Chromophoric structures for lanthanide chelates
The present application discloses novel lanthanide chelate designs (Formula (I) and Formula (III)) having fluorenyl-, fluorenylethynyl, 9H-carbazolyl-, 9H-carbozolylethynyl-, dibenzothiophenyl-, dibenzothiophenylethynyl-, dibenzofuranyl or dibenzofuranylethynyl pyridine chromophores around an emitting lanthanide ion, e.g. an europium(III) ion. The three-membered ring chromophores exhibit high molar absorptivity and luminescence with lanthanide ions. The application also discloses a detectable molecule comprising a biospecific binding reactant useful in bioaffinity based binding assay, luminescent lanthanide chelating ligands, as well as a solid support conjugated with the chelates.
1. A luminescent lanthanide chelate comprising one or more chromophoric moieties of the formula (I):
wherein:
X is selected from the group consisting of —S—, —O—, —NR 1 —, —CR 1 R 2 —, >C═O, and >C═N—O—R 1 ;
R 1 and R 2 each independently are selected from the group consisting of hydrogen, —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1- 6 NHR 5 , —(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 2 ) 1-6 N(R 5 ) 2 , —(CH 2 ) 1-6 N + (CH 3 ) 2 R 5 , —(CH 1-6 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OCH 3 , —(CH 2 ) 1-6 NHC(═O)R 5 , —(CH 2 ) 1-6 NCH 3 C(═O)R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 2 ) 1-6 C(═O)NEtR 5 , —(CH 2 ) 1-6 C(═O)N(R 5 ) 2 , —(CH 2 ) 1-6 NHC(═O)NHR 5 , —(CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O)R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 , —COR 5 , —CO(CH 2 ) 1-6 NHR 5 , and —CO(CH 2 ) 1-6 NCH 3 R 5 , wherein R 5 is selected from the group consisting of hydrogen, C 1-12 -alkyl, —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 N + (CH 3 ) 2 (CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , a hydrophilic group, a reactive group Z, an oligopeptide, a polypeptide and a nucleotide;
R 3 and —CH 2 —R 4 each independently are selected from the group consisting of —CH 2 —N(CH 2 COOH) 2 , —CH 2 —N(CH 2 COO − ) 2 , —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1-6 NHR 5 , —(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 2 ) 1-6 N(R 5 ) 2 , —(CH 2 ) 1-6 OH, —(CH 2 ) 1- 6 OR 5 , —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OCH 3 ,—(CH 2 ) 1-6 SH, —(CH 2 ) 1-6 SR 5 , —(CH 2 ) 1-6 NHC(═O)R 5 , —(CH 2 ) 1-6 NCH 3 C(═O)R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 2 ) 1-6 C(═O)NEtR 5 , —(CH 2 ) 1-6 C(═O)N(R 5 ) 2 , —(CH 2 ) 1-6 NHC(═O)NHR 5 , —(CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O)R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 , —(CH 2 ) 1-3 O(CH 2 ) 1-3 -Chr, and —(CH 2 ) 1-3 S(CH 2 ) 1-3 -Chr; or R 3 and R 4 each independently are selected from the group consisting of —COOH, —COO − , —PO 3 H 2 , —PO 3 2− , —P(CH 3 )O 2 H, —P(CH 3 )O 2 − , —P(Ph)O 2 H, —P(Ph)O 2 − , —COR 5 , —CO(CH 2 ) 1-6 NHR 5 , and —CO(CH 2 ) 1-6 NCH 3 R 5 , wherein R 5 is selected from the group consisting of —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , and —(CH 2 ) 1-6 PO 3 2− , a hydrophilic group, a reactive group Z, an oligopeptide, a polypeptide and a nucleotide;
or R 3 and —CH 2 —R 4 are independently —(CH 2 ) 1-3 N(R 8 )—(CH 2 ) 1-3 —Chr, wherein R 8 is selected from —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1-6 NHR 5 , —(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 2 ) 1-6 N(R 5 ) 5 , —(CH 2 ) 1-6 NHC(═O)R 5 , —(CH 2 ) 1-6 NCH 3 C(═O)R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 2 ) 1-6 NHC(═O)NHR 5 , —(CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O)R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 , —COR 5 , —CO(CH 2 ) 1-6 NHR 5 , —CO(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-10 OH, —(CH 2 ) 1-10 OR 5 , —(CH 2 ) 1-10 NH 2 , —(CH 2 ) 1-10 NHR 5 , —(CH 2 ) 1-10 NCH 3 R 5 , —(CH 2 ) 1-10 SH, and —(CH 2 ) 1-10 SR 5 wherein R 5 is selected from —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , and a reactive group Z, and wherein Chr represents another chromophoric moiety;
R 6 is selected from the group consisting of hydrogen —Cl, —Br, —F, —I, —CH 3 , —(CH 2 ) 1-6 OH, —(CH 2 ) 1-6 OCH 3 —CF 3 , —CN, —NO 2 , —OH, —O(CH 2 ) 1-6 OH, —O(CH 2 ) 1-6 OCH 2 , —O(CH 2 ) 1-6 COOH, —O(CH 2 ) 1-6 COO − , —SCH 3 , —S(CH 2 ) 1-6 OH, —S(CH 2 ) 1-6 OCH 2 , —S(CH 2 ) 1-6 COOH, —S(CH 2 ) 1-6 COO − , —CONH 2 , —CONHCH 3 , —CON(CH 3 ) 2 , —CONH(CH 2 ) 1-6 OH, —CONHCH(CH 2 OH) 2 , —CONHC(CH 2 OH) 3 , —NHCOCH 3 , —NHCO(CH 2 ) 1-6 OH, —NHCO(CH 2 ) 1-6 COOH, —NHCO(CH 2 ) 1-6 COO − , a reactive group Z, and a hydrophilic group; wherein the hydrophilic group is selected from the group consisting of monosaccharides, disaccharides, —(CH 2 ) 1-6 CH 2 OH, —CH(CH 2 OH) 2 , —C(CH 2 OH) 3 —(CH 2 ) 1-3 —O—(CH 2 CH 2 O) 0-5 —H, —(CH 2 ) 1-3 —O—(CH 2 CH 2 O) 0-5 —C 1-4 -alkyl, —O—(CH 2 CH 2 O) 1-6 —H, and —O—(CH 2 CH 2 O) 1-6 —C 1-4 —alkyl,
wherein the reactive group Z is selected from the group consisting of azido(—N 3 ), alkynyl (—C≡CH), alkylene (—CH═CH 2 ), amino (—NH 2 ), aminooxy (—O—NH 2 ), carboxyl(—COOH), aldehyde (—CHO), hydrazide (—CONHNH 2 ), mercapto (—SH),maleimido, activated derivatives of maleimido, isocyanato (—NCO), isothiocyanato (—NCS), diazonium (—N + N), bromoacetamido, iodoacetamido, pyridyl-2-dithio, and 6- substituted 4-chloro-1,3,5-triazin-2-ylamino,
wherein a spacer is or is not included in a reactive group Z or a hydrophilic group and, when included, comprises one to five moieties, each moiety being independently selected from the group consisting of a phenylene, an alkylene containing 1-10 carbon atoms, an ethylenediyl (—C═C—), an ether (—O —), a thioether (—S—), a disulfide (—S—S—), an amide (—C(═O)—NH—, —NH—C(═O)—, —C(═O)—NCH 3 — and —NCH 3 —C(═O)—), a thiourea (—NH—C(═S)—NH—), and a triazole; and
Ln 3+ is a lanthanide ion.
2. The luminescent lanthanide chelate according to claim 1 , wherein X is —CR 1 R 2 —.
3. The luminescent lanthanide chelate according to claim 1 , wherein R 1 and R 2 each independently are selected from the group consisting of hydrogen, —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —CH 2 CONHCH 2 COOH, —CH 2 CONHCH 2 COO − , —CH 2 CON(CH 2 COOH) 2 , —CH 2 CON(CH 2 COO − ) 2 , —COCH 2 NHCH 2 COOH, —COCH 2 NHCH 2 COO − , —COCH 2 N(CH 2 COOH) 2 , and —COCH 2 N(CH 2 COO − ) 2 .
4. The luminescent lanthanide chelate according to claim 1 , wherein the luminescent lanthanide chelate is selected from the following formulae
5. A lanthanide chelating ligand comprising one or more chromophoric moieties of the formula (II)
wherein X is selected from the group consisting of —S—, —O—, —NR 1 —, —CR 1 R 2 —, >C═O, and >C═N—O—R 1 ;
R 1 and R 2 each independently are selected from the group consisting of hydrogen, —(CH 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1- 6 NHR 5 , —(CH 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 1-6 N (R 5 ) 2 , —(CH 2 ) 1-6 N+(CH 3 ) 2 R 5 , —(CH 1-6 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OCH 3 , —(CH 2 ) 1-6 NHC(═O)R 5 , —(CH 2 ) 1-6 NCH 3 C(═O)R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 1-6 C(═O)NEtR 5 , —(CH 2 ) 1-6 C(═O)N(R 5 ) 2 , —(CH 1-6 NHC(═O)NHR 5 , —(CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O) R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 , —COR 5 , —CO(CH 1-6 NHR 5 , and —CO(CH 1-6 NCH 3 R 5 , wherein R 5 is selected from the group consisting of hydrogen, C 1-12 -alkyl, —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 −, —(CH 2 ) 1-6 N + (CH 3 ) 2 (CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , a hydrophilic group, a reactive group Z, an oligopeptide, a polypeptide and a nucleotide;
R 3 and —CH 2 —R 4 each independently are selected from the group consisting of —CH 2 —N(CH 2 COOH) 2 , —CH 2 —N(CH 2 COO − ) 2 ,—(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1- 6 NHR 5 , —(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 2 ) 1-6 N(R 5 ) 2 , —(CH 2 ) 1-6 OH, —(CH 2 ) 1-6 OR 5 , —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OH, —(CH 2 CH 2 O) 1-4 CH 2 CH 2 OCH 3 , —(CH 2 ) 1-6 SH, —(CH 2 ) 1-6 SR 5 , —(CH 2 ) 1-6 NHC(═O) R 5 , —(CH 2 ) 1-6 NCH 3 C(═O) R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 2 ) 1-6 C(═O)NEtR 5 , —(CH 2 ) 1-6 C(═O)N(R 5 ) 2 , —(CH 2 ) 1-6 NHC(═O)NHR 5 , —(CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O) R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 , —(CH 2 ) 1-3 O(CH 2 ) 1-3 —Chr, and —(CH 2 ) 1-3 S(CH 2 ) 1-3 -Chr; or R 3 and R 4 each independently are selected from the group consisting of —COOH, —COO − , —PO 3 H 2 , —PO 3 2− , —P(CH 3 )O 2 H, —(CH 3 )O 2 − , —P(Ph)O 2 H, —(Ph)O 2 − , —COR 5 , —CO(CH 2 ) 1-6 NHR 5 , and —CO(CH 2 ) 1-6 NCH 3 R 5 , wherein R 5 is selected from the group consisting of —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO) 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , a hydrophilic group, a reactive group Z, an oligopeptide, a polypeptide and a nucleotide;
or R 3 and —CH 2 —R 4 are independently —(CH 2 ) 1-3 N(R 8 )—(CH 2 ) 1-3 —Chr, wherein R 8 is selected from —(CH 2 ) 1-6 COOH , —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , —(CH 2 ) 1-6 NHR 5 , —(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-6 NEtR 5 , —(CH 2 ) 1-6 N(R 5 ) 2 , —(CH 2 ) 1-6 NHC(═O)R 5 , —(CH 2 ) 1-6 NCH 3 C(═O) R 5 , —(CH 2 ) 1-6 C(═O)NHR 5 , —(CH 2 ) 1-6 C(═O)NCH 3 R 5 , —(CH 2 ) 1-6 NHC(═O)NHR 5 , —CH 2 ) 1-6 NHC(═S)NHR 5 , —(CH 2 ) 1-6 C(═O) R 5 , —(CH 2 ) 1-6 —C 6 H 4 —R 5 ,—COR 5 , —CO(CH 2 ) 1-6 NHR 5 , —CO(CH 2 ) 1-6 NCH 3 R 5 , —(CH 2 ) 1-10 OH, —CH 2 ) 1-10 OR 5 , —CH 2 ) 1-10 NH 2 , —(CH 2 ) 1-10 NHR 5 , —(CH 2 ) 1-10 NCH 3 R 5 , —(CH 2 ) 1-10 SH, and —(CH 2 ) 1-10 SR 5 wherein R 5 is selected from —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , and a reactive group Z, and wherein Chr represents another chromophoric moiety
R 6 is selected from the group consisting of hydrogen —Cl, —Br, —F, —I, —CH 3 , —(CH 2 ) 1-6 OH, —(CH 2 ) 1-6 OCH 3 —CF 3 , —CN, —NO 2 , —OH, —O(CH 2 ) 1-6 OH, —O(CH 2 ) 1-6 OCH 2 , —O(CH 2 ) 1-6 COOH, —O(CH 2 ) 1-6 COO − , —SCH 3 , —S(CH 2 ) 1-6 OH, —S(CH 2 ) 1-6 OCH 2 , —S(CH 2 ) 1-6 COOH, —S(CH 2 ) 1-6 COO − , —CONH 2 , —CONHCH 3 , —CON(CH 3 ) 2 , —CONH(CH 2 ) 1-6 OH, —CONHCH(CH 2 OH) 2 , —CONHC(CH 2 OH) 3 , —NHCOCH 3 , —NHCO(CH 2 ) 1-6 OH, —NHCO(CH 2 ) 1-6 COOH, —NHCO(CH 2 ) 1-6 COO − , a reactive group Z, and a hydrophilic group; wherein the hydrophilic group is selected from the group consisting of monosaccharides, disaccharides, —CH 2 ) 1-6 CH 2 OH, —CH(CH 2 OH) 2 , —C(CH 2 OH) 3 —(CH 2 ) 1-3 —O—(CH 2 CH 2 O) 0-5 —H, —(CH 2 ) 1-3 —O—(CH 2 CH 2 O) 0-5 —C 1-4 -alkyl, —O—(CH 2 CH 2 O) 1-6 —H, and —O—(CH 2 CH 2 O) 1-6 —C 1-4 -alkyl,
wherein the reactive group Z is selected from the group consisting of azido (—N 3 ), alkynyl (—C≡CH), alkylene (—CH═CH 2 ), amino (—NH 2 ), aminooxy (—O—NH 2 ), carboxyl (—COOH), aldehyde (—CHO), hydrazide (—CONHNH 2 ), mercapto (—SH), maleimido, activated derivatives of maleimido, isocyanato (—NCO), isothiocyanato (—NCS), diazonium (—N + N), bromoacetamido, iodoacetamido, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino,
wherein a spacer is or is not included in a reactive group Z or a hydrophilic group and, when included, comprises one to five moieties, each moiety being independently selected from the group consisting of a phenylene, an alkylene containing 1-10 carbon atoms, an ethylenediyl (—C═C—), an ether (—O—), a thioether (—S—), a disulfide (—S—S—), an amide (—C(═O)—NH—, —NH—C(═O)—, —C(═O)—NCH 3 — and —NCH 3 —C(═O)—), a thiourea (—NH—C(═S)—NH—), and a triazole.
6. The luminescent lanthanide chelate according to claim 1 , wherein R 5 is C 1 -C 6 -alkyl.
7. The luminescent lanthanide chelate according to claim 3 , wherein R 1 and R 2 each independently are —CH 2 —COOH, —CH 2 —COO − , —CH 2 CON(CH 2 COOH) 2 , and —CH 2 CON(CH 2 COO − ) 2 .
8. The luminescent lanthanide chelate according to claim 1 , wherein R 1 and R 2 are each independently —(CH 2 CH 2 O) 2 CH 2 CH 2 OCH 3 .
9. The luminescent lanthanide chelate according to claim 1 , wherein Ln 3+ is Eu 3+ .
10. The luminescent lanthanide chelate according to claim 1 , wherein R 3 and —CH 2 —R 4 each independently are selected from the group consisting of —CH 2 —N(CH 2 COOH) 2 , —CH 2 N(CH 2 COO − ) 2 , and —CH 2 ) 1-3 N(R 8 )—(CH 2 ) 1-3 -Chr, wherein R 8 is selected from the group consisting of —(CH 2 ) 1-6 COOH, —(CH 2 ) 1-6 COO − , —(CH 2 ) 1-6 SO 3 H, —(CH 2 ) 1-6 SO 3 − , —(CH 2 ) 1-6 —O—PO 3 H 2 , —(CH 2 ) 1-6 —O—PO 3 2− , —(CH 2 ) 1-6 PO 3 H 2 , —(CH 2 ) 1-6 PO 3 2− , and reactive group, or wherein R 3 and R 4 each independently are selected from the group consisting of —COOH, —COO − , —PO 3 H 2 , —PO 3 2− , —P(CH 3 )O 2 H, —P(CH 3 )O 2 − , —P(PH)O 2 H, and —P(Ph)O 2 − .