The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond timeresolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 ± 0.4 ps to the accessory bacteriochlorophyll B. State P+B-decays with a time constant of 0.9 ± 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H-to the quinone QA within 220 ± 40 ps. According to present knowledge, an electron is transferred upon light absorption from the P along branch A to the quinone QA (8)(9)(10)(11)(12)(13) Materials and Experimental TechniquesRCs from two strains ofRb. sphaeroides, the wild-type strain (ATCC 17023) and the carotenoid-free strain (R 26.1), were isolated as described (39). The measurements were performed at room temperature (297 K) in cuvettes with a 1-mm path length with stirring. The concentration of the samples was adjusted to OD8w values between 0.5 and 1.0 mm' A synchronously pumped unidirectional ring dye laser (42) generated pulses with a duration of 60 fs at a wavelength of 860 nm. The energy of single pulses was increased to 20 lJ by a three-stage dye amplifier (repetition rate, 10 Hz). Each of these pulses was split into two parts. (i) The excitation pulse was focused to a 0.5-mm spot providing an energy density of not more than 100 uJ/cm2 in the sample. In the probed volume, -7% of the RCs were excited per pulse. This low excitation density prevents double excitation of the RC and related nonlinear processes. The exciting pulse, centered at 860 nm, had a bandwidth of <15 nm (full width at half-maximum). No spectral components of the exciting pulse were detected at wavelengths of <840 nm. Thus only the lowest electronic level ofP at 860 nm was excited. (ii) The probing pulse passed an adjustable delay line and was focused onto a 1-mm-thick jet of ethylene glycol to generate a femtosecond white-light pulse. A 10-to 20-nm-wide portion of this continuum was selected by means of a special dispersion compensating monochromator (43). The energy of the probing pulses was <7 p.J/cm2.

The primary light-induced charge separation in reaction Centers of Rkfobacter sphueroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 mn could only be explained by a kinetic model which uses three time constants. This fmding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone.

The sequence-specific recognition of double-helical DNA by oligodeoxyribonucleotide-directed triple-helix formation is limited mostly to purine tracts. Within the geometric constraints of the phosphate-deoxyribose position of a purine-purine-pyrimidine triple-helical structure, model building studies suggested that the deoxyribonucleoside 2'-deoxynebularine (dN) might form one specific hydrogen bond with cytosine (C) or adenine (A) of Watson-Crick cytosine-guanine (CG) or adenine-thymine (AT) base pairs. 2-Deoxynebularine (dN) was incorporated by automated methods into purine-rich oligodeoxyribonucleotides. From affinity cleavage analysis, the stabilities of base triplets within a purine.purine.pyrimidine (Pu.Pu.Py) triple helix were found to decrease in the order N.CG approximately N.AT >> N.GC approximately N.TA (pH 7.4, 37 degrees C). Oligodeoxyribonucleotides containing two N residues were shown to bind specifically within plasmid DNA a single 15 base pair site of the human immunodeficiency virus genome containing two CG base pairs within a purine tract. This binding event occurs under physiologically relevant pH and temperature (pH 7.4, 37 degrees C) and demonstrates the utility of the new base. Quantitative affinity cleavage titration reveals that, in the particular sequence studied, an N.CG base triplet interaction results in a stabilization of the local triple-helical structure by 1 kcal.mol-1 (10 mM NaCl, 1 mM spermine tetrahydrochloride, 50 mM Tris-acetate, pH 7.4, 4 degrees C) compared to an A.CG base triplet mismatch.

No abstract

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as 125I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with K(i) values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.