Substrates for beta-lactamase and uses thereof

A compound of formula I:

• wherein:
• one of X and Y is a fluorescent donor moiety or a membrane-permeant derivative thereof selected from the group consisting of bismanes, acridines, isoindoles, cyanines and a moiety of formula V;
• and the other is a quencher moiety, an acceptor fluorophore moiety or a membrane-permeant derivative thereof selected from the group consisting of bismanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides, aminophthalimides, amino benzofurans, aminoquinolines, dicyanohydroquinones, cyanine, nitrodyes, cyano derivatives, quinones, dicyanovinyl and tricyanovinyl dyes, indoanilines, indigo dyes, anthraquinones, polymethines, di-and triphenylmethane dyes, indamines and a structure of formula LVI;
• R′ is selected from the group consisting of H, lower alkyl, —(CH2)nOH, —(CH2)nCOOR″ and═NOJ, wherein each n is independently 0 or an integer from 1 to 5 and J is —H, —CH3, —CH2COOH, —CHMeCOOH and —CMe2COOH;
• R″ is selected from the group consisting of —H, physiologically acceptable metal and ammonium cations, —CHR2OCO(CH2)nCH3, —CHR2OCOC(CH3)3, acylthiomethyl, acyloxy -alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, betamorpholinoethyl, dialkylaminoethyl, dialkylaminocarbonyloxymethyl, in which R2 is selected from the group consisting of —H and lower alkyl;
• A is selected from the group consisting of S, O, SO, SO2 and CH2;
• Z′ is a linker for X;
• and
• Z″ is a linker for Y.

A method of preparing a fluorescent substrate for β-lactamase, said method comprising:

• providing a first fluorescent molecule having a first-reactive functional group, a second fluorescent molecule having a second-reactive finctional group and a cephalosporin compound having the formula:
• wherein G and Q are reactive functional groups and R0 is selected from the group consisting of H and alkyl, optionally substituted with 1-2 aryl groups;
• and
• contating the cephalosporin compound with the first fluorescent molecule and the second fluorescent molecule to form the fluorescent substrate, wherein the first-reactive functional group reacts with G and the second-reactive finctional group reacts with Q.