Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid. Immunoreactivity of free C-terminal epitope with specific antibodies.
A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N2, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide N2-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide.
Hansen
P R
PR
Chemistry Department, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Flyge
H
H
Holm
A
A
Lauritzen
E
E
Larsen
B D
BD
D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D008956:Models, Chemical; D015394:Molecular Structure; D010455:Peptides / Q000378:metabolism*; D010782:Photolysis; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013830:Thiadiazoles / Q000378:metabolism*; D014269:Trifluoroacetic Acid / Q000494:pharmacology
C105329:1,2,3-thiadiazole-4-carboxylic acid; D000906:Antibodies; D003432:Cross-Linking Reagents; D000939:Epitopes; D010455:Peptides; D013830:Thiadiazoles; D000804:Angiotensin II; D012710:Serum Albumin, Bovine; D014269:Trifluoroacetic Acid