α-d-Glucopyranosyl-(1→2)-[6-O-(l-tryptophanyl)-β-d-fructofuranoside]

Round 1

Reviewer 1 Report

In connection with previous studies, the authors report an amino acid glycoside that exhibit antitrypanosomal activity against Trypanosoma brucei brucei. 

This a new compound of potential pharmacological interest.

Accordingly, it is publishable in Molbank after minor revision of the English wording.

Author Response

The manuscript has been reviewed according to the recommendations of the first reviewer.

Reviewer 2 Report

The authors reported on the isolation of a new compound from the fermentation extracts produced by the strain Mycobacterium sp. BRS2A-AR2.

The topic is interesting and well within the aims and scopes of the journal.

Yet, I think some modifications and improvements in the manuscript are necessary and so I can not recommend its acceptance it in its present form.

These things are listed below one by one:

TITLE:

- The presented title is not appropriate. Please modify it adding some important details such as the fact that this compound is new, that it was isolated from the bacterium etc.

ABSTRACT:

- Please write the complete botanical name of the plant species i.e. Rhizophora racemosa G.Mey.

- “Spectroscopy and spectrometry aided chemical profiling of fermentation extracts produced by the 19 strain led to the isolation of the new compound, O¹⁰-(α-D-Glucopyranosyl-(1→2)-β-D-fructofuranoside)-L-tryptophan (1)”. This sentence is poorly written. Please modify it.

- The denomination Trypanosoma brucei brucei is not correctly written. Please mention that the second term is eventually a subspecies.

- Please write the complete botanical name of the species i.e. Coptis japonica (Thunb.) Makino.

- “When tested against Trypanosoma brucei brucei, 1 (IC₅₀ 11.25 μM) was just as effective as Coptis japonica (IC₅₀ 8.20 μM)” Please write something more for this point for those who are not acquainted with this subject. For example, what is Trypanosoma brucei brucei? Then what does this compound is as effective as Coptis japonica in? Then why Coptis japonica? All these things may attract the reader more.

KEYWORDS:

- Please modify a little the keywords. For example insert that you found a new compound, that you used several spectroscopic techniques for its identification, that you tested it against Trypanosoma brucei brucei. This part is very important since this is what readers read as first thing.

INTRODCUTION:

- “Glycosylated natural products contain sugars in the form of polysaccharides, oligosaccharides and monosaccharides which are in turn bound to other well-known medicinally important natural products” This sentence is quite misleading. Please rephrase it.

- Page 2 line 45: “increased activity”? In what?

- “We have collected several mangrove plants including Conocarpus erectus, Laguncularia racemosa and Rhizophora racemosa” All these plant species must be written with their complete botanical names.

- “From these samples, we have isolated many cultivable strains of endophytic microbes…” What are these? Please cite some at least.

- “against the neglected tropical parasites Trypanosoma brucei brucei, Leishmani donovani, Leishmania major, Schistosoma mansoni and Trichomonas mobilensis.” All these parasites must be written with their complete names. Moreover, the right term is Leishmania donovani.

- Lines 68-77: All this part is not written in the same format as the others. Please level it with the rest. Moreover, I deem that all this part would be better to be found in the experimental part. At most, you can say that you isolated a new compound.

- I think a brief discussion about the morphology of the plant and the etiology of the bacterium must be written. Again this is for readers who do not know this plant species and this bacterium.

- Figure 1 should be presented in the Results and discussion section. Moreover, the structure you presented is not much in accordance with the name of the compound. The terminology O- is used for saccharides forming ether bonds while here you have an ester bond. Please consider it.

RESULTS AND DISCUSSION:

- “The very stable compound 1” How do you know this compound is very stable? Did you perform stability tests on it? If so, which ones?

- Table 1: Please put 1H-NMR data before ¹³C-NMR ones. Moreover, you numbered NH₂ with 11. For coherence, please cite this everywhere you write about this functional group.

- Please consider to modify the numbering of the sugars putting primes to fructose as suggested from your structure.

-  “The IC₅₀ value of 11.25 μM calculated for 1 was almost as effective as that of the routine laboratory standard Coptis japonica (IC₅₀ 8.20 μM) which is known to contain the alkaloid berberine” Why is berberine so important? Please explain. Moreover, after the first time you cite a plant or bacterium species in the main text, you can write them using the initial of the genus such as for C. japonica.

- Lines 145-148: The phrase is right but quite uneasy to read. Please consider to split it up.

EXPERIMENTAL SECTION:

- (δ_H 3.33, 2.50 and δ_C 39.52 ppm in DMSO-d6). Please write the multiplicity of the proton peaks and to what they refer to since 3.33 actually refers to methanol present as stabilizer in DMSO-d6.

- Please give more details about the HR-MS analysis and, in particular, about the used ion source.

- Please provide more details about the collection site of the plant with geographical coordinates and altitude.

- “extracted sequentially and alternatively with MeOH and CH₂Cl₂.” What do you mean with sequentially and alternatively?  

- “The total crude extract was subjected to a modification of Kupchan’s solvent partitioning process”. Please explain this process and why this was necessary.

- Please explain clearly why you hydrolyzed compound 1.  

CONCLUSION:

- Line 242: The name of the plant species should be written in Italics.

- “Compound 1, exhibited potent  antitrypanosomal effects when tested against our laboratory strain of Trypanosoma brucei brucei” What do you mean with our? Where did you get it from exactly?

REFERENCES:

- Reference 3: 115 is not the page. It is the issue. Please write the pages.

- Reference 8: 7 is not the page. It is the issue. Please write the pages.

- Reference 15: 39 is not the page. It is the issue. Please write the pages.

- Reference 20. Missing pages.

- Reference 25: Please specify all the number of the pages i.e. 440-448.

- Reference 36: 592 is not the page. It is the issue. Please write the pages.

- Reference 37: 233 is not the page. It is the issue. Please write the pages.

- Reference 39: 721 is not the page. It is the issue. Please write the pages.  

- Reference 44: 36 is not the page. It is the issue. Please write the pages.

SUPPLEMENTARY:

- The retention time you wrote in the text for compound 1 does not correspond to the chromatogram present (20 min vs 14.59). Please check it.  

- The arrow for the MS spectrum must be addressed to the corresponding peaks of the proton and sodium adducts.

- Figure S2 is useless since you did not discuss about it in the text.

- Figure S3: The name tortomycoglycoside is not present anywhere in the text. I noticed you used this name for all the spectra. Please, you have to write somewhere that you are using a trivial name for compound 1.

- Figures S6-S10: Eliminate the term NMR from the captions of the spectra. Moreover, eliminate also 2D from the caption of figure S9.

Author Response

ABSTRACT:

- Please write the complete botanical name of the plant species i.e. Rhizophora racemosa G.Mey. Done

- “Spectroscopy and spectrometry aided chemical profiling of fermentation extracts produced by the 19 strain led to the isolation of the new compound, O¹⁰-(α-D-Glucopyranosyl-(1→2)-β-D-fructofuranoside)-L-tryptophan (1)”. This sentence is poorly written. Please modify it. Done

- The denomination Trypanosoma brucei brucei is not correctly written. Please mention that the second term is eventually a subspecies. Done

- Please write the complete botanical name of the species i.e. Coptis japonica (Thunb.) Makino. Done

- “When tested against Trypanosoma brucei brucei, 1 (IC₅₀ 11.25 μM) was just as effective as Coptis japonica (IC₅₀ 8.20 μM)” Please write something more for this point for those who are not acquainted with this subject. For example, what is Trypanosoma brucei brucei? Then what does this compound is as effective as Coptis japonica in? Then why Coptis japonica? All these things may attract the reader more. Done

KEYWORDS:

- Please modify a little the keywords. For example insert that you found a new compound, that you used several spectroscopic techniques for its identification, that you tested it against Trypanosoma brucei brucei. This part is very important since this is what readers read as first thing. What the reviewer describes here cannot go into keywords. The abstract and introduction do justice to this.

INTRODCUTION:

- “Glycosylated natural products contain sugars in the form of polysaccharides, oligosaccharides and monosaccharides which are in turn bound to other well-known medicinally important natural products” This sentence is quite misleading. Please rephrase it. Done

- Page 2 line 45: “increased activity”? In what? Done. Increased activity = bioactivity

- “We have collected several mangrove plants including Conocarpus erectus, Laguncularia racemosa and Rhizophora racemosa” All these plant species must be written with their complete botanical names. Done

- “From these samples, we have isolated many cultivable strains of endophytic microbes…” What are these? Please cite some at least. Done

- “against the neglected tropical parasites Trypanosoma brucei brucei, Leishmani donovani, Leishmania major, Schistosoma mansoni and Trichomonas mobilensis.” All these parasites must be written with their complete names. Moreover, the right term is Leishmania donovani. Done

- Lines 68-77: All this part is not written in the same format as the others. Please level it with the rest. Moreover, I deem that all this part would be better to be found in the experimental part. At most, you can say that you isolated a new compound. Done. A little overview of the whole isolation and characterization process suffice here.

- I think a brief discussion about the morphology of the plant and the etiology of the bacterium must be written. Again this is for readers who do not know this plant species and this bacterium. Done.

- Figure 1 should be presented in the Results and discussion section. Moreover, the structure you presented is not much in accordance with the name of the compound. The terminology O- is used for saccharides forming ether bonds while here you have an ester bond. Please consider it. Done. Compound has been named an ester instead of an ether.

RESULTS AND DISCUSSION:

- “The very stable compound 1” How do you know this compound is very stable? Did you perform stability tests on it? If so, which ones? Done. Changed wording.

- Table 1: Please put 1H-NMR data before ¹³C-NMR ones. Moreover, you numbered NH₂ with 11. For coherence, please cite this everywhere you write about this functional group. Done Putting 1H-NMR data in the table before 13C-NMR data will be quite strange and cumbersome. I reserve that, this should stay the same. Yes, if that functional group is mentioned we will refer to it as 11.

- Please consider to modify the numbering of the sugars putting primes to fructose as suggested from your structure.

-  “The IC₅₀ value of 11.25 μM calculated for 1 was almost as effective as that of the routine laboratory standard Coptis japonica (IC₅₀ 8.20 μM) which is known to contain the alkaloid berberine” Why is berberine so important? Please explain. Moreover, after the first time you cite a plant or bacterium species in the main text, you can write them using the initial of the genus such as for C. japonica. Done

- Lines 145-148: The phrase is right but quite uneasy to read. Please consider to split it up. Done

EXPERIMENTAL SECTION:

- (δ_H 3.33, 2.50 and δ_C 39.52 ppm in DMSO-d6). Please write the multiplicity of the proton peaks and to what they refer to since 3.33 actually refers to methanol present as stabilizer in DMSO-d6. Done

- Please give more details about the HR-MS analysis and, in particular, about the used ion source. Done

- Please provide more details about the collection site of the plant with geographical coordinates and altitude. Done. However, we have already published the collection sites in the previous paper.

- “extracted sequentially and alternatively with MeOH and CH₂Cl₂.” What do you mean with sequentially and alternatively? Done. 

- “The total crude extract was subjected to a modification of Kupchan’s solvent partitioning process”. Please explain this process and why this was necessary. Done

- Please explain clearly why you hydrolyzed compound 1. Done 

CONCLUSION:

- Line 242: The name of the plant species should be written in Italics. Done

- “Compound 1, exhibited potent  antitrypanosomal effects when tested against our laboratory strain of Trypanosoma brucei brucei” What do you mean with our? Where did you get it from exactly? Done

REFERENCES:

- Reference 3: 115 is not the page. It is the issue. Please write the pages.

- Reference 8: 7 is not the page. It is the issue. Please write the pages.

- Reference 15: 39 is not the page. It is the issue. Please write the pages.

- Reference 20. Missing pages.

- Reference 25: Please specify all the number of the pages i.e. 440-448.

- Reference 36: 592 is not the page. It is the issue. Please write the pages.

- Reference 37: 233 is not the page. It is the issue. Please write the pages.

- Reference 39: 721 is not the page. It is the issue. Please write the pages.  

- Reference 44: 36 is not the page. It is the issue. Please write the pages Done All references done.

SUPPLEMENTARY:

- The retention time you wrote in the text for compound 1 does not correspond to the chromatogram present (20 min vs 14.59). Please check it.  Done Chromatogram in the supplementary is HRESI-LCMS. Retention time quoted in the paper is for Semi-prep HPLC. These two are not the same. 

- The arrow for the MS spectrum must be addressed to the corresponding peaks of the proton and sodium adducts. Done

- Figure S2 is useless since you did not discuss about it in the text. DI have referred to it in main text. It stays.

- Figure S3: The name tortomycoglycoside is not present anywhere in the text. I noticed you used this name for all the spectra. Please, you have to write somewhere that you are using a trivial name for compound 1. Done. The name has been put in the text.

- Figures S6-S10: Eliminate the term NMR from the captions of the spectra. Moreover, eliminate also 2D from the caption of figure S9. Done

Cheers!

Round 2

Reviewer 2 Report

The authors presented a revised version of a manuscript I had previously reviewed.

This has been well improved but I still have got a few concerns and I recommend a further minor revision.

These concerns are listed below one by one:

- My query: The denomination Trypanosoma brucei brucei is not correctly written. Please mention that the second term is eventually a subspecies.

Your answer: Done

My reply: What you wrote is not totally correct. Please write the part as follows: Trypanosoma brucei subsp. brucei. Do write this also at line 73.

ABSTRACT:

- : Line 29: Please write the relative part as follows: T. brucei subsp. brucei.

INTRODUCTION:

- The description are fine and well presented but I deem they were not inserted in the right position of the manuscript. Please insert them elsewhere in this same section, for example the plant description at line 70 maybe renouncing to cite the other species.

RESULTS AND DISCUSSION:

- I still have some concerns about the IUPAC name of your compound. You did not write the linkage between fructose and tryptophan.

EXPERIMENTAL SECTION:

- (δ_H 2.50 (1H, p, J_HD = 1.97Hz). Please eliminate the J value here. It is not important.

Author Response

My reply: What you wrote is not totally correct. Please write the part as follows: Trypanosoma brucei subsp. brucei. Do write this also at line 73. This has been done.

ABSTRACT:

- : Line 29: Please write the relative part as follows: T. brucei subsp. brucei. This has also been done.

INTRODUCTION:

- The description are fine and well presented but I deem they were not inserted in the right position of the manuscript. Please insert them elsewhere in this same section, for example the plant description at line 70 maybe renouncing to cite the other species. Added paragraphs have been re-positioned.

RESULTS AND DISCUSSION:

- I still have some concerns about the IUPAC name of your compound. You did not write the linkage between fructose and tryptophan. The name has been revised further to: α-D-Glucopyranosyl-(1→2)-β-D –fructofuranoside 6-L-tryptophan ester 

EXPERIMENTAL SECTION:

- (δ_H 2.50 (1H, p, J_HD = 1.97Hz). Please eliminate the J value here. It is not important. Done