Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a B ackbone- I nstalled S plit I ntein- A ssisted L igation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L–Cfa^N and L–Cfa^C) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.     

Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under‐utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high‐yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)‐free protein eglin C.     

Comparison of multiple gene assembly methods for metabolic engineering

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a SfiI-based ligation method. SfiI ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the SfiI method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique SfiI restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The SfiI method consists of three steps: first a SfiI linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring SfiI linkers on SfiI digestion; second, the linkers are attached to the desired genes by cloning them into SfiI linker vectors; third, the genes flanked by the three-base linkers, are released by SfiI digestion. In the final step, genes of interest are joined together in a simple one-step ligation.     

One‐Pot N2C/C2C/N2N Ligation To Trap Weak Protein–Protein Interactions

Weak transient protein–protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity‐induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N‐terminus to N‐terminus) and C2C (C‐terminus to C‐terminus) ligation approaches. By using N2C (N‐terminus to C‐terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t_1/2 of 4.8 min and near quantitative conversion. The Ypt1‐GDI conjugate revealed that binding of Ypt1 G‐domain causes opening of the lipid‐binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.     

Near-Quantitative Preparation of Short Single-Stranded DNA Circles

Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 μM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.     

Recent developments in peptide ligation independent of amino acid side-chain functional group

Chemical synthesis of peptides and proteins has evolved into an indispensable tool for chemical biology. Peptide ligation is a straightforward technique for joining two short peptide fragments together via a native peptide bond to afford a larger natural peptide or protein. However, the junction sites are limited to several specific amino acids because most peptide ligations involve participation of the side-chain functional groups of the junction-site amino acids. To overcome such intrinsic limitations, “general” peptide ligations which do not rely on the side-chain functional group have been developed. This review summarized the recent developments in peptide ligations that are independent of side-chain functional group of ligation-junction-site amino acid.     

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho-Dithiophenols

A bioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho-dithiophenols (DT) is presented. Double nucleophilic substitutions of ortho-dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in fluorescence together with release of the other functional molecules. Three cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates. The CQ-DT bioorthogonal reactions can be applied for the bioorthogonal ligations, bioorthogonal cleavages, and trans-tagging of proteins, and show advantages of readily accessible unnatural orthogonal groups, appealing reaction kinetics (k₂≈1.3 m ⁻¹ s⁻¹), excellent biocompatibility of orthogonal groups, and high stability of conjugates. This complements previous bioorthogonal reactions and is a new route for protein-fishing applications and in-gel fluorescence analysis.     

Destabilizing universal linkers for signal amplification in self-ligating probes for RNA

Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4-5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays.     

Design of thiol-containing amino acids for native chemical ligation at non-Cys sites

<正>Protein chemical synthesis usually relies on the use of native chemical ligation that couples peptide thioester with a Cys-peptide.A limitation of this method is the difficulty of finding an appropriate Cys ligation site in many synthetic targets.To overcome this problem,the ligation-desulfurization approach has been developed.This approach involves the use of a thiol-containing amino acid as the ligation partner.After the sequence assembly is completed,the thiol group is removed through a desulfurization reaction to generate the standard amino acids.Currently this strategy has been applied to the ligations at a number of amino acids including Ala,Phe,Val,Lys,Thr,Leu,Pro and Gln.The present article reviews the design and synthesis of these thiol-containing amino acids for native chemical ligation at non-Cys sites.     

Theoretical investigations on the thiol–thioester exchange steps of different thioesters

As the rate-determining step in native chemical ligation reactions, the thiol–thioester exchange step is important in determining the efficiency of the ligations of peptides. In the present study, systematic theoretical calculations were carried out on the relationships between the structure of different thioesters and the free energy barriers of the thiol–thioester exchange step. According to the calculation results, the thiol–thioester exchange step is disfavored by the steric hindrance around the carbonyl center, while the electronic effect(i.e. conjugation and hyper-conjugation effects) becomes important when the steric hindrance is insignificant.     

Potassium Acyltrifluoroborate (KAT) Ligations are Orthogonal to Thiol‐Michael and SPAAC Reactions: Covalent Dual Immobilization of Proteins onto Synthetic PEG Hydrogels

The covalent immobilization of peptides, proteins, and other biomolecules to hydrogels provides a biologically mimicking environment for cell and tissue growth. Bioorthogonal chemical reactions can serve as a tool for this, but the paucity of such reactions and mutual incompatibilities limits the number of distinct molecules that can be introduced. We now report that the potassium acyltrifluoroborate (KAT ) amide‐forming ligation is orthogonal to both thiol‐Michael and strain promoted azide alkyne cycloadditions (SPAAC ) and the requisite functional groups – KAT s and hydroxylamines – are stable and compatible to hydrogel formation, protein modification, and post‐assembly immobilization of biomolecules onto hydrogels. In combination these ligations enables stepwise covalent protein immobilization of multiple BSA ‐derivatives onto the hydrogel scaffold regardless of the order of addition.     

Site selection in tandem arrays of metal-binding domains

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical TFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(II) was found to be 15 +/- 10 microM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(II) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.     

Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S-Benzylidene Thioacetals

Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.     

Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.     

Enzymatic Fluoromethylation as a Tool for ATP-Independent Ligation

S-adenosylmethionine-dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme-catalyzed F-methylation of carboxylate substrates produces F-methyl esters that readily react with N- or S-nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site-specific protein modification and native chemical ligation.     

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins**

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.     

无痕施陶丁格连接反应机理的理论研究

叠氮化合物和膦硫酯的无痕施陶丁格连接反应是一种有效生成酰胺键的方法. 通过密度泛函方法研究了无痕施陶丁格连接的反应机理. 计算结果表明反应的决速步为反应的第一步, 即膦进攻叠氮的端基氮, 形成膦基叠氮化物的过程. 膦亚胺中间体形成之后, 向不同方向转化(酰基迁移后水解或者直接水解)的难易程度决定了最终产物的组成. 对几种不同偶联试剂介导的无痕施陶丁格连接反应的计算结果, 都与实验数据相吻合.     

Enhancement of sortase A-mediated protein ligation by inducing a β-hairpin structure around the ligation site

A Staphylococcus aureus transpeptidase, sortase A (SrtA), catalyzes selective peptide/protein ligations that have been applied to cell imaging and protein engineering, while the ligations do not proceed to completion due to their reversibility. We successfully enhanced SrtA-mediated protein ligation through the formation of a β-hairpin around the ligation site.     

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C‐ and N‐termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo‐, regio‐, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C‐terminal salicylaldehyde esters to peptides with N‐terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one‐pot ligation‐desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216‐311), PD‐1 (192‐288) and Eglin C.