CA1281335C - Chiral two-phase system and method for resolution of racemic mixtures and separation of diastereomers - Google Patents

Abstract

ABSTRACT

A chiral two-phase system for resolution of racemic mixtures, or for separation of diastereomers, is dis-closed. The system comprises two immiscible liquid phases and one or more enantioselectively binding chiral components, each of which is substantially in one of said phases.
Also disclosed is a method for chiral resolution of racemic mixtures, or for separation of diastereo-mers. Use is here made of the fact that different enantiomers are partitioned differently between the phases in the above-mentioned two-phase system in that one of the enantiomers is selectively bound to one of the chiral components which is substantially in one of said phases.

Description

CHIRAL TWO-PHASE SYSTEM AND METHOD FOR RESOLVTION
OF RACEMIC MIXTURES AND SEPARATION
OF DIASTEREOMERS

The invention relates to a system and a method for chiral resolution of racemic mixtures and for separation of diastereomers.
The pharmaceutical industry generally makes great demands on the optical purity of the pharmaceutical preparations. However, the synthesis of the drug fre-quently results in a relatively low optical purity, and the resulting product must be enriched by the right enantiomer by some method of separation.
An attractive method in the context is the direct chiral resolution based on HPLC technique (see for example reference 1). However, because of the low capacity of these systems, the use is restricted to the analytical scale, and therefore there is need in the art for a system which is suitable for upscaling and can be used for preparative purposes.
The invention aims at providing a chiral two-phase system for resolution of racemic mixtures, or for separation of diastereomers, said system being cha-racterised in that it comprises two immiscible liquidphases and one or more enantioselectively binding chiral components, each of which is substantially in one of said phases.
Furthermore, the invention aims at providing a method for chiral resolution of racemic mixtures, or for separation af diastereomers, said method being characterised in that the different enantiomers or diastereomers are partitioned differently between the phases of a chiral two-phase system comprising two immiscible liquid phases and one or more enantio~
selectively binding chiral components, each of which is substantially in one of said phases, by selectively 33~;

binding one of said enantiomers or diastereomers to one of the chiral components.
The two-phase system according to the invention thus is a liquid/liquid/two-phase system for direct chiral resolution. The phase system consists of two immiscible liquid phases which may consist either of two immiscible solvents or of two different poly-mers mixed with water. Examples of immiscible solvents are water and butanol. Examples of different polymers which in mixture with water provide two immiscible phases are a polysaccharide (such as dextran, Aquaphase) and a polyalcohol (polyethylene glycol = PEG).
Phase systems of this type are capable of resolv-ing chiral components, such as proteins, carbohydrates, crownethers and amino acids, or derivatives thereof.
By selecting suitable conditions in the two-phase system, such a chiral componènt can be placed to almost 100~ in one phase, whereas other components partition themselves more equally between the two phases.
By selecting the chiral component such that it selectively binds one enantiomer in a racemic mix-ture or one component in a mixture of diastereomers, this enantiomer will lie substantially within one phase.
After that, the two phases may be separated either by extraction or by means of a multistage process, such as countercurrent distribution.
Examples of carbohydrates that may be used as chiral components in the system according to the in-vention are cyclodextrin and cellulose. As chiral amino acids, use may be made of D- and/or L-proline, and in that case one phase may contain a D-proline derivative and the other phase a L-proline derivative.
The invention will now be described in more detail, reference being had to the following nonrestrictive Examples.

~ ' ~2~ 3S

Example 1 The protein BSA (bovine serum albumin) is used as chiral component for separation of D,L-tryptophan.
The protein was located in the bottom phase of a PLG/
dextran system.
_hemicals and ~hase system_ _ _ _ _ _ _ _ _ _ Dextran 40 was obtained ~rom Pharmacia (Uppsala, Sweden), and polyethylene glycol PEG 6000 (now re-named PEG 8000) from Union Carbide (New York, USA).
The composition of the phase system was: 10% (w/w) Dextran 40, 7% (w/w) PEG 6000, 0.1 M sodium chloride and 50 mM sodium carbonate buffer (pH 9.2). Bovine serum albumin (6.5 g) (Sigma, No. A-3912, fraction V) was added per 100 g phase system. The albumin-con-taining phase system was shaken carefully and thenleft overnight in cold store (4C). D- and L-tryptophan had been obtained from Sigma and L-(side chain-2,3-3H)--tryptophan, specific activity: 50 ~Ci per nmol, from Amersham International ~U.K.).
Countercurrent_distribution An automatic countercurrent distribution equipment with 60 cavities was used (5, 6). To cavities Nos.
1-59, 0.79 ml of respectively top and bottom phase was added, and cavity No. 0 was filled with 0.79 ml bottom phase and the sample is (9 ~mol of each enan-tiomer of tryptophan) dissolved in 0.79 ml top phase.
The shaking time was 40 s, and the partition time 12 min. After 60 transfers (4C) the contents of the cavities were collected in a fraction collector. The top phase of every third fraction was diluted to give a suitable absorbance at 280 nm. The radioactivity both in the top phase and in the total system was measured in a beta-scintillation counter. The scin-tillation liquid was Lumagel (Lumac, 6372 AD Schaes-berg, The Netherlands)._easuring the binding of L-try~tophan to serum albumin_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ In fractions Nos. 0-5 the radioactivity of the 12~33~

top phase and of the total phase system was measured, and the difference therebetween gave the radioactivity of the bottom phase. From the partition coefficient of L-tryptophan, the amount of free L-tryptophan in the bottom phase was calculated. Since -the total radio-activity of the bottom phase was known, the amount of L-tryptophan bound to serum albumin could be cal-culated (for further details, see Fig. 2 in reference 7).
_esults and discussion_ _ _ _ _ _ _ _ _ The partition of serum a:Lbumin depends on, inter alia, the molecular weight of the polymers and the ion content. Here, a phase system was sought in which serum albumin has a low partition coefficient, and this is made possible with dextran of low molecular weight (8) and sodium chloride as the dominating salt.
In this system, 95~ of the serum albumin were in the bottom phase, and the free tryptophan partitioned more equally between the phases (partition coeffi-cient: 1.2).
When the enantiomers were applied separately in the countercurrent distribution, the profiles accord-ing to Fig. 1 were obtained. The enantioselectivity is obvious: the L-enantiomer was retained more in the bottom phase than the D-enantiomer (GL=0.13, GD=0.39).
A separation factor (GD/GL) of 3.1 was obtained. It should therefore be possible to obtain several optically pure fractions.
Similar results were obtained on application of the racemic mixture (Fig. 2). No resolution of the racemate was obtained when separation was carried out without serum albumin. A minor proportion of tritium-labelled L-tryptophan was added, and the major proportion resulted in a peak in the same location as the unlabelled L-form according to Fig. 1. However, part of the tritium labelling was found in fractions Nos. 20-40, presumabLy because of labelled impurities (degradation products) in the tritium-labelled L-tryptophan which was employed L3~i and which was 2 years old.
By determining the partition of the tritium-labelled L-tryptophan in fractions Nos. 0-5, the concentration of free and bound L-tryptophan in the bottom phase, containing serum albumin, could be calculated. A Scat-chard plot of these data is shown in Fig. 3. An asso-ciation constant of 2.9 104 M 1 was obtained, which is well in agreement with published values obtained by other methods (2, 9-12). The number of binding sites was 0.4 (i.e. less than 1), which is to be expected according to other studies (2r 10-12). The low number of binding sites in some studies may be due to the fact that fatty acids are present in the BSA prepara-tion. Since the serum albumin in this study was prac-tically free from fatty acids, a more likely explanationis an inhibition by the phase system polymers. Poly-ethylene glycol resembles decanol which has been found to reduce the binding of tryptophan to bovine serum albumin (2).
Example 2 As chiral component, use is made of cellulose in solid form which was located in the bottom phase of a dextran-PEG-phase system and used for separation of D,L-tryptophan.
Ch_micals and ~hase system Dextran 40 (Pharmacia) and Poly(ethylene glycol) PEG 8000 (Union Carbide, New York). The composition of the phase system was: 10% (w/w) Dextran, 7% (w/w) PEG, 0.5% (w/w) Imidazole and 0.1 M sodium citrate, pH 8Ø 7.5 g cellulose were added per 100 g phase system. The phase sys-tem was shaken carefully and then left overnight in cold store (4C). D- and L-trypto-phan had been obtained from Sigma.
_ountercurrent distribution_ _ _ _ _ _ _ _ _ _ _ _ A 60-cavity automatic countercurrent distribution equipment was used (5). To cavities Nos. 1-59, 0.79 ml of respectively top and bottom phase was added. Cavity 33~i No. 0 was filled with 0.79 ml bottom phase and the sample (0.40 ~mol of each enantiomer) dissolved in 0.79 ml top phase. Shaking was conducted for 40 s, and the partition time was 20 min. After 60 transfers in cold store, the contents oE each cavity were col-lected in 60 test tubes.
nalysis_and_results The absorbance at 280 nm on every other top phase was measured directly (Fig.4).
Example 3 As chiral component, use was made of the protein BSA (bovine serum albumin) in an Aquaphase-PEG-phase system for separation of R,S-methylsulfinyl benzoic acid on a semipreparative scale.
Ch_micals and ~hase system Aquaphase (Perstorp) and Poly(ethylene glycol)PEG
8000 (Union Carbide, New York). The composition of the phase system was: 14~ (w/w) Aquaphase, 5~ (w/w) PEG, 40 mM NaCl and 20 mM sodium phosphate, pH 5.3.
7.0 g bovine serum albumin (Sigma) were added per 100 g phase system. The phase system was shaken carefully and then left overnight in cold store (4C).
Countercurrent_distr_butlon Use was made of an automatic countercurrent di-- 25 stribution equipment comprising 60 cavities (5). To cavities Nos. 1~59, 0.79 ml of respectively top and bottom phase was added. The cavity No. 0 was filled with 0.79 ml bottom phase and the sample (R,S-methyl-sulfinyl benzoic acid, 17 mg, 92 ~mol) dissolved in 30 0.79 ml top phase. Shaking was conducted for 40 s, and the partition time was 20 min. After 60 transfers in cold store, the contents of each cavity were col-lected in 60 test tubes.
Anal~sis_and_results Part of the top phase in every other tube was diluted to a suitable absorbance 225 nm (Fig. 5).
0.3 ml of the top phase of fractions Nos. 10-13 was combined and diluted with part of 40 mM NaCl, 20 mM
sodium phosphate, pH 5.3, whereupon the optical rotation was measured in a Perkin-Elmer 141 polarimeter. Fraetions Nos. 14-17, 18-20, 21-24, 25-27, 28-30 and 31-33 were combined, diluted and measured in the same manner.
The results are shown in Table 1.

~ D
Fractions Nos. 10-13 4.30 Fractions Nos. 14-7 3.80C
Fractions Nos. 18-20 3.85C
Fractions Nos. 21-24 4.48~C
15 Fractions Nos. 25-27 4.02C
Fractions Nos. 28-30 4.19C
Fractions Nos.31-33 4.22C
Example 4 As chiral component, use was made of beta-cyelo-dextrin coupled to Aquaphase (Perstorp) which was partitioned in an Aquaphase/PEG-phase system for sepa-ration of R,S-terbutaline.
Ch_mieals and ~hase system Aquaphase (Perstorp), Poly(ethylene glycol)PEG
8000 (Union Carbide, New York), p-toluene sulfonyl chloride (Sigma), 1,4-diamino butane (Janssen, ~elgium), B-cyclodextrin (Stadex AB, Sweden).
Synthesis of_p-toluene_sulfonyl-B-cyclodextrln(~-CD-OTs) 25 g (22 mmol) B-cyclodextrin (washed with di-ethyl ether and dried over P2O5) are dissolved in 100 ml dry pyridine, the solution is saturated with N2 and placed in an ice bath. 4.18 g (22 mmol) p-toluene-sulfonyl chloride are dissolved in 20 ml dry pyridine, the solution is saturated with N2 and placed in an ice 35 bath. The two solutions are mixed and caused to react at room temperature overnight. The solution is evaporated, and the resulting tough oil is recrystallised from 3~i distilled water. The product is dried over P2O5. Yield 25.9 g. Elementary analysis gave C: 36.9-37.1%, H:
5.36-5.37%, N: 1.06-1.08%, S: 0.38%. Thus, the product contains 6.95% (w/w) pyridine, and about every sixth ~-cyclodextrin unit is tosylated. Rf 0.80 (Silica-platten, Merck in CHC13-MeOH 9-1).
Synthesis of_p-toluene_sulfonyl-_qua~hase(Aq~h-OTs) 10 g (61 mmol) Aquaphase (dried over P2O5) are slurried in 75 ml dry pyridine, the mixture is satu-rated with N2 and placed in an ice bath. 22 g (116mmol) p-toluenesulfonyl chloride are dissolved in 75 ml dry pyridine, the solution is saturated with N2 and placed in an ice bath. The components are mixed, and the mixture is slowly agitated at room temperature for 3 days. A minor undissolved residue remains which is filtered off, whereupon the solution is evaporated.
The resulting semicrystalline mass is treated with 500 ml 0.5 M sodium phosphate buffer, pH 7, for some hours, whereupon the product is obtained. The sub-stance is washed carefully with distilled water andthen dried over P2O5. Yield 16 g. Elementary analysis gave C: 28.2-28.5%, H 2.81-2.88~, N: 0.70-0.72~, S: 6.01-6.16%. (Elementary analysis of Aquaphase gave C: 42.6-42.8~, H: 6.60-6.65.) The product thus contains 4-61% (w/w) pyridine, and every other carbohydrate monomer is tosylated. Rf 0.00-0.10, no traces of p-to-luenesulfonyl chloride (Silicaplatten, Merck in CHC13-MeOH
9-1 and CHC13-MeOH-HOAc-H2O 6-4-1-1).
Synthesis of_l,4-dia_ino-butane-_quaphase(H2N-(CH2)4-NH-_qph) 15 g (60 mmol) Aqph-OTs (above) are added in batches to 60 ml 1,4-diamino butane. After the addition, the solution is heated to 80C for 4 hours and is then left overnight at room temperature. The solution is evaporated, the remainder is diluted with 150 ml distilled water, and pH is adjusted -to 7 with 6 M HC1.
The neutralised solution is dialysed against 3 x 20 1 ~213~335 g distilled water. Freeze drying gave 2.6 g product.
Elementary analysis gave C: 43.2-43.3%, H: 6.85-6.97%, N: 7.28-7.59%.Thus, every other carbohydrate monomer has been provided with an amino spacer. Rf 0.00-0.10 (Silicaplatten,Merck in CHC13-MeOH 9-1 and CHC13-MeOH-HOAc-H2O 6-4-1-1), no traces of 1,4-diamino butane.
Synthesis of_~-CD-NH-(CH2)4~NH~_qPh 2,6 g (13 mmol) H2N-(CH2)4-NH-Aqph (above~ are dissolved together with 15 g (actually 12 mmol tosylated ~-cyclo dextrin) ~-CD-OTs (above) containing 11 mmol pyridine and 95 ml DMF. After agitation at room tempera-ture overnight, 0.8 ml triethyl amine is added. Coupl-ing is allowed to continue for 6 days at room tempera-ture, and finally heating is effected to 80C during 4 hours. The solution is evaporated, and the remainder is diluted with 50 ml distilled water, pH is adjusted with 1 M HCl to 7. The solution is dialysed against 3 x 20 1 distilled water. Freeze drying gave 3.9 g product. Elementary analysis gave C: 42.3-42.6%, H:
6.26-6.27%, N: 2.24-2.32%. Thus, at least every third carbohydrate monomer has been provided with a ~-cyclo-dextrin unit (actually 0.4 mol ~-cyclodextrin units/mol carbohydrate monomer). Rf 0.00-0.10 (Silicaplatten, Merck in CHC13-MeOH 9-1 and CHC13-MeOH-HOAc-H2O 6-4-1-1), ~5 ninhydrin positive reaction left. Composition of the phase system: 14% (w/w) Aquaphase, 2.5% (w/w) ~-CD-NH--(CH2)4-NH-Aqph, 5% PEG, 0.1 M Li2SO4, 6.25 mM sodium citrate, pH 6Ø The phase system was shaken carefully and left overnight in cold store (4C).
Countercurrent_distr_bution Use was made of an automatic countercurrent distri-bution equipment with 60 cavities (5). To cavities Nos. 1-59, 0.79 ml of respectively bottom and top phase was added, cavity No. 0 was filled with 0.79 ml bottom phase and the sample (4.5 ~mol R,S-terbutaline) dissolved in 0.79 ml top phase. Shaking was conducted for 40 s, and the distribution time was 20 min. After 60 transfers in cold store, the contents of the respec-tive cavities were collected in 60 test tubes.
_nalysis_and_results The absorbance at 280 nm on the top phase of every third Eraction was measured (Fig. 6).
Example 5 As chiral component, use was made of L-proline (Pro) coupled to Aquaphase (Perstorp) which was parti-tioned in an Aquaphase/PEG-phase system and used for separation of D,L-tryptophan.
Chemicals and ~hase system Aquaphase (Perstorp), Polyethylene glycol PEG
8000 (Union Carbide, New York), p-toluene sulfonyl chloride (Sigma), Proline (Sigma). Composition of the phase system: 5% (w/w) Aquaphase-Pro, 10% (w/w) Aquaphase, 5% (w/w) PEG 8000, 0.1 M NaCl.
Synthesis of_p-toluene_sulfo yl-Aqua~hase _Aqph-OTs) 10 g (61 mmol) Aquaphase (dried over P2O5) is slurried in 75 ml dry pyridine, the mixture is satu~
rated with N2 and placed in an ice bath. 22 g (116 mmol) p-toluene sulfonyl chloride are dissolved in 75 ml dry pyridine, the solution is saturated with N2 and placed in an ice bath. The components are mixed, and the mixture is agitated slowly at room temperature for 3 days. A minor undissolved residue remains which is filtered off, and the solution is evaporated. The resulting semicrystalline mass is treated with 500 ml 0.5 M sodium phosphate buffer, pH 7, for several hours during which the product is obtained. The substance is washed carefully with distilled water and then dried over P2O5. Yield 16 g. Elementary analysis gave C: 28.2-28.5%, H: 2.81-2.88%, N: 0.70-0.72%, S:
6.01-6.16%. Elementary analysis of A~uaphase gave C: 42.6-42.8%, H: 6.60-6.65%). Thus, the product contains 4.61% (w/w) pyridine. Every other carbohydrate monomer is tosylated. Rf 0.00-0.10, no traces of p-toluene sulfonyl chloride (Silicaplatten, Merck in CHC13-MeOH

~;~8~L335i 9-1 and CHC13-MeOH-HOAc-H2O 6-4-1-1).
Synthesis of_A~uaphase-Pro 10 g (40 mmol) Aqph-OTs and 7 g (60 mmol) L-pro-line are slurried in 100 ml DMF under nitrogen gas.
6 g triethyl amine are added, and the mixture is heated to 80C for 10 hours, whereupon it is left overnight at room temperature. AEter filtration and evaporation, the substance is dissolved in 150 ml water and neutra-lised, followed by freeze drying. The product is dried over P2O5. Rf 0.4 (Silicaplatten, Merck in CHC13-MeOH--HOAc-H2O: 6-4-1-1). Positive ninhydrin reaction.
Countercurrent_dlstrlbutlon Use was made of an automatic countercurrent distri-bution equipment having 60 cavities (5). To cavities Nos. 1-59, 0.79 ml of respectively bottom and top phase was added, cavity No. 0 was filled with 0.79 ml bottom phase and the sample ~5 mg D,L-tryptophan) dissolved in 0.79 ml top phase. Shaking was conducted for 40 s, and the distribution time was 20 min. After 60 transfers in cold store, the respective cavity contents were collected in 60 test tubes.
_nalysis and results_ _ _ _ _ _ _ The absorbance at 280 nm on the top phase of every third fraction was measured (Fig. 7).
Example 6 In modification of Example 5, D-proline coupled to PEG was added to the PEG phase. Thus, L-proline-Aqua-phase is in the bottom phase, and D-proline-PEG in the top phase. Due to opposed selectivity, there is thus obtained a more efficient separation if Example 5 is followed in other respects. D-proline-PEG is simply coupled to PEG via tosyl or tresyl activation.
Our results indicate that liquid/liquid/two-phase systems may be used for direct chiral resolution of racemic mixtures if an enantioselectively binding component is included in the phase system. These systems may be used both for analytical and for preparative ':

33~i purposes. Since liquid/liquid distribution is readily upscaledr these phase systems are especially interesting for large scale resolution of racemic mixtures.

,:. .
: ' . .

.

33Si REFERENCES
1. S. Allenmark, J. Biochem. Biophys. Meth., 9 (1984) 1-25.
2. P.-A. Albertsson, Partition of Cell Particles and Macromolecules, Wiley, New York, 1971.
3. P.-A. Albertsson, B. Andersson, C. Larsson and H.-E. ~kerlund, Meth. Biochem. Anal., 28 (1982) 115-150.
4. P.-~. Albertsson, Meth. Biochem. Anal., 29 (1983) 1-24.
5. V.P. Shanbhag, R. Sodergard, H. Carstensen and P.-A. Albertsson, J. Ster. Biochem., 4 (1973) 537.

6. L. Backman, J. Chromatogr., 196 (1980) 207-216.

7. G.F. Fairclough, Jr and J.S. Fruton, Biochemistry, 5 (1966) 673-683.

8. T.P. King and M. Spencer, J. Biol. Chem., 245 (1970) 6134-6148.

9. V.J. Cunningham, L. Hay and H.B. Stoner, Biochem., J., 146 (1975) 653-658.

-. . : .,

Claims (9)

1. Chiral two-phase system for resolution of racemic mixtures, or for separation of diastereomers, said system being characterised in that it comprises two immiscible liquid phases and one or more enantioselectively binding chiral components, each of which is substantially in one of said phases.

2. Two-phase system as claimed in claim 1, characterised in that the phases consist of two different polymers mixed with water or consist of two immiscible solvents.

3. Two-phase system as claimed in claim 1, characterised in that one phase comprises polyethylene glycol and the other comprises a polysaccharide.

4. Two-phase system as claimed in claim 1, characterised in that the chiral components are selected from one or more of a protein, a carbohydrate, and an amino acid, or derivatives thereof.

5. Two-phase system as claimed in claim 4, characterised in that the protein is an albumin.

6. Two-phase system as claimed in claim 4, characterised in that the carbohydrate is cyclodextrin and/or cellulose.

7. Two-phase system as claimed in claim 4, characterised in that the amino acid is D- and/or L-proline.

8. Two-phase system as claimed in any one of claim 1-3, characterised in that one phase contains a D-proline derivative and the other phase a L-proline derivative as chiral components.

9. Method for chiral resolution of racemic mixtures, or for separation of diastereomers, characterised in that the different enantiomers or diastereomers are partitioned differently between the phases of a chiral two-phase system comprising two immiscible liquid phases and one or more enantioselectively binding chiral components, each of which is substantially in one of said phases, by selectively binding one of said enantiomers or diastereomers to one of the chiral components.