N-aminopeptides as peptidonucleic acids building blocks : A HF/DFT and AIM study

Abstract

Introduction

Material & methods

Energetical results

AIM analysis

Conclusions

Abstract

In our laboratory, peptidic synthesis is carried on on N-aminopeptides in order to synthesize peptidonucleic acids based on an N-aminopeptidic backbone. In the present study, peptidonucleic acids building blocks have been optimized at the B3LYP/6-31G(d,p)//HF/STO-3G level in various conformations and their electronic density has been studied by means of the AIM methodology, in order to assess intramolecular bonding and thus discuss the stability of the conformers. The studied oligomers are built from two nucleic bases linked by an amido arm to an N-aminopeptidic backbone. It is shown that trans configuration of peptidic linkages is favoured energetically and that a supplementary amino group in the peptidic backbone tends to destructurate the helical structure.

Introduction

Two compared backbones of DNA (left) and deltapeptidic PNA (right)

Peptidonucleic acids (PNA) are DNA mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or RNA), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA, RNA (or PNA) oligomers. Usually, PNA are built on a delta peptide backbone, in order to optimize tha spacing between two stacked nucleic bases. In our laboratory, peptidic synthesis is carried on on N-aminopeptides, in order to synthesize peptidonucleic acids based on an N-aminopeptidic backbone.

deltapeptidic backbone(left) and N-aminopeptidic backbone (right)

In the present study, we want to model the interactions between the N-aminopeptidic backbone and the amide linkages used to serve as “arms” bearing the nucleic bases, in order to assess the stability of a peptidonucleic acid building block, with regard to its conformational freedom.

PNA-DNA duplex showing the DNA part in sticks and the PNA part in balls and sticks

To do this, we extracted a PNA-DNA duplex structure from the PDB database, erased the DNA moiety of the structure, changed the deltapeptidic backbone to a N-aminopeptidic one, and then optimized the geometry of the macromolecule at the molecular mechanics level, with every nucleic base frozen to its initial geometry in order to mimic a complex with a DNA moiety. It is important to note that this molecular mechanics optimization is only a preliminary study as the N-N bond is very poorly defined by molecular mechanics.

The molecular mechanics optimized N-amino PNA structure showing frozen parts (in green)

The configurational position (cis or trans) of every peptidic link was then assessed and different assemblies of monomers (the moieties linking one nucleic base to another through two arms linked to the peptidic backbone) were defined according to their cis or trans configuration. Four kinds of building blocks were thus defined, and then optimised at the B3LYP/6-31G(d,p)//HF/STO-3G level of theory and their energies compared. To find reasons for energetical differences, an AIM study of the electronic density of the optimized structures has then been carried on, focussing on the intramolecular hydrogen bonding network.

A monomer building block showing the four peptidic linkages in cis or trans configuration
(note that the backbone central peptidic link is always trans)
In this case, the peptidic links from upper left (thymine) to down left (cytosine) are
trans then cis then trans then trans. It is thus named :
Thy-trans-cis-trans-trans-Cyt

Admittedly, the present study does not model the role of the solvent around this typical biomolecular system. The idea is to get hints about intrinsic reasons for conformational stability of the studied building blocks, as opposed to external reasons from the solvent or other influences.

Material & methods

Structures of PNAs moieties were extracted from the PDB database, from the 1NR8 Xray diffraction structure. This tructure is a PNA-DNA duplex, so first of all, the DNA moiety has been removed. The peptidonucleic backbone was then changed from deltapeptidic to N-aminopeptidic, with two possibilities : An N-amino group on every peptidic link of the backbone (thereafter referred to as “with NH2”), or an N-amino group on one out of two peptidic links (thereafter refrred as “without NH2”). These structures were then optimised using molecular mechanics (AMBER forcefield, default options, within the Hyperchem 7.5 program) with the constraint of all nucleic bases frozen to their initial positions.

From these two optimized structures, eight (four each) building blocks monomers were extracted corresponding to different configurations (cis or trnas) of the five peptidic links encountered in a monomer : two peptidic links in the arms, linking the nucleic bases to the backbone, and three peptidic links in the backbone, two out of three bearing an N-amino group and an arm, and a central one (always trans) bearing (“with NH2”) or not (“without NH2”) an N-amino group.

Quantum calculations on these building blocks at the HF and B3LYP level of theory were performed using the Gaussian 03 suite of programs. The 6-31G(d,p) basis set was used for B3LYP single point energies, calculated on structures previously optimised at the HF level with the STO-3G basis set .

The AIM analysis has been performed with the AIM 2000 code, with all default options. Integration of atomic properties over the atomic basins have been performed in natural coordinates, with a tolerance of 10 -4 per integration steps.

Thy-trans-cis-trans-trans-Cyt
without NH2

Thy-trans-trans-trans-trans-Cyt
without NH2

Thy-trans-trans-cis-trans-Cyt
without NH2

Thy-trans-cis-cis-trans-Cyt
without NH2

Thy-trans-cis-trans-trans-Cyt
with NH2

Thy-trans-trans-trans-trans-Cyt
with NH2

Thy-trans-trans-cis-trans-Cyt
with NH2

Thy-trans-cis-cis-trans-Cyt
with NH2

Energetical results

AIM analysis

In order to assess the intramolecular interactions that may be responsible for conformational energy differences, an AIM topological analysis of the electronic density has been carried on on the six most stable considered building blocks. Out of the B3LYP/6-31G(d,p) wavefunction, the molecular graph and bond critical points have been assessed. Then each intramolecular interaction, as revealed by a bond path between two non covalently bound atoms, has been quantified by means of its density and laplacian at the bond critical point. On these criteria, the interaction has been classified as a (weak or strong) hydrogen bond or not.

AIM Molecular graph of Thy-trans-cis-trans-trans-Cyt without NH2
small red point represent bond critical points ; gray lines represent bond paths
Out of the five non covalent bond paths, only two are retained as efficient hydrogen bond,
after analysis of the density and laplacian at the bond critical point.

Below are the representations of the two most stable building blocks without NH2, showing the revealed intramolecular hydrogen bonds. The Thy-trans-cis-trans-trans-Cyt presents two hydrogen bonds : one N-H...O=C very strong and one C-H...O=C weak. The Thy-trans-trans-trans-trans-Cyt presents only one hydrogen bond : a weak C-H...O=C. The difference in energy between the two configurations is small (2 kcal/mol) but the most stable building block is the Thy-trans-trans-trans-trans-Cyt one, so we must admit that an all trans configuration is favoured energetically, even though it possesses less intramolecular favourable interactions.

Thy-trans-cis-trans-trans-Cyt presents two hydrogen bonds : one N-H...O=C very strong and one C-H...O=C weak (left)
Thy-trans-trans-trans-trans-Cyt presents only one hydrogen bond : a weak C-H...O=C (right)

Then we present the three most stable building blocks with NH2. The Thy-trans-cis-trans-trans-Cyt shows one strong NH2...O=C hydrogen bond and one weak C-H...O=C one. The Thy-trans-trans-trans-trans-Cyt also possesses one strong NH2...O=C hydrogen bond and one weak C-H...O=C one. Lastly, the Thy-trans-cis-cis-trans-Cyt has one strong NH2...N hydrogen bond (even though the hydrogen bond acceptor is an amidic nitrogen). In this case, the most stable conformation is NOT the all trans one, by far. To explain this, we can notice that for the two least stable conformations, a strong hydrogen bond involves the backbone free NH2 and an hydrogen bond acceptor from the backbone itself. This means that these interactions do not contribute to the stabilization of the building block, as defined in our study (with nucleic bases stacked as in a duplex) but tend to move the bases away from each other. This is not the case for Thy-trans-cis-trans-trans-Cyt, which is the most stable configuration.

Thy-trans-cis-trans-trans-Cyt shows one strong NH2...O=C hydrogen bond and one weak C-H...O=C one (left)
Thy-trans-trans-trans-trans-Cyt possesses one strong NH2...O=C hydrogen bond and one weak C-H...O=C one (middle)
Thy-trans-cis-cis-trans-Cyt has one strong NH2...N hydrogen bond (right)

Conclusions

We tried to find reasons why there were differences in stability between PNA building blocks. In this prospect, we analysed the intramolecular hydrogen bond network of the various conformers of the studied building blocks.

We found :

•  that an all trans configuration of the peptidic links is energetically favoured even though the intramolecular network of hydrogen bonds may be disfavoured. This is especially the case when comparing the two most stable conformers of the “without NH2” building blocks. They have approximately the same energy even though the al trans conformer lacks a strong hydrogen bond.

•  that this view is not valid when referring to building blocks “with NH2”. The extra amino group in the peptidic backbone is involved in extra hydrogen bonds that may tend in certain cases to disrupt the building block by driving the bases away from each other and thus destabilizing the structure. In this case, the most stable structure is the one that does not present such a hydrogen bond.

Friedrich Biegler-König and the University of Bielefeld for making available the AIM2000 program, CINES for providing time and space for calculations, are kindly acknowledged.

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