The multicomponent approach to N-methyl peptides: Total synthesis of antibacterial (-)-viridic acid and analogues

Article abstract of DOI:10.3762/bjoc.8.234

Two syntheses of natural viridic acid, an unusual triply N-methylated peptide with two anthranilate units, are presented. The first one is based on peptide-coupling strategies and affords the optically active natural product in 20% overall yield over six steps. A more economical approach with only four steps leads to the similarly active racemate by utilizing a Ugi four-component reaction (Ugi-4CR) as the key transformation. A small library of viridic acid analogues is readily available to provide first SAR insight. The biological activities of the natural product and its derivatives against the Gram-negative bacterium Aliivibrio fischeri were evaluated.

Full text of DOI:10.3762/bjoc.8.234

The multicomponent approach to N-methyl peptides:
total synthesis of antibacterial (–)-viridic acid
and analogues
Ricardo A. W. Neves Filho1, Sebastian Stark1,2, Bernhard Westermann1,2
and Ludger A. Wessjohann*1,2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 2085–2090.
1Department of Bioorganic Chemistry, Leibniz Institute of Plant
Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany, Tel: +49
345 5582 1301, Fax: +49 345 5582 1309 (Address for
correspondence) and 2Martin-Luther-University Halle-Wittenberg,
Institute of Organic Chemistry, Kurt-Mothes-Str. 2, 06120 Halle
(Saale), Germany
doi:10.3762/bjoc.8.234
Received: 10 August 2012
Accepted: 30 October 2012
Published: 28 November 2012
This article is part of the Thematic Series "Antibiotic and cytotoxic
peptides".
Email:
Ludger A. Wessjohann* - wessjohann@ipb-halle.de.
Guest Editor: N. Sewald
* Corresponding author
© 2012 Neves Filho et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
antibiotic; anticancer; Gram negative bacteria; natural product;
peptide coupling; peptides; peptoid; toxin; Ugi reaction
Abstract
Two syntheses of natural viridic acid, an unusual triply N-methylated peptide with two anthranilate units, are presented. The first
one is based on peptide-coupling strategies and affords the optically active natural product in 20% overall yield over six steps. A
more economical approach with only four steps leads to the similarly active racemate by utilizing a Ugi four-component reaction
(Ugi-4CR) as the key transformation. A small library of viridic acid analogues is readily available to provide first SAR insight. The
biological activities of the natural product and its derivatives against the Gram-negative bacterium Aliivibrio fischeri were evalu-
ated.
Introduction
Viridic acid (1) is a tetrapeptide produced by several fungi of sible for the toxicity of this extract due to its metal-chelating
the genus Penicillium, including P. viridicatum, P. nordicum, properties [6]. Later, this (putative) mycotoxin was also found
and P. aurantiogriseum among others [1-4]. It was first isolated in cultures of P. nordicum cultivated on cheese agar medium.
from the basic fraction of the chloroform/methanol extract of The crude extracts from these cultures displayed pronounced
P. viridicatum Westling [5], and it was assumed to be respon- cytotoxicity in a MTT assay on an undisclosed cell line [7].
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Albeit that these two reports on the toxicity of extracts approach. In this endeavor we envisioned two routes. The first
containing constituent 1 were very intriguing, no further bio- one uses improved peptide-coupling protocols, leading to the
logical screening of the pure substance has been performed to natural enantiomer (Scheme 1) [11]. The blueprint of the syn-
date. The connection between compound 1 and the bioactivity thesis was planned as a bidirectional sequence from the least- to
of the extract containing it is thus purely correlative, i.e., specu- the most-reactive amine (NMe-Val < H-Ant-OBn < H-Gly) to
lative. No causal relationship between the compound and the yield the protected tetrapeptide 2 (Scheme 1a). Alternatively, a
MTT results is proven.
multicomponent (MCR) approach based on a Ugi four-compo-
nent reaction (Ugi-4CR) of dipeptide 3, isobutyraldehyde,
The structure of compound 1 was determined as the peptide methylamine and isonitrile 4 as the key transformation was
N(Me)2Ant-Gly-(N-Me)Val-Ant (Ant = anthranilic acid) in envisioned to yield racemic viridic acid (±)-1 (Scheme 1b) [12].
1986, based on a series of degradation experiments, NMR, and Besides furnishing the desired natural product in only four
IR measurements as well as a first total synthesis [5]. Thus, it steps, the MCR approach allows a ready access to analogues
was revealed that 1 belongs to the small group of natural endowed with a proteolysis-resistant peptoid moiety [13].
peptides that contain anthranilic acid residues in the peptidic Recently, we demonstrated that chemically more stable peptoid
backbone [8-10]. Furthermore, to the best of our knowledge, analogues of tubulysins, named tubugis, present cytotoxicity
viridic acid is unique in its N-terminus, which bears a N,N- against cancer cell lines comparable to the native natural prod-
dimethyl anthranilic amide moiety of still unknown biosyn- uct [14]. Thus, it was hoped that some viridic acid analogues
thetic origin. The previously reported synthetic strategy toward readily accessible by MCR may also display enhanced bio-
1 was based upon a series of peptide couplings employing DCC logical activity or at least stability.
reagent [5]. The necessity of difficult-to-perform peptide
couplings with phenyl carboxylates and N-methylated Results and Discussion
amino groups demanded harsher than usual conditions, upon The 2-CR approach based on the peptide coupling of Boc-Gly
which the desired viridic acid (1) was obtained in just 5% and NMe-Val-OMe in the presence of EDCl and HOAt gave the
overall yield.
central dipeptide 5 in 73% yield (Scheme 2) [15].The use of
even more activating coupling reagents, such as HATU and
The lack of sufficient amounts of isolated materials from BOP, was also investigated and resulted in increased formation
natural sources did not allow for reliable bioactivity tests, and of side products [11]. After saponification of intermediate 5 to
the demand for higher quantities and derivatives of viridic acid dipeptide 6, the latter was coupled with benzyl anthranilate.
(1), required the development of a more efficient and milder This reaction was particularly challenging due to the very poor
Scheme 1: Retrosynthetic strategies for (−)-viridic acid (1) and analogues by (a) a bidirectional peptide-coupling sequence, and (b) a novel MCR
approach.
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Scheme 2: Reactions and conditions: (a) Boc-Gly-OH, EDCI, HOAt, TEA, CH2Cl2, rt, 20 h, 73%. (b) LiOH, THF/H2O (1:1), rt, 3 h, 97%. (c) Benzyl
anthranilate, EEDQ, CHCl3, rt, 20 h, 51%. (d) TFA, CH2Cl2, rt, 5 h. quant. (e) 8, NMM, ethyl chloroformate, CHCl3, rt, 20 h, 85%. (f) H2, 10% Pd/C,
MeOH, rt, 16 h, 92%.
reactivity of this combination [10]. All attempts to perform this mediate was hydrogenated to afford the dipeptide acid 3
coupling with carbodiimides, HBTU, HATU, and PyBroP failed quantitatively. An alternative multicomponent approach to
or resulted in very low conversions. The addition of a catalytic dipeptide 3 requires the use of ammonia or an ammonia equiva-
amount of DMAP to the carbodiimide-mediated reactions im- lent, such as 2,4-dimethoxybenzylamine (DMB-NH2), as amino
proved the conversions, but resulted in severe racemization. The component, formaldehyde as oxo-component, and a convertible
best result was obtained by employing N-ethoxycarbonyl-2- isonitrile [22-25]. Although many convertible isonitriles are
ethoxy-1,2-dihydroquinoline (EEDQ) as coupling reagent, reported in the literature [24,26], the recently developed
which gave the optically active tripeptide 7 in 51% yield 4-isocyanopermethylbutane-1,1,3-triol (IPB) was chosen due to
[16,17]. Intermediate 7 was converted into amine 8 under acidic its ease of preparation, better reactivity, and mild conversion
conditions, and coupled directly to N,N-dimethylanthranilic acid conditions [27]. The Ugi-4CR involving carboxylic acid 9,
(9). It was already reported that carbodiimide-mediated DMB-NH2, formaldehyde and IPB resulted in intermediate 11
couplings involving aromatic carboxylic acids such as 9 can in 35% yield. A tandem DMB group cleavage/pyrrole-forma-
result in the formation of N-acylurea adducts through competi- tion sequence under acidic conditions followed by saponifica-
tive N–O rearrangement [18,19]. In order to overcome this tion afforded the desired carboxylic acid 3 in 21% yield over
problem the mixed-carbonate method was used [20]. The reac- the two steps (Scheme 3). The MCR approach to building block
tion resulted in the desired optically active key intermediate 2 in 3 with two convertible components gives lower yields
85% yield. Its hydrogenation afforded the desired (−)-viridic compared to the classical amide formation, but it carries the
acid (1) in quantitative conversion and >92% isolated yield, i.e., diversity-generating ability inherent in Ugi-4CRs, and the
in 20% overall yield. Analytical data such as the HRMS, potential to synthesize derivatives where classical methods are
1H NMR, melting point, and optical rotation of synthetic com- less suitable.
pound 1 are consistent with the data reported for the natural
substance (Scheme 2) [5].
The key Ugi-4CR combining isobutyraldehyde, methylamine
(12a), dipeptidic carboxylate 3 and anthranilic isonitrile 4a, to
In order to more rapidly access derivatives for biological form the final tetrapeptide backbone was performed by using
activity screens, we decided to investigate the suitability of a standard protocols, with imine preformation in methanol, to
MCR approach utilizing the Ugi reaction. Due to the character give 13a in 51% yield. Finally, saponification of 13a afforded
of the Ugi-4CR, the racemate of viridic acid and congeners is the racemic viridic acid (±)-1 in 83% yield. Attempts to
easily available, and assaying with (±)-1 can give an estimation improve the MCR yield with conventional or microwave
of the relevance or effect of the configuration of the asym- heating, or by employing trifluoroethanol or DMF as solvents
metric center on the biological activity. With this goal in mind, resulted in poor conversions, with competitive Passerini reac-
the Ugi-4CR between methylamine, isobutyraldehyde, dipep- tion in the latter case [28,29]. With the general process in place,
tide 3, and the anthranilate-derived isonitrile 4a was planned the MCR approach was employed to generate a library of syn-
(Scheme 1) [21]. For the synthesis of the N-terminal dipeptide thetic derivatives of 1 with the hope of gaining a first glimpse of
3, amino acid 9 was coupled with benzyl glycinate in the pres- structure–activity relationships (SAR), and to give hints for
ence of ethyl chloroformate to give 10 in 88% yield. This inter- further applications, as for example for attachment points for
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Scheme 3: Reactions and conditions: (a) H-Gly-OBn·HCl, NMM, ethyl chloroformate, CHCl3, 18 h, rt, 88%. (b) H2, 10% Pd/C, MeOH, rt, 16 h, 98%.
(c) 2,4-Dimethoxybenzylamine, formaldehyde, IPB, MeOH, rt, 18 h, 35%. (d) TFA, CH2Cl2, 0 °C to rt, 48 h, then LiOH, THF/H2O (1:1), rt, 3 h, 21%
(over two steps). (e) MeOH, rt, 20 h, 51–70%. (f) LiOH, THF/H2O, rt, 8 h, 81–91%. (g) KOH, MeOH/H2O (3:1), rt, 8 h, 70%.
probe design and experiments [30,31]. Thus, methylamine was etc. This is in accordance with earlier results, where also no or
substituted by 12b–f in the key Ugi-4CR to yield the intermedi- only negligible diastereoselection could be achieved [35,36].
ates 13b–f (55–70% yields) [16], which afforded the desired Saponification of the menthyl ester 13g afforded the racemic
peptoid analogues 14b–f after saponification. Compared to viridic acid (1) again in 70% yield (Scheme 3) [37].
simple peptides, N-alkylated ones (peptoid moieties) allow
different low-energy conformations, and contrary to common To our knowledge no biological screening of pure (−)-viridic
belief they are more restricted in conformational space [32]. acid or its analogues has been performed, and due to the poten-
Moreover, they commonly possess a higher lipophilicity and tial of natural peptides and peptoids as antibacterial agents
protease stability, and this combination seems to improve their [13,38-47], it was decided to investigate their activity against
antibiotic properties (Scheme 3) [13].
the Gram-negative bacterium Aliivibrio fischeri [48]. Com-
pounds (−)-1 and (±)-1 were the most active ones with
Since the MCR results in racemates, we decided to investigate IC50 values of 45.0 ± 4.4 and 38.4 ± 5.8 μM, respectively. In
the applicability of a chiral auxiliary MCR approach for the this test system, derivatives 14b–f displayed IC50 values above
asymmetric synthesis of viridic acid (1). The Ugi-4CR is not 100 μM and can be considered as inactive. Although (−)-viridic
specifically prone to asymmetric induction, but at least some acid (1) was isolated thirty years ago, this is the first unam-
auxiliaries are known to result in preferential formation of a dia- biguous report concerning its biological activity. Based upon
stereoisomer [33,34]. Therefore, the isonitrile 4b was synthe- the results presented above, it seems that the configuration of
sized from menthyl anthranilate. The Ugi-4CR of 4b in analogy the stereogenic center has almost no influence on the antibac-
to the reaction of 4a with methylamine (12a) gave the desired terial effect of 1. The lack of activity of the derivatives 14b–f is
peptoid–peptide adduct 13g in 47% yield, albeit as a 1:1 difficult to rationalize without knowing the target, but it demon-
diastereomeric mixture. Unfortunately, even a separation of the strates that the size of the group attached to the nitrogen of the
epimers failed by using thin- or thick-layer or conventional Val residue has a clear influence. This fact suggests that 1 does
column chromatography or HPLC, under varied conditions of not act just by engaging bacterial membranes as most antibac-
different column types, methods, mobile-phase compositions, terial peptides do [49], but that it may bind to a specific target.
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Acknowledgements
The authors acknowledge support from the state of Saxony-
Anhalt (MK-LSA, WZW project “Lipopeptide”). We thank
Dr. Jürgen Schmidt and Ms. Annett Werner for HRMS and
HPLC support, respectively. R.A.W.N.F. is grateful to Brazilian
funds from CNPq for a Ph.D. fellowship.
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