[61] PMR characteristics of folic acid and analogs

[61]

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even during extraction procedures, ~8,~2,33 although an occasional serum will undergo autoxidation if improperly handled. Ascorbic acid should not be used as a substitute for sodium ascorbate, since 5 mg/ml will lower the serum pH to 5.1 (personal communication). The values obtained for folates when assayed as described correlate well with the hematologic findings and the L. casei diagnostic groups. These assays serve as a rapid, inexpensive, and sensitive clinical tool aiding in the diagnosis of folate deficiency and in the detection of unsaturated folate binders. a2 j. L i n d e m a n s , J. Van Kapel, and J. Abels, Clin. Chim. Acta 65, 15 (1975). aa j. K e r k a y , C. Coburn, and D. M c E v o y , Am. J. Clin. Pathol. 68, 481 (1977).

[61] P M R

Characteristics of Folic Acid and Analogs By MARTIN POE

Nuclear magnetic resonance (NMR) studies of small organic molecules are useful in structural determinations. The most popular nuclei for study are 13C and 1H. 13C NMR has the advantage of looking directly at the most important element in organic chemistry. 1H NMR, better known as proton magnetic resonance (PMR), is more widely used because it is about 104 times more sensitive. Pteridines like folic acid are not particularly well suited for PMR studies because the protons on the pteridine moiety are few or too similar. Nevertheless, PMR is such a powerful tool in the analysis of organic compounds that it has been widely used anyway. 1 There have also been useful laC studies. 2,a PMR studies were crucial in establishing the 7,8dihydro structure of natural dihydrofolate4 and the conformation of the tetrahydropyrazine ring of tetrahydrofolate? Preparation of Solutions for P M R Spectroscopy Spectrometer Selection. Three classes of PMR spectrometers are commercially available. The low-field PMR spectrometers that operate 1 M. Poe, J. Biol. Chem. 248, 7025 (1973). z j. A. L y o n , P. D. Ellis, a n d R. B. Dunlap, Biochemistry 12, 2425 (1973). a W. Frick, R. Weber, and M. Viscontini, Helv. Chim. Acta 57, 2658 (1974). 4 E. J. Pastore, Ann. N.Y. Acad. Sci. 186, 43 (1971). 5 A. Dieffenbacher, R. Mondelli, and W. V. Phillipsborn, Heir. Chim. Acta 49, 1355 (1966).

METHODS IN ENZYMOLOGY,VOL, 66

Copyright © 1980by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3

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[61]

at 60 MHz are suitable for studies on fully aromatic folates and for samples of 25 mg or larger. Reduced folates are better studied with intermediate-field instruments that operate at 90 or 100 MHz, or on highfield instruments. To examine the protons of the tetrahydropyrazine ring of tetrahydrofolates, it is necessary to use high-field instruments that operate at 220, 270, 300, or 360 MHz. Most applications of PMR to folates use intermediate- or high-field computer-controlled spectrometers for whose operation expert help is usually needed. Amount of Sample. Although PMR is intrinsically poor in sensitivity, modern PMR spectrometers have partially overcome this problem. An overnight run using a 300 MHz PMR spectrometer in the Fourier transform mode can give a useful PMR spectrum of 3/xg of a folate. Solutions containing 0.01 M folate, 2.25 mg in 0.5 ml, give useful spectra in a few minutes. The sample is not destroyed during measurement. When 0.1-l mg of a folate is available, dissolve it in 0.35 ml solvent and accumulate data for up to several hours. For 3-100 /xgm of sample, dissolve the sample in 50 /zl solvent and use a spherical insert for overnight accumulation. More than 2.5 mg folate in 0.5 ml 0.1 M NaOD will give a usable PMR spectrum in 10 min or less of data accumulation. For published work, about 1 mg of an NMR reference compound such as (CHa)4NC1 or sodium-3-trimethylsilyl[2,2,3,3-2D]propionate (Merck Sharp & Dohme, Montreal, Canada) should be included. Sample Containers and Volume. Most PMR spectrometers use cylindrical glass NMR tubes 5 mm in diameter (OD) and 7 in. long which are commercially available from a number of suppliers, including Wilmad Glass in Vineland, New Jersey. Typically 0.5 ml of a solution is analyzed, but as little as 50/~1 may be used with special procedures. Solvent. Folates have only a limited solubility in most solvents. It is best to run PMR of folates in deuterated solvents so that the PMR resonances of the solvent do not obscure the folate resonances. A good choice of solvents is 0.1 M NaOD in D20. Solid folates are often prepared in the acid form, the form which is least soluble in D20. Of course, deuterated water exchanges the protons bound to nitrogen and oxygen atoms, making them nonobservable. For use with dihydro- and tetrahydrofolates, the dilute base should be bubbled with dry N2 before use, and the solutions should be prepared the day of use. Pteridines not containing glutamate moieties and "nonclassical" folate antagonists like trimethoprim and pyrimethamine are fairly soluble in CFaCOOD. Impurities are often less troublesome in PMR spectroscopy than in other analytical techniques. Resonance positions in PMR solutions are affected by pH,

[61]

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temperature, and solute concentration, 1,4 so these conditions should be noted for each spectrum run. Conditions for P M R Data Accumulation For solutions of folates prepared as described above, the conditions used for routine PMR spectra of organic compounds are suitable. Listed in the following are the conditions the author uses on a Varian SC-300 PMR spectrometer for folate solutions in D20 at 25°: Tube OD, 5 mm Spin rate, 35 rps Deuterium lock Rf frequency, 300,250,200 Hz Spectra width, 3000 Hz Acquisition time, l sec Pulse width, 5/~sec Number of transients, 100 Spectra are accumulated in the Fourier transform mode of the spectrometer with the sensitivity artificially enhanced by use of an exponential weighting of the free induction decay with a time constant of - 1 sec. Analysis of P M R Spectra of Folates In the discussion that follows, the IUPAC-IUB 6 numbering system and structural formula for folates given in Fig. I will be used. In the use of PMR spectra for purity analysis and structural identification, it is necessary to have resonance assignments. Present in Fig. 2 are PMR spectra of folate and two folate analogs, methotrexate and aminopterin, each at 0.010 M in 0.1 M NaOD and at 25°. Aminopterin is 4-deoxy-4aminofolate and methotrexate is N-10-methylaminopterin. The lowest field resonance at the left corresponds to the C-7 proton. The next two resonances, each a doublet, are part of an AA'BB' quartet, with the lowfield pair corresponding to the two protons at C-2' and C-6', and the upfield pair corresponding to the two protons at C-Y and C-5'. Next, and immediately up-field from the water resonance, is a resonance corresponding to the two protons at C-9. Next up-field is a doublet of doublet with spin-spin splittings of 8.8 and 4.4 Hz, corresponding to the proton on the a-carbon of the glutamate moiety, split by unequal coupling to the two/3-carbon protons. Next up-field in the methotrexate spectrum is a 6 Commission on Biochemical Nomenclature, IUPAC-IUB Tentative Rules, J. Biol. Chem. 241, 2987 (1966).

486

[61]

PTER1DINES, ANALOGS, AND PTERIN COENZYMES

COOH I

O

COOH

H2

N

N

F1G. I. Numbering system used for folates. This is folate (pteroylglutamate).

resonance corresponding to the methyl protons of the N-10-methyl group. The resonances between 2 and 2.5 ppm correspond to the four protons on the/3- and y-carbons of the glutamate moiety on the three compounds. The two protons on the glutamate y-carbon are part of an asymmetric triplet slightly to low-field of the complex multiplets corresponding to the two /3-carbon protons. Despite free rotation the E-carbon protons are nonequivalent due to the adjacent chiral center. A summary of the i

I

I

I

i

I

i

I

I

I

I

I

t

I

I

1

Aminopterin

'

'

i

I

t

J "1

J

I

Folate

._Jl I0

L

I

8

i

I

t

I

6

4

t

I

2

0

Ppm FlG. 2. The 300 MHz PMR spectrum of" three f'olates in 0.1 M N a O D at 25 ° and at 10 raM. Chemical shift scales in ppm downfield from internal (CH3)~Si(CD~)~COONa. HDO resonance at about 4.9 ppm not shown.

[61]

PMR

OF FOLIC ACID AND ANALOGS

487

assignments and the chemical shifts of the assigned resonances in ppm (Hz down-field from the reference resonance divided by 300 MHz) under these conditions is given in the table. The PMR spectra of 7,8-dihydrofolate, (-+), /-5,6,7,8-tetrahydrofolate, and (-+),/-5-methyl-5,6,7,8-tetrahydrofolate in 0.1 M N a O D at 25 ° and near 0.01 M are shown in Fig. 3. The protons on the p-aminobenzoylL-glutamate moieties on these three compounds are at about the same positions as in folate. The carbon-bound protons on the pyrazine ring of the three reduced compounds are near 3.5 ppm. The two stereoisomers for the tetrahydrofolates have essentially the same PMR spectrum. In 7,8-dihydrofolate the protons on C-9 are at 4.04 ppm while the two C-7 protons are at 3.98 ppm. The PMR spectrum of dihydrofolate also has a small resonance at 8.63 ppm, which shows that some folate is present. The two small resonances near 4 ppm in the PMR spectrum of tetrahydrofolate show that some dihydrofolate is present, and the two triplet

T

T

7

~

F

r

r

r

7-"

N- 5- Methyltetrohydrofolate

~ ~ T e t r a h y d r ° f ° l a t e

Dihydrofolate

I0

8

6

4

2

0

Ppm FIG. 3. The 300 MHz PMR spectrum of (-+), l-5-methyl-5,6,7,8-tetrahydrofolate, (+_+_),l5,6,7,8-tetrahydrofolate, and 7,8-dihydrofolate in 0. ! M NaOD at 25° and at |3.6, 9.0, and 10.0 r a M , r e s p e c t i v e l y . C h e m i c a l s h i f t s a s in F i g . 2.

488

PTERIDINES,

ANALOGS,

AND

PTERIN

[61]

COENZYMES

03

i

e-

% <

r-

O

O

Z Z

Z © ©

©

6= N N

6~

t~

._= t~

r~

O Z

_=

[61]

P M R OF FOLIC ACID AND ANALOGS

489

resonances at 3.88 and 2.90 ppm correspond to the methylene protons of 2-mercaptoethanol. In the two tetrahydrofolates, the five protons on C6, C-7, and C-9 are very close in chemical shift. In tetrahydrofolate, due to the chiral center at C-6, the two C-7 protons at 3.52 and 3.21 ppm are nonequivalent despite conformational averaging of their position, the C6 proton is at 3.43 ppm, and the C-9 protons at 3.34 ppm. From analysis of the proton spin-spin coupling constants, 7 it has been shown that the tetrahydropyrazine ring of tetrahydrofolate is a roughly equal mixture of two half-chair conformations, one with the C-6 proton axial and the other with the C-6 proton equatorial. A similar conformational analysis 8 of the tetrahydropyrazine ring of 5-methyltetrahydrofolate suggested that the ring is in a half-chair conformation with the methyl group at N-5 and the methylene group C-9 t r a n s and with C-6-H in an equatorial position. The chemical shifts of the assigned resonances of the reduced folates depicted in Fig. 3 are also given in the table. Stereochemical Applications of P M R Studies of Folates There are a number of features in the PMR spectra of folates that give insight into the conformation of these compounds in solution, specifically the chemical shifts, spin-spin splittings, and relaxation times T1 and T2. The dependence of the quantities upon pH, temperature, and concentration give insight into molecular geometry, protonation sites, and ring geometry. Sites of protonation in folates may be observed directly in CFzCOOH 3"5 or indirectly in DzO by using the principle that protons bonded to carbon atoms immediately adjacent to a site of protonation exhibit larger down-field shifts in their chemical shift upon protonation than those protons remote from the site. 9 Those carbons one bond away from a site of protonation will have their protons change chemical shift roughly 0.3-0.9 ppm upon protonation while those three bonds away will shift less than 0.05 ppm. F'olates exhibit relatively strong, noncovalent self-association in water, with association constants as low as 11 mM. 1 This is probably due to the hydrophobic and highly aromatic character of theirp-aminobenzoyl and pteridine moieties. Through a study of dependence of chemical shifts upon concentration, the change in chemical shift upon self-association can be measured. The change can be related to a molecular geometry of M. Poe and K. Hoogsteen,J. Biol. Chem. 253, 543 (1978). 8 M. Poe, O. D. Hensens, and K. Hoogsteen,J. Biol. Chem. (in press). 9 M. Poe, J. Biol. Chem. 252, 3724(1977).

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[62]

the self-associated form by theoretical calculations. It has been proposed 1 that folates exist in a ~'stretched-out" configuration in solution with the pteridine and p-aminobenzoyl rings roughly coplanar, both in the monomeric and self-associated form. As mentioned in the preceding section, analysis of the spin-spin splittings for the protons on the tetrahydropyrazine ring of tetrahydrofolates can provide information about the conformation of the ring, provided assignments can be made.

[62] A s s a y o f U n c o n j u g a t e d Fluids and Tissues

P t e r i d i n e s in B i o l o g i c a l with Crithidia I

By HERMAN BAKER, OSCAR FRANK, ANNA SHAPIRO,

and S. H. HUTNER A dietary requirement of the trypanosomatid insect parasite Crithidia fasciculata was met by unphysiologically high concentrations of folic acid or, potently, by a virtually folate-free liver fraction. 2 The factor was an acid-stable, new, unconjugated pteridine, biopterin [2-amino-6(L-erythro-l',2'-dihydroxypropyl)pterin] a (Fig. 1). The active unconjugated pteridines are biopterin, di- and tetrahydrobiopterin products, and, to a much lesser extent, L-neopterin and several congeners. 4 All Trypanosomatidae ("hemoflagellates") have the requirement, 5 and so does a Para m e c i u m , n Crithidia assays were detailed by Dewey and Kidder 7 and Guttman. s Biopterin is a cofactor for the formation of tyrosine from phenylalanine, for hydroxylations leading from tyrosine to dopa and from tryptophan to serotonin and melatonin, and quite likely for as yet unelucidated 1 Aided in part by grants from Hoffmann La-Roche, Inc., Nutley, New Jersey and Takeda Chemical Industries, Ltd., Osaka, Japan (to H.B.); AM15137 (to S.H.H.); and Public Health Service (N.I.H.) grant GRSFR 05586 (to F. S. Cooper, Haskins Labs.). 2 j. Cowperthwaite, M. M. Weber, L. Packer, and S. H. Hutner, Ann. N.Y. Acad. Sci. 56, 972 (1953); H. A. Nathan and J. Cowperthwaite, J. Protozool. 2, 37 (1955). 3 E. L. Patterson, H. P. Broquist, A. M. Albrecht, M. H. V. Saltza, and E. L. R. Stokstad, J. Am. Chem. Soc. 77, 3167 (1955). 4 H. Rembold and W. L. Gyure, Angew. Chem., Int. Ed. Engl. 11, 1061 (1972). 5 S. H. Hutner, H. Baker, O. Frank, and D. Cox, in "Biology of Nutrition" (R. N. Fienes, ed.), p. 85. Pergamon, Oxford, 1972. A. T. Soldo and G. A. Godoy, Biochim. Biophys. Acta 362, 521 (1974). 7 V. C. Dewey and G. W. Kidder, this series, Vol. 18 [174]. a H. N. Guttman, Anal. Microbiol. 2, 457 (1972).

METHODS IN ENZYMOLOGY. VOL. 66

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3