Terphenyl-Based Bak BH3 alpha-helical proteomimetics as low-molecular-weight antagonists of Bcl-xL

We describe a general method for the mimicry of one face of an alpha-helix based on a terphenyl scaffold that spatially projects functionality in a manner similar to that of two turns of an alpha-helix. The synthetic scaffold reduces the flexibility and molecular weight of the mimicked protein secondary structure. We have applied this design to the development of antagonists of the alpha-helix binding protein Bcl-x(L). Using a sequential synthetic strategy, we have prepared a library of terphenyl derivatives to mimic the helical region of the Bak BH3 domain that binds Bcl-x(L). Fluorescence polarization assays were carried out to evaluate the ability of terphenyl derivatives to displace the Bcl-x(L)-bound Bak peptide. Terphenyl 14 exhibited good in vitro affinity with a K(i) value of 0.114 muM. These terphenyl derivatives were more selective at disrupting the Bcl-x(L)/Bak over the HDM2/p53 interaction, which involves binding of the N-terminal alpha-helix of p53 to HDM2. Structural studies using NMR spectroscopy and computer-aided docking simulations suggested that the helix binding area on the surface of Bcl-x(L) is the target for the synthetic ligands. Treatment of human embryonic kidney 293 (HEK293) cells with terphenyl derivatives resulted in the disruption of the binding of Bcl-x(L) to Bax in intact cells.     

Rational design of new class of BH3-mimetics as inhibitors of the Bcl-xL protein

The Bcl-2 family of proteins plays an important role in the intrinsic pathway of cell apoptosis. Overexpression of pro-survival members of this family of proteins is often associated with the development of many types of cancer and confers resistance against conventional therapeutic treatments. Accordingly, antagonism of its protective function has emerged as an encouraging anticancer strategy. In the present work, we use a pharmacophore for describing interaction between the BH3 domain of different pro-apoptotic members and the pro-survival protein Bcl-x(L) in order to identify new lead compounds. In the strategy followed in the present work, the pharmacophore was derived from molecular dynamics studies of different Bcl-x(L)/BH3 complexes. This pharmacophore was later used as query for 3D database screening. Hits obtained from the search were computationally assessed, and a subset proposed for in vitro testing. Two of the 15 compounds assayed were found able to disrupt the Bcl-x(L)/Bak(BH3) complex with IC(50) values in the lower micromolar range. Finally, docking studies were performed to explore the binding mode of these compounds to Bcl-x(L) for further modifications.     

Relaxation of the rigid backbone of an oligoamide-foldamer-based α-helix mimetic: identification of potent Bcl-xL inhibitors

By conducting a structure-activity relationship study of the backbone of a series of oligoamide-foldamer-based α-helix mimetics of the Bak BH3 helix, we have identified especially potent inhibitors of Bcl-x(L). The most potent compound has a K(i) value of 94 nM in vitro, and single-digit micromolar IC(50) values against the proliferation of several Bcl-x(L)-overexpressing cancer cell lines.     

Evaluation of diverse α/β-backbone patterns for functional α-helix mimicry: analogues of the Bim BH3 domain

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x(L) and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β(3)-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β(3)-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β(3)-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-x(L), affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.     

Molecular Determinants of Bim(BH3) Peptide Binding to Pro-Survival Proteins

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.     

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.     

Molecular dynamics study of peptide segments of the BH3 domain of the proapoptotic proteins Bak, Bax, Bid and Hrk bound to the Bcl-xL and Bcl-2 proteins

Overexpression of Bcl-2 and Bcl-xL proteins, both inhibitors of apoptosis or programmed cell death, is related to the generation and development of several types of cancer as well as to an elevated resistance to chemotherapeutic treatments. Given that synthetic peptide fragments of the BH3 domain are capable to bind to both proteins and induce apoptosis in cell-free systems and HeLa cells, small molecule non-peptide mimics of these peptides can be considered as a new therapeutic strategy for the treatment of diseases associated to a deficient apoptosis or resistant to the treatments with chemotherapeutic drugs. This strategy is supported by experimental evidences about the death of transformed cells and sensibilization of tumoral cells by the inhibition of the antiapoptotic proteins Bcl-2 and Bcl-xL. In the current work, these proteins complexed with X(16BH3), where X designates the proapoptotic proteins Bak, Bax, Bid and Hrk, have been modeled in order to establish a pharmacophoric hypothesis that must be present in any ligand capable of binding with the antiapoptotic proteins Bcl-2 and Bcl-xL. The pharmacophore is also used to explain the structural features of a set of new small molecule inhibitors of these antiapoptotic proteins.     

Reactions of 26-iodopseudodiosgenin and 26-iodopseudodiosgenone with various nucleophiles and pharmacological activities of the products

26-Iodopseudodiosgenin (8) and 26-iodopseudodiosgenone (9) were reacted with various nucleophiles (KSCN, KOCN, NaCN, NaN(3) and various amines) to give pseudodiosgenin derivatives (4, 12, 16-20, 26) and pseudodiosgenone derivatives (5, 13, 21-25, 27), respectively. The reactions of 8 and 9 with KOCN gave the elimination products (10) and (11), respectively. The reaction of 9 with NaCN gave 5alpha,26- (14) and 5beta,26-dicyanocholestan-3-one (15). The reaction of 8 with NaN3 gave triazepine derivative (30), while that of 9 gave 26-azidopseudodiosgenone (31). Compound 31 was converted into triazepine derivative (32) by heating at 120 degrees C. The cytotoxicity of the pseudodiosgenins and pseudodiosgenones on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Pseudodiosgenins 2, 4, 12 and 30 showed strong cytotoxic activity (IC50 values: 2.6+/-0.3-6.7+/-1.4 microM), as did pseudodiosgenones 3, 5, 11, 13, 21-25 and 27 (IC50 values: 1.3+/-0.3-6.4+/-0.3 microM) toward HCT 116 cells. Pseudodiosgenins 12, 16 and 30 (IC50 values: 1.2+/-0.7-2.2+/-0.6 microM) and pseudodiosgenones 22, 23, 25 and 27 (IC50 values: 0.6+/-0.1-2.5+/-0.3 microM) were highly cytotoxic to Hep G2 cells. Compounds 3 and 27 showed efficient antibacterial activity (MIC: 15.6, 10.4 microg/ml) and (MIC: 7.8, 15.6 microg/ml) against Bacillus subtilis and Staphylococcus aureus, respectively.     

NMR solution structure of a photoswitchable apoptosis activating Bak peptide bound to Bcl-xL

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x(L). This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states.     

The study of the interaction of a model alpha-helical peptide with lipid bilayers and monolayers

We studied the interaction of the alpha-helical peptide acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air-water interface. The interaction of L(24) with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K(c)=2.4+/-0.6 microM(-1). The peptide L(24) an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E(radial). The decrease of E(radial) with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure-area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the alpha-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide-lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.     

Terpenoids with a new skeleton and novel triterpenoids with anti-inflammatory effects from Garcinia subelliptica

Three novel phloroglucinol derivatives, garcinielliptones F (1), H (3), and I (4), and two novel terpenoids, garcinielliptones G (2) and J (5), with a new skeleton have been isolated from the seeds of Garcinia subelliptica. Their structures, including relative configurations, were elucidated by spectroscopic methods and computer-generated molecular modeling. Compound 1 showed potent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils that had been stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). This effect was concentration-dependent with IC(50) values of 26.9+/-2.6 and 20.0+/-1.3 microM, respectively. Compound 1 also showed a potent concentration-dependent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with fMLP/CB, with an IC(50) value of 17.0+/-0.9 microM. Compound 4 showed a potent inhibitory effect on NO production in culture media of N9 cells in response to lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) in a concentration-dependent manner with an IC(50) value of 7.4+/-0.2 microM.     

Mechanistic study of the SmI2/H2O/amine-mediated reduction of alkyl halides: amine base strength (pKBH+) dependent rate

The kinetics of the SmI(2)/H(2)O/amine-mediated reduction of 1-chlorodecane has been studied in detail. The rate of reaction is first order in amine and 1-chlorodecane, second order in SmI(2), and zero order in H(2)O. Initial rate studies of more than 20 different amines show a correlation between the base strength (pK(BH+) of the amine and the logarithm of the observed initial rate, in agreement with Bronsted catalysis rate law. To obtain the activation parameters, the rate constant for the reduction was determined at different temperatures (0 to +40 degrees C, DeltaH++ = 32.4 +/- 0.8 kJ mol(-1), DeltaS++ = -148 +/- 1 J K(-1) mol(-1), and DeltaG++(298K) = 76.4 +/- 1.2 kJ mol(-1)). Additionally, the (13)C kinetic isotope effects (KIE) were determined for the reduction of 1-iododecane and 1-bromodecane. Primary (13)C KIEs (k(12)/k(13), 20 degrees C) of 1.037 +/- 0.007 and 1.062 +/- 0.015, respectively, were determined for these reductions. This shows that cleavage of the carbon-halide bond occurs in the rate-determining step. A mechanism of the SmI(2)/H(2)O/amine-mediated reduction of alkyl halides is proposed on the basis of these results.     

Novel and anti-inflammatory constituents of Garcinia subelliptica

Four novel phloroglucinol derivatives, garcinielliptones A (1), B (2), C (3), D (4), a novel triterpenoid, garcinielliptone E (5), and three known compounds were isolated from the seeds of Garcinia subelliptica. The structures, including relative configurations, were elucidated by means of spectroscopic data. Known compounds garsubellin A (6) and garcinielliptin oxide (7) showed potent inhibitory effects on the release of beta-glucuronidase, and beta-glucuronidase and histamine, respectively, from peritoneal mast cells stimulated with compound 48/80 in a concentration-dependent manner with IC(50) values of 15.6+/-2.5, and 18.2+/-3.6 and 20.0+/-2.7 microM, respectively. Compound 7 showed potent inhibitory effects on the release of beta-glucuronidase and lysozyme from neutrophils stimulated with formyl-Met-Leu-Phe(fMLP)/cytochalasin B (CB) in a concentration-dependent manner with IC(50) values of 15.7+/-3.0 and 23.9+/-3.2 microM, respectively. Compound 7 also showed potent inhibitory effect on superoxide formation from neutrophils stimulated with fMLP/CB also in a concentration-dependent manner with an IC(50) value of 17.9+/-1.5 microM.     

Theoretical study on the mechanism of the gas-phase reaction of diborane(3) anion with carbon disulfide

The complex potential energy surface of the gas-phase reaction of HB(H)BH- with CS2 to give three low-lying products [B2H3S]- + CS, [BH2CS]- + HBS, and [BH3CS] + BS-, involving nine [B2H3CS2]- isomers and 12 transition states, has been investigated at the CCSD(T)/6-311++G(d,p)/B3LYP/6-311++G(d,p) level. Our calculations are in harmony with the recent experimental and theoretical results, and reveal some new bonding and kinetic features of this reaction system. Our theoretical results may help the further identification of the products [BH2CS]- + HBS and [BH3CS] + BS- and may provide useful information on the chemical behaviors of other electron-deficient boron hydride anions.     

Practical, catalytic, asymmetric synthesis of beta-lactones via a sequential ketene dimerization/hydrogenation process: inhibitors of the thioesterase domain of fatty acid synthase

The recent finding that the FDA-approved antiobesity agent orlistat (tetrahydrolipstatin, Xenical) is a potent inhibitor of the thioesterase domain of fatty acid synthase (FAS) led us to develop a concise and practical asymmetric route to pseudosymmetric 3,4-dialkyl-cis-beta-lactones. The well-documented up-regulation of FAS in cancer cells makes this enzyme complex an interesting therapeutic target for cancer. The described route to 3,4-dialkyl-beta-lactones is based on a two-step process involving Calter's catalytic, asymmetric ketene dimerization of acid chlorides followed by a facial-selective hydrogenation leading to cis-substituted-beta-lactones. Importantly, the ketene dimer intermediates were found to be stable to flash chromatography, enabling opportunities for subsequent transformations of these optically active, reactive intermediates. Subsequent alpha-epimerization and alpha-alkylation or acylation led to trans-beta-lactones and beta-lactones bearing alpha-quaternary carbons, respectively. Several of the ketene dimers and beta-lactones displayed antagonistic activity (apparent Ki in the low micromolar range) in competition with a fluorogenic substrate toward a recombinant form of the thioesterase domain of fatty acid synthase. The best antagonist, a simple phenyl-substituted cis-beta-lactone 3d, displayed an apparent Ki (2.5 +/- 0.5 microM) of only approximately 10-fold lower than that of orlistat (0.28 +/- 0.06 microM). In addition, mechanistic studies of the ketene dimerization process by ReactionView infrared spectroscopy support previous findings that ketene formation is rate determining.     

Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation

It has recently been found that Pluronics (block copolymers of ethylene oxide, EO, and propylene oxide, PO) favor the permeability and accumulation of anthracycline antibiotics, for example doxorubicin (Dox), in tumor cells. In an effort to understand these results, the interaction of EO(2)/PO(32)/EO(2) (Pluronic L61) with unilamellar egg yolk vesicles (80-100 nm in diameter) was examined. A partition coefficient K(p)=[Pl](membrane)/[Pl](water)=45 was determined. This corresponds to adsorption of about 20 polymer molecules to the surface of each vesicle in a 20 microM polymer solution. Despite this rather weak adsorption, Pluronic has a substantial effect upon the transmembrane permeation rate of Dox and upon the phospholipid flip-flop rate within the bilayers. Thus, the Dox permeation rate increases threefold and the flip-flop rate increases sixfold in 20 microM Pluronic. The two rates increase linearly with the amount of adsorbed polymer. The obvious ability of Pluronics to increase the mobility of membrane components may have important biomedical consequences.     

Molecular dynamics simulations of pro-apoptotic BH3 peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein Bcl-X<Subscript>L</Subscript>

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the intrinsic pathway of apoptosis. Interactions between specific anti- and pro-apoptotic Bcl-2 proteins determine the fate of a cell. Anti-apoptotic Bcl-2 proteins have been shown to be over-expressed in certain cancers and they are attractive targets for developing anti-cancer drugs. Peptides from the BH3 region of pro-apoptotic proteins have been shown to interact with anti-apoptotic Bcl-2 proteins and induce biological activity similar to that observed in parent proteins. However, the specificity of BH3 peptides derived from different pro-apoptotic proteins differ for different anti-apoptotic Bcl-2 proteins. In this study, we have investigated the relationship between the stable helical nature of BH3 peptides and their affinities to Bcl-X_L, an anti-apoptotic Bcl-2 protein. We have carried out molecular dynamics simulations of six BH3 peptides derived from Bak, Bad and Bim pro-apoptotic proteins for a period of 50 ns each in aqueous medium. Due to the amphipathic nature of BH3 peptides, the hydrophobic residues on the hydrophobic face tend to cluster together in all BH3 peptides. While this process resulted in a complete loss of helical structure in 16-mer Bak and 16-mer Bad wild type peptides, stabilizing interactions in the hydrophilic face of the BH3 peptides and capping interactions helped to maintain partial helical character in 16-mer Bad mutant and 16-mer Bim peptides. The latter two 16-mer peptides exhibit higher affinity for Bcl-X_L. Similarly the longer BH3 peptides, 25-mer Bad and 33-mer Bim, also resulted in smaller and stable helical fragments and their helical conformation is stabilized by interactions between residues in the solvent-exposed hydrophilic half of the peptide. The stable nature of helical segment in a BH3 peptide can be directly correlated to its binding affinity and the helical region encompassed the highly conserved Leu residue. We propose that upon approaching the hydrophobic groove of anti-apoptotic proteins, a longer helix will be induced in high affinity BH3 peptides by extending the smaller stable helical segments around the conserved Leu residue in both N- and C-terminal regions. The results reported in this study will have implications in developing peptide-based inhibitors for anti-apoptotic Bcl-2 proteins.     

Discovery of novel inhibitors of anti-apoptotic Bcl-2 proteins derived from Bim BH3 domain

The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2 family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5 (K_D=9.48 nmol/L for Bcl-2) and SM-6 (K_D=0.08 nmol/L for Bcl-x_L), which exhibited improved binding affinity compared with the lead Bim (K_D=16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-x_L, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.     

Quantitative trace-level speciation of arsenite and arsenate in drinking water by ion chromatography

We describe an improved method for the determination of inorganic arsenic in drinking water. The method is based on comprehensive optimization of the anion-exchange ion chromatographic (IC) separation of arsenite and arsenate with post-column generation and detection of the arsenate-molybdate heteropoly acid (AMHPA) complex ion. The arsenite capacity factor was improved from 0.081 to 0.13 by using a mobile phase (2.0 mL min(-1)) composed of 2.5 mM Na2CO3 and 0.91 mM NaHCO3 (pH 10.5). A post-column photo-oxidation reactor (2.5 m x 0.7 mm) was optimized (0.37 microM potassium persulfate at 0.50 mL min(-1)) such that arsenite was converted to arsenate with 99.8 +/- 4.2% efficiency. Multi-variate optimization of the complexation reaction conditions yielded the following levels: 1.3 mM ammonium molybdate, 7.7 mM ascorbic acid, 0.48 M nitric acid, 0.17 mM potassium antimony tartrate, and 1.0% (v/v) glycerol. A long-path length flow cell (Teflon AF, 100-cm) was used to measure the absorption of the AMHPA complex (818 +/- 2 nm). Figures of merit for arsenite/arsenate include: limit of detection (1.6/0.40 microg L(-1)): standard error in absorbance (5.1 x 10(-3)/3.5 x 10(-3)); and sensitivity (2.9 x 10(-3)/2.2 x 10(-3) absorbance units per ppb). Successful application of the method to fortified surface and ground waters (100 microL samples) is also described.