Two-colour light activated covalent bond formation

We introduce a photochemical bond forming system, where two colours of light are required to trigger covalent bond formation. Specifically, we exploit a visible light cis/trans isomerization of chlorinated azobenzene, which can only undergo reaction with a photochemically generated ketene in its cis state. Detailed photophysical mapping of the reaction efficiencies at a wide range of monochromatic wavelengths revealed the optimum irradiation conditions. Subsequent small molecule and polymer ligation experiments illustrated that only the application of both colours of light affords the reaction product. We further extend the functionality to a photo reversible ketene moiety and translate the concept into material science. The presented reaction system holds promise to be employed as a two-colour resist.

I find this study quite fascinating and think this is a significant step forward in this field. A dual wavelength induced reaction with both upstream reactions being reversible is extremely promising. The study is conducted well, and I support the conclusions drawn by the authors. What I think could use some improvement is the presentation. There seem to some mistakes in some graphs (see comments below) but even without them it is sometimes too hard to understand what is presented in detail. Especially for a broad audience as expected for Nature Comm the authors should reassess if it is possible to present the information in a more straight forward and intuitive way.
From the side of experiments, I would be highly interested in seeing a polymer-polymer coupling reaction. This is always a great benchmark for such a coupling reactions. Even if this shows no quantitative coupling, this isn't a severe disadvantage for this study in my opinion. It would just show the limits of what can be achieved with this particular system. Also in order to use this reaction for 3D printing, I don't think its fast enough at this point. Again, I don't see this as a problem for the current study but a short coming that should be addressed in the manuscript (or disproven by an actual 3d printing experiment).
Overall, I think this manuscript could be accepted after major revision.
Comments: Scheme 1 looks very nice but could be more informative. For instance, for someone who isn't familiar with this kind of cycloaddition, its relatively hard to make out what is reacting with each other in what way. Perhaps adding simplified reaction mechanism (similar to the one in the SI) that ignores all the residues that are not essential to the cycloaddition would help. After all, there would be enough space for that. I think especially when targeting a broad audience this would be helpful. Scheme 1 will be the item which every reader will look at first so it should be easier to understand the chemistry based on it. Figure 1A: I am not sure what the insert is. Is this switching rate? If so the axis title is missing and its unintuitive that the axis numbering is on the left side (abs. in the full plot) and not factored (10-3) as the right y axis. Also, the 550 nm, which marks the beginning of the orange area in the full plot should also be in the insert (maybe including the orange area?) Other than that its hard to make out what exactly happens at 375nm . A zoom in on that region would be much more interesting. At the moment it looks like this negligible switching at 275 is in the same order of magnitude as what happens at 550 nm.
"As expected, this wavelength corresponds to the isosbestic point of the absorbance spectra of the trans and cis isomer." Is this referring to Figure S39/40? If so please refer to it in the text.
"Whilst a tuneable laser was vital in identifying these wavelength regions, subsequent studies revealed that the orthogonality was maintained when commercial LEDs (1,max = 385 nm and 2,max = 625 or 650 nm) were employed." please link to data (in SI?) Something is wrong in Figure 2. If the peak assignment in the NMR is correct than the trans A1 is doing nothing under red light and isomerization happens at 380 nm. On the "blue" side: K1a is unreactive under 380 nm and reacts at 650 nm. The scheme and the discussion states it reverse. Please clarify. Is the order just messed up?
Naming of substitution in Figure 2A is confusing. 1 should be "X =1", 2 should be "X=2" and then PEG-X2 should be "X=PEG" after that logic and sample name for conjugate would then be cis-APEG ?
Anyway, it took far too long to figure out what the authors mean. There is certainly a better, more intuitive way to do this. Figure 2D: the shift in the SEC looks nice. It would be really interesting to see what happens when both molecules are conjugated to a polymer. Then the coupling could be followed by SEC and would give a nice insight into how good this reaction is performing. This would be even more interesting for the final system shown in Figure 3A! A relatively easy experiment would be taking the cross linker from the resin synthesis with 2 equivalents of the PEG conjugated azobenzene and look at the product in SEC. This would show how quantitative the reaction works when confronted with such a sterically difficult problem. Figure 3E and following: please provide an axis with numbers for the free floating mass spec. Also the x axis doesn't seem to have the right numbers. The spectra should be an isotopic pattern, but the assigned m/z values have only a 0.01 change? Same problem in the respective SI graph.
Then, it seems that the reaction is not quantitative form the NMR (Neither the isomerization not the ketene formation and consequently also not the cycloaddition. The full MS 8Figrue S51 I think should be provided in higher resolution (regarding the plot, thinner lines etc) to see the remaining precursor distribution if there is one. All distributions should be assigned as well Figure 3D: what is the second isotopic pattern when PEGtrans A2 is irradiated at 385 nm? Some minor spelling mistakes: "formaulated" on page 8; a "." after the reference in the introduction. Some double space's.
As stated, overall I support this study and think it will be a very nice piece for Nature Comm after some revisions.
Response: As confusion around labelling was raised by both reviewers, we have carefully attended to the matter and simplified the structure labelling. Each structure has now been given a unique identifier, eliminating the reference to 'PEG' structures. We trust that the modified naming convention is more intuitive to follow. Figure 2D: the shift in the SEC looks nice. It would be really interesting to see what happens when both molecules are conjugated to a polymer. Then the coupling could be followed by SEC and would give a nice insight into how good this reaction is performing. This would be even more interesting for the final system shown in Figure 3A! A relatively easy experiment would be taking the cross linker from the resin synthesis with 2 equivalents of the PEG conjugated azobenzene and look at the product in SEC. This would show how quantitative the reaction works when confronted with such a sterically difficult problem.

Response:
We thank the reviewer for their helpful suggestion and have undertaken the suggested experiment. Specifically, we have synthesized a PEG containing the diazoketene endgroup (termed K5 in the SI), and conduct the two-colour induced ligation with the PEG containing the azobenzene endgroup (A3). Our result indicates a very efficient coupling, with the SEC trace of the product clearly shifting towards larger molecular weights. We have included an additional brief discussion in the manuscript to describe the additional result: "Ultimately, we tested the polymer coupling between PEG-azobenzene A3 and a triethylene glycol ketene K5 to determine the effect of the ligation. The SEC-analysis (refer to supporting information, Figure S60) clearly shows an increase in molecular weight without the appearance of any shoulder towards the starting distribution indicating quantitative ligation." Comments: Figure 3E and following: please provide an axis with numbers for the free floating mass spec. Also the x axis doesn't seem to have the right numbers. The spectra should be an isotopic pattern, but the assigned m/z values have only a 0.01 change? Same problem in the respective SI graph.

Response:
We thank the reviewer for the very valuable comment and agree that the presented spectra 2E might have been confusing without further description. Therefore, we included the adjusted isotopic pattern and added the requested axis. In addition, we now included the explanation for the small m/z change during the end-group modification: "Upon end-group modification, the polymer distribution of A3 is expected to show a mass change corresponding to the molecular mass of K2. Since the molecular weight of K2 (M = 132.0575 g/mol) equals three times the molecular weight of ethylene glycol repeating unit (M = 132.0786 g/mol) the mass change is minor. With size exclusion chromatography coupled with high resolution mass spectrometry (SEC-HRMS) it is still possible to determine the change of isobaric masses by comparing their isotopologues ( Figure 2E, zoom-in). A representative section of the obtained mass distribution is displayed (refer to supporting information, Figure S55-57) with the mass increase of ΔmK2 schematically indicated. " Comments: Then, it seems that the reaction is not quantitative form the NMR (Neither the isomerization not the ketene formation and consequently also not the cycloaddition. Response: This is a good observation and we agree that neither the switching nor the ligation reaction is quantitative. In the NMR experiments we didn't intent to drive the reaction to full conversion to prevent side reactions. Additionally, the isomerization, similar to other isomerization reactions, has its equilibrium at 70-80% cis-isomer. Furthermore, in Section 2.8 of the supporting information we determined the isolated yield of the photoreaction (79%) to show that the reaction is not quantitative. Despite this, by implementing the reaction on the materials scale, we demonstrate that a fully quantitative reaction is not required for our intended application.

Comments:
The full MS 8Figrue S51 I think should be provided in higher resolution (regarding the plot, thinner lines etc) to see the remaining precursor distribution if there is one. All distributions should be assigned as well Response: Figure S51 was adjusted and replaced as requested. For the assignments of the residual distributions, we want to point the reviewer's attention to the following Figures S52, 53. Here, we provided detailed information regarding all observed distribution based on simulated and matched spectra. Generally, we want to point out that mass spectrometry cannot be used to obtain quantitative information. Depending

CRICOS No. 00213J
on the ionisation strength of starting material and product small impurities can appear severe if they are charged easily but do not represent the real amount. Figure 3D: what is the second isotopic pattern when PEGtrans A2 is irradiated at 385 nm?

Comments:
Response: The isotopic pattern emerging when the polymer is irradiated with 385 and 625 nm and with only 385 nm is a side reaction of the reactive ketene with the polymer. Since it does not match with the calculated pattern of the product or the starting material, the structure is unknown. However, we do not believe the side reaction is problematic or surprising since we do observe side reactions on the small molecule scale already. Furthermore, we cannot estimate the amount, which could be very minor, of the impurities since mass spectrometry is not a quantitative method, as described previously.
Comments: Some minor spelling mistakes: "formaulated" on page 8; a "." after the reference in the introduction. Some double space's.

Response:
Thank you for bringing this to our attention. The error has been corrected.

Comments:
As stated, overall I support this study and think it will be a very nice piece for Nature Comm after some revisions.
Response: Again, we thank the reviewer for their positive assessment and trust they will find out revisions satisfactory.