Capillary electrophoresis study of systemin peptides spreading in tomato plant.

Systemin (Sys) is an 18-aa plant peptide hormone involved in the regulation of plant's defensive response. Sys is considered as a fast-spreading systemic wound signal. We developed a simple and rapid CE method to monitor the spreading of Sys peptides through tomato plant. A 1,2,3-triazole-linked AZT-systemin conjugate was designed as a model to study the possibility of translocating small cargo molecules 3'-Azido-2',3'-dideoxythymidine by systemin. The Sys peptides (Sys, N-propiolyl Sys, and AZT-systemin conjugate) were injected into the stem and leaves of mature tomato plant. Its transportation throughout the plant tissue was traced by CE. The peptides were clearly visible in the crude tomato exudates and an optimum separation was achieved in 25 mM phosphate "buffer" at pH 2.5 and a voltage of 20 kV using uncoated fused silica capillary. CE analysis showed that Sys peptides are well separated from tomato plant exudates ingredients and are stable in tomato stem and leaf exudates for up to 24 h. CE study revealed that the Sys peptides are effectively spreading throughout tomato stem and leaves and the peptides could be directly detected in the crude plant matrixes. The translocation was strongly inhibited by sodium azide. The results showed that the established CE method can be used to characterize plant peptides spreading under plant physiological conditions.

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

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.

Synthesis and hybridization studies of a new CPP-PNA conjugate as a potential therapeutic agent in atherosclerosis treatment.

The objective of this study was to design and synthesize a new CPP-PNA conjugate that would be able to penetrate endothelial cells, bind STAT1 mRNA and thereby block the activity of STAT1 (the Signal Transducer and Activator of Transcription 1), which is important in cases of vessel inflammation. In the course of the study, the TAMRA-PTD-4- Hal(traziole-Gly-PNA)-conjugate was successfully synthesized using a specific 1,3-dipolar Huisgen cycloaddition reaction known as a "click reaction". The hybridization properties of the conjugate to complementary hSTAT1 mRNA and hSTAT1 ssDNA fragments was verified by capillary electrophoresis (CE). Studies have shown that the attachment of a fluorescence-labeled peptide to a PNA sequence via a 1,2,3-triazole ring did not alter the binding properties of the PNA to the complementary hSTAT1 mRNA or hSTAT1 ssDNA fragments maintaining similar binding affinity. Furthermore, the data obtained suggest that the use of such a conjugate to modulate the activity and expression of STAT1 could provide a new therapeutic strategy for atherosclerosis treatment.