Selective Proton-Mediated Transport by Electrogenic K+-Binding Macrocycles
Round 1
Reviewer 1 Report
The manuscript concerns electrogenic ionophores selective for K ions and characterises their ion transport ability.
It is written to a good standard and well presented. I have no changes to suggest.
Author Response
Thanks to reviewer 1 to consider our manuscript.
Reviewer 2 Report
This manuscript presents an interesting proposal on cation transport selectivity modulation by simple isomeric variations on a C8 alkyl moiety linked to a classical crown ether scaffold. The overall work seems interesting and the preliminary conclusions are, in principle, appealing. However, the whole thesis of the manuscript cannot be fairly assessed since the molecular characterization is incomplete for the new compounds and the data included in this regard does not match with the chemical structures claimed by the authors. For instance, elemental analysis, HR-MS, 2D NMR are not included, whereas 1H spectra in SI are only partially included and number of protons are not in agreement with molecular formulas. In addition, the authors did not include neither any micrograph nor any sort of structural characterization for their vesicles containing the self-assembled ionophores, so that their conclusions could be more strongly supported.
Author Response
Thanks for the comment from this reviewer. We noticed the mistake and replaced the wrong characterization with the correct data.
We put the wrong characterization information for the compound 1, it is actually referring to another compound we previously reported which bears no aromatic ring. We have replaced the wrong characterization with the correct data. We are sorry about this mistake.
Due to the limitation of our lab, we could not provide data from elemental analysis, HR-MS, 2D NMR. The data obtained from NMR and ESI MS for structurally simple compounds are still in accord with the proposed formula.
The structural caracterization of the macrocyclic channels within vesicles cannot be detected by any micrography. the obtained vesicles of 100 nm diamteter are characterized by DLS. Some labbeling can be also used to detected the amount of the compounds in the membrane. However in the absence of any precipitate we consider that the studied compounds with a low solubility in water are completely embedded in the membrane.
Reviewer 3 Report
In this manuscript, synthetic compounds are reported that are able to transport Na+ and K+ across the membranes of LUVs. The structures are based on crown-ethers for the recognition of the cations and alkylureas to form superstructures in the membranes. The way the octyl chain is connected is varied and this was found to result in differences in rates of cation transport and selectivities, as studied by the comparison of rates between Na+ and K+ transport in presence of protonophore FCCP.
The novelty of this work is limited considering that experiments as reported by the same authors in Ref 25 have been repeated with small variations in the octyl groups. Although the observed selectivity for K+ over Na+ in the presence of FCCP is remarkable (two-fold larger than in their already published systems), this manuscript does not explain why such a selectivity was obtained, which makes it difficult to translate the observations from this manuscript to any other transport system.
Selectivities are commonly calculated from the ratio of EC50 values, which in this case would only give a value ~5.
A comparison between the performance of their systems and the well-known cationophore valinomycin in the same experiments is absent.
A serious problem of this manuscript is that the Figures showing the structure of compound 1 and the text describing its synthesis do not match the characterization information given, both in Section 2.3 and in the SI (Figures S1 and S2), which makes the reader doubt what structure the authors have actually used in this work.
The term “ionophore” is most commonly used for small compounds acting via a mobile carrier mechanism, such as valinomycin and monensin. While it can refer to compounds acting as channels, this manuscript uses the notions of ionophores and channels in a rather confusing way. Such as on p1 line 29-30, where it is suggested that macrocyclic crown-ethers are to be connected (covalently or via H-bonding) to serve as carriers, which is not clear. Similarly, on p1 lines 42-44 the effect of alkyl groups on ionophores is discusses, referring exclusively to compounds acting as mobile carriers and not to channels, while the authors suggest that their compounds form channels throughout the manuscript, to then introduce the notion of potential dimers on page 8 line 237, without showing any experimental work to distinguish the different mechanisms.
Minor issues are for instance the absence of any concentrations in Figures S9-S11 and the absence of a description on how the compounds are added to LUVs (volume and concentrations, in what solvent).
Author Response
In this paper, we first translated the fluorescence change to pH change using a equation from calibration adopted from reference (J. Am. Chem. Soc. 2003 125(10):2840-2841.), and eventually using pH change during first seconds versus time to obtain the rate.
While I do agree that EC50 value is a very important parameter in terms of evaluating potency of the facilitating ion transport, it is also worth noting that the time to obtain the value of fractional activity for calculating EC50 is essential and often not the same between different literatures. For example, if we chose the values at 500s as the fractional activities from the data we had, the EC50 would have been smaller. On the other hand, while the standard deviation may be bigger during faster transport, the initial rate is free from the influence of the time frame we chose for calculation. Therefore, we believe the initial rate is also a good parameter to compare.
We are sorry that we are short of this comparison due to the restriction of our current condition. From our assessment, the valinomycin has lower EC50 and higher selectivity than our ionophores.
Thanks for the comment from this reviewer concerning the compound 1: We noticed the mistake and replaced the wrong characterization with the correct data.
Many thanks to the reviewer for this discussion. We fixed the term used in these sentences by using macrocycles inside ionophores and changed al over the manuscript. “On p1 line 29-30”, most of the examples we referred were reported as channels. On p1 lines 42-44 we used alkyl ureido-macrocycles. The term of dimers and selectivity issues has been cited from references 13 and 15 in the literature and the experimental work was already described there.
In Figures S9-S11, the solvent is CDCl3. And the concentrations are from 0.01 to 0.16 M for all three compounds as shown by Figure 2.
Round 2
Reviewer 2 Report
After a careful revision of this second version of the manuscript, I did not find this work suitable for publication in its current form. My main concern is that the characterization is not complete and the authors only argued that they do not have the equipment in their institution to make it (?) but only 1H and 13C NMR and low resolution mass spectroscopy are not enough as proof of the identity of new compounds as in any chemistry related journal asks authors to do. Therefore, I do not recommend its publication in the journal, at least in this form. I strongly ask the authors to at least make the HR-MS and/or EA characterizations to complete the work properly.
Author Response
Response: We are very sorry that the actual period of the year has been considered for an update of the Mass spec service in our University. We had the chance right now to have HR-MS spectra in another university and we are providing these data in the manuscript and supporting information -Figure S26. We hope this would be accepted by this referee as satisfactory for our compounds characterization.
Reviewer 3 Report
The largest problem with the manuscript, which was the inconsistency between the structure and the characterization data of compound 1, has been solved.
However, other issues are not or only partially solved:
Concentrations were added to Figures S9-11 “(0.01 to 0.16 M, from bottom to top)”, but when comparing the intensities of the signals with that of the solvent, it appears more likely that the concentrations are the other way around (with the highest concentration at the bottom and the lowest at the top). Information on how the compounds are added to LUVs (volume and concentrations, in what solvent) is still absent. I appreciate that the authors have replaced the confusing use of “ionophores” and “carriers” by other terms. However, K+-macrocycles brings a new confusion (as if the macrocycles is composed of potassium cation). Instead, I would recommend using “K+-binding macrocycles” or a similar phrase in title and abstract. Also the phrases “ionophore-mediated” on pages 7 and 8 should be replaced for consistency. Page 1 line 43 now talks about “alky ureido-macrocycles”, while referring to compounds that are not macrocyclic. The authors compare their system with valinomycin in their response, but such a comparison should be included in the article itself to give the readers the ability to see to what extent these synthetic systems can approach the performance of valinomycin.Author Response
The largest problem with the manuscript, which was the inconsistency between the structure and the characterization data of compound 1, has been solved.
However, other issues are not or only partially solved:
Concentrations were added to Figures S9-11 “(0.01 to 0.16 M, from bottom to top)”, but when comparing the intensities of the signals with that of the solvent, it appears more likely that the concentrations are the other way around (with the highest concentration at the bottom and the lowest at the top).
Response: The reviewer is right. We corrected this
Information on how the compounds are added to LUVs (volume and concentrations, in what solvent) is still absent.
Response: this information is now described in page 5: Then, after addition of 1, r2, s2, 3 in the bilayer membrane via injection of 20 μL of compound aliquots from stock 10 mM DMSO solutions, see the final concentration values (% mol of the compound / mol of lipid) in hill plot analyses in Fig S12-S25), an external pH gradient was created by addition of NaOH.
I appreciate that the authors have replaced the confusing use of “ionophores” and “carriers” by other terms. However, K+-macrocycles brings a new confusion (as if the macrocycles is composed of potassium cation). Instead, I would recommend using “K+-binding macrocycles” or a similar phrase in title and abstract.
Response: We agree; modified
Also the phrases “ionophore-mediated” on pages 7 and 8 should be replaced for consistency.
Response: We agree; replaced
Page 1 line 43 now talks about “alky ureido-macrocycles”, while referring to compounds that are not macrocyclic.
Response: We agree; corrected with carriers
The authors compare their system with valinomycin in their response, but such a comparison should be included in the article itself to give the readers the ability to see to what extent these synthetic systems can approach the performance of valinomycin.
Response: We agree; we introduced the following on page 5
Compound 1 is the most active, as it has the lowest EC50 for both Na+ (10.6 mol%,) and K+ (4.1 mol%), much lower than its isomers following the transport activity sequence of 1 > s2 > 3 within the µM concentration range. Compared with Valinomicin (EC50K+=5 pM) they are several orders of magnitude for K+ activity. All the Hill coefficients are above 2, indicative of a formation of self-assembled aggregates, containing more than two molecules of macrocycles, which transport cooperatively the cations, contrarely to Valinomycin acting as a carrier with Hill number = 1
