Ternary Copper(II) Complexes in Solution Formed With 8-Aza Derivatives of the Antiviral Nucleotide Analogue 9-[2-(Phosphonomethoxy)Ethyl]adenine (PMEA)

Me al Based D ugs Vol. 7, N . 6, 2000 TERNARY COPPER(II) COMPLEXES IN SOLUTION I21 FORMED WITH 8-AZA DERIVATIVES OF THE ANTIVIRAL NUCLEOTIDE ANALOGUE 9- [2-(PHOSPHONOMETHOXY)ETHYL] ADENINE (PMEA) Raquel B. G6mez-Coca ,9-, La isa E. Kapinos , An onin Ho13 3, Rosa io A. Vilaplana , F ancisco Gonz ilez-Vilchez and Helmu Sigel *1 Ins i u e o Ino ganic Chemis y, Uni e si y o Basel, Spi als asse 51, CH-4056 Basel, Swi ze land <[email p o ec ed]> 2 Ino ganic Chemis y Depa men , Facul y o Chemis y, Uni e si y o Se ille, E-41071 Se ille, Spain Ins i u e o O ganic Chemis y and Biochemis y, Academy o Sciences, CZ- 16610 P ague, Czech Republic Dedica ed o he memo y o P o esso Ma c Leng, an ou s anding scien is and iend Abs ac The s abili y cons an s o he mixed-ligand complexes o med be ween Cu(A m) 2+, whe e A m 2,2’- bipy idine (Bpy) o 1,10-phenan h oline (Phen), and he dianions o 9-[2-(phosphonome hoxy)e hyl]-8-aza- adenine (9,8aPMEA) and 8-[2-(phosphonome hoxy)e hyl]-8-azaadenine (8,8aPMEA) (bo h also abb e ia ed as PA 2-) we e de e mined by po en iome ic pH i a ions in aqueous solu ion (25 C; I 0.1 M, NaNO3). All ou e na y Cu(A m)(PA) complexes a e conside ably mo e s able han co esponding Cu(A m)(R-PO3) species, whe e R-PO- ep esen s a phosph(on)a e ligand wi h a g oup R ha is unable o pa icipa e in any kind o in e ac ion wi hin he complexes. The inc eased s abili y is a ibu ed o in amolecula s ack o ma ion in he Cu(A m)(PA) complexes and also o he o ma ion o 5-membe ed chela es in ol ing he e he oxygen p esen in he -CH2-O-CH2-POZ3 esidue o he azaPMEAs. A quan i a i e analysis o he in amolecula equilib ia in ol ing h ee s uc u ally di e en Cu(A m)(PA) species is ca ied ou . Fo example, abou 5% o he Cu(Bpy)(8,8aPMEA) sys em exis wi h he me al ion solely coo dina ed o he phosphona e g oup, 14% as a 5-membe ed chela e in ol ing he -CH2-O-CH2-PO3 z- esidue, and 81% wi h an in amolecula s ack be ween he 8-azapu ine moie y and he a oma ic ings o Bpy. The esul s o he o he sys ems a e simila hough wi h Phen a o ma ion deg ee o abou 90% o he in amolecula s ack is eached. The exis ence o he s acked species is also p o en by spec opho ome ic measu emen s. In addi ion, he Cu(A m)(PA) complexes may be p o ona ed, leading o Cu(A m)(H;PA) + species o which i is concluded ha he p o on is loca ed a he phosphona e g oup and ha he complexes a e mainly o med by a s acking adduc be ween Cu(A m) 2+ and H(PA)-. Conclusions ega ding he biological p ope ies o hese azaPMEAs a e sho ly indica ed. 1. INTRODUCTION Nucleo ides and hei me al ion complexes play a key ole in all aspec s o me abolism and he e o e, a emp s o exploi nucleo ide analogues as d ugs a e old (e.g.[3l).4.Among he analogue, s wi h biological p ope ies 9-[2-(phosphonome hoxy)e hyl]adenine (PMEA), an analogue o (2- deoxy)-adenosiae 5’-monophosoha e [(d)AMp2-], is a mos ema kable one; i exhibi s an i i al, 581 cy os a ic [9,11 and an ia h i ic [ l] e ec s. Conside ing he b oad biological ac i i y o PMEA, i is no su p ising ha many de i a i es ha e been syn hesized and s udied, [2] and i is now clea ha in o de o be an i i ally ac i e, PMEA and i s de i a i es mus be phospho yla ed in he cell o he diphospha e (PMEApp 4-) and his is hen ecognized by DNA polyme ases as a subs a e and inco po a ed in o he g owing nucleic acid chain which is e mina ed he ea e . [3’4] Knowing ha polyme ases depend on he p esence o me al ions [15’16] and ha he nucleoside 5’- iphospha es mus be p esen as complexes (mos ly Mg2+) [7] we ha e been s udying complexes o PMEA [8"24] and a mechanism o ac ion has been p oposed ecen ly [25’261 in which he co ec loca ion o wo me al ions a he iphospha e chain o achie ing a desi ed eac ion is emphasized. [26] In o he wo ds, he co ec o ien a ion o he nucleoside 5’- iphospha e o i s analogue in he ac i e-si e ca i y o he enzyme is c ucial. [26] One o he ways in which he co ec ancho ing p ocess o a subs a e in he ac i e si e o an ’z7] enzyme can be achie ed, is ia s acking in e ac ions o he nucleobase esidue, e.g. wi h an indole moie y o a yp ophan uni . [28] Fo his eason we became in e es ed in he PMEA ela i es, 9-[2-(phosphonome hoxy)e hyl]-8-azaadenine (9,8aPMEA) and 8-[2-(phosphonome hoxy)e hyl]-8- azaadenine (8,8aPMEA) (see Fig. 1), and he ques ion was: Do he s acking p ope ies o PMEA, 9,8aPMEA and 8,8aPMEA di e ? To his end we measu ed he s abili ies o he mixed iigand Cu(A m)(PA) complexes whe e A m 2,2’-bipy idine (Bpy) o 1,10-phenan h oline (Phen) and 2 2 PA- 9,8aPMEA- o 8,8aPMEA 2-. These Cu(A m)(PA) complexes can old such ha he 313 Helmu Sigel e al. Te na y Coppe (II) Complexes in Solu ion[I,2] Fo med wi h 8-Aza De i a i es o he An i i al Nucleo ide Analogue 9-[2-(Phosphonome hoxy)e hyl)Adenine(PMEA) a oma ic ings o Bpy o Phen can in e ac wi h he 8-azaadenine esidues. In ac , Bpy and Phen ha e p o en e y help ul as indica o s o e alua ing he s acking capabili ies o a oma ic esidues in me al ion complexes. [21 A s acking in e ac ion o he indica ed kind should be e lec ed in an enhanced o e all complex s abili y.p,29,l Indeed, he esul s ob ained p o e such enhanced s abili ies and hese a e compa ed now wi h hose ob ained ea lie pI o he co esponding Cu(A m)(PMEA) complexes. NH2 NH2 1N PMEA2- 9’8aPMEA2- N I 8’8aPMEA2- 7# 9 -O -O 3 o /CH2 o /CH2 -0 -O--P--C / C" -O--P--C / C" | c/Oc/CH2 H2 H2 H2 H2 -O--P-- 0 0 II H:, 0 Figu e 1. Chemical s uc u es o he dianions o 9-[2-(p.hosphonome hoxy)e hyl]adenine (PMEA2-), 9-[2-(phosphonome hoxy)e hyl]-8-azaadenine (9,8aPMEA -) and 8-[2-(phosphonome hoxy)e hyl]-8- azaadenine (8,8aPMEA2-). The h ee nucleo ide analogues a e also abb e ia ed as PA 2-. 2. MATERIALS AND METHODS 2.1. Ma e ials Two old p o ona ed 9-[2-(phosphonome hoxy)e hyl]-8-azaadenine, i.e. H2(9,8aPMEA) , and i s 8- isome 8-[2-(phosphonome hoxy)e hyl]-8-azaadenine, H2(8,8aPMEA) , we e syn hesized by alkyla ion o 8- azaadenine wi h a syn hon ca ying he s uc u al ea u es o he equi ed side chain. [32] 2,2’-Bipy idine, 1,10- phenan h oline monohyd a e, and he ni a e sal s o Na + and Cu 2+ (all p o anal si) we e om Me ck AG, Da ms ad , FRG. All he o he eagen s we e iden ical wi h hose used p e iously pal and all solu ions o he po en iome ic pH i a ions we e p epa ed wi h ul apu e CO2- ee wa e as desc ibed, phi 2.2. Po en iome ic pH Ti a ions The appa a us o he po en iome ic pH i a ions, he calib a ion p ocedu e, he compu e s, and he calcula io_n me hods used now a e he_same as in [24]. The s abili y cons an s hm (H.9 8aP4ZA) and K (9 8aP4ZA), whe e M e+ Cu.(Bpy) 2+ o Cu(Phen) 2+, we e de e mined by i _ a ing 30 m_L o aq6ous 0.83 mM [: O3, 0.4 2+ 2+ mM 9,8aPMEA -, and 4.4 mM o 2.2 mM Cu /A m (i.e., Cu :A m:PA 11:11:1 o 5.5:5.5:1) unde N2 (25 C; I 0.1 M, NaNO3) wi h mL 0.03 M NaOH. Each i a ion was epea ed in he absence o ligand and he di e ences in NaOH consump ion be ween such a pai o i a ions we e used o he calcula ions. The condi ions o he measu emen s wi h 8,8aPMEA we e iden ical wi h hose gi en abo e o 9,8aPMEA. I may be added ha he acidi y cons an s K2(PA) and K IH(PA o H2(PA) and H(PA)-, l l + espec i ely, and he s abili y cons an s /Cu(H.PA, and/Cu,PA o he bina y Cu(H;PA) and Cu(PA) ....... I331 complexes we e de e mined unde he co esponding condi ions. Fu he mo e, he abo e condi ions a e simila o hose desc ibed in [24]. Unde he gi en expe imen al condi ions he o ma ion o he Cu(A m) 2+ complexes is+ p ac ically comple e [341 (in ag eemen he ewi h, i a ions o solu ions wi h HNO3 and HNO3 plus Cu /A m we e iden ical in he lowe pH ange) and he e o e, he e alua ion o he i a ion da a o he e na y complexes could be done in he way desc ibed p e iously o bina y complexes. [241 The Cu(Bpy)+/9,8aPMEA 5.5:1 and 11:1 sys ems we e e alua ed in he pH ange 3.4-5.3, eaching o ma ion deg ees o abou 4.5 and 8% o Cu(Bpy)(H;9,SaPMEA) + and 64 o 78% o Cu(Bpy)(9,SaPMEA), espec i ely. Fo he Cu(Phen)Z+/9,SaPMEA 5.5:1 and l:l sys ems da a we e collec ed in he pH anges 3.4-5.0 and 3.4-4.8, espec i ely, eaching o ma ion deg ees o abou 8 and 14% o Cu(Phen)(H;9,SaPMEA) + and abou 61 o 66% o Cu(Phen)(9,SaPMEA). The uppe limi s o he e alua ed pH anges we e always de e mined by he beginning o he hyd olysis o he Cu(A m)a species. Simila ly, he Cu(Bpy)2+/8,SaPMEA 5.5:1 and 11:1 sys ems we e e alua ed be ween p+H 3.6-5.3 and 3.5-5.3, espec i ely, wi h o ma ion deg ees o abou 7 and 12% o _Cu(Bpy)(H;8,SaPMEA) and abou 62 o 76% o Cu(Bpy)(8,SaPMEA), espec i ely. Fo he Cu(Phen)e+/8,SaPMEA 5.5:1 and l:l sys ems da a we e collec ed in he pH anges 3.6-5.0 and 3.5-5.0, espec i ely, eaching o ma ion deg ees o abou 12 and 19% o Cu(Phen)(H;8,SaPMEA) + and 60 o 73% o Cu(Phen)(8,SaPMEA), espec i ely. The calcula ed s abili y cons an s showed no dependence on pH o on he excess o Cu-+/A m employed. The inal esul s o he cons an s a e in each case he a e ages o he e alua ions o i e independen pai s o i a ions. Howe e , due o he low o ma ion deg ees eached o he monop o ona ed M(H;PA) +/- species, he s abili y cons an s gi en o hese complexes mus be conside ed as es ima es. These es ima es we e u he subs an ia ed by compa isons wi h he known [181 alues o he Cu(A m)(H;PMEA) + H complexes by aking he di e en acidi y cons an s (Kz(pA)) in o accoun . 314 Me al Based D ugs Vol. 7, N . 6, 2000 2.3. Spec opho ome ic Measu emen s The UV-Vis spec a o he Cu2+/Phen/9,8aPMEA o 8,8aPMEA sys ems we e eco ded in aqueous solu ion and 1-cm cells wi h a Va ian Ca y 3C spec opho ome e connec ed o an IBM-compa ible desk compu e (OS/2 sys em) and an EPSON s ylus 1500 p in e . The pH o he solu ions was adjus ed by do ing wi h ela i ely concen a ed NaOH and measu ed wi h a Me ohm 713 pH me e using a Me ohm 6.204.100 glass elec ode. Fu he de ails a e gi en in he legend o Figu e 4 in Sec ion 3.5. 3. RESULTS AND DISCUSSION All po en iome ic pH i a ions (25 C; I 0.1 M, NaNO), he esul s o which a e summa- ized below, we e ca ied ou wi h a ligand concen a ion o 0.4 mM and a CuZ+/A m concen a- ions equal o o below 4.4 mM. Unde hese condi ions sel -s acking o he ligands is negligibly small as has been shown [81 o PMEA; he same applies o he sel -associa ion o Cu(Phen) +. [3] This means, he sel -associa ion is negligible o any o he eac an s unde he p esen expe imen- al condi ions and he esul s gi en below ce ainly e e o monome ic species. 3.1. De ini ion o he Equilib ium Cons an s The ligands 9,8aPMEA 2- and 8,8aPMEA 2-, abb e ia ed as PA 2- (Fig. 1), may bind wo p o ons a he phosphona e g oup and one a N1 o he adenine moie y. F om H(PMEA) + he i s p o on is eleased om he -P(O)(OH)2 esidue [] wi h pKa 1.2 and he same may be su mised o he o he H(PA) + species. Hence, o he p esen wo k only he elease o he p o on om he + (N 1)H si e, ollowed by he one om he -P(O)z(OH)- g oup need o be conside ed: H2(PA) +/- H(PA)- + H + (la) /H2(PA) [H(PA)-] [H+]/[H2(PA) +] (1 b) H(PA)- PA 2- + H + (2a) /H(PA) PA2-] [H+]/[H(PA) -] (2b) Indeed, he expe imen al da a o he po en iome ic pH i a ions o he M2+/pA sys ems, whe e M 2+ Cu 2+, Cu(Bpy) 2+ o Cu(Phen) 2+, can be ully desc ibed by conside ing he acidi y cons an s o Ha(PA) +/- (eqs (1) and (2)) and he ollowing equilib ia (3) and (4), M 2+ + H(PA)- , M(H;PA) + (3a) KM(H;PA) [M(H;PA)+]/([M 2+] [H(PA)-]) (3b) M 2+ + PA 2- : M(PA) (4a) /(PA) [M(PA)]/([ M2+] [pA2-]) (4b) p o ided he e alua ion o he da a is es ic ed o he pH ange below he beginning o he o ma- ion o hyd oxo complexes which was e iden om he i a ions o M 2+ wi hou ligand. I should be no ed ha in o mulas like M(H;PA) + he H + and he PA 2- a e sepa a ed by a semicolon o acili a e eading, ye hey appea wi hin he same pa en heses o indica e ha he p o on is a he ligand wi hou de ining i s loca ion. Equilib ia (3a) and (4a) a e connec ed ia equilib ium (5a), and he co esponding acidi y cons an (eq. (5b)) may be calcula ed wi h equa ion (6): M(H;PA) + M(PA) + H + ]-IM(H;PA [M(PA)] [H+]/[M(H;PA) +1 (5a) (5b) P/M(H;PA) P/H(PA) + log KMMM(H;PA)- log KMMM(PA) (6) The equilib ium cons an s acco ding o equa ions (3), (4), and (5) a e lis ed in columns 2, 3, and 4 o Table 1, espec i ely. The acidi y cons an s o he ligands (see oo no e "a" in Table 1) and he s abili y cons an s o he bina y Cu(H;PA) + and Cu(PA) complexes will be discussed in a di e en con ex . [33] He e we concen a e on he p ope ies o he e na y complexes. 315 Helmu Sigel e al. Te na y Coppe (II) Complexes in Solu ion[I,2] Fo med wi h 8-Aza De i a i es o he An i i al Nucleo ide Analogue 9-[2-(Phosphonome hoxy)e hyl)Adenine(PMEA) Table 1. Loga i hms o he S abili y Cons an s o he Te na y Cu(A m)(H;PA) + (eq. (3)) and Cu(A m)(PA) (eq. (4)) Complexes as De e mined by Po en iome ic pH Ti a ions in Aqueous Solu ion, Toge he wi h he Nega i e Loga i hms o he Acidi y Cons an s (eqs (5) and (6)) o he Cu(A m)(H;PA) + Species a 25 C and I 0.1 M (NaNO3) a’b M /M(PA) M(PA) log KM(H;PA log P/(H;PA) A log KCu/A m/PA d Cu(9,8aPMEA) [33] 0.95+ 0.25 3.98 + 0.04 3.8 + 0.25 Cu(Bpy)(9,8aPMEA) 1.4 + 0.25 4.56 + 0.06 3.7 + 0.3 0.58 + 0.07 Cu(Phen)(9,8aPMEA) 1.7 + 0.25 4.81 + 0.06 3.7 + 0.3 0.83 + 0.07 Cu(8,8aPMEA) [33] 1.3 + 0.25 3.68 + 0.06 4.4 + 0.25 Cu(Bpy)(8,8aPMEA) 1.7 + 0.25 4.49 + 0.05 4.0 + 0.3 0.81 + 0.08 Cu(Phen)(8,SaPMEA) 2.0 + 0.25 4.79 + 0.07 4.0 + 0.3 1.11 + 0.09 a The acidi y cons an s o H2(9,8aPMEA) +/- a e pgHH2 9 8aPMEA 2.73 + 0.02 and pKHH(9,8aPWWA 6.85 + 0.02; hose o H2(8,8aPMEA) + a e pKHH2(8 8aPMEA)-" 3.56’4’ 0.02 ahd pKHH(8 8aPMEA)- 6.79 + 0.Ol. 3"3],b b The e o s gi en a e h ee imes’ he s anda d e o o he me.n alue o he sum o he p obable sys ema ic e o s, whiche e is la ge . The e o limi s o he de i ed da a (columns 4 and 5) we e calcula ed acco ding o he e o p opaga ion a e Gauss. The alues in his column a e es ima es (see Sec ion 2.2) d S abili y cons an di e ences calcula ed acco ding o eq.’(9). 3.2. On he S uc u e o he Monop o ona ed Te na y Cu(A m)(H;PA) + Complexes The analysis o po en iome ic pH i a ions only yields he amoun and dis ibu ion o he species o a ne cha ged ype; i.e., u he in o ma ion is equi ed o loca e he binding si es o he p o on and he me al ion in Cu(A m)(H;PA) + species. A compa ison o he acidi y cons an s o H2(9,8aPMEA) +, P/H2(9,8aPMEA) 2.73 an.d P/H(9,8aPMEA)= 6.85, wi h pKCu(A m)(H;9,8aPMEA) 3.7 (Table 1) o he Cu(A m)(H;9,8aPMEA)* complexes e eals ha he p o on in hese complexes mus be loca ed a he phosphona e g oup, since me al ion coo dina ion mus gi e ise o an acidi ica ion, [36,371 which amoun s o A pK a pKIH(9,8aPMEA)- pKCu(A m)(H;9,8a+PMEA) (6.85 4- 0.02) --(3.7 + 0.3) 3.15 + 0.3 in he p esen case. In he Cu(A m)(H;8,SaPMEA) species he p o on is also clea ly bound a he phosphona e g oup hough he acidi ica ion due o he me al ion migh be somewha less p onounced: A pK a PH(8,SaPMEA)- P/Cu(A m)(H;8,8aPMEA) (6.79 +/- 0.01) (4.0 +- 0.3)=2.8+0.3. Whe e is he Cu(A m) 2+ uni loca ed? In p inciple, he e a e wo possibili ies" One, whe e Cu(A m) 2/ is s acked wi h he pu ine sys em o H(PA)-, designa ed as [Cu(A m)/(H;PA)]s, and ano he one, whe e Cu(A m) 2+ is coo dina ed ei he o he N1/N7 si es o he adenine esidue (see [24]), [H;PA’Cu(A m)]a+de, o o he phosphona e g oup which al eady_ ca ies he p o on. Howe e , he o ma ion o his la e species wi h bo h he p o on and Cu(A m) + a he phosphona e g oup is unlikely, in ag eemen wi h p e ious conclusions. [241 Hence, we a e le wi h he species [H;PA’Cu(A m)]a+de and [Cu(A m)/(H’PA)] +, s and we ha e o conside he in amolecula equilib ium (7): [H; PA.Cu(A m)]a+de [Cu(A m)/(H;PA)]s (7) An e alua ion ollowing exac ly he ou e desc ibed in [2] leads o he conclusion ha o all ou Cu(A m)(H;PA) + sys ems he s acked species in equilib ium (7) domina e wi h o ma ion deg ees o mo e han 70%, mos likely being be ween 80 and 95%. As one migh expec , he o ma ion deg ee o [Cu(Phen)/(H;PA)]s is abou 10% la ge han he one o [Cu(Bpy)/(H;PA)]s. 3.3. P oo o an Inc eased S abili y o he Mixed Ligand Cu(A m)(PA) Complexes [38391 The s abili y o mixed-ligand complexes may be quan i ied by conside ing equilib ium (8a); he co esponding equilib ium cons an is calcula ed wi h equa ion (9). Cu(A m) 2+ + Cu(PA) .... Cu(A m)(PA) + Cu 2+ 10 zx log KCu/A m/P A [Cu(A m)(PA)] [Cu 2+] [Cu(A m) 2+ [Cu(PA)] (8a) (8b) A2u(A m) --log/C(PA) A log KCu/A m/P A log "Cu(A m)(PA) (9) 316 Me al Based D ugs Vol. 7, N . 6, 2000 Acco ding o he gene al ule o complex s abili ies, K > K?, equilib ium (8a) is expec ed o be on he le side wi h nega i e alues o A log KCu/A m/P A, in ag eemen wi h s a is ical conside- a ions, [38’39] i.e., A log Kcu/s a is -0.5. [39] F om e alues lis ed in column 5 o Table i is e iden ha equilib ium (8) is signi ican ly displaced o he igh side. Mo e impo an , howe e , is a compa ison wi h he esul s ob ained o he dianion o phosphonome hoxye hane (PME:-), CH3CH2-O.CH2-PO-, which e lec s he p ope ies o he side chain o 9,8aPMEA 2- and 8,8aPMEA’-- (Fig. 1). Indeed, hese esul s, A log KCu/B ) /PME 0.13 + 0.04 and A log Kcu/Phen/PM E 0.17 + 0.05,[311a e conside ably smalle han he alue s o A log KCu/A m/P A o he Cu(A m)(PA) complexes, which means ha he adenine esidue con ibu es o he s abili y o he Cu(A m)(PA) species. This compa ison hus p o ides he i s clea hin o he occu ence o an in amolecula s acking in e ac ion in hese la e men ioned complexes. Ano he way o e alua e he inc eased s abili y o e na y Cu + complexes, independen ly o he p ope ies o he bina y Cu(9,8aPMEA) and Cu(8,8aPMEA) species, es s on he p e iously es ablished [2’311 s aigh -line co ela ions o log KUu( mm) RPO e sus p/ po) plo s (eqs (10) c )( 3) and (11)), whe e R-PO- ep esen s phospha e monoes e o phosphona e lgands in which he esidue R is unable o in e ac wi h Cu(A m)Z/: log Ko u(Bpy) 0.465 x Cu(Bpy)(R-PO3) P/H(R-PO3) + 0.009 (10) log zCu(Phen) "’Cu(Phen)(R-PO3) 0.465 x PKH(R.PO3 + 0.018 (11) The e o limi s o log s abili y cons an s calcula ed wi h gi en p/,, alues and equa ions H(R-PO3 (10) and (1 1) a e +0.07 and +0.06 (3) log uni s, espec i ely, in he pH ange 5-8.[ 311 The e e ence lines as de ined by equa ions (10) and (11) a e shown in Figu e 2, whe e he s abili y cons an s log Ko u(A m) e sus he acidi y cons an s pK o he 9,8aPMEA and Cu( m)(P 8,8aPMEA species a e also pAl o e, oge he wi h he co esponding da ’a l] o he Cu(A m)(PME) sys ems. All hese da a poin s a e abo e hei e e ence lines, p o ing an inc eased complex s abili y which mus mean [41 ha aside om 2+ he Cu(A m) -phosphona e coo dina ion u he in e ac ions ake place. The e ical dis ances in Figu e 2 be ween he da a poin s due o Cu(A m)(9,8aPMEA), Cu(A m)(8,8aPMEA) and Cu(A m)(PME) and he e e ence lines a e a measu e o he ex en o he in amolecula in e ac ions in hese complexes and hey can be de ined acco ding o equa ion (12) (in his case, PA 2- also ep esen s PME2-) log ACu/A m/P A log ,.’Cu(A m) log ,.’Cu(A m) ’Cu(A m)(PA) a"Cu(A m)(PA)op (12a) k,-Cu(A m) log k,Cu(A m) log ’"Cu(A m)(PA)exp l "Cu(A m)(PA)calcd (12b) u(A m) u(A m) The exp essions log (eq (12b)) and log Kc u A m PA o (eq (12a)) a e synonymous /u(A m)(PA)calcd p because he calcula ed alue equals he s abili y cons an o he lopen’ isome , Cu(A m)(PA)o,, n 2 2+ which only a -PO3-/Cu(A m ) in e ac ion occu s. The i s e m on he igh hand side in equa ion (12) is he expe imen ally de e mined s abili y cons an (e . (4)), whe eas a alue o log K(2 /!. A m), can be calcula ed wi h he acidi y cons an P/’"PA’ and he s aigh -line equa ions (1--0u V(l]ai"As indica ed abo e, such a calcula ed alue ha { i ies he s abili y o he open isome . The ligand PME 2- o e s o me al ions he phosphona e g oup o coo dina ion, bu an in e - ac ion wi h he e he oxygen is also possible as has epea edly been p o en. [822’41’421 This gi es ise o 5-membe ed chela es and he e o e equilib ium (13a) needs o be conside ed" H O ROCP, 0,, %"M2+ R-- 0,,,, H 0 %M2" (13a) 317 Helmu Sigel e al. Te na y Coppe (II) Complexes in Solu ion[I,2] Fo med wi h 8-Aza De i a i es o he An i i al Nucleo ide Analogue 9-[2-(Phosphonome hoxy)e hyl)Adenine(PMEA) K [Cu(A m)(PME)c]/[Cu(A m)(PME)op] (13b) The dimensionless equilib ium cons an K (eq. (13b)) is calcula ed acco ding o equa ion (14), K 10 lg aCu/A m/PME (14) 5,0- 4,8- 4.6- ’E 4.4- OO 4.2 4.0- 3.8 3.6 O 3.4 E z m 2.8 2.6 2.4- 2.2 8,8aPMEA2- O0 9,8aPMEA 2" , PME 2- "’,Cu(Bpy)(R.PO3) (Phen)(R-PO 3) 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 H pKHH(R.PO3) o PKH(PA Figu e 2. E idence o an enhanced s abili y o he e na y Cu(A m)(9,8aPMEA) Cu(A m)(8,SaPMEA) ((C),O), and Cu(A m)(PME) (A,,) complexes based on he ela ionship be ween log /,,_ u( Ann u(Ann)( -PO3) o log "Cu(Ann)(PA)and PK(R-PO3) o pKIH(pA) in aqueous solu ion a I 0.1 M (NaNO 3) and 25 C. The plo ed da a o 9,8aPMEA and 8,8aPMEA a e om Table and hose o PME om [31]. The ASu(Ann) wo e e ence lines ep esen he log Cu(Ann)( .po3) e sus P/(-PO3) ela ionship o e na y Cu(A m)(R-PO3) complexes (eqs (10) and (11)); R-PO- symbolizes phosphona es o phospha e monoes e s in which he g oup R is unable o unde go any kind o hyd ophobic, s acking o o he ype o in e ac ions, i.e. ligands like D- ibose 5-monophospha e, me hanephosphona e o e hanephosphona e. [31 The b oken line holds o A m Bpy and he solid line o A m Phen. Bo h s aigh lines ep esen he si ua ion o e na y complexes wi hou an in amolecula ligand-ligand in e ac ion. The e ical do ed lines emphasize he s abili y di e ences om he e e ence lines; hey equal log ACu/A m/P A as de ined in equa ion (12). and now, knowing K, he pe cen age o he closed isome , Cu(A m)(PME)c, in equilib ium (13a) can be ob ained wi h equa ion (15)" % Cu(A m)(PME)c 100. K/(I + KI) (15) 318 Me al Based D ugs Vol. 7, N . 6, 2000 Table 2. Quan i ica ion o he S abili y Inc ease ia loe ZCu/A m/P A (eq. (12)) o he Cu(A m)(PA) Complexes, whe e PA 9,8aPMEA 2-, 8,8aPMEA 2- o PME "-, and A m Bpy o Phen, Toge he wi h he Ex en o he In amolecula Chela e Fo ma ion (eq. (13)) in he Cu(A m)(PME) Species [311 in Aqueous Solu ion a 25 C and I 0.1 M (NaNO3) a log 1o Cu(A m)(PA) Cu(Ann) b Cu(Ann Kia Cu(Ann)(PA)exp l Cu(Ann)(PA)calcd log ACu/Ann/P A %Cu(A m)(PME)cl Cu(Bpy)(9,8aPMEA) 4.56 + 0.06 Cu(Phen)(9,8aPMEA) 4.81 + 0.06 3.19 + 0.07 1.37 + 0.09 3.20 + 0.06 1.61 + 0.08 Cu(Bpy)(8,8aPMEA) 4.49 + 0.05 3.17 + 0.07 1.32 + 0.09 Cu(Phen)(8,8aPMEA) 4.79 + 0.07 3.18 + 0.06 1.61 + 0.09 Cu(Bpy)(PME) [31] 3.86 + 0.03 3.27 + 0.07 0.59 + 0.08 2.89 + 0.68 74 + 5 Cu(Phen)(PME) 31] 3.90 + 0.04 3.28 + 0.06 0.62 + 0.07 3.17 + 0.69 76 + 4 Fo he e o limi s see oo no e ’b’ o Table 1. These alues a e om column 3 in Table 1. These cons an s we e calcula ed wi h eqs (10)o (11) and he H pK(pA alues gi en in oo no e ’a’ o Table 1. See eqs (13b) and (14). Calcula ed acco ding o equa ion (15). F om he esul s gi en in he lowe pa o Table 2 i is e iden ha he closed isome o Cu(A m)(PME) is an impo an species wi h a o ma ion deg ee o abou 75%. Na u ally, he o - ma ion o he co esponding isome in ol ing he e he oxygen is also o be expec ed (see Fig. 1) o he Cu(A m)(9,8aPMEA) and o Cu(A m)(8,8aPMEA) sys ems and we designa e i as Cu(A m)(PA) o. Howe e , he log ACu/A m/P A alues lis ed in column 4 o Table 2 a e by abou 0.7 o log uni la ge o he la e men ioned complexes han o he Cu(A m)(PME) species and his mus mean ha in he sys ems wi h 9,8aPMEA and 8,8aPMEA, nex o Cu(A m)(PA)op and Cu(A m)(PA)l/o, a hi d isome mus occu which in ol es he adenine esidue. The ligands 9,8aPMEA z- and 8,8aPMEA 2- o e only wo such possibili ies" The phosphona e-coo dina ed Cu(A m) 2+ o ms (i) a mac ochela e wi h one o he ni ogens o he adenine esidue, o (ii) an in amolecula s ack be ween he a oma ic ing sys ems o Bpy/Phen and he adenine moie y. Tha he i s possibili y is no o ele ance has been discussed in de ail o 3’-deoxa-PMEA, [21 and he same a gumen s also apply he e, whe eas o he second possibili y in ol ing in amolecula s acks, many examples exis . [2,28,30,31,39’40’43] Hence, he addi ional enhanced complex s abili y may be a ibu ed indeed o in amolecula s ack o ma ion. Applica ion o space- illing molecula models e eals ha he adenine esidue o he 9,8aPMEA o 8,8aPMEA ligands, which a e equa o ially chela ed o Cu(A m) 2+ ia he phospho- ha e g oup and he e he oxygen, canno s ack well wi h he a oma ic ings o he also equa o ially coo dina ed A m; a subs an ial and s ain- ee o e lap o he a oma ic sys ems is only possible i he e he oxygen is no equa o ially coo dina ed o Cu e+. This la e si ua ion is depic ed in Figu e 3 o 9,8aPMEA. Howe e , om he molecula models i is also e iden ha an apical e he oxygen coo dina ion and simul aneous s ack o ma ion would be compa ible wi h each o he in he Cu(A m)(PA) species wi h PA 2- 9,8aPMEA o 8,8aPMEA. Hence, he e a e a ious in amole- cula ly s acked Cu(A m)(PA) species possible including hose wi h somewha di e en o ien a ions o he a oma ic ings owa d each o he . As he e is a p esen no way o dis inguish hese a ious isome s and con o me s om each o he , we ea all he s acked species oge he and designa e hem as Cu(A m)(PA)s . The sum o he abo e easonings hen gi es ise o he equilib ium scheme (16), whe e he pu e phosphona e-coo dina ed isome is designa ed as Cu(A m)(PA)op. I is e iden ha he uppe b anch o his equilib ium scheme e lec s equilib ium (13a) while he lowe b anch e lec s he s acking in e ac ion (Fig. 3). Cu(A m)(PA)c,o Cu(A m) 2+ + PA 2- Cu(A m)(PA)op Cu(A m)(PA). (16) 319 Helmu Sigel e al. Te na y Coppe (lI) Complexes in Solu ion[I,2] Fo med wi h 8-Aza De i a i es o he An i i al Nucleo ide Analogue 9-[2-(Phosphonome hoxy)e hyl)Adenine(PMEA) H2 NV7 x /,OH ’ H2! O Figu e 3. Ten a i e and simpli ied s uc u e o a Cu(Phen)(9,8aPMEA) species wi h an in amolecula s ack. The o ien a ion o he a oma ic ings may a y among he s acked species; such a s acked complex in solu ion should no be conside ed as being igid. 3.4. E alua ion o he In amolecula Equilib ia In ol ing Th ee Di e en Cu(A m)(PA) Species Based on he equilib ium scheme (16) he co esponding equilib ium cons an s can be de ined as gi en in equa ions (17)-(19): log "R2u(A m)(PA)opk’Cu(A m) [Cu(A m)(PA)op]/([Cu(A m)2+] [pA2-]) (17) KI/o [Cu(A m)(PA)cl/O]/[Cu(A m)(PA)op] (18) Kl/s [Cu(A m)(PA)s ]/[Cu(A m)(PA)op] (19) Wi h hese de ini ions he expe imen ally accessible equilib ium cons an (4b) can be e o mula ed as equa ion (20): [20a,31] [Cu(A m)(PA)] (4b) (A m)(PA) [Cu(A m)2+ [pA2_] ([Cu(A m)(PA)op] + [Cu(A m)(PA)cl/O] + [Cu(A m)(PA)s ]) [Cu(A m) 2+ [PA 2- /(Cu(A m) k-Cu(A m) + Kl/s " "’Cu(A m)(PA)op k,Cu(A m) + KI/O "’Cu(A m)(PA)op "xCu(A m)(PA)op (20a) (20b) k’Cu(A m) "’Cu(A m)(PA)op (1 + KI/0 + Kl/s ) (20c) F om he e one a i es easily [31 a equa ion (21), whe e Cu(A m)(PA)in / o e e s o he sum o all he species wi h an in amolecula in e ac ion: Cu(A m) Cu(A m)(PA) 10 Ig ACu/A m/PA (21 a) K Kl/ o ,Cu(g m) "’Cu(A m)(PA)op 320 Me al Based D ugs Vol. 7, N . 6, 2000 [Cu(A m)(PA)in / (21 b) /’(i Kl/ o [Cu(A m)(PA)op [Cu(A m)(PA)cl/O] + [Cu(A m)(PA)s ] [Cu(A m)(PA)op (21c) KI/0 + Ki/s (2 d) In hose ins ances whe e he s acked species do no o m, he abo e equa ions educe o he wo- isome p oblem ea ed in equa ions (13) and (14). I is e iden ha /I Kl/ o acco ding o equa- ion (21a) can be calcula ed ia he alues log ACu/A m/P A as de ined by equa ion (12) and lis ed in he uppe pa o column 4 in Table 2. Table 3. ln amolecula Equilib ium Cons an s o he Fo ma ion o he Th ee Di e en ly S uc u ed Cu(A m)(PA) Species Shown in he Equilib ium Scheme (16), Toge he wi h he Pe cen ages in Which These Species Occu in Aqueous Solu ion a 25 C and I 0.1 M (NaNO3) a No. Cu(A m)(PA) log ACu/Ann/l:, A g ’= KI/ o %Cu(A m)(PA)in / o %Cu(A m)(PA)op a Cu(Bpy)(9,8aPMEA) 2a Cu(Phen)(9,8aPMEA) 1.37 + 0.09 22.44 + 4.86 95.73 + 0.88 4.27 + 0.88 1.61 + 0.08 39.74 + 7.50 97.55 +/- 0.45 2.45 + 0.45 3a Cu(Bpy)(8,8aPMEA) 1.32 + 0.09 19.89 + 4.33 4a Cu(Phen)(8 SaPMEA) 1.61 + 0.09 39.74 + 8.44 No. Cu(A m)(PA) K I/O Ki/s 95.21 + 0.99 4.79 + 0.99 97.55 + 0.51 2.45 +/- 0.51 %Cu(A m)(PA)el/o b %Cu(A m)(PA)s b Cu(Bpy)(9,SaPMEA) 2.89 +/- 0.68 19.55 + 4.91 2b Cu(Phen)(9,SaPMEA) 3.17 +/- 0.69 36.57 +/- 7.53 3b Cu(Bpy)(8,SaPMEA) 2.89 +/- 0.68 17.00 +/- 4.38 4b Cu(Phen)(8,8aPMEA) 3.17 + 0.69 36.57 +/- 8.47 12.3 +/- 3.9 83.4 +/- 4.0 7.8 +/- 2.2 89.8 +/- 2.2 13.8 +/- 4.3 81.4 +/- 4.4 7.8 + 2.3 89.8 + 2.4 a The alues lis ed in he hi d column o he uppe pa a e om he ou h column in he uppe pa o Table 2. The alues o K’ Ki/ o ollow om eq. (21a) and %Cu(A m)(PA)in / o is calcula ed analogously o eq. (15). The alues gi en in he six h column o %Cu(A m)(PA)op ollow om 100 %Cu(A m)(PA)in / o . The cons an s o K o in column 3 o he lowe pa a e om column 5 in he lowe pa o Table 2 ( o he co esponding jus i ica ion[ 311 see also ex in Sec ion 3.4); wi h eq. (21d) and he now known alues o K’ and KI/0 ha o Kl/s may be calcula ed (column 4 in he lowe pa ). All e o limi s co espond o h ee imes he s anda d de ia ion (3); hey we e calcula ed acco ding o he e o p opaga ion a e Gauss. b These alues we e calcula ed ia eq. (18) wi h K o and %Cu(A m)(PA)op. The alues o %Cu(A m)(PA)s ollow om he di e ence %Cu(A m)(PA)in / o - %Cu(A m)(PA)cl/o (c . eqs (21b) and (2 c)); %Cu(A m)(PA)s may also be calcula ed ia eq. (19) wi h K s and %Cu(A m)(PA)op. The esul s a e he same o bo h calcula ion me hods ye he e o limi s a e unde s andably la ge o he second me hod (da a no shown). The esul ing K alues a e gi en in he ou h column o he uppe pa o Table 3 and hey allow o calcula e he concen a ions o he open isome s, Cu(A m)(PA)op. To be able o calcula e he o ma ion deg ee o he species ha o m he i e-membe ed chela e wi h he e he oxygen, i.e. Cu(A m)(PA)c/O (eq. (18)), we made he jus i ied assump ion ha Cu(A m)(PME)c (Sec ion 3.3; Table 2) I31 and Cu(A n)(PA)cl/O ha e he same s abili y, i.e. ha he equilib ium cons an K o o Cu(A m)(PA)cVO equals he co esponding alue (= K) o Cu(A m)(PME)c. Knowledge o K and K/o pe mi s now o calcula e K/s by using equa ion (21d) and hence he o ma ion deg ee o he Cu(A m)(PA)s species. Finally, he di e ence be ween 100 and he sum o he pe cen ages o Cu(A m)(PA)op and Cu(A n)(PA)c O will, o cou se, also esul in % Cu(A m)(PA)s and Ki/s . The esul s o hese Calcula ions a e summa ized in he lowe pa o Table 3. Conside ing he equilib ium scheme (16) and he co esponding esul s summa ized in Table 3 se e al conclusions a e e iden : (i) All h ee s uc u ally di e en species a e o med in app eciable amoun s in he Cu(Phen)(9,8aPMEA) and Cu(Phen)(8,SaPMEA) sys ems. (ii) The s acked species (Fig. 3) clea ly domina e, eaching o ma ion deg ees o abou 80 o 90%. (iii) Consequen ly, he o ma ion deg ee o he i e- nembe ed chela es in ol ing he e he oxygen is supp essed, oughly speaking o abou 10%, compa ed wi h he app oxima ely 75% p esen in he Cu(A n)(PME) sys ems (c Table 2). 321