Amide Bond Rotation Research Articles

Cover Feature: Exploration of N‐Arylation of Backbone Amides as a Novel Tool for Conformational Modification in Peptides (Chem. Eur. J. 40/2023)

N-aryl amide bonds for peptide conformation: In the universe of peptides,theN-aryl amide bond, with its large steric and electronic effects and ability to restrain amide bond rotation, is represented as a new star. By employing N-arylation as a backbone amide modification strategy in peptides, we could unravel the different worlds of distinct and predominantly homogeneous conformations in cyclic hexa- and cyclic pentapeptides containing one or two N-aryl amide bonds. More information can be found in the Research Article by A. Dangi and U. Kiran Marelli (DOI: 10.1002/chem.202300753).

Synthesis and Structural Properties of Adamantane-Substituted Amines and Amides Containing an Additional Adamantane, Azaadamantane or Diamantane Moiety.

Introduction of adamantane moieties on diamondoids such as adamantane, 2‐azaadamantane or diamantane by amide formation and reduction to the corresponding amine was performed in a straightforward and easy way by amidation under Schotten–Baumann conditions and reduction with BH3 ⋅ THF. The obtained amides and amines were studied in terms of structural properties towards the perspective of transformation into nanodiamonds. Crystal structure and dynamic NMR experiments of the most crowded amide obtained gave structural insights into the effect of bulkiness and steric strain on out‐of‐planarity of amide bonds (16.0°) and the kinetics and thermodynamics of amide bond rotation (ΔG≠ 298K=11.5–13.3 kcal ⋅ mol−1).

Role of solvent dielectric constant on the enthalpy-entropy compensation in the hindered amide bond rotation

The purpose of this investigation is to determine if enthalpy-entropy compensation (EEC) is a valid concept when the experiments are performed on a set of molecules in multiple solvents. Dynamic nuclear magnetic resonance (DNMR) experiments are performed on a series of analogs derived from N, N-diethyl-m-toluamide (DEET) in six different solvents of varying dielectric constant from toluene‑d8 (ε = 2.38) to dimethyl sulfoxide-d6 (ε = 46.7). The thermodynamic parameters of the restricted rotation to the solvent dielectric constant do not have a straightforward correlation relationship between them. However, the activation enthalpy and entropy of more than sixty different experimental measurements indicate that enthalpy-entropy compensation (EEC) is independent of the solvent condition with a compensation temperature of 287.81 ± 3.95 K. The results, therefore, support the broader role of EEC on the partial amide bond dynamics in the DEET analogs over the temperature range studied.

Polarity effects in 4-fluoro- and 4-(trifluoromethyl)prolines.

Fluorine-containing analogues of proline are valuable tools in engineering and NMR spectroscopic studies of peptides and proteins. Their use relies on the fundamental understanding of the interplay between the substituents and the main chain groups of the amino acid residue. This study aims to showcase the polarity-related effects that arise from the interaction between the functional groups in molecular models. Properties such as conformation, acid–base transition, and amide-bond isomerism were examined for diastereomeric 4-fluoroprolines, 4-(trifluoromethyl)prolines, and 1,1-difluoro-5-azaspiro[2.4]heptane-6-carboxylates. The preferred conformation on the proline ring originated from a preferential axial positioning for a single fluorine atom, and an equatorial positioning for a trifluoromethyl- or a difluoromethylene group. This orientation of the substituents explains the observed trends in the pKa values, lipophilicity, and the kinetics of the amide bond rotation. The study also provides a set of evidences that the transition state of the amide-bond rotation in peptidyl-prolyl favors C4-exo conformation of the pyrrolidine ring.

Validation of Enthalpy-Entropy Compensation Mechanism in Partial Amide Bond Rotation.

The concept of enthalpy–entropy compensation (EEC) is one of the highly debated areas of thermodynamics. The conformational change due to restricted double-bond rotation shows a classic two-site chemical exchange phenomenon and has been extensively studied. Fifty-four analogs of N,N-diethyl-m-toluamide (DEET) as a model system were synthesized to study the thermodynamics of the partial amide bond character using nuclear magnetic resonance (NMR) spectroscopy. Line-shape analysis as a function of temperature is used to estimate the chemical exchange. Eyring analysis was then used to convert the chemical exchange rates to determine the transition state enthalpy and entropy of the molecules. The experimental design follows selective variations that perturb one aspect of the molecular system and its influence on the observed thermodynamic effect. The results of the study demonstrate that amide bond resonance in analogs of DEET follows an EEC mechanism. Simple modifications made to DEET’s structural motif alter both the enthalpy and entropy of the system and were limited overall to a temperature compensation factor, Tβ = 292.20 K, 95% CI [290.66, 293.73]. We suggest EEC as a model to describe the kinetic compensation seen in chemical exchange phenomena in analogs of DEET.

Transannular Hydrogen Bonding in Planar‐Chiral [2.2]Paracyclophane‐Bisamides

AbstractA series of [2.2]paracyclophane‐bisamide regioisomers and alkylated comparators were designed, synthesized, and characterized in order to better understand the transannular hydrogen bonding of [2.2]paracyclophane‐based molecular recognition units. X‐Ray crystallography shows that transannular hydrogen bonding is maintained in the solid‐state, but no stereospecific self‐recognition is observed. The assignment of both transannularly and intermolecularly hydrogen bonded N−H stretches could be made by infrared spectroscopy, and the effect of transannular hydrogen bonding on amide bond rotation dynamics is observed by 1H‐NMR in nonpolar solvents. The consequences of transannular hydrogen bonding on the optical properties of [2.2]paracyclophane is observed by comparing alkylated and non‐alkylated pseudo‐ortho 4,12‐[2.2]paracyclophane‐bisamides. Finally, optical resolution of 4‐mono‐[2.2]paracyclophane and pseudo‐ortho 4,12‐[2.2]paracyclophane‐bisamides was achieved through the corresponding sulfinyl diastereoisomers for circular dichroism studies. Transannular hydrogen bonding in [2.2]paracyclophane‐amides allows preorganization for self‐complementary intermolecular assembly, but is weak enough to allow rapid rotation of the amides even in nonpolar solvents.

Density Functional Studies on Secondary Amides: Role of Steric Factors in Cis/Trans Isomerization.

Cis/trans isomerization of amide bonds is a key step in a wide range of biological and synthetic processes. Occurring through C-N amide bond rotation, it also coincides with the activation of amides in enzymatic hydrolysis. In recently described QM studies of cis/trans isomerization in secondary amides using density functional methods, we highlighted that a peptidic prototype, such as glycylglycine methyl ester, can suitably represent the isomerization and complexities arising out of a larger molecular backbone, and can serve as the primary scaffold for model structures with different substitution patterns in order to assess and compare the steric effect of the substitution patterns. Here, we describe our theoretical assessment of such steric effects using tert-butyl as a representative bulky substitution. We analyze the geometries and relative stabilities of both trans and cis isomers, and effects on the cis/trans isomerization barrier. We also use the additivity principle to calculate absolute steric effects with a gradual increase in bulk. The study establishes that bulky substitutions significantly destabilize cis isomers and also increases the isomerization barrier, thereby synergistically hindering the cis/trans isomerization of secondary amides. These results provide a basis for the rationalization of kinetic and thermodynamic properties of peptides with potential applications in synthetic and medicinal chemistry.

Probing Solvation-Induced Structural Changes in Conformationally Flexible Peptides: IR Spectroscopy of Gly3H+·(H2O).

IR predissociation spectroscopy of the Gly3H+(H2O) complex formed inside of a cryogenic ion trap reveals how the flexible model peptide structurally responds to solvation by a single water molecule. The resulting one-laser spectrum is quite congested, and the spectral analyses were assisted by both H2O/D2O substitution and IR-IR double resonance spectroscopy, revealing the presence of two contributing isomers and extensive anharmonic features. Comparisons to structures found via a systematic computational search identified the geometries of these two isomers. The major isomer, with all trans amide bonds and protonation on the terminal amine, represents ∼90% of the overall population. It noticeably differs from the unsolvated Gly3H+, which exists in two isomeric forms: one with a cis amide bond and the other with protonation on an amide C═O. These results indicate that interactions with just one water molecule can induce significant structural changes, i.e., cis- trans amide bond rotation and proton migration, even as the clustering occurs within an 80 K cryogenic ion trap. Calculations of the isomerization pathways further reveal that the binding energy of the water molecule provides sufficient internal energy to overcome the barriers for the observed structural changes, and the minor solvation isomer results from a small fraction of the ions being kinetically trapped along one of the pathways.

Unexpected Resistance to Base-Catalyzed Hydrolysis of Nitrogen Pyramidal Amides Based on the 7-Azabicyclic[2.2.1]heptane Scaffold.

Non-planar amides are usually transitional structures, that are involved in amide bond rotation and inversion of the nitrogen atom, but some ground-minimum non-planar amides have been reported. Non-planar amides are generally sensitive to water or other nucleophiles, so that the amide bond is readily cleaved. In this article, we examine the reactivity profile of the base-catalyzed hydrolysis of 7-azabicyclo[2.2.1]heptane amides, which show pyramidalization of the amide nitrogen atom, and we compare the kinetics of the base-catalyzed hydrolysis of the benzamides of 7-azabicyclo[2.2.1]heptane and related monocyclic compounds. Unexpectedly, non-planar amides based on the 7-azabicyclo[2.2.1]heptane scaffold were found to be resistant to base-catalyzed hydrolysis. The calculated Gibbs free energies were consistent with this experimental finding. The contribution of thermal corrections (entropy term, –TΔS‡) was large; the entropy term (ΔS‡) took a large negative value, indicating significant order in the transition structure, which includes solvating water molecules.

Synthesis and biological evaluation of pyrimidine derivatives as novel human Pin1 inhibitors

Pin1 (Protein interacting with NIMA1) is a cis–trans isomerase and promotes the amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1 might be a novel strategy for developing anticancer agents. Herein, a series of pyrimidine derivatives were synthesized and their Pin1 inhibitory activities were evaluated. Among them, four compounds (2a, 2f, 2h and 2l) displayed potent inhibitory activities against Pin1 with IC50 values lower than 3 µM. This series of pyrimidine-based inhibitors presented time-dependent inhibition against Pin1. The structure–activity relationships on the 2-, 4- and 5-positions of the pyrimidine ring were analyzed in details, which would facilitate further exploration of new Pin1 inhibitors.

Synthesis of novel norsufentanil analogs via a four-component Ugi reaction and in vivo, docking, and QSAR studies of their analgesic activity.

Novel substituted amino acid tethered norsufentanil derivatives were synthesized by the four-component Ugi reaction. Norsufentanil was reacted with succinic anhydride to produce the corresponding carboxylic acid. The resulting carboxylic acid has undergone a multicomponent reaction with different aldehydes, amines, and isocyanides to produce a library of the desired compounds. In all cases, amide bond rotation was observed in the NMR spectra. In vivo analgesic activity of the synthesized compounds was evaluated by a tail flick test. Very encouraging results were obtained for a number of the synthesized products. Some of the synthesized compounds such as 5a, 5b, 5h, 5j, and 5r were found to be more potent than sufentanil, sufentanil citrate, and norsufentanil. Binding modes between the compounds and mu and delta-opioid receptors were studied by molecular docking method. The relationship between the molecular structural features and the analgesic activity was investigated by a quantitative structure-activity relationship model. The results of the molecular modeling studies and the in vivo analgesic activity suggested that the majority of the synthesized compounds were more potent than sufentanil and norsufentanil.

Amide rotation trajectories probed by symmetry.

Amide rotation of peptidyl-prolyl fragments is an important factor in backbone structure organization of proteins. Computational studies have indicated that this rotation preferentially proceeds through a defined transition-state structure (syn/exo). Here, we complement the computational findings by determining the amide bond rotation barriers for derivatives of the two symmetric proline analogues, meso and racemic pyrrolidine-2,5-dicarboxylic acids. The rotations around these residues represent syn/exo-syn/exo and anti/endo-syn/exo hybrid transition states for the meso and racemic diastereomer, respectively. The rotation barriers are lower for the former rotation by about 9 kJ mol-1 (aqueous medium), suggesting a strong preference for the syn/exo (clockwise) rotation over the anti/endo (anticlockwise) rotation. The results show that both hybrid rotation processes are enthalpically driven but respond differently to solvent polarity changes due to the different transition state dipole-dipole interactions.

Water Model Tuning for Improved Reproduction of Rotational Diffusion and NMR Spectral Density

A water model for molecular simulation was optimized to improve the reproduction of translational and rotational diffusion of pure water and proteins. The SPC/E(b) model was developed from the original SPC/E model with a slight increase of the O-H bond length of 1%. This tuning has significantly improved the translational and rotational diffusion when compared to the experimental values, whereas only small changes were observed in the other thermodynamic properties examined. The overall tumbling correlation times (τ(p)) from ubiquitin, protein G, bovine pancreatic trypsin inhibitor, and barstar C42/80A were successfully reproduced using the SPC/E(b) model. Calculated site-specific spectral densities of the main chain amide bond rotation in ubiquitin and protein G were in good agreement with those derived from nuclear magnetic resonance reduced spectral density mapping. The SPC/E(bT) model was also developed with temperature-dependent bond-length tuning to facilitate reproduction of the experimental τ(p) around room temperature.

Solution Structures of the Prototypical 18 kDa Translocator Protein Ligand, PK 11195, Elucidated with 1H/13C NMR Spectroscopy and Quantum Chemistry

Eighteen kilodalton translocator protein (TSPO) is an important target for drug discovery and for clinical molecular imaging of brain and peripheral inflammatory processes. PK 11195 [1a; 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide] is the major prototypical high-affinity ligand for TSPO. Elucidation of the solution structure of 1a is of interest for understanding small-molecule ligand interactions with the lipophilic binding site of TSPO. Dynamic (1)H/(13)C NMR spectroscopy of 1a revealed four quite stable but interconverting rotamers, due to amide bond and 2-chlorophenyl group rotation. These rotamers have been neglected in previous descriptions of the structure of 1a and of the binding of 1a to TSPO. Here, we used quantum chemistry at the level of B3LYP/6-311+G(2d,p) to calculate (13)C and (1)H chemical shifts for the rotamers of 1a and for the very weak TSPO ligand, N-desmethyl-PK 11195 (1b). These data, plus experimental NMR data, were then used to characterize the structures of rotamers of 1a and 1b in organic solution. Energy barriers for both the amide bond and 2'-chlorophenyl group rotation of 1a were determined from dynamic (1)H NMR to be similar (ca.17 to 18 kcal/mol), and they compared well with those calculated at the level of B3LYP/6-31G*. Furthermore, the computed barrier for Z to E rotation is considerably lower in 1a(18.7 kcal/mol) than in 1b (25.4 kcal/mol). NMR (NOE) unequivocally demonstrated that the E rotamer of 1a is the more stable in solution by about 0.4 kcal/mol. These detailed structural findings will aid future TSPO ligand design and support the notion that TSPO prefers to bind ligands as amide E-rotamers.

The amide bond rotation controlled by an unusual C–H···O hydrogen bonding that favors the 1,4-phenyl radical migration

Experimental evidence in support of the presence of a weak C–H···O hydrogen bonding that favors 1,4-phenyl radical migration in a chiral amide is provided.

An Inversion Recovery NMR Kinetics Experiment

A convenient laboratory experiment is described in which NMR magnetization transfer by inversion recovery is used to measure the kinetics and thermochemistry of amide bond rotation. The experiment utilizes Varian spectrometers with the VNMRJ 2.3 software, but can be easily adapted to any NMR platform. The procedures and sample data sets in this article will enable instructors to use inversion recovery as a laboratory activity in applied NMR classes and provide research students with a convenient template with which to acquire inversion recovery data on research samples.

Acceleration of Amide Bond Rotation by Encapsulation in the Hydrophobic Interior of a Water-Soluble Supramolecular Assembly

The hydrophobic interior cavity of a self-assembled supramolecular assembly exploits the hydrophobic effect for the encapsulation of tertiary amides. Variable-temperature (1)H NMR experiments reveal that the free energy barrier for rotation around the C-N amide bond is lowered by up to 3.6 kcal/mol upon encapsulation. The hydrophobic cavity of the assembly is able to stabilize the less polar transition state of the amide rotation process. Carbon-13 labeling studies showed that the (13)C NMR chemical shift of the carbonyl resonance increases with temperature for the encapsulated amides, which suggests that the assembly is able to favor a twisted form of the amide.

Development of a polarizable intermolecular potential function (PIPF) for liquid amides and alkanes.

A polarizable intermolecular potential function (PIPF) employing the Thole interacting dipole (TID) polarization model has been developed for liquid alkanes and amides. In connection with the internal bonding terms of the CHARMM22 force field, the present PIPF-CHARMM potential provides an adequate description of structural and thermodynamic properties for liquid alkanes and for liquid amides through molecular dynamics simulations. The computed heats of vaporization and liquid density are within 1.4% of experimental values. Polarization effects play a major role in liquid amides, which are reflected by an increase of 1.5 to 1.8 D in molecular dipole moment for primary and secondary amides. Furthermore, the computed polarization energies contribute to the total intermolecular interaction energy by 6 to 24%. The ability of the PIPF-CHARMM force field to treat protein backbone structures is tested by examining the potential energy surface of the amide bond rotation in N-methylacetamide and the Ramachandran surface for alanine dipeptide. The agreement with ab initio MP2 results and with the original CHARMM22 force field is encouraging, suggesting the PIPF-CHARMM potential can be used as a starting point to construct a complete polarizable force field for proteins.

Transesterification and amide cis–trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc–Asp(Dpa)–OBzl

The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions.

Enterobactin Protonation and Iron Release: Structural Characterization of the Salicylate Coordination Shift in Ferric Enterobactin1

The siderophore enterobactin (Ent) is produced by many species of enteric bacteria to mediate iron uptake. This iron scavenger can be reincorporated by the bacteria as the ferric complex [Fe(III)(Ent)](3)(-) and is subsequently hydrolyzed by an esterase to facilitate intracellular iron release. Recent literature reports on altered protein recognition and binding of modified enterobactin increase the significance of understanding the structural features and solution chemistry of ferric enterobactin. The structure of the neutral protonated ferric enterobactin complex [Fe(III)(H(3)Ent)](0) has been the source of some controversy and confusion in the literature. To demonstrate the proposed change of coordination from the tris-catecholate [Fe(III)(Ent)](3)(-) to the tris-salicylate [Fe(III)(H(3)Ent)](0) upon protonation, the coordination chemistry of two new model compounds N,N',N''-tris[2-(hydroxybenzoyl)carbonyl]cyclotriseryl trilactone (SERSAM) and N,N',N''-tris[2-hydroxy,3-methoxy(benzoyl)carbonyl]cyclotriseryl trilactone (SER(3M)SAM) was examined in solution and solid state. Both SERSAM and SER(3M)SAM form tris-salicylate ferric complexes with spectroscopic and solution thermodynamic properties (with log beta(110)() values of 39 and 38 respectively) similar to those of [Fe(III)(H(3)Ent)](0). The fits of EXAFS spectra of the model ferric complexes and the two forms of ferric enterobactin provided bond distances and disorder factors in the metal coordination sphere for both coordination modes. The protonated [Fe(III)(H(3)Ent)](0) complex (d(Fe)(-)(O) = 1.98 A, sigma(2)(stat)(O) = 0.00351(10) A(2)) exhibits a shorter average Fe-O bond length but a much higher static Debye-Waller factor for the first oxygen shell than the catecholate [Fe(III)(Ent)](3)(-) complex (d(Fe)(-)(O) = 2.00 A, sigma(2)(stat)(O) = 0.00067(14) A(2)). (1)H NMR spectroscopy was used to monitor the amide bond rotation between the catecholate and salicylate geometries using the gallic complexes of enterobactin: [Ga(III)(Ent)](3)(-) and [Ga(III)(H(3)Ent)](0). The ferric salicylate complexes display quasi-reversible reduction potentials from -89 to -551 mV (relative to the normal hydrogen electrode NHE) which supports the feasibility of a low pH iron release mechanism facilitated by biological reductants.