Phototriggered DNA phosphoramidate ligation in a tandem 5'-amine deprotection/3'-imidazole activated phosphate coupling reaction.

We report the preparation and use of an N-methyl picolinium carbamate protecting group for applications in a phototriggered nonenzymatic DNA phosphoramidate ligation reaction. Selective 5'-amino protection of a modified 13-mer oligonucleotide is achieved in aqueous solution by reaction with an N-methyl-4-picolinium carbonyl imidazole triflate protecting group precursor. Deprotection is carried out by photoinduced electron transfer from Ru(bpy)(3)(2+) using visible light photolysis and ascorbic acid as a sacrificial electron donor. Phototriggered 5'- amino oligonucleotide deprotection is used to initiate a nonenzymatic ligation of the 13-mer to an imidazole activated 3'-phospho-hairpin template to generate a ligated product with a phosphoramidate linkage. We demonstrate that this methodology offers a simple way to exert control over reaction initiation and rates in nonenzymatic DNA ligation for potential applications in the study of model protocellular systems and prebiotic nucleic acid synthesis.

Interactions between catalysts and amphiphilic structures and their implications for a protocell model.

One of the essential elements of any cell, including primitive ancestors, is a structural component that protects and confines the metabolism and genes while allowing access to essential nutrients. For the targeted protocell model, bilayers of decanoic acid, a single-chain fatty acid amphiphile, are used as the container. These bilayers interact with a ruthenium-nucleobase complex, the metabolic complex, to convert amphiphile precursors into more amphiphiles. These interactions are dependent on non-covalent bonding. The initial rate of conversion of an oily precursor molecule into fatty acid was examined as a function of these interactions. It is shown that the precursor molecule associates strongly with decanoic acid structures. This results in a high dependence of conversion rates on the interaction of the catalyst with the self-assembled structures. The observed rate logically increases when a tight interaction between catalyst complex and container exists. A strong association between the metabolic complex and the container was achieved by bonding a sufficiently long hydrocarbon tail to the complex. Surprisingly, the rate enhancement was nearly as strong when the ruthenium and nucleobase elements of the complex were each given their own hydrocarbon tail and existed as separate molecules, as when the two elements were covalently bonded to each other and the resulting molecule was given a hydrocarbon tail. These results provide insights into the possibilities and constraints of such a reaction system in relation to building the ultimate protocell.

Nucleobase mediated, photocatalytic vesicle formation from an ester precursor.

We report the use of photoinduced electron transfer to drive reductive cleavage of an ester to produce bilayer-forming molecules; specifically, visible photolysis in a mixture of a decanoic acid ester precursor, hydrogen donor molecules, and a ruthenium-based photocatalyst that employs a linked nucleobase (8-oxo-guanine) as an electron donor generates decanoic acid. The overall transformation of the ester precursor to yield vesicles represents the use of an external energy source to convert nonstructure forming molecules into amphiphiles that spontaneously assemble into vesicles. The core of our chemical reaction system uses an 8-oxo-G-Ru photocatalyst, a derivative of [tris(2,2'-bipyridine)-Ru(II)](2+).

Experimental and computational investigation of the uncatalyzed rearrangement and elimination reactions of isochorismate.

The versatile biosynthetic intermediate isochorismate decomposes in aqueous buffer by two competitive pathways, one leading to isoprephenate by a facile Claisen rearrangement and the other to salicylate via elimination of the enolpyruvyl side chain. Computation suggests that both processes are concerted but asynchronous pericyclic reactions, with considerable C-O cleavage in the transition state but relatively little C-C bond formation (rearrangement) or hydrogen atom transfer to the enolpyruvyl side chain (elimination). Kinetic experiments show that rearrangement is roughly 8-times more favorable than elimination. Moreover, transfer of the C2 hydrogen atom to C9 was verified by monitoring the decomposition of [2-(2)H]isochorismate, which was prepared chemoenzymatically from labeled shikimate, by (2)H NMR spectroscopy and observing the appearance of [3-(2)H]pyruvate. Finally, the isotope effects obtained with the C2 deuterated substrate are in good agreement with calculations assuming pericyclic reaction mechanisms. These results provide a benchmark for mechanistic investigations of isochorismate mutase and isochorismate pyruvate lyase, the enzymes that respectively catalyze the rearrangement and elimination reactions in plants and bacteria.

Isochorismate pyruvate lyase: a pericyclic reaction mechanism?

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated at C2 as a substrate. Monitoring the reaction by 2H NMR spectroscopy revealed that the label is quantitatively transferred from C2 to C9, producing stoichiometric amounts of [3-2H]pyruvate as product. Moreover, the deuterium kinetic isotope effect of 2.34 +/- 0.08 on kcat indicates that C-H cleavage is significantly rate limiting. Consistent with these data, hybrid density functional theory (HDFT) calculations at the Becke3LYP/DZ+(2d,p) level of theory predict a concerted but highly asynchronous pericyclic transition structure, in which carbon-oxygen bond cleavage is more advanced than hydrogen atom transfer from C2 to C9; the calculated 2H isotope effect of 2.22 at C2 is in excellent accord with the experimental value. Together, these findings indicate that IPL should be added to the small set of proteins that are known to catalyze pericyclic reactions. They also raise the possibility that enzymes, such as chorismate pyruvate lyase, salicylate synthase, 4-amino-4-deoxychorismate lyase, and anthranilate synthase, which accelerate formally similar reaction steps, may also exploit pericyclic mechanisms.

Isotope effects on the enzymatic and nonenzymatic reactions of chorismate.

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.

Polysiloxane-bound ligand accelerated catalysis: a modular approach to heterogeneous and homogeneous macromolecular asymmetric dihydroxylation ligands.

Polysiloxane acts as a modular scaffold for macromolecular reagent development. Two separate components were covalently integrated into one material, one constituent provided reagent functionality, the other modulated solubility. In particular cinchona alkaloid based ligands used in the osmium tetroxide catalyzed asymmetric dihydroxylation (AD) reaction were covalently attached to commercially available polysiloxane. To enhance the reactivity of these polymeric ligands, multifunctional reagents were designed to include both the cinchona alkaloid and an alkoxyethylester solubilizing moiety providing random co-polymers. While the mono-functional materials led to heterogeneous conditions, the bi-functional polymers resulted in homogeneous reaction mixtures. Although both reagent types provided diol products with excellent yield and selectivity (>99% ee in nearly quantitative yield) the homogeneous analog has nearly twice the reactivity. Every repeat unit in the heterogeneous material was functionalized along the polysiloxane backbone while approximately half of these sites contained ligand in the homogeneous version. This approach led to macromolecular catalysts with high loadings of ligand and therefore materials with very low equivalent weights. Although these polymers are highly loaded they do maintain reactivity on a par with their free ligand counterpart. Using straightforward purification techniques (i.e. precipitation, simple filtration, or ultrafiltration) these polymeric ligands were easily separated from diol product and reused multiple times. Polysiloxane is a viable support for the catalysis of AD reactions and may provide a generally useful backbone for other catalytic systems.

Reactivity of Enol Carbonates with Ozone.

Several dienes of varying steric bulk containing an enol carbonate have been synthesized and reacted selectively with ozone at the isolated double bonds. Rate measurements have been made for ozonolysis in a series of substituted cyclohexenes to demonstrate the unusually slow reactivity of the enol carbonate. Proton and carbon NMR chemical shifts have been presented to show that the enol carbonate is not particularly electron deficient in its double bond. Calculation of partial charges from the Mulliken population analysis shows good correlation with the NMR data. The results suggest a carbonate association with ozone that slows the rate of carbon-carbon bond cleavage.