Chromatographic Separation and Purification of Folic Acid Analogs VINCENT T. OLIVER10 Nafional Cancer Institute, National lnstifutes o f Health, Bethesda,
b The study of the physiological disposition of several folic acid antagonists of current interest in cancer chemotherapy necessitated the development of a generally applicable method for the quantitative separation and analysis of mixtures of closely related folic acid analogs. This has been accomplished by linear gradient elution with pH 8 phosphate buffer from DEAE ion exchange columns,
S
of the physiological disposition of folic acid analogs in animal and microbiological systems have been impeded by the instability and inhomogeneity of these compounds. I n addition, a generally applicable method has hitherto been unavailable for the quantitative wparation and analysis of mixtures of closely related folic acid analogs that differ by as little as one functional group as a result of deamination, osidation, demethylation, etc. Although the separation and identification of mixtures of pteridines has been achieved by paper chromatography (4) and column electrophoresis ( I S ) , the methods described are not practical for the purification and resolution of folic acid analogs on the preparative scale of milligram or gram quantities. Columnar chromatography with cellulose (6) or ion exchange resins (7, 8, I S , 14) has been useful only to the extent of purification of a single compound or a mixture of a few compounds, an exception being the Dowex 1 chloride (401, crosslink) resin employed by Heinrich et al. ( 2 ) for the resolution of mixtures of certain folic acid analogs or free pteridines. However, as stated by the authors, the method is limited by the low solubility of pteridines in hydrochloric acid used as the eluent and the acid lability of compounds such as citrovorum factor (CF). I n view of the limitations of the aforementioned methods, trial of cellulose ion exchanger was begun in this laboratory. The merits of cellulose ion exchange chromatography in the separation and identification of proteins are well known (9). This report describes the chromatographic separation, purification, and analysis of folic acid anaTUDIES
Md.
logs on diethylaminoethylcellulose (DEAE) anion exchange columns by a linear gradient elution with phosphate buffer. While this work was in progress, Toennies and Phillips (IO) and Usdin (11) reported a similar technique for the resolution of folateactive materials from human blood cells on triethylaminoethylcellulose (TEAE) columns. However. the majority of these compounds were formyl derivatives of mono- and diglutamates of folic acid and tetrahydrofolic acid. In the present study, folic acid analogs, some differing only slightly in structure, were resolved quantitatively in both micro and preparative scales from various admixtures. EXPERIMENTAL
Reagents. Stock solutions of 0.01 and 0.4M reagent grade sodium phosphate buffer (pH 8) were prepared. D E A E cellulose (on SolkaFloc, 0.8 meq. per gram, obtained from California Corp. for Biochemical Research), of 100- to 250-mesh, was prepared batch-mise by successive washings with 1 N sodium hydroxide, water, 1N hydrochloric acid, water, 1 N sodium hydroxide, and water until p H 7 to 8. The cellulose was finally washed with 0.01M phosphate buffer (pH 8) and stored in the cold. Apparatus. Fractionations were carried out on a n automatic Gilson Medical Electronics Model V15square fractionator. Borosilicate glass columns, 1-cm. inner diameter and 25 to 30 cm. in length, were used for the adsorbent. Columns of greater cross-sectional area were used for the chromatography of more than 25 mg. of material. Transmittance was recorded on Varian Associates Model G-10 graphic recorder. A Model 14 Cary recording spectrophotometer was used for spectral determinations. Procedure. I n a typical experiment, a dilute suspension of the adsorbent was added to a column and packed by air pressure gradually increased to 10 p.s.i. Five to 25 mg. of a single compound or a n admixture of compounds of unknown purity was dissolved in a minimal amount of 0.01M phosphate buffer (pH 8) and allowed to run into a column of washed DEAE. The column was attached to an automatic fraction collector
with a 254-mp light source to monitor continually the transmittance of the eluent and plot the per cent transmittance on a rectilinear recorder. The material was then separated into its individual components by a linear gradient elution with p H 8 phosphate buffer of increasing molarity. The mixing chamber contained 500 ml. of 0.OlM phosphate buffer and the reservoir contained 500 ml. of 0.4M phosphate buffer. The rate of flow of the eluent was adjusted to approximately 0.5 ml. per minute with a screw clamp placed on polyethylene tubing connected to the end of the column and 5-ml. fractions were collected. All chromatographic operations were carried out at room temperature and in subdued light to protect the compounds from any possible photochemical effects (3). The peak tubes, as shown by the per cent transmittance plot, were pooled and the ultraviolet absorption spectrum (230 to 400 mp) of each peak was measured by a recording spectrophotometer. The purity of a single compound was determined by the per cent of its absorbance a t a given wave length recovered following chromatography. The chromatographic fractions were usually suitable for microbiological assay procedures following appropriate dilutions. Removal of the phosphate salt was effected by concentration of the various fractions by evaporation in vacuo followed by adsorption and deadsorption of the compounds on charcoal (8,lW). RESULTS
Table I lists the folic acid analogs employed in the present study. The approsimate phosphate buffer concentrations for elution from DEAE arc also shown. I n some cases, when a compound was chromatographed in the presence of other folic acid analogs, these elution values changed slightly. The purity of the starting compounds ranged from 40 to 97%. Chromatographic patterns of some of the compounds studied are shown in Figure 1 ( A to E ) , in which the per cent transmittance has been plotted against the volume and corresponding molarity of phosphate buffer eluent. I n all cases, repeated chromatography of the major peak gave a single peak VOL. 33, NO. 2, FEBRUARY 1961
263
Table 1.
Folic Acid Analogs
(Compounds were obtained from Lederle Laboratories, Division of American Cyanamid Co.) Approximate Buffer Molarity for Elution from Compounda DEAE N-(p-[(2-Amino-5-formyl-5 67t?-t,etrahydro-Phydroxy-k
’’
pteridiny1)methylaminol benzoyl ] glutamic acid (citrovorum 0.03 factor, CF; folinic acid-SF) N- { p -[ (2,4Diamino-6-pteridinylmethyl)amino] benzoy1)alanine 0.04 N-{ p - [ ( 2 PDiamino-6-pteridinylmethyi)methylamino] benzoyl } glutamic acid (methotrexate, MTX) 0.05
N - ( p - [1-(2,4Diamino-& pteridinyl)ethyl] methylaminobenzoyl }glutamic acid ( Adenopterin)
0.06
1
N - ( 4[2,4Diamino-6-pteridinylmethyl)methylamino]-3chlorobenzoy1)glutamic acid (chloromethotrexate, MCM) 0.06 N- (3-Bromo-4[(2,4-diamino-6pteridinylmethy1)methyl-
amino]benzoyl}glutamic acid (bromomethotrexate)
0.07
N- { 3,5-Dichloro-4-[(2,4diamho6pteridinylmethyl)methyl-
amino]benzoyl}glutamic acid (dichloromethotrexate, DCM) 0.07 N- {3-Bromo-5-chloro-4-[(2,4&amino-6-pteridinylmethyl ) methylamino] benaoyl 1 glutamic acid (3’-bromo-5’-chloromethotrexate) 0.08
N-{ p- [ (2,4-Diamino-6-pteridinylmethyl)amino] benzoyl} aspartic acid
0.09
N- ( p -[ (2,4Diamino-6-pteridinylmethyl)amino] benzoyl ] glutamic acid (aminopterin) 0.09
N - { p - [ (2-Amino-Phydroxy-6pteridiny1methyl)methyl-
amino]benzoyl ]glutamic acid (methopterin)
N - (p-[(2,4Dihydroxy-6-pteridinylmethyl)amino]benroyl ] glutamic acid (2-deamino-2hydroxypteroylglutamic acid)
N-{3,5-Dichlor0-4-[(2-amino-P
0.14
0.16
hydrox 6pteridinylmethyl) amino] tensoy1 ]glutamic acid (dichloropteroylglutamic acid) 0.18 N - [N-(N- { p - [ (2,4-Diamino-6pteridinylmethy1)aminol benzoyl 1- y-glutamy1)-7-glutamyl] glutamic acid (A-teropterin) 0.20 N-( p [( 2-Amino-4-hydroxy-6gteridin ylmeth yl)amino]enzoy1)glutamic acid (pteroylglutamic acid, PGA) 0.21 a The full chemical and generic names are listed; however, generic names are employed throughout the text.
264
ANALYTICAL CHEMISTRY
with quantitative recovery and thus ruled out the possibility of alteration of the material by either the cellulose anion exchanger or the ultraviolet light of the monitor. Mixtures of the various analogs were made up with the purpose of studying the effect of the substituent groups on the chromatographic behavior and elution sequence. Figure 1,A represents the pattern obtained when 5 mg. of aminopterin was chromatographed on DEAE according to the technique described above. This sample of aminopterin, several years old, was estimated t o be 40% pure. The major contaminant, appearing as the last peak on the chromatogram, mas spectrally identified as pteroylglutamic acid and represented approximately 50% of the total impurities. The compounds appearing before the elution of aminopterin were not identified but had spectral characteristics of pteridines, as evidenced by peaks in the 360- t o 370-mp and 260to 280-mp regions. A recently prepared sample of aminopterin was chromatographed and estimated t o be of 80 to 85% purity. As much as 1 gram of aminopterin was purified without loss of resolution through the use of a 5 x 30 em. column of DEAE. In this case, however, a discontinuous elution procedure was employed to minimize the volume of eluting buffer. To remove the impurities which elute before aminopterin] 0.07M phosphate buffer was used. The volume and concentration employed did not remove the pteroylglutamic acid from the column. In the chromatography of 5 mg. of methotrexate (MTX) on DEAE (Figure I,B), the major contaminant was identified as methopterin. This hydrolysis product, appearing as the last peak on the chromatogram, accounted for two thirds of the total impurities. Methotrexate, prepared several years ago, was estimated to be 85% pure. The unidentified first and second peaks had spectral characteristics of pteridines. Chromatography of 5 mg. of dichloromethotrexate (DCM) resulted in approximately a quantitative yield of the pure analog. Only a trace of the hydrolysis product, X- { 315-dichloro-4 [(2amino - 4 - hydroxy - 6 - pteridinylmethy1)methylamino ] benzoyl 1 glutamic acid, was detectable in this drug. Notably absent was chloromethotrexate (MCM), reported ( 1 ) to be a contaminant of early samples of DCM. Figure 1,C represents the chromatographic resolution into its individual components of a mixture of 3 mg. each of D C M and MCM. A less complete separation was obtained in the chromatography of a mixture of D C M and bromomethotrexate. It was not possible to resolve into its individual
components a mixture of D C M and 3’ - bromo - 5’ chloromethotrexate on DEAE. Phosphate buffer of relatively high ionic strength was required to elute Camino folic acid analogs with two or more glutamic acid groups. On the other hand, phosphate buffer of relatively low ionic strength was required for the elution of 9-methyl or N1O-metliyl substituted derivatives of the Pamino folic acid analogs. This was illustrated in part by the chromatographic pattern (Figure 1,D) of a mixture of 3 mg. each of A-denopterin, aminopterin, and A-teropterin. Similarly] for separation of methopterin, a N1o-methylpteroylglutamic acid analog, from PGA, phosphate buffer of lower ionic strength was required for its elution. Folic acid analogs generally appeared to follow the elution sequence shown in Figure 1,E describing the resolution of a mixture of one milligram each of CF. MTX, DCM, aminopterin] methopterin, 2 - deamino - 2 - hydroxypteroylglutamic acid, dichloropteroylglutamic acid, and PGA. Jt appears that structurally related folic acid analogs possess similar elution properties. With increasing molarity of phosphate buffer, 2,4diamino compounds elute before 2-amino-Phydroxy compounds.
-
DISCUSSION
I n the present experiments reproducible results were obtainable without rigorous control of temperature and p H required by some of the previously cited chromatographic systems. For example, phosphate buffer of either p H 7 or 8 could be employed to achieve maximum purification and separation. The selection of p H 8 phosphate buffer was actually determined by the ease of solubilization of the compounds a t this pH. The stability of the folic acid analogs on the cellulose anion exchanger was also a n advantage in the present system. This was evidenced by the fact that repeated chromatography of the main peak failed to introduce any artifact, As expected, particle size of the cellulose affected the resolution. Use of the larger 50- to 100-mesh size cellulose resulted in a significant loss of resolution. Resolution was restored by the reduction of the flow rate of the phosphate buffer eluent. Further studies are being carried out on the chromatographic behavior of other pteridines and folic acid antagonists of biological interest. Preliminary results of the application of the DEAE anion exchange chromatographic system to the isolation of urinary metabolites of M T X and DCM
,
AM/NOP TERN
t
i l
80 -L
01
90 --?
02
MOLARITY
OF
03
04
PHOSPHATE BUFFER I PH 61 i
IC 0
t.-_ -
0
~
+
I00
Cl ___ __
.
200
-__-_
MOLARITY
300
OF
400
0 2- - - PHOSPhATE
500
-
03
SUFFER
600
+----+----i
700
02 4
IpH 8 )
600
900
1000
VOLLME (rnl) Cl TROVOPUM FAC TOR
2-DEAMINO-2-H YDROX YP TEROYL GL U TAMIC ACID
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OT c
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Figure 1 . Elution of various folic acid analogs on a DEAE cellulose column Mg. of eoch compound in load
A.
5
E. 5 c. 3 D. E. 01
0
100
200
1
02
MOLARITY
100
3
OF
PHOSPHATE BUFFER l p H 8 ) 1
300
400
500
600
700
800
900
IO00
VOLUME (ml.1
in mice and humans have been reported from this laboratory (6). The high resolving power for pteridines of DEAE cellulose chromatography should render this technique particularly useful for the study of biological systems in which complex mixtures of pteridines commonly occur. ACKNOWLEDGMENT
The author expresses his appreciation t o Dorthea Thomasson and Marvin Michael for their technical assistance,
and to T i Li Loo for many helpful discussions. LITERATURE CITED
(1) Angier, R. B., Curran, W. V., J . A m . Chem. Soc. 81,2814 (1959). (2) Heinrich, M. R., Dewey, V. C., Kidder, G. W., J . Chromatog. 2, 296 (1959). (3) Jukes, T. H. Science 120, 324 (1954). (4) Kwietny, Bergmann, F., J . Chromatog. 2, 162 (1959). (5) Oliverio, V. T., Loo, T.L., Proc. A m . Assoc. Cancer 3,140 (1960).
A.,
(6) Sakami, W., Knowles, R., Science 129,274 (1959). (7) Silverman, M., Ebaugh, F. G., Jr.,
. l'1,41 (1957): (14) Zakrzewski, S. F., Nichol, C. A., J. Biol. Chem. 213, 697 (1955). RECEIVEDfor review August 2, 1960. Accepted October 4, 1960.
VOL. 33, NO. 2, FEBRUARY 1961
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