Rigid Peptide Macrocycles from On‐Resin Glaser Stapling

Peptide macrocycles are widely utilized in the development of high affinity ligands, including stapled α‐helices. The linear rigidity of a 1,3‐diynyl linkage provides an optimal distance (7 Å) between β‐carbons of the i,i+4 amino acid side chains, thus suggesting its utility in stabilizing α‐helical structures. Here, we report the development of an on‐resin strategy for an intramolecular Glaser reaction between two alkyne‐terminated side chains by using copper chloride, an essential bpy‐diol ligand, and diisopropylethylamine at room temperature. The efficiency of this ligation was illustrated by the synthesis of (i,i+4)‐, (i,i+5)‐, (i,i+6)‐, and (i,i+7)‐stapled BCL‐9 α‐helical peptides using the unnatural amino acid propargyl serine. Overall, this procedurally simple method relies on inexpensive and widely available reagents to generate low molecular weight 23‐, 26‐, 29‐, and 32‐membered peptide macrocycles.     

X-ray Crystallographic Structure of α-Helical Peptide Stabilized by Hydrocarbon Stapling at i,i + 1 Positions

Hydrocarbon stapling is a useful tool for stabilizing the secondary structure of peptides. Among several methods, hydrocarbon stapling at i,i + 1 positions was not extensively studied, and their secondary structures are not clarified. In this study, we investigate i,i + 1 hydrocarbon stapling between cis-4-allyloxy-l-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled side chains and structure of the olefin-tethered amino acids, E- or Z-selectivities were observed during the ring-closing metathesis reaction (E/Z was up to 8.5:1 for 17–14-membered rings and up to 1:20 for 13-membered rings). We performed X-ray crystallographic analysis of hydrocarbon stapled peptide at i,i + 1 positions. The X-ray crystallographic structure suggested that the i,i + 1 staple stabilizes the peptide secondary structure to the right-handed α-helix. These findings are especially important for short oligopeptides because the employed stapling method uses two minimal amino acid residues adjacent to each other.     

Linear Aliphatic Dialkynes as Alternative Linkers for Double‐Click Stapling of p53‐Derived Peptides

We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double‐click stapling of p53‐based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53‐activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.     

Recent Advances in Late-Stage Construction of Stapled Peptides via C−H Activation

Stapled peptides have been widely applied in many fields, including pharmaceutical chemistry, diagnostic reagents, and materials science. However, most traditional stapled peptide preparation methods rely on prefunctionalizations, which limit the diversity of stapled peptides. Recently, the emergence of late-stage transition metal-catalyzed C−H activation in amino acids and peptides has attracted wide interest due to its robustness and applicability for peptide stapling. In this review, we summarize the methods for late-stage construction of stapled peptides via transition metal-catalyzed C−H activation.     

Strategy and Effects of Polyproline Peptide Stapling by Copper(I)-Catalyzed Alkyne–Azide Cycloaddition Reaction

Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC), we successfully prepared stapled polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on polyproline helix II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate polyproline conformation for various applications.     

Improving the Binding Affinity of in‐Vitro‐Evolved Cyclic Peptides by Inserting Atoms into the Macrocycle Backbone

Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure. Here, we proposed to improve the affinity of a phage‐selected bicyclic peptide inhibitor of coagulation factor XII (FXII) by screening variants with one or two carbon atoms inserted into different positions of the backbone, and thus tapping into a structural space that was not sampled by phage display. Two mutants showed 4.7‐ and 2.5‐fold improved K_i values. The better one blocked FXII with a K_i of 1.5±0.1 nm and inhibited activation of the intrinsic coagulation pathway (EC_2x 1.7 μm) . The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in‐vitro‐evolved cyclic peptides.     

Bis-azobenzene crosslinkers for photocontrol of peptide structure

Crosslinkers that undergo large changes in length upon photoisomerization can produce large conformational changes, and thereby functional changes, in biomolecules. We have designed and synthesized extended and rigid bis‐azobenzene crosslinkers: 4,4′‐bis(4‐(2‐chloroacetamido)phenyl)diazenylbiphenyl (BPDB) and the water‐soluble sulfonated analogue 4,4′‐bis(4‐(2‐chloroacetamido)phenyl)diazenylbiphenyl‐2,2′‐disulfonate (BPDBS). These photoswitches can produce end‐to‐end distance changes of a minimum of ≈5 Å and a maximum of ≈23 Å upon trans/cis isomerization. They have high absorption coefficients (45–60 000 M ^?1 cm^?1) and can produce up to ≈80 % cis isomers under favorable conditions. The photoswitching behavior of BPDBS‐crosslinked peptides was found to be highly dependent on the crosslinker attachment site. Upon UV irradiation (365 nm), significant decreases in α‐helix content were observed for peptides that were crosslinked with BPDBS through Cys residues at i,i+19, and i,i+21 positions. In contrast, large increases in α‐helix content were exhibited by i,i+11 crosslinked peptides. BPDBS thus constitutes a particularly bright and effective photoswitch for biomolecule photocontrol.     

Segregation of resin‐bound peptides during solid‐phase synthesis

To compare the segregation ability of 1,4‐butanediol dimethacrylate‐crosslinked polystyrene (BDDMA‐PS) and divinylbenzene‐crosslinked polystyrene (DVB‐PS), a set of difficult sequence peptides characterized by high‐arithmetic‐average β‐sheet stabilizing potential (SP_β) and low‐stepwise arithmetic average random coil conformational parameter (P_c*) were synthesized on both supports (～ 2 mmol Cl g^?1) under identical conditions. The yield and purity of the peptides obtained from BDDMS‐PS resin were higher than from DVB‐PS resin. The synthetic efficiency of the new support was found to be its ability to suppress the aggregation of growing peptide chains by β‐sheet formation. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1717–1723, 2002     

Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α₃β₁ (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α₃β₁.     

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization

Aminoimidazole carboxamide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein–protein interaction with an interface of over 5000 Ų, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small‐molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K_i of 17 μM ), identified by high‐throughput screening. The active motif of the cyclic peptide is identified as an arginine‐tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K_i value of 84 μM . A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K_i value of 685 nM , a 25‐fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose‐dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.     

Two‐Face,Two‐Turn α‐Helix Mimetics Based on a Cross‐Acridine Scaffold: Analogues of the Bim BH3 Domain

The design of a cross‐acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two‐face, two‐turn α‐helix is described. Docking studies and 2D ¹H,¹⁵N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl‐1 and Bcl‐2 proteins (K_i 0.079 and 0.056 μM , respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.     

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group

An i−i+4 or i−i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i−i+4 constrained peptide adopts a 3₁₀-helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.     

Tailor‐Made Designer Helical Peptides that Induce Mitochondrion‐Mediated Cell Death without Necrosis

Managing protein–protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side‐chain‐crosslinked helical peptides based on the binding domain of a pro‐apoptotic protein (Bad) that induces programed cell death. The peptide showed high helical content and bound to its target, Bcl‐X_L, more strongly than its non‐crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl‐X_L localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70 % of HeLa cells died by the apoptosis pathway (without evidence of necrosis).     

Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study

Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. We highlight the decisive impact of the intrinsic properties of the crosslinks on cell permeability rather than the helical contents of the peptides in model amphipathic sequences targeting estrogen receptor–coactivator interaction. We envision this finding to shed further light on the chemical optimization of stapled α-helical peptides or macrocyclic cell-penetrating peptides for enhanced cell penetration.     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain‐to‐side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non‐hydrogen‐bonded amide groups to solvent. Here, we develop a “capped‐strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end‐capping interactions. We have designed a ten‐residue capped‐strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X‐ray crystallography revealed a novel quaternary structure with implications for foldamer design.     

A Bimane-Based Peptide Staple for Combined Helical Induction and Fluorescent Imaging

The thiol-selective fluorescent imaging agent, dibromobimane, has been repurposed to crosslink cysteine- and homocysteine-containing peptides, with the resulting bimane linker acting as both a structural constraint and a fluorescent tag. Macrocyclisation was conducted on nine short peptides containing two cysteines and/or homocysteines, both on-resin and in buffered aqueous solution, to give macrocycles ranging in size from 16 (i,i+2) to 31 (i,i+7) atoms. The structures were defined by CD, NMR structure calculations by using Xplor-NIH, NMR secondary shift and J_HαNH analyses to reveal helical structure in the i,i+4 ( 1 , 2 ), and i,i+3 ( 5 ) constrained peptides. Cellular-uptake studies were conducted with three of the macrocycles. Subsequent confocal imaging revealed punctate fluorescence within the cytosol indicative of peptides trapped in endocytic vesicles. These studies demonstrate that dibromobimane is an effective tool for defining secondary structure within short peptides, whilst simultaneously introducing a fluorescent tag suitable for common cell-based experiments.     

Synthesis and properties of an insoluble chitosan resin modified by azamacrocycle copper(II) complex for protein hydrolysis

We reported the synthesis process and the proteolytic ability of a novel crosslinked chitosan resin modified by azamacrocycle copper complex (CMCR). Characterization of CMCR showed that the Cu(II)Cyclen complex had successfully bound to the resin matrix covalently. Its proteolytic activity toward peptide bonds was evaluated by investigating the hydrolysis of bovine serum albumin in heterogeneous media. The maximum‐observed pseudo‐first‐order rate constant (k_obs) was achieved under 60°C, pH 6.0, where protein was hydrolyzed into smaller peptides and amino acids confirmed by SDS–PAGE and ninhydrin reaction. The results indicated that the hydrolyzing efficiency delivered by CMCR was 4 × 10⁵ times higher than that of spontaneous hydrolysis. CMCR also featured a desirable stability for chelating Cu²⁺ ions, which paved the way for its feasible recovery process and good reusability. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci, 2013     

Novel Peptide‐Heterocycle Hybrids: Synthesis and Preliminary Studies on Calpain Inhibition

New peptidic compounds, having peptide chains linked to bi‐ and tricyclic heterocycles (peptide‐heterocycle hybrids), have been synthesized. The heterocyclic components are derivatives of partially reduced isoquinoline and pyrido[1,2‐b]isoquinoline bearing α,β‐unsaturated carbonyl functionalities. The heterocyclic compounds have been used as acylating agents in coupling reactions with short N‐unprotected peptides. Based on our interest on potential calpain inhibitors, we have used short (2–4 amino acids) peptides with hydrophobic amino acids of the two enantiomeric series. We report preliminary studies on the inhibition of calpain, with some compounds having IC₅₀ values in the nanomolar range.