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Review
Peer-Review Record

Application and Suggestions of Morpholine Ring as a Lysosome Targeting Group

by Xuelian Liu, Ximeng Zhang, Yinghong Han, Xingrui Li * and Jinyao Li *
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Submission received: 10 April 2025 / Revised: 8 May 2025 / Accepted: 17 May 2025 / Published: 21 May 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Xuelian Liu et al. in the submitted manuscript “Application and suggestions of morpholine ring as a lysosome targeting group” provide the extensive review of literature describing lysosome targeted probes based on morpholino moiety which are applicable for a wide range of fluorescence assays and microscopy. The authors provide reference to 179 literature sources and provide 177 chemical structures of the described lysosome probes. The manuscript is well structured consisting of four main sections which highlight structures of the fluorescent probes, a particular focus is directed at colocalization with well characterized lysosomal probes. In addition, the authors are highlighting applications of lysosomal targeted sensors detecting: pH, viscosity, polarity, reactive oxygen species (ROS), various metal ions (Zn²⁺, Cu²⁺, Fe³⁺), sulfides, biothiols (GSH, Cys), formaldehyde, HNO, NO. Furthermore, the authors mention lysosomal probes for enzyme activity detection (cathepsins, galactosidases, esterases, nitroreductases) and the highlight derivatives useful for photodynamic therapy. The structure-activity relationships are discussed highlighting dual localization probes (endoplasmic reticulum and mitochondria).

Overall, the manuscript is a well written and interesting to read. Thus, I am happy to recommend this manuscript to be accepted for publishing in Chemistry (MDPI publisher) after a minor revision to improve the clarity of the text:

Minor points:

  1. Currently, the manuscript is lacking clarity and systematic summary of the probes. The reader would benefit if parameters discuss in the text would be summarized in the tables. Please include tables for each group of the discussed probes. They should contain photophysical parameters (absorbance and emission wavelengths, quantum yields if available), colocalization coefficients with known lysosomal markers. For sensors please indicate which type of analyte they are detecting. pH sensors should have indicated pKa.
  2. Please name each figure accordingly to what is presented in the figure. Indicate if it is lysosomal sensor or dual purpose probes. Do not use general names of the figures “The structure of fluorescent probes XXX and YYY”.
  3. Please expand structure–activity relationships discussion by including a separate section. This section should include description: how hydrophobicity influences targeting, effects of charge state (cationic vs neutral), role of linker flexibility/length and off-targeting issues.
  4. Sensor description would benefit from the mechanisms of sensing. Please include them into the figure showing structures of sensors. pH sensors should have indicated protonization sites. Metal sensors should have indicated chelating groups. Enzyme sensors should have indicated fragments recognized by corresponding enzymes.
  5. Please add “Future Directions” section. It should include possible future modifications and applications of lysosomal probes. Please highlight current limitation and indicate possible ways for improvements.
Comments on the Quality of English Language

Improve grammar and clarity in the following sections: abstract and conclusion.

Author Response

Minor points:

 

  1. Currently, the manuscript is lacking clarity and systematic summary of the probes. The reader would benefit if parameters discuss in the text would be summarized in the tables. Please include tables for each group of the discussed probes. They should contain photophysical parameters (absorbance and emission wavelengths, quantum yields if available), colocalization coefficients with known lysosomal markers. For sensors please indicate which type of analyte they are detecting. pH sensors should have indicated pKa.

Response:Thank you. Your suggestions have made our article more organized. We have summarized the photophysical parameters (absorbance and emission wavelengths, quantum yields, and colocalization coefficients with known lysosomal markers) for each set of probes using a table (Appendix. Photophysical parameters of the probes). However, after careful consideration, we did not include it in the article.

The main reasons are as follows:

  1. The optical parameters (λabs, λem, Φfl) of fluorescent probes are highly dependent on the environment, especially solvent, pH, and temperature.

2.Most probes themselves are non-fluorescent and only generate fluorescent products after reacting with specific substances. Only fluorescent molecules exhibit emission spectra.

Therefore, we believe it is not rigorous to uniformly label the λabs (nm), λem (nm), and [ϕfl] of the probes without considering these factors.

 

Table 1 Optical properties of 1a (original reference is named 2a) and 1b (original reference is named 2a) 1.

Table 2. Photophysical properties of the probes 2

Ly-Tet is probe 2(Ly-Tet) in this review

Ly-Pyr is the fluorescent product of probe 2 (Ly-Tet).

 

 

  1. Please name each figure accordingly to what is presented in the figure. Indicate if it is lysosomal sensor or dual purpose probes. Do not use general names of the figures “The structure of fluorescent probes XXX and YYY”.

Response:Thank you for the reminder. We have named each image after please based on what is shown in the image.

 

  1. Please expand structure–activity relationships discussion by including a separate section. This section should include description: how hydrophobicity influences targeting, effects of charge state (cationic vs neutral), role of linker flexibility/length and off-targeting issues.

Response: Thank you for your advice. We have added this section to the manuscript.

 

  1. Sensor description would benefit from the mechanisms of sensing. Please include them into the figure showing structures of sensors. pH sensors should have indicated protonization sites. Metal sensors should have indicated chelating groups. Enzyme sensors should have indicated fragments recognized by corresponding enzymes.

Response:Thank you for your advice. We have labeled the reaction sites in the structure of the fluorescent probe.

 

  1. Please add “Future Directions” It should include possible future modifications and applications of lysosomal probes. Please highlight current limitation and indicate possible ways for improvements.

Response:Thanks again for your suggestion. We have added this section to the manuscript.

 

 

 

Refs:

 

  1. Bertman, K. A.; Abeywickrama, C. S.;  Baumann, H. J.;  Alexander, N.;  McDonald, L.;  Shriver, L. P.;  Konopka, M.; Pang, Y., A fluorescent flavonoid for lysosome detection in live cells under "wash free" conditions. J. Mater. Chem. B 2018, 6 (31), 5050-5058.
  2. Liu, S.; Su, H.;  Bu, L.;  Yan, J.;  Li, G.; Huang, J., Fluorogenic probes for mitochondria and lysosomes via intramolecular photoclick reaction. Analyst (Cambridge, U. K.) 2021, 146 (4), 1369-1375.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the author systematically summarizes and organizes up to 177 different lysosome targeting probe containing morpholine ring, based on their structures and functions. The general strategies for lysosome targeting are clearly introduced as background, followed by a functional categorization of the probes by their function, like imaging, pH detection, reactive oxygen species detection, etc. Additionally, the author includes a thorough analysis of the relationship between chemical structure and in vivo function. Overall, this review is informative, well-organized, and valuable to the field. This manuscript is recommended to be published after including and addressing the below listed comments with minor corrections.

1) In section 2.1, the author summarized the property of NIMCn as: "When n=2-10, all compounds can be localized in lysosome..." For clarity and accuracy, this should be revised to "When n=2-6, 8, 10, all compounds can be localized in lysosome...", since the original literature only made linker with n=2-6, 8, 10.

2) Several abbreviations are introduced without being defined when they are firstly mentioned. For improved clarity and reader-friendliness, please define all abbreviations at first mention, including “ROS” (reactive oxygen species), “PET” (photoinduced electron transfer), and “ICT” (intramolecular/internal charge transfer).

3) Minor typo in line 272: "Probes 75- were morpholine-modified rhodamine derivatives." Please revise.

4) Minor mistakes in Figure 4: The "()2" should be on the carbon chain for probe 68 and 71. Probe 89 has Br and N overlap. Please revise.

5) Minor typo in line 343: "The N atom on imine and morpholine of ..." There is no imine in this probe. Please revise.

6) Minor mistakes in Figure 6, the "()2" should be on the carbon chain for probe 116. Probe 123 should have the acrylate on the coumarin OH. Please revise.

7) In section 2.8, the author discusses lysosome probes for their polarity detection. However, probes 144 and 145 were discussed about their viscosity monitoring ability in this section. Would it be more appropriate to discuss probes 144 and 145 under Section 2.7, which covers viscosity?

8) Minor typo in line 458, "Probes XX and XX were prepared by introducing a morpholine..." Please revise.

Author Response

1)    In section 2.1, the author summarized the property of NIMCn as: "When n=2-10, all compounds can be localized in lysosome..." For clarity and accuracy, this should be revised to "When n=2-6, 8, 10, all compounds can be localized in lysosome...", since the original literature only made linker with n=2-6, 8, 10.
Response:Thank you very much! We have revised it.
2)     Several abbreviations are introduced without being defined when they are firstly mentioned. For improved clarity and reader-friendliness, please define all abbreviations at first mention, including “ROS” (reactive oxygen species), “PET” (photoinduced electron transfer), and “ICT” (intramolecular/internal charge transfer).
Response:Thank you for your suggestion. We have revised it.
3)    Minor typo in line 272: "Probes 75- were morpholine-modified rhodamine derivatives." Please revise.
Response:Thank you for your reminder. We have made the change in the article.
4)    Minor mistakes in Figure 4: The "()2" should be on the carbon chain for probe 68 and 71. Probe 89 has Br and N overlap. Please revise.
Response:Thank you for your reminder. We have made the change in the article.
5)    Minor typo in line 343: "The N atom on imine and morpholine of ..." There is no imine in this probe. Please revise.
Response:Sorry for the inaccurate description, we have corrected it.
6)    Minor mistakes in Figure 6, the "()2" should be on the carbon chain for probe 116. Probe 123 should have the acrylate on the coumarin OH. Please revise.
Response:Thank you for your reminder. We have corrected it.
7)    In section 2.8, the author discusses lysosome probes for their polarity detection. However, probes 144 and 145 were discussed about their viscosity monitoring ability in this section. Would it be more appropriate to discuss probes 144 and 145 under Section 2.7, which covers viscosity?
Response:Thank you again. We have made changes in the article.
8)     Minor typo in line 458, "Probes XX and XX were prepared by introducing a morpholine..." Please revise.
Response:Thank you for your reminder. We have corrected it.

Author Response File: Author Response.pdf

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