Nucleotides part XXX Chemical synthesis of adenylyl‐(2′ → 5′)‐adenylyl‐(2′ → 5′)‐8‐azidoadenosine, and activation and photoaffinity labelling of RNase L by [³²P]p5′A2′p5′A2′p5′N₃⁸A

Abstract

The chemical synthesis of adenylyl-(2-5')-adenylyl-(2-5')-8-azidoadenosine (15) was performed by the phosphotriester approach. Enzymatic phosphorylation of 15 by [y -3ZP]ATP led to the corresponding labelled 5'-monophosphate 16. Photoinsertion of 16 took place on UV irradiation by covalent cross linking to a protein of M, 80 K known to be RNase L. Radiobinding and core-cellulose assays as well as photoaffinity labelling experiments with 16 are described.

Characterization of a 2′,5′-Oligoadenylate (2–5A)-dependent 37-kDa RNase L

“…In previous photolabeling studies, we have used 2-5A azido photoprobes to specifically identify the different forms of RNase L in interferon-treated L929 cell extracts (21). We have also demonstrated that the activation of the 80-kDa RNase L in interferon-treated L929 cells extracts by an 8-azido-2-5A photoaffinity probe was comparable to the RNase L activity measured in the presence of p 3 A 3 using core (2-5A)-cellulose assays (16,21). Core (2-5A)-cellulose assays were employed in the current study to determine 2-5A-dependent RNase activity in healthy control and CFS PBMC extracts.…”

Nucleosides. Part LIX. The 2‐(4‐nitrophenyl)ethylsulfonyl (Npes) Group: A New Type of Protection in Nucleoside Chemistry

“…In an analogous manner reacted the 5'-0-(monomethoxytrity1)-(63), 5'- Much efforts were finally invested in the direct sulfonation of the base-protected 5'-0 -(mono-or dimethoxytrity1)ribonucleosides 59-77 hoping that a regioselective or at least a predominant formation of the corresponding 2'-0-[2-(4-nitrophenyl)ethylsulfonyl] derivatives would take place. Unfortunately, mixtures of the three possible products, the 2',3'-di-, 2'-mono-, and 3'- found already in former studies with N6-benzoyl-5'-0 -(dimethoxytrityl)adenosine [43]. The separation into the pure components by chromatographic methods was mostly a very tedious procedure and was paid with loss of material.…”

“…Since the sulfonate function is a good leaving group, this finding is not surprising due to the fact that the intramolecular displacement reaction seems to be of similar rate as the bond cleavage by p-elimation (+ 66). The value of the 2-(4-nitrophenyl)ethylsulfonyl group for sugar-OH protection in oligonucleotide synthesis was already demonstrated [42] [43] and may be an interesting alternative for special, multifunctional syntheses.…”

Synthesis, Characterization, and Biological Properties of 8-Azido- and 8-Amino-Substituted 2‘,5‘-Oligoadenylates

“…Several 8-substitued analogues of 2−5A were prepared and their biological activities were evaluated. 2−5A analogues with 8-bromo, , 8-hydroxy, 8-azido, , and 8-methyl groups in the 2‘-terminal residue have been reported to have strong RNase L binding and activating ability, equal to or stronger than that of naturally occurring 2−5A, while the corresponding 2−5A analogues bearing fully 8-substituted adenosine showed 10- to 100-fold reduction of the biological activities. On the other hand, 2−5A analogues containing an 8-mercapto or 8-hydroxypropyl 9 group have been reported to possess very low or no RNase L binding and activating abilities.…”

“…On the other hand, 2−5A analogues containing an 8-mercapto or 8-hydroxypropyl 9 group have been reported to possess very low or no RNase L binding and activating abilities. The difference in their biological activities was partly explained by the difference in the syn-anti conformation around the base-glycosydic bonds of 2−5A. − However, the detailed mechanism of 2−5A and its congener to bind and activate RNAse L is still unknown. Among the 8-substituted adenosine 2−5A analogues, 8-azidoadenosine analogue is very interesting, because it has strong biological activities despite its bulky azido group at the 8-position, and it can be useful as a photoaffinity labeling agent for RNase L to study the binding mechanism .…”