Conor C. G. Scully

Metal clips that induce unstructured pentapeptides to be α-helical in water

Short peptides corresponding to protein helices do not form thermodynamically stable helical structures in water, a solvent that strongly competes for hydrogen-bonding amides of the peptide backbone. Metalloproteins often feature metal ions coordinated to amino acids within hydrogen-bonded helical regions of protein structure, so there is a prospect of metals stabilizing or inducing helical structures in short peptides. However, this has only previously been observed in nonaqueous solvents or under strongly helix-favoring conditions in water. Here cis-[Ru(NH(3))(4)(solvent)(2)](2+) and [Pd(en)(solvent)(2)](2+) are compared in water for their capacity as metal clips to induce alpha-helicity in completely unstructured peptides as short as five residues, Ac-HARAH-NH(2) and Ac-MARAM-NH(2). More alpha-helicity was observed for the latter pentapeptide and, when chelated to ruthenium, it showed the greatest alpha-helicity yet reported for a short metallopeptide in water (approximately 80%). Helicity was clearly induced rather than stabilized, and the two methionines were 10(13)-fold more effective than two histidines in stabilizing the lower oxidation state Ru(II) over Ru(III). The study identifies key factors that influence stability of an alpha-helical turn in water, suggests metal ions as tools for peptide folding, and raises an intriguing possibility of transiently coordinated metal ions playing important roles in native folding of polypeptides in water.

Development of the Direct Suzuki–Miyaura Cross-Coupling of Primary B-Alkyl MIDA-boronates and Aryl Bromides

The development of a palladium-catalyzed sp(3)-sp(2) Suzuki-Miyaura cross-coupling of B-alkyl-N-methyliminodiacetyl (B-alkyl MIDA) boronates and (hetero)aryl bromides is reported. This transformation is tolerant of a variety of functional groups (F, NO2, CN, Cl, COCH3, and CHO). B-Alkyl MIDA boronates allow an efficient cross-coupling reaction directed toward the synthesis of unsymmetrical methylene diaryls as well as alkylated arenes in good to excellent yields.

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.

Conformational modulation of in vitro activity of cyclic RGD peptides via aziridine aldehyde-driven macrocyclization chemistry.

Here, we demonstrate a conjugation strategy whereby cyclic RGD-containing macrocycles are prepared using aziridine aldehydes, isocyanides, and linear peptides, followed by conjugation to a cysteamine linker. Our method involves site-selective aziridine ring-opening with the nucleophilic sulfhydryl group of cysteamine. Fluorescein was then efficiently conjugated to the primary amine of cysteamine by NHS-chemistry. This strategy may be expanded to provide easy access to a wide variety of fluorescent dyes or radiometal chelators. Modeling studies showed that aziridine aldehyde cyclization chemistry stabilized the RGD motif into the required bioactive conformation and that this cyclization chemistry modulated the geometry of macrocycles of different residue lengths. In vitro studies showed that cPRGDA and cPRGDAA both selectively bound to α(V)β(3)-overexpressing U87 glioblastoma cells, and that cPRGDA had a better binding affinity compared to cPRGDAA. The improved binding affinity of cPRGDA was attributed to the fixed Pro-C(α)-Asp-C(α) distance surrounding the stabilized RGD motif in cPRGDA.

Oxadiazole grafts in peptide macrocycles

Synthetic methods that provide control over macrocycle conformation and, at the same time, mitigate the polarity of peptide bonds represent valuable tools for the discovery of new bioactive molecules. Here, we report a macrocyclization reaction between a linear peptide, an aldehyde and (N-isocyanimino)triphenylphosphorane. This process generates head-to-tail cyclic peptidomimetics in a single step. This method is tolerant to variation in the peptide and aldehyde components and has been applied for the synthesis of 15-, 18-, 21- and 24-membered rings. The resulting peptide macrocycles feature a 1,3,4-oxadiazole and a tertiary amine in their scaffolds. This non-canonical backbone region acts as an endocyclic control element that promotes and stabilizes a unique intramolecular hydrogen-bond network and can lead to macrocycles with conformationally rigid turn structures. Oxadiazole-containing macrocycles can also display a high passive membrane permeability, an important property for the development of bioavailable peptide-based therapeutics. Controlling macrocycle conformation represents a powerful tool for the construction of new bioactive molecules. Now, peptide-based macrocycles bearing a 1,3,4-oxadiazole moiety grafted into their backbone have been synthesized via a new cyclization approach. The resulting cyclic products exhibit conformationally rigid turn structures (stabilized through intramolecular hydrogen bonding) that can display passive membrane permeability.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Site-specific integration of amino acid fragments into cyclic peptides.

The concept of site-specific integration of fragments into macrocyclic entities has not yet found application in the realm of synthetic chemistry. Here we show that the reduced amidicity of aziridine amide bonds provides an entry point for the site-specific integration of amino acids and peptide fragments into the homodetic cyclic peptide architecture. This new synthetic operation improves both the convergence and divergence of cyclic peptide synthesis.

Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor

Human anaphylatoxin C3a, formed through cleavage of complement protein C3, is a potent effector of innate immunity via activation of its G protein coupled receptor, human C3aR. Previously reported short peptide ligands for this receptor either have low potency or lack receptor selectivity. Here we report the first small peptide agonists that are both potent and selective for human C3aR, derived from structure-activity relationships of peptides based on the C-terminus of C3a. Affinity for C3aR was examined by competitive binding with (125)I-labeled C3a to human PBMCs [corrected], agonist versus antagonist activity measured using fluorescence detection of intracellular calcium, and general selectivity monitored by C3a-induced receptor desensitization. An NMR structure for an agonist in DMSO showed a beta-turn motif that may be important for C3aR binding and activation. Derivatization produced a noncompetitive and insurmountable antagonist of C3aR. Small molecule C3a agonists and antagonists may be valuable probes of immunity and inflammatory diseases.

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.