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

2. 2 Outline I.Introduction to spider silks and silk structure II.Biosynthetic methods to produce silk protein analogs III.Chemical methods to synthesize silk-like polymers IV.Applications V.Conclusions

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

4. 4 The classic strong synthetic fiber Material Strength (GPa) Elasticity (%) Energy to break (J/kg) Dragline Silk 1.1 354 x 10 5 Kevlar 3.6 53 x 10 4 Rubber 0.001 6008 x 10 4 Nylon, type 6 0.07 2006 x 10 4 Fiber axis Kevlar®: Dupont (1960s) Uses - Bulletproof vests and helmets - Automobile brake pads - Ropes and cables - Aerospace components Lewis, R. Chem. Rev. 2006, 106, 3762-3774. Vollrath, F.; Knight, D.P. Nature 2001, 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. 5 Spider silks have potential in many applications Surgical sutures Scaffolds for tissue engineering Biomedical applications Parachutes High strength ropes/cables Fishing line Technical and industrial applications Ballistics

6. 6 Forced silking to obtain silk fibers Spiders are anesthetized with CO 2 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. JOM 2005, 57.

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

8. 8 Primary structure of spider dragline silk Hinman, M.B.; Jones, J. A.; Lewis, R. TIBTECH 2000, 18, 374-379. Vollrath, F.; Knight, D. P. Nature 2001, 410, 541-548. Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87. QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGQGGRGAGQN SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAGQGGRGAGQN SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG SSLRSAAAAASAASAGS 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

9. 9 Antiparallel and parallel  -sheet structure Poly(alanine) segment Rotondi, K. S.; Gierasch, L. M. Biopolymers 2005, 84, 13-22. Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237. N-terminus C-terminus N-terminus C-terminus N C N C N C N C

10. 10 Solid state 13 C-NMR and FT-IR spectroscopy Marcotte, I.; van Beek, J. D.; Meier, B. H. Macromolecules 2007, 40, 1995-2001. Simmons, A.; Ray, E.; Jelinski, L.W. Macromolecules 1994, 27, 5235-5237. Dong, Z.; Lewis, R.; Middaugh, C. R. Arch. Biochem. Biophys. 1991, 1, 53-57. 13 C-NMR chemical shifts (ppm) 13 C-labeled Alanine Wavenumber (cm -1 ) 170016001500 0.1550 0.2800 0.4050 Absorbance 1691 1666 1637 1612 Infrared spectrum of silk from Nephila clavipes Amide I (antiparallel  -sheet)  -carbon  -carbon Anti-parallel β-sheet Parallel β-sheet α-helix Ala C  α-helix Ala C  Ala C C=O  -sheet 20.115.1 48.752.5 171.9176.5 Infrared wavelengths (cm -1 ) 1630, 1685 1630, 1645 1650, 1560

11. 11 Proposed secondary structure and mode of elasticity 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. Poly(Ala) modules form anti-parallel β-sheets (~30-40%) Glycine-rich, amorphous regions are thought to be helical Disordered chain region Strain Crystalline region with  -sheet structure

12. 12 Synthetic approaches to spider dragline silk BiosynthesisChemical Synthesis Protein sequences

13. 13 Two biosynthetic routes to spidroin proteins Vendrely, C.; Scheibel, T. Macromol. Biosci. 2007, 7, 401-409. Altman, G.H. et al. Biomaterials 2003, 24, 401-416. Synthetic DNA Spider cDNA Flexibility with host Protein fibers Reverse transcription Eukaryotic host (insect cells) Spider silk protein sequences/mRNA Gene design Nephila clavipes

14. 14 Expression of spider silk cDNA in mammalian cells Lazaris, A. et al. Science 2002, 295, 472-476. Dragline silk gene sequence from A. diadematus Gene sequence inserted into expression vector Transformation of vector in mammalian cells protein synthesis Protein purification, and characterization Protein: MW ~ 60-140 kDa Fiber diameter ~ 40 μm Yield ~ 37 mg/L Mechanical Properties: Protein sampleToughness (MJ/m 3 ) Modulus (GPa) Elasticity (%) Strength (GPa) ADF-3 A. diadematus dragline 851343.40.26 13010301.1

15. 15 Recombinant expression of synthetic silk genes DNA fragment 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. AGQGGYGGLGSQG-------------------------------------------- AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGG AGQG-------GLGSQGA---------- GQGAGAAAAAA----GG AGQGGYGGLGSQGAGRG-----GQGAGAAAAAA---GG Spidroin 1 analog: DP-1B [ ] n=8-16 Ligate 8 or 16 DNA fragments DNA duplex Hybridize complementary strands Premature termination with expression in E. coli High MW polymers from yeast Transform in Escherichia coli Insert gene into plasmid vector Or transform in yeast Protein fibers 1 g/L Protein fibers 300 mg/L 170 nm diameter fibers

16. 16 Summary of biosynthetic pathways Biosynthetic Method Advantages Disadvantages Spider Silk cDNA Difficulty with protein purification (aggregation) 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 Flexibility with expression host Truncated syntheses in many hosts

17. 17 Synthetic approaches to spider dragline silk BiosynthesisChemical Synthesis Protein sequences

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

19. 19 Synthesis of silk-like polymers: “Lego” approach Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. Linkers  -sheet nucleation center Peptide sequence (GAGA) + A B +

20. 20 Synthesis of the building blocks Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. Wagner, G.; Feigel, M. Tetrahedron 1993, 49, 10831-10842.

21. 21 Spectroscopic evidence for the required phenoxathiin template 34 Flexible linear peptide Peptides with phenoxathiin template Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. 12 3424 cm -1 3336 cm -1 3a 3b 4 3407 cm -1 3342 cm -1 3415 cm -1

22. 22 Polymerization of the building blocks Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. Interfacial Polymerization Solution Polymerization Monomer A Monomer B Copolymer AB “Nylon Rope Trick” 22

23. 23 Polymerization results % Yield – Interfacial: % Yield – Solution: M n (Solution) (g/mol): PDI: 57 60 19,100 2.08 50 56 20,600 1.79 46 39 17,400 1.54 82 67 20,200 1.79 M n = average molecular weight of sample PDI = distribution of molecular weights in a sample Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. Spider silk: M n = ~ 605,000 g/mol (Sp1+Sp2) PDI = 1.05 P1P2P3P4

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

25. 25 Phenoxathiin template with ethylene glycol linkers 57% yield M n = 22,400 PDI=1.72 60% yield M n = 14,000 PDI = 2.4 Interfacial: Solution: 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. 26 13 C-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. 27 Changes in interfacial polymer after annealing above T g Solution polymerization Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym. Chem. 2000, 38, 352-366. 2nd derivative Raw 1647 cm -1 1628 cm -1 1635 cm -1 1683 cm -1 Raw 2nd derivative 1647 cm -1 1633 cm -1 1683 cm -1 InitialPost Annealing Polymerization procedure affects structure Heating above T g enhances  -sheet content in interfacial polymer

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

29. 29 Non-templated polymeric dragline silk mimics Generic polymer structure peptide [ ] soft linker x ~ 4 or 6 n ~ 13 QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGQGGRGAGQN SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG GLGGYGGQGAGGAAAAAGQGGRGAGQN SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG SSLRSAAAAASAASAGS Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87. Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.

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

31. 31 Polymer P1410±35 750±156 Modulus (MPa) Tensile strength (MPa) Elongation at break (%) 13.0±1.4 14.2±2.7 22.9±13.6 5.4±1.7P2 Silk (N. clavipes) Mechanical properties: 22,0001,100 34 Mechanical properties of the polymer fibers FT-IR and 13 C-NMR indicate formation of anti-parallel  -sheets P1: x~4, n~13 P2: x~6, n~13 Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.

32. 32 Synthesis of silk-like multiblock copolymers containing flexible alkyl linkers 3 cycles Yao, J. et al. Macromolecules 2003, 36, 7508-7512. Yield: 70% MW (viscosity): 44,900

33. 33 Multiblock copolymers with poly(isoprene) as the “soft” linker Zhou, C. et al. Biomacromolecules 2006, 7, 2415-2419. n = 31 (M n =2200) n = 72 (M n =5000)

34. 34 13 C-NMR and FT-IR characterization of the polymers Zhou, C. et al. Biomacromolecules 2006, 7, 2415-2419. P1 Chemical shift (ppm) P1 P2 P1 Wavenumber (cm -1 ) Absorbance 1655 1643 1630 176 171 52 48 18 P2 P1: n = 31 P2: n = 72

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

36. 36 Atom transfer radical polymerization (ATRP) of silk-like triblock copolymers M n (GPC): 11.5 kDa PDI: 1.29 M n (GPC): 4.6 kDa PDI: 1.17 Ayres, L. et al. Biomacromolecules 2005, 6, 825-831.

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

38. 38 Silk-like polymers without peptide motifs Soft segmentHard segment Endcapped macrodiol Macrodiol James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer 2006, 47, 3073-3082. % Hard segment: P1 = 26% P3 = 43% P2 = 33% P4 = 47%

39. 39 Mechanical properties of poly(urethane) polymers Polymer P1 Tensile strength (MPa) Elongation at break (%) 587 460 14.9 18.1 72.5 200P2 Modulus (MPa) 447 23.6 156P3 202 18.2 198P4 65.1 59.2 Toughness (MJ/m 3 ) 77.4 31.6 34 1,100 22,000Spider dragline silk160 James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer 2006, 47, 3073-3082. Cuniff, P.M. et al. Polym. Adv. Tech. 2003, 5, 401-410. Soft segmentHard segment P1 = 26% P3 = 43% P2 = 33% P4 = 47%

40. 40 Living polymerization of peptide monomers - Forms  -sheets - Control over MW of peptide blocks - Low PDI 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 Summary of chemical synthetic pathways

41. 41 Applications for spider dragline silk: Tissue Engineering 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. Light micrograph of artificial nerve construct Artificial nerve grafts: Artificial ligaments: - Silks promote proliferation of bone marrow cells - High tensile strengths could restore knee function immediately - Nerve cells attach and grow on spider silk fibers - Nerve construct composed of pig venules, filled with cells seeded on silk fibers

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

43. 43 BioSteel® Lazaris, A. et al. Science 2002, 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. - 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

44. 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. 45 Acknowledgments Professor David Lynn Lynn Group Members: Jingtao Zhang Xianghui Liu Chris Jewell Nat Fredin Bin Sun Mike Kinsinger Eric Saurer Ryan Flessner Shane Bechler Practice talk attendees: Lauren Boyle Claire Poppe Julee Byram Becca Splain Alex Clemens Katherine Traynor Richard Grant Matt Windsor Margie Mattmann