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Antibody-Catalyzed Phosphate Triester Hydrolysis.

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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.
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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
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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-
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