Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification

A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre‐packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP‐IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on‐line through a 6‐way switching valve to an HPLC apparatus containing an analytical or a semi‐preparative chromatographic column. The synthesis of five 6‐modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP‐IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP‐catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min⁻¹) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52–89%; <10 mg) where sample handling was minimized. To date, AhPNP‐IMER has retained completely its activity upon 50 reactions in 10 months.

     

核苷合成技术的研究进展

介绍了核苷的类别、用途。叙述了核苷合成技术进展，重点为酶法生物合成法；评述了各种方法的优缺点。认为微生物酶（细胞）合成核苷的前景乐观，该法反应简单、污染小、成本低。但合成核苷时要注意保持菌体的高活力和稳定性．并解决合成产物的分离纯化问题。     

An Expedient Synthesis of Flexible Nucleosides through Enzymatic Glycosylation of Proximal and Distal Fleximer Bases

The structurally unique “fleximer” nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor–ligand recognition and function. Recently they have been shown to have low-to-sub-micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex-bases) has been successfully coupled to both ribose and 2′-deoxyribose sugars by using the N-deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno-expanded tricyclic heterocyclic base, as well as several distal and proximal flex-bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein.     

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

The enzymatic transglycosylation of 2,6‐dichloropurine (26DCP) and 6‐chloro‐2‐fluoropurine (6C2FP) with uridine, thymidine and 1‐(β‐D ‐arabinofuranosyl)‐uracil as the pentofuranose donors and recombinant thermostable nucleoside phosphorylases from G. thermoglucosidasius or T. thermophilus as biocatalysts was studied. Selection of 26DCP and 6C2FP as substrates is determined by their higher solubility in aqueous buffer solutions compared to most natural and modified purines and, furthermore, synthesized nucleosides are valuable precursors for the preparation of a large number of biologically important nucleosides. The substrate activity of 26DCP and 6C2FP in the synthesis of their ribo‐ and 2′‐deoxyribo‐nucleosides was closely similar to that of related 2‐amino‐ (DAP), 2‐chloro‐ and 2‐fluoroadenines; the efficiency of the synthesis of β‐D ‐arabinofuranosides of 26DCP and 6C2FP was lower vs. that of DAP under similar reaction conditions. For a convenient and easier recovery of the biocatalysts, the thermostable enzymes were immobilized on MagReSyn^® epoxide beads and the biocatalyst showed high catalytic efficiency in a number of reactions. As an example, 6‐chloro‐2‐fluoro‐(β‐D ‐ribofuranosyl)‐purine ( 9 ), a precursor of various antiviral and antitumour drugs, was synthesized by the immobilized enzymes at 60 °C under high substrate concentrations (uridine:purine ratio of 2:1, mol). The synthesis was successfully scaled‐up [uridine (2.5 mmol), base (1.25 mmol); reaction mixture 50 mL] to afford 9 in 60% yield. The reaction reveals the great practical potential of this enzymatic method for the efficient production of modified purine nucleosides of pharmaceutical interest.

     

Spectral Unmixing-Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases

The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC-like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.     

Comparative Analysis of Enzymatic Transglycosylation Using E. coli Nucleoside Phosphorylases: A Synthetic Concept for the Preparation of Purine Modified 2′-Deoxyribonucleosides from Ribonucleosides

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2′-deoxynucleosides, was performed. We demonstrated that maximal yields of 2′-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2′-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction’s output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2′-deoxyribonucleosides, starting from the corresponding ribonucleosides.     

Synthesis of 2′‐Deoxynucleosides by Transglycosylation with New Immobilized and Stabilized Uridine Phosphorylase and Purine Nucleoside Phosphorylase

Multimeric uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) of Bacillus subtilis have been expressed from genes cloned in Escherichia coli, purified, characterized, immobilized and stabilized on solid support. A new immobilization strategy has been developed for UP onto Sepabeads coated with polyethyleneamine followed by cross‐linking with aldehyde‐dextran. PNP has been immobilized onto glyoxyl‐agarose. At pH 10 and 45 °C these derivatives catalyzed the transglycosylation of 2′‐deoxyuridine to 2′‐deoxyguanosine in high yield (92%). Under the same conditions the not immobilized enzymes were promptly inactivated.     

General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations

The biocatalytic synthesis of natural and modified nucleosides with nucleoside phosphorylases offers the protecting-group-free direct glycosylation of free nucleobases in transglycosylation reactions. This contribution presents guiding principles for nucleoside phosphorylase-mediated transglycosylations alongside mathematical tools for straightforward yield optimization. We illustrate how product yields in these reactions can easily be estimated and optimized using the equilibrium constants of phosphorolysis of the nucleosides involved. Furthermore, the varying negative effects of phosphate on transglycosylation yields are demonstrated theoretically and experimentally with several examples. Practical considerations for these reactions from a synthetic perspective are presented, as well as freely available tools that serve to facilitate a reliable choice of reaction conditions to achieve maximum product yields in nucleoside transglycosylation reactions.     

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4–10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10⁶ for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.     

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Microwave Accelerated Transglycosylation of Rutin by Cyclodextrin Glucanotransferase from Bacillus sp. SK13.002

Rutin was subjected to intermolecular transglycosylation assisted with microwave irradiation using cyclodextrin glucanotransferase (CGTase) produced from Bacillus sp. SK13.002. Compared with the conventional enzymatic method for rutin transglycosylation (without microwave irradiation), microwave-assisted reaction (MAR) was much faster and thus more efficient. While the conventional reaction took dozens of hours to reach the highest conversion rate of rutin and yield of transglycosylated rutin, MAR of rutin transglycosylation completed within only 6 min providing almost the same conversion rate of rutin and yield of products consisting of mono-, di-, tri-, tetra-, penta-glucosylated rutins. The optimum transglycosylation conditions for microwave irradiation were 40 °C and 60 W with the reaction system consisting mainly of the mixture of 0.3 g rutin (0.49 mmol) pre-dissolved in 15 mL methanol, 1.8 g maltodextrin in 15 mL of 0.2 M sodium acetate buffer (pH 5.5) and CGTase (900 U). Results from this study indicated that MAR could be a potentially useful and economical technique for a faster and more efficient transglycosylation of rutin.     

Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX “thumb” were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.     

HPLC测定海马中核苷和核苷酸的含量

为建立一种同时测定海马中5种核苷(胞苷、尿苷、肌苷、鸟苷和腺苷)和5种核苷酸(CMP、UMP、IMP、GMP和AMP)含量的方法,本研究采用HPLC,以Accalaim C30(2.1 mm×150 mm,3μm)为分析柱,以水(A)-100 mmol/L NH4OAC溶液(p H=5.0)(B)-乙腈(C)为流动相,梯度洗脱,流速0.4 m L/min,检测波长254 nm,柱温25℃。结果显示,5种核苷和5种核苷酸的浓度均在0.02~10 mg/L范围内线性关系良好。平均加样回收率在88.38%~96.30%(n=3)之间。本法能够快速、简便地测定海马中主要核苷和核苷酸的含量,为其质量控制提供保证。     

Synthesis of Chiral Cyclopropyl Carbocyclic Purine Nucleosides via Asymmetric Intramolecular Cyclopropanations Catalyzed by a Chiral Ruthenium(II) Complex

The synthesis of chiral cyclopropyl carbocyclic purine nucleoside analogues via the highly enantioselective intramolecular cyclopropanation reactions has been reported. With a chiral ruthenium(II)‐phenyloxazoline complex as the catalyst, cyclopropyl carbocyclic purine nucleoside analogues containing three contiguous stereocenters were obtained with up to 99% yield and 99% ee. Furthermore, a chiral cyclopropyl carbocyclic adenosine nucleoside having anti‐BLV activity could be synthesized in a concise manner using this strategy.

     

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1_GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1_GTEPG.     

Enantiocontrolled Preparation of ϒ-Substituted Cyclohexenones: Synthesis and Kinase Activity Assays of Cyclopropyl-Fused Cyclohexane Nucleosides

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a–e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs.     

N(2)-乙酰基-7-[(2-乙酰氧乙氧基)甲基]鸟嘌呤的转糖基作用

研究了N(2 ) 乙酰基 7 [(2 乙酰氧乙氧基 )甲基 ]鸟嘌呤通过转糖基作用生成其9位异构体N(2 ) 乙酰基 9 [(2 乙酰氧乙氧基 )甲基 ]鸟嘌呤的条件 ,转化率为 5 5 %。     

A Conformational Mimetic Approach for the Synthesis of Carbocyclic Nucleosides as Anti‐HCV Leads

Computer‐aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti‐hepatitis C virus (HCV) agents. Conformational analyses were carried out on 6‐amino‐1H‐pyrazolo[3,4‐d]pyrimidine (6‐APP)‐based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA‐dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6‐APP analogues were prepared and evaluated for anti‐HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC₅₀ value of 6.6 μM as a preliminary anti‐HCV lead.     

Adamantane-Substituted Purine Nucleosides: Synthesis,Host–Guest Complexes with β-Cyclodextrin and Biological Activity

Purine nucleosides represent an interesting group of nitrogen heterocycles, showing a wide range of biological effects. In this study, we designed and synthesized a series of 6,9-disubstituted and 2,6,9-trisubstituted purine ribonucleosides via consecutive nucleophilic aromatic substitution, glycosylation, and deprotection of the ribofuranose unit. We prepared eight new purine nucleosides bearing unique adamantylated aromatic amines at position 6. Additionally, the ability of the synthesized purine nucleosides to form stable host–guest complexes with β-cyclodextrin (β-CD) was confirmed using nuclear magnetic resonance (NMR) and mass spectrometry (ESI-MS) experiments. The in vitro antiproliferative activity of purine nucleosides and their equimolar mixtures with β-CD was tested against two types of human tumor cell line. Six adamantane-based purine nucleosides showed an antiproliferative activity in the micromolar range. Moreover, their effect was only slightly suppressed by the presence of β-CD, which was probably due to the competitive binding of the corresponding purine nucleoside inside the β-CD cavity.