Synthesis and Pharmacological Actions of 5 Adenylmethylenediphos

Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1959 Synthesis and Pharmacological Actions of 5' Adenylmethylenediphosphonate: An Analog of APT James Wendell Flesher Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Medicine and Health Sciences Commons Recommended Citation Flesher, James Wendell, "Synthesis and Pharmacological Actions of 5' Adenylmethylenediphosphonate: An Analog of APT" (1959) Dissertations 568 https://ecommons.luc.edu/luc_diss/568 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons For more information, please contact ecommons@luc.edu This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License Copyright © 1959 James Wendell Flesher SYiltHfSIS MID PHAJVtf\C£'JLOOlr.A.L J\!rflnm Ul~ S A':D~laU1l::TllfLFJJErIPHOOPH1)NA'i'E A}; AJ!,U.OO A1P bI' Jamoa ft'.~ll l"lHOOr A d1uel'tat1on Suta1 ttod to t,he Faoul ot the Ol'oouatG School of Loyola !jn1~ty in Partial Ful!'1.11l'llOnt th~ Requ.iJ!lIl!rn(!H'lt& for the Degree ot Decto!' of Ph1lo8opl~ ot The autbor was b!CIrn on June 24, 192$ in Chicago nUnois I wh;:%'e he had h1s prtu.ry and eecond.a.ry fttifueatlon He spent three years in t1" Anq L"ld Air ?oroe during World "'jar II Ue took M.e E'.S degree in 191&9 frClll Northftu1iern University From 19$0 to 19$1 he 'ffU • R• •arch technioian at U" Institute of t'adlobiology and 31opt4"51ctf, U%d.veralty hie ~-:rad1late or Chicago In 19>1, he began work at ! c:ry'ola, and wu awarded l'Wter of Soience deuee in June 19SL From 19$1 to 19S5 he wu a control and refining chemist for lever arothere 1955 C~ in the F;dible Fate and 0118 dlvi.ion Tn he bee rU8M'Cb teahniclan inc tbe DepartMnt of' Biological Cheal.try, Universit.y of 1111n!lu He 18 a mtmber of tr e Alllerican A.S8OC iatl em tar the Advaooe_nt of' SeilJl'lOe 111 ! wish to expreu ntf far help received t:lVer' 'fo 'Or Y If the appl'G01atl~ c~ ~t to a mltlber of individuals or tJ-J investigation for hi continu.ed i.nt.erest and encourage- ment as adviser, to Dr T C 1"fyen tor directing the eh$!dca.l 9t1.1'c1108, and to M!lherS of the fJepartMnt or ?ha'nlr.acology for valu.a'ble conoerning these st11diee, the author 15 deeply grateful 1" sU!!,r;_tit)Q8 • phosphatee • • • H:tpothuia tor the ~cb&n~ cf act.lon of adenoa1M der1vat1vee • • • • • • 'Abe l."'lP01"t.ancs of armlQg6 ot natural metaboliWe • • to~olo'l1 Analogs of nucleoside phoepbatos • It Bta~nt of the probleD D BackRl"OW'ld of the problem • " • • of adenoei!w Introduct.ion w t.llt' p~{)logy ) It ' It • " • • e • • 10 • 11 ae1rl anaJ.ogfJ of nucleoside pnOliJpnaW8 s l>hoepllOnic Properties oonpU"ed with phosphates \")i G !!etbod r>t appJ:"Oach A.?~ol~ 1cal mood t,.:, UW problem • • •• • 12 13 methods ~ure l."'eBp1rato1"1 eh~ ~)hO$'f:'honatos 1 and @loctroeardiograph1c ," ~th ""1\.016 e:t"'tecta • •• • • • • • • ) Striated l'!fw.H31o ef'fect ~., • •• • • 'I " Ih };r1cUtatlng ~b~ prep~t.ton • • • • • B Method of may for Hemk:tnase iitud1ea • • • • C Syntbet10 Pbosphon1a acid malog of Ii TP (tƠPe n} ã ã ?hoaphom.c acid analog of um l (type I) ) ChaNcttmation and datem1nnt1on of propert1f'4 2l 22 of ATP analog • • • • • • • • • • • • 22 III RESULTS J\ Pha.mAcologto 'i'he ~()1'" .3 rotJpOrlSfJ 1, erf~ot on th8 ~~1rat1on • • • 'i'he eiectrocardto"rAPh:1c ohlmp& in the cat • v '\ ?~ nrrect on 1so1n:l>~d rabbi t il&UFI1 'i1~ (ltt't'et on the tib1n11a anticus preparation • • Nictitating ~brano preparaGion • • • •• Inhibl t.inn of heXokinasll1 Y.'f\'Actton by analog • • • • • • •• s n 44 Sh 56 S8 C ,fynt hetic P~t1,on and Pl'tl'IXI!'t1es of S· adenyl'M8thylen.ed1phoa~te PN'pal'ation of urid1na S' tbylened1pb~bonate •• IV • • • Pbarmaeologto • • • • • • Synt.het1e • • • • • • • • • 'DISCUSS!ON OF lU:SUL1'S • • • • • • • • • • • • • •'" • 61 6It 61 •• 61 12 7S 77 Aj:':F1\NDrx • • • • • • • • • • • • • at A lJ v •• • • · • 10 • • II · • I II COKPARUFm >,F THE D9t~DIA'll: li)fRLSSJR AC7IVI'1'lES OF VJillIOUS aJRIJ"), rLRrlA'tIVf~; IN 1'if' CAT • • • • • " IN m:.oon P':{n;r;UR IN A CA'T AN1SS',('H!''!'IZID;: WI:m Nj,}U)U'rAr IHT11AVb nous n~JlJJ'rrl)N ;~}i' All' Aim S AJENILt 7::tYr1',llEfIFH,l)f'IiUNA m " " •• " FAI.1~ lUi'''l''~,:tl III IV v• VI 27 lJtCR'.IiSt: IN MJX1};; f-1i:f>SbtJID IN OATS A1ID DUOS AtiF 5·t'Hl~T!imD HI'£11 Nl:lWTAL I'Jt~(fR IUl'!tAVF!!!ij{, 1l4JhC'i'! INJF t1'UE Al.!E1i·!l3!Nt PHOSPIiAT1:S Am} Al'a,V::O • • • • 26 VAS~Ji:f;Htr,0S:'ll Ri:,f;·.OJ£; 'If' A!'1P AliJl{i; :Ja IN cmW.Uu\T.Fm lfi17l4 H£,:,un ~,"1I?rI11K1;'lf'if.)tau 1£ m L~ THE CAT " " 3h (I" 91P, If \1M, JiJ111'1: Aun !N OCMU!U,\'1!ON WITU l'"~·:,~t'«U~U'H(%Wit)NIC ACID, q uM, (it FYROP.!Fl')i"HAtl!; III 'llIE CAT " • • " • • • 35 V/~ot:t.Wm:SSRlSSOR Rl:5POlIm va~us Ttl!:: TJJG !~a::i' A,j''A''1J't Al'Ơ"' Alit AJiAl'J.'Xl ã ã • , , • , •• 2.S , ) Ot.JMPARJS01t ,J}" T"l£ KO'lliiCI (Hf' 'l1:lE AriALOO AIID ATP lIWORF; Al4L ,JU;"'u,R 'Hit INY~'RA~H(JUS llUEC'rION f)}'" A'.rn.Ofm& , 32 CJf'ri~ARI.EO~£lF ram POt.J:',t>r.Y f,.nr T'~ IiNALOl A1~ ~ ATP l'£f'onr: AIID AF1'Uf 'il~ IlffRAVIDUUS· !N~1ION OF mOOTI01iOS , • • • • , 31 s F.ESP1Jf l\'rn'RY ftJi!) VASaU~l'R ~':~ Rt!"I:O.!'l.il', Al'1,,}';R lUJU; t 'IOt 0 "1l!o1o.g1eal standardisat.ion" » -a Ued., J2tI.29 adl N.T AGad Ozford Medical Publicati_ Uurlbert and V R Potter 195'2 Anion uc.hange cbrccuii.ographT J aiel Chem 1S161 717 of ac1dfll of the citric acid cycle 77 - 18 catt.tarow, A and B S,chepart,a 19$1 Bloch.ai.try, _.conci edition W B Sauader COIIp8D1' Cla1"ke, D A., J Dava11, F S PblUr- and G B Brown 1952 lnayaatl0 deaII1nation and vuodepreuOl" trect of acienOline anal• • J 'Phana Expt Ther., 106, 291 - 1936 The plv8101og1cal 8Ot1 ,1t7 £1" Ph7alo1 R ,., Jel or nucleio acid aDd it' deriva- 292 Jlr\JJ'7, A N and A S~orl11 1929 The phy8iologioal activity of adenine oOllPO\IDds with ,,.cUl0 reference to t.he1r action up_ the u.n heart J Pbplo1., ~ 213 E_l1n J K and Vi '.ldbel'l 1,48 8ptemio effect of ATP and Cbeaotb., J* 273 Srit J Pbaral Euler, H., U S Iul.er and F 5ehlenk 19laO tUX' Pbarmakolope del' Cos, •• Aota Pbyeiol $oand., 1, 19 - IIf1ng, P L and F Sob1.nIt 191.) Some ~olog1oa.l actlema of acleni thlometbyl J Pbarta ~~ Ther., 161 , pent nt 'eldberg, W and C Heb'b 191.8 The at111Ulatlng action or pht'JIpbate oOllpoundlJ Oft the pertueed superior oervical ganglion of the oat J Pb1e101., !2II 210 192; Dfltennnation of total acid 801ut>le pnOffphoroue as doacribed in ~, Qatar , SUIBIIN'IOA, p S81, 12th edition 111.ak18tOl1 C0IIlPfUlT F1.skwSubbarow, GaddWlt, J H and P ,Holtz 1933 Tbe local.iH.tion at t.he action of dl'Up on the pulllonary " of cop and oaw J Pbplo1., III 139 Gllle1pl., J H 19lh The biologioal 1gnificance of the llDkape in adeno,:lne v1phOftpho.r1c acid J Ph;ysiol., 50 345 - 19 Gornall, A a., C J Baraill and tI I Davld 19b8 net.el'llinAtion or aerua proteina by means of t1'- biuret reaction J Biol Chela., 7Sl .m Often, U N and H n Staner 19$0 The 8iolopcal Aotiou 01 the Aden1De Nucleotide B X Imrie & C~ }litchi• • 0., E }laren, t 'alco and B VandGrwerrf Ae Cl••• Soc., BOIkln, F new York, 1947 Abatraote N.T c 19$6 SOMe ob4iIeJ"Y.tiona coneerain;: the bioolMmdc:al inert Mth,ylpho.phon1c: acid and 1aopr!pYl _thylph08pbonic aclda Can J 81ochelll and Pl\Y'8'101., 743 or al rill, 19S5 H i "Introduction to ChelI1cal PhanlaOology By R Barl_ 1thorana, G G 19S4 Carbod11l1dd v novel synthes1s of ADP, ATf and P, pt diadenoalne pyrophoaphate J Am Chem Soc., 76 lSl7 it_alopoN' n If 19S0 "OrgaMPhCl8phorous C_ponnde"'O 19lah raomerint1.on - J X•• Cn Soc,., 66, 109 or - Wiley It C~, H.I tri-alJql pbcaphatee Kopparv-1, T and A G K'aroSlllU' 19h9 f!:aeri.ne-llke activity of ad.ql1e acid and of water loluble AT' A•• J Phyeiol., Yft Sl6 - 1'Arr1ne, P A and H S Tipon 19.34 Preparation of [Jl:)noacet·one urldine J Blol Cham., lC6 113 ~ Lohmann, K 1929 tfuer die Pyl"ophoBphatet,.8ktiol'l 1.111 ~~U8kel lIJa\urwisa., !I 62L Marcou, E 19)1 Sur l'adenosine et san action hypotensIve COI'af,It Rend Soc Mol., 109 188 - Martell, E E and G Schwar.enback 19S6 Aden08ine ph08phat.e and tripboepba\ ala Caplexbildnel" fur CalciUlll urad kpe:1.u Hely Chim Acta, 39 69 McDowall, 11: S J ~chich, g., - 19L4 Quoted frQlll Bie18cnowsky et aI, 19h6 D A CIU"lr and i S Philip' on teolated int.••tine and utel'U8 19Sh Etteot of' adenOlline anal J Pbarm Espt Ther 111e 3lS - t.ttorrell, and R M Bock Abetl"acW Am Chem Soo., 126th 1Aeetlng, Hew York D1vis1on or Biological Chemistry, p 44 c 19.30 StudieD uber i)l"gan38cbe.n Pboephorrel"blD4ungen Wylen, Dl •••rtatiorh tlprNJula Parikh, J R and A 3ur." 195$ Analoge of nucleotld I ThwophylU",lalkyl phoephon1o acids and relate4 c~ J AMI' Chea Soo., 2)86 n' 19S1 ADalop or nucleoM.du II Pbosphonate awn on 1"1 boee and glucopyn.nose purIne cieri vativee Parikh, J R•• M E Woltt and A.Bur~ J • AMI' Chem Sao., Pauliq, L Rud, M., 19h8 The :'tiIture 12' 2718 or the Chu10al Sonde Cor Il UDivers1t.y Pros A Skff'Ot"d and l H TMrp 1955 The potentiatlon r4 t.he action of adel'u.:tne on the gu1_ pig heart by Ml.tbaln J Pbana Esp Ther lllu 119 a .cw, r and 51f1owko-elien 1916 Some replariti_ il1 the 8ert of phoephoroue cumta1rdng _ida Zbur ObMbei Kbim.!2t 22)) (C.A.,at A 1810,) Schm:ldt 1928 m~r fermentative ?bPiol el-*il., !Z2t lid Scftatdt E., and M s-rel.dtu' Ann 8) ma 19S1 tltM.lI1n:1~ 1m Muskel Zeltaebr r Zur rentltn1a AUphatiseher Carbodiim1de Schwanl, ~f and T c ltren 19S8 Simple micrometr1c cONItant-pH _\hod tor eocur e~ , Anal." ~hem., 1.2' 11S0 ., 19SO Die Pyro und die llnt.ervhC*pbonaure Vergleich mit orpni8cban DipfioophOQN.\U"en ~onat.sob., 81, 2al 8obwanellblllCk, G and J 2.uro Skipper, H 11: arr - 19S3 A rev!cnu On the IIIJOhar11tIta of action ot oertain tnp_ anti oancer agent CIA R".,l1' ShS Salth, R V anrt R A AJ.bert., 19$6 The apparent, atabil!ty conatant.8 of ionic CIIIIPla•• ot 'Varieu adcmceine ph08phatea with biTal@lnt 06t1_ J AlIeI' Cbft eoc., 18c 2316 - Smith, R 1I and R A Alberty 19;6 Tbe apparent tIItabilit.y conetanta of ionic oO!llPl .u of 'Var1~ adenoe1ne ph_pbatea with moncwalent 0_\1_ Todd, A i., I lrumd, V M Clark and R %1 nall 19$2 Nucleotic1 tv A ynthee13 o.f U2"idine $' P1l"ophoephate~ a breakdO'llm product of the o~ "urid.1ne-Upheephate glueose" J Chela Soo., 266S 81 Wqne, E J t J f Ooodwin and ~} ;1toner 1949 The effect .r AtP OIl the e1eatroov-rdiegram or man and al'dala Ueart Journal, U SSe - 1Jedd, A•.~~ 19)1 The actIon of adenosine and Oenai!l related sube\anoo OIl the coronary flow of t.be peS"fused rabbits' heart J Fhana., !l.)S, :v., D M P:apientl.(1,~t L ~r andtli' S fluabourgw 19S1 Cot!'.ln.l.l"y dilator action or the adenine-A'fP Mri J PhaI'm Ixp 'tilbury, M ,her -109 2SS Weo1ley, D • 118., I w 1952 B US4 A St.udy or AntimetaboUt "The chatrl.81'1l t)f • , !l:dited by ~,f'iF.1rlJ1 'I and Olau, B lip! f , 19)1 Dle Cb8l:lt:aehe Arch exp Path ~.t"" de: Phan., Jobn W11e, aI3d SODe Irlo • aottont' Jobu ftopldna h"_ "depressor18ct.en Subetanaft dee B1u 160 S19 1) Materials tor tJw pnparaUoa of the A'1'P anal._ :r!~!!I'lt"fb!!PE!!!Jh The ~ 01 Xoaolopott (l9S.J) I.tlacI to _tntat.n a SO a1 'be - ~ of tftetb¥l phoepbS.te Sev :l ackll\lou port1on of t.rietJ\7'l pboaphtte to ubla1_ dlpbospbonate to 'rhla Wft at whan tM apcmt.llneou 1"ef1 ~ 1no the 71e14 r4 te .l_~ '1i' _~p.bge!e !!If Nylen' method (19)0) of eater ~ JtOd1t1ec:l b.Y ret1wd ng " of tar vith J&l of water, S 1111 of ooncctn.t.e4 IEl ad 10 a1 of ,l.ao1al _tic &01d Pol I' amlQ"lc~ Prepared by the _tbod of Sehid.clt (lgJl) 2) Material8 tor the preptU"at.1on "or the UDP ana.log ",!£Mllver -b:\tmGd1Rlel!9!M1:! !b1a cOlJlpOUnCl wu lw 1,' prrepued _ deaor1be4 Wyle (1930>~t !ER.r2Pl11fi1M ~ Tb1a ~ wu prepart,d deecr1be4 tr IArfine and T1p8on (193b) It, I' ~~!1d1ne s:!A1ne ~t tofZ!!te :, lAW1ne and 't'tpaon (19lb)' if M!R:l'loda 2' a ~~ ~114Hl! ~ PrePM'8d after t.m.na 'H.pItoB (19.3b)' and 8, ,) ruritieation of obaraoter1.aatlon of rl:~.~' M!nZ~!t21maed1za mona~ -th11tme41phoaphonaw 'Were J"e'l!'!Oftd b.Y 'SO and nne milliliter of uter1a1 88 troatad w:!th em_ pnoiplt&te vas pun down and ~ aden7lmethflenedtpboepbate 'The ~ l7oPh111aed to dl'J1'MU YaCNllm eubl1matlon at dUeol, in water 5' rraoticne -of Sf -8Ofl- ~ formate va • portion of the ftI1!ldua1 II) lid wu th1e eolutton eonta.1n1ng )0 !II or bart acetate 1n a centrJ.hp tube "'_had wtt.h tbrM portic:mtl of vatel' !he It wu 4!uol'ntd 1n a Mln~ ot 0.1 n tel at SOC I oentrituced tree f'roft a u ~ ot wt,lub.le !'!lAter-tat, and rerprec1p1taW at pH bT addition ot 0.1 RaOH ~'b1s loRd waa uuhed WI th th'Ne portions ot vater, then tiona ot ~ ethanol, then with etheP, and dJto1ed tor 12 hove YieU If ma baJIt_ tmltate vu A portion YU trltur'4ted ~oved paper ehrcmatogra This n\h 1% ~\JM 'tJ1 th two por- OWl' ',os at 0.1 nltat the b,y centf'1tupt1on and the aupomataftt aam1Ded on ~~ produced lingle spot in &odl_ hJdro- 11ft phoepbate ($%)-1 .1 alooho1, rtt 0.81 · Anaq't1oal data~, 1nd1ca- ted OOMitlerebl oontaminatlon, probab~ td ttl barium -tl\Yl ~attJ Anal oalc • to!- Cll~/>12R~(~O)6: I, 1.9; P 10.5 H, 6.9, P 11.' Found Anal calc tt1r phofsphate-Pf total P ( _ below), 11) Foundt 1".15 t:1tmm m111111 ~ of solution ecntaining 0.53 mJ~ ot product, deteM.necl opt1oal denelV ~ts, (:tlooM the f'Ovu col.Ul\'4'l after l"'8II1O'fal ot the _- !!tent fonu.te) was adjuated w pH with 11 Iel (ca, 10 ~ and , tnated in • centrifuge tube "ith 1.0 g of b8i.nc requ1Nd) aoit.~.had i40r1te-A unt1l r I optScal defttd."" ~t.a a' 6':> on the eu.pernata Ind1cated that ,., 9O'J' of tlw J'lUC1eoUde del"1vatlft had adsorbed a total ot lao Itl of _ The}{ori te wu vubecl wi 1A tl'Ifte ~l to Oft IIn¥ pho8phm.o 1111 till _ttwl .~ Uld, ad the pl'DCJwt\ 11M \beD -lutld 1d\b • total 0' 100 111 ot 10$ W'J"lcl1ne 1ft )0$ etMnol 1ft etcht porUona _ _ ndtaced " \he ~SMd 101'1 , , .olaU.Dt11~ p., tU me bart ot a '!'be ethaol rw- to reaove h7 oop1o -1ta aol1d• • J'O.f- t10D (16).> d1aacl 1A tal of _tel> and preCipitated JOMlb1e w1 t.h at _ !be 0' PJ!"H1p1ta w:ebacl am\ ~1w4 1ft 0.1 N Hlp (ote) and a u ôtMpla-lƠ ã 111 a _11 _01IIIt ~ o.t 1_o1»14tt _ter181 eeni' r1.f'1.laM ott The a'fIPWftAtant ted witJt • few c!npI of ao1w ~_ • 6.s to tate and no :l eo ~ added unittl tbe pH adJ- 'h Pft01p1\ate , eolleoW c:enUlf\lcaUcm, vaahe4 w1tb , ~ 111fFl port4_ of atw, ponto_ of ethanol ($) and thea etbW 0.3 _ tor tit hot8 nei~,I18 (1$) AMl Calod r_ dft.ecl at ., Cu~O~s'3~("t0)6' 8, l~J It 8.2 ~, ~ ot tate Jrp'tlft ).OJ If, 1.' " 10.S !!!Il~J!I!l!!!\18 !'!~•• -tav'1 u.~ of thu _tiled oen~ tube 1$.t1J B, 0, lb.9, BMP1ea Atter preltai.11a17 ~1:a aold ad AMP bad uta:bl.iJlhed the weN trl'eeW 1n " vith _ 0.011 fJ\Il1Ur1o ac14 (SwlO ml) ott ro.s 10.) !h2'~b9!eI!' vUb 0, and v_heel wi tIl t.hree tntpeftIat.arJ\ and d • Wft S M1 d11w" to ~nB 15 111 and t.he ban_ ot water SO 111 in a whnttrio nl- -n oombinld t1Mk and ali- as quote taken tor t.be ~tit.lO of phoephate-pb.phor-ou.a andf total phoephOPOU8 or 'or t.b8 detenll.nation a.s _1 or SN aultvlo acid w pboaptlate-phoepncrolll, III ali.quot pl_ refluxed cwern1.gbt., them cooled and V_felTed a 2S al Tolw.t;rlc rlUk tor color deYel.opaenft , the deteftd.ut1on at t.okl-pbaepborot18, a ) al aliquot was tnatGd with 2.$ III of 1ft a dlpat.l_ tube tJte wat b011ed ott I and t.ba a total or tbr boun '1 or eix pOJ'\lona of 3~ 8&tDple At t.be nd bo1W ror or the 4tpeti_ acId cU.pned t lV'drapa peraa.de (two \bree dhpI eacb) addefl during thi_ period \0 \lOA sa auU'uric S III at OOllpl.e\e _ til" added and the ••batt 10 IdAUtell t.o bJ'drol7M pol;rpb_phatM pree_ and ton ooolM 8ncl vanatened to a 2S III '1'01'-'1'10 t1aek tor color deY.l Color 1fU &Welope4, t_ each of t.he abOM NUIp_ att.er the , ooed- of r18k and 3ubbarca u d.eaonbe4 by Hawk, " and SUBa8ftCIQ (191.7) The -.plea read ml a 8eobman DO peet.rophot.~er at 660 aJJ .' t.otal P ration wu thue :PI 0&104 Pboapbate-h £1 ,be pboapbata- by tbe ratio of tbe Wo ruct1 t.ot.&l P, 1.) Found 1.) reat tC' -labi1ephoepnoroue8 • Bari_ was remG9'H trOll a sarrple of the bariWl salt, by t.l'eatmeut w:lth dilute aulturlc acid and oent.rifugation, and the aupernatant we anal1Hd tor "lablle t:boephorouaW (phoephate-phoaphoroua 01 • 10 at- "u with N Hel at lOOo) b7 the ~ of Allen (1940) 11~ by th1a proced.m"th In _ in 10 pboapbate alternative pr.fK1un, , 111 al.:14uot ide.sal \0 that tak_ t phOlpbate-ph.pbcIJ'OU8 deteninat10ll tr••t ~ and t,otal-phosphoroua detend.nat1ona 10 phoapbate l1berate4 by tht proceduN !tt1!:o1z!18 With 111.0f! A S ag • ,18 of the bart_ ealt tnatecl with "7 cumtl"ttugat,1 h.S 111 of 0.02 If auU'ur10 _id &ad tt-A bari_ eultate nMlW_ O.S of 10 J odium l\Ydrarlde wu added \0 the clear s~ an4 \1' 801- !.an heated in a water bat,h at ~ t tw lloar'II, thea -utnJJ.Md with ooa.oentrated 001 aDd eDa1ned CD paper ebr.atopo_ (diad_ phoe- pbate (SJ)-1 .1 aloohol) apot id• •loal with that of lUP, It 0.72 peared t.ogether with • _.11_ spot IdentIcal wl'th that of _ _• • ~U,t.l- of '.&ow,AbeOl'baM:y Illda BIr1_ the ba1._ ••1t, With dl1ute eultu:l1.o acId Q aUquot wu dil\fted pbate (pH 1.0) ",0 10 111 with Is Ca.tt) WU _1 or aupla and ,.be co ~ naak A S fill and 111 '8l 1_ pboe- o.0Sb5 « pGr l1ter the mQlar oalcuu.ted trOl'l the equation 1'be "orb&ncy (M) of the sample at 260 anal1ttcal data, 8eL In a typical SO: id in a vohlMtJoio to gift a tiMl oonocmtnt1an of abeOl"banay 1Qda S.lD ItO.lJO t?ca naplM of bari_ aulfate cleMrndnaticm the 0.1_ p1'tlClpltated h'0lI a npeJ"ftl.tant and h.tnp ' " " diluted to ~ ap- and IN O.97S, the ooocontrat1on 18 the molecu1al" weich' o.OS16 sr- t.h1 • ,ari_nt the war abaorl:Nmcy lftda tound to be pw' 15.8 X trOll liter In »' The in molar absorDancy lndltx for AMP, ADP, and ATP 18 1$.1& • lal at pH 7.0 (r,J~ll and Book, 19$4) Neutralization suiv.lent and Rl vall.lel! Samplee of t.he bu'i_ a.lt III> treat.ed with • aUght o;ee at \ube and the barium eultate apUD d~" 0.4 (08 >-10 !'I aulf'U1'1.c acid in a oontr1tup 'Ute bari_ 8ultate bed with \bnte porti_ of ••ter, and the supemat.&ttt anti washing tltrned with 0.5' N bOR in 0.2 If Nail (total volume all a.n,., the method of ll~ (19S1) \Wing the tlt7at.ion apparatus de8cribed bySchnrta and., (l9S7) Il'l det.ona1Mtion 1.8) !I€ of bart_ aal,., required 0.0191.1 ot 0.li7 • ad b-_ • mloromet,g driven ayrinp, to t1tr'ate ODe 14 group \lV.t., neutr&li.at1on equivalent, 86b Aaalpl (.mao and or the tltratiOtl c indloated 1.S ( mdary Found 870 &ppaftnt pI ,.1_ of la.1 phoephaM) ~ , tr~ of potua1U11l h) Preparation SIlT tr1beMTl '.QH, deliver ItaCla ab_ • l.arae 41 uid a1de .inc to· ~,_ tbat • tAg cOllpla U' t -cl the etnngt,h of biMini, Wadi", t,n1cal tKtuivalGJl\ 0&104 tor coe A \ltratiOl'l oU!"t'e in 0.1 I 101 and in IeI~nt t d t.he & oarmot be oalculated trOll We date t the loa for tlle aulog 18 not kn.a at tntermed1at.es tor Mt~1'lened.1 pbo&phOMte, the ar,yn~li8 of IlOP a.l.,_ key 1nte:rmed1ate 1a toM ab fIIDt- the81., wu pntpanKl by 1'l:1nodebeMylat,10n III tetrabenITl MtJ:\f1eDed1pho1pGoaate oompouacl prepared by reaction or bensyl brora1de on tetru11 ".1- MZ ,11 , ~P!!l1 ~!l~~ A st1rN4 ~ of ~ ",leGadlpbcep!lOMte (loa 0.0166 U), 90 111 of dl"7 tbl~ aDd 1S 111 {11.6 i, 0.l21 V} of belllSYl bt'Clldd.e "U retl'" t rn "- atlY breed_ w tl1t~ ott ·and bed ,~~l til' With " ' - , a1 of the benaane nth u:p1l"at1.on, tbo dON' ott at 0.1 _ With batb ~l"@ture of et._ ad t.be et.l~ elution 1fU 1tfi ~l brOllf.dll 4lft1 The residual 011 1IP 1I\1.$bed -era1 t~ with d ~ olD1., theft with watf4t I then with ••tnrated odi_ chlortde t1 1l1' dried _or 'bie ••Uel, in and o1a\1., ~~~ 10eU auUate \0 a j.ll;y l.11N oUd SO III of benaeMj sa there 111&1 adde