Serge Zaretsky

Small heterocycles in multicomponent reactions.

Benjamin H. Rotstein,†,‡ Serge Zaretsky,† Vishal Rai,†,§ and Andrei K. Yudin*,† †Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario Canada, M5S 3H6 ‡Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, and Department of Radiology, Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114, United States Department of Chemistry, Indian Institute of Science Education and Research (IISER) Bhopal, Indore By-pass Road, Bhauri, Bhopal 462 066, MP India

Exocyclic Control of Turn Induction in Macrocyclic Peptide Scaffolds

Macrocyclic peptides have found wide utility across scientific disciplines that range from medicinal chemistry to materials science. In biological function-directed applications, macrocycles often need to have two attributes: conformational stability and cellular permeability. Of particular significance are stable peptide scaffolds that adopt structurally homogenous conformations that can mimic protein surface epitopes. Careful control of conformational properties enables exquisite selectivity for therapeutically relevant targets such as signaling proteins, immune system regulators, neurotransmitters, and enzymes. Due to their size, polarity, and number of rotatable bonds, most macrocyclic peptides possess little, if any, cellular permeability. Amide N-methylation, a strategy inspired by the orally bioavailable macrocyclic undecapeptide cyclosporine A, has produced encouraging results. Seven of eleven peptide bonds in cyclosporine A are N-methylated. Thus, the dominant conformation of cyclosporine A in hydrophobic media is characterized by minimal polar surface exposure, which allows the molecule to pass across the cell membrane. However, applying extensive N-methylation as a general approach to improve the cellular permeability of cyclic peptides presents significant synthetic challenges. As a result, alternative methods of modulating conformational preferences of cyclic peptides are in demand. The strategy introduced in this paper hinges upon maximizing the number of internal hydrogen bonds by recruiting an exocyclic amide group as a key hydrogen bond partner. In this report, we show that control over cyclic peptide conformation by using exocyclic amides can lead to the formation of well-defined types of b-turns and results in improved cellular permeability. The strategy also allows for further “lockdown” of the molecule by rational design of a covalent linkage that minimizes the number of rotatable bonds and accurately recreates the desired secondary structure elements. We recently reported the macrocyclization of peptides with amphoteric aziridine aldehydes, which operate in conjunction with isocyanides. In the course of these studies, we noted that the strategic role of the isocyanide component was to provide an exocyclic amide in all products of our reaction. We hypothesized that the proximity of the exocyclic carbonyl moiety to the backbone could have a significant effect on the structure of the peptide. Due to the absence of natural amino acids equipped with an a-amide group, this strategy is without precedent in cyclic peptide chemistry. At the outset, we focused our attention on 18-membered rings present in the structures of cyclic hexapeptides. These molecules have found wide utility as biological probes. For example, potent analogues of somatostatin, cyclic hexapeptides with anti-inflammatory, antiangiogenic, and antitumor properties have been reported. The propensity of 18membered cyclic peptide rings to incorporate b-turns into their structure is the main reason for sustained interest in this class of molecules. b-Turns are defined by a reverseturn of the peptide backbone in a four residue segment (residues i to i+3), with less than 7 separation between the a carbons of the i and i+ 3 residues. As the conformational stability goal hinged on comparison to known protein bturn structures, we sought to have b-turn-inducing residues, such as Pro and Gly, in our molecules. Cyclized Pro-Gly-Leu-Gly-Phe compound 1 (Figure 1a) was an ideal fit and while NMR spectroscopic analysis of 1 and of the isobutyl substituted analogue 2 showed sharp peaks for all N H s and Ca H s, the VT (variable temperature) NMR spectra did not indicate any internal hydrogen bonding. Acyl aziridines exhibit greater flexibility than regular amides because of the sp nature of the aziridine nitrogen atom. The resulting flexibility of the peptide backbone prompted us to explore nucleophilic aziridine ring opening. We hypothesized that the regular amide present in the product of ring opening might show more constrained behavior (Figure 1a, 3). Compound 3 was synthesized by a telescopic cyclization from linear Pro-Gly-Leu-Gly-Phe, aziridine aldehyde 4, and tert-butyl isocyanide, followed by aziridine ring opening with thiobenzoic acid and desulfurization with Raney-Ni (Figure 1b) in 14 % overall yield after HPLC purification. Gratifyingly, a crystal of the ring-opened macrocycle 3 was grown in chloroform. The crystal structure of 3 (Figure 2a) showed the peptide adopting an amphiphilic conformation of a polar-bottom face with a hydrophobic cap [a] S. Zaretsky, Dr. C. C. G. Scully, Prof. Dr. A. K. Yudin Davenport Research Laboratories, Department of Chemistry University of Toronto 80 St. George Str., Toronto, ON, M5S 3H6 (Canada) E-mail : ayudin@chem.utoronto.ca [b] Dr. A. J. Lough X-ray Laboratory, Department of Chemistry University of Toronto 80 St. George Str., Toronto, ON, M5S 3H6 (Canada) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303453. It contains all experimental procedures and spectroscopic data.

Passive Membrane Permeability of Macrocycles Can Be Controlled by Exocyclic Amide Bonds

We have developed a strategy for synthesizing passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intramolecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multicomponent-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and d-amino acids to produce passively permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles.

Bending Rigid Molecular Rods: Formation of Oligoproline Macrocycles

Bent but not broken: cyclic oligoprolines are accessed in a reaction that effectively bends rigid oligoproline peptides (see scheme; TBDMS=tert-butyldimethylsilyl). The stitching is accomplished during macrocyclization enabled by aziridine aldehydes and isocyanides. Molecular modeling studies suggest that electrostatic attraction between the termini of the linear peptide is pivotal for macrocyclization. The macrocycles were studied by circular dichroism with a polyproline II structure being observed in larger macrocycles.

Solid-Phase Parallel Synthesis of Functionalised Medium-to-Large Cyclic Peptidomimetics through Three-Component Coupling Driven by Aziridine Aldehyde Dimers

The first solid-phase parallel synthesis of macrocyclic peptides using three-component coupling driven by aziridine aldehyde dimers is described. The method supports the synthesis of 9- to 18-membered aziridine-containing macrocycles, which are then functionalized by nucleophilic opening of the aziridine ring. This constitutes a robust approach for the rapid parallel synthesis of macrocyclic peptides.

Stereocontrolled Disruption of the Ugi Reaction toward the Production of Chiral Piperazinones: Substrate Scope and Process Development

The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.

Shifting the energy landscape of multicomponent reactions using aziridine aldehyde dimers: a mechanistic study.

A multicomponent reaction between an aziridine aldehyde dimer, isocyanide, and l-proline to afford a chiral piperazinone was studied to gain insight into the stereodetermining and rate-limiting steps of the reaction. The stereochemistry of the reaction was found to be determined by isocyanide addition, while the rate-limiting step was found to deviate from traditional isocyanide-based multicomponent reactions. A first-order rate dependence on aziridine aldehyde dimer and a zero-order rate dependence on all other reagents have been obtained. Computations at the MPWPW91/6-31G(d) level supported the experimental kinetic results and provide insight into the overall mechanism and the factors contributing to stereochemical induction. These factors are similar to traditional isocyanide-based multicomponent reactions, such as the Ugi reaction. The computations revealed that selective formation of a Z-iminium ion plays a key role in controlling the stereoselectivity of isocyanide addition, and the carboxylate group of l-proline mediates stereofacial addition. These conclusions are expected to be applicable to a wide range of reported stereoselective Ugi reactions and provide a basis for understanding the related macrocyclization of peptides with aziridine aldehydes.

Bicycle synthesis through peptide macrocyclization using aziridine aldehydes followed by late stage disulfide bond installation

We present a method that can be applied to generate medium-sized peptidomimetic macrocycles equipped with disulfide bonds. The reaction hinges on amphoteric aziridine aldehydes and their ability to bridge the ends of linear peptides in the presence of isocyanides. Aziridine aldehyde dimers enable the initial cyclization, which is followed by site-specific aziridine ring-opening with sodium azide. Subsequent to that, thallium-induced oxidative disulfide bond formation furnishes the final product. The NMR characterization of the molecules obtained using this method indicates that conformationally well-behaved systems are readily accessible. The site-specific introduction of azide functionality should open the doors to subsequent functionalization using well-established protocols.

Rational Design of Calpain Inhibitors Based on Calpastatin Peptidomimetics

Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by natures hint that a β-turn loop within calpastatin forms a broad interaction around calpains active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

Macrocyclic Templates for Library Synthesis of Peptido-Conjugates

Cyclic peptides have wide utility in the biological sciences. As conformationally locked analogs of the parent linear peptides, they possess greater stability under physiological conditions and increased binding affinity for their targets. As investigations of biological processes often require reporter molecules and functional readouts, chemical probes are commonly appended with functional groups that allow for conjugation to biological entities. Herein we describe the functionalization of cyclic peptides prepared via aziridine aldehyde-mediated macrocyclization. These cyclic peptides contain an aziridine ring that can be further functionalized by ring opening with nucleophiles. We report on the methodology used to produce a cyclic peptide analog of Pro-Gly-Leu-Gly-Phe with either azido or sulfhydryl functionality.