‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4-linked 1,2,3-triazole AZT-systemin conjugate

Abstract

The Cu(I) catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was applied for a nucleoside-peptide bioconjugation. Systemin (Sys), an 18-aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N-propynoic acid functionalized analog (Prp-Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp-Sys with 3'-azido-2',3'-dideoxythymidine (AZT), a model cargo molecule. 1,4-Linked 1,2,3-triazole AZT-Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by-products. Under typical separation conditions of phosphate 'buffer' at low pH and uncoated fused bare-silica capillary, an increasing peak intensity assigned to triazole-linked AZT-Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE-ESI-MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs.

Capillary electrophoresis study of systemin peptides spreading in tomato plant

“…Because of the lack of strong chromophores in the molecule, low abundance, and a complex matrix of plant exudates, identification and characterization of Sys behavior in tomato plant is a challenging task. CE has been previously applied to the study of Sys in tomato plant . But, CE method has not been used to characterize the peptide spreading through plant tissues.…”

“…Sys and its Prp‐Sys were synthesized following the standard Fmoc procedure of the solid phase peptide synthesis method. Details of the synthesis were described elsewhere . AZT‐Sys conjugate was synthesized using Prp‐Sys and AZT as substrates of the copper‐catalyzed alkyne‐azide cycloaddition (click reaction).…”

“…AZT‐Sys conjugate was synthesized using Prp‐Sys and AZT as substrates of the copper‐catalyzed alkyne‐azide cycloaddition (click reaction). Details of the “click” conjugation were described previously . The peptides structures and selected physicochemical properties are shown in Fig.…”

“…Recently, the trend has been to use CE as a new analytical method to characterize phytocompounds including plant hormones . Although peptides were one of the first biomolecules studied by CE, plant peptides including Sys had been characterized to a small extent .…”

Recent developments in capillary and microchip electroseparations of peptides (2013–middle 2015)

“…In another studies, CE has been employed to monitor tetrapeptide (Arg‐Gly‐Asp‐Ser) grafting onto antimicrobial and biocompatible chitosan film and to analyze the nucleoside‐peptide bioconjugation, particularly the click chemistry synthesis of 1,4‐linked 1,2,3‐triazole 3’‐azido‐2’,3’‐dideoxythymidine–systemin conjugate . ACE has been applied to in vitro evaluation of potential complexation between bovine insulin and BSA .…”