Green Strategies for the Preparation of Enantiomeric 5–8-Membered Carbocyclic β-Amino Acid Derivatives through CALB-Catalyzed Hydrolysis

Candida antarctica lipase B-catalyzed hydrolysis of carbocyclic 5–8-membered cis β-amino esters was carried out in green organic media, under solvent-free and ball-milling conditions. In accordance with the high enantioselectivity factor (E > 200) observed in organic media, the preparative-scale resolutions of β-amino esters were performed in tBuOMe at 65 °C. The unreacted β-amino ester enantiomers (1R,2S) and product β-amino acid enantiomers (1S,2R) were obtained with modest to excellent enantiomeric excess (ee) values (ees > 62% and eep > 96%) and in good chemical yields (>25%) in one or two steps. The enantiomers were easily separated by organic solvent/H2O extraction.


Introduction
Interest in enantiomeric carbocyclic β-amino acids has greatly increased in recent years due to their utility in synthetic chemistry and drug research [1,2] and their pharmacological properties. For instance, both cispentacin and icofungipen exhibit antifungal activity [3][4][5][6][7][8][9]. They can be used as building blocks for the synthesis of modified peptides and self-organizing foldameric structures with increased activity and stability [8,10]. Therefore, the large number of publications about their synthesis, including those using enzymatic methods, is not surprising. As an example, an efficient direct enzymatic method through β-lactam ring cleavage was devised [11]. CALB-catalyzed hydrolysis of cyclopentane, cyclohexane, and cyclohexene skeletons bearing cis and trans β-amino esters in iPr 2 O has also been published for the first time [12]. Among recent developments, implementation of green approaches enables rather attractive techniques that can carry out enantioselective reactions for the preparation of β-amino acid enantiomers. For instance, on the principle that the best solvent is no solvent, a solvent-free enzymatic method was developed through CALB-catalyzed hydrolysis of β-lactams at 70 • C to afford enantiopure β-amino acids [13]. Furthermore, in recent years, sustainable synthetic chemistry under novel mechanochemical conditions with the use of ball milling has proved to be an efficient and useful method [14][15][16][17][18][19][20]. In particular, mechanochemistry has left its mark on the road to green synthesis due to the reusability of catalysts [21][22][23][24][25][26][27]. In this regard, groundbreaking research on the concept of sustainable biocatalysis, combined with mechanochemical forces and enantioselective synthesis of biologically active molecules through mechanoenzymatic kinetic resolution of racemic compounds, has been developed [28][29][30][31][32][33]. In a noteworthy study, Perez-Venegas et al., demonstrated the employment of ball milling for liquid-assisted grinding (LAG) enzymatic resolution of N-benzylated-β 3 -amino esters yielding enantioenriched N-benzylated-β 3 -amino acids [34].
In order to explore the enzyme reusability, the hydrolysis of ethyl cis 2-aminocyclohexanecarboxylate 8 was carried out with CALB that had already been used in 1, 2 or 3 cycles ( Table 2). The reaction rate was progressively decreased while the enantiomeric excess of the product appeared unaffected. This observation suggests the possibility of reusing enzyme.  [40,41]. d c = ee s /(ee s + ee p ) [42].
In view of earlier results on β-lactam ring opening under solvent-free conditions [13], the hydrolysis of cis 6-membered amino ester 8 was performed in the presence of 30 mg CALB without added H 2 O. The reaction was completed without the addition of H 2 O, since the H 2 O present in enzyme preparation (<5%) was sufficient for the hydrolysis at 65 • C ( Table 3, entry 2). When the reaction was carried out at room temperature (23 • C) (E = 45, entry 1) or at higher temperatures of 70 and 80 • C (E = 13, 11, entries 3, 4) a significant decrease in E was observed. On the basis of these data, 65 • C was selected as the optimum temperature.  [40,41]. d c = ee s /(ee s + ee p ) [42].
When increasing the amount of enzyme from 20 to 30 mg, both the conversion and enantioselectivity increased considerably, while reducing to 10 mg was accompanied by a drop in both conversion and E (Table 5, entries 1, 2 vs. Table 4, entry 5).  [40,41]. d c = ee s /(ee s + ee p ) [42].

Preparative-Scale Resolutions of cis 7-9, and 13
The preparative-scale resolution of ethyl cis 2-aminocyclohexanecarboxylate 8 under the optimized conditions of the investigated strategies was performed ( Table 6). The resolution in tBuOMe was carried out in one step. However, when attempting this at larger scale, for reasons of economy, a low (substrate: enzyme 1: 4.5) ratio was employed, which maintained excellent enantioselectivity and achieved reasonable reaction time. The reaction was stopped at 50% conversion by filtering off the enzyme (entry 1). The filtered enzyme was washed with EtOAc. The solvent was evaporated to yield unreacted β-amino ester (1R,2S)-8. The filtered enzyme was washed with hot distilled H 2 O, then evaporation of the filtrate yielded the crystalline product β-amino acid (1S,2R)-15. The resolutions under solvent-free conditions (entry 2) and using ball milling (entry 3) were performed in two steps The reactions were stopped when ee p > 96% (conv. < 50% under-run step) by adding tBuOMe to the reaction mixtures and filtering off the enzyme. The filtered enzyme was washed with hot distilled H 2 O. Evaporation of the filtrate yielded the crystalline product β-amino acid (1S,2R)-15. The repeated enzymatic reactions were stopped when ee s > 98% (conv. > 50% over-run step). The filtered enzyme was washed with EtOAc. Evaporation of the filtrate yielded the unreacted β-amino ester (1R,2S)-8.  [40,41]. f c = ee s /(ee s + ee p ) [42].
The best combination of conversion and enantioselectivity was observed in the reaction carried out in tBuOMe (conv. 50%, E > 200, after 23 h, entry 1). Therefore, preparative-scale hydrolysis of ethyl cis 2-aminocyclopentaneecarboxylate 7, ethyl cis 2-aminocycloheptanecarboxylate 9, and ethyl cis 2-aminocyclooctanecarboxylate 13 was performed in tBuOMe in the presence of CALB at 65 • C (Table 7). It is noteworthy that the same substrate: enzyme ratio (1: 4.5) was applicable in the large-scale hydrolysis of 7 but, due to the slow reaction rate observed in small-scale reactions, resolution of substrates with bigger cycles 9 and 13 necessitated a higher ratio of substrate: enzyme (1: 7.5). As the reactions progressed, the ee p values of product amino acid enantiomers 14-17 started to decrease, while the ee s values of unreacted esters 7-9 and 13 increased (data not shown). In order to obtain enantiopure amino acid products, the hydrolysis was performed in two steps, namely, once under-run (conv. < 50%) then over-run (conv. > 50%) conditions (Experimental Section).

Determination of Absolute Configurations
The

Procedure for the Synthesis of 7-9 and 13
The synthesis of racemic ethyl-2-aminocycloalkanecarboxylates 7-9 and 13 was carried out according to methods reported previously (the only exception is the synthesis of β-lactam 6), starting from 50 mmol cycloalkane [35][36][37][38]. 1 H-and 13 C-NMR as well as HRMS data on the enantiomeric derivatives were found to be similar to those for the racemates [12,[35][36][37][38][39]46,47]. Seven-membered β-lactam 6 was synthesized with a slightly modified literature procedure used for the synthesis of 4, 5, and 11 [38], as follows. CSI (4.42 g, 31 mmol, 1.0 equiv) was added dropwise over 60 min to neat cycloheptene (3.0 g, 31 mmol, 1.06 equiv) at 78 • C (keeping the reaction temperature as close to 78 • C as possible). After the addition was complete, the mixture was cooled to room temperature over a period of 60 min and then stirred at that temperature for 18 h. The reaction mixture was added dropwise to a stirred suspension of ice water (170 mL), Na 2 SO 3 (17 g), and NaHCO 3 (51 g) over a period of 20 min. The mixture was warmed to 23 • C and stirred at this temperature for 20 min followed by adding CH 2 Cl 2 (50 mL) and stirring for an additional 5 min. The solids were collected by vacuum filtration, rinsed sequentially with water (2 × 10 mL) and CH 2 Cl 2 (2 × 100 mL), and then discarded. The organic layer was separated from the filtrate and the aqueous layer was extracted with CH 2 Cl 2 (3 × 25 mL). The combined organic phases were dried over Na 2 SO 4 , filtered, and were concentrated under reduced pressure to afford 6 (3.33 g, 84% yield) as a pale solid.

Derivatization Process
Double derivatization of β-amino acids was performed by adding a saturated solution of CH 2 N 2 in Et 2 O dropwise to the MeOH (20 µL) aliquot until a yellow color persisted [Caution! the derivatization with diazomethane should be performed under a well-ventilated hood]. The next acylation step was carried out with Ac 2 O (15 µL) and a mixture of DMAP and pyridine (15 µL) in the same test tube, where the color immediately disappeared. Then the double-derivatized samples were analyzed by GC [40,41]. Derivatization of β-amino esters were performed in a single step by adding Ac 2 O and a mixture of DMAP/pyridine to the sample solution.

Conclusions
Efficient enzymatic strategies have been developed for the enzymatic resolution of 5-8-membered carbocyclic β-amino esters through hydrolysis in green organic media, under solvent-free conditions and using ball milling. In view of the best E, preparativescale resolutions were performed in tBuOMe at 65 • C, resulting in the desired enantiomeric unreacted β-amino esters (1R,2S)-7-9, 13, and product β-amino acids (1S,2R)-14-17 with high ee p values (>96%). Easy separation of the enantiomers could be achieved since the unreacted β-amino esters were soluble in organic solvent and the product β-amino acids in H 2 O. To the best of our knowledge, the lipase-catalyzed hydrolysis of 7-and 8-membered carbocyclic β-amino esters was described for the first time.
Author Contributions: E.F. and M.P. planned and designed the project. S.S. and T.F. performed the syntheses and characterized the synthesized compounds. S.S., E.F. and M.P. prepared the manuscript for publication. All authors discussed the results and commented on the manuscript. All authors have read and agreed to the published version of the manuscript.