Facile, Expeditious and Cost-effective Preparation of N-Phthaloyl (S)-Amino Acids and Their in silico Activities against Staphylococcus Aureus

Exploration of cost-effective and eco-friendly synthetic processes that offer high yields is a Selecting suitable protecting in this exploration. In this N -phthaloyl amino acids are significant intermediates in a variety of chemical and biological process. ease of of the phthaloyl moiety it for masking the primary amino group, 2 without the complications of side reactions associated with the strong nucleo-philicity of the nitrogen atom in its unmasked state. the synthetic routes -protection -protection of values, confirming that the reaction occurs with retention of configuration. Comparative molecular docking assessments of 3a-f and sulbactam inside the active cavity of the b -lactamase of Staphylococcus aureus (PDB ID: 3BLM) suggest that these compounds may be potential b -lactamase inhibitors and thus have antibacterial activities against S. aureus.

solvents. [25][26][27][28][29][30][31] The fruit juice of citrus lemon is an effective natural catalyst, containing mainly water, carbohydrates, ascorbic acid and citric acid. 32 Lemon juice is about 3-7% citric acid, with pH between 2 and 3. Microwave assisted (MW, mv) reactions support the goals of green chemistry because they generally are high yielding in short reaction times and have been demonstrated to facilitate synthesis under solvent-free reaction conditions. 10 In the present work, we introduce a green high-yielding approach for the N-phthaloylation of (S)-amino acids (Scheme 1) and make note of the fact that the optical rotations of products 3a-f show good agreement with literature values, indicating that there is no racemization.
In optimizing our procedure, we investigated the N-protection of (S)-amino acids (1a-f) under a variety of experimental conditions, and the results are shown in Table 2.
In each case, we were guided by the practical concerns of achieving the best yields and lowest racemization and made no attempt to examine every permutation of conditions. Under MW, when the power of the microwave was tuned to medium high (500-800W), the reaction tended to completion within a few minutes with excellent yield (Entry 2, 95-98%). Higher MW power led to decomposition, and lower MW power gave incomplete conversion. Reactions with conventional heating were also explored as an alternative method, bearing in mind the precedent for keeping the temperature below 135 C, as high temperatures will lead to racemization. 5 At 130-135 C, excellent (95-98%) yields were achieved in 15 minutes (Table 2, entry 9). Under lemon juice catalysis, N-protection did not occur under ambient conditions, not even with overnight stirring or grinding the reactants ( Table 2, entries [10][11]. The N-phthaloylation of chiral amino acids in the presence of acetic acid was also tried under these conditions ( Table 2, entries 5-7), but yields and reaction times were less favorable.
As it is the most significant organic acid in lemon juice (3-7%), we assume that citric acid is responsible for catalyzing the entire N-protection reaction. To investigate the likely participation of this triprotic acid during the reaction (Scheme 2), we carried out our procedure in the presence of an aqueous solution of commercially available citric acid at the same concentration and volume. The results obtained from commercial citric acid were found to be consistent with those obtained from natural juice ( Table 2, entries  7 and 13). The specific rotation of the N-protected derivatives 3a-f, obtained from MW and conventional heating was found consistent with the literature values (Table 3). 2 These findings support the conclusion that (S)-amino acids did not suffer racemization under either set of conditions.
Other spectroscopic techniques provided consistent information towards the structures of chiral phthalides 3a-f. This was indicated by the appearance of IR signals for C ¼ O (both acid and amide) and OH groups near at 1730 and 3470 cm À1 , respectively, and by k max, in the absorption spectrum near 312 nm.  Lemon juice Ã overnight ambient stirring --12 Citric acid # 14 -MW Medium high 97 Ã 0.1 mL, #1.5 mL, high ¼ 800-1000 W, medium high ¼ 500-800, medium ¼ 400-500 W, low ¼ 100-400 W, # 0.1 mL of 7% aq. solution is used for the N-protection of (S)-leucine. Scheme 2. Possible mechanism of formation of N-phthaloyl S-amino acids.
The splitting patterns of 1 H-NMR of all N-protected derivatives showed a multiplet of four aromatic protons at approx. d H 7.80-7.90 ppm belong to isoindoline aromatic cycle. The aliphatic H signals of 3a and 3b were observed as doublets at d H 1.65 and 0.88/1.14 ppm, respectively. The doublets at d H 0.92 (J ¼ 6.8 Hz) and 0.94 (J ¼ 6.8 Hz) ppm, multiplets at d H 1.46, 1.93 and 2.31 ppm in the NMR of 3c indicated the presence of the isobutyl group. The methylthio protons of 3d gave singlet signal at d H 2.04 ppm. The 1 H NMR spectra of compounds 3e and 3f showed aromatic doublet and multiplet signals in the range of d H 6.95-7.60 ppm. The LR-EIMS of the N-phthaloyl (S)-amino acids showed the molecular ion peak [M] þÁ . Compounds 3b-d contain c-hydrogens and undergo Mc-Lafferty rearrangement, therefore revealing an intense peak at m/z ¼ 205 amu.
To probe the potential antibacterial activities of 3a-f, we used in silico methods. In the field of structure based drug design, the assessment of ligand-receptor complexes is very helpful. 33,34 We compared the structure and stereochemistry of 3a-f with sulbactam ( Figure 1), a Class A b-lactamase inhibitor. The relevant chiral carbon (C-2 in both cases) has the S configuration, making sulbactam an appropriate choice for comparative docking studies with 3a-f. We performed docking studies of these chiral molecules inside the active cavity of the b-lactamase of Staphylococcus aureus Table 3. Reported and calculated values of specific rotation, yield and spectroscopic data of 3a-f.

Reported
[a] 20    Table 5). The standard drug, sulbactam, with binding energy À5.6 kcal.mol À1 , docked with the target through hydrogen bonding with SER70 and the GLN237 residue at a distance of 3.29Å from SER70. It was observed that all the title compounds, except 3f, exhibited hydrogen bonding with SER70 of the active site, with binding energies (BE) less than that of sulbactam. Compound 3a exhibited BE À5.8 kcal.mol À1 , docked with residues SER70, ASN132, GLU166 and GLN237 through hydrogen bonding. This binding energy is comparable with that of sulbactam. Modifying the R-chain as shown in structures 3b-3d further reduces the BE value. In addition to H-bond interactions, the compounds 3b-3d also displayed p-alkyl interactions (Figures 2-3). The modifications shown in compounds 3e and 3f further decrease the binding energy. The compound 3e showed the highest docking energy (-6.9 kcal.mol À1 ) and displayed four hydrogen bonding interactions with SER70, SER130, ASN170 and SER235. Finally, the molecule 3f showed good docking energy (-6.8 kcal.mol À1 ) with four hydrogen bonds, but no interaction was observed with the SER70 residue. The results are generally supportive of the idea that the title compounds are predicted to have antibacterial activity.
In conclusion, we have developed a simple and green approach for the protection of (S)-amino acids under solvent free conditions. We found that (S)-amino acids do not undergo racemization in the protection reactions. The specific rotation values and characterization data by UV-VIS, IR, 1 H-NMR and LR-EIMS of 3a-f were consistent with the previously reported values, confirming that the reaction occurs with retention of configuration. Comparative molecular docking assessments of 3a-f and sulbactam inside the active cavity of the b-lactamase of Staphylococcus aureus (PDB ID: 3BLM) suggest that these compounds may be potential b-lactamase inhibitors and thus have antibacterial activities against S. aureus.

Experimental section
All chemicals were purchased from Sigma Aldrich Chemical Co. Melting points were determined via the open capillary method and are uncorrected. Thin layer chromatography (TLC) was performed to check the extant of reaction in which silica gel 60 F 254 and EtOAc or EtOH were used as stationary and mobile phases respectively. Ninhydrin was used as luminous indicator. An AP-300 Polarimeter was used for determination of specific rotations. A Prestige 21 Spectrophotometer was used to record the IR spectra. A MAT 312 instrument was used to record LR EIMS. Proton NMR spectra were recorded   on a Bruker (400 MHz) spectrometer. For microwave irradiation, a Dawlance DW-MD4N instrument was used (1000 W). For catalyst preparation, fresh lemons were cut and pressed to obtain the juice. The crude juice was successively filtered through muslin cloth and filter paper. The clear filtrate (having pH 3.2-3.5) was used as the catalyst.

Method A (microwave mediated reaction)
A mixture of phthalic anhydride (1.1 eq) and the appropriate (S)-amino acid (0.10 g) in a small amount of lemon juice (1-2 drops, 0.1 mL) was irradiated for 5-12 min in a closed flask, the time being signified by TLC in EtOAc or EtOH. The cold reaction mixture was dissolved in methanol, filtered and the residue recrystallized from a mixture of methanol and water (7:3 ratio) to furnish pure crystals of 3a-f.

Method B (conventional heating method)
A mixture of the appropriate (S)-amino acid (0.10 g) and phthalic anhydride (1.3 eq) in the presence of lemon juice (1-2 drops, 0.1 mL) was heated at 130-135 C in an oil bath for 15-20 min, as indicated by TLC in EtOAc or EtOH. After the completion of reaction, the products were purified by crystallization as above to afford pure crystals of 3a-f.

Molecular docking
Molecular docking of compounds 3a-f and the reference standard (Sulbactam) with active site 3BLM was achieved using the AutoDock Vina docking algorithm in the program 1-Click Docking (Mcule Inc., Palo Alto, CA, USA). The binding site center was identified as default, with the Cartesian coordinates (X: 2.533, Y: À10.807 and Z: À9.408). The simulation was RUN with a maximum hit of 1000. We selected the docking pose with the most negative docking score corresponding to the highest binding power, and hydrogen bonding was analyzed by Biovia Discovery Studio 2020 (Systemes Dassault, 2016). Details are available in the Supplementary Materials of the online version of this article and from the corresponding author upon request.