Human carnitine biosynthesis proceeds via (2S,3S)-3-hydroxy-N ε-trimethyllysine† †Electronic supplementary information (ESI) available: Synthesis procedures, assay conditions, NMR assignments and spectra, and MS analyses. See DOI: 10.1039/c6cc08381a Click here for additional data file.

The stereochemistry of human trimethyllysine hydroxylase was determined to be (2S,3S)-3-hydroxy-N ε-trimethyllysine by comparison to asymmetrically synthesised (2S,3R)-3-hydroxy-N ε-trimethyllysine.


Amino acid analysis
Samples were prepared as described for NMR analyses and subsequently freeze-dried. Freeze-dried samples were resuspended in borate buffer pH 9.0 and derivatised by 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC) in acetonitrile at 25°C according to the AccQ-Tag TM Ultra Derivatisation Protocol (Waters, USA). LC-MS analyses were performed as described 2 using a Waters Acquity ultra performance liquid chromatography system coupled to a Xevo ® G2-S QTof mass spectrometer equipped with an electrospray ionisation source (Waters, USA). Gradient conditions for separation were carried out as described in the AccQ-Tag TM Ultra Derivatisation Protocol (Waters, USA). Extracted ion chromatograms were smoothed to the mean (number of smooths, 2; smooth window channels, 3), and total ion current chromatograms were further baseline subtracted (polynomial order, 1; below curve %, 40; tolerance, 0.01).

Figure S1A 1 H NMR spectrum of the Mosher's esters resulting from the reaction of formamide (9) with (S)-Mosher's acid.
The signals used in the stereochemical assignment are enlarged. As yet, separation of the mixture of stereoisomers has been unsuccessful by HPLC or crystallisation, hence the Mosher's method was used.

Synthesis General Considerations
All chemicals, reagents, and solvents were obtained from Sigma-Aldrich (Dorset, UK) and used without further purification. HPLC grade solvents were used for reactions, chromatography, and work-ups. Aqueous solutions were made using de-ionized water. Thin layer chromatography (TLC) was carried out using Merck (Darmstadt, Germany) silica gel 60 F254 TLC plates. TLC visualisation was carried out under UV light and stained with one of three stains; ninhydrin, potassium permanganate, or anisaldehyde. Chromatographic purifications were carried out using a Biotage ® (Uppsala, Sweden) Isolera One or Biotage ® SP4 flash purification system, using Biotage ® pre-packed SNAP columns. Reactions were monitored using an Agilent (Cheshire, UK) 1200 series, 6120 quadrupole LC-MS system, and a Merck Chromolith ® Performance RP-18 HPLC column. Deuterated solvents were obtained from Sigma-Aldrich, and 1D and 2D NMR spectra were obtained on a Bruker AVIII HD 400 (400 MHz), Bruker AVII 500 (500 MHz) with a 13 C cryoprobe, and/or Bruker AVIII 700 (700 MHz) with an inverse TCI cryoprobe. All signals are described in δ ppm with multiplets being denoted as singlet, doublet, triplet, quartet, and multiplet using the abbreviations s, d, t, q, and m, respectively. Chemical shifts were referenced CH2Cl2 (30 ml). The aqueous layer was washed with CH2Cl2 (2 x 30 ml); the organic layers were combined, dried over MgSO4, then filtered. After evaporation of the solvent in vacuo, the crude reaction mixture was loaded onto a silica column and purified using flash column chromatography with an isocratic gradient (50:50 EtOAc/cyclohexane). After collecting the appropriate fractions, the solvent was removed in vacuo to yield dibenzyl-4,4-diethoxybutan-1amine as a thin, colourless oil (21.8 g, 63.9 mmol, 89%).  ii)

N-[(9R)-10,11-dihydrocinchonan-9-yl]-2-(diphenylphosphino)benzamide
The procedures of Sladojevich et al. 5 and Cassani et al. 6 were used. Cinchonine 7 (3.0 g, 10.2 mmol) and triphenylphosphine (3.2 g, 12.2 mmol) were suspended in dry THF (51 ml). The mixture was cooled in an ice bath to 0°C, after which diisopropyl azodicarboxylate (2.41 ml, 12.2 mmol) was added. Diphenyl phosphoryl azide (2.64 ml, 12.2 mmol) was then dissolved in dry THF (21 ml); the resultant solution was added dropwise to the reaction mixture. Upon removing the ice bath, the reaction mixture was allowed to warm to room temperature and stirred overnight. The resultant solution was heated for 2 h at 50°C, after which the volatiles were removed in vacuo. The resultant crude mixture was dissolved in CH2Cl2 (150 ml), and 4M hydrochloric acid was added. The aqueous phase was removed, and solid NaHCO3 was then added to the aqueous phase until the pH was ~10 (litmus paper). The basic aqueous phase was then extracted with CH2Cl2 (3 x 100 ml), dried over MgSO4, the CH2Cl2 was then removed in vacuo to give the desired azide in 94% crude yield.
The crude azide was then dissolved in MeOH (50 ml), and 10% Pd/C (750 mg) was added. The reaction flask was placed under a H2 atmosphere by fitting the vessel with a septum, H2 balloon, and syringe. The flask was evacuated and filled with H2 three times, then stirred at room temperature at 1 atm overnight. The reaction mixture was then filtered through a short pad of Celite ® ; volatiles were removed in vacuo to yield the crude amine in apparent quantitative yield as a yellow oil, which was used without further purification.

HN
200-400 (protonated form) resin that had been washed with H2O. Crude (11) was then dissolved in a minimal amount of H2O and loaded onto the column, followed by washing with at least 3 column volumes of water. The product was eluted with 3% (v/v) NH4OH in MeOH to give purified (11) as a clear oil (Fig. S6A-B