Intramolecular Hydrogen Bonding Involving Organic Fluorine: NMR Investigations Corroborated by DFT-Based Theoretical Calculations

molecules

Review Intramolecular Hydrogen Bonding Involving Organic Fluorine: NMR Investigations Corroborated by DFT-Based Theoretical Calculations

Sandeep Kumar Mishra and N. Suryaprakash *

NMR Research Centre, Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India; [email protected] * Correspondence: [email protected]; Tel.: +80-2293-7344 or +80-2293-3300 or +91-9845124802; Fax: +91-2360-1550

Academic Editor: Steve Scheiner Received: 31 January 2017; Accepted: 2 March 2017; Published: 7 March 2017

Abstract: The combined utility of many one and two dimensional NMR methodologies and DFT-based theoretical calculations have been exploited to detect the intramolecular hydrogen bond (HB) in number of different organic fluorine-containing derivatives of molecules, viz. benzanilides, hydrazides, imides, benzamides, and diphenyloxamides. The existence of two and three centered hydrogen bonds has been convincingly established in the investigated molecules. The NMR spectral parameters, viz., coupling mediated through hydrogen bond, one-bond NH scalar couplings, physical parameter dependent variation of chemical shifts of NH protons have paved the way for understanding the presence of hydrogen bond involving organic fluorine in all the investigated molecules. The experimental NMR findings are further corroborated by DFT-based theoretical calculations including NCI, QTAIM, MD simulations and NBO analysis. The monitoring of H/D exchange with NMR spectroscopy established the effect of intramolecular HB and the influence of electronegativity of various substituents on the chemical kinetics in the number of organic building blocks. The utility of DQ-SQ technique in determining the information about HB in various fluorine substituted molecules has been convincingly established.

Keywords: NMR spectroscopy; intramolecular hydrogen bond; organic ﬂuorine

1. Introduction

1.1. Weak Molecular Interactions The presence of molecular interactions in Nature cannot be ignored. The existence of various forms of matter, such as, solids and liquids is mainly due to the presence of intermolecular interactions. One can safely make a statement that the world would be a uniform ideal gas in the absence of intermolecular interactions. The existence of intermolecular interactions is reflected at the molecular level, viz., the thermodynamic non-ideal-gas behavior arising due to vapor pressure, viscosity, virial coefficients, absorption, and superficial tension [1]. The molecular interactions are non-covalent and are inherently electrostatic in nature. These forces could be attractive, repulsive or the combination of attractive as well as repulsive between or within the molecules. They could also be between non-bonded atoms. Intermolecular interactions play predominant role in many fields, such as, conformation of biomolecules, drug design, etc. Kaplan classified the intermolecular interactions on the basis of distance between the interacting objects [2]. In the classification, there are three main ranges for molecular forces depending on the

Molecules 2017, 22, 423; doi:10.3390/molecules22030423 www.mdpi.com/journal/molecules Molecules 2017, 22, 423 2 of 44

Moleculesinteratomic 2017, 22 distances, 423 (R) for an interatomic potential (V). For range I the short distances are deﬁned2 of 41 where the potential is repulsive in nature and the electronic exchange is due to the overlapped wheremolecular the potential electronic is repulsive shells. The in nature range and II representsthe electronic the exchange intermediate is due distancesto the overlapped with the molecular van der electronicWaals minimum, shells. The which range arises II represents due to the the balance intermediate between distances repulsive with and the attractive van der Waals forces. minimum, Range III whichrepresents arises the due large to the distances balance with between negligible repulsive electronic and attractive exchange forces. where Range the intermolecular III represents forcesthe large are distancespredominantly with negligible attractive. electronic The representation exchange ofwh rangeere the of molecularintermolecular forces forces is illustrated are predominantly in Figure1a. attractive.Depending The on representation the nature, the of various range of molecular molecular interactions forces is illustrated can also bein Figure classiﬁed 1a. asDepending given in theon theFigure nature,1b. the various molecular interactions can also be classified as given in the Figure 1b.

FigureFigure 1. 1. ((aa)) The The classification classiﬁcation of intermolecular interactionsinteractions onon thethe basis basis of of interatomic interatomic distances distances for for a atypical typical interatomic interatomic potential. potential. Where WhereR Ris is thethe distancedistance betweenbetween the centers of masses masses of of the the molecules, molecules, V is is the the Lennard-Johns Lennard-Johns potential potential and and the the graph graph of V of as V the as function the function of R is of called R is Le callednnard-Johns Lennard-Johns model potentialmodel potential graph; graph;(b) Schematic (b) Schematic illustration illustration of some of some of known of known molecular molecular interactions. interactions. Some Some other other molecularmolecular interactions interactions that that are are not not listed listed in in the sch schemeeme may also be possible. This This review review is is mainly mainly focusedfocused on on the the intramolecular intramolecular HB HB hence hence hydr hydrogenogen bonding is highlighted in the scheme.

The change in molecular interactions are reflected in physical, chemical and biological phenomenon, such as, phase transitions of water (ice to water to vapor or vice versa), protein folding and unfolding and separation of DNA strands, RNA unfolding, etc. Such processes do not fall under chemical reactions. All the weak molecular interactions have their specific significance and govern the properties of a substrate it may be pointed out that the intermolecular interactions cannot be measured directly by any experiment.

Molecules 2017, 22, 423 3 of 44

The change in molecular interactions are reflected in physical, chemical and biological phenomenon, such as, phase transitions of water (ice to water to vapor or vice versa), protein folding and unfolding and separation of DNA strands, RNA unfolding, etc. Such processes do not fall under chemical reactions. All the weak molecular interactions have their specific significance and govern the properties of a substrate it may be pointed out that the intermolecular interactions cannot be measured

Moleculesdirectly 2017 by any, 22, 423 experiment. 3 of 41 Among all the weak molecular interactions described in Figure1, except for the hydrogen bond (HB),Among usually all pertain the weak to molecular intermolecular interactions interactions. described Onin Figure the other 1, except hand, for thethe hydrogen HB can be bond detected (HB), usuallywithin thepertain molecule to intermolecular or between theinteractions. two or more On the molecules. other hand, This the review HB can is focusedbe detected on thewithin studies the moleculecarried out or by between authors’ the laboratory two or more on the molecules. rare type This of intramolecular review is focu hydrogensed on the bonding studies (HB)carried involving out by authors’organic ﬂuorine.laboratory on the rare type of intramolecular hydrogen bonding (HB) involving organic fluorine.

1.2. Hydrogen Hydrogen Bond The idea of HB was first ﬁrst suggested by Huggins [3–6] [3–6] in 1919, and was further described by Larimer and Rodebush in 1920 [7]. [7]. HB is an interaction which occurs between an atom containing a lone pair of of electrons electrons (a (a Lewis Lewis base) base) and and a a hydrogen hydrogen atom atom which which is is bonded bonded to to an an electronegative electronegative atom atom (e.g., N, O,O, SS oror F)F) throughthrough a a covalent covalent bond. bond. In In a a HB, HB, the the Lewis Lewis base base plays plays the the role role of aof HB a HB acceptor acceptor (A) (A)and and the the electronegative electronegative atom atom bonded bonded to to proton proton is is called called the the HB HB donor donor (D). (D). TheThe hydrogenhydrogen bonding interaction is schematically depicted in Figure2 2..

Figure 2. 2. TheThe pictorial pictorial illustration illustration of of hydrogen hydrogen bond bond inte interaction,raction, where where HB HB acceptor/donor acceptor/donor can canbe F, be O, F, N,O, N,or S or atom S atom in the in the molecule. molecule. Polarization Polarization of electron of electron and and exposure exposure of ofpositive positive proton proton on oneither either side side is shownis shown schematically. schematically. The ﬁgure and the associated text were adapted from [8].

Hydrogen is the only atom that easily participate in the HB. This is due to the fact that hydrogen Hydrogen is the only atom that easily participate in the HB. This is due to the fact that hydrogen atom can form covalent sigma bonds with electronegative atoms like F, N, O and S, etc., where the atom can form covalent sigma bonds with electronegative atoms like F, N, O and S, etc., where the electron of 1s shells participates in the covalent bond. Since the more electronegative atom has tendency electron of 1s shells participates in the covalent bond. Since the more electronegative atom has tendency to pull the shared electron pair towards it, in the covalent bond between hydrogen and electronegative to pull the shared electron pair towards it, in the covalent bond between hydrogen and electronegative atom the bonding electrons moves away from hydrogen nucleus. Due to this the positive charge gets atom the bonding electrons moves away from hydrogen nucleus. Due to this the positive charge gets exposed from the back side (the opposite side to the bonded partner). This exposed positive nucleus exposed from the back side (the opposite side to the bonded partner). This exposed positive nucleus attracts the partial negative charge of lone pair of the other nucleus. On the other hand, the atoms attracts the partial negative charge of lone pair of the other nucleus. On the other hand, the atoms other than proton having non-bonding electrons in the inner shell shields the nucleus precluding it other than proton having non-bonding electrons in the inner shell shields the nucleus precluding it from getting exposed in this manner. from getting exposed in this manner. Importance of Hydrogen Bond Importance of Hydrogen Bond Hydrogen bonding is an important non-covalent interaction encountered most often in Hydrogen bonding is an important non-covalent interaction encountered most often in chemistry chemistry and biology [8–11]. Voluminous information is available on the self-assembly of molecules and biology [9–12]. Voluminous information is available on the self-assembly of molecules driven driven by HB [12–24]. HB interaction is responsible for the three-dimensional conformation of proteins, synthetic foldamers [25–33], peptide catalysis, peptidomimetic design [34–39], double helical structure of DNA [40,41], unique properties of water [42], etc. Thus, in-depth knowledge of HB interaction aids in understanding the chemical properties of many organic and bio-macromolecules [43–53].

1.3. Organic Fluorine in the HB A large fraction of the information available in the literature on intramolecular HB pertains to the motifs of the type O···H-N and N···H-N [54–58]. It is well known that organic fluorine hardly ever accepts HB. Dunitz and co-workers have even published a paper entitled “organic fluorine hardly

Molecules 2017, 22, 423 4 of 44 by HB [13–25]. HB interaction is responsible for the three-dimensional conformation of proteins, synthetic foldamers [26–34], peptide catalysis, peptidomimetic design [35–40], double helical structure of DNA [41,42], unique properties of water [43], etc. Thus, in-depth knowledge of HB interaction aids in understanding the chemical properties of many organic and bio-macromolecules [44–54].

1.3. Organic Fluorine in the HB A large fraction of the information available in the literature on intramolecular HB pertains to the motifs of the type O···H-N and N···H-N [55–59]. It is well known that organic fluorine hardly ever accepts HB. Dunitz and co-workers have even published a paper entitled “organic fluorine hardly ever accepts hydrogen bonds” [60–63]. Nevertheless, a few reports are available on the participation of organic fluorine in the HB in the solution state [64,65]. In foldamers and benzanilides, NMR and X-ray crystallographic studies have also been reported on the N-H···F-C HB [66,67]. Recently DFT theoretical studies have been carried out on organofluorine systems and through space J couplings have been 1h also reported [68,69]. The first NMR spectroscopic observation of through space ( JFH) coupling revealed the information about the involvement of organic fluorine in the HB in the solution state [70]. The Limbach group have reported numerous examples where not only organic fluorine but other halogens also participate in the intermolecular HB [71–74]. Subsequently this field of research has seen phenomenal growth. Furthermore, the utility of organofluorine molecules as drugs, agrochemicals, biomaterials and also in molecular imaging is well documented [75,76]. Organic fluorine also has enormous importance in molecular association applications, such as crystal engineering [77–79] and in the design of the functional materials [80]. Consequent to the binding nature of fluorinated molecules with enzyme active sites, intermolecular hydrogen bonds of the type X-H···F-C (X = O, N) are highly significant, especially in bio-inorganic and medicinal chemistry [81–85]. Despite all its importance in various branches of chemistry and biology, there is limited exploration of organic fluorine-involved HBs, attributed mainly to the fact flourine hardly ever accepts intramolecular HB [86–90]. The present review summarizes the work carried out by authors’ laboratory in recent years, taken directly from their published work.

1.4. NMR Spectroscopic Techniques for the Detection of Hydrogen Bond NMR spectroscopy has proven as a powerful tool in deriving information about HB. Monitoring of the chemical shift as a function of variation in the physical parameters, such as, dilution, temperature is a general technique adopted for gathering information about the HB. The other important parameter that can provide the unambiguous evidence about the existence of HB is through space coupling between two NMR active nuclei mediated by HB. Many of these parameters can be measured by various one and two dimensional NMR experiments.

1.5. Theoretical Calculations The information derived by NMR spectroscopic analysis will conclusively evident if it is also corroborated by theoretical calculations. For such a purpose, various DFT-based theoretical calculations can be carried out. The commonly employed theoretical methods are brieﬂy introduced below.

1.5.1. Non-Covalent Interaction (NCI) Calculations For the detection of non-covalent interactions in real space, which is dependent on the electron density and its derivatives, NCI [91] calculations are performed. They provide a strong representation of the steric repulsion, van der Waals interactions and the HBs. The calculations yield a large positive gradient of the reduced density gradient (RDG), and the RDG values will be small and approach to zero in the density tail (i.e., regions far from the molecule, where the electron density exponentially decays to zero). This is the condition for both the covalent and non-covalent bonding regions respectively. There will be a strong correlation of electron density (ρ(r)) with the weak interactions in the corresponding regions. The correlations for the HB are negative and positive for the steric effect. On the other hand, Molecules 2017, 22, 423 5 of 44

for van der Waals interaction the ρ(r) values are always small [91] (near to zero). The calculated grid points are plotted for a deﬁned real space function, sign(λ2(r))ρ(r), as function 1 and reduced density gradient (RDG) as function 2.

1.5.2. Atoms in Molecules (AIM) Calculations For in depth understanding of the molecular properties influenced by weak HBs the interaction energy must be known. The topology analysis technique has been reported and is cited as “atoms in molecules” (AIM) theory. It is also cited as “the quantum theory of atoms in molecules” (QTAIM) [92–96] which is dependent on the quantum observables (electron density ρ(r) and the energy densities). In topology analysis, the critical points (CPs) are those points where gradient norm of function value is zero (except at infinity). According to the negative eigenvalues of the Hessian matrix of the real space function [92–96] CPs can be of four types. Out of these the (3, −1) CP is called the bond critical point (BCP). The value of the real space function at the BCP has a great significance. For example, the bond strength and bond type respectively are related closely to the value of electron density (ρ(r)) 2 2 and the sign of Laplacian of electron density (∇ ρ(r)) at BCPs [92–96]. The sign of (∇ ρ(r)) at BCP is important in discriminating shared-shell (covalent bond (−ve)) and closed-shell (ionic, van der 2 Waals interaction, and HB (+ve)). AIM calculations yield the magnitudes of ρ(r) and signs of ∇ ρ(r) for BCP of HBs of interest. At corresponding (3, −1) critical points (rcp) the gradients of electron density (ρ(r)) gets vanished. The energy of HB (EHB) is directly related to potential energy density (V(r)) by straightforward relationship EHB = V(rbcp)/2 [97]. The EHB of X···HX type HB can be calculated.

1.5.3. Natural Bond Orbitals (NBO) Per-Olov Löwdin introduced the concept of natural orbitals in 1955, to describe the unique set of orthonormal 1-electron functions which are intrinsic to the N-electron wavefunction. NBO is calculated where the electron density could be the maximum. The general sequence of localized orbital is: (1) natural atomic orbitals (NAO); (2) natural hybrid orbitals (NHO); (3) natural bonding orbitals (NBO) and (4) natural (semi-) localized molecular orbitals (NLMO). These localized sets are considered as intermediate between basic atomic orbitals (AO) and molecular orbitals (MO). For the calculation of the electron density distribution in individual atoms and in bonds formed between atoms, NBOs are frequently utilized in computational chemistry. NBOs provide the percentage of highest possible electron density and most possibility for “natural Lewis structure” of ψ. During the formation of HB, the electron transfer from the HB acceptor (lone pairs (lp)) to the anti-bonding orbital (σ*) of the H atom takes place. NBO analysis is proved to be a powerful technique to obtain the detailed information of lp transfer.

1.5.4. Molecular Dynamics (MD) The idea of molecular dynamics (MD) was first proposed in the 1950s [98]. MD is a computational method for studying the motion of atoms, groups, or molecules. MD studies give dynamic information about the atoms or group of atoms or molecules that interact for a period of time. During MD simulations, mostly the trajectories of atoms/molecules are stabilized where forces and potential energies among the particles are calculated by molecular mechanics force fields or interatomic potentials. MD is also called statistical mechanics by numbers and Laplace’s vision of Newtonian mechanics of predicting the dynamics by animating nature's forces and revealing the information of molecular motion at an atomic scale. MD simulations are also found be very powerful in the detection and quantification of the percentage of different conformers of a molecule where there exists a freely rotating group. Molecules 2017, 22, 423 6 of 44

2. Studies on Benzanilides Isomers of fluorinated benzanilides belong to the class of molecules where the cooperative interplay of weak interactions is highly significant in building their higher analogues, called driven foldamers, supra-molecular clusters and bulk moieties such as dendrimers [87,99,100]. Hence these molecules have drawn great attention in structural chemistry. Furthermore, the C-F bond is longer, has opposite bond polarity, stronger than C-H bond and makes the molecule more resistant towards metabolic degradation. As a consequence, the families of these molecules have important biological applications as voltage dependent potassium channel openers [101–104]. Especially while modeling the higher analogs of benzanilides, three centered N-H···F HBs provide a new approach for the formation of structural features via strong binding effects and induced chirality upon complexation with chiral L-tyrosine-derived ammonium ions [87]. Hence extensive studies have been carried out on the intramolecular HB interactions in the fluorine-substituted derivatives of benzanilides [105].

2.1. NMR SpectroscopicMolecules 2017, 22, 423 Experimental Findings 6 of 41

NMR2.1. spectroscopy NMR Spectroscopic can beExperimental used to distinguishFindings between labile free and hydrogen bonded protons, possessing differentNMR spectroscopy exchange can rates, be used even to distinguish in complex between molecules labile free and if their hydrogen exchange bonded protons, rates are within the NMR timepossessing scale different [106–111 exchange]. Hence rates, information even in complex about molecules hydrogen if their exchange bonding rates can are be within derived the from the NMR spectralNMR parameterstime scale [105–110]. [71,73 Hence,112– information115]. Investigations about hydrogen have bonding thus can been be derived carried from out the on NMR a set of four derivativesspectral of ﬂuorinated parameters benzanilides,[70,72,111–114]. Investigations whose chemical have thus structures been carried are reportedout on a set in Figureof four 3, where derivatives of fluorinated benzanilides, whose chemical /structures are reported in Figure 3, where there there the ﬂuorinethe fluorine substitution substitution at at positions positions X and/or X and/or X/ has been X hassystematically been systematically altered. altered.

Figure 3. Chemical structures with numbering of interacting spins of fluorine-substituted benzanilides. Figure 3. Chemical structures with numbering of interacting spins of ﬂuorine-substituted benzanilides. The 400 MHz 1H-NMR spectra of molecules 1, 2, 3 and 4 respectively (top trace to bottom trace) in the 1 The 400 MHzsolventH-NMR CDCl3. The spectra peak assignments of molecules have1 ,been2, 3 madeand 4basedrespectively on multiple (top quantum trace (MQ)-single to bottom trace) in the solventquantum CDCl3 .(SQ) The correlation peak assignments experiments. have The expans beenions made for basedamide onprotons multiple in each quantum molecule (MQ)-singleare quantum (SQ)depicted correlation by arrows experiments. (reproduced from The [103]). expansions for amide protons in each molecule are depicted by arrows (reproduced from [104]). Unambiguous assignment of aromatic 1H resonances have also been made employing the higher quantum correlation experiments for filtering of spectrum corresponding to different spin systems [115–117]. The systematic study by employing diverse one and two dimensional NMR experiments revealed information about the weak interactions in all the investigated fluorine-substituted derivatives of benzanilides at ambient conditions.

2.1.1. Detection of Spin-Spin Couplings Mediated by Hydrogen Bonds NH proton signals are either singlets (in molecules 1, 2) or a partially resolved doublet (in molecules 3 and 4). The peak separation of the partially resolved doublet is about 16 Hz. These partially resolved doublets might be due to the proton exchange between two distinguishable chemical environments

Molecules 2017, 22, 423 7 of 44

Unambiguous assignment of aromatic 1H resonances have also been made employing the higher quantum correlation experiments for ﬁltering of spectrum corresponding to different spin systems [116–118]. The systematic study by employing diverse one and two dimensional NMR experiments revealed information about the weak interactions in all the investigated ﬂuorine-substituted derivatives of benzanilides at ambient conditions.

2.1.1. Detection of Spin-Spin Couplings Mediated by Hydrogen Bonds NH proton signals are either singlets (in molecules 1, 2) or a partially resolved doublet

(inMolecules molecules 2017, 22, 3423and 4). The peak separation of the partially resolved doublet is about 167 of Hz. 41 These partially resolved doublets might be due to the proton exchange between two distinguishable chemicalor due to environmentsthe existence of or long due torange the existencescalar couplin of longgs. rangeThe particular scalar couplings. type of interaction The particular responsible type of interactionfor this can be responsible identified forby either this can two-dimensional be identiﬁed byexchange either two-dimensionalspectroscopy (EXSY), exchange or 1H-{19 spectroscopyF} or 19F-{1H} (EXSY),experiments. or 1H-{ The19 collapseF} or 19F-{ of 1theH} NH experiments. doublets in The both collapse 1H-{19F} ofor the19F-{ NH1H} spectra doublets in the in bothmolecules1H-{19 3F} and or 194, F-{is conclusively1H} spectra in evident the molecules from Figure3 and 4. 4This, is conclusively unambiguously evident established from Figure that the4. This doublet unambiguously detected for establishedNH proton is that due the to the doublet coupling detected between for NH proton proton and is due 19F. toSuch the a coupling large coupling between value NH of protonNH proton and 19 5 4 5 is F.very Such unlikely a large for coupling 5JF(5)H(11) value, 4JF(6)H(11) of NH, 5JH(11)H(1) proton and is very 4JH(11)H(10) unlikely [115–117] for J F(5)H(11)and has, JthusF(6)H(11) been, JattributedH(11)H(1) and to 4 19 theJH(11)H(10) coupling[116 between–118] and NH has proton thus beenand 19 attributedF atom mediated to the coupling through between hydrogen NH bond proton bridges and betweenF atom mediated19F and 1H. through hydrogen bond bridges between 19F and 1H.

Figure 4. The 400 MHz19 19F,19 19F-{11H} and 11H-{1919F} NMR spectra of molecules 2, 3 and 4 in CDCl3. Figure 4. The 400 MHz F, F-{ H} and H-{ F} NMR spectra of molecules 2, 3 and 4 in CDCl3.

2.1.2. Simultaneous Detection of Two throughthrough SpaceSpace CouplingsCouplings / In the molecules where XX andand XX/ areare fluorine, ﬂuorine, the the coupling coupling of of NH proton with both the fluorine ﬂuorine 1 atoms, even even if if it it exists, exists, could could not not be bedetected detected in the in theone onedimensional dimensional H spectrum.1H spectrum. The excessively The excessively broad 14 broadNH peak NH due peak to due quadrupolar to quadrupolar N nucleus14N nucleus prevented prevented the visualizatio the visualizationn of small of smallcouplings, couplings, if any. if The any. 14 TheN decoupling14N decoupling achieved achieved by systematic by systematic variation variation of the of power the power and andfrequency frequency of second of second irradiating irradiating RF RFover over a wide a wide range range circumvented circumvented this thisproblem problem of quadrupolar of quadrupolar broadening broadening and andresulted resulted in sharp in sharp NH NHsignals signals as reported as reported in Figure in Figure 5A. 5A. Nearly eight-fold reduction in line width of NH peak in 4 (reduced from 16 Hz to 2 Hz), resulted in a distinct doublet of doublet (dd) with measurable couplings of 17.7 Hz (1hJN-H…F(X/)) and 3.7 Hz. Similarly, a doublet for molecules 3, (16 Hz), and a doublet (3.6 Hz pertaining to 1hJN-H…F(X/)) for the NH proton of molecule 2 were detected at ambient temperature. Another interesting feature observed was the significant narrowing of NH signal on lowering the temperature (Figure 5B, molecule 4). This is because of self-decoupling of NH proton with 14N, at very low temperatures. However, this fact is not clearly obvious from the 1H-{14N} spectrum reported in Figure 5B as the decoupling at very low temperatures has very little effect on the line width due to the strengthening of the bifurcated HB at this temperature. Due to the slightly perturbed structural conformation, X/(F) is not in close proximity with the amide proton. Thus in this case the hydrogen bond could not be detected by NMR as predicted by single crystal X-ray diffraction studies.

Molecules 2017, 22, 423 8 of 44 Molecules 2017, 22, 423 8 of 41

Molecules 2017, 22, 423 8 of 41

1 1 14 FigureFigure 5. 5.( A(A)) The The1 H (left side) 1H-{H-{14N}N} (right (right side) side) NMR NMR spectrum spectrum of NH of NH proton proton for the for molecules the molecules 2, 3 and2, 1 1 14 3 and4 respectively4 respectively at 298 at K. 298 (B) K.The (B H) The and1 HH-{ andN}1 H-{NMR14N} spectrum NMR spectrumof NH proton of NH for protonmolecule for 4 moleculeat 230 K. 4 at 230 K. 2.1.3. Temperature and Solvent Induced Perturbations

NearlyThe investigated eight-fold reduction molecules in were line widthsubjected of NH to temperature peak in 4 (reduced variation from over 16 the Hz range to 2 Hz), of 320–220 resulted K 1h … 1h / inin a the distinct CDCl doublet3 solvent. of doubletThe chemical (dd) with shift measurable of NH protons couplings (δNH) ofand 17.7 JNH Hz (F wereJN-H ···monitoredF(X )) and and 3.7 Hz.their 1h / Similarly,dependency a doublet on the for temperature molecules 3 is, (16plotted Hz), in and Fi agure doublet 6A for (3.6 all Hz the pertaining investigated to molecules.JN-H···F(X )) for the NH proton of molecule 2 were detected at ambient temperature. Another interesting feature observed was the signiﬁcant narrowing of NH signal on lowering the temperature (Figure5B, molecule 4). This is because of self-decoupling of NH proton with 14N, at very low temperatures. However, this fact is not clearly obvious from the 1H-{14N} spectrum reported in Figure5B as the decoupling at very low temperatures has very little effect on the line width due to the strengthening of the bifurcated HB at this temperature. Due to the slightly perturbed structural conformation, X/(F) is not in close proximityFigure with 5. (A the) The amide 1H (left proton. side) 1H-{ Thus14N} (right in this side) case NMR the spectrum hydrogen of NH bond proton could for not the bemolecules detected 2, 3 by and NMR as predicted4 respectively by single at 298 crystal K. (B) The X-ray 1H and diffraction 1H-{14N} NMR studies. spectrum of NH proton for molecule 4 at 230 K.

2.1.3.2.1.3. TemperatureTemperature andand SolventSolvent InducedInduced PerturbationsPerturbations TheThe investigatedinvestigated moleculesmolecules werewere subjectedsubjected toto temperaturetemperature variationvariation overover thethe rangerange ofof 320–220320–220 KK in the CDCl solvent. The chemical shift of NH protons (δ ) and 1h1hJ ···… were monitored and their in the FigureCDCl3 36. solvent. (A) Variation The chemicalof chemical shift shift of of NHNH protonsprotons (δ NH(NHδNH) with) and temperature NHJNH FF were for allmonitored the investigated and their dependencydependencymolecules. onon theThethe temperaturetemperaturesquares, circles, isis plottedupperplotted triangles inin FigureFigure and6 A6Ainverted for for all all triangles the the investigated investigated correspond molecules. molecules. to molecules 1, 2, 3

and 4 respectively; (B) The variation of 1hJN-H...F(5), with temperature for molecules 3 and 4. Squares pertain to molecules 3 and triangles correspond to molecule 4.

HB gets strengthened on lowering the temperature and hence results in deshielding in the 1H-NMR peaks as reported in Figure 6A. Another interesting observation is the change in 1hJN-H…F(X). It varied from 17.03 (220 K) to 15.72 (320 K) Hz and 17.71 Hz (220 K) to 15.94 Hz (320 K) respectively for molecules 3 and 4, as reported in Figure 6B, which provides clear and straightforward evidence in the favor of HB.

the B3LYP/6-311+G**Figure 6. (A) Variation basis of set chemical [118]. Self-Consistentshift of NH protons Isodensity (δNH) with Polarized temperature Continuum for all the Model investigated (SCI-PCM) Figure 6. (A) Variation of chemical shift of NH protons (δNH) with temperature for all the investigated has been utilized to create the uniform CHCl3 solvent environment [119]. Optimized molecular molecules.molecules. TheThe squares,squares, circles,circles, upperupper trianglestriangles andand invertedinverted trianglestriangles correspondcorrespond toto moleculesmolecules1 1,,2 2,,3 3 geometries along with HBs are reported 1hin Figure 7. andand 44respectively; respectively; ( B(B)) The The variation variation of of1h J JN-H...F(5), ,with with temperature temperature for for molecules 3 andand 44.. SquaresSquares The theoretically observed HB distancesN-H...F(5) involving N-H…F(X/) are in close agreement with the pertainpertain toto moleculesmolecules3 3and andtriangles trianglescorrespond correspond to to molecule molecule4 4. . X-ray structures [104]. HB gets strengthened on lowering the temperature and hence results in deshielding in the 1H-NMR peaks as reported in Figure 6A. Another interesting observation is the change in 1hJN-H…F(X). It varied from 17.03 (220 K) to 15.72 (320 K) Hz and 17.71 Hz (220 K) to 15.94 Hz (320 K) respectively for molecules 3 and 4, as reported in Figure 6B, which provides clear and straightforward evidence in the favor of HB.

2.2. Theoretical Calculations For the fluorinated oligomers ground state geometry optimizations, have been carried out. The geometry optimization was performed to enumerate the intramolecular N-H…F distances by employing the B3LYP/6-311+G** basis set [118]. Self-Consistent Isodensity Polarized Continuum Model (SCI-PCM) has been utilized to create the uniform CHCl3 solvent environment [119]. Optimized molecular geometries along with HBs are reported in Figure 7. The theoretically observed HB distances involving N-H…F(X/) are in close agreement with the X-ray structures [104].

Molecules 2017, 22, 423 9 of 44

HB gets strengthened on lowering the temperature and hence results in deshielding in the 1 1h H-NMR peaks as reported in Figure6A. Another interesting observation is the change in JN-H . . . F(X). It varied from 17.03 (220 K) to 15.72 (320 K) Hz and 17.71 Hz (220 K) to 15.94 Hz (320 K) respectively for molecules 3 and 4, as reported in Figure6B, which provides clear and straightforward evidence in the favor of HB.

2.2. Theoretical Calculations For the ﬂuorinated oligomers ground state geometry optimizations, have been carried out. The geometry optimization was performed to enumerate the intramolecular N-H···F distances by employing the B3LYP/6-311+G** basis set [119]. Self-Consistent Isodensity Polarized Continuum Model (SCI-PCM) has been utilized to create the uniform CHCl3 solvent environment [120]. Optimized Moleculesmolecular 2017 geometries, 22, 423 along with HBs are reported in Figure7. 9 of 41

Molecules 2017, 22, 423 9 of 41

Figure 7. OptimizedOptimized structures structures of fluorinated fluorinated benzanilides visualized using Molden-4.7: HB parameters were measured measured by by their their optimization optimization (Molden (Molden Control) Control) in the vicinity in the vicinityof F…H-N of HB F··· distancesH-N HB and distances angles. and angles. 3. CF3 Substituted Benzanilides The theoretically observed HB distances involving N-H···F(X/) are in close agreement with the Extending the study another series of CF3 substituted benzanilides have been investigated [120]. The X-raychemical structures structures [105 of]. the investigated molecules are reported in Figure 8. In this series of molecules, the unambiguousFigure evidence 7. Optimized for structures the engagement of fluorinated of benzanilides CF3 group visualized in N-H···F-C using Molden-4.7: type HB, HB in parametersthe different CF3 3. CF3 Substituted Benzanilides … substitutedwere derivatives measured by has their been optimization observed (Molden in Control)a low inpolarity the vicinity solvent of F H-N CDCl HB distances3. It is andwell angles. known that C(SpExtending3)-F fluorine the is studya better another acceptor series than of C(Sp CF32substituted)-F and C(Sp)-F benzanilides fluorine [121]. have beenTo understand investigated the [ 121role]. 3. CF3 Substituted Benzanilides Theof CF chemical3 group in structures HB and in of structural the investigated chemistry molecules an extensive are utility reported of NMR in Figure experimental8. In this techniques, series of includingmolecules, variableExtending the unambiguous temperature the study another and evidence dilution series of for studies,CF the3 substituted engagement has been benzanilides made. of CFThe have3 firstgroup been example investigated in N-H of ···theF-C [120]. engagement type The HB, chemical structures of the investigated molecules are reported in Figure 8. In this series of molecules, the ofin the differentCF3 group CF in3 substitutedthe intramolecular derivatives HB hasof the been type observed C-F···H-N in ahas low been polarity reported solvent [121–127]. CDCl3. The It is unambiguous evidence for the engagement of CF3 group in N-H···F-C type HB, in the different CF3 wellNMR known findings that have C(Sp been3)-F addition fluorineally is substantiated a better acceptor by molecular than C(Sp dynamics2)-F and (MD) C(Sp)-F simulations. fluorine [122]. substituted derivatives has been observed in a low polarity solvent CDCl3. It is well known that To understand the role of CF group in HB and in structural chemistry an extensive utility of C(Sp3)-F fluorine is a better acceptor3 than C(Sp2)-F and C(Sp)-F fluorine [121]. To understand the role NMR experimental techniques, including variable temperature and dilution studies, has been made. of CF3 group in HB and in structural chemistry an extensive utility of NMR experimental techniques, The firstincluding example variable of the temperature engagement and ofdilution the CF studies,3 group has in been the made. intramolecular The first example HB of of the the typeengagement C-F···H-N has beenof the reported CF3 group [122 in– 128the ].intramolecular The NMR findings HB of the have type been C-F···H-N additionally has been substantiated reported [121–127]. by molecular The dynamicsNMR (MD) findings simulations. have been additionally substantiated by molecular dynamics (MD) simulations.

Figure 8. Chemical structure of benzanilide (1) and its trifluoromethyl derivatives 5–8.

3.1. NMR Experimental Findings

In the 1H-NMR spectrum the NH proton of molecule 5 displayed a doublet with a separation of 16.7 Hz [120].Figure ThisFigure 8. isChemical attributed 8. Chemical structure tostructure 1hJFH of (coupling of benzanilide benzanilide mediated ( 1(1)) andand itsits through trifluoromethyl triﬂuoromethyl HB) between derivatives derivatives NH 5–8 5proton. –8. and the fluorine of ring a. Collapsing of this doublet to a singlet in the 1H{19F} experiment unambiguously 3.1. NMR Experimental Findings established that it is mediated through HB, confirming the involvement of F in HB. Analogous to benzanilidesIn thediscussed 1H-NMR in spectrum the previous the NH section, proton evenof molecule in this 5 molecule displayed the a doublet excessive with broadening a separation ofof NH 1h signal 16.7was Hz observed, [120]. This due is attributed to quadrupole to JFH (couplingrelaxation mediated by 14N, throughmay prevent HB) between the precise NH proton measurement and the of 1 19 small fluorinecouplings, of ring if any, a. Collapsing that may ofbe this hidden doublet within to a thesinglet line in width. the H{ InF} circumventing experiment unambiguously such problems, established that it is mediated through HB, confirming the involvement of F in HB. Analogous to a 2D 15N-1H HSQC experiment has been carried out where 15N is present in its natural abundance. benzanilides discussed in the previous section, even in this molecule the excessive broadening of NH The correspondingsignal was observed, spectrum due tois quadrupolereported in relaxation Figure 9. by 14N, may prevent the precise measurement of 1h 2h 15 1 Thesmall two couplings, types of if any,couplings that may mediated be hidden through within theHB, line Jwidth.FH and In circumventingJFN are reflected such in problems, the N- H- HSQCa spectrum. 2D 15N-1H TheHSQC signs experiment of couplings has been decided carried on out the where basis of15N the is presentrelative in slopes its natural of the abundance. displacement vectorsThe assuming corresponding 1hJFH to spectrum be negative is reported [104]. in Figure 9. The two types of couplings mediated through HB, 1hJFH and 2hJFN are reflected in the 15N-1H- HSQC spectrum. The signs of couplings decided on the basis of the relative slopes of the displacement vectors assuming 1hJFH to be negative [104].

Molecules 2017, 22, 423 10 of 44

3.1. NMR Experimental Findings In the 1H-NMR spectrum the NH proton of molecule 5 displayed a doublet with a separation of 1h 16.7 Hz [121]. This is attributed to JFH (coupling mediated through HB) between NH proton and the ﬂuorine of ring a. Collapsing of this doublet to a singlet in the 1H{19F} experiment unambiguously established that it is mediated through HB, conﬁrming the involvement of F in HB. Analogous to benzanilides discussed in the previous section, even in this molecule the excessive broadening of NH signal was observed, due to quadrupole relaxation by 14N, may prevent the precise measurement of small couplings, if any, that may be hidden within the line width. In circumventing such problems, a 2D 15N-1H HSQC experiment has been carried out where 15N is present in its natural abundance. TheMolecules corresponding 2017, 22, 423 spectrum is reported in Figure9. 10 of 41

15 1 1 FigureFigure 9. 400 MHz two-dimensional two-dimensional 15N-N-H-HSQCH-HSQC spectrum spectrum of ofmolecule molecule 5 in5 thein the CDCl CDCl3, depicting3, depicting the thethrough-space through-space couplings. couplings. The The measured measured co couplingupling strengths strengths are are identified identiﬁed by by a −dd and and their values havehave alsoalso beenbeen reported.reported.

3.2. Theoretical Calculations 1h 2h The two types of couplings mediated through HB, JFH and JFN are reflected in the 15 1 N- ToH-HSQC ascertain spectrum. the presence The signsof weak of couplingsmolecular decidedinteractions on the established basis of theby relativeNMR experiments slopes of the in 1h displacementthese molecules, vectors the DFT assuming based theoreticalJFH to be negativecalculatio [105ns ].have been carried out. Gaussian09 has been used at B3LYP/6-311+g (d,p) level of theory [118] for full-geometry optimizations of the molecules 5–8. 3.2. Theoretical Calculations With the presence of three fluorine atoms in the CF3 group there are various possible ways of interactions,To ascertain suchthe as, presencetwo centered, of weak three-centered, molecular interactions or four-centered established HBs. To by derive NMR the experiments quantitative in theseinformation molecules, about the HBs, DFT quantum based theoretical chemical calculationscalculations haveand MD been simulations carried out. have Gaussian09 been carried has been out. usedFor calculation, at B3LYP/6-311+g the geometry (d,p) levelmeasurement of theory criteria [119] for have full-geometry been used optimizationsin which HB is of represented the molecules as 5N-H···F,–8. With where the presence N is the of donor three atom fluorine and atoms F is the in acceptor the CF3 groupatom. thereIf the aredistance, various r between possible N ways and of F interactions,atom is less than such 3.5 as, Å two and centered, the angle three-centered, ∠NHF (θ) is greater or four-centered than 120°, then HBs. an To HB derive exists the between quantitative the N informationand F atoms. about The most HBs, probable quantum distance, chemical P(r), calculations and appropriate and MD angle, simulations P(θ), are have calculated been carried for all out.the Forfluorine calculation, atoms in the each geometry of these measurementfour molecules. criteria The percentage have been of used occurrence in which of HBvarious is represented types of HBs as N-Hhas been···F, wherecalculated N is from the donorthe MD atom simulations. and F is theA snap acceptorshot of atom. the formation If the distance, of HB and r between their percentage N and F ◦ atomof the is occurrence less than 3.5 for Å the and molecule the angle 6 is∠ NHFreported (θ) isin greater Figure than10. 120 , then an HB exists between the N and F atoms. The most probable distance, P(r), and appropriate angle, P(θ), are calculated for all the fluorine atoms in each of these four molecules. The percentage of occurrence of various types of HBs has been calculated from the MD simulations. A snapshot of the formation of HB and their percentage of the occurrence for the molecule 6 is reported in Figure 10.

Figure 10. (a,b) Snapshot of HB formation in molecule 6, along with their percentage of occurrence, obtained from MD simulations. Blue colored dashed lines show H-bonds; (c,d) Probability distributions of angles and distances, respectively.

The calculations reveal that the percentage of occurrence in a situation when there is no HB is 56.27%, whereas the occurrence of a two-centered HB is 42.60% (Figure 10a), and the occurrence of a

Molecules 2017, 22, 423 10 of 41

Figure 9. 400 MHz two-dimensional 15N-1H-HSQC spectrum of molecule 5 in the CDCl3, depicting the through-space couplings. The measured coupling strengths are identified by a−d and their values have also been reported.

3.2. Theoretical Calculations To ascertain the presence of weak molecular interactions established by NMR experiments in these molecules, the DFT based theoretical calculations have been carried out. Gaussian09 has been used at B3LYP/6-311+g (d,p) level of theory [118] for full-geometry optimizations of the molecules 5–8. With the presence of three fluorine atoms in the CF3 group there are various possible ways of interactions, such as, two centered, three-centered, or four-centered HBs. To derive the quantitative information about HBs, quantum chemical calculations and MD simulations have been carried out. For calculation, the geometry measurement criteria have been used in which HB is represented as N-H···F, where N is the donor atom and F is the acceptor atom. If the distance, r between N and F atom is less than 3.5 Å and the angle ∠NHF (θ) is greater than 120°, then an HB exists between the N and F atoms. The most probable distance, P(r), and appropriate angle, P(θ), are calculated for all the fluorine atoms in each of these four molecules. The percentage of occurrence of various types of HBs hasMolecules been2017 calculated, 22, 423 from the MD simulations. A snapshot of the formation of HB and their percentage11 of 44 of the occurrence for the molecule 6 is reported in Figure 10.

FigureFigure 10. 10. ((aa,,bb)) Snapshot Snapshot of of HB HB formation formation in in molecule molecule 66, ,along along with with their their perc percentageentage of of occurrence, occurrence, obtainedobtained from from MD MD simulations. simulations. Blue Blue colored colored dashed dashed lines lines show show H-bonds; H-bonds; ( (cc,,dd)) Probability Probability distributions distributions ofof angles angles and and distances, distances, respectively. respectively.

The calculations reveal that the percentage of occurrence in a situation when there is no HB is MoleculesThe 2017 calculations, 22, 423 reveal that the percentage of occurrence in a situation when there is no11 HB of 41 is 56.27%, whereas the occurrence of a two-centered HB is 42.60% (Figure 10a), and the occurrence of a 56.27%, whereas the occurrence of a two-centered HB is 42.60% (Figure 10a), and the occurrence of abifurcated bifurcated HB HB is ismuch much less less and and is is 1.13% 1.13% (Figure (Figure 10b).10b). Figure Figure 10c,d10c,d contain the HB geometricalgeometrical parameters obtained obtained by by MD MD simulations. simulations. It has It has been been observed observed that thatall the all three the three F atoms F atoms in the inCF the3 group CF3 grouphave equal have probability equal probability of forming of forming HBs, as HBs, P(r) asand P(r) P( andθ) are P( θsame) are for same three for F three atoms F atoms (Figure (Figure 10c,d). 10 c,d). For molecule 5, where a single fluorinefluorine isis substitutedsubstituted onon ringring “a”“a” andand thethe CFCF33 group on the ring “b”,“b”, therethere areare fivefive differentdifferent possibilitiespossibilities ofof fluorinefluorine gettinggetting involved in the HB formation and also a possibility when there is no HB. All such possibilities with their percentage of occurrence determined by MD simulations are reported in Figure 1111..

Figure 11. Various possibilities of HB formation for molecule 5, with their % of occurrence obtained Figure 11. Various possibilities of HB formation for molecule 5, with their % of occurrence obtained from the MD simulations (HB is shown by blue line). from the MD simulations (HB is shown by blue line).

A very interesting result has been derived from the 19F{1H} NMR spectrum of this molecule, A very interesting result has been derived from the 19F{1H} NMR spectrum of this molecule, which exhibited doublet and quartet patterns for CF3 and CF groups, respectively. The corresponding which exhibited doublet and quartet patterns for CF3 and CF groups, respectively. The corresponding spectrum is reported in Figure 12. Fluorine atoms of CF and CF3 groups in molecule 5 are separated by eight covalent bonds and the observed doublet peak with separation of 5.7 Hz is quite large. Thus the scalar couplings between the fluorine atoms, mediated through covalent bonds is quite unlikely. It can be safely attributed to the interaction between CF and CF3 groups mediated through HB of the type F…H…F (2hJFF).

Figure 12. 376.7 MHz 19F{1H} NMR spectrum of molecule 5 showing 2hJFF through-space coupling.

The 2D-19F-19F COSY and J-resolved experiments also reflected the cross peaks pertaining to 2hJFF [120]. The strengths of HB for each fluorine atom present in all the molecules have also been calculated using atomistic molecular dynamics. The histogram of the HB formation H(r,θ) i.e., as a function of the distance, r between donor and acceptor atoms and θ the angle between donor-hydrogen- acceptor atoms, reported in Figure 13, have been calculated at 300 K for molecule 7, from the 50 ns molecular dynamics simulation trajectory.

Molecules 2017, 22, 423 11 of 41 bifurcated HB is much less and is 1.13% (Figure 10b). Figure 10c,d contain the HB geometrical parameters obtained by MD simulations. It has been observed that all the three F atoms in the CF3 group have equal probability of forming HBs, as P(r) and P(θ) are same for three F atoms (Figure 10c,d). For molecule 5, where a single fluorine is substituted on ring “a” and the CF3 group on the ring “b”, there are five different possibilities of fluorine getting involved in the HB formation and also a possibility when there is no HB. All such possibilities with their percentage of occurrence determined by MD simulations are reported in Figure 11.

Figure 11. Various possibilities of HB formation for molecule 5, with their % of occurrence obtained from the MD simulations (HB is shown by blue line).

Molecules 2017, 22, 423 12 of 44 A very interesting result has been derived from the 19F{1H} NMR spectrum of this molecule, which exhibited doublet and quartet patterns for CF3 and CF groups, respectively. The corresponding spectrumspectrum isis reportedreported inin FigureFigure 1212.. FluorineFluorine atomsatoms ofof CFCF andand CFCF33 groupsgroups inin moleculemolecule 55 areare separatedseparated byby eight covalent covalent bonds bonds and and the the observed observed doublet doublet peak peak with with separation separation of 5.7 of Hz 5.7 is Hz quite is quitelarge. large.Thus Thusthe scalar the scalarcouplings couplings between between the fluorine the ﬂuorine atoms, atoms,mediated mediated through through covalent covalent bonds is bondsquite unlikely. is quite unlikely.It can be Itsafely can beattributed safely attributed to the interaction to the interaction between CF between and CF CF3 groups and CF mediated3 groups mediatedthrough HB through of the … … 2h 2h HBtype of F theH typeF ( FJFF···).H ···F( JFF).

Figure 12. 376.7 MHz 1919F{1H}1 NMR spectrum of molecule 5 showing 2hJFF2h through-space coupling. Figure 12. 376.7 MHz F{ H} NMR spectrum of molecule 5 showing JFF through-space coupling.

The 2D-19F-19F COSY and J-resolved experiments also reflected the cross peaks pertaining to The 2D-19F-19F COSY and J-resolved experiments also reﬂected the cross peaks pertaining 2hJFF [120]. The strengths of HB for each fluorine atom present in all the molecules have also been to 2hJ [121]. The strengths of HB for each ﬂuorine atom present in all the molecules have also calculatedFF using atomistic molecular dynamics. The histogram of the HB formation H(r,θ) i.e., as a been calculated using atomistic molecular dynamics. The histogram of the HB formation H(r,θ) function of the distance, r between donor and acceptor atoms and θ the angle between donor-hydrogen- i.e., as a function of the distance, r between donor and acceptor atoms and θ the angle between acceptor atoms, reported in Figure 13, have been calculated at 300 K for molecule 7, from the 50 ns donor-hydrogen-acceptor atoms, reported in Figure 13, have been calculated at 300 K for molecule 7, Moleculesmolecular 2017 dynamics, 22, 423 simulation trajectory. 12 of 41 from the 50 ns molecular dynamics simulation trajectory.

FigureFigure 13.13. F(r,θθ)) forfor N-HN-H···F1-C···F1-C H-bond occurring in moleculemolecule 77..

TheThe freefree energyenergy landscapelandscape ofof eacheach atomatom involvedinvolved inin thethe HBHB calculationcalculation cancan bebe performedperformed fromfrom thisthis histogramhistogram usingusing formulaeformulae givengiven inin thethe EquationEquation (1):(1):

F(r,θ) = −kBT ln(2πr2sinθH(r, θ)) (1) F(r,θ) = −kBT ln(2πr sinθH(r, θ)) (1) For any fluorine atom participating in the HB with N atom, F(r,θ) has two minima corresponding to twoFor states, any ﬂuorine one with atom HB participatingand other with in theno HBHB. withIn the N free atom, energy F(r,θ) landscape has two minima for molecule corresponding 7 given toin twoFigure states, 13, onefor N-H···F with HB1-C and the other minima with on no the HB. left-hand In the free side energy is for landscapeHB (where for r moleculeand θ satisfy7 given the inconditions Figure 13 for, for HB) N-H and··· theF1-C other the minima belongs on the left-hand to a situation side where is for HBthere (where is no HB. r and Theθ strengthsatisfy the of conditionsHB has also for been HB) calculated and the other for the minima molecules belongs 2–5 to [120] a situation using Equation where there (2): is no HB. The strength of HB has also been calculated for the molecules 2–5 [121] using Equation (2): Ehb = Fhbmin (r, θ) − Fno−hbmax (r, θ) (2) hb no−hb hb E = F (r, θ) − F (r, θno)−hb (2) where F min (r, θ) is the minima hbof hydrogenmin bonded statemax and F max (r, θ) is the maxima for no hydrogen bonded state.

4. Studies on Hydrazides Hydrazides are organic compounds that share a common functional group with a N-N covalent bond where at least one of the four substituents should be an acyl group [128]. Different derivatives of hydrazides have proven to be extremely important as, reagents in organic synthesis [129], anti-tumor medicine [130,131], and also in the cytotoxic functioning [132]. In combination with other cmedicines, the derivatives of hydrazides are utilized for the treatment or prevention of tuberculosis [133].

4.1. N,N-Diacyl Substituted Derivatives of Hydrazides The hydrazides provide a possibility to synthesize a variety of derivatives with different combinations of substituted acyl group(s) of interest. The different derivatives of hydrazides have been synthesized and characterized using one and two dimensional multinuclear NMR experiments, and ESI mass spectrometry. The procedure for synthesis of these molecules and the NMR spectral analysis have been reported [134]. The NMR experiments reveal the existence of weak intramolecular interactions in all the investigated derivatives of hydrazides [134]. The NMR derived HB information has been further ascertained by DFT [135,136] based NCI [90], and QTAIM [91–95] calculations.

4.1.1. NMR Spectroscopic Detection One of the NMR spectral parameters that provides information on the HB is the variation of chemical shift under different experimental conditions. In the reported work [134] the different derivatives of hydrazide, 9–18, generically named 2-X-N-(2-X’)benzohydrazides, whose basic chemical structures and their site specific substituents, reported in Figure 14, have been investigated. The disubstituted molecules are classified into two categories, one where X = X’ (9–11 and 18) and the other where X ≠ X’ (12–17).

Molecules 2017, 22, 423 13 of 44

hb no−hb where F min (r, θ) is the minima of hydrogen bonded state and F max (r, θ) is the maxima for no hydrogen bonded state.

4. Studies on Hydrazides Hydrazides are organic compounds that share a common functional group with a N-N covalent bond where at least one of the four substituents should be an acyl group [129]. Different derivatives of hydrazides have proven to be extremely important as, reagents in organic synthesis [130], anti-tumor medicine [131,132], and also in the cytotoxic functioning [133]. In combination with other cmedicines, the derivatives of hydrazides are utilized for the treatment or prevention of tuberculosis [134].

4.1. N,N-Diacyl Substituted Derivatives of Hydrazides The hydrazides provide a possibility to synthesize a variety of derivatives with different combinations of substituted acyl group(s) of interest. The different derivatives of hydrazides have been synthesized and characterized using one and two dimensional multinuclear NMR experiments, and ESI mass spectrometry. The procedure for synthesis of these molecules and the NMR spectral analysis have been reported [135]. The NMR experiments reveal the existence of weak intramolecular interactions in all the investigated derivatives of hydrazides [135]. The NMR derived HB information has been further ascertained by DFT [136,137] based NCI [91], and QTAIM [92–96] calculations.

4.1.1. NMR Spectroscopic Detection One of the NMR spectral parameters that provides information on the HB is the variation of chemical shift under different experimental conditions. In the reported work [135] the different derivatives of hydrazide, 9–18, generically named 2-X-N-(2-X’)benzohydrazides, whose basic chemical structures and their site speciﬁc substituents, reported in Figure 14, have been investigated. The disubstituted molecules are classiﬁed into two categories, one where X = X’ (9–11 and 18) and the Moleculesother where 2017, 22 X, 6=423X’ (12–17). 13 of 41

Figure 14. TheThe chemical structures of 2-X-N-(2-X’) benzohydrazide derivatives; ( (a)) symmetrically substitutedsubstituted molecules and ( b) asymmetrically substituted molecules.

For discarding any possibility of dimerization or self-aggregation, if any, 2D DOSY [137,138] For discarding any possibility of dimerization or self-aggregation, if any, 2D DOSY [138,139] experiments, ESI-MS analysis and dilution studies have been carried out. Solvent titration experiments, ESI-MS analysis and dilution studies have been carried out. Solvent titration experiment [63,64,139] has been employed to understand the weak interactions, such as, intra- and experiment [64,65,140] has been employed to understand the weak interactions, such as, intra- and intermolecular HB, to compare their relative strengths of interaction and also to evaluate the effect of intermolecular HB, to compare their relative strengths of interaction and also to evaluate the effect monomeric water on HB, which is absorbed from the atmosphere. The variation in the chemical shift of of monomeric water on HB, which is absorbed from the atmosphere. The variation in the chemical NH proton as a function of temperature (300–220 K) is compared for all the investigated molecules shift of NH proton as a function of temperature (300–220 K) is compared for all the investigated in Figure 15b. On lowering the temperature, the NH peak of all investigated molecules except one molecules in Figure 15b. On lowering the temperature, the NH peak of all investigated molecules NH proton peak of molecule 13 is showed downfield shift due to the strengthening of HB. The except one NH proton peak of molecule 13 is showed downﬁeld shift due to the strengthening of HB. unusual behaviour of this NH proton of the molecule 13 has been attributed to the switching of this The unusual behaviour of this NH proton of the molecule 13 has been attributed to the switching of molecule to another possible conformer on lowering the temperature. The possibility of such a this molecule to another possible conformer on lowering the temperature. The possibility of such a switching phenomenon is illustrated in Scheme 1. switching phenomenon is illustrated in Scheme1.

Scheme 1. Two different possible stable conformations of molecule 13, (a) at higher temperature; and (b) at lower temperature.

The relative strength of intramolecular HBs has been qualitatively derived by titration with dimethylsulphoxide (DMSO) solvent [134]. The observed variation in the chemical shifts as a function of the incremental addition of DMSO-d6 is plotted for the molecules 9–17, in Figure 15c. Disruption of intramolecular HB [104] by the solvent DMSO results in the deshielding of NH protons. On the contrary for the NH protons of molecule 11, and one of the two NH protons of asymmetric molecules, 15–17 the high field shift was detected on addition of DMSO-d6. This is due to the fact that these NH protons involved in HB with OMe group are relatively stronger than the DMSO interaction. The high field shift of these protons is attributed to an equilibrium which is stabilized between intra- and intermolecular hydrogen bonded species. Another strong evidence in the favour of organic fluorine involved intramolecular HB is the detection of through space couplings between the NH proton and fluorine [40,140–142]. The 19F coupled and decoupled 1H spectrum of molecule 9 in the solvent CDCl3, and the 19F coupled spectrum in the solvent DMSO-d6 are given in Figure 16. The NH peak of the molecule 9 is a doublet with a separation of 12.75 Hz (Figure 16a). This doublet collapsed into a singlet in the 1H{19F) experiment confirming the presence of the coupling between 1H and 19F (Figure 16c). The doublet also collapsed to a singlet in a high polarity solvent DMSO-d6 (Figure 16b).

Molecules 2017, 22, 423 13 of 41

Figure 14. The chemical structures of 2-X-N-(2-X’) benzohydrazide derivatives; (a) symmetrically substituted molecules and (b) asymmetrically substituted molecules.

For discarding any possibility of dimerization or self-aggregation, if any, 2D DOSY [137,138] experiments, ESI-MS analysis and dilution studies have been carried out. Solvent titration experiment [63,64,139] has been employed to understand the weak interactions, such as, intra- and intermolecular HB, to compare their relative strengths of interaction and also to evaluate the effect of monomeric water on HB, which is absorbed from the atmosphere. The variation in the chemical shift of NH proton as a function of temperature (300–220 K) is compared for all the investigated molecules in Figure 15b. On lowering the temperature, the NH peak of all investigated molecules except one NH proton peak of molecule 13 is showed downfield shift due to the strengthening of HB. The unusual behaviour of this NH proton of the molecule 13 has been attributed to the switching of this Moleculesmolecule2017 to, 22another, 423 possible conformer on lowering the temperature. The possibility of such14 of 44a switching phenomenon is illustrated in Scheme 1.

Scheme 1. Two different possible stable conformations of molecule 13,(, (a) at higher temperature; and (b) at lower temperature.

The relative strength of intramolecular HBs has been qualitatively derived by titration with The relative strength of intramolecular HBs has been qualitatively derived by titration with dimethylsulphoxide (DMSO) solvent [134]. The observed variation in the chemical shifts as a function dimethylsulphoxide (DMSO) solvent [135]. The observed variation in the chemical shifts as a function of the incremental addition of DMSO-d6 is plotted for the molecules 9–17, in Figure 15c. Disruption of the incremental addition of DMSO-d is plotted for the molecules 9–17, in Figure 15c. Disruption of intramolecular HB [104] by the solvent6 DMSO results in the deshielding of NH protons. On the of intramolecular HB [105] by the solvent DMSO results in the deshielding of NH protons. On the contrary for the NH protons of molecule 11, and one of the two NH protons of asymmetric molecules, contrary for the NH protons of molecule 11, and one of the two NH protons of asymmetric molecules, 15–17 the high field shift was detected on addition of DMSO-d6. This is due to the fact that these NH 15–17 the high field shift was detected on addition of DMSO-d . This is due to the fact that these protons involved in HB with OMe group are relatively stronger than6 the DMSO interaction. The high NH protons involved in HB with OMe group are relatively stronger than the DMSO interaction. field shift of these protons is attributed to an equilibrium which is stabilized between intra- and inter- The high field shift of these protons is attributed to an equilibrium which is stabilized between intramolecular hydrogen bonded species. and intermolecular hydrogen bonded species. Another strong evidence in the favour of organic fluorine involved intramolecular HB is the Another strong evidence in the favour of organic fluorine involved intramolecular HB is the detection of through space couplings between the NH proton and fluorine [40,140–142]. The 19F detection of through space couplings between the NH proton and fluorine [41,141–143]. The 19F 1 19 coupled and decoupled 1H spectrum of molecule 9 in the solvent CDCl3, and the 19F coupled spectrum coupled and decoupled H spectrum of molecule 9 in the solvent CDCl3, and the F coupled spectrum in the solvent DMSO-d6 are given in Figure 16. The NH peak of the molecule 9 is a doublet with a in the solvent DMSO-d are given in Figure 16. The NH peak of the molecule 9 is a doublet with a separation of 12.75 Hz 6(Figure 16a). This doublet collapsed into a singlet in the 1H{19F) experiment separation of 12.75 Hz (Figure 16a). This doublet collapsed into a singlet in the 1H{19F) experiment confirming the presence of the coupling between 1H and 19F (Figure 16c). The doublet also collapsed confirming the presence of the coupling between 1H and 19F (Figure 16c). The doublet also collapsed to a singlet in a high polarity solvent DMSO-d6 (Figure 16b). toMolecules a singlet 2017 in, 22 a, 423 high polarity solvent DMSO-d6 (Figure 16b). 14 of 41

Figure 15. (a, b) VariationVariation inin thethe chemicalchemical shiftsshifts ofof NHNH protonproton as aa functionfunction of temperaturetemperature and the volume of DMSO-DMSO-d66 respectively for the molecules 9–17. The initial concentration was 10 mM in thethe

solvent CDCl3..( (aa)) The The DMSO- DMSO-dd6 6waswas incrementally incrementally added added to to an an initial initial volume volume of of450 450 µLµ inL inCDCl CDCl3, at3, at298 298 K; K;(b ()b The) The temperature temperature was was varied varied from from 300 300 to to 220 220 K. K. The The molecules molecules 99––1717 areare identified identiﬁed by the symbols givengiven inin thethe inset.inset.

Figure 16. 400 MHz 1H-NMR spectrum of molecule 9, (a) in CDCl3; (b) 1H-NMR spectrum in DMSO-d6

and (c) 1H{19F} NMR spectrum in CDCl3.

The 3JHH and 4hJFH couplings, if any, are not detectable due to the symmetry of the molecule. For detection of these couplings the symmetry of the molecule has to be broken. For such a purpose 2D 1H-15N HSQC experiment has been carried out for the molecule 9, where 15N is present in its natural abundance. The HSQC spectrum is reported Figure 17A where all the possible seven couplings, 1JNH, 2hJFN, 2JNH, 3hJFN, 1hJFH, 4hJFH and 3JHH has been measured from this spectrum. The observation of through space couplings of significant strengths, such as 1hJFH, 2hJFN 3hJFN and 4hJFH, provided strong and direct evidence for the involvement of organic fluorine in the intramolecular HB. In the solvent DMSO, except for 1JNH, all the other couplings mediated through HB disappeared as reported in Figure 17B. The relative signs of all the couplings were determined with respect to coupling c marked in Figure 17B. From this it was inferred that 1hJFH is negative. The comparison of 1JNH [143,144] of molecules 9–17 with unsubstituted molecule 18, provided ample evidence that the nature of HBs in derivatives of hydrazides are predominantly covalent in nature. The close proximity of NH proton with the fluorine in fluorinated molecules is detected by 2D HOESY (hetero-nuclear Overhauser effect spectroscopy) experiment. This also provided evidence in favor of intramolecular HB.

Molecules 2017, 22, 423 14 of 41

Figure 15. (a, b) Variation in the chemical shifts of NH proton as a function of temperature and the volume of DMSO-d6 respectively for the molecules 9–17. The initial concentration was 10 mM in the

solvent CDCl3. (a) The DMSO-d6 was incrementally added to an initial volume of 450 µL in CDCl3, at Molecules2982017 K; (,b22) ,The 423 temperature was varied from 300 to 220 K. The molecules 9–17 are identified by the15 of 44 symbols given in the inset.

1 11 Figure 16. 400 MHz H-NMRH-NMR spectrum of molecule 9,(, (aa)) inin CDClCDCl33;(; (b) H-NMR spectrum in DMSO-DMSO-dd66 1 1919 and ( c) H{H{ F}F} NMR NMR spectrum spectrum in in CDCl CDCl33. .

3 4h The 3JHH and 4hJFH couplings, if any, are not detectable due to the symmetry of the molecule. For The JHH and JFH couplings, if any, are not detectable due to the symmetry of the molecule. For detection of these couplings the symmetry of the molecule has to be broken. For such a purpose 2D detection of these couplings the symmetry of the molecule has to be broken. For such a purpose 2D 1H-15N HSQC experiment has been carried out for the molecule 9, where 15N is present in its natural 1H-15N HSQC experiment has been carried out for the molecule 9, where 15N is present in its natural 1 abundance. The HSQC spectrum is reported Figure 17A where all the possible seven couplings,1 JNH, abundance. The HSQC spectrum is reported Figure 17A where all the possible seven couplings, JNH, 2hJFN, 2JNH, 3hJFN, 1hJFH, 4hJFH and 3JHH has been measured from this spectrum. The observation of through 2hJ , 2J , 3hJ , 1hJ , 4hJ and 3J has been measured from this spectrum. The observation FN NH FN FH FH HH 1h 2h 3h 4h space couplings of significant strengths, such as JFH, JFN JFN1h and 2hJFH, provided3h strong4h and direct of through space couplings of signiﬁcant strengths, such as JFH, JFN JFN and JFH, provided evidence for the involvement of organic fluorine in the intramolecular HB. In the solvent DMSO, strong and direct evidence for the involvement of organic ﬂuorine in the intramolecular HB. In the 1 except for JNH, all the other1 couplings mediated through HB disappeared as reported in Figure 17B. solvent DMSO, except for JNH, all the other couplings mediated through HB disappeared as reported The relative signs of all the couplings were determined with respect to coupling c marked in inMolecules Figure 2017 17B., 22, 423 15 of 41 Figure 17B. From this it was inferred that 1hJFH is negative. The comparison of 1JNH [143,144] of molecules 9–17 with unsubstituted molecule 18, provided ample evidence that the nature of HBs in derivatives of hydrazides are predominantly covalent in nature. The close proximity of NH proton with the fluorine in fluorinated molecules is detected by 2D HOESY (hetero-nuclear Overhauser effect spectroscopy) experiment. This also provided evidence in favor of intramolecular HB.

Figure 17. (A) 800 MHz 1H-115N-HSQC15 (NH-coupled) spectrum of molecule 9 in CDCl3; (B) the chemical Figure 17. (A) 800 MHz H- N-HSQC (NH-coupled) spectrum of molecule 9 in CDCl3;(B) the chemicalstructure structureof the molecule of the 9 molecule with marking9 with ofmarking couplings of determined couplings determinedand their magnitudes; and their magnitudes;(C) 400 MHz 1H-15N-HSQC1 spectrum15 (NH-coupled) in DMSO-d6. (C) 400 MHz H- N-HSQC spectrum (NH-coupled) in DMSO-d6.

4.1.2. NCI Plot The relative signs of all the couplings were determined with respect to coupling c marked in 1h 1 FigureThe 17 B.weak From molecular this it was interactions inferred thatestablishedJFH is by negative. NMR studies The comparison have also been of JcorroboratedNH [144,145] ofby moleculestheoretical9 –DFT17 with [135,136] unsubstituted optimized molecule structure18 ,based provided calculations. ample evidence The calculation that the nature of noncovalent of HBs in interaction (NCI) has been shown to be very useful technique for the visualization of weak interactions [90]. The calculated grid points are plotted for a defined real space function, sign(λ2(r))ρ(r), as function 1 and reduced density gradient (RDG) as function 2 and also the color filed isosurfaces for all the investigated molecules. Figure 18 gives these plots for the molecule 9.

Figure 18. (a) The plot of sign(λ2(r))*ρ(r) as function 1 v/s the RDG as function 2; and (b) coloured isosurface plot (green color denotes weak H-bond and red color stands for steric effect) for molecule 9.

There are three spikes on the left hand side of Figure 18a (i.e., sign(λ2(r))*ρ(r) is negative) denoting three HBs, viz., N-H···F, N-H···O and C-H···O. These three HBs can be observed in Figure 18b as green coloured isosurfaces. The red colour in isosurface plot (Figure 18b) represents the steric hindrance arising from phenyl ring of the molecule and other HB mediated rings. This is seen as four spikes on the right hand side (i.e., sign(λ2(r))*ρ(r) is positive) of Figure 18a. Similar results have been derived for all other investigated molecules [134].

Molecules 2017, 22, 423 15 of 41

Molecules 2017, 22, 423 16 of 44

derivativesFigure 17. of ( hydrazidesA) 800 MHz 1 areH-15 predominantlyN-HSQC (NH-coupled) covalent spectrum in nature. of molecule The close 9 in CDCl proximity3; (B) the of chemical NH proton with thestructure ﬂuorine of the in molecule ﬂuorinated 9 with molecules marking isof detectedcouplingsby determined 2D HOESY and (hetero-nuclear their magnitudes; Overhauser (C) 400 MHz effect spectroscopy)1H-15N-HSQC experiment. spectrum (NH-coupled) This also provided in DMSO evidence-d6. in favor of intramolecular HB.

4.1.2. NCI Plot The weakweak molecularmolecular interactions established by NMR studies have also been corroborated by theoreticaltheoretical DFTDFT [[135,136]136,137] optimizedoptimized structurestructure basedbased calculations.calculations. The calculationcalculation ofof noncovalentnoncovalent interactioninteraction (NCI) has beenbeen shownshown toto bebe veryvery usefuluseful techniquetechnique forfor thethe visualizationvisualization ofof weakweak interactionsinteractions [[90].91]. TheThe calculatedcalculated gridgrid pointspoints areare plottedplotted forfor aa deﬁneddefined realrealspace space function, function, sign( sign(λλ2(r)2(r)))ρρ(r)(r),, as functionfunction 11 andand reducedreduced density density gradient gradient (RDG) (RDG) as as function function 2 and2 and also also the the color color ﬁled filed isosurfaces isosurfaces for allfor theall the investigated investigated molecules. molecules. Figure Figure 18 gives 18 gives these these plots plots for thefor moleculethe molecule9. 9.

2(r) (r) Figure 18.18. ((aa)) TheThe plotplot ofof sign(sign(λλ2(r))*ρ(r) asas function 1 v/s the the RDG RDG as function 2; and (b)) colouredcoloured isosurfaceisosurface plot (green color denotes weak H-bond and and red red color color stands stands for for steric steric effect) effect) for for molecule molecule 99. .

There are three spikes on the left hand side of Figure 18a (i.e., sign(λ2(r))*ρ(r) is negative) denoting There are three spikes on the left hand side of Figure 18a (i.e., sign(λ2(r))*ρ(r) is negative) denoting three HBs, viz., N-H···F, N-H···O and C-H···O. These three HBs can be observed in Figure 18b as green three HBs, viz., N-H···F, N-H···O and C-H···O. These three HBs can be observed in Figure 18b as green coloured isosurfaces. The red colour in isosurface plot (Figure 18b) represents the steric hindrance coloured isosurfaces. The red colour in isosurface plot (Figure 18b) represents the steric hindrance arising from phenyl ring of the molecule and other HB mediated rings. This is seen as four spikes on arising from phenyl ring of the molecule and other HB mediated rings. This is seen as four spikes on the right hand side (i.e., sign(λ2(r))*ρ(r) is positive) of Figure 18a. Similar results have been derived for the right hand side (i.e., sign(λ )*ρ is positive) of Figure 18a. Similar results have been derived for all other investigated molecules2(r) [134].(r) all other investigated molecules [135].

4.1.3. Atoms in Molecules (AIM) Calculations

2 The magnitudes of ρ(r), signs of ∇ ρ(r) and potential energy density (V(r)) at corresponding (3, −1) BCP (bond critical points) (rcp) for HBs of interest have been calculated using QTAIM calculations [92– 96]. The value of (V(r)) used in the EHB = V(rbcp)/2 [97] for the calculation of energy of HB (EHB) of X···HX type. The calculated EHB of different HBs in all investigated molecules ranged between −3 to −8.6 Kcal/mol [135]. The 1H-NMR spectra were simulated using the GIAO [146] and CSGT [146] methods. It is observed that in most of the cases there are more than one conformers for the molecules. Thus the calculated chemical shift values of NH protons are not in complete agreement with the experimental data. The values observed from CSGT method are comparable with the experimental results.

4.2. N, N Acyl-Phenyl Substituted Derivatives of Hydrazides In this work another series of hydrazide derivatives has also been synthesized and investigated, where the formation of ﬁve and six membered rings mediated through HBs is possible. The general chemical structure is given in the Figure 19. Molecules 2017, 22, 423 16 of 41 Molecules 2017, 22, 423 16 of 41 4.1.3. Atoms in Molecules (AIM) Calculations 4.1.3. Atoms in Molecules (AIM) Calculations The magnitudes of ρ(r), signs of ∇2ρ(r) and potential energy density (V(r)) at corresponding (3, −1) BCP The magnitudes of ρ(r), signs of ∇2ρ(r) and potential energy density (V(r)) at corresponding (3, −1) BCP (bond critical points) (rcp) for HBs of interest have been calculated using QTAIM calculations [91–95]. The (bond critical points) (rcp) for HBs of interest have been calculated using QTAIM calculations [91–95]. The value of (V(r)) used in the EHB = V(rbcp)/2 [96] for the calculation of energy of HB (EHB) of X···HX type. The value of (V(r)) used in the EHB = V(rbcp)/2 [96] for the calculation of energy of HB (EHB) of X···HX type. The calculated EHB of different HBs in all investigated molecules ranged between −3 to −8.6 Kcal/mol [134]. calculated EHB of different HBs in all investigated molecules ranged between −3 to −8.6 Kcal/mol [134]. The 1H-NMR spectra were simulated using the GIAO [145] and CSGT [145] methods. It is The 1H-NMR spectra were simulated using the GIAO [145] and CSGT [145] methods. It is observed that in most of the cases there are more than one conformers for the molecules. Thus the observed that in most of the cases there are more than one conformers for the molecules. Thus the calculated chemical shift values of NH protons are not in complete agreement with the experimental calculated chemical shift values of NH protons are not in complete agreement with the experimental data. The values observed from CSGT method are comparable with the experimental results. data. The values observed from CSGT method are comparable with the experimental results. 4.2. N, N Acyl-Phenyl Substituted Derivatives of Hydrazides 4.2. N, N Acyl-Phenyl Substituted Derivatives of Hydrazides In this work another series of hydrazide derivatives has also been synthesized and investigated, In this work another series of hydrazide derivatives has also been synthesized and investigated, where the formation of five and six membered rings mediated through HBs is possible. The general whereMolecules the2017 formation, 22, 423 of five and six membered rings mediated through HBs is possible. The general17 of 44 chemical structure is given in the Figure 19. chemical structure is given in the Figure 19.

Figure 19. The chemical structures of N0 ′-phenylbenzohydrazide and its halo derivatives. The dotted FigureFigure 19. TheThe chemical structures of N′-phenylbenzohydrazide-phenylbenzohydrazide and and its its halo halo derivatives. The The dotted dotted lines indicate the HB. lineslines indicate indicate the the HB. HB. The main focus of this work is to determine the conformations of the fluorinated hydrazide TheThe main main focus focus of of this work is to determine the conformations of the fluorinated fluorinated hydrazide derivatives and thereby understanding the effect of HB of the type F/CF3…H (N). derivativesderivatives and and thereby thereby understanding the effect of HB of the type F/CF33…···HH (N). (N). 4.2.1. Spectroscopic Experimental Observations 4.2.1.4.2.1. Spectroscopic Spectroscopic Experimental Experimental Observations Observations For initiating the study, the 1H-119F HOESY19 experiments have been carried out for all the investigated ForFor initiating initiating the the study, study, the 1 theH-19FH- HOESYF HOESY experiments experiments have been have carried been out carried for all the out investigated for all the molecules and the strong correlation in 1H-19F HOESY spectrum1 19 is observed where fluorine (X’) in the moleculesinvestigated and molecules the strong andcorrelation the strong in 1H- correlation19F HOESY in spectrumH- F HOESY is observed spectrum where isfluorine observed (X’) wherein the phenyl ring B established correlation with NH(1) proton whereas fluorine in the phenyl ring A (X) phenylfluorine ring (X’) B in established the phenyl correlation ring B established with NH correlation(1) proton whereas with NH fluorine(1) proton in whereasthe phenyl fluorine ring A in (X) the established correlation with NH(2) proton. From the obtained correlations, the conformations of all the establishedphenyl ring correlation A (X) established with NH correlation(2) proton. From with the NH obtained(2) proton. correlat Fromions, the the obtained conformations correlations, of all the synthesized molecules have been derived and 1H-19F HOESY spectrum for1 the19 molecule 20 is reported synthesizedconformations molecules of all the have synthesized been derived molecules and 1H- have19F HOESY been derived spectrum and forH- the moleculeF HOESY 20 spectrum is reported for in Figure 20. inthe Figure molecule 20. 20 is reported in Figure 20.

Figure 20. 400 MHz two dimensional 1H-19F HOESY spectrum of molecule 20. The detection of strong Figure 20. 400 MHz two dimensional 11H-1919F HOESY spectrum of molecule 20. The detection of strong Figurecross peaks 20. 400 convincingly MHz two dimensional establishedH- the Fcorrelations HOESY spectrum between of moleculeNH(1) proton20. The and detection F and also of strong NH(2) cross peaks convincingly established the correlations between NH(1) proton and F and also NH(2) crossproton peaks and convincinglyF. The cross peaks established were also the observed correlations for phenyl between protons NH(1) aproton1 and b1 and. The F mixing and also time NH used(2) proton and F. The cross peaks were also observed for phenyl protons a1 and b1. The mixing time used protonin the andexperiment F. The cross was peaks450 ms. were also observed for phenyl protons a1 and b1. The mixing time used in the experiment was 450 ms. in the experiment was 450 ms.

After arriving at the conformations of the molecules, the further studies were directed to derive the information about the weak molecular interactions (HB) that validate the proposed conformations. The titration experiment with CDCl3 did not show any significant change in the chemical shift of NH protons confirming the absence of any intermolecular interactions. The peak at 1.54 ppm corresponding to monomeric water remain constant indicating the absence of hydrizide-water interaction [146]. For discarding the possibility of dimerization or aggregation, if any, DOSY experiment has been carried out with the mixture of 1:1 molar ratio of molecules 20 and 25 in the solvent CDCl3, which have different diffusion coefficients. The 1H-NMR spectra for the investigated molecules have been acquired at different temperatures whereMolecules the2017 HB, 22 ,gets 423 strengthened on lowering of temperature and results in deshielding of the18 NH of 44 proton participating in HB. The corresponding plots of temperature dependent variation of chemical shifts of NH protons are reported in the Figure 2121A,B.A,B. In addition to this the incremental additions of DMSO-d6 resulted in the deshielding of NH proton providing information about the strength of of DMSO-d6 resulted in the deshielding of NH proton providing information about the strength of HB. The perturbation on the chemical shifts of NH proton (δNH) induced by DMSO-d6 are given in HB. The perturbation on the chemical shifts of NH proton (δNH) induced by DMSO-d6 are given in Figure 21C,D.21C,D. The temperature coefficient coefﬁcient is directly proportional to the strength of HB. On the the other other hand, the ΔδNH/ΔVDMSO has inverse relation to the strength of HB. Hence mere visual inspection of hand, the ∆δNH/∆VDMSO has inverse relation to the strength of HB. Hence mere visual inspection theof the graphs graphs given given in inFigure Figure 21 21 provides provides information information about about the the comparative comparative strengths strengths of of HB HB in in the studied molecules.

Figure 21. TheThe variation of the chemical shift of; (A)) NHNH(1)(1);;( (B)) NH (2) protonsprotons with with temperature; temperature; (C) NH (1) andand ( (DD)) NH NH(2)(2) protonsprotons with with the the increm incrementalental addition addition of of DMSO- DMSO-d6 forfor the the molecules molecules 19–25 inin thethe solvent CDCl33 at 298 K. The colorcolor codecode forfor thethe moleculesmolecules are are given given as as an an inset inset in in Figure Figure 21 21C,C, and and is issame same for for Figure Figure 21 21A–D.A–D.

The direct evidence about the presence of HB can be obtained by deriving the coupling between twotwo NMR active active nuclei nuclei which which are are participating in in the the formation formation of of HB. HB. In In order to overcome the broadening (due to quadrupolar 14NN nucleus) in the NMR spectrum, thethe 2D2D 1515N-1HH HSQC spectra have beenbeen recorded recorded for for all theall investigatedthe investigated molecules molecules where where15N is in15N natural is in abundance.natural abundance. The expanded The expanded NH(2) 15region1 of 15N-1H HSQC spectrum of molecule 20 in CDCl3 is given in Figure 22 which NH(2) region of N- H HSQC spectrum of molecule 20 in CDCl3 is given in Figure 22 which provides provides values1h of 1hJ2hFH and 2hJNF. These1h 1hJFH and2h 2hJNF detected in the solvent CDCl3, disappeared in values of JFH and JNF. These JFH and JNF detected in the solvent CDCl3, disappeared in the solvent DMSO-d6. This unequivocally established that these are coupling arising from through the solvent DMSO-d6. This unequivocally established that these are coupling arising from through space interactions.

Molecules 2017, 22, 423 19 of 44 Molecules 2017, 22, 423 18 of 41

Figure 22. 400 MHz coupled 1515N-1H-HSQC1 spectra of molecule 20 in the solvents (A) CDCl3 and (B) Figure 22. 400 MHz coupled N- H-HSQC spectra of molecule 20 in the solvents (A) CDCl3 and DMSO-d6. The measured couplings and their signs are reported in the Figure 22A. (B) DMSO-d6. The measured couplings and their signs are reported in the Figure 22A.

15 1 (2) Furthermore, in the 2D 15N- H1 HSQC spectrum of molecule 25, the NH peak appeared as a Furthermore, in the 2D N- H HSQC spectrum of molecule 25, the NH(2) peak appeared as quartet in CDCl3 while it appeared as a singlet in DMSO solvent indicating that the couplings observed a quartet in CDCl3 while it appeared as a singlet in DMSO solvent indicating that the couplings in CDCl3 are the through space contribution [134]. The 1JNH scalar coupling is another important NMR observed in CDCl are the through space contribution [135]. The 1J scalar coupling is another parameter for extracting3 the information about the nature of HB [40,144,147].NH There is a significant important NMR parameter for extracting the information about the nature of HB [41,145,148]. There is increase in 1JNH(1) and 1JNH(2) values in halo/methoxy substituted molecules compared to the a significant increase in 1J and 1J values in halo/methoxy substituted molecules compared unsubstituted molecule ofNH(1) hydrazide,NH(2) confirming that the corresponding HBs are predominantly to the unsubstituted molecule of hydrazide, confirming that the corresponding HBs are predominantly electrostatic in nature. electrostatic in nature. 4.2.2. Theoretical Calculations 4.2.2. Theoretical Calculations The observed NMR experimental results have been corroborated by IR and QTAIM [91–95], The observed NMR experimental results have been corroborated by IR and QTAIM [92–96], NCI [90] and the conformational study by using relaxed potential energy scan. The chemical structures NCI [91] and the conformational study by using relaxed potential energy scan. The chemical structures of all the synthesized molecules have been optimized using G09 program with B3LYP/6-311G** level of of all the synthesized molecules have been optimized using G09 program with B3LYP/6-311G** level of theory by taking chloroform as a solvation medium. The molecular structures were also optimized with theory by taking chloroform as a solvation medium. The molecular structures were also optimized with larger basis set like aug-cc-pVTZ and 1H-NMR spectra were simulated using GIAO [145] method for larger basis set like aug-cc-pVTZ and 1H-NMR spectra were simulated using GIAO [146] method for optimized structures (both by 6-311G** and aug-cc-pVTZ basis sets) in chloroform solvation medium. optimized structures (both by 6-311G** and aug-cc-pVTZ basis sets) in chloroform solvation medium. Additional information has been derived by IR and QTAIM an NCI analysis. The C=O and N-H Additional information has been derived by IR and QTAIM an NCI analysis. The C=O and N-H stretching frequencies in the IR spectra of all the investigated molecules showed blue shift, providing stretching frequencies in the IR spectra of all the investigated molecules showed blue shift, providing an evidence for the existence of HB [148]. Positive sign for Laplacian of electron density (∇2ρ) at the an evidence for the existence of HB [149]. Positive sign for Laplacian of electron density (∇2ρ) at the NH(1)…X’ [148] indicates that in all the molecules the type of interaction is HB. NH(1)···X’ [149] indicates that in all the molecules the type of interaction is HB. The NCI index detects the weak interactions in real space based on the electron density and its The NCI index detects the weak interactions in real space based on the electron density and its derivatives. Using Multiwfn program, the grid points have been calculated and plotted for the two derivatives. Using Multiwfn program, the grid points have been calculated and plotted for the two functions: sign(λ2)*ρ as function 1, and reduced density gradient (RDG) as function 2. Both these functions: sign(λ2)*ρ as function 1, and reduced density gradient (RDG) as function 2. Both these functions have the information about HB. The color filled isosurface graphs show the presence of functions have the information about HB. The color filled isosurface graphs show the presence of H(N)…X–C and C=O…H(N)…X–C type HBs in the investigated molecules. These calculations further H(N)···X–C and C=O···H(N)···X–C type HBs in the investigated molecules. These calculations further supported the NMR observations. supported the NMR observations.

RelaxedRelaxed Potential Potential Energy Scan

In NMR study, the NH(2) proton of molecule 25 appeared as a quartet in 11H-1515N HSQC spectrum. In NMR study, the NH(2) proton of molecule 25 appeared as a quartet in H- N HSQC spectrum. To understandunderstand thethe reasonreason behind behind the the observation observation of of quartet quartet the the relaxed relaxed potential potential energy energy scan scan has beenhas beenperformed performed using using B3LYP/6-311G** B3LYP/6-311G** level level of of theory theory at at ambient ambient temperature temperature for for thethe F28–C27–C5–H12F28–C27–C5–H12 dihedral angle through which the internal rotation of the CF3 group was confirmed (Figure 23). The dihedral angle through which the internal rotation of the CF3 group was conﬁrmed (Figure 23). calculated energy barrier for the CF3 internal rotation is about 2.31 kcal/mol. Because of such a low The calculated energy barrier for the CF3 internal rotation is about 2.31 kcal/mol. Because of such a energy barrier CF3 group appeared as a quartet in NMR spectrum. low energy barrier CF3 group appeared as a quartet in NMR spectrum.

Molecules 2017, 22, 423 20 of 44 MoleculesMolecules 2017 2017, ,22 22, ,423 423 1919 of of 41 41

Figure 23. Relaxed potential energy surface Scan for the internal rotation of the CF3 group. The scan Figure 23. Relaxed potential energy surface Scan for the internal rotation of the CF3 group. The scan waswas performed performed in in 28 28 steps steps with with increm incrementsincrementsents of of 5° 55°◦ in in the the dihedral dihedral angle. angle.

The relaxed potential energy surface scan has also been carried out for the molecule 20 using The relaxed potential energy surface scan scan ha hass also been carried out for the molecule 20 using B3LYP/6-311G** level of theory at ambient temperature in 36 steps with 10° increment in the dihedral B3LYP/6-311G** level level of of theory theory at at ambient ambient temperature temperature in 36 steps steps wi withth 10° 10◦ incrementincrement in in the the dihedral dihedral angle (−180° to +180°) in order to get the information about the internal rotation of the phenyl ring angle ( −180°180 ◦toto +180°) +180◦ )in in order order to to get get the the information information ab aboutout the the internal internal rotation rotation of of the the phenyl ring through a single bond. From Figure 24 it is clear that the proposed conformation has lower energy throughthrough a a single bond. From From Figure Figure 2424 itit is is clea clearr that the proposed conformation has has lower lower energy energy over the other. over the other.

FigureFigure 24.24. RelaxedRelaxedRelaxed potentialpotential potential energyenergy energy surfacesurface surface scanscan scan forfor for thethe the inin internalternalternal rotationrotation rotation ofof of phenylphenyl ringring throughthrough aa singlesinglesingle bond bond in in molecule molecule 20 20. .Selected Selected Selected dihedral dihedral dihedral angles; angles; angles; ( ( (AA)) N26–C10–C13–H14 N26–C10–C13–H14 ( ( BB)) N26–N27–C4–C5. N26–N27–C4–C5.

5.5. StudiesStudies Studies onon on DerivativesDerivatives Derivatives ofof ImidesImides TheThe imidesimides are are the thethe diacyldiacyl derivativesderivatives of ofof ammonia ammoniammoniaa or oror the thethe primary primaryprimary amines aminesamines [129 [128].[128].]. The TheThe importance importanceimportance of ofofimide imideimide derivatives derivativesderivatives is found isis foundfound in many inin manymany ﬁeld of fieldfield daily ofof interest dailydaily interest suchinterest as, such highsuch strengthas,as, highhigh electrically strengthstrength electricallyelectrically conductive conductiveconductivepolymers [ 150polymerspolymers–152], synthetic[149–151],[149–151], applications syntheticsynthetic applicatioapplicatio [153], medicinalnsns [152],[152], activitymedicinalmedicinal [154 activityactivity], as ionic [153],[153], ﬂuids asas ionic [ionic155], fluidsfluidsin pharmacology [154],[154], inin pharmacologypharmacology [156] and as [155][155] synthetic andand asas precursors syntheticsynthetic precursorsprecursors [157] are well [156][156] documented. areare wellwell documented.documented. The NH The linkerThe NHNH of linkerlinkeran imide ofof an providesan imideimide ample providesprovides scope ampleample to synthesize scopescope toto different syntsynthesizehesize derivatives differentdifferent with derivativesderivatives the desired withwith substitution thethe desireddesired on substitutionsubstitutionacyl group(s). onon Several acylacyl group(s).group(s). derivatives SeveralSeveral of imides derivativesderivatives whose of chemicalof imidesimides structures whosewhose chemicalchemical are given structuresstructures in the Figure areare givengiven 25 have inin thethebeen Figure Figure synthesized 25 25 have have bybeen been using synthesized synthesized microwave by by assistedusing using micr micr methodowaveowave [158 assisted assisted] and characterizedmethodmethod [157] [157] and byand extensive characterized characterized utility by by of extensiveextensiveNMR techniques utilityutility ofof and NMRNMR ESI-HRMS techniquestechniques spectrometry andand ESI-HRMSESI-HRMS [159]. spectrometryspectrometry [158].[158].

Molecules 2017, 22, 423 20 of 41

Molecules 2017, 22, 423 21 of 44 Molecules 2017, 22, 423 20 of 41

Figure 25. The chemical structures of the derivatives of 2-X-N-(2-X’-benzoyl)benzamide.

5.1. Information Derived by NMR Spectroscopy

The NMR spectroscopic derived information on intramolecular HB in these molecules has been unequivocallyFigure 25. Thesupported chemical by structures Density of Function the derivatives Theory ofof 2-X-2-X-DFTNN -(2-X’-benzoyl)benzamide.-(2-X’-benzoyl)benzamide.[135,136] based NCI [90,159], and QTAIM [91–95] calculations. 5.1. Information Derived by NMR Spectroscopy 5.1. InformationThe solvent Derived titration by experiments NMR Spectroscopy [62,63,140] have been performed on the molecules 26–32 in the solventThe CDCl NMRNMR3 to spectroscopic spectroscopicdistinguish the derivedderived intra- and informationinformation intermolecular on intramolecularintramolecular HB and to monitor HB inthe thesethese effect moleculesofmolecules atmospheric hashas beenmonomeric unequivocally water on supported HB. It is observed by Density that Function there is no Theory change DFT in the [[113635,136] chemical,137] based shift NCI of NH [[90,159],91 ,160proton], andand as QTAIMwell as the [[91–95]92 –residual96] calculations.calculations. water peak position in the deuterated solvent [158]. These observations discarded any possibilityThe solvent of titration titrationintermolecular experiments experiments HB, aggregation, [62,63,140] [63,64,141 have]dimerization have been been performed performed or water-imide on onthe the interaction.molecules molecules 26 –2632– in32 thein thesolvent solventDMSO- CDCl CDCld36 to solvent distinguish3 to distinguish titration the intra- has the beenand intra- inter- employed and molecular inter- to molecularderive HB and the to HB monitorqualitative and tothe monitor effectinformation of theatmospheric effect on the of atmosphericmonomericrelative strengths water monomeric onof intramolecularHB. water It is observed on HB. HB It isthat on observed therethe molecules is that no change there 26 is– 32in no. the changeThe chemical excessive in the shift chemical deshielding of NH shift proton of NH as protonwellproton as astheis wellobserved residual as the wateras residual a peakconsequence water position peak ofin position thethe dedisruterated inuption the deuterated solvent of intramolecular [158]. solvent These [159 observationsHB]. Thesedue to observations the discarded strong discardedanyinteraction possibility any with possibilityof DMSO.intermolecular ofThe intermolecular upfield HB, aggregation, shift HB,for aggregation,NH dimerization peak with dimerization or the water-imide addition orwater-imide ofinteraction. DMSO [158] interaction. in the moleculeDMSO- 6, indicateddd66 solventsolvent that titration titration in this has hasparticular been been employed employedmolecule to, derive theto deriveintramolecular the qualitative the qualitative HB information formed information between on the relativeoxygenon the strengthsrelativeatom of thestrengths of methoxy intramolecular of groupintramolecular and HB NH on the proton HB molecules on might the molecules to26 –be32 stronger. The 26 excessive–32 than. The its excessive deshieldinginteraction deshielding with of NH DMSO proton of [134]. NH is observedprotonThe strength is asobserved a of consequence HB getsas a increasedconsequence of the disruption on loweringof the of disr intramolecular theuption temperature. of intramolecular HB dueExcessive to the strongdeshieldingHB due interaction to theof the strong with NH DMSO.interactionproton is The observed with upﬁeld DMSO. on shift lowering forThe NH upfield the peak temperature withshift thefor additionNHdue topeak the of withdisplacement DMSO the [ 159addition] inof thehydrogen of molecule DMSO bonded [158]6, indicated protonin the thatmoleculetowards in this the 6, particular indicated HB acceptor. molecule,that Thisin this is the particularan intramolecular evidence molecule in favor HB, the of formed intramolecular betweenoxygen HBHB formed[160–162]. atom between of The the methoxychemical oxygen groupatomshift ofof and NHthe NH methoxyprotons proton asgroup mighta function and to NH be of strongerproton temperat might thanure to(over its be interaction stronger the range than with of its298–220 DMSO interaction K) [135 for ].with molecules The DMSO strength 26[134].–32 of HBTheare getscontainedstrength increased ofin HBFigure on gets lowering 26A. increased the temperature. on lowering Excessivethe temperature. deshielding Excessive of the NHdeshielding proton isof observed the NH onproton loweringThe is changeobserved the temperaturein on FH lowering coupling due the value to temperature the on displacement lowering due tothe ofthe temperature hydrogen displacement bonded has of also hydrogen proton been detected. towards bonded theprotonSuch HB a acceptor.towardsvariation the is This possible HB is acceptor. an only evidence whenThis inis the an favor spin evidence of polarization intramolecular in favor is of transmitted intramolecular HB [161– 163between]. HB The [160–162]. two chemical NMR The active shift chemical ofnuclei NH protonsshiftmediated of NH as through a protons function HB. as of a temperature Thefunction variation of (overtemperat in the ure rangecoupling (over of 298–220 theconstant range K) of(through for 298–220 molecules space) K) for26 – as32molecules area function contained 26– 32of inaretemperature Figure contained 26A. is in reported Figure 26A. in the Figure 26B, for the molecules 26 and 29–32. The change in FH coupling value on lowering the temperature has also been detected. Such a variation is possible only when the spin polarization is transmitted between two NMR active nuclei mediated through HB. The variation in the coupling constant (through space) as a function of temperature is reported in the Figure 26B, for the molecules 26 and 29–32.

(a) (b)

Figure 26. Variation of (a) chemical shifts of NH protons as a function of temperature forfor thethe moleculesmolecules 26–32 andand ((bb)) throughthrough spacespace mediatedmediated HFHF couplingcoupling constantconstant as aa functionfunction of temperaturetemperature for thethe molecules 26 andand 2929––3232.. TheThe moleculesmolecules areare identiﬁedidentified byby thethe symbolssymbols givengiven inin thethe inset.inset. TheThe initialinitial concentration was 10 mM(a) in CDCl3. (b) concentration was 10 mM in CDCl3. Figure 26. Variation of (a) chemical shifts of NH protons as a function of temperature for the molecules The26–32 change and (b) in through FH coupling space mediated value on HF lowering coupling theconsta temperaturent as a function has alsoof temperature been detected. for the Such a variationmolecules is possible26 and 29– only32. The when molecules the spin are polarizationidentified by the is transmittedsymbols given between in the inset. two The NMR initial active nuclei concentration mediated through was 10 mM HB. in The CDCl variation3. in the coupling constant (through space) as a function of temperature is reported in the Figure 26B, for the molecules 26 and 29–32.

Molecules 2017, 22, 423 22 of 44

TheMolecules GIAO 2017 [146, 22,] 423 and CSGT [146], DFT methods of NMR simulation have been used21 for of 41 chemical shifts calculation and it has been observed that the CSGT method is giving the values that are in close MoleculesThe 2017 GIAO, 22, 423 [145] and CSGT [145], DFT methods of NMR simulation have been used for chemical21 of 41 agreementshifts with calculation the experimentally and it has been observed that values. the CSGT method is giving the values that are in close It isagreement obviousThe GIAO with that [145] the the andexperimentally molecule CSGT [145], 2-methoxy- observed DFT methods values.N’-(2-methoxybenzoyl)benzohydrazide of NMR simulation have been used for chemical labelled as 11 whoseshiftschemical calculationIt is obvious structure and that it thehas molecule isbeen given observed 2-methoxy- in Figure that Nthe 27’-(2-methoxybenzoyl)benzohydrazide ,CSGT does method not show is giving any typethe values of self-dimerization labelledthat are asin 11close or aggregationagreementwhose [ 135chemical with], even the structure experimentally in 20 mM is given solution. observed in Figure The values. 27,1H-DOSY does not [show138, 139any] type NMR of experimentself-dimerization has or therefore It is obvious that the molecule 2-methoxy-N’-(2-methoxybenzoyl)benzohydrazide1 labelled as 11 been carriedaggregation out for [134], a mixture even in of20 1:1mMmolar solution. ratio The (20 H-DOSY mM ﬁnal [137,138] solution) NMR ofexperiment molecules has11 thereforeand 26 in the whosebeen chemicalcarried out structure for a mixture is given of 1:1 inmolar Figure ratio 27, (20 does mM finalnot showsolution) any of typemolecules of self-dimerization 11 and 26 in the or solvent CDCl3 and the corresponding spectrum is1 reported in Figure 27. aggregationsolvent CDCl [134],3 and even the correspondingin 20 mM solution. spectrum The is H-DOSYreported in[137,138] Figure 27. NMR experiment has therefore been carried out for a mixture of 1:1 molar ratio (20 mM final solution) of molecules 11 and 26 in the solvent CDCl3 and the corresponding spectrum is reported in Figure 27.

Figure 27. 500 MHz 1H-DOSY NMR spectrum of 20 mM solution of the mixture of molecules 11 and Figure 27. 500 MHz 1H-DOSY NMR spectrum of 20 mM solution of the mixture of molecules 11 and 26 at a 1:1 molar ratio in CDCl3. 26 at a 1:1 molar ratio in CDCl3. FigureThe different27. 500 MHz diffusion 1H-DOSY coefficients NMR spectrum detected of for 20 mMthese solution two molecules of the mixture discarded of molecules the possibility 11 and of self or cross dimerization. The triplet pattern was detected for the NH peak of the molecule 26 with The different26 at a 1:1 molar diffusion ratio in coefficients CDCl3. detected for these two molecules discarded the possibility the separation of 13.03 Hz between adjacent peaks. This triplet collapses into a singlet in 1H{19F} of self or cross dimerization. The triplet pattern was detected for the NH peak of the molecule 26 experimentThe different confirming diffusion the coefficients presence detectedof coupling for thesebetween two 1moleculesH and 19F. discarded This has thebeen possibility further of with the separation of 13.03 Hz between adjacent peaks. This triplet collapses into a singlet in 1H{19F} selfascertained or cross dimerization. by acquiring Thethe spectrumtriplet pattern in a highwas detectedpolarity solvent for the DMSO-NH peakd6 whichof the resultedmolecule in 26 the with 1 19 experimentthecollapsing separation confirming of tripletof 13.03 the to presenceaHz singlet between confirming of coupling adjacent that betweenpeaks. this coupling ThisH triplet andis mediated collapsesF. This through has into been HB.a singlet further in ascertained1H{19F} 1 15 by acquiringexperimentThe the visualization confirming spectrum of inthe coupling a highpresence became polarity of morecoupling solvent prominent between DMSO- in the d1 H62D which andH- N19F. resultedHSQC This NMR has in experiment thebeen collapsing further of and the corresponding spectrum for molecule 26 is reported in Figure 28. The 1H-15N HSQC spectrum tripletascertained to a singlet by confirming acquiring the that spectrum this coupling in a high is mediated polarity solvent through DMSO- HB. d6 which resulted in the of the same molecule in the solvent DMSO-d6 is reported in Figure 28c. In the solvent DMSO, except Thecollapsing visualization of triplet of to coupling a singlet confirming became more that prominentthis coupling in is the mediated 2D 1H- through15N HSQC HB. NMR experiment for 1JNH, all the other couplings disappeared, giving strong and unambiguous evidence that the and the correspondingThe visualization spectrum of coupling for moleculebecame more26 is prominent reported in in the Figure 2D 1H- 2815.N The HSQC1H- 15NMRN HSQC experiment spectrum measured couplings 1hJFH and 2hJFN in the solvent CDCl3 are mediated through HB. The 1H-15N HSQC and the corresponding spectrum for molecule 26 is reported in Figure 28. The 1H-15N HSQC spectrum of the samespectra molecule of all the in theother solvent fluorine DMSO- containingd6 is inve reportedstigated in molecules Figure 28 c.also In exhibited the solvent through DMSO, space except for 1 of the same molecule in the solvent DMSO-d6 is reported in Figure 28c. In the solvent DMSO, except JNH, allcouplings the other of couplings different magnitudes. disappeared, giving strong and unambiguous evidence that the measured for 1JNH1h, all the other2h couplings disappeared, giving strong and unambiguous1 evidence15 that the couplings JFH and JFN in the solvent CDCl3 are mediated through HB. The H- N HSQC spectra measured couplings 1hJFH and 2hJFN in the solvent CDCl3 are mediated through HB. The 1H-15N HSQC of all the other fluorine containing investigated molecules also exhibited through space couplings of spectra of all the other fluorine containing investigated molecules also exhibited through space differentcouplings magnitudes. of different magnitudes.

Figure 28. Cont.

Figure 28. Cont. Figure 28. Cont.

Molecules 2017, 22, 423 23 of 44 Molecules 2017, 22, 423 22 of 41 Molecules 2017, 22, 423 22 of 41

Figure 28. 800 MHz spectrum of molecule 26 in CDCl3 (a) 1H-1 15N-HSQC15 (NH-coupled) spectrum; (b) Figure 28. 800 MHz spectrum of molecule 26 in CDCl3 (a) H- N-HSQC (NH-coupled) spectrum; Figure 28. 800 MHz spectrum of molecule 26 in CDCl3 (a) 1H-15N-HSQC (NH-coupled) spectrum; (b) (theb) thechemical chemical structure structure of molecule of molecule 1 and1 measuredand measured couplings couplings with their with signs; their ( signs;c) 400 MHz (c) 400 1H- MHz15N- the chemical structure of molecule 1 and measured couplings with their signs; (c) 400 MHz 1H-15N- 1HSQC15 spectrum (NH-coupled) in DMSO-d6. H- N-HSQC spectrum (NH-coupled) in DMSO-d6. HSQC spectrum (NH-coupled) in DMSO-d6.

1 The 1JNH values of the molecules 26, 27 and 29–32 are substantially smaller than the molecule 28, The 1J values of the molecules 26, 27 and 29–32 are substantially smaller than the molecule 28, providingThe JNHstrongNH values and of unambiguous the molecules evidence26, 27 and that 29 –the32 arenature substantially of HBs in smaller the derivatives than the moleculeof imides 28 is, providing strong and unambiguous evidence that the nature of HBs in the derivatives of imides is providing strong and unambiguous evidence that the nature of HBs in the 1derivatives19 of imides is predominantly of the covalent type [143,144] The strong correlations in the 2D1 H-19F HOESY [163–165] predominantly of the covalent type [144,145] The strong correlations in the 2D 1H- 19F HOESY [164–166] experimentspredominantly for of the the molecules covalent type26 and [143, 29144]–32 establishedThe strong correlationsthe close proximity in the 2D between H- F HOESY NH and [163–165] F atoms experiments for the molecules 26 and 29–32 established the close proximity between NH and F atoms experiments for the molecules1 1926 and 29–32 established the close proximity between NH and F atoms in these molecules. The 2D 1H- 19F HOESY spectrum of the molecule 26 is reported in the Figure 29. in these molecules. The The 2D 2D 1H-H-19F FHOESY HOESY spectrum spectrum of ofthe the molecule molecule 2626 is isreported reported in inthe the Figure Figure 29. 29 .

Figure 29. 376.5 MHz 2D 1H-19F HOESY spectrum of the molecule 26 in CDCl3. Figure 29. 376.5 MHz 2D 11H-1919F HOESY spectrum of the molecule 26 in CDCl3. Figure 29. 376.5 MHz 2D H- F HOESY spectrum of the molecule 26 in CDCl3. 5.2. Theoretical Calculations 5.2. Theoretical Calculations The DFT [135,136] optimized structure calculations have been carried out to ascertain the NMR observations.The DFT The [[135,136]136 ,DFT137] optimizedcalculations structure have been calculations performed have with been B3LYP/6-311+g carried out to (d,p) ascertain level theof theory NMR observations.with the chloroform The DFT as calculationsthe solvation have medium. beenbeen Th performedperformede energy withwithminimized B3LYP/6-311+gB3LYP/6-311+g structures (d,p) were level confirmed of theory by withharmonic the chloroform vibrational as frequency. the solvationsolvation The wave medium. function TheThe energyfiles for minimized QTAIM, and structures NCI studies were and confirmedconfirmed simulated by harmonic1H-NMR spectra vibrational using frequency. CSGT [145] The were wave ge functionnerated from filesfiles foroptimized QTAIM, coordinates. and NCI studies and simulated 1 1H-NMR spectra using CSGT [145] [146] were generatedgenerated from optimized coordinates. 5.2.1. Conformational Study 5.2.1. Conformational Study For all the investigated imides maximum of 3–4 major conformations are possible due to ring flip asFor shown allall thethe in investigated investigatedthe Figure 30. imides imides maximum maximum of of 3–4 3–4 major major conformations conformations are are possible possible due due to ring to ring flip asflip shown as shown in the in Figurethe Figure 30. 30.

Molecules 2017, 22, 423 24 of 44 Molecules 2017, 22, 423 23 of 41

Molecules 2017, 22, 423 23 of 41

FigureFigure 30. The30. The possible possible conformers conformers of of the the investigatedinvestigated im imideide molecules, molecules, arising arising due due to the to ring the ringflip. ﬂip.

OutFigure of the 30. fourThe possiblepossible conformers conformers of thethe cis-cisinvestigated and trans-trans imide molecules, conformers arising were due to optimized the ring flip. and the Out of the four possible conformers the cis-cis and trans-trans conformers were optimized and the difference {(cis-cis) – (trans-trans)} of minimum energy has been taken. The energy difference ΔEcis-trans differencevariedOut {( fromcis-cis of the approximately)–( fourtrans-trans possible − conformers)}2 to of − minimum11 Kcal/mol the cis-cis energy among and has trans-transall beenthe investigated taken. conformers The imide energy were molecules optimized difference and and∆ it Ethe iscis-trans variedalsodifference from found approximately {(thatcis-cis the) – energy (trans-trans− 2of to cis-cis)}− of11 minimumconformers Kcal/mol energy amongis always has all beenlower the taken. investigated than theThe trans-transenergy imide difference that molecules are Δ dueEcis-trans and to it is also foundthevaried presence thatfrom the approximatelyof HB. energy of cis-cis −2 to conformers−11 Kcal/mol is among always all lower the investigated than the trans-trans imide moleculesthat are and due it is to the presencealso offound HB. that the energy of cis-cis conformers is always lower than the trans-trans that are due to Relaxedthe presence Potential of HB. Energy Scan Relaxed Potential Energy Scan RelaxedThe Potentialfindings ofEnergy cis-cis Scan and trans-trans energies of the conformers has been further supported by Therelax ﬁndingspotential ofenergycis-cis scans.and Relaxedtrans-trans potentialenergies ener ofgy thesurface conformers for the internal has been rotation further of the supported phenyl by The findings of cis-cis and trans-trans energies of the conformers has been further supported by relaxring potential through energy single bond scans. has Relaxed been performed. potential The energy rotation surface of 360° for (−180–0–+180) the internal through rotation single of the bond phenyl relax potential energy scans. Relaxed potential energy surface for the internal rotation of the phenyl ring throughwas scanned single in 20 bond steps has with been 18° performed.of rotational segments. The rotation The ofscanned 360◦ ( −graph180–0–+180) of energy through(kcal/mol) single ring through single bond has been performed. The rotation of 360° (−180–0–+180) through single bond versus dihedral angle (θ°) is reported◦ in Figure 31. From the graph, it is clear that the energy of the bondwas was scanned scanned in in 20 20 steps steps with with 18° 18 ofof rotational rotational segments. segments. The The scanned scanned graph graph of energy of energy (kcal/mol) (kcal/mol) cis conformer is lower◦ than the other, and the maximum is for the trans conformation. versusversus dihedral dihedral angle angle (θ )(θ is°) reported is reported in in Figure Figure 31 31.. From From the the graph, graph, itit is clear that that the the energy energy of of the the cis conformercis conformer is lower is thanlower the than other, the other, and theand maximumthe maximum is for is for the thetrans transconformation. conformation.

Figure 31. Relaxed potential energy surface for the internal rotation of the phenyl ring through single bond. The dihedral angle and rotation direction are highlighted in the structure of molecule 26. FigureFigure 31. Relaxed 31. Relaxed potential potential energy energy surface surface for for thethe internal rotation rotation ofof the the phenyl phenyl ring ring through through single single bond.5.2.2. bond.Non The Covalent dihedralThe dihedral angleInteraction angle and and rotation (NCI) rotation Calculations direction direction areare highlighted in in the the structure structure of molecule of molecule 26. 26.

5.2.2.The Non grid Covalent points Interaction based on (NCI)NCI [90] Calculations calculations have been plotted for a defined real space 5.2.2.function, Non Covalent sign(λ2(r) Interaction)ρ(r), as function (NCI) 1 and Calculations reduced density gradient (RDG) as function 2 and color filed isosurfacesThe grid are points plotted based for onthe NCIinvestigated [90] calculations molecules. have The been NCI plotted studies for provideda defined thereal visualspace Theinformationfunction, grid pointssign( aboutλ2 based(r) )weakρ(r), as oninteractions function NCI [91 1 ]and and calculations reduced repulsions density have in the beengradie molecules plottednt (RDG) [158]. for as a function deﬁned 2 real and spacecolor filed function, sign(λisosurfaces2(r))ρ(r), as are function plotted 1 and for reducedthe investigated density gradientmolecules. (RDG) The asNCI function studies 2 andprovided color ﬁledthe isosurfacesvisual are plotted5.2.3.information Atoms for the aboutin Molecules investigated weak interactions (AIM) molecules. Calculations and repulsions The NCI in studies the molecules provided [158]. the visual information about weak interactions and repulsions in the molecules [159]. (r) ∇2 (r) (r) 5.2.3.The Atoms magnitudes in Molecules of ρ (AIM), signs Calculationsof ρ and potential energy density (V ) at corresponding (3, −1) BCP (bond critical points) (rcp) for HBs of interest have been calculated using QTAIM calculations 5.2.3. Atoms in Molecules (AIM) Calculations2 [91–95].The The magnitudes value of (ofV(r) ρ)(r) used, signs in ofthe ∇ EρHB(r) and= V(r potentialbcp)/2 [96] energy for the density calculation (V(r) )of at energycorresponding of HB (E (3,HB) − of1) BCP (bond critical points) (rcp) for HBs2 of interest have been calculated using QTAIM calculations TheX···HX magnitudes type. The calculated of ρ(r), signs EHB ofof ∇differentρ(r) and HBs potential in all the energy investigated density molecules (V(r)) at correspondingvaried from −2 to (3, −1) −[91–95].8.56 Kcal/mol The value [158]. of The(V(r) )bond used paths in the for E HB(3, = − 1)V(r BCPsbcp)/2 were[96] forgenerated the calculation for the investigated of energy of molecules HB (EHB )for of BCP (bond critical points) (rcp) for HBs of interest have been calculated using QTAIM calculations [92– visualizationX···HX type. andThe the calculated molecular EHB model of different for the moleculeHBs in all 26 the containing investigated BCPs moleculesand bond pathvaried is reportedfrom −2 into 96]. The value of (V(r)) used in the EHB = V(rbcp)/2 [97] for the calculation of energy of HB (EHB) of Figure−8.56 Kcal/mol 32. [158]. The bond paths for (3, −1) BCPs were generated for the investigated molecules for X···HX type. The calculated EHB of different HBs in all the investigated molecules varied from −2 to visualization and the molecular model for the molecule 26 containing BCPs and bond path is reported in −8.56Figure Kcal/mol 32. [159]. The bond paths for (3, −1) BCPs were generated for the investigated molecules for visualization and the molecular model for the molecule 26 containing BCPs and bond path is reported in Figure 32. Molecules 2017, 22, 423 25 of 44 Molecules 2017, 22, 423 24 of 41

Molecules 2017, 22, 423 24 of 41

FigureFigure 32. The32. The visualization visualization of of BCPs BCPs andand bondbond paths of of HB HB for for the the molecule molecule 26 plotted26 plotted using using the the Multiwfn software. Dots represent the CPs and thin bar represents the HB interactions. Multiwfn software. Dots represent the CPs and thin bar represents the HB interactions. Figure 32. The visualization of BCPs and bond paths of HB for the molecule 26 plotted using the 6. StudiesMultiwfn on Diphenyloxamidessoftware. Dots represent the CPs and thin bar represents the HB interactions. 6. Studies on Diphenyloxamides TheFigure existence 32. The visualizationof three centered of BCPs C=Oand …bondH(N) paths…X-C of HB HB involvingfor the molecule organic 26 plottedfluorine, using and the other 6. Studies on Diphenyloxamides ThehalogensMultiwfn existence in the software. ofderivatives three Dots centered repres of diphenyloxamideent the C=O CPs··· andH(N) thin ··· hasbarX-C reprbeen HBesents explored involving the HB byinteractions. organicNMR spectroscopy ﬂuorine, and and other halogensquantum inThe the theoreticalexistence derivatives ofstudies. three of diphenyloxamide Thecentered diphenyloxamide C=O…H(N) has …de beenX-Crivatives exploredHB involving are the by basic NMR organic units spectroscopy fluorine,of foldamers, and and whereother quantum 6. Studies on Diphenyloxamides theoreticalthehalogens formation studies. in the of three Thederivatives centered diphenyloxamide of HB diphenyloxamide contributes derivatives to th ehas stable been are and theexplored rigid basic structure. unitsby NMR ofThe foldamers,spectroscopy possible mode where and of the formationthree-centeredquantumThe of threeexistencetheoretical HB centered formationof studies. three HB The centeredC=O contributes diphenyloxamide…H(N) C=O…X-C… toH(N) the(where… de stableX-Crivatives X HB= andCF involving 3are, rigidF, the Cl, basic structure. Brorganic and units I) fluorine,ofin The foldamers,the possible investigated and whereother mode of molecules,halogensthe formation in along the of threewithderivatives centeredtheir chemical of HB diphenyloxamide contributes structures to are th egivenhas stable been in and Figure explored rigid 33. structure. by NMR The spectroscopy possible mode and of three-centered HB formation C=O···H(N)···X-C (where X = CF3, F, Cl, Br and I) in the investigated quantumthree-centered theoretical HB formation studies. The C=O diphenyloxamide…H(N)…X-C (where derivatives X = CF are3, F, the Cl, basic Br and units I) ofin foldamers, the investigated where molecules, along with their chemical structures are given in Figure 33. themolecules, formation along of three with centeredtheir chemical HB contributes structures to are th egiven stable in and Figure rigid 33. structure. The possible mode of three-centered HB formation C=O…H(N)…X-C (where X = CF3, F, Cl, Br and I) in the investigated molecules, along with their chemical structures are given in Figure 33.

Figure 33. Chemical structures of N,N-diphenyloxamide (33) and its derivatives 34–40. The dotted line3 indicate the HBs. Figure 33. Chemical structures of N,N-diphenyloxamide (33) and its derivatives 34–40. The dotted Figure 33. N,N 33 34 40 6.1. Experimentalline3 indicateChemical Observation the HBs. structures by NMR of Spectroscopy-diphenyloxamide ( ) and its derivatives – . The dotted line3 indicate the HBs. Figure 33. Chemical structures of N,N-diphenyloxamide (33) and its derivatives 34–40. The dotted Initial studies were focused on the concentration dependence of NH chemical shift (δNH) for all 6.1. Experimentalline3 indicate Observationthe HBs. by NMR Spectroscopy the molecules and it was observed that δNH remained unaltered for all the molecules, discarding the 6.1. Experimental Observation by NMR Spectroscopy presenceInitial of studiesany type were of intermolecularfocused on the interactioconcentrationns [166]. dependence Compared of toNH the chemical unsubstituted shift (δ NHmolecule) for all 6.1. Experimental Observation by NMR Spectroscopy Initial33the the molecules chemical studies and wereshifts it was of focused NH observed protons on the that are concentration δobservedNH remained to be unaltered dependence more deshielded for all of the NH for molecules, the chemical different discarding shift substituted (δNH the) for all presence of any type of intermolecular interactions [166]. Compared to the unsubstituted molecule the moleculesmolecules,Initial and 34studies–38 it, wasindicating were observed focused the participation on that theδ NHconcentrationremained of NH proton dependence unaltered in weak for of molecular NH all thechemical molecules, interactions shift (δ discarding[166].NH) for The all the presencedeshieldingthe33 the molecules of chemical any typeof andNH shifts of itchemical intermolecularwas of NH observed protonsshift on that loweringare interactions δ observedNH remained the temperatureto [167 be unaltered ].more Compared deshielded due for toall tothe the thefor strengthening molecules, the unsubstituted different discarding in substituted the molecule HB the is 33 molecules, 34–38, indicating the participation of NH proton in weak molecular interactions [166]. The the chemicalevidentpresence from of shifts any Figure oftype NH 34A. of protonsintermolecular are observed interactio tons be[166]. more Compared deshielded to the for unsubstituted the different molecule substituted 33deshielding the chemical of NH shifts chemical of NH protonsshift on arelowering observed the totemperature be more deshielded due to the for strengthening the different insubstituted the HB is molecules, 34–38, indicating the participation of NH proton in weak molecular interactions [167]. molecules,evident from 34 –Figure38, indicating 34A. the participation of NH proton in weak molecular interactions [166]. The The deshieldingdeshielding of of NH NH chemical chemical shift shift on on lowering lowering the thetemperature temperature due to due the to strengthening the strengthening in the HB in theis HB is evidentevident from from Figure Figure 34 34A.A.

Figure 34. The variation of the chemical shift of NH proton (A) with temperature; (B) with the

incremental addition of DMSO-d6 to the solution containing 200 µL of CDCl3. The 10 mM concentration atFigure 298 K 34. for Thethe molecules,variation of33 –the38 haschemical been initiallyshift of takenNH protonfor both ( Athe) withstudies. temperature; (B) with the incremental addition of DMSO-d6 to the solution containing 200 µL of CDCl3. The 10 mM concentration at 298 K for the molecules, 33–38 has been initially taken for both the studies. Figure Figure 34. 34.The The variation variation of of the the chemical chemical shift of of NH NH proton proton (A ()A with) with temperature; temperature; (B) with (B) withthe the incremental addition of DMSO-d6 to the solution containing 200 µL of CDCl3. The 10 mM concentration incremental addition of DMSO-d6 to the solution containing 200 µL of CDCl3. The 10 mM concentration at 298 K for the molecules, 33–38 has been initially taken for both the studies. at 298 K for the molecules, 33–38 has been initially taken for both the studies.

Molecules 2017, 22, 423 26 of 44 Molecules 2017, 22, 423 25 of 41

The high polarity solvent DMSO-d6 induced perturbation of δNH is reported in Figure 34B. The The high polarity solvent DMSO-d6 induced perturbation of δNH is reported in Figure 34B. larger value of ΔδNH/ΔVDMSO in molecule 33 compared to the molecules, 34–38, is giving an indication The larger value of ∆δNH/∆VDMSO in molecule 33 compared to the molecules, 34–38, is giving an that the probable three centered H-bond is relatively stronger than the two centered one. indicationthat the probable that the three probable centered three H-bond centered is H-bondrelatively is relativelystronger than stronger the two than centered the two one. centered one. The 2D 15N-1H HSQC experiments for all the investigated molecules have been carried out, TheThe 2D2D15 N-N-1HH HSQC HSQC experiments experiments for for all theall the investigated investigated molecules molecules have beenhave carriedbeen carried out, where out, 15 15 1 15where 15N is in natural abundance.15 The1 15N-1H HSQC spectrum of molecule 38 in CDCl3 yielded a N is in natural abundance. The N- H HSQC spectrum of molecule 38 in CDCl3 yielded a quartet quartet for the NH proton (reported in Figure 35B) implying that the rotation of CF3 group is very for the NH proton (reported in Figure 35B) implying that the rotation of CF3 group is very fast unlike 15 1 fast unlike in the earlier reported studies15 [79].1 The 15N-1H HSQC spectrum of molecule 34 in the in the earlier reported studies [80]. The N- H HSQC spectrum of molecule 34 in the solvent CDCl3, solvent CDCl3, reported in Figure 35A, yielded a doublet for the NH proton. reportedsolvent CDCl in Figure3, reported 35A, yielded in Figure a doublet 35A, yielded for the a NHdoublet proton. for the NH proton.

Figure 35. (A,B) 1515N-1 H-HSQC spectra of the molecules 34 and 38 respectively, in CDCl . The molecular Figure 35. (A,B) 15N-1H-HSQC spectra of the molecules 34 and 38 respectively, in CDCl33. The molecular structuresstructures andand thethe measuredmeasured couplings couplings have have also also been been reported. reported.

1515 11 TheThe quartetquartet inin 15N- 1H HSQC spectrum of molecule 3838 whichwhich waswas observedobserved inin thethe solventsolventCDCl CDCl33 collapsedcollapsed to to the the singlet singlet in the in solventthe solvent DMSO- DMSO-d6 reportedd6 reported in Figure in 36 FigureB. This unambiguously36B. This unambiguously established theestablished existence the of HBexistence in the of molecule HB in the38 .molecule 38.

Figure 36. (A,B) 15N-115H HSQC1 spectra of the molecules 34 and 38 respectively, in DMSO-d6. FigureFigure 36. (A 36.,B()A ,N-B) HN- HSQCH HSQC spectra spectra of the ofmolecules the molecules 34 and34 38and respectively,38 respectively, in DMSO- in DMSO-d6. d6.

The molecule 34 in solvent DMSO-d6 yielded a doublet as reported in Figure 36A, with the The molecule 34 in solvent DMSO-d6 yielded a doublet as reported in Figure 36A, with the JHF = 0.85 Hz and JNF = 1.25 Hz, whereas in CDCl3 these values were JHF = 2.9 Hz and JNF = 0.4 Hz, JHF = 0.85 Hz and JNF = 1.25 Hz, whereas in CDCl3 these values were JHF = 2.9 Hz and JNF = 0.4 Hz, respectively. But it is observed that the relative slopes of the displacement vectors of the cross sections in the solvents (DMSO-d6 or CDCl3) are opposite (Figures 35A and 36A). As the HB gets ruptured in

Molecules 2017, 22, 423 26 of 41 the high polar solvents, the couplings observed in the DMSO-d6 are considered as through bond couplings and the observed coupling in the solvent CDCl3 is considered as through space coupling. Hence the contribution from the HB alone turns out to be −3.75 Hz. As far as signs of JNF is concerned, it has to be negative. These observations gave a strong evidence for the presence of HB in the molecule 34. The close proximity between fluorine atom and MoleculesNH protons2017, 22has, 423 also been confirmed by the detection of cross peak in the 2D 1H-19F HOESY spectra27 of 44 of molecules 34 and 38. This is additional support for the presence of intramolecular HB between F and NH proton [166]. An increase in the 1JNH relative to molecule 33, is observed in molecules 34–36 respectively. But it is observed that the relative slopes of the displacement vectors of the cross sections and 38, indicating that the HB in these molecules is predominantly an electrostatic in nature [143,144]. in the solvents (DMSO-d6 or CDCl3) are opposite (Figures 35A and 36A). As the HB gets ruptured On the other hand, 1JNH decreases in molecule 37, hence the nature of HB is predominantly covalent in the high polar solvents, the couplings observed in the DMSO-d are considered as through bond in this molecule [40]. 6 couplings and the observed coupling in the solvent CDCl3 is considered as through space coupling. Hence6.2. Theoretical the contribution Studies from the HB alone turns out to be −3.75 Hz. As far as signs of JNF is concerned, it has to be negative. These observations gave a strong evidenceThe forobserved the presence experimental of HB in results the molecule have been34. The also close corroborated proximity betweenby DFT ﬂuorinebased theoretical atom and NHcalculations, protons hassuch also as, beenQTAIM conﬁrmed [91–95],by Natural the detection Bond Orbital of cross (NBO) peak [159] in the and 2D NCI1H-19 [90].F HOESY spectra of molecules 34 and 38. This is additional support for the presence of intramolecular HB between F 6.2.1. QTAIM Calculations 1 and NH proton [167]. An increase in the JNH relative to molecule 33, is observed in molecules 34–36 and 38The, indicating magnitude that of the electron HB in thesedensity molecules (ρ) and is predominantlysign of Laplacian an electrostaticof electron indensity nature [(144∇2ρ),145 are]. 1 Onexamined the other for hand,the determinationJNH decreases of inHB. molecule For such37 a, hencepurpose the BCPs nature and of bond HB is paths predominantly for intramolecular covalent inHBs this are molecule detected [ 41for]. the investigated molecules and are reported in Figure 37. The observed electron density values fall in the expected range (0.0102–0.0642 a.u.) for HBs [167]. 6.2. Theoretical Studies The positive sign of Laplacian of electron density (∇2ρ) at the (3, −1) BCPs indicates the type of interactionsThe observed that belong experimental to the HBs. results On the have basis beenof electron also corroborated density, the proton by DFT acceptance based theoretical trend for calculations,organic halogens such as,in the QTAIM intramolecular [92–96], Natural five membered Bond Orbital N-H (NBO)...X follows [160] and the NCIpattern [91]. Br > Cl > I, as detected from the chemical shift difference between the molecules 34–37 [166] compared to that of 6.2.1.molecule QTAIM 33. This Calculations trend is further supported by the ρ(NH...X) values [166]. The obtained electron ...... densityThe values magnitude are about of electron 0.015, density 0.020 and (ρ) and0.017 sign for of NH LaplacianX, NH ofO electron and CH densityO, respectively. (∇2ρ) are examinedBy using forthese the values determination in the bonding of HB. energy For such (BE) a purposeequation; BCPs {BE (kj/mol) and bond = 777 paths × ρ for (in intramoleculara.u.) − 0.4}, the HBsBEs are ...... detectedestimated for to thebe around investigated 11, 15 molecules and 13 kJ andfor NH are reportedX, NH O in and Figure CH 37O .HB interactions, respectively.

Figure 37. Molecular graphs, contour plots and AIM calculated bond paths for the molecules 33–38. Figure 37. Molecular graphs, contour plots and AIM calculated bond paths for the molecules 33–38. Dotted lines indicate the intramolecular HBs. Green and red dots are bond critical points (BCP) and Dotted lines indicate the intramolecular HBs. Green and red dots are bond critical points (BCP) and ring critical points (RCP), respectively. Electron density is plotted as contours in the blue curves in ring critical points (RCP), respectively. Electron density is plotted as contours in the blue curves in the planethe plane of molecules. of molecules.

6.2.2. NBO Analysis The observed electron density values fall in the expected range (0.0102–0.0642 a.u.) for HBs [168]. The positiveFor HB formation, sign of Laplacian electron of transfer electron from density the elec (∇2tronρ) at rich the (3,region−1) of BCPs HB indicatesacceptor to the the type anti- of interactionsbonding orbital that (σ belong*) of the to HB the donor HBs. takes On the place. basis This of electronhas been density, studied theby the proton Natural acceptance Bond Orbital trend for organic halogens in the intramolecular ﬁve membered N-H...X follows the pattern Br > Cl > I, as detected from the chemical shift difference between the molecules 34–37 [167] compared to that of molecule 33. This trend is further supported by the ρ(NH...X) values [167]. The obtained electron density values are about 0.015, 0.020 and 0.017 for NH...X, NH...O and CH...O, respectively. By using these values in the bonding energy (BE) equation; {BE (kj/mol) = 777 × ρ (in a.u.) − 0.4}, the BEs are estimated to be around 11, 15 and 13 kJ for NH...X, NH...O and CH...O HB interactions, respectively. Molecules 2017, 22, 423 28 of 44

6.2.2. NBO Analysis

MoleculesFor 2017 HB, 22, formation, 423 electron transfer from the electron rich region of HB acceptor27 to of the 41 anti-bonding orbital (σ*) of the HB donor takes place. This has been studied by the Natural Bond Orbital (NBO)(NBO) analysis analysis and and found found that that the the values values of of lp(O) lp(O) →→ σσ*(N-H)*(N-H) were were always always greater greater than than the the lp(O) lp(O) →→ σσ*(C-H)*(C-H) values. values. Hence Hence the the N-H N-H…···OO bonds bonds are are concluded concluded to to be be stronger stronger than than the the C-H C-H…···OO bonds. bonds.

6.2.3.6.2.3. NCI NCI Analysis Analysis QTAIMQTAIM calculations and and NBO analysis did not show thethe intramolecularintramolecular BCPBCP forfor N-HN-H···…FF interactioninteraction in in molecule molecule 22 whichwhich contradicts contradicts the the NMR observations. Another Another powerful powerful method, method, NCI NCI indexindex has has been been used used to to visualize visualize the the weak weak interaction interaction which which showed showed the the presence presence of of HB HB in in molecule molecule 3434,, which which is is in in agreement agreement with with NMR NMR observations. observations. In In Fi Figuregure 3838 (left(left side), side), the the calculated calculated grid grid points points areare plotted plotted for for the the two functions: sign( sign(λλ22)*)*ρρ asas function function 1 1 and and reduced reduced density density gradient gradient (RDG) (RDG) as as functionfunction 2 by MultiwfnMultiwfn program.program. Color Color ﬁlled filled isosurface isosurface graphs graphs are are plotted plotted using using these these grid grid points points and andare reportedare reported in the in rightthe right side side of the of Figurethe Figure 38. 38.

FigureFigure 38. 38. TheThe plot plot of of function function 1 1 (sign( (sign(λλ22)*)*ρρ values)values) on on X-axis X-axis vs vs function function 2, 2, the the reduced reduced density density gradientgradient (RDG) (RDG) on on YY-axis.-axis. (left (left hand hand side), side), colored colored isosurface isosurface plots plots in in which which Green Green colour colour denotes denotes weakweak HB HB and and red red color stands for steric effect. Labels Labels 33–38 represent molecules 33–38, respectively.

ForFor the the molecules molecules 3535,, 3636,, and and 3737,, there there are are three three spikes spikes on on the the left-hand left-hand side side which which denote denote three three HBsHBs namely namely N-H N-H…···X,X, N-H N-H…O··· andO and C-H C-H…O··· (whereO (where X = Cl, X = Br, Cl, and Br, andI), these I), these three three bonds bonds can be can also be seen also inseen the in colored the colored isosurface isosurface plots plotsreported reported on the on right the rightside of side the of Figure the Figure 38. For 38 X. For= F, Xall = three F, all threebonds bonds exist andexist the and spikes the spikes corresponds corresponds to two to HBs two are HBs overlappe are overlapped,d, these thesethree threeHBs HBsalso alsoare clearly are clearly seen seenin the in isosurfacesthe isosurfaces plot 2. plot For 2. X For = CF X3 =, the CF four3, the spikes four spikes have been have seen been that seen are that also are visible also in visible isosurfaces in isosurfaces plot 6. plot 6. 6.2.4. Relaxed Potential Energy Scan 6.2.4. Relaxed Potential Energy Scan It may be pointed out that in the 15N-1H-HSQC NMR spectrum of molecule 38 the NH group 15 1 appearedIt may as bea quartet pointed due out to that fast inrotation the N-of CFH-HSQC3 group NMRat the ambient spectrum temp of moleculeerature. Relaxed38 the NH potential group energyappeared scan as for a quartet the H9–C5–C16–F2 due to fast rotation dihedral of CF angle3 group was at performed the ambient to temperature.derive the information Relaxed potential about theenergy energy scan barrier for the of H9–C5–C16–F2 internal rotation dihedral of CF3 anglegroup. was This performed is reported to in derive the Figure the information 39. about the energy barrier of internal rotation of CF3 group. This is reported in the Figure 39.

Figure 39. Relaxed potential energy surface for the internal rotation of CF3 group at B3LYP/6-311G** level of calculation. The dihedral angle H9–C5–C16–F2 has been selected for the scan.

The barrier for this internal rotation was calculated as 2.15 kcal/mol using B3LYP/6-311G** level of theory and the energy barriers that can be observed in NMR is in between the 7–24 kcal/mol. This was the reason for observation of quartet for NH group.

Molecules 2017, 22, 423 27 of 41

(NBO) analysis and found that the values of lp(O) → σ*(N-H) were always greater than the lp(O) → σ*(C-H) values. Hence the N-H…O bonds are concluded to be stronger than the C-H…O bonds.

6.2.3. NCI Analysis

QTAIM calculations and NBO analysis did not show the intramolecular BCP for N-H…F interaction in molecule 2 which contradicts the NMR observations. Another powerful method, NCI index has been used to visualize the weak interaction which showed the presence of HB in molecule 34, which is in agreement with NMR observations. In Figure 38 (left side), the calculated grid points are plotted for the two functions: sign(λ2)*ρ as function 1 and reduced density gradient (RDG) as function 2 by Multiwfn program. Color filled isosurface graphs are plotted using these grid points and are reported in the right side of the Figure 38.

Figure 38. The plot of function 1 (sign(λ2)*ρ values) on X-axis vs function 2, the reduced density gradient (RDG) on Y-axis. (left hand side), colored isosurface plots in which Green colour denotes weak HB and red color stands for steric effect. Labels 33–38 represent molecules 33–38, respectively.

For the molecules 35, 36, and 37, there are three spikes on the left-hand side which denote three HBs namely N-H…X, N-H…O and C-H…O (where X = Cl, Br, and I), these three bonds can be also seen in the colored isosurface plots reported on the right side of the Figure 38. For X = F, all three bonds exist and the spikes corresponds to two HBs are overlapped, these three HBs also are clearly seen in the isosurfaces plot 2. For X = CF3, the four spikes have been seen that are also visible in isosurfaces plot 6.

6.2.4. Relaxed Potential Energy Scan

It may be pointed out that in the 15N-1H-HSQC NMR spectrum of molecule 38 the NH group appeared as a quartet due to fast rotation of CF3 group at the ambient temperature. Relaxed potential energyMolecules scan2017, 22for, 423 the H9–C5–C16–F2 dihedral angle was performed to derive the information 29about of 44 the energy barrier of internal rotation of CF3 group. This is reported in the Figure 39.

3 FigureFigure 39. 39. RelaxedRelaxed potential potential energy energy surface for the internal rotation of CF3 group at B3LYP/6-311G** levellevel of of calculation. calculation. The The dihedral dihedral angle angle H9–C5–C16–F2 H9–C5–C16–F2 has has been been selected for the scan.

The barrier for this internal rotation was calculated as 2.15 kcal/mol using B3LYP/6-311G** level The barrier for this internal rotation was calculated as 2.15 kcal/mol using B3LYP/6-311G** level of theory and the energy barriers that can be observed in NMR is in between the 7–24 kcal/mol. This of theory and the energy barriers that can be observed in NMR is in between the 7–24 kcal/mol. This was the reason for observation of quartet for NH group. was the reason for observation of quartet for NH group.

7. Utility of H/D Exchange for Study of HB The presence of HBs affects the release rate of labile hydrogen(s) which is/are participating in the reaction. There are number of chemical reactions in which the transfer of one or more protons (viz., hydride ions or hydrogen atoms) takes place in the rate determining step [41,43–46] The reaction kinetics depend on several factors, which also include the strength of intramolecular HB and the electronic effects caused by the substituents. The NMR spectroscopy is found to be a powerful technique which is widely employed for understanding the protein conformation, and dynamics [47–53] in aqueous media can be utilized to monitored by hydrogen/deuterium (H/D) exchange.

7.1. Factors Affecting the H/D Exchange The rate of H/D exchange in a particular molecule can be affected by the inter- and the intramolecular HBs. There will be a rapid exchange of the proton attached to nitrogen with the labile protons of the solvent. The mechanism of H/D exchange has been reported earlier in the derivatives of amides [54]. In the non-deuterated solvents, the proton exchange does not reflect in the NMR spectrum. On the other hand, in the presence of a labile deuterium-containing solvent the exchange takes place with the deuterium of the solvent molecules. This will have significant effect on the time dependent variation in the NMR signals intensities and permits the determination of H/D exchange rate. If the deuterated solvent is used in excess compared to the substrate, then the rate of exchange follows the pseudo first order kinetics. It is well known that the rate of H/D exchange is not only dependent on the strength of the intramolecular hydrogen bond, but also dependent on the electronic effect of the substituents. The amides and amines are the basic building blocks for the synthesis of heterocyclic and linear nitrogen containing compounds. Consequent to significant difference in electronegativity (EN) and size among halogens, the strength of hydrogen bonds, steric hindrance and electronic effects [169] by these groups would also be substantially different. Hence the electronic effect, size effect and the effect of organic fluorine involved intramolecular HB on H/D exchange using 1H-NMR spectroscopic techniques has also been explored in the different halo substituted anilines and benzamides [170]. The chemical structures of all the investigated molecules are reported in Figure 40. Molecules 2017, 22, 423 28 of 41

7. Utility of H/D Exchange for Study of HB The presence of HBs affects the release rate of labile hydrogen(s) which is/are participating in the reaction. There are number of chemical reactions in which the transfer of one or more protons (viz., hydride ions or hydrogen atoms) takes place in the rate determining step [40,42–45] The reaction kinetics depend on several factors, which also include the strength of intramolecular HB and the electronic effects caused by the substituents. The NMR spectroscopy is found to be a powerful technique which is widely employed for understanding the protein conformation, and dynamics [46– 52] in aqueous media can be utilized to monitored by hydrogen/deuterium (H/D) exchange.

7.1. Factors Affecting the H/D Exchange The rate of H/D exchange in a particular molecule can be affected by the inter- and the intramolecular HBs. There will be a rapid exchange of the proton attached to nitrogen with the labile protons of the solvent. The mechanism of H/D exchange has been reported earlier in the derivatives of amides [53]. In the non-deuterated solvents, the proton exchange does not reflect in the NMR spectrum. On the other hand, in the presence of a labile deuterium-containing solvent the exchange takes place with the deuterium of the solvent molecules. This will have significant effect on the time dependent variation in the NMR signals intensities and permits the determination of H/D exchange rate. If the deuterated solvent is used in excess compared to the substrate, then the rate of exchange follows the pseudo first order kinetics. It is well known that the rate of H/D exchange is not only dependent on the strength of the intramolecular hydrogen bond, but also dependent on the electronic effect of the substituents. The amides and amines are the basic building blocks for the synthesis of heterocyclic and linear nitrogen containing compounds. Consequent to significant difference in electronegativity (EN) and size among halogens, the strength of hydrogen bonds, steric hindrance and electronic effects [168] by these groups would also be substantially different. Hence the electronic effect, size effect and the effect of organic fluorine involved intramolecular HB on H/D exchange using 1H-NMR spectroscopic techniques has also been explored in the different halo substituted anilines and benzamides [169]. Molecules 2017, 22, 423 30 of 44 The chemical structures of all the investigated molecules are reported in Figure 40.

FigureFigure 40. 40. TheThe general general chemical chemical structures structures of of investigat investigateded halo halo substituted substituted be benzamidesnzamides and and anilines. anilines. ForFor ( (aa)) and and ( (bb)) X X = = F, F, Cl, Cl, Br, Br, I I and and H H and and for for ( (cc),), ( (dd),), ( (ee),), and and ( (ff)) X X = = F, F, Cl Cl and and H. H.

For all investigated molecules the 5 mM stock solution was prepared on the volume scale of 5 mL in For all investigated molecules1 the 5 mM stock solution was prepared on the volume scale of the fresh CDCl3 solvent and the H-NMR spectra1 have been obtained using 450 µL of this stock solution. 5 mL in the fresh CDCl3 solvent and the H-NMR spectra have been obtained using 450 µL of To this solution 50 µL of CD3OD was added with the marking time as zero, resulting in the final this stock solution. To this solution 50 µL of CD OD was added with the marking time as zero, substrate concentration of less than 5 mM. This 3was well below the concentrations where the resulting in the final substrate concentration of less than 5 mM. This was well below the concentrations possibility of any aggregation and intermolecular interaction is observable. The added methanol where the possibility of any aggregation and intermolecular interaction is observable. The added created a 10% methanol:chloroform solution, with the final methanol concentration of 2.47 M, which methanol created a 10% methanol:chloroform solution, with the final methanol concentration of 2.47 M, ensured pseudo-first-order kinetics. The NMR spectra were acquired at every two minutes’ interval which ensured pseudo-first-order kinetics. The NMR spectra were acquired at every two minutes’ until the amino proton signal intensity submerged within the baseline noise. A distinct non- interval until the amino proton signal intensity submerged within the baseline noise. A distinct exchangeable aromatic proton peak was used as an internal integration reference. Rate constants and non-exchangeable aromatic proton peak was used as an internal integration reference. Rate constants correspondingMolecules 2017, 22 ,half-lives 423 were determined from the slope of a nonlinear least squares fit to the graph29 of 41 and corresponding half-lives were determined from the slope of a nonlinear least squares fit to the graphof A(t) of= A(o) A(t) =exp A(o)(−kt),exp rather(−kt) than, rather estimating than estimating the values the at values the extended at the extended time. The time. absolute The intensity absolute intensityof Y intercept of Y interceptwas taken was 1 at taken zero 1 time, at zero and time, this and value this was value used was to used normalize to normalize with respect with respect to the toremaining the remaining hydrogen. hydrogen. The X-intercept The X-intercept was the was time. the To time. ensure To ensure reproducibility reproducibility the experiments the experiments have havebeen beenrepeated repeated on a different on a different sample sample at different at different time. time.

7.2. Exchange Mechanism

On addition of CD3OD the labile protons undergo exchange with the hydroxy deuterium of the solvent molecule. This time dependent phenomenon followedfollowed the first ﬁrst order reaction kinetics and the intensity of the labile protons was systematicallysystematically reduced. The mechanism of exchange is pictorially illustrated in Figure 4141..

Figure 41. 41. TheThe mechanism mechanism of ofH/D H/D exchange exchange of labile of labile protons protons with the with hydroxy the hydroxy cdeuterium cdeuterium of the solvent of the solvent molecule. The rate of deuteration is fast due to the high concentration of CD OD compared molecule. The rate of deuteration is fast due to the high concentration of CD3OD compared3 to substrate. to substrate. To get qualitative information about the strength of intramolecular HBs, and electronic effect the differentTo get halo qualitative substituted information benzamide about molecules the strength were also of intramolecularinvestigated. The HBs, intensity and electronic of the peak effect at thezero different time is taken halo substitutedas 100%. The benzamide other spectra molecules were acquired were also after investigated. adding 50 The µL intensityof CD3OD of to the 450 peak µL solution. The plot of integral areas of NH2 peaks as a function of time, for unsubstituted and ortho- halosubstituted benzamides (2-fluoro, 2-chloro, 2-bromo, 2-iodo) are compared in Figure 42. Similar results for meta- and para-substituted derivatives have also been obtained [169].

Figure 42. The plot of integral intensities of the NH2 peaks v/s time (in mins) for unsubstituted and ortho-halosubstituted benzamides. The molecules are identified by the symbols given in inset.

The nature of the substituents dictated the H/D rate constants and they were different for different substituents. It has been observed that when all the physical parameters were kept unchanged for any of the investigated molecules, the results could be reproduced with high degree of accuracy within the experimental error [169]. There is a perfect match of graphical plots obtained from integral intensity of the labile protons peaks as a function of time, carried out for different samples at different times for 2-fluorobenzamide. This is clearly evident from Figure 43.

Molecules 2017, 22, 423 29 of 41 of A(t) = A(o) exp(−kt), rather than estimating the values at the extended time. The absolute intensity of Y intercept was taken 1 at zero time, and this value was used to normalize with respect to the remaining hydrogen. The X-intercept was the time. To ensure reproducibility the experiments have been repeated on a different sample at different time.

7.2. Exchange Mechanism

On addition of CD3OD the labile protons undergo exchange with the hydroxy deuterium of the solvent molecule. This time dependent phenomenon followed the first order reaction kinetics and the intensity of the labile protons was systematically reduced. The mechanism of exchange is pictorially illustrated in Figure 41.

Figure 41. The mechanism of H/D exchange of labile protons with the hydroxy cdeuterium of the solvent

molecule. The rate of deuteration is fast due to the high concentration of CD3OD compared to substrate.

Molecules 2017, 22, 423 31 of 44 To get qualitative information about the strength of intramolecular HBs, and electronic effect the different halo substituted benzamide molecules were also investigated. The intensity of the peak at atzero zero time time is taken is taken as 100%. as 100%. The other The other spectra spectra were wereacquired acquired after adding after adding 50 µL 50of CDµL3 ofOD CD to 3450OD µL to 450solution.µL solution. The plot The of plotintegral of integral areas of areas NH2 of peaks NH2 aspeaks a function as a function of time, of for time, unsubstituted for unsubstituted and ortho and- orthohalosubstituted-halosubstituted benzamides benzamides (2-fluoro, (2-ﬂuoro, 2-chloro, 2-chloro, 2-bromo, 2-bromo, 2-iodo) 2-iodo)are compared are compared in Figure in 42. Figure Similar 42. Similarresults for results meta for- andmeta para- and-substitutedpara-substituted derivatives derivatives have also have been also obtained been obtained [169]. [170].

Figure 42. The plot ofof integralintegral intensitiesintensities ofof thethe NHNH22 peakspeaks v/sv/s time (in mins) for unsubstituted and ortho-halosubstituted benzamides. The molecules are identiﬁedidentified by the symbolssymbols givengiven inin inset.inset.

The naturenature of of the the substituents substituents dictated dictated the H/Dthe H/ rateD constantsrate constants and they and were they different were different for different for substituents.different substituents. It has been It observed has been that observed when all thethat physical when all parameters the physical were keptparameters unchanged were for kept any ofunchanged the investigated for any molecules, of the investigated the results molecules, could be reproduced the results withcould high be reproduced degree of accuracy with high within degree the experimentalof accuracy within error [the170 ].experimental There is a perfect error match[169]. Th ofere graphical is a perfect plots match obtained of fromgraphical integral plots intensity obtained of thefrom labile integral protons intensity peaks of as the a function labile protons of time, peaks carried as out a function for different of time, samples carried at different out for timesdifferent for samples at different times for 2-fluorobenzamide. This is clearly evident from Figure 43. 2-ﬂuorobenzamide.Molecules 2017, 22, 423 This is clearly evident from Figure 43. 30 of 41

Figure 43. The integral intensity of NH2 peaks v/s time (in minutes) for 2-fluorobenzamide plotted Figure 43. The integral intensity of NH2 peaks v/s time (in minutes) for 2-ﬂuorobenzamide plotted from two experiments carried out at different times intervals are represented by two different from two experiments carried out at different times intervals are represented by two different symbols, shownsymbols, in shown the inset. in the inset.

8. Multiple Quantum (MQ) NMR for the Detection of Intramolecular HBs 8. Multiple Quantum (MQ) NMR for the Detection of Intramolecular HBs In deriving the evidence for C-F…H-N HB it is very important to know the precise magnitudes In deriving the evidence for C-F···H-N HB it is very important to know the precise magnitudes and also the relative signs of 1hJFH, 3hJFH, 2hJFN and 1JNH [65,69,170]. When three or more NMR active and also the relative signs of 1hJ , 3hJ , 2hJ and 1J [66,70,171]. When three or more NMR active spins are coupled among themselvesFH theFH magnitudesFN NHand signs of the couplings between the passive spins are coupled among themselves the magnitudes and signs of the couplings between the passive spins cannot be determined from the one-dimensional spectrum of any one of the coupled nuclei. spins cannot be determined from the one-dimensional spectrum of any one of the coupled nuclei. 2h Consequently, to obtain 2hJFN in organofluorine molecules several experimental strategies have been Consequently, to obtain JFN in organoﬂuorine molecules several experimental strategies have been reported where 15N is either isotopically labelled [111,170,171] or unlabelled [69]. reported where 15N is either isotopically labelled [112,171,172] or unlabelled [70]. 8.1. The Pulse Sequence

Figure 44. The pulse Sequence for DQ-SQ and ZQ-SQ experiments. For DQ and ZQ excitation, I and S spins are 1H and 15N of NH group. And the pulses were selective on these two spins. The phases of the

pulses φ1 and φR are x, −x, −x, x. The phase of all the remaining pulses are x. The gradients ratio G1:G2 employed are 50:55 for DQ-SQ and 50:45 for ZQ-SQ experiments. The τ delay for all the DQ-SQ and ZQ-

SQ experiments and also 15N-1H HSQC experiments was optimized for 1/2JNH (JNH is taken to be 90 Hz).

Molecules 2017, 22, 423 30 of 41

Figure 43. The integral intensity of NH2 peaks v/s time (in minutes) for 2-fluorobenzamide plotted from two experiments carried out at different times intervals are represented by two different symbols, shown in the inset.

8. Multiple Quantum (MQ) NMR for the Detection of Intramolecular HBs

In deriving the evidence for C-F…H-N HB it is very important to know the precise magnitudes and also the relative signs of 1hJFH, 3hJFH, 2hJFN and 1JNH [65,69,170]. When three or more NMR active spins are coupled among themselves the magnitudes and signs of the couplings between the passive spins cannot be determined from the one-dimensional spectrum of any one of the coupled nuclei. Consequently, to obtain 2hJFN in organofluorine molecules several experimental strategies have been reported where 15N is either isotopically labelled [111,170,171] or unlabelled [69].

8.1. The Pulse Sequence

The utilization of two dimensional 15N-1H correlation (HSQC) type experiments [172,173] gives information on the magnitudes of 1hJFH, 3hJFH, 2hJFN and 1JNH but fails to provide the signs of the passive couplings. Hence the multiple quantum NMR experimental methodology has been employed Molecules 2017, 22, 423 32 of 44 [174,175]. The application of two dimensional heteronuclear 15N-1H double quantum-single quantum (DQ-SQ) and zero quantum-single quantum (ZQ-SQ) correlation experiment, where 15N is present in 8.1. Theits natural Pulse Sequence abundance has been exploited for the determination of relative signs and magnitudes of the couplings among 1H, 19F and 15N, involved in hydrogen bonding [169]. The pulse sequence Theutilized utilization for DQ-SQ of twoand ZQ-SQ dimensional experiments15N-1 isH well correlation known and (HSQC) is reported type in experiments the Figure 44. [173 ,174] gives 1h 3h 2h 1 informationThe onMQ the experiments magnitudes have of beenJFH carried, JFH out, onJFN differentand J NHfluorinebut failssubstituted to provide derivatives the signs of of the passivebenzamide couplings. in solution Hence state in the a low multiple polarity quantum solvent CDCl NMR3. The experimental magnitudes and methodology signs of through has been employedspace [and175 ,176long]. range The application coupling interactions of two dimensional among fluorine, heteronuclear hydrogen15 N-and1 Hnitrogen double nuclei quantum-single in its natural abundance have been used to extract direct evidence for the existence of non-linear and/or quantum (DQ-SQ) and zero quantum-single quantum (ZQ-SQ) correlation experiment, where 15N three centered intra-molecular C-F...H-N type HB. The chemical structures and 1D 1H-NMR spectra is present in its natural abundance has been exploited for the determination of relative signs and obtained for the investigated molecules are given in the Figure 45. 1 19 15 magnitudesSubsequently of the couplings the theoretically among derivedH, F results and [63]N, involvedhave been inemployed hydrogen in conjunction bonding [170 with]. NMR The pulse sequencefindings utilized to draw for the DQ-SQ direct and and unambigu ZQ-SQ experimentsous conclusions is on well the knownintra-molecular and is reportedC-F…H-N type in the HB. Figure 44.

FigureFigure 44. The 44. The pulse pulse Sequence Sequence for for DQ-SQ DQ-SQ and and ZQ-SQZQ-SQ experiments. experiments. For For DQ DQ and and ZQ ZQexcitation, excitation, I and I S and S 1 15 spinsspins are 1 areH andH and15N N of of NH NH group. group. And the the pulses pulses were were select selectiveive on these on these two spins. two spins.The phases The of phases the of pulses φ1 and φR are x, −x, −x, x. The phase of all the remaining pulses are x. The gradients ratio G1:G2 the pulses φ1 and φR are x, −x, −x, x. The phase of all the remaining pulses are x. The gradients ratio employed are 50:55 for DQ-SQ and 50:45 for ZQ-SQ experiments. The τ delay for all the DQ-SQ and ZQ- G1:G2 employed are 50:55 for DQ-SQ and 50:45 for ZQ-SQ experiments. The τ delay for all the DQ-SQ SQ experiments and also 15N-1H HSQC experiments was optimized for 1/2JNH (JNH is taken to be 90 Hz). and ZQ-SQ experiments and also 15N-1H HSQC experiments was optimized for 1/2J (J is taken NH NH to be 90 Hz).

The MQ experiments have been carried out on different ﬂuorine substituted derivatives of benzamide in solution state in a low polarity solvent CDCl3. The magnitudes and signs of through space and long range coupling interactions among ﬂuorine, hydrogen and nitrogen nuclei in its natural abundance have been used to extract direct evidence for the existence of non-linear and/or three centered intra-molecular C-F...H-N type HB. The chemical structures and 1D 1H-NMR spectra obtained for theMolecules investigated 2017, 22, 423 molecules are given in the Figure 45. 31 of 41

1 FigureFigure 45. 45. ChemicalChemical structures structures and andexpanded expanded NH regions NH of regions the 1D ofH-NMR the 1Dspectra1H-NMR in CDCl3spectra of 2- in fluorobenzamide (41) 2-fluoro-N-(2-fluorophenyl)benzamide (42) and 2-fluoro-N-phenyl-benzamide (43). CDCl3 of 2-fluorobenzamide (41) 2-fluoro-N-(2-fluorophenyl)benzamide (42) and 2-fluoro-N-phenyl- 8.2.benzamide C-F…N-H (43 Hydrogen). Bond in 2-Fluorobenzamide

The two broad singlets with a separation of nearly 145 Hz observed in 1H-NMR spectrum of molecule 41, are attributed to two non-equivalent protons (δNH(1) > δNH(2)) [111]. The excessive broadening of the 1H spectrum due to quadrupolar 14N relaxation prevented the determination of through space and through bond couplings, if any. For visualization of such couplings if present, the heteronuclear 15N-1H DQ-SQ correlation experiments have been carried out using the pulse sequence given in Figure 36 [176,177]. The 15N-1H DQ-SQ correlated spectrum is reported in Figure 37, where DQ coherence evolve at the algebraic sum of the couplings between active (1H, 15N) and passive (19F) spins. Nearly equal 1JNH of each amide proton permitted the simultaneous excitation and detection of two different 15N-1H DQ spectra and are identified by the groups marked N-H(1) and N-H(2) in Figure 46.

Figure 46. 500 MHz 15N-1H DQ-SQ spectrum of molecule 41 in the solvent CDCl3.

For N-H(1) DQ excitation, H(1) and 15N are the active spins and H(2) and 19F are passive spins, while for N-H(2) DQ, H(2) and 15N are the active spins and H(1) and 19F are passive spins. The SQ dimension yields the normal spectrum of four coupled spins, viz., 15N, 19F and two NH protons. The nuclei involved in the DQ coherence flip simultaneously and can be treated as a single spin, also

Molecules 2017, 22, 423 31 of 41

Molecules 2017, 22, 423 33 of 44

Subsequently the theoretically derived results [64] have been employed in conjunction with NMR

ﬁndingsFigure to45. draw Chemical thedirect structures and and unambiguous expanded NH conclusions regions of onthe the1D intra-molecular1H-NMR spectra in C-F CDCl···H-N3 of type2- HB. fluorobenzamide (41) 2-fluoro-N-(2-fluorophenyl)benzamide (42) and 2-fluoro-N-phenyl-benzamide (43). 8.2. C-F···N-H Hydrogen Bond in 2-Fluorobenzamide

… 8.2. C-FTheN-H two Hydrogen broad singlets Bond in with2-Fluorobenzamide a separation of nearly 145 Hz observed in 1H-NMR spectrum of moleculeThe two41 broad, are singlets attributed with to a separa two non-equivalenttion of nearly 145 protons Hz observed (δNH(1) in 1>H-NMRδNH(2))[ spectrum112]. The of molecule excessive 1 14 41broadening, are attributed of theto twoH non-equivalent spectrum due toprotons quadrupolar (δNH(1) > δNH(2)N) relaxation [111]. The preventedexcessive broadening the determination of the 1H of spectrumthrough due space to quadrupolar and through 14 bondN relaxation couplings, prevented if any. Forthe visualizationdetermination of of such through couplings space and if present, through the 15 1 bondheteronuclear couplings, ifN- any.H For DQ-SQ visualization correlation of experimentssuch couplings have if pres beenent, carried the heteronuclear out using the 15 pulseN-1H sequenceDQ-SQ 15 1 correlationgiven in Figureexperiments 36[ 177 have,178]. been The carriedN- H out DQ-SQ using correlated the pulse spectrum sequenceis given reported in Figure in Figure 36 [176,177]. 37, where 1 15 19 TheDQ 15 coherenceN-1H DQ-SQ evolve correlated at the algebraicspectrum sum is reported of the couplings in Figure between 37, where active DQ ( coherenceH, N) and evolve passive at the ( F) 1 algebraicspins. Nearly sum of equalthe couplingsJNH of eachbetween amide active proton (1H, permitted15N) and passive the simultaneous (19F) spins. Nearly excitation equal and 1JNH detection of each 15 1 amideof two proton different permittedN- H the DQ simultaneous spectra and excitation are identiﬁed and detection by the groups of two marked different N-H(1) 15N-1H and DQ N-H(2) spectra in andFigure are identified 46. by the groups marked N-H(1) and N-H(2) in Figure 46.

15 1 FigureFigure 46. 46. 500500 MHz MHz 15N-N-1H HDQ-SQ DQ-SQ spectrum spectrum of ofmolecule molecule 4141 in inthe the solvent solvent CDCl CDCl3. 3.

ForFor N-H(1) N-H(1) DQ DQ excitation, excitation, H(1) H(1) and and 15N15 Nare are the the active active spins spins and and H(2) H(2) and and 19F19 areF are passive passive spins, spins, whilewhile for for N-H(2) N-H(2) DQ, DQ, H(2) H(2) and and 1515NN are are the the active active spins spins and and H(1) H(1) and and 1919F Fare are passive passive spins. spins. The The SQ SQ dimensiondimension yields yields the the normal normal spectr spectrumum of four of four coupled coupled spins, spins, viz., viz.,15N, 1519FN, and19F two and NH two protons. NH protons. The nucleiThe nucleiinvolved involved in the in DQ the coherence DQ coherence flip simultaneously ﬂip simultaneously and andcan canbe treated be treated as asa single a single spin, spin, also also called as super spin [179]. The 19F and 1H being passive spins, the four possible spin states yield four 15 1 1h 2h 2 1 transitions. The N- H(1) DQ coherence gives the parameters JFH(1), JFN, JH(1)H(2) and JNH(2) 15 1 3h 2h 2 1 while N- H(2) DQ coherence yields JFH(2), JFN, JH(1)H(2) and JNH(1). The marked separations 1 1 in the SQ dimensions of Figure 37 yield couplings (in Hz); a = JNH(1) (90.5), c = JNH(2) (89.2) and 2 i = JH(1)H(2) (3.05 Hz). The separations in DQ dimension provide magnitudes of the couplings (in Hz), 1 2 1 2 1h 2h 3h g = JNH(1) + JH(1)H(2) (93.5), e = JNH(2) + JH(1)H(2) (92.2), f = JFH(1) + JFN (3.8) and h = JFH(2) + 2h 1h JFN (10.4). The displacement of F2 cross sections yield respectively (in Hz), b = JFH(1) (11.2) and 3h d = JFH(2) (3.0). Opposite directions of F1 displacement vectors at δH(1) and δH(2), denoted by tilted 1h 3h 1 1 arrows, conﬁrm opposite signs of JFH(1) and JFH(2) [178,180]. The signs of JNH(1) and JNH(2) are negative because of the convention that one bond scalar coupling between two nuclei of opposite signs of magnetic moments is negative. The algebraic combination of parameters obtained from the 1h 3h 1h DQ spectrum established that the relative signs of JFH(1) and JFH(2) are opposite, that of JFH(1), 2h 1 2 JFN and JNH are same (negative), JH(1)H(2) is positive. Thus, the magnitudes and relative signs of the couplings among all the three nuclei could be obtained from a single 2D NMR experiment. This information can be utilized for ascertaining the existence of C-F···H-N hydrogen bonding, if any, Molecules 2017, 22, 423 32 of 41 called as super spin [178]. The 19F and 1H being passive spins, the four possible spin states yield four transitions. The 15N-1H(1) DQ coherence gives the parameters 1hJFH(1), 2hJFN, 2JH(1)H(2) and 1JNH(2) while 15N-1H(2) DQ coherence yields 3hJFH(2), 2hJFN, 2JH(1)H(2) and 1JNH(1). The marked separations in the SQ dimensions of Figure 37 yield couplings (in Hz); a = 1JNH(1) (90.5), c = 1JNH(2) (89.2) and i = 2JH(1)H(2) (3.05 Hz). The separations in DQ dimension provide magnitudes of the couplings (in Hz), g = 1JNH(1) + 2JH(1)H(2) (93.5), e = 1JNH(2) + 2JH(1)H(2) (92.2), f = 1hJFH(1) + 2hJFN (3.8) and h = 3hJFH(2) + 2hJFN (10.4). The displacement of F2 cross sections yield respectively (in Hz), b = 1hJFH(1) (11.2) and d = 3hJFH(2) (3.0). Opposite directions of F1 displacement vectors at δH(1) and δH(2), denoted by tilted arrows, confirm opposite signs of 1hJFH(1) and 3hJFH(2) [177,179]. The signs of 1JNH(1) and 1JNH(2) are negative because of the convention that one bond scalar coupling between two nuclei of opposite signs of magnetic moments is negative. The algebraic combination of parameters obtained from the DQ spectrum established that the relative signs of 1hJFH(1) and 3hJFH(2) are opposite, that of 1hJFH(1), 2hJFN and 1JNH are same (negative), 2 JH(1)H(2)Molecules is positive.2017, 22, 423 Thus, the magnitudes and relative signs of the couplings among all the three nuclei34 of 44 could be obtained from a single 2D NMR experiment. This information can be utilized for ascertaining the existence of C-F…H-N hydrogen bonding, if any, utilizing the theoretical results [63]. Nevertheless,utilizing the for theoretical unequivocally results [ascertaining64]. Nevertheless, the relative for unequivocally signs of 1hJFH(1) ascertaining, 3hJFH(2) and the 2hJ relativeFN, the 15 signsN-1H of 1h 3h 2h 15 1 15 1 zeroJ FH(1)quantum, JFH(2) 15N-and1H ZQ-SQJFN, the correlationN- H zero experiment quantum hasN- alsoH ZQ-SQbeen carried correlation out using experiment the identical has also pulsebeen sequence carried out with using appropriate the identical gradient pulse ratio sequence and phases with appropriate of the pulses. gradient The ZQ ratio coherence and phases evolves of the 1 15 atpulses. the algebraic The ZQ coherencedifference evolves of the atcouplings the algebraic between difference active of the(1H, couplings 15N) and between passive active (19F) ( spins.H, N) 19 Consequentand passive to ( theF) spins.opposite Consequent signs of magnetic to the opposite moments signs of ofexcited magnetic spins moments the coherence of excited evolved spins as the algebraiccoherence sums evolved [180]. asThis algebraic resulted sums in the [181 enha]. Thisnced resulted separation in the along enhanced the ZQ separation dimension along of ZQ-SQ the ZQ spectrumdimension given of ZQ-SQin Figure spectrum 47. given in Figure 47.

Figure 47. 500 MHz 15N-1H ZQ-SQ spectrum of 41 in CDCl . Figure 47. 500 MHz 15N-1H ZQ-SQ spectrum of 41 in CDCl3. 3

1h 2h 3h It Itis isevident evident that that signs signs of of1hJFH(1)JFH(1) andand 2hJFN areJFN oppositeare opposite to 3hJFH(2) to . TheJFH(2) similar. The similarstudies studies have also have been also carriedbeen carriedout for other out for molecules other molecules and the obtained and the obtainedresults established results established the presence the of presence fluorine ofinvolved ﬂuorine intramolecularinvolved intramolecular HB. HB.

9. 9.Conclusions Conclusions TheThe combined combined NMR NMR experimental experimental observations observations and and various various DFT DFT based based theoretical theoretical calculations calculations revealedrevealed the the existence existence of ofintramolecular intramolecular HBs HBs in di infferent different organofluorine-substituted organofluorine-substituted derivatives derivatives of differentof different classes classes of ofmolecules, molecules, viz. viz. benzanilides, benzanilides, hydrazides, hydrazides, imides, imides, benzamides, benzamides, and and diphenyloxamides.diphenyloxamides. The The existence existence of ofmore more than than one one conformer conformer has has also also been been established established in in several several moleculesmolecules by by adopting adopting diverse diverse NMR NMR experimental experimental st strategies.rategies. In In many many examples examples the the solvent solvent titration titration 1 andand 1H HDOSY DOSY techniques techniques are areemployed employed to discount to discount any possibility any possibility of self- of or self- cross-dimerization. or cross-dimerization. The variableThe variable temperature temperature and solvent and solvent titration titration studie studiess provided provided the thevaluable valuable information information about about the the 1 19 existenceexistence of ofHB, HB, and and their their relative relative strengths. strengths. The The 1H-19H-F HOESYF HOESY experiment experiment is utilized is utilized to extract to extract the informationthe information about about the spatial the spatial proximity proximity and andpossib possibilityility of intramolecular of intramolecular HB, HB, as aswell well as asfor for the the determination of different conformers. Through space coupling between two NMR active nuclei, where the spin polarization is transmitted through HB of diverse strengths has been detected in several fluorine containing molecules. The 2D 1H-15N HSQC experiment aided in the measurement of coupling strengths and their relative signs. The weak molecular interactions established by NMR studies have also been corroborated by theoretical DFT based structure calculations. The NCI analysis served as a very sensitive tool for the detection of non-covalent interactions. NCI analysis provides the visual evidence for the presence of bi and trifurcated HBs in some of the molecules. For the calculation of strengths of different HBs in the investigated molecules the QTAIM calculation is found to be very powerful tool. The Laplacian of electron density sign is used to discriminate the HBs from the covalent bonds. The relaxed potential energy scan has been performed for some of the molecules to determine the free rotation energy of CF3 group and the energies of different possible conformers for a molecule. The quantum calculations and classical MD simulations have supported the various possible ways of intramolecular HB formation with fluorine atom. The use of H/D exchange rates obtained by Molecules 2017, 22, 423 35 of 44 conventional 1H-NMR spectroscopic studies confirms the presence or absence of intramolecular HBs, and permitted the calculation of their relative strengths. The effect of inherent electronic and steric effects is also successfully correlated with the relative rates of H/D exchange in the various halo substituted derivatives of anilines and benzamides. The utilization of heteronuclear 15N-1H DQ-SQ and ZQ-SQ correlation experiment, in isotopically unlabeled systems for the detection of organic fluorine involved intramolecular HB and unequivocal determination of relative signs and magnitudes 1h 2h of both hydrogen bond and covalent bond mediated through scalar couplings such as JFH, JFN and 3h JFH has been convincingly demonstrated.

10. Computational Methods and the Employed Programs For DFT calculations Gaussian G09 [182] suite of programs has been used. The B3LYP/6-311G** and aug-cc-pVTZ level of theories and basis sets were used to optimize the structures of the molecules. To ensure that all optimized structures are global minima, harmonic vibrational frequency calculation has been performed at the same level of theories and all the real frequencies indicated that optimized geometry is minimum at potential energy surface. The MD simulations were performed using NAMD simulation package [183]. The general AMBER force field (GAFF) along with torsion parameters and partial charges obtained from quantum calculations in vacuum. The long range electrostatic interactions were calculated with the Particle Mesh Ewald (PME) method [184]. Constant pressure-temperature (NPT) simulation is performed followed by constant volume-temperature (NVT) simulation. AIMAll [185] and multiwfn programs were used for QTAIM calculations. Multiwfn [186] program was used for NCI plots. Colour filled isosurface graphs have been plotted by VMD [187] program. Specific combination of methods for theoretical calculations was used for particular and individual series of molecules, the specific information is available with the native articles. Molden 4.7 has been used as a visualization software for Gaussian outputs [188].

Acknowledgments: NS gratefully acknowledges the generous financial support by the Science and Engineering Research Board (SERB), New Delhi (Grant Number: EMR/2015/002263). Conflicts of Interest: The authors declare no conflict of interest.

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