Practical synthesis of DOPA derivative for biosynthetic production of potent antitumor natural products, saframycins and ecteinascidin 743

Article abstract of DOI:10.2174/157017811799304160

A practical synthetic route of DOPA derivative 2, which should be useful for direct biosynthetic production of potent antitumor natural products, saframycins and ecteinascidin 743 was established. The developed strategy features i) easy-to-handle reactions without special care upon both dryness and inert atmopsphere, and ii) the facile HPLC-free purification of 2 via recrystallization enabling scalable synthesis of 2.

Full text of DOI:10.2174/157017811799304160

686
Letters in Organic Chemistry, 2011, 8, 686-689
Practical Synthesis of DOPA Derivative for Biosynthetic Production of
Potent Antitumor Natural Products, Saframycins and Ecteinascidin 743
Kohei Torikai*, Takayoshi Saruwatari#, Tatsuya Kitano#, Takuya Sano, Atsuko Nakane,
Hiroshi Noguchi and Kenji Watanabe
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,
Suruga-ku, Shizuoka 422-8526, Japan
Received August 16, 2011: Revised September 21, 2011: Accepted September 21, 2011
Abstract: A practical synthetic route of DOPA derivative 2, which should be useful for direct biosynthetic production of
potent antitumor natural products, saframycins and ecteinascidin 743 was established. The developed strategy features i)
easy-to-handle reactions without special care upon both dryness and inert atmopsphere, and ii) the facile HPLC-free
purification of 2 via recrystallization enabling scalable synthesis of 2.
Keywords: Biosynthesis, 3,4-dihydroxyphenylalanine, DOPA, ecteinascidin 743, non-natural amino acid, saframycin.
INTRODUCTION
and if 2 were fed from outside the system, enough supply of
SMs would be realized. Herein, we report practical and
scalable synthesis of amino acid 2 to tolerate the feeding
experiments.
Saframycins (SMs), produced by Streptomyces and
various soil bacteria as well as marine vertebrates such as
ascidians and sponges, are potent antitumor antibiotics [1]. In
particular, a highly potent SM analog, ecteinascidin 743 (ET-
743) [2], has recently been in use as an anticancer drug
against soft-tissue sarcoma [3]. ETs share the central
pentacyclic tetrahydroisoquinoline core with SMs, except for
the oxidation state of their terminal rings and the additional
ten-membered lactone bridge found in ET-743. Due to the
short supply from natural resources, the production of ET-
743 should depend on a semi-synthesis including 21
synthetic steps [4]. In order to facilitate the direct
biosynthetic production of SMs including ETs, unremitting
bioinfomatic analyses were carried out, and it was found that
SMs are biosynthesized from L-alanine, glycine and two
RESULTS AND DISCUSSION
Synthesis of DOPA derivative 2 commenced with N-t-
butoxycabonyl (N-Boc) tyrosine
8 according to the
Schmidt’s report [8], one of the most expeditious and easy-
to-handle methods to date [9] (Scheme 2). Aldehyde 9 was
prepared by Reimer-Tiemann formylation [10a] and the
subsequent esterification of N-Boc tyrosine 8 as reported
previously [8, 10b]. Although we attempted the conversion
of 9 into iodobenzene 10 by the action of I2 and H2O2, the
reaction could not be reproduced even in refluxing ethanol.
Hence, we decided to set a robust and reproducible route for
the two-steps-introduction of iodine as follows; (i) NaBH4
reduction of aldehyde 9 into the corresponding alcohol [9a],
and (ii) iodination of the resulting alcohol with I2/H2O2
combination. As a result, probably due to the fact that
electron-withdrawing formyl group was converted to
electron-donating hydroxymethyl group, electrophilic
iodination proceeded smoothly to afford 11 in good yield
(69% for two steps).
molecules
derivative
of
3,4-dihydroxyphenylalanine
(DOPA)
1
[5] through dual Pictet-Spengler (PS)
mechanism [6] (Scheme 1). Briefly, tetrahydroisoquinoline
core is constructed by the following three steps; i) Schiff
base is formed between DOPA derivative 1 and dipeptidic
aldehyde 3, generated from glycine and L-alanine by the aid
of non-ribosomal polypeptide synthetases (NRPSs); ii) PS
cyclization occurs to give 4; and iii) enzymatic region is
reductively eliminated to afford aldehyde 5, which is
involved in the same sequence to furnish SMs and ETs
through intermediates 6 and 7. Thus, to develop an
engineered perpetual SM-producing system, we cloned
necessary biosynthetic gene clusters for SMs and expressed
them in model creatures, however, it was unsuccessful to
detect the production of SMs even by mass spectrometric
analysis [7]. We envisaged that one of the reasons would be
insufficient amount of endogenous non-natural amino acid 1,
Our next task was to oxidize alcohol 11 into Schmidt’s
intermediate 10 with MnO2 under argon atmosphere,
however, our endeavor was wasteful only to observe the
decomposition of substrate 11. We postulated that o-
hydroxybenzaldehyde structure of 10 would be unstable
even to mild heterogeneous oxidant under inert atmosphere,
therefore, methylation of phenolic hydroxy group was first
performed. After selective methylation of 11 using methyl
iodide and potassium carbonate (92%), the resultant o-
methoxybenzylalcohol derivative 12 was subjected to MnO2
oxidation. As expected, the reaction successfully furnished
the desired aldehyde 13 in excellent yield with spot-to-spot
manner (93%).
*Address correspondence to this author at the Department of Chemistry,
Graduate Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan; Tel/Fax: +81-92-642-3913;
E-mail: torikai@chem.kyushu-univ.jp
#These authors contributed equally to this work.
1875-6255/11 $58.00+.00
© 2011 Bentham Science Publishers

Practical Synthesis of DOPA Derivative
Letters in Organic Chemistry, 2011, Vol. 8, No. 10
Me
687
Me
O
O
NRPS
H
N
HO2C
NH2
HO2C
NH2
+
H
X
N
R
H
O
Y
3: R=C13H27
O
Pictet-Spengler
(PS) reaction
Me
OH
OMe
O
1: X=OH, Y=NH2
2: X=OH, Y=NH2·TFA
Me
X
NH
NH
O
X
MeO
OH
PS
NH
Me
O
Me
O
HN
OH
HN
OH
Me
N
H
C13H27
Me
N
C13H27
H
OMe
O
OMe
O
6: X=S-Enzyme
7: X=H
4: X=S-Enzyme
5: X=H
OMe
PS
O
Me
O
HO
O
OMe
Me
H
NH
Me
MeO
HO
Me
N
O
Me
O
AcO
S
N
H
N
MeO
O
Me
N
O
CN
Me
NH
O
O
O
OH
Ecteinascidin 743
Saframycin A
Scheme 1. Hypothetical Biosynthesis of Saframycin A and Ecteinascidin 743.
The final stage of this synthesis, that is, methylation of
iodide 13 by Stille protocol using Pd2(dba)3·CHCl3 and
Me4Sn (14, 82%), followed by saponification, Dakin
oxidation of 14 into DOPA derivative 15 and deprotection to
give 2, was all successful by the process of Schmidt’s report.
However, their reported route seemed not to be suitable for
scale-up, for HPLC purification after the final step was
inevitable.
CONCLUSION
In conclusion, by improving the Schmidt’s method, we
have established a practical synthetic route of DOPA
derivative 2, which should be useful for biosynthetic
production of saframycins and ecteinascidins, potent
antitumor antibiotics. The developed route features higher
reproducibility, more facile handling, and much better
scalability than the original method to tolerate the feeding
experiments. Moreover, this improved route without needs
of setting dry, inert, and cold (< 0 °C) conditions should
open the door to access amino acid 2 for researchers in
biosynthetic community who are not so familiar with organic
synthesis. Further studies toward totally biosynthetic
production of saframycins and their analogs with potent
biological activities, that are available only at low yields or
produced by difficult-to-culture organisms, are now in
progress in our laboratory.
To construct scalable and non-laborious HPLC-free
system, we pursued clean conditions to afford 2, as well as
facile
purification
technique.
After
considerable
experimentation, we found that (i) removal of Boc group
with trifluoroacetic acid (TFA) in the presence of
dimethylsulfide as cation scavenger to avoid side reactions,
probably including Friedel-Crafts type t-butylation of
electron-rich aromatic ring, and (ii) recrystallization of crude
TFA salt from diethyl ether/methanol (10:1) furnished the
desired product 2 with excellent purity [11].

688 Letters in Organic Chemistry, 2011, Vol. 8, No. 10
Torikai et al.
I
OH
OH
OH
O
ref. 8
OH
b,c
BocHN
CO2H
BocHN
CO2Et
BocHN
CO2Et
8
11
9
e
I
a
d
I
OMe
OH
OH
O
BocHN
CO2Et
BocHN
CO2Et
12
10
f
Me
R
Me
OMe
OH
OMe
O
OMe
OH
h,i
j
TFA.H2N
CO2H
BocHN
CO2Et
BocHN
CO2H
13: R=I
14: R=Me
2
15
g
Scheme 2. Reagents and Conditions: (a) I2, 30% H2O2 aq, EtOH, rt, 115 h, no reaction; (b) NaBH4, EtOH, 0 °C, 20 min, 78%; (c) I2, H2O2,
EtOH, rt, 1.5 h, 88%; (d) MnO2, CH2Cl2, rt, 1 h, decomposed; (e) MeI, K2CO3, acetone, reflux, 15 h, 92%; (f) MnO2, Na2SO4, CH2Cl2, rt, 25
min, 93%; (g) Pd2(dba)3·CHCl3, Me4Sn, NMP, 70 °C, 71 h, 82%; (h) 4 M LiOH aq, THF, 0 °C, 3.5 h, then H2O2, rt, 41 h; (i) 4 M
NH3/MeOH, MeOH, rt, 3.5 h; (j) TFA, Me2S, CH2Cl2, rt, 3.5 h, then recrystallization from diethyl ether/methanol (10:1), 42% (over 3 steps).
tunicate Ecteinascidia turbinate. J. Org. Chem., 1990, 55, 4512-
4515.
ACKNOWLEDGEMENTS
We are grateful to Kiyofumi Wanibuchi in our laboratory
for spectroscopic analyses. This work was supported by
grants from The Naito Foundation (to K.T.), Japan Society
for the Promotion of Science, Grant-in-Aid for Young
Scientists (B) (No. 21780106) (to K.W.) and Industrial
Technology Research Grant Program in 2009 (No.
09C46001a) from New Energy and Industrial Technology
Development Organization (NEDO) of Japan (to K.W.). This
work was also supported in part by Kato Memorial
Bioscience Foundation (to K.W.), by The Sumitomo
Foundation (to K.W.) and by Takeda Science Foundation (to
K.W.). This paper is dedicated to the memory of Dr. Tetsuo
Shiba (Professor Emeritus of Osaka University, Japan), who
contributed enormously to our knowledge of amino acid and
peptide chemistry.
[3]
[4]
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[5]
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Practical Synthesis of DOPA Derivative
Letters in Organic Chemistry, 2011, Vol. 8, No. 10
689
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28
[11]
Data of 2: Mp. 233-236 °C; [ꢀ]D -6.1 (c 0.13, H2O); 1H NMR
(400 MHz, CD3OD) ꢀ 7.55 (1H, s), 7.34 (1H, s), 3.82 (4H, m), 3.14
[10]
(2H, m), 2.32 (3H, s); 13C NMR (100 MHz, CD3OD)ꢀ 173.2,
170.2, 158.7, 136.8, 134.4, 132.4, 130.9, 127.2, 61.9, 57.1, 37.3,
24.8, 16.2 ppm; IR (KBr) 3431, 2989, 2361, 2341, 1695, 1610,
1481, 1396, 1265, 1220, 1139, 1011, 897, 807 cm-1.