Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid.

Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.

Enantioselective Cascade Biocatalysis for Deracemization of Racemic ß-Amino Alcohols to Enantiopure (S)-ß-Amino Alcohols by Employing Cyclohexylamine Oxidase and ω-Transaminase.

Optically active ß-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of ß-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34 U mg-1 protein) and excellent enantioselectivity toward the tested ß-amino alcohols. By using purified ArCHAO, a wide range of racemic ß-amino alcohols were resolved, (S)-ß-amino alcohols were obtained in >99 % ee. Deracemization of racemic ß-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-ß-amino alcohols in excellent conversion (78-94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50 mM (6.859 g L-1 ) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.

Microbial production of 4-amino-1-butanol, a four-carbon amino alcohol.

4-Amino-1-butanol (4AB) serves as an important intermediate compound for drugs and a precursor of biodegradable polymers used for gene delivery. Here, we report for the first time the fermentative production of 4AB from glucose by metabolically engineered Corynebacterium glutamicum harboring a newly designed pathway comprising a putrescine (PUT) aminotransferase (encoded by ygjG) and an aldehyde dehydrogenase (encoded by yqhD) from Escherichia coli, which convert PUT to 4AB. Application of several metabolic engineering strategies such as fine-tuning the expression levels of ygjG and yqhD, eliminating competing pathways, and optimizing culture condition further improved 4AB production. Fed-batch culture of the final metabolically engineered C. glutamicum strain produced 24.7 g/L of 4AB. The strategies reported here should be useful for the microbial production of primary amino alcohols from renewable resources.

Reshaping the substrate binding region of (R)-selective ω-transaminase for asymmetric synthesis of (R)-3-amino-1-butanol.

(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher kcat/Km value and 9.4 °C higher [Formula: see text] than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L-1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.

High throughput solid-phase screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic ß-amino alcohols.

OBJECTIVES: To screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic ß-amino alcohols. RESULTS: A new strain named Arthrobacter sp. TYUT010-15 with the (R)-selective deamination activity of cyclic ß-amino alcohol has been isolated from nature via a high throughput solid-phase screening method. The reaction conditions of TYUT010-15 were optimized. Using the resting cell of TYUT010-15 as the catalyst, kinetic resolution of trans-2-aminocyclopentanol, trans-2-aminocyclohexanol and cis-1-amino-2-indanol was carried out to afford (1S, 2S)-trans-2-aminocyclopentanol, (1S, 2S)-trans-2-aminocyclohexanol and (1R, 2S)-cis-1-amino-2-indanol in > 99% ee and 49.6-50% conversion. Four aromatic ß-amino alcohols and two amines were also resolved, (S)-ß-amino alcohols and (R)-amines were obtained in > 99% ee. Preparation experiment was conducted with 200 mM (23.2 g L-1) racemic trans-2-aminocyclohexanol, yielding the desired (1S, 2S)-trans-2-aminocyclohexanol in 40% isolated yield, > 99% ee and 5.8 g L-1 d-1 space time yields. CONCLUSIONS: This study provides a high throughput solid-phase method for screening of bacteria with cyclic amino alcohol deamination activity and a first example for practical preparation of chiral cyclic ß-amino alcohol by Arthrobacter sp. TYUT010-15.

Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum ω-transaminase alone or combined with native transketolase.

Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration or robustness to fluctuating pH, osmolarity and biocontaminant load in feedstocks. The methylotrophic yeast Komagataella phaffii is an attractive alternative to Escherichia coli for whole cell biocatalysis due to its genetic tractability and capacity to grow to up to 60% wet cell weight by volume. We sought to exploit high cell density K. phaffii to intensify whole-cell chiral amino-alcohol (CAA) biosynthesis. We engineered two novel K. phaffii GS115 strains: one by inserting a Chromobacterium violaceum ω-transaminase CV2025 transgene, for strain PpTAmCV708, and a second strain, PpTAm-TK16, by also inserting the same CV2025 transgene plus a second transgene for a native transketolase. At high cell density, both strains tolerated high substrate concentrations. When fed three low cost substrates, 200 mM glycolaldehyde, 200 mM hydroxypyruvate and 150 mM methylbenzylamine, PpTAm-TK16 whole cells achieved 0.29 g L-1 hr-1 space-time yield of the acetophenone by-product, a 49-fold increase of the highest levels reported for E. coli whole cells harboring the equivalent pathway. When fed only the low-cost substrate, 150 mM methylbenzylamine, strain PpTAmCV708 achieved a 105-fold increase of reported E. coli whole cell biocatalysis performance, with a space-time yield of 0.62 g L-1 hr-1 of the CAA, 2-amino-1,3,4-butanetriol (ABT). The rapid growth and high biomass characteristics of K. phaffii were successfully exploited for production of ABT by whole-cell biocatalysis at higher levels than the previously achieved with E. coli in the presence of the same substrates.

Design, synthesis, and structure-activity relationship studies of l-amino alcohol derivatives as broad-spectrum antifungal agents.

To discover broad spectrum antifungal agents, two strategies were applied, and a novel class of l-amino alcohol derivatives were designed and synthesized. 3-F substituted compounds 14i, 14n, 14s and 14v exhibited excellent antifungal activities with broad antifungal spectra against C. albicans and C. tropicalis, with MIC values in the range of 0.03-0.06 µg/mL, and against A. fumigatus and C. neoformans, with MIC values in the range of 1-2 µg/mL. Notably, Compounds 14i, 14n, 14s and 14v also displayed moderate activities against fluconazole-resistance strains 17# and CaR that were isolated from AIDS patients. Moreover, only compounds in the S-configuration showed antifungal activity. Preliminary mechanistic studies showed that the potent antifungal activity of compound 14v stemmed from inhibition of C. albicans CYP51. Compounds 14n and 14v were almost nontoxic to mammalian A549 cells, and their stability in human plasma was excellent.

Efficient biosynthesis of (R)-3-amino-1-butanol by a novel (R)-selective transaminase from Actinobacteria sp.

(R)-3-amino-1-butanol is a key intermediate of Dolutegravir for the treatment of HIV/AIDS and its green and efficient biosynthesis has attracted a great deal of attention. Transaminases are currently used as promising biocatalyst for the synthesis of chiral amines. However, many transaminases have (S)-specificity and (R)-selective transaminases were less exploited and studied, making the production of (R)-amines remain challenging. In this study, a novel transaminase from Actinobacteria sp. (As-TA) was obtained and applied for the biosynthesis of (R)-3-amino-1-butanol by transferring the amino group from isopropylamine to 4-hydroxy-2-butanone. After optimization of the reaction condition and using a substrate fed-batch strategy, the conversion of 100, 200, 300, 400 and 500 mM 4-hydroxy-2-butanone reached 100%, 94.9%, 86.1%, 76.1% and 70.9%, respectively. (R)-3-amino-1-butanol with a maximum yield of 29.6 g/L and 99.9% e.e. value was obtained. This was the first time demonstrating the successful biosynthesis of (R)-3-amino-1-butanol with transaminase as biocatalyst and the obtained As-TA enriched the enzyme pool of transaminase with (R)-specificity.

Designing of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Production of 1,2-Amino Alcohols from Epoxides.

Optically pure 1,2-amino alcohols are highly valuable products as intermediates for chiral pharmaceutical products. Here we designed an environmentally friendly non-natural biocatalytic cascade for efficient synthesis of 1,2-amino alcohols from cheaper epoxides. A redesignated ω-transaminase PAKω-TA was tested and showed good bioactivity at a lower pH than other reported transaminases. The cascade was efficiently constructed as a single one-pot E. coli recombinant, by coupling SpEH (epoxide hydrolase), MnADH (alcohol dehydrogenase), and PAKω-TA. Furthermore, RBS regulation strategy was used to overcome the rate limiting step by increasing expression of MnADH. For cofactor regeneration and amino donor source, an interesting point was involved as that a cofactor self-sufficient system was designed by expression of GluDH. It established a "bridge" between the cofactor and the cosubstrate, such that the cofactor self-sufficient system could release cofactor (NADP+) and cosubstrate (l-Glutamine) regenerated simultaneously. The recombinant E. coli BL21 (SGMP) with cofactor self-sufficient whole-cell cascade biocatalysis showed high ee value (>99%) and high yield, with 99.6% conversion of epoxide ( S)-1a to 1,2-amino alcohol ( S)-1d in 10 h. It further converted ( S)-2a-5a to ( S)-2d-5d with varying conversion rates ranging between 65-96.4%. This study first provides one-step synthesis of optically pure 1,2-amino alcohols from ( S)-epoxides employing a synthetic redox-self-sufficient cascade.

Enantioselective synthesis of enantiopure ß-amino alcohols via kinetic resolution and asymmetric reductive amination by a robust transaminase from Mycobacterium vanbaalenii.

Chiral ß-amino alcohols are very important chiral building block for preparing bioactive compounds for use in pharmaceutical and fine chemical industries. Synthesis of chiral ß-amino alcohols by transaminase is big challenging due to the strict substrate specificities and very low activity of the enzyme. In this work, a (R)-selective ω-transaminase (MVTA) from Mycobacterium vanbaalenii was employed as a biocatalyst for the first time for the synthesis of chiral ß-amino alcohol via kinetic resolution and asymmetric reductive amination. The enzyme was purified and characterized. Kinetic resolution of a set of racemic ß-amino alcohols including two cyclic ß-amino alcohols by MVTA was demonstrated, affording (R)-ß-amino alcohols, (1S, 2S)-trans-2-aminocyclopentanol and (1R, 2S)-cis-1-amino-2-indanols in >99% ee and 50-62% conversion. Asymmetric reductive amination of three α-hydroxy ketones (10-300 mM) by MVTA was conducted, (S)-ß-amino alcohols were obtained with >99% ee and 80-99% conversion. Preparation experiment for the reductive amination of 200 mM 2-hydroxyacetophenone by the resting cells of recombinant E. coli (MVTA) was proceeded smoothly and product (S)-2-amino-2-phenylethanol was obtained with 71% isolated yield, >99% ee and 68.6 g/L/d volumetric productivity. The current research proved that the MVTA is a robust enzyme for the preparation of chiral ß-amino alcohol with high volumetric productivity.

Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach.

Chiral amino alcohols are compounds of pharmaceutical interest as they are building blocks of sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Due to the challenges of chemical synthesis we recently developed two TK-TAm reaction cascades using natural and low cost feedstocks as substrates: a recycling cascade comprising of 2 enzymes and a sequential 3-step enzyme cascade yielding 30% and 1% conversion, respectively. In order to improve the conversion yield and aid the future host strain engineering for whole cell biocatalysis, we used a combination of microscale experiments and statistical experimental design. For this we implemented a full factorial design to optimise pH, temperature and buffer type, followed by the application of Response Surface Methodology for the optimisation of substrates and enzymes concentrations. Using purified enzymes we achieved 60% conversion for the recycling cascade and 3-fold improvement using the sequential pathway. Based on the results, limiting steps and individual requirements for host cell metabolic integration were identified expanding the understanding of the cascades without implementing extensive optimisation modelling. Therefore, the approach described here is well suited for optimising reaction conditions as well as defining the relative enzyme expression levels required for construction of microbial cell factories.

Construction of Rhodococcus expression vectors and expression of the aminoalcohol dehydrogenase gene in Rhodococcus erythropolis.

NADP+-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 produces double chiral aminoalcohols, which are used as pharmaceuticals. However, the genetic manipulation of Rhodococcus strains to increase their production of such industrially important enzymes is not well studied. Therefore, I aimed to construct Rhodococcus expression vectors, derived from the Rhodococcus-Escherichia coli shuttle vector pRET1102, to express aadh. The plasmid pRET1102 could be transformed into many actinomycete strains, including R. erythropolis. The transformation efficiency for a species closely related to R. erythropolis was higher than that for other actinomycete strains. Promoters of various strengths, hsp, 1200rep, and TRR, were obtained from Gram-positive bacteria. The activity of TRR was stronger than that of hsp and 1200rep. The aadh-expressing plasmid pRET1172 with TRR could be transformed into many actinomycete strains to increase their AADH production. The Rhodococcus expression vector, pRET11100, constructed by removing aadh from the pRET1172 plasmid may be useful for bioconversion.

Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator, via phosphorylation and dephosphorylation in humans.

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes. 2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 µM to 1060 µM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate. 3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 µM to 32.1 µM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes. 4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

One pot simultaneous preparation of both enantiomer of ß-amino alcohol and vicinal diol via cascade biocatalysis.

OBJECTIVES: To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-ß-amino alcohols to enantiopure vicinal diol and ß-amino alcohol. RESULTS: An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10-60 mM R, S-ß-amino alcohol to (R)- and (S)-diol and (R)- and (S)-ß-amino alcohol in 90-99% ee with 50-52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40-42% isolated yield from racemic 2-amino-2-phenylethanol. CONCLUSIONS: This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an ß-amino alcohol.

Rationally designed benzopyran fused isoxazolidines and derived ß2,3,3-amino alcohols as potent analgesics: Synthesis, biological evaluation and molecular docking analysis.

Based on structure activity analysis of morphine related opiates, we have synthesized some novel benzopyran fused isoxazolidines (2a-e) and derived conformationally constrained ß2,3,3-amino alcohols (3a-e), which were evaluated in vivo for their anti-nociceptive activity through acetic acid induced writhing test (peripheral) and formalin induced algesia (central). Results showed that, compound 2a possesses significant opioid agonist activity. Further, molecular docking analysis reveals that compound 2a binds to δ-opioid receptor (DOR) with comparatively better D-score than to µ (MOR) and κ (KOR) receptors. Compound 2a did not show any toxicity up to a 2000 mg kg-1 dose.

A high-throughput microtiter plate assay for the discovery of active and enantioselective amino alcohol-specific transaminases.

Chiral vicinal amino alcohols are important chiral building blocks and intermediates in the pharmaceutical industry. The transaminase (TAm) catalyzed kinetic resolution of racemic amino alcohols provides a straightforward approach to access these important compounds. This study describes the development of a novel microtiter plate assay to screen vicinal amino alcohol-specific TAms using a tetrazolium red-based colorimetric assay to monitor the rate of α-hydroxy ketone formation at 510 nm. This approach is the first to determine the Michaelis-Menten parameters for a recombinant TAm (PpbauA) from Pseudomonas putida NBRC14164. The corresponding Vmax and KM values for both enantiomers of 2-amino-1-propanol and 2-amino-1-butanol were obtained, and the calculated kinetic E-factors of PpbauA toward 2-amino-1-propanol and 2-amino-1-butanol are 3 (S) and 6 (R), respectively. The method is sensitive and exhibits low level background coloration. Moreover, this method can be used to detect transaminase activity and enantioselectivity toward amino alcohols in a high-throughput format. Additionally, this simple method is compatible with the most widely used (R)- and (S)-selective transaminases and may be a broadly applicable tool for screening transaminases from a transaminase mutant library.

Enantioselective rhodium/ruthenium photoredox catalysis en route to chiral 1,2-aminoalcohols.

A rhodium-based chiral Lewis acid catalyst combined with [Ru(bpy)3](PF6)2 as a photoredox sensitizer allows for the visible-light-activated redox coupling of α-silylamines with 2-acyl imidazoles to afford, after desilylation, 1,2-amino-alcohols in yields of 69-88% and with high enantioselectivity (54-99% ee). The reaction is proposed to proceed via an electron exchange between the α-silylamine (electron donor) and the rhodium-chelated 2-acyl imidazole (electron acceptor), followed by a stereocontrolled radical-radical reaction. Substrate scope and control experiments reveal that the trimethylsilyl group plays a crucial role in this reductive umpolung of the carbonyl group.

Multi-step biocatalytic strategies for chiral amino alcohol synthesis.

Chiral amino alcohols are structural motifs present in sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Their chemical synthesis presents several challenges in establishing at least two chiral centres. Here a de novo metabolic pathway using a transketolase enzyme coupled with a transaminase enzyme has been assembled. To synthesise this motif one of the strategies to obtain high conversions from the transaminase/transketolase cascade is the use of hydroxypyruvate (HPA) as a two-carbon donor for the transketolase reaction; although commercially available it is relatively expensive limiting application of the pathway on an industrial scale. Alternately, HPA can be synthesised but this introduces a further synthetic step. In this study two different biocatalytic strategies were developed for the synthesis of (2S,3R)-2-amino-1,3,4-butanetriol (ABT) without adding HPA into the reaction. Firstly, a sequential cascade of three enzymatic steps (two transaminases and one transketolase) for the synthesis of ABT from serine, pyruvate and glycolaldehyde as substrates. Secondly, a two-step recycling cascade where serine is used as donor to aminate erythrulose (catalysed by a transketolase) for the simultaneous synthesis of ABT and HPA. In order to test the novel pathways, three new transaminases are described, two ω-transaminases able to accept a broad range of amine acceptors with serine as amine donor; and an α-transaminase, which showed high affinity towards serine (KM: 18mM) using pyruvate as amine acceptor. After implementation of the above enzymes in the biocatalytic pathways proposed in this paper, the two-step recycling pathway was found to be the most promising for its integration with E. coli metabolism. It was more efficient (10-fold higher conversion), more sustainable and cost-effective (use of low cost natural substrates and only two enzymes), and the reaction could be performed in a one-pot system.

RNA/aTNA chimeras: RNAi effects and nucleases resistance of single and double stranded RNAs.

The RNA interference pathway (RNAi) is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic L-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the L-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3'/5'-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B (ApoB). Further studies on the activation of the innate immune system showed that L-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.

From amino alcohol to aminopolyol: one-pot multienzyme oxidation and aldol addition.

In this work, the successful coupling of enzymatic oxidation and aldol addition reactions for the synthesis of a Cbz-aminopolyol from a Cbz-amino alcohol was achieved for the first time in a multienzymatic one-pot system. The two-step cascade reaction consisted of the oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by chloroperoxidase from the fungus Caldariomyces fumago and aldol addition of dihydroxyacetone phosphate to Cbz-glycinal catalyzed by rhamnulose-1-phosphate aldolase expressed as a recombinant enzyme in Escherichia coli, yielding (3R,4S)-5-{[(benzyloxy)carbonyl]amino}-5-deoxy-1-O-phosphonopent-2-ulose. Tools of enzymatic immobilization, reactor configurations, and modification of the reaction medium were applied to highly increase the production of the target compound. While the use of soluble enzymes yielded only 23.6 % of Cbz-aminopolyol due to rapid enzyme inactivation, the use of immobilized ones permitted an almost complete consumption of Cbz-ethanolamine, reaching Cbz-aminopolyol yields of 69.1 and 71.9 % in the stirred-tank and packed-bed reactor, respectively. Furthermore, the reaction production was 18-fold improved when it was catalyzed by immobilized enzymes in the presence of 5 % (v/v) dioxane, reaching a value of 86.6 mM of Cbz-aminopoliol (31 g/L).