Control of electron transfer in supramolecular systems.

The fluorescence quantum yield of zinc porphyrin (ZnP) covalently linked to 9,10-bis(phenylethynyl)anthracene (AB) is strongly dependent upon the solvent properties. The bichromophoric system ZnP-AB exhibits 'normal' zinc porphyrin fluorescence in solvents that cannot coordinate to the central zinc atom. In contrast, if a Lewis base, such as pyridine, is added to a sufficiently polar solvent, the fluorescence is significantly quenched. Picosecond transient absorption measurements, in conjunction with fluorescence quenching and cyclic voltammetric measurements, suggest that the quenching mechanism is intramolecular electron transfer from ZnP to AB. The charge separated state. ZnP*+-AB*-, has a lifetime of not more than 220 ps before recombining. If a secondary electron acceptor, iron(III) porphyrin (FeP), is covalently connected to the AB unit, a second electron transfer from AB*- to FeP occurs and the charge separated state, ZnP*+-AB-FeP*-, has a lifetime of at least 5 ns. This demonstrates that electron transfer might be sensitively tuned (switched on) by specific solvent effects.

Bridge-dependent electron transfer in porphyrin-based donor-bridge-acceptor systems.

Photoinduced electron transfer in donor-bridge-acceptor systems with zinc porphyrin (or its pyridine complex) as the donor and gold(III) porphyrin as the acceptor has been studied. The porphyrin moieties were covalently linked with geometrically similar bridging chromophores which vary only in electronic structure. Three of the bridges are fully conjugated pi-systems and in a fourth, the conjugation is broken. For systems with this bridge, the quenching rate of the singlet excited state of the donor was independent of solvent and corresponded to the rate of singlet energy transfer expected for a Förster mechanism. In contrast, systems with a pi-conjugated bridging chromophore show a solvent-dependent quenching rate that suggests electron transfer in the Marcus normal region. This is supported by picosecond transient absorption measurements, which showed formation of the zinc porphyrin radical cation only in systems with pi-conjugated bridging chromophores. On the basis of the Marcus and Rehm-Weller equations, an electronic coupling of 5-20 cm(-)(1) between the donor and acceptor is estimated for these systems. The largest coupling is found for the systems with the smallest energy gap between the donor and bridge singlet excited states. This is in good agreement with the coupling calculated with quantum mechanical methods, as is the prediction of an almost zero coupling in the systems with a nonconjugated bridging chromophore.

Enhanced intersystem crossing in donor/acceptor systems based on zinc/iron or free-base/iron porphyrins.

The deactivation pathways of the singlet excited state of a series of zinc or free-base donor porphyrins covalently linked by a bridge to a paramagnetic iron(III) chloride porphyrin acceptor have been studied. These donor-bridge-acceptor systems all share a similar geometry (25 A donor-acceptor center-to-center distance), but the bridges vary in electronic structure. In previously reported investigations of zinc/iron porphyrin systems, the fluorescence quenching of the donor has predominantly been assigned to electron transfer. However, for the porphyrin systems studied in this paper, we show that the dominant deactivation channels are enhanced intersystem crossing and singlet energy transfer. In both series, the intersystem crossing rate (S1-->T1) of the donor moiety is almost doubled in the presence of a paramagnetic high-spin metal-porphyrin acceptor. The significant spectral overlap of the donor fluorescence and acceptor absorption in both series allows for efficient singlet energy transfer (Forster mechanism). Furthermore, the bridging chromophores mediate energy transfer and the enhancement is inversely dependent upon the energy gap between the donor and bridge excited states. Although Marcus theory predicts thermodynamically favorable electron transfer to occur in the systems investigated, the quenching rate constants were found to be independent of solvent polarity, and no charge-separated state could be detected, indicating very small electronic coupling for electron transfer.

Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis.

We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.

Adenovirus type 41 lacks an RGD alpha(v)-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells.

Human adenovirus (Ad) types 2, 3 and 12 are known to interact with cell surface integrins alpha(v)beta(3) and alpha(v)beta(5) through an RGD motif carried by the penton base. This interaction is thought to augment virus entry after initial contact between the fiber and specific receptor(s). Ad40 and Ad41 are the only members of the human subgroup F adenoviruses. The penton base protein sequence of one Ad40 strain is known to carry the motif RGAD rather than RGD, suggesting that not all human adenoviruses use the above integrins for cell entry. We confirmed that different genomic variants of Ad40 all carry an RGAD motif on the penton base, and found that the Ad41 prototype and several other genomic variants of Ad41 carry the motif IGDD in place of RGAD or RGD. This region is most likely exposed on the Ad41 particle, but attempts to block Ad41 infectivity using a homologous peptide were unsuccessful. Infectivity of an Ad41 preparation as measured by fluorescent focus assay in A549 cells was highly dependent on the length of the adsorption period, indicating that fiber-mediated attachment is inefficient in these cells. Moreover, Ad41 virions adsorbed for 1 h were internalized in a semi-linear fashion over 8 h. This inefficient uptake may be a direct consequence of independence of subgroup F adenoviruses from alpha(v)beta(3) and alpha(v)beta(5) integrin-mediated endocytosis. Ad40 and Ad41 may thus have lost or may never have developed a dependence on the penton base RGD motif for entry.

Detection of adenoviruses in stools from healthy persons and patients with diarrhea by two-step polymerase chain reaction.

The use of the polymerase chain reaction (PCR) for detection of human adenoviruses in diluted stool samples was investigated. Two sets of nested primers, including primers specific for the hexon-coding region and for the E1B region of enteric adenoviruses (EAd), were assessed by two-step amplification. The primers constitute two different PCR systems designed for the detection of adenoviruses belonging to all six subgenera (A-F), and the two EAds Ad40 and Ad41, respectively. In a two-step PCR mediated amplification a single virus particle was detected when the two sets of general hexon primers or EAd specific primers were used. Earlier results from PCR detection of adenoviruses in stool from children suffering from diarrhea gave indications that adenovirus particles are commonly shed in stools without being identified as the cause of illness [Allard et al.: Journal of Clinical Microbiology 28:2659-2667, 1990]. Therefore, the general and the EAd specific PCR assays were assessed on 150 stool specimens from three groups including 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea. When the two sets of general hexon primers were used, 25 of the 50 specimens from the healthy children (mean age 21 months) were found positive by two-step PCR amplification. Nine of the 50 specimens from the healthy adults (mean age 32 years) were found positive whereas 12 of the 50 specimens from sick adults (mean age 31 years) gave amplification products, using the two sets of general hexon primers in a nested fashion. None of the 150 specimens were found to be positive by two-step PCR amplification using the two sets of EAd-specific primers.(ABSTRACT TRUNCATED AT 250 WORDS)