QUARTERLY Communication Conformational properties of N-acetyl-L-alanine

Ab initio/DFT analysis of the conformational properties of free Ac-Ala-NMe(2) (N-acetyl-L-alanine-N',N'-dimethylamide) in terms of the N-H.O, N-H.N, C-H.O hydrogen bonds and C(delta+) = O(delta-) dipole attractions was performed. The Ala residue combined with the C-terminal tertiary amide prefers an extended conformation and that characteristic of the (i + 1)th position of the betaVIb turn. These can be easily remodelled into a structure compatible with the (i + 1)th position of the betaII/betaVIa turn. The residue has also the potential to adopt the conformation accommodated at both central positions of the betaIII/betaIII' turn or the (i + 1)th position of the betaI/beta'I turn.

N-Alkylation of biologically active peptides can result in analogues with improved pharmacological properties, such as resistance to enzymatic degradation, receptor selectivity, enhanced potency and bioavailability, and sometimes in conversting an agonist into an antagonist (Gilon et al., 2003).Although these structural modifications of the peptide back-bone exert a powerful influence on peptide bioactivity, the consequences of the N-substitution for peptide conformation have not been thoroughly examined so far.The literature provides a systematic conformational theoretical analysis of only a few diamide and triamide models (Möehle & Hofmann, 1995;1998).They show remarkable differences as compared with the corresponding standard peptide units, caused by the limited or lost capacity to form conventional hydrogen bonds.The authors notice the conformers stabilised with N-H•••O hydrogen bonds, and the presence of those without such a bond is as a rule neglected.However, recent reports indicate weaker hydrogen bonds (Kim & Friesner, 1997;Malone et al., 1997;Vargas et al., 2000;2001) and non-covalent attractions between two C d+ = O d-dipoles not mediated by a hydrogen atom (Maccallum et al., 1995;Allen et al., 1998) as important factors in stabilising the energy-minimised structures.The latest report on the most often studied peptide model Ac-Ala-NHMe (N-acetyl-L-alanine-N¢methylamide) (Vargas et al., 2002) for the first time uses not only the N-H•••O but also the weaker N-H•••N and C-H•••O hydrogen bonds as key to the energetics.In this work, we present a detailed ab initio and DFT study of the conformational properties of Ac-Ala-NMe 2 (N-acetyl-L-alanine-N¢,N¢-dimethylamide) in terms of all the contacts mentioned above and compare the results obtained with those on related molecules.

METHODS
The conformational properties were examined on a free molecule using the GAUSSIAN 98 package (Frisch et al., 1998).To generate the (f,y) potential energy surface, 576 structures calculated at the ab initio HF/6-31G*// HF/3-21G level were used.In each structure, all geometrical parameters were fully relaxed, except for the constrained torsion angles f and y.Values of these angles were chosen by using a step size of 15°, within the range from -180°to 180° (Head-Gordon et al., 1991).The minima observed on the surface were then subjected to full geometry optimisation at the DFT/B3LYP/6-31+G** level, which should enable correct prediction of the stability order of the minima calculated (Vargas et al., 2002), followed by a second derivative analysis (frequency), which proved all of them to be minima.The geometrical parameters of the corresponding energy-minimised conformers were then further discussed.
The accessible conformational space of the molecule studied was assumed on the basis of the close resemblance between the Ramachandran contact map and the energy contours map within the limit of 5.0 kcal × mol -1 (Ramachandran & Sasisekharan, 1968), as is also applied elsewhere (Zimmerman et al., 1977;Herzberg & Moult, 1991).The space was calculated by way of the Surfer 8 programme using the radial basis function as a gridding method.
As the overall conformational profiles of modified peptide models can differ from those of common peptide models, we describe the energy-minimised conformers of the investigated molecule by the general short hand letter notation introduced by Zimmerman ( Zimmerman et al., 1977).

Figure 1 depicts the landscape and contour
Ramachandran plot for Ac-Ala-NMe 2 , obtained at the HF/6-31G*//HF/3-21G level, accompanied by the conformers corresponding to seven minima fully re-optimised at the B3LYP/6-31+G** level.These conformers along with the energy information and selected geometric parameters are listed in Table 1. Figure 1 and Table 1 also present the comparative data for the Ac-L/DL-Ala-NMe 2 crystal conformers (Rzeszotarska et al., 2002).Tables 2 and 3 collect the B3LYP/6-31+G** conformer geometric parameters, respectively, of the hydrogen bonds, based on Steiner's criteria (Steiner, 2002), and of dipole attractions, based on Allen's criteria (Allen et al., 1998).
Calculations revealed two low-energy conformers E and C as well as five high-energy conformers G, A, A*, D* and F*.The lowest-energy conformer E (f, y = -154E, 160E)  .The dashed energy contours are drawn every 0.5 kcal × mol -1 to 5.0 kcal × mol -1 as the limit between the allowed and disallowed regions of Ramachandran diagram (Herzberg & Moult, 1991).¨DFT/B3LYP/6-31+G** minima.+ Solid state conformers.b (•••••) Hydrogen bond, (uqqqt) dipole attraction in the conformers.For geometric parameters of those interactions see Tables 2 and 3.The departure from planarity of peptide bond is fully described by the twisting parameter t and the out-of-plane parameters c C and c N (Winkler & Dunitz, 1971).The values of these parameters for the C-terminal tertiary amide in all conformers of Ac-Ala-NMe 2 essentially do not differ from the corresponding averages for the standard peptide bond (Mac-Arthur & Thornton, 1996).However, the value of the parameter c N for conformer G and c C for conformer A* are outstanding, and seem to reflect some internal attractions.The high value of the former results from the The accessible (f,y) conformational space of the Ac-Ala-NMe 2 molecule, based on a 5.0 kcal × mol -1 cut-off (Zimmerman et al., 1977;Herzberg & Moult, 1991), constitutes 18% of the total space, and is located almost entirely in the top-left corner of the map, in the flat region around conformers E and C. The conformer HF, found in the solid state of Ac-L/DL-Ala-NMe 2 (Rzeszotarska et al., 2002), but not detected in the free molecule at the B3LYP/6-31+G** level, also falls in this shallow area.

DISCUSSION
The two lowest-energy conformers E and C of the Ac-Ala-NMe 2 molecule each contain the N-HqqqO hydrogen bond and the C a -HqqqO one.The former bond is a conventional interaction commonly accepted to play the dominant role in determining the relative stability of peptide conformation.The latter has been recently shown to contribute considerably to the molecular energetics.For instance, when the hydrogen donor is made more acidic by the presence of electron-withdrawing groups, the C a -HqqqO interaction can increase in strength to half of that of an N-HqqqO hydrogen bond (Vargas et al., 2000).The Ac-Ala-NMe 2 molecule displays also five other conformers G, A, A*, D*, and F*, high in en-ergy.The energetic gap between structure C and G is very large, viz.DE G-C amounts to as much as 6.5 kcal × mol -1 .The conformers of this group are stabilised by neither N-HqqqO nor C a -HqqqO hydrogen bonds.Nevertheless, they are clearly observable on the conformational map and exist due to C-HqqqO and N-HqqqN hydrogen bonds as well as dipole C=OuqqqtC=O interactions.The latter two contacts seem to be crucial for conformational stabilisation.However, because they are much weaker than the N-HqqqO or C a -HqqqO hydrogen bonds, conformers G, A, A*, D*, and F* have much higher energy than conformers E and C, and are not so easily accessible for the Ac-Ala-NMe 2 molecule.Hence, one may conclude that the studied free molecule heavily prefers the extended conformation (conformer E) and the conformation typical of the (i + 1)th position of the bVIb turn (conformer C).Moreover, when the Ac-Ala-NMe 2 molecule is involved in a condensed phase, e.g.crystal (Rzeszotarska et al., 2002), where some intermolecular forces operate, it readily adopts the conformation H/F located in a quite near neighbourhood of conformers E and C in the large flat region of the easily accessible conformational space (Fig. 1).The low-energy conformer H/F has been also found in the crystal of tBuCO-Ala-(Me)Ala-NHiPr and that of tBuCO-Ala-D -(Me)Ala-NHiPr, for their N-terminal Ala residue.Both these triamides assume a b-turn structure with this residue at its (i +1)th position.The former molecule adopts the bVIa-turn (f 1 , y 1 , f 2 , y 2 = -66°, 137°, -113°, 48°, respectively), and the latter adopts bII-turn (f 1 , y 1 , f 2 , y 2 = -61°, 129°, 99°, -23°, respectively) (Vitoux et al., 1986).The Ala residue combined with a C-terminal tertiary amide can be expected to accept also, although much less readily, a conformation (conformer A/A*) compatible with both central positions of the bIII/bIII¢ turn or the (i + 1)th position of the bI/bI¢ turn.
These results for Ac-Ala-NMe 2 compare well with the theoretical conformational profile of the triamide molecule Ac-Ala-NAla-NHMe (Möehle & Hofmann, 1998) (acc.to Gilon et al., 2003, NAla = N-methylglycine).The f, y torsion angles found for its N-terminal Ala residue in the peptide conformers correspond to those for the six conformers H/F, E, C, A*, A, and C* of the Ac-Ala-NMe 2 molecule, and there is a good agreement between the energetics of these two sets of conformers.Those conformers of Ac-Ala-NAla-NHMe molecule, which are the counterparts of the conformers H/F, E and C of the Ac-Ala-NMe 2 molecule are of low energy, and those related to conformers A*, A, and C* are of much higher energy.As seen, even the high-energy conformers found for the small diamide Ac-Ala-NMe 2 molecule can be adopted by the C-terminally methylated Ala residue in a larger system.So, the weak interactions within this residue can contribute to the energy of a given conformer stabilised by a strong N-H•••O hydrogen bond(s) of a longer range.
Comparison of the conformational pattern of Ac-Ala-NMe 2 (E, C, G, A, A*, D*, F*, -in the given energy order) to that of the standard analogue, Ac-Ala-NHMe (C, E, C*, B, A*, G) (Vargas et al., 2002), enables one to estimate the influence of the N¢-methylation.Five conformers of the Ac-Ala-NMe 2 molecule, E, C, G, A*, and D*, have their counterparts among the Ac-Ala-NHMe conformers.Conformer A has no counterpart in Ac-Ala-NHMe that assumes conformer B instead.Conformer F*, although observed for Ac-Ala-NHMe in earlier reports (Head-Gordon et al., 1991;Jalkanen & Suhai,, 1996) The Ala residue combined with the C-terminal tertiary amide distinctly prefers the extended conformation as well as that characteristic of the (i + 1)th position of the bVIb turn.Both are stabilised first of all by the C 5 type N-HqqqO hydrogen bond and C a -HqqqO one.They lie in the flat region of the potential energy surface and can easily remodel their torsion angles into ones compatible with the (i + 1)th position of the bII/bVIa turn, when influenced by other forces stabilising a larger system.This is documented by X-ray crystal structures ( Vitoux et al., 1986;Rzeszotarska et al. 2002) as well as suggested by the theoretical analysis on a triamide system (Möehle & Hofmann, 1998).The studied residue has the potential to adopt also the conformations typical of both central positions of the bIII/bIII¢ turn or the (i + 1)th position of the bI/bI¢ turn, however, at much higher energy cost.These conformations exist due to weak internal contacts.The stronger of the contacts manifest themselves in the departure from planarity of the tertiary amide bond.

Figure 1 .
Figure 1.Landscape and contour representation of the f, y potential energy surface of the Ac-Ala-NMe 2 molecule a together with its seven conformers b after optimisation at the B3LYP/6-31+G** level of theory.a The map was calculated at the HF/6-31G*//HF/3-21G level of theory in vacuo.Geometry optimisations were performed on a 15°grid.The solid energy contours are drawn every 1 kcal × mol -1 is stabilised by the conventional N-HqqqO hydrogen bond and by two weaker C-HqqqO bonds.The second lowest-energy conformer C (f, y = -101E, 113E) contains also the conventional N-HqqqO hydrogen bond, however, with the geometry far away from the optimal parameters, and, in addition, three C-HqqqO bonds.The remaining conformers have much higher energy.Conformer G (f, y = -158E, -47E), the third in the energy order is N-HqqqN and C-HqqqO hydrogen-bonded.Conformers A (f, y = -67E, -36E) and A* (f, y = 50E, 53E) are stabilised by the C-HqqqO hydrogen bond and the sheared parallel dipole C=OuqqqtC=O interaction.Conformer D* (f, y = 114E, -60E) has two C-HqqqO hydrogen bonds.Finally, the highest-energy conformer F* (f, y = 76E, 165E) is stabilised by the C-HqqqO hydrogen bond and the slightly sheared antiparallel motif involving a pair of dipole C=OuqqqtC=O interactions.The HF/ 6-31G*//HF/3-21G potential energy surface also shows minimum C*.However, it was not confirmed by the full optimisation and this region belongs to conformer D*.
for the X-H•••A contacts (X = N, C; A = O, N) in which H•••A £ 3.2 C and ÐX-H•••A > 90°acc.to Steiner (2002).Distances are given in C. Angles are given in degrees.a, b and N denote C a for the N-HqqqO bond are: HqqqO = 2.809, NqqqO = 2.958, ÐN-HqqqO = 88.4,ÐC=OqqqH = 73.4N-HqqqN contact and the high value of the latter reveals the exceptionally short dipole C=OuqqqtC=O distance.

Table 3 .
Structural parameters for the internal C=OuqqqtC=O dipole interactions in the B3LYP/6-31+G** conformers of the Ac-Ala-NMe 2 molecule a a Data presented only for the C=OuqqqtC=O contacts in which HqqqA £ 3.6 C acc. to Allen et al. (1998).Distances are given in C. Angles are given in degrees.N,C Denote the N-terminal and C-terminal carbonyl group.b As given by Allen et al. (1998).
, was not confirmed by higher level methods.The N¢-methylation does not influence much the geometry of conformers E and G, which are stabilised by the C 5 type N-H•••O and N-H•••N hydrogen bond, respectively.However, N¢-methylation markedly affects other conformers.Conformers C and D* are shifted toward top-left and bottom-right corners of the map with respect to the corresponding conformers of Ac-Ala-NHMe.Those of Ac-Ala-NHMe are stabilised mainly by the relatively strong C 7 type N-H•••O hydrogen bond, whereas in those of Ac-Ala-NMe 2 , this hydrogen bond cannot exist.In consequence, they are stabilised by various weaker hydrogen bonds including the weak C 5 hydrogen bond within conformer C. Similarly, the conformer A* of Ac-Ala-NHMe is stabilised mainly by the C 7 type N-HqqqO hydrogen bond whereas that of Ac-Ala-NMe 2 by the weaker dipole C=OuqqqtC=O interactions.All this features cause that Ac-Ala-NMe 2 suffers more restrictions of its conformational space than Ac-Ala-NHMe.The accessible conformational space is small (18%), and located almost entirely in the top-left corner of the map, in the flat region around the conformers E and C, which are N-H•••O hydrogen-bonded.In contrast, all conformers of Ac-Ala-NHMe, except conformer G, are stabilised by the C 7 or C 5 type N -H•••O hydrogen bond and therefore the conformational space accessible for this molecule is much greater (about 33%) (Ramachandran & Sasisekharan, 1968).CONCLUSION N-Alkylation of peptides is a powerful approach to modify their biological profiles.However, its conformational consequences are not sufficiently recognised.This work presents a detailed ab initio and DFT study of the conformational properties of the Ac-Ala-NMe 2 molecule (N-acetyl-L-alanine-N¢,N¢-dimethyl-amide) in terms of the conventional N-H•••O hydrogen bond as well as the weaker N-H•••N and C-H•••O interactions and non-covalent C d+ = O d-dipole attractions.