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An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Department of Pharmaceutical ChemistrySchool of Pharmacy, Addis Ababa UniversityAddis AbabaEthiopia.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
2010 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 16, no 3, 167-176 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, the cleavage of the protected peptide and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies-the Mobius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a "one pot" reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II.

Place, publisher, year, edition, pages
2010. Vol. 16, no 3, 167-176 p.
Keyword [en]
Cyclotides, Microwave chemistry, Fmoc-SPPS, Circular proteins, Cystine knot, Native chemical ligation
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-134899DOI: 10.1007/s10989-010-9221-0ISI: 000281682600007OAI: oai:DiVA.org:uu-134899DiVA: diva2:374063
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2017-05-11Bibliographically approved
In thesis
1. Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
Open this publication in new window or tab >>Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations.

Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used.

Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 139
Keyword
cyclotides, cystine knot, protein engineering, NMR, solid phase, disulfide rich, ultra-stable, microprotein
National Category
Pharmaceutical Sciences
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-145749 (URN)978-91-554-8003-5 (ISBN)
Public defence
2011-03-25, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-03-04 Created: 2011-02-10 Last updated: 2011-05-04
2. Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity
Open this publication in new window or tab >>Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects.

The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels.

A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides’ biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds.

Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 218
Keyword
cyclotide, cyclic cystine knot, evolution, peptide synthesis, chemical ligation, QSAR, Viola, Violaceae, phylogeny
National Category
Mathematics Organic Chemistry Physical Chemistry Evolutionary Biology
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-292668 (URN)978-91-554-9604-3 (ISBN)
Public defence
2016-06-10, B/C4:301, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-24 Created: 2016-05-06 Last updated: 2016-06-15

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Gunasekera, SunithiAboye, Teshome LetaGöransson, Ulf

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