Reactivity of UNCAs with Grignard reagents

Summary The reactivity of Grignard reagents with UNCAs (Urethane N-protected Carboxyanhydrides of Amino acids) is described. We observed that, depending on the method of addition of the organometallic compounds, the reaction proceeded differently: (i) when the UNCA was added to the Grignard reagent, we obtained a mixture of five different products which were all identified; and (ii) when the organometallic reagent was added to the UNCA, we also obtained a mixture of the same products but in different proportions, with the major component corresponding to the urethane N-protectedN-acyl amino acid derivative.     

Highly diastereoselective synthesis of enantiopure naphthylaminoalcohols with analgesic properties

The diastereoselective synthesis via Grignard reaction of enantiopure analgesic naphthylaminoalcohols has been performed. The chiral racemic key intermediate 3-dimethylamino-2-methyl-1-(naphthalen-2-yl)propan-1-one and enantiomers were prepared and transformed into the desired compounds by addition of the organometallic reagent. The chemical characterization of all diastereoisomers was accomplished by 1H NMR and HPLC analyses and the absolute configuration assigned by CD spectroscopy. The in vitro and in vivo profile has also been evaluated.     

Use of triphenylmethyl (trityl) amino protecting group in the synthesis of ketomethylene analogues of peptides

The Grignard reagents of 2-(2-bromoethyl)-1,3-dioxane and 2-(2-bromoethyl)-1,3-dioxolane readily reacted with the 2-thiopyridyl ester of N-triphenylmethyl-L-leucine to give the ketone adducts 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxane (8a) and 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxolane (8b) in near quantitative yield. When 1 equiv. of the Grignard reagent of 2-(2-bromoethyl)-1,3 dioxane was used, the desired ketone adduct 8a was formed slowly but quantitatively. In contrast, when 2 equiv. of the Grignard reagent were used, the formation of ketone 8a was instantaneous. The triphenylmethyl protecting group was easily removed from 8a using dilute acid to give the amino ketone 2-[3-oxo-4(S)-amino-6-methylheptyl]-1,3-dioxane oxalate salt (9). This material served as a useful intermediate in the synthesis of the ketomethylene analogues of the peptides, Z-Pro-Leu-Gly-OH and Leu-Gly-Val-Phe-OCH3.     

Prostaglandins and congeners V. (1) Synthesis of d1-prostaglandin E1, d1-11-deoxyprostaglandin E1, and d1-15-hydroxy-9-oxo-13-cis-prostenoic acid via conjugate addition of 3-trityloxy-1-octenylmagnesium bromides

d1-Prostaglandin E₁ and d1-11-deoxyprostaglandin E₁ are conveniently synthesized via the copper (I) catalyzed conjugate addition of the Grignard reagent prepared from 3-trityloxy-$\text{trans}$-1-octenyl bromide to the appropriate cyclopentenone precursor. The Grignard reagent also afforded the synthesis of a novel structure, d1-15-hydroxy-9-oxo-13-$\text{cis}$-prostenoic acid.     

Determination of the Positional-Species Composition of Plant Reserve Triacylglycerols by Partial Chemical Deacylation

To determine the positional-species composition (PSC) of the sunflower oil triacylglycerols (TAGs), these TAGs were separated in a native state by preparative TLC on alumina and subjected to a partial chemical deacylation with the Grignard reagent under anhydrous conditions. The rac-1,2-diacylglycerols (DAGs) formed in this reaction were identified by comparing their TLC mobilities with the respective standard separated from the products of enzymatic hydrolysis of lecithin. Subsequently, the DAGs as their borate complexes were isolated from the reaction mixture by preparative TLC on silica gel. The quantitative fatty acid composition of both the DAGs and the initial TAGs was determined by capillary GLC. The PSC of TAGs calculated from the data obtained using a computer program was consistent with the data on sunflower TAG composition reported by other researchers using an independent method. Thus, the technique developed here makes it possible to obtain reliable data on the concentration of all TAG positional species in the plant tissue.     

Chemical synthesis of (24R)-24,25-dihydroxy[26,27-3H]vitamin D3 of high specific activity

Chemical synthesis of (24R)-24,25-dihydroxy-[26,27-3H]vitamin D3, and its 24-epimer has been devised that allows introduction of 3H at the terminal step of the synthesis. The epimeric mixture is derivatized as the tris(trimethylsilyl) ethers and resolved by high-performance liquid chromatography. The product has a specific activity of 178 Ci/mmol and is fully active in binding to the rat plasma vitamin D binding protein and in the elevation of serum calcium levels of vitamin D deficient rats. The synthesis begins with the readily available 3 beta-hydroxy-5-cholenic acid methyl ester and involves a Pummerer rearrangement, introduction of the delta 7, irradiation, and isolation of the 26,27-dinor-25-carboxylic acid methyl ester of vitamin D3. This compound is then treated with a Grignard reagent containing 3H (80 +/- 10 Ci/mmol).     

Studies on Menthol Derivatives

(–)-Menthyl carbinol (1-(R)-methyl-3-(R)-hydroxymethyl-4-(S)-isopropylcyclohexane) (4) was prepared stereospecifically in good yield by treatment of formaldehyde with the Grignard reagent from (–)-menthyl chloride (2), which was prepared from (–)-menthol-(1-(R)-methyl-3-(R)-hydroxy-4-(S)-isopropylcyclohexane) (1) by chlorination using the Lucas reagent (HCl+ZnCl₂). The configuration of 4 was assigned by the chemical method.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Copper-catalyzed divergent kinetic resolution of racemic allylic substrates

When a racemic mixture is fully consumed the products may still be enantiomerically enriched. In particular, the regiodivergent kinetic resolution is a process in which a single chiral catalyst or reagent reacts with a racemic substrate to form regioisomers possessing an opposite configuration on the newly-formed stereogenic centers. This review reports the major advances in the field of the copper-catalyzed regiodivergent and stereodivergent kinetic resolution of allylic substrates with organometallic reagents. The chiral recognition matching phenomena found with particular allylic substrates with the absolute configuration of the chiral catalyst allows in some cases an excellent control of the regio- and stereoselectivity, sheding some light on the so-called "black-box" mechanism of a copper-catalyzed asymmetric allylic alkylation.     

A new efficient synthesis of efaroxan

The key step of the synthesis of efaroxan was the dihydrobenzofuran ring formation involving an intramolecular cyclization of the tertiary alcohol intermediate with the fluoroaromatic moiety in basic medium. This carbinol was prepared according to two routes, either from reaction of a benzyl Grignard reagent with an alpha-ketoester, or from a Darzens condensation.     

Synergism among three purified cellulolytic components of Clostridium thermocellum

Abstract Three clostridial cellulases viz. a hydrophilic cellobiohydrolase (CBH3), a hydrophobic endoglucanase (EG1), and an aggregate-forming hydrophilic endoglucanase (EG5), all purified from recombinant strains of Escherichia coli , were used in different combinations to reconstitute the synergistic effect during cellulose hydrolysis. EG1 and EG5 were weakly active on crystalline cellulose, if added separately or together in the reaction mixture. However, when CBH3 was added to the reaction mixture, its hydrolytic activity was increased to 1.8-fold in the presence of EG1 and EG5. A further increase in the activity from 1.8 to 2.2-fold was observed when calcium and dithiothreitol were added to the reaction mixture containing all three enzymes and filter paper as substrate. The synergistic effect remained unaffected even when EG1 was replaced by its 33-amino acid C-terminal deleted variant BL35. BL35 was less active compared to EG1, but was equally hydrophobic as EG1. These results suggest that the hydrophobic interaction between cellulolytic components and/or with the crystalline substrate is important for positive synergistic effect.     

A three-component Fischer indole synthesis

This protocol describes a three-component approach to multiply-substituted indoles from nitriles, organometallic reagents and arylhydrazine hydrochloride salts. The condensation of organolithium or Grignard reagents with nitriles produces metalloimines, which under acidic conditions and in the presence of arylhydrazines lead to arylhydrazones, the starting materials for the Fischer indole reaction. Combining this approach with the Fischer indole reaction produces indoles in an efficient, one-pot process. The procedure takes approximately 20 h to complete: 3 h for metalloimine formation, 15 h for the Fischer indole reaction and 2 h for isolation and purification.     

Preparation of polymer-supported benzyllithium reagents

Cross-linked polystyrenes (2, 8, and 11) possessing pendant double bonds conjugated with benzene ring were prepared by both polymerization method and chemical modification of pre-formed polymers. These double bonds were readily lithiated with n-butyllithium to give the benzyllithium species on the polymer. The polymer supported benzylic anions (3 and 12) are a versatile polymeric organometallic reagent that reacted with various kinds of electrophiles.     

Enantioselective vinylation of aldehydes with the vinyl Grignard reagent catalyzed by magnesium complex of chiral BINOLs

Enantioselective vinylation of aldehydes via direct catalytic asymmetric Grignard reaction of aldehdyes and the vinyl Grinard reagent is a long‐standing challenge. This work demonstrated that the magnesium (S)‐3,3′‐dimethyl BINOLate enantioselectively catalyze the direct vinylation of aldehydes with the deactivated vinylmagnesium bromide by bis(2‐[N,N′‐dimethylamino]ethyl) ether (BDMAEE) in the addition of n‐butylmagnesium chloride. The highest ee of 63% was achieved up to date.     

Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino Acid Inhibition

Lemna minor grown in sterile culture on a minerals-sucrose medium can utilize as nitrogen source, in order of increasing growth rate: ammonia, nitrate, a mixture of glutamic and aspartic acids plus arginine, or a balanced mixture of amino acids (hydrolyzed casein). Maximum growth is found with nitrate plus hydrolyzed casein.Many synthetic mixtures of amino acids are unable to support growth. Many single amino acids are inhibitory, and when added (at 2 mm or less) to cultures, growing in the presence of nitrate, cause a decrease in growth rate or even death of the plants (e.g. with alanine, valine, methionine or leucine). Some of these inhibitory effects are also found when the amino acid is added to cultures growing on ammonia or hydrolyzed casein. Arginine was the only amino acid of those tested which gave a marked stimulation of growth when added to cultures growing with inorganic nitrogen.The rapid rate of growth, sterile nature of tissue, decreased biological variation of samples containing many plants and ability to utilize different culture media make this an attractive organism for studies on higher plant metabolism.     

Detection of vesicular lipoproteins in lecithin:cholesterol acyltransferase-deficient plasma by 1H-NMR spectroscopy

The proton NMR spectra of the N-methyl choline region of normal and lecithin:cholesterol acyltransferase (LCAT)-deficient lipoproteins and of egg yolk phosphatidylcholine-cholesterol 55:45 (mol %) vesicle mixtures have been examined in the presence and absence of manganous sulfate as a line-broadening reagent. Manganous ions quenched all of the signal arising from normal lipoproteins and only part of the vesicle signal corresponding to the outer monolayer. There was no net loss of vesicular phospholipid when vesicles were added to normal lipoproteins and as little as 5% (or 100 micrograms) of the vesicular phospholipid could be detected and quantitated in the mixture of lipoproteins. Similar experiments performed on plasma lipoproteins from an LCAT-deficient patient indicated that 42% of the phospholipid was associated with vesicular lipoproteins. These experiments demonstrate that this technique can be used to detect and quantify small amounts of vesicular structures directly in a mixture of micellar lipoproteins.     

A rapid and direct method for the quantitative determination of tryptophan in the intact protein

1. A method is given for the quantitative determination of free tryptophan or tryptophan in the intact protein by treating with ninhydrin in a mixture of formic acid and hydrochloric acid (reagent b), for 10min at 100 degrees C. Glycyltryptophan was used as a standard for the determination of tryptophan in the intact protein. The extinction at 390nm was linear in the range 0.05-0.5mumol for free tryptophan (in7120) and 0.05-0.30mumol for glycyltryptophan (in15400). 2. Free tryptophan in the presence of protein may be determined by treating with ninhydrin in a mixture of acetic acid and 0.6m-phosphoric acid (reagent a) for 10min at 100 degrees C, the extinction being linear for tryptophan in the range 0.05-0.9mumol. N-Terminal tryptophan peptides also give the typical yellow product on treatment with reagent a. 3. Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others. A mean tryptophan content of 11.25 (s.e.m. +/-0.08) mumol/100mg of protein was found in rat brain during development from 1 to 82 days after birth.     

Increased uniformity in the response of the coomassie blue G protein assay to different proteins

Coomassie blue G dye-based protein assays are exceptionally convenient because of their simplicity, sensitivity, speed, and resistance to interfering chemicals, notably reducing agents and most buffers. A major problem with the assay is the variation in response to different proteins. The addition of NaOH to the protein assay reagent reduced the variation in the response of this assay to different proteins. In addition, the sensitivity of the assay is increased. The NaOH can be added either in a separate step to solubilize cells or membranes or directly to the reagent. Linear standard curves were obtained when the log of the absorbance was plotted against the log of the protein quantity.     

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli.

The methylesterase enzyme from Bacillus subtilis was compared with that from Escherichia coli. Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH. When attractants were added to a mixture of B. subtilis MCPs and E. coli methylesterase, the rate of demethylation was enhanced. Conversely, when attractants were added to a mixture of E. coli MCPs and B. subtilis methylesterase, the rate of demethylation was diminished. These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation. These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology.