Ser-Pro isomerization is likely a key regulatory step to spindle formation during the G2-to-M transition of mitosis. To investigate this natural phenomenon, a native Ser-Pro dipeptide will be incorporated into full-length Bora protein as a model for installing cis- and trans-locked Ser-Pro alkenes. For in vitro transcription-translation, full-length tRNA will be enzymatically misacylated with substrate 1 by Flexizyme dFx, a ribozyme. HBF4-Ser-Pro-O-3,5-dinitrobenzyl ester 1 was synthesized in 4 steps and 79% overall yield. Alternatively, truncated tRNA will be aminoacylated with Ser-Pro-pdCpA 2 by T4RNA ligase. The coupling was optimized with a model reaction of Boc-Pro-OCH2CN with TBS-adenosine in 44% yield. Boc-Ser(tBu)-Pro-OCH2CN, was synthesized in 3 steps and 77% overall yield, coupled to dinucleotide, pdCpA, and deprotected with HBF4-Et2O to give chemical misacylation substrate, 2. Reagent HBF4-Et2O was newly developed for the mild deprotection of Boc and tBu from aminoacyl dinucleotides for chemical misacylation of tRNA.

Two methods for misacylation of tRNA with ser-pro for in vitro-transcription translation

ACS Spring Meeting 2021

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