Hi,

I have an issue synthesizing a phosphopeptide. I'm not phosphorylating, just using phosphorylated residue (Fmoc-Ser(PO(OBzl)OH)-OH).

I tried 2 different synthesis protocols.

1. Resin used was Rink Amide AM. Deprotection with 20% 4-methylpiperidine in DMF for 30 min. 5 equivalents HOBt and Fmoc-residue in DMF were added to resin. 5 equiv. of DICl were added. Incubation 1h for regular residues, O/N for phosphoresidue. Final cleavage with 95% TFA 2.5% water 2.5% TIPS for 90 min. Filtered, Precipitated with ether. It was really difficult to precipitate it, the peptide is acidic so I added Tris and also dissolved it in mixture of water and ACN (tried different ratios), MS showed there is no peptide there. This way I am able to get my peptide but when I resuspend it it's very gel-like and it already caused some issues with HPLC during purification and HPLC tech guy told me to never run it again, so I guess I'd like some help with what might be causing this gellification and how can I resuspend it successfully. Even when it seems resuspended after few hours it solidifies again.

2. Resin used was Rink Amide MBHA. Deprotection with 20% 4-methylpiperidine in DMF for 30 min. 5 equivalents HBTU and Fmoc-residue in DMF were added to resin. 10 equiv. of DIPEA were added. Incubation 1h for regular residues, O/N for phosphoresidue. Next the same as above. During synthesis when I incubated with phosphoresidue a brown precipitate started forming, I had to change the vessel because it completely clogged it, I don't know what this precipitate is but I guess that's the issue with this method. What could it be and how do I prevent it?

Thanks!

Kasia Radziwon