Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase

Abstract

During the total chemical synthesis of the water‐soluble globular Haemophilus Influenzae DNA ligase (Hin‐Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance‐improving strategies, such as installation of a terminal/side‐chain Arg tag or O‐acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

“…One effective solution to improve the solubility of very poorly soluble proteins is our removable backbone modification (RBM) strategy,w hich enables the installation of RBM groups into backbone amides and increases the solubility by both the addition of as olubilizing tag and the disruption of aggregation to effectively facilitate the chemical synthesis of anumber of membrane proteins. [21] By the RBM strategy,we synthesized the S-palm SLN(L 25,RBM ,C 9,palm ) 2 with aRBM tag at Leu25 using the reported protocol. [22] Thec rude RBMmodified peptide was found to be well soluble,b ut its crude RP-HPLC profile was too complex to identify any target peptide (Figure 1c).…”

Discovery, synthesis, and optimization of teixobactin, a novel antibiotic without detectable bacterial resistance

“…63 widely used in synthetic protein chemistry by different labs. [71][72][73][74][75][76][77][78][79] They have found increasing applications in the synthesis of various proteins and peptides, including posttranslationally modified proteins, customdesigned proteins, protein-based probes, membrane proteins, and cyclic peptides etc. [80][81][82][83][84] Different from the linear synthetic strategy developed by the Payne group, the Li group achieved the first convergent total synthesis of teixobactin through Ser ligation mediated "6 + 5" strategy (Figure 13).…”

DIC/Oxyma‐based accelerated synthesis and oxidative folding studies of centipede toxin RhTx

“…Compared with traditional racemic inhibitors such as HOBt, Oxyma has the higher coupling efficiency, lower risk of explosion, and lower rate of racemization during peptide coupling reaction 21 . The N , N ′‐diisopropylcarbodiimide (DIC)/Oxyma coupling system has been demonstrated to be compatible with microwave‐assisted SPPS and reduce racemization under high temperature (from 70°C to 90°C) 27–29 . Strikingly, the DIC/Oxyma system with low synthetic cost exhibited efficient coupling efficiency in both manual and automated syntheses 20,21,28 .…”

“…21 The N,N 0 -diisopropylcarbodiimide (DIC)/Oxyma coupling system has been demonstrated to be compatible with microwave-assisted SPPS and reduce racemization under high temperature (from 70 C to 90 C). [27][28][29] Strikingly, the DIC/Oxyma system with low synthetic cost exhibited efficient coupling efficiency in both manual and automated syntheses. 20,21,28 Nevertheless, the ideal reaction molar ratio of DIC/Oxyma condensation system is less studied.…”