Re: [ccp4bb] A question about Cys and fluoro benzene ring

[CRAIG A BINGMAN]

As others have suggested, it looks very much like a substitution at the 
fluorine position.  I might add that high resolution electrospray mass spec 
should be very useful in this case, because I suspect that this adduct will  
survive sample preparation, and it would provide strong and orthogonal 
experimental evidence that a chemical reaction had occurred at that position, 
specifically that fluorine had been lost during the reaction. To make your life 
easier, I’d definitely run an unreacted protein sample as a control (subjecting 
it to the full experimental procedure for creating the reaction.) The mass 
resolution of electrospray is amazing, but in many cases there are 
complications to the spectrum from associated ions. You definitely want the 
control spectrum for the unreacted protein to be from exactly the same solution 
conditions as your experimental sample.

> On Aug 23, 2017, at 10:01 AM, Cheng Zhang  wrote:
> 
> Hi everyone,
> 
> We recently got a structure of a receptor bound to a ligand. The ligand has a 
> fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
> receptor. The density for the ligand and the Cys seems to suggest a covalent 
> bond. However, I don't know if a covalent bond is chemically possible. Also, 
> I believe Cys is rarely involved in cation-pi interactions? Any suggestions 
> for placing the Cys and the fluoro methyl benzene ring?
> 
> Thanks! 
> 
> Cheng
> 
> 
> 
> -- 
> -
> Cheng Zhang

[ccp4bb] AW: [ccp4bb] A question about Cys and fluoro benzene ring

[Herman . Schreuder]

Dear Cheng,

You could check whether you get irreversible inhibition with your compound, 
which could be a sign of a covalent link with the protein.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Savvas 
Savvides
Gesendet: Mittwoch, 23. August 2017 21:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] A question about Cys and fluoro benzene ring

Dear Cheng,

it is possible that the sulfhydryl of the cysteine might have reacted with the 
methyl fluoro benzyl group via nucleophilic aromatic substitution. Fluorine is 
not the best leaving group among the halides but it would certainly allow this 
especially if the ligand-protein incubation time was sufficient and if the the 
pH of the reaction is above 7. This would get the Cys-SH closer to the 
cysteinyl moiety (S:-). The pKa of the Cys-SH group is just under 8.5 and in 
your case it might even be lower depending on local structure environment, 
resulting in an even better nucleophile.

Your electron density indeed suggests that the sulfur from the Cys-SH is 
covalently bonded to the benzyl ring and this would happen at the carbon that 
originally carried the fluorine. You will have to remodel your ligand, keeping 
in mind that the fluorine will no longer be part of your ligand model. The 
methyl group will of course be retained.

It might also be useful to try to confirm the reactivity of the ligand of 
interest with the protein (reminiscent of a suicide or covalent inhibitor 
against cysteine proteases?) via some kind of an orthogonal method (e.g. mass 
spectrometry). This would even tell you if the particular cysteine in your 
structure is truly the relevant covalent target. In this way you can even 
compare washed and dissolved crystals of your protein-ligand complex with the 
complex in solution.

best wishes
Savvas


---
Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.vib.be_en_research_scientists_Pages_Savvas-2DSavvides-2DLab.aspx=DQMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=v3UeZrHd-kKi-opvnW6bdc5D-MnNO-NhlxCt1Yok2bI=CGxCcJFHGdrRun7SpR3EoYieHZvMVcmj-ykaijcp90w=>




On 23 Aug 2017, at 17:01, Cheng Zhang 
<chengzh1...@gmail.com<mailto:chengzh1...@gmail.com>> wrote:

Hi everyone,

We recently got a structure of a receptor bound to a ligand. The ligand has a 
fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
receptor. The density for the ligand and the Cys seems to suggest a covalent 
bond. However, I don't know if a covalent bond is chemically possible. Also, I 
believe Cys is rarely involved in cation-pi interactions? Any suggestions for 
placing the Cys and the fluoro methyl benzene ring?

Thanks!

Cheng



--
-
Cheng Zhang

Re: [ccp4bb] A question about Cys and fluoro benzene ring

[Savvas Savvides]

Dear Cheng,

it is possible that the sulfhydryl of the cysteine might have reacted with the 
methyl fluoro benzyl group via nucleophilic aromatic substitution. Fluorine is 
not the best leaving group among the halides but it would certainly allow this 
especially if the ligand-protein incubation time was sufficient and if the the 
pH of the reaction is above 7. This would get the Cys-SH closer to the 
cysteinyl moiety (S:-). The pKa of the Cys-SH group is just under 8.5 and in 
your case it might even be lower depending on local structure environment, 
resulting in an even better nucleophile.

Your electron density indeed suggests that the sulfur from the Cys-SH is 
covalently bonded to the benzyl ring and this would happen at the carbon that 
originally carried the fluorine. You will have to remodel your ligand, keeping 
in mind that the fluorine will no longer be part of your ligand model. The 
methyl group will of course be retained.

It might also be useful to try to confirm the reactivity of the ligand of 
interest with the protein (reminiscent of a suicide or covalent inhibitor 
against cysteine proteases?) via some kind of an orthogonal method (e.g. mass 
spectrometry). This would even tell you if the particular cysteine in your 
structure is truly the relevant covalent target. In this way you can even 
compare washed and dissolved crystals of your protein-ligand complex with the 
complex in solution.

best wishes
Savvas


---
Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 





> On 23 Aug 2017, at 17:01, Cheng Zhang  wrote:
> 
> Hi everyone,
> 
> We recently got a structure of a receptor bound to a ligand. The ligand has a 
> fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
> receptor. The density for the ligand and the Cys seems to suggest a covalent 
> bond. However, I don't know if a covalent bond is chemically possible. Also, 
> I believe Cys is rarely involved in cation-pi interactions? Any suggestions 
> for placing the Cys and the fluoro methyl benzene ring?
> 
> Thanks! 
> 
> Cheng
> 
> 
> 
> -- 
> -
> Cheng Zhang