US4220722A
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Method for conjugating to polyamino compounds employing haloacyl groups and compositions prepared thereby
Assignee: Siemens Healthcare Diagnostics GmbH Germany
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Filing: 1978-02-10
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Granted: 1980-09-02
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Expires: 1998-02-10
Abstract
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.
Inventors
- 👤 Gerald L. Rowley
- 👤 Danton Leung
- 👤 Prithiphal Singh
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 19:26
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/535; Y; Y10; Y10S; Y10S435/00; Y10S435/961; Y10S435/964; Y10S436/00; Y10S436/815; Y10S436/816; Y10S436/823; Y10S530/00; Y10S530/806
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
150 words
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.
Claim 2
Independent Claim
150 words
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.
Claim 3
Dependent Claim
15 words
A method according to claim 1, wherein said bromoacetyl functionality is introduced as N-bromoacetyl glycine.
Claim 4
Dependent Claim
14 words
A method according to claim 1, wherein said poly(amino acid) compound is an enzyme.
Claim 5
Dependent Claim
15 words
A method according to claim 4, wherein said bromoacetyl group is introduced as N-bromoacetyl glycine.
Claim 6
Independent Claim
125 words
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.
Claim 7
Independent Claim
125 words
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.
Claim 8
Dependent Claim
13 words
A method according to claim 6, wherein said poly(amino acid) is an enzyme.
Claim 9
Dependent Claim
12 words
A method according to claim 7, wherein said enzyme is a dehydrogenase.
Claim 10
Dependent Claim
34 words
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.
Claim 11
Dependent Claim
14 words
A method according to claim 9, wherein said mercapto containing compound is an alkaloid.
Claim 12
Dependent Claim
14 words
A method according to claim 9, wherein said mercapto containing compound is an aminoglycoside.
Claim 13
Independent Claim
141 words
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).
Claim 14
Independent Claim
141 words
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).
Claim 15
Dependent Claim
13 words
A method according to claim 12, wherein said poly(amino acid) is an enzyme.
Claim 16
Dependent Claim
19 words
A method according to claim 13, wherein said enzyme is a dehydrogenase and said bromoacetyl compound is N-bromoacetyl glycine.
Claim 17
Dependent Claim
12 words
A method according to claim 14, wherein said aminoglycoside is a gentamicin.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-09-02 | 1984-03-03 | 1984-09-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-09-02 | 1988-03-03 | 1988-09-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-09-02 | 1992-03-03 | 1992-09-02 | $7700.00 | Window Closed / Lapsed |