A scalable synthesis of (+)-coronafacic acid

A facile, efficient, and scalable synthesis of optically pure coronafacic acid by resolution of racemic coronafacic acid obtained using an improved version of Watson's method has been developed. By optimizing the boron-mediated aldol reaction of Watson, we were able to prepare 2.1 g of racemic coronafacic acid. This was coupled with (S)-4-isopropyl-2-oxazolidinone to give a mixture of diastereomeric coronafacyl oxazolidinones, which were readily separable by silica-gel column chromatography to give 630 mg of optically pure (+)-coronafacic acid.     

Method to optimize high-pressure, multicomponent gas mixing.

By use of the equations derived herein, a method is outlined to determine the optimum filing sequence and to obtain the maximum possible pressure when two or more pure high-pressure gases are to be transferred to a receiver cylinder in order to prepare a multicomponent gas mixture. The method is valid for any number of gas components, originating from high-pressure storage cyclinders of arbitrary size and pressure and for a receiver cylinder to contain initially one or more of the component gases. Percentage concentrations within 1% of desired are easily obtained with this method.     

Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate.     

Progress of cassava mosaic virus disease and whitefly vector populations in single and mixed stands of four cassava varieties grown under epidemic conditions in Uganda

Progress curves of cassava mosaic virus disease (CMD) and populations of the whitefly vector (Bemisia tabaci) were assessed using four cassava varieties grown alone and as a random mixture in two experiments established under epidemic conditions at a site near Kampala in southern Uganda. There were significant differences in final CMD incidence and in the areas under the disease progress curves between varieties when grown alone and as a mixture in both experiments. Variety Ebwanateraka had the highest incidence and SS4 the lowest, even though it supported the largest populations of adult whiteflies. The overall incidence of CMD in the mixture was similar to that in pure stands of the partially resistant Nase 2 and greater than in the resistant Migyera and SS4. Compared to pure stands, incidence of CMD in each component of the mixture was reduced significantly only in Ebwanateraka, whereas vector populations were less only in SS4 and Nase 2. On several observation dates the actual incidence of CMD and populations of adult whiteflies in the mixture were significantly less than expected values estimated from the results for the four varieties when each was grown alone. A highly significant positive relationship was established for each variety between peak populations of adult whitefly and leaf area index at the time. The implications of the findings and the scope for future research on the use of varietal mixtures for the management of CMD are discussed.     

Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein.

We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1. Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix. Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields. Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled. ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B. Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient. The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports.     

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.     

Synthesis and properties of nucleoside derivatives acylated by chemically stable 2-(trimethylsilyl)benzoyl group

We report the synthesis and properties of nucleoside derivatives acylated by 2-(trimethylsilyl)benzoyl (TMSBz) that proved to be extremely stable under basic conditions when introduced into the 5′-hydroxyl group of thymidine, the 4-amino group of deoxycytidine and the 2′-hydroxyl group of uridine. In particular, 2′-O-TMSBz-uridine could be isolated and was more stable in pyridine, while it isomerized in CH₂Cl₂ in the presence of Et₃N to yield a mixture of the 2′-O- and 3′-O-acylated species.     

The resolution of acyclic P‐stereogenic phosphine oxides via the formation of diastereomeric complexes: A case study on ethyl‐(2‐methylphenyl)‐phenylphosphine oxide

As an example of acyclic P‐chiral phosphine oxides, the resolution of ethyl‐(2‐methylphenyl)‐phenylphosphine oxide was elaborated with TADDOL derivatives, or with calcium salts of the tartaric acid derivatives. Besides the study on the resolving agents, several purification methods were developed in order to prepare enantiopure ethyl‐(2‐methylphenyl)‐phenylphosphine oxide. It was found that the title phosphine oxide is a racemic crystal‐forming compound, and the recrystallization of the enantiomeric mixtures could be used for the preparation of pure enantiomers. According to our best method, the (R)‐ethyl‐(2‐methylphenyl)‐phenylphosphine oxide could be obtained with an enantiomeric excess of 99% and in a yield of 47%. Complete racemization of the enantiomerically enriched phosphine oxide could be accomplished via the formation of a chlorophosphonium salt. Characterization of the crystal structures of the enantiopure phosphine oxide was complemented with that of the diastereomeric intermediate. X‐ray analysis revealed the main nonbonding interactions responsible for enantiomeric recognition.     

Efficient resolution of venlafaxine and mechanism study via X‐ray crystallography

Numbers of resolving factors were investigated to improve resolution of venlafaxine 1 . An effective resolving agent, O,O′‐di‐p‐toluoyl‐(R, R)‐tartaric acid 2 , was screened using similar method of ‘Dutch resolution’ from tartaric acid derivatives. The resolution efficiency was up to 88.4%, when the ratio of rac‐ 1 and 2 was 1:0.8 in THF with little water (10:1 v/v). Enantiomerically pure venlafaxine was prepared with 99.1% ee in 82.2% yield. The chiral resolution mechanism was first explained through X‐ray crystallographic study. One diastereomeric salt with well solubility forms a columnar supramolecular structure as the acidic salt (R)‐ 1 · 2 , while the other diastereomeric salt with less solubility forms a multilayered sandwich supramolecular structure by enantio‐differentiation self‐assembly as the neutral salt 2(S)‐ 1 · 2 . The water molecules play a key role in the optical resolution, as indicated by the special structures of the diastereomeric salts.     

Quantitative estimation of 3'5' cyclic AMP phosphodiesterase using anion exchange resin in a batch process.

An improved method for the preparation of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) is described which includes: enzymes from Chlorella for PAPS synthesis; conversion of ATP to AMP after PAPS formation with hexokinase (EC 2.7.1.1) and myokinase (EC 2.7.4.3); and separation of PAPS on DEAE-Sephadex using triethylammonium bicarbonate buffers. Any specific activity can be obtained by using appropriate concentrations of carrier-free ³⁵S and nonradioactive sulfate in the incubations. Between 300 and 2000 μmol of PAPS per batch can be obtained depending on the scale of the preparation. The PAPS is over 95% pure radiochemically and shows only one ultraviolet-absorbing spot on paper electrophoresis at pH 5.8. Adenosine 5′-phosphosulfate (APS) is prepared by incubating PAPS with a 3′-nucleotidase (EC 3.1.3.6) from rye grass. Quantitative conversion of PAPS to APS is obtained, and the APS is purified by column chromatography in the same manner as for PAPS. The APS obtained is better than 95% pure radiochemically and shows only one uv-absorbing spot on paper electrophoresis at pH 5.8.     

A Preparation of Cow's Late Colostrum Fraction Containing αs1-Casein Promoted the Proliferation of Cultured Rat Intestinal IEC-6 Epithelial Cells

The asymmetric epoxidation of (±)-methyl (2Z,4E)-1′,4′-dihydroxy-α-ionylideneacetates is described for the preparation of chiral abscisic acid. A conventional Shapless kinetic resolution of (±)-1′,4′-cis-dihydroxyacetate with diethyl l-tartarate and then two simple steps of conversion gave (S)-abscisic acid, which was also obtained by the combination of (±)-1′,4′-trans-dihydroxyacetate with diethyl d-tartarte. Finally, (S)-abscisic acid was obtained in a 25% overall yield from the racemic mixture.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

An Unusual Dephosphorylation Reaction

Abstract

In the course of our work to prepare l¹, Z²-seco-nucleotides, an unusual dephosphorylation reaction took place. When compound 1 was subjected to transfer hydrogenation conditions, the expected product was not obtained. Instead, a crystalline solid that proved not to have phosphorus was isolated in 78% yield. ¹H and ¹³C nmr analyses confirmed the hydrogenolysis of the benzyl groups as well as the loss of the phenyl esters. with elemental analytical data suggested that the structure of the compound could be either the 2′,5′-anhydro (2, R[dbnd]H) or 3′,5′-an- hydro derivative 3. precluded the use of spectroscopic analyses to prove the struc-ture. However, based on our experience with related compounds, structure 2 was favored, and we set out to prove it by another synthetic route.     

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.     

Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture

The flavonolignan isosilybin (1) is one of the silybin congeners contained in the silymarin complex, which is isolated from the seeds of the milk thistle (Silybum marianum). A number of recent studies have demonstrated that isosilybin is probably the most potent anticancer agent found in silymarin. It occurs as a mixture of two diastereoisomers, A and B. Lipase Novozym 435 was found to allow the preparative production of both optically pure isosilybin A and B in a diastereoisomeric purity of over 95%. A preparatory method based on the enzymatic resolution and other purification methods has been developed, enabling multigram amounts of both optically pure isosilybins A (1a) and B (1b) to be obtained.     

Identification and biological activity of peptides containing a partially benzyloxycarbonylated L-arginine on their amino terminus (author's transl)]

Nomega-Z-L-Arg-L-Phe-L-Phe was shown to be the antibiotically active compound in a peptide mixture which was obtained by treating Z3-L-Arg-L-Phe-L-Phe with hydrogen bromide/trifluoroacetic acid or 4N HBr/glacial acetic acid, respectively. Identification of this compound was achieved by thin-layer chromatography, enzymatic digestion and autobiograms with fungi. The pure Nomega-Z-L-Arg-L-Phe-L-Phe was not the only compound with antibiotic qualities; generally it could be said that all peptides with the sequence Nomega-Z-L-Arg-X-L-Phe (X might be any amino acid) are antibiotically active. All of them are antagonized by L-aspartic acid and asparagine in the crossstrip test (on fungi). The antibiotical activity of all these peptides must be due to the Nomega-Z-L-Arg-residue provided that it is coupled to a dipeptide X-L-Phe, or to an aromatic system (e.g. L-Phe or benzyl amine).     

Failure of leukotriene B4 to translocate calcium across phosphatidylcholine membranes

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogenously at 3 microM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.     

Purification and crystallization of creatine kinase from rabbit skeletal muscle

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.     

Enzymatic and Microbial Preparation of d-Xylulose from d-Xylose

A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized.