Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Abstract

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host-pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein we decribe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors enabling the enzymatic synthesis of Pse5Ac7Ac containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot-two-enzyme process using biosynthetic enzymes. Finally screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac terminated glycosides. Figure 1 Nonulosonic acid structures; the pseudaminic acid Pse5Ac7Ac 1, and sialic acid Neu5Ac 2. Scheme 2 Chemical synthesis of 6-deoxy-L-AltdiNAc 4. a) i) NaOMe, MeOH. ii) Diphenyl borinate, DIPEA, BzCl, CH 3 CN, 80% over 2steps. b) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) NaN 3 , DMF, 110 °C, 58% over 2-steps. c) NaOMe, MeOH, 95%. d) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) TBANO 2 , CH 3 CN, 70 °C, 46% over 2-steps. e) i) PMe 3 , NaOH, THF, 60 °C. ii) Ac 2 O, Pyridine, 69% over 2-steps. f) NaOMe, MeOH, 81%. g) CAN, CH 3 CN:H 2 O (4:1 v/v), 94%, α:β 1:3. Scheme 1 Biosynthesis of CMP-Pse5Ac7Ac 3 via the key hexose intermediate 6-deoxy-ʟ-AltdiNAc 4.

Azide‐Functionalized Derivatives of the Virulence‐Associated Sugar Pseudaminic Acid: Chiral Pool Synthesis and Labeling of Bacteria

“…Several glycoconjugates containing a number of Pse derivatives that differ in their N5 or N7 substitutions have been identified to date in various drug‐resistant pathogens, and a connection between the Pse and virulence has been demonstrated. [ 16 , 17 ] Chemical probes for identification of Pse‐expressing bacteria are therefore highly appealing and can pave the way for anti‐virulence agents that inhibit Pse biosynthesis.…”

Facile and Scalable Route to Access Rare Deoxy Amino Sugars for Nonulosonic Acid Aldolase Biosynthesis

“…Previous studies on inversion reactions in the synthesis of 2,4-di-azido-2,4-di-deoxy- α - L -Rha recommended harsh conditions (110°C in DMF/HMPA); however, the yield was not satisfactory (58%/69%), and no detailed explanations were given ( Sanapala and Kulkarni, 2016a ; Flack et al, 2020 ). In our study, using 3- O -acetyl- α - L -Fuc 1 as reactant and TBAN 3 (5.0 equiv.×2) in the double inversion process resulted in a mixture of 2,4-di-azido-3- O -acetyl-2,4,6-trideoxy- α - L -Glu 2 and 2,4-di-azido-3- O -acetyl-2,4-di-deoxy- α - L -Rha 3 ( Table 1 , Entry 1, NMR ratio 51 / 49 , 2 : 3 ).…”

“…To prepare di-NAc sugars to obtain nonulosonic acid precursors, we need to synthesize 6-deoxy- L -AltdiNA 40 ( Scheme 4 ). Fascione’s group used the Lattrell–Dax reaction ( Guo and O'Doherty, 2007 ; 2008 ) with β -Rha 34 as the substrate and found that the epimerization product 35 was obtained in moderate yield in the TBANO 2 /acetonitrile system (46%) ( Flack et al, 2020 ). They also found α -Rha 32 was inefficient for the reaction.…”

“… Solvent-dependent nitrite-mediated carbohydrate epimerization. a (a) Tf 2 O, pyridine, and DCM, -20°C, 2 h; (ii) TBANO 2 and toluene, r.t., 6 h. (b) Tf 2 O, pyridine, and DCM, 0°C; (ii) TBANO 2 and CH 3 CN, 70°C ( Flack et al, 2020 ). …”

“…Kulkarni and co-workers developed a general and divergent protocol to access differentially protected rare deoxy amino L-sugar building blocks that can be used as glycosyl donors or acceptors in the assembly of complex bacterial glycans in one-pot double serial or double parallel displacements (stereocenter inversion) of 1,3-diol f L -sugars by the NaN 3 /DMF system ( Emmadi and Kulkarni, 2013b ; a ; c ; 2014 ; Clark et al, 2016 ; Sanapala and Kulkarni, 2016b ; a ; Behera and Kulkarni, 2018 ; Emmadi and Kulkarni, 2018 ). Based on this chemical strategy, Fascione and co-workers established an efficient chemoenzymatic method to prepare pseudaminic acid, which is a nonmammalian sugar present on the cell surface of a number of bacteria, including Pseudomonas aeruginosa , Campylobacter jejuni , and Acinetobacter baumannii ( Flack et al, 2020 ).…”