Light-induced primary amines and o-nitrobenzyl alcohols cyclization as a versatile photoclick reaction for modular conjugation

The advent of click chemistry has had a profound impact on many fields and fueled a need for reliable reactions to expand the click chemistry toolkit. However, developing new systems to fulfill the click chemistry criteria remains highly desirable yet challenging. Here, we report the development of light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) as a photoclick reaction via primary amines as direct click handle, to rapid and modular functionalization of diverse small molecules and native biomolecules. With intrinsic advantages of temporal control, good biocompatibility, reliable chemoselectivity, excellent efficiency, readily accessible reactants, operational simplicity and mild conditions, the PANAC photoclick is robust for direct diversification of pharmaceuticals and biorelevant molecules, lysine-specific modifications of unprotected peptides and native proteins in vitro, temporal profiling of endogenous kinases and organelle-targeted labeling in living systems. This strategy provides a versatile platform for organic synthesis, bioconjugation, medicinal chemistry, chemical biology and materials science.

transformed Rho-o-Nitrosobenzaldehyde species toward primary amine residues occurred outside the mitochondria organelles. It is important to add an extra channel via immuno-staining of mitochondrial antigens to identify the specificity of the PANAC photo-cross-linking methods. The fixed cell lysate could also be collected for subsequent in-vitro analysis to examine the selectivity. In addition, during the fluorescent mitochondrial targeted imaging experiments, a control group should be supplemented (-UV-365, then remove noncovalent probes via fixation and washing) to verify that in dark environment the o-nitrobenzyl alcohol reagents does not proceed any undesired labeling toward other endogenous substances inside live cells after extensive washing.
Collectively, based on current development of the PANAC photo-cross-linking chemistry and the progress made in this article, it is not recommended that this manuscript could be published in the Nature Communications, unless all the concerns could be fully resolved.
Reviewer #3 (Remarks to the Author): Guo and co-workers report a light-induced reaction between primary amines and o-nitrobenzyl alcohols (O-NBA). Based on previous reports that o-nitrobenzyl alcohol could react with amines under UV activation to form indazolones, they found that electron-withdrawing amide group substituent could increase the reaction efficiency of o-nitroenzyl alcohol. With this O-NBA amide derivatives, they have conducted amine compound and peptide modification, native protein bioconjugation, protein profiling and in vivo protein labeling. The experiments are well executed and are very solid. There are some issues to be addressed: 1) the authors spent a page discuss click chemistry, and tried the best to link the chemistry in this manuscript with click chemistry. This reviewer does not see the point to do so and does not agree with such connection. 2) there are many reactions in the literature which could rapidly label/modify amine for bioconjugation. Should cite them. 3) page 7, the PANAC product stability comparing with maleimide-type conjugates is between apple and orange. There are other amine bioconjugation products which are very stable. 4) In Figure 3, only HPLC conversions are provided. The isolated yields of some reactions, if not all, should be given. Based on the HPLC profiles, there are some small peaks. Isolated yields would be more accurate to reflect how well these reactions went. 5) For labeling nanobody-HER2, protein aggregation degree should be provided. 6) In supporting information, there are shoulder peaks for the PANAC products, such as Figures S10-C/S11-d/S12-d/S34-d, what are they? 7) In supporting information, some B-BNA showed two peaks, such as in Figure  S15/16/17/18/19/20. Why?
Chen et.al. reported a photo-initiated cyclization reaction of primary amine and o-nitrobenzyl alcohols. The reaction is actually the conjugation reaction of primary amines with aryl-nitroso generated from photolysis of o-nitrobenzyl alcohols upon UV irradiation. As mentioned in Figure 2A and 2B, this UV-triggered fast cyclization reaction has been reported (J. Org. Chem. 2018, 83, 15493;RSC Adv. 2019, 9, 13249) and applied to the lysine-selective photo-crosslinking to capture protein-protein interaction. The authors screen different o-NBA substrates, then they found that o-NBA amide proceeded fast photolysis as well as the following cyclization with primary amine, which took around 30~40 minutes in total to accomplish. The reaction showed good selectivity to primary amine in the lysine side chain. The reaction seemed to have good compatibility with terminal alkynes and azide, which are functionalities used in CuAAC. Lysine-specific modification of peptides or proteins was demonstrated using this reaction. The authors also showed preliminary applications of this reaction in endogenous kinase profiling and mitochondria-targeted labeling. In general, the work is of good quality and important to the filed of bioconjugation, but the following issues still need to be addressed: 1. The importance of CuAAC and SPAAC as click reaction is largely originated from the bioorthogonality of the reactions. However, this reaction can not be bioorthogonal since lysine is abundant in biological systems. In this sense, Figure 1 was not very proper. I would suggest the authors add some introduction to other fast and biocompatible ligation reactions, such as the CBT chemistry. Besides, comparison of this reaction to known amine-involved bioconjugation method with respect to their efficiency in protein labeling is necessary.
Response: We are honestly grateful for the comments from this reviewer. We agree that rg[\f eXTVg\ba Vbh_W abg UX bioorthogonal since lysine is abundant in biological systemss+ Ca YTVg) we demonstrated the light-induced PANAC reaction as a photoclick reaction in our manuscript.
We have modified the Figure 1 in our revised manuscript as reviewer suggested, and also added some introduction and have cited reference about other fast and biocompatible ligation reactions such as CBT-click reaction (ref. 19-21 ), tyrosine-click reaction (ref. 22-23 ), amine bioconjugations (ref. 24-26) and Sulfur(VI) fluoride exchange (SuFEx) chemistry (ref. 7 ). We have added these citations to the reference part in our revised manuscript.
The comparison of the PANAC photoclick to known amine-involved bioconjugation methods had been discussed in the discussion part. Please see the W\fVhff\ba cTeg) rCCC+ <l VbageTfg) T`baZ jX__-established spontaneous primary amine conjugation approaches 43 , highly amine-reactive electrophiles (e.g. activated ester, aryl sulfonyl fluoride) are probably too prone to non-enzymatic or enzymatic hydrolysis with reduced conjugation efficiency, during in vitro conjugations or during the process into cells as chemical biology probes 27,28 .s ;aW rCO+ Several frequently used amine (or lysine)-reactive electrophiles cross-react with other nucleophiles (e.g. nucleophilic chains of amino acids, O, N-nucleophiles, GSH) giving promiscuous reactivity. We show here that o-NBA derived reactants are able to overcome several aforementioned challenges in complex biological environments, such as background labeling from spontaneous conjugations or cross-reactivity with other nucleophiles, and reduced efficiency from hydrolysis 27,28 +s 2. The photo-initiation condition may add some advantages to the reaction if spatial control of the reaction can be demonstrated in a well-designed experiment. The importance of temporal control of bioconjugation reactions is not as significant as spatial control, since the other reaction substrate can be introduced to the system with temporal resolution.
Supplementar y Fig. 9 | Schematic illustration of spatial labeling of live cells by PANAC photo-click reaction. a, Incorporation of the o-NBA moiety (compound S40) to the HER2 specific nanobody (sdAb-HL C, Bioconjugate Chem. 2014, 25, 979-988) at C-terminal to produce (sdAb-HL C-o-NBA). b, The structure of the FITC compound. c, The HER2 specific nanobody (sdAb-HL C-o-NBA) labeled with FI TC-NH 2 via PANAC photo-click reaction on the surface of SK-Br-3 cells.
Labeling with spatial control was demonstrated simply by shielding part of the cells from UV-365 nm light irradiation (bottom panel) or with light-activation for 10 min (top panel). After labeling, cells were washed by PBS wash for 3 times.
And cell imaging using confocal fluorescence. LVT_X UTe7 .-y`+ 3. Original NMR spectra of the products listed in Figure 3 should be privided in supplementary data.
Response: We are grateful to reviewer for this comment. We have added the Original NMR spectraof the products listed in Figure 3 to the revised Supplementary Information on page #87-109.
4. The authors mentioned kinase profiling using the probe containing the o-NBA structure captured much more kinase species than the work using the probe containing the diazirine photo-affinity moiety. It is hard to understand since the carbene species generated from photolysis of diazirine should have much higher chance to form covalent bonds with adjacent groups. The authors may need to comment on this.
Response: The highly reactive carbene intermediate photogenerated from diazirine moiety, which can be readily quenched with water in complex biological environment, or convert to the less reactive species, resulting in short half-life (nanosecond to microsecond range). This event will lead to lower crosslinking efficiency (Angew. Chem. Int. Ed. 2018, 57, 14350). While our o-nitrosobenzaldehyde intermediate has a long half-life ( Fig. 2d) and is selective for amine groups, which is not easy to be quenched by water, so o-NBA structure captures more kinase substrates than diazirine photo-affinity moiety.
5. The mitochondria-targeted labeling was not really a spatial control realized by the photo-controllable reaction.
More convincing results to show the advantage of this reaction to spatial labeling may need photo-irradiation confined to subcellular level.
Response: We are grateful to reviewer for this suggestion. We have demonstrated the spatial labeling of live cells by PANAC photo-click reaction with pre-tagged o-NBA moiety which was further labeled with FITC-NH 2 via the PANAC photo-click reaction on the surface of SK-Br-3 cells (please see details in response to issue 2 of this reviewer). We further demonstrated the spatial covalent labeling of engineered human histone H2B which localizes at the nucleolus, via PANAC photo-click reaction (Supplementary Fig. 13-15).
We could not get access to the photo-irradiation confined to subcellular level with UV light at this stage. As spatial control of subcellular labeling, we have demonstrated the mitochondria-targeted labeling with spatial control simply by shielding part of the cells from UV-365 nm light irradiation ( Supplementary Fig. 12) (J. Am. Chem. Soc. 2018, 140, 14542-14546;J. Am. Chem. Soc. 2017, 139, 8090-8093;Nat. Chem. 2011, 3, 256-259.). We further demonstrated the spatial covalent labeling of engineered human histone H2B which localizes at the nucleolus, via PANAC photo-click reaction ( Supplementary Fig. 13). The red fluorescent protein (mKate2) was fused to the C-terminus of H2B as red fluorescent marker. The unnatural amino acid (o-NBAK) was site-specifically incorporated at the site of 175 of mKate2 of the fusion protein (H2B-mKate2-175TAG) via expansion of the genetic code (please see our previous research, ref 41, Chem. 201941, Chem. , 5, 295541, Chem. -2968. The fusion protein (H2B-mKate2-175TAG) was expressed in live 293T cells, then the cells were labelled with FITC-NH 2 via PANAC reaction upon light-activation. After labeling, the unreacted FITC-NH 2 was removed by washing, and then imaged with confocal microscopy. Supplementar y Fig. 13 | Schematic illustration of site-specific labeling of H2B-mKate2-175-o-NBAK protein which localizes at the nucleolus, via PANAC photo-click reaction with temporal and spatial control.
After the light-induced PANAC reaction of the FITC-NH 2 with o-NBAK, the FITC signals colocalized with mKate2 signals, suggesting the site-specific labelling of histone H2B ( Supplementary Fig. 14).
Supplementar y Fig. 15 | a, with unnatural amino acid o-NBAK was added to the growth medium 1h prior transfection at a final concentration of 1 mM. b, without unnatural amino acid o-NBAK. HEK293T cells were transfected with pcDNA3.1-H2B-mKate2 K175TAG HEK293T (red channel) and pNEU-hMbPylRS-4xU6M15 plasmids. After cultured for 24 h, HEK293T cells were fixed, stained with Hoechest 33342 (2 yg/mL, blue channel) for 15 min, incubated with FITC-NH 2 (20 yM, green channel) for 15 min without light-activation+ LVT_X UTe7 / y`+ We have added these results (13)(14)(15) to the revised Supplementary Information, and discussed the spatial control of the PANAC conjugations in the discussion part of the revised manuscript on the page of #18, with the sentence as: rAlternatively, o-NBA-related handles could be in principle incorporated in native biomolecules such as proteins (Supplementary Fig. 9-12, 13-15) 18, 41 , followed by exogenous primary-amine probe labeling in spatial and temporal control in vitro or in living systems, which would greatly expand the power of this light-induced PANAC conjugation 9 .s Reviewer #2 (Remar ks to the Author ): Pros.: 1. Via systematic optimization, the developed PANAC reaction now has higher yields for various substrates including drugs targets, peptides and protein substrates via in-vitro chemical modification under different UV irradiation conditions. The selectivity of PANAC reaction toward primary appears to be pretty good. After UV irradiation, the o-nitrosobenzaldehyde amide 3 showed negligible reactivates toward alcohols, phenols, carboxylic acids, and nitrogen-containing heterocycles groups in the presence of primary amine. But there is no 2. In this paper, the introduction of amide groups on para-position of o-nitrobenzyl alcohols has increased the reaction efficacy of the well-established PANAC reaction toward solvent exposed primary amine groups, demonstrating a useful photo-cross-linking tool for studying the pharmacological properties of payload molecules via covalent tagging to native primary amines in biological systems. Since the primary amines are key and abundant residues on biomolecules in live cells, it is obvious that this type of PANAC photo-cross-linking reagents hardly show any bioorthogonality.
Response: We are grateful to Reviewer for this comment. In fact, primary-amine-containing molecules are abundant and widespread in synthetic chemistry, biological systems and materials science (Annu. Rev. Biochem. 2019, 88, 365-381;ACS Comb. Sci. 2015, 17, 600-607). PX TZeXX g[Tg rthis type of PANAC photo-cross-linking reagents hardly show any bioorthogonalitys. Indeed, we have demonstrated the light-induced PANAC reaction as a photoclick reaction via diverse applications in our manuscript. Thus, the PANAC photoclick chemistry provides versatile platform for modular conjugation of multiple functional motifs and primary amines '' one of the most abundant functional groups as straightforward click handle, conferring expanding the click chemistry toolkit. Cons.: 1. The novelty of this PANAC reaction is not prominent, it is a continuous study derived from previously research work reported by the same group in which the o-nitrobenzyl alcohol is specifically incorporated on the desired residues of target proteins (Ref. 31). The PANAC reaction was early reported in 2011(Chem. Commun., 2011 for covalent photo-assembly of gold nanoparticles, and has been utilized for bio-material modifications with decent spatiotemporal resolution (e.g. Chem. Mater. 2019, 31, 4710l4719, ACS Macro Lett. 2016, 5, 19l23, Angew. Chem. Int. Ed. 2012, 51, 9181h9184, Adv. Mater. 2016. The photon quantum efficiency and the reaction rate constant of the second imine formation step have been reported (Ref. 28), but it is not investigated carefully in this article; The choice of light wavelength for triggering the photo-transformation of 2 were also investigated previously (Ref. 29, J. Org. Chem. 2018, 83, 24, 15493-15498).
However, when applying to live cell studies as a bio-conjugation chemistry, the authors ignored the photo-toxicity of using 365 nm UV irradiation and potential cytotoxicity caused by extensively blocking of vital primary amines.
By contrast, in our research, the PANAC photo-click reaction provides the stable cyclization pr oducts (indazolones) which are different compared to those products in bio-material modifications. The PANAC product was hydrolytically and thermally stable in buffer conditions (please see, Supplementary Fig. 2). On the other hand, the imine/hydrazone/oxime bonds are reversible in nature (ACS Macro Lett. 2016, 5, 19-23). With respect to these factors, the PANAC photo-click reaction is different in mechanism to those imine/hydrazone/oxime ligations.
We have added the Supplementary Fig. 21 into the revised Supplementary Information. And have added the sentence rWe wondered if light irradiation conditions might cause sufficient harm to cells. Fortunately, cell availability assay showed that the short time of 365 nm UV irradiation employed in the PANAC reactions as well as in the presence of (1yM or 4 yM) o-NBA derived probes, did not noticeably affect cell proliferation of MDA-MB-468 cells ( Supplementary Fig. 21).s into the revised manuscript on page #13.
We agree with reviewer that (potential cytotoxicity caused by extensively blocking of vital primary amines). We will investigate more details about the potential cytotoxicity of PANAC photo-click reaction in vivo rather than in cells in our future research, as this reviewer kindly suggested.
2. The most important characteristic of a photo-click reaction is the control of when and where to induce covalent bond formation between a pair of bioorthogonal groups (azide-alkyne, tetrazole-alkene, azide-cyclopropenone), not native groups, e.g. primary amine. However, even the developed PANAC reactions does not appear to have spatial controllability which probably is due to the long life-time (Fig. 2d) and insufficient reactivity of o-nitrosobenzaldehyde amide 3 toward primary amine, resulting in its diffusion and nonrestrictive labeling in the cellular environment. The unsatisfied bimolecular reaction rate was also reflected as the higher yields of desired indazolone 4 after prolonged incubation up to 30 mins (Fig. 2c, 2e and 2f without indication of reaction time). It is essential to demonstrate the spatial covalent labeling for a photo-cross-linking tool.
Response: We are grateful to reviewer for this comment. As the concept of click chemistry was introduced by Sharpless and their colleagues, click chemistry refers to a class of reactions that satisfy certain characteristics, such as modularity, operational simplicity (e.g. be insensitive to oxygen or water), reliable selectivity, high yields, good reaction rate and diversity of the reactants (Angew. Chem. Int. Edit. 2001, 40, 2004-2021Angew. Chem. Int. Edit. 2013, 52, 5930-5938).
Generally, the click chemistry mainly includes two kinds of reactions in bioconjugations. The first type is the bioorthogonal click reactions with pre-incorporation of exogenous click handles (such as azide-alkyne, tetrazole-alkene, azide-cyclopropenone). The second type is the efficiently chemoselective labeling reactions based on native residues of biomolecules, such as 2-cyanobenzothiazole (CBT) and N-terminal cysteine click reaction ( We agree with this reviewer that iIt is essential to demonstrate the spatial covalent labeling for a photo-cross-linking tool.j Our previous research (Chem. 2019, 5,2955-2968, Genetically encoded residue-selective photo-crosslinker to capture protein-protein interactions in living cells) has showed the light-induced indazolone formation with spatial control for photo-crosslinking of protein-protein interactions. In addition, we have designed more experiments to demonstrate the spatial covalent labeling of the photo-induced PANAC reaction as follows:  Chem. Soc. 2018, 140, 14542-14546;J. Am. Chem. Soc. 2017, 139, 8090-8093;Nat. Chem. 2011, 3, 256-259.) Supplementar y Fig And cell imaging using confocal fluorescence. LVT_X UTe7 .-y`+ 2). Spatial covalent labeling of histone pr otein H2B at the nucleolus via PANAC photo-click reaction (please see the following results in Supplementary Fig. 13-15) We further demonstrated the spatial covalent labeling of engineered human histone H2B which localizes at the nucleolus, via PANAC photo-click reaction ( Supplementary Fig. 13). The red fluorescent protein (mKate2) Fig. 17).
We have dded the Supplementary Fig. S16-S17 into the revised Supporting Information. And have added the sentence as rwith a quantum yield of 0.52 ( Supplementary Fig. 16-17) 38 , the photogenerated intermediate aryl-nitroso 3s into the revised manuscript on page #7. vitro proteins or peptides labeling or in vivo applications. (ref 7, Chem. Soc. Rev. 2019, 48, 4731-4758;ref 27, ACS Chem. Biol. 2017, 12, 1478-1483. Thirdly, in the research of (Bioconjugate Chem. 2017, 28, 1422v1433) as reviewer mentioned, the dialdehyde click chemistry for amine bioconjugation is in a spontaneous manner without the temporal control. In our research, we have not only showed the applications with primary amines such as drug molecules (Fig. 3) or peptides (Fig. 4) or proteins (Fig. 5) in vitro, but also demonstrated the cell-based and temporally controlled applications, such as profiling of endogenous kinases and organelle-targeted labeling in living systems (Fig. 6). By contrast, the spontaneous manner of the click reactions for native biomolecules remains challenging when applied into living systems, since the reactions would initiate in extracellular environment or during the process approaching cellular targets once certain clickable functional groups are in proximity to each other, thus with increased background labeling from spontaneous conjugations or with reduced efficiency from hydrolysis. The success of the applications via PANAC reaction in living systems mainly based on the intrinsic advantage of temporal control, where the o-NBA derived reactants are inert before light activation, thus serve as general masked reactants in living systems. The advantages of temporal control of photo-click reactions are also discussed in introduction part and discussion part of our manuscript.
Overall, with the unique mechanistic pathway (addition/cyclization/dehydration/tautomerization, Figure 2a Response: We are grateful to reviewer for this comment. We calculated the mitochondrial area (Fig. 6e, top or bottom panel, red color) and nuclear area of (Fig. 6e, top or bottom panel, blue color) by ImageJ software, respectively. Base on the calculation in Supplementary Fig. 19 (left, data from Fig. 6e), jX TZeXX j\g[ g[\f eXi\XjXe g[Tg rthere was a significant expansion of the Rho-o-NBA signals (Fig. 6e, bottom panel, red color) in comparing with the Rho-o-NBA channel without the photo-irradiation (Fig. 6e, top panel, red color)

s+
The r eason for the [significant expansion of the Rho-o-NBA signals (Fig. 6e, top panel vs bottom panel, r ed DOLOQ&\ probably resulted from the fixation and extensive washing procedures in the experiment (Fig. 6e, bottom panel, red color). In that experiment, we want to validate that the covalent bond was forming with Rho-o-NBA probe with (+ UV-365 nm) light activation, therefore, we fixed and washed the cells to remove unreacted Rho 123 for intended comparison (Fig. 6e, bottom panel, Rho-o-NBA vs Rho 123 cells staining). However, of note, for regular live cell imaging, due to good cell uptake and subcellular distribution (Fig. 6e, top panel) of Rho-o-NBA probe, it is no necessary to fix cells for imaging or dynamic mitochondrial tracking in living mammalian cells.
Therefore, we have added the note rSignal expansion was observed compared to live cell imaging (Fig. 6e, red color), which probably resulted from the fixation and washing procedures in the experiment, see Supplementary Fig. 19-20 for details.s into the revised manuscript in the figure legends of Fig.6. iIn addition, during the fluorescent mitochondrial targeted imaging experiments, a control group should be supplemented (-UV-365, then remove noncovalent probes via fixation and washing) to verify that in dark environment the o-nitrobenzyl alcohol reagents does not proceed any undesired labeling toward other endogenous substances inside live cells after extensive washing.j The control group (-UV-365, then remove noncovalent probes via fixation and washing) was displayed in supplementary Fig. S8, which proves the reaction will not proceed in dark environment, as the reviewer suggested.
This result was provided in our last manuscript, as described in the sentence Tf rGbeXbiXe) g[XfX eXTVg\ba-based covalent labeling of the mitochondria is light-dependent event (Supplementary @\Z+ 5's+ iCollectively, based on current development of the PANAC photo-cross-linking chemistry and the progress made in this article, it is not recommended that this manuscript could be published in the Nature Communications, unless all the concerns could be fully resolved.j Response: We have tried our best to resolve all of these concerns as reviewer mentioned, and we hope this reviewer will kindly agree with us. We are honestly grateful for all of these comments and great suggestions from this reviewer.

Reviewer #3 (Remarks to the Author):
Guo and co-workers report a light-induced reaction between primary amines and o-nitrobenzyl alcohols (O-NBA).
Based on previous reports that o-nitrobenzyl alcohol could react with amines under UV activation to form indazolones, they found that electron-withdrawing amide group substituent could increase the reaction efficiency of o-nitroenzyl alcohol. With this O-NBA amide derivatives, they have conducted amine compound and peptide modification, native protein bioconjugation, protein profiling and in vivo protein labeling. The experiments are well executed and are very solid. There are some issues to be addressed: 1. The authors spent a page discuss click chemistry, and tried the best to link the chemistry in this manuscript with click chemistry. This reviewer does not see the point to do so and does not agree with such connection.
Response: We are grateful to reviewer for this comment. We have revised the introduction part and the Figure 1, please see the revised manuscript for details.
2. There are many reactions in the literature which could rapidly label/modify amine for bioconjugation. Should cite them.
3. Page 7, the PANAC product stability comparing with maleimide-type conjugates is between apple and orange.
There are other amine bioconjugation products which are very stable.
Response: We thank Reviewer for this comment. We have switched g[\f fXagXaVX r@\aT__l) ghe PANAC product was hydrolytically and thermally stable in buffer conditions ( Supplementary Fig. 2), while certain conjugates (e.g. maleimide-type) are prone to elimination 21 .s tb r@\aT__l) ghe PANAC product was hydrolytically and thermally stable in buffer conditions (Supplementary Fig. 2).s 4. In Figure 3, only HPLC conversions are provided. The isolated yields of some reactions, if not all, should be given. Based on the HPLC profiles, there are some small peaks. Isolated yields would be more accurate to reflect how well these reactions went.
Response: We are grateful to reviewer for this comment. We have obtained several isolated yields of products.
These results were added to the revised manuscript in Figure 3.
Small peaks on the HPLC profiles are by-products derived from the photogenerated reactive intermediates. We have identified the probable structures of light-induced by-products of o-NBA derivatives referred to the literature (Angew. Chem. Int. Ed. 2012, 51, 6502-6505), which were provided in Supplementary Figure 28. We used 4 equivalents of o-BNA derivatives to the primary amines in light-induced PANAC reaction, and we also found the excessive intermediates after reaction time. Therefore, these small peaks form the by-products of the photogenerated reactive intermediates probably have no relevance with amine reactants, and they will not affect the reaction yields.
Response: For light-induced PANAC covalent labeling of lysines on the nanobody-HER2 with o-NBA-alkyne, there was no protein aggregation found. However, in the following CuAAC reaction of alkyne with azide (Fig 5a), with the present of copper ions and additives in the reaction system, it caused partial (~20%) protein aggregation and precipitation, based on the measurement of the protein concentration of the nanobody-HER2. We added this data in figure legends of Fig.5, in the sentence as rin CuAAC, partial (~20%) modified nanobody-HER2 protein aggregation and precipitation.s On the other hand, the GE-SEC column analysis showed that the modified nanobody-HER2 obtained after desalting had no dimerization and multimerization according to the retention time of the monomer nanobody-HER2 (13.7 kDa) and the reference protein e-Chymotrypsinogen A (25.6 kDa)

6.
In supporting information, there are shoulder peaks for the PANAC products, such as Figures S10-C/S11-d/S12-d/S34-d, what are they?
Response: We thank Reviewer for this comment. The PANAC products (indzalones) contain multiple functional groups showing complex acidity and basicity. The acidity of the mobile phase with additives (0.01% formic acid in H 2 O, and 0.01% formic acid in CH 3 CN) for UPLC are not enough to make these samples completely free or ionized, so they appear as shoulders in the UPLC-MS spectrum analysis.
Therefore, we reduced sample loading, meanwhile, increased the acidity of the mobile phase (0.05% TFA in H 2 O and CH 3 CN) and changed flow method of partial samples to obtain a symmetrical peak under the same reaction conditions. We The author has addressed most of my comments. The revised manuscript was of improved quality and I would recommend it for publication in Nat. Commun. One minor concern is about the relative anount of the two substrates shown in Fig. 2f and Figure 3. Figure 2f showed the use of excess amount of lysine derivative and primary amine substrate, Figure 3 used amine at 0.5mM and o-NBA derivatives at 2 mM. Does that mean that the yields were calculated based on different substates but all could be very high?
Reviewer #2 (Remarks to the Author): Based on the revised data, the PANAC reactions displayed decent selectivity to primary amines on the side chain of lysine residues over others. The bimolecular reaction rate constant of PANAC photo-click reaction is also decent and comparable to those of CuAAC, tetrazole photo-click and certain tetrazines in their early generations, but is not enough for highly spatial-resolved applications. The authors also showed the low photo-toxicity of o-NBA reagents by using 365 nm UV irradiation at very low dose (1 or 4 µM). With more supplementary data provided, the PANAC photo-reaction investigated in this article was proved to be a photo-crosslinking method, which has been utilized recently. This research is based on previously discovered photo-reaction with slight modification of the substituents to improve the performance for bio-conjugation. Innovation of this study is not enough for publication in Nature Communication, but the results are sufficient to support this chemistry as a photo-conjugation method. However, there are still several issues that must be addressed before further consideration.
Although there were reports in the previous research that exposure to 365 nm for 60 minutes had no significant effect on cell viability or leading to apoptosis. The authors should choose 20 minutes or longer time to test the photo-toxicity instead of 10 min, because the irradiation time was more than 10 minutes in the live cell surface labeling or mitochondria tracking experiments, e.g. in the supplementary Fig. 10  As for photo-conjugation toward small molecule conjugations, drug modifications and peptide cyclizations, the PANAC photo-reaction were triggered by 365 nm UV light and then usually require incubation at 25 °C for 30 min to complete the conjugation, because the secondary reaction rate is not fast enough. But for live cell studies in supplementary Fig. 10 or Fig. 11, there was no information on whether it is necessary to incubate for certain time after irradiation for a successful completion of the PANAC bio-conjugation. If the important incubation was no longer needed for this reaction in live cell application, why is it also necessary for small molecule and peptides? Please indicate.
Upon irradiation of 365 nm UV for 10 min with partial shielding, the temporal and spatial labeling performance of PANAC reactions on live cells surface was demonstrated in supplementary Fig. 11. However, it is better to have cells presented cross the dotted line where the spatial resolution on single cell could be explained on the edge of exposure area (e.g. J. Am. Chem. Soc. 2018, 140, 14542-14546;J. Am. Chem. Soc. 2017,139,8090-8093). Currently, the images in supplementary Fig. 11 are not convincing. Why don't show those images in the maintext since it is a characteristic nature of a photo-click reaction?
For the experimental investigation and calculation of the reaction rate constant of PANAC, the bimolecular rate constant k2 value derived from a single concentration of Cbz-Lys-OMe was not rigorous enough for a photo-click type reaction. The authors should plot various concentration of Cbz-Lys-OMe versus observed apparent first-order rate to derive the k2. The deviation of the linear fitting curve, especially in the initial stage of the photo-conversion, was not negligible. Please add more data points and explain.
In the live cell study via mitochondria targeting Rho-o-NBA, confocal images from the Rho-o-NBA channels did not show obvious changes under conditions with (lower row) or without phototriggering (upper row) when cells are still alive, indicating the rhodamine probe most likely was enriched and well-confined in the mitochondria organelles, reflecting its nature for probing mitochondria. However, these phenomena do not represent the reactivity and specificity of the PANAC reaction for blocking the primary amine inside mitochondria. The authors claim that the significant expansion was probably caused by fixation and extensive washing. On the contrary, the fixation procedure disrupts the membrane integrity of mitochondria and washing may cause the diffusion of the released intermediate Rho-o-Nitrosobenzaldehyde, leading to non-specific conjugations toward primary amines outside the mitochondria with expanded fluorescence signal readout (Fig. 6, Supplementary Fig. 19). This is probably the evidence of incomplete reaction of Rho-o-Nitrosobenzaldehyde intermediate. Although I advised the authors to clarify the point of specificity via an immune-staining of mitochondria via fluorescent anti-body because the cells were fixed, it seems there was no direct answer to this important question. Alternatively, longer incubation time after photo-irradiation for live cell experiments might solve this problem! Reviewer #3 (Remarks to the Author): The previous comments raised have been addressed.

Point-By-Point Reply to the Comments of Reviewer #1 (from the second review round)
Reviewer #1 (Remarks to the Author): The author has addressed most of my comments. The revised manuscript was of improved quality and I would recommend it for publication in Nat. Commun. One minor concern is about the relative anount of the two substrates shown in Fig. 2f and Figure 3. Figure 2f showed the use of excess amount of lysine derivative and primary amine substrate, Figure 3 used amine at 0.5mM and o-NBA derivatives at 2 mM. Does that mean that the yields were calculated based on different substates but all could be very high?
Response: We appreciate the positive comments from this reviewer.
In fact, we always used the excess amount of o-NBA derivatives in Figure 2f and Figure 3. The yields are all very high in Figure 2f and Figure 3.
In Figure 3, we used o-NBA derivatives at 2 mM and primary amines at 0.5 mM, thus the ratio of (o-NBA derivatives) to (primary amines) also is 4.
Point-By-Point Reply to the Comments of Reviewer #2 (from the second review round) Reviewer #2 (Remarks to the Author): Based on the revised data, the PANAC reactions displayed decent selectivity to primary amines on the side chain of lysine residues over others. The bimolecular reaction rate constant of PANAC photo-click reaction is also decent and comparable to those of CuAAC, tetrazole photo-click and certain tetrazines in their early generations, but is not enough for highly spatial-resolved applications. The authors also showed the low photo-toxicity of o-NBA reagents by using 365 nm UV irradiation at very low dose (1 or 4 µM). With more supplementary data provided, the PANAC photo-reaction investigated in this article was proved to be a photo-crosslinking method, which has been utilized recently. This research is based on previously discovered photo-reaction with slight modification of the substituents to improve the performance for bio-conjugation. Innovation of this study is not enough for publication in Nature Communication, but the results are sufficient to support this chemistry as a photo-conjugation method. However, there are still several issues that must be addressed before further consideration.  Chem. Soc. 2004, 126, 4581-4595; Photochemical reaction mechanisms of 2-nitrobenzyl compounds: 2-nitrobenzyl alcohols form 2-nitroso hydrates by dual proton transfer, Photochem. Photobiol. Sci., 2005, 4, 33-42). In these excellent papers, Jakob Wirz and colleagues measured the absorption of o-nitrobenzyl alcohols compounds, and also used 365 nm light source for the activation of o-nitrobenzyl alcohols. In addition, Mark J.
Kurth and colleagues also found 365 nm light source is best for activation of the o-nitrobenzyl alcohols (o-NBA), and used the 365 nm light source for photochemical preparation of indazolones (please see reference: J. Org. Chem. 2018, 83, 15493-15498).
Last time, we had measured and showed the absorption spectra of o-NBA amide 1 in Supplementary Fig.   17 showed weak absorption at 365 nm ( Supplementary Fig. 17), the power density from 365 nm light source is enough, and probably more suitable for activation of the o-nitrobenzyl alcohol derivatives (o-NBA amides), when applied the PANAC photo-click reaction in vitro for biological samples or in biological environment.
As the reviewer suggested, we had checked the emission spectra of the 365 nm light source used in this study. The spectrum of lighting systems for this PANAC photo-click reactions ranges from 350 to 390 nm. The Supplier of hand-held UV lamp: ZF-7A, portable UV lamp, Shanghai Gucun Electron Optic Instrument Factory,

China.
As an additional control experiment, we also used the Kessil PR160, UVA LED 370nm lamp (Kessil PR160-370, the spectrum of lighting systems ranges from 355 to 400 nm) as light source, this 370nm light source also provided very high to excellent yields for the PANAC photo-click reactions, at identical conditions.
3. As for photo-conjugation toward small molecule conjugations, drug modifications and peptide cyclizations, the PANAC photo-reaction were triggered by 365 nm UV light and then usually require incubation at 25 °C for 30 min to complete the conjugation, because the secondary reaction rate is not fast enough. But for live cell studies in supplementary Fig. 10 or Fig. 11, there was no information on whether it is necessary to incubate for certain time after irradiation for a successful completion of the PANAC bio-conjugation. If the important incubation was no longer needed for this reaction in live cell application, why is it also necessary for small molecule and peptides? Please indicate.
Response: We are grateful for the comments from this reviewer. As we showed in Figure 2e, the PANAC photo-reaction provides high yields (e.g. about 80% yield, 7 min) after hundreds seconds of light activation 30 min) of the reaction mixture is better to give increasing yields (up to 98% yield).
With these results in hand, to get even higher yields of the PANAC photo-reactions, we chose a further incubation for the small molecule conjugations, drug modifications and peptide cyclizations. If without the further incubation for some small molecule conjugations, the yields will be a little (about 20%) lower as provided in our pilot experiments. This is why the PANAC photo-reactions were triggered by 365 nm UV light, and then usually require incubation at 25 °C for 30 min for the experiments of small molecule and peptides.
Overall, only to get even higher yields, the further incubation was used for the conjugation experiments of small molecule and peptides.
In our pilot experiments of labeling of live cells on the cell surface, we found the signal intensity is very strong compared to background, thus the signal intensity is enough for cell imaging using confocal fluorescence. This is why there was no further incubation after irradiation for the live cell studies in Response: We are honestly grateful for the comments from this reviewer. We had already showed the spatial labeling of biomolecules via the PANAC photo-click reaction under biological environment such as: 1) Temporal and spatial labeling of live cells on the cell surface (please see the results in Supplementary Fig. 10); 2) Spatial covalent labeling of histone protein H2B at the nucleolus via PANAC photo-click reaction (please see the results in Supplementary Fig. 13-15).
These results already demonstrated the spatial labeling of biomolecules on the scale of single cell or subcellular level, via the PANAC photo-click reaction under biological environment as the reviewers suggested in the last review comments. We could not get access to the spatial resolution on single cell scale with the photo-irradiation cross the single cell for the supplementary Fig. 11, at this stage.
According to the length limits and figure limits of this article content, we chose those images as supplementary information. In addition, we had discussed these results as spatial labeling of biomolecules via the PANAC photo-click reaction under biological environment, in the discussion part of our manuscript.
5. For the experimental investigation and calculation of the reaction rate constant of PANAC, the bimolecular rate constant k2 value derived from a single concentration of Cbz-Lys-OMe was not rigorous enough for a photo-click type reaction. The authors should plot various concentration of Cbz-Lys-OMe versus observed apparent first-order rate to derive the k2. The deviation of the linear fitting curve, especially in the initial stage of the photo-conversion, was not negligible. Please add more data points and explain.
Response: We are honestly grateful for the comments from this reviewer. We have got results of additional four concentrations ( In addition, we also have added additional data points of different reaction times such 40s/ 50s/ 70s / 80s. Therefore, the revised curves have nine different reaction time points (10s/ 20s/ 30s/ 40s/ 50s/ 60s/ 70s/ 80s/ 90s) to measured and calculated the reaction rate constant of the PANAC photo-click reaction with each concentration of Cbz-Lys-OMe, via LC-MS ( Supplementary Fig. 18). In these conditions, the deviation of the linear fitting curves were revised and improved.
Based on these additional results and improvement, we have plotted various concentration of Cbz-Lys-OMe versus observed apparent first-order rate to derive the K 2 . Thus, the revised reaction rate constant of the PANAC photo-click reaction is (k 2 = 87.4 M -1 s -1 ) by LC-MS, please see the revised Supplementary Fig. 18.
6. In the live cell study via mitochondria targeting Rho-o-NBA, confocal images from the Rho-o-NBA channels did not show obvious changes under conditions with (lower row) or without photo-triggering (upper row) when cells are still alive, indicating the rhodamine probe most likely was enriched and well-confined in the mitochondria organelles, reflecting its nature for probing mitochondria. However, these phenomena do not represent the reactivity and specificity of the PANAC reaction for blocking the primary amine inside mitochondria. The authors claim that the significant expansion was probably caused by fixation and extensive washing. On the contrary, the fixation procedure disrupts the membrane integrity of mitochondria and washing may cause the diffusion of the released intermediate Rho-o-Nitrosobenzaldehyde, leading to non-specific conjugations toward primary amines outside the mitochondria with expanded fluorescence signal readout (Fig. 6, Supplementary Fig. 19). This is probably the evidence of incomplete reaction of