Asparaginyl endopeptidases (AEP) are ideal for peptide and protein labeling. Its pairing with a simple chemical reaction significantly lowers the amount of label needed for effective bioconjugation.

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.

Abuse of opiate medications has reached epidemic proportions, and elective total hip arthroplasty (THA) and total knee arthroplasty (TKA) typically require outpatient use of narcotic medications. This survey sought to determine opiate-prescribing habits of members of the American Association of Hip and Knee Surgeons (AAHKS) for patients undergoing primary THA and TKA. An 11-question online survey was developed to evaluate current prescribing habits for opiate and nonopiate medications prescribed after primary THA and TKA. An invitation to complete the survey was e-mailed to 2698 orthopedic surgeons using an AAHKS listserv. Surgeons' demographic information and their prescribing habits of opiate and nonopiate medications postdischarge were recorded. Data were examined using descriptive statistics, chi-square, and multivariate logistic regression. Responses were received from 325 of 2698 (12.1%) AAHKS members. Significant variation in the type of opiate prescribed and the number of pills dispensed was observed. Higher surgical volume and less years in surgical practice were associated with a higher number of opiate pills prescribed after THA and TKA. There were no statistically significant associations between opiates prescribed and use of an ambulatory surgery center or presence of departmental guidelines. Although THA and TKA are relatively standardized procedures performed nationwide, significant variability exists among surgeons regarding postdischarge opiate- and nonopiate-prescribing habits. There is a need for greater standardization to create a unified, evidence-based, and safe regimen for the postoperative period while reducing the opiate burden in the surrounding community. [ Orthopedics . 2019; 42(6):361–367.]

Tryptophan is frequently found on the surface of membrane-associated proteins that interact with the lipid membrane. However, because of their multifaceted interactions, it is difficult to pinpoint the structure-activity relationship of each tryptophan residue. Here, we describe the use of racemic protein crystallography to probe dedicated tryptophan interactions of a model tryptophan-rich bacteriocin aureocin A53 (AucA) by inclusion and/or exclusion of potential ligands. In the presence of tetrahedral anions that are isosteric to the head group of phospholipids, distinct tryptophan H-bond networks were revealed. H-bond donation by W40 was critical for antibacterial activity, as its substitution by 1-methyltryptophan resulted in substantial loss of activity against bacterial clinical isolates. Meanwhile, exclusion of tetrahedral ions revealed that W3 partakes in formation of a dimeric interface, thus suggesting that AucA is dimeric in solution and dissociated to interact with the phosphate head group in the presence of the lipid membrane. Based on these findings, we could predict the tryptophan residue responsible for activity as well as the oligomeric state of a distant homologue lacticin Q (48%).

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effective and affordable electrophile, 2-formyl phenylboronic acid (FPBA), to yield a non-reactive cyclic byproduct. In the presence of FPBA our model reaction proceeds with ~95% yield using only 1.2 equivalent of substrate, whereas the yield remains at ~50% in the absence of this additive. This “quenching” approach enables protein labeling at both the N- and C-termini ranging from 75 to 85% (five examples). The simplicity and versatility of this quenching approach will enhance the future use of AEPs in protein bioconjugation.

Total chemical protein synthesis provides access to entire D-protein 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 D-enantiomers facilitating mirror-image replication and transcription of L-DNA. 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 on non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.