Deoxyribonucleoside phosphoramidites

Polystyrene-supported deoxyribonucleoside-3 phosphoramidites (160), or analogous compounds bound to the support via a piperazine... 

Figure 6.7. Solid-Phase Synthesis of a DNA Chain by the Phosphite Triester Method. The activated monomer added to the growing chain is a deoxyribonucleoside 3 -phosphoramidite containing a DMT protecting group on its 5 oxygen atom, a p -cyanoethyl ( p CE) protecting group on its 3 phosphoryl oxygen, and a protecting group on the base.

The development of deoxyribonucleoside phosphoramidite derivatives for the synthesis of oligodeoxyribonucleotides was first described by Beaucage and Caruthers (1) in 1981. The conceptual basis of this methodology emerged as a modification of the phos-phorodichloridite coupling procedure reported earlier by Letsinger and CO workers (2). Essentially, the approach involved the reaction of the protected deoxyribonucleosides la-d (Fig. 1) withchloro-(A(iV-dimethyl-amino) methoxyphosphine (2) in the presence of iV,iV-diiso-propylethylamine. The rapid reaction yielded the deoxyribonucleoside phosphoramidites 3a-d (Fig. 1), which were isolated by precipitation and stored as dry powders. 

The landmark of the phosphoramidite approach entailed the conversion of the relatively stable deoxyribonucleoside phosphoramidite derivatives to reactive intermediates suitable for oligonucleotide synthesis. Specifically, the interaction of lV,iV-dimethy laniline hydrochloride with 3a-d (Fig. 2) generated the corresponding deoxyribonucleoside chlorophosphites 4a-d (Fig. 2), which upon reaction with 3 -0-levulinylthymidine afforded the (3 —> 5 )-dinucleoside phosphite triesters 5a-d (Fig. 2) in near quantitative yields (1). The hygroscopic nature of tertiary amine hydrochlorides became an impediment to reliable oligonucleotide synthesis, since anhydrous conditions... 

The popularity of the deoxyribonucleoside phosphoramidites 8a-d (Fig. 3) in the automated synthesis of oligonucleotides has nonetheless been hampered by the thiolate treatment required for the removal... 

An alternate strategy to the synthesis of deoxyribonucleoside phosphoramidites was reported by Beaucage (14) and coworkers (15). The approach involved the reaction of properly protected deoxyribonucleo-sides (la-d Fig. 5) with i>w-(pyrrolidino)methoxyphosphine chemo-selectively activated by 4,5-dichloroimidazole. P-NMR spectroscopy indicated that the corresponding deoxyribonucleoside phosphoramidites (9a-d Fig. 3) were generated in yields exceeding 86% within 10 min. Without further purification, these monomers were immediately activated with 1/f-tetrazole and applied to the synthesis of a deoxyribo-nucleotide (22 mer) (14,15). This approach eliminated the problems... 

In this chapter, the preparation of monochlorophosphoramidite and phosphorodiamidite derivatives and their application toward a general synthesis of deoxyribonucleoside phosphoramidites will be delineated from representative literature procedures. The synthetic protocols provided herein should allow the preparation of a myriad of deoxyribonucleoside phosphoramidite monomers having any modification of the following entities nucleobases, nucleobase-protecting... 

To facilitate the design of deoxyribonucleoside phosphoramidite analogs, information regarding the activation of deoxyribonucleoside phosphoramidites will be provided to emphasize the importance imparted by the steric bulk of the substituents attached to the phosphorus center and to specific nucleobases on the coupling rates. The capping and oxidation steps of the synthetic cycle will also be discussed in relation to the potential formation of side products during oligonucleotide synthesis. 

This methodology has been applied to the synthesis of various monofunctional phosphitylating reagents useful in the synthesis of deoxyribonucleoside phosphoramidites. A selected number of them are listed in Table 1. 

Chlorophosphoramidite Derivatives Useful in the Synthesis of Deoxyribonucleoside Phosphoramidites... 

The facile preparation of deoxyribonucleoside phosphoramidites from the selective activation of phosphorodiamidites by N.N-diiso-propylammonium tetrazolide (16) or 1/f-tetrazole (77) provides an excellent tool for the incorporation of new phosphate-protecting groups during oligonucleotide synthesis. Several phosphorodiamidites have recently been prepared, and a few of them are shown in Table 2. 

General Procedure for the Preparation of Deoxyribonucleoside Phosphoramidites from Chloro-(NfN-Dialkylamino)alkoxyphosphines... 

The activation of deoxyribonucleoside phosphoramidites by IH- tetrazole and their application to the solid-phase synthesis of oligo-deoxyribonucleotides will not be described in detail. This standard technology is outlined in Note 2,... 

The mechanism of activation of deoxyribonucleoside phosphoramidites by IH-tetrazole has recently attracted considerable attention. It has been argued that the protonation of the phosphoramidite function by l f-tet azole was rapid and followed by the reversible and slower formation of a phosphorotetrazolide intermediate (39). It is to be noted that relative to 5-(p-nitrophenyl)- IH-tetrazole (55), 1 -hydroxy-benzotriazole (24), / /-methylanilinium trifluoroacetate (40), iV-methyl-anilinium trichloroacetate (41), 5-trifluoromethyl-l//-tetrazole (23), A-methylimidazole hydrochloride (23), and )V-methylimidazole-trifluoromethane sulfonate (42), which have also been tested as activators, l/f-tetrazole still remained the most commonly used reagent for the activation of deoxyribonucleoside phosphoramidites. 

Interestingly, the aqueous iodine oxidation of 2-cyano-l,l-dimethylethyl deoxyribodinucleoside phosphite or < -methylbenzyl-deoxyribodinucleoside phosphite prepared from deoxyribonucleoside phosphoramidite intermediates resulted in the formation of the corresponding phosphate diesters (60). Furthermore, Caruthers et al. (32)... 

Hamamoto, S. and Takaku, H (1986) New approach to the synthesis of deoxyribonucleoside phosphoramidite derivatives. Chem, Lett 1401-1404... 

Among sugar modified phosphoramidite derivatives prepared this year are 3, 5 -ethano-2 -deoxyribonucleoside derivatives (118), the 4, 6 -methano carbocyclic thymidine derivative (119), a 2 -0-methyl-6,3 -ethanouridine derivative (120), (R)- and (S)-3, 4 -seco-thymidine derivatives, e.g. (121), and arabino derivatives (122). The first three are conformationally rigid nucleoside analogues, and were made in order to prepare oligonucleotide analogues which bind better... 

In the phosphite triester synthesis (phosphoramidite synthesis), an efficient coupling reaction takes place between the 3 -hydroxyl group of the chain and 5 -DMTr-(A -acy-lated)-deoxyribonucleoside 3 -0-(MA -diisopropylamino)phosphite. 

The influence of diastereomeric ratios of deoxyribonucleoside phos-phoramidites on the synthesis of phosphorothioate oligonucleotides has been investigated. Almost diastereomerically-pure phosphoramidites were found to give an almost equal mixture of diastereomers of a monophosphorothioate decamer. A facile synthetic route to dimeric phosphorothioate building blocks 222 has been developed. Dinucleoside phosphite triesters were obtained in a one-pot procedure by sequential coupling to PCI3 of protected nucleosides with... 

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