Optimization of Enzymatic Synthesis of D-Glucose-Based Surfactants Using Supported Aspergillus niger Lipase as Biocatalyst
Round 1
Reviewer 1 Report
This manuscript describes the elaboration of the reaction conditions and characterization of lipase-catalyzed acylation of glucose. This reviewer recommends the acceptance by “Chemistry,” if the following comments are suitably considered and the manuscript is adequately revised.
Abstract: The name of solvent should be described “abbreviation” (“original full name”), according to the styles in the text. The abstract is independent document from the main text.
Page 1, paragraph 1, line 35. The references 7 and 8 are not suitable for citation. Please carefully read ref. 10 and cite proper revies for 7 and 8.
Page 3. Do not use tert-but. Please change tert-BuOH throughout the text.
Line 121 enzymatic synthesis: There is no description for the quantity of substrate (# g, # mmol), and no description of isolated yield, especially in large-scale preparative synthesis.
Page 4, line 141. The NMR spectrum of the product is problematic. In DMSO-d6, the OH protons should be clearly observed. The non-equivalent protons at C-6 substituted with acetoxy group are not clear, just mentioned as multiplet protons. Judged by 4.76 (d, J = 2.4 Hz, 1H), the product seems to be pure a-anomer. The 13C NMR spectrum also suggested the formation of a-anomer exclusively. Please show authentic spectra with proper citation of literatures. The starting material, glucose is a mixture of a,b-anomers. What was the destination of the b-anomer in the mixture of stereoisomers in substrate?
Page 10, line 353 The authors claimed that the addition of DMSO lowered the site-selectivity judged by HPLC analysis. Detailed spectra of such byproducts, however, are not shown. Please confirm of the stability of product in DMSO toward the disproportionation of the primary product, 6-acylated derivative.
Moderate editing of English language required, in addition to the check of abbreviation of the name of reagents.
Author Response
We would like to thank the two reviewers for their thorough evaluation and fair comments. Our point-by-point responses to the comments received are summarized below with the reviewer's original question italicized, and our response in bold. We have carried out the modifications requested. Changes in manuscript are highlighted in yellow.
Abstract: The name of solvent should be described “abbreviation” (“original full name”), according to the styles in the text. The abstract is independent document from the main text.
As requested, we now corrected this point in abstract and added original full name for DMSO and 2MeTHF3one.
Page 1, paragraph 1, line 35. The references 7 and 8 are not suitable for citation. Please carefully read ref. 10 and cite proper revies for 7 and 8.
References 7, 8 have been changed to more pertinent ones. Reference 10 has also been changed to a reference not specific to flow biocatalysis.
Page 3. Do not use tert-but. Please change tert-BuOH throughout the text.
The document has been amended as requested.
Line 121 enzymatic synthesis: There is no description for the quantity of substrate (# g, # mmol), and no description of isolated yield, especially in large-scale preparative synthesis.
We have now added this information. For example, line 124: (0.18 g, 1 mmol, 1 eq.). Concerning the quantity of product, we specified the quantity of monoester isolated after purification in initial conditions line 140: 0.125 g, 34.6 % isolated yield for initial synthesis conditions in 2M2B/DMSO.
Page 4, line 141. The NMR spectrum of the product is problematic. In DMSO-d6, the OH protons should be clearly observed. The non-equivalent protons at C-6 substituted with acetoxy group are not clear, just mentioned as multiplet protons. Judged by 4.76 (d, J = 2.4 Hz, 1H), the product seems to be pure a-anomer. The 13C NMR spectrum also suggested the formation of a-anomer exclusively. Please show authentic spectra with proper citation of literatures. The starting material, glucose is a mixture of a,b-anomers. What was the destination of the b-anomer in the mixture of stereoisomers in substrate?
OH protons are actually visible and are now assigned, in accordance with the literature integrated into the characterization section ([19, 33, 37]).
As far as α/β-anomers are concerned, this is correct. We have now amended this point by a α/β anomers mixture: 5.08 (m, 1H) and 4.90 (d, J = 8.7 Hz, 1H).
Concerning 13C NMR spectrum, as document 1 shows, the spectrum displays 2 peaks characteristic of the α/β anomers mixture of 6-O-lauroyl-D-glucopyranose. The peak assignment has therefore been amended in the characterization of the product, line 144 and 3 references ([19, 33, 37]) have been added. The final product is therefore a α/β anomers mixture of 6-O-lauroyl-D-glucopyranose.
Document 1: 13C NMR spectrum for the 6-O-lauroyl-D-glucopyranose synthesis.
Page 10, line 353 The authors claimed that the addition of DMSO lowered the site-selectivity judged by HPLC analysis. Detailed spectra of such byproducts, however, are not shown. Please confirm of the stability of product in DMSO toward the disproportionation of the primary product, 6-acylated derivative.
This is an interesting point, and we agree with this comment as the stability of the 6-acylated derivative in DMSO has not been studied here. DMSO is described as a disproportionating agent, but these studies do not mention sugar esters. This point is however discussed line 379.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this work the authors report on the synthesis of D-glucose lauric ester using an Aspergillus niger lipase. Reaction conditions were optimized considering the effect of different parameters including the addition of DMSO as co-solvent that led to higher yields. In subsequent investigation DMSO was efficiently replaced by greener solvent 2-methyltetrahydrofuran-3-one addition resulting in good performance in the transesterification reaction toward the desired product.
I think that the article is quite interesting, and the data reported fully support the related discussion, thus I suggest that the article could be accepted after addressing some minor corrections:
1) I would suggest the authors to revise the abstract were they stated that “This lipase appeared to be highly selective for 6-O-lauroyl-D-glucopyranose. However, the addition of DMSO as a co-solvent led to a loss of selectivity.” In fact, also according to the result reported the addition of DMSO led to an increase in yield. This sentence should be revised or clarifies.
2) In several figures there is the presence of one or more *. I am not sure what these signs stand for please clarify it in the article text and also in the figure captions.
3) When reporting the temperature values, it should be 70 °C not 70°C. This should be revised in all the text as sometimes there is a space and in some other cases there is not.
Author Response
Answer to Reviewer #2 comments and queries
We would like to thank the two reviewers for their thorough evaluation and fair comments. Our point-by-point responses to the comments received are summarized below with the reviewer's original question italicized, and our response in bold. We have carried out the modifications requested. Changes in manuscript are highlighted in yellow.
1) I would suggest the authors to revise the abstract were they stated that “This lipase appeared to be highly selective for 6-O-lauroyl-D-glucopyranose. However, the addition of DMSO as a co-solvent led to a loss of selectivity.” In fact, also according to the result reported the addition of DMSO led to an increase in yield. This sentence should be revised or clarifies.
We fully agree with this comment and have reworded this sentence as follows: “However, the addition of DMSO (dimethyl sulfoxide) as a co-solvent displays a duality, increasing yields but leads to a loss of selectivity.”. We have added the notion of duality to the use of DMSO, line 18.
2) In several figures there is the presence of one or more *. I am not sure what these signs stand for please clarify it in the article text and also in the figure captions.
We have now added the meaning of the * in the figure legends. For example for figure 3 “Results are expressed as the mean ± SD. Statistical significance was determined by Two-way ANOVA Tukey’s test. ****P < 0.0001.”
3) When reporting the temperature values, it should be 70 °C not 70°C. This should be revised in all the text as sometimes there is a space and in some other cases there is not.
The document has been amended as requested.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
This reviewer confirmed the authors' response to the reviewing opinion and revised manuscript, and recommends the acceptance by "Chemistry". If you have opportunity to revise further, tert-butanol should be tert-butyl alcohol or 2-methyl-2-propanol. The carbon parent skeleton "tertiary butane" does not exist and tertiary butyl group is allowed.
Please further revise the English for the manuscript by yourselves.
