Macrocyclic Control in Helix Mimetics - Chemical Reviews (ACS

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Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

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Macrocyclic Control in Helix Mimetics Danielle A. Guarracino,* Jacob A. Riordan, Gianna M. Barreto, Alexis L. Oldfield, Christopher M. Kouba, and Desiree Agrinsoni

Chem. Rev. Downloaded from pubs.acs.org by BETHEL UNIV on 05/02/19. For personal use only.

Department of Chemistry, The College of New Jersey, Ewing, New Jersey 08628, United States ABSTRACT: The α-helix is the most commonly found natural secondary structure in proteins and is intrinsic to many protein−protein interactions involved in important biological functions. Novel peptides designed to mimic helices found in nature employ a variety of methods to control their structure. These approaches are significant due to potential applications in developing new therapeutic agents and materials. Over the years, many strategies have emerged to influence, initiate, and propagate helical content in short, synthetic peptides. Early innovations used the natural macrocycle tether of disulfide bond formation, metal-mediated or lactam group addition as a means to prompt helical formation. These examples have been applied to a host of peptides as inhibitors toward relevant diseases including cancer, viral and bacterial infection. In the most recent decades, hydrocarbon bridges to “staple” peptides across side chains or hydrogen bond surrogates in the backbone of peptides have been effective in producing biologically functional, helical peptidomimetics with non-natural elements, increased protease resistance and potency in vitro and in vivo. Modern methods expand and elaborate these, with applications of functional peptides from both synthetic and recombinant origins. Overall, efforts persist using these strategies to create peptides with great biological potential and a better understanding of the control of helical structure in protein folding.

CONTENTS 1. Introduction 2. Natural Cycles and Disulfide-Constrained Mimetics 2.1. Position of the Disulfide Bond 2.2. Substitutions and Derivatives 2.3. Stability in the Context of a Functional Protein 2.4. Supporting Applications of Inhibitors 3. Traditional Macrocycle Helix Stabilization 3.1. Metal-Mediated Bridges 3.1.1. Using Metal Ions to Control Helicity in Multi-Helical Complexes and CoiledCoils 3.1.2. Metal Mediation in Short Peptide Helices 3.2. Lactam Bridged Helices 3.2.1. Synthesis of Lactam Bridged Peptides 3.2.2. Traditional Use of Lactam Bridges in Constraining Helicity 3.2.3. Unique Structural Considerations with Lactam Bridges 3.2.4. Lactam Bridged Peptide Libraries 3.2.5. Lactam Bridged Peptides in an Array of Biological Applications 4. Hydrocarbon Bridged Side-Chain Crosslinks “Staple” Helicity in Mimetics 4.1. Background and Synthesis 4.2. Initial Applications of Hydrocarbon Bridged Peptides 4.3. Hydrocarbon Staples in Anticancer Applications © XXXX American Chemical Society

4.4. Applications of Hydrocarbon Staples in Other Biologically Relevant Systems 4.5. Uses of Hydrocarbon Stapled Peptides in Gene Expression and Stem Cells 5. Hydrogen Bond Surrogates Constrain the Peptide Backbone into an Organized Helix 5.1. Background about Hydrogen Bond Surrogates (HBS) 5.2. Synthesis of Peptides Constrained by Hydrogen Bond Surrogates 5.3. Structural Characterizations of HBS α-Helical Peptides 5.4. Use of HBS Helical Constraints in Key Biological Applications 5.5. Current Work and the Future of HBS Constrained α-Helical Peptides 6. Constrained Peptides with Non-Natural Backbones 6.1. Synthetic Strategies and Characterization of Stapled β-Peptides 6.2. Biological Functionality of Stapled β-Peptides 7. Some Additional Methods for Constraining Peptide Helices 7.1. Alkanediyl Tethers

B C C D D E E F

F H I I I J K L

Q R S S T U V W X X Y Z Z

M M Special Issue: Macrocycles

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Received: October 9, 2018

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DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 7.2. Photoinduced 1,3-Dipolar Cylcoaddition Stapling 7.3. Triazole-Stapled Peptidomimetics from “Click” Chemistry 7.4. Thiol-Ene Coupling Approach to Helical Tethers 7.5. Other Two-Component Stapling Techniques 7.5.1. Photoswitchable Two-Component Azobenzene Staples 7.5.2. “Double-Click” Stapling and Its Applications 8. toward Synthetic Helical Proteins 9. Concluding Remarks Author Information Corresponding Author ORCID Author Contributions Notes Biographies Acknowledgments References

Review

To elucidate the role of the α-helix, methods have been developed to stabilize and mimic the structure to emulate and control specific helical functions. This review will look at the development of helix mimetic peptides that contain various forms of macrocyclic constraints to preorganize and regulate the helical structure obtained. In peptidomimetics, α-helical conformations are obtained by conformational rigidity and often endow proteolytic stability to otherwise vulnerable molecules with protein-like functionality.8 Short peptide stretches outside the context of a greater protein rarely retain any conformation even if the sequence was contributing to a helix in a PPI. Peptides are therefore susceptible to proteolytic degradation and can lose their ability to target a protein surface, which limits their abilities in drug discovery or biochemical mechanism elucidation.9 Much work in the field of peptidomimetics has focused on stabilizing the α-helix or mimicking the domain with non-natural scaffolds.10 There are three general categories of approach for initializing helical formation: helical foldamers, helical surface mimetics, and helix stabilization.8,9 Helical foldamers, such as β-peptides and peptoids, use unnatural backbones and amino acid analogs and derivatives to create polymers that adopt conformations similar to the αhelix in structure.11 Surface mimetics use small molecule scaffolds that display functional groups reminiscent of those projected by natural amino acids in the i, i+4, and i+7 positions along the α-helix they are mimicking.12 Helix stabilization methods use covalent and noncovalent techniques to impose a helical structure along a stretch of amino acids in a peptide sequence. Once the peptide is formed, these methods are used to maintain the stability of its structure. This review focuses on this latter kind of helical control, the use of covalent macrocycles that are prevalent in controlling such conformations and many of these methods rely on cross-links that preorganize helical orientations. The helix−coil transition theory for peptides describes the energetic cost to organize three consecutive amino acids residues into a helical conformation and the limits of short, unstructured peptides to spontaneously assume secondary structure.13 Peptides of ten or fewer amino acid residues generally have a low helical nucleation probability. However, propagation of helix formation along the chain after initial nucleation is actually much faster, as determined in theoretical folding models.2 Studies of helical formation attempt to explain the ability of short peptides to fold into helices, the stability of the fold once initiated, and the propensity of specific sequences for propagating helical structure in an already initiated helix. Such studies are applicable for understanding the relationship between protein folding and function. In this review, we will discuss methods of macrocyclization in peptides to promote structure, preorganizing a helix to overcome the nucleation barrier. Such techniques allow for facile nucleation, lowering the energy barrier for the origination of helicity. Macrocycles in helix mimetics explore potential applications in stability, binding, conformation analysis, cell permeability, and therapeutics. We will begin by discussing natural disulfide bond formation in peptides and how it can be used to tether specific helical fragments. Traditional laboratory methods for cyclizing peptides to facilitate helical structure such as lactam ring and metal ionmediated helical constraints, and their applications, will be explained. Following this, two methods that use hydrocarbonstapling techniques will be described; those that constrain the side-chains of key positions on a peptide into a ring to nucleate helicity, and those that instead use a backbone “staple” as a

AA AA AB AC AC AC AC AD AE AE AE AE AE AE AE AE

1. INTRODUCTION The α-helix, first defined by Pauling, is the most abundant secondary structure in natural proteins and its prevalence is fundamental to understanding the process of protein folding.1,2 Defined by its generally right handed structure, the α-helix has approximately 3.6 amino acid residues per turn, with a vertical pitch of 5.4 Å. The key interaction is the hydrogen bond formed between the backbone carbonyl oxygens in the “ith” position and amide hydrogens i+4 residues away. This hydrogen bonding pattern contributes to a macrodipole from the positioning of the carbonyl oxygens, pointing C-terminally across the stretch of helix.3 The α-helix is influential in the function of a majority of proteins found in the cell and while helices in the protein core help stabilize tertiary structures, helices exposed on the surface are commonly found at the interface between proteins and bear intrinsic bioactive residues.4 When mis-regulated, helixpromoted interactions can have deleterious consequences and are implicated in a host of diseases. Understanding protein− protein interactions (PPIs) is paramount due to their involvement in so many key biological processes (e.g., gene expression, cell growth, nutrient uptake, intercellular communication, and apoptosis).5 Despite containing a finite set of building blocks, proteins are diverse and complex macromolecules with folded, three-dimensional structures that aid in their function.6 PPIs are problematic targets for therapeutic development as proteins tend to interact through large, shallow interfaces which are not amenable to distinct small molecule binding.4,7 PPIs mediated by α-helices have a more clearly defined interface and are open to ligand discovery methods.7 Efforts to understand protein folding, to develop nextgeneration pharmaceuticals, to comprehend the mechanisms of disease, and the function of helices in biological processes necessitate the study of α-helices and the control of their structures. New strategies using peptide-based compounds that resemble part of one PPI partner, displaying a subset of amino acids that contribute to the binding interface, can greatly impact existing PPIs and their roles. Studies with peptides bearing constraints that help them fold into helices have resulted in compounds with a well-defined chemical functionality.7 B

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Disulfide bonds tethering helices that are found in nature. A. Representation of bee venom peptide apamin indicating the two crossed disulfide bonds (PDB: 3IUX).17 B. Peptidomimetic of estrogen receptor modulator 1 bound to estrogen receptor a, indicating the i, i+3 disulfidelinked bridge (PDB: 1PCG).20 C. Peptide EcAMP1 from kernels of barnyard grass Echinochloacrus-galli seeds, indicating the hairpin-like connection of α-helices as well as the pair of disulfide bonds connecting the helices (PDB: 2L2R).27 Figure created by author from data in refs 17, 20, and 27.

structure, many early studies utilized the bee venom peptide apamin as a model system (Figure 1A).17 Apamin serves as an excellent scaffold for examining disulfide bond formation as a theoretical method of stabilization. As a short peptide neurotoxin, Apamin can cross the blood brain barrier and contains helical structure. It bears a crossed disulfide pattern comprised of two disulfide bonds linking the C-terminal helix to an N terminal loop. Studies have found that Apamin undergoes only small conformational changes on exposure to 6 M guanidinium chloride and retains its helical structure at temperatures as high as 70 °C and pH values as low as 2.18 Therefore, the use of Apamin as a general scaffold is only limited by the necessity of both cysteine pairs. Replacement of either pair with a pair of alanines showed a marked decrease in helicity when evaluated by circular dichroism (CD). Additionally, Apamin with an i to i+7 disulfide tether was more effective at inducing helicity than an i to i+3 tether.17 This positioning of the disulfide proved paramount to understanding disulfide control of helicity.

hydrogen bond surrogate to initiate helical structure. The application of hydrocarbon-stapling in non-natural foldamers to maintain helical-type structure is also explored. Other methods gaining interest include alkanediyl tethers, photoinduced 1,3dipolar cycloaddition stapling, 1,3 dipolar cycloaddition (“click chemistry”) tethering a macrocycle across a helix using a 1,4 substituted 1,2,3-triazole between side chains, and the use of a thiol−ene connection between cysteine side chains to stabilize helices. Several two-component stapling techniques are described as they highlight a new direction in the field that is amenable for expressed or natural peptide modifications. Finally, applications of these techniques in developing fully synthetic, helical proteins highlights an exciting new pathway for macrocyclic helical constraints. We conclude with a brief discussion of such future directions, visiting the challenges each of the various techniques still possess.

2. NATURAL CYCLES AND DISULFIDE-CONSTRAINED MIMETICS Disulfide bonds are nature’s way of tethering different parts of a protein. They are formed upon the oxidation of proximal thiol groups of cysteine amino acid residues resulting in a covalent bond. Disulfide bond formation plays an important role in protein folding and the stability of tertiary and quaternary structures, usually in proteins found outside of the cytosol’s reducing environment.14 In protein folding, the improvement of protein stability by introduction of a disulfide bond is intrinsically linked to its location in the protein; the most effective disulfide bonds are those introduced at the hydrophobic core without any disruption in protein structure.15 Disulfide bonds stabilize a folded protein by lowering the entropy of the unfolded protein and predisposing it toward folding. In fact, disulfide coupled folding is highly cooperative; polypeptides in isolation are often unstructured but adopt stable secondary and tertiary structures upon forming one or many linking disulfide bonds across chains. The rate at which disulfide bonds can form is dependent on the proximity of the cysteine residues and the probability of their reaction relies on the necessity of the secondary, tertiary, and quaternary structures that result.16 Early synthesis of isolated α-helices utilized nature’s disulfide bond formation. To properly characterize the role of disulfide bonds in successfully promoting and sustaining α-helical

2.1. Position of the Disulfide Bond

In general, many factors contribute to the stability of a disulfide bond and its ability to restrict conformational freedom in stabilizing an α-helix. One of the initial points of interest in earlier studies involved the relative position of cysteine residues with respect to one another. If the number of intervening amino acids is large (n ≥ 4), there is considerable conformational flexibility possible within the loop. However, if the number of amino acids separating the two Cys residues is small (n < 4), the cyclic structures formed will have significantly less conformational freedom and well-defined geometries may be obtained for the cyclic disulfide segments.19 Despite this, many studies have found effective use of disulfide bonds between the i and i+4 or i and i+7 positions, while more rarely in the i and i+3 position. One such experiment tested the capability of short peptide drug candidates to inhibit a protein−protein interaction between the estrogen receptor and a coactivator. A particular helical region of the coactivator binds to the nuclear receptor, and inhibiting this interaction is key in examining the role of the estrogen receptor in breast cancer. Evidence suggests that nonapeptides containing a disulfide bridge are able to adopt a helical conformation when binding to the groove within estrogen receptor α (Nuclear receptor subfamily 3, group A, C

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

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ranging from 0 to 60 °C was measured for several eight residue peptides bearing a single disulfide bridge between residues i and i +7.22 In addition, it was discovered that the incorporation of D and L enantiomers of an acetaminomethyl cysteine (Acm(Cys) derivative, from mercaptohexanoic acid (Figure 2) at the same

member 1) despite a lack of structure in water. When presented with helical-inducing solvent trifluoroethanol (TFE), the peptide was able to adopt a helical conformation. Compared to other bridges, an i, i+3 disulfide-linked analog, H-Lys-cyclo(DCys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln−NH2 (peptidomimetic estrogen receptor modulator 1), bound to estrogen receptor α with a Ki of 25 nM, significantly better than the i, i+4 bridged cyclic amide (Figure 1B). The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy.20 In another study, an interchain disulfide bond was introduced to a de novo two stranded α-helical coil at varying positions across a peptide.21 Structure and stability was determined by circular dichroism in the absence and presence of guanidine hydrochloride. In general, disulfide inclusion effected the total helicity. It was concluded that the disulfide bond should be introduced in a hydrophobic region to improve stability of the fold while minimizing disruptions to existing structure. Also, greater perturbances in helicity occurred when the disulfide bond was placed at nonterminal positions and closer to the center of the coil.21 Changes to cysteine, such as using derivatives or unnatural forms of the amino acid, can be linked to specific patterns in disulfide bond formation and can affect overall helicity.

Figure 2. S-Acetamidomethyl-protecting group on cysteine (ACM).22 Figure created by author from data in ref 22.

positions of an α-helix, respectively, could lead to intramolecular disulfide bond formation with little perturbation of helix conformation.22 Further in the study, peptides containing different enantiomers showed a large increase in α-helicity upon oxidation, including the transformation of a peptide that had negligible helicity in the Acm-derivatized form. Moreover, the fact that two 16 amino acid peptides studied showed high helical content at 0 °C in their oxidized forms suggested that the disulfide bridge not only locked two turns of the helix but also efficiently propagated the helicity to neighboring residues.22 In another example, a novel 18 residue peptide named NTH18 with the sequence Ac-D-L-Q-C-A−I−K-C-R-A-G-E-P-AQ−C−N-C-NH 2 , using Apamin as a framework, was developed.18 Here, the N terminus was stabilized by two disulfide bonds crossed to the C terminus. When the secondary structure was compared to Apamin, a similar fold was adopted by NTH-18; however, there was an inversion in the arrangement of residues relative to Apamin, while the crossed disulfide structure was properly obtained. Using computer modeling, the simple reversal was deemed to have an unstable helix, so amino acid replacements were made without adjusting the position of the cysteine residues. The resultant peptide still showed high stability among a wide pH-range and high temperatures, similar to that of Apamin, concluding that the stabilization of α-helices can even be promoted utilizing a crossed disulfide pattern.18 These experiments on short peptide mimetics paved the way for work involving stabilizing helices in a larger protein context.

2.2. Substitutions and Derivatives

Molecular modeling work has shown that incorporation of a mixture of D and L enantiomeric forms of cysteine into disulfide positions has an effect on overall helicity.22 Disulfides with Dcysteine in the i position but the L enantiomer at the i+7 position demonstrated a loss of helicity surrounding the D-amino acid, agreeing with the observation that D-amino acids destabilize helical peptides.17 D and L enantiomers at specific positions in the formation of disulfide bonds can actually improve helicity, as well. In an experiment which examined the binding of small peptides to the estrogen receptor α, mimetics with amide linkages were compared to disulfide bonds to observe which subsequently cyclized peptide had better binding ability. The results showed that compounds with either linkage could adopt a helical-like shape, although it appeared that the better fit was retained by the disulfide bridge(H-Lys-D-Cys-Ile-Leu-Cys-ArgLeu-Leu-Gln-NH2), especially with the combination of D-Cys and L-Cys.20 This finding was consistent with prior modeling and structural reports that support the D,L cysteine motif as the better helical mimetic. From previous research, however, it has been shown that while the D-Cys, L-Cys bridge exhibits helical character it does not form the ideal α-helix. When an i, i+3 link was examined in A−V-S-E-C-Q-L−C-H-D-K-G-NH2, with differing chirality for the first cysteine in position 5, the results for the D,L motif showed helical structure present for amino acids from position 2 through 9. This helical structure contained abnormal φ and ψ angles which was caused by the presence of DCys; however, the short stretch of amino acids did have the ability to initiate the formation of a type of helix.23 It was evident, however, that despite nucleation actual helical presence was influenced by the environment in which the peptide was folding, depending greatly on the solvent used (e.g., aqueous or micellar). Therefore, the use of D with L substitution of Cys residues can improve helicity; however, the extent by which this works is context dependent. The molecule Apamin inspired the development of related peptides with high helicity. In the earliest work using Apamin as a model, the stability of helical content across temperatures

2.3. Stability in the Context of a Functional Protein

As discussed, helices that have been induced by the incorporation of disulfide bonds have shown folded stability under varying temperatures and pH levels. To test this in the context of a larger protein, the enzyme Trichoderma reesei endo1,4-xylanase II (XYNII; family 11) was specifically mutated to incorporate a disulfide bond at the N-terminus of an α-helical portion (S110C−N154C). The subsequent enzyme showed improved thermal stability from 1 min half-life to a 14 min halflife at 65 °C. While not native to this specific protein’s structure, the disulfide linkage is commonly found among other stable xylanases.24 The presence of the disulfide coupled to other weakly stabilizing mutations was shown to have a synergistic effect on the enzyme stability. Mutations that incorporated a disulfide bridge by replacing a serine and asparagine, respectively, to cysteine were combined with a substitution of a histidine or tyrosine at a key glutamine at the C-terminus of the α-helix. These changes increased the half-life of the enzyme at 65 °C from 14 to 63 min, which, overall, showed tolerance of a 10− 20 °C higher temperature than the wild-type enzyme, indicating a retention of structure for longer than expected.24 However, a decrease in enzyme activity was seen at varying pH levels above D

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

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Table 1. Correlations between Helicity and Activity for HIV-126 activity (IC50, μM)

% helictiy name

sequence

0% TFE

50% TFE

CCR5 cellsb

CXCR4 cellsb

C28Aa C28Ba C28B1c Ac-C28B1c

WEREIDNYTDYIYDLLEKSQTQQEKNEK-NH2 EIDNYTDYIYDLLEKSQTQQEKNEKELL-NH2 EIDdCYTCYIYDLLEKSQTQQEKNEKELL-NH2 Ac-EIDdCYTCYIYDLLEKSQTQQEKNEKELL-NH2

16.8 13.9 16.4 18.9

54.2 70.8 75.3 70.6

8.299 ± 1.442 2.472 ± 0.452 0.615 ± 0.0973 0.0924 ± 0.0109

0.0541 ± 0.0072

a

Peptides are templates derived from HIV-1 C-peptide bRepresents two different cell strains for the HIV-1 infectivity assay. cdC and C indicate presence of disulfide bond.

and below 6. Having a strong α-helical structure may have increased the rigidity of the enzyme enough to disturb the active site, therefore subsequent catalytic activity. Low and high pH conformational changes may be needed to adjust the structure for its subsequent function.24 While the stabilizing effects of disulfide bonds appear to be stronger, there is evidence that the rigidity of the bridge in an α-helix in the context of B. circulans xylanase can work both for and against stabilization. Using disulfide bonds to initiate helicity comes with a variety of results, from increased structure to better affinity for a protein target, depending on the environment of the helix. This method and the information gleaned from these studies can be utilized in the development of helical peptides as inhibitors.

structurally diverse, and generally positively charged. A novel peptide, EcAMP1, isolated from kernels of barnyard grass Echinochloacrus galli seeds, forms a disulfide stabilized α-helical hairpin structure in aqueous solution which is unique in the realm of naturally occurring antimicrobial peptides.27 This 37 amino acid peptide with sequence GSGRGSCRSQCMRRHEDEPWRVQECVSQCRRRRGGGD had an unusual cysteine spacing with a pair of disulfide bonds connecting C7−C29 and C11−C25. The structure (Figure 1C), as determined by COSY, NOESY, and TOCSY 2D NMR studies, involved a hairpin-like connection of α-helices, which are also linked by a pair of disulfide bonds connecting each helix.27 EcAMP1 showed strong antifungal action toward members of the Fusarium genus, with EC50 values of 1−10 μM (F. solani and F. graminearum with ∼4 μM).27 Fluorescently labeled peptide was introduced to specific fungal spores to examine the mode of action in confocal laser scanning microscopy experiments. This antimicrobial peptide proved to have multiple targets. It appeared that first the peptide bound one of the abundant components (could be a lipid or glycan, depending if the plasma membrane or cell wall, respectively) of the F. solani surface, accumulating there. In phase two, the peptide was internalized and taken up by the fungi to accrue in the vesicles, which may have led to the peptide finding an intramolecular target. This was the first report of an antimicrobial with a mode of action this complex, as most usually only disrupt the microbial membrane.27 The unique disulfide bonded peptide structure appears to be intrinsically linked to this novel activity. The EcAMP1 helical hairpin can serve as a scaffold for future studies of novel antimicrobial peptide design. Understanding how disulfide bonds are used in nature and their application in the development of peptide inhibitors, and in the context of larger proteins, originated the field of macrocyclic control of helicity. However, disulfides contain intrinsic vulnerability due to the redox nature of their bond formation. Thus, to develop strongly stable tethers that induce helicity but have a versatility of context, other methods proved necessary.

2.4. Supporting Applications of Inhibitors

There are many examples of helices involved in important PPIs implicated in diseases. The HIV-1 gp41 protein contains a domain made up of the fusion peptide with disulfide-bonded loops across a six helix bundle.25 This bundle forms post fusion when a trimeric coiled coil of the gp41 heptad repeat (HR1) region packs against three helices in the HR2 region in an antiparallel fashion.25 This brings the viral membrane (tethered at one end of gp41) in close proximity to the host cell membrane (tethered at the other end), which then causes membrane fusion, allowing HIV virus to enter the cell. Peptide inhibitors to HIV fusion, developed over the last 20 years, have shown a correlation between inhibitory effectivity and increasing αhelical character. As there is evidence that the presence of disulfide bonds helps to stabilize and propagate α-helices throughout regions of a peptide, structure−activity relationships were explored with a designed peptide based on the HIV-1 gp41 C-peptide, which inhibits HIV-1 activity. Two 28mer peptide sequences were explored: W-E-R-E-I-D-N-Y-T-D-Y-I-Y-D-L-LE-K-S-Q-T-Q-Q-E-K-N-E-K-NH2 (C28A) and E-I-D-N-Y-TD-Y−I-Y-D-L-L-E-K-S-Q-T-Q-Q-E-K-N-E-K-E-L-L-NH 2 (C28B) with D-Cys or L-Cys substitutions at several positions with i, i+3 separation.26 They positioned the Cys residues from close proximity to the C-terminus all the way to closer to the Nterminus. The resultant peptides were examined for helical content using CD spectroscopy and for activity by monitoring the inhibition of the HIV-1/MuLV pseudotype infection in target cells. All peptides with D-Cys and L-Cys showed over 90% disulfide bond formation. When a disulfide bond was introduced near the N-terminus in C28A there was no effect on activity or helicity; however in C28B, a higher helicity and activity was detected. However, for both peptides, when a C-terminal disulfide bond was introduced, a decrease in helicity was detected which coincided with a subsequent decrease in helicity.26 The correlating results between helicity and activity for the peptides described can be seen in Table 1. In another study, disulfide bonds in helical antimicrobial peptides were examined. Antimicrobial peptides are small,

3. TRADITIONAL MACROCYCLE HELIX STABILIZATION By stabilizing helical secondary structure, synthetic peptides are capable of gaining conformational endurance, proteolytic stability, and even the protein-like functionality that is key when designing special classes of therapeutic drugs. Synthetic methods of peptide macrocyclization provide an elegant way to target biomolecular interfaces with appreciable specificity and functionality.28 Therefore, considering the importance of macrocyclic syntheses, the focus now remains on devising the most effective technique of peptide macrocyclization and its applications in creating helical peptides.28 E

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 3. Metal mediated cyclizations. A. Metal-ion mediated dimerization of peptide methyl esters, causing a cyclodimerization after the metal center first is coordinated.29,30 B. Alkali metal ions promoting macrocyclization bringing N- and C-termini together.31 C. Ag+ ions coordinating C-terminal thioesters and N-terminal amines, giving stable ring structures.32 Figure created by author from data in ref 29−31.

cytochrome cb562 (cyt cb562).33 This cytochrome is a 4-helix bundle heme containing protein. Zn(II) exhibits a large binding affinity for the di-His motifs on cyt cb562, and metal coordination controls the self-organization with little to no predisposition from specific protein−protein contacts. With rapid ligand exchange, kinetically trapped protein−protein complexes were avoided and multimeric His4-cb562 structures formed upon Zn(II) addition.33 Aggregates of cb562 helices dissolved upon changing the pH to below 6 and EDTA treatment, demonstrating that Zn-His coordination was intrinsic to protein multimerization. At high Zn levels, multiple modes of inter-protein coordination were likely, and NMR and sedimentation velocity experiments demonstrated that in the absence of the metal the only protein species present were linear monomers.33 Conversely, in the presence of Zn(II) cations, the sedimentation velocity indicated the desired 16-helix peptide. As a result of metal addition, self-organization of the macrocycle cyt cb562 was therefore highly stabilized via a helix-bundle fold (Figure 4). Further, the rigid cylindrical shape of cyt cb562 can be used as a basis to construct even larger, helix stabilized proteins. The strong binding potencies of metal ligands can further be applied to the application of metal ligand bridges in the formation of macrocyclic proteins. In the de novo design of macrocyclic peptides with α-helical structure, metal mediated bridges can provide folded structures resistant to variations in pH, temperature, ligand binding, and even covalent modification. Several considerations must be made in the design of de novo peptides exhibiting specific dimeric coiled coil structure. The heptad repeat sequence is usually designated by letters a-g. Hydrophobic residues are generally placed at the a and d positions, which face each other making a hydrophobic core of interacting helices which drives coiled-coil formation by burying the hydrophobics.34−36 The e and g positions of the heptad repeat typically participate in ionic interactions which influence the stability of coil forma-

3.1. Metal-Mediated Bridges

Metal-mediated bridges have demonstrated the ability to enclose peptides into a macrocycle, providing stability without deterring the peptide from its targeted functionality and intended use.8 Inspiration for the use of metals linking side chains and inducing macrocyclization comes from naturally occurring cyclic peptides including valinomycin and many strong ionophores. These naturally found cyclic peptides construct complexes with metallic cations in vivo. In early applications of metal-ion mediation of cyclization, symmetric cyclic tetrapeptides were constructed via metal-ion-mediated dimerization of peptide methyl esters (Figure 3A).29,30 It was imperative that the metal center first was coordinated prior to cyclodimerization, which allowed ring sizes of 12- to 18residues.30 Alkali metal ions were shown to promote macrocylization by binding carbonyl and amide groups at the Cterminus of peptides, inducing a turn structure in linear peptides, allowing N- and C-termini to be brought in close proximity (Figure 3B).31 Ag+ ions provided entropic and enthalpic activation for macrocyclization of linear peptide thioesters through coordination of C-terminal thioesters and N-terminal amines (Figure 3C).32 The Ag+ ion can facilitate an acyl-transfer, which leads to either an individual cyclization of peptides or bringing together a mixture. These examples all led to particularly stable ring structures through metal-mediated macrocyclization, and provided a firm basis for metal induction of α-helical secondary structure. 3.1.1. Using Metal Ions to Control Helicity in MultiHelical Complexes and Coiled-Coils. To engineer novel compounds that interface with protein−protein interactions, the construction of peptides with α-helical secondary structure is vital. Metal−ligand interactions have the strength and selectivity to direct peptide structural control. An example of this concept applied in a macromolecular manner, a Zn-mediated bridge was used to form a 16-helix arrangement of four copies of F

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX

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toward the folded state, removing the peptide from the unfolded/folded equilibrium.39 After this, the second metal ion binds and the final, folded structure is achieved. It seems, based on CD and NMR results, that the first metal ion binding changes the structure to increase the binding ability of the peptide to the subsequent ion, leading to cooperative folding.39 This may be a result of the first metal ion decreasing the dynamic flexibility of the folded peptide. The Gla2Nx example could be used as a means of reversibly assembling any two molecules attached to the coiled-coil chains, as a switch for some activity that needs the coiled-coil, or even a novel way to carry specific metals (i.e., those used in radioimaging) for diagnostic and/or therapeutic benefit.39 Recall that HIV-1 uses a six-helix bundle viral fusion macromolecule of intercalating heptad repeats (HRs) 1 and 2 to fuse with the host cell.25 The coiled-coil repeats of the three HR1 helices are potential targets to anti-HIV peptide-derived therapeutics, especially those that resemble helices thus forming a similar, resultant bundle that would preclude HR2 from binding. To study and screen such compounds, a model for HR1 is necessary, as the three stranded coiled-coil from gp41 is unstable when excised from the buried, intact protein.40 One such design of a stable, trimeric helical core used the metal ionbinding bidentate ligand, 2,2′-bipyridyl(bpy) attached to the Nterminus of a 31 residue peptide that contained HR1 residues from gp41.40 When treated with Fe2+ or Ni2+ the tris-bipyridyl metal complex formed, stabilizing the coiled-coil structure. When treated with Fe2+, the complex turned magenta with an absorbance maximum at 545 nm. Therefore, UV, CD, and NMR spectroscopy were used to characterize the system to recognize that the three helix bundle was formed with proper helical induction. Metal ion-binding was detected by a red shift of the 291 nm π−π* band and metal−ligand transfer band for Ni2+ or Fe2+ and CD results showed 87−90% helicity upon metal treatment.40 Additionally, fluorescence spectroscopy utilizing FRET quenching was established for a known C-peptide from the HR2 region of gp41, appended with a dansyl fluorophore on a terminal cysteine residue.40 Dansyl absorbs at 340 nm and emits at 542 nm, therefore quenching of the signal by Fe(II)-bpy would signify close proximity of the HR2-based peptide with the bpy-appended HR1 model. A Kd of 0.4 ± 0.09 μM was found between the C-peptide and Fe(II)-bpy-HR1 moiety, which was within error of characterized binding of C-peptide with other similar coiled coil model HR1 systems in the literature.40 This provided an elegant use of metal binding to initiate coiled-coil structure and its potential for screening anti-HIV drugs using absorbance and quenching.

Figure 4. Zn(II)-mediated 16-helical arrangement of cytochrome cb562, four-helix heme containing protein, indicating the histidine and aspartate coordination of the metal ion (PDB: 2QLA).33 Figure created by the author from data in ref 33.

tion.21,37,38 By specifically orienting the g of one helix with the e of another, designated e’, a stable interaction is achieved and can be propagated five residues down the chain (i, i’+5). However, eg’ (i to i’+2) interactions are often blocked by large hydrophobic residues in the d position. Using a metal ion to induce the coiledcoil folding transition by incorporating high-affinity metalbinding sites at the e and g positions allows negatively charged side chains to repel one another unless involved in the preferred metal-bound, folded structure. This came to fruition in a disulfide-bridged two-stranded coiled-coil Gla2Nx protein, which incorporated γ-carboxyglutamate (Gla) side chains in the key e and g positions (Figure 5).39 This peptide was almost completely unfolded at pH 7 and 20 °C but when treated with a small amount of lanthanide ion, it became highly helical (∼60% in a TFE containing solution by circular dichroism). The electronic configuration of the metal ion used significantly affected the interaction between the ligand residues and the metal. Yb3+ and La3+ have no electrons in the (n-1) d orbitals and are considered comparable with Ca2+ in this respect, lending toward more ionic versus covalent character.39 However, Gla2Nx has a much higher affinity for the trivalent lanthanides over calcium, which is thought to be from the higher cation charge. Gla2Nx appears to model a two-state process of folding. When the first metal ion is added, the peptide binds and shifts

Figure 5. Depiction of two-stranded coiled-coil Gla2Nx protein with the x-carboxyglutamate residues in e and g positions which coordinate metal ions when introduced, inducing helicity and coiled-coil pairing.39 Figure created by author from data in ref 39. G

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Figure 6. Solid phase reaction scheme representations showing original and more recent use of lactams to enclose peptide macrocylces. A. General scheme for original methods of solid-phase synthesis of lactam-bridge peptides using Boc and Fmoc orthogonal protection and OFm-ester for the lactam components.46 B. General scheme for more recent method of lactam-bridged peptide formation using Fmoc/tert-butyl methods for solid phase synthesis and Mtt and OPip protection on the lactam components.46 Figure created by author from data in ref 46.

Unstructured peptides can be converted to 3- and 4-turn αhelices with the use of palladium, and the helicity is well-defined throughout the peptide, including the N- and C-termini. The stable macrocycle, [Pd(en)(HxxxH)]2+ where x represented other residues than histidine in the sequence, had regioselective coordination and the close proximity of the i, and i+4 histidines on a helical turn allow for chelation of cis-positions.41 A suggested limitation was that these peptides were more capable of maintaining their helical structures in aprotic rather than protic solvents. This may be indicative of the location of the helices, as HxxxH motifs are usually buried on the hydrophobic interior of proteins. Overall, it was implied that metal ions can help “template” protein folding, and that once helices become longer, or further effects such as hydrophobic interactions occur upon folding, the metal is no longer required to drive thermodynamic stability, and are displaced. Overall, transition metals Ni2+, Zn2+, Cd2+, and Cu2+ can enhance helicity of small peptides in aqueous solutions by forming stable complexes with histidines, cysteines and even amino-diacetic acid residues incorporated in the chains.42−44 These do so by forming macrocyclic complexes between the

These examples illustrate the use of metal ions in controlling helicity in larger protein systems. However, for the direct development of small, helical peptide therapeutics, or other agonists and antagonists, the method needs applicability in shorter sequences. 3.1.2. Metal Mediation in Short Peptide Helices. Short peptides comprised of less than 15 residues cannot easily adopt α-helical structure without preorganization, therefore metals can be used to create bridges that nucleate secondary structure in such limited sequences. As mentioned previously, stabilizing structure in small helices can have many uses, including investigations in protein folding, creation of artificial proteins, and aiding in the design of inhibitors or mimics to protein function.41 Small peptides containing the Zn-binding region of the protein thermolysin, with 4−6 histidine residues along one side of a helix, made use of the ion-binding propensity of the imidazole ring. These peptides were reacted with Pd(en)(ONO2)2 and showed evidence through 2D-NMR, CD, and NMR analysis of molecular flexibility in solution (NAMFIS) of the resultant helicity.41 Coordination of metal ions at the i, i+4 histidine residues led to significant helicity in water and DMF. H

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metal and multiple side chains. Remarkably, the use of alkali metals (such as Li+, Na+, K+, Rb+, and Cs+) in association with short peptides can transform random coils into rigid helices, without the use of metal-chelating side chains on the peptide. With “oxyphilic” metal ions, the carboxy terminus can become “capped” which then allows favorable interaction between the helix macrodipole and the metal ion, leading to helical nucleation.45 Using high-resolution ion mobility measurements and molecular dynamics simulations, it was shown that chains of alanine that are 6−20 residues were able to form helices upon treatment with alkali metals in a chain-length-dependent manner. For more than 12 residues, peptides showed helical propagation upon treatment with the metal ions from the molten globule state, with Li+ as an exception as the random state was preferred, possibly due to better self-solvation of its higher charge density.45 Therefore, metal mediation of helicity can play an active role both in the formation of macrocyclic interactions across side chain residues and also in stabilizing the backbone. Through these interactions, and the use of metal ionic bonding, secondary structure is stabilized across a range of helices. Challenges remain, however, in that metals ion oxidation state, concentration, and solubility (given many helices are in hydrophobic environments) can prove tricky when balancing the necessity of stable α-helices over a range of conditions. Other methods using more traditional covalent tethering need to be explored.

cycles with up to 34-atom rings in monocyclic and dicyclic peptide products.46 Peptide chains bearing more than one lactam ring often result in low yields when the second cyclization is considered. The synthesis of dicyclic lactam-bridged peptides by strictly solid-phase methods is less reliable than for the monocyclic case.46 In recent approaches, a Rink acid resin can be used, allowing for the synthesis of a protected peptide by Fmoc/ tert-butyl methods, with incorporation of acid-labile 4methyltrityl (Mtt) lysine protection and a 2-phenylpropyl ester (OPip) group on a key aspartate. After cleavage from the resin, two separate solution-phase cyclizations can occur to provide a bicyclic lactam peptide (Figure 6B).46 These methods can be applied to a range of short peptides with constrained secondary structure. 3.2.2. Traditional Use of Lactam Bridges in Constraining Helicity. The most common peptide analog connects a lactam bridge across i and i+4 amino acids residues. Incorporation of lactam bridges increases the overall helical conformation of the peptide and selective introduction of side chain cross-links can enhance and stabilize the secondary structure of even short peptides. The use of a lactam bridge brings definitive advantages to short peptide structures, including the ease of synthesis, resistance to proteolysis, and insensitivity to the redox conditions of biological media, to which disulfide bonds are vulnerable.47 In early studies, helical peptides with lactam bridges were evaluated with NMR and CD spectroscopy, showing specific cross-links between aspartate and lysine in the i and i+3 positions of a peptide with sequence H2N-GDTLKEQVQEELLSEQVKDELKAG-COOH.47 This peptide showed evidence of two short α-helices connected by a nonhelical segment and a turn and was significant for its ability to mimic a helical domain of Apolipoprotein E. Given its helical form, the peptide demonstrated specific binding of low-density lipoprotein (LDL) to cells via a receptor other than the LDLreceptor.47 When the spacing of the cross-link was changed to i, i +4, the peptide contained helicity along its entire length but lost biological activity. It seemed the central “interruption” of structural order in the helix was necessary for biological activity, and an i, i+4 lactam bridge with a possible end-capping effect from a strategically placed serine recovered activity.47 This showed that lactam cross-linked peptides can adopt bioactive conformations. In other early work, lactams between aspartate and lysine in a cyclic human growth hormone releasing factor (GRF residues 1−29) analog were found to propagate a long α-helical segment that was confirmed in aqueous solution, using CD as well as molecular dynamics calculations based on NOE-distance constraints from 2D NMR.48 The physical constraint increased helical conformation and also potency of the peptide.48,49 In more detail, the separation of glutamate and lysine based lactam bridges spaced i and i+4 apart increased helical content of synthetic amphiphathic peptides of the sequence Ac-EIEALKKEIEALKK-NH2.49 Inserting two lactam bridges at both the N and C-termini brought the peptide to a highly helical conformation which greatly exceeded that of an unconstrained counterpart.49 The peptides contained a 3,4 repeat of isoleucine and leucine that is often found in coiled-coils and the possibility of peptide−peptide associations was uncovered by concentration dependent molar ellipticity as observed by CD. It appeared that a lactam-containing peptide associated as a dimer, as per size exclusion chromatography, with a length as low as 14 residues. Monomeric peptides showed high helical content with

3.2. Lactam Bridged Helices

In another method for stabilizing α-helical structure using macrocycles, cyclic amides can form between the amine functionality on a lysine residue with the carboxy of a glutamate or aspartate. As amide bonds are chemically inert to most natural conditions, the resultant “lactam” can be used to stabilize helical conformations in peptides and substitute for disulfide bonds which are subject to exchange, even at neutral pH. Using a lactam as a mild conformational constraint often links amino acid residues at positions in the peptide chain with 2−6 intervening unconstrained residues.46 This locks the α-helical structure in its conformation and can be applied to impose secondary structure on many lengths of peptide used for a variety of functions. 3.2.1. Synthesis of Lactam Bridged Peptides. Introducing a lactam ring into a peptide presents an interesting synthetic challenge. A wide range of protecting groups for amines and carboxylic acids, which are either highly selectable or have orthogonal cleavage properties, allow for facile preparation of macrocyclic peptides with lactam bridge constraints. A solutionphase approach to synthesis appears most successful due to the higher control over the synthetic process as each step can be directly monitored for its progress.46 For solid-phase synthesis, which is preferred for most modern peptide work, 9-fluorenyl methyoxycarbonyl (Fmoc) methods can be used with standard cleavage of protecting groups not participating in cyclization, and the solid support can be removed using trifluoroacetic acid. t-butoxycarbonyl (Boc) and benzyl chemistry can be used for side-chain protection, with Fmoc-amine and OFm-ester protecting groups on the specific side-chains that form the lactam bridge (Figure 6A). Coupling of the bridge uses benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and other phosphonium-ion coupling agents to increase yields and decrease carcinogenic side products. Successful linkage of amine and carboxylic acid groups on side chains with 3−6 intervening residues created macroI

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Table 2. Lactam Constrained Peptides, Helicity, Antimicrobial Activity, and Bacterial Permeability51 % helictiy

name

sequencea

490lac 491lac 492lac 493lac

KWKSFIKNLEKVLKPGGLLSNIVTSL KWKSFIKNLEKVLKKGPILANLVSIV KWKEFIKKLTTAVKKVLTTGLPALIS KWKKFIKELQKVLAPGGLLSNIVTSL

MIC (mg/mL)

0% TFE 50% TFE P. aeruginosab 6 37 66 10

46 92 93 45

6.2 50 50 12.5

E. colib 1.5 12.5 3.1 1.5

permeability

S. C. epidermidis albicans 12.5 >100 50 12.5

>200 >100 >100 >100

outer membrane I50 (μg/mL)

inner membrane ONPG (units)c

0.6 5.6 4.0 3.0

2.5 1.9 3.7 2.4

a Bold residues indicate the position of the lactam bridge. bWild type strains. c1 unit of activity is equal to 0.7 mmol ONPG (ortho nitrophenyl galactoside) hydrolyzed per min per mg cells.

least reduction in activity.51 In the one case where the lactam bridge did increase efficacy, it had reduced α-helicity in aqueous and hydrophobic media, despite the lactam ring. However, it was shown that the lactam bridged peptides had a greater ability to permeabilize the cytoplasmic membranes of bacterial cells when compared to the other peptides (Table 2).51 It seems that what is called the “helican parameter”, which roughly correlates to a low helicity in aqueous solution but ability to adopt α-helicity in a membrane-like environment, correlates with antibacterial activity.51 Cyclic analogs of neuropeptide Y (NPY), with either a free or acetylated N-terminus and the first nine residues removed, were developed containing a single Lys-Asp lactam bridge.52 Linear and cyclic versions were compared via CD studies, which indicated self-association and oligomerization among the peptides. Monomeric linear peptides showed a helical preference for those without the truncation and N-terminal cap, indicating that the first nine residues were involved in the pancreatic polypeptide helical fold.52 However, the lactam bridge in the capped and truncated peptide stabilized helicity to 51% and had little stabilizing effects on the free N-terminal peptide (21% helicity), indicating that helicity from lactam bridging may work differently than helical control in nonbridged peptides. When used in brain receptor binding assays, the cyclic and linear capped and truncated peptides were equipotent with IC50 values in the low nanomolar range, with the cyclic version much more potent in a rat vas deferens assay.52 Overall, the studies showed that including the Lys-Asp lactam bridge did foster helicity, especially in peptides that otherwise suffered a loss of helicity if kept linear, and aided in their biological activity. 3.2.3. Unique Structural Considerations with Lactam Bridges. Constraining peptides with derivatives of lactam bridges has contributed more information to the field of helical secondary structure control. The use of para-substituted amino acid derivatives of benzene was used to link pairs of side chains in the i, i+7 position (3 and 10 on a model peptide) in an amphiphilic, 14-residue peptide and directly compared to a peptide containing lactam constraints across Lys-Asp pairs in the i, i+4 positions.53 The most effective bridge used a 4(aminomethyl)phenylacetic acid residue (AMPA) linked by amide bonds between a non-natural (S)-2,3-diaminopropionic acid residue (Dap) and an aspartate, which showed the same amount of helical control as Lys-Asp pairs (Figure 7A).53 The synthesis of the AMPA substituted peptides occurred in good yield using standard solid phase methods and orthogonal protecting groups for the macrocyclization steps during elongation, comparable to the lactam-containing peptides. The Lys-Asp lactam bridged peptide showed 41% helicity at 25 °C in 100% aqueous buffer, with a jump to 64% in 50% TFE at the

calculated helicity based on molar ellipticity at 222 nm suggesting 100% helicity at 20 °C, and NOEs at 5 °C comparable to those at 25 °C, indicating stability.49 Having the lactam bridges at the termini may enhance dimerization, providing a nonpolar face that can undergo hydrophobic interactions. Overall, early on, the use of lactam bridges was shown to stabilize peptide secondary structure and can serve as a model for protein folding, with potential in the study of protein− protein interactions as well as the design of helical peptides with biological activities. Incorporation of lactam bridges across Lys and Asp or Lys and Glu residues with i and i+4 spacing became a common way to bridge peptides and influence their helical structure. Coupling these bridges with amine and carboxy-containing side chains forming salt bridges to further promote helicity helped achieve additional stabilization. Using a GRF analog comprised of a hybrid sequence from rodents and humans, it was found that peptides with greater helical content were more biologically active.50 The most promising candidate for drug development had constrained helicity by lactam bridging, incorporated the bioactive core from human GRF, was resistant to chemical and enzymatic degradation and also amenable to large-scale recombinant synthesis.50 In another experiment, a series of hybrids of moth cecropin and bee melittin peptides were constrained using a lactam bridge between glutamate and lysine in positions of i and i+4 at a variety of places across the sequence in an effort to create peptides with antibacterial effects.51 The lactam bridges were highly efficacious in inducing and stabilizing helicity across the peptide structures however there were several sequence-based effects. The lactam bridge was connected Glu to Lys in nearly all of the peptides, with one notable exception where the orientation switched from Lys to Glu. Generally, Lys to Glu bridges are considered helix destabilizing in aqueous conditions but can adopt helical structure in 50% trifluoroethanol (TFE).51 Typically, Glu to Lys oriented bridges are stabilizing across aqueous and hydrophobic media. All but one of the Glu-Lys bridged peptides were over 90% helical in 50% TFE (the exception bore the lactam constraint near a flexible hinge region of proline and glycines), whereas the Lys-Glu showed only 45% helicity in identical conditions.51 The presence of TFE is thought to resemble a membrane-mimicking environment, and the absence of strong helicity in aqueous buffer converting to high helicity in TFE conditions indicates that such peptides would become helical upon introduction to the bacterial membrane they are targeting. Overall, the lactam bridge led to reduced antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Candia albicans as compared with linear peptides, between 2 and 8-fold, but the peptide with the Lys-Glu reversed bridge orientation showed the J

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of peptide for melanoma imaging with the possibility of future therapeutic designs. An investigation of a synthetic, heterodimeric peptide linked by two lactam bridges considered a range of characteristics: dual orientation of the amide bond, varying bridge length, and the direction of the lactam. Here, a lactam bridge was employed as a cysteine disulfide bond isostere in preparation of an analog of insulin-like peptide 3 (INSL3).55 INSL3 is a hormone that plays a vital role in testicular descent during sexual development and is heterodimeric with a 26 residue A-chain and 31-residue B chain. It contains the disulfide bonding pattern characteristic of the insulin/relaxin superfamily with one intra-A-chain and two between the A and B chains, stabilizing the final peptide structure. Synthesis of the bis-lactam INSL3 analogues used a combination of semiorthogonal amine and acid protecting groups with a non-fragment-based, microwave assisted, solid phase peptide synthesis, which replaced one or both of the interchain disulfides with lactams.55 The lactam-containing peptides were shown to have significant RXFP2 receptor binding affinity in the nanomolar concentration range. The monolactam was found to have an affinity of 8.35 ± 0.11 nM and the bis-lactam 7.92 ± 0.12 nM.55 When compared to native INSL3, the lactam analogues had 10- and 12-fold better affinity and 1.7- and 4.6-fold greater affinity when compared to an INSL3 peptide with a truncated (for simplified assembly) Cterminus of the A-chain.55 The monolactam reduction in affinity, as compared to the bis-lactam and the truncated analogue, was most likely due to subtle changes in conformation by the mixture of disulfide and amide bonds across the chains, as there is a different bond length for each, and the electronic and steric effects of the lactam versus disulfide.55 These model peptides indicated that bis-lactam bridge modifications to insulin-like peptides hold promise for the incorporation into novel peptides with similar structures and functions. Using two lactams or two different macrocyclic strategies (i.e., lactam and disulfide) may be beneficial for future peptide designs for other targets. 3.2.4. Lactam Bridged Peptide Libraries. Lactam bridges in a peptide may not only be used as a “staple” to stabilize α-helix secondary structure, but also as a strategy to generate small cyclic peptide libraries.55 In an early study, two model systems were used: one containing a native coiled-coil of two amphipathic helices wrapped around each other with a lefthanded super twist, and another with a coiled-coil stabilized by lactam bridges at i, and i+4 at the N- and C-termini, to stabilize helicity to enhance dimerization (Figure 7B).56 For comparison, the hydrophobic 3,4 repeat of the lactam bridge peptide was substituted with all alanines to decrease the possibility of dimerization, in an additional peptide. For all of the peptides studied, five sites were chosen on the hydrophilic face of the amphiphathic helix for “randomization” in library design.56 The coiled-coil sequence was derived from a zinc finger library and incorporated into the random positions and the ability of the resultant peptide to inhibit binding of a particular Zn finger peptide to its receptor determined using an enzyme-linked immunosorbent assay (ELISA).56 Using CD spectroscopy, it was found that both the native and lactam bridged coiled−coiled peptides were predominantly αhelical in aqueous solution, and nearly 100% helical in 50% TFE.56 Two-dimensional NMR experiments showed that the residues contained in the lactam bridges were tightly constrained into helical conformation. Both the native and lactam bridged coiled coil peptides showed concentration dependence of

Figure 7. Indicates where lactam linkages, and other covalent linkages, are places in individual and across dimeric helices. A. Indicates 4(aminomethyl)phenylacetic acid linkage through amide bonds in comparison to a peptide that has traditional Lys-Asp pairs.53 B. Depiction of a coiled-coil arrangement of two amphipathic helices indicating lactam bridges at i and i+4 arrangements in the b (b′) e (e′) positions.56 Figure created by author from data in refs 53 and 56.

same temperature.53 Likewise, the AMPA bridge-containing peptide was 47% helical with a jump to 62% in identical conditions.53 Overall, the AMPA-containing peptide was more resistant to heat denaturation, showing promise for the future development of highly stabilized α-helical peptides using small molecules in macrocyclic constraints, such as the modern twocomponent systems described below in section 7. The incorporation of organic moieties into helical peptides can be used to probe interesting biological phenomena that rely on helicity to dictate interaction. The effect of DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting by a peptide and the pharmacokinetics of this radiolabeled, lactam bridged peptide based on melanocyte stimulating hormone (MSH) was studied.54 Using a lactam bridge in the peptide Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPheArg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)], with the DOTA group attached to the Lys in the macrocycle, built upon prior work with 111 In-labeled α-MSH peptides containing a lactam bridge. These peptides were used as imaging probes for melanoma detection and were stable both in vivo and in vitro, which was attributed to the lactam bridge cyclization. Incorporating the DOTA metal chelator for radiolabeling produced high receptor-mediated melanoma uptake in B16/F1 melanoma-bearing mice, with primary and pulmonary metastases visualized using the peptide as an imaging agent.54 Having DOTA inside the cycle instead of as an N-terminal tag, showed 0.6 nM MC1 receptor binding affinity, high tumor uptake, prolonged tumor retention, and low uptake in non-target organs.54 Shifting the DOTA from the Nterminus to the lactam bridged ring increased uptake and retention by melanoma, which shed light into the use of this class K

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helicity and a dimerization induced increase in helicity.56 The lactam stabilized coiled coil had a much lower dissociation constant than that of the native coiled coil peptide, which appeared to come from the lactam bridges maintaining their fold even at low peptide concentrations, serving as nucleation sites for initiation of both helicity and dimerization. This was further evidenced by results from urea denaturation which indicated the native coiled-coil peptide went through a smooth transition, while the lactam bridged peptide showed low cooperativity for the unfolding transition with a less cooperative folding conversion due to decreased peptide surface exposure.56 Helical peptides with disulfide bonds also show similar behavior due to residues within the constraint maintaining partial helicity as residues outside of the constraint relax to a random coil. The lactam bridges thus conferred a loss of conformational entropy in the unfolded state and increased the overall stability of the fold.56 After characterization of the coiled-coil scaffolds, testing occurred to see if the spatial arrangement of the consensus residues properly mimicked the specific HFVQH Zn finger peptide binding to monoclonal antibody IgAC5.56 The coiledcoil peptides were shown to inhibit the Zn finger peptide in a dose dependent manner, with the lactam bridged peptide comparable to the wild type Zn finger and much more effective at inhibition than the non-bridged coiled coil. At lower peptide concentrations, less of the non-bridged coiled coil would be dimeric and thus not fully folded, as compared with the lactambridged peptide.56 The single-stranded peptide with lactam bridges and alanine substitutions was also dose dependent, as its folded helical structure contributed to its activity. However, while more active than the non-constrained coiled-coil peptide, the monomeric peptide was much less effective than the lactam bridged coiled-coil. Therefore, the lactam bridged peptide had the most stable helical structure, with a strong coiled-coil dimeric structure, and was most effective against its biological target. This model is promising for the creation of peptide libraries where a stabilized coiled-coil can serve as a template for conformational stability with specificity “dialed in” through key placement of residues. Further design may be warranted to show that the restricted nature of the lactam bridged α-helix is ideal for molecular modeling and the design of such peptide mimetics. In similar work, standard allyl/alloc methods and solid phase techniques were used to create small libraries of cyclic peptides containing lactam bridges.57 Excellent yields were obtained and cyclization of the lactam ring occurred through an Asp-Lys pair in the i, i+7 positions achieved in orthogonal conditions on solid support. The preparation of a library of 16 novel cyclic peptides, including 7 residue melanotropin analogues and 24 amino acid cyclic glucagon analogues, was achieved and continuing work on these compounds paves the way for the creation of parallel libraries of similarly constrained peptides with helical content.57 3.2.5. Lactam Bridged Peptides in an Array of Biological Applications. There are many ways in which lactam bridges can be applied to helical peptides of interest, as were discussed through numerous examples above. To expand on this, various peptides continue to be developed, incorporating lactam bridges in novel ways to target proteins involved in diseases. C-peptides, based on the HR2 region of gp41 of HIV have much potential as fusion inhibitors, however do not generally maintain high helicity. Creating constrained Cpeptides using a lactam-styled cross-link between two residues separated in sequence, but close spatially when the peptide is

folded into a helix, connected two glutamic acid residues in the i, i+7 positions using a diaminoalkane group.58 This tether proved to be a flexible cross-linker with ease in synthesis as well as effective helical stabilization.58 A cell−cell fusion experiment showed that the cross-linked peptide was a potent fusion inhibitor with an IC50 around 35 μM, displaying measurable inhibitory activity in a luciferase-based viral infectivity assay.58 NMR spectroscopy confirmed that the cross-linked C-peptide bound to the hydrophobic pocket of IQN17, a soluble peptide model of the HIV gp41-HR1 pocket fused to a GCN4 leucine zipper.58 Distinct resonances in a fingerprint region that corresponded to the hydrophobic pocket Trp residues showed changes prior to and after introduction of the cross-linked peptide. A 1.9-Å resolution X-ray crystal structure of the crosslinked peptide with IQN17 displayed the peptide bound to the gp41 region of the model, binding in the hydrophobic pocket in the same manner as the original, unconstrained C-peptide.58 The cross-link itself showed an inherent flexibility in the averaged structure. Isothermal titration calorimetry (ITC) was performed to determine the binding of the cross-linked peptide to IQN17, in comparison with other peptides less structured or more locked into helical arrangements. The most potent inhibitor, the cross-linked peptide, showed a Kd of 1.2 μM, whereas peptides at extremes (more or less structure than the cross-linked peptide) showed weaker binding affinities.58 A large gain in enthalpy was balanced by a loss of binding entropy; however, the cross-linked peptide showed a midlevel enthalpy− entropy balance and proved the best at inhibition. The ability of the cross-linked peptide to adopt the helical conformation necessary to bind the gp41 hydrophobic pocket was countered by its flexibility to assume the proper structure. Reduction in conformational entropy allowed proper orientation and interaction with the helix bundle.58 This has implications for the future design of constrained peptides as a balancing act between flexibility and structure when targeting a protein of interest. In another experiment, glucagon-like peptide-1 (GLP-1), an incretin released from intestinal L-cells in response to nutrient ingestion, was chosen as a peptide to mimic using the lactamstyle bridges.59 GLP-1 interacts with receptors in various organs, including pancreatic β-cells, and is involved in many biological functions such as glucose-dependent insulin secretion, insulin gene transcription promotion, β-cell proliferation initiation, neogenesis, and inhibition of β-cell apoptosis.59 Due to these varied roles, GLP-1 has potential as a therapeutic for treating type 2 diabetes, however, it is highly susceptible to enzyme degradation which compromises the effectiveness of its clinical applications. The N-terminal region of GLP-1 is essential for receptor activation, yet the C-terminal region more strongly contributes to receptor binding.59 There are two α-helices within the structure of GLP-1 and to determine the importance of secondary structure on receptor interaction, a cyclization scanning study was performed, fixing the α-helical structure by introducing a lactam bridge between Glu and Lys residues (or Lys-Glu orientation) i, and i+4 positions apart.59 The lactams were incorporated in one (monocyclic), two (bicyclic), or three (tricyclic) positions in various locations from the N- to Cterminus. “Locking” the helical conformation across the peptide with lactam rings was thought to contribute to a high binding affinity of the peptide to its receptor and provide protection from proteases by preorganizing the helix. All of the GLP-1 analogues with one lactam bridge between Glu (i) and Lys (i+4) exhibited high efficacy in receptor L

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activation and bore greatly stabilized helical segments.59 The introduction of two lactam bridges resulted in comparable or moderate increases in potency due to the increased stabilization of the receptor-bound conformation. It was also found that the orientation of the lactam bridges do not have a significant effect on the peptide’s efficacy, although a slight preference was found for Glu-Lys versus Lys-Glu. C-terminal cyclization stabilized the peptides against dipeptidyl peptidase-IV (DPP-IV), and those peptides with two lactam bridges, simultaneously at the Cterminus, promoted a 3-fold increase in stability.59 Peptides with two lactam bridges showed protection against degradation by neutral endopeptidase 24.11 (NEP 24.11), regardless of the position across the peptide from N- to C-termini. Remarkably, the tricyclic peptide was the most highly constrained GLP-1 analogue and was exceptionally stable against both proteases with the highest enzyme stability and potency.59 Each of the classes of constrained peptides provided insight for the development of peptides with long-acting in vivo activity and this work serves as a model for the development of molecular probes for imaging pancreatic β-cells. In another example, lactam bridging constraints were used to gain insight into the receptor-bound conformation of secretin, stabilizing a receptor antagonist. The secretin receptor is a member of the family of B of G protein-coupled receptors (GPCR).60 Secretin is a 27-residue carboxyamidated linear peptide produced in the small intestines and secreted in response to acid, stimulating subsequent secretions that help regulate the duodenal pH. The carboxy terminus is the most important peptide region for binding affinity to its GPCR, and the amino terminus is essential for receptor selectivity and activation.60 When bound in the hydrophobic receptor membrane, secretin adopts a conformation containing two helical segments. The use of lactam bonds to constrain secretin analogues discerned which conformations of secretin are associated with the peptide-binding cleft at the receptor’s amino terminus and which led to the most effective receptor antagonists. A series of truncated secretin (5−27) analogues were developed with the lactam bridge at different positions.60 A single lactam bridge was employed between Glu and Lys residues 3−4 positions apart to stabilize the α-helical structure. Of 11 analogues, the one with the lactam bridge from position 16 to 20 showed significantly increased binding affinity and inhibition of secretin-stimulated biological activity, with an IC50 value of 19 ± 6 nM, while the parent peptide (secretin 5− 27) had an IC50 of 410 ± 59 nM, and was unable to fully displace all saturable radioligand binding even up to 1 μM.60 Subsequently, two full-length secretin analogues containing the lactam bridge in the 16 and 20 positions were created and sustained the same binding affinity and biological activity as natural secretin. Molecular mechanics simulations of the unbound peptide showed that the most active peptides were also highly α-helical, with the shorter, original analogue having slightly more favorable van der Waals, hydrogen bond, and electrostatic energies.60 These results ultimately contributed to the creation of more effective secretin antagonists. Similar lactam-constraining studies were also performed with other GPCR targets, as many ligands can benefit from stabilizing helical structure in the peptides’ midregion and carboxyl terminus, which interact with the peptide-binding cleft of GPCRs. In recent research, the insulin superfamily of peptides (mentioned above for INSL-3) was investigated to better understand how to create isosteres that substitute the inherent

disulfide bonds. Insulin peptides have three disulfide bonds (one intra-A chain and two interchain) that aid in the conformation and functionality of the peptides. By inserting isosteres of the naturally found disulfide bonds, including lactam rings (as well as dicarba, and cystathionine bonds) several novel insulin-like peptides were developed that contained intra-A-chain disulfide isosteres with improved properties, including increased receptor selectivity and greater in vivo half-lives due to the stability against disulfide reductases.61 Generally, the binding affinity for the insulin receptor, the secondary structure of the peptide, and stability in human serum were maintained, however indications were that a thioether mimic was closer to the disulfide bridge than other isosteres, including lactam bridges.61 It is commonly understood that removal of any of the three disulfide bonds in insulin causes tremendous loss of both biological activity and secondary structure. Therefore, functional isosteres in novel analogues will aid in uncovering the structure−functional relationships of INSLs but also help future therapeutic development, especially in novel diabetes drugs for use when refrigeration is limited.61 The lactam bridge as a substitute for disulfides and its stable, conformation-locking employment in a variety of peptides provides and enhances α-helical secondary structure. Lactam bridges continue to be studied, although new methods of constraint have entered the field of macrocyclic helical peptide mimetics. These techniques can promote helicity but institute greater conformational flexibility as well as more versatility in synthesis.

4. HYDROCARBON BRIDGED SIDE-CHAIN CROSSLINKS “STAPLE” HELICITY IN MIMETICS As mentioned, current work is focused on designing short helical peptide mimetics with unique tether that could potentially function as inhibitors of interactions between proteins and eventually become therapeutics.62 The application of short peptides as therapeutics is difficult, as discussed, because peptides generally lack defined structures and subsequently lose natural binding behavior when taken out of their physiological context. Consequently, short peptides exposed to proteolytic enzymes can be degraded and remain unable to pass through target cell membranes. Modifying the short peptide to maintain an α-helical conformation, even in isolation, has been shown to overcome the obstacles associated with stability, binding, and intracellular access. A well-defined, modern method available for maintaining and reinforcing α-helical conformation utilizes a hydrocarbon bridge as a link between the side chains of neighboring amino acid residues on a peptide.62 To facilitate the process, two neighboring amino acids on the same face of the α-helix would have olefin functional groups on their side chains catalytically joined via a covalent bond. The resulting structure is essentially fixed in an α-helical arrangement with greater potential for biological function. 4.1. Background and Synthesis

To properly design an α-helical peptide mimetic, a small molecular scaffold must contain the desired functional groups in a specific spatial orientation that emulates the helical arrangement and has the potential to modify protein−protein interactions.63 Protease degradation and instabilities most commonly occur when a peptide is in an extended conformation, so increased helical stabilization as a result of carbon−carbon bridge formation can lead to an overall increase in peptide stability and potency in vivo. In a helix mimetic with a M

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Figure 8. Depiction of olefin ring-closing metathesis via a catalyst and hydrogenation reagents occurring across side chains of a peptide, forming a carbon−carbon bridge. The residues represent either L-serine or L-homoserine O-allyl ethers that were previously incorporated into the peptide through standard synthetic techniques.64 Figure created by author from data in ref 64.

Figure 9. Examples of the residues (R, S, and a doubly substituted residue with various native R groups and the alkyl chain) that comprise the carbon− carbon bridge prior to olefin metathesis. Additionally, a ribbon structure indicates the placement of these bridges across the helix.65 Figure created by author from data in ref 65.

unbranched terminal olefin groups; these residues were either Lserine or L-homoserine O-allyl ethers, which were incorporated into the peptide through standard solution-phase peptide synthesis (Figure 8). The peptide with the olefin-containing residues was treated with the RCM catalyst and exposed to catalytic hydrogenation reagents to generate a hydrocarbon bridged peptide helix in high yield.64 CD spectroscopy and X-ray crystallography confirmed a helical arrangement of the peptide; however, it was mostly 310 with little α-helical signature.64 Prior to carbon−carbon bridge formation the peptide was already arranged in a helical conformation. The incorporation of the carbon−carbon bridge appeared to add rigidity to the helical structure, which could potentially lead to increased stability and proteolytic resistance; however, the replacement of the i and i+4 Ala residues with the tethered residues induced the backbone to predominately transform to a 310-helix from the α-helix.64 These results indicated that macrocyclic peptide helices could be obtained through a relatively straightforward synthetic procedure, and more control would be needed to keep α-helicity.

hydrocarbon bridged side chain cross-link, a macrocycle forms because the cross-link attaches at two points along a synthetic peptide backbone. This arrangement confers both stability and increased hydrophobic character that manifests itself in different applications as explored below. It was previously established that helix stabilization could occur if a bridge was created between the i and i+4 amino acid residues in a helical peptide, but initial discoveries focused on noncovalent bridge connections.64 Such connections, however, are not favorable because they are not as stable as covalent bridges and are unable to consistently confer protease resistance and biological activity because of a peptide’s potential loss of helical structure. The Grubbs olefin metathesis catalyst [(PCy3)2Cl2RuCHPh] is capable of initiating the formation of a carbon−carbon bridge connecting specific olefin-containing amino acid side chains via a ring-closing metathesis (RCM) reaction (Figure 8).64 A hydrophobic peptide model system [Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe] was previously shown, through X-ray crystallography, to adopt an α-helical conformation in the solid state.64 The use of α-aminoisobutyric acid (Aib) is known to stabilize 310 and α-helical character in peptides. The i (Ala) and i+4 (Ala) residues in the model peptide were replaced with non-natural amino acids containing

4.2. Initial Applications of Hydrocarbon Bridged Peptides

Expanding on this foundation, there was a need for cross-links that not only stabilized existing helices but also promoted helical N

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formation and stability within a peptide.65 Various cross-link lengths, attachment sites, and stereochemical properties were screened to probe helix-stabilization potential. Both R and S stereochemistries were tested at the site of the substituted αcarbons and the alkyl bridge length was modified. The cross-link lengths in the analysis varied from 6-carbons to 12-carbons. When designing non-natural amino acids to incorporate into the peptide, residues containing a D-configuration were avoided because of their known helix-destabilization properties, but α,αdisubstituted residues (adding an α-methyl group, as in Aib) were incorporated for their helix-promoting propensities.65 The C-peptide sequence from RNase A was selected as a model system because of its partial helicity in aqueous environments, which would allow observation of both increases and decreases of helicity following the addition of various non-natural residues.65 Furthermore, the cross-links were attached between the i and i+4 residues (one turn of the helix) or the i and i+7 residues (two turns of the helix). Results indicated that increasing the bridge length (larger macrocyclic cross-link) led to a higher success rate in the RCM reaction, but interestingly, rates increased dramatically with only a slight increase in length.65 Results from CD spectroscopy concluded that adding two α,α-disubstituted residues to the model system increased its α-helical content. A bridge linking an R-residue at i and an Sresidue at i+7 with 11 carbons in the metathesized cross-link exhibited the greatest amount of helix stabilization (Figure 9).65 Trends showed that cross-links 9-carbons or 10-carbons in length were too short to allow the formation of a stable helix, but a 12-carbon cross-link was too long because it did not impart the necessary structural constraints. By examining the cross-link length, attachment sites, and stereochemical properties, the cross-link can be optimized to provide the most stable helical mimetic (Table 3). Implementation of this information into functional peptides is useful, albeit there will be context dependence.

antiapoptotic BCL-2 proteins are overexpressed, or BH3containing proteins are nonfunctional, sequestered, or mutated, programmed cell death is decreased which can lead to cancer. Designing a short peptide-based therapeutic to mimic the BH3 domain is difficult as it would lose much of its α-helical structure once in solution. Using the Grubbs ruthenium catalyst, a hydrocarbon bridge can be formed between the olefin containing α,α-disubstituted non-natural amino acids to stabilize the α-helix of a BH3 mimicking peptide, “stapling” it into the proper helical conformation.66 Apart from the two nonnatural amino acids that serve as anchors for the hydrocarbon bridge, initial designs of the peptide contained the natural BH3 domain sequence. The non-natural amino acid substitutions were added flanking three (in positions i, i+4) or six (in positions i, i+7), amino acids from the BID BH3 domain, so that the reactive olefin residues would be on the same face of the helix, when formed (Table 4).66 Table 4. Compound, Sequence, and Percent α-Helicity of Hydrocarbon Stapled Peptides Used As Mimetics for BH3 Domains and the p53 Activation Domain66,68

Table 3. Hydrocarbon Stapled Peptides Indicating Staple Position, Stereochemistry, Percentage of Staple Closure, and Change in α-Helical Content Following Insertion65 cross-link

% conversion by RCM

Ri, Si+7 (8-carbons) Ri, Si+7 (9-carbons) Ri, Si+7 (10-carbons) Ri, Si+7 (11-carbons) Ri, Si+7 (12-carbons) Si, Si+4 (6-carbons) Si, Si+4 (7-carbons) Si, Si+4 (8-carbons) Ri, Ri+4 (6-carbons) Ri, Ri+4 (7-carbons) Ri, Ri+4 (8-carbons)

0 51 77 >98 >98 0 68 >98 0 17 >98

change in helical content

a

21% decrease 12% decrease 26% increase 13% increase

compound

sequencea

% helicity

BID BH3 SAHBA SAHBA(G→E) SAHBB SAHBC SAHBD p53WT SAH-p53−1 SAH-p53−2 SAH-p53−3 SAH-p53−4 SAH-p53−5 SAH-p53−6 SAH-p53−7 SAH-p53−8 SAH-p53−8F19A UAH-p53−8

EDIIRNIARHLAQVGDSNleDRSIW EDIIRNIARHLAS5VGDS5NleDRSIW EDIIRNIARHLAS5VEDS5NleDRSIW EDIIRNIS5RHLS5QVGDSNleDRSIW EDIIRNIAS5HLAS5VGDSNleDRSIW EDIIRNIARR5LAQVGDS8NleDRSIW Ac-LSQETFSDLWKLLPEN-NH2 Ac-LSQETFSDR8WKLLPES5−NH2 Ac-LSQER8FSDLWKS5LPEN-NH2 Ac-LSQR8TFSDLWS5LLPEN-NH2 Ac-LSQETFR8DLWKLLS5EN-NH2 Ac-LSQETFR8NLWKLLS5QN-NH2 Ac-LSQQTFR8NLWRLLS5QN-NH2 Ac-QSQQTFR8NLWKLLS5QN-NH2 Ac-QSQQTFR8NLWRLLS5QN-NH2 Ac-QSQQTAR8NLWRLLS5QN-NH2 Ac-QSQQTFR8NLWRKKS5QN-NH2

15.7 ± 0.3 87.5 ± 0.3 77.8 ± 0.6 85.5 ± 1.3 59.7 ± 6.5 35.6 ± 1.8 11 25 10 12 59 20 14 36 85 39 36

S5 and R8 refer to the amino acid residues in Figure 9

Degradation assays showed that the BH3 mimetic experienced enhanced protease resistance due to the shielding effect of the stapled conformation on the amide backbone.66 Furthermore, CD spectroscopy showed that the BID BH3 domain itself was only approximately 16% helical in aqueous solution, whereas the most active stapled peptides were in the 86−88% helicity range. Various assays were performed to assess the BH3 mimetic’s ability to penetrate leukemia cells and selectively trigger apoptosis leading to cancer cell death. Using Jurkat T cell leukemia cells and a fluorescent tag on the BH3 mimetic, it was determined that the mimetic was able to penetrate the cancer cell membrane.66 Comparatively, BH3 without an added hydrocarbon bridge was ineffective, which showed that the hydrocarbon staple in the BH3 mimetic increased the peptide’s lipophilicity and associated ability to penetrate the membrane. Moreover, the BH3 mimetic increased apoptosis within the cancer cells and limited cancer cell proliferation.66 Studies conducted using mice bearing human leukemia xenografts showed a consistent suppression of cancer growth when compared to nonstapled BH3 domain, alone.66 This comprehensive application of hydrocarbon bridged peptides provided

1% decrease 4% increase

2% increase

4.3. Hydrocarbon Staples in Anticancer Applications

Unlike hydrogen-bond, lactam, or disulfide linked bridges, the hydrocarbon bridge stabilizes a helix mimetic in such a way that allows it to withstand harsh in vivo proteolytic conditions and penetrate cell membranes while maintaining bioactivity.63 The use of α,α-disubstituted non-natural amino acids in small helical peptide mimetics can be applied to the development of in vivo therapeutics.66 The BH3 domain of the BID protein from the BCL-2 protein family is α-helical in vivo and responsible for activating the apoptotic pathway in human cells. When O

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Figure 10. Shows hydrocarbon bridged peptides as helix mimetics bound in protein surfaces. A. Representation of a hydrocarbon bridged helical peptide mimetic, modeled after a known coactivator, bound to the estrogen receptor (PDB: 2YJD).71 B. Representation of the stapled peptide based on the membrane-proximal external region of HIV-1 glycoprotein gp41 bound to the antigen-binding region of 4E10 through the tethered α-helix and a phosphate (PDB: 4NGH).75 Figure created by author from data in refs 71 and 75.

neutral side chains. The stapled p53 mimetic contained point mutations at both the nuclear export and ubiquitylation sites along its chain and generated a peptide with the most successful combination of cell permeability, structural stability, high affinity for hDM2, and ability to initiate apoptosis, decreasing cancer cell viability.68 The successful interaction between this helical mimetic and the protein hDM2 further highlights the therapeutic potential of the hydrocarbon bridge containing peptides. The oncogenic mutation, E545K, which occurs at a specific αhelical domain (referred to as p110α) of phosphatidylinositol 3kinase α (PI3Kα), leads to an unregulated interaction between p110α and insulin receptor substrate 1 (IRS1). The p110α-IRS1 interaction is a major contributor to the growth of colon cancer cells.71 A previously synthesized 18-residue all-hydrocarbon [i, i +4] stapled peptide potently blocked the intracellular xenograft tumor growth formed by p1101[E545K]-expressing colon cancer cells after direct injection.72 This stapled peptide, however, lacked a high percentage of α-helicity and had insufficient proteolytic stability, in part caused by “loose” residues near the N- and/or C-terminus. These residues were not considered vital for peptide function and are not within the limits of the hydrocarbon staple.71 Therefore, derivatives of the initial peptide mimetic extended the staple length from [i, i+4] to [i, i+7] but ensured that the key lysine at position 545 was preserved.71 In addition, 3 residues were removed from both the N- and C-termini to simplify the peptide’s structure. The final 14-residue peptide, CH3CONH-EIT(K545)R8EKDFLWS5HRCONH2, had 17% more α-helical structure than the firstgeneration peptide (up to ∼65% in aqueous solution) and exhibited significantly greater proteolytic stability.71 When examining the stapled peptide’s ability to deactivate intracellular AKT phosphorylation at Ser473, a vital process derived from the p110α[E545K]-IRS1 interaction in colon cancer cells, the stapled peptide was capable of readily crossing the cell membrane and potently switching off the process.71 Additional structural analyses are desired to optimize the stapled peptide as a novel anticolon cancer therapeutic. These developments can be broadened to create unique hydrocarbon-stapled peptidomimetics that target a broader range of biologically relevant systems.

direct evidence of their potential as therapeutics and useful manipulators of biological pathways. One of the most common defects in human cancer is an inability to properly regulate levels of the transcription factor p53 by deletion, mutation or hDM2 overexpression.67,68 Transcription factor p53 initiates cell cycle arrest and apoptosis in response to DNA damage and cellular stress.68−70 The ubiquitin protein ligase hDM2 controls p53 levels by forming a tight complex with the p53 protein. When hDM2 is overexpressed in many human cancers, its tumorigenic potential increases as it inhibits p53-mediated transactivation of apoptosis.68,70 The proteins interact via an α-helical domain on p53 and a hydrophobic cleft on hDM2 and have served as a model system for decades in the design and development of αhelical peptide mimetics with therapeutic potential. Using the RCM method with ruthenium catalyst to create a hydrocarbon bridge between α,α-disubstituted amino acids on a p53mimicking peptide, the constrained macrocycle was used to promote α-helicity in the design of peptide-based anticancer therapeutics. The α-helical, 16-residue transactivation domain of p53 was used as a basis for the designs and the non-natural substitutions were anchored at i, and i+7 positions at four varying locations within the peptide, as can be seen in Table 4, along with the helicities determined by CD spectroscopy in aqueous solution. The synthetic residues (R8 and S5, Figure 9) at the i and i+7 positions on the peptide backbone had long alkyl chains that were connected to form the hydrocarbon bridge via the olefin metathesis technique. CD spectroscopy confirmed that α-helicity increased with the addition of the hydrocarbon bridge and the affinity for hDM2 was greater for those more highly helical peptides.68 The hydrocarbon stabled p53 mimetics were capable of entering cells and reactivating the p53 tumor suppressor cascade that was disabled following the overexpression of hDM2.68 The mimetics bound to the high levels of hDM2, preventing hDM2 from neutralizing p53 transactivation activity, and inducing the transcription of p53-responsive genes. Peptide variants containing asparagine and glutamine substitutions in place of aspartic and glutamic acids, respectively, were capable of passing through the cancer cell membranes at a much higher rate. These substitutions increased positive charge on the peptide as a whole by replacing negatively charged amino acids with those bearing P

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4.4. Applications of Hydrocarbon Staples in Other Biologically Relevant Systems

positions and brought together using the Grubbs ruthenium catalyst.64,76 As the peptide is lengthy, two staples were incorporated, and specific bridge locations were chosen to limit the disruption of critical hydrophobic interfaces in the peptide. Results showed that the double-stapled peptide was 24 times more stable in chymotrypsin than the unmodified peptide and 4 times more stable than an equivalent single-stapled peptide.76 The doubly stapled peptide also demonstrated a halflife approximately 200-fold longer than the unstapled T649 when exposed to acidic conditions and pepsin and was 80% intact after pepsin exposure at pH 2 for greater than 12 hours. Interestingly, it was determined that the physical presence of the two covalent hydrocarbon staples, not only the α-helicity or residue hydrophobicity, was responsible for the increased protease resistance of the peptide. While the double-stapled peptide exhibited only moderate α-helicity (36%) which was lower than a singly stapled (63%) but higher than the unmodified peptide (13%), it was the most optimized in terms of proteolytic resistance and antiviral potency. The doublestapled peptide had an IC50 of approximately 2.5 nM in a luciferase-based HIV-1 infectivity assay and had the most potent antiviral activity against neutralization-resistant virus (IC50 ≈ 16 nM), outperforming enfuvirtide (IC50 > 300 nM) and the unmodified peptide (IC50 ≈ 44 nM). Additionally, the doublestapled peptide exhibited an enhanced duration of systemic exposure in mice, a better ability to maintain effectiveness when faced with viral enfuvirtide resistance mutations, and improved systemic levels following oral absorption in mice.76 Doublestapling was effective at improving all of the anticipated shortcomings of using a long peptide as a therapeutic and proved that the structural technique should be investigated further. In related work, a hydrocarbon stapled peptide was developed that showed ability to bind to specific neutralizing human antibodies. Antibodies will deactivate pathogens through specific peptide sequences, and often vaccines will present such structures to increase the recruitment of neutralizing antibodies. HIV-1 has been intractable in vaccine development and an effective vaccine would need to initiate and recruit broadly neutralizing antibodies to intercept the virus prior to penetration of the host cell.77 In the gp41 fusion structure of HIV-1 glycoprotein, there is a membrane-proximal external region (MPER) downstream to HR2 that contains a sequence recognized by several neutralizing human antibodies including 4E10. A 13-residue portion of MPER has been shown to adopt an α-helical conformation upon binding 4E10 Fab (antigenbinding fragment).78 Designed stapled peptides with stabilized α-helices containing the MPER sequence of gp41 were extraordinarily protease resistant and bound to 4E10 with high affinity. Using a competitive ELISA, the hydrocarbon stapled peptides were compared with an unmodified MPER peptide (amino acids 662−683). An i, i+3 stapled peptide yielded 60fold enhancement of binding over the unmodified peptide and indicated that the staple’s location needed to balance helical stabilization with preservation of the binding epitope and key residues.77 Structural analysis of the stapled MPER peptide complexed with 4E10 revealed that the i, i+3 bridge was maintained on a non-interacting surface to allow the peptidebinding interface to persist. A phosphate tether was added to the MPER design, through a Dab (diaminobutyric acid)-Glyphospho-Ser linker, as early structures indicated that a phosphate ion was incorporated into the antigen-binding region of 4E10.77 By adding the tether into the stapled-MPER design,

In addition to their application as therapeutics against cancer, hydrocarbon bridged helical peptide mimetics have the potential to influence a variety of biological systems (i.e., the endocrine system, the immune system, viral infectivity). The estrogen receptor (ER) is a member of the steroid hormone receptor class of the nuclear receptor superfamily and is responsible for binding estrogen to modulate associated gene expression involved in areas such as reproduction, central nervous system function, bone density and immunity.73,74 The ER has been targeted in several diseases including breast and endometrial cancers as well as osteoporosis. Two isomeric forms of the ER exist, types α and β, and are structured around a variable Nterminal transactivation domain, a well-conserved DNA binding domain, and a C-terminal ligand binding domain (LBD). Estrogens bind to the LBD inducing conformational changes in the ER, homodimerization, and binding to specific promoter DNA sequences to activate gene expression. The binding of estrogen alone is not enough to elicit a response, however, because a coactivator protein is also required to bind the complex.73,74 The coactivator binds at the LBD, as well, via αhelices. Hydrocarbon bridged helical peptide mimetics have the potential to regulate the estrogen receptor because of their biologically active α-helical conformation that mimics the estrogen receptor coactivator. The hydrocarbon bridged mimetic was modeled after a known coactivator peptide and the bridge was positioned to conserve receptor contacts. To study the structure of the peptide, the bridged mimetic produced well-diffracting crystals due to its rigidity, which allowed for its utility in structure and binding studies (Figure 10A). The nonpolar character of the hydrocarbon bridge appeared to increase the favorable hydrophobic protein−protein interaction in the coactivator binding pocket of the ER.73 The preorganized nature of the bridged mimetics resulted in a higher association rate with the estrogen receptor because of the lower entropic cost of binding. The location of the hydrocarbon bridge and its overall rigidity generated unexpected consequences when binding this highly targeted cellular receptor; the hydrophobic character of the bridge must be considered for its potential to disrupt natural protein−protein interactions, therefore contributing additional potency. Peptide therapeutics are often ineffective because they easily lose their bioactive structure when taken out of the context of a protein and are highly susceptible to proteolysis. Enfuvirtide is a 36-residue peptide, the first fusion inhibitor shown to block HIV-1 entry in human cells.75 As mentioned in prior sections, HIV-1 uses the six-helix bundle of structural heptad repeats 1 and 2 to fuse with the host cell.25 Enfuvirtide mimics the HR2 domain of the gp41 envelope glycoprotein, however its limited application is attributed to poor in vivo stability and lack of oral bioavailability, due to its peptide chain.75,76 T649 peptides contain residues from HIV-1 gp41 that stabilize the six helix bundle and show improved antiviral potency when compared to enfuvirtide.76 Using T649 as a model, the variant T649v was prepared that became the first application of hydrocarbon double-stapling to fortify natural helical structure in an anti-HIV directed peptide therapeutic. Double stapling was conducted to improve stabilization, protease resistance, antiviral efficacy, and oral absorption.76 The “staple” was inserted at specific locations at both the N- and C-termini of the peptide by incorporating two sets of the synthetic residue (S)-2-((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-hept-6-enoic acid at the i and i+4 Q

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the α-helical binding site and phosphate-binding regions of 4E10 were simultaneously accessed (Figure 10B).77 This showed that hydrocarbon stapling of peptides is a useful approach in generating peptide-based structures for eliciting the response of neutralizing antibodies for HIV-1 which can perhaps be applied to other viral targets. Expanding on these biological investigations, new research into stapled peptides and their potential in other areas beyond protein target binding includes their use as vehicles in RNA interference and regulating stem cell pluripotency. This work is ongoing and presents an interesting challenge for the future of the technique.

important model system for protein−protein interactions and can be targeted with hydrocarbon stapled peptides.83 Traditionally, an α-methyl group was utilized when synthesizing synthetic residues for hydrocarbon staple formation to promote helix stabilization.65 This process, however, can limit the peptide sequences that can be stapled, decrease solubility, and eliminate beneficial side chain interactions. Instead, the retention of the natural side chain in the stapling residue can circumvent these issues.83 An amino acid that contains both the native side chain and a group for stapling would introduce the ability to staple to a residue without sacrificing its natural functional group. The synthetic route for this unique, dually functioned residue generated product at greater than 95% enantiopurity.83 Modified amino acids that were tested included Leu, Met, Ser, Tyr, Lys, Arg, and Phe, each containing their native side chain with a stapling group at the α-carbon (Figure 9). The hydrocarbon stapled sequence was based on a 14-residue portion of Axin, Ac-PESILDEHVQRVMK-NH2 and staples were added in an i, i+4 arrangement, cyclized using Grubbs RCM catalyst in the usual manner.64 Transcriptional activation as a result of the hydrocarbon bridged peptide releasing βcatenin from its protein complex was monitored using a luciferase assay.83 Results from reporter expression showed a 3fold enhancement of potency following introduction of the stapled peptide, as compared to the wild type peptide. Additionally, the stapled peptide exhibited greater protease resistance and cell permeability than the native protein sequence.83 It was determined that the Met residue contributed to a strong interaction with β-catenin, the Asp residue was responsible for strong salt bridge formation, and the Lys residue contributed to an important hydrogen bond. These results taken together indicated that incorporating natural side chains into the stapling positions on peptides applied as inhibitors, protein− protein interactions of novel importance could be probed from optimized binding of the stapled sequences. The future directions of hydrocarbon stapled α-helical peptides are widespread as there is increasing interest in peptide therapeutics in the drug market. Peptides provide an excellent starting point for new drug designs and offer greater specificity that ensures safety, tolerability, and efficacy in vivo. Hydrocarbon stapled peptides offer enhanced protection from enzymatic cleavage, greater specificity in protein−protein interactions as a result of the highly conserved α-helix, and levels of cell permeability that have not been recorded in related peptide therapeutics. The greatest advantage of hydrocarbon stapled peptides is the drastic increase in oral bioavailability that the peptides confer as a therapeutic, which provides a clinical advantage with ease of use for patients. Hydrocarbon bridged helical peptide mimetics have diverse potential as therapeutics and as probes for chemical interactions. Applications in cancer, transport, and the endocrine system emphasize the hydrocarbon bridged mimetic’s ability to target protein−protein interactions that previously precluded such intervention. Compared to more reactive stabilizing bridges such as those containing disulfide or lactam linkages, the hydrocarbon bridge offers a level of stability, rigidity, and helicity that is unique. Furthermore, the increased level of protease resistance, potency, and cell permeability highlights the hydrocarbon bridge as a powerful therapeutic tool. Work continues on stapled peptides which retain the recognition residues at the site of the staple. In one particular study, a reduction in binding between a stapled peptide based on the BH3 domain of pro-apoptotic Bim protein and antiapoptotic, oncogenic BCl-2 family proteins was

4.5. Uses of Hydrocarbon Stapled Peptides in Gene Expression and Stem Cells

Short interfering RNAs (siRNAs) are capable of regulating gene expression through a silencing mechanism and have potential applications in the treatment of genomic diseases and tumorigenic disorders. Current siRNA delivery methods using lipids lead to harmful accumulation of siRNA in organs, undesirable immune response, and toxicity.79 Utilizing hydrocarbon stapled Lys-, Leu-, and His-rich peptides as vehicles for siRNA delivery is a favorable approach due to their stability, cell membrane permeability, and binding affinity to RNA.80 Lys- and Leu-rich peptides were targeted because of their history in noncovalent delivery of siRNA. Cell penetrating peptides can complex with siRNA through ionic interactions between the negatively charged RNA and positive lysine residues. Monomeric 16-residue α-helical stapled peptides were developed that exhibit an optimal balance between binding affinity of siRNA, release of siRNA in the cytosol, and overall cell penetrating ability.80 The bridging residues (R)-2-(7′-octenyl)alanine (R8) and (S)-2-(4′-pentenyl)alanine (S5) were placed at the i and i+7 positions in multiple locations throughout the 16-residue peptide for RCM to commence.80 The stEK peptide (staple between residue 8 and 15; LKKLLKLR8KKLLKLS5G) showed the greatest degree of cell penetration (>70%) via flow cytometry, greatest degree of hydrophobicity via HPLC, and greatest percent α-helical content (75%) via CD spectroscopy.80 Despite the large degree of penetration, the stEK peptide-siRNA complex disappointingly delivered approximately 20% gene silencing RNA to the abundant gene target CypB. It was determined, however, that the peptide-siRNA complex was getting trapped in endosomes, so histidine residues were incorporated into the stEK peptide. His residues have a pKa value similar to that of endosomes and, thus, once trapped inside the endosome they can undergo changes in protonation state to allow siRNA release.80 The new peptide, LKH-stEK (sequence: LKHLLHLR8KHLLKLS5G), exhibited greater than 90% knockdown of target genes at a 1:50 molar ratio of siRNA to peptide.80 This highlighted the immense potential of hydrocarbon stapled peptides as siRNA delivery vehicles and emphasized the importance of the proper balance between cell penetrating ability and binding affinity to siRNA to ensure optimal delivery. The Wnt/β-catenin signaling pathway is responsible for regulating stem cell pluripotency and cell fate during development. When the pathway is in the ON-state, Wnt causes the dissociation of β-catenin from a cytoplasmic protein complex including Axin, which allows β-catenin to stimulate transcriptional activity. In the OFF-state, β-catenin stays bound to the cytoplasmic protein complex leading to the phosphorylation of β-catenin and subsequent ubiquitination and proteasome degradation.81,82 The β-catenin-Axin interaction serves as an R

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observed.84 Enhancing the helicity of the BimBH3 peptide with a hydrocarbon staple appeared to work against its affinity for the “pro-survival” proteins, as indicated by cellular uptake and measurements taken from cytochrome c release from the mitochondria (directly assessing the process of apoptosis).84 The design of such peptides is highly important, as the staple may interact with the target and the rest of the peptide, disrupting the key binding interfaces but also interactions within the peptide itself.84 The peptide discussed was a “weakened-bydesign” construct for HSQC-NMR studies, with suboptimal characteristics that could hinder both stabilization and cellular uptake (i.e., modifications were for a particular structural study), thus showing that not all stapled peptides are alike, even with a conserved BH3 sequence.62 Even a construct that is very potent in vitro may prove a lack of activity in certain cell studies, such as the mouse embryo fibroblasts analyzed in the case of BimBH3, and yet in others (e.g., hematologic cancer cells), the apoptotic pathway may be properly activated.62,84 These conflicting results indicate the importance of choosing the proper sequence and testing the proper biological properties to determine the activity of these novel compounds, moving forward.62 One of the early challenges of this technique was managing the use of staples across side chains, thus sacrificing α-helical side chain identities for use in the tether. Newer techniques have found ways to circumvent this in the creation of macrocyclic helical peptides.

Figure 11. Hydrogen bond surrogate in a helix. A. Depiction of the hydrogen bond along the backbone of the α-helix, a side-chain crosslinked α-helix, and the hydrogen bond surrogate (HBS) α-helix, replacing the carbonyl oxygen of the ith amino acid and the amide of the i+4th amino acid.86 B. Representation of the olefin groups that replaced the carbonyl oxygen of the ith and the amide of the i+4th amino acid hydrogen bond and their covalent bond in the HBS helix representation.85 Figure created by author from data in refs 85 and 86.

5. HYDROGEN BOND SURROGATES CONSTRAIN THE PEPTIDE BACKBONE INTO AN ORGANIZED HELIX As has been described, low molecular weight helix mimetics that can participate in selective interactions with proteins are advantageous in the development of new drugs.85 Molecules with such inhibitory potential are key in the realm of experimental molecular biology and biochemistry and pave the way for novel therapeutics.8,86 Many studies, including those described herein, have been performed to construct stabilized and preserved helical conformations in short peptides, establishing peptide-based oligomers with conformational constraint, proteolytic stability and a desired array of protein-like functions.8 Helix stabilization that overcomes the fundamental nucleation barrier, limits flexibility and helps to initiate and propagate helicity is invaluable.85 The concept of the hydrogen bond surrogate, inducing a conformational constraint along the backbone of a short peptide, is a highly efficient method as compared to other helical stabilizing strategies and will thus be described. Its employment of a macrocycle across the backbone, and not through side chain substitution, presents a unique alternative that does not sacrifice peptide functionality across its residues.

intrinsically restricts the stability of the short α-helices.85 Hydrogen bond surrogates preorganize amino acid residues to initiate helix formation, constraining short peptides allowing them to have stable structures and allow for targeted interactions.8 The nucleation constant, σ, refers to the organization of three consecutive amino acid residues in an αhelical turn and is usually very low (≪ 1) in unconstrained analogues, disfavoring helix formation.8 The HBS approach allows for a nucleation constant of about one, as a result of the efficiency and stability of the HBS method.85 By preorganizing the amino acid residues in the first turn of an α-helix, the HBS approach overwhelms the basic nucleation disposition and successfully initiates helix formation and preservation.9 The synthetic design principles of the HBS approach suggest that an accurate α-turn imitator is critical for helix nucleation and the initiation of helix formation.88 HBS methodology is favorable since placing the cross-link along the backbone of the chain on the inside of the helix excludes side chains, leaving solvent-exposed molecular recognition surfaces unblocked.85 Since the helical constraint is internal, any protein-binding properties of the now-helical peptide’s side-chains is not compromised.85 HBS α-helices contain a carbon−carbon bond from a ring-closing metathesis reaction which results in replacing the N-terminal main chain hydrogen bond between the carbonyl oxygen of the ith amino acid and the amide of the i+4th amino acid (Figure 11A).85,89 The replacement of a main chain hydrogen bond is only necessary at one location within the α-helix in order to organize the rest of the chain.

5.1. Background about Hydrogen Bond Surrogates (HBS)

According to the helix−coil transition theory, α-helices comprised of 10 or fewer amino acids are expected to be fairly unfolded due to a low nucleation probability.87 The hydrogen bond surrogate (HBS) approach has advantages over other stabilizing strategies by focusing on improved initiation of nucleation. A limitation of the hydrocarbon bridged side-chain “staples” is that side-chain functionality is generally sacrificed to nucleate stable helical conformations, which makes the modified side chains unavailable for molecular recognition and blocking at least one face of the constructed helix (Figure 11A).86 The HBS approach is based on the helix−coil transition theory of peptides: the energetically demanding organization of three successive amino acids constructed into a helical orientation S

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Scheme 1. Process of Creating Hydrogen Bond Surrogate Peptides Includes Building a Peptide on Rink Amide Resin Using Standard Techniques, Including Incorporation of a Bis-Olefin at the N-Terminus and N-Substituted Amine on Residue Threea

a

Microwave irradiation and Grubbs catalyst close the hydrocarbon staple.85

Scheme 2. Synthesis of Hydrocarbon Stapled Peptidesa

a A. O-allyl serines placed in the i and i+3 positions were added to the β-peptide prepared using standard solid phase techniques. A mixture of trifluoroethanol (TFE) and dichloromethane in the presence of Hoveyda-Grubbs generation II catalyst promoted the closing of the ring to form the staple.110 B. RCM of bis olefin-containing β-peptides using Grubbs II ruthenium catalyst in dichloromethane to give the carbon tether, followed by hydrogenation for the alkyl chain.111 C. β3-Ser (O-allyl) residues incorporated into positions along one face of the β-peptide helix and treated with the First Generation Grubbs catalyst for ring closing into the carbon tether.103.

5.2. Synthesis of Peptides Constrained by Hydrogen Bond Surrogates

mentioned, the connection of these groups through the backbone instead of side chains removes any encumbrances on the helix surface, increasing α-helix stability.86 To mimic the CO of the ith amino acid residue and the NH of the i+4th amino acid residue hydrogen bond as closely as possible, a covalent bond of the type CX···Y−N is created, where X and Y would be part of the i and the i+4 amino acids, respectively (Figure 11B).85 Previously employed stabilization methods for

Hydrogen bond surrogate α-helices can be synthesized from commercially available amino acids or their simple derivatives and do not require enantiomerically pure preparation.86 The synthetic method of RCM for bis-olefin peptides (Scheme 1) brings together the CO and NH of the i and i+4 residues beginning at the N-terminus of the peptide (Figure 11B).85 As T

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Figure 12. Structure of HBS helix and scheme of disulfide-linked HBS. A. High resolution crystal structure of a short HBS α-helix with the RCMmediated macrocycle displayed (CIF: 675526) and an HBS a-helix overlaid with an ideal α-helix, adapted with permission from ref 9. Copyright 2008 Elsevier. B. Scheme to represent the disulfide-linked HBS helices through oxidation, with the N-terminal tripeptide sequence nucleating and preorganizing the peptide into an α-helix. Figure created by author from data in ref 2.

angles expected for a fully hydrogen-bonded short α-helix.9 The backbone conformation of the HBS α-helix in the crystal structure superimposes onto the backbone conformation of a model α-helix with an RMS difference of 0.75 Å (Figure 12A).9,85 This supports the hypothesis that stable helices can be obtained by the HBS approach, verifying that the HBS α-helix structure shows a high degree of conformational stability.85 A peptide in the conformation of an α -helix would show particular NOE cross peaks between key atoms of i, i+1; i, i+3, and i, i+4 positions, and each of those were observed in the spectrum from NOESY 2D NMR spectroscopy of the HBS αhelices. The nuclear Overhauser correlations were used to assign 1 H NMR resonances and the backbone proved to be similar to that of an idealized α-helix.85 Even the final residues at the Cterminus provided NOE signals that were detectable, indicating that the ends of the helix did not start fraying. Results from CD spectroscopy on the HBS α-helices showed, on average, 65−70% helicity, meanwhile unconstrained analogues were unstructured.85,86 HBS α-helices preserve this helical conformation even at 85 °C, revealing a remarkable amount of thermal stability, consistent with the theoretical predictions for a nucleated helix.9 The HBS approach was intended to afford prenucleated helices such that the nucleation constant, σ, would be close to 1, indicating a well-folded structure, and these experiments have shown this to be true.9 Structural characterization results taken together, the HBS approach provides peptides that are highly helical, and support nucleation and preservation of the helical conformation in many conditions. The HBS method can effectively stabilize α-helical conformations in biologically relevant sequences, resulting in their resistance to proteolytic degradation as compared to unconstrained analogues.86 Proteolytic cleavage is one of the main limitations to the in vivo effectiveness of peptides as pharmaceuticals; moreover, proteases are known to bind their substrates in linear or beta-strand conformations, and peptides locked into helical conformations have a greater resistance to proteolytic degradation.86 In a particular example, HBS helices that mimic BH3 domains were compared with the linear BH3 domain of the Bak peptide, in a stability assay in the presence of

inducing α-helicity in peptides mostly utilized side chain crosslinks through hydrocarbon staples, lactam, disulfide, and metalmediated bridges to initiate helical formation.89 Early versions used a hydrazine bridge to imitate the backbone hydrogen bond, but the result were weak in comparison and the carbon−carbon covalent bond developed in the HBS approach truly stabilizes helical structures.89 The bis-olefin residues are added to the growing chain of amino acids using standard solid-phase peptide synthesis; the peptide is then converted to the corresponding HBS α-helices by treatment with the metathesis catalyst.85 In early experiments, standard ring-closing metathesis did not generate a significant amount of product; this lead to the evaluation of multiple RCM catalysts and reaction conditions.85 It was discovered that conventionally heating the commonly used Grubbs catalyst was unsuccessful in yielding the necessary macrocycle; meanwhile, successful RCM synthesis of resinbound bis-olefins was only possible with the second-generation Grubbs catalysts such as Grubbs II and Hoveyda-Grubbs II (Scheme 2).85,90 With the use of these second-generation catalysts and exposure to microwave irradiation, RCM production was optimized, and gaining access to HBS macrocyles was achieved with shorter reaction times and high yields.85,90 Typically, the trans alkene product was obtained. The RCM-mediated synthetic method is favorable for HBS α-helices because it works well with standard resins and commercially available amino acid derivatives, allows inclusion of any amino acid residue at any position in the helix, is compatible with solid phase and produces the final product on resin with no extra modifications required except cleavage and purification.13,85,90,91 The HBS approach can stabilize α-helical conformations in short (7−12 amino acid) peptide sequences.9 5.3. Structural Characterizations of HBS α-Helical Peptides

The adopted conformation and stability of the hydrogen bond surrogate α-helices have been extensively analyzed through 2D NMR and CD spectroscopy.9 A high resolution (1.15 Å) crystal structure of a short HBS α- helix showed that the RCMmediated macrocycle accurately reproduced the conformation of a canonical α-helix.9 It was found that all of the i and i+4 C O and NH hydrogen bonding partners fell within distances and U

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trypsin.86 All of the sequences had a specific arginine residue after which trypsin was expected to cleave; however, it was positioned two residues away from the macrocycle in the HBS helices. One HBS helix was cleaved 30-fold slower than the unconstrained control peptide.86 The increase in “locked” helicity of the HBS helices resulted in further decreases of initial velocity of trypsin cleavage. Overall, stabilities of the HBS peptides ranged from 2-fold to sixty-fold more stable against proteolysis when compared to the unfolded, unconstrained peptide.86 Along with increased stability against proteolytic degradation, the HBS approach allows for internal constraint of the α-helix maintaining side-chain functionality and thus permits the peptides to bind to a protein target with high affinity compared to unconstrained peptides.86

to IQN17, mentioned earlier as a soluble model of the gp41 HR1 three-helix bundle.58 The unconstrained peptide had a Kd for IZN17 of around 37 μM, whereas the best HBS helices had estimated Kd values of 90%. Both CD spectroscopy and NOESY 2D NMR experiments were used to characterize and determine the X

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Figure 13. Helical net diagrams depicting the β-peptide scaffold. A. Helical net diagram of the original β-peptide that emulates the α-helical domain of p53. B. Helical net diagrams depicting the replacement of key residues with a diether or hydrocarbon based bridge, “stapling” the β-peptides that mimic p53 using RCM techniques.103 Figure created by author from data in ref 103.

hydrocarbon bridges in positions two and five (taking the place of an isoleucine pair) were 4- to 8-fold worse than the unbridged versions. Molecular modeling in silico suggested that a steric clash of the hydrocarbon bridge with the hDM2 surface was present in the two-five position but not in the four-seven arrangement.103 Uptake of the bridged β-peptides was monitored using flow cytometry. β-Peptides with either the diether or hydrocarbon bridges in the four and seven position were more efficiently taken up by cells whereas the β-peptides with bridges in positions two and five displayed much poorer cell uptake, much like the unbridged versions.103 Localization of the β-peptides upon cell uptake were analyzed by confocal microscopy and HeLa cells were treated to visualize recycling endosomes and nuclei. Once again, β-peptides with the bridges, regardless whether diether or hydrocarbon, between positions four and seven showed a widespread distribution across endosomes, nuclear and cytosolic compartments, whereas those with bridges in the two and five positions were not distributed as well.103 Resultantly, a correlation can be made between affinity for hDM2, cellular uptake and distribution. A subtlety exists between increases in stabilization of artificial helical structure and overall biological function of peptidomimetics: the proximity of a staple to key residues in protein− protein interactions can have major influences on potency and effect. Continuing studies using unnatural backbones and macrocyclic stabilizing effects on helicity would be needed to apply these compounds in vivo. Controlling and influencing the helicity of short peptides using macrocyclization is a technique still being honed and developed in novel ways in natural, αpeptides, as well.

to form a complex salt bridge and a face of β3-Val residues to influence hydrophobic packing through intercalation of the nonpolar groups, and provide amphiphilicity to the molecule. The β3-Tyr was added for spectrophotometric detection for concentration determination and β3-Ala as a place holder. CD spectroscopy results obtained in Tris buffer indicated that the introduction of the diether bridge did not significantly increase or decrease the extent of 14-helical structure.103 The strategy was then applied toward a known β-peptide that mimicked the α-helical domain of p53 and bound hDM2 with tight affinity. The original β-peptide (Figure 13A) contained a version of the critical Phe, Trp, Leu motif intrinsic to p53 recognition of hDM2, presenting β3-Phe, β3-trifluoromethyl phenylalanine, and β-Leu on one face of the peptide. The non-natural substitute for Trp had previously been shown to facilitate favorable binding affinity to hDM2.106 The other two faces of the original βpeptide have the ornithine-glutamate complex salt bridge and a face of β3-isoleucines with a similar effect to the β3-valines mentioned above. In the stapled designs, either one pair of isoleucines or one salt bridging pair was replaced with the staple, respectively (Figure 13B). The variants included either the (Oallyl)-β3-Ser for a diether bridge or an (S)-3-aminooct-7-eonic acid) to generate a fully hydrocarbon bridge (Figure 13B). All of the bridged β-peptides were moderately more 14-helical than the unbridged versions when compared using CD spectroscopy. 6.2. Biological Functionality of Stapled β-Peptides

Using the stapled β-peptides designed to target hDM2, described above, several in vitro studies as well as cellular uptake and localization studies succeeded. First, a direct binding fluorescence polarization study, where β-peptides were tagged with a small fluorophore molecule, showed that diether or hydrocarbon bridges between positions four and seven (taking the place of a salt bridge) bound hDM2 2-fold better (Kd = 50− 100 nM versus Kd = 100−200 nM) than the unbridged analogues.103 However, the β-peptides with both diether or Y

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Scheme 3. Various Synthetic Methods for Constraining Helicesa

a

A. Constrained peptides synthesized by orthogonal protection, coupling of an alkanediamine linker and selective deprotection followed by coupling to provide a crosslink.17 3B. Standard solid phase methods used, with alkene and tetrazole-modified lysine incorporation, followed by UV irradiation allowing for coupling to complete the tethered ring.116 3C. Peptides included azide and alkyne functionalities which cyclized into a tetrazole ring upon treatment with copper sulfate and ascorbic acid in 2:1 H2O/t-BuOH, allowing for tethers along the peptide helix.119 3D. The thiol−ene stapling system, allowing a diene of unspecified length to couple two cysteine thiols in NMP to cyclize through the tether.122.

7. SOME ADDITIONAL METHODS FOR CONSTRAINING PEPTIDE HELICES Many new and diverse methods have been established as the field continues to grow and the inhibitor and the therapeutic potential of peptidomimetics expands, as well as broader biological applications.

solid-phase techniques; and (4) the helicity must stand the test of varying solvents, a pH range, and temperatures.17 Linear peptides were synthesized using standard Merrifield methods and the constraints were made by the removal of the 9fluorenylmethyl ester from glutamate residue three, coupling with alkanediamine, followed by the removal of the allyl ester from glutamate residue 10 and cyclization (Scheme 3A).17 Completed peptides were cleaved from the resin with HF and purified.17 Peptide characterization with 2D 1H NMR using TOCSY and ROSEY spectra indicated through ROE crosspeaks from residues i and i+3 that the peptides were highly helical. This was confirmed by CD spectroscopy showing 84% helicity for the bridged peptide as compared to 20% for the unbridged control, at 7 °C in an aqueous based buffer.17 The bridged peptide also retained its conformation under thermal denaturing conditions. The amide tether in i and i+7 was generally successful for inducing α-helicity, and allowed for

7.1. Alkanediyl Tethers

Using the α-helical bee venom peptide apamin, with its two disulfide bonds, specifically the C-terminal helical region, as well as a peptide derived from the C-peptide of RNase A as model helical sequences allowed for the development of a unique amide based tether with several established criteria.17 These different conditions included (1) the presentation of random amino acids so as to not limit the amino acid identities in the sequence; (2) a short peptide of less than 20 residues must maintain helicity approaching 100% in water at room temperature; (3) the synthetic process must be straightforward and compatible with Z

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charged stapled peptides showed moderate, reproducible activity with 1.6-fold increases in luciferase activation.116 Overall, the unique stapling strategy, taken together with other forms of sequence control, led to moderately active peptide inhibitors of both MDM2 and MDMX with abilities quantified in vitro and in cells. This strategy, with its clean use of photoinducible tethering in its synthesis, could prove useful and sustainable for the creation of helical peptides with biological function. The cross-link that results from this work is reminiscent of “click” chemistry and the use of triazole-staples as a versatile method of tethering residues across a helix.

surrounding variability of the residue in short 12−13 residues peptides.17 This new type of tether resembles the lactams discussed above; however, it introduces a longer, more flexible cross-link that, with further applications, could allow for biologically active helices and a closer look at helix formation, propagation and stability. However, simplifying synthetic pathways to stapling is also considered a goal for the field and using orthogonal and photoinducable protection a unique method. 7.2. Photoinduced 1,3-Dipolar Cylcoaddition Stapling

A particular study, using phage display, indicated that a peptide dual inhibitor (PDI) of the p53-MDM2/MDMX protein− protein interactions had the sequence LTFEHYWAQLTS.115,116 However, the initial peptide was not cell permeable and improvements to its design sought to increase peptide cell entry with retention of the dual inhibitory activity. A photoinduced 1,3-dipolar cycloaddition stapling reaction that would bring together tetrazole and alkene moieties on lysine residues in varying positions could lead to enhanced cell permeability, as prior studies in 310 helical model peptides showed improved physiochemical properties.116,117 By using photoinduction to prompt macrocyclization, versus metal catalysis or other chemical catalytic means, a facile, green, and direct method was used with few side products produced. Three possible positions for stapling were identified: two in the i, i+4 position where there had been a Glu-Asp salt bridge pair or a His-Gln pair, and one i, i+7 position between a Glu and Thr. For synthesis, alkene and tetrazole-modified lysines were prepared first and then used in standard solid phase peptide synthesis. After resin cleavage, and purification, linear peptides were brought up in acetonitrile and water and exposed to UV irradiation at 302 nm for 2 h (Scheme 3B). Using an ELISA, the stapled peptides were compared with PDI, and the peptide with residues four and eight cross-linked showed comparable activities against both MDM2 and MDMX, whereas the other i, i+4 cross-linked peptide, between residues five and nine, showed a 4-fold drop in activity from MDM2 and 3-fold drop against MDMX.116 However, the i, i+7 stapled peptide had a 7-fold increase in MDM2 activity and slight improvement to MDMX activity, in vitro. These results indicated that the placement of this tether was key in determining activity and more than likely target-specific. To examine the efficacy of the peptides in cells, positive charges were added to the peptides to improve their cellular uptake. The remaining nonessential residues (His-5, Ala-8, Gln-9, and Thr11) of PDI were examined and replaced with either one or two arginines, which did cause a 2−6 fold drop in activity against MDM2, with small changes against MDMx.116 Monitoring the cellular uptake of the peptides was performed using fluorescence microscopy using the intrinsic fluorescence of the pyrazoline cross-link as a tag. The i, i+7 peptide that was most active in vitro was not cell-permeable when neutral in charge, however when a +1 charge was added a punctuated fluorescence pattern was seen, indicating peptide uptake by a pinocytotic mechanism with peptides trapped in endosomes.116 Peptides with +2 charges had more diffusive patterns indicating escape from the endosomes. One particular stapled peptide with a continuous positive “patch” in an i, i+4 direction gave a bold and uniform fluorescence distribution in the cytoplasm.116 Linear peptides, even with added positive charge, were not taken up by cells. In vivo activity utilized a p53-dependent luciferase reporter system. Linear control peptides were inactive whereas all positively

7.3. Triazole-Stapled Peptidomimetics from “Click” Chemistry

The Wnt/β-catenin signaling pathway, described above, is important in tumorigenesis in several cancers, including colon, prostate, and melanoma, by helping to regulate the cell cycle and apoptosis.118 Activation of β-catenin requires a supercomplex to form, including an interaction through its large binding groove to a specific 25-residue helical segment of B-cell lymphoma 9 (BCL9).119 Using a Huisgen 1,3-dipolar cycloaddition reaction to generate triazole-stapled BCL9-based α-helical peptides, the design of potent and metabolically stable, helical peptides resulted. The Cu(I)-mediated reaction (often called a “click” reaction) was used to generate a 1,4-substituted 1,2,3-triazole linker between side chain azido and alkynyl groups at the i and i +4 positions, balancing high yields with mild reaction conditions and biocompatibility.120,121 For single stapled peptides, two residues in the BCL9 helical sequence that were central, solvent exposed and uninvolved in recognition of β-catein were chosen to bear the residues for stapling. Using computational methods, it was shown that the least amount of distortion of the α-helical backbone was found with an L-azido-norleucine (L-Nle(εN3) and D-propargylglycine (D-Pra) and early designs used both LNle(εN3) in the i position and either D- and L-Pra in the i+4 spot (Scheme 3C).119 For synthesis, Fmoc-L-Lys-OH was converted to the azide and D- or L-Pra included in the solid phase peptide synthesis. Cyclization using CuSO4 and ascorbic acid in a 2:1 H2O/t-BuOH system worked well promoting the click reaction (Scheme 3C).119 A linear version that did not receive treatment with the catalyst had a binding affinity for β-catenin half that of the wild type BCL9 24-residue peptide. Meanwhile, the triazolestapled peptides were two to 4-fold more potent than the wild type, for L-Pra or D-Pra, respectively.119 CD spectroscopy indicated that the triazole-stapled peptide with all L-amino acids was 66% helical in PBS buffer as compared to the approximately 45% helical wild type peptide. Interestingly, the stapled peptide with D-Pra in the i+4 position showed 90% helicity, an unexpected result as non-natural stereochemistry is thought to break helicity.119 Shortening the linker by a carbon resulted in a loss of affinity for β-catenin and a reduction in helical structure, whereas lengthening the linker, also by one carbon, demonstrated weaker affinity and no helix stabilization.119 Even shifting the triazole linkage by one atom reduced binding affinity and negatively affected solubility. Shifting the location of the triazole greatly perturbed helical structure, inferring that the dipole of the triazole may influence the amount of helicity.119 Reversing the position for Nle(εN3) versus Pra had no effect in the all-L version but in the L-D pair, DPra in the i position had 6-fold weaker affinity.119 Gleaning information from these optimization trials, a double stapled peptide of longer length was developed to improve binding affinity, helical content and metabolic stability. The two AA

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Scheme 4. Two-Component Stapling Techniquesa

a

A. Azobenzene-stapled across two cysteine residues using an acetamide group. Treatment with light promotes the trans to cis transformation which promotes helicity.127 B. A dialkynyl linker “clicked” across two azidoornithines through a triazole ring.126,130 C. Cross-linking of two cysteines (in a peptide with a fluorescent moiety at the N-terminus) using arylation with perfluoroaryl groups in mild conditions, allowing for regioselective disubstitution in both single and biphenyl systems.132,133

tion.122,123 As mentioned above, disulfide bonds in nature are formed from oxidation of thiol groups, typically under enzyme mediated biological control in the presence of a catalytic amount of base.124 In eukaryotes, these disulfide bonds form on proteins that are sequestered in the lumen of the rough endoplasmic reticulum, or in the mitochondrial intermembrane space, away from the reducing environment of the cytosol, limiting the ubiquitous use of disulfides in proteins throughout a biological system. Therefore, a synthetic system that makes use of the naturally found thiols on proteins, or short, expressed peptides, in a two-component staple could build on the groups found in nature with a more robust way to control helicity. In a twocomponent thiol−ene stapling system, first a short peptide with sequence YCKEACAL was produced with multiple unprotected functional groups, incubated with a specific diene in the presence of 2,2-dimethoxy-2-phenylacetophenone (DMPA) in NMP producing 90% yield of the cyclized product (Scheme 3D).122 The coupling of the thiol−ene was selective in the presence of amines, alcohols, and carboxylic acids.122 This process was then used on a larger sequence, a peptide analogue from prior literature that had hydrocarbon stapling as described above, shown to inhibit the Wnt signaling pathway.125 In a direct comparison to the stapled mimetic (Axin) with alkenyl-amino acids in positions i/i+4, an unstapled version was synthesized replacing the two unnatural amino acids for the staple with cysteines, which was then reacted with a diene (of two possible

staples were placed adjacent on two helical turns, with a full turn between them, with reversed orientation to eliminate possible “mixed” staples. The L-Nle(εN3)/D-Pra combination gave a 2− 3-fold improvement in binding affinity as compared to the best singly stapled version, and helicity of greater than 95% by CD.119 Cell media from actively cultured colon cancer cells was used as a test for resistance to proteolytic degradation. The doublestapled peptides were 60−75% stable and did not degrade after more than 90 min of treatment whereas the wild-type peptide had only 10% remaining in the same amount of time.119 Having two strategically placed staples in a longer peptide allows for the potential of future peptide inhibitor development with possibilities in cell permeability and true in vivo elaboration. Having two synthetic staples, however, presents a unique challenge to the ease of preparation, whereas a dual-tethering method amenable to derivatizing naturally found or expressed peptides could expand such possibilities. 7.4. Thiol-Ene Coupling Approach to Helical Tethers

Many of the methods described have incorporated unnatural amino acids into a synthetic peptide chain, yet a strategy that could bypass the necessity of peptide synthesis would have potential as a modification to recombinantly expressed peptides.122 A thiol−ene click reaction could modify existing cysteines, which are generally found in low abundance in proteins, using specificity to olefins and quick transformaAB

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lengths, Scheme 3D).122 The final peptides were examined by CD spectroscopy; an unstapled version and the seven-carbon linker version had low α-helicity; however, the eight-carbon linker peptide had a high helicity on par with the Axin mimetic in the literature.122 Next, the process was used in the creation of a p53 mimetic for MDM2, using another hydrocarbon stapled peptide as a model and replacing an i/i+7 pair of unnatural amino acids with cysteines. Again, two possible lengths of dienes were used to cyclize the peptides at the cysteine residues. CD spectra showed all of the stapled peptides had a significant αhelicity over the unstapled control peptides, with direct similarities between the hydrocarbon stapled and thiol−ene stapled peptides. To verify the comparison between hydrocarbon versus thiol− ene staples, the peptides were tested in an ELISA to quantify the extent of inhibition of the p53-MDM2 interaction.126 The thiol−ene stapled peptide was as effective as the hydrocarbon stapled mimetic, showing IC50 values around 50 nM. In a cell viability assay, p53 wild-type and p53 null colorectal carcinoma cells were used and the thiol−ene peptides were compared with hydrocarbon stapled mimetics. Both forms of the constrained peptides selectively induced apoptosis in the p53 wild-type cells but not in the p53 null cells. It was clear that thiol−ene stapled peptides, created from native, unprotected peptides, could recapitulate the structure and activity achieved by hydrocarbon stapled mimetics. This opens the potentiality of future peptides with biological functions that have controlled helicity but can be naturally expressed. Several other methods that promote helicity through macrocyclization using tethers through dual-component systems, removing the need to incorporate unnatural amino acids in the peptide synthesis, are being developed.

receptor protein was controlled by simple photoswitching the azobenzene macrocyclic linker. This provides great versatility, by adding the staple across natural cysteine residues and moderating the biological effects of the compound using light. In addition to ease of preparation and adaptability, methods that apply two-component stapling across a large repertoire of peptides bearing residues that are amenable to a variety of linkers presents another great direction in the field. 7.5.2. “Double-Click” Stapling and Its Applications. Creation of a synthetic library of peptides with varying staples conferring different properties to the peptide upon helix formation is an important direction of two-component stapling. Using dialkynyl linkers in an i, i+7 “double-click” method, two azidoornithine residues were brought together with a diethynylbenzene in a peptide with a sequence based on p53 (Scheme 4B).127,131 The peptide had a high affinity to MDM2 in vitro using fluorescence polarization and isothermal calorimetry measurements but initially lacked natural cellular uptake in a reporter assay.131 Modifications of the dialkynyl linker to contain positively charged Arg residues allowed for cellular uptake of the peptide and its activation of p53 in the cell; clearly the linker can bestow an array of properties on the intervening peptide, while also working to control inherent helicity.132 By using click chemistry, the bioorthogonal copper reaction allows for a single synthetic step without the need for optimization. This technique can then be applied in a more general way; using such methods to introduce tethers across cysteines in peptides of varying length and beyond a single chain. Methods that append cysteines in such a manner can be applied to promote helicity and attach several chains leading to the development of de novo helical proteins.

7.5. Other Two-Component Stapling Techniques

8. TOWARD SYNTHETIC HELICAL PROTEINS As has been discussed, there are many methodologies for building on reactive cysteines across peptides. Cross-linking two or three available cysteines using arylation with perfluoroaryl groups uses mild conditions and does not require the cysteines to be modified synthetically.133,134 By coupling cysteine thiols to perfluorinated substrates (single and biaryl systems) the peptides exclusively became disubstituted in a regioselective manner. Only the 1,4 substitution of the benzene ring was noted, lending evidence to rationalizations of steric hindrance at the 2,5-ortho sites and activation of the 4-para-sites of the aromatic ring by thioether formation (Scheme 4C).134 The sulfur is thought to stabilize negative charge as a result of the first substitution, leading to the increased probability of the second thiol substitution occurring.134 This was found in both the single and biphenyl systems (Scheme 4C). The first peptide designs using this type of tether were developed to bind the C-terminal domain of HIV-1 capsid assembly polyprotein C-CA, a target previously used with peptides bearing the hydrocarbon staples previously discussed above.134 The perfluoro-linker did contribute some unexpected signal upon treatment with CD; however, the stapling was still found to provide a CD signature indicative of an α-helix. The perfluoroaryl cross-linked peptides showed enhanced proteolytic stability when compared to unstapled peptides, when tested against trypsin and chymotrypsin.134 When tested in a surface-plasmon resonance (SPR) assay with immobilized CCA, the cross-linked peptides showed better binding affinity than those nonhelical counterparts, when evaluated at 5 μM concentrations.134 Upon labeling the peptides with FITC fluorescence dye, it was seen that the thiourea moiety allowed

While the thiol−ene method above relates to recombinantly expressed peptide modifications, there is much recent work on two-component stapling methods that utilize a bifunctional linker compound in synthetic methods, stapling two non-natural amino acids on a peptide, leading to many potential combinations, including inter- and intramolecular tethering.127 The major advantage these synthetic two-component systems is the use of diverse stapling linkage combinations without the need to introduce non-native amino acids into the backbone of the peptide.127 7.5.1. Photoswitchable Two-Component Azobenzene Staples. Having a photoswitchable group that uses UV/visible light to control helicity in a tethered peptide is an interesting recent direction, and based on the orientation of the appended groups, allows for tunable conformational changes that can be taken inside biological systems. This was achieved by an azobenzene-based staple with 2-iodoacetamide groups introduced in the i, i+7 positions to cysteine residues (Scheme 4A).128 Prior to treatment with light, the moiety has a trans conformation and after irradiation at 380 nm for 5 min cisazobenzene is obtained and the peptide becomes helical, as found using CD spectroscopy.128 This method of using switchable azobenzene stapling in the i, i+4 (or 7, or 11) positions was applied to peptides that bind Bcl-XL, with evidence that switching “on” helicity allows binding to the protein target.127,129 A similar use in peptides that regulate clathrin-mediated endocytosis target an intrinsic protein, the AP2 complex, and were not just successful in vitro, showing reversible affinity upon photoswitching, but also cell-permeability.127,130 In cellular studies, endocytosis of the transferrin AC

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helices that interact in longer protein-based coiled coils, such tertiary structure mimetics could be useful inhibitors when applied to protein−protein interactions involving macrocyclic interfaces in nature.135 Working toward the goal of synthetic proteins, macrocyclic constraints can contribute to stabilization of helices in “mesosized” proteins (i.e., those that lie between the size of small peptides and conventional proteins).136 Using Rosetta and Tertiary Motifs (TERMS) computational modeling to design small proteins with multivalent cross-links, the most helical proteins were ascertained, using hydrophobicity at a small nonpolar core. After in silico optimization, the CovCore proteins (short for “covalent core”) were prepared by chemical synthesis. The three-helix structure of CovCore Protein 3H1 was previously unseen in nature.136 A 1, 3, 5-trimethyl benzene group formed three thioether bonds across each individual helix, tethering them together into a helical “cycle,” nucleating a small hydrophobic core at the center of the structure.136 Including xylyl and mesityl cross-links provided covalent constraint to coax the helical fold, but also to form the hydrophobic core of the individual peptide chains into a distinct tertiary structure not easily found.136 Due to the modular nature of the cross-links, they were fully compatible with display technology and highthroughput screening and the further development of such proteins with “dialed-in” function presents an exciting future direction. Both solution NMR and X-ray crystallography confirmed their structures as unique, helical based, and easily accessible.136 Predicting the overall fold of these mesosized proteins using the CovCore template and building in residues capable of recognizing another protein target will allow for computational design of a new era of molecules that can behave as potential inhibitors of protein−protein interactions.

for easy labeling (not seen in the hydrocarbon stapling method), and the perfluoroaryl cross-linked peptides were significantly taken up by HEK293T cells, localized in both endosomes and the cytoplasm, as opposed to nonstapled peptides.134 On the heels of these favorable results, the novel staple was applied to one helix among many on a prior engineered affibody.134 This antibody mimetic was previously identified as having a 1 nM affinity toward human epidermal growth factor 2 (HER2) receptor. By stapling one of the helices, the study tested whether the methodology would be tolerated in the total chemical synthesis of proteins. Native chemical ligation was used to adjoin each of the three helical fragments and then the perfluoroaryl cross-link applied to the C-terminal peptide. CD spectra showed that the staple did not alter the overall protein helical signature and thermal denaturation values remained consistent.134 Similar binding between the stapled affibodies to the HER2 receptor (using SPR) was observed when compared to the nonstapled version, lauding this as the first example of staple incorporation into a peptide as part of a larger, synthetic protein without significant changes made to the functional properties of the protein.134 As described in earlier sections, coiled coils are a multimeric motif found in many protein complexes used for biological processes. Emulating coiled coil interactions in protein complexes would have significant inhibitor potential, but the process of creating stabilized helical bundles under discrete control is still being actively investigated. Several strategies that began with simple dimeric helices in synthetic sequences used the concept of replacing an ionic bond with a covalent one, thus instituting macrocyclic intervention to control helical content in helical bundles. Using a disulfide bond at the a and d′ heptad repeat positions along with a covalent linker at the e and e’ positions heightened overall conformational stability.135 Strategies to hold together short helical dimers could be applied to longer chains in the design of synthetic, helically constrained proteins. There were three different approaches, using a model sequence that incorporated favorable hydrophobic residues at the a and d positions and ionic interactions intra-and acrosschains.135 These strategies included: (1) the use of HBS helices in each singular peptide of a dimer to control for individual helicity; (2) a covalent bond using bis-triazole linkers formed by copper-catalyzed azide−alkyne “click” cycloaddition to link interchain helices together, replacing e and e′ ionic interactions in the heptad repeat; and (3) a disulfide bond between chains at the hydrophobic a and d′ positions for further tethering.135 When considering the bis-triazole bridges of various lengths, incorporation of amino acids azidolysine or azidohomoalanine contributed most to eventual bundles of highest helicity.135 CD analysis revealed high helicity confirmed by 1D NMR using TOCSY and NOESY techniques. This success was applied to a native protein coiled coil that mimics the Nervy homology two (NHR2) domain of a specific AML1ETO transcription factor complex which interacts with the NHR2-binding motif (N2B) of E-proteins.135 This complex is important in leukemogenesis, and NHR2 uses a dimeric, antiparallel coiled-coil to interact with N2B. In the resultant bundles formed, placing a disulfide bond farthest away from the triazole linker gave the largest helical stability of the protein complex, shown by CD spectroscopy.135 Such doubly cross-linked helices displayed high conformational stability and also portrayed highest affinity when tested in an established fluorescence polarization assay using fluoresceinlabeled N2B peptide binding and compared with the native NHR2 coiled coil. By using these principles to design stable

9. CONCLUDING REMARKS As the field of helically controlled peptidomimetics continues to grow, many methods will be developed that utilize tethers, linkages, and bridges of non-natural origins to help organize and propagate folding stability. The folded α-helix remains the most sought after conformation for peptides as helices are at the forefront of protein−protein interactions that play key roles in many biological functions in the cell. Disulfide bond formation forms macrocycles in many proteins found in nature and their use in short peptides to initiate helicity introduced the first examples of peptidomimetics using macrocyclic restraint to promote helicity. The challenge with using disulfide bonds in synthetic peptides was their vulnerability to reduction, especially when taken in a cellular context. As other methods developed, metal-mediated macrocycles to generate helices was established as particularly successful in coiled-coil dimer formation. While promising, the challenge associated with these methods is the use of metal ions that would be nontoxic for use in cells, and the remaining constraints of pH, oxidation, and reducing conditions, as well as concentration. Lactam rings through side chains also prompted the folding and association of several helices into bundles, however the loss of flexibility by the use of amide bond formation remained challenging. The peptides displaying the greatest protease resistance, as well as potent function as inhibitors in vitro and in vivo, contained the hydrocarbon staples and hydrogen surrogate backbones from using ring-closing metathesis and unnatural amino acid substitutions, relying on bioorthogonal syntheses. Hydrocarbon staples use side chain tethering, thus the installation in a peptide must take into account the loss of these positions to the staple, therefore AD

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limiting what side chains can be used for recognition of a protein target surface. The HBS method liberates the side chains, but the unnatural backbone requires an involved synthesis and its versatility to different side chain substitutions in the region of the tether is still being studied. The hydrocarbon stapling strategy was also successful in applications toward foldamers with completely unnatural backbones, in the control of alternative helical structures that resemble α-helices and in application to successful inhibitors that transverse the cell membrane. The challenge of foldamers remains the use of an unnatural backbone requiring detailed synthesis in large enough quantity that must be tolerated by the cell for transport. Additional methods described highlight the varied and promising directions of the field. Improvements to safer “click” chemistry reagents, and the biological tolerance of the two-component stapling techniques will pave the way for future successes. The ability to make a universal system that is neither sequence nor context dependent but allows for discrete helical initiation and control while maintaining the important residues for molecular recognition remains a challenge. In this manner, such peptides could become the next wave of therapeutics due to their balance of toxicity, amphiphilicity, ease of creation, ability to fold, and the larger range of functionalities that can be installed alongside-chain recognition.137 Peptides have much potential for high selectivity and strong activity, large chemical diversity, and, by using strategic macrocycle inclusion, improved metabolic and proteolytic stability as well as bioavailability.138,139

department. He graduated as a member of the college honors program. His research interests include applications of CRISPR in oncology, the brain-gut connection, and high-throughput screening of chemical compounds for drug discovery. Gianna M. Barreto is a fourth year undergraduate student at The College of New Jersey studying chemistry. She will graduate in the spring of 2019 with a B.S. in Chemistry, ACS-certified, with research. She is a member of the national chemistry honor society, Gamma Sigma Epsilon, within the chemistry department. She is interested in research involving biological chemistry as well as research that enhances and formulates potential improvements within medicine. She began research with Dr. Guarracino during a mentored undergraduate research program in 2017 at TCNJ and continues research with her throughout the academic semesters. She plans to become a nurse and enter an accelerated BSN program after graduation and then to further her education by becoming a nurse practitioner in the near future. Alexis L. Oldfield is a first year Ph.D. student at Cornell University. She is studying biological and biomedical sciences with a concentration in cellular and molecular medicine. In 2018, she received her B.S. in Chemistry from The College of New Jersey, ACS certified with research. During the summer of 2016, she participated in The College of New Jersey’s Mentored Undergraduate Research Program under Dr. Danielle Guarracino, where her work involved finding a peptide inhibitor for thrombosis. In the summer of 2017, she worked in the Sample and Discovery Management division at Merck where she used mass spectrometry to identify targets and their purity. Her research interests include computer based drug design and the use of protein− protein interactions to target disease.

AUTHOR INFORMATION

Christopher M. Kouba is a current undergraduate student at The College of New Jersey graduating in May 2019 with a B.S. in Applied Mathematics and B.S. in Chemistry, ACS certified with research. He began research with Dr. Guarracino during the summer mentored undergraduate research program in 2017 at TCNJ and continues research with her as well as independent research in mathematical biology with Dr. Jana Gevertz. He will initially be working for Johnson Matthey as a technical commercial manager and after experience, he plans to serve in the Air Force before attending medical school to pursue a career as a pediatric electrophysiologist.

Corresponding Author

*Tel: (609) 771-2416. E-mail: [email protected]. ORCID

Danielle A. Guarracino: 0000-0001-6775-6564 Author Contributions

All of the authors contributed to the completion of the manuscript. Notes

Desiree Agrinsoni is an undergraduate Chemistry student at The College of New Jersey. During the semester, she is a research assistant for Dr. Danielle A. Guarracino’s research group through which she carries out cyclic peptide synthesis in efforts to inhibit a protein− protein interaction that leads to blood clot formation. From June 2018August 2018, she conducted research in an NSF funded summer research experience for undergraduates (REU) in Dr. Paramjit Arora’s laboratory at New York University. There she handled work with intrinsically disordered proteins in efforts to target a protein−protein interaction that is responsible for tumor formation. She is expected to receive her B.S. in Chemistry in 2020, ACS certified with research. Postgraduation, she intends to pursue a Ph.D. in chemistry; her research interests are in biological chemistry.

The authors declare no competing financial interest. Biographies Danielle A. Guarracino is an Associate Professor at The College of New Jersey. She received a B.A. in Biological Sciences with a concentration in Biochemistry and a double major in Chemistry from Cornell University in 2002, with distinction in all subjects. She then continued her studies at Yale University where she worked for Alanna Schepartz in bioorganic chemistry and chemical biology and received her M.S. in 2004 and Ph.D. in 2008, optimizing beta-peptide structure and function with applications to a novel viral target. From 2008-2010 she performed postdoctoral research at New York University with Paramjit Arora working on oligooxopiperazines as inhibitors to a protein−protein interaction involved in cancer. She joined the chemistry department at The College of New Jersey in 2010. Her research interests include peptidomimetics, cyclic peptide mimicry and synthesis, and controlling short helical structure of alpha- and beta-peptides using sequence design.

ACKNOWLEDGMENTS The authors thank The College of New Jersey, Department of Chemistry for continued support and to fellow colleagues and students for advice and support.

Jacob A. Riordan is a medical student (M.D.) at Cooper Medical School of Rowan University. He received an ACS-with-research certified B.S. in Chemistry from The College of New Jersey (TCNJ) in 2018. He attended TCNJ as a merit scholar and was the recipient of The Philip Dumas Memorial Award for the Top Overall Senior in the Chemistry

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AI

DOI: 10.1021/acs.chemrev.8b00623 Chem. Rev. XXXX, XXX, XXX−XXX