dnp rmn des solides - bruker · innovation with integrity dnp rmn des solides préparation des...

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


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


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


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


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


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


• Once the powder is a wet paste add a bit more dry powder so it remains a paste but is no longer visibly wet




• Pack the paste-like sample into the rotor


Sample Packing: The Modified Funnel

Funnel

Sapphire Rotor


• Fits in Eppendorf tube and sticky sample can be spun down in a centrifuge

Sample Packing: The Modified Funnel


Sample Sealing with Soft Silicone Rubber Plugs

Soft silicone rubber plug Capillary Depth tool



• Seating silicone plug in rotor with depth tool

Excess solvent



• Silicone rubber plug is self sealing when the capillary is removed


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.


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


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


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


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


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


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


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


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!


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


-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


• Endogenous radical Native sample


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



• 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


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


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


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)


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

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