Isosteric and fluorescent DNA base pair formed by 4-amino-phthalimide and 2,4-diaminopyrimidine: melting, structure, and THz polar solvation dynamics.

An artificial base pair in the center of a duplex DNA oligomer, formed by 2,4-diaminopyrimidine and fluorescent 4-aminophthalimide C-nucleosides, is characterized spectroscopically, with a view towards its use in femtosecond solvation dynamics. Quantum-chemical calculations predict H-bonding energy equivalent to A:T. UV-vis absorption spectra provide insight into local melting at the 4-aminophthalimide modification site. Increase of temperature to nearly the melting temperature of the duplex leads to better hybridisation of the fluorescent nucleoside, contrary to native base pairs. This unusual observation is explained by the NMR solution structure of the duplex. Two conformations are adopted by the artificial pair due to backbone constraints, having either two or one interbase hydrogen bonds. In the latter, hydrogen bonding sites remain accessible for water solvation. The time-resolved dynamic Stokes' shift of 4-aminophthalimide fluorescence is consistent with that of a mixture of a slow and fast species. From the observations, the optimal linkage between 4-aminophthalimide and 2-deoxyribose for fitting into the duplex B-DNA structure is deduced.

Ultrafast energy transfer and structural dynamics in DNA.

Ultrafast structural dynamics concomitant to excitation energy transfer in DNA has been studied using a pair of pyrene-labeled DNA bases. The temporal evolution of the femtosecond pump-probe spectra reveals the existence of two electronic coupling pathways, through-base stack and through-space, which lead to excitation energy transfer and excimer formation even when the labeled DNA bases are separated by one AT base pair. The electronic coupling which mediates through-base stack energy transfer is so strong that a new absorption band arises in the excited-state absorption spectrum within 300 fs. From the analysis of time-dependent spectral shifts due to through-space excimer formation, the local structural dynamics and flexibility of DNA are characterized on the picosecond and nanosecond time scale.

Direct observation of radical intermediates in protein-dependent DNA charge transport.

Charge migration through the DNA base stack has been probed both spectroscopically, to observe the formation of radical intermediates, and biochemically, to assess irreversible oxidative DNA damage. Charge transport and radical trapping were examined in DNA assemblies in the presence of a site-specifically bound methyltransferase HhaI mutant and an intercalating ruthenium photooxidant using the flash-quench technique. The methyltransferase mutant, which can flip out a base and insert a tryptophan side chain within the DNA cavity, is found to activate long-range hole transfer through the base pair stack. Protein-dependent DNA charge transport is observed over 50 A with guanine radicals formed >10(6) s(-1); hole transport through DNA over this distance is not rate-limiting. Given the time scale and distance regime, such protein-dependent DNA charge transport chemistry requires consideration physiologically.

Synthetic active site analogues of heme-thiolate proteins. Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase.

In view of recent results from different sources, the reaction mechanisms of two heme-thiolate proteins, cytochrome P450cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H2O2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl+ donor chlorinating activated C-H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

7-Pyrrolidinyl- and 7-piperidinyl-5-aryl-pyrrolo[2,3-d]pyrimidines--potent inhibitors of the tyrosine kinase c-Src.

7-Heterocyclyl-5-aryl-pyrrolo[2,3-d]pyrimidines represent a new class of highly potent and selective inhibitors of the tyrosine kinase pp60(c-Src).

DNA-Bound peptide radicals generated through DNA-mediated electron transport.

Flash-quench experiments were carried out to explore peptide/DNA electron-transfer reactions. DNA-bound [Ru(phen)(2)(dppz)](3+) (phen = 1,10-phenanthroline; dppz = dipyridophenazine) and [Ru(phen)(bpy')(dppz)](3+) [bpy' = 4-(4'-methyl-2, 2'-bipyridyl)valerate], generated in situ by flash-quench methodology, are powerful ground-state oxidants, capable of oxidizing guanine or tyrosine intercalated in DNA. In flash-quench experiments with mixed-sequence oligonucleotides in the presence of Lys-Tyr-Lys, transient absorption spectroscopy yielded a spectrum with a sharp maximum at 405 nm assigned to the tyrosine radical. Experiments with poly(dG.dC) suggested the intermediacy of the guanine radical, since the rise of the 405 nm signal occurred with the same kinetics as the disappearance of the guanine radical, as monitored at 510 nm. In oligonucleotide duplexes containing [Ru(phen)(bpy')(dppz)](2+) tethered at one end, damage to distant guanines was observed by gel electrophoresis, consistent with the mobility of the electron hole through the DNA duplex; the presence of the peptide did not inhibit but instead altered the distribution of guanine damage. Covalent adducts of the DNA and Lys-Tyr-Lys were detected as final irreversible products of this peptide-to-DNA electron-transfer chemistry by mass spectrometric and enzymatic digestive analysis. From these different assays and comparison of reactions of Lys-Trp-Lys and Lys-Tyr-Lys, the reactivity of the DNA-bound tyrosine radical was found to differ considerably from that of the tryptophan radical. These results establish that Lys-Tyr-Lys and Lys-Trp-Lys can participate in long-range electron-transfer reactions through the DNA from a distinct binding site. On that basis, proposals for functional roles for these peptide radicals may be considered.

Identification of intermediates in the catalytic cycle of chloroperoxidase.

BACKGROUND: Chloroperoxidase (CPO) is the most versatile of the hemethiolate proteins, catalyzing the chlorination of activated C-H bonds and reactions reminiscent of peroxidase, catalase, and cytochrome P450. Despite 30 years of continuous efforts, no intermediates of the enzyme's catalytic cycle have been identified except for compound I. Thus, in the absence of conclusive evidence it is generally believed that the halogenation of substrates proceeds by means of 'free HOCI' in solution. RESULTS: The pH profile of chloroperoxidase from Caldariomyces fumago revealed a new active-site complex that can be detected only at pH 4.4. According to ultra-violet (UV) spectroscopy, and by comparison with suitable enzyme models, this intermediate is the HOCl adduct of the iron(III) protoporphyrin(IX). Inactivation of chloroperoxidase by diethyl pyrocarbonate, which interrupts the proton shuttle by modification of the distal histidine, led to the formation of the -OCl adduct of the iron complex, which was identified by comparison with a corresponding active site analogue. CONCLUSIONS: The availability of enzyme models of heme-thiolate proteins allowed the identification by UV spectroscopy of both the -OCl adduct and the HOCl adduct of the iron(III) protoporphyrin(IX) of chloroperoxidase. The existence of these previously elusive intermediates suggests that the chlorination catalyzed by CPO, and its corresponding active site analogue, proceeds by Cl+ transfer from the HOCl adduct to the substrate bound in the distal pocket of the enzyme.

Analogues of disaccharides and glycosides containing a cyclic guanidinium structure show varying inhibitory effects on glycoside hydrolases.

By condensation of 1,3-diamino-2,4-(R)-O-benzylidene-1,3-dideoxy-D-erythritol (3) and 1,3-diamino-2,4-di-O-benzyl-1,3-dideoxy-D-threitol (4) with methyl 2,3,6-tri-O-benzyl-4-deoxy-4-iso-thiocyanato-beta-D-glucopyranosid e (9) the (1-->4)-linked disaccharide analogues 4-deoxy-4-[(4R,5S)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6-tetrahydropyri midin-2- yl[amino-alpha,beta-D-glucopyranose hydrochloride (15) and 4-deoxy-4-[(4R,5R)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6-tetrahydropyri midin- 2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (18) were synthesized. By the same reaction sequence, using 3 and methyl isothiocyanate, the glycoside analogue (4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-methylamino-1,4,5,6- tetrahydropyrimidine hydrochloride (20) was obtained. All compounds possess in their 'glyconic' moiety the flat guanidinium group, mimicking a glucopyranosyl cation. Together with the previously synthesized (1-->6)-linked disaccharide analogues 6-deoxy-6-[(4R,5S)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6- tetrahydropyrimidin-2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (1) and 6-deoxy-6-[(4R,5R)-5-hydroxy-4-(hydroxy-methyl)-1,4,5,6- tetrahydropyrimidin-2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (2), a possible inhibitory effect on the action of alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, and beta-D-galactosidase was investigated. All compounds, except 20 with alpha-D-glucosidase where no inhibition could be detected, showed either competitive or mixed competitive inhibition with all enzymes. The effects of the disaccharide analogues were generally weaker as compared to the effect of the previously synthesized configurationally related nitrophenyl glycoside analogues (4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-(p-nitrophenyl)amino-1,4,5,6- tetrahydropyrimidine hydrochloride (21) and (4R,5R)-5-hydroxy-4-(hydroxymethyl)-2-(p-nitrophenyl)amino-1,4,5,6- tetrahydropyrimidine hydrochloride (22). On the basis of experimental results, different binding hydrochloride (22). On the basis of experimental results, different binding modes of competitive inhibitors to the active site of corresponding enzymes are discussed.