reagents of general formula i , wherein group x is selected from either nh 2 , nhch 3 and nhoh , and wherein group y is o , are derived from either 4 - aminosalicylic acid or 5 - aminosalicylic acid . as illustrated in fig1 - or 5 - aminosalicylic acid is first esterified to afford either methyl 4 - aminosalicylate or methyl 5 - aminosalicylate , respectively . the ester is neutralized and then amidated by reaction with an amine selected from either ammonia , methylamine and hydroxylamine , to afford either a 4 - or 5 - aminosalicylamide of general formula ix , wherein group x is selected from either nh 2 , nhch 3 and nhoh . the compound of general formula ix is next condensed with an activated carboxylic acid preferably selected from , but not limited to , succinic anhydride , methyl succinyl chloride , maleic anhydride , n - methoxycarbonylmaleimide , 3 - bromopropionyl chloride , 3 - iodopropionyl chloride , iodoacetyl chloride , bromoacetyl chloride and chloroacetyl chloride , to afford a compound of general formula x , wherein group r is preferably selected from , but not limited to , either ch 2 ch 2 cooh , ch 2 ch 2 cooch 3 , ch = chcooh , ch 2 ch 2 br , ch 2 ch 2 i , ch 2 i , ch 2 br and ch 2 cl , and wherein group x is as was previously defined . compounds of general formula x , wherein group r is selected from either ch 2 i and ch 2 br ( fig1 a , 1 ), are useful as thiol reactive reagents suitable for appending a phenylboronic acid complexing moiety to a bioactive species having pendant thiol groups . compounds of general formula x , wherein group x is oh , and wherein group r is ch 2 i , have been prepared previously for use as enzyme inhibitors ( holbrook , j . j ., roberts , p . a . and wallis , r . b . ( 1973 ) biochem . j ., 133 , 165 - 171 ). compounds of general formula x , wherein group r is ch = chcooh ( fig1 a , 2 ), may be further functionalized by ring closure to afford a thiol reactive maleimide reagent suitable for appending a phenylboroic acid complexing moiety to a bioactive species having pendant thiol groups . compounds of general formula x , wherein group r is ch 2 ch 2 cooch 3 ( fig1 a , 3 ), may be further functionalized by reaction with hydrazinc hydrate to afford a hydrazide reagent of general formula x , wherein r is ch 2 ch 2 conhnh 2 , suitable for appending a phenylboronic acid complexing moiety to a bioactive species having pendant aldehyde groups ( which result from periodate oxidation of carbohydrate residues ). compounds of general formula x , wherein group r is selected from either ch 2 ch 2 br and ch 2 ch 2 i ( fig1 ), may be further functionalized by reaction with potassium thiolacetate to afford an intermediate which , upon deprotection , affords a thiol containing compound of general formula x , wherein group r is ch 2 ch 2 sh . the thiol containing compound of may be activated by reaction with a reagent preferably selected from , but not limited to 2 , 2 &# 39 ;- dithiodipyridine , 4 , 4 &# 39 ;- dithiodipyridine and 2 , 2 &# 39 ;- dithiodi ( 3 - nitropyridine ), to prepare an activated disulfide containing reagent suitable for appending a phenylboronic acid complexing moiety to a bioactive species having pendant thiol groups , through a cleavable disulfide bond . compounds of general formula x , wherein group x is selected from either nh 2 and nhch 3 , and wherein group r is ch 2 ch 2 cooh ( fig1 a , 5 ), may be further functionalized by reaction with dicyclohexylcarbodiimide ( dcc ) and a reagent preferably selected from , but not limited to , either n - hydroxysuccinimide ( nhs ) and n - hydroxysulfosuccinimide ( snhs ), to afford an activated ester reagent suitable for appending a phenylboronic acid complexing moiety to a bioactive species having pendant amine groups . activated esters of general formula x , wherein group x is selected from either nh 2 and nhch 3 , and wherein group r is ch 2 ch 2 co -- nhs , are useful synthetic intermediates for the preparation of reagents of general formula i , wherein group z is comprised of an alkyl chain or polyethyleneglycol chain , of greater than 6 carbon equivalents in length ( as illustrated in fig1 a ). compounds of general formula x , wherein group x is nhoh , can not be routinely employed to prepare reagents having activated ester moieties , due to problems which arise when the carboxylic acid group is activated in the presence of both nhs and the benzohydroxamic acid group associated with the phenylboronic acid complexing moiety . this represents a limitation , due to the popularity of n - hydroxysuccinimide reagents , and the fact that only reagents of general formula i , wherein group x is selected from either nhoh and nhoch 3 , form stable complexes of general formulas v , vi and vii , over a broad ph range . to overcome this limitation , an alternate synthetic route is employed to prepare activated ester reagents of general formula i , wherein group x is nhoch 3 , and group y is o ( fig2 ). 4 - aminosalicylic acid or 5 - aminosalicylic acid is condensed with methyl succinyl chloride to afford a compound of general formula x , wherein group x is oh , and wherein group r is ch 2 ch 2 cooch 3 . subsequent reaction with n , n - carbonyldiimidazole ( cdi ) followed by addition of methoxylamine hydrochloride , affords a compound of general formula xi , wherein group x is nhoch 3 . alkaline hydrolysis of the carboxylic acid ester group affords a compound having a free carboxylic acid group . subsequent activation of the carboxylic acid group by reaction with dcc and a reagent preferably selected from , but not limited to , either nhs and snhs , affords an activated ester reagent of general formula xii , wherein group x is nhoch 3 , suitable for appending a phenylboronic acid complexing moiety to a bioactive species having pendant amine groups . n - hydroxysuccinimidyl esters of general formula xii , wherein group x is nhoch 3 , are useful synthetic intermediates for the preparation of reagents of general formula i , wherein group z is comprised of an alkyl chain or polyethyleneglycol chain , of greater than 6 carbon equivalents in length . compounds of general formula xii , wherein group x is nhoch 3 , may be further functionalized by reaction with a reagent preferably selected from , but not limited to 6 - aminohexanoic acid , 4 - aminobutanoic acid , n - tert - butoxycarbonyl - 1 , 6 - diaminohexane ( n - boc - 1 , 6 - diaminohexane ) and n - boc - 1 , 4 - diaminobutane , to afford , after removal of the boc protecting group , if required , a compound having an extended spacer and either a terminal carboxylic acid group ( fig2 ), or a terminal amine group ( fig2 )). the aforementioned reagents , having pendant carboxylic acid groups , are useful for the preparation nhs ester , snhs ester and hydrazide containing reagents having long spacers , which are useful to overcome steric hindrance know to be associated with biological macromolecules of high molecular weight . similarly , the aforementioned reagents , having pendant amine groups , are useful for the preparation iodoacetamide , maleimide and activated disulfide containing compounds having long spacers . additionally , the aforementioned reagents having either pendant carboxylic acid groups or pendant amine groups groups are useful synthetic intermediates for the preparation of solid - phase supports . reagents of general formula ii , wherein group x is selected from either nh 2 , nhch 3 and nhoh , and wherein group y is o , are derived from either 4 - or 5 - aminosalicylic acid in a manner analogous to that employed to prepare reagents of general formula i . a compound of general formula ix , prepared as previously described , is condensed with an activated dicarboxylic acid preferably selected from , but not limited to , succinyl chloride , adipoyl chloride , adiptic acid diisobutylcarbonate , suberoyl chloride , 3 , 3 &# 39 ;- dithiopropionyl chloride , 3 , 6 , 9 - trioxaundecanedioyl chloride and 3 , 6 , 9 - trioxaundecanedioic acid diisobutylcarbonate , to afford a compound of general formula ii , wherein group x is selected from either n 2 , nhch 3 and nhoh , wherein group y is o , and wherein group z is selected from , but not limited to , either ( ch 2 ) 2 , ( ch 2 ) 4 , ( ch 2 ) 6 , ( ch 2 ) 2 ss ( ch 2 ) 2 and ch 2 ( och 2 ch 2 ) 2 och 2 , respectively . reagents of general formula ii , wherein group x is nhoch 3 , and wherein group y is o , are prepared by an alternate synthetic route , in a manner analogous to the preparation of reagents of general formula i , wherein group x is nhoch 3 . either 4 - or 5 - aminosalicylic acid is condensed with an activated dicarboxylic acid preferably selected from , but not limited to , succinyl chloride , adipoyl chloride , adiptic acid diisobutylcarbonate , suberoyl chloride , 3 , 3 &# 39 ;- dithiopropionyl chloride and 3 , 6 , 9 - trioxaundecanedioyl chloride and 3 , 6 , 9 - trioxaundecanedioic acid diisobutylcarbonate , to afford a compound of general formula ii , wherein group x is oh , wherein group y is o , and wherein group z is selected from , but not limited to , either ( ch 2 ) 2 , ( ch 2 ) 4 , ( ch 2 ) 6 , ( ch 2 ) 2 ss ( ch 2 ) 2 and ch 2 ( och 2 ch 2 ) 2 och 2 , respectively ( fig3 a ). subsequent reaction of a compound of general formula ii , wherein group x is oh , wherein group y is o , and wherein group z is selected from , but not limited to , either ( ch 2 ) 2 , ( ch 2 ) 4 , methoxylamine hydrochloride , affords a compound wherein group x is nhoch 3 , and wherein groups y and z are as were previously defined . bioconjugates of general formulas v , vi and vii may be prepared in buffered aqueous or aqueous / organic solutions . preferred buffers include acetate , citrate , phosphate , carbonate and diglycine . borate and tris buffers should be avoided due to their ability to complex with the phenylboronic acid complexing moiety and phenylboronic acid moiety , respectively . the bioconjugate is formed within 1 to 15 minutes at room temperature . the reaction is insensitive to variations in ionic strength over the range 0 . 01 to 2 molar . stability of the conjugate increases with increasing temperature , limited only by the volatility of the buffer . the addition of organic solvents including acetonitrile , methanol , ethanol , isopropanol , butanol , n , n - dimethylformamide and dimethylsulfoxide serves to further stabilize conjugates . chaotropic reagents including urea , guanidine hydrochloride and formamide also serve to further stabilie conjugates . once formed , bioconjugates are stable upon removal of water , and can be purified by lyophilization . the stability of the bioconjugate at a given ph is determined by substituent groups x and y . for compounds of general formulas i and ii , individuals skilled in the art will recognize that a number of tautomeric and conformational isomers exist in equilibrium as illustrated in fig4 ( bioactive species omitted for clarity ). each of the isomers depicted in fig4 may contribute to phenylboronic acid complexation as illustrated in fig5 ( bioactive species omitted for clarity ). for all substituent groups x and y , except when both x and y are o , conjugates are stable in buffered alkaline aqueous solutions over the approximate ph range 8 . 5 to 11 . 5 . phenylboronic acid complexes of general formulas v , vi and vii , wherein both groups x and y are o , represent a novel situation in which conjugates are stable in buffered acidic aqueous solutions of approximate ph less than 4 . 5 . this range of ph stability results from the requirement that group x must posses an acidic hydrogen if a stable conjugate is to form . as illustrated in fig6 ( bioactive species omitted for clarity ), conjugates will only form at low ph where the salicylic acid moiety is protonated . phenylboronic acid complexes of general formulas v , vi and vii , wherein group x is selected from either noh and noch 3 , and wherein group y is o , represent a novel situation in which bioconjugates are stable in buffered aqueous solutions over the approximate ph range 2 . 5 to 11 . 5 . the wide range of ph stability is thought to result from the presence of a coplanar 1 , 3 - diol complexing moiety associated with the enol form of 2 - hydroxybenzohydroxamic acid . as illustrated in fig7 ( bioactive species omitted for clarity ), the enol tautomer affords a stable complex with both neutral phenylboronic acid , under acidic conditions , and phenylboronate anion , under alkaline conditions , due to the stability of the six - membered cyclic ester ( wherein r &# 39 ; is selected from either h and ch 3 ). absolute methanol ( 100 ml ) and fresh concentrated h 2 so 4 are carefully combined in a 250 ml round botton flask with continuous stirring ( exothermic ). 4 - aminosalicylic acid ( 10 . 0 grams , 65 . 4 mmol ) is added to produce a dark solution , which is heated under reflux for 6 hours . the product is allowed to cool and then concentrated on a rotary evaporator to approximately half the original volume . at this point a solid precipitate appears . the concentrate is poured into 400 ml of water , and the resulting suspension titrated to approximately ph 3 , by stepwise addition of 5n naoh ( to ph 6 . 5 ), followed by solid na 2 co 3 , with evolution of co 2 gas . the concentrate was chilled on ice , and the resulting precipitate collected by filtration . the filtrate is washed with cold water , and then dried in vacuo over naoh pellets , to afford 9 . 6 grams ( 88 % yield ) of methyl 4 - aminosalicylate , a pale lavendar powder ( m . p . 115 °- 117 ° c .). the structure was confirmed by 1 h nmr spectrometry in d 6 - acetone . naoh ( 4 . 0 grams ) in 16 ml of water is carefully added to hydroxylamine hydrochloride ( 2 . 8 grams , 40 mmol ) and 20 grams of ice . after dissolution , na 2 so 3 ( 0 . 4 grams ) is added , followed by methyl 4 - aminosalicylate ( 3 . 35 grams , 20 mmol ). the resulting solution is stirred for up to 24 hours at room temperature , the course of the reaction is monitored every few hours by reverse phase hplc . the resulting solution is chilled on ice and acidified by addition of 25 % h 2 so 4 . a precipitate is first formed at approx . ph 6 . the ph is finally adjusted to approx . ph 4 , and the pale tan precipitate collected by filtration . the product is dried in vacuo over p 2 o 5 , to afford 3 . 0 grams ( 89 % yield ) of 4 - amino - 2 - hydroxybenzohydroxamic acid ( m . p . 180 °- 181 ° c .). the structure was confirmed by 1 h nmr spectrometry in d 6 - dmso . 4 - amino - 2 - hydroxybenzohydroxamic acid is a key synthetic intermediate in the preparation of reagents of both general formulas i and if , wherein group x is nhoh , and wherein group y is o . reagents having the 2 - hydroxybenzohydroxamic acid moiety can form bioconjugates which are stable in buffered aqueous solutions over the approximate ph range 2 . 5 to 11 . 5 . preparation of an aldehyde reactive phenylboronic acid complexing reagent of general formula i to an ice - cooled , stirred solution of 4 - amino - 2 - hydroxybenzohydroxamic acid ( 8 . 4 grams , 0 . 05 mol ), prepared as described above , in 150 ml of water containing nahco 3 ( 7 . 0 grams , 0 . 02 mol ), 3 - carbomethoxypropionyl chloride ( 9 . 0 grams , 0 . 06 mol ) is added dropwise over 15 min . after stirring at 0 °- 5 ° c . for 1 hr ., the solution is acidified with cold 6n hcl . the precipitate is collected and dried in vacuo over naoh pellets , to afford 13 . 5 grams ( 96 % yield ) of crude n - 4 -( 3 - carbomethoxypropionamido )- 2 - hydroxy - benzohydroxamic acid , which is utilized without further purification . to a solution of n - 4 -( 3 - carbomethoxypropionamido )- 2 - hydroxybenzohydroxamic acid ( 10 grams , 0 . 035 mol ) in 50 ml of methanol is added hydrazinc hydrate ( 12 ml ). the reaction is allowed to proceed overnight at room temperature , and the product is filtered and washed with ether . the product is recrystallized twice from dimethylformamide to afford 7 . 3 grams ( 78 % yield ) of n - 4 ( 3 - hydrazidopropionamido )- 2 - hydroxybenzohydroxamic acid . glycoproteins , and particularly antibodies , may be conjugated with an aldehyde reactive phenylboronic acid complexing hydrazide reagent after treatment of the protein with from 5 to 20 mm sodium meta periodate ( naio 4 ), in from 0 . 1 to 0 . 5m sodium acetate buffer at ph 5 to 6 , containing up to 0 . 2m sodium chloride , at 0 ° c ., for from 30 minutes to 4 hours . the excess periodate is removed by dialysis or desalting , and the activated protein , having pendant adjacent aldehyde moieties resulting from periodate oxidation of carbohydrate residues having adjacent coaxial 1 , 2 - diol moities , is condensed with the hydrazide reagent , for from 1 to 24 hours at room temperature , to afford a semiconjugate having pendant phenylboronic acid complexing moities covalently appended to the protein throught a shiff base ( an imine ) type linkage . the stability of the linkage to the protein may be increased , if desired , by mild sodium cyanoborohydride reduction of the schiff base to the corresponding alkylamine . it is important to note that sodium meta periodate oxidation of a glycoprotein activates the protein toward reaction with a hydrazide type reagent while simultaneously removing most naturally occuring phenylboronic acid complexing sites ( coaxial 1 , 2 - diols ) associated with glycoproteins . preparation of a thiol reactive phenylboronic acid complexing reagent of general formula i to an ice - cooled , stirred solution of s - aminosalicylamide ( 10 . 0 grams , 0 . 073 mol ), in 300 ml of water containing nahco 3 ( 42 . 0 grams , 0 . 5 mol ), iodoacetyl chloride ( 18 . 4 grams , 0 . 09 mol ) is added dropwise over 15 min . after stirring at 0 °- 5 ° c . for 1 hr ., the solution is acidified with cold 6n hcl . the precipitate is collected and dried in vacuo over naoh pellets , to afford 21 . 3 grams ( 96 % yield ) of crude 5 -( iodoacetamido ) salicylamide . proteins containing disulfide bonds ( cystine residues ) or cysteine residues may be modified with a thiol reactive phenylboronic acid complexing reagent , such as 5 -( iodoacetamido )- salicylamide . disulfide bonds are first reduced , if required , by reaction with 2 - mercaptoethanol or dithiothreitol , in alkaline aqueous solution which has been throughly degassed . the excess reducing reagent is removed by dialysis or desalting , and the protein reacted with the alkylating reagent in neutral aqueous solution , for 1 hour at 4 ° c ., to afford a semiconjugate having pendant phenylboronic acid complexing moities covalently appended to the protein . upon completion of the reaction , the excess reagent is removed by desalting . preparation of an amine reactive phenylboronic acid complexing reagent of general formula i to an ice - cooled , stirred solution of 4 - aminosalicylic acid ( 7 . 7 grams , 0 . 05 mol ), prepared as described above , in 150 ml of water containing nahco 3 ( 7 . 0 grams , 0 . 02 mol ), 3 - carbomethoxypropionyl chloride ( 9 . 0 grams , 0 . 06 mol ) is added dropwise over 15 min . after stirring at 0 °- 5 ° c . for 1 hr ., the solution is acidified with cold 6n hcl . the precipitate is collected and dried in vacuo over naoh pellets , to afford 11 . 9 grams ( 89 % yield ) of crude n - 4 -( 3 - carbomethoxy - propionamido ) salicylic acid , which is utilized without further purification . to a vigorously stirred solution of n - 4 -( 3 - carbomethoxypropionamido ) salicylic acid ( 10 . 0 grams , 0 . 036 mol ) in 50 ml of tetrahydrofuran is added stepwise 1 , 1 &# 39 ;- carbonyldiimidazole ( 5 . 84 grams , 0 . 036 mol ) and methoxylamine hydrochloride ( 3 . 0 grams , 0 . 036 mol ). the vessel is fitted with a drying tube and the reaction vigorously stirred for 30 min . at room temperature . imidazolium hydrochloride , which separated during the course of the reaction , is removed by filtration . the filtrate is concentrated on a rotary evaporator to afford an amber oil , which is dissolved in 10 ml of warm tetrahydrofuran , and then added to 150 ml of 2n h 2 so 4 is precipitate is collected by filtration , washed with 2n h 2 so 3 , washed with water , and then dried overnight in vacuo over naoh pellets to afford 10 . 0 grams ( 94 % yield ) of n - 4 -( 3 - carbomethoxy - propionamido )- 2 - hydroxy - o - methylbenzohydroxamic acid . n - 4 -( 3 - carbomethoxypropionamido )- 2 - hydroxy - o - methylbenzohydroxamic acid ( 7 . 4 grams , 0 . 025 mol ) is dissolved in 25 ml of 0 . 2n methanolic lioh . the solution is stirred overnight at room temperature under nitrogen . methanol is removed on a rotary evaporator , and the residue dissolved 150 ml of water . acidifation of the solution to approximately ph 2 with 2n h 2 so 4 is followed by extraction into 100 ml of ether . a second extration into ether is followed by drying of the combined ether extracts over anhydrous na 2 so 4 . the product is concentrated on a rotary evaporator and then dried overnight in vacuo over p 2 o 5 , to afford 6 . 28 grams ( 89 % yield ) of n - 4 - succinamido - 2 - hydroxy - o - methylbenzohydroxamic acid . n - 4 - succinamido - 2 - hydroxy - o - methylbenzohydroxamic acid ( 5 . 65 grams , 0 . 02 mol ) is dissolved in 50 ml of hot dimethylformamide and allowed to cool to room temperature . to the stirred solution is added n - hydroxysuccinimide ( 2 . 3 grams , 0 . 02 mol ) followed by a fleshly prepared solution of dicyclohexylcarbodiimide ( 4 . 1 grams , 0 . 02 mol ) in 10 ml of dimethyl - formamide . the resulting suspension is stirred overnight at room temperature . dicyclohexylurea is filtered from solution , and the filtrate concentrated on a rotary evaporator to a minimum volume . the residue is precipitated with ether , and the precipitate collected by filtration , washed with ether , washed with 2 - propanol , and then dried briefly in vacuo over p 2 o 5 , to afford 5 . 6 grams ( 74 % yield ) of n - 4 - succinamido - 2 - hydroxy - o - methlybenzohydroxamic acid succinimidyl ester . the product is stored in a freezer at - 15 ° c . proteins may be conjugated with amine reactive phenylboronic acid complexing reagents by reaction with the side - chain ε - amino groups of lysine residues , to afford a semiconjugate having pendant phenylboronic acid complexing moities covalently appended to the protein through stable amide bonds . mildly alkaline aqueous buffers , in the ph range 7 . 8 to 8 . 8 , should be employed so as to insure that the amino group is unprotonated , while minimizing hydrolysis of the nhs ester . primary amine containing buffers including tris and glycine must be avoided , so as to avoid cross - reactivity . solid - phase supports having pendant primary amine moities may be functionalized , in an analogous manner , by reaction with phenylboronic acid complexing reagents to afford solid - phase supports having pendant phenylboronic acid complexing moities . 4 - amino - 2 - hydroxybenzohydroxamic acid ( 6 . 4 grams , 0 . 038 mol ) is dissolved in 50 ml of dry dichloromethane . triethylamine ( 5 . 3 ml , 0 . 038 mol ) is added followed by the dropwise addition of a solution of 3 , 6 , 9 - trioxaundecanedioyl chloride ( 5 . 0 grams , 0 . 019 mol ) in 50 ml of dry dichloromethane , over a period of 2 hours . triethylammonium hydrochloride is removed by filtration , and the flitrate washed with water ( 2 × 100 ml ), saturated nahco 3 ( 2 × 100 ml ), saturated nacl ( 100 ml ), and dried over anhydrous na 2 so 4 . the solvent is removed on a rotary evaporator and the residue recrystallized from methanol ( 100 ml ) to afford 6 . 0 grams ( 61 % yield ) of colorless crystals . the structure was confirmed by 1 h nmr spectrometry in d 6 - dmso . proteins which have been conjugated to phenylboronic acid may be cross - linked by the action of reagents of general formula ii . this process is particularly useful for the preparation of protein aggregates , which are useful for modifying the properties of protein stability and solubility . additionally , enzyme aggregates , prepared by cross - linking , are useful for amplifying the sensitivity of elisa . this principle is exploited in the avidin - biotin system by the use of avidin - biotin complexes ( called abc complexes ), in which a biotinylated enzyme is first employed to form a high molecular weight complex with avidin coy cross - linking ), prior to introduction of the complex into an elisa for the high sensitivity detection of a biotinylated antibody .