Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid immunoreactivity of free c-terminal epitope with specific antibodies

TY - JOUR

T1 - Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid immunoreactivity of free c-terminal epitope with specific antibodies

AU - Hansen, Paul R.

AU - Flyge, Hanne

AU - Holm, Arne

AU - Lauritzen, Edgar

AU - Larsen, Bjarne Due

PY - 1996/12/1

Y1 - 1996/12/1

N2 - A new heterobifunctional cross-linking reagent, l,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 hydrogenexchange. 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 (All) was conducted. The photoreactive peptide A-4carbonyl-l,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 Cterminal Pro-Phe epitope of photoconjugated All with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-All abs), although antibodies against both the C- and -terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AH, angiotensin I (AI), only bound to anti-All abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide.

AB - A new heterobifunctional cross-linking reagent, l,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 hydrogenexchange. 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 (All) was conducted. The photoreactive peptide A-4carbonyl-l,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 Cterminal Pro-Phe epitope of photoconjugated All with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-All abs), although antibodies against both the C- and -terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AH, angiotensin I (AI), only bound to anti-All abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide.

KW - Angiotensin Ii

KW - Antibodies

KW - Bovine serum albumin

KW - Capture competition ELISA

KW - L,2,3-thiadiazole-4-carboxylic acid

KW - Photochemical conjugation

KW - Photoprobe

KW - Solid-phase peptide synthesis

UR - http://www.scopus.com/inward/record.url?scp=0029817065&partnerID=8YFLogxK

M3 - Journal article

C2 - 8836769

AN - SCOPUS:0029817065

SN - 0367-8377

VL - 47

SP - 419

EP - 426

JO - International journal of protein research

JF - International journal of protein research

IS - 6

ER -