Antibacterial and ATP Synthesis Modulating Compounds from Salvia tingitana

A surface extract of the aerial parts of Salvia tingitana afforded a nor-sesterterpenoid (1) and eight new sesterterpenoids (2–-9), along with five known sesterterpenoids, five labdane and one abietane diterpenoid, one sesquiterpenoid, and four flavonoids. The structures of the new compounds were established by 1D and 2D NMR spectroscopy, HRESIMS, and VCD data and Mosher’s esters analysis. The antimicrobial activity of compounds was evaluated against 30 human pathogens including 27 clinical strains and three isolates of marine origin for their possible implications on human health. The methyl ester of salvileucolide (10), salvileucolide-6,23-lactone (11), sclareol (15), and manool (17) were the most active against Gram-positive bacteria. The compounds were also tested for the inhibition of ATP production in purified mammalian rod outer segments. Terpenoids 10, 11, 15, and 17 inhibited ATP production, while only 17 inhibited also ATP hydrolysis. Molecular modeling studies confirmed the capacity of 17 to interact with mammalian ATP synthase. A significant reduction of ATP production in the presence of 17 was observed in Enterococcus faecalis and E. faecium isolates.

T he most evident challenge to the treatment of several infectious diseases is the increasing rate of bacterial resistance to several antibiotics. 1 The development of new drugs has decreased alarmingly; in the past half century, only a few new classes of antibiotics have entered the clinic, and the elaboration of novel therapies is urgently required. 2−4 The lipophilic extracts of plant surfaces were shown to possess antimicrobial activities, 5−7 due to the secretion of defense compounds onto the cuticular layer. 8−10 In a search for diterpenoids from Salvia species with activity against multidrug-resistant human clinical strains, 11−13 the aerial parts of Salvia tingitana Etl. (Lamiaceae) were investigated. The species is an aromatic woody perennial shrub originating from North Africa and the Middle East and is now cultivated as an ornamental plant in different parts of the world. 14 S. tingitana could be extinct in North Africa, 15 and the only known recent collection is from Saudi Arabia. 16 For a long time the taxonomic interpretation of the species was not clear, 15−17 but recent studies defined S. tingitana as a distinct species and separate from S. sclarea and other Salvia species that in the past have been considered as related to it. 14 Herein we report the isolation and structure elucidation of compounds obtained from the CH 2 Cl 2 -soluble extract of the plant surface and their antimicrobial activity. The microbial species selected for the study were mainly Gram-positive species, belonging to the Staphylococcus and Enterococcus genera. Staphylococci and methicillin-resistant-Staphylococci (MRS), particularly Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), are normally present on human skin and mucosa. They are responsible for a wide range of mild to life-threatening infections. MRSA are considered to be major pathogens for humans, causing hospital-and community-acquired conditions, such as sepsis, pneumonia, skin and soft tissue infections, endocarditis, and many other serious ailments. 18,19 In addition, MRSE, due to their ability to produce biofilms, can generate difficult-to-eradicate infections, like those occurring on prostheses, on intravenous catheters, or in carriers of cardiac valvular lesions. 19 For their relevant etiological role in several clinical settings, Enterococcus faecium and S. aureus have been included in a particular group of drugresistant pathogens, acronymically referred to as "ESKAPE" (E. faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), against whom the search for new curative antibacterial agents has become critically urgent. 20 The Gram-positive Enterococcus genus includes facultative anaerobic species that can normally inhabit the human intestine as commensals. After dispersion in the hospital environment they can survive in the wards for long periods and may easily contaminate patients and the surface of medical equipment. 21 E. faecium and E. faecalis, the two most relevant clinical species, are characterized by high levels of intrinsic and acquired antibiotic resistance, mainly expressed toward β-lactams and glycopeptides such as vancomycin (VRE). 21,22 They are involved in several nosocomial conditions, including urinary tract infections, as well as in serious bacteraemias, endocarditis, and meningitis. 21 In addition, these compounds were investigated for the modulation of ATP synthase activity. ATP synthase is associated directly or indirectly with various human diseases, 23,24 and the search for natural and synthetic inhibitors of this protein complex may generate new lead compounds, 25−27 including new antimicrobial agents. 24,28 Modulation of ATP synthesis has been described as the underlying principle for the activity of various compounds against multidrug-resistant mycobacteria, Gram-positive pathogens 25,29 including S. aureus and Streptococcus pneumoniae, 30 and fungi. 31 ATP synthase is known to be conserved from bacteria to mitochondria and chloroplasts. 28,32,33 Similar overall structures of ATP synthase monomers or dimers have been described in nonrelated organisms such as prokaryotes, yeasts, and mammalian species. 23 The possibility of selective inhibition of the bacterial enzyme by modulation of specific bacterial subunits is at present considered at the base of pathogen ATP synthases as potential drug targets, 32 and thus of the so-called sixth antibiotic target space. 23,34 Given that the overall structure and energy transduction mechanism of the Ftype ATP synthases are well conserved from bacteria to mammalians, 35−38 purified mammalian rod outer segments (OS) were used as a subcellular system, allowing the rapid assay of the modulating action of the isolated compounds on the ectopic F o F 1 -ATP synthase. 39 −41 In fact, the OS are composed of a stack of membranous disks, naturally sealed vesicles expressing the molecular machinery for the complete oxidation of glucose, thereby comprising the tricarboxylic acid cycle, 42 and the five complexes of respiration. 43,44 This could provide some indication of a possible correlation between the antibacterial activity and the modulation of the ATP synthase, in view of deeper investigations. Docking, molecular dynamics (MD) simulation studies, and ligand−protein binding energy evaluations were used to analyze the interaction of the most active compound with ATP synthase. Finally, its effect on ATP production in bacterial cells was evaluated.

■ RESULTS AND DISCUSSION
The lipophilic extract of the plant surface of S. tingitana afforded one nor-sesterterpenoid (1), eight new sesterterpenoids (2−9), and five known sesterterpenoids, along with other known compounds including five labdane and one abietane diterpenoid, one sesquiterpenoid, and four flavonoids. H COSY and 1D TOCSY measurements allowed establishment of the spin systems C-1− C-3, C-5−C-7, C-9−C-12, and C-14−C-16. The NMR data and the index of hydrogen deficiency indicated that the structure was tricyclic. The deshielded shift of C-4 (δ C 73.4), C-3 (δ C 42.0), and C-5 (δ C 59.7) suggested the presence of an oxygenated group at C-4, confirmed by the HMBC correlations of H 2 -2/C-4, H 2 -3/C-4, H-5/C-4, and H 3 -24/C-4. These data, and the comparison with related sesterterpenoids, led to the conclusion that 1 was a C-23 norsesterterpenoid. The location of a hydroxy group at C-6 was confirmed by the HMBC correlations of H-5/C-6, H-6/C-5, and H 2 -7/C-6. The presence of an α,β-unsaturated butenolide moiety 46,47 at C-15 was inferred on the basis of the H-18 resonance, the C-19 carbonyl resonance (δ C 172.6), the HMBC correlations of H 2 -15/C-17, H-16/C-18, H-18/C-16, H-18/C-17, H-18/C-19, and H 3 -20/C-16 and C-18, and the long-range COSY coupling between CH 3 -20 and H-18. The NOESY correlations between H-6, H 3 -22, H 3 -24, and H 3 -25 and the correlation of H-5 with H-9 indicated a trans-junction of the decalin system and a β-orientation of H-6 and CH 3 -24. The E configuration of the Δ 13 (14) double bond was established from the 13 C chemical shift of C-21. 48 Thus, the structure of compound 1 was defined as (13E)-4α,6α,8α-trihydroxylabd-13 (14),17(18)-dien-16,19-olide. Only a few nor-sesterterpenoids have been reported from species of Salvia. 49 (Table 1) closely resembled those of the methyl ester of salvileucolide, 52 with the exception of the presence of a methylene group at C-6 (δ H 1.35, 1.51; δ C 23.7) instead of a hydroxymethine. This was confirmed by the HMBC correlations of H 2 -6/C-5, C-7, C-8, C-10 and H 2 -7/C-6. J values of H-5 (δ H 1.77, dd, J = 12.0, 2.3 Hz) and NOESY data confirmed the same relative configuration as for the methyl ester of salvileucolide. 52,53 The NOESY correlations between H-5 and H-9 and between H 3 -22, H 3 -24, and H 3 -25 were consistent with a β-orientation of Me-22, Me-24, and Me-25 and with a trans-ring junction of the decalin system. Thus, compound 2 and its relative configuration were identified as (13E)-8α-hydroxy-23-carboxymethyllabd-13 (14), 17 Table 2) showed similarities with 2, except for the presence of a 6,23-epoxide moiety, supported by HMBC correlations of H-6 (δ C 73.6, C-6 and δ H 3.72, ddd, J = 11.4, 11.3, 4.4 Hz) with C-4 and C-23 and HMBC correlations of H-23 (δ C 112.6, C-23 and δ H 4.43, s) with C-6. The relative configuration at C-4, C-5, C-6, C-9, and C-10 was established considering the coupling constants of H-5 (δ H 1.49, d, J = 11.4 Hz), H-6 (δ H 3.72, ddd, J = 11.4, 11.3, 4.4 Hz), and H-9 (δ H 1.09, t, J = 5.2, 5.2 Hz) and the NOESY correlations of H-6 with H 3 -22, H 3 -24, and H 3 -25 and of H-5 with H-9 and H 3 -OMe/C-23. The E geometry of the Δ 13 double bond was inferred from the 13 C chemical shift of C-21. Thus, compound 5 was identified as (13E)-8α-hydroxy-23α-O-methyl-23,6α-epoxylabd-13 (14) (Table  3) closely resembled those of 5, with the exception of the presence of a hydroxy group at C-23 (δ H 4.43, s, H-23; δ C 111.1) instead of a methoxy group in 5. The location was corroborated by an HMBC correlation of H 3 -24/C-23. ROESY correlations of H-6 with H 3 -22, H 3 -24, and H 3 -25 and of H-23 with H 3 -24 and the 13 C chemical shift of C-21 confirmed the same relative configuration as for compound 5. Thus, the structure of 7 and its relative configuration were defined as (13E)-8α,23α-dihydroxy-23,6α-epoxylabd-13 (14),17(18) (Table 3) exhibited 25 carbon resonances corresponding to six methyl, eight methylene, one hydroxymethylene (δ C 58.4), four methine, one oxymethine (δ C 70.5), four quaternary carbons, and one oxygenated tertiary carbon (δ C 74.3). Two of the four degrees of hydrogen deficiency were accounted for by two double bonds (Table 3). Thus, the compound contained only two rings. This was corroborated by the absence of a carbonyl signal at C-19 as in 1 (δ H 4.18, dd, J = 12.6, 6.8 Hz and 4.04, dd, J = 12.6, 6.9 Hz, H 2 -19; δ C 58.4, C-19) and by the chemical shifts of H-16 (δ H 4.49, t, J = 6.9, 6.9 Hz) and C-16 (δ C 70. In cases where enough material was available, the absolute configurations were studied by vibrational circular dichroism (VCD) and complemented by a Mosher ester analysis to determine the configuration at C-15 for compounds 3 and 6. After derivatization of the C-15 carbinol moiety with methoxytrifluoromethylphenylacetic acid (MTPA, Mosher's reagent), the 1 H NMR chemical shifts of the resulting diastereomeric esters were compared. 55 VCD spectra of compounds 2, 3, 6, and 7 were recorded and compared to their calculated spectra at the B3LYP/6-31+G(d,p) level of theory ( Figures S15, S23, S47, and S56, Supporting Information). Similarity indices SimVA (vibrational absorption) and SimVCD were calculated with VCD SpecTech 57 using scaling factors between 0.8 and 1.2 ( Figures  S15, S23, S47, and S56, Supporting Information). The scaling factor corresponding to the maximal value of SimVA calculated for all configurations was used to plot the calculated spectra.  for (4R,5R,6S,8R,9R,10S,16R,23S). Therefore, no assignment was possible. Since the results from the VCD data of compound 6 conflicted with those for the Mosher esters, VCD data were thoroughly reviewed. It was found that most of the vibrational bands originated from the decalin system. The stereocenters at C-15 and C-16 did not impact the VCD spectra to an extent enabling the determination of the absolute configuration. Therefore, VCD was not considered suitable for this type of compounds. Owing to a lack of material, a hydrolysis of the lactone rings followed by a Mosher ester analysis was not possible. For compounds 3 and 6, comparison of the NMR data with those from reported congeners 46 indicated the (16S) configuration. For compounds 2 and 7, the configuration could be 16R, as previously described. 47,58 Hence  (24), 68 and cirsimaritin (25). 69 The extract, the n-hexane-insoluble and -soluble portions, and 13 semipurified fractions (I a −VI a and I b −VII b ) were tested against 12 representative clinical strains (Table S1, Supporting Information). The total extract showed MIC values of 128 μg/ mL on S. aureus, S. epidermidis, E. faecium, and E. faecalis strains, while MICs > 128 μg/mL were found against the two other Gram-positive species (Streptococcus agalactiae and Streptococcus pyogenes), the four Gram-negative bacterial strains (Escherichia coli, Proteus mirabilis, Moraxella catarrhalis, and K. pneumoniae), and the two fungi (Candida albicans and Candida glabrata). Likewise, the n-hexane-insoluble and -soluble portions and the 13 semipurified fractions showed some activity against the Gram-positive strains but were inactive against the three Gram-negative bacteria and the two Candida strains (Table S1, Supporting Information). Nineteen compounds out of the 25, isolated in suitable quantities for biological assays, were analyzed for antibacterial activity by determining MIC values on a panel of 30 microbial clinical strains, mainly Gram-positive pathogens, belonging to several clinically relevant species of Staphylococcus and the Enterococcus genera. As depicted in Table 4, interesting results were obtained especially for Staphylococci and Enterococci, while MIC values above 128 μg/mL were obtained for S. agalactiae, S. pyogenes, the four Gram-negative species, and the two mycetes (data not shown). Interestingly, the antimicrobial activities observed were often uniform among the bacterial species and independent from the resistance patterns of the several isolates to classic antibiotics. Sclareol (15) and manool (17) displayed the lowest MIC values among the other pure compounds. Sclareol was active against several species of Staphylococcus and Enterococcus of clinical interest with very uniform MIC values, ranging from 32 to 64 μg/mL (Table 4). Manool, on the contrary, was particularly powerful against Enterococcus, reaching MIC values of 4 μg/mL on several species isolates (Table 4). Although we could not demonstrate any significant effects of these two labdane diterpenoids against the selected aerobic Gram-negative species, Souza and colleagues 70 reported antimicrobial activity of the same compounds also against a few Gram-negative periodontal bacteria, probably because these organisms were endowed with an anaerobic metabolism. Moreover, we could not confirm any significant antimicrobial activity of manool against S. aureus as reported by Ulubelen, 71 probably because of the clinical origin and the multidrug-resistant characteristics of the strains of Staphylococcus we employed.
For sclareol (15), manool (17), the methyl ester of salvileucolide (10), and salvileucolide-6,23-lactone (11), the mechanism of action on the most clinically relevant and susceptible bacteria (S. aureus, S. epidermidis, E. faecium, and E. faecalis) was investigated. Time killing curves for representative resistant and multiresistant isolates are shown in Figure 2 and Figure S74 (Supporting Information). The four compounds were found to be bacteriostatic, as they prevented the growth of the starting inoculum or produced a decrease of bacterial count by 1 to 2 orders of magnitude within 24 h.
The n-hexane-insoluble and -soluble fractions, the 13 semipurified fractions (I a −VI a and I b −VII b ), and the 19 pure compounds were investigated for the modulation of ATP synthase activity. Resveratrol, a known inhibitor of rod OS ATP synthase, was used as a positive control. 40 The data were verified by one-way ANOVA (performed in MATLAB 2019a), and differences among groups evaluated with the Bonferroni test (p < 0.05) ( Figures S75 and S76, Supporting Information). The extract and most semipurified fractions showed activity ( Figure S75, Supporting Information). ANOVA singled out five groups among the 19 compounds. The group with the most interesting activity comprised compounds 10, 11, 15, and 17, which showed an inhibition of ATP production of 60%, 79%, 70%, and 60%, respectively. The differences within this group were not statistically significant. The obtained data, showing inhibition of ATP production in the OS ( Figure S76, Supporting Information) by the four pure compounds, could suggest an inhibitory action on the oxidative phosphorylation. 23,40,72 The inhibition of ATP hydrolysis activity was also evaluated, as some plant metabolites were shown to be able to hinder also the clockwise rotation, which causes the reversal activity of the enzyme. 39,73 The effects of extract, fractions, semipurified fractions, and pure compounds on this activity are shown in Figures S77 and S78  Journal of Natural Products pubs.acs.org/jnp Article concentration of 80 μg/mL, manool (17) inhibited ATP hydrolysis by 92%. ANOVA analysis showed that this compound had a mode of action that was distinctly different from the others ( Figure S78). The ability to inhibit also ATP hydrolysis activity could indicate that the modulating effect on ATP synthesis is not merely due to membrane uncoupling. 38,39 To evaluate how 17 could interact with ATP synthase, docking, MD simulation studies, and ligand−protein binding energy evaluations were carried out. The analysis focused on the F 1 catalytic domain of the protein (F 1 -ATPase), since several plant compounds such as resveratrol, piceatannol, and quercetin are known to interact with F 1 -ATPase, inhibiting ATP synthesis and hydrolysis. Indeed, X-ray structures of bovine F 1 -ATPase in complex with these compounds revealed a common binding site located among the α and β subunits of the protein, constituting the crown domain, and the C-terminal tip of the γ-subunit that is known to rotate inside the crown domain in association with ATP synthesis and hydrolysis. These ligands are thus supposed to inhibit ATP synthase activity by impeding this rotation, thus disrupting the catalytic machinery of the protein. 39 For this reason, manool was docked into the X-ray structure of bovine F 1 -ATPase in complex with quercetin (PDB code 2JJ2) 39 using a thorough AUTODOCK 74 procedure that produced good results in both virtual screening and pose prediction studies. 75,76 The docking protocol generated 200 different docking solutions, which were clustered based on their reciprocal root-mean square (RMSD) deviation using a threshold of 2.0 Å (see Experimental Procedures for details), thus producing a total of three different clusters of poses. The three corresponding ATPasemanool complexes were studied through a 30 ns MD simulation protocol in order to evaluate the stability of the binding modes predicted by docking. The results were analyzed in terms of RMSD of the ligand disposition during the simulation with respect to its coordinates in the starting complex. The analysis highlighted a high stability for pose 3, in which the ligand maintained an average RMSD of about 1.9 Å during the whole simulation ( Figure S79, Supporting Information). On the contrary, the other two binding poses predicted by docking did not show enough stability. In both cases the ligand moved considerably from its initial binding disposition, as demonstrated by the high RMSD of its coordinates with respect to the starting pose that reached values around 9 ̵ 10 Å. On the basis of these results, we could already consider both pose 1 and 2 as unreliable binding dispositions with respect to pose 3. However, in order to evaluate the different binding modes from an energetic point of view, relative binding free energy evaluations were performed on all three ATPase-manool complexes with the aim of identifying the most energetically reliable binding mode. 77 Ligand−protein binding energies were calculated using the molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) method 78 on the MD trajectories relative to the last 15 ns of simulation (Table S2, Supporting Information). The analysis clearly confirmed the reliability of pose 3, whose estimated ligand−protein binding affinity (−21.4 kcal/mol) exceeded by about 9−12 kcal/mol those evaluated for pose 1 and pose 2 (Table S2, Supporting Information). Figure 3 shows the minimized average structure of F 1 -ATPase complexed with the manool in binding mode 3, as obtained from the last 15 ns of MD simulation. Owing to its lipophilic nature, the ligand predominantly forms hydrophobic interactions with the binding site residues. The bicyclic core of the Table 4. continued Journal of Natural Products pubs.acs.org/jnp Article ligand strongly interacts with P320, as well as with V276, T318, and Q330, while its lateral chain shows lipophilic interactions with A278, V279, A293, and I263, constituting a small subpocket together with K260, E264, and E292. Interestingly the terminal vinyl group of manool shows an NH−π interaction with the backbone nitrogen of A278. 79 Despite the fact that the polar portion of the ligand is limited to its hydroxy group, this moiety is able to establish strong H-bonds with both K260 and E264 that account for a non-negligible contribution to the total ligand−protein binding energy (Table  S2, Supporting Information) and probably promotes the stability of the binding pose by providing the ligand with a good anchoring point. The whole docking/MD simulation and ligand−protein binding energy evaluation protocol was also validated using the reference X-ray structure of bovine F 1 -ATPase in complex with quercetin (PDB code 2JJ2). The bound ligand was first subjected to a self-docking study using the same docking protocol employed for manool; in this case, two different clusters of poses were generated. The two corresponding ATPase-quercetin complexes were evaluated through the MD protocol and analyzed in terms of RMSD of the ligand disposition during the simulation with respect to its coordinates in the starting complex. As shown in Figure S80, pose 2 showed strong stability, with an average RMSD of about 1.5 Å, while pose 1 diverged from the initial docking solution of about 6 Å. The binding free energy evaluation performed on the two complexes confirmed pose 2 as the most reliable binding mode from both the qualitative and quantitative point of view (Table S3). The binding mode predicted for quercetin by our computational protocol was very similar to the experimental disposition of the ligand ( Figure S81), with an RMSD between the two binding modes of around 2 Å. These results confirmed the reliability of the whole computational workflow applied for predicting the binding mode of manool into F 1 -ATPase. Finally, the ATP production in the presence or absence of manool (17) by E. faecalis MB 1 (VRE) and E. faecium MB 152 (VRE) was assessed in a whole-cell assay, where the bacteria were supplied with nutrients, after an incubation of 2 h. This timing was chosen, as the duplication time of Enterococcus spp. is around 30 min. 80 A significant reduction of the ATP amount of bacterial cells was observed in E. faecium (inhibited by 30%) (Figure 4). The data on ability to regenerate ATP of bacteria are to be considered with caution, as the pool of steady state ATP is dependent on many processes (glycolysis, substratelevel phosphorylation, oxidative phosphorylation, nutrient uptake systems) affecting its consumption and production. Our results show that a correlation between the in vivo antibacterial effect and the modulation of ATPase activity could be hypothesized for manool (17), and this could deserve further study.  ). An optical low-pass filter (<1800 cm −1 ) in front of the photoelastic modulator was used to enhance the signal/noise ratio. Solutions of 5−9 mg in 150 μL of CDCl 3 were prepared and measured in a transmission cell equipped with CaF 2 windows and a 200 μm spacer. Artifacts were eliminated by subtracting the VCD spectrum of the pure solvent (reference) from the VCD spectrum of the compound. For both the sample and the reference, ca. 24 000 scans at 4 cm −1 resolution were averaged. FTIR spectra were recorded as films or KBr pellets on a PerkinElmer System 2000 instrument (PerkinElmer). NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe at 300 K and a Bruker DRX-400 spectrometer. All 2D NMR spectra were acquired in CDCl 3 , and standard pulse sequences and phase cycling were used for TOCSY, COSY, ROESY, NOESY, HSQC, and HMBC spectra. The NMR data were processed using UXNMR software. The ROESY spectra were acquired with t mix = 400 ms. HRESIMS data were acquired in the positive ion mode by an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The Orbitrap mass analyzer was calibrated according to the manufacturer's directions by using a mixture of caffeine, methionine-arginine-phenylalanine-alanine-acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621. Data were collected and analyzed using the software provided by the manufacturer. MPLC chromatography was performed on a spot liquid chromatography system (Armen Instrument, Saint Ave, France) with normal phase Si60 Cartridges Supervarioflash and LiChroprep RP-18 ( Extraction and Isolation. Fresh aerial parts (10.3 kg) of S. tingitana were immersed in CH 2 Cl 2 for 20 s as previously described, 82 to afford 103.0 g of exudate. The exudate was partitioned with nhexane to afford an n-hexane-soluble (85.8 g) and an n-hexaneinsoluble portion (17.7 g) (see Supporting Information for details).  To a solution of 6 (5.0 mg, 11 μmol) in dry CH 2 Cl 2 (400 μL), in a reactive-vial, were subsequently added pyridine (4.5 μL, 56 μmol) and (R)-(−)-MTPA-Cl (8.3 μL, 45 μmol). The progress of the reaction was monitored by TLC analysis, by eluting with a solvent composed of hexanes and EtOAc in a 1:1 ratio. The mixture was left overnight (no trace of the starting material was present), and the reaction was quenched by addition of 400 μL of distilled water. The water layer was extracted three times with 2.0 mL of Et 2 O. The organic layer was dried with anhydrous solid MgSO 4 and concentrated in vacuo. The crude reaction mixture contained the (S)-MTPA ester of 6. The same procedure was repeated in the presence of (S)-(+)-MTPA-Cl.
Computational Methods. Conformational analysis was performed with Schrodinger MacroModel 9.8 (Schrodinger, LLC, NY, USA) employing the OPLS2005 (optimized potential for liquid simulations) force field in CHCl 3 for VCD calculations. The five conformers with the lowest energy were selected for geometrical optimization and energy calculation applying DFT with the Becke's nonlocal three-parameter exchange and correlation functional and the Lee−Yang−Parr correlation functional level (B3LYP), using the 6-31G+(d,p) basis set and the SCRF method with the CPMC model for solvation with the Gaussian 09 program package. Vibrational frequencies (given as wavenumbers in cm −1 ), rotator strength (Rstr), IR intensity (IRinten), and dipole strength (Rstr) were calculated in CHCl 3 with B3LYP/6-31+G(d,p). VCD curves were obtained on the basis of rotator strengths with a bandwidth of 7 cm −1 using CDspecTech v22.0. 83,84 VCD spectra were calculated from the spectra of individual conformers according to their contribution calculated by Boltzmann weighting. Comparison was done visually as well as by calculation of similarity indices (SimVA, SimVCD), which were generated by VCDspecTech v22.0. 57 The SimVCD values were plotted against the scaling factors of the x axis, and graphs compared between the different stereoisomers.
Statistical Analysis. All determinations were done in triplicate, and the results reported as mean ± standard deviation (SD). Data were considered statistically significant at p ≤ 0.05. The null hypothesis of equality in action for all compounds was tested with one-way ANOVA. 85 In all cases the null hypothesis was rejected, and Journal of Natural Products pubs.acs.org/jnp Article Ewald electrostatics and periodic boundary conditions, 96 while a cutoff of 10 Å was employed for the nonbonded interactions and the SHAKE algorithm was used to keep rigid all bonds involving hydrogens. Binding Energy Evaluation. Relative binding free energy evaluations were performed using AMBER 14. The trajectories extracted from the last 15 ns of each simulation were used for the calculation, for a total of 150 snapshots (at time intervals of 100 ps). van der Waals, electrostatic, and internal interactions were calculated with the SANDER module of AMBER 14, whereas the Poisson− Boltzman method was employed to estimate polar energies through the MM-PBSA module of AMBER 14 as previously reported. 75,77 Gas and water phases were represented using dielectric constants of 1 and 80, respectively, while nonpolar energies were calculated with the MOLSURF program. The entropic term was considered as approximately constant in the comparison of the ligand−protein energetic interactions.