On the Use of Orthoformates as an Efficient Approach to Enhance the Enzymatic Enantioselective Synthesis of (S)-Ibuprofen
Round 1
Reviewer 1 Report
In this article the authors describe the effectiveness of the combination between an organic solvent system mixture (isooctane and cosolvents) with orthoformates (for trapping the water released and generating the nucleophile in situ) during enzymatic kinetic resolution of rac-ibuprofen using immobilized lipase B of Candida antarctica as biocatalyst.
However, throughout the manuscript several typographical errors were found that must be corrected before approval for publication:
1) Line 5, delete the first “and”
2) Line 133, change whit to with.
3) Line 138, delete 1.
4) Line 156, 1,4-dioxane entry 4 change to entry 5
5) Line 157, MTBE, Entry 5 change to entry 4
6) Line 176, E red, left change to right and EF green, right change to left.
7) Line 200 Table 2, change 1. for 2.
8) Line 281 Table 3 change to Table 4.
On the another hand, it is not clear if the data reported in Figure 5 are the authors' experimental values or were taken from Zdun and Jose (paragraphs 178-191).
Additionally, in the references 80-84, the following citation must be included:
Solvent‐Free Lipase‐Catalyzed Transesterification of Alcohols with Methyl Esters Under Vacuum‐Assisted Conditions
GK Sánchez‐Muñoz, MA Ortega‐Rojas, L Chavelas‐Hernández, ...
ChemistrySelect 7 (30), e202202643
In addition to reference 68, should be included:
One-Pot Lipase-Catalyzed Enantioselective Synthesis of (R)-(−)-N-Benzyl-3-(benzylamino)butanamide: The Effect of Solvent Polarity on Enantioselectivity
MA Ortega-Rojas, JD Rivera-Ramírez, CG Ávila-Ortiz, E Juaristi, ...
Molecules 22 (12), 2189
Thermodynamically controlled chemoselectivity in lipase-catalyzed aza-Michael additions
JD Rivera-Ramírez, J Escalante, A López-Munguía, A Marty, E Castillo
Journal of Molecular Catalysis B: Enzymatic 112, 76-82
Author Response
REVIEWER #1
In this article the authors describe the effectiveness of the combination between an organic solvent system mixture (isooctane and cosolvents) with orthoformates (for trapping the water released and generating the nucleophile in situ) during enzymatic kinetic resolution of rac-ibuprofen using immobilized lipase B of Candida antarctica as biocatalyst.
However, throughout the manuscript several typographical errors were found that must be corrected before approval for publication:
1) Line 5, delete the first “and”
Done
2) Line 133, change whit to with.
Done
3) Line 138, delete 1.
The foot text of Table 1 has been corrected. The reviewer was right, and the “1” has been deleted.
4) Line 156, 1,4-dioxane entry 4 change to entry 5
Corrected: Now, the text is the following: “On the other hand, a very slow reaction with a very little increase in terms of enantioselectivity was observed in case of 1,4-dioxane (Entry 5). The reaction was faster in presence of tert-butyl methyl ether (MTBE, Entry 4) compared to those.”
5) Line 157, MTBE, Entry 5 change to entry 4
Corrected, see above.
6) Line 176, E red, left change to right and EF green, right change to left.
We thanks the reviewer for their accurate revision. In fact, the caption of Figure 4 could be misunderstood. Now it has been corrected to clearly differentiate between part A and Part B of the figure.
7) Line 200 Table 2, change 1. for 2.
The foot text of Table 2 has been corrected. Thanks again for their revision
8) Line 281 Table 3 change to Table 4.
Tables are now correctly mentioned in the revised text.
On the another hand, it is not clear if the data reported in Figure 5 are the authors' experimental values or were taken from Zdun and Jose (paragraphs 178-191).
Figure 5 is showing our experimental data. We have corrected that paragraph to clarify this point: “Interestingly, Jose et al. [68] described the deleterious effect (both in conversion and enantioselectivity) of polar solvents (including DCM ) in the direct esterification of rac-ibuprofen with EtOH catalysed by this same enzyme; thus, as this effect is not observed in our system using TEOF for the release of EtOH, our esterification method seems to proceed through different pathways. Figure 5 shows the progress curves (% conversion in black; % eeS in red) obtained for the esterification using TEOF in 100% isooctane and in the mixture using 20% DCM; this last reaction was selected for further studies”.
Additionally, in the references 80-84, the following citation must be included:
Solvent‐Free Lipase‐Catalyzed Transesterification of Alcohols with Methyl Esters Under Vacuum‐Assisted Conditions. GK Sánchez‐Muñoz, MA Ortega‐Rojas, L Chavelas‐Hernández, ChemistrySelect 7 (30), e202202643
Included (new ref. #87)
In addition to reference 68, should be included:
One-Pot Lipase-Catalyzed Enantioselective Synthesis of (R)-(−)-N-Benzyl-3-(benzylamino)butanamide: The Effect of Solvent Polarity on Enantioselectivity. MA Ortega-Rojas, JD Rivera-Ramírez, CG Ávila-Ortiz, E Juaristi, Molecules 22 (12), 2189
Included (new ref. #72)
Thermodynamically controlled chemoselectivity in lipase-catalyzed aza-Michael additions. JD Rivera-Ramírez, J Escalante, A López-Munguía, A Marty, E Castillo. Journal of Molecular Catalysis B: Enzymatic 112, 76-82
Included (new ref. #71)
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dear Authors,
The paper presents new results in a much-studied field. The conclusions drawn are correct. However, there are many errors in the communication that require correction and clarification.
Line 65 dot is missing.
In the first table, the definition of ees (remaining (S)-ibuprofen enantiomeric purity) must be given.
In the first table, the solvent data in lines 4 and 5 have been swapped.
Please provide the exact definition and calculation method of E and EF in the article. (The reference is not enough.)
The representation of the ee value of the experiment in pure isooctane in Figure 5 is completely wrong. (The 24-hour data is 69%, the figure shows 1%.)
The experimental parameters given in Table 3 are not consistent with those described in the experimental section. Which one is true?
Line 259 "Figure 7" instead of "Figure 6".
Line 281 "Table 4" instead of "Table 3".
In Figure 8, the lines are quite different from the given points. Basically, the standard deviation of the points is quite large. This is especially true for the standard condition. Why is the ee curve of this series of measurements missing? Were the measurements performed once or are they from several parallel experiments?
The substrate amount (concentration) is missing from the conditions given in the footnote of Table 5. This part is not properly specified in the experimental part either, and there is a contradiction between them.
Author Response
REVIEWER #2
Dear Authors,
The paper presents new results in a much-studied field. The conclusions drawn are correct. However, there are many errors in the communication that require correction and clarification.
Line 65 dot is missing.
Included
In the first table, the definition of ees (remaining (S)-ibuprofen enantiomeric purity) must be given.
Included
In the first table, the solvent data in lines 4 and 5 have been swapped.
This has been corrected in the text, so that now the new text fits with the entries in Table 1
Please provide the exact definition and calculation method of E and EF in the article. (The reference is not enough.)
The definition of the metrics has been included in the footnote of Table 1. The enantiomeric ratio is quite popular and well known, while EF is not that common. In any case, to explain the parameters, the paragraph has been modified as follows: “To measure enantioselectivity, two parameters were used (see Section 4.2); the most popular one is the Enantiomeric Ratio, the ratio between the specificity constants (kcat/KM, also called kinetic efficiency) for both enantiomers [74,75]. This parameter, although commonly used and accepted, is not easy to be straightforwardly visualized, as it involves logarithmic scales ( see Table 1 footnotes; as a rule of thumb, E values below 15 are inacceptable for practical purposes, being moderate to good in the range of 15–30, and excellent above this value [76]). On the other hand, the Enantiomeric Factor (EF), as reported by López-Belmonte et al. [26], represents a more intuitive metric. This parameter is defined as the correlation between the observed enantiomeric excess and the theoretical enantiomeric excess obtained at the experimental conversion if only the fast reacting enantiomer would have been converted (formula shown in Table 1 footnote). Thus, EF is comprised between 0 and 1; the closer to 1, the higher the enantioselectivity.
The representation of the ee value of the experiment in pure isooctane in Figure 5 is completely wrong. (The 24-hour data is 69%, the figure shows 1%.)
Reviewer is right, there was a severe mistake in the data, now corrected in new Figure 5. For pure isooctane, ee data is 75%, as shown in Table 1. The value of ee = 69% is for 80/20 isooctane/DCM.
The experimental parameters given in Table 3 are not consistent with those described in the experimental section. Which one is true?
Once again reviewer is right, we want to thank them for their precise and detailed revision of the manuscript. The mistake was ours because of a sluggish copy/paste protocol. Now data in footnote of Table 3 are according to those in experimental section.
Line 259 "Figure 7" instead of "Figure 6".
This has been corrected in the text
Line 281 "Table 4" instead of "Table 3".
Also corrected
In Figure 8, the lines are quite different from the given points. Basically, the standard deviation of the points is quite large. This is especially true for the standard condition. Why is the ee curve of this series of measurements missing? Were the measurements performed once or are they from several parallel experiments?
The ee of standard reaction conditions (in blue) has been included. Initially, it hadn’t been included for clarifying the plot, as this value (as well as conversion for standard conditions) had already been plotted in Figure 5.
It is true that for standard conditions it seems to be a relatively higher standard deviation compared to other plots. As commented in the text, experimental data were adjusted to single exponential growing model using the program INRATE implemented inside SIMFIT fitting package. For the fitting of standard conditions data to the exponential model, R2 was 0.9781, based on an unweighted sum of squares SSQ = 9.5935E+01. For the other plots on Figure 8, R2 were higher (0.9925 for 5 mg/mL, 0.9999 for 15 mg/mL). Esterification under standard conditions was repited twice, obtaining similar results (R2 = 0.984), although for straight comparison with the other experiments in Fig. 8 only one of those similar ruins was plotted.
The substrate amount (concentration) is missing from the conditions given in the footnote of Table 5. This part is not properly specified in the experimental part either, and there is a contradiction between them.
Footnote of Table 5 has been modified, as well as the experimental section 3.3.5. Hope that now it can be more understandable.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Dear Author,
The requested corrections have been implemented.
There are still three typos left in it.
Line 134 "section 3.3.1" instead of "section 4.3.1"
Line 147 "Section 3.2" instead of "section 4.2"
Line 331 "down to 0,53" instead of "down to 0,63" (If the data in Table 5 is correct.)
Please fix these in the final version.
