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Peptide materials have a wide array of functions, from tissue engineering and surface coatings to catalysis and sensing. Tuning the sequence of amino acids that comprise the peptide modulates peptide functionality, but a small increase in sequence length leads to a dramatic increase in the number of peptide candidates. Traditionally, peptide design is guided by human expertise and intuition and typically yields fewer than ten peptides per study, but these approaches are not easily scalable and are susceptible to human bias. Here we introduce a machine learning workflow-AI-expert-that combines Monte Carlo tree search and random forest with molecular dynamics simulations to develop a fully autonomous computational search engine to discover peptide sequences with high potential for self-assembly. We demonstrate the efficacy of the AI-expert to efficiently search large spaces of tripeptides and pentapeptides. The predictability of AI-expert performs on par or better than our human experts and suggests several non-intuitive sequences with high self-assembly propensity, outlining its potential to overcome human bias and accelerate peptide discovery.

De novo designed peptides self-assembled on a graphite surface to produce highly robust and active electrocatalysts and promote peroxidation reactions with activities that rival those of natural enzymes in both single and multi-substrate reactions.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins

Peptide materials have a wide array of functions from tissue engineering, surface coatings to catalysis and sensing. This class of biopolymer is composed of a sequence, comprised of 20 naturally occurring amino acids whose arrangement dictate the peptide functionality. While it is highly desirable to tailor the amino acid sequence, a small increase in their sequence length leads to dramatic increase in the possible candidates (e.g., from tripeptide = 20^3 or 8,000 peptides to a pentapeptide = 20^5 or 3.2 M). Traditionally, peptide design is guided by the use of structural propensity tables, hydrophobicity scales, or other desired properties and typically yields

The self-assembly of short peptides gives rise to versatile nanomaterials capable of promoting efficient catalysis. We have shown that short, seven-residue peptides bind hemin to produce functional catalytic materials which display highly efficient peroxidation activity, reaching a catalytic efficiency of 3×10(5) m(–1) s(–1). Self-assembly is essential for catalysis as non-assembling controls show no activity. We have also observed peroxidase activity even in the absence of hemin, suggesting the potential to alter redox properties of substrates upon association with the assemblies. These results demonstrate the practical utility of self-assembled peptides in various catalytic applications and further support the evolutionary link between amyloids and modern-day enzymes.

The self-assembly of short peptides into catalytic amyloid-like nanomaterials has proven to be a powerful tool in both understanding the evolution of early proteins and identifying new catalysts for practically useful chemical reactions. Here we demonstrate that both parallel and antiparallel arrangements of β-sheets can accommodate metal ions in catalytically productive coordination environments. Moreover, synergistic relationships, identified in catalytic amyloid mixtures, can be captured in macrocyclic and sheet-loop-sheet species, that offer faster rates of assembly and provide more complex asymmetric arrangements of functional groups, thus paving the way for future designs of amyloid-like catalytic proteins. Our findings show how initial catalytic activity in amyloid assemblies can be propagated and improved in more-complex molecules, providing another link in a complex evolutionary chain between short, potentially abiotically produced peptides and modern-day enzymes.

Interactions between multiple functional groups are key to catalysis. Previously, we reported synergistic interactions in catalytic amyloids formed by mixtures of heptameric peptides that lead to significant improvements in esterase activity. Herein, we describe the in-depth investigation of synergistic interactions within a family of amyloid fibrils, exploring the results of functional group interactions, the effects of chirality and the use of mixed enantiomers within fibrils. Remarkably, we find that synergistic interactions (either positive or negative) are found in the vast majority of binary mixtures of catalytic amyloid-forming peptides. The productive arrangements of functionalities rapidly identified by mixing different peptides will undoubtedly lead to the development of more active catalysts for a variety of different transformations.

Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.

The field of protein design has grown enormously in the past few decades. In this review we discuss the minimalist approach to design of artificial enzymes, in which protein sequences are created with the minimum number of elements for folding and function. This method relies on identifying starting points in catalytically inert scaffolds for active site installation. The progress of the field from the original helical assemblies of the 1980s to the more complex structures of the present day is discussed, highlighting the variety of catalytic reactions which have been achieved using these methods. We outline the strengths and weaknesses of the minimalist approaches, describe representative design cases and put it in the general context of the de novo design of proteins.

Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pK(a).

We have shown that de novo designed peptides self-assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self-assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion-like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.

Self-assembly of short de novo designed peptides gives rise to catalytic amyloids capable of facilitating multiple chemical transformations. We show that catalytic amyloids can efficiently hydrolyze paraoxon, which is a widely used, highly toxic organophosphate pesticide. Moreover, these robust and inexpensive metal-containing materials can be easily deposited on various surfaces, producing catalytic flow devices. Finally, functional promiscuity of catalytic amyloids promotes tandem hydrolysis/oxidation reactions. High efficiency discovered in a very small library of peptides suggests an enormous potential for further improvement of catalytic properties, both in terms of catalytic efficiency and substrate scope.

Self-assembly of molecules often results in new emerging properties. Even very short peptides can self-assemble into structures with a variety of physical and structural characteristrics. Remarkably, many peptide assemblies show high catalytic activity in model reactions reaching efficiencies comparable to those found in natural enzymes by weight. In this review, we discuss different strategies used to rationally develop self-assembled peptide catalysts with natural and unnatural backbones as well as with metal-containing cofactors.

Enzymes require many, often hundreds, of amino acid residues arranged in a protein fold to promote catalysis. Now, self-assembly of a single amino acid — phenylalanine — in the presence of zinc is shown to form supramolecular structures that promote hydrolysis better than natural enzymes on a weight basis.

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, β-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with β-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with β-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the β-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing β-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the β-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

Activity of allosteric protein catalysts is regulated by an external stimulus, such as protein or small molecule binding, light activation, pH change, etc., at a location away from the active site of the enzyme. Since its original introduction in 1961, the concept of allosteric regulation has undergone substantial expansion, and many, if not most, enzymes have been shown to possess some degree of allosteric regulation. The ability to create new catalysts that can be turned on and off using allosteric interactions would greatly expand the chemical biology toolbox and will allow for detection of environmental pollutants and disease biomarkers and facilitate studies of cellular processes and metal homeostasis. Thus, design of allosterically regulated protein catalysts represents an actively growing area of research. In this paper, we describe various approaches to achieving regulation of catalysis.

We employed a minimalist approach for design of an allosterically controlled retroaldo- lase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca 21 ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (~2000-fold) and the change in the apparent pKa of the active lysine residue.

Biosynthetic incorporation of beta-(1-azulenyl)-l-alanine, an isostere of tryptophan, is reported using a tryptophan auxotroph expression host. The azulene moiety introduced this way in proteins features many attractive spectroscopic properties, particularly suitable for in vivo studies.

This mini review gives an overview over different design approaches and methodologies applied in rational and semirational enzyme engineering. The underlying principles for engineering novel activities, enantioselectivity, substrate specificity, stability, and pH optimum are summarized.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When β-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel β-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.

The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N–O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)–N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes., The majority of enzymatic Kemp elimination reactions proceed via a well-established acid-base mechanism. Here, the authors show that cytochrome P450 is able to metabolize the leflunomide drug via a redox Kemp elimination, offering new insights into enzyme catalysis.

It only takes one mutation: a strategically placed single mutation in a non-enzymatic protein scaffold produced AlleyCat, a small, allosterically regulated catalyst of Kemp elimination. In only 7 rounds of directed evolution enzymatic efficiency of the original 74 amino acid residue catalyst was improved more than 220-fold to achieve kcat value higher than that of catalytic antibodies for the same reaction, still preserving allosteric regulation.

We recently reported that a computationally designed catalyst nicknamed AlleyCat facilitates C-H proton abstraction in Kemp elimination at neutral pH in a selective and calcium-dependent fashion by a factor of approximately 100,000 (Korendovych et al. in Proc. Natl. Acad. Sci. USA 108:6823, 2011). Kemp elimination produced a colored product that can be easily read out, thus making AlleyCat a catalytically amplified metal sensor for calcium. Here we report that metal-binding EF-hand motifs in AlleyCat could be redesigned to incorporate trivalent metal ions without significant loss of catalytic activity. Mutation of a single neutral residue at position 9 of each of the EF-hands to glutamate results in almost a two orders of magnitude improvement of selectivity for trivalent metal ions over calcium. Development of this new lanthanide-dependent switchable Kemp eliminase, named CuSeCat EE, provides the foundation for further selectivity improvement and broadening the scope of the repertoire of metals for sensing. A concerted effort in the design of switchable enzymes has many environmental, sensing, and metal ion tracking applications.

This review focuses on recent progress in noncomputational methods to introduce catalytic function into proteins, peptides, and peptide assemblies. We discuss various approaches to creating catalytic activity and classification of noncomputational methods into rational and combinatorial classes. The section on rational design covers recent progress in the development of short peptides and oligomeric peptide assemblies for various natural and unnatural reactions. The section on combinatorial design describes recent advances in the discovery of catalytic peptides. We present the future prospects of these and other new approaches in a broader context, including implications for functional material design.

Nature facilitates chemical transformations with exceptional selectivity and efficiency. Despite a tremendous progress in understanding and predicting protein function, the overall problem of designing a protein catalyst for a given chemical transformation is far from solved. Over the years, many design techniques with various degrees of complexity and rational input have been developed. Minimalist approach to protein design that focuses on the bare minimum requirements to achieve activity presents several important advantages. By focusing on basic physicochemical properties and strategic placing of only few highly active residues one can feasibly evaluate in silico a very large variety of possible catalysts. In more general terms minimalist approach looks for the mere possibility of catalysis, rather than trying to identify the most active catalyst possible. Even very basic designs that utilize a single residue introduced into nonenzymatic proteins or peptide bundles are surprisingly active. Because of the inherent simplicity of the minimalist approach computational tools greatly enhance its efficiency. No complex calculations need to be set up and even a beginner can master this technique in a very short time. Here, we present a step-by-step protocol for minimalist design of functional proteins using basic, easily available, and free computational tools.

Binding of inorganic anions, carboxylic acids, and tetraalkylammonium carboxylates by macrocyclic compounds of different size was studied by NMR in DMSO-d6. It has been shown that at least a 15-membered ring is necessary for successful recognition of fluoride. Larger macrocycles were shown to bind HSO4(-), H2PO4(-), Cl(-), and carboxylic acid salts. Effects of binding topicity are discussed. The 30-membered macrocycles 4 and 4m selectively bind substrates that are size- and shape-complementary: maximum binding is observed for dicarboxylic acids and dicarboxylates with four-carbon chains, and the binding constant for association of fumaric acid and 4 is ca. 5 orders of magnitude higher than that of maleic acid. The 30-membered macrocycle 4m showed selectivity toward alpha-ketocarboxylic acids. Secondary amino groups were not crucial for binding of fluoride to the macrocycles; however, they proved to be very important for selectivity and strength of carboxylic acid binding. The X-ray structure of the adduct of 4 and nitrobenzoic acid confirmed the guest H-bonding with both the amide and the secondary amino groups of the 30-membered macrocyclic host.

A new family of dicopper(I) complexes [CuI2RL](X)2(R = H, 1X, R =tBu, 2X and R = NO2, 3X, X = CF3SO3, ClO4, SbF6, or BArF, BArF = [B{3,5-(CF3)2C6H3}4]-), whereRL is a Schiff-base ligand containing two tridentate binding sites linked by a xylyl spacer, has been prepared and characterized, and its reaction with O2has been studied. The complexes were designed with the aim of reproducing structural aspects of the active site of type 3 dicopper proteins; they contain two three-coordinate copper sites and a rather flexible podand ligand backbone. The solid-state structures of 1ClO4, 2CF3SO3, 2ClO4, and 3BArF·CH3CN have been established by single-crystal X-ray diffraction analysis. 1ClO4adopts a polymeric structure in the solid state while 2CF3SO3, 2ClO4, and 3BArF-CH3CN are monomeric. The complexes have been studied in solution by means of1H and19F NMR spectroscopy, which put forward the presence of dynamic processes. 1-3BArF and 1-3CF3SO3in acetone react rapidly with O2to generate metaestable [CuIII2(μ-O)2(RL)]2+1-3(O2) and [CuIII2(μ-O)2(CF3SO3)(RL)]+1-3(O2)(CF3SO3) species, respectively, that have been characterized by UV-vis spectroscopy and resonance Raman analysis. Instead, reaction of 1-3BArF with O2in CH2Cl2results in intermolecular O2binding. DFT methods have been used to study the chemical identities and structural parameters of the O2adducts, and the relative stability of the CuIII2(μ-O)2form with respect to the CuII2(μ- η2:η2-O2) isomer. The reaction of 1X, X = CF3SO3and BArF, with O2in acetone has been studied by stopped-flow UV-vis exhibiting an unexpected very fast reaction rate (k = 3.82(4) ± 103M-1s-1, ΔH‡ = 4.9 ± 0.5 kJ·mol-1, ΔS‡ = -148 ± 5 J·K-1·mol-1), nearly 3 orders of magnitude faster than in the parent [CuI2(m-XYLMeAN)]2+. Thermal decomposition of 1-3(O2) does not result in aromatic hydroxylation. The mechanism and kinetics of O2 binding to 1X (X = CF3SO3and BArF) are discussed and compared with those associated with selected examples of reported models of O2-processing copper proteins. A synergistic role of the copper ions in O2binding and activation is clearly established from this analysis Financial support from MCYT of Spain through Projects CTQ2006-05367/BQU to M.C., CTQ2005-08797-C02-01 to M.S., and CTQ2006-01080 to T.P.; from NIH (GM-38767 to L.Q.); and from the US Department of Energy, Office of Basic Energy Sciences (DEFG02-06ER15799 to E.R.-A.). A.C. and L.G. thank MEC for Ph.D. grants

β-(1-Azulenyl)-L-alanine (AzAla) can be incorporated into the influenza A virus M2 proton channel. AzAla's sensitivity to the protonation state of the nearby histidines and the lack of environmental fluorescence dependence allow for direct and straightforward determination of histidine pKa values in ion channels.

[Structure: see text] Binding of fluoride anion as well as carboxylic acid tetraalkylammonium salts by macrocyclic compounds of different size was studied by NMR in DMSO-d6. It has been found that at least a 15-membered ring is necessary for successful recognition of fluoride. Larger macrocycles obtained in a [2+2] cyclization were shown to bind dicarboxylic acid salts. Effects of binding topicity are discussed.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, β-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study we investigated the effect of replacing Trp with β-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with β-(1-azulenyl)-L- alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the β-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing β-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the β-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

We demonstrated that β-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

A series of complexes with [Fe(II)(2)(mu-OH)(2)] cores has been synthesized with N3 and N4 ligands and structurally characterized to serve as models for nonheme diiron(II) sites in enzymes that bind and activate O(2). These complexes react with O(2) in solution via bimolecular rate-limiting steps that differ in rate by 10(3)-fold, depending on ligand denticity and steric hindrance near the diiron center. Low-temperature trapping of a (mu-oxo)(mu-1,2-peroxo)diiron(III) intermediate after O(2) binding requires sufficient steric hindrance around the diiron center and the loss of a proton (presumably that of a hydroxo bridge or a yet unobserved hydroperoxo intermediate). The relative stability of these and other (mu-1,2-peroxo)diiron(III) intermediates suggests that these species may not be on the direct pathway for dioxygen activation.

A new, stable iron(III) complex with a pentadentate amide-containing macrocyclic ligand was prepared and fully characterized. The complex adopted a pentagonal-bipyramidal geometry, where an equatorial plane is occupied by the pyridine nitrogen, two deprotonated amide nitrogens, and two secondary amines from the macrocycle, and two axial positions are available for monodentate ligand (chloride anion or solvent molecule) coordination. The rigid, planar iron-amide building blocks are linked in a three-dimensional network via a system of hydrogen bonds, with the shortest Fe-Fe separation of 8.02 A. The coordination of strongly electron-donating, negatively charged deprotonated amide groups resulted in expected stabilization of a high oxidation state of iron (the redox potential of the Fe(III)L/Fe(II)L couple, -0.57 V vs SCE). In contrast to the majority of the iron complexes with polydentate amide ligands, the pentagonal-bipyramidal geometry of the macrocyclic complex described in this work affords a high-spin configuration of the central metal ion (room-temperature magnetic moment is 5.84 micro(beta)). Variable-temperature iron-57 Mössbauer spectroscopy and ac and dc magnetization studies indicate slow paramagnetic relaxation and a crossover to long-range antiferromagnetic order at Tapproximately 3.2 K.

Three complexes of benzo-15-crown-5 (B15C5) with protonated primary amines [PhCH 2 NH 3 (B15C5)](ClO 4 ), [ p -C 6 H 4 (CH 2 NH 3 ) 2 (B15C5) 2 ](ClO 4 ) 2 , and [(CH 2 ) 4 (NH 3 ) 2 (B15C5) 2 ](SCN) 2 were isolated and studied in acetonitrile solutions by NMR, and in the solid state by X-ray crystallography. In all complexes, one B15C5 molecule was bound with each R-NH 3 + moiety with characteristic small separation of 1.84–1.86 A between the nitrogen of the R-NH 3 + group and the O 5 mean plane of the crown residue. No sandwich-type complexes with a 1:2 R-NH 3 + /B15C5 stoichiometry were observed. Binding affinities of B15C5 in acetonitrile were similar for all ammonium cations studied: K 1 =550±10 M −1 for [PhCH 2 NH 3 ] + ; K 1 =1100±100 and K 2 =400±30 M −1 for [ p -C 6 H 4 (CH 2 NH 3 ) 2 ] 2+ ; and K 1 =1100±100 and K 2 =300±30 M −1 for [H 3 N(CH 2 ) 4 NH 3 ] 2+ . The complexation is primarily enthalpy-driven (Δ H °=−4.9±0.5 kcal/mol, Δ S °=−3.8±1.0 eu for PhCH 2 NH 3 + –B15C5), as determined by variable temperature 1 H NMR titrations.

An artificial esterase with no known natural structural analogues has been formed via the homo-heptameric self-assembly of a designed peptide. This esterase represents the first report of a functional catalytic triad rationally engineered into a de novo protein framework.

— Reductive amination of 4-formyl[benzo-15-crown-5] with sodium cyanoborohydride in the NH 4 Ac/MeOH mediumfollowed by acid addition and extraction with CHCl 3 unexpectedly lead to the isolation of the salt (B15C5-CH 2 -NH 3 ) + -(H 3 BCNBH 2 CN) − with an unusual dicyanodiborohydride anion. The self-complementary 4-ammoniomethyl[benzo-15-crown-5]cation was found to exist as a supramolecular dimer in the solid state, acetonitrile solution, and gas phase as revealed by X-raycrystallography, NMR and mass spectrometry, respectively. © 2003 Elsevier Science Ltd. All rights reserved. Self-complementary monofunctionalized crown-ethershave proved to be useful building blocks forsupramolecular architecture. 1–5 Stoddart and co-work-ers successfully explored systems based on protonatedsecondary ammonium or 4,4-dipyridinium functionali-ties threaded through large ( 24-membered) crownethers. 2–4 Very recently, we 5 and others 6 reported crys-talline dimers obtained from simple self-complementaryaminomethylcrown ethers (benzo-18-crown-6 andcrown-5, respectively). The ‘non-threaded’ dimer basedon a well-known host–guest motif, namely benzo-18-crown-6 and R-NH

Template condensation between 2,6-dicarbonylpyridines and a tripodal tetramine tris(3-aminopropyl)amine in the presence of copper(II) or nickel(II) yielded macrocyclic complexes that contained a coordinated primary amine as an appended functional group. The derivatives of 2,6-diformylpyridine contained labile azomethine bonds susceptible to water or alcohol addition, and were isolated in the form of crystallographically characterized hemiaminals or O-ethyl hemiaminals. The nickel(II) hemiaminal complex produced a macrocyclic product upon reduction with sodium borohydride, and the structures of the two complexes had very similar ligand conformations. The derivatives of 2,6-diacetylpyridine formed stable Schiff-base macrocycles with rigid, nearly planar structures in which azomethine bonds were conjugated with the pyridine ring. The steric strain was relieved upon heating of the complexes in water–alcohol media. The products were determined by the coordination requirements of the metal ions: the copper(II) underwent a CN double bond shift, forming a more flexible five-coordinate macrocyclic complex, whereas the nickel(II) compound underwent a partial ring-opening hydrolysis, giving rise to a pseudo-octahedral six-coordinate species. The unusual isomerization of the copper(II) complex was accompanied by H/D exchange at CH-bonds, and was facilitated by alcohol addition. The mechanism of isomerization is similar to B6-catalyzed transamination reactions (D.E. Metzler, M. Ikawa and E.S. Snell. J. Am. Chem. Soc., 1954, 76, 648). The corresponding four-coordinate copper(II) complex lacking a pendant arm was more thermally stable than its five-coordinate counterpart, and underwent partial double-bond migration under harsh conditions (prolonged heating at reflux in water–ethanol mixtures at pH > 8.5).

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.