The anomeric Pudovik rearrangement

O-GlcNAc transferase (OGT) is a biologically ubiquitous enzyme that promotes the 2-acetamido-2-deoxy-β-D-glucosylation of serine and threonine residues of a wide variety of nuclear and cytoplasmic proteins (Figure 1).i Despite the importance of OGT in myriad biological processes and disease states, there is a dearth of OGT inhibitors for use as probes of its action and effects. As a component of our search for OGT inhibitors, we examined the phosphorylation of 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-1-thio-α-D-glucopyranose (1), and have uncovered a new, anomeric, version of the Pudovik rearrangement of phosphothioites to phosphonothioates. Figure 1 Glycosyl transfer to serine/threonine catalyzed by O-GlcNAc transferase (OGT) The reactions of 1 with O,O-dialkyl (N,N-diisopropyl)phosphoramidites under acidic activation with tetrazole (Table 1), followed by oxidation with tert-butylhydroperoxide,ii,iii,iv,v gave good to poor yields of the desired O,O-dialkyl-S-glycosylphosphothioates 2a-c, as a function of the O-alkyl substituent (allyl, methyl, or benzyl, respectively). These products were characterized by their mass, 1H NMR (JH-1/H-2 ~ 5 Hz), 13C NMR and 31P NMR (diagnostic signal near 24 ppm) spectra. In addition, 2 was accompanied by varying amounts of the rearranged O,O-dialkyl-P-glycosyl-phosphonothioates 3a-c (Table 1, entries 1–3). These products were characterized by their mass, 1H NMR, 13C NMR and 31P NMR (diagnostic signal near 88 ppm) spectra. The alpha configurations of 3 were indicated by their large trans-diaxial H-2/P couplings (e.g., 31 Hz for 3c, whereas the beta isomer should show ~10 Hz).vi Products 3a-c reflect a Pudovik rearrangement (Scheme 1), wherein the phosphothioite S-substituent (here, glucopyranosyl) has migrated to phosphorus. This reaction, which may be viewed as a version of the Arbuzov rearrangement, has been previously studied by A. N. Pudovik and M. A. Pudovik and their coworkers,vii,viii,ix,x,xi,xii,xiii,xiv as well as by others.xv,xvi,xvii,xviii The Arbuzov rearrangementxix itself encompasses a broad family of phosphite to phosphonate transformations initiated variously by alkylation, heat, light, or radicals. Scheme 1 A prototypal Pudovik rearrangement Table 1 S-Phosphorylations with O,O-dialkyl N,N-diisopropylphosphoramidites Most prior examples of the Pudovik rearrangement occur upon exposure of the phosphothioite to oxygen or air, and are postulated to be radical processes.13 Accordingly, the reaction of 1 with O,O-dibenzyl (N,N-diisopropyl)phosphoamidite was repeated, but with the radical inhibitor 2,6-di-tert-butyl-4-methylphenol (BHT) added during the oxidation step. The result (entry 4) was an increase in the percentage of phosphothioate 2c, and less rearrangement to 3c. Very likely the O-allyl substituents in entry 1 also inhibit radical propagation, accounting for the preferred formation of 2a. In contrast, the Pudovik rearrangement product 3c was formed almost exclusively when the oxidation was promoted by the radical initiator combinationxx of triethylborane and air (entry 5). Modifications in the promoterxxi and the oxidation conditions allowed the isolation of the phosphorylation product 2c in good yield to the exclusion of the rearrangement (entry 6, NMB = N-methylbenzimidazole). The oxidant m-chloroperbenzoic acid probably operates by a polar mechanism, minimizing formation of 3c. Alternatively, the S-glycosylphosphothioate product 4 was prepared without oxidation by treating 1 directly with base and diethyl chlorophosphate (Scheme 2). The latter reaction failed in the O,O-dibenzyl version; instead, benzyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-1-thio-α-D-glucopyranoside was formed in 27% yield as the result of thiolate S-benzylation. Scheme 2 Direct phosphorylation of mercaptan 1 The formation of phosphonothioates 3 can be accommodated by the radical chain process shown in Scheme 3. Initial S-phosphitylation of 1 to give the intermediate phosphothioite 5 is followed, upon addition of initiator, by a radical initiation step consisting of the removal of the phosphothiyl fragment 7 and formation of the 3,4,5-O-triacetyl-2-acetamido-2-deoxy-D-glucopyranosyl anomeric radical 6.xxii Subsequent and analogous reaction of 6 with 5 in a propagation step provides product 3 and regenerates 6. The alpha anomeric stereochemistry of 3 reflects the tendency (with ample precedent) of D-glucopyranosyl anomeric radicals such as 6 to react with almost exclusive alpha stereoselectivity.xxiii Scheme 3 Radical chain mechanism for the Pudovik rearrangement of 5 A radical fragmentation/recombination mechanism has been advanced for the Pudovik rearrangement;13 to our knowledge the stereochemical consequences at the migrating carbon have not previously been investigated. Application of the reaction conditions of entry 5 to the phosphitylation/oxidation of the corresponding beta anomeric mercaptan 8 led, after chromatography, to the array of products shown in Scheme 4. The alpha phosphothioate 3c matched the compound obtained from 1. The corresponding beta phosphonothioate, which if formed should have eluted near 3c, was neither isolated nor detected by 31P or 1H NMR analysis of the crude reaction mixture or chromatography fractions. The beta phosphodithioate 9 was characterized by its 1H (with COSY analysis), 13C, 31P, and mass spectra.xxiv Its beta configuration was established by the large H-1/H-2 coupling (10 Hz). The oxazoline 10 and 3,4,5-tri-O-acetyl-2-acetamido-1,5-anhydro-2-deoxy-D-glucitol 11 were identified by spectroscopic comparison with the known compounds. Scheme 4 Phosphitylation/oxidation/rearrangement of the beta mercaptan 8 Scheme 5 shows pathways that rationalize the formation of 3c, 9, 10, and 11 from 8. Following phosphitylation of 8 analogous to that of 1, the phosphothioite 13 reacts with the radical initiator to produce the same 3,4,5-O-triacetyl-2-acetamido-2-deoxy-D-glucopyranosyl anomeric radical 6. The analogous chain propagation step involving 6 and 13 likewise produces the alpha phosphonothioate 3c (the Pudovik rearrangement product). To the extent that starting mercaptan 8 remains, reaction of 6 with 8 gives thiyl radical 14 by hydrogen atom transfer, and a chain propagation step involving 14 and 13 leads to the phosphodithioate 9. Dithio products analogous to 9 have been isolated previously from Pudovik rearrangement reaction mixtures, particularly when excess mercaptan is present.4,11 The other (reduced) product from the latter process has not been reported, but in this case can be isolated in the form of tri-O-acetyl-2-acetamido-1,5-anhydro-2-deoxy-D-glucitol 11. While 9 can come from unreacted 8, an additional reductive pathway (e. g., 6 –> 11) is likely present to account for the fact that there is formed more 11 than 9. Scheme 5 Proposed pathways for the beta mercaptan phosphitylation/oxidation Some of the (P-oxidized) phosphothioate 12 probably formed in competition with 3c, as has been noted in other systems,4,11,18 and in the entries in Table 1. Circumstantial evidence for 12 was found in the crude reaction mixture: 31P NMR analysis showed a phosphothioate peak at 25.1 ppm (compare 2c at 24.4), and the mass spectrum featured a prominent signal at m/z 646, which corresponds to [M+Na]+ for 12. Instead of 12, however, oxazoline 10 was isolated in significant amounts, even though it was not present in the crude product mixture (e. g., no H-4 of 10 at or near 4.81 ppm). Oxazoline 10 could be formed by acid-promoted cyclization of 12 during chromatography. The formation of 3c, independent of the stereochemistry of the precursor phosphothioite (5 or 13) and to the apparent exclusion of the beta stereoisomer, provides strong evidence that the same anomeric radical (6) is the intermediate in both reactions. Formation of the reduction product 11 is also rationalized by invoking 6 as the intermediate. The differences in the phosphitylation/oxidation reactions of 1 and 8 merit comment. Although the reaction conditions are very similar, the relative rates of initial phosphitylation differ, with alpha mercaptan 1 being qualitatively more reactive according to TLC analysis. The relative rates of conversion of the phosphothioites 5 and 13 to the anomeric radical 6 may also differ, although the related 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl anomeric radical is formed from both the alpha and beta anomeric chloride precursors at about the same rate (Cl abstraction by tributyltin radical).xxv Finally, autooxidation at phosphorusxxvi of 5 and 13 to the respective phosphothioates 2c and 12 (competing with formation of 6) may occur at different rates. A faster rate of oxidation of 13 would account for the formation of more oxidized product 12 (and hence 10) compared with 2c under these conditions.xxvii