O benzylhydroxylamine

Sahaquine 5 was synthesized in four reaction steps (Fig. 1) adapted from the synthetic approach of Zhang et al^{45 (link)}. Details of these steps are provided in the Supplementary Information. The first step included amide bond formation between mono-methyl glutarate (1, Sigma-Aldrich, St. Louis, MO, USA) and primaquine (Sigma-Aldrich, St. Louis, MO, USA), with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, Alfa Aesar, Thermo Fisher, Kandel, Germany) as a coupling agent and N,N-diisopropylethylamine (DIEA, Alfa Aesar, Thermo Fisher, Kandel, Germany) as a base. The prepared product 2 was further hydrolyzed with lithium hydroxide (Sigma-Aldrich, St. Louis, MO, USA) and gave carboxylic acid 3. In the next step, 3 was coupled with O-benzylhydroxylamine (Sigma-Aldrich, St. Louis, MO, USA) in the presence of HATU/DIEA and yielded O-benzylhydroxamic acid 4, which was deprotected by catalytic hydrogenation and gave the target compound 5 (sahaquine). All reactions proceeded at room temperature.

Using frozen, fasting samples, a 100 μL aliquot of human plasma was spiked with a 10 μL mixture of amino acid or acylcarnitine internal standard mixture and treated with bleach (to inactivate HIV particles) to a final concentration of 10% bleach. Samples were treated with 800 μL of ice-cold methanol and centrifuged to pellet precipitated protein. Amino acids were derivatized by drying down 100 μL of methanolic extract and reconstituting the sample with 80 μL of borate buffer (Waters Corp., Milford, MA) and 20 μL of MassTrak AAA Reagent Powder dissolved in MassTrak AAA Reagent Diluent (Waters Corp., Milford, MA) in a 96-well plate. Acylcarnitines were derivatized by reconstituting the dried methanolic extract in 100 μL of 0.2 M O-benzylhydroxylamine (Sigma–Aldrich, St. Louis, MO) and 10 μL of 2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma–Aldrich, St. Louis, MO).