Uridine -, crystal

The residue crystallizes in ether and yields about 600 mg of (3-3, 5 -di-p-toluyl-2 -desoxy-5-iodo-uridine which is recrystallized from toluene. The product Is obtained as colorless crystals, soluble in chloroform and pyridine, sparingly soluble In acetone, benzene ether and alcohol. Insoluble in water, MP 193°C. 

In this context, it is interesting to note that the first synthesis of 2, 3 -0,0-cyclic phosphorothioate 22a was reported by Eckstein in 1968 [25], He also isolated pure Rp diastereomer by fractional crystallization of the triethylammonium salts [26] and used it as reference to determine the absolute configurations of the other phosphorothioate analogues [27], 2, 3 -0,0-Cyclic H-phosphonate 20a was used as a key substrate for the synthesis of uridine 2, 3 -0,0-cyclic boranophosphate 27. Silylation of H-phosphate 20a gave the phosphite triester 25 (two diastereomers). Its boronation, with simultaneous removal of the trimethylsilyl group, was achieved by its reaction with borane-A.A-diisopropylethylamine complex (DIPEA-BH3). 

Structural studies on Ago proteins revealed that the so-called MID domain binds the 5 end of the guide strand (39,40). Because of its central localization in the Ago protein, this domain has been named MID domain. Crystal co-structures and Kj measurements of the MID domain in combination with all four nucleotides at the 5 end revealed that uridine (U) binds with the highest affinity to the MID domain, adenosine (A) with a slightly reduced affinity, and cytosine (C) and guanosine (G) with more than tenfold less affinity (41). Therefore, siRNA guide (antisense) strands should ideally contain a U or A at the 5 end. C and G should be avoided. For the passenger (sense) strand 5 end, C and G should be selected in order to minimize strand incorporation (Fig. la). Based on our own unpublished data the nucleotide specificity is not only a tool to manipulate strand selection but siRNA strands with U or A at the 5 end show also a higher absolute affinity for Ago proteins and therefore are more likely to be potent siRNAs (see Notes 2 and 3). 

From extensive analysis of recombinant proteins, and the crystal structure of A. thaliana protein, detailed reaction mechanisms have been proposed. The ANS reaction likely proceeds via stereospecific hydroxylation of the leucoanthocyanidin (flavan-3,4-cA-diol) at the C-3 to give a flavan-3,3,4-triol, which spontaneously 2,3-dehydrates and isomerizes to 2-flaven-3,4-diol, which then spontaneously isomerizes to a thermodynamically more stable anthocyanidin pseudobase, 3-flaven-2,3-diol (Figure 3.2). The formation of 3-flaven-2,3-diol via the 2-flaven-3,4-diol was previously hypothesized by Heller and Forkmann. The reaction sequence, and the subsequent formation of the anthocyanidin 3-D-glycoside, does not require activity of a separate dehydratase, which was once postulated. Recombinant ANS and uridine diphosphate (UDP)-glucose flavonoid 3-D-glucosyltransferase (F3GT, sometimes... 

Potentially the most interesting crystal structure solved is that of the complex with uridine vanadate (16.40),... 

The site B, has been established by three-dimensional electron density maps for UpcA, 3 -CMP, 3 -UMP, 2 -CMP, d-TpT, and 4-thio uridine. The site B2 has similarly been demonstrated for the adenine in UpcA (526a), 3 -AMP, 5 -AMP, 3 5 A > p, and ATP. The two sites can be occupied simultaneously or independently by 3 -AMP and 3 -CMP with no strong interaction. The Bj site has also been demonstrated for 3 -CMP and 3 -UMP in the DNP-Lys 41 derivative. Electron density has appeared at the B2 site in high concentrations of pyrimidines and in the inferred digestion products of UpU in the DNP-Lys 41 crystals. 

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