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Conformation of Pyrene Labeled Amylose Studied with Fluorescence Blob Model

Conformation of Pyrene Labeled Amylose Studied with Fluorescence Blob Model. Name: Lu Li Supervisor: Prof. Jean Duhamel. Introduction: Organization of the starch granule. Branched polymer –Amylopectin α -1,4-glucan with 1,6-glycosidic branches containing ~23 anhydroglucose units

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Conformation of Pyrene Labeled Amylose Studied with Fluorescence Blob Model

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  1. Conformation of Pyrene Labeled Amylose Studied with Fluorescence Blob Model Name: Lu Li Supervisor: Prof. Jean Duhamel

  2. Introduction: Organization of the starch granule Branched polymer –Amylopectin α-1,4-glucan with 1,6-glycosidic branches containing ~23 anhydroglucose units ( 2×106 anhydroglucose units) Linear α-1,4-glucan – Amylose (200 to 2000 anhydroglucose units)

  3. Introduction: Organization of the starch granule Branched polymer –Amylopectin α-1,4-glucan with 1,6-glycosidic linked branches containing 20-30 anhydroglucose units Linear α-1,4-glucan – Amylose (200 to 2000 anhydroglucose units)

  4. Amylose conformation In aqueous solution, amylose forms a random coil based on the consistent results obtained by optical rotation, intrinsic viscosity [η], light scattering, and sedimetation measurements However, despite much experimental work done on dilute solutions of amylose, its conformation in DMSO is still a matter of controversy Random coil: Intrinsic viscosity versus weight-average molecular weight (Everett et al.1,Banks et al.2, Nakanishi et al.3) Helix: Intrinsic viscosity versus weight-average molecular weight (Cowie4, Fujii et al.5) NMR (St-Jacques et al6, Norman et al.7, Jane et al.8) Amylose precipitated from DMSO into a nonsolvent adopts a helical conformation based on crystallographic studies9 1. Everett, W.W.; Foster, J. F. J. Am. Chem. Soc.1959, 81, 3464. 2. Banks, W.; Greenwood, C. T. Carbohydr. Res.1968, 7, 414. 3. Nakanishi, Y.; Norisuye, T.; Teramoto, A. Marcomolecules1993, 26, 4220. 4. Cowie, J. M. G. Macromol. Chem. Phys. 1961, 42, 230. 5. Fujii, M.; Honda, K.; Fujita, H. Biopolymers 1973, 12, 1177 6. St-Jacques, M.; Sundararajan, P. R.; Taylor, K.J.; Marchessault, R. H. J. Am. Chem. Soc. 1976, 98, 4386 8. Jane, J. L.; Robyt, J. F. Carbohydr. Res. 1985, 140, 21 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287 4 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287

  5. Amylose conformation In aqueous solution, amylose forms a random coil based on the consistent results obtained by optical rotation, intrinsic viscosity [η], light scattering, and sedimetation measurements However, despite much experimental work done on dilute solutions of amylose, its conformation in DMSO is still a matter of controversy • Experimental difficulties in the accurate determination of Mw • Weak dependence of intrinsic viscosity on molecular weight2 1. Everett, W.W.; Foster, J. F. J. Am. Chem. Soc.1959, 81, 3464. 2. Banks, W.; Greenwood, C. T. Carbohydr. Res.1968, 7, 414. 3. Nakanishi, Y.; Norisuye, T.; Teramoto, A. Marcomolecules1993, 26, 4220. 4. Cowie, J. M. G. Macromol. Chem. Phys. 1961, 42, 230. 5. Fujii, M.; Honda, K.; Fujita, H. Biopolymers 1973, 12, 1177 6. St-Jacques, M.; Sundararajan, P. R.; Taylor, K.J.; Marchessault, R. H. J. Am. Chem. Soc. 1976, 98, 4386 8. Jane, J. L.; Robyt, J. F. Carbohydr. Res. 1985, 140, 21 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287 9. Jeannette, N.; Putaux, J. L.; Bail, P.L.; Buleon, A. Macromolecules 2003, 33, 227 5 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287

  6. Amylose conformation However, despite much experimental work done on dilute solutions of amylose, its conformation in DMSO is still a matter of controversy The two conformations are expected to exhibit very different internal dynamics Amylose (Helix) Characterize the internal dynamics of amylose in DMSO Amylose (Random coil) Pyrene excimer formation 6 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287

  7. Amylose conformation The two conformations are expected to exhibit very different internal dynamics Amylose (Helix) Amylose (Random coil) Characterize the internal dynamics of amylose in DMSO As a comparison, poly(methyl acrylate) (PMA) was studied in DMSO. Pyrene excimer formation Flexible polymer with Tg equal to 12 ˚C. Synthesized by Shiva Farhangi 7 7. Cheetham, N. W. H.; Tao, L. Carbohydr. Polym. 1998, 35, 287

  8. k diff n h + Py + Py Py* + Py (PyPy)* t t M E Pyrene-labeled polymers and chain stiffness

  9. SyntheticRouteforLabelingAmylosewithPyrenebutyricAcid DIC,DMAP + DMSO+DMF DMAP:Dimethylaminopyridine DIC: Diisopropylcarbodiimide Procedure • Amylosewasdissolved intoamixtureofDMSOandDMF(3:1); pyrenebutyricacidandDMAPwereadded • Themixturewasputinanicebath,andDICwasaddeddropwise • Thereactionwasleftatroomtemperaturefor48hoursunderN2inthedark • The reaction mixture was precipitated into methanol three times to get rid of free pyrene This method was developed by Wei Yi.

  10. Steady-State fluorescence results PMA in DMSO Amylose in DMSO

  11. Steady-State fluorescence results

  12. Time-Resolved Fluorescence Spectroscopy Excimer formation by diffusion λex = 344 nm Coupled Monomer quenching λem = 510 nm λem = 375 nm Coupling of the kinetics of monomer quenching and excimer formation allows for global analysis of the monomer and excimer decays; improves accuracy.

  13. Py Py * Py Py Py Py Py Py The Fluorescence Blob Model (FBM) • FBM Parameters: • kblob: Excimer formation rate constant in a blob containing one excited and one ground-state pyrene • <n>: Average number of ground-state pyrenes per blob • kexch: Exchange rate constant ofground-state pyrenes between two blobs • Nblob: The blob size in number of monomer units • <kblobNblob>: Excimer formation rate constant

  14. Energy Transfer 294 nm Characterizationof dynamics of SNPs using pyrenefluorescences Side chain length Side chain length Backbone flexibility Backbone flexibility ■ PMMA C1 □ PBMA C4 ● POMA C8 ○ PLMA C12 1-x x 1-x x 1-x x 1-x x Farhangi, S.; Weiss, H.; Duhamel, J. Macromolecules2013, 46, 9738 14 Wavelength / nm

  15. Time-Resolved Fluorescence Spectroscopy PMA (degassed) PMA (aerated) PMA (aerated) PMA (degassed) Amylose (aerated) Amylose (degassed) Amylose (degassed) Amylose (aerated) • Nblob remains constant with pyrene content • Within experimental error, increasing the monomer lifetime from ~90 ns to ~135 ns by degassing does not lead to a larger blob size for both amylose and PMA • The overall average <Nblob> value for amylose is 11±2 • The overall average <Nblob> value for PMA is 45±2 • <kblob×Nblob> for amylose is found to be 0.16 ± 0.01ns-1 • <kblob×Nblob> for PMA is found to be 0.50 ± 0.05 ns-1

  16. Py *Py Py *Py FBM results Tg=~ 73 ˚C Tg=~ 12 ˚C PMA is flexible Amylose is rigid *Py Py <Nblob> =45 monomers <Nblob> =11 monomers <kblobNblob> = 0.50±0.05 ns-1 <kblobNblob> = 0.16±0.01 ns-1 *Py Py

  17. Py *Py Py *Py FBM results Tg=~ 73 ˚C Tg=~ 12 ˚C PMA is flexible Amylose is rigid 1 monomer = 2 chain atoms 1 monomer = 5 chain atoms *Py Py <Nblob> =90±4 chain atoms <Nblob> =55±10 chain atoms <kblobNblob> = 0.80±0.05 ns-1 <kblobNblob> = 1.0±0.1 ns-1 *Py Py Py-PMA and Py-Amylose form excimer with a similar efficiency. The unexpectedly large value of <kblob×Nblob> for amylose cannot be explained by a higher mobility of the amylose backbone which can be only explained amylose adopting a helical conformation in DMSO.

  18. An illustration of the ability of two pyrene groups to overlap using Hyperchem Two pyrene groups separated by 9 Glucose units Good overlap Two pyrene groups separated by 34 Glucose units No overlap The conformation of amylose is optimized from the default structure provided by Hyperchem

  19. An illustration of the ability of two pyrene groups to overlap using Hyperchem The conformation of amylose is optimized from the crystal structure of 7-fold amylose provided by Nishiyama et. al. Two pyrene groups separated by 1 Glucose Good overlap Two pyrene groups separated by 22 Glucose No overlap Nishiyama, Y.; Mazeau, K.; Morin, M.; Cardoso, M. B.; Chanzy, H.; Putaux, J. Macromolecules 2010, 43, 8628

  20. An illustration of the ability of two pyrene groups to overlap using Hyperchem Two pyrene groups separated by 1 Glucose Good overlap Two pyrene groups separated by 6 Glucose No overlap The orientation of an individual glucose unit changes along linear chain. The two pyrene groups have to be attached on different glucose units to minimize the effect of configuration.

  21. An illustration of the ability of two pyrene groups to overlap using Hyperchem The orientation of an individual glucose unit changes along linear chain. The two pyrene groups have to be attached on different glucose units to minimize the effect of orientations.

  22. Comparison of the results obtained by blob model analysis and Hyperchem simulation The blob model analysis suggests that pyrene can probe its surrounding area which consists of 11±2 glucose units. 9-fold helix 7-fold helix A good overlap between two pyrenes is identified by one pyrene having at least 7C-atoms covered by a second pyrene. Hyperchem simulation indicates that the number of glucose units that an excited pyrene can reach equals 11 regardless of the type of helix generated by amylose.

  23. Comparison of the results obtained by blob model analysis and Hyperchem simulation The blob model analysis suggests that pyrene can probe its surrounding area which consists of 11±2 glucose units. If amylose is a random coil in DMSO, <Nblob> must be 7 or less. That is contrary to the results of fluorescence analysis. => Amylose must adopt a helical conformation in DMSO.

  24. FBM results-Fraction of aggregation Amylose (degassed) Amylose (aerated) PMA (degassed) PMA (aerated) • The molar fraction fEOrepresents prestacked pyrene dimers before excitation • fEO for Py-Amylose is much higher than that for a same pyrene content but pyrene should be much more spread out along Py-Amylose compared to Py-PMA and thus generate much less pyrene aggregation Pyrene units attached on amylose are located in a very compact environment Amylose forms a helical structure in DMSO

  25. Summary • A series of pyrene labeled amylose samples were synthesized and their chain dynamics in DMSO was characterized by steady-state and time-resolved fluorescence • <Nblob> value for amylose is 11±2 <Nblob> value for PMA is 45±2 • <kblob×Nblob> for amylose is found to be 0.16 ± 0.01 ns-1 <kblob×Nblob> for PMA is found to be 0.50 ± 0.05 ns-1 Considering the size of the monomer units, in terms of chain atoms • <kblob×Nblob> for amylose is found to be 0.80± 0.05 ns-1 • <kblob×Nblob> for PMA is found to be 1.0 ± 0.1 ns-1 • The unexpectedly large value of <kblob×Nblob> for amylose can be only explained amylose adopting a helical conformation in DMSO • The results from the molecular mechanics optimization of the amylose helices point to a blob made of 2×5+1 = 11 glucose units, in close agreement the <Nblob>value of 11 ± 2 obtained by FBM analysis of the Py-Amylose constructs Amylose must adopt a helical conformation in DMSO

  26. Acknowledgements Lab members: Duhamel Group Gauthier Group Project colleagues: Wei Yi Joanne Fernandez Ryan Amos Magda Karski Duncan Li Imran Khimji Alexander Ip Ziyi Sun Howard Tsai Damin Kim Bowen Zheng 26

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