Amino compounds analysis

The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatogra phed on sephadex LH-20 in 0.001 M HCl. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val - 6.0 5.6 (calcd. 6 6). Cycll2ation in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reaaions the characterization of the final product should, of course, be more elaborate. 

LOV MOLECULAR WEIGHT MODEL COMPOUNDS. The mechanisms of radiation effects on polymers are frequently investigated by studies of low molecular weight model compounds. Analysis of the chemical reactions is much easier than with high molecular weight polymers. Thus, N-acetyl amino acids can be studied as model compounds for poly(amino acid)s and hence for proteins. 

There are several compounds that will react with amino acids to give coloured or fluorescent products and as a result can be used in qualitative or quantitative methods. Fluorimetric methods are gaining in popularity and offer some important advantages over absorption spectrophotometry for amino acid analysis. 

Problems such as diffusional limitations and the analysis of catalyst composition occur with solid-phase catalysts. Much work has been done on diffusion in bound enzymes (for reviews, see 24 and 88). In our work we used ninhydrin, which is a reagent ideal for surface analysis amino acid analysis is used wherever possible. Amine depletion as followed by ninhydrin is not exact, but some quantitative guides are obtained. Certainly synthetic catalysts must be made with bonds other than amide bonds and components other than those compounds that are detectable on the amino acid analyzer. 

Compound 16 (2.1 g, 0.48 mmol) was treated with TFA (30 mL) as described above and the product was converted into the HC1 salt, as shown in the synthesis of 1 or 9. The HC1 salt and 13 (0.79 g, 0.50 mmol) were both dissolved in DMF (50mL). After adding HODhbt (81.6mg, 0.50mmol), the soln was chilled to —10 °C, and EDC (free base, 91.5 pL, 0.50 mmol) was added slowly with stirring. The entire mixture was allowed to react for 16 h at rt. After confirming the completion of the reaction by TLC, the product was precipitated by addition of excess chilled H20, the precipitates were collected by filtration, and washed successively with H20 and Et20. The product was further purified by reprecipitation from MeOH with Et20, and dried yield 2.6 g (92%). Formation of the desired product was confirmed by amino acid analysis. 

Compound 29 (2.44 g, 1.4 mmol) was dissolved in TFA (50 mL) at —10 °C and the soln allowed to react for 1 h at rt. The soln was concentrated to a residue, which was then triturated with 5.2 M HC1 in dioxane (0.4 mL, 2.1 mmol) and the residue precipitated with Et20 to give a powder. The product was washed with Et20 and dried. The entire product and 41 (3.0 g, 1.47 mmol) were dissolved in DMF (100 mL), and HODhbt (0.24 g, 1.47 mmol) was then added. EDC (free base, 0.27 mL, 1.47 mmol) was added to the soln with stirring at —10 °C, and the mixture allowed to react at rt for 8h. The product was precipitated by adding an excess of H20, the precipitate collected by filtration, washed successively with MeOH, EtOAc, and hexane, and then dried yield 4.5 g (87%) TLC (Merck Kieselgel 60F-254) R 0.41 (CHC13/ 80% aq AcOH/TFE 6 1 1). Formation of the desired product 42 was confirmed by amino acid analysis. 

Sephadex G-25. The product 59 was further purified by semipreparative HPLC yield 2.3 mg (42%) the compound was characterized by amino acid analysis and FAB-MS. 

The bis-disulfide bridged human insulin 59 (l.Omg, 0.17pmol) in TFA (0.6mL) was treated with Me-SiCl3 (5 pL 250 equiv) in the presence of PhS(0)Ph (0.7 mg, 20 equiv) at 25 °C for 15 min. NH4F (3 mg) was added to the mixture, and the solvent was removed in under reduced pressure. The residue was dissolved in 50% AcOH (1 mL) and the soln was gel-filtered on Sephadex G-25. The product 42 was further purified by semipreparative HPLC yield 0.6mg (61%) the synthetic human insulin was characterized by amino acid analysis and FAB-MS, it exhibited identical chromatographic and biological properties as a reference compound. 

The peptide was synthesized on TentaGel S RAM resin (lg, 0.25 mmol loading). Cleavage from the resin and deprotection was performed with TFA/H20/anisole/TIS (88.5 5 5 1.5, 20 mL) for 2 h at rt and the crude product was precipitated with tBuOMe/hexane (2 1) yield of crude product 160 mg. The relatively low yield (28%) is due to reattachment as determined by amino acids analysis of the resin hydrolysate (40% reattachment). The title compound was obtained by preparative HPLC (linear gradient of 0.1% aqTFA/0.08% TFAin MeCN from 90 10 to 40 60 in 105min) yield 40mg (10%) ESI-MS (m/z) Found 2543.2. Calcd for CjcBHisil bjCb Sej 2542.2. 

For the analytical characterization of sulfated tyrosine peptides, spectroscopic methods as well as amino acid analysis and, more recently, mass spectrometry are employed. In Table 2 the spectroscopic data of tyrosine 0-sulfate are compared to those of the related sulfonic acid derivatives as possible byproducts in the chemical sulfation of the tyrosine or tyrosine peptides.[361 In the course of the synthesis of tyrosine 0-sulfate peptides and, particularly in the final deprotection step, desulfation may occur which limits the characterization of the final compounds in terms of quantitative identification of the tyrosine 0-sulfate. This is achieved by amino acid analyses of basic hydrolysates of the sulfated tyrosine peptides or preferably by analyses of the enzymatic hydrolysates with aminopeptidase M or leucine-aminopeptidase. 

Azetidine-2-carboxylic acid (2) like proline gives an intense blue color with sodium nitroprusside in 10% acetaldehyde solution in the presence of sodium carbonate. 98,99 Upon usual acid hydrolysis (6M HC1, 110 °C, 24 h or more) as required for amino acid analysis, azetidine-2-carboxylic acid is completely decomposed, yielding mainly homoserine lactone, as well as other ninhydrin-positive compounds. 87,89,99 To enable an accurate quantification of this imino acid, azetidine-2-carboxylic acid peptides should be hydrolyzed by alkali (5M barium hydroxide, at 100 °C for 18 h 89 or 2 M sodium hydroxide at 110°C for 22h 100 ). There are extensive NMR spectroscopic data available 100-104 and the absolute configurations of A-acetyl-L-azetidine-2-carboxylic acid 105 and A-terf-butoxycarbonyl-L-azetidine-2-car-boxylic acid 106 have been determined by X-ray analysis. 

V Barkhold, AL Jensen. Amino acid analysis determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal Biochem 177 318-322, 1989. 

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