US 5,397,695 A · Attachment of compounds to polymeric particles using carbamoylonium compounds and a kit containing same

Abstract

Useful materials for diagnostic tests, affinity chromatography, enzymatic reactions and immunoassays are prepared by covalently attaching reactive compounds containing reactive amino or sulfhydryl groups to polymeric particles having pendant carboxyl groups on the outer surfaces. Such reactive compounds include biologically reactive species, such as enzymes, polypeptides and proteins. This attachment is carried out using carbamoylonium compounds which react with the carboxyl groups to form intermediate reactive groups which then react with the amino or sulfhydryl groups to form a covalent linkage between particle and reactive compound. A kit comprises polymeric particles having carboxyl groups on the outer surfaces, and a carbamoylonium compound.

A method for attaching a reactive amine- or sulfhydryl-containing compound to polymeric particles comprising:

A. contacting (1) an aqueous suspension of polymeric particles having pendant carboxyl groups on the surface thereof with (2) a carbamoylonium compound to produce reactive intermediate polymer particles having pendant intermediate reactive groups, and
B. contacting the reactive intermediate polymer particles produced in step A with a reactive amine- or sulfhydryl-containing compound having a reactive amine or sulfhydryl group, respectively, which reacts with said intermediate reactive groups to form a covalent linkage between said particles and said reactive compound,
wherein said carbamoylonium compound has the structure:
##STR4## wherein Z represents the atoms necessary to complete a substituted or unsubstituted 5- or 6-membered heterocyclic aromatic ring,
m and n are independently 0 or 1,
R.sup.1 and R.sup.2 are independently of each other, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted aralkyl, or R.sup.1 and R.sup.2 together represent the atoms necessary to complete a substituted or unsubstituted piperidine, piperazine or morpholine ring,
R.sup.3 is a hydrogen atom, a substituted or unsubstituted alkyl, or the group ##STR5## wherein A represents the polymerized vinyl backbone of a homo- or copolymer formed from one or more ethylenically unsaturated polymerizable compounds such that the molecular weight of said homo- or copolymer is greater than about 1000,
R.sup.4 is a hydrogen atom, a substituted or unsubstituted alkyl, or when Z represents the atoms necessary to complete a pyridinium ring and n is 0, R.sup.4 is selected from the following groups:
(a) --NR.sup.6 --CO--R.sup.7 wherein R.sup.6 is hydrogen or substituted or unsubstituted alkyl, R.sup.7 is hydrogen, substituted or unsubstituted alkyl or --NR.sup.8 R.sup.9 wherein R.sup.8 and R.sup.9 are independently hydrogen or substituted or unsubstituted alkyl,
(b) --(CH.sub.2).sub.q --NR.sup.10 R.sup.11 wherein R.sup.10 is --CO--R.sup.12, R.sup.11 is hydrogen or substituted or unsubstituted alkyl, R.sup.12 is hydrogen, substituted or unsubstituted alkyl or --NR.sup.13 R.sup.14 wherein R.sup.13 is substituted or unsubstituted alkyl or substituted or unsubstituted aryl, R.sup.14 is hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, and q is 1 to 3.
(c) --(CH.sub.2).sub.r --CONR.sup.15 R.sup.16 wherein R.sup.15 is hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, R.sup.16 is hydrogen or substituted or unsubstituted alkyl, or R.sup.15 and R.sup.16 together represent the atoms necessary to complete a 5- or 6-membered aliphatic ring and r is 0 to 3, ##STR6## wherein R.sup.17 is hydrogen, substituted or unsubstituted alkyl, Y is oxy or --NR.sup.19 -, R.sup.18 is hydrogen, substituted or unsubstituted alkyl, --CO--R.sup.20 or --CO--NHR.sup.21 wherein R.sup.19, R.sup.20 and R.sup.21 are independently hydrogen or substituted or unsubstituted alkyl, and t is 2 or 3, and
(e) --R.sup.21 X'.sup..THETA. wherein R.sup.21 is substituted or unsubstituted alkylene, and X'.sup..THETA. is a covalently bonded anionic group so as to form an inner salt group with the pyridinium ring,
R.sup.5 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted aralkyl, provided that m is 0 when the nitrogen atom to which R.sup.5 is bound is attached to remainder of the ring through a double bond, and
X is an anion, and v is 0 or 1, provided that it is 0 only when R.sup.4 is --R.sup.21 X'.sup..THETA..

11 more elements

Attachment of compounds to polymeric particles using carbamoylonium compounds and a kit containing same

Life of this patent

Attachment of compounds to polymeric particles using carbamoylonium compounds and a kit containing same

Life of this patent

Abstract

Claims27