SPPS cyclic peptides

Bipyrrole alkaloids, cyclic peptides, quinones, macrolides, polyethers Vertebrata Osteichthyest, 22,000 spp. 

Ascidians are marine filter feeders with a rich natural products chemistry that five commonly associated with symbiotic bacteria (88, 89, 109). A well-studied symbiosis consists of photosynthetic Prochloron spp. cyanobacteria that occur in ascidians of the family Didemnidae (110). Prochloron spp. also can be found in bacterial mat structures of stromatoliths (111) but so far have not been detected outside of such structured environments. From didemnid ascidians, numerous cytotoxic cyclic peptides of the patellamide group (Fig. 5) were isolated (109, 112, 113). Mechanical separation of the Prochloron sp. symbiont from its host Lissoclinum patella and subsequent genome sequencing revealed a set of biosynthetic genes that after transfer into E. coli enabled this bacterium to produce two different patellamides (114). The genes also were identified in an independent study by screening a library of Prochloron sp. DNA... 

To extend the scope of NCL, efforts have been made to introduce modifications so as not to depend on a Cys site. Cys residues can be selectively desulfurized to Ala [124], even in the presence of Cys(Acm) and Met [125]. Homocysteine and selenocysteine also serve as ligation sites, and can then be converted into Met [126] and into Ala or didehydroalanine, respectively [127]. Recently, ligation at Phe sites has also been accomplished [128]. The use of auxiliaries that are removed after ligation has been extensively studied [129-134]. NCL has also been applied to SPPS [135, 136], and to the synthesis of cyclic peptides [137-139], In another extension of the technique, proteins have been prepared by tandem ligation [135, 140], and expressed protein ligation [141]. Recently, NCL has been applied to the synthesis of glycoproteins [104, 142-144]. 

Given the potential use of cyclic peptides as therapeutic agents [147-149], the synthesis of these compounds has been the object of extensive studies since the first development of SPPS. Cyclization increases proteolytic resistance and may result in enhanced biological activity compared to their linear counterparts [150, 151]. Cyclic peptides consist of distinct types of linkage (i) in the most common type, the N- and C-termini are joined ( head-to-tail ) (ii) a side-chain is linked to the C- or N-terminus (iii) two side-chains are joined (side-chain-to-side-chain). The linkage is usually an amide bond but can also be a disulfide or another type of functionality. 

Keywords Acyl migration Acyl transfer Chemical ligation Computational studies Cyclic peptides Glycopeptides Neoglycoconjugates SPPS... 

The most successful method of fragment condensation for the synthesis of polypeptides and proteins in solution phase is NCL, reported by Dawson for the first time in 1994 [7]. This was a significant contribution because NCL overcomes one of the main limitations of solid phase peptide synthesis (SPPS), namely the production of long peptide sequences (>50 amino acid residues) [1, 3, 29]. NCL may be used in both solution and solid phases solution phase NCL has been used for the synthesis of small peptides and cyclic peptides [30-32] whereas SPPS is more widely applied in polypeptide and protein synthesis. 

Conventional synthesis of lactam-bridged cyclic peptides have in the past utilized the efficient assembly of the linear sequence by SPPS and upon cleavage from the solid support, cyclization of the semi-protected peptide is then carried out in solution. This method requires a dilute medium to minimize dimerization as a result of intermolecular condensation of the linear sequences (81). As a consequence, the reaction rate and yield are drastically reduced. 

Figure 3 summarizes the steps involved in cyclic peptide synthesis and these are described in more detail in Subheadings 3.1 and 3.2 for Boc and Fmoc methods, respectively. Carry out all procedures at room temperature and in a well-ventilated fume hood unless otherwise specified. The amounts of reagents are given based on a 0.5 mmol scale synthesis, which typically yields >10 mg of final product after purification. These procedures have been used in our laboratory to synthesize several classes of disulfide-rich cyclic peptides, including SFTI-1 (14 amino acids, one disulfide bond), cyclic Vcl.l (22 amino acid, two disulfide bonds), kalata B1 (29 amino acids, three disulfide bonds), MCoTI-II (34 amino acids, three disulfide bonds), and cyclic chlorotoxin (43 amino acids, four disulfide bonds) (Fig. 4). The procedures are described for manual synthesis but are also applicable to automated SPPS.

Redox-sensitive resin 24 designed for solid-phase peptide synthesis (SPPS) [29] was prepared from commercially available 2,5-dimethylben-zoquinone in seven steps [30] and loaded to a support via a Wittig reaction. Release of the peptide occurs using two sequential mild conditions, reduction with NaBH4 followed by TBAF-catalyzed cyclic ether formation (Scheme 8) which provide orthogonality to acid sensitive reactions. 

The commercial availability of protected /V-methyl amino acids [(Me)Xaa] of many proteinogenic amino acids (as well as other V-alkyl amino acids), the availability of procedures for the synthesis of protected V-alkyl analogues of all the protein amino acids, and the availability of synthetic procedures for site-selective alkylation during SPPS (see Section 10.1.2.1) allows the alkylation of nearly all peptide bonds in a given parent peptide. The synthesis of a series of V-alkyl peptide analogues based on the sequence of a given bioactive peptide (linear or cyclic) in which each peptide bond is successively alkylated and evaluation of the biological activity of this series will be called herein A-alkyl-scan (for example Me-scan, Et-scan, etc.)... 

An A -methyl-amino-oxy amino acid has also been incorporated into peptides using standard SPPS procedures. Reaction of these peptides with native reducing sugars yields neoglycopeptides via a chemoselective reaction with the amino-oxy side chains, maintaining the linked sugars in their cyclic conformations and... 

T Nishizawa, M Asayama, K Fujii, K Herada, M Shirai. Genetic analysis of the peptide synthetase genes for the cyclic heptapeptide microcystin in Microcystis spp. J Biochem (Tokyo) 126 520-529, 1999. 

Prochloron spp. synthesize patellamides using a bacteriocin-like process. " The final patellamide structures are directly encoded on a precursor peptide, PatE. This linear, ribosomally encoded peptide must be modified and cleaved to yield heterocyclized, macrocyclized patellamides. In particular, the patellamides must be cut out of the precursor peptide from both their C- and N-termini, a property not yet observed in any other cyclic... 

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