Acid-promoted DNA-cleaving activities and total synthesis of varacin C

Article abstract of DOI:10.1021/ja020531i

The first total synthesis of the antibiotic varacin C has been accomplished. In addition, it has been demonstrated that this antibiotic exhibits potent antitumor activity and is capable of causing efficient DNA cleavage under acidic conditions. Copyright

Full text of DOI:10.1021/ja020531i

Published on Web 11/02/2002
Acid-Promoted DNA-Cleaving Activities and Total Synthesis of Varacin C
Alex H. F. Lee, Jian Chen, Dongsheng Liu, Thomas Y. C. Leung, Albert S. C. Chan,* and Tianhu Li*
Open Laboratory of Chirotechnology of the Institute of Molecular Technology for Drug DiscoVery and Synthesis
and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic UniVersity,
Hung Hom, Kowloon, Hong Kong
Received April 12, 2002
The antibiotic varacin C (3, Scheme 1),1 a new member of the
varacin2 family, was discovered in the Far Eastern ascidian Polycitor
sp. by Makarieva and associates in 1995. The unprecedented trithiol
oxide ring contained in varacin C makes it quite different from
any other previously identified microbial metabolite. With the aim
of clarifying the mode of action of varacin C at the molecular level,
a program was initiated recently in our laboratories directed at the
total synthesis and examination of the chemical and biological
properties of this antibiotic. We now report the first total synthesis
of varacin C and show that this molecule can cause efficient DNA
cleavage under acidic conditions. Given that the extracellular pH
in most tumor cells is lower than that in normal tissue3 and that
antibiotics which possess acid-triggered functionalities are very rare
in nature, this newly discovered mode of action of varacin C might
have significant implications in tumor chemotherapy.4
Scheme 1 illustrates our total synthesis of varacin C. The
synthesis begins with 1, prepared by a slight modification of the
earlier procedures of Davidson and co-workers.5 Installation of the
trithiol oxide functionality was achieved by reduction of 1 with
LiAlH4 followed by seven further reactions6 in which the overall
yield of the total synthesis was 24% (Scheme S1).
The DNA-cleaving activity of varacin C was examined by
monitoring its effectiveness in converting circular supercoiled DNA
(form I) to the corresponding circular nicked form (form II). Plasmid
pBR322 was accordingly incubated with varacin C in different
buffer solutions (pH values 5.0-7.5). As shown in Figure 1A,
varacin C caused single-stranded DNA cleavage effectively at pH
5.0 (41%, lane 2), 5.5 (47%, lane 4), and 6.0 (39%, lane 6), but
not at higher pH: [pH 7.0 (23%, lane 10) and pH 7.5 (12%, lane
Figure 1. Thiol-dependent cleavage of DNA by varacin C. Assays were
performed in 50 mM sodium phosphate containing 10% (v/v) acetonitrile
12)]. Moreover, even a low concentration of varacin C (50 nM)
could generate a detectable amount of form II DNA at pH 5.5 (lane
4, Figure S1, Supporting Information).
and 0.5 µg of supercoiled pBR322 DNA in the presence or absence of
varacin C and activating reagents (20 µL). (A) pH dependence of DNA
cleavage by varacin C. The concentrations of varacin C and 2-mercapto-
ethanol in the mixtures (37 °C, 2 h) corresponding to lanes 2, 4, 6, 8, 10,
and 12 were 5 and 500 µM, respectively. (B) Activation of varacin
C-promoted DNA cleavage by different nucleophiles. Concentrations of
all the added nucleophiles in the mixtures (pH 5.5, 37 °C, 6 h, and 5 µM
varacin C) were 1 mM. (C) Inhibitory effects of radical scavengers and
nucleophiles on DNA cleavage by varacin C. Concentrations of varacin C
and 2-mercaptoethanol in mixtures (pH 5.5, 37 °C, 3 h) corresponding to
lanes 2-11 were 5 and 500 µM, respectively. Lane 1: DNA alone; lane 2:
standard DNA-cleaving reaction without added inhibitors; lane 3: 53 µg/
mL catalase; lane 4: 10 mM pyridine; lane 5: 10 mM 4-methylaniline;
lane 6: 1 M methanol; lane 7: 1 M ethanol; lane 8: 80 mM DTT; lane 9:
100 mM mannitol; lane 10: 2.5 mM DETAPAC; lane 11: 80 µg/mL SOD;
lane 12: 200 mM Tiron.
The activating course of the DNA cleavage by varacin C was
next examined by adding different types of thiols to the corre-
sponding reaction mixtures. As shown in Figure 1B, both gluta-
thione and cysteine were capable of triggering varacin C-mediated
DNA cleavage as effectively as 2-mercaptoethanol (lanes 4 and
6). However, when 2-mercaptoethanol was replaced with phenol
or aniline (lanes 8 and 10), DNA cleavage did not occur. In addition,
the DNA-cleavage by varacin C did not take place when thiol was
omitted from the reaction (lane 1).
With the aim of identifying the nature of the reactive species
responsible for the DNA cleavage by varacin C, the inhibitory
effects of certain nucleophiles and free radical scavengers were
investigated. As shown in Figure 1C, addition of pyridine or
4-methylaniline to the reaction mixture did not slow the rate of the
reactions (lanes 4 and 5), a result which could be indicative of the
absence of an active electrophilic species produced in the DNA-
cleaving process. However, the rate of this DNA-cleaving reaction
was reduced drastically by the addition of the known superoxide
radical (O2•-) scavenger dithiothreitol (DTT) (lane 8) and Tiron
(lane 12).7 In addition, catalase (an enzyme that reduces the
concentration of hydrogen peroxide in solution)8,9 (lane 3) ex-
hibited significant inhibitory effects on the DNA-cleavage reac-
tion. Moreover, this DNA-cleaving process was inhibited by the
* Corresponding author. E-mail: bctli@polyu.edu.hk.
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J. AM. CHEM. SOC. 2002, 124, 13972-13973
10.1021/ja020531i CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1a
a Reagents and conditions: (a) i. LiAlH4, Et2O, reflux; ii. HCl(aq). (b) (Boc)2O, TEA, THF, reflux, 52%. (c) i. Li/NH3; ii. HCl(aq), 100%. (d) Me2SnCl2,
KOH/H2O/EtOH, 94%. (e) SOCl2, THF, 72%. (f) CH3CN, hν, 75%. (g) TFA, CH2Cl2, 92%.
Scheme 2
varacin C can be promoted by an acidic environment. Tumor cells
possess a lower extracellular pH than normal cells, which is an
intrinsic feature of the tumor phenotype and caused by alterations
either in acid export or in clearance of extracellular acid.3
Pharmaceutical agents may be particularly desirable in cancer
Table 1. Cytotoxic Property of Varacin C toward Certain Cancer
chemotherapy if they undergo selective activation by the acidic
Cell Lines
microenvironments around tumor tissue.4 Accordingly, we hope that
cell type
cell line
IC50 (nM) varacin C
IC50 (nM) doxorubicin
the mode of action of varacin C described in this report may
stimulate the design of a new generation of acid-activated, tumor-
activated prodrugs.
colon cancer
prostate cancer
breast cancer
bladder cancer
lung cancer
renal cell
HT-29
PC-3
MDA231
UMUC3
PACA2
A549
11.9
2.4
2.9
8.4
7.2
41.4
41.8
3.5
2.2
17.1
2.4
Acknowledgment. We thank The Hong Kong Polytechnic
University ASD Fund and the Hong Kong UGC Areas of Excel-
lence Fund (Project No. AoE P/10-01) for financial support of this
study and Drs. Alan Nadin (Merck, UK) and Jinyou Xu (Merck,
U.S.A.) for helpful discussions.
48.2
24.9
carcinoma
A4982LM
46.0
efficient hydroxyl radical (OH•) scavengers methanol (lane 6),
ethanol (lane 7), and mannitol (lane 9).8,9 Besides these oxygen
radical scavengers, the metal chelator diethylenetriaminepentaacetic
acid (DETAPAC) also inhibited the varacin C-promoted DNA-
cleavage reaction (lane 10). DETAPAC is known to sequester
adventitious traces of transition metals, thereby preventing them
from catalyzing the conversion of peroxide to hydroxyl radicals.8,9
On the basis of these observations, we suggest tentatively that
varacin C in concert with a thiol leads to the conversion of
molecular oxygen to hydrogen peroxide, which is then further
converted to the DNA-cleaving hydroxyl radical by a trace-metal-
dependent Fenton reaction (Scheme 2).10 This proposed mechanism
is analogous to the mode of action of antibiotic leinamycin11
established by Gates and associates earlier.
It should be pointed out that addition of superoxide dismutase
(SOD) (an enzyme which decomposes superoxide)8,12 to the reaction
mixture did not slow the rate of the DNA-cleavage reaction (lane
11, Figure 1C). This observation does not rule out the possibility
that superoxide radical is involved in the DNA-cleaving process
by varacin C. This is because the hydrogen peroxide produced by
SOD from superoxide could itself lead to DNA cleavage through
a Fenton reaction in which a thiol serves as the reducing reagent,
a phenomenon observed in the DNA-cleaving reaction by leinama-
cin.11 Further characterization of the mechanism by which varacin
C cleaves DNA is still in progress.
The cytotoxic activity of varacin C toward seven different human
cancer cell lines was evaluated by determining the concentration
(IC50) of the antibiotic at which 50% of cell proliferation was
inhibited, and comparing these values with data obtained for
doxorubicin in the same cell lines. As shown in Table 1, varacin C
is more potent than the clinically used antitumor agent doxorubicin
in cell lines of colon cancer, prostate cancer, human breast cancer,
lung cancer, and carcinoma, exhibiting IC50 values against these
cell lines in the range 2.4-48.2 nM.
In conclusion, our studies demonstrate for the first time that
varacin C is capable of causing DNA cleavage effectively. This
may explain the observed potent cytotoxic activities of varacin C
(see Table 1) and its antifungal and antimicrobial activities against
Candida albicans and Bacillus subtilus.1 Most significantly, the
current work reveals that the rate of this DNA-cleaving course by
Supporting Information Available: Experimental details of the
synthesis of varacin C, characterization data for all compounds, Figure
S1, and Scheme S1 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
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