Liquid-Phase Synthesis of 2'-Methyl-RNA on a Homostar Support through Organic-Solvent Nanofiltration.

Due to the discovery of RNAi, oligonucleotides (oligos) have re-emerged as a major pharmaceutical target that may soon be required in ton quantities. However, it is questionable whether solid-phase oligo synthesis (SPOS) methods can provide a scalable synthesis. Liquid-phase oligo synthesis (LPOS) is intrinsically scalable and amenable to standard industrial batch synthesis techniques. However, most reported LPOS strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris. Precipitation can be difficult to develop and control on an industrial scale and, because many precipitations would be required to prepare a therapeutic oligonucleotide, we contend that this approach is not viable for large-scale industrial preparation. We are developing an LPOS synthetic strategy for 2'-methyl RNA phosphorothioate that is more amenable to standard batch production techniques, using organic solvent nanofiltration (OSN) as the critical scalable separation technology. We report the first LPOS-OSN preparation of a 2'-Me RNA phosphorothioate 9-mer, using commercial phosphoramidite monomers, and monitoring all reactions by HPLC, (31)P NMR spectroscopy and MS.

Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

Beyond PEG2000: synthesis and functionalisation of monodisperse PEGylated homostars and clickable bivalent polyethyleneglycols.

A new strategy to access highly monodisperse, heterobifunctional linear polyethylenglycols (PEGs) has been designed. This was built around unidirectional, iterative chain extension of a 3-arm PEG homostar. A mono-(4,4'-dimethoxytriphenylmethyl) octagol building block, DmtrO-EG8-OH, was constructed from tetragol. After six rounds of chain extension, the monodisperse homostar reached the unprecedented length of 56 monomers per arm (PEG2500). The unique architecture of the synthetic platform greatly assisted in facilitating and monitoring reaction completion, overcoming kinetic limitations, chromatographic purification of intermediates, and analytical assays. After chain terminal derivatisation, mild hydrogenolytic cleavage of the homostar hub provided heterobifunctional linear EG56 chains with a hydroxyl at one end, and either a toluene sulfonate, or a tert-butyl carboxylate ester at the other. A range of heterobifunctional, monodisperse PEGs was then prepared having useful cross-linking functionalities (-OH, -COOH, -NH2, -N3) at both ends. A rapid preparation of polydisperse PEG homostars, free of multiply cross-linked chains, is also described. The above approach should be extendable to other high value oligomers and polymers.

Understanding the relative affinity and specificity of the pleckstrin homology domain of protein kinase B for inositol phosphates.

Protein kinase B (PKB) is a serine/threonine kinase that plays a key role in the phosphoinositide 3-kinase (PI3K) pathway-one of the most frequently activated proliferation pathways in cancer. In this pathway, PKB is recruited to the plasma membrane by direct interaction of its pleckstrin homology (PH) domain with the inositol phosphate head-group of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] or phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)]. This recruitment is a critical stage in the activation of PKB, whose downstream effectors play important roles in cell survival, proliferation and growth. It is therefore of great interest to understand PKB's mode of binding, as well as its specificity and affinity for different phosphoinositides. We have used a total of 3 µs of molecular dynamics (MD) simulations to better understand the interactions of the PKB PH domain with the inositol phosphate head-groups of phosphoinositides involved in the PI3K pathway. Our computational models successfully mirror PKB's in vivo selectivity for 3-phosphorylated phosphoinositides. Furthermore, the models also help to rationalize unexpected in vitro data in which inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] binds with a relatively high affinity to the PKB PH domain, despite its parent lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] being known not to bind in vivo. With the support of computational simulations, we propose that when not bonded to a phosphatidate tail Ins(1,4,5)P(3) binds in an orientation in which its inositol ring is flipped with respect to the 3-phosphorylated inositol phosphate ligands and its parent lipid.

A theoretical investigation of inositol 1,3,4,5-tetrakisphosphate.

This paper describes the parameterization of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)] for use in molecular dynamics (MD) simulations. For this theoretical investigation, eleven isomers of Ins(1,3,4,5)P(4), with different levels and arrangements of protonation, have been considered. Herein we report accurate quantum mechanics (QM) calculations offering a detailed description of the energetic and structural properties of the Ins(1,3,4,5)P(4) isomers and subsequent development of parameters for these isomers for application in the AMBER force field. QM calculations were employed to geometry optimize the Ins(1,3,4,5)P(4) isomers, using the DFT-B3LYP level of theory in gas phase. In subsequent steps, charge parameters were generated for each isomer. These charge parameters, plus assigned atom-types from the AMBER ff99SB force field, were then applied to the optimized isomers for energy minimization in AMBER. The quality of the parameters was evaluated by comparing the structural, energetic and spectroscopic properties of the Ins(1,3,4,5)P(4) isomers between the QM geometry optimization stage, from which the parameters were generated, and the energy minimization stage, in which the parameters were applied. The results were shown to be in strong qualitative agreement between these stages, suggesting good quality parameters have been obtained. Additionally, adaptations to the gas phase protocol, investigating the use of the MP2 method for the geometry optimization stage and GAFF atom-types for the energy minimization stage, were tested. These results confirmed the initial protocol applied was the most appropriate. Calculations for the Ins(1,3,4,5)P(4) isomers were also carried out in the presence of implicit solvent, allowing comparison and validation of the theoretical calculations with experimental data. The computed energetic properties of the Ins(1,3,4,5)P(4) isomers were assessed against their experimental probabilities based on (31)P-NMR titration data. The computational and experimental results were shown to be in strong agreement, with the lower energy isomers corresponding to those more probable. This paper reports a clearly-defined algorithmic approach to generate parameters for the highly charged Ins(1,3,4,5)P(4) ligand, permitting their use in future MD studies.

Synthesis of 4-C-alkyl inositol 1,4,5-trisphosphates and 1,3,4,5-tetrakisphosphates.

The preparation of 2,3,6-O-tribenzyl- and 2,6-O-dibenzyl-myo-inositols with beta-primary, secondary, and tertiary 4-C-alkyl or aryl groups is reported. Five of these novel polyols are elaborated to 4-C-alkyl Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) analogues. Regio- and stereoselective introduction of 4-C-alkyl or aryl substituents proceeded via a 4-exo-methylene oxide. Subsequent regioselective reduction of an orthobenzoate provided a divergent method to access both InsP(3) and InsP(4) precursors. Previously unreported phosphorylation of the tertiary hydroxyl and global deprotection afforded novel analogues that retain their full complement of polar and charged binding features.

Synthesis of unsaturated phosphatidylinositol 4,5-bisphosphate and analogues.

A new approach for the synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is described, compatible with unsaturated fatty acid esters, as well as phosphorothioate and acetylenic analogues. This strategy depends on masking the phosphate charges with base-labile cyanoethyl esters, and the hydroxyls of the target with mild acid-labile protecting groups. A two-step basic then acidic global unblocking of orthogonal protecting groups provides the target lipid. A xanthenylidene acetal was used for key temporary protection of the 4,5-diol, and the 6-O was protected with a 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) acetal.

Regioselective deprotection of orthobenzoates for the synthesis of inositol phosphates.

Synthetic myo-inositol 1,4,5-triphosphate, Ins(1,4,5)P(3), and myo-inositol 1,3,4,5-tetraphosphate, Ins(1,3,4,5)P(4), continue to be valuable in biological studies. Inositol orthoesters have proved an important class of intermediate to access these compounds. We investigated the ability of steric bulk from a 4-O protecting group to direct DIBAL-H reduction of inositol orthobenzoates to generate the natural Ins(1,4,5)P(3) precursor 2,3,6-O-tribenzyl myo-inositol. Introduction of an equatorial 4-C-methyl group imparts totally selective reduction and we report the synthesis of novel 4-C-methyl-Ins(1,4,5)P(3) and 4-C-methyl-Ins(1,3,4,5)P(4).

The pH-dependence of lipid-mediated antimicrobial peptide resistance in a model staphylococcal plasma membrane: A two-for-one mechanism of epithelial defence circumvention.

The mechanisms of membrane defence by lysylphosphatidylglycerol (LPG), were investigated using synthetic biomimetic mono- and bilayer models of methicillin resistant S. aureus ST239 TW, based on its lipid composition in both pH 7.4 (28% LPG) and pH 5.5 (51% LPG) cultures. These models incorporated a stable synthetic analogue of LPG (3adLPG) to facilitate long-duration biophysical studies, which were previously limited by the lability native LPG. Both increased 3adLPG content and full headgroup ionization at pH 5.5, increased bilayer order and dampened overall charge, via the formation of neutral ion pairs with anionic lipids. Ion pair formation in air/liquid interface lipid monolayers elicited a significant condensing effect, which correlated with the inhibition of subphase-injected magainin 2 F5W partitioning. In fluid phase lipid vesicles, increasing the proportion of 3adLPG from 28 to 51 mol% completely inhibited the adoption of the membrane-active α­helical conformation of the peptide, without the need for full headgroup ionization. Neutron reflectivity measurements performed on biomimetic PG/3adLPG fluid floating bilayers, showed a significant ordering effect of mild acidity on a bilayer containing 30 mol% 3adLPG, whilst peptide binding/partitioning was only fully inhibited in a bilayer with 55 mol% 3adLPG at pH 5.5. These findings are discussed with respect to the roles of LPG in resistance to human epithelial defences in S. aureus and the continued evolution of this opportunistic pathogen's virulence.

Author Correction: Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving.

In the version of this Article originally published, the authors inadvertently cited ref. 10 in two places in the first paragraph. They would like to clarify that it should not have been cited in the sentence that starts "Polymer chemists have employed strategies such as single monomer insertion..." as it mistakenly implied that the IEG+ method described in ref. 10 could not produce unimolecular polymers; it can do so, as was demonstrated in ref. 10. The authors would also like to clarify that ref. 10 should not have been cited in the sentence that starts "Moreover, solid-phase synthesis is generally difficult to scale up...", as it implied that ref. 10 uses solid-phase synthesis; it does not, and is a purely liquid-phase process. The citation of ref. 10 has now been removed from these two sentences, but has been included elsewhere in the first two paragraphs of the Article as follows. In the first paragraph, at the end of the sentence "In iterative synthesis, specific monomers are added one at a time, or as multiples, to the end of a growing polymer chain, then reaction debris is separated from the chain extended polymer, and the cycle is repeated using the next monomer in the sequence10-12."; this sentence has been further amended to indicate multiple monomers can also be added. The reference has also been added to the end of the first sentence of the second paragraph, which starts "Consequently, liquid-phase iterative synthetic methods...", and in the third sentence of that paragraph, which now starts "For example, Johnson10, Whiting....".

Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving.

Synthetic chemists have devoted tremendous effort towards the production of precision synthetic polymers with defined sequences and specific functions. However, the creation of a general technology that enables precise control over monomer sequence, with efficient isolation of the target polymers, is highly challenging. Here, we report a robust strategy for the production of sequence-defined synthetic polymers through a combination of liquid-phase synthesis and selective molecular sieving. The polymer is assembled in solution with real-time monitoring to ensure couplings proceed to completion, on a three-armed star-shaped macromolecule to maximize efficiency during the molecular sieving process. This approach is applied to the construction of sequence-defined polyethers, with side-arms at precisely defined locations that can undergo site-selective modification after polymerization. Using this versatile strategy, we have introduced structural and functional diversity into sequence-defined polyethers, unlocking their potential for real-life applications in nanotechnology, healthcare and information storage.

Specific N-terminal protein labelling: use of FMDV 3C pro protease and native chemical ligation.

We report an effective strategy for generating N-terminal cysteinyl proteins by proteolytic cleavage using the enzyme 3C pro, suitable for a wide range of applications via native chemical ligation.

Regulation of PLCß2 by the electrostatic and mechanical properties of lipid bilayers.

Phosphoinositide-specific phospholipase C (PLC) is an important family of enzymes constituting a junction between phosphoinositide lipid signaling and the trans-membrane signal transduction processes that are crucial to many living cells. However, the regulatory mechanism of PLC is not yet understood in detail. To address this issue, activity studies were carried out using lipid vesicles in a model system that was specifically designed to study protein-protein and lipid-protein interactions in concert. Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation. Furthermore, for the first time, the relationships between PLC activity and substrate presentation in lipid vesicles of various sizes, as well as lipid composition and membrane mechanical properties, were analyzed. PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

Protein sulfenation as a redox sensor: proteomics studies using a novel biotinylated dimedone analogue.

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

Why are silyl ethers conformationally different from alkyl ethers? Chair-chair conformational equilibria in silyloxycyclohexanes and their dependence on the substituents on silicon. The wider roles of eclipsing, of 1,3-repulsive steric interactions, and of attractive steric interactions.

An NMR study of the diaxial/diequatorial chair equilibrium in a range of silylated derivatives of trans-1,4- and trans-1,2-dihydroxycyclohexane is reported and discussed with a view to explaining unusually large populations of chair conformations with axial substituents, noted previously for some monosilyloxycyclohexanes and in some silylated sugars. X-ray diffraction studies of three bis-triphenylsilyloxycyclohexanes are reported and show both axial and equatorial silyloxy groups with the exocyclic bonds eclipsed. Eclipsing is also suggested by molecular mechanics (MM3) calculations on such derivatives. Both axial and equatorial tertiary silyl groups have 1,3-repulsive interactions with whatever substituents or hydrogen atoms are at the two adjacent equatorial positions, and these are relieved by rotation toward the eclipsed conformation of the exocyclic C-O bond. The three substituents on silicon interact attractively with the nine atoms at the 3, 4, and 5-positions of the cyclohexane ring and calculations suggest that these stabilizing interactions are significantly greater in the axial than in the equatorial conformation. An equatorial C-OSiR(3) bond with one or two equatorial neighbors has a restricted potential energy well that becomes much broader when the bond is axial without any equatorial neighbors in the alternative chair. Adjacent silyl groups in the 1,2-disubstituted series interact in a stabilizing way overall in all conformations, this being particularly marked in the diaxial conformation of the more complex ethers. These factors lead to unusually large axial populations.

Synthesis of orthogonally protected lanthionines.

Synthetic approaches to the lantibiotics, a family of thioether-bridged antimicrobial peptides, require flexible synthetic routes to a variety of orthogonally protected derivatives of lanthionine 1. The most direct approaches to lanthionine involve the reaction of cysteine with an alanyl beta-cation equivalent. Several possibilities exist for the alanyl beta-cation equivalent, including direct activation of serine under Mitsunobu conditions: however, the low reactivity of sulfur nucleophiles in the Mitsunobu reaction has previously precluded its use in the synthesis of the lantibiotics. We report here a new approach to the synthesis of protected lanthionine, using a novel variant of the Mitsunobu reaction in which catalytic zinc tartrate is used to enhance the nucleophilicity of the thiol. In the course of these studies, we have also demonstrated that the synthesis of lanthionine from trityl-protected beta-iodoalanines is prone to rearrangement, via an aziridine, to give predominantly trityl-protected alpha-iodo-beta-alanines, and hence norlanthionines, as the major products.