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A simple method for preparation of methyl-labelled (—) carnitine
552 BBA
SHORTCOM3'lUNICATIOKS
533QO
A simple method for preparation of methyl-labellled (-1 (-)Cartinine acid metabolism.
carnitine
and its esters have proved to be useful toois in studies on fatty Labelled carnitine is commercially available in its racemic form
only. Our previous labelling method1 also gave a mixture of the two isomers, and the biologically inactive labelled (+) carnitine will interfere in many experiments. We have therefore (-)carnitine
worked from
out a simple method
the commercially
method is much simpler than a previously of labelled ( -)carnitine2. Sodium benzthiolate
nonradioactive
published
was made from thiophenol
and stored over P,O, until used3. 0.5 g (3.1 mmoles) of (-)carnitine ceutical Factory,
for the preparation
available
procedure
of methyl-labelled (-)carnitine.
This
for the preparation
(Fluka AC, Buchs, Switzerland)
hydrochloride
(gift from Otsuka Pharma-
Osaka, Japan} was dissolved in zo ml of propanol.
The solution was
saturated with dry HCl gas and refluxed for IO min. To ensure complete esterification the HCl saturation and the refluxing was repeated once. The propylcarnitine was taken to dryness on a rotating vacuum evaporator and dissolved in 50 ml of butanone. z g (IS mmoles) of sodium benzthiolate were added, and the mixture was refluxed under nitrogen
for 24 h while stirred with a magnetic
stirrer. The solvent was then
evaporated and the residue dissolved in 20 ml of 0.5 M NaOH and heated under reflux for 2 h. After acidification with HCI the excess of benzthiolate was removed as thiophenol vacuum
by extraction evaporator,
with diethyl
ether. The water was removed
and the (-)p-hydroxy-y-dimethylaminobutyric
in a rotating acid hydro-
chloride was freed from the sodium chloride by taking it up in a small volume methanol. The hydrochloride was then crystallized by addition of ethylacetate.
of
The yield of the synthesis was 67%. Thin-layer chromatography on silica gel (Kieselgel H “Merck”) with phenol-water (90/30, w/v) as solvent showed, after exposure to iodine vapour, one main spot with RF 0.5. A very weak spot corresponding to carnitine (RF 0.6) could be seen under ultraviolet light. Mass spectrometry verified the identity of the compound (Fig. I). The small residual amount of carnitine was too small to give any of the characteristic mass peaks of carnitine4. The melting point measured on a Kofler Micro Hot Stage (Reichert, Austria) was sharp at 87-88”. (The Radiochemical Center, The methylation with i3H-CH,] methyliodide Amersham,
England) was performed as follows: The ampoule containing the L3H-CH,j,
methyliodide (25 mC, approximately 0.3 mmoles) was opened after cooling of the lower end of the ampoule on solid carbon dioxide to minimize evaporation. Half a mI of cold methanol was added to the cooled ampoule followed by 0.5 mmoies of i-j/% hydroxy-y-dimethylaminobutyric acid hydrochloride and 2 mmoles of NaOH in z ml of 50% methanol. The ampoule was tightly stoppered with a rubber stopper and left at room temperature over night. The content of the ampoule was then neutralized with HCl and chromatographed twice on a column of Dowex SOW-H+ (2ooo400 mesh), approximately I cm x 50 cm, previously washed with I .5 M HCl and water. The column was eluted with 1.5M HCl. The [3H-CH,] (-) carnitine was eluted between 150 and 200 ml. The excess of unlabeled P-hydroxy-y-dimethylaminobutyric acid was eluted just in front of the carnitine. The fractions containing the radioactive carnitine were B&him.
Biophys.
Acta,
218 (1970)
55C-jjq
SKORT COMMUNICATIONS C”3,
553
?H
N-CHzCH;CH2-COOH
CH3/j
too-
1 ’
1 : &
j :
ve Fig. I. Mass spectrum of the synthesized ~-~~droxy-y”dimethyla~inobu~r~c acid hydrochloride. The analysis was performed on a Varian CH-7 instrument with a direct inlet system. Probe temperature, 100’. Electron energy, 70 eV. Hydrochloric acid is immediately split off from the molecule, and the molecular peak at m/e 147 corresponds to the free base. evaporated
to a crystalline
mass in vacua, The yield of carnitine was about 80 per
cent (calculated
on the basis of radioactivity). The radiopurity of the [3H-CH,] carnitine was checked in the thin-layer chromatographic system above. No radioimpurities were detected. The radiopurity was
also checked in an enzymic exchange reaction with (-)propionylc~tine catalyzed with carnitine acetyltransferase (E.C. 2.3.1.7) (Boehringer and Soehne, Mannheim,
Germany). Table I shows that almost exactly twice as much radioactivity was recovered in propionylcarnitine from the added [sH-Cl& (-)carnitine as from previously sy=thesized labelled (f ) carnitine. Thus no racemization had taken place. TABLE I INCORPOKATKJN OF (-) [3H-CHg]~~~~~~~~~ CARNITINE
BY CARNITINE
AND OF DL-[*H-CH,]CARNITINE ACETYLTRANSPERASE
INTO (-)
PROPIONYL-
Unlabelied (--)carnitine (I ;&mole), (-)propionylcarnitine (I ,Nmole), approximately z PC of the synthesized radioactive carnitine (less than 30 nmoles), CoA (approximately 0.1 pmole) and carnitine acetyltransferase (0.2 units) were incubated in I ml of 0.1 M Tris buffer (pH 7.4) for 30 min at 37”. (previous experiments had shown that isotope equilibrium was reached in less than I5 min). The reaction was stopped by addition of z ml of ethanol. Precipitated protein was removed by centrifugation and the supernatant evaporated to dryness under a stream of air. The residue was dissolved in 0.1 ml of 50~/~ methanol and samples were chromatographed on thin-layer silica gel with chloroform-methanol-water-concentrated ammonia-formic acid (55/5o/Io/7.5/2.5, by vol.) to separate carnitine and propionylcarnitine. The distribution of radioactivity was measured by scraping bands of the silicic acid into counting vials, and after addition of scintillation solution the radioactivity was measured in a Packard liquid scintillation spectrometer.
Labelled cavxziti%e added
--
;&; --
0/Oofradiactivzty recovaVed in carnitirLe propionylcarnitine 73.3 49-g
26.7 50.2
I 2 3 4
K. R. NORUM AND J. ?&33M[ER,J. Bid. Chew., 242 ($967) 1744‘ S. LINDSTEDT AND 6. LINDSTEDT, Aykiv Kemi, 22 (1964)93, D. J. JENDES, I. HANIN AND S. I. LAMB, Anal. Chewa. 40 (x968) 125. G. HVISTENDAHL, K. UNDXEIM AND J. QREMER, OY~. Mass Spccbowz.,in the press.
Received Biochinz.
October Biophys.
7th, 1970
Ada,
218 (1970) 552-554
SHORTCOM3'lUNICATIOKS
533QO
A simple method for preparation of methyl-labellled (-1 (-)Cartinine acid metabolism.
carnitine
and its esters have proved to be useful toois in studies on fatty Labelled carnitine is commercially available in its racemic form
only. Our previous labelling method1 also gave a mixture of the two isomers, and the biologically inactive labelled (+) carnitine will interfere in many experiments. We have therefore (-)carnitine
worked from
out a simple method
the commercially
method is much simpler than a previously of labelled ( -)carnitine2. Sodium benzthiolate
nonradioactive
published
was made from thiophenol
and stored over P,O, until used3. 0.5 g (3.1 mmoles) of (-)carnitine ceutical Factory,
for the preparation
available
procedure
of methyl-labelled (-)carnitine.
This
for the preparation
(Fluka AC, Buchs, Switzerland)
hydrochloride
(gift from Otsuka Pharma-
Osaka, Japan} was dissolved in zo ml of propanol.
The solution was
saturated with dry HCl gas and refluxed for IO min. To ensure complete esterification the HCl saturation and the refluxing was repeated once. The propylcarnitine was taken to dryness on a rotating vacuum evaporator and dissolved in 50 ml of butanone. z g (IS mmoles) of sodium benzthiolate were added, and the mixture was refluxed under nitrogen
for 24 h while stirred with a magnetic
stirrer. The solvent was then
evaporated and the residue dissolved in 20 ml of 0.5 M NaOH and heated under reflux for 2 h. After acidification with HCI the excess of benzthiolate was removed as thiophenol vacuum
by extraction evaporator,
with diethyl
ether. The water was removed
and the (-)p-hydroxy-y-dimethylaminobutyric
in a rotating acid hydro-
chloride was freed from the sodium chloride by taking it up in a small volume methanol. The hydrochloride was then crystallized by addition of ethylacetate.
of
The yield of the synthesis was 67%. Thin-layer chromatography on silica gel (Kieselgel H “Merck”) with phenol-water (90/30, w/v) as solvent showed, after exposure to iodine vapour, one main spot with RF 0.5. A very weak spot corresponding to carnitine (RF 0.6) could be seen under ultraviolet light. Mass spectrometry verified the identity of the compound (Fig. I). The small residual amount of carnitine was too small to give any of the characteristic mass peaks of carnitine4. The melting point measured on a Kofler Micro Hot Stage (Reichert, Austria) was sharp at 87-88”. (The Radiochemical Center, The methylation with i3H-CH,] methyliodide Amersham,
England) was performed as follows: The ampoule containing the L3H-CH,j,
methyliodide (25 mC, approximately 0.3 mmoles) was opened after cooling of the lower end of the ampoule on solid carbon dioxide to minimize evaporation. Half a mI of cold methanol was added to the cooled ampoule followed by 0.5 mmoies of i-j/% hydroxy-y-dimethylaminobutyric acid hydrochloride and 2 mmoles of NaOH in z ml of 50% methanol. The ampoule was tightly stoppered with a rubber stopper and left at room temperature over night. The content of the ampoule was then neutralized with HCl and chromatographed twice on a column of Dowex SOW-H+ (2ooo400 mesh), approximately I cm x 50 cm, previously washed with I .5 M HCl and water. The column was eluted with 1.5M HCl. The [3H-CH,] (-) carnitine was eluted between 150 and 200 ml. The excess of unlabeled P-hydroxy-y-dimethylaminobutyric acid was eluted just in front of the carnitine. The fractions containing the radioactive carnitine were B&him.
Biophys.
Acta,
218 (1970)
55C-jjq
SKORT COMMUNICATIONS C”3,
553
?H
N-CHzCH;CH2-COOH
CH3/j
too-
1 ’
1 : &
j :
ve Fig. I. Mass spectrum of the synthesized ~-~~droxy-y”dimethyla~inobu~r~c acid hydrochloride. The analysis was performed on a Varian CH-7 instrument with a direct inlet system. Probe temperature, 100’. Electron energy, 70 eV. Hydrochloric acid is immediately split off from the molecule, and the molecular peak at m/e 147 corresponds to the free base. evaporated
to a crystalline
mass in vacua, The yield of carnitine was about 80 per
cent (calculated
on the basis of radioactivity). The radiopurity of the [3H-CH,] carnitine was checked in the thin-layer chromatographic system above. No radioimpurities were detected. The radiopurity was
also checked in an enzymic exchange reaction with (-)propionylc~tine catalyzed with carnitine acetyltransferase (E.C. 2.3.1.7) (Boehringer and Soehne, Mannheim,
Germany). Table I shows that almost exactly twice as much radioactivity was recovered in propionylcarnitine from the added [sH-Cl& (-)carnitine as from previously sy=thesized labelled (f ) carnitine. Thus no racemization had taken place. TABLE I INCORPOKATKJN OF (-) [3H-CHg]~~~~~~~~~ CARNITINE
BY CARNITINE
AND OF DL-[*H-CH,]CARNITINE ACETYLTRANSPERASE
INTO (-)
PROPIONYL-
Unlabelied (--)carnitine (I ;&mole), (-)propionylcarnitine (I ,Nmole), approximately z PC of the synthesized radioactive carnitine (less than 30 nmoles), CoA (approximately 0.1 pmole) and carnitine acetyltransferase (0.2 units) were incubated in I ml of 0.1 M Tris buffer (pH 7.4) for 30 min at 37”. (previous experiments had shown that isotope equilibrium was reached in less than I5 min). The reaction was stopped by addition of z ml of ethanol. Precipitated protein was removed by centrifugation and the supernatant evaporated to dryness under a stream of air. The residue was dissolved in 0.1 ml of 50~/~ methanol and samples were chromatographed on thin-layer silica gel with chloroform-methanol-water-concentrated ammonia-formic acid (55/5o/Io/7.5/2.5, by vol.) to separate carnitine and propionylcarnitine. The distribution of radioactivity was measured by scraping bands of the silicic acid into counting vials, and after addition of scintillation solution the radioactivity was measured in a Packard liquid scintillation spectrometer.
Labelled cavxziti%e added
--
;&; --
0/Oofradiactivzty recovaVed in carnitirLe propionylcarnitine 73.3 49-g
26.7 50.2
I 2 3 4
K. R. NORUM AND J. ?&33M[ER,J. Bid. Chew., 242 ($967) 1744‘ S. LINDSTEDT AND 6. LINDSTEDT, Aykiv Kemi, 22 (1964)93, D. J. JENDES, I. HANIN AND S. I. LAMB, Anal. Chewa. 40 (x968) 125. G. HVISTENDAHL, K. UNDXEIM AND J. QREMER, OY~. Mass Spccbowz.,in the press.
Received Biochinz.
October Biophys.
7th, 1970
Ada,
218 (1970) 552-554