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  1. Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber: Spider Dragline Silk Maren E. Buck Lynn Group 5/3/2007

  2. Outline • Introduction to spider silks and silk structure • Biosynthetic methods to produce silk protein analogs • Chemical methods to synthesize silk-like polymers • Applications • Conclusions

  3. Large diameter egg Case fiber (Tubuliform) Tubuliform Aggregate Flagelliform Minor ampullate Capture Spiral (Flagelliform) Pyriform Glue coating (Aggregate silk) (?) Acini- form Major ampullate Wrapping and egg case fiber (aciniform) Web reinforcement (Minor ampullate 1 and 2) Pyriform silk (?) Dragline (major ampullate 1 and 2) Spiders spin 6 different fibers Vollrath, F. J. Biotechnol. 2000, 74, 67-83. Hu, X. et al. Cell. Mol. Life Sci.2006, 63, 1986-1999.

  4. The classic strong synthetic fiber Kevlar®: Dupont (1960s) Uses - Bulletproof vests and helmets - Automobile brake pads - Ropes and cables - Aerospace components Fiber axis Strength (GPa ) Energy to break (J/kg ) Material Elasticity (%) 5 Dragline Silk 35 4 x 10 1.1 4 Kevlar 5 3 x 10 3.6 4 Rubber 600 8 x 10 0.001 4 Nylon, type 6 200 6 x 10 0.07 Lewis, R. Chem. Rev.2006, 106, 3762-3774. Vollrath, F.; Knight, D.P. Nature2001, 410, 541-548. Tanner, D.; Fitzgerald, J.A.; Phillips, B.R. Angew. Chem. Int. Ed. Engl. Adv. Mater.1989, 5, 649-654. Kubik, S. Angew. Chem. Int. Ed.2002, 41, 2721-2723.

  5. Spider silks have potential in many applications Biomedical applications Surgical sutures Scaffolds for tissue engineering Technical and industrial applications High strength ropes/cables Ballistics Parachutes Fishing line

  6. Forced silking to obtain silk fibers Spiders are anesthetized with CO2 and secured ventral side up Silk is pulled from the spinneret, attached to a reel, and drawn at a specified speed Work, R. W.; Emerson, P. D. J. Arachnol. 1982, 10, 1-10. Elices, M.; Perez-Rigueiro, J.; Plaza, G. R.; Guinea, G. V. JOM2005, 57.

  7. Spiders are highly developed fiber “spinners” Lumen Duct Spidroin secretion Fiber alignment Spinneret Tail Funnel Duct 1 mm Lewis, R. Chem. Rev.2006, 106, 3762-3774. Dicko, C.; Vollrath, F.; Kenney, J.M. Biomacromolecules 2004, 5, 704-710.

  8. QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGQGGRGAGQN SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAGQGGRGAGQN SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG SSLRSAAAAASAASAGS Primary structure of spider dragline silk • Fibrous protein composed of Spidroin 1 (MaSp1) and Spidroin 2 (MaSp2) • - Sequences highly conserved • - Repetitive stretches of poly(Ala) and (GlyGlyXaa)n sequences • (Xaa = Tyr, Leu, Gln) • - MW of MaSp1 ~ 275-320 kDa; Sp1+Sp2 ~ 700-750 kDa Repeating sequence of MaSp1 Hinman, M.B.; Jones, J. A.; Lewis, R. TIBTECH2000, 18, 374-379. Vollrath, F.; Knight, D. P. Nature2001, 410, 541-548. Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science1996, 271, 84-87.

  9. Antiparallel and parallel -sheet structure C N N-terminus C-terminus N-terminus C-terminus C N C N C-terminus N-terminus Poly(alanine) segment N-terminus C-terminus C N Rotondi, K. S.; Gierasch, L. M. Biopolymers2005, 84, 13-22. Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237.

  10. Solid state 13C-NMR and FT-IR spectroscopy 1637 0.4050 1612 1666 1691 Absorbance 0.2800 0.1550 1700 1600 1500 Wavenumber (cm-1) 13C-NMR chemical shifts (ppm) Infrared wavelengths (cm-1) -sheet α-helix Anti-parallel β-sheet 1630, 1685 Ala C 48.7 52.5 Parallel β-sheet 1630, 1645 Ala C 20.1 15.1 α-helix 1650, 1560 Ala CC=O 171.9 176.5 -carbon Infrared spectrum of silk from Nephila clavipes -carbon 13C-labeled Alanine Amide I (antiparallel -sheet) Marcotte, I.; van Beek, J. D.; Meier, B. H. Macromolecules2007, 40, 1995-2001. Simmons, A.; Ray, E.; Jelinski, L.W. Macromolecules1994, 27, 5235-5237. Dong, Z.; Lewis, R.; Middaugh, C. R. Arch. Biochem. Biophys.1991, 1, 53-57.

  11. Proposed secondary structure and mode of elasticity • Poly(Ala) modules form anti-parallel β-sheets (~30-40%) • Glycine-rich, amorphous regions are thought to be helical Crystalline region with -sheet structure Strain Disordered chain region Kubik, S. Angew. Chem. Int. Ed.2002, 41, 2721-2723. Van Beek, J. D.; Hess, S.; Vollrath, F. Meier, B. H. Proc. Nat. Acad. Sci. 2002, 99, 10266-10271.

  12. Synthetic approaches to spider dragline silk Protein sequences Biosynthesis Chemical Synthesis

  13. Two biosynthetic routes to spidroin proteins Eukaryotic host (insect cells) Nephila clavipes Reverse transcription Spider cDNA Spider silk protein sequences/mRNA Protein fibers Gene design Synthetic DNA Flexibility with host Vendrely, C.; Scheibel, T. Macromol. Biosci.2007, 7, 401-409. Altman, G.H. et al.Biomaterials2003, 24, 401-416.

  14. protein synthesis Protein: MW ~ 60-140 kDa Fiber diameter ~ 40 μm Yield ~ 37 mg/L Expression of spider silk cDNA in mammalian cells Transformation of vector in mammalian cells Protein purification, and characterization Dragline silk gene sequence from A. diadematus Gene sequence inserted into expression vector Mechanical Properties: Protein sample Toughness (MJ/m3) Modulus (GPa) Elasticity (%) Strength (GPa) 85 13 43.4 0.26 ADF-3 A. diadematus dragline 130 10 30 1.1 Lazaris, A. et al.Science2002, 295, 472-476.

  15. Recombinant expression of synthetic silk genes Ligate 8 or 16 DNA fragments Spidroin 1 analog: DP-1B [ AGQGGYGGLGSQG-------------------------------------------- AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGG AGQG-------GLGSQGA---------- GQGAGAAAAAA----GG AGQGGYGGLGSQGAGRG-----GQGAGAAAAAA---GG DNA fragment ] n=8-16 Hybridize complementary strands Transform in Escherichia coli Protein fibers 300 mg/L Insert gene into plasmid vector Protein fibers 1 g/L DNA duplex Or transform in yeast 170 nm diameter fibers Premature termination with expression in E. coli High MW polymers from yeast Fahnestock, S. R.; Irwin, S. L. Appl. Microbiol. Biotechnol.1997, 47, 23-32. Stephens, S.J. et al. Mat. Res. Soc. Symp. Proc.2003, 774, 2.3.1-2.3.10. Fahnestock, S. R.; Bedzyk, L. A. Appl. Microbiol.Biotechnol.1997, 47, 33-39. O’Brien, J. P.; Fahnestock, S. R.; Termonia, Y.; Gardner, K. H. Adv. Mater.1998, 10, 1185-1195.

  16. Summary of biosynthetic pathways Biosynthetic Method Advantages Disadvantages Difficulty with protein purification (aggregation) Spider Silk cDNA Produce proteins most like native silk High MW polymers are readily attainable Eukaryotic hosts are expensive Synthetic DNA Polymer structure can be tuned based on DNA sequence used Truncated syntheses in many hosts Flexibility with expression host

  17. Synthetic approaches to spider dragline silk Protein sequences Biosynthesis Chemical Synthesis

  18. Chemical approaches to synthesizing silk-like polymers Poly(Ala) blocks - PEG linker - Alkyl linkers Lego approach (-sheet template) - Rigid or short linkers - Long, flexible linkers Protein structure and properties Living polymerization of peptide monomers Non-peptide polymers

  19. Synthesis of silk-like polymers: “Lego” approach Linkers -sheet nucleation center Peptide sequence (GAGA) + A + B Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

  20. Synthesis of the building blocks Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876. Wagner, G.; Feigel, M. Tetrahedron1993, 49, 10831-10842.

  21. Spectroscopic evidence for the required phenoxathiin template 3424 cm-1 3336 cm-1 3407 cm-1 3415 cm-1 3a 3342 cm-1 3b 4 Flexible linear peptide 1 2 Peptides with phenoxathiin template 3 4 Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

  22. Polymerization of the building blocks Interfacial Polymerization “Nylon Rope Trick” Monomer A Copolymer AB Monomer B Solution Polymerization 22 Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

  23. Polymerization results P1 P2 P3 P4 57 50 46 82 % Yield – Interfacial: 60 56 39 67 % Yield – Solution: 19,100 20,600 17,400 20,200 Mn (Solution) (g/mol): 2.08 1.79 1.54 1.79 PDI: Mn = average molecular weight of sample PDI = distribution of molecular weights in a sample Spider silk: Mn = ~ 605,000 g/mol (Sp1+Sp2) PDI = 1.05 Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

  24. FT-IR characterization of the polymer structure 1: 2: 1645 cm-1 Polymer 1 or 2 Polymer 2 Polymer 1 1645 cm-1 Peptide 1 Peptide 2 Peptide 1 or 2 Winningham, M.J.; Sogah, D.Y. Macromolecules. 1997, 30, 862-876.

  25. Phenoxathiin template with ethylene glycol linkers Interfacial: 57% yield Mn = 22,400 PDI=1.72 Solution: 60% yield Mn = 14,000 PDI = 2.4 Rathore, O.; Winningham, M. J.; Sogah, D.Y. J. Polym. Sci: Part A, Polym Chem.2000, 38, 352-366. Dattagupta, N.; U.S. Patent 4,968,602; 1990.

  26. 13C-NMR spectra suggest -sheet structure Interfacial polymerization Solution polymerization Total -sheet content: - Interfacial polymerization: 40% - Solution polymerization: 80% Spider silk -sheet content: 30-40% Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym Chem.2000, 38, 352-366.

  27. Changes in interfacial polymer after annealing above Tg • Polymerization procedure affects structure • Heating above Tg enhances -sheet content • in interfacial polymer Solution polymerization Initial Post Annealing 1647 cm-1 Raw 1647 cm-1 Raw 1683 cm-1 2nd derivative 2nd derivative 1683 cm-1 1635 cm-1 1633 cm-1 1628 cm-1 Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym. Chem.2000, 38, 352-366.

  28. Poly(Ala) blocks - PEG linker - Alkyl linkers Poly(Ala) blocks - PEG linker - Alkyl linkers Lego approach (-sheet template) - Rigid or short linkers - Long, flexible linkers Protein structure and properties Living polymerization of peptide monomers Non-peptide polymers Chemical approaches to synthesizing silk-like polymers

  29. QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGQGGRGAGQN SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAGQGGRGAGQN SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG SSLRSAAAAASAASAGS Non-templated polymeric dragline silk mimics [ ] soft linker peptide Generic polymer structure x ~ 4 or 6 n ~ 13 Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science1996, 271, 84-87. Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

  30. Synthesis of triblock copolymers with poly(Ala) Water-soluble fraction (46%) + Water-insoluble fraction (54%) P1: x~4, n~13; 75% yield P2: x~6, n~13; 69% yield Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

  31. Mechanical properties: Modulus (MPa) Tensile strength (MPa) Elongation at break (%) Polymer 22.9±13.6 P1 410±35 13.0±1.4 750±156 14.2±2.7 P2 5.4±1.7 Silk (N. clavipes) 22,000 1,100 34 Mechanical properties of the polymer fibers P1: x~4, n~13 P2: x~6, n~13 FT-IR and 13C-NMR indicate formation of anti-parallel -sheets Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

  32. Synthesis of silk-like multiblock copolymers containing flexible alkyl linkers Yield: 70% MW (viscosity): 44,900 3 cycles Yao, J. et al. Macromolecules2003, 36, 7508-7512.

  33. Multiblock copolymers with poly(isoprene) as the “soft” linker n = 31 (Mn=2200) n = 72 (Mn=5000) Zhou, C. et al. Biomacromolecules2006, 7, 2415-2419.

  34. P1: n = 31 P2: n = 72 1630 1643 P1 18 P2 1655 48 171 176 Absorbance 52 P1 P2 P1 Chemical shift (ppm) Wavenumber (cm-1) Zhou, C. et al. Biomacromolecules2006, 7, 2415-2419. 13C-NMR and FT-IR characterization of the polymers

  35. Poly(Ala) blocks - PEG linker - Alkyl linkers Lego approach (-sheet template) - Rigid or short linkers - Long, flexible linkers Protein structure and properties Living polymerization of peptide monomers Living polymerization of peptide monomers Non-peptide polymers Chemical approaches to synthesizing silk-like polymers

  36. Atom transfer radical polymerization (ATRP) of silk-like triblock copolymers Mn (GPC): 4.6 kDa Mn (GPC): 11.5 kDa PDI: 1.17 PDI: 1.29 Ayres, L. et al. Biomacromolecules2005, 6, 825-831.

  37. Poly(Ala) blocks - PEG linker - Alkyl linkers Lego approach (-sheet template) - Rigid or short linkers - Long, flexible linkers Protein structure and properties Living polymerization of peptide monomers Non-peptide polymers Non-peptide polymers Chemical approaches to synthesizing silk-like polymers

  38. Silk-like polymers without peptide motifs Endcapped macrodiol Macrodiol % Hard segment: P1 = 26% P3 = 43% P2 = 33% P4 = 47% Soft segment Hard segment James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer2006, 47, 3073-3082.

  39. Modulus (MPa) Toughness (MJ/m3) Elongation at break (%) Tensile strength (MPa) Polymer 587 14.9 72.5 65.1 P1 18.1 P2 460 200 59.2 23.6 P3 447 156 77.4 18.2 P4 202 198 31.6 1,100 Spider dragline silk 34 22,000 160 Mechanical properties of poly(urethane) polymers Soft segment Hard segment P1 = 26% P3 = 43% P2 = 33% P4 = 47% James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer2006, 47, 3073-3082. Cuniff, P.M. et al. Polym. Adv. Tech.2003, 5, 401-410.

  40. Summary of chemical synthetic pathways Lego approach (-sheet template) - Forms -sheets - Brittle, non-fibrous Poly(Ala) blocks - Forms -sheets - Produces fibers; not as strong as native silk Non-peptide polymers - Self-assembles into fibers - High elasticity, low strength Living polymerization of peptide monomers - Forms -sheets - Control over MW of peptide blocks - Low PDI

  41. Applications for spider dragline silk: Tissue Engineering Artificial nerve grafts: - Nerve cells attach and grow on spider silk fibers - Nerve construct composed of pig venules, filled with cells seeded on silk fibers Light micrograph of artificial nerve construct Artificial ligaments: - Silks promote proliferation of bone marrow cells - High tensile strengths could restore knee function immediately Allmeling, C.; Jokuszies, A.; Reimers, K.; Kall, S.; Vogt, P. M. J. Cell. Mol. Med.2006, 10. 1-8. Altman, G. H. et al. Biomaterials 2003, 24, 401-416.

  42. Spider silks have potential in many applications Biomedical applications Surgical sutures Scaffolds for tissue engineering Technical and industrial applications High strength ropes/cables Ballistics Parachutes Fishing line

  43. BioSteel® - Genetically modified goats produce silk in mammary glands • Silk is spun from the goats’ milk • Extrusion through “spinnerets” produces fibers • Aqueous spinning process is environmentally friendly • - Anticipated uses: • Surgical sutures • Adhesives • Fishing line • Body armor/military applications Lazaris, A. et al.Science2002, 295, 472-476. Karatzas, C. N.; Turcotte, C. 2003, PCT Int. Appl. WO03057727. Karatzas, C. 2001, PCT Int. Appl. WO0156626. Islam, S. et al. 2004, U.S. Pat. 20040102614.

  44. Conclusions - Spiders can spin fibers with exceptional strength, elasticity, and toughness - Biosynthetic methods have generated fibers with structure and properties approaching those of native silks - Chemists can use spider silk as a model to design new fibers and materials with silk-like properties - Silk-spinning processes must be optimized in order for commercialization to occur

  45. Acknowledgments Professor David Lynn Practice talk attendees: Lauren Boyle Claire Poppe Julee Byram Becca Splain Alex Clemens Katherine Traynor Richard Grant Matt Windsor Margie Mattmann Lynn Group Members: Jingtao Zhang Xianghui Liu Chris Jewell Nat Fredin Bin Sun Mike Kinsinger Eric Saurer Ryan Flessner Shane Bechler