Amino acids solution preparation

Charge a burette with the deprotonated amino acid solution prepared in step 3. This is your titrant. 

Unlabeled amino acid solution (10 x stock) See Table 3.2.6. These amino acids, which are included in the natural L-amino acid kit (ICN Biomedical, 100586), except for the two alkaline amino acids (cystine and tyrosine), are mixed into a 100-ml volumetric flask. Then, 10 ml of L-glutamine (200 mM, Sigma G7513 must be in solution before use) and 50 ml water are added and the solution stirred until all components are dissolved. For L-cystine and L-tyrosine, the indicated amount is added to a 10-ml volumetric flask and the pH adjusted to > 8.0 by adding 2N NaOH drop by drop until the amino acids are dissolved. Then water is added to the 10-ml mark and the solution transferred to a 100-ml flask to complete the solution. Aliquots of 10 ml are prepared using 15-ml conical screw-top tubes and stored at -80°C. 

Freshly made amino acid solutions are recommended for peptide synthesis, although stock solutions of /V -Fmoc-AAs (A-fluorenylmethoxycar-bonyl a-amino acids) with HOBt in DMF (/V,/V-dimethylformamide) can be stored at 4° for a week. Since the synthesis of a hexapeptide or octapep-tide library usually takes only 2-3 days, all the amino acid solutions may be prepared before the synthesis. A 3-fold excess of Aa-Fmoc-AAs is needed in each couphng step to ensure completion of the coupling reaction. We have been using the following equations to calculate the amount of necessary reagents. 

Preparation of the activated amino acid solutions can be performed according to two different methods. 

One method involves the use of preactivated Fmoc-protected amino acids (e.g., pentafluorophenyl ester). This method has the advantage that only one reagent is necessary since preparation of the amino acid solutions is very simple and the likelihood of mistakes is low. One disadvantage is the higher price of the amino acid derivatives, but due to the small amount of activated amino acids used the absolute difference falls in the range of a few dollars for synthesis of an entire peptide membrane array. Another disadvantage lies in the fact that activated esters are only commercially available for the standard amino acids. 

Prepare a 0.9 M solution of HOBt in amine-free NMP. Dissolve the Fmoc-amino acids or desired protected building blocks with the HOBt-solution to a concentration of 0.45 M. Except for the arginine derivatives, these solutions can be stored at -20°C for at least a week. Each day, prepare a 20% mixture of DIC in amine-free NMP. Use the fresh amino acid/HOBt solution every day and discard the previous one. To these solutions add 20% DIC/NMP at a ratio of 3 1 (e.g., 75 pL amino acid solution and 25 p,L DIC mixture prepared fresh every day) (see Note 3). 

Coupling of activated amino acids The stepwise buildup of peptides starts from the C-terminus. Deliver the prepared corresponding activated amino acid solutions to the corresponding positions on the membrane with the desired volume. Repeat the spotting after 20 min (see Note 7). 

TMS derivatives of amino acids were also combined with other procedures and some difficulties were thus avoided. N-TMS-methyl and -ethyl esters of most protein amino acids were prepared by the action of TMSDEA on alkyl esters of amino acids and were chromatographed on methylsilicone stationary phases [246], Their retention times were found to be 15—20% lower than those of the corresponding TMS derivatives. Despite having an additional step in comparison with direct silylation, the procedure was applied by Hardy and Kerrin [259] to the GC analysis of twenty protein amino acids, including Hypro and CysH. Amino acids were esterified with a 3 N HC1 solution in n-butanol at 150°C for 15 min with subsequent silylation with BSTFA for 90 min at the same temperature. Acetonitrile and methylene chloride were used as solvents for the silylation. In the former solvent double derivatives of Gly and Lys (bis- and tris-) were produced, whereas in the latter the less silylated form only was produced. As Arg, in contrast to direct silylation, also leads to one peak in this instance, methylene chloride is recommended as the silylation solvent. The separation of all twenty amino acids was achieved on a simple column with 2% of OV-7 on GLC-110 textured glass beads (100—120 mesh). 

The symmetric anhydrides of BOC-amino acids were prepared by adding 1.565 mL (10 mmol) DIC to a stirred solution of 15 mmol BOC-amino acids in 10 mL DCM. The solution was then added to H-Gly-Leu-Leu-aminomethylpoly-styrene resin previously derivatized with the linker 7. Solid DMAP (approx 50 mg) was then added to the solution and shaking continued for 3 h. The resin was then filtered and washed with DMF (4x12 mL) and then DCM (4x12 mL). 

Diazotization of 2-aminopyrazine with nitrous acid in dilute or concentrated sulfuric acid gave 2 hydroxypyrazine (to 67% yield) (86, 477, 720, 818). Many such conversions have been described, mostly using nitrosylsulfuric acid in concentrated sulfuric acid solution. Preparations of hydroxypyrazines from the aminopyrazines are summarized as follows 2-hydroxy-3-methylpyrazine (sodium nitrite in concentrated sulfuric acid-acetic acid) (681), 2Jiydroxy-3,5-dimethylpyrazine (aqueous nitrous acid, then at 60°) (524), 3-hydroxy-2,5-dimethylpyrazine (477), 2,5-diethyl-3-hydroxypyrazine (aqueous nitrous acid) (478), 2-hydroxy-6-phenyl-pyrazine (365a), 24iydroxy-3,5-diphenylpyrazine (nitrous acid in N hydrochloric acid) (524), 3-hydroxy-2,5-diphenylpyrazine (282), 2-s-butyl-3-hydroxy-5-isobutyl-pyrazine (93), 5TS-butyl-3 hydroxy-2-isobutylpyrazine (92, 536), 2,5-di-s-butyl-3-hydroxypyrazine (89, 720), 3-hydroxy-2-isobutyl-5-isopropylpyrazine (103, 525), 2,3-dihydroxypyrazine (from 2 amino-3-hydroxypyrazine) (757, 1055) and its... 

Membrane-controUed devices, responsive to the concentration of external amines and amino acids, are prepared by polymerizing 2-hydroxy-dependent ethylmethacrylate and then reacting the polymer with 3,5-dinitrobenzenoylchloride to attach 3,5-di-nitrobenzoate groups to the polymer [48]. Upon addition of amine substances to the external solution a charge-transfer complex forms, which increases the permeabihty of the membrane. 

Since the introduction of parenteral nutrition in hospital care the potential microbiological risks associated with the manufacture, preparation, and administration of these products have abated but not disappeared (133,152). Fatal infectious complications still occur. The parenteral nutrition mixture is a good growth medium for microorganisms, more conducive to microbial growth than glucose or amino acid solutions. Storage of mixtures... 

Fig. 20. Apparatus used for preparation of derivatives. Amino acid solution is placed in tube A and propanol-HCl in the funnel. Dry nitrogen is passed through the apparatus as required. Reproduced from Coulter and Hann (Cll) with permission.

Procedure The apparatus shown in Fig. 20 is used for esterification. About 0.1 ml of amino acid solution or protein hydrolyzate is placed in tube A (Quickfit MF 24/0, capacity ca. 2 ml) and quickly dried in a vigorous stream of dry nitrogen (dried by passing through a molecular sieve grade 4A, Union Carbide). A convenient amount for preparation of standards is 10 mole of each amino acid but 10 to 10" mole is satisfactory for unknowns. After drying, the nitrogen is shut off ca. 0.4... 

A suitable aliquot part of amino acid solution is evaporated to dryness in a 2-dram vial. Then 100 /il of reagent mixture (prepared by mixing 5 ml of tetrahydrofuran, 2 ml of BSA, and 5 drops of chlorotrimethyl-silane in a 2-dram vial with a foil-lined screw cap) is added. The vial is sealed with a foil-lined screw cap and warmed on the front edge of a water bath (in a fume cupboard) for 1 minute. Appropriate aliquot parts of this mixture are then injected into the gas chromatograph. 

Fmoc amino acids required to synthesize desired peptide. Prepare 200 mM amino acid solutions by dissolving 20 mmol Fmoc-protected amino acids in DMF to final volume of lOOmL. 

Several vitamins are known to be photolabile, and the photochemical stability of these compounds is influenced by TPN composition. The photochemical stability depends on composition of the amino acid solutions as well as the presence of lipids in the preparations (i.e., the formation of emulsions). Photochemical decomposition of the hpophihc vitamin A is reduced in admixtures containing lipids, possibly due to diffusion of the vitamin into the lipophilic phase. On the other hand, the hydrophilic vitamin riboflavin is protected by emulsification, probably because the opaque emulsion will reduce the optical transmission of the preparation to some extent (Smith et al., 1988). However, emulsification protects neither the water-soluble vitamin C nor the lipohilic vitamins A and K1 from photochemical degradation, which illustrates the complexity of photochemical reactions in heterogeneous media (Smith et. al., 1988 Billionrey et al., 1993). 

Food industry and medical supplies Edible salt production from seawater,23 demineralization of whey,24 recovery of amino acids from fermentation liquor,25 separation of amino acids,26 preparation of lactic acid27, gluconic acid28 amino acids,29 etc. from their salts, stabilization of grape juice30 and pre-treatment of wine,31 deacidification of sour orange juice,32 desalination of soups, desalination of soybean sauce,33 continuous fermentation in the presence of electrodialysis,34 de-ionization of sugar solution.35... 

The coupling reaction in peptide synthesis involves the reaction of an activated amino acid with a free amino group on the solid support. Activated amino acids are generally unstable in solution and must be prepared just before delivery to the solid support. The preparation of the activated amino acid solution ranges from simply dissolving a solid preactivated derivative to on-line reaction, isolation, and dissolution of the activated derivative. 

Few medicines based on boron are known, in general boric acid or a boronic acid serve to esterify an a-diol or an o-diphenol. This is the case for the emetic antimony borotartrates of the ancient pharmacopoeias, for the injectable catecholamine solutions, for tolboxane, which is close to meprobamate and which was commercially available as a tranquillizer some decades ago, and also for the phenylboronic esters of chloramphenicol. Boro-mycine was the first natural product containing boron. It is a complex between boric acid and a polyhydroxylated tetradentate macrocycle. Some boronic analogues of amino acids were prepared as chymotrypsine and elastase inhibitors. The most important medical use of derivatives of boron derivatives is the treatment of some tumours by neutron capture therapy, " the problem here being to ensure a sufficient concentration of the product in the tumour being treated. 

Use the software to calculate the amounts and volumes of amino acid solutions required. Weigh out the amino acids for the current day s work in labelled tubes and dissolve them in the required volume of DMF. Prepare the working volumes of TBTU and NMM solutions these should be stable for the duration of the synthesis. 

The commercial amino acid preparations do not contain carbohydrates and amino acids in the right proportions needed in infant nutrition. Mixing of amino acid solutions and carbohydrate preparations prior to use is therefore necessary. The electrolyte patterns are also far from ideal, necessitating further additions. The registered preparations do not, of course, contain phosphorus or calcium in significant amounts, since they are primarily designed for use in adult patients. 

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