Controlling Segment Solubility in Large Protein Synthesis

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

“…However, NCL has several challenges that limit its scope in chemical protein synthesis (CPS). , First, NCL requires high peptide concentrations, typically ≥1 mM, at neutral pH for efficient ligation. Unfortunately, peptide segments are often too insoluble to reach these ideal concentrations, even in denaturing conditions . While many groups have developed strategies to increase peptide solubility (e.g., incorporating temporary solubilizing tags − and using buffers containing hexafluoro-2-propanol , or ionic liquids), these do not overcome the inherent issue of requiring high concentrations.…”

“…Unfortunately, peptide segments are often too insoluble to reach these ideal concentrations, even in denaturing conditions . While many groups have developed strategies to increase peptide solubility (e.g., incorporating temporary solubilizing tags − and using buffers containing hexafluoro-2-propanol , or ionic liquids), these do not overcome the inherent issue of requiring high concentrations. Additionally, the availability of suitable Cys or Ala ligation junctions is limited for many synthesis projects, forcing the selection of suboptimal segments for NCL. , Although alternative thiol-containing amino acids, such as penicillamine (thiol derivative of Val), can be used as junctions to increase access to alternative ligation strategies, these often suffer from slow ligation rates. − In cases where limited suitable junctions exist, the use of sterically hindered thioesters may be required (e.g., Thr, Ile, and Val). − Even under ideal NCL conditions, such thioesters suffer from long reaction times that often result in loss of peptide because of competing side reactions, creating complex HPLC purifications that further lower yields.…”

“…Unfortunately, peptide segments are often too insoluble to reach these ideal concentrations, even in denaturing conditions. 29 While many groups have developed strategies to increase peptide solubility (e.g., incorporating temporary solubilizing tags 29−32 and using buffers containing hexafluoro-2-propanol 33,34 or ionic liquids 35 ), these do not overcome the inherent issue of requiring high concentrations. Additionally, the availability of suitable Cys or Ala ligation junctions is limited for many synthesis projects, forcing the selection of suboptimal segments for NCL.…”

Enhancing native chemical ligation for challenging chemical protein syntheses

“…Two general strategies have emerged to address these challenges. Poorly soluble peptides can be functionalized with solubilizing groups that enable optimal NCL concentrations to be reached (see recent reviews [14,[17][18][19]). Alternatively, as covered in this review, otherwise suboptimal NCL junctions can be rescued using chemical auxiliaries, selenium-based chemistry, or templating.…”

A cysteine-specific solubilizing tag strategy enables efficient chemical protein synthesis of difficult targets

“…10 Therefore, hydrophilic, semipermanent group linked to peptide segments is oen used as an effective strategy to enhance solubility, especially in streamlining subsequent chemical ligation reactions. 11 Numerous solubilizing tags and removal strategies have been reported, providing invaluable tools for chemical protein synthesis. [12][13][14][15][16] On the other hand, synthesizing and implementing these solubilizing tags create unique applicability constraints and limitations depending on the sequence, reactive groups, and removal conditions.…”