Patent DE-20023789-U1 (DE)
Title: Novel synthetic intermediate compounds useful for preparing modified peptides and peptide conjugates which are useful in biosensors for detecting the location of cellular proteins in living cells
IPC: C07K14/435
Filed: 2000-09-29
Abstract: A compound (I) which is a synthetic intermediate (a synthon) useful for preparing modified peptides, is new. A compound (I) which is a synthetic intermediate (a synthon) useful for preparing modified peptides. (I) Has the structure of formula (A). [Image] R 1>hydrogen or amino protecting group; R 2>hydrogen or carboxy protecting group; and R : an organic radical comprising one or more aminooxy groups. Independent claims are also included for the following: (1) a peptide (IIa) comprising a backbone and one or more aminooxy groups, provided the peptide is not glutathione and the peptide has at least one aminooxy group that is not part of a group NH 2-O-CH 2-C(=O)- positioned at the N-terminus of the peptide or that is not part of a group -C=N-O-CH 2-C(=O)- that is in the backbone; (2) a peptide (IIb) comprising a backbone and one or more secondary aminooxy groups, provided the peptide having an aminooxy group that is not part of an oxime (C=N-O-) in the backbone; (3) a peptide conjugate (III) having the structure of formula (B); (4) identifying (IV) an optimal position for replacement of a functional molecule on a peptide having a peptide backbone and a known activity, by making a series of peptide conjugates, each peptide conjugate having the same amino acid sequence and the same functional molecule, where the functional molecule is linked at a different location along the backbone of every peptide conjugate in the series, and observing which functional molecule location does not substantially interfere with the known activity of the peptide; (5) a polypeptide biosensor (V) which comprises (III); (6) a polypeptide biosensor (VI) comprising a polypeptide capable of binding a GTP-activated Rho GTPase protein, where the polypeptide is operatively linked to a functional molecule; (7) a fusion protein (VII) comprising a biologically active Rho GTPase protein domain operatively linked to a fluorescent protein by (III), where the Rho GTPase protein domain is capable of binding GTP and forming an activated GTPase:GTP complex; (8) detecting binding of an antibody to an antigen, by reacting an antibody comprising (III) with an antigen or vice versa and detecting an antibody-antigen complex; (9) a fluorescent compound (VIII) of the formula (C); (10) a peptide biosensor comprising (VIII); (11) a protein, polypeptide, peptide, antibody or its fragment, or a nucleic acid attaching linked to (VIII); (12) attaching a biosensor to a cellular protein within a living cell, by providing the living cell with a biosensor capable of binding to a tag on the cellular protein, where the tag is a peptide segment that has been fused to the cellular protein expressed by the living cell; (13) a nucleic acid (IX) encoding the tag fused to above cellular protein; (14) a nucleic acid (X) encoding (VII); (15) an isolated vector comprising (IX); (16) an expression vector capable of expressing (VII) or the tag fused to the cellular protein as above; and (17) a cell comprising the above vector. [Image] R 6>peptide or polypeptide; X : a direct bond or a linking group; R 7>hydrogen, 1-6C alkyl, amino protecting group, or a radical comprising one or more aminooxy groups; Y : direct bond or a linking group; and D : a functional molecule. [Image] m : 1-3; n : 0-5; R 8>, R 11>, R 12>CO, SO 2, C=C(CN) 2, S, O or C(CH 3) 2; R 13>alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability; and R 9>and R 10>alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.