D‐Peptide and D‐Protein Technology: Recent Advances, Challenges, and Opportunities**

Abstract

Total chemical protein synthesis provides access to entire Dprotein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L-and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of nonproteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

Design and Synthesis of Monobody Variants with Low Immunogenicity

“…We have focused on developing mirror-image monobodies for novel protein therapeutics to overcome this immunogenicity shortcoming. Mirror-image peptides ( d -peptides) and proteins ( d -proteins) are expected to have favorable pharmacokinetic and safety profiles because the sequences consisting entirely of d -amino acids are less susceptible to proteolytic degradation by endogenous peptidases . The less efficient processing of d -peptide- or d -protein-based biologics in antigen-presenting cells (APCs) may avoid antigen presentation on major histocompatibility complex (MHC) molecules for T cell recognition, leading to less ADA generation via T cell activation …”

Development of convergent hybrid phase ligation for efficient and convenient total synthesis of proteins

“…Treatment with methoxylamine afforded the "ring-opened" Cys product suitable for use in subsequent ligation cycles (15)(16)(17)(18). For documentation of these in initial syntheses, ligation progress was also monitored by RP-HPLC (Figure 4b) and SDS-PAGE (see ESI); however, these analyses consume significantly more material than mass spectrometric analysis, and are less sensitive than MALDI-TOF MS for detection of unreacted material.…”

“…[2][3][4]6,13] Moreover, total chemical protein synthesis provides full control of protein composition, and thus can overcome inherent limitations of biological synthesis, for example generating naturally absent mirror-image D-proteins which have potential research and therapeutical applications. [14,15] However, due to the size limitation of peptides prepared by SPPS, [10] multiple rounds of NCLs are often required to achieve synthesis of full-length proteins. While the ligation reaction itself is typically efficient, intermediate purification steps are unavoidable for either sequential or convergent solution phase strategies, which often leads to significant yield losses that compound over each round.…”

Chemically Synthetic d-Sortase Enables Enzymatic Ligation of d-Peptides

“…Mirror-image proteins ( d -proteins) comprised of d -amino acids possess potentials in the creation of mirror-image life, racemic protein crystallography, and discovery of nonproteolytic d -peptide ligands, enabling applications in mirror-image synthetic biology, structural biology, and biomedicine studies. − Owing to the development of chemical ligation reactions such as hydrazide-based native chemical ligation, − chemical synthesis can afford d -proteins with customized sequences and functional groups, and present a reliable and robust route for d -protein engineering and drug discovery . Nonetheless, the applicability of the totally chemical synthesis is restricted by the requirement of specialized equipment and reagents, and the size limit for synthetic proteins …”

“…1−10 Owing to the development of chemical ligation reactions such as hydrazide-based native chemical ligation, 11−13 chemical synthesis can afford D-proteins with customized sequences and functional groups, and present a reliable and robust route for D-protein engineering and drug discovery. 14 Nonetheless, the applicability of the totally chemical synthesis is restricted by the requirement of specialized equipment and reagents, and the size limit for synthetic proteins. 15 Enzymatic protein ligation using Sortase A has been widely investigated for the production of homogeneously functional proteins such as antibody−drug conjugates.…”