Fmoc-Compatible and C-terminal-Sequence-Independent Peptide Alkyl Thioester Formation Using Cysteinylprolyl Imide.

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.

Cysteinylprolyl imide (CPI) peptide: a highly reactive and easily accessible crypto-thioester for chemical protein synthesis.

Native chemical ligation (NCL) between the C-terminal peptide thioester and the N-terminal cysteinyl-peptide revolutionized the field of chemical protein synthesis. The difficulty of direct synthesis of the peptide thioester in the Fmoc method has prompted the development of crypto-thioesters that can be efficiently converted into thioesters. Cysteinylprolyl ester (CPE), which is an N-S acyl shift-driven crypto-thioester that relies on an intramolecular O-N acyl shift to displace the amide-thioester equilibrium, enabled trans-thioesterification and subsequent NCL in one pot. However, the utility of CPE is limited because of the moderate thioesterification rates and the synthetic complexity introduced by the ester group. Herein, we develop a new crypto-thioester, cysteinylprolyl imide (CPI), which replaces the alcohol leaving group of CPE with other leaving groups such as benzimidazolidinone, oxazolidinone, and pyrrolidinone. CPI peptides were efficiently synthesized by using standard Fmoc solid-phase peptide synthesis (SPPS) and subsequent on-resin imide formation. Screening of the several imide structures indicated that methyloxazolidinone-t-leucine (MeOxd-Tle) showed faster conversion into thioester and higher stability against hydrolysis under NCL conditions. Finally, by using CPMeOxd-Tle peptides, we demonstrated the chemical synthesis of affibody via N-to-C sequential, three-segment ligation and histone H2A.Z via convergent four-segment ligation. This facile and straightforward method is expected to be broadly applicable to chemical protein synthesis.

Simultaneous and Traceless Ligation of Peptide Fragments on DNA Scaffold.

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.

Hybridization-sensitive fluorescent oligonucleotide probe conjugated with a bulky module for compartment-specific mRNA monitoring in a living cell.

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.