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.

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

Pseudaminic acids (Pses) are a group of non‐mammalian nonulosonic acids (nulOs) that have been shown to be an important virulence factor for a number of pathogenic bacteria, including emerging multidrug‐resistant ESKAPE pathogens. Despite their discovery over 30 years ago, relatively little is known about the biological significance of Pse glycans compared with their sialic acid analogues, primarily due to a lack of access to the synthetically challenging Pse architecture. Recently, however, the Pse backbone has been subjected to increasing synthetic exploration by carbohydrate (bio)chemists, and the total synthesis of complex Pse glycans achieved with inspiration from the biosynthesis and subsequent detailed study of chemical glycosylation by using Pse donors. Herein, context is provided for these efforts by summarising recent synthetic approaches pioneered for accessing Pse glycans, which are set to open up this underexplored area of glycoscience to the wider scientific community.