# US 12,000,846 B2 · Click-mass spectrometry of alkyne-labeled compounds

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- Number: US 12,000,846 B2
- Document ID: US12000846B2
- Status: Granted
- Kind: B2
- Application number: 17/254,922
- Filed: 2019-06-28
- Published: 2021-09-02
- Granted: 2024-06-04
- Earliest claimed: 2018-06-28
- Assignees: Rheinische Friedrich-Wilhelms-Universitaet Bonn
- Applicants: Rheinische Friedrich-Wilhelms-Universitaet Bonn
- Inventors: Christoph Thiele
- Web page: https://patentgrep.com/patent/US12000846B2

## CPC

- C07B 59/00: Introduction of isotopes of elements into organic compounds {; Labelled organic compounds per se}
- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- C07C 247/04: being saturated
- C07D 249/04: 1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
- C07D 487/04: Ortho-condensed systems
- G01N 1/4022: {by thermal techniques; Phase changes}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- C07B 2200/05: Isotopically modified compounds, e.g. labelled
- G01N 2001/4027: {evaporation leaving a concentrated sample}
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)

## Abstract

Nitrogen containing compounds may have formulas (I), (II) and (IV), and such compounds may be suitable for detecting alkyne group containing organic compounds by mass spectrometry. Furthermore, methods may also include synthesizing these compounds, detecting of organic compounds containing the specific compounds, uses of the compounds in mass spectrometry for determining enzyme activity or monitoring the lipid metabolism in a cell, and a kit which contains at least one of these compounds and at least one internal standard.

## Claims

### Claim 1

A compound comprising formula (I)
wherein R1 to R3 are independently selected from substituted or unsubstituted linear C1-C10 alkyl, substituted or unsubstituted branched C3-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, wherein the linear, branched alkyl, or cycloalkyl groups can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; or
wherein R1 is a free valence, and R2 and R3 form a 4 to 7-membered cyclic hydrocarbon ring, wherein the 4 to 7-membered cyclic hydrocarbon ring can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; or
wherein R1 and R2 are each methyl and R3 is ethyl;
wherein n is 2;
wherein 1 to 4 X are -D and the remaining X are —H;
wherein A− is an anion;
wherein each N in the compound can independently be 15N; and wherein each C in the compound can independently be 13C; and
wherein if R1 to R3 are substituted, the substituent is independently selected from -D, —F, —19F, —OH, or —OD.

### Claim 2 (depends on 1)

A compound comprising formula (II)
wherein R1 to R3 are independently selected from substituted or unsubstituted linear C1-C10 alkyl, substituted or unsubstituted branched C3-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof, and wherein if R1 to R3 are substituted the substituent is independently selected from -D, —F, —19F, —OH, or —OD; or
wherein R1 is a free valence, and R2 and R3 form a 4 to 7-membered cyclic hydrocarbon ring, wherein the 4 to 7-membered cyclic hydrocarbon ring can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof;
wherein n is 2;
wherein 1 to 4X are -D and the remaining X are —H;
wherein m ranges from 1 to 3; wherein each N in the compound can independently be 15N; and wherein each C in the compound can independently be 13C;
wherein the compound of formula (II) is obtained by reacting a compound of formula (I) according to claim 1 with an organic compound of formula (III)
wherein R4 is derived from an organic compound; and wherein m ranges from 1 to 3.

### Claim 3 (depends on 2)

A compound of formula (IV)
wherein R4 is derived from an organic compound;
wherein 1 to 4X are -D and the remaining X are —H;
wherein m ranges from 1 to 3;
wherein n ranges from 1 to 4;
wherein each N in the compound can independently be 15N; and
wherein each C in the compound can independently be 13C;
wherein the compound of formula (IV) is obtained by treating a compound of formula (II) according to claim 2 in a mass spectrometer.

### Claim 4 (depends on 1)

A method for the detection of an organic compound, wherein the method comprises:
reacting in a solvent at least one compound of formula (III) with at least one compound of formula (I) as defined in claim 1; wherein the reaction occurs in the presence of catalytic amounts of Cu(I) to form a compound of formula (II);
optionally purifying the compound of formula (II) by chromatography or liquid-liquid extraction; and
detecting the compound of formula (II), formula (IV), or combinations thereof;
wherein formula (III) comprises:
wherein R4 is derived from an organic compound, and wherein m ranges from 1 to 3;
wherein formula (II) comprises:
wherein R1 to R3, X, A−, N, and C are defined the same as R1 to R3, X, A−, N, and C of formula (I) in claim 1;
wherein formula (IV) comprises:
wherein R4 is derived from an organic compound; and wherein X, A−, N, and C are defined the same as X, A−, N, and C of formula (I) in claim 1.

### Claim 5 (depends on 4)

The method according to claim 4, wherein
the at least one compound of formula (I) is provided in excess over the at least one compound of formula (III); and wherein the method further comprises evaporating the solvent to concentrate the reaction mixture and aid in complete reaction of the at least one compound of formula (I) with the at least one compound of formula (III) to form the compound of formula (II).

### Claim 6 (depends on 4)

The method of claim 4, wherein one or more of the following occurs:
the at least one compound of formula (III) is derived from a lipid selected from the group consisting of saturated fatty acids, unsaturated fatty acids, glycerolipids, glycerophospholipids, lysophosphoglycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, carotenoids, waxes, and polyketides;
the method further comprises adding a Cu(I) salt or complex to the reaction mixture;
the method further comprises adding a Cu(I) salt or complex, wherein the Cu(I) salt or complex is selected from the group consisting of CuI, CuBr, CuCl, CuOTf*C6H6, [Cu(NCCH3)4], and Cu[acetonitrile]4BF4, Cu[acetonitrile]4PF6, and combinations thereof;
the method further comprises evaporating the solvent of the reaction mixture to increase the concentration of the at least one compound of formula (II);
the method further comprises evaporating the solvent of the reaction mixture to increase the concentration of the at least one compound of formula (I) to a concentration from 50 to 5000 μM;
the method further comprises providing the at least one compound of formula (I) at a concentration range from 40 to 100 μM;
the chromatography is selected from the group consisting of HPLC, LC, gel chromatography, Solid Phase Extraction (SPE), TLC, SMART, and combinations thereof;
the liquid-liquid extraction is performed with a mixture of CHCl3/MeOH/H2O;
further comprising drying the compound obtained after the liquid-liquid extraction and dissolving the residue in isopropanol/methanol/H2O comprising ammonium acetate;
the detection occurs by mass spectrometry;
wherein the method further comprises extracting the at least one compound of formula (III) from a cell, a tissue, an organ, a whole organism, or a biological fluid prior to reacting the at least one compound of formula (III) with the at least one compound of formula (I).

### Claim 7 (depends on 4)

The method according to claim 4, wherein one or more of the following occurs:
the compound of formula (III) is derived from a lipid selected from the group consisting of oleate, palmitate, cholesterol, cholesterol ester, cardiolipin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, mono-, di, and triacylglycerol, sphingosine and sphinganine;
the method further comprises extracting the at least one compound of formula (III) from a cell, a tissue, an organ, a whole organism or a biological fluid reacting the at least one compound of formula (III) with the at least one compound of formula (I);
the method further comprises extracting the at least one compound of formula (II) from a cell, a tissue, an organ, a whole organism, or a biological fluid prior to reacting the at least one compound of formula (III) with the at least one compound of formula (I); wherein the cell, the tissue, the organ, the whole organism, or the biological fluid has been incubated with at least one compound of formula (I) and at least one compound of formula (III), wherein the at least one compound of formula (III) is derived from a compound selected from the group consisting of glycerolipids and glycerophospholipids containing at least one terminal alkyne group, terminal alkyne cholesterol derivatives, omega alkyne oleates, omega alkyne palmitates, omega alkyne sphinganins, omega alkyne sphingosines, omega alkyne fatty acids, and omega alkyne unsaturated fatty acids.

### Claim 8 (depends on 4)

The method according to claim 4, wherein
at least two different compounds of formula (I) are included in the reaction mixture, wherein at least one of the compounds of formula (I) comprises one or more substituents independently selected from -D, —F, —19F, OH, OD, and combinations thereof; or
at least two different compounds of formula (VII) are used,
wherein at least one of the compounds of formula (VII) comprises one or more substituents independently selected from -D, —F, —19F, OH, OD, and combinations thereof.

### Claim 9 (depends on 1, 4)

The method according to claim 4, wherein
at least two different isotopically labeled compounds are present in the reaction mixture; wherein one of the at least two different isotopically labeled compounds is a compound of formula (I); or
at least two different isotopically labeled compounds are present in the reaction mixture;
wherein one of the at least two different isotopically labeled compounds is a compound comprising formula (VII):
wherein:
R1 to R3, X, A−, N, and C are defined the same as R1 to R3, X, A−, N, and C of formula (I) in claim 1.

### Claim 10 (depends on 1)

A method for producing a compound of formula (I) according to claim 1; wherein:
a compound according to formula (V) is reacted with NR1R2R3 in an organic solvent to give the compound according to formula (I);
wherein the compound of formula (V) comprises:
wherein X is independently selected from —H, -D, —F, —19F, —OH, —OD;
n ranges from 1 to 4;
Ms is a leaving group;
wherein each N is 15N, and wherein each C is 13C;
wherein R1, R2, and R3 are independently selected from substituted or unsubstituted linear C1-C10 alkyl, substituted or unsubstituted branched C3-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; or wherein R1 is a free valence and R2 and R3 form a 4 to 7-membered cyclic hydrocarbon ring, wherein the 4 to 7-membered cyclic hydrocarbon ring comprises one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; and wherein N is 15N;
or
a compound according to formula (V) is reacted with NR1R3H in an organic solvent to give a compound according to formula (VI), wherein
R1 and R3 are independently selected from substituted or unsubstituted linear C1-C10 alkyl, substituted or unsubstituted branched C3-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; or wherein R1 and R3 form a 4 to 7-membered cyclic hydrocarbon ring, wherein the 4 to 7-membered cyclic hydrocarbon ring comprises one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; and wherein N is 15N;
wherein formula (VI) comprises:
wherein X, R1 and R3, and n are as defined above, and wherein each N is optionally 15N; and wherein each C is optionally 13C; and wherein the compound of formula (VI), if R1 and R3 are not a 4 to 7-membered cyclic hydrocarbon ring is subsequently reacted with R2Y, wherein
R2 is independently selected from substituted or unsubstituted linear C1-C10 alkyl, substituted or unsubstituted branched C3-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl group comprises one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; and
—Y is a halogen group.

### Claim 11 (depends on 1)

A kit for the detection of alkyne-labeled compounds comprising or consisting of:
at least one compound according to formula (I) of claim 1;
at least one internal standard of alkyne-labeled compounds according to formula (III);
wherein formula (III) comprises:
wherein R4 is derived from an organic compound, and wherein m ranges from 1 to 3; and
optionally at least one Cu(I) salt or complex.

### Claim 12

A compound comprising formula (I)
wherein R1 and R2 are each methyl and R3 is ethyl;
wherein n is 2;
wherein each X is independently selected from —H, -D, —F, —19F, —OH, —OD;
wherein A− is an anion; and
wherein each N in the compound can independently be 15N; and wherein each C in the compound can independently be 13C.

### Claim 13 (depends on 12)

A method for producing a compound of formula (I) according to claim 12, wherein:
a compound according to formula (V) is reacted with NR1R2R3 in an organic solvent to give the compound according to formula (I);
wherein the compound of formula (V) comprises:
wherein X is independently selected from —H, -D, —F, —19F, —OH, —OD;
wherein n is 2;
Ms is a leaving group;
wherein each N is 15N, and/or wherein each C is 13C;
wherein R1 and R2 are each methyl and R3 is ethyl and can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof; and wherein N is 15N;
or
a compound according to formula (V) is reacted with NR1R3H in an organic solvent to give a compound according to formula (VI), wherein
R1 is methyl and R3 is ethyl and can contain one or more -D atoms, one or more 13C carbon atoms or a combination thereof; and wherein N is 15N
wherein formula (VI) comprises:
wherein X, R1 and R3, and n are as defined above, and wherein each N is optionally 15N; and wherein each C is optionally 13C; and wherein the compound of formula (VI), is subsequently reacted with R2Y,
wherein R2 is independently selected from substituted or unsubstituted methyl and can contain one or more -D atoms and a 13C carbon atoms or a combination thereof; and
—Y is a halogen group.

### Claim 14 (depends on 2)

The compound of formula (II) according to claim 2, wherein each N is 15N, and/or wherein each C is 13C.

### Claim 15 (depends on 3)

The compound of formula (IV) according to claim 3, wherein each N is 15N, and/or wherein each C is 13C.

### Claim 16 (depends on 12)

A compound comprising formula (II)
wherein R1 and R2 are each methyl and R3 is ethyl and can contain one or more -D atoms, one or more 13C carbon atoms, or combinations thereof, and wherein if R1 to R3 are substituted the substituent is independently selected from -D, —F, —19F, —OH, or —OD;
wherein n is 2;
wherein each X is independently selected from —H, -D, —F, —19F, —OH, —OD;
wherein m ranges from 1 to 3; wherein each N in the compound can independently be 15N;
and wherein each C in the compound can independently be 13C;
wherein the compound of formula (II) is obtained by reacting a compound of formula (I) according to claim 12 with an organic compound of formula (III)
wherein R4 is derived from an organic compound; and wherein m ranges from 1 to 3.