Total synthesis of proximicin A - C and synthesis of new furan-based DNA binding agents

Article abstract of DOI:10.1021/ol901003p

The total synthesis of the natural occurring polyamides proximicin A-C (3-5) has been accomplished. A short and efficient synthesis of a thus far unknown 4-amino-2-furan carboxylic acid was developed. Furthermore, this unique heterocyclic γ-amino-acid was

Full text of DOI:10.1021/ol901003p

ORGANIC
LETTERS
2009
Vol. 11, No. 13
2804-2807
Total Synthesis of Proximicin A-C and
Synthesis of New Furan-Based DNA
Binding Agents
Falko E. Wolter,† Kathrin Schneider,† Brian P. Davies,† Elke R. Socher,‡
Graeme Nicholson,§ Oliver Seitz,‡ and Roderich D. Su¨ssmuth*,†
Institut fu¨r Chemie, TU Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany,
Institut fu¨r Chemie, HU Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany,
and Institut fu¨r Chemie, Eberhard-Karls-UniVersita¨t Tu¨bingen, Auf der Morgenstelle 18,
72076 Tu¨bingen, Germany
suessmuth@chem.tu-berlin.de
Received May 8, 2009
ABSTRACT
The total synthesis of the natural occurring polyamides proximicin A-C (3-5) has been accomplished. A short and efficient synthesis of a
thus far unknown 4-amino-2-furan carboxylic acid was developed. Furthermore, this unique heterocyclic γ-amino-acid was used for the synthesis
of a new class of AT-selective DNA-binding agents derived from the natural products combining structural features of the proximicins with
those from the known DNA-binding natural products netropsin (1) and distamycin (2).
The polyamide antibiotics netropsin (1) and distamycin (2)
synthesized by Streptomyces were arguably the first natural
products for which AT-selective DNA binding was demon-
strated (Figure 1).1,2 Characteristic features of peptides 1 and
2 are the N-methyl pyrrol core amino acid and the modifica-
tion with charged amidino or guanidino functions. Using
these structures as a molecular scaffold, a considerable
number of synthetic DNA ligands has been derived from
these natural products.2 A lot of effort has been made
incorporating various other heterocyclic amino acids into the
peptide backbone.2b,f
(1) (a) Finlay, A. C.; Hochstein, F. A.; Sobin, B. A.; Murphy, F. X.
J. Am. Chem. Soc. 1951, 73, 341–343. (b) Arcamone, F.; Penco, S.; Orezzi,
P.; Nicolella, V.; Pirelli, A. Nature 1964, 203, 1064–1065. (c) Zimmer, C.;
Wahnert, U. Prog. Biophys. Mol. Biol. 1986, 47, 31–112.
† TU Berlin.
‡ HU Berlin.
§ Eberhard Karls Universita¨t Tu¨bingen.
10.1021/ol901003p CCC: $40.75
Published on Web 06/11/2009
 2009 American Chemical Society

on the synthesis, cytotoxicity, and DNA binding of netropsin-
proximicin-hybrids combining the N-methyl pyrrol amino
acid of netropsin with N- and C-terminal residues derived
from the proximicins.4 In this contribution, we present the
first total synthesis of the proximicins (3-5) and examine
the influence of structural modifications of analogues (6-12)
with a furan amino acid core on DNA-binding.
A considerable number of syntheses toward variously
substituted furans have been reported to date, assembled
either by condensation and metal-catalyzed cyclization of
linear precursors or by directly performing substitutions at
the furan ring. However, methods for preparation of 2,4-
disubstituted furans are rare.5 Our attempts were directed at
a three step synthesis of the central core amino acid of the
proximicins beginning with commercially available 3-fural-
dehyde (13), which was selectively lithiated at the C-5
position (Scheme 1).6 Subsequent trapping of the lithiated
Scheme 1
.
Total Synthesis of Proximicin A (3), B (4), and C
(5)
Figure 1. Structures of netropsin (1), distamycin (2), proximicin
A-C (3-5), and of newly designed potential minor groove binders
(6-12). Structures of compounds capable to act as DNA-binding
agents are marked with a green check.
Recently, proximicin A, B, and C (3-5) composed of a
dipeptide of a 2,4-disubstituted furan amino acid were
isolated from a marine actinomycete of the genus Verruco-
sispora (Figure 1).3 Besides the apparent structural similari-
ties of the proximicin core to netropsin (1) and distamycin
(2) the central dipeptide of 4-amino-furan-2-carboxylic acids
has neither been reported for natural products nor from
synthetic studies. In proximicins the charged residues of (1)
and (2) are formally replaced by an N-terminal methyl
carbamate and C-terminal amides, respectively. These struc-
tural differences of proximicins apparently lead to a change
of the molecular target.3a Remarkably, it was found through
cell cycle analysis and the expression level of cell cycle
regulating proteins (p53, p21, and cyclinE) that, in contrast
to distamycin (2), the proximicins address the G0/G1 phase
of the cell cycle.3a Previous experimental work has reported
species with methyl chloroformate led to derivative 14. The
aldehyde 14 was oxidized under mild conditions to the
corresponding carboxylic acid (15) which was further
converted by a Curtius rearrangement to the central 2,4-
disubstituted furan amino acid in a suitably protected form
(16) in good yields. Removal of the Boc-group and introduc-
tion of the methyl carbamate functionality led to 18. The
methyl ester was easily saponified and the resulting free acid
was coupled without further purification to previously
synthesized H-Fu-OMe•HCl (17) with HOAt/EDCI to yield
Meoc-Fu-Fu-OMe (19) After saponification of dipeptide 19
(2) (a) Bailly, C.; Chaires, J. B. Bioconjug. Chem. 1998, 9, 513–538.
(b) Dervan, P. B.; Poulin-Kerstien, A. T.; Fechter, E. J.; Edelson, B. S.
Top. Curr. Chem. 2005, 253, 1–31. (c) Lown, J. W. J. Mol. Recognit. 1994,
7, 79–88. (d) Wemmer, D. E. Annu. ReV. Biophys. Biomol. Struct. 2000,
29, 439–461. (e) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215–2235.
(f) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122,
6382–6394. Incorporation of a 2,5-disubstituted furan amino acid: (g) Lee,
M.; Krowicki, K.; Shea, R. G.; Lown, J. W.; Pon, R. T. J. Mol. Recogn.
1989, 2, 84–93.
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doi: 10.1016/j.bmcl.2009.04.042.
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Wong, H. N. C. Pure Appl. Chem. 2005, 77, 139–143. (b) Kirsch, S. F.
Org. Biomol. Chem. 2006, 4, 2076–2080. (c) Keay, B. A. Chem. Soc. ReV.
1999, 28, 209–215. (d) Jeevanandam, A.; Ghule, A.; Ling, Y. C. Curr.
Org. Chem. 2002, 6, 841–864. (e) Hou, X. L.; Cheung, H. Y.; Hon, T. Y.;
Kwan, P. L.; Lo, T. H.; Tong, S. Y.; Wong, H. N. C. Tetrahedron 1998,
54, 1955–2020. (f) Gilchrist, T. L. J. Chem. Soc., Perkin Trans. 1 1999,
2849–2866. (g) Brown, R. C. D. Angew. Chem., Int. Ed. 2005, 44, 850–
852.
(3) (a) Schneider, K.; Keller, S.; Wolter, F. E.; Roeglin, L.; Beil, W.;
Seitz, O.; Nicholson, G.; Bruntner, C.; Riedlinger, J.; Fiedler, H.-P.;
Su¨ssmuth, R. D. Angew. Chem., Int. Ed. 2008, 47, 3258–3261. (b) Fiedler,
H.-P.; Bruntner, C.; Riedlinger, J.; Bull, A. T.; Knutsen, G.; Goodfellow,
M.; Jones, A.; Maldonado, L.; Pathom-Aree, W.; Beil, W.; Schneider, K.;
Keller, S.; Su¨ssmuth, R. D. J. Antibiot. 2008, 61, 158–163.
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Chem. 1992, 57, 3126–3131.
Org. Lett., Vol. 11, No. 13, 2009
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the free acid 19a was used for the syntheses of the three
proximicins A-C. Ammonolysis of an OBt-ester led to
proximicin A (3), whereas HOAt/EDCI-mediated coupling
with the biogenic amines tyramine and tryptamine furnished
proximicin B (4) and C (5), respectively. The synthetic
proximicins were identical in all aspects to natural samples.
Previous concepts have amply addressed the variation of
heterocycles, their multiple alignment and N-terminal and
C-terminal substitutions in order to evaluate preconditions
for DNA-binding and to address sequence specificity.2 We
applied these concepts to the synthesis of seven compounds
(6-12, Figure 1) containing the 4-amino-furan-2-carboxylic
acid in order to set these proximicin analogues in context
with previously reported heterocyclic polyamide antibiotics
regarding DNA-binding capabilities.2 These new furan-based
derivatives can be subdivided into three groups: (a) prox-
imicin homologues (6-8) containing a third furan amino acid
(multiple alignment), (b) derivatives with basic C-terminal
side chains (9-10), and (c) furan analogues of netropsin and
distamycin (11-12). As a basic side chain in 9-12, a
dimethylpropylamine (Dp) residue was used instead of the
amidine group found in netropsin (1) or distamycin (2). This
was mainly chosen to avoid the variable yielding Pinner
reaction and for the overall ease of synthesis. This substitu-
tion is admissable with regard to DNA-affinity and
selectivity.2f,7 Because of the substitution pattern of the
central furan amino acid, all designed derivatives were
expected to show selectivity for an AT-rich DNA sequence.2a
The synthesis of the polyamides 6-10 begins with the
universal dipeptide building block 19 (Scheme 2). Saponi-
A-C were introduced, yielding the tripeptide homologues
6-8. Coupling of dimethylpropylamine led to tripeptide 10.
The synthesis of furan-containing netropsin analogue 11
starts with the introduction of a Boc-protected glycyl residue
at the N-terminus of 16 yielding 21 (Scheme 3). Then 23
Scheme 3. Synthesis of Furan Analogue (11) of Netropsin
was assembled from 21 by a sequence of C-terminal
deprotection and peptide coupling steps. The Boc-group of
23 was cleaved and guanidylation with Goodman’s reagent
yielded 24, which was deprotected and purified by anion
exchange chromatography to yield 11.8
The synthesis of 12, a furan analogue of distamycin,
followed in principle the same route as described for 10,
but started with Boc-Fu-OMe (16) (Scheme 4). After
assembly of the tripeptide composed of three furan units and
a dimethylaminopropyl side chain, the Boc-group of 27 was
cleaved and subsequent formylation using N-formyl imida-
zole provided 12.
Scheme 2. Synthesis of Proximicin Homologues (6-8) and of
Derivatives with a Charged Side Chain (9, 10)
The interaction of 6-12 with DNA was investigated by
melting analysis of a GC-rich (5′-catggccatg-3′) and an AT-
rich (5′-cgcaaatttgcg-3′) oligonucleotide (Table 1). In ac-
cordance with the literature, netropsin (1) and distamycin
(2) had a stabilizing effect on the AT-rich oligonucleotide.1c
The derivatives 7 and 10-12 also had a stabilizing effect
on the melting of the DNA of the AT-rich sequence (Table
1) as observed by an increased TM value. As expected, 6-12
had no effect on the GC-rich sequence (see Supporting
Information).
fication of the methyl ester and direct use of the free acid
for introduction of either another furan amino acid or of a
basic dimethylpropylamine (Dp) residue leads to 20 or to
derivative 9 with a charged side chain. Upon saponification,
tripeptide 20 served as the source for the synthesis of further
derivatives. C-terminal residues characteristic of proximicin
To more extensively evaluate the DNA-binding activity
of 6-12 binding constants were determined (Table 1) using
an ethidium bromide displacement assay.9 Binding constants
(8) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org. Chem.
1998, 63, 3804–3805.
(7) (a) Taylor, J. S.; Schultz, P. G.; Dervan, P. B. Tetrahedron 1984,
40, 457–465. (b) Yamamoto, Y.; Kimachi, T.; Kanaoka, Y.; Kato, S.;
Bessho, K.; Matsumoto, T.; Kusakabe, T.; Sugiura, Y. Tetrahedron Lett.
(9) (a) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick,
M. P. J. Am. Chem. Soc. 2001, 123, 5878–5891. (b) Satz, A. L.; Bruice,
T. C. Bioorg. Med. Chem. 2000, 8, 1871–1880.
1996, 37, 7801–7804
.
2806
Org. Lett., Vol. 11, No. 13, 2009

systems with three repetitive furan rings exemplified by 10
and 12. Additionally, the N-terminal modification of 10 and
12 with a methylcarbamate or a formyl group does not
significantly alter the molecule properties in terms of DNA-
binding or binding constants. Moreover, the results obtained
for 11 (Table 1) imply that an additional charge at the
N-terminus can compensate for the lack of one furan with
regard to DNA-binding affinity. Absence of the N-terminal
charge, as represented by 9 results in a breakdown of DNA-
binding.
Scheme 4. Synthesis of Furan Analogue (12) of Distamycin
The stabilizing effect on AT-rich DNA of the furan
analogues of netropsin and distamycin, 11 and 12, are clearly
lower than the effect of netropsin (1) and distamycin (2)
(Table 1). The primary difference in 1 and 11 or in 2 and 12
is the furan heterocycle. The replacement of the C-terminal
amidine by a dimethylaminopropylamine is expected to have
a neglectable influence on the binding affinity.2f Hence, the
lower stabilizing effect might be due to the different
electronic structures of the two heterocyclic cores resulting
in altered stacking interactions. Additionally, the N-methyl
pyrrol of netropsin or distamycin is bulky and nonpolar and
in proximity to the anionic backbone of the DNA. In contrast,
the furan ring bears a free electron pair, which might lead
to a repulsive interaction whith the DNA backbone.
obtained for netropsin (1) and distamycin (2) were compa-
rable to literature values.9a The binding constants for new
derivatives 6-12 show a good correlation with the data found
by DNA melting analysis (Table 1). A significant binding
affinity was found for 10, 11, and 12, which also show the
highest ∆TM values among the synthesized compounds.
From the proximicin homologues 6-8 only compound 7
had a DNA-binding effect with a ∆TM of 5 °C (Table 1)
indicating that C-terminal modifications exert a distinguishing
influence. Tyramine derivative 7 showed the best interaction
compared to the sterically more demanding tryptamine
derivative 8, or amide derivative 6 putatively lacking the
establishment of hydrophobic interactions (Figure 1, see
binding-mode of 1).2a,10
In summary, we developed a short and efficient synthesis
for a novel heterocyclic amino acid in an orthogonally
protected manner, facilitating the first total synthesis of
proximicin A-C. Furthermore, this amino acid was used for
the first time as a building block in the synthesis of
polyamides capable of interacting with AT-rich DNA. Thus,
the stock of five-membered heterocyclic amino acids as
scaffolds for minor groove binders was expanded and a novel
element of diversity was added for generating a new class
of DNA binding agents. Further work is being done using
these synthetic methods to generate novel proximicin deriva-
tives to investigate mode of action and to identify the
molecular target of the proximicins.
The introduction of the C-terminal dimethylaminopropyl-
amine leads to an increased DNA-binding activity for
Table 1. DNA Melting Analysis and Determination of Binding
Constants (Ka) to an AT-Rich Oligonucleotide (cgcaaatttgcg) of
6-12 and Comparison with Netropsin (1) and Distamycin (2)
Acknowledgment. This work was supported by the DFG
(project SU 239-5/1) and from the European Commission
(project ACTINOGEN, sixth framework, LSHM-CT-2004-
005224).
b
compound
∆TM (°C)a
Ka (× 106 M-1
)
1
2
6
7
8
9
10
11
12
19
15
2
5
0
1
8
9
8
280 ( 100
190 ( 40
2.5 ( 0.8
1.5 ( 0.2
0.44 ( 0.03
0.63 ( 0.09
27 ( 10
Supporting Information Available: Experimental pro-
cedures and characterization of all new compounds. DNA
melting analysis and determination of binding constants by
ethidium bromide assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
31 ( 7
24 ( 5
OL901003P
a Difference in TM of oligonuleotide cgcaaatttgcg and after adding 6
µM of compound 1, 2, 6-12 to a 2 µM solution of the oligonucleotide.
b Nonlinear analysis of ethidium bromide displacement titration curve.
(10) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376–1380.
Org. Lett., Vol. 11, No. 13, 2009
2807