Reply to the 'Comment on "Theoretical study of the NO3 radical reaction with CH2ClBr, CH2ICl, CH2BrI, CHCl2Br, and CHClBr2"' by C. J. Nielsen and Y. Tang, Phys. Chem. Chem. Phys., 2022, 24, DOI: 10.1039/D2CP03013F.

In this Reply, we answer the main argument raised in the Comment about the energy of the NO3 radical and its influence in the reaction profiles of the reaction of the NO3 radical with CH2ClBr, CH2ICl, CH2BrI, CHCl2Br, and CHClBr2 by C. J. Nielsen and Y. Tang. The optimized geometry of the NO3 radical has been obtained using 49 DFT functionals: 26 functionals predict a minimum with D3h symmetry and 23 with C2v symmetry. The former functionals have been used to calculate the thermodynamic values of three reactions (X + HNO3 → XH + NO3, X= OH, CH3 and CCl3) and compared with experimental data. Those functionals with smaller errors have been used to recalculate the barriers of the reaction of NO3 with CH2ClBr, CH2ICl, CH2BrI, CHCl2Br, and CHClBr2. The results show differences of 10.5 kJ mol-1 when compared to those obtained with the M08HX functional.

The Chemistry of Mercury in the Stratosphere.

Mercury, a global contaminant, enters the stratosphere through convective uplift, but its chemical cycling in the stratosphere is unknown. We report the first model of stratospheric mercury chemistry based on a novel photosensitized oxidation mechanism. We find two very distinct Hg chemical regimes in the stratosphere: in the upper stratosphere, above the ozone maximum concentration, Hg0 oxidation is initiated by photosensitized reactions, followed by second-step chlorine chemistry. In the lower stratosphere, ground-state Hg0 is oxidized by thermal reactions at much slower rates. This dichotomy arises due to the coincidence of the mercury absorption at 253.7 nm with the ozone Hartley band maximum at 254 nm. We also find that stratospheric Hg oxidation, controlled by chlorine and hydroxyl radicals, is much faster than previously assumed, but moderated by efficient photo-reduction of mercury compounds. Mercury lifetime shows a steep increase from hours in the upper-middle stratosphere to years in the lower stratosphere.

Theoretical study of the NO3 radical reaction with CH2ClBr, CH2ICl, CH2BrI, CHCl2Br, and CHClBr2.

The potential reaction of the nitrate radical (NO3), the main nighttime atmospheric oxidant, with five alkyl halides, halons (CH2ClBr, CH2ICl, CH2BrI, CHCl2Br, and CHClBr2) has been studied theoretically. The most favorable reaction corresponds to a hydrogen atom transfer. The stationary points on the potential energy surfaces of these reactions have been characterized. The reactions can be classified into two groups based on the number of hydrogen atoms in the halon molecules (1 or 2). The reactions with halons with only one hydrogen atom show more exothermic profiles than those with two hydrogen atoms. In addition, the kinetics of the reaction of NO3 + CH2BrI was studied in much higher detail using a multi-well Master Equation solver as a representative example of the nitrate radical reactivity against these halocarbons. These results indicate that the chemical lifetime of the alkyl halides would not be substantially affected by nitrate radical reactions, even in the case of NO3-polluted atmospheric conditions.

Photochemistry of oxidized Hg(I) and Hg(II) species suggests missing mercury oxidation in the troposphere.

Mercury (Hg), a global contaminant, is emitted mainly in its elemental form Hg0 to the atmosphere where it is oxidized to reactive HgII compounds, which efficiently deposit to surface ecosystems. Therefore, the chemical cycling between the elemental and oxidized Hg forms in the atmosphere determines the scale and geographical pattern of global Hg deposition. Recent advances in the photochemistry of gas-phase oxidized HgI and HgII species postulate their photodissociation back to Hg0 as a crucial step in the atmospheric Hg redox cycle. However, the significance of these photodissociation mechanisms on atmospheric Hg chemistry, lifetime, and surface deposition remains uncertain. Here we implement a comprehensive and quantitative mechanism of the photochemical and thermal atmospheric reactions between Hg0, HgI, and HgII species in a global model and evaluate the results against atmospheric Hg observations. We find that the photochemistry of HgI and HgII leads to insufficient Hg oxidation globally. The combined efficient photoreduction of HgI and HgII to Hg0 competes with thermal oxidation of Hg0, resulting in a large model overestimation of 99% of measured Hg0 and underestimation of 51% of oxidized Hg and ∼66% of HgII wet deposition. This in turn leads to a significant increase in the calculated global atmospheric Hg lifetime of 20 mo, which is unrealistically longer than the 3-6-mo range based on observed atmospheric Hg variability. These results show that the HgI and HgII photoreduction processes largely offset the efficiency of bromine-initiated Hg0 oxidation and reveal missing Hg oxidation processes in the troposphere.

Photodissociation Mechanisms of Major Mercury(II) Species in the Atmospheric Chemical Cycle of Mercury.

Mercury is a contaminant of global concern that is transported throughout the atmosphere as elemental mercury Hg0 and its oxidized forms HgI and HgII . The efficient gas-phase photolysis of HgII and HgI has recently been reported. However, whether the photolysis of HgII leads to other stable HgII species, to HgI , or to Hg0 and its competition with thermal reactivity remain unknown. Herein, we show that all oxidized forms of mercury rapidly revert directly and indirectly to Hg0 by photolysis. Results are based on non-adiabatic dynamics simulations, in which the photoproduct ratios were determined with maximum errors of 3%. We construct for the first time a complete quantitative mechanism of the photochemical and thermal conversion between atmospheric HgII , HgI , and Hg0 compounds. These results reveal new fundamental chemistry that has broad implications for the global atmospheric Hg cycle. Thus, photoreduction clearly competes with thermal oxidation, with Hg0 being the main photoproduct of HgII photolysis in the atmosphere, which significantly increases the lifetime of this metal in the environment.

Gas-Phase Photolysis of Hg(I) Radical Species: A New Atmospheric Mercury Reduction Process.

The efficient gas-phase photoreduction of Hg(II) has recently been shown to change mercury cycling significantly in the atmosphere and its deposition to the Earth's surface. However, the photolysis of key Hg(I) species within that cycle is currently not considered. Here we present ultraviolet-visible absorption spectra and cross-sections of HgCl, HgBr, HgI, and HgOH radicals, computed by high-level quantum-chemical methods, and show for the first time that gas-phase Hg(I) photoreduction can occur at time scales that eventually would influence the mercury chemistry in the atmosphere. These results provide new fundamental understanding of the photobehavior of Hg(I) radicals and show that the photolysis of HgBr increases atmospheric mercury lifetime, contributing to its global distribution in a significant way.

Photoreduction of gaseous oxidized mercury changes global atmospheric mercury speciation, transport and deposition.

Anthropogenic mercury (Hg(0)) emissions oxidize to gaseous Hg(II) compounds, before deposition to Earth surface ecosystems. Atmospheric reduction of Hg(II) competes with deposition, thereby modifying the magnitude and pattern of Hg deposition. Global Hg models have postulated that Hg(II) reduction in the atmosphere occurs through aqueous-phase photoreduction that may take place in clouds. Here we report that experimental rainfall Hg(II) photoreduction rates are much slower than modelled rates. We compute absorption cross sections of Hg(II) compounds and show that fast gas-phase Hg(II) photolysis can dominate atmospheric mercury reduction and lead to a substantial increase in the modelled, global atmospheric Hg lifetime by a factor two. Models with Hg(II) photolysis show enhanced Hg(0) deposition to land, which may prolong recovery of aquatic ecosystems long after Hg emissions are lowered, due to the longer residence time of Hg in soils compared with the ocean. Fast Hg(II) photolysis substantially changes atmospheric Hg dynamics and requires further assessment at regional and local scales.

A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug.

Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.

Definition and determination of the triplet-triplet energy transfer reaction coordinate.

A definition of the triplet-triplet energy transfer reaction coordinate within the very weak electronic coupling limit is proposed, and a novel theoretical formalism is developed for its quantitative determination in terms of internal coordinates The present formalism permits (i) the separation of donor and acceptor contributions to the reaction coordinate, (ii) the identification of the intrinsic role of donor and acceptor in the triplet energy transfer process, and (iii) the quantification of the effect of every internal coordinate on the transfer process. This formalism is general and can be applied to classical as well as to nonvertical triplet energy transfer processes. The utility of the novel formalism is demonstrated here by its application to the paradigm of nonvertical triplet-triplet energy transfer involving cis-stilbene as acceptor molecule. In this way the effect of each internal molecular coordinate in promoting the transfer rate, from triplet donors in the low and high-energy limit, could be analyzed in detail.

Synthesis and photophysics of novel biocompatible fluorescent oxocines and azocines in aqueous solution.

The spectroscopic properties in water solution of the different prototropic forms of the strongly fluorescent hemiacetal 4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]benzofuran-5(4H)-one (1a, monardine), the aza analogue 4,9-dihydroxy-3,4-dihydro-1H-4,11a-methanobenzofuro[2,3-d]azocin-5(2H)-one (2a, azamonardine) and the respective 2-carboxyl derivatives (1b, 2b) have been studied by experimental and quantum-chemical methods. Monardine and carboxymonardine are the major products of new fluorogenic, room-temperature reactions of hydroxytyrosol or salvianic acid in aqueous solution, respectively, and present unique photophysical properties. Near neutral pH (pKa = 7.2) monardine switches from a weakly emitting, UV-absorbing (382 nm) neutral species to a VIS-absorbing (426 nm), blue emitting (464 nm) anion form, with a fluorescence quantum yield ϕF = 1 and single-exponential decay τF = 2.74 ns. This binary-like spectroscopic change from the neutral to the anionic form was interpreted based on time-dependent density functional theory (TDDFT) calculations as due to (i) the reversal of (n,π*) and (π,π*) lowest-lying singlet excited states, and (ii) a change in the triplet-state distribution accompanying monardine ionization which may abolish de-excitation via intersystem crossing. A similar fluorogenic reaction takes place with catecholamines such as dopamine and DOPA, to yield fluorescent azocines 2a and 2b which, depending on pH, may be present as cationic, neutral or anionic species. TDDFT computations of these forms were also carried out to assign the corresponding excitation transitions and emission properties. Besides the analytical interest of the fluorogenic reactions, the photochemical stability and biocompatibility of the bright-dark pH-controlled molecular switches 1a and 1b may facilitate novel labels and probes to be developed for superresolution fluorescence microscopy.

Edelfosine and miltefosine effects on lipid raft properties: membrane biophysics in cell death by antitumor lipids.

Edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-phosphocholine) and miltefosine (hexadecylphosphocholine) are synthetic alkylphospholipids (ALPs) that are reported to selectively accumulate in tumor cell membranes, inducing Fas clustering and activation on lipid rafts, triggering apoptosis. However, the exact mechanism by which these lipids elicit these events is still not fully understood. Recent studies propose that their mode of action might be related with alterations of lipid rafts biophysical properties caused by these lipid drugs. To achieve a clear understanding of this mechanism, we studied the effects of pharmacologically relevant amounts of edelfosine and miltefosine in the properties of model and cellular membranes. The influence of these molecules on membrane order, lateral organization, and lipid rafts molar fraction and size were studied by steady-state and time-resolved fluorescence methods, Förster resonance energy transfer (FRET), confocal and fluorescence lifetime imaging microscopy (FLIM). We found that the global membrane and lipid rafts biophysical properties of both model and cellular membranes were not significantly affected by both the ALPs. Nonetheless, in model membranes, a mild increase in membrane fluidity induced by both alkyl lipids was detected, although this effect was more noticeable for edelfosine than miltefosine. This absence of drastic alterations shows for the first time that ALPs mode of action is unlikely to be directly linked to alterations of lipid rafts biophysical properties caused by these drugs. The biological implications of this result are discussed in the context of ALPs effects on lipid metabolism, mitochondria homeostasis modulation, and their relationship with tumor cell death.

Drug uptake, lipid rafts, and vesicle trafficking modulate resistance to an anticancer lysophosphatidylcholine analogue in yeast.

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy.

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 µm/0.4-0.6 µm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking.

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles.

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis.

Defeating Leishmania resistance to miltefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: a proof of mechanism for trypanosomatid chemotherapy.

Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful against leishmaniasis, is especially active on the visceral form of the disease. Resistance mechanisms are almost exclusively associated to dysfunction in HePC uptake systems. In order to evade the requirements of its cognate receptor/translocator, HePC-resistant Leishmania donovani parasites (R40 strain) were challenged with constructs consisting of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide (CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter and kill both promastigote and intracellular amastigote forms of the R40 strain. Intracellular release of HePC by reduction of the disulfide-based conjugate was confirmed by means of double tagging at both the CPP (Quasar 670) and HePC (BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the metabolically more stable thioether conjugate retained substantial activity. The disulfide conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally resistant to HePC. Our results provide proof-of-mechanism for the use of CPP conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as the possibility to extend chemotherapy into other parasites intrinsically devoid of membrane translocation systems.

Structure and formation of the fluorescent compound of Lignum nephriticum.

The intense blue fluorescence of the infusion of Lignum nephriticum (Eysenhardtia polystachya), first observed in the sixteenth century, is due to a novel four-ring tetrahydromethanobenzofuro[2,3-d]oxacine which is not present in the plant but is the end product of an unusual, very efficient iterative spontaneous oxidation of at least one of the tree's flavonoids.

Synthesis of BODIPY-labeled alkylphosphocholines with leishmanicidal activity, as fluorescent analogues of miltefosine.

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved.

Synthesis and biological evaluation of fluorescent leishmanicidal analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite mechanisms.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains.

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.