Notes - Improved Synthesis of DL-Carnitine Hydrochloride

Huber J.E., Raushel J., Hirsch M., Zong G., Shi W.

Bredikhin A.A., Bredikhina Z.A., Zakharychev D.V., Samigullina A.I., Gubaidullin A.T.

Chiral 4-benzoylamino-3-hydroxybutyric acid (1) was recognized in 1930 as the first example of “anomalous racemates” (correct to say, anomalous conglomerates), that is, specific addition compounds formed by different enantiomers in unequal ratio. Through the comparative (racemic against homochiral samples) inspection of the IR spectra, single crystal X-ray diffraction, PXRD analysis, and solubility data we have found that this substance forms normal racemic compound in the solid state, and must be excluded from the very short list of anomalous conglomerates. At the same time homo-1 is dissolved in 25 times better than rac-1, and this feature belongs to another interesting and rare type, namely, “anticonglomerates”. Some of the reasons for this behavior are discussed.

Huber J.E., Raushel J.

Pandey R.K., Kumar R.

In a mixed solvent system of acetonitrile and methanol, titanium silicate molecular sieve, TS-1, having MFI topology, efficiently catalyses the epoxidation of allyl chloride to the corresponding epichlorohydrin in excellent yields using dilute hydrogen peroxide (45%) as an oxidizing agent.

Huber J.E.

Rebouche C.J.

The Caco-2 cell culture system was used as a model to investigate the mechanism of carnitine absorption in human small intestinal epithelium, and to determine if valproic acid inhibits this process in the model system. The hypotheses tested were: Carnitine is absorbed by a mechanism not involving carrier-mediated transport; and valproic acid specifically inhibits carnitine absorption. Results of the investigation were consistent with exclusively passive, paracellular absorption of carnitine across the Caco-2 cell monolayer. Saturable and structure-specific intracellular accumulation of carnitine from the apical medium was observed, but was independent of the process of absorption. At high concentration (10 mmol/L), both sodium valproate and its straight-chain analog sodium octanoate inhibited cellular accumulation of carnitine from the apical medium, but enhanced transmonolayer passage of carnitine from the apical to the basal medium. Lower concentrations of these organic acid salts (0.1 or 1 mmol/L) did not affect cellular accumulation of carnitine, but at 1 mmol/L concentration, they slightly enhanced transmonolayer flux. Paradoxically, cells cultured for 5 days in the presence of sodium valproate or sodium octanoate accumulated carnitine at a faster rate than cells cultured in the absence of these compounds. It is concluded that carnitine is absorbed across the Caco-2 monolayer by a passive, paracellular route that is not inhibited by sodium valproate.

Gross C.J., Henderson L.M., Savaiano D.A.

Uptake and metabolism of L-carnitine, D-carnitine and acetyl-L-carnitine were studied utilizing isolated guinea-pig enterocytes. Uptake of the D- and L-isomers of carnitine was temperature dependent. Uptake of L-[14C]carnitine by jejunal cells was sodium dependent since replacement by lithium, potassium or choline greatly reduced uptake. L- and D-carnitine developed intracellular to extracellular concentration gradients for total carnitine (free plus acetylated) of 2.7 and 1.4, respectively. However, acetylation of L-carnitine accounted almost entirely for the difference between uptake of L- and D-carnitine. About 60% of the intracellular label was acetyl-L-carnitine after 30 min, and the remainder was free L-carnitine. No other products were observed. D-Carnitine was not metabolized. Acetyl-L-carnitine was deacetylated during or immediately after uptake into intestinal cells and a portion of this newly formed intracellular free carnitine was apparently reacetylated. L-Carnitine and D-carnitine transport (after adjustment for metabolism and diffusion) were evaluated over a concentration range of 2-1000 microM. Km values of 6-7 microM and 5 microM, were estimated for L- and D-carnitine, respectively. Ileal-cell uptake was about half that found for jejunal cells, but the labeled intracellular acetylcarnitine-to-carnitine ratios were similar for both cell populations. Carnitine transport by guinea-pig enterocytes demonstrate characteristics of a carrier-mediated process since it was inhibited by D-carnitine and trimethylaminobutyrate, as well as being temperature and concentration dependent. The process appears to be facilitated diffusion rather than active transport since L-carnitine did not develop a significant concentration gradient, and was unaffected by ouabain or actinomycin A.

Broquist H.P.

This chapter discusses the assay procedure, purification, and properties of the S-adenosylmethionine: ɛ-N-lysine methyltransferase. The initial extract for the assay is prepared from Neurospora crassa 33933. The methyllysine transferase assay measures the formation of radioactive methylated lysine derivatives following incubation of appropriate lysine substrates with [methyl-3H]AdoMet. The purification procedure is also tabulated in the chapter. The purification scheme yields a highly purified protein that is homogeneous following column chromatography, polyacrylamide gel electrophoresis, and ultracentrifugation in which lysine methyltransferase activity for Reactions A, B, and C remains relatively constant. S-Adenosylmethionine: ɛ- N-L-lysine methyltransferase as prepared herein from N. crassa is devoid of protein methylase III activity; but N. crassa contains a cytochrome c-specific proteinlysine methyltransferase. The molecular weight of the lysine methyltransferase is estimated to be 22,000 based on sedimentation equilibrium and molecular filtration data and 24,000 based on amino acid analysis assuming two methionine residues per mole protein. The protein appears to be devoid of subunit structure based on observations from sedimentation equilibrium analysis of the protein in 6 M guanindine hydrochloride and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both carnitine and trimethyllysine repress synthesis of lysine methyltransferase in growing cultures of N. crassa.

Rebouche C.J.

This chapter discusses the synthesis of carnitine precursors and related compounds. L-Carnitine contains a quaternary nitrogen atom which is formed biosynthetically early in the pathway. Thus several intermediates in the pathway are quaternary amines which can readily be synthesized from the commercially available parent primary amines. These intermediates include ɛ-N-trimethyl-L-lysine (from α-N-acetyl-L-lysine), γ-N-trimethylaminobutyraldehyde (from 4-aminobutyraldehyde diethyl acetal), and γ-butyrobetaine (from γ- aminobutyric acid). Other compounds of interest which are synthesized by this general method include δ-N-trimethylaminovaleric acid (from δ-aminovaleric acid) and ɛ-N-trimethylaminocaproic acid (from ɛ-aminocaproic acid). Another carnitine precursor, β-hydroxy-ɛ-N-trimethyl-L-lysine, is prepared by condensation of γ-N-trimethylaminobutyraldehyde with glycine in the presence of potassium carbonate and copper sulfate, yielding a diastereomeric mixture. Carnitine is synthesized, as a racemic mixture, by several related methods. For example, DL-carnitine is prepared by reaction of epichlorhydrin with NaCN followed by condensation with trimethylamine, and subsequent hydrolysis. Neurospora crassa (but apparently not in mammals) ɛ-N-methyl-L-lysine and ɛ-N-dimethyl-L-lysine are direct precursors of L-carnitine. The latter compound is prepared by reductive methylation of α-N-acetyl- L-lysine with formaldehyde and H2/Pd catalyst. α-Keto-ɛ-N-trimethylaminocaproic acid, a catabolic product of ɛ-N-trimethyl- L-lysine, is synthesized enzymatically by reaction of ɛ-N-trimethyl- L-lysine with commercial L-amino acid oxidase. The chapter describes the representative synthetic procedures for ɛ-N- Trimethyl-L-lysine, ɛ-N-[methyl-14C]DimethyI-L-lysine, β-Hydroxy-ɛ-N-[methyl-3H]trimethyl-L-lysine, γ-[1-14C]Butyrobetaine, and L-[methyl-3H] Carnitine.

Comber R.N., Brouillette W.J.

Rebouche C.J., Mack D.L.

Dropsy E.P., Klibanov A.M.

An enzymatic method for the preparative resolution of racemic carnitine (whose L-isomer and its acyl-derivatives have numerous therapeutical applications) has been developed. It is based on our finding that electriceel acetylcholinesterase hydrolyzes the D- but not the L-isomer of acetylcarnitine. (Another cholinesterase tested, horse serum butyrylcholinesterase, is also stereospecific and hydrolyzes only the L-isomer of butyrylcarnitine.) Acetylcholinesterase, covalently attached to alumina, was employed for the resolution of D,L-carnitine; the latter was first chemically acetylated, then stereoselectively hydrolyzed with the immobilized enzyme, and finally the acetyl-L-carnitine and D-carnitine produced were separated by ion-exchange chromatography. Gram quantities of D,L-carnitine were thereby resolved.

Gross C.J., Henderson L.M.

The process by which L- and D-carnitine are absorbed was investigated using the live rat and the isolated vascularly perfused intestine. A lumenal dose of 2-6 nmol in the perfused intestine resulted in less than 5% transport of either isomer to the perfusate in 30 min. The L-isomer was taken up by the intestinal tissue about twice as rapidly as the D-isomer by both the perfused intestine (52.8% and 21.6%, respectively) and the live animal (80% and 50%, respectively) in 30 min. After 1 h 90% of the L-carnitine had accumulated in the intestinal tissue and was released to the circulation over the next several hours. Accumulation of D-carnitine reached a maximum of 80% in 2 h and release to the circulations was similar to that of L-carnitine. Uptake of both L-[14C]carnitine and acetyl-L-[14C]carnitine was more rapid in the upper jejunal segment than in other portions of the small intestine. Acetylation occurred in all segments, resulting in nearly 50% conversion to this derivative in 5 min. Increasing the dose of L-carnitine reduced the percent acetylation. The uptake of both isomers was a saturable process and high concentrations of D-carnitine, acetyl-L-carnitine and trimethylaminobutyrate inhibited L-carnitine uptake. In the live animal after 5 h, the distribution of isotope from L-[14C]carnitine and D-[3H]carnitine differed primarily in the muscle where 29.5% of the L-carnitine and 5.3% of the D-carnitine was found and in the urine where 2.9% of the L-carnitine and 7.1% of the D-carnitine was found. The renal threshold for L-carnitine was 80 microM and for D-carnitine 30 microM, in the isolated perfused kidney. Approx. 40% of the L-carnitine but none of the D-carnitine excreted in the urine was acetylated. L-Carnitine and D-carnitine competed for tubular reabsorption.

Wood G.W., Collacott R.J.

Intramolecular methyl transfer has been observed previously in pyrolysis electron impact mass spectra of complex ammonioalkanecarboxylates. We present here field desorption mass spectra results of more simple N,N,N-trimethylammoniocarboxylate hydrochloride salts and their N,N,N-perdeuterotrimethylammonium analogs in which we observe methyl transfer. We demonstrate that mechanisms for this and other fragmentation and rearrangement processes are dependent on anode heating current. Addition of the protonating agent p-toluenesulfonic acid suppresses most ions except the protonated molecular ion.

Daveluy A., Parvin R., Pande S.V.

Radioactive γ-butyrobetaine was prepared by quaternization of γ-aminobutyric acid with tritiated methyl iodide under conditions giving high yields with respect to both the above precursors. Part of the product was passed through a column of ion-retardation resin and gave radioactive γ-butyrobetaine of good purity. The remainder was converted to (−)-carnitine stoichiometrically by employing a 50–60% ammonium sulfate fraction of rat liver supernatant as the source of γ-butyrobetaine hydroxylase (EC 1.14.11.1). Successive column chromatographies on a cation exchanger and ion-retardation resins then gave radioactive (−)-carnitine of good purity in high yield.