Synthesis and Gas Chromatography/Mass Spectrometry Analysis of Stereoisomers of 2-Hydroxy-3-methylpentanoic Acid

Orval A. Mamer

Introduction

2-Hydroxy-3-methylpentanoic acid (HMPA) is a familiar metabolite of isoleucine commonly found in normal human urine, plasma, and other fluids. In disorders affecting branched-chain amino acid metabolism, such as branched-chain ketoaciduria, HMPA is frequently elevated in these fluids, sometimes by two orders of magnitude or more. HMPA is formed by the reduction of 2-keto-(3S)-methylpentanoic acid [(3S)-KMPA], which is the transamination product of L-isoleucine in the degradative pathway of that amino acid.1,2 The chiral center of KMPA is spontaneously racemized in solution at high pH through keto–enol tautomerism1 (Fig. 1).

Having two chiral centers, HMPA exists in four isomeric forms (Fig. 2): (2R)-hydroxy-(3R)-methylpentanoic acid [(2R,3R)-HMPA], (2S)-hydroxy- (3S)-methylpentanoic acid [(2S,3S)-HMPA], (2R)-hydroxy-(3S)-methylpentanoic acid [(2R,3S)-HMPA], and (2S)-hydroxy-(3R)-methylpentanoic acid [(2S,3R)-HMPA]. Each of these may be synthesized in pure form by conventional diazotization of the isomer of 2-amino-3-methylpentanoic acid having the corresponding steric configuration and trivial or common names of D-isoleucine, L-isoleucine, D-alloisoleucine, and L-alloisoleucine, respectively. All four of these amino acids are commercially available in relatively pure form.

Synthesis

General

All solutions are aqueous unless stated otherwise, reactions are carried out in a fume hood to reduce exposure to hazardous vapors, organic chemicals are from Sigma-Aldrich Canada (Oakville, ON, Canada), and inorganic materials are purchased from local suppliers and are used as received.

Gas Chromatography/Mass Spectrometry Analysis

Analyses are run on a Hewlett-Packard (Palo Alto, CA) model 5988A gas chromatograph/mass spectrometer (GC/MS) equipped with a 5890A gas chromatograph fitted with a 30 m × 0.25 mm (i.d.) fused silica column having a 0.25-μm dimethylsilicone coating. The helium flow rate is set at 1 ml/min. The injector port is set at 250° and configured for splitless injection of 1-μ1 aliquots of the trimethylsilyl (TMS) derivative mixtures. After a 1-min hold at 70° for septum purging, the column is programmed at 5°/min to 200°. The interface and ion source temperatures are set at 270 and 200°, respectively. Ionization is by electron impact at 70 eV, and scanning is over the range 100 to 400 Da.

Racemic 2-Hydroxy-3-methylpentanoic Acid

Fully racemic HMPA may be conveniently synthesized by the sodium borohydride reduction of KMPA racemized in solution at high pH. The sodium salt of KMPA (150 mg, 1 mmol; Sigma-Aldrich) is dissolved in 5 ml of water, a few drops of 2 N sodium hydroxide are added to raise the pH to pH > 12, and 50 mg of sodium borohydride is added with stirring until dissolution is complete. The resulting solution is held in a water bath at 60° for 2 hr, cooled in an ice–water bath, and then cautiously acidified to pH < 2 with 2 N hydrochloric acid. (Caution: Vigorous evolution of hydrogen gas occurs.) The acidified solution is saturated with sodium chloride and extracted three times with volumes of diethyl ether equal to the aqueous phase. The ether extracts are combined, treated with 200 mg of anhydrous sodium sulfate to remove water, and evaporated to an oil (approximately 100-mg yield) under a warm, dry nitrogen stream. The oil may be crystallized from an ethyl acetate–petroleum ether solvent pair if desired, although this is not necessary for further work.

A few micrograms of the product oil is converted to the TMS derivative in a mixture of 25-μl volumes of anhydrous pyridine and N,O-bis(trimethylsily)trifluoroacetamide (BSTFA) in a capped autoinjector vial, heated at 60° for 20 min, and analyzed by GC/MS as described above. The relevant portion of the resulting chromatogram is reproduced in Fig. 3A.

Two unequal peaks are found for racemic HMPA made from fully racemized KMPA. Although it is not possible to separate by chromatography enantiomers in achiral stationary phases, diastereomers that are not mirror images of each other are frequently easily separable, as they have different chemical and physical properties. Sodium borohydride reduction is expected to produce less of a racemic (2S,3R)- and (2R,3S)-HMPA enantiomeric pair than of a (2S,3S)-HMPA, (2R,3R)-HMPA pair.1 This is borne out in the resulting chromatogram. The mass spectra of the two peaks are reproduced in Fig. 4A and B, and cannot be distinguished reliably.

L-2-Hydroxy-3-methylpentanoic Acid

Sodium L-2-hydroxy-3-methylvalerate (50 mg, 0.3 mmol; Sigma) is dissolved in 2 ml of water, which is then acidified, extracted, isolated, and derivatized as described above. The resulting mixture when analyzed by GC/MS produces a single peak (Fig. 3B) having the same retention time as the second eluting peak in Fig. 3A and the mass spectrum shown in Fig. 4C. Again, the mass spectrum of this isomer is indistinguishable from those of the racemate. Because (3S)-KMPA is probably not racemized after its biosynthesis from L-isoleucine, reduction to HMPA, presumably by a lactate dehydrogenase (LDH) isozyme, will leave the S-chirality at the 3-carbon unaffected. LDH reductions of 2-keto acids produce S-chirality at the 2-carbon. This assignment of “L-HMPA” as (2S,3S)-HMPA is consistent then with its elution with the retention time of peak 2 in the racemate (Fig. 3).

2-Hydroxy-3-methylpentanoic Acid Stereoisomers

HMPA isomers may be conveniently synthesized in pure form by diazotization of the corresponding amino acid in dilute perchloric acid. Replacement of the amino group by a hydroxyl group is known to occur with retention of configuration about the 2-carbon atom, owing to anchiomeric participation of the neighboring carboxyl function1 (Fig. 5).

L-Isoleucine (100 mg, 0.75 mmol is dissolved in 50 ml of cold (0°) 0.2 N perchloric acid. To this is added a cold solution (0°) of sodium nitrite (1.4 g, 20 mmol) in 20 ml of water with rapid stirring. Stirring is continued while the mixture is allowed to warm to room temperature and the evolution of gas subsides (approximately 0.5 hr). The resulting solution is raised to the boiling point for a few minutes, cooled to room temperature, and saturated with sodium chloride. Ether extracts are made and dried as described above, and the product is isolated similarly to yield 75 mg of (2S,3S)-HMPA as an oil. This product is also analyzed as the TMS derivative by GC/MS.

The three other stereoisomers, (2R,3R)-HMPA, (2R,3S)-HMPA, and (2S,3R)-HMPA, are synthesized in a similar manner from D-isoleucine, D-alloisoleucine, and L-alloisoleucine, respectively.

All four of the stereoisomers have mass spectra indistinguishable from each other.1 Relative retention times are: (2S,3S)- and (2R,3R)-HMPA coelute after (2R,3S)- and (2S,3R)-HMPA, which also coelute.

Resolution of Four Stereoisomers in Racemic Mixture

The four stereoisomers may be resolved by introducing a third chiral center (Fig. 6). For this purpose, 20 mg (0.2 mmol) of (S)-(+)-2-methylbutanoic acid is dissolved in 10 ml of toluene, 75 μl of thionyl chloride (1 mmol) is added, and the resulting solution is heated at reflux for 2 hr. While still warm, the toluene solution is reduced in volume under reduced pressure to approximately 3 ml and then added to 10 mg of racemic HMPA produced as described above and dissolved in 15 ml of toluene. A few drops of pyridine are added, and the resulting mixture is heated at reflux...