Photochemical Probes Research Articles

Photocleavage of Lysozyme by Cobalt(III) Complexes

Photochemical reagents that cleave proteins at specific sites (photoproteases) are useful for studying protein structure and protein-ligand interactions. PolyammineCo(III) complexes are tested here as photochemical probes to cleave proteins. Irradiation of a mixture of lysozyme, a model protein, and polyammineCo(III) complexes resulted in the facile cleavage of the peptide backbone. Photocleavage yielded two fragments of molecular weights 10.6 and 3.7 kDa, and these masses sum to the molecular mass of lysozyme (14.3 kDa). No cleavage was detected in the absence of the metal complex, in the dark, or upon irradiation at wavelengths of >420 nm. The photocleavage yield increased with irradiation time and with the concentrations of the metal complex and the protein. N-terminal sequencing of the 10.6 kDa fragment indicated residues that are identical to the N-terminus of lysozyme, and sequencing of the 3.7 kDa fragment indicated Val-Ala-Trp-Arg, an internal sequence of lysozyme. From the known primary sequence of lysozyme and the sequencing data, the cleavage site was assigned to Trp108-Val109. Molecular modeling indicates that the observed cleavage site is within few angstroms from the proposed metal binding site at Glu35-Asp52. This is the first report of the successful photocleavage of proteins, with high selectivity, by transition metal complexes. This novel observation can facilitate the rational design of transition metal complexes for the photochemical footprinting of metal binding sites on proteins.

Local compressibilities in insulin as determined from pressure tuning hole burning experiments and MD simulations

We present a hole burning study on insulin in a glycerol-water solvent by using the intrinsic amino acid tyrosine as a photochemical probe. The focus of the experiments is on the comparative pressure response of the spectral holes for insulin in its native state, in its chemically denatured state and for tyrosine in the glycerol-water solvent. From an analysis of the color effect of the pressure response, we can identify two different spectral ranges characterized by a markedly different sensitivity to pressure. We conclude that at least two tyrosines (or two groups of tyrosines) out of the eight in the insulin dimer are photochemically labeled, and that they are characterized by markedly different compressibilities, namely 0.08 and 0.13 GPa(-1), respectively. An interesting observation concerns the compressibility in the unfolded state: It is significantly lower and closer to the value measured for the pure tyrosine molecule in a glycerol-water solvent. In contrast to the native state, the response of the various tyrosines in the unfolded state to pressure variations is quite uniform. The experiments are compared with MD simulations of monomeric insulin at ambient temperature. The computational results show that the local compressibilities around the different tyrosines vary significantly and that they strongly depend on whether just the first shell of molecules or the first two shells are included in the local volume.

Combinatorial chemical reengineering of the alpha class glutathione transferases.

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.

Detection of residual acidic groups in several poly(N‐alkyl methacrylate)s using photophysical and photochemical probes

AbstractThis work describes three different methods for detecting acidic groups copolymerized in poly(methyl methacrylate), poly(ethyl methacrylate), and poly(n‐butyl methacrylate) chains using molecular spectroscopy. The first was based on the shift of the tautomeric equilibrium of 4‐dimethylaminoazobenzene by acidic groups that modify the absorption band in the UV/vis spectra. We also show that the acidic groups present in the polymer influenced the anti‐syn photoisomerization reaction of this dye. Further, a mercury–dithizonate complex was completely bleached when sorbed in poly(n‐alkyl methacrylate) matrices containing acidic groups. Finally, Nile Blue A was used as a spectrophotometric probe to quantify the amount of acidic groups in these polymers. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 830–838, 2004

Chemistry of bifunctional photoprobes. 6. Synthesis and characterization of high specific activity metalated photochemical probes: development of novel rhenium photoconjugates of human serum albumin and fab fragments.

Functionalization of perfluoro aryl azides by bifunctional chelating agents (BFCAs) capable of forming high specific activity complexes with (99m)Tc (for gamma-imaging) and (188)Re (for radiotherapy) is described. The synthesis of multidonor BFCAs containing N(2)S(2), N(4), and N(3)S donor groups containing imidazole, pyridine, and pyrazine functionalities that may be important for tuning the pharmacokinetic parameters is also described. Functionalization of perfluoro aryl azides at various sites on BFCAs yields novel bifunctional photolabile chelating agents (BFPCAs) that are useful for covalent attachment to biomolecules. A representative Re-BFPCA 8a in a model solvent, diethylamine, proceeded to give a high yield of intermolecular NH insertion product without the decomplexation of the metal ion from 8a. All products originated from the photolysis of 8a in diethylamine are characterized by analytical techniques, and a plausible mechanism of formation of different photolytic products is suggested. The high yield of intermolecular NH insertion of Re-BFPCA 8a is extended to labeling of human serum albumin (HSA) and Fab fragments under aqueous conditions. The photolabeling technology developed here offers a new way to attach diagnostically and therapeutically useful radiotracers (e.g., (99m)Tc, (188)Re) to Fab fragments for potential noninvasive imaging and therapy of cancer.

Synthesis of a [14C]-labelled photoactivatable cyclic tetrapeptide: [carbonyl-14C]-4-benzoylbenzoyl MeSer1-tentoxin

The synthesis of a new photochemical probe for labelling tentoxin 1 binding sites in the soluble part of chloroplast F0F1 ATPsynthase is described. [Carbonyl-14C]-4-benzoylbenzoyl MeSer1-tentoxin 6 was synthesised by coupling MeSer1-tentoxin 2 with [carbonyl-14C]-4-benzoylbenzoic acid 3, itself obtained by coupling [1-14C]-benzoyl chloride with the dilithio derivative of 4-bromobenzoic acid (9 ). Photolysis of the probe in methanolic solution occurs readily upon irradiation with UV light at 366 nm. Copyright © 2000 John Wiley & Sons, Ltd.

Probing Nucleic Acid Structure with Shape‐Selective Rhodium and Ruthenium Complexes

In this unit, transition metal complexes are used as photochemical probes for the structure of RNA and DNA. The transition metal ion provides a rigid substitutionally inert framework and an octahedral geometry for ligand coordination. The complexes can be constructed to define shapes, symmetries, and functionalities that complement those of the nucleic acid target. Complex formation is easily detected by light-induced nucleic acid cleavage. The modular construction of the complexes makes it possible to generate probes to examine a wide variety of structural characteristics of nucleic acids.

A photophysical study of the interactions in poly(styrene-4-sulphonate)-n-butylvinylether block and random copolymers

Interactions between the moieties responsible for the conformations and hydrophobic microdomains in poly(styrene-4-sulphonate) (PSS) and its copolymers with n-butylvinylether (BVE) were studied by their emission spectra and the lifetimes of the phenyl groups and pyrene used as a photochemical probe. The emission spectra of PSS shows bands due to dimers and higher aggregates as well as the characteristic excimer band. At low concentrations, the random copolymers have spectra similar to that of the free monomer, whereas the block copolymers have spectra like that of PSS. At higher concentrations, the random copolymer also shows these excimer bands, due to interchain interactions. Results from the emission of pyrene prove that the behaviour of the copolymers with approximately 40% BVE seems to be relatively independent of having random or block configurations. Except at low concentrations (<0.05 g/dl), where the block copolymer already has a conformation with “stable” hydrophobic microdomains, both types of copolymers behave similarly. There is an initial aggregate equilibrium between individual chains and aggregates, associated with a relocation of the probes. At higher concentrations, both copolymers suffer a severe change in conformation, due to the formation of “stable” hydrophobic microdomains, resulting from interchain interactions. In both cases the lifetimes of pyrene are of the order of 240 ± 10 ns.

Synthesis, UV-visible and electrochemical studies of lipophilic and hydrophilic lanthanide(III) bis(porphyrinates)

In a recent work we described the synthesis and some physicochemical properties of lanthanide(III) monoporphyrinate complexes. The increasing interest in cationic and/or anionic metalloporphyrins due to their biocompatibility with human tissues, their application as intercalating agents or as photochemical probes and NMR imaging agents, prompted us to extend our research to the synthesis and physico-chemical study of hydrophilic lanthanide double-deckers. We present herein the synthesis of four new lanthanide(III) lipophilic 5,10,15,20-tetrapyridyl-bis (porphyrinates) and their easy conversion (upon methylation) to the corresponding water soluble double-deckers. Electrochemical studies were carried out using different polarity media while the optical properties of the complexes were studied in organic and aqueous media.

Synthesis and Enzymatic Phosphorylation of a Photoactivatable Dolichol Analogue

The synthesis of the photochemical probes 6 and 7 is described. These photoprobes are analogues of dolichol and dolichol phosphate, obligatory intermediates in the N-linked glycosylation pathway in the endoplasmic reticulum. The synthesis of 6 and 7 follows a new strategy. It involves the sequential alkylation of a monoterpenoid hydroxysulfonyl dianion with allyl chlorides. The photoreactive group, a 3-(trifluoromethyl)-3-aryldiazirine, was connected to the hydroxylated methyl group of the β-prenyl unit of the fully assembled polyprenyl chain. The photoactivatable dolichol analogue 6 is a substrate for dolichol kinase from yeast membranes, an essential enzyme involved in the N-linked glycosylation pathway.

Targeting the tat-binding site of bovine immunodeficiency virus TAR RNA with a shape-selective rhodium complex

The Tat-binding site of the bovine immunodeficiency virus TAR RNA hairpin has been targeted by Rh(phen) 2phi 3+ (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine), a photochemical probe of RNA tertiary structure. The primary site cleaved by the rhodium complex, upon photoactivation, is U24, a base which participates in the novel base triple (with bases A13 and U10) characteristic of this folded RNA. Δ-Rh(phen) 2phi 3+ binds to this site with an affinity of 2 × 10 6 M −1. Upon mutation of U24 and A13 to A24 and U13, respectively, so that the RNA oligomer is unable to form the base triple, site-specific cleavage by the rhodium complex is abolished. Moreover, as determined through rhodium photocleavage, at a concentration of 20 μM, Rh(phen) 2phi 3+ inhibits specific binding of BIV-Tat peptide (2 μM) to its target site. Thus the rhodium complex, in matching its shape to the opened major groove of the properly folded RNA, specifically targets its site and is able to compete for its target with the BIV-Tat peptide.

Application of Vitamin K3 as a Photochemical Fluorescence Probe in the Determination of Nucleic Acids

ABSTRACT An in situ photochemical fluorescence probe method for the determination of nucleic acids with vitamin k3(VK3) as the photochemical fluorescence probe was developed for the first time. It was based on the conversion of VK3 into an intensively fluorescent product on irradiating with UV radiation. The photochemical reaction is decelerated by nucleic acids. The determination can be carried out by measuring the fluorescence intensity at a fixed time. The calibration graph was linear in the range of 0– 1.5 μg/ml for CT DNA and 0–2.0μg/ml for yeast RNA, the limit of detection was 10 ng/ml for CT DNA and 26 ng/ml for yeast RNA. The kinetic behaviour of the photochemical reaction and the effects of some experimental conditions were investigated and discussed in detail. CT DNA could be determined in the presence of 40%(w/w) yeast RNA and yeast RNA was determined when the content of CT DNA in synthetic samples was below 6%(w/w).

Dynamics of solubilization of naphthalene and pyrene into dodecylammonium trifluoroacetate micelles

A photochemical probe was used to determine the rate constant for a solubilizate molecule to escape from its parent micelle, where the quencher molecules for the excited probe are located exclusively outside of the micelle. Stern–Volmer kinetics of the solubilizates, naphthalene and pyrene, in dodecylammonium trifluoroacetate micelles were analyzed at 35°C in order to determine their exit (k-) and entry rate constants (k+) from and to the micelle. The ion Ag+, with the same charge as the micelle, was used as the quencher for the excited probes. The values of k- (k+) were determined to be 6.11×106 s-1 (2.25×1012 d mol-1 s-1) and 1.39×106 s-1 (1.51×1013 d mol-1 s-1) for naphthalene and pyrene, respectively, from analysis of the Stern–Volmer quenching ratio vs. the quencher concentration in micellar solutions. Analysis using the initial slope of the curve led to slightly lower values for the rate constants. The spontaneous decay rate constants for the excited probes in the aqueous bulk and solubilized in the micelles and the quenching rates of excited probes by the quencher were also determined from this analysis. The same experiments were also conducted with anthracene, but the final kinetic results were not discussed as they could not be readily explained.

A novel host molecule p-[1-(4-hydroxyphenyl)-1-methylethyl]calix[8]arene. Synthesis and complexation properties in non-aqueous polar solution

A novel deep-cavity calixarene, p-[1-(4-hydroxyphenyl)-1-methylethyl]calix[8]arene 1 was synthesized and its complexation with aromatic compounds was examined. Reaction of p-[1-(4-methoxyphenyl)-1-methylethyl]phenol 2 with paraformaldehyde under various conditions only resulted in p-[1-(4-methoxyphenyl)-1-methylethyl]calix[8]arene 3, and no corresponding calix[4]arene and calix[6]arene were detected. Removal of the methyl in the ether groups of 3 afforded 1 in 80% yield. Solubilization, fluorescence and photochemical probe studies demonstrated that 1 forms inclusion complexes with a variety of aromatic compounds in polar non-aqueous solutions.

Mechanistic Studies on the Dual Reaction Pathways of Singlet Excited Dibenzoyl(methanato)boron Difluoride (DBMBF2): Reactions of the Excimer and Exciplexes

The mechanism of the interaction of singlet excited dibenzoyl(methanato)boron difluoride (*DBMBF2 = *A) with olefins and substituted benzenes (SB) was investigated by the determination of effective quenching distances (Reff) from fluorescence monitors in acetonitrile as analyzed by the Smoluchowski−Collins−Kimball equation and by solvent effects on the dichotomy of the formation of cycloadducts and [4 + 2] dimers from 1,3-cyclohexadiene (CHD) and 1,3-cyclooctadiene (1,3-COD). The correlation of Reff with directly demonstrates that the reactants have to get closer to affect electron transfer from these substrates as the driving force become more positive and coalesce on exciplexes. Both photophysical and photochemical probes converge on the partition of encounter pairs to the SSRIP (the ket pathway which leads to radical ion reactions and nonfluorescent) and exciplexes (the kex pathway which leads to cycloadducts and fluorescent species); the former pathway was promoted by low redox energies of donor−accep...

A Photochemical Probe for Single Electron Transfer in Nucleophilic Aliphatic Substitution: Evidence for Geminate Radical Coupling in the Solvent Cage

A Photochemical Probe for Single Electron Transfer in Nucleophilic Aliphatic Substitution: Evidence for Geminate Radical Coupling in the Solvent Cage

STRUCTURE‐SPECIFIC BINDING and PHOTOSENSITIZED CLEAVAGE OF BRANCHED DNA THREE‐WAY JUNCTION COMPLEXES BY CATIONIC PORPHYRINS

The interactions of cationic porphyrins with DNA oligonucleotides that form branched, three-way junction complexes (TWJ) were investigated using native gel electrophoresis, absorption spectroscopy and photochemical probing using DNA sequencing techniques. Meso-tetra(para-N- trimethylaniliniumyl)porphine (TMAP), meso-tetra(4-N-methylpyridiniumyl)porphine (T4MPyP) and meso-tetra(3-N-methylpyridiniumyl)porphine(T3MPyP) were found to bind more tightly to DNA TWJ than to DNA duplexes. The binding to the junction DNA persists at high ionic strength, conditions that greatly decrease porphyrin binding affinity to duplex DNA. THe TWJ DNA binding sites of TMAP and T4MPyP were localized to the junction region based on the observation of site- and structure-specific, porphyrin-sensitized photodamage to guanosine residues flanking the junction region.

Identification of Transmembrane Domains of the Red Cell Calcium Pump with a New Photoactivatable Phospholipidic Probe

The membrane-associated regions of the human erythrocyte Ca 2+ pump were investigated by hydrophobic photolabeling. Purified Ca 2+ pump was reconstituted in asolectin vesicles loaded with [ 3H]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Sec. 115, 3458-3474, 1993). After photolysis and SDS-PAGE analysis, a significant light-dependent labeling of the Ca 2+ pump was found. Controlled proteolysis of the photoadduct with trypsin or protease V8 followed by SDS-PAGE and immunoblotting yielded individual labeled fragments. The labeling pattern indicated the existence of three sequential clusters of transmembrane regions, consistent with the current model for the topography of this enzyme.

Photochemical probes of intramolecular electron and energy transfer

Photochemically reactive systems have been developed which allow the quantitative assessment of free energy/rate correlations for intramolecular excited state electron and energy transfer (ET and E NT, respectively). Preliminary results are described on systems which contain the (diimine)Re I(CO) 3-chromophore covalently linked to either an electron donor or an (excited state) energy acceptor. The electron donor and the energy acceptor units undergo rapid chemical reaction upon being oxidized (via photoinduced ET) or photochemically excited (by intramolecular E NT). Quantitative examination of the photophysics and photochemical reaction quantum yields in these molecules allows evaluation of the rates of intramolecular ET and E NT.

Photochemical probes of conformational mobility in liquid crystals. The photochemistry of β-phenyl-4-methoxypropiophenone in the nematic and smectic B phases of CCH-n and OS-nm liquid crystals

The photochemistry of β-phenyl-4-methoxypropiophenone (1) in the isotropic and liquid crystalline (nematic and smectic B) phases of trans, trans-4′-alkyl-(1,1′-bicyclohexyl)-4-carbonitrile (CCH-n) and trans-4-alkylcyclohexyl-trans-4′-alkylcyclohexylcarboxylate (OS-nm) mesogens has been investigated using nanosecond laser flash photolysis techniques. Solubility limits of 1 as a function of temperature in the smectic phases have been rigorously determined by 2H NMR spectroscopy, using α-, methoxy-, and β-phenyl-deuterated analogs of the ketone. Triplet decay of 1 in the smectic (Crystal B) phase of CCH-4 is multiexponential, suggesting that the ketone is solubilized in a distribution of conformations in the highly-ordered liquid crystalline lattice. The average triplet lifetime of 1 at various temperatures throughout the smectic phase is about a factor of three longer than that in homologous nematic and isotropic phases at the same temperature. The differences in the Arrhenius parameters for triplet decay in the smectic and nematic/isotropic phases are very small, however. The present results for 1 in the smectic phase of the CCH-n mesogens correct the interpretation of previously reported data obtained with higher concentration mixtures, and afford an accurate indication of the true effects of smectic B liquid crystalline order on the conformation-dependent triplet state behavior of this molecule. In OS-nm liquid crystals, triplet decay follows clean first order kinetics in all phases, but the variations in Arrhenius dependence with solvent phase are similar to those observed in the CCH-n liquid crystals. Key words: smectic, liquid crystals, 2H NMR, nanosecond laser flash photolysis, intramolecular triplet quenching, photochemistry.