Side-chain conformations for selected backbone conformations of N-acetyl-l -isoleucine-N-methylamide and N-acetyl-l -nor-isoleucine-N-methylamide. An exploratory ab initio study

Journal of Molecular Structure (Theochem) 548 (2001) 21±37

www.elsevier.com/locate/theochem

Side-chain conformations for selected backbone conformations of N-acetyl-l-isoleucine-N-methylamide and N-acetyl-l-norisoleucine-N-methylamide. An exploratory ab initio study M.N. Barroso a, E.S. Cerutti a, A.M. RodrõÂguez a,*, E.A. JaÂuregui a, O. Farkas b, A. Perczel b, R.D. Enriz a a

Department of Chemistry, National University of San Luis, Chacabuco 915 5700, San Luis, Argentina b Institute of Organic Chemistry, Eotvos University, P.O. Box 32, H-1117, 112 Budapest, Hungary Received 24 November 2000; accepted 27 December 2000

Abstract The Ramachandran or backbone potential energy surface (PES) of N-acetyl-l-isoleucine-N-methylamide has been explored using the a,a side-chain conformation. The side-chain conformational PES were generated with ®xed backbone conformations: gl and bl. The bl side-chain PES of the isoleucine derivative was compared to that of the nor-isoleucine derivative. q 2001 Elsevier Science B.V. All rights reserved. Keywords: l-isoleucine residue; Side-chain orientation; Isoleucine residue; Nor-isoleucine residue; Side-chain±backbone interactions; Ab initio MO study; Peptide conformations

1. Introduction Isoleucine has an apolar side-chain and hence it is analogous to alanine, valine and leucine. However, isoleucine is the only one of the four amino acids with an aliphatic side-chain that has a stereocentre at the b-carbon. This carbon has an R-absolute con®guration. Isoleucine is an isomer of leucine and a higher homologue of valine. This also implies that the isoleu* Corresponding author. E-mail addresses: [email protected] (M.N. Barroso), [email protected] (E.S. Cerutti), [email protected] (A.M. RodrõÂguez), gchasse@®xy.org (E.A. JaÂuregui), [email protected] (O. Farkas), [email protected] (A. Perczel), [email protected] (R.D. Enriz).

cine side-chain can reach further out than that of valine. For this reason, isoleucine in a protein may have not only a structural but also a functional role to play. The true functional role of isoleucine is still to be explored for a full understanding. To illustrate its importance, we may cite a few interesting cases. For example, in neuropsychiatric genetics, DNA sequences [1] are analysed for certain proteins in schizophrenic patients, which may play a role in brain function and malfunction. Such studies included the serotonin 1F(5-HT1F) receptor that had a rare sequence variant, which is characterized by a single base substitution (T ! A) in the third position of codon 261 encoding isoleucine. Isoleucine has been used also in NMR structure determination. Incorporating partially deuterated

0166-1280/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0166-128 0(01)00355-4

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M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

isoleucine

2180 # c # 1808 …1†

helps in the structural assignment [2] when 15N, 13C, 2 D-enriched amino acids are used to produce proteins biologically. Also 13C NMR studies on model compounds such as Gly±Ile±Gly suggested that the side-chain motion of hydrophobic residues helps to drive the early stages of protein folding [3]. In the case of isoleucine, the side-chain may be mimicked by 2-methylbutane (I). One of the branched terminal CH3 groups in I corresponds to the a-carbon of N-acetyl-l-isoleucine-N-methylamide (II). Note that the branching carbon is achiral in I but it has R absolute con®guration in II:

2180 # x i # 1808

…2b† 1#i#4

…2c†

A modular numbering system has been used as shown in II, which allows easy extension of the backbone to oligopeptide chain. 2.2. Molecular computations Computations were performed on I and II using Gaussian 98 [5] at the RHF/3-21G [6] level theory. The total energies are given in hartrees, the relative energies and stabilization energies are give in kilocalories per mole using the conversion factor: 1 hartree ˆ 627:5095 kcal mol21 : 2.3. Stabilization energies An isodesmic reaction (3), where R ˆ CHMe±Et;

was used to calculate the stabilization energies (Eq. (4)) with respect to either the gl or to the bl backbone conformation of N- and C-protected glycine [7,8]:

2. Methods 2.1. Conformational analysis Torsional angles were speci®ed (Eq. (2)) within 2180 and 1808 for both backbone …f; c† and sidechain …x 1 ; x2 † conformations in accordance with the IUPAC±IUB [4] recommendation:

MeCONH±CH2 ±CONHMe 1 CH3 ±R

2180 # f # 1808

The stabilization energy (DEstabil) is calculated as

…2a†

reference conformation gl or bl

! MeCONH±CHR±CONHMe 1 CH3 ±H conformation X

…3†

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

23

Fig. 1. PES cross-sections of the PES associated with 2-methyl-n-butane Me±(x 1)±CHMe±(x 2)±CH2 ±(x 3)±Me.

follows:

Table 1. The conformational potential energy curve (PEC) computed at the HF/3-21G level of theory is shown in Fig. 1. Due to the internal symmetry, two conformers (a and g 2) are energetically degenerate since these two structures are enantiomeric. The

DEstabil ˆ {E‰MeCONH±CHR±CONHMeŠX 1 E‰CH3 ±HŠ} 2 {E‰MeCONH±CH2 ±CONHMeŠgl 1 CH3 ±RŠ}

or bl

(4)

Table 1 De®nition for key dihedral angles for isobutane (I) and N-acethyl-lisoleucine-N-methylamide (II) Dihedral angle

3. Results and discussion 3.1. Side-chain model: isopentane Isopentane (2-methyl-n-butane) (I) may be used to mimic the side-chain of isoleucine (II) under ideal conditions. Since the methyl rotations may be ignored due to the fact that the CH3 group has only one unique orientation, only one torsional angle needs to be varied. This is labelled as x 2 in I and de®ned in

x1 x2 x3 x4 v0 v1 f c

Molecule Isobutane (I)

N-acethyl-lisoleucine-Nmethylamide (II)

H 1 ±C 2 ±H 3 ±C 4 H 1 ±C 2 ±C 4 ±C 5 C 2 ±C 4 ±C 5 ±C 7 C 2 ±C 4 ±C 5 ±C 6 ± ± ± ±

N 1 ±C 2 ±C 7 ±C 8 C 2 ±C 7 ±C 8 ±C 9 C 7 ±C 8 ±C 9 ±H 10 C 2 ±C 7 ±C 11 ±H 12 C 21 ±C 20 ±N 1 ±C 2 C 2 ±C 3 ±N 26 ±C 27 C 20 ±N 1 ±C 2 ±C 3 N 1 ±C 2 ±C 3 ±N 26

24

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

Table 2 Optimized torsional angle, total and relative energies for the conformational minima of 2-methylbutane, Me2CH±CH2Me (I) as computed at the RHF/3-21G level of theory Initial x 2

Optimized x 2

Emin (hartree)

DE (kcal mol 21)

60 180 260

62.66 2172.29 265.71

2195.2505694 2195.2520747 2195.2520745

0.94 0.00 0.00

third conformer (g 1) is higher in energy since the three CH3 groups are close to each other. As may be seen in Fig. 1, the pro-chiral R and pro-chiral S hydrogens (labelled as HR and HS) are equivalent, therefore enantiotopic, only in the g 1 conformation but they are diastereotopic in the a and g 2 conformations. The results of the geometry optimizations are shown in Table 2. 3.2. Peptide model: N-acetyl-isoleucine-Nmethylamide With the exception of proline [9], nine backbone conformations are expected to exist for every amino acid residue [10±12] as can be seen in Fig. 2A. However, not all nine legitimate conformers appear as energy minima on the Ramachandran potential energy surface (PES) [10±12] of most peptides. Very often the al and 1l conformations are annihilated [10±12]. As far as side-chain conformers are concerned, the PES or potential energy hypersurface

(PEHS) depends on the number of torsional modes as well as the type of functionality the side-chain contains. The situation is relatively simple for hydrocarbon side-chains. However, the number of rotating bonds predetermines the dimensionality of the potential energy (hyper) surface. For isoleucine (II), only two torsional angles are required since there is only one unique methyl orientation. The topology of the side-chain PES for isoleucine (II) is shown in Fig. 2B. Such topology, in general, is valid for most double rotors. In the ®rst phase of this part of our study, we explored the backbone or Ramachandran PES keeping the side-chain in a, a orientation (Eq. (5)). In the subsequent phase of this part of our work, we explored the side-chain PES keeping the backbone in either gl (Eq. (6)) or bl (Eq. (7)) conformation: E ˆ Ea;a …f; c†

…5†

E ˆ E gl …x1 ; x2 †

…6†

E ˆ Ebl …x1 ; x2 †

…7†

The geometry optimized minima for the above three surfaces are summarized in Tables 3±5. The following observations can be made from the data presented in Table 3: (i) Considering the Ramachandran PES, the al and 1l conformations, which are usually annihilated, are also missing here.

Fig. 2. Topology of the backbone or Ramachandran (A) and a double rotor side-chain (B) PES as cross-sections of a PEHS of four independent variables: Ex3 ˆsyn ˆ E…f; c; x 1 ; x2 †: Such a PEHS is applicable for all trans-peptide bond …v0 ˆ v1 ˆ 1808† containing isoleucine residue in a peptide chain.

2173.22

2176.28

265.82

263.30 263.28 267.21 260.39 261.21

x3

f

57.69

287.99

65.30 2135.19 63.68 60.58 55.52 74.19 64.54 75.10 67.08 2157.35

x4

72.95

136.43 40.35 149.63 257.26 257.01

c

2176.27

177.42 172.95 2161.44 175.10 174.64

v0 179.70 179.69 2179.82 2178.36 2179.58 Not found Not found 2177.319 Not found

v1

2606.5773364

0.21

24.95

21.88 1.60 6.18 22.54 3.69

gl

DE (kcal mol 21)

25.616

22.54 0.94 5.52 21.88 3.03

bl

DEstabil (kcal mol 21)

Relative energy a

2606.5724302 3.29 2606.5668882 6.77 2606.5595934 11.34 2606.5734868 2.74 2606.5635545 8.86

(Emin (hartree)

Total energy

a The global minimum corresponds to bl(g 2, g 2) conformation having 2606.5776747 hartree total energy. This value is taken a reference value, corresponding to relative energy 0.00 kcal mol 21.

2167.48 2167.61 2176.11 2162.11 2163.75

2174.20 2161.80 2179.53 2171.19 2168.40

bl ad 1d gd dd al 1l gl dl

x2

x1

(backbone)

Initial conformation Optimized parameters

Table 3 All backbone conformations of N-acetyl-l-isoleucine-N-methylamide for selected side-chain conformations optimized at RHF/3-21G level of theory

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37 25

45.55 63.09 72.91 179.92 2176.28 2159.96 280.30 269.80 268.75

61.10 2168.76 259.58 59.91 2173.22 255.35 65.945 2171.25 266.94

263.48 265.26 250.77 263.32 265.82 252.13 263.29 265.36 255.38

x3 53.45 53.48 56.29 63.25 57.69 66.04 53.99 50.28 52.33

x4 287.61 286.53 285.85 287.80 287.99 288.04 286.07 286.40 286.70

f 67.65 66.24 62.95 74.25 72.95 79.16 65.77 63.96 64.06

c 2175.15 2174.24 2173.18 2176.45 2176.27 2177.55 2174.93 2174.68 2174.94

v0 2178.98 2179.00 2179.67 2177.28 2177.32 2176.47 2178.94 2179.52 2179.49

v1 2606.5700812 2606.5763949 2606.5734051 2606.5764925 2606.5773364 2606.5745981 2606.5741786 2606.5769448 2606.5772574

Emin (hartree)

Total energy

4.76 0.80 2.68 0.74 0.21 1.93 2.19 0.45 0.26

20.40 24.36 22.49 24.43 24.95 23.24 22.97 24.71 24.91

gl

DE (kcal mol 21)

21.06 25.03 23.15 25.09 25.62 23.90 23.64 25.37 25.57

bl

DEstabil (kcal mol 21)

Relative energy a

a The global minimum corresponds to bl(g 2,g 2) conformation having 2606.5776747 hartree total energy. This value is taken as a reference value, corresponding to relative energy 0.00 kcal mol 21.

g a g2 g1 a g2 g1 a g2

1

1

g g1 g1 a a a g2 g2 g2

x1

x2

x1

x2

Optimized parameters

Initial parameters

Table 4 Optimized torsional angles, total energy values, relative energies and stabilization energies computed at the RHF/3-21G level of theory, for side-chain conformational minima of Nacetyl-l-isoleucine-N-methylamide in its (gl) backbone conformation

26 M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

g1 a g2 g1 a g2 g1 a g2

g1 g1 g1 a a a g2 g2 g2

Not found 64.08 78.56 2177.01 2174.20 Not found 263.88 267.47 269.16

x1

x2

x1 264.27 249.79 260.04 263.30 254.18 262.34 249.07

90.20 2165.23 261.27

x3

2165.08 259.04 66.77 2167.48

x2

Optimized parameters

Initial parameters

61.31 53.09 52.29

51.47 52.36 70.05 65.30

x4

2137.57 2140.90 2137.35

2162.96 2163.84 2136.89 2135.19

f

164.59 163.95 161.94

156.69 159.73 141.54 136.43

c

172.75 173.76 173.76

178.88 178.83 176.79 177.42

v0

177.69 177.64 177.253

178.64 178.46 178.86 179.70

v1

2606.5704826 2606.5749176 2606.5776747

2606.5748555 2606.5730294 2606.5716212 2606.5724302

Emin (hartree)

Total energy

4.51 1.73 0.00

1.77 2.91 3.80 3.29

20.65 23.44 25.17

23.40 22.25 21.37 21.87

gl

DE (kcal mol 21)

21.31 24.10 25.83

24.06 22.91 22.03 22.53

bl

DEstabil (kcal mol 21) Relative energy

Table 5 Optimized torsional angles, total energy values, relative energies, and stabilization energies, computed at the RHF/3-21G level of theory, for the conformational minima of N-acetyll-isoleucine-N-methylamide in its (bl) backbone conformation

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37 27

28

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

Fig. 3. Six backbone conformers located on the Ramachandran PES.

(ii) An additional conformation dl, which is usually an energy minimum on the Ramachandran PES now does not represent a stable structure. (iii) All backbone conformations tolerate the a, a side-chain conformation.

All in all, isoleucine is a rather typical amino acid with respect to those already studied. The current database, which may provide the basis for comparison includes the following N- and C-protected amino acids containing a trans-peptide bond: Gly [10±18], Ala [10±18], Val [19], Phe [20,21] and

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

29

Fig. 4. Nine conformers located on the side-chain PES for the gl backbone conformation. [Note, the gl(a,a) conformation is already shown in Fig. 3].

Ser [22±24]. Preliminary studies have been published on Pro [9], Asp [25], Asn [26], Cys [27] and Sec (selenocysteine) [28]. A number of observations can be made from the

data given in Tables 4 and 5: (i) All side-chain orientations occur in the gl backbone conformation on the E ˆ E…x1 ; x2 † side-chain

30

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

Fig. 5. Seven conformers located on the side-chain PES for the bl backbone conformation. [Note the bl(a,a) conformation is already shown in Fig. 3].

PES. In other words, none of the side-chain orientations was repulsive enough to annihilate the gl backbone conformation. (ii) Even though some side-chain orientations were annihilated (i.e. g 1g 1, ag 1 and ag 2) at the bl back-

bone conformation, the global minimum came from this family namely the bl (g 2, g 2) conformation. Relative energies measure the collective stabilizing and destabilizing interactions originating both from

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

the backbone and the side-chain. As far as the backbone is concerned, the stabilizing interactions occur only in the gl and gd and to some extent, in the bl conformation. These stabilizing interactions are of the CyO: ! H±N type of hydrogen bonding. The side-chain, of course, may interact with the backbone. If the side-chain has a polar group such as ±CH2 ±OH, ±CH2 ±SH, ±CH2 ±COOH, ±CH2 ± COO 2, ±CH2 ±CONH2 and the like, then extensive side-chain±backbone interactions are possible. All of these lead to overall stabilization. The analysis of these structures reveals what type of interactions are responsible for the stabilization of the conformers that do, in fact, occur on the PEHS. In contrast to that, when the side-chain is apolar, such as ±CHMe2 (valine), ±CH2 ±CHMe2 (leucine) and ±CHMe±CH2Me (isoleucine), only repulsive interactions are possible. In such cases, the conformers, which are destabilized to such an extent that they are annihilated from the conformational PES, are structurally more interesting than those that actually do occur on the PES. Of course, from the biomedical point of view, the non-annihilated stable structures are still the signi®cant conformers of study. In the present case, no side-chain conformer has disappeared when the backbone was of gl conformation. However, two side-chain orientations were not tolerated when the backbone conformation was bl. The annihilated conformations were close neighbours

31

as follows:

…8†

Consequently, it seems reasonable that the steric repulsion is originating from the side-chain±backbone proximity. Indeed, this was the case when we examined the 27 geometries. Their structures, arranged according to the topology of their corresponding PESs, are shown in Figs. 3±5. 3.3. Peptide model: N-acetyl-nor-isoleucine-Nmethylamide One of the dif®culties we have in assessing the repulsive interaction involving the side-chain is related to the branching at its b-carbon. Simply, we may consider the side-chain as ±CHMeEt. It may well be that the Et group is more dominant than the Me group since it is longer. However, we cannot separate the two effects because they always occur together. For this reason, it was necessary to study the simpler system n-butane (III) as side-chain model for the norisoleucine residue within N-acetyl-nor-isoleucine-Nmethylamide (IV):

g1 a g2 g1 a g2 g1 a g2

g1 g1 g1 a a a g2 g2 g2

Not found 59.05 57.88 2175.43 2174.62 Not found 257.37 258.16 259.98

x1

x2

x1 260.75 258.05 265.12 259.70 257.80 258.52 252.28

97.46 173.27 262.99

x3

2177.91 293.88 65.44 174.94

x2

Optimized parameters

Initial parameters

2139.88 2141.90 2133.55

2165.40 2161.59 2162.51 2163.10

f

163.32 163.99 162.82

169.02 163.86 154.29 154.81

c

173.49 174.43 173.70

178.14 178.43 179.98 179.95

v0

176.99 176.98 176.88

178.14 177.11 178.26 178.28

v1

2567.7526328 2567.7551371 2567.7566297

2567.7575510 2567.7540052 2567.7539239 2567.7551572

Emin (hartree)

Total energy

3.09 1.51 0.58

0.00 2.22 2.28 1.50

21.56 23.33 24.26

24.84 22.62 22.57 23.34

gl

DE (kcal mol 21)

22.22 23.99 24.92

25.50 23.28 23.23 24.00

bl

DEstabil (kcal mol 21) Relative energy

Table 6 Optimized torsional angles, total energy values, relative energies, and stabilization energies, computed at the RHF/3-21G level of theory, for the conformational minima of N-acetyll-nor-isoleucine-N-methylamide in its (bl) backbone conformation

32 M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

The geometry optimization results of compound IV in its bl-backbone conformation are summarized in Table 6. If the Me group played no steric role at all, then the two sets of DE values given in Table 5 for leucine and Table 6 for isoleucine would be very close to each other. However, the data show that some of them are quite different. Even their global minima are not the same. A correlation of the two sets of values is shown in Fig. 6. In three of the structures (g 2g 2, g 2a and g 1g 2), the average deviation (Eq. (9)) between the two sets of points is only 10.12 kcal mol 21: DDE ˆ …20:58 1 0:26 1 0:69†=3 ˆ 0:12

…9†

This suggests that in these side-chain conformations (when the backbone is bl) the b-Me group does not play a signi®cant repulsive role. However, in the case of the other three structures (g 1a, aa and g 2g 1) the average deviation (Eq. (10)) is 1.66 kcal mol 21: DDE ˆ …11:77 1 1:79 1 1:42†=3 ˆ 1:66

…10†

It is therefore very likely that in these side-chain orientations (while the backbone is bl), the b-Me group does exert a repulsive interaction, which may be estimated in the vicinity of 1.5 kcal mol 21 on an average. (Note 1:66 2 0:12 ˆ 1:54†: In other

Fig. 6. Correlation of DEisoleucine and DEnor-isoleucine values.

33

side-chain orientations, the CH3 group plays no significant role in generating a steric effect. 3.4. Stabilization energies We are now in a position to compare the stabilization energy that the two side-chains (for isoleucine and nor-isoleucine) exert on the backbone. These are shown graphically in Fig. 7. Of course, the two sets of results do not parallel each other since there is a difference in the order of the minima as was seen in Fig. 6. However, if we compare the global minima, we can see that isoleucine is stabilized slightly more, by 0.33 kcal mol 21 than nor-isoleucine. A comparison with other amino acids is shown in Fig. 8. 3.5. Side-chain folding Side-chain folding is de®ned by the side-chain torsional angles x 1, x 2, x 3 and x 4. However, the consequence of folding is the induction of point Table 7 Values of side-chain C±H bond length of the pro-chiral CH2 moieties as in 2-methylbutane and N-acetyl-l-isoleucine-N-methylamide in their various conformations 2-Methylbutane

N-acetyl-l-isoleucine-Nmethylamide

x2

C 5 ±HS

C 5 ±HR

C 8 ±HS

C 8 ±HR

x1

x2

Backbone

g1

1.0863

1.0863

1.0855 1.0857 1.0845 1.0857

1.0783 1.0861 1.0733 1.0879

g2 g2 a g1

g1 g1 g1 g1

bl gl gl gl

a

1.0861

1.0866

1.0823 1.0809 1.0798 1.0763 1.0827 1.0772 1.0787 1.0827 1.0877 1.0861

1.0862 1.0872 1.0868 1.0866 1.0844 1.0892 1.0836 1.0833 1.0790 1.0851

a a a a a a g1 g2 g1 g2

a a a a a a a a a a

gd bl gl 1d dd ad bl bl gl gl

g

1.0866

1.0869

1.0836 1.0861 1.0872 1.0766 1.0851

1.0859 1.0859 1.0843 1.0857 1.0854

g2 g1 a g1 g2

g2 g2 g2 g2 g2

bl bl gl gl gl

34

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

Fig. 7. A comparison of DEstabil(gl) values obtained for isoleucine (II) and nor-isoleucine (IV).

M.N. Barroso et al. / Journal of Molecular Structure (Theochem) 548 (2001) 21±37

35

Fig. 8. A comparison of DEstabil(gl) values obtained for various amino acid residues.

Table 8 Values of side-chain C±H bond length of the pro-chiral CH2 moieties as in n-butane and N-acetyl-nor-isoleucine-N-methylamide in their various conformations n-Butane

N-acetyl-nor-isoleucine-N-methylamide

x2

C3 ±HR

C3 ±HS

C4 ±HR

C4 ±HS

C7 ±HR

C7 ±HS

C8 ±HR

C8 ±HS

x1

x2

Backbone

g1

1.0856

1.0859

1.0856

1.0844

1.0849 1.0852

1.0847 1.0817

1.0847 1.0826

1.0816 1.0848

g2 g1

g1 a

bl bl

a

1.0858

1.0858

1.0858

1.0858

1.0823 1.0854

1.0845 1.0856

1.0830 1.0828

1.0870 1.0824

a g2

a a

bl bl

g2

1.0859

1.0856

1.0859

1.0856

1.0850 1.0857

1.0820 1.0854

1.0834 1.0831

1.0847 1.0856

g1 g2

g2 g2

bl bl

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chirality by axis chirality. Such consequences can be ªmeasuredº or ªassessedº in terms of the variation of C±H bond lengths. In the case of pro-chiral CH2, the different values for C±HR and C±HS are very useful indicators [29]. However, in the case of isoleucine there is a second stereocentre, in addition to the a-carbon at Scon®guration. This stereocentre is in the side-chain and it is due to the attached Me-group to the b-carbon leading to a R-con®guration. Consequently, we must start with a simpler system: n-butane (III), which mimics the side-chain of the nor-isoleucine (IV) residue. Our computed results are shown in Tables 7 and 8 for the isoleucine and nor-isoleucine residues, respectively. However, even the simpler system has proved to be too complicated. The only conclusion that emerges in the amino acids (II and IV), all C±H bonds are different in comparison to the model side-chains (I and II). This suggests that in all the conformers investigated, each and every carbon in the side-chain becomes an induced stereocentre. Acknowledgements This work was supported by grants from Universidad Nacional de San Luis (UNSL) and Consejo Nacional de Investigaciones Cientõ®cas y teÂcnicas (CONICET) of Argentina. R.D. Enriz is a carrier researcher of CONICET. References [1] D. Shimron-Abarbane, H. Harms, J. Erdmann, M. Albus, W. Maier, M. Rietschel, J. Korner, B. Weigent, E. Franzek, T. Sander, M. Knapp, P. Propping, M.M. Northen, Am. J. Med. Genet. 67 (1996) 225±228. [2] K.H. Gardner, L.E. Kay, J. Am. Chem. Soc. 119 (1997) 7599± 7600. [3] D.V. Mikhailov, L. Washington, A.M. Voloshin, V.A. Daragan, K.H. Mayo, Biopolymers 49 (1999) 373±383. [4] IUPAC±IUB Commission on Biochemical Nomenclature, Biochemistry 9 (1970) 3471. [5] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A. Montgomery, Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam, A.D. Daniels, K.N. Kudin, M.C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y.

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[29] G.A. Chasse, M.L. Mak, E. Deretey, Z. Szeckely, A. Perczel, R.D. Enriz, I.G. Csizmadia, Conformationally induced stereocentres in small achiral single rotor molecules, submitted for publication.