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Chemistry Forums for Students => Organic Chemistry Forum => Topic started by: crystalguy on December 12, 2018, 10:56:15 AM

Title: Water soluble version of Cystine
Post by: crystalguy on December 12, 2018, 10:56:15 AM
Cystine is poorly soluble in water.  It is essentially two Cysteine amino acids linked through their side chains by a thioether. 

I would like to ask how Cystine can be made more water soluble without drastically modifying its structure.  Eg. Can the sulfur atoms in the thioether link be replaced by another atom that would have a similar chemistry and size but make it more soluble in water? 

What about addition of more polar moieties to either the amino or carboxy ends of the individual amino acids. I would have thought that at around neutral pH since both the amino and carboxy are ionized (i.e. zwitterionic form) that they would be sufficient to make Cystine as a whole, more soluble in water, but apparently not. So what minor changes could be made here to make Cystine more water soluble?

Thanks in advance for any suggestions.
Title: Re: Water soluble version of Cystine
Post by: wildfyr on December 12, 2018, 12:41:59 PM
If you modify the atoms On cystine it is no longer cystine.

In such a small bioactive molecule, any change will have strong effects on function.

Cysteine can be reduced to two cystines for better solubility and they won't rejoin immediately.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 12, 2018, 05:08:27 PM
If you modify the atoms On cystine it is no longer cystine.

In such a small bioactive molecule, any change will have strong effects on function.

Cysteine can be reduced to two cystines for better solubility and they won't rejoin immediately.

I don't need it to be exactly Cystine, just to mimic it, for example as is done in so many drugs where they mimic protein substrates but are unreactive. A more specific example, phosphoserine can be mimicked with the amino acid glutamic acid - not exactly, but the size and charge are similar.

Can't use Cysteine, because the it won't bind, need a Cystine mimic. Any ideas for modification that can be made that will retain similar properties (not exactly of course) but is more soluble.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 12, 2018, 05:12:08 PM
I suppose I'm looking for advice from chemistry experts that can offer insight into what chemical modifications are feasible to make.

For example, what about linking Serine amino acids by their side chain via the link (-O-O-) like (-S-S-) in Cystine.  Is this chemically feasible? 
Title: Re: Water soluble version of Cystine
Post by: vikram on December 12, 2018, 09:54:39 PM
If you are searching for similar structure with water solubility, then Serine would be one of the choice.-SH is replaced by -OH
Title: Re: Water soluble version of Cystine
Post by: Babcock_Hall on December 13, 2018, 04:45:45 AM
crystalguy,

If you link two serine atoms in the way that you suggest, it would create a peroxide functional group, which has certain chemical properties that might be undesirable in your application.  Also, the bond lengths and bond angles would not be identical.
Title: Re: Water soluble version of Cystine
Post by: rolnor on December 13, 2018, 06:58:04 AM
A sulphur atom can be replaced by two methylene groups, you can replace the S-S link with CH2CH2CH2CH2. I would be interesting to look at this in a molecular modeling software.
Maybe its better with three methylenes, CH2CH2CH2.
If this gets you better solubility is not certain.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 15, 2018, 06:29:55 AM
If you are searching for similar structure with water solubility, then Serine would be one of the choice.-SH is replaced by -OH

the only problem is that it needs to be in the context of Cystine, i.e. -O-O- vs. -S-S-, not -OH vs. -SH
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 15, 2018, 06:30:55 AM
A sulphur atom can be replaced by two methylene groups, you can replace the S-S link with CH2CH2CH2CH2. I would be interesting to look at this in a molecular modeling software.
Maybe its better with three methylenes, CH2CH2CH2.
If this gets you better solubility is not certain.

That seems like a good idea, but unfortunately, I doubt -CH2- groups would improve the solubility.
Title: Re: Water soluble version of Cystine
Post by: wildfyr on December 15, 2018, 07:02:14 AM
What is the desired usage?
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 16, 2018, 03:01:01 PM
What is the desired usage?

To have it act as a substrate mimic for a protein it binds to.
Title: Re: Water soluble version of Cystine
Post by: hypervalent_iodine on December 16, 2018, 05:06:52 PM
What is the desired usage?

To have it act as a substrate mimic for a protein it binds to.

Do you know exactly how it binds to this protein? What you are looking for is a bioisosteric replacement, but it is difficult to be able to suggest one without knowing more information, since bioisosteres are heavily context-dependant.

Is the cysteine part of a larger molecule? Is the intended application for use in some sort of medicine? Why is it important that it be very water soluble?
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 17, 2018, 09:06:35 AM
What is the desired usage?

To have it act as a substrate mimic for a protein it binds to.

Do you know exactly how it binds to this protein? What you are looking for is a bioisosteric replacement, but it is difficult to be able to suggest one without knowing more information, since bioisosteres are heavily context-dependant.

Is the cysteine part of a larger molecule? Is the intended application for use in some sort of medicine? Why is it important that it be very water soluble?

No clue how it binds.  The protein is a channel that actually transports cystine across a membrane.  The Cystine is standalone, it's the result of degradation of disulphide bond containing proteins.  The intended application is to study the binding of Cystine in complex with the protein to get an idea of where and how it binds. The solubility needs to be improved because we need to add large quantities to small volumes, and presumably its binding affinity is quite weak. Having an improved solubility Cystine mimic, we can just throw in loads of it to drive the binding without it precipitating out of solution.
Title: Re: Water soluble version of Cystine
Post by: hypervalent_iodine on December 17, 2018, 10:00:30 AM
What is the desired usage?

To have it act as a substrate mimic for a protein it binds to.

Do you know exactly how it binds to this protein? What you are looking for is a bioisosteric replacement, but it is difficult to be able to suggest one without knowing more information, since bioisosteres are heavily context-dependant.

Is the cysteine part of a larger molecule? Is the intended application for use in some sort of medicine? Why is it important that it be very water soluble?

No clue how it binds.  The protein is a channel that actually transports cystine across a membrane.  The Cystine is standalone, it's the result of degradation of disulphide bond containing proteins.  The intended application is to study the binding of Cystine in complex with the protein to get an idea of where and how it binds. The solubility needs to be improved because we need to add large quantities to small volumes, and presumably its binding affinity is quite weak. Having an improved solubility Cystine mimic, we can just throw in loads of it to drive the binding without it precipitating out of solution.

In that case, I would say that you are stuck using cysteine. As was mentioned above, any molecular changes you could make to it would render it no longer cysteine, and using such a molecules to make conclusions about how cysteine bonds would be a bit dodgy, IMO.

What method are you using for co-crystallisation? If you use the hanging drop method, there’s no reason you couldn’t dissolve the cysteine in DMSO. There are also other ways to acoustically shoot molecules into crystals that may be of assistance.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 17, 2018, 11:29:54 AM
What is the desired usage?

To have it act as a substrate mimic for a protein it binds to.

Do you know exactly how it binds to this protein? What you are looking for is a bioisosteric replacement, but it is difficult to be able to suggest one without knowing more information, since bioisosteres are heavily context-dependant.

Is the cysteine part of a larger molecule? Is the intended application for use in some sort of medicine? Why is it important that it be very water soluble?

No clue how it binds.  The protein is a channel that actually transports cystine across a membrane.  The Cystine is standalone, it's the result of degradation of disulphide bond containing proteins.  The intended application is to study the binding of Cystine in complex with the protein to get an idea of where and how it binds. The solubility needs to be improved because we need to add large quantities to small volumes, and presumably its binding affinity is quite weak. Having an improved solubility Cystine mimic, we can just throw in loads of it to drive the binding without it precipitating out of solution.

In that case, I would say that you are stuck using cysteine. As was mentioned above, any molecular changes you could make to it would render it no longer cysteine, and using such a molecules to make conclusions about how cysteine bonds would be a bit dodgy, IMO.

What method are you using for co-crystallisation? If you use the hanging drop method, there’s no reason you couldn’t dissolve the cysteine in DMSO. There are also other ways to acoustically shoot molecules into crystals that may be of assistance.

The protein won't bind Cysteine, it does not transport cysteine.  It only transports Cystine.  Actually getting a small molecule mimic is more than just trying to understand where and how it binds, it's to stabilize the protein as well to coax it to crystallize - this is very commonly done.

Yes hanging drop.  Can't use DMSO, too much of it will cause the protein to unfold.  Maybe can get away with less than 1% DMSO, but that won't dissolve Cystine.
Title: Re: Water soluble version of Cystine
Post by: Babcock_Hall on December 17, 2018, 11:33:12 AM
I am assuming that you are using cystine and not a peptide containing cystine.  The solubility of amino acids depends upon pH.  What pH is your experiment, and can it be changed?
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 17, 2018, 11:46:59 AM
I am assuming that you are using cystine and not a peptide containing cystine.  The solubility of amino acids depends upon pH.  What pH is your experiment, and can it be changed?

Yes, Cystine only.  The pH needs to be around 7 to 8 for the protein to behave well.  I realize at very acidic conditions Cystine is more soluble, but that would kill the protein.

Need to add the Cystine at concentrations approaching 1 mM.
Title: Re: Water soluble version of Cystine
Post by: Babcock_Hall on December 17, 2018, 11:48:33 AM
I would predict that cystine would be least soluble at its pI, which should be near 6.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 17, 2018, 11:51:59 AM
I would predict that cystine would be least soluble at its pI, which should be near 6.

Yes true, but even at pH 8 it's not very soluble unfortunately.
Title: Re: Water soluble version of Cystine
Post by: hypervalent_iodine on December 17, 2018, 12:21:54 PM
Cysteine was an autocorrect typo on my part, apologies. I would still be very concerned that making any changes to your cystine molecule will change or abrogate binding in ways that you can’t predict (because you don’t know about how it binds). Especially if the changes you are making are for increasing solubility, since this would normally mean making the molecule more polar, and polarity changes like that could drastically alter binding. IMO, any results you might get from such a molecule would not really be that meaningful in terms of what you are trying to look at.

Do you know how much more cystine you can get into solution at 1% DMSO and how much excess you will have compared to enzyme concentration? It might be enough to get crystals. Have you been able to get crystals of the protein without cystine bound? I assume you probably have. Do you know if the structure changes when it binds to cystine, and could you possibly do any in silico modelling to get an idea of where it might bind?
Title: Re: Water soluble version of Cystine
Post by: Babcock_Hall on December 17, 2018, 03:24:33 PM
https://pubs.acs.org/doi/pdf/10.1021/je9501853
"Solubilities of l-Cystine, l-Tyrosine, l-Leucine, and Glycine in Aqueous Solutions at Various pHs and NaCl Concentrations"

I imagine that you have seen this already.
Title: Re: Water soluble version of Cystine
Post by: crystalguy on December 30, 2018, 01:38:08 PM
Cysteine was an autocorrect typo on my part, apologies. I would still be very concerned that making any changes to your cystine molecule will change or abrogate binding in ways that you can’t predict (because you don’t know about how it binds). Especially if the changes you are making are for increasing solubility, since this would normally mean making the molecule more polar, and polarity changes like that could drastically alter binding. IMO, any results you might get from such a molecule would not really be that meaningful in terms of what you are trying to look at.

Do you know how much more cystine you can get into solution at 1% DMSO and how much excess you will have compared to enzyme concentration? It might be enough to get crystals. Have you been able to get crystals of the protein without cystine bound? I assume you probably have. Do you know if the structure changes when it binds to cystine, and could you possibly do any in silico modelling to get an idea of where it might bind?

Yes you're right, the changes could be deleterious.  But it really is a trial and error process.  The main goal though is to get crystals of the protein, regardless of what the small molecule is doing which is minimally just stabilizing the protein/'tightening' it up. 

At 1% DMSO, we cannot get enough Cystine into solution because the affinity is quite weak, in the micromolar range.  We cannot get crystals of the protein alone and have no idea where it binds on the protein.