Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations.

Multienzymatic cascade reactions are a powerful strategy for straightforward and highly specific synthesis of complex materials, such as active substances in drugs. Cross-inhibitions and incompatible reaction steps, however, often limit enzymatic activity and thus the conversion. Such limitations occur, e.g., in the enzymatic synthesis of the biologically active sialic acid cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). We addressed this challenge by developing a confinement and compartmentalization concept of hydrogel-immobilized enzymes for improving the efficiency of the enzyme cascade reaction. The three enzymes required for the synthesis of CMP-Neu5Ac, namely, N-acyl-d-glucosamine 2-epimerase (AGE), N-acetylneuraminate lyase (NAL), and CMP-sialic acid synthetase (CSS), were immobilized into bulk hydrogels and microstructured hydrogel-enzyme-dot arrays, which were then integrated into microfluidic devices. To overcome the cytidine triphosphate (CTP) cross-inhibition of AGE and NAL, only a low CTP concentration was applied and continuously conveyed through the device. In a second approach, the enzymes were compartmentalized in separate reaction chambers of the microfluidic device to completely avoid cross-inhibitions and enable the use of higher substrate concentrations. Immobilization efficiencies of up to 25% and pronounced long-term activity of the immobilized enzymes for several weeks were realized. Moreover, immobilized enzymes were less sensitive to inhibition and the substrate-channeling effect between immobilized enzymes promoted the overall conversion in the trienzymatic cascade reaction. Based on this, CMP-Neu5Ac was successfully synthesized by immobilized enzymes in noncompartmentalized and compartmentalized microfluidic devices. This study demonstrates the high potential of immobilizing enzymes in (compartmentalized) microfluidic devices to perform multienzymatic cascade reactions despite cross-inhibitions under continuous flow conditions. Due to the ease of enzyme immobilization in hydrogels, this concept is likely applicable for many cascade reactions with or without cross-inhibition characteristics.

Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives.

Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might not be appropriate for an antimicrobial drug.

Discovery of Novel Nucleotide Prodrugs with Improved Potency Against HCV Variants Carrying NS5B S282T Mutation.

Resistant HCV variants carrying NS5B S282T mutation confer reduced sensitivity to sofosbuvir, the sole marketed NS5B polymerase inhibitor. On the basis of the finding that 2'-α-F-2'-ß-C-methylcytidine 5'-triphosphate (8) was more potent than sofosbuvir's active metabolite on inhibition of both wild-type and S282T mutant polymerase, a dual-prodrug approach has been established. Twenty-nine phosphoramidates with N4-modified cytosine were designed, synthesized, and evaluated for anti-HCV activity. The results showed that compounds 4c-4e and 4m (EC50 = 0.19-0.25 µM) exhibited comparable potency to that of sofosbuvir (EC50 = 0.15 µM) on inhibition of wild-type replicons. Notably, 4c (EC50 = 0.366 µM) was 1.5-fold more potent than sofosbuvir (EC50 = 0.589 µM) on inhibition of S282T mutant replicons. In vitro metabolic studies disclosed the possible metabolic pathways of 4c. The toxicity study results indicated a good safety profile of 4c. Together, 4c-4e and 4m hold promise for drug development for the treatment of HCV infection, especially the resistant variants with NS5B S282T mutation.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids.

Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved 31P phosphorus nuclear magnetic resonance (31P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. 31P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

A novel cytarabine crystalline lipid prodrug: hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate for proliferative vitreoretinopathy.

PURPOSE: The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection. METHODS: Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-ß-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 µM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01). CONCLUSIONS: The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes.

Unique self-anhydride formation in the degradation of cytidine-5'-monophosphosialic acid (CMP-Neu5Ac) and cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) and its application in CMP-sialic acid analogue synthesis.

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.

Chemical and enzymatic stability of amino acid derived phosphoramidates of antiviral nucleoside 5'-monophosphates bearing a biodegradable protecting group.

Ribavirin and 2'-O-methylcytidine 5'-phosphoramidates derived from L-alanine methyl ester bearing either an O-phenyl or a biodegradable O-[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] or O-[3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl] protecting group were prepared. The kinetics of the deprotection of these pro-drugs by porcine liver esterase and by a whole cell extract of human prostate carcinoma was studied by HPLC-ESI-MS/MS. The 3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl and 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl groups were readily removed releasing the l-alanine methyl ester phosphoramidate nucleotide, the deprotection of the 3-(acetyloxymethoxy) derivative being approximately 20 times faster. The chemical stability of the 2'-O-methylcytidine pro-drugs was additionally determined over a pH range from 7.5 to 10.

Synthesis and biological activity of a gemcitabine phosphoramidate prodrug.

A gemcitabine (2',2'-difluorodeoxycytidine, dFdC) phosphoramidate prodrug designed for the intracellular delivery of gemcitabine 5'-monophosphate was synthesized. The prodrug was about an order of magnitude less active than gemcitabine against wild-type cells, and the nucleoside transport inhibitor dipyridamole reduced prodrug activity. The prodrug was more active than gemcitabine against two deoxycytidine kinase-deficient cell lines. The results suggest that the prodrug is a potent growth inhibitor that can bypass dCK deficiency at higher drug concentrations.

Synthesis and characterization of a novel liver-targeted prodrug of cytosine-1-beta-D-arabinofuranoside monophosphate for the treatment of hepatocellular carcinoma.

Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC) due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor and high conversion in other tissues. These characteristics lead to poor efficacy, high toxicity, and a drug class associated with an unacceptable therapeutic index. Cyclic 1-aryl-1,3-propanyl phosphate prodrugs selectively release the monophosphate of a nucleoside (NMP) into CYP3A4-expressing cells, such as hepatocytes, while leaving the prodrug intact in plasma and extrahepatic tissues. This prodrug strategy was applied to the monophosphate of the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). Compound 19S (MB07133), in mice, achieves good liver targeting compared to araC, generating >19-fold higher cytarabine triphosphate (araCTP) levels in the liver than levels of araC in the plasma and >12-fold higher araCTP levels in the liver than in the bone marrow, representing a >120-fold and >28-fold improvement, respectively, over araC administration.

Synthesis and evaluation of S-acyl-2-thioethyl esters of modified nucleoside 5'-monophosphates as inhibitors of hepatitis C virus RNA replication.

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.

Synthesis and evaluation of phosphoramidate amino acid-based inhibitors of sialyltransferases.

Several phosphoramidate analogues of CMP-N-acetylneuraminic acid were prepared for evaluation as inhibitors of alpha-2,3- and alpha-2,6-sialyltransferase. Central to the synthesis was the oxidative coupling of an amino acid ester with an H-phosphonate to construct the phosphoramidate linkage. All compounds synthesized were weak inhibitors of both of the sialyltransferases as determined by an HPLC-based inhibition assay.

Novel method for enzymatic synthesis of CMP-NeuAc.

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.

Synthesis and characterization of an anomeric sulfur analogue of CMP-sialic acid.

alpha-2,3-Sialyltransferase catalyzes the transfer of sialic acid from CMP-sialic acid (1) to a lactose acceptor. An analogue of 1 was synthesized in which the anomeric oxygen atom was replaced with a sulfur atom (1S). The key step in the synthesis of 1S was a tetrazole-promoted coupling of a cytidine-5'-phosphoramidite with a glycosyl thiol of a protected sialic acid. Compounds 1 and 1S were characterized for their activity in a sialyl transfer assay. The rate of solvolysis in aqueous buffer of analogue 1S was 50-fold slower than that of 1. Analogue 1S was found to be substrate for alpha-2,3-sialyltransferase. The K(m) of 1S was just 3-fold higher than that of 1, while the k(cat) of 1S was 2 orders of magnitude lower compared to 1.

Design of fluorogenic substrates for continuous assay of sialyltransferase by resonance energy transfer.

Glycosyltransferases are important synthetic enzymes for the construction of naturally occurring glycoconjugates as well as for the design of neoglycoconjugates. The assay methods currently available for these enzymes require tedious and time-consuming procedures for separation of products and do not permit continual assay of enzyme activities. As a set of convenient fluorogenic substrates for continuous monitoring of sialyltransferase activities, we designed and synthesized a novel CMP-Neu5Ac derivative with a naphthylmethyl group at the C-9 position and N-acetyllactosamine derivative containing a dansyl group at the terminal position of aglycon. In such substrates, the emission peak of the naphthylmethyl group (lambdaem = 340 nm) of the glycosyl donor is successfully overlapped with the excitation peak due to the dansyl group (lambdaex = 335 nm) of the glycosyl acceptor. A coupling reaction of these two substrates catalyzed by rat liver 2,6-sialyltransferase caused an increase of dansyl fluorescence (lambdaem = 525 nm) and a decrease of naphthylmethyl fluorescence on the basis of resonance energy transfer between two fluorescence probes. The substrates presented here permit continuous fluorescent monitoring of enzymatic sugar combining reactions. Actually, using this time course of enzymatic reactions, kinetic constants of rat liver 2,6-sialyltransferase against glycosyl donor substrates were estimated to be Km = 4.85 microM and Vmax. = 0.119 micromol/min, respectively. This strategy allows precise and efficient analyses of enzyme kinetics not possible with the conventional assay methods for the glycosyltransferases that usually require separation of products from the reaction mixture.

Prodrugs of Ara-CMP and Ara-AMP with a S-acyl-2-thioethyl (SATE) biolabile phosphate protecting group: synthesis and biological evaluation.

The bis(S-pivaloyl-2-thioethyl) phosphotriesters of Ara-C and Ara-A were synthesized as potential bioreversible mononucleotide prodrugs. Some N- and O-acylated derivatives were also prepared with the aim to modify the lipophilicity of the title pronucleotides. Compounds were tested for their antitumor/antiviral activity against a variety of tumor cells and viruses.

Enzymatic synthesis of Kdn oligosaccharides by a bacterial alpha-(2-->6)-sialyltransferase.

Synthesis of CMP-deaminoneuraminic acid (CMP-beta-D-Kdn) and its enzymatic transfer reaction using bacterial alpha-(2-->6)-sialyltransferase were examined. CMP-beta-D-Kdn was prepared from methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonate (2) in 24% overall yield. Enzymatic synthesis of Kdn oligosaccharide with CMP-beta-D-Kdn (10.2 mumol), methyl beta-D-lactosaminide (7, 8.1 mumol) and purified sialyltransferase (80 munits) afforded Kdn-alpha-(2-->6)-Gal-beta-(1-->4)-GlcNAc-beta-1-OMe in 77% yield.