Carbohydrate Based Molecular Logic: Beyond Binary Solutions

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1 Carbohydrate Based Molecular Logic: Beyond Binary Solutions Adam B. Braunschweig New York University / University of Miami AFOSR Program Review January 2013

NEW INSIGHTS STATUS QUO Carbohydrate Nanotechnology: Hierarchical Assemblies and Information Processing with Oligosaccharide Host-Guest Systems Adam B.

developed to explore how carbohydrates convey biological information.

molecular information processing. Synthetic Lectins bind primarily to glucose derivatives with weak affinity and poor selectivity and do not offer insights into biological activity.

2 NEW INSIGHTS STATUS QUO Carbohydrate Nanotechnology: Hierarchical Assemblies and Information Processing with Oligosaccharide Host-Guest Systems Adam B. Braunschweig, New York University / University of Miami New binding conjugates that can distinguish between monosaccharides that differ only by the orientation of a single hydroxyl group have been developed to explore how carbohydrates convey biological information. Carbohydrate Recognition Cell-surface oligosaccharides contain higher information density than any other molecule, but the lack of selective binding conjugates limits their utility in the context of molecular information processing. Synthetic Lectins bind primarily to glucose derivatives with weak affinity and poor selectivity and do not offer insights into biological activity.. Hierarchical self-assembly in nature is a combination of autonomous motion and carbohydrate recognition, but their interactions are not understood. Proteins and Bacteria α-man bound to a PBP. Periplasmic binding proteins (PBPs) combine flexible dimers to achieve high monosaccharide in the second binding event, and these proteins suggest new strategies for developing synthetic lectins. Flagellated bacteria combine autonomous motion and saccharide binding to direct feeding and predatorprey interactions. So these need to be studied together. MAIN ACHIEVEMENT: Established a new synthetic lectin architecture that recognizes β-mannose selectively over other monosaccharides that differ only by the orientation of a singly hydroxyl group by using multivalency and cooperativity. ConA binding to a mannose array. Measured diffusion of autonomous swimmers and determined how curvature and flipping control their spread. Lays the foundation for understanding how autonomous motion and carbohydrate-recognition control the organization of cells and microorganisms. Combined new molecular printing method Polymer Pen Lithography with covalent surface reactions to control density and feature size of carbohydrates in molecular arrays. Several different surface reactions have been explored to maximize binding. Impact Achieved Developed a new synthetic receptor that binds b-mannosides preferentially and displays concentration dependent selectivity. Braunschweig, Chem. Sci Combined Polymer Pen Lithography with covalent surface reactions to control density and feature size in carbohydrate arrays. Braunschweig, Small, Described a new model that explains the diffusivity of active nanorod suspensions. Braunschweig, PRL, Research Goals. Create a set of synthetic lectins that can rapidly distinguish between the 10 common monosaccharides. Develop a new molecular printing protocol based on Polymer Pen Lithography that can vary the surface density of saccharides on a surface. Utilize carbohydrate-synthetic lectin binding in solution and on surfaces in the context of new molecular memory, logic, and other information processing technologies. Apply the subtleties of multivalent carbohydrate binding in autonomous molecular machines. New concepts in molecule based memory, logic, and information transduction systems. QUANTITATIVE IMPACT END-OF-PHASE GOAL

3 Program Goals Year 1 Develop new carbohydrate-binding synthetic hosts that can distinguish between monosaccharides that vary only by the orientation of a single hydroxyl group Print carbohydrates on surfaces using molecular printing techniques combined with carbohydrate-compatible surface chemistry Year 2 Study effects of synthetic lectins on sugar conformational equilibration Optimize an efficient route to new synthetic lectin scaffolds Quantify surface binding of lectins and synthetic lectins as a function of carbohydrate surface chemistry and density EPANDED GOAL: Study motion of autonomous swimmers Year 3 Harness multivalent carbohydrate binding on surfaces to control and study the movements of autonomous nanostructures Templated assembly of complex hierarchical nanostructures Multiplexed carbohydrate printing with control over density, orientation, and pattern Utilize synthetic lectin-carbohydrate recognition for information processing

4 Progress Towards Y2 Goals Developed new synthetic carbohydrate receptor that utilizes cooperativity to achieve selectivity for b-mannose and that displays concentration driven switching, which provides important insights into how carbohydrates are used to convey information and direct self-assembly in natural systems. Explored and optimized new surface reactions to print carbohydrate onto surfaces with nanoscale feature dimensions to understand how the display influences binding with lectins. Reported a new model to explain the autonomous motion of catalytic Au-Pt nanorods in H 2 O 2 (aq) solutions. This is a critical first step in understanding how autonomous motion and chemical recognition work synergistically to control selfassembly and hierarchical organization in biological systems.

5 Scientific and Technological Transitions in Y2 Sent mannose-selective carbohydrate receptor to the Coutrot group at Montpelier. Sent data on graphene functionalization to the Houk group at UCLA. Patent Applications. Braunschweig, A. B.; Bian, S.; He, J.; Schesing, K. B. Nanoreactor Polymer Pen Lithography Non- Provisional Patent Application Braunschweig, A. B.; Zhong, X.; Bian, S.; Schesing, K. B. Polymer Tips Non-Provisional Patent Application Braunschweig, A. B.; Rieth, S.; Miner, M. R. Carbohydrate Specific Receptors Non-Provisional Patent Application

Luis M. Campos Department of Chemistry, Columbia University Phys. Rev. Lett. 2013, In Press.

6 Dispersion of Self-Propelled Rods Prof. Michael J. Shelley and Prof. Jun Zhang Courant Institute of Mathematical Sciences, NYU Collaborators Materials for Polymer Pen Lithography Prof. Luis M. Campos Department of Chemistry, Columbia University Phys. Rev. Lett. 2013, In Press. Diels-Alder Reactions on Graphene Prof. Ken Houk Department of Chemistry, UCLA J. Poly. Sci. A Chem. 2013, In Press. Carbohydrate Molecular Machines Prof. Frédéric Coutrot Université Montpellier Glycorotaxanes

7 Publications [5] Zhong, X.; Bailey, N. A.; Schesing, K. B.; Bian, S.; Campos, L. M.;* Braunschweig, A. B.* Materials for the Preparation of Polymer Pen Lithography Tip Arrays and a Comparison of their Printing Properties Journal of Polymer Chemistry A: Polymer Chemistry, 2013, In Press. [4] Takagi, D.;* Braunschweig, A. B.; Zhang, J.; Shelley, M. J. Dispersion of self-propelled rods undergoing fluctuation-driven flips Physics Review Letters, 2013, In Press. [3] Rieth, S.; Miner, M. R.; Chang, C. M.; Hurlocker, B.; Braunschweig, A.B.* Saccharide Receptor Achieves Concentration Dependent Mannoside Selectivity Through Two Distinct Cooperative Binding Pathways Chemical Science, 2013, 4, [2] Bian, S.; Schesing, K. B.; Braunschweig, A. B.* Matrix-Assisted Polymer Pen Lithography Induced Staudinger Ligation Chemical Communications, 2012, 48, [1] Bian, S.; He, J.; Schesing, K. B.; Braunschweig, A. B.* Polymer Pen Lithography (PPL) Induced Site-Specific Click Chemistry for the Formation of Functional Glycan Arrays Small, 2012, 8, Presentations [3] American Chemical Society National Meeting, Philadelphia, PA Bian, S., Braunschweig, A. B.* Using Polymer Pen Lithography to create the new covalent bonds on surfaces with submicrometer feature diameters [2] American Chemical Society National Meeting, Philadelphia, PA Rieth, S., Braunschweig, A. B.* [1] Center for the Chemistry of Integrated Systems, Northwestern University Branschweig, A. B.* A Mannose-Selective Synthetic Receptor With a Unique Cooperative Binding Mechanism (Invited Lecture)

8 Why is this transformational research? Sugars are considered untargetable because of structural similarities and cross specificities of sugar binding proteins. Potential to directly read saccharide expression on a cell surface. New recognition systems for sensors or hierarchical assembly. Accessing the massive information potential of carbohydrates would be a major step forward in molecular information processing systems (logic, memory, cryptography). Distinguishing between complex saccharides that may differ in structure only by the orientation of a single hydroxyl group is a challenge for which supramolecular chemists have not developed a viable strategy. Understanding how autonomous motion and molecular recognition work synergistically could revolutionize our understanding of biological networks. Why is this evolutionary research? Existing molecular printing technologies are employed to print carbohydrates and vary their surface densities to mimic carbohydrate cell-surface expression.

9 Logic on the Cell Surface: Glycoconjugates Sugar-Lectin (Monovalency) How is information encoded with Sugars? Molecular Recognition Patterning and Spacing Density (Multivalency / Cooperativity) Sugar-Sugar Sugar-Lectin (Multivalency) Cell Essentials of Glycobiology; Varki, A et al. Cold Spring Harbor Press, NY, 1999.

10 Chemical Information Density Based on the 10 mammalian monosaccharides. OH OH OH OH HO O NHAc O H 3 C HO OH OH OH O O O OH Size = 3 Chain length = 2 Branch Point = 1 HO O NHAc O H 3 C HO OH OH OH O O O OH O OH OH O NHAc O HO OH OH Size = 5 Chain length = 4 Branch Point = 1 O OH Average glycoconjugate contains 8 monosaccharides and 1 branch point. Seeberger, P.H. et al. ACS Chem. Biol. 2007, 2, 685.

11 Stoddart, et al. Chem. Eur. J. 2006,12, 261. Beyond Binary Molecular Logic Multistate Carbohydrate Driven Logic System Bistable Molecular Logic Bio-based Logic Synthetic Bistable Rotaxanes Streptavidin Labelled Bead Biotin Labeled Unit K a > M -1 (irreversible)

12 Lebrilla, C. B. et al. Curr. Opin. Chem. Biol. 2009, 13, Seeberger, P.H. et al. ACS Chem. Biol. 2007, 2, 685. Programming in Molecular Informational Complexity: Shallow Wells Free Rotation Flexible Hinge Monosaccharide Targets D-Sialic Acid α-d-sia L-Fucose α-l-fuc D-Galactose α-d-gal N-Acetylglucosamine β-d-glcnac Hydrogen Bonding Groups The dynamic receptor possesses multiple recognition units and conformational flexibility so it can rearrange to bind monosaccharides. Upon recognition, conformational restrictions induce cooperative binding with tightly bound substrates. D-Mannose α-d-man D-Glucose β-d-glc >75% O- and N-glycans are D-GlcNac (32%), D- Gal (25%), and D-Man (19%) Three monosaccharides dominate the nonreducing terminus: D-Sia, D-Fuc, D-Gal

VT 1 H NMR of β-man: Receptor Complex T ( C) -63 H k H k H a H e H e H a H b H j/j H h/h /i/i OH 2 H 1 H c/c /d/d /f/f /g/g H 2/6/6 /7/7 H 3 H5 OH 3 OH 4 H 4 OH 6-49 -38-26 -9 2 13 24 10.

13 VT 1 H NMR of β-man: Receptor Complex T ( C) -63 H k H k H a H e H e H a H b H j/j H h/h /i/i OH 2 H 1 H c/c /d/d /f/f /g/g H 2/6/6 /7/7 H 3 H5 OH 3 OH 4 H 4 OH δ (ppm) 1 H NMR, 400 MHz, CDCl 3, 12.0 mm Receptor, 24 mm β-man At low temperature a C 2 symmetric complex forms Braunschweig, et al. Chem. Sci. 2013, 4,

5 α-glcnac, 5 C δ 2:1 Fitting Equation for NMR (fast exchange) observed = [H]δ H + K [H][G]δ + K K 1 HG 1 [H] t

14 NMR Titrations Reveal a New Equilibrium δ (ppm) δ (ppm) 3.9 K 1 + K dimer Model K 1 + K 3 + K dimer Model 3.7 β-man, 5 C 3.5 H 7' K 1 K dimer β-man H 3 H 5 H Molar Equiv. of Receptor K α-glcnac, 5 C δ 2:1 Fitting Equation for NMR (fast exchange) observed = [H]δ H + K [H][G]δ + K K 1 HG 1 [H] t [H][G] Braunschweig, et al. Chem. Sci. 2013, 4, δ HG H 7 H 7' H Molar Equiv. of Receptor

15 Van t Hoff Plots To Measure ΔH o and ΔS o Braunschweig, et al. Chem. Sci. 2013, 4,

Pathway Selectivity in Second Binding Event n-octyl pyranoside

preference toward any pyranoside during the first association

16 Pathway Selectivity in Second Binding Event n-octyl pyranoside Log K a β-gal β-glcnac β-man β-glc α-gal α-glcnac α-man α-glc Log K 1 Log K 3 CDCl 3, 25 C The receptor does not show preference toward any pyranoside during the first association process (K 1 ). A 30:1 preference for β-man: β-man is seen in K 3. Braunschweig, et al. Chem. Sci. 2013, 4,

17 Binding Constants (K) and Cooperativity (a) Pyranoside Log K 1 Log K 2 α (K 1 /K 2 ) Log K 3 α-glc 2.75 ± β-glc 3.16 ± α-man 2.73 ± ± β-man 2.32± ± ± 0.02 α-gal 2.18 ± β-gal 2.59 ± α-glcnac 2.53 ± β-glcnac 2.65 ± CDCl 3, 298 K

Energy (kcal mol -1 ) Enthalpy-Entropy Compensation Keeps Selectivity Low and Wells

β-glc β-man -5-5 -10-15 -20-25 ΔH TΔS -10-15 -20-25 CDCl 3, T = 25 C Enthalpy and entropy

similar increases in entropy, thus the host is able to reorganize itself upon binding to

For K 3 there is a large difference in activation parameters between β-man and b-glc,

18 Energy (kcal mol -1 ) Enthalpy-Entropy Compensation Keeps Selectivity Low and Wells Shallow 0 α-glc α-glcnac α-man β-glc K 1 K 3 H + G HG HG + H H 2 G β-man β-glcnac β-gal 0 β-glc β-man ΔH TΔS CDCl 3, T = 25 C Enthalpy and entropy values were determined from van t Hoff plots For K 1, increase in enthalpy occurs with similar increases in entropy, thus the host is able to reorganize itself upon binding to various monosaccharides (enthalpyentropy compensation). For K 3 there is a large difference in activation parameters between β-man and b-glc, indicating significantly more non-covalent contacts form with β-man than b-glc. ΔG ΔG Braunschweig, et al. Chem. Sci. 2013, 4,

19 Concentration Dependent Pathway Selectivity Concentration dependent selectivity is unprecedented in synthetic receptors and is a direct result of the cooperative binding mechanism. Similar mechanism in natural lectins could help explain transport and surface binding where ligand density could drive recognition. Braunschweig, et al. Chem. Sci. 2013, 4,

Cooperativity Drives the Dominant Equilibria [Output] Cooperativity 1 : Mannose ΔS o K1 = 57 eu S. Shinkai, M. Ikeda, A. Sugasaki, and M. Takeuchi, Acc. Chem. Res., 2001, 34, 494 503.

20 Cooperativity Drives the Dominant Equilibria [Output] Cooperativity 1 : Mannose ΔS o K1 = 57 eu S. Shinkai, M. Ikeda, A. Sugasaki, and M. Takeuchi, Acc. Chem. Res., 2001, 34, Positive Cooperativity K 2 > K 1 φ 1 2 : Mannose N H O C H π Non-Cooperative K 2 = K 1 ΔS o K1 = 54 eu [Input] 2D NMR / B3LYP 6-31G(d,p) Braunschweig, et al. Chem. Sci. 2013, 4,

21 1:2 Host:β-Man C 2 Symmetric Structure Braunschweig, et al. Chem. Sci. 2013, 4,

22 Selectivity From Additional Contacts 8 noncovalent contacts β-mannose 13 noncovalent contacts β-glucose Models suggest that the 2:1 β-mannose complex is more compact than the corresponding Glucose complex. C-H π Interaction Structure explains cooperativity and Hydrogen Bond thermodynamics. Braunschweig, et al. Chem. Sci. 2013, 4,

23 2:1 Host:β-Man Cage Complex Braunschweig, et al. Chem. Sci. 2013, 4,

Science 2008, 321, 1658. Use in Carbohydrate Recognition Test new chemistries for immobilizing carbohydrates onto different surfaces.

24 Molecular Printing Polymer Pen Lithography 160 μm Advantages of PPL High throughput over large area, low cost. Force and contact time dependent feature size. Compatible with various delicate molecular inks and substrates. Mirkin, C.M. et al. Science 2008, 321, Use in Carbohydrate Recognition Test new chemistries for immobilizing carbohydrates onto different surfaces. Study surface density and feature size effects as feature sizes approach the nanoscale. Under stand how patterning and gradients affect trajectories of autonomous swimmers. Mirkin, C. A., et al., Nat. Chem. 2009, 1, 353.

25 Exploring Different Surface Chemistries to Control Carbohydrate Surface Display Approach Carbohydrate Compatible Surface Chemistries Cu I Catalyzed Azide-Alkyne Cycloaddition (Cuaac) PPL Tips Braunschweig, A.B.*, Small, 2012, 8, Staudinger Ligation Coat with PEG Print Braunschweig, A. B.* Chem. Commun. 2012, 48, Thiol-Ene React Radical Polymerization Rinse

vs. Dwell time 1/2 50 µm 200 µm Nikon Eclipse Ti, λ ex = 532-587

26 Fluorescent Patterns Produced by CuAAC Matrix-Assisted PPL Intensity Profile Magnified Image of 11*11 Array Feature Diameter vs. Dwell time 1/2 50 µm 200 µm Nikon Eclipse Ti, λ ex = nm, λ obs = nm Braunschweig, A. B.* et al., Small, 2012, 8,

Variable Scan Rate CV Scan rate (v/s) 0.

27 Redox Active Patterns Produced by CuAAC Matrix-Assisted PPL AFM Topography Variable Scan Rate CV Scan rate (v/s) µm Scan Rate vs. Current Scan Rate vs. Current Pt counter electrode, Ag/AgCl reference electrode in 1 M HClO 4 electrolyte solution Braunschweig, A. B.* et al., Small, 2012, 8,

28 Site-Specific Staudinger Ligation Matrix-Assisted PPL Intensity Profile Fluorescent Microscope Image Feature Diameter vs. Dwell time 1/2 Nikon Eclipse Ti, λ ex = nm, λ obs = nm Braunschweig, A. B.* et al., Chem. Comm., 2012, 48,

29 Redox Active Patterns by Staudinger Ligation Matrix-Assisted PPL Variable Scan Rate CV Optical Microscope Image Pt counter electrode, Ag/AgCl reference electrode in 1 M HClO 4 electrolyte solution Scan Rate vs. Current Braunschweig*, et al. Chem. Comm. 2012, 48,

Autonomous Nanoswimmers AuPt Nanorods are models for the motions of self-propelled microorganisms, but dynamics governed by physics (viscous forces and thermal fluctuations).

30 Autonomous Nanoswimmers AuPt Nanorods are models for the motions of self-propelled microorganisms, but dynamics governed by physics (viscous forces and thermal fluctuations). If these dynamics could be understood, then logic behind their motion could be used to understand how autonomous motion and hierarchical assembly work together for hierarchical assembly. Mechanism: Electric field, generated by ions arising from two redox reactions, drives the ions and induces fluid flow. Models still do not exist that accurately predic their motion and dispersion. Mallouk et al. J. Am. Chem. Soc. 2004, 124,

curvature, and these defects dominate their motion.

31 Synthesis of AuPt Nanorods Calibration Curves 1um 1μm Rods inevitably have minor imperfections such as curvature, and these defects dominate their motion. 1um Braunschweig, et al. Phys. Rev. Lett. 2013, In Press.

because of thermal fluctuations Fast rods change direction coherently because

32 Trajectories Over 15s H 2 O 2 Fuel Concentration C=3% Speed ~8μm/s H 2 O 2 Fuel Concentration C=25% Speed ~40μm/s Slow rods change direction randomly because of thermal fluctuations Fast rods change direction coherently because of underlying deterministic motion Braunschweig, et al. Phys. Rev. Lett. 2013, In Press.

CW and CCW orbits Thermal energy KBT can flip the rods

33 Systematic Turns and Spontaneous Flips Speed ~40μm/s Slightly curved rods have two stable states of moving in CW and CCW orbits Thermal energy KBT can flip the rods Accumulated angle Braunschweig, et al. Phys. Rev. Lett. 2013, In Press.

34 Mean Square Displacement (MSD) Saturates Diffusive behavior after t ~ 1s Diffusivity saturates with speed! Braunschweig, et al. Phys. Rev. Lett. 2013, In Press.

35 Conclusions A new receptor that can reorganize in response to different monosaccharides has been prepared and the binding to an array of monosaccharides has been fully characterized by 1 H NMR and ITC. The receptor is highly selective for Mannnose over all other monosaccharides that were tested. This receptor achieves selectively through a unique cooperative binding mechanism that can be explained through 2D NMR in combination with molecular modeling that explain the selectivity and cooperativity, and suggest new structures that will improve binding affinity. To access multivalent binding interactions, a new molecular printing strategy that sitespecifically immobilizes organic molecules through glycan-tolerant bioorthogonal reaction the Staudinger Ligation and the Cuaac to achieve sub-micrometer features with control over ligand density over cm 2 areas. Following optimization of printing and reaction conditions, a glycan microarray with control over ligand density and orientation was printed successfully and shown to demonstrate biological activity through binding with ConA. The motion of autonomous swimmers has be described with a new mathematical model, and these swimmers will be combined with glycan patterns to understand hierarchical biological assembly and information networks.

36 Acknowledgements University of Miami New York University Kevin Schesing Shudan Bian Dr. Stephen Rieth Dr. Daisuke Takagi Molecular Design Institute Brian Schmatz Matthew Miner Hugh DeLong Young Investigator Award #: FA Carbohydrate Nanotechnology

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AFRL-OSR-VA-TR-2014-0279 Carbohydrate Nanotech Hierarchical Assemblies of Oligosaccharide m Braunschweig UNIVERSITY OF MIAMI 09/17/2014 Final Report DISTRIBUTION A: Distribution approved for public release.