COMMUNICATIONS Table 3. C ~,pulyiiicri/:itioiiofTMDA and erhene uith t u o zirconocene.MA0 catalysts :I1 50 ( ' I,r]. Antibody-Catalyzed Phosphate Triester Hydrolysis** Jonathan S. Rosenblum, Lee-Chiang Lo, Tingyu Li, Kim D. Janda,* and Richard A. Lerner* Dedicated 10 the nzernor.~of James D. Boin ( ' r C ' , Molar r.itio olTMDA:ethene. , A : Activity in kgpolinyer permol mctallocene pcr hour. %I,,: .\vcrage molecular weight determined froin viscosity, Incorp [:I1 Inc~,rpi~i-:itioii 01 'IMDA ing point in p;irenlhcses. in the copolymer. T,: Glass transition tempcrature. melt- copolymers than for DMON/ethene copolymers. At 50°C the r l value for the TMDAlethene copolymerization is 15.6, the r 2 value is 0.06. and r 1 x r z is 0.94 (cf. Table 3). The results presented here indicate that the performance of different metallocenes in the copolymerization of ethene and cyclooletins strongly depends on the nietallocene used. The copolymers produced by the zirconocene 3 feature the highest incorporation rates of bulky comonomers. Phosphate ester hydrolysis has challenged biologists and chemists for many years. While attention has focused on phosphate mono- and diesters since these have the best understood biological properties, phosphate triesters are also biologically relevant. The significance of phosphotriesters in biology ranges from their essential role in the adaptive response in E. coli''] to their use as prodrugs for nucleoside analog pharmaceuticals.['. 31 Derivatives of phosphate triesters are used as insecticides and are potent biological warfare agents. This class of compounds includes sarin. isopropyl(methyl)phosphonotluoridate, which was recently used in a deadly bombing in a Japanese subway.["] Although there are phosphotriesterases in nature. their natural substrates are not known.[51 0 0 II I- F - O O N O 2 E t OOEt F 0 - P - CH3 IF 0 II E10-P-CN I Received: April 7. 1995 [Z 7875 IE] (ierman Lession : Aii,qc~w C'heni. 1995. 107, 2469- 2471 Paraoxon Keywords: catalysis . copolymerizations mers Sandwich complexes [ I ] W. K n i i i i n s h ~ .K.Spichl. M a k i - o n i o l . Chon. 1989, IYO. 515-526: W. Kaminsky. A . Bark. R. Steigcr. .1. Mol. Cuial. 1992. 74, 109 119: S. Collins. D. G. Ward. .I A i i i . C'/iwii. .SM. 1992. 114, 5460 5462. S. Collins. W. M. Kelly. D. A. Holden. M ~ i ~ , r o i i i o l [ , ~ u /1992. c.\ 25. 1780- 1785: W. Kaminsky, A. Noll. f'o/~wi H u / / . 1993. 31. 175 1x2: H. Cherdron. M:J. Brekner. F. Osan. A i i ~ wMukro. i > i o / ('/win 1994, 223. 121 133. [21 (i 1)all'Asta. ' L f u A r i i i i i d . Cli~wi.1972. 1.54. 1 19: G. Natta. G . Dall'Asta. G. Miiuanti. Iii:.vn.. Cli~vii.1964. 76.765 -772. Ai?gim.Chcrii. h l . Ed. Eirgf. 1964. 3. 733 720. T. Siigine. A Mizuno. T. Takatn. Mukriimo/. ('hrni. 1992. 193. I I : ( ' Mehlcr, W. Risae. Mokron?o/. (%tin. RupidCoiiiinnii 1992. 13. : I ) . S. Breslow. Pro,q. Po/wii. &i. 1993. I S . 1141 1195: Ref. [9a]. [ 3 ] W. Spalcck. f i w c ~ f i ,High ~i Ciicni. 1993. 14. 44-48; R. Mulhaupt. N m l i ~Clirrii. . / [ , ( / I , /Lib 1993. 41. 1341 ~ 1 1 5 1 . 141 1. .,\. Ewcn. I. Haspc\lagh. J. L Atuood. H. Zhang, J An?. Clicni. S k . 1987. IOV. 6544 0545: A . Razivi. J. Ferrnra. J Orpaiionier. Chmi. 1992. 435. 2993 10 [S] A Winter. .I Kohrniann. M. Antberg. V. Dolle, W. Spaleck (Hoechst AG). 1)F-H 3907Yh5. 1990 [C'hc.nr. A/r.~tr..1991, 114, 1651031 [ O ] M L. H (ii-ceii. N. Ishihara, J. Chom SOI..Dullon Purl.\. 1994. 657-665. N. ~ \ h i I i a f i ~,tfm , r o i i d Siwip. 1995. KY. 553 -562. 171 W KaiiiinAy. C ' u l u / , l i h r 1994. 30. 257 -271: H.-H. Brintzinger. D. Fischer. K Miilliaupt. 8 . Rieger, R . Waymouth. .4igeti. C/iwi. 1995, 107. 1255 1283: I < ' l w i I ? . Iiil.E d Eii,q/. 1995, 34. 1143-1170. illoyl.nphic details for 3 . crqstnlluation from toluene: monoclinic: space P 3 , ii.%=4.1i=12.35X(?),h=13.969(3),~ =13.482(3).~~=111.74(2). I = 21hl.H(X) 4'. L I . , , ~ , , = 1 . 5 5 6 g ~ i n - ~5377 : reflections measured: 4985 of thcin indepcndcnt. range0 = 2.3G27.55 :temperature 153 K : number ofrefincd pnranicler\ 292. reflection to parameter iatio 17.07. least squares method K = 13.0301. thffractometer Hilger & Watts ( Y 290). Further details of the cr>\t;il \ t i iic'tiirc invcrtigaiion [nay be obtained from the Fachinform;itioiiszentruiii Karl~riihc,D-76344 Eggenstein-Leopoldsh~~fen (Germany). on quoting tlic dcpobitori number CSD-401676. (91 i t ) ( i . M Ikiiedikt. H L. C;oodall. N. S. Marchant. L. F.Rhodes. N c i v J C'IIFIPI. 1994. ltY. 11)s 114: b) G . H. Llinas. R. 0. Day. M. D. Rausch. J. C. W. Chicn. ~~l,~~iii,iill,~l~ 1993. l l / l ~ ,12. \ 12x3 -~ 1288. LlO] I I< M;i1.'i>. t M L . J i l i i i . C'11c.m.Siw. 1944. 66. 1594 - 1601. M.Finem ~ i i i i i 5. . I ) Ros\. ,I. P o / i . i i i . &;. 1950. 5 . 259 -262. I1 1 1 W. K;iiiiin\ky. A . Bark. M . Arndt. , M u k m i i d . Clirm ~Murriiin(dS i v i p . 1991. ~ 4-.x3 9: Sartn Tabun cycloolefin copolyIn addition to their biological importance. phosphate esters present fundamental chemical issues relating to their unique hydrolytic stability['] and involvement of a pentacoordinate transition state for hydrolysis. Antibodies catalyzing the hydrolysis of phosphate triesters would be interesting from biological and chemical points of view. Such antibodies could be used to interfere with the response of E. coli to mutagenic methylating agents, activate prodrugs (carboxylate ester prodrugs have been hydrolyzed by catalytic antibodiesf7.81) or treat o r prevent organophosphate neurotoxin poisoning.[', '1 Such catalysts should also help define the structural and electrostatic requirements of the transition states for phosphate ester hydrolysis. Herein we describe an antibody capable of catalyzing phosphate triester hydrolysis. The pentacoordinate transition state for base-promoted phosphate di- and triester hydrolysis arises from attack of hydroxide and leaving group extrusion in-line on the apical axis (Scheme 1). Whereas the tetrahedral intermediate of carboxylate ester hydrolysis is accurately mimicked by many functionalities,[". 1 2 ] pentavalent centers are rare in organic chemistry (for recent exceptions see refs. [I 3.1 41). Additionally. many pentavalent species are either toxic o r unstable. decreasing their utility as haptens for immunization. Conversely. the unique electrostatic features of the transition state anticipated on the [*] Prof. Dr. K. D. Janda, Prof. Dr. R. A. Lerner. J S. Ro\enhlum. Dr L.-C Lo. Dr. T. Li The Scripps Research Institute Departments of Chemistry and Molecular Biology 10666 N. Torrey Pines Road, La Jolla, CA 92037 (USA) Telehx: Int. code + (619)554-6068 [**I We acknowledge support from the Alfred P. S h i n Foundation. Ihe National Institutes of Health (GM48351 to K. D. J.). and the National Science Foundation (predoctoral fellowship to J. S. R.). We thank Ping Fan and Tami Danon fhr expert technical support and Dr. Dee-Hua Huang I'm iirsistaiicc with obtaining " P N M R spectra. COMMUNICATIONS OR^ OH f +8R3 Me 0 Scheme 1 Mechanism of phosphate triester hydrolysis emphasizing structure and electrosriitics of the transition state. Numbers refer to bond order in thc case of base-catalyLcd puraoxon hydrolysis 1271. reaction coordinate are more easily mimicked. As such it is of interest to determine if a pentavalent species is a necessary feature of the hapten. or if addressing the electrostatic features of the transition state alone are sufficient to yield an efficient catalyst. We have screened antibodies which recognize either an amine oxide-containing hapten or a quaternary amine-containing hapten for phosphotriesterase activity. Charged haptens are expected to induce antibodies that affect catalysis by either stabilization of a polar transition state o r by elicitation of a reactive side chain that may act as a general base.[t5-'71Hapten I contains an amine oxide whose dative bond overlaps with the scissile bond of 3 and the P=O bond of 4. Any positive charge developing at the phosphorus center would be stabilized by anionic functionalities in the antibody combining site, induced by the positive pole of the amine oxide hapten 1. The negative pole of the amine oxide corresponds to the developing negative charge on the oxygen atom of the leaving group in the transition state for the hydrolysis of 3, or to a partial negative charge on the phosphoryl oxygen atom in the transition state for the hydrolysis of 4 (Scheme2). As a result of regiospecific induction of countercharge, it may be possible. by comparing the hydrolysis of 3 and 4 by antibodies to 1, to determine which of the partial negative charges in the transition state need to be stabilized. Hapten 2 is a close analog of 1. differing only in the substitution of a quaternary amine for the atnine oxide functionality of l.['81This hapten is expected to induce anionic but not cationic character in the antibody binding site and as such may help to further deconstruct the important charge parameters for transition state stabilization. Twenty-five antibodies to 1 and 17 antibodies to 2"" were screened for their ability to catalyze the hydrolysis of 3 and 4. Five antibodies to each hapten were catalytic. One antibody to 1, Txl -4C6."'I was particularly efficient and was studied fiirther. Reaction of Txl-4C6 with 3 and 4 followed Michaelis-Menten kinetics (Fig. 1, Table 1). Antibody-catalyzed reactions were stoichiometrically inhibited by the hapten, could be saturated by Substrdte and displayed turnover. Antibody Txl-4C6 bound hapten 1 with a Kd of 0 . 6 7 as ~ ~ determined by quenching of intrinsic antibody fluorescence by the hapten. The rate of the uncatalyzed hydrolysis of 3 is slower than that of 4. since 3 is stabilized by the generalized anomeric Scheme 2. Structural formulas of the haptens I and 2 and substrates 3 2nd 4. Arrows indicate orientation of attacking hydroxide for in-line triester hydrolysis. R = linkage to protein (for immunization). R = NHCOCH, (for inhibition studies). v 61x1 51x1 t 4(x' 'I:: A -0.2 0.2 1I[S] (pM-') - Fig. 1 . Lineweaver. Burke plot of antibody Txl-4Ch hydrolysis of 3 (0) and 4 (0). S = substrate. V,, = initial rate. Table I . Kinetic parameters for Txl-4C6-catalyzed hydrolysis of compounds 3 and 4. 3 4 2.77 x 1 0 - 5 8.77 x 10- 1.01 x 10-2 1.X5 x 3.57 18.7 COMMUNICATIONS Interestingly. Tx I-4C6 was able to effect a reversal of intrinsic reactivity. catalyzing the hydrolysis of 3 more so than 4. X ,,,,L.,,(3)ik,,,,'J4)= 0.316; kc.t,(3)/k,..l(4) = 5.46. This reversal may be derived from either ground-state stabilization" '1 or a tighter- geomctrical fit of the transition state for the hydrolysis of 3 than that of4. This differential accomodation can be rationali7ed in light ofthe structure ofthe eliciting hapten I (Scheme 2). For substrate 3. the apical axis of the pentavalent phosphorus at the truisition state overlaps with the axial orientation of the N - 0 bond in l.rl''l thus stabilizing a mechanism in which the leaving group departs from a subtended angle of 180 relative to the thrcc interacting atoms. By contrast. antibodies to ainine oxide hapten I would be expected to polarize the P=O bond for substrate 4. As 3 is ii better substrate for Txl-4C6. we suggest that polarization of the scissile bond for a concerted reaction is iiiore prolitablc than polarization of the P=O bond which may fiivor ;I two-step reaction involving pseudorotation."2' Antibody Tx 1 -4C6 has a limited substrate range. It does not bind. and therefore does not hydrolyze, paraoxon. Likewise. Txl-4C6 is not able to hydrolyze carboxylate esters related to 3 and 4. One ofthe powerful aspects of antibody catalysis is the ability to address selected features of the transition state of any chemical reaction. Her-c. we have demonstrated this principle and achieved phosphate ester hydrolysis by an antibody that was programmed to accomodatc only the electrostatics of the transition state of the reaction. The best antibody catalysts were induced by amine oxide I . yet 2 also induced several catalysts, indicating that catalysis may be achieved by the singular event of lowering the energy of a developing positive charge in the transition state. But, as expected, better catalysis is achieved when additional features of the transition state, such as the developing negative charge at the leaving-group oxygen is addressed. In order to generate inore efficient catalysts it will now be necessary to keep the electrostatic features of the antibody in place and address the more difficult geometric parameters. I n this way we can learn about catalysis by building an enzyme piece by piece. Furthermore, through the development of antibodies we could explore whether the hydrolysis of phosphate diesters can be Facilitated through ;i "triester-like'' mechanism,12" 241 in which the initial step is the protonation of the phosphate anion oxygen. E.vpc~iinlcrlllilProcdrrc> 3. 4 . .All\ i,lii/iiin o/ (Ircrlid r . ~ i ) p ~ ~ i ~ i ~ l i ~ l i ~ i i ~[XI: ~ i n ~To i l ~ai isolution i ~ i t i ~ of diethyl isopi-~)p~lidencin.iloi~~tc (10.0 g. 50 minol) in anhydrous ether (20 m L ) w a s slouly added phcnylmagnesium bromide (50inL. SOinmol, 1 . 0 in ~ T H F ) at 0 C . The reaction u i i \ kept a t 0 C for 30 m i n . then heated to reflux for 1 h. T h e reaction m i x t u ~ cw i s conccntratcd. pourcd into ice-cold dlluted H,SO,, and extracted three time\ nit11FtOAc. 711s combined EtOAc solution b a s concentrated and the 1.4-addition product ptirilied ( 3 8 g. 28%) hy silica gel column chromatography eluted wirh Iicx;ine,EtOAc (8.1 ). L i A 1 / l 4 w d i i i ~ / ; o i( ~J / /hi, i / i i ~ \ r e r T h e diestcr ( 1 3 4 g) obtained w a s dissolved in anhybq slow addition of LiAIH, (0.37 g). T h e rcaction drour ethcr (42 ~ L I(;,llowed . wv;is kcpt .it r o o i n tempcrature overnight. Usual workop ofl'cred diol in 3 5 % yield. ' H N M K ( 3 0 0 MI+/. C W I , ) : d =7.34 7.27 (m. 4 H : aromatic). 7.21-7.16 (m. 1 H.;inmatic).? 7X(dd.J=10.4.3.7Hr.~H).3.70(dd.J=10.4.9XHr.ZH).2.15 (in. I HI. 2 00 ih\. 2 H : O H / . / ~ l i ~ j , ~ / i / i ~ , i - i l i i o/ r i ~/lir J / i ( l i d : T h e d i d (145 mg) in T H F (2 m L ) was lidded d r o p w s e t o ii wlution of pyridiiie ( I33 gLJ and p-nitrophenylphosphorodichloridatc (210 mg) in TClt ( 5 m L ) . The mixture wiis stiri-cd at room temper:iture for 2 h, then conccntriitcd. l l i e dcsired products 3 ( 7 0 mg. 2 5 % ) and 4 (72 mg. 26%) were separated b) s i l i c : ~gel column chromatography eluted with hexaneiEtOAc ( 2 ' 1 ) Comp ~ ~ i 3i :dA' = 0 27. ' H N M R (300 MHz. CDCI,). 0 = X.22 (d. J = 9.2 H7. 2 H : ;irom,itic). 7.39 7.24 (m. 7 H : aromatic). 4.32 (dd. J = 10.9.4.9 Hz, 2 H ) . 4.26 (dd. J = 1 0 9 . 4.9 Hd. 2 H). 2.68 (in. 1 H ) . 1.38 (5. 6 H : C H , ) : "P N M R (300MHz. C.lX I , hclcroiiiiclc,irdecoupled): (7 = - 13.37. C o m p o u n d 4 : R, = 0.20. ' H N M R I300 MH/. C ' I K I , ) h = 8.22 (d, J = 9.2 Hz. 2 H: aromatic), 7.38- 7.22 (in. 7 H : aroni;itic). 4.48 4.76 (in. 4 H ) , 2.31 (m, 1 H ) . 1.47 (5. 6 H , CH.J. "P N M R 1300 MI I/. C'EX.1, hctcronucleal- decoupled): 6 = - 12.29. The stereochemistry of 3 and 4 w a s determined hy comparing their R, v'ilues. 'H and "P N M R spectra with those of similar compounds from the literature 1261. ,4.i.1oi 1 . All reactions were Jbllowed sjxctroplioloinetric~ili~ :it 30 C. lblloi\ ingp-iii- trophenolate production at 402 nm. The extinction coefticient dcterinincd under the reaction conditions was 1.80 x 1 0 ' ! ~ ' cm-'. Typically. solution\ of SOmM Bicinc. pH 8.S M i t h or uithout 5 p I 4C6 were equilibr;i~eda t t h e re:iction conditions for 30 min.Reactions were initiated by addition of ii constant voliiinc of \iirioiis concentriitioiis ofthe substrate i n DMSO T h e final DMSO concentialion w a s 0.175k1. The reactions Here follofied thr approximately Z h. allcr whicli tiine lesh than ten percent of the substrate had hecn consumed. Kccc~ved M a ) 2. 1995 Reviaed version: Jul) 11. I995 [Z7949IE] Gcrman version: .41i,qiw <'/liwi. 19Y5. 107. 2448-2450 Keywords: catalytic antibodies * electrostatic interactions drolyses phosphates transition states - - * hy- [ I ] B. M. Saget. G. C'. Walker. P r o i . N a i l . . A c a d S c i . L,SA 1994. Y I . CJ730-9734.  C. Perigaud. A. M. Aubertin. S. BenKaria. H. Pelicnno. J. la Girardet. C; Maury. G . Gosselin. A . Kirn. J. L. Imb;ich. Bioi,hcwi. / ' l ~ ~ i r n i ~ i u1994. ~ l . 415'. 1 1 14. [ 3 ] A. Numane. C . Gouyette. M . P. Fillion. Ci. Fillion. T. Ilii!nli-Dinh, .I M i d C'hrni. 3 1992. 35, 3039 3044  N D. KristoC Tliirc .Yiw YorX T;irrc.v. 1995. A I . A6 [S] S. R . Caldwell. J. R. Newcomh. K. A. Schlccht. F. M Raushel. B i i ~ d i ~ m i , \ ~ ~ 1991, 30, 7438-7444.  F. H. We\theiiner. S w n w 1987. 3 . 5 , 1173 1178. (71 H. Miyashita. Y. 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Korpic\. .I d m . (%m. Soi.. 1Y54. 76. 1911 1913. [XI 1.. D. Hall. R . B. Malcolm. Con. J, <'hcrii 1972. X i , 2102 2111). [?7] S. R. Caldwell. F. M. Raushel. P. M . Weiss. W. W Clelaiid. Biocliomsiri. 1991. 30. 7444 7450.