Detection of triplet repeat sequences in the double-stranded DNA using pyrene-functionalized pyrrole-imidazole polyamides with rigid linkers

Methods for sequence-specific detection in double-stranded DNA (dsDNA) are becoming increasingly useful and important as diagnostic and imaging tools. Recently, we designed and synthesized pyrrole (Py)-imidazole (Im) polyamides possessing two pyrene moieties, 1, which showed an increased excimer emission in the presence of (CAG)(12)-containing oligodeoxynucleotides (ODN) 1 and 2. In this study, we synthesized bis-pyrenyl Py-Im polyamides with rigid linkers 2, 3, and 4 to improve their fluorescence properties. Among the conjugates, 2 showed a marked increase in excimer emission, which was dependent on the concentration of the target ODN and the number of CAG repeats in the dsDNA. Unlike conjugate 1, which has flexible linkers, the excimer emission intensity of 2 was retained at over 85%, even after 4h. Py-Im polyamides have the potential to be important diagnostic molecules for detecting genetic differences between individuals.     

Fluorescent phosphonate labels for serine hydrolases. Kinetic and spectroscopic properties of (7-nitrobenz-2-oxa-1,3-diazole)aminoalkyl methylphosphonofluoridates and their conjugates with acetylcholinesterase molecular forms

The synthesis, kinetic, and spectral characterization of (7-nitrobenz-2-oxa-1,3-diazole)aminoethyl and (7-nitrobenz-2-oxa-1,3-diazole)aminopentyl methylphosphonofluoridate are described. These homologous organophosphorous agents contain the environmentally sensitive 7-nitrobenz-2-oxa-1,3-diazole chromophore. They inhibit acetylcholinesterase from Torpedo at rates exceeding 10(7) M-1 min-1 to form long-lived conjugates with one chromophore/80-kilodalton subunit. The intensity, position, and line width of the absorption spectra of the conjugates and reactivation kinetics in the presence and absence of the bisquaternary oxime 1,1'-trimethylene-bis(4-formylpyridinium bromide) dioxime indicate that these agents form conjugates in which the NBD-aminoalkyl moieties experience distinctive microscopic environments within the active center. NBD-aminoethyl methylphosphono-acetylcholinesterase undergoes oxime-induced as well as spontaneous reactivation at rates that are 3.6 and 35 times faster, respectively, than the corresponding rates measured for the NBD-aminopentyl conjugate. Hence, reactivation exhibits a marked dependence on structure of the methylphosphonate. Fluorescence emission at wavelengths greater than 520 nm is highly quenched and exhibits quantum efficiencies of less than 5%. Absorption maxima for the covalent NBD-aminoethyl methylphosphono-acetylcholinesterase appear at 475-480 nm while those for the corresponding NBD-aminopentyl methylphosphono-acetylcholinesterase appear at 485-490 nm. Bandwidths of the absorption maxima are substantially broader for the acetylcholinesterase adduct with NBD-aminoethyl methylphosphonofluoridate (3870 cm-1) than for the enzyme adduct with NBD-aminopentyl methylphosphonofluoridate (2870 cm-1). The CD spectrum of NBD-aminopentyl methylphosphono-acetylcholinesterase shows optical activity coincident with the shape and position of the absorption spectrum. In contrast, in addition to optically active transitions at the absorption maxima, the CD spectrum of NBD-aminoethyl methylphosphono-acetylcholinesterase shows intense optical activity at 430 nm, a wavelength region coincident with the region of spectral broadening. The spectral properties of alpha-chymotrypsin conjugates formed by reaction with the two probes are different, and the respective spectra differ also from those observed for the acetylcholinesterase conjugates. These results indicate that there is a reciprocal relationship between the structure of the probe and the structure of the active center.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bis-pyrene-labeled oligonucleotides: sequence specificity of excimer and monomer fluorescence changes upon hybridization with DNA

The design, synthesis, and properties of a new pyrene excimer-forming probe of DNA have been described. 2,2-(Aminomethyl)propanediol was converted by the reaction with 1-pyrenebutylic acid to bis-pyrene-modified propanediol as a fluorescent non-nucleosidic linker. The bis-pyrene-modified linker can be incorporated via phosphoramidite chemistry into the 5'-terminal or internal positions of oligonucleotides (ODNs). The terminally modified ODNs showed almost similar affinity for complementary DNA when compared with the corresponding unmodified ODNs. The duplexes containing the bis-pyrene in the main chain exhibited higher melting temperatures relative to the corresponding duplexes containing propanediol linker at the same position. The UV and CD spectral studies indicate that the stacking interactions between the pyrene and DNA bases occur in the internally modified duplex and do not in the terminally modified duplex. The bis-pyrene modified linker itself displays excimer (E at 480 nm) and monomer (M at 380 nm) emission in a quantum yield (QY) of 0.17 and the E/M intensity ratio of 15. Incorporation of this linker into the terminal or internal positions of ODNs reduced the QY (0.003-0.009) and the E/M ratio (0.3-0.8). While small changes in the QY and E/M ratio was obtained in binding of the internally labeled ODNs to DNA, up to 27-fold increase in the QY and 17-fold increase in the E/M ratio was observed upon hybridization of the terminally labeled ODNs with DNA. The excimer and monomer fluorescence changes were found to be sensitive to a mismatch base present in the target DNA. The bis-pyrene-modified ODNs thus provide a sequence-sepcific fluorescent probe of DNA.     

Spectrofluorometric measurements of the dispersion state of pyrenedodecanoic acid and its uptake by cultured cells and liposomes

Pyrene dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene is covalently linked, showed a considerable increase in fluorescence when the probe was introduced into a hydrophobic environment. Also, when closely packed in an aggregate, an energy transfer between two adjacent molecules of pyrene occurred, resulting in a shift of the peak of the emission spectrum from 378 nm ('monomeric') to 475 nm ('excimeric'). These two respective properties were utilized for the following: (a) A spectrofluorometric measurement of the critical micellar concentration (CMC) of the pyrene fatty acid, defined as the concentration at which the 475 nm emission peak appeared as a consequence of the aggregation of P12 molecules in aqueous solution to form micelles; the CMC of P12 was found to be in the range of 1 to 2 microM. (b) The penetration of P12, from an aqueous solution or dispersion, into unilamellar phospholipid vesicles was determined by monitoring the increase of the fluorescence at 378 nm. The fluorescence increase was time-dependent and proportional to the respective concentrations of P12 or phospholipid vesicles. Substituting the neutral phosphatidylcholine with the negatively-charged phosphatidylserine vesicles resulted in a slower rate as well as lesser total uptake of P12. (c) The uptake of P12 by cells was accompanied by an increase in the monomeric fluorescence emission intensity. Using cells in suspension, this could be followed continuously in a spectrofluorometer equipped with a recorder. The uptake was found to be time-dependent and proportional to P12 concentration.     

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure

Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λ_max 382 nm) and a broad emission peak with λ_max 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.     

Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. V. Effect of the structure of the target DNA. Quantitative photomodification]

A sensitized photomodification of several single-stranded target DNAs by binary systems of oligonucleotide conjugates complementary to the adjacent regions of DNA was performed. One of the conjugates contained a sensitizer (pyrene, anthracene, or 1,2-benzanthracene), and another conjugate contained a photoreagent 4-azidotetrafluorobenzalhydrazone. The sensitized photomodification is initiated by irradiation at 365-580 nm due to effective energy transfer from the excited sensitizer to the photoreagent in a complementary complex of the binary system with the target DNA where the sensitizer and photoreagent are brought sterically together. Conditions for the quantitative photomodification of a single-stranded DNA by the binary system of oligonucleotide conjugates were found. The maximum degree of photomodification depends on the number of guanosine residues in the (pG)n sequence of the target DNA at the modification site: at n = 1 the yield of covalent adducts was 62-68%, at n = 2, 75-82%, and at n = 4, 98-99%.     

Preparation and evaluation of ODN conjugates with polycation comb-type copolymer.

Oligodeoxynucleotide (ODN) conjugates with the polylysine comb-type copolymer having an ability to promote and stabilize duplex and triplex DNA formation were prepared. 5'-Aminated ODN was succinylated with succinic anhydride. The resulting ODNs having carboxyl terminus were coupled with epsilon-amino groups of the comb-type copolymer using water soluble carbodiimide. The conjugate free from unconjugated ODNs was obtained by gel permeation chromatography. The resulting conjugate maintains ability to form duplex and triplex DNA as estimated by melting curve analysis. Both specificity and stability of the triplex DNA formation were increased by employing the ODN-copolymer conjugates compared to those with their mixture.     

PAH biotransformation in terrestrial invertebrates--a new phase II metabolite in isopods and springtails

Soil-living invertebrates are exposed to high concentrations of contaminants accumulating in dead organic matter, such as polycyclic aromatic hydrocarbons (PAHs). The capacity for PAH biotransformation is not equally developed in all invertebrates. In this paper, we compare three species of invertebrates, Porcellio scaber (Isopoda), Eisenia andrei (Lumbricidae) and Folsomia candida (Collembola), for the metabolites formed upon exposure to pyrene. Metabolic products of pyrene biotransformation in extracts from whole animals or isopod hepatopancreas were compared to those found in fish bile (flounder and plaice). An optimized HPLC method was used with fluorescence detection; excitation/emission spectra were compared to reference samples of 1-hydroxypyrene and enzymatically synthesized conjugates. Enzymatic hydrolysis after fractionation was used to demonstrate that the conjugates originated from 1-hydroxypyrene. All three invertebrates were able to oxidize pyrene to 1-hydroxypyrene, however, isopods and collembolans stood out as more efficient metabolizers compared to earthworms. In contrast to fish, none of the invertebrates produced pyrene-1-glucuronide as a phase II conjugate. Both Collembola and Isopoda produced significant amounts of pyrene-1-glucoside, whereas isopods also produced pyrene-1-sulfate. A third, previously unknown, conjugate was found in both isopods and springtails, and was analysed further using electrospray and atmospheric pressure chemical ionisation mass spectrometry. Based on the obtained mass spectra, a new conjugate is proposed: pyrene-1-O-(6"-O-malonyl)glucoside. The use of glucose-malonate as a conjugant in animal phase II biotransformation has not been described before, but is understandable in the microenvironment of soil-living invertebrates. In the earthworm, three other pyrene metabolites were observed, none of which was shared with the arthropods, although two were conjugates of 1-hydroxypyrene. Our study illustrates the great variety of the still unexplored metabolic diversity of invertebrate xenobiotic metabolism.     

Pyrene eximer mapping in cultured fibroblasts by ratio imaging and time-resolved microscopy

The kinetics of pyrene eximer formation provide a measure of lateral diffusibility in bilayer membranes. Swiss 3T3 fibroblasts were labeled with pyrene, pyrenedecanoic acid (PDA) and 1,3-bis(1 pyrene) propane (BPP) by incubation in the presence of Pluronic F127. Single-cell emission spectra obtained by epifluorescence microscopy (excitation 350 nm) with photodiode array detection showed monomer (380-420 nm) and eximer (475 nm) peaks. The eximer-to-monomer fluorescence ratio (E/M) increased with increasing temperature and loading time. Time-resolved microscopy studies of fibroblasts labeled with PDA for 15 min gave monomer and eximer lifetimes of 101 and 78 ns, respectively, with a monomer-to-eximer conversion rate of 0.02 ns-1. E/M ratio images were obtained with a microchannel plate intensifier and CCD camera at 350-nm excitation and 405 +/- 5 nm (monomer) and greater than 470-nm (eximer) emission wavelengths. E/M ratios of PDA showed spatial variation across the cell with highest ratios at the peripheral plasma membrane. These results establish the methodology to label cells with pyrene eximer-forming probes and to image eximer distributions in membranes of intact cultured cells. Eximer-to-monomer fluorescence ratios are sensitive to maneuvers that alter the membrane physical state and should be of utility in examining the cellular regulation of membrane fluidity.     

Fluorescent tandem phycobiliprotein conjugates. Emission wavelength shifting by energy transfer.

A fluorescent tandem phycobiliprotein conjugate with a large Stokes shift was prepared by the covalent attachment of phycoerythrin to allophycocyanin. The efficiency of energy transfer from phycoerythrin to allophycocyanin in this disulfide-linked conjugate was 90%. A distinctive feature of this phycocyanin conjugate is the wide separation between the intense absorption maximum of phycoerythrin (epsilon = 2.4 x 10(6) cm-1 M-1 at 545 nm) and the fluorescence emission maximum of allophycocyanin (660 nm). Energy transfer from a donor to a covalently attached acceptor can be used to adjust the magnitude of the Stokes shift. Tandem phycobiliprotein conjugates can be used to advantage in fluorescence-activated cell sorting, fluorescence microscopy, and fluorescence immunoassay analyses.     

Benzo(a)pyrene oxidation, conjugation and disposition in the isolated perfused rabbit lung: role of the glutathione S-transferases

The isolated perfused rabbit lung metabolised 7--11 % of 20 mumol of [14C]-benzo(a)pyrene added in the perfusion medium in 1 h. The major metabolite formed was 3-hydroxybenzo(a)pyrene, both free (30--40 % of the total metabolites) and conjugated (4 % of total metabolites). Quinones comprised 15 % of the total and metabolism at the 9, 10 position accounted for a further 10 %. Forty per cent of the water-soluble metabolites was chromatographically identical to the glutathione conjugate of benzo(a)pyrene 4,5-oxide. Sulphate and glucuronide conjugates were formed in small but detectable amounts, principally from phenols, but also from dihydrodiols. After 1 h the more water-soluble conjugates had diffused from the lung into the perfusion medium, but the majority (60--90 %) of the metabolic products were still concentrated within the lung. The lung's limited ability to conjugate its major metabolites of benzo(a)pyrene with sulphuric or glucuronic acid, coupled with slow elimination of the products formed, particularly dihydrodiols may contribute to the susceptibility of this organ to polycyclic aromatic hydrocarbon-induced carcinogenesis.     

Conformational differences between aged and nonaged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin as detected by optical spectroscopy

Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.     

Fluorescent ligands for the hemagglutinin of influenza A: synthesis and ligand binding assays

Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a delta-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low microM range. Binding was accompanied by approximately 4.5-fold fluorescence enhancement for the dansyl conjugate 1 and approximately 3-fold fluorescence quenching for the pyrene conjugate 3.     

A sulfhydryl-reactive ruthenium (II) complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.     

Pyrene excimer fluorescence as a probe for parallel G-quadruplex formation

The formation and properties of G-quadruplex structures from short single-strand oligonucleotide conjugates possessing two to four guanines and a 5'-terminal pyrenebutanol are reported. The 4-G conjugate forms a stable G-quadruplex under low or high potassium ion concentrations, whereas the 3-G conjugate forms a stable G-quadruplex only in the presence of high potassium. The 2-G conjugate fails to form a stable G-quadruplex even at low temperature and high potassium concentration. Both pyrene monomer and excimer fluorescence are observed for the G-quadruplex structures, whereas only monomer fluorescence is observed for the single-strand conjugates. Thus, pyrene excimer fluorescence can be used as a probe for the formation of G-quadruplex structures. The excimer/monomer intensity ratios for the G-quadruplex structures are dependent upon both the temperature and potassium or lithium salt concentration. The salt effect is attributed to a change in the structure of the hydrophobic pyrene chromophores, which are assembled on the 5'-face of the G-quadruplex as a consequence of electrostriction.     

2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.     

Design of a new fluorescent probe: Pyrrole/imidazole hairpin polyamides with pyrene conjugation at their γ-turn

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py–Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py–Im polyamides (1–5). We evaluated the steady state fluorescence of the synthesized conjugates (1–5) in the presence and absence of oligodeoxynucleotides 5′-CGTATGGACTCGG-3′ (ODN 1) and 5′-CCGAGTCCATACG-3′ (ODN 2) and observed a distinct increase in emission at 386 nm with conjugates 4 and 5. Notably, conjugate 5 that contains a β-alanine linker had a stronger binding affinity (K_D = 1.73 × 10^?8 M) than that of conjugate 4 (K_D = 1.74 × 10^?6 M). Our data suggests that Py–Im polyamides containing pyrene fluorophore with a β-alanine linker at the γ-turn NH₂ position can be developed as the competent fluorescent DNA-binding probes.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged α-chloroaldehyde

We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selective interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of α-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared for the synthesis of photoresponsive α-chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitrobenzyl)acetal group of a PCA-thymidine conjugate was completely removed by UV irradiation at 365nm (400mW/cm(2)) for 1min. Photo-cross-linking studies revealed that PCA-ODN selectively reacted with the target nucleotides having an adenine or a cytosine moiety at the frontal position of the α-chloroaldehyde group.     

Conformational change at the active center of serine-proteases upon their binding to alpha 2-macroglobulin

The inhibition rates and spectral characteristics of 2 probes specific for the active-site serine residue of proteases were examined for evidence of conformational change of the proteases upon their binding to alpha 2-macroglobulin (alpha 2M). Elastase, chymotrypsin, trypsin, and plasmin were reacted with (7-nitrobenz-2-oxa-1,3-diazole) aminoethyl- and aminopentyl methylphosphonofluoridate. The inhibition rate constants depend on the chain length of the aminoalkyl moiety of the probe and range from 10(5) to 10(4) M-1 min-1 for elastase and chymotrypsin. They are significantly modified when the proteases are stoichiometrically bound to alpha 2M. The absorption maximum of the chromophore appears in the range of 460-470 nm and 475-480 nm for the aminoethyl- and aminopentyl- conjugates, respectively. The fluorescence emission is maximal around 530 nm with a low quantum yield of about 3%. These spectral characteristics are altered in different ways by the covalent or non-covalent binding mode of the protease to alpha 2M. Finally, the CD spectrum of the NBD aminoethyl and aminopentyl elastase and chymotrypsin conjugates exhibits intense optical activity in the absorbing band of the NBD-moiety. These chiral properties are greatly altered upon binding of the protease to alpha 2M. All these results strongly suggest a conformational change in the protease at its active center upon its binding to alpha 2M; this conformational change could be taken into account to explain the alteration of the catalytic properties of the alpha 2M-bound proteases.