CO Photorelease Research Articles

Light Response of Three Water-Soluble MnI PhotoCORMs: Spectroscopic Features and CO Release Investigation

Currently, there is great interest in the study of water-soluble metal compounds capable of releasing carbon monoxide, due to their potential therapeutic use. This paper reports the synthesis, spectroscopic characteristics and CO release properties of three water-soluble manganese(I) carbonyl compounds [Mn(aaz)(CO)3]Br (1; where aaz = 6-amino-6-methylperhydro-1,4-diazepine), [Mn(Me2aaz)(CO)3]Br (2; where Me2aaz = 6-amino-1,4,6-trimethyl-1,4-diazacycloheptane) and [Mn(tacn)(CO)3]Br (3; where tacn = 1,4,7-triazacyclononane), that can act as photoCORMs (where CORM is carbon monoxide-releasing molecule). The main aim was to propose a mechanism for the CO release. Compounds 1-3 are capable of releasing carbon monoxide when exposed to light (λ385 and λ410) and thus, act as photoCORMs. The formation of a biscarbonyl intermediate was identified during the photo-release process at λ385 and the quantum yields and rates of CO release were determined. The proposed CO release mechanisms involve two steps, where photo and redox processes take place, and compounds 1 and 2 exhibit a slightly different mechanism from compound 3. A good understanding of the CO photo-release mechanism is very important with regard to the development of more efficient compounds, particularly those intended for medical applications.

Synthesis and structures of photoactive manganese-carbonyl complexes derived from 2-(pyridin-2-yl)-1,3-benzothiazole and 2-(quinolin-2-yl)-1,3-benzothiazole.

PhotoCORMs (photo-active CO-releasing molecules) have emerged as a class of CO donors where the CO release process can be triggered upon illumination with light of appropriate wavelength. We have recently reported an Mn-based photoCORM, namely [MnBr(pbt)(CO)3] [pbt is 2-(pyridin-2-yl)-1,3-benzothiazole], where the CO release event can be tracked within cellular milieu by virtue of the emergence of strong blue fluorescence. In pursuit of developing more such trackable photoCORMs, we report herein the syntheses and structural characterization of two MnI-carbonyl complexes, namely fac-tricarbonylchlorido[2-(pyridin-2-yl)-1,3-benzothiazole-κ2N,N']manganese(I), [MnCl(C12H8N2S)(CO)3], (1), and fac-tricarbonylchlorido[2-(quinolin-2-yl)-1,3-benzothiazole-κ2N,N']manganese(I), [MnCl(C16H10N2S)(CO)3], (2). In both complexes, the MnI center resides in a distorted octahedral coordination environment. Weak intermolecular C-H...Cl contacts in complex (1) and Cl...S contacts in complex (2) consolidate their extended structures. These complexes also exhibit CO release upon exposure to low-power broadband visible light. The apparent CO release rates for the two complexes have been measured to compare their CO donating capacity. The fluorogenic 2-(pyridin-2-yl)-1,3-benzothiazole and 2-(quinolin-2-yl)-1,3-benzothiazole ligands provide a convenient way to track the CO release event through the `turn-ON' fluorescence which results upon de-ligation of the ligands from their respective metal centers following CO photorelease.

Effects of Azole Drugs on Conformational Dynamics and Energetics Associated with Diatomic Ligand Photorelease and Migration within Bacterial Flavohemoglobins

Bacterial flavohemoglobins (fHbs) are a widely distributed class of proteins which are though to protect microbes from nitrosative stress due to the immune response of a host organism via nitric oxide dioxygenase (NOD) activity. There has been interest lately in developing fHbs as targets for antibiotics due to their ubiquity in bacteria and the discovery of inhibition of NOD activity of fHbs by azole-based drugs which bind with high affinity (<12 μM). To determine how azole drugs affect fHbs we have used photoacoustic calorimetry (PAC) and transient absorption spectroscopy (TA) to calculate enthalpy and volume changes (ΔH and ΔV) and kinetic parameters for CO photorelease and rebinding to fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus (HMPSa) in the presence and absence of the drugs ketoconazole and miconazole. We observe that binding of drugs to fHbs results in a more exothermic ΔH for CO photorelease and faster bimolecular rebinding of CO. We also use classical molecular dynamics (cMD) to characterize ligand migration pathways present in FHP. We have conducted cMD simulations of CO-FHP WT and FHP WT containing free CO molecules using a locally enhanced sampling (LES) algorithm. We have carried out a 10 ns simulation of CO-FHP WT and a 30 ns simulation of the LES-enabled CO:FHP WT. We observe dissociation of 18 of 20 LES-enhanced CO molecules from the distal heme pocket. The primary escape pathways are between the CD-loop and heme-propionate groups and through a pathway exiting the protein matrix near the N-terminal end of the A-helix. These pathways are close to the locations of ketoconazole and miconazole binding predicted by AutoDock Vina, suggesting that drug binding inhibits NOD activity by closing migration pathways for O2 and NO.

A Theranostic Two-Tone Luminescent PhotoCORM Derived from Re(I) and (2-Pyridyl)-benzothiazole: Trackable CO Delivery to Malignant Cells.

A Re(I) carbonyl complex derived from 2-(2-pyridyl)-benzothiazole (pbt), [Re(H2O)(CO)3(pbt)](CF3SO3) (1), rapidly releases CO under low-power UV illumination. CO photorelease from 1 is accompanied by a change in luminescence from orange to deep blue. These two distinct luminescence signals have been successfully employed to track (a) the entry of the pro-drug 1 into cancer cells and (b) the end of the CO (drug) delivery step within the target.

Revisiting oxo-centered carbonyl-triruthenium clusters: investigating CO photorelease and some spectroscopic and electrochemical correlations

We synthesized and characterized a series of oxo-centered carbonyl-triruthenium complexes with the general formula [Ru3O(CH3COO)6(L)2(CO)], where L = 2,6-dimethylpyrazine (dmpz) (1), isonicotinamide (adpy) (2), 4-acetylpyridine (acpy) (3), 3-methylpyridine (3-pic) (4), 4-methylpyridine (4-pic) (5), 4-tert-butylpyridine (4-tbpy) (6), 4-(dimethyl)aminopyridine (dmap) (7), or 4-aminopyridine (ampy) (8); we also investigated the photoreactivity of these complexes. Single-crystal X-ray diffraction helped to elucidate the structures of 1·H2O, 7·C2H4Cl2, and 8. The unit cell of 8 is composed of four cluster units; the hydrogen bonds between the amino groups of the terminal ligand of a neighboring molecule and the oxygen atoms of CO or acetate bridging ligands hold these cluster units together. The spectroscopic (NMR, UV-visible, and IR) and the electrochemical properties (cyclic voltammetry) of these complexes correlated with the ancillary ligands in terms of their σ-donating and π-accepting characteristics. The molecular orbital and the electronic localized description of the [Ru3O]-CO unit helped to rationalize the correlations. The photoreactivity of compounds 1-8 was investigated by laser excitation at 377 nm. Given the CO photorelease quantum yields, σ-donor ligands and aqueous medium (more polar) stabilized the charge-transfer excited state that culminated in CO photosubstitution, leading to higher Φ values.

Role of Ionic Strength and the Bohr Effect in Modulating Thermodynamic Profiles Associated with Co Escape in Rice Non-Symbiotic Hemoglobin 1

Rice hemoglobin (rHb1) is a type 1 non-symbiotic hemoglobin (nsHb1), which is found in plants and which is evolutionarily related to leghemoglobins and truncated hemoglobins. It is structurally similar to vertebrate heme proteins, containing Fe-protoporphyrin IX and the classic 3-over-3 globin fold. Unlike other hexacoordinate heme proteins found in plants, the ligand binding properties of nsHb1 proteins make it unlikely to be involved in nitrogen fixation or oxygen transport. We have characterized ligand migration upon CO photorelease from rHb1 under various conditions in order to elucidate functional role(s) of rHb1 in vivo. Using photoacoustic calorimetry, we have determined thermodynamic parameters (ΔH and ΔV) for CO photorelease from wild type rHb1 and a distal histidine to leucine (H74L) mutant taking place within ∼ 5 µs after ligand photo-dissociation. Under stripped conditions, we observe a strong temperature dependence of the observed parameters. Photorelease of CO from the wild type protein below 16°C is associated with ΔH = 28.7 ± 2.2 kcal mol−1 and ΔV = 4.3 ± 0.4 mL mol−1, while above 16°C we observe ΔH = 9.7 ± 3.4 kcal mol−1 and ΔV = 0.4 ± 0.7 mL mol−1. This temperature dependence is not observed in the H74L mutant, in the presence of 500 mM NaCl, or at pH 6. We have also determined rate constants and quantum yields for bimolecular rebinding of CO using transient absorption spectroscopy, both of which increase with temperature. The contributions of electrostriction, proton uptake by Bohr groups, oligomerization state, and the distal residue of rHb1 to the observed kinetic and thermodynamic parameters will be discussed. We will also discuss ligand migration pathways and protein dynamics upon CO photorelease in rHb1.

Photodelivery of CO by designed PhotoCORMs: correlation between absorption in the visible region and metal-CO bond labilization in carbonyl complexes.

The therapeutic potential of photoactive CO-releasing molecules (photoCORMs) have called for close examination of the roles of the ligand(s) and the central metal atoms on the overall photochemical labilization of the metal-CO bonds. Along this line, we have synthesized four metal complexes, namely, [MnBr(azpy)(CO)3 ] (1), [Mn(azpy)(CO)3 (PPh3 )]ClO4 (2), [ReBr(azpy)(CO)3 ] (3), and [Re(azpy)(CO)3 (PPh3 )]ClO4 (4), derived from 2-phenylazopyridine. These complexes were characterized by spectroscopic and crystallographic studies. Although both 1 and 3 exhibit strong metal-to-ligand charge-transfer bands in the 500-600 nm region, only 1 photoreleases CO upon illumination with visible light. Results of theoretical studies were used to gain insight into this surprising difference. Strong spin-orbit coupling (prominent in heavy metals) appears to promote intersystem crossing to a triplet state in 3, a step that discourages CO release upon illumination with visible light. Slow release of CO from 2 and 4 also indicates that strong σ-donating ligands, such as Br(-) , accelerate the rate of CO photorelease relative to π-acid ligands, such as PPh3 .

Differences in the CO photolability of cis- and trans-[RuCl2(azpy)(CO)2] complexes: Effect of metal-to-ligand back-bonding

Reaction of [RuCl2(CO)3]2 with 2-phenylazopyridine (azpy) in methanolic solution affords cis-[RuCl2(azpy)(CO)2] (1) and trans-[RuCl2(azpy)(CO)2] (2) depending on the temperature of the reaction mixture. In both complexes, the two CO ligands are cis to each other. Competition in π backbonding labilizes CO trans to the azo-N (N3) in 2 and increases the rate of CO photorelease of 2 (the trans isomer) compared to 1 (the cis isomer).

Syntheses, Structures, and Properties of New Manganese Carbonyls as Photoactive CO-Releasing Molecules: Design Strategies That Lead to CO Photolability in the Visible Region

The unusual role of CO as a signaling molecule in several physiological pathways has spurred research in the area of synthesizing new CO-releasing molecules (CORMs) as exogenous CO donors. Auxiliary control on CO delivery can be achieved if CO can be released under the control of light. To synthesize such photoactive CORMs (photoCORMs) with the aid of smart design, a series of manganese carbonyls have been synthesized with ligands that contain extended conjugation and electron-rich donors on their frames. Five such photoCORMs, namely, [Mn(pimq)(CO)(3)(MeCN)]ClO(4) (1, where pimq = (2-phenyliminomethyl)quinoline), [Mn(qmtpm)(CO)(3)(MeCN)]ClO(4) (2, where qmtpm = 2-quinoline-N-(2'-methylthiophenyl) methyleneimine), [Mn(qmtpm)(CO)(3)Br] (3) [Mn(pmtpm)(CO)(3)(MeCN)]ClO(4) (4, where pmtpm = 2-pyridyl-N-(2'-methylthiophenyl)methyleneimine), and [Mn(pmtpm)(CO)(3)Br] (5), have been synthesized and structurally characterized. These designed carbonyls readily release CO upon exposure to light (400-550 nm). The apparent CO release rates and quantum yield values at 509 nm (φ(509)) of the photoCORMs increase steadily with rise in conjugation in the ligand frame and inclusion of a -SMe group. Addition of Br(-) as an ancillary ligand also improves the CO-donating capacity. Results of density functional theory (DFT) and time dependent DFT (TDDFT) studies indicate that low energy metal-to-ligand charge transfer (MLCT) transitions from Mn-CO bonding into ligand-π orbitals lead to reduction of M-CO(π*) back-bonding and loss of CO from these photoCORMs. Inclusion of -SMe and Br(-) in the coordination sphere attenuates the energies of the HOMO and LUMO levels and causes further enhancement of CO photorelease. Collectively, the results of this work demonstrate that new photoCORMs with excellent sensitivity to visible light can be synthesized on the basis of smart design principles.

Impact of the Internal Disulfide Bond on Structural Dynamics in Vertebrates Hexacoordinate Hemeoglobins

There are two new additions to the vertebrate globins family, neuroglobin and cytoglobin, which exhibit bis-hystidyl hexacoordination. Mainly expressed in neurons, neuroglobin (Ngb) has a neuro-protective role under hypoxic-ischemic insults; whereas cytoglobin (Cygb) expression is no tissue-specific and this protein was proposed to protect cells against oxidative stress. To understand the impact of the disulfide bond on structural dynamics in Ngb and Cygb, we determined kinetics and thermodynamics for CO photo-release and rebinding to both proteins in the presence and absence of the disulfide bond (Ngbred and Cygbred). Photoacoustic calorimetry (PAC) data indicate that CO photo-dissociation from Ngb and Ngbred is monophasic (τ<50 ns) leading to an endothermic enthalpy change (ΔH∼20 kcal mol−1) and the volume change is ∼3 times larger for hNgb than hNgbred (ΔV=4.4±0.3 mL mol−1). Interestingly, biphasic kinetics were observed for CO migration from the heme pocket of Cygb with τ1<50 ns and an additional τ2∼200 ns at 20 oC. Cygbred produces monophasic kinetics with τ<50 ns suggesting that the disulfide bond strongly modulates the ligand migration pathway. CO photo-release from Cygb first leads to a volume expansion (ΦΔV1= 4.2 ± 1.9 mL mol−1) and endothermic enthalpy change (ΦΔH1= 23.3 ± 9.1 kcal mol−1), followed by a smaller volume expansion (ΦΔV2= 3.7 ± 1.5 mL mol−1) and exothermic enthalpy change (ΦΔH2= −11.8 ± 7.0 kcal mol−1). Upon disruption of the disulfide bond, CO photo-dissociation from Cygbred produces a prompt endothermic enthalpy change (ΦΔH= 6.3 ± 0.4 kcal mol−1) and a small volume expansion (ΦΔV= 1.8 ± 0.8 mL mol−1). These results suggest different mechanisms of ligand migration among vertebrate hexacoordinate hemoglobins as well as a larger impact of the disulfide bond on mechanism of ligand migration in Cygb than in Ngb.

Time-Resolved Enthalpy and Volume Changes for Co-Photorelease from Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a heme protein catalyzing the first and rate limiting step in the metabolism of L-tryptophan via the kynurenine pathway. The enzyme is responsible for the insertion of dioxygen into the indole ring generating l-formyl-kynurenine. However, the mechanisms underlying ligand binding and the resulting protein conformational changes leading to the reaction products are not well understood. Photoacoustic calorimetry was utilized to probe conformational dynamics associated with CO photo-release from the ferrous form of IDO in the presence and absence of L-tryptophan.

Structural Dynamics in Chloroperoxidase: A Photoacoustic Study

Chloroperoxidase (CPO) is the most versatile heme containing enzyme that exhibits peroxidase, catalase, halogenase and monooxigenase activities. The heme iron coordination resembles to P450s enzymes whereas the composition of the distal pocket is more polar, containing Glu183 hydrogen bounded to His 105 and the heme pocket is connected with the surrounding solvent by two hydrophobic channels. To investigate the ligand-protein interactions in CPO we have employed time-resolved photoacoustic calorimetry and transient absorption study. Unlike other peroxidases, the CO photo-dissociation from CPO can be described as a two step process with distinct volume and enthalpy changes. The photodissociation of Fe-CO bond is associated with negligible volume change (V = 1 ± 8 mL mol−1 ) and a positive enthalpy change (H = 48 ± 34 kcal mol−1). Subsequent CO escape from the protein matrix is characterized by a positive volume change (25 ± 8 mL mol−1) and insignificant enthalpy change (−2 ± 33 kcal mol−1) and occurs with a lifetime of ∼ 20 ns. Similar thermodynamic parameters were found in D2O at pD 4.6 but the CO photo-release occurs two times slower (∼ 50 ns) than in phosphate buffer. Presence of substrates (halides or cyclopentanedione) did not show a significant impact on the thermodynamic profiles associated with the ligand dissociation.

Dynamics And Energetics Associated With Ligand Photodissociation From Co Bound Chloroperoxidase

Chloroperoxidase (CPO) is the most versatile heme peroxidase that can function as peroxidase, catalase, halogenase/dehalogenase and monooxigenase. The active site of CPO strongly resembles that of cytochrome P450 since the heme iron in both proteins is coordinated with a Cys axial ligand. Two hydrophobic channels that connect the heme binding cavity with the surrounding are likely to provide ligand access into the active site. At alkaline pH the protein undergoes a conformational transition resulting in the population of six coordinated heme iron with the histidine residue in the position of the sixth ligand. Here we report the results of the photoacoustic study of ligand dissociation from CPO. The ligand photodissociation and migration out of the protein matrix occurs within 50 ns and no additional kinetics was determined (detected?) between 50 ns and 5 μs. At acidic pH ligand dissociation is associated with a positive volume change (ΦΔV = 10.9 ± 0.9 mL mol−1) and enthalpy change (ΦΔH = 24 ± 5 kcal mol−1), strongly indicating that the ligand release from the protein matrix does not result in significant changes in protein conformation and CO is likely to migrate through one of the hydrophobic channels. On the other hand, the thermodynamic parameters associated with CO photorelease at pH 10 are significantly smaller than those determined at acidic pH (ΦΔV = 3.2 ± 1.1 mL mol−1 and ΦΔH = -12.1 ± 5.3 kcal mol−1) due to changes in heme coordination upon CO dissociation.

Time Resolved Thermodynamics of Fast Protein Folding in Cytochrome c

One of the earliest studies of fast protein folding involved the photolysis of CO bound Cytochorme c in the presence of 4.5 M Gdn-HCl HCl, pH 7.0. These studies revealed fast coordination of non-native His and Met residues resulting in a ‘frustrated’ folding pathway resulting from non-native coordination. These early events occur on timescales of less than 10 μs. Similar fast folding can be initiated at lower Gdn-HCl at pHs above 9.5. Under these conditions the non-native coordination occurs with time constants of ∼ 300 ns and ∼ 3 μs at 22 dg C. Previous time resolved CD spectra suggest that 8% of the native like secondary structure forms in < 1 μs (Goldbeck et al., PNAS (1999), 96, 2782). Here, photoacoustic calorimetry (PAC) has been utilized to probe the thermodynamics associated with fast folding and non-native ligand coordination in CO-Cytochrome c ta pH 12.7 and 350 mM Gdn-HCl. At temperatures below 18 ° C the PAC signals indicate multi-phasic kinetics that can not be fit to simple exponential sums suggesting a distribution of conformations in the presence of CO. The integrated thermodynamics (integrated thermodynamics for processes occurring in < ∼ 20 μs) give an enthalpy change of 5±2 kcal/mol and a molar volume change of −0.5±0.4 mL/mol. Taking into account the enthalpy and volume changes associated with CO photo-release from the heme, an enthalpy change of - 12 kcal/mol and volume change of -6 mL/mol is obtained for the 8% folding as well as non-native His and Met coordination. These results will be discussed within the context of the protein funnel mechanism for fast folding in cytochorme c.

Characterization of Carbon Monoxide Photodissociation from FeIILPO with Photoacoustic Calorimetry

Lactoperoxidase belongs to a family of mammalian peroxidases that catalyze the oxidation of halides and small organic molecules in the presence of H2O2. We have used photoacoustic calorimetry to characterize thermodynamic parameters associated with ligand dissociation from bovine milk lactoperoxidase. Upon CO photorelease, a prompt (tau < 50 ns) exothermic volume contraction (DeltaH = -20 +/- 7 kcal mol-1 and DeltaH = -2 +/- 1 mL mol-1) was measured at pH 7.0 and 4.0, whereas an endothermic expansion (DeltaH = 30 +/- 13 kcal mol-1 and DeltaV = 9 +/- 2 mL mol(-1)) was observed at pH 10.0 and 7.0 in the presence of 500 mM NaCl. We attribute the observed volume and enthalpy changes to electrostriction arising from changes in the charge distribution associated with a reorganization of the heme binding pocket upon ligand dissociation. It is likely that cleavage of the Fe-CO bond is accompanied by distortion of a salt bridge between Arg557 and the heme propionate group, resulting in the observed electrostriction due to changes in charge distribution.