Frame Shifts Affect the Stability of Collagen Triple Helices.

Collagen model peptides (CMPs), composed of proline-(2S,4R)-hydroxyproline-glycine (POG) repeat units, have been extensively used to study the structure and stability of triple-helical collagen─the dominant structural protein in mammals─at the molecular level. Despite the more than 50-year history of CMPs and numerous studies on the relationship between the composition of single-stranded CMPs and the thermal stability of the assembled triple helices, little attention has been paid to the effects arising from their terminal residues. Here, we show that frame-shifted CMPs, which share POG repeat units but terminate with P, O, or G, form triple helices with vastly different thermal stabilities. A melting temperature difference as high as 16 °C was found for triple helices from 20-mers Ac-OG[POG]6-NH2 and Ac-[POG]6PO-NH2, and triple helices of the constitutional isomers Ac-[POG]7-NH2 and Ac-[GPO]7-NH2 melt 10 °C apart. A combination of thermal denaturation, circular dichroism and NMR spectroscopic studies, and molecular dynamics simulations revealed that the stability differences originate from the propensity of the peptide termini to preorganize into a polyproline-II helical structure. Our results advise that care must be taken when designing peptide mimics of structural proteins, as subtle changes in the terminal residues can significantly affect their properties. Our findings also provide a general and straightforward tool for tuning the stability of CMPs for applications as synthetic materials and biological probes.

Mechanistic Investigation of the Nickel-Catalyzed Metathesis between Aryl Thioethers and Aryl Nitriles.

Functional group metathesis is an emerging field in organic chemistry with promising synthetic applications. However, no complete mechanistic studies of these reactions have been reported to date, particularly regarding the nature of the key functional group transfer mechanism. Unraveling the mechanism of these transformations would not only allow for their further improvement but would also lead to the design of novel reactions. Herein, we describe our detailed mechanistic studies of the nickel-catalyzed functional group metathesis reaction between aryl methyl sulfides and aryl nitriles, combining experimental and computational results. These studies did not support a mechanism proceeding through reversible migratory insertion of the nitrile into a Ni-Ar bond and provided strong support for an alternative mechanism involving a key transmetalation step between two independently generated oxidative addition complexes. Extensive kinetic analysis, including rate law determination and Eyring analysis, indicated the oxidative addition complex of aryl nitrile as the resting state of the catalytic reaction. Depending on the concentration of aryl methyl sulfide, either the reductive elimination of aryl nitrile or the oxidative addition into the C(sp2)-S bond of aryl methyl sulfide is the turnover-limiting step of the reaction. NMR studies, including an unusual 31P-2H HMBC experiment using deuterium-labeled complexes, unambiguously demonstrated that the sulfide and cyanide groups exchange during the transmetalation step, rather than the two aryl moieties. In addition, Eyring and Hammett analyses of the transmetalation between two Ni(II) complexes revealed that this central step proceeds via an associative mechanism. Organometallic studies involving the synthesis, isolation, and characterization of all putative intermediates and possible deactivation complexes have further shed light on the reaction mechanism, including the identification of a key deactivation pathway, which has led to an improved catalytic protocol.

Impact of solvent interactions on 1H and 13C chemical shifts investigated using DFT and a reference dataset recorded in CDCl3 and CCl4.

1H and 13C chemical shifts of 35 small, rigid molecules were measured under standardized conditions in chloroform-d and in tetrachloromethane. The solvent change mainly affects carbon shifts of polar functional groups. This difference due to specific interactions with CDCl3 cannot be adequately reproduced by DFT calculations in implicit solvent. The new dataset provides an accurate basis for the validation and calibration of shift calculations, especially with respect to improved solvent models.

Efficient affinity ranking of fluorinated ligands by 19F NMR: CSAR and FastCSAR.

Fluorine NMR has recently gained high popularity in drug discovery as it allows efficient and sensitive screening of large numbers of ligands. However, the positive hits found in screening must subsequently be ranked according to their affinity in order to prioritize them for follow-up chemistry. Unfortunately, the primary read-out from the screening experiments, namely the increased relaxation rate upon binding, is not proportional to the affinity of the ligand, as it is polluted by effects such as exchange broadening. Here we present the method CSAR (Chemical Shift-anisotropy-based Affinity Ranking) for reliable ranking of fluorinated ligands by NMR, without the need of isotope labeled protein, titrations or setting up a reporter format. Our strategy is to produce relaxation data that is directly proportional to the binding affinity. This is achieved by removing all other contributions to relaxation as follows: (i) exchange effects are efficiently suppressed by using high power spin lock pulses, (ii) dipolar relaxation effects are approximately subtracted by measuring at two different magnetic fields and (iii) differences in chemical shift anisotropy are normalized using calculated values. A similar ranking can be obtained with the simplified approach FastCSAR that relies on a measurement of a single relaxation experiment at high field (preferably > 600 MHz). An affinity ranking obtained in this simple way will enable prioritizing ligands and thus improve the efficiency of fragment-based drug design.

Connecting the conformational behavior of cyclic octadepsipeptides with their ionophoric property and membrane permeability.

Cyclic octadepsipeptides such as PF1022A and its synthetic derivative emodepside exhibit anthelmintic activity with the latter sold as a commercial drug treatment against gastrointestinal nematodes for animal health use. The structure-permeability relationship of these cyclic depsipeptides that could ultimately provide insights into the compound bioavailability is not yet well understood. The fully N-methylated amide backbone and apolar sidechain residues do not allow for the formation of intramolecular hydrogen bonds, normally observed in the membrane-permeable conformations of cyclic peptides. Hence, any understanding gained on these depsipeptides would serve as a prototype for future design strategies. In previous nuclear magnetic resonance (NMR) studies, two macrocyclic core conformers of emodepside were detected, one with all backbone amides in trans-configuration (hereon referred as the symmetric conformer) and the other with one amide in cis-configuration (hereon referred as the asymmetric conformer). In addition, these depsipeptides were also reported to be ionophores with a preference of potassium over sodium. In this study, we relate the conformational behavior of PF1022A, emodepside, and closely related analogs with their ionophoric characteristic probed using NMR and molecular dynamics (MD) simulations and finally evaluated their passive membrane permeability using PAMPA. We find that the equilibrium between the two core conformers shifts more towards the symmetric conformer upon addition of monovalent cations with selectivity for potassium over sodium. Both the NMR experiments and the theoretical Markov state models based on extensive MD simulations indicate a more rigid backbone for the asymmetric conformation, whereas the symmetric conformation shows greater flexibility. The experimental results further advocate for the symmetric conformation binding the cation. The PAMPA results suggest that the investigated depsipeptides are retained in the membrane, which may be advantageous for the likely target, a membrane-bound potassium channel.

Structural and functional diversity in Listeria cell wall teichoic acids.

Wall teichoic acids (WTAs) are the most abundant glycopolymers found on the cell wall of many Gram-positive bacteria, whose diverse surface structures play key roles in multiple biological processes. Despite recent technological advances in glycan analysis, structural elucidation of WTAs remains challenging due to their complex nature. Here, we employed a combination of ultra-performance liquid chromatography-coupled electrospray ionization tandem-MS/MS and NMR to determine the structural complexity of WTAs from Listeria species. We unveiled more than 10 different types of WTA polymers that vary in their linkage and repeating units. Disparity in GlcNAc to ribitol connectivity, as well as variable O-acetylation and glycosylation of GlcNAc contribute to the structural diversity of WTAs. Notably, SPR analysis indicated that constitution of WTA determines the recognition by bacteriophage endolysins. Collectively, these findings provide detailed insight into Listeria cell wall-associated carbohydrates, and will guide further studies on the structure-function relationship of WTAs.

Conformational Properties of a Peptidic Catalyst: Insights from NMR Spectroscopic Studies.

Peptides have become valuable as catalysts for a variety of different reactions, but little is known about the conformational properties of peptidic catalysts. We investigated the conformation of the peptide H-dPro-Pro-Glu-NH2, a highly reactive and stereoselective catalyst for conjugate addition reactions, and the corresponding enamine intermediate in solution by NMR spectroscopy and computational methods. The combination of nuclear Overhauser effects (NOEs), residual dipolar couplings (RDCs), J-couplings, and temperature coefficients revealed that the tripeptide adopts a single predominant conformation in its ground state. The structure is a type I ß-turn, which gains stabilization from three hydrogen bonds that are cooperatively formed between all functional groups (secondary amine, carboxylic acid, amides) within the tripeptide. In contrast, the conformation of the enamine intermediate is significantly more flexible. The conformational ensemble of the enamine is still dominated by the ß-turn, but the backbone and the side chain of the glutamic acid residue are more dynamic. The key to the switch between rigidity and flexibility of the peptidic catalyst is the CO2H group in the side chain of the glutamic acid residue, which acts as a lid that can open and close. As a result, the peptidic catalyst is able to adapt to the structural requirements of the intermediates and transition states of the catalytic cycle. These insights might explain the robustness and high reactivity of the peptidic catalyst, which exceeds that of other secondary amine-based organocatalysts. The data suggest that a balance between rigidity and flexibility, which is reminiscent of the dynamic nature of enzymes, is beneficial for peptidic catalysts and other synthetic catalysts.

Conformational Analysis of an Antibacterial Cyclodepsipeptide Active against Mycobacterium tuberculosis by a Combined ROE and RDC Analysis.

Griselimycin (GM) and methylgriselimycin (MGM), naturally produced by microorganisms of the genus Streptomyces, are cyclic depsipeptides composed of ten amino acids. They exhibit antibacterial activity against Mycobacterium species by inhibiting the sliding clamp of prokaryotic DNA polymerase III and are therefore considered as potential anti-tuberculosis drugs. The difference between the peptides is the presence of l-(R)-4-methyl-proline in MGM instead of l-proline in GM at position 8 of the amino acid sequence. Methylation increases both metabolic stability and activity of MGM compared to GM. To get deeper insight into the structure-activity relationship, the solution structure of the cyclic part of MGM was determined using rotating-frame nuclear Overhauser effect (ROE) distance restraints and residual dipolar couplings (RDC). The structure of MGM in solution is compared to the structure of GM in a co-crystal with DNA polymerase III subunit beta. As a result, a highly defined structural model of MGM is obtained, which shows related characteristics to the bound GM.

Gastric and Postgastric Processing of 13C Markers Renders the 13C Breath Test an Inappropriate Measurement Method for the Gastric Emptying of Lipid Emulsions in Healthy Adults.

Background: Breath tests (BTs) present an alternative gastric-emptying (GE) measure. However, their efficacy in the measurement of the GE rate of lipid emulsions (LEs) is unknown.Objective: The objective of this work was to investigate the validity of 13C BTs as a measure of fat GE rate in LEs.Methods: The lipophilic 13C octanoate (OCC) BT marker was investigated for fat GE with the hydrophilic 13C sodium acetate (ACC) and the triglyceride 13C trioctanoin (TCC) markers as comparators. Data from 2 randomized studies were combined [50 healthy participants; 25 men, mean ± SD age: 23 ± 2.8 y; mean ± SD body mass index (in kg/m2): 22.4 ± 1.7]. Each participant was given either an acid-stable LE (LE1) or an acid-unstable LE (LE4) at each visit. Twenty-three participants underwent simultaneous MRI. The effect of LEs on 13CO2 excretion profiles was determined. The BT half-emptying times (BT T50) were validated with the MRI half-emptying time of the ingested fat volume (MRI T50).Results: The effect of LEs on 13CO2 excretion depended on the properties of the 13C marker. T50 for OCC was shorter by 98 min for LE1 than for LE4 (P < 0.001). Other markers showed either no LE dependency or a longer T50 for LE1 than for LE4. No difference in T50 between OCC and ACC was detected in LE1. In LE4, the T50 was longer by 154 min (P < 0.0001). There was some concordance between MRI T50 and OCC BT T50 for LE1 (rc = 0.7). No other marker showed any concordance with fat GE. 13C-Nuclear magnetic resonance in vitro findings were compatible with changes in the kinetics of phase transfer of OCC dependent on its protonation state.Conclusions: The structure of fat present in the stomach affects 13CO2 excretion. The chemical properties of the 13C marker and their gastric and postgastric interaction with fat renders 13CO2 excretion an inappropriate measure of LE emptying in healthy adults. This trial was registered at clinicaltrials.gov as NCT02226029 and NCT02602158.

The use of ene adducts to study and engineer enoyl-thioester reductases.

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.

RDC-enhanced structure calculation of a ß-heptapeptide in methanol.

Residual dipolar couplings (RDCs) are a rich source of structural information that goes beyond the range covered by the nuclear Overhauser effect or scalar coupling constants. They can only be measured in partially oriented samples. RDC studies of peptides in organic solvents have so far been focused on samples in chloroform or DMSO. Here, we show that stretched poly(vinyl acetate) can be used for the partial alignment of a linear ß-peptide with proteinogenic side chains in methanol. 1 DCH , 1 DNH , and 2 DHH RDCs were collected with this sample and included as restraints in a simulated annealing calculation. Incorporation of RDCs in the structure calculation process improves the long-range definition in the backbone of the resulting 314 -helix and uncovers side-chain mobility. Experimental side-chain RDCs of the central leucine and valine residues are in good agreement with predicted values from a local three-state model. Copyright © 2016 John Wiley & Sons, Ltd.

Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes.

The pyridine nucleotides NADH and NADPH (NAD(P)H) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P)H and the corresponding substrate is lacking for many enzymes. Here we present evidence for a new mechanism that operates during enzymatic hydride transfers using crotonyl-CoA carboxylase/reductase (Ccr) as a case study. We observed a covalent ene intermediate between NADPH and the substrate, crotonyl-CoA, using NMR, high-resolution MS and stopped-flow spectroscopy. Preparation of the ene intermediate further allowed direct access to the catalytic cycle of other NADPH-dependent enzymes-including those from type II fatty acid biosynthesis-in an unprecedented way, suggesting that formation of NAD(P)H ene intermediates is a more general principle in catalysis.

Stereoselective Organocatalyzed Synthesis of α-Fluorinated ß-Amino Thioesters and Their Application in Peptide Synthesis.

α-Fluorinated ß-amino thioesters were obtained in high yields and stereoselectivities by organocatalyzed addition reactions of α-fluorinated monothiomalonates (F-MTMs) to N-Cbz- and N-Boc-protected imines. The transformation requires catalyst loadings of only 1 mol % and proceeds under mild reaction conditions. The obtained addition products were readily used for coupling-reagent-free peptide synthesis in solution and on solid phase. The α-fluoro-ß-(carb)amido moiety showed distinct conformational preferences, as determined by crystal structure and NMR spectroscopic analysis.

Substitution of proline32 by α-methylproline preorganizes ß2-microglobulin for oligomerization but not for aggregation into amyloids.

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human ß2-microglobulin (ß2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a ß2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]ß2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type ß2m, [MePro32]ß2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type ß2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]ß2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial ß2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of ß2m amyloid structures.

Homochiral [2]Catenane and Bis[2]catenane from Alleno-Acetylenic Helicates - A Highly Selective Narcissistic Self-Sorting Process.

Homochiral strands of alternating alleno-acetylenes and phenanthroline ligands (P)-1 and (P2)-2, as well as their corresponding enantiomers, selectively assemble with the addition of silver(I) salt to yield dinuclear and trinuclear double helicates, respectively. Upon increasing the solvent polarity, the dinuclear and trinuclear helicates interlock to form a [2]catenane and bis[2]catenane, bearing 14 chirality elements, respectively. The solid-state structure of the [2]catenane reveals a nearly perfect fit of the interlocked strands, and the ECD spectra show a significant amplification of the chiroptical properties upon catenation, indicating stabilization of the helical secondary structure. Highly selective narcissistic self-sorting was demonstrated for a racemic mixture consisting of both short and long alleno-acetylenic strands, highlighting their potential for the preparation of linear catenanes of higher order.

Chiroptical detection of nonchromophoric, achiral guests by enantiopure alleno-acetylenic helicages.

Enantiopure alleno-acetylenic ligands assemble diastereoselectively upon the addition of a zinc(II) salt to form triple-stranded helicates, which provide a sufficiently large helical cage ("helicage") for the encapsulation of guests. The inclusion complexation of heteroalicycles is confirmed by ROESY and DOSY NMR spectroscopy and quantified in (1) H NMR binding titrations. The ECD spectra of the helicates, which showed strong Cotton effects and exciton coupling, were found to be extremely sensitive to the nature of the guest molecules. Consequently, a series of nonchromophoric, achiral guests of different sizes as well as regioisomers (1,3- and 1,4-dioxane) became distinguishable on the basis of their induced CD (ICD) spectra. Molecular dynamics (MD) simulations show the adaptability of the cavity size to individual guest molecules and support the selective ICD output. Particularly high affinity towards 1,4-dioxane allowed its selective detection at parts-per-million (ppm) levels in aqueous solutions.

Donor-acceptor (D-A)-substituted polyyne chromophores: modulation of their optoelectronic properties by varying the length of the acetylene spacer.

A series of donor-acceptor-substituted alkynes, 2 a-f, was synthesized in which the length of the π-conjugated polyyne spacer between the N,N-diisopropylanilino donor and the 1,1,4,4-tetracyanobuta-1,3-diene (TCBD) acceptor was systematically changed. The effect of this structural change on the optoelectronic properties of the molecules and, ultimately, their third-order optical nonlinearity was comprehensively investigated. The branched N,N-diisopropyl groups on the anilino donor moieties combined with the nonplanar geometry of 2 a-f imparted exceptionally high solubility to these chromophores. This important property allowed for performing INADEQUATE NMR measurements without (13) C labeling, which, in turn, resulted in a complete assignment of the carbon skeleton in chromophores 2 a-f and the determination of the (13) C-(13) C coupling constants. This body of data provided unprecedented insight into characteristic (13) C chemical shift patterns in push-pull-substituted polyynes. Electrochemical and UV/Vis spectroscopic studies showed that the HOMO-LUMO energy gap decreases with increasing length of the polyyne spacer, while this effect levels off for spacers with more than four acetylene units. The third-order optical nonlinearity of this series of molecules was determined by measuring the rotational averages of the third-order polarizabilities (γrot ) by degenerate four-wave mixing (DFWM). These latter studies revealed high third-order optical nonlinearities for the new chromophores; most importantly, they provided fundamental insight into the effect of the conjugated spacer length in D-A polyynes, that can be exploited in the future design of suitable charge-transfer chromophores for applications in optoelectronic devices.

Mechanistic Investigation of the Nickel-Catalyzed Transfer Hydrocyanation of Alkynes.

The implementation of HCN-free transfer hydrocyanation reactions on laboratory scales has recently been achieved by using HCN donor reagents under nickel- and Lewis acid co-catalysis. More recently, malononitrile-based HCN donor reagents were shown to undergo the C(sp3)-CN bond activation by the nickel catalyst in the absence of Lewis acids. However, there is a lack of detailed mechanistic understanding of the challenging C(sp3)-CN bond cleavage step. In this work, in-depth kinetic and computational studies using alkynes as substrates were used to elucidate the overall reaction mechanism of this transfer hydrocyanation, with a particular focus on the activation of the C(sp3)-CN bond to generate the active H-Ni-CN transfer hydrocyanation catalyst. Comparisons of experimentally and computationally derived 13C kinetic isotope effect data support a direct oxidative addition mechanism of the nickel catalyst into the C(sp3)-CN bond facilitated by the coordination of the second nitrile group to the nickel catalyst.

Redox-switchable resorcin[4]arene cavitands: molecular grippers.

Diquinone-based resorcin[4]arene cavitands that open to a kite and close to a vase form upon changing their redox state, thereby releasing and binding guests, have been prepared and studied. The switching mechanism is based on intramolecular H-bonding interactions that stabilize the vase form and are only present in the reduced hydroquinone state. The intramolecular H-bonds were characterized using X-ray, IR, and NMR spectroscopies. Guests were bound in the closed, reduced state and fully released in the open, oxidized state.

Designing fluorinated cinchona alkaloids for enantioselective catalysis: controlling internal rotation by a fluorine-ammonium ion gauche effect (φ(NCCF)).

The C9 position of cinchona alkaloids functions as a molecular hinge, with internal rotations around the C8-C9 (τ(1)) and C9-C4' (τ(2)) bonds giving rise to four low energy conformers (1; anti-closed, anti-open, syn-closed, and syn-open). By substituting the C9 carbinol centre by a configurationally defined fluorine substituent, a fluorine-ammonium ion gauche effect (σ(C-H) → σ(C-F)*; F(δ-)⋅⋅⋅N(+)) encodes for two out of the four possible conformers (2). This constitutes a partial solution to the long-standing problem of governing internal rotations in cinchonium-based catalysts relying solely on a fluorine conformational effect.