# Non-Enzymatic Formation of \(N\)-acetylated Amino Acid Conjugates in Urine
## 3 Results
### Molecular Modelling to Investigate the Non-Enzymatic Formation of \(N\)-AAA Conjugates  
Molecular modelling was used to investigate a newly proposed reaction mechanism for the non-enzymatic formation of \(N\)-AAA conjugates (Figure 7). Due to cost constraints, only the reaction mechanism for the simplest structural analogue, \(N\)-Ac-Ala formed from pyruvate, was calculated. R1 and R2 in Figures 6 and 7 thus represent only a methyl group, but the principles could also be applied to other 2-keto acids. The hydronium ion was selected to represent Bronsted acid. A TS was found for the protonation of pyruvate.  
Figure 5: Separation of racemic amino acid via GC–MS. The compound list is as follows: (a) L-Ala, (b) D-Ala, (c) L-Val, (d) D-Val, (e) the di-tBDMS ester of Ala, (f) L-Nva, (g) D-Nva, (h) L-Leu, (i) D-Leu, (j) L-alle, (k) D-alle and L-Ile, (l) D-Ile and (m–q) the di-tBDMS esters of Val, Nva, Leu, alle, and Ile, respectively. The y-axis (abundance) is scaled to 100% of (p), and the x-axis (retention time in minutes) is cropped to between 15.5 and 25.3 min to show only the section of interest.  
Figure 6: Branched-chain amino acid enantiomer composition of the case study (pink peaks) and a control overlay (red line). As indicated in the image, blue peaks are extracted ion chromatograms (from the case study) of the amino acid enantiomers.  
The results in Figure 8 indicated a slightly higher \(\rm E_{a}\) compared to experimental \(\rm E_{a}\) values reported in the literature [33; 34; 35; 36; 37; 38; 39; 40; 41; 42]. However, considering the energy requirements of enzymatic reactions, the \(\rm E_{a}\) obtained for the non-enzymatic formation of \(N\)-Ac-Ala fell within an acceptable range for an in vivo non-enzymatic reaction. It is worth noting that \(N\)-AAA conjugates may also form in vitro under higher temperature conditions during sample preparation. A higher \(\rm E_{a}\) slows down the reaction rate but does not prevent the reaction from occurring [43]. The trace amounts of \(N\)-AAA conjugates observed in the non-enzymatic in vitro experiment align with the slower reaction rate. [PERSON] et al. [31] also reported slow hydrolysis during \(N\)-oxyl-Gly formation, which could have contributed to the low yields observed.  
Figure 8: Energy profile for the non-enzymatic formation of \(N\)-Ac-Ala from pyruvate and ammonia. To simplify the figure, each alpha-numeral represents the most significant reaction intermediate in a balanced pool of reaction intermediates. The orange lines represent the reaction path of the second pyruvate molecule, starting with a transition state (TS) during protonation and ending with the condensation reaction with #G2 to form #K1. These values are intentionally slightly offset to show the blue line underneath.  
Figure 7: Newly proposed reaction mechanism for the non-enzymatic formation of \(N\)-AAA conjugates, constructed from molecular modelling observations. The red, light blue, and dark blue groups correlate to the net reaction shown. The reaction steps are numbered in green, while reaction intermediates are labelled with grey letters preceded by a hash symbol. The symbol ‘HX’ indicates the Brensted acid, \(\rm H_{3}0^{+}\). Where coloured arrows are used (pink and orange), the reaction may proceed via two separate steps.