I need some advise in relation with a particular peptide synthesis
that I will probably need to run incorporating Ser, Tyr, Glu and Asp
amino acids without protection at the side chains. The reason for that is
that the peptides should be enriched in 13C isotope at the carbonyl
positions of those amino acids (for solid state NMR measurements), and the
apropriate N-terminus and side chain protected derivatives are not
commercially available or are very expensive (around $20000 per gram in
the case of L-aspartic acid 1-13C, 99% atom N-FMOC beta-O-t-butyl ester).
I am planning to buy the free amino acids, prepare the N-FMOC derivatives
and use them without side chain protection. My guess is that in the case
of Tyr or Ser, the decrease in the yield of the peptide would not be very
dramatic. It will not be the same for unprotected Asp and Glu, but still
I might obtain some final correct product (they are close to the
N-terminus end of the peptide), that could be purified
(hopefully) by HPLC and identified by MS and high resolution liquid NMR.
As I am not an expert in peptide synthesis I would appreciate any
comment about that. First of all, if it does make sense to try
that. And second, if there is some literature or anybody has
experience with optimized protocols in case that side protection is
not available (what could be the case in the early days of SPPS, I
guess).
We use an ABI SPP synthesizer, model 413 A, with FastMOC chemistry
in the 0.1mmol scale. This chemistry uses HBTU/HOBt/DIEA activation of the
N-protected aa. HBTU/HOBt in DMF and some amount of NMP is added into the
cartridge to solubilize the amino acid, and the mixture is transferred
to the reaction vessel where DIEA is added for in situ activation
and coupling (in around 9 minutes). Standard FMOC and BOC chemistry could
be used as well, but we have no experience with that.
The sequence of the peptide is as follows:
Acetyl-FGSDYEDRYYRENMHRYPNQ-OH
*****
the asterisks mark the amino acids that probably need to be used without
side chain protection. In a given synthesis, only two of the aa (not the
5 of them at once) would be used unprotected, that is, I do not plan to
synthesize the peptide with five amino acids in a row without protection,
only two of them, although different pairs in each synthesis.
Specific questions that might help me are:
- does the existence of two free carboxylates (in Asp and Glu) makes
the activation reaction with HBTU/HOBt not working properly? I assume
that simply both carboxyls will be activated.
- I also assume that a given proportion of the Asp or Glu amino
acids will couple through their alfa-carboxyl to the peptide chain
attached to the resin, while another proportion will do through the side
chain carboxyl. Is there any simple step that could be used to
"deactivate" the remaining activated carboxyls after coupling and prior
to the additon of the next activated amino acid?
Thank you very much for reading the message and for any comment
about that!
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Francisco Blanco phone: 301 402 4687
National Institutes of Health fax: 301 496 0825
Laboratory of Chemical Physics, NIDDKD blanco@speck.niddk.nih.gov
9000 Rockville Pike, Building 5, room 406
Bethesda, MD 20892 0520, USA