Design, Synthesis, and Conformational Analysis of Oligobenzanilides as Multifacial α-Helix Mimetics

The design, synthesis, and conformational analysis of an oligobenzanilide helix mimetic scaffold capable of simultaneous mimicry of two faces of an α-helix is reported. The synthetic methodology provides access to diverse monomer building blocks amenable to solid-phase assembly in just four synthetic steps. The conformational flexibility of model dimers was investigated using a combination of solid and solution state methodologies supplemented with DFT calculations. The lack of noncovalent constraints allows for significant conformational plasticity in the scaffold, thus permitting it to successfully mimic residues i, i+2, i+4, i+6, i+7, and i+9 of a canonical α-helix.


Contents
: Geometrical properties of the two crystallographically independent form of 16 calculated from the X-ray crystal structure.
163.6 52.9 40.6 3-R a -156.7 -48.2 -38.9   Figure S4: Experimental and simulated 1 H NMR spectra of dimer 16 at the slow exchange limit. Inlets show anisochronicity observed for methylene protons 2-Hα and 2-Hα' (blue) and methyl doublets 2-Hγ', 2-Hδ' and 1-Hβ' (red). DOI  All calculations were performed with ultrafine grid (integral=grid=ultrafine) and without restriction on symmetry (No Symm). All transition states were characterized by normal coordinate analysis revealing precisely one imaginary mode corresponding to the intended reaction. Vibrational frequency calculations only were performed using 6-311++G(d,p) basis set for selected transition states as per Table S9.
The geometry of the molecular structure determined by X-Ray diffraction was used as a starting point to build a molecular structure where all alkyl groups have been replaced by methyl groups. Geometry optimisation of this molecule model led to compound I anti/cis (S7) which will be the reference (∆G 298 = 0 kcal/mol) and the starting point of the following calculations. Full coordinates for all the stationary points are available on the data repository at DOI: 10.14469/hpc/5171.

Amide bond rotation (dihedral angle ω1 rotation):
A relaxed scan of the amide dihedral angle ω1(36 x 10 • ) was performed using I anti/cis as starting point. The total energy (kcal/mol) was plotted against the dihedral angle ω1 and show a series of minima and maxima as per Figure S9. Maxima were subjected to transition state optimisation. Minima were deduced from the corresponding TS (either using IRC calculations or by moving atoms along the negative frequency of the TS).     Figure S15: Rotation of the amide bond only leading to a cis/trans conformational exchange from a syn' conformation via transition state I-TS3 (∆G 298 = 14.4 kcal/mol) from a syn' conformation.  Transition states I-TS4 and I-TS5 were found with the O-methoxy of the benzamide not   'passing' underneath the amide moiety and thus not leading to a syn/anti interconversion, contrary to I-TS1 and I-TS6 (described in Figure S10 and Figure S19). It appears that the O-methoxy 'moves away' to allow the carbonyl rotation.    Figure S18: Rotation of the amide bond only leading to a cis/trans conformational exchange from a syn conformation via transition state I-TS5 (∆G 298 = 16.2 kcal/mol).
To cover every possibility, from the geometry of I-TS1 corresponding to an anti/cis ↔ syn/trans conformational exchange, I-TS6 corresponding to a syn/cis ↔ anti/trans conformational exchange was calculated and found to be ∆G 298 = 12.4 kcal/mol.  and I-TS4, respectively). Depending on the orientation of the Ar-N moiety regarding the carbonyl of the amide group, rotation of the amide bond will allow the carbonyl group to push the O-methoxy substituent of the Ar-N and induce concerted rotations (as per I-TS1 and III-TS6).

Ar-C(O) rotation (dihedral angle ω2 rotation)
A relaxed scan of the Ar-C(O) dihedral angle ω2 (36 x 10 • ) was performed using an extended conformation (II) as starting point. The total energy (kcal/mol) was plotted against the dihedral angle ω2 and show a series of minima and maxima as per Figure S20. Maxima were subjected to transition state optimisation. Minima were deduced from the corresponding TS by moving atoms along the negative frequency.  A relaxed scan of the Ar-C(O) dihedral angle ω2 (36 x 10 • ) was performed using I syn/trans as starting point. The total energy (kcal/mol) was plotted against the dihedral angle ω2 and shows a series of minima and maxima as per Figure S22. Maxima were subjected to transition state optimisation. Minima were deduced from the corresponding TS by moving atoms along the negative frequency. Figure S22 shows that Ar-C(O) bond rotations in trans conformations occur with energy barriers below 10 kcal/mol on the PES (I anti/cis as reference with ∆G 298 = 0 kcal/mol).  Does not induce cis/trans conformational exchange (no concerted amide bond rotations).   Figure S23: Ar-C(O) dihedral rotation energy profile in trans-conformation (from relaxed scan as per Figure S22).

Ar-N roation (dihedral angle ω3 rotation)
From I anti/cis , a relaxed scan of the Ar-N dihedral angle (72 x 10 • ) was performed. The total energy (kcal/mol) was plotted against the dihedral ω3 and show a series of minima and maxima as per Figure S24. Maxima were subjected to transition state optimisation followed by IRC calculations to confirm the identity of the TS. Both ends of the IRC were subjected to optimisation and the output geometries were found in good accordance with the minima found in the scan (excepted when notified). As deduced form IRC, III-TS5 was found to directly lead to III anti/cis . III anti/cis and the corresponding III-TS6 (as noted in Figure S24) were calculated but were not included in the reaction profile.    Figure S25: Ar-N dihedral angle ω3 rotation energy profile (as per Figure S24).

Name
The transition state corresponding to a solely dihedral rotation of the Ar-N bond in constrained cis conformation has not been found upon explicit optimization of the geometries of the maxima from the above restricted scan. This is likely due to the steric bulk induced by the ortho substituent of the Ar-N. Thus, rotation about the Ar-N bond either trigger the amide bond rotation and lead to a trans conformation (as per I-TS1 G298=12.

Boltzmann Distributions:
The population distributions of the extended vs folded conformations were calculated based on their relative energy via amide, Ar-N or concerted rotations using the following assumptions: • The population difference is a function of two dihedral angles (Ar-N and amide).
• Take X-ray crystal structure as minimum energy conformation.
• Entropies of the conformers are similar.
Calculations were performed as follows: The probability that state Ei is occupied is; The constant C can be found by summing over all P i , which should give unity-the probability that some state is occupied. Thus, 1 = C P i , or C = 1 z , where Z = P i is called the partition function. Each state of the system is represented in Z by its Boltzmann factor.
Boltzmann factor at 298 K and at standard state (1 M) is; The resulting population distribution is;  High resolution mass spectrometry (HRMS) data were acquired by the Imperial College To a stirring solution of amine (1 eq.) in anhydrous dichloromethane (10 mL/g) was added anhydrous triethylamine (1.2 eq.) under a nitrogen atmosphere. The reaction mixture was cooled to 0°C and di-tert-butyl dicarbonate (1 eq.) was added portion-wise over 10 min. The reaction mixture was warmed to room temperature and stirred for 16 h under a nitrogen atmosphere. The reaction was followed by TLC (ninhydrin). The reaction mixture was diluted with dichloromethane (50 mL/g). The organic layer was washed with 0.1 M hydrochloric acid (10 mL/g x2), water (10 mL/g) and brine (10 mL/g), dried (MgSO 4 ) and concentrated in vacuo. A stirring solution of alcohol (1 eq.) in anhydrous dichloromethane (10 mL/g) was cooled to 0°C under a nitrogen atmosphere. DessMartin periodinane (1.2 eq.) was added portion-wise over 5 min. The reaction mixture was stirred at 0°for 15 min, then warmed to room temperature slowly. The reaction was followed by TLC (dinitrophenylhydrazine).

Procedure B (Oxidation of alcohol)
Upon completion, the reaction mixture was diluted with diethyl ether (100 mL/g) and 10% sodium thiosulphate solution (25 mL/g) and saturated sodium bicarbonate solution (25 ml/g) were added. The resulting suspension was stirred rapidly until the precipitate was fully dissolved. The layers were separated and the aqueous layer was extracted with diethyl ether (2 x 50 mL/g). The organic layers were combined and washed with 10% sodium thiosulphate solution (2 x 20 mL/g), saturated sodium bicarbonate solution (2 x 20 mL/g) and brine (20 mL/g), dried (MgSO 4 ) and concentrated in vacuo. The product was verified by 1 H NMR and carried forward without further purification. (2.5 eq.) was suspended in anhydrous tetrahydrofuran (20 mL/g) and the alcohol (1.1 eq.) was added dropwise at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at 0°C for 15 min. 3-fluoro-4-nitrobenzoic acid (1 eq.) was added portion-wise over 10 min at 0°C with rapid stirring. The reaction mixture was stirred at 0°C for 15 min, then warmed to room temperature slowly. The reaction was followed by TLC (bromocresol green) and LC-MS. Upon completion, saturated NH 4 Cl (10 mL/g) was added and the reaction mixture was poured into ethyl acetate (50 mL/g). The organic layer was washed with 1 M hydrochloric acid (20 mL/g x 3), water (20 ml/g) and brine (20 ml/g). The organic layer was dried (MgSO 4 ), filtered and concentrated in vacuo.

18-24 h
Procedure adapted from Murphy et al. 2 To a stirred solution of methyl-3-hydroxy-4nitrobenzoate in (1 eq.) and potassium carbonate (5 eq.) in dimethylformamide (10 mL/g) was added bromide (1.5 eq.). The reaction mixture was warmed to 50°C under a nitrogen atmosphere. The reaction was followed by TLC (potassium permanganate) and LC-MS.
Upon completion, the reaction mixture was cooled to room temperature, poured into water (20 mL/g) and extracted with ethyl acetate (3 x 100 mL/g). The combined organic fractions were washed with 5% lithium chloride (20 mL/g x 2), water (20 mL/g x 2) and brine (20 mL/g), dried (MgSO 4 ) and concentrated in vacuo. tetrahydrofuran (20 mL/g, 1:1, v/v) was added 10 % sodium hydroxide solution (10 mL/g) and the reaction mixture heated to 40°C. The reaction mixture was stirred for 16 h and followed by TLC (ninhydrin and bromocresol green) and LC-MS. Further equivalents of alcohol and sodium hydride were added if required. Upon completion, the organic solvents were removed in vacuo, the residue dissolved in water (20 mL/g) and acidified to pH 4 with conc. hydrochloric acid. The precipitate was extracted with dichloromethane (50 mL/g x 3), the combined organic extracts washed with water ( 20 mL/g x 2) and brine (20 mL/g x 1), dried (MgSO 4 and concentrated in vacuo. Procedure F (Hydrogenation -Pd/C) The reaction mixture was stirred gently and followed by TLC (ninhydrin) and LC-MS.
Upon completion, the reaction was filtered through a celite plug and concentrated in vacuo.
The residue was taken up in dichloromethane and washed with water ( 20 mL/g x2) and brine (20 mL/g x 1), dried (MgSO 4 and concentrated in vacuo. In the majority of cases, the product was of sufficient purity to take forward without further purification. Procedure G (Reductive Amination) eq) in anhydrous chloroform (10 mL/g) was added dropwise to a solution of secondary aniline (1 eq) and sodium hydrogen carbonate (1.2 eq) in anhydrous chloroform (20 mL/g).
The reaction mixture was stirred at 50°C under a nitrogen atmosphere and followed by TLC (ninhydrin). Upon completion, the reaction was concentrated and the crude reaction mixture was dissolved in dichloromethane (50 mL/g). The organic layer was washed with 1 M hydrochloric acid (20 mL/g x3) and brine (20 mL), dried (MgSO 4 , filtered and concentrated in vacuo. Purification by column chromatography afforded the desired product.  The pre-activated monomer solution was added to the resin and stirred gently at room temperature for 16h under a nitrogen atmosphere. The reaction mixture was transferred to a syringe and filtered under water vacuum. The resin was washed (Section ) and dried under a flow of nitrogen. Remaining reactive sites on the resin were acetylated with acetic anhydride (5% in DMF, v/v, 10 mL/g, 45 min). The resin was washed and stored under reduced pressure in a desiccator. Exact resin loading was calculated via UV-Vis spectroscopy (vide infra).

Variable Temperature NMR Spectroscopy
The barrier to rotation about the Ar-N axis (ΔG), the rate of enantiomerisation (k ) and the half-life of racemisation (t 1/2 ) were calaculated using the equations presented by Sandstrom. 7 If two chemically equivalent nuclei are exchanged by an intermolecular process (e.g. A B), the observed NMR spectrum is a function of the difference in resonance frequencies (Δν A -Δν B ) and the rate of exchange (k ) ( Figure S36).  Figure S36: The exchange regimes observed in a reversible, unimolecular process . At the slow exchange limit, the rate of chemical exchange is much slower than the NMR timescale and so the NMR spectrum consists of two resonances. At the coalesence temperature, the rate of chemical exchange is approximately equal to the NMR timescale and so a single broad resonance is observed in the NMR spectrum. When the rate of chemical exchange is fast compared to the NMR timescale, the NMR spectrum appears as a single resonance at the mean of the chemical shifts observed in the slow exchange regime. This is known as the fast exchange limit.
At the coalesence temperature, assuming an equal population of conformers, the lifetime of a conformation is equal to: Assuming first-order kinetics, the rate constant (k ) is inversely proportional to the lifetime τ: Therefore, at the coalesence temperature, the rate of exchange between the two species is equal to: k = π∆ν o √ 2 (for uncoupled signals) (4) k = π (∆ν o ) 2 + 6(J AB ) 2 2 (for coupled signals) These approximate values of k were refined using gNMR v5.0 (https://home.cc.umanitoba. ca). The Gibbs Free Energy of Activation (ΔG ‡ ) is related to the rate constant by the Eyring equation; where κ is the transmision coefficient (assumed to be equal to 1 in most cases), k b is the Boltzmann constant, T is the temperature, h is the Planck constant and R is the gas constant.
Therefore, substituting the value for k into the following formula gives the value of ΔG ‡ in kJ/mol; ∆G ‡ = 0.01914 × T c × (10.319 + log 10 ( T c k )) (7) a Barrier to bond rotation at coalescence temperature. b t 1/2 = ln2/2k -corresponds to the rate of racemisation. k is the rate constant for enantiomerisation of the amide. c Thermodynamic parameters for 2 could not be determined due to spectral crowding. .

X-ray Crystallography
The X-ray crystal structure of 16 The structure of 16 was found to contain two crystallographically independent molecules (3-A and 3-B) in the asymmetric unit. The OH and NH hydrogen atoms on O14A, N26A, O14B and N26B were all located from ΔF maps and refined freely subject to OH and NH distance constraints of 0.90Å.