- Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents
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Penicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis, immobilised as a cross-linked enzyme aggregate (CLEA), catalysed the synthesis of ampicillin in water-miscible organic solvents at low water concentrations. Below 4% water (v/v) no reaction was observed, showing the crucial role of water in maintaining the activity of penicillin acylase. The initial value of S/H was strongly affected by the nature of the solvent, but the effect of the water content was slight in the studied range of 4 to 15%. A reaction in acetonitrile containing 8% water afforded ampicillin in up to 86% yield.
- Van Langen, Luuk M.,Oosthoek, Natasja H. P.,Van Rantwijk, Fred,Sheldon, Roger A.
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- Penicillin Acylase-Catalyzed Solid-State Ampicillin Synthesis
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The ability of immobilized penicillin acylase from E. coli to retain a remarkable catalytic activity in solid-state systems has been demonstrated. Stabilization of immobilized penicillin acylase by inorganic salt hydrates allowed us to exploit nearly the whole catalytic activity of the enzyme at a very low water content. Using this technique, enzymatic synthesis of ampicillin in solid-state systems was performed with high yields (up to 70% starting from equimolar mixture of reagents) and rates comparable to the corresponding values in homogeneous solutions and heterogeneous systems, "aqueous solution-precipitate". Peculiarities of the enzymatic solid-state acyl transfer process, such as absence of the clear-cut maximum on the ampicillin accumulation curves and dependence of the synthetic efficiency on the enzyme loading, have been observed. The space-time yield of solid-state enzymatic ampicillin synthesis was shown to be up to ten times higher compared to the homogeneous solutions and heterogeneous "aqueous solution-precipitate" systems.
- Youshko,Svedas
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- Efficient enzymatic synthesis of ampicillin using mutant Penicillin G acylase with bio-based solvent glycerol
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To fulfill the industry demand of ampicillin enzymatic synthesis, immobilized mutant Penicillin G acylase and bio-based solvent glycerol were employed at high substrate concentration and low acyl donor/nucleophile ratio. After process optimization, good yield and low operation costs were achieved.
- Deng, Senwen,Ma, Xiaoqiang,Sun, Ming,Wei, Dongzhi,Su, Erzheng
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- An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase
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Penicillin G acylase (PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm, specific surface area of 128.2 m2/g and saturate magnetization of 6.4 emu/g. The 96.7% ampicillin yield with 1.60 of the synthesis/hydrolysis (S/H) ratio from 6-aminopenicillanic acid (6-APA) and D-(-)-alpha-phenylglycine methyl ester (D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol, where only 82.1% yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions. The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.
- Xue, Ping,Song, Xiao Dan,Cao, Xue Rong
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- A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases
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Research involving α/β hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/β hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a ‘hit’ or a ‘miss,’ in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/β hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.
- Paye, Mariétou F.,Rose, Harrison B.,Robbins, John M.,Yunda, Diana A.,Cho, Seonggeon,Bommarius, Andreas S.
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- IMMORTALIZED STEM CELL, COMPOSITIONS, PREPARATIONS AND USES THEREOF
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The purpose of the present invention is to provide immortalized stem cells, which produce a growth factor capable of regenerating various kinds of tissues that have been damaged by a variety of causes, and a method for producing the aforesaid immortalized stem cells. Another purpose is to provide a medicinal composition and a medicinal preparation for restoring damaged tissues, and a method for the percutaneous absorption of a culture supernatant. Provided are immortalized stem cells that are obtained by isolating stem cells selected from the group consisting of mammalian mesenchymal cells, an embryo at the early stage of the development and somatic cells, first culturing the cells to give first stage culture cells, transferring four kinds of genes into the first stage culture cells to give transgenic cells, and selecting the desired immortalized stem cells from among the transgenic cells using the expression of STRO-1 as an index.
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- Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin
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α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of kcat of 72.5 s-1 and KM of 1.1 mM. The synthesis parameters α, βo, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, βo = 42.8 M-1, and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.
- Blum, Janna K.,Bommarius, Andreas S.
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experimental part
p. 21 - 28
(2010/12/19)
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- Apparatus And Methods For Delivering A Plurality Of Medicaments For Management Of Co-Morbid Diseases, Illnesses Or Conditions
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A method and apparatus for delivering a plurality of medicaments in a single delivery vehicle for the management of co-morbid diseases, illnesses and conditions. The present invention provides a novel delivery process for many medicaments. Medicaments may be encapsulated and stored separately within a larger capsule until the time of ingestion, consumption, or the like. Benefits of the present invention include maintaining separation of distinct ingredients within a single capsule and the capability to control the time release of multiple ingredients within the capsule.
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- Combinatorial improvement of bifunctional drug properties
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A method is provided for improving at least one pharmacokinetic property and maintaining or improving affinity of a therapeutic upon administration to a host. In the method, one administers to the host an effective amount of a bifunctional compound of less than about 5000 Daltons comprising the therapeutic or an active derivative, fragment or analog thereof and a recruiter ligand moiety. The recruiter ligand moiety binds to at least one biomoiety. The bifunctional compound has at least one modulated pharmacokinetic property upon administration to the host and equivalent or greater affinity for a target of the therapeutic as compared to a free drug control that comprises the therapeutic. In addition, the overall drug efficacy is improved by the steric bulk of the bifunctional complexed with the recruited biomoiety.
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- Method of using deuterated calcium channel blockers
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Therapeutic methods and compositions using deuterated enriched 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid 3-ethyl 5-methyl ester and other deuterated dihydropyridine compounds are described. The deuterated compounds exhibit enhanced efficacy in blocking calcium channels over non-deuterated dihydropyridines.
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- Chemical reactivity of penicillins and cephalosporins. Intramolecular involvement of the acyl-amido side chain
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The rate of degradation of 6-epi-ampicillin in acidic, neutral, and alkaline aqueous solutions was followed at 35 °C and an ionic strength of 0.5 mol dm-3 (KCl) by high-performance liquid chromatography (HPLC) and spectrophotometric assays. Pseudo-first-order rate constants were determined in a variety of buffer solutions, and the overall pH-rate profile was obtained by extrapolation to zero buffer concentration. The hydrolysis of 6- epi-ampicillin is subject to acid and hydroxide-ion catalysis and, for a penicillin, an unusual pH-independent reaction. Intramolecular general base- catalyzed hydrolysis by the side chain amido group is proposed to explain the enhanced rate of neutral hydrolysis of 6-epi-ampicillin and cephalosporins. The β-lactam of 6-epi-ampicillin also undergoes intramolecular aminolysis by nucleophilic attack of the 6-α side chain amino group to give a stable piperazine-2,5-dione derivative. The low effective molarity for intramolecular aminolysis of only 40 M is partly attributed to the unfavorable trans to cis isomerization about the 6-amide side chain required for ring closure. Theoretical calculations show that the intramolecular aminolysis of 6-epi-ampicillin nucleophilic attack occurs from the α-face of the β-lactam ring with an activation energy of 14.4 kcal/mol.
- Llinas, Antonio,Vilanova, Bartolome,Frau, Juan,Munoz, Francisco,Donoso, Josefa,Page, Michael I.
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p. 9052 - 9060
(2007/10/03)
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- Penicillin acylase in the industrial production of β-lactam antibiotics
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Immobilized penicillin acylase is a biocatalyst suitable for the kinetically controlled industrial synthesis of semi-synthetic antibiotics in aqueous environments. Amoxiciliin and ampicillin are obtained by condensing 6-aminopenicillanic acid with the amide or ester of D-( - )-4-hydroxyphenyIglycine and D-( - )phenylglycine, respectively. Similarly, the cephalosporin antibiotics cefadroxil and cephalexin can be obtained from 7-aminodesacetoxycephalosporanic acid.
- Brugging, Alle,Roos, Eric C.,De Vroom, Erik
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p. 128 - 133
(2013/09/08)
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- Enhancement of the efficacy of nifedipine by deuteration
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A method of enhancing the efficiency and increasing the duration of action of drugs (e.g. dihydropyridines and anti-bacterials) and particularly of nifedipine and penicillins wherein one or more hydrogen atoms are deuterated and wherein the deuterated drug has unexpectedly improved properties when used in much lower concentrations than unmodified drug. A method for determining the identity and bioequivalency of a new drug is also disclosed wherein the molecular and isotope structure of a new drug is determined by isotope ratio mass spectrometry and compared with the molecular and isotope structure of a known human drug.
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- Ester prodrugs of ampicillin tailored for intracellular accumulation
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Seven new ester prodrugs of ampicillin with hydrolysis half-lives ranging from 65 to 308 min were synthesized. The cellular accumulation of two of them (in J774 mouse macrophages) and their activities against intracellular Staphylococcus aureus were determined in comparison with the pivaloyloxymethylester of ampicillin (pivampicillin) and ampicillin. The esters accumulated extensively and were more active than ampicillin in this in vitro system.
- Fan,Paternotte,Vermander,Li,Beaujean,Scorneaux,Dumont,Osinski,Claesen,Tulkens,Sonveaux
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p. 3107 - 3112
(2007/10/03)
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- Prodrug derivatives of carboxylic acid drugs
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Novel ester derivatives of carboxylic acid medicaments of formula (I), wherein R--COO--represents the acyloxy residue of a carboxylic acid drug or medicament, n is an integrer from 1 to 3, and R1 and R2 are the same or different and are selected from a group consisting of an alkyl, an alkenyl, an aryl, an aralkyl, a cycloalkyl and which group may be unsubstituted or substituted, or R1 and R2 together with the N forms a 4-, 5-, 6- or 7-membered heterocyclic ring, which in addition to the nitrogen atom may contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur and which heterocyclic group may be substituted. These compounds are highly biolabile prodrug forms of the corresponding carboxylic acid compounds and are highly susceptible to undergoing enzymatic hydrolysis in vivo whereas they are highly stable in aqueous solution. The novel derivatives are less irritating to mucosa than the parent carboxylic acids and may provide an improved bio-availability of the drugs.
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- Extremely Selective and Mild Cleavage of β-Haloalkyl Groups by Cobalt(I)phthalocyanine Anion in Semisyntheses of β-Lactam Antibiotics
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Cleavage of β-haloalkyl groups can be performed extremely selectively by cobalt(I)phthalocyanine anion under very mild conditions, qualifying it best in the chemistry of highly sensitive β-lactam antibiotics.This is demonstrated in penicillin and cephalosporin semisyntheses.
- Eckert, Heiner
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p. 1715 - 1724
(2007/10/02)
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- Pharmaceutical tablet, pharmaceutical granulate and process for their preparation
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A pharmaceutical tablet containing an amphoteric beta-lactam antibiotic, microcrystalline or micro fine cellulose or a mixture of both and a second disintegrant, being low-substituted hydroxypropylcellulose, which fully disintegrates in water within 60 seconds. When swallowed it shows a bioavailability as good as a pharmacy prepared suspension of the antibiotic. The tablet is compressed from a mixture containing a new granulate which is prepared from the antibiotic substance, microcrystalline and/or micro fine cellulose and water only. Such tablets can also be prepared by using other known tablet disintegrants as the second disintegrant.
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- A novel senile amyloid precursor protein and an antibody specific for the same
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A protein comprising a specific amino acid sequence including at least 55 amino acids and having a protease inhibiting activity is disclosed. Further, an antibody specific for the protein, a DNA coding for the protein, a complementary DNA to the DNA and RNA's corresponding to these DNA's, and human chromosomal DNA fragment coding for the protein are also disclosed. The protein, antibody, DNA RNA and human chromosomal DNA fragment of the present invention are useful for diagnosis of dysbolism in the central nervous system, including senile dementia Alzheimer's type.
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- β-Lactam antibiotic esterification process using methoxymethyl methane sulfonate
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Disclosed is an improved process for esterifying carboxylic acids, particularly 3-carboxylic acid groups of penicillins and 4-carboxylic acid groups of cephalosporins, to form methoxymethyl esters. Replacement according to the present process of the conventional halomethyl methyl ether esterifying agent with methoxymethyl mesylate avoids the carcinogenicity problem of the prior art reagent while still giving good yields of high quality product.
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- Protection of functional groups during reaction and their subsequent restoration
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In the process for preparing an organic compound of the formula in which X is an amino group, a hydroxyl group or a carboxyl group, and A' is the remainder of the molecule, from an organic compound of the formula in which A is the remainder of the molecule which can undergo reaction to form A', by converting A -- X into a compound of the formula in which Z is --NH--, --O-- or a direct C--C bond, and R is a radical of the formula STR1 IN WHICH Y is a direct C--C single bond, the --CH=CH-- group or an arylene group, R1 to R4 each independently is hydrogen, halogen or an alkyl, aryl, aralkyl, alkoxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl or cycloalkylaminocarbonyl radical, or R1 + r2 and R3 + R4 each independently completes a 5- or 6-membered carbocyclic ring, or R1 and R3 conjointly with the grouping --C--Y--C-- forms a carbocyclic ring with 5 or 6 carbon atoms, and Hal is halogen, Thereby to protect X, then converting A -- Z -- COOR into a compound of the formula and then treating the compound A' -- Z -- COOR to restore the group X, the improvement which comprises effecting the treatment of the compound A' -- Z -- COOR with an alkali metal compound of a complex of monovalent cobalt. The process is applicable particularly to aminocarboxylic acids including intermediates from various stages of the synthesis of penicillins and cephalosporins.
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- Process for preparation of alkali metal salts of ampicillin
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A process for the preparation of an alkali metal salt of ampicillin, especially the sodium salt, which comprises adding to an aqueous suspension of ampicillin, at a temperature not exceeding about 4° C, an aqueous solution of an equimolar or somewhat lesser amount of an alkali metal base, filtering the resulting solution until sterile, freezing the filtrate, and lyophilizing the frozen filtrate.
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- Intermediates for producing semi-synthetic cephalosporins
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Novel 6-APA, 7-ACA and 7-ADCA derivatives are described which comprise phosphorylated derivatives of 6-APA, 7-ACA, or 7-ADCA and the corresponding acylated derivatives thereof. The novel compounds are prepared by the reaction of 6-APA, 7-ACA, 7-ADCA or a salt thereof with a phosphorus halide in the presence of an acid acceptor and subsequently acylating the thus formed phosphorylated compound, to form a phosphorylated acylated derivative which upon hydrolysis with water splits off the protective group(s) to provide the corresponding semi-synthetic penicillin or cephalosporin having useful antibacterial activity.
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