Knottins can be obtained either
- By extraction from natural sources
[Favel et al, 1989;
Saether et al, 1995;
Hernandez et al, 2000;
Cemazar et al, 2006].
- By chemical synthesis.
- As expressed recombinant proteins
[Camarero et al, 2007;
Krause et al, 2007;
Cui et al, 2007;
Kimura et al, 2006;
Kimura & Camarero, 2005;
Ji et al, 2005;
Schmoldt et al, 2005;
Kratzner et al, 2005;
Escoubas et al, 2003;
Chen et al, 1992].
Synthesis of Bracelet cyclotide Cycloviolacin O2
The oxidative folding conditions used for kalata B1 appeared to produce
only low yields of Bracelet cyclotides, i.e. cyclotides without the
cis-Proline found in loop 'e' of Moebius cyclotides.
A new improved strategy for the synthesis of Bracelet cycloviolacin O2
has been reported which uses Fmoc instead of BOC peptide chemistry and optimized
conditions for the oxidative folding step (detergents, redox reagents, DMSO)
[Leta Aboye et al., 2008].
15N labeled cyclotides
Complete labeling of cyclotides has been obtained by growing the cyclotide-bearing
plant (Oldenlandia affinis) on nitrogen-free agar media supplemented with
[Mylne & Craik, 2008].
The thia zip cyclization
The so-called thia zip reaction has proved very efficient for cyclization
of disulfide-rich knottins.
One efficient way to produce peptide cyclization is based on a transthioesterification
between an N-terminal cysteine and a C-terminal thioester that forms an intermediate
α-amino thiolactone that rearrange itself via an S,N-acyl isomerization into
a C-to-N cyclic peptide.
When there are internal thiols in the sequence between the N- and C-termini, there is a
cascade of thiol-thiolactone exchanges, starting from the cysteine closest to the C-terminus,
that eventually leads to the largest cyclic thiolactone where the irreversible S,N-acyl
isomerization can take place. This entire process, which is called the thia zip cyclization
(see scheme below), has been largely used to synthesize cyclic knottins
[Tam & Lu, 1998;
Tam & Yu, 1997;
Daly et al, 1999].