3- Amino-4-hydroxy cinnamic acid

Peptides were sequenced using either an Applied Biosystems Model 470 or 477 or a Hewlett Packard GIOOOA protein sequencer, each equipped with narrow bore RP-HPLC for on-line analysis of the PTH-amino acids. Mass analysis of peptides was performed using matrix-assisted laser desorption/ionization mass spectrometry on a KRATOS MALDl 111 with a-cyano-4 hydroxy-cinnamic acid as the matrix. 

Figure 6. MALDI mass spectrum of fraction 13 from RP-HPLC. The H,K-ATPase-enriched vesicles were trypsinized and centrifuged to separate supernatant from pellet. The supernatant was subjected to RP/HPLC and individual fractions collected and subjected to MALDI/MS. The MALDI mass spectrum (reflectron-ion mode) was obtained using a-cyano-4-hydroxy cinnamic acid as a matrix (Panel A). The signals were assigned to a-subunit peptides (Table 2). The signal at m/z 1798, indicated by an arrow was next subjected to PSD-analysis. The PSD-spectrum of MH+ 1798.4 is shown in Panel B. Only the peaks for the b and y fragment ions are labeled. The deduced amino acid sequence is shown at the top of the panel.

Amino-3-hydroxy-cinnamic acid. This material was prepared according to the above procedure in 65% yield m.p. 169-170 . 

The aromatic amino acid L-phenylalanine (primary metabolite) is directed into the phe-nylpropanoid pathway leading to hydroxy-cinnamic acids, lignin and flavonoids by the activity of L-phenylalanine ammonia-lyase (PAL), which brings about its nonoxidative deamination yielding ammonia and tvans-cinnamic acid (Fig. 1). PAL is one of the most studied plant enzymes, and its crystal structure has recently been solved [2]. PAL is related to the histidine and tyrosine ammonia-lyases of amino acid catabolism. A class of bifunctional PALs found in monocotyle-donous plants and yeast can also deaminate tyrosine [3]. A single His residue is responsible for this switch in substrate preference [3, 4]. All three enzymes share a unique MIO (4-methylidene-imidazole-5-one) prosthetic group at the active site. This is formed auto-catalytically from the tripeptide Ala-Ser-Gly by cyclization and dehydration during a late... 

Carbostyril.—If in the Baeyer and Drewsen synthesis cinnamic acid is used instead of cinnamic aldehyde the or//fo-amino derivative, by loss of water, yields a hydroxy quinoline known as carbostyril. 

Lobeline.—The results of feeding experiments with DL-[2- C]lysine and dl-[2- Clphenylalanine in Lobelia inflata have shown that these amino-acids are both specific precursors for the alkaloid lobeline (13). In further experiments, DL-[3- C]phenylalanine, [3- C]cinnamic acid, and [3- C]-3-hydroxy-3-phenylpropionic acid [as (9)] have been found to be specific precursors for lobeline (13). These results are consistent with the anticipated pathway " to lobeline illustrated in Scheme 2, with benzoylacetic acid (10) as the intermediate which couples with A -piperideine to give the intermediate (11). The probability of 3-hydroxy-3-phenylpropionic acid (9) being an intermediate in lobeline biosynthesis is increased by the isolation of this acid from L. inflata ... 

Experiments with tritium and C-labelled phenylalanine samples established that this amino-acid was incorporated intact as a Cg unit (with loss of C-1). Support for a pathway proceeding via cinnamic acid and hydroxylated derivatives and benzoylacetic acid derivatives was provided in further experiments with cinnamic acid, ferulic acid/isoferulic acid, and the (easily hydrolysed) ethyl esters of benzoylacetic acid and piperonylacetic acid. As far as they go the results suggest that a mixed pathway is in operation with introduction of hydroxy- and methylenedioxy-groups on more than one intermediate. 

The conversion of phenylalanine, a C-6—C-3 precursor, to the C-6—C-1 unit of the Amaryllidaceae alkaloids requires the formal loss of two carbon atoms from the side chain of the amino acid as well as the introduction of at least two oxygenated substituents into the aromatic ring. The results shown in the latter part of Table III emphasize the specificity of the C-6—C-1 precursor. Benzaldehyde, -hydroxybenzal-dehyde, isovanillin, and protocatechuic acid are not incorporated to any appreciable extent into the alkaloids, while cinnamic, -hydroxy-cinnamic and caffeic acids, protocatechuic aldehyde, and 3-hydroxy-4-methoxy-i C-A-methyl-i4( ., enzylamine readily become part of the C-6—C-1 unit. 

Fig. 1. A generalized scheme showing the kinds of secondary products that arise from the aromatic amino acids in higher plants. Several similarities are found in fungi and bacteria some fungi produce alkaloids ftom tryptophan and lignin-like materials from phenylalanine. Plant pathogenic fungi produce cinnamate and para and meta hydroxy phenyl-acetate from phenylalanine. Certain bacteria produce antibiotics and fluorescent pigments from metabolites in the shikimate pathway. Microorganisms are not known to produce coumarin, substituted coumarins, flavonoids and isoflavonoids.

AT-phenylpropenoyl amino acids (2-17) have been identified as the key contributors to the astringent taste of non-fermented cocoa beans and cocoa products. Besides the already known (E) -N- [ 3, 4 -dihydroxycinnamoyl-3-hydroxy-L-tyrosine (known as clovamide), (E)-Ar-(4Chydroxycinnamoyl)-L-tyrosine (deoxy-clovamide) and (E)-AT-(3, 4 -dihydroxycinnamoyl)-L-tyrosine, seven additional amides derived from cinnamic, 4-coumaric, caffeic and feruHc acids, namely, (- -)-(E)-N-(cinnamoyl)-L-aspartic acid, (-f)-( )-N-(4 -hydroxycinnamoyl)-L-aspartic add, (- -)-( )-iV-(3, 4 -dihydroxydnnamoyl)-L-aspartic add, (-l-)-(E)-N-(4 -hydroxy-3 methoxydnnamoyl)-L-aspartic acid, (-)-( )- /-(4 -hydroxydnnamoyl)-L-glutamic add, (-)-( )-N-(3, 4 -di-hydroxycinnamoyl)-L-glutamic add and (-)-( )-N-(4 -hydr-oxycinnamoyl)-3-hydroxy-L-tyrosine, were recently identified. 

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