A new crypto-thioester, cysteinylprolyl imide (CPI) peptide, offers a practical synthetic pathway and reliable reaction rate to be successfully applied to chemical protein synthesis.
Peptide ligation is an indispensable step in the chemical synthesis of target peptides and proteins that are difficult to synthesize at once by a solid-phase synthesis. The ligation reaction is generally conducted with two peptide fragments at a high aqueous concentration to increase the reaction rate; however, this often causes unpredictable aggregation and precipitation of starting or resulting peptides due to their hydrophobicities. Here, we have developed a novel peptide ligation strategy harnessing the two intrinsic characteristics of oligodeoxynucleotides (ODNs), i.e., their hydrophilicity and hybridization ability, which allowed increases in the water solubility of peptides and the reaction kinetics due to the proximity effect, respectively. Peptide−ODN conjugates that can be cleaved to regenerate native peptide sequences were synthesized using novel lysine derivatives containing conjugation handles and photolabile linkers, via solid-phase peptide synthesis and subsequent conjugation to 15-mer ODNs. Two complementary conjugates were applied to carbodiimide-mediated peptide ligation on a DNA scaffold, and the subsequent DNA removal was conducted by photoirradiation in a traceless fashion. This DNA scaffold-assisted ligation resulted in a significant acceleration of the reaction kinetics and enabled ligation of a hydrophobic peptide at a micromolar concentration. On the basis of this chemistry, a simultaneous ligation of three different peptide fragments on two different DNA scaffolds has been conducted for the first time.
Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes.
We report an Fmoc-compatible
and external-thiol-free method of
peptide C-terminus thioesterification with cysteinylprolyl imide.
The newly synthesized structure, i.e., cysteinylprolyl-thiazolidinone,
provided high conversion and sequence-independent fast kinetics (90
min) in the diketopiperazine thioester formation under relatively
mild conditions: pH 6.0, 37 °C. Employing this thioesterification
method, we synthesized histone H3.2 bearing K56 acetylation.
We report an
Fmoc-compatible and external-thiol-free method of peptide C-terminus
thioesterification with cysteinylprolyl imide. The newly synthesized structure,
i.e., cysteinylprolyl-thiazolidinone, provided high conversion and
sequence-independent fast kinetics (90 min) in the diketopiperazine thioester
formation under relatively mild conditions: pH 6.0, 37 °C. Employing this thioesterification method, we
synthesized histone H3.2 bearing K56 acetylation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.