Study of Chiral Center Effect on CaLB-Catalyzed Hydrolysis of (±)-1-(Acetoxymethyl)-3, 4, 5-methylpyrrolidin-2-ones
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The MS describes the synthesis of 1-(acetoxymethyl)-3-, 4-, 5-methylpyrrolidin-2-ones and their enzymatic resolution through CALB-catalyzed hydrolysis. Rather low enantioselectivities (20, 1, 6) characterized the transformations in 2M2B solvent. Therefore, no practical use from the point of view of the synthesis of the desired enantiomers. Unfortunately no further optimization (eg. enzyme screening, examination of solvents effect on E), nor an adequate analytical method devised to follow the progress of the enzymatic reaction and to calculate ee, conv. and E. The ring cleavage of the lactams has not been investigated. However, the CALB-catalyzed ring cleavage of racemic β- and γ-lactams are known in the literature. All together I can not suggest acceptance of the MS (Synthesis and Enzymatic Resolution of (±)-3-, 4-, 5-methylpyrrolidin-2-ones by CaLB) for publication in Catalysts.
Remarks 1. The Introduction part is lacking, it should be completed (CALB-catalyzed ring cleavage of γ-lactams resulting the ring opened γ-amino acids and unreacted γ-lactams)
2. Contrary to the claim “Also, the resolution of γ-lactams by hydrolysis is complicated since the enzymes that have been reported so far, capable of hydrolysis are difficult to obtain, such as ENZA-1 and 71 ENZA-20 [18]” (rows 70-72) the CALB-catalyzed enantioselective ring cleavage of N-Boc protected and unprotected racemic γ-lactams and N-hydroxymethyl γ-lactam have been described
3. The possible ring cleavage of γ-lactams should be investigated
4. In the frame of optimization, an enzyme screening should be performed and the solvent effect on E also investigated
5. An adequate analytical method should be devised to follow the progress of the enzymatic reaction and to calculate ee, conv. and E.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Dear Authors,
in the introduction you write that Candida antarctic lipase B (CaLB) is commercially available at a low cost, both in its free and immobilized form, the latter is easy to use and recycle.
Have you tried to recycle the enzyme? Does it work well or does some kind of deactivation take place?
In this work did you use free form or immolized form?
Fig.3: It would be useful near the type of reaction to insert the corresponding reference.
line 87: γ2-, γ3, and γ4-Lactams (±)-1-3, were obtained using the methodology described by Escalante et al. us [20]
line 172: In these calculations, it is possible to know the affinity enzyme-substrate through interactions with amino acid residues, substrate orientation within the catalytic site, etc. Please detail “etc" or remove from the text.
line 225: γ-nitroaliphatic γ-Nitroaliphatic methyl esters synthesis
About the Experimental Section:
- For each purification by column chromatograpy, please, report the eluent mixture and the solvents ratio in the eluent mixture.
- Please, for each synthesys near the yield report the amount (g or mg) of the obtained product.
Nitro group reduction: report the reaction time and not a time range.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Interesting article regarding the production of lactams via enzymatic resolution using a lipase. IN general, the article is well written and contains significant experimental results for chemical synthesis and biotransformations, providing a good computational discussion rationalizing the obtained results. There are aspects that need to be to highly improved, in fact, it is unclear to me how the enantiomeric excess and enantioselectivity were measured. It seems that the hydrolyzed products (4, 5 and 6) enantiomeric excess were not measured, however this is very simple after derivatization into the corresponding esters (7, 8 and 9):
1. Revise the abstract, it is unclear:
- “Several chemical and biocatalytic methods have been described for chiral γ-lactams syntheses, however, only one method has been reported for γ4-lactam resolution, while γ2- and γ3-lactams have not been reported...”
- “On the other hand, the resolution of γ-lactams by hydrolysis is complicated since the enzymes that have been reported so far, capable of hydrolysis, are difficult to obtain…”
2. Revise the introduction:
- Figure 1 or 2: Include the general structure of γ2, γ3 and γ4 lactams for a clearer understanding
- “Pd-catalyzed” instead of “Palladium-catalyzed”
- “Rh and Ru” instead of “Rhodium and Ruthenium”
- “Candida antarctic lipase B (CaLB)” instead of “Candida antarctica lipase B (CaLB)” Also use italic font for the microorganism name. Same in Section 3.
3. Revise results and discussion:
- Section 2.1:
o Add weight and volumes for reactants
o “p-formaldehyde” instead of “paraformaldehyde”
o Is there any reason for the low yield achieve for lactam 8? (30%)
o Scheme 1: “Ac2O” instead of “Acetic anhyd.”
- Section 2.2.:
o Use “methyl tert-butyl ether (MTBE)” instead of “2M2B” (same for Scheme 2 and discussion
o Scheme 2 contains a mistake: it must read (S)-4 and not (S)-7 for the hydrolyzed product. Also be careful with that in the Table 1 because (S-)7 is wrong
o Legend of Scheme 2: Use bold font for compound 7
o Regarding the CalB action, why the terms “assisted” is used and not “catalyzed”?
o “chiral HPLC” instead of “quiral HPLC”
o Table 1: do not use decimal figures for enantiomeric excess values because the analytical technique has a ±1% error
o Table 1: For NMR conversion indicate the steps before the measurement: filtration, extraction and solvent distillation?
o Table 1 is unclear to me: what is “(S-)7 (%) [α]” and “ee”. The authors must claim the enantiomeric excess of both substrate and product by HPLC (or after derivatization), and then somewhere in the text discuss the absolute configuration assignment. This will simplify the Table 1 understanding.
o Scheme 3: (S)-10 must be (S)-5. Same for the table, and please avoid in the table 2 the inclusion of optical rotation values, just focus on enantiomeric excess measurements of compounds 5 and 8
o Table 3: Please avoid in the table the inclusion of optical rotation values, just focus on enantiomeric excess measurements of compounds 5 and 8
o Table 4: I would not use a ratio for NMR conversion for instance, 68:32. Please indicate if this is 68 or 32, but not a ratio
4. Revise materials and method section: Add weights and volumes for all reactants included in this section (not only mmol or volumes for some of them)
5. Revision conclusion section: “γ4-lactam” instead of “γa-lactam”
6. Reference section: no need to add issue numbers, they are in some of them, but not in others.
7. Supporting information: section “HPLC chiral”, the wavelength used ins 250 nm but in the main text a 216 nm is claimed.
Comments on the Quality of English Language
The English language is fine
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors I still do not recommend accepting this article (Synthesis and Enzymatic Resolution of (±)-3-, 4-, 5-methylpyrrolidin-2-ones by CALB) for publication in Catalysts, because despite the rather low enantioselectivities (20, 1, 6) of the enzymatic reactions (CALB-catalyzed hydrolysis of 1-(acetoxymethyl)-3-, 4-, 5-methylpyrrolidin-2-ones), it was not further optimized the reaction conditions, in order to increase the E. Therefore, no practical use from the point of view of the synthesis of the desired enantiomers. No adequate analytical methods devised to follow directly the progress of the enzymatic reactions and to calculate ee, conv. and E. Furthermore, it was not investigated the possible ring cleavage of the lactams. However, the CALB-catalyzed ring cleavage of racemic γ-lactams via CALB in the presence of H2O are well known from the literature.
Comments for author File: Comments.pdf
Author Response
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