An economical synthesis of famciclovir.

An economical synthesis of famciclovir from N-2-acetyl-7-benzylguanine by a novel regioselective alkylation with the diester cyclopropane compound was developed.     

An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates

Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.     

An economical wireless cavity-nest viewer

ABSTRACT.   Inspection of cavity nests and nest boxes is often required during studies of cavity-nesting birds, and fiberscopes and pole-mounted video cameras are sometimes used for such inspection. However, the cost of these systems may be prohibitive for some potential users. We describe a user-built, wireless cavity viewer that can be used to access cavities as high as 15 m and with entrance diameters as small as 2.5 cm. System components include a wireless camera, boom with near-infrared lighting system, hand-held television, and telescoping pole. The unit can be assembled in about one day with commonly available tools, and the total cost was about $850 (US). Our cavity-viewing system was used to monitor the nests of Western Bluebirds ( Sialia mexicana ) and proved to be reliable and effective. During two field seasons, we attempted to check the status of nests 928 times and were able to determine the nesting stage 901 times (97%). For those willing to assemble their own units, our cavity viewer permits safe, direct examination of cavity nests or open nests for investigators with limited budgets.     

An economical 20 litre bench-top fermenter

We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400-600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20-37 degrees C) and pH (range 7.0-7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD(600)) of 30-32, compared with OD(600) 4-6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source

In this study, we describe the development of a cost effective and highly productive cell-free protein synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3 mg/mL of protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this cell-free synthesis technique a promising alternative for more efficient protein production.     

An economical minichamber for immunohistochemical incubation

A simple and economical "slide-minichamber" method for incubating tissue sections with antisera in immunohistochemical (peroxidase-antiperoxidase) staining procedures is described. The technique requires only materials routinely used in the laboratory. The method permits prolonged incubation of tissue sections with antiserum at 4 degrees C or at room temperature, use of small quantities of antiserum, and simultaneous incubation of two tissue sections with the same small quantity of antiserum, thereby allowing use of very dilute antisera and conservation of antisera when availability is limited.     

Design and synthesis of novel carbocyclic versions of 2'-spirocyclopropyl ribonucleosides as potent anti-HCV agents

The discovery of 2'-spirocyclopropyl-ribocytidine as a potent inhibitor of RNA synthesis by NS5B (IC(50) = 7.3 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of carbocyclic versions of 2'-spiropropyl-nucleosides from cyclopentenol 6. Spirocyclopropylation of enone 7 was completed by using (2-chloroethyl)-dimethylsulfonium iodide and potassium t-butoxide to form the desired intermediate 9a. The synthesized nucleoside analogues, 18, 19, 26, and 27, were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. The synthesized cytosine nucleoside 19 showed moderate anti-HCV activity (IC(50) = 14.4 μM).     

The novel economical synthesis and antimicrobial activity of a trithiocarbonate derivative

The compound diethyl 2,2′-(thiocarbonyl-bis(sulfanediyl))-diacetate 4 belongs to the trithiocarbonate class containing a trithiocarbonate function group flanked by ethyl acetate. In this procedure, a novel economic synthesis route to obtain compound 4 is described. This compound proved to possess broad-spectrum antimicrobial activity both in vitro and in vivo, and could be used as a lead compound. It is worth mentioning that this compound has been patented [No. US 9,988,348 B1; date of patent: June 5, 2018].     

Synthesis of phosphono-pyrimidine and -purine ribonucleosides

The reaction of the lithiated species of 2', 3', 5'-tri-O-protected uridines and 6-chloropurine ribonucleoside with diethyl chlorophosphate provided the corresponding diethyl phosphonate derivatives (III, IV and VIII). The Arbuzov-reaction of 2', 3', 5'-tri-O-protected 4-chloro-2(1H)-pyrimidinone ribonucleoside with triethyl phosphite afforded the diethyl phosphonate derivative (VI).     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

An economical mobile intensive care unit.

A mobile intensive care unit is an important extension of a regional intensive treatment service. The treatment and monitoring of critically ill patients can be maintained during transfer for specialist investigation or treatment. Such units can be brought into service with low construction and running costs.     

The synthesis of chiral glycerides starting from D- and L-serine.

A method for synthesizing chiral glycerides starting from L- or D-serine is described. Optically-active serine (both enantiomers are commerically available) was transformed into glyceric acid by stereospecific diazotization. The configuration at carbon atom 2 was maintained during the reaction. The glyceric acid was then converted into optically pure isopropylideneglycerol - which is an important intermediate in the synthesis of mono-, di- and triglyderides - by esterification followed by acetalization with acetone and reduction with lithium aluminium hydride. Reaction of this intermediate with triphenylphosphine in tetrachloromethane followed by acid-catalysed hydrolysis and dehydrohalogenation provided optically-active glycidol (2,3-epoxy-1-propanol). The epoxy ring of an ester of glycidol and a fatty acid was then opened stereospecifically with retention of configuration by heating the glycidol ester in the presence of a second fatty acid and a catalyst. This yielded a chiral 1,3-diglyceride which could be converted into a chiral triglyceride.     

Biscoumarin: new class of urease inhibitors; economical synthesis and activity

A variety of biscoumarins (1-21) with variable substituents at C-11 were synthesized with an improved method and evaluated as urease inhibitors. The synthesized compounds showed varying degree of urease inhibitory activity ranging from 15.06-91.35 microM. The size and electron donating or withdrawing effects of substituents influenced the activity, which lead to the urease inhibitors.     

Chemoselective deprotection of alpha-indole and imidazole ribonucleosides

A series of 2 ',3 '-isopropylidene and 5 '-trityl-protected alpha-indole and alpha/beta-benzimidazole and imidazole ribonucleosides were deprotected with different acids. Selectivity was achieved for 5 '-versus 2 ',3 '- deprotection by using formic acid in the alpha-indole ribonucleoside series. Treatment of alpha-indole ribonucleosides with a mixture of formic acid and ether at room temperature afforded 2 ',3 '-deprotected alpha-ribonucleosides, whereas treatment of the alpha-benzimidazole ribonucleosides with the same acid afforded the 5 '-deprotected ribonucleoside without any 2 ', 3 '-deprotected products. The structures of these ribonucleosides were elucidated with 2D (NOESY, COSY, and HMQC) NMR spectroscopy.     

Structure of periodated ribonucleosides and ribonucleotides]

Periodate oxidation converted adenosine, guanosine, cytidine, and uridine into the corresponding dialdehydes. No free dialdehydes are present in solution, but several hydrated species occur (i.r. and n.m.r.). In the solid state, the dialdehydes are completely polymerized. The m.s. molecular peaks corresponding to the free daildehydes were of very low intensity. The c.d spectra of adenosine- and cytidinedialdehyde showed heavily diminished Cotton effects in comparison to the parent nucleosides. For uridine-dialdehyde, the signal intensity of the strongest transition was diminished by about one half, indicating a looser structure allowing free rotation of the base. By contrast, the c.d. signal of guanosine-dialdehyde was increased, indicating self-association of oligomers. The nucleoside-dialdehydes gave, with hydrazides, morpholine derivatives, which posses rigid, stable structures (c.d).