The Diamino Acids

The separation and estimation of the three compounds—arginine, histidine, lysine—is carried out by the method described by Kossel and Kutscher in 1900, which was slightly modified in 1903 by Kossel and Patten. It is based upon the earlier work of Drechsel, Hedin and Kossel, and depends upon the precipitation of arginine and histidine as their silver salts, and of lysine by phosphotungstic acid, and then by picric acid. 

As described by Kossel, Kutscher and Patten the methojd is as follows — 

About 25 to 50 grammes of protein are hydrolysed by boiling with 

The liquid is now neutralised with baryta, and barium nitrate is added, so long as a precipitate of barium sulphate is formed this is filtered off and washed. The filtrate is concentrated to 300 c.c. and treated with silver nitrate, as before, till a test drop gives a yellow colour with baryta when this occurs it is exactly neutralised with baryta, and from a burette small quantities of baryta are added till the silver salt of histidine is completely precipitated this is determined by taking out a drop when the precipitate has settled and testing with ammoniacal silver solution if a precipitate easily soluble in excess of ammonia be formed, when the two liquids come together, histidine is still present and more baryta water must be added, until it is completely thrown out, when it is filtered off, stirred up with water, again filtered off and washed out. 

The filtrate containing the arginine is saturated with baryta, and the precipitate of the silver salt of arginine, so obtained, is stirred up with baryta, filtered off and washed till free from nitric acid. It is then suspended in water containing sulphuric acid and decomposed with hydrogen sulphide. The filtrate and washings from the precipitate  

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By this method of extracting the esters from their hydrochlorides, neither that of tyrosine, which remains behind combined with alkali, nor those of the diamino acids, which are soluble with difficulty in ether, are obtained. This is advantageous for the subsequent process of separation, but the method has the disadvantage that the whole quantity of esters is not taken up by the ether on account of their destruction by the alkali. In order to avoid their loss, the mass of carbonate is treated with excess of hydrochloric acid and evaporated down, the potassium... 

Only in a few cases has this compound been isolated from the products of hydrolysis of proteins, since its separation is extremely laborious. It can only be effected after all the other amino acids have been removed by crystallisation and by the ester method, and after the diamino acids have been precipitated by phosphotungstic acid. From the last mother-liquors it is obtained by crystallisation, and is best identified in the form of its /8-naphthalene sulpho-derivative. 

The separation and estimation of the two main groups of amino acids can be carried out in one experiment, instead of separately as described. The protein is hydrolysed by sulphuric acid, the tyrosine, cystine and diaminotrioxydodecanic acid are removed by crystallisation, and the diamino acids are precipitated by phosphotungstic acid. From this precipitate they are obtained by decomposition with baryta, and they are then separated by means of their silver compounds by Kossel, Kustcher and Patten s method. The filtrate from the phosphotungstic acid precipitate is freed from the excess of phosphotungstic acid by means of baryta, and the solution is treated by Fischer s ester method for the monoamino acids. 

The combination of the two processes is generally only carried out when the amount of protein available is limited they require very different quantities of material thus, the diamino acids can be determined in 25 to 50 grammes of protein with considerable accuracy, whereas the monoamino acids can only be determined with fair accuracy when 250 to 500 grammes of protein can be used. On the whole, it is not... 

The presence of diamino acids in all proteins led Kossel to suppose that there was a protamine nucleus (t.e, of diamino acids) in all proteins the more recent work, especially that by Osborne and Clapp on the gliadins, where the diamino acids are present in such small amounts, though it supports the theory, yet suggests that proteins may exist in which it is not present, more especially if the view of Emil Fischer be taken that all the proteins we know, even the crystalline ones, are still mixtures of several proteins. The isolation of complexes containing only diamino acids from proteins, where they are combined together, will be the only proof of a protamine nucleus in a protein molecule. 

The synthesis of the diamino acids, in comparison with that of the monoamino acids, is very much more difficult and has only been achieved within the last few years. 

The —COOH of this D-Ala is linked to the free —NH2 of the diamino acid in another chain... 

When the mononitro-acid is nitrated, using mixed acid, a 78 per cent, yield of this acid results. It crystallises from boiling water in very small needles, and from glacial acetic acid in bunches of small, prismatic needles. Reduction in the usual way yields the diamino-acid, which separates in small, rhomb-shaped plates. The diamino-acid may be reduced to the oxide. 

Dinitrodiphenylarsinic acid (p. 175) yields the diamino-acid when reduced with hot ferrous hydroxide solution, 15 grams of the dinitro-acid giving 6 to 7 grams of the free diamine. It crystallises in pale, reddish plates, soluble in dilute hydrochloric acid, the addition of concentrated add predpitating the hydrochloride in crystalline form. If the reduction is effected with iron powder, the principal product is not the diamino-add, but an oxide,... 

Diamino-4-hydroxyphenylarsinic acid, required for this preparation, is obtained by reducing the 8 5-dinitro-aeid with sodium h.yFormic acid is allowed to react with the diamino-acid at room temperature for twenty-four hours, the formamido-acid being produced. This forms short, colourless, sp< ar-shaped needles, which commence to decompose rapidly at about 200° C., but are unmeltcd at 275° C. The acid is sparingly soluble in cold water, soluble in dilute aqueous sodium hydroxide, methyl or ethyl alcohol, insoluble in most other organic solvents, and decomposed by hychloric acid. The sodium salt has been prepared. 

Topliss reductively cyclized JV-(2-nitro-4-chlorophenyl)-JV-inethyl-2-benzo-ylbenzamide to dibenzo[b,/]-l,4-diazocine 10(67JMC642 68USP3409608). Cyclodehydration of N-(2-aminoethyl)-lV-2-(4-chlorobenzoyl)benzylben-zenesulfonamide afforded tetrahydro-2,5-benzodiazocine II (67JOC3270 70USP3496164). Cyclization of the diamino acids 12 using dicyclohexyl-carbodiimide (DCC) resulted in formation of benzodiazocines 13. Com-... 

Saunders and Sprake cyclized the amino ester and acid 14 and obtained the dibenzo[h,/]-l,4-diazocine 15. Dehydrosulfonation of 15 (R = alkyl) afforded ring-contracted products (see Section IV,C)(72JCS(P1)1964). Cycli-zation of the diamino acid 16 using DCC and triethylamine resulted in formation of 1,4-diazocinone 17. This compound was also prepared by a ring-expansion route (see Section II,B,4) (74JOC1710). 

Massa et al. cyclized pyrrolidines 19 in DCC to obtain the pyrrolidino-1,4-benzodiazocinediones 20 (81FES425). When Thompson reacted succinic anhydride with 2,3-diaminopyridine, the diamino acid 21 was obtained. This compound could be cyclodehydrated to 22 in acetic anhydride however, succinic and phthalic anhydride reacted with a diaminopyridine to afford diazocines directly (see Section II,C,1) (86JHC1545). 

Synthetic P-sulfonopeptide-based receptors 9 and 10 were prepared using the diamino acid derivative 8 (Fig. 7.6) and screened for binding against an encoded library containing ca. 25 000 tripeptides [42]. 

These transformations of amino acid derivatives increases our interest in these compounds prepared by Curtius and his pupils, and gives an impulse to their further study, especially as formaldehyde is such an important compound in the synthesis of sugars by plants, and as the diamino acids and diamines occur as products of decomposition of proteins by enzymes and bacteria, although according to our present knowledge they are not formed in nature in this manner. 

As shown in Scheme 12.95, the methylcarbamate was removed with hydrogen bromide (HBr) in acetic acid to form hydrobromide salt. When the amino hydrobromide salt was heated in acetic acid, a bromolactam was generated from the presumed aziridine intermediate and then treatment of the bromolactam with lithium azide in dimethylformamide produced a cis azidolactam, which then yielded the corresponding amine on reduction. Hydrolysis of the lactam with aqueous barium hydroxide ([Ba(OH)2] then produced the diamino acid, and addition of phosgene (COCI2) in aqueous sodium bicarbonate (NaHCOs) produced bisnorbio-tin, which was isolated as the corresponding methyl ester. 

L-Hydroxyproline has been isolated from gelatin, hemoglobin and other proteins by other procedures but few, if any, of the products were proved to be pure. L-Hydrox3rproline was first isolated by Fischer (281) by crystallization from a gelatin hydrolyzate from which glycine had been removed as its ethyl ester hydrochloride and the diamino acids as their phosphotungstates. Other workers (1, 3, 183, 729) have followed this procedure. Methods have been described for the isolation of hydroxyproline by means of its picrate (466), cadmium chloride complex (441),... 

When two cysteine residues are oxidized they become united by a disulphide (-S-S-) bond to form the diamino acid cystine. The disulphide bond is a covalent bond and is found in some proteins, e.g. insulin (page 53). It is very important since it serves to form stable cross-linkages between different polypeptide chains or different parts of the same chain. 

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