Synthesis and Photolysis Properties of a New Chloroquine Photoaffinity Probe.

A new chloroquine-derived photoaffinity probe has been prepared by a convergent synthesis from derivative of 4,7-dichloroquinoline and N1,N1-diethyl-N4-methylpentane. The features of this probe are a unique 3-azido photolabel, the pyridine ring of the quinoline, and the presence of a secondary amine at the 4-position of the quinoline. These features, particularly the 4-amino methylation, prevent triazole formation through combination of the 3-azide and the 4-amine. This undergoes facile cleavage with exposure to a medium-pressure mercury lamp with a 254 nm excitation wavelength. Trapping of the nitrene byproduct is accomplished with its reaction with N-phenylmaleimide as its cycloazidation product. The structure of a ring-opened DBU amine has been structurally characterized.

Coordination Chemistry of the Parent Dithiocarbamate H2NCS2-: Organometallic Chemistry and Tris-Chelates of Group 9 Metals.

Two tris-chelate complexes of cobalt and rhodium and two complexes of Ru(II) of dithiocarbamate, [S2CNH2]-, were synthesized. The complexes were spectroscopically characterized by IR, NMR, UV-vis, and MS and structurally characterized by X-ray diffraction. The structural features of the rhodium complex were compared to those of other tris-chelate Rh(III) dithiocarbamate complexes and are characterized by a change in the ground-state geometry in comparison to expected octahedral tris-chelate complexes. This was confirmed both experimentally by X-ray diffraction and theoretically using DFT calculations. The inversion barriers of Rh(Bz2dtc)3, Ir(Bz2dtc)3, and Rh(Et2dtc)3 were determined using VT-NMR in DMSO. These barriers were found to be surprisingly low for heavy group 9 elements of d6 tris-chelate complexes: values of 16.7, 17.1, and 16.4 kcal/mol were calculated, respectively. By comparing structural features, we are able to determine that the activation barrier for the inversion of stereochemistry of Rh(H2dtc)3 must have a similarly low value. A modified version of the Bailar twist involving an intermediate with C3h geometry was proposed as the mechanism of inversion.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

2,3,5-Metallotriazoles: Amphoteric Mesoionic Chelates from Nitrosoguanidines.

Soluble nitrosoguanidine- and N-methylnitrosoguanidine-based metallotriazole complexes of ruthenium(II) monocarbonyls have been prepared and characterized. Both nitrosoguanidines prove to be strong chelates with the formally π-accepting nitroso nitrogen binding cis to carbon monoxide and a π-donating amide trans to the CO. The resulting ensemble consists of ruthenium examples of 1-metallo-2,3,5-triazoles. The ruthenium coordination sphere is completed by anions, either H-, Cl-, or Ph-, trans to the nitroso group as well as two mutually trans PPh3 groups. The π-donating amide group is formally sp2 hybridized with a planar nitrogen to give a strongly bound five-membered chelating anion. Together, these results illustrate the remarkable potential for the nitrosoguanidinates as a family of new metal chelates.

Quantification of local zinc and tungsten deposits in bone with LA-ICP-MS using novel hydroxyapatite-collagen calibration standards.

Tungsten has recently emerged as a potential toxicant and is known to heterogeneously deposit in bone as reactive polytungstates. Zinc, which accumulates in regions of bone remodeling, also has a heterogenous distribution in bone. Determining the local concentrations of these metals will provide valuable information about their mechanisms of uptake and action. A series of bone (BN), 7:3 hydroxyapatite:collagen (HC), and hydroxyapatite (HA) standards were spiked with tungsten and zinc and used as calibration standards for laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis of bone tissue. The analytical performance of these standards was studied and validated at different step sizes using NIST SRM 1486 Bone Meal. The effect of matrix-matched calibration was assessed by comparing the calibration with BN and HC standards, which incorporate both inorganic and organic components of bone, to that of HA standards. HC standards were found to be more homogenous (RSD < 10%) and provide a linear calibration with better accuracy (R2 > 0.994) compared to other standards. The limits of detection for HC at a 15 µm step size were determined to be 0.24 and 0.012 µg g-1 for zinc and tungsten, respectively. Using this approach, we quantitatively measured zinc and tungsten deposits in the femoral bone of a mouse exposed to 15 µg mL-1 tungsten for four weeks. Localized concentrations of zinc (942 µg g-1) and tungsten (15.7 µg g-1) at selected regions of enrichment were substantially higher than indicated by bulk measurements of these metals.

Topical combination of meldonium and N-acetyl cysteine relieves allodynia in rat models of CRPS-1 and peripheral neuropathic pain by enhancing NO-mediated tissue oxygenation.

Local microvascular dysfunction and consequent tissue ischemia/hypoxia contribute to the symptoms of complex regional pain syndrome (CRPS) and peripheral neuropathic pain. As nitric oxide (NO) is a key regulator of microvascular blood flow, compounds that increase it are potentially therapeutic for these pain conditions. This led us to hypothesize that the topical administration of drugs that modulate local tissue NO levels can alleviate the pain of CRPS and peripheral neuropathic pain. We investigated the anti-allodynic effect of a combination of two NO-modulating drugs: meldonium and N-acetylcysteine (NAC). An equimolar topical formulation of the two drugs was tested on chronic post-ischemic pain (CPIP), a rat model of CRPS, as well as chronic constriction injury (CCI) of the sciatic nerve and chemotherapy-induced painful neuropathy (CIPN), rat models of peripheral neuropathic pain. Topical meldonium-NAC produced significant anti-allodynia in CPIP, CCI, and CIPN rats. Moreover repeated application of topical meldonium-NAC produced an increase in the duration of anti-allodynia in the CPIP and CCI rats. While pre-treatment with an NO synthase inhibitor attenuated the anti-allodynic effects of meldonium-NAC, 30-min hyperbaric oxygen treatment combined with a non-effective dose of meldonium-NAC produced significant anti-allodynic effects in CPIP rats. Both experiments implicated NO in the drug combination's anti-allodynic effects. To ascertain the role played by changes in local tissue NO, we performed a quantification of plantar muscle NO in CPIP rats after hind paw topical treatment with meldonium-NAC and revealed significantly increased plantar muscle NO levels in drug-treated rats. The drug combination also reversed the reduction in tissue oxygenation normally observed in CPIP hind paws. In addition to introducing a novel topical treatment for mechanical allodynia in CRPS and peripheral neuropathic pain, this work showcases the analgesic potential of locally targeting microvascular dysfunction and tissue ischemia/hypoxia in these conditions, with emphasis on the role of NO.

Addressing K/L-edge overlap in elemental analysis from micro-X-ray fluorescence: bioimaging of tungsten and zinc in bone tissue using synchrotron radiation and laser ablation inductively coupled plasma mass spectrometry.

Synchrotron radiation micro-X-ray fluorescence (SR-µXRF) is a powerful elemental mapping technique that has been used to map tungsten and zinc distribution in bone tissue. However, the heterogeneity of the bone samples along with overlap of the tungsten L-edge with the zinc K-edge signals complicates SR-µXRF data analysis, introduces minor artefacts into the resulting element maps, and decreases image sensitivity and resolution. To confirm and more carefully delineate these SR-µXRF results, we have employed laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to untangle the problem created by the K/L-edge overlap of the tungsten/zinc pair. While the overall elemental distribution results are consistent between the two techniques, LA-ICP-MS provides significantly higher sensitivity and image resolution compared with SR-µXRF measurements in bone. These improvements reveal tissue-specific distribution patterns of tungsten and zinc in bone, not observed using SR-µXRF. We conclude that probing elemental distribution in bone is best achieved using LA-ICP-MS, though SR-µXRF retains the advantage of being a non-destructive method with the capability of being paired with X-ray techniques, which determine speciation in situ. Since tungsten is an emerging contaminant recently found to accumulate in bone, accurately determining its distribution and speciation in situ is essential for directing toxicological studies and informing treatment regimes. Graphical abstract Tungsten and zinc localization and uptake in mouse femurs were imaged by synchrotron radiation, left, and by laser ablation ICP-MS, right. The increased resolution of the LA-ICP-MS technique resolves the problem of the overlap in tungsten's L-edge and zinc's K-edge.

Hydrating the Bispropionate Notch in Malaria Pigment: A New Structural Motif in the Iron(III)(deuteroporphyrin) Dimer.

Treating deuterohemin, chloro(deuteroporphyrinato)iron(III), with a non-coordinating base in DMSO/methanol allows for the isolation of [(deuteroporphyrinato)iron(III)]2 , deuterohematin anhydride (DHA), an analogue of malaria pigment, the natural product of heme detoxification by malaria. The structure of DHA obtained from this solvent system has been solved by X-ray powder diffraction analysis and displays many similarities, yet important structural differences, to malaria pigment. Most notably, a water molecule of solvation occupies a notch created by the propionate side chains and stabilizes a markedly bent propionate ligand coordinated with a long Fe-O bond, and a carboxylate cluster associated with water molecules is generated. Together, these features account for its increased solubility and more open structure, with an increased porphyrin-porphyrin separation. The IR spectroscopic signature associated with this structure also accounts for the strong IR band at 1587 cm-1 seen for many amorphous preparations of synthetic malaria pigment, and it is proposed that stabilizing these structures may be a new objective for antimalarial drugs. The important role of the vinyl substituents in this biochemistry is further demonstrated by the structure of deuterohemin obtained by single-crystal X-ray diffraction analysis.

{Fe(NO)(2)(9) Dinitrosyl Iron Complex Acting as a Vehicle for the NO Radical.

To carry and deliver nitric oxide with a controlled redox state and rate is crucial for its pharmaceutical/medicinal applications. In this study, the capability of cationic {Fe(NO)2}9 dinitrosyl iron complexes (DNICs) [(RDDB)Fe(NO)2]+ (R = Me, Et, Iso; RDDB = N,N'-bis(2,6-dialkylphenyl)-1,4-diaza-2,3-dimethyl-1,3-butadiene) carrying nearly unperturbed nitric oxide radical to form [(RDDB)Fe(NO)2(•NO)]+ was demonstrated and characterized by IR, UV-vis, EPR, NMR, and single-crystal X-ray diffractions. The unique triplet ground state of [(RDDB)Fe(NO)2(•NO)]+ results from the ferromagnetic coupling between two strictly orthogonal orbitals, one from Fe dz2 and the other a π*op orbital of a unique bent axial NO ligand, which is responsible for the growth of a half-field transition (ΔMS = 2) from 70 to 4 K in variable-temperature EPR measurements. Consistent with the NO radical character of coordinated axial NO ligand in complex [(MeDDB)Fe(NO)2(•NO)]+, the simple addition of MeCN/H2O into CH2Cl2 solution of complexes [(RDDB)Fe(NO)2(•NO)]+ at 25 °C released NO as a neutral radical, as demonstrated by the formation of [S5Fe(NO)2]- from [S5Fe(µ-S)2FeS5]2-.

Solution and Solid State Correlations of Antimalarial Drug Actions: NMR and Crystallographic Studies of Drug Interactions with a Heme Model.

Solution NMR has been used in tandem with a diamagnetic non-iron heme model compound as a simple and effective tool to rapidly probe the structures of the bound complexes formed between the metalloporphyrin and antimalarial drugs from the 4-aminoquinoline, 4-methylenehydroxylquinoline, and 8-aminoquinoline subfamilies. The ability of gallium(III) protoporphyrin IX to mimic heme chemistry is exploited. The 4-aminoquinolines quinacrine and amodiaquine and two novel 3-halo chloroquine analogues are found to bind to the metalloporphyrin through hydrogen-bonding and stacking interactions, while halofantrine and the 4-methylenehydroxylquinolines, quinine and mefloquine bind through the alcohol group of the drug. In each case, detailed structural information is available from the NMR assessment. The mefloquine model is confirmed crystallographically. The 8-aminoquinoline primaquine does not interact strongly. These tools show promise for future applications in assessing antimalarials in preclinical development for heme-binding drug targets.

Generation of a Mn(IV)-Peroxo or Mn(III)-Oxo-Mn(III) Species upon Oxygenation of Mono- and Binuclear Thiolate-Ligated Mn(II) Complexes.

A thiolate-bridged binuclear complex [PPN]2[(MnII(TMSPS3))2] (1, PPN = bis(triphenylphosphine)iminium and TMSPS3H3 = (2,2',2″-trimercapto-3,3',3″-tris(trimethylsilyl)triphenylphosphine)), prepared from the reaction of MnCl2/[PPN]Cl and Li3[TMSPS3], converts into a mononuclear complex [PPN][MnII(TMSPS3)(DABCO)] (2) in the presence of excess amounts of DABCO (DABCO = 1,4-diazabicyclo[2.2.2]octane). Variable temperature studies of solution containing 1 and DABCO by UV-vis spectroscopy indicate that 1 and 2 exist in significant amounts in equilibrium and mononuclear 2 is favored at low temperature. Treatment of 1 or 2 with the monomeric O2-side-on-bound [PPN][MnIV(O2)(TMSPS3)] (3) produces the mono-oxo-bridged dimer [PPN]2[(MnIII(TMSPS3))2(µ-O)] (4). The electrochemistry of 1 and 2 reveals anodic peak(s) for a MnIII/MnII redox couple at shifted potentials against Fc/Fc+, indicating that both complexes can be oxidized by dioxygen. The O2 activation mediated by 1 and 2 is investigated in both solution and the solid state. Microcrystals of 2 rapidly react with air or dry O2 to generate the Mn(IV)-peroxo 3 in high yield, revealing a solid-to-solid transformation and two-electron reduction of O2. Oxygenation of 1 or 2 in solution, however, is affected by diffusion and transient concentration of dioxygen in the two different substrates, leading to generation of 3 and 4 in variable ratios.

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO.

Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

Surface Characterization of Hematin Anhydride: A Comparison between Two Different Synthesis Methods.

During the intraerythrocytic stage of malaria, the parasite digests hemoglobin and aggregates the released heme as an insoluble crystalline material called hemozoin. This detoxification step is an excellent drug target for developing new antimalarials, which can bind to hemozoin surface to inhibit further growth. Although the bulk crystalline properties of hemozoin are well-known, the surface properties remain poorly defined. Here, we use a combination of spectroscopic and adsorption techniques to study the surface of synthetic hemozoin, hematin anhydride, produced by two different methods. We show that the two synthetic methods produce crystals with major differences, such as the amount of water adsorbed on the surface and surface carboxylate groups. These results imply that the methodology to produce hematin anhydride affects its surface reactivity; this information needs to be considered whenever hematin anhydride is used as a model to study host immune response or to design new antimalarials.

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum.

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum.

Stabilizing and Activating Nitrogen Catenates.

The remarkably stable catenated hexa-nitrogen chain in bis(benzotriazene-4-one) is structurally, theoretically, and spectroscopically characterized to illustrate the durability of the central N-N bond in this hexaazo chain. The reactions of this species illustrate the potential of these nitrogen catenates for the preparation of other condensed heterocycles, such as bispyrazolones, by thermal nitrogen exclusion or by trapping the single ring-opened Dimroth intermediates. In these latter reactions, 2-naphtholate anion condenses with bis(benzotriazene-4-one) to trap and retain a zwitterionic diazonium intermediate as an isolated diazo product, whereas transition metals ring effect ring-extrusion of dinitrogen from the Dimroth intermediate to generate chelating σ-aryls. The catenated nitrogen species can be stabilized by incorporating strong formal sp(2)-sp(2) N-N σ bonds with orthogonal orientations. Extending these stabilization and activation principles may allow these types of nitrogen catenates to be useful synthons for other polyaza species.

Nitric Oxide Catalysis of Diazene E/Z Isomerization.

Nitric oxide is an efficient catalyst for the cis-trans (E/Z) isomerization of diazenes. We compare the effect of room temperature solutions bearing low concentrations of nitric oxide, nitrogen dioxide, or oxygen on the rate of cis-trans isomerization, CTI, of the alkene bond in stilbene and on the azo double bond in azobenzene, as well as in four azo derivatives as measured by UV-vis spectroscopy. These rate enhancements can be as large as 3 orders of magnitude for azobenzene in solution. A mechanism is proposed where catalysis is promoted by the interaction of the nitric oxide with the diazene nitrogen lone pairs. Density functional theory, B3LYP/6-311++g** suggests that the binding of NO to the diazene should be weak and reversible but that its NO adduct has an E/Z isomerization barrier of 7.5 kcal/mol.

Plasmodium products contribute to severe malarial anemia by inhibiting erythropoietin-induced proliferation of erythroid precursors.

Low reticulocytosis, indicating reduced red blood cell (RBC) output, is an important feature of severe malarial anemia. Evidence supports a role for Plasmodium products, especially hemozoin (Hz), in suppressed erythropoiesis during malaria, but the mechanism(s) involved remains unclear. Here, we demonstrated that low reticulocytosis and suppressed erythropoietin (Epo)-induced erythropoiesis are features of malarial anemia in Plasmodium yoelii- and Plasmodium berghei ANKA-infected mice, similar to our previous observations in Plasmodium chabaudi AS-infected mice. The magnitude of decreases in RBC was a reflection of parasitemia level, but low reticulocytosis was evident despite differences in parasitemia, clinical manifestation, and infection outcome. Schizont extracts and Hz from P. falciparum and P. yoelii and synthetic Hz suppressed Epo-induced proliferation of erythroid precursors in vitro but did not inhibit RBC maturation. To determine whether Hz contributes to malarial anemia, P. yoelii-derived or synthetic Hz was administered to naive mice, and the development of anemia, reticulocytosis, and RBC turnover was determined. Parasite-derived Hz induced significant decreases in RBC and increased RBC turnover with compensatory reticulocytosis, but anemia was not as severe as that in infected mice. Our findings suggest that parasite factors, including Hz, contribute to severe malarial anemia by suppressing Epo-induced proliferation of erythroid precursors.

The novel arsenical darinaparsin is transported by cystine importing systems.

Darinaparsin (Dar; ZIO-101; S-dimethylarsino-glutathione) is a promising novel organic arsenical currently undergoing clinical studies in various malignancies. Dar consists of dimethylarsenic conjugated to glutathione (GSH). Dar induces more intracellular arsenic accumulation and more cell death than the FDA-approved arsenic trioxide (ATO) in vitro, but exhibits less systemic toxicity. Here, we propose a mechanism for Dar import that might explain these characteristics. Structural analysis of Dar suggests a putative breakdown product: dimethylarsino-cysteine (DMAC). We show that DMAC is very similar to Dar in terms of intracellular accumulation of arsenic, cell cycle arrest, and cell death. We found that inhibition of γ-glutamyl-transpeptidase (γ-GT) protects human acute promyelocytic leukemia cells (NB4) from Dar, but not from DMAC, suggesting a role for γ-GT in the processing of Dar. Overall, our data support a model where Dar, a GSH S-conjugate, is processed at the cell surface by γ-GT, leading to formation of DMAC, which is imported via xCT, xAG, or potentially other cystine/cysteine importing systems. Further, we propose that Dar induces its own import via increased xCT expression. These mechanisms may explain the enhanced toxicity of Dar toward cancer cells compared with ATO.

Activation of nitrogen Brønsted acids: synthesis and reactivity of a new class of nitrogen acid complexes.

The nitrogen acids RC(O)NHNO2, N-nitroamide, R = CH3 (1), C2H5 (2) and N-nitrocarbamate, R'OC(O)NHNO2, R' = CH3 (3), C2H5 (4) are a class of primary N-nitrocarboxamide compounds that oxidatively add to trans-Ir((I))(Cl)(N2)(PPh3)2 to give six-coordinate Ir((III))(η(2)-(NO2)-nitrogen acid)(H)(Cl)(PPh3)2 complexes 5-8. Unexpected fluxional behavior of the complexes in solution is observed by (1)H NMR spectroscopy. Reaction intermediates of the oxidative addition reactions were also observed and monitored using (31)P and (1)H NMR and solution IR spectroscopies. Complex 5 reacts with methyl triflate in CH3CN to generate bis(acetonitrile) complex (9) from a net loss of the nitrogen acid anion. P(CH3)2Ph reacts with 5 to give phosphine-substituted and P(CH3)2Ph addition isomers (10). Reactivity studies of 5 with CO gave metastable CO adduct isomer 11, which loses CO on prolonged standing in solution.

Soluble meso and deuteroporphyrin analogs of the malaria pigment hematin anhydride.

Two soluble heme analogs of the insoluble malaria pigment hematin anhydride (HA, or ß-hematin), [Fe(III)(protoporphyrin)]2, with either mesoporphyrin (MHA) or deuteroporphyrin (DHA) are characterized by elemental analysis, SEM, IR spectroscopy, electronic spectroscopy, paramagnetic 1H NMR spectroscopy and solution magnetic susceptibility. While prior single crystal and X-ray powder diffraction results indicate all three have a common propionate linked dimer motif, there is considerable solid state variation in the conformation. This is associated with enhanced solubility of MHA and DHA. As with HA, DHA undergoes thermally promoted reversible hydration/dehydration in the solid state. Solution 1H NMR studies of DHA suggest a high spin dimeric structure with the porphyrin methyls distributed between two isomers which are also present in the solid state. These soluble iron(III)porphyrin dimers allow for the first direct solution studies by NMR and UV-Vis spectroscopies of these key species. Taken together the results illustrate the importance and utility of varying the substituents on the periphery of the porphyrin for studying heme aggregation and malaria pigment formation.