Systems and methods for producing hyperpolarized materials - Patents.com

Abstract

This disclosure describes hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.This disclosure describes methods for producing hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.This disclosure describes precursor compounds for use in producing hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 164,585, filed March 23, 2021, U.S. Provisional Patent Application No. 63 / 260,631, filed August 27, 2021, and U.S. Provisional Patent Application No. 63 / 266,986, filed January 21, 2022, each of which is incorporated by reference in its entirety for all purposes.

[0002] The disclosed embodiments relate generally to the production of hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications. [Background technology]

[0003] Parahydrogen-induced polarization (PHIP) is a low-cost, high-throughput method for polarizing metabolites for hyperpolarized (HP) magnetic resonance imaging (MRI). Parahydrogen-induced polarization with a side arm (PHIP-SAH) can be used to polarize metabolites, such as acetate molecules. However, existing PHIP-SAH polarization approaches may not be suitable for preclinical or clinical HP MRI applications. Summary of the Invention

[0004] In some embodiments, the present disclosure provides a composition comprising a compound of formula (I): [ka] In the formula, Z is: 1 H (proton), 2or (ii) a carbon-carbon double bond (-C=C-) substituted to include H (deuterium), or a combination thereof, wherein R1 comprises a parahydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biorelevant imaging agent comprising a non-hydrogen nuclear spin.

[0005] In some embodiments, the present disclosure provides a composition comprising a compound of formula (II): [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 A composition is described that comprises a biorelevant imaging agent comprising a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or combinations thereof, where H* is hydrogen with spin order derived from the para-hydrogen, R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a non-hydrogen nuclear spin.

[0006] In some embodiments, the present disclosure provides a composition comprising: (i) a biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (ii) a compound of formula (III): [ka] In the formula, Z″ is: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2The present invention describes compositions in which R1′ is a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or combinations thereof, R1′ comprises a para-hydrogen induced polarization (PHIP) transfer moiety, and R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine.

[0007] In some embodiments, the present disclosure provides a composition comprising: (i) a hyperpolarized biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (ii) a compound of formula (IV): [ka] In the formula, Z is: 1 H (proton), 2 or (ii) a carbon-carbon triple bond (-C≡C-), substituted to include H (deuterium), or a combination thereof; R1' comprises a parahydrogen induced polarization (PHIP) transfer moiety; and R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine.

[0008] In some embodiments, the disclosure describes compounds of formula I, II, III, or IV that include a PHIP transfer moiety. In some embodiments, the PHIP transfer moiety includes an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon. In some embodiments, the PHIP transfer moiety includes *CR4R5, *CR4Y, *C=Y, or any deuterated version thereof, where *C is 12 C or 13 C carbon isotope, R and R are each independently 1 H, 2 H, 3H, linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups, and Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from: spin 1 / 2 atoms, and heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the PHIP transfer moiety comprises *CR6R7-*CR8R9, or any deuterated version thereof, and *C is 12 C or 13 C is a carbon isotope,

[0009] R6, R7, R8, and R9 are each independently 1 H, 2 H, 3 H, straight, branched, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups. In some embodiments, the PHIP transfer moiety comprises *CH2, *CH2-*CH2, *CHY, *C=Y, or any deuterated version thereof, where *C is 12 C or 13 is a carbon isotope of C, and Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from: spin 1 / 2 atoms, and heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the spin 1 / 2 atoms are 1 H, 13 C. 15 N, 19 F, or 31P. In some embodiments, the PHIP transfer moiety comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz). In some embodiments, Z comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz).

[0010] In some embodiments, the disclosure describes compounds of formula I, II, III, or IV, where R2 comprises a solubilizing moiety. In some embodiments, R2 comprises a hydrophobic and / or organophilic moiety. In some embodiments, R2 comprises an organic solubilizing moiety. In some embodiments, R2 comprises a hydrophilic and / or organophilic moiety. In some embodiments, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine. In some embodiments, R2 comprises or is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

[0011] In some embodiments, the present disclosure describes a compound of Formula I, Formula II, Formula III, or Formula IV that comprises a biologically relevant imaging agent. In some embodiments, the biologically relevant imaging agent is a compound of Formula R 10 C(═O)X—, where R 10 is selected from linear, branched or cyclic C1-C10 alkyl groups, where one or more C atoms are optionally substituted with C=C, CO, COH, CNH2, COOH, CH2COOH, CONH2, OC(=O), and X is NR 11 , S, and O, wherein R11 teeth, 1 H, 2 H, 3 H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butyl carbonate, and benzyl. In some embodiments, the biologically relevant imaging agent is selected from pyruvate, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbic acid, α-ketoglutarate, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids. In some embodiments, the biologically relevant imaging agent comprises pyruvate. In some embodiments, the biologically relevant imaging agent comprises lactate. In some embodiments, the biologically relevant imaging agent comprises α-ketoglutarate.

[0012] In some embodiments, the compositions of Formula I, II, III, or IV have a solubility in water of less than 50 millimolar (mM). In some embodiments, the compositions of Formula I, II, III, or IV have a solubility in organic solvents (e.g., acetone, ethanol, chloroform, and toluene) of less than 50 millimolar (mM).

[0013] In some embodiments, reacting the composition of Formula I with para-hydrogen provides a chemical yield of para-hydrogenated product of at least 30%.

[0014] In some embodiments, the compositions of the present disclosure are for use in a parahydrogen induced polarization (PHIP) process.

[0015] In some embodiments, the present disclosure describes a method for preparing a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the method comprising: (a) providing a composition comprising a compound of formula (I): [ka] In the formula, Z is: 1 H (proton),2 H (deuterium), or combinations thereof, wherein R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biorelevant imaging agent comprising a non-hydrogen nuclear spin; and (b) hydrogenating the double or triple bond in the compound of formula I with para-hydrogen to form a para-hydrogenated derivative of the compound of formula I, wherein the para-hydrogenated derivative has the structure of formula (II): [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 H (deuterium), or combinations thereof, where H* is hydrogen having spin order derived from para-hydrogen, R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (c) applying a polarization transfer waveform to the non-hydrogen nuclear spins to transfer nuclear spin order from at least one H* in the compound of formula II, thereby forming a derivative of formula II having a hyperpolarized biologically relevant imaging agent.

[0016] In some embodiments, the disclosure provides a method for preparing a hyperpolarized biologically relevant imaging agent, or a pharma- ceutically acceptable salt thereof, the method comprising: (a) providing a composition comprising a compound of formula (II): [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 and (b) applying a polarization transfer waveform to the non-hydrogen nuclear spins to transfer nuclear spin order from at least one H* in the compound of formula II, thereby forming a derivative of formula II having a hyperpolarized biologically relevant imaging agent.

[0017] In some embodiments, the method comprises hydrolyzing the derivative of formula II to produce (i) a hyperpolarized biologically relevant imaging agent comprising non-hydrogen nuclear spins, and (ii) a compound of formula (III): [ka] In the formula, Z″ is: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2and a compound, wherein R1' comprises a parahydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or a combination thereof, R1' comprises a parahydrogen induced polarization (PHIP) transfer moiety, and R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine. In some embodiments, the method further comprises washing the hyperpolarized biologically relevant imaging agent one or more times with an organic solvent. In some embodiments, the non-hydrogen nuclear spins have a non-hydrogen nuclear spin polarization of greater than 10% after the washing step.

[0018] In some embodiments, the present disclosure describes a hyperpolarized biologically relevant imaging agent, or a pharma- ceutically acceptable salt thereof, produced by the methods of the present disclosure.

[0019] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory but are not restrictive of the disclosed embodiments, as claimed. [Brief description of the drawings]

[0020] The accompanying drawings, which form a part of this specification, illustrate certain embodiments and, together with the description, serve to explain certain principles and features of the disclosed embodiments.

[0021] [Figure 1] 1 illustrates a first exemplary process for producing a polarized biologically relevant imaging agent, according to various embodiments. [Diagram 2] 1 illustrates a second exemplary process for producing a polarized biologically relevant imaging agent, according to various embodiments. [Figure 3A] 1 shows an exemplary proton (1H) nuclear magnetic resonance (NMR) spectrum corresponding to 4-((2-oxopropanoyl)oxy)but-2-ynoate methyl, according to various embodiments. [Figure 3B] 1 shows an exemplary 13C NMR corresponding to 4-((2-oxopropanoyl)oxy)but-2-ynoate methyl according to various embodiments. [Figure 4A] 1 shows an exemplary 1H NMR spectrum corresponding to isopropyl 4-((2-oxopropanoyl)oxy)but-2-ynoate, in accordance with various embodiments. [Figure 4B] 1 shows an exemplary 13C NMR corresponding to isopropyl 4-((2-oxopropanoyl)oxy)but-2-ynoate, according to various embodiments. [Figure 5A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate, in accordance with various embodiments. [Figure 5B] 1 shows an exemplary 13C NMR corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate, according to various embodiments. [Figure 6A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate, in accordance with various embodiments. [Figure 6B] 1 shows an exemplary 13C NMR corresponding to tert-butyl 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate according to various embodiments. [Figure 7A] 1 shows an exemplary 1H NMR spectrum corresponding to 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate in accordance with various embodiments. [Figure 7B] 1 shows an exemplary 13C NMR corresponding to 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate according to various embodiments. [Figure 8A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate-4-d, in accordance with various embodiments. [Figure 8B]1 shows an exemplary 13C NMR corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate-4-d, according to various embodiments. [Figure 9A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)pent-2-ynoate, in accordance with various embodiments. [Figure 9B] 1 shows an exemplary 13C NMR corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)pent-2-ynoate, according to various embodiments. [Figure 10A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)-4-phenylbut-2-ynoate, in accordance with various embodiments. [Figure 10B] 1 shows an exemplary 13C NMR corresponding to tert-butyl 4-((2-oxopropanoyl)oxy)-4-phenylbut-2-ynoate, in accordance with various embodiments. [Figure 11] 1 shows an exemplary 1H NMR spectrum corresponding to benzhydryl 4-((2-oxopropanoyl)oxy)but-2-ynoate, in accordance with various embodiments. [Figure 12A] 1 shows an exemplary 1H NMR spectrum corresponding to 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate according to various embodiments. [Figure 12B] 1 shows an exemplary 13C NMR corresponding to 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate according to various embodiments. [Figure 13A] 1 shows an exemplary 1H NMR spectrum corresponding to 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-oxopropanoate according to various embodiments. [Figure 13B] 1 shows an exemplary Carbon-13 (13C) NMR corresponding to 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-oxopropanoate according to various embodiments. [Figure 14A]1 shows an exemplary 1H NMR spectrum corresponding to 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate according to various embodiments. [Figure 14B] 1 shows an exemplary 13C NMR corresponding to 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate according to various embodiments. [Figure 15A] 1 shows an exemplary 1H NMR spectrum corresponding to 4-(2,2-dichloroacetoxy)but-2-ynoate methyl according to various embodiments. [Figure 15B] 1 shows an exemplary 13C NMR corresponding to 4-(2,2-dichloroacetoxy)but-2-ynoate methyl according to various embodiments. [Figure 16A] 1 shows an exemplary 1H NMR spectrum corresponding to tert-butyl 4-acetoxybut-2-ynoate, in accordance with various embodiments. [Figure 16B] 1 shows an exemplary 13C NMR corresponding to tert-butyl 4-acetoxybut-2-ynoate, according to various embodiments. [Figure 17A] FIG. 1 shows an exemplary H NMR spectrum corresponding to para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate having two protons H* with spin order derived from para-hydrogen) according to various embodiments. [Figure 17B] FIG. 1 shows an exemplary H NMR spectrum corresponding to para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (i.e., tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate with 2 H*) in accordance with various embodiments. [Figure 18A] 1 shows an exemplary 1H NMR spectrum corresponding to para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate having 2 H*) according to various embodiments. [Figure 18B] FIG. 1 shows an exemplary H NMR spectrum corresponding to para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate (i.e., 4-oxo-4-phenylbut-2-en-1-yl 2-oxopropanoate having two H*) according to various embodiments. [Figure 19A] 1 shows an exemplary 13C NMR spectrum corresponding to 200 mM para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate having 2 H*) according to various embodiments. [Figure 19B] FIG. 1 shows an exemplary C NMR spectrum corresponding to 200 mM para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (i.e., tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate with 2 H*) in accordance with various embodiments. [Figure 20A] 1 shows an exemplary 13C NMR spectrum corresponding to 200 mM para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate having 2 H*) according to various embodiments. [Figure 20B] 1 shows an exemplary C NMR spectrum corresponding to 200 mM para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate (i.e., 4-oxo-4-phenylbut-2-en-1-yl 2-oxopropanoate having 2 H*) according to various embodiments. [Figure 21] FIG. 1 shows an exemplary C NMR spectrum corresponding to 133 mM para-hydrogenated methyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (i.e., methyl 4-((2-oxopropanoyl)oxy)but-2-enoate with 2 H*) in accordance with various embodiments. [Figure 22A]FIG. 1 shows an exemplary H NMR spectrum corresponding to para-hydrogenated 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate (i.e., 1-(4-(tert-butoxy)-4-oxobut-2-en-1-yl) 5-ethyl 2-oxopentanedioate having two H*) according to various embodiments. [Figure 22B] 1 shows an exemplary C NMR spectrum corresponding to 70 mM para-hydrogenated 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate (i.e., 1-(4-(tert-butoxy)-4-oxobut-2-en-1-yl) 5-ethyl 2-oxopentanedioate having 2 H*) according to various embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Exemplary embodiments will now be described in detail and discussed with reference to the accompanying drawings. Unless otherwise defined, technical and / or scientific terms have the meanings commonly understood by those skilled in the art. The disclosed embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosed embodiments. It will be understood that other embodiments may be utilized and changes may be made without departing from the scope of the disclosed embodiments. Thus, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0023] Recent research in the fields of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) has demonstrated that NMR and MRI signals associated with various biorelevant contrast agents can be enhanced by orders of magnitude using various so-called hyperpolarization techniques. This signal enhancement allows for improved spectroscopic analysis of the biorelevant contrast agents as they are metabolized by various tissues at different locations in the body. Analysis of metabolic information determined by such spectroscopic imaging can allow for non-invasive determination of the health of tissues in the body. For example, abnormal metabolism of a biorelevant contrast agent can indicate disease, such as cancer, at several locations in the body.

[0024] Existing techniques for hyperpolarized biorelevant contrast agents include dissolution dynamic nuclear polarization (DNP), parahydrogen induced polarization (PHIP), PHIP side chain hydrogenation (PHIP-SAH), and signal amplification by reversible exchange (SABRE). In PHIP-SAH, a precursor of the biorelevant contrast agent is reacted with parahydrogen to form a parahydrogenated derivative of the precursor. Spin order is then transferred from the added proton via the parahydrogenation reaction to the nucleus of interest (such as the carbon-13 nucleus) contained within the biorelevant contrast agent. The parahydrogenated derivative of the precursor is cleaved (e.g., hydrolyzed) to obtain the hyperpolarized biorelevant contrast agent. The biorelevant contrast agent is then purified and used in NMR or MRI procedures. In some embodiments, the precursor can include a biorelevant contrast agent attached to a side arm that contains at least one unsaturated bond (e.g., at least one carbon-carbon double bond or at least one carbon-carbon triple bond) suitable for reaction with parahydrogen. However, previous precursors have used side arms that may not allow for the production of biorelevant contrast agents with clinically relevant polarization, concentration, volume, or purity. Such behavior may be related to poor solubility of the precursor in organic solvents (where para-hydrogen is highly soluble), poor yield in the reaction between the unsaturated bond and para-hydrogen, or a variety of other factors. Thus, there is a need for new PHIP-SAH precursors that produce hyperpolarized biorelevant contrast agents with clinically relevant polarization, concentration, volume, or purity.

[0025] The disclosed embodiments include systems and methods for producing biologically relevant imaging agents with clinically relevant polarization, concentration, volume, and purity. The disclosed embodiments provide technical improvements in polarizing biologically relevant imaging agents in solution. These technical improvements support increased biologically relevant imaging agent concentrations and degrees of biologically relevant polarization.

[0026] Hyperpolarization and Parahydrogen As used in this disclosure, hyperpolarization describes a state in which the absolute value of the difference between a population of spin states (e.g., nuclear spin states, proton spin states, etc.) in one state (e.g., spin up) and a population of spin states in another state (e.g., spin down) exceeds the absolute value of the corresponding difference at thermal equilibrium.

[0027] Parahydrogen may be used as a polarization source consistent with disclosed embodiments. Parahydrogen is a form of molecular hydrogen in which two proton spins are in a singlet state, as described herein. The disclosed embodiments are not limited to a particular method of generating parahydrogen. Parahydrogen may be formed in gaseous or liquid form. In some embodiments, parahydrogen is generated in gaseous form by flowing hydrogen gas at low temperature through a chamber with a catalyst (e.g., iron oxide or another suitable catalyst). The hydrogen gas may contain both parahydrogen and orthohydrogen. The low temperature may bring the hydrogen gas to thermodynamic equilibrium in the chamber, increasing the population of parahydrogen.

[0028] The disclosed embodiments are not limited to a particular parahydrogen production or use location. Parahydrogen may be produced at a first location and then transported to a second location for use. In some embodiments, the first location is a chamber that may be part of a container, bottle, holder, or other area capable of holding a gas or liquid. Such a chamber may be maintained at a suitable pressure or temperature. In some embodiments, the first location is a physical location such as a room, a laboratory, a particular warehouse, a hospital, or other location where parahydrogen is produced.

[0029] The disclosed embodiments are not limited to a particular parahydrogen transport method. The parahydrogen produced may be transported in a chamber that may be different from the chamber in which the parahydrogen was produced. The chamber that transports the parahydrogen gas may be maintained at a suitable pressure or temperature that allows it to be transported by vehicle or person. Transporting the parahydrogen may involve moving the parahydrogen from one container to a different container. Transporting the parahydrogen may involve moving the parahydrogen within the same location, such as from one part of a room to another part of a room. Transporting the parahydrogen may involve moving the parahydrogen from one room of a building to a different room of the same building, or to a nearby building. Transporting the parahydrogen may involve moving the parahydrogen to a different location in another part of the same city, or to a different city. Transporting the parahydrogen may involve bringing the parahydrogen near a polarizer, an NMR device, or an MRI device. Transporting the parahydrogen may involve packaging or transporting the parahydrogen in a suitable container.

[0030] In some embodiments, the population difference between two spin states is the difference between the populations of the two spin states divided by the total population of the two spin states. The population difference can be expressed as a fractional population difference or a percentage population difference. In some embodiments, the fractional population difference is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more, up to about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less, or within a range defined by any two of the foregoing values.

[0031] Hydrogen gas may exhibit a population difference between proton spin states at thermal equilibrium that significantly exceeds the population difference between the proton spin states. Parahydrogen may have a large population difference between either the singlet spin state and the triplet spin state. In the case of Iz1Iz2, for example, there is a large population difference between the spin states |↑>|↓> and the spin states |↑>|↑>. The population difference of the proton spin states may be at least about 0.1 (e.g., a 10% difference in spin states - 55% of the parahydrogen molecules in a sample are in the singlet state and 45% are in the triplet state), 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more, up to about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less, or within a range defined by any two of the foregoing values.

[0032] Bio-related contrast agents Disclosed embodiments include systems and methods for producing and utilizing biorelevant imaging agents with clinically relevant polarization, concentration, volume, or purity. In some embodiments, the method is for preparing an NMR material. In some embodiments, the NMR material is suitable for use in NMR or MRI operations. In some embodiments, the NMR material increases the NMR or MRI signal and signal-to-noise ratio (SNR). In some embodiments, the NMR material is suitable for use in solution NMR spectroscopy. In some embodiments, the NMR material is a chemical compound. In some embodiments, the NMR material is a metabolite (e.g., a molecule with biorelevance, such as an amino acid, sugar, derivatives thereof), such as a metabolite suitable for use in NMR metabolomics applications. In some embodiments, the NMR material is suitable for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the NMR material is used in an NMR probe to investigate transient effects where high signal enhancement due to hyperpolarization is required, such as proton exchange between water and biomolecules. In some embodiments, the NMR material is a small molecule or metabolite suitable for injection into cells, tissues, or organisms for detection in MRI scans. In some embodiments, the NMR material is introduced into the chamber for further analysis by NMR or MRI operations. In some embodiments, the NMR material is introduced into the chamber for further analysis by NMR or MRI operations. 2 H) or carbon-13 ( 13 C) concentrated in atoms.

[0033] Consistent with the disclosed embodiments, the NMR material may include a bio-relevant contrast agent. In some embodiments, the bio-relevant contrast agent may be suitable for use in NMR or MRI operations. In some embodiments, the bio-relevant contrast agent may increase the NMR or MRI signal or signal-to-noise ratio (SNR). In some embodiments, the bio-relevant contrast agent may be suitable for use in solution NMR spectroscopy. In some embodiments, the bio-relevant contrast agent may be a metabolite (e.g., a molecule with bio-relevance, such as an amino acid, sugar, derivatives thereof, etc.), such as a metabolite suitable for use in NMR metabolomics applications. In some embodiments, the bio-relevant contrast agent is used for perfusion or contrast-enhanced imaging in MRI scans. In some embodiments, the bio-relevant contrast agent may be suitable for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the bio-relevant contrast agent is used for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the bio-relevant contrast agent may be used in NMR probes to investigate transient effects where high signal enhancement due to hyperpolarization is required, such as proton exchange between water and biomolecules. In some embodiments, the biorelevant imaging agent may be a small molecule or metabolite suitable for injection into a cell, tissue, or organism for detection in an MRI scan. In some embodiments, the biorelevant imaging agent may be introduced into the chamber for further analysis by NMR or MRI operation. In some embodiments, the biorelevant imaging agent may be one or more 2 H or 13 It is enriched in C atoms.

[0034] In some embodiments, the biologically relevant imaging agent is pyruvic acid, lactic acid, α-ketoglutaric acid, bicarbonate, fumaric acid, urea, dehydroascorbic acid, glutamic acid, glutamine, acetic acid, dihydroxyacetone, acetoacetic acid, glucose, ascorbic acid, zymonate, alanine, fructose, imidazole, nicotinamide, nitroimidazole, pyrazinamide, isoniazid, conjugate acids of any of the above, natural and unnatural amino acids, esters thereof, or any of the foregoing. 2 H, 13 C, or nitrogen-15 ( 15In some embodiments, the biologically relevant imaging agent includes pyruvate, lactate, α-ketoglutarate. In some embodiments, the biologically relevant imaging agent includes pyruvate. In some embodiments, the biologically relevant imaging agent includes lactate. In some embodiments, the biologically relevant imaging agent includes α-ketoglutarate (e.g., ethyl α-ketoglutarate).

[0035] In some embodiments, the biologically relevant imaging agent comprises at least one non-hydrogen nuclear spin. In some embodiments, the non-hydrogen nucleus comprises at least one spin 1 / 2 atom. In some embodiments, the non-hydrogen nuclear spin is 13 C or 15 N. In some embodiments, the biologically relevant imaging agent is at least partially isotopically labeled with non-hydrogen nuclear spins. In some embodiments, the biologically relevant imaging agent is at least partially enriched in non-hydrogen nuclear spins compared to an analog of the biologically relevant imaging agent that characterizes the non-hydrogen nuclear spins at their natural abundance. In some embodiments, the biologically relevant imaging agent is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to about 99%, 98%, 97%, 96%, The enrichment may be such that the non-hydrogen nuclear spins are characterized by an abundance of 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or an abundance within a range defined by any two of the foregoing values.

[0036] In some embodiments, the non-hydrogen nuclear spins are NMR inactive (i.e., spin 0) nuclei of an analog of a biologically relevant imaging agent that characterizes the non-hydrogen nuclear spins at their natural abundance (e.g., 12 C or quadrupolar (i.e., spin >1 / 2) nuclei (e.g., nitrogen-14, 14N) at their natural abundance. 13 The pyruvic acid analogue that characterizes C is one of the Cs of the structure H3C-C*(=O)-C*OOH, with approximately 98.9% 12 C and about 1.1% 13 As a biorelevant imaging agent, pyruvate may contain one or both C* at any abundance as described herein. 13 To include C, instead 13 As used herein, *C and C* refer to 12 C or 13 As another example, carbon can be any of the C carbon isotopes at its natural abundance: 15 The analogue of urea that features N has the structure H2N*-C(=O)-*NH2 with approximately 99.6% N*. 14 N and about 0.4% 15 As a biorelevant imaging agent, urea may contain one or both N* in any abundance as described herein. 15 To include N, instead 15 As used herein, *N and N* refer to 14 N or 15 Describes nitrogen, which can be any of the nitrogen isotopes of N.

[0037] Bio-relevant contrast agent precursors In some embodiments, the present disclosure describes a precursor (i.e., precursor compound) that includes a biologically relevant imaging agent and a side arm. In some embodiments, the biologically relevant imaging agent is covalently attached to the side arm. In some embodiments, the biologically relevant imaging agent is attached to the side arm via a transfer moiety, such as a PHIP transfer moiety, that is part of the side arm. The side arm can be para-hydrogenated using para-hydrogen (e.g., by mixing the precursor and para-hydrogen). In some embodiments, the hydrogenation produces an Iz1Iz2 order, which is a lower energy state between |↑>|↓>, |↓>|↑>, or a singlet spin order of two hydrogen spins, depending on whether the hydrogenation is performed in a low or high magnetic field.

[0038] In some embodiments, the precursor is selected such that after hydrogenation and other optional chemical reactions, the biorelevant contrast agent is suitable for use in hyperpolarized NMR or MRI applications. In some embodiments, additional chemical reactions after hydrogenation can be used to separate the biorelevant contrast agent from the precursor. Such additional chemical reactions can include, for example, cleavage of the side arm of the precursor by hydrolysis. For example, the biorelevant contrast agent can be a metabolite molecule such that the precursor can be a derivative of the metabolite molecule, the derivative having the general chemical structure of Formula I. The biorelevant contrast agent can be polarized using the PHIP-SAH method (i.e., para-hydrogenation of the side arm and subsequent polarization transfer to the biorelevant contrast agent). After hydrogenation and polarization transfer, the bond in the precursor (e.g., an ester bond) can be hydrolyzed to produce a polarized biorelevant contrast agent and a separate side arm element.

[0039] As used herein, hydrolysis is defined as the cleavage of a molecule via a nucleophilic substitution reaction involving the addition of the element water. Hydrolysis can also be carried out under anhydrous conditions in the presence of hydroxide ions.

[0040] Consistent with the disclosed embodiments, precursors of the general chemical form presented in Formula I can be used as precursors for PHIP-SAH. After hydrogenation of such precursors, two PHIP-SAHs exhibiting spin order are obtained. 1 The H spin is close (e.g., no more than 3, 4, or 5 bonds away) from the target carbon or nitrogen on the metabolite, as described herein. 13 C concentrated or 15 In some embodiments, the 13 C or 15 Originating from N spin and parahydrogen 1A high J-coupling between at least one of the H spins is achieved. In some embodiments, the J-coupling is achieved at least about 0.1 Hertz (Hz), 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, or more, up to about 10 Hz, 9 Hz, 8 Hz, 7 Hz, 6 Hz, 5 Hz, 4 Hz, 3 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, or less, or within a range defined by any two of the aforementioned values. For example, in some embodiments, the J-coupling may be from 1 Hz to 2 Hz, 1 Hz to 3 Hz, 1 Hz to 4 Hz, 1 Hz to 5 Hz, 1 Hz to 6 Hz, 1 Hz to 7 Hz, 1 Hz to 8 Hz, 1 Hz to 9 Hz, 1 Hz to 10 Hz, 2 Hz to 3 Hz, 2 Hz to 4 Hz, 2 Hz to 5 Hz, 2 Hz to 6 Hz, 2 Hz to 7 Hz, 2 Hz to 8 Hz, 2 Hz to 9 Hz, 2 Hz to 10 Hz, 3 Hz to 4 Hz, 3 Hz to 5 Hz, 3 Hz to 6 Hz, 3 Hz to 7 Hz, 3 Hz to 8 Hz, Hz, 3Hz-9Hz, 3Hz-10Hz, 4Hz-5Hz, 4Hz-6Hz, 4Hz-7Hz, 4Hz-8Hz, 4Hz-9Hz, 4Hz-10Hz, 5Hz-6Hz, 5Hz-7Hz, 5Hz-8Hz, 5Hz-9Hz, 5Hz-10Hz, 6Hz-7Hz, 6Hz-8Hz, 6Hz-9Hz, 6Hz-10Hz, 7Hz-8Hz, 7Hz-9Hz, 7Hz-10Hz, 8Hz-9Hz, 8Hz-10Hz, or 9Hz-10Hz. Such J-couplings are 13 This may enable efficient polarization of the C spins.

[0041] The present invention relates to compounds of formula I, II, III, and IV, their tautomers, deuterated derivatives of these compounds, and their tautomers, salts thereof, and to compounds having a tautomer structure at one or more sites within the molecule. 13 C or 15Novel precursors are disclosed, including N-enriched derivatives (which can then be subjected to hyperpolarization), as well as the subsequent generation of precursors given by general formulas I, II, III, and IV.

[0042] Precursor of Formula I In some embodiments, the precursor comprises a compound of formula I. Formula I includes the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z is selected from the group consisting of (i) and (ii) N-enriched derivatives. 1 H (proton), 2 H (deuterium), or combinations thereof (e.g., -C 1 H=C 1 H-, -C 1 H=C 2 H-, -C 2 H=C 2 In some embodiments, R1 comprises a carbon-carbon double bond (-C=C-) substituted to include a substituted aryl group (-H-), or (ii) a carbon-carbon triple bond (-C≡C-). In some embodiments, R1 comprises a parahydrogen induced polarization (PHIP) transfer moiety as described herein. In some embodiments, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In some embodiments, R3 comprises a biorelevant imaging agent as described herein. In formula I, all moieties to the right of the R3-R1 bond (i.e., -R1-Z-(C=O)-R2) may be collectively referred to as a side arm.

[0043] In some embodiments, the compound of formula I has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more, and up to about 1,00 0 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the aforesaid values.

[0044] In some embodiments, the compound of formula I is at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more in an organic solvent (e.g., acetone, ethanol, chloroform, toluene). in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0045] In some embodiments, the compound of formula I comprises 4-((2-oxopropanoyl)oxy)but-2-ynoate methyl. In some embodiments, the compound of formula I comprises 4-((2-hydroxypropanoyl)oxy)but-2-ynoate methyl. In some embodiments, the compound of formula I comprises 5-((4-methoxy-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0046] In some embodiments, the compound of formula I comprises isopropyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises isopropyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 5-((4-isopropoxy-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0047] In some embodiments, the compound of formula I comprises tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0048] In some embodiments, the compound of formula I is tert-butyl 4-((2-oxopropanoyl-1- 13 C) oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises tert-butyl 4-((2-hydroxypropane-1- 13 C l)oxy)but-2-ynoate. In some embodiments, the compound of formula I is 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid 13 Contains C acid.

[0049] In some embodiments, the compound of formula I comprises 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-hydroxypropanoyl-1-13C)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 5-((4-((2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)oxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0050] In some embodiments, the compound of formula I comprises tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate-4-d. In some embodiments, the compound of formula I comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate-4-d. In some embodiments, the compound of formula I comprises 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl-1-d)oxy)-4,5-dioxopentanoic acid.

[0051] In some embodiments, the compound of formula I comprises tert-butyl 4-((2-oxopropanoyl)oxy)pent-2-ynoate. In some embodiments, the compound of formula I comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)pent-2-ynoate. In some embodiments, the compound of formula I comprises 5-((5-(tert-butoxy)-5-oxopent-3-yn-2-yl)oxy)-4,5-dioxopentanoic acid.

[0052] In some embodiments, the compound of formula I comprises tert-butyl 4-((2-oxopropanoyl)oxy)-4-phenylbut-2-ynoate. In some embodiments, the compound of formula I comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)-4-phenylbut-2-ynoate. In some embodiments, the compound of formula I comprises 5-((4-(tert-butoxy)-4-oxo-1-phenylbut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0053] In some embodiments, the compound of formula I comprises benzhydryl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises benzhydryl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 5-((4-(benzhydryloxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0054] In some embodiments, the compound of formula I comprises 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-oxo-4-phenylbut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 4,5-dioxo-5-((4-oxo-4-phenylbut-2-yn-1-yl)oxy)pentanoic acid.

[0055] In some embodiments, the compound of formula I comprises 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 4,5-dioxo-5-((4-oxo-4-(phenyl-d5)but-2-yn-1-yl)oxy)pentanoic acid.

[0056] In some embodiments, the compound of formula I comprises tert-butyl 4-acetoxybut-2-ynoate. In some embodiments, the compound of formula I comprises 1-(4-(Tert-butoxy)-4-oxobut-2-yn-1-yl)5-ethyl 2-oxopentanedioate.

[0057] In some embodiments, the compound of formula I comprises 4-(2,2-dichloroacetoxy)but-2-ynoate methyl.

[0058] In some embodiments, the compound of formula I comprises trityl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises trityl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula I comprises 4,5-dioxo-5-((4-oxo-4-(trityloxy)but-2-yn-1-yl)oxy)pentanoic acid.

[0059] In some embodiments, the compound of formula I comprises 4-(diphenylamino)-4-oxobut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-(diphenylamino)-4-oxobut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 5-((4-(diphenylamino)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0060] In some embodiments, the compound of formula I comprises 4-(diisopropylamino)-4-oxobut-2-yn-1-yl 2-oxopropanoic acid salt. In some embodiments, the compound of formula I comprises 4-(diisopropylamino)-4-oxobut-2-yn-1-yl 2-hydroxypropanoic acid salt. In some embodiments, the compound of formula I comprises 5-((4-(diisopropylamino)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0061] In some embodiments, the compound of formula I comprises 4-oxopent-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-oxopent-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 4,5-dioxo-5-((4-oxopent-2-yn-1-yl)oxy)pentanoic acid.

[0062] In some embodiments, the compound of formula I comprises 4-oxo-4-(pyridin-2-yl)but-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-oxo-4-(pyridin-2-yl)but-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 4,5-dioxo-5-((4-oxo-4-(pyridin(27yridine)-2-yl)but-2-yn-1-yl)oxy)pentanoic acid.

[0063] In some embodiments, the compound of formula I comprises 4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula I comprises 4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula I comprises 5-((4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0064] Hydrogenated precursor of formula II In some embodiments, the compound of formula I is para-hydrogenated (i.e., modified via addition of a para-hydrogenation proton across Z via a hydrogenation reaction between formula I and para-hydrogen) as described herein. In some embodiments, para-hydrogenation of the compound of formula I gives rise to a compound of formula II. Formula II encompasses the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z' is selected from the group consisting of (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that has been substituted to include H (deuterium), or combinations thereof (e.g., -CH2H*-CH2H*-, -CHDH*-CH2H*-, -CD2H*-CH2H*-); or (ii) 1 H (proton), 2H (deuterium), or a combination thereof. In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (i.e., a hydrogen atom or proton added across a carbon-carbon double bond or carbon-carbon triple bond Z via a hydrogenation reaction between a compound of formula I as described herein and para-hydrogen). In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (e.g., prior to polarization transfer). In some embodiments, R1 comprises a PHIP transfer moiety as described herein. In some embodiments, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In some embodiments, R3 comprises a biorelevant imaging agent as described herein. In formula II, all moieties to the right of the R3-R1 bond (i.e., -R1-Z'-(C=O)-R2) may be collectively referred to as a para-hydrogenated side arm.

[0065] In some embodiments, the compound of formula II has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more, and up to about 1,0 In some embodiments, the compound has a solubility in water of 00 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the aforesaid values.

[0066] In some embodiments, the compound of formula II is at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more in an organic solvent (e.g., acetone, ethanol, chloroform, toluene). in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0067] In some embodiments, when the composition of Formula I reacts with parahydrogen, the chemical yield (e.g., the chemical yield of the compound of Formula II) is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, up to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, when the composition of formula I is reacted with parahydrogen, the chemical yield is between 30% and 35%, 30% and 40%, 30% and 45%, 30% and 50%, 30% and 55%, 30% and 60%, 30% and 65%, 30% and 70%, 30% and 75%, 30% and 80%, 30% and 85%, 30% and 90%, 30% and 95%, 35% and 40%, 35% and 45%, 35% and 50%, 35% and 55%, 35% and 60%, 35% and 65%, 35%~70%, 35%~75%, 35%~80%, 35%~85%, 35%~90%, 35%~95%, 40%~45%, 40%~50%, 40%~55%, 40%~60%, 40%~65%, 40%~70%, 40%~75%, 40%~80%, 40%~85%, 40%~90%, 40%~95%, 45%~50%, 45%~55%, 45%~60%, 45%~65%, 45%~70%, 45%~75%, 45%~80%, 45 %~85%, 45%~90%, 45%~95%, 50%~55%, 50%~60%, 50%~65%, 50%~70%, 50%~75%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 55%~60%, 55%~65%, 55%~70%, 55%~75%, 55%~80%, 55%~85%, 55%~90%, 55%~95%, 60%~65%, 60%~70%, 60%~75%, 60%~80%, 60% ~85%, 60%~90%, 60%~95%, 65%~70%, 65%~75%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 70%~75%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 80%~85%, 80%~90%, 80%~95%, 85%~90%, 85%~95%, or 90%~95%.

[0068] Cleaved precursor of formula III In some embodiments, the compound of formula II is cleaved (e.g., hydrolyzed) as described herein. In some embodiments, the compound of formula II is cleaved (e.g., hydrolyzed) as described herein to provide a side arm compound and a corresponding biorelevant imaging agent. In some embodiments, cleavage of the compound of formula II results in a compound of formula III and a corresponding biorelevant imaging agent as described herein. Formula III encompasses the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z″ is selected from the group consisting of (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 H, or a combination thereof. In some embodiments, R1' comprises a PHIP transfer moiety as described herein. In some embodiments, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In formula III, all of the moieties R1-Z''-(C=O)-R2 may be collectively referred to as a cleaved sidearm or a hydrolyzed sidearm.

[0069] In some embodiments, the compound of formula III has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more, and up to about 1,0 In some embodiments, the compound has a solubility in water of 00 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the aforesaid values.

[0070] In some embodiments, the compound of formula III is dissolved in at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more of an organic solvent (e.g., acetone, ethanol, chloroform, toluene). in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0071] Formula IV Side Arm In some embodiments, a biorelevant imaging agent and a side arm, such as a side arm compound of formula IV, are conjugated as described herein to form a precursor compound, such as a compound of formula I. Formula IV includes the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z is selected from the group consisting of (i) and (ii) N-enriched derivatives. 1 H (proton), 2 H (deuterium), or combinations thereof (e.g., -C 1 H=C 1 H-, -C1 H=C 2 H-, -C 2 H=C 2 In some embodiments, R1 comprises a carbon-carbon double bond (-C=C-) substituted to include a carbon-carbon double bond (-C=C-), or (ii) a carbon-carbon triple bond (-C≡C-). In some embodiments, R1 comprises a parahydrogen induced polarization (PHIP) transfer moiety as described herein. In some embodiments, R2 comprises a solubilizing moiety as described herein. In some embodiments, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine. In some embodiments, complexation of a compound of formula IV with a biorelevant imaging agent results in a compound of formula I as described herein.

[0072] PHIP moving part In some embodiments, the PHIP transfer moieties described herein can transfer one or more para-hydrogenated protons H* (e.g., side-arm H*) to one or more non-hydrogen nuclear spins of a biologically relevant imaging agent (e.g., one or more of the biologically relevant imaging agents as described herein). 13 C or 15 The PHIP transfer moiety comprises a chemical moiety configured to permit or enhance polarization transfer from a para-hydrogenated proton H* in the side arm of the compound of formula II to a non-hydrogen nuclear spin of the corresponding biologically relevant imaging agent of the compound of formula II after the para-hydrogenation reaction between the compound I and para-hydrogen. In some embodiments, the PHIP transfer moiety permits or enhances polarization transfer from a para-hydrogenated proton H* in the side arm of the compound of formula II to a non-hydrogen nuclear spin of the corresponding biologically relevant imaging agent of the compound of formula II after the para-hydrogenation reaction between the compound I and para-hydrogen.

[0073] In some embodiments, the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

[0074] In some embodiments, the PHIP transfer moiety comprises a chemical moiety in the form of *CR4R5, *CR4Y, *C=Y, or any deuterated version thereof. 12 C or 13 C is a carbon isotope. In some embodiments, R and R are each independently selected from: 1 H, 2 H, 3 H, linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups. In some embodiments, Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the spin 1 / 2 atoms are selected from: 1 H, 13 C. 15 N, 19 F, and 31 P. In some embodiments, 15 N may be substituted with a nitro group, an amine group, an amide group, or an imine group. 31 P may be substituted with one or more keto groups, one or more nitro groups, one or more amine groups, one or more amide groups, or one or more imine groups.

[0075] In some embodiments, the PHIP transfer moiety comprises a chemical moiety of the form *CR6R7-*CR8R9, or any deuterated version thereof. 12 C or 13 C is a carbon isotope. In some embodiments, R6, R7, R8, and R9 are each independently selected from: 1 H, 2 H, 3H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

[0076] In some embodiments, the PHIP transfer moiety comprises a chemical moiety in the form of *CH2, *CH2-*CH2, *CHY, *C=Y, or any deuterated version thereof. 12 C or 13 is a carbon isotope of C. In some embodiments, Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the spin 1 / 2 atoms are selected from: 1 H, 13 C. 15 N, 19 F, and 31 P.

[0077] In some embodiments, the compositions described herein include a first J-coupling, J, between a spin ½ atom described herein and a non-hydrogen nuclear spin described herein. 12 In some embodiments, the compositions described herein include a second J-coupling J between a spin 1 / 2 atom described herein and a para-hydrogenated proton H* described herein. 13 In some embodiments, the compositions described herein include a third J-coupling J between a non-hydrogen nuclear spin as described herein and a para-hydrogenated proton H* as described herein. 23 In some embodiments, J 12 and / or J 13 J 23 In such cases, the PHIP transfer moiety may permit or enhance polarization transfer.

[0078] In some embodiments, the PHIP transfer moiety induces J-coupling between one or both of *H nuclear spins having non-hydrogen nuclear spins of at least about 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz or more, up to about 10 Hz, 9 Hz, 8 Hz, 7 Hz, 6 Hz, 5 Hz, 4 Hz, 3 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz or less, or induces J-coupling with non-hydrogen nuclear spins within a range defined by any two of the foregoing values. For example, in some embodiments, the J-coupling may be from 1 Hz to 2 Hz, 1 Hz to 3 Hz, 1 Hz to 4 Hz, 1 Hz to 5 Hz, 1 Hz to 6 Hz, 1 Hz to 7 Hz, 1 Hz to 8 Hz, 1 Hz to 9 Hz, 1 Hz to 10 Hz, 2 Hz to 3 Hz, 2 Hz to 4 Hz, 2 Hz to 5 Hz, 2 Hz to 6 Hz, 2 Hz to 7 Hz, 2 Hz to 8 Hz, 2 Hz to 9 Hz, 2 Hz to 10 Hz, 3 Hz to 4 Hz, 3 Hz to 5 Hz, 3 Hz to 6 Hz, 3 Hz to 7 Hz, 3 Hz to 8 Hz, Hz, 3Hz to 9Hz, 3Hz to 10Hz, 4Hz to 5Hz, 4Hz to 6Hz, 4Hz to 7Hz, 4Hz to 8Hz, 4Hz to 9Hz, 4Hz to 10Hz, 5Hz to 6Hz, 5Hz to 7Hz, 5Hz to 8Hz, 5Hz to 9Hz, 5Hz to 10Hz, 6Hz to 7Hz, 6Hz to 8Hz, 6Hz to 9Hz, 6Hz to 10Hz, 7Hz to 8Hz, 7Hz to 9Hz, 7Hz to 10Hz, 8Hz to 9Hz, 8Hz to 10Hz, or 9Hz to 10Hz.

[0079] R2 units In some embodiments, the R2 groups described herein include optionally substituted hydrocarbons, alkoxy groups, primary amines, secondary amines, or tertiary amines. In some embodiments, the R2 groups described herein include optionally substituted hydrocarbons, alkoxy groups, primary amines, secondary amines, or tertiary amines that function as solubilizing moieties. In some embodiments, the R2 groups described herein include solubilizing moieties. In some embodiments, the solubilizing moieties include any chemical moiety configured to permit or enhance the solubility of a compound, such as any of the compounds of formula I, II, III, and / or IV, in a solution in which a parahydrogenation reaction or a cleavage (e.g., hydrolysis) reaction is performed. In some embodiments, the solubility enhancement is measured for variants of the compounds of formula I, II, III, or IV that utilize one or more protons in place of the R2 group. In some embodiments, the solubility enhancement is measured for variants of the compounds of formula I, II, III, or IV that utilize a methyl group as the R2 group.

[0080] In some embodiments, the solubilizing moiety comprises a hydrophobic moiety or an organophilic moiety. In some embodiments, the solubilizing moiety comprises an organic solubilizing moiety. For example, in some embodiments, the solubilizing moiety comprises a hydrophobic moiety, an organophilic moiety, or an organic solubilizing moiety. In some embodiments, the solubilizing moiety comprises a hydrophilic moiety or an organophilic moiety.

[0081] In some embodiments, the R2 group comprises or is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide. In some embodiments, the substituted phenyl group is selected from fluorobenzene, chlorobenzene, bromobenzene, iodobenzene, toluene, cumene, ethylbenzene, styrene, ortho-xylene, meta-xylene, para-xylene, phenol, benzoic acid, benzaldehyde, acetophenone, methyl benzoate, anisole, aniline, nitrobenzene, benzonitrile, benzamide, benzenesulfonic acid, naphthalene, and anthracene.

[0082] R3 groups In some embodiments, the R3 group described herein comprises a biorelevant imaging agent. In some embodiments, the biorelevant imaging agent has the formula R4C(=O)X-. In some embodiments, R4 is selected from a linear, branched, or cyclic C1-C10 alkyl group, where one or more C atoms are optionally substituted with CO, COOH, CH2COOH, CONH2, OH, amino (NR'R''), one or more halogen atoms, one or more haloalkyl groups, or one or more carbocycles, where the carbocycles are optionally substituted with one or more aliphatic or aromatic rings optionally substituted with one or more functional groups. In some embodiments, X is selected from NR''' and O. In some embodiments, R', R'', and R''' are each independently 1 H, 2 H, 3H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzyl, tert-butylcarbonate, and benzyl. In some embodiments, the R3 group comprises any biologically relevant imaging agent described herein.

[0083] In some embodiments, the R3 group comprises at least one non-hydrogen nuclear spin. In some embodiments, the non-hydrogen nucleus comprises at least one spin 1 / 2 atom. In some embodiments, the non-hydrogen nuclear spin is 13 C or 15 N. In some embodiments, the R3 group is at least partially isotopically labeled with non-hydrogen nuclear spins. In some embodiments, the R3 group is at least partially enriched in non-hydrogen nuclear spins compared to an analog of the R3 group that characterizes the non-hydrogen nuclear spins at their natural abundance. In some embodiments, the R3 group is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to about 99%, 98%, 97%, 96%, 95 .... %, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less abundance, or an abundance within a range defined by any two of the aforesaid values.

[0084] In some embodiments, the non-hydrogen nuclear spins are characterized as NMR inactive (i.e., spin 0) nuclei of an analog of an R3 group that characterizes the non-hydrogen nuclear spins at their natural abundance, as described herein (e.g., 12 C or quadrupolar (i.e., spin >1 / 2) nuclei (e.g., 14In some embodiments, the non-hydrogen nuclear spin is located no more than about one or two chemical bonds from the carbonyl (C=O) carbon in the R3 group.

[0085] Parahydrogenation Consistent with the disclosed embodiments, a precursor of a biorelevant imaging agent (such as a compound of formula I as described herein) may be para-hydrogenated by combining the precursor, para-hydrogen, and a hydrogenation catalyst. The disclosed embodiments are not limited to a particular method of generating the para-hydrogenated precursor. In some embodiments, the precursor is added to a mixture containing para-hydrogen. In some embodiments, para-hydrogen gas is added to a solution containing the precursor (e.g., para-hydrogen gas may be bubbled into such a solution). In hydrogenating the precursor, para-hydrogen may create a preferred population of lower energy states between Iz1Iz2 order, |↑>|↓>, |↓>|↑>, or singlet spin order on the two hydrogen spins in the precursor.

[0086] The precursor can have unsaturated bonds (such as unsaturated carbon-carbon double bonds or unsaturated carbon-carbon triple bonds) that can be hydrogenated by parahydrogen gas. After combination of the precursor with parahydrogen, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the precursor, up to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the precursor, or a percentage of the precursor that is within a range defined by any two of the foregoing values, can be hydrogenated.

[0087] In some embodiments, the para-hydrogenated precursor has a population difference in the para-hydrogenated proton spin state of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, the population difference is between 10% and 15%, 10% and 20%, 10% and 25%, 10% and 30%, 10% and 35%, 10% and 40%, 10% and 45%, 10% and 50%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, 20% and 25%, 20% and 30%, 20% and 35 ... 0%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%. In some embodiments, the population difference is between a spin state that includes a para-hydrogenated proton and another nuclear spin, e.g., a spin state that includes an additional proton on the compound. In some embodiments, the para-hydrogenated precursor includes a side chain and the para-hydrogenated spin may be located on the side chain.

[0088] In some embodiments, the concentration of the hydrogenation catalyst during hydrogenation is at least about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or more, up to at about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, or less, or within a range defined by any two of the aforesaid values.

[0089] Disclosed embodiments may include methods implemented by the disclosed systems for producing a hyperpolarized biologically relevant contrast agent. The disclosed methods may include mixing (e.g., by a mixing mechanism) a solution including a precursor to the biologically relevant contrast agent and a hydrogenation catalyst. The mixing mechanism may be a device for introducing, holding, and facilitating a blend, mixture, or solution of two or more materials. In some embodiments, the mixing mechanism is disposed within a chamber and mixing occurs within the chamber. In some embodiments, the solutions are mixed at a location remote from the chamber. The solution can be at least about 1 milliliter (ml), 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, or more in volume, or up to 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, or less in volume, or within a volume range defined by any two of the foregoing values.

[0090] In some embodiments, the mixing mechanism is a gas-liquid exchange mechanism. For example, the gas-liquid exchange mechanism can be a bubbler or a diffusion system. In some embodiments, the mixing mechanism includes a membrane adapted to allow diffusion of molecular hydrogen. In some embodiments, the mixing can be performed using a spray chamber, where the solution is sprayed into a chamber filled with pressurized parahydrogen.

[0091] In some embodiments, the catalyst is a molecule, complex, or particulate system that catalyzes hydrogenation. In some embodiments, the catalyst comprises a homogeneous metal catalyst, such as a rhodium complex or a ruthenium complex. The rhodium complex may be used to prepare and activate the precursor molecule and parahydrogen. In some embodiments, the heterogeneous metal catalyst is attached to a nanoparticle.

[0092] Various embodiments of the present disclosure describe introducing a solution including a precursor of a biorelevant imaging agent and a hydrogenation catalyst into a chamber configured to hold the solution during polarization transfer. In some embodiments, the solution is mixed in the chamber. In some embodiments, the solution is hydrogenated in the chamber. In some embodiments, the chamber is within a magnetic shield (e.g., a mu metal shield). The magnetic shield can reduce the effects of the Earth's magnetic field (or other external magnetic fields) and allows for modulation of the amplitude of a low-level magnetic field applied to the solution. Thus, placing the solution in the chamber can include placing the solution in a magnetic shield.

[0093] As described herein, in some embodiments, para-hydrogenation occurs prior to polarization transfer (e.g., before modulating the amplitude of a magnetic field applied to a solution, etc.). In some embodiments, para-hydrogenation occurs during polarization transfer. For example, para-hydrogen may be combined with the solution (e.g., flowed or bubbled through the solution) during modulation of the amplitude of the magnetic field.

[0094] In some embodiments, the parahydrogen gas is combined with the solution in the hydrogenation chamber at a pressure. The pressure may be at least about 10 bar, 15 bar, 20 bar, 30 bar, 50 bar, or more, and may be up to about 50 bar, 30 bar, 20 bar, 15 bar, 10 bar, or less, or within a range defined by any two of the preceding values. In some embodiments, the parahydrogen is combined with the solution in a metal chamber that can withstand the pressure. The parahydrogen may be combined with the solution over a time interval (or dissolution of the parahydrogen may occur in less than a time interval). The time interval can be up to about 90 seconds, 60 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, or less, at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, or more, or within a range defined by any two of the foregoing values. In some embodiments, hydrogenation is performed or occurs within the time interval.

[0095] Polarization transfer using radio frequency waveforms In some embodiments, the concentration of the precursor in the solution prior to polarization transfer is at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1,000 mM, or more, up to at most about 1,000 mM, 900 mM, 800 mM, 700 mM, 600 mM, 500 mM, 400 mM, 300 mM, 200 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or less, or within a range defined by any two of the foregoing values. The volume of the solution may be at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1000 ml, 2000 ml, or more, up to about 2000 ml, 1 The volume may be 000ml, 900ml, 800ml, 700ml, 600ml, 500ml, 400ml, 300ml, 200ml, 100ml, 90ml, 80ml, 70ml, 60ml, 50ml, 40ml, 30ml, 20ml, 10ml, 9ml, 8ml, 7ml, 6ml, 5ml, 4ml, 3ml, 2ml, 1ml or less, or within a range defined by any two of the aforesaid values.

[0096] Various embodiments of the present disclosure describe the application of a polarization transfer magnetic perturbation intended to generate a magnetic field around a solution (e.g., around a solution comprising Formula II as described herein). In some embodiments, the magnetic field is at least about 0.1 Gauss (G), 0.2G, 0.3G, 0.4G, 0.5G, 0.6G, 0.7G, 0.8G, 0.9G, 1G, 2G, 3G, 4G, 5G, 6G, 7G, 8G, 9G, 10G, 20G, 30G, 40G, 50G, 60G, 70G, 80G, 90G, 100G, 200G, 300G, 400G, 500G, 600G, 700G, 800G, 900G, 1,000G, 2,000G, 3,000G, 4,000G, 5,000G, 6,000G, 7,000G, 8,000G, 9,000G, 10,000G, 20,000G, 30,000G, 40,000G, 50,000G, 60,000G, 70,000G, 80,000G, 90,000G, 100,000G, 200,000G or more, up to a maximum of approximately 200 ,000G, 100,000G, 90,000G, 80,000G, 70,000G, 60,000G, 50,000G, 40,000G, 30,000G, 20,000G, 10,000G, 9,000G, 8,000G, 7,000G, 6,000G, 5,000G, 4,000G, 3,000G, 2,000G, 1,000G, 900G, 800G, 700G, 600G, 500G , 400G, 300G, 200G, 100G, 90G, 80G, 70G, 60G, 50G, 40G, 30G, 20G, 10G, 9G, 8G, 7G, 6G, 5G, 4G, 3G, 2G, 1G, 0.9G, 0.8G, 0.7G, 0.6G, 0.5G, 0.4G, 0.3G, 0.2G, 0.1G, or less, or within a range defined by any two of the preceding values. In some embodiments, the magnetic field has a strength of 0.1G to 200,000G around the solution. The magnetic perturbation may be generated by an electromagnet or a permanent magnet. The magnetic field may be applied to the sample in a pulse or continuous wave (CW). The magnetic perturbation may be static or time-varying.

[0097] The signal generator may be configured to generate one or more radio frequency (RF) waveforms that may be applied to the sample to transfer polarization. The signal generator may include another computing unit, processor, controller, associated memory, PC, computer services, or any device that can perform computational operations using inputs and generate outputs. In some embodiments, the RF coil may emit or "apply" a pulse sequence that includes the first RF waveform. In some embodiments, the RF coil may have one or more channels. A channel may be a path for an RF signal. At least one channel may be provided for each different type of NMR spectroscopy. In some embodiments, 1 H has at least one channel, 2 H, 13 C. 15 N, 19 F, and 31 P has at least one channel. For example, the first RF waveform is generated by one or more radio frequency coils (RF coils) disposed around the sample. 1 In some embodiments, the second RF waveform may be applied to the H channel of the RF coil. 13 In some embodiments, 1 H channel and 13 The RF waveform on the C channel is configured to apply a polarization transfer sequence such as PH-INEPT, Goldman's sequence, S2M, S2hM, SLIC, ADAPT, or ESOTERIC.

[0098] In some embodiments, the RF waveform is configured to support polarization transfer even in the presence of large proton full width at half maximum (FWHM). Such RF waveforms can include pulse sequences that can include tens to hundreds of RF pulses. The sequences can be configured such that the pulses protect against the deleterious effects of magnetic field inhomogeneities on polarization transfer.

[0099] In some embodiments, the polarization pulse sequence can be used to separate two non-equivalent ions, e.g., when the chemical shift difference is greater than the J-coupling between them. 1H hydride spins. ESOTHERIC may, for example, be a pulse sequence suitable for polarization transfer in this regime.

[0100] In some embodiments, the pulse sequences are equivalent when, for example, the chemical shift difference is smaller than the J-coupling between them. 1H hydrogen spins. Such pulse sequences are configured to transfer spin order from the H hydrogen spins. T, 9mT, 10mT, 20mT, 30mT, 40mT, 50mT, 60mT, 70mT, 80mT, 90mT, 100mT, 200mT, 300mT, 400mT, 500mT, 600mT, 700mT, 800mT, 900mT, 1,000mT, 2,000mT, 3,000mT, 4,000mT, 5,000mT, 6,000mT or more, up to about 6,000mT, 5,000mT, 4,000mT, 3,000mT, 2,000mT, 1,000mT, 900mT, 800mT, 700mT, 600mT, 500mT, 400mT, 300mT, 200mT , 100mT, 90mT, 80mT, 70mT, 60mT, 50mT, 40mT, 30mT, 20mT, 10mT, 9mT, 8mT, 7mT, 6mT, 5mT, 4mT, 3mT, 2mT, 1mT , 0.9mT, 0.8mT, 0.7mT, 0.6mT, 0.5mT, 0.4mT, 0.3mT, 0.2mT, 0.1mT, 0.09mT, 0.08mT, 0.07mT, 0.06mT, 0.05mT, 0.04mT, 0.03mT, 0.02mT, 0.01mT, or less, or within a range defined by any two of the preceding values. Examples of such sequences may be Goldman's sequence (M. Goldman, H. Johannesson, CR Phys. 2005, 6, 575-581, which is incorporated herein by reference in connection with pulse sequence configurations for transferring spin order), singlet to heteronuclear magnetization (S2hM) sequences, or other sequences used in singlet NMR (e.g., ADAPT, SLIC, etc.).

[0101] In some embodiments, the magnetic shielding can be configured to withstand a magnetic field applied to the solution of at least about 0mG, 0.1mG, 0.2mG, 0.3mG, 0.4mG, 0.5mG, 0.6mG, 0.7mG, 0.8mG, 0.9mG, 1mG, 2mG, 3mG, 4mG, 5mG, 6mG, 7mG, 8mG, 9mG, 10mG, 20mG, 30mG, 40mG, 50mG, 60mG, 70mG, 80mG, 90mG, 100mG, or more, up to about 100mG. The magnetic shield is configured to maintain a magnetic field applied to the solution of less than or equal to 0.5 G, 90 mG, 80 mG, 70 mG, 60 mG, 50 mG, 40 mG, 30 mG, 20 mG, 10 mG, 9 mG, 8 mG, 7 mG, 6 mG, 5 mG, 4 mG, 3 mG, 2 mG, 1 mG, 0.9 mG, 0.8 mG, 0.7 mG, 0.6 mG, 0.5 mG, 0.4 mG, 0.3 mG, 0.2 mG, 0.1 mG, or a magnetic field within a range defined by any two of the preceding values. The magnetic shield may maintain the magnetic field strength in the polarization chamber at such amplitudes during application of the polarizing waveforms to the one or more radio frequency coils.

[0102] Consistent with disclosed embodiments, an RF waveform may be applied to a solution containing a para-hydrogenated precursor.

[0103] Polarization transfer using magnetic field modulation In some embodiments, the polarization transfer magnetic perturbation is performed within a magnetic shield (e.g., a mu-shield, etc.) to achieve a homogeneous low magnetic field. The magnetic shield is designed to provide a low magnetic field in the microtesla (μT) range, below the Earth's magnetic field. 13The low magnetic field allows for the performance of polarization transfer to the C nuclear spins. The low magnetic field may be about 0 mG, 0.1 mG, 0.2 mG, 0.3 mG, 0.4 mG, 0.5 mG, 0.6 mG, 0.7 mG, 0.8 mG, 0.9 mG, 1 mG, 2 mG, 3 mG, 4 mG, 5 mG, 6 mG, 7 mG, 8 mG, 9 mG, 10 mG, 20 mG, 30 mG, 40 mG, 50 mG, 60 mG, 70 mG, 80 mG, 90 mG, 100 mG, or more, up to about 100 mG, 90 mG , 80mG, 70mG, 60mG, 50mG, 40mG, 30mG, 20mG, 10mG, 9mG, 8mG, 7mG, 6mG, 5mG, 4mG, 3mG, 2mG, 1mG, 0.9mG, 0.8mG, 0.7mG, 0.6mG, 0.5mG, 0.4mG, 0.3mG, 0.2mG, 0.1mG, or less, or within a range defined by any two of the aforesaid values.

[0104] In such a magnetic field, the polarization is determined by the proton spin and 2 H, 13 C. 15 N, 19 F, and 31 The polarization transfer is performed by utilizing a level pseudocrossing (LAC) between the spin species of interest, including P, and other spin species of interest. In some embodiments, the magnetic field can be tuned to a specific field strength for the LAC. In various embodiments, the magnetic field strength can be adjusted in time to enable robust polarization transfer in larger volumes of samples. For example, the magnetic field strength can be swept through the LAC conditions. Alternatively or additionally, the sample can be physically moved within the magnetic field. Such modulation can relax constraints on magnetic field homogeneity and magnetic field offset. Thus, robust polarization transfer can be performed in larger volumes and with greater efficiency. Furthermore, relaxing constraints on magnetic field homogeneity and magnetic field offset can enable the use of less complex, accurate, or expensive polarization systems.

[0105] The lower limit of the magnetic field modulation may be at least about -10μT, -9μT, -8μT, -7μT, -6μT, -5μT, -4μT, -3μT, -2μT, -1μT, -0.9μT, -0.8μT, -0.7μT, -0.6μT, -0.5μT, -0.4μT, -0.3μT, -0.2μT, -0.1μT, or more, up to about -0.1μT, The value may be -0.2μT, -0.3μT, -0.4μT, -0.5μT, -0.6μT, -0.7μT, -0.8μT, -0.9μT, -1μT, -2μT, -3μT, -4μT, -5μT, -6μT, -7μT, -8μT, -9μT, -10μT, or less, or within a range defined by any two of the aforesaid values. The upper modulation limit can be at least about 0.1 μT, 0.2 μT, 0.3 μT, 0.4 μT, 0.5 μT, 0.6 μT, 0.7 μT, 0.8 μT, 0.9 μT, 1 μT, 2 μT, 3 μT, 4 μT, 5 μT, 6 μT, 7 μT, 8 μT, 9 μT, 10 μT, or more, up to but not including about 10 μT, 9 μT, 8 μT, 7 μT, 6 μT, 5 μT, 4 μT, 3 μT, 2 μT, 1 μT, 0.9 μT, 0.8 μT, 0.7 μT, 0.6 μT, 0.5 μT, 0.4 μT, 0.3 μT, 0.2 μT, 0.1 μT, or less, or within a range defined by any two of the foregoing values.

[0106] The magnetic field may be applied to a volume of at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1,000 ml, 2,000 ml, or more, and up to about 2,000 ml, 1,000 ml, 90 The modulation may have such amplitude over a volume of 0 ml, 800 ml, 700 ml, 600 ml, 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml or less, or a volume within a range defined by any two of the foregoing values. The modulation may be performed over a duration. The duration may be at least about 100 milliseconds (ms), 200 ms, 300 ms, 400 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1,000 ms, 2,000 ms, 3,000 ms, 4,000 ms, 5,000 ms, 6,000 ms, 7,000 ms, 8,000 ms, 9,000 ms, 10,000 ms, 20,000 ms, 30,000 ms, 40,000 ms, 50,000 ms, or more, up to about 50,000 ms. The time may be within a range defined by any two of the foregoing values, or less than or equal to 40,000 milliseconds, 30,000 milliseconds, 20,000 milliseconds, 10,000 milliseconds, 9,000 milliseconds, 8,000 milliseconds, 7,000 milliseconds, 6,000 milliseconds, 5,000 milliseconds, 4,000 milliseconds, 3,000 milliseconds, 2,000 milliseconds, 1,000 milliseconds, 900 milliseconds, 800 milliseconds, 700 milliseconds, 600 milliseconds, 500 milliseconds, 400 milliseconds, 300 milliseconds, 200 milliseconds, 100 milliseconds, or less, or less than or equal to 40,000 milliseconds, 30,0 ...

[0107] Thus, the rate of change of the amplitude of the magnetic field is at least about 0.01 μT / s, 0.02 μT / s, 0.03 μT / s, 0.04 μT / s, 0.05 μT / s, 0.06 μT / s, 0.07 μT / s, 0.08 μT / s, 0.09 μT / s, 0.1 μT / s, 0.2 μT / s, 0.3 μT / s, 0.4 μT / s, 0.5 μT / s, 0.6 μT / s, 0.7 μT / s, sec, 0.8 μT / sec, 0.9 μT / sec, 1 μT / sec, or more, up to about 1 μT / sec, 0.9 μT / sec, 0.8 μT / sec, 0.7 μT / sec, 0.6 μT / sec, 0.5 μT / sec, 0.4 μT / sec, 0.3 μT / sec, 0.2 μT / sec, 0.1 μT / sec, or less, or within a range defined by any two of the foregoing values. The upper limit of the rate of change of the amplitude of the magnetic field may be determined by the capabilities of the equipment used to perform the sweep.

[0108] In some embodiments, when the magnetic field is within the upper and lower limits disclosed above, the spatial deviation of the magnetic field across the volume during modulation is less than about half (or a quarter, or an eighth, or a tenth) of the amplitude of the magnetic field. For example, when the magnetic field strength is less than 2 μT (or greater than −2 μT), the spatial deviation of the magnetic field across the volume during modulation can be less than 1 uT. As an additional example, when the magnetic field strength is less than 10 μT (or greater than −10 μT), the spatial deviation of the magnetic field across the volume during modulation can be less than 5 μT. The spatial deviation can be measured, for example, by taking at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more spatially randomly sampled or equally spatially distributed measurements of the magnetic field within the volume and calculating the standard deviation of the sampled magnetic field measurements. Such homogeneity can be achieved within a large homogeneous magnetic shield, for example, by having a large piercing solenoid through the magnetic shield, or by using a large Helmholtz coil with a large homogeneous region to generate the magnetic field amplitude modulation. In some embodiments, the modulation is a sweep of the magnetic field. In some embodiments, the magnetic field amplitude modulation includes a non-adiabatic jump, a monotonic amplitude change, or a combination thereof.

[0109] In some embodiments, following the polarization transfer step, a biologically relevant imaging agent (e.g., 13 C or 15 The non-hydrogen nuclear spins of N) have a nuclear spin polarization of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or a polarization that is within a range defined by any two of the foregoing values. For example, in some embodiments, following the polarization transfer step, the non-hydrogen nuclear spins of the biologically relevant contrast agent may be at least 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to 35%, 20% to 45%, 20% to 50%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 100%, 20% to 150%, 20% to 250%, 20% to 300%, 20% to 350%, 20% to 400%, 20% to 5 ... having a nuclear spin polarization of 0% to 30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to 35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to 45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to 50%, or 45% to 50%.

[0110] In some embodiments, this polarization is achieved for solution volumes of about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml or more, up to about 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml or less, or for volumes within a range defined by any two of the foregoing values.

[0111] In some embodiments, after polarization transfer, a portion of the population difference in the para-hydrogenated proton spin state is determined by the target of the biologically relevant imaging agent (e.g., 13 C or 15 N) is transferred to the polarization of the nuclear spins, which may be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, this portion is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 20% and 25%, between 20% and 30%, 20%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%.

[0112] In some embodiments, the magnetic field modulation includes a non-adiabatic jump of the magnetic field. The non-adiabatic jump may be performed for a magnetic field where a level pseudo-crossing occurs involving proton spins and non-proton spins. Considering the J-coupling between the nuclear spins in the system, this value may be analytically calculated or identified by plotting the energy levels of the Hamiltonian for different magnetic fields and identifying the LAC. In some embodiments, the duration for which the magnetic field amplitude is in the LAC condition is at most about 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 0.9 seconds, 0.8 seconds, 0.7 seconds, 0.6 seconds, 0.5 seconds, 0.4 seconds, 0.3 seconds, 0.2 seconds, 0.1 seconds, or less, at least about 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, or more, or within a range defined by any two of the aforementioned values.

[0113] In some embodiments, modulating the amplitude of the magnetic field comprises varying the magnetic field amplitude monotonically (or monotonically over each of a limited number of intervals, such as 1-10 increasing intervals and / or 1-10 decreasing intervals). In some embodiments, modulating the amplitude of the magnetic field comprises linearly varying the amplitude of the magnetic field. The initial magnetic field amplitude of the sweep, the end magnetic field amplitude, and the total duration of the sweep may be optimized for the target molecule. In some embodiments, the magnetic field amplitude during the sweep is within a lower and upper limit. The lower limit can be at least about -2μT, -1μT, -0.9μT, -0.8μT, -0.7μT, -0.6μT, -0.5μT, -0.4μT, -0.3μT, -0.2μT, -0.1μT or more, up to about -0.1μT, -0.2μT, -0.3μT, -0.4μT, -0.5μT, -0.6μT, -0.7μT, -0.8μT, -0.9μT, -1μT, -2μT or less, or within a range defined by any two of the foregoing values. The upper limit can be at least about 0.1 μT, 0.2 μT, 0.3 μT, 0.4 μT, 0.5 μT, 0.6 μT, 0.7 μT, 0.8 μT, 0.9 μT, 1 μT, 2 μT, or more, up to about 2 μT, 1 μT, 0.9 μT, 0.8 μT, 0.7 μT, 0.6 μT, 0.5 μT, 0.4 μT, 0.3 μT, 0.2 μT, 0.1 μT, or less, or within a range defined by any two of the foregoing values. In some embodiments, the duration of the modulation is at least about 100 ms, 200 ms, 300 ms, 400 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1,000 ms, 2,000 ms, 3,000 ms, 4,000 ms, 5,000 ms, 6,000 ms, 7,000 ms, 8,000 ms, 9,000 ms, 10,000 ms, or more, up to about 10 ,000 ms, 9,000 ms, 8,000 ms, 7,000 ms, 6,000 ms, 5,000 ms, 4,000 ms, 3,000 ms, 2,000 ms, 1,000 ms, 900 ms, 800 ms, 700 ms, 600 ms, 500 ms, 400 ms, 300 ms, 200 ms, 100 ms, or less, or within a range defined by any two of the foregoing values. In some embodiments, the rate of amplitude change varies along the amplitude profile.In some embodiments, the constant adiabatic sweep is calculated by selecting a specific subpopulation of level pseudocrossings of the spin system. In some embodiments, the magnetic amplitude modulation comprises a combination of non-adiabatic jumps, monotonic amplitude modulation, and rate of change sign reversals.

[0114] Purification and Separation In some embodiments, the precursor may be selected or designed such that, following hydrogenation and other potential chemical reactions, one of the products is a biorelevant imaging agent usable in hyperpolarized NMR or MRI applications. In some embodiments, the biorelevant imaging agent is generated through additional chemical reactions following hydrogenation. Such additional chemical reactions may include cleaving a side arm of the molecule (e.g., cleavage of a compound of formula II as described herein) to form a biorelevant imaging agent and a side arm (e.g., a compound of formula III as described herein), for example, by hydrolysis. After hydrogenation and polarization transfer, the para-hydrogenated precursor (e.g., a compound of formula II as described herein) may be cleaved to generate a hyperpolarized biorelevant imaging agent.

[0115] The volume of solution containing the biorelevant contrast agent after cleavage (and the concentration of the biorelevant contrast agent produced) may depend on the volume of solution used for polarization transfer and the concentration of the precursors in that solution. Exemplary ranges of solution volumes and precursor concentrations are described herein. As a further specific example, at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, or more of solution containing at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or more of the biorelevant contrast agent may be produced.

[0116] Consistent with disclosed embodiments, following polarization transfer and cleavage of the precursor to generate the hyperpolarized biologically relevant contrast agent, the properties of the solution containing the hyperpolarized biologically relevant contrast agent may be modified to induce precipitation of the hyperpolarized biologically relevant contrast agent.

[0117] Such precipitation can allow separation of the hyperpolarized biorelevant contrast agent from other substances in the solution (e.g., sidearm fragments). The precipitate can form crystals, amorphous solid particles, polycrystalline materials, etc. After precipitation, at least a fraction of the solid hyperpolarized biorelevant contrast agent (or hyperpolarized precursor) can be separated from the solution and other substances in the solution. For example, the mixture of the precipitate and the solution can be filtered to remove particles. The filtered precipitate can be washed using a second solvent. The second solvent can be selected to remove residues of the original solvent and other substances in the solution without completely dissolving the filtered precipitate. In some embodiments, the cleaning step occurs for up to about 300 seconds, 200 seconds, 100 seconds, 90 seconds, 80 seconds, 70 seconds, 60 seconds, 50 seconds, 30 seconds, 20 seconds, 10 seconds, at least about 10 seconds, 20 seconds, 30 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 200 seconds, 300 seconds, or more, or for a period within a range defined by any two of the foregoing values.

[0118] Precipitation can be achieved with acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyceride (diethylene glycol dimethyl ether), 1,2-dimethoxyethane (glyceride, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, and glyceryl. The polarization and precipitation steps may be carried out using aqueous solutions or solutions containing organic solvents such as hexane, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphoric triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidone (NMP), nitromethane, pentane, petroleum ether (ligroin), 1-propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, triethylamine, water, deuterium oxide, o-xylene, m-xylene, p-xylene, etc. In some embodiments, an organic solvent is used for the polarization and precipitation steps. For example, parahydrogen may be more soluble in a range of organic solvents than in aqueous solutions. Thus, hydrogenation may occur more efficiently in such solvents. Thus, PHIP may occur in a solution formed with an organic solvent. In some embodiments, an aqueous solution is used for the hydrogenation and polarization transfer. In some embodiments, the aqueous solution may be mixed with a miscible organic solvent prior to precipitation of the hyperpolarized biorelevant imaging agent.

[0119] In some embodiments, precipitation of the biorelevant contrast agent can be induced by changing the pH of the solvent. In organic solvents, certain biorelevant contrast agents (e.g., carboxylic acids, pyridines, etc.) can be soluble at concentrations suitable for polarization using PHIP. However, salts of these biorelevant contrast agents can be highly insoluble. For example, such salts may be at least about 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, 0.09 mM, 0.08 mM, 0.07 mM, 0.06 mM, 0.05 mM, 0.04 mM, 0.03 mM, 0.02 mM, 0.01 mM, or less, at least about 0.01 mM, 0.02 The organic solution may be insoluble at a concentration of 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.04 mM, 0.05 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM or more, or at a concentration within a range defined by any two of the aforementioned values. The organic solution may contain a concentration of the biologically relevant contrast agent suitable for hyperpolarization using PHIP. After polarization, the pH of the organic solution may be altered to induce precipitation of a salt of the biologically relevant contrast agent. In aqueous solutions, salts of certain biorelevant contrast agents (e.g., fumarate, glutamate, etc.) are more soluble than the acidic forms. Thus, the aqueous solution may contain a concentration of the biorelevant contrast agent suitable for hyperpolarization using PHIP. After polarization, the pH of the aqueous solution may be lowered to induce precipitation of the acidic form of the biorelevant contrast agent.

[0120] Consistent with disclosed embodiments, the change in pH can be induced by the addition of an acidic or basic molecule to the solution, such as sodium chloride or sodium hydroxide. In some embodiments, the change in pH can be induced by mixing the solution with another solution having a substantially different pH. In some embodiments, the precipitation resulting from the change in pH occurs in up to about 100 seconds, 90 seconds, 80 seconds, 70 seconds, 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, or less, at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, or more, or within a period of time defined by any two of the foregoing values.

[0121] In some embodiments, precipitation of the biologically relevant imaging agent may be induced without changing the pH of the solution, which would induce the hyperpolarized biologically relevant imaging agent to change between salt and acid forms.

[0122] In some embodiments, the hydrogenation and polarization transfer occurs in a first solution having a first solvent. The biorelevant contrast agent may have high solubility in the first solvent. Precipitation may be induced by mixing the first solution with a second solvent to form a second solution. The second solvent may be selected such that the solubility of the biorelevant contrast agent in the second solution is low enough to initiate precipitation.

[0123] In some embodiments, the temperature of the solution is changed, thereby decreasing the solubility of the hyperpolarized molecules and initiating precipitation. In most solvents, the solubility decreases as the temperature is decreased. The change in temperature to the desired temperature with lower solubility can be carried out in a period of at most about 100 seconds, 90 seconds, 80 seconds, 70 seconds, 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second or less, within at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds or more, or within a range defined by any two of the aforementioned values. For example, the maximum molar fraction of acetic acid in n-heptane is 0.935 at 14.8° C. and only 0.02 at −29.2° C., and is lower at lower temperatures. The temperature selected for precipitation may be selected as a temperature above the freezing point of the solvent.

[0124] In some embodiments, the surface area of ​​the solution is increased to induce nucleation of a precipitate. This can be accomplished, for example, by spraying the solution through a nozzle to create very small droplets with a high surface area. In some embodiments, microcrystalline seeds (which may be of a biorelevant contrast agent or another biocompatible compound) are added to the solution to induce precipitation.

[0125] In some embodiments, the pressure of the solution is changed, thereby decreasing the solubility of the hyperpolarized compound and initiating precipitation.

[0126] In some embodiments, the concentration of the hyperpolarized biologically relevant contrast agent is increased above its solubility limit, thereby inducing precipitation without decreasing the solubility level of the compound in the solvent. This can be achieved, for example, by adding unpolarized molecules of the biologically relevant contrast agent or by evaporating a certain volume of the solvent, thereby increasing the concentration of the biologically relevant contrast agent above its solubility limit.

[0127] In some embodiments, the parameters of the polarization of PHIP are optimized to exceed the solubility limit of the biorelevant imaging agent in its acid or salt form, even at the expense of achieving a lower polarization for high concentration, e.g., This can be achieved by starting with a high concentration of the precursor for hydrogenation and choosing a long hydrogenation time that can reduce the polarization due to relaxation, in order to achieve a high biorelevant imaging agent concentration.

[0128] In some embodiments, precipitation is accelerated by the addition of mechanical energy or improved mixing of the mixture for a certain duration, which can be done, for example, by applying ultrasound to the mixture through an ultrasound solicitor, or by mechanical or magnetic mixing of the sample. In some embodiments, the additional mixing or introduction of mechanical energy is performed for at least about 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or more, up to about 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 0.9 seconds, 0.8 seconds, 0.7 seconds, 0.6 seconds, 0.5 seconds, 0.4 seconds, 0.3 seconds, 0.2 seconds, 0.1 seconds, or less, or for a period within a range defined by any two of the foregoing values.

[0129] In some embodiments, precipitation is induced by a chemical reaction involving the hyperpolarized bio-relevant contrast agent. The hyperpolarized bio-relevant contrast agent may react with another compound or in response to an external stimulus, such as electromagnetic radiation (e.g., ultraviolet radiation). The product of this reaction may have reduced solubility compared to the hyperpolarized bio-relevant contrast agent, thereby inducing precipitation. For example, the external stimulus may modify the structure of the hyperpolarized bio-relevant contrast agent, thereby reducing the solubility of the hyperpolarized bio-relevant contrast agent. Following re-dissolution of the precipitate, additional reactions may be carried out to produce the desired end product (e.g., a bio-relevant contrast agent, an NMR material, etc.).

[0130] In some embodiments, hydrolysis of the precursor induces precipitation. In some embodiments, the solvent is selected such that the precursor is more soluble than the biorelevant contrast agent. The concentration of the precursor in the solution may be selected such that after cleavage of the side chains and generation of the biorelevant contrast agent, the biorelevant contrast agent precipitates from the solution. In some such embodiments, cleavage is initiated by changing the pH of the solution. For example, cleavage may be initiated by adding a base (e.g., sodium hydroxide or another suitable base). In some embodiments, the solution may be formed with an organic solvent and cleavage is carried out under basic conditions. After cleavage, the poorly soluble biorelevant contrast agent undergoes rapid precipitation while retaining its polarization. The same solution may be used for hydrogenation, polarization transfer, and precipitation, or separate solvents may be mixed into the solutions used for hydrogenation and polarization transfer.

[0131] In some embodiments, precipitation of the precursor occurs before cleavage. Such embodiments may be preferred when the precursor is more stable than the biorelevant contrast agent. Precipitation of the precursor may be induced by modifying the pH of the solution such that the solubility of the precursor is reduced, or by mixing in a solution or compound that reduces the solubility of the precursor. In such embodiments, cleavage of the precursor may be performed after redissolution of the precursor in a solvent. The precursor may be filtered and washed as described herein to remove other substances present in the original solution, such as hydrogenation catalysts. The precursor may then be reacted (e.g., by cleaving the side chain) to form the biorelevant contrast agent. The biorelevant contrast agent may be separated from other reaction products by liquid-liquid extraction or by an additional precipitation step, consistent with the precipitation methods described herein. In some embodiments, precipitation may occur after the generation of the biorelevant contrast agent from the precursor. Such precipitation may be performed according to the methods described herein.

[0132] In some embodiments, the conversion of spin order to polarization occurs before the compound is solidified and precipitated, while in other embodiments, the conversion occurs after redissolution of the crystals with a biologically relevant imaging agent.

[0133] In some embodiments, several steps of precipitation, washing, and redissolution are carried out. This may be advantageous in further purifying the biorelevant contrast agent, increasing the relaxation time of the precipitation, or further separating the polarization and cleavage steps. For example, the first precipitation may be used to wash the catalyst, while the second precipitation is a crystalline form with a longer relaxation time and may be used for transportation. In some embodiments, the first precipitation is of the ester of the compound of formula II after hydrogenation and polarization transfer, and the second precipitation is carried out after cleavage.

[0134] Consistent with disclosed embodiments, the precipitation (and optionally washing) step may separate the hyperpolarized biologically relevant imaging agent from other substances in the original solution (e.g., catalyst, original solvent, reaction products, etc.) For example, a majority of the hydrogenation catalyst present in the original solution may be retained in the original solution after precipitation of the biologically relevant imaging agent. In some embodiments, the precipitate (optionally after washing) contains at most about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less of hydrogenation catalyst, at least about 0.001%, The hydrogenation catalyst may be retained in an amount of 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more, or an amount of hydrogenation catalyst that is within a range defined by any two of the foregoing values.Similarly, the precipitate may contain at most about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less cleavage by-products (e.g., side arms or other residues of cleavage, at least about 0.001% of the cleavage by-products). %, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the cleavage byproducts, or an amount of the cleavage byproducts that is within a range defined by any two of the foregoing values, may be retained.

[0135] In some embodiments, after precipitation and separation from the original solvent (and potential transport of the hyperpolarized precipitate), the precipitate is redissolved in a solvent (e.g., for use as an agent in hyperpolarized NMR or MRI). The solvent may be a biocompatible solvent, such as an aqueous solution. In some embodiments, the redissolution may be performed in up to about 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, or less, at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, or more, or for a period of time that is within a range defined by any two of the foregoing values.

[0136] In some embodiments, after reconstitution, the biorelevant imaging agent may be present at a higher concentration than that used during PHIP polarization. In some embodiments, the concentration of the hyperpolarized biorelevant imaging agent after reconstitution is at least about 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more, up to about 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, or less, or within a range defined by any two of the aforementioned values. Thus, the concentration of the precursor or biorelevant imaging agent during polarization may be independent of the concentration of the molecule in the injection solution. The concentration of the precursor or biorelevant imaging agent during polarization may be selected for efficient polarization transfer. In some embodiments, the polarization concentration is less than the concentration of the hyperpolarized biologically relevant imaging agent after redissolution. For example, the polarization concentration can be at least about 300 mM, 200 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or less, up to about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 300 mM, or more, or within a range defined by any two of the foregoing values.

[0137] In some embodiments, a precipitate sample exhibits a polarization of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or a polarization within a range defined by any two of the foregoing values. For example, in some embodiments, the precipitate sample may be 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50 ... The polarization may be 0%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%.

[0138] After resolubilization of the particles, the concentration of the catalyst, precursor, or cleavage byproduct is at most about 1 μM, 900 nanomolar (nM), 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or less, It may be at least about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, or more, or within a range defined by any two of the foregoing values. In some embodiments, the purity of the hyperpolarized biologically relevant imaging agent after reconstitution is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, up to about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or less, or within a range defined by any two of the preceding values. In some embodiments, at least a fraction of the hyperpolarized compound is separated from cleaved side chains or other reaction by-products, if present.

[0139] transportation Consistent with the disclosed embodiments, the polarization transfer and use of the biorelevant contrast agent can occur at different locations. In some embodiments, the precursor (in the solution used for polarization transfer, or as a precipitate) is transported to another location after precipitation. In some embodiments, the biorelevant contrast agent (in the solution used for polarization transfer, as a precipitate, or redissolved) is transported to another location. The disclosed embodiments are not necessarily limited to any particular transport distance or duration. Instead, the maximum distance or duration can be determined based on the target molecule, the original degree or polarization, the final degree of polarization required, and the transport conditions. In some embodiments, the precipitate is transported at least 1 meter with a suitable transport device.

[0140] Consistent with disclosed embodiments, the transport device may be configured to transport a sample of a precursor or a biologically relevant contrast agent. The transport device may be arranged and configured to transport one or more samples simultaneously. The transport device may include a transport chamber configured to receive one or more samples. The transport device may be configured to maintain the transport chamber within a predetermined temperature range and a predetermined magnetic field strength. The transport device may be configured to maintain one or more samples in a magnetic field of at least about 10G, 20G, 30G, 40G, 50G, 60G, 70G, 80G, 90G, 100G, 200G, 300G, 400G, 500G, 600G, 700G, 800G, 900G, 1,000G, or more, up to about 1,000G, 900G, 800G, 700G, 600G, 500G, 400G, 300G, 200G, 100G, 90G, 80G, 70G, 60G, 50G, 40G, 30G, 20G, 10G, or less, or in a magnetic field within a range defined by any two of the foregoing values.

[0141] A permanent magnet or electromagnet included in the transport device can provide the magnetic field. In some embodiments, the permanent magnet or electromagnet is shielded to reduce the strength of the magnetic field outside the transport device. The transport device can also include a cooling system. The cooling system can be configured to maintain the sample at a predetermined temperature or within a predetermined temperature range during transport. For example, the cooling system can be configured to maintain the sample at a temperature below 270K, below 80K, or below 4K. In some embodiments, the transport device is configured to maintain the sample at approximately liquid nitrogen temperature. The transport device can include thermal insulation between the cooling system and the exterior of the transport device to minimize heat exchange with the external environment. In some embodiments, the cooling system is configured to maintain the temperature of the sample using a cold gas flow. In some embodiments, the cooling system is configured to maintain the temperature of the sample using a cooling liquid. In some embodiments, the transport device includes a dewar that provides cooling for the sample. To distribute the hyperpolarized sample over long distances, the container can be transported by standard transport vehicles such as planes, trains, trucks, cars, and ships.

[0142] In some embodiments, the hyperpolarized precipitate particles are transported in a transport device. In some embodiments, the relaxation time of the hyperpolarized precipitate particles in the transport device is at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or more, up to about 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, or less, or within a range defined by any two of the foregoing values.

[0143] Generation of polarized biorelevant contrast agents 1 illustrates a first exemplary process 100 for producing a polarized biologically relevant imaging agent, according to various embodiments. In the illustrated example, process 100 includes providing a composition comprising a compound of formula I at step 110. In some embodiments, the compound of formula I is provided by: (i) 1 H (proton), 2 or (ii) a carbon-carbon triple bond (-C≡C-), substituted to include H (deuterium), or a combination thereof; wherein R1 group comprises a PHIP transfer moiety as described herein; R2 group comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety, as described herein; and R3 group comprises a biorelevant imaging agent, as described herein.

[0144] In step 120, the double or triple bond in the compound of formula I is hydrogenated with para-hydrogen to form a para-hydrogenated derivative of the compound of formula I, the para-hydrogenated derivative being a compound having the structure of formula II. In some embodiments, the compound of formula II is a compound having the structure of formula II, comprising: (i) 1 H (proton), 2 Para-hydrogenated carbon-carbon single bonds (-CH*-CH*-) substituted to contain H (deuterium), or combinations thereof; or (ii) 1 H (proton), 2 The compound of formula I comprises Z', which is a para-hydrogenated carbon-carbon double bond (-CH*=CH*-), substituted to include H (deuterium), or combinations thereof, where H* is hydrogen with spin order derived from para-hydrogen, the R1 group comprises a PHIP transfer moiety as described herein, the R2 group comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety as described herein, and the R3 group comprises a biorelevant imaging agent as described herein. In some embodiments, the compound of formula I is hydrogenated with para-hydrogen using the hydrogenation processes described herein.

[0145] In step 130, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of formula II to any non-hydrogen nuclear spins in the biologically relevant imaging agent of the compound of formula II, as described herein, thereby forming a derivative of the compound of formula II having a hyperpolarized biologically relevant imaging agent. In some embodiments, the nuclear spin order is transferred using any of the polarization transfer processes described herein.

[0146] 2 illustrates a second exemplary process 200 for producing a polarized biologically relevant imaging agent, according to various embodiments of the present disclosure. In the illustrated example, process 200 includes providing a composition comprising a compound of formula II at step 210. In some embodiments, formula II is represented by the formula: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that is substituted to include H (deuterium), or a combination thereof; or (ii) as described herein, 1 H (proton), 2 The compound includes a Z' group which is a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or combinations thereof, where H* is a hydrogen with spin order derived from para-hydrogen, the R1 group includes a PHIP transfer moiety as described herein, the R2 group includes an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety, as described herein, and the R3 group includes a biorelevant imaging agent, as described herein.

[0147] In step 220, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of Formula II to any non-hydrogen nuclear spins in the biologically relevant imaging agent of the compound of Formula II, as described herein, thereby forming a derivative of the compound of Formula II having a hyperpolarized biologically relevant imaging agent.

[0148] In step 230, the derivative compound of formula II is hydrolyzed to form a composition comprising a hyperpolarized biologically relevant imaging agent and a separate side arm compound of formula III. In some embodiments, the compound of formula III is represented by (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that is substituted to include H (deuterium), or a combination thereof; or (ii) as described herein, 1 H (proton), 2 H, or combinations thereof; the R1' group comprises a parahydrogenated carbon-carbon double bond (-CH*=CH*-), as described herein; and the R2 group comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, as described herein, or a solubilizing moiety.

[0149] In step 240, the hyperpolarized biologically relevant contrast agent is washed one or more times with an organic solvent. In some embodiments, the non-hydrogen nuclear spins in the biologically relevant contrast agent have a non-hydrogen spin polarization after the washing step of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or within a range defined by any two of the foregoing values.

[0150] In some embodiments, process 100 or process 200 includes one or more additional steps or operations. In some embodiments, process 100 or process 200 omits one or more steps or operations. In some embodiments, one or more steps or operations of process 100 are combined with one or more steps or operations of process 200. In some embodiments, all steps or operations of process 100 and process 200 are combined to obtain a complete process for producing a hyperpolarized imaging agent from a precursor having the structure of Formula I.

[0151] Enumerated Embodiments The foregoing non-limiting embodiments disclosed herein include the following:

[0152] Embodiment 1. A composition comprising a compound of formula (I), [ka] In the formula, Z is: 1 H (proton), 2 a carbon-carbon double bond (-C=C-) substituted to include H (deuterium), or a combination thereof; or (ii) a carbon-carbon triple bond (-C≡C-), wherein R1 comprises a parahydrogen induced polarization (PHIP) transfer moiety; R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine; and R3 comprises a biologically relevant imaging agent comprising a non-hydrogen nuclear spin.

[0153] Embodiment 2. A composition comprising a compound of formula (II), [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2A composition comprising a biologically relevant imaging agent comprising a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or combinations thereof, where H* is hydrogen with spin order derived from the para-hydrogen, R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a non-hydrogen nuclear spin.

[0154] Embodiment 3. A composition comprising: (i) a biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (ii) a compound of formula (III): [ka] In the formula, Z″ is: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 A composition comprising a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H, or a combination thereof, R1' comprises a para-hydrogen induced polarization (PHIP) transfer moiety, and R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine.

[0155] Embodiment 4. A composition comprising: (i) a hyperpolarized biologically relevant imaging agent containing non-hydrogen nuclear spins; and (ii) a compound of formula (IV), [ka] In the formula, Z is: 1 H (proton), 2or (ii) a carbon-carbon triple bond (-C≡C-), substituted to include H (deuterium), or a combination thereof, wherein R1' comprises a parahydrogen induced polarization (PHIP) transfer moiety, and R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine.

[0156] Embodiment 5. The composition of any one of embodiments 1-4, wherein the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

[0157] Embodiment 6. The PHIP transfer moiety comprises *CR4R5, *CR4Y, *C=Y, or any deuterated version thereof, where *C is 12 C or 13 C carbon isotope, R and R are each independently 1 H, 2 H, 3 The composition according to any one of the preceding embodiments, wherein Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from H, linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups, and Y is selected from spin 1 / 2 atoms and heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups.

[0158] Embodiment 7. The PHIP transfer moiety comprises *CR6R7-*CR8R9, or any deuterated version thereof, and *C is 12 C or 13 C carbon isotope, and R6, R7, R8, and R9 are each independently 1 H, 2 H, 3The composition of any one of embodiments 1-5, wherein the alkyl group is selected from H, linear, branched, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

[0159] Embodiment 8. The PHIP transfer moiety comprises *CH2, *CH2-*CH2, *CHY, *C=Y, or any deuterated version thereof, wherein *C is 12 C or 13 The composition according to any one of the first to fifth embodiments, wherein Y is a carbon isotope of C and Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen or haloalkyl groups.

[0160] Embodiment 9. A spin 1 / 2 atom is 1 H, 13 C 15 N, 19 F, or 31 The composition of embodiment 6 or 8, wherein the compound is selected from P.

[0161] Embodiment 10. The composition of any one of embodiments 1-9, wherein the PHIP transfer moiety comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz).

[0162] Embodiment 11. A composition described in any one of embodiments 1 to 10, wherein Z comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz).

[0163] Embodiment 12. A composition according to any one of embodiments 1 to 11, wherein R2 comprises a solubilizing moiety.

[0164] Embodiment 13. A composition according to any one of embodiments 1 to 12, wherein R2 comprises a hydrophobic and / or organophilic moiety.

[0165] Embodiment 14. The composition of embodiment 13, wherein R2 comprises an organic solubilizing moiety.

[0166] Embodiment 15. A composition according to any one of embodiments 1 to 12, wherein R2 comprises a hydrophilic and / or organophilic moiety.

[0167] Embodiment 16. The composition of any one of the preceding embodiments, wherein R2 comprises or is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

[0168] Embodiment 17. The biologically relevant imaging agent is represented by the formula R 10 C(═O)X—, where R 10 is selected from linear, branched or cyclic C1-C10 alkyl groups, one or more C atoms are optionally substituted with C=C, CO, COH, CNH2, COOH, CH2COOH, CONH2, OC(=O), and X is NR 11 , S, and O, wherein R 11 but, 1 H, 2 H, 3H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butyl carbonate, and benzyl.

[0169] Embodiment 18. The composition of any one of embodiments 1 to 17, wherein the biologically relevant imaging agent is selected from pyruvic acid, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbic acid, α-ketoglutaric acid, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids.

[0170] Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the composition has a solubility in water of less than 50 millimolar (mM).

[0171] Embodiment 19a. The composition of any one of embodiments 1-18, wherein the composition has a solubility in an organic solvent (e.g., acetone, ethanol, chloroform, toluene) of less than 50 millimolar (mM).

[0172] Embodiment 20. The composition of any one of embodiments 1-19a, wherein reacting the composition with parahydrogen provides a chemical yield of parahydrogenated product of at least 30%.

[0173] Embodiment 21. The composition of any one of embodiments 1 to 20, for use in a parahydrogen induced polarization (PHIP) process.

[0174] Embodiment 22. A method for preparing a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the method comprising: (a) providing a composition comprising a compound of formula (I): [ka] In the formula, Z is: 1 H (proton),2 H (deuterium), or combinations thereof, wherein R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biorelevant imaging agent comprising a non-hydrogen nuclear spin; and (b) hydrogenating the double or triple bond in the compound of formula I with para-hydrogen to form a para-hydrogenated derivative of the compound of formula I, wherein the para-hydrogenated derivative has the structure of formula (II): [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include at least one H, H (deuterium), or combinations thereof, where H* is hydrogen having spin order derived from para-hydrogen, R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (c) applying a polarization transfer waveform to the non-hydrogen nuclear spins to transfer nuclear spin order from at least one H* in the compound of formula II, thereby forming a derivative of formula II having a hyperpolarized biologically relevant imaging agent.

[0175] Embodiment 23. A method for preparing a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the method comprising: (a) providing a composition comprising a compound of formula (II): [ka] In the formula, Z′ is: 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include at least one H, H (deuterium), or combinations thereof, where H* is hydrogen having spin order derived from para-hydrogen, R1 comprises a para-hydrogen induced polarization (PHIP) transfer moiety, R2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, and R3 comprises a biologically relevant imaging agent comprising non-hydrogen nuclear spins; and (b) applying a polarization transfer waveform to the non-hydrogen nuclear spins to transfer nuclear spin order from at least one H* in the compound of formula II, thereby forming a derivative of formula II having a hyperpolarized biologically relevant imaging agent.

[0176] Embodiment 24. Hydrolysis of a derivative of formula II to obtain (i) a hyperpolarized biologically relevant imaging agent containing non-hydrogen nuclear spins, and (ii) a compound of formula (III), [ka] In the formula, Z″ is: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 and a compound in which R is a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H, or a combination thereof, R comprises a para-hydrogen induced polarization (PHIP) transfer moiety, and R comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine.

[0177] Embodiment 25. The method of embodiment 24, further comprising washing the hyperpolarized biologically relevant imaging agent one or more times with an organic solvent.

[0178] Embodiment 26. The method of embodiment 25, wherein the non-hydrogen nuclear spins have a non-hydrogen nuclear spin polarization of greater than 10% after the washing step.

[0179] Embodiment 27 The method of any one of embodiments 22-26, wherein the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

[0180] Embodiment 28. The PHIP transfer moiety comprises *CR4R5, *CR4Y, *C=Y, or any deuterated version thereof, where *C is 12 C or 13 C carbon isotope, R and R are each independently 1 H, 2 H, 3 27. The method of any one of embodiments 22-26, wherein Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from H, linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups, and Y is selected from spin 1 / 2 atoms and heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups.

[0181] Embodiment 29. The PHIP transfer moiety comprises *CR6R7-*CR8R9, or any deuterated version thereof, and *C is 12 C or 13 C carbon isotope, and R6, R7, R8, and R9 are each independently 1 H, 2 H, 3The method of any one of embodiments 22-26, wherein the alkyl group is selected from H, linear, branched, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

[0182] Embodiment 30. The PHIP transfer moiety comprises *CH2, *CH2-*CH2, *CHY, *C=Y, or any deuterated version thereof, wherein *C is 12 C or 13 The method of any one of embodiments 22-26, wherein Y is a carbon isotope of C and Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups.

[0183] Embodiment 31. A spin 1 / 2 atom is 1 H, 13 C. 15 N, 19 F, or 31 The method of embodiment 28 or 30, wherein said at least one of said first and second terminal ends is selected from P.

[0184] Embodiment 32. The method of any one of embodiments 22-31, wherein the PHIP transfer moiety comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz).

[0185] Embodiment 33. The method of any one of embodiments 22-32, wherein Z or Z' comprises at least one atom that is J-coupled with a non-hydrogen nuclear spin of at least 0.1 Hertz (Hz).

[0186] Embodiment 34. The method of any one of embodiments 22 to 33, wherein R2 comprises a solubilizing moiety.

[0187] Embodiment 35. The method of any one of embodiments 22 to 34, wherein R2 comprises a hydrophobic and / or organophilic moiety.

[0188] Embodiment 36. The method of embodiment 35, wherein R2 comprises an organic solubilizing moiety.

[0189] Embodiment 37. The method of any one of embodiments 22 to 33, wherein R2 comprises a hydrophilic and / or organophilic moiety.

[0190] Embodiment 38. The method of any one of embodiments 22 to 37, wherein R2 comprises or is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

[0191] Embodiment 39. The biologically relevant imaging agent has the formula R 10 C(═O)X—, where R 10 is selected from linear, branched or cyclic C1-C10 alkyl groups, one or more C atoms are optionally substituted with C=C, CO, COH, CNH2, COOH, CH2COOH, CONH2, OC(=O), and X is NR 11 , S, and O, wherein R 11 but, 1 H, 2 H, 3H, and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butylcarbonate, and benzyl.

[0192] Embodiment 40. The method of any one of embodiments 22 to 39, wherein the biologically relevant imaging agent is selected from pyruvate, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbic acid, α-ketoglutaric acid, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids.

[0193] Embodiment 41. A hyperpolarized biologically relevant imaging agent, or a pharma- ceutically acceptable salt thereof, produced by the method according to any one of embodiments 22 to 40. EXAMPLES

[0194] Example 1 - NMR Procedure NMR spectrum, 1 H is 400.13MHz, and 13 The spectra were recorded on a Bruker Avance Neo 400 MHz spectrometer operating at 100.61 MHz for C. Dichloromethane (CHCl3) was used. 1 H NMR experiments, while deuterated dichloromethane (CDCl3) was used as the solvent. 13 C was used as the solvent for the 1C NMR experiments. NMR chemical shifts (δ) are reported in ppm. 1 H and 13 For C spectra, the solvent signal was internally calibrated [ 1 H NMR: δ(CHCl3) 7.26; 13 C NMR: δ(CDCl3) 77.16] was used. 13C NMR spectra were recorded in proton decoupling mode. Flash chromatography was performed on a Biotage® Selekt Flash Purification System using a Biotage® Sfar HC Duo column as the solid phase and a mixture of cyclohexane and ethyl acetate as the mobile phase. Column chromatography was performed on silica gel (silica 60, 63-200 μm, Macherey-Nagel) using a mixture of cyclohexane and ethyl acetate as the mobile phase.

[0195] Example 2 - General synthetic procedure Synthesis of esters with a,b-unsaturated side arms [ka] Synthesis of side arms 1a-g Alcohols 1a, 1c, 1d, and 1g were prepared according to published procedures (1a: T. Yoshinao, K. Masanari, T. Shuji, K. Sigeru, Y. Zenichi, Bull. Chem. Soc. Jpn. 1994, 67, 2838-2849; 1c: BRBlank, IPAndrews, O. Kwon, ChemCatChem 2020, 12, 4352-4372; 1d: GCTsui, K. Villeneuve, E. Carlson, W. Tam, Organometallics 2014, 33, 3847-3856; 1g: L. Pauli, R. Tannert, R. Scheil, A. Pfaltz, Chem. Eur. J. 2015, 21, 1482-1487, each of which is incorporated herein by reference in connection with the methods for the synthesis of alcohols 1a, 1c, 1d, and 1g. Alcohol 1b was prepared by starting from tetrahydro-2-(2-propynyloxy)-2H-pyran and isopropyl chloroformate, following the procedure used to synthesize 1a, followed by deprotection of the THP group with pyridinium p-toluenesulfonate. The synthesis of 1c-d9 with selective deuteration of the tert-butyl group was carried out by using di-tert-butyl bicarbonate-d instead of di-tert-butyl bicarbonate.18 The selective monodeuteration of 1c-d1 was carried out according to the following reaction scheme: [ka]

[0196] Alcohol 1e was prepared according to a published one-step synthesis from tert-butyl propiolate and benzaldehyde (A. Kondoh, R. Ozawa, M. Terada, Chem. Lett. 2019, 48, 1164-1167, which is incorporated herein by reference in connection with the method for synthesizing alcohol 1e). Alcohol 1f was prepared by starting from tetrahydro-2-(2-propynyloxy)-2H-pyran via carboxylation using CO2, followed by esterification with benzylol under Mitsunobu conditions according to known procedures in the literature (O. Mitsunobu, Y. Yamada, Bulletin Chem. Soc. Japan 1967, 40(1), 2380-2382, which is incorporated herein by reference in connection with the method for synthesizing alcohol 1f) and THP cleavage with 1c. Alcohol 1g-d5 was prepared according to 1g using benzoyl chloride-d5 instead of benzoyl chloride.

[0197] Synthesis of esters 2-4 with a,b-unsaturated side arms General Procedure A: The target carboxylic acids (1.0-2.0 equiv.) were dissolved in anhydrous tetrahydrofuran (THF, 200-350 mmol), the corresponding alcohols 1a-g were added, and the solution was cooled to 0 °C in an ice / water bath. At this temperature, pyridine (4.0-5.0 equiv.) was added dropwise over 5 min, followed by methanesulfonyl chloride (1.2-2.0 equiv.) (or p-toluenesulfonyl chloride dissolved in THF) added dropwise over 10 min. The mixture was stirred until all of the corresponding alcohols 1a-g were consumed (2-5 h at 0 °C if methanesulfonyl chloride and pyruvic acid are used in high excess, and up to 18 h at ambient temperature if only a slight excess of methanesulfonyl chloride or p-toluenesulfonyl chloride is used). 1 H NMR). During the reaction, a colorless precipitate formed in all cases. After the reaction was complete, the reaction mixture was poured into a stirred mixture of 0.5N aqueous HCl (same volume as the solvent of the reaction) and diethyl ether (same volume as the solvent of the reaction). The phases were separated, the organic phase was collected, and the aqueous phase was washed with diethyl ether (2×200 mL). The combined organic phase was washed with saturated aqueous NaHCO3 (100 mL) followed by brine (100 mL). The organic phase was dried over Na2SO4 and the volatile components were removed under reduced pressure (800→1 mbar, 40° C. water bath). The crude product was purified by flash or column chromatography.

[0198] General Procedure B: The corresponding alcohols 1a-g (1.0 equiv.) were dissolved in anhydrous THF (200 mmol) and cooled to -78 °C in an acetone / dry ice bath. To the clear mixture, anhydrous triethylamine (NEt3, 1.0 equiv.) was added in one portion, followed by the dropwise addition of the target carboxylic acid chloride (1.0 equiv.) over 3 min. After complete addition of the acid chloride, the cooling bath was removed and the mixture was allowed to warm to ambient temperature. The reaction was quenched by pouring the suspension into a stirred mixture of 0.5 N aqueous HCl (same volume as the solvent in the reaction) and diethyl ether (same volume as the solvent in the reaction). The phases were separated, the organic phase was collected, and the aqueous phase was washed with diethyl ether (2 x 200 mL). The combined organic phase was washed with saturated aqueous NaHCO3 (100 mL), followed by brine (100 mL). The organic phase was dried over Na2SO4 and the volatile components were removed under reduced pressure (800 → 1 mbar, 40 °C water bath). The crude product was purified by flash chromatography if necessary.

[0199] Example 3-Methyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (2a) [ka] Structure 2a was prepared from alcohol 1a (7.08 grams (g), 62.1 millimoles (mmol)), pyruvic acid (10.9 g, 124 mmol, 2.0 equivalents (eq.)), pyridine (25 mL, 310 mmol, 5.0 eq.), and methanesulfonyl chloride (9.6 mL, 124 mmol, 2.0 eq.) in THF (300 mL) at 0° C. for 4 hours (h). Purification was performed via column chromatography to yield 2a as a colorless, slightly viscous liquid (7.1 g, 38 mmol, 61% yield).

[0200] FIG. 3A shows an exemplary 1 H NMR (400 MHz, CDCl3) spectrum is shown. The spectrum showed peaks at d = 2.52 (single (s), integrated signal: 3H, corresponding to CH3), 3.80 (s, 3H, OCH3), and 4.96 (s, 2H, OCH2).

[0201] FIG. 3B illustrates an exemplary 13 The C NMR (101 MHz, CDCl3) spectrum shows peaks at d = 26.78 (CH3), 52.84 (OCH3), 53.05 (OCH2), 78.61 (C≡C), 79.52 (C≡C), 153.00 (COOCH3), 159.36 (COO), and 190.23 (C≡O).

[0202] Example 4 - Isopropyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (2b) [ka] Structure 2b was prepared from alcohol 1b (6.36 g, 40.7 mmol), pyruvic acid (7.16 g, 81.4 mmol, 2.0 equiv), pyridine (16.4 mL, 203 mmol, 5.0 equiv), and p-toluenesulfonyl chloride (15.5 g, 81.4 mmol, 2.0 equiv) in THF (200 mL) following general procedure A at 0° C. for 2 h and at ambient temperature for 15 h. Purification via flash chromatography afforded 2b as a yellowish slightly viscous liquid (5.7 g, 25 mmol, 62% yield).

[0203] FIG. 4A shows an exemplary 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 1.28 (doublet (d), 3 J H,H =6.29Hz, 6H, 2×CH3), 2.50(s, 3H, CH3), 4.94(s, 2H, OCH2), and 5.09(hept, 3 J H,H = 6.26 Hz, 1H, OCH).

[0204] FIG. 4B is an exemplary embodiment corresponding to structure 2b. 13The C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 21.61 (2 × CH3), 26.79 (CH3), 52.93 (OCH2), 70.64 (OCH), 78.62 (C≡C), 79.33 (C≡C), 152.15 (COO t It showed peaks at 159.43 (Bu), 159.43 (COO), and 190.25 (C=O).

[0205] Example 5 - Tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (2c) [ka] Structure 2c was prepared from alcohol 1c (30.0 g, 192 mmol), pyruvic acid (33.8 g, 384 mmol, 2.0 equiv), pyridine (77.0 mL, 954 mmol, 5.0 equiv), and methanesulfonyl chloride (30.0 mL, 388 mmol, 2.0 equiv) in THF (500 mL) following general procedure A at 0° C. for 3 h. Purification via flash chromatography afforded 2c as a yellowish slightly viscous liquid (33 g, 146 mmol, 76% yield).

[0206] FIG. 5A shows an exemplary 1 1 H NMR (400 MHz, CDCl3) spectrum. The NMR spectrum shows d=1.49 (s, 9H, t It showed peaks at 2.50 (s, 3H, CH3), 2.50 (s, 3H, CH3), and 4.93 (s, 2H, OCH2).

[0207] FIG. 5B is an exemplary embodiment corresponding to structure 2c. 13 C NMR (101 MHz, CDCl3) spectrum. The NMR spectrum shows d = 26.81 (CH3), 27.97 (3 × CH3), 53.02 (OCH2), 76.78 (C≡C), 80.21 (C≡C), 84.36 (OC- t Bu), 151.59 (COO t It showed peaks at 159.47 (Bu), 159.47 (COO), and 190.32 (C=O).

[0208] Example 6 - Tert-butyl 4-((2-oxopropanoyl-1- 13 C) Oxy)but-2-ynoate (2c- 13 C) [ka] Structure 2c- 13 C( 13 C-labeled 2c) was reacted with alcohol 1c (4.40 g, 28.1 mmol), pyruvate-1- 13 2c- was prepared according to general procedure A from C (5.00 g, 56.1 mmol, 2.0 equiv), pyridine (11.3 mL, 140 mmol, 5.0 equiv), and methanesulfonyl chloride (4.34 mL, 56.1 mmol, 2.0 equiv) in THF (80 mL) at 0° C. for 4 h. Purification was achieved via flash chromatography to give 2c- as a colorless, slightly viscous liquid (4.50 g, 19.8 mmol, 70% yield). 13 This gave rise to C.

[0209] FIG. 6A shows the structure 2c- 13 An example corresponding to C 1 1 H NMR (400 MHz, CDCl3) spectrum. The NMR spectrum shows d=1.49 (s, 9H, t Bu), 2.50(d, 3 J C、H = 1.58Hz, CH3), and 4.93(d, 3 J C、H = 3.54 Hz, 2H, OCH2).

[0210] FIG. 6B shows the structure 2c- 13 An example corresponding to C 13 C NMR (101 MHz, CDCl3) spectrum. The NMR spectrum shows d = 26.91 (d, 2 J C,C =17.08Hz, CH3), 28.07(3×CH3), 53.12(d, 2 J C、C =2.58Hz, OCH2), 76.76(d, 3 J C,C=2.51Hz, C≡C), 80.35(C≡C), 84.49(OC- t Bu), 151.69 (COO t Bu), 159.56 (COO), and 190.39 (d, 1 J C、C = 67.07, C = O).

[0211] Example 7-2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-oxopropanoyl-1- 13 C) Oxy)but-2-ynoate (2c-d9- 13 C) [ka] Structure 2c-d 9- 13 C(9× deuterated and 13 C-labeled structure 2c) was synthesized by the reaction of alcohol 1c-d9 (5.50 g, 33.2 mmol), pyruvate-1- 13 Prepared according to general procedure A from C (5.00 g, 56.1 mmol, 1.7 equiv), pyridine (11.3 mL, 140 mmol, 4.2 equiv), and methanesulfonyl chloride (4.3 mL, 56.1 mmol, 1.7 equiv) in THF (130 mL) at 0 °C for 5 h. Purification was achieved via flash chromatography to give 2c-d as a colorless, slightly viscous liquid (5.08 g, 21.6 mmol, 65% yield). 9- 13 This gave rise to C.

[0212] FIG. 7A shows structures 2c-d 9- 13 An example corresponding to C 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 2.48 (d, 3 J C,H = 1.56Hz, CH3), and 4.91(d, 3 J C,H = 3.55 Hz, 2H, OCH2).

[0213] Figure 7B shows 2c-d9- 13 An example corresponding to C 13 C NMR (101 MHz, CDCl3) spectrum. The NMR spectrum shows d = 26.62 to 27.41 (multiplet (m), 3 × CD3), 26.84 (d, 2 J C,C =17.06Hz, CH3), 53.06(d, 2 J C,C =2.68Hz, OCH2), 76.76(d, 3 J C,C =2.45Hz, C≡C), 80.27(C≡C), 83.99(OC- t Bu), 151.64 (COO t Bu), 159.50 (COO), and 190.35 (d, 1 J C,C = 67.13, C=O).

[0214] Example 8 - Tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate-4-d (2c-d1) [ka] Structure 2c-d1 (1x deuterated structure 2c) was prepared from alcohol 1c-d1 (14.0 g, 89.0 mmol), pyruvic acid (15.8 g, 178 mmol, 2.0 equiv), pyridine (36.0 mL, 445 mmol, 5.0 equiv), and methanesulfonyl chloride (13.8 mL, 178 mmol, 2.0 equiv) in THF (400 mL) following general procedure A at 0°C for 1 h and at ambient temperature for 3 h. Purification via flash chromatography afforded 2c-d1 as a yellowish slightly viscous liquid (13.2 g, 58.1 mmol, 65% yield).

[0215] FIG. 8A shows an exemplary 1 1 H NMR (400 MHz, CDCl3) spectrum. The NMR spectrum shows d=1.49 (s, 9H, t Bu), 2.51 (s, 3H, CH3), and 4.92 (triplet (t), 2 JH,D = 2.34 Hz, 2H, OCH2).

[0216] FIG. 8B shows an exemplary 13 The C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 26.66 (CH3), 27.84 (3 × CH3), 52.66 (t, 1 J C,D =23.56Hz, OCHD), 76.78(C≡C), 80.00(C≡C), 84.19(OC- t Bu), 151.46 (COO t It showed peaks at 159.36 (Bu), 159.36 (COO), and 190.24 (C=O).

[0217] Example 9 - Tert-butyl 4-((2-oxopropanoyl)oxy)pent-2-ynoate (2d) [ka] Structure 2d was prepared from alcohol 1d (4.83 g, 28.4 mmol), pyruvic acid (5.00 g, 56.8 mmol, 2.0 equiv), pyridine (11.5 mL, 142 mmol, 5.0 equiv), and methanesulfonyl chloride (4.40 mL, 56.8 mmol, 2.0 equiv) in THF (80 mL) following general procedure A at 0° C. for 3 h and ambient temperature for 1 h. Purification via flash chromatography afforded 2d as a yellowish slightly viscous liquid (4.85 g, 20.2 mmol, 71% yield).

[0218] FIG. 9A shows an exemplary 1 H NMR (400 MHz, CDCl3) spectrum. The NMR spectrum shows d=1.48 (s, 9H, t Bu), 1.64(d, 3 J H,H = 6.80 Hz, OCHCH3), 2.49 (s, 3H, CH3), and 5.58 (quartet (q), 3 J H、H = 6.79 Hz, 1H, OCHCH3).

[0219] FIG. 9B is an exemplary embodiment corresponding to structure 2d. 13 The C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 20.35 (OCHCH3), 26.77 (CH3), 27.97 (3 × CH3), 61.87 (OCHCH3), 78.58 (C≡C), 80.62 (C≡C), 84.21 (OC- t Bu), 151.82 (COO t It showed peaks at 159.26 (Bu), 159.26 (COO), and 190.75 (C=O).

[0220] Example 9 - Tert-butyl 4-((2-oxopropanoyl)oxy)-4-phenylbut-2-ynoate (2e) [ka] Structure 2e was prepared from alcohol 1e (5.81 g, 25.0 mmol), pyruvic acid (4.40 g, 50.0 mmol, 2.0 equiv), pyridine (10.7 mL, 125 mmol, 5.0 equiv), and methanesulfonyl chloride (3.87 mL, 50.0 mmol, 2.0 equiv) in THF (100 mL) following general procedure A at 0° C. for 1.5 h and at ambient temperature for 2.5 h. Purification via flash chromatography afforded 2e as a yellowish viscous liquid (5.00 g, 16.5 mmol, 66% yield).

[0221] FIG. 10A shows an exemplary 1 H NMR (400 MHz, CDCl3) spectrum. The NMR spectrum is d=1.50 (s, 9H, t Bu), 2.48(s, 3H, CH3), 6.57(s, 1H, OCHPh), 7.40-7.43(m, 3H, H Ph ), and 7.53-7.56 (m, 2H, H Ph ) showed a peak.

[0222] FIG. 10B illustrates an exemplary 13C NMR (101 MHz, CDCl3) spectrum. The NMR spectrum shows d = 26.98 (CH3), 28.09 (3 × CH3), 67.23 (OCHPh), 78.88 (C≡C), 80.69 (C≡C), 84.51 (OC- t Bu), 128.26(C Ph ), 129.18(C Ph ), 130.09(C Ph ), 134.23(C Ph ), 151.86(COO t It showed peaks at 159.16 (Bu), 159.16 (COO), and 190.56 (C=O). Example 10-Benzylhydryl 4-((2-oxopropanoyl)oxy)but-2-ynoate (2f) [ka]

[0223] Structure 2f was prepared from 1f (1.60 g, 6.00 mmol) following general procedure B to give 2f as a colorless viscous oil (1.92 g, 5.70 mmol, 95% yield).

[0224] FIG. 11 shows an exemplary 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 2.51 (s, 3H, CH3), 4.97 (s, 2H, OCH2), 6.94 (s, 1H, OCH), and 7.28-7.38 (m, 10H, H Ph ) showed a peak.

[0225] Example 11-4-Oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate (2g) [ka] Structure 2g was prepared from 1g (2.49 g, 15.5 mmol) following general procedure B purified by flash chromatography to give 2g-d5 as a slightly orange solid (1.86 g, 8.06 mmol, 52% yield).

[0226] FIG. 12A shows an exemplary 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 2.54 (s, 3H, CH3), 5.11 (s, 2H, OCH2), 7.48-7.52 (m, 2H, H Ph ), 7.61-7.66(m, 1H, H Ph ), and 8.09-8.11 (m, 2H, H Ph ) showed a peak.

[0227] FIG. 12B illustrates an exemplary 13 The C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 26.81 (CH3), 53.24 (OCH2), 84.65 (C≡C), 85.56 (C≡C), 128.78 (C Ph ), 129.65(C Ph ), 134.65(C Ph ), 136.03(C Ph ), 159.59 (COO), 176.91 (C(O)Ph), and 190.34 (C=O).

[0228] Example 12 - 4-Oxo-4-(phenyl-d5)but-2-yn-1-yl 2-oxopropanoate (2g-d5) [ka] Structure 2g-d5 was prepared from 1g-d5 (4.66 g, 28.2 mmol) according to general procedure B to give 2g-d5 as a brownish solid (6.1 g, 25.9 mmol, 92%).

[0229] FIG. 13A shows an exemplary 1 H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum showed peaks at d = 2.49 (s, 3H, CH3) and 5.09 (s, 2H, OCH2).

[0230] FIG. 13B shows an exemplary 13C NMR (101 MHz, CDCl3) spectrum. The NMR spectrum shows d = 26.79 (CH3), 53.23 (OCH2), 84.62 (C≡C), 85.55 (C≡C), 128.24 (t, 1 J C,D =24.58Hz, C Ph ), 129.23(t, 1 J C,D =24.58Hz, C Ph ), 134.14(t, 1 J C,D =22.45Hz, C Ph ), 135.83(C Ph ), 159.54 (COO), 176.91 (C(O)Ph), and 190.35 (C=O).

[0231] Example 13-1-(4-(Tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate (3c) [ka] Structure 3c was purified from alcohol 1c (0.92 g, 5.9 mmol), mono-ethyl a-ketoglutarate (1.02 g, 5.85 mmol, 1.0 equiv), pyridine (1.88 mL, 23.3 mmol, 4.0 equiv) and methanesulfonyl chloride (543 μL, 7.02 mmol, 1.2 equiv) in THF (40 mL) following general procedure A at 0° C. for 5 h and 7° C. for 40 h. Purification afforded 3c as a colorless viscous liquid (1.40 g, 4.48 mmol, 77% yield) containing 20% ​​of 1c, as esterification was not quantitative and 3c and 1c have similar retention times via flash chromatography.

[0232] FIG. 14A shows an exemplary 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d=1.25 (t, 3 J H,H =7.14Hz,3H, OCH2CH3), 1.49(s, 9H, t Bu), 2.68(t,3 J H,H =6.49Hz, 2H, CH2CH2), 3.17(t, 3 J H,H =6.36Hz, 2H, CH2CH2), 4.14(q, 3 J H,H = 7.14 Hz, 2H, OCH2CH3), and 4.94 (s, 2H, OCH2).

[0233] FIG. 14B shows an exemplary 13 The C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum shows d = 14.26 (OCH2CH3), 27.87 (CH2CH2), 28.06 (3×CH3), 28.10 (CH2CH2), 53.16 (OCH2), 61.15 (OCH2CH3), 76.74 (C≡C), 80.37 (C≡C), 84.46 (OC- t Bu), 151.69 (COO t It showed peaks at 159.33 (Bu), 159.33 (COO), 171.95 (COOEt), and 191.27 (C=O).

[0234] Example 14-Methyl 4-(2,2-dichloroacetoxy)but-2-ynoate (4a) [ka] Structure 4a was prepared from 1a (4.00 g, 35.0 mmol) according to general procedure B to give 4a as a colorless liquid (7.72 g, 34.3 mmol, 98% yield).

[0235] FIG. 15A shows an exemplary 1 H NMR (400 MHz, CDCl3) spectrum is shown. The NMR spectrum showed peaks at d = 3.80 (s, 3H, OCH3), 4.97 (s, 2H, OCH2), and 5.99 (s, 1H, CHCl2).

[0236] FIG. 15B illustrates an exemplary 13C NMR (101 MHz, CDCl3) spectrum is shown. The NMR spectrum showed peaks at d = 53.19 (OCH2), 54.07 (OCH3), 63.60 (CHCl2), 79.05 (C≡C), 79.11 (C≡C), 153.08 (COOCH3), and 163.75 (COO).

[0237] Example 15-Tert-butyl 4-((acetoxy)but-2-ynoate (5c) [ka] Structure 5c was prepared from 1c (5.50 g, 35.2 mmol) according to general procedure B and purified via flash chromatography to give 5c as a colorless liquid (5.72 g, 28.8 mmol, 82% yield).

[0238] Figure 16A shows an exemplary 1H NMR (400 MHz, CDCl3) spectrum corresponding to structure 5c. The NMR spectrum shows δ = 1.49 (s, 9H, t It showed peaks at 2.10 (s, 3H, CH3) and 4.76 (s, 2H, OCH2).

[0239] Figure 16B shows an exemplary C NMR (101 MHz, CDCl) spectrum corresponding to structure 5c. The NMR spectrum shows δ = 20.68 (CH), 28.08 (3xCH), 51.50 (OCH), 78.59 (C≡C), 79.32 (C≡C), 84.16 (OC- t Bu), 151.96 (COO t It showed peaks at 170.03 (Bu) and 170.03 (COO).

[0240] Example 16 - Improved hydrogenation efficiency FIG. 17A shows an exemplary 1H NMR (400 MHz, CDCl3) spectrum corresponding to para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate with two H*). The para-hydrogenation reaction between 3-phenylprop-2-yn-1-yl 2-oxopropanoate and para-hydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar para-hydrogen pressure for 10 seconds at 60°C. The reaction forms the previously known 3-phenylallyl 2-oxopropanoate with two H*, where the * symbol indicates a molecule containing a proton originating from para-hydrogen. In the example shown, the NMR signal marked with the * symbol corresponds to a para-hydrogen molecule added across the carbon-carbon triple bond of 3-phenylprop-2-yn-1-yl 2-oxopropanoate. 1 Based on the 1 H NMR spectrum, the yield of the para-hydrogenation reaction was determined to be about 29%.

[0241] FIG. 17B illustrates an exemplary tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate corresponding to para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate (i.e., tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate having two H*). 1 The H NMR (400 MHz, CDCl3) spectrum is shown. The parahydrogenation reaction between this new molecule and parahydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar parahydrogen pressure for 10 seconds at 60°C. The reaction forms tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate with two H*, where the * symbol indicates a molecule containing a proton originating from parahydrogen. In the example shown, the NMR signal marked with the * symbol corresponds to a parahydrogen molecule added across the carbon-carbon triple bond of tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. 1 Based on the 1 H NMR spectrum, the yield of the para-hydrogenation reaction was determined to be about 85%.

[0242] As shown in Figures 17A and 17B, the para-hydrogenation reaction between tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate and para-hydrogen resulted in a significantly higher yield than the para-hydrogenation reaction between 3-phenylprop-2-yn-1-yl 2-oxopropanoate and para-hydrogen.

[0243] FIG. 18A shows an exemplary 1H NMR (400 MHz, CDCl3) spectrum corresponding to para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate with two H*). The para-hydrogenation reaction between 3-phenylprop-2-yn-1-yl 2-oxopropanoate and para-hydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar para-hydrogen pressure for 5 seconds at 45° C. The reaction forms the previously known 3-phenylallyl 2-oxopropanoate with two H*, where the * symbol indicates a molecule containing a proton originating from para-hydrogen. In the example shown, the NMR signal marked with the * symbol corresponds to a para-hydrogen molecule added across the carbon-carbon triple bond of 3-phenylpropargylpyruvic acid. 1 Based on the 1 H NMR spectrum, the yield of the para-hydrogenation reaction was determined to be about 21%.

[0244] FIG. 18B illustrates an exemplary compound corresponding to para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate (i.e., 4-oxo-4-phenylbut-2-en-1-yl 2-oxopropanoate having two H*). 11 H NMR (400 MHz, CDCl3) spectrum is shown. The parahydrogenation reaction between 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate and parahydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar parahydrogen pressure for 5 seconds at 45°C. The reaction forms 4-oxo-4-phenylbut-2-en-1-yl 2-oxopropanoate with two H*, where the * symbol indicates a molecule containing a proton originating from parahydrogen. In the example shown, the NMR signal marked with the * symbol corresponds to a parahydrogen molecule added across the carbon-carbon triple bond of 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate. 1 Based on the 1 H NMR spectrum, the yield of the para-hydrogenation reaction was determined to be about 44%.

[0245] As shown in Figures 18A and 18B, the para-hydrogenation reaction between 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate and para-hydrogen gave a significantly higher yield than the para-hydrogenation reaction between the known pyruvic acid derivative, -phenylallyl 2-oxopropanoate, and para-hydrogen.

[0246] Example 17 - Improved 13 C polarization FIG. 19A shows an exemplary 3-phenylallyl 2-oxopropanoate salt corresponding to 200 mM para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate salt with 2 H*) in acetone-d6. 13 17A. Para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate was formed using the procedure described herein with respect to FIG. 17A. In the example shown, para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate was about 9.8% 13 It was determined that the ion beam had a polarization of 1.0.

[0247] FIG. 19B is an exemplary reaction mixture corresponding to 200 mM para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (i.e., tert-butyl 4-((2-oxopropanoyl)oxy)but-2-enoate with two H*) in acetone-d6. 13 17C NMR (101 MHz, CDCl3) spectrum is shown. Para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate was formed using the procedure described herein with respect to FIG. 17B. In the example shown, para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate was about 17.5% 13 It was determined that the ion beam had a polarization of 1.0.

[0248] As shown in Figures 19A and 19B, para-hydrogenated tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate resulted in significantly higher polarization than para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate. It should be noted that para-hydrogenated 3-phenylpropargylpyruvate represents one of the current gold standard precursors for generating hyperpolarized pyruvic acid via PHIP-SAH. Thus, the compositions described herein may enable the production of pyruvic acid and other biorelevant imaging agents with significantly enhanced polarization compared to known precursors.

[0249] FIG. 20A shows an exemplary 200 mM para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate (i.e., 3-phenylallyl 2-oxopropanoate having two H*) in acetone-d6. 13 18A. Para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate was formed using the procedure described herein with respect to FIG. 18A. In the example shown, para-hydrogenated 3-phenylprop-2-yn-1-yl 2-oxopropanoate was about 13.9% 13 It was determined that the ion beam had a polarization of 1.0.

[0250] FIG. 20B shows an exemplary 200 mM para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate salt (i.e., 4-oxo-4-phenylbut-2-en-1-yl 2-oxopropanoate salt having two H*). 13 18C NMR (101 MHz, CDCl3) spectrum is shown. Para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate was formed using the procedure described herein with respect to FIG. 18B. In the example shown, para-hydrogenated 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate was about 10.3% 13 It was determined that the ion beam had a polarization of 1.0.

[0251] FIG. 21 shows an exemplary 133 mM para-hydrogenated methyl 4-((2-oxopropanoyl)oxy)but-2-ynoate (i.e., methyl 4-((2-oxopropanoyl)oxy)but-2-enoate with two H*). 13 C NMR (101 MHz, CDCl3) spectrum is shown. The parahydrogenation reaction between this new molecule and parahydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar parahydrogen pressure for 10 seconds at 60°C. The reaction forms methyl 4-((2-oxopropanoyl)oxy)but-2-enoate with two H*, where the * symbol indicates a molecule containing a proton originating from parahydrogen. In the example shown, the parahydrogenated methyl 4-((2-oxopropanoyl)oxy)but-2-ynoate has a 17.2% 13 It was determined that the ion beam had a polarization of 1.0.

[0252] Example 18 - Hyperpolarized α-Ketoglutarate FIG. 22A illustrates an exemplary compound corresponding to para-hydrogenated 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate (i.e., 1-(4-(tert-butoxy)-4-oxobut-2-en-1-yl) 5-ethyl 2-oxopentanedioate having two H*). 1 1 H NMR (400 MHz, CDCl3) spectrum is shown. The parahydrogenation reaction between 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate and parahydrogen was carried out in the presence of 1 mol% [Rh(dppb)(COD)]BF4 at 10 bar parahydrogen pressure for 10 seconds at 60°C. The reaction forms 1-(4-(tert-butoxy)-4-oxobut-2-en-1-yl) 5-ethyl 2-oxopentanedioate with two H*, where the * symbol indicates a molecule containing a proton originating from parahydrogen. In the example shown, the NMR signal marked with the * symbol corresponds to a parahydrogen molecule added across the carbon-carbon triple bond of 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate.

[0253] FIG. 22B illustrates an exemplary 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate salt (i.e., 1-(4-(tert-butoxy)-4-oxobut-2-en-1-yl) 5-ethyl 2-oxopentanedioate salt having two H*) at 70 mM in acetone-d6. 13 22A. Para-hydrogenated 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate was formed using the procedure described herein with respect to FIG. 22A. In the example shown, para-hydrogenated 1-(4-(tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate was found to be approximately 12.8% 13 It was determined that the ion beam had a polarization of 1.0.

[0254] The foregoing description is presented for purposes of illustration, and is not exhaustive or limited to the precise forms or embodiments disclosed. Modifications and adaptations of the embodiments will be apparent from consideration of the specification and practice of the disclosed embodiments. For example, while the described implementations include hardware, systems and methods consistent with the present disclosure can be implemented using both hardware and software. Furthermore, while certain components are described as being coupled together, such components may be integrated with each other or distributed in any suitable manner.

[0255] Furthermore, while exemplary embodiments are described herein, the scope includes any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., aspects across various embodiments), adaptations, or alterations based on this disclosure. The elements of the claims should be interpreted broadly based on the language used in the claims, and not limited to the examples described herein or in the filing of the application, which examples should be interpreted as non-exclusive. Furthermore, the steps of the methods of the present disclosure can be modified in any manner, including reordering steps, or inserting or deleting steps.

[0256] The features and advantages of the present disclosure are apparent from the detailed specification, and therefore, the appended claims are intended to cover all systems and methods that fall within the true spirit and scope of the present disclosure. As used herein, the indefinite articles "a" and "an" mean "one or more." Similarly, the use of a plural word does not necessarily imply a plurality, unless ambiguous in a given context. Furthermore, since numerous modifications and variations will readily result from the study of this disclosure, it is not desired to limit the exact structure and operation of the present disclosure to that shown and described. Thus, all suitable modifications and equivalents may be utilized to fall within the scope of the present disclosure.

[0257] As used herein, unless specifically stated otherwise, the term "or" includes all possible combinations unless impracticable. For example, if it is stated that a component may include A or B, the component may include A, or B, or A and B, unless specifically stated otherwise or impracticable. As a second example, if it is stated that a component may include A, B, or C, the component may include A, or B, or C, or A and B, or A and C, or B and C, or A and B and C, unless specifically stated otherwise.

[0258] The embodiments may be further described using the following clauses.

[0259] Other embodiments will be apparent from consideration of the specification and practice of the embodiments disclosed herein. It is intended that the specification and examples be considered as examples only, with a true scope and spirit of the disclosed embodiments being indicated by the following claims.

Claims

1. A composition comprising a compound of formula (I), 【Chemistry 1】 wherein Z contains (i) a carbon-carbon double bond (-C=C-) that is substituted to contain 1H (proton), 2H (deuterium), or a combination thereof, or (ii) a carbon-carbon triple bond (-C≡C-); R 1 comprises a parahydrogen induced polarization (PHIP) transfer moiety; R 2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine; R 3 comprises a biologically relevant imaging agent that is at least partially isotopically labeled with said non-hydrogen nuclear spins; The PHIP moving part is *CH 2 , *CH 2 -*CH 2 , *CHY, *C=Y, *CR ４ R ５ , *CR ４ Y, *C=Y, *CR 6 R 7 -*CR 8 R 9 or any deuterated version thereof; *C is １２ C or １３ C is a carbon isotope, R ４ and R ５ However, each independently, １ H. ２ H. ３ H, linear, branched, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups; Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups; A composition wherein R 6 , R 7 , R 8 , and R 9 are each independently selected from 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

2. A composition comprising a compound of formula (I), 【Chemistry 2】 wherein Z contains (i) a carbon-carbon double bond (-C=C-) that is substituted to contain 1H (proton), 2H (deuterium), or a combination thereof, or (ii) a carbon-carbon triple bond (-C≡C-); R 1 comprises a parahydrogen induced polarization (PHIP) transfer moiety; R 2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine; R 3 comprises a biologically relevant imaging agent that is at least partially isotopically labeled with said non-hydrogen nuclear spins; A composition wherein the PHIP transfer moiety comprises at least one atom that is J-coupled to said non-hydrogen nuclear spins of at least 0.1 Hertz (Hz), or Z comprises at least one atom that is J-coupled to said non-hydrogen nuclear spins of at least 0.1 Hertz (Hz).

3. A composition comprising a compound of formula (I), 【Chemistry 3】 wherein Z contains (i) a carbon-carbon double bond (-C=C-) that is substituted to contain 1H (proton), 2H (deuterium), or a combination thereof, or (ii) a carbon-carbon triple bond (-C≡C-); R 1 comprises a parahydrogen induced polarization (PHIP) transfer moiety; R 2 comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine; R 3 comprises a biologically relevant imaging agent that is at least partially isotopically labeled with said non-hydrogen nuclear spins; The biorelevant imaging agent comprises a compound of formula RC(=O)X-, where R4 is selected from a linear, branched or cyclic C1-C10 alkyl group and one or more C atoms are selected from C=C, CO, COH, CNH ２ , COOH, CH ２ COOH, CONH ２ , optionally substituted with OC(=O), and X is NR １１ , S, and O, wherein R5 is １ H. ２ H. ３ H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butylcarbonate and benzyl.

4. The composition of claim 1, wherein the PHIP transfer moiety comprises at least one atom that is J-coupled to the non-hydrogen nuclear spins of at least 0.1 Hertz (Hz), or Z comprises at least one atom that is J-coupled to the non-hydrogen nuclear spins of at least 0.1 Hertz (Hz).

5. The composition of claim 1, wherein R 2 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

6. The composition of claim 1, wherein the biologically relevant imaging agent comprises a compound of formula R 10 C(═O)X—, where R 10 is selected from a linear, branched, or cyclic C1-C10 alkyl group, one or more C atoms are optionally substituted with C═C, CO, COH, CNH 2 , COOH, CH 2 COOH, CONH 2 , OC(═O), and X is selected from NR 11 , S, and O, where R 11 is selected from hydrogen and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butylcarbonate, and benzyl.

7. The composition of claim 1, wherein the biorelevant contrast agent is selected from pyruvic acid, glutamate, glutamine, lactic acid, acetic acid, acetoacetic acid, zymonate, alanine, fructose, fumaric acid, bicarbonate, urea, dehydroascorbic acid, α-ketoglutaric acid, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids.

8. The composition of claim 1, wherein the composition has a solubility in water of less than 50 millimolar (mM).

9. The composition of claim 1, wherein the composition has a solubility in an organic solvent of less than 50 millimolar (mM), and the organic solvent is acetone, ethanol, chloroform, or toluene.

10. The composition of claim 1, wherein reacting the composition with parahydrogen results in a chemical yield of parahydrogenated product of at least 30%.

11. The composition of claim 2, wherein the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

12. The method of claim 1, wherein the PHIP transfer moiety comprises *CH2, *CH2-*CH2, *CHY, *C=Y, *CR4R5, *CR4Y, *C=Y, *CR6R7-*CR8R9, or any deuterated version thereof; *C is a carbon isotope of 12C or 13C; R 4 and R 5 are each independently selected from 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups; Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups; 3. The composition of claim 2, wherein R 6 , R 7 , R 8 , and R 9 are each independently selected from 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

13. The composition of claim 2, wherein R 2 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

14. The composition of claim 2, wherein the biologically relevant imaging agent comprises a compound of formula R 10 C(═O)X—, where R 10 is selected from a linear, branched, or cyclic C1-C10 alkyl group, one or more C atoms are optionally substituted with C═C, CO, COH, CNH 2 , COOH, CH 2 COOH, CONH 2 , OC(═O), and X is selected from NR 11 , S, and O, where R 11 is selected from hydrogen and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzoxy, tert-butylcarbonate, and benzyl.

15. The composition of claim 2, wherein the biorelevant contrast agent is selected from pyruvic acid, glutamate, glutamine, lactic acid, acetic acid, acetoacetic acid, zymonate, alanine, fructose, fumaric acid, bicarbonate, urea, dehydroascorbic acid, α-ketoglutaric acid, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids.

16. The composition of claim 2, wherein the composition has a solubility in water of less than 50 millimolar (mM).

17. The composition of claim 2, wherein the composition has a solubility in an organic solvent of less than 50 millimolar (mM), and the organic solvent is acetone, ethanol, chloroform, or toluene.

18. The composition of claim 2, wherein reacting the composition with parahydrogen results in a chemical yield of parahydrogenated product of at least 30%.

19. The composition of claim 3, wherein the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

20. The method of claim 19, wherein the PHIP transfer moiety comprises *CH2, *CH2-*CH2, *CHY, *C=Y, *CR4R5, *CR4Y, *C=Y, *CR6R7-*CR8R9, *CR10R11, or any deuterated version thereof; *C is a carbon isotope of 12C or 13C; R 6 and R 7 are each independently selected from 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups; Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups; 4. The composition of claim 3, wherein R 8 , R 9 , R 10 , and R 11 are each independently selected from 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

21. The composition of claim 3, wherein the PHIP transfer moiety comprises at least one atom that is J-coupled to the non-hydrogen nuclear spins of at least 0.1 Hertz (Hz), or Z comprises at least one atom that is J-coupled to the non-hydrogen nuclear spins of at least 0.1 Hertz (Hz).

22. The composition of claim 3, wherein R 2 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide.

23. The composition of claim 3, wherein the biorelevant contrast agent is selected from pyruvic acid, glutamate, glutamine, lactic acid, acetic acid, acetoacetic acid, zymonate, alanine, fructose, fumaric acid, bicarbonate, urea, dehydroascorbic acid, α-ketoglutaric acid, dihydroxyacetone, glucose, ascorbic acid, and their conjugate acids.

24. The composition of claim 3, wherein the composition has a solubility in water of less than 50 millimolar (mM).

25. The composition of claim 3, wherein the composition has a solubility in an organic solvent of less than 50 millimolar (mM), and the organic solvent is acetone, ethanol, chloroform, or toluene.

26. The composition of claim 3, wherein reacting the composition with parahydrogen results in a chemical yield of parahydrogenated product of at least 30%.

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