# US 7,745,377 B2 · Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation

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- Number: US 7,745,377 B2
- Document ID: US7745377B2
- Status: Granted
- Kind: B2
- Application number: 11/122,795
- Filed: 2005-05-05
- Published: 2005-12-29
- Granted: 2010-06-29
- Earliest claimed: 2000-07-13
- Assignees: The Scripps Research Institute
- Inventors: Klaus M. Hahn; Alexei Toutchkine; Rajeev Muthyala; Vadim Kraynov; Steven J. Bark; Dennis R. Burton; Chester Chamberlain
- Web page: https://patentgrep.com/patent/US7745377B2

## CPC

- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C07K 1/13: Labelling of peptides
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying the optimum probe attachment site. Biosensors are provided having environmentally sensitive dyes that can locate specific biomolecules within living cells and detect chemical and physiological changes in those biomolecules as the living cell is moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, the environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound. New fluorophores whose fluorescence responds to environmental changes are also provided that have improved detection and attachment properties, and that can be used in living cells, or in vitro.

## Claims

### Claim 1

A polypeptide biosensor comprising a polypeptide and a fluorescent dye, wherein said polypeptide is linked by a direct bond or a linking group to said fluorescent dye, and
wherein said fluorescent dye on said biosensor comprises a compound of the formula:
each m is separately an integer ranging from 1-3;
n is an integer ranging from 0 to 5;
R8, R11 and R12 are separately CO, SO2, C═C(CN)2, S, O or C(CH3)2;
each R13 is hydrogen, alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability;
each R9 and R10 is separately hydrogen, an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.

### Claim 2 (depends on 1)

The polypeptide biosensor of claim 1 wherein said polypeptide biosensor comprises a protein binding domain of p21 activated kinase 1.

### Claim 3 (depends on 1)

The polypeptide biosensor of claim 1 wherein when said polypeptide sensor is bound to a GTP-activated Rho GTPase protein having a fluorophore on said GTP-activated Rho GTPase protein, said fluorescent dye on said polypeptide biosensor undergoes fluorescence resonance energy transfer with said fluorophore, and
wherein said fluorophore on said GTP-activated Rho GTPase protein is Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Red Fluorescence Protein (RFP) or enhanced GFP (EGFP).

### Claim 4 (depends on 3)

The polypeptide biosensor of claim 3 wherein said Rho GTPase protein is Rae or cdc42.

### Claim 5 (depends on 1)

The polypeptide biosensor of claim 1, wherein the fluorescent dye on the polypeptide biosensor comprises a compound of the formula:

### Claim 6 (depends on 5)

The polypeptide biosensor of claim 5, wherein R10 is an alkyl group with 1 to 6 carbon atoms.

### Claim 7

A peptide biosensor comprising a peptide and a fluorescent dye, said peptide linked by a direct bond or a linking group to said fluorescent dye, and wherein said fluorescent dye on the peptide biosensor comprises a compound of the formula:
each m is separately an integer ranging from 1-3;
n is an integer ranging from 0 to 5;
R8, R11 and R12 are separately CO, SO2, C═C(CN)2, S, O or C(CH3)2;
each R13 is hydrogen, alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability;
each R9 and R10 is separately hydrogen, an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.