Bruce Eaton

Expanding the Chemistry of DNA for in Vitro Selection

Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.

Selection of biomolecules capable of mediating the formation of nanocrystals.

Biopolymers in the biosphere are well known to mediate the formation of a wide array of inorganic materials, such as bone, shells, lenses, and magnetic particles to name a few. Recently, in vitro experiments with biopolymers such as peptides, RNA, and DNA have shown that templating by these macromolecules can yield a variety of materials under mild reaction conditions. The primary sequence of the biopolymer can be viewed as a proteomic or genomic signature for the templating of an inorganic material from defined metal precursors and reaction conditions. Together with the rapid advances in inorganic particle synthesis by other combinatorial methods, these bioinspired in vitro materials experiments may provide additional insights into possible inorganic materials yet to be discovered and subsequently synthesized by conventional methods. Some of the concepts important to understanding the crystallization phenomena occurring during biopolymer mediation are discussed. A simple kinetic model is provided in the context of known biopolymer-mediated inorganic crystallizations.

The RNA World: Functional Diversity in a Nucleoside by Carboxyamidation of Uridine

Abstract A facile method for the 5-carboxyamidation of protected uridines is described allowing synthesis of an array of uridines with diverse functionality.

Synthesis of 2′-Deoxyuridine Nucleosides with Appended 5-Position Carbonyl Cross-Linking Groups

Abstract A simple modification of Stille type carbonylative coupling conditions resulted in high yield reactions giving new carbonyl appended 2′-deoxyuridine derivatives useful for chemical cross-linking.

Investigation of a transition metal-assisted retro Diels-Alder reaction used in the synthesis of transition metal S2O complexes

Abstract Treatment of 4,5-diphenyl-3,6-dihydro-1,2-dithiin-1-oxide with coordinatively unsaturated transition metal complexes resulted in the production of S2O complexes and 2,3-diphenylbutadien. A transition metal-assisted retro Diels-Alder mechanism has been proposed for this reaction. A series of IR and 1H NMR experiments and MNDO calculations on 4,5-diphenyl-3,6-dihydro-1,2-dithiin-1-oxide have been performed to gain additional information about this transformation.

Palladium Catalysis in the Synthesis of 8-Position modified Adenosine, 2′-Deoxyadenosine and Guanosine

Abstract Adenosine and guanosine analogs with 8-position vinyl and aryl groups were prepared by palladium catalyzed cross-coupling of organostannanes with 8-bromopurine nucleosides. The reaction conditions and catalyst composition were improved so that both vinyl and aryl modifications could be made by a general procedure.

Synthesis of N,N-Dialkylaniline-2′-deoxyuridine Conjugates for DNA-Mediated Electron Transfer Studies

Abstract Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2′-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2′-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O4-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.

Addition of primary amines to conjugated allenyl aldehydes and ketones

Allenyl imines have been prepared by condensation of allenyl aldehydes with primary amines.

Carboxyamidation of Purine Nucleosides: New Secondary 8-Carboxamidopurine Nucleosides

Abstract Palladium catalyzed carboxyamidation at the 8-position of 8-bromoadenosine and 8-bromoguanosine nucleosides is a versatile reaction, which allows primary, secondary, heterocyclic, aromatic mine and amino acids to be incorporated into purine nucleosides.