Tryptophyl groups

Glazer and Smith (1960, 1961a) point out that there is no correlation between the content of tyrosyl and tryptophyl groups of the several proteins they studied, and the magnitude of the 2300-2350 A denaturation... 

Lyophilized porcine pancreatic elastase exhibited denaturation during storage at 40°C and 75% relative humidity in which oxidation of tryptophyl groups was probably involved.823 Reducing sugar impurities in mannitol used as an excipient824 induced solid-state oxidative degradation of a cyclic heptapeptide. 

The difference spectrophotometric technique (38) has been applied to P. notatum cellulase at pH 1.9 and 2.4 relative to native cellulase. Only a minor acid difference spectrum was obtained, while after heat denatura-tion a much more pronounced spectrum was observed. It was possible to evaluate from these measurements that a striking structural change has occurred in the environment of the tryptophyl groups upon heat denaturation. It may be concluded from these data that tryptophyl groups are buried in the interior of the native protein. 

Investigation of the Tryptophyl Groups. The structure and function of the cellulase from P. notatum has been studied by chemical modification and solvent accessibility (37). 

Figure 18. Experimental (solid circles) and calculated (solid line) energy-transfer efficiency from excited tryptophyl groups in the tetramer of avidin to the pyrenylalanine-linked biotin bound to the avidin tetramer.

Figure 19. Three-dimensional arrangement of tryptophyl groups in avidin tetramer complexed with a single pyrenylalanine-linked biotin.

Some physical and functional properties of casein modified by the covalent attachment of amino acids are given in Table IX. Despite extensive modification, the relative viscosities of 2% solutions of the modified proteins did not change significantly, with the exception of aspartyl casein which was more viscous. There was some decrease in the solubilities of aspartyl casein and tryptophyl casein as compared with the casein control. It is anticipated that adding some 11.4 tryptophyl residues per mole of casein would decrease the aqueous solubility of the modified protein. However the results with aspartyl casein are unexpected. The changes in viscosity, solubility, and fluorescence indicate that aspartyl casein is likely to be a more extended molecule than the casein control. There was a marked decrease in the fluorescence of aspartyl casein and tryptophyl casein (see Table IX). The ratios of the fluorescences of acetylmethionyl casein to methionyl casein and t-BOC-tryptophyl casein to tryptophan casein were 1.20 and 2.01, respectively, indicating the major effects that these acyl groups have on the structure of the casein. 

Modification of tryptophan residues. Various indole derivatives are reduced to 2,3-dihydro compounds by borane-pyridine in ethanolic 20% hydrochloric acid. This reduction fails completely with tryptophan itself and gives only low yields (15-55%,) with N-acyltryptophans. However, use of CF3COOH as solvent leads to the desired 2,3-dihydro compounds in yields of 85-95%. This is a useful method for selective modification of tryptophyl residues in peptides. The imidazole group is partially decomposed under these conditions, but other groups encountered in peptides are stable. 

Figure 11 shows the result of this experiment on a solution of 5 mM N-acetyl tryptophan and 0.2 mM 3-N-carboxy-methyl lumiflavin, hereafter simply called flavin (see Figure 10). Positive enhancements can be observed for the aromatic C-2, C-4 and C-6 protons, while the CH2 group shows emission. This polarization pattern corresponds with a tryptophyl radical in which the electron spin is delocalized over the aromatic ring. It can further be noted that almost no flavin polarization is present in the difference spectrum. Figure 11c (weak lines are present at 2.6 and 4.0 ppm). This is due to cancellation of recombination and escape polarization as will be discussed in Section 5. The mechanism of the photoreaction undoubtedly involves triplet flavin (17). Since 1-N-methyl tryptophan shows similar CIDNP effects, the primary step most probably is electron transfer to the photo-excited flavin. This is also supported by a flash photolysis study by Heelis and Phillips (18). The nature of the primary step in the photoreactions with amino acids is important in view of the interpretation of "accessibility" of an amino acid side chain in a protein as seen by the photo-CIDNP method. This question is therefore the subject of further study.

In the previous examples, Martin and his coworkers utilized the intramolecular Diels-Alder reaction to construct the DE-ring system of the penta-cyclic targets and then, following the strategy of Woodward, introduced the tryptophyl moiety followed by C-ring closure. The Martin group has synthesized other members of the yohimbine family by routes which do not adhere... 

In this reserpine synthetic route (Scheme 3.21), Diels-Alder reaction of dihydropyridine 133 (100) and methyl a-acetoxyacrylate 134 afforded iso-quinuclidenes 135 and 136 (36). Addition of lithium r-butyl acetate to 135 afforded the )8-ketoester 137 which was converted to the corresponding enol ester 138. When 138 was refluxed in xylenes, the cis-fused hexahydroiso-quinoline 139, in which the reserpine C(15), C(16), and C(20) stereocenters are in place, formed in good yield. Hydrogenation of the enamine function in 139 followed by reduction of the enol ester afforded 140 which has the C(17)-methoxy group with the required relative stereochemistry. Acylation of ketone 140 yielded the C(18), C(19) enol ester which was hydrogenated stereoselectively to produce 141 in which the C(18) stereochemistry is set. Deprotection of the nitrogen function followed by tryptophylation afforded 142 which was then cyclized to provide the anticipated isoreserpine diol 143... 

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