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(R)-b-Phenylalanine

Biosynthesis of (R)-b-Phenylalanine in Taxus brevifolia The phenylisoserine side-chain of taxol in Taxus brevifolia originates from (R)-b-phenylalanine 41 [53, 54]. Floss and coworkers elucidated the stereochemistry of the phenylalanine 2,3-aminomutase reaction in Taxus brevifolia in detail by incubating cell-free extracts of T. brevifolia with a series of isotopically labeled phenylalanines [55]. The configuration of b-phenylalanine 41 was established as R by HPLC comparison of the (1S )-camphanate methyl ester of the aminomutase reaction product with authentic samples. The configuration of 41 corresponds to that of Winterstein's acid (see below). Incubation with (S)-[2-15N,ring-2H5]phenylalanine generated, according to results from GC-MS analysis of the N-benzoyl methyl ester derivative, exclusively (R)-[3-15N,ring-2H5]-b-phenylalanine, indicating a strictly intramolecular shift of the amino group. Incubation experiments with (2S,3R)-[ring,3-2H6]phenylalanine and (2S,3S)-[ring,3-2H6]phenylalanine revealed that the pro-3R hydrogen atom remains at C3 whereas the pro-3S hydrogen atom of 40 migrates to position 2 in 41 (Scheme 1.6.12). In this regard, the phenylalanine 2,3-aminomutase differs from the lysine 2,3-aminomutase in C. subterminale SB4; the mutase in T. brevifolia might therefore constitute a new type of aminomutase.

phenylalanine H. ^NHg

2,3-aminomutase

L-/J-phenylalanine (41) Scheme 1.6.12. Biosynthesis of (R)-b-phenylalanine 41 in Taxus brevifolia.

L-a-phenylalanine (40)

L-a-phenylalanine (40)

Although there are no reports of isolation of the enzyme and its cofactor requirements, the involvement of cobalamin seems unlikely, because it is generally accepted that plants do not contain B12.

Biosynthesis of (R)-b-Phenylalanine in Taxus baccata The biosynthesis of the (R)-3-(dimethylamino)-3-phenylpropionic acid (Winterstein's acid) moiety of taxine A [56] and taxine B [57] in Taxus baccata has been reported by Haslam and coworkers [58]. They investigated whether b-phenylalanine is generated by an aminomutase or by addition of ammonia to cinnamic acid. Their findings provide evidence for the action of a phenylalanine 2,3-aminomutase in T. baccata, because (2S)-[2-14C]phenylalanine is incorporated 10 to 100 times more effectively into the Win-terstein's acid moiety than either [2-14C]- or [3-14C]cinnamic acid. The stereochemistry of Winterstein's acid was determined to be R [59]. According to Haslam [58], the aminomutase reaction in T. baccata is combined with loss of 91% of the radioactivity if the pro-3R was labeled with tritium and loss of 33% of the pro-3S hydrogen atom if that atom was labeled, giving a hint that the pro-3S hydrogen is retained on C-3 (Scheme 1.6.13). This conclusion would, however, be in contradiction to results from the aminomutase in T. brevifolia [55]. Reinvestigation is therefore desirable to clarify whether different types of aminomutase are present in the closely related species T. baccata and T. brevifolia.

Scheme 1.6.13. Biosynthesis of (R)-b-phenylalanine 41 in Taxus baccata according to Haslam.

1.6.3.8 b-Tyrosine

Biosynthesis of (S)-b-Tyrosine in Bacillus brevis Vm4 b-Tyrosine 43 is a constituent of the peptide antibiotics edeine A and B [60] obtained from cultures of Bacillus brevis Vm4. b-Tyrosine is derived from a-tyrosine 42 by use of a tyrosine 2,3-aminomutase [61]. The purified enzyme has properties fundamentally different from those of all other aminomutases so far mentioned. It requires ATP and Mg2+ ions, but no other cofactors.

The stereochemistry of 43 was established to be S by comparing the optical rotation of an authentic sample derived from (R)-b-tyrosine hydrochloride with b-tyrosine hydrochloride isolated from edeine A and B [62]. Incubation experiments with [15N]tyrosine revealed that the [15N]amino group in b-tyrosine is lost, suggesting a reaction mechanism similar to that of an ammonia lyase [62]. This finding is supported by further incubation experiments with (2RS,3R)-[3-3H]tyrosine and (2RS,3S)-[3-3H]tyrosine in combination with (2RS)-[3-14C]tyrosine. Determination of the 3H/14C ratios of the isolated b-tyrosine 43 leads to the conclusion that the pro-3S hydrogen is lost in the course of the reaction whereas the pro-3R hydrogen is retained (Scheme 1.6.14). A similar incubation experiment with (2S)-[2-3H]tyrosine proceeds with a loss of most of the tritium label from C2.

Biosynthesis of (R)-b-Tyrosine and (R)-b-Dopa in Cortinarius violaceus Fruit bodies of the higher fungus Cortinarius violaceus produce (R)-b-dopa 44 [63]. The biosyn-

Scheme 1.6.14. Biosynthesis of (S)-b-tyrosine in Bacillus brevis Vm4.

thesis of (R)-44 was investigated by in-vivo feeding experiments mainly with fluorine-labeled precursors [64]. The incorporation was monitored by GC-MS analysis of the pertrimethylsilyl derivatives of b-dopa. The latter is synthesized in the mushroom by hydroxylation of b-tyrosine 43, as demonstrated by incorporation of (S)-3-fluorotyrosine and (RS)-3-fluoro-b-tyrosine; incorporation of (RS)-6-fluoro-b-dopa was not observed, suggesting the presence of a tyrosine 2,3-aminomutase. The stereochemistry of b-tyrosine and b-dopa was determined to be R by GC-MS comparison of a Mosher amide derivative of natural 44 with a corresponding authentic sample. Administration of (rac)-3-fluoro[15N]tyrosine showed that the shift of the amino group proceeds with retention of the nitrogen, indicating the involvement of a mutase acting similarly to the lysine 2,3-aminomutase. This deduction was confirmed by feeding of 3-fluoro-[3',3'-2H2]tyrosine resulting in formation of 5-fluoro-[2',3'-2H2]-b-dopa, and revealed an (at least partial) intramolecular transfer of a hydrogen from C3 to C2 (Scheme 1.6.15).

Scheme 1.6.15. Biosynthesis of (R)-b-tyrosine and (R)-b-dopa in Cortinarius violaceus.

These experiments do not, nevertheless, enable conclusions to be drawn about which of the hydrogen atoms at C3 is transferred to C2. It therefore remains an open question whether the mutase of the mushroom resembles more the phenylalanine 2,3-aminomutase from Taxus brevifolia or the lysine 2,3-aminomutase from Clostridium subterminale SB4. Because of the lack of any evidence of the occurrence of B12 in higher fungi [65], involvement of B12 in the aminomutase reaction is unlikely.

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