I should have read Jan Lukszo's message more carefully. In the peptides we
synthesized with N-terminal Acm cysteine, our objective was to prepare
dimeric, disulfide-linked, cyclized peptides; not cyclic peptides with a
free N-terminal thiol for coupling to KLH. In our case, we compared the
iodine and chlorosilane-sulfoxide methods for removing the Acm group, but
both of these methods oxidize cysteine to the disulfide and do not give the
free thiol. Incidentally, we found that the chlorosilane method gave
higher yields of dimeric peptide, and did not appear to interfere with or
scramble the existing disulfide bonds.
Latif
At 03:01 PM 5/18/98 +1000, you wrote:
>Latif
>
>I would be interested to know how the Acm group was removed without
>comprimising or scrambling the already intact disulfide. Currently we make
>such peptides by fmoc chemistry and use the fmocCys(MeOBzl)-OH derivative as
>the nondisulfide Cys. This requires of course HF cleavage which I would
>prefer not to do as anything but a 15 minute cleavage time "junks up" the
>peptide substantially.
>
>Regards
>
>Denis Scanlon
>
>AuspepP/L
>
>>
>>Jan:
>>
>>We have made peptides by fmoc chemistry that contain a disulfide bond and
>>an N-terminal cys using the approach you outlined, except that the internal
>>cysteines (those participating in the disulfide) were Trt protected and the
>>N-terminal cysteine was Acm protected. Following synthesis and cleavage
>>(the Trt groups come off), the peptides were air oxidized to form the
>>disulfide, followed by removal of the Acm protecting group from the
>>N-terminal cys.
>>
>>There are a couple of good reviews on cys protecting groups and related
>>techniques:
>>
>>Annis et al. Methods in Enzymology 289:198, 1997
>>Andreu et al.Methods in molecular biology 35:91, 1994
>>
>>Latif
>>
>>
>>
>>At 10:15 AM 5/15/98 -0400, you wrote:
>>>Dear ABRFers,
>>>
>>>We have received a request to synthesize a peptide and to make a
peptide/KLH
>>>conjugate
>>>involving a short (15 residue), cyclic (disulfide bridge) peptide with an
>>>extra (3rd)
>>>Cysteine, at the N-terminus, for coupling to KLH. The N-term Cys was just
>>>added to the
>>>sequence to help in coupling to the carrier, as the presence of internal
Glu
>>>and a couple of Lys residues (and absence of Tyr) in the sequence would
>>>probably
>>>eliminate alternative coupling schemes (coupling through an internal AA not
>>>an option).
>>>While it was suggested to us that we could pursue a scheme involving a
>>synthesis
>>>of peptide with 2 internal Cys(Acm) and free N-term Cys, coupling of this
>>>peptide
>>>to KLH and deblocking/oxidation of the conjugate, we would like to hear
>>>some opinions
>>>and suggestions from those of you who dealt with such or similar problems.
>>>
>>>Jan Lukszo
>>>Dr. Jan Lukszo
>>>Peptide Analysis and Synthesis
>>>NIH/NIAID/SBS
>>>Twinbrook II
>>>12441 Parklawn Drive
>>>Rockville, MD 20852
>>>phone:(301)496-3786
>>>fax:(301)480-2618
>>>E-Mail: lukszo@fcrfv2.ncifcrf.gov
----------------------------------------
A. Latif Kazim, Ph.D.
Biopolymer Facility
Science-4
Roswell Park Cancer Institute
Elm & Carlton Sts.
Buffalo, NY 14263
E-mail: kazim@sc3101.med.buffalo.edu
Phone: (716) 845-8923
Fax: (716) 845-7621