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1. WO1991016336 - PEPTIDES FLUOROGENES ET LEUR UTILISATION POUR DETERMINER LES ACTIVITES ENZYMATIQUES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]
C L A I M S

1. A fluorogenic peptide exhibiting intramolecular quenching having the general formula

F-A-Q-Y-j^ or Y2-Q-A-F

wherein Q is a fluorescence quenching substituted amino acid residue

- NH - CH - CO -


wherein R R2, R3, R. and R- are independently selected from hydrogen, hydroxy, alkyl, alkoxy, amino, nitro, amido, halogen, aryl or aralkyl, with the proviso that at least one of R1 to R,- is hydroxy and at least one is nitro,

n is an integer from 0 to 20, preferably 0 to 4,

F is a fluorescent group having a maximum emission within the absorption band for the quenching residue Q,

A is a spacing entity selected from amino acid residues and peptide residues and may include a non-peptide molecular entity, being linked to the fluorescent group F via an amide bond or ester bond.

Y_. is OH, NH , an amino acid residue, a peptide residue or a C-terminal protective group,

Y2 is H, an amino acid residue, a peptide residue or an N- terminal protective group.

2. A peptide according to claim 1 having the general formula

F1-A1-Tyr(N02)-Y1

wherein F. is a fluorescent group having a maximum emission of from 400 to 460 nm, A. is an amino acid residue or a peptide residue linked to the group F via a peptide bond, and Y_. is as defined above.

3. A peptide according to claim 1 or 2, wherein F or F- is ABz ( anthraniloyl ) .

4. A peptide according to any of claims 1 to 3, wherein Y- is OH or an amino acid residue which is attachable to a solid phase or a peptide residue having a C-terminal amino acid residue which is attachable to a solid phase.

5. A peptide according to any of claims 1 to 4, wherein A or A. is an amino acid residue or a peptide residue comprising 2-25 amino acids.

6. A peptide according to any of claims 1 to 5, wherein A includes a mono- or oligosaccharide entity.

7. Substrate for determination of enzymatic activities comprising one or more fluorogenic peptides according to claims 1-6.

8. Process for the detection or determination of enzymatic activities in a sample, c h a r a c t e r i z e d by incubating the sample with one or more substrates according to claim 7 under conditions favourable to enzymatic activities, determining and comparing the fluorescence emitted before and after the incubation.

AMENDED CLAIMS
[received by the International Bureau
on 6 September 1991 (06.09.91) ;
original claim 1 amended; other claims unchanged ( 3 pages) ]

1. ( Amended ) A fluorogenic peptide exhibiting intramolecular quenching having the general formula

F-A-Q-Y.. or Y2-Q-A-F

wherein Q is a fluorescence quenching substituted amino acid residue



NH

wherein R R2, R3, R. and R- are independently selected from hydrogen, hydroxy, alkyl, alkoxy, amino, nitro, amido, halogen, aryl or aralkyl, with the proviso that at least one of R., to R5 is hydroxy and at least one is nitro,

wherein X in a spacing group which may be absent or selected from any combination of -CO-NH-, -CO-0-, -S-, S-S-, -0-, -NH- or -C,H4~, and n and m are integers from 0 to 10, F is a fluorescent group having a maximum emission within the absorption band for the quenching residue Q,

A is a spacing entity selected from amino acid residues and peptide residues and may include a non-peptide molecular entity, being linked to the fluorescent group F via an amide bond or ester bond,

Y-. is OH, NH,.,, an amino acid residue, a peptide residue or a C-terminal protective group,

Y„ is H, an amino acid residue, a peptide residue or an N-terminal protective group.

2. A peptide according to claim 1 having the general formula

F1-A1-Tyr(N02 )-Y

wherein F- is a fluorescent group having a maximum emission of from 400 to 460 nm. A., is an amino acid residue or a peptide residue linked to the group F via a peptide bond, and Y is as defined above.

3. A peptide according to claim 1 or 2 , wherein F or F.. is ABz ( anthraniloyl ) .

4. A peptide according to any of claims 1 to 3, wherein Y-. is OH or an amino acid residue which is attachable to a solid phase or a peptide residue having a C-terminal amino acid residue which is attachable to a solid phase.

5. A peptide according to any of claims 1 to 4 , wherein A or A^ is an amino acid residue or a peptide residue comprising 2-25 amino acids.

6. A peptide according to any of claims 1 to 5, wherein A includes a mono- or oligosaccharide entity.

7. Substrate for determination of enzymatic activities comprising one or more fluorogenic peptides according to claims 1-6.

8. Process for the detection or determination of enzymatic activities in a sample, c h a r a c t e r i z e d by incubating the sample with one or more substrates according to claim 7 under conditions favourable to enzymatic activities, determining and comparing the fluorescence emitted before and after the incubation.