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

The scope of proteins accessible to total chemical synthesis via native chemical ligation (NCL) is often limited by slow ligation kinetics. Here we describe Click-Assisted NCL (CAN), in which peptides are incorporated with traceless "helping hand" lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles. The resulting strain-promoted alkyne-azide cycloaddition (SPAAC) increases their effective concentration to greatly accelerate ligations. We demonstrate that copper(I) protects DBCO from acid-mediated rearrangement during acidic peptide cleavage, enabling direct production of DBCO synthetic peptides. Excitingly, triazole-linked model peptides ligated rapidly and accumulated little side product due to the fast reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in one pot. CAN is a useful method for overcoming challenging ligations involving sterically hindered junctions. Additionally, CAN is anticipated to be an important stepping stone toward a multisegment, one-pot, templated ligation system.

Aligator: A computational tool for optimizing total chemical synthesis of large proteins.

The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

Generation of Potent and Stable GLP-1 Analogues Via "Serine Ligation".

Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of "serine ligation" for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.

Enhancing native chemical ligation for challenging chemical protein syntheses.

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.