The macrocycle of leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product

Article abstract of DOI:10.1016/j.bmcl.2012.04.003

Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property.

Full text of DOI:10.1016/j.bmcl.2012.04.003

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3791–3794
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The macrocycle of leinamycin imparts hydrolytic stability
to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product
Santhosh Sivaramakrishnan a, Leonid Breydo a, Daekyu Sun c, Kent S. Gates a,b,
⇑
a Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, United States
b Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, United States
c Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, BIO5 Institute, Room 102, 1657 E. Helen Street, Tucson, AZ 85721, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the gener-
ation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin
reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to
the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing
property.
Received 6 February 2012
Revised 21 March 2012
Accepted 2 April 2012
Available online 12 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
DNA cleavage
DNA damage
The propensity to undergo intracellular bioactivation is an
important property of many DNA-damaging natural products.1–5
Characterization of these processes is relevant to both medicinal
chemistry and toxicology as it has the potential to reveal novel
and chemically interesting strategies for the intracellular genera-
tion of biologically active reactive intermediates.6 The interior of
cells is relatively rich in thiols7 and reaction with intracellular
thiols represents a common means for unmasking DNA-damaging
intermediates from natural products. For example, calicheamicin,
dynemicin, neocarzinostatin, acylfulvenes related to the
illudins, myrocin C, 2-crotonyloxymethyl-2-cycloalkeneones,
lissoclinotoxin A, varacin, thiarubrin C, and some analogues of
mitomycin C can be activated by reactions with thiols.8–24
Leinamycin (1) is a thiol-activated, Streptomyces-derived sec-
ondary metabolite that displays potent activity against human
cancer cell lines.25–29 Leinamycin contains a unique 1,2-dithio-
lan-3-one 1-oxide heterocycle that serves as its ‘thiol sensing’ unit.
Reaction of thiols with this moiety initiates rearrangement of
leinamycin into a DNA-alkylating episulfonium ion 4 and converts
the attacking thiol into a persulfide 2 that can mediate generation
of reactive oxygen species (Scheme 1).17,30–44 The 1,2-dithiolan-3-
one 1-oxide heterocycle is hydrolytically labile; however, in the
context of leinamycin this reaction is slow relative to the thiol-
mediated activation of the natural product.45 Indeed, it seems
likely that resistance to hydrolysis coupled with high thiol-reactivity
combine to allow efficient and selective bioactivation of leinamy-
cin inside cells.
O
O
OH
OH
O
C7
O
C3'
C6
C8
C4'
SR
S
S
C3
OH
H
OH
H
H
H
S2'
SO–
SR
S1'S+
O–
O
C1
N
N
O
N
H
N
H
S
S
1
:Nu-DNA
O
OH O
CH3
CO2
O
–
O
S
H
N
S+
OH
H
OH
H
H
HO
N
H
O
N
H
O
N
H
S
RSS–
S
3
4
2
Nu-DNA
CH3
CO2
O
–
S
OH
H
H
HO
N
H
O
N
H
S
⇑
Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754.
E-mail address: gatesk@missouri.edu (K.S. Gates).
Scheme 1. Thiol-activated DNA alkylation by leinamycin.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.003

3792
S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3791–3794
Cl
CO2Et
O
O
t-BuOK,THF,
0 o
CO2Et
C
Na, EtOH
O
PhCH2SH
HO
HO
O
OH
Ti(i-OPr)4
THF
–
CO2
S
Ph
Na, liq. NH3
THF
O
OH
DCC, DMAP
THF,RT
OH
SH
Figure 1. A representative plot showing the decomposition of
buffered solution (5 (70 M); MOPS (250 mM, pH 7); MeCN (25% v/v); 24 °C). The
disappearance of 5 was monitored by HPLC using a C18 reverse phase column
(Varian microsorb-MV 100 Å pore size, 5 m particle size, 250 mm length, 4.6 mm
5 in aqueous
O
l
S
l
1.H2S, TEA, –78oC
diam.) eluted with acetonitrile/water (35:65) at a flow rate of 1.0 mL/min.
2. FeCl3, MeOH
HO
HO
O
O
HO
O
O
O
3
4
at 19.1 ppm. The methyl group at C5 with a cis relationship to the
C4 methyl appears at 20.1 ppm and the methyl group at C5 with a
trans relationship to the C4 methyl appears at 17.1. Literature
precedents indicate that a methyl group with a cis relationship to
the oxygen of an adjacent sulfoxide will appear upfield relative
to a methyl group in the trans orientation.49,53 Thus, our data
showing that the shielded, upfield methyl at C5 is trans to the C4
methyl, suggested that, in the major isomer, the methyl group at
C5 which has a cis relationship to the sulfoxide oxygen also has a
cis relationship to the hydroxyl group at C4. This is consistent with
structure 5 for the major isomer. Thus, we were able to assign the
major isomer as 5, in which the 4-hydroxyl group is cis to the sulf-
oxide oxygen, and the minor isomer as 6, in which the 4-hydroxyl
group is trans to the sulfoxide oxygen.54 In the oxidation leading to
5 and 6, hydrogen bonding may direct dimethyldioxirane to
the same face as the hydroxyl substituent leading to favored
production of 5 during the synthesis.55 For the remainder of this
Letter, 5 will be referred to as the cis isomer and 6 as the trans
isomer.
With the cis and trans isomers of leinamycin’s thiol-sensing unit
in hand, we examined their stability at 24 °C in an aqueous buf-
fered solution containing the compound (5 or 6, 70 lM), MOPS buf-
fer (250 mM, pH 7), and acetonitrile (25% v/v). Disappearance of
the compounds was monitored by HPLC over the course of three
half-lives and the data fit to a first-order decay process to deter-
mine apparent rate constants and half-lives for the decomposition
process (Fig. 1).56
The apparent rate constants for the decomposition of 5 and 6 un-
der these conditions were measured at kobs = 13.2 0.01 ꢀ 10ꢁ3 minꢁ1
(t1/2 = 53 min) and kobs = 6.2 0.6 ꢀ 10ꢁ3 minꢁ1 (t1/2 = 112 min),
respectively (Table 1). The decomposition rate of 6 increases with
increasing buffer concentration (kobs = 1.33 0.02 ꢀ 10ꢁ2 minꢁ1 at
100 mM MOPS, kobs = 1.5 0.2 ꢀ 10ꢁ2 minꢁ1 at 300 mM MOPS, and
kobs = 1.9 0.2 ꢀ 10ꢁ2 minꢁ1 at 500 mM MOPS). While the contribution
of buffer is significant, at buffer concentrations used in our studies the
buffer-independent reaction predominates. Near the physiological pH
range, the rate at which 6 decomposes increases with increasing pH
(kobs = 8.7 ꢀ 10ꢁ3 minꢁ1 at pH 6.5, kobs = 1.5 ꢀ 10ꢁ2 minꢁ1 at pH 7.0,
and kobs = 4.7 ꢀ 10ꢁ2 minꢁ1 at pH 7.5; all at 300 mM MOPS). This mir-
rors the effect of pH observed previously on the stability of leinamycin
in sodium phosphate buffer containing 10% methanol.57 The pH effects
are consistent with literature precedent indicates that hydroxide rather
than water is the kinetically relevant species involved in the hydrolysis
of thiosulfinates and thioesters.58–60 We find that the decomposition of
leinamycin under the same conditions used for the 5and 6above occurs
2
5
S
S
1
S
S
S
+
O–
+
S
acetone
O–
5
6
Scheme 2. Synthesis of 1,2-dithiolan-3-one 1-oxides related to leinamycin.
To better understand the chemical events underlying the bioac-
tivation of leinamycin, we examined the inherent reactivity of the
thiol-sensing unit found in the natural product. Accordingly, we
synthesized this fragment of leinamycin via a modification of the
route described by Pattenden and Shuker (Scheme 2).46 The pub-
lished route employing NaSH for the epoxide ring-opening was
ineffective in our hands, perhaps due to the notoriously impure
nature of commercially available NaSH reagents.47 Instead, we
used benzyl mercaptan as a protected hydrogen sulfide surrogate.
In addition, we employed dimethyldioxirane for the final oxidation
step rather than m-CPBA.48 The final oxidation reaction gave two
major products and proton NMR analysis suggested that these
were a 4:1 mixture of two diastereomers. These compounds were
separable by thin layer chromatography and column chromatogra-
phy on silica gel. The major isomer showed three methyl reso-
nances at 1.74, 1.37, and 1.19 ppm, while resonances for the
methyl groups in the minor isomer appeared at 1.58, 1.62, and
1.28 ppm. HMBC experiments were used to unambiguously assign
the chemical shifts of the 4-methyl groups as the resonances at
1.37 ppm and 1.58 ppm for the major and minor isomer, respec-
tively, via observation of three-bond coupling between methyl
hydrogens and the carbonyl carbon. Literature precedents indicate
that the proton NMR resonance of a methyl group in the cis orien-
tation relative to a sulfoxide oxygen in a five-membered ring will
be shifted downfield relative to a trans methyl group.49–52 Thus,
the NMR results immediately suggested that the major isomer
has the methyl group in the 4-position in a trans relationship to
the sulfoxide oxygen as shown in 5 (Scheme 2). Consistent with
this assignment, an NOE experiment on the major isomer revealed
a much stronger through space interaction of the 4-methyl substi-
tuent with the 5-methyl group that displays a resonance at
1.19 ppm (trans to the sulfoxide oxygen) than with the 5-methyl
group at 1.74 ppm (cis to the sulfoxide oxygen). Analysis of
13C-chemical shifts further supported the structural assignment.
An HMQC experiment, along with the HMBC and NOE experiments
described above, allowed us to assign the chemical shifts of each
methyl group in the major isomer. The methyl group at C4 appears

S. Sivaramakrishnan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3791–3794
3793
Table 1
Rate constants for 1, 5, and 6
Compound
Conditions
kobs (minꢁ1
)
t1/2
53 min
112 min
27 h
5 No thiol
6 No thiol
1 No thiol
5 + Thiol
6 + Thiol
1 + Thiol
5 (70
6 (70
1 (70
5 (70
5 (70
5 (70
l
l
l
l
l
l
M); MOPS (250 mM, pH 7); MeCN (25% v/v); 24 °C
M); MOPS (250 mM, pH 7); MeCN (25% v/v); 24 °C
M); MOPS (250 mM, pH 7); MeCN (25% v/v); 24 °C
M); GSH (700
M); GSH (700
M); GSH (700
13.2 0.01 ꢀ 10ꢁ3
6.2 0.6 ꢀ 10ꢁ3
0.408 0.001 ꢀ 10ꢁ3
24.3 0.1 ꢀ 10ꢁ2
18.3 0.2 ꢀ 10ꢁ2
6.67 0.01 ꢀ 10ꢁ2
l
l
l
M) MOPS (300 mM, pH 7); MeCN (25% v/v); 24 °C
M) MOPS (300 mM, pH 7); MeCN (25% v/v); 24 °C
M) MOPS (300 mM, pH 7); MeCN (25% v/v); 24 °C
2.8 min
3.8 min
10.4 min
with a rate constant of kobs = 0.408 0.001 ꢀ 10ꢁ3 minꢁ1 (t1/2 = 27 h).
or C30—reactions that may be suppressed by the macrocyclic
substituent.
This value is generally consistent with a previous measurement of
leinamycin’s stability (100
l
M 1 in HEPES, 250 mM, pH 7, 24 °C, con-
Previous work established three mechanisms by which the mac-
rocycle of leinamycin may facilitate efficient DNA alkylation. First,
the Z,E-5-(thiazol-4-yl)-penta-2,4-dienone portion of the macrocy-
taining no organic co-solvent) that gave a rate constant of 0.72 ꢀ 10
ꢁ3 minꢁ1 (t1/2 = 16.1 h).45,61 Clearly, the 1,2-dithiolan-3-one 1-oxide
heterocycle, when embedded in the context of the natural product, en-
joys remarkably increased stability compared to the simple analogues 5
and 6.
We next examined the reaction of 5, 6, and leinamycin (70
with the biological thiol glutathione (GSH, 700 M) in MOPS buffer
(300 mM, pH 7) containing acetonitrile (25% v/v). The pseudo-first-
cle presents a slightly twisted p-surface that confers non-covalent
DNA-binding properties to the natural product.32,64,65 Second, the
hydroxyl group at C8 of the macrocycle engages the episulfonium
ion 5 in a reversible thia-Payne reaction that may stabilize the epis-
ulfonium ion against hydrolytic destruction.30 Third, the conforma-
tionally rigid macrocycle may accurately position the C6–C7 alkene
for efficient reaction with the electrophilic sulfur of 3 in the gener-
ation of the alkylating agent 4 (Scheme 1).66 The work presented
here establishes an additional role for the macrocyclic portion of
leinamycin in facilitating efficient thiol-triggered alkylation of
cellular DNA. The macrocyclic portion of leinamycin imparts
substantial aqueous stability to the 1,2-dithiolan-3-one 1-oxide
without compromising its ability to act as a thiol-sensing unit.
Thus, the 18-membered macrocycle of leinamycin enables efficient
and selective bioactivation of the natural product in the thiol-rich
environment found inside cells.
lM)
l
order rate constants for the disappearance of 5 and 6 under these
conditions were measured at kobs = 24.3 0.1 ꢀ 10ꢁ2 minꢁ1 (t1/2
=
2.8 min) and kobs = 18.3 0.2 ꢀ 10ꢁ2 minꢁ1 (t1/2 = 3.8 min), respec-
tively. From these values, one can estimate second-order rate
constants of 344 Mꢁ1 minꢁ1 for 5 and 261 Mꢁ1 minꢁ1 for 6. The
apparent rate constant for the reaction of leinamycin with GSH
under these conditions was kobs = 6.67 0.01 ꢀ 10ꢁ2 minꢁ1
(t1/2 = 10.4 min, 95 Mꢁ1 minꢁ1). This is reasonably close to the
value of 10.4 Mꢁ1 sꢁ1 (624 Mꢁ1 minꢁ1) reported previously for
the reaction of leinamycin with GSH (in HEPES buffer, 50 mM, pH
7, 24 °C, containing no organic co-solvent).45
The natural product leinamycin was isolated as the single ste-
reoisomer shown in Scheme 1, with a trans relationship between
the C40-OH group and the S10-sulfinyl oxygen.62 Our work with 5
and 6 show that the naturally-occurring trans isomer is approxi-
mately two times more stable than the cis isomer in aqueous buf-
fered solution. However, differences in the hydrolytic stability of
the cis/trans isomers 5 and 6 are subtle compared to the dramatic
effect that leinamycin’s 18-membered macrocycle brings to the
stability of the natural product in aqueous solution. Leinamycin
is approximately 30 times more stable than 5 and 15 times more
stable than 6 against decomposition in aqueous buffer. It is inter-
esting to consider potential mechanisms by which the macrocycle
stabilizes leinamycin against decomposition in aqueous solution.
Based upon computational analysis of small model systems, Wu
Acknowledgment
We are grateful to the National Institutes of Health for support
of this work (CA83925 and 119131).
References and notes
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!
rꢂS10 interaction between the
amide carbonyl in the macrocycle and the S10-sulfinyl group of
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by ꢃ6 kcal/mol.63 Alternatively, or in addition, the macrocycle of
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of leinamycin. The solution structure of leinamycin deserves
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0.28 mmol) in acetone (0.5 mL) in an ice bath was added freshly prepared
dimethyldioxirane (dropwise using
a pipette). The disappearance of the
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starting material was closely monitored by TLC (5:1 hexane/ethyl acetate)
and the reaction was complete in about 5 min. Flash column chromatography
on silica gel eluted with ethyl acetate/dichloromethane/hexane (1:3:4) gave
the diastereomers 5 and 6 as colorless solids (50 mg total, 90%). Compound 5:
Rf = 0.36; 1H NMR (300 MHz, CDCl3) d 1.19 (3H, s, C5-CH3 trans to sulfoxide
oxygen), 1.37 (3H, s, C4-CH3), 1.74 (3H, s, C5-CH3 cis to sulfoxide oxygen); 13C
NMR (CDCl3, 62.9 MHz) d 201.8 (C@O), 84.9 (C4), 66.7 (C5), 20.1 (C5-CH3 trans
to sulfoxide oxygen), 19.1 (C4-CH3), 17.1 (C5-CH3 cis to sulfoxide oxygen);
HRMS: (EI) calcd for C6H10O3S2 [M+Li]+ 201.0225, found 201.0215. Compound
6: Rf = 0.24 1H NMR (300 MHz, CDCl3) d 1.27 (3H, s), 1.58 (3H, s), 1.62 (3H, s),
2.9 (1H, s, OH); 13C NMR (CDCl3, 62.9 MHz) d 201.9, 84.7, 65.9, 24.2, 18.8, 16.8;
HRMS: (EI) calcd for C6H10O3S2 [M+Na]+ 216.9963, found 216.9962.
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