Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber: Spider Dragline Silk...

Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber:

Spider Dragline Silk

Maren E. Buck

Lynn Group

5/3/2007

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

3Vollrath, 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 eggCase fiber (Tubuliform)

Aggregate TubuliformFlagelliform

Pyriform

Minor ampullate

Major ampullate

4

The classic strong synthetic fiber

Material Strength (GPa) Elasticity (%) Energy to break (J/kg)

Dragline Silk 1.1 35 4 x 105

Kevlar 3.6 5 3 x 104

Rubber 0.001 600 8 x 104

Nylon, type 6 0.07 200 6 x 104

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

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

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. JOM 2005, 57.

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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

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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.

QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGAGQGGRGAGQS

SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGQGGRGAGQNSQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQSSQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQSSQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG

GLGGYGRQGAGGAAAAAAGAGQGGRGAGQSNQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAGQGGRGAGQNSQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG

GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQSSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG

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

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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

N-terminus

C-terminus

C-terminus

N-terminus

N-terminus

C-terminus

C-terminus

N C

NC

N C

N C

10

Solid state 13C-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.

13C-NMR chemical shifts (ppm)

13C-labeledAlanine

Wavenumber (cm-1)

1700 1600 15000.1550

0.2800

0.4050

Ab

sorb

an

ce

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-helixAla C

α-helix

Ala C

Ala CC=O

-sheet

20.1 15.1

48.7 52.5

171.9 176.5

Infrared wavelengths (cm-1)

1630, 1685

1630, 1645

1650, 1560

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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

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Synthetic approaches to spider dragline silk

Biosynthesis Chemical Synthesis

Protein sequences

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 withhost

Protein fibers

Reverse transcription

Eukaryotic host (insect cells)

Spider silk protein sequences/mRNA

Gene design

Nephila clavipes

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Expression of spider silk cDNA in mammalian cells

Lazaris, A. et al. Science 2002, 295, 472-476.

Dragline silk gene sequencefrom A. diadematus

Gene sequence inserted intoexpression 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 sample Toughness(MJ/m3)

Modulus(GPa)

Elasticity(%)

Strength(GPa)

ADF-3

A. diadematus dragline

85 13 43.4 0.26

130 10 30 1.1

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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--------------------------------------------AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGGAGQG-------GLGSQGA---------- GQGAGAAAAAA----GGAGQGGYGGLGSQGAGRG-----GQGAGAAAAAA---GG

Spidroin 1 analog: DP-1B[

]n=8-16

Ligate 8 or 16DNA fragments

DNA duplex

Hybridize complementary

strands

Premature termination with expression in E. coli

High MW polymers from yeast

Transform inEscherichia coli

Insert gene into plasmid vector

Or transform inyeast

Protein fibers1 g/L

Protein fibers300 mg/L

170 nm diameter fibers

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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

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Synthetic approaches to spider dragline silk

Biosynthesis Chemical Synthesis

Protein sequences

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Chemical approaches to synthesizing silk-like polymers

Poly(Ala) blocks - PEG linker - Alkyl linkers

Protein structure and properties

Non-peptidepolymers

Living polymerization of peptide monomers

Lego approach(-sheet template) - Rigid or short linkers - Long, flexible linkers

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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 +

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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.

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Spectroscopic evidence for the requiredphenoxathiin template

3 4

Flexible linear peptide

Peptides with phenoxathiin template


1 2

3424 cm-1

3336 cm-1

3a

3b

4

3407 cm-1

3342 cm-13415 cm-1

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Polymerization of the building blocks


Interfacial Polymerization

Solution Polymerization

Monomer A

Monomer B

Copolymer AB

“Nylon Rope Trick”

22

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Polymerization results

% Yield – Interfacial:

% Yield – Solution:

Mn (Solution) (g/mol):

PDI:

576019,1002.08

505620,6001.79

463917,4001.54

826720,2001.79

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

P1 P2 P3 P4

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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 1Peptide 2

Polymer 2

1645 cm-1

1645 cm-1

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Phenoxathiin template with ethylene glycol linkers

57% yieldMn = 22,400PDI=1.72

60% yieldMn = 14,000PDI = 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.

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13C-NMR spectra suggest -sheet structure

Interfacial polymerization

SolutionpolymerizationTotal -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.

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Changes in interfacial polymer after annealing above Tg

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-11635 cm-11683 cm-1

Raw

2nd derivative

1647 cm-1

1633 cm-1

1683 cm-1

Initial Post Annealing

• Polymerization procedure affects structure

• Heating above Tg enhances -sheet content in interfacial polymer

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Non-peptidepolymers




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Non-templated polymeric dragline silk mimics

Generic polymer structurepeptide

[

]soft linker

x ~ 4 or 6n ~ 13

QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGAGQGGRGAGQS

SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGQGGRGAGQNSQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQSSQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQSSQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG

GLGGYGRQGAGGAAAAAAGAGQGGRGAGQSNQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAGQGGRGAGQNSQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG

GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQSSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG

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.

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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% yieldP2: x~6, n~13; 69% yield

+

31

Polymer

P1 410±35

750±156

Modulus(MPa)

Tensile strength(MPa)

Elongationat break (%)

13.0±1.4

14.2±2.7

22.9±13.6

5.4±1.7P2

Silk (N. clavipes)

Mechanical properties:

22,000 1,100 34

Mechanical properties of the polymer fibers

FT-IR and 13C-NMR indicate formation of anti-parallel -sheets

P1: x~4, n~13P2: x~6, n~13

Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.

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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

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Multiblock copolymers with poly(isoprene) as the “soft” linker

Zhou, C. et al. Biomacromolecules 2006, 7, 2415-2419.

n = 31 (Mn=2200)n = 72 (Mn=5000)

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13C-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)

Ab

so

rba

nc

e

1655

1643 1630

176171

52

48

18

P2

P1: n = 31P2: n = 72

35




Non-peptidepolymers




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Atom transfer radical polymerization (ATRP) of silk-like triblock copolymers

Mn (GPC): 11.5 kDaPDI: 1.29

Mn (GPC): 4.6 kDaPDI: 1.17

Ayres, L. et al. Biomacromolecules 2005, 6, 825-831.

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Non-peptidepolymers



Non-peptidepolymers

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Silk-like polymers without peptide motifs

Soft segment Hard 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%

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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/m3)

77.4

31.6

34 1,100 22,000Spider dragline silk 160

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 segment Hard segment

P1 = 26% P3 = 43%P2 = 33% P4 = 47%

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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

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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

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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

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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’ milkExtrusion through “spinnerets” produces fibersAqueous spinning process is environmentally friendly

- Anticipated uses:

Surgical suturesAdhesivesFishing lineBody armor/military applications

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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

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Acknowledgments

Professor David Lynn

Lynn Group Members:Jingtao ZhangXianghui LiuChris JewellNat FredinBin SunMike KinsingerEric SaurerRyan FlessnerShane Bechler

Practice talk attendees:Lauren BoyleClaire PoppeJulee ByramBecca SplainAlex ClemensKatherine TraynorRichard GrantMatt WindsorMargie Mattmann

Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber: Spider Dragline Silk...

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