Regioselective formation of RNA strands in the absence of magnesium ions.

The oligomerization of ribonucleotides can produce short RNA strands in the absence of enzymes. This reaction gives one of two regioisomeric phosphodiester linkages, a 2',5'- or a 3',5'-diester. The former, non-natural linkage is detrimental for duplex stability, and is known to form preferentially in oligomerizations occurring in homogeneous solution with preactivated nucleotides in the presence of magnesium cations. We have studied ribonucleotide oligomerization with in situ activation, using NMR as monitoring technique. Unexpectedly, the known preference for 2',5'-linkages in the oligomerization of AMP was reversed in the absence of magnesium ions at slightly basic pH. Further, oligomerization was surprisingly efficient in the absence of Mg2+ salts, producing oligomers long enough for duplex formation. A quantitative systems chemistry analysis then revealed that the absence of magnesium ions favors the activation of nucleotides, and that the high concentration of active species can compensate for slower coupling. Further, organocatalytic intermediates can help to overcome the unfavorable regioselectivity of the magnesium-catalyzed reactions. Our findings allay concerns that RNA may have been difficult to form in the absence of enzymes. They also show that there is an efficient path to genetic material that does not require mineral surfaces or cations known to catalyze RNA hydrolysis.

2'/3' Regioselectivity of Enzyme-Free Copying of RNA Detected by NMR.

The RNA-templated extension of oligoribonucleotides by nucleotides produces either a 3',5' or a 2',5'-phosphodiester. Nature controls the regioselectivity during RNA chain growth with polymerases, but enzyme-free versions of genetic copying have modest specificity. Thus far, enzymatic degradation of products, combined with chromatography or electrophoresis, has been the preferred mode of detecting 2',5'-diesters produced in enzyme-free reactions. This approach hinges on the substrate specificity of nucleases, and is not suitable for in situ monitoring. Here we report how 1 H NMR spectroscopy can be used to detect the extension of self-templating RNA hairpins and that this reveals the regioisomeric nature of the newly formed phosphodiesters. We studied several modes of activating nucleotides, including imidazolides, a pyridinium phosphate, an active ester, and in situ activation with carbodiimide and organocatalyst. Conversion into the desired extension product ranged from 20 to 90 %, depending on the leaving group. Integration of the resonances of H1' protons of riboses and H5 protons of pyrimidines gave regioselectivities ranging from 40:60 to 85:15 (3',5' to 2',5' diester), but no simple correlation between 3',5' selectivity and yield. Our results show how monitoring with a high-resolution technique sheds a new light on a process that may have played an important role during the emergence of life.

Formamides as Lewis Base Catalysts in SN Reactions-Efficient Transformation of Alcohols into Chlorides, Amines, and Ethers.

A simple formamide catalyst facilitates the efficient transformation of alcohols into alkyl chlorides with benzoyl chloride as the sole reagent. These nucleophilic substitutions proceed through iminium-activated alcohols as intermediates. The novel method, which can be even performed under solvent-free conditions, is distinguished by an excellent functional group tolerance, scalability (>100 g) and waste-balance (E-factor down to 2). Chiral substrates are converted with excellent levels of stereochemical inversion (99 %→≥95 % ee). In a practical one-pot procedure, the primary formed chlorides can be further transformed into amines, azides, ethers, sulfides, and nitriles. The value of the method was demonstrated in straightforward syntheses of the drugs rac-Clopidogrel and S-Fendiline.