Aqueous reaction mixtures

The D- -)-o -aminobenzylpenicillin may then be recovered from the aqueous reaction mixture by concentration to small volume and recovering the product by filtration. However, due to the fact that anhydrous D- -)-a-aminobenzylpenicillin is soluble in water to the extent of about 20-25 mg/ml at 20°-25°C, it is very difficult to recover the product in high yields. Furthermore, the recovered D- -)-a-aminobenzylpenicillin may be obtained in the form of a monohydrate. The monohydrates as well as the dihydrates) of D- -)-a-amino-benzylpenicillin possess poor biological stability. 

A thermally stable NHase from Comamonas testosteroni 5-MGAM-4D (ATCC 55 744) [22] was recombinantly expressed in Escherichia coli, and the resulting transformant cells immobilized in alginate beads that were subsequently chemically cross-linked with glutaraldehyde and polyethylenimine. This immobilized cell catalyst (at 0.5 % dew per reaction volume) was added to an aqueous reaction mixture containing 32wt% 3-cyanopyridine at 25 °C, and a quantitative conversion to nicotinamide was obtained. The versatility of this catalyst system was further illustrated by a systematic study of substrates, which included... 

SATA is freely soluble in many organic solvents. In use, it is typically dissolved as a stock solution in DMSO, DMF, or methylene chloride, and then an aliquot of this solution is added to an aqueous reaction mixture containing the protein to be modified. 

M2C2H is slightly soluble in aqueous solutions, reportedly having a maximal solubility of 3.2 mg/ml in 0.1 M sodium acetate at pH 5.5. It is also soluble in organic solvents, which allows for the preparation of concentrated stock solutions to be made prior to addition of a small aliquot to an aqueous reaction mixture. The crosslinker is particularly stable in acetonitrile. 

Benzophenone-4-iodoacetamide is water-insoluble and should be pre-dissolved in DMF or another organic solvent prior to adding an aliquot to an aqueous reaction mixture. Stock solutions may be prepared and stored successfully if protected from light. 

Fluorescein-5-maleimide is slighdy soluble in aqueous solutions above pH 6 ( lmM concentration). It may be dissolved in DMF at higher concentrations and a small addition of this solution made to an aqueous reaction mixture to initiate labeling. Do not exceed 10 percent DMF in the reaction buffer to avoid protein precipitation. At pH 8, the reagent has an extinction coefficient at 490nm of about 78,000M 1 cm-1. 

TRITC is relatively insoluble in water, but it can be dissolved in DMF or DMSO as a concentrated stock solution prior to its addition to an aqueous reaction mixture. The isothiocyanate group is reasonably stable in aqueous solution for short periods, but will degrade by hydrolysis. TRITC also is more stable to photobleaching than FITC (Section 1, this chapter), and its absorption and emission spectra are less sensitive to environmental conditions, such as plT. It is best, however, to use only fresh reagent for modification purposes. Storage should be done under desiccated conditions, protected from light, and at -20°C. 

Br-BODIPY 493/503 is insoluble in aqueous reaction mixtures, but may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small amount to a buffered solution. Coupling to sulfhydryl-containing molecules is rapid, leading to the formation of a thioether linkage. The reaction may be done in 50mM sodium borate, 5mM EDTA, pH 8.3. An important consideration is to protect the iodoacetyl derivative from light which may generate iodine and reduce the reactivity of the probe. 

Biotin-BMCC is insoluble in water and must be dissolved in an organic solvent prior to addition to an aqueous reaction mixture. Preparing a concentrated stock solution in DMF or DMSO allows transfer of a small aliquot to a buffer reaction. The upper limit of biotin-BMCC solubility in DMSO is approximately 33 mM or 17 mg/ml. In DMF, it is only soluble to a level of about 7 mM (4 mg/ml). Upon addition of an organic solution of the reagent to an aqueous environment (do not exceed 10 percent organic solvent in the aqueous medium to prevent protein precipitation), biotin-BMCC may form a micro-emulsion. This is normal and during the course of the reaction, the remainder of the compound will be driven into solution as it couples or hydrolyzes. 

Modification of (strept)avidin with Texas Red sulfonyl chloride may be done similarly, except the fluorophore is first dissolved in acetonitrile prior to addition to the aqueous reaction mixture. 

Dg]-butadiene monoepoxide, 8, has been synthesized2 by treating the water solution (pH 5.5) of magnesium monoperoxyphthalate hexahydrate at room temperature with [Dg]-1,3-butadiene at 1 atmosphere in 94% yield after 50 min reaction time. Under these conditions less than 1% of butadiene diepoxide has been formed as determined by GC/MS. The concentration of the [Dg]-butadiene monoepoxide in the aqueous reaction mixture at various reaction times has been determined by selective ion monitoring of ions with mjz... 

Another crystallization technique is used when the isolation of a highly water-soluble compound in its salt form is required from aqueous reaction mixtures. This technique takes advantage of the common-ion effect and is based on the le Chatelier s principle, which states that, if, to a system in equilibrium, a stress is applied, the system will react so as to relieve the stress. Thus, in aqueous solutions, the solubility of the compound in salt form can be reduced by adding large amoimts of a common ion which is more soluble than the salt of the compoimd. 

Product (in aqueous reaction mixture) Product (in organic solvent)... 

The practical preparation of melamine-formaldehyde resins is done under the same conditions as for urea-formaldehyde resins. Melamine is at first insoluble in the aqueous reaction mixture but dissolves completely as the condensation proceeds. Because of the greater stability of the N-hydroxymethylmel-amines compared with the corresponding urea compounds the reaction can easily be followed by titration of the unconverted formaldehyde with sodium hydrogen sulfite (see Sect. 4.1.4.1). 

The product is extracted from the reaction mixture by two extractions with ether. The aqueous reaction mixture is then saturated with sodium chloride and again extracted with ether. The combined ether extracts are washed in turn with two portions of dilute acetic acid and one portion of a 10% calcium chloride solution. Finally the solution is dried over calcium chloride. Low-boiling solvents and impurities are separated by evaporation followed by cautious distillation up to 100° (30 mm Hg). The residual product weighs 213-228 gm (75-80%), m.p. 21°-22°C. 

The Solvay process depends on the fact that of all the possible combinations of ions present in the aqueous reaction mixture (NH +, Na+, Cl , HCO ) sodium bicarbonate is the least soluble and hence precipitates out first under the proper concentration conditions. The facts are summarized in the following table, which shows percentage concentrations of the salts in lOOg of a saturated aqueous solution. 

The diethyl ether extractions are carried out in the reaction vessel in the following manner. Diethyl ether (50 mL) is added by syringe to the aqueous reaction mixture and the two-phase mixture is vigorously stirred, still under argon, for a few minutes. The layers are allowed to separate and the organic layer is quickly cannulated into the nickel solution. The procedure is repeated twice. 

A water-soluble analog to SMPB, sulfosuccinimidyl-4-(p-maleimidophenyl)butyr-ate (sulfo-SMPB), contains a negatively charged sulfonate group that lends considerable hydrophilicity to the molecule (Pierce). Sulfo-SMPB may be added directly to aqueous reaction mixtures without prior dissolution in organic solvent. Concentrated stock solutions made in water should be dissolved quickly and used immediately to prevent hydrolysis of the NHS ester. 

The sulfo-NHS ester of sulfo-SBED is negatively charged and provides a degree of water solubility (about 5 mM maximum concentration) for the entire molecule. Limited water solubility is all that can be expected due to the large size of the trifunctional, most of it consisting of relatively hydrophobic structures. However, the reagent is much more soluble in organic solvents such asDMF (170 mM) andDMSO (125 mM). Concentrated stock solutions may be prepared in these solvents prior to addition of a small aliquot to an aqueous reaction mixture. 

---