Modeling predicts facile release of nitrite but not nitric oxide from the thionitrate CH3SNO2 with relevance to nitroglycerin bioactivation

Abstract

Nitroglycerin is a potent vasodilator in clinical use since the late 1800s. It functions as a prodrug that is bioactivated by formation of an enzyme-based thionitrate, E-Cys-NO₂. This intermediate reportedly decomposes to release NO and NO₂^- but their relative yields remain controversial. Hence, we determined barriers for NO and NO₂^- production from the model thionitrate, CH₃SNO₂, using comprehensive high-level quantum chemistry calculations [CCSD(T)//MP2/aug-cc-pVTZ]. We find that the sulfenyl nitrite, CH₃SONO, readily releases NO on (S)O-N bond homolysis but CH₃SONO formation from CH₃SNO₂ either by S-NO₂ bond homolysis or concerted rearrangement faces prohibitively high barriers (ΔH_calc/ΔH^‡_calc > 42 kcal/mol). Dramatically lower barriers (ΔH^‡_calc ~ 17-21 kcal/mol) control NO₂^- release from CH₃SNO₂ by gas-phase hydrolysis or nucleophilic attack by OH^- or CH₃S^- on the sulfur atom within the C-S-NO₂ molecular plane. Moreover, attack by either anion along the S-NO₂ bond results in barrierless NO₂^- release (ΔH^‡_calc ~ 0 kcal/mol) since a σ-hole (i.e., area of positive electrostatic potential) extends from this bond. Consistent with our high-level calculations, ALDH2 and GAPDH, enzymes implicated in nitroglycerin bioactivation via an E-Cys-NO₂ intermediate, catalyze mainly or exclusively NO₂^- release from the prodrug.

Keywords: Bioactivation; Nitric oxide; Nitrite; Nitroglycerin; Thionitrate; Vasodilation.

Fig. 1

Proposed pathways for thionitrate (RSNO₂) decomposition with NO₂⁻ or NO release.^,,– Hydrolysis by H₂O or OH⁻ (activated water) or thiolysis by R–S⁻ (low-molecular-weight thiolate or protein-based cysteinate) releases NO₂⁻. Homolysis of the S–N bond produces highly reactive radicals that can recombine to give RSONO (sulfenyl nitrite), which undergoes (S)O–N bond homolysis to release ^•NO and RS^•O radicals. Recombination of these radicals can additionally produce RS(O)NO (sulfinyl nitrite). Alternatively, concerted rearrangement of RSNO₂ directly generates RSONO. In this work we examine these pathways for R = CH₃.

Fig. 2

Gas-phase thionitrate molecular geometries optimized with MP2/aug-cc-pVTZ. The S–N bond length (green font; Ả), H/C–S–N angle (blue font; degrees, °) and the partial charge on the sulfur atom (black font) are marked on each structure. Note that in each structure the H/C–S–NO₂ atoms are in the same plane (molecular plane), and the barrier for rotation around the S–N bond is ~ 8 kcal/mol (see text). Nucleophiles in the molecular plane can attack the sulfur atom along the S–N bond or at angles roughly perpendicular to this bond either trans (top attack) or cis (bottom attack) to the R-group. Electrostatic repulsion with the sulfur lone pairs inhibits attack by nucleophiles perpendicular to the molecular plane (Fig. S1, Supporting Information). O1 and O2 designate the oxygen atoms trans and cis to the H atom and R group, respectively.

Fig. 3

Modeling CH₃SNO₂ homolysis and concerted rearrangement to CH₃SONO. Geometries are optimized with MP2/aug-cc-pVTZ in the gas phase and the vertical axis represents MP2/aug-cc-pVTZ electronic energies relative to those of the reactants. Note that values of ΔH_calc (or ΔH^‡_calc for the transition state, TS) calculated with CCSD(T)//MP2/aug-cc-pVTZ in the gas phase and in PCM-water (in parentheses) are given on the graph. The bond lengths and bond angles marked on the structures are in Ả and degrees (°), respectively.

Fig. 4

Modeling CH₃SNO₂ hydrolysis by one water molecule. Attack by H₂O in the C-S-NO₂ molecular plane (a) trans (top) and (b) cis (bottom) to the methyl group (see Fig. 2). The transition-state structures TS2-t and TS2-b are shown to the left of the graphs. Geometries are optimized with MP2/aug-cc-pVTZ in the gas phase and the vertical axis represents MP2/aug-cc-pVTZ electronic energies relative to the reactants. Note that the ΔH_calc (ΔH^‡_calc for TS) values listed in the graphs are those calculated with CCSD(T)//MP2/aug-cc-pVTZ in the gas phase and in PCM-water (in parentheses). Energies/enthalpies in kcal/mol, and the bond lengths and bond angles marked on the structures are in Ả and degrees (°), respectively.

Fig. 5

Transition states in modeling CH₃SNO₂ hydrolysis by two and three water molecules in the gas phase. Water attack by two and three water molecules in the C–S–NO₂ molecular plane trans (top) to the methyl group (see Fig. 2) gives TS3-t and TS4-t, respectively. The ΔH^‡_calc values (kcal/mol) indicated under the structures are calculated with CCSD(T)//MP2/aug-cc-pVTZ. The bond lengths and bond angles marked on the structures are in Ả and degrees (°), respectively.

Fig. 6

Modeling the reaction of CH₃SNO₂ with OH⁻ in PCM-water. Attack by the OH⁻ nucleophile in the C–S–NO₂ molecular plane (a) trans (top) and (b) cis (bottom) to the methyl group (see Fig. 2) to give transition states TS5-t and TS5-b, respectively. (c) OH⁻ attacks along the S–N bond with the O_(OH-₎–S distance (Å) decreasing in the intermediates from 3.8 in A5-I to 2.3 in the pseudo transition state B5-I at − 11.9 kcal/mol and 1.6 in C5-I but no transition state was found. Geometries are optimized with MP2/aug-cc-pVTZ(PCM-water), and the vertical axis represents MP2/aug-cc-pVTZ(PCM-water) electronic energies (kcal/mol) relative to the reactants. Note that ΔH_calc (or ΔH^‡_calc for TS) values calculated with CCSD(T)//MP2/aug-cc-pVTZ(PCM-water) are listed in parenthesis on the graphs in all three panels. The bond lengths and bond angles marked on the structures are in Ả and degrees (°), respectively.

Fig. 7

Modeling the reaction of CH₃SNO₂ with CH₃S⁻. Attack by the CH₃S⁻ nucleophile in the C–S–NO₂ molecular plane (a) trans (top) to the methyl group (see Fig. 2) to give transition state TS6-t. (b) CH₃S⁻ attacks along the S–N bond with the S–S distance (Å) decreasing in the intermediates from 3.5 in A6-I to 2.7 in the pseudo transition state B6-I at − 7.9 kcal/mol and 2.0 in C6-I (Table S6) but no transition state was found. Geometries are optimized with MP2/aug-cc-pVTZ(PCM-water), and the vertical axis represents MP2/aug-cc-pVTZ(PCM-water) electronic energies (kcal/mol) with respect to the reactants. Note that the ΔH_calc (ΔH^‡_calc for TS) values calculated with CCSD(T)//MP2/aug-cc-pVTZ(PCM-water) are listed in parenthesis on the graph in both panels. The bond lengths and bond angles marked on the structures are in Ả and degrees (°), respectively.

Fig. 8

Active site of GAPDH and ALDH2. (a) Catalytic Cys152 is converted to Cys–NO₂ on reaction of GAPDH with GTN. Glu317 is positioned to activate a water molecule for nucleophilic attack on Cys–NO₂ but Cys156 is too far away to attack Cys152. (b) Catalytic Cys302 is converted to Cys–NO₂ on reaction of ALDH2 with GTN. Either Cys301 or Cys303 is positioned to attack Cys–NO₂ and Glu268 also activates a water molecule for possible hydrolysis. Distances (black font) in Å.