Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions. 1 , 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly₅ on the peptidoglycan of bacterial cell walls by sortase A. 1 The enzyme is an important anti‐virulence and anti‐infective drug target for resistant strains of Gram‐positive bacteria. 2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science. 3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase A_ΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size‐exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.     

Proximity‐Based Sortase‐Mediated Ligation

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site‐specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity‐based sortase‐mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag–SpyCatcher peptide–protein pair. By expressing the target protein with SpyTag C‐terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher–SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.     

A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full‐length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε‐amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site‐specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site‐specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.     

Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

The enzyme‐mediated site‐specific bioconjugation of a radioactive metal complex to a single‐chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single‐chain variable fragment. The antibody fragment scFv_anti‐LIBS targets ligand‐induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron‐emitting isotope ⁶⁴Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site‐specific enzyme‐mediated conjugation and should be broadly applicable to other metal complexes and proteins.     

Site‐Specific N‐Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide

An efficient ligase with exquisite site‐specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase‐mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N‐terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.     

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

Enzyme‐mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N‐terminally labeled with lithocholic acid (LA)—an inexpensive bile acid that exhibits strong binding to β‐cyclodextrin (βCD). Capture and recycling of the LA‐Pro‐SrtA 7M conjugate was achieved using βCD‐modified sepharose resin. The LA‐Pro‐SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.     

Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy

Bioorthogonal, chemoselective ligation methods are an essential part of the tools utilized to investigate biochemical pathways. Specifically enzymatic approaches are valuable methods in this context due to the inherent specificity of the deployed enzymes and the mild conditions of the modification reactions. One of the most common strategies is based on the transpeptidation catalyzed by sortase A derived from Staphylococcus aureus. The procedure is well established and a wide variety of applications have been published to date. Here, implementations of sortase A, which range from protein labeling using fluorescence dyes and the preparation of cyclic proteins to the modification of entire cells, are summarized. Furthermore, there is a focus on the optimization approaches established to solve the drawbacks of sortase‐mediated transpeptidation.     

Making and breaking peptide bonds: protein engineering using sortase

Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.     

Directed evolution of sortase A mutants with altered substrate selectivity profiles

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.     

Electrochemical Functional‐Group‐Tolerant Shono‐type Oxidation of Cyclic Carbamates Enabled by Aminoxyl Mediators

An electrochemical method has been developed for α‐oxygenations of cyclic carbamates by using a bicyclic aminoxyl as a mediator and water as the nucleophile. The mediated electrochemical process enables substrate oxygenation to proceed at a potential that is approximately 1 V lower than the redox potential of the carbamate substrate. This feature allows for functional‐group compatibility that is inaccessible with conventional Shono oxidations, which proceed by direct electrochemical substrate oxidation. This reaction also represents the first α‐functionalization of non‐activated cyclic carbamates with oxoammonium oxidants.     

Double Suzuki cross‐coupling reaction of pyrimidine boronic acid: synthesis of new versatile dielectrophile

The double Suzuki cross‐coupling reaction has successfully been applied for the synthesis of 5,5′‐(5‐butoxy‐1,3‐phenylene)bis(2‐chloropyrimidine) with two reactive chloro groups and an alkoxy side chain starting from 2‐chloropyrimidin‐5‐ylboronic acid and 1,3‐dibromo‐5‐butoxybenzene. The reactivity of this dielectrophile was tested by reaction with aniline and phenol, a nitrogen and oxygen nucleophile, respectively. The new dielectrophile would further provide an ideal platform for the construction of large hetero‐atom bridged macrocycles for desired properties and functions in supramolecular and material chemistry. Copyright © 2012 John Wiley & Sons, Ltd.     

Transition‐Metal‐Free Formal Decarboxylative Coupling of α‐Oxocarboxylates with α‐Bromoketones under Neutral Conditions: A Simple Access to 1,3‐Diketones

A transition‐metal‐free formal decarboxylative coupling reaction between α‐oxocarboxylates and α‐bromoketones to synthesize 1,3‐diketone derivatives is presented. In this reaction, a broad scope of substrates can be employed, and neither a metal‐based reagent nor an additional base is required. DFT calculations reveal that this reaction proceeds through a coupling followed by decarboxylation mechanism and the α‐bromoketone unprecedentedly serves as a nucleophile under neutral conditions. The rate‐determining step is an unusual hydrogen‐bond‐assisted enolate formation by thermolysis.     

Magnesium Halide‐promoted Ring‐opening Reaction of Cyclic Ether in the Presence of Phosphine Halide

A new route to the direct preparation of H‐phosphinate esters has been explored. The ring‐opening reaction of cyclic ether (tetrahydrofuran or tetrahydropyrane) was carried out with magnesium halide in the presence of phosphine halide (PRCl₂ or PCl₃). The process is straightforward and all the reagents are relatively cheap and readily available. Magnesium halide‐mediated THF ring‐opening (S_N2@C) and the subsequent S_N2@P elementary reactions that giving rise to the intermediate of haloalkyl phosphinates have been discussed based on our experimental findings ( Path I : S_N2@C−+S_N2@P). Another possible route, the direct S_N2 between THF (nucleophile) and phosphine halide (electrophile) that followed by THF ring opening by halide dissociated from phosphine halide ( Path II: S_N2@P−+S_N2@C), was also proposed. However, path II is the least likely reaction path because neutral THF is not a good nucleophile. H‐phosphinate esters could be readily available in the subsequent hydrolysis process. Considering the ionic bond strength in magnesium halides and the nucleophilicity of halides dissociated from MgX₂ in protic solvents like water, MgBr₂ is recommended for ring‐opening reactions of cyclic ethers.     

Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments

The site‐specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self‐quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site‐specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide‐modified fluorescent single‐domain antibody fragment or an intact immunoglobulin produced in a sortase‐catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single‐domain and full‐sized antibodies without deleterious effects on antigen binding.     

Nucleophile‐Dependent Regio‐ and Stereoselective Ring Opening of 1‐Azoniabicyclo[3.1.0]hexane Tosylate

1‐[(1R)‐(1‐Phenylethyl)]‐1‐azoniabicyclo[3.1.0]hexane tosylate was generated as a stable bicyclic aziridinium salt from the corresponding 2‐(3‐hydroxypropyl)aziridine upon reaction with p‐toluenesulfonyl anhydride. This bicyclic aziridinium ion was then treated with various nucleophiles including halides, azide, acetate, and cyanide in CH₃CN to afford either piperidines or pyrrolidines through regio‐ and stereoselective ring opening, mediated by the characteristics of the applied nucleophile. On the basis of DFT calculations, ring‐opening reactions under thermodynamic control yield piperidines, whereas reactions under kinetic control can yield both piperidines and pyrrolidines depending on the activation energies for both pathways.     

Nucleophile‐Initiated Catalytic and Multicomponent Reactions

A number of well‐known reactions, proceed through the intermediacy of dipolar/zwitterionic species generated via the addition of a neutral nucleophile with an unsaturated electrophile. A mechanistic understanding of these reactions was made possible by seminal contributions of Huisgen. The design of novel reactions based on such dipolar species was, however, not pursued in detail for a long time. Our efforts to exploit various reactivity profiles available for the zwitterionic/dipolar intermediates have resulted in the discovery of a large number of novel, convenient protocols to access a wide variety of products. The nucleophilic initiators may participate in the reaction or play a mediating role depending upon the nature of nucleophile, its quantity and the reaction conditions. In a majority of these transformations two electrophilic components, that would normally be inert towards each other, are combined by the intermediacy of a nucelophile. A brief summary of such nucleophile‐initiated novel reactions that were developed in our research group are described. Reactions involving a variety of nucleophiles such as phosphines, pyridine, quinoline, isoquinoline, isocyanides, dimethoxycarbene and N‐heterocyclic carbenes (NHCs) are discussed.     

Silylative Cyclopropanation of Allyl Phosphates with Silylboronates

A potassium‐bis(trimethylsilyl)amide‐mediated cyclopropanation of allyl phosphates with silylboronates has been developed. Unlike the reported copper‐catalyzed allylic substitution reactions, the nucleophile selectively attacks at the β‐position of the allylic substrates under the present reaction conditions. The mechanism of this process has also been investigated, thus indicating the involvement of a silylpotassium species as the active nucleophilic component.     

A New Reactivity Mode for the Diazo Group: Diastereoselective 1,3‐Aminoalkylation Reaction of β‐Amino‐α‐Diazoesters To Give Triazolines

A novel mode of reactivity for the diazo group, the 1,3‐addition of a nucleophile and an electrophile to the diazo group, has been realized in the intramolecular aminoalkylation of β‐amino‐α‐diazoesters to form tetrasubstituted 1,2,3‐triazolines. The reaction exhibited a broad scope, good functional group tolerance, and excellent diastereoselectivity. In addition, a new Au‐catalyzed intramolecular transannulation reaction of the obtained propargyl triazolines to give pyrroles has been discovered.     

Synthesis of 5‐aryl and 5‐amidocamptothecins

A variety of 5‐aryl‐(20S)‐camptothecin derivatives were synthesized by the reaction of 5‐hydroxy‐(20S)‐camptothecin with aromatic hydrocarbons under Friedel‐Craft reaction conditions in moderate to good yield as diastereomeric pairs. The methodology was then extended for the synthesis of 5‐amido‐(20S)‐camptothecin derivatives by reacting 5‐hydroxy‐(20S)‐camptothecin with alkyl and aryl nitriles under Ritter type reaction conditions. The reaction is presumed to proceed through an iminium ion intermediate under Friedel Craft and Ritter type reaction condition, which is further trapped by nucleophile present in the reaction medium. J. Heterocyclic Chem., 00 , 00 (2011).     

Palladium‐Catalyzed Heck‐Type Cross‐Couplings of Unactivated Alkyl Iodides

A palladium‐catalyzed, intermolecular Heck‐type coupling of alkyl iodides and alkenes is described. This process is successful with a variety of primary and secondary unactivated alkyl iodides as reaction partners, including those with hydrogen atoms in the β position. The mild catalytic conditions enable intermolecular C? C bond formations with a diverse set of alkyl iodides and alkenes, including substrates containing base‐ or nucleophile‐sensitive functionality.