Chlorophylls and Wasielewski M. R. and Svec W. A., 1980. Syn- Chlorophyllasc. J. Biol. Chem.. 267. 22043-2204'7. thesis of- covalently linked dirncric derivatives ot' Fiedor L., (iorman A. A., Hamblett I., Rosenbach- Chlorophyll a, Pyrochlorophylla. Chlorophyll D. and Belkin V., Salomon Y. and Scherz A., 1993. A Bacteriochlorophylla. J. Org. Chent..45, 1969-1974. pulsed laser and pulse radiolysis study o1'amphiphilic Willstaedter R. and Stoll A., 1913. Untersut'hungeniiber Chlorophyll dcrivatives with PDT activity toward Chlrtrcphtll. Springer.Berlin. malignant melanoma.Photochem. Photobiol., 58, -506- Zaks A., 1990.Enzymes in or-eanicsolvents. ln Bioctrtttlt"stt 5lt. .for indu.ttr\'.Dordick .1.S., ed..Plenum Press, New York, Hafga5 J., 1992. ln Biottttalysts in orgtmic .t'rn1lreri.s. l6l-180. Elsevier'.Am:lcrdam. Zuber H, and Brunisholz R. A., 1991. Structurc and Hoff A. J. and Amesz J., 1991. Visible absorption functiono1' antenna polypeptides and Chlorophyll-protein spectroscopy of chlorophylls. In ChLoropft.r'//s,Scheer complexes:principles and variability. In Chlorophtlls, H.. ed.. CRC Press.Boca Raton. 123-738. SchcerH.. ed.. CRC Press.Boca Raton. 627-703.

Plunt Phr.siol.Bkx'lrcnt.