L.J. Wigley et al. / Phytochemistry 67 (2006) 561–569
569
Cannell, R.J.P., Dawson, M.J., Hale, R.S., Hall, R.M., Nobel, D., Lynn,
S., Taylor, N.L., 1993. The squalestatins, novel inhibitors of squaline
10 mg, 6-chlorotryptamine (Sigma) 10.6 mg and DL-5-
methyltryptophan (Aldrich) 10 mg, were ꢁtrickle-fedꢀ, each
in 25 ml sterile distilled water, to separate culture flasks
of production medium, 100 ml, frequently between 40
and 69 h of the fermentation, which continued until 96 h.
The ethyl acetate broth extracts and methanol cell extracts
were purified by TLC, and compounds co-chromatograph-
ing with 1, 2 and other selected bands were eluted from the
silica and investigated by CIMS. Accurate mass measure-
ments were obtained for any important ions in the spectra
that did not correspond to either 1 or 2.
synthase produced by
a species of Phoma 1V. Preparation of
fluorinated squalestatins by directed biosynthesis. Journal of Antibi-
otics 46, 1381–1389.
Enger-Valk, B.E., Heyneker, H.L., Oosterbaan, R.A., Pouwells, P.H.,
1980. Construction of new cloning vehicles with genes of the
tryptophan operon of Escherichia coli as genetic markers. Gene 9,
69–85.
Goddijn, O.J.M., De Kam, R.J., Zanetti, A., Schilperoort, R.A., Hoge,
J.H.C., 1992. Auxin rapidly down-regulates transcription of the
tryptophan decarboxylase gene from Cantharanthus roseus. Plant
Molecular Biology 18, 1113–1120.
Gutierrez, S., Diez, B., Montenegro, E., Martin, J.F., 1991. Character-
isation of the Cephalosporium acremonium pcbAB gene encoding a-
aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide
synthetase: Linkage to the pcbC gene as a cluster of early cephalo-
sporin biosynthetic genes and evidence of multiple functional domains.
Journal of Bacteriology 173, 2354–2365.
Henrickson, J.B., Goschke, R., Rees, R., 1964. Total synthesis of the
calycanthaceous alkaloids. Tetrahedron 20, 565–579.
Kirby, G.W., Shah, S.W., Herbert, E.J., 1969. Biosynthesis of chimonan-
thine from [2-3H] tryptophan and [2-3H] tryptamine. Journal of the
Chemical Society (C), 1916–1919.
3.2.10. Probing for un-methylated communesins with 14C-
tryptophan following ethionine addition to fermentations
DL-Ethionine (Sigma), 50 mg in 10 ml sterile distilled
water, was added to a 100 ml fermentation at 38 h. 10 ml
water was added to a control fermentation. 30 min later
14C-tryptophan (1 lCi) in 1 ml sterile water was added to
each culture. Fermentations were harvested at 72 h and
the broth extracted with ethyl acetate. Extract was chro-
matographed and the TLC plate autoradiographed for
one month.
Kleinkauf, H., von Dohren, H., 1990. Nonribosomal biosynthesis of
peptide antibiotics. European Journal of Biochemistry 192, 1–15.
Maniatis, T., Fritsch, E.L., Sambrook, J., 1989. Molecular cloning, a
laboratory manual. Cold Spring Harbor, New York.
Mantle, P.G., Shipston, N.F., 1987. Temporal separation of steps in the
biosynthesis of verruculogen. Biochemistry International 14, 1115–
1120.
Acknowledgements
Matthews, S., Jandu, S.K., Leatherbarrow, R.J., 1992. 13C NMR study of
the effects of mutation on the tryptophan dynamics in chymotrypsin
inhibitor 2: correlations with structure and stability. Biochemical
Journal 32, 657–662.
Noe, W., Mollenschott, C., Berlin, J., 1984. Tryptophan decarboxylase
from Catharanthus roseus cell suspension cultures: purification,
molecular and kinetic data of the homogeneous protein. Plant
Molecular Biology 3, 281–288.
Numata, A., Takahashi, C., Ito, Y., Takada, T., Kawai, K., Usami, Y.,
Matsumura, E., Imachi, M., Ito, T., Hasegawa, T., 1993. Commune-
sins, cytotoxic metabolites of a fungus isolated from a marine alga.
Tetrahedron Letters 34, 2355–2358.
We thank R. Shepherd and J. Barton (Chemistry Depart-
ment, Imperial College) for obtaining NMR and MS data,
G. Goodwin (Pfizer, UK) for synthesis of indole-N-(13C-
methyl) tryptophan, and Prof. C. Moody, Loughborough
University, UK, for confirming our tryptophan synthesis.
We thank BBSRC for a research studentship (LJW) in col-
laboration with Pfizer Central Research (Sandwich, Kent,
UK) and acknowledge the advice of Dr. M. Haxell (Pfizer,
UK) concerning communesin fermentations.
Robinson, Sir R., Teuber, H.J., 1954. Calycanthine and calycanthidine.
Chemistry and Industry, 783–784.
References
Songstad, D.D., De Luca, V., Brisson, N., Kurz, W.G.W., Nessler, C.L.,
1990. High levels of tryptamine accumulation in transgenic tobacco
expressing tryptophan decarboxylase. Plant Physiology 94, 1410–
1413.
Taylor, D.C., Wightman, F., 1987. Metabolism of D,L-chloro-phenylala-
nines by phenylalanine aminotransferase isozymes purified from
bushbean shoots. Phytochemistry 26, 1279–1288.
Wigley, L.J. 1995. Biosynthesis of communesin alkaloids. Ph D thesis,
University of London.
Yamada, S., Shioiri, T., Itaya, T., Hara, T., Matsueda, P., 1965. Ind.-N-
alkylation of tryptophan and synthesis of 1-alkyltryptophan hydraz-
ides. Chemical and Pharmaceutical Bulletin 13, 88–93.
Adjibade, Y., Weniger, B., Quirion, J.C., Kuballa, B., Cabalion, P.,
Anton, R., 1992. Dimeric alkaloids from Psychotria fosteriana.
Phytochemistry 31, 317–319.
Aerts, R.J., Alarco, A.-M., De Luca, V., 1992. Auxins induce tryptophan
decarboxylase activity in radicles of Catharanthus seedlings. Plant
Physiology 100, 1014–1019.
Beck, J., Ripka, S., Siegner, A., Schiltz, E., Schweizer, E., 1990. The
multifunctional 6-methylsalicylic acid synthase gene of Penicillium
patulum. Its gene structure relative to that of other polyketide
synthases. European Journal of Biochemistry 192, 487–498.