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Innovation with Integrity
DNP RMN des Solides Préparation des échantillons et Applications
30ème Réunion Utilisateurs RMN, 29 et 30 novembre 2016, Paris
Fabien AUSSENAC, Applications RMN
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Solid State DNP Overview
• Samples preparation for DNP exp. – Biological samples – Materials samples – Tools
• DNP Applications – Biological samples – Materials – Small organic molecules – Polymers
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
B0
mwaves 100 K
Solid-State Dynamic Nuclear Polarization (DNP)
• Transfer polarization from unpaired
electron spins to nuclear spins
ge >> gn
• Driven by microwave irradiation close
to the EPR frequency
H2O/ glycerol
H2O/ glycerol
DNP/NMR
H2O/ glycerol
H2O/ glycerol
H2O/ glycerol H2O/
glycerol
H2O/ glycerol
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
General Sample Preparation for Biological Solids
Soluble Biomolecules
• Solution of AMUPol (or TOTAPOL) in Glycerol-d8, D2O, and H2O
• Prep. solvent solution 60:30:10 (vol:vol:vol) Glycerol-d8:D2O:H2O for 10 mM TOTAPOL
• Stock solution (0.3 ml) - 0.18 ml Glycerol, 0.09 ml D2O, 0.03 ml H2O
• Weigh ~ 2.2 mg of AMUPOL or 1.2 mg of TOTAPOL and dissolve in D2O/H2O mixture
• Add glycerol-d8 and mix well
• Dissolve sample in enough radical solution to pack in ~25 ml rotor
• Many radicals are hygroscopic – warm vial to RT before opening and limit exposure to ambient conditions
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Preparation for Membrane Proteins
Membrane proteins and fibrils
• More complicated to distribute radical homogeneously
• Some samples can be simply mixed/suspended with a small volume of radical stock solution
• The majority are typically centrifuged from a radical solution
• Start with higher radical concentration so final is ~10 mM
• Samples with large amounts of lipids the concentration of glycerol may be reduced as the lipids offer some cryo-protecting properties
• Exact details of DNP solvent solution (pH, buffer, salt, etc.) may be adjusted for each sample but nitroxides can degrade at low pH
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Preparation for Material or Surface Science Samples
Solvent options
• When solvent/radical mixture is just wetting the surface of material glycerol:water can be a poor option due to viscosity
• Systems compatible with H2O, a 10 mM biradical solution with 90:10 (vol:vol) D2O:H2O or DMSO:D2O:H2O 78:14:8 (wt:wt:wt) can be tried
• For non-aqueous systems 10 mM TEKPOL can be used with 1,1,2,2-tetrachloroethane (TCE)
• Start with a mass of solid sample approximately equal to a full rotor
Lesage A. et al., J. Am. Chem. Soc., 2010, 132(44), 15459
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Preparation by Wet Impregnation Technique
• Slowly add solvent to powder with mixing until it becomes a wet paste
• For many materials the surface or pores sorb solvent
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
• Once the powder is a wet paste add a bit more dry powder so it remains a paste but is no longer visibly wet
Sample Preparation by Wet Impregnation Technique
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Preparation by Wet Impregnation Technique
• Pack the paste-like sample into the rotor
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Packing: The Modified Funnel
Funnel
Sapphire Rotor
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
• Fits in Eppendorf tube and sticky sample can be spun down in a centrifuge
Sample Packing: The Modified Funnel
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Sealing with Soft Silicone Rubber Plugs
Soft silicone rubber plug Capillary Depth tool
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Sealing with Soft Silicone Rubber Plugs
• Seating silicone plug in rotor with depth tool
Excess solvent
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Sealing with Soft Silicone Rubber Plugs
• Silicone rubber plug is self sealing when the capillary is removed
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Sample Sealing with Teflon Plugs
• 3.2 mm Teflon Insert (1.8 mm length) can be used as a top spacer or to tightly pack powder/impregnated powder or thick membrane protein samples.
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Inserting Zirconia Cap
• Unlike plastic caps the zirconia cap should be “loose” and can be inserted and removed with finger tips
• Do not twist cap and avoid using the cap puller as the ceramic cap is delicate and easy to break
Insert cap with fingers Inspect cap for damage Mark rotor and get ready to start experiments
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Solid State DNP Applications Areas
Solid State DNP
Biological Samples - Proteins - Peptides
- Membranes proteins - Fibrils
- Cell walls - …
Materials - Nanoparticles
- Catalysts - Mesoporous samples
- Bio-Materials - Functionalized silica
- …
Small Organic
Molecules - Pharmaceutical
applications
Polymers - Natural
- Synthetic
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
DNP Applications Biological Samples
DNP Biological Samples
• Amyloid fibrils • Membrane proteins - Bacteriorhodopsin - Photorhodopsin
• Channel proteins - KscA potassium channel
- M2 proton channel
• Peptides - Vaccines
- Self-assembled
• Cell walls - plants
- bacteria • Neurotoxin bound to acethylcholine receptor
Mainly 13C and 15N detection Multidimensional experiments
Structural biology for highly complex environments
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Takahashi H. et al., J. Am. Chem. Soc. 2013, 135, 5105
Selective DNP-enhanced NMR of cell-wall polymers in intact cells
2D 13C-13C correlation
4h30
• Enhancement of cell wall PG in intact cells • Absolute DNP gain: 24 (600 in times!)
Peptidoglycan (PG)
Bacillus subtilis bacterial cell
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
DNP gain: 17 Less than 2h for 2D PISEMA!
[5x15N]-hF19W(3mg) in POPC with PyPol-C16
15N chemical shift
1H
-15N
dip
ola
r co
up
ling
0
Oriented Biological Membranes Static DNP
Salnikov et al., J. Phys. Chem. B, 2015, 119, 14574
40-fold enhancement compared to room temperature => 1600-fold time save
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
DNP Applications Materials
DNP Materials
Detection: 13C, 29Si, 15N, 119Sn, 27Al, 17O, 89Y 2D experiments
Characterization of the materials at the molecular level
• Field of applications Photonic, sensors, bio-material, catalyst, drug delivery,
separation processes, medical imaging
• Functionalized materials
• Mesoporous silica, alumina
• Nanoparticles
• Metal Organic Framework (MOFs)
• Hybrid organic-silica materials
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Dynamic Nuclear Polarization Oxygen-17 detection
16O (99.757 %, I = 0), 17O (0.038 %, I = 5/2), 18O (0.205 %, I = 0) • Very few studies of 17O at natural abundance • Need medium/very high field, large sample volume and/or extended
experiment times to often obtain poor S/N.
• Oxygen is ubiquitous in chemistry and biology • Development of methods for their characterization
is of fundamental importance
Oxides Minerals
Organic molecules
Biomolecules Supramolecular networks
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Dynamic Nuclear Polarization Mg(OH)2
1H/17O DNP Oxygen-17 detection at natural abundance
1H/17O CP-MAS Mg(OH)2 at natural abundance!
(20 mM bCTbK in C2Cl4H2, 400 MHz)
Further enhancement of 270 obtained with
QCPMG
Blanc F. et al, J. Am. Chem. Soc., 2013, 135, 2975
O H
Mg
Only 10 min
with DNP!
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Mesoporous silicon nanomaterials PSi
EPR spectrum of TCSi sample • Carbon radicals: pyrolyzed polymers,
diamonds films, silicon carbide (SiC)
• Thermal carbonization PSi
(TCPSi): stabilization of the SiC layer on the surface
• Presence of endogenous radicals embedded in the surface layer of TCPSi materials
Riikonen A. et al., J. Phys. Chem. C, 2015, 119, 19272
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
-6
-4
-2
0
2
4
6
8
10
399 399.5 400 400.5 401
1H
DN
P S
ign
al E
nh
an
ce
me
nt
1H Frequency (MHz)
Mesoporous silicon nanomaterials TCPSi
• Porous silicon samples TCPSi • Average size pore 17nm • Particle size 25-75mm
• Field dependence curve • Positive and negative optimum • Cross Effect for 1H spins • DNP gain: 8.5
1H field dependence curve of TCPSi
• No addition of polarizing agent!
DNP gain: 8.5
• Optimization of DNP gain • Sweep field experiment
Riikonen A. et al., J. Phys. Chem. C, 2015, 119, 19272
• Endogenous radical Native sample
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
1H-13C CP-MAS NMR spectra of TCPSi sample a) at 108 K with microwave irradiation at 263 GHz to
induce DNP gain (8 192 scans)
b) at 298 K (50 734 scans ) * Spinning side bands
Mesoporous silicon nanomaterials TCPSi
• Porous silicon samples TCPSi • Average size pore 17nm • Particle size 25-75mm • Endogenous radicals
• 13C enhanced DNP spectrum • With DNP: 14h (a) • Without DNP: 3 days (b) • DNP gain: 8.5 • 129 ppm: aromatic C=C • 11.4 and 29 ppm: Different silicon carbides Polytypes SiC
Riikonen A. et al., J. Phys. Chem. C, 2015, 119, 19272
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
• Fast characterization of drug formulation components
• Rapid natural abundance 13C and 15N correlation spectra in drug formulations
• Determination of crystalline vs. amorphous API
• API-excipient interactions observed by correlation spectra
• High DNP enhancement and sensitivity for compounds with a long proton T1
Rossini A. et al., J. Am. Chem. Soc., 2014, 136, 2324
DNP-enhanced Characterization of Pharmaceutical Formulations
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
DNP-enhanced Characterization of Pharmaceutical Formulations
● DNP allows fast characterization of components of drug formulations
● DNP allows rapid collection of 2D 1H-15N correlation spectra in natural abundance
● Interactions between the drug molecule and matrix can be probed
Rossini A. et al., J. Am. Chem. Soc., 2014, 136, 2324
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Functional Synthetic Polymers
Polyethylene oxide (PEO) Macromonomers
Polystyrene (PS)
PS-b-PEO-b-PS samples with distinct morphologies (surfactants, templates)
• NMR signals from chain-ends are intrinsically diluted • High S/N is required for detection, especially for large MW samples • Chain-ends can be involved in further reactions, including polymerization
reactions, leading eventually to so-called “smart” materials with complex macromolecular architectures
Ouari O. et al., ACS Macro Lett., 2013, 2, 715
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Ouari O. et al., ACS Macro Lett., 2013, 2, 715
Analysis of Functional Polymers PolyStyrene (PS)
13C CP-MAS spectra DNP on/off of PS polymer (Mn 13500 g.mol-1) 0.5% mass of bCTbK (polarizing agent, bi-radical)
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris
Solid State DNP at 263 to 527 GHz
Solid State DNP
Biological Samples - Proteins
- Membranes proteins - Fibrils
- …
Materials - Nanoparticles
- Catalysts - Mesoporous samples
- Bio-Materials - Functionalized silica
- …
Small Organic
Molecules - Pharmaceutical
applications
Polymers - Natural
- Synthetic
• Higher sensitivity and reduction of acquisition time
• Many nuclei detected: 13C, 15N, 29Si, 31P, 27Al,119Sn, 17O, 89Y
• DNP opens new field of applications for NMR
30ème Réunion Utilisateurs RMN – 29-30 novembre 2016 – Maison de la Chimie, Paris 9. Dezember 2016 32 © Copyright Bruker Corporation. All rights reserved
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