A Convenient Asymmetric Synthesis of a Β-amino Ester with Additional Functionalization as a Precursor for Peptide Nucleic Acid (pna) Monomers

We report the asymmetric synthesis of di-3-pentyl


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 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.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].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 γand 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.
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].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 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].
Our attempts to obtain ester 17 by oxidation of dioxolane 14 were unsuccessful, since when compound 14 was treated with PDC and t BuOOH in dichloromethane (DCM) [14] for long periods of time or with with Oxone® and wet Al 2 O 3 in CHCl 3 [15] only starting material was recovered, and treatment with O 3 [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.

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 O 3 or NaClO 2 ; 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 1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 at 200 and 400 MHz ( 1 H) or 50 and 100 MHz ( 13 C) 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 cm 2 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 KMnO 4 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 CaH 2 and dichloromethane (DCM) from KOH.

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 H 2 O was added.The resulting solution was warmed to r.t and added to a mixture of NaHCO 3 and Na 2 SO 4 in ether.It was then filtered through Celite ® and concentrated in vacuo to give 13 (60 mg, 69%); IR (film) ν (cm   (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 H 2 O (1 mL) was added and the reaction mixture was extracted with Et 2 O.The organic layer was washed with H 2 O and brine.It was then dried over Na 2 SO 4 and filtered.Evaporation of the solvent followed by flash chromatography on silica gel (95:5 DCM-MeOH) gave 11 (36 mg, 73%).

Figure 1 .
Figure 1.Structure of DNA, PNA and modified PNAs.