Phosphoramidates formation

Conjugation via Carbodiimide Reaction with the 5 Phosphate of DNA (Phosphoramidate Formation)... 

Protection of the amino function of adenine, guanine, or cytosine may be necessary to prevent phosphoramidate formation during a phosphorylation. In rarer cases (especially with guanine) it may also serve the purpose of introducing hydrophobicity in an otherwise too polar nucleoside. 

With familiarity of the various protecting groups available for the hydroxyl and base amino functions, it is possible to prepare nucleoside intermediates with reactive groups available only for the formation of a 3, 5 -phospho-diester linkage. Unwanted reactions (3, 3 - or 5, 5 -phosphodiester formation, phosphoramidate formation, etc.) cannot possibly occur with the selection of appropriate intermediates. A representative example would be the condensation between adenosine and uridine ... 

Dialkyl dimethyl phosphoramidites (16) react with j8-propiolactone to give the phosphoramidate (17) and the phosphonate (18), A kinetic study suggests a mechanism involving initial attack of phosphorus at saturated carbon to give (17), while a four-centred transition state (19) is invoked to explain the formation of (18). 

The phosphoramide, Me(Et0)P(0) NEt P(0)(0Et)2, independently synthesised by the route shown, was obtained only on heating the phosphazene at 165—175 °C for several hours. P N.m.r. and i.r. spectroscopy confirmed these findings and were used to show that the formation of XCH2p(0)(OR) N=PR R 2 was never accompanied by isomerization. 

Carbodi-imides are used to mediate the formation of amide linkage betwen a carboxylate and an amine or phosphoramidate linkages between a phosphate and an amine [12]. The following is essentially the method of Rockwood [13] and is modified to give a phospho-diester link between the terminal monophosphate of the oligonucleotide and the hydroxyl group of 2-hydroxyethyl disulfide (HEDS) [14]. 

The reaction of 151 with methanol to give dimethyl phosphate (154) or with N-methylaniline to form the phosphoramidate 155 and (presumably) the pyrophosphate 156 complies with expectations. The formation of dimethyl phosphate does not constitute, however, reliable evidence for the formation of intermediate 151 since methanol can also react with polymeric metaphosphates to give dimethyl phosphate. On the other hand, reaction of polyphosphates with N-methylaniline to give 156 can be ruled out (control experiments). The formation of 156 might encourage speculations whether the reaction with N,N-diethylaniline might involve initial preferential reaction of monomeric methyl metaphosphate via interaction with the nitrogen lone pair to form a phosphoric ester amide which is cleaved to phosphates or pyrophosphates on subsequent work-up (water, methanol). Such a reaction route would at least explain the low extent of electrophilic aromatic substitution by methyl metaphosphate. 

Reaction of the (S)-amino alcohol 171 with A-(2-bromoethyl)phosphoramidic dichloride or aryl phosphonodichloridates 154 in the presence of triethylamine led to the formation of a single diastereomer in each case of 1,3,2 oxazaphospholidine-2-ones 172a-e (taking into consideration that in the 31P-NMR spectra only one singlet in the range 6.49-2.45 ppm was observed) (Scheme 48) [79],... 

The formation of a phosphorimidazolide intermediate provides better reactivity toward amine nucleophiles than the EDC phosphodiester intermediate if EDC is used without added imidazole. The EDC phosphodiester intermediate also has a shorter half-life in aqueous conditions due to hydrolysis than the phosphorimidazolide. Although EDC alone will create nucleotide phosphoramidate conjugates with amine-containing molecules (Shabarova, 1988), the result of forming the secondary phosphorimidazolide-activated species is increased derivatiza-tion yield over carbodiimide-only reactions. 

Fig. 9.14. Metabolic activation of phosphoramidic acid diester prodrugs 9.79 of stavudine (and analogous nucleosides). Carboxylesterase-mediated hydrolysis of the terminal carboxy-late is followed by spontaneous cyclization-elimination with formation of a pentacyclic mixed-anhydride species. The latter hydrolyzes rapidly to the corresponding phosphoramidic acid monoester, which can then be processed to stavudine 5 -monophosphate.

Based on the same strategy, Denmark and coworkers developed a vinylogous aldol reaction using enolate activation with a catalyst derived from SiCl4 and dimeric phosphoramide 47 [24,25]. This strategy relies on the observation that not all Lewis acid - Lewis base interactions diminish the Lewis acidity [26-28]. Due to the formation of a pentacoordinated silicon cation (48), both the enolate and the substrate can be assembled in a closed transition state, giving rise to the observed high selectivities (Scheme 19) [29,30]. 

Two patterns are possible in the activation mechanism by simple chiral Lewis base catalysts. One is through the activation of nucleophiles such as aUyltrichlorosilanes or ketene trichlorosilyl acetals via hypervalent silicate formation using organic Lewis bases such as chiral phosphoramides or A-oxides. " In this case, catalysts are pure organic compounds (see Chapter 11). The other is through the activation of nucleophiles by anionic Lewis base conjugated to metals. In this case, transmetal-lation is the key for the nucleophile activation. This type of asymmetric catalysis is the main focus of this section. 

An intriguing feature is that the previously unknown bisindoles 154 display atropisomerism as a result of the rotation barrier about the bonds to the quaternary carbon center. With the use of A-triflyl phosphoramide (1 )-41 (5 mol%, R = 9-phenanthryl), bisindole 154a could be obtained in 62% ee. Based on their experimental results, the authors invoke a Brpnsted acid-catalyzed enantioselective, nucleophilic substitution following the 1,2-addition to rationalize the formation of the bisindoles 154 (Scheme 65). 

In the same year, Enders and coworkers reported an asymmetric one-pot, two-step synthesis of substituted isoindolines 159 in the presence of chiral A-triflyl phosphoramide (R)-Ae (10 mol%, R = d-NO -C H ) (Scheme 67) [87]. The cascade was triggered by a Brpnsted acid-catalyzed aza-Friedel-Crafts reaction of indoles 29 and A-tosyliminoenoates 160 followed by a DBU-mediated aza-Michael cyclization of intermediates 161 to afford the isoindolines 159 in high yields (71-99%) and short reaction times (10 min to 4 h) along with good enantioselectivities (52-90% ee). Longer reaction times (16 h to 10 days) caused increasing formation of the bisindole byproduct 162 (Scheme 68) along with amplified optical purity of isoindolines 159. 

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