Iodine Oxidation of S-Trityl- and S-Acetamidomethyl-cysteine-peptides Containing Tryptophan: Conditions Leading to the Formation of Tryptophan-2-thioethers

Cystine peptides are conveniently prepared from S-acetamidomethyl- or S-trityl-protected cysteine derivatives by direct oxidation with iodine. Since the reaction proceeds through the formation of sulfenyl iodides, these highly reactive groups may substitute the indole ring of tryptophan residues, resulting in the formation of 2-thioethers. During the synthesis of the peptide hormone somatostatin, we investigated this possible side reaction. By-products of the tryptophan-2-thioether type can be produced under conditions which lead to a marked retardation of the disulfide bond formation. The largest amount of these compounds were formed when the oxidation was carried out in 90% aqueous trifluoroethanol. In model peptides in which tryptophan and cysteine residues were separated by 1 to 4 glycine residues, the ring size of the resulting thioether exerted a strong influence on the yield: in peptides with 1 and 2 glycines, only dimeric disulfides were formed. Incorporation of 3 and 4 glycine residues gave thioethers in yields of about 40% and 70% respectively. Conversely, under normal conditions of iodine oxidation, when disulfides are rapidly formed from the S-acetamidomethyl- or S-tritylcysteine residues, tryptophan-2-thioethers are produced only in insignificant amounts or not at all.     

Chemical Synthesis of Human Insulin‐Like Peptide‐6

Human insulin‐like peptide‐6 (INSL‐6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL‐6 utilizing fluorenylmethyloxycarbonyl‐based (Fmoc) solid‐phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation‐sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine‐mediated S‐acetamidomethyl (Acm) deprotection and the second utilized iodine‐free, sulfoxide‐directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL‐6 and will further improve synthetic access to other multiple‐disulfide‐containing peptides with oxidation‐sensitive residues.     

Alternative Strategies for the Fmoc Solid-Phase Synthesis of O4-Phospho-L-tyrosine-Containing Peptides

A number of biologically relevant O⁴-phospho-L-tyrosine-containing peptides have been synthesized by either the global phosphorylation of the side-chain-unprotected L-tyrosine moiety in presynthesized resin-bound peptides or alternatively by the incorporation of suitably protected O⁴-phospho-L-tyrosine building blocks in the continuous-flow method of Fmoc solid-phase peptide synthesis. Different phosphate-protecting groups have been applied.     

Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes

Flax cyclic peptides (orbitides, linusorbs (LOs)) [1–8‐NαC],[1‐MetO₂]‐linusorb B1 ([MetO₂]‐LO1) and [1–9‐NαC],[1‐MetO₂]‐linusorb B2 ([MetO₂]‐LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]‐LO1 and [Met]‐LO2 analogues containing activated groups (?CN, ?COOEt, and ?NH₂) in the form of methionine, methionine (S)‐oxide, and methionine (S,S)‐dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu³⁺ yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths.     

Liquid Adsorption Chromatography on Preparative Scale of Protected Synthetic Peptides

Abstract

This paper reports the purification of synthetic protected peptides on a preparative scale by means of adsorption chromatography on silica gel 60 columns.

The protected peptides described were precursors of a free asymmetrical cystine peptide corresponding to the insulin sequence A^18–21-B^19–26 and were obtained in analytically pure form. Selection of solvent systems for isocratic or stepwise elution depended largely on R_F data obtained from thin-layer chromatograms which were used for monitoring and optimizing synthetic reactions.     

Metal Binding Ability of Small Peptides Containing Cysteine Residues

The Cd(II)-, Pb(II)-, Ni(II)- and Zn(II)-complexes of small terminally protected peptides containing CXXX, XXXC, XCCX, CX_nC (n=1–3) sequences have been studied with potentiometric, UV/Vis and CD spectroscopic techniques. The cysteine thiolate group is the primary binding site for all studied metal ions, but the presence of a histidyl or aspartyl side chain in the molecule contributes to the stability of the complexes. For two-cysteine containing peptides the (S⁻,S⁻) coordinated species are formed in the physiological pH range and the stability increases in the Ni(II)<Zn(II)<Pb(II)<Cd(II) order. As a conclusion, the inserting of −CXXC− sequence into the peptide makes the synthesis of peptides with high selectivity to toxic Cd(II) or Pb(II) ion possible. In addition, the spectroscopic characterization of these complexes can contribute to the discovery of the exact binding site and binding mode of longer peptides mimicking the biologically important proteins.     

Eco‐Friendly Combination of the Immobilized PGA Enzyme and the S‐Phacm Protecting Group for the Synthesis of Cys‐Containing Peptides

Enzyme‐labile protecting groups have emerged as a green alternative to conventional protecting groups. These groups introduce a further orthogonal dimension and eco‐friendliness into protection schemes for the synthesis of complex polyfunctional organic molecules. S‐Phacm, a Cys‐protecting group, can be easily removed by the action of a covalently immobilized PGA enzyme under very mild conditions. Herein, the versatility and reliability of an eco‐friendly combination of the immobilized PGA enzyme and the S‐Phacm protecting group has been evaluated for the synthesis of diverse Cys‐containing peptides.     

A novel approach to the regioselective synthesis of a disulfide-linked heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor

We describe a novel acetamidomethyl to S-pyridinyl exchange that is used for the synthesis of a multi-disulfide-linked and constrained heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor (BDNF). This simple and effective method should be readily transferable to the synthesis of similar disulfide-linked heterodimeric peptides, as well as being of general utility for the synthesis of peptides bearing multiple cystine frameworks.     

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.     

Stapled Peptides with γ‐Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction

“Stapled” peptides are typically designed to replace two non‐interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ‐position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm ) replaces isoleucine 689 with an S‐γ‐methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm ). Through X‐ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S‐γ‐methyl peptide minimizes the syn‐pentane interactions between the α‐ and γ‐methyl groups.     

Synthesis of insulin fragments with disulfide bridges between intact chains A20-B19

The synthesis of six insulin fragments is described, in which various sequences of the two chains are linked by the disulfide bridge between A²⁰ and B¹⁹. The fragments in question are: A^20–21–B^19–21, A^20–21–B^18–21, A^20–21–B^17–21, A^19–21–B^19–21, A^16–21–B^18–21 and A^20–21–B^12–21. In order to build up the simpler fragments the disulfide bridge was established by oxidation with iodine of two S-trityl cysteine peptides in which the carboxyl and amino groups were protected by the t-butyl and t-butyloxycarbonyl residue. From the mixture obtained the unsymmetrical cystine peptide was separated in all cases from the two symmetrical ones by counter-current distribution. In the synthesis of the more complex fragments advantageous use was made of smaller unsymmetrical fragments prepared as above but having one amino group protected by the N-trityl residue. After selective elimination of this group it was possible to lengthen the peptide chain at this position. The free peptides were obtained by removal of the protecting groups with strong acids, in particular concentrated hydrochloric acid. While in this deprotecting step the disulfide bond was stable, conditions are discussed under which disproportionation was observed. None of the six synthetic insulin fragments showed activity in stimulating rat adipose tissue to convert ¹⁴C-labelled glucose to CO₂ in vitro.     

人参寡肽的合成

使用溶液和有机磷肽缩合试剂DEPBT合成了从人参中分离鉴定的三个寡肽,N,N'－双－(γ-谷氨酰甘氨酰)胱氨酸1,N,N'－双-γ－谷氨酰胱氨酰甘氨酸2,N-γ-谷氨酰胱氨酰-双-甘氨酸3,以及γ-谷氨酰甘氨酰半胱氨酸。4.产物经离子交换树脂柱或HPLC纯化,用质谱、核磁共振谱、氨基酸分析进行了验证。     

Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type adenylylated amino acid derivatives

Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of condensation of an N-nonprotected adenosine derivative and Fmoc-Ser/Thr/Tyr-OAllyl using allyl-N,N-diisopropylchlorophosphoramidite and subsequent oxidation with m-chloroperbenzoic acid gave phosphotriester-type AMPylated Ser/Thr/Tyr derivatives. After Pd(0)-mediated deprotection of allyl groups, the resulting phosphodiester-type AMPylated Ser/Thr/Tyr derivatives were successfully incorporated into peptides by standard Fmoc solid phase peptide synthesis without significant side reactions including dehydroalanine formation.     

Synthesis of Cyclic Peptides in SPPS with Npb-OH Photolabile Protecting Group

Significant efforts have been made in recent years to identify more environmentally benign and safe alternatives to side-chain protection and deprotection in solid-phase peptide synthesis (SPPS). Several protecting groups have been endorsed as suitable candidates, but finding a greener protecting group in SPPS has been challenging. Here, based on the 2-(o-nitrophenyl) propan-1-ol (Npp-OH) photolabile protecting group, a structural modification was carried out to synthesize a series of derivatives. Through experimental verification, we found that 3-(o-Nitrophenyl) butan-2-ol (Npb-OH) had a high photo-release rate, high tolerance to the key conditions of Fmoc-SPPS (20% piperidine DMF alkaline solution, and pure TFA acidic solution), and applicability as a carboxyl-protective group in aliphatic and aromatic carboxyl groups. Finally, Npb-OH was successfully applied to the synthesis of head–tail cyclic peptides and side-chain–tail cyclic peptides. Moreover, we found that Npb-OH could effectively resist diketopiperazines (DKP). The α-H of Npb-OH was found to be necessary for its photosensitivity in comparison to 3-(o-Nitrophenyl)but-3-en-2-ol (Npbe-OH) during photolysis-rate verification.     

A Multicomponent Conjugation Strategy to Unique N‐Steroidal Peptides: First Evidence of the Steroidal Nucleus as a β‐Turn Inducer in Acyclic Peptides

Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N‐substituent of an internal amide bond is reported. The approach comprises the one‐pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N‐steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head‐to‐tail cyclization to produce chimeric cyclopeptide–steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three‐dimensional structure of an acyclic N‐steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a β‐turn secondary structure even in the all‐s‐trans isomer. This report provides the first evidence of the steroid skeleton as β‐turn inducer in linear peptide sequences.     

Chemo-Enzymatic Synthesis of Optically Active Amino Acids and Peptides

The industrial alkaline protease, alcalase, is stable and active in a high concentration of organic solvents and useful as a biocatalyst for (i) diastereoselective hydrolysis of peptide esters and preparation of racemization-free peptides; (ii) selective incorporation of esters of D-amino acid into peptides in t-butanol via a selective hydrolysis of esters of D,L-amino acid, followed by using the unhydrolyzed D-esters as a nucleophile in a kinetically controlled peptide bond formation; (iii) resolution of esters of amino acid in 95% t-butanol/5% water, followed by saponification of the unreacted esters to offer both enantiomers with high yield and optical purity; (iv) completely resolve amino-acid esters with high yield and optical purity via in situ racemization of the unreacted antipode catalyzed by pyridoxal 5-phosphate; (v) cryobioorganic synthesis of peptides with increased yields 15%–40% of peptide bond formation by reaction at 5 °C instead of 25–30 °C of a kinetically controlled enzymatic reaction in alcohols.     

HPLC Separation of Geometric Isomers of Carbamyl Peptides

Abstract

Carbamylated peptides that were studied showed much improved resolution on C-18 reversed phase HPLC columns compared to the parent peptides. A number of dipeptides were carbamylated with ethyl-, n-propyl- or isopropyl isocyanates. The three carbamyl derivatives of each dipeptide could easily be resolved. Carbamylated dipeptides with reversed amino acid sequences were also easily separated. Methionine-enkephalin, leucine-enkephalin, Angiotensins I, II and III and substance P were carbamylated with isocyanates derived from certain carcinostatic 2-chloroethyl nitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1-(2-chloroethyl)-3-(cis-4-hydroxycyclohexyl)-1-nitrosourea (cis-4-hydroxy-CCNU) and 1-(2-chloroethyl)-3-(trans-4-hydroxycyclohexy1)-1-nitroso urea (trans-4-hydroxy-CCNU) gave carbamylated derivatives of each peptide and each mixture of derivatives from a single parent peptide could be resolved. Conditions were found in each case whereby baseline resolution of the corresponding cis-4- and trans-4-hydroxycyclohexylcarbamyl peptides was attained. Cyclohexyl-carbamyl peptides were easily separated from the corresponding peptides from hydroxy-CCNUs. Potential applications are discussed. carbamylation in the expression of antitumor activity or toxicity to normal tissues is still not very clear. A number of DNA polymerases (10, 11), DNA ligase (12), glutathione reductase (13), Serine proteases (14, 15) and tubulin polymerization (16) have been shown to be inhibited by carbamylation by nitrosoureas or isocyanates.

The question of whether carbamylation by CENUs and their metabolites is selective to certain enzymes is an important one because nitrosoureas such as CCNU and Methyl-CCNU are converted to antitumor-active metabolites (8, 17, 18). If it is found that the individual metabolites target different proteins, it could have important applications in chemotherapy and drug design.

In order to determine whether such selectivity of carbamylation exists, it was required that analytical methodology be developed that would permit separation of peptides with the same sequence but with different carbamyl groups. These studies offer hope that if peptides with up to 11 aminoacids are carbamylated by CCNU or the 4-hydroxy-CCNUs, one may be able to separate them by HPLC. This limit may be extended if one need only separate cyclohexyl carbamyl peptides from mixtures of geometric and positional isomers of hydroxycyclohexyl carbamyl peptides as might occur in the cell.     

On the relation between polypeptide structure and spredptogenic activity. 1. Peptides of cystine

The authors propose for the characterization of strepogenic substances of known composition the specific activity, i. e. the number of WOOLLEY units per μmole. Starting from L -leucyl-L -cystinyl-L -leucyl-L -valyl-L -glutamic acid (of very high strepogenine activity, 400 WOOLLEY units per mg, 230 WOOLLEY units per μmole) seven peptides have been synthesized by suppression and/or replacement of amino acid residues. A study of the relation between activity and structure of these peptides shows that:

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N,O-Benzylidene Acetal Dipeptides (NBDs) Enable the Synthesis of Difficult Peptides via a Kinked Backbone Strategy

Proteins with highly hydrophobic regions or aggregation-prone sequences are typically difficult targets for chemical synthesis at the current stage, as obtaining such type of peptides via solid-phase peptide synthesis requires sophisticated operations. Herein, we report N,O-benzylidene acetal dipeptides (NBDs) as robust and effective building blocks to allow the direct synthesis of difficult peptides and proteins via a kinked backbone strategy. The effectiveness and easy accessibility of NBDs have been well demonstrated in our chemical syntheses of various challenging peptides and proteins, including chemokine, therapeutic hormones, histone, and glycosylated erythropoietin.     

Structure and Reactivity of Lithium Enolates. From Pinacolone to Selective C-Alkylations of Peptides. Difficulties and Opportunities Afforded by Complex Structures

The chemistry of lithium enolates is used to demonstrate that complex structures held together by noncovalent bonds (“supramolecules”) may dramatically influence the result of seemingly simple standard reactions of organic synthesis. Detailed structural data have been obtained by crystallographic investigations of numerous Li enolates and analogous derivatives. The most remarkable features of these structures are aggregation to give dimers, tetramers, and higher oligomers, complexation of the metal centers by solvent molecules and chelating ligands, and hydrogen-bond formation of weak acids such as secondary amines with the anionoid part of the enolates. The presence in nonpolar solvents of the same supramolecules has been established by NMR-spectroscopic, by osmometric, and by calorimetric measurements. The structures and the order of magnitude of the interactions have also been reproduced by ab-initio calculations. Most importantly, supramolecules may be product-forming species in synthetic reactions of Li enolates. A knowledge of the complex structures of Li enolates also improves our understanding of their reactivity. Thus, simple procedures have been developed to avoid complications caused by secondary amines, formed concomitantly with Li enolates by the common methods. Mixtures of achiral Li enolates and chiral Li amides can give rise to enantioselective reactions. Solubilization by LiX is observed, especially of multiply lithiated compounds. This effect is exploited for alkylations of N-methylglycine (sarcosine) CH₂ groups in open-chain oligopeptides. Thus, the cyclic undecapeptide cyclosporine, a potent immunosuppressant, is converted into a THF-soluble hexalithio derivative (without epimerization of stereogenic centers) and alkylated by a variety of electrophiles in the presence of either excess lithiumdiisopropyl amide or of up to 30 equivalents of lithium chloride. Depending on the nature of the LiX additive, a new stereogenic center of (R) or (S) configuration is created in the peptide chain by this process. A structure-activity correlation in the series of cyclosporine derivatives thus available is discussed.