Method for conjugating to polyamino compounds employing haloacyl groups and compositions prepared thereby

• US 4,220,722 A
• Filed: 02/10/1978
• Issued: 09/02/1980
• Est. Priority Date: 02/10/1978
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syva Co.
• Inventors: Rowley, Gerald L., Leung, Danton, Singh, Prithiphal
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US05/876,772
• Time in Patent Office: 935 Days
• Field of Search: 195/63, 195/68, 195/DIG. 11, 260/112 R, 424/12, 435/7, 435/177, 435/174, 435/188
• US Class Current: 435/188
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

Methods and compositions are provided for conjugating a wide variety of compounds, particularly polyfunctional compounds, having a mercapto group, either naturally present or synthetically introduced, to a polyamino compound, particularly a polypeptide (including proteins). The method employs a haloalkylcarbonyl compound, which is conjugated to one or more of the amino groups under mild acylating conditions. This is followed by combining the acylated polyamino compound with a mercapto containing compound, whereby the halogen is displaced by the sulfur of the mercapto group to form a stable thioether linkage. The resulting conjugates, depending on the compounds involved, can find uses in immunoassays, as hapten-antigen conjugates for the production of antibodies, and as ligand analog enzyme conjugates for use as reagents in controlling the distribution of substitution of a mercapto compound to a polyamino compound.

Claims

1. A method for covalently linking a poly(amino acid) compound being substantially free of active mercapto groups and having a plurality of amino functionalities to a mercapto compound, which comprises:

covalently linking at least one α

-bromoacetyl containing compound to said poly(amino acid) through a non-oxocarbonyl group, including the nitrogen and sulfur analogs thereof, to form amide linkages or the nitrogen or sulfur analogs respectively, by combining said α

-bromoacetyl containing compound with said poly(amino acid) in an inert polar solvent at a pH in the range of about 6.5 to 9 at a temperature in the range of about -5°

to 40°

C. to provide an α

-bromoacetyl functionalized poly(amino acid); and

combining said α

-bromoacetyl functionalized poly(amino acid) compound with said mercapto compound under mild conditions in a polar medium at a pH of about 6.5 to 8.5 to form a thioether by displacement of said bromo with the sulfur of said mercapto.

2. A method according to claim 1, wherein said bromoacetyl functionality is introduced as N-bromoacetyl glycine.

3. A method according to claim 1, wherein said poly(amino acid) compound is an enzyme.

4. A method according to claim 4, wherein said bromoacetyl group is introduced as N-bromoacetyl glycine.

5. A method for covalently linking a poly(amino acid) having a plurality of amino functionalities and being substantially free of active mercapto groups to a mercapto compound which comprises:

combining a bromoacetyl functionality containing compound having a non-oxocarbonyl functionality with said poly(amino acid) to form through said non-oxocarbonyl functionality amide linkages, in an aqueous solvent at a pH of about 6.7 to 9 and at a temperature in the range of about -5°

to 25°

C. to provide a bromoacetyl functionalized poly(amino acid);

combining said bromoacetyl functionalized poly(amino acid) with a mercapto containing compound in an aqueous solvent at a pH of from about 7 to 8 at a mild temperature to form a thioether by displacement of said bromo with the sulfur of said mercapto.

6. A method according to claim 6, wherein said poly(amino acid) is an enzyme.

7. A method according to claim 7, wherein said enzyme is a dehydrogenase.

8. A method according to claim 8, wherein said mercapto compound is a hapten containing an hydroxyl or amino functionality having said mercapto group linked to said hydroxyl or amino functionality through a linking group.

9. A method according to claim 9, wherein said mercapto containing compound is an alkaloid.

10. A method according to claim 9, wherein said mercapto containing compound is an aminoglycoside.

11. A method for conjugating an aminoglycoside to a poly(amino acid) having a plurality of amino functionalities and being substantially free of active mercapto groups which comprises:

acylating said aminoglycoside with a sulfur functionality containing carboxylic acid compound;

modifying the sulfur functionality as required to provide a mercaptan to form a mercapto-substituted aminoglycoside;

acylating said poly(amino acid) with a bromoacetyl containing carboxylic acid compound to form a bromoacetyl substituted poly(amino acid) in an inert polar medium at a pH in the range of about 6.5 to 9 and at a temperature in the range of about -5°

to 40°

C.;

combining said mercapto-substituted aminoglycoside with said bromoacetyl substituted poly(amino acid) at a pH of from about 7 to 8 at a mild temperature, whereby said mercapto group displaces said bromo group to form a thioether to provide an aminoglycoside conjugated poly(amino acid).

12. A method according to claim 12, wherein said poly(amino acid) is an enzyme.

13. A method according to claim 13, wherein said enzyme is a dehydrogenase and said bromoacetyl compound is N-bromoacetyl glycine.

14. A method according to claim 14, wherein said aminoglycoside is a gentamicin.