CLICK-MASS SPECTROMETRY OF ALKYNE-LABELED COMPOUNDS

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

1. A compound comprising formula (I) ##STR00028## wherein R.sup.1 to R.sup.3 are independently selected from substituted or unsubstituted linear C.sub.1-C.sub.10 alkyl, substituted or unsubstituted branched C.sub.3-C.sub.10 alkyl, substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more .sup.13C carbon atoms, or combinations thereof; or wherein R.sup.1 is a free valence, and R.sup.2 and R.sup.3 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 .sup.13C carbon atoms, or combinations thereof; wherein n ranges from 1 to 4; wherein each X is independently selected from —H, -D, —F, —.sup.19F, —OH, —OD; wherein A.sup.− is an anion; wherein each N is .sup.15N; and wherein each C is .sup.13C.

2. A compound comprising formula (II) ##STR00029## wherein R.sup.1 to R.sup.3 are independently selected from substituted or unsubstituted linear C.sub.1-C.sub.10 alkyl, substituted or unsubstituted branched C.sub.3-C.sub.10 alkyl, substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more .sup.13C carbon atoms, or combinations thereof; or wherein R.sup.1 is a free valence, and R.sup.2 and R.sup.3 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 .sup.13C carbon atoms, or combinations thereof; wherein n ranges from 1 to 4; wherein each X is independently selected from —H, -D, —F, —.sup.19F, —OH, —OD; wherein m ranges from 1 to 3; wherein each N is .sup.15N; and wherein each C is .sup.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) ##STR00030## wherein R.sup.4 is derived from an organic compound; and wherein m ranges from 1 to 3.

3. A compound of formula (IV) ##STR00031## wherein R.sup.4 is derived from an organic compound; wherein X is independently selected from —H, -D, —F, —.sup.19F, —OH, —OD, wherein m ranges from 1 to 3; wherein n ranges from 1 to 4; wherein each N is .sup.15N; and wherein each C is .sup.13C; wherein the compound of formula (IV) is obtained by treating a compound of formula (II) according to claim 2 in a mass spectrometer.

4-6. (canceled)

7. 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: ##STR00032## wherein R.sup.4 is derived from an organic compound, and wherein m ranges from 1 to 3; wherein formula (II) comprises: ##STR00033## wherein R.sup.1 to R.sup.3, X, A-, N, and C are defined the same as R.sup.1 to R.sup.3, X, A-, N, and C of formula (I) in claim 1; wherein formula (IV) comprises: ##STR00034## wherein R4 is derived from an organic compound; and wherein X, A-, N, and C of formula (I) in claim 1.

8. The method according to claim 7, 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).

9. The method of claim 7, 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*C.sub.6H.sub.6, [Cu(NCCH.sub.3).sub.4], and Cu[acetonitrile].sub.4BF.sub.4, Cu[acetonitrile].sub.4PF.sub.6, 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 CHCl.sub.3/MeOH/H.sub.2O; further comprising drying the compound obtained after the liquid-liquid extraction and dissolving the residue in isopropanol/methanol/H.sub.2O 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).

10. The method according to claim 7, 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 sphinganin; 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.

11. The method according to claim 7, 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, —.sup.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, —.sup.19F, OH, OD, and combinations thereof.

12. The method according to claim 7, 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): ##STR00035## wherein: R.sup.1 to R.sup.3, X, A-, N, and C are defined the same as R.sup.1 to R.sup.3, X, A-, N, and C of formula (I) in claim 1.

13. A method for producing a compound of formula (I) according to claim 1; wherein: a compound according to formula (V) is reacted with NR.sup.1R.sup.2R.sup.3 in an organic solvent to give the compound according to formula (I); wherein the compound of formula (V) comprises: ##STR00036## wherein X is independently selected from —H, -D, —F, —.sup.19F, —OH, —OD; n ranges from 1 to 4; Ms is a leaving group; wherein each N is .sup.15N, and wherein each C is .sup.13C; wherein R.sup.1, R.sup.2, and R.sup.3 are independently selected from substituted or unsubstituted linear C.sub.1-C.sub.10 alkyl, substituted or unsubstituted branched C.sub.3-C.sub.10 alkyl, substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more .sup.13C carbon atoms, or combinations thereof; or wherein R.sup.1 is a free valence and R.sup.2 and R.sup.3 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 .sup.13C carbon atoms, or combinations thereof; and wherein N is .sup.15N; or a compound according to formula (V) is reacted with NR.sup.1R.sup.3H in an organic solvent to give a compound according to formula (VI), wherein R.sup.1 and R.sup.3 are independently selected from substituted or unsubstituted linear C.sub.1-C.sub.10 alkyl, substituted or unsubstituted branched C.sub.3-C.sub.10 alkyl, substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl groups comprise one or more -D atoms, one or more .sup.13C carbon atoms, or combinations thereof; or wherein R.sup.1 and R.sup.3 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 .sup.13C carbon atoms, or combinations thereof; and wherein N is .sup.15N; wherein formula (VI) comprises: ##STR00037## wherein X, R.sup.1 and R.sup.3, and n are as defined above, and wherein each N is optionally .sup.15N; and wherein each C is optionally .sup.13C; and wherein the compound of formula (VI), if R.sup.1 and R.sup.3 are not a 4 to 7-membered cyclic hydrocarbon ring is subsequently reacted with R.sup.2Y, wherein R.sup.2 is independently selected from substituted or unsubstituted linear C.sub.1-C.sub.10 alkyl, substituted or unsubstituted branched C.sub.3-C.sub.10 alkyl, substituted or unsubstituted C.sub.3-C.sub.10 cycloalkyl; wherein the linear, branched alkyl, or cycloalkyl group comprises one or more -D atoms, one or more .sup.13C carbon atoms, or combinations thereof; and —Y is a halogen group.

14. 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: ##STR00038## 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0151] The accompanying drawings serve to afford an understanding of various embodiments. The drawings illustrate embodiments and together with the description serve to elucidate same. Further embodiments and numerous advantages from among those intended are evident directly from the following detailed description. The elements and structures shown in the drawings are not necessarily illustrated in a manner true to scale with respect to one another.

[0152] FIG. 1 Detection of alkyne-labeled compounds by click reaction with trialkylammonium salts (here C171) and subsequent mass spectrometry, whereby a stable cyclic fragment ion and a neutral trialkylamine group are formed and measured.

[0153] FIG. 2 Chemical synthesis strategies to provide compounds of formula (I). The following abbreviations have been used: Ms=mesylate, DMF=dimethylformamide, PCC=pyridinium chlorochromate, THP=tetrahydropyran, AcCI=acetyl chloride, DMEA=N,N-dimethyl-N-ethylamine, MEA=N-methyl-N-ethylamine, DHP=dihydropyran, and THF=tetrahydrofuran.

[0154] FIG. 3 General degradation pattern of measured neutral lipids (mono-, di-, triacylglycerols, sterol ester, ceramides), simple fatty acids and derivatives thereof (ester, amide, hydroxyl or keto fatty acids) after click reaction with C171 and subsequent mass spectrometry at ionization energies of 30 to 35 eV.

[0155] FIG. 4 Degradation pattern of phosphatidylcholine after click reaction with C171 and subsequent mass spectrometry by ionization energies of 30 to 35 eV. The head group and the trialkylammonium group are both eliminated uniformly as neutral losses (neutral loss of head group+73.09 Da (trialkylamine)).

[0156] FIG. 5 Preferred compounds according to formula (I) (click reagents C171 and C175-XX).

[0157] FIG. 6 Mass distribution of samples a) to d), after click reaction with different compounds of formula (I) and subsequent detection by mass spectrometry, showing the general concept of multiplexing.

[0158] FIG. 7 Multiplexed analysis of alkyne-labeled lipids in hepatocytes: Mouse hepatocytes were grown in single wells of a multiwall plate for 2 h in the presence of alkyne-palmitate and in the absence or presence of different inhibitors of DGAT1 or DGAT2, or a combination thereof. Cells were washed to remove the alkyne-palmitate and lipids were extracted by CHCl.sub.3/MeOH along with addition of a mixture of various internal standards. Lipid extracts were reacted with C175-73, -75, -76, or -77. Afterwards, the samples reacted with different C175-XX reagents were pooled and analyzed together by tandem mass spectrometry. 7A) MS2 spectrum at the mass of the internal standard for triacylglycerol TG(48:3), demonstrating the equal amount and response in the four samples. 7B) MS2 spectrum at the mass of triacylglycerol TG(49:4), which was synthesized by the cells, demonstrating the different effects of DGAT inhibitors on the amount of this TG in the sample.

[0159] FIG. 8A Complete mass spectrum of hepatocytes grown for 2 h with alkyne-palmitate before click reaction.

[0160] FIG. 8B Complete mass spectrum of hepatocytes grown for 2 h with alkyne-palmitate after click reaction.

DETAILED DESCRIPTION

Examples

[0161] Chemical Synthesis

[0162] The strategy for synthesis of C171 (N-1-(4-azidobutyl)-N-ethyl-N-dimethylammonium salt) and the C175-XX series is depicted in FIG. 2. There are two different synthesis routes. The first started with commercially available butyrolactone 1, butanediol 2, or its bis-methansulfonate 3. It was reacted with sodium azide to the azidobutylmesylate 4. The second route started with mono-tetrahydropyran (THP) butanediol 7 (Petroski, Synth. Commun., 2006, 33, 18, 3251-3259) or the THP-protected semialdehyde 6 (Petroski, Synth. Commun., 2006, 33, 18, 3251-3259). After activation to the mesylate 8 and reaction with sodium azide the protective group was removed to obtain 4-azido-1-butanol 10. Upon activation with MsCl, the routes converge at the azidobutylmethansulfonate 4. Depending on the needs, 4 was directly reacted with dimethylethylamine to give the trialkylammonium product 5, or sequentially reacted with methylethylamine to intermediate 11, followed by methylation with methyl iodide to give product 5. The latter reaction serves to introduce heavy isotopes into the alkylammonium head group, whereas two reductions with complex borodeuteride (1.fwdarw.2, 6.fwdarw.7) were used to introduce deuterium atoms into the butanediol moiety (see FIG. 2). d4-Butanediol can be obtained commercially.

Example 1: Synthesis of C171

[0163] 4-Azidobutan-1-ol (10): To a stirred solution of 20 mmol (3.48 g) mono-THP butanediol 7 and 3.0 g (30 mmol) trimethylamine in 100 mL CH.sub.2Cl.sub.2 was added at 0° C. to a solution of 22 mmol (2.52 g) methanesulfonylchloride in 8 mL CH.sub.2Cl.sub.2. After 2 h of stirring, the reaction was quenched with 10 mL 5% citric acid in water. The aqueous layer was removed, the organic phase washed with water and brine, dried over sodium sulfate and evaporated to obtain 4.7 g of the mono-THP mesylate 8 as a viscous liquid that was used without further purification. Four gram of 8 were stirred for 16 h at 60° C. with a solution of 3 g (46 mmol) NaN.sub.3 in 30 mL DMF. After addition of 50 mL ethyl acetate and 50 mL hexane, the mixture was extracted with 2×30 mL water and 50 mL brine, dried over sodium sulfate and the solvent was removed to give 3 g (14 mmol) of protected azidobutanol 9. Thin layer chromatography (TLC) analysis (hexane/ethyl acetate 1/1) showed complete conversion with two minor impurities. For removal of the THP-group, the material was dissolved in 100 mL methanol plus 0.6 mL acetyl chloride. After 5 min, the solvent was removed in vacuo and the residue purified on silica column (hexane/ethyl acetate 1/1) to obtain 1.4 g (12.2 mmol) product. NMR (400 MHz in CDCl.sub.3): 1.7 ppm (m, 4H, 2, 3-CH.sub.2), 3.34 ppm (t, J=6.5 Hz, 2H, 4-CH.sub.2), 3.70 ppm (t, J=6.1 Hz, 2H, 1-CH.sub.2).

[0164] 4-Azido-1-(methylsulfonyloxy)butane (4): Alcohol 10 (1.4 g) was reacted with 1.8 g triethylamine and 1.6 g methanesulfonyl chloride in 60 mL CH.sub.2Cl.sub.2 to obtain 2.2 g of 4. NMR (400 MHz in CDCl.sub.3): 1.74 ppm (m, 2H, 3-CH.sub.2), 1.87 ppm (m, 2H, 2-CH.sub.2), 3.04 ppm (s, 3H, S—CH.sub.3), 3.38 ppm (t, J=6.6 Hz, 2H, 4-CH.sub.2), 4.28 ppm (t, J=6.1 Hz, 2H, 1-CH.sub.2).

[0165] At this point, the mesylate 4 was either directly reacted with N-ethyl-N,N-dimethylamine to give the non-isotope labeled reagent C171 (same strategy as used for the synthesis of C175-73) or was reacted with N-ethyl-N-methylamine to give nor-methyl-C171. This was used to introduce isotope labels into the alkylammonium group by reaction with accordingly labeled methyl iodide (i.e. the synthesis of C175-75, -76, and -77).

Example 2: N-Ethyl-N-methyl-1-(4-azidobutyl)amine (11), nor-methyl-C171)

[0166] A solution of 100 μL (140 mg, 0.7 mmol) 4 and 200 μL (140 mg, 2.4 mmol) N-ethyl-N-methylamine in 0.5 mL THF was heated in a closed tube under argon for 6 h at 60° C. The solvent was removed in a stream of argon. The residue was dissolved in 3 mL CH.sub.2Cl.sub.2, the solution was extracted with 1 mL 1 M NaOH and washed with 2 mL water. The organic phase was collected, dried over sodium sulfate and evaporated to give 11 (100 mg, 0.6 mmol), which can be used for further synthesis without purification (see FIG. 2). NMR (400 MHz in CDCl.sub.3): 1.07 ppm (t, J=7.2 Hz, 3H, ethyl-CH.sub.3), 1.60 ppm (m, 4H, 2, 3-CH.sub.2), 2.23 ppm (s, 3H, methyl-CH.sub.3), 2.38 ppm (t, J=7.3 Hz, 2H, ethyl-CH.sub.2, 1-CH.sub.2), 2.44 ppm (q, J=7.2 Hz, 2H, ethyl-CH.sub.2), 3.45 ppm (t, J=6.6 Hz, 2H, 4-CH.sub.2).

Example 3: C171

[0167] A solution of 400 μL (560 mg, 2.9 mmol) 4 and 400 μL (270 mg, 3.7 mmol) N-ethyl-N,N-dimethylamine in 1 mL CH.sub.2Cl.sub.2 was heated in a closed tube under argon for 6 h at 60° C. TLC control showed complete conversion. The solvent was removed in a stream of argon and the residue precipitated and washed with ether (3×6 mL) and hexane (2×6 mL). The solvent was removed in vacuo to give C171 (570 mg, 2.15 mmol) as its mesylate salt.

[0168] NMR (400 MHz in CDCl.sub.3): 1.38 ppm (t, J=7.3 Hz, 3H, ethyl-CH.sub.3), 1.67 ppm (m, 2H, 3-CH.sub.2), 1.83 ppm (m, 2H, 2-CH.sub.2), 2.69 ppm (s, 3H, S—CH.sub.3), 3.21 ppm (s, 6H, methyl-CH.sub.3), 3.45 ppm (m, 6H, ethyl-CH.sub.2, 1-CH.sub.2, 4-CH.sub.2), 3.45 ppm (m, 2H+q, 4H, J=7.3 Hz, 1-CH.sub.2+ethyl-CH.sub.2).

[0169] Subsequently, the mesylate was exchanged against the BF.sub.4.sup.−-anion. C171-mesylate salt (570 mg) was dissolved in 10 mL 50% MeOH. The solution was loaded on a 5 mL column of Amberlite A26 (purchased in the OH.sup.−-form, washed sequentially with 8 vol. each of 50% MeOH, 1 M NaOH, water, 1 M NH.sub.4BF.sub.4, water, 50% MeOH) and eluted with 50% MeOH. The first 1.5 mL flow-through were discarded and the following 20 mL pooled to obtain a 100 mM solution of C171-BF.sub.4 that was used for click reactions.

Example 4: C175-73

[0170] 1 g (10.6 mmol), 2,3-d4-butanediol (Cambridge Isotopes) was reacted with two equivalents of methanesulfonylchloride as described above to give d4-1,4-bis(methylsulfonyloxy)butane in quantitative yield. To minimize risk from handling this potentially dangerous compound, the entire amount was dissolved in 30 mL DMF and stirred with 825 mg (1.2 eq.) NaN.sub.3 for 16 h at 60° C. After standard workup (see above), 2,3-d4-4 was isolated by chromatography on silica gel (hexane/ethyl acetate 2/1) to give 700 mg product. Reaction with dimethylethylamine (see C171) yielded 760 mg C175-73 as the mesylate salt (see FIG. 2).

Example 5: C175-75

[0171] Sodiumborodeuteride (1 g, 24 mmol) and 2.06 g (24 mmol) LiBr were dissolved in 20 mL THF and stirred for 20 min at 65° C. Gammabutyrolactone (2 mL, 26 mmol) were added and stirring continued at 60° C. under argon for 16 h. Then 300 μL CH.sub.3OD and 1 mL D20 were added subsequently and stirring was continued for 5 min, followed by addition of 20% HCl until the mixture was acidic. The upper, organic phase was collected, the lower phase extracted with 2×5 mL THF and the combined THF phases evaporated, which resulted in a viscous liquid and large amounts of solid, presumably boric acid and related compounds. The entire material was loaded onto a short silica column and extracted with CHCl.sub.3/MeOH/H.sub.2O 30/60/10. The solvent was removed to obtain 2 g of viscous, impure material. For further purification, the butanediol was converted to its mono-THP ether according to the procedure of Petroski (Petroski, Synth. Commun., 2006, 33, 18, 3251 to 3259) and added 2 mL 0.1 M HCl and 25 mmol dihydropyran in 60 mL DCM, followed by stirring for 96 h. The mixture was neutralized with saturated NaHCO.sub.3, the CH.sub.2Cl.sub.2 phase was collected, dried and the solvent evaporated. The residue was purified on silica gel with hexane/ethyl acetate 1/1 to obtain 710 mg d2-7 mono-THP ether along with 1 g of the di-protected diol. The d2-7 was converted to d2-4 as outlined above to give 3.5 mmol (680 mg) product, which was reacted with a 3-fold excess of ethylmethylamine in 2 mL THF for 6 h at 60° C. After addition of 10 mL water and 15 mL 1M NaOH, the mixture was extracted with 3×30 mL CH.sub.2Cl.sub.2, the extracts were combined and the solvent removed in vacuo. The residue was dissolved in 2 ml THF and treated with 5 mmol (700 mg, 313 μL) CHD.sub.2I (Sigma-Aldrich) in 2 mL THF. After 1 h of stirring at 50° C., ether (5 mL) was added, the upper phase discarded and the lower phase washed with ether and hexane to give C175-75 as the iodide salt (see FIG. 2).

Example 6: C175-76

[0172] A solution of 210 mg (5.1 mmol) NaBD.sub.4 in 15 mL 0.1N NaOH in D.sub.2O was added in one batch to a stirred solution of 2.5 g (14.5 mmol) 6 in 35 mL THF. After 5 min, TLC control showed complete conversion to the alcohol. Brine (10 mL) was added, the THF phase collected and the water phase re-extracted with 10 mL ethyl acetate. Organic phases were combined, washed with brine, dried and evaporated. The residue was purified on silica (hexane/ethyl acetate/trimethylamine, 50/50/1) to give 2.0 g (11.4 mmol) 4-d1-7. After activation with MsCl (2 g 7, 1.43 g MsCl, 1.7 g triethylamine, 50 mL CH.sub.2C.sub.12, to give 2.88 g 4-d1-8), subsequent reaction with sodium azide (2.3 g in 30 mL DMF to give 2.24 g 4-d1-9), removal of THP, activation with MsCl (1.35 g 4-d1-9, 1.7 g ethylmethylamine in 3 mL THF at 60° C. for 3 h to yield 1.53 g 4-d1-11. This was reacted with a 3-fold excess of CD.sub.3I in THF to give 2.5 g of C175-76 as the iodide salt (see FIG. 2).

Example 7: C175-77

[0173] To prepare .sup.13CD.sub.3I from .sup.13CD.sub.3OH, the procedure of Olah et al. (Olah et al. Angew. Chem. Int. Ed. Engl., 1979, 18, 612 to 614) was used. To a stirred solution of .sup.13CD.sub.3OH (128 mg, 4 mmol) and sodium iodide (600 mg, 4 mmol) in 4 mL dry acetonitrile was slowly added 500 μL (430 mg, 4 mmol) chlorotrimethylsilane. After 30 min of stirring, the tube was centrifuged for 5 min at 500 g and the supernatant was transferred into a tube that contained a solution of 470 mg (3 mmol) of 11 in 1 mL THF. The mixture was stirred for 4 h at room temperature. The major part of the solvent was evaporated in a stream of argon to leave about 1.5 mL, the product was precipitated as its iodide salt by addition of 6 mL ether and the precipitate washed with ether (3×6 mL) to give 750 mg of C175-77 as an iodide salt (see FIG. 2).

[0174] Purification for all C175 reagents: The raw material (600-1500 mg) was dissolved in 10 mL 50% MeOH and passed over a column of 5 mL Amberlyst A15 (purchased in the H.sup.+-form, washed with 10 vol. 50% MeOH, 10 vol. 1 M NH.sub.3 in H.sub.2O, 10 vol. 1 M ammonium formate in 50% MeOH and 10 vol. 50% MeOH). The column was washed with 50% MeOH until absorbance of 280 nm and conductivity returned to the baseline. The product was eluted with 40 mL 1 M ammonium formate in 50% MeOH. The eluates were evaporated at a final vacuum of 1.5 mbar at 70° C. until all remnants of ammonium formate had disappeared, leaving the formate salts of the reagents as viscous liquids. These formates were analyzed by 1H-NMR:

[0175] C175-73: (400 MHz in CDCl.sub.3): 1.40 ppm (t, J=7.3 Hz, 3H, ethyl-CH.sub.3), 3.17 ppm (s, 6H, methyl-CH.sub.3), 3.42 ppm (2×s, 4H, 1-CH.sub.2, 4-CH.sub.2), 3.48 ppm (q, J=7.3 Hz, 2H, ethyl-CH.sub.2).

[0176] C175-75: (400 MHz in CDCl.sub.3): 1.41 ppm (t, J=7.3 Hz, 3H, ethyl-CH.sub.3), 1.67 ppm (m, 2H, 3-CH.sub.2), 1.84 ppm (m, 2H, 2-CH.sub.2), 3.14 ppm (s, 1H, methyl-CD.sub.2H), 3.17 ppm (d, J=1.0 Hz, 3H, methyl-CH.sub.3), 3.46 ppm (m, 4H, ethyl-CH.sub.2, 1-CH.sub.2, 4-CH.sub.2). C175-76: (400 MHz in CDCl.sub.3): 1.41 ppm (t, J=7.3 Hz, 3H, ethyl-CH.sub.3), 1.67 ppm (m, 2H, 3-CH.sub.2), 1.84 ppm (m, 2H, 2-CH.sub.2), 3.17 ppm (s, 3H, methyl-CH.sub.3), 3.45 ppm (m, 5H, ethyl-CH.sub.2, 1-CH.sub.2, 4-CH.sub.2).

[0177] C175-77: (400 MHz in CDCl.sub.3): 1.40 ppm (t, J=7.3 Hz, 3H, ethyl-CH.sub.3), 1.67 ppm (m, 2H, 3-CH.sub.2), 1.84 ppm (m, 2H, 2-CH.sub.2), 3.20 ppm (d, J=3.6 Hz (couples to .sup.13C of the .sup.13CD.sub.3-methyl), 3H, methyl-CH.sub.3), 3.45 ppm (m, 6H, ethyl-CH.sub.2, 1-CH.sub.2, 4-CH.sub.2).

[0178] Anion exchange: For use of the C175 reagents in the click reaction, the formate was exchanged against the BF.sub.4.sup.−-anion. This was done to avoid possible interference of the formate with the Cu(I)-ions in the click reaction and possible adduct formation in MS. Since the Cu(I) is supplied as the BF.sub.4.sup.−-salt anyway, the formate was exchanged against BF.sub.4.sup.−. Each reagent (220 mg, 1 mmol) was dissolved in 3 mL 50% MeOH. The solution was loaded on a 5 mL column of Amberlite A26 (purchased in the OH.sup.−-form, washed sequentially with 8 vol. each of 50% MeOH, 1 M NaOH, water, 1 M NH.sub.4BF.sub.4, water, 50% MeOH) and eluted with 50% MeOH. The first 1.5 mL flow-through were discarded and the following 10 mL pooled to obtain 100 mM solutions of C175-XX.sup.+BF.sub.4.sup.−, which were used for subsequent click reactions.

[0179] For MS analysis of isotopic purity, the reagents were diluted to 10 μL in MeOH and analyzed in MS1 and MS2. First, isotopic composition was examined and it was found that all reagents showed a major peak at the expected nominal mass m/z 175 plus the expected first isotope peak at m/z 176 with about 8% intensity relative to the monoisotopic peak. In addition, peaks at m/z 174 were found, indicating incomplete replacement of hydrogen by deuterium, with relative intensities ranging between 1.5% (C175-77) and 6.5% (C175-75). Subsequently, all 12 isotope peaks were analyzed in MS2 to determine the distribution of isotopes between the dimethylethylamino group and the butanediol moiety. The C175 reagents show a uniform fragmentation with neutral loss of nitrogen plus the dimethylethylamine group, leaving the cation of the butanediol-N, likely in a cyclic form. From the pattern of these ions, the isotope composition of each of the C175 reagents was calculated in detail. For all the C175-XX reagents, the fraction of the monoisotopic peak with the correct NL of the alkylamine group is between 86 and 91%. In the first isotope peaks at m/z 176, both NL of XX and of XX+1 were found, indicating that the .sup.13C, which gives rise to the m/z 176 peaks, distributes as expected over both the butanediol and the alkylamine moiety. In contrast, for the peaks of m/z 174 the defect (missing deuterium) was found to localize mostly to the butanediol moiety, with the exception of C175-77, which has an unlabeled butanediol and therefore a small deuterium defect in the alkylamine group. These data enable correction of the primary NL mass spectra for the specific properties of each of the four reagents.

[0180] Quantitative Internal Standards

[0181] Alkyne-labeled standards were synthesized in a 10-50 mg scale using standard lipid synthesis techniques. All compounds were purified using silica gel chromatography, dissolved at 1 mg/mL in methanol (PC, PA) or 2-propanol and mixed to obtain a standard mix stock solution, which subsequently was calibrated against two unlabeled quantitative standard mixtures.

TABLE-US-00001 TABLE 1 Alkyne-labeled standards, their synthesis method and their calculated and measured masses (m/z). Calculated and measured Standard name Synthesis masses (m/z) PC(16:0/a18.3) Acylation of LPC (16:0) [M + H].sup.+ using EDC/DMAP meas: 756.551, calc: 756.554 PA(a17:2/17:1) Acylation of sn-glycerol-3 [M + H].sup.+ diethylphosphate and meas: 669.451, subsequent deprotection calc: 669.450 with bromotrimethylsilane (Gaebler et al., J. Lipid Res., 2013, 54, 8, 2282-2290) Cer(d18:1/a18:3) Acylation of sphingosine [M + H].sup.+ with a 18:3-NHS meas: 560.502, calc: 560.504 TG(14:0/17:1/a17:2) Acylation of DG [M + NH.sub.4].sup.+ (14:0/17:1) using meas: 818.721, EDC/DMAP calc: 818.723 CE(a18:3) Acylation of cholesterol [M + NH.sub.4].sup.+ using EDC/DMAP meas: 664.602, calc: 664.603 DG(a17:2/15:0) Acylation of MG(a17:2) [M + NH.sub.4].sup.+ using EDC/DMAP meas: 582.508, calc: 582.509 TG(a17:2/15:0/a19:3) Acylation of [M + NH.sub.4].sup.+ DG(a17:2/15:0) using meas: 856.735, EDC/DMAP calc: 856.739

Example 8: Multiplexing

[0182] Multiplexed analysis of alkyne-labeled lipids in hepatocytes was performed. Mouse hepatocytes were grown in single wells of a multiwall plate for 2 h in the presence of alkyne-palmitate and in the absence or presence of different inhibitors of DGAT1 or DGAT2, or a combination thereof. Cells were washed to remove the alkyne-palmitate and lipids were extracted by CHCl.sub.3/MeOH along with addition of a mixture of various internal standards. Lipid extracts were reacted with C175-73, -75, -76, or -77. Afterwards, the samples reacted with different C175-XX reagents were pooled and analyzed together by tandem mass spectrometry. FIG. 7A shows a MS2 spectrum at the mass of the internal standard for triacylglycerol TG(48:3), demonstrating the equal amount and response in the four samples. FIG. 7B shows a MS2 spectrum at the mass of triacylglycerol TG(49:4), which was synthesized by the cells, demonstrating the different effects of DGAT inhibitors on the amount of this TG in the sample. The complete mass spectrum of hepatocytes grown for 2 h with alkyne-palmitate before and after click reaction are shown in FIG. 8A (before) and 8B (after).

Advantages Over Known Analysis Methods

[0183] The compounds and methods and uses associated therewith, have many advantages over known analysis methods:

[0184] The reaction of the alkyne-labeled compound of formula (III) with the click reagent of formula (I) results in a mass increase of the resulting molecule of formula (II). For example, in case of C171, the click reagent has a mass of 171.16 Da. During mass spectrometry, the mass of the measured compound shifted by 171 Da. Thereby, labeled lipids can be shifted in a nearly empty field of the spectra, whereby the analysis of data and the separation of signals is improved (see FIG. 8A/8B).

[0185] Because of the four nitrogen atoms in the compound according to formula (II), the compound identification in mass spectra is improved.

[0186] Fragmentation of compounds of formula (II) during mass spectrometry is surprisingly uniform. Using low to medium ionization energies, no fragmentation of the triazol ring of formula (II) occurs. Only a neutral loss of the trialkylammonium group takes place. It is assumed, without being bound to any theory, that the compound of formula (II) forms a stable cyclic product of formula (IV) after separation of the trialkylammonium group.

[0187] Preferably, with ionization energies of 10 to 50 eV, more preferably of 25 to 40 eV, most preferably of 30 to 35 eV, the neutral loss is the only fragmentation of compounds of formula (II) and the fragmentation is nearly complete. With higher ionization energies, further fragmentations can take place, but preferably, a degradation of the triazol ring does not occur (see fragmentation pattern of FIGS. 3 and 4). With too low ionization energies, the fragmentation can be incomplete and not consistent.

[0188] With phosphatidylcholine, the head group and the trialkylammonium group are both eliminated uniformly as neutral losses (neutral loss of head group+73.09 Da) with ionization energies of 30 to 35 eV (see FIG. 4). Based on the neutral loss, the corresponding phosphatidylcholine can be identified. If C171 would set free a charged reporter ion, the identification and differentiation in MS/MS would not be possible.

[0189] The permanent positive charge of the compound of formula (II) leads to an improved ionization and signal strength of measured lipids (see FIG. 8B).

[0190] Typical Procedure for Utilizing C171:

1) Metabolic labeling of the usually living biological test system with an alkyne-labeled precursor, e.g. a fatty acid. Alternatively, all known alkyne-labeled compounds can be used such as lipids, nucleic acids, amino acids, peptides or proteins.
2) Finishing the labeling and subsequent extraction of the labeled metabolites, optionally addition of alkyne-labeled internal standard lipids for a later absolute quantification.
3) Click reaction with C171 in the presence of Cu(I), preferably in an organic solvent containing Cu(I)-tetrafluoroborate.
4) For lipids: Removal of reagents by chloroform/methanol/water 2-phase-distribution. Lipids are present the chloroform phase, ionic compounds are present the water/methanol phase.
5) Drying of the chloroform phase and dissolving the residue in a suitable organic solvent mixture for use in mass spectrometry, preferably isopropanol/methanol/water mixtures with addition of ammonium acetate are used.
6) MS and MS/MS analysis: The preferred form of analysis of lipids is a so-called shotgun-analysis of the unseparated mixture by direct infusion into the mass spectrometer (e.g. commercially available Thermo Q-Exactive Plus with a HESI ion source). The lipid sample was sprayed into the mass spectrometer by a standard syringe pump for 10 to 30 min with 2 to 10 μL/min. In this time, MS1 spectra (generally 300-1200 Da) and subsequently MS/MS spectra were measured program-controlled over the whole range (steps of 1 Da, selection window of 1 Da, charge+1) and subsequently MS/MS spectra of 300 to 700 Da were measured (steps 0.5 Da, selection window 0.7 Da, charge+2). The measured data set contains MS1 and MS/MS data for each positive ion of the selected measuring range, which enables the later broad evaluation of each lipid class
7) For evaluation of the measurements, the software LipidXplorer (3) was used, which is platform independent and available free of charge. With this software, it is possible to define search parameters in so-called MFQL files, which work with a molecular search logic, with which the data set can be searched.

[0191] MFQL files can be set up for every lipid class. Phosphatidylcholine and triacylglycerol give, in addition to the usual signals with charge of +1, also signals with charge of +2. In case of phosphatidylcholine, these signals occur because of the stable charge of the head group. In case of triacylglycerol, it occurs because of the introduction of two labeled fatty acids in the triacylglycerol molecule. These species can be detected and quantified by corresponding MFQL files, if corresponding spectra were measured during data acquisition. Reading and searching of a data set of 10 samples with usual search parameters and 20 MFQL files needs approximately 20 minutes and detects 150 to 500 labeled species, when labeled with 16-heptadecynoic acid, also called “Click-Palmitate”.

[0192] Absolute quantification of lipid amounts in a sample is possible, using internal standards. Preferably, a mixture of synthetic internal standards, which are alkyne-labeled lipids of known concentrations with unnatural fatty acid patterns, can be added to the sample, preferably during lipid extraction. Intensities of the samples can be compared to intensities of the internal standards to calculate the present concentrations. The usual internal standard mixture contains the most important lipid classes (MAG, DAG, TAG, Cer, CE, PA, PC). The extension of the mixture by new developed and synthesized standards is possible. In one embodiment, the internal standard mixture contains lipid classes CE, Cer, (MAG), DAG, TAG, double-labeled TAG, PA, PC, PE, PI and PS.

[0193] The lower detection limit for most of the lipid classes is at approximately 0.02 pmol per species in the complete sample, which is a low concentration in comparison to other methods, especially unlabeled analysis methods. The reduction of the sample concentration is possible due to the improved ionization of the permanent charge of C171.

[0194] Multiplex-Analysis: C171 has a flexible basic structure, which allows the formation of different variants. Heavy isotopes (-D, -.sup.13C, -.sup.15N) can be integrated in parts of the molecule, whereby the same nominal mass, but different neutral losses occur (see Table 2). Labeled lipid samples, which are clicked with different reagents (for example variants C175-XX, see FIG. 5), can be pooled and analyzed in combination. Thereby, experimental noise due to variations in sample handling, spray fluctuations, ionization suppression or fluctuations in fragmentation is reduced. Furthermore, the comparability of samples is improved and the measurement time reduced.

[0195] The difference of the Cl 75-XX procedure and the procedure with C171 is that the four samples of C175-XX are pooled after click reaction (see FIG. 6).

[0196] C175-77 and C175-73 to 76 differ in their mass by 2.87 mDa. This difference is not recognized by MS1, whereby only one peak is present (see FIG. 6). Only in MS2, after neutral loss of the C175-reagents, the single peak is separated into four peaks, which contain information about the amount of precursors in the single samples (see FIG. 6).

[0197] After syntheses of C175 reagents, the content of monoisotopic reagent is in the range of 87 to 91%. 9 to 13% are different impurities, which result from .sup.13C content and incomplete substitution with -D. As a result, multiplex peaks have complex disorders in the range of 3 to 5% of intensity. Since this disorder occurs in all samples uniformly, the error can be eliminated by relative standardization.

[0198] By utilization of an internal standard, the error can be corrected and total quantification is possible.

TABLE-US-00002 TABLE 2 Overview of preferred compounds (C171 and C175-XX) according to formula (I) and their corresponding masses before and after fragmentation (neutral loss) by mass spectrometry. [00027] Nominal mass Exact mass NL mass Remaining mass C.sub.4H.sub.11 N C.sub.4H.sub.8 N.sub.3 171 171.16097 73.08915 98.07182 C.sub.4H.sub.11 N C.sub.4H.sub.4D.sub.4 N.sub.3 175 175.18553 73.08915 102.09693 C.sub.4H.sub.9D.sub.2 N C.sub.4H.sub.6D.sub.2 N.sub.3 175 175.18553 75.10170 100.08438 C.sub.4H.sub.8D.sub.3 N C.sub.4H.sub.7D.sub.1 N.sub.3 175 175.18553 76.10798 99.07810 C.sub.3.sup.13C.sub.1H.sub.8D.sub.3 N C.sub.4H.sub.8 N.sub.3 175 175.18260 77.11133 98.07182

TABLE-US-00003 TABLE 3 Neural loss of click reagents (C171, C175-XX) bound to different labeled lipid samples (PA, PC, PE, PG, PI, PS, MAG, DAG, TAG, Cer, free fatty acids). NL of head NL after click reaction with group C171 C175-73 C175-75 C175-76 C175-77 PA 97.977 171.066 171.066 173.079 174.085 175.088 PC 183.066 256.155 256.155 258.168 259.174 260.177 PE 141.019 214.108 214.108 216.121 217.127 218.130 PG 172.014 245.103 245.103 247.116 248.122 249.125 PI 260.030 333.119 333.119 335.132 336.138 337.141 PS 185.009 258.098 258.098 260.111 261.117 262.120 MAG, DAG, 0 73.089 73.089 75.102 76.108 77.111 TAG, Cer, free fatty acids

[0199] Evaluation of Data

1. The intensities of the four peaks can be compared to each other, whereby relative amounts can be calculated.
2. If internal standards are added to the samples during lipid extraction, total amounts of the sample precursors can be calculated.

[0200] Quantitative Alkyne-Labeled and Deuterium-Labeled Internal Standards

TABLE-US-00004 TABLE 4 Alkyne-labeled and deuterium-labeled standards, their synthesis method and their calculated and measured masses (m/z). Calculated and measured Standard name Synthesis masses (m/z) PA(a17:2-15:1-d8) Sequential acylation of [M + NH.sub.4].sup.− sn-glycerol-3 diethylphosphate meas: 668.510 with alkyne-palmitate calc: 668.510 (aPal) and FA15:1-d8 and subsequent deprotection with bromotrimethylsilane (see Gaebler et al., J. Lipid Res. 54, 2281-2290 (2013)) PC(a17:2-15:1-d8) Reaction of PA(a17:2-15:1-d8) [M + H].sup.+ with choline tosylate in meas: 736.571 pyridine/trichloroacetonitrile calc: 736.573 PE(a17:2-15:1-d8) PLD-catalyzed reaction of [M + H].sup.+ PC(a17:2-15:1-d8) with meas: 694.525 ethanolamine (see Gaebler et calc: 694.526 al., J. Lipid Res. 54, 2281-2290 (2013)) PS(a17:2-15:1-d8) Reaction of PA(a17:2-15:1-d8) [M + H].sup.+ with Boc-Ser(OH)-tBu in meas: 738.513 pyridine/trichloro- calc: 738.5155 acetonitrile and subsequent deprotection in TFA/DCM PI(a17:2-15:1-d8) Sequential acylation of [M + NH.sub.4].sup.+ glycerol-phospho-(2,3:4,5- meas: 830.561 dicyclohexylidene-6O- calc: 830.563 methoxymethyl-inositol, see Aneja, U.S. Pat. No. 7,977,497B2 (2011)) followed by deprotection using bromotrimethylsilane in DCM. dhCer Acylation of alkyne- [M + H].sup.+ (alkyne-d18:2/15:1-d8) sphinganine (Gaebler et al., meas: 528.523 J. Lipid Res. 54, 2281-2290 calc: 528.523 (2013) with FA15:21-d8-NHS DAG Acylation of MG(a17:2) with [M + NH.sub.4].sup.+ (a17:2/15:1-d8) FA15:1-d8 using EDC/DMAP meas: 588.543 calc: 588.544 TAG Acylation of DG(a17:2/15:1-d8) [M + NH.sub.4].sup.+ (a17:2/15:1-d8/16:0) with palmitic acid using meas: 826.774 EDC/DMAP calc: 826.773 TAG Acylation of DG(a17:2/15:1-d8) [M + NH.sub.4].sup.+ (a17:2/15:1-d8/a17:2) with aPal using EDC/DM AP meas: 826.774 calc: 826.773 CE(a17:2-d7) Acylation of commercial [M + NH.sub.4].sup.+ cholesterol-d7 with meas: 659.647 aPal using EDC/DMAP calc: 659.647

Exemplary Synthesis of Deuterium-Labeled Fatty Acids

[0201] Pentadec-11c-enoic acid (12,13,14,15-D8, FA15:1-D8)

[0202] D10-Butanol (2 g) and 5 g powdered molecular sieve was treated with 2 equivalents of pyridinium chlorochromate in 100 ml dichloromethane to give d8-butanal that was isolated by destillation. 1-tetrahydropyranyl-oxy-undecanyl-triphenylphosphonium bromide was reacted with one equivalent of lithiumhexamethylenedisilazane in THF and the resulting Wittig reagent reacted with the d8-butanal to give THP-protected pentadec-11c-en-1-ol (12,13,14,15-D8). The THP group was removed by treatment with HCl in dry methanol and the free alcohol was oxidized with H.sub.2SO.sub.4/CrO.sub.3 in acetone to give the final product.