## 3 Results and Discussion
### Mechanism of Antibacterial Activity  
[PERSON] _et al_. [13, 15] investigated the MIC to bacteria and fungi of 20 compounds based on the \(\beta\)-nitrostyrene scaffold to bacteria and fungi and found that they were broadly antimicrobial, with greater activity overall against Gram-positive bacteria and fungi. Whilst it was established from SAR studies that their lipophilic and the nucleophilic addition of thiol groups to the nitroolefin are the key physicochemical features, their antimicrobial mechanisms are not well understood.  
Various \(\beta\)-nitrostyrene derivatives are used as tyrosine kinase inhibitors with applications in potent antiplatelet activity [16, 17]. Furthermore, pronounced substituent effects were observed. Specific mono- and di-substitutions on the aromatic ring of \(\beta\)-nitrostyrene tended to increase the anti-platelet activity and decrease the cytotoxic activity [17]. 3,4-Methylenedioxy-\(\beta\)-nitrostyrene (MNS) blocks the assembly of NLRP3 inflammasome by the inhibition of NLRP3 ATPase activity [18, 19]. The nitrovinyl group is essential for the inhibitory activity of MNS.  
The enzyme 4-oxalocrotonate tautomerase (4-OT), which contains an amino-terminal proline (Pro1), promiscuously catalyses the asymmetric Michael-type additions of aldehydes and ketone donors to \(\beta\)-nitrostyrene to yield \(\gamma\)-nitroaldehydes, with 89% enantiomeric excess [20]. Significantly, this Michael addition reaction has been designed and performed _in vivo_ in resting _E. coli_ BL21 whole  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Strain** & **A-1** & **I** & **D** & **F** \\ \hline _Staphylococcus aureus_ ATCC 29213 & 32 & 32 & 32 & 32 \\ _Bacillus subtilis_ ATCC 6633 & 16 & 16 & 16 & 32 \\ _Enterococcus faecalis_ ATCC 29212 & 64 & 128 & 128 & 256 \\ _Escherichia coli_ ATCC 25922 & 512 & 256 & 256 & 128 \\ _Candida albicans_ ATCC 10231 & 32 & 32 & 32 & 32 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Assay 3: MICs of halogenated derivatives **I**, **D** and **F** compared against **A-1**.  
cells expressing 4-OT. This implies that the \(\beta\)-nitrostyrene used in the context as a reactant/substrate had no antibacterial effect on the host _E. coli_ BL21 cells [21].  
[PERSON] and [PERSON] [22] proposed that their nitroalkenyl aromatic compounds act as tyrosine mimetics, thereby inhibiting protein tyrosine phosphatases and interfering with downstream cell signaling in microorganisms. They proposed that \(\beta\)-nitrostyrene acts as a reversible inhibitor of this enzyme by interaction and formation of a covalent complex via nucleophilic attack of PTP1B cysteine on the \(\beta\)-nitro group at the catalytic site. However, sulphur ylides, like thiol nucleophiles, are found to undergo Michael addition to a wide range of \(\beta\)-nitrostyrenes [23, 24, 25, 26]. The known reactivity of \(\beta\)-nitrostyrenes with sulfhydryl groups is consistent with these observations. A more attractive mechanistic proposal is that the conjugated nitroalkene is a good Michael acceptor of nucleophiles owing to the non-covalent binding capability of the nitro group with PTP1B, as shown in Scheme 2 below.  
**Scheme 2.** Modified Michael addition mechanism: _E_-\(\beta\)-nitrostyrene-PTP1B binding complex and inhibition mechanism.  
Furthermore, in contrast to what was reported by [PERSON] and [PERSON], research published by [PERSON] _et al._[27] and supported from our work [12, 13] found that the antibacterial activity was substantially improved by the addition of a \(\beta\)-methyl group, as shown by the MIC comparisons in Table 3. The \(\beta\)-methyl group would be expected to enhance the above mechanism [12].  
Table 3 shows the enhanced antibacterial activity (MIC values) of Compound **I** by the introduction of the \(\beta\)-methyl group to 4-F-nitrostyrene [12]. The values are the geometric means of two determinations.