Spiroligomer-Based Macrocycles for Atomically Precise Membranes.

Here, we report a new class of peptidomimetic macrocycles with well-defined three-dimensional structures and low conformational flexibility. They are assembled from fused-ring spiro-ladder oligomers (spiroligomers) by modular solid-phase synthesis. Two-dimensional nuclear magnetic resonance confirms their shape persistency. Triangular macrocycles of tunable sizes assemble into membranes with atomically precise pores, which exhibit size and shape-dependent molecular sieving towards a series of structurally similar compounds. The exceptional structural diversity and stability of spiroligomer-based macrocycles will be explored for more applications.

Metal Cation-Binding Mechanisms of Q-Proline Peptoid Macrocycles in Solution.

The rational design of foldable and functionalizable peptidomimetic scaffolds requires the concerted application of both computational and experimental methods. Recently, a new class of designed peptoid macrocycle incorporating spiroligomer proline mimics (Q-prolines) has been found to preorganize when bound by monovalent metal cations. To determine the solution-state structure of these cation-bound macrocycles, we employ a Bayesian inference method (BICePs) to reconcile enhanced-sampling molecular simulations with sparse ROESY correlations from experimental NMR studies to predict and design conformational and binding properties of macrocycles as functional scaffolds for peptidomimetics. Conformations predicted to be most populated in solution were then simulated in the presence of explicit cations to yield trajectories with observed binding events, revealing a highly preorganized all-trans amide conformation, whose formation is likely limited by the slow rate of cis/trans isomerization. Interestingly, this conformation differs from a racemic crystal structure solved in the absence of cation. Free energies of cation binding computed from distance-dependent potentials of mean force suggest Na+ has a higher affinity to the macrocycle than K+, with both cations binding much more strongly in acetonitrile than water. The simulated affinities are able to correctly rank the extent to which different macrocycle sequences exhibit preorganization in the presence of different metal cations and solvents, suggesting our approach is suitable for solution-state computational design.

Metal-Binding Q-Proline Macrocycles.

We introduce the efficient Fmoc-SPPS and peptoid synthesis of Q-proline-based, metal-binding macrocycles (QPMs), which bind metal cations and display nine functional groups. Metal-free QPMs are disordered, evidenced by NMR and a crystal structure of QPM-3 obtained through racemic crystallization. Upon addition of metal cations, QPMs adopt ordered structures. Notably, the addition of a second functional group at the hydantoin amide position (R2) converts the proline ring from Cγ-endo to Cγ-exo, due to steric interactions.

Development of Spiroligomer-Peptoid Hybrids.

Creating functional macromolecules that possess the diversity and functionality of proteins poses an enormous challenge, as this requires large, preorganized macromolecules to facilitate interactions. Peptoids have been shown to interact with proteins, and combinatorial libraries of peptoids have been useful in discovering new ligands for protein binding. We have created spiroligomer-peptoid hybrids that have a spirocyclic core that preorganizes functional groups in three-dimensional space. By utilizing spiroligomers, we can reduce the number of rotatable bonds between functional groups while increasing the stereochemical diversity of the molecules. We have synthesized 15 new spiroligomer monomer amines that contain two stereocenters and three functional groups (67-84% yields from a common hydantoin starting material) as well as a spiroligomer trimer 25 with six stereocenters and five functional groups. These 16 amines were used to synthesize five first-generation spiroligomer-peptoids hybrids.

One-Pot Synthesis of Chiral, Spirocyclic 4-Hydantoin-Proline Derivatives for Incorporation into Spiroligomer-Based Macromolecules.

Derivatives of 4-hydantoin-proline have been synthesized via a direct two-step alkylation method. This method is valuable in the development of applications of N,N'-disubstituted hydantoin bearing α-amino acids by improving yields, reducing the time and number of steps required to synthesize these substituted molecules, and enabling late stage functionalization of spiroligomer termini. Over 20 unique electrophiles have been tested, highlighting the inherent versatility of this chemistry.

Synthesis of Spiroligomer-Containing Macrocycles.

We demonstrate the synthesis and characterization of the solution conformations of a collection of functionalized spiroligomer-based macrocycles. These macrocycles contain 14 independently controllable stereocenters and four independently controllable functional groups on a highly preorganized scaffold. These molecules are being developed to display complex, preorganized surfaces for binding proteins and to create enzyme-like active sites. In this work, we demonstrate the convergent synthetic approach to this new class of macrocycles and demonstrate that the conformational properties of these molecules can be changed by altering the configuration stereocenters within the backbone.

Acceleration of an aromatic Claisen rearrangement via a designed spiroligozyme catalyst that mimics the ketosteroid isomerase catalytic dyad.

A series of hydrogen-bonding catalysts have been designed for the aromatic Claisen rearrangement of a 1,1-dimethylallyl coumarin. These catalysts were designed as mimics of the two-point hydrogen-bonding interaction present in ketosteroid isomerase that has been proposed to stabilize a developing negative charge on the ether oxygen in the migration of the double bond.1 Two hydrogen bond donating groups, a phenol alcohol and a carboxylic acid, were grafted onto a conformationally restrained spirocyclic scaffold, and together they enhance the rate of the Claisen rearrangement by a factor of 58 over the background reaction. Theoretical calculations correctly predict the most active catalyst and suggest that both preorganization and favorable interactions with the transition state of the reaction are responsible for the observed rate enhancement.

Architectural spiroligomers designed for binuclear metal complex templating.

The first structurally, spectroscopically, and electronically characterized metal-spiroligomer complexes are reported. The binuclear [M2L2](4+) ions (M = Mn, Zn) are macrocyclic "squares" and are characterized by X-ray diffraction, (1)H and (13)C NMR, electronic absorption, emission, and mass spectroscopies. The manganese complex contains two spin-independent Mn(II) ions and is additionally characterized using EPR and CD spectroscopies and CV.

A spiroligomer α-helix mimic that binds HDM2, penetrates human cells and stabilizes HDM2 in cell culture.

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

Spiroligozymes for transesterifications: design and relationship of structure to activity.

Transesterification catalysts based on stereochemically defined, modular, functionalized ladder-molecules (named spiroligozymes) were designed, using the "inside-out" design strategy, and mutated synthetically to improve catalysis. A series of stereochemically and regiochemically diverse bifunctional spiroligozymes were first synthesized to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacted with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7 × 10(3)-fold faster than the background reaction with a benzyl alcohol. Two trifunctional spiroligozymes were then synthesized that combined a urea with the pyridine and alcohol to act as an oxyanion hole and activate the bound acyl-spiroligozyme intermediate to enable acyl-transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k(1)/k(uncat) of 2.2 × 10(3) and k(2)/k(uncat) of 1.3 × 10(2). Quantum mechanical calculations identified the four transition states of the catalytic cycle and provided a detailed view of every stage of the transesterification reaction.

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Hydrophobic substituent effects on proline catalysis of aldol reactions in water.

Derivatives of 4-hydroxyproline with a series of hydrophobic groups in well-defined orientations have been tested as catalysts for the aldol reactions. All of the modified proline catalysts carry out the intermolecular aldol reaction in water and provide high diastereoselectivity and enantioselectivity. Modified prolines with aromatic groups syn to the carboxylic acid are better catalysts than those with small hydrophobic groups (1a is 43.5 times faster than 1f). Quantum mechanical calculations provide transition structures, TS-1a(water) and TS-1f(water), that support the hypothesis that a stabilizing hydrophobic interaction occurs with 1a.

Solid-phase synthesis of functionalized bis-peptides.

We demonstrate the first solid-phase synthesis of highly functionalized bis-peptides. Bis-peptides are ladder oligomers composed of stereochemically pure, cyclic bis-amino acids joined by substituted diketopiperazine linkages. They have a shape-programmable backbone that is controlled by controlling the stereochemistry and sequence of the monomers within each oligomer. Functionalized bis-peptides are assembled using a new amide bond forming reaction (acyl-transfer coupling) that we have previously developed and a novel activation strategy that allows the sequential formation of penta- and hexa-substituted diketopiperazines from extremely hindered N-alkyl-alpha,alpha-disubstituted amino acids. We present mechanistic evidence that acyl-transfer coupling is competitive with direct acylation in the formation of hindered amide bonds. We also detail the synthesis of four functionalized bis-peptides, and that by combining bis-peptides with amino acids through diketopiperazine linkages, bis-peptides can mimic the display of residues i, i+4, i+7 of an alpha-helical peptide.

Synthesis of hexa- and pentasubstituted diketopiperazines from sterically hindered amino acids.

Steric hindrance assists in the formation of hindered diketopiperazines using acyl-transfer coupling. In acyl-transfer coupling, the carboxylate of an unprotected N-alkylamino acid attacks an active ester to form a transient anhydride that undergoes an O,N acyl transfer to form a tertiary amide. If the active ester is part of an N-alkylamino acid it will form a diketopiperazine. It is demonstrated here that acyl-transfer coupling can assemble highly functionalized bis-peptides bearing a functional group on every monomer.

Synthesis of a carboxylate functionalized bis-amino acid monomer.

The synthesis of the first functionalized bis-amino acid monomer proAc(2S3S4R) 1 that carries an acetyl side chain is presented. This monomer was incorporated into oligomer 3 and the solution phase structure was determined by using two-dimensional nuclear magnetic resonance. The solution structure confirmed the intended connectivity and stereochemistry of the oligomer. This first functionalized bis-amino acid represents a milestone toward functionalized bis-peptide nanostructures for catalytic, molecular recognition, and nanotechnology applications.

Experimental evidence for water mediated electron transfer through bis-amino acid donor-bridge-acceptor oligomers.

This work compares the photoinduced unimolecular electron transfer rate constants for two different solute molecules (D-SSS-A and D-SRR-A) in water and DMSO solvents. The D-SSS-A solute has a cleft between the electron donor and acceptor units, which is able to contain a water molecule but is too small for DMSO. The rate constant for D-SSS-A in water is significantly higher than that for D-SRR-A, which lacks a cleft, and significantly higher for either solute in DMSO. The enhancement of the rate constant is explained by an electron tunneling pathway that involves water molecule(s).

Exploiting an inherent neighboring group effect of alpha-amino acids to synthesize extremely hindered dipeptides.

The creation of highly hindered peptides that contain combinations of non-natural N-alkyl amino acids and N-alkyl-alpha,alpha-disubstituted amino acids presents a formidable challenge. Hindered, non-natural amino acids are of interest because they import resistance to proteolysis and unusual conformational properties to peptides that contain them. Toward a solution to this problem, we describe a new approach to creating extremely hindered dipeptides that is operationally simple and uses mild conditions and commercially available amino acids. The approach reduces the need for protecting groups and yields urethane-protected dipeptide acids that can be used as building blocks in the synthesis of larger peptides. We propose that the reaction proceeds through a previously unexploited intramolecular O,N-acyl transfer pathway.

Shape-programmable macromolecules.

Proteins catalyze specific chemical reactions and carry out highly selective molecular recognition because they adopt well-defined three-dimensional structures and position chemically reactive functional groups in specific constellations. Proteins attain these well-defined structures through the complex process of protein folding. We seek to emulate these protein functions by constructing macromolecules that are easier to engineer by avoiding folding altogether. Toward that goal, we have developed an approach for the synthesis of macromolecules with programmable shapes. As described in this Account, we have constructed synthetic building blocks called bis-amino acids that we then couple through pairs of amide bonds to create water-soluble, spiroladder oligomers (bis-peptides) with well-defined three-dimensional structures. Bis-peptides use the conformational preferences of fused rings, stereochemistry, and strong covalent bonds to define their shape, unlike natural proteins and synthetic foldamers, which depend on noncovalent interactions and an unpredictable folding process to attain structure. Using these bis-amino acid monomers, we have built and characterized a number of bis-peptide nanostructures. We also constructed a molecular actuator that undergoes a large change in conformation under the control of metal exchange; the first application of bis-peptides. We are currently developing further approaches to functionalize bis-peptides as scaffolds to present well-defined constellations of functional groups. Such macromolecules could facilitate multifunctional catalysis and molecular recognition and lead to nanoscale molecular devices.

Observation of contraction and expansion in a bis(peptide)-based mechanical molecular actuator.

A novel, bis(peptide) based molecular actuator (1) has been synthesized. It is demonstrated to undergo contraction and expansion controlled by the addition and removal of Cu2+; this is demonstrated by the direct observation of a change in hydrodynamic properties by using sedimentation analysis and size exclusion chromatography. The molecule undergoes a large change in sedimentation coefficient, axial ratio, and size exclusion chromatography elution time when it binds copper. The demonstration of a controlled change in the mechanical properties of 1 make it a good starting point for the development of molecular devices that will harness changes in molecular shape and size to create molecular devices such as sensors or valves.

Distance distributions of end-labeled curved bispeptide oligomers by electron spin resonance.

We demonstrate the synthesis of a series of spin-labeled curved oligomers to determine their end-to-end lengths and distance distributions using electron spin resonance. We synthesize shape-persistent macromolecules from conformationally restricted, asymmetric monomers that are coupled through pairs of amide bonds to create water-soluble, spiro-ladder oligomers with well-defined three-dimensional structures. We synthesized seven different macromolecules, each containing eight monomers but differing in the sequence to create macromolecules with different curved shapes. The ends of the oligomers were labeled with nitroxide spin probes, and double electron-electron resonance (DEER) electron spin resonance (ESR) experiments were carried out to obtain quantitative information about the shapes and flexibility of the oligomers. The most probable end-to-end distance of the oligomers ranges from 23 to 36 A, a range of length that we previously accessed by assembling rod-like homo-oligomers that contain 4-8 bisamino acid monomers. The relative distances measured for the oligomers confirm that, by varying the sequence of an oligomer, we are able to control its shape. The shapes of the ESR-derived population distributions allow us to compare the degree of shape persistence and flexibility of spiro-ladder oligomers to other well-studied nanoscale molecular structures such as p-phenylethynylenes.