Re: PepSyn: N-epsilon Lys derivatization
Dan Brune (DBrune@asu.edu)
Tue, 09 Feb 1999 11:17:34 -0700 (MST)
Dear Jeni,
I had good luck with the Lys(Mtt) derivative using PAL-PEG-PS resin
obtained from PerSeptive Biosystems (now part of Perkin Elmer Biosystems).
The linker here is a 4-aminomethyl-3,5-dimethoxyphenoxy group. The
cleavage solution that I used was also somewhat different from yours, and
consisted of 1% trifluoroacetic acid in dichloromethane with 3%
triisopropylsilane as a scavenger. This is basically the method
recommended in the Novabiochem catalog. As recommended by Anita Hong, this
was preceeded by solvent exchange to replace the DMF solvent with CH2Cl2.
My synthesis was done on a Milligen Biosearch 9050 synthesizer, which uses
continuous flow through a resin-packed column. Solvent exchange was done
simply by flowing CH2Cl2 through the column using the sandard conditions
(flow rate and duration) for DMF washes after piperidine deprotection.
After solvent exchange, the deprotecting solvent was then flowed through
the column (or at least this was the plan - in actuality, a lot of air got
in the line while trying to pump the deprotection solvent through the
column, so I wound up taking the column off of the synthesizer and shooting
the deprotection reagent through the column with a syringe. As noted by
Anita Hong, the Mtt gave a bright yellow color as it was cleaved off the
resin, so I continued to pulse 1% TFA with 3% TIS in DCM through the column
with the syringe until I no longer observed this yellow color. I should
add that the yellow color was transient - over a period of a couple
minutes, it disappeared, probably due to reaction of the Mtt carbonium ion
with TIS.) After reconnecting the column to the instrument, the synthesis
was completed, with good results. The product was a 29 amino acid
polypeptide with a dye molecule added to a Lys at the C-terminus. The dye
was added after adding and deprotecting the Lys, and served as the side
chain protecting group for that Lys during the rest of the synthesis.
Although I have no experience with using the Dde group for Lys
protection, I noticed that Novabiochem in their discussion of Allyl
protection notes some interference from the hydrazine used for removal of
the Dde group. They say that the problem is thought to be due to a small
amount of diazine in the hydrazine, which can reduce the double bond in the
allyl group (p. S36 in the 1999 catalog), and note that the problem can be
overcome by including allyl alcohol in the hydrazine reagent. I do not
know if reduction of one or both NO2 groups in the Dnp group on your
peptide could account for the 50 Da loss in mass during your synthesis, but
the thought occurs to me that you might similarly be able to inhibit the
destruction of that group during the hydrazine treatment by including an
excess of dinitrophenol or a Dnp-labelled amino acid in the hyudrazine
cleavage solution as a scavenger for diazine or whatever else is causing
the problem (assuming that it is not the hydrazine itself). I mention this
speculation mainly since you said that you already have some of the resin
from your synthesis still available.
Best wishes,
Dan Brune
Dept. of Chemistry & Biochemistry
Arizona State University