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Title:
CHIRAL TWO-PHASE SYSTEM AND METHOD FOR RESOLUTION OF RACEMIC MIXTURES AND SEPARATION OF DIASTEREOMERS
Document Type and Number:
WIPO Patent Application WO/1987/000165
Kind Code:
A1
Abstract:
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 diastereomers. 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.

Inventors:
Sellergren
B�rje, Ekberg
Bj�rn, Albertsson
Per-aoke, Mosbach
Klaus
Application Number:
PCT/SE1986/000315
Publication Date:
January 15, 1987
Filing Date:
June 27, 1986
Export Citation:
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Assignee:
Sellergren
B�rje, Ekberg
Bj�rn, Albertsson
Per-aoke, Mosbach
Klaus
International Classes:
C07B57/00; C07C67/00; C07C313/00; C07C319/28; C07C323/62; C07D209/20; (IPC1-7): C07B57/00
Other References:
Methods of Biochemical Analysis, Volume 28 (1982) 115-150, P-A ALBERTSSON et al. "Phase Partition" see pages 117, third paragraph and 122, third paragraph.
Journal of Chromatography, Vol 196 (1980) 207-216, L BACKMAN "Protein-Protein Interactions Studied by Counter-Current Distribution" see Summary and page 208, lines 7-9.
Anal Chem Vol 56 (1984) 1152-1155, TOSHIFUMI TAKEUCHI et al "Enantioselective Solvent Extraction of Nentral DL-Amino Acids in Two-Phase Systems Containing N-n-Alkyl-1-Proline Derivatives and Copper (II) Ion".
Journal of Chromatography Vol 333 (1) (1985) 211-214 EKBERG B et al "Direct Chiral Resolution in an Aqueons Two-Phase System Using the Counter Current Distribution Principle".
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Claims:
CLAIMS
1. Chiral twophase system for resolution of race¬ mic mixtures, or for separation of diastereomers, said system" being c h a r a c t e r i s e d 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. Twophase system as claimed in claim 1, c h a ¬ r a c t e r i s e d in that the phases are polymer based or consist of two immiscible solvents.
3. Twophase system as claimed in claim 1, c h a ¬ r a c t e r i s e d in that one phase is polyethylene glycolbased and the other is polysaccharidebased.
4. Twophase system as claimed in one or more of claims 13, c h a r a c t e r i s e d in that the chiral components are a protein and/or a carbo¬ hydrate and/or an amino acid, or derivatives thereof.
5. Twophase system as claimed in claim 4, c h a ¬ r a c t e r i s e d in that the protein is an albumin.
6. Twophase system as claimed in claim 4, . c h a r a c t e r i s e d in that the carbohydrate is cyclo dextrin and/or cellulose.
7. Twophase system as claimed in claim 4, 'c h a ¬ r a c t e r i s e d in that the amino acid is D and/or Lproline.
8. Twophase system as claimed in one or more of claims 13, c h a r a c t e r i s e d in that • one phase contains a Dproline derivative and the other phase a Lproline derivative as chiral components.
9. Method for chiral resolution of racemic mixtures, or for separation of diastereomers, c h a r a c t e r ¬ i s d in that the different enantiomers or diastereo¬ mers are partitioned differently between the phases of a chiral twophase 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.
Description:
CHIRAL TWO-PHASE SYSTEM AND METHOD FOR RESOLUTION 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 liquid phases 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

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 component 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.

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 PEG/ dextran system.

Chemi_ca^ls_ and johase system

Dextran 40 was obtained from 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 rtiM 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 then left overnight in cold store (4°C). D- and L-tryptophan

3 had been obtained from Sigma and L-(side chain-2,3- H)-

-tryptophan, specific activity: 50 μCi per nmol, from Amersham International (U.K.). Countercurr_ent_d_is^r_ibut_ion

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 μ ol 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 (4 C) 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).

Measurin the_ bind nc of_L__try£tophan to serum albumin In fractions Nos. 0-5 the radioactivity of the

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). Resul_t£ and discussion The partition of serum albumin 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 GD=0.39). A separation factor (G-VG-.) 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

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 • 10 4 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 (2, 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 explanation is 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. hernicals ^ ^nd p_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 8.0. 7.5 g cellulose were added per 100 g phase system. The phase system was shaken carefully and then left overnight in cold store (4°C). D- and L-trypto¬ phan had been obtained from Sigma. Coun ercurrent_dis r^butipn 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

No. 0 was filled with 0.79 ml bottom phase and the sample (0.40 μmol 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 of each cavity were col¬ lected in 60 test tubes. Analysis 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. Che_micalj3 arid p_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 (4 C). Counte Curr_en_t_di ^ s^r_ibuti_on

Use was made of an automatic countercurrent di- 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 μmol) 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 of each cavity were col¬ lected in 60 test tubes. Analysis 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. Fractions 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.

TABLE 1

22 α D

Fractions Nos. 10-13 4 . . 30 '

Fractions Nos. 14-7 3 . . 80 ' 5 C

Fractions Nos. 18-20 3 . . 85 ' 5 C

Fractions Nos. 21-24 4 . . 48 ' 5 C Fractions Nos. 25-27 4. . 02 ' 'C

Fractions Nos. 28-30 4 , . 19 ' 5 C

Fractions Nos.31-33 4 . . 22 ' 'C

Example 4

As chiral component, use was made of beta-cyclo- dextrin coupled to Aquaphase (Perstorp) which was partitioned in an Aquaphase/PEG-phase system for sepa¬ ration of R,S-terbutaline.

Chemi^cals ^ and p_hase_ system

Aquaphase (Perstorp), Poly(ethylene glycol)PEG 8000 (Union Carbide, New York), p-toluene sulfonyl chloride (Sigma), 1,4-diamino butane (Janssen, Belgium),

8-cyclodextrin (Stadex AB, Sweden).

S_ynthesis_ o_fj . t __.l_l e _l e _ s __l_L 0 _lY_L~_i~__Y£l£^_i x __ r __. n X β __CD-OT ) 25 g (22 mmol ) B-cyclodextrin (washed with di- ethyl ether and dried over P 2 °5 ^ are dissolved in 100 ml dry pyridine, the solution is saturated with 2 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 ~ and placed in an ice 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

distilled water. The product is dried over -P 2 °5* ^i-^- 1 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 β-cyσlodextrin unit is tosylated. R f 0.80 (Silica¬ platten, Merck in CHCl 3 -MeOH 9-1).

S_ynthes_is_ of_p_^toluene_sulf ny^-Aquap_hase(Aq£h__OTsJ_ 10 g (61 mmol) Aquaphase (dried over are slurried in 75 ml dry pyridine, the mixture is satu- rated with 2 and placed in an ice bath. 22 g (116 mmol) p-toluenesulfonyl chloride are dissolved in 75 ml dry pyridine, the solution is saturated with N 2 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 and then dried over P 2 0 5 - 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. R f 0.00-0.10, no traces of -p-to¬ luenesulfonyl chloride (Silicaplatten, Merck in CHCl.-.-MeOH 9-1 and CHCl-.-MeOH-HOAc-H 2 0 6-4-1-1). ΣSynthesis_ of_l_,_4__dlamino__butane-Aquap_hase(H 2 N-J_CH 2 -NH- AqEhJ_

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 80°C 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

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. R . - 0.00-0.10 (Silicaplatten,Merck in CHCl 3 -MeOH 9-1 and CHCl- j -MeOH- HOAc-H 2 0 6-4-1-1), no traces of 1,4-diamino butane. Synthes_is_ of_β-CD-NH-(CH 2 1 4 __NH-Aqp_h

2,6 g (13 mmol) H 2 -(CH 2 ) 4 ~NH-Aqph (above) are dissolved together with 15 g (actually 12 mmol tosylated 3-cyclo dextrin) β-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 80 C during 4 hours. The solution is evaporated, and the remainder is diluted with 50 ml distilled water, pH is adjusted with 1 M HC1 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 β-cyclo- dextrin unit (actually 0.4 mol -cyclodextrin units/mol carbohydrate monomer). R^ 0.00-0.10 (Silicaplatten, Merck in CHCl--MeOH 9-1 and CHCl 3 -MeOH-HOAc-H-,0 6-4-1-1), ninhydrin positive reaction left. Composition of the phase system: 14% (w/w) Aquaphase, 2.5% (w/w) β-CD-NH- -(CH 2 ) 4 -NH-Aqph, 5% PEG, 0.1 M Li 2 S0 4 , 6.25 mM sodium citrate, pH 6.0. The phase system was shaken carefully and left overnight in cold store (4°C). Countercurr nt_d_istributi.on

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 μmol 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. Analysis and results

The absorbance at 280 nm on the top phase of every third fraction 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. Che_micals ^nd p_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. S_ynthesi£5 of_p^t£luene_suljEonyl ^ -Aqua£hase_ J[_Aoj?h-OTs)

10 g (61 mmol) Aquaphase (dried over P 2 0 5 ) is slurried in 75 ml dry pyridine, the mixture is satu- rated with N 2 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 N 2 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 P 2 0.-. 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%). Thus, the product contains 4.61% (w/w) pyridine. Every other carbohydrate monomer is tosylated. R f 0.00-0.10, no traces of p-toluene sulfonyl chloride (Silicaplatten, Merck in CHCl-.-MeOH

9-1 and CHCl 3 -MeOH-HOAc-H-,0 6-4-1-1). Synthes_is_ of Aquaphase-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 80 C for 10 hours, whereupon it is left overnight at room temperature. After filtration and evaporation, the substance is dissolved in 150 ml water and neutra¬ lised, followed by freeze drying. The product is dried over P 2 O ς . R f 0.4 (Silicaplatten, Merck in CHCl 3 -MeOH- -HOAc-H 2 0: 6-4-1-1). Positive ninhydrin reaction. Coun;tercurrent_di.stribution

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. Analysis 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

purposes. Since liquid/liquid distribution is readily upscaled, these phase systems are especially interesting for large scale resolution of racemic mixtures.

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. Akerlund, Meth. Biochem. Anal., 28 (1982) 115-150.

4. P.-A. 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.