Tryptophan, dimethylallylation

The ergoline alkaloids are derived from L-tryptophan, dimethylallyl pyrophosphate (D 6) and the methyl group of L-methionine. 

Heinstein PF, Lee SI, Floss HG (1971) Isolation of dimethylallylpyrophosphate tryptophan dimethylallyl transferase from the ergot fungus Claviceps spec.). Biochem Biophys Res Commun 44 1244-1251... 

The enzyme responsible for condensation of L-tryptophan and DMAPP is dimethylallylpyrophosphate L-tryptophan dimethylallyl transferase (DMAT synthase). It has been isolated by the group of Floss and these investigators also purified it to homogeneity and performed the first characterisations (Heinstein et al., 1971 Lee et ai, 1976). Initially, the enzyme was reported to be a monomeric... 

Ergot Alkaloids.—4-(yy-Dimethylallyl)tryptophan (122) is the first intermediate beyond tryptophan in ergot alkaloid biosynthesis. Chanoclavine-I (127) is the first tricyclic base (cf. Vol. 10, p. 26, and ref. 2). Recently, (124 labels as shown) has been found to be a very efficient and intact precursor for elymoclavine (128).45 The high level of incorporation indicates that (123) is a probable intermediate situated between (122) and (127). The decarboxylation product (125) was not utilized for biosynthesis, so, although decarboxylation of (123) is required for the conversion of (123) into (127), either it is intimately associated with ring-closure or an imine that is related to (126) is involved. 

Shibuya, M., Chou, H.-M., Fountoulakis, M., Hassam, S., Kim, S.-U., Kobayashi, K., Otsuka, H., Rogalska, E., Cassady, J.M. and Floss, H.G. (1990) Stereochemistry of the isoprenylation of tryptophan catalysed by 4-( Y, Y-dimethylallyl)tryptophan synthase from Claviceps, the first pathway-specific enzyme in ergot alkaloid biosynthesis. /. Am. Chem. Soc., 112, 297-304. 

The biosynthetic pathway to the ergoline nucleus proceeds through 4-dimethylallyl tryptophan (4-DMAT), chanoclavine-I, agroclavine, and lysergic acid. Two cis, trans isomerizations occur one before chanocla-vine-I and the other before agroclavine, as shown by experiments with [2- C]-mevalonic acid and [Z-CH3]-4-DMAT (Fig. 36). The peptide unit is derived from a combination of three amino acids, one of which is always proline. Several genera in the plant family Convolvulaceae Rivea, Ipomoea, etc.) also produce ergot alkaloids. 

Mould Metabolites.—Intermediates on the presumed biosynthetic pathway from tryptophan to echinulin and its analogues continue to be isolated. The introduction of a reversed isoprene unit at C-2 leads to L-analyl-2-(l,l-dimethylallyl)-L-tryptophan anhydride (31), a compound which has already been synthesized, and which has now been shown to be a constituent of Aspergillus chevalieri (Mangin) Thom et Church IFO 4090. The dehydro-derivative, neoechinulin A (32), recently isolated from A. amstelodami, has now been shown to occur in A. ruber A... 

Tryptophan and its relative indolylpyruvic acid (3.42) have been shown to precursors of hinnuliquinone (3.41), which is a pigment of Nodulisporium hin-nuleum. Typical of many fungal indoles in which alkylation by a dimethylallyl or isopentenyl group has occurred, mevalonate was also a precursor. However, the stage at which prenylation of a monomer or a dimer took place was unclear. Asterriquinone (3.43) from Aspergillus terreus and cochliodinol (3.44) from Chaetomium cochliodes are similar metabolites. Fission of the hydroxyquinone in the latter followed by lactonization leads to cochliodinone (3.45) in a sequence that is similar to that which inter-relates the terphenyls and pulvinic acids described in Chapter 7. 

The early steps in the ergot alkaloid biosynthetic pathway are outlined in Fig. 1. The first determinant and rate-limiting step is the prenylation of tryptophan to 4-(y,y-dimethylallyl)tryptophan (DMAT), catalyzed by dimethy-lallyl-diphosphate L-tryptophan dimethylallyltransferase (DMAT synthase EC 2.5.1.34) (Heinstein et al., 1971 Gebler and Poulter, 1992). The prenyl group for the DMAT synthase reaction is provided in the form of dimethylallyl diphosphate (DMAPP), which is derived from mevalonic acid. After the formation of DMAT, the free amino group of this intermediate is N-methylated with a methyl group donated by S-adenosylmethionine (AdoMet). The N-methylated DMAT is then converted into chanoclavine I by closure of the... 

Figure 1 Key intermediate and enzymes from the first committed step in ergot alkaloid biosynthesis through the synthesis of lysergic acid. Simple amides of lysergic acid and ergopeptines are covered in Figs 2 and 3, respectively. Abbreviations DMAPP = dimethylallyl pyrophosphate DMAT = dimethylallyl tryptophan AdoMet = S-adenosyl-methionine.

Although several previous reports claimed that the enzyme had been purified, Gebler and Poulter (1992) appear to have been the first to fully characterize the activity of the purified DMAT synthase. The enzyme was purified from Claviceps fusiformis ATCC 26245 [erroneously annotated in type specimen collections as a C. purpurea strain (Pazoutova and Tudzynski, 1999)]. The monomeric size was estimated at 53 kDa, and by gel filtration analysis the native enzyme was determined (at 105 kDa) to be a homodimer. Unlike other prenyltransferases, no metal ion requirement has been noted. However, when assayed in a buffer with 4 mM Ca2+, the purified protein gave a specific activity of 500 nmol/min/mg, essentially the same as with 4 mM Mg2+, but approximately twice that of the measured without added divalent cations and with the chelator EDTA included in the assay buffer. These divalent metal cations eliminated negative cooperativity of substrate binding observed both for dimethylallyl diphosphate and L-tryptophan, indicating that Ca2+ and Mg2+ probably had allosteric effects. In buffer with 4 mM MgCl2 the KM for dimethylallyl diphosphate was 8 jlM, and the KM for L-tryptophan was 12 xM. The enzyme product was authenticated by mass spectrometry, UV spectrometry, and -NMR. 

Gebler JC, Poulter CD. Purification and characterisation of dimethylallyl tryptophan synthase from Claviceps purpurea. Arch Biochem Biophys 296 308-313, 1992. 

The tryptophan tetramic acid (63) has been isolated from Penicillium cyclo-pium it thus seems certain that the biosynthetic sequence leading to cyclopiazonic acid involves C-4 dimethylallylation of (63), i.e. that the formation of the tetramic acid unit from tryptophan and acetoacetate occurs first. 

Another group of tryptophan-proline-derived fungal metabolites having a 3,3-dimethylallyl (normal isopentenyl) unit at position 2 of the indole ring is the... 

Amauromine, a dimeric indole alkaloid with hypotensive vasodilator properties, was isolated from Amauroascus species (Gymnoascales, Ascomycotina) (267) and Penicillium verrucosum (26S). The structure of this metabolite was determined by chemical and spectroscopic investigations and confirmed by a total synthesis by Takase et aL 268). The symmetrical diketopiperazine made up of two modified tryptophans, each bearing a 1,1-dimethylallyl group at C-3, was synthesized as shown briefly in Scheme 45. The two inverted prenyl groups were introduced simultaneously by a thio-Claisen rearrangement reaction through the sulfonium salt. 

The synthesis of dimethylallyltryptophan (132) by a crude extract of Claviceps purpurea from tryptophan and dimethylallyl pyrophosphate recorded earlier has been reported again recently. In addition to (132), the formation of (133) was observed. (The latter compound, with unspecified stereochemistry around the double bond, has also been isolated from a C. purpurea culture ). It was found further that both (132) and (133) could act as precursors for lysergic acid amides in C. paspali cultures. Both (133) and its (Z)-isomer have been found to act as precursors for elymoclavine (137) but not chanoclavine-I (138) or agro-clavine (136), which are considered to be normal intermediates in elymoclavine biosynthesis.It may be concluded, however, from the combined evidence, that elymoclavine, lysergic acid, and related compounds may normally be formed along an alternative pathway via these allylic hydroxy-compounds. 

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