Hi ,
This summary is for Nicola and anybody else who did not see all the helpul
suggestions I recieved.
Radhika.
City of Hope ,Duarte, CA.
*************************************************************************
Hi ABRF ers,
Hope somebody can help me with these questions.
a) Is there a convenient reference with all the amino acid UV absorbances?
b) what is the the generally accepted quantity of sample(peptide) that can be
injected on a analytical column(4.6x250mm) . Over what absorbance level( in mAu)
is it considered to be "over loading "the column.
Radhika
Most of the amino acids do not have a UV absorbance of use (at >190 nm). Most
people use 210 nm as a compromise, the AMIDE bonds absorb there (not lamda max).
The more amides (i.e. the bigger the peptide) the more signal. As you go
further into the UV, the solvents themselves have too much absorbance to pick up
the peptides. Of course there is some signal for some of the sidechain groups
(mainly for Trp at about 280 nm, also less for Tyr at about 280, small for Phe
and Cys around 260).
I would ask the maker of the column for loading, it depends on more than the
size.
********************************************************************************
****************************
Hi Everyone,
Just wondering if there are any lipopetide generators out there. I have been
having problems dissolving Palmitic acid in 1:1, 1M DIEA and 0.5M HBTU/HOBt (
Pioneer activators). I get a brown coagulated mass, that dissolves after some
warming up in a water bath. When I add the solution to the resin however it
coagulates up too, but does liquefy on warming up. I am using the normal mole
excess that is recommended and doing the coupling manually.
How would you generate symmetric anhydride of the Palmitic acid and what
activators would be used for that coupling.
On a related note. has anybody used DMF for analytical HPLC seperations and
what wavelength would you monitor the run. We do have a PDA.
Hope I can get some hits on these questions.
Radhika,
We do not use the Pioneer, but routinely add palmitic acid (mono) to our
peptides in a 10X molar excess to resin (0.02 mM). 0.2 mM palmitic acid (A)
is dissolved a 0.20 mM HOBt/NMP solution. 0.2 mM DIC(B) is also prepared in
NMP. The resin in 200 uL NMP is mixed with 450 uL solution A and 150 uL
solution B for 40 min (first coupling) and repeated again (second coupling).
You can scale the reaction to your conditions. You may need more time for
the couplings, check with ninhydrin after each coupling.
Danny
Dear Radhika,
no suggestions regarding the palmitoylation issue, however, as to the
second point there is a paper published by Fernando Albericio and
Ernest Giralt that may be of help: "Convergent solid-phase peptide
synthesis. 12. Chromatographic techniques for the purification of
protected peptide segments", Int J Pept Protein Res 1995
Aug;46(2):119-33. They describe modified reversed-phase
chromatographic techniques in which DMF was added to the water and
acetonitrile mixtures used as eluents.
Luisa
Dear Radhika,
As I remember, fatty acids are well soluble in methylene chloride, but
do not chill the solution during activation. I had the experience of
obtaining succinimide ester of stearic acid with DCC, think any
coupling reagent should work. From the colleagues' experience, some
palmitoylated peptides elute from RP columns and some don't. I'm not
sure adding DMF would help, perhaps, trying less hydrophobic phases
(C1, C2...) or using direct phase would help (I purified my
lipopeptides on conventional glass columns with silica gel using
RP-HPLC only for analysis).
Hope this helps
Vladimir
This archive was generated by hypermail 2b29 : Mon Feb 14 2000 - 16:05:59 EST