Protonation sites and dissociation mechanisms of t-butylcarbamates in tandem mass spectrometric assays for newborn screening

Abstract

Structures of tert-butylcarbamate ions in the gas-phase and methanol solution were studied for simple secondary and tertiary carbamates as well as for carbamate-containing products and internal standards for lysosomal enzyme assays used in newborn screening of a α-galactosidase A deficiency (Fabry disease), mucopolysaccharidosis I (Hurler disease), and mucopolysaccharidosis II (Hunter disease). The protonation of simple t-butylcarbamates can occur at the carbonyl group, which is the preferred site in the gas phase. Protonation in methanol solution is more favorable if occurring at the carbamate nitrogen atom. The protonation of more complex t-butylcarbamates occurs at amide and carbamate carbonyl groups, and the ions are stabilized by intramolecular hydrogen bonding, which is affected by solvation. Tertiary carbamates containing aminophenol amide groups were calculated to have substantially greater gas-phase basicities than secondary carbamates containing coumarin amide groups. The main diagnostically important ion dissociation by elimination of 2-methylpropene (isobutylene, i-C(4)H(8)) and carbon dioxide is shown by experiment and theory to proceed in two steps. Energy-resolved collision-induced dissociation of the Hurler's disease enzymatic product ion, which is a coumarin-diamine linker-t-butylcarbamate conjugate (3a(+)), indicated separate energy thresholds for the loss of i-C(4)H(8) and CO(2). Computational investigation of the potential energy surface along two presumed reaction pathways indicated kinetic preference for the migration of a t-butyl hydrogen atom to the carbamate carbonyl resulting in the isobutylene loss. The consequent loss of CO(2) required further proton migrations that had to overcome energy barriers.

Figure 1

Chemical structures of neutral t-butylcarbamates.

Figure 2

Energy-resolved collision-induced dissociation of 3⁺ at nominal collision gas pressures of (top) 5.2 × 10⁻⁴ and (bottom) 1.3 ×10⁻⁴ Torr. Black circles: m/z 377; black squares: m/z 321; blue triangles: m/z 277. Inset shows the enlarged section of fragment ion relative intensities between 0.1 and 1.0 eV.

Figure 3

B3LYP/6-31+G(d,p)-optimized structures of ion tautomers of 1⁺ and 2⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.

Figure 4

B3LYP/6-31+G(d,p)-optimized structures of ion tautomers of 3⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.

Figure 5

B3LYP/6-31+G(d,p)-optimized structures of ion tautomers of 4⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.

Figure 6

B3LYP/6-31+G(d,p)-optimized structures of ion tautomers of 5⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.

Figure 7

(a) B3-MP2/6-311++G(2d,p) potential energy surface for dissociations of 3a⁺ and (b,c) RRKM kinetics of elimination of i-C₄H₈. (b) Rate constants for elimination through TS1 (k₁) and TS3 (k₃); (c) Calculated fractions of non-dissociating 3a⁺ at the indicated reaction times: Black circles: 1 ms; black squares: 100 μs; blue circles: 42 μs.

Scheme 1

Loss of i-C₄H₈ and CO₂ from protonated t-butylcarbamates.

Scheme 2

Pathway A for dissociations of ion 3a⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.

Scheme 3

Pathway B for dissociations of ion 3a⁺. The atoms are color-coded as follows: Green = C; red = O; blue = N, gray = H.