Conformationally controlled, thymine-based α-nucleopeptides

Article abstract of DOI:10.1039/b822789f

Rigid peptide backbones and backbone-to-side chain H-bonds permit the design of α-nucleopeptides with known 3D-structure; thymine-thymine base pairing is also observed.

Full text of DOI:10.1039/b822789f

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Conformationally controlled, thymine-based a-nucleopeptidesw
Piero Geotti-Bianchini,ab Marco Crisma,a Cristina Peggion,a Alberto Bianco*b
and Fernando Formaggio*a
Received (in Cambridge, UK) 18th December 2008, Accepted 30th March 2009
First published as an Advance Article on the web 30th April 2009
DOI: 10.1039/b822789f
Rigid peptide backbones and backbone-to-side chain H-bonds
permit the design of a-nucleopeptides with known 3D-structure;
thymine–thymine base pairing is also observed.
The synthesis of Z-L-AlaT-OH was achieved according to
literature procedures,6,11 starting from the Na-protected L-Ser
b-lactone. In our hands, the method described in ref. 11 gave less
racemisation, as proved by the higher optical rotation values
measured. This finding was confirmed by comparing the HPLC
chromatograms of two samples of Z-(Aib-L-AlaT-Aib)2-OtBu
(Fig. 1), synthesised from nucleoamino acids prepared according
to two different methods (ESI, Fig. S1 and S2).w
RNA interference (RNAi) approaches are currently widely
investigated for gene therapy applications.1 The goal is to
design molecules able to prevent the translation of undesirable
proteins upon binding to specific mRNA. To this aim, synthetic
oligonucleotides are of little use because of their limited
resistance to enzymatic hydrolysis and their poor ability to
cross biological membranes.
Due to the low reactivity of Aib in peptide bond formation,
particularly when two or more residues of this type occur
consecutively, the nucleopeptides were synthesised in solution.
The sterically hindered peptide bond between Z-L-AlaT-OH
and H-Aib-OtBu was successfully formed by means of the
very effective 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
(EDC)/7-aza-1-hydroxy-1,2,3-benzotriazole (HOAt) coupling
method.12 Catalytic hydrogenolysis of Z-L-AlaT-Aib-OtBu,
followed by acylation with the symmetrical anhydride from
Z-Aib-OH yielded the protected tripeptide Z-Aib-L-AlaT-
Aib-OtBu. The hexapeptide Z-(Aib-L-AlaT-Aib)2-OtBu was
prepared in satisfactory yield by condensing the 5(4H)-oxazolone
from Z-Aib-L-AlaT-Aib-OH (generated in turn by acidolysis
of its tert-butyl ester) with H-Aib-L-AlaT-Aib-OtBu (obtained
by catalytic hydrogenolysis of its Na-protected precursor) in
DMF at room temperature.
To overcome these drawbacks, different solutions have been
proposed, mostly based on phosphate and/or sugar modifications,
while preserving the natural nucleobases. Examples are the
recently proposed homo-oligomers of nucleoside-derived
amino acids2 or the classic peptide nucleic acids (PNAs).3
The latter have gained much attention as they are insensitive
to phosphatase hydrolytic actions, while retaining (or even
improving) polynucleotide complexation properties. Chiral
PNA4 and oxy-PNA5 enjoy interesting applications, although
their low ability to permeate membranes enables so far only
in vitro uses. A polyamide-based alternative to PNAs is
represented by nucleopeptides,6,7 i.e. peptides constituted
by nucleobase-containing amino acids.8 In the case of
a-nucleopeptides, the backbone perfectly matches that of a
natural a-peptide, thus allowing for an easier prediction of
their 3D-structure.7,9 This observation stimulated us to
undertake a research program aimed at building a-nucleopeptides
with predictable and well-defined spatial distributions of their
nucleobases.
A conformational NMR investigation was performed on
Z-Aib-L-AlaT-Aib-OtBu in CDCl3 solution. The presence of
sequential NH–NH cross-peaks in the ROESY spectrum
(Fig. 2, left) suggests that this nucleopeptide adopts a folded
conformation in this solvent. The chemical shift behaviour of
the four NH protons upon DMSO addition (Fig. 2, right)
indicates solvent-shielding for the a-NHs of AlaT2 and Aib3.
Whereas this result is expected for the Aib3 NH if involved in
a b-turn, it is somehow surprising for the AlaT a-NH.
To better understand this behaviour we carried out an IR
absorption analysis in CDCl3 solution in the concentration
By taking advantage of our long-standing experience in
the synthesis and 3D-characterisation of conformationally
constrained a-amino acids and peptides, particularly those
based on Ca-tetrasubstituted a-amino acids,10 we designed
and synthesised short, b-turn- or helix-forming, a-nucleopeptides.
Specifically, alanyl–thymine (AlaT, Fig. 1) residues were
inserted into host, homo-peptide stretches of a-aminoisobutyric
acid (Aib, Fig. 1). The primary structure of the longest
peptide, a hexamer (Fig. 1), was designed to allow the
alignment of two nucleobases on the same face of the helical
molecule.
range 10–0.1 mM. The NH stretching region (3500–3200 cmꢀ1
,
amide A) allows one to discriminate between free (solvated)
and H-bonded amide NHs (vibration frequency above and
below 3400 cmꢀ1, respectively).13 Self-association is observed
at high peptide concentration. At the lowest concentration, the
a Institute of Biomolecular Chemistry, Padova Unit, CNR,
Department of Chemistry, University of Padova, 35131, Padova,
Italy. E-mail: fernando.formaggio@unipd.it
b
´
CNRS, Institut de Biologie Moleculaire et Cellulaire, Laboratoire
d’Immunologie et Chimie Therapeutiques, 67084, Strasbourg, France.
´
E-mail: a.bianco@ibmc.u-strasbg.fr
w Electronic supplementary information (ESI) available: Synthesis,
spectroscopic and X-ray data. CCDC 714103. For ESI and crystallo-
graphic data, see DOI: 10.1039/b822789f
Fig.
1 Chemical structures of Aib, L-AlaT and the longest
nucleopeptide synthesised.
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Fig. 2 Portion of the ROESY spectrum (left) of Z-Aib-L-AlaT-Aib-OtBu
(1 mM in CDCl3 solution) and chemical shift dependence (right) of its NH
protons upon addition of increasing amounts of DMSO.
spectrum of the nucleo-tripeptide shows three bands centred at
about 3430, 3385 and 3350 cmꢀ1 (ESI, Fig. S3).w To facilitate
data interpretation, we also synthesised the corresponding
nucleo-tripeptide allylated at the N(3)H of the thymine ring.
Its spectrum (ESI, Fig. S4)w displays only two bands, at about
3430 cmꢀ1 and 3350 cmꢀ1, respectively. Therefore, the band at
about 3385 cmꢀ1 in the spectrum of Z-Aib-L-AlaT-Aib-OtBu
is likewise due to the nucleobase NH. In addition, the absence
of aggregation in the allylated tripeptide suggests a fundamental
role of the nucleobase in the self-association process.
The band at about 3350 cmꢀ1 in the spectrum of Z-Aib-L-
AlaT-Aib-OtBu and its allylated analogue might be associated,
at least in part, with the Aib3 NH, H-bonded in a b-turn, as it
is present also in the spectrum of Z-Aib-L-Ala-Aib-OMe14
(ESI, Fig. S4).w Furthermore, the H-bonded band is much
more intense for the allylated AlaT tripeptide than for the
Ala analogue. As inferred from the NMR analysis, this
observation suggests that the a-NH of AlaT2 as well is
intramolecularly H-bonded to a significant extent.
Fig.
3
Top: X-ray diffraction structure of Z-Aib-(D,L)-AlaT-
Aib-OtBu with atom numbering. The L-enantiomer is shown. H-atoms
have been omitted for clarity. Bottom: crystal packing mode.15
Intra- and intermolecular H-bonds are represented by dashed lines.
clearly assigned by NMR in CHCl3 solution (see below),
whereas we were unable to unambiguously draw the same
conclusion for the tripeptide.16
Thymine–thymine association appears to be a common
tendency in these nucleopeptides, not just limited to enantiomeric
pairs as in the crystal state. Indeed, we often observed in the
mass spectra of AlaT-containing nucleopeptides small, but
significantly intense, peaks at m/z = [2M + H]+, indicative of
relevant dimer stability (ESI, Fig. S6).w
Luckily enough, we were able to obtain a single crystal of
Z-Aib-(D,L)-AlaT-Aib-OtBu, suitable for X-ray diffraction
15
analysis.wz The peptide backbone is folded into a type-I
(I0) b-turn (Fig. 3, top). This outcome, not unexpected for
Aib-rich peptides,10 is in agreement with the IR absorption
and NMR analyses in solution. However, besides the intra-
molecular H-bond (from the Aib3 NH to the Z carbonyl)
The IR spectra of the hexapeptide Z-(Aib-AlaT-Aib)2-OtBu
at different concentrations show remarkable contributions
from intermolecular H-bonds (ESI, Fig. S5).w However at
the lowest concentration examined (0.05 mM), the strong
H-bonded N–H band is largely due to intramolecularly
bonded NHs. Taking also into account the position of this
band (3324 cmꢀ1), we can tentatively conclude that the
hexapeptide folds into a helical structure.
stabilizing the b-turn,
a second intramolecular H-bond
between the a-NH of AlaT2 and the C(2)QO of its thymine
moiety is observed. This finding, explaining also the IR and
NMR results described above, is of particular significance
because such a backbone-to-side chain H-bond possibly
reduces the nucleobase mobility, thus allowing for a fine
tuning of its spatial arrangement.
Preliminary CD data show that in MeOH solution
the thymine absorption band (B270 nm, induced CD) is
about twice as intense in the hexapeptide (two thymines) as
compared to the tripeptide (one thymine). However, in CHCl3,
the solvent used for the IR absorption and NMR analyses, the
same band in the hexapeptide is about five times as intense as
that of the tripeptide (ESI, Fig. S7).w We are inclined to
attribute the observed phenomenon to a stronger propensity
The crystal state packing mode (Fig. 3, bottom) reveals an
additional interesting feature: dimerisation through thymine–
thymine base pairing. This behaviour is probably responsible
for the remarkable tendency of these nucleopeptides to self-
associate, as observed also in our IR absorption analysis.
Unfortunately, only for the hexamer was a dimeric structure
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the dimer and the highly deshielded NHs (ESI, Table S4)w of
the thymine rings. By combining this information with
the presence of inter-chain, thymine–thymine cross peaks
(ESI, Table S5),w it appears that our helical hexa-nucleopeptide
forms a thymine-mediated parallel dimer.
To summarise, interesting features of these new nucleo-
peptides are: (i) the markedly rigid peptide backbones forming
b-turn or helices, depending on main-chain length, (ii) the
backbone-to-side chain H-bond reducing the nucleobase
mobility and (iii) the strong tendency to form nucleo-
base-mediated dimers. Altogether, we believe that these
features will allow us to design nucleopeptides with a precise
spatial distribution of their nucleobases.2 Such structured
nucleopeptides might be able to force complementary
polynucleotide chains to adopt unusual conformations, thus
influencing their biological functions.8
We are currently extending to the other nucleobases the
approach here illustrated for thymine.
This work was funded by a grant of the ‘‘Universita
italo-francese/Universite franco-italienne’’ (VINCI 2005) to
P.G.-B.
Fig. 4 NHi - NHi+1 sequential NOEs in the ROESY spectrum of
Z-(Aib-L-AlaT-Aib)2-OtBu (5 mM in CDCl3). For clarity, only the
residue numbers of the two molecules (one is primed) are indicated
(see ESI for details).w
Notes and references
z Crystal data for Z-Aib-(D,L)-AlaT-Aib-OtBu: C28H39N5O8;
M
= 573.64, monoclinic, space group P21/n, a = 12.341(3),
to nucleobase-mediated self-association17 of the hexapeptide,
occurring in solvents of low polarity. However, more extensive
experimental and theoretical investigations are needed to fully
understand this phenomenon.
b = 17.198(4), c = 14.434(3) A, b = 93.36(6)1, V = 3058.2(12) A3,
T = 293(2) K. 5075 reflections measured, 4537 independent reflections
(Rint = 0.031), R1 = 0.0515 [F Z 4s(F)], wR2 = 0.1756 (F2 all data),
goodness-of-fit on F2 = 1.131.
The 1H NMR analysis of the hexa-nucleopeptide in
CDCl3 solution not only confirms the IR and CD findings
on self-aggregation, but also suggests the formation of a
dimer. At 1 mM concentration, the spectrum shows twice as
many NH signals (sharp peaks) as expected (ESI, Fig. S8,
trace a),w three of them resonating at unusually high frequency
(d 4 11 ppm). Interestingly, upon DMSO addition (ESI, Fig. S8,
traces b–e)w the NH signals do not shift at all; instead their
intensity decreases as a new set of signals (with the expected
number of peaks) grows. A reasonable explanation for
these results is that in neat CDCl3 solution a single type of
non-centrosymmetric dimeric structure is formed, whereas
DMSO addition causes the dimer to dissociate into two
monomeric peptides. The NH protons in the dimer are
strongly H-bonded, while in the monomer part of them are
exposed to the solvent and shift to lower fields when DMSO
concentration increases.
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2D-NMR homo- and hetero-correlated experiments, carried
out in CDCl3 solution, allowed us to assign all proton
resonances (ESI, Table S4)w and to confirm this hypothesis:
the signals in the 1D spectrum belong indeed to two distinct
hexapeptide molecules. In the ROESY spectrum two sets of
sequential NHi - NHi+1 NOEs are observed (Fig. 4), thus
indicating that both molecules adopt a helical structure.18
This finding suggests that the nucleobases protrude from the
same side of the peptide helix, as expected for residues at
i, i + 3 positions in Aib-rich 310-helical peptides. Indeed, intra-
chain thymine–thymine connectivities are found (ESI, Table S5).w
Such nucleobase alignment strongly favours cooperative
H-bond mediated interactions, as proved by the stability of
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15 Despite numerous attempts, we could grow single crystals only
from a sample containing about 6% of Z-Aib-D-AlaT-Aib-OtBu.
For this reason, two enantiomeric tripeptides (one containing
L-AlaT and the other D-AlaT) are present in the crystal cell.
16 400 MHz NMR spectra were recorded, at 298 K, for various
CDCl3 solutions of Z-Aib-L-AlaT-Aib-OtBu, at concentrations up
to the solubility limit (about 100 mg mLꢀ1).
17 A. Weiß and U. Diederichsen, Eur. J. Org. Chem., 2007, 5531.
18 K. Wuthrich, NMR of Proteins and Nucleic Acids, Wiley,
New York, 1986, p. 162.
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3180 | Chem. Commun., 2009, 3178–3180