Synthesis of Glycosidic (β-1′′→6, 3′ and 4′) Site Isomers of Neomycin B and Their Effect on RNA and DNA Triplex Stability

Round  1

Reviewer 1 Report

This manuscript is a nice example of how painstaking experimental work is required to shed light on the structure-activity relationship of the interaction of aminoglycoside antibiotics and nucleic acids. The authors synthesized glycosidic regioisomers of neomycin B by "dissecting" the neomycin azide into glycosyl donors and acceptors. Synthesizing the novel regioisomers they have studied their effects on duplex and triplex stabilization with selected DNA and RNA samples. A marked stabilization was found in each case and it was concluded that the carbohydrate core and spatial orientation of their amino groups do not play a significant role for sufficient discrimination between the affinities of individual regioisomers. The experimental work is excellent and very thorough.

This manuscript can be published almost as it is. Minor improvements are suggested by changing some compound names, e.g. the first meaningful character in compound names should be capitalized: "5,6,3’-tri-O-acetyl-1,3,2’,6’-tetra-azidoneamine" should be  "5,6,3’-Tri-O-acetyl-1,3,2’,6’-tetra-azidoneamine" etc.; sometimes hyphens are missing from the compund names: "1,3,2’,6’,2’’’,6’’’ hexa-azido-neobiosamine-(beta-1’’-> 3’) neamine" should be "1,3,2’,6’,2’’’,6’’’-Hexa-azido-neobiosamine-(beta-1’’-> 3’) neamine" etc.

It is of absolutely minor importance and serves only as an advice to the authors that there are a number of odourless thiols that can be used in synthetic work, e.g. in thioglycoside formation:

Kumar, R. K.; Cole, D. L.; Ravikumar, V. T., 2-Methyl-5-tert-butylthiophenol--an odorless deprotecting reagent useful in synthesis of oligonucleotides and their analogs. Nucleosides Nucleotides Nucleic Acids 2003, 22, (4), 453-460.

Node, M.; Kajimoto, T., Development of Odorless Organosulfur Reagents and Asymmetric Reaction Using Odorless Thiols. Heteroatom Chemistry 2007, 18, 572-583.

Despras, G.; Urban, D.; Vauzeilles, B.; Beau, J.-M., One-pot synthesis of D-glucosamine and chitobiosyl building blocks catalyzed by triflic acid on molecular sieves. Chemical Communication 2014, 50, 1067-1069.

Author Response

Cf. the enclosed authors' response.

Author Response File: Author Response.docx

Reviewer 2 Report

Aminoglycosides are best known as antibiotics binding to the A-site of the small ribosomal subunit. In addition, they are important ligands for a multitude of other relevant RNA targets. Synthetic methods giving access to isomers of natural aminoglycosides, therefore, are highly welcome.

In the present manuscript the chemical transformation of neomycin B into three constitutional isomers is reported. All synthetic steps are well described. The characterization of intermediates and of all final products is sound.

The second part of the manuscript compares the affinities of neomycin and the synthetic analogs to double or triple stranded RNA and DNA by measuring the corresponding melting points. Although the effects were in the same range for all isomers, some distinct differences could be observed. I can imagine that even higher selectivities may be seen in future experiments with less regularly structured RNAs containing bulges, loops or pseudoknots.

To summarize: This is a sound, well conducted study leading to rather ambitious target compounds. It should be accepted for print almost unchanged.

Some minor errors can be corrected in a minute:

Line 63-64: 2,5,3’,4’-tetra-O-acetyl, not 2,5,2’3’,4’-tetra-O-acetyl

Line 64: 1,3,2’,6’-tetraazido, not 1,3,2’,6’-diazido neamine.

Line 66: 2,5,3’,4’-tetra-O-acetyl, not 2,5,2’3’,4’-tetra-O-acetyl

Table 1. The meaning of T_m3 and T_m2 is well explained in the text but not in the very short legend of Table 1.

Line 139: “Consistently with the previous findings (affinity of neomycin follows the trend:”  Accidental parenthesis?

Line 142: "DeltaT_m2 = +11.1 – 18.6°C vs. +0.7 – 1.9 °C". The highest increase of T_m2 for DNA is 2.3, not 1.9°C according to Table 1.

Line 146: "DeltaT_m = +19°C, +17.4°C...."     +18.6°C can be found in Table 1 but not +19°C.

Author Response

Cf. the enclosed authors' response.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript is well written but there are some errors in the materials and methods section that need to be corrected before it it ready for publication. 

Careful review of the NMR spectra data reveals some peculiarities, for ex. 

1) 5.48(dd,1H,J=4.2Hz,both)

A doublet of doublet with identical coupling constants would be a triplet. Thus, the splitting pattern of these peaks is not clear.

This repeats multiple times in the NMR data of other compounds as well.

2)  5.05(dd,1H,J=2.9&2.8Hz

         The coupling constants are so close as to be virtually identical which would, again, result in a triplet. Were the authors able to achieve this resolution with a 500 MHz NMR?

3)  5,23 (d, 1H, J = 4.5 Hz)

 Authors mislabel the chemical shift as 5,23 instead of 5.23. This happens at multiple places in the NMR spectra data.

As NMR data is a crucial piece of information to confirm identity of the products obtained, accurate analysis is critical. It is advised that the authors correct the various mistakes in the NMR spectral data. Only then can the manuscript be ready for publication.

Author Response

Cf. the enclosed authors' response.

Author Response File: Author Response.pdf

Round  2

Reviewer 3 Report

The authors have made the required changes that make the manuscript ready for publication. 

Author Response

All corrections have now been done (i.e. dd:s changed to t). We agree that this depends on the view. dd:s with the same coupling constant could be defined as pseudo-triplets and they are now corrected to triplets.

Respectfully,

Pasi Virta