The binding behaviours between cyclopentanocucurbit[6]uril and three amino acids

Binding behaviours between cyclopentanocucurbit[6]uril (CyP6Q[6]) and three amino acids have been investigated by means of X-ray crystallography, proton nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. The results showed that CyP6Q[6] forms a 1 : 2 inclusion complex with glycine, but 1 : 1 complexes with both leucine and lysine. Whereas the carboxyl group of glycine can enter the interior of the cavity of CyP6Q[6], only the alkyl chains of leucine and lysine can enter this cavity. Interestingly, leucine can adopt two different self-assembly modes upon its interaction with cucurbituril, depending on the external conditions, whereas glycine and lysine do not exhibit such behaviour.

engages in hydrogen bonds with another amino acid. A discussion about this point should be added to the main text. 5. The cif file of complex 3 includes hydrogen atoms bound to both oxygens of the carboxylic acid group in the amino acid molecule. However, the images in the main text and SI do not show both hydrogens. The cif file should be fixed to show the correct modeling of the carboxylic acid group. 6. In the discussion regarding the NMR titration of the cucurbituril with L-Lys (following Fig 6), the authors state that the alpha proton and all other alkyl chain protons shift upfield. However, in Fig 7, the alpha protons appear to shift downfield, analogous to what is observed in the titration with L-Leu. Thus, this suggests that the acid group remains outside the cavity, which is in agreement with the crystal structures.
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Dear Dr Ma:
Title: The Binding Behaviors between Cyclopentanocucurbit[6]uril and Three Amino Acids Manuscript ID: RSOS-202120 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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RSC Associate Editor: 2 Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This manuscript is clearly presented with an interesting study of 3 amino acids showing significant binding in the cavity of, as the authors point out the 'immature' cyclopentanocucurbit[6]uril, with only a few example guests having being studied. The results are relevant and given that there is limited binding data for other comparable sized cucurbituril examples would suggest a greater significance of a binding advantage demonstrated for CyP6Q [6]. The solid state evidence also provide a good comparison to the NMR results. This referee feels that the work should be published.

Reviewer: 2
Comments to the Author(s) Ma and coworkers describe the crystal structures, NMR titrations, and isothermal titration calorimetry experiments for a cyclopentyl cucurbituril with three amino acids. The crystal structures are generally in agreement with the NMR data, and glycine behaves differently than lysine and leucine. The manuscript is interesting and I recommend publication after the following points are addressed by the authors.
1. In experimental section 2.4 (ITC), the volume of the aliquot is given in parentheses. For clarity, the authors should indicate if this is the volume per aliquot that is added or the total volume injected over the course of the experiment. 2. The last sentence of section 2.4 (ITC) states that the data was analyzed after the first two unwanted data points were deleted. The authors should clarify the reason why these data points were deleted. 3. Is there a reason cadmium salts were used in complexes 1 and 2, while zinc was used for complexes 3 and 4? This point should be clarified in the experimental section. 4. It is interesting to note that the carboxylic acid groups of the amino acids adopt a syn conformation in complex 1, an anti conformation in complex 2 and 4. In the anti conformation, the acid engages in hydrogen bonds with the cucurbituril, while in the syn conformation, it engages in hydrogen bonds with another amino acid. A discussion about this point should be added to the main text. 5. The cif file of complex 3 includes hydrogen atoms bound to both oxygens of the carboxylic acid group in the amino acid molecule. However, the images in the main text and SI do not show both hydrogens. The cif file should be fixed to show the correct modeling of the carboxylic acid group. 6. In the discussion regarding the NMR titration of the cucurbituril with L-Lys (following Fig 6), the authors state that the alpha proton and all other alkyl chain protons shift upfield. However, in Fig 7, the alpha protons appear to shift downfield, analogous to what is observed in the titration with L-Leu. Thus, this suggests that the acid group remains outside the cavity, which is in agreement with the crystal structures.

See Appendix A.
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Dear Dr Ma:
Title: The Binding Behaviors between Cyclopentanocucurbit[6]uril and Three Amino Acids Manuscript ID: RSOS-202120.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. Thank you very much for your valuable suggestions on this article. We have revised the manuscript and sent the revised manuscript for your consideration as a research paper to be published in Royal Society Open Science. Uploaded please find a manuscript entitled "The Binding Behaviors between Cyclopentanocucurbit[6]uril and Three Amino Acids".We have studied reviewer's comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments. Following are our responses (in BOLD type) to the Reviewers' comments.

RSC Associate Editor: 1
Comments to the Author: Both referees have expressed interest in this work. Through my own reading, I feel the manuscript is of high quality and will likely attract some interest. Reviewer 2 does raise a few valid concerns that should be addressed by the authors. I would also like to point out that one of the reviewers has raised a question with regard to how the NMR data of one of the complexes was interpreted. Such an interpretation may impact the This referee feels that the work should be published.
Response: Thank you for agreeing to our paper. We hope to use a lot of examples in future study.

Reviewer: 2
Comments to the Author(s) Ma and coworkers describe the crystal structures, NMR titrations, and isothermal titration calorimetry experiments for a cyclopentyl cucurbituril with three amino acids.
The crystal structures are generally in agreement with the NMR data, and glycine behaves differently than lysine and leucine. The manuscript is interesting and I recommend publication after the following points are addressed by the authors.
1. In experimental section 2.4 (ITC), the volume of the aliquot is given in parentheses.
For clarity, the authors should indicate if this is the volume per aliquot that is added or the total volume injected over the course of the experiment.
Response: This is a very good suggestion, We have made corresponding changes in the manuscript.
2. The last sentence of section 2.4 (ITC) states that the data was analyzed after the first two unwanted data points were deleted. The authors should clarify the reason why these data points were deleted.
Response: According to the reviewer's suggestion, we have made the following changes to the section: Considering that the top of the syringe is easy to mix in air bubbles, the data were analyzed with ORIGIN 8.0 software using an independent model，after deleting the first two unwanted data points.
3. Is there a reason cadmium salts were used in complexes 1 and 2, while zinc was Response: We would like to apologize for our carelessness. We have modified and replaced the cif file as required.
6. In the discussion regarding the NMR titration of the cucurbituril with L-Lys (following Fig 6), the authors state that the alpha proton and all other alkyl chain protons shift upfield. However, in Fig 7, the alpha protons appear to shift downfield, analogous to what is observed in the titration with L-Leu. Thus, this suggests that the acid group remains outside the cavity, which is in agreement with the crystal structures.

Response: Thank you very much for your pertinent suggestions on this article.
We have tried our best to make the corresponding changes based on your valuable suggestions in the text. The details are as follows: Similarly to L-Leu, the α proton signals of L-Lys shift downfield, while the signals of the other alkyl chain protons shift upfield. This suggests that its alkyl chain enters the cavity of the CyP6Q[6], while the acid group remains outside the cavity, forming a structure similar to that of butanediamine@CyP6Q[6], [15] indicating that its alkyl chain enters the cavity of the host due to the interaction of an extra amino group of L-Lys at other portal of the host.
We would like to express our great appreciation to you and reviewers for comments on our paper. Looking forward to hearing from you.
Thank you and best regards.