Inositol Derivatives Research Articles

Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

The chemoselective O-glycosylation of alcohols in the presence of a phosphate diester and its application to the synthesis of oligomannosylated phosphatidyl inositols

The glycosylation of hydroxyl groups in the presence of a phosphate diester and its use in the synthesis of oligomannosylated phosphatidiyl inositols are reported. Using a catalytic amount of TMSOTf as an activator for the glycosidation of glycosyl imidates in the presence of a primary alcohol and a phosphate diester provided the desired glycoside along with a glycosyl phosphnate. Complete glycosylation of the primary alcohol required a stoichiometric amount of TMSOTf or TBSOTf. We next examined an application of the method to the synthesis of the phosphatidylinositol mannosides, which are components in the mycobacterial cell wall envelope. Glycosylation of the primary alcohol at the C6 position of a 2,6-dimannosyl myo-inositol core containing a diacylated phosphatidyl lipid with glycosyl imidiates proceeded smoothly to provide multimannosylated inositol derivatives in good yields. However, the 4,6 diol mannoside was a poor acceptor in this reaction.

Preparation of azide biosynthetic surrogates of myo-inositol

As a prelude to biomolecular incorporation studies, practical routes to a series of four regioisomeric azido-deoxy derivatives of inositol that mimic the natural myo-stereochemistry are described. Starting from commercially available myo-inositol, the regioselective and stereoselective introduction of azide functionality was achieved at the C-2, C-3, C-4, and C-5 positions via azide displacement of the corresponding O-sulfonates of suitably protected scyllo-, chiro-, epi-, and neo-inositols, respectively. Notably, a final one-pot acetolysis method conveniently allowed for rapid access to pentaacetate azido-deoxy inositols. Investigations on the metabolic incorporation of these myo-inositol azide surrogates in both acetate and free alcohol forms are in progress.

Synthesis and in vitro anticancer activity evaluation of novel bioreversible phosphate inositol derivatives

The chemistry and biology of phosphorylated inositols have become intense areas of research during the last two decades due to their involvement in various cellular signaling processes. However, the metabolic instability by phosphatases or kinases and poor penetration make it difficult to become a drug used in the clinic. The bioreversible protection technique can enhance membrane penetration characteristics and increase the stability of phosphorylated inositols against enzymatic degradation and is applied widely in drug discovery and development. In this paper, we described the design and synthesis of 22 bioreversible phosphotriester inositols, along with the initial antitumor activity results. Most compounds exhibited significant cytotoxic activity against human cancer cell lines A549, MDA-MB-231 and HeLa, but lower cellular toxicity on normal cell MCF10A in comparison with Cisplatin. These compounds can be used as probes to study the mechanism of intracellular signal transduction mediated by phosphate inositol or as leads of phosphate inositol drugs in the clinic.

Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

ChemInform Abstract: Myo‐Inositol 1,3‐Acetals as Early Intermediates During the Synthesis of Cyclitol Derivatives

AbstractReview: 80 refs.

Enhanced biomass and lipid production by supplement of myo-inositol with oceanic microalga Dunaliella salina

We investigated the effects of inositols, which are well-known plant growth-promoting agents, on the growth of the oceanic microalga Dunaliella salina. Of the four inositol derivatives tested (myo-inositol, scyllo-inositol, d-chiro-inositol, and l-chiro-inositol), myo-inositol (MI) showed the greatest growth-promoting effect in a concentration-dependent manner. The yield of biomass from the alga cultured with 500 mg L−1 of MI was 1.48-times that of the control culture. No significant effect of MI on the total carotenoid content was observed, but neutral lipid content was significantly increased, 1.34-times greater than the control. MI also influenced the fatty acid methyl ester composition, with the levels of linoleic, linolenic, and linolelaidic acids significantly higher than those of the control culture. To the best of our knowledge, this is the first demonstration that MI promotes the growth of a marine microalga. Our results suggest that MI has potential for enhancing the efficiency of biofuel production by D. salina through growth promotion and increasing lipid productivity.

Myo-Inositol 1,3-acetals as early intermediates during the synthesis of cyclitol derivatives

Synthetic sequences starting from commercially available myo-inositol necessarily involve protection–deprotection strategies of its six hydroxyl groups. Several strategies have been developed/attempted over the last several decades leading to the synthesis of naturally occurring phosphoinositols, their analogs, and cyclitol derivatives. Of late, myo-inositol 1,3-acetals, which can be obtained by the reductive cleavage of myo-inositol orthoesters have emerged as early intermediates for the synthesis of phosphorylated and other inositol derivatives. This mini-review is an attempt to illustrate the economy and convenience of using myo-inositol 1,3-acetals as early intermediates during syntheses from myo-inositol.

The chiro-Inositols and Related Cyclitols as Chiral Monomers for Polymerization: Expansion of a Family of Chiral Polymers

Three different synthetic approaches to three different classes of oligomeric and polymeric inositol and inositol-like derivatives are presented: Grubbs cross-metathesis polymerization, ­Lewis acid catalyzed epoxide opening, and head-to-tail Diels–Alder polymerization. All three approaches lead to the formation of oligomers and polymers. The oligomers/polymers formed range from three (low-molecular-weight fraction of Diels–Alder polymerization) to 60 (Lewis acid catalyzed polymerization) repeats of inositol or inositol-like units. The molecular weights of the polymers were characterized by gel-permeation chromatography (GPC), MALDI, and NMR end-group analysis.

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3' end processing, through the regulation of SA production, is a key component of plant immune responses.

4-Hydroxyphenylacetic acid derivatives of inositol from dandelion (Taraxacum officinale) root characterised using LC–SPE–NMR and LC–MS techniques

The combination of hyphenated techniques, LC–SPE–NMR and LC–MS, to isolate and identify minor isomeric compounds from an ethyl acetate fraction of Taraxacum officinale root was employed in this study. Two distinct fractions of 4-hydroxyphenylacetic acid derivatives of inositol were isolated and characterised by spectroscopic methods. The 1H NMR spectra and MS data revealed two groups of compounds, one of which were derivatives of the di-4-hydroxyphenylacetic acid derivative of the inositol compound tetrahydroxy-5-[2-(4-hydroxyphenyl)acetyl] oxycyclohexyl-2-(4-hydroxyphenyl) acetate, while the other group consisted of similar tri-substituted inositol derivatives. For both fractions the derivatives of inositols vary in the number of 4-hydroxyphenylacetic acid groups present and their position and geometry on the inositol ring. In total, three di-substituted and three tri-substituted 4-hydroxyphenylacetic acid inositol derivates were identified for the first time along with a further two previously reported di-substituted inositol derivatives.

Recent progress in the synthesis of GPIs and GPI-anchored proteins

Glycosylphosphotidylinositol (GPI) attachment to the protein C-terminus is a common posttranslational modification in eukaryotic species. GPIs help anchor proteins onto the out leaflet of cell membranes. GPI-anchored proteins play an important role in various biological and pathological processes. To study GPIs and GPI-anchored proteins in detail, it is necessary to access these molecules in homogeneous form and sufficient quantities, which is difficult to achieve via natural product isolation. As a result, chemical and chemoenzymatic synthesis has become a potent approach to obtain GPIs and GPI-anchored proteins for various studies. This paper reviews the recent progress in the synthesis of GPI anchors and GPI-anchored proteins by mainly focusing on the strategies developed for the preparation of optically pure, position-differentiated inositol derivatives, the key intermediates in GPI synthesis, for natural GPI anchor assembly, for diversity-oriented synthesis of functionalized GPI derivatives, and for peptide/protein and GPI anchor coupling.

Chemical constituents from Inula wissmanniana and their anti-inflammatory activities

Two new inositol derivatives, 4-acetate-1,2,3,5-tetrakis(2-methyl-2-butenoate) inositol (1) and 3-acetate-1,2,4,5-tetrakis(2-methyl-2-butenoate) inositol (2), one new flavonolignan 23-o-acetylsilychristin A (3), and one new thymol derivative 3-hydroxy-4-(2-hydroxy-1-methylethyl)benzoic acid (4), along with 13 known compounds, including ten flavonolignans (5-14) and three thymol derivatives (15-17) were isolated from the aerial part of Inula wissmanniana. The structures of new compounds were elucidated by detailed spectroscopic analysis, including HRESIMS and 2D-NMR spectroscopic data. All the compounds were isolated from this plant for the first time. The anti-inflammatory activities of the compounds were tested against LPS-induced NO production in RAW 264.7 macrophages.

Regioselective opening of myo-inositol orthoesters: mechanism and synthetic utility.

Acid hydrolysis of myo-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-O-acyl myo-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-O-acyl inositol 1,3,4,5,6-pentakisphosphates and myo-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-O-C(O)CD2R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester myo-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-O-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments.

Synthesis of Mercaptobenzothiazole and Mercaptobenzimidazole Condensed with Inositol Derivatives

This work describes the synthesis of inositol derivatives condensed with 2‐mercaptobenzothiazole or 2‐mercaptobenzimidazole, potential antimicrobial agents. These compounds were prepared by ring opening of epoxide intermediate (2S,3R‐epoxy‐1‐O‐methyl‐ l‐chiro‐inositol and 2R,3S‐epoxy‐1‐O‐methyl‐ l‐chiro‐inositol), which were obtained from l‐quebrachitol (1‐O‐methyl‐chiro‐inositol). Microwave irradiation was used to promote the condensation reaction.

The NifA-RpoN Regulon of Mesorhizobium loti Strain R7A and Its Symbiotic Activation by a Novel LacI/GalR-Family Regulator

Mesorhizobium loti is the microsymbiont of Lotus species, including the model legume L. japonicus. M. loti differs from other rhizobia in that it contains two copies of the key nitrogen fixation regulatory gene nifA, nifA1 and nifA2, both of which are located on the symbiosis island ICEMlSymR7A. M. loti R7A also contains two rpoN genes, rpoN1 located on the chromosome outside of ICEMlSymR7A and rpoN2 that is located on ICEMlSymR7A. The aims of the current work were to establish how nifA expression was activated in M. loti and to characterise the NifA-RpoN regulon. The nifA2 and rpoN2 genes were essential for nitrogen fixation whereas nifA1 and rpoN1 were dispensable. Expression of nifA2 was activated, possibly in response to an inositol derivative, by a novel regulator of the LacI/GalR family encoded by the fixV gene located upstream of nifA2. Other than the well-characterized nif/fix genes, most NifA2-regulated genes were not required for nitrogen fixation although they were strongly expressed in nodules. The NifA-regulated nifZ and fixU genes, along with nifQ which was not NifA-regulated, were required in M. loti for a fully effective symbiosis although they are not present in some other rhizobia. The NifA-regulated gene msi158 that encodes a porin was also required for a fully effective symbiosis. Several metabolic genes that lacked NifA-regulated promoters were strongly expressed in nodules in a NifA2-dependent manner but again mutants did not have an overt symbiotic phenotype. In summary, many genes encoded on ICEMlSymR7A were strongly expressed in nodules but not free-living rhizobia, but were not essential for symbiotic nitrogen fixation. It seems likely that some of these genes have functional homologues elsewhere in the genome and that bacteroid metabolism may be sufficiently plastic to adapt to loss of certain enzymatic functions.

Chemoselective alcoholysis/acetolysis of trans-ketals over cis-ketals and its application in the total synthesis of the cellular second messenger, d-myo-inositol-1,4,5-trisphosphate

The involvement of natural phosphoinositols in various cellular signalling processes and the use of synthetic inositol derivatives in catalysis, supramolecular chemistry, natural product synthesis etc. gave momentum to myo-inositol chemistry. The presence of six secondary hydroxyl groups necessitates efficient protection-deprotection strategies for the synthesis of inositol derivatives. An important strategy for the initial protection of myo-inositol is the di-ketalization, which gives a mixture of three diketals, each having both cis-fused and trans-fused ketals. It is important to have methodologies either to selectively hydrolyze one of the two ketals or to convert one of the two acid labile ketals to an orthogonal base labile protecting group. By exploiting the difference in strain between trans-ketals and cis-ketals, we developed two operationally simple, high yielding methodologies for the chemoselective hydrolysis/acetolysis of trans-ketals (both isopropylidene and cyclohexylidene) of inositols, leaving the cis-ketal undisturbed, using cheap and easily preparable H2SO4-silica as the catalyst. Also, terminal ketal moieties of carbohydrates and acyclic polyols could be selectively hydrolyzed/acetolyzed leaving the internal ketals intact. The use of methanol as the solvent leads to chemoselective alcoholysis but the use of DCM and acetic anhydride leads to chemoselective acetolysis. Applying this methodology, a short synthesis of D-myo-inositol-1,4,5-trisphosphate has been achieved.

H2SO4-silica: an eco-friendly heterogeneous catalyst for the differential protection of myo-inositol hydroxyl groups

There is enormous interest in myo-inositol derivatives as they serve as precursors for the synthesis of several biologically important phosphoinositols, natural products, catalyst, supramolecular architectures etc. However the presence of six secondary hydroxyl groups of similar reactivity warrants protection of inositol hydroxyl groups for effective synthesis. Acid catalyzed protection of inositol hydroxyl groups as orthoesters or ketals are the most commonly used protecting strategy in inositol chemistry. Traditionally, homogeneous acid catalysts such as para-toluene sulfonic acid (p-TSA) or camphorsulfonic acid (CSA) are used for these transformations. While the reversible nature of these reactions necessitates the catalyst removal, aqueous work up cannot be employed for their removal as the products are water soluble. We have circumvented this problem by using H2SO4-silica as the solid supported catalyst, which can be removed by filtration, for these transformations. Treatment of myo-inositol with trialkylorthoesters in presence of H2SO4-silica under normal conditions resulted in esterification at the least reactive hydroxyl group (C2-OH) giving exclusively the corresponding 2-O-acyl-myo-inositol. By doing the reaction in a rotary evaporator under reduced pressure resulted in the formation of the corresponding orthoesters, wherein three hydroxyl groups of inositol are protected simultaneously. We could synthesize different myo-inositol orthoesters 6–10 in excellent yields by this method. Monoketaliziation of myo-inositol with one equivalent of 1,1-dimethoxycyclohexane or 2,2-dimethoxypropane in presence of H2SO4-silica under similar conditions resulted in the simultaneous protection of 1-OH and 2-OH giving the 1,2-O-cyclohexylidene-myo-inositol (11) or 1,2-O-isopropylidene-myo-inositol (12) in excellent yields. Also, diketalization of myo-inositol with 2,2-dimethoxypropane gave three diketals namely (±)-1,2:4,5-di-O-isopropylidene-myo-inositol (17), (±)-1,2:5,6-di-O-isopropylidene-myo-inositol (18) and (±)-1,2:3,4-di-O-isopropylidene-myo-inositol (19) in considerable yields. Similarly when dimethoxycyclohexane was used, the dicyclohexylidene derivatives (±)-1,2:4,5-di-O-cyclohexylidene-myo-inositol (20), (±)-1,2:5,6-di-O-cyclohexylidene-myo-inositol (21) and (±)-1,2:3,4-di-O-cyclohexylidene-myo-inositol (22), were obtained in good yield. While the yields of 1,2:3,4-di-O-alkylidene-myo-inositols are negligibly small by other known methods, interestingly, our method give these diketals in good yields and hence can be exploited synthetically. Thus by using a cheap, eco-friendly, easy-to-make and easy-to-handle H2SO4-silica as the catalyst, we could tune the conditions to make mono-protected, di-protected, tri-protected or tetra-protected inositol derivatives. These strategies can be applied for the economic synthesis of various key intermediates of inositol for various purposes.

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo-Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer's disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.