in order that the invention may be better understood , preferred embodiments now are described by way of example only , with reference to the accompanying reaction schemes and diagrams . in one preferred form of the invention , methyl ecgonine ( compound 9 ) or 2β - methoxycarbonyltropan - 3β - ol may be transformed into the cocaine - based immunogen ( compounds 12 , 14 ) by a sequence of chemical reactions portrayed in fig2 b . phosphorylation of benzyl 4 - bromophenylacetate ( compound 6 ) under nickel chloride catalysis ( formation of compound 8 via intermediate compound 7 ) is of particular importance since it is the crucial step in the reaction scheme not previously known in the art . in all other aspects , the conditions of reactions performed follow protocols generally established in synthetic organic chemistry and any other convenient procedure . a number of methods known to those skilled in the art may be adapted to follow quantitatively the hydrolysis of cocaine to 2β - methoxycarbonyltropan - 3β - ol and benzoic acid by reaction scheme 1 shown in fig2 a . one such method utilizes capillary electrophoresis and detection at λ = 200 nm , as described in detail in example 9 below and illustrated in fig5 . thus , it is possible to quantitatively measure the hydrolysis of cocaine by either following the loss of cocaine itself , or appearance of the cocaine breakdown products benzoic acid and 2β - methoxycarbonyltropan - 3β - ol . the activity of catalytic antibodies and catalytic antisera can be directly related to the activity of naturally occurring esterases , comprising 104 - cholinesterase , or control sera . protein conjugates 12a , b and 14a , b ( see fig2 b ) of the analogs of transition state for the hydrolysis of the cocaine benzoyl ester are used as immunogens in mice and rabbits . rabbits provide large volume of antisera and mice provide the potential for generating monoclonal antibodies by conventional hybridoma technology . the esterase activity directed against cocaine benzoyl ester of purified antibodies from either control or immunized rabbits , and from selected hybridomas , was assayed by capillary electrophoresis as described hereinabove . the conjugates 14a , b were endowed with this cocaine esterase activity . the following examples are used to illustrate the present invention . they should not be construed as limiting it in any way . all parts and percentages are by weight unless otherwise indicated . all abbreviations and acronyms have the standard meanings in the art . melting points were determined on a reichert - thermovar melting point apparatus and are not corrected . optical rotations were measured with a perkin - elmer polarimeter ( model 243 b ) at 26 ° c . 1 h and 13 c nmr spectra were recorded at 300 . 13 mhz ( 75 . 47 mhz , 13 c ) or 500 . 15 mhz ( 125 . 04 mhz 13 c ) with bruker spectrometers at the nmr spectroscopy laboratory , carbohydrate research centre , university of toronto . spectra were obtained at 20 ° c . either in cdcl 3 or cd 3 od containing a trace of tms ( 0 ppm , 1 h and 13 c ) as internal standard . fast atom bombardment mass spectra ( fab - ms ) were recorded with a vg analytical zab - se instrument at the mass spectrometry laboratory , carbohydrate research centre , university of toronto . high resolution mass spectrometry ( hrms ) is used for exact mass measurements . thin - layer chromatography ( tlc ) was performed on silica 60f ( merck ) plastic plates and visualized by spraying with 50 % aqueous sulphuric acid and heating at 200 ° c . silica gel ( 230 - 400 mesh , toronto research chemicals ) was used for flash chromatography . all solvents and reagents used were reagent grade . this example illustrates the preparation of benzyl 4 - bromophenylacetate ( compound 6 , fig2 b ). to a suspension of 4 - bromophenylacetic acid ( 2 . 150 g , 10 mmol ) and benzyl alcohol ( 2 . 5 ml ) in dry dichloromethane ( 20 ml ) was added dicyclohexylcarbodiimide ( 2 . 5 g ) at 0 ° c . the mixture was allowed to warm up to room temperature , and stirred overnight . after dilution with dichloromethane ( 250 ml ), the solution was washed with water , dried over sodium sulphate , and dichloromethane was evaporated to give an oily residue . this residue was subsequently subjected to flash chromatography on silica gel using hexane / ethyl acetate ( 9 : 1 ) to give pure compound 6 in 88 % yield ( 3 . 81 g ). 1 h nmr ( cdcl 3 ): 7 . 47 - 7 . 42 ( m , 2h ), 7 . 31 - 7 . 40 ( m , 5h ) 7 . 15 - 7 . 17 ( m , 2h ), 5 . 16 ( s , 2h ), 3 . 61 ( s , 2h ). this example illustrates the preparation of benzyl 4 -( diethylphosphonyl -) phenylacetate ( compound 7 , fig2 b ). triethylphosphite ( 10 ml ) was added dropwise to a mixture of compound 6 ( 7 . 51 g , 20 mmol ) and nickel chloride ( 0 . 5 g ) heated at 160 ° c ., and the reaction continued to be heated to this temperature for additional 3 hours . then the mixture , cooled to room temperature , was diluted with dichloromethane ( 250 ml ) and filtered through a celite bed . the filtrate was washed with water , dried over sodium sulphate and evaporated to dryness . the oily residue was purified by flash chromatography on silica gel using ethyl acetate to give pure compound 7 ( 7 . 11 g , 81 % yield ). 1 h nmr ( cdcl 3 ): 7 . 86 - 7 . 77 ( m , 2h ), 7 . 45 - 7 . 42 ( m , 2h ), 7 . 30 - 7 . 40 ( m , 5h ), 5 . 15 ( s , 2h ), 4 . 10 - 4 . 20 ( m , 6h ), 3 . 70 ( s , 2h ), 1 . 38 ( t , j = 6 hz 9h ). exact mass measurement ( ei ): for c 18 h 23 o 5 p calc . 362 . 1283 , found 362 . 1267 . this example illustrates the preparation of 2β -( methyloxycarbonyl -) tropan - 3β - yl 4 -( benzyloxycarbonylmethyl -) phosphonate ( compound 10 , fig2 b ). a mixture of compound 7 ( 2 . 80 g , mmol ) and trimethylsilyl bromide ( tmsbr ; 2 . 01 g ) was stirred under argon overnight at room temperature . the excess tmsbr was removed in vacuo , and to the residue was added imidazole ( 25 mg ) and oxalyl chloride ( 1 ml ) and the resulting solution was stirred overnight at room temperature . then both the solvent and volatiles were evaporated in vacuo and the resulting crude dichloride 8 ( fig2 b ) was diluted with pyridine ( 10 ml ) and added to a solution of triazole ( 1 . 0 g ) in pyridine ( 20 ml ) and stirred for 30 minutes . then ecgonine methyl ester ( compound 9 , fig2 b ; 1 . 81 g ) was added to this solution and the reaction mixture was stirred for 1 hour at room temperature . the reaction mixture was diluted with 1m teabc buffer ( 50 ml ) and the resulting solution was extracted with chloroform ( 7 × 50 ml ). the combined chloroform extracts were evaporated to dryness in vacuo to give compound 10 in 71 % yield ( 2 . 98 ). 1 h nmr ( cdcl 3 ): 7 . 77 - 7 . 62 ( m , 27 . 41 - 7 . 12 ( m , 7h ), 5 . 11 ( s , 2h ), 4 . 50 ( m , 1h ), 3 . 70 ( s , 3h ), 3 . 55 ( s , 2h ), 2 . 40 ( s , 3h ). 1 h nmr ( d 2 o ): 7 . 80 - 7 . 70 ( m , 2h ), 7 . 50 - 7 . 41 ( m , 7h ), 5 . 21 ( s , 2h ), 4 . 62 - 4 . 59 ( m , 1h ), 4 . 02 ( d , j = 6 . 7 hz , 1h ), 3 . 95 ( d , j = 7 hz , 1h ), 3 . 88 ( s , 3h ), 3 . 72 ( s , 2h ), 3 . 10 ( dd , j = 9 hz , 2hz , 1h ), 2 . 80 ( s , 3h ). exact mass measurement ( fab ): for c 25 h 31 no 7 p calc . 468 . 1838 , found 488 . 1819 . this example illustrates the preparation of 2β -( methyloxycarbonyl -) tropan - 3β - yl 4 -( methylcarboxyl -) phosphonate ( compound 11 , fig2 b ). to a solution of the compound 10 ( 750 mg ) in glacial acetic acid ( 15 ml ) was added pd / c ( 10 %; 100 mg ) and the mixture was hydrogenated in a parr instrument overnight . then the mixture was diluted with dichloromethane ( 250 ml ), the solution was filtered through a celite bed , and the filtrate was evaporated to dryness in vacuo to give compound 11 ( 700 mg ). 1 h nmr ( d 2 o ): 7 . 73 - 7 . 66 ( m , 2h ), 7 . 45 - 7 . 40 ( m , 2h ), 4 . 65 - 4 . 58 ( m , 1h ), 4 . 10 ( d , j = 7 . 20 , 1h ), 3 . 95 ( d , j = 6 . 80 , 1 h ), 3 . 77 ( s , 3h ), 3 . 755 ( s , 2h ), 3 . 49 ( dd j = 11 , 1 . 5 hz , 1h ), 2 . 80 ( s , 3h ). exact mass measurement ( fab ): for c 18 h 24 no 7 pna calc . 420 . 1188 , found 420 . 1157 . this example illustrates the preparation of protein conjugates 12a and 12b ( fig2 b ). compound 11 ( 30 mg ), klh ( 30 mg ) and dimethylamino - propyl - 3 - ethylcarbodiimide hydrochloride ( 20 mg ) was dissolved in water ( 20 ml ) and adjusted to ph = 5 by teabc buffer . after stirring this solution at room temperature for 48 hours , it was filtered through ym - 30 ( amicon ) membrane filter using 3 × 10 ml distilled water . the membrane filter was decanted with distilled water 3 × 10 ml , and the aqueous solution after lyophilization to dryness gave protein conjugate 12a . using bsa instead of klh , protein conjugate 12b was obtained . this example illustrates the preparation of protein conjugates 14a and 14b ( fig2 b ). a solution of 6 - aminocaproic acid ( 3 . 0 g ), klh ( 500 mg ) and dimethylamino - propyl - 3 - ethylcarboiimide hydrochloride ( 1 . 0 g ) in water ( 50 ml ), adjusted to ph = 5 by teabc buffer , was stirred for 48 hours . then it was filtered through ym - 30 ( amicon ) membrane filter using 3 × 10 ml distilled water . the membrane filter was decanted with distilled water 3 × 10 ml , and the aqueous solution after lyophilization to dryness gave 6 - aminocaproyl klh ( compound 13a , fig2 b ). using bsa ( 0 . 5 g ) instead of klh , compound 13b , fig2 b , was obtained . a reaction of free carboxyl - containing compound 11 ( 70 mg ) and 6 - aminocaproyl klh ( compound 13a ) ( 50 mg ) according to the procedure used to synthesize 12a and 12b ( as described in example 5 ), gave protein conjugate 14a . under identical conditions , compound 11 reacted with 6 - aminocaproyl bsa ( compound 13b ) to give protein conjugate 14b . antibodies were purified from rabbit sera using protein a sepharose chromatography . briefly , serum was diluted 1 / 10 with 50 mm tris / 150 mm nacl ( ph = 8 . 6 ) and loaded slowly onto a protein a sepharose column . after all the material was loaded , the column was washed with 3 column volumes of 50 mm tris / 150 mm nacl ( ph = 8 . 6 ). bound antibodies were eluted with 50 mm sodium acetate / 150 mm nacl buffer ( ph = 3 . 5 ) after the column had been washed sequentially with 50 mm sodium phosphate / 150 mm nacl buffer ( ph = 7 . 0 ) and 50 mm sodium citrate / 150 mm nacl buffer ( ph = 5 . 5 ). eluted antibodies were dialyzed against 5 mm sodium borate buffer ( ph = 8 . 3 ) and quantified using extinction coefficient of 1 . 43 at 280 nm for a 0 . 1 % solution ( 1 mg / ml ). elisa assays were used to determine by titration the level of hapten - specific antibody in the sera of immunized animals . ninety - six well elisa plates ( nunc - maxisorp ) were coated with 100 μl of 10 μg / ml hapten - conjugate in 20 ml sodium carbonate buffer ( ph = 9 . 6 ) overnight ( 16 hours ). excess reactants were washed away with 0 . 05 % tween 80 in phosphate buffer saline ( ph = 7 . 2 ; pbs / tween ) using a corning plate washer . residual protein - binding sites on the elisa plates were blocked by coating the wells with 1 % low - fat milk in pbs ( ph = 7 . 2 ) for 30 minutes , and washing the plates again . diluted [ 1 / 8 → 1 / 16 , 384 with pbs , ph = 8 . 0 ] sera ( 50 μl ) were added to the wells and incubated at 37 ° c . for 1 hour . then wells were washed 3 × with pbs / tween and stained with either goat anti - rabbit or goat anti - mouse igg ( as appropriate ) conjugated to alkaline phosphatase ( diluted 1 / 1 , 000 from commercial stock with 1 % low - fat milk in pbs ). plates were incubated at 37 ° c . for one hour and washed 3 × with pbs / tween and then stained with substrate ( p - nitrophenyl phosphate ) at 1 mg / ml in 100 mm diethanolamine with 5 mm mgcl 2 added . plates were read at 405 nm using a titertek multiskan elisa plate reader . the animals immunized with protein conjugate 14a ( klh long linker ) or with protein conjugate 12a ( klh short linker ) were assayed against protein conjugate 14b ( bsa short linker ); likewise animals immunized with either protein conjugate 14b ( bsa long linker ) or protein conjugate 12b ( bsa short linker ) were assayed against 14a protein conjugate ( klh long linker ). capillary electrophoresis was used to monitor the breakdown of cocaine by separately monitoring the presence of cocaine and its breakdown products , methyl ecgonine and benzoic acid , in the reaction mixture . using a beckman p / ace system 2100 capillary electrophoresis apparatus and a fused silica column ( 57 cm long ) with an internal diameter of 75 μm , all samples were analyzed in either 5 mm or 100 mm borate buffer ( ph = 8 . 3 ). specimens of cocaine ( 0 . 5 mm ) were incubated at room temperature with either horse serum ψ - cholinesterase ( 25 units ; sigma chemicals ), or purified rabbit antibodies . material was loaded onto the column using low ( 0 . 5 psi ) pressure injection ( 4 seconds long ) and separated under influence of 24 kv at 25 ° c . peaks were read at 200 nm and analyzed using beckman system gold software ( version 7 . 01 ). this example shows the effect of immunization of mice with the different hapten carrier conjugates . twenty balb / c mice ( female ; 4 - 6 weeks old ) were each immunized subcutaneously with 50 μg of hapten - conjugates ( cf . table 1 below ) in pbs ( ph = 7 . 2 ; 25 μl ) emulsified with an equal volume of freund &# 39 ; s complete adjuvant on day 0 . all animals were boosted intraperitoneally with an equivalent amount of the corresponding hapten - conjugate emulsified this time with freund &# 39 ; s incomplete adjuvant on day 28 . all animals were bled from the retro - orbital plexus on day 42 ( 2 weeks after boosting ) and sera were tested for hapten - specific antibodies by elisa . the same four different hapten - conjugates , as were used with the rabbits , were tested in each of four different groups of five animals each as summarized in table 1 below . fig3 shows graphically the results of the determination by elisa titration analysis of the level of anti - cocaine analog activity responses in individual mice , with each mouse being indicted by a separate symbol , to immunization by one of the different hapten - carrier conjugates . the upper panel a illustrates the anti - hapten response to the klh - long ( conjugate 14a ) and klh - short ( conjugate 12a ) conjugates assayed on the bsa - long ( conjugate 14b ) conjugate . the lower panel b illustrates the anti - hapten response to the bsa - long ( conjugate 14b ) and bsa - short ( conjugate 12b ) conjugates assayed on the klh - long ( conjugate 14a ) conjugate . collectively , the data presented in fig4 indicate that the cocaine analog hapten - protein carrier conjugates are more effective at inducing anti - hapten responses when the analog is conjugated to the carrier protein by the long linker . this example shows the effect of immunization of rabbits with the different hapten carrier conjugates . eight new zealand white rabbits ( female ; 2 . 5 kg each ) were immunized in three sites ( one subcutaneous , two intramuscular ) with a hapten - conjugate ( 500 μg ) in pbs ( ph = 7 . 2 ; 250 μl ), emulsified with an equal volume of freund &# 39 ; s complete adjuvant on day 0 . all animals were boosted with an equivalent amount of the corresponding hapten - conjugate emulsified this time with freund &# 39 ; s incomplete adjuvant , again in three different sites ( one subcutaneous , two intramuscular ) on day 28 . all animals were bled from the marginal ear vein on day 42 ( two weeks after boosting ) and sera were tested for hapten specific antibodies by elisa . four different hapten - conjugates were tested , each in two rabbits , as summarized in the following table 1 : table 1______________________________________hapten - conjugate recipients______________________________________14a ( klh long linker ) rabbits # 1 & amp ; 2 ; five mice12a ( klh short linker ) rabbits # 3 & amp ; 4 ; five mice14b ( bsa long linker ) rabbits # 5 & amp ; 6 ; five mice14b ( bsa short linker ) rabbits # 7 & amp ; 8 ; five miceunimmunized control rabbit # 9 ; five mice______________________________________ this example illustrates an assay by capillary electrophoresis of the degradation of cocaine by the enzyme ψ - cholinesterase . capillary electrophoresis was used to monitor the rate of enzymic degradation by 25 units of horse ψ - cholinesterase ( sigma ) of 0 . 5 mm cocaine at 25 ° c . in borate buffer ph 8 . 3 into products of the ester hydrolysis , namely methyl ecgonine and benzoate ( scheme 1 , fig2 a ), by measuring the diminishing area under the cocaine peak and the growing area under benzoate peak . the results obtained are shown in the upper panel a of fig5 . a representative tracing of the capillary electrophoresis pattern for one of these time points illustrated in the lower panel b of fig5 . this example illustrates an assay by capillary electrophoresis of the degradation of cocaine by antibodies from the hapten - conjugate - immunized rabbits . a capillary electrophoresis analysis of the degradation of cocaine by purified rabbit antibodies isolated from a control unimmunized rabbit and rabbits immunized by the bsa - long conjugate ( conjugate 14b ), as in example 8 above . the results obtained are shown in fig6 . the upper panel a shows the rate of degradation of 0 . 5 mm cocaine at 24 ° c . in borate buffer ph 8 . 3 for two immunized rabbits and that the breakdown was significantly greater for such rabbits than for the breakdown of cocaine alone in buffer . in the lower panel b , the immunized rabbit antibodies were also more effective than antibodies from an unimmunized animal , which was not significantly different from buffer alone . these data show that the animals immunized with the bsa - long conjugates of the cocaine analog possessed antibodies which were able to catalyze the breakdown of cocaine . while the present invention has been described with reference to specific embodiments thereof , it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention . in addition , many modifications may be made to adapt a particular situation , material or composition of matter , process , process step or steps , or then present objective to the spirit of this invention without departing from its essential teachings . 1 . f . i . carroll and a . h . lewin , in emerging technologies and new directions in drug abuse research ( r . s . rapaka , a . makriyannis , and m . j . kuhar , eds . ), nida research monograph # 112 , superintendent of documents , u . s . government printing office , washington d . c . 1991 , 284 - 299 . 2 . k . d . janda , s . j . benkovic , and r . a . lerner , science 244 , 437 - 440 ( 1989 ). 3 . s . j . pollack , p . hsiun , and p . g . schultz , j . am . chem . soc . 111 , 5961 - 5962 ( 1989 ). 4 . n . s . chandrakumar , c . c . carron , d . b . meyer , p . m . beardsley , s . a . nash , l . l . tam , and m . rafferty , bioorg . med . chem . lett . 3 , 309 - 312 ( 1993 ). 5 . d . w . landry , k . zhao , g . x .- q . yang , m . glickman , t . m . georgiadis , science 259 , 1899 - 1901 ( 1993 ). 6 . m . y . meah , d . l . skea , w . a . corrigall , b . h . barber , and j . j . krepinsky , unpublished observations .
methods are described for the rapid synthesis in satisfactory yield of methyl ecgonine phenylphosphonates as analogues of transition states for the hydrolysis of the benzoyl ester of an ecgonine derivative , namely cocaine , and their linking to carrier proteins , for the purpose of using them as immunogens . the resulting immunogens elicit the formation in experimental animals of antibodies able to promote the hydrolysis of cocaine . both these catalytic anti - cocaine antibodies and the immunogens themselves are potentially useful for the treatment of individuals seeking to avoid the pharmacological effects of cocaine and in diagnostic applications .