Radical compounds and methods of using thereof
Disclosed are methods for performing dynamic nuclear polarization using the polarizing agents described herein. In general, the methods involve (a) providing a frozen sample in a magnetic field, wherein the frozen sample includes a polarizing agent described herein and an analyte with at least one spin half nucleus; (b) polarizing the at least one spin half nucleus of the analyte by irradiating the frozen sample with radiation having a frequency that excites electron spin transitions in the polarizing agent; (c) optionally melting the sample to produce a molten sample; and (d) detecting nuclear spin transitions in the at least one spin half nucleus of the analyte in the frozen or molten sample. In certain embodiments, the polarizing agents can be peptide-based. In these embodiments, the polarizing agents can be readily prepared by solid-phase peptide synthesis.
1. A method comprising steps of:
providing a frozen sample in a magnetic field, wherein the sample includes a polarizing agent and an analyte with at least one spin half nucleus;
polarizing the at least one spin half nucleus of the analyte by irradiating the frozen sample with radiation having a frequency that excites electron spin transitions in the polarizing agent;
optionally melting the frozen sample to produce a molten sample; and
detecting nuclear spin transitions in the at least one spin half nucleus of the analyte in the frozen or molten sample,
wherein the polarizing agent comprises a compound defined by Formula I
wherein
L represents a direct bond or a linking group;
R 1 selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, C 1-12 alkylcarbonyl, C 1-12 alkoxycarbonyl, C 1-12 alkylcarbamyl, di(C 1-12 -alkyl)carbamyl, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 2 is selected from the group consisting of hydrogen, hydroxy, —OR 11 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, amino, C 1-12 alkylamino; di(C 1-12 -alkyl)amino, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 3 and R 4 are independently selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; or R 3 and R 4 , together with the carbon atom to which they are attached, form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ring each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 5 and R 6 are independently selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; or R 5 and R 6 , together with the carbon atom to which they are attached, form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ring each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 7 and R 8 are independently selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; or R 7 and R 8 , together with the carbon atom to which they are attached, form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ring each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 9 and R 10 independently selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; or R 9 and R 10 , together with the carbon atom to which they are attached, form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ring each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 11 is selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; and each R X , when present, are each independently selected from OH, NO 2 , CN, halo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, cyano-C 1-3 alkyl, HO-C 1-3 alkyl, amino C 1-6 alkylamino, di(C 1-6 alkyl)amino, thio, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, carboxy, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, aminosulfonyl, C 1-6 alkylaminosulfonyl, di(C 1-6 alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 alkylaminosulfonylamino, di(C 1-6 alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino.
2. The method of claim 1 , further comprising a step of freezing a sample within a magnetic field to provide the frozen sample in a magnetic field.
3. The method of claim 1 , wherein the method does not include a step of melting the sample to produce a molten sample and, in the step of detecting, the nuclear spin transitions in the at least one spin half nucleus of the analyte in the frozen sample are detected by solid-state NMR.
4. The method of claim 1 , wherein the method does include a step of melting the sample to produce a molten sample and, in the step of detecting, the nuclear spin transitions in the at least one spin half nucleus of the analyte in the molten sample are detected by liquid-state NMR.
5. The method of claim 1 , wherein the analyte is a protein or nucleic acid.
6. The method of claim 1 , further comprising repeating the freezing, polarizing, melting and detecting steps at least once.
7. The method of claim 1 , wherein the method does include a step of melting the sample to produce a molten sample and, in the step of detecting, the nuclear spin transitions in the at least one spin half nucleus of the analyte in the molten sample are detected by MRI.
8. The method of claim 7 , wherein the spin half nucleus of the analyte has a T 1 relaxation time of at least 6 seconds at 310 K in D 2 O in a magnetic field of 7 T.
9. The method of claim 8 further comprising a step of administering at least a portion of the molten sample that includes the analyte to a subject before the step of detecting.
10. The method of claim 1 , wherein the at least one spin half nucleus has a γ-value smaller than that of 1 H and the step of polarizing further comprises irradiating the frozen sample with radiation having a frequency that causes cross -polarization between a 1 H nucleus present in the sample and the at least one spin half nucleus of the analyte.
11. The method of claim 10 , wherein the at least one spin half nucleus is a 13 C nucleus, a 15 N nucleus, a 19 F nucleus, or a 1 H nucleus.
12. The method of claim 1 , wherein in the optional step of melting, the frozen sample is exposed to radiation having a wavelength of less than about 100 μm.
13. The method of claim 1 , wherein the magnetic field has a strength in the range of about 0.1 T to about 30 T.
14. The method of claim 1 , wherein the radiation has a frequency in the range of about 2.8 GHz to about 840 GHz.
15. The method of claim 1 , wherein the compound comprises three or more radicals.
16. The method of claim 1 , wherein the compound is defined by Formula IA
wherein
L represents a direct bond or a linking group;
R 1 selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, C 1-12 alkylcarbonyl, C 1-12 alkoxycarbonyl, C 1-12 alkylcarbamyl, di(C 1-12 -alkyl)carbamyl, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 2 is selected from the group consisting of hydrogen, hydroxy, —OR 11 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, amino, C 1-12 alkylamino; di(C 1-12 -alkyl)amino, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 11 is selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; and
each R X , when present, are each independently selected from OH, NO 2 , CN, halo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, cyano-C 1-3 alkyl, HO-C 1-3 alkyl, amino C 1-6 alkylamino, di(C 1-6 alkyl)amino, thio, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, carboxy, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, aminosulfonyl, C 1-6 alkylaminosulfonyl, di(C 1-6 alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 alkylaminosulfonylamino, di(C 1-6 alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino.
17. The method of claim 1 , wherein the compound is defined by Formula IB
wherein
L represents a direct bond or a linking group;
R 1 selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, C 1-12 alkylcarbonyl, C 1-12 alkoxycarbonyl, C 1-12 alkylcarbamyl, di(C 1-12 -alkyl)carbamyl, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 2 is selected from the group consisting of hydrogen, hydroxy, —OR 11 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, amino, C 1-12 alkylamino; di(C 1-12 -alkyl)amino, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; R 11 is selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups; and
each R X , when present, are each independently selected from OH, NO 2 , CN, halo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, cyano-C 1-3 alkyl, HO-C 1-3 alkyl, amino C 1-6 alkylamino, di(C 1-6 alkyl)amino, thio, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, carboxy, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, aminosulfonyl, C 1-6 alkylaminosulfonyl, di(C 1-6 alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 alkylaminosulfonylamino, di(C 1-6 alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino.
18. The method of claim 1 , wherein the compound is defined by Formula IC
wherein
L represents a direct bond or a linking group;
R 1 selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, C 1-12 alkylcarbonyl, C 1-12 alkoxycarbonyl, C 1-12 alkylcarbamyl, di(C 1-12 -alkyl)carbamyl, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 2 is selected from the group consisting of hydrogen, hydroxy, —OR 11 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, amino, C 1-12 alkylamino; di(C 1-12 -alkyl)amino, amino acid, poly(amino acid), and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 11 is selected from group consisting of C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, and poly(alkylene oxide), each optionally substituted with 1, 2, 3, or 4 independently selected R X groups;
R 12 is selected from the group consisting of OH, NO 2 , CN, halo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, cyano-C 1-3 alkyl, HO-C 1-3 alkyl, amino C 1-6 alkylamino, di(C 1-6 alkyl)amino, thio, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, carboxy, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, aminosulfonyl, C 1-6 alkylaminosulfonyl, di(C 1-6 alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 alkylaminosulfonylamino, di(C 1-6 alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino, C 3-10 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C 3-10 cycloalkyl-C 1-4 alkylene, 4-10 membered heterocycloalkyl-C 1-4 alkylene, 6-10 membered aryl-C 1-4 alkylene, 5-10 membered heteroaryl-C 1-4 alkylene, and poly(alkylene oxide); and
each R X , when present, are each independently selected from OH, NO 2 , CN, halo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, cyano-C 1-3 alkyl, HO-C 1-3 alkyl, amino C 1-6 alkylamino, di(C 1-6 alkyl)amino, thio, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, carboxy, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, aminosulfonyl, C 1-6 alkylaminosulfonyl, di(C 1-6 alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 alkylaminosulfonylamino, di(C 1-6 alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino.
19. The method of claim 1 , wherein L represents a direct bond.
20. The method of claim 1 , wherein L comprises one or more amino acid residues.
21. The compound of claim 20 , wherein L is defined by the structure below
wherein for each occurrence in L, R 14 is H and R 13 is selected from one of the following
or R 13 and R 14 , together with the atoms to which they are attached, form a five-membered heterocycle defined by the structure below
and
m is an integer selected from 1, 2, 3, 4, 5, and 6.
22. The method of claim 21 , wherein m is 1 or 2.
23. The method of claim 20 , wherein L comprises a serine residue, a threonine residue, an asparagine residue, a glutamine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid residue, or any combination thereof.
24. The method of claim 1 , wherein R 1 is H, an acetyl group, or an amino acid residue selected from the group consisting of a serine residue, a threonine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a cysteine residue, a 2,2,6,6-tetramethyl-N -oxyl-4-amino-4-carboxylic acid residue, and a glutamic acid residue.
25. The method of claim 1 , wherein R 2 comprises a hydroxy group or an amino acid residue selected from the group consisting of a serine residue, a threonine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a cysteine residue, a 2,2,6,6-tetramethyl-N -oxyl-4-amino-4-carboxylic acid residue, and a glutamic acid residue.
26. The method of claim 1 , wherein the compound is one of the following:
27. The method of claim 12 , wherein in the optional step of melting, the frozen sample is exposed to radiation having a wavelength in the range of about 0.5 μm to about 50 μM.