@László

You may consider 2 options:

1: Redo the Fmoc protection on the vacuum-dried mixture (if you have enough Fmoc-OSu remaining) to convert the mixture completely to Fmoc-Cys(Propargyl)-OH. 

2: Purify the desired product by extraction and chromatography

Does the product mixture dissolve in dichloromethane (CH2Cl2)? If so, you may try to remove the free amine (without Fmoc protection) by extracting with 1 molar aqueous hydrochloric acid (1 M HCl) from the product mixture dissolved in CH2Cl2. More than likely, the CH2Cl2 will be at the bottom layer in the separation funnel. Extract the organic solution at least 5 times with 1 M HCl(aq) (2X volume with respect to the organic solution), then dry with a brine wash (saturated NaCl, 2X volume) and sodium sulfate (remove by decanting or filtering). If there is still too much (>5% by NMR) H-Cys(Propargyl)-OH remaining, then perform column chromatography on normal-phase silica gel using a MeOH/CH2Cl2 gradient (1:19 to 1:9 v/v of MeOH/CH2Cl2). The free-amine should have a lower Rf than the Fmoc-product (confirm by UV lamp and ninhydrin or Dragendorff staining). If you plan to use the Fmoc-Cys(Propargyl)-OH for solid-phase peptide synthesis, having

@László

You may consider 2 options:

1: Redo the Fmoc protection on the vacuum-dried mixture (if you have enough Fmoc-OSu remaining) to convert the mixture completely to Fmoc-Cys(Propargyl)-OH. 

2: Purify the desired product by extraction and chromatography

Does the product mixture dissolve in dichloromethane (CH2Cl2)? If so, you may try to remove the free amine (without Fmoc protection) by extracting with 1 molar aqueous hydrochloric acid (1 M HCl) from the product mixture dissolved in CH2Cl2. More than likely, the CH2Cl2 will be at the bottom layer in the separation funnel. Extract the organic solution at least 5 times with 1 M HCl(aq) (2X volume with respect to the organic solution), then dry with a brine wash (saturated NaCl, 2X volume) and sodium sulfate (remove by decanting or filtering). If there is still too much (>5% by NMR) H-Cys(Propargyl)-OH remaining, then perform column chromatography on normal-phase silica gel using a MeOH/CH2Cl2 gradient (1:19 to 1:9 v/v of MeOH/CH2Cl2). The free-amine should have a lower Rf than the Fmoc-product (confirm by UV lamp and ninhydrin or Dragendorff staining). If you plan to use the Fmoc-Cys(Propargyl)-OH for solid-phase peptide synthesis, having

prep-HPLC cost so high， it‘s better wash with acid solution HCl or H3PO4 to remove free amine derivatives.

Chromatography... 

Before purification I would try to redo the Fmoc protection to improve the yield.

Some tips on making it go to completion that may help:

Make sure that your Fmoc-OSu is not 'off' - it can hydrolyse over time if not stored properly.

Use an organic solvent to keep the Fmoc-OSu in solution (eg. ~50% THF).

Use a slight excess (~1.2x) of Fmoc-OSu over the amine.

Check the pH periodically after adding Fmoc-OSu - it can drop significantly as the reaction progresses (keep it at ~8-9 using a mild base).

When complete, remove some of the organic solvent (not to dryness). Extract excess Fmoc-OSu with EtOAc (not very soluble in Et2O) - your product should remain in the aqueous phase as long as it is slightly basic. Acidify the aqueous phase with aq. HCl and extract your product with EtOAc or CH2Cl2 then do the acid wash (+chromatography if required) as per Lael's answer.

Good luck!

I would try stirring the mixture in acetonitrile. In my Hands S-acylcysteines were insoluble in acetonitrile and I expect that free S-propargylcystein will be insoluble in acetonitrile. However, focus protected one will dissolve. Good luck.

Dear colleagues!

Acetonitrile dissolved most of the remaining S-prop-cysteine, a white solid remained, after filtration I added a bit of chloroform, that dissolved a yellow impurity (The solution was yellow, we did not check what it was), filtered a white solid. I washed the white solid with 5% HCl.

NMR showed a mixture of D and L S-propargyl-cysteine protected with Fmoc. This is not uncommon while protecting Cysteine with Fmoc. This seems to be okay for most preparative purposes, however we will check its purity via HRMS.

Your ideas and suggestions helped solve this, I am very grateful.

Thank you!