Restricted amide rotation with steric hindrance induced multiple conformations

Chemical Physics Letters 689 (2017) 148–151

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Research paper

Restricted amide rotation with steric hindrance induced multiple conformations V.V. Krishnan a,b,⇑, Salvador Vazquez a, Kalyani Maitra a, Santanu Maitra a,⇑ a b

Department of Chemistry, California State University, Fresno, CA 93740, United States Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Davis, CA 95616, United States

a r t i c l e

i n f o

Article history: Received 5 August 2017 In final form 4 October 2017 Available online 9 October 2017 Keywords: Dynamic NMR Chemical exchange Restricted amide rotation Steric hindrance

a b s t r a c t The CAN bond character is dependent directly upon the resonance-contributor structure population driven by the delocalized nitrogen lone-pair of electrons. In the case of N, N-dibenzyl-ortho-toluamide (oDBET), the molecule adopts subpopulations of conformers with distinct NMR spectral features, particularly at low temperatures. This conformational adaptation is unique to o-DBET, while the corresponding meta- and para- forms do not show such behavior. Variable-temperature (VT) NMR, two-dimensional exchange spectroscopy (EXSY), and qualitative molecular modeling studies are used to demonstrate how multiple competing interactions such as restricted amide rotation and steric hindrance effects can lead to versatile molecular adaptations in the solution state. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction The origin of the rotational barrier around the CAN bond of amides is associated with its partial double bond character, which arises from resonance interaction between the lone pair of electrons on the nitrogen atom and the carbonyl p system. Dynamic nuclear magnetic resonance spectroscopy (DNMR) is often the chosen technique to investigate the partial double bond characteristics of the amide bond. With experimental demonstration using NMR spectroscopy in N, N-dimethylformamide, [1,2], there is sustaining interest in the field [3–10]. In the case of N, N-diethyl-o-toluamide (o-DEET), we recently demonstrated that the steric interactions between the methyl group at the ortho- position and the two N-ethyl groups modulate the restricted rotation of the amide bond kinetics [11]. In particular, the steric hindrance induced by the methyl group introduces an additional third higher-energy barrier by altering both the enthalpic and entropic contributions to rotational energy barrier as determined by DNMR measurements. The energy barrier to the restricted amide rotation is primarily determined by the pbond order of the CAN bond, and therefore the electronic effect of substituent groups on either side of the bond will influence the rotational barrier. The third high-energy conformation can ⇑ Corresponding authors at: Department of Chemistry, California State University, Fresno, CA 93740, United States. E-mail addresses: [email protected], [email protected] (V.V. Krishnan), [email protected] (S. Maitra). https://doi.org/10.1016/j.cplett.2017.10.013 0009-2614/Ó 2017 Elsevier B.V. All rights reserved.

be eliminated either by moving the methyl group to the meta/para positions of DEET [11] or by reducing the size of the nitrogen side-chain from the ethyl to a methyl group [12]. We explored the kinetic aspects of N, N-dibenzyl-o-toluamide (o-DBET), where the ethyl group is replaced by a benzyl group. The motivation behind this series of molecules (ortho, meta and para forms of DBET) is to investigate how the molecules adapt to competing interactions between restricted amide rotation, steric hindrance, increased size of the side-chain, as well as the relative juxtaposition of the two benzyl groups and the phenyl ring in the molecule. Variable temperature NMR experiments performed over a temperature range (1 °C–55 °C) did provide characteristic DNMR band shapes for the m-DBET and p-DBET molecules. However, in the case of o-DBET, to our surprise, the conformational states divided into two distinct sets of populations. The DNMR results show these are two independent sets of molecular conformations introduced due to the combination of geometric constraints induced by both the methyl group at the ortho position as well as the relative juxtaposition of the aromatic moieties present in the two benzyl groups. The presence of the two sets of conformational states is demonstrated experimentally using twodimensional exchange spectroscopy (EXSY), particularly at low temperatures. Thermodynamic parameters are estimated using variable temperature NMR experiments, and the molecular mechanics calculations further elucidate the complex conformational energy landscape for o-DBET in comparison with m-DBET and p-DBET.

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2. Materials and methods

For each value of a calculated, the dihedral angle b was changed
180° to +180° in steps of 5 °C, leading a matrix of 33  33.

2.1. Synthesis All chemicals were purchased from Sigma Aldrich or MERCK and were used as received without further purification. OrthoDBET: N, N-dibenzyl-o-methyl benzamide: To a solution of o-toluic acid (2.08 g, 15.3 mmol) in methylene chloride (28 mL), a catalytic amount of DMF (2 drops) was added with stirring. Freshly distilled thionyl chloride (2.3 mL, 31.0 mmol) was added to the flask, and the resulting solution was gently refluxed for two hours. The reaction mixture was concentrated under reduced pressure to remove excess thionyl chloride using a warm water bath (55 °C). The crude acid chloride was redissolved in methylene chloride (28 mL), and dibenzyl amine (5.88 mL, 30.6 mmol) was carefully added to the flask, dropwise, over the course of 2–3 min (Note: exothermic, produces gaseous HCl). The solution was allowed to stir at room temperature overnight. The organic layer was washed with water (30 mL) followed by 5% HCl (30 mL), 10% NaOH (30 mL), finally with brine (30 mL), and dried with anhydrous Na2SO4. The solution was filtered and concentrated under reduced pressure to yield a granular, slightly yellow solid before being dissolved in a minimum amount of hot hexanes. The solution was allowed to cool, and crystals were collected and dried to yield 1.98 g (41% overall) of the pure N, N-dibenzyl-o-toluamide. Each DEET analog was synthesized by following the above method and purified by crystallization (hot hexanes) or column chromatography (10% ethyl acetate in hexanes and 230–400 mesh silica gel) as required. Approximately 10 mg of each sample was dissolved in CDCl3 (total volume of 600 lL) for the NMR experiment. Each NMR tube was glass-sealed by first applying the freeze-thaw technique using liquid nitrogen to expel any dissolved air/oxygen, followed by sealing them using a small butane torch.

2.2. NMR spectroscopy All the NMR experiments were performed in a 400 MHz (1H resonance frequency) VNMRS spectrometer (Varian-Agilent) and using a one-NMR probe. The probe temperature was calibrated using MeOH [13]. The probe temperature was varied from 1 °C to 55 °C (in steps of 3 °C). One-dimensional, variable-temperature experiments were performed with 16 transients over 16 K complex points after calibrating the 90° pulse at each temperature. Samples were equilibrated for 20 min at each temperature, and a relaxation delay of 30 s was used between the transients. WinDNMR [14,15] was used to estimate the exchange rates (kex s
1). A three-site exchange model was used to fit the variable temperature line shape data of o-DBET, and a two-site exchange model was used to fit for m-DBET and p-DBET molecules. The constant line width of 6 Hz was used for all the fits. In the three-site model with a slow exchange between the third spin to first two spins was assumed. It is necessary to adopt a three-site model for o-DBET, due to chemical shift overlap between the spins in the up-field part of the exchange spectrum. The activation energy was estimated following the Eyring analysis (exchange rates vs. inverse of temperature in K). Two-dimensional exchange spectroscopy (EXSY) [16] was performed at three different temperatures (1 °C, 25 °C, and 55 °C) using a standard Nuclear Overhauser Effect Spectroscopy (NOESY) pulse sequence [17] using the procedure described previously [11]. Molecular mechanics calculations were performed using Avogadro as a function of two dihedral angles: a (CACACAO/ Aromatic-CO) and b (OACANAC) [18]. The dihedral angle a was changed from
180° to +180° in steps of 5 °C. The calculations can only be considered qualitative or at most semi-quantitative.

3. Results Fig. 1 shows the variable temperature NMR experiments of the ortho (o), meta (m) and para (p) – DBET molecules over the temperature range recorded by the variable temperature, between 1 °C and 51 °C. At low temperature (1 °C) the chemical shifts of the chemically exchanging methylene protons at the two sites are of m-DBET are at 5.45 ppm and 5.15 ppm, which reaches an intermediate exchange region at the high temperature (51 °C) (Fig. 1, panel m). The p-DBET also shows a similar chemical exchange behavior with the two distinct sites at 5.65 ppm and 5.38 ppm at low temperature (1 °C) and a coalesced single peak at higher temperatures (51 °C) (Fig. 1, panel p). At high temperature (51 °C), o-DBET molecule shows a broad resonance (5.32 ppm) representing an intermediate exchange and a sharp resonance (4.77 ppm) similar to a third high-energy conformation observed previously in the case o-DEET [11]. Upon lowering the temperature, a complex peak pattern emerges (Fig. 1, panel o). When the probe temperature reaches approximately below 10 °C, in addition to the splitting of downfield resonance (5.78 ppm), the upfield peak shows a complex line shape (centered at 4.81 ppm). The two distinct set of exchange peaks from o-DBET are seen in the two-dimensional exchange spectrum (Fig. 2) recorded at 1 °C with a mixing time of 300 ms. Two sets of spin systems identified are as follows: an (AX)2 spins system (dAX = 400.2 Hz and JAX = 12 Hz) and an (AB)2 spin system (dAB = 56.2 Hz and JAB = 16 Hz). These NMR parameters are determined by simulating the 1D NMR spectrum in the absence of chemical exchange (Fig. S1). Contribution from zero-quantum coherences in the EXSY experiment is expected to be significantly reduced due to long mixing time (300 ms) and the effect of solvent viscosity over the temperature range (Chloroform viscosity 0.699 mPa s at 0 °C–0.389 mPa s at 60 °C) is also expected to be insignificant [11]. Increasing the temperature alters the kinetics of the sub-spin systems separately (Fig. 1). Fig. 3 shows variable temperature NMR experiments of o-DBET (red lines) along with the corresponding line shape fitted using a three-site exchange model (black lines). At temperatures below 11 °C, a three-site model was unable to reproduce the line shape (Fig. 3). In contrast, the temperaturedependence of the m-DBET and p-DBET spectra follows the characteristic features corresponding to a two-site chemical exchange process. Fig. 4 shows the Eyring analysis plot (ln (kex/T) vs. 1000/

o

m

p 51°C

ppm

1°C

Fig. 1. Variable temperature DNMR spectra. (o) Temperature dependence of the NMR line shapes of the methylene protons in N, N-dibenzyl-o-toluamide (o-DBET, black), (m) N, N-dibenzyl-m-toluamide (m-DBET) and N, N-diethyl-m-toluamide (m-DEET, red) and (p) N, N-dibenzyl-p-toluamide (p-DBET, blue). The stacked plot for each molecule was recorded from 1 °C to 51 °C and in steps of 2 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

V.V. Krishnan et al. / Chemical Physics Letters 689 (2017) 148–151

ln kex (s-1)

150

1000/T (K-1)

ppm Fig. 2. Two-dimensional exchange spectroscopy (EXSY) spectrum of N, N-dibenzylo-toluamide (o-DBET) at 1 °C. Methylene region of EXSY spectrum shoes two distinct sets exchanging two-spin systems as marked by the dotted squares.

Fig. 4. Eyring analysis between the natural logarithm of reaction rate to temperature (exchange rate/Temperature) derived from the line shape analysis vs. inverse temperature. Black, red and blue symbols represent data from o-DBET, m-DBET, and p-DBET, respectively. The continuous line shows the linear best fit and the dashed lines correspond to prediction at 95% confidence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Intensity (Arbitrary Units)

(DHz = 49.8 ± 1.4 kJ/mol and DSz =
3.8 ± 0.1 J/mol K, adjusted R2 0.97), m- DBET (DHz = 85.8 ± 1.5 kJ/mol and DSz =
4.8 ± 0.9 J/mol

51°C

1788s-1

233s-1

25°C

49°C

1412s-1

204s-1

23°C

47°C

1193s-1

187s-1

21°C

45°C

1106s-1

154s-1

19°C

43°C

900s-1

134s-1

17°C

41°C

710s-1

110s-1

15°C

39°C

590s-1

60s-1

13°C

37°C

552s-1

29s-1

11°C

35°C

500s-1

÷2

9°C

33°C

455s-1

÷2

7°C

31°C

408s-1

÷2

5°C

29°C

336s-1

÷2

3°C

K, adjusted R2 0.98) and p- DBET (DHz = 73.5 ± 0.8 kJ/mol and DSz =
4.5 ± 0.5 J/mol K, adjusted R2 0.98). The results of the qualitative molecular mechanics calculations, performed as a function of the two independent dihedral angles a and b, for all three DBET molecules are presented in Fig. S2. Structures optimized using Gaussian and dihedral angles of the starting structures are arbitrarily assigned to zero, (a, b = 0; left lower corners of Fig. S2). The o-DBET molecule shows high-energy conformations with both a and b close to 270° (red patches of Fig. S2, panel o) with the corresponding low energy conformations at 120° ± 90°. Concerning b in o-DBET, high-energy conformations are found between
120° and 270°. Although the relative conformational landscape between the three molecules is similar, the semi-quantitative calculations of the energy barrier between the low and high energy conformations in the case of o-DBET is much higher (a120° (270°) and b270°). The conformational energy landscape as a function of the dihedral angles for the o-DBET has several high-energy ridges as result of the complex interaction between molecular structure involving the ortho-methyl group on the ring and the relative bulkiness of the phenyl rings.

27°C

308s-1

÷2

1°C

4. Discussion & conclusions

100 Hz Fig. 3. Line shape analysis of N, N-dibenzyl-o-toluamide (o-DBET). The variable temperature 1D experimental NMR spectra (red lines) of o-DBET are fit to a threespin exchange model (black lines). The probe temperature and the corresponding fitted values of the exchange rates are marked in each panel. The three spin exchange model fails to reproduce the line shape at temperatures below 11 °C. The small peaks are due to unidentified impurities. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

T) for o-DBET (black symbols, three-site model), m-DBET (red symbols, two-site model) and p-DBET (blue symbols, two-site model). Linear fit (95% confidence prediction intervals plotted as dashed lines of Fig. 4) was used to determine the activated enthalpy (DHz ) and entropy (Dsz ) of the exchange mechanisms: o-DBET

The changes in the number, shape, and multiplicity of chemical shifts observed in the NMR spectra of chiral organic compounds can be caused by restricted single bond rotation. The restricted rotation alleviates molecular symmetry causing atropisomerism (non-superimposable mirror images without chiral center) and generating diastereotopicity (non-superimposable, non-mirror image stereoisomers). The presence of two enantiomers or atropisomers (enantiomers or non-superimposable mirror images caused by restricted rotation) cannot be detected by conventional one-dimensional NMR spectrum. However, severe symmetry alteration resulting from restricted bond rotation can lead to the formation of diastereotopic protons with different chemical shifts detectable by 1D NMR spectroscopy. The greater number of signals and complexity of the peak shape and enhanced multiplicity are signs of the existence of diastereotopic protons. Variabletemperature (VT) NMR studies are ideal for capturing this phe-

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nomenon as the degree of single bond rotation varies proportionately with temperature changes: diastereotopicity of the methylene (CH2) group at low temperatures that is lost due to the increased rotation at higher temperatures [11,19]. For the meta and para versions of the DBET molecules, the restricted amide rotation follows the conventional expectations, two-site exchange mechanism that systematically goes through a coalescence with increasing temperature (Fig. 1). In contrast, for the o-DBET at high temperatures, the NMR spectrum resembles that of an apparent three site exchange, but lowering the sample temperature (<10 °C) indicates the presence of two distinct set of populations in each set (Fig. 1). The conglomerates of these sub-structures do not interact with each other as evidenced by cross peak pattern in the EXSY spectrum (Fig. 2). The final band shape of the two independently exchanging sets of spins can be fit to an (AX)2 and (AB)2 spin systems (Fig. S1). The chemical shift separation between the exchanging spins (Dm) is approximately 120 Hz for both the m-DBET and p-DBET, while the o-DBET shows an Dm of 400 Hz for the weakly coupled spin pair and 56 Hz for the corresponding strongly coupled spins (Figs. 1 and S1). It is important to note spectral integration for the spin system fits the total number of protons in each molecule, and the relative chemical shifts of methyl group (singlet) resonance (2.34 ppm) for all the molecules. Variable temperature NMR experiments used to determine the thermodynamic parameters (Figs. 3 and 4) do not show a large difference in the rotational barriers between the three molecules studied. Reaction enthalpy of m- DBET is the largest of the three ((DHz = 85.8 ± 1.5 kJ/mol), following the p- DBET (DHz = 73.5 ± 0.8 kJ/mol) and o-DBET (DHz = 49.8 ± 1.4 kJ/mol) while the entropic contributions are similar,
3.8 to
4.8 J/mol K. Further exploration of the conformational landscape of these molecules determined using qualitative molecular mechanics calculations (Fig. S2) show that the overall shape of the landscape is similar with the relative high and low energy barriers are distinctly highest in the o- DBET, followed by m- DBET and p- DBET. Simplification of energy landscape estimated as a function of the dihedral angles and the experimental determination of the energy barrier suggest that steric effect introduced by the methyl group are systematically reduced as the substitution moves from ortho, meta and para positions. We have shown that the o-DBET efficiently adapts to pair of independent set of molecules when forced with an alteration in the chemical structural changes. In comparison with o-DEET [11], the energy barrier for the o-DBET is perhaps higher to overcome the restricted amide rotation due to competing steric hindrance in between the o-methyl group and the side chain phenyl rings. Therefore, the molecule adopts sub-populations where independently each population is optimal within the constraints provided. This unique conformational arrangement by the o-DBET is perhaps the simple solution that these molecules prefer. Chemical exchange studies provide fundamental knowledge on the physical chemistry of the molecules, and it is an area of continuing interest to the research community [3–5,20–22]. Though restricted amide-rotation is one of the well-studied systems in the literature, a simple system such as o-DBET does provide surprising results that do not follow the convention.

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Acknowledgements Thanks to C Cortney for critical reading of the paper. SV was supported in part by a graduate fellowship from National Science Foundation (NSF Award # 1059994) and faculty initiated student research award from College of Science and Mathematics. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cplett.2017.10.013. References [1] H.S. Gutowsky, C.H. Holm, Rate processes and nuclear magnetic resonance spectra. 2. Hindered internal rotation of amides, J. Chem. Phys. 25 (6) (1956) 1228–1234. [2] W.D. Phillips, Restricted rotation in amides as evidenced by nuclear magnetic resonance, J. Chem. Phys. 23 (7) (1955) 1363–1364. [3] A.D. Bain, Chemical exchange in NMR, Prog. Nucl. Magn. Reson. Spectrosc. 43 (3–4) (2003) 63–103. [4] A.D. Bain, G.A. Webb (Ed.), Chemical exchange, in: Annual Reports on NMR Spectroscopy, vol 63, 2008, pp. 23–48. [5] A.D. Bain, J. Fisher (Ed.), Dynamic NMR, in: Modern NMR Techniques for Synthetic Chemistry, 2015, pp. 15–61. [6] M. Oki, Applications of dynamic NMR spectroscopy to organic chemistry, in: Methods in Stereochemical Analysis, VCH Publishers, Deerfield Beach, FL, vol. xii, 1985, pp. 423. [7] C.L. Perrin, T.J. Dwyer, Application of 2-dimensional NMR to kinetics of chemical exchange, Chem. Rev. 90 (6) (1990) 935–967. [8] M. Pons, O. Millet, Dynamic NMR studies of supramolecular complexes, Prog. Nucl. Magn. Reson. Spectrosc. 38 (4) (2001) 267–324. [9] W.E. Stewart, T.H. Siddall, Nuclear magnetic resonance studies of amides, Chem. Rev. 70 (5) (1970) 517–551. [10] K. Umemoto, K. Ouchi, Hindered internal-rotation and intermolecular interactions, Proc. Ind. Acad. Sci. Chem. Sci. 94 (1) (1985) 1–119. [11] V.V. Krishnan, W.B. Thompson, K. Maitra, S. Maitra, Modulations in restricted amide rotation by steric induced conformational trapping, Chem. Phys. Lett. 523 (27) (2012) 124–127. [12] S.C. Vazquez, Substituent effects on double bond character: NMR studies of steric and electronic effects in DEET analogs as a model system/by Salvador Cesar Vazquez, in: Chemistry, California State University, Fresno, College of Science and Mathematics, Henry Madden Library, 2014. [13] A.L. Van Geet, Calibration of methanol nuclear magnetic resonance thermometer at low temperature, Anal. Chem. 42 (6) (1970) 679–680. [14] N. Bampos, A. Vidal-Ferran, Understanding NMR multiplet structure with WinDNMR, J. Chem. Educ. 77 (1) (2000) 130–133. [15] H.J. Reich, WinDNMR: Dynamic NMR Spectra for Windows, JCE Software, 1995. [16] J. Jeener, B.H. Meier, P. Bachmann, R.R. Ernst, Investigation of exchange processes by 2-dimensional NMR-spectroscopy, J. Chem. Phys. 71 (11) (1979) 4546–4553. [17] A. Kumar, R.R. Ernst, K. Wüthrich, A two-dimensional nuclear overhauser enhancement (2D NOE) experiment for the elucidation of complete proton– proton cross-relaxation networks in biological macromolecules, Biochem. Bioph. Res. Co. 95 (1980) 1–6. [18] M.D. Hanwell, D.E. Curtis, D.C. Lonie, T. Vandermeersch, E. Zurek, G.R. Hutchison, Avogadro: an advanced semantic chemical editor, visualization, and analysis platform, J. Cheminform. 4 (1) (2012) 17. [19] B.L. Jensen, R.C. Fort, Molecular mechanics and variable-temperature H-1 NMR studies on N, N-diethyl-m-toluamide - an undergraduate NMR and molecular modeling experiment, J. Chem. Educ. 78 (4) (2001) 538–540. [20] V.V. Krishnan, N. Murali, Radiation damping in modern NMR experiments: progress and challenges, Prog. Nucl. Magn. Reson. Spectrosc. 68 (2013) 41–57. [21] M. Ababneh-Khasawneh, B.E. Fortier-McGill, M.E. Occhionorelli, A.D. Bain, Solvent effects on chemical exchange in a push-pull ethylene as studied by NMR and electronic structure calculations, J. Phys. Chem. A 115 (26) (2011) 7531–7537. [22] Z. Szalay, J. Rohonczy, Monte Carlo simulation of NMR lineshapes in chemically exchanging spin systems, Prog. Nucl. Magn. Reson. Spectrosc. 56 (2) (2010) 198–216.