Development of Trityl Group Anchored Solubilizing Tags for Peptide and Protein Synthesis

Recently, solubilizing tag methods (Trt-K and Trt-R method) were developed for the challenging synthesis of peptides/proteins by means of native chemical ligation. In this system, the solubilizing tag can be attached to the Cys side chain by simply mixing the tag-introducing reagent under acidic conditions. The tagged peptides/proteins exhibited high water solubility thanks to the introduction of redundant oligo-Lys/Arg. In the final reaction, the tag can be quickly and cleanly detached by a standard deprotection reaction with trifluoroacetic acid. Herein, the development and application of these methods are described.     

A reversible protection strategy to improve Fmoc-SPPS of peptide thioesters by the N-Acylurea approach

C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully or semisynthetic proteins. However, the efficient generation of C-terminal thioesters by Fmoc solid-phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on the deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc SPPS. Here, we demonstrate that this approach results in the formation of side products through the over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which can be purified and converted in situ to thioester under ligation conditions. This method is compatible with the automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves the synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.     

1,6‐hexanediol diacrylate‐crosslinked polystyrene: Preparation,characterization, and application in peptide synthesis

A copolymer of 1,6‐hexanediol diacrylate (HDODA) and styrene was prepared by a suspension polymerization method. The resin was characterized by infrared and carbon‐13 cross‐polarization magic‐angle spin (¹³C CP‐MAS) spectroscopy. The topology of the resin was examined by scanning electron microscopy (SEM). The polymer swells extensively in common solvents used for peptide synthesis. The resin exhibited chemical stability even in neat trifluoroacetic acid. The applicability of the new resin was demonstrated by synthesis of Val‐Ala‐Val‐Ala‐Ala‐Gly, Gln‐Val‐Gly‐Gln‐Val‐Glu‐Leu‐Gly, and Val‐Gln‐Ala‐Ala‐Ile‐Asp‐Tyr‐Ile‐Asn‐Gly. Comparative synthetic studies showed that the new resin is superior to divinylbenzene (DVB)‐based resin in the case of the synthesis of hydrophobic peptide sequences. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 1290–1296, 2003     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

A broadly applicable one‐pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)‐catalyzed reaction (CEPS/CLIPS/CuAAC; “triple‐C”) locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head‐to‐tail by using the peptiligase variant omniligase‐1 (CEPS). Subsequent ligation–cyclization with tetravalent (T4_1/2) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF‐T4_1/2) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero‐bifunctional variants (UF/FU‐T4_1/2), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the “bifunctional tetracyclic peptide” inhibitor concept.     

Combination of Thiol‐Additive‐Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol‐additive‐free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin‐related peptide and neuroendocrine regulatory peptide‐2 (NERP‐2) by this method. We confirmed that both conventional alkyl thioester and thioester‐equivalent N‐acyl‐N′‐methyl‐benzimidazolinone (MeNbz) can be adopted as thioester components for thiol‐additive‐free NCL of multi‐Cys‐containing peptides.     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

N‐Glycosylation with Synthetic Undecaprenyl Pyrophosphate‐Linked Oligosaccharide to Oligopeptides by PglB Oligosaccharyltransferase from Campylobacter jejuni

The oligosaccharyltransferase PglB from Campylobacter jejuni catalyses the N‐glycosylation reaction with undecaprenyl‐pyrophosphate‐linked Glc₁GalNAc₅Bac₁ (Und‐PP‐Glc₁GalNAc₅Bac₁). Experiments using chemically synthesized donors coupled to fluorescently tagged peptides confirmed that biosynthetic intermediate Und‐PP‐Bac₁ and Und‐PP‐GalNAc₂Bac₁ are transferred efficiently to the Asn residue in the consensus sequence (D/E‐X′‐N‐X‐T/S, X′,X≠P). The products were analyzed in detail by tandem MS to confirm their chemical structures.     

Tailored Synthesis of 162‐Residue S‐Monoglycosylated GM2‐Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library

A synthetic protocol for the preparation of 162‐residue S‐monoglycosylated GM2‐activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one‐pot/N‐to‐C‐directed sequential ligation strategy using readily accessible middle N‐sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1–31)] with the N‐terminal cysteinyl prolyl thioester [GM2AP (32–67)] proceeded smoothly to yield the 67‐residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as “tailored synthesis” for the construction of a GM2AP library.     

Native Chemical Ligation Combined with Desulfurization and Deselenization: A General Strategy for Chemical Protein Synthesis

Of the many approaches proposed to generalize the native chemical ligation approach for protein synthesis, the simple procedure of global desulfurization of peptide thiols has become the most widely adopted. In this review, the development of the native ligation–desulfurization strategy is described, focusing on the conversion of Cys to Ala following ligation at N-terminal Cys residues. Subsequent variations on this theme have broadened the scope to other natural amino acids including Phe, Leu, Val, and Lys, and even non-native peptide linkages such as isopeptide bonds on lysine side chains. Using insights from both selenocysteine–peptide side reactions and radical initiated desulfurization procedures, a new method for the selective deselenization of peptides containing both selenocysteine and cysteine residues has been developed. Together, these approaches represent a robust and flexible methodology for the synthesis of complex polypeptides without the use of protecting groups.     

Designing amino-based ionic liquids for improved carbon capture: One amine binds two CO2

Generally, amine group captures CO₂ according to 2:1 or 1:1 stoichiometry. Here, we report a kind of improved carbon capture using amino-functionalized ionic liquids (ILs) through 1:2 stoichiometry. A serial of amino-functionalized ILs various with basicity and steric hindrance of anion were designed, prepared, and applied in CO₂ capture. Through a combination of absorption experiment, quantum chemical calculation, spectroscopic investigation and calorimetric method, the results indicated that one amine group could bind two CO₂ through proton transfer (PT) process and intramolecular hydrogen bond formation, which leading to enhanced capacity that breaks through equimolar. The basicity and steric hindrance of anion play a significant role in promoting amine group to capture two CO₂. [P₆₆₆₁₄]₂[Asp] with dual anion was further designed and synthesized to promote PT process, which showed high capacity of 1.96 mol/mol IL at 30°C and 1 atm as well as excellent reversibility. © 2018 American Institute of Chemical Engineers AIChE J, 65: 230–238, 2019     

Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylaseappeared by differential labeling to be in the active site.To test their functions, they were replaced by mutagenesis withAsn, Gln and Gln respectively, and kinetic parameters and pHdependencies of all enzyme forms were determined. Glu179 –Gln glucoamylase was not active on maltose or isomaltose, whilethe k_cat for maltoheptaose hydrolysis decreased almost 2000-foldand the K_M was essentially unchanged from wild-type glucoamylase.The Glu180 – Gln mutation drastically increased the K_Mand moderately decreased the k_cat with maltose and maltoheptaose,but affected isomaltose hydrolysis less. Differences in substrateactivation energies between Glu180 – Gln and wild-typeglucoamylases indicate that Glu180 binds D-glucosyl residuesin subsite 2. The Asp176 – Asn substitution gave moderateincreases and decreases in K_M and k_cat respectively, and thereforesimilar increases in activation energies for the three substrates.This and the differences in subsite binding energies betweenAsp176 – Asn and wild-type glucoamylases suggest thatAsp176 is near subsite 1, where it stabilizes the transitionstate and interacts with Trp120 at subsite 4. Glu179 and Asp176are thus proposed as the general catalytic acid and base ofpK_a 5.9 and 2.7 respectively. The charged Glu180 contributesto the high pK_a value of Glul79. Received May 25, 1989; accepted October 19, 1989.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

Chemical Synthesis of A Pore‐Forming Antimicrobial Protein,Caenopore‐5, by Using Native Chemical Ligation at a Glu‐Cys Site

The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore‐5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore‐forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid‐phase peptide synthesis and native chemical ligation. No γ‐linked by‐product was observed despite the use of a C‐terminal Glu‐thioester. The folding of the synthetic protein was confirmed by ¹H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore‐5. The permeabilisation activities of the protein and of shortened analogues were evaluated.     

Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges

An alkaline, mesophilic endo-1,4-ß-glucanase fromalkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilizedby replacement of both Asn179 and Asp194 with lysine by site-directedmutagenesis. Structural remodeling of the mutant enzyme newlygenerated by the double mutation suggested that Glu175     

Solution Structure and Constrained Molecular Dynamics Study of Vitamin B12 Conjugates of the Anorectic Peptide PYY(3–36)

Vitamin B₁₂–peptide conjugates have considerable therapeutic potential through improved pharmacokinetic and/or pharmacodynamic properties imparted on the peptide upon covalent attachment to vitamin B₁₂ (B₁₂). There remains a lack of structural studies investigating the effects of B₁₂ conjugation on peptide secondary structure. Determining the solution structure of a B₁₂–peptide conjugate or conjugates and measuring functions of the conjugate(s) at the target peptide receptor may offer considerable insight concerning the future design of fully optimized conjugates. This methodology is especially useful in tandem with constrained molecular dynamics (MD) studies, such that predictions may be made about conjugates not yet synthesized. Focusing on two B₁₂ conjugates of the anorectic peptide PYY(3–36), one of which was previously demonstrated to have improved food intake reduction compared with PYY(3–36), we performed NMR structural analyses and used the information to conduct MD simulations. The study provides rare structural insight into vitamin B₁₂ conjugates and validates the fact that B₁₂ can be conjugated to a peptide without markedly affecting peptide secondary structure.     

Macromolecular sorbent materials for urea capture

Three types of chitosan–glutaraldehyde (Chi–Glu) crosslinked copolymer materials were prepared at various Chi–Glu weight ratios (i.e., 1 : 0.0835, 1 : 0.334, and 1 : 0.585) and variable reaction times. The corresponding Chi–Glu copolymer materials were imbibed in CuSO₄ solution to yield impregnated materials in the form of copolymer/Cu(II) complexes. The copolymer materials were characterized using FTIR spectroscopy and thermogravimetry analysis. Urea sorption isotherms were obtained in aqueous solution at 295 K and pH 7 with pristine chitosan, Chi–Glu copolymers (i.e., 1 : 0.0835 and 1 : 0.585), and the corresponding Chi–Glu/Cu(II) complexes. The concentration of unbound urea was monitored indirectly using a colorimetric method with p‐dimethylaminobenzaldehyde. The equilibrium adsorption data were analyzed using the Sips isotherm model. The uptake of urea with pristine chitosan was 4.7% w/w, whereas Chi–Glu copolymers display increased sorption (Q_m = 10.6–17.1% w/w) with increasing glutaraldehyde content. Urea sorption is further enhanced (Q_m = 16.3–26.4% w/w) for copolymer Chi–Glu/Cu(II) complexes. The preparation of Chi–Glu copolymers at various conditions illustrates that the sorption capacity and molecular recognition of urea can be systematically tuned via crosslinking and the formation of copolymer/Cu(II) complexes, and these results are related to a previously reported study (Shimizu and Fujishige, J. Biomed. Mater. Res. 1983, 17, 597). © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Linear and Cyclic Depsipeptidomimetics with β‐Lactam Cores: A Class of New αvβ3 Integrin Receptor Inhibitors

The α_vβ₃ integrin receptor plays an important role in tumor metastasis and tumor‐induced angiogenesis. The inhibition of this receptor with diverse ligands, antibodies, or cyclic peptides is a promising research field for the treatment of a variety of tumors. The replacement of Phe‐(Me)Val dipeptide by a β‐lactam ring in Cilengitide has led to new products that show higher inhibitory activity than the parent cyclopeptide. In particular, substitution of a peptide bond β‐lactam‐NH‐Asp linkage by a β‐lactam‐O‐Asp ester linkage increases the activity of the new cyclodepsipeptide. In the same way it has been found that open‐chain compounds of the form Asp‐β‐lactam‐Arg can interact with the receptor and inhibit its activity moderately. The integrin inhibitory activity of the synthesized compounds has been established by using the CGH array, a method that appears to be a more reliable trial than the classical adhesion test.     

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions

The role of electrostatic interactions between the ionizableAsp158 and the active site thiolate-imidazolium ion pair ofsome cysteine proteinases has been the subject of controversyfor some time. This study reports the expression of wild typeprocaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants fromEscherichia coli. Purification of autocatalytically maturedenzymes yielded sufficient fully active material for pH (k_cat/K_m)profiles to be obtained. Use of both uncharged and charged substratesallowed the effects of different reactive enzyme species tobe separated from the complications of electrostatic effectsbetween enzyme and substrate. At least three ionizations aredetectable in the acid limb of wild type caricain and the Gluand Asn mutants. Only two pK_a, values, however, are detectablein the acid limb using the Ala mutant. Comparison of pH activityprofiles shows that whilst an ionizable residue at position158 is not essential for the formation of the thiolate-imidazoliumion pair, it does form a substantial part of the electrostaticfield responsible for increased catalytic competence. Changingthe position of this ionizable group in any way reduces activity.Complete removal of the charged group reduces catalytic competenceeven further. This work indicates that hydronations distantto the active site are contributing to the electrostatic effectsleading to multiple active ionization states of the enzyme.     

Separation of L‐aspartic acid and L‐glutamic acid mixtures for use in the production of bio‐based chemicals

BACKGROUND: Amino acids are promising feedstocks for the chemical industry due to their chemical functionality. They can be obtained by the hydrolysis of potentially inexpensive protein streams such as the byproduct of biofuel production. However, individual amino acids are required before they can be used for the further production of chemicals. Here, the separation of L‐aspartic acid (Asp) and L‐glutamic acid (Glu) mixture, which can be isolated from protein hydrolysis solutions at low pH or from electrodialysis of complex amino acid mixtures, was studied. RESULTS: Glu was converted into L‐pyroglutamic acid (pGlu) which can be separated from the mixture of Asp and Glu due to its higher solubility in water. The conversion was carried out under aqueous or melt conditions. Under aqueous conditions, the conversion was studied as a factor of time, temperature and the amount of Glu. The conversion was specific with high yield and not effected by Asp. After pGlu was separated from Asp and residual Glu by solubility difference, it can be transferred back to Glu through hydrolysis. CONCLUSION: The conversion of Glu to pGlu is specific and can be applied to separation Asp and Glu for their use in the production of bio‐based chemicals. Copyright © 2012 Society of Chemical Industry