!"# $ % %&#' (!!) *+*, (!&) -.++/.00+ 1 22 211234/+-/ 77 To my mother and father List of papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Aboye, T. L., Johan, K. R., Gunasekera, S., Bruhn, J. B., El- Seedi, H., Göransson, U. (2011) Discovery, synthesis, and structural determination of a toxin-like disulfide-rich peptide from the cactus Trichocereus pachanoi. Manuscript. II Aboye, T. L., Clark, R. J., Craik, D. J., Göransson, U. (2008) Ultra-stable peptide scaffolds for protein engineering: Synthsis and folding of the circular cystine knotted cyclotide cycloviola- cin O2. ChemBioChem, 9(1): 103–113 III Park, S., Gunasekera, S., Aboye, T. L., Göransson, U. (2010) An efficient approach for the total synthesis of cyclotides by microwave assisted Fmoc-SPPS. International Journal of Pep- tide Research and Therapeutics, 16(3): 167-176 IV Aboye, T. L., Clark, R. J., Burman, R., Roig, M. B., Craik, D. J., Göransson, U. (2011) Interlocking disulfides in circular pro- teins. Toward efficient oxidative folding of cyclotides. Antioxi- dants & Redox Signaling, 14(1): 77-86 V Aboye, T. L., Burman, R., Göransson, U. Design, synthesis, structural and biological evaluation of backbone engineered cy- clotides. Manuscript Reprints were made with permission from the respective publishers. Contents 1 Introduction ............................................................................................ 13 1.1 Serendipitous discovery of knotted microproteins ......................... 13 1.2 Structure of cystine knotted microproteins ..................................... 14 1.3 Stability and biological activities .................................................... 19 2 A hybrid of solid and solution phase synthesis of microproteins .......... 20 2.1 Linear chain assembly .................................................................... 20 2.2 Native chemical ligation/cyclization .............................................. 23 2.3 Synthesis of activated precursors for NCL/cyclization .................. 24 2.4 Oxidative folding: locking in the native state ................................. 28 2.5 Chemical and biomimetic engineering ........................................... 28 2.6 Biomolecular engineering ............................................................... 29 3 Aims of the present study ....................................................................... 31 4 Synthesis and structural determination of a novel disulfide-rich peptide (Paper I) .................................................................................................. 32 4.1 Discovery of an acyclic disulfide-rich peptide ............................... 32 4.2 Solid phase synthesis and oxidative folding ................................... 33 4.3 Solution NMR structural determination ......................................... 35 5 Fmoc-based synthesis and oxidative folding of bracelet, cycloviolacin O2 (Papers II and III) ................................................................................... 38 5.1 The bracelet cyclotides ................................................................... 38 5.2 Synthesis of linear chain and protected peptides ............................ 38 5.3 Peptide cyclization using peptidyl α-thioester ............................... 39 5.4 Optimization of oxidative folding conditions ................................. 40 5.5 NMR structural studies and disulfide bond determination ............. 43 5.6 Conclusion ...................................................................................... 44 6 Folding pathways to lock in the native disulfide bonds (Paper IV) ...... 45 6.1 Oxidative folding of cyclic cystine knot microproteins ................. 45 6.2 Quantification of heterogeneous folding intermediates .................. 45 6.3 The diversity of cyclotide folding pathways .................................. 48 7 From native to engineered macrocyclic cystine knotted peptides (Paper V) ................................................................................................. 50 7.1 Backbone engineering of the cyclotide scaffold ............................. 50 7.2 Rational design strategy using backbone spacers ........................... 51 7.3 Synthesis, biological and structural evaluation of mer-cyclotides . 52 7.4 Conclusion ...................................................................................... 58 8 Concluding Remarks .............................................................................. 59 9 Summary of popular Science ................................................................. 61 10 Acknowledgments ................................................................................ 63 11 References ............................................................................................ 66 Abbreviations MeCN acetonitrile Boc tert-butyloxycarbonyl CCK cyclic cystine knot CKM cystine knot microprotein COSY correlated spectroscopy CyO2 cycloviolacin O2 Dbz 3,4-diaminobenzoic acid DCM dichloromethane DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethylsulfoxide DQF-COSY double quantum filtered correlated spectroscopy DTT dithiothreitol E-COSY exclusive correlation spectroscopy ESI-MS electrospray mass spectrometry Fmoc 9-fluorenylmethoxycarbonyl GFK growth factor cystine knot GSH reduced glutatione GSSG oxidized glutatione HATU (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) HBTU O-(1-benzotriazolyl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HF hydrogen fluoride HOBt N-hydroxybenzotriazole ICK inhibitor cystine knot KB1 kalata B1 MBHA methylbenzhydrylamine MeOH methanol MPAA 4-mercaptophenylacetic acid Nbz N-acylbenzimidazolone NCL native chemical ligation NEM N-ethylmaleimide NMR nuclear magnetic resonance NOESY nuclear Overhauser effect spectroscopy Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PyBOP benzotriazole-1-yl-oxy- trispyrrolidinophosphoniumhexafluorophosphate R fully reduced peptide RP-HPLC reversed phase high performance liquid chromatography SPPS solid phase peptide synthesis t-Bu tert-butyl TCEP tris(2-carboxyethyl)phosphine TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra- fluoroborate) TFA trifluoroacetic acid TFE 2,2,2-trifluoroethanol TIPS triisopropylsilane TOCSY total correlation spectroscopy TRIS tris(hydroxymethyl)aminomethane Trt trityl 2D, 3D two, three-dimensional 1SS, 2SS, 3SS isomers containing One-, two-, or three- disulfide bond Amino acids residues and polymeric building blocks 1 Introduction 1.1 Serendipitous discovery of knotted microproteins Peptides regulate most physiological processes, acting as endocrine signals, neurotransmitters or growth factors. They are used therapeutically in diverse areas including neurology, endocrinology and hematology (Vlieghe, 2010; Edwards, 1999). Due to their advantageous features of high specificity and low toxicity, peptides are considered drugs of the future. In recent years, naturally occurring cystine-knotted peptides attracted great interest (Sommerhoff, 2010; Gunasekera, 2008b) because of their higher stability, manifested by high resistance to protease degradation, and rigidity (Gunasekera, 2008b; Colgrave and Craik, 2004). Most potential pep- tide drug leads are composed of amino acids in linear chains with open ends that are targets for proteolytic enzymes, leading to short plasma half-life times. This feature, coupled with their poor absorption due to their generally hydrophilic nature, results in poor oral bioavailability, making them unsuita- ble for oral drug therapies (Vagner, 2008; Werle and Bernkop-Schnürch, 2006). Cyclization has been used to reduce susceptibility to degradation of peptide drugs (Clark, 2010) and when combined with oxidative folding, it has been found to stabilize peptides in harsh environments by forming rigid, compact molecules (Clark, 2011; Werle and Bernkop-Schnürch, 2006; Colgrave and Craik, 2004). For example, cyclic cystine-knotted peptides show greater stability than their solely cystine-knotted counterparts (Colgrave and Craik, 2004; Daly and Craik, 2000). The exceptional properties of such peptides first came to notice through the serendipitous discovery of a bioactive agent in indigenous medicine used in the Congo (Zair) region of Africa (Gran, 1973b). During a Red Cross relief mission in Zaire in the 1960s, the Norwegian physician noted that dur- ing labor, women sipped a decoction made by boiling leaves of the plant Oldenlandia affinis, to accelerate uterine contractions and facilitate child- birth. In the 1970s, the active ingredient was determined to be a peptide, named kalata B1 after the traditional name for the native medicine, 'kalata- kalata' (Gran, 1973c; a). The stability of the peptide during boiling and oral ingestion (and thus resistance to high temperature and gastrointestinal tract enzymes) indicated that it has oral bioavailability, which is unusual