Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of a membrane protein with two transmembrane regions.

A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.     

Synthesis of a Glycosylated Peptide Thioester by the Boc Strategy and Its Application to Segment Condensation

The synthesis of a chitobiosylated peptide thioester by the t-butoxycarbonyl (Boc) strategy is demonstrated. Boc-Asn carrying benzyl-protected chitobiose was introduced during application of the Boc mode solid-phase method. HF treatment of the resulting protected peptide resin gave the desired chitobiosylated peptide thioester. This thioester was used to prepare the peptide sequence derived from extracellular matrix metalloproteinase inducers (emmprin) (34-94), (34-118) and (22-118) by the thioester segment condensation method. The conformation of these glycopeptides is characterized by circular dichroism (CD) spectral measurement.     

Synthesis of a glycosylated peptide thioester by the Boc strategy and its application to segment condensation

The synthesis of a chitobiosylated peptide thioester by the t-butoxycarbonyl (Boc) strategy is demonstrated. Boc-Asn carrying benzyl-protected chitobiose was introduced during application of the Boc mode solid-phase method. HF treatment of the resulting protected peptide resin gave the desired chitobiosylated peptide thioester. This thioester was used to prepare the peptide sequence derived from extracellular matrix metalloproteinase inducers (emmprin) (34-94), (34-118) and (22-118) by the thioester segment condensation method. The conformation of these glycopeptides is characterized by circular dichroism (CD) spectral measurement.     

Polypeptide synthesis by the thioester method

A novel method for polypeptide synthesis, in which partially protected peptide thioesters are used as building blocks, has been developed. Partially protected peptide thioesters are easily prepared by solid-phase methodology. The thioester moiety is converted to an active ester in the presence of a silver compound such as AgNO(3) or AgCl and an active ester component such as 1-hydroxybenzotriazole or 3,4-dihydro-3-hydro-4-oxo-1,2, 3-benzotriazine. Segment condensation can be accomplished using partially protected peptide segments. The consecutive condensation of the partially protected peptide segments is realized by the selective removal of the 9-flourenylmethoxycarbonyl group, for terminal amino protection, after segment condensation has been achieved. In this method, large peptide segments can easily be used. Thus, the products obtained by the thioester method can be separated from by-products by reverse phase high performance liquid chromatography, even when no purification process was performed during the prior segment condensation procedures. This indicates that proteins that have no specific features such as enzymatic or biological activities can be obtained after isolation, solely based on their chromatographic profiles. Thus, the thioester method will provide a new basis for protein studies including phosphorylated and glycosylated polypeptides.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N-backbone cyclic peptide chemistry.

Protected Nalpha-(aminoallyloxycarbonyl) and Nalpha-(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of Nalpha-backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and alpha,beta-unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side-chain, resulting in fully orthogonal trifunctional building units for the solid-phase peptide synthesis of small cyclic analogs of peptide loops (SCAPELs). Nalpha-amino groups of building units were protected by Fmoc, functional side-chains were protected by t-Bu/Boc/Trt and N-alkylamino or N-alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large-scale solid-phase peptide synthesis. These building units have significant advantage in the synthesis of peptido-related drugs.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Fmoc-based chemical synthesis and selective binding to supercoiled DNA of the p53 C-terminal segment and its phosphorylated and acetylated derivatives.

The C-terminal domain of p53 comprises a linker, the tetramerization domain and the regulatory domain, and contains at least seven sites of potential post-translational modification. An improved strategy was developed for the synthesis of large peptides that contain phosphorylated amino acids and p53(303-393), a 91-amino acid peptide, and three post-translationally modified derivatives were synthesized through the sequential condensation of three partially protected segments. Peptide thiolesters were prepared using the sulfonamide-based 'safety-catch' resin approach and employing Fmoc-based solid-phase peptide synthesis. At the N-terminus of the middle building block, a photolabile protecting group, 3,4-dimethoxy-6-nitrobenzyloxycarbonyl, was incorporated to differentiate the N-terminal amino group from the side-chain amino groups. Two sequential couplings were accomplished following this protection strategy. The synthetic products, p53(303-393) and its phosphorylated or acetylated derivatives, exhibited the ability to bind specifically to supercoiled DNA, which is one of the characteristics of this domain.     

Chemical synthesis of proteins in solution

Development of a novel strategy suitable for the solution synthesis of proteins is described, wherein the entire molecule is assembled from fully protected segments in the size range of about 10 residues. Each segment is designed so as to have a common structure of Boc-peptide-OPac (Pac: phenacyl) and all of the side-chain functional groups are protected by Bzl-based groups. New types of solvent systems are described for dissolving fully protected segments in which the segment condensation reactions in solution can be carried out smoothly. After removal of the Boc or Pac group, the segments are coupled together to obtain the entire sequence using the 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide/3, 4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine method. The side-chain protecting groups are then removed by HF and the liberated peptide is subjected to folding reactions to obtain the native conformation. Applying the strategy, the 123-residue human angiogenin, the 121-residue human midkine, the 136-residue human pleiotrophin, and the 238-residue Aequoria green fluorescent protein were synthesized successfully.     

2-Chloro-1-methylpyridinium iodide. A suitable reagent for peptide synthesis

2-Chloro-1-methylpyridinium iodide is a coupling reagent for peptide synthesis. By its use different peptides were synthesized with protected di- and trifunctional amino acids. While urethan-protected amino acids react free of racemization, the fragment condensation needs N-Hydroxysuccinimide, as shown by the Young test.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on a photolysable anchoring linkage

Several 3-nitro-4-(N-protected aminomethyl)benzoic acids; with protection provided by tert.-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl (Tfa), dithiasuccinoyl (Dts), or phthaloyl (Phth), have been prepared by reproducible routes. Synthesis of Dts-handle 6 illustrates some particularly novel and efficient chemistry, and is preferred over more intricate routes to Boc-handle 3 and Fmoc-handle 4. The five handles were each evaluated for their application to the synthesis of peptide amides. Coupling onto amino-functionalized supports provided a general starting point for peptide chain assembly. The handle amino function was deblocked (Boc, Fmoc, Dts), the C-terminal residue was coupled as its N alpha-protected free acid, and ultimately the ortho-nitrobenzylamide anchorage linkage was cleaved photolytically to give the corresponding amide. Starting with handles 3, 4, and 6, several free and protected peptide amides were synthesized.     

Synthesis of peptide nucleic acid FRET probes via an orthogonally protected building block for post-synthetic labeling of peptide nucleic acids at the 5-position of uracil

We report the design and synthesis of an orthogonally protected peptide nucleic acid (PNA) building block, Fmoc-PNA-U'-(Dde)-OH, and its use in the construction of PNA FRET probes. This building block allows for the post-synthetic attachment of reporter groups to the amino group attached to the 5-position of uracil (U) following selective deprotection of the Dde group. We illustrate the use of this building block for the synthesis of a series of FAM Cy5 donor acceptor pairs and their ability to detect a target DNA sequence.     

A new amino acid derivative with a masked side-chain aldehyde and its use in peptide synthesis and chemoselective ligation.

A new amino acid derivative with a diol side-chain, L-2-amino-4,5-dihydroxy-pentanoic acid (Adi), has been prepared from L-allylglycine by suitable protection, for use in peptide synthesis, as Fmoc-L-Adi(Trt)2. This building block enables the introduction of a side-chain aldehyde at any position in a given peptide sequence without use of specialized side-chain protection schemes. The aldehyde is revealed by mild oxidation with sodium periodate, circumventing the problematic release of reactive peptidic aldehydes in TFA solution. Peptides with aldehyde side-chains are useful for chemo-selective ligation, reacting selectively with oxyamines to yield oxime links, while all other peptide functions can be left unprotected. The utility of the new building block has been demonstrated by the synthesis of peptide dimers and a cyclo-peptide.     

A convenient incorporation of conformationally constrained 5,5-dimethylproline into the ribonuclease A 89-124 sequence by condensation of synthetic peptide fragments.

The presence of l-5,5-dimethylproline (dmP) within an amino acid sequence results in the formation of an X-dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C-terminal 36-residue peptide, corresponding to the 89-124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5- and 31-residue peptide fragments, which were synthesized by solid-phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-dmP-OH pentapeptide (residues 89-93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate was found to be superior to bromo-tris-pyrrolidino-phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the -Tyr(tBu)-dmP- peptide bond in solution as well as on a resin.     

Semisynthesis of human ghrelin: condensation of a Boc-protected recombinant peptide with a synthetic O-acylated fragment

The creation of peptide using a combination of recombinant expression and chemical synthesis can be a powerful tool for the production of a wide variety of polypeptides modified by phosphorylation, glycosylation, etc. We have developed a new method for the preparation of a recombinant peptide with a free N(alpha)-amino group and protected N(epsilon)-amino groups, and have used this method in the semisynthesis of human ghrelin. Ghrelin, a natural ligand for growth hormone secretagogue receptor, is a 28-residue peptide with an essential n-octanoyl modification on Ser3. A 7-residue N-terminal fragment of ghrelin containing the octanoyl modification was prepared by Fmoc chemistry. In the preparation of it, all reactions were performed on the 2-chlorotrityl resin. Additionally, TBDMS and tBu turned out to be the most effective protection groups for the Ser3 and the Ser2, Ser6, respectively. For preparation of a 21-residue C-terminal fragment, we established a two-step protease processing method for the partially protected segment. A recombinant precursor peptide was Boc protected and subsequently cleaved using two distinct proteases, OmpT and Kex2. The peptides were then coupled to each other and, after deprotection, resulted in fully active human ghrelin.     

The Use of Crown Ethers in Peptide Chemistry – V. Solid-phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non-covalent Protecting Groups

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N^α -amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N^α -complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly⁵² to Leu⁷² (21-mer) and Pro³³ to Leu⁷² (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product had been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.