Conformational Control of Macrocycles by Remote Structural

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

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Conformational Control of Macrocycles by Remote Structural Modification Focus Review

Solomon D. Appavoo, Sungjoon Huh, Diego B. Diaz, and Andrei K. Yudin*

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Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario, Canada M5S 3H6 ABSTRACT: The conformational analysis of macrocycles is a complex and challenging problem. There are many factors that contribute to this complexity. These include a large number of degrees of freedom, transannular interactions such as hydrogen bonds and hydrophobic interactions, and a range of steric interactions, along with ring strain effects. To a greater extent than within acyclic molecules, these interactions within macrocycles are coupled such that changing one dihedral angle can significantly affect other dihedral angles, further complicating the situation. However, this coupling of bond rotations and transannular interactions enables the transmission of three-dimensional information from one side of a macrocycle to the other. Making relatively small structural modifications to a macrocycle can result in local conformational changes that propagate along the ring to affect distal structural features. The factors that control how such changes can propagate are poorly understood, and it is difficult to predict which modifications will result in significant conformational reorganizations of remote regions of a macrocycle. This review discusses examples where small structural modifications to macrocyclic scaffolds change the conformational preferences of structurally remote regions of the ring. We will highlight evidence provided for conformational changes triggered by remote substituents and explanations of how these changes might occur in an effort to further understand the factors that control such phenomena.

CONTENTS 1. Introduction 2. Conformational Analysis of Macrocycles 2.1. Experimental Analysis of Macrocycle Conformation 2.2. Computational Modeling of Macrocycle Conformation 3. Structural Modifications That Result in Remote Conformational Changes 3.1. Changes in Stereochemistry of Backbone Stereocenters 3.2. Addition of Exocyclic Functionality 3.3. Incorporation of Heterocycles into the Macrocyclic Backbone 3.4. Addition of Alkyl Groups to Backbone Atoms of a Macrocycle 3.5. Changes in Ring Size 3.6. Oxidation or Reduction of Ring Functionality 4. Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

© XXXX American Chemical Society

1. INTRODUCTION The three-dimensional preorganization of binding elements afforded by macrocycles is useful for the development of biologically active ligands. The majority of macrocyclic drugs are cyclic peptides or macrolides.1 There are over 100 macrocyclic drugs on the market, and extensive efforts are u n d e r wa y t o de v e l op n e w m a c r o c y c l i c m o da li ties.2−5Cyclization of a linear precursor can diminish the entropic penalties associated with the formation of a protein/ ligand complex by reducing the amount of unfavorable conformations that the ligand can adopt.6,7 Macrocycles are particularly well-suited for targeting complex protein−protein interactions (PPIs),8,9 which is a challenge with small molecules because they are not well suited to interact with the shallow and relatively featureless binding surfaces that typify PPIs. There are several reported examples of macrocyclic ligands being used to successfully interrogate PPIs such as the Ras−effector interaction10 and NADH-dependent dimerization of the C-terminal binding protein transcriptional repressor,11 for which small molecule programs have been unsuccessful. A review of macrocycle/protein structural data has indicated that macrocycles have a tendency to bind protein surfaces faceon, maximizing their surface area and forming extensive networks of interactions that are unattainable to small

A C C D E E O P T U V W X X X X X X X

Special Issue: Macrocycles Received: December 3, 2018

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DOI: 10.1021/acs.chemrev.8b00742 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Methods and spectroscopic techniques commonly utilized in conformational analysis of macrocycles.

molecules.12 Many PPIs involve a loop of one protein that binds to a concave surface of its partner. The geometrical features of such protein loops can be approximated using synthetic macrocycles.13 As a result, selectivity between PPIs with similar binding surfaces can be achieved by subtle modifications of ligand conformation and the resultant arrangement of binding elements.14,15 Although macrocycles are conformationally restricted, they can still be flexible and exist as ensembles of low energy conformations in a given environment. Notably, the predominant conformation in solution is not necessarily the same as the protein-binding conformation of a macrocycle.16 It has been shown that, in some cases, the population of the bioactive conformation in solution can be as low as 4%.17 A study of free and targetbound cysteine-rich animal venom peptides, whose binding surfaces are made up of disulfide stabilized macrocyclic loops, found that peptides such as α-cobrotoxin and fasciculin-II underwent loop reorganization upon target engagement.18 Accordingly, the relative energies and kinetic barriers between conformations are critical to a macrocycle’s capacity to engage a biological target.19,20 Conformation is also important in determining the physical properties of macrocycles. Increases in the amount of polar binding elements can render larger molecules inadequate for acceptable bioavailability under physiological conditions.21 Nevertheless, there are several important examples of macrocycles that break the traditional rules for drug-likeness but still display significant bioavailability.22 Conformation plays a critical role in the behavior of these molecules.23 In some instances, low barriers between hydrophobic conformations with polar groups “buried” in the macrocycle and hydrophilic conformations with polar groups exposed to the solvent lead to the emergence of chameleonic properties that allow macrocycles to change shape during permeation of biological membranes.24 In other cases, stabilization of a single conformation leads to improved permeability, either through masking of polar groups25 or targeting of a highly specific transport mechanism.26,27 An understanding of the structural factors that influence the relative energies of macrocycle conformations and the kinetic barriers that separate them has long been recognized as critical to optimize macrocycle biological activity via chemical modification.28

The conformational analysis of macrocycles can be a complex and challenging problem. There are several factors that contribute to this complexity such as a large number of degrees of freedom, noncovalent transannular interactions such as hydrogen bonds and hydrophobic interactions, and a range of steric interactions, along with ring strain effects.29 To a greater extent than in the case of acyclic molecules, such interactions within macrocycles are coupled such that changing one dihedral angle can significantly affect other dihedral angles, further complicating conformational analysis. This coupling of bond rotations and transannular interactions is of particular interest to those who attempt to design macrocycles with specific conformations through transmission of three-dimensional (3D) information from one side of a macrocycle to the other. Making relatively small structural modifications to a macrocycle can result in local conformational changes that propagate along the ring to affect distal structural features. Barriers to specific bond rotations such as E/Z amide isomerization30 and biaryl atropisomerism31 can be significantly higher when the bond is incorporated into a macrocyclic backbone. The factors that control the propagation of such changes, such as the way in which bond rotations are coupled, are poorly understood. This makes it difficult to predict which modifications might result in significant conformational reorganizations of remote regions of a macrocycle. As a consequence, the development of structure−activity relationships (SAR) for macrocycles can be significantly complicated. There is a possibility that substitutions intended to probe the SAR of a macrocycle can result in a backbone reorganization that changes the 3D display of other pharmacophoric groups. In several instances, minor structural modifications of macrocyclic scaffolds have resulted in unexpected changes in binding affinity.32−34 This review discusses examples where small structural modifications to macrocyclic scaffolds change the conformational preferences of remote regions of the ring. For the purposes of this review, a change of at least three bonds away is classified as remote. We will highlight evidence provided for remote conformational changes triggered by substituents and explanations of how these changes might occur to further understand the factors that control such phenomena. B


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2. CONFORMATIONAL ANALYSIS OF MACROCYCLES

protons from the solvent with bulky alkyl groups can also change chemical shift/temperature coefficients.21,50 In order to separate the influences of intramolecular hydrogen bonding from shielding by bulky groups, long-range HNCO experiments on 13C and 15N labeled samples can be used to identify intramolecular hydrogen bonds.51 Alternatively, intramolecular hydrogen bonding can be assessed by relative changes in chemical shifts upon addition of strong hydrogen bonding solvents such as TFE or weak hydrogen bonding solvents such as CDCl3.52,53 The chemical shifts of protons involved in intramolecular hydrogen bonds change less significantly than those of protons exposed to solvent. However, such effects can be difficult to evaluate since changes in the hydrogen bonding capacity of a solvent can also result in changes of intermolecular hydrogen bonding and, therefore, a conformational reorganization of the macrocycle. As an alternate approach, H/D exchange rates can be measured upon addition of D2O and used to assess intramolecular hydrogen bonding.54 Slow rates of exchange indicate that protons are involved in intramolecular hydrogen bonds or shielded from the solvent. However, H/D exchange rates can also be dependent on pH and must be interpreted carefully, as there are examples where results from variable temperature (VT) 1H NMR and H/D exchange rates suggest that different amide protons are interacting with the solvent.47 In the case of macrocycle conformations that interconvert on the NMR time scale, the exchange of individual nuclei between conformations can be interrogated by saturationtransfer55 or 2D-exchange56 spectroscopy. Some of the most important data for solution-phase conformational analysis of macrocycles is derived from through-space dipolar couplings represented by NOEs. The intensity of NOESY and ROESY cross-peaks can be related to internuclear distances via an r6 relationship.57 This provides a set of interproton distances that enable the generation of effective molecular models. NOEderived distances reflect an average of conformations interconverting faster than the NMR time scale. 13 C NMR spin−lattice relaxation times can provide information on the relative segmental motion of different carbons in a macrocycle.58 13C NMR chemical shifts are highly sensitive to the chemical nature of the carbon atom, and changes in macrocycle conformation result in observable changes in the chemical environment around each carbon atom. For instance, the chemical shift of the prolyl γ-carbon may be used to determine if an X-Pro peptide linkage is in a cis or trans configuration.59−62 Carbons are deshielded when eclipsed with a vicinal substituent,63 and changes in 13C chemical shifts are sensitive enough for the determination of subtle conformational changes.64 Circular dichroism (CD) can be used to analyze secondary structural characteristics of polypeptide fragments within macrocycles.65 While NMR primarily measures the properties of individual groups within a macrocycle with conformational effects resulting in perturbations from the spectra of common small molecules, optical activity depends uniquely on the asymmetric array of chromophores and interacting polar groups as a whole. If a correspondence between a secondary structural type and CD spectral features can be established, the presence of such spectral features can be used to infer about the presence of the corresponding secondary structural type.66 However, CD spectral data has been shown to be in disagreement with NMR data for several small peptide based macrocycles.67 IR (infrared) spectroscopy can also provide

2.1. Experimental Analysis of Macrocycle Conformation

The first step in understanding the conformational preferences of a given macrocycle involves spectroscopic analysis (Figure 1). X-ray crystallography can provide a detailed picture of static conformation in the solid phase; however, crystal packing can significantly affect conformation, and caution is required when attempting to rationalize a conformation adopted in solution using crystallographic data.35 Nuclear magnetic resonance (NMR) spectroscopy is routinely used for the conformational analysis of macrocycles in solution. Resonances can be rapidly assigned with the help of modern two-dimensional NMR techniques.36 For example, amino acid residues in cyclic peptides can be sequenced using the TOCSY-HMBC sequential backbone walk method.37 Once peaks have been assigned, 1H NMR spectra can provide many critical pieces of evidence.38,39The appearance of multiple peaks and peak broadening are indicative of multiple conformations exchanging on the NMR time scale.40 In this regard, NMR observations arise from ensembles of interconverting conformations,41−45 and one must avoid viewing NMR-derived data as describing a single conformation. The conformation of a macrocycle is defined by various dihedral angles of the bonds in the ring backbone. Homonuclear vicinal (3J) coupling constants can be related to dihedral angles via the Karplus equation. However, there are several caveats when analyzing dihedral angles with 3J-coupling constants. The Karplus equation should be modified based on the substituent involved, and special expressions derived from experimental data must be utilized for certain bond types.46 Additionally, most measured coupling constants can correspond to four possible solutions to the Karplus equation. Finally, values that approach the mean (approximately 7.5 Hz) can either be interpreted as arising from free mobility (i.e., an averaging of coupling constants of rotamers) or a from stabilized dihedral angle that results in a coupling constant of 7.5 Hz. Therefore, 3J-coupling analysis should be used in conjunction with other methods when assessing macrocycle flexibility. Relative chemical shifts provide information about differences in the chemical environment of similar protons in different regions of a macrocycle. For example, large chemical shift differences between amino acid residues in a polypeptide are indicative of a rigid structure because each residue adopts a distinct chemical environment.47 Flexible polypeptides tend to display small differences between the chemical shifts of various residues. Transannular interactions, such as intramolecular hydrogen bonds, are often observed in macrocyclic scaffolds. A variety of NMR techniques can be used to evaluate if intramolecular hydrogen bonding is occurring in a given macrocycle. Intermolecular hydrogen bonds, especially those with solvent, dissociate with increasing temperature, resulting in significant changes in the chemical shifts of the corresponding proton resonances. However, the decrease in entropy afforded by intramolecular hydrogen bonding means that intramolecular hydrogen bonds are not as affected by changes in temperature, and low chemical shift/temperature coefficients are indicative of strong intramolecular hydrogen bonding.48,49 Nevertheless, it is important not to overinterpret chemical shift/temperature coefficients as arising mainly from intramolecular hydrogen bonding, as several studies have indicated that sterically shielding exchangeable amide NH C


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The most popular conformational search methods for macrocycles involve molecular mechanics (MM) approaches, which use a set of classical equations and associated parameters called a force field. The corresponding parameters are derived from experimental results or quantum mechanical (QM) calculations. Energy calculations are combined with systematic or stochastic methods such as Monte Carlo searches to generate new molecular geometries.85 Molecular dynamics (MD) simulations can also be used to explore the local conformational space around a starting geometry; however, they require significantly more computer time and are slow to cross kinetic barriers between local energy minima. Userfriendly interfaces allowed chemists to evaluate macrocycle conformations and relative energies, enabling significant early contributions to the field of macrocyclic stereocontrol.86 However, a recent study of such methods has concluded that they are often unable to reproduce the experimentally determined low energy conformation for a macrocycle.84 The ring constraint leads to high energy barriers between different metastable conformations.87−89 A range of distance geometry90 and inverse kinematics91 approaches that utilize less intensive energy evaluations have been found to excel at generating a diverse set of macrocycle conformations. However, they typically are not designed to provide reliable thermodynamic insights.92,93 Newer kinematics-based algorithms can characterize the conformational energy landscape more accurately.94 Application of modern MM force field-based methods with enhanced sampling techniques such as meta-dynamics, replica exchange MD, accelerated MD, and low mode MD show highly promising results for characterization of structural ensembles and the energy surfaces accessible to a macrocycle.95−97 Particularly exciting is the utility of these new methods for accurate evaluation of energy maxima and the corresponding barriers to conformational interconversion. QM calculations such as density functional theory (DFT) calculations on macrocycles can be used to calculate the energy of a single conformation. QM calculations are used to predict chemical shifts and coupling constants for a given macrocycle conformation.98−101 They can be used to further optimize the geometry of conformations found by classical methods or incorporated as secondary energy minimization steps after a force field has been applied during a conformational search.102 The majority of conformational energy calculations correspond to enthalpies rather than free energies, as they calculate the energy of a single set of coordinates. Scoring functions approximate entropy using a weighted sum of the molecule’s rotatable bonds for linear molecules.103 However, such approximations may not hold true for macrocycles as they are subject to additional and often unpredictable rotational constraints. This serves as a reminder that it is important to view macrocycles as ensembles of flexible conformations whose conformational entropy can be difficult to predict. Factors such as the root mean squared deviation (RMSD) of ring atom coordinates among a cluster of conformers that are close in energy can be utilized to qualitatively predict the entropy of a given conformational ensemble. Statistical cluster analysis can also provide insight into conformational entropy.104 Experimentally derived constraints can be combined with computational modeling techniques to more accurately predict the conformation of a macrocycle. The importance of solutionphase conformations to biological activity means that chemists rely heavily on solution NMR data for conformational analysis

information about the secondary structural features of peptide fragments within a macrocycle.68 The amide bond shows several strong IR absorbances whose fine structure depends on the various types of H-bonding patterns in a peptidic macrocycle and their relative amounts. In some cases, macrocycles can aggregate in solution to form nanostructures, complicating the spectroscopic analysis of the macrocycles. Such behavior can be evaluated using CD, IR, and T2 relaxation times.69 Structures for lossless ion manipulations (SLIM)70 is an ion-mobility mass spectrometry technique that can distinguish conformational differences between compounds using small amounts of unpurified samples.71 Changes in physical properties can be indirect indicators of the conformational differences between structurally similar macrocycles, as the conformation of a macrocycle determines its exposed polar surface area. Differences in properties such as LogD and high-performance liquid chromatography (HPLC) retention time between configurational isomers can imply that the two molecules adopt similar or very different conformations with different amounts of solvent exposed polar surface area.72−74 Differences in chemical reactivity often arise from conformational differences.75 This enables the use of changes in selectivity in the assessment of conformational differences between macrocycles with similar structures. Site-selective reactivity of amides in peptide macrocycles can be influenced by the relative exposure of different amides, which is dependent on the overall conformational preferences of the macrocycle.76,77 Facial selectivity is dependent on the conformation of the surrounding parts of the molecule, i.e., concave vs convex face.78 This has been shown to effect highly diastereoselective transformations in medium and large rings, and the concept is known as macrocyclic stereocontrol.79 Differences in reactivity between macrocycles can suggest conformational changes. When inferring conformational changes using differences in reactivity it is important to remember that selectivity reflects the conformational preferences of the transition state for reactions under kinetic control, which does not necessarily resemble the ground state conformation. 2.2. Computational Modeling of Macrocycle Conformation

In order to conduct the conformational analysis of a macrocycle, it is necessary to generate a three-dimensional model of the molecule in a given solvent environment. For macrocycles, tens to thousands of kinetically distinct conformations can be found within 10 kcal/mol of the global minima.80 To generate conformers of a macrocycle, computational methods utilize a sampling algorithm that acts on the conformational degrees of freedom to generate new molecular geometries and an energy model that evaluates the relative stability of each conformer. The various combinations of sampling algorithms and energy models have been comprehensively reviewed.81−83 The criteria for an effective search include the speed, number, and diversity of conformers generated, and the ability to accurately identify the global energy minimum and/or an experimentally determined conformation.84 Additionally, identifying dynamic conformational behavior requires a sampling algorithm that can cross barriers separating local energy minima. A high diversity of conformers generated often arises as a result of applying an algorithm with unrealistically low barriers between conformations. D


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Figure 2. Structures of phepropeptin C (1) and its proline-epimeric isomer (2) and their physicohemical and ADME properties (top). Solution structure of phepropeptin C (1) in CDCl3 and NMR data of its amide protons in CDCl3 and DMSO (bottom).

authors often use different force fields, search algorithms, and simulation times. Sometimes intramolecular hydrogen bonds determined using the spectroscopic methods mentioned above are also utilized as restraints during force field based conformational searches.119 In other instances they are used only to check the validity of the final structure.59 For a complete characterization of macrocycle conformation, a combination of techniques is required. A holistic approach with an appreciation of the limitations and assumptions inherent to each method of analysis enables a better appreciation for the conformational preferences of a macrocycle.65 With these tools in hand, one can proceed to assessing the changes in macrocycle conformation.

when trying to identify biologically relevant conformations. Of particular importance is the combination of NOE-derived and 3 J-coupling torsional restraints with conformational search methods. Several strategies for determining solution structures of macrocycles have been reported.56 These involve a conformational search with experimentally determined distance and torsion restraints. The restraints can then be removed as the low-energy conformer is subjected to further simulations. The low-energy conformers generated by the second simulation are then compared with experimentally determined distances to check the validity of the structures.59 One must keep in mind that NMR data represents an ensemble of interconverting conformers. The Boltzmann populations of conformers are determined by their relative free energies and in most cases the solution conformation of a macrocycle cannot be accurately represented by a single structure.105 Sometimes the constraints do not match with the conformer generated by a search,106−111 or they match with multiple conformers.50 Determining the relative contributions of each conformer can therefore be challenging. Attempts to solve this problem involve relaxing the NMR-derived limitations on a single conformer,112 generating a family of conformers that do not violate the NMR limitations as a whole,113,114 or utilizing independent energy calculations with a statistical weighting function.115 The NMR Analysis of Molecular Flexibility in Solution (NAMFIS) method uses statistical weighting to fit a conformational ensemble to a given set of NMR-derived restraints.116 A conformational ensemble is generated by an unrestrained conformational search. NAMFIS then varies the mole fraction of each conformer from the input ensemble until the best possible fit with the experimental restraints is achieved. Since NAMFIS does not rely on energy calculations, inaccurate evaluation of conformer populations due to long-range interactions or inappropriate force-fields is avoided.117 In some cases, NAMFIS indicates conformations that are not identified using restrained conformational searches.118 There is no standardized method to determine the solution structures of a macrocycle, and

3. STRUCTURAL MODIFICATIONS THAT RESULT IN REMOTE CONFORMATIONAL CHANGES Herein, we offer a collection of examples where relatively small modifications to macrocyclic scaffolds change the conformational preferences of remote regions of the ring. We have only included examples with spectroscopic evidence of a conformational change. We have generated visual models using Avogadro120 for each of the following examples to clearly illustrate important conformational changes. These models do not necessarily correspond exactly to structures published in the corresponding reports, but we have endeavored to carefully reproduce the key conformational features. 3.1. Changes in Stereochemistry of Backbone Stereocenters

Phepropeptins A−D are macrocyclic 18-membered hexapeptides containing two D-amino acid residues at opposing sides of the ring. These molecules were isolated from Streptomyces sp. MK600-cF7.121 They display modest inhibition of mouse liver proteasome, and their structures were determined by analysis of 1D and 2D NMR experiments and confirmed by synthesis. Lokey and co-workers prepared a series of phepropeptins and their proline-epimeric isomers (epiphepropeptins)122 and determined several physiochemical and ADME properties. MDCK monolayer permeability assays were used to assess cell E


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Figure 3. Lowest-energy calculated NMR structures of 4 and 3 showing different hydrogen bonding interactions between the two epimers.The intramolecular H-bonds are represented as dotted black lines.

permeability. The epiphepropeptins showed 2−4-fold slower permeability than their natural congeners (Figure 2). Epimer 1 had a permeation rate of 40 × 10−6 cm/s, while epimer 2 had a permeation rate of 15 × 10−6 cm/s. Surprisingly, both epimers displayed similar aqueous solubility, with 1 at 0.06 mg/mL and 2 at 0.07 mg/mL. The authors hypothesized that each isomer had a different capacity to adopt lipophilic conformations and used NMR experiments in a high dielectric (DMSO) and low dielectric (CDCl3) solvents. VT 1H NMR of 1 in CDCl3 indicated that four of the amide protons were involved in intramolecular hydrogen bonds and only the Phe2-NH was solvent-exposed. A solution structure in CDCl3 was determined using unrestrained MD simulations. The conformation from the unrestrained MD that displayed the lowest deviation from the ROESY-derived interproton distances and 3Jcoupling-derived dihedral angles was selected as representative of the solution conformation (Figure 2). The solution structure also indicated that the Phe-NH was the only amide proton exposed to the solvent. VT 1H NMR of 2 in CDCl3 indicated only one amide NH was involved in an intramolecular hydrogen bond. Limited solubility of the 2 in CDCl3 prevented peak assignment and further characterization. Because 1 adopted a more lipophilic conformation in CDCl3 but displayed similar aqueous solubility to 2, the authors concluded that 1 could also adopt a hydrophilic conformation in high dielectric solvent. VT 1H NMR of 1 in DMSO indicated that only the Leu5-NH proton was shielded from solvent. Surprisingly, the solution structure determined using NMR restraints from the DMSO spectra showed additional intramolecular hydrogen bonds with the Phe2- and Leu5-NH protons, which were involved in γ-turns, making them less accessible to the solvent. The authors proposed that the

balance of membrane permeability and water solubility displayed by 1 could be explained by the ability of 1 to equilibrate between two conformations depending on the solvent environment. The difference in stereochemistry between 1 and 2 results in major reorganization of the remote hydrogen bonding pattern, which significantly affects physical properties. The local steric interactions around the stereocenter propagate throughout the ring and affect the overall flexibility of the backbone and the macrocycle’s ability to rearrange in different solvents. The change in stereochemistry influences not only the ground state conformational preferences but also the kinetic barriers that control conformational behavior upon switching solvents. Cyclic tetrapeptides are known to be hydrolytically unstable and conformationally heterogeneous in water due to strain embedded in the 12-membered ring with all amides in the trans geometry. Fairlie and co-workers have demonstrated that increasing ring size to 13 by incorporation of a β-amino acid (βhPhe, β-S-homophenylalanine) makes cyclic tetrapeptides both conformationally homogeneous in water and easier to make because of a reduction in overall ring strain.123 A range of 12- and 13-membered rings, including 3 and 4, were prepared (Figure 3). Each diastereomer displayed a single set of resonances, indicating conformational homogeneity. The authors commented that separation of the D-Pro from the βhPhe by one residue was required to cooperatively affect conformational stability. VT 1H NMR suggested that the AsuNH participated in hydrogen bonding in both 3 and 4. Both βhPhe and Asu residues displayed 3JNH‑CαH couplings over 9 Hz for 3, while only the βhPhe residue did so in the case of 4. These coupling constants were used to derive dihedral restraints, which were subsequently utilized along with ROE F


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Figure 4. Backbone solution structure of α3β cyclic tetrapeptide epimers. The change in macrocycle conformation and intramolecular hydrogen bonding interactions is due to the stereochemistry of the glycosyl amino acid being changed. The intramolecular H-bonds are represented as dotted black lines.

Figure 5. Structure of sanguinamide B (7) and its analogues. The stereocenter of R group affects the conformation of amide adjacent to the proline as determined by the difference in 13C NMR chemical signals of β-C and γ-C of proline.

three L-α-amino acid residues to generate macrocycles such as 5 and 6 (Figure 4). 1H NMR spectra of these two compounds in DMSO showed sharp proton resonances, suggesting a single major conformer in solution on the NMR time scale in each case. VT 1H NMR of 6 suggests that the Phe3-NH and Met4NH are involved in intramolecular hydrogen bonds. The 3 JNH‑CαH values of 6.2 and 9.3 Hz for Phe2 and Phe3, respectively, in compound 6 are characteristic of a Type I βturn in which Phe2 adopts the i+2 position. In addition, strong NOE signals between Phe2-NH/Phe3-NH, Phe3-NH/Met4NH, and Met4-NH/Phe2-CαH and restrained MD simulations support the β-turn in this position (Figure 4). A 3JNH‑CαH value of 9.2 Hz was observed for Phe3 of 5, while all other 3 JNH‑CαH couplings approached the random coil value of approximately 7 Hz. Phe3-NH/Caa1-CαH, Phe3-NH/Phe2CαH, and Caa1-NH/Met4-CαH displayed strong NOE crosspeaks for 5, suggesting the change in stereochemistry from 6 resulted in a major conformational reorganization in remote regions of the macrocycle. VT studies suggested Phe3-NH and Caa1-NH of 5 were involved in intramolecular hydrogen bonds. Restrained MD simulations indicated a γ-turn conformation for 5 as illustrated (Figure 4). These results

cross-peak intensity derived distances as restraints in a dynamic annealing and energy minimization protocol to generate solution structures for 3 and 4 (Figure 3). The Asu-NH is involved in intramolecular hydrogen bonds with the Phe-CO in 4 and the βhPhe-CO in 3. The distal change in the stereochemistry of Asu influences the orientation of the βhPheNH six atoms away. In 4, the carbonyl group is perpendicular to the plane of the macrocycle, while in 3, the transannular hydrogen bond with the Asu-NH causes it to become parallel with the plane of the macrocycle. The authors suggest these changes occur in order to maintain a gauche relationship between the Pro-CO and the Cα-Cβ bond of the Asu residue. This 1,3 pseudoallylic strain interaction is on the opposite side of the macrocycle from the amide that changes its relative orientation. This change in orientation is likely mediated by the formation of the transannular hydrogen bond. As part of an another research program to generate conformationally homogeneous cyclic tetrapeptides, Ampapathi and co-workers have reported the synthesis and conformational analysis of 13-membered α3β cyclic tetrapeptides.124 Glycosyl-substituted β-amino acids (Caa) with either R or S configuration were incorporated into cyclic peptides containing G


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Figure 6. Structure of apicidin (10) and its analogues containing 1,4- and 1,5-disubstituted triazole (top). NMR-derived solution structure of the triazole analogues (bottom). The structure 11 has 1,4-disubstituted triazole and adopted an all-trans-amide conformation. In the case of 12 and 13, the 1,5-disubstituted triazole enforced a cis−trans−trans−trans conformation with L-Leu but changed to a cis−trans−cis−trans conformation upon stereoinversion to a D-Leu.

show that changing β-amino acid stereochemistry in 13membered rings results in profound reorganization of the remote hydrogen bonding pattern and change in the type of turn displayed. The large steric bulk of the glycosyl group is likely a contributing factor, as it promotes a significant reorganization of the Caa residue to avoid any local strain interactions. The resulting changes in Caa residue conformation appear to propagate throughout the rest of the ring, affecting the distal turn geometry. Sanguinamide B (7) is a 24-membered cyclic octapeptide derived macrocycle isolated from Hexabranchus sanguineaus.125 This 24-membered ring contains two thiazole residues and one oxazole residue. The first total synthesis of 7 was completed by McAlpine and co-workers, who identified three macrocyclic conformations with unique prolyl amide configuration around Pro1 and Pro2 (trans/trans, trans/cis, and cis/cis).126 In an effort to generate conformationally restricted analogues of 7, the authors have reported the synthesis of several analogues (Figure 5).127 In compounds 8 and 9, the Val residue of 7 has been substituted with an L-Phe of D-Phe residue, respectively, which varied in stereochemistry of the new residue (Figure 5). Both 8 and 9 adopted two conformations that were separable by HPLC. The authors utilized a temperature gradient in their 1 H NMR analysis to find the temperature at which the peaks were sharpest and then used 2D experiments to assign all proton and carbon resonances. The difference in chemical shifts between β- and γ-carbon (ΔδCβγ) values for each Pro residue allowed the authors to assign either a cis or trans conformation to proline residues, with cis-Pro amide having a higher value. A set of isolated conformers for 8 and 9 adopted

a similar conformation with a cis/cis arrangement for Pro1 and Pro2. However, the other isolated conformers of 8 and 9 differ, with 8 containing a trans/cis arrangement, while 9 adopting a cis/trans linkage. The variable stereocenter is remote from both Pro residues, one being 4 atoms away and the other 11 atoms away. The distal change in stereochemistry results in local strain interactions which propagate to the other side of the macrocycle via bond rotations, resulting in amide isomerization. It is notable that both Pro residues are separated by an oxazole/thiazole linker, which contains only sp2 atoms. The rigidity of this segment may enable the phenylalanine stereochemistry to affect the Pro 2 amide configuration at such a long distance. Apicidin (10) is a 12-membered cyclic tetrapeptide containing a pipecolic acid residue and an amino acid with an exocyclic ketone side chain (Aoda). It was originally isolated from the fermentation broth of Fusarium sp. ATCC 74289.128 The compound displays antiprotozoal activity through the nanomolar inhibition of Apicomplexan histone deacetylase. An all-trans-amide conformation of 10 is known to predominate in solution.129 However, several groups have reported the appearance of a minor cis-amide-containing conformation in DMSO. Ghadiri and co-workers synthesized analogues in which the pipecolic acid residue was replaced with a 1,4- or 1,5-disubstituted 1,2,3-triazole residue, which mimics trans- or cis-amide, respectively (Figure 6).130 In this way, the authors were able to probe the binding affinity of distinct cis and trans conformations. 1H NMR spectra of 11, 12, and 13 indicated distinct conformations on the NMR time scale. 2D NMR experiments and distance geometry calculations enabled H


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Figure 7. Structures of 15 and 16 illustrating the change in intramolecular hydrogen bonding.

Figure 8. Structure of Acpca incorporated RGD cyclic peptide (center). NMR derived solution of 17 illustrating how the stereochemistry of the Acpca group influences the conformational rigidity of the macrocycles by changing the capacity to form an intramolecular H-bond. Relative to 17, epimer 18 was shown to be conformationally flexible.

while 16 has a Tma (trans-methyl Azy) residue. Broad peaks in the 1H NMR spectra indicated that both 15 and 16 are flexible in solution. Strong NOEs between Phe-CαH/Gly-CαH protons and Leu-CαH/Cma-CαH for 15 indicated that both amide linkages adopt a cis conformation. The lack of CαH to CαH NOEs for the other residues suggest the amides are in trans conformations. 16 displayed the same pattern of NOE cross-peaks, suggesting similar amide conformations as 15. VT 1 H NMR of 15 and 16 indicated that the Phe-NH is involved in an intramolecular hydrogen bond in both compounds, while Leu-NH is involved in hydrogen only in 15. This suggests that the overall conformation of 16 is more flexible than 15. Although the variable stereocenter is exocyclic to the macrocyclic ring and is remote (6 atoms) from the Leu-NH, it changes the degree of intramolecular hydrogen bonding for Leu-NH. To obtain low energy conformations of both macrocycles, NOE restrained MD annealing simulations followed by a DFT-based optimization were used. While the resulting structures confirmed that the amide bond configurations of 15 and 16 were the same, 15 adopted a folded global conformation, while 16 adopted an extended global conformation. The nitrogen pyramid for 15 was measured at 35°, while for 16, it was less steep at 27°. The steeper nitrogen pyramid angle for 15 indicated reduced lone pair delocalization and more N−C single bond character for the amide, suggesting that the acyl aziridine of 15 should be more flexible than that

the generation of NOE-based solution structures for these molecules (Figure 6). The 13-membered ring 11 displayed an all-trans-amide conformation. The 12-membered analogue 12, which differs by the substitution of Leu for Ile and triazole substitution, also displayed an all trans-amide conformation. However, the orientations of the Ala-NH and Trp-NH in 12 were flipped relative to the plane of the macrocycle. 13 is a diastereomer of 12, that differs only by the stereochemistry of the Leu residue. In contrast to 12, 13 contains a cis-amide at the Aoda residue. This amide bond is distal from the change in stereochemistry, which implies that local strain interactions around the triazole residue are transmitted to the opposite side of the macrocycle. 12-membered ring 14 also closely resembles 12, differing only by the substitution of Ile for Leu. Surprisingly, the 1H NMR spectrum of 14 indicates that it is conformationally heterogeneous with several conformations interconverting slowly on the NMR time scale. The movement of the methyl group to the β-carbon places the triazole in proximity with the methyl group, where a 1,3-allylic strain could contribute to the conformational destabilization of the molecule. As part of an effort to understand the effects of acyl aziridine incorporation on the conformation and reactivity of cyclic tetrapeptides, Yudin and co-workers have reported the conformational analysis of 12-membered macrocycles 15 and 16 (Figure 7).131 15 contains a Cma (cis-methyl Azy) residue, I


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Figure 9. Structure of chiral sulfoxide cross-linked macrocycle (center). The crystal structure of the R-stereoisomer of the sulfoxide (19) induces alpha helicity in the macrocycle (left); however, the S-stereoisomer of the sulfoxide (20) generates a random coil (right). The H-bonds are represented as dotted black lines.

Figure 10. (a) Structure of thioether-tethered cyclic peptide. The stereochemistry of the methyl group influences helicity (b) Representative structure from REMD simulations of peptide 23 showing that R-stereochemistry stabilizes the helical conformation. (c) α-Helix segment of 23.

carboxyl group trans to the amino group, possesses no defined secondary structure with all three 3JNH‑CαH-coupling around 7.0 Hz, suggesting conformational averaging of the macrocyclic backbone. There is a hydrogen bond between the Acpa hydroxyl group and Asp carboxylate in 18. The macrocyclic backbone must twist to accommodate the new exocyclic hydrogen bond, resulting in dissociation of the previous hydrogen bond. Additionally, the binding of 17 to integrin receptor is 6 times tighter than 18, which suggests that the well-defined secondary structure and a flexible side chain conformation are key elements for biological activity. The research highlights that change in stereochemistry of the Acpca group can alter the intramolecular hydrogen bonding network of the entire macrocyclic backbone. Side-chain cross-linking or “stapling” is a well-known strategy to induce α-helicity in peptides.134 To understand the effects of chirality in the cross-linking segment on the overall conformation of the stapled peptide, Li and co-workers have reported the synthesis and conformational analysis of short peptides with chiral sulfoxide-containing cross-links (Figure 9).135 20-membered rings 19 and 20 were generated by oxidation of the corresponding thioethers and were separable by HPLC, giving an initial indication of conformational differences in solution. CD spectroscopy indicated that

of 16 because of a lower barrier to bond rotation. This is surprising as the VT NMR data suggests that 16 is more flexible. Overall, these results are significant because they illustrate that structural changes expected to indicate increased flexibility can actually be observed when VT 1H NMR indicates reduced flexibility. Small molecular templates can be grafted into peptides to induce secondary structure that is favorable for biological recognition.132 The Casiraghi group investigated γ-aminocyclopentane carboxylic acid (Acpca) platforms to induce secondary structure in the Arg-Gly-Asp (RGD) tripeptide sequence and evaluated the binding affinity of the analogues toward αvβ3 and αvβ5 integrin.133 The conformations of the analogues were determined through NOESY, VT NMR, 3 JNH‑CαH-coupling, dynamic simulated annealing, and energy minimization protocol to generate a family of low energy conformers (Figure 8). 17 has an Acpca residue containing a cyclopentatane with amide and amino functionality in a 1,3 cis relationship. 17 possesses an inverse γ-turn that is stabilized by an Acpca-NH and Gly-CO hydrogen bond (Acpca amide temperature coefficient = −2.0 ppb/K). There is no hydrogen bonding occurring between the hydroxyl group and Asp and Arg side chain functionalities, which allows for the side chains to have conformational flexibility. In contrast, 18, which has its J


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Figure 11. (a) Structure of cyclophane-like macrocycle with 1,4-disubstituted 1,2,3- triazole from CuAAC reaction. (b) Superimposed X-ray structure of macrocycle 25 and 26. Stereoinversion of the methyl group adjacent to the amide causes a change in the orientation of the triazole in the macrocycle.

−38° to −53°). To further their understanding of the conformational differences between the two diastereomers, the Li group performed replica exchange molecular dynamics (REMD). The simulation for the S-diastereomer indicated no significant secondary structure, which was in line with the CD results. Additional simulations on the peptide without the methyl group were conducted to show that the conformation it adopts is not helical. The change in tether stereochemistry resulted in a change in overall macrocycle conformation. In this nonhelical conformation, a methyl group can be added in the S-configuration with no steric interference. However, the addition of a methyl group in the R-configuration leads to destabilization of the nonhelical conformation to the preferred α-helical structure. This destabilization is due to steric interaction between the R-methyl group and the Ala2-CO that disallows the nonhelical conformation. To relieve this clash, the tether rotates the methyl group and oxygen away from each other, resulting in a reorganization of the entire macrocycle backbone and remote hydrogen bonding network. James and co-workers have reported the synthesis and characterization of a series of drug-like cyclophane macrocycles.142 These molecules were generated by an intramolecular copper-catalyzed azide/alkyne cycloaddition and featured a (1R,2S)-ephedrine fragment or the corresponding diastereomeric fragment. 24-(S) was cyclized to generate the 12-membered macrocycle 25 in 73% yield, while the diastereomer 26 was generated from 24-(R) in 40% yield. The structures of these macrocycles were determined by X-ray crystallography. DFT geometry optimizations of the crystal structures enabled the calculation of strain energies. The strain energy of the 12-membered ring system was determined to be insignificant. The crystal structure of 25 indicated the triazole hydrogen is pointing down relative to the amide carbonyl, while for diastereomer 26 the orientation of the triazole hydrogen relative to the macrocyclic ring plane and amide carbonyl is flipped in the opposite direction (Figure 11). The authors found it difficult to rationalize why inversion of the distal stereocenter resulted in flipping of the triazole ring. The relative enthalpies of the “up” and “down” conformations of 25 and 26 were calculated. For 25 the “down” conformation was observed in the crystal structure and the “up” conformation was generated only in silico. For 26 the up conformation was observed in the crystal structure. The “up”-form is 2.5 kcal/mol higher for 25 while the “up”-form is 1.1 kcal/mol for 26. The authors suggest that the energy difference for 26 is low and the energetic cost of assuming the “down”-arrangement is compensated by crystal packing forces. This example illustrates

19 has a significant degree of α-helicity, while 20 adopts a random coil. Small 3JNH‑CαH values (2 mM SDS. These results were an initial indication that 50 changes conformation in a lipophilic environment, while 51 does not. H/D exchange studies indicated that Leu1-NH and Leu4-NH exchange was slow (t1/2> 700 min) in CDCl3 for both compounds. In DMSO, the exchange rates for 51 are slow; however, the exchange is much more rapid (t1/2 < 60 min) for 50. These results indicated that both 50 and 51 display intramolecular hydrogen bonding in a hydrophobic environment, while only 51 retains the intramolecular hydrogen bonding in a hydrophilic environment. Solution structures for both macrocycles in CDCl3 and DMSO were determined using NOE-derived distance restraints and Hbonding restraints derived from the H/D exchange experiments. The structures were determined using a dynamic simulated annealing protocol in a geometric force field.163 The structures in CDCl3 are similar for both 50 and 51, having two hydrogen bonds between Leu1-Leu4 and adopting an antiparallel β-sheet connected by type I β-turns at each end (Figure 18). 51 displays a similar conformation in DMSO with the Leu1-Leu4 hydrogen bonding still intact, consistent with the H/D exchange data (Figure 18). 50 adopts a significantly different conformation in DMSO, forming a ring-shaped structure with N-methyl groups and carbonyls perpendicular to the plane of the ring (Figure 18). Unrestrained MD simulations of the solution structures from DMSO displayed significantly higher backbone RMSDs for 50 than 51, consistent with increased flexibility of 50. Overall, these results indicated that substitution of N-Me- D -Leu for D -Pro significantly impacts the capacity of remote regions of the macrocycle to rearrange from a stabilized β-sheet to a flexible ring-shaped structure upon changing solvents. The Pro

conformations was used as the input for NAMFIS analysis. The NAMFIS analysis suggests that the NMR data for 49 describes a conformational ensemble with only 29% β-hairpin character (Figure 17). In contrast, for 48, NAMFIS analysis indicated 74% β-hairpin character. Temperature/chemical shift coefficients of the backbone amide protons of 48 also suggest an intramolecular hydrogen bonding pattern characteristic of the β-hairpin conformation. The magnitudes of the 3J scalar couplings are also in agreement with a β-hairpin conformation. To investigate if a transannular Cl-OMe halogen bonding interaction was occurring, the temperature/chemical shift coefficients of the OMe protons in 49 and 48 were compared. The chemical shift of nuclei in the vicinity of an equilibrium process are assumed to show a comparably high temperature dependence, because the equilibrium constants describing dynamic processes have a temperature-dependent entropy component. The OMe protons of 48 have a temperature dependence of 0.32 ppm/K, while those of 49 show a temperature dependence of only 0.07 ppm/K, suggesting the OMe of 48 is more likely to be involved in a dynamic process such as halogen bond formation. DFT calculations suggest a 4.4 kJ/mol contribution from the halogen bond interaction to the β-hairpin stabilization. This study illustrates how an exocyclic modification can affect the conformational preferences of the entire macrocyclic backbone if there is a possibility for remote stabilizing transannular interactions. 3.3. Incorporation of Heterocycles into the Macrocyclic Backbone

Conformational flexibility is speculated to affect passive membrane permeability and oral bioavailability of cyclic peptides.161 Fairlie and co-workers have prepared the 18membered cyclic hexapeptides 50 and 51 to investigate this idea.162 The D-Pro residue of 51 was replaced with a N-Me-DLeu residue in 50 in an attempt to increase the flexibility of the macrocycle. CD spectra were measured in increasing P


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Figure 19. Ring opening of the endocyclic aziridine of 52 stabilizes the macrocycle conformation and enables the exocyclic amide to coordinate a network of intramolecular hydrogen bonds in 53.

Figure 20. Incorporation of an oxadiazole and amine into the macrocyclic backbone of 55 to generate 54 resulting in stabilization of the remote hydrogen bonding network and β-turn.

authors explained that the apparent decrease in flexibility from 52 to 53 is due to the conversion of the acyl aziridine to a regular amide. They reasoned that the increased flexibility is due to the increased sp3 nature of the aziridine nitrogen and the decreased resonance donation of the nitrogen lone pair causing the rotational barrier around the N−C bond of acyl aziridine to be lower than that of the amide.167 Without a stabilized backbone in this region, the exocyclic amide is unable to coordinate the network of intramolecular hydrogen bonds across the whole ring. In addition, without ring opening of the aziridine, there is no linker-NH to engage in the transannular hydrogen bond observed for 53. These results illustrate that a change in the hybridization of a ring atom in a macrocycle can have a significant impact on conformation flexibility if the modification is positioned close to key conformational control elements capable of making remote interactions. The authors also determined the X-ray crystal structure of 53. In the crystal structure, the Pro residue assumes the ith position of a β-turn. Like the solution structure, the exocyclic amide-CO is hydrogen bonding with the LeuNH. The authors contrasted this structure with 16-membered Pro-containing homodetic cyclic hexapeptide crystal structures. In the homodetic macrocycles, the Pro residue adopts the i+1 position. The homodetic macrocycles lack functionality capable of forming the remote 10-membered hydrogen-

residues affect the kinetic barrier to reorganize the conformation of distal macrocyclic backbone functionality. In an effort to modulate the conformational preferences of peptide macrocycles, Yudin and co-workers have utilized unnatural residues incorporated into the peptide backbone as strategic linkers to control hydrogen bonds.164 Compound 52 has an endocyclic acyl aziridine. Analysis of the 1H NMR spectrum of 52 in DMSO indicated a single conformation on the NMR time scale (Figure 19). However, high temperature/ chemical shift coefficients for the amide protons indicated a lack of internal hydrogen bonding. Ring opening of the acyl aziridine with thiobenzoic acid and reduction with Raney-Ni produced 18-membered macrocycle 53. VT NMR of 53 revealed that the Leu-NH, linker-NH, and exocyclic amide-NH all have low temperature/chemical shift coefficients, indicating a stabilized network of intramolecular hydrogen bonds. The solution structure of 53 was determined using ROESY and 3Jcoupling derived restraints in a dynamic simulated annealing and energy minimization protocol.165,166 This analysis of 53 identified two families of conformers in agreement with the experimental data. One family involves a hydrogen bond between the linker-NH and the Gly2-CO, while the other involves a hydrogen bond between the exocyclic amide-CO and the Leu3-NH. In both cases, the exocyclic amide-NH is hydrogen bonding with the endocyclic pyrrolidine. The Q


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Figure 21. Structure of stylostatin 1 (56) and its solution structure (top). Incorporation of 6-membered lactam (green) into stylostatin 1 (56) to generate 57 results in conformational destabilization and loss of intramolecular hydrogen bonding (bottom).

analysis of the solution and crystal structures of 54 indicated a 4.08 kcal/mol interaction between the reduced amide nitrogen and the Gly2-NH. The oxadiazole oxygen and Gly2-NH contribute an additional 1.05 kcal/mol of stabilization. The authors have reported a further investigation of the properties of this motif and have found that it reliably nucleates distal βturns regardless of ring size.169 pH titrations where the chemical shift of a reporter proton was monitored revealed an inflection point which indicated that the reduced amide had a pKaH of 1.98, which is remarkably low for ammonium cation. A series of control compounds indicated that while some of the reduction in pKaH arises from the electron withdrawing properties of the oxadiazole, a significant portion can be attributed to restriction in the macrocyclic environment. It is likely that the reduction in pKaH correlates to the energetic cost of stabilizing the remote hydrogen bonding pattern. The cycloheptapeptide stylostatin 1 (56) is a 21-membered macrocycle isolated from Stylotella sp170 and is known to inhibit the growth of lymphocytic leukemia cell lines. The NOE-derived solution structure of 56 indicated a single conformation with two β-turns at Ser-Leu and Ile-Pro and a cis conformation at the Ile-Pro amide linkage. Diez and coworkers have reported the incorporation of a 3-aminohydroxylactam residue into the backbone of 56 to generate 57 (Figure 21).171 The 3-amino-hydroxylactam residue is known to induce β-turns in linear peptides.172,173 The 1H NMR spectrum of 57 in DMSO indicated the presence of two stable conformers on the NMR time scale. Analyzing the ΔδCβγ values for both conformers of 57 indicated the two

bonded ring which is present for the endocyclic amide of 53, and it is possible this results in the change of distal β-turn positioning. Yudin and co-workers also reported a cyclization reaction which generates cyclic peptides where one amino acid residue is replaced with an endocyclic amine and an oxadiazole (Figure 20).168 To investigate the conformational implications of inserting this motif into a cyclic peptide backbone, the authors compared the 18-membered macrocycles 54 and 55. Compound 55 is a homodetic cyclic peptide, while in compound 54 the Ala has been replaced with a reduced amide/oxadiazole residue. 1H NMR spectra indicated several slowly interconverting conformations for 55. However, 54 displays a single conformation on the NMR time scale. VT 1H NMR indicated that only the Leu3-NH of 55 is involved in an intramolecular hydrogen bond. Meanwhile, in compound 54, Gly2-NH and Phe5-NH were shown to be involved in intramolecular hydrogen bonds. NOE distances and 3Jcoupling were then used in combination with unrestrained MD simulation to determine the solution structure of 54. The solution structure of 54 shows a β-turn with Gly2 at the i position where the Gly2-NH engages in an intramolecular hydrogen bond with the reduced amide and the oxadiazole. The crystal structure of 54 displays a high similarity with the solution structure. The change from Ala to reduced amide/ oxadiazole results in significant changes in the remote intramolecular hydrogen bonding pattern, imparts conformational stability to the macrocycle, and may be responsible for nucleating the distal β-turn. Natural bonding orbital (NBO) R


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Figure 22. Incorporation of a thiazole into the backbone of 59 results in remote stabilization of the ProB amide and increases in lipophicity and membrane permeation.

Figure 23. Structures of apratoxin A and B. Removal of backbone N-methyl from apratoxin A (60) to generate apratoxin B (61) resulting in the formation of a transannular hydrogen bond and remote cis/trans amide isomerization.

conformations corresponded to the cis- and trans-amide at the Ile-Pro linkage. The Ile-Pro amide is 9 atoms from the 3aminohydroxylactam, which induces a distal change in conformation. To investigate the intramolecular hydrogen bonding network of these macrocycles, the change in the chemical shifts of the amide protons with a gradient of DMSO in CDCl3 was determined. For 56, only the side chains AsnNH and Ile-NH were found to interact with the solvent. Yet, for 57, all amide protons were determined to interact with the solvent. Interestingly, in CDCl3, only the cis conformation of 57 was observed, and the ratio of the trans conformation increased with higher DMSO concentration. For 56, only the cis isomer was observed, indicating to the authors that 57 is more flexible than 56 and is sensitive to changes in solvent.

Temperature/chemical shift coefficients of the amide protons of 56 indicated four intramolecular hydrogen bonds at the Phe, Ala, Leu, and Asn residues. For the cis conformation of 57, the Ala-NH hydrogen bond is maintained. The Phe-NH is in equilibrium between a hydrogen bonded and non-hydrogen bonded state, and the Leu-NH hydrogen bond is no longer present. Heating led to a significant loss of the resolution of amide protons of the trans conformation of 57 to the point where reliable assignments could not be made. These results indicated that the cis conformation of 57 is less stable than that of 56, which allows for the slow isomerization of the Ile-Pro amide linkage when changing solvents. The conformational destabilizing effect of the 3-aminohydroxylactam is somewhat surprising, as increased degrees of cyclization should rigidify S


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Figure 24. Addition of a methyl group to the macrocyclic backbone of 62 to generate 63 stabilizing the biaryl conformation.

(Figure 23). NMR analysis of 60 and 61 indicated that they had the same conformation in the 3,7-dihydroxy-2,5,8,8tetramehtylnonanoic acid region, as evidenced by only minor differences in the 1H NMR chemical shifts of that portion. However, the spectrum of 61 shows two conformers in a 3:1 ratio and a change in the chemical shifts between 60 and 61 for the amino acid region. A strong ROE between H14 and H18 was observed in 61, which indicated a cis-amide bond between Tyr(OMe) and MeAla that is not observed in 60. Distance and dihedral angle restrained MD studies produced structures which indicated that upon N-demethylation of 60 trans to cis amide isomerization occurrs. The Tyr(OMe)-MeAla amide which undergoes cis/trans isomerization is 5 atoms from the site of demethylation. The hydrogen bond between the moCys-CO and Ile-NH is likely important to the isomerization since bringing the hydrogen bond donor and acceptor together could increase the ring strain of this region of the macrocycle, making the cis-amide configuration geometrically preferred. Richardson and co-workers have reported the design and synthesis of the 12-membered macrocycle lorlatinib (62) as a potent inhibitor of anaplastic lymphoma kinase.176 The bioactive conformation of 62 was determined by cocrystallization (Figure 24). The molecule was found to exist as a single atropisomer at the biaryl linkage. The authors also prepared the desmethyl analogue of loratinib, 63. This macrocycle was prepared as a mixture of two enantiomeric atropisomers which could be separated by chiral supercritical fluid chromatography (SFC) but racemized after several weeks at room temperature. Further studies utilizing VT HPLC determined that the barrier to atropisomerization for 63 was 24.6 kcal/mol.177 Heating of 62 did not result in any isomerization. Minimized models of the observed atropisomer of 62 and the second potential diastereomer indicated that the methyl group of 62 results in 1,3-allylic strain which destabilizes the second conformation by 8.5 kcal/mol. The methyl group is 5 atoms from the biaryl linkage, and it effectively stabilizes a single atropisomer. It is unclear if this effect is due to the change in the isomerization barrier or is simply the result of a change in thermodynamics, where the large energy difference would drive the equilibrium completely to the bioactive conformation, meaning only a

the cyclic peptide backbone as it does within a linear peptide. The loss of the Leu-NH and its capacity to form distal, transannular hydrogen bonds may be significant. These results are important as they illustrate that structural elements that induce stabilized conformations in linear peptides can have the opposite effect in cyclic peptides and that the arrangement of a motif relative to other key structural features is critical. Over the course of an effort to optimize the passive membrane permeability and oral bioavailability of 21membered thiazole containing cyclic heptapeptide sanguinamide A (58), Fairlie and co-workers have reported the synthesis and 1H NMR analysis of the homodetic cyclic heptapeptide 59 (Figure 22).174 The 1H NMR spectrum of 59 in DMSO displays two sets of resonances arising from the cis/ trans isomerization at Ile5-Pro6. The authors did not conduct further conformational analysis of 59. Replacement of the Ala with a thiazole in 58 results in stabilization of a single conformer with a trans-amide at Ile5-Pro6, which is 6 atoms away. H/D exchange rates and chemical shift/temperature coefficients indicate a stabilized hydrogen bonding network for 58. The solution conformation of 58 was determined using a simulated annealing protocol with NOE-derived distance restraint. The solution structure indicates two transannular hydrogen bonds between the Ala2-NH and Ile5-CO along with the Ile5-NH and Ala2-CO. It is likely that stabilization of the intramolecular hydrogen bonding is a factor in the isomerization of the Ile5-Pro6 amide bond upon incorporation of the distal thiazole. 59 has an experimental logP of 1.7, while 58 has an experimental logP of 2.5. The difference between the two is striking as the calculated logP values differ by only 0.3. This indicates that the increased lipophilicity of 58 is a result of changes in the conformational preferences of the macrocycle and not simply from reducing the amount of H-bond donors in the molecule by one. 3.4. Addition of Alkyl Groups to Backbone Atoms of a Macrocycle

Apratoxin A (60) and B (61) are 25-membered cyclodepsipeptides that were isolated from the marine cyanobacterium Lyngba majuscule by Moore and Paul.175 These compounds were found to be cytotoxic against certain cancer cell lines in vitro and differ by a N-methyl group on the Ile T


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Figure 25. Methylene insertion into the backbone of 64 to generate 65 resulting in stabilization of the remote cis-amide conformation.

Figure 26. Ring expansion and reduction of the enamide functionality of callyearin A (66) to generate 67 results in destabilization of the intramolecular hydrogen bonding network and conformational interconversion.

amide (>9:1). This research shows how insertion of a backbone methylene can enable distal control over the cis/ trans-amide isomerization in macrocycles. Callyaerin A (66) is a proline-rich cyclic peptide isolated from Indonesian marine sponge Callyspongia aerizusa in 2015 by the Proksch group.181 It is a potent inhibitor of M. tuberculosis (MIC100 of 6.0 μM) with low cyctoxicity against human cells (IC50 > 10 μM). The Brimble group developed the first total synthesis of callyaerin A as well as investigated the conformational effect of the (Z)-2,3-diaminoacrylamide (DAA) bridging motif (Figure 26).182 VT analysis of the native callyaerin A indicated two strong hydrogen bonds from DAA and Ile2-NH (−1.1 ppb/K for both). It was also suggested that two additional hydrogen bonds from Ile4-NH and Leu5-NH (−4.0 and −2.7 ppb/K respectively) stabilized the conformation. For comparative purposes, Asp was used to replace the DAA motif callyaerin A, which increased the ring size of the macrocycle. Asp-containing callyaerin A (67) has a more complex 1H NMR spectra. The amide resonances exceeded the number of amide protons, suggesting multiple amide rotamers and conformations that were in slow exchange. As temperature was increased during the VT study, peaks became broader, suggesting that conformational exchange between conformations was accelerated. However, 67 still exhibited broad peaks at 80 °C and required higher temperatures for fast exchange. 66 adopts a single conformer stabilized by two to four hydrogen bonds while the 67 adopts multiple conformations. This suggests the DAA motif can stabilize a remote hydrogen

single conformation will be observed despite a low kinetic barrier. 3.5. Changes in Ring Size

The Doi group pursued the design and synthesis of apratoxin A (60) mimetics that had better synthetic accessibility and stability with comparable potency to the parent compound.178 To do so, the authors replaced the thiazoline moiety with an amino acid linker, since modifications in this region have been shown to maintain cytotoxicity and increase in vivo stability.179,180 NMR studies on the modified apratoxin A analogs were conducted to investigate their conformations. Analysis of the 4-aminobutanoic acid linker-containing macrocycle 64 showed two sets of chemical shifts in a 1:1 ratio suggesting that two conformers are present. For one of the conformations, a strong ROE between H14 and H18 was observed, indicating a cis-amide bond between Tyr(OMe) and MeAla. Similar NMR spectra for (E)-dehydroapratoxin A (an analogue that also exists in two conformers of cis- and transamide) and 64 confirmed the presence of both cis−trans conformers (Figure 25). Despite the differences in amide bond geometry, the conformation of the Dtrena region is similar between both conformers of 64, as indicated by the 3JH−H− coupling in that region. Molecular modeling of 64 showed that the trans-amide conformation resembled apratoxin A (60), which mainly adopts a trans-amide, and that cis-amide conformation resembled apratoxin B (61), which mainly adopts a cis-amide. Interestingly, the shortening of the linker to 3-aminopropanoic acid (65) increased the ratio of cis to trans U


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Figure 27. Insertion of methylene into the backbone of 68 to generate 69 results in reorganization of the hydrogen bonding network and remote isomerization of the Pro amide.

Figure 28. Aromatization of thiazoline of tawicyclamide B (70) to form 71 resulting in conformational reorganization and isomerization of remote Pro amide.

bonding pattern by rigidifying the entire macrocyclic backbone of callyaerin A. Sewald and co-workers have investigated the conformations of several cyclic peptides containing single β-amino acid residues.183 These studies were conducted to characterize the capacity of β-amino acid to nucleate a γ-turn in various structural environments. During the course of their investigations, they prepared the 15-membered pentapeptide 68 and 16-membered analogue 69 where the asparagine residue has been converted into a β-Asn residue (Figure 27). The conformations of these macrocycles were analyzed in DMSO. Experimental distances derived from NOE spectra were used for restrained MD simulations. The results of the restrained MD were clustered based on torsional angle similarity to provide final structural proposals. These structures were then validated by comparison with experimental 3J-coupling constants and temperature/chemical shift coefficients of amide as well as by further unrestrained MD simulations. 68 adopts a classical cyclic pentapeptide conformation with a γturn around the Asp residue and a Type I β-turn around Pro and Leu. Strong NOEs were observed between the Ser-CαH and the Pro-CδH. Temperature/chemical shift coefficients for

Leu-NH (−2.0 ppb/K) and Asp-NH (−1.8 ppb/K) support the postulated arrangement of turns. Exchange of Asn to β-Shomoasparagine (βhAsn) to generate 69 results in a reorganization of the backbone conformation. The remote peptide bond between Ser and Pro changes to cis configuration for 69, as supported by strong NOE between Ser-CαH and Pro-CαH and the 13C chemical shifts. The Pro of 69 adopts the i+2 position of a Type VIa β-turn, while the βhAsn residue was found in the central position of a so-called Ψψ-turn. The insertion of a methylene group results in isomerization of an amide 7 atoms away and a change in turn placement and subtype. These conformational changes are likely mediated by a relief of ring strain. 3.6. Oxidation or Reduction of Ring Functionality

Tawicyclamide B (70) is a cyclic peptide that possesses thiazole and thiazoline amino acid residues.184 It was isolated from the ascidian Lissoclinum patella by the Clardy group, and its structure was elucidated by NMR spectroscopy (1H NMR, 13 C NMR, COSY, ROESY) and X-ray crystallography (Figure 28). The crystal structure of tawicyclamide B indicated a cisamide bond between Pro and Val. The cis-amide bond is thought to be stabilized by weak hydrogen bonding between V


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Figure 29. Reduction of the backbone amide of 72 to generate 71 resulting in a change of the hydrogen bonding capacity of the NH and subsequent reorganization of the remote hydrogen bonding pattern.

the Val-CO of the cis-amide and the Leu-NH. During the structure elucidation, oxidation of the thiazoline to a thiazole to generate 71 was used to confirm the heterocycle’s presence in the macrocycle. Both X-ray crystallography and NOE analysis of the natural product and oxidized derivative indicated that oxidation had significant conformational effects on the molecule, including isomerization of the Pro-Val amide 11 atoms away from the site of oxidation. NOE derived distances were used as restraints for an energy minimization and MD protocol to generate solution structures of 71. Additional evidence for the isomerization of this amide bond came from analysis of the ΔδCβγ of Pro. The authors noted that upon oxidation of the thiazoline ring to a thiazole a π to π* interaction can stabilize the rotation of the adjacent carbonyl to become coplanar with the ring. The authors suggest this contributes to the drastic conformational changes observed upon oxidation. This example illustrates how low energy stereoelectronic effects can also stabilize significant conformational reorganization of a macrocycle. As part of continued efforts to modulate the conformation of the RGD motif in cyclic peptides for the development of integrin inhibitors, Kessler and co-workers have reported the synthesis and conformational analysis of the 15-membered reduced amide containing cyclic pseudopentapeptide 71.185 The conformation of 71 was compared with corresponding allamide cyclic pentapeptide 72, whose conformational analysis has been discussed previously (Figure 14). 72 adopts a conformation with a type II′ β-turn and γ-turn. The D-Phe residue adopts the i+1 position of the type II′ β-turn (Figure 29). NMR analysis of the trifluoroacetate salt of 71 was conducted in DMSO/water (8:2). Resonances were assigned using 2D techniques. VT 1H NMR revealed a high temperature/chemical shift coefficient of −7.5 ppb/K for the Arg-NH, which is typical of a solvent exposed amide proton. A small temperature/chemical shift coefficient measured for the AspNH indicated its involvement in an intramolecular hydrogen bond. These coefficients, along with significant separation of the chemical shifts of diastereotopic geminal protons were assumed to indicate low probability for conformational averaging on the 1H NMR time scale. NOE and J-coupling derived restraints were used during MD simulations in DMSO and water. These results produced an ensemble of highly similar equilibrium conformations (Figure 29), which are significantly different from that of 72. The Arg of 71 adopts the i+1 position of a type II′ β-turn, with a hydrogen bond between the Asp-CO and the Val-NH. The D-Phe adopts the i +1 position of a γ-turn, stabilized by a hydrogen bond between

the D-Phe-CO and the ammonium of the protonated reduced amide. The substitution of the D-Phe amide for a reduced amide results in a complete reorganization of the backbone structure. This was surprising to the authors, as the reduced amide has less steric restraints and could easily fulfill the structural requirements of a type II′ β-turn centered on the DPhe. However, the capacity of the ammonium to form hydrogen bonds with the D-Phe-CO appears to override the typical preference of the D-residue to nucleate a β-turn and stabilized a γ-turn instead. This results in the reorganization of the remote γ-turn at the Gly residue to a β-turn. Spatola and Ma have also reported a highly similar reorganization of a cyclic pentapeptide backbone upon incorporation of a reduced amide.186 These results highlight the significance of local transannular hydrogen bonds as a driving force for distal conformational reorganization.

4. CONCLUSIONS The goal of this review has been to illustrate that relatively minor structural modifications can result in conformational reorganization of remote regions of a macrocyclic backbone. For instance, alteration in backbone stereochemistry can lead to coupled bond rotations that can result in remote changes in conformation. Modification of exocyclic functionality can lead to conformational changes through transannular interactions. Changes in the oxidation states of atoms comprising macrocyclic backbones can alter ring flexibility. Incorporation of heterocycles tends to planarize the macrocyclic backbone and bring about additional reduction in the conformational degrees of freedom. The location of a structural modification relative to the existing features such as stereocenters, unsaturated functionality, and hydrogen bond donors/acceptors is a critical consideration when attempting to understand how conformational changes are propagated throughout the ring. Most examples in this review rely on computational analysis to build models of potential conformers, which can be challenging if cooperative behavior between bond rotations is at play. We hope that the examples highlighted in this review illustrate the complex nature of the relationship between structure and conformation in macrocycles and will inform further efforts to control macrocycle properties through rational modification. This is particularly important for attempts to derive meaningful structure/activity relationships. W


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AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Andrei K. Yudin: 0000-0003-3170-9103 Notes

The authors declare no competing financial interest. Biographies Solomon Appavoo received his B.S. degree in chemistry from Carleton University (Ottawa, Canada) in 2016, where he carried out research with Professor Jeffrey Manthorpe on the design of kinase inhibitors. In 2016 he joined the research group of Profesor Andrei K. Yudin at the University of Toronto, where he is currently pursuing a Ph.D. degree in chemistry. His research interests involve the synthesis and conformational analysis of medicinally relevant organic molecules. Sungjoon Huh was born in Changwon, South Korea, and raised in Edmonton, Alberta, Canada. He then moved to Vancouver, British Columbia, where he completed his B.Sc. at the University of British Columbia with a major in chemistry, where he worded under the supervision of Dr. Chris Orvig and Dr. David M. Perrin. In September 2017, Sean joined the Yudin lab as a graduate student with interests in macrocycle synthesis. Diego B. Diaz was born in Toronto, Canada, in 1992. He received his B.Sc. (Hons) degree in 2014 at the University of Toronto Mississauga, where he carried out research work in the laboratories of Professor Ulrich Fekl and Professor Patrick T. Gunning. In 2014 he joined the research group of Professor Andrei K. Yudin at the University of Toronto and is currently working towards his Ph.D. degree in organic chemistry. His main research interests are in organic/biological/medicinal chemistry and asymmetric catalysis. Andrei K. Yudin received his undergraduate degree at Moscow State University in 1992. He subsequently worked in the laboratories of G. K. S. Prakash and the Nobel Laureate George A. Olah at USC, where he obtained his Ph.D. in 1996. In 1998, following postdoctoral training in the laboratory of the Nobel Laureate K. Barry Sharpless at the Scripps Research Institute, Professor Yudin started his independent career at the University of Toronto. He became an Associate Professor in 2002, which was followed by promotion to the rank of a Full Professor in 2007. In addition to his University of Toronto appointment, Professor Yudin is an Associate Editor for Chemical Science. His research addresses the fundamental challenges of synthetic organic chemistry.

ACKNOWLEDGMENTS The Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged for financial support of this work. Yike Zhou (UCLA) is thanked for his assistance with the editing process. We would also like to thank the members of the Yudin group for helpful discussions and assistance with the editing process. S.D.A. and D.B.D. also thank NSERC for CGS-D and PGS-D funding, respectively. REFERENCES (1) Giordanetto, F.; Kihlberg, J. Macrocyclic drugs and clinical candidates: what can medicinal Chemists Learn from their properties? J. Med. Chem. 2014, 57, 278−295. (2) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The exploration of macrocycles for drug discovery − an underexploited structural class. Nat. Rev. Drug Discovery 2008, 7, 608−642. X


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Conformational Control of Macrocycles by Remote Structural

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