(NOTE: An [*] indicates a change from last year ... · Reference: Najac et al, Sci Rep 6:31397...

(NOTE: An [*] indicates a change from last year) Formulation of 13C-labelled compounds for dissolution-DNP Updated 3/22/2017 Table of Contents 1) GLASSINGANDHIGHCOMPOUNDCONCENTRATION 22) STABILITYOFCOMPOUNDFORMULATIONS 2

II. TRITYLRADICALS 31) STORAGEANDHANDLING 32) STRUCTURESANDMOLECULARWEIGHTS 3

III. RECIPES 41) METABOLISMPROBES 4A. [13CN]PYRUVICACID 4B. [1-13C]LACTICACID 6C. [1-13C]ALANINE 7D. [5-13C]GLUTAMINE 8E. [6-13C]ARGININE 10F. [1-13C]2-OXOKETOGLUTARICACID(ΑKG) 11G. [1-13C]2-OXOISOCAPROICACID(KIC) 12H. [13C4]Α-KETOBUTYRATE 13I. [1-13C]BUTYRICACID 14J. [2-13C]FRUCTOSE 15K. [2-13C]DIHYDROXYACETONE 162) PERFUSIONPROBES 17A. [13C]UREA 17B. [1-13C,D8]BIS-1,1-(HYDROXYMETHYL)CYCLOPROPANE(HP001) 18C. [1-13C,D9]TERT-BUTANOL 193) EXTRACELLULARPHPROBES 20A. SODIUM[13C]BICARBONATE 20B. [1-13C]1,2-GLYCEROLCARBONATE(GLC) 22C. [AMIDE-13C,15N]N-(2-ACETAMIDO)-2-AMINOETHANESULFONICACID(ACES) 24D. [2-13C,D10]DIETHYLMALONICACID(DEMA) 25E. [2-13C,D4]CYCLOPROPANE-1,1-DICARBOXYLICACID(CPDA) 264) METALIONPROBES 27A. *[1-13C]CYSTEINE 27B. *[1-13C2]IMINODIACETICACID(IDA) 285) REDOXPROBES 29A. [1-13C]DEHYDROASCORBICACID(DHA) 29B. *[1,3-13C2]ACETOACETATE(ACAC) 30NECROSISPROBES 32A. [1,4-13C2]FUMARICACID 32

General Remarks

1) GlassingandhighcompoundconcentrationThe highest levels of nuclear spin polarization and MR signal are attained when the 13C concentration is as high as possible. The ideal compound formulations therefore are small molecule liquids prepared neat. If needed, the compounds must be formulated with appropriate (co)solvents and at a concentration such that they reliably form an amorphous glass when cooled rapidly. Glassing is critical to achieve high levels of polarization.

2) StabilityofcompoundformulationsThe various compounds are stable to various degrees and the radicals are light sensitive. Formulated compounds are best stored frozen and protected from light. Gadolinium(III) chelates, e.g. Dotarem / Gd-DOTA, are relatively unstable at low pH, such as when dissolved in pyruvic acid, and will dissociate to free Gd3+ ions when in liquid state. This can have a significant negative impact on the T1 relaxation rate upon dissolution, which results in the hyperpolarized signal decaying faster. To summarize:

• Protect from light exposure whenever possible • Do not let compound formulations sit at room temperature for extended periods

II. Trityl Radicals

1) StorageandhandlingThe stabilized trityl radicals used as the source of spin polarization are costly and must be stored and handled properly. They are best stored long term cold, dry and protected from light, e.g. in a jar containing a dessicating agent in the freezer. To prevent the buildup of water condensation, allow them to warm to room temperature before handling. The trityl radicals are typically light, lyophilized solids of various shades of green. To reduce their tendency to cling to surfaces when handling them, it helps to use an antistatic gun to dissipate static electricity on the plasticware and weighing containers.

2) StructuresandmolecularweightsThe structures and molecular weights of the radicals:

R = Name Formula Molecular Weight -CH2CH2OH OX-63 C52H60Na3O18S12 1426.77 -CH2CH2OCH3 “GE Trityl” / AH-

111501 C64H84Na3O18S12 1595.09

-CH3 Finland C40H36Na3O18S12 1066.46

S

SS

S

SS

SSS

S

SS

CO2Na

CO2NaNaO2C

R RRR

RRR

R

RR

RR

III. Recipes

1) MetabolismProbes

a. [13Cn]pyruvicacid

i. [1-13C]pyruvic acid

(Note: this is for a large batch; scale down as needed) Materials 130 mg GE-Trityl 6.0 g (~4.9 ml) [1-13C]pyruvic acid (Aldrich: 677175, CIL: CLM-8077)

MW: 89.05 g/mol; density 1.281 14.4 µl Dotarem / 500 mM Gd-DOTA Protocol:

1. Set sonicator bath to 40°C. 2. Remove pyruvic acid from freezer and allow to warm to room temperature. 3. Weigh GE-Trityl and transfer to 15 mL conical plastic centrifuge tube. 4. Add pyruvic acid to tube. 5. Dissolve GE-Trityl with a combination of vortex mixing and bath sonication. 6. Add Gd-DOTA to 1.5 mM 7. Test buildup on HyperSense & adjust radical conc. as needed 8. Pack into 200 µl aliquots and store in -80 freezer

Typical build-up in HyperSense OX21 (NMR Lab) (2014.04.23)

• Vol. / mass 24 µl / 30.2 mg • µwave freq. 94.080 GHz • Maximum 94000 • Time constant 935 s

ii. [2-13C]pyruvic acid Materials & Protocol

• as for [1-13C] pyruvic acid

13CO

HO

O

• [2-13C] pyruvic acid (Aldrich # 692670) Typical build-up in HyperSense OX21 (NMR Lab) (2014.10.06)

• Vol. / mass 7.5 µl / 9.5 mg • µwave freq. 94.073 GHz • Maximum 2300 • Time constant

iii. [1,2-13C2]pyruvic acid

Materials & Protocol

• as for [1-13C]pyruvic acid • [1,2-13C2]pyruvic acid (Aldrich # 721298)


• Vol. / mass 60 µl / 78.0 mg • µwave freq. 94.097 GHz (unswept) • Maximum 116160 • Time constant 1073 s

b. [1-13C]lacticacid

Materials 250 mg [1-13C] L-lactic acid (Aldrich # 738778, MW 91.06; density 1.2 g/mL) 20 µL Millipore water 4.4 mg OX063 trityl radical 0.4 µL Gd-DOTA Protocol

1. Set sonicator bath to 50°C 2. Charge centrifuge tube with lactic acid and water 3. Add OX063, sonicate for 30 minutes 4. Add Gd-DOTA, vortex and invert to ensure full mixing


• Vol. / mass • µwave freq. • Maximum • Time constant

c. [1-13C]alanine

Reference: modified from Dave Wilson’s prep Materials 350 mg [1-13C] alanine (CIL # CLM-116-1 MW 90.09 g/mol) 250 µl 18.9 M NaOH 100 µl DMSO 22 mg OX63 3.7 µl 50 mM Gd-DOTA (prepare fresh from 500 mM stock) ~6.0 M Alanine ~7.4 M NaOH 0.3 mM Gd-DOTA Protocol

1. Set sonicator bath to ~50°C. 2. Charge microcentrifuge tube with [1-13C]alanine 3. Add NaOH solution 4. Dissolve in sonicator bath with occasional vortex mixing 5. Add weighed OX63 to tube & dissolve with sonication & vortex mixing 6. Add DMSO & mix 7. Add Gd-DOTA & mix


• Vol. / mass 90 µl / 112.4 • µwave freq. 94.092 GHz • Maximum 27759 • Time constant 2945 s

13C

NH2

O

HO

d. [5-13C]glutamine

Note: To increase its solubility, the sodium salt of glutamine is prepared. The resulting glutamine slowly reacts to produce [5-13C] pyroglutamate, which resonates very closely to [5-13C] glutamate. This reaction is minimized by using the minimum NaOH necessary to get the glutamine into solution (0.9 eq), keeping the prep cold, and storing it aliquotted in the -80. Alternatively, the glutamine can be prepared as a cesium salt in an anhydrous DMSO prep; however, toxic Cs+ must be removed via ion exchange prior to in vivo injection. Both preps are described herein.

i. Na-glutamine *Reference: adapted from recipe from C. Cabella; J. Mag. Res. 2013, 232:45-52 *Materials (Makes 200 µL of prep; scale as needed) 109.0 mg [5-13C]-L-glutamine (CIL # CLM-1166-PK, MW 147.07 g/mol) 3.5 M 47.4 µL Millipore water 75.6 µL NaOH, 10 M 3.5 M 4.9 mg OX063 radical 16.2 mM 8 µL 50 mM Gd-DOTA 1.95 mM Protocol

1. Keep sonicator bath at room temperature 2. Charge microcentrifuge tube with water and NaOH 3. Add glutamine, mix & chill on dry ice 4. Add OX63, sonicate to dissolve 5. Add Gd-DOTA and mix 6. Store aliquots in -80



NH2

13CO

H2N

O

OH

ii. Cs-glutamine Reference: adapted from recipe from C. Cabella; J. Mag. Res. 2013, 232:45-52 Materials (Makes 400 µL of prep; scale as needed) 165.1 mg [5-13C]-L-glutamine (CIL # CLM-1166-PK, MW 147.07 g/mol) 188.6 mg CsOH monohydrate 10.8 mg OX063 radical 1.72 µL 500 mM Gd-DOTA 215.5 µL DMSO Protocol

1. Keep sonicator bath at room temperature 2. Charge amber glass bottle with OX63 and DMSO, sonicate to dissolve 3. Charge prep tube with CsOH monohydrate, using antistatic gun during

a. Work quickly and cap tube after – CsOH is hygroscopic 4. Transfer DMSO mixture to prep tube, spin down, vortex/sonicate to mix 5. Add Gd-DOTA to tube, vortex to mix 6. Store in -80



e. [6-13C]arginine

Reference: Najac et al, Sci Rep 6:31397 Materials (Makes ~220 µL of prep; scale as needed) 160 mg [6-13C]-Arginine (CIL: CLM-2070) 3.4 M 100 µL 7.2x10-2 M Trizma® base solution (Sigma: T1503) 4.77 mg OX-63 (Oxford Instruments) 15 mM 6.69 µL Dotarem / 50mM Gd-DOTA 1.5 mM Protocol:

1. Set sonicator bath at 50 °C 2. Prepare 7.2x10-2 M Trizma® base solution: 18.17 mg in 2mL dH2O3 3. Weight 160 mg [6-13C]-Arginine and transfer to 2 mL Eppendorf 4. Add 100 uL Trizma® base solution to [6-13C]-Arginine 5. Mix and place in water bath with sonication for 30 minutes 6. Add OX-63 7. Dissolve with a combination of vortex mixing and bath sonication (if needed) 8. Add Gd-DOTA 9. Store in -80 °C until use

Typical build-up in HyperSense OX21 (NMR Lab, 6/16/15):

• Volume/weight: 74 mg • µwave frequency: 94.067 MHz • Maximum: 2800 • Time constant: 2400 s

f. [1-13C]2-oxoketoglutaricacid(αKG)

Reference: Nat Comm. 2013; 4:2429 Materials 100 mg [1-13C]-2-oxoglutaric acid (Aldrich # 704334 MW 147.11 g/mol) 33 µl Water 12 µl Glycerol 2.2 mg OX63 0.9 µl 50 mM Gd-DOTA (prepare fresh from 500 mM stock) [1-13C]-2-oxoglutaric acid at ~5.8 M Protocol

1. Set sonicator bath to ~40°C. 2. Weigh OX63 & αKG and transfer to microcentrifuge tube 3. Add water 4. Dissolve with vortex mixing & bath sonication 5. Add Gd-DOTA & mix 6. Test on HyperSense; adjust radical concentration if needed & store aliquots in

-80

Typical build-up in HyperSense OX21 (NMR Lab) (2011.07.14 prep)


13CO

HO

O

OHO

g. [1-13C]2-oxoisocaproicacid(KIC)

Reference: Materials 300 µl [1-13C]-2-oxoisocaproic acid (from sodium salt; MW 131.14 g/mol) 53 µl Glycerol 7.2 mg OX63 (14 mM) 2.8 µl 50 mM Gd-DOTA (prepare fresh from 500 mM stock) (0.4 mM) (KIC conc ~6.9 M (neat: 8.1 M)) (preparation of [1-13C]-2-oxoisocaproic acid from sodium [1-13C]-2-oxoisocaproate) to 1.0 g sodium [1-13C]-2-oxoisocaproate (CIL # CLM-2093-0; MW 153.12 g/mol),

• add ~10 ml water • add 200 µl concentrated H2SO4 (pH goes to ~1) • Extract w/ 3 x 25 mL diethyl ether (using separatory funnel) • Dry combined organic extracts with MgSO4 • Filter & remove ether by rotary evaporation • Yields 810 mg pale oil with faint isovaleric acid odor

Protocol

1. Set sonicator bath to ~40°C. 2. Weigh OX63, transfer to microcentrifuge tube. 3. Add KIC via micropipettor 4. Dissolve OX63 with vortex mixing & bath sonication 5. Add glycerol (helps to trim pipette tip to enlarge orifice diameter) 6. Add Gd-DOTA 7. Test on HyperSense & store aliquots in -80



13CO

HOO

h. [13C4]α-ketobutyrate

Materials (Makes 1 mL of prep; scale as needed) 350 mg [13C4] α-ketobutyrate, sodium salt (CIL # CLM6164, MW 128.04) 0.5 mL Millipore water 630 mg (0.5 mL) Glycerol 21.4 mg OX063 trityl radical 2 µL Gd-DOTA Protocol

1. Set sonicator bath to ~45 °C. 2. Charge centrifuge tube with water and glycerol, invert and vortex until

homogeneous 3. Add α-ketobutyrate and OX063, dissolve by inverting, vortexing, and sonicating

for 15 minutes 4. Add Gd-DOTA, vortex and invert repeatedly to fully mix

Typical build-up in HyperSense OX9 (Surbeck Lab) (2015.01.26 prep)

• Vol. / mass 60 mg / 45 µL • µwave freq. 94.100 GHz • Maximum 9401 • Time constant 2051 s

i. [1-13C]butyricacid

Reference: Magn Res Med 2014; 71(5):1663-9 Materials: (Makes ~100 µL of prep; scale as needed) 76.6 µL [1-13C]butyric acid (CIL: CLM-9666, MW=89.1 g/mol, 8.2 M density=1 g/mL) 13.4 µL DMSO (Sigma: D2438) 1.47 mg OX063 (Oxford Instruments) 10 mM 10 µL Dotarem / 10mM Gd-DOTA 1 mM Protocol (to minimize smell): 1. Transfer 13.4 µL of DMSO to 2 mL ependorf 2. Weigh and add OX063 3. Add Gd-DOTA 4. In the chemical hood, add butyric acid 5. Store in -80 °C until use Typical build-up in HyperSense OX21 (6/10/16): • Volume/weight: 30 µL/ mg • µwave frequency: 94.077MHz • Maximum: 2800 • Time constant: 2000 s

j. [2-13C]fructose

Reference: J Am Chem Soc. 2009; 131(48): 17591–17596. Adapted from Kayvan Keshari’s recipe Materials 974 mg [2-13C]fructose (MW 181.15 g/mol) 400 µl water 27 mg OX63 (14 mM) 0.66 µl 500 mM Gd-DOTA (0.3 mM) (fructose conc ~4.0 M) Protocol

1. Set sonicator bath to ~50°C. 2. Weigh Fructose & OX63, transfer to microcentrifuge tube. 3. Add water 4. Dissolve with vortex mixing & bath sonication 5. Add Gd-DOTA 6. Test on HyperSense & store aliquots frozen



13CO

OHHO

OH

OHHO

k. [2-13C]dihydroxyacetone

Reference: J Biochem. 2014; 289: 35859-35867. Materials (Makes 700-750 uL of prep; scale as needed) 500 mg [2-13C]DHAc-dimer (99% 13C, Aldrich #767891, 182.14 g/mol) 343 ul 2:1 water:DMSO 1.38 ul 500 mM Gd-DOTA 21 mg OX63-trityl DHAC at 8M, 15mM trityl radical, 1.0mM Gd-DOTA. Protocol

1. Set sonicator bath to ~40ºC 2. Make a stock of solvent with 300 ul water and 150 ul DMSO 3. Weigh DHAC and load into 1.5 ml eppendorf 4. Add water:DMSO and dissolve by bath sonication (5 min) & vortex mixing 5. Weigh and add OX63. Dissolve by bath sonication (2 min) & vortex mixing 6. Add Gd-DOTA and vortex mix it

To obtain a 80mM solution after dissolution: polarize 40ul and dissolve in 4ml of 1xPBS (pH=7.4) Typical build-up in HyperSense (3T Lab)

• Vol./mass 40 ul / 46mg • uwave freq. 94.95 GHz • Maximum 17K • Time constant ~1500s

2) PerfusionProbes

a. [13C]urea

Reference: adapted from recipe from Dave Wilson; J Mag. Res. 2010, 205(1):141-7 (Note: this is for a large batch (~8 ml); scale down as needed) Materials 3100 mg Urea-13C (Aldrich # 603430; MW 61.05 g/mol) 7150 mg Glycerol 216 mg OX63 3.2 µl Dotarem / 500 mM Gd-DOTA (Final urea concentration ~ 6.4 M) Protocol

1. Set sonicator bath to ~60°C. 2. Charge 15 ml Falcon tube with urea & glycerol 3. Dissolve with a combination of vortex mixing and bath sonication with heat. 4. Add trityl & dissolve with sonication & heat (It works well to add the weighed

radical to a fresh tube and add the urea / glycerol solution to it.) 5. Add Gd-DOTA and mix 6. Test on HyperSense and adjust radical / urea-glycerol conc. as needed. 7. Make 400 µl aliquots and store long term in -80


• Vol. / mass 27.5 µL / 34.4 mg • µwave freq. 94.066 GHz • Maximum 6120 • Time constant 2980 s

H2N13CO

NH2

b. [1-13C,D8]bis-1,1-(hydroxymethyl)cyclopropane(HP001)

Reference: from Jan Henrik Ardenkjaer-Larsen ~ 28% water 20 mM OX63 1.2 mM Dotarem Materials 250 mg HP001 (1,1-bis(hydroxy-d2-methyl)-[1-13C]-2,3-d4-cyclopropane)

(MW 111.06 g/mol,) 90 µl Water 11 mg OX63 0.7 µl 500 mM Gd-DOTA HP001 concentration ~7.2 M Protocol

1. Weigh OX63 & transfer to microcentrifuge tube 2. Add water & dissolve with bath sonication & vortex mixing 3. Weigh in HP001 4. Add Gd-DOTA 5. Mix & test on HyperSense; adjust radical concentration if needed & store

aliquots in -20 Typical build-up in HyperSense OX21 (NMR Lab) (2010.05.12 prep)

• Vol. / mass 100 µl / 110.8 mg • µwave freq. 94.093 GHz • Maximum 63806 • Time constant 1518 s (a little on the fast side – ~2000 s has higher polarization)

13C OHHO

DD DD

DDD

D

c. [1-13C,D9]tert-butanol

Materials (Makes 1 mL of prep; scale as needed) 390 mg (0.5 mL) [1-13C, D9] tert-butanol (Aldrich # 679860, MW 83.98) 630 mg (0.5 mL) Glycerol 16.0 mg Finland trityl radical 2 µL 500 mM Gd-DOTA Protocol

1. Set sonicator bath to ~45°C. 2. Charge centrifuge tube with tert-butanol and glycerol, invert and vortex until

homogeneous 3. Add Finland radical, vortex and invert to dissolve

a. If necessary, immerse in water bath 4. Add Gd-DOTA, vortex and invert to fully mix 5. Store in 200 µL aliquots in -80

Typical build-up in HyperSense OX9 (Surbeck Lab) (2012.11.07 prep)

• Vol. / mass 10 mg / 10 µL • µwave freq. 94.100 GHz • Maximum 1300 • Time constant 3051 s

3) ExtracellularpHProbes

a. Sodium[13C]bicarbonate

Note: While heating of bicarbonate in glycerol is necessary to promote solubility, it also generates linear-chain adducts of bicarbonate and glycerol. These adducts are visible in hyperpolarized spectra as two peaks ~1.5 ppm upstream of the bicarbonate resonance, typically in a 1:10 ratio (upstream:downstream) and separated from each other by 0.45 ppm. Two alternate recipes are included here to try.

i. With heat gun, no Gd-DOTA Reference: adapted from recipe from Dave Wilson; J Mag. Res. 2010, 205(1):141-7 Materials (Makes ~800 µl of bicarbonate in glycerol and 200 uL of prep; scale as needed) 135.0 mg Sodium 13C-bicarbonate (Aldrich # 372382, MW 85.01 g/mol) 1099 mg Glycerol 5.7 mg OX063 radical Protocol

1. Charge small glass bottle with bicarbonate, crush with scoopula 2. Charge bottle with glycerol, on top of bicarbonate 3. Use heat gun to heat bicarbonate to bubbling (will not get all into solution)

a. Solution will turn slight yellow – avoid further color change 4. Transfer to Eppendorf tube, spin down for 15 minutes at 3000 rpm 5. Transfer supernatant to new glass bottle for storage at -20 6. Charge new glass bottle with OX063 7. Add 264.2 mg (~200 uL) of bicarbonate in glycerol to bottle, quickly heat/vortex

into solution Typical build-up in HyperSense OX21 (NMR Lab) (2014.12.05)

• Vol. / mass ~30 µl / 37.6 mg • µwave freq. 94.060 GHz • Maximum 1300 • Time constant 2360 s

Na+

13CHO

O

O-

ii. With sonication, Gd-DOTA Materials (Makes ~7.5 ml of 1.69 M solution in glycerol; scale as needed) 1080 mg Sodium 13C-bicarbonate (CIL # CLM-441-1; MW 85.01 g/mol) 8780 mg Glycerol (For 1mL) 23.2 mg OX63 (23.2 mg/ml; 16 mM) 2 µl 100 mM Gd-DOTA (prepare fresh from 500 mM stock) to 0.2 mM

1. Set sonicator bath to ~60°C. 2. Charge 15 ml Falcon tube with urea & glycerol 3. Dissolve with a combination of vortex mixing and bath sonication with heat. 4. Add trityl & dissolve with sonication & heat (It works well to add the weighed

radical to a fresh tube and add the urea / glycerol solution to it.) 5. Add Gd-DOTA and mix 6. Test on HyperSense and adjust radical / urea-glycerol conc. as needed. 7. Make 400 µl aliquots and store long term in -80

Protocol (Glycerol solutions are much easier to transfer when they’re hot and not so viscous. It also helps to trim the end of a pipette tip to transfer.)

1. Set sonicator bath to ~60°C. 2. Weigh sodium 13C-bicarbonate & transfer to 15 mL Falcon tube 3. Weigh glycerol into 15 mL Falcon tube 4. Dissolve with a combination of vortex mixing and bath sonication with heat. (can

take up to a couple hours with occasional vortexing) 5. Add OX63, or charge separate tube with OX63 and add glycerol-bicarbonate 6. Add Gd-DOTA and mix 7. Test on HyperSense and adjust radical / glycerol-bicarbonate conc. as needed. 8. Store in freezer



b. [1-13C]1,2-glycerolcarbonate(GLC)

Notes: This compound is a precursor for HP 13C-bicarbonate, which is formed via base-catalyzed hydrolysis immediately after dissolution. When dissolving using a HyperSense system, care must be taken in order to ensure full and rapid hydrolysis without excessive loss of CO2 and/or polarization. The amounts of base and acid required for hydrolysis and neutralization should be determined empirically by loading unlabeled GLC and performing mock dissolutions with the HyperSense system, measuring the pH of the resulting solution afterward. While heating is necessary for full hydrolysis, excessive heating has been found to cause permanent reductions in HP 13C-bicarbonate T1. This may be related to metal oxide formation and precipitation from EDTA complexes at the very high pH values required for hydrolysis.

Reference: Korenchan et al, Chem. Comm. 2016, 52:3030-33 Materials: (Makes ~900 uL of prep; scale up/down as needed) 1250 mg 1,2-glycerol carbonate 11.9 M (CIL, catalog #CLM-9675, MW 119.09 g/mol) 18.7 mg OX063 radical 15 mM (or 20.9 mg GE trityl radical 15 mM) 2.7 µL 100 mM Ho-DOTA 0.3 mM Protocol:

1. Weigh out radical in prep tube a. Use antistatic gun to prevent loss of radical

2. Add glycerol carbonate to tube, vortex, spin down in microcentrifuge 3. Sonicate for 10 minutes at room temperature 4. Add Ho-DOTA, mix well 5. Aliquot into 300 uL aliquots, store at -80 °C

Typical build-up in Testbed polarizer (NMR Lab) (5/18/15 prep) • Vol. / mass 38.5 uL/54.0 mg • µwave freq. 94.095 GHz – same as pyruvic acid • Maximum 13.5k (no background, after 3+ hours)

• Time constant 3200 ± 400 s Polarization + dissolution protocol, HyperSense polarizer:

1. Weigh out 70.0 ± 0.2 mg of GLC prep in HyperSense cup, load into polarizer, start polarization

2. Wash HyperSense dissolution system with 1 syringe of 100 mM TRIS buffer + 0.3 mM EDTA, pH 7.8, dry

3. Wash out all flasks, syringe tips, etc. with 0.3 mM EDTA solution prior to use 4. Weigh out 96.0 ± 0.2 mg of NaOH solution, 50-52% wt, transfer to pear-shaped

flask 5. Weigh out 560 ± 5 mg of 1.5 M HCl in 3 mL syringe, place with syringe tip at

bottom of pear-shaped flask 6. Weigh out dissolution buffer: 3.5 g of deionized water + 0.3 mM EDTA in 5 mL

syringe 7. Set up heat gun + room-temperature water cooling bath 8. Inject dissolution buffer into bomb, place end of dissolution line in flask, start

dissolution procedure a. Good to use an injection nozzle to keep dissolution from spraying

throughout flask: cut top half of 3 mL syringe off, attach to Luer lock port connected to tubing that fits over the dissolution line from the HyperSense

9. When dissolution is ejected, heat with heat gun for 10 s at max power, inject HCl into dissolution over ~2-3 s, swirl-mix, place in cooling bath, draw up into syringe for injection

c. [amide-13C,15N]N-(2-Acetamido)-2-aminoethanesulfonicacid(ACES)

Note: The carbamide moiety of ACES will hydrolyze over time in the presence of

strong base. Therefore, it is recommended that the prep is kept at -80 °C when not in immediate use. In addition, the OX063 radical should be prepared at a stock concentration in DMSO (200 mg/mL) and mixed with the ACES prep in the cup just prior to hyperpolarization. The prep should be copolarized with urea to provide a chemical shift reference for pH quantification.

Reference: Flavell et al, Chem. Comm. 2015, 51:14119-22 Materials: (Makes ~800 uL of prep; scale up/down as needed) 600 mg [amide-13C,15N] ACES (MW 184.2 g/mol) ~2.8 M 300 uL 10 M NaOH ~2.66 M (0.95 eq) 0.9 uL 500 mM Gd-DOTA 0.5 mM OX063 radical in DMSO, 200 mg/mL 20 mM (final, in prep) Protocol:

1. Weigh out ACES in glass bottle 2. Add NaOH, use light heat gun or sonication until fully dissolved 3. Aliquot into 200 uL aliquots, store at -80 °C

Before polarization: 4. Add 43 uL of ACES to cup 5. Add 7 uL of OX063 in DMSO to cup, mix by pipetting 6. Freeze compound in LN2, add 20 uL of urea prep on top of frozen sample

Typical build-up in HyperSense OX9 polarizer (Surbeck Lab) • Vol. / mass 50 uL • µwave freq. 94.084 GHz (~0.010 GHz lower than pyruvate) • Maximum 5.5k • Time constant 1660 s

d. [2-13C,D10]diethylmalonicacid(DEMA)

Reference: Korenchan et al, Analyst 2017; DOI: 10.1039/C7AN00076F Materials: (Makes ~800 uL of prep; scale up/down as needed) 515.4 mg [2-13C,D10]DEMA (MW 171.2 g/mol) ~3.8 M 514.7 mg N,N-dimethylacetamide 16.7 mg OX063 radical 15 mM 3.2 uL 500 mM Gd-DOTA 2 mM Protocol:

1. Preheat sonicator bath to 40 °C 2. Weigh out DEMA in prep tube 3. Add N,N-dimethylacetamide to tube, sonicate at 40 °C for 10 minutes 4. Weigh out OX063 radical on weigh paper 5. Add OX063 to tube, sonicate at 40 °C for 10 minutes 6. Add Gd-DOTA, vortex to mix 7. Aliquot into 200 uL aliquots, store at -80 °C

Typical build-up in testbed polarizer (11/1/16 prep) • Vol. / mass 25 uL / 25.1 mg • µwave freq. 94.105 (~10 MHz higher than pyruvate) • Maximum 1500 • Time constant 2250 s

e. [2-13C,D4]cyclopropane-1,1-dicarboxylicacid(CPDA)

Reference: Korenchan et al, Analyst 2017; DOI: 10.1039/C7AN00076F Materials: (Makes ~600 uL of prep; scale up/down as needed) 299.0 mg [2-13C,D4]CPDA (MW 171.2 g/mol) ~4.0 M 328.0 mg Dimethyl sulfoxide (DMSO) 13.0 mg OX063 radical 15 mM Protocol:

1. Preheat sonicator bath to 40 °C 2. Weigh out DEMA in prep tube 3. Add DMSO to tube, sonicate at 40 °C for 10 minutes 4. Weigh out OX063 radical on weigh paper 5. Add OX063 to tube, sonicate at 40 °C for 10 minutes 6. Aliquot into 200 uL aliquots, store at -80 °C

Typical build-up: not available

4) MetalIonProbes

a. [1-13C]cysteine

Note: The compound is assumed to dimerize over time in basic conditions, forming

cysteine (this has not been rigorously tested, although it leads to an undesired 13C peak in the NMR spectrum). Therefore, it is recommended that the prep is made fresh immediately prior to use. The prep may also be able to be stably kept at -80 °C when not in immediate use, but this has not been verified yet.

Reference: Wang et al, Chem Eur J 2019; DOI: 10.1002/chem.201902771 Materials: (Makes ~100 uL of prep; scale up/down as needed) 30.5 mg [1-13C]cysteine (CIL #CLM-3852-PK,

MW 122.16 g/mol) ~2.3 M 31.25 µL Hydrochloric acid, 4N ~1.15 M (0.5 eq) 61 mg Glycerol 3.14 mg OX063 radical 20 mM Protocol:

1. Weigh out Cys in glass bottle 2. Weigh out OX063 on weigh paper, add to bottle 3. Add HCl to bottle 4. Add glycerol to bottle 5. Heat with heat gun for about 5 seconds, then vortex to mix 6. (optional) Sonicate at room temperature for 5 minutes 7. Store at -80 °C or use immediately

Typical build-up in HyperSense polarizer (3/2/20 prep) • Vol. / mass 110 mg • µwave freq. 94.081 (~2 MHz lower than pyruvate) • Maximum 2300 • Time constant 1227 ± 30 s

b. [1-13C2]iminodiaceticacid(IDA)

Reference: Wang et al, Chem Eur J 2019; DOI: 10.1002/chem.201902771 Materials: (Makes ~80 uL of prep; scale up/down as needed) 27.6 mg [1-13C2]iminodiacetic acid (MW 135.10 g/mol) ~1.7 M 40 µL Deuterium oxide (D2O) 20 µL Dimethyl sulfoxide (DMSO) 1.9 mg OX063 radical 15 mM Protocol:

1. Weigh out IDA in bottle 2. Weigh out OX063 radical on weigh paper, add to bottle 3. Add D2O to bottle 4. Add DMSO to bottle 5. Heat for about 5 seconds using heat gun, then vortex to mix 6. (optional) Sonicate at room temperature for 5 minutes 7. Store at -80 °C

Typical build-up in HyperSense polarizer (2/7/18 prep) • Vol. / mass 72 mg • µwave freq. 94.080 (~3 MHz lower than pyruvate) • Maximum 1050 • Time constant 1480 ± 38 s

5) RedoxProbes

a. [1-13C]dehydroascorbicacid(DHA)

Reference: adapted from recipe from Kayvan Keshari; PNAS 2011, 108(46):606-11 Materials (Makes ~600 uL of prep; scale up or down as needed) 240 mg [1-13C] dehydroascorbic acid dimer (Aldrich # Q50980) 2.2 M 374.8 mg (400 uL) Dimethylacetamide (DMA) 14.0 mg OX063 radical 15 mM Protocol

a) Preheat sonication bath to 40 °C b) Charge glass bottle with DHA dimer c) Add DMA, sonicate for 5-10 minutes to dissolve d) Add OX063 radical, sonicate for 10 minutes to dissolve e) Store as 200 µl aliquots in -80

b. [1,3-13C2]acetoacetate(AcAc)

Note: This compound is not commercially available, but can be generated from [1,3-

13C2]ethyl-acetoacetate under conditions of strong base prior to formulating. Note: These procedures have not been validated, as they are not based upon actual lab

notes but from the published paper. Reference: Najac et al, Scientific Reports 2019; DOI: 10.1038/s41598-019-39677-2 Materials: (Makes ~100 µL of prep; scale up/down as needed) To generate AcAc sodium salt: 33.1 mg [1,3-13C2]ethyl-acetoacetate (Sigma #485640, MW 132.14 g/mol) 7.9 M 218.4 µL Water 4000 µL Sodium hydroxide, 1 M solution To formulate sodium AcAc for hyperpolarization: 31.6 mg Sodium [1,3-13C2]acetoacetate (MW 126.06 g/mol, from previous steps) ~2.4 M 25 µL Water 75 µL Dimethyl sulfoxide (DMSO) 2.1 mg OX063 radical 15 mM 1 µL 100 mM Gd-DOTA 1 mM Protocol: To generate AcAc sodium salt:

1. Mix together ethyl-AcAc and water in glass bottle to obtain 1 M solution 2. Add 1 M NaOH solution, incubate at 37° C for 24 hours 3. Place sample in dry ice box to freeze, then immediately lyophilize for 24 hours

To formulate sodium AcAc for hyperpolarization: 4. Mix together water and DMSO in a separate tube 5. Add to lyophilized sodium AcAc, vortex to mix 6. Weigh out OX063 radical on a small piece of weigh paper 7. Add radical to mixture, sonicate at room temperature for 5 minutes 8. Add Gd-DOTA, vortex to mix 9. Store at -80 °C

Typical build-up in HyperSense polarizer

• Time constant 1480 ± 38 s

NecrosisProbes

a. [1,4-13C2]fumaricacid

Reference: after Dave Wilson’s prep Materials 1400 mg [1,4-13C2]fumaric acid (CIL # CLM-4450-0 MW 118.07 g/mol,) 2200 mg DMSO 62 mg GE-Trityl 7.25 µl 500 mM Gd-DOTA Fumaric acid at 4.1 M Protocol

1. Set sonicator bath to ~40°C. 2. Weigh fumaric acid & load into 15 ml plastic conical tube 3. Add DMSO and dissolve by bath sonication & vortex mixing 4. Add Trityl & dissolve by bath sonication & vortex mixing 5. Add Gd-DOTA, mix, test buildup, adjusting radical conc. if needed 6. Make aliquots & store in freezer

Typical build-up in HyperSense OX21 (NMR Lab) (2009-10-05 prep)

• Vol. / mass 35 mg • µwave freq. 94.090 GHz • Maximum 24800 • Time constant 1106 s

13C13C

O

OHO

HO

(NOTE: An [*] indicates a change from last year ... · Reference: Najac et al, Sci Rep 6:31397...

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