Diazeniumdiolate Ions as Leaving Groups in Anomeric Displacement Reactions: A Protection-Deprotection Strategy for Ionic Diazeniumdiolates Brett M. Showalter, ² Melissa M. Reynolds, ‡ Carlos A. Valdez, ² Joseph E. Saavedra, § Keith M. Davies, | John R. Klose, ⊥ Gwendolyn N. Chmurny, ⊥ Michael L. Citro, § Joseph J. Barchi, Jr., # Scott I. Merz, ¶ Mark E. Meyerhoff, ‡ and Larry K. Keefer* ,² Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109, Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, Department of Chemistry, George Mason UniVersity, Fairfax, Virginia 22030, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, Frederick, Maryland 21702, and Michigan Critical Care Consultants Inc., Ann Arbor, Michigan 48103 Received July 7, 2005; E-mail: keefer@ncifcrf.gov Carbohydrates substituted at the anomeric position with nitric oxide-releasing diazeniumdiolate ions are a promising new class of prodrug, having been reported to be stable at physiological pH but readily hydrolyzed by glycosidases, as in eq 1. 1 Thus, Wu et al. have introduced them as a means of selectively generating bioactive nitric oxide in cell types that possess the appropriate enzymatic activity. 1 We have discovered a second, synthetically and mechanistically significant feature of their chemistry: an unexpected sensitivity to base- (but not acid-) induced cleavage of the anomeric substituent. Hydrolysis of glucoside 1 was found to proceed as outlined in eq 2. The observed reactivity is described by a rate expression of the form k obs ) k o + k OH - [OH - ]. The hydrolysis rate displayed a linear dependence on [OH - ] between pH 12.9 and 14.0. The mean value of k OH - was 5.3 × 10 -3 M -1 s -1 . Diazeniumdiolate ion 2 was confirmed as a primary product in 0.1 M sodium hydroxide, with clean isosbestic behavior being observed as the starting material’s peak at 228 nm gave way to that of 2 at 250 nm. 2 Hydrolysis of 1 in acid was very slow. The mean value of k o , (7.8 ( 2.4) × 10 -7 s -1 , obtained from NO generation rates followed over 8 days by chemiluminescence 2 at 37 °C and pH values of 7.4, 5.6, and 2.6, was almost 800-fold slower than that in 0.1 M NaOH. Rate constants estimated from NO release profiles at pH 5.6 (citrate) and 7.4 (phosphate) showed the hydrolysis rate to be only marginally (ca. 50%) slower than that at 2.6 (citrate). Inverse pH rate behavior was also observed for the hydrolysis of the -fluoro 3,4 and -p-nitrophenoxy 5 glucosides. The half-life of 1 was >25-fold shorter than that for the p-nitrophenoxide, as measured by following the decrease in the respective anomeric proton NMR signals. That of the fluoro analogue proved to be too fast to follow under these conditions, bracketing the nucleofugality of the diazeniumdiolate as being between those of fluoride and p-nitrophenoxide. Rate constants (k obs ) for dissociation of the diazeniumdiolate, the fluoride, and the p-nitrophenoxide were 8.8 × 10 -5 , >1.2 × 10 -3 , and 3.3 × 10 -6 s -1 , respectively, at 37 °C and pH 12.4. Base-induced hydrolysis of -D-glucosyl fluoride and related saccharides that have a potentially nucleophilic C-2 substituent trans to a good leaving group at the anomeric position has been rationalized 4-7 as proceeding via anchimeric assistance by a C-2 heteroatom as shown in eq 3. According to this mechanism, the substrates are postulated to convert initially to glycal oxide 3, an intermediate whose oxirane ring is rapidly opened by attack of the C-6 hydroxyl group at C-1 to form 1,6-anhydroglucose (4), as outlined in eq 3. Consistent with this prediction, 4 was the major product of all three pH 12.4 reactions, with glucose being produced in low yield in each case. There was no evidence for conversion of glucose to 4 under these conditions, indicating that 4 was indeed a primary product of these reactions. The above-described similarities between anomerically substi- tuted 2, fluoride, and p-nitrophenoxide suggest that diazeniumdiolate ions can be considered halide-like, but there are important differ- ences that may make them uniquely useful. For example, ion 2 ² Laboratory of Comparative Carcinogenesis. ‡ University of Michigan. § Basic Research Program. | George Mason University. ⊥ Laboratory of Proteomics and Analytical Technologies. # Laboratory of Medicinal Chemistry. ¶ Michigan Critical Care Consultants Incorporated. Published on Web 09/24/2005 14188 9 J. AM. CHEM. SOC. 2005, 127, 14188-14189 10.1021/ja054510a CCC: $30.25 © 2005 American Chemical Society