Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase

Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [(32)P]3a or of [(32)P]3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.     

Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg).

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.     

ARGINYL-tRNA SYNTHETASE FROM Escherichia coli AFFINITY LABELING WITH 3''''-OXIDIZED tRNA~(Arg)

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehydederivative of tRNA~(Arg) (tRNA_(ox)~(Arg)) resulted in the complete inactivation of the ATP-PPi ex-change and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gelelectrophoresis of the ArgRS-tRNA_(ox)~(Arg) covalent complexes indicated that two bands simulta-neously appeared on the gel parallel with inactivation corresponding to different higher mo-lecular weights. This result was different from that of the other aminoacyl-tRNA synthetaselabeling systems as previously reported. Upon the ribonuclease treatment of the modifiedArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities wererecovered. During the whole process of labeling and RNase treatment, the two activities ofthe enzyme were closely associated.     

Designing systems for a multiple use of light signals.

The photochemical and photophysical behavior of two dendrimers consisting of a benzophenone core and branches that contain dimethoxybenzene units has been investigated. Such dendrimers can undergo a variety of photochemical and photophysical processes, namely: 1) quenching of the fluorescence and phosphorescence of the dimethoxybenzene units by energy transfer to the benzophenone core (antenna effect), 2) direct and sensitized phosphorescence (and delayed fluorescence) of the benzophenone core, 3) hydrogen abstraction by the triplet excited state of the benzophenone core from solvent molecules, 4) intramolecular hydrogen abstraction by the triplet excited state of the benzophenone core from the dendrimer branches, 5) quenching of the phosphorescence and hydrogen abstraction reaction of the benzophenone core by energy transfer to terbium ions and dioxygen; 6) conversion of the UV light absorbed by the dendrimer branches into visible (Tb3+) or near infrared (O2) emission via the sequence of processes 1) and 5). The results obtained emphasize the great potential of suitably designed dendrimers for a multiple use of light signals.     

Chymotrypsin immobilization on epoxy monolithic silica columns: development and characterization of a bioreactor for protein digestion

The preparation and optimization of a new monolithic chymotrypsin bioreactor for online protein digestion is described. Silica monolithic supports have been activated with epoxide functionalities following an optimized in situ procedure and used for covalent immobilization of chymotrypsin in one-step reaction under different conditions. A total of four bioreactors were prepared and characterized in terms of the amount of immobilized enzyme and apparent active units by using a standard substrate, N-benzoyl-L-tyrosine p-nitroanilide (BTPNA). The stability of the bioreactors was evaluated and the morphology of the support after immobilization and use was studied by SEM analysis. The proteolytic activity of the optimized chymotrypsin bioreactor was evaluated using HSA as a model protein by online coupling of the bioreactor with LC-ESI-MS. With the online protocol, complete protein digestion in 120 min was achieved with a sequence coverage of 97.3%.     

Affinity labeling of a single guanine bulge

We have developed a conceptually new method for the selective labeling of duplex DNA containing a guanine bulge with a masked form of fluorescent 2-amino-1,8-naphthyridine. A naphthyridine derivative 2 tethering DNA-alkylating epoxide was synthesized from (S)-epichlorohydrin and naphthyridine derivative 1, which selectively binds to the guanine bulge duplex. HPLC analysis of the labeling reaction of bulge duplex d(GTT GTGTTG GA)/d(CAA CA A ACC T) (TGT/A_A) with 2 showed a formation of 2-TGT adduct for the guanine bulge. The reaction proceeded for the guanine bulge and a reduced efficiency for the cytosine bulge, but not at all for adenine and thymine bulges. The site of covalent bond formation in 2-TGT was unambiguously identified at the guanine two bases away from the bulge by the use of MALDI-TOF MS analysis of the oligomer fragments produced by strand scission. The labeling reaction was also observed for the guanine bulge flanking two G-C base pairs (CGC/G_G), producing a 2:1 adduct (2.2-CGC). Upon hydrolysis of 2-TGT and 2.2-CGC with concentrated hydrogen chloride, a release of fluorescent 2-aminonaphthyridine from the adduct was clearly detected, verifying a concept of an affinity labeling of the guanine bulge with a masked fluorescent chromophore. The affinity labeling targeting of the guanine bulge is a conceptually novel method for the postsynthetic labeling of DNA. Hybridization, to the target sequence, of a probe DNA possessing one extra guanine especially between two cytosines provides a unique site for the labeling by masked fluorophore 2. The technique may have broad application in genetic typing without using a conventional synthesis of fluorescent-labeled DNA oligomers.     

Photochemical Labeling: Can Photoaffinity Labeling be Differentiated from Site-Directed Photochemical Coupling?

Site-directed photochemical labeling is a methodology designed to irreversibly and specifically label, through the action of light, a ligand binding site of a biological mac-romolecule. Photoaffinity labeling, a widely used site-directed labeling methodology, uses photosensitive ligand analogs generally obtained after chemical modification of the ligand by introducing an appropriate photoactivata-ble moiety. This methodology can be applied to natural ligands showing inherent photosensitivity, without any additional modification, and which can be linked efficiently to their receptor binding site by direct photoac-tivation. The emergence of an alternative methodology that links nonphotosensitive ligands to their receptors has raised the question of their potential use and their mechanisms of photocoupling. This article presents a series of examples that are meant to compare the general characteristics of the different site-directed labeling reactions and proposes distinct photochemical activation processes between photoaffinity labeling and site-directed photochemical coupling reactions. We suggest in particular that the former is necessarily a ligand-mediated activation process while the latter might involve a receptor-mediated mechanism.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

Stepwise Versus Concerted Mechanisms in General‐Base Catalysis by Serine Proteases

General‐base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp‐His‐Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser‐His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pK_a values of catalytic residues were QM calculated by the MD‐QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pK_a values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.     

Attachment of Polymer Films to Aluminium Surfaces by Photochemically Active Monolayers of Phosphonic Acids

Summary: We describe a simple photochemical process that allows the covalent attachment of a variety of different polymers at room temperature onto aluminium surfaces. The system is based on a photoreactive benzophenone derivative that is bound to aluminium surfaces by a phosphonic acid anchor. The synthesis of the phosphonic acid is described and the immobilization of this compound is studied by X‐ray photoelectron and FT‐IR spectroscopy. After deposition of the polymeric coating, UV light illumination at 365 nm, and solvent extraction of the substrate, polymer monolayers are obtained that are chemically bound to the surface.

Covalent attachment of polymers to the aluminium‐bound benzophenone phosphonic acid.     

Non-specific binding in the affinity chromatography of chymotrypsin

The strong and specific binding of chymotrypsin on chromatographic columns containing agarose substituted with N--amino Caproyl- -tryptophan methyl ester is abolished when the -amino groups on the surface of the enzyme are reacted with acetic anhydride. Because the catalytic properties of the acetylated chymotrypsin are identical to those of the underivatized enzyme, it is concluded that the high affinity of chymotrypsin for this column is not due solely to biospecific inhibitor binding, which is by itself very weak, but requires reinforcement through weak non-specific interactions with the column support. It is postulated that these non-specific interactions include electrostatic interactions between agarose matrix and positively charged lysyl residues on the enzyme. The results demonstrate for the first time that residues on the surface of an enzyme not associated with its active site can play an important role in some chromatographic systems previously thought to be based on purely biospecific interactions.     

PHOTOCHEMICAL REACTIONS OF AROMATIC KETONES WITH NUCLEIC ACIDS AND THEIR COMPONENTS I—. PURINE AND PYRIMIDINE BASES AND NUCLEOSIDES*.

Abstract— The photochemical reactions of benzophenone and acetophenone with purine and pyrimidine derivatives in aqueous solutions have been investigated by flash photolysis and steady-state experiments. Upon excitation of these two ketones in aqueous solutions, two transient species are observed: molecules in their triplet state and ketyl radicals. The triplet state lifetimes are 65 μsec for benzophenone and 125 μsec for acetophenone. The ketyl radicals disappear by a second order reaction, controlled by diffusion. In the presence of pyrimidine derivatives, the triplet state is quenched and the ketyl radical concentration is decreased without any change in its kinetics of disappearance. Ketone molecules in their triplet state react with purine derivatives leading to an increase in the yield of ketyl radicals due to H-atom abstraction from the purines. Steady-state experiments show that benzophenone and acetophenone irradiated in aqueous solution at wavelengths longer than 290 nm undergo photochemical reactions. The rate of these photochemical reactions is increased in the presence of pyrimidine derivatives and even more in the presence of purine derivatives. Following energy transfer from the triplet state of benzophenone to diketopyrimidines, cyclobutane dimers are formed. The energy transfer rate decreases in the order orotic acid > thymine > uracil. Benzophenone molecules in their triplet state can also react chemically with pyrimidine derivatives to give addition photoproducts. All these results are discussed with respect to photosensitized reactions in nucleic acids involving ketones as sensitizers.     

DEVELOPMENT OF A PLAQUE REDUCTION ASSAY AND APPLICATION TO THE STUDY OF PSORALEN-DAMAGED DNA

Abstract— We have developed a procedure called a plaque reduction assay to assess the biological activity of duplex circular DNA modified by covalent adduct formation with psoralen derivatives. The replicating form (RF) of bacteriophage DNA modified by photochemical addition of a psoralen derivative was introduced into bacterial cells using the CaCI₂ transfection method. The transfected cells. plated upon a confluent lawn of cells permissive for the bacteriophage in the inoculum, provided a measure of the reduction in infectivity of the RF DNA which resulted from its covalent modification. Use of this assay is illustrated in studies which screened and compared the activities of several recently synthesized psoralen derivatives. We describe two new compounds. β-(8-psoralenoxy)-ethanol and β-(8-psoralenoxy)ethylamine that are significantly more active than either 8-methoxypsoralen or trioxsalen in the biological assay     

HYDROGEN ABSTRACTION FROM LIPIDS BY TRIPLET STATES OF DERIVATIZED BENZOPHENONE PHOTOSENSITIZERS

Laser photolysis techniques have been used to characterize the reactivity of triplet state lipoidal benzophenone derivatives toward fatty acids and glycerides in benzene solution. The reactivities of benzophenone-4-heptyl-4'-pentanoic acid (BHPA) toward fatty acid compounds having different configurations of olefinic bonds have been determined. The rates of hydrogen abstraction are found to be lower when compared with similar measurements using benzophenone alone. However, the contribution of physical quenching of the triplet derivative by double bonds also appears to be slightly lower than that found with benzophenone itself. The hydrogen abstraction efficiencies of three other benzophenone derivatives toward linoleic acid in benzene have also been measured. For benzophenone incorporated into a fatty acid molecule, there is a limited relationship between structure and photoreactivity. Finally, these sensitizers have been incorporated into mixed SDS/linoleate micelles to determine the effects of molecular organization on photochemical behavior of the sensitizer and lipid.     

On Penicillin‐Binding Protein 1b Affinity‐Labeling Reagents

The synthesis of some 3‐aryl‐3‐(trifluoromethyl)3H‐diazirine and benzophenone‐based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity‐labeling experiments were carried out.     

Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.     

One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.     

Preparation of anhydrochymotrypsin diol silica as selective adsorbent for affinity chromatography of peptides with aromatic amino acids at C-termini

Summary Anhydrochymotrypsin (AHC), a catalytically inert derivative of chymotrypsin in which the serine-residue active site has been converted chemically to a dehydroalanine residue, was immobilized on diol silica by activation with trifluoroethanesulfonyl chloride. A AHC-diol-silica column was used for high-performance affinity chromatographic separation of peptides with aromatic amino acids at their C-termini from other peptides. Faster separations were achieved.     

Intramolecular interactions in the triplet excited states of benzophenone-thymine dyads

Time-resolved and product studies on the synthesized dyads 1 and 2 have provided evidence that the benzophenone-to-thymine orientation strongly influences intramolecular photophysical and photochemical processes. The prevailing reaction mechanism has been established as a Paterno-Büchi cycloaddition to give oxetanes 3-6; however, the ability of benzophenone to achieve a formal hydrogen abstraction from the methyl group of thymidine has also been evidenced by the formation of photoproducts 7 and 8. These processes have been observed only in the case of the cisoid dyad 1. Adiabatic photochemical cycloreversion of the oxetane ring is achieved upon direct photolysis to give the starting dyad 1 in its excited triplet state. The photobiological implications of the above results are discussed with respect to benzophenone-photosensitized damage of thymidine.     

Photolysis of Regioisomeric α,α-Diphenyl-Substituted Diazotetrahydrofuranones: Primary and Secondary Photochemical Processes

The formation of C–H insertion products in the course of direct photolysis of α,α-diphenylsubstituted diazo ketones of the tetrahydrofuran series was rationalized by secondary photochemical processes which give rise to benzophenone acting as a sensitizer. Triplet excited states of diazo ketones generated by the action of benzophenone are capable of undergoing bimolecular transformations.