N-Substituted Valiolamine Derivatives as Potent Inhibitors of Endoplasmic Reticulum α-Glucosidases I and II with Antiviral Activity.

Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.

One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate.

2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

Design, synthesis and biological evaluation of new Myo-inositol derivatives as potential RAS inhibitors.

Ras is a small family of GTPases that control numerous cellular functions like cell proliferation, growth, survival, gene expression, and is closely engaged in cancer pathogenesis. The ras-targeted methodology entails a holy grail in oncology. Nevertheless, there are no specific molecules reported targeting the same, although it is a known oncogene for more than three decades. In this study, we have designed and synthesized new phosphate derivatives of Myo-inositol to inhibit the oncogenic KRAS pathway in breast cancer cells, which has been validated by cellular and theoretical studies. The synthesized compound 1b (C2-O-phosphate derivative of Myo-inositol 1,3,5-orthobenzoate) inhibited the downstream signaling pathway of oncogenic KRAS, RAF/MEK/ERK. Furthermore, we also found that this compound induced necrosis/apoptosis and causes cell cycle arrest. This class of molecules may work as a potential inhibitor of breast cancer caused by a mutation in KRAS and its downstream proteins. Though the efficacy of the molecules is in the micromolar scale, they have not been explored previously for RAS inhibition. Impressive preliminary results are presented in this article which could be further explored for its detailed biological studies to get better candidates as RAS inhibitors.

Synthesis and evaluation of Nα,Nε-diacetyl-l-lysine-inositol conjugates as cancer-selective probes for metabolic engineering of GPIs and GPI-anchored proteins.

Two myo-inositol derivatives having an Nα,Nε-diacetyl-l-lysine (Ac2Lys) moiety linked to the inositol 1-O-position through a self-cleavable linker and a metabolically stable 2-azidoethyl group linked to the inositol 3-O- and 4-O-positions, respectively, were designed and synthesized. The Ac2Lys moiety blocking the inositol 1-O-position required for GPI biosynthesis was expected to be removable by a combination of two enzymes, histone deacetylase (HDAC) and cathepsin L (CTSL), abundantly expressed in cancer cells, but not in normal cells, to transform these inositol derivatives into biosynthetically useful products with a free 1-O-position. As a result, it was found that these inositol derivatives could be incorporated into the glycosylphosphatidylinositol (GPI) biosynthetic pathway by cancer cells, but not by normal cells, to express azide-labeled GPIs and GPI-anchored proteins on cell surfaces. Consequently, this study has established a novel strategy and new molecular tools for selective metabolic labeling of cancer cells, which should be useful for various biological studies and applications.

Synthesis of Bradyrhizose from d-Glucose.

We describe the synthesis of the unusual bicyclic sugar bradyrhizose in 14 steps and a 6% overall yield from d-glucose. The synthesis involves the elaboration of a trans-fused carbocyclic ring onto the preexisting glucopyranose framework followed by adjustment of the oxidation levels. Key steps include radical extension of the glucopyranose side chain, ring closing metathesis, allylic oxidation, Luche reduction, hydroxy-directed epoxidation, and acid-catalyzed epoxide opening at the more substituted position.

Production of scyllo-Inositol: Conversion of Rice Bran into a Promising Disease-Modifying Therapeutic Agent for Alzheimer's Disease.

scyllo-Inositol (SI) is one of the inositol stereoisomers, rare in the nature, and expected as a promising disease-modifying therapeutic agent for Alzheimer's disease. On the other hand, myo-inositol (MI) is another inositol stereoisomer most abundant in nature and thus supplied from agricultural byproducts including rice bran. Bacillus subtilis was genetically modified in its inositol metabolism and phytase secretion, to develope the bioconversion processes to produce SI from rice bran. Phytase, an enzyme that degrades phytate in rice bran into MI, was secreted in a B. subtilis strain with the optimized signal peptide. Another B. subtilis strain was constructed with the constitutive and simultaneous overexpression of IolG and IolW, which are the two inositol dehydrogenases responsible for the conversion, to demonstrate an efficient conversion of MI into SI with a rate up to 10 g/L/48 h. In order to devise further elevation in the conversion efficiency, we attempted to improve the substrate uptake by overexpressing iolT for the major MI transporter. In addition, Escherichia coli pntAB encoding the membrane-bound transhydrogenase was introduced aiming at enhanced supply of NADPH required for the rate-limiting IolW reaction. These additional modifications successfully elevated the conversion efficiency with an improved rate up to almost 30 g/L/48 h. Together with the improved phytase secretion, technological infrastructure for social implementation of SI production from rice bran is on the way.

A synthetic heparin mimetic that allosterically inhibits factor XIa and reduces thrombosis in vivo without enhanced risk of bleeding.

BACKGROUND: Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long-standing hypothesis has been that allosterism originating from heparin-binding site(s) on coagulation enzymes is a promising approach to yield safer agents. OBJECTIVES: To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk. METHODS: We employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work. RESULTS: Sulfated chiro-inositol (SCI), a non-saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin-binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway-based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum-dose-tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl3 -induced arterial and thromboplastin-induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 µg/animal, which matched enoxaparin at 2500 µg/animal. CONCLUSIONS: Overall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.

Total Synthesis of (+)-Lycoricidine and Conduramine B-1, ent-C-1, C-4, D-1, ent-F-1, and ent-F-4, and Formal Synthesis of (-)-Laminitol: a C2-Symmetric Chiral-Pool-Based Flexible Strategy.

A facile and diversity-oriented synthetic strategy toward aminocyclitol natural products from inexpensive C2-symmetric l-tartaric acid was developed. The pivotal epoxide was used as a common intermediate to accomplish eight diverse target molecules in six to eleven steps. Various allyl-amine-type conduramines were synthesized in a diastereoselective manner. Heck arylation was explored to construct a phenanthridone ring in a concise synthesis of (+)-lycoricidine. In addition, a highly efficient formal synthesis of (-)-laminitol was developed.

Facile synthesis of (-)-vibo-quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem.

(-)-vibo-Quercitol (VQ: 1L-1,2,4/3,5-cyclohexanepentol), a form of deoxyinositol, is an alternative chiral building block in the synthesis of bioactive compounds to control diabetes. In this study, an adenosine triphosphate-free in vitro synthetic enzymatic biosystem composed of five enzymes (including one enzyme for NADH regeneration) was constructed to produce VQ from maltodextrin in one-pot. After optimization of reaction conditions, 7.6 g/L VQ was produced from 10 g/L maltodextrin with a product yield (mol/mol) of 77%, and 25.3 g/L VQ with a purity of 87% was produced from 50 g/L maltodextrin through simple scaling up of this nonfermentative enzymatic biosystem. Therefore, this study provides an economical and environmentally friendly method for the envisioned quercitol biosynthesis.

Total Synthesis of Sialyl Inositol Phosphosphingolipids CJP-2, CJP-3, and CJP-4 Isolated from Feather Star Comanthus japonica.

The first total synthesis of three echinodermatous sialyl inositol phosphosphingolipids, which exhibit unusual neuritogenic activity in the absence of nerve growth factor, are reported. Highlights of the syntheses include 9- O-methylation on sialic acid, inter-residual amide bond formation between sialic acid residues, and highly stereo- and regioselective sialylation of inositol. A key phosphodiester linkage between the mono-, di-, and trisialyl inositols and ceramide was formed at a late state employing the phosphoramidite method.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

Syntheses of the plant auxin conjugate 2-O-(indole-3-acetyl)-myo-inositol IAInos.

The plant hormone conjugate 2-O-(indole-3-acetyl)-myo-inositol (IAInos) has been selectively prepared for the first time by two routes from myo-inositol. One of the syntheses depended upon the construction of the 3-indoleacetyl group by a Fischer indole synthesis on an unreactive axial hydroxyl group, while the other via a direct acylation of the equatorially orientated hydroxy group created by conformational constraint of the cyclohexane ring. The latter synthesis produced IAInos in 5 steps and 29% overall yield.

Stereoselective synthesis of new rac-quercitols containing hydroxymethyl groups as glucosidase inhibitors.

Stereoselective and efficient synthesis of hydroxymethyl-substituted rac-quercitols (13-15) was achieved, starting from cis-furan (Kobayashi, 2008) with photooxygenation reaction, which is readily available by the reduction of cis-phtalic anhydride. α- and ß-Glucosidase enzyme activity of the target molecules was evaluated and good inhibitor activity was seen. One- and two-dimensional NMR spectroscopy, IR spectroscopy and X-ray crystallography were utilized in the structure characterization of products.

Synthesis of C-Glycoinositols from C-Glycosylcrotylstannanes.

A strategy for the synthesis of C-pseudodisaccharides that centers on the reaction of a C-linked crotyltin and a substituted pent-4-enal and a ring-closing metathesis-alkene dihydroxylation sequence on the derived crotylation products is illustrated in the preparation of analogues of the insulin modulatory inositol galactosamine-ß-(1 → 4)-3-O-methyl-d- chiro-inositol (ß-INS-2). The modularity of this approach and versatility of the pivotal crotylation products make this a potentially general methodology for diverse libraries of C-glycoinositols.

Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.

The synthesis of perdeuterated and 13C enriched myo-inositol is presented. Myo-inositol and its derivatives are of interest as substrates for enzymes producing phosphorylated species with regulatory functions in many organisms. Its utility in monitoring real-time phosphorylation by myo-inositol-3-kinase is illustrated using dynamic nuclear polarization (DNP) to enhance NMR observation.

Synthesis and preliminary in vivo evaluation of new [18F]fluoro-inositols as Positron Emission Tomography radiotracers.

This study describes the synthesis and radiosynthesis of eight new [18F]fluoro-inositol-based radiotracers in myo- and scyllo-inositol configuration. These radiotracers are equipped with a propyl linker bearing fluorine-18. This fluorinated arm is either on a hydroxyl group, i.e. O-alkylated inositols, or on the cyclohexyl backbone, i.e. C-branched derivatives. To modulate lipophilicity, inositols were synthesized in acetylated or hydroxylated form. Automated radiosynthesis was performed on the AllInOne module and the radiotracers were produced in good radiochemical yields (15-31.5% dc). Preliminary in vivo preclinical evaluation of these eight [18F]fluoro-inositols as Positron Emission Tomography (PET) imaging agents in a breast tumour-bearing mouse model was performed and compared with [18F]-2-fluoro-2-deoxy-d-glucose ([18F]FDG). Amongst the different inositols, [18F]myo-2 showed the highest tumour uptake 2.34±0.39%ID/g, revealing the potential of this tracer for monitoring breast cancer.

Synthesis of (±)-myo-inositol 4-methylenephosphonate via Rh-Catalyzed hydrogenation of vinylphosphonate.

Phosphatidylinositol phosphate (PIP) synthetase is a promising target for the development of new anti-mycobacterium compounds. We previously reported that myo-inositol 1-methylenephosphonate showed inhibitory activity against PIP synthetase. Herein, we report the synthesis of unprotected myo-inositol 4-methylenephosphonate, a constitutional isomer of myo-inositol 1-methylenephosphonate and found that the stereoselective hydrogenation of vinylphosphonate proceeded via Rh catalysis.

Synthesis and conformational analysis of a simplified inositol-model of the Streptococcus pneumoniae 19F capsular polysaccharide repeating unit.

Carbohydrate mimics have been studied for a long time as useful sugar substitutes, both in the investigation of biological events and in the treatment of sugar-related diseases. Here we report further evaluation of the capabilities of inositols as carbohydrate substitutes. The conformational features of an inositol-model of a simplified repeating unit corresponding to the capsular polysaccharide of Streptococcus pneumoniae 19F has been evaluated by computational analysis, and compared to the native repeating unit. The inositol mimic was synthesized, and its experimental spectroscopic data allowed for verification of the theoretical results.

C-5a-substituted validamine type glycosidase inhibitors.

A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for ß-galactosidases as well as ß-glucosidases.

Synthesis and biological activities of d-chiro-inositol analogues with insulin-like actions.

d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease.