Blog

[16]) by simply TLC, to afford triol8in 83% yield. the exocyclic side chain. Among the phosphate containing appendices, 2-aminoethyl phosphodiester (PEtn) groups have been found at positions 3, 4, 6, and 7 NSC 228155 of L, D-Hep, and the group of Oscarson has successfully prepared 4-O-, 6-O-, and 7-O-substituted (2-aminoethyl)phosphate monoheptosides as well as various 3-O-and 6-O-PEtn substituted LPS oligosaccharides to unravel the structural basis for cross-reactive antibodies againstNeisseria meningitidisandHaemophilus influenzae, respectively [1013]. Recently, the structure of an antigen-binding fragment (Fab) from the bactericidal monoclonal antibody LPT3-1 complexed to an inner core octasaccharide fragment ofN. meningitidishas been solved, which had been isolated via KOH treatment from the bacterial lipooligosaccharide [14]. The isolation protocol, however , leads to hydrolysis of the phosphoethanolamine units. As the 3-O-PEtn substituent is present in ~70% ofN. meningitidisstrains and constitutes a relevant epitope for the neutralizing antibodies, chemical synthesis NSC 228155 is needed to provide material for binding and crystallographic studies [15]. For this purpose, we have set out to access both 3-O- and 4-O-substituted heptosides starting from a common intermediate with a minimum number of protecting group manipulations. == Results and discussion == The previously reported 6, 7-O-TBDPS protected heptoside1served as a versatile precursor for the introduction of the PEtn moiety via intermediate 2, 3-orthoester formation as shown for the synthesis of 4-O-monophosphate derivatives [16]. In our hands, a three step sequence performed in Cd24a a one-pot reaction could be elaborated to give a fair yield of the phosphotriester derivative5(Scheme1). First, the reaction of1with,, -triethoxytoluene (2) in the presence of camphorsulfonic acid (CSA) led to the intermediate orthoester3, which was followed by the application of the phosphoramidite procedure with [2-(benzyloxy-diisopropylamino-phosphanyloxy)ethyl]-carbamic acid benzyl ester (4) promoted by 1H-tetrazole, and the ensuing oxidation of the resulting phosphite withmeta-chloroperbenzoic acid (mCPBA) [1719]. Since the phosphorylated orthoester5was present as a mixture of four diastereoisomers, the product mixture was then separated into individual components to exclude the presence of potential impurities in the subsequent deprotection steps. MPLC separation allowed the isolation of a 1: a few mixture of the phosphorylatedendoorthobenzoates5a, 5bandexo-isomers6a, 6bin 56% overall yield for three steps, followed by further HPLC separation of the phosphate diastereomers; no attempts, however , for assignment of the stereogenic center at phosphorus were undertaken. Assignment of theexo/endoconfiguration was based on the high-field shift of theexo-oriented OCH2group at a few. 30 ppm compared to the corresponding low-field shifted signal of theendo-isomer at 3. 80 ppm [20]. Next, theendo/exo-orthoester derivatives5aand6a(representing one of the diastereomeric forms on phosphorus) were subjected NSC 228155 to acid-promoted orthoester opening, which produced the homogeneous 2-O-benzoyl derivative7in 91% yield. Compound7is equipped with an orthogonal protecting group pattern which allows access to chain elongation at position a few, as well as at the exocyclic side-chain positions. Removal of the 1, 1, a few, 3-tetraisopropyl-1, 3-disiloxane-1, 3-diyl group was achieved by treatment of7with triethylamine trifluoride (TREAT). The reaction had to be monitored until full removal of the monofluorinated silyl intermediate (Ref. [16]) by TLC, to afford triol8in 83% yield. Hydrogenation of8was uneventful and gave the phosphodiester9in 93% yield. Cleavage of the benzoyl ester under Zempln transesterification conditions was sluggish but eventually provided the 4-O-PEtn derivative10in NSC 228155 good yield. Optical rotation values and13C NMR data matched the previously reported data of10, which, however , had been synthesized via a different route based onH-phosphonate coupling chemistry [10]. The orthoester approach was then applied for the synthesis of the 3-O-substituted derivative16(Scheme2). 1was subjected to CSA-promoted orthoester formation with 1, 1, 1-trimethoxyethane to give 2, 3-O-orthoacetate11, which was not isolated but directly converted into the 2-O-acetate12in 71% yield. The structure of ester12was readily assigned on the basis of the low-field shifted H-2 signal at 5. 02 ppm. Based on previous evidence that a hydroxyl group adjacent to an axial one in NSC 228155 acis-vicinal diol is more reactive, and that the 4-OH group in amanno-pyranoside is much less reactive, a direct regioselective phosphorylation was expected to directly lead to the 3-O-substituted phosphoester, thereby avoiding additional protecting group manipulations [21, 22]. Thus, phosphorylation of diol12using4and 1H-tetrazole was followed by oxidation withmCPBA. The 3-O-substituted derivative13could then be separated from additional phosphorylated species by chromatography, and was isolated.