Biomolecular Synthesis and applications of secondary amine derivatives of (+)-dehydroabietylamine in chiral molecular recognition †

(+)-Dehydroabietylamine ( 1a ), the novel derivatives ( 2a – 6a ) and their NTf 2 salts ( 1b – 6b ) were tested as chiral NMR solvating agents for the resolution of enantiomers of the model compound Mosher ’ s acid ( 7 ) and its n -Bu 4 N salt ( 8 ). Best enantiomeric discrimination of 7 was obtained using bisdehydroabietyl-amino- N 1 , N 2 -ethane-1,2-diamine ( 6a ), and of 8 using N -(dehydroabietyl)-2-(dehydroabietylamino)ethana-minium bis((tri ﬂ uoromethyl)-sulfonyl)-amide ( 6b ). For the maximal resolution of enantiomers of 8 , 1.0 eq. of 6b were needed. However, 0.5 eq. of 6a su ﬃ ced for the maximal resolution of enantiomers of 7 . Enantiomeric excess studies were successfully conducted using 6a and 6b . The capability of 6a and 6b to recognize the enantiomers of various α -substituted carboxylic acids and their n -Bu 4 N salts were examined. Best resolutions were observed for aliphatic and aromatic carboxylic acids bearing an electronegative α -substituent. Now the ee studies on such non-aromatic carboxylic acids are also feasible.


Introduction
Analytical enantiomeric purity determinations are of great interest particularly in organic, medicinal and biological chemistry, where stereocontrol is important. Compared to commonly used techniques such as HPLC, NMR is more versatile, and the development of more sensitive instruments has made it a potential competitor for the traditional methods in chiral recognition. As NMR can provide fast and easy enantiomeric excess (ee) measurements (up to 94-99% ee), 1 it is the ideal tool for quick ee determinations when developing new asymmetric synthesis or when studying the efficacy of a new chiral catalyst. Also, if the enantiomeric resolution cannot be efficiently performed with traditional methods, NMR can be used as an alternative. 2 In NMR, two general methods to investigate the enantiomeric purity of a compound may be applied. One is to use an enantiomerically pure chiral derivatising agent to produce two diastereomers. However, this method is time consuming and also may cause concerns of kinetic resolution and racemization. The second, more convenient and faster method is provided by chiral solvating agents (CSAs) where the resolving ability is based on the supramolecular complexation between two enantiomers of a chiral guest and a chiral host. 3, 4 Since complex formation is strongly dependent on interactions between a host and a guest, CSAs may contain hydrogen bond acceptor and donor groups (such as -NH 2 , -OH, -COOH), aromatic functionality for π-π stacking, and ionic and dipolic groups for ion-ion, dipole-dipole and ion-dipole interactions. 4,5 However, the functional groups of the host and guest are not solely responsible for an efficient enantiomeric discrimination, but also the deuterated solvent used, concentration, molar ratio of host and guest, temperature and the anion (if present) of the chiral host 6 have an effect on the resolution. 3 The non-ionic and ionic CSAs are often derived from chiral natural compounds such as amino acids, menthol or mandelic acid. These compounds are generally not only chiral but also contain suitable functionalities for complex formation.
In this study we have used the readily available softwood resin derivative (+)-dehydroabietylamine 7 (1a, Scheme 1) as a starting material to create novel CSAs for the enantiomeric resolution of racemic carboxylic acids by NMR. As only few of the reported amine based CSAs are ionic, and it being thought advisable to see if ionic functionalities might provide better resolution, protonated forms of our amines were also tested.

Results and discussion
Five secondary amine derivatives 2a-6a of 1a were prepared and converted along with 1a to bis(trifluoromethane)-sulfoni-mide (NTf 2 ) salts (1b-6b) to give CSAs with ionic functionality. NTf 2 was chosen for a counter anion as it has been found to provide better enantiomeric resolution with an ionic CSA. 7,8 Compounds 3a-6a were readily prepared by an expedient one step reaction with suitable alkyl bromides in a microwave reactor (Scheme 1). Compound 2a was synthesized in two steps by the conversion of 1a to formamide followed by reduction. Physical data of the compounds are listed in Table 1, showing that when the amines are converted to NTf 2 salts the melting points increase and the optical rotations decrease.
The ability of compounds 1-6a and b to recognize the chirality of ionic and non-ionic racemic carboxylic acids was examined using Mosher's acid 7 and its n-Bu 4 N salt 8. The effect of concentration of CSA was also investigated, as according to literature, the magnitude of non-equivalence (Δδ) increases when the concentration of host is higher than that of the guest. 3,4 CDCl 3 was chosen as solvent for the experiments because it is known that polar solvents can solvate ions and protic solvents may interfere in hydrogen bond formation which are important for complex formation. 9 NMR experiments were performed by taking 0.5 mL (1.0 eq., 22.0 mM) of a stock solution containing 7 or 8 (guest) and dissolved CSA (host) (1.0 or 2.0 eq.). Both 1 H and 19 F NMR spectra were recorded (Table 2). Results indicate that 1-6a and b form a dia-stereometric salt pair with the model compound (7 or 8) as no resolution was detected between a non-ionic CSA (1a-6a) and 8, or an ionic CSA (1b-5b) and 7. This is most probably caused by the lack of suitable interactions in forming a salt pair. However, the ionic 6b resolved the non-ionic 7 presumably because the former has a non-ionic amine group to be protonated by 7 and is therefore able to resolve the enantiomers of 7. Due to the poor solubility of 6b in CDCl 3 the resolution of 7 under 2 : 1 conditions could not be determined. No significant increase in the chemical shift difference between R and S enantiomers (Δδ) was detected when the concentration of host was doubled. In some cases the increase of concentration even led to a decrease in Δδ. This was especially notable in the case of compounds 6a and 6b. Among the non-ionic CSAs, 1a, 2a and 6a resolved the enantiomers of 7 highly efficiently. Especially 6a worked exceptionally well (host : guest ratio 1 : 1) both in 1 H NMR (0.14 ppm, 71.8 Hz) and in 19 F NMR (0.045 ppm, 21. 2 Hz). The extent of resolution decreased both in 1 H NMR and in 19 F NMR when the molar ratio was increased to 2 : 1. For the resolution of 8 the corresponding NTf 2 salts 1b, 2b and 6b gave best results. In this case the highest resolution was obtained with 6b both in 1 H NMR (0.16 ppm, 81.1 Hz) and in 19 F NMR (0.076 ppm, 35.8 Hz). As in the case of 6a, an increase in concentration lowered Δδ in 1 H NMR; however, in 19 F NMR Δδ was increased to 0.32 ppm (149. 9 Hz).
As compounds 6a and 6b gave the best results, their enantiomeric discrimination ability in NMR was further investigated. To find out how much guest is needed for maximum resolution and to obtain information about the composition of complex (e.g. 2 : 1 vs. 1 : 1 complex), the guest 7 (0.5 mL, 2.0 mM) was titrated with host 6a (46.6 mM). Due to the poor solubility of 6b in CDCl 3 , titration was performed in an opposite manner compared to 6a (i.e., titrating a 2.0 mM solution of host 6b with a 46.6 mM solution of guest 8) (Fig. 1). Titration results from both 1 H and 19 F NMR spectra indicate that the maximal resolution with host 6a occurs at the point where the molar ratio of host and guest was 0.5 : 1 (0.200 ppm,  . Commercially available CSAs are often expensive, and the ability of 6a to resolve enantiomers at the host : guest molar ratio 0.5 : 1 is a clear improvement as a minimum of 1.0 eq. (and in some cases an excess up to 24 eq.) of host is needed to obtain a maximal resolution. 3 Since Δδ between the enantiomers of 7 (and of 8) decreased in 1 H NMR when the concentration of 6a (or 6b) increased, it is assumed that the supramolecular complexation pattern changes when the concentration of host increases.
The suitability of CSAs 6a and 6b for enantiomeric excess (ee) NMR measurements was tested with 7 and 8. Both 6a and 6b can be used to detect the enantiomeric composition of samples with excellent reliability ( Fig. 2a and b; see ESI 4.1 and 4.2 †).
The resolution of racemic α-substituted carboxylic acids or their n-Bu 4 N salts by 6a and 6b, respectively, is presented in Table 3 and ESI 5.1 and 5.2. † CSAs 6a and 6b discriminate best carboxylic acids having an electronegative atom (e.g. O, N, Br) at the α-position (11-15a and b). Such aromatic or nonaromatic carboxylic acids were discriminated equally well. This is a major improvement as it has been suggested that the presence of an aromatic ring is necessary for good signal separation. 10 In any case the resolution of non-aromatic carboxylic acids, especially using amine based CSAs, has been largely neglected. 4,9,11 The carboxylic acids 9a and b, 10a and b were discriminated by the corresponding host only moderately (1-7 Hz). This may be due to the lack of suitable interactions between the host and guest. This, however, is not a problem for ee determination as certain specialized NMR experiments are now available and can be used when the multiplet resolution Table 2 The magnitude of non-equivalence (Δδ) between R and S enantiomers of racemic Mosher's acid (7) and its n-Bu 4 N salt (8)  a Could not be measured since 6b was not soluble in CDCl 3 at high concentrations. nd (no resolution was detected). needs improvement or when the overlapping of CSA and substrate is an issue. 12 Compound 6b resolves the enantiomers of 9-12b better than its non-ionic form 6a resolves those of compounds 9-12a, indicating that stronger interaction can be obtained between the ionic CSA and ionic substrate. This can be used to advantage if the resolution of non-ionic CSA and substrate is not sufficient. CSA 6a resolves the enantiomers of 13-15a better than the corresponding ionic CSA 6b those of 13-15b. Interestingly 6a resolved the prochiral CH 2 hydrogens in compound 9a. Since CSAs are usually able to resolve α-substituted carboxylic acids at the chiral α-site 13 only, this is an additional advantage.

Conclusions
A number of derivatives (2a-6a) of (+)-dehydroabietylamine (1a) and their NTf 2 salts (1b-6b) were prepared for use in chiral molecular recognition studies, the syntheses being carried out by highly expedient microwave techniques. The ability of the CSAs 1-6a and b to resolve racemic 7 and 8 was examined by 1 H and 19 F NMR. 6a showed excellent discrimination ability for 7 and its corresponding NTf 2 salt 6b for 8. Optimum conditions for enantiomeric discrimination with 6a and 6b were determined by titration. 6b gives best results at a 1 : 1 host : guest molar ratio whereas 6a gave best resolution at a 0.5 : 1 host : guest molar ratio. This is a useful result since usually at least 1.0 eq. of CSA are needed for maximum resolution. 6a and 6b are highly useful in ee determination as well.
In resolving various α-substituted racemic carboxylic acids using 6a or 6b, best results were given by acids bearing an electronegative α-substituent. In general, acids bearing or lacking an aromatic moiety performed equally well. For carboxylates, somewhat better results were obtained when ionic CSA 6b was used for the resolution than when using 6a for non-ionic sub-strates. In future, the applicability of compounds 6a and 6b in resolution by NMR will be further investigated, and the development of new (+)-dehydroabietylamine based CSAs are being continued.

Materials and methods
All reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise stated. Dehydroabietylamine was purchased (Sigma Aldrich) as 60% grade and purified by a method described in the literature 14

Synthesis of chiral solvating agents
Purification of (+)-dehydroabietylamine 1a. 60% (+)-dehydroabietylamine (42.0 g) was dissolved in toluene (70.0 mL) and acetic acid (9.65 g) in toluene (30.0 mL) was slowly added. The salt was let to crystallize in fridge. The product was filtered and washed with hexane.  (+)-Dehydroabietylformamide (2.01 g, 6.41 mmol, 1.0 eq.) in THF (20 mL) was added dropwise to a flask containing LiAlH 4 suspension (0.26 g, 6.74 mmol, 1.05 eq.) in THF (15 mL) at 0°C, refluxed for 6 h and let to cool to rt. MeOH was added to reaction mixture, which was stirred for 10 min.  a 11.0 μL of a 46.6 mM 6a solution was added to 0.5 mL of a 2.0 mM solution of the analyte studied, to give an 0.5 : 1 host : guest molar ratio. b 22.5 μL of a 46.6 mM solution of the analyte studied was added to 0.5 mL of a 2.0 mM solution of 6b, to give a 1 : 1 host : guest molar ratio. c Peak overlapped with host peaks; nd (no resolution was detected).
General procedure for preparation of secondary amines under microwave radiation (+)-Dehydroabietylamine (1.0 eq.), 1-bromoethane (1.05 eq.) and Na 2 CO 3 (0.6 eq.) were added to a microwave tube with isopropanol (in the case of 6a (+)-dehydroabietylamine (2.0 eq.), 1,2-dibromoethane (1.0 eq.) and Na 2 CO 3 (1.0 eq.) were used). The reaction mixture was microwave irradiated (110 W at 110°C) for 2 h. The solvent was evaporated and the residue triturated with ether, filtered and mixed with Et 2 O. The separated phases and organic phase was washed with water until neutral. The organic phase was dried over Na 2 SO 4 and filtered, the solvent evaporated and the product dried under vacuum.

General procedure of NTf 2 salts
Primary amine 1a or secondary amine 2a-6a (1.0 eq.) was dissolved to DCM (0.5 mL). HNTf 2 (1.0 eq.) was added at 0°C. Reaction mixture was stirred for 1 h at rt. The layers were separated and the organic phase washed with water (3 × 2.0 mL). The organic solvent was evaporated and product dried in vacuum.

Synthesis of guests
N-Acetylation of phenylalanine was performed according to literature. 16 Preparation of [NBu 4 ] + salts was performed by adding tetrabutylammoniumhydroxide (1.0 M in MeOH, 1.0 eq.) to racemic acid (1.0 eq.) in MeOH. After stirring for 1-3 h, the solvent was evaporated and product was dried in vacuum.