A bioinspired and biocompatible ortho-sulfiliminyl phenol synthesis

Synthetic methods inspired by Nature often offer unique advantages including mild conditions and biocompatibility with aqueous media. Inspired by an ergothioneine biosynthesis protein EgtB, a mononuclear non-haem iron enzyme capable of catalysing the C–S bond formation and sulfoxidation, herein, we discovered a mild and metal-free C–H sulfenylation/intramolecular rearrangement cascade reaction employing an internally oxidizing O–N bond as a directing group. Our strategy accommodates a variety of oxyamines with good site selectivity and intrinsic oxidative properties. Combining an O–N bond with an X–S bond generates a C–S bond and an S=N bond rapidly. The newly discovered cascade reaction showed excellent chemoselectivity and a wide substrate scope for both oxyamines and sulfenylation reagents. We demonstrated the biocompatibility of the C–S bond coupling reaction by applying a coumarin-based fluorogenic probe in bacterial lysates. Finally, the C–S bond coupling reaction enabled the first fluorogenic formation of phospholipids, which self-assembled to fluorescent vesicles in situ.


Reviewers' comments:
Reviewer #1 (Remarks to the Author): In this work, Zhao and colleagues develop a bioinspired ortho-sulfiliminyl phenol synthesis using oxyamines and sulfenylation reagents. It's a clever strategy and the authors have thoroughly explored and described reaction scope. Furthermore, they highlight some interesting applications, including the modification of substrates in bacterial lysates and the synthesis of phospholipid vesicles. Their fluorogenic scheme is very interesting and adds impact to work as there is tremendous interest in coupling methods that lead to fluorogenic products. While they demonstrate their method for turning on a coumarin, the general scheme of unmasking a phenol function group should also be applicable to the turn-on of other fluorophores such as fluoresceins etc.
Overall, this work should be of interest to a general audience.

Response
We thank the reviewer for this nice comment.
Reviewer #2 (Remarks to the Author): Zhao and co-workers report the metal-free synthesis of ortho sulfilimine substituted phenols via a very interesting rearrangement of N-phenoxy acetamides that is triggered by a sulfenylating agent (a source of S+). The work builds nicely upon the authors previous reports of reactions of N-phenoxy acetamides (refs 31-33). The manuscript begins with the optimisation and discovery of the best sulfenylating agent.
Following this, the scope of the reaction is assessed. The scope of the N-phenoxy acetamides is reasonable, with the reaction being amenable to variation in the phenoxy and amide moieties. The phenoxy portion can contain weakly electron donating alkyl groups, electron withdrawing esters, and halides substitution. However, a key example is missing: an electron-donating group such as OMe. I ask the authors to try to add one more example to the scope with OMe substituted in the phenoxy portion. When phenoxy moiety is meta substituted, i.e two inequivalent ortho CHs, then 1:1 mixtures result.
The scope with respect to the group introduced through the sulfenylating agent has apparently been investigated based on the SI and manuscript text but Table 2 is   missing and instead Table 1 appears again.
The authors' ambitions in applying the method are particularly commendable, and they nicely demonstrate the robustness of their approach by applying the reaction under biocompatible conditions and in the synthesis of a fluorescent phospholipid.
This makes the paper of interest to a wider audience, for example in chemical biology.
The reaction contains a very interesting rearrangement and is unusual (a good thing).
Therefore I feel that the authors should comment on the mechanism with reference to the literature and by performing some simple experiments: 1) can intermediate B be isolated/observed to prove its structure?; and 2) what results does a cross-over experiment yield between differently substituted intermediates B. This will shed light on the mechanism.
The SI file is in good shape.
For these reasons, the paper is certainly suitable for publication in Nature Communications after the additional changes are also made: Question 1: However, a key example is missing: an electron-donating group such as OMe. I ask the authors to try to add one more example to the scope with OMe substituted in the phenoxy portion.

Response
We thank the reviewer for this valuable advice. We have subjected the 4-OMe substituted N-phenoxyacetamide to the C-S coupling reaction, the desired product was obtained in 88% yield. The corresponding product was added to Table 1  Question 2: The scope with respect to the group introduced through the sulfenylating agent has apparently been investigated based on the SI and manuscript text but Table 2

Response
We thank the reviewer for this kind suggestion. We have now added the correct Table   2 in the manuscript.  and CsOAc (1 eq.) in MeOH at room temperature for 3 h. (c) HR-MS spectrum of (a).

Revisions Made
(d) HR-MS spectrum of (b).

Computational details
All the calculations were performed with the Gaussian09 suite of programs. 5 Geometry optimization and energy calculations were conducted with B3LYP. 6 LANL2DZ + d (0.289) basis set with ECP was used for I and 6-31G (d) basis set was used for atoms. 7-9 To verify the stationary points as minima or transition states, vibration frequency calculation at the same level of theory was performed for each structure. The zero-point energy, thermal energy, entropy, and free energy were also derived from vibration frequency. Single point energies were calculated at the B3LYP-D 10 /SDD 11 -6-311++G (d, p) level. A solvent correction for methanol at 298 K was calculated by using SMD solvation model. 12 As shown in Fig. 2a Table 6). These results indicated that the acidity of the N-H is important for this reaction and the N-H bond might be deprotoned by the base to initiate the following reaction. 13 The N-sulfenylation step (TS1) was found to be the rate-determining step (RDS).
By examining the structure of TS1, we envisioned that the energy barrier of nucleophilic substitution process could be influenced by the steric hindrance of the N-substituted thiophthalimides. DFT calculation revealed the activation free energy of the reaction between INT1 and N-t-butylthiophthalimide was indeed 8 kcal/mol higher (TS1') than that of N-ethylthiophthalimide, which implied that N-sulfenylation was unlikely to occur for the bulky N-t-butylthiophthalimide. The experiment was then conducted and confirmed the computational results ( Supplementary Fig. 37b).
The calculation results speculated that the reaction process containing two main stages: (1) N-sulfenylation.

Response
We thank the reviewer for this suggestion. We have replaced sulfenylation formation with C-S bond formation and sulfoxidation.

Revisions Made
(Please refer to the abstract) Inspired by an ergothioneine biosynthesis protein EgtB, a mononuclear non-heme iron enzyme capable of catalyzing the sulfenylation formation C-S bond formation and sulfoxidation, we discovered a mild and metal-free C-H sulfenylation/intramolecular rearrangement cascade reaction employing an internally oxidizing O-N bond as a directing group.

Response
We thank the reviewer for this valuable advice. We have removed those phrases and made the following revision.

Revisions Made
(Please refer to the abstract) Our strategy accommodates a variety of oxyamines with good site selectivity, redox versatility, imine-donating capacity, and easy cleavability. and intrinsic oxidative property.

Question 6:
Page 1, line 21. A compound number (1o) should not be mentioned in the abstract.

Response
We thank the reviewer for this valuable suggestion. We have removed the compound number 1o.

Revisions Made
(Please refer to the abstract) We demonstrated the biocompatibility of the C-S bond coupling reaction by applying a coumarin-based fluorogenic probe 1o in bacterial cell lysates.

Response
We thank the reviewer for those valuable advices. We have corrected those spelling mistakes and made the following revisions.

Revisions Made
Page 1, paragraph 1. The key step in its biosynthesis pathway is the mononuclear non-heme iron enzyme EgtB-catalyzed sulfenylation formation between γ-glutamyl cysteine and N-α-trimethyl histidine (TMH), involving a sulphur sulfur transfer step and an oxygen transfer step (Fig. 1a).
Page 2, paragraph 4. At the outset of this study, compounds 1 with those bonds were firstly screened to couple with a thionating reagent N-ethylthiophthalimide 2a under previously reported metal catalyzed conditions for similar reactions (Fig. 2a).

Response
We thank the reviewer for this valuable advice. We wanted to show that these are parallel bond formations: both processes have C-S bond formation and S=X bond formation. Here we kindly ask the reviewer to allow us to leave the comparison between Figure 1a and 1b since it was the inspiration of our work. However, if the reviewer insists, we will remove this comparison.

Question 9:
Page 2, line 45. "….may undergo an electrophilic addition to the ortho-position of the X-N directing group" -this needs to be rewritten. The directing group does not have a benzene ring.

Response
We thank the reviewer for this valuable advice. We have made the following revision.

Revisions Made
(Please refer to page 2, paragraph 3)

Response
We thank the reviewer for this valuable advice. The bond between iron and nitrogen is dashed line (a coordination bond). We have redrawn the Figure 1 and made the following revision.

Response
We thank the reviewer for this valuable advice. We have removed the comments on how products were characterized and made the following revision.

Revisions Made
(Please refer to page 3, paragraph 4; page 4, paragraph 7) The structure of 3aa was confirmed by NMR spectroscopy, HRMS and X-ray crystallography ( Supplementary Information, Fig. 30). A bond length of 1.67Å is characteristic of the S=N bond. 47 The structure of 3ab was confirmed by NMR spectroscopy, HRMS and X-ray crystallography (Supplementary, Fig. 31).

Question 12:
Page 4. In Figure 2, please use another abbreviation for the leaving group on sulfur: M is normally used to represent a metal.
Page 4. In Figure 2, (a) the structure of 3aa must be given here. (b) a compound number for the product must be given.

Response
We thank the reviewer for this valuable advice. We have replaced the M with R as the abbreviation for the leaving group, and showed the structure of 3aa in Figure 2a, added the compound number 3af in Figure 2b. The revision is as follows.

Response
We thank the reviewer for this valuable advice. We have removed the Table 1a from the main scheme. Question 14:

Revisions Made
Page 6 lines 94-107. As structures 1 are not shown in all cases I think it is best to refer to product structures 3 when talking through the scope and FG tolerance.

Response
We thank the reviewer for this valuable advice. We have made the following revision.

Revisions Made
(Please refer to page 3 and 4, paragraph 6 and 7) To probe the scope of the transition metal-free cascade C-S and S=N bond formation, we examined a series of oxyamide substrates (Table 1). Replacing the acetyl group with a bulkier pivaloyl (1b) or a benzoyl (1c) group only slightly decreased the yield to 80% (3ba) and 83% (3ca), respectively. It is worth noting that for substrate 1c, the sulfilimine substitution occurred exclusively at the ortho-position of the phenoxyamide moiety instead of the benzamide moiety (3ca), which indicated the stronger directing ability of the oxyamide group for sulfenylation. Substitutions on the phenoxy side of 1 had little impact on the yield. Electron-donating groups (1d, 1e, 1g,   1m 3da, 3ea, 3ia, 3la), electron-withdrawing groups (1f 3ha), as well as halogen groups (1h~1l 3fa, 3ga) were well tolerated, which afforded substituted sulfilimines in 85% to 92% yield. The C-S bond formation proceeded exclusively at the site ortho to the acetylaminoxy group. Therefore, for substrate 1 with two different ortho-sites (1g, 1j~1l), two regioisomers with ratio almost 1:1 were produced (3ja:3jaʹ, 3ka:3kaʹ, 3ma:3maʹ, 3na:3naʹ). Fusion of a benzene ring as in the substrate of naphthalene (1n) did not affect the reaction yield but resulted in high regioselectivity, which only functionalized the ortho C-H at C-1 position, resulting in a 2-naphthol derivative (3oa).
Question 15: Page 8. Figure 3. Why does the coumarin have a letter rather than a compound number?

Response
We thank the reviewer for this valuable advice. We have replaced the coumarin P with the compound number 3pa and made the following revision.