Methods

Computational.Gas-phase geometries of polynitroaromatic neutrals and radical anions were fully optimized at the restricted Hartree-Fock (RHF) and half-electron restricted Hartree-Fock (HE) levels, respectively, using the semiempirical Austin Model 1 (16) (AM1) Hamiltonian. The radical anions showed excessive spin contamination at the unrestricted Hartree-Fock (UHF) level, so this formalism was not further explored. Aqueous solvation effects were accounted for by reoptimization using the AM1-Solvation Model 2 (SM2) methodology. (17) The Fock operator of the SM2 model includes electronic and electric polarization effects, as well as localized non-electrostatic effects associated with interactions between the solute and the solvent. (17-19) Relaxation of the wavefunction in the presence of the solvent reaction field can significantly alter the molecular electronic and geometric structure, (20,21) especially in aromatic systems. (22-25)

Electrostatic potentials were calculated on the 0.002 isodensity surface from partial atomic point charges, i.e.,

              V(r) = Sum_over_atoms_k (qk / | r - rk | )                 (1)

Charges qk were calculated using the Class IV Charge Model 1A (CM1A) method, which maps AM1 Mulliken charges to high quality partial atomic charges that closely reproduce both experimental dipole moments and also partial atomic charges derived from fitting to high quality electrostatic potential surfaces. (26) Solvated potential surfaces were derived from CM1 mappings of AM1-SM2 partial charges (which differed considerably from AM1 gas-phase Mulliken charges).

To assess the reliability of the AM1 Hamiltonian for certain conformational issues, additional studies were carried out on the radical anions of nitrobenzene and 2- nitrotoluene at the restricted open-shell Hartree Fock (ROHF) level. Correlation effects were also included to second order in perturbation theory (ROMP2) in single-point calculations at the ROHF geometries. The 6- 31G* (27-29) and cc- pVDZ (30) basis sets were used. When exploring specific conformational coordinates, all other degrees of freedom were fully optimized.

Semiempirical calculations were carried out with a locally modified version 4.5 of AMSOL (31). Ab initio calculations were carried out with the Gaussian92/DFT program suite. (32) Visualization of the electrostatic potential surfaces was accomplished using version 3.0 of the SPARTAN program. (33)

Experimental:

Materials. Standard grade chemicals were purchased from a variety of comercial sources and used without further purification. Stock solutions (0.01M) of the aromatic compounds were prepared in MeOH and routinely monitored for degradation. Stock solutions of 0.01 M jugalone (JUG) and 1.0 M Na2S were prepared in MeOH and ultrapure (18 megaohm/cm) water, respectively, and stored in serum bottles sealed with Bellco butyl rubber stoppers.

Reduction Reactions. Reductions of substituted-nitroaromatics by Na2S were performed in anaerobic culture tubes sealed with Hungate butyl rubber stoppers. All reactions were carried out in 50 mM phosphate buffer at pH 7.0 with the exception of 2,4-dinitrophenol where a 5 mM phosphate solution was buffered to pH 2.5 to ensure predominance of the conjugate acid (i.e., the phenol). A 10 mL volume of buffer solution was de-oxygenated by bubbling a H2/N2 (5%/95%) gas mixture through the solution for 20 min. Appropriate amounts of the Na2S stock solution and jugalone were added to the buffer and allowed to equilibrate for at least 12 hours. Reactions were initiated by spiking with 50 micro-L of the nitroaromatic stock solution for an initial concentration of 50 micro-M. Aliquots were removed via syringe and analyzed by HPLC for the parent compound and the reduction products. (See Table 2)

The reductions of four compounds were also performed in sediment-water slurries. Sediment slurries were collected from a eutrophic pond in the vicinity of Athens, GA and were wet-sieved through a 1-mm mesh. While stirring, aliquots of the slurry were added to serum bottles and then crimp sealed with butyl rubber stoppers. The aqueous volume of the slurry was estimated and an appropriate volume of nitroaromatic stock solution was added to achieve an approximate 50 micro-M initial concentration. At selected sampling times, a representative sample aliquot was withdrawn and centrifuged at 14,000 rpm.. The supernatant was analyzed for the analytes by HPLC. (See Table 3)

Analytical Methods. HPLC ananlyses were performed on a Gilson 305 gradient HPLC equipped with a variable wavelength UV detector (Applied Biosystems). Analyses were run on C18 (phenomenex ODS-30) or polymer (Hamilton PRP1) reverse phase columns typically under isocratic conditions (ACN and H2O). For analysis of 2,4-dinitrophenol and phenolate, a mixture of ACN and 5 mM pH 2.5 phosphate buffer was used.

Product yields were calculated by dividing the moles of product formed by the moles of parent compound reacted. For the majority of the reactions, product yields are reported for approximately 50% of the parent compound reacted. Despite the fact that for most compounds there was no further reduction to the diamino product, this arbitrary 50%-cutoff was chosen to ensure that the initial products were not lost to further reduction. In a few cases formation of the primary reduction products was very low due to either slow kinetics or competing reaction pathways. Product yields were then reported for the best yields obtained; this was typically from earlier reaction times when loss of the parent compound from competing reaction pathways was minimal.

Table 2. 1-Substituted-2,4-dinitrobenzene compounds, experimental conditions, and the distribution and total yield of ortho and para reduction products. 

Compound                  [HS-] [Juga] %ortho   %para  %yield

                         micro M micro M



2-amino-4,6-dinitrotoluene   500         100      0      16

bromo-2,4-dinitrobenzene    1000         100      0       6
     
2-bromo-3,5-dinitroaniline  5000         100      0      89

                            1000         100      0      90

chloro-2,4-dinitrobenzene    100          86     14      14

                             500    5     89     11      39

2,4-dinitroaniline          4500   10                    NRb

2,4-dinitroanisole          5000         100      0      86

2,4-dinitrophenolatec       5000   10     98      2      99

                            5000                         NRb

2,4-dinitrophenolate        5000         100      0      52

                            5000    5     99      1      90

2,4-dinitrotoluene          5000          12     88      62

                             500   25     80     92      80

2,4,6-trinitrotoluene       1000           0    100      74

                             500   20      0    100       9
a Jugalone (5-hydroxy-1,4-naphthoqhinone) b No reaction under the experimental conditions. c Reaction performed at pH 2.5. All other reactions performed at pH 7.0

Table 3. Distribution of ortho and para regio isomer products from the reduction of 1-substituted-2,4-dinitrobenzene compounds in reducing Cherokee Park sediments. 

Compound                    %ortho     %para     %yield



bromo-2,4-dinitrobenzene       +          ?      



2-bromo-3,5-dinitroaniline     99         1         75



chloro-2,4-dinitrobenzene      65         35        27



2,4,6-trinitrotoluene          1          99        73

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