CA2027022A1 - Compounds useful for measuring low levels of sulfonylureas by immunoassay - Google Patents

Abstract

COMPOUNDS USEFUL FOR MEASURING
LOW LEVELS OF SULFONYLUREAS BY IMMUNOASSAY
Abstract of the Disclosure Sulfonylurea carboxylic acids, their protein conjugates, and polyclonal antibodies produced in response to these conjugates are useful, particularly in kit form, for measuring the presence of very low levels of sulfonylurea herbicides and their derivatives in an enzyme-linked immunosorbent assay.

Description

TITLE ~A-8811-A
COMPOUNDS USEFUL FOR MEASURING
LOW LE~EL5 OF SULFONYLUREAS BY IMMUNOASSAY

~ackqr~und o the In~entiGa Field of ~he InventiQn This invention pertains to certain sulfonylurea carbo~ylic acids which have been found useful in an enzyme-linked i~munoabsorbent assay (ELISA) technique for determining the presence of even very low levels of sulfonylurea compounds in ground water and other matrices. This invention also concerns protein conjugates of sulfonylurea carbo~ylic acids, antibodies produced in response thereto and an ELISA
kit for determining the presence of sulfonylureas at low levels.

State ~f the Alt Each of the following publications describes sulfonylureas whose presence can be detected by the improved ELISA method of this invention or conjugated to proteins according to the teachings herein and employed in said ELISA:-U.S. 4,394,506 U.S. 4,394,506 - U.S. ~,481,029 EP~ 87,7B0 EP 95,925 EPA 161,211 -- U.S. 4,435,206 -- Japan 63~166j803A
U.S. 4,522,64S U.S. 4,684,395 U.S. 4,420,325 EP 87,780A.
DE 3,016,831 discloses synthesis of conju~ates of simple peptides and other organic compounds, but not sulfonylurea-derived compounds.
3S The ELISA procedure is a well-known method for detecting pesticide resi~ues in the parts per billion range in soil or water; Wie et al., J. Aaric. Food Chem., ~2, (1984) pages 1294 to 1301.
Kell~y et al., J.~Aq~ic. Food Chem., 33, (1985) pages 962 to 965 disclose a sulfonylurea immunoassay using an ELISA techni~ue.
Synthesis of conjugates often used in immunoassays is described in "The Handbook of E~perimental Immunology,~ 4th Ed., Vol. I, NImmunochemistry~, Weir, Ed. Chapter 3, ~Haptens and Carriers," Makela et al., Blackwell Scientific Publications, Ogford, 1986.
An ELISA technique relies on the specific interaction of the antibody molecule with substances called antigens. The introduction of an antigen into the body of a vertebrate animal triggers the animal's immune system to generate antibodies that will bind the antigen. To be an effective antigen, a molecule must have two attributes. It must have a site that can bind to the cell-surface antibody of a virgin B
cell and it must have a site that promotes cell-cell interactions between various cells of the immune system. Smaller molecules (haptens) such as the sulfonylureas seldom have both of these attributes and are generally nct good antigens. To overcome this problem, haptens are generally covalently linked to proteins, called carriers, which provide the sites that promote cell-cell interactions in the immune system. The immunized animal~s response to such hapten-carrier-protein antigens is polyclonal. Other relevant factors in an ELISA techni~ue will be described hereafter in relation to this invention.
Immunoassays for pesticides have been in the literature for a number of years (Van Emon et al., Analytical Methods for Pesticides and Plant Growth Regulators, Vol. XCII, 1389, pp. 217 to 263). Many analytical methods have been developed for clinical monitoring of pesticide e~posure, primary among them are the Enzyme Immunoassays (EIA). Because pesticides are small molecules, usually of molecular weights between 100 and lO00, they alone do not induce the immune response and mus~ be attached to larger proteins. Essential ~o the design of these methods has been the hapten which resembles the compound of interest but which contains an additional functional group for the attachment of the hapten to the protein; and the immunogen, which must be lS selected to give optimum immune response and antigen recognition.
Su~mar~ of thQ Invention This ir,vention concerns certain of the compounds of Formula I, protein conjugates of the compounds of Formula I, and the polyclonal antibodies produced by the e2posure of mammals to the conjugates.
This invention also concerns an ELISA method for measuring very low concentrations of sulfonylureas (the "analytes~), e.g., from less than 10 up to lO,000 picograms per milliliter (pg/mL).
This invention also concerns kits for the measurement of sulfonylureas comprising coating-conjugate-coated solid phase and sufficient guantities of premeasured first antibody, second (labeled) antibody, enzyme substrate, and control samples.
The compounds of this invention are herbicidal sulfonylureas sr derivatives, with a carbo$ylate group added to any methyl, methylene or methine carbon o said sulfonylurea that results in a water-stable compound, said carbo~ylate being tethered with a 0-3 atom chain wherein the chain is made up of carbon atoms, and, if the tether is 2 or three atoms, one can be a sulfur, nitrogen or o~ygen atom.
Compounds useful in the improved ELI5A method of this invent~on comprise compounds of Formula I, including their agriculturally suitable salts:

Il .

R

wherein J is ~ R ~

~-1 J-2 J-3 ~' ~e ' R

J_~ J-5 G is H or M02C(alkyl)nI.;
n is 0 or 1;
alkyl is 1 to 3 carbon atoms optionally substituted with one or two of halogen, methyl, metho~y, or methylthio;
L is O; S, NR5, -~3(CYO)_ or a direct bond;
R, R4 and R5 are independently H or CH3;
E is a single bond or CH2;
R is H~ Cl to C3 alkyl, Cl to C3 haloalkyl, halogen, nitro, Cl to C3 alko~y, SO2NRaRb, CONR~Rb, Cl to C3 alkylthio, C1 to C3 alkylsulfinyl, C1 to C3 alkylsulfonyl, CH2CN, CN, C02RC, Cl to C3 haloalko2~y, Cl to C3 haloalkylthio, C2 to C4 alko~yalkyl, C3 to C4 alko~yalko~y, C2 to C~,~ alkylthioalkyl, CH2N3, NRdRe, or Q;
R2 is H, Cl to C3 alkyl, Cl to C3 haloalkyl, halogen, nitro, Cl to C3 alkoxy, Cl to C3 alkylthio, CN, Cl to C3 haloalko~y, or C2 to C4 alko2~yalkyl;
Ra is H, Cl to C4 alkyl, C2 to C3 cyanoal3cyl, 2 5 methol~y o r etho2~y;
Rb is H, Cl to C4 alkyl or C3 to C4 alkenyl; or Ra and Rb may be taken together as -(CH2)3-, (CH2)4-~ ~CH2~)5- or CH2cH2ocH2cHz-;
Rc is Cl to C4 alkyl, C3 to C4 alkenyl, C3 to C4 alkynyl, C2 to C4 haloalkyl, C2 to C3 cyanoalkyl, C5 to C6 cycloalkyl, C4 to C7 cycloalkylalkyl or C2 to C4 allco:cyalkyl;
Rd and Re are independently H or Cl to C2 alkyl;
Q is a saturated 5- or 6-membered ring containing one heteroatom selected f rom O, S, or N, tetrazole optio~ally substituted with Cl-C3 alkyl, or an unsaturated 5- or 6-membered ring containing 1 to 3 heteroatoms selected from 0-1 S, 0-1 0 or 0-3 N and when Q is an unsaturat~d 5- or 6-membered ring, it may optionally be substituted by one or more groups selected from Cl to C4 alkyl, halogen, C3 to C4 alkenyl, Cl to C3 alko~y, C1 to C3 alkylthio, C3 to C4 alkenylthio, Cl to C2 haloalkoxy or Cl to C3 haloalkylthio;

A i9 (E~ Z

X is H, Cl to C4 alkyli Cl to C4 alko~y, Cl to C4 haloalko~y, Cl to C4 haloalkyl, halogen, C2 to C5 alkoxyalkyl, C2 to C5 alko~yalkoxy, amino, C1 to C3 alkylamino, di~Cl to C3 alkyl)amino or C3 to C5 cycloalkyl: or Cl to C4 alkyl, C1 to C4 alko~y or Cl to C4 haloalko~y substituted on the alkyl, alko~y or haloalko~y group with C02M;
Y is H, Cl to C4 alkyl, C1 to C4 alko~y, Cl to C4 haloalko~y, C2 to C5 alkosyalkyl, C2 to C5 alko~yalko~y, amino, Cl to C3 alkylamino, di(Cl to C3 alkyl)amino, or Cl to C4 alkyl;
M is H or an alkali or alkaline earth metal salt;
R3 is H or Cl to C3 alkyl;
Z is CH, N or CCO2M; and El is a direct bond or CH2 Compounds of this invention are those of Formula I wherein:
(i) when G is not H and when L is a direct bond, then n ~ l;
(ii) when G is H, then J is J-l, J-4 or J-5;
(iii) when G is H and ~ is J-4 or J-5, then X
is Cl to C4 alkyl, Cl to C4 alko~y, or C
to C4 haloalko~y substituted with CO2M;
and ~iv) when G is H, and J is J-1, then E is CH2, and X is Cl to C4 alkyl, Cl to C4 alkoxy, or Cl to C4 haloalko~y substituted with C02M .

The preferred ELISA method of this invention employs compounds of the following formulae:

oc~

~I~'CN~C 1- ( 1 ) ~N~

c~

~6 NH NH~ ~2`CXH ~2) ~
,,N~

Cl ~,Jl~,,;~ ~4) OC~ C~) a ' ;~ cH3)2 H~OC 1 ~) (6) Cl ~H3 ( ) :

~[~ ~H3 C8 ~OC

2 0 _~ ( 9 ~ ' ' ` .
~00 ~H~

~H2CH3 OOC:H3 10 ~oc~NH~}l (11) ooc}~

$~ HzSO2 ~ ( 12) O~CH3 ocH2co2H

~H~SO~N~ONH~ N

"C~CH~
CO2~

The conjugates of this invention comprise the compounds of Formula I bound, through a carboxyl group, to proteins that are capable of eliciting an immune response in at least one mammal selected from: mice, rabbits, goats, dogs, horses, sheep, guinea pigs, chickens, hamsters, and rats. Any protein or molecule whose introduction into an animal can lead to T-cell proliferation and differentiation can be used as a carrier protein or its equivalent.
E3amples of proteins useful in immunogen synthesis are keyhole limpet hemocyanin (KLH~, pumpkin seed globulin (PSG), marijuana seed globulin ~MSG~, ovalbumin (OVA), or serum albumin from cows (BSA~, rabbits, or mice.
The ~oating conjugates of this invention are independently characterized by their ability to adhere to thP solid phase Pmployed in Ste~ B of the method described hereafter and their ability to comple2 with the antibody of Step A. By "coating conjugate" is- meant a ~apten chemically conjugated to a protein, sometimes called Ucoating antigenn.
Preferred coating conjugates are compounds selected from 1 to 13 covalently linked by methods described herein to keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin. Other useful proteins will suggest themselves to one skilled in the art.
The antibodies of the invention are polyclonal antibodies produced by mammals in response to the injection of sulfonylurea-protein conjugates described above. These antibodies bind with high affinity to the conjugate described herein and to the sulfonylurea that is the subject of the ELISA.
The improved enzyme-linked immunosorbent assay for detecting the presence of a sulfonylurea in an unknown sample comprises the steps:
(A) forming a comple~ of the sulfonylurea with an e~cess of a first antibody of known concentration, (B) comple~ing the unbound first antibody from Step A with a coating conjugate adhered to a solid phase, (C) binding a labeled antibody to the antibody-conjugate comple2 of B, and (D) determining the presence and the amount of sulfonylurea in the unknown sample ~y measuring the amount of unbound antibody in Step A with reference to controls which comprise known concentrations of the sulfonylurea;
wherein the improvement comprises (i) employing a conjugate of this invention as an immunogen to generate the antibody in Step A; and (ii) employing the same or a different conjugate of this invention as coating conjugate in Step B.

This invention also concerns a test kit, comprising the ingredien~s necessary to run an ELISA
S measurement on target sulfonylureas. The test kit has these components:
(i) an antibody to the sulfonylurea in the unknown;
tii) a solid phase having a coating conjugate O bound to it;
(iii) a labeled a~tibody that recognizes antibody (i);
(iv) a developer that develops color in the presence of a label; and ~v) controls comprising at least one known concentration of the sulfonylurea and one containing no sulfonylurea; whereby the components cooperate so that i, ii and iii are contacted with each other and, upon addition of iv, develop a color which indicates, upon comparison to the controls, the presence and concentration of the target sulfonylurea.
Using the procedures and the kit described herein, the following sulfonylurea herhicides can be detected:
2-chloro-N-t(4-metho~y-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzenesulfonamide ~chlorsulfuron) methyl 2-t[~[(9,6-dimethyl-2-pyrimidinyl)amino]-carbonyl]amino~sulfonyl]benzoate (sulfometuron methyl) methyl 2- E [ [ t ( 4-metho~y-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate (metsulfuron methyl) 2-[[N-(4-metho~y-6-methyl-1,3,5-triazin-2-yl)-N-methylamino]carbonyl]amino~sulfonyl]benzoic acid, methyl ester (tribenuron methyl) ethyl 2-[~[[(4-chloro-6-methosy-2-pyrimidinyl), amino~carbonyl]amino]sulfonyl~benzoate (chlorimuron etnyl~
2-[[(4-ethoxy-6-methylami~o-1,3,5-triazin-2-yl~aminocarbonyl~aminosul~onyl~benzoic acid, methyl ester (ethametsulfuron methyl) 2-[[(4,6-dimetho~y-1,3,5-triazin-2-yl)aminocarbonyl]-aminosulfonyl]-4-~2,2,2-trifluoroetho~y)benzoic acid, ethyl ester 4-chloro-2-[t(4-metho~y-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]aminosulfonyl]benzoic acid, -isopropyl ester 3-[[[[(4-metho~y-6-methyl-1,3,5-triazin-2-yl)amino]-~arbonyl]amino]sulfonyl]-2-thiophene carbo~ylic acid, methyl ester (thifensulfuron methyl) 0 methyl 2-[[[t(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]amino]sulfonyl]methylbenzoate (bensulfuron methyl) 2-tt(4,6-dimetho~ypyrimidin-2-yl)aminocarbonyl]-aminosulfonyljN,N-dimethyl-3-pyridinecarbo~amide 5 2-[[(4,6-dimetho~ypyrimidin-2-yl))aminocarbonyl]-aminosulfonyl~-3-pyridinecarbo~ylic acid, methyl ester N-t(4,6-dimetho~ypyrimidin-2-yl))amino~rbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide 0 N-t(4,6-dimethosypyrimidin-2-yl))aminocarbonyl]-2,3-dihydro-2-methyl-b~nzo~b)thiophene-7-sulfonamide, 1,1 dio~ide 2-tttt~4,6-bis(difluorometho~y)-Z-pyrimidinyl]-amino]carbo~yl]amino]sulfonyl]benzoic acid, methyl ester ethyl 5-t3-(4,6-dimethoxYPYrimidin-2-Yl)ureido-sulonyl] 1-methylpyrazole-4-carboxylate N-[(6-methoxy-4-methyl-1,3~5-triazin-2-yl)amino~
carbonyl]-2-(2-chloroetho~y)benzene sulfonamide N-[(4~6-dimetho2y-l~3~s-triazin-2-yl)amino-carbonyl3 2-(2-methosyetho~y)benzenesulfonamide N-l(4,6-dimetho~ypyrimidin-2-yl)-amino]carbonyl]-3-trifluoromethyl-2-pyridinesulfonamide.
Preferred sulfonylurea target pesticides are chlorsulfuron, bensulfuron methyl, metsulfuron methyl and chlorimuron ethyl. The method for using the kit to detect the target sulfonylurea comprises contacting components i, ii, iii and iv and com~aring the color that is developed to controls, v, thereby determinin~ the presence and concentration of the target sulfonylurea.

D~ils of the Inv~ntion ~ynthe$is Compounds of Formula I can be synthesized by reaction of sulfonamides with the phenyl ester of the appropriate carbamic acid in the presence of an equimolar quantity of a tertiary amine base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as shown in Equation 1.
~uation 1 ~ 1) DBU
JS2NH2 PhOC~-A 2~ H30 Sulfonamides of Formula II and sulfonyl chlorides of Formula III are defined below:

R1 ~ Rl ~ Oz D

OzD

D = NH2 or Cl D = NH2 or Cl G Rl R~ S02D ~02l~1HZ

D = N~ or Cl Rl G
~E50aNHa 3~
II D, NH2;
III 1) ~ Cl.

Sulfonamides of Pormula II can be conv~rted to homologated sulfonamides of Formula IY by procedures such as those shown in Eguation 1 according to the methods reviewed in ~eier and Zeller, ngew._~h~m~
Inc. Ed. Enal., 1~, (1975) pages 32 to 43; or Equation 2 according to the methods reviewed in Johnson, "Ylid Chemistry, n Academic Press, New York, 1966; or by other methods known to one skilled in the art.
Equ~tion 1 SC12 CH2N2 ~92 H2N02S-Ar-L(alkyl)nC02H or ~COCl)2 /
II ~
H2No2s-Ar-L(alkyl)ncH2co2H (1) IV

Equation 2 tH]
H2N02S-Ar-L~alkyl)~C02H ~ H2NO~S-Ar-L(alkyl)nCHO
Il Ph P~CH(CH2) C02H
3 ~ : H2~02S-Ar-L(alkyl)nC~CH(CH2)nC02H (2) [H]
H2No2s-Ar-L(alkyl)n-~H2cH2(cH2~nco~H

where n . 0, 1.
Sulfonamides of Formula II can also be prepared as shown in Eguations 3 and 4 according to the methods described in Patai, "Chemistry of the Ether Linkage Interscience," New York, 1967 and Reid, ~Organic Chemistry of Bivalent Sulfur, n Vol. l, Chemical Publishing Company, ~ew ~ork, 1958, or by other methods known to one skilled in the art.
Equation 3 Base H2N02SArSH ~ BrCH2(alkyl)nC02H ~

H2NO2SArS- ~ lkyl)nCO2H ~3) Equa~iQn 4 Base H2NO2SArOH + BrCH2(alkyl)nCO2H ~ ~

H2NO2SArO-CH2~alkyl)nCO2H (4) II
Sulfonamides of Formuia II can also be prepared from suitably activated aromatics by the methods shown in Equation~5 such as those reviewed in Zoltewicz, To~. Curr. ~hem., ~ 1975) page 33 or by other methods known to one skilled in the art.
Equation 5 Ho2c-(alkyl)nL
~2NO2S-Ar-halogen _ H2No2s-Ar-L(alkyl)nco2H (5) Sulfonamides of Formula II can also be prepared by the methods shown in Equation 6 ~where L is O, NH
or S) or by other methods known to one skilled in the art.

Equa~ion 6 ( ~ o , H2NO2S-Ar-LH

O

H2NO2S~Ar-L-c(O)ccH2)nc~2c~H (6) The preparation of sulfonamides from sulfonyl chlorides is widely reported in the literature; for reviews see Hawking et al., ~The Sulfonamides, n Lewis and Co., London, 1950 and Northey, ~The Sulfonamides and Allied Compounds,~ Reinhold Publishing Corp., New York, 1948.
Additionally, primary sulfonamides, such as those of Formula II, can be formed by removal of an N-~-butyl protecting grQup from the corresponding secondary sulfonamide with trifluoroacetic acid, Catt et al~, J. Orq. Chem., 39 (1974), 566 or polyphosphoric acid, Lombardino, ~ _QIs__Çh~m~
(1971), 1~43.
The requisite sulfonyl chlorides of Formula III
can be synthesized by known methods or with slight modifications thereof, by one skilled in the art.
Several representative teachings are listed below.

- Aromatic nitro groups can be transformed into sulfonyl chlorides by reduction, diazotization and coupling with sulfur dio~ide/cupric chloride as taught in U.S. Patent 4,310,346.
European Publication No. 9~,821 discloses the displacement of aromatic halides with thiolate anions and subsequent o~idative chlorination to yield sulfonyl chlorides.
Halogen-metal e~change of aromatic halides or proton-metal e~change of aromatics followed by quenching with sulfur dio~ide gives sulfinate salts.
these salts yield sulfonyl chlorides upon reaction with N-chlorosuccinimide as taught in U.S. Patent 4,481,029. Directed proton-metal exchange of aromatic compounds has been reviewed by Gschwend and Rodriguez, Or~. ~eactions, 26 (1979), 1. Directed lithiation of aryl-N-t-butylsulfonamides is described by Lombardino, J. Orq. Ch~m., ~6 (1971), 1843. Also, aryllithiums may be converted directly to arylsulfonyl chlorides with sulfuryl chloride as described in Bhattacharya et al., J. Chem. Soc. C., (1968), 1265.
Electrophilic chlorsulfonation of an aromatic ring to give a sulfonyl chloride is well known in the literature. This technique works best for alkyl aryl ethers and alkyl aromatics. Its application is described by Huntress et al., J~ Am. Chem. Soc., 62 (1940), 511 to 514 and 603 to 604.
Transformation of phenols to sulfonyl chlorides can be accomplished by the formation of a thiocarbamate, rearrangement, hydrolysis and o~idative chlorination as described by Newman et al., ~. OrqL Chem., ~1 (1966), 3980.

PROCEDVRE A

3-rr(4-Metho~y-6-methyl-1,3 5-tri2~in-2-Yl~amino-~arbonyllamino5~1fonYllbenzoi~_~çid (C~mDound 9) To a solution of 2.00 g (9.95 mmol) of 3-(aminosulfonyl)benzoic acid tPrePared by chlorosulfonation of benzoic acid according to Thurber et al., ~ et. Chem., 19, (1982) 961 and reaction with ammonia] and 2.59 g (9.95 mmol) of phenyl 4-methoxy-6-methyl-1,3,5-triazin-2-yl-carbamate in 50 mL of acetonitrile was added 2.97 mL
(14.9 mmol) of 1,8-diazabicyclo[5.4.0~undec-7-ene dropwise. After 1 hour, 50 mL of water and 25 mL of 1~ HCl were added. The resulting solid was isolated by filtration and was dried to give l.S g of the title compound in 90% purity (contaminated with the starting sulfonamide). A 0.5 9 sample of this material was purified by trituration with 2-propanol to give 0.4 g of a white solid; m.p. 169 to 172C.

~EDU~
3-Chloro-4 r r ~4-methoxy-6-methYl-1.3.5-triazin-2-Yl!-aminocarbonYllamino ~l~onYllbenzoic (Com~ound 8) To a solution of 2.00 g (8.5 mmol) of 4~aminosulfonyl-3-chlorobenzoic acid and 2.20 9 (8.5 mmol) of phenyl 4-methoxy-6-methyl-1,3,5-triazin-2-yl-carbamate in 45 mL of acetonitrile was added 2.54 mL
(17 mmol) of 1,8-diazabicyclot5.4.0]undec-7-ene dropwise. The reaction mi2ture was stirred for 2 hours. 45 mL of water was added. Upon the dropwise addition of 25 mL of 1~ HCl, a precipitate formed.
The solid was filtered.off and dried to give 2.05 g of the title compound as a white solid; m.p. 132 to 13SC.

PROCEDURE ~

2-Amino~ benzenedicarbo~xlic açid l-methYl ~ster A solution of 7.0 g of 2-nitro-1,4-benzene-dicarbo~ylic acid, l-methyl ester (prepared according lS to Ger. Offen 3,001,695, C.A. ~:13252Sw) and 3.S g of 10% palladium on carbon was placed under 50 psig of hydrogen for 16 hours. The solution was filtered. The filtrate was evaporated to give 5.3 g of the title compound as a yellow solid; m.p. 164 to 167C.

PROCEDU~E ~

2-(t-Butylaminosulfonyl)~ benzenedicarbo~ylic acid. l-methy1 esteL
The compound of Procedure C, ?..0 9 ~10.9 mmol) was slurried in 5 mL of acetic acid. With cooling, 20 mL of concentrated HCl was added. The reaction mi~ture was cooled to -10C and a solution of 0.B3 9 (12 mmol) of sodium nitrite was added dropwise. The r~action mi~ture was stirred 20 minutes.
This solution was poured into a flask containing 50 mL of acetic acid, 0.5 g CuCl and 4 mL
of liquified sulfur dio~ide at 0C. After stirring 2 hours, the reaction was e~tracted with dichloro-methane. The organic layer was dried and all of the volatiles were removed with a rotary evaporator.

The residue was dissolved in 150 mL of dichloromethane and cooled to -7BC. 8 mL of S t-butylamine was added dropwise. The reaction was allowed to warm to room temperature. It was washed with water, dried and the solvent was removed with a rotary evapora~or. This procedure gave 2.3 g of the title compound as a tan solid; m.p. 165 to 171C.
PRO~EDURE E

2~(AminosulfQnYl)-l,q-benzenedicarbox~lic acid, l-methvl ester To 2.0 g of the material from Procedure D was added 20 mL of trifluoroacetic acid. The solution was stirred for 2 hours. The volatiles were removed with a rotary evaporator. The residue was triturated with l-chlorobutane to give 1.5 g of the title compound as a tan solid; m.p. 211 to 216~C.

PROCEDURE F

2-r r ~4-Metho~Y-~m~hvl-1,3,5-triazin-2-Yl)amino-carbonyllaminQ~ulfQn~Ll-1,4-benzen~dicarbo~ylic, l-meth~l ester (Compoun~ 3) By the procedure given in Procedure B, 1.00 g of the material of Procedure E was converted to 1.25 g of the title compound as a white solid; m.p. 165 to 169C(d).

pROCEPURE G

~-r r (4-~thoxy-6-(methylamino)-1,3,5-tLiazin-2~
aminocarbonyllamino$ul~onv~ ,4~~en~enedicarbo~Ylic acid, l-methyl e~ter (Compound lQ) By the procedure given in Procedure ~, 200 mg of the material from Procedure E was reacted with 235 mg of phenyl 4-etho~y-6-methylamino-1,3,5-triazin-2-yl-carbamate to give 250 mg of the title compound as a white solid; m,p, 202 to 205C, P~Q~pURE H

2-rr(4.6-~ çthvl-1,3.5-triazin-2-yl)aminQcarbQnyll-amino~ulfQnyll-l,~-benzenedicarbo~Ylic acid, l-methyl ester (CQm~o~nd 11) By the procedure in Procedure B, 200 mg of the material from Procedure E was reacted with 190 mg of phenyl 4,6-dimethylpyrimidin-2-yl-carbamate to give 120 mg of the title compound as a white solid; m.p, 145 to 150C, PROCEDURE I
2-r r (4-Chloro-6-metho~ypyrimiain-2-yl)aminocarbonvll-ama~os~L~onyll-1.4-benz~nedicarbo~ylis~acid.
l~eth~l ester (~om~ound 4~

~y the procedure given in Procedure ~, 1,0 g of 2-(aminosulfonyl)-1,4-benzenedicarboxylic acid, l-ethyl ester and 1,03 g of phenyl 4-chloro-6-metho~y-pyrimidin-~-yl-carbamate were reacted to give 90 mg of the title compound as a white solid; m.p. 125 to 128C.
~ROCE~RE J
Phenylmethyl ~-(4~aminQ-6-metho~y~ .5-triazin-2-Ylo~y~-pro~anoat~

A 33 g (83 mmol) portion of 60% sodium hydride in mineral oil was washed with he~anes and 75 mL of THF was added. After cooling to 0C, 15 g ~83 mmol~
of phenylmethyl 2-hydro~ypropanoate was added dropwise. After 1 hour at room temperature, the reaction mi~ture was cooled to 0C. 7 9 ~94 mmol) of 4-chloro-6-metho~y-pyrimidin-2-yl-amine was added portionwise over a 15 minute period. After stirring 16 hours at room temperature, dichloromethane was added to the reaction. It was washed with water, dried and the volatiles weIe removed with a rotary evaporator.
Half of the residue was purified by flash chromatography to give 5.5 g of the title compound as a glass, which solidified upon standing; m.p. 87 to 92C.
P~OCEDURE K

l-Phenylmethyl 2-r~-ph~nvlcar~amoyl)-4-~mino-6-metho~y-l 3,5-~riazi~-2-ylo~yl~ropa~oate To a solution of 5.0 9 (17 mmol) of the material from Pro~edure J in 90 mL THF at -78C 2.9 mL (19 mmol) of phenylchloroformate was added. 38 mL
(38 mmol) of a 1~ solution of lithium bis(trimethylsilyl)amide was added dropwise over 30 minutes. After stirring for 15 minu~es at -78C, the reaction was allowed to warm to -25C. 2.2 mL o~
acetic acid was added. The reaction mi~ture was poured into ice/water, made acidic with acetic acid and extracted with ether. The organic layer was dried and the solvent was removed with a rotary evaporator.
The residue was purified by flash chromatography to give 4.34 9 of the title ~ompound as a gummy white solid.
IR (XBr) 1755, 1790 cm~l.
lH-NMR (CDC13) ~ 1.64 (d, 3), 3.93 (s, 3), 5.17 (d, 2), 5.35 (q, 1), 7.27 (m, 10), 7.9 (s, 1).
PRQ~EDURE L
MethYl 2-r r r r4-(1-carbo~yetho~y~-6-methoxY-1.3,5-triazin-2-YllaminQcarbonyllaminosul~onyll-methYllkenzoate (Comsou~d l~

By the procedure given in Procedure B, 1.77 g (4.4 mmol) of the material from ~rocedure ~ was reacted with 1.0 g (4.4 mmol) of methyl 2-aminosulfonylmethylbenzoate. No precipitate was obtained so the reaction was e2tracted with dichloromethane. The organic layer was dried and the organics were removed with a rotary evaporator.
Half of the residue was dissovled in 40 mL oE
ethanol. One gram of 10% palladium on carbon was added. The reaction mi~ture was placed under 50 psig of hydrogen for 2 hours. After filtration the volatiles were removed with a rotary evaporator. The residue was triturated with l-chlorobutane to give 200 mg of the title compound as a white solid; m.p.
152C(d).

p~OCEDURE
2-r4-r r r3-r (~imeth~amino)ca~bon~ PY~idinYl=
s~lonyLlaminocarhonYllaminoL~-metho~y~
trLazin-2-Ylosylpropanoic acid ~Com~ound 1) By the procedure given in Procedure B, 1.22 9 (2.90 mmol) of the material from Procedure X and 0.66 9 (2.9 mmol) 2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide was reacted to give 80 mg of the title compound as a white solid; m.p. 135 to 140C.

Using Procedures A to ~ described above, the compounds ;n Tables 1 to 8 can be prepared.
General_FQLmula for Tables 1-8 R
Table 1 ~ O2NH~NI ~ ~

R Y

O X

Rl ~
Table 2 II

2 0 Table 3 ~SOzNHCNH ~Z

III

3 0 Table 4 ~502N~lCNH ~(~z R3 y IV

~bl~ 5 $~~ ~Z
~3 Rl ~,NE~CON~I~((~Z
l~ble 6 b~ y VI

Rl R~ X

3 OzN~ Z

VII

~ ~ I I ~ N ~

Table 8 y VIII

M~lting points (in C) of compounds footnoted in the Tables are as follows with the number before the / indicating the footnote number: 1/165 to 169(d), 2/202 to 205, 3/125 to 128, 4J145 to 150, 5/207 to 212, 6/157 to 158, 7/147 to 148, 8/130(d), 9~163 to 164(d), 10~142 to 145(d), 11/154 to 157, 12/177 to 178td), 13/142 to 144~d), 14/142 to ls4(d)~
15/207:to 209(d), 16/173 to 175, 17/155 to 157td), 18/162 to 164(d), 19/155 to 156, 20~135 to 137, ~1/148 to 150, 22/143 to-145, 23/200 to 202, 24/l9S
to 199, 25/192 to 194, 26/17~ to 182, 27/139 to 142, 28/167 ~o 171, 29~145 ~o lS0, 30/165, 31~132 to 136, 32~156 to 158, 33~15Ztd), 34fl76 to 182, 35/182 to 187~d), 36~161 to 162.5, 37~142 to 145, 38~195 to 197, 39~198 to 201, 40/132 to 135, 41/135 to 140 and 42/169 to 172.

~L~

9~aeral FQrmula I

~2 ~ ~ ~ Y

H H H 3-CO2H ~ CH3 OC~3 N~42 H H H 3-CO2H ~ CH3 OCH3 CH
H H H 3-CO2H - OCH3 OC~3 N
H H H 3-CO2H ~ OCH3 OCH3 CH

H H H 3-CO2H ~ C~3 CH3 CX
H H H 3-CO2H - Cl OCH3 CH

H H H 4-CO2H ~ C~3 OCH3 N
H 2-Cl H 4-CO2H ~ CH3 OC~3 CH
H 2-CO2CH3 H S-CO2~ ~ C~3 OCH3 N(l) H 2-CO2CH3 H 5-CO2H ~ CH3 OCH3 CH
H 2-CO2CH3 H 5-CO2H ~ OCH3 OCH3 N
H 2-CO2CH3 H 5-CO2H ~ OC~3 OCH3 CH
H 2-CO2CH3 ~ 5-CO2H - NHCH3 OCH2CH3 N ( 2 ) H 2-CO2CH2CH3 H 5-CO2H - Cl OCH3 CH(3) CH3 2-CO2C~3 5-CO2H ~ OCH3 OCH3 CH
H H H 3-CX2CO2~ CH3 OC~3 N
H ~ H 4-OCH2C02H ~ OC~3 OC~3 CH
R 502N ( CH3) 2 H 5-scH2(cH3)co2H OC~3 OCH3 CH
H H H S-NC(O)CH2CH2CO2H - OC~3 OCH3 CH
H 2-CO2CH3 ~ 5-CO2H ~ CH3 CH3 CH(4) CH3 2-CO2CH3 H 5-CO2~ 3 oc~3 N
H H B 2-CO2~ C~3 OC~3 CH(5) X H H 2-CO2H ~ CH3 OCH3 N(6) CH3 H H 2-CO2H ~ CH3 OCH3 ~(7) H H H 2-CO2H ~ CH3 CH3 CH(8) H H H 2-CO2H - Cl OCH3 CH(~3 l ~2 X H H 2-C02H _ OCH2CH3 0CH2CH3 CH(10) H H H 2-CO2H - NHCH3 OCH2CH3 N(ll) H H H 2-OCH2CO2H CH3 CH3 CB(12) H H H 2 CH2C02~ CH3 CH3 N~13 H H H 2-CH2C02~ CH3 OCH3 N~14~
H H H 2-CH2CO2H CH3 OCH3 CH(15) H H H 2-C~2C2H - OCH3 OC~3 CH(16) H H H 2-CHzCO2~ - OCH3 OCH3 N~17~
H H H 2-CH2CO2H CH3 CH3 CH(18) H H H 2-CO2H - OCH3 OCH3 CH'l9' H 6-Cl H 2-SCH(CH3)CO2H - CH3 OCH3 CH(20) H 6-C1 H 2-SCH(CH3)CO2H - OC~3 OC~3 C~(21) H 6-Cl H 2-scH(cH3)co2H - CH3 OCH3 N(22) H 6-OCH3 H 3-CO2H - OC~3 OCH3 CH(23) H 6-OCH3 H 3-CO2X - CH3 OC~3 ~24) H 4_Ci 6-C1 3-CO2H - CH3 CH3 CH(25) H 4-C1 6-C1 3-C02H - CH3 OCH3 CH(26) H 4-C1 6-C1 3-CO2H - OCH3 OCH3 CH(27) H 4-C1 6-C1 3-CO2H - CH3 OCH3 N(28) H 4-C1 6-C1 3-CO2H - OCH3 OCH3 N~29~
B 4-C1 6-C1 3-CO2H - Cl OCH3 C~(30) H 2-C1 8 4-CO2H. - CH3 OCH3 N~31~
H 5-Cl H 2-CO2H CH3 OC~3 ~(32) H 2-CO2CH3 H H ~H2 O Q (CH3)COZH OCH3 N(33) H 2-Cl H 4-CO2H - OCH(CH3)2 OCH(CH3)2 N(34) H 2-Cl H 4-CO2H - H H CH
H 2-CO2CH3 H H CH2 OCH2CO2H OC~3 CH

B H H 4-CO2H - Cl OCH3 CH
H S-CH3 H 2-COzH - N(CH3)2 OC~2CF3 N

<: :

T~BLE 2 Ge~eral F~rmula II ~where ~2 = ~

X ~ Z

H 5-C2~ C~3 OC~3 H 5_CO2H C~3 OC~3 C~
2-C2CH3 5~C2~ OCH3 OC~3 C~
2~S2N(CH3)2 5_SCH2CO2H CH3 OCH3 N
H 4_CO2H CH3 OCH3 CH(35) H 2-CO2H CH3 OCH3 N(36) H 2~C2H OCB3 CH3 CH(37) H 4_CO2H OCH3 OC~3 CH(38 Genera1 FQrmU1a III (whese 22 = HL

~1 G ~ Y

H 4_CO2H CH3 CH3 N
H . . 5 CO2H C~3 CH3 CH
3_CO2CH3 5~SCH2C2H CH3 OCH3 N

~ARLE ~
Geueral_FQrmula IV ~wh~r~ B2-=- H) G ~ ~ Y

H 3_CO2H CH3 OCH3 OCN3 CH
4_CO2CH3 3-C2H CH3 OCH3 ~CH3 C~
4-(2-methyl- 3_CO2H CH3 OC~3 OC~3 CH
tetra~ol-5-yl) 3s ~1 Q B3 4-C02CH3 H CH3 OCH3 OCH~CH3)C02H N
4-(2-methyl- H CH3 OCH3 OCH(CH3)C02H N
tetr~201-5-yl) 4-(2-methyl- H CH3 CH3 OCHzC02H CH
tetra~ol-S-yl) 4-(2-ethyl- 3-CO~H CH3 OCH3 OCH3 CH
tetrazol-5-yl) 4-~2-ethyl- H CH3 OCH3 OCH~CH3)C02H N
tetra~ol-5-yl 4-(2-ethyl- H CH3 CH3 CH2c2H CH
tetra~ol-5-yl H 4-C02H CH3 OCH3 C~3 CH(39) H 4-OCH2C02H CH3 OCH3 OC~3 CH

~BLE 5 Ge~eral Formula V (where ~ = H) ~1 G R3 X Y Z

s-Co2CH3 3-OC~2C~3 CH3 OC~3 OCH3 CH
3-CH3 5-C02H CH3 Cl OCH3 CH(40) TABL~ 6 General Formula VI (where RZ _ ~
~1 G~3 ~ ~ Z

Çeneral Formula VII

~1 R2 E

3-CON(CH3)2 ~ OCH3 OcH(cH3)co2~ N(41) 3-CON(CH3)~ ~ - C~3 OC~2CO2~ CH
3-C(O)C~3 H C~2 OCH3 OCH(c~3)co2H N
3-SO2CH2CH3 H ~ OCH3 OCH(cH3)co2H N
3-50zCH2CH3 H ~ c~3 C~2c2H CH

TA~L~ 8 Ge~eral Formula VlII

~1 B2 ~ X z 2-CO2C~3 H OCH3 OcH(c~3)co2~ N
2-CO2CH3 H CH3 OCH2CO2~ CH

~ynthesis of Coniuq~tes The procedure for synthesis of protein conjugates with the sulfonylurea carbo~ylic acld ~hereafter referred to as the analyte) entails activation by means of a carbo~yl~activating reagent (CAR) such as 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or disuccinimidyl carbonate followed by reaction of the activated carbo~ylic acid so obtained with the amines (lysines~
of a protein. This process results in the attachment of the analyte to the protein by means of a hydrolytically stable amide bond.
If the analyte does not contain a carbo~yl group but contains either an amine or thiol, it can be converted to a carbogylic acid derivative as a first step in the synthesis without the necessity of isolating the derivative. Primary and secondary amines react rapidly and quantitatively with succinic anhydride in an appropriate solvent (dimethylforma-mide, dimethylsulfo~ide) and the carboxylic acid thus generated can be activated and coupled in the normal manner. An analyte molecule containing a thiol group can be treated with N-succinimidyl bromoacetate and thus converted directly to a derivative in which the carbo~yl group is already activated.
If the analyte contains both amine a~d carbo~ylic acid groups,-it may be necessary to block the amine to prevent formation of insoluble polymers during activation of the carbo~ylic acid. For permanent blocking, the acetyl group can be used. If it is necessary that the amine group be free in the final conjugate (to improve the changes of obtaining a specific antibody, for e~ample), block with a trifluoroacetyl group, which can be removed by treating the analyte protein conjugate with 1 M
piperidine or oth0r nucleophilic amine.
S There are many CARs co~mercially available, but 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydro~hloride (ECDI) and disuccinimidyl carbonate ~DSC) are especially useful. ~CDI can be used in either aqueous or organic solvents. Normally, as a first step, one reacts the desired analyte carbo~ylic acid in either aqueous or organic solvent with ECDI.
In water, the ma~imum rate of reaction occurs at pH 4 to 6, in anhydrous solvents, adjustment of acidity is not necessary because of the acidic nature of both the carboxylic acid and the ECDI hydrochloride. The second step consists of combining the activated acid solution and a solution of the desired amine (or protein) in an appropriate solvent. Reaction of the activated carbo~ylic acid with the amine is ~ catalyzed:

N~CH2CH3 H~ ~CH2CH3 1l n RCOOH + C ~--~ R-C-O-IC Isourea(active) ~ CH2CH2CH2N(cH3)2 / C 2 2 2~( 3)2 Cl- / Cl-R-C~ ~CH2CH3 ~ H2N Protein C~O N-AcylureaSinert) H O , 2 2CH2N(CH3)2 . R-C-NH -Protein Cl ECDI reacts with amines very slowly. In some cases reported in the literature, ECDI is simply added to a premi~ed solution of the carbo~ylic acid .

and amine so that activation and coupling take place simultaneously. This should not be done in the synthesis of analyte-protein conjugates because proteins also contain carbozyl groups (aspartate and glutamate), which compete with the analyte carboxylic acid and lead to extensive crosslinking of the protein. A further disadvantage of this procedure arises from the activation step being acid-catalyzed and the subsequent coupling reaction being base-catalyzed; conditions cannot be optimized for both reactions simultaneously.
An additional disadvantage of ECDI activation is that the activated intermediate, an acylisourea, rearranges to an N-acylurea, which is completely inert (see equations above~. To circumvent this problem, a mi~ture of analyte carbo~ylic acid, ECDI
and N-hydroxysuccinimide (NHS) can be r~acted together so that the acylisourea formed is immediately converted to the N-succinimidyl ester, which is stable.
DSC is a relatively new reagent that reacts directly with a carbo~ylic acid to form the corresponding N-hydroxysuccinimide ester. The reaction proceeds rapidly when catalyzed by tertiary amines (triethylamine, pyridine). Carbon dioside and N-hydro~ysuccinimide are innocuous by-products:

~o ,4 o ~ o o~ o RCOOH+ ~ N-O-C-O-N ~ R-C-O-N ~ ~ CO2 + ~ -OH
¦ H2N-Protein R
R-C-NH-Protein DSC is unlike carbodiimides and 2-fluors-1-methylpyridinium salts in two important aspects. It hydrolyzes rather rapidly, and it reacts very rapidly with primary and secondary amines to form carbamates. Therefore, activation of the analyte carbo~ylic acid in an anhydrous, aprotic, water-miscible solvent (dimethylformamide, dimethylsulfo~ide, l-methyl-2-pyrrolidinone) must be carried out as a separate first step. Unblocked reactive amines should not be present in the analyte.
Bovine serum albumin (BSA~ is commonly u~ed as -- -an immunogenic carrier protein and as a protein for the synthesis of coating conjugates. Its molecular weight is 67,000 and it contains 59 available lysines. Conjugates are prepared by activating an analy~e (hapten) carbosylic acid with 1.1 equivalents of DSC in solvent ~dimethylformamide, dimethylsulfo~ide, or l-methyl-2-pyrroli~inone) in the presence of 2 equivalents of triethylamine. The mi~ture is allowed to react for one hour after addition of the triethylamine. This solution is then mi~ed with a solution of BSA in buffer, pH 8 to 9.
The buffer must not contain amino acids or ammonium salts. The coupling reaction i5 complete within 3 hours.
Becallse of such factors as varying DSC quality ~resulting in incompleté activation of the hapten carbo~ylic acid), possible side reactions, and/or competing hydrolysis of the succinimidyl ester formed, quantitative coupling of hapten to protein does not occur.
It is believed that BSA containing an average of 10 to 15 attached haptens will function best as an immunogen. When the number of hapten molecules introduced into a protein or other polyamine is less than the number of amine groups available, the conjugate obtained is a mixture of products of varying degrees of substitution.
~ eyhole limpet hemocyanin (~LH) (Sigma Chemical Co., St. Louis, MO) is about 90% soluble in 0.15 ~
sodium bicarbonate at pH 8.l and nearly ~ompletely soluble in O.lS M HCO3-/CO3~ buffer at pH lO. Any insoluble material should be removed by centrifugation before a~dition of the activated hapten.
KLH is believed to contain about 20 reactive lysines per 100 kD. In immuno~en synthesis with activated hapten carbo~ylic acids, use at least 60 moles of activated hapten per lO0 kD of ~LH. The conjugate solutions are generally dialyzed first against several changes o 0.15 M sodium bicarbonate, then against either phosphate buffer or, if the ~2 conjugate is to be lyophilized, against deionized water. The conjugate remains soluble in spite of the changes in pH and ionic strength. Once lyophilized, however, the conjugate is difficult to dissolve again. Dissolution is aided ~y mois~ening the dry conjugate with a few yL of ethanol -- this e~pels air and facilitates wetting -- and using a buffer with as high a pH as possible.
Pumpkin seed globulin (PSG) and marijuana seed globulin (edestin) are available from Sigma. They are ine~pensive and function well as carrier proteins for immunogen synthesis. By definition, globulins are proteins that are not soluble in pure water or weak salt solutions but are soluble in fairly concentrated salt solutions. PSG is particularly useful and is completely soluble in buffers containing at least 0.5 M NaCl. Our sample consists of a major component eluting in the 150,000 MW range on a GF 450a column, and a minor component eluting in the 500,000 MW range. It is estimated that PSG
contains about 20 reactive lysines per 100 kD, the same as ~LH.
~5 Although native PSG is completely soluble in high-salt buffers, it becomes insoluble when the amine groups are conjugated. Precipitation of conjugate begins shortly after ~he activated hapten carbo~ylic acid is mi~ed with the PSG solution and continu~s during dialysis against low-salt buffers and distilled water. The conjugate has generally been isolated by lyophilization of the suspension, but simple centrifugation might also be satisfac~ory.
The commercial samples of edestin that were used were only about 50% soluble in salt solutions.
~destin is probably isolated from marijuana seeds that have been heated to destroy viability, which process can severely denature ~he protein.
5 Nevertheless, the soluble portion of edestin may be satisfactory for immunogen syn~hesis. Conjugates are insoluble.

Proteins fQ~ Coatina Con7uqate syn~h~L~
As used here, the term "coating conjugate"
signifies a reagent used to bind antianalyte antibody from sera or other liquid medium to a solid support.
The first step in this operation is attachment of the coating conjugate itself to the support. Attachm~nt may be through physical adsorption (on polystyrene, or e~ample) or by chemical bonding through appropriate functional groups on both the conjugate and the support. If the coating conjugate is to be adsorbed to polystyrene, a protein known to adsorb tenaciously and resist displacement by detergent and othèr proteins should be chosen for synthesis of the conjugate. If the coating conjugate is to be chemically bonded to the support (this is typically done through residual lysine groups), the protein chosen must have a sufficient number of lysines to allow both the initial attachment of an ade~uate number of analyte hapten molecules and the subsequent attachment of the resulting conjugate to a functionalized support.
It is sometimes possible to use the same conjugate as both an immunogen and a coating antigen. However, this approach is not generally used for screening sera for the presence of antianalyte antibodies since it would pick up concomitant antibodies against various peptide segments in the carrier protein and greatly increase the number of false positives.
S Bovine serum albumin (BSA~ and ovalbumin are proteins especially preferred for synthesis of coating conjugates. Conjugates remain soluble, even when a substantial fraction of the available lysine groups have been coupled to hydrophobic haptens, and are suitable for attachment to supports by either adsorption or by chemical bonding through residual lysines. After adsorption of the coating conjugate to polystyrene, it is generally necessary to block the plate with another protein such as BSA or milk protein (casein), or polyvinylpyrrolidone, to prevent subsequent adsorption of nonspecific antibodies.

~ynthesis Q~ ~nti~n-Protein ~on~uqates The PKa of a peptidic lysine residue is lO.S.
Theoretically, this is the pH at which the concentrations of protonated and unprotonated amine groups are equal, or, better, the pH at which any given amine group is unprotonated 50% of the time.
Since it is the unprotonated amine that reacts with an activated carbo~ylic acid, it is advantageous to carry out conjugations in basic solution.
Bicarbonate/carbonate buffer systems have proved - satisfactory. In any case, the system must not contain amino acids, ammonium salt~ or any reactive primary or secondary amine other than that present on the protein being conjugated.
Special care should be taken to protect carbozyl-activating reagents [1-~3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride (ECDI) and disuccinimidyl carbonate (DSC~ from atmospheric moisture. Purchase small quantities and store the bottles in the refrigerator in a plastic jar containing a calcium carbonate drying agent. Two or - 5 three hours before a sample is needed, remove the jar from the refrigerator and allow it to warm to room temperature before opening. Open ~he jar, then the bottle, and quickly transfer somewhat more material than will be needed to another container.
Immediately close the bottle and re~urn it to the jar and to the refrigerator: New bottles should be dated when opened; discard unus~d material 3 months after opening.

S~nthesis of Com~ound 12-KLH CQniuaate OCH~
N~
S102NEICONH ~H2COOE~

~OOCH3 (12) The depicted Compound 12 was selected for use in the coating conjugate used to detect the sulfonyl-urea, bensulfuron methyl. However, the conjugation procedure described here is a model for ~ynthesis of conjugate from any analyte carbo2ylic acid that is soluble in and not affected by either DMF, DMSO, l-methylpyrrolidinone, or other water-miscible, inert solvent. In this E~ample, DMF is used as a sol~ent in the activation step because sulfonylureas are known to slowly degrade in ~MSO when e~posed to light. The conjugation is carried out at pH 8 to avoid hydrolysis of the methyl ester group present in Compound 12. If the desired analyte carboxylic acid lacks base-sens.;tive groups, it is advantageous to use hi~her pH to improve the solubility of ~LH. The molecular weight of KLH is very high. For this reason, it dissolves very slowly, even in alkaline buffers.
Stoichiometry: 60 moles of Compound 12 per 100 kD of KLH. (For purposes of calculation, assume that the KLH is 100% pure and completely soluble).
DSC/Compound 13 mole ratio ~ 1.10.

KLH Solution: 150 mg of KLH (Sigma) was weighed into a 40 mL, polycarbonate centrifuge tube. A magnetic stirring bar was added and 15.0 mL of 0.15 M sodium bicarbonate was added ~pH - 8.1). The mi~ture was stirred for about one hour at room temperature, then an additional 10.0 mL of 0.15 M sodium bicarbonate was added. The mi~ture was stirred at room temperature for 3 more hours. The sample was stored in a refrigerator overnight and removed from the refrigerator and stirred sn additional hour the nest day. The magnetic stirrer bar was withdrawn and the sample centrifuged. The supernatant liquid was stored in a 2-ounce bottle in the refrigerator for later use in conjugate synthesis.

D~Ç ~Qck Solutîon: This solution is made up immediately beore use, that is, after Compound 12 has been weighed out (below). The values indicated here for DSC and total ~eiyht of solution are guides. It is not necessary to get these exact values. Actual values should be recorded to the precision indicated.
Disuccinimidyl carbonate (~SC; MW ~ 256.2;
51.00 mg) was weighed directly into a 4 mL, screw-capped vial. Dry DMF was added to bring the total weight to 3.400 grams, was agitated in the vial until the DSC had completely dissolved. The density of the solution was detected by weighing a 1000 ~L
sample (discard this sample). A typical value is O.9589 g/mL. From the actual weights obtained, the concentration of DSC in mmoles/~L was calculated:

51.00 ~ 0.9589 DSC conc. , . 5.614 ~ 10 5 mmoles/~L.
256.2 ~ 3400 Compound 2 (40.90 mg; 9 000 ~ 10-2 mmoles) was weighed directly into a 4 mL, screw-capped, vial. A
magnetic-stirrer bar was added, and 9.900 ~ 10-2 mmoles of DSC were added (1763 ~L3. The mixture was stirred until the Compound 12 had dissolved. 20 ~L
of triethylamine was added, and stirring continued at room temperature for e~actly one hour.
At the end of the one hour activation period, the activated Compound 12 soluti~n was quickly transferred to the rapidly-stirred ~LH solution. The solution was transferred back and forth between the two containers to effect quantitative mi~;ng. (No precipitate should form during this step.3 The sample was stored in the refri~erator overnight. The sample was dialyzed a~ainst 3 changes of 0015 M
sodium bicarbonate (2 liters each), then, ~a) if the conjugate is to be lyophilized, against 3 changes of deionized water, or (b) if the conjugate is to be aliquoted and stored frozen, against 3 changes of phosphate buffer. The average yield of lyophilized conjugate is 120 to 130 mg. If the dialyzed conjugate is not lyophilized, the appro~imate conjugate concentration can be obtained by dividing 125 mg by the final volume of the dialyzate.

EX~
Synthesis o~ ~ompo~nd l~-BSA Co~ e COOH NH~::H3 ~ 02NXCONH ~ H2CH3 CH30~0 ( 1 O) The secondary amine present in this molecule is so weakly nucleophilic that it is not considered necessary to block it. The conjugate is intended for use as an immunogen. The object is to attach 10 to 15 molecules of the Compound 10 to each BSA molecule.
Stoichiometry: Compound 10/BSA mole ratio 30; DSC/Compound 10 ratio ~ 1.1.

BSA Solution: Dissolve 100 mg of BSA in 10.0 mL of 0.15 M sodium bicarbonate, pH 8.1.

Compound 10 (20.78 mg; 4.753 ~ 1o~2 mmoles~ was weighed directly into a 1 mL reaction vial. A
magnetic stirring bar was added and an amount of DSC
stock solution was pipetted in that contains S.503 x 10 2 mmoles of DSC, here, (5.503 x 10-2)/(5.614 ~
10-5) . 896 ~L. The mi~ture was stirred until the Compound 11 had dissolved; 10 ~ of triethylamine was added, and stirring continued at room temperature for exactly one hour.
At the end of the one-hour activation period, the activated Compound 10 solution was quickly transferred to the rapidly-stirred BSA solution. The solution was transferred back and forth between the two containers to effect quantitative mi~ing. (No precipitate should form during this step.) The sample was stored in the refrigerator overnight. The sample was dialyzed against 3 changes of 0.15 M
sodium bicarbonate, then against 3 changes of phosphate buffer (2 liters each change). The conjugate concentration was estimated by divi~ing 90 mg by the final volume of dialyzate (that is, assume that 10% of the protein is lost during the synthesis process). The solution was aliquoted into suitable sample sizes and stored frozen until ready to immunize.

E~AMPLE ~
Synthesis of Compound l-OVA Coniu~ats cCX3~27 [~O~N~CON~ CH-COOH
CE~3 cl~

This conjugate is intended for use as a coating antigen. The object is to attach 7 to 10 molecules of the Compound 1 to each ovalbumin molecule.
Stoichiometry: Compound l/ovalbumin mole ratio ~ 20; DSC/Compound 1 ratio = 1.1.

Ovalbumi~ SolutLQn: Dissol~e 100 mg of ovalbumin in 10.0 mL of 0.15 M sodium bicarbonate, pH 8.1.

. Compound 1 (20.78 mg; 4.446 ~ 10-2 mmoles) was weighed directly into a 1 mL reaction vial. A
magnetic stirring bar was added and that amount of DSC stock solution was pipetted that contains 4.a91 x 10-2 mmoles of DSC, here, (4~a9l ~ 10-2)/~5.614 ~
10-5) ~ 871 ~L. The mi~ture was stirred until the Compound 1 had dissol~ed. 10 ~L of triethylamine was added, and stirring continued at room temperature for exactly one hour.

At the end of the one-hour activation period, the activated Compound 1 solution was quickly - 5 transferred to the rapidly-stirred ovalbumin solution. The solution was transferred back and forth between the two containers to ~ffect quantitative mi~ing. (No precipitate should form during this step.~ The sample was stored in the refrigerator overnight. The sample was dialyzed against 3 changes of 0.15 M sodium bicarbonate, then against 3 changes of phosphate bu~fer (2 liters each change). The conjugate concentration was estimated by dividing 90 mg by the final volume of dialyzate (that is, assume that 10% of the protein is lost during the synthesis process). Antimicrobial agents were added to the solution before storage.

~XA~P~E 4 Pre~ara~ion of the OVA con~uaate of ComPound 9 To a solution of 0.331 g (0.82 mmol) of Compound 9 in 10 mL of dry DMSO was added 0.252 9 (0.98 mmol~ of N,N'-disuccinimidyl carbonate.
Triethylamine (0.5 g) was added, and the resulting solution was diluted to 134.98 g with DMSO and allowed to stir for 1 hour.
A solution of OVA (Sigma-Grade III) was prepared b~ dissolving 0.750 g of O~A in 0.15 M
sodium bicarbonate ~adjusted to pH 9.2 with 1~ NaOH) to a total weight of 75.00 9. To a 5.00 9 aliquot of this solution was added 0.281 g of the activated sulfonylurea solution.

Vsing the procedure describ0d above, the following sulfonylureas were conjugated with the following proteins, a~d purified by dialysis.

BSA ~LH PSG
Bovine Keyhole Pumpkin Serum OVA ~impet Seed CMPD Al~umun Ovalbumin Hemo~anin ~lo~ulin 102b g 3b 8b X X X
9a X x lob X X
15 lzb X X

_ a. Made as described in E~ample 4.
b. Made as described in E~ample 3.

~NrI~ODY PRO~UCTIO~
Vallejo et al., J. Aqric. Food Chem., ~Q, ~1982), pages 572 to 580 studied different hapten structures for parathion. He concluded that nthe determinant groups of the small molecule must be preserved" and that "the hapten's determinant groups must not be masked~ for'antibody production. One format used for pesticide immunoassays has utilized a solid phase with hapten bound to it (coating conjugate) for capture of antibodies not bound to free compound. The quantification of the captured antibody is used to determine the original concentration of compound in the original aqueous sample Hapten structures were further e~plored by Wie et al., ~ ~g~ic, Foo~ Chem., 32, (1984), pages 1294 to 1301 to develop an assay for diflubenzuron.
Their main purpose was to show that sensitive assays could be achieved by using a coating antigen of differsnt structure than the immunizing hapten.
Thus, they demonstrated that some sensitivity could be formed by shifting linker arm position, and that lack of specificity cou~d be achieved by changing functional groups.
Van Emon et al., Analytiçal ~ethods ~or Pesticides and Plant ~rowth ReauLators, Vol. XVII, (1989), pages 217 to 263 state that the point of attachment of hapten to protein should occur away from any suspected antigenic determinants~ to insure proper antigenic response for developing specific antibodies. They also state that to develop a compound class specific assay the hapten structure for immunization is important, particularly preserving the common antigenic determinants of the --related compounds.

~ P$E_~

Rabhit Immunization Protocol for Gom~ound 3 Three New Zealand white rabbits were immunized with a conjugate prepared by coupling the Compound 3 to keyhole limpet hemocyanin (KLH) using the schedule set out in Table 9. Where the use of an adjuvant is indicated, 0.5 mL of the adjuvant was mised with 0.5 mL of a solution or suspension of the conjugate in phosphate buffered saline to form an emulsion.

Day of Amount Immunizatiun (m~ Route ~ ~n~

o 1 . scl CFP~2 0.025 IM3 IFA4 120 0.025 IV5 none 150 0.1 IV none 350 0.5 IV none 3~0 0.5 IV none 410 0.5 IV none 440 0.5 IV none 470 0 5 Iv none 1. subcutaneously 2. complete Freund's adjuvant 3. intramuscularly 4. incomplete Freund's adjuvant 5. intravenously Antiserum was collected on the seventh, ninth and twelfth days following each boost from days 350 to 470.

~.~

S ~abbit Imm~nL~ation ProtocQl f~ ~QmPound 4 Three New Zealand white rabbits were immunized wi~h a conjugate prepared by coupling the Compound 4 to K~H using ~he schedule set out in Tables lOa and b. Where the use of an adjuvant is indicated, 0 . 5 mL of the adjuvant was mi~ed with D . 5 mL of a solution or suspension of the conjugate in phosphate buffered saline to form an emulsion.

TA~LE LOa Day of Amount Immunization (ma) ~Q~te Ad;uvant SC IFA

6 0 1 ~;C IFA

220 0.025 IV none 0 . 025 IM IFA
290 0 . 025 IV none 0 . 025 IM IFA
320 0 . 05 IV none O.OS TM IFA
350 O.OS IV none 0.05 IM IFA
380 0 . 05 IV none 0.05 IM IFA
410 0 . 05 IV none 0.05 IM IFA
440 0 . 05 IV none 0 . 05 IM IFA

Antiserum was collected twenty days after the 35 boost on day 66 and on the fourteenth day following each boost from days 220 to 440.

~k Day of Amount - 5I~ununiz~tion ~ma) Rout~ ~liuvant IM
14 0 . S SCIFA
0. 5 IM
21 0 . 2 IVnone 220 0 . 025 IVnone 0 . 025 IMIFA
290 0 . 025 IVnone 0 . 025 IMIFA
320 0 . 05 SCIFA
0 . 05 IMIFA
350 0 . 05 SCIFA
0.05 IMIFA
380 0 . 05 SCIFA
0 . 05 IMIFA
410 0 . 05 SCIFA
0.05 IMIFA
440 0 . OS SCIFA
0 . 05 IMIFA

Antiserum was collected on the fourteenth day following each boost from ~ays 220 to 440.

~E~ ' 5Rab~it I~munization ~rQto~ol fo~ Cgmp~und 10 Three New Zealand white rabbits were immunized with a conjugate prepared by coupling the Compound 10 to ~LH using the schedule set out in Table 11. Where the use of an adjuvant is indicated, 0.5 mL of the adjuvant was mi2ed with 0.5 mL of a solution or suspension of the conjuga~e in phosphate buffered saline to form an emulsion.

lS TABLE 11 Day of Amount Immunization (ma) Rou~ ~diuva~
0 0.5 IM CFA
0 5 IV none 180 0.5 IV none 210 0.5 IV none 240 O.S IV none 270 0~5 IV none 300 0.5 IV none 330 0.5 IV none 360 0.5 IV none 390 0.5 IV none 420 O.S IV none ~ -Antiserum was collected on the fourteenth day following each boost from day 4~ to day 164 and on the seventh, ninth and eleventh days following each boost from days 210 to ~20.
Using the method described in E~amples 5 to 7, antisera to the following conjugates were produced:

Example ~o~ Coniuaate 8 Compound 8 - BSA
9 Compound 8 - RLH
Compound 13 - XLH

METHOD OF MEASURING SULFO~YLUREA CONCENT~ATIONS

GE~ERAL P~OCEDURE
Optimization of First Antibody Titer ~nd CoatLn~
Coniu~ate Concen~ration .
Optimum starting concentrations for the reagents in this immunoassay are determined with a 2S checkerboard assay. In this assay, ~decreasing concentrations of coating antigen are coated on a plate in one direction, for instance 10 ~g/mL across the first row, 1 ~g/mL across the second row, and on down. Increasing dilutions of antisera are added to the plate in the other direction, for instance a dilution of 100 down the first column of wells, 1000 down the second column, etc. In this manner, the binding of each concentration of antisera to each concentration of coating antigen is determined at a fi~ed time point. The combination of antisera and coating conjugate concentrations giving a half-ma~imal (O.D. ~ 1,0) reading after addltion of second antibody and substrate is optimum. Two or three combinations can be chosen for the nest step, generation of an inhibition curve.
The optimum concentration of the antisera of Example 9 and coating conjugate ovalbumin-Compound 9 was determined with this checkerboard assay as described ne~t. Coating conjugate solution was made consisting og the conjugate dissolved in coating buffer at 10, 1 and 0.1 and 0.01 ~g~mL
concentrations. The coating antigen solutions (200 ~L) were pipetted into polypropylene microtiter wells such that each concentration ran across the plate in a row with decreasing concentrations from top to bottom. Peripheral wells were not used because of the occasional variability of results.
The plates were incubated at 4C for 16 hours, and dried. Plates were washed with lX PBS-Tween~
(PBST) three times in a plate washer and dried by hitting the plate 3-4 times upside-down against a paper towel to remove water droplets from the sides of the wells. Aliquots (200 ~L) of serial two-fold dilutions of antisera in lX PBST with 0.5% BSA and 0.1~ gelatin starting at a dilution of 200 and ending at 204,800, were added to wells from left to right across the plate. P~at~s were incu~ated 1.5 hours at room temperature and then washed again with lX PBST
as described above. Goat anti-rabbit IgG antibody labeled with alkaline phosphatase was dissolved in lX
PBST with 0.5% BSA and 0.1% gelatin at a dilution of 1:5000 and 200 ~L was added to each well. Plates were incubated at room temperature for one hour.
After washing the plates, the para-nitrophenyl phosphate substrate was dissolved (1 mg/mL) in substrate buffer and 200 ~L was added to the microtiter wells. The plates were incubated at room temperature for a total of 40 minutes and read at 405 nm. Optimal titer and coating antigen concentrations wer determined by those concentrations reaching an absorbance of 1.0 O.D. units after 20 minutes incubation and corresponding to 50% inhibition of the enzyme reaction.
The optimum antiserum dilution was determined to be 1:50,000; and the optimum coating conjugate concentration was determined to be 0.1 ng/mL.
The above procedure was carried out for Compound 8-OVA and Compound 8-~SA coating conjugates. It was found that 0.1 yg/ml concentration with 25,000 titer antibody to Compound 8-KLH immunogen was optimum.
The same procedure was carried out for Compound 13-OVA against antisera from E~ample 10 e~cept that 1.O ~g/mL coating concentration was determined against 200,000 titer antisera as optimal.

Standard PreParation:
Chlorsulfuron, 99.7% pure analytical standard ~E. I. du Pont de Nemours and Co., Inc., Agricultural Products Department, Wilmington, Delaware, 19880) was used to make up the standard curve. 10 mg of chlorsulfuron was weighed and placed into a 100-mL
volumetric flask, then dissolved in 100 mL methylene chloride. This standard is stable for several months in the refrigerator and is used to make all working standards during the procedure.

Standard Curve PreDaration:
50 yL of the 100 ~g/mL standard from above was transferred into the bottom of a lS ~ 85 mm culture tube and air dried 5 to 10 minutes until all the solvent was evaporated. 5 mL of lx PBS/Protein was added, covered with paraffin film and vortexed vigorously. It was reconstituted at least 1/2 hour with occasional vorte~ing. Standard gave the following concentrations: 100 ng/mL, 12.5 ng/mL, 3.1 ng~mL, 0.8 ng/mL, 0.2 ng/mL, 0.05 ng/mL, 0.Dl ng/mL, 0.003 ng/mL and 0.~008 ng/mL. A final volume of 1.8 mL of each was added to-a small 15 ~ 85 mm glass disposable culture tube. 0.2 mL of 10~ PBS/Protein (0.5~ ~SA, 0.1% gelatin) was added to the tube for a final volume of 2.0 mL.
Fresh 1:500 dilution antisera stock was made by adding anti-chlorsulfuron antisera #127 from E~ample 9 and PBS/Protein. 20 ~L of the 1:500 titer antisera was added to all tubes e~cept the reagent blank. It is most important that the antisera stock not touch the glass before the solution to ensure reproducible amounts of antisera entering each tube. The tubes were gently vorte~ed, covered with a strip of paraffin film making sure each tube was sealed against the film, and incubated overnight on the bench at room temperature.
(i) Pl~t~P~Paration: Coating conjugate was made up by adding 12.5 ~L of the 0.2 mg/mL Compound 9-OVA per 25 mL PBS/~ro~ein. Using a 12-channel pipetter, 200 ~L was added to every well of a microtiter plate, covered, sealed in a plastic bag and incubated 4C overnight. Blocking was done ~he ne~t day. The coated plates were washed three times with 12 PBST and dried. To block the plates, 200 ~L
of 3% BSA was added to each well ~ith a 12-channel pipetter. The plates were incubated 2 hours at room temperature.

~ !

.

(ii) Tube Addition: The plate was washed and dried by the same procedure as described above. 200 ~L was added to three wells for each standard sample tube with a repeater pipette. For e~ch tube O.2 mL
was added into four wells, tips changed and another tube added to another four wells. Care was taken to prevent splashing of contents from the wells. The plate was incubated a~ room temperature 1 hour.
(iii) Second ~n~ibody Addition: The plates were washed and dried. Fresh second antibody was prepared as described above. Then 200 ~L per well was added to the plate. The plate was allowed to incubate 1 hour at room temperature.
(iv) Enzyme Substrate Addition: The plate was washed and dried, and 200 ~L of 1 mg/mL substrate solution was added to each well. The plate was incubated at room temperature and read. The plate was read between 1.0 to 2.0 O.D. If stopping the reaction was necessary, 50 ~L of stop solution (lON
NaOH) was added to each well.
Using the above procedure, the following measurements were made:

.

Detection E~. . Coating Limit(a) 5 ~Q~ Pesticide I~munQ~n ~on~uaate ~a/mL2 11 chlorsulfuron Compound Compound 10 8-~H 8-OVA
(1:50000) SO.l yg/mL) 10 1~ chlorsulfuron Compound Compound 50 8-~LH 8-OVA
(1:25000) (O.l~g/mL) 13 metsulfuron Compound Compound 25 methyl 3-KLH 3-OVA
(1:100,000) (1.2 ~g/mL) 14 metsulfuron Compound Compound 10 methyl 3-KLH 9-OVA
(1:100,000) (1.2 ~g/mL~
15 bensulfuron Compound Compound 10 methyl 13-OVA 13-RLH
16 chlorimuron Compound Compound 25 ethyl 4-KLH 7-O~A

. _ (a) The detection limit is the minimum concentration of pesticide needed to produce 1S% inhibition of absorbance relative to negative control.

The assay can be applied to any aqueous water sample includin~ aqueous soil e~tract. Two e~amples ~ollow, L~KE W~TE~
A standard curve was generated as described in Procedure I. Triplicate 1.8 mL aliquots of lake water were placed in 15 ~ 85 mm disposable culture tubes. Then, 0.2 mL 10X P~S/Protein was added as described above followed by Z0 ~L of 500 titer antisera stock as described. The assay steps were followed e~actly as described in sec~ions (i)-(iv) and the Flow Laboratories Titercalc software using a four parameter logistic calculated the concentration of chlorsulfuron in the water samples versus the standard curve. A detection limit of 10 pg/mL was obtained, wherein the detection limit is as defined with respect to E~amples 11 to 16.

SOIL SAMP~ES
Ten grams of soil were e~tracted with 20 mL of 0.2 M ammonium bicarbonate buffer using either a probe sonicator at low wattage for 3 minutes or overnight tumbling. The slurry was centrifuged at 5,000 9 for 15 minutes to pellet the solids.
Aliquots of 1.8 mL volume of the supernatant are used directly in the assay. The standard curve was generated as described above e~cept that e~tract from untreated soil was used instead of water. Sample aliquots from treated soil of 1.8 mL volume were taken in duplicate and run against the standard curve. A 25 pg~mL detection limit was attainable in the soil extract.

Claims (20)

1. A compound having the formula I

wherein J is , , , , , G is H or MO2C(alkyl)nL;
n is 0 or 1;
alkyl is 1 to 3 carbon atoms optionally substituted with one or two of halogen, methyl, methoxy, or methylthio;
L is O, S, NR5, -N(C=O)- or a direct bond;
R, R4 and R5 are independently H or CH3;
E is a single bond or CH2;
R1 is H, C1 to C3 alkyl, C1 to C3 haloalkyl, halogen, nitro, C1 to C3 alkoxy, SO2NRaRb, CONRaRb, C1 to C3 alkylthio, C1 to C3 alkylsulfinyl, C1 to C3 alkylsulfonyl, CH2CN, CN, CO2RC, C1 to C3 haloalkoxy, C1 to C3 haloalkylthio, C2 to C4 alkoxyalkyl, C3 to C4 alkoxyalkoxy, C2 to C4 alkylthioalkyl, CH2N3, NRdRe, or Q;
R2 is H, C1 to C3 alkyl, C1 to C3 haloalkyl, halogen, nitro, C1 to C3 alkoxy, C1 to C3 alkylthio, CN, C1 to C3 haloalkoxy, or C2 to C4 alkoxyalkyl;
Ra is H, C1 to C4 alkyl, C2 to C3 cyanoalkyl, methoxy or ethoxy;
Rb is H, C1 to C4 alkyl or C3 to C4 alkenyl; or Ra and Rb may be taken together as -(CH2)3-, -(CH2)4-, -(CH2)5- of -CH2CH2OCH2CH2-;
Rc is C1 to C4 alkyl, C3 to C4 alkenyl, C3 to C4 alkynyl, C2 to C4 haloalkyl, C2 to C3 cyanoalkyl, C5 to C6 cycloalkyl, C4 to C7 cycloalkylalkyl or C2 to C4 alkoxyalkyl;
Rd and Re are independently H or C1 to C2 alkyl;
Q is a saturated 5- or 6-membered ring containing one heteroatom selected from O, S, or N, tetrazole optionally substituted with C1-C3 alkyl, or an unsaturated 5- or 6-membered ring containing 1 to 3 heteroatoms selected from 0-1 S, 0-1 O or 0-3 N and when Q is an unsaturated 5- or 6-membered ring, it may optionally be substituted by one or more groups selected rom C1 to C4 alkyl, halogen, C3 to C4 alkenyl, C1 to C3 alkoxy, C1 to C3 alkylthio, C3 to C4 alkenylthio, C1 to C2 haloalkoxy or C1 to C3 haloalkylthio;

;

X is H, C1 to C4 alkyl, C1 to C4 alkoxy, C1 to C4 haloalkoxy, C1 to C4 haloalkyl, halogen, C2 to C5 alkoxyalkyl, C2 to C5 alkoxyalkoxy, amino, C1 to C3 alkylamino, di(C1 to C3 alkyl)amino or C3 to C5 cycloalkyl; or C1 to C4 alkyl, C1 to C4 alkoxy or C1 to C4 haloalkoxy substituted on the alkyl, alkoxy or haloalkoxy group with CO2M;
Y is H, C1 to C4 alkyl, C1 to C4 alkoxy, C1 to C4 haloalkoxy, C2 to C5 alkoxyalkyl, C2 to C5 alkoxyalkoxy, amino, C1 to C3 alkylamino, di(C1 to C3 alkyl)amino, or C1 to C4 alkyl;

M is H or an alkali or alkaline earth metal salt;
R3 is H or C1 to C3 alkyl;
Z is CH, N or CCO2M; and E1 is a direct bond or CH2; wherein:
(i) when G is not H and when L is a direct bond, then n = 1;
(ii) when G is H, then J is J-1, J-4 or J-5;
(iii) when G is H and J is J-4 or J-5, then X
is C1 to C4 alkyl, C1 to C4 alkoxy, or C1 to C4 haloalkoxy substituted with CO2M;
and (iv) when G is H, and J is J-1, then E is CH2, and X is C1 to C4 alkyl, C1 to C4 alkoxy, or C1 to C4 haloalkoxy substituted with CO2M.

2. A sulfonylurea-protein conjugate comprising a sulfonylurea of the formula I

wherein J is , , , , , G is H or MO2C(alkyl)nL;
n is 0 or 1;
alkyl is 1 to 3 carbon atoms optionally substituted with one or two of halogen, methyl, methoxy, or methylthio;
L is O, S, NR5, -N(C=O)- or a direct bond;
R, R4 and R5 are independently H or CH3;
E is a single bond or CH2;
R1 is H, C1 to C3 alkyl, C1 to C3 haloalkyl, halogen, nitro, C1 to C3 alkoxy, SO2NRaRb, CONRaRb, C1 to C3 alkylthio, C1 to C3 alkylsulfinyl, C1 to C3 alkylsulfonyl, CH2CN, CN, CO2RC, C1 to C3 haloalkoxy, C1 to C3 haloalkylthio, C2 to C4 alkoxyalkyl, C3 to C4 alkoxyalkoxy, C2 to C4 alkylthioalkyl, CH2N3, NRdRe, or Q;
R2 is H, C1 to C3 alkyl, C1 to C3 haloalkyl, halogen, nitro, C1 to C3 alkoxy, C1 to C3 alkylthio, CN, C1 to C3 haloalkoxy, or C2 to C4 alkoxyalkyl;

Ra is H, C1 to C4 alkyl, C2 to C3 cyanoalkyl, methoxy or ethoxy;
Rb is H, C1 to C4 alkyl or C3 to C4 alkenyl; or Ra and Rb may be taken together as -(CH2)3-, -(CH2)4-, -(CH2)5- or -CH2CH2OCH2CH2-;
Rc is C1 to C4 alkyl, C3 to C4 alkenyl, C3 to C4 alkynyl, C2 to C4 haloalkyl, C2 to C3 cyanoalkyl, C5 to C6 cycloalkyl, C4 to C7 cycloalkylalkyl or C2 to C4 alkoxyalkyl;
Rd and Re are independently H or C1 to C2 alkyl;
Q is a saturated 5- or 6-membered ring containing one heteroatom selected from O, S, or N, tetrazole optionally substituted with C1-C3 alkyl, or an unsaturated 5- or 6-membered ring containing 1 to 3 heteroatoms selected from 0-1 S, 0-1 O, or 0-3 N and when Q is an unsaturated 5- or 6-membered ring, it may optionally be substituted by one or more groups selected from C1 to C4 alkyl, halogen, C3 to C4 alkenyl, C1 to C3 alkoxy, C1 to C3 alkylthio, C3 to C4 alkenylthio, C1 to C2 haloalkoxy or C1 to C3 haloalkylthio;

;

X is H, C1 to C4 alkyl, C1 to C4 alkoxy, C1 to C4 haloalkoxy, C1 to C4 haloalkyl, halogen, C2 to C5 alkoxyalkyl, C2 to C5 alkoxyalkoxy, amino, C1 to C3 alkylamino, di(C1 to C3 alkyl)amino or C3 to C5 cycloalkyl; or C1 to C4 alkyl, C1 to C4 alkoxy or C1 to C4 haloalkoxy substituted on the alkyl, alkoxy or haloalkoxy group with CO2M;
Y is H, C1 to C4 alkyl, C1 to C4 alkoxy, C1 to C4 haloalkoxy, C2 to C5 alkoxyalkyl, C2 to C5 alkoxyalkoxy, amino, C1 to C3 alkylamino, di(C1 to C3 alkyl)amino, or C1 to C4 alkyl;
M is H or an alkali or alkaline earth metal salt;
R3 is H or C1 to C3 alkyl;
Z is CH, N or CCO2M; and E1 is a direct bond or CH2; and a protein.

3. A conjugate according to Claim 2 wherein the protein is selected from the group pumpkin seed globulin, keyhole limpet hemocyanin, marijuana seed globulin, ovalbumin and bovine serum albumin.

4. A conjugate according to Claim 3 wherein the compound is selected from the group of compounds (1) through (13) and the protein is selected from the group pumpkin seed globulin, keyhole limpet hemocyanin, ovalbumin and bovine serum albumin.

5. A conjugate according to Claim 4 wherein the compound is selected from the group of compounds (2), (3), (4), (8), (10) and (13) and the protein is keyhole limpet hemocyanin.

6. A conjugate according to Claim 4 wherein the compound is selected from the group of compounds (8), (9) and (10) and the protein is bovine serum albumin.

7. A conjugate according to Claim 4 wherein the compound is selected from the group of compounds (8), (9) and (13) and the protein is ovalbumin.

8. A conjugate according to Claim 9 wherein the compound is compound (8) and the protein is pumpkin seed globulin.

9. Antibodies to the conjugate of any one of Claims 2 through 8.

10. Antibodies to the conjugate of any one of Claims 2 through 8 produced in rabbits.

11. An improved enzyme-linked immunosorbent assay for detecting the presence of a sulfonylurea in an unknown sample comprising the steps:
(A) forming a complex of the sulfonylurea with an excess of a first antibody of known concentration, (B) complexing the unbound first antibody from Step A with a coating conjugate adhered to a solid phase, (C) binding a labeled antibody to the antibody-conjugate comples of B, and (D) determining the presence and the amount of sulfonylurea in the unknown sample by measuring the amount of unbound antibody in Step A with reference to controls which comprise known concentrations of the sulfonylurea;
wherein the improvement comprises (i) employing a conjugate according to Claim 2 as an immunogen to generate the antibody in Step A; and (ii) employing the same or a different conjugate according to Claim 2 as coating conjugate in Step B.

12. An assay according to Claim 11 employing a compound selected from the group of compounds (1) to (13) conjugated with a protein selected from keyhole limpet hemocyanin, pumpkin seed globulin, marijuana seed globulin, ovalbumin and bovine serum albumin.

13. An assay according to Claim 11 wherein the antibody employed in Step A is produced in a rabbit.

14. An assay according to Claim 12 employing a compound selected from at least one member of the group (1), (2), (3), (4), (8) and (13) conjugated with a protein selected from at least one member of the group pumpkin seed globulin, keyhole limpet hemocyanin, ovalbumin and bovine serum albumin.

15. An assay according to Claim 11 for measuring the presence of a sulfonylurea selected from the group chlorsulfuron, metsulfuron methyl, chlorimuron ethyl and bensulfuron methyl.

16. An assay according to Claim 14 wherein the antibody employed in Step A is produced in a rabbit.

17. A kit useful for measuring the presence of a target sulfonylurea in an unknown sample comprising the components:
(i) an antibody to the sulfonylurea in the unknown;
(ii) a solid phase having a coating conjugate bound to it;
(iii) a labeled antibody that recognizes antibody (i);
(iv) a developer that develops color in the presence of a label; and (v) controls comprising at least one known concentration of the sulfonylurea and one containing no sulfonylurea; whereby the components cooperate so that i, ii and iii are contacted with each other and, upon addition of iv, develop a color which indicates, upon comparison to the controls, the presence and concentration of the target sulfonylurea.

18. A test kit according to Claim 17 comprising an antibody to a target sulfonylurea selected from the group chlorsulfuron, bensulfuron methyl, metsulfuron methyl and chlorimuron ethyl.

19. A method for using the kit according to Claim 17 or Claim 18 to detect a target sulfonylurea comprising contacting components i, ii, iii and iv and comparing the color that is developed to controls, v, thereby determining the presence and concentration of the target sulfonylurea.

20. A compound according to Claim 1 wherein J
is J-1, J-2, J-3 or J-4; E1 is a direct bond and Y is methoxy, ethoxy, methyl, methylamino or chloro.