New photoreactive cleavable reagents with (trifluoromethyl)diazirine group

Photoreactive cross-linking reagents that simultaneously contain a trifluoromethyldiazirine and an o-nitrobenzyl groups were synthesized for the first time. Photochemical properties of the reagents were studied, and the possibility of separate activation of the diazirine group and o-nitrobenzyl linker was shown.     

Controlled release of DNA/polyamine complex by photoirradiation of a solid phase presenting o-nitrobenzyl ether tethered spermine or polyethyleneimine

Various gene transfer and automated/monitorized analytical applications require the controlled release of nucleic acid. A solid phase with spermine or polyethyleneimines (PEI, 600 MW) tethered by o-nitrobenzyl linkages was synthesized with polyethylene oxide beads (ArgoGel-NH(2)). The photolysis of test compound O-2-nitrophenethyl O,O-diethyl phosphate or solid phase with o-nitrobenzyl group as synthetic linker was completely degradable with photoirradiation at 365 nm for 10-18 min at 3.5 mW/cm(2). DNA binding with polyamine of the solid phase and releasing of DNA/polyamine were monitored by UV measurement and gel electrophoresis. The potential exists to employ a DNA-loaded solid phase for spatially, temporally, or dose-controlled release of DNA, at extracellular or intracellular sites.     

Studies on transfer ribonucleic acids and related compounds. XXXII. Synthesis of ribonucleotides corresponding to residues 1-5 and 6-10 of tRNAfMet from E. coli and their base conversion analogs.

E. Coli tRNAfMet fragments, C-G-C-G-Gp (bases 1-5), U-G-C-G-Gp (base 1 transition, analog) pG-G-C-G-Gp (base 1 transversion analog) and pG-G-s4U-G-Gp (bases 6-10) were synthesized by triester methods using 2'-O-(o-nitrobenzyl) nucleotides including a 3',5'-bisphosphorylated guanosine derivative. The s4U containing pentanucleotide was derived from the pG-G-C-G-Gp by treatment with liquid hydrogen sulfide.     

Photoresponsive poly(S-(o-nitrobenzyl)-L-cysteine)-b-PEO from a L-cysteine N-carboxyanhydride monomer: synthesis, self-assembly, and phototriggered drug release

A photoresponsive S-(o-nitrobenzyl)-l-cysteine N-carboxyanhydride (NBC-NCA) monomer was for the first time designed, and the related poly(S-(o-nitrobenzyl)-l-cysteine)-b-poly(ethylene glycol) (PNBC-b-PEO) block copolymers were synthesized from the ring-opening polymerization (ROP) of NBC-NCA in DMF solution at 25 °C. Their molecular structures, physical properties, photoresponsive self-assembly, and drug release of PNBC-b-PEO were thoroughly investigated. The β-sheet conformational PNBC block within copolymers presented a thermotropic liquid crystal phase behavior, and the crystallinity of PEO block was progressively suppressed over the PNBC composition. The characteristic absorption peaks of these copolymers at about 310 and 350 nm increased over UV irradiation time and then leveled off, indicating that the o-nitrobenzyl groups were gradually photocleaved from copolymers until the completion of photocleavage. The PNBC-b-PEO copolymers self-assembled into spherical nanoparticles in aqueous solution, presenting a photoresponsive self-assembly behavior, together with a size reduction of nanoparticles after irradiation. The anticancer drug doxorubicin can be released in a controlled manner by changing the light irradiation time, which was induced by gradually photocleaving the PNBC core of nanoparticles. This work provides a facile strategy not only for the synthesis of photoresponsive polypeptide-based block copolymers but also for the fabrication of photoresponsive nanomedicine potential for anticancer therapy.     

Synthesis and NMR study of ribo-oligonucleotides forming a hammerhead-type RNA enzyme system.

We designed a hammerhead-type ribozyme system which consists of three RNA fragments and synthesized the component and related ribo-oligonucleotides by the solid-phase phosphoramidite method using o-nitrobenzyl groups for 2'-hydroxyl protection. Improved conditions for photolytic removal of the o-nitrobenzyl groups are described. Imino proton NMR spectra of the ribozyme complex were measured. The resonances for the hydrogen-bonded imino protons were assigned by comparison with the spectra of model complexes which contain the base sequences of one or two of the stem regions. The results suggest that the complex indeed forms a hammerhead structure and the loop regions take an ordered conformation in the absence of magnesium ions.     

Synthesis and properties of 5'-triphosphoryl 2'-5' oligoadenylates (2-5A), and a general method for synthesis of 3', 5'-bisphosphorylated oligonucleotides.

2'-5'-Linked oligoadenylic acid 5'-triphosphates (2-5A) having chain lengths of 2-4 have been synthesized by polymerization of 3'-O-(o-nitrobenzyl)-N-benzoyladenosine 5'-phosphate followed by 5'-triphosphorylation via the imidazolidates. A large scale preparation of 5'-O-phosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine was performed by the phosphotriester method using 5'-O-monomethoxytrityl-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate and 5'-O-phosphorodianilido-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate as intermediates. The trimer was also triphosphorylated by the imidazolide method. CD spectra for 5'-mono and triphosphorylated 2'-5' adenylates were measured as well as their UV hypochromicities. This triester method was also applied to the synthesis of 3',5'-bisphosphorylated protected oligoadenylic acids with natural 3'-5' linkages which could be used for further condensations to yield 5'-phosphorylated polynucleotides.     

Photocleavage of o-nitrobenzyl ether derivatives for rapid biomedical release applications

The externally controlled cleavage of covalently linked prodrugs, proteins, or solid-phase formulation vehicles offers potential advantages for controlled drug or gene delivery. A series of o-nitrobenzyl ester compounds (1-8) were synthesized to allow a systematic study of photolability. The o-nitrobenzyl ester was strictly required for photolability, while imido esters were not photolabile. The degradation kinetics of 1-o-phenylethyl ester was an order of magnitude faster than that of o-nitrobenzyl ester. Tosylate, phosphate, and benzoate derivatives of 1-o-nitrophenylethyl displayed similar photolability (>80% decomposition within 10 min at 3.5 mW/cm2 at 365 nm). O-o-Nitrobenzyl O',O'-diethyl phosphate displayed the fastest decomposition at photoirradiation condition (3.5 mW/cm2, 365 nm) suitable for biological systems. We report the synthesis and photo-decomposition of 1-o-nitrophenylethyl derivatives amenable for the creation of photolabile prodrugs or formulation particles for drug depots, DNA condensation, or tissue engineering applications.     

A new method for 3''-labelling of polyribonucleotides by phosphorylation with RNA ligase and its application to the 3''-modification for joining reactions.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.     

Synthesis and properties of RNA with catalytic activity

Ribooligonucleotides, which form the self-cleavage domain of a satellite RNA of tobacco ringspot virus, were synthesized by the solid-phase phosphoramidite method using o-nitrobenzyl groups for 2'-hydroxyl protection. The oligomers were obtained in quantities sufficient for NMR measurement. Specific cleavage at an expected site was observed when the three RNA fragments were mixed in the presence of magnesium ions.     

Studies on transfer ribonucleic acids and related compounds. XL. Synthesis of an eicosaribonucleotide corresponding to residues 35-54 of tRNAfMet from E. coli.

An E. coli tRNAfMet fragment [C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-U-C-G-G (bases 35-f54)] containing the anticodon triplet has been synthesized by the phosphotriester method involving protected oligonucleotide blocks. Di- or tri-nucleotide blocks were prepared by condensation of 2'-O-(o-nitrobenzyl) nucleotide derivatives and used for the synthesis of pentanucleotide blocks. The 5'-hydroxy, heterocyclic amino and internucleotide linkage were protected with monomethoxytrityl, acyl and p-chlorophenyl groups, respectively. The 3'-phosphates of the pentanucleotides, except for the GUCGG block where 2'-O-benzoyl 3'-O-(o-nitrobenzyl) N-isobutyrylguanosine was used, were protected with p-chlorophenyl and anilido groups. The anilido groups were removed by treatment with isoamyl nitrite and the 3'-phosphodiesters of resulting pentamers were activated with mesitylenesulfonyl nitrotriazolide to give protected decanucleotides in yields of 61-89%. The two decanucleotides were condensed similarly to yield the protected eicosanucleotide in a yield of 59%. The product was deblocked and purified by ion-exchange chromatography on DEAE-Sephadex A-25 and characterized by enzymatic hydrolysis after labelling the 5'-end by phosphorylation using polynucleotide kinase and [gamma-32P]ATP.     

Solid phase synthesis of oligoribonucleotides using o-nitrobenzyl protection of 2''-hydroxyl via a phosphite triester approach.

The hepta and undecaribonucleotide were synthesized on a controlled pore glass beads using o-nitrobenzyl protection of 2'-hydroxyls via a phosphite approach. By using 5-p-nitrophenyltetrazole for the activation of nucleoside-phosphoramidite, the condensation reaction was carried out very rapidly (2.5 min). The time required for one cycle was only 16 min. The hepta-(UACUAAC) and undecaribonucleotides (GUAUGUUAAUA) were obtained in yields of 28 and 17% respectively from the original resin.     

Synthesis and properties of photolabile caged phosphotriester derivatives of dinucleoside phosphates

Dinucleoside phosphates that harbor phosphate groups transiently blocked (caged) by o-nitrobenzyl or o-nitroveratryl residues were synthesized. It was shown that the conditions of the UV-induced deprotection largely depend on the nature of the protective group. The phosphotriesters obtained were resistant toward snake venom phosphodiesterase and nucleases of the cellular extract. The synthesis of the dinucleoside phosphates containing a photolabile group preceded the incorporation of the modified blocks into extended oligonucleotides by the phosphoramidite method.     

Parallel solid-phase synthesis of a model library of 7alpha-alkylamide estradiol derivatives as potential estrogen receptor antagonists.

The C17-THP derivative of 7alpha-(11-azidoundecanyl)-estradiol (4) was synthesized and coupled to an aminomethyl resin via a photolabile o-nitrobenzyl linker. Reduction of the azide by the Staudinger reaction to its corresponding amine followed by acylation using four activated NFmoc protected amino acids gave a first level of diversity. Subsequent deprotection of the Fmoc followed by a second acylation with five activated carboxylic acids produced, after photocleavage, a model library of twenty antiestrogen-related 7alpha-alkylamide estradiol derivatives in acceptable overall yields and very good purities.     

Solid phase synthesis of oligoribonucleotides using the o-nitrobenzyl group for 2''-hydroxyl protection and H-phosphonate chemistry.

Oligoribonucleotides with chain length of 7, 11, 15, 17, 24 and 34 were synthesized on long chain alkylamine controlled pore glass beads (LCA-CPG) using o-nitrobenzyl protection of 2'-hydroxyls via a H-phosphonate approach either manually or by using an automatic synthesizer. The oligoribonucleotides were obtained in yields of 0.6 0.6-20%, based on initial nucleoside bound to the LCA-CPG support.     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

Photoenhancement of transfection efficiency using novel cationic lipids having a photocleavable spacer

New cationic lipids having an o-nitrobenzyl moiety as a photocleavable spacer between its hydrophilic and hydrophobic region were synthesized. To improve the efficiency of transfection with lipoplexes, after transfecting the cationic lipid aggregate/DNA complex, photoirradiation was performed. Photochemical decomposition of lipids would not only make the vector's membrane unstable to facilitate the fusion with endocytic vesicles, but also promote dissociation of cationic lipid-DNA complex, thus aiding the escape of DNA from the endocytic vesicles. Using a luciferase gene as a model, we show that UV irradiation of photoresponsive lipoplex-treated COS-1 cells induces a substantial increase in the efficiency of transfection. Herein, we show a novel photoresponsive gene delivery system.     

Biotinylated photocleavable polyethylenimine: capture and triggered release of nucleic acids from solid supports

A biotinylated photocleavable polyethylenimine (B-PC-PEI) was designed and synthesized for the capture and controlled release of nucleic acids from solid supports. B-PC-PEI was synthesized via a three-step reaction process and verified by 1H NMR and mass spectrometry. In aqueous solution, the o-nitrobenzyl group within B-PC-PEI was efficiently cleaved by 5 min of 365 nm light exposure from a distance of 20 cm (9 mW/cm2). When coupled to streptavidin-coated beads, the PEI domain of Cy5-labeled B-PC-PEI was released by 365 nm light exposure. In contrast, a Cy5-labeled biotinylated PEI (B-PEI) was used as a control and negligible fluorescence loss was observed. Cy5-labeled siRNA was electrostatically captured to streptavidin-coated beads preabsorbed with B-PC-PEI or B-PEI, and flow cytometry demonstrated significant loss of fluorescence from the bead surface after 5 min of light exposure only for B-PC-PEI, demonstrating controlled release of siRNA from the bead surface. Finally, the release of the Cy5-labeled siRNA into the supernatant was quantified. The release of Cy5-siRNA into the supernatant was significantly greater after 5 min of light exposure for B-PC-PEI/streptavidin beads compared to 0 min exposure and remained unchanged for B-PEI/streptavidin beads. B-PC-PEI facilitates capture and triggered release of surface-tethered nucleic acids with light exposure and is fully compatible with streptavidin-based applications.     

Photolabile protecting groups for an acetylcholine receptor ligand. Synthesis and photochemistry of a new class of o-nitrobenzyl derivatives and their effects on receptor function

Two compounds have been synthesized that feature a photosensitive o-nitrobenzyl moiety attached directly to the carbamate nitrogen of carbamoylcholine. The well-characterized acetylcholine analogue, carbamoylcholine, was released from these derivatives in response to laser light pulses at wavelengths between 300 and 355 nm. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis. The yield of carbamoylcholine molecules per photon absorbed was 0.25. A short-lived photochromic intermediate in the photolysis reaction was detected by laser flash photolysis. A single laser flash induced an instantaneous increase in absorbance at 406 nm, followed by a first-order decay to products, with a half-time of 0.07 ms for one of the compounds [N-[1-(2-nitrophenyl)ethyl]carbamoylcholine iodide] in aqueous buffers at pH 7 and 23 degrees C. Decay rates and quantum yields depended on the nature of the substituent on the protecting group. Evidence is presented in support of the conclusion that the transient species is an aci-nitro intermediate that decays directly to carbamoylcholine and therefore determines its rate of release. The photosensitive carbamoylcholine derivatives activated the nicotinic acetylcholine receptor only after photolysis, as determined by 86Rb+ flux measurements with membrane vesicles prepared from Torpedo californica and Electrophorus electricus. Before photolysis, the compounds interacted weakly with the acetylcholine-binding sites as shown by competitive inhibition of acetylcholine-stimulated flux at high concentrations. The compounds did not induce receptor desensitization at a significant rate. The new compounds afford several major advantages over other photoactivatable acetylcholine analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of a photocleavable cross-linker and its application on tunable surface modification and protein photodelivery

A heterobifunctional photocleavable cross-linker based on an o-nitrobenzyl ester moiety was synthesized. The cross-linker has N-hydroxysuccinimidyl and disulfide groups attached at each end and thus can anchor a protein to a gold-coated substrate surface. Steady-state spectroscopic studies suggest that the cross-linker undergoes a clean C-O fragmentation upon irradiation with a quantum yield of 0.1. Consequently, immobilized proteins (such as avidin or antibodies) on a substrate surface can be released efficiently (>95%) under UV irradiation (lambda > 300 nm) without degrading the protein functionality. We also demonstrated protein delivery via bioconjugation of protein molecules to a gold-coated atomic-force microscope (AFM) tip. When the proteins are photoreleased from the AFM tip, they are delivered to the substrate surface as protein clusters of uniform size. This has been confirmed using both AFM and fluorescence microscopy. The application of bioconjugation in this study opens a new avenue for tunable surface modification and controllable protein delivery in studies of biological systems on the nanometer scale.     

DNA primase-DNA polymerase alpha assembly from mouse FM3A cells. Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.