Devices, systems and methods for the synthesis and purification of hyperpolarized metabolites from parahydrogen

Abstract

The present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites. The devices, systems, and methods of making hyperpolarized metabolite can be made in a continuous manner, which can be advantageous when collecting an MRI image of a subject. The present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite. The separation can be performed quickly and faster than other methods. The present disclosure also provides for contrast agents and methods of imaging using contrast agents.

Description

ATTORNEY DOCKET NO. 222112-2510DEVICES, SYSTEMS AND METHODS FOR THE SYNTHESIS AND PURIFICATION OF HYPERPOLARIZED METABOLITES FROM PARAHYDROGENCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application entitled “A DEVICE FOR CONTINUOUS-FLOW PARAHYDROGEN INDUCED HYPERPOLARIZATION OF METABOLITES IN AQUEOUS SOLUTION” and having Serial No. 63 / 682,431 , filed August 13, 2024, which is herein incorporated by reference in its entirety.

[0002] In addition, this application claims the benefit of U.S. Provisional Application entitled “DEVICES, SYSTEMS AND METHODS FOR THE SYNTHESIS AND PURIFICATION OFHYPERPOLARIZED METABOLITES FROM PARAHYDROGEN” and having Serial No. 63 / 760,322, filed February 19, 2025, which is herein incorporated by reference in its entirety.FEDERAL SPONSORSHIP

[0002] This invention was made with government support under Grant Number 2108306, awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] Magnetic Resonance Imaging (MRI) based on proton magnetic resonance is a non-ionizing technique with high anatomic contrast. Unfortunately, the presence of proton background signals (water / fat) and the limited 1H chemical shift range render it unsuitable for chemically selective molecular imaging. Thus, there is a need in the art to address these and other issues.SUMMARY

[0004] The present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites. The devices, systems, and methods of making hyperpolarized metabolite can be made in a continuous manner, which can be advantageous when collecting an MRI image of a subject. The present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite. The separation can be performed quickly and faster than other methods. The present disclosure also provides for contrast agents and methods of imaging using contrast agents.ATTORNEY DOCKET NO. 222112-2510

[0005] The present disclosure provides for continuous flow hydrogenation reactor devices, comprising: a hydrogenation reactor, wherein hydrogenation reactor is configured to continuously form a first mixture from a precursor solution and parahydrogen; a spin order transfer device, wherein the spin order transfer device is in fluidic communication with the hydrogenation reactor and is configured to receive the first mixture, wherein a hyperpolarized metabolite is produced in the spin order device from the first mixture, wherein a hyperpolarized metabolite solution includes the hyperpolarized metabolite; a reagent introduction device in fluidic communication with the spin order transfer device, wherein one or more reagents are added to the hyperpolarized metabolite solution to form a modified hyperpolarized metabolite liquid; one or more membrane separator devices in fluid communication with the spin order transfer device and the reagent introduction device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase, wherein each of the one or more membrane separator devices includes one or more membrane separators; and a collection system in fluidic communication with the membrane separator device, wherein the collection system receives the hyperpolarized metabolite aqueous solution.

[0006] The present disclosure provides for methods for making a hyperpolarized metabolite aqueous solution, comprising: providing a modified hyperpolarized metabolite solution, wherein the modified hyperpolarized metabolite solution comprising a hyperpolarized metabolite, a solvent, and one or more reagents; separating the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase, wherein the separation is performed using a membrane separator device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase; a collecting the hyperpolarized metabolite aqueous solution.

[0007] The present disclosure provides for contrast agents, comprising the hyperpolarized metabolite as described herein and produced using the method as described herein.

[0008] The present disclosure provides for methods for detecting a disease state associated with abnormal concentration or abnormal activity of a precursor compound metabolite in a subject, the method comprising: (a) administering the contrast agent as described herein to the subject; and (b) detecting the contrast agent in the subject.ATTORNEY DOCKET NO. 222112-2510

[0009] The present disclosure provides for methods that employ a hydrophilic membrane for liquid / vapor separation for continuous flow stripping of residual volatile organics dissolved in the aqueous buffer containing the hyperpolarized metabolite.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0011] Fig. 1A illustrates a flow-chemistry process for synthesis of hyperpolarized metabolites incorporating hydrogenation of the ester precursor with pH2 in acetone spin order transfer (SOT), hydrolysis with NaOD, phosphate buffering, mixing with methyl t-butyl ether (MTBE), continuous-flow liquid-liquid separation and continuous-flow stripping of residual volatile organic compounds using a liquid-vapor membrane separator. Fig. 1 B illustrates a diagram showing the operating flow path of the concentric annular liquid-liquid separator. Fig. 1C illustrates a cross-sectional diagram of Zaiput Flow Technologies’ patented membrane separator, which can provide continuous separation of an immiscible phase (liquid-liquid or gas-liquid) by leveraging differences in wetting properties of the liquids onto a porous membrane.

[0012] Fig. 2 illustrates specific and generalized chemical structures of unsaturated side-arm ester precursors to which the devices, systems, and methods for the synthesis and purification of hyperpolarized metabolites from parahydrogen.

[0013] Fig. 3A illustrates timing diagrams for coherence transfer after adiabatic transport of ethyl or allyl ester adducts of pH2 from strong to weak coupling followed by selective INEPT or MINERVA NMR pulse sequences. Fig. 3B illustrates hyperpolarized NMR spectra for APd, selectively deuterated allyl pyruvate ester (preliminary data), where a 13C signal enhancement of 14,500 was observed, corresponding to a spin polarization of 12% (24% for APd2).

[0014] Fig. 4 illustrates numerical density matrix simulations of 13C polarizations resulting from application of MINERVA or INEPT coherence transfer pulse sequences to various hydrogenation adduct molecules after PASADENA or ALTADENA preparation.

[0015] Fig. 5 illustrates comparison of the droplet size distribution resulting from passage of a liquid through either an ultrasonically vibrating nozzle or a conventional spray injection nozzle.

[0016] Fig. 6A illustrates a representative flow separation scheme combining three Zaiput membrane separation devices, each fitted with a hydrophobic membrane, where the water / acetone / metabolite solution is mixed with an extraction solvent (e.g., MTBE) before the first stage, and the liquid emerging from non-wetting port is mixed with fresh extractionATTORNEY DOCKET NO. 222112-2510 solvent after each consecutive separation device. Residual volatile organic compounds are removed using a solvent stripping method such as the one described in part C.

[0017] Fig. 6B illustrates an alternative counter-current configuration using three stages of membrane separation which provides optimal extraction efficiency using a fixed volume of extraction solvent (MTBE).

[0018] Fig. 6C illustrates a method for stripping of residual dissolved extraction solvent (e.g., MTBE) and acetone or other volatile compounds in the aqueous phase by mixing with nitrogen gas to form a biphasic gas / liquid mixture. After diffusion of the volatile organic compounds into the gas phase, the vapor is removed by a membrane separation device. In the scheme shown, where a hydrophilic membrane is employed, the vapor is the non-wetting phase, and the aqueous phase containing the metabolite is the wetting phase.

[0019] Fig. 7 illustrates preliminary results demonstrating the similar performance in the extraction of acetone from a 1 :1 v / v acetone / water mixture containing 200 mM Na pyruvate into methyl t-butyl ether (MTBE) using (a) conventional gravimetric extraction (dashed curves) and (b) flow separation using a Zaiput SEP-10 device fitted with a hydrophobic membrane (solid curves). Syringe pump flow rates of 3 ml / min for MTBE and 1.5 ml / min for the 1 :1 v / v acetone / water mixture were used. The liquids were mixed using an IDEX mixing tee fitted with a 10 pm frit followed by a 1 m section of 1 / 16 in (O.D.) green PEEK tubing.

[0020] Fig. 8A illustrates a generalized experimental flow chemistry system incorporating spin order transfer in NMR #1 and spectroscopic analysis in NMR #2. Following the multi-stage liquid-liquid (LL) extraction of the organic solvent, residual volatile organic solvents (VOCs) are removed from the aqueous phase by mixing with nitrogen gas, diffusion of the VOCs into the nitrogen gas phase, and vapor-liquid (VL) separation using a hydrophilic membrane at elevated temperature.

[0021] Fig. 8B illustrates separation of the hyperpolarized metabolite dissolved in the aqueous phase from the original using a hydrophobic membrane. Removal of the dispersing medium by diffusion across the membrane fosters coalescence of the dispersed phase, shown in blue.

[0022] Fig. 9A and 9B illustrate the 300 MHz 1H NMR spectra acquired after hydrolysis of ethyl pyruvate and extraction into the aqueous phase by either batch or continuous-flow separation processes. Fig. 9A illustrates the pre- and post-hydrolysis spectra obtained after addition of aqueous sodium carbonate solution to the ethyl pyruvate solution (in acetone-d6). Fig. 9B illustrates spectra of the aqueous fraction after addition of aqueous base and separation by either gravimetric (orange spectrum) or continuous-flow LL separation using a Zaiput SEP- 10 device fitted with a hydrophobic membrane.

[0023] Fig. 10 illustrates schemes 1-3.ATTORNEY DOCKET NO. 222112-2510

[0024] Figs. 11 A and 11 B illustrate gravimetric extraction of acetone from water-acetone mixture under various conditions. The initial volume ratio of solvent to water to acetone was roughly 4:1 :1 . Fig. 12A illustrates the residual organic solvent amounts (mass fractions) after extraction with different solvents at room temperature. Anisole performs best. Fig. 11 A illustrates extraction with anisole at different temperatures. The performance improves with increasing temperature. An extrapolation (dashed black line) after two simulated extractions is overlaid on the plot.

[0025] Figure 12 illustrates hydrolysis of allyl acetate under various conditions. All samples were vigorously mixed twice to ensure reaching chemical equilibrium. (A) Comparison of heterogeneous hydrolysis of allyl acetate (i.e. , ester and base initially in different phases) in various organic solvents with homogeneous hydrolysis in acetone, which is miscible with water at all compositions. Pentane performed best, but the yield is still below 20%. (right) Heterogeneous hydrolysis of allyl acetate in cyclohexane-water mixture. Increasing the volume ratio between aqueous and organic phase greatly im- proved hydrolysis yield up to 50%. Note that NaOD as a base performed better than Na CO .no match of homogeneous hydrolysis in acetone-water mixtures.DETAILED DESCRIPTION

[0026] The present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites. In an aspect, the devices, systems, and methods of making hyperpolarized metabolites can be made in a continuous manner. The present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0028] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value inATTORNEY DOCKET NO. 222112-2510 that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0030] The present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites. The present disclosure also provides for contrast agents and methods of imaging using contrast agents.

[0031] Definitions:

[0032] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a heteronucleus,” “a metabolite,” or “a parahydrogen atom,” include, but are not limited to, mixtures or combinations of two or more such heteronuclei, metabolites, or parahydrogen atoms, and the like.

[0033] The term “heteronucleus” refers to any atomic nucleus other than the proton; e.g., 13C, 15N, 31 P, 19F, 2H, or 29Si.

[0034] The term “polarization” refers to the difference in fractional populations of two spin states (for example, the spin-up and spin-down quantum states of the proton, denoted Polarization of a spin-1 / 2 particle is defined as: where A'^ andA'^ are the numbers of particles in the spin-up and spin-down states, respectively (See Fig. 1D).

[0035] The term “hyperpolarization” refers to a non-Boltzmann thermal equilibrium nuclear spin order resulting in nuclear magnetic resonance signal enhancement.

[0036] The phrase “hyperpolarized fluid sample” refers to a liquid or gas containing target molecules hosting hyperpolarized nuclear spins.

[0037] The phrase “precursor solution” refers to a liquid solvent or solvent mixture containing a catalyst compound or catalyst nanoparticles together with the hydrogenation substrate molecules (precursor compound) that incorporate at least one unsaturated chemical moiety (e.g. a double bond, a triple bond, a carbonyl group, or a hydroxy group) to which one or more protons of H2 may be transferred, or alternatively, that becomes hyperpolarized through a spin order transfer mechanism, e.g. by Signal Amplification by Reversible Exchange (SABRE), Surface Waters are Magnetized from Parahydrogen (SWAMP), Nuclear Exchange Polarization by Transposing Unattached Nuclei (NEPTUN), chemical exchange of hyperpolarized protons, or through sharing of parahydrogen spinATTORNEY DOCKET NO. 222112-2510 order to other protons or heteronuclei via spin-spin couplings, or spin exchange mediated by passage through level anti-crossings that enhance the mixing of the spin states of the nuclei.

[0038] The term “parahydrogen” refers to the metastable spin isomer of dihydrogen with proton spins in a singlet state that is antisymmetric with respect to permutation of the two protons. For simplicity, the term parahydrogen will in some cases, depending on the context, also refer to dihydrogen gas that is only partially enriched in the parahydrogen spin isomer content relative to normal hydrogen, which is about 25% parahydrogen and about 75% orthohydrogen (the triplet state, which is symmetric with respect to permutation of the two protons).

[0039] The term “heteronuclei” will refer to the spin-1 / 2 isotopes other than the proton, including carbon-13, nitrogen-15, fluorine-19, and phosphorus-31.

[0040] “INEPT” or “insensitive nuclei enhancement by polarization transfer” refers to a signal enhancement method used in nuclear magnetic resonance spectroscopy and magnetic resonance imaging. An INEPT pulse sequence can be used to transfer nuclear spin polarization from protons, including, but not limited to, atoms originating in parahydrogen, to a heteronucleus.

[0041] “MRI” refers to magnetic resonance imaging, the processing of collecting the spatial distribution of nuclear spins in an object, specimen, or patient.

[0042] “MINERVA” or “(Maximizing Insensitive Nuclear Enhancement Reached Via para-hydrogen Amplification)” refers to a coherence transfer pulse sequence for converting bilinear spin order generated by the PASADENA, ALTADENA, or LACADENA effects into hyperpolarized magnetization of a heteronucleus.

[0043] “Side Arm” refers to an unsaturated molecule containing a double or triple bond that is attached by an ester linkage to another molecule, which may be a carboxylic acid (or carboxylate) metabolite. The side arm provides the chemical unsaturation necessary for incorporation of parahydrogen by chemical hydrogenation reaction. Examples of side arms are provided in Scheme 1 (Fig. 10).

[0044] “Side Arm Hydrogenation” (SAH) refers to hydrogenation of the double or triple bond of the side arm. After spin order transfer of parahydrogen spin order to one or more nuclear spins of the metabolite, the side arm is removed by chemical hydrolysis reaction by addition of aqueous base or acid to yield the hyperpolarized molecule or metabolite of interest, and in this way the spent side arm is removed.Discussion

[0045] The present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites. In an aspect, the devices, systems, and methods of making hyperpolarized metabolite can be made in a continuous manner, which is advantageousATTORNEY DOCKET NO. 222112-2510 when collecting an MRI image of a subject (e.g., animal, mammal (e.g., dog, cat), human,). The present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite. The separation can be performed quickly. For example, the separation can be performed in about 5-15 seconds per extraction, which is between 1x and 10x, 2x to 10x, or 4x to 10x faster than other methods. The present disclosure also provides for contrast agents and methods of imaging using contrast agents. Additional details are provided in the Examples and Attachment.

[0046] Nuclear spin hyperpolarization (HP) techniques have ushered in a new era of chemically selective magnetic resonance imaging (MRI) and localized nuclear magnetic resonance (NMR) spectroscopy. By inducing high-field NMR signal enhancements that can exceed five orders of magnitude, metabolites and other biomolecules become visible despite their low in vivo concentrations, thereby enabling disease detection and treatment response monitoring without exposure to ionizing radiation. Parahydrogen (pH2) is a convenient source of singlet nuclear spin order that can be rapidly transformed into MRI-observable proton hyperpolarization through symmetry-breaking hydrogenation chemistry. For metabolites like pyruvic acid, the requisite unsaturation for pH2 addition can be incorporated by synthesis of the vinyl or propargyl ester, as well as those shown in Fig. 2. After hydrogenation with pH2, the non-equilibrium spin order is transferred to the carbonyl 13C to render, after hydrolytic cleavage, a hyperpolarized molecule that was not directly producible as the product of pairwise hydrogenation. After hydrogenation and cleavage of the side arm, the hyperpolarized metabolite can be separated from the solvent quickly and efficiently. The metabolite NMR signals obtained from nuclear spins in this hyperpolarized state can be many orders of magnitude stronger than the signals obtained when the nuclear spins are initially at thermal equilibrium.

[0047] The present disclosure provides for a continuous flow hydrogenation reactor. The continuous flow hydrogenation reactor will first be described generally and then in more detail. The continuous flow hydrogenation reactor can include a hydrogenation reactor, a spin order transfer device, a reagent introduction system, one or more membrane separator devices, and a collection device. Fig. 6A in the Example section illustrates an embodiment of the reactor.

[0048] In an aspect, the hydrogenation reactor is configured to continuously form a first mixture from precursor solution (e.g., a catalyst, precursor compound, a solvent) and parahydrogen. The parahydrogen can be introduced in one or more ways, for example using a spray injection nozzle as described herein. The hydrogenation reactor can be configured as a homogeneous hydrogenation device or system or a heterogeneousATTORNEY DOCKET NO. 222112-2510 hydrogenation device or system. Examples of each of these are described in the Example section.

[0049] The hydrogenation reactor is in fluidic communication with a spin order transfer device. In an aspect, the spin order transfer device is configured to receive the first mixture. The spin order transfer device is configured to form a hyperpolarized metabolite from the first mixture, where a hyperpolarized metabolite solution includes the hyperpolarized metabolite. The hyperpolarization is described herein and in the Examples.

[0050] In an aspect, a reagent introduction device is in fluidic communication with the spin order transfer device. One or more reagents are added to the hyperpolarized metabolite solution to form a modified hyperpolarized metabolite solution. The one or more reagents can include acetone, an aqueous base or acid, a phosphate buffer, and a methyl t- butyl ether (MTBE) solvent. Additional details regarding the reagents are described below and in the Examples.

[0051] In an aspect, a membrane separator device (or a plurality of membrane separator devices in serial connection) is in fluid communication with the spin order transfer device and the reagent introduction device. The membrane separator device includes one or more membrane separators. The membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase. Additional details regarding the membrane separator device are provided herein and in the Examples.

[0052] In an aspect, a collection system is in fluidic communication with the membrane separator device(s). The collection system receives the hyperpolarized metabolite aqueous solution. In an aspect, the collection system can independently collect the organic solvent phase, which is kept separate from the hyperpolarized metabolite aqueous solution.

[0053] Each of the components of the continuous flow hydrogenation reactor can be in fluidic communication as provided using tubing, flow meters, flow controllers, in-line pumps, syringe pumps, back-pressure regulators, check-valves, pneumatic switching valves, manual valves, and the like.

[0054] In regard to the membrane separator device, the membrane separator(s) can be a liquid-liquid membrane separator that separates the organic solvent(s) from the modified hyperpolarized metabolite aqueous solution by phase separation using differential surface wetting. In an aspect, the membrane separator includes a membrane having hydrophobic surface with pores extending from one side of the membrane to the other side of the membrane so that fluid can pass through the membrane; optionally, wherein the fluid is the organic non-polar solvent phase. Alternatively, the membrane separator includes a membrane having hydrophilic surface with pores extending from one side of the membraneATTORNEY DOCKET NO. 222112-2510 to the other side of the membrane so that fluid can pass through the membrane, optionally, wherein the fluid is the hyperpolarized metabolite aqueous solution. The membrane separator device can include a pressure system or backpressure regulator that maintains a pressure differential across the membrane. The pressure differential is adjusted to maximize the fluid transport through the pores to produce the separation, while preventing unwanted breakthrough of the aqueous phase through the membrane. Additional details regarding the membrane separator device are provided in the Example.

[0055] In an aspect, the membrane separator device(s) is configured to separate the organic solvent from the hyperpolarized metabolite solution to form a purified aqueous solution of the hyperpolarized metabolite (also referred to as the “hyperpolarized metabolite aqueous solution”) and a separate solution containing organic solvents (e.g., MTBE, acetone), the spent side arm alcohol, and catalyst residues. The liquid-liquid membrane separation can be performed about 1 to 10, about 2 to 10, about 4 to 10, about 6 to 10, about 8 to 10, or about 10 times faster than a continuous flow hydrogenation reactor having a gravimetric separation device (all else being equal with only the substitution of the gravimetric separation device for the membrane separator device). In particular, the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase in about 2 to 15 seconds per extraction.

[0056] In an aspect, the spin order transfer device can include comprising an adiabatic transport tube, wherein the adiabatic transport tube is configured to receive the first mixture from the hydrogenation chamber.

[0057] In an aspect, the spin order transfer device can include one of the following: an NMR spectrometer configured for field cycling of the static magnetic field or radiofrequency field; an NMR spectrometer configured for coherence transfer radiofrequency pulse sequences; or an NMR spectrometer configured for adiabatic field cycling in conjunction with coherence transfer radiofrequency pulse sequences; or an NMR spectrometer configured for amplitude ramps and / or frequency sweeps of the radio-frequency fields

[0058] Now having described the continuous flow hydrogenation reactor in general, methods of the present disclosure are described in general, where additional details are provided herein.

[0059] The present disclosure provides for a method for making a purified aqueous solution of hyperpolarized metabolites. The method includes providing a modified hyperpolarized metabolite solution. The modified hyperpolarized metabolite solution comprising a hyperpolarized metabolite (the solute), a solvent, and one or more reagents. In an aspect, the method provides for the hyperpolarized metabolite solution and the firstATTORNEY DOCKET NO. 222112-2510 mixture as described herein, where the first mixture and hyperpolarized metabolite solution are processed to form the modified hyperpolarized metabolite solution.

[0060] The method includes separating the organic solvent phase from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a separated organic solvent phase. The separation is performed using a membrane separator device as described above and herein. The membrane separator device is configured to continuously receive the liquid mixture including the hyperpolarized metabolite molecule, the hydrogenated side-arm adduct, water, salts and buffers, and organic solvents. The membrane separates this mixture into a modified hyperpolarized metabolite in aqueous solution and the non-polar organic solvent phase. Then the method provides for separately collecting the hyperpolarized metabolite in aqueous solution as well as the solvent phase.

[0061] The present disclosure also provides for a contrast agent including the hyperpolarized metabolite described herein as well as the hyperpolarized metabolite produced using the methods described herein.

[0062] In an aspect, the present disclosure provides for methods that employ a hydrophilic membrane for liquid / vapor separation for continuous flow stripping of residual volatile organics (e.g., acetone, MTBE, or a combination thereof) dissolved in the aqueous buffer containing the hyperpolarized metabolite. In an aspect, the membrane strictly separates liquid and gas phases and neither chemical reactions nor accumulation of solvents or solutes occurs inside the membrane pores. In an aspect, a carrier gas (e.g., nitrogen, N2) is used that does not appreciably dissolve in water (and water does not appreciably diffuse into the gas). Therefore, the volatile organic solvents (e.g., MTBE and acetone) diffuse from water into the gas phase during their contact in the biphasic gas / liquid mixture, and then the gas is removed from the membrane separator device through the nonwetting outlet port, while the aqueous buffer containing the hyperpolarized metabolite is collected from the wetting port of the separator device with the hydrophilic membrane installed.

[0063] Now having described the devices, systems, and methods generally, additional details are provided.

[0064] In another aspect, the hydrogenating can conducted in a magnetic field of from about 0 to 1 pT, or 1 pT to 100 pT, or 100 pT to 2 T, 0.25 to 2 T, or of about 0, 0.25, 0.5, 0.75, 1 , 1.25, 1.5, 1 .75, or about 2 T, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.

[0065] In another aspect, the hydrogenation reaction can include contacting the precursor compound and the supply of parahydrogen with a hydrogenation catalyst. In one aspect, the hydrogenation catalyst can be selected from or include Pt, Pd, Cu, Au, Ag, Rh, Ru, Ir, Ni, Sn, Co, Zn, Ce, Ti, Al, Fe, Si, or any combination thereof. In some aspects theATTORNEY DOCKET NO. 222112-2510 hydrogenation catalyst can be in nanoparticle form or in liquid form, as a suspension, dispersion, a colloid, or emulsion.

[0066] In one aspect, the precursor compound can be an unsaturated ester of a Cn (n>0) carboxylic acid, where n can be 2 to 20, 2 to 10, 2 to 8, 2 to 6, 4 to 10, or 4 to 20. In another aspect, the unsaturated ester can be a vinyl ester or a propargyl ester. In one aspect, the precursor compound can be vinyl acetate, propargyl pyruvate, or any combination thereof. In one aspect, one or more protons of the precursor compound are replaced with deuteron(s) or R-groups to reduce or eliminate homonuclear spin couplings with the parahydrogen sourced protons.

[0067] In an aspect, at least one heteronucleus of the precursor compound can be a carbon-13 atom, nitrogen-15, phosphorous-31 , or fluorine-19. In a further aspect, at least one heteronucleus of the precursor compound can be a carbon atom double bonded to an oxygen atom. In some aspects, the precursor compound can further include at least one deuterium.

[0068] In one aspect, in the disclosed method, the ester can be hydrolyzed after hydrogenating the precursor compound.

[0069] In an aspect, spin is transferred from the first parahydrogen atom or the second parahydrogen atom or simultaneously from both hydrogen atoms to the third hydrogen atom using adiabatic passage through one or more anti-crossings of the nuclear spin energy levels. Further in this aspect, adiabatic passage can be accomplished via exposing the precursor compound, after hydrogenation to form the precursor adduct molecule, to a continuously increasing magnetic field until the detection magnetic field is reached, with a strength greater than about 0.25 T.

[0070] In one aspect, spin can be transferred from the third hydrogen atom to at least one heteronucleus using a suitable coherence transfer pulse sequence, e.g., selective or non-selective insensitive nuclei enhancement by polarization transfer (INEPT) or the radio frequency pulse sequence known as MINERVA.

[0071] In one aspect, the process is at least 10% efficient, about 10 % to 80%, about 80% to 100%, 80% to 99%, 80% to 90%, about 90 to 100%. Also disclosed herein are precursor compounds including at least one hyperpolarized heteronucleus produced by the disclosed method or other spin order transfer method as well as contrast agents including the precursor compounds.

[0072] In an aspect, the hydrogenation reactor can include an ultrasonic nozzle. A precursor solution can be introduced to the surface of the ultrasonic nozzle, where the ultrasonic nozzle is configured to produce droplets of the precursor solution. In one aspect, the droplets of the precursor solution have an average diameter of from about 1 to about 50 pm, or of about 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pm, or a combination of anyATTORNEY DOCKET NO. 222112-2510 of the foregoing values, or a range (e.g., about 5 to 50 m, about 15 to 45 pm, about 5 to 20 pm, etc.) encompassing any of the foregoing values. In another aspect, the droplets can have a droplet distribution range of about 25 pm or less. In one aspect, the precursor solution can be introduced using a syringe pump configured to deliver an amount of the precursor solution to the surface of the ultrasonic nozzle.

[0073] In one aspect, parahydrogen gas can be introduced into a flowing stream of liquid precursor solution containing the dissolved precursor compound and dissolved or suspended catalyst using an Telfon™ AF 2400 (amorphous fluoroplastic resin, or similar) gas permeable membrane.

[0074] In one aspect, parahydrogen gas can be introduced by merging it with a stream of the liquid precursor solution containing the dissolved precursor compound using a mixing chamber or mixing tee connected to a check valve and a back-pressure regulator. The mixing chamber or mixing tee may be followed by a section of tubing that is sufficiently long to allow thorough mixing and dissolution of the gas into the liquid precursor solution.

[0075] In one aspect, the hydrogenation reaction is catalyzed by a solid, insoluble heterogeneous catalyst material by passing the precursor solution containing the dissolved substrate molecule and dissolved parahydrogen through a packed bed reactor containing the solid catalyst. In such continuous-flow heterogeneous hydrogenation reactions, the parahydrogen gas is introduced by bubbling the parahydrogen through a reservoir of the precursor solution or by passing the precursor solution through a Telfon™ AF 2400 (amorphous fluoroplastic resin, or similar) gas permeable membrane.

[0076] In one aspect, the fluid sample is a homogeneous fluid including the precursor compound and a catalyst. In another aspect, the fluid sample is a heterogeneous fluid including the precursor compound and a particle that includes a catalyst. In one aspect, the catalyst can be a Group VIII, IB, or I IB transition metal-based catalyst including at least two different metals, or may not include a metal. In still another aspect, the catalyst can include at least one of Pt, Pd, Cu, Au, Ag, Rh, Ru, Ir, Ni, Sn, Co, Zn, Ce, Ti, Al, Fe, Si, O, or any combination thereof in the form of an intermetallic compound or an alloy. The particle may be of homogeneous composition or it may have a core-shell structure, where the core of the particle is of one composition, not necessarily catalytically active, and the outer layer with a different composition that exposes catalytically active sites to the solution.

[0077] In one aspect, the catalyst can be a nanoparticle having a diameter of a single metal atom to about 0.5 nm, or 0.5 nm to 1 nm, or 1 nm to 10 nm, or 10 nm to about 500 nm, or of about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 nm, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values. In some aspects, the catalyst can be in liquid form. In one aspect, the catalyst canATTORNEY DOCKET NO. 222112-2510 be a Rh compound, cluster, or nanoparticle, or a Rh alloy, that is supported, tethered, or ligand stabilized in solution, or any combination thereof.

[0078] In another aspect, the precursor compound can be a metabolite or a derivative thereof. In one aspect, the metabolite or derivative thereof can be administered in vitro or to a subject in vivo. In some aspects, the metabolite or derivative thereof can be an unsaturated ester of a C1 to C4 carboxylic acid or C1 to C8 carboxylic acid or C1 to C12 carboxylic acid. In another aspect, the unsaturated ester can be a vinyl ester or a propargyl ester. In one aspect, the metabolite or derivative thereof can be vinyl acetate, propargyl pyruvate, or any combination thereof. In one aspect, at least one heteronucleus of the precursor compound is a carbon-13, nitrogen-15, phosphorous-31 , or fluorine-19 atom, or in general, any spin-1 / 2 isotope

[0079] In one aspect, in the disclosed device or system, the precursor solution can be introduced over a range of speeds, where a specific introduction speed can be selected prior to introducing the precursor solution, and where the introduction speed of the precursor solution is correlated to an amount of hyperpolarization in the fluid sample. In this way, the device or system can operate in a continuous manner.

[0080] In one aspect, disclosed herein is a method for detecting a disease state associated with abnormal concentration or abnormal activity of a precursor compound metabolite in a subject, the method including at least the steps of:(a) administering the contrast agent as described herein or the fluid sample as described herein to the subject; and(b) detecting the contrast agent or the target molecule in the subject.

[0081] In some aspects, the method further includes the step of administering one or more additional contrast agents to the subject, wherein the one or more additional contrast agents can be administered sequentially or simultaneously with the contrast agent or target molecule. In some aspects, detecting is accomplished using magnetic resonance imaging.

[0082] In general, a hyperpolarized metabolite solution can be made by exposing a precursor solution (e.g., including a solvent, precursor compound, and a catalyst) to parahydrogen. The interaction and / or chemical reaction between the molecules in the precursor solution and the parahydrogen in the presence of an appropriate homogeneous or heterogeneous catalyst can produce a hyperpolarized metabolite solution that includes hyperpolarized protons or heteronuclei on the precursor compounds (e.g., metabolites).

[0083] In general, the method of making the hyperpolarized metabolite solution can include making droplets of a particular size having a particular (e.g., narrow) size distribution using ultrasonic energy. The droplets, which have a very high surface area compared to those produced by bubbling or non-ultrasonic spray-injection methods, are then exposed to gaseous parahydrogen. The precursor compound, catalyst, and parahydrogen interactATTORNEY DOCKET NO. 222112-2510 and / or react to form the hyperpolarized fluid sample, where a portion (about 50 to 100%) of the precursor compounds are hyperpolarized. In an aspect, the precursor compound acquires one or more magnetized protons or hydrogen nuclei from parahydrogen by chemical exchange. In another aspect, the spin order of parahydrogen is transferred to the spins in a precursor compound without any chemical hydrogenation of the target molecule, as occurs in the hyperpolarization phenomena commonly known as SABRE (Signal Amplification by Reversible Exchange), SWAMP (Surface Waters Are Magnetized from Parahydrogen), NEPTUN (Nuclear Exchange Polarization by Transposing Unattached Nuclei), or proton chemical exchange, as in the literature methods referred to as PHIP-X or PHIP-RELAY.

[0084] In an embodiment, the reaction chamber can be operated at a temperature of about 25 °C to 300 °C and a pressure between about 1 bar to 100 bar. In an aspect, the reaction chamber includes an ultrasonic nozzle that has a surface. The precursor solution can be delivered to the surface of the ultrasonic nozzle. An ultrasonic nozzle may include a titanium horn body, a crystal / ceramic element with piezoelectric properties protected within a stainless-steel rear and front housing. Piezoelectric elements can include crystalline materials such as quartz, gallium orthophosphate, langasite, lithium tantalate, lithium niobate. Alternatively, piezoelectric ceramics that have been reported are barium titanate, potassium niobate, sodium tungstate, and the most commonly used lead zirconate titanate. Active and ground electrodes on the faces of the piezoelectric elements allow for an electrical connection to an ultrasonic generator. A liquid feed tube is situated through the titanium horn body to the tip of the atomizing surface. The atomizing surface shape can be conical, focused, or flat depending on the desired spray pattern. The production of the droplets results from the inverse piezoelectric effect, where high frequency (acoustic) sound waves are converted to mechanical energy to produce a vibrating surface. Transduction of the vibrations into the liquid phase produces standing waves in the liquid precursor solution and the amplitude of the surface increases until the liquid film becomes unstable and collapses into a fine mist of uniformly sized droplets. The ultrasonic nozzle can operate at a frequency ranging from about 20 kHz to 3 MHz to produce droplets of the precursor solution having diameters between 1 and 50 microns, depending on the operating frequency of a given nozzle. The high surface-to-volume ratio of droplets formed by the ultrasonic nozzle favors rapid diffusion of gaseous parahydrogen across the liquid / gas interface and into the interior of the liquid droplet within a relatively short timescale. The droplet diameter and size distribution can be tailored to maximize the hydrogenation reaction rate, conversion, and resultant hyperpolarization level in the collected fluid sample. The hydrogenation device can have a volume sufficient to produce the desired volume of hyperpolarized fluid sample for in- vivo use, for example. In this regard, the hydrogenation device has a volume of about 1 toATTORNEY DOCKET NO. 222112-251015 ml_. The hydrogenation device can be made of materials such as stainless steel, aluminum, polysulfone, vespel, polytetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), or polyether ether ketone (PEEK). In an aspect, the ultrasonic nozzle is located at the top of the hydrogenation device and a collection vessel is located at the bottom of the hydrogenation device to collect the droplets as they move through the reaction chamber. The hydrogenation device can have a length of about 2 to 6 inches and a width or diameter of about 1 to 3 inches.

[0085] The parahydrogen is introduced to the reaction chamber using a gas introduction system that is in gaseous communication therewith. The gas introduction system can be configured to controllably introduce parahydrogen into the reaction chamber. The gas introduction device can include appropriate equipment to acquire (if part of a different system) and / or flow the parahydrogen to the reaction chamber. For example, the gas introduction system can include tubing, flow valves, pressure gauges, pressure regulators, syringe pumps, in-line pumps, HPLC pumps, peristaltic pumps, thermocouples, flow meters, and the like to control introduction into the holding vessel through the inlet port.

[0086] In an aspect, the sample can be a homogeneous fluid including the solvent, the precursor compound, and a catalyst, where the catalyst is dissolved in the fluid. In another aspect, the sample, the sample is a heterogeneous fluid including the precursor compound and a particle comprising a catalyst, where the catalyst is insoluble in the fluid or is in the form of a solution, suspension, colloid, or emulsion of nanoparticles.

[0087] In an aspect, the fluid can be an aprotic solvent. The aprotic solvent can include: dioxane, nitromethane, acetonitrile, acetone, dichloromethane, dimethylformamide (DMF), dimethylsulfoxide (DMSO) or a combination thereof. In an aspect, the aprotic solvent can be a perdeuterated and partially deuterated form of each of the solvents listed above or herein. In an embodiment, the fluid is perdeuterated or partially deuterated water and is diluted in the aprotic solvent. In an embodiment, the fluid is biphasic, including immiscible polar and non-polar liquids.

[0088] In an aspect, the amount of catalyst in the precursor solution and in each droplet should be sufficient to provide enough active sites to accommodate hyperpolarization of the desired number of target molecules. Ideally, the total number of active sites of the catalyst in the volume of precursor solution should be sufficient to allow complete conversion of all precursor compounds to hyperpolarized molecules. Concentrations of precursor compounds, parahydrogen, and catalyst are appropriate for hyperpolarization either by exchange of one or more magnetized protons from adsorbed parahydrogen or by non-hydrogenative mechanisms. In the case of catalyst nanoparticles, the amount of catalyst required can depend on the particle size, as the surface to volume ratio scales as 1 / r, where r is theATTORNEY DOCKET NO. 222112-2510 particle radius (assuming a spherical particle shape), the surface composition, and the rate of exchange, as well as the type of catalyst.

[0089] When a range is expressed, a further aspect includes from one particular value and / or to other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0090] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0091] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.EXAMPLES

[0092] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviationsATTORNEY DOCKET NO. 222112-2510 should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1

[0093] Magnetic Resonance Imaging (MRI) is a non-ionizing technique with superior anatomic contrast compared to traditional clinical imaging techniques, such as positron emission tomography (PET). Unfortunately, the presence of strong proton background signals (water / fat) and the limited chemical shift range of 1 H MRI render it unsuitable for chemically selective molecular imaging of metabolic activity. Despite its major inadequacies, which include exposure to ionizing radiation, cost, resolution, lack of anatomical detail, and limited availability (radioactive 18F is produced in cyclotrons and has a relatively short halflife of 110 min), 18F PET is in widespread clinical use to detect anomalous metabolic uptake of fluorodeoxyglucose. MRI based on carbon-13 (13C) would offer the requisite chemical specificity for imaging metabolic dysfunction, but magnetic field polarized 13C transitions are too weak to be detected due to the unfavorable Boltzmann distribution and low physiological concentrations. The sensitivity limitation can be overcome by hyperpolarized MRI which can intensify 13C NMR signals by more than five orders of magnitude, 1-3 and parahydrogenbased hyperpolarization is faster, cheaper, and more reliable than competing hyperpolarization techniques while providing comparable molar polarization levels.

[0094] To further advance the broad applicability of hyperpolarized MRI, the present disclosure provides for a new methodology, assisted by specifically designed catalyst materials, for continuous synthesis of parahydrogen-hyperpolarized metabolites in aqueous buffer. The present disclosure concerns the recent advances enabling pH2-based hyperpolarization of discrete batches of [1-13C] pyruvate and other metabolites. While the batch synthesis of hyperpolarized metabolites is largely a solved problem, continuous-flow hyperpolarization requires new methods for flow chemistry, spin order transfer, and liquidliquid extraction. This challenging but important goal will overcome a major limitation of existing batch hyperpolarization methods; specifically, the MRI image after injection must be acquired on a timescale defined by the spin relaxation time (T1) of the tracer, necessitating the use of fast-imaging pulse sequences. Continuous intravenous administration would replenish the hyperpolarization and facilitate signal averaging and k-space sampling over extended acquisition periods, as in conventional MRI. The present disclosure provides devices and methods for continuous-flow synthesis of hyperpolarized [1-13C] pyruvate, as envisaged in Fig. 1A and 1 B, at a molar polarization level comparable to state-of-the-art batch production protocols, affording further gains in sensitivity and resolution of hyperpolarized 13C MRI.ATTORNEY DOCKET NO. 222112-2510

[0095] Fig. 1 A illustrates a flow-chemistry process for synthesis of hyperpolarized metabolites incorporating hydrogenation of the ester precursor with pH2 in acetone spin order transfer (SOT), hydrolysis with NaOD, phosphate buffering, mixing with methyl t-butyl ether (MTBE), continuous-flow liquid-liquid separation and continuous-flow stripping of residual volatile organic compounds using a liquid-vapor membrane separator.

[0096] Fig. 1 B illustrates a diagram showing the operating flow path of the concentric annular liquid-liquid separator. The organic solvent is the wetting phase, the aqueous solution of the hyperpolarized metabolite is the non-wetting phase. Pper = permeate channel back pressure; Pret = retentate channel back pressure.

[0097] Fig. 1C illustrates a cross-sectional diagram of Zaiput Flow Technologies’ patented membrane separator, which can provide continuous separation of an immiscible phase (liquid-liquid or gas-liquid) by leveraging differences in wetting properties of the liquids onto a porous membrane. The aqueous phase consists of the hyperpolarized metabolite in the form of a carboxylate ion, while the wetting phase consisting of MTBE and acetone, is removed using a hydrophobic membrane

[0098] Hydrogen has two spin isomers: parahydrogen (pH2) and orthohydrogen (oH2). In pH2, spins of the two hydrogen nuclei are antiparallel (opposite), leading to a singlet state with total spin l=0, while in orthohydrogen, the spins are parallel (same direction), in a triplet state, with 1=1 . The density operator for a sample of H2 with pH2 mole fraction xpis ppara= 1 / 4 - 1 / 3 (4xp- 1) • I2, where is the ith spin operator. Owing to the Pauli principle, pH2 and oH2 are associated with symmetric and antisymmetric rotational levels, respectively, 4 and because the energy splitting between the lowest two rotational levels is approximately 170 K, significant para-enrichment occurs even at moderately low temperatures. At 77 K, xp« 0.5, while at 20 K, still above the normal boiling point of H2, xp« 0.99. After flowing the gas over an ortho-para interconversion catalyst (e.g., iron oxide), the para-enriched gas can be warmed to ambient temperature where the enrichment can persist for weeks.

[0099] Parahydrogen-Based Hyperpolarization (PHIP)

[0100] Parahydrogen-based hyperpolarization, or PHIP, is induced by symmetrybreaking interactions that transform the scalar spin order into NMR-observable spin order. In hydrogenative PHIP, the protons in a pH2 molecule add to magnetically inequivalent sites across an unsaturated bond in a substrate molecule, as shown in Scheme 1 (Fig. 10). PASADENA and ALTA-DENA are the two most common variants of hydrogenative PHIP. In PASADENA, 23, 24 hydrogenation is performed at high magnetic field in the weak coupling regime of the parahydrogen-sourced proton pair. For an isolated proton pair, such as in d3- ethyl ester (Scheme 1A, Fig. 10),ATTORNEY DOCKET NO. 222112-2510Pf = ~4~ Iz- z2 where xp= 1 (PASADENA) (1)

[0101] Single quantum PASADENA transitions, consisting of pairs of antiphase doublets centered at each chemical shift, are stimulated by a TT / 4 RF pulse. In ALTADENA, hydrogenation is performed at a low magnetic field in the strong coupling regime where the singlet state is conserved. Adiabatic transport to high field then yields a density operator with Zeeman operator terms in addition to the IzlIz2bilinear operator seen in Eq. (1):(ALTADENA) (2)

[0102] The different operator forms of spin order in Eq (1) and (2) are a key to the versatility of the spin order transfer (SOT) protocol (Section

[0118] ).

[0103] Side-Arm Hydrogenation

[0104] Side-arm hydrogenation (SAH) provides a way of incorporating pH2 into vital metabolites (e.g., pyruvate) that are not directly synthesizable as products of hydrogenation.5, 6 After hydrogenation, the pH2 spin order is transferred via the J coupling across the ester linkage to the carbonyl 13C. Both propargylic and vinylic side arms (Scheme 1. Fig. 10) have been demonstrated.5-13 Vinyl affords the advantage of stronger J couplings (three or four bonds) to the 1-13C carbonyl, but these compounds are relatively unstable and challenging to synthesize. Propargyl esters, in contrast, are stable at ambient temperature, and their triple bonds afford higher rates of hydrogenation. Disadvantageously, the pH2 sourced protons in allyl ester adducts are also further from the 13C target, and so the J-couplings are quite small.

[0105] The processes described herein are applicable to precursors with the specific and generalized chemical structures show in Fig. 2.

[0106] FIG. 2 illustrates specific and generalized chemical structures of unsaturated side-arm ester precursors to which the devices, systems, and methods for the synthesis and purification of hyperpolarized metabolites from parahydrogen, as described herein, are applicable.

[0107] After SOT in an organic solvent, hydrolysis of the ester is induced by addition of aqueous acid or base, yielding the 13C hyperpolarized carboxylic acid. This is followed by addition of a buffer to restore the solution to neutral pH. In the literature, gravimetric phase separation is employed for extraction of the carboxylate species into the aqueous phase.14- 16 Volatile organic solvents can also be removed by evaporation under vacuum.14, 17

[0108] Existing Spin Order Transfer (SOT) Methods

[0109] For context, the current approaches to SOT in SAH-PHIP experiments are briefly reviewed here.

[0110] Magnetic field cycling. Aime et al. used magnetic field cycling for SOT in the first SAH-PHIP 13C NMR experiments.5 In this method, the adduct molecule containing theATTORNEY DOCKET NO. 222112-2510 target heteronucleus is subjected to adiabatic rapid passage through a level anti-crossing (LAC) at sub- zT fields.18 Reported polarization levels of a few percent are not competitive with more recent SOT methods due to dilution of spin order with ancillary protons and other factors. Selective deuteration is not beneficial as relaxation losses are incurred by strong coupling to the rapidly relaxing deuterons at the low fields where the LAC occurs.

[0111] Adiabatic passage through a rotating frame LAC. The SOT method employed in Nagel (Ref. 3, Advanced Science 2023, 10 (30), 2303441) uses adiabatic RF field sweeps at / iT static field.19 Dilution of pH2 spin order due to state mixing with the methylene protons is overcome by deuteration of the propargylic side-arm (Scheme 2, Fig. 10). Cross- relaxation to 2H is suppressed by performing the RF sweeps at a high enough static magnetic field for deuterons and protons to be weakly coupled. In Ref. 3, propargylic esters are employed because of their chemical stability, ease of synthesis, and higher rates of hydrogenation compared to vinyl esters20,21. Disadvantageously, this process method relies on a long- range 1 H-13C J-coupling of only 0.4 Hz, which sets a correspondingly long timescale for SOT. Nevertheless, an impressive 20% 13C polarization level was achieved for purified [1- 13C] pyruvate.

[0112] RF Pulse Sequences. Various coherence transfer pulse sequences harvesting PASADENA spin order (Eq. 1) have appeared in the literature for both ethyl and allyl ester side-arms. The pH2 sourced protons in allylic side-arms are further away (4 or 5 bonds) from the 13C target than in vinylic side-arms, and the longer-range couplings are too weak to mediate efficient transfer in a single coherence transfer step.22 More efficient are the two- step relayed coherence transfer sequences.7, 17,20,23 ESOTHERIC.17 which is mediated by 13C-13C coupling and thus requires two-site 13C enrichment, has achieved pre-cleavage 13C polarizations of up to 8.9% in a cinnamyl pyruvate ester.23 PH-RELAY, where coherence transfer is relayed via the methylene proton, produced a modest 13C polarization of 7% in the 3-d3-1-d-buten-2-yl [1-13C] pyruvate ester despite a theoretical efficiency close to 100%. MINERVA, a recently introduced pulse sequence specific to vinyl ester hydrogenation under PASADENA conditions, 17 has produced batches of per-deuterated pyruvate ethyl ether with a claimed polarization of 60% 13C.15

[0113] Scheme 2, Fig. 10, illustrates the SOT method employed in Nagel relies on the 0.4 Hz coupling in this deuterated allyl ester adduct

[0114] Extraction and Purification of Hyperpolarized Metabolites

[0115] Extraction of hyperpolarized metabolites into aqueous buffer is the final step in SAH-PHIP. Literature protocols employ gravimetric phase separation and / or vacuum evaporation, depending on the miscibility of the chosen organic solvent with water. In all published protocols, a significant fraction of the 13C hyperpolarization is lost to relaxation during extraction. For the most commonly employed catalyst, [Rh(COD)dppb]BF4, acetoneATTORNEY DOCKET NO. 222112-2510 affords higher rates of hydrogenation, but disadvantageously, acetone is miscible in water. However, it can be rapidly removed by vacuum evaporation at elevated temperature.

[0116] In the process employed in Ref. 3, hydrogenation is performed in acetone, and after addition of aqueous NaOD and phosphate buffer, MTBE is added to sequester acetone, the spent side-arm, and catalyst residues. The propargyl ester used in this process exhibits fast and complete hydrogenation within 5 s after the start of pH2 bubbling. The CH2 group (Scheme 2, Fig. 10) is fully deuterated for improved retention of pH2 singlet order. The spent side-arm following hydrolysis is hydrophobic by design and partitions into the organic phase. After gravimetric separation, the hyperpolarized metabolite is washed one or more times with additional MTBE, and residual acetone and MTBE is removed by vacuum evaporation. Despite significant relaxation losses during the purification, Nagel demonstrated 13C polarizations of up to 20% in aqueous solutions of =70-160 mM [1-13C] pyruvate in volumes of up to 2ml_. This performance can be regarded as a benchmark for comparison to our continuous-flow process.

[0117] Aspects for the present disclosure provide for devices, systems, and methods for continuous flow synthesis of 13C hyperpolarized pyruvate and other metabolites in aqueous buffer that combine, in series: (i) efficient continuous-flow hydrogenation reactor technology; (ii) efficient spin order transfer for vinyl or propargyl esters, and (iii) novel liquid-liquid membrane wetting separator technology for continuous extraction of hyperpolarized metabolites into a biocompatible aqueous buffer. The steps in the flow chemistry were presented in Fig. 1A.

[0118] Efficient Spin Order T ransfer from Propargyl Esters.

[0119] While our continuous-flow process can incorporate any of the known spin order transfer (SOT) methods, we anticipate certain advantages of using our versatile and efficient SOT technique in view of its broad applicability to both vinylic and propargylic esters, with the specific or generalized chemical structures shown in Fig. 2. The technique combines adiabatic rapid passage from the weak to strong homonuclear coupling regime among the protons followed by the application of a coherence transfer pulse sequence. The versatility stems from the occurrence of both linear and bilinear terms in the ALTADENA density operator (Eq. 2).24 For allyl ester adducts with the generalized structure APd2 in Scheme 3 (Fig. 10), spin exchange with the initially unpolarized methylene proton is induced by adiabatic passage through a level anti-crossing (LAC) induces. This sets up an ideal initial condition for subsequent coherent transfer to the carbonyl 13C via a stronger J-coupling. After adiabatic passage through the LAC (referred to as LACADENA24), the ALTADENA density operator is obtained but with H1 and H3 swapped:ATTORNEY DOCKET NO. 222112-2510

[0120] Fig. 10 illustrates Scheme 3. Scheme 3 illustrates the density matrix simulations of the 1H spin polarizations for hydrogenation with pH2 at low field (strong coupling) followed by adiabatic transport to high field for deuterated vinyl and propargyl esters. The high H3 polarization in APd2 derives from adiabatic passage through a LAC.24

[0121] As illustrated in Fig. 3A., ALTADENA affords flexibility in the choice of coherence transfer pathways. The MINERVA pulse sequence operates on the bilinear terms (fzl / z2in EPd3, or fz27z3in APd2) while sINEPT operates on the Zeeman operators (i.e., 7Z1- lz2in EPd3 or 7z2- iz3in APd2). In the preliminary experimental spectra shown in Fig. 3B, a preliminary 13C spin polarization of P(13C) = 12 % was observed for APd (without any optimization), corresponding to an anticipated polarization of 24 % for APd2, a promising initial result. Fig. 4 presents Spinach25 density matrix calculations of P(13C) for allyl and ethyl adducts. For EPd3 MINERVA achieves P(13C) > 95 % for either ALTADENA or PASADENA preparation. Similar theoretical performance is obtained for sINEPT after ALTADENA preparation. Calculations for APd2 indicate P(13C) = -90% for the LACADENA- sINEPT protocol. Selective 1 H excitation is also beneficial for carboxylic acids bearing protons with appreciable coupling to the carbonyl 13C (e.g., acetate). In APd, where only H5 is deuterated, adiabatic passage through the LAC divides the terminal proton polarization between the pair of H3 protons in the allyl CH2 group, thereby reducing the efficiency of INEPT by a factor of two, 21 ,26,27 as confirmed by numerical simulations. Our hybrid method will be compared with literature SOT pulse sequences.

[0122] Continuous-Flow Spin Order T ransfer

[0123] The ALTADENA / LACADENA preparation of the spin system is inherently compatible with flow conditions, as adiabatic passage from low field (c.a. 0.05 mT) to high field (ca. > 10 mT) occurs as the fluid flows from the reactor at low field to the RF detection coil in the NMR probe at high field. The flow rate and tubing diameter are selected to fulfill the adiabaticity criterion to the greatest possible extent while minimizing spin relaxation. The coherence transfer pulse sequence will be applied to the liquid entering the RF coil of the NMR probe. The pulse sequence will be applied repeatedly using a recycle time selected to match the residence time in the RF coil of the NMR flow cell. Flow rate is the key parameter. While a high flow rate minimizes spin relaxation losses, a flow rate that is too high will cause the residence time in the NMR coil to be too short for completion of the coherence transfer step, which is dictated by J^H-ISC - An interrupted-flow mode of operation can also be envisaged, where the flow is briefly halted during the application of the coherence transfer pulse sequence to prevent fluid from leaving the NMR flow cell before coherence transfer is complete.ATTORNEY DOCKET NO. 222112-2510

[0124] Fig. 3 A illustrates timing diagrams for coherence transfer after adiabatic transport of ethyl or allyl ester adducts of pH2 from strong to weak coupling followed by selective INEPT or MINERVA NMR pulse sequences. Fig. 3B. Hyperpolarized NMR spectra for APd (preliminary data), where a 13C signal enhancement of 14,476 was observed, corresponding to a spin polarization of 12% (24% for APd2).

[0125] Fig. 4 illustrates the density matrix simulations of 13C polarizations for MINERVA or INEPT coherence transfer pulse sequences applied to various adducts after PASADENA or ALTADENA preparation. Losses due to spin relaxation are neglected. AP=allyl pyruvate, APd=allyl pyruvate-d, APd2=allyl pyruvate-d2, EPd3=ethyl pyruvate-d3 (structures shown in Scheme 3, Fig. 10). Deuteration improves the efficiency across the AP series.

[0126] High Performance Continuous-Flow Hydrogenation Reactors

[0127] Hydrogenative PHIP experiments are typically initiated by bubbling of pH2 gas through a capillary tube immersed in the reactant solution. This is simple and effective, but not well-suited for chemical kinetics or quantification of PHIP signal enhancement due to changing reactant and product concentrations, ongoing accumulation of product molecules, and deactivation of dissolved catalysts. Estimation of the signal enhancement in a batch reactor is prone to large errors as the PHIP and thermally polarized signals are difficult to isolate for equal numbers of product molecules. Thus, batch mode experiments are not well- suited for quantitative comparison of SOT methods. Continuous-flow PHIP is ideal for systematic study and quantification of conversion, signal enhancement, and SOT efficiency because the initial condition of each experiment is identical, there is no accumulation of products, and fresh catalyst solution is employed in each trial. However, continuous flow PHIP requires a continuous-flow reactor. An ultrasonic spray injection reactor suitable for dissolved or suspended catalysts;28-31 This reactor design is scalable and integrable into continuous or interrupted flow SAH-PHIP processes.

[0128] Ultrasonic Spray Injection Hydrogenation Reactor. Fig. 5 compares the droplet size distributions for a Sono-Tek™ ultrasonic nozzle, vibrating at F = 180 kHz with conventional pressure-driven spray injection.

[0129] Fig. 5 illustrates the reactor for homogeneous hydrogenation. Comparison of the droplet size distributions produced by ultrasonic spray injection at a nozzle frequency of 180 kHz (green) and conventional spray injection

[0130] For the ultrasonic nozzle, droplet size is modeled by Lang’s equation, where median droplet size D50 decreases proportional to 1 / F2 / 3. The higher interfacial area of smaller droplets affords faster dissolution of pH2 gas and hence more efficient hydrogenation. Moreover, ultrasonic spray injection is nearly isobaric, which avoids the evaporative cooling that will lower reaction rates. In our article, 32 an ultrasonic nozzle wasATTORNEY DOCKET NO. 222112-2510 fitted to a 5 mL cylindrical chamber pressurized to 5 bars with pH2. Droplets of the hydrogenation adducts accumulate at the funnel-shaped bottom of the chamber. This spray injection reactor can be operated in continuous flow, interrupted flow, or batch hydrogenation modes.

[0131] Continuous-Flow Liquid-Liquid Separation

[0132] Traditional gravimetric phase separation for extraction of the aqueous phase containing the hyperpolarized metabolites and removal of organic solvents, including acetone and MTBE, which is the method employed in the batch SAH-PHIP process of Nagel, is not well-suited to a flow chemistry process where rapid extraction is required to minimize spin relaxation of the hyperpolarized molecules or metabolites. The present disclosure provides a means for liquid-liquid separation (LLS) (See Fig. 1B and Fig. 1C) of hyperpolarized molecules based on differences in wetting properties onto a porous hydrophobic membrane.33-40 The process in Fig. 1A relies on continuous removal of the organic solvent to yield an aqueous solution of hyperpolarized metabolite(s). While continuous liquid-liquid extraction (phase separation) can be achieved using gravimetric settling vessels, 38 a more rapid phase separation can be achieved by leveraging differential surface wetting. In such separators the organic phase preferentially wets a porous hydrophobic surface such as PTFE, while the aqueous phase preferentially wets a hydrophilic surface such as glass or stainless steel or remains in the bulk phase.41 Aspects of the present disclosure utilize a microporous PTFE membrane for the removal of organic solvent from an emulsion with an aqueous phase in which the hyperpolarized molecules are preferentially dissolved. The separation step is performed immediately after injection of an aqueous base or other aqueous hydrolysis catalysis into the hyperpolarized fluid and restoration of neutral pH by mixing with a buffer.

[0133] Continuous-flow liquid-liquid and liquid-gas separation devices incorporating a porous PTFE membrane are commercially available from Zaiput Flow Technologies, Inc. [see Fig. 1 C], These separators provide continuous separation of immiscible phases by leveraging differences in wetting properties of the liquids onto a porous membrane. Both hydrophilic and hydrophobic membranes are available. The membrane pores are filled with the wetting phase, and a pressure differential is maintained between the two sides of the membrane. This pressure differential is finely adjusted by an internal pressure controller to apply just enough pressure to “push” the wetting phase through without forcing the nonwetting phase through the pores. An aspect of this technology is that it exploits differences in wettability and surface forces to accomplish separation; hence, the device can even separate liquids with the same density. Applications include radioisotope purification, 33 continuous separation and purification of chemical species, 34 and on-demand flow production of pharmaceuticals.35-37 These devices are suitable for use in a multi-stageATTORNEY DOCKET NO. 222112-2510 extraction configuration, in a counter- current configuration or in series, as shown in Fig. 6A- C for three stages The schemes can be readily modified to include additional separation stages or fewer stages, as needed to achieve the desired final purity.

[0134] Fig. 6 illustrates a three stage in-series configuration of Zaiput membrane separation devices, each fitted with a hydrophobic membrane, where the water / acetone / metabolite solution is mixed with MTBE before the first stage, and the liquid emerging from non-wetting port is mixed with fresh MTBE after each consecutive separation device. Residual volatile organic compounds are removed using a solvent stripping method such as the one described in part C of this figure. Fig. 6B. Alternative counter-current configuration using three stages if membrane separation which provides optimal extraction efficiency using a fixed volume of extraction solvent (MTBE). Fig. 6C. Method for stripping of residual dissolved MTBE and acetone or other volatile compounds in the aqueous phase by mixing with nitrogen gas to form a biphasic gas / liquid mixture. After diffusion of the volatile organic compounds into the gas phase, the vapor is removed by a membrane separation device. In the scheme shown, a hydrophilic membrane is installed in the separation device, the vapor is the non-wetting phase, and the aqueous phase containing the metabolite is the wetting phase.

[0135] Multistage Liquid Liquid Extraction (LLE) is a process where multiple extraction steps are repeated in order to increase the recovery of a product. This process is required when, due to a small partition coefficient, the recovery in a single extraction step is insufficient. LLE can be performed with a “countercurrent extraction” scheme that provides the smallest consumption of extractant. In this scheme, the aqueous raffinate from one stage is fed to a former stage as a feed while the organic phase is moved in the opposite direction. Hence, even if the recovery of product in each stage is small, the overall system can achieve a high level of recovery. In multistage extraction, selectivity of the extraction and process yield are decoupled as the yield depends on the number of extraction stages used. As the number of stages increases, extraction efficiency increases, while still using the same amount of organic solvent that would be used if only one batch extraction stage was performed.

[0136] The parameters for selecting an appropriate membrane are the interfacial tension between the two phases and the viscosity of the permeating phase. In general, the lower the interfacial tension, the smaller the required pore size. For our complex mixture (e.g., acetone, MTBE, phosphate buffer, base (for instance, NaOH or sodium carbonate), and the metabolite in the form of carboxylate ions (for instance, Na-Pyruvate or Na-acetate), the interfacial tension of the non-polar component (MTBE) provides an initial approximation (9.8 mN / m) for the mixture.ATTORNEY DOCKET NO. 222112-2510

[0137] Water soluble salts (for example, NaCI, Na2CO3, phosphate buffers) can increase the interfacial tension, resulting in improved liquid-liquid phase separation and improved membrane separation. Indeed, our experimental results conclusively demonstrate that separation by the Zaiput membrane device is improved by the addition of sodium carbonate salt to the acetone / water / MTBE mixture, resulting in lower mass fraction of acetone and MTBE in the aqueous phase. This effect is closely related to the “salting-out” effect that occurs when the solubility of a nonelectrolyte in water decreases after adding salt.

[0138] A metabolite molecule which is present in the form of a sodium carboxylate salt can, at sufficiently high concentration, improve the removal of acetone from the aqueous phase in the separation of a mixture of MTBE, acetone, and water, thus increasing the purity of the aqueous phase and lowering the final concentration of acetone in the extracted aqueous phase.

[0139] While the addition of salts (e.g. NaCI, Na2CO3) can improve separation, the salt concentration in an aqueous solution of the hyperpolarized metabolite must not exceed established limits for safe in-vivo administration. To circumvent this limitation, higher concentrations of metabolite, which is present in the form of a sodium carboxylate salt, can be hyperpolarized. The high concentration of the sodium carboxylate species will increase the partitioning of acetone into the organic phase, without the need for addition of other salts (e.g., NaCI, Na2CO3) that cannot be readily removed from the aqueous solution, and the final concentration of acetone in the aqueous phase after gravimetric or membrane separation will be reduced. After separation, the concentration of sodium pyruvate can be reduced by dilution with additional aqueous buffer to obtain the desired concentration that is safe for in- vivo administration.

[0140] Preliminary data comparing the efficacy of extraction by conventional gravimetric phase separation and flow separation using a Zaiput SEP- 10 device fitted with a hydrophobic membrane (900 nm pore size) are presented in Fig. 5. Three extractions were performed by both methods, with and without 200 mM Na pyruvate. In both methods, successive extractions were run using fresh MTBE. After three extractions, gravimetric and flow separation yielded similar amounts of residual acetone: 3.8 wt% and 2.5 wt%, respectively. Loss of Na pyruvate into the organic phase was negligible. Notably, the flow separation is complete after only 13 s per extraction (including the plug flow mixing time), while the gravimetric separation required 1 - 2 minutes per extraction to allow complete settling

[0141] Fig. 7 illustrates preliminary results that demonstrate the similar performance in the extraction of acetone from a 1 :1 v / v acetone / water mixture containing 200 mM Na pyruvate into methyl t-butyl ether (MTBE) using (a) conventional gravimetric extraction (dashed curves) and (b) flow separation using a Zaiput SEP-10 device fitted with aATTORNEY DOCKET NO. 222112-2510 hydrophobic membrane (solid curves). Syringe pump flow rates of 3 ml / min for MTBE and 1.5 ml / min for the 1 :1 v / v acetone / water mixture. The liquids were mixed using an IDEX mixing tee fitted with a 10 pm frit followed by a 1 m section of 1 / 16 in green PEEK tubing to promote plug flow mixing

[0142] The residual acetone and MTBE organic solvents that remain dissolved in the aqueous phase after gravimetric or membrane separation must be removed prior to in-vivo administration. In batch mode process, the organic solvents are removed by evaporation at elevated temperature (c.a. 60 °C) and reduced pressure.

[0143] We introduce methods suitable for continuous flow stripping of residual (< 5 wt%) volatile organics (acetone, MTBE) from the aqueous buffer containing the hyperpolarized metabolite that employ either a hydrophobic or hydrophilic membrane. The membrane strictly separates liquid and gas phases and neither chemical reactions nor accumulation of solvents or solutes occurs inside the membrane pores. This method uses nitrogen as a carrier gas that does not dissolve in water. Furthermore, water does not diffuse into nitrogen. Therefore, the volatile organic solvents (MTBE and acetone) diffuse from water into the gaseous nitrogen phase during their contact in the biphasic gas / liquid mixture.

[0144] The first implementation for removing residual organic solvents (acetone and MTBE) uses a concentric annular liquid-liquid phase separator such as the one illustrated in Fig. 1B. The interface between liquid and gas phases at the membrane pore entrance is flat, and the device operates isothermally. The tube-in-tube device is comprised of concentric annular channels. The device may be fabricated using SwagelokTM fittings without adhesives or O-ring seals. A similar tube-in-tube device for dissolution and degassing of hydrogen has been demonstrated using Dupont Teflon AF2400 tubing, a porous PTFE membrane material.

[0145] A second method for stripping residual volatile organic solvents (acetone, MTBE) from the aqueous solution of hyperpolarized metabolites (as carboxylate species) uses a Zaiput membrane device (such as the Zaiput model SEP- 10) that is suitable for gasliquid separations. Mixing of the acetone / MTBE / water mixture with N2 gas to form a biphasic mixture is followed by diffusion of the volatile organics into the gas phase followed by removal of the aqueous phase from the mixture by its selective wetting and transport through a hydrophilic porous membrane. The purified aqueous phase containing the hyperpolarized metabolite exits the device through the wetting side port. The nitrogen gas laden with acetone and MTBE vapors emerges from the non-wetting port of the device, as described in the SEP-10 instruction manual. By carrying out the mixing with N2 at elevated temperature, the vapor pressure of the volatile solvents can be increased, which increases the density of the organic solvents in the gas phase, thereby increasing the effectiveness of stripping the organics from the aqueous phase.ATTORNEY DOCKET NO. 222112-2510

[0146] Process Integration

[0147] A block diagram of an embodiment of an experimental setup is shown in Fig. 8A. Arbitrary mixtures of dissolved (or dispersed) catalyst, precursor, and solvent can be prepared by control of the infusion pump rates, enabling kinetics studies of the flow chemistry and achieving desired molar polarization. Reactants are continuously infused into the reactor (ultrasonic spray injection or planar gas diffusion device) at a target flow rate of 5 ml / min. Dissolved catalyst can be removed using a commercially available silica gel supported chelating agent (SiliCycle, Inc.). Next, the pH2 adducts flow adiabatically into the NMR #1 where the coherence transfer pulse sequence is applied. At a flow rate of 5 ml / min, the residence time in the active volume of the NMR flow cell is approximately 6 s. After completion of the SOT step, pumps A, B, and C inject an aqueous base (e.g., NaOD, to induce hydrolysis), phosphate buffer, and MTBE solvent, respectively. After mixing with MTBE, acetone and the hydrophobic side-arm partition into the organic phase, while the hyperpolarized carboxylate remains in the aqueous phase. The emulsion is then separated into organic and aqueous fractions using a Zaiput model SEP-10 liquid-liquid separator which can operate at flow rates up to 10 ml / min. “NMR #2” refers to our Varian VNMRS 400 MHz NMR spectrometer fitted with a Varian flow probe which will be used to analyze the final composition and polarization of the emergent metabolite solution. This setup will facilitate the systematic studies necessary to develop a comprehensive numerical model of the overall process. A powerful feature of this experimental setup is its modularity, allowing the various system parameters to be isolated and separately characterized. The flow chemistry can be studied without hyperpolarization to establish the reaction conditions and chemical kinetics leading to complete conversion and hydrolysis. Similarly, the hyperpolarization and SOT steps can be independently characterized. Finally, the performance of LLS will be studied as a function of flow rate, relative fractions of aqueous and organic phases, solute concentrations, and membrane back-pressure. Isolating the various parameter subspaces will assist the development of a complete numerical model and a rational strategy to maximize the molar polarization in aqueous buffer.

[0148] Fig. 8A illustrates experimental flow chemistry system with SOT (NMR #1) and spectroscopic analysis (NMR #2). Following the multi-stage liquid-liquid (LL) removal of the organic phase, residual volatile organic solvents (VOCs) are removed from the aqueous phase by mixing with nitrogen gas, diffusion of the VOCs into the nitrogen gas phase, and vapor-liquid (VL) separation using a hydrophilic membrane at elevated temperature. Fig. 8B. Separation of the hyperpolarized metabolite in aqueous buffer using a hydrophobic membrane. Removal of the dispersing medium fosters coalescence of the dispersed phase, shown in blue.ATTORNEY DOCKET NO. 222112-2510

[0149] The results of nuclear magnetic resonance (NMR) spectroscopy experiments comparing traditional batch mode hydrolysis of ethyl pyruvate with gravimetric extraction of pyruvate with continuous-flow hydrolysis with membrane separation are presented in Fig. 9. Fig. 9A shows the 300 MHz 1 H NMR spectrum of the initial solution of ethyl pyruvate in acetone-d6, in blue, and the spectrum obtained after the addition of aqueous base (Na2CO3 in D2O), in orange. The amount of added base was deliberately limited to ensure that the hydrolysis of ethyl pyruvate would be incomplete, in order to leave some fraction of the initial ester in the acetone / water solution. The peaks labelled HAand HArefer to the ethyl pyruvate ester precursor and free pyruvate methyl group signals, respectively. Also visible are the ethyl ester side-arm and ethanol signals resulting from hydrolysis. Fig. 9B shows the 300 MHz 1 H NMR spectra after mixing and extraction of the partially hydrolyzed solution in acetone-d6 / D20 after a single-pass through the SEP- 10 continuous-flow membrane separator device (blue spectrum) or after traditional gravimetric extraction with a single mixing of MTBE. The presence of strong pyruvate methyl signals (labelled HA) in the blue and orange spectra with similar intensities shows that the efficacy of the two separation methods is similar.

[0150] Fig. 9A and 9B illustrate 300 MHz 1H NMR spectra comparing the hydrolysis of ethyl pyruvate and extraction into the aqueous phase by batch and continuous-flow processes. Fig. 9A illustrates the pre- and post-hydrolysis spectra obtained after addition of aqueous sodium carbonate solution to the ethyl pyruvate solution (in acetone-d6). Fig. 9B illustrates the spectra of the aqueous fraction after addition of aqueous base and single stage separation by gravimetric (orange spectrum) or continuous-flow Zaiput SEP- 10 device with the hydrophobic membrane installed.References

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[0191] (41) Karlberg, B.; Thelander, S. Extraction Based on the Flow-InjectionPrinciple. Anal Chim Acta 1978, 98 (1), 1-7. https: / / doi.org / 10.1016 / s0003-2670(01)83231-1.

[0192] (42) Hale, W. G.; Zhao, T. Y.; Choi, D.; Ferrer, M.-J. J.; Song, B.; Zhao, H.;Hagelin-Weaver, H. E.; Bowers, C. R. Toward Continuous-Flow Hyperpolarisation of Metabolites via Heterogenous Catalysis, Side-Arm-Hydrogenation, and Membrane Dissolution of Parahydrogen. ChemPhysChem 2021 , 22 (9), 822-827. https: / / doi.Org / 10.1002 / cphc.2021001 19.

[0193] (43) Zhao, T. Y.; Lapak, M.; Ferrer, M.-J.; Zhao, H.; Behera, R. K.; Hale,W. G.; Huang, W.; Hagelin-Weaver, H. E.; Bowers, C. R. Hyperpolarization Ad-Infinitum: A Closed-Loop, Continuous-Flow System for Hyperpolarization of Catalyst-Free Metabolites via Heterogeneous Catalysis. In 63rd Experimental NMR Conference; Orlando, Florida, 2022.

[0194] Example 2Hydrogenation Reaction ConditionsContinuous-Flow Spray Injection Reactor Operation

[0195] Operation of the ultrasonic spray injection reactor developed in our lab has been limited to the batch mode hydrogenations. That is, injection of the unsaturated precursor solution into the chamber pressurized with para-enriched hydrogen and acquisition of NMR spectra were performed sequentially in time. In this invention, spray injection hydrogenation will be performed as a continuous flow process. Using the setup described previously, acetone solution of the unsaturated ester precursor (e.g., propargyl acetate) and Rh(cod) catalyst will be infused through the ultrasonic nozzle and into the parahydrogen pressurized reaction chamber while controlling the rate of outflow of the product liquid collected at the bottom of the chamber using either a second syringe pump or a liquid flow controller. The reaction temperature, catalyst concentration, and infusion rate are the operating parameters that can be varied to optimize the rate of production of the desired hydrogenation products (e.g. allyl acetate). The ultrasonic frequency of the nozzle can also be adjusted to vary the droplet size and hence the liquid / gas interfacial surface area.Solvent Composition

[0196] After demonstrating the effective operation of the spray injection catalytic reactor in continuous flow mode using acetone as the solvent, the rate of hydrogenation in pure acetone will be compared to the rates in other pure and mixed solvent systems. While PHIP has been observed in many different solvents, acetone is most common due to the high solubility of hydrogen in this solvent (3.85 mM / bar)

[0019] and the fact that acetone canATTORNEY DOCKET NO. 222112-2510 coordinate to the rhodium catalyst and enhance its activity.

[0018] , Hydrogen is also highly soluble in cyclohexane (3.87 mlWbar)

[0020] , but this non-polar solvent does not coordinate to the rhodium complex, which is essential to maintaining the high catalytic activity and stability. Similarly, Duckett et al. [7] added a small amount (~ 100 mM) of DMSO as a cosolvent in methanol, which greatly increases the activity of the catalyst. Here we claim that a co-solvent with acetone may also be beneficial for hydrogenative PHIP. The activity and pairwise selectivity of the Rh(dppb) catalyst will be examined in mixtures with cyclohexane, DMSO, or other added ligands, and hydrogenation rate with co-solvent will be compared to the rate in pure acetone.

[0197] If the addition of a cosolvent is found to improve catalytic activity and pairwise selectivity to levels comparable to acetone, hydrogenation in the co-solvent system will be tested in the ultrasonic spray injection reactor and the operating parameter systematically varied to optimize conversion to the desired product.Workup and Purification

[0198] Acetone as Hydrogenation Solvent

[0199] Acetone is an outstanding solvent for hydrogenation of unsaturated ester precursors using the Rh(dppb) catalyst. After hydrogenation, aqueous base and buffer solutions are added to induced hydrolysis and restore the pH, respectively. Since acetone is miscible with water at all compositions, another organic solvent (immiscible with water) is required for extraction of acetone from the aqueous solution. The process described in Nagel employed MTBE to extract acetone.

[0014] Based on our preliminary data, we claim that anisole (and perhaps other solvents with similar polarity) is a better solvent for extracting acetone from acetone-water mixtures, as seen in Figure 12a. Moreover, the efficacy of extraction improves with increasing temperature. Extrapolation shows that at 60 °C, two successive extractions can reduce the mass fraction of the residual acetone from 45% to 2.5%. We claim that the acetone fraction may be further reduced by exploiting the salting-out effect. In our method, continuous extraction using anisole at 60 °C is assisted by the presence of salt. Given the low target metabolite (e.g. pyruvate) concentration, our strategy is to start with a high salt concentration to achieve sufficient removal of organic the solvents. Prior to in-vivo injection, the solution is diluted in waterto the appropriate physiological level.

[0200] Despite the effectiveness of extraction, the 25000 ppm of residual acetone is far greater than the 5000 ppm limit specified by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH)

[0031] , Small amounts of acetone can be removed by evaporation while mixing with nitrogen and heating, allowing acetone to migrate to the gas phase as demonstrated in the processes described in publications by Gldggler et al. and authors of Nagel. Alternatively, we claim that acetone can be removed by ultrasonic spray injection into vacuum or into a nitrogen atmosphere to increase the surface area and expedite evaporation.ATTORNEY DOCKET NO. 222112-2510

[0201] Alternative Solvents

[0202] If we find that the performance of the Rh(dppb) catalyst in a co-solvent system (such as cyclohexane- acetone) can match hydrogenation in acetone, it would greatly simplify purification, as the solubility of cyclohexane (55 ppm) is much lower than ICH’s limit of 3370 ppm

[0031] , rendering multi-stage extraction unnecessary. The SEP-10 membrane device was shown to be able to remove emulsion of organic solvents, ensuring low organic solvent content, making residual solvent stripping unnecessary. These simplifications could reduce the extraction time by up to 40 s, thereby reducing the relaxation losses by about 20%.

[0203] However, a significant drawback of using a solvent immiscible with water is the low hydrolysis yield as the aqueous base does not readily encounter the ester in the organic phase, especially for the hydrophobic esters used in our invention. Specifically, the best yield of heterogeneous hydrolysis is less than 25% of homogeneous hydrolysis (acetone), as shown in Figure 13a. However, this problem can be partly addressed by increasing the volume of aqueous phase. As shown in Figure 13b, increasing the volume ratio of organic to aqueous phases to 1 :8 resulted in an 8-fold increase in the hydrolysis yield, which is impressive given the aqueous phase is safe for injection without any further processing. By comparison, in results published by Nagel, an overall yield of only 14.5% was demonstrated

[0014] , The next step will be to implement this reaction with subsequent membrane wetting separation to demonstrate continuous heterogeneous hydrolysis.

[0204] Catalyst Scavenging

[0205] Homogeneous rhodium compounds are toxic, and their concentration must be reduced to the ICH’s limit of 10 pg / day

[0032] , While extraction using non-miscible organic solvents may remove most of the residual rhodium, we claim that commercially available functionalized silica with thiol and amine groups that sequester dissolved Rh can further reduce the residual Rh concentration in the final product liquid. The efficacy of catalyst scavenging by extraction and silica adsorption under the conditions of our process will be evaluated by flowing a solution of only the catalyst through the system. The catalyst’s four phenyl groups give strong NMR signal, allowing easy quantification. If the NMR SNR is too low, ICP-MS can be used to accurately determine the rhodium content.References

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Claims

ATTORNEY DOCKET NO. 222112-2510CLAIMSWhat is claimed is:

1. A continuous flow hydrogenation reactor device, comprising: a hydrogenation reactor, wherein hydrogenation reactor is configured to continuously form a first mixture from a precursor solution and parahydrogen; a spin order transfer device, wherein the spin order transfer device is in fluidic communication with the hydrogenation reactor and is configured to receive the first mixture, wherein a hyperpolarized metabolite is produced in the spin order device from the first mixture, wherein a hyperpolarized metabolite solution includes the hyperpolarized metabolite; a reagent introduction device in fluidic communication with the spin order transfer device, wherein one or more reagents are added to the hyperpolarized metabolite solution to form a modified hyperpolarized metabolite liquid; one or more membrane separator devices in fluid communication with the spin order transfer device and the reagent introduction device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase, wherein each of the one or more membrane separator devices includes one or more membrane separators; and a collection system in fluidic communication with the membrane separator device, wherein the collection system receives the hyperpolarized metabolite aqueous solution.

2. The reactor of claim 1 , wherein the membrane separator is a liquid-liquid membrane wetting separator that separates the solvent from the modified hyperpolarized metabolite solution by phase separation using differential surface wetting.

3. The reactor of claim 2, wherein the membrane separator includes a membrane having hydrophobic surface with pores extending from one side of the membrane to the other side of the membrane so that fluid can pass through the membrane, optionally, wherein the fluid is the solvent phase; or wherein the membrane separator includes a membrane having hydrophilic surface with pores extending from one side of the membrane to the other side of the membrane so that fluid can pass through the membrane, optionally, wherein the fluid is the solvent phase.ATTORNEY DOCKET NO. 222112-25104. The reactor of claim 3, wherein the membrane separator device includes a pressure system that produces a pressure differential across the membrane, wherein the pressure system operates to flow a fluid through the pores of the membrane.

5. The reactor of any one of claims 1-4, wherein the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase about 1-10 times faster than a continuous flow hydrogenation reactor having a gravimetric separation device, where the only difference between reactors is the substitution of the gravimetric separation device for the membrane separator device.

6. The reactor of any one of claims 1-5, wherein the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase in about 10 to 15 seconds per extraction.

7. The reactor of any one of claims 1 -6, wherein the spin order transfer device comprising an adiabatic transport tube, wherein the adiabatic transport tube is configured to receive the first mixture from the hydrogenation chamber.

8. The reactor of any one of claims 1-7, wherein the spin order transfer device comprises one of the following: an NMR spectrometer configured for field cycling of the static magnetic field or radiofrequency field; an NMR spectrometer configured for coherence transfer radiofrequency pulse sequences; or an NMR spectrometer configured for adiabatic field cycling in conjunction with coherence transfer radiofrequency pulse sequences.

9. The reactor of any one of claims 1-8, wherein the collection system is configured to introduce the hyperpolarized metabolite to a subject.

10. A method for making a hyperpolarized metabolite aqueous solution, comprising: providing a modified hyperpolarized metabolite solution, wherein the modified hyperpolarized metabolite solution comprising a hyperpolarized metabolite, a solvent, and one or more reagents; separating the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase, wherein the separation is performed using a membrane separator device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution andATTORNEY DOCKET NO. 222112-2510 separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase; and collecting the hyperpolarized metabolite aqueous solution.11 . The method of claim 10, wherein the one or more reagents comprises one or more of the following: an aqueous base, a phosphate buffer, and a methyl t-butyl ether (MTBE) solvent12. The method of any one of claims 10-11 , wherein the membrane separator is a liquidliquid membrane wetting separator that separates the solvent from the modified hyperpolarized metabolite solution by phase separation using differential surface wetting.

13. The method of claim 12, wherein the membrane separator includes a membrane having hydrophobic or a hydrophilic surface with pores extending from one side of the membrane to the other side of the membrane so that fluid can pass through the membrane, optionally, wherein the fluid is the solvent phase.

14. The method of claim 13, wherein the membrane separator device includes a pressure system than produces a pressure differential across the membrane, wherein the pressure system operates to flow a fluid through the pores of the membrane.

15. The method of any one of claims 10-11 and 13-14, wherein the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase about 10 times faster than a continuous flow hydrogenation reactor having a gravimetric separation device, where the only difference between reactors is the substitution of the gravimetric separation device for the membrane separator device.

16. The method of any one of claims 10-11 and 13-14, wherein the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase in about 10 to 15 second per extraction.

17. A contrast agent, comprising the hyperpolarized metabolite of claims 1-9 and produced using the method of claims 10-16.ATTORNEY DOCKET NO. 222112-251018. A method for detecting a disease state associated with abnormal concentration or abnormal activity of a precursor compound metabolite in a subject, the method comprising:(a) administering the contrast agent of claim 17 to the subject; and(b) detecting the contrast agent in the subject.

19. The method of claim 18, wherein the detecting is accomplished using magnetic resonance imaging.

20. A method that employs a hydrophilic membrane for liquid / vapor separation for continuous flow stripping of residual volatile organics dissolved in the aqueous buffer containing the hyperpolarized metabolite.21 . The method of claim 20, wherein the residual volatile organic includes acetone, methyl t-butyl ether (MTBE), or a combination thereof.

22. The method of any one of claims 20-21 , wherein the membrane strictly separates liquid and gas phases and neither chemical reactions nor accumulation of solvents or solutes occurs inside the membrane pores.

23. The method of any one of claims 20-21 , wherein a carrier gas is used that does not appreciably dissolve in water.

24. The method of claim 23, wherein the carrier gas is nitrogen, N2.

25. The method of claim 23, wherein water does not appreciably diffuse into the gas.

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