Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Protein-liposome conjugates using cysteine-lipids and native chemical ligation

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.     

Synthesis of N-terminally linked protein and peptide dimers by native chemical ligation

Dimerization can be utilized to double the molecular weight of proteins and peptides and potentially increase their avidity of binding to target receptors. These dimerization effects may be utilized to increase in vivo half-lives in a manner similar to PEGylation and may also improve biological activity. In this paper, we report a new strategy for the synthesis of N-terminally linked protein and peptide homodimers utilizing native chemical ligation to conjugate a short dithioester linker to the N-terminal cysteines of protein and peptide monomers to form dimers in a single step. This strategy is general and has been applied to the production of dimers from three recombinantly expressed polypeptides, the IgG binding domain Protein G, an HIV entry inhibitor peptide C37H6, and human interleukin-1 receptor antagonist (IL-1ra). The biological activities of the C37H6 and IL-1ra dimers produced by these methods were retained or even slightly increased when compared to their corresponding monomers.     

Total chemical synthesis of human psoriasin by native chemical ligation

Human psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is richly expressed in keratinocytes of patients suffering from psoriasis. To date, the exact physiological function of psoriasin abundant in many human cell types remains unclear. A recent report by Schr?der and colleagues suggests that psoriasin, purified from human stratum corneum extracts, selectively kills Escherichia coli by sequestering Zn(2+) ions essential for bacterial growth, indicative of an important role in innate immune defense against microbial infection. We chemically synthesized the N-terminally acetylated psoriasin of 100 amino acid residues using solid phase peptide synthesis in combination with native chemical ligation. More than 140 mg of highly pure and correctly folded synthetic psoriasin was obtained from a single synthesis on a 0.25 mmol scale. Analysis of synthetic psoriasin by size exclusion chromatography showed that the protein forms a homodimer in solution. Circular dichroism analysis indicated that the alpha-helicity of psoriasin increases by more than 20% in the presence of CaCl(2) or ZnCl(2), suggesting a metal ion binding induced conformational change. Circular dichroism based titration further established that the synthetic protein binds two Ca(2+) and two Zn(2+) ions per dimer, in agreement with the published structural findings. Importantly, the ability of the synthetic protein to kill E. coli and the inhibition of the killing by ZnCl(2) is comparable to that of psoriasin isolated from its natural source. The robust synthetic access to large quantities of human psoriasin should facilitate studies of its biological functions as well as its mode of action.     

Investigation toward multi-epitope vaccine candidates using native chemical ligation

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.     

Synthesis by native chemical ligation and characterization of the scorpion toxin AmmTx3

The scorpion toxin AmmTx3 is a specific blocker of K_v4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.     

Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins

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Synthesis of an O‐acyl isopeptide by using native chemical ligation in an aqueous solvent system

O‐Acyl isopeptides, in which the N‐acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O‐acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O‐acyl isopeptide of 34‐mer pyroGlu‐ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu¹‐ADan(1‐21)‐SCH₂CH₂SO₃^?Na⁺ (3) and Cys²²‐O‐acyl isopeptide (4), in which the amino group of the Ser²⁹ residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O‐acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O‐to‐N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O‐acyl isopeptide, (ii) HPLC purification as the O‐acyl isopeptide form, and (iii) O‐to‐N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

Surface PEGylation via native chemical ligation

Native chemical ligation (NCL) is an emerging chemoselective chemistry that forms an amide bond by trans-thioesterification followed by intramolecular nucleophilic rearrangement between thioester and cysteine. The reaction is simple, occurs in a mild aqueous solution, and gives near-quantitative yields of a desired product. Since the first report in 1994, most studies involving the use of NCL have focused on the total synthesis of proteins to address fundamental questions pertaining to many aspects of protein science, such as folding, mirror images, and site-specific labeling of proteins, but applications of the NCL reaction for other areas remain largely unexplored. Herein, we present a facile strategy for surface immobilization of poly(ethylene glycol) (PEG) utilizing the NCL reaction. Surface immobilization of PEG (i.e., PEGylation) plays a key role in preventing nonspecific protein adsorption on surfaces, which is crucial in a wide variety of medical devices. Using cysteine-PEG and thioester-containing phosphonic acid conjugates, we achieved efficient surface PEGylation on titanium surfaces. Ellipsometry, goniometry, and X-ray photoelectron spectroscopy (XPS) unambiguously confirmed the presence of PEGs, which provided nonfouling effects of surfaces. This study indicates that the NCL reaction will be a useful toolkit for surface bioconjugation and functionalization.     

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.     

The first semi-synthetic serine protease made by native chemical ligation

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure-function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.     

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation

In Alzheimer’s disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231 + pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.     

Synthesis of lucifensin by native chemical ligation and characteristics of its isomer having different disulfide bridge pattern

The antimicrobial 40‐amino‐acid‐peptide lucifensin was synthesized by native chemical ligation (NCL) using N‐acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid‐phase synthesis is imcompatible. Two models of ligation were developed: [15 + 25] Ala‐Cys and [19 + 21] His‐Cys. The [19 + 21] His‐Cys method gives lower yield because of the lower stability of 18‐peptide‐His‐Nbz‐CONH₂ peptide, as suggested by density functional theory calculation. Acetamidomethyl‐deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1?6], [C3?5], and [C2?4], which limits its ability to form α‐helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one‐pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Monitoring of native chemical ligation on solid substrate by surface plasmon resonance

During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well-suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide-bradykinin thioester-was carried out and monitored with a custom-built SPR apparatus. Solid-phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4-mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide-mercaptophenylacetic acid thioester. SPR curves indicate typical first-order kinetics with t(1/2) of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester.     

Expeditious chemical synthesis of ubiquitinated peptides employing orthogonal protection and native chemical ligation

Ubiquitination-the attachment of ubiquitin to a protein target-is involved in a wide range of cellular processes in eukaryotes. This dynamic posttranslational modification utilizes three enzymes to link, through an isopeptide bond, the C-terminal Gly of ubiquitin to the lysine side chain from a protein target. Progress in the field aiming at deciphering the role of ubiquitination in biological processes has been very dependent on the discovery of the enzymatic machinery, which is known to be very specific to each protein target. Chemical approaches offer a complementary route to the biochemical methods to construct these conjugates in vitro in order to assist in unraveling the role of ubiquitination on protein function. Herein is presented a novel method for the rapid synthesis of ubiquitinated peptides employing solid-phase peptide to generate the critical isopeptide linkage. Using these tools, several ubiquitinated peptides derived from known ubiquitinated proteins were prepared. Among them is the ubiquitinated C-terminal fragment of H2B, which can be used in the synthesis of monoubiquitinated H2B. For the first time, we systematically assessed the effect of the length of the ubiquitinated peptides on the UCH-L3 activity and found that peptides of up to ～20 residues are preferred substrates.     

Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.