Transition State Conformations Research Articles

Opposing Auxiliary Conformations Produce the Same Torquoselectivity in an Oxazolidinone-Directed Nazarov Cyclization

Most applications of chiral oxazolidinone auxiliaries in asymmetric synthesis operate through a common set of stereocontrol principles. That is, the oxazolidinone is made to adopt a specific, coplanar conformation with respect to the prochiral substrate, and reaction occurs preferentially at whichever stereoheterotopic face is not blocked by the substituents on the oxazolidinone. In contrast to these principles, we report here the discovery of an alternative mechanism of oxazolidinone-based stereocontrol that does not require coplanarity and is driven instead by allylic strain. This pathway has been uncovered through computational studies of an asymmetric Nazarov cyclization. Chiral oxazolidinone auxiliaries provide essentially complete control over the torquoselectivity of ring closure and the regioselectivity of subsequent deprotonation. Density functional theory calculations (M06-2X//B3LYP) reveal that in the transition state of 4π electrocyclic ring closure, the oxazolidinone ring and the cyclizing pentadienyl cation are distorted from coplanarity in a manner that gives two transition state conformations of similar energy. These two conformers are distinguished by a 180° flip in the auxiliary orientation such that in one conformer the oxazolidinone carbonyl is oriented toward the OH of the pentadienyl cation (syn-conformer) and in the other it is oriented away from this OH (anti-conformer). Surprisingly, both conformations induce the same sense of torquoselectivity, with a 3-5 kcal/mol preference for the C5-β epimer of the ring-closed cation. In both conformations, the conrotatory mode that leads to the C5-α epimer is disfavored due to higher levels of allylic strain between the oxazolidinone substituent and adjacent groups on the pentadienyl cation (R(4) and OH). The excellent torquoselectivities obtained in the oxazolidinone-directed Nazarov cyclization suggest that the allylic strain-driven stereoinduction pathway represents a viable alternative mechanism of stereocontrol for reactions of sterically congested substrates that lie outside of the traditional coplanar (N-acyloxazolidinone) paradigm.

Ground State Conformational Preferences and CH Stretch–Bend Coupling in a Model Alkoxy Chain: 1,2-Diphenoxyethane

1,2-Diphenoxyethane (C6H5-O-CH2-CH2-O-C6H5, DPOE) is a flexible bichromophore in which the two phenyl rings are separated from one another by an -O-CH2-CH2-O- chain with five flexible dihedral angles about which hindered rotation can occur. As such, it is a phenyl capped analog of dimethoxyethane (DMOE), which has served as a model compound for development of force fields for polyethylene glycol (PEG). The ground state conformational energy landscape of DPOE is explored using a combination of single-conformation spectroscopy of the jet-cooled molecule and calculations of the conformational minima and transition states. In the experimental UV spectrum, ultraviolet hole-burning establishes the presence of just two conformations with significant population in the supersonic jet expansion. Fluorescence dip infrared (FDIR) spectroscopy is used to record infrared spectra of the two conformers in the alkyl CH stretch, CH bend, and CO stretch regions. When compared with harmonic vibrational frequency calculations, the two isomers are determined to be of C2h and C2 symmetry, and labeled ttt and tgt to denote the three central dihedrals as trans or gauche. Infrared population transfer spectroscopy is used to determine fractional abundances for the two conformers (f(ttt) = 0.53 ± 0.01; f(tgt) = 0.47 ± 0.01). Relaxed potential energy curves along the three nonequivalent dihedral angles are used to map out the shape of the potential energy landscape that leads to these preferences. The Fermi resonance in the alkyl CH stretch spectrum is successfully modeled using a recently developed methodology [Buchanan et al., J. Chem. Phys. 2013, 138, 064308] employing a reduced dimension Hamiltonian. The scissor overtones couple to the CH2 symmetric stretch and only indirectly to the asymmetric stretch through symmetric stretch/asymmetric stretch coupling. The presence of the oxygen atoms in the chain shifts the CH scissor overtones to higher frequencies than in pure alkyl chains, qualitatively changing the spectral consequences of the Fermi resonance, with the scissor overtones now appearing as the highest frequency bands in the spectrum. The spectra are contrasted with those in 1,2-diphenylethane, a close analog with a very different appearance to its CH stretch spectrum, in which the scissor overtones appear as the lowest frequency bands.

ChemInform Abstract: Chemoselectivity of [4 + 2] Cycloaddition in N‐Maleyl‐ and N‐Allyl‐2,6‐difurylpiperidin‐4‐ones.

On the basis of a transition state conformation analysis, an attempt was made to explain the high chemoselectivity of intramolecular [4 + 2] cycloaddition in 3-alkyl-2,6-difuryl-N-maleylpiperidin-4-ones. It was shown that the thermal Diels–Alder reaction in these piperidine derivatives takes place through the “boat” conformation and leads to the formation of hydrogenated 1-alkyl-4-(2-furyl)-2H-8,10a-epoxy-pyrido[2,1-a]isoindol-2-ones. The alternative regioisomers, 3-alkyl-4-(2-furyl)-2H-8,10a-epoxy-pyrido[2,1-a]isoindol-2-ones, are hardly formed at all. At the same time, the intramolecular Diels–Alder reaction in the isostructural 3-alkyl-N-allyl-2,6-difurylpiperidin-4-ones, takes place non-regioselectively from the “chair” conformation.

Ratcheted molecular-dynamics simulations identify efficiently the transition state of protein folding

The atomistic characterization of the transition state (TS) is a fundamental step to improve the understanding of the folding mechanism and the function of proteins. From a computational point of view, the identification of the conformations that build out the transition state is particularly cumbersome, mainly because of the large computational cost of generating a statistically sound set of folding trajectories. Here we show that a biasing algorithm, based on the physics of the ratchet-and-pawl, can be used to approximate efficiently the transition state. The basic idea is that the algorithmic ratchet exerts a force on the protein when it is climbing the free-energy barrier, while it is inactive when it is descending. The transition state can be identified as the point of the trajectory where the ratchet changes regime. Besides discussing this strategy in general terms, we test it within a protein model whose transition state can be studied independently by plain molecular dynamics simulations. Finally, we show its power in explicit-solvent simulations, obtaining and characterizing a set of transition-state conformations for Acyl-Coenzyme A-Binding Protein (ACBP) and Chymotrypsin Inhibitor 2 (CI2).

Hückel and Möbius Bond-Shifting Routes to Configuration Change in Dehydro[4n+2]annulenes

Computational investigation of the potential energy surfaces of dehydro[10]- and dehydro[14]annulenes revealed that mechanisms involving Hückel and Möbius π-bond shifting can explain the observed or proposed configuration change reactions. Unlike the case of annulenes, in which bond-shift midpoints correspond to transition states, for transformations of dehydroannulenes with Δtrans = 0, "hidden" Hückel bond shifts occur on the side of an energy hill, on the way to a cumulenic, purely conformational transition state. For example, interconversion between CTCCTC-dehydro[14]annulene (1a) and CCTCTC-dehydro[14]annulene (2a) has a CCSD(T)/cc-pVDZ//BHLYP/6-31G* barrier of 18.7 kcal/mol, consistent with experimental observations, and proceeds via a conformational transition state, with Hückel π-bond shifts occurring both before and after the transition state. However, when Δtrans = 1, a true Möbius π-bond shift transition state was located. The isomerization of CCTC-dehydro[10]annulene (10) to CCCC-dehydro[10]annulene (11) occurs by an initial "hidden" Hückel bond shift, followed by passage through a Möbius bond-shift transition state to 11, with an overall barrier of 29.8 kcal/mol at the CASPT2(12,12)/cc-pVDZ//(U)BHLYP/6-31G* level of theory. This is the lowest energy pathway between 10 and 11, in contrast to a cyclization/ring-opening route via a bicyclic allene described in previous reports.

A Transition-State Interaction Shifts Nucleobase Ionization toward Neutrality To Facilitate Small Ribozyme Catalysis

One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca(1)a(2)X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca(1)a(2)X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors.

Synthesis of α-trifluoromethylated amides by Eschenmoser–Claisen-type rearrangement of allylic alcohols

A new, mild method to prepare α-trifluoromethylated amides bearing an additional trans-configured double bond in γ-position is described. Treatment at room temperature of fluorinated and non-fluorinated allylic alcohols 1 and 2 with the 1,1,3,3,3-pentafluoropropene–diethylamine adduct (PFPDEA) in the presence of triethylamine as base gave the products of [3,3]-sigmatropic Eschenmoser–Claisen-type rearrangements with good yields and excellent diastereoselectivity due to chair-like conformations of transition states. Starting with enantiomerically enriched allylic alcohols chirality transfer from carbon 3 of the allylic system to carbon 2 of the final α-trifluoromethyl carboxamides was observed. This methodology was applied to both simple and more complex, including terpenic, allylic alcohols and might be developed to an alternative strategy to the well-known electrophilic α-trifluoromethylation of carbonyl compounds.

Chemoselectivity of [4 + 2] cycloaddition in N-maleyl- and N-allyl-2,6-difurylpiperidin-4-ones

On the basis of a transition state conformation analysis, an attempt was made to explain the high chemoselectivity of intramolecular [4 + 2] cycloaddition in 3-alkyl-2,6-difuryl-N-maleylpiperidin-4-ones. It was shown that the thermal Diels–Alder reaction in these piperidine derivatives takes place through the “boat” conformation and leads to the formation of hydrogenated 1-alkyl-4-(2-furyl)-2H-8,10a-epoxy-pyrido[2,1-a]isoindol-2-ones. The alternative regioisomers, 3-alkyl-4-(2-furyl)-2H-8,10a-epoxy-pyrido[2,1-a]isoindol-2-ones, are hardly formed at all. At the same time, the intramolecular Diels–Alder reaction in the isostructural 3-alkyl-N-allyl-2,6-difurylpiperidin-4-ones, takes place non-regioselectively from the “chair” conformation.

Stepwise Diels–Alder: More than Just an Oddity? A Computational Mechanistic Study

We have employed hybrid DFT and SCS-MP2 calculations at the SMD-PCM-6-311++G(2d,2p)//6-31+G(d) level to investigate the relationship between three possible channels for forming a Diels-Alder adduct from a highly nucleophilic diene and moderately to highly electrophilic dienophiles. We discuss geometries optimized using the B3LYP and M06-2X functionals with the 6-31+(d) basis set. The transition states and intermediates are characterized on the basis of geometric and electronic properties, and we also address the possibility of predicting detectability of a zwitterionic intermediate based on its relative stability. Our results show that a conventional Diels-Alder transition state conformation yields intermediates in all four investigated cases, but that these are too short-lived to be detected experimentally for the less activated reactants. The stepwise trans pathway, beginning with a conjugate addition-like transition state, becomes increasingly competitive with more activated reactants and is indeed favored for the most electrophilic dienophiles. Addition of a trans diene leads to a dead-end as the trans intermediates have insurmountable rotation barriers that prohibit formation of the second bond, unless another, heterocyclic intermediate is formed. We also show that introduction of a hydrogen bond donating catalyst favors a stepwise pathway even for less activated dienophiles.

Catalytic Transitions in the Human MDR1 P-Glycoprotein Drug Binding Sites

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

The Subunit B-Dimer of the F1-ATPase Influences Conformational Transitions During Catalysis

The F1FO-ATP synthase, the enzyme responsible for most of ATP generated in biological systems, is a highly asymmetric enzyme complex. Part of the asymmetry stems from interactions of the nucleotide binding subunits α and β with the central rotor subunit gamma. Subunit gamma transfers the energy of the proton-driven rotation of the FO subunit c ring to subunit β, and induces conformational changes that drive ATP release during synthesis. We hypothesize that interactions with the external stalk subunit b-dimer will introduce additional asymmetry to the enzyme complex that may influence catalytic transitions. To investigate our hypothesis, we substituted two tyrosines in subunit β at amino acid positions 331 and 354 for cysteines in an otherwise cysteine-less enzyme. These tyrosines were shown previously to be part of the catalytic and non-catalytic sites, respectively. Using the cysteine-less enzyme as a control, our experiments showed that trapping efficiency of the cysteine-less protein as well as both mutants using AlFx- in what is considered to be a transition-state like conformation was influenced by the presence of soluble b2. Reactivation of trapped enzyme occurred in presence of excess of MgATP and was also influenced by the presence of soluble b2. In separate experiments using site-specific spin labeling and two different spin labels as reporter groups, we observed changes in the conformation of both the catalytic and non-catalytic sites during trapping. In each case, the presence of subunit b-dimer resulted in changes in the conformational transitions. Experiments are underway to localize the binding site of the subunit b-dimer in either the catalytic or the non-catalytic cleft of the ATPase.

“Lethal Synthesis” of Fluorocitrate by Citrate Synthase Explained through QM/MM Modeling

The term “lethal synthesis” was coined for enzymatic formation of fluorocitrate, but this classic problem of enzyme stereoselectivity remains poorly understood. Here, we show that high-level ab initio quantum mechanics/ molecular mechanics (QM/MM) modeling can accurately capture this enzymatic enantioselectivity and the results provide detailed insight into its origin. Citrate synthase (CS) performs the first reaction in the citric acid cycle: the formation of citrate from oxaloacetate and acetate in the form of acetyl-CoA. When fluoroacetyl-CoA (from fluoroacetate) is used as a substrate instead of acetyl-CoA, 2fluorocitrate is formed, which inhibits aconitase, the next enzyme in the citric acid cycle. This process is responsible for the lethal toxicity of fluoroacetate to humans and other mammals. The fluorocitrate enantiomer that is predominantly formed by CS, (2R,3R)-fluorocitrate (Figure 1), is the same enantiomer that specifically inhibits aconitase. The only (semi-)quantitative experimental study published to date indicates that the minor product of the enzymatic formation of fluorocitrate, (2S,3R)-fluorocitrate, amounts to 2–3% of the major product. Using transition-state theory, this translates to a difference in activation free energy (DDG ) of 2.06– 2.30 kcalmol . The causes of this selectivity remain uncertain. We have previously modeled the two initial reaction steps for the natural substrates, proton abstraction from acetylCoA and condensation with oxaloacetate (OAA), in CS with high-level QM/MM methods. For the reaction with fluoroacetyl-CoA (FaCoA), the distinction between enantiomers is made in the proton-abstraction step, where either an Eor a Z-enolate is formed. Here, we show that 1) the calculated relative energy of the enolates accurately predicts the experimentally observed enantiospecificity and 2) the enantiospecificity is mostly due to the inherent energy difference of the reacting species. A model of the enzyme with OAA and FaCoA bound was built for QM/MM simulation, with the Asp375 side chain from Cb, the methylthioester part of FaCoA, and OAA in the QM region. QM/MM molecular dynamics indicated that preE and pre-Z conformations in the CS active site can be sampled in the same trajectory. Five different approximate transition-state conformations for both E-enolate and Zenolate formation were generated by QM/MM umbrella sampling molecular dynamics (see the Supporting Information). These conformations were used to perform high-level QM/MM modeling of proton abstraction, using the established reaction coordinate r= d(OAsp375H) d(CFaCoAH). Geometries were optimized at the B3LYP/6-31+G(d)//MM level and energies calculated at the SCS-MP2/aug-cc-pVDZ// MM level. This describes the reaction of CS with acetyl-CoA accurately, in agreement with local coupled-cluster QM/MM results. The QM/MM energy profiles for enolate formation show correctly that formation of the E-enolate is preferred (Figure 2). Boltzmann-weighted energy differences between the E and Z profiles are 1.80 and 2.05 kcalmol 1 for the activation energy and reaction energy in this step, respectively. Entropy contributions for Eand Z-enolate formation are expected to be (nearly) identical (supported by AM1/MM free energy profiles, see the Supporting Information). The Figure 1. Conversion of fluoracetyl-CoA to fluorocitrate by CS.

Stereochemical Differentiation in the Simmons-Smith Reaction for Cyclopropanated Glucopyranose Derivatives as Molecular Probes for Glycosidases

Glucopyranose analogues carrying a bicyclo[4.1.0]heptane framework and the diastereomer of the cyclopropane moiety were synthesized as the unit for molecular probes to mimic the unstable transition state conformation of the glucopyranose ring in enzymatic hydrolysis. The synthesis features differentiation of the α- and β-stereoselectivity in cyclopropanation of the corresponding cyclohexene derivative.

Formation of molecular hydrogen from protonated 9,10-dihydroanthracene: Is the ejected H 2 rotationally and vibrationally excited?

The infrared multiple-photon dissociation (IRMPD) spectrum of protonated 9,10-dihydroanthracene ([DHA+H] +, m/ z 181) has been recorded using an infrared free electron laser. Protonation was accomplished by electrospray ionization with subsequent mass-selection and trapping in a Fourier transform ion cyclotron mass spectrometer. IR-induced fragment ions were observed at m/ z 179, 166, and 165. Density functional calculations (B3LYP/6-311++G(d,p)) of the infrared spectra of the two possible protonated isomers of DHA showed that the observed IRMPD spectrum is best fit to a mixture of the two isomers. Potential energy surfaces for the loss of atomic and molecular hydrogen from the aliphatic carbons of [DHA+H] + have been calculated. The lowest energy barriers are for loss of H 2. After H 2 ejection, stabilization of the remaining fragment occurs by hydrogen migration from one of the aliphatic carbons to the bare ejection site. In all cases the stabilized fragment is computed to be 9-hydroanthracene. The IRMPD spectrum of the m/ z 179 fragment has been recorded and is shown to correspond closely both to the calculated and previously recorded IRMPD spectrum of ionic 9-hydroanthracene. The highly asymmetric transition state conformation of the to-be-formed H 2 and the remaining fragment is highly suggestive of rotational, vibrational, and, possibly, translational excitation of the ejected H 2. Evidence for such excitation from astronomical observations that show the close proximity of PAHs and H 2 in certain interstellar objects and that show H 2 rotational excitation, which has been difficult to explain via either collisional activation or UV pumping, is reviewed.

Direct Experimental Evidence for the High Chemical Reactivity of α‐ and β‐Xylopyranosides Adopting a 2,5B Conformation in Glycosyl Transfer

The effect of a (2,5)B boat conformation on xyloside reactivity has been investigated by studying the hydrolysis and glycosylation of a series of synthetic xyloside analogues based on a 2-oxabicyclo[2.2.2]octane framework, which forces the xylose analogue to adopt a (2,5)B conformation. The locked β-xylosides were found to hydrolyze 100-1200 times faster than methyl β-D-xylopyranoside, whereas the locked α-xylosides hydrolyzed up to 2×10(4) times faster than methyl α-D-xylopyranoside. A significant rate enhancement was also observed for the glycosylation reaction. The high reactivity of these conformers can be related to the imposition of a (2,5)B conformation, which approximates a transition state (TS) boat conformation. In this way, the energy penalty required to go from the chair to the TS conformation is already paid. These results parallel and support the observation that the GH-11 xylanase family force their substrate to adopt a (2,5)B conformation to achieve highly efficient enzymatic glycosidic bond hydrolysis.

Syntheses of cellotriose and cellotetraose analogues as transition state mimics for mechanistic studies of cellulases

Cellotriose and cellotetraose analogues carrying cyclohexene rings were developed as molecular probes which are expected to mimic the transition state conformation of hydrolysis by cellulases. The cyclohexene ring was placed at the pyranose ring being expected to locate the −1 subsite of the enzyme. In order to evaluate these probes, sulfur derivatives of cellotriose and cellotetraose were also synthesized as the enzyme tolerant analogues which mimic the stable conformations of the natural cellulose. The binding assays using differential scanning calorimetry revealed that introduction of the cyclohexene ring is effective to the complexation with an endoglucanase, NCE5 from Humicola insolens.

Substituent effects on the reduction of 2–alkylcyclohexanones by LiAlH4: an investigation of conformational equilibria and transition states

AbstractTransition state (TS) structures for the reduction of 2‐Me and 2‐i‐Pr‐cyclohexanone by LiAlH4 were optimized by density functional theory (B3LYP/6‐31G(d,p)). Four TS structures corresponding to axial and equatorial attacks by LiAlH4 were located for each ketone conformer. Electronic potential maps were used to investigate the substituent electronic effect on the TS stabilization. The uneven carbonyl orbital distribution in the LUMO (π*) was also analyzed. Reduction stereoselectivity was shown to depend on both the ketone conformational ratio and on the reaction TS. Copyright © 2010 John Wiley & Sons, Ltd.

Constrained proper sampling of conformations of transition state ensemble of protein folding

Characterizing the conformations of protein in the transition state ensemble (TSE) is important for studying protein folding. A promising approach pioneered by Vendruscolo et al. [Nature (London) 409, 641 (2001)] to study TSE is to generate conformations that satisfy all constraints imposed by the experimentally measured φ values that provide information about the native likeness of the transition states. Faísca et al. [J. Chem. Phys. 129, 095108 (2008)] generated conformations of TSE based on the criterion that, starting from a TS conformation, the probabilities of folding and unfolding are about equal through Markov Chain Monte Carlo (MCMC) simulations. In this study, we use the technique of constrained sequential Monte Carlo method [Lin et al., J. Chem. Phys. 129, 094101 (2008); Zhang et al. Proteins 66, 61 (2007)] to generate TSE conformations of acylphosphatase of 98 residues that satisfy the φ-value constraints, as well as the criterion that each conformation has a folding probability of 0.5 by Monte Carlo simulations. We adopt a two stage process and first generate 5000 contact maps satisfying the φ-value constraints. Each contact map is then used to generate 1000 properly weighted conformations. After clustering similar conformations, we obtain a set of properly weighted samples of 4185 candidate clusters. Representative conformation of each of these cluster is then selected and 50 runs of Markov chain Monte Carlo (MCMC) simulation are carried using a regrowth move set. We then select a subset of 1501 conformations that have equal probabilities to fold and to unfold as the set of TSE. These 1501 samples characterize well the distribution of transition state ensemble conformations of acylphosphatase. Compared with previous studies, our approach can access much wider conformational space and can objectively generate conformations that satisfy the φ-value constraints and the criterion of 0.5 folding probability without bias. In contrast to previous studies, our results show that transition state conformations are very diverse and are far from nativelike when measured in cartesian root-mean-square deviation (cRMSD): the average cRMSD between TSE conformations and the native structure is 9.4 Å for this short protein, instead of 6 Å reported in previous studies. In addition, we found that the average fraction of native contacts in the TSE is 0.37, with enrichment in native-like β-sheets and a shortage of long range contacts, suggesting such contacts form at a later stage of folding. We further calculate the first passage time of folding of TSE conformations through calculation of physical time associated with the regrowth moves in MCMC simulation through mapping such moves to a Markovian state model, whose transition time was obtained by Langevin dynamics simulations. Our results indicate that despite the large structural diversity of the TSE, they are characterized by similar folding time. Our approach is general and can be used to study TSE in other macromolecules.

Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.