Triostin A derived hybrid for simultaneous DNA binding and metal coordination

Article abstract of DOI:10.1007/s00726-010-0764-3

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7] triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.

Full text of DOI:10.1007/s00726-010-0764-3

Amino Acids (2011) 41:449–456
DOI 10.1007/s00726-010-0764-3
ORIGINAL ARTICLE
Triostin A derived hybrid for simultaneous DNA binding
and metal coordination
•
Eike-F. Sachs Andre Nadler Ulf Diederichsen
•
´
Received: 19 May 2010 / Accepted: 20 September 2010 / Published online: 22 October 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract The natural product triostin A is known as an
antibiotic based on specific DNA recognition. Structurally,
a bicyclic depsipeptide backbone provides a well-defined
scaffold preorganizing the recognition motifs for bisinter-
calation. Replacing the intercalating quinoxaline moieties
of triostin A by nucleobases results in a potential major
groove binder. The functionalization of this DNA binding
triostin A analog with a metal binding ligand system is
reported, thereby generating a hybrid molecule with DNA
binding and metal coordinating capability. Transition metal
ions can be placed in close proximity to dsDNA by means
of non-covalent interactions. The synthesis of the nucleo-
base-modified triostin A analog is described containing a
propargylglycine for later attachment of the ligand by
click-chemistry. As ligand, two [1,4,7]triazacyclononane
rings were bridged by a phenol. Formation of the proposed
binuclear zinc complex was confirmed for the ligand and
the triostin A analog/ligand construct by high-resolution
mass spectrometry. The complex as well as the respective
hybrid led to stabilization of dsDNA, thus implying that
metal complexation and DNA binding are independent
processes.
Introduction
DNA binding peptides have received considerable attention
´
as promising drug candidates (Vazquez et al. 2003). Valu-
able lead structures in drug design are derived from peptidic
natural products. Numerous methods exist for peptide
modification with respect to sequence, backbone or side
chains, also incorporating non-proteinogenic amino acids
(Gante 1994). Next to actinomycin D as one of the most
prominent examples of a DNA binding peptidic natural
product used in anti-cancer therapy (Farber 1966; Lewis
1972), the quinoxaline antibiotics echinomycin, thiocora-
line, and triostin A (1) are well established (Lee and Waring
1978; Waring and Wakelin 1974). Transcription and rep-
lication are efficiently blocked by these sequence-specific
double strand DNA bisintercalators, thus possessing
antibiotic and cytotoxic activity (Katagiri et al. 1975). The
disulfide-bridged depsipeptide backbone of triostin A pro-
vides a rigid scaffold preorganizing two quinoxaline inter-
˚
calators in a distance of 10.5 A. Therefore, bisintercalation
of dsDNA spanning a dinucleotide is entropically favored
(Addess and Feigon 1994). In earlier studies, the pep-
tide scaffolds of triostin A, des-N-tetramethyltriostin A
(2, TANDEM) (Ciardelli and Olsen 1977), and the peptide
analog aza-TANDEM (3) (Dietrich and Diederichsen 2005)
were used as rigid templates for functional modification in
order to generate a new class of DNA binders (Fig. 1). As
part of our studies to change the DNA binding mode of
triostin A and its derivatives from bisintercalation to spe-
cific major groove recognition, the rigid templating depsi-
peptide scaffold is used to organize nucleobases or other
recognition moieties instead of the native quinoxaline
moieties (Lorenz and Diederichsen 2004).
Keywords Amino acids ꢀ Click-chemistry ꢀ
DNA recognition ꢀ Metal binding ꢀ Triostin A
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-010-0764-3) contains supplementary
material, which is available to authorized users.
´
Eike-F. Sachs ꢀ Andre Nadler ꢀ U. Diederichsen (&)
Institut fu¨r Organische und Biomolekulare Chemie,
¨
Georg-August-Universitat Gottingen, Tammannstr. 2,
37077 Gottingen, Germany
¨
With the intention to use the DNA recognition unit for
guiding a functional metal complex in proximity to the
¨
e-mail: udieder@gwdg.de
123

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Eike-F. Sachs et al.
Fig. 1 Structures of triostin A (1), TANDEM (2), aza-TANDEM (3), and hybrid of the nucleobase substituted aza-TANDEM 4 with a ligand
capable of forming binuclear transition metal complexes
oligonucleotide backbone, synthesis of a hybrid molecule
was intended. Comparable examples for the use of the
hybrid approach in drug design are the combination of a
DNA binding motif with a cell penetrating peptide (Langer
et al. 2001) or a cytotoxic warhead (Xie et al. 1996; Jia
et al. 1999; Baraldi et al. 1998). We synthesized a conju-
gate of a triostin A derivative as DNA binder and a ligand
capable of forming binuclear transition metal complexes.
Such a hybrid molecule 4 was expected to retain its DNA
binding properties, and additionally, being able to recruit
loosely bound copper and zinc ions from cellular pools.
Positioning a binuclear metal center in close proximity of
DNA strands can be considered as an opportunity to gen-
erate, e.g., reactive oxygen species (ROS), which ulti-
mately cause cytotoxic DNA damage (Sensi et al. 1999;
Manzl et al. 2004).
derivative was ligated with an azide-functionalized ligand
composed of two [1,4,7]triazacyclononane (TACN) moie-
ties and a bridging phenol. Similar ligands have been
shown to form binuclear zinc(II) and copper(II) complexes
(Rossi et al. 1998) and to catalyze hydrolysis (O’Donoghue
et al. 2006). Two metal ions are likely to be arranged in a
˚
distance of 3.4 A which is well suitable for cooperative
processes.
Materials and methods
General remarks
The solvents used were of the highest grade available.
Dichloromethane and triethylamine were distilled from
calcium hydride prior to use. DMF was purchased dry and
A common strategy for the attachment of ligands to
peptides is based on the incorporation of artificial amino
acid building blocks by solid phase peptide synthesis
˚
stored over molecular sieves (4 A). All reagents were of
analytical grade and used without further purification.
NMR spectra were recorded with a Varian Unity 300 or
Varian INOVA-600 instrument. Chemical shifts are refer-
enced to the residual solvent peaks of [D6]DMSO (1H:
d = 2.49 ppm; 13C: d = 39.5 ppm) or CDCl3 (1H:
d = 7.24 ppm; 13C: d = 77.0 ppm). ESI-MS data were
measured with a LCQ Finnigan spectrometer. HRMS data
were determined with a Bruker APEX-Q IV 7T spec-
trometer. Chromatographic separations were performed
using silica gel 60 (0.040–0.063 mm, Merck). HPLC sep-
¨
(Dirscherl and Konig 2008; Levadala et al. 2004; Cheng
et al. 2005; Damian et al. 2010). Bipyridine- and phenan-
throline-substituted amino acids or functionalized lysine
and tyrosine derivatives are reported (Jiang et al. 2005;
Iranzo et al. 2007). In addition, site-specific coupling of
ligand systems with amino acid side chains can be achieved
by disulfide formation (Su et al. 2006), C–C coupling
reactions (Albrecht et al. 2003) or copper-mediated [3 ? 2]
cycloaddition of an azide to an alkyne (click-chemistry)
(Best 2009; Nadler et al. 2009). Click-chemistry can be
applied especially on sterically hindered or conformational
rigid systems. Therefore, it was used for the ligation of our
triostin A analog with the ligand providing construct 4 as a
chimera. A propargylglycine containing the aza-TANDEM
¨
arations were carried out with a Pharmacia Akta Basic
using J’sphere ODS-H80 RP-C18 columns for analytical
samples (250 9 4.6 mm, S-4 lm, 8 nm, 1 mL min-1) and
for preparative samples (250 9 20 mm, S-4 lm, 8 nm,
10 mL min-1) with a linear gradient of A (0.1% TFA in
123

Triostin A derived hybrid for simultaneous DNA binding and metal coordination
451
H2O) to B (MeCN/H2O, 9:1 ? 0.1% TFA). Analytical
thin-layer chromatography was performed using Merck
silica gel 60 F254 precoated aluminum plates. Visualiza-
tion was achieved with UV light (254 nm) or by dyeing
with ninhydrin (3% in ethanol).
[M ? H]?. HRMS (ESI): calcd. for C40H68BrN6O9
([M ? H]?): 855.42257, found: 855.42230.
4-Bromo-2,6-bis-([1,4,7]triazacyclononane-1-yl-methyl)-
phenol (16)
Di-tertbutoxycarbonyl-[1,4,7]triazacyclononane (BBT, 12)
A solution of ligand 15 (48 mg, 56 lmol) in TFA/
DCM = 1:1 (2 mL) was stirred at rt for 1 min. The mix-
ture was evaporated to dryness in vacuo and purified by
HPLC (preparative, 0–60% MeOH ? 0.1% TFA in
30 min, Rt = 21.61 min) to yield a yellowish solid
(21.7 mg, 85%). 1H-NMR (300 MHz, [D6]DMSO):
d = 2.80–2.87 (m, 8 H, TACN-2,20,9,90-H2), 3.06–3.15 (m,
8 H, TACN-3,30,8,80-H2), 3.43–3.51 (m, 8 H, TACN-
5,50,6,60-H2), 3.80 (s, 4 H, CH2), 7.43 (s, 2 H, Ar-H), 9.13
(s br, 4 H, NH) ppm. 13C-NMR (150 MHz, [D6]DMSO):
d = 42.8 (TACN-2,20,9,90-CH2), 43.9 (TACN-3,30,8,80-
CH2), 48.4 (TACN-5,50,6,60-CH2), 52.9 (CH2), 110.5 (Ar-
CBr), 126.9 (Ar-CCH2), 133.4 (Ar-CH), 153.9 (Ar-COH)
ppm. HRMS (ESI): calcd. for C20H35BrN6O ([M ? H]?):
455.2128, found: 455.2120.
Dry Et3N (5.18 mL, 37.1 mmol) was added to a cooled
solution (-5°C) of TACN (2.40 g, 18.6 mmol) in dry
DCM (60 mL). A solution of Boc2O (tert-butyloxycar-
bonyl anhydride, 8.10 g, 37.1 mmol) in dry DCM (20 mL)
was added over a period of 1 h and stirred for 30 min at
-5°C. All volatile components were removed in vacuo and
the residue was dissolved in MeOH/EtOAc = 1:8 and
purified by flash chromatography (4 9 18 cm, MeOH/
EtOAc = 1:8) to yield a yellow oil (3.28 g, 54%). 1H-
NMR (300 MHz, CDCl3): d = 1.45 (s, 18 H, Boc-CH3),
2.87–2.97 (m, 4 H, 2, 9-H2), 3.17–3.32 (m, 4 H, 3, 8-H2),
3.39–3.52 (m, 4 H, 5, 6-H2) ppm. MS (ESI, MeOH):
m/z (%) = 330 (100) [M ? H]?, 352 (10) [M ? Na]?.
4-Bromo-2,6-di-(bromomethyl)-phenol (14)
4-Azido-2,6-bis-(di-tertbutoxycarbonyl-
[1,4,7]triazacyclononane-1-yl-methyl)-phenol (6)
HBr in acetic acid (33 wt%, 37.0 mL, 0.21 mol) was
added to 4-bromo-2,6-dihydroxy-phenol (13, 10.0 g,
42.9 mmol) at 0°C and stirred for 20 min at 0°C. The
solution was allowed to warm to rt and was stirred for
another 24 h at rt. Water (50 mL) was added and the
precipitate was filtered off, washed with water (29
40 mL) and dried in vacuo to yield 92% (14.2 g,
39.6 mmol) of compound 14 which was used for the
synthesis of 15 without further purification.
An argon-saturated solution of ligand 15 (0.52 g, 0.60
mmol) and dimethylethylenediamine (64.9 lL, 0.60 mmol)
in EtOH/H2O = 7:3 (50 mL) was added to sodium azide
(0.12 g, 1.80 mmol), sodium ascorbate (0.36 g, 1.80 mmol)
and copper iodide (0.35 g, 1.80 mmol) and refluxed for
24 h. The mixture was filtered over silica, the filtrate
evaporated to dryness and dried in vacuo to yield a
brown-yellow solid (0.34 g, 70%). 1H-NMR (300 MHz,
[D6]DMSO, 100°C): d = 1.44 (s, 36 H, Boc-CH3),
2.66–2.77 (m, 8 H, TACN-6,60,8,80-H2), 3.24–3.31 (m, 8 H,
TACN-5,50,9,90-H2), 3.38–3.45 (m, 8 H, TACN-2,20,3,30-
H2), 3.75 (s, 4 H, CH2), 6.83 (s, 2 H, Ar-H) ppm. MS
(ESI, MeOH): m/z (%) = 818 (100) [M ? H]?, 840
(25) [M ? Na]?. HRMS (ESI): calcd. for C40H68N9O9
([M ? H]?): 818.51345, found: 818.51336.
4-Bromo-2,6-bis-(di-tertbutoxycarbonyl-
[1,4,7]triazacyclononane-1-yl-methyl)-phenol (15)
Potassium carbonate (1.01 g, 7.30 mmol) was added to a
solution of 4-bromo-2,6-bis-bromomethyl-phenol (1.25 g,
3.48 mmol), BBT 12 (2.40 g, 7.30 mmol) in dry MeCN
(25 mL) and stirred for 16 h at 60°C. The remaining solid
was separated by filtration and the filtrate evaporated to
dryness. The residue was purified by flash chromatography
(4 9 20 cm, EtOAc/pentane = 1:1) to yield a yellow solid
Cyclo[b-D-Dap(adenine-9-yl-acetyl)-L-Ala-L-Cys-L-[1-(4-
hydroxy-3,5-bis-[1,4,7]triazacyclonane-1-yl-methyl-
phenyl)-1H-[1,2,3]triazole-4-yl]-Gly-b-D-Dap
1
(2.42 g, 81%). H-NMR (300 MHz, [D6]DMSO, 100°C):
(thymine-1-yl-acetyl)-L-Ala-L-Cys-L-Val]-disulfide (4)
d = 1.44 (s, 36 H, Boc-CH3), 2.67–2.74 (m, 8 H, TACN-
6,60,8,80-H2), 3.25–3.30 (m, 8 H, TACN-5,50,9,90-H2), 3.40
(s, 8 H, TACN-2,20,3,30-H2), 3.75 (s, 4 H, CH2), 7.22 (s, 2
H, Ar-H) ppm. 13C-NMR (150 MHz, [D6]DMSO, 100°C):
d = 27.7 (Boc-CH3), 48.5 (TACN-CH2), 52.3 (TACN-
CH2), 55.4 (CH2), 78.4 (Boc-C), 109.3 (Ar-CBr), 126.0
(Ar-CCH2), 130 (Ar-CH), 154.2 (Boc-CO), 154.5
(Ar-COH) ppm. MS (ESI, MeOH): m/z (%) = 855 (100)
A solution of 2,6-lutidine in dry DMF (5 mL, 8 mM) was
saturated with argon; cyclopeptide 5 (0.5 mg, 0.4 lmol),
ligand 6 (0.9 mg, 1.1 lmol), [CuIP(OEt)3] (catalytic)
and
tris-(1-benzyl-1H-[1,2,3]triazole-4-yl-methyl)amine
(TBTA) (catalytic) were placed in a 0.5 mL Eppendorf cap
equipped with a magnetic stirrer. 2,6-Lutidine in dry DMF
(100 lL, 0.8 mmol) was added under argon. The mixture
123

452
Eike-F. Sachs et al.
was stirred for 5 days at rt. The solution was evaporated
to dryness in vacuo and the residue was dialyzed for
7 h (ZelluTrans/Roth V-Series, MWCO 1000, H2O/
MeCN = 1:1) and purified by HPLC (analytical, 50–100%
B in 30 min, Rt = 12.2 min) to yield a yellowish solid of
the Boc- and Z-protected intermediate. HRMS (ESI): calcd.
for C90H130N26O23S2 ([M ? 2H]2?): 1003.46163, found:
1003.46029; calcd. for C90H131N26O23S2 ([M ? 3H]3?):
669.31018, found: 669.31026. For Boc and Z deprotection,
the intermediate was dissolved in TFA (2 mL) and eth-
anedithiol (0.1 mL) was added. The solution was stirred for
48 h at rt, and afterward, evaporated to dryness in vacuo.
The residue was purified by HPLC (analytical, 12–28% B,
Rt = 13.6 min) to yield target compound 4 as yellowish
solid (0.1 mg, 16%). MS (ESI, MeOH): m/z (%) = 368 (49)
aza-TANDEM derivative 5 and the azide-functionalized
ligand system 6 were synthesized (Scheme 1). The phenol-
bridged bis-TACN ligand was substituted with an azide in
the para position with respect to the phenolic hydroxyl
group in order to achieve a spatial separation of the metal
binding site and the triazole moiety formed during the
click-reaction. Thus, involvement of the triazole ring as
nitrogen donor in metal coordination should be avoided.
The aza-TANDEM scaffold required alkyne function-
alization at a position not being essential for DNA recog-
nition. Therefore, in peptide 5, a valine from the original
triostin A sequence that is well suited for side chain
modifications (Addess and Feigon 1994) was replaced by
propargylglycine. Side chain functionalization to hybrid 4
was now feasible using click-chemistry with no need for
further protecting groups (Tornøe et al. 2002).
[M ? 4H]4?
,
491 (100) [M ? 3H]3?
,
736 (73)
[M ? 2H]2?. HRMS (ESI): calcd. for C62H91N26O13S2
([M ? H]?): 1471.6700, found: 1471.6689.
Synthesis of the nucleobase-functionalized bicyclic
aza-TANDEM scaffold 5
[Zn2(4-Bromo-2,6-bis-(di-tertbutoxycarbonyl-
[1,4,7]triazacyclononane-1-yl-methyl)-phenol)] (17)
The octapeptide 7 was synthesized by manual Fmoc-SPPS
according to an already reported protocol (Dietrich and
Diederichsen 2005) with modifications concerning cou-
pling conditions and amino acid sequence (Scheme 2). The
coupling of Fmoc-protected propargylglycine was achieved
by coupling with 1-hydroxy-7-aza-benzotriazole (HOAt)
and O-(7-azabenzotriazolyl)-tetramethyluronium hexa-
fluorophosphate (HATU) using three equivalents of amino
acid. Resin loading before and after coupling was deter-
mined by UV spectroscopy to confirm quantitative con-
version (Gude et al. 2002).
To a solution of 16 in H2O (5 mM, 10 lL), a solution of
ZnSO4 in H2O (4.8 mM, 20.8 lL) was added and the
reaction mixture agitated for 10 min at rt. HRMS (ESI):
calcd. for C20H34BrN6O5SZn2 ([M ? SO4]?): 677.00722,
found: 677.00732.
[Cu2(4-Bromo-2,6-bis-(di-tertbutoxycarbonyl-
[1,4,7]triazacyclononane-1-yl-methyl)-phenol)] (18)
To a solution of 16 in H2O (5 mM, 10 lL), a solution of
CuSO4 in H2O (4.8 mM, 20.8 lL) was added and the
reaction mixture stirred for 10 min at rt. HRMS (ESI):
calcd. for C20H34BrN6O5SCu2 ([M ? SO4]?): 675.00813,
found: 675.00919.
Orthogonal protection of the two D-diaminopropionic
acid (D-Dap) a-amino functionalities was required as dif-
ferent nucleobases were attached after macrocyclization.
Boc and benzyloxycarbonyl (Z) protecting groups were
chosen both being orthogonal to the Fmoc-SPPS protocol.
The linear peptide was obtained in an overall yield of 87%.
Formation of the disulfide bridge (26%) and macrocycli-
zation yielded the triostin analog scaffold 8 (30%)
according to a reported protocol (Dietrich and Diederich-
sen 2005).
[Zn2(Cyclo[b-D-Dap(adenine-9-yl-acetyl)-L-Ala-L-Cys-L-
[1-(4-hydroxy-3,5-bis-[1,4,7]triazacyclonane-1-yl-methyl-
phenyl)-1H-[1,2,3]triazole-4-yl]-Gly-b-D-Dap
(thymine-1-yl-acetyl)-L-Ala-L-Cys-L-Val]-disulfide)] (19)
For nucleobase functionalization with thymine and ade-
nine derivatives by amide formation, substituted acetic
acids, (thymine-1-yl)acetic acid (9) and (N6-Z-adenine-1-
yl)acetic acid (10) were prepared as reported by Nielsen and
co-workers (Dueholm et al. 1994). The Boc-protecting
group of D-Dap in peptide 8 was cleaved under mild con-
ditions in order to preserve Z protection. Subsequent cou-
pling of (thymine-1-yl)acetic acid was achieved with HOAt,
diisopropylcarbodiimide (DIC) and N-methylmorpholine
(NMM) providing nucleopeptide 11 in 47% yield. Cleavage
of the Z group and subsequent coupling of (N6-Z-adenine-
9-yl)acetic acid with bromo-tris-pyrrolidino-phosphonium
To a solution of 4 in H2O (0.68 mM, 10 lL), a solution of
ZnSO4 in H2O (1.36 mM, 10 lL) was added and the reac-
tion mixture agitated for 10 min at rt. HRMS (ESI): calcd.
for C64H92N26O17S2Zn2 ([M ? 2HCOO]2?): 844.2576,
found: 844.2569.
Results and discussion
To enable click-chemistry as the ligation step generating
the hybrid compound 4, the propargylglycine containing
123

Triostin A derived hybrid for simultaneous DNA binding and metal coordination
453
OH
N
N
Boc
Boc
N
N
OH
N
N
Boc
N
Boc
N3
+
N
N
6
HN
NH
Z
N
N
HN
N
NH2
N
H
H
N
N
N
N
N
N
O
O
N
O
O
H
H
N
1. [CuIP(OEt)3], TBTA
lutidine, DMF
O
N
N
H
NH
N
NH
N
H
N
H
N
H
HN
O
2. TFA, EDT
HN
O
S
S
O
O
S
S
O
O
O
O
O
O
O
16% (2 steps)
O
NH
O
NH
N
N
H
N
H
N
H
H
HN
HN
O
HN
N
O
N
O
N
H
O
N
H
O
HN
5
4
O
Scheme 1 Ligation of the aza-TANDEM scaffold 5 with ligand 6 by means of copper-mediated [3 ? 2] cycloaddition
O
Acm
S
H
N
1. I2, 80% aq. HOAc
rt, 1 h (26%)
O
Z
N
H
NH
O
O
S
HN
O
O
H
H
N
H
H
HN
O
N
Z
N
H2N
N
S
S
O
O
N
H
Boc
N
N
COOH
N
H
H
H
2. DIC, HOAt, NMM,
DCM/DMF = 9/1
Boc
O
NH
O
O
O
N
O
NH
O
H
N
H
HN
rt, 48 h (30%)
Acm
N
H
7 (87%)
8
1. TFA/DCM = 1/1
2. DIC, HOAt, NMM,
DCM/DMF = 9/1,
O
H
1. TFA/thioanisole (9/1)
2. PyBroP, DIEA,
DMF, rt, 48 h, (37%, 2 steps)
N
O
N
NH
H
N
Z
rt, 48 h (47%, 2 steps)
H
HN
O
5
O
S
S
O
O
Z
O
O
HN
O
N
NH
O
H
H
HN
N
N
N
O
N
O
HN
N
H
O
N
N
N
HN
10
9
COOH
11
HOOC
Scheme 2 Synthesis of the propargylglycine-functionalized aza-TANDEM analog 5
hexafluorophosphate (PyBroP) yielded 37% of the click
precursor 5. Reaction details and analytical data concern-
ing the peptide synthesis can be found in the supporting
information.
15. The required azide for the click-reaction was obtained
by mild halogen azide exchange employing dim-
ethyldiethyldiamine and copper iodide (Andersen et al.
2005). In order to provide an additional ligand for metal
binding studies, compound 15 was Boc deprotected to yield
the TACN derivative 16 (Scheme 3).
Synthesis of the ligand system 6 and assembly
of the hybrid compound 4
Copper-mediated [3 ? 2] cycloaddition was applied in
order to generate the desired hybrid molecule 4 containing
the triostin A analog recognition unit and the binuclear
metal binding site. For ligation of the azide containing
ligand 6 to the alkynyl side chain of cyclopeptide 5, 2,6-
lutidine was employed as base, the copper(I) complex
[CuIP(OEt3)] as catalytic precursor and TBTA as ligand for
the in situ generated active copper complex (Chan et al.
2004). The crude product was dialyzed. Subsequent
deprotection of the TACN and adenine moieties was
achieved with TFA and ethanedithiol (EDT) as scavenger
(Scheme 1). After HPLC purification target compound 4
The design of the ligand system attached to the triostin A
scaffold allows for binuclear metal complexation employ-
ing two TACN moieties bridged by a phenol. The phenol
derivative 13 was prepared by hydroxymethylation with
formaldehyde and KOH starting from 4-bromophenol.
Subsequently, it was converted into the corresponding 2,6-
dibromomethyl compound 14 (92%) by treatment with
HBr. Attachment of two BBT moieties (12) obtained from
TACN by Boc protection of two amino functions (Kimura
et al. 2001) was achieved in 81% yield to give compound
123

454
Eike-F. Sachs et al.
H
N
OH
OH
H
N
OH
MeCN, K2CO3,
60 °C, 16 h (81%)
Et3N, Boc2O, DCM
-5 °C, 30 min (54%)
33% HBr, EtOAc
rt, 24 h (92%)
HO
Br
Br
N
N
HN
NH
Boc
Boc
Br
Br
12
14
13
OH
Boc
Boc
N
N
N
N
dimethylethylenediamine,
N
N
Br
sodium azide, sodium ascorbate,
copper iodide, EtOH/H2O (7:3),
reflux, 24 h (70%)
Boc
Boc
TFA/DCM = 1/1,
rt, 1 min (85%)
15
OH
OH
Boc
Boc
N
N
N
N
HN
N
N
NH
N
N
N
N
N3
Br
H
H
Boc
Boc
16
6
Scheme 3 Synthesis of the metal chelating building block 6 and unprotected ligand 16
Fig. 2 High-resolution MS of complex 19 (left) in comparison with the respective isotopic distribution obtained by calculation (right)
was obtained in 16% yield (over two steps) and charac-
terized by high-resolution mass spectrometry.
two zinc ions can be deduced from the isotope pattern
observed in the high-resolution mass spectrum, which is in
accordance with the calculated spectrum for the binuclear
zinc complex (Fig. 2). No higher aggregates or complexes
containing one or three zinc ions were observed, thereby
suggesting that transition metal ions are solely coordinated
in the binding pockets formed by the TACN nitrogen atoms
and the phenolic hydroxyl group.
Metal binding and DNA recognition
First evidence for metal coordination was obtained with
ligand 16 by generation of zinc and copper complexes. The
binuclear zinc complex 17 and the respective copper
complex 18 were obtained from a 2:1 mixture of metal
sulfate and ligand 16 as indicated by high-resolution mass
spectrometry. The hybrid molecule 4 was also shown to
form a binuclear zinc complex 19. The incorporation of
Temperature-dependent UV spectroscopy was used to
probe for interaction between the hybrid 4 and dsDNA
¨
(Porschke 1971). The melting temperature of the DNA
0
0
stem loop 5 CGCGATACGCAAAAAGCGTATCGCG3
123

Triostin A derived hybrid for simultaneous DNA binding and metal coordination
455
to directed DNA damaging and the decoration of the helical
dsDNA with metal centers for catalytic purposes are
applications that are currently under investigation.
Acknowledgments Financial support from the Deutsche Fors-
chungsgemeinschaft (Di 542/7-1 and the IRTG 1422 ‘Metal Sites in
Biomolecules’) is gratefully acknowledged.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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