Toward an Ideal Synthesis of Oligonucleotides: Development of a Novel Phosphoramidite Method with High Capability

Several novel methods for reliably carrying out the oligonucleotide synthesis via the phosphoramidite strategy have been developed. The first is the method using allyloxycarbonyl and allyl groups for the protection of nucleoside bases and internucleotide linkages, respectively. These protectors can be cleanly removed under almost neutral conditions with a mixture of an organic Pd(0) complex such as Pd[P(C 6 H 5 ) 3 ] 4 or Pd 2 [(C 6 H 5 CH w CH) 2 CO] 3 • CHCl 3 , and P(C 6 H 5 ) 3 in the presence of a nucleophile such as butylammonium formate, diethylammonium hydrogencarbonate, diethylammonium carbonate, or dimedone. A method for the facile oxidation of nucleoside phosphites to phosphates has been developed; this is performed under non-aqueous and non-basic conditions using 2-butanone peroxide in dichloromethane, t -butyl hydroperoxide in toluene, or bis(trimethylsilyl) peroxide in dichloromethane. The combined use of the N -allyloxycarbonyl/ P–O -allyl-protected method and anhydrous/non-basic oxidation is extremely useful for the synthesis of nucleotide derivatives sensitive to moisture and/or bases, producing them in higher yield and at a greater level of purity than the existing methods using acyl or aroyl and cyanoethyl groups for the protection of nucleoside bases and internucleotide linkages, respectively, and iodine in aqueous pyridine for the oxidation. Further, acid/azole complexes serving as highly useful activators of phosphoramidites have been invented. The reagents include imidazolium triflate, N -(phenyl)imidazolium triflate, benzimidazolium triflate, and N -(methyl)benzimidazolium triflate, which allow higher-yield synthesis of oligonucleotides than conventionally employed 1 H -tetrazole and related reagents. For example, synthesis of oligoribonucleotides via the allyloxycarbonyl/allyl-protected strategy has been carried out more efficiently using N -(phenyl)imidazolium triflate as a promoter than 5-(ethylthio)-1 H -tetrazole, which has been found to be the most effective promoter among those currently available for oligoribonucleotide synthesis. Further, imidazolium triflate has opened the door to the extremely convenient and efficient synthesis of oligodeoxyribonucleotides without nucleoside-base protection. This method is obviously advantageous over N -protected methods because it eliminates tedious operations and the use of superfluous reagents required for the introduction and removal of the N -protecting group; risks of decomposition and loss of product by protection and deprotection are also avoided. Facile liquid-phase preparation of short oligomers has also been developed; this is successfully accomplished using stoichiometric amounts of building blocks by the aid of an equimolar amount of azolium reagent or a catalytic amount of 5-( p -nitrophenyl)-1 H -tetrazole. These two methods are beneficial from an economical point of view, particularly in large-scale synthesis.