A Photo-Enzymatic Cascade to Transform Racemic Alcohols into Enantiomerically Pure Amines

This manuscript reported the one-pot   two-step approach for conversion of convert racemic alcohols to enantiopure   amines. The proposed photo-enzymatic cascade showed good conversion   efficiency and product yields. The current manuscript is well organized and   written. Taking into account the quality and scope of the journal, I would   recommend the acceptance of this manuscript to publish in ‘Catalysts’.

Herewith I have added few comments that   will help authors to improve the current review article: 

Thank you for the   suggestions!

The authors should   discuss the stability of recombinant transaminases over higher concentration   of amines. Is there any change in the final product by varying transaminases   concentrations?

We apologize for not   having elaborated more on this in the original manuscript. Following the   reviewer’s suggesting, we have added a passage dealing with activity- and   stability issues of the enzymes used.

The authors should   discuss the continuous conversion of amines with immobilized enzymes. Is it possible   to reuse of enzymes in the current approach?

The reviewer is   absolutely right with his/her comment here! We agree that a continuous   reaction setup e.g. in a flow chemistry approach may also be a solution to   the inactivation of the biocatalysts by the photoexcited photocatalysts.

A comment was added   to the manuscript.

The author   should consider the effect of pH of enzyme reaction solution and temperature   on conversion efficiency.

We apologize for not   having stated this clearly in the original manuscript. These factors had been   investigated and are now discussed more clearly in the revised manuscript.

Comments   and Suggestions for Authors

In this manuscript, the authors use sodium anthraquione-2-sulfonate   (SAS) to mediate aerobic oxidation alcohol and transaminase to catalyze the   enantiospecific reductive amination of the intermediate. When the process was   switched to one-pot two-step from one-pot one-step, the productivity was   significantly improved. And this method was applied to synthesize many   amines. The results sound good. However, there are a lot of mistypes in the   manuscript.

The authors should examine and correct the   text carefully, not limit in the following comments.

1) Line 26

No dot after square bracket.

2) Line 48

Add one space before square bracket.

We apologize for the   somewhat careless writing of the original manuscript. We have carefully   revised the manuscript eliminating the typos.

3) Only substrates’ structure was given in   Scheme 1. It is better to show all products’ structure (R- and S-), which can   be include in supplementary part.

Following the   reviewer’s advice, a figure showing the structures of all reagents has been   added to the supporting information.

4) Line 76

No short line between “sodium” and   “antraquione”.

5) Line 96

What is “5c”? If it is a product, it should   be bold.

6) Line 103

No short line between “pot” and “one”.

7) Line 126

“5a”   should has parentheses.

No short line between “pot” and “one”.

8) Line 149

Equation Y, delete one parentheses in   denominator.

9) Line 151

Is this "GC" same as abbreviation   of "gas chromatography"?

10) Line 180

Add “and” before square bracket.

11) Line 194

Same as Line 180.

12) Line 201

Add space between “1mM”.

13) Line 202

Add space to “2sulfonate” and “1M”.

We apologize for the   somewhat careless writing of the original manuscript. We have carefully   revised the manuscript eliminating the typos.

Comments   and Suggestions for Authors

The authors describe a process in which primary   and secondary alcohols are step-wise converted to amines via photooxidation   followed by reductive amination by a biocatalyst. While the process is highly   novel and of great interest to chemists needing to make enantiopure amines,   this reviewer is not convinced that it is in any way is or replaces a   Mitsonobu reaction. The only similarity between the two processes is that the   starting material is an alcohol and therefore the title of the manuscript is   horribly misleading. This reviewer recommends major revisions and I will   detail my comments:

Following the   reviewer’s suggestion we have refrained from comparing our reaction scheme   with the established Mitsonobu   reaction.

1) The entire introduction will need to be   reworked in order to be consistent with the overall significance of the   research results. It is not a Mitsonobu reaction. It is an alternative, at   best.

Changed as suggested   by the reviewer. The comparison with the Mitsonobu reaction was removed.

2) Change all occurrences of "one pot   one-step" and similar statements to one-pot, one-step."

Changed as   suggested.

3) Line 69: Define "E-factor."

Changed as suggested.

4) Figure 1: do you have error bars to   represent on the bar graph?

The referee is   right, that the experiments shown in Figure 1 have not been performed as   duplicates. A note has been added.

5) Where were the enzymes obtained?

All enzymes have   been prepared in-house via recombinant expression in Escherichia coli. A note   to the respective section in the supporting information has been added.

6) Line 92: typo, change   "staring" to "starting."

Changed as   suggested.

7) Line 133 and 233: try to avoid   colloquial phrase like "to our delight."

Changed as   suggested.

Title   of Table 2 is Reductive amination of chiral substrates. If the substrates are   ketones, this title should be modified to “prochiral substrates” instead of   chiral substrates.

Changed as suggested   by the reviewer.

Compound   4b should be hexanal instead of 2-hexanone. 4b is not prochiral   substrate.

Changed as suggested   by the reviewer.

In   Table 3, compound 3b (2-hexanone) may produce chiral amine as a   product.

Changed as suggested   by the reviewer.

In some experiments, the   authors should show the isolated yield of the product.

We fully agree with   the reviewer that this would have been desirable.  However, we hope that the reviewer will   understand that the aim of this study was to establish the cascade itself and   to provide a proof-of-concept for this new cascade. Further studies will   focus on the preparative application and upscaling.

Comments   and Suggestions for Authors

Introduction:   you have discussed a lot about the oxidation step, you could extend the   enzymatic part by providing examples of transaminases and their applications.

The state-of-the art   about the biocatalytic transamination has been discussed in more details in   the introduction. We have mentioned: 1) the main applications of   transaminases in (bio)chemical manufacturing; 2) the use of traditional amine   donor (e.g., alanine) and the common methods to shift unfavourable   equilibria; 3) the use of alternative amine donor, listing the advantage   (improved equilibrium) but also the disadvantages; 3) a more clear   explanation why isopropylamine was selected as amine donor.

Selected, important   references were added to give a complete and balanced overview of the   state-of-the art in the field.

L-26 The point after the reference is not   necessary

Changed as   suggested.

Results and discussion: you have   chosen only rac-1-phenyl ethanol as a model reaction. Is it possible   that different results are obtained in the oxidation of hydroxyl groups using   different substrates?  In my opinion it   would be interesting to screen all the substrates (1a-10a) with homogeneous   and heterogeneous catalyst.

The reviewer is   certainly right that a broader screening of photocatalyst-alcohol   combinations would have been interesting (possibly yielding more active   combinations).

However, we hope   that the reviewer will understand that the aim of this study was to establish   the cascade itself and not strive for the ‘optimal’ reaction system. Further   experiments will focus on the optimisation of this system which will   certainly entail the suggestions by the reviewer!

L-92 what do you mean for staring material?

Changed as   suggested.

L-102 Please specify how control reactions   have been set up.

The original   statement was indeed misleading. Here we refer to further characterisation of   the reaction to identify the main limitations. This has been corrected.

Needles to mention   that we have performed the necessary negative control reactions (leaving out   either the photocatalyst or performing the reactions in the dark) and that   they indeed yielded no product formation.

Materials   and method:

L-199   you only talk about 1-phenyl ethanol. Do you follow the same protocol for all   the

substrates?

It has been   clarified in the revised version.

L-213   The first reaction mixture was in water, do you diluted with NaPI 10 mM?   Please specify this point.

It has been   clarified in the revised version. NaPi buffer (100 mM) was used. The final   concentration of the NaPi was 50 mM.

Supplementary   materials:

Figure S31 in   the graf you reported 1a formula instead of 2a formula.

Corrected.