Synthesis, characterization and electrode adsorption studies of porphyrins coordinated to ruthenium(II) polypyridyl complexes

Two new porphyrins, meso-tris-3,4-dimethoxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3,4DMPP) and meso-tris-3-methoxy-4-hydroxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3M4HPP), and their ruthenium analogs obtained by coordination of [Ru(bpy)₂Cl]⁺ groups (where bpy = 2,2′-bipyridine) to the pyridyl nitrogens have been synthesized and studied by electronic absorption spectroscopy, cyclic voltammetry and spectroelectrochemistry. These ruthenated porphyrins couple Ru chromophores to porphyrins containing electroactive meso-substituents. The highest energy electronic absorption for the ruthenated complexes is assigned as a bpy(π) → bpy(π*) intraligand charge transfer while the next lowest energy electronic absorption is assigned as Ru(dπ) → bpy(π*) metal-to-ligand charge transfer (MLCT) transition. The Ru^III/II couples occur at approximately 0.95 V versus the SHE reference electrode in acetonitrile solutions. The first oxidation of the porphyrin is localized on the 3,4-dimethoxyphenyl and 3-methoxy-4-hydroxyphenyl substituents, respectively. Electroactive surfaces result from adsorption of these compounds onto glassy carbon electrodes followed by anodic cycling in acidic media.     

Photoluminescence and electrogenerated chemiluminescence of a bis(bipyridyl)ruthenium(II)-porphyrin complex

The photoluminescence (PL) and electrogenerated chemiluminescence (ECL) of [H₂(MPy3,4DMPP)Ru(bpy)₂Cl](PF₆), where H₂MPy3,4DMPP = meso-tris-3,4-dimethoxyphenyl-mono-(4-pyridyl)porphyrin and bpy = 2,2′-bipyridine, are reported in acetonitrile. The compound has a complex absorbance spectrum with bands characteristic of both the porphyrin and ruthenium moieties. PL emission maxim are observed at 655 nm when excited at the maximum absorption intensity corresponding to the porphyrin Soret π → π^∗ band, and around 600 nm when excited at wavelengths corresponding to Ru(dπ)-bpy (π^∗) MLCT transition. The photoluminescence efficiency (?_em) of the 655 nm emission is 0.039 and that of the free porphyrin is 0.69 compared to at 0.042.[H₂(MPy3,4DMPP)Ru(bpy)₂Cl](PF₆) displays complex electrochemical behavior, with one electrochemically reversible Ru^II-Ru^III oxidation and two quasi-reversible waves at more cathodic potentials corresponding to the porphyrin moiety. Oxidative ECL was generated using the coreactant tri-n-propylamine (TPrA). ECL efficiencies (?_ecl) were 0.14 for [H₂(MPy3,4DMPP)Ru(bpy)₂Cl]⁺ and 0.099 for H₂MPy3,4DMPP using as the standard (?_ecl = 1). ECL intensity was linear with respect to concentration from 1 to 0.001 μM.The ECL intensity peaks at potentials corresponding to oxidation both the ruthenium and porphyrin moieties as well as TPrA, indicating that multiple pathways for formation of the excited state are possible. However, an ECL spectrum shows a band similar in energy and shape to that of the Soret emission (655 nm for the PL and 656 nm for the ECL, respectively), indicating the same excited state is formed in each experiment.     

On the elusive, non-emissive yet reactive, upper excited states of [(4,4′-bpy)Re(CO)3(dppz)]PF6 (dppz = dipyridil[3,2-a:2′3′-c]phenazine)

Gradual solvation of [(4,4′-bpy)Re^I(CO)₃(dppz)]⁺ (dppz = dipyridil[3,2-a:2′3′-c]phenazine) by water molecules causes a quenching of the emission in accordance with Perrin’s model of spheres. The calculated radius of the sphere, r = 2.6 ± 0.2 ?, is therefore very close to the distance from the Re center to the oxygen atom of the CO ligands, i.e., l = 2.73 ?. In addition, excited state reactions with TEA produce [(4,4′-bpy⁻)Re^I(CO)₃(dppz)] and [(4,4′-bpy)Re^I(CO)₃(dppz⁻)]. This experimental observation is inconsistent with the formation of the products in the lowest lying and emissive dppz-centered ³ππ* excited state. Jablonski schemes based on the participation of excited states other than the lowest ³ππ* excited state are proposed.     

Tris-ruthenium(II)/copper(II) multimetallic porphyrin: Synthesis, characterization, DNA binding and supercoiled DNA photocleavage studies

The porphyrin, meso-5-(pentafluorophenyl)-10, 15, 20-tris(4-pyridyl)porphyrin has been used to synthesize two new metalloporphyrin complexes. Insertion of copper(II) into the porphyrin center gives the copper(II) porphyrin. Coordination of three [Ru(bipy)₂Cl]⁺ moieties (where bipy = 2,2′-bipyridine) to the pyridyl nitrogens of the copper(II) porphyrin gives the target complex. Electronic transitions associated with the copper(II) porphyrin and the triruthenium copper(II) porphyrin include an intense Soret band and a less intense Q-band in the visible region of the spectrum. An intense π-π∗ transition in the UV region associated with the bipyridyl groups and a metal to ligand charge transfer (MLCT) band appearing as a shoulder to the Soret band are observed for the ruthenated copper(II) porphyrin. Electrochemical properties associated with the multimetallic complex include a redox couple in the cathodic region with E_1/2 = −0.86 V versus Ag/AgCl attributed to the porphyrin and a redox couple in the anodic region E_1/2 = 0.88 V versus Ag/AgCl due to the Ru^III/II couple. DNA titrations indicate the triruthenium copper(II) porphyrin interacts with DNA potentially through a groove binding mechanism. Irradiation of aqueous solutions of the target complex and supercoiled DNA at a 10:1 base pair to complex ratio with visible light above 400 nm indicates that the complex causes nicking of the DNA helix.     

Synthesis, characterization and DNA interactions of 5,15-(4-pyridyl)-10,20-(pentafluorophenyl)porphyrin coordinated to two [Ru(bipy)2Cl] groups

A new porphyrin 5,15-(4-pyridyl)-10,20-(pentafluorophenyl)porphyrin (H₂DPDPFPP) and its diruthenium(II) analog ([trans-H₂(DPDPFPP)Ru₂(bipy)₄Cl₂(PF₆)₂]) have been synthesized and characterized. Electronic transitions associated with the porphyrin consist of an intense Soret band near 400 nm and four Q-bands from 500 nm to 650 nm. Coordination of two [Ru(bipy)₂Cl]⁺ groups, where bipy = 2,2′-bipyridine, to the pyridyl nitrogens of the porphyrin give additional electronic transitions associated with the bipy orbitals and metal to ligand charge transfer (MLCT) transitions associated with the Ru(II) and bipy orbitals. Reversible redox couples in the cathodic region occur at E_1/2 = −0.74 V and −1.21 V versus Ag/AgCl reference which are shifted to more positive potentials when the porphyrin is coordinated to the Ru(II) groups. Gel electrophoresis studies with linearized pUC18 indicate an interaction between the metallated porphyrin and DNA which is confirmed by UV/Vis titrations with calf thymus (CT) DNA giving a binding constant of ca. 10⁵ M⁻¹. When buffered, pH 7, solutions of circular plasmid DNA containing the ruthenium porphyrin are irradiated with a 50 W tungsten lamp cleavage of the DNA is observed.     

A convenient synthesis of isocyclam and [16]aneN4 and the photophysics of their dicyanochromium(III) complexes

The syntheses of the tetraazamacrocyclic ligands 1,4,7,11-tetraazacyclotetradecane (isocyclam) and 1,5,9,13-tetraazacyclohexadecane ([16]aneN₄) in two steps starting from the corresponding tetraamine and diethylmalonate is reported. The trans-dicyanochromium(III) complexes, trans-[Cr(isocyclam)(CN)₂]PF₆ and trans-[Cr([16]aneN₄)(CN)₂]PF₆ have also been prepared. Both are ²E_g emitters with 0-0 band emission wavelengths at 721.2 and 704.8 nm, respectively. The isocyclam complex has a room temperature excited state lifetime of 147 μs in aqueous solution which increases to 215 μs upon macrocyclic N-H deuteration, whereas the corresponding lifetime of the [16]aneN₄ complex is 25 μs and is unaffected by macrocyclic N-H deuteration. The implications of the temperature dependence of the excited state lifetimes are also presented.     

Meso-[{Ru(phen)2}2(μ-bpm)]: A high-affinity DNA bulge probe {bpm = 2,2′-bipyrimidine; phen = 1,10-phenanthroline}

The binding of the stereoisomers of [{Ru(Me₂bpy)₂}₂(μ-bpm)]⁴⁺, [{Ru(phen)₂}₂(μ-bpm)]⁴⁺ and [{Ru(Me₂phen)₂}₂(μ-bpm)]⁴⁺ (Me₂bpy = 4,4′-dimethyl-2,2′-bipyridine; bpm = 2,2′-bipyrimidine; phen = 1,10-phenanthroline; Me₂phen = 4,7-dimethyl-1,10-phenanthroline) to a tridecanucleotide d(CCGAGAATTCCGG)₂ which contains a single adenine bulge site, and four control dodecanucleotides, have been studied using a fluorescence intercalator displacement (FID) assay. The meso isomer of [{Ru(phen)₂}₂(μ-bpm)]⁴⁺ showed the strongest binding to the bulge-containing tridecanucleotide. In order to gain a greater understanding of the basis of the higher affinity exhibited by the meso isomer towards the bulge sequence, a ¹H NMR study of the binding of the two enantiomers (ΔΔ and ΛΛ) of rac-[{Ru(phen)₂}₂(μ-bpm)]⁴⁺, and the, meso (ΔΛ) diastereoisomer, to the tridecanucleotide d(CCGAGAATTCCGG)₂ was carried out. The NMR results suggest that the meso isomer binds selectively at the bulge site in the tridecanucleotide minor groove, but closer to the 3′-direction and with less structural perturbations of the groove than the ΔΔ and ΛΛ isomers. The results of this study confirm that dinuclear ruthenium complexes have excellent potential as DNA bulge probes, and meso-[{Ru(phen)₂}₂(μ-bpm)]⁴⁺ in particular has a high affinity (1 × 10⁶ M⁻¹) and selectivity for a single adenine bulge site.     

Low-lying electronic states of the ferrous high-spin (S=2) heme in deoxy-Mb and deoxy-Hb studied by highly-sensitive multi-frequency EPR

The low-lying electronic states of the ferrous high-spin heme in deoxy-myoglobin (deoxy-Mb) and deoxy-hemoglobin (deoxy-Hb) were probed by multi-frequency electron paramagnetic resonance (MFEPR) spectroscopy. An unexpected broad EPR signal was measured at the zero magnetic field using cavity resonators at 34-122 GHz that could not be simulated using any parameter sets for the S = 2 spin Hamiltonian assuming spin quintet states in the ⁵B₂ ground state. Furthermore, we have observed novel, broad EPR signals measured at 70-220 GHz and 1.5 K using a single pass transmission probe. These signals are attributed to the ferrous high-spin heme in deoxy-Mb and deoxy-Hb. The resonant peaks shifted to a higher magnetic field with increasing frequency. The energy level separation between the ground singlet and the first excited state at the zero magnetic field was directly estimated to be 3.5 cm^− 1 for deoxy-Hb. For deoxy-Mb, the first two excited singlet states are separated by 3.3 cm^− 1 and 6.5 cm^− 1, respectively, from the ground state. The energy gap at the zero magnetic field is directly derived from our MFEPR for deoxy-Mb and deoxy-Hb and strongly supports the theoretical analyses based on the Mössbauer and magnetic circular dichroism experiments.     

Mixed-valency with cyanides as terminal ligands: Diruthenium(III,II) complexes with the 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine bridge and variable co-ligands (CN vs. bpy or NH3)

New diruthenium complexes (PPN)₄[(NC)₄Ru(μ-bptz)Ru(CN)₄], (PPN)₄1, and [(bpy)₂Ru(μ-bptz)Ru(CN)₄], 2, (PPN⁺ = bis(triphenylphospine)iminium; bptz = 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine; bpy = 2,2′-bipyridine), were synthesised and characterised by spectroscopic and electrochemical techniques. The comproportionation constant K_c = 10^7.0 of the mixed-valent species [(NC)₄Ru(μ-bptz)Ru(CN)₄]³⁻ as obtained by oxidation of 1⁴⁻ in CH₃CN is much lower than the K_c = 10^15.0 previously detected for [(H₃N)₄Ru(bptz)Ru(NH₃)₄]⁵⁺, reflecting the competition between CN⁻ and bptz for the π-electron density of the metals. Comparison with several other bptz-bridged diruthenium(II,III) complexes reveals an approximate correlation between K_c and the diminishing effective π acceptor capacity of the ancillary terminal ligands. In addition to the intense MLCT absorption at λ_max = 624 nm, the main IVCT (intervalence charge transfer) band of 1³⁻ was detected by spectroelectrochemistry at λ_max = 1695 nm (in CH₃CN; ε = 3200 M⁻¹ cm⁻¹). The experimental band width at half-height, Δν_1/2 = 2700 cm⁻¹, is slightly smaller than the theoretical value Δν_1/2 = 3660 cm⁻¹, calculated from the Hush approximation for Class II mixed-valent species. In agreement with comparatively moderate metal-metal coupling, the mixed-valent intermediate 1³⁻ was found to be EPR silent even at 4 K. The unsymmetrical mixed-valent complex [(bpy)₂Ru^II(μ-bptz)Ru^III(CN)₄]⁺, obtained in situ by bromine oxidation of 2 in CH₃CN/H₂O, displays a broad NIR absorption originating from an IVCT transition at λ_max = 1075 nm (ε ≈ 1000 M⁻¹ cm⁻¹, Δν_1/2 ≈ 4000 cm⁻¹). In addition, the lifetime of the excited-state of the mononuclear precursor complex [Ru(bptz)(CN)₄]²⁻ was measured in H₂O by laser flash photolysis; the obtained value of τ = 19.6 ns reveals that bptz induces a metal-to-ligand electronic delocalisation effect intermediate between that induced by bpy and bpz (bpz = 2,2′-bipyrazine) in analogous tetracyanoruthenium complexes.     

Electronic and vibrational analysis of porphyrazine liquid-crystalline structure: Toward photochemical phase switching

The electronic and vibrational Raman spectra of octa-substituted (R = -SC₁₀H₂₁) Co- and Cu-porphyrazines are reported in their solid-state, mesophase, and isotropic liquid forms, as well as in THF solution. Their electronic spectra are composed of traditional Soret (CuS10 = 355 nm, CoS10 = 347 nm) and lower energy Q-bands (CuS10 = 669 nm, CoS10 = 639 nm), as well as a weaker, functionality-specific sulfur n → porphyrin π^∗ feature (CuS10 = 500 nm; CoS10 = 447 nm). In contrast to the broad Q-band for CoS10 in all three neat phases, the lower energy analogue for CuS10 is markedly sharper in the microcrystalline state, but similarly broadens in the mesophase, indicative of long range macrocycle π-π interactions that persist even into the liquid state. The resonance (λ = 647 nm) and off-resonance (λ = 785 nm) Raman spectra of these materials in each phase exhibit four diagnostic vibrations; the C_α-N_m stretch (∼1540-1553) cm⁻¹, C_β-C_β stretch (∼1450 cm⁻¹), C_α-C_β-N_p stretch (∼1300-1315 cm⁻¹), and C_α-C_β stretch (∼1070 cm⁻¹). For CoS10, these vibrations systematically shift to lower energy upon melting, while those for CuS10 collapse to degenerate sets. The differences in the electronic and vibrational profiles as a function of temperature suggest that the mesophase structure is governed by strong axial Co-S interactions for CoS10 which template macrocycle π-π stacking, while for CuS10 the same contacts exist, but they are phase dependent and markedly weaker. These inter-porphyrazine interactions are, therefore, responsible for the distinct differences in the melting and clearing temperatures of their respective mesophases. Finally, based on these diagnostic spectroscopic signatures, a photo-thermal, phase-switching mechanism is demonstrated with λ = 785 nm excitation at reduced temperatures, leading to the ability to spectrally monitor and phase change with a single photon source.     

Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana

The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F₀) and closed (F_M) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F_V = F_M − F₀, over F_M, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F_V/F_M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F_V/F_M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F₀ and F_M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo.     

The multichromophore approach: A case of temperature controlled switching between single and dual emission in Ru(II) polypyridyl complexes

A new series of ruthenium(II) complexes, based on 4′-(9-anthryl)-2,2′:6′,2″-terpyridine (an-tpy), have been synthesized from the Ru(III) precursor (an-tpy)RuCl₃ (4). These new Ru(II) complexes, [(an-tpy)Ru(tpy-pm-R)] (R = H, 3a; Cl, 3b; phenyl, 3c; p-bromophenyl, 3d), have extended π-conjugation through the 5′-substituted pyrimidyl group, and have been characterized by analytical and spectroscopic methods, and X-ray single crystal structure determination for 3b and 3d. Luminescence lifetime measurements have shown that the anthryl chromophore greatly increases room temperature (r.t.) excited-state lifetime, even though it is not directly connected to the ligand involved in the metal-to-ligand charge transfer (³MLCT) emitting state. This result demonstrates that an equilibrium is established between the anthryl triplet state (³an) and the ³MLCT state even though the two chromophores are physically separated by more than one nanometer. At low temperature in rigid matrix, such equilibrium does not take place and emission arises from both ³an and ³MLCT excited states. The temperature dependence of the excited-state equilibration induces a temperature switch from single (room temperature) to dual (77 K) emission behavior.     

Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor

Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 × 10⁶ M⁻¹ s⁻¹ for k_a and 3.15 × 10⁻³ s⁻¹ for k_d. These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k_a, k_d, and K_D as 8.48 × 10⁵ M⁻¹ s⁻¹, 1.7 × 10⁻³ s⁻¹, and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.     

Modulation of trans-to-cis photoisomerization and photoluminescence of 1,2-bis(4-pyridyl)ethylene or 4-styrylpyridine coordinated to fac-tricarbonyl(5-chloro-1,10-phenathroline)rhenium(I)

Photochemical and photophysical properties of fac-[Re(CO)₃(Clphen)(trans-L)]⁺ complexes, Clphen = 5-chloro-1,10-phenathroline and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy, were investigated to complement the understanding of intramolecular energy transfer process in tricarbonyl rhenium(I) complexes having an electron withdrawing group attached to polypyridyl ligands. These new compounds were synthesized, characterized and the photoisomerization quantum yields were accurately determined by ¹H NMR spectroscopy. The true quantum yields for fac-[Re(CO)₃(Clphen)(trans-bpe)]⁺ were constant (Φ = 0.55) at all investigated irradiation wavelengths. However, for fac-[Re(CO)₃(Clphen)(trans-stpy)]⁺, similar true quantum yields were observed only at higher energy irradiation (Φ_{313 nm} = 0.53 and Φ_{365 nm} = 0.57), but it decreased significantly at 404 nm (Φ = 0.41). These results indicated different deactivation pathways for the trans-stpy complex photoisomerization. Quantum yields decreased as the ³IL_trans-L and ³MLCT_Re→NN excited states become closer and the behavior was discussed in terms of the excited state energy gaps. Additionally, luminescence properties of photoproducts, fac-[Re(CO)₃(Clphen)(cis-L)]⁺, were also investigated in different environments to analyze the relative energy of the ³MLCT_Re→Clphen excited state for each compound.     

Ultrafast excited-state dynamics of photoisomerizing complexes fac-[Re(Cl)(CO)3(papy)2] and fac-[Re(papy)(CO)3(bpy)] (papy = trans-4-phenylazopyridine)

The character and dynamics of low-lying electronic excited states of the complexes fac-[Re(Cl)(CO)₃(papy)₂] and fac-[Re(papy)(CO)₃(bpy)]⁺ (papy = trans-4-phenylazopyridine) were investigated using stationary (UV-Vis absorption, resonance Raman) and ultrafast time-resolved (visible, IR absorption) spectroscopic methods. Excitation of [Re(Cl)(CO)₃(papy)₂] at 400 nm is directed to ¹ππ^∗(papy) and Re → papy ¹MLCT excited states. Ultrafast (?1.4 ps) intersystem crossing (ISC) to ³nπ^∗(papy) follows. Excitation of [Re(papy)(CO)₃(bpy)]⁺ is directed to ¹ππ^∗(papy), ¹MLCT(papy) and ¹MLCT(bpy). The states ³nπ^∗(papy) and ³MLCT(bpy) are then populated simultaneously in less then 0.8 ps. The ³MLCT(bpy) state decays to ³nπ^∗(papy) with a 3 ps time constant. ³nπ^∗(papy) is the lowest excited state for both complexes. It undergoes vibrational cooling and partial rotation around the -NN- bond, to form an intermediate with a nonplanar papy ligand in less than 40 ps. This species then undergoes ISC to the ground state potential energy surface, on which the trans and cis isomers are formed by reverse and forward intraligand papy rotation, respectively. This process occurs with a time constant of 120 and 100 ps for [Re(Cl)(CO)₃(papy)₂] and [Re(papy)(CO)₃(bpy)]⁺, respectively. It is concluded that coordination of papy to the Re center accelerates the ISC, switching the photochemistry from singlet to triplet excited states. Comparison with analogous 4-styrylpyridine complexes (M. Busby, P. Matousek, M. Towrie, A. Vl?ek Jr., J. Phys. Chem. A 109 (2005) 3000) reveals similarities of the decay mechanism of excited states of Re complexes with ligands containing -NN- and -CC- bonds. Both involve sub-picosecond ISC to triplets, partial rotation around the double bond and slower ISC to the trans or cis ground state. This process is about 200 times faster for the -NN- bonded papy ligand. The intramolecular energy transfer from the ³MLCT-excited ^∗Re(CO)₃(bpy) chromophore to the intraligand state of the axial ligand occurs for both L = stpy and papy with a comparable rate of a few ps.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

Molecular sensing behavior of the first Mn(I)-compound of di-2-pyridylketone-p-nitrophenylhydrazone (dpknph), fac-[Mn(CO)3(dpknph)Br]

The reaction between [Mn(CO)₅Br] and di-2-pyridylketone-p-nitrophenylhydrazone (dpknph) in diethyl ether under ultrasonic conditions gave fac-[Mn(CO)₃(dpknph)Br] in good yield. Optical and thermodynamic measurements on fac-[Mn(CO)₃(dpknph)Br] in non-aqueous polar solvents revealed reversible interconversion between two intense charge transfer absorption bands due to π-π* (dpk), followed by dpk → nitro intraligand charge transfer transition (ILCT), mixed with metal ligand charge transfer transition (MLCT) due to . In non-polar solvents, a single absorption band appeared. Extinction coefficients of 46 200 ± 2000 and 28 400 ± 2000 M⁻¹ cm⁻¹ were calculated in DMSO for the low- and high-energy electronic states of fac-[Mn(CO)₃(dpknph)Br] using excess NaBF₄. Changes in enthalpy (ΔH^ø) of +14.0 and −12.1 kJ mol⁻¹, entropy (ΔS^ø) of +28.65 and −64.30 J mol⁻¹ K⁻¹, and free energy (ΔG^ø) of +5.48 and +7.08 kJ mol⁻¹ at 298 K were calculated for the interconversion between the high and low energy electronic states of fac-[Mn(CO)₃(dpknph)Br]. These results allow for the use of these systems (fac-[Mn(CO)₃(dpknph)Br] and surrounding solvent or solute molecules) as optical sensors for a variety of physical and chemical stimuli that include metal ions. Group 12 metal ions in concentrations as low as 1.00 × 10⁻⁹ M can be detected and determined using fac-[Mn(CO)₃(dpknph)Br] in dmso in the presence and absence of NaBH₄.     

Effects of steric constraint on chromium(III) complexes of tetraazamacrocycles, 4: Comparison of the trans-difluoro-complexes of tet a, 1,4-C2-cyclam, and 1,11-C3-cyclam

The synthesis and characterization of the trans-difluorochromium(III) complexes of the constrained macrocyclic ligands 1,4-C₂-cyclam = 1,4,8,11-tetraazabicyclo[10.2.2]hexadecane and 1,11-C₃-cyclam = 1,4,8,11-tetraazabicyclo[9.3.3]heptadecane is reported. Only trans complexes are formed, and the structures of both trans-[Cr(1,4-C₂-cyclam)F₂]ClO₄ and trans-[Cr(1,11-C₃-cyclam)F₂]ClO₄ are presented. The photochemical and photophysical behavior of the 1,4-C₂-cyclam and 1,11-C₃-cyclam complexes is compared with that of the corresponding tet a (C-meso-5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane) complex. The constraint imposed by the additional bridging groups of 1,4-C₂-cyclam and 1,11-C₃-cyclam distorts the complexes away from both octahedral symmetry and centrosymmetry, as evidenced by the bond angles that deviate from ideal 90° and 180° values and by a significant distortion of the Cr(N₄) plane. This reduction in symmetry correlates with a monotonic increase in the extinction coefficients in going from the tet a to the 1,11-C₃-cyclam, to the 1,4-C₂-cyclam complex. These three complexes also exhibit large variations in their aqueous room-temperature excited state behavior; namely, the lifetimes of the ²T_1g (O_h) excited states are 30, 60, and 1.0 μs for the tet a, 1,11-C₃-cyclam, and 1,4-C₂-cyclam complexes, respectively. Studies of the excited-state lifetime of these complexes in acidified H₂O/dimethyl sulfoxide over the temperature range between −30 and +95 °C suggest that the 1,4-C₂-cyclam complex accesses a temperature-dependent relaxation mechanism at significantly lower temperatures than do the tet a or 1,11-C₃-cyclam complexes. The emission behavior of these complexes is also solvent-dependent, consistent with established theories that the degree of splitting of the emitting ²T_1g (O_h) state varies with the hydroxylic nature of the solvent.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3₁₀-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min⁻¹ (Tyr), 0.09 min⁻¹ (Arg, β-turns), and 1.88 × 10⁻³ min⁻¹ (β-sheets and 3₁₀-helix); and for TGF-α 0.91 min⁻¹ (Tyr), 0.27 min⁻¹ (Arg, β-turns), and 1.41 × 10⁻⁴ min⁻¹ (β-sheets). The time constants for AR 0.47 min⁻¹ (Tyr), 0.04 min⁻¹ (Arg), and 1.00 × 10⁻⁴ min⁻¹ (buried 3₁₀-helix, β-turns, and β-sheets); for HB-EGF 0.89 min⁻¹ (Tyr), 0.14 min⁻¹ (Arg and 3₁₀-helix), and 1.00 × 10⁻³ min⁻¹ (buried 3₁₀-helix, β-sheets, and β-turns); and for epiregulin 0.16 min⁻¹ (Tyr), 0.03 min⁻¹ (Arg), and 1.00 × 10⁻⁴ min⁻¹ (3₁₀-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.