UCSF Hyperpolarized MR Seminar Summer 2019, Lecture #3 "Sample Preparation" Lecturer: Dave Korenchan Sponsored by the NIH/NIBIB-supported UCSF Hyperpolarized MRI Technology Resource Center (P41EB013598) https://radiology.ucsf.edu/research/labs/hyperpolarized-mri-tech

1. Fantastic Preps and How to Combine
Them
Basics, Tips, and Tricks of HP Chemistry
9/19/2019
Dave Korenchan
Department of Radiology and Biomedical Imaging
(not me)

2. Learning Goals
 Identify the requirements of a good hyperpolarized (HP) agent
 Describe the essential elements of a HP formulation, as well as properties that
are favorable to hyperpolarization + dissolution
 Outline the general approach to formulating a new compound
 Describe the process of quality assurance for a new batch of formulated
compound
 Understand new approaches to improving polarization, buildup time, etc. of HP
formulations by adjusting the probe molecule, radical, solvent, glassing, and/or
electron relaxation agent
By the end of this lecture, students should be able to:
9/19/2019Basics, Tips, and Tricks of HP Chemistry – Dave Korenchan2

3. 13C Hyperpolarization: The Pivotal Players
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Albers et al. Cancer Res, 2008.
Chen et al. Cancer Res, 2017.
Lauetal.Magn
ResonImag,2016.
Chaumeil et al. Neuroimage, 2012.
Milshteyn et al. Magn Reson Imag, 2017.
Urea
What makes pyruvic acid and urea
good HP agents???
Glycolysis Perfusion

4. Identifying Successful HP Agents: Desirable Properties
Biological
1. Biologically relevant
2. Rapid conversion (+ cellular transport)
3. Tolerable at 10-250 mM
Chemical
4. Long solution-state T1 nucleus (> 30 s)
5. Large ∆CS upon conversion (> 1 ppm)
6. High liquid concentration (> 2 M)
7. High chemical stability
Measures glycolysis
Fast cell uptake, conversion in
cytosol
Phase I: 230 mM w/ no adverse effects
C1 pyruvic acid @ 3 T: T1 ~ 60 s
Pyruvate-lactate: ~12 ppm
Neat pyruvic acid: 14.4 M
Stable
9/19/2019Basics, Tips, and Tricks of HP Chemistry – Dave Korenchan4
Agent Requirements Why pyruvic acid nails it

5. Basics of Dissolution Dynamic Nuclear Polarization (d-DNP)
9/19/2019Basics, Tips, and Tricks of HP Chemistry – Dave Korenchan5
B0 = 3-10 T, T = 1.4-0.8 K
Microwaves
transfer
polarization
from
electrons to
nuclei
Rapidly dissolve

6. Anatomy of a HP Prep
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Stable radical
(contains a lone electron)
HP agent
(13C-enriched)
Glass at 0.8-1.4 K
(homogeneous distribution)
Solvent
(dissolves high [agent])
Glassing agent
(disrupts crystallization)
Electron relaxation agent
(improves polarization)

7. Anatomy of a HP Prep: The Radical
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• Lone electron delocalized by resonance structures -> stability
• Tune solubility by changing –R groups
• Radical is both polarization source and relaxation pathway for 13C nuclei!
‒ Typical concentration optimum: 15-20 mM
Radical
concentration
Observed solid-state buildup
Too little Slow buildup + lower max
signal
Too much Fast buildup + lower max signal

8. Dimethylacetamide
(DMA)
Anatomy of a HP Prep: The Solvent
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• Must be nontoxic (somewhat….)
‒ Other options for low-toxicity solvents?
Water
Glycerol
Dimethyl sulfoxide
(DMSO)
Propylene glycol?
Ethanol?

9. Dimethylacetamide
(DMA)
Anatomy of a HP Prep: The Glassing Agent
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• Solvents often disrupt crystal formation upon freezing -> glassing agents
‒ More viscous (eg. glycerol) tend to glass better
• Can combine in order to optimize concentration + glassing (ex. glycerol-H2O)
Glycerol
Dimethyl sulfoxide
(DMSO)
Propylene glycol?
Sugars? (trehalose)
How do you know if it glasses, anyhow???
- We’ll see in the lab – stay tuned!!

10. Anatomy of a HP Prep: The Electron Relaxation Agent
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What the heck?? Why do we want this?
• Short e- T1 -> recover polarization faster -> more efficient transfer to nuclei ->
higher polarization
• If too concentrated -> shorten 13C T1 too! (both solid glass & liquid dissolution)
Gadolinium chelate (Gd-DOTA)
e- 13C
MW
MW
T1,e-

11. Gln
Glu
pGlu
 High concentration (> 2 M)
• Higher polarization (generally)
• Larger dissolution concentrations
 Radical soluble up to 15-20 mM
• Poor solubility may compromise
polarization
 Forms a good glass at 0.8-1.4 K
• Radical homogeneously distributed
 Agent stable at high concentration
• Byproduct formation: complicates
spectral excitation/resolution,
reduces desired peak signal, may
contribute nonenzymatic conversion
 Low toxicity
Formulation Requirements
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Gln
Glu pGlu
degradation
glutaminase
(GLS)

12. Designing New Agent Formulations
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Step 1: Research!
 Chemical structure
 Chemical properties
New HP agent (assume biological criteria fulfilled/likely to succeed):
• Liquid at room temperature?
• Water solubility
• Ionization properties (introducing net charge
improves water solubility)
• Stability (degradation conditions?)
• Functional groups (13C’s away from H’s, close
to site(s) of conversion)
• Molecular weight (larger MW -> shorter T1s)

13. Designing New Agent Formulations (cont’d)
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Step 2: Test different formulations
 First, maximize concentration
New HP agent (assume biological criteria fulfilled/likely to succeed):
Online at:
https://radiology.ucsf.edu/sites/radiolo
gy.ucsf.edu/files/wysiwyg/research/HM
TRC/dess_training/2017_prep-
chemistry_plan.pdf

14. Designing New Agent Formulations (cont’d)
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Step 2: Test different formulations
 Then, test glassing
• Add glassing agents if
necessary
New HP agent (assume biological criteria fulfilled/likely to succeed):

15. Designing New Agent Formulations (cont’d)
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Step 3: Test T1, polarizeability of best formulation
1. Formulate agent (no 13C enrichment) with
radical
2. Polarize 50-100 µL at pyruvate frequency (just
a guess!) on HyperSense
3. Dissolve once solid-state signal plateaus, do
dynamic 13C NMR (10-20° pulse-acquire)
4. Identify spectral peaks, measure T1 value(s)
New HP agent (assume biological criteria fulfilled/likely to succeed):
If good signal + T1, THEN you can drop some $$ for the 13C-labeled compound

16. Designing New Agent Formulations (cont’d)
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Step 4: Measure polarization of 13C prep
1. Formulate 13C-labeled agent
2. Sweep 50-100 µL on HyperSense polarizer
• Identify MW frequency for max buildup
3. Polarize 20-50 µL on HyperSense polarizer
4. Dissolve, measure dynamic 13C-NMR
spectra
• TR = 3 s, 5-20° tip angle
• Calculate T1 (use tip angle correction:
multiply nth spectrum by sec(α)n-1)
1. Destroy remaining HP signal (90° pulses),
acquire thermal 13C-NMR spectrum
• TR = 5 * (measured T1), 90° tip angle
• Calculate (back-calculated) polarization,
PHP:
New HP agent (assume biological criteria fulfilled/likely to succeed):
Thermal polarization
1st HP spectrum
Good % polarization values (back-calc): > 15%

17. Large Batch Production: Quality Assurance (QA)
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Scale up production once formulation is
(somewhat) optimized, reproducible
 Good to perform QA on small sample
QA Steps:
1. Formulate compound, take ~100 µL for QA
2. Sweep on 50-100 µL (once every 2-3 months)
3. Polarize 20-50 µL @ max frequency, dissolve
4. Obtain HP + thermal spectra as before, calculate
T1 and % polarization
5. Compare with previous results:
• Solid-state buildup max + time constant
• Solution-state T1 + % polarization
• Dissolution pH
6. Adjust large batch if numbers off
• Add more solvent, radical, Gd-DOTA, etc.
Observation Potential Cause
Buildup too fast (too slow) Too much (too little) radical in prep
Solution T1 too low Too much Gd-DOTA in prep
Dissolution pH outside
range
Incorrect prep concentration
Sweep freq significantly off Wrong amount of Gd-DOTA in prep

18. Advanced Prep-Making: Modifying the HP Agent
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Precursor approach to improve %
polarization:
 Polarize precursor, rapid breakdown after
dissolution
• May work for smaller molecules
• Polarization loss during breakdown
Deuteration to improve T1:
 Deuteration of sites 1-2 bonds away from 13C
label
• T1 gains may be more pronounced at
lower field (less CSA contributing to T1)
• Can be difficult/expensive
Lee et al. Chem Commun, 2014.
Korenchan et al. Chem Commun, 2016.
Gln T1s, 9.4 T:
1H: 15 s
2H: 33 s (Really?)
Qu et al. Acad Radiol, 2011.

19. Advanced Prep-Making: Modifying the Radical
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Narrow-line radicals
 Narrower EPR line -> higher polarization
 BDPA: narrower EPR line than trityl
• Less stable….
 Can also mix with trityl (biradical approach)
Removable radicals
 Impregnate microcrystalline HP agent
(hydrophilic) with solution containing radical
(hydrophobic)
 Dissolve: radical and HP agent phase-
separate
• Reduce paramagnetic relaxation via
radical
Ji et al. Nat Commun, 2017.
BDPA

20. Advanced Prep-Making: Modifying the Solvent/Glassing
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13C-labeled solvents
 Labeling solvent ([13C]DMSO) with NaPyr:
faster buildup, but similar max polarization
• Speeds up passing of polarization
outwards from radical
Glassing w/o glassing agents
 Inverse Leidenfrost phenomenon: cryogen
gas surrounds sample, slows cooling -> bad
glassing
 Freeze atomized prep in nonvolatile fluid:
glass! (Problem: isopentane contamination)
Lama et al. NMR Biomed, 2015.Lumata et al. Phys Med Biol, 2011.

21. Advanced Prep-Making: Modifying the e- Relaxing Agent
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Ho-DOTA vs. Gd-DOTA
 Ho-DOTA: similar % pol gains to Gd-DOTA,
but less 13C T1 reduction
Gordon et al. Magn Reson Med, 2012. Kiswandhi et al. Phys Chem Chem Phys, 2016.
[1-13C]sodium acetate,
aqueous solution
Ho

22. Takeaways and Final Thoughts
 Currently: very much an art, with some educated guessing
• Need for more scientific rigor? (eg. calorimetry for glassing)
• New paradigms for formulations? (eg. eliminating solvents + glassing
agents)
• Formulation strategies likely to co-evolve with polarization technology
 Important to keep biocompatibility in mind!
• Need for medical collaboration + expertise
• Borrow from pharmaceuticals
Formulating compounds for HP: essential for enabling new agents
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23. (Happy prep-making!!)