## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Homanuclear \({}^{1}\)H-\({}^{1}\)H Correlation and Total Correlation Spectroscopies  
Two-dimensional (2D) \({}^{1}\)H-\({}^{1}\)H correlation spectroscopy (COSY) is a rapid and convenient confirmatory strategy to employ for the relatively simple confirmation of directly coupled C1-H and C2-H nuclei in a range of salivary metabolites, and most especially if one or both of the resonances involved are located within crowded or overcrowded spectral regions such as the 0.80-1.50, 1.80-2.50, or 2.90-4.20 ppm sectors. For example, the technique is valuable for verifications of the identities of the amino acids glutamate and glutamine in human saliva, together with the bacterial catabolites _n_- and _iso_-butyrates. However, the \({}^{1}\)H-\({}^{1}\)H TOCSY spectroscopic technique permits the transfer of magnetization from a single or magnetically equivalent group (2 or 3) of \({}^{1}\)H nuclei covalently bonded to one specific carbon atom (C1), to one or more magnetically distinct \({}^{1}\)H nuclei within regions of two or more carbon positions further along a molecular chain, and remote from the C1 location (i.e., those at the C3, C4, C5 positions, in addition to the C2 one, etc. ), most notably for the long side-chains of selected amino acid residues such as branched-chain ones (valine, isoleucine, etc. ), the lengthy 'backbones' of salivary bacterial-derived organic acid anions such as _n_-butyrate and 5-aminovalerate, or the malodorous amine putrescine. A brief guide to these C1, C2, C3, etc., labels is provided in Figure 5. Hence, for such TOCSY spectra, much additional molecular structural information over that deduced from the facile direct 3-bond coupling of C2 \({}^{1}\)H proton(s) to the C1-borne \({}^{1}\)H nucleus, as observed with the \({}^{1}\)H-\({}^{1}\)H COSY pulse sequence, is derivable. Indeed, our previous studies have reported on applications of the TOCSY technique as a means of aiding preliminary assignments made from the 1D \({}^{1}\)H NMR profiles of HSS samples and for overcoming spectral resonance overlap issues therein [3]. From such investigations, it was confirmed that this technique was of much value for the seeking and verification of the identities of complete molecular chains of relevant salivary biomolecules, and the unambiguous assignment of their \({}^{1}\)H signals. The advent of quantitative 2D NMR spectroscopy as applied to recent metabolomics studies [128; 129] may serve to facilitate the determination of salivary concentrations of metabolites with resonances that are not readily electronically integratable in the 1D profiles acquired in view of spectral signal superimposition or crowding phenomena. Typical \({}^{1}\)H-\({}^{1}\)H TOCSY profiles of typical WMS supernatant samples are shown in Figure 6, and these display strong connectivities between the \({}^{1}\)H signals of a wide range of salivary biomolecules. Figure 5: Molecular structures of valine, isoleucine, and 5-aminovalerate. For the purpose of TOCSY spectral interpretations, C1-Cn labels for each structure are arbitrary, although for this diagram they should be read from left-to-right. For valine, the 2 x \(\gamma\)-CH\({}_{3}\), \(\beta\)-CH, and \(\alpha\)-CH protons represent the C1A and C1B, C2 and C3 ones, respectively; for isoleucine, the \(\delta\)-CH\({}_{3}\), \(\gamma\)-CH\({}_{2}\), \(\beta\)-CH\({}_{3}\)-CH(CH\({}_{3}\)), and \(\alpha\)-CH protons represent the C1, C2, C3, C4A, and C4B ones, respectively; and for 5-aminovalerate, the \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and \(\delta\)-CH\({}_{2}\) groups represent the C1, C2, C3, and C4 ones, respectively. 2D \({}^{1}\)H-\({}^{1}\)H COSY spectra reveal connectivities between a specified nucleus and its nearest neighboring ones only, for example between adjacent C1 and C2 nuclei, between C2 and C1 plus C3 nuclei in 5-aminovalerate.