The emergence of halophilic evolutionary patterns from a dynamic combinatorial library of macrocyclic pseudopeptides

Article abstract of DOI:10.1039/c2cc37869h

The increase of the ionic strength amplifies the species bearing acidic side chains from a bio-inspired dynamic combinatorial library of macrocyclic pseudopeptides, in close resemblance to the evolution observed for the proteins of halophilic microorganisms.

Full text of DOI:10.1039/c2cc37869h

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The emergence of halophilic evolutionary patterns
from a dynamic combinatorial library of macrocyclic
pseudopeptides†
Cite this: Chem. Commun., 2013,
49, 487
Received 30th October 2012,
Accepted 16th November 2012
Joan Atcher, Alejandra Moure and Ignacio Alfonso*
DOI: 10.1039/c2cc37869h
www.rsc.org/chemcomm
The increase of the ionic strength amplifies the species bearing at the surface of halophilic proteins at physiological pH.
acidic side chains from a bio-inspired dynamic combinatorial library Although the effect is still not fully understood, it has been
of macrocyclic pseudopeptides, in close resemblance to the evolu- suggested that hydrated ions can interact with the surface
tion observed for the proteins of halophilic microorganisms.
residues to stabilize the folded conformation,9 the solvent-
accessible surface area being a key parameter.10
Dynamic combinatorial libraries (DCLs) are formed by mixtures
Taking this phenomenon as bio-inspiration, we hypothesized
of components able to reversibly exchange under thermo- that simple dynamic chemical systems would display evolutionary
dynamic control, their composition being dictated by the stability trends in parallel with those observed for the biological evolution
of the members within the mixture.1 Thus, the presence of an in halophilic microorganisms. To investigate that, we designed a
external stimulus can operate as a trigger to induce changes in minimalistic DCL of pseudopeptidic macrocycles constructed by
the dynamic mixture, which makes the library evolve toward a the connection of simple building blocks containing pertinent
better adapted constitution.2 Accordingly, dynamic combina- information in the amino acid side chains (Scheme 1). Our design
torial chemistry (DCC) has proved to be a very powerful approach was based on a C2-symmetric scaffold with two arms, each formed
for the preparation of new receptors, catalysts, sensors, potential by an amino acid residue with a mercaptoacetyl moiety attached to
drugs and responsive materials.1–3 However, more recently, the N-terminus (1a–d). The central aromatic m-phenylenediamine
a holistic view of DCLs has emerged within the frame of systems allows HPLC-UV quantification and confers rigidity to the systems
chemistry.4 Since the DCLs are adaptive complex chemical in a pre-organized conformation for macrocyclization. The amino
entities that operate as a whole, they are a model benchmark5 acid side chains permit the introduction of chemical diversity with
for understanding the evolutionary processes present in nature, protein-like structural information, whereas the two thiol ends
despite the mechanisms of chemical and genetic evolution being
different. Among all the evolutionary processes observed in
nature, the one undergone by extremophiles is especially
appealing, since it has allowed them to survive under very
extreme conditions.6 Halophilic archea are extremophiles that
thrive in highly saline environments such as natural salt lakes.
They have managed to skip osmotic shock by increasing the salt
concentration inside the cell. Accordingly, their bio-molecular
machinery has evolved to be stable and fully functional in this
hypersaline medium.7 Interestingly, the characteristic features
associated to the surface of the halophilic proteins include a
large increase in Glu and Asp acids and a decrease in the overall
hydrophobic content.8 This trend concentrates anionic charges
Department of Biological Chemistry and Molecular Modelling, IQAC-CSIC,
Jordi Girona 18-26, E-08034, Barcelona, Spain. E-mail: ignacio.alfonso@iqac.csic.es;
Fax: +34 932 045 904; Tel: +34 934 006 100
† Electronic supplementary information (ESI) available: Synthetic procedures,
full spectroscopic and analytical data, control experiments, and molecular
modelling details. See DOI: 10.1039/c2cc37869h
Scheme 1 Schematic representation of the molecular diversity generated within
the DCL of macrocyclic pseudopeptides obtained by the oxidation of building
blocks 1a–d.
c
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was amplified by the salt in all the sub-libraries containing this
building block, while the sub-library made of only 1b + 1c was
insensitive to the salt content. We also checked that the salt was
operating under thermodynamic control (ESI†). From the data
obtained in the different experiments and by making use of the
DCLSim17 software, we estimated that the dimer [1a–1a] was
stabilized in 1 M NaCl by about DDG = À2.6 kJ molÀ1. Regarding
the trimers, the salt induced the increase of those joining two or
three Glu building blocks. Accordingly, the trimers [1a–x] (with
x = 1b or 1c) showed an amplification factor of 1.4–1.6 while
[1a–1a], which was barely detected in the absence of salt, showed a
fourfold increase of its concentration in 2 M NaCl. Actually, the
trimer/dimer proportion in a mixture formed by the oxidation of
1a alone was also strongly affected by the presence of salt, leading
to a tenfold increase of the corresponding equilibrium constant in
favour of the trimer (ESI†). Thus, very remarkably, the hypersaline
environment induces the evolution of our DCL toward the ampli-
fication of the macrocycles containing a concentrated number of
acidic residues, in a clear parallelism with the evolution of the
halophilic organisms. The same experiment using different salts
like KCl or NaNO3 rendered practically identical amplification
factors than NaCl (ESI†) implying the ionic strength as the driving
force for the amplification.15 Moreover, the corresponding DCL
generated at pH 2.5 was insensitive to the presence of salt (ESI†).
In spite of the difficulty of reaching the equilibrium composition at
such a low pH,18 it seemed evident that the salt-induced amplifica-
tion of the glutamic species requires the anionic side chains.
In order to better understand the process at the molecular
Fig. 1 (A, B) HPLC traces of the [1a + 1b + 1c] DCL (A) alone and (B) in the
presence of 1 M NaCl. (C) Amplification factors of the macrocyclic dimers
versus the NaCl concentration.
establish the DCL by thiol-disulfide and disulfide exchange level, we performed the structural characterization of the [1a–1a]
equilibria in aqueous solution at neutral pH (Scheme 1).11,12
dimer. Molecular modelling rendered important differences
Within this design, we mixed the corresponding building blocks depending on the charge of the molecule (Fig. 2 and ESI†). These
derived from Glu (1a, used as a probe containing an anionic amino differences can be explained considering the H-bonding patterns:
acid at neutral pH), Gln (1b, as an isostructural non-charged the tetraanion (B) sets eight intramolecular H-bonds in a figure
congener) and Ser (1c, as a highly soluble derivative) at 2 mM each eight folding shape,19 whereas the neutral species (A) shows an
in 20 mM aqueous phosphate buffer at pH 7.5 (containing 25% unfolded conformation with only four H-bonds. 1H NMR and CD
DMSO13 to ensure total dissolution). The oxidation of the thiols to techniques provided experimental evidence of these conforma-
1
the corresponding macrocyclic compounds was monitored by HPLC tions (ESI†). Several H NMR signals changed upon increasing
(Fig. 1A). Once the system reached the equilibrium composition,14 the pH from 2.5 to 7.5, which are consistent with the deprotona-
it was subsequently analyzed by HPLC coupled with ESI-MS, allowing tion of the side chains and the concomitant formation of the
the unambiguous assignment of the species formed in the DCL H-bonded folded conformation. The corresponding NMR spectra
(ESI†). All the possible macrocyclic dimers (six) and trimers (ten) of the isostructural non-charged Gln derivative [1b–1b] showed
were detected, the library being dominated by the dimers (Fig. 1A).
Interestingly, when generating the same DCL in the presence
of a high salt concentration,15 we observed remarkable differ-
ences (Fig. 1B). The composition of the library changed following
a pattern in agreement with the amplification of the glutamate
rich species. This effect was evident by plotting the salt-induced
amplification factors (area of the peak in the presence of salt
over the area of the peak in the absence of salt) of the dimers at
different salt concentrations (Fig. 1C). We observed an increase
of the [1a–1a] homodimer (up to B60% increase), a B20%
decrease of the heterodimers containing 1a and the increase
(B20%) of the corresponding dimers exclusively made of 1b
and/or 1c. This pattern corresponds to the effect of the single
amplification of the [1a–1a] dimer. We additionally confirmed
Fig. 2 (A, B) Proposed conformations for [1a–1a] in the neutral (A) and in the
this trend by dynamic deconvolution:16 the macrocycle [1a–1a] tetraanionic (B) forms (non-polar H-atoms have been omitted).
c
488 Chem. Commun., 2013, 49, 487--489
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DCLs of macrocyclic pseudopeptides induces the amplification of
the members concentrating a large number of acidic side chains.
This behaviour has a remarkable resemblance with the natural
evolution of the proteins of halophilic organisms for surviving in
hypersaline media. Our findings show the utility of DCLs for the
experimental modelling of the molecular evolutionary trends
observed in nature, with foreseen implications in the prebiotic
chemistry and in the understanding of the origin of life.20
This work was supported by the Spanish Ministry of Economy
and Competitiveness (MINECO, CTQ2009-14366-C02-02 project).
J.A. thanks CSIC for personal financial support (JAE-predoc
fellowship). We thank Prof. Sijbren Otto for providing us with
the DCLSim software.
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c
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