A convenient asymmetric synthesis of a β-amino ester with additional functionalization as a precursor for peptide nucleic acid (PNA) monomers

Article abstract of DOI:10.3390/11060435

We report the asymmetric synthesis of di-3-pentyl (3S,αS,7E)-3-N- benzyl-N-α-methylbenzylamino-dec-7-enedioate (9), which contains the correct functionalization to produce δ-amino acid derivatives to be used as monomers for Peptide Nucleic Acid (PNA) formation With this aim, thymine-pentanoic acid 15 and some of its ester derivatives were obtained, their reactivity was studied and the noteworthy ethyl ester 12 was quantitatively produced by transesterification of methyl ester 11, thus paving the way for the synthesis of the thymine-containing amino ester IV, which has been designed as a building block for a Nucleic-Acid analog with a chiral, flexible peptide backbone

Full text of DOI:10.3390/11060435

Molecules 2006, 11, 435-443
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
A Convenient Asymmetric Synthesis of a β-amino Ester with
Additional Functionalization as a Precursor for Peptide Nucleic
Acid (PNA) Monomers
Narciso M. Garrido*, David Díez, Sara H. Domínguez, M. Rosa Sánchez, Mercedes García and
Julio G. Urones
Departamento de Química Orgánica, Universidad de Salamanca, Plaza de los Caídos 1-5, 37008,
Salamanca, Spain; Tel. (+34) 923-294474, Fax (+34) 923-294574
* Author to whom correspondence should be addressed; e-mail: nmg@usal.es
Received: 8 June 2006; in revised form: 13 June 2006 / Accepted: 15 June 2006 / Published: 21 June
2006
Abstract: We report the asymmetric synthesis of di-3-pentyl (3S,αS,7E)-3-N-benzyl-N-
α-methylbenzylamino-dec-7-enedioate (9), which contains the correct functionalization
to produce δ-amino acid derivatives to be used as monomers for Peptide Nucleic Acid
(PNA) formation With this aim, thymine-pentanoic acid 15 and some of its ester
derivatives were obtained, their reactivity was studied and the noteworthy ethyl ester 12
was quantitatively produced by transesterification of methyl ester 11, thus paving the
way for the synthesis of the thymine-containing amino ester IV, which has been designed
as a building block for a Nucleic-Acid analog with a chiral, flexible peptide backbone
Keywords: Peptide nucleic acids (PNA), thymine derivatives, transesterification,
asymmetric synthesis, β-amino acids.
Introduction
Recently, the chemistry of nucleic-acid analogs (Figure 1, DNA/RNA I) has gained considerable
attention due to their potential use as antisense or antigene agents [1]. Among the known
oligonucleotide analogues [2,3], acyclic N-(2-aminoethyl)-glycyl peptide nucleic acids (Figure 1,
aegPNA II), are found to be very good mimics of DNA/RNA hybrids, and their stability towards
proteases and nucleases has generated interest in medicinal chemistry [3]. A great deal of work is

Molecules 2006, 11
436
currently being undertaken in this field [4]. Leumann et al. [5] decided to investigate the influence of
higher flexibility of the amide backbone (relative to PNA) of the base–pairing properties with
complementary DNA and RNA (Figure 1, δaa PNA III). They performed the synthesis of the
monomer building block IV (Figure 1), related to the repeating monomeric δ-amino acid in III,
containing the nucleobase thymine.
Figure 1. Structure of DNA, PNA and modified PNAs.
HN
H
O
HN
Base
O
Base
N
Base
O
O
H
O
O H(OH)
H
O
-O P O
O
BocHN
N
NH
HN
HN
Base
O
O
O
Base
N
O
Base
COOEt
O
O H(OH)
monomer IV
aegPNA II
δaa PNA III
DNA/RNA I
We have demonstrated the asymmetric synthesis of the monoaddition products 5 and 6 by chiral
lithium amide [(R)-1] Michael addition to diendioate esters 2 and 3 respectively (Scheme 1) [6]. While
addition to (E,E)-octa-2,6-diendioate gave the cyclopentane adduct through a domino reaction initiated
by an asymmetric Michael addition, followed by a 5-exo-trig intramolecular cyclization [7], addition
to the (E,Z)-counterpart gave the monoaddition adduct 4, as it is known that lithium amide does not
produce addition to (Z)-α,β-unsaturated esters [8].
Scheme 1.
Ph
Ph
Ph
Ph
N
Ph
N
COOR
Ph
COOR
COOR
KOtBu
N
Li
ROOC
(R)-1
ROOC
COOR
4
5
2
R = CH(CH2CH3)2
Ph
Ph
NHBoc
CH2
Ph
N
Ph
N
X
COOR
Li
COOR
COOR
n
ROOC
(R)-1
COOR
n = 1; V: γ-aminoacid derivate
n = 3; VI: ε-aminoacid derivate
3
6

Molecules 2006, 11
437
The monoaddition product 6 is obtained instead by treating the acceptor 3 with an equimolecular
amount of the lithium amide, as an excess of amide produces the diaddition adduct [6]. We envisaged
that degradation of the remaining alkenes in these compounds and subsequent transformation of the
ester group would make them precursors of γ- and ε-amino acid derivatives, V and VI respectively.
Davies et al. [9] has recently published a comprehensive review in this area of chemistry covering the
scope, limitation and synthetic applications of the use of enantiomerically pure lithium amides as
homochiral ammonia equivalents in conjugate addition reactions. We present here the synthesis of
monoaddition adduct 9 precursor of IV, and the study of the reactivity of thymine-pentanoic acid and
derivatives, with similar functionality, in order to apply the results to the synthesis of PNA monomers.
Results and Discussion
Whereas (E)-α,β-unsaturated esters are very susceptible to conjugate addition, their (Z)-
counterparts are highly susceptible to γ-deprotonation [8,9]. This suggested a strategy towards the
monoadduct 9 from di-3-pentyl (E,Z)-deca-2,8-diendioate 7. Addition of 7 to an excess (3 equiv.) of
(S)-1 generated the (E)-β,γ-unsaturated monoadduct 9, [α]D26=-6.9 (c 0.93, CHCl3), in 70% yield after
work-up and > 95% de [6], as expected from α-protonation of the intermediate dienoate 8 (Scheme 2).
The absolute configuration at C(3) within (3S,αS)-9 relative to the N-α-methylbenzyl stereocentre is
assigned by analogy with previous authenticated models developed to explain the stereoselectivity
observed during addition of lithium amide (S)-1 to α,β-unsaturated acceptors [10].
Scheme 2.
Ph
Ph
O
Ph
Ph
N
H
Ph
N
COOR
COOR
BocHN
N
NH
Ph
N
COOR
COOR
Li
O
(S)-1
-O
ROOC
COOEt
70%
OR
monomer IV
δ-aminoacid derivate
9
7
8
We propose the monoadduct 9 as a precursor of the the δ-aminoacid monomer IV, via
transformation of the ester group and degradation of the remaining double bond in 9 to an ester, further
work within our group haven proven that the double bond can easily be transformed into an aldehyde,
the corresponding dioxolane derivative or the related methyl ester [11]. We envisaged performing
some reactions once the thymine nucleobase is attached, so we decided to synthesize the methyl
thymine-pentanoate 11 and undertake the reactions shown in Scheme 3. It is of great interest to
achieve, with the nucleobase attached, the transesterification process from methyl or other alkyl ester
to the ethyl one, which is the ester group in the compound IV described in the literature [5], in order to
ascertain the optical rotation and thereby the enantiomeric excess produced over the whole process.
Nevertheless, the reactions shown in scheme 3, and the preparation of thymine pentanoic acid 15 are

Molecules 2006, 11
438
of importance on their own, as the homologous thymine acetic acid has already been used to form an
amide bond with a diamino acid derivative to furnish a chiral peptide nucleic acid monomer [12].
Esterification of 5-bromovaleric acid (10) with diazomethane and subsequent treatment with
thymine in basic media produces methyl ester 11 (62%), which upon transesterification by treatment
with HCl( in EtOH yields ethyl ester 12 quantitatively.
g)
Scheme 3.
MeOOC
EtOOC
i,ii
iii
N
N
3
3
HOOC
Br
3
99%
62%
O
N
H
O
O
N
H
O
12
10
11
vi
99%
iv
v
xii
69%
73%
O
O
3
HOOC
OHC
N
N
N
3
72%
vii
3
O
N
O
O
N
O
O
N
H
O
viii
H
H
14
15
13
ix
ó x
ó xi
i
80%
O
MeOOC
HO
N
3
O
N
3
11
+
O
N
O
O
N
O
Me
H
16
17
ratio: 1/1
i: CH2N2; ii: thymine, K2CO3, Bu4NI; iii: HCl(g), EtOH; iv: DIBAL-H; v: PDC, MeOH;
vi: KOH/MeOH; vii: ethane-1,2-diol, p-TsOH; viii. NaClO2, NaH2PO3; ix: PDC, tBuOOH;
Oxone®, wet Al2O3 ; xi: O3; xii: NaOH, EtI, HMPA.
Reaction of ester 11 with DIBAL-H produces aldehyde 13 (69%) which upon further treatment
with ethane-1,2-diol and p-TsOH produces the acetal compound 14 in 72% yield. When aldehyde 13
was treated with different oxidant reagents such as aq. NaClO2 only degradation products were
obtained. However, PDC oxidation in MeOH [13] gave methyl ester 11 in good yield (73%).
Our attempts to obtain ester 17 by oxidation of dioxolane 14 were unsuccessful, since when
compound 14 was treated with PDC and tBuOOH in dichloromethane (DCM) [14] for long periods of
time or with with Oxone® and wet Al2O3 in CHCl3 [15] only starting material was recovered, and
treatment with O3 [16] produced a complex mixture.
Hydrolysis of 11 with KOH/MeOH 2M gave a quantitative yield of 15, which upon treatment with
diazomethane yielded a 1:1 mixture of esters 11 and 16 in 80% yield. Compound 16 is a methyl ester
where the nitrogen of the thymine group has been alkylated. When acid 15 was subjected to treatment
with EtI in NaOH and HMPA it did not produce the ethyl ester 12.

Molecules 2006, 11
439
Conclusions
We have demonstrated an efficient strategy for the asymmetric synthesis of β-amino ester 9 by
asymmetric Michael addition of (S)-1 to the (E) double bond and γ-deprotonation of the (Z) double
bond in the (E,Z)-deca-2,8-diendioate ester 7. Reactivity assays on the nucleobase containing the
model thymine-pentanoic acid and derivatives showed that they are prone to degradation upon
treatment with oxidants such as O3 or NaClO2; the oxidation of dioxolane 14 to produce ester 17,
needs further study. On the other hand, the aldehyde and acid functions in 13 and 11 can be easily
interchanged; the diazomethane esterification of the acid function in compound 15 is competitive with
nitrogen methylation of the thymine moiety, but interestingly, quantitative transesterification of methyl
ester 11 to produce ethyl ester 12 was achieved, paving the way to obtain the required ester in the
preparation of the previously synthesized δ-amino acid IV. The application of this strategy for the
preparation of δ-amino acid monomer IV is currently under investigation in our laboratory and will be
published in due course.
Experimental Section
General
13
1H-NMR and C-NMR spectra were recorded in CDCl3 at 200 and 400 MHz (1H) or 50 and 100
MHz (13C) on Varian 200 VX and Bruker DRX 400 instruments, respectively. Multiplicities were
determined by DEPT experiments. IR spectra were recorded using a BOMEM 100 FTIR
spectrophotometer. Optical rotations were determined using a Perkin-Elmer 241 polarimeter in a 1 dm
cell and are given in units of 10-1 deg cm2 g-1. Concentrations are quoted in g per 100mL. The electron
impact (EI) mass spectra were run on a VG-TS 250 spectrometer using a 70 eV ionizing voltage.
HRMS were recorded using a VG Platform (Fisons) spectrometer using Chemical Ionization
(ammonia as gas) or Fast Atom Bombardment (FAB) techniques. Thin layer chromatography (tlc) was
performed on aluminum sheets coated with 60 F254 silica. Sheets were visualized using iodine, UV
light or 1% aqueous KMnO4 solution. Column chromatography (CC) was performed with Merck silica
gel 60 (70- 230 mesh). Solvents and reagents were generally distilled prior to use: DMF from CaH2
and dichloromethane (DCM) from KOH.
Preparation of di-3-pentyl (3S,αS,7E)-2-N-benzyl-N-α-methylbenzylamino-dec-7-enedioate (9)
n-BuLi (1.6 M, 0.41 mL, 0.66 mmol) was added to a stirred solution of (S)-N-benzyl-N-α-methyl-
benzylamine (145 mg, 0.69 mmol) in THF (3 mL) at –78°C and the mixture stirred for 30 minutes
prior to the addition of a solution of 7 (76 mg, 0.22 mmol) in THF (1 mL) at –78°C. After 80 minutes,
saturated aqueous NH4Cl solution was added and the resulting solution warmed to r.t., partitioned
between DCM (3 x 50 mL) and brine and dried over Na2SO4. Concentration followed by flash
chromatography on silica gel (95:5 Hex-EtOAc) gave 9 (85 mg, 70%); IR (film) ν (cm-1): 2971, 2880,
1732, 1458, 1260, 1157, 1117, 968; 1H-NMR (400 MHz): 0.84 (6H, t, J= 7.5 Hz, CH(CH2CH3)2), 0.89
(6H, t, J= 7.5 Hz, CH(CH2CH3)2), 1.34 (3H, d, J= 7.0 Hz, C(α)Me), 1.50 (4H, m, H-4 and H-5), 1.57
(8H, m, CH(CH2CH3)2), 1.98 (4H, m, H-2 and H-6), 3.01 (2H, d, J= 5.1 Hz, H-9), 3.36 (1H, m, H-3),

Molecules 2006, 11
440
3.49 (1H, d, J= 13.9 Hz, NCHAHPh), 3.81 (1H, d, J= 13.9 Hz, NCHHBPh), 3.82 (1H, q, J= 7.0 Hz,
C(α)H), 4.68(1H, quin, J= 5.4 Hz, CH(CH2CH3)2), 4.77 (1H, quin, J= 5.4 Hz, CH(CH2CH3)2), 5.54
13
(2H, m, H-7 and H-8), 7.21-7.41 (10 H, m, Ar-H); C-NMR (50 MHz): 9.5 (4 CH3, CH(CH2CH3)2),
20.5 (CH3, C(α)Me), 26.3 (2CH2, CH(CH2CH3)2), 26.5 (2CH2, CH(CH2CH3)2), 26.6 (CH2, C-5), 32.4
(CH2, C-4), 33.2 (CH2, C-6), 36.7 (CH2, C-2), 38.5 (CH2, C-9), 50.1 (CH2, NCH2Ph), 53.7 (CH, C-3),
58.3 (CH, C(α)H), 76.6 (CH, CH(CH2CH3)2), 77.0 (CH, CH(CH2CH3)2), 122.0 (CH, C-7), 134.4 (CH,
C-8), 126.6-128.2 (10CH, Ar-C), 141.8 (C, Cipso), 142.9 (C, Cipso), 172.6 (2C, COOR); EIMS m/z (%):
549 (M+, 10), 462 (8), 352 (40), 248 (15); HRMS (EI): C35H51NO4, M+ requires 549.382; found
549.383;
[
α
]
26 = -6.9 (c= 0.93, CHCl3).
D
Preparation of 1-(4’-methoxycarbonylbutyl)-thymine (11)
5-Bromovaleric acid 10 (300 mg, 1.6 mmol) was treated with a solution of gaseous CH2N2 in ether
and stirred for 3 hours. Evaporation of the solvent gave methyl-5-bromo-pentanoate (300 mg, 96%);
IR (film) ν (cm-1): 2953, 1740, 1437, 1364, 1260, 1206, 1173, 1125, 1035; 1H-NMR (200 MHz): 1.60-
13
1.95 (4H, m, H-3 and H-4), 2.27 (2H, m, H-2), 3.33 (2H, m, H-5), 3.59 (3H, s, COOMe); C-NMR
(50 MHz): 23.6 (CH2, C-3), 32.1 (CH2, C-2), 32.2 (2CH2, C-4 and C-5), 51.7 (CH3, COOMe), 173.6
(C, COOMe); EIMS m/z (%): 196 (M+), 167 (15), 165 (15), 137 (12), 115 (100), 101 (45), 89 (40). To
a solution of thymine (479 mg, 4.6 mmol) in DMF (400 mL), K2CO3 (317 mg) and Bu4NI (124 mg)
were added and the reaction mixture was stirred for 30 minutes at 0ºC and at 65ºC for another 30
minutes. To this solution the previously synthesized methyl-5-bromopentanoate (1.53 mmol) was
added and the mixture was stirred at 65ºC overnight. The reaction mixture was then cooled to r.t. and
extracted with EtOAc. The organic layer were washed with water and dried over Na2SO4.
Concentration followed by flash chromatography on silica gel (95:5 DCM-MeOH) gave 11 (228 mg,
1
62%); IR (film) ν (cm-1): 3190, 3057, 2955, 1720, 1684, 1458, 1362, 1204, 1082; H-NMR (200
MHz): 1.61-1.80 (4H, m, H-2’ and H-3’), 1.91 (3H, s, Me), 2.36 (2H, t, J= 8.0 Hz, H-4’), 3.67 (3H, s,
13
COOMe), 3.70 (2H, t, J= 8.0 Hz, H-1’), 6.98 (1H, s, H-6), 9.10 (1H, m, NH); C-NMR (50 MHz):
12.1 (CH3, Me), 21.5 (CH2, C-3’), 28.3 (CH2, C-2’), 33.2 (CH2, C-4’), 47.8 (CH2, C-1’), 51.4 (CH3,
COOCH3), 110.4 (C, C-5), 140.6 (CH, C-6), 151.2 (C, C-2), 164.8 (C, C-4), 173.4 (C, COOCH3);
EIMS m/z (%): 240 (M+, 52), 209 (42), 180 (25), 140 (39), 126 (62), 110 (38), 96 (100); HRMS (EI):
C11H16O4N2 requires 240.111; found 240.113.
Preparation of 1-(4’-ethoxycarbonylbutyl)-thymine (12)
A solution of 11 (17 mg, 0.07 mmol) in EtOH (5 mL) at 0ºC was treated with a stream of gaseous
HCl till the mixture was saturated. The solution was stirred overnight at r.t., and the solvent was then
removed under vacuum to give 12 (19 mg, 99%); IR (film) ν (cm-1): 3185, 3057, 2955, 1724, 1684;
1H-NMR (200 MHz): 1.24 (3H, t, J= 6.8 Hz, CO2CH2CH3), 1.60-1.80 (4H, m, H-3’ and H-2’), 1.91
(3H, s, Me), 2.35 (2H, m, H-4’), 3.70 (2H, m, H-1’), 4.12 (2H, q, J= 6.8 Hz, CO2CH2CH3), 6.98 (1H, s,
H-6), 9.22 (1H, s, NH); 13C-NMR (50 MHz): 12.3 (CH3, Me), 14.2 (CH3, CO2CH2CH3), 21.7 (CH2, C-
3’), 28.4 (CH2, C-2’), 33.2 (CH2, C-4’), 48.2 (CH2, C-1’), 60.2 (CH2, CO2CH2CH3), 110.7 (C, C-5),

Molecules 2006, 11
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140.4 (CH, C-6), 150.8 (C, C-2), 164.3 (C, C-4), 173.0 (C, CO2CH2CH3); HRMS (EI): C12H18O4N2
requires 254.127; found 254.127.
Preparation of 1-(5’-oxopentyl)-thymine (13)
To a solution of 11 (99 mg, 0.41 mmol) in DCM (4 mL) at –78ºC, DIBAL-H (0.9 mL, 1.0 M) was
added. The reaction mixture was stirred for 1 hour at -40ºC and then H2O was added. The resulting
solution was warmed to r.t and added to a mixture of NaHCO3 and Na2SO4 in ether. It was then
filtered through Celite® and concentrated in vacuo to give 13 (60 mg, 69%); IR (film) ν (cm-1): 3187,
3048, 2947, 1684, 1472, 1362, 1258, 1221, 1094; 1H-NMR (200 MHz): 1.62-1.81 (4H, m, H-2’ and H-
3’), 1.90 (3H, s, Me), 2.52 (2H, t, J= 8.0 Hz, H-4’), 3.70 (2H, t, J= 8.0 Hz, H-1’), 6.98 (1H, s, H-6),
9.35 (1H, s, NH), 9.77 (1H, s, CHO).
Preparation of 1-(4’-[1,3]dioxolan-2-yl-butyl)-thymine (14)
p-TsOH and ethane-1,2-diol were added to a solution of 13 (69 mg, 0.33 mmol) in dry benzene.
The reaction mixture was refluxed in a Dean Stark apparatus for 12 hours. The solution was cooled
and then H2O (1 mL) was added, extracted with Et2O and the organic layer washed with NaHCO3 and
brine, dried and concentrated in vacuo to give 14 (60 mg, 72%); IR (film) ν (cm-1): 3196, 3059, 2953,
1
1688, 1472, 1362, 1221, 1177, 1130; H-NMR (200 MHz): 1.62-1.85 (4H, m, H-2’, H-3’ and H-4’),
1.91 (3H, s, Me), 3.69 (2H, t, J= 8.0 Hz, H-1’), 3.83 (2H, m, OCH2CH2O), 3.95 (2H, m, OCH2CH2O),
13
4.85 (1H, t, J= 8.0 Hz, H-5’), 6.97 (1H, s, H-6), 8.82 (1H, s, NH); C-NMR (50 MHz): 12.5 (CH3,
Me), 21.0 (CH2, C-3’), 29.1 (CH2, C-2’), 33.7 (CH2, C-4’), 48.6 (CH2, C-1’), 65.0 (CH2, OCH2CH2O),
65.1 (CH2, OCH2CH2O), 104.3 (CH, C-5’), 110.8 (C, C-5), 140.7 (CH, C-6), 150.9 (C, C-2), 164.4 (C,
C-4); EIMS m/z (%): 254 (M+, 5), 182 (12), 153 (10), 126 (14), 96 (19), 73 (100); HRMS (EI):
C12H18O4N2 requires 254.1266; found 254.1298.
Preparation of 1-(4’-methoxycarbonyl-butyl)-3-methylthymine (11)
To a solution of 13 (50 mg, 0.21 mmol) in DMF (1 mL) and MeOH (0.05 mL) was added PDC
(474 mg, 126 mmol) at r.t. and the mixture was stirred for 20 hours, then H2O (1 mL) was added and
the reaction mixture was extracted with Et2O. The organic layer was washed with H2O and brine. It
was then dried over Na2SO4 and filtered. Evaporation of the solvent followed by flash chromatography
on silica gel (95:5 DCM-MeOH) gave 11 (36 mg, 73%).
Preparation of 1-(4’-carboxybutyl)-thymine (15)
Compound 11 (30 mg, 0.12 mmol) was added to the solution of KOH (0.1 mL) and MeOH (2M)
and was stirred at r.t. for 8 hours, then H2O was added to the reaction mixture and the solution was
treated with 2M HCl to pH = 2 and extracted with Et2O. The organic extracts were washed with water
to give, after drying and concentration, compound 15 (28 mg, 99%); IR (film) ν (cm-1): 3600, 3185,
1
3057, 2928, 1684, 1474, 1418, 1362, 1260; H-NMR (200 MHz): 1.61-1.82 (4H, m, H-3’ and H-4’),

Molecules 2006, 11
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13
1.90 (3H, s, Me), 2.40 (2H, t, J= 6.8 Hz, H-2’), 3.72 (2H, t, J= 6.8 Hz, H-1’), 7.04 (1H, s, H-6); C-
NMR (50 MHz): 14.6 (CH3, Me), 25.4 (CH2, C-3’), 30.8 (CH2, C-2’), 31.9 (CH2, C-4’), 49 (CH2, C-1’),
113.8 (C, C-5), 145.6 (CH, C-6), 150.9 (C, C-2), 169.4 (C, C-4), 179.5 (C, COOH); EIMS m/z (%):
244 ((M+NH4)+, 38), 226 (M+, 8), 209 (35), 126 (70), 110 (40), 96 (100). Compound 15 (10 mg, 0.05
mmol) was dissolved in a solution of gaseous CH2N2 in ether and stirred for 30 minutes at r.t.
Concentration followed by flash chromatography on silica gel (95:5 DCM-MeOH) gave 16 (5 mg,
1
40%) and 11 (4 mg, 40%); Compound 16: H-NMR (200 MHz): 1.67-1.69 (4H, m, H-3’ and H-4’),
1.93 (3H, s, Me), 2.36 (2H, t, J= 8.0 Hz, H-2’), 3.34 (3H, s, Me-N), 3.65 (3H, s, COOMe), 3.70 (2H, t,
J= 8.0 Hz, H-1’), 7.16 (1H, s, H-6).
Acknowledgements
The authors wish to thank Junta de Castilla y Leon and F. S. E. for financial support (SA045A05)
and Spanish MEC (CTQ2005-06813/BQU).
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