Synthesis and photophysical properties of light-harvesting arrays comprised of a porphyrin bearing multiple perylene-monoimide accessory pigments

We present the synthesis and characterization of new light-harvesting arrays containing two, four, or eight perylene-monoimide accessory pigments attached to a zinc porphyrin. Each perylene is substituted with one or three 4-tert-butylphenoxy substituents. A 4,3'- or 4,2'-diarylethyne linker joins the perylene N-imide position and the porphyrin meso-position, affording divergent or convergent architectures, respectively. The architectures are designed to provide high solubility in organic media and facile perylene-to-porphyrin energy transfer, while avoiding charge-transfer quenching of the excited porphyrin product. For the array containing four perylenes per porphyrin in both nonpolar (toluene) and polar (benzonitrile) media and for the array containing eight perylenes per porphyrin in toluene, the photoexcited perylene-monoimide dye (PMI) decays rapidly ( approximately 3.5 ps) and predominantly (>or=90%) by energy transfer to the zinc porphyrin to form the excited zinc porphyrin (Zn), which has excited-state characteristics (lifetime, fluorescence yield) comparable (within approximately 10%) to those of the isolated chromophore. For the array containing eight perylenes in benzonitrile, PMI decays approximately 80% by energy transfer (forming Zn) and approximately 20% by hole transfer (forming PMI- Zn+); Zn subsequently decays approximately 20% by electron transfer (also forming PMI- Zn+) and approximately 80% by the normal routes open to the porphyrin monomer (intersystem crossing, internal conversion, fluorescence). In addition to rapid and efficient perylene-to-porphyrin energy transfer, the broad blue-green to yellow absorption of the perylene dyes complements the blue absorption of the porphyrin, resulting in excellent light harvesting across a significant spectral region. Collectively, the work described herein identifies multiperylene-porphyrin arrays that exhibit suitable photochemical properties for use as motifs in larger light-harvesting systems.     

Oligo-2'-deoxyribonucleotides containing uracil modified at the 5-position with linkers ending with guanidinium groups

We report here the synthesis and binding studies of oligo-2'-deoxyribonucleotides (ODNs) containing 2'-deoxyuridines, modified at the 5-position by linkers ending with either one or two guanidinium groups. Comparison was made with ODNs containing 2'-deoxyuridines modified at the 5-position with linkers ending with either two or one amino groups. One or two modified 2'-deoxyuridines were incorporated into pyrimidine strands, and their influence on the stability of duplex (with both DNA and RNA targets) and triplex structures was studied. The strongest stabilization was obtained with modified ODNs containing guanidinium groups. This result confirms that the reduction of the global negative charge number on one strand is an important parameter in the stability of duplex and triplex structures.     

Porphyrin-DNA conjugates: porphyrin induced adenine-guanine homoduplex stabilization and interduplex assemblies

DNA has found widespread uses as a nanosized scaffold for assembly of patterned multichomophoric nanostructures. Herein we report the synthesis, self-assembly, stability, and spectroscopic studies of short alternating non-self-complementary DNA sequences 5'-(dGdA)(4) and 5'-(dAdG)(4) with non-charged tetraarylporphyrins covalently linked to the 5' position of deoxyadenosine or deoxyguanosine via a phosphate or amide linker. The linker, the metal in the porphyrin coordination center, and the neighboring nucleobase have very distinct effects on the duplex formation of porphyrin-deoxyguanosine-deoxyadenosine oligodeoxynucleotides. At ionic strength between 5 mM and 40 mM, free base trispyridylphenylporphyrin appended to the 5' termini of 5'-(dAdG)(4) oligonucleotide via short non-polar amide linker served as a hydrophobic molecular cap inducing deoxyadenosine-deoxyguanosine antiparallel homoduplex. At ionic strength of ≥60 mM, the free base porphyrin functioned as a molecular 'glue' and induced the formation of porphyrin-DNA inter-homoduplex assemblies with characteristic tetrasignate CD Cotton effects in the porphyrin Soret band region. When the porphyrin cap was covalently attached to 5' position of deoxyguanosine or deoxyadenosine via charged phosphate linker, no significant deoxyadenosine-deoxyguanosine hybridization was observed even at elevated ionic strengths.     

Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple oligonucleotides

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.     

Synthesis and optical properties of polyphenylene dendrimers based on perylenes

A series of polyphenylene-dendronized perylenes have been synthesized, and their physical and mesoscopic properties have been investigated. The attached polyphenylene dendrons have significant effects on the physical properties of the perylenes. They increase the solubility of perylenes in common organic solvents, suppress significantly the aggregation of the perylene core, and lead to red-shifted absorption and emission. The polyphenylene dendrons give rise to a strong absorption band in the UV region and exhibit efficient intramolecular energy transfer to the perylene moiety. The functionalization of perylenes with polyphenylene dendrons allows the preparation of films by spin-coating.     

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Photoregulation of DNA triplex formation by azobenzene

Formation and dissociation of DNA triplex are reversibly photoregulated by cis <--> trans isomerization of the azobenzene tethered to the third strand. When the azobenzene takes the trans from, a stable triplex is formed. Upon the isomerization of trans-azobenzene to its cis form by UV light irradiation (300 < lambda < 400 nm), however, the modified oligonucleotide is removed from the target duplex. The triplex is re-formed on photoinduced cis --> trans isomerization (lambda > 400 nm). The photoregulating activity significantly depends on the position of azobenzene in the third strand, as well as on the geometric position (meta or para) of its amido substituent. For m-amidoazobenzene, the photoregulation is the most effective when it is tethered to the 5'-end of the third strand. However, p-amidoazobenzene should be introduced into the middle of the strand for effective regulation. In the optimal cases, the change of T(m) of the triplex, caused by the cis <--> trans isomerization of azobenzene, is greater than 30 degrees C. UV-visible and CD spectroscopy, as well as computer modeling studies, clearly demonstrate that the trans-azobenzene intercalates between the base pairs in the target duplex and thus stabilizes the triplex by stacking interactions. On the other hand, nonplanar cis-azobenzene destabilizes the triplex due to its steric hindrance against the adjacent base pairs.     

Probing binding preferences of DNA and RNA: backbone chirality of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids to differentiate DNA versus RNA selective binding

Subtle differences in RNA and DNA duplex geometry could be sensed by the changed stereochemistry at 3'-amino function in the 5-atom thioacetamido linker of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids modified oligomers. In contrast to the preferred N-type sugar conformations for either 3'- ribo- or xylo amino nucleosides, predominant S-type sugar conformations were found in the dimers. Although the CD spectral differences for the dimer blocks were found to be identical for those found in phosphodiester linked ribo/xylo dimers, the 5-atom thioactamido linker could reverse the RNA binding selectivity to DNA binding selectivity by the change in configuration at the 3'-amino-substituted sugar.     

DNA oligomers having a diazapyrenium dication (DAP2+); synthesis and DNA cleavage activities.

Aiming at the creation of functionalized antisense DNA oligomers possessing site-selective DNA cleaving activity, viologen and a related compound, diazapyrenium dication (DAP2+), were selected and introduced into oligodeoxyribonucleotides as a functionalized molecule. The conjugation of these functionalized molecules with DNA proceeded smoothly by using standard H-phosphonate chemistry. A part of the DAP(2+)-tethered DNA oligomers was synthesized by a combination of solid support method and liquid phase technique. Viologen-tethered DNA oligomers showed no significant activity toward DNA cleavage in spite of their characteristic ESR spectra. On the other hand, it was observed that the DAP(2+)-tethered DNA oligomers formed more stable duplexes with their complementary strands than the corresponding wild type, and these molecules effectively cleaved the complementary strands at the specific site of 2-3 bases away from the modified phosphoramidate linkage. The effect of position and length of the linker arm on the selectivity in the cleavage reaction was also investigated, and it was found that introduction at the 3'- or 5'-end phosphate site is more favorable, probably due to duplex stabilization.     

Duplex and triplex formation of mixed pyrimidine oligonucleotides with stacking of phenyl-triazole moieties in the major groove

5-(1-Phenyl-1,2,3-triazol-4-yl)-2'-deoxycytidine was synthesized from a modified CuAAC protocol and incorporated into mixed pyrimidine oligonucleotide sequences together with the corresponding 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine. With consecutive incorporations of the two modified nucleosides, improved duplex formation with a complementary RNA and improved triplex formation with a complementary DNA duplex were observed. The improvement is due to π-π stacking of the phenyl-triazole moieties in the major groove. The strongest stacking and most pronounced positive influence on thermal stability was found in between the uridine analogues or with the cytidine analogue placed in the 3' direction to the uridine analogue. Modeling indicated a different orientation of the phenyl-triazole moieties in the major groove to account for the difference between the two nucleotides. The modified oligonucleotides were all found to be significantly stabilized toward nucleolytic degration.     

Perylene-3,4:9,10-tetracarboxylic acid bisimide dye as an artificial DNA base surrogate

A phosphoramidite of the perylene bisimide dye was synthesized as a DNA building block that allows incorporation of this chromophore as an artificial nucleoside surrogate either at the 5'-terminus or at internal positions of duplex DNA. The internally incorporated perylene bisimide chromophore shows strong interactions with the DNA base stack; the 5'-terminally attached perylene bisimide is able to induce dimerization of two whole DNA duplexes.     

Unmediated by DNA electron transfer in redox-labeled DNA duplexes end-tethered to gold electrodes

Electron transfer (ET) between gold electrodes and redox-labeled DNA duplexes, immobilized onto the electrodes through the alkanethiol linker at the 3'-end and having internal either methylene blue (MB) or anthraquinone (AQ) redox labels, was shown to depend on the redox label charge and the way the redox label is linked to DNA. For loosely packed DNA monolayers, the conjugation of the positively charged MB to DNA through the long and flexible alkane linker provided ET whose kinetics was formally governed by the diffusion of the redox label to the negatively charged electrode surface. For the uncharged AQ label no ET signal was detected. The conjugation of AQ to DNA through the short and more conductive acetylene linker did not provide the anticipated DNA-mediated ET to the AQ-moiety: ET appeared to be low-efficient if any in the studied system, for which no intercalation of AQ within the DNA duplex occurred. The ET communication between the electrode and AQ, built in DNA through the acetylene linker, was achieved only when Ru(NH(3))(6)(3+) molecules were electrostatically attached to the DNA duplex, thus forming the electronic wire. These results are of particular importance both for the fundamental understanding of the interfacial behavior of the redox labeled DNA on electrodes and for the design of biosensors exploiting a variation of ET properties of DNA in the course of hybridization.     

A versatile modification of on-column oligodeoxynucleotides using a copper-catalyzed oxidative acetylenic coupling reaction

We report herein a versatile postsynthetic modification of on-column oligodeoxynucleotides (ODNs) using a copper-catalyzed oxidative acetylenic coupling reaction. Hexamers supported on resins via a methylamino-modified linker were prepared, and on-column modifications of ODNs were examined. ArgoPore resin proved to be the best choice for the modification, and introduction of functional molecules, such as anthraquinone, biotin, and fluorescein, resulted in good yields at not only the 5'-terminal but also the internal 3'-end of the ODNs. This method is applicable to the modification of 12mer ODN consisting of a random sequence. The resulting ODN9 possessing fluorescein at its 5'-terminal acts as a non-RI primer for primer extension assays using the Klenow fragment.     

Color Bricks: Building Highly Organized and Strongly Absorbing Multicomponent Arrays of Terpyridyl Perylenes on Metal Oxide Surfaces

Terpyridine‐substituted perylenes containing cyclic anhydrides in the peri position were synthesized. The anhydride group served as an anchor for assembly of the terpyridyl‐crowned chromophores as monomolecular layers on metal oxide surfaces. Further coordination with Zn²⁺ ions allowed for layer‐by‐layer formation of supramolecular assemblies of perylene imides on the solid substrates. With properly selected anchor and linker molecules it was possible to build high quality structures of greater than ten successive layers by a simple and straightforward procedure. The prepared films were stable and had a broad spectral coverage and high absorbance. To demonstrate their potential use, the synthesized dyes were employed in solid‐state dye‐sensitized solar cells, and electron injection from the perylene antennas to titanium dioxide was observed.     

Intercalation of ethidium into triple-strand poly(rA).2poly(rU): a thermodynamic and kinetic study

The kinetics and equilibria of the interaction of ethidium bromide (EB) with the triple-stranded RNA, poly(rA).2poly(rU), have been investigated by stopped-flow, absorption, fluorescence, and circular dichroism methods; to properly assess the effect of the third strand on the polymer molar properties, molar volumes, adiabatic compressibilities, and heats of melting have also been measured for both poly(rA).2poly(rU) and poly(rA).poly(rU). The melting experiments reveal that ethidium tends to destabilize the triplex, whereas it stabilizes the duplex; however, the triplex/ethidium system in 0.1 M NaCl is stable below 37 degrees C. The static titrations reveal that one ethidium ion binds every three base triplets of the polymer; on the basis of the excluded-site model, this feature suggests intercalation, as in the duplex, but the binding affinity for the triplex is weaker compared to that for the duplex. The kinetic experiments displayed a two-phase behavior, which was rationalized assuming the sequence D + S right arrow over left arrow DS(I), DS(I) + S right arrow over left arrow DS(II) + S (D = drug, S = site), the second step involving direct transfer of the drug between strands. Comparison with the duplex/EB system reveals that the additional strand of poly(U), present in the triplex, hinders the formation of the intermediate complex DS(I), while stabilizing the structure of the final DS(II) complex by hampering the partial slipping out of the dye from the triplex cavity.     

Synthesis of new OBAN's and further studies on positioning of the catalytic group

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety. OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one placed internally and the other at the at the 5'-end of the oligonucleotide. The key step in the synthesis of the OBAN systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences, in the presence of Zn(II) ions. Differences in efficiency between these and previously reported systems are discussed.     

Dipyrido[3,2-a:2′,3′-c]phenazine-Tethered Oligo-DNA: Synthesis and Thermal Stability of Their DNA⋅DNA and DNA⋅RNA Duplexes and DNA⋅DNA⋅DNA Triplexes

Dipyrido[3,2-a:2′,3′-c]phenazine (dppz) derivatives were conjugated to 9-mer and 18-mer DNA (ODN) at a site without nucleobase, either at the 5′- or 3′-end or at a internucleotide position, via linkers of 7, 12, or 18 atoms lengths. These dppz-linked ODNs were synthesized using novel backbone glycerol phosphoramidites: Glycerol, serving as artificial nucleoside without nucleobase, was modified to amines 10 , 23 , and 24 , which were suitable for the subsequent key reaction with dppz-carboxylic acid 3 (Schemes 2 and 3). The products of these reactions (see 5 – 7 ) were then transformed to the standard phosphoramidite derivatives (see 27 , 29 , and 30 ) or used for loading on a CPG support (see 28 , 31 , and 32 ). The dppz-modified ODNs were subsequently assembled in the usual manner using automated solid-phase DNA synthesis. The 9-mer ODN-dppz conjugates 35 – 43 were tested for their ability to form stable duplexes with target DNA or RNA strands (D11 ( 60 ) or R11 ( 61 )), while the 18-mer ODN-dppz conjugates 48 – 56 were tested for their ability to form stable triplexes with a DNA target duplex D24⋅D24 ( 62 ) (see Tables 1 and 2). The presence of the conjugated dppz derivative increases the stability of DNA⋅DNA and DNA⋅RNA duplexes, typically by a ΔT_m of 7.3 – 10.9° and 4.5 – 7.4°, respectively, when the dppz is tethered at the 5′- or 3′-terminal (Table 2). The dppz derivatives also stabilize triplexes when attached to the 5′- or 3′-end, with a ΔT_m varying from 3.8 – 11.1° (Table 3). The insertion of a dppz building block at the center of a 9-mer results in a considerably poorer stability of the corresponding DNA⋅DNA duplexes (ΔT_m=0.5 to 4.2°) and DNA⋅RNA duplexes (ΔT_m=−1.5 to 0.9°), while the replacement of one interior nucleotide by a dppz building unit in the corresponding 8-mer ODN does not reveal the formation of any duplex at all. Different types of modifications in the middle of the 18-mer ODN, in general, do not lead to any triplex formation, except when the dppz derivative is tethered to the ODN through a 12-atom-long linker (Entry 9 in Table 3).     

Molecular layer-by-layer self-assembly of water-soluble perylene diimides through pi-pi and electrostatic interactions

A layer-by-layer deposition process has been carried out for two oppositely charged water-soluble perylene diimide dyes without the use of intervening polyelectrolyte layers. The strong pi-pi interactions between the perylene moieties help stabilize the layers and simultaneously diminish the fluorescence quantum yield of the array without strongly affecting the absorption or fluorescence spectra. There is an alternation of fluorescence intensity according to which perylene species is on the outer layer, which is interpreted as the effect of facile energy transfer between the perylenes.     

Interaction between 3-(p-tolylamino)-1,5-azulenequinone and the deoxyguanosine residue in various oligonucleotides upon photolysis.

Eight single-stranded oligodeoxyribonucleotides 32P-labeled at the 5'-end were synthesized; they were annealed with the complementary oligodeoxyribonucleotides to form the corresponding double-stranded helices. These duplexes possessed standard Watson-Crick base pairs, locally perturbed sites of a base mismatch, or a bulge. Further, 5'-32P-labeled oligodeoxyribonucleotides with a hairpin loop were also synthesized. Cleavage of these single- and double-stranded oligodexyribonucleotides selectively at the deoxyguanosine residue was accomplished by use of 3-(p-tolylamino)-1,5-azulenequinone 1 upon irradiation with 350 nm UV light. The single strands were cleaved more efficiently than the double-helices. For the helices containing a deoxyguanosine residue at a bulge, at a hairpin loop or toward the end, the cleaving efficiency was increased. Computation results indicate that two possibilities exist for agent 1 to form two "Watson-Crick type" hydrogen bonds with guanine in single-stranded oligodeoxyribonucleotides; yet, only one possibility exists in duplexes.