I am using cystamine (free based) to conjugate to a phosphate group on an oligonucleotide. I tried several keyword searches on Google for neutralizing the dihydrochloride at the ends of the cystamine. But, I am unable to find a protocol. I had once found a procedure for the process, but I did not reference it and hence I am unable to find it again.

The procedure I followed was:

  1. Dissolve the cystamine.2HCl in water.

  2. Transfer to a separation funnel and add chloroform.

  3. To this add 1N NaOH.

  4. After shaking vigorously collect the chloroform layer which contains the cystamine.

  5. Repeat extraction couple of times using more chloroform.

  6. Pool fractions - dry with anhyd. MgSO4 - transfer to an RBF.

  7. Evaporate on a RotaVap

  8. Dry on schlenk line and store in an inert environment at 4 deg. C.

However, the extraction was not very efficient. Can anyone refer a protocol for the same?

Thanks a lot for your help!!!!

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    $\begingroup$ Two things come to mind: cysteamine is water soluble so you may need to salt out your aq phase and also 1M NaOH may be strong enough to deprotonate the thiol. I would suggest adjusting the pH to close to 7 and adding salt before trying the organic extraction. $\endgroup$ – Waylander Oct 19 '17 at 9:37
  • $\begingroup$ Thanks for the tip. I will attempt your method to see if there is any change in the result. But, I have done an NMR on the extracted "cystamine" and it shows a clean spectra with an intact disulfide bond. $\endgroup$ – Somdeb Oct 19 '17 at 21:16
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    $\begingroup$ Perhaps go with NaHCO3 sat $\endgroup$ – Alchimista Oct 19 '17 at 21:22
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    $\begingroup$ First off, I would always use methylene chloride ahead of chloroform, especially under basic conditions. You can 'pep' up your extraction by adding a small amount of a third solvent that dissolves in both water and your extracting solvent, and also dissolves your compound to some extent. Methanol, ethanol or THF normally spring to mind. $\endgroup$ – Beerhunter Oct 21 '17 at 9:44
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    $\begingroup$ @Somdeb you said yourself the extraction wasn't efficient. Either switch extraction solvent, augment your extraction solvent or hamper your water solubility. I have no feel for the solubility without data. If polarity is an overriding factor, increase it for your organic phase. I don't mean add so much solvent you get one phase. Start with 5-10%. $\endgroup$ – Beerhunter Oct 23 '17 at 16:41

Maybe the MgSO4 is absorbing your product. You may want to try another drying agent Try Na2SO4, zeolites, or azeotropic distillation (e.g. with benzene). Maybe NaOH will work, but it may react with cysteamine.

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