Efficient Synthesis of New Fluorinated β-Amino Acid Enantiomers through Lipase-Catalyzed Hydrolysis

An efficient and novel enzymatic method has been developed for the synthesis of β-fluorophenyl-substituted β-amino acid enantiomers through lipase PSIM (Burkholderia cepasia) catalyzed hydrolysis of racemic β-amino carboxylic ester hydrochloride salts 3a–e in iPr2O at 45 °C in the presence of Et3N and H2O. Adequate analytical methods were developed for the enantio-separation of racemic β-amino carboxylic ester hydrochlorides 3a–e and β-amino acids 2a–e. Preparative-scale resolutions furnished unreacted amino esters (R)-4a–e and product amino acids (S)-5a–e with excellent ee values (≥99%) and good chemical yields (>48%).


Introduction
In recent years, enantiomerically pure β-aryl-substituted β-amino acids have been intensively investigated due to their pharmacological significance, unique and remarkable biological properties [1], their utility in synthetic chemistry [2], and drug research [3]. Therefore, this class of compounds has been documented as a crucial scaffold in the design and synthesis of conceivable pharmaceutical drugs. For instance, 3-amino-3-phenylpropionic acid, which is a key pharmaceutical building block, is present in anticancer agents, such as Taxol [4]. It can also find application as a fundamental component in the synthesis of novel antibiotics [5] and analgesic endomorphine-1 analogue tetrapeptides [6].
On the other hand, tremendous achievements in the development of fluorinated amino acid drugs verified the high importance of this type of compounds in pharmaceutical chemistry. It is known that the occurrence of fluorine in biologically active natural compounds is extremely low. In turn, the number of fluorine-containing drugs on the market is rising continuously. The reasons are the unique characteristics of the fluorine atom in terms of its high electronegativity and the polarity of a carbon-fluorine bond [7,8]. Thus, incorporation of fluorine into β-amino acids has gained increasing attention in recent decades. For example, Januvia (sitagliptin phosphate) acts as an antidiabetic agent via inhibition of dipeptidyl peptidase IV [9], whereas (±)-Eflornithine was used for the treatment of trypanosomiasis [10] and against facial hirsutism in women [11]. [15,16]. Lipase-catalyzed methods for the resolution of both cyclic [17] and acyclic [18] β-amino carboxylic esters through hydrolysis are known in the literature. Various enzymatic procedures have been developed by our research group for the preparation of biologically active β-aryl-substituted, β-heteroaryl-substituted, and β-arylalkyl-substituted β-amino acid enantiomers through enantioselective (E > 200) hydrolysis of the corresponding β-amino carboxylic esters both in H2O or in an organic solvent catalyzed by lipase (Pseudomonas cepacia) PS [19][20][21]. Catalyzed kinetic and dynamic kinetic resolution of β-amino carboxylic esters or their hydrochloride salts with tetra-hydroisoquinoline and tetra-hydro-β-carboline skeleton through hydrolysis have been performed. Catalysts used include Candida antarctica lipase B (CAL-B) (in aqueous NH4OAc buffer at pH 8.5 and or in iPr2O in the presence of 1 equiv of H2O), Alcalase (in borate buffer at pH 8), and lipase PS (in iPr2O with 4 equiv of added H2O) [22][23][24][25].
Herein, in view of the importance of fluorinated β-amino acids, our aim was to synthesize (±)-βamino carboxylic ester hydrochloride salts 3a-e (Scheme 1), then to devise a suitable enzymatic protocol for the synthesis of new fluorinated β-amino acids via enantioselective hydrolysis of 3a-e (Scheme 2) and provide an adequate characterization of the enantiomeric products.
(Porcine pancreatic lipase), and CAL-B (Table 1, entries 2-5) showed activity in enzymatic hydrolysis. However, with the exception of lipase AK affording an eep value of 75% and a moderate E (8) (19% conversion in 10 min, entry 3), low reactivities and low enantio-selectivities were achieved (entries 2, 4, and 5). It is noteworthy that PPL catalyzed the reaction with opposite enantio-selectivity. Lipase PSIM, in contrast, provided an E value of 108 (entry 1) and, consequently, it was selected for further studies.
Next, we analyzed the effect of solvent on enantio-selectivity and reaction rate. Very different E and reaction rate data were observed in the green solvents tested ( Table 2). The hydrolytic reactions of 3a in the ether-type solvents were rapid (conv. 52%, 51%, and 54% after 10 min, E = 59, 113, and 27, entries 1, 2, and 4), while, in EtOAc, the reaction proceeded relatively slowly with low enantioselectivity (conv. 11%, after 10 min, E = 3, entry 3). On the basis of our earlier results [33], the reaction was also performed under solvent-free conditions when, in harmony with our earlier observation, a reasonable enantioselectivity (E 74) was observed in addition to a rapid transformation (conv. 49% after 10 min, entry 5). For the reason of economy (taking into account that 2-Me-THF is the most expensive selected solvent), despite the highest E (113), iPr2O, with a slightly lower E (108), but as significantly less expensive was identified as the most suitable solvent.
In order to follow up the progress of the reaction while maintaining high enantioselectivity, it was wise to slow down the reaction. When the reaction temperature decreased from 45 °C to 25 °C, both the reaction rate and enantio-selectivity for the hydrolysis of 3a clearly decreased (30% conv. in 10 min, E = 48 vs. 48% conv. in 10 min, E = 108, Table 1, entry 1). To our surprise, the fastest reaction was achieved at 3 °C with the highest degree of conversion (50% in 10 min) and an E value of 134. In order to collect more information, we decided to carry out the reaction with different enzyme concentrations at 3 °C. As shown in Table 3, there was no significant difference in the reaction rates, when the enzyme concentration decreased from 10 to 5 or 2 mg mL -1 (~50% conv. in 10 min reaction time, entries 1-3). In contrast, E dropped significantly when the reaction was performed with a 2-mg mL -1 enzyme (entry 3). Since both high E and satisfactory reaction rate were attained at 45 °C, we Scheme 2. Enzymatic kinetic resolution of (±)-3a-e through a hydrolytic procedure.

Synthesis of Ethyl 3-Amino-3-Arylpropanoate Hydrochloride Salts (±)-3a-e
Racemic β-amino acids (±)-2a-e were synthesized by modified Rodionov synthesis through the reaction of the corresponding aldehydes with malonic acid in the presence of NH 4 OAc in EtOH at a reflux temperature (Scheme 1) [31,32]. Subsequently, the β-amino carboxylic ester hydrochloride salts (±)-3a-e were prepared with yields ranging from 76% to 98% by esterification of the corresponding β-amino acids in the presence of SOCl 2 in EtOH.

Preliminary Experiments
On the basis of the results achieved on the enzyme-mediated enantioselective hydrolysis of β-amino carboxylic esters [17,18], the hydrolysis of model compound (±)-3a (Scheme 2) was conducted with 5 equiv of Et 3 N and 0.5 equiv of H 2 O in the presence of 30 mg mL -1 enzyme in iPr 2 O at 45 • C (Table 2, entry 1). In the frame of enzyme screening, lipase AY (Candida rugosa), lipase AK, PPL (Porcine pancreatic lipase), and CAL-B (Table 2, entries 2-5) showed activity in enzymatic hydrolysis. However, with the exception of lipase AK affording an ee p value of 75% and a moderate E (8) (19% conversion in 10 min, entry 3), low reactivities and low enantio-selectivities were achieved (entries 2, 4, and 5). It is noteworthy that PPL catalyzed the reaction with opposite enantio-selectivity. Lipase PSIM, in contrast, provided an E value of 108 (entry 1) and, consequently, it was selected for further studies.
Next, we analyzed the effect of solvent on enantio-selectivity and reaction rate. Very different E and reaction rate data were observed in the green solvents tested ( Table 1). The hydrolytic reactions of 3a in the ether-type solvents were rapid (conv. 52%, 51%, and 54% after 10 min, E = 59, 113, and 27, entries 1, 2, and 4), while, in EtOAc, the reaction proceeded relatively slowly with low enantioselectivity (conv. 11%, after 10 min, E = 3, entry 3). On the basis of our earlier results [33], the reaction was also performed under solvent-free conditions when, in harmony with our earlier observation, a reasonable enantioselectivity (E 74) was observed in addition to a rapid transformation (conv. 49% after 10 min, entry 5). For the reason of economy (taking into account that 2-Me-THF is the most expensive selected solvent), despite the highest E (113), iPr 2 O, with a slightly lower E (108), but as significantly less expensive was identified as the most suitable solvent.  [34]. d c = ee s /(ee s + ee p ) [35].  [34]. d c = ee s /(ee s + ee p ) [35].
In order to follow up the progress of the reaction while maintaining high enantioselectivity, it was wise to slow down the reaction. When the reaction temperature decreased from 45 • C to 25 • C, both the reaction rate and enantio-selectivity for the hydrolysis of 3a clearly decreased (30% conv. in 10 min, E = 48 vs. 48% conv. in 10 min, E = 108, Table 2, entry 1). To our surprise, the fastest reaction was achieved at 3 • C with the highest degree of conversion (50% in 10 min) and an E value of 134. In order to collect more information, we decided to carry out the reaction with different enzyme concentrations at 3 • C. As shown in Table 3, there was no significant difference in the reaction rates, when the enzyme concentration decreased from 10 to 5 or 2 mg mL -1 (~50% conv. in 10 min reaction time, entries 1-3). In contrast, E dropped significantly when the reaction was performed with a 2-mg mL -1 enzyme (entry 3). Since both high E and satisfactory reaction rate were attained at 45 • C, we decided to use this optimal reaction temperature for preparative-scale reactions. Additionally, a set of preliminary experiments was performed in order to determine the influence of enzyme concentration on the reaction rate ( Table 4). The reaction rate for the hydrolysis of (±)-3a clearly increased as the concentration of enzymes was increased. The highest reaction rate was observed with a 40-mg mL -1 enzyme (entry 5). However, for a satisfactory reaction time (the time needed to reach 50% conversion), the use of a 30-mg mL -1 enzyme (Table 2, entry 1) was selected for preparative-scale resolutions. Table 3. Effect of enzyme concentration in the hydrolysis of (±)-3a a .

General Methods
Lipase PSIM and lipase AK were from Amano Pharmaceuticals and lipase AY was from Fluka. PPL and CAL-B immobilized on acrylic resin were purchased from Sigma (Budapest, Hungary). Substituted benzaldehydes were from Sigma-Aldrich. Triethylamine was from Merck. Solvents of the highest analytical grade were from Sigma-Aldrich. Optical rotations were measured with a Perkin-Elmer 341 Polarimeter. 1 H-NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer. Melting points were determined on a Kofler apparatus (see the Supplementary Materials). The enantiomeric excess ee values for the unreacted β-amino carboxylic ester and the β-amino acid enantiomers produced were determined by GC equipped with a Chirasil-L-Val column after double derivatization [34] with (i) diazomethane [Caution: the derivatization with diazomethane should be performed under a well-working hood] and (ii) acetic anhydride in the presence of 4-dimethylaminopyridine and pyridine [90 • C for 10 min → 170 • C (temperature rise 20 • C min −1 ), 10

General Procedure for the Syntheses of Racemic β-Amino Acids 2a-e
Compounds 2a-e were synthesized based on the modified Rodionov synthesis [31,32] through condensation of the corresponding aldehydes 1a-e (2 g) with malonic acid (1 equiv) in the presence of NH 4 OAc (2 equiv) in EtOH under reflux for 8 h [19]. The resulting white crystals were filtered off and washed with acetone and then they were recrystallized from H 2 O and acetone. 3.3. General Procedure for the Syntheses of Racemic β-Amino Carboxylic Ester Hydrochloride Salts 3a-e SOCl 2 (1.05 equiv) was added to 30 mL of EtOH at a temperature kept under -15 • C with saline ice. To this solution, 2a-e (1 g) were added at once. The mixture was stirred at 0 • C for 30 min, then at room temperature for 3 h, and, finally, heated under reflux for 1 h. The solvent was evaporated off under reduced pressure and the resulting white 3a-e. HCl salts were recrystallized from EtOH and Et 2 O.