Attempted synthesis of iminoquinone-cysteine adducts: a protected cysteine-RA-1351 adduct was made, characterized using LCMS and 1H NMR, but C13 missing signals

[qxsml]

Please scroll down to my next comment for the successful reactions.

The reactions shown in this comment are unsuccessful.

The above reaction was attempted, on a 20 mg scale.

Below is its t.l.c. with different reaction lengths. First column: iminoquinone starting material; second column: N-acetyl cysteine methyl ester; third column: co-spot; fourth column: the reaction solution.

The Rf 0.6 spot gradually disappeared overnight after comparing the first and second tlc.

The top spot on the third tlc was isolated as a major product and its NMR is shown below: Its structure is to be further confirmed, but it's clearly the quinone part of the starting material. This molecule doesn't fly in the LCMS and it has a low boiling point. This result suggesting that the cysteine adducts may not be stable under such reaction conditions.

The Rf 0.6 spot eluted together with the adjacent spot after Biotage purification, and they are minor products. Further purification isolated TLC clean products but their NMR is still not clean.

I attempted to analyse their crude NMR by comparing the spectra obtained.

By comparing these spectra, I can roughly tell which peak belongs to which compound for fraction 2 (the higher running spot), as shown in the below spectrum. The integration seems not accurate, presumably due to the quantity of compound being extremely low.

After comparing this spectrum with the H NMR of the iminoquinone starting material (as shown below), it can be concluded that the aromatic ring is unaffected.

Next the LCMS of the corresponding spot was obtained. The result suggest that the higher running spot might be the one that shown below. However, I'm trying to get more material to confirm.

The structure of the Rf0.6 (the lower running spot) spot is still not known.

Further investigation, including a control reaction by heating iminoquinone in DMF without cysteine, a reaction of using base, is in progress.

[mattodd]

Good work. I would definitely just prep HPLC all this if the biotage can't purify things. If not, I'd bring the important spots down the plate to RF 0.3 or so, rather than 0.6, since you get better separation then. If biotage doesn't cut it, manual column it, but then you may as well prep HPLC it.

Gut feeling? If the SH was doing a Michael addition on the ring and forming the big adduct (the structure of which you show next to your prep TLC) then you'd expect a lower RF spot, no? What if the SH is attacking the sulfur and kicking out the, what, nitrosophenol. Would that match what you're seeing? Is that a known thing, or silly?

...and you wonder why the SH doesn't just react with its own ester, at 40 degrees for a long time.

[qxsml]

Attempted synthesis of iminoquinone-cysteine has been tricky. So I decided to do a time-course NMR study of the reaction in organic solvent. I used DMSO-d6 instead of DMF-d7 as it's readily available.

I studied the reaction with and without base. I choose an organic base (won't have solubility issue), Et3N, because it's common and its signals can be easily identified.

Cysteine reacted and a major set of signals was observed. Iminoquinone1351 was also reacted, and it seems the signals of the CH of the quinone ring at ~6.4 ppm gradually disappeared and formed a new signal at ~6.3 ppm.

The reaction without base showed no change after 20h, so I'm not putting its spectra here. I then heated the reaction at 40 degree. It seemed cysteine reacted and formed a major set of signals which are different from the reaction with base. The signal of the CH of the quinone was also disappeared, but it doesn't look like a sole new compound was formed.

So, I decided to conduct a reaction with base and try to isolate the product.

I put up a reaction in a 50 mg scale, in DMF, and in the meanwhile, I decided to check whether iminoquinone1351 is stable in DMSO-d6 with base but without the presence of cysteine. The spectrum surprised me that the iminoquinone is not stable under such conditions, and the CH and CH3 on the quinone ring are disappeared. But I decided to purify the 50 mg reaction and isolate the cysteine product anyway. Below is the NMR spectrum of the isolated product and its proposed structure.

It quite surprised me that there's exactly 1 equiv. of DMF mixed in it. But its chemical shifts would have changed if it binds with other things, right? Here all three signals matches literature data. The other signals look OK and its LCMS showed clean spectra at 208,254,280 nm wavelength and correct mass.

I can confirm it is the major cysteine product by comparing its H NMR with the spectra obtained in the time-course NMR study.

However, I encountered problems with the intensity of carbon signals. The carbon signals of the CH on the ring were missing, although, there are strong correlations between the proton signal at 6.3 ppm (assigned as CH on the ring) and the carbon signals at 126 and 130 ppm on HSQC, but the intensity of both signals on the C13 spectrum is very low. And one of the proposed proton signals (2.1, 2.4 ppm) of CH3 on the ring correlated with the same carbon signal as the proton signal of acetyl CH3 but the other one correlated with a very weak carbon signal at 19 ppm.

From the HMBC spectrum (as shown below), I can see the correlation between the proton signal at 6.3 ppm (assigned as CH on the ring) correlate with a carbon signal at 153 ppm (assigned as a tertiary carbon on the ring), which has very strong intensity, and with carbon signals at 126 and 130 ppm (assigned as CH3), which have very weak intensity.

All these information confused me. So far I'm not convinced that the proposed structure is correct.

[mattodd]

Hi @qxsml - any updates here, i.e. more data on the isolated material and its yield? The iminoquinone S/M has geometric isomers that do not quickly interconvert, giving two peaks for the iminoquinone H's, correct? Whereas the putative product with the N-S bond does not? Is that implied by that 2H singlet for those protons in the NMR?

There was an unresolved issue to do with the low intensity 13C peak - might that just be a weak signal, rather than missing? Note that for any proven structure we're going to need a sense of its yield, just to be clear that we're looking at the major product, rather than some byproduct. Either than means a mass yield, or it means showing that it's the only thing formed when you monitor the crude by NMR.

The new NMR peaks you're seeing in the timecourse experiment, where a new pair of para-substituted aryl doublets appear. I'm assuming this is for 4-chlorobenzenesulfonate, and that the ppm/J values match those in the lit for that material?

[qxsml]

Hi @qxsml - any updates here, i.e. more data on the isolated material and its yield? The iminoquinone S/M has geometric isomers that do not quickly interconvert, giving two peaks for the iminoquinone H's, correct? Whereas the putative product with the N-S bond does not? Is that implied by that 2H singlet for those protons in the NMR?

There was an unresolved issue to do with the low intensity 13C peak - might that just be a weak signal, rather than missing? Note that for any proven structure we're going to need a sense of its yield, just to be clear that we're looking at the major product, rather than some byproduct. Either than means a mass yield, or it means showing that it's the only thing formed when you monitor the crude by NMR.

The new NMR peaks you're seeing in the timecourse experiment, where a new pair of para-substituted aryl doublets appear. I'm assuming this is for 4-chlorobenzenesulfonate, and that the ppm/J values match those in the lit for that material?

Hi @mattodd ,

The lit. data is in CDCl3, ~7.3 and 7.7 ppm, but the fact that the new pair of para-substituted aryl doublets (timecourse experiment) is identical to the new pair of para-substituted aryl doublets (in the control, where no cysteine, just 1351+base+DMSO-d6) tells us this is the 4-chlorobenzenesulfonate.

[qxsml]

Hi @qxsml - any updates here, i.e. more data on the isolated material and its yield? The iminoquinone S/M has geometric isomers that do not quickly interconvert, giving two peaks for the iminoquinone H's, correct? Whereas the putative product with the N-S bond does not? Is that implied by that 2H singlet for those protons in the NMR?

There was an unresolved issue to do with the low intensity 13C peak - might that just be a weak signal, rather than missing? Note that for any proven structure we're going to need a sense of its yield, just to be clear that we're looking at the major product, rather than some byproduct. Either than means a mass yield, or it means showing that it's the only thing formed when you monitor the crude by NMR.

The new NMR peaks you're seeing in the timecourse experiment, where a new pair of para-substituted aryl doublets appear. I'm assuming this is for 4-chlorobenzenesulfonate, and that the ppm/J values match those in the lit for that material?

I obtained clean NMR in CDCl3, still, proton spectrum is great, but C13 missing signals.

Still, I can see correlation between the proton at 6.3 ppm with Carbons at 127 and 131 ppm, but no signals on Carbon spectrum.

[qxsml]

I also ran the sample at a higher concentration on LCMS, still single signal on different wavelength, indicating it's clean.