# Non-Enzymatic Formation of \(N\)-acetylated Amino Acid Conjugates in Urine
###### Abstract  
Unknown \(N\)-acylated amino acid (\(N\)-AAA) conjugates have been detected in maple syrup urine disease (MSUD) and other inborn errors of metabolism (IEMs). This study aimed to elucidate the mechanism behind the formation of urinary \(N\)-AAA conjugates. Liquid-liquid extraction was employed to determine the enantiomeric composition of \(N\)-AAA conjugates, followed by liberation of conjugated amino acids through acid hydrolysis. Gas chromatography-mass spectrometry (GC-MS) was used to separate amino acid enantiomers. In vitro experiments were conducted to test the non-enzymatic formation of \(N\)-AAA conjugates from 2-keto acids and ammonia, with molecular modelling used to assess possible reaction mechanisms. Adequate amounts of \(N\)-AAA conjugates were obtained via organic acid extraction without concurrent extraction of native amino acids, and hydrolysis was complete without significant racemisation. GC-MS analysis successfully distinguished amino acid enantiomers, with some limitations observed for L-isoleucine and D-alloisoleucine. Furthermore, investigation of racemic \(N\)-AAA conjugates from an MSUD case confirmed its non-enzymatic origin. These findings highlight the value of employing chiral strategy and molecular modelling to investigate the origin of unknown constituents in biological samples. Additionally, these conjugates warrant further investigation as potential factors contributing to MSUD and other IEMs. \(N\)-acylated amino acid conjugates; non-enzymatic reactions; maple syrup urine disease; inborn errors of metabolism; racemic resolution; density functional theory (DFT) +
Footnote †: journal: Accepted: 29 August 2023  
MSUD (OMIM: 248600) is an autosomal recessive disorder characterised by impaired metabolism of branched-chain amino acids (BCAAs) due to various mutations in the branched-chain \(\alpha\)-keto acid dehydrogenase complex (BCKDH) [8]. The condition exhibits a range of phenotypes, from mild thiamine-responsive forms to severe classical MSUD. Metabolic profiling of MSUD typically reveals elevated levels of BCAAs [9; 10], particularly alloisoleucine, along with their corresponding 2-keto acids (substrates of BCKDH), such as 2-keto-3-methylvaleric acid, 2-keto-isoavaleric acid, and 2-keto-isoocarpic acid, as well as their 2-hydroxy acid analogues [10; 11]. Additionally, elevated levels of ammonia, \(N\)-acetyl BCAA (\(N\)-Ac BCAA), \(N\)-lactyl BCAA conjugates, and BCAA conjugation of 2-hydroxyisovaleric acid have also been observed in some MSUD cases [6; 7; 12; 13]. [PERSON] [7] conducted research on the induced metabolism of MSUD and identified several metabolites (Figure 1). These include trace amounts of (1) decarboxylated amines (2) 2-hydroxy BCAA conjugates (3) \(N\)-lactyl conjugates, (4) \(N\)-acetyl conjugates, and (5) hydantoin-like metabolites. Additionally, [PERSON] [7] identified three variant cases of MSUD that presented with \(N\)-AAA conjugates derived from the BCAAs valine (Val), leucine (Leu) and isoleucine (Ile), including \(N\)-isobutyryl-, \(N\)-isovaleryl, and \(N\)-2-methylbutyryl conjugates. These findings have been subsequently confirmed [6]. Of these metabolites, \(N\)-AAA conjugates are carboxamides formed through the conjugation of an acyl group to an amino acid via a peptide bond [14]. \(N\)-AAA conjugates play a role as intermediates in both primary and alternative metabolism, participating in urea cycle regulation [15], endo- and exogenous \(N\)-acetylated protein degradation [16], and the myelination of neurons by providing the precursor for myelin lipid biosynthesis, acetate, upon hydrolysis [17]. Furthermore, during Gly-mediated biotransformation, glycine \(N\)-acyltransferase (GLYAT) (Enzyme Commission number [EC]: 2.3.1.13) catalyses the conjugation of Gly to various acyl-coenzyme A (CoA) substrates. These biotransformation products are frequently observed in inherited metabolic diseases.