I have made a lot of phosphopeptides and have tried most of the methodologies.
The Novabiochem reagents, mono-benzyl protection)[D with mono-benzyl protection work very well. I have
made up to 26mers with up to three phosphates. I use HATU/HOAt/NMM, couple
for 1 hour. This is using a perseptive Biosystems 9050 synthesiser. It is
true as others have commented, you can get aggregation after incorporation
of the ser phosphate, but in most cases I get a crude purity of 50%-90% and
HPLC purification normally separates the deletion sequences. I also find
phosphothreonine, although a dropin deprotection peak height is often
observed a few residues after its incorporation, this does not result in
significant deletion sequences being formed and crude purities are in
line with peptides of similar natures without phosphate incorporation.
I would suggest increasing the deprotection time after incorporation of
the phosphate residue. On the Perseptive Biosystems 9050, the default
is 7min. i normally increase to 10-12min which seems to be adequate.
Dr Graham
Bloomberg
Dept. Biochemistry
Medical School
University of Bristol
Bristol BS8 1TD
UK
E-mail G.B.Bloomberg@bristol.ac.uk