Diketopiperazines as advanced intermediates in the biosynthesis of ecteinascidins

Article abstract of DOI:10.1016/S0040-4020(00)00249-0

The diketopiperazines of phenylalanine, tyrosine and DOPA have been synthesized from ¹4C-labeled amino acids and tested as intermediates in the biosynthesis of the ecteinascidins. Biosynthetic experiments were performed using an enzyme preparat

Full text of DOI:10.1016/S0040-4020(00)00249-0

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3303±3307
Diketopiperazines as Advanced Intermediates in the Biosynthesis
of Ecteinascidins
*
Shanti Jeedigunta, Joann M. Krenisky and Russell G. Kerr
Department of Chemistry and Biochemistry, Center for Molecular Biology and Biotechnology, 777 Glades Road,
Florida Atlantic University, Boca Raton, FL 33431-0991, USA
Received 29 January 2000; accepted 21 March 2000
AbstractÐThe diketopiperazines of phenylalanine, tyrosine and DOPA have been synthesized from 14C-labeled amino acids and tested as
intermediates in the biosynthesis of the ecteinascidins. Biosynthetic experiments were performed using an enzyme preparation of the tunicate
Ecteinascidia turbinata, the source of the ecteinascidins. Tyrosine and DOPA diketopiperazines were both transformed to the ecteinascidins
whereas the diketopiperazine of phenylalanine was not modi®ed. It is now apparent that the biosynthesis of the ecteinascidins is initiated by
the condensation of tyrosine to its cyclic dipeptide followed by a subsequent oxidation to the diketopiperazine of DOPA. q 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
ascidins are structurally related to the safracins4 and safra-
mycins5 from microbes as well as the renieramycins6 and
xestomycin7 from sponges.
The ecteinascidins are a family of tetrahydroisoquinoline
alkaloids isolated from the tunicate Ecteinascidia
turbinata.1 Ecteinascidin 743 (ET 743) is a candidate for
the treatment of several human cancers, in particular lung
cancer and melanoma. Ecteinascidins have been shown to
kill tumor cells in vitro,2 inhibit tumor growth in vivo,2
suppress allograft rejection,2b diminish host reactions to
tissue grafts and inhibit lymphocyte proliferation.3 The
ecteinascidins have two mechanisms of action: they are
unique alkylating agents and also disaggregate microtubule
complexes without affecting tubulin itself. The ectein-
As initially suggested by Rinehart and co-workers,1b an
inspection of the structure of ecteinascidin 743 (1) (Scheme
1) suggests that this metabolite could be derived from inter-
mediates 2 and 3. (It is, however, conceivable that inter-
mediate 2 reacts ®rst with cysteine [or b-mercaptopyruvic
acid] and then with dopamine rather than with intact 3.)
Pentacyclic intermediate 2 appears to be derived from a
diketopiperazine of either phenylalanine (4), tyrosine (5) or
DOPA (6), likely via intermediates 7 and 8 (Scheme 2).
Scheme 1. Possible biosynthetic origin of ecteinascidin 743.
Keywords: biosynthesis; alkaloids; marine metabolites; antitumor compound.
* Corresponding author. Tel.: 11-561-297-3356; fax: 11-561-297-2759; e-mail: rkerr@acc.fau.edu
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00249-0

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S. Jeedigunta et al. / Tetrahedron 56 (2000) 3303±3307
Scheme 2. Potential diketopiperazine intermediates in ecteinascidin biosynthesis.
Results and Discussion
alanine, tyrosine and DOPA with a cell-free extract of E.
turbinata.
We recently reported in vitro conditions to evaluate the
biosynthetic transformation of putative ecteinascidin
precursors and demonstrated the conversion of labeled
amino acids to ecteinascidin 743.8 A cell-free extract of E.
turbinata afforded 3000 dpm ET 743 when incubated with
tyrosine (5.5£106 dpm) and 2100 dpm ET 743 when incu-
bated with cysteine (11£106). Herein, we report the results
of incubating radiolabeled diketopiperazines of phenyl-
Diketopiperazines 4, 5 and 6 were prepared by standard
methods.9 While 4 and 5 are known compounds, 6 had not
been previously reported. The syntheses of 4 and 5
proceeded as outlined in Jung's report9 and proved to be
applicable to both medium scale and 1 mg scale prepara-
tions. Conditions were established to perform this synthesis
on a 1 mg scale to de®ne appropriate techniques for the
Scheme 3. Initial steps of the biosynthesis of ecteinascidin 743.

S. Jeedigunta et al. / Tetrahedron 56 (2000) 3303±3307
Table 1. Incorporation of labeled precursors into ecteinascidin 743
3305
Radiolabeled
precursor
Total radioactivity
used (dpm)
Recovered radioactivity
in ET 743 (dpm)
Conversion
(%)
[U±14C] Tyrosine
[U±14C] Phe DKP
[U±14C] Tyr DKP
[U±14C] DOPA DKP
1.21£107
2,000
Background
11,530
0.016
0
1.4
6£105
8£105
4£105
39,960
11.4
synthesis of radiolabeled compounds required for the
biosynthetic studies.
activity of the ecteinascidin HPLC peak was at least 100
times that of the eluent immediately prior to, and immedi-
ately following, this fraction.
In general, the methyl ester of the amino acid was
condensed with an equimolar amount of t-BOC protected
amino acid using DCC. The acyclic dipeptide was then
treated with formic acid to afford the cyclic dipeptide. The
medium scale reactions produced cyclic dipeptides 4 and 5
in yields of 93 and 96%, respectively. The synthesis of the
diketopiperazine of DOPA (6) had not been previously
reported and in fact, to our knowledge, there are no reports
describing the incorporation of DOPA into peptides.10 Inter-
estingly, we found that 6 could be readily prepared in an
overall yield of 52% if all transformations were performed
under argon. NMR analysis of this diketopiperazine
revealed the presence of small amounts of dicyclohexylurea,
a byproduct of the coupling reaction, which proved dif®cult
to remove without degradation of product.
Diketopiperazine 6 afforded ET 743 with a radiochemical
yield of 11.4%, signi®cantly higher than that from 5 (Table
1). The radiochemical transformation of both diketopiper-
azines was at least two orders of magnitude greater than that
for tyrosine as is to be expected for committed biosynthetic
intermediates. These data indicate that the diketopiperazine
of tyrosine (5) is the ®rst committed intermediate in ectein-
ascidin biosynthesis and suggest that prior to any further
cyclization, 5 is oxidized to 6. It is conceivable that these
diketopiperazines are not the actual intermediates but
instead are transformed to the ecteinascidins through a
reduction to the aminol. Recent analysis of putative peptide
synthetases SafB and SafA involved in saframycin
biosynthesis suggest that a linear tetrapeptide is a pre-
cursor.12 It has been further suggested that this tetrapeptide
is likely to be reductively released to afford Ala-Gly-Tyr-
Tyr-CHO prior to an intramolecular cyclization to a
heminaminal structure.13 To address this possibility in
ecteinascidin biosynthesis, we examined the conversion of
5 to 6 in our enzyme preparation of E. turbinata. This oxi-
dation was con®rmed from an analysis of the tyrosine
diketopiperazine incubation mixture. Addition of 6 as
`cold carrier' to the quenched incubation of labeled 5 facili-
tated the puri®cation of radioactive DOPA diketopiperazine
(4300 dpm),14 providing strong support for the direct
involvement of diketopiperazines 5 and 6 in ecteinascidin
biosynthesis.
The 14C-labeled diketopiperazines 4, 5 and 6 were prepared
from 50 mCi each of phenylalanine ethyl ester, tyrosine and
DOPA. Radiolabeled tyrosine and DOPA esters are not
commercially available and their methyl esters were synthe-
sized by the treatment of tyrosine with acidic 2,2-dimethoxy
propane.11 The cyclic dipeptides were puri®ed by vacuum
chromatography over ODS, followed by repeated RPHPLC
using an acetonitrile/water (0.1% TFA) gradient to afford
radiochemically pure material.
All biosynthetic experiments were performed using a cell-
free extract of E. turbinata. We found that the activity of
these enzyme preparations was greatly dependent upon the
handling of the organism following the collection. Optimum
results were obtained when the tunicate was immediately
¯ash frozen in liquid nitrogen upon collection. The frozen
samples were then transported to the laboratory at 2808C
and maintained at this temperature. The tunicate (250 g)
was then ground to a ®ne powder in a chilled mortar and
pestle, added to a phosphate buffer (500 mL) at pH 7.7
containing DTT, EDTA and leupeptin, centrifuged at
10,000 g and the supernatant stored in 30 mL aliquots at
2808C.
We have provided evidence that the biosynthesis of the
ecteinascidins is initiated by the condensation of tyrosine
to its cyclic dipeptide followed by a subsequent oxidation
to the diketopiperazine of DOPA (Scheme 3). Further
experiments directed at identifying the fate of 6 are
currently underway.
Experimental
General
To assess the viability of the cell-free extract, an aliquot was
incubated with [U±14C] tyrosine (2.5 mCi), a known ectein-
ascidin precursor, as previously described.8 Recovery of ET
743 with an activity of 2000 dpm (0.017% conversion) indi-
cated a viable extract. As indicated in Table 1, diketopiper-
azines 5 and 6 were both transformed to ET 743 while 4 was
not modi®ed. Radiochemical purity of ET 743 was estab-
lished by repeated HPLC analysis. The extremely trace
quantities of this metabolite precluded degradative work
and recrystallization to constant speci®c activity. In all
cases in which radioactive product is reported, the radio-
1H NMR spectra were recorded on a Unity Inova Varian 500
instrument at 500 MHz. IR spectra were obtained on a
Mattson 4020 instrument. HPLC puri®cations were
performed on either a PE Series 410 pump equipped with
a PE 325 detector or an HP 1090 instrument. Incubations
were performed at 278C in a Labline 4628 Environmental
shaker at 220 rpm. Radioactivity was measured using an
LKB Wallac 1219 Rackbeta scintillation counter. All
solvents used were Optima grade and the phenylalanine
methyl ester, tyrosine methyl ester, DOPA methyl ester,

3306
S. Jeedigunta et al. / Tetrahedron 56 (2000) 3303±3307
EDTA, BSA, DTT and leupeptin were purchased from
Sigma Chemical Co. Radiolabeled amino acids were
purchased from American Radiolabeled Chemicals Inc.
2 h. The mixture was concentrated and directly puri®ed by
RPHPLC using a Vydac column (4.6£150 mm2), moni-
toring 230 nm with a linear gradient of 75% H2O (0.1%
TFA): 25% CH3CN to CH3CN over 20 min to afford
2.4£106 dpm of 14C-cyclo-(l-Phe-l-Phe) (4).
Collection of animal material and preparation of the
cell-free extract
Synthesis of cyclo-(l-Tyr-l-Tyr) (5). The medium scale
synthesis of cyclo-(l-Tyr-l-Tyr) was performed as
described for cyclo-(l-Phe-l-Phe). The overall yield was
96% and the melting point and IR, 1H NMR and 13C
NMR were in accord with literature values.9a
Colonies of E. turbinata were collected at a depth of 0.1±
0.5 m in a mangrove community in Long Key, Florida. The
freshly collected tunicate was quickly cleaned of extraneous
material, ¯ash frozen, and ground to a ®ne powder in a
chilled mortar and pestle. This powder (250 g) was added
to 500 mL of a phosphate buffer at pH 7.7 containing DTT
(290 mg), EDTA (550 mg), BSA (375 mg) and leupeptin
(0.5 mg). The mixture was vortexed for 5 min and then
centrifuged at 10,000 g and the supernatant stored in
30 mL aliquots at 2808C.
The radiochemical synthesis of 5 was performed as
described for 4 to afford 2.0£106 dpm. [14C±U] Tyrosine
methyl ester was synthesized as follows: a solution of tyro-
sine (50 mCi) in 1.5 mL 2,2-dimethoxy propane was treated
with 100 mL of HCl (12N) and the mixture stirred for 18 h.
The reaction mixture was passed through a column of silica
and concentrated to dryness.
Synthesis of cyclo-(l-Phe-l-Phe) (4)
Synthesis of cyclo-(l-DOPA-l-DOPA) (6). The medium
scale synthesis of cyclo-(l-DOPA-l-DOPA) was performed
as described for cyclo-(l-Phe-l-Phe) in an overall yield of
52% with the exception that all transformations were
performed under argon: IR (KBr pellet) 3404, 3320,
Medium scale reaction conditions: To a solution of
l-phenylalanine methyl ester hydrochloride (323 mg) and
N-t-BOC-l-phenylalanine (397 mg) in 3 mL DMF and
12 mL acetonitrile at 08C was added triethylamine
(210 mL) followed by DCC (309 mg). The mixture was
stirred at 08C for 2 h and allowed to stand at this temperature
for 12 h. The dicyclohexyl urea was ®ltered and washed
with EtOAc. The combined ®ltrate was evaporated under
nitrogen to afford a gummy residue, which was partitioned
between EtOAc and H2O. The organic layer was washed
sequentially with 0.5N HCl, H2O, 0.5N NaHCO3 and
brine, and then dried over sodium sulfate, and evaporated
to afford a white solid. Puri®cation by ¯ash chromatography
(70% EtOAc/hexane) gave a colorless foam of N-(tert-
butoxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl
ester. This dipeptide was stirred with formic acid (20 mL)
at ambient temperature for 2 h and subsequently evaporated.
The residue was dissolved in sec-butanol (40 mL) and
toluene (10 mL) and the solution re¯uxed for 2 h. The
mixture was concentrated and puri®ed by ¯ash chroma-
tography over ODS using a step gradient of H2O (0.1%
TFA), H2O (0.1% TFA)/CH3CN 8:2, H2O (0.1% TFA)/
CH3CN 6:4, H2O (0.1% TFA)/CH3CN 4:6. Final puri®-
cation with RPHPLC using an Altex Ultrasphere column
(10 mm£25 cm), monitoring 230 nm with a linear gradient
of 75% H2O (0.1% TFA): 25% CH3CN to CH3CN over
20 min afforded cyclo-(l-Phe-l-Phe) (4) in an overall
1671 cm21 1H NMR (d6-DMSO, 500 MHz) d 8.7 (s,
;
OH), 8.1 (br. s, NH), 6.55 (dd, 2 H, Jˆ8.5, 1.5 Hz), 6.51
(br. s, 2 H), 6.36 (d, 2 H, Jˆ8.0 Hz), 4.15 (m, 2 H), 2.75
(ddd, 2 H, Jˆ4 Hz), 1.9 (m, 2 H); HRFABMS calcd for
C18H19N2O6 (M11)1 359.1238, found 359.1243.
The radiochemical synthesis of 6 was performed (under
argon) as described for 4 to afford 6.0£105 dpm.
Incubation of 14C-labeled tyrosine and DKPs
The 14C-labeled precursor was added to 30 mL of the cell-
free extract forti®ed with 1 mg each of ATP, NADH and
NADPH and the mixture incubated at 200 rpm at 278C for
24 h. The incubations were quenched by shaking with
30 mL EtOAc, which was subsequently removed in vacuo
and the aqueous mixture dried by lyophilization. The ectein-
ascidins were puri®ed by a reversed phase (C-18) vacuum
¯ash chromatography using a step gradient of H2O (0.1%
TFA), H2O (0.1% TFA)/CH3CN 8:2, H2O (0.1% TFA)/
CH3CN 6:4, H2O (0.1% TFA)/CH3CN 4:6. The third
fraction was concentrated to dryness and subjected to
1
yield of 93%. Melting point, IR, H NMR and 13C NMR
RPHPLC puri®cation using
a C-18 Vydac column
were in accord with literature values.9a
(4.6£250 mm2) and monitoring 230 nm with a mobile
phase of H2O (0.1% TFA)/CH3CN 82:18.
Radiochemical synthesis: To a solution of l-phenylalanine
ethyl ester hydrochloride (50 mCi) and N-t-BOC-l-phenyl-
alanine (1.1 mg) in 50 mL DMF and 450 mL acetonitrile at
08C was added triethylamine (10 mL) followed by DCC
(1 mg). The mixture was stirred at 08C for 2 h and allowed
to stand at this temperature for 12 h. The dicyclohexyl urea
was ®ltered and washed with EtOAc. The residue was
washed sequentially with 400 mL each of 0.5N HCl, H2O,
0.5N NaHCO3 and brine and the organic layer evaporated
under nitrogen. The dipeptide was stirred with formic acid
(2 mL) at ambient temperature for 2 h and subsequently
evaporated. The residue was dissolved in sec-butanol
(1.5 mL) and toluene (1 mL) and the solution re¯uxed for
Acknowledgements
Financial support for this research was provided by a
National Sea Grant award (R/LR-MB-6) and the American
Cancer Society (RPG-97-170-01). The staff of the Keys
Marine Laboratory is gratefully acknowledged for pro-
viding ®eld support. A. Wright (Harbor Branch Oceano-
graphic Inst.) is acknowledged for the generous gift of a
sample of ecteinascidin 743. We thank S. Kerr for assistance
with the preparation of cell-free extracts. Mass spectra were

S. Jeedigunta et al. / Tetrahedron 56 (2000) 3303±3307
3307
6. He, H.-Y.; Faulkner, D. J. J. Org. Chem. 1989, 54, 5822.
recorded at the University of Florida and their assistance is
acknowledged.
7. Gulavita, N. K.; Scheuer, P. J.; De Silva, E. D. Abstracts, Indo-
United States Symposium on Bioactive Compounds from Marine
Organisms, Goa, India, 1989; p 28.
References
8. Kerr, R. G.; Miranda, N. F. J. Nat. Prod. 1995, 58, 1618±1621.
9. (a) Jung, M. E.; Rohloff, J. C. J. Org. Chem. 1985, 50, 4909±
4913. (b) Nitecki, D. E.; Halpern, B.; Westley, J. W. J. Org. Chem.
1967, 33, 864±866.
1. (a) Wright, A. E.; Forleo, D. A.; Geewananda, P. G.; Gunasekera,
S. P.; Koehn, F. E.; McConnell, O. J. J. Org. Chem. 1990, 55,
4508±4512. (b) Rinehart, K. L.; Holt, T. G.; Fregeau, N. L.;
Stroh, J. G.; Keifer, P. A.; Sun, F.; Li, L. H.; Martin, D. G.
J. Org. Chem. 1990, 55, 4512±4515.
10. Solid-phase peptide synthesis. In Methods in Enzymology;
Fields, G. B., Ed.; Academic: Orlando, 1997; Vol. 289.
11. Rachele, J. R. J. Org. Chem. 1963, 17, 2898.
12. Pospiech, A.; Bietenhader, J.; Schupp, T. Microbiology 1996,
142, 741±746.
2. (a) McCumber, L. J.; Trauger, R.; Sigel, M. M. Int. Symp. Fish
Biologics 1981, 49, 289±294. (b) Lichter, W.; Wellham, L. L.; Van
der Werf, B. A.; Middlebrook, R. E.; Sigel, M. M. In Food-drugs
from the Sea: Proceedings; Worthen, L. R., Ed.; Marine Tech-
nology Society: Washingnton, D.C., 1972; pp 7±127.
3. Lichter, W.; Lopez, D. M.; Wellham, L. L.; Sigel, M. M. Proc.
Soc. Exp. Mar. Biol. Med. 1975, 150, 475±478.
13. (a) Ehmann, D. E.; Gehring, A. M.; Walsh, C. T. Biochemistry,
1999, 38, 6171±6177. (b) Konz, D.; Marahiel, M. A. Chem. Biol.
1999, 6, 39±48.
14. The diketopiperazines are not present in isolable quantities in
the extract of E. turbinata and thus the recovered activity repre-
sents a high level of speci®c activity.
4. Ikeda, Y.; Matsuki, H.; Ogawa, T.; Munakata, T. J. Antibiot.
1983, 36, 1284.
5. Trowitzssch, W.; Irschik, H.; Reichenbach, H.; Wray, V.; Ho¯e,
G. Liebigs Ann. Chem. 1988, 475.