Jonathan Ellman

US Patent:

6680178, Jan 20, 2004

Filed:

May 25, 2001

Appl. No.:

09/866132

Inventors:

Jennifer L. Harris - San Diego CA
Bradley J. Backes - San Diego CA
Jonathan A. Ellman - Oakland CA
Charles S. Craik - San Francisco CA

Assignee:

The Regents of the University of California - Oakland CA

International Classification:

C12Q 137

US Classification:

435 23, 435 24, 435 34, 435 4, 530324, 530350, 530334, 530345, 525 50, 525 541, 549288, 702 22, 702 30

Abstract:

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed.

US Patent:

7629437, Dec 8, 2009

Filed:

Oct 15, 2003

Appl. No.:

10/686884

Inventors:

Jennifer L. Harris - San Diego CA, US
Bradley J. Backes - San Diego CA, US
Jonathan A. Ellman - Oakland CA, US
Charles S. Craik - San Francisco CA, US

Assignee:

The Regents of the University of California - Oakland CA

International Classification:

C12Q 1/00
C12Q 1/37
C07K 5/00
C07K 7/00

US Classification:

530300, 530324, 530334, 530345, 530350, 435 4, 435 23, 435 24, 435 34, 525 50, 525 541, 549288

Abstract:

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques.