Tryptophan and derivatives

Little or no tryptophan will usually be found in protein hydrolysates prepared with acid in the usual manner. Mercaptans added to the 6 N HCl usually increase the yields of tryptophan (Matsubara and Sasaki 1969), and substitution of p-toluenesulfonic acid (with added 3-(2-aminoethyl) indole) in place of the HCl has been used to good advantage (Liu and Chang 1971). 

At the present time the hydrolytic methods of choice for tryptophan analyses appear to be with methanesulfonic acid in the presence of added 3-(2-aminoethyl) indole (see Moore 1972) or with NaOH in the presence of starch (Hugh and Moore 1972). Both methods will be described below, but it should be noted here that hydrolysis with NaOH has the advantage that carbohydrate in the protein sample does not affect the yields of tryptophan as it does in the acid hydrolysates. Other methods of tryptophan analysis involving spectrophotometry (Goodwin and Morton 1946 Edelhock 1967), titration with N-bromo-succinimide (Spande and Witkop 1967), or the formation of colored derivatives (Spies and Chambers 1948 Barman and Koshland 1967  

Spies 1967 ScofTone et al. 1968) may be used to give initial estimates or verification of tryptophan content. It should also be possible to obtain good tryptophan analyses after complete enzymic hydrolysis ( 2.11). 

Hydrolysis of proteins with methanesulfonic acid is carried out in the usual type of hydrolysis tube with 4 N methanesulfonic acid, containing 0.2% 3-(2-aminoethyl) indole, at 115°C for 24 hr (Liu see Moore 1972), The acid mixture is available in sealed vials from Pierce Chemical Corp. The usual precautions to remove oxygen should be followed. Since the acid is not volatile, it is necessary partially to neutralize the cooled hydrolysate with an equal volume of 3.5 N NaOH and dilute with water to a volume 5 times the volume of acid used for hydrolysis. The procedure recommends the use of 1 ml of the methanesulfonic acid mixture (for about 2-5 mg of protein), which would result in 5 ml of diluted hydrolysate. Since 1-ml aliquots are analyzed (see below), only 20 % of the protein hydrolyzed is utilized for each analysis. However, where the amount of protein available for hydrolysis is limited, it should be possible to scale down the amounts of materials used to levels such as 0.2 ml of the acid mixture for 0.5 mg of protein if appropriately smaller hydrolysis tubes are used. 

Aliquots of 1.0 ml of the diluted hydrolysates are analyzed on the short column (Beckman analyzer), eluted at 50 ml per hour with a buffer prepared by diluting the usual pH 5.3 buffer to give a final Na  

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McLaren, J. A., The reaction of tryptophan and derivatives with some 1,3-dicarbonyl compounds, Aust. ]. Chem., 30, 2045, 1977. 

Scheme 5 One-step IV-tert-prenylation of tryptophan and derivatives, enzyme-catalysed (CdpNPT cyclic dipeptide iV-prenyl transferase from Aspergillus fumigatus) [41] and Pd-catalysed [42], compared with an earlier pathway...

Many biologically active polypeptides have been shown to possess CD bands in the wavelength region above 270 nm. In peptide hormones and antibiotics and in non-conjugate proteins these bands are due to tryptophan and tyrosine residues, the only chromophores which absorb in this spectral region. Because of the weakness and spectral overlap of these Cotton effects, their structure is usually imperfectly resolved. However, in recent years some progress in analyzing the fine structure of the CD spectra of tryptophan and derivatives has been reported (7 9). 

The first objective was the conversion of L-tryptophan into a derivative that could be converted to pyrroloindoline 3, possessing a cis ring fusion and a syn relationship of the carboxyl and hydroxyl groups. This was achieved by the conversions shown in Scheme 1. A critical step was e. Of many variants tried, the use of the trityl group on the NH2 of tryptophan and the t-butyl group on the carboxyl resulted in stereospecific oxidative cyclization to afford 3 of the desired cis-syn stereochemistry in good yield. 

The ethyl ester of the tetrahydrocarbolinecarboxylic acid derived from dZ-tryptophan and acetaldehyde has been resolved into its four stereoisomers. ... 

The reactions of tryptophan and of tryptamine derivatives with formaldehyde require special comment. Whereas tryptamine and its 5- and 7-methoxy and i id-A-methyl and -ethyl derivatives react... 

The second line of circumstantial evidence quoted in support of this hypothesis is the ready formation of l,2,3,4-tetrahydro-/3-carboline derivatives under pseudo-physiological conditions of temperature, pH, and concentration. Tryptamine and aldehydes, trypt-amine and a-keto acids, and tryptophan and aldehydes condense at room temperature in a Pictet-Spengler type intramolecular Mannich reaction in the pH range 5.2-8.0 (cf. Section III, A, 1, a). It was argued that experiments of this type serve as models for biochemical reactions and may be used in evidence. 

The optically active 1,3-/ram-substitutcd tetrahydro-/f-carboline 3 is obtained predominantly on treatment of the imine 2, derived from L-tryptophan and isovaleraldehyde, with A-benzyl -oxycarbonyl-L-prolyl chloride145. 

Since tryptophan (and its decarboxylation product, tryptamine) serve as precursors in many synthetic and biosynthetic routes to /J-carbolincs, it is not surprising that C-1 of the /J-carbolinc ring is the most common site of substitution (as it is the only ring atom of the /J-carbolinc ring system not derived from tryptophan). Indeed, this is the only site of substitution for many /J-carboline natural products. Two examples of naturally occurring /J-carbolines substituted only at C-1 which possess antitumor activity are manzamine A and manzamine C (Fig. 2). Owing to its greater simplicity and nearly equal antitumor activity, most initial synthetic efforts were directed toward manzamine C [11,12]. 

Shikimates, which include phenylalanine, tyrosine, tryptophan, and their derivatives, are represented by many aromatic natural products, including hydroquinones found inbrownalgae such as Sargassum (Segawaand Shirahama 1987). Flavonoids are a structural class of shikimates found in plants, including isoflavonoids or neo-flavonoids, as is the y-pyrone (coumarin) core structure (Knaggs 2003). 

Unusual amino acids include a class of unnatural a-amino acids such as phenylalanine, tyrosine, alanine, tryptophan, and glycine analogs, and f)-amino acid analogs containing 1,2,3,4-tetrahydroisoquinoline, tetraline, l,2,3,4-tetrahydro-2-carboline, cyclopentane, cyclohexane, cyclohexene, bicyclo[2.2.1]heptane or heptene skeletons. Different selectors were exploited for the separation of unusual amino acids, most of the production being made by Peter and coworkers teicoplanin [41, 56, 84, 90, 93, 124, 141-144], ristocetin A [33, 94, 145, 146], and TAG [56, 147]. Enantiomeric and diastereomeric separations of cyclic -substituted a-amino acids were reported by other authors on a teicoplanin CSP [88, 89], Ester and amide derivatives of tryptophan and phenylalanine were recently analyzed on a Me-TAG CSP [58],... 

Tryptophan and its derivatives can be characterized as existing in three possible tautomers the indole form, the indolenine form (hydrogen atom at position 3), and the cyclic form. In neutral solvents only the indole form exists NMR studies indicate no evidence for the other two isomers (60JA2184). However, when dissolved in 85% phosphoric acid or TFA, N-methoxycarbonyltryptophan exists as a pyrrolo[2,3-i]indole derivative 119 (78JA5564 81T1487). Although the tricyclic compound is stable in the crystalline form, it reverts to the starting material when it dissolves in acetic acid or methanolic HCl. 

For the preparation of 3,4-dihydro-/3-carbolines the Bischler-Napieralski reaction is widely used (510R74). Since this reaction requires rather drastic conditions, A-acetyl tryptophan and its analogs yielded l-methyl-/8-carboline (harman) rather than l-methyl-3,3-dihydro-j8-carboline-3-carboxylic acid derivatives owing to accompanying decarboxylation and aromatization (41JCS153 50JA2962). 

Dopamine is the decarboxylation product of DOPA, dihydroxyphenylalanine, and is formed in a reaction catalysed by DOPA decarboxylase. This enzyme is sometimes referred to as aromatic amino acid decarboxylase, since it is relatively non-specific in its action and can catalyse decarboxylation of other aromatic amino acids, e.g. tryptophan and histidine. DOPA is itself derived by aromatic hydroxylation of tyrosine, using tetrahydrobiopterin (a pteridine derivative see Section 11.9.2) as cofactor. 

Biogenic amines arise from amino acids by decarboxylation (see p. 62). This group includes 4-aminobutyrate (y-aminobutyric acid, GABA), which is formed from glutamate and is the most important inhibitory transmitter in the CNS. The catecholamines norepinephrine and epinephrine (see B), serotonin, which is derived from tryptophan, and histamine also belong to the biogenic amine group. All of them additionally act as hormones or mediators (see p. 380). 

True alkaloids derive from amino acid and they share a heterocyclic ring with nitrogen. These alkaloids are highly reactive substances with biological activity even in low doses. All true alkaloids have a bitter taste and appear as a white solid, with the exception of nicotine which has a brown liquid. True alkaloids form water-soluble salts. Moreover, most of them are well-defined crystalline substances which unite with acids to form salts. True alkaloids may occur in plants (1) in the free state, (2) as salts and (3) as N-oxides. These alkaloids occur in a limited number of species and families, and are those compounds in which decarboxylated amino acids are condensed with a non-nitrogenous structural moiety. The primary precursors of true alkaloids are such amino acids as L-ornithine, L-lysine, L-phenylalanine/L-tyrosine, L-tryptophan and L-histidine . Examples of true alkaloids include such biologically active alkaloids as cocaine, quinine, dopamine, morphine and usambarensine (Figure 4). A fuller list of examples appears in Table 1. 

From L-tryptophan, the serotonin synthesis pathway also begins. Serotonin is 5-hydroxytryptamine. It is derived from L-tryptophan, which at first is simply hydroxylated to 5-hydroxy-L-tryptophan, and subsequently to the serotonin (Figure 39). Structurally, serotonin is also a 5-HT monoamine neurotransmitter. 

The occurrence of alanine in proteins was first shown by Schutzen-berger, who did not actually identify his product with the synthetical one Weyl in l88i obtained it as a decomposition product of silk and showed that his preparation was similar in properties to Strecker s synthetical alanine. He thus established it as a constituent of a protein molecule. The researches of Emil P ischer have shown that alanine is a constant constituent of all proteins. It is worthy of note that of the eighteen definitely determined units of a protein molecule, six of them, namely, isoleucine, phenylalanine, tyrosine, serine, histidine and tryptophane, are derivatives of a-aminopropionic acid. 

A. Ellinger und C. Flamand. tiber synthetisch gewonnenes Tryptophan and einige seiner Derivate. Zeit. physiol. Chem., 1908, 55, 8-24. 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

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