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A simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate
ANALYTICAL
122, 70-78 (1982)
BIOCHEMISTRY
A Simple Procedure
for the Synthesis of High Specific Activity (6S)-5Formyltetrahydrofolate’
RICHARD G.MORAN Laboratory
of Cellular
Tritiated
AND PAUL D. COLMAN
and Biochemical Pharmacology, Division of Hematology/Oncology, of Los Angeles, Los Angeles, California 90054
Childrens
Hospital
Received October 28, 1981 The 5position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as Sformyltetrahydrofolate (leucovorin). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folatedepleted Ll210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) [3H](6S)-5-formyltetrahydrofolate in high yield. Experiments with [‘%]formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-SFormyltetrahydrofolate, labeled in the formyl group with 14C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utilization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.
A growth factor for the folate-requiring bacterium Pediococcus cerevesiae, previously called Leuconostoc citrovorum (that was, hence, named citrovorum factor or CF)’ was originally isolated from liver (1) and, thereafter, shown to have the structure of 5-formyL5,6,7,Wetrahydropteroylglutamic acid (5-CHO-H,PteGlu) (2). Subsequently, the enzymatically active diastereoisomer of this compound was shown to have the S chirality at the 6-carbon (3). The
6R,S mixture of diastereoisomers has been called folinic acid or leucovorin and the calcium salt of this mixture is commercially available. Folinic acid is usually found to be the most potent source of folates for the support of the growth of mammalian cells in culture due to its stability in aqueous solution and its affinity for the reduced folate membrane transport system in such cells (4-6). For instance, growth supporting concentrations of folic acid are approximately 100 times higher than equieffective concentrations of folinate (7-9). Moreover, folinic acid has been shown to reverse the toxicity of antifols, such as methotrexate (MTX), to cultured mammalian cells and to animals (8, lo- 12). This latter effect is the basis for clinical protocols involving “high-dose MTX-CF rescue” in which supralethal doses of MTX are administered to patients with some types of neoplasms and the potentially lethal cytotoxicity
’ This investigation was supported by Grants CA27146 and CA27605 from the National Institutes of Health, DHEW. * Abbreviations used: HQteGlu, tetrahydrofolate; H,PteGlu, dihydrofolate; 5-CHO-HQteGlu, 5-formyltetrahydrofolate; 5-CHa-H4PteGlu, 5-methyltetrahydrofolate; 5,10-CH=H,PteGlu, 5,10-methenyltetrahydrofolate; IO-CHO-H,PteGlu, IO-formyltetrahydrofolate; PteGlu, folic acid; MTX, methotrexate. EDC, I-ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl; DEAE-cellulose, diethylaminoethylcellulose; CF. citrovorum factor; &ME, 2-mercaptoethanol. 0003-2697/82/070070-09$02.00/0 Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
70
SYNTHESIS
OF TRITIATED
5-FORMYLTETRAHYDROFOLATE
of MTX to the stem cells of the gut and bone marrow is terminated by the delayed administration of leucovorin ( 13). Several synthetic approaches to folinic acid have been published including the catalytic reduction of folic acid in concentrated formic acid (2,14), the direct formylation of tetrahydrofolate and conversion of the product to folinic acid by treatment with alkali at elevated temperatures (15), and the hydrogenation of lo-formylfolate at elevated temperatures ( 16,17). Recently, a convenient synthesis of folinate was reported involving the reaction of tetrahydrofolate with methyl formate in dimethylsulfoxide under mild conditions (18). The synthesis of labeled and unlabeled (6S)-5-CHO-H,PteGlu has been reported using enzymatically reduced tetrahydrofolate as starting material (19,20). In addition, a unique synthetic approach to (6S)-5-CHO-H,PteGlu has appeared in which folic acid is first reduced using dihydrofolate reductase and then formylated specifically at the 5-position with purified horse liver N-formylglutamate: tetrahydrofolate transformylase (21). It has previously been reported that watersoluble carbodiimides promote the coupling of compounds containing a carboxyl function to the N-5 position of 6,7-dimethyltetrahydropterin (22,23). In this report, we describe the analogous, facile, carbodiimideinduced formylation of (6S)-H,PteGlu and the utilization of this convenient reaction for the synthesis of unlabeled as well as high specific activity tritiated (6S)-5-CHOH,PteGlu and [ “C]formyl-labeled compound. MATERIALS
AND METHODS
DEAE-cellulose was purchased from Eastman Chemical, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl (EDC) from Sigma Chemical Company, reagent-grade ammonium acetate from Baker Chemicals, [‘4C]formic acid (lo-20 mCi/mmol) and [ 3’,5’,7,9-3H]folic acid (38-44 Ci/mmol)
71
were from New England Nuclear and Amersham, respectively. RPM1 1640 and dialyzed fetal calf serum were obtained from Grand Island Biological Company, Inc. H,PteGlu was made by reduction of PteGlu (Sigma) with sodium dithionite and was purified by repeated recrystallizations (24). (6s) H,PteGlu was prepared by reduction of H,PteGlu using L. casei/MTX dihydrofolate reductase as previously described (25). (6&Y)-5-CHO-H,PteGlu (folinic acid) and (6R,S)-5-CH,-H,PteGlu were purchased from Sigma Chemical Company, the former was used in these experiments without purification. (6&S)-5-CH,-H,PteGlu was a tan to brown powder as purchased and was purified by chromatography on a 4.9~cm2 X 30-cm column of DEAE-cellulose (containing 40 g of DEAE-cellulose) that was eluted with a convex gradient of ammonium acetate (0.01-1.5 M). When the peak tubes were pooled and lyophilized, a pure white, fluffy powder was obtained. Labile folates were stored at -25°C in foil-covered ampules that were sealed under dry N2. loCHO-H,PteGlu was made from 5-CHOH,PteGlu immediately before use by first adjusting a concentrated solution to pH 1 for 30 min, then carefully adjusting the pH to 6.5 with NaOH. Dihydrofolate reductase was purified from sonicates of L. casei/MTX by (NH4)$04 precipitation and chromatography on phosphocellulose as described by others (27). All other materials were of the highest grade available. Chromatographic columns were prepared and equilibrated as described previously (9). Cell culture. Mouse L1210 leukemia cells (26) were maintained as previously described (9). They were depleted of intracellular folates by 2-3 weeks of continuous growth on RPM1 1640 medium that was formulated with 32 PM hypoxanthine, 5.6 PM thymidine, and 10% dialyzed fetal calf serum (9). To determine the ability of a compound to act as a source of folate cofactors, folate-depleted L 12 10 cells were washed
72
MORAN
AND
once with phosphate-buffered saline, suspended in RPM1 1640 medium formulated without folic acid but with 10% dialyzed fetal calf serum and were distributed into Corning 25cm* T flasks (10 ml/flask containing 3-l 1 X lo4 cells per ml). Various concentrations of the compound were added to duplicate flasks, the flasks were incubated for 72 h and the cell number was determined using a Coulter counter Model B. Reaction of (bS)-H,PteGlu with formic acid. In a typical reaction, 177 pmol of (6S)H,PteGlu and 20 mmol of reagent-grade formic acid were dissolved in a total of 10 ml of 50 mM phosphate containing 1% @mercaptoethanol (P-ME); the final pH was 3.6. EDC (41.7 mg) was added and allowed to dissolve; the mixture was incubated for an additional 10 min at room temperature. The solution was shell-frozen and lyophilized, the dry residue was dissolved in 10 ml of 0.01 M ammonium acetate and the resultant solution was applied to a 4.9-cm* x 30cm column of DEAE-cellulose that had been equilibrated with ammonium acetate (9). The column was eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate with a mixer volume of 2 liters; all buffers contained 1% P-ME. The single main peak that was eluted from this column was pooled and lyophilized; this peak contained 74-80% of the material that absorbed at 285 nm and that was eluted from the column. This reaction was also run in vigorously degassed buffer without P-ME with the same results. EDC could be added either as a powder or as a freshly prepared, concentrated solution. In earlier experiments, the reaction product from 1- to 60-pmol reactions was purified by chromatography on a 0.64-cm’ X 75-cm column of Sephadex G-25. In 50 mM phosphate, pH 6.0, 5-CHO-H4PteGlu was found to elute from this column significantly after 3H20 (K,, = 1.85) as has been observed by others (19,28). However, the 5CHO-H,-PteGlu peak eluted from this column was often broad and the separation from salt depended upon the concentration
COLMAN
of salt in the sample. This technique was subsequently abandoned in favor of DEAEcellulose chromatography (9) which proved more reproducible. Synthesis of [:‘H](dS)S-CHO-H,PteGlu. In a representative reaction, 100 &i of [ 3’5’,7,9-3H]PteGlu (38-44 Ci/mmol, approximately 2.5 nmol) was incubated with 35 munit of L. casei/MTX dihydrofolate reductase (4.1 IU/mg) and 15 nmol of NADPH in a total volume of 100 ~1 of 50 mrvf phosphate, pH 7, containing 50 mM /3ME. After a IO-min incubation at 37°C 900 ~1 of 100 mrvr formic acid containing 50 mM phosphate and 1% b-ME was added, the pH was adjusted to 4-5 and 5 mg of EDC was added. After 10 additional min at room temperature, the reaction mixture was purified on a 0.64-cm* X 50-cm column of DEAEcellulose (containing 5.0 g of DEAE-cellulose) that was eluted with a convex gradient of ammonium acetate (0.01 to 1.5 M) with a mixer volume of 475 ml; chromatography buffers also contained 1% @-ME. In some experiments, folates used as chromatographic markers (0.5-1.5 mg) were added before sample was applied to the top of a column. The concentration of eluting salt was determined by measurement of the conductivity of suitable dilutions of the effluent; conductivity was found to be a linear function of ammonium acetate concentration on the range of lo-80 mM. Reaction offolates with [‘ICI formic acid. Approximately 1 pmol of a folyl compound was incubated with 100 pmol of [ “C]formic acid ( 1 &i) and 5 mg EDC in a total volume of 1 ml of 50 mM phosphate, pH 3.6, containing 1% &ME. In some experiments, the reaction mixture was added to a 0.64-cm* X 35-cm column of DEAE-cellulose after 10 min incubation and eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate; mixer volume was 475 ml for these separations. In these experiments, the stoichiometry between H,PteGlu and formate was determined by spectrophotometric determination of the amount of 5-CHO-H,PteGlu,
SYNTHESIS
OF TRITIATED
73
5FORMYLTETRAHYDROFOLATE
assuming a millimolar extinction coefficient of 37.2 at 285 nm (29). The amount of [ “C]formate was determined by scintillation counting in RIA-Solve II cocktail (Research Products International, Inc.); corrections for counting efficiency were made by the h-number method using a Beckman LS 7500 scintillation counter. In other experiments, folate-bound radioactivity was separated from [ i4C]formate by fractionation of reaction mixes on minicolumns of DEAE-cellulose. To prepare these minicolumns, DEAE-cellulose (8 g) was stirred for 15 min in 0.1 N NaOH (400 ml), then packed to a height of 4 f 0.25 cm in Pasteur pipets that were plugged with a minimal amount of cotton. Plastic Dispo transfer pipets (Scientific Products, catalog no. P5214-10) were most convenient for this purpose: if the plastic bulb was cut at the top, the pipet would hold 4.5 ml of eluting buffer in the reservoir that was formed. Prior to use, each minicolumn was washed with 10 ml each of water, 2.5 M ammonium acetate, and water followed by 4 ml of 1% pME. Each reaction mixture was washed onto a minicolumn with 1 ml of 10 mM ammonium acetate that was 1% in P-ME and the columns were eluted with 17 ml of this same solution to remove excess [ “C]formate. Then, each column was sequentially eluted with 3 ml of ammonium acetate at 0.7, 1.0, 1.25, and 1.50 M, all containing 1% ,&ME. Elution washes were collected directly into scintillation vials, shell-frozen, and lyophilized. The dry vials were hydrated with 1 ml of HZ0 and 9 ml of scintillation cocktail and radioactivity was determined by scintillation counting. The radioactivity in each wash was corrected for the residual tritium found in minicolumn eluates of triplicate blank (i.e., no folate added) reaction mixtures; the counts per minute reported in Table 1 are the sum of those found in the 0.7, 1.0, and 1.25 M washes. No radioactivity was found in the 1.50 M washes. The absorbancy corresponding to 5-CHO-H,PteGlu, 5-CH3H,PteGlu, and H,PteGlu was found in the
0.7 M wash while that corresponding to PteGlu and H,PteGlu was found in the 1.0 and 1.25 M washes. Calculation of yields. The yield of 5CHO-H,PteGlu formation from H,PteGlu was calculated by spectrophotometric determination of the amount of H,PteGlu used assuming ~292 = 19.1 mM-’ cm-’ (29-3 1) in 0.1 N HCl and by spectrophotometric determination of the amount of 5-CHO-H,PteGlu in the pooled chromatographic fractions assuming +85 = 37.2 rnrv-’ cm-’ at neutral pH (29). The yield of [3H]5-CHO-H4PteGlu produced from 3H-PteGlu was calculated from the ratio of radioactivity in the pooled chromatographic fractions to that originally added to the reaction mix. RESULTS
Large-Scale H,PteGlu
Synthesis of (6S)-5-CHO-
The reaction of enzymatically reduced H,PteGlu with 100 mM formic acid in the presence of EDC was found to proceed rapidly with the appearance of a compound with the spectrum of 5-CHO-H,PteGlu (pH 7, x max = 286) (Fig. 1A). The crude reaction mixture was conveniently purified either by chromatography on Sephadex G-25, as previously reported by others (19,27) or by chromatography on DEAE-cellulose eluted with a convex gradient of ammonium acetate (9). Rechromatography of the purified material allowed an estimate of the purity of the product to be in excess of 95% (Fig. 1B). This product was converted into a compound with a spectrum identical to that of 5,10CH=H,PteGlu (pH 0, X,,, = 288,346) by treatment with 1.0 N HCl for 1 h (Fig. 1A). The ratio of absorbance of the product at 285 in neutral solution to that of the acidtreated product at 345 in 1.0 N HCl was 1.43, in agreement with the published extinction coefficients of 5-CHO-H,PteGlu and 5,10-CH=H,PteGlu under these conditions (37.2 and 26.3, respectively) (29-31) from which the expected ratio of 1.41 can
14
MORAN
AND COLMAN
be calculated. The neutral and acidic spectra observed on the unfractionated reaction mixture were identical to those seen with the chromatographically purified product. However, the mean yield calculated for three preparations starting with (6S)-H,PteGlu on the range of l-200 pmol was 54 + 2%. When the EDC-catalyzed formylation of H,PteGlu was run in the presence of [‘4C]formic acid and the product was purified by DEAE-cellulose column chromatography, the 14C-labeled product chromatographed as a peak with the spectrum of 5CHO-HJ%eGlu; this uv-absorbing product was found to have a ratio of 14C to Azss equivalent to approximately 1 mol of formate per mol of 5-CHO-H,PteGlu; this ratio was constant through the chromatographic peak (the mean ratio for tubes 70-
0.75
2
I ^ 1.
.
f
/t
'*?0.6
A
..
* fj :: 4
Q?I ,I L:L
0.50
..
0.25
7
8.
;
260
290
;
;
320
350
Wavrlen6th
Fraction
Fraction
,
:
360
410
, nm
Numbrr
FIG. 1. (A) Spectral characteristics of the product of EDC-promoted formylation of H,PteGlu. The ultraviolet absorption spectrum of the purified product was determined at pH 7 (0) and after standing for 1 h in 1 N HCl (0). (B) Purity of reaction product. The formylation product was purified on a column of Sephedex G-25, lyophilized, and an aliquot was rechromatographed on a 0.64-cm2 X 35-cm column of DEAE-cellulose that was eluted with a linear gradient of ammonium acetate (0.05 to 2.5 M) (9) to determine purity. The fractions contained 3.5 ml.
Number
FIG. 2. Chromatography of 14C product on DEAEcellulose. (A) A reaction mixture containing 100 amol of [“Clformic acid and 1 pmol of H,PteGlu was absorbed onto a 0.62~cm* X 50-cm column of DEAE-cellulose 10 min after the addition of 5 mg of EDC and the column was eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate. The folate peak eluted from the column shown in A was pooled, lyophilized, and rechromatographed on DEAE-cellulose under identical conditions as described for A without further treatment (B) or following 1 h in 1 N HCl (C). The symbols are: Absorbance at 285 nm (O), radioactivity (0) salt concentration (m), ratio of [ ‘%]formate to A2s5, (A). The ratio of ‘%I to A2s5 is expressed as mol of 14C per mol of 5-CHO-H.,PteGlu by correcting the raw data for counting efficiency, specific activity of the formic acid and the extinction coefficient of S-CHO-H,PteGlu at 285 nm. The fractions contained 3.3 ml. Radioactive peaks 1 and 2 reached 22,000 and 500,000 cpm/ml, respectively.
75 was 1.24; in a replicate experiment this ratio was 1.17) (Fig. 2A). Likewise, when this 14C-labeled product was acidified to cy-
SYNTHESIS TABLE REACTION
OF TRITIATED I
OF['%]FORMICACID FOLATE COMPOUNDS
WITH VARIOUS
Counts per minute Compound (6S)-H1PteGlu (6R,S)&CHO-H,PteGIu H,PteGlu PteGlu (6R.S)-5-CH,-H,PteGlu
Expt I 3378
129 5 118 -110
Expt 2 5193
49 155 -34 94
Percentage H,PteGlu
of
100 2.4 1.6 1.4 -0.7
Nore. Triplicate reaction mixtures of each folate compound (0.7-1.0 wmol/tube.) were incubated for 10 min at room temperature with 26 pmol of EDC and 100 pmol(1 pCi) of formic acid in 50 rnM phosphate containing I % &ME; the final reaction mix was pH 3.6. Each reaction mixture was chromatographed on a 4-cm Pasteur pipet column of DEAE-cellulose as described under Materials and Methods. The values listed represent the mean cpm eluted with 0.7-1.25 M ammonium acetate, corrected for the amounts of radioactivity found in the eluate from columns run on reaction mixes incubated without folate compounds. Three reactions were run for each compound in each experiment.
clize any SCHO-H,PteGlu, a new peak appeared in the chromatogram at the position of lo-CHO-H,PteGlu, as would be expected at the pH of this column (6.5) if the original compound was 5-CHO-HSPteGlu (Fig. 2C). It is of interest that the reaction product was not quantitatively converted to 1O-CHOH,PteGlu under these conditions. However, the material that chromatographed at the location of 5-CHO-H,PteGlu after acidification had a ratio of absorbance at 285 nm to 14C that was not significantly different from that of the original reaction product (mean ratio for tubes 71-75 = 1.33) (Fig. 2). The concentration of ammonium acetate that eluted the reaction product (Fig. 2B) and the compound to which it was converted by acid treatment were identical to those that elute 5-CHO-H,PteGlu and lo-CHOH,PteGlu under these chromatographic conditions, namely, 0.48 and 0.34 M, respectively (9). Specificity
75
SFORMYLTETRAHYDROFOLATE
of This Reaction
Under these conditions, [ “C]formic acid reacted with H4PteGlu but did not react ap-
preciably with PteGlu or H,PteGlu implying that the reactivity of N-5 of the folate moiety requires a fully reduced pyrazine ring (Table 1). The lack of reactivity of 5-CHOH,PteGlu and 5-CH3-H,PteGlu under these conditions indicates that the formylation of N-5 of H,PteGlu is highly favored over attack at any other position in this ring system, e.g., N- 10. Furthermore, these data indicate that further formylation of the 5-CHOH,PteGlu product of reaction of formate with H,PteGlu is quite limited under these conditions. Biological
Activity
The product of the EDC-catalyzed formylation of (6S)-H,PteGlu was found to support the growth of folate-depleted L1210 cells in the absence of an added source of folates to the same extent as commercial leucovorin (Fig. 3). However, half-maximal growth of these cells was observed at a concentration of commercial (6R,S)-5-CHOH,PteGlu that was twice that required for the synthetic (6S)-5-CHO-H,PteGlu product made by this EDC-promoted formylation (Table 2). Synthesis
of [‘H](6S)-5-CHO-H,PteGlu
The synthesis of high specific activity (6S)-H,PteGlu that is catalyzed by L. casei/
FOLINIC
ACID , nM
FIG. 3. Support of the growth of folate-depleted mouse L1210 cells by (6R,S)-SCHO-H,PteGlu (0) and the EDC-promoted formylation product of HQteGlu (i.e., by (6S)-5-CHO-H,PteGlu) (0). For details, see text.
76
MORAN TABLE
AND
2
SUPPORT OF THE GROWTH OF Ll210 CELLS BY SYNTHETIC (6S)-5-FORMYLTETRAHYDROFOLATE ECx, (M x IO-+) Experiment 1 2 3
Mean f SD
Commercial 2.32 2.55 1.73 2.20 f 0.42
Synthetic
Ratio
1.20 1.21 1.02 1.14 * 0.11
2.10 1.70 1.91 + 0.20
1.93
Note. The growth of Ll210 mouse leukemia cells was observed following a 72-h incubation of 3-I 1 X lo’ folate-depleted cells/ml in RPM1 1640 medium constituted without folic acid and supplemented with 10% exhaustively dialyzed fetal calf serum (9) and various concentrations of 5-CHO-H,PteGlu that was either purchased from Sigma Chemical Company or was prepared as described in the text. The concentration of each compound that supported a half-maximal increase in cell number during this period (EC,) was determined graphically as in Fig. 3.
MTX dihydrofolate reductase proceeds rapidly under our reaction conditions; timecourse experiments (data not shown) revealed that the reduction of [3H]PteGlu was complete within 15 min. [3H]H,PteGlu produced in this reaction is easily purified by DEAE-cellulose chromatography (Fig. 4A). However, intermediate purification of 3H,PteGlu prior to formylation has not been found necessary. The synthesis of C3H](6S)5-CHO-H,PteGlu was easily accomplished by adding EDC and formic acid directly to the reaction tube containing [ )H]H,PteGlu which had been enzymatically generated in situ from [ ‘H]PteGlu during a IO-min preincubation. The final product was then purified by DEAE-cellulose chromatography (Fig. 4B). The yield of high specific activity (38-45 Ci/mol) [3H](6S)-5-CHO-H&eGlu found in four preparations has been 55 & 14% relative to the [3H]PteGlu starting material. The yields appeared to be primarily affected by the purity of [ 3H]PteGlu used as starting material; yields as high as 75% have been attained with freshly received [ 3H]PteGlu. However, formylation of [ ‘H]HoPteGlu was quantitative under these conditions; the radioactivity that cochromatographed with 5-CHO-H,PteGlu after
COLMAN
formylation (Fig. 4B) was 98% of that which cochromatographed with H,PteGlu before formylation (Fig. 4A). In addition, the amount of radioactive formylation product that chromatographed with 1O-CHOH,PteGlu was negligible under these conditions (Fig. 4B). The kinetics of this formylation reaction have been studied by analysis of reaction mixes for H,PteGlu utilizing a newly developed assay that is sensitive to subpicomole quantities of this compound (32). In the absence of EDC, H,PteGlu was not consumed. However, after addition of EDC, reaction occurs rapidly and was essentially complete within 10 min but was usually not linear with time (data not shown). For quantitative consumption of HdPteGlu, the concentration of
” u
25
50
Fraction
75
100
0
Number
FIG. 4. Chromatography of ‘H-reaction intermediates on DEAE-cellulose. A sample of a reaction mixture containing 100 pCi of [‘H]PteGlu was chromatographed after 10 min incubation with 35 mU dihydrofolate reductase (A) and after an additional 10 min incubation with 900 nmol of formic acid and 5 mg of EDC (B). For details, see text. Chromatographic conditions were the same as in the experiment shown in Fig. 2. The folate markers that were mixed with sample immediately prior to chromatography were: (6&S)-IO-CHOH,PteGlu (I ), p-aminobenzoylglutamate (2), (6R,S)-5CHO-HQteGlu (3). (LR)-H,PteGlu (4), and PteGlu (5). Absorbance at 285 nm is indicated by (-), radioactivity by (0 0). Fractions contained 3.6 ml.
SYNTHESIS
OF TRITIATED
S-FORMYLTETRAHYDROFOLATE
formic acid had to be at or above 100 mM; 30 and 80% of the initial amounts of H,PteGlu remained unreacted after 1 h if formic acid was present at 50 and 10 mM, respectively. DISCUSSION
The reactivity of the Sposition of H,PteGlu toward carbodiimide-induced formyiation appears closely analogous to the unusual reactivity of 6,7-dimethyl-5,6,7,8tetrahydropterin in similar reactions (22,23). The formylation is specific for a tetrahydro reduction level and does not seem to proceed if the N-5 position is blocked (Table 1). Presumably, other carboxylic acids would react with the N-5 position of H,PteGlu under appropriate conditions. This reaction offers promise for the synthesis of other 5-substituted tetrahydrofolyl compounds as well as for the synthesis of affinity columns containing tetrahydrofolate linked to an insoluble matrix via the N-5 position. It should be noted that intermolecular condensations of two molecules of H,PteGlu via coupling of the a or y carboxyls of the glutamic acid with the 5-nitrogen were minimized in this work simply by using a substantial molar excess of formic acid. This method for the synthesis of 5-CHOH,PteGlu has significant advantages over other methods published to date. The ease, simplicity, and high yields obtainable are definite advantages. The formylation per se is specific and quantitative and proceeds rapidly under mild conditions. In addition, the products formed have the high degree of purity necessary for biological experiments; unlabeled (6S)-5-CHO-H,PteGlu prepared by this technique has a purity > 95% (Fig. 1B) as is the case with (6S)-5-CHOH,PteGlu (Fig. 2B) and (6S)-5-[3H]CHOH,PteGlu (data not shown). However, the fact that precipitation steps and other similar procedures are not used for the purification of product from reaction mixtures allows radioactive compound to be made without dilution of the specific activity of the
77
[ 3H]PteGlu that is presently commercially available. The yields reported here should probably be considered as minimal estimates. Thus, although the yield of production of moderate quantities (200 pmol) of 5-CHO-H4PteGlu is here reported as 54 f 2%, 5-CHOH,PteGlu accounted for 75-80s of the ultraviolet-absorbing species eluted from the DEAE-cellulose columns used to purify the product. Likewise, the yield of [ ‘HIS-CHOH,PteGlu obtainable from a new batch of [3H]PteGlu was 75% and, in fact, formylation of [ 3H]H,PteGlu proceeded quantitatively (Fig. 4). The labeled compounds made with this procedure should be quite useful in the delineation of the fate of the 5-formyl group during the entry of folinic acid into the intracellular folate pools and in the study of the mechanism of reversal of methotrexate cytotoxicity by reduced folate cofactors. ACKNOWLEDGMENTS We thank Natalie Rucker for her invaluable assistance in some of these experiments, Drs. August Pellino and Peter Danenberg for their critical evaluation of the manuscript, and Patrice Johnston for typing the manuscript. Note added in proof The extent of reaction of formate with H,PteGlu in the presence of EDC was found to be highly dependent on pH. Reaction was maximal at pH 3-3.6 and sharply declined above this pH range.
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MORAN
AND COLMAN
liuk, R. L., Combepine, G., Gaumont,Y., and Montgomery, J. A. (1979) Biochem. Pharmacol. 28,2993-2997. Chello, P. L., and Bertino, J. R. (1973) Cancer Rex 33, 1898-1904. Hakala, M. T., Zakrzewski, S. F., and Nichol, C. A. (1961) J. Biol. Chem. 236, 952-958. Moran, R. G., Zakrzewski, S. F., and Werkheiser, W. C. (1975) J. Biol. Chem. 251, 3569-3575. Goldin, A., Mantel, N., Greenhouse, S. W., Vendetti, J. W., and Humphreys, S. R. (1954) Cancer Res. 14, 43-48. Ensminger, W. D., Grindey, G. B., and Hoglind, J. A. (1979) Advan. Cancer Chemother. 1, 61109. Borsa, J., and Whitmore, G. F. (1969) Mol. Phormacol. 5, 303-3 17. Bertino, J. R. (1977) Semin. Oncol. 4, 203-216. Brockman, J. A., Roth, B., Broquist, H. P., Hultquist, M. E., Smith, J. M., Fahrenbach, M. J., Cosulich, D. B., Parker, R. P., Stokstad, E. L. R., and Jukes, T. H. (1950) J. Amer. Chem. Sot. 72,4325-4326. Zakrzewski, S. F., and Sansone, A. M. (1980) in Methods in Enzymology (D. B. McCormick and L. D. Wright, eds.), Vol. 66, pp. 731-733, Academic Press, New York. Shive, W., Bardos, T. J., Bond, T. J., and Rogers, L. L. (1950) J. Amer. Chem. Sot. 72, 28172818. Flynn, E. H., Bond, T. J., Bardos, T. J., and Shive, W. (1951)J. Amer. Chem. Sot. 73, 1979-1982. Khalifa, E., Ganguly, A. N., Bieri, J. H., and Viscontini, M. (1980) Helv. Chim. Acta 63, 25542558.
19. Nixon, P. F., and Bertino, J. R. (1971) Anal. Biochem. 43, 162- 172. 20. Nixon, P. F., and Bertino, J. R. (1980) in Methods in Enzymology (D. B. McCormick and L. D. Wright, eds.), Vol. 66, pp. 661-663. 21. Friedkin, M., Plante, L. T., Crawford, E. J., and Crum, M. (1975) J. Biol. Chem. 250, 56145621. 22. Cotton, R. G. H. (1974) FEBS Lett. 44, 290-292. 23. Cotton, R. G. H., and Jennings, I. G. (1978) Eur. J. Biochem. 85, 357-363. 24. Blakley, R. L. ( 1960) Nature (London) 188, 23 l232. 25. Moran, R. G., Spears, C. P., and Heidelberger, C. (1979) Proc. Nat. Acad. Sci. USA 76, 14561460. 26. Moore, G. E., Sandberg, A. A., and Ulrich, K. (1966) J. Nat. Cancer Inst. 36, 405-413. 27. Sharma, R. K., and Kisliuk, R. L. (1975) Biochem. Biophys. Res. Commun. 64, 648-655. 28. Shin, Y. S., Buehring, K., and Stokstad, E. L. R. (1972) J. Biol. Chem. 247, 7266-7269. 29. Uyeda, K., and Rabinowitz, J. C. (1965) J. Biol. Chem. 240, 1701-1710. 30. Blakley, R. L. (1969) The Biochemistry of Folic Acid and Related Pteridines, American Elsevier, New York. 31. Rabinowitz, J. C. (1970) in The Enzymes (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 2, 2nd Ed., pp. 185-252, Academic Press, New York. 32. Moran, R. G. (1982) Proc. Amer. Assoc. Cancer Res., in press.
122, 70-78 (1982)
BIOCHEMISTRY
A Simple Procedure
for the Synthesis of High Specific Activity (6S)-5Formyltetrahydrofolate’
RICHARD G.MORAN Laboratory
of Cellular
Tritiated
AND PAUL D. COLMAN
and Biochemical Pharmacology, Division of Hematology/Oncology, of Los Angeles, Los Angeles, California 90054
Childrens
Hospital
Received October 28, 1981 The 5position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as Sformyltetrahydrofolate (leucovorin). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folatedepleted Ll210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) [3H](6S)-5-formyltetrahydrofolate in high yield. Experiments with [‘%]formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-SFormyltetrahydrofolate, labeled in the formyl group with 14C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utilization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.
A growth factor for the folate-requiring bacterium Pediococcus cerevesiae, previously called Leuconostoc citrovorum (that was, hence, named citrovorum factor or CF)’ was originally isolated from liver (1) and, thereafter, shown to have the structure of 5-formyL5,6,7,Wetrahydropteroylglutamic acid (5-CHO-H,PteGlu) (2). Subsequently, the enzymatically active diastereoisomer of this compound was shown to have the S chirality at the 6-carbon (3). The
6R,S mixture of diastereoisomers has been called folinic acid or leucovorin and the calcium salt of this mixture is commercially available. Folinic acid is usually found to be the most potent source of folates for the support of the growth of mammalian cells in culture due to its stability in aqueous solution and its affinity for the reduced folate membrane transport system in such cells (4-6). For instance, growth supporting concentrations of folic acid are approximately 100 times higher than equieffective concentrations of folinate (7-9). Moreover, folinic acid has been shown to reverse the toxicity of antifols, such as methotrexate (MTX), to cultured mammalian cells and to animals (8, lo- 12). This latter effect is the basis for clinical protocols involving “high-dose MTX-CF rescue” in which supralethal doses of MTX are administered to patients with some types of neoplasms and the potentially lethal cytotoxicity
’ This investigation was supported by Grants CA27146 and CA27605 from the National Institutes of Health, DHEW. * Abbreviations used: HQteGlu, tetrahydrofolate; H,PteGlu, dihydrofolate; 5-CHO-HQteGlu, 5-formyltetrahydrofolate; 5-CHa-H4PteGlu, 5-methyltetrahydrofolate; 5,10-CH=H,PteGlu, 5,10-methenyltetrahydrofolate; IO-CHO-H,PteGlu, IO-formyltetrahydrofolate; PteGlu, folic acid; MTX, methotrexate. EDC, I-ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl; DEAE-cellulose, diethylaminoethylcellulose; CF. citrovorum factor; &ME, 2-mercaptoethanol. 0003-2697/82/070070-09$02.00/0 Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
70
SYNTHESIS
OF TRITIATED
5-FORMYLTETRAHYDROFOLATE
of MTX to the stem cells of the gut and bone marrow is terminated by the delayed administration of leucovorin ( 13). Several synthetic approaches to folinic acid have been published including the catalytic reduction of folic acid in concentrated formic acid (2,14), the direct formylation of tetrahydrofolate and conversion of the product to folinic acid by treatment with alkali at elevated temperatures (15), and the hydrogenation of lo-formylfolate at elevated temperatures ( 16,17). Recently, a convenient synthesis of folinate was reported involving the reaction of tetrahydrofolate with methyl formate in dimethylsulfoxide under mild conditions (18). The synthesis of labeled and unlabeled (6S)-5-CHO-H,PteGlu has been reported using enzymatically reduced tetrahydrofolate as starting material (19,20). In addition, a unique synthetic approach to (6S)-5-CHO-H,PteGlu has appeared in which folic acid is first reduced using dihydrofolate reductase and then formylated specifically at the 5-position with purified horse liver N-formylglutamate: tetrahydrofolate transformylase (21). It has previously been reported that watersoluble carbodiimides promote the coupling of compounds containing a carboxyl function to the N-5 position of 6,7-dimethyltetrahydropterin (22,23). In this report, we describe the analogous, facile, carbodiimideinduced formylation of (6S)-H,PteGlu and the utilization of this convenient reaction for the synthesis of unlabeled as well as high specific activity tritiated (6S)-5-CHOH,PteGlu and [ “C]formyl-labeled compound. MATERIALS
AND METHODS
DEAE-cellulose was purchased from Eastman Chemical, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl (EDC) from Sigma Chemical Company, reagent-grade ammonium acetate from Baker Chemicals, [‘4C]formic acid (lo-20 mCi/mmol) and [ 3’,5’,7,9-3H]folic acid (38-44 Ci/mmol)
71
were from New England Nuclear and Amersham, respectively. RPM1 1640 and dialyzed fetal calf serum were obtained from Grand Island Biological Company, Inc. H,PteGlu was made by reduction of PteGlu (Sigma) with sodium dithionite and was purified by repeated recrystallizations (24). (6s) H,PteGlu was prepared by reduction of H,PteGlu using L. casei/MTX dihydrofolate reductase as previously described (25). (6&Y)-5-CHO-H,PteGlu (folinic acid) and (6R,S)-5-CH,-H,PteGlu were purchased from Sigma Chemical Company, the former was used in these experiments without purification. (6&S)-5-CH,-H,PteGlu was a tan to brown powder as purchased and was purified by chromatography on a 4.9~cm2 X 30-cm column of DEAE-cellulose (containing 40 g of DEAE-cellulose) that was eluted with a convex gradient of ammonium acetate (0.01-1.5 M). When the peak tubes were pooled and lyophilized, a pure white, fluffy powder was obtained. Labile folates were stored at -25°C in foil-covered ampules that were sealed under dry N2. loCHO-H,PteGlu was made from 5-CHOH,PteGlu immediately before use by first adjusting a concentrated solution to pH 1 for 30 min, then carefully adjusting the pH to 6.5 with NaOH. Dihydrofolate reductase was purified from sonicates of L. casei/MTX by (NH4)$04 precipitation and chromatography on phosphocellulose as described by others (27). All other materials were of the highest grade available. Chromatographic columns were prepared and equilibrated as described previously (9). Cell culture. Mouse L1210 leukemia cells (26) were maintained as previously described (9). They were depleted of intracellular folates by 2-3 weeks of continuous growth on RPM1 1640 medium that was formulated with 32 PM hypoxanthine, 5.6 PM thymidine, and 10% dialyzed fetal calf serum (9). To determine the ability of a compound to act as a source of folate cofactors, folate-depleted L 12 10 cells were washed
72
MORAN
AND
once with phosphate-buffered saline, suspended in RPM1 1640 medium formulated without folic acid but with 10% dialyzed fetal calf serum and were distributed into Corning 25cm* T flasks (10 ml/flask containing 3-l 1 X lo4 cells per ml). Various concentrations of the compound were added to duplicate flasks, the flasks were incubated for 72 h and the cell number was determined using a Coulter counter Model B. Reaction of (bS)-H,PteGlu with formic acid. In a typical reaction, 177 pmol of (6S)H,PteGlu and 20 mmol of reagent-grade formic acid were dissolved in a total of 10 ml of 50 mM phosphate containing 1% @mercaptoethanol (P-ME); the final pH was 3.6. EDC (41.7 mg) was added and allowed to dissolve; the mixture was incubated for an additional 10 min at room temperature. The solution was shell-frozen and lyophilized, the dry residue was dissolved in 10 ml of 0.01 M ammonium acetate and the resultant solution was applied to a 4.9-cm* x 30cm column of DEAE-cellulose that had been equilibrated with ammonium acetate (9). The column was eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate with a mixer volume of 2 liters; all buffers contained 1% P-ME. The single main peak that was eluted from this column was pooled and lyophilized; this peak contained 74-80% of the material that absorbed at 285 nm and that was eluted from the column. This reaction was also run in vigorously degassed buffer without P-ME with the same results. EDC could be added either as a powder or as a freshly prepared, concentrated solution. In earlier experiments, the reaction product from 1- to 60-pmol reactions was purified by chromatography on a 0.64-cm’ X 75-cm column of Sephadex G-25. In 50 mM phosphate, pH 6.0, 5-CHO-H4PteGlu was found to elute from this column significantly after 3H20 (K,, = 1.85) as has been observed by others (19,28). However, the 5CHO-H,-PteGlu peak eluted from this column was often broad and the separation from salt depended upon the concentration
COLMAN
of salt in the sample. This technique was subsequently abandoned in favor of DEAEcellulose chromatography (9) which proved more reproducible. Synthesis of [:‘H](dS)S-CHO-H,PteGlu. In a representative reaction, 100 &i of [ 3’5’,7,9-3H]PteGlu (38-44 Ci/mmol, approximately 2.5 nmol) was incubated with 35 munit of L. casei/MTX dihydrofolate reductase (4.1 IU/mg) and 15 nmol of NADPH in a total volume of 100 ~1 of 50 mrvf phosphate, pH 7, containing 50 mM /3ME. After a IO-min incubation at 37°C 900 ~1 of 100 mrvr formic acid containing 50 mM phosphate and 1% b-ME was added, the pH was adjusted to 4-5 and 5 mg of EDC was added. After 10 additional min at room temperature, the reaction mixture was purified on a 0.64-cm* X 50-cm column of DEAEcellulose (containing 5.0 g of DEAE-cellulose) that was eluted with a convex gradient of ammonium acetate (0.01 to 1.5 M) with a mixer volume of 475 ml; chromatography buffers also contained 1% @-ME. In some experiments, folates used as chromatographic markers (0.5-1.5 mg) were added before sample was applied to the top of a column. The concentration of eluting salt was determined by measurement of the conductivity of suitable dilutions of the effluent; conductivity was found to be a linear function of ammonium acetate concentration on the range of lo-80 mM. Reaction offolates with [‘ICI formic acid. Approximately 1 pmol of a folyl compound was incubated with 100 pmol of [ “C]formic acid ( 1 &i) and 5 mg EDC in a total volume of 1 ml of 50 mM phosphate, pH 3.6, containing 1% &ME. In some experiments, the reaction mixture was added to a 0.64-cm* X 35-cm column of DEAE-cellulose after 10 min incubation and eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate; mixer volume was 475 ml for these separations. In these experiments, the stoichiometry between H,PteGlu and formate was determined by spectrophotometric determination of the amount of 5-CHO-H,PteGlu,
SYNTHESIS
OF TRITIATED
73
5FORMYLTETRAHYDROFOLATE
assuming a millimolar extinction coefficient of 37.2 at 285 nm (29). The amount of [ “C]formate was determined by scintillation counting in RIA-Solve II cocktail (Research Products International, Inc.); corrections for counting efficiency were made by the h-number method using a Beckman LS 7500 scintillation counter. In other experiments, folate-bound radioactivity was separated from [ i4C]formate by fractionation of reaction mixes on minicolumns of DEAE-cellulose. To prepare these minicolumns, DEAE-cellulose (8 g) was stirred for 15 min in 0.1 N NaOH (400 ml), then packed to a height of 4 f 0.25 cm in Pasteur pipets that were plugged with a minimal amount of cotton. Plastic Dispo transfer pipets (Scientific Products, catalog no. P5214-10) were most convenient for this purpose: if the plastic bulb was cut at the top, the pipet would hold 4.5 ml of eluting buffer in the reservoir that was formed. Prior to use, each minicolumn was washed with 10 ml each of water, 2.5 M ammonium acetate, and water followed by 4 ml of 1% pME. Each reaction mixture was washed onto a minicolumn with 1 ml of 10 mM ammonium acetate that was 1% in P-ME and the columns were eluted with 17 ml of this same solution to remove excess [ “C]formate. Then, each column was sequentially eluted with 3 ml of ammonium acetate at 0.7, 1.0, 1.25, and 1.50 M, all containing 1% ,&ME. Elution washes were collected directly into scintillation vials, shell-frozen, and lyophilized. The dry vials were hydrated with 1 ml of HZ0 and 9 ml of scintillation cocktail and radioactivity was determined by scintillation counting. The radioactivity in each wash was corrected for the residual tritium found in minicolumn eluates of triplicate blank (i.e., no folate added) reaction mixtures; the counts per minute reported in Table 1 are the sum of those found in the 0.7, 1.0, and 1.25 M washes. No radioactivity was found in the 1.50 M washes. The absorbancy corresponding to 5-CHO-H,PteGlu, 5-CH3H,PteGlu, and H,PteGlu was found in the
0.7 M wash while that corresponding to PteGlu and H,PteGlu was found in the 1.0 and 1.25 M washes. Calculation of yields. The yield of 5CHO-H,PteGlu formation from H,PteGlu was calculated by spectrophotometric determination of the amount of H,PteGlu used assuming ~292 = 19.1 mM-’ cm-’ (29-3 1) in 0.1 N HCl and by spectrophotometric determination of the amount of 5-CHO-H,PteGlu in the pooled chromatographic fractions assuming +85 = 37.2 rnrv-’ cm-’ at neutral pH (29). The yield of [3H]5-CHO-H4PteGlu produced from 3H-PteGlu was calculated from the ratio of radioactivity in the pooled chromatographic fractions to that originally added to the reaction mix. RESULTS
Large-Scale H,PteGlu
Synthesis of (6S)-5-CHO-
The reaction of enzymatically reduced H,PteGlu with 100 mM formic acid in the presence of EDC was found to proceed rapidly with the appearance of a compound with the spectrum of 5-CHO-H,PteGlu (pH 7, x max = 286) (Fig. 1A). The crude reaction mixture was conveniently purified either by chromatography on Sephadex G-25, as previously reported by others (19,27) or by chromatography on DEAE-cellulose eluted with a convex gradient of ammonium acetate (9). Rechromatography of the purified material allowed an estimate of the purity of the product to be in excess of 95% (Fig. 1B). This product was converted into a compound with a spectrum identical to that of 5,10CH=H,PteGlu (pH 0, X,,, = 288,346) by treatment with 1.0 N HCl for 1 h (Fig. 1A). The ratio of absorbance of the product at 285 in neutral solution to that of the acidtreated product at 345 in 1.0 N HCl was 1.43, in agreement with the published extinction coefficients of 5-CHO-H,PteGlu and 5,10-CH=H,PteGlu under these conditions (37.2 and 26.3, respectively) (29-31) from which the expected ratio of 1.41 can
14
MORAN
AND COLMAN
be calculated. The neutral and acidic spectra observed on the unfractionated reaction mixture were identical to those seen with the chromatographically purified product. However, the mean yield calculated for three preparations starting with (6S)-H,PteGlu on the range of l-200 pmol was 54 + 2%. When the EDC-catalyzed formylation of H,PteGlu was run in the presence of [‘4C]formic acid and the product was purified by DEAE-cellulose column chromatography, the 14C-labeled product chromatographed as a peak with the spectrum of 5CHO-HJ%eGlu; this uv-absorbing product was found to have a ratio of 14C to Azss equivalent to approximately 1 mol of formate per mol of 5-CHO-H,PteGlu; this ratio was constant through the chromatographic peak (the mean ratio for tubes 70-
0.75
2
I ^ 1.
.
f
/t
'*?0.6
A
..
* fj :: 4
Q?I ,I L:L
0.50
..
0.25
7
8.
;
260
290
;
;
320
350
Wavrlen6th
Fraction
Fraction
,
:
360
410
, nm
Numbrr
FIG. 1. (A) Spectral characteristics of the product of EDC-promoted formylation of H,PteGlu. The ultraviolet absorption spectrum of the purified product was determined at pH 7 (0) and after standing for 1 h in 1 N HCl (0). (B) Purity of reaction product. The formylation product was purified on a column of Sephedex G-25, lyophilized, and an aliquot was rechromatographed on a 0.64-cm2 X 35-cm column of DEAE-cellulose that was eluted with a linear gradient of ammonium acetate (0.05 to 2.5 M) (9) to determine purity. The fractions contained 3.5 ml.
Number
FIG. 2. Chromatography of 14C product on DEAEcellulose. (A) A reaction mixture containing 100 amol of [“Clformic acid and 1 pmol of H,PteGlu was absorbed onto a 0.62~cm* X 50-cm column of DEAE-cellulose 10 min after the addition of 5 mg of EDC and the column was eluted with a 0.01 to 1.5 M convex gradient of ammonium acetate. The folate peak eluted from the column shown in A was pooled, lyophilized, and rechromatographed on DEAE-cellulose under identical conditions as described for A without further treatment (B) or following 1 h in 1 N HCl (C). The symbols are: Absorbance at 285 nm (O), radioactivity (0) salt concentration (m), ratio of [ ‘%]formate to A2s5, (A). The ratio of ‘%I to A2s5 is expressed as mol of 14C per mol of 5-CHO-H.,PteGlu by correcting the raw data for counting efficiency, specific activity of the formic acid and the extinction coefficient of S-CHO-H,PteGlu at 285 nm. The fractions contained 3.3 ml. Radioactive peaks 1 and 2 reached 22,000 and 500,000 cpm/ml, respectively.
75 was 1.24; in a replicate experiment this ratio was 1.17) (Fig. 2A). Likewise, when this 14C-labeled product was acidified to cy-
SYNTHESIS TABLE REACTION
OF TRITIATED I
OF['%]FORMICACID FOLATE COMPOUNDS
WITH VARIOUS
Counts per minute Compound (6S)-H1PteGlu (6R,S)&CHO-H,PteGIu H,PteGlu PteGlu (6R.S)-5-CH,-H,PteGlu
Expt I 3378
129 5 118 -110
Expt 2 5193
49 155 -34 94
Percentage H,PteGlu
of
100 2.4 1.6 1.4 -0.7
Nore. Triplicate reaction mixtures of each folate compound (0.7-1.0 wmol/tube.) were incubated for 10 min at room temperature with 26 pmol of EDC and 100 pmol(1 pCi) of formic acid in 50 rnM phosphate containing I % &ME; the final reaction mix was pH 3.6. Each reaction mixture was chromatographed on a 4-cm Pasteur pipet column of DEAE-cellulose as described under Materials and Methods. The values listed represent the mean cpm eluted with 0.7-1.25 M ammonium acetate, corrected for the amounts of radioactivity found in the eluate from columns run on reaction mixes incubated without folate compounds. Three reactions were run for each compound in each experiment.
clize any SCHO-H,PteGlu, a new peak appeared in the chromatogram at the position of lo-CHO-H,PteGlu, as would be expected at the pH of this column (6.5) if the original compound was 5-CHO-HSPteGlu (Fig. 2C). It is of interest that the reaction product was not quantitatively converted to 1O-CHOH,PteGlu under these conditions. However, the material that chromatographed at the location of 5-CHO-H,PteGlu after acidification had a ratio of absorbance at 285 nm to 14C that was not significantly different from that of the original reaction product (mean ratio for tubes 71-75 = 1.33) (Fig. 2). The concentration of ammonium acetate that eluted the reaction product (Fig. 2B) and the compound to which it was converted by acid treatment were identical to those that elute 5-CHO-H,PteGlu and lo-CHOH,PteGlu under these chromatographic conditions, namely, 0.48 and 0.34 M, respectively (9). Specificity
75
SFORMYLTETRAHYDROFOLATE
of This Reaction
Under these conditions, [ “C]formic acid reacted with H4PteGlu but did not react ap-
preciably with PteGlu or H,PteGlu implying that the reactivity of N-5 of the folate moiety requires a fully reduced pyrazine ring (Table 1). The lack of reactivity of 5-CHOH,PteGlu and 5-CH3-H,PteGlu under these conditions indicates that the formylation of N-5 of H,PteGlu is highly favored over attack at any other position in this ring system, e.g., N- 10. Furthermore, these data indicate that further formylation of the 5-CHOH,PteGlu product of reaction of formate with H,PteGlu is quite limited under these conditions. Biological
Activity
The product of the EDC-catalyzed formylation of (6S)-H,PteGlu was found to support the growth of folate-depleted L1210 cells in the absence of an added source of folates to the same extent as commercial leucovorin (Fig. 3). However, half-maximal growth of these cells was observed at a concentration of commercial (6R,S)-5-CHOH,PteGlu that was twice that required for the synthetic (6S)-5-CHO-H,PteGlu product made by this EDC-promoted formylation (Table 2). Synthesis
of [‘H](6S)-5-CHO-H,PteGlu
The synthesis of high specific activity (6S)-H,PteGlu that is catalyzed by L. casei/
FOLINIC
ACID , nM
FIG. 3. Support of the growth of folate-depleted mouse L1210 cells by (6R,S)-SCHO-H,PteGlu (0) and the EDC-promoted formylation product of HQteGlu (i.e., by (6S)-5-CHO-H,PteGlu) (0). For details, see text.
76
MORAN TABLE
AND
2
SUPPORT OF THE GROWTH OF Ll210 CELLS BY SYNTHETIC (6S)-5-FORMYLTETRAHYDROFOLATE ECx, (M x IO-+) Experiment 1 2 3
Mean f SD
Commercial 2.32 2.55 1.73 2.20 f 0.42
Synthetic
Ratio
1.20 1.21 1.02 1.14 * 0.11
2.10 1.70 1.91 + 0.20
1.93
Note. The growth of Ll210 mouse leukemia cells was observed following a 72-h incubation of 3-I 1 X lo’ folate-depleted cells/ml in RPM1 1640 medium constituted without folic acid and supplemented with 10% exhaustively dialyzed fetal calf serum (9) and various concentrations of 5-CHO-H,PteGlu that was either purchased from Sigma Chemical Company or was prepared as described in the text. The concentration of each compound that supported a half-maximal increase in cell number during this period (EC,) was determined graphically as in Fig. 3.
MTX dihydrofolate reductase proceeds rapidly under our reaction conditions; timecourse experiments (data not shown) revealed that the reduction of [3H]PteGlu was complete within 15 min. [3H]H,PteGlu produced in this reaction is easily purified by DEAE-cellulose chromatography (Fig. 4A). However, intermediate purification of 3H,PteGlu prior to formylation has not been found necessary. The synthesis of C3H](6S)5-CHO-H,PteGlu was easily accomplished by adding EDC and formic acid directly to the reaction tube containing [ )H]H,PteGlu which had been enzymatically generated in situ from [ ‘H]PteGlu during a IO-min preincubation. The final product was then purified by DEAE-cellulose chromatography (Fig. 4B). The yield of high specific activity (38-45 Ci/mol) [3H](6S)-5-CHO-H&eGlu found in four preparations has been 55 & 14% relative to the [3H]PteGlu starting material. The yields appeared to be primarily affected by the purity of [ 3H]PteGlu used as starting material; yields as high as 75% have been attained with freshly received [ 3H]PteGlu. However, formylation of [ ‘H]HoPteGlu was quantitative under these conditions; the radioactivity that cochromatographed with 5-CHO-H,PteGlu after
COLMAN
formylation (Fig. 4B) was 98% of that which cochromatographed with H,PteGlu before formylation (Fig. 4A). In addition, the amount of radioactive formylation product that chromatographed with 1O-CHOH,PteGlu was negligible under these conditions (Fig. 4B). The kinetics of this formylation reaction have been studied by analysis of reaction mixes for H,PteGlu utilizing a newly developed assay that is sensitive to subpicomole quantities of this compound (32). In the absence of EDC, H,PteGlu was not consumed. However, after addition of EDC, reaction occurs rapidly and was essentially complete within 10 min but was usually not linear with time (data not shown). For quantitative consumption of HdPteGlu, the concentration of
” u
25
50
Fraction
75
100
0
Number
FIG. 4. Chromatography of ‘H-reaction intermediates on DEAE-cellulose. A sample of a reaction mixture containing 100 pCi of [‘H]PteGlu was chromatographed after 10 min incubation with 35 mU dihydrofolate reductase (A) and after an additional 10 min incubation with 900 nmol of formic acid and 5 mg of EDC (B). For details, see text. Chromatographic conditions were the same as in the experiment shown in Fig. 2. The folate markers that were mixed with sample immediately prior to chromatography were: (6&S)-IO-CHOH,PteGlu (I ), p-aminobenzoylglutamate (2), (6R,S)-5CHO-HQteGlu (3). (LR)-H,PteGlu (4), and PteGlu (5). Absorbance at 285 nm is indicated by (-), radioactivity by (0 0). Fractions contained 3.6 ml.
SYNTHESIS
OF TRITIATED
S-FORMYLTETRAHYDROFOLATE
formic acid had to be at or above 100 mM; 30 and 80% of the initial amounts of H,PteGlu remained unreacted after 1 h if formic acid was present at 50 and 10 mM, respectively. DISCUSSION
The reactivity of the Sposition of H,PteGlu toward carbodiimide-induced formyiation appears closely analogous to the unusual reactivity of 6,7-dimethyl-5,6,7,8tetrahydropterin in similar reactions (22,23). The formylation is specific for a tetrahydro reduction level and does not seem to proceed if the N-5 position is blocked (Table 1). Presumably, other carboxylic acids would react with the N-5 position of H,PteGlu under appropriate conditions. This reaction offers promise for the synthesis of other 5-substituted tetrahydrofolyl compounds as well as for the synthesis of affinity columns containing tetrahydrofolate linked to an insoluble matrix via the N-5 position. It should be noted that intermolecular condensations of two molecules of H,PteGlu via coupling of the a or y carboxyls of the glutamic acid with the 5-nitrogen were minimized in this work simply by using a substantial molar excess of formic acid. This method for the synthesis of 5-CHOH,PteGlu has significant advantages over other methods published to date. The ease, simplicity, and high yields obtainable are definite advantages. The formylation per se is specific and quantitative and proceeds rapidly under mild conditions. In addition, the products formed have the high degree of purity necessary for biological experiments; unlabeled (6S)-5-CHO-H,PteGlu prepared by this technique has a purity > 95% (Fig. 1B) as is the case with (6S)-5-CHOH,PteGlu (Fig. 2B) and (6S)-5-[3H]CHOH,PteGlu (data not shown). However, the fact that precipitation steps and other similar procedures are not used for the purification of product from reaction mixtures allows radioactive compound to be made without dilution of the specific activity of the
77
[ 3H]PteGlu that is presently commercially available. The yields reported here should probably be considered as minimal estimates. Thus, although the yield of production of moderate quantities (200 pmol) of 5-CHO-H4PteGlu is here reported as 54 f 2%, 5-CHOH,PteGlu accounted for 75-80s of the ultraviolet-absorbing species eluted from the DEAE-cellulose columns used to purify the product. Likewise, the yield of [ ‘HIS-CHOH,PteGlu obtainable from a new batch of [3H]PteGlu was 75% and, in fact, formylation of [ 3H]H,PteGlu proceeded quantitatively (Fig. 4). The labeled compounds made with this procedure should be quite useful in the delineation of the fate of the 5-formyl group during the entry of folinic acid into the intracellular folate pools and in the study of the mechanism of reversal of methotrexate cytotoxicity by reduced folate cofactors. ACKNOWLEDGMENTS We thank Natalie Rucker for her invaluable assistance in some of these experiments, Drs. August Pellino and Peter Danenberg for their critical evaluation of the manuscript, and Patrice Johnston for typing the manuscript. Note added in proof The extent of reaction of formate with H,PteGlu in the presence of EDC was found to be highly dependent on pH. Reaction was maximal at pH 3-3.6 and sharply declined above this pH range.
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