An in vitro-in vivo sequential cascade for the synthesis of iminosugars from aldoses

Here, we report a chemoenzymatic approach for the preparation of a small panel of biologically important iminosugars from readily available aldoses. Our approach involves an in vitro transaminase-mediated amination of aldoses in combination with an in vivo selective oxidation of the chemically protected transaminase products, using Gluconobacter oxydans DSM 2003 whole cells. Chemically catalysed deprotection and reduction steps afford a selection of valuable iminocyclitols.

2][3] These small molecules are known inhibitors of several carbohydrate processing enzymes, but many have failed to reach the clinic due to insufficient potency or lack of selectivity. 46][7][8] The absence of efficient synthetic routes for the preparation of structurally diverse derivatives has been highlighted as one of the key contributing factors to the limited development of second-generation iminosugars as therapeutics. 4A robust biocatalytic route for the preparation of libraries of natural iminosugars and their derivatives has not yet been developed and has the potential to (i) greatly simplify and streamline their synthesis, (ii) allow access to derivatives that are currently inaccessible and (iii) provide a sustainable route from readily available carbohydrates.
The direct amination and subsequent selective oxidation of readily available aldoses, such as glucose, mannose, and fructose represents an ideal approach for the synthesis of chiral aminopolyols that can be readily converted to iminosugars and circumvents the need for petrochemical-derived feedstocks.We recently reported that transaminases (TAs) could be used to introduce amine functionality into aldoses to access a variety of aminopolyols. 9A study showing activity on ketoses was reported shortly after. 10Such activity had not been previously observed towards monosaccharides which exist predominantly in their cyclic form at equilibrium, where the carbonyl functionalities are predominantly masked.This opened a range of new synthetic possibilities for the preparation of high-value targets from chiral pool building blocks by using combinations of enzymes.Please do not adjust margins Please do not adjust margins G. oxydans DSM 2003 has been previously reported to display activity towards aminopolyol substrates; analogous to those accessible from aldoses using TAs. 12 Similar to galactose oxidases and aldolases, G. oxydans has been shown to only accept aminopolyols where the amine functionality is protected. 14,152][13] We sought to explore the substrate scope of G. oxydans DSM 2003 towards additional aminopolyols and develop methodology to enable a chemoenzymatic approach for the preparation of iminosugars, combining a TA-mediated amination with the oxidising capabilities of G. oxydans (Figure 1C).
To explore the substrate scope of G. oxydans DSM 2003, the aldoses 2-deoxy-D-ribose (1), D-arabinose (2), D-ribose (3), Dmannose (4), 2-deoxy-D-galactose ( 5), D-lyxose ( 6) and D-xylose (7) were selected, and the corresponding aminopolyols were chemically synthesized and N-Cbz-protected, to provide potential substrates 8-14 (Table 1).Whole-cell biotransformations were performed on a 13-15 mM scale and analysed by NMR to determine conversion.1D and 2D NMR experiments were carried out to ascertain the site of oxidation.The N-Cbz-aminopolyols 8-12 were quantitatively converted to the oxidised products 15-19.Selective oxidation to the corresponding ketone proceeds regioselectively, at position C4 for the pentose derivatives 8-10, and at position C5 for hexosederived 11 (Table 1).The oxidised products were isolated in good to excellent yields (purification was not optimised), with different ratios of linear and cyclic anomers observed.In general, the linear structures were characterised by a distinctive carbonyl peak around 212 ppm in the 13 C NMR spectrum, while the cyclic anomers contained hemiaminal peaks at approximately 90 ppm, where a mixture of anomers is most evident for 15.The oxidised aminopolyols 16 and 17 exist predominantly in linear form, whereas 18 is only detectable as the cyclic acetal (see SI for more details).The cyclisation of 5membered Cbz-protected aminopolyols has previously been reported. 15While 12 was quantitatively converted by G. oxydans, oxidation took place at position C3 and product 19 was in equilibrium with its cyclic anomer.However, 19 proved to be unstable, resulting in a poor isolated yield of 15%, and was therefore not carried forward to the next step.The corresponding unprotected aminopolyols of 1-7 were also tested (data not shown).As expected, these unprotected derivatives were either not oxidised by the organism, or where oxidation occurred, it is thought that the product suffered from instability. 16aving established activity on a small panel of substrates, our focus turned to optimising the reaction conditions of the preparative scale biotransformations, focusing on substrates 8-11.It is possible that more than one G. oxydans enzyme is responsible for catalysing these transformations and therefore Reaction conditions: 20 mL reaction mixture containing substrate (13-15 mM), resting G. oxydans DSM 2003 cells (100 mg mL -1 wet weight), H2O (20 mL), pH 6.8, 16 h, 280 rpm, 30 °C.c] n.d.d] Compound exists predominantly in its cyclic form at equilibrium -the linear form is presented here to show the site of oxidation clearly.
the effect of varying the temperature and substrate concentration for each substrate was evaluated (Figure 2).In general, the organism maintained high activity over a wide range of temperatures.Next, the effect of substrate concentration on conversion was evaluated by running the transformations at concentrations between 5-50 mM, while keeping the concentration of wet cells constant (100 mg mL -1 ).The cells converted all four substrates quantitatively to the corresponding oxidation products 15-18 at 20 mM (8 and 9) or 25 mM (10 and 11).Conversions of up to 90% and 77% were also achieved at 30 mM and 50 mM, respectively.While it is clear that reaction temperatures between 20-40 °C have little impact on the conversion of 8-11 by G. (Figure 2), we observed that the organism was metabolising the oxidised products at 20 °C, as complete conversion was achieved, but isolated yields were often low (Table 2, entries 1-3).Additionally, the prolonged reaction time (>48 h) led to an almost complete disappearance of the product.Interestingly, the optimum growth temperature for G. oxydans is between 25-30 °C, while temperatures above 37 °C are known to inhibit growth.Bacterial growth and metabolism can be controlled by adjusting the temperature or pH or by applying osmotic pressure.We investigated the effect that performing biotransformations at 35 °C had on the isolated yields (Table 2, entries 5-8).Quantitative conversions were achieved for substrate 11, while 8-10 were converted in 90-95% based on analysis.The oxidised products were purified using silica gel column chromatography in very good yields (65-89%) and the products isolated in multi-milligram (70-138 mg).This minor temperature modification had a very positive effect on the biotransformations and may be a strategy that could be explored for other whole-cell systems, where substrates and/or products are being metabolised.The next step involved concurrent deprotection, cyclisation and reduction using Pd/C as the hydrogenation catalyst.Upon deprotection, the amine reacts at the newly oxidised carbonyl site (C4 or C5) to form a polyhydroxylated cyclic imine with the elimination of water.The imine is reduced affording the target iminocyclitols 24-29 in 87-93% yield (Scheme 1/Table 3).The cyclic imines of 11 and 16 undergo a stereoselective reduction, yielding the single diastereoisomers, 29 and 24, respectively.In the case of the oxidised aminopolyols 8 and 10, the reduction is non-stereoselective and a mixture of diastereoisomers (25 -28) is isolated (Table 3).The purification of diastereomeric mixtures was not undertaken in this work and is likely to be a challenge, due to the highly polar nature of these compounds.2D NMR experiments (HSQC, HMBC, COSY, and NOESY) were used to structurally and stereochemically characterise each of the iminosugar isomers (see ESI).
While the need to protect the aminopolyols currently prevents the design of a one-pot cascade, we were still keen to demonstrate that biologically important iminosugars could be readily prepared from monosaccharides using a chemoenzymatic approach.D-aldoses 1-4 were selected, which are precursors of the natural iminosugar products 2hydroxymethyl-3-hydroxypyrrolidine 25 (CYB-3), DAB 24, 1,4dideoxy-1,4-imino-D-ribitol 27 (DRB), and L-1deoxygulonojirimycin 29 (L-gulo-DNJ).The commercially available transaminase ATA-256 was used to convert these aldoses to the corresponding aminopolyols (Table 3).Biotransformations were carried out using 50-200 mM of substrate and 4 eq. of IPA as the amine donor.
Conversions were analysed by 1 H NMR spectroscopy using our previously reported method.Conversions ranged from 62-86%, and purification was carried out using ion-exchange column chromatography (Dowex 50WX8 resin, H + form).The transaminase-derived aminopolyols were then N-Cbz-protected before being subjected to G. oxidation.Excellent conversions and isolated yields were achieved with this microbial oxidation and the oxidised products were treated with Pd/C under an atmosphere of hydrogen, to afford the target iminosugars in excellent yield.

Conclusions
Our work has highlighted G. oxydans DSM 2003 as a useful whole-cell catalyst for the selective oxidation of N-Cbz protected aminopolyols derived from aldoses, with an expanded substrate range.We demonstrated that via a fourstep sequential cascade, featuring a biocatalytic transamination and oxidation, a range of valuable iminosugars could be accessed in excellent yields.While efforts are ongoing to find biocatalysts that can oxidise aminopolyols without the need for protecting group manipulations, this work takes an important step towards developing a robust approach for the preparation of valuable iminosugars. [a] Step

Figure 1 .
Figure 1.The chemo-enzymatic synthesis of piperidine and pyrrolidine iminosugars from aldoses.A) The structural diversity of natural and synthetic iminosugars is shown.Iminosugars with known biological activity are highlighted in blue.B) Previously reported syntheses of DNJ 11 , DAB 12 and GNJ 13 from aldoses feature a key microbial oxidation by Gluconobacter oxydans.C) This work expands the methodology and substrate scope via a two-enzyme, four-step sequence.Aldoses are shown in open chain conformation.a. J. Kuska, F. Taday, School of Chemistry, University Park, Nottingham, NG7 2RD, UK b.J. Ryan, K. Yeow and E. O'Reilly, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.*Email: elaine.oreilly@ucd.ieElectronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here].See DOI: 10.1039/x0xx00000x

Figure 2 .
Figure 2. Analytical scale biotransformations to optimise temperature and substrate concentration for substrates 8-11.Reaction conditions for temperature optimisation (left): 1 mL reaction mixture containing 10 mM substrate, resting cells of G. oxydans DSM

Table 3 .
The cascade applied to 1, 3 and 4 to yield iminosugars CYB-3 and DRB as a mixture of diastereomers, and L-gulo-DNJ 29 as a single diastereomer.