细胞穿透环肽

细胞穿透环肽能够穿透细胞膜,特异性地靶向细胞内靶标,并具有较好的体内稳定性,因而具备很好的成药性,并受到研究者越来越多的关注.目前,主要有两种方式获得细胞穿透环肽,即从天然产物中获得和对已有的环肽或线性肽进行化学修饰.本文主要对上述两方面进行简要综述,重点介绍两类细胞穿透环肽天然产物以及通过化学修饰方法得到细胞穿透环肽的策略,并探讨细胞穿透环肽的结构-活性关系及其细胞穿透机理.     

Cell‐Permeable Peptides Containing Cycloalanine Residues

We present here an efficient alternative to N‐methylation for the purpose of morphing protein‐binding peptides into more serum‐stable and cell‐permeable compounds. This involves the incorporation of a cycloalanine (CyAla) into a peptide in a way that avoids difficult coupling steps. We demonstrate the utility of this chemistry in creating a cell‐permeable derivative of a high‐affinity HIV Rev protein‐binding peptide.     

Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR‐derived structures, amide H–D exchange rates, and temperature‐dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N‐methylation.     

An Efficient Method for the In Vitro Production of Azol(in)e‐Based Cyclic Peptides

Heterocycle‐containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine‐derived enzymes and substrates obtained from a family of ribosomally produced and post‐translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non‐native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6–9 residues representing 11 out of the 20 canonical amino acids.     

Synthesis of Biaryl‐Bridged Cyclic Peptides via Catalytic Oxidative Cross‐Coupling Reactions

Biaryl‐bridged cyclic peptides comprise an intriguing class of structurally diverse natural products with significant biological activity. Especially noteworthy are the antibiotics arylomycin and its synthetic analogue G0775, which exhibits potent activity against Gram‐negative bacteria. Herein, we present a simple, flexible, and reliable strategy based on activating‐group‐assisted catalytic oxidative coupling for assembling biaryl‐bridged cyclic peptides from natural amino acids. The synthetic approach was utilized for preparing a number of natural and unnatural biaryl‐bridged cyclic peptides, including arylomycin/G0775 and RP 66453 cyclic cores.     

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.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Stereoselective Fluorination Alters the Geometry of a Cyclic Peptide: Exploration of Backbone‐Fluorinated Analogues of Unguisin A

New methods for enhancing the efficiency of peptide cyclization, and for fine‐tuning the conformations of cyclic peptides, are valuable from a drug development perspective. Herein stereoselective fluorination is investigated as a new strategy for achieving these goals. Four vicinal difluorinated analogues of the natural cyclic heptapeptide unguisin A have been efficiently synthesized. The analogues are found to adopt dramatically different secondary structures, controlled by the fluorine stereochemistry.     

Emerging Methods and Design Principles for Cell‐Penetrant Peptides

Biomolecules such as antibodies, proteins, and peptides are important tools for chemical biology and leads for drug development. They have been used to inhibit a variety of extracellular proteins, but accessing intracellular proteins has been much more challenging. In this review, we discuss diverse chemical approaches that have yielded cell‐penetrant peptides and identify three distinct strategies: masking backbone amides, guanidinium group patterning, and amphipathic patterning. We summarize a growing number of large data sets, which are starting to reveal more specific design guidelines for each strategy. We also discuss advantages and disadvantages of current methods for quantifying cell penetration. Finally, we provide an overview of best‐odds approaches for applying these new methods and design principles to optimize cytosolic penetration for a given bioactive peptide.     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Molecular Pom Poms from Self‐Assembling α,γ‐Cyclic Peptides

The hierarchical self‐assembly properties of a dimer‐forming cyclic peptide that bears a nicotinic acid moiety to form molecular pom‐pom‐like structures are described. This dimeric assembly self organizes into spherical structures that can encapsulate small organic molecules owing to its porosity and it can also facilitate metal deposition on its surface directed by the pyridine moiety.     

Phosphorylation‐Responsive Membrane Transport of Peptides

Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation‐responsive membrane transport systems. Dye‐efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell‐penetrating peptides, with EC₅₀ values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label‐free kinase assay.     

Synthesis and Conformational Analysis of Cyclic Homooligomers from Pyranoid ε‐Sugar Amino Acids

New pyranoid ε‐sugar amino acids were designed as building blocks, in which the carboxylic acid and the amine groups were placed in positions C2 and C3 with respect to the tetrahydropyran oxygen atom. By using standard solution‐phase coupling procedures, cyclic homooligomers containing pyranoid ε‐sugar amino acids were synthesized. Conformation analysis was performed by using NMR spectroscopic experiments, FTIR spectroscopic studies, X‐ray analysis, and a theoretical conformation search. These studies reveal that the presence of a methoxy group in the position C4 of the pyran ring produces an important structural change in the cyclodipeptides. When the methoxy groups are present, the structure collapses through interresidue hydrogen bonds between the oxygen atoms of the pyran ring and the amide protons. However, when the cyclodipeptide lacks the methoxy groups, a U‐shape structure is adopted, in which there is a hydrophilic concave face with four oxygen atoms and two amide protons directed toward the center of the cavity. Additionally, we found important evidence of the key role played by weak electrostatic interactions, such as the five‐membered hydrogen‐bonded pseudocycles (C₅) between the amide protons and the ether oxygen atoms, in the conformation equilibrium of the macrocycles and in the cyclization step of the cyclic tetrapeptides.     

Conformational Studies on Peptides of α‐Aminoxy Acids with Functionalized Side‐Chains

Peptides of homochiral α‐aminoxy acids of nonpolar side chains can form a 1.8₈‐helix. In this paper, we report the conformational studies of α‐aminoxy peptides 1 , 2 , 3 , which have functionalized side chains, in both nonpolar and polar solvents. ¹H NMR, XRD, and FTIR absorption studies confirm the presence of the eight‐membered‐ring intramolecular hydrogen bonds (the N‐O turns) in nonpolar solvents as well as in methanol. CD studies of peptides 1 , 2 , 3 in different solvents indicate that a substantial degree of helical content is retained in methanol and acidic aqueous buffers. The introduction of functionalized side chains in α‐aminoxy peptides provides opportunities for designing biologically active peptides.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Chirality and Template‐Mediated Induction of Helical Preferences in Achiral β‐Peptides

This study describes chirality‐ or template‐mediated helical induction in achiral β‐peptides for the first time. A strategy of end capping β‐peptides derived from β‐hGly (the smallest achiral β‐amino acid) with a chiral β‐amino acid that possesses a carbohydrate side chain (β‐Caa; C‐linked carbo β‐amino acid) or a small, robust helical template derived from β‐Caas, was adopted to investigate folding propensity. A single chiral (R)‐β‐Caa residue at the C‐ or N‐terminus in these oligomers led to a preponderance of right‐handed 12/10‐helical folds, which was reiterated more strongly in peptides capped at both the C‐ and N‐terminus. Likewise, the presence of a template (a 12/10‐helical trimer) at both the C‐ and N‐terminus resulted in a very robust helix. The propagation of the helical fold and its sustenance was found in a homo‐oligomeric sequence with as many as seven β‐hGly residues. In both cases, the induction of helicity was stronger from the N terminus, whereas an anchor at the C terminus resulted in reduced helical propensity. Although these oligomers have been theoretically predicted to favor a 12/10‐mixed helix in apolar solvents, this study provides the first experimental evidence for their existence. Diastereotopicity was found in both the methylene groups of the β‐hGly moieties due to chirality. Additionally, the β‐hGly units have shown split behavior in the conformational space to accommodate the 12/10‐helix. Thus, end capping to assist chiralty‐ or template‐mediated helical induction and stabilization in achiral β‐peptides is a very attractive strategy.     

β‐Turn Analogues in Model αβ‐Hybrid Peptides: Structural Characterization of Peptides Containing β2,2Ac6c and β3,3Ac6c Residues

The effect of gem‐dialkyl substituents on the backbone conformations of β‐amino acid residues in peptides has been investigated by using four model peptides: Boc‐Xxx‐β^2,2Ac₆c(1‐aminomethylcyclohexanecarboxylic acid)‐NHMe (Xxx=Leu ( 1 ), Phe ( 2 ); Boc=tert‐butyloxycarbonyl) and Boc‐Xxx‐β^3,3Ac₆c(1‐aminocyclohexaneacetic acid)‐NHMe (Xxx=Leu ( 3 ), Phe ( 4 )). Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed conformations about flanking single bonds. The crystal structure of Boc‐Leu‐β^2,2Ac₆c‐NHMe ( 1 ) established a C₁₁ hydrogen‐bonded turn in the αβ‐hybrid sequence. The observed torsion angles (α(?≈?60°, ψ≈?30°), β(?≈?90°, θ≈60°, ψ≈?90°)) corresponded to a C₁₁ helical turn, which was a backbone‐expanded analogue of the type III β turn in αα sequences. The crystal structure of the peptide Boc‐Phe‐β^3,3Ac₆c‐NHMe ( 4 ) established a C₁₁ hydrogen‐bonded turn with distinctly different backbone torsion angles (α(?≈?60°, ψ≈120°), β(?≈60°, θ≈60°, ψ≈?60°)), which corresponded to a backbone‐expanded analogue of the type II β turn observed in αα sequences. In peptide 4 , the two molecules in the asymmetric unit adopted backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopted an unfavorable backbone conformation, with the energetic penalty being offset by a favorable aromatic interaction between proximal molecules in the crystal. NMR spectroscopy studies provided evidence for the maintenance of folded structures in solution in these αβ‐hybrid sequences.     

Three‐Residue Turns in α/β‐Peptides and Their Application in the Design of Tertiary Structures

A new three‐residue turn was serendipitously discovered in α/β hybrid peptides derived from alternating C‐linked carbo‐β‐amino acids (β‐Caa) and L ‐Ala residues. The three‐residue β‐α‐β turn at the C termini, nucleated by a helix at the N termini, resulted in helix‐turn (HT) supersecondary structures in these peptides. The turn in the HT motif is stabilized by two H bonds—CO(i?2)–NH(i), with a seven‐membered pseudoring (γ turn) in the backward direction, and NH(i?2)–CO(i), with a 13‐membered pseudoring in the forward direction (i being the last residue)—at the C termini. The study was extended to generalize the new three‐residue turn (β‐α‐β) by using different α‐ and β‐amino acids. Furthermore, the HT motifs were efficiently converted, by an extension with helical oligomers at the C termini, into peptides with novel helix‐turn‐helix (HTH) tertiary structures. However, this resulted in the destabilization of the β‐α‐β turn with the concomitant nucleation of another three‐residue turn, α‐β‐β, which is stabilized by 11‐ and 15‐membered bifurcated H bonds. Extensive NMR spectroscopic studies were carried out to delineate the secondary and tertiary structures in these peptides, which are further supported by molecular dynamics (MD) investigations.     

Effect of β3‐Amino Acids on the Performance of the Peptidic Catalyst H‐dPro‐Pro‐Glu‐NH2

The effect of β³‐amino acids on the conformation and catalytic performance of the peptidic catalyst H‐d Pro‐Pro‐Glu‐NH₂ was investigated. Analogues of the peptidic catalyst bearing instead of the α‐amino acids the respective β³‐amino acids were prepared and their reactivity and stereoselectivity was investigated in conjugate addition reactions of aldehydes to nitroolefins. Additional computational studies provided insights into the preferred conformations of the peptidic catalysts. The results show that conformational flexibility at the N‐terminus has a severe effect on the stereoselectivity but is tolerated at the C‐terminus.     

Late‐Stage Functionalization of Peptides and Cyclopeptides Using Organozinc Reagents

We report a new late‐stage functionalization of small peptides and cyclopeptides relying on the Negishi cross‐coupling of readily prepared iodotyrosine‐ or iodophenylalanine‐containing peptides with aryl‐, heteroaryl‐, and alkylzinc pivalates or halides. In silico and in vitro determinations of membrane permeability parameters of the modified cyclopeptides showed that in most cases, the solubility was improved by the introduction of polar pyridyl units while the cell‐membrane permeability was maintained.