De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Visual paper-based sensor for the highly sensitive detection of caffeine in food and biological matrix based on CdTe-nano ZnTPyP combined with chemometrics.

Caffeine naturally occurs in tea and cocoa, which is also used as an additive in beverages and has pharmacological effects such as refreshing, antidepressant, and digestion promotion, but excessive caffeine can cause harm to the human body. In this work, based on the specific response between nano zinc 5, 10, 15, 20-tetra(4-pyridyl)-21H-23H-porphine (nano ZnTPyP)-CdTe quantum dots (QDs) and caffeine, combined with chemometrics, a visual paper-based sensor was constructed for rapid and on-site detection of caffeine. The fluorescence of QDs can be quenched by nano ZnTPyP. When caffeine is added to the system, it can pull nano ZnTPyP off the surface of the QDs to achieve fluorescence recovery through electrostatic attraction and nitrogen/zinc coordination. The detection range is 5 × 10-11~3 × 10-9 mol L-1, and the detection limit is 1.53 × 10-11 mol L-1 (R2 = 0.9990) (S/N = 3). The paper-based sensor constructed exhibits good results in real samples, such as tea water, cell culture fluid, newborn bovine serum, and human plasma. Therefore, the sensor is expected to be applied to the rapid instrument-free detection of caffeine in food and biological samples.Graphical abstract.

Multifunctional Hf/Mn-TCPP Metal-Organic Framework Nanoparticles for Triple-Modality Imaging-Guided PTT/RT Synergistic Cancer Therapy.

BACKGROUND: Recent studies have validated and confirmed the great potential of nanoscale metal-organic framework (NMOF) in the biomedical field, especially in improving the efficiency of cancer diagnosis and therapy. However, most previous studies only utilized either the metal cluster or the organic ligand of the NMOF for cancer treatments and merely reported limited theranostic functions, which may not be optimized. As a highly designable and easily functionalized material, prospective rational design offers a powerful way to extract the maximum benefit from NMOF for cancer theranostic applications. MATERIALS AND METHODS: A NMOF based on hafnium (Hf) cluster and Mn(III)-porphyrin ligand was rational designed and synthesized as a high-performance multifunctional theranostic agent. The folic acid (FA) was modified on the NMOF surface to enhance the cancer targeting efficacy. The proposed "all-in-one" FA-Hf-Mn-NMOF (fHMNM) was characterized and identified using various analytical techniques. Then, in vitro and in vivo studies were performed to further explore the effects of fHMNM both as the magnetic resonance imaging (MRI)/computed tomography (CT)/photoacoustic imaging (PAI) contrast agent and as the photothermal therapy (PTT)/radiotherapy (RT) agent. RESULTS: A tumour targeting multifunctional fHMNM was successfully synthesized with high performance for MRI/CT/PAI enhancements and image-guided PTT/RT synergistic therapy properties. Compared with the current clinical CT and MR contrast agents, the X-ray attenuation and T1 relaxation rate of this integrated nanosystem increased 1.7-fold and 3-5-fold, respectively. More importantly, the catalase-like Mn(III)-porphyrin ligand can decompose H2O2 into O2 in tumour microenvironments to improve the synergistic treatment efficiency of PTT and RT. Significant tumour growth inhibition was achieved in mouse cancer models without obvious damage to the other organs. CONCLUSION: This work highlights the potential of fHMNM as an easily designable material for biomedical applications, could be an effective tool for in vivo detection and subsequent treatment of tumour.

Fluorescence-enhanced covalent organic framework nanosystem for tumor imaging and photothermal therapy.

Fluorescent dyes, as a key factor in fluorescence imaging, usually exhibit a low signal-to-noise ratio (SNR) due to the limited loading capacities of delivery systems (usually less than 10.0 wt%) and their uncontrolled release. Herein, we developed a type of pH-responsive nanoplatform (MnO2/ZnCOF@Au&BSA) based on a zinc porphyrin covalent organic framework (COF), in which the zinc porphyrin (ZnPor) loading rate is 22.5 wt%. At pH = 7.4, the interlinked ZnPor in the assembly state did not show a fluorescence signal ("off" state). Together with the pH-triggered disintegration of ZnCOF in tumor cells (pH = 5.5), the scattered ZnPor displayed an obvious fluorescence signal recovery ("on" state). Simultaneously, the shed BSA-coated gold nanoparticles ingeniously caused the fluorescence signal to be further amplified through the metal-enhanced fluorescence effect, which was about 3.0-fold higher in vivo than in the free ZnPor group. Combined with the excellent photothermal therapy effect by the nanoplatform itself with the tumor inhibition rate of 79.5%, this nanosystem effectively solves the problem of low loading capacities and imaging SNR by traditional delivery systems, and successfully develops the potential of COFs for fluorescence imaging, achieving the purpose of integration of diagnosis and treatment.

Monocrotophos detection with a bienzyme biosensor based on ionic-liquid-modified carbon nanotubes.

Acetylcholinesterase (AChE) biosensor technology is widely applied in the detection of organophosphate pesticides in agricultural production via the inhibition of AChE activity by organophosphates. However, the AChE electrode has some drawbacks, such as low stability and high overpotential. Combining the advantages of multiwalled carbon nanotubes (MWCNTs) and ionic liquids, we constructed a novel bienzyme electrode [Cl/iron porphyrin (FePP)-modified MWCNTs/AChE/glassy carbon electrode], which included AChE and mimetic oxidase FePP. In this electrode, FePP is covalently bound to the AChE carrier via ionic liquid for increased electrode sensitivity and stability. Under optimal conditions, this novel biosensor has a monocrotophos detection limit of 3.2 × 10-11 mol/L and good recovery of 89-104%. After 5 weeks of storage at 4 °C, the oxidation current was 97.8% of its original value. The biosensor has high stability and sensitivity for monocrotophos detection and is a promising device for monitoring food safety. Graphical abstract The complete synthesis process of Cl/FePP-MWCNTs/AChE/GCE.

Let There Be Light: Targeted Photodynamic Therapy Using High Aspect Ratio Plant Viral Nanoparticles.

The development of plant viral nanoparticles (VNP) loaded with different molecular versions of a photodynamic drug is described. Specifically, tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) are developed as drug carriers that encapsulate the monocationic, dicationic, tricationic, and tetracationic versions of a porphyrin-based photosensitizer drug (Zn-Por). While TMV has been extensively explored for various nanotechnology applications, this is the first study investigating TMGMV for medical applications. Light-activated cancer cell killing of Zn-Por-loaded VNPs is studied in vitro using melanoma and cervical cancer models. Native and nucleolin-targeted VNP drug carriers are developed and their efficacy assessed. A fivefold increase in cancer cell killing is observed using nucleolin-targeted TMV loaded with tricationic Zn-Por and displaying the nucleolin-specific F3 peptide.

Synthetic Iron Porphyrins for Probing the Differences in the Electronic Structures of Heme a3, Heme d, and Heme d1.

A variety of heme derivatives are pervasive in nature, having different architectures that are complementary to their function. Herein, we report the synthesis of a series of iron porphyrinoids, which bear electron-withdrawing groups and/or are saturated at the ß-pyrrolic position, mimicking the structural variation of naturally occurring hemes. The effects of the aforementioned factors were systematically studied using a combination of electrochemistry, spectroscopy, and theoretical calculations with the carbon monoxide (CO) and nitric oxide (NO) adducts of these iron porphyinoids. The reduction potentials of iron porphyrinoids vary over several hundreds of millivolts, and the X-O (X = C, N) vibrations of the adducts vary over 10-15 cm-1. Density functional theory calculations indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π*-acceptor orbital energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to why cytochrome c containing nitrite reductases and cytochrome cd1 containing nitrite reductases follow different mechanistic pathways of nitrite reduction. This study also helps to rationalize the choice of heme a3 and not the most abundant heme b cofactor in cytochrome c oxidase.

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes.

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

The Eponymous Cofactors in Cytochrome P460s from Ammonia-Oxidizing Bacteria Are Iron Porphyrinoids Whose Macrocycles Are Dibasic.

The enzymes hydroxylamine oxidoreductase and cytochrome (cyt) P460 contain related unconventional "heme P460" cofactors. These cofactors are unusual in their inclusion of nonstandard cross-links between amino acid side chains and the heme macrocycle. Mutagenesis studies performed on the Nitrosomonas europaea cyt P460 that remove its lysine-heme cross-link show that the cross-link is key to defining the spectroscopic properties and kinetic competence of the enzyme. However, exactly how this cross-link confers these features remains unclear. Here we report the 1.45 Å crystal structure of cyt P460 from Nitrosomonas sp. AL212 and conclude that the cross-link does not lead to a change in hybridization of the heme carbon participating in the cross-link but rather enforces structural distortions to the macrocycle away from planarity. Time-dependent density functional theory coupled to experimental structural and spectroscopic analysis suggest that this geometric distortion is sufficient to define the spectroscopic properties of the heme P460 cofactor and provide clues toward establishing a relationship between heme P460 electronic structure and function.

Apigenin-7-O-glucoside oxidation catalyzed by P450-bioinspired systems.

Apigenin-7-O-glucoside (A7G) is the main flavonoid of Bidens gardneri Bak., a Brazilian plant with wide application in folk medicine. Despite the popular use of this plant, its biological effects are not completely known. This work tested the 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin iron(III) and manganese(III) chloride (Fe(TFPP)Cl and Mn(TFPP)Cl), and Jacobsen's catalyst as P450-bioinspired catalysts for A7G oxidation by different oxidants (PhIO, H2O2, m-CPBA, and t-BuOOH). Up to nine different products were detected by HPLC analysis; Reactions with metalloporphyrin/PhIO systems afforded high catalytic conversions (58-89%). In spite of providing smaller product yields, the metalloporphyrin/H2O2 systems led to superior product distribution. Fe(TFPP)Cl yielded the highest A7G conversion rates (79-93%) with the four different oxidants tested herein. In the presence of PhIO, the oxidative profile of the manganese catalysts was very close to the oxidative profile of Fe(TFPP)Cl. However, in medium containing peroxide, the reactivity of the manganese catalysts was lower as compared to the reactivity of Fe(TFPP)Cl. Reactions with Fe(TFPP)Cl/oxidant systems were analyzed by UPLC-MS; up to thirteen compounds were detected. A7G oxidation catalyzed by Fe(TFPP)Cl yielded seven compounds. Three other compounds had m/z profile compatible with the profile of the A7G metabolites. The A7G oxidation assays performed in the presence of P450-bioinspired catalysts demonstrated their great catalytic potential toward A7G. The present results may be useful to many areas of knowledge and to the research and development of numerous chemical and phamarcological processes, especially in terms of drug design, biological assays, and applications in medicinal chemistry.

An Evolved Orthogonal Enzyme/Cofactor Pair.

We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.

Role of Spin States in Nitric Oxide Binding to Cobalt(II) and Manganese(II) Porphyrins. Is Tighter Binding Always Stronger?

Binding of nitric oxide (NO) to metalloporphyrins and heme groups is important in biochemistry while challenging to describe accurately by density functional theory (DFT) calculations. Here, the structural and thermochemical aspect of NO binding to Co(II) and Mn(II) porphyrins is investigated by DFT and DFT-D (dispersion-corrected) calculations, supported by reliable coupled-cluster methodology (CCSD(T)), and critically correlated with the experimental data. It is argued that whereas the bonding of NO to Co(II) porphyrin is a simple radical recombination, the bonding of NO to Mn(II) porphyrin is accompanied by a crossing of spin states. For this reason, the spin-state conversion energy contributes to the Mn-NO bond energy, and the paradigmatic correlation between bond length and bond energy is violated for the considered nitrosyl complexes: the Mn-NO bond is (structurally) shorter by ∼0.2 Å, albeit (energetically) weaker by a few kcal/mol, compared with the Co-NO bond. Moreover, none of the many tested DFT methods can reproduce the Co-NO and Mn-NO bond energies simultaneously, except for calculations with B3LYP*-D3, TPSSh-D3, and M06-D3 methods supplemented with the proposed spin-state energy correction (to compensate for an error on the calculated spin-state conversion energy). The results of this study are important to appreciate the role of spin-state changes in ligand binding properties of heme-related models. They also highlight the need for accurate calculations for correct interpretation of experimental data, including the qualitative structure-energy relationship.

Resonant inelastic X-ray scattering on ferrous and ferric bis-imidazole porphyrin and cytochrome c: nature and role of the axial methionine-Fe bond.

Axial Cu-S(Met) bonds in electron transfer (ET) active sites are generally found to lower their reduction potentials. An axial S(Met) bond is also present in cytochrome c (cyt c) and is generally thought to increase the reduction potential. The highly covalent nature of the porphyrin environment in heme proteins precludes using many spectroscopic approaches to directly study the Fe site to experimentally quantify this bond. Alternatively, L-edge X-ray absorption spectroscopy (XAS) enables one to directly focus on the 3d-orbitals in a highly covalent environment and has previously been successfully applied to porphyrin model complexes. However, this technique cannot be extended to metalloproteins in solution. Here, we use metal K-edge XAS to obtain L-edge like data through 1s2p resonance inelastic X-ray scattering (RIXS). It has been applied here to a bis-imidazole porphyrin model complex and cyt c. The RIXS data on the model complex are directly correlated to L-edge XAS data to develop the complementary nature of these two spectroscopic methods. Comparison between the bis-imidazole model complex and cyt c in ferrous and ferric oxidation states show quantitative differences that reflect differences in axial ligand covalency. The data reveal an increased covalency for the S(Met) relative to N(His) axial ligand and a higher degree of covalency for the ferric states relative to the ferrous states. These results are reproduced by DFT calculations, which are used to evaluate the thermodynamics of the Fe-S(Met) bond and its dependence on redox state. These results provide insight into a number of previous chemical and physical results on cyt c.

A nanoscale bio-inspired light-harvesting system developed from self-assembled alkyl-functionalized metallochlorin nano-aggregates.

Self-assembled supramolecular organization of nano-structured biomimetic light-harvesting modules inside solid-state nano-templates can be exploited to develop excellent light-harvesting materials for artificial photosynthetic devices. We present here a hybrid light-harvesting system mimicking the chlorosomal structures of the natural photosynthetic system using synthetic zinc chlorin units (ZnChl-C6, ZnChl-C12 and ZnChl-C18) that are self-aggregated inside the anodic aluminum oxide (AAO) nano-channel membranes. AAO nano-templates were modified with a TiO2 matrix and functionalized with long hydrophobic chains to facilitate the formation of supramolecular Zn-chlorin aggregates. The transparent Zn-chlorin nano-aggregates inside the alkyl-TiO2 modified AAO nano-channels have a diameter of ∼120 nm in a 60 µm length channel. UV-Vis studies and fluorescence emission spectra further confirm the formation of the supramolecular ZnChl aggregates from monomer molecules inside the alkyl-functionalized nano-channels. Our results prove that the novel and unique method can be used to produce efficient and stable light-harvesting assemblies for effective solar energy capture through transparent and stable nano-channel ceramic materials modified with bio-mimetic molecular self-assembled nano-aggregates.

A spectroscopic investigation of the interaction between c-MYC DNA and tetrapyridinoporphyrazinatozinc(II).

The c-MYC gene plays an important role in the regulation of cell proliferation and growth and it is overexpressed in a wide variety of human cancers. Around 90% of c-MYC transcription is controlled by the nuclease-hypersensitive element III1 (NHE III1), whose 27-nt purine-rich strand has the ability to form a G-quadruplex structure under physiological conditions. Therefore, c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Here, the interaction of water-soluble tetrapyridinoporphyrazinatozinc(II) with 27-nt G-rich strand (G/c-MYC), its equimolar mixture with the complementary sequence (GC/c-MYC) and related C-rich oligonucleotide (C/c-MYC) is investigated. Circular dichroism (CD) measurements of the G-rich 27-mer oligonucleotide in 150 mM KCl, pH 7 demonstrate a spectral signature consistent with parallel G-quadruplex DNA. Furthermore, the CD spectrum of the GC rich oligonucleotide shows characteristics of both duplex and quadruplex structures. Absorption spectroscopy implies that the complex binding of G/c-MYC and GC/c-MYC is a two-step process; in the first step, a very small red shift and hypochromicity and in the second step, a large red shift and hyperchromicity are observed in the Q band. Emission spectra of zinc porphyrazine are quenched upon addition of three types of DNA. According to the results of spectroscopy, it can be concluded the dominant binding mode is probably, outside binding and end stacking.

Complementary strategies for developing Gd-free high-field T1 MRI contrast agents based on Mn(III) porphyrins.

Mn(III) porphyrin (MnP) holds the promise of addressing the emerging challenges associated with Gd-based clinical MRI contrast agents (CAs), namely, Gd-related adverse effect and decreasing sensitivity at high clinical magnetic fields. Two complementary strategies for developing new MnPs as Gd-free CAs with optimized biocompatibility were established to improve relaxivity or clearance rate. MnPs with distinct and tunable pharmacokinetic properties can consequently be constructed for different in vivo applications at clinical field of 3 T.

Classification of tea category using a portable electronic nose based on an odor imaging sensor array.

A developed portable electronic nose (E-nose) based on an odor imaging sensor array was successfully used for classification of three different fermentation degrees of tea (i.e., green tea, black tea, and Oolong tea). The odor imaging sensor array was fabricated by printing nine dyes, including porphyrin and metalloporphyrins, on the hydrophobic porous membrane. A color change profile for each sample was obtained by differentiating the image of sensor array before and after exposure to tea's volatile organic compounds (VOCs). Multivariate analysis was used for the classification of tea categories, and linear discriminant analysis (LDA) achieved 100% classification rate by leave-one-out cross-validation (LOOCV). This study demonstrates that the E-nose based on odor imaging sensor array has a high potential in the classification of tea category according to different fermentation degrees.

Chlorophyll J-aggregates: from bioinspired dye stacks to nanotubes, liquid crystals, and biosupramolecular electronics.

Among the natural light-harvesting (LH) systems, those of green sulfur and nonsulfur photosynthetic bacteria are exceptional because they lack the support of a protein matrix. Instead, these so-called chlorosomes are based solely on "pigments". These are self-assembled bacteriochlorophyll c, d, and e derivatives, which consist of a chlorophyll skeleton bearing a 3(1)-hydroxy functional group. Chemists consider the latter as an essential structural unit to direct the formation of light-harvesting self-assembled dye aggregates with J-type excitonic coupling. The intriguing properties of chlorosomal J-type aggregates, particularly narrow red-shifted absorption bands, compared with monomers and their ability to delocalize and migrate excitons, have inspired intense research activities toward synthetic analogues in this field. The ultimate goal of this research field is the development of (opto-)electronic devices based on the architectural principle of chlorosomal LH systems. In this regard, the challenge is to develop small, functional building blocks with appropriate substituents that are preprogrammed to self-assemble across different length scales and to emulate functions of natural LH systems or to realize entirely new functions beyond those found in nature. In this Account, we highlight our achievements in the past decade with semisynthetic zinc chlorins (ZnChls) as model compounds of bacteriochlorophylls obtained from the naturally most abundant chlorin precursor: chlorophyll a. To begin, we explore how supramolecular strategies involving π-stacking, hydrogen bonding, and metal-oxygen coordination can be used to design ZnChl-based molecular stack, tube, and liquid crystalline assemblies conducive to charge and energy transport. Our design principle is based on the bioinspired functionalization of the 3(1)-position of ZnChl with a hydroxy or methoxy group; the former gives rise to tubular assemblies, whereas the latter induces stack assemblies. Functionalization of the 17(2)-position with esterified hydrophilic or hydrophobic chains, dendron-wedge substituents, and chromophores having complementary optical properties such as naphthalene bisimides (NBIs) is used to modulate the self-assembly of ZnChl dyes. The resulting assemblies exhibit enhanced charge transport and energy transfer abilities. We have used UV/vis, circular dichroism (CD), fluorescence spectroscopy, and dynamic light scattering (DLS) for the characterization of these assemblies in solution. In addition, we have studied assembly morphologies by atomic force microscopy (AFM), scanning tunneling microscopy (STM), transmission electron microscopy (TEM), and cryogenic-TEM. Crystallographic techniques such as powder X-ray and solid-state NMR have been used to explain the precise long- and short-range packing of dyes in these assemblies. Finally, functional properties such as charge and energy transport have been explored by pulse radiolysis time-resolved microwave conductivity (PR-TRMC), conductive AFM, and time-resolved fluorescence spectroscopy. The design principles discussed in this Account are important steps toward the utilization of these materials in biosupramolecular electronics and photonics in the future.

A nonclinical safety assessment of MnTE-2-PyP, a manganese porphyrin.

Manganese (III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP or BMX-010; CASRN 219818-60-7) is a manganese porphyrin compound developed as a potential drug substance for use as a radioprotective and for the ex vivo treatment of cells, tissues, and organs intended for transplantation. In preparation for an investigational new drug filing, a full good laboratory practice nonclinical safety assessment was conducted in order to evaluate the safety of MnTE-2-PyP and included the performance of in vitro genotoxicity studies, local tissue tolerance evaluation, safety pharmacology core battery studies, and single- and repeat-dose intravenous (iv) toxicity studies in mice and monkeys. The MnTE-2-PyP was determined not to be genotoxic or hemolytic, did not demonstrate flocculation or elicit adverse pharmacologic effects on respiration, the central nervous system (CNS), and had limited transitory effects on the cardiovascular system only at levels well above the therapeutic target dose. The intended iv clinical solution did not cause venous irritation in rabbits. The no observed adverse effect level (NOAEL) in mice was determined to be 10 mg/kg/day after 18 consecutive days of bolus iv dosing once daily in the morning. The NOAEL in monkeys after 14 days of bolus iv dosing in the morning was determined to be 5 mg/kg/day. At doses relevant to clinical use in humans, neither study revealed any indication of any specific target organ toxicity, including the classic heme porphyrin kidney, liver, CNS, or cardiac toxicities, or manganese toxicity. Mortality seen shortly after dosing in individual animals at higher doses was not accompanied by any organ or clinical pathology indications, suggesting a functional pharmacological-mediated effect. Based on the results of these studies, a conservative safe initial starting clinical dose of 5.0 mg (0.083 mg/kg in a 60 kg adult) was proposed for the initiation of human trials. Because of patent life issues, use of MnTE-2-PyP as a transplantation aid or radioprotective agent is not currently being pursued past the preclinical stages. It serves as a model for the clinical development of this class of drugs.

Synthesis of carbazole-based selenaporphyrin via annulation.

Cu(I)-mediated alkoxylation of doubly 1,3-butadiyne-bridged carbazole dimer 1, followed by acid-catalyzed cyclization, provided furan-bridged carbazole dimer 3, while annulation reaction of 1 with selenium in the presence of hydrazine monohydrate provided selenophene-bridged carbazole dimer 5a. Oxidation of isophlorin 5a afforded carbazole-based selenaporphyrin 5b, which possessed distinct aromaticity and produced intensified and red-shifted absorption bands in the near-IR region.