Synthesis of 3-O- and 4-O-(2-aminoethylphosphono) derivatives of methyl l-glycero-α-d-manno-heptopyranoside

Abstract Phosphoethanolamine derivatives of the bacterial saccharide l-glycero-d-manno-heptose have been prepared using a phosphoramidite-based coupling reaction at position 4 of a side-chain-protected 2,3-O-orthoester methyl heptoside and at position 3 of a 3,4-diol heptoside, respectively. Global deprotection afforded the corresponding 2-aminoethylphosphodiester derivatives as substrates for crystallographic and binding studies with lectins and antibodies targeting the inner core structure of bacterial lipopolysaccharides. Graphical abstract


Introduction
The outer membrane of the cell wall of Gram-negative bacteria harbors higher carbon sugars as characteristic components, which occur in the inner core region of bacterial lipopolysaccharides (LPS), but have also been detected in capsular polysaccharides (CPS) [1]. Heptoses of the L-glycero-D-manno configuration (L,D-Hep), in particular, constitute a structurally conserved domain in Enterobacteriaceae, such as in Escherichia coli, Salmonella or Yersinia, and are common LPS core determinants in the genera Haemophilus, Pseudomonas, Helicobacter, or Neisseria [2,3]. Heptoses contribute toward many biomedically important interactions with the complement system, antibodies and lectins, and these features have been substantiated by recent data from crystallographic and glycan array studies [4][5][6][7][8][9]. These binding interactions and specificities are further modulated by additional phosphate substituents in the pyranose ring as well as at the exocyclic side chain.
Among the phosphate containing appendices, 2-aminoethyl phosphodiester (PEtn) groups have been found at positions 3, 4, 6, and 7 of L,D-Hep, and the group of Oscarson has successfully prepared 4-O-, 6-O-, and 7-Osubstituted (2-aminoethyl)phosphate monoheptosides as well as various 3-O-and 6-O-PEtn substituted LPS oligosaccharides to unravel the structural basis for crossreactive antibodies against Neisseria meningitidis and Haemophilus influenzae, respectively [10][11][12][13]. Recently, the structure of an antigen-binding fragment (Fab) from the bactericidal monoclonal antibody LPT3-1 complexed to an inner core octasaccharide fragment of N. meningitidis has 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% of N. meningitidis strains and constitutes a relevant epitope for the neutralizing antibodies, chemical synthesis 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 heptoside 1 served 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 a one-pot reaction could be elaborated to give a fair yield of the phosphotriester derivative 5 (Scheme 1). First, the reaction of 1 with a,a,a-triethoxytoluene (2) in the presence of camphorsulfonic acid (CSA) led to the intermediate orthoester 3, 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 with metachloroperbenzoic acid (mCPBA) [17][18][19]. Since the phosphorylated orthoester 5 was 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 1:3 mixture of the phosphorylated endo orthobenzoates 5a, 5b and exoisomers 6a, 6b in 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 the exo/endo configuration was based on the high-field shift of the exo-oriented OCH 2 group at 3.30 ppm compared to the corresponding low-field shifted signal of the endo-isomer at 3.80 ppm [20].
Next, the endo/exo-orthoester derivatives 5a and 6a (representing one of the diastereomeric forms on phosphorus) were subjected to acid-promoted orthoester opening, which produced the homogeneous 2-O-benzoyl derivative 7 in 91% yield. Compound 7 is equipped with an orthogonal protecting group pattern which allows access to chain elongation at position 3, as well as at the exocyclic side-chain positions. Removal of the 1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl group was achieved by treatment of 7 with triethylamine trifluoride (TREAT). The reaction had to be monitored until full removal of the monofluorinated silyl intermediate (Ref. [16]) by TLC, to afford triol 8 in 83% yield. Hydrogenation of 8 was uneventful and gave the phosphodiester 9 in 93% yield. Cleavage of the benzoyl ester under Zemplén transesterification conditions was sluggish but eventually provided the 4-O-PEtn derivative 10 in good yield. Optical rotation values and 13 C NMR data matched the previously reported data of 10, which, however, had been synthesized via a different route based on H-phosphonate coupling chemistry [10].
The orthoester approach was then applied for the synthesis of the 3-O-substituted derivative 16 (Scheme 2). 1 was subjected to CSA-promoted orthoester formation with 1,1,1-trimethoxyethane to give 2,3-O-orthoacetate 11, which was not isolated but directly converted into the 2-Oacetate 12 in 71% yield. The structure of ester 12 was readily assigned on the basis of the low-field shifted H-2 signal at 5.02 ppm. Based on previous evidence that a Scheme 1 hydroxyl group adjacent to an axial one in a cis-vicinal diol is more reactive, and that the 4-OH group in a mannopyranoside is much less reactive, a direct regioselective phosphorylation was expected to directly lead to the 3-Osubstituted phosphoester, thereby avoiding additional protecting group manipulations [21,22]. Thus, phosphorylation of diol 12 using 4 and 1H-tetrazole was followed by oxidation with mCPBA. The 3-O-substituted derivative 13 could then be separated from additional phosphorylated species by chromatography, and was isolated as a diastereomeric mixture in 32% yield.
The structural assignment of 13 was based on the 1 H-31 P correlated HMBC spectrum, which showed the connectivity of H-3 to the phosphate unit, as well as on the low-field shifted H-3 signal at 4.21 ppm. Similar to 6, phosphotriester 13 was then treated with TREAT to give triol 14 in 70% yield, which was hydrogenated in the presence of 10% Pd-C in MeOH containing 1% acetic acid. The addition of the acid was needed to prevent formation of N-methylated products [23]. Removal of the 2-O-acetyl group was carried out by reaction of 15 with triethylamine in aqueous MeOH and furnished the deprotected zwitterionic glycoside 16 in 63% yield [24]. The structures of 10 and 16 were fully confirmed by their 13 C NMR data, which showed a characteristic downfield shift of the respective carbons C-4 and C-3, respectively, involved in the phosphodiester linkage as well as heteronuclear J C,P coupling interactions of C-4 and C-5 for 9 and C-3 and C-4 for 16 (Table 1). Similar J C,P coupling interactions were also seen for the ethanolamine fragments. These assignments were further supported by 1 H-31 P HMBC connectivity data.