ANALYTICAL

BIOCHEMISTRY

The Preparative

9,

48-50 (1979)

Electrochemical

MAGGIE E. SALEM, Department

of Child

Health,

Reduction

GARRY

University

P. LEWIS,

of Sydney,

Sydney.

of Pteroylpentaglutamate AND PETER B. ROWE’ New

South

Wales,

2050, Australia

Received November 28, 1978 A method is described for the electrochemical reduction of pteroylpentaglutamate results in high yields of the fully reduced tetrahydro derivative.

As a result of the extreme sensitivity of tetrahydrofolic acid to air oxidation many research groups prepare this folate derivative immediately prior to use. The methods used for the reduction of folic acid include catalytic hydrogenation over platinum, chemical reduction with agents such as dithionite, or enzymatic reduction with dihydrofolate reductase (1). Although the last method produces the biologically active diastereoisomet-, these methods generally have certain disadvantages including (i) the inability to effectively monitor the course of the reduction reaction which may result in low yields either from incomplete reduction or from the breakdown of tetrahydrofolic acid and (ii) the contamination of the end product by a variety of salts. These objections are particularly relevant when reducing polyglutamate derivatives of folic acid which are extremely expensive and not available in large quantities. The reduction technique described here obviates many of these problems and gives a high yield of fully reduced derivatives.

which

ducibly high product yield. Strict attention was paid to temperature control in the esterification of the first glutamate residue to the Merrifield resin. The oil bath temperature was slowly increased to 82°C at a rate of lO”C/h and never exceeded 86°C. This resulted in a 60% substitution, i.e., 1.2 mEq glutamate/g of resin. Coupling efficiency was significantly improved by increasing the molar excess of N-methylmorpholine from 2.5- to 2.75-fold in the formation of the substituted glutamate anhydride and from 1.8to 2. l-fold in the formation of the trifluoroacetylpteroate anhydride. In addition the reaction vessel was designed to allow smooth efficient mixing for long time periods. With these modifications and precautions a typical synthesis of pteroylpentaglutamate gave a 55% yield (expressed in terms of the amount of glutamate originally substituted onto the resin) of a product which was 92% pure. Triethylamine bicarbonate buffer (1 .O M) was prepared by adding 140 ml freshly distilled triethylamine to 860 ml of distilled, degassed water and titrating to pH 9.25 with carbon dioxide at 4°C. MATERIALS AND METHODS Pteroylpentaglutamate (50 pmol) was disPteroylpentaglutamate was synthesized solved in 15 ml of 0.5 M triethylamine biby a modification of the solid-phase method carbonate, pH 9.25, in a polarographic repreviously described (2). These modificaaction vessel (Metrohm EA 996-20). As iltions were essential to achieve a repro- lustrated in Fig. 1 a magnetically stirred mercury pool constituted the working elec’ To whom communications should be addressed at trode with additional reference (platinum) Institute of Child Health, Royal Alexandra Hospital and auxiliary (graphite) electrodes. Nitrofor Children, Camperdown, N.S.W., 2050, Australia. 0003-2697/79/l 1004%03$02.00/0 Copyright 0 1979 by Academic Press. Inc. All rights of reproducuon in any form reserved.

48

PREPARATIVE

ELECTROCHEMICAL

FOLATE

49

REDUCTION

FIG. 1. Apparatus for electrochemical reduction, PS, potentiostat-regulated power supply; C, polarographic reaction vessel; Hg, mercury pool working electrode; G, graphite auxilliary (output) electrode; Pt, platinum reference (input) electrode: Nz, nitrogen gas inlet; S, stirring bar.

gen gas was gently bubbled through the solution for 10 min prior to the application of current and maintained over the surface of the solution throughout the reaction. Voltage was applied via a potentiostatregulated power supply (McKee-Pederson Model MP-1026 A) in a step-wise manner as illustrated in Fig. 2 until the maximum (-950 mV) was attained after 3.5 h. This was maintained for another 20 h at which time current flow was terminated and 2-mercaptoethanol was added to a final concentration of 0.2 M. Samples were removed throughout the reduction process for analysis by ultraviolet spectroscopy, anion-exchange high performance liquid chromatography (hplc)2 (3) and by polarography (Metrohm Polarecord E 506). The solution was rapidly freeze-dried, removing the relatively volatile buffer, yielding a fine white powder. Based on spectroscopic and HPLC data the final yield was of the order of 96% with respect to the starting material with no detectable breakdown of the polyglutamate chain.

Qualitative studies on the electrochemical reduction of folate to dihydro- and tetrahydrofolate and of 5, lo-methenyltetrahydrofolate to 5, lo-methylenete~ahydrofolate and subsequently to S-methyl tetrahydrofolate have been briefly reported by Kwee and Lund (4) using somewhat different conditions with respect to the buffer employed and the voltage stepping procedure. The gradual increase in voltage was essential to prevent breakdown of the folate molecule. If the voltage was rapidly raised to the maximum level approximately 20 to 25% of the folate was cleaved at the C9-NlO bond producing p-aminobenzoylglutamate

DlSCUSSlON

Electrochemical reduction is a technique whereby small quantities of valuable pteroylpolyglutamate derivatives can be quantitatively reduced to their tetrahydro form. * Abbreviation used: HPLC, high performance liquid chromatography.

t

0'

I.0

t 2-O TIME (kits)

3.0

4~0

FIG. 2. Graphical representation of time-related voltage steps during reduction.

50

SALEM,

LEWIS, AND ROWE

and, presumably, 6-methyl-7,8-dihydropterin. While any one of the variety of stepping techniques could be used, the point to be made is that the voltage must be increased slowly. Although we do not have any evidence to support the hypothesis, it is likely that the cleavage of the C9-NlO bond results from protonation of the NIO of 7,&dihydrofolate (4). Studies with the enzyme formyltetrahydrofolate synthetase confirmed that the reduced folate consisted of an equal mixture of the two diastereoisomers (5). Kwee and Lund have indicated that the relative proportions of the diastereoisomers could be modified by alterations in the reducing conditions but have not, as yet, revealed any details (4). We have attempted, without success, the electrochemi~~ reduction of lO-formy~olate, a readily synthesized highly stable folate derivative, in an effort to bypass the isolation step necessary for the synthesis of 5,10-methenyltetrahydrofolate. For some, as yet unexplained, reason lo-formylfolate breaks down under a wide range of electrochemical reducing conditions. It is evident, however, that a scaled-up

version of this reduction procedure could economically provide supplies of biologically active polyglutamate derivatives of folic acid for research purpose. ACKNOWLEDGMENTS These studies were supported by grants from the Australian Department of Health, the National Health and Medical Research Council of Australia, and the N.S.W. State Cancer Council. The authors are extremely grateful to Dr. Henning Lund and Dr. Sianette Kwee of the University of Aarhus, Denmark, for their advice and assistance with this study.

REFERENCES 1. Blakley, R. L. (1%9) The Biochemistry of Folic Acid and Related Pteridines, pp. 78-82, North Holland, Amsterdam. 2. Silink, M., Reddel, R. R., Bethel, M., and Rowe, P. B. (1975) J. Biol. Chum. 250, 5982-5994. 3. Lewis, G. P., and Rowe, P. B. (1979) Anal. Biothem.,

93,91-W.

Kwee, S., and Lund, H. (1979) in Chemistry and Biology of Pteridines (Kisliuk, R. L., Brown, G. M., eds.), pp. 247-252, Elsevier, Amsterdam/New York. 5. Lewis, G. P., Salem, M. E., and Rowe, P. B. (1978) in Chemistry and Biology of Pteridines (Kisliuk, R. L., Brown, G. M., eds.), pp. 441-442, Elsevier, Amsterda~New York.

4.

The preparative electrochemical reduction of pteroylpentaglutamate.

ANALYTICAL BIOCHEMISTRY The Preparative 9, 48-50 (1979) Electrochemical MAGGIE E. SALEM, Department of Child Health, Reduction GARRY Univer...
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