download report - Istituto Pasteur

P a r t i c i p a n t s :
Bruno Botta, Claudio Villani, professors; Ilaria
D’Acquarica, Marco Pierini, researchers; Alessia Ciogli,
Deborah Subissati, post-doc fellows; Laura Nevola,
PhD student; Giovanna Cancelliere, technician.
Report of activity
One of the greatest current challenges in modern
bioanalysis is to gather some insights into the understanding
of biochemical regulation and communication
processes, most of which are based on non-covalent
interactions between proteins and a variety of
binding partners. Proteins and other macromolecules
can associate with one another, and they can bind to
low-molecular-weight ligands. Such interactions are
fundamental for signalling, enzymatic activity regulation,
immune response processes, and many other
functions in living systems. In addition, these binding
events play a key role in drug action mechanisms.
There are numerous techniques currently available to
detect the presence of binding interactions and for
the determination of dissociation constants. For
example, affinity chromatography involves the immobilization
of either a protein target or a ligand on a
column, such that binding partners are diversely
retained. In the present project, the rational design of
systems featuring molecular recognition abilities was
focused on the surface linking of suitable proteins or
ligands on solid supports. These materials are the
cores of high performance liquid chromatography
(HPLC), one of the most used techniques for the
rapid and effective analysis of complex mixtures of
biomolecules (such as proteins, polynucleotides, and
polysaccharides) in biofluids, and for the isolation of
suitable targets at preparative scale. A relatively
recent advance in chromatographic devices is the
development of monolithic supports based on either
silica or polymer skeletons. These self-supporting
columns do not require frits, have a continuous
Principal investigator: Francesco Gasparrini
Professor of Organic Chemistry
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49912776; Fax: (+39) 06 49912780
francesco.gasparrini@uniroma1.it
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Molecular recognition in biomolecules - AREA 4
Molecular and enantioselective recognition by receptors
and proteins studied in the gas phase, in free solution
and at solid-liquid interfaces
bimodal porosity that leads to high-plate numbers,
and have high through-pore volumes, which provide
low back-pressure and hence increased flow-rates relative
to bead columns. Together, these factors lead to
shorter separation times and more efficient separations.
In this context, we prepared new polymerbased
monolithic HPLC columns by means of gamma
radiation-induced polymerization of monomers
directly inside the final vessels (columns). For this
purpose, a mixture of monomers and cross-linkers
was irradiated in the presence of suitable solvents,
directly inside the chromatographic column whose
internal walls have adequately been pre-activated and
functionalized. Activation was achieved by pre-treatment
with a silane containing methacryloyl groups
(the “grafting to the wall” process), or by introducing
azo-groups onto the internal walls of the tubular
support (“grafting from the wall” approach). These
fragments are able to generate radical species that
trigger the polymerization from the wall of the tubular
support inwards, creating an “active” functionalization
of the internal surfaces.
In the way to find a new class of synthetic receptors
endowed with the ability of recognizing complex
frames such as dipeptide chains, we attached four dipeptide
loops differing for composition and stereochemistry
to the feet of cone resorc[4]arene octamethyl
ethers through their terminal amino groups. The
resulting N-linked peptidoresorc[4]arenes were shown
to recognize the homologue dipeptides by means of
hydrogen bonds, both in solution and in the gas phase.
The complexes appeared to be quite stable, since hosts
and guests could not be separated by column chromatography.
Complexation phenomena were investigated
by NMR methods, including DOSY experiments,
for the detection of translational diffusion.
Heteroassociation constants of 2030 and 186 M -1 were
obtained by the Foster-Fyfe method for one homochiral
complex and the corresponding heterochiral, respectively,
the latter being comparable to the self-association
constant of the dipeptide itself.