Regioselective Approach to Phosphatidylinositol 3,5-Bisphosphates: Syntheses of the Native Phospholipid and Biotinylated Short-Chain Derivative

A selective bis-silylation of 1D-O-TBDPS-myo-inositol leads to a 1,3,5-trisubstituted inositol, which can be advanced to the headgroup of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. A mild, regioselective method for construction of the diacylglycerol moiety containing differing fatty acid chains, including the naturally occurring lipids, was developed. Their union in the synthesis of the cell-signaling molecule PI(3,5)P2 containing the sn-1-stearoyl and sn-2-arachidonoyl groups is described. The methodology was also used to generate dioctanoyl-PI(3,5)P2 and a previously unreported biotin-PI(3,5)P2 conjugate, which was coupled to neutravidin beads and used to pull down PI(3,5)P2-binding proteins from the cytosolic extract of adrenal neurosecretory cells. We report the specific pull-down of the PI(3,5)P2-binding protein svp1p, a known PI(3,5)P2 effector involved in membrane trafficking

The Dimethoxyphenylbenzyl Protecting Group: An Alternative to the <i>p</i>‑Methoxybenzyl Group for Protection of Carbohydrates

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)­benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH₂Cl₂ and 3,4-(methylenedioxy)­toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and <i>p</i>-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, <i>p</i>-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)­benzyl ether as an effective and robust alternative to <i>p</i>-methoxybenzyl as a protecting group for alcohols

Resolution of Orthogonally Protected <i>myo</i>-Inositols with Novozym 435 Providing an Enantioconvergent Pathway to Ac₂PIM₁

Orthogonally protected chiral <i>myo-</i>inositol derivatives are important intermediates for higher order <i>myo</i>-inositol-containing compounds. Here, the use of the immobilized enzyme Novozym 435 to efficiently catalyze the acetylation of the 5<i>R</i> configured enantiomer of racemic 1,2-<i>O</i>-isopropylidene-<i>myo</i>-inositols possessing chemically and sterically diverse protecting groups at O-3 and O-6 is described. The resolutions were successful with allyl, benzyl, 4-bromo-, 4-methoxy-, 4-nitro-, and 4-(3,4-dimethoxyphenyl)­benzyl, propyl, and propargyl protection at O-6 in combination with either allyl or benzyl groups at O-3. Bulky protecting groups slow the rate of acetylation. No reaction was observed for 3,6-di-<i>O</i>-triisopropylsilyl-1,2-<i>O</i>-isopropylidene-<i>myo</i>-inositol. The utility of this methodology was demonstrated by the first reported synthesis of an Ac₂PIM₁ (<b>9</b>), which used both enantiomers of the resolved 3-<i>O</i>-allyl-6-<i>O</i>-benzyl-1,2-<i>O</i>-isopropylidene-<i>myo</i>-inositol in a convergent synthesis

The Rapid and Facile Synthesis of Oxyamine Linkers for the Preparation of Hydrolytically Stable Glycoconjugates

The synthesis of a number of <i>N</i>-glycosyl-<i>N</i>-alkyl-methoxyamine bifunctional linkers is described. The linkers contain an <i>N</i>-methoxyamine functional group for conjugation to carbohydrates and a terminal group, such as an amine, azide, thiol, or carboxylic acid, for conjugation to the probe of choice. The strategy for the linker synthesis is rapid (3–4 steps) and efficient (51–96% overall yield), and many of the linkers can be synthesized using a three-step one-pot strategy. Moreover, the linkers can be conjugated to glycans in excellent yield and they show excellent stability toward hydrolytic cleavage

Synthesis and Structure of Phosphatidylinositol Dimannoside

(R)-Tuberculostearic acid (2) was synthesized in seven steps from (S)-citronellol (5). The carbon chain of 2 was assembled by copper-catalyzed cross coupling of (S)-citronellol tosylate (6) and hexylmagnesium bromide; subsequent ozonolysis and reaction with 6-benzyloxyhexylmagnesium bromide furnished alcohol 10. Functional group manipulation afforded (R)-2 in 49% overall yield from 5. DCC coupling of (R)-2 with 3-O-benzyl-1-O-palmitoyl-sn-glycerol (16), followed by hydrogenolytic removal of the benzyl group and treatment with benzyl bis(diisopropyl)phosphoramidite, afforded phosphoramidite 20. Tetrazole-mediated coupling of 20 with PIM1 head group 21 gave 22, and subsequent debenzylation afforded phosphatidylinositol mono-mannoside, PIM1 (23). Similarly, coupling of 20 and 24 and removal of the benzyl protecting groups gave PIM2 (1c). Both 23 and 1c have a clearly defined acylation pattern, which was confirmed by mass spectrometry, with sn-1 palmitoyl and sn-2 tuberculostearoyl groups on the glycerol moiety. Both 23 and 1c were shown to modulate the release of the pro-inflammatory cytokine, IL-12, in a dendritic cell assay

Physicochemical and Biological Characterization of Synthetic Phosphatidylinositol Dimannosides and Analogues

Native phosphatidylinositol mannosides (PIMs), isolated from the cell wall of Mycobacterium bovis, and synthetic PIM analogues have been reported to offer a variety of immunomodulating properties, including both suppressive and stimulatory activity. While numerous studies have examined the biological activity of these molecules, the aim of this research was to assess the physicochemical properties at a molecular level and correlate these characteristics with biological activity in a mouse model of airway eosinophilia. To accomplish this, we varied the flexibility and lipophilicity of synthetic PIMs by changing the polar headgroup (inositol- vs glycerol-based core) and the length of the acyl chains of the fatty acid residues (C0, C10, C16, and C18). A series of six phosphatidylinositol dimannosides (PIM2s) and phosphatidylglycerol dimannosides (PGM2s) were synthesized and characterized in this study. Langmuir monolayer studies showed that surface pressure–area (π–A) isotherms were greatly influenced by the length of the lipid acyl chains as well as the steric hindrance and volume of the headgroups. In aqueous solution, lipidated PIM2 and PGM2 compounds were observed to self-assemble into circular aggregates, as confirmed by dynamic light scattering and transmission electron microscopic investigations. Removal of the inositol ring but retention of the three-carbon glycerol unit maintained biological activity. We found that the deacylated PGM2, which did not show self-organization, had no effect on the eosinophil numbers but did have an impact on the expansion of OVA-specific CD4+ Vα2Vβ5 T cells

Synthesis and Mass Spectral Characterization of Mycobacterial Phosphatidylinositol and Its Dimannosides

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM₂, AcPIM₂, and Ac₂PIM₂) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)­(19:0/16:0)-PIM₂) was achieved from (±)-1,2:4,5-diisopropylidene-d-<i>myo</i>-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the <i>p</i>-(3,4-dimethoxyphenyl)­benzyl protecting group for its use at the <i>O</i>-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of <i>M</i>. <i>bovis</i> BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract

Second Generation Transition State Analogue Inhibitors of Human 5‘-Methylthioadenosine Phosphorylase

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of spermidine and spermine is 5‘-methylthioadenosine (MTA). In humans MTA is processed by 5‘-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not accumulate. Products of the MTAP reaction (adenine and 5-methylthio-α-d-ribose-1-phosphate) are recycled to S-adenosylmethionine, the precursor for polyamine synthesis. Potent inhibitors of MTAP might allow the build-up of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis and/or reduce S-adenosylmethionine levels. We recently reported the design and synthesis of a family of potent transition state analogue inhibitors of MTAP. We now report the synthesis of a second generation of stable transition state analogues with increased distance between the ribooxocarbenium ion and purine mimics. These compounds are potent inhibitors with equilibrium dissociation constants as low as 10 pM. The first and second generation inhibitors represent synthetic approaches to mimic early and late features of a dissociative transition state

Phosphatidylinositol Mannoside Ether Analogues: Syntheses and Interleukin-12-Inducing Properties

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol dimannoside ether analogues 2 and 3 and evaluate their interleukin-12 (IL-12)-inducing properties along with dipalmitoyl PIM2 (1) in an in vitro bovine dendritic cell assay. Both synthetic PIM analogues and synthetic dipalmitoyl PIM2 (1) were effective at enhancing IL-12 production by immature bovine dendritic cells. Unexpectedly, ether analogue 2 was significantly more active than dipalmitoyl PIM2 (1) which indicates that modified PIM compounds can be strongly immunoactive and may have significant adjuvant activities

Image_1_Distinct Dysfunctional States of Circulating Innate-Like T Cells in Metabolic Disease.TIF

The immune system plays a significant role in controlling systemic metabolism. Innate-like T (ILT) cells in particular, such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cell receptor expressing cells, have been reported to promote metabolic homeostasis. However, these different ILT cell subsets have, to date, been generally studied in isolation. Here we conducted a pilot study assessing the phenotype and function of circulating MAIT, iNKT, and Vδ2+ T cells in a small cohort of 10 people with obesity and type 2 diabetes (T2D), 10 people with obesity but no diabetes, and 12 healthy individuals. We conducted phenotypic analysis by flow cytometry ex vivo, and then functional analysis after in vitro stimulation using either PMA/ionomycin or synthetic agonists, or precursors thereof, for each of the cell-types; use of the latter may provide important knowledge for the development of novel therapeutics aimed at activating human ILT cells. The results of our pilot study, conducted on circulating cells, show clear dysfunction of all three ILT cell subsets in obese and obese T2D patients, as compared to healthy controls. Importantly, while both iNKT and Vδ2+ T cell dysfunctions were characterized by diminished IL-2 and interferon-γ production, the distinct dysfunctional state of MAIT cells was instead defined by skewed subset composition, heightened sensitivity to T cell receptor engagement and unchanged production of all measured cytokines.</p