Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.
Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada.
Bryan
B
Simard
John L. Holmes Mass Spectrometry Facility, University of Ottawa, Marion Hall, Ottawa, Ontario, Canada.
Gleb G
GG
Mironov
Department of Oncology, Division of Medical Physics, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada.
Boguslaw
B
Tomanek
Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, British Columbia, Canada.
Frank C J M
FC
van Veggel
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada.|Human Health Therapeutics, National Research Council of Canada, 1200 Montreal Rd., Ottawa, Ontario, Canada.
Abedelnasser
A
Abulrob
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D002234:Carbodiimides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*
D000906:Antibodies; D002234:Carbodiimides; D005456:Fluorescent Dyes; D010455:Peptides; D008239:Lysine
N-terminal; activatable; conjugation; fluorescence; quencher; site-specific; smart probe; targeted