Efficient Enzymatic Routes for the Synthesis of New Eight-Membered Cyclic β-Amino Acid and β-Lactam Enantiomers

Efficient enzymatic resolutions are reported for the preparation of new eight-membered ring-fused enantiomeric β-amino acids [(1R,2S)-9 and (1S,2R)-9] and β-lactams [(1S,8R)-3, (1R,8S)-3 (1S,8R)-4 and (1R,8S)-7], through asymmetric acylation of (±)-4 (E > 100) or enantioselective hydrolysis (E > 200) of the corresponding inactivated (±)-3 or activated (±)-4 β-lactams, catalyzed by PSIM or CAL-B in an organic solvent. CAL-B-catalyzed ring cleavage of (±)-6 (E > 200) resulted in the unreacted (1S,8R)-6, potential intermediate for the synthesis of enantiomeric anatoxin-a. The best strategies, in view of E, reaction rate and product yields, which underline the importance of substrate engineering, are highlighted.

In addition to conventional resolution methods for the preparation of enantiomeric β-amino acids and β-lactams, enzymatic strategies have also been described [18][19][20]. Our research group has also devised a number of efficient enzymatic kinetic and sequential kinetic resolution processes (acylations, deacylations and hydrolyses). Most of these methods have been reviewed [21][22][23].
Molecules 2017, 22 A primary aim of this work was to devise adequate enzymatic strategies for the preparation of valuable new enantiomeric eight-membered carbocyclic β-lactam and β-amino acid derivatives.

Scheme 2.
Lipase-catalyzed O-acylation of (±)-4. When the acylation was performed at higher temperatures, the reaction rate increased with the concomitant decrease in E (entries 2 and 3 vs. 1). As the amount of VB was increased from 2 to 10 equiv., the reaction rate increased while E apparently decreased (entry 4 vs. 3). The addition of a catalytic amount of Et 3 N and Na 2 SO 4 resulted in a clear increase in E (entry 5 vs. 4).
To further increase the E values, acyl donors, such as 2,2,2-trifluoroethyl butyrate, vinyl acetate (VA), ethyl acetate (EtOAc) and acetic anhydride (Ac 2 O) were tested (entries 6-9). Unfortunately, none of the acyl donors tested exerted any beneficial influence on the reaction course. Several solvents have also been tested. When iPr 2 O was replaced by tBuOMe, practically no change was observed in the reaction course (entry 15 vs. 4). The same high E but somewhat lower reaction rate was observed in acetone (entry 17), while the best E and fastest reaction were detected in toluene (entry 16). Finally the environmentally less harmful iPr 2 O was selected as solvent.
In further studies, we have probed our very recent results found about the ring cleavage of specially activated lactams [28], where the activating group underwent to a traceless, in situ degradation. Accordingly, the ring cleavage of (±)-4 was attempted with H 2 O in the presence of CAL-B and benzylamine to capture formaldehyde in iPr 2 O at 60 • C (Scheme 4, Table 2, entry 3). Excellent ee (>99%) characterized formed amino acid (1R,2S)-9 at a conversion close to 50%. The ring cleavage of regioisomeric (±)-6 was also carried out under the same conditions (entry 4), and the same high ee (>98%) for amino acid (1R,2S)-10 and unreacted lactam (1S,8R)-6, potential intermediate in the synthesis of enantiomeric anatoxin-a was observed. In good accordance with the earlier observation that the hydroxymethyl group activates the ring cleavage of lactams, racemic 4 and 6 underwent ring cleavage much faster than their corresponding inactivated counterparts (entry 3 vs. 1 and 4 vs. 2).

Scheme 4.
Lipase-catalyzed two-step transformation of (±)-4 and (±)-6. On the basis of the preliminary results, preparative-scale resolutions of racemic 3, 4 and 6 were performed under the optimized conditions (footnotes to Table 3). The results are reported in Table 3 and Experimental Section.      On the basis of the preliminary results, preparative-scale resolutions of racemic 3, 4 and 6 were performed under the optimized conditions (footnotes to Table 3). The results are reported in Table 3 and Experimental Section.
On the basis of the preliminary results, preparative-scale resolutions of racemic 3, 4 and 6 were performed under the optimized conditions (footnotes to Table 3). The results are reported in Table 3 and Experimental Section.

Typical Small-Scale Enzymatic Experiments
Racemic β-lactam in an organic solvent (0.05 M solution, 1 mL) was added to the lipase tested (30 mg mL −1 ). The tested acyl donor (2 or 10 equiv.) in acylations or H 2 O (1 equiv.) in hydrolyses was added. The mixture was shaken at −15 • C, 2-3 • C, 30 • C or 60 • C. The progress of the reaction was followed by taking samples from the reaction mixture at intervals and analysing them by using a gas chromatograph equipped with a chiral column. The ee values for the unreacted β-lactam enantiomers were determined directly on a Chromopack Chiralsil-Dex CB column [retention times are given in min;

Acidic Hydrolyses to β-amino Acid Hydrochlorides
When the enantiomeric lactam at issue (0.2 mmol) was treated with 18% aqueous HCl (5 mL) at reflux temperature, the desired amino acid hydrochloride was obtained, as follows: (

Conclusions
A highly efficient CAL-B (lipase B from Candida antarctica)-catalyzed ring-cleavage reaction (E > 200) of 9-azabicyclo[6.2.0]dec-6-en-10-one [(±)-3)] with 1 equiv. of H 2 O in iPr 2 O at 60 • C resulted new eight-membered ring-fused β-lactam (1S,8R)-3 and β-amino acid (1R,2S)-9 (ee ≥ 99%). Even more efficient two-step transformation was carried out for the ring cleavage of activated lactams (±)-4 with 1 equiv. of H 2 O, in the presence of CAL-B using 1 equiv. of benzylamine in iPr 2 O at 60 • C. The ring cleavage of regioisomeric (±)-6, carried out under the same conditions resulted unreacted (1S,8R)-6, potential intermediate for the synthesis of enantiomeric anatoxin-a. These results underline the importance of substrate engineering, since faster reactions were clearly detected when activated lactams vs. their inactivated counterparts were reacted. Advantages of these reactions are the spontaneous degradation of N-hydroxymethyl groups and easy product separation by organic solvent-H 2 O extraction.
An indirect enzymatic method, in view of the synthesis of enantiomeric β-amino acids, has also been devised, through a relatively fast acylation of N-hydroxymethyl 9-azabicyclo[6.2.0]dec-6-en-10-one [(±)-4)] with 10 equiv. of VB mediated by lipase PSIM (Burkholderia cepacia) using a catalytic amount of Et 3 N and Na 2 SO 4 in iPr 2 O at 30 • C (E = 114). This route is somewhat less efficient and, in fact, it is a longer procedure to prepare amino acid enantiomers. However