THE Q-D-CONFIGURATION OF THE RIBOSYL NICOTINAMIDE PORTION OF DIPHOSPHOPYRIDINE NUCLEOTIDE

Transcription

1 THE Q-D-CONFIGURATION OF THE RIBOSYL NICOTINAMIDE PORTION OF DIPHOSPHOPYRIDINE NUCLEOTIDE R. U. LEMIEUX AND J. W. LOWN~ Department of Chemistry, University of Alberta, Edmonton, Alberta Received October 9, 1962 ABSTRACT The configurations of the anomeric N-(tetra-O-acetyl-~-glucopyranos~I)-3-carboxamidopyridium bromides were established by nuclear magnetic resonance (n.m.r.) spectroscopy. The p-d-anomer was found to undergo deacetylation without anomerization. This compound on oxidation with periodate has been shown (M. Viscontini and E. Hiirzeler-Jucker. Helv. Chim. Acta, 39, 1620 (1956)) to yield a different product than did a synthetic N-(D-ribofuranosy1)-3-carboxamidopyridinium chloride. Therefore, the anomeric configuration of the latter compound must be a-d. Since its anomer was used (N. A. Hughes, G. W. Kenner, and A. Todd. J. Chem. Soc (1957)) to synthesize diphosphopyridine nucleotide (DPN), the configuration of the anomeric center in this portion of the coenzyme must be p-d. Optical rotatory dispersion curves are reported for DPN, its component nucleotides, and the abovementioned D-glucosides. Because of its great biological importance, considerable interest attaches to the structure and configuration of diphosphopyridine nucleotide. The gross structure is well known and has been confirmed by synthesis (1,2). However, the anomeric configuration of the ribosyl nicotinamide portion has remained an open question. The currently held view that the configuration is P-D rests on the following work by Todd and his coworkers (2). The anomeric N-(D-ribofuranosy1)-3-carboxamidopyridinium salts were known, the a-anomer having a specific rotation (D-line of sodium) of +4g0 (3) and the p-form -28" (1). DPN was synthesized (2) from a mixture of the a- and P-anomers of N-(D-ribofuranosy1)-3-carboxamidopyridinium chloride assumed to be present in a 1:4 ratio from arguments based on optical rotation. Since purified synthetic DPN was obtained in about 20% yield and had an active coenzyme content of 90%, it- could be concluded that it had formed from the levorotatory D-ribofuranoside which was assigned to the 0-series of anomers on the basis of Hudson's rules of isorotation (4). It was tentatively assumed to possess the 1,2-trans-configuration by analogy to the structure - optical activity correlations now well established for 0-glycofuranosides (5). However, it has been recently shown (6) that pyrimidine deoxyribofuranosides may have rotations opposite to those expected on the basis of these rules. It has also been observed2 that the tri-0-acetyl-2-deoxy-2-iodo derivatives of the fi-~-gl~~~pyran~~yl and a-d-mannopyranosyl pyridinium perchlorates have specific rotations of +157", +29S0, +604", +1270, and for the /3-D-isomer and of -4", -4", -92", -380, and -650" for the a-d-compound at wavelengths of 590,450,350,300, and 280 mp, respectively. Although these compounds are not anomers, as was found for thymidine and its a-anomer (B), the optical rotatory dispersion trends are definitely opposite to those which would be expected on the basis of Hudson's rules of isorotation. Thus, it can be anticipated that the anomers for these pyranosides, which have an absorbing group attached directly to the anomeric center, map well provide further exceptions to Hudson's rules of isorotation. Clearly, therefore, care must be exercised in the application of these empirical rules for structure - optical activity correlations. As it was pointed out by Todd et al. (2), the conclusion, 'University of Alberta Postdoctoral Fellow, Unpublished results by R. U. Lemieux and A. R. Morgan. Canadian Journal of Chemistry. Volume 41 (1963) 889 I

2 890 CANADIAN JOURNAL OF CHEMISTRY. VOL reached by Viscontini and Hiirzeler-Jucker (3) to the effect that the D-ribofuranosyl nicotinamide with rotation +49" possesses the 0-configuration, is defective in several respects and can be neglected. As will be seen below, the assignment of the a-configuration to a synthetic D-glucopyranosyl nicotinamide was erroneous and the convention proposed for the assignment of the a- and 0-designations to N-glycosides which is based on the sign of rotation of the product of oxidation by periodate should be abandoned. There exists no direct evidence for the assignment of configuration on the basis of optical rotation for N-glycosides wherein the nitrogen is quaternized. It is now well established (7, 8) that the n.m.r. signal for the anomeric hydrogen often can be used to unequivocally establish anomeric configurations, since the anomeric hydrogen is generally considerably shifted from the envelope of other C-H resonances and the spin-spin splitting can be related to dihedral bond angle by employing the Karplus relation (9). The purpose of this communication is to demonstrate such an application in the determination of the configurations of the anomeric D-gl~~~pyran~~yl derivatives of nicotinamide. Published information (2, 3) could then be used to correlate the configurations of these compounds to the natural D-ribofuranosyl nicotinamide. The N-(tetra-O-acetyl-~-glucopyranosyl)-3-carboxamidopyridinium bromides were prepared as described by Haynes and Todd (10). The product (I) which formed in preponderance could be purified simply by recrystallization. The mother liquors provided a residue the n.m.r. spectrum of which required the material to be a roughly equimolar mixture of I and its anomeric form. Repeated partition chomatography and fractional crystallization provided a pure compound (11) with the n.m.r. spectrum expected for the - anomer of I. Compound I, [a], -20.5", provided the n.m.r. spectrum in Fig. 1A. The doublet of intensity one at 6.46 p.p.m. has a spacing of 9 c.p.s. Since the anomeric proton forms part of a strongly coupled system of protons extending around the pyranose ring, one may legitimately question whether the observed spacing reflects the true coupling constant between the protons at the 1- and 2-positions. In fact, such a spacing is influenced by "second order" effects (11, 12) so that, for example, if the protons at the 1,2- and 3- positions form a strongly coupled set, the spacing of the proton is dependent upon the ratio of the chemical shift between the 2- and 3-protons and their coupling constant. The spacing will approximate the true coupling constant more closely the larger the ratio becomes. If this system is regarded as an AMX then the theory of the system (13) states that the separation of the centers of gravity of the two main groups of lines comprising the X branch corresponds to JAX. Therefore, an estimate of the centers of gravity of the two branches of the doublet for the anomeric center may be safely regarded as a minimum value of J1,z. This is an important consideration when doublet spacings are used to decide anomeric configuration. In the case of compound I, it is clear that Jl-2 is approximately 9 c.p.s. Since the compound has a six-membered ring, this value requires the 1- and 2-hydrogens to define a dihedral angle of about 180" (9). Therefore, the compound possesses the 0-D-configuration as shown in Fig. 1A. Compound 11, [a], +22.1, provided the n.m.r. spectrum shown in Fig. 1B. The doublet of intensity one at 7.06 p.p.m. can be assigned to the anomeric hydrogen. The spacing of 3 c.p.s. requires a dihedral angle of about 60" and the substance therefore possesses the a-d-gluco-configuration. Compounds I and I1 were deacetylated to afford the 0- and a-anomers (I11 and IV, respectively) for N-(D-glucopyranosy1)-3-carboxamidopyridinium bromide. The n.m.r. spectra for these compounds are in obvious agreement with the structures shown in Fig. 2.

3 LEMIEUX AND LOWN: CONFIGURATION I I I I I I I I I FIG. 1. Nuclear magnetic resonance spectra measured in deuterium oxide and the chemical shifts expressed in p.p.m. from the signal for tetramethylsilane as external reference. A. N-(Tetra-0-acetyl-8-Dglucopyranosy1)-3-carboxamidopyridinium bromide (I). B. N-(Tetra-0-acetyl-a-D-glucopyranosy1)-3- carboxamidopyridinium bromide (11). Thus, the deacetylations proceeded without complication and the configuration of the products are those shown in Fig. 2. It is well established (7) that, for anomeric structures, barring the presence of unusually large screening effects, the axial anomeric proton resonates at higher field than does the equatorial proton. The greater shielding of the axial proton most likely arises from the diamagnetic anisotropy of C-C and C-0 bonds in gauche relationship (7, 14). The above configurational assignments are consistent with this finding. The n.m.r. spectra of DPN and DPNH are shown in Fig. 3. One of the doublets of intensity one at about 6 p.p.m. in the spectrum for DPN can be assigned to the anomeric proton of the ribose residue which is bonded to the pyridine base since one of these signals disappeared on reduction to DPNH. The anomeric configuration cannot be assigned directly from the observed spacing (4 or 5 c.p.s.) since coupling constants of this

4 892 CANADIAN JOURNAL OF CHEMISTRY. VOL. 41, 1963 I I I I I I I I I % DO DO H E Dse ONH, I ' I DOH: '- FIG. 2. Nuclear magnetic resonance spectra measured in deuterium oxide and the chemical shifts expressed in p.p.m. from the signal for tetramethylsilane as external reference. A. N-(B-D-Glucopyranosyl)-3- carboxamidopyridinium bromide (111). B. N-(a-D-Glucopyranosy1)-3-carboxamidoppridinium bromide (IV). magnitude can arise from structures wherein the 1- and 2-hydrogens define dihedral angles of either about 40" or 140". The 40' angle is readily possible for the hydrogens in either cis or trans relationships on a flexible five-membered ring (15). In fact, for cisneighboring hydrogens, coupling constants in the range JoO = 8 to J46' = 3 C.P.S. can be reasonably expected. IThen the configurational relationship is trans, the coupling constants can fall in the range J16e0 S 9 to J740 S 0.2 C.P.S. Thus, a direct assignment of configuration can only be made when the coupling constant is very small (in view of the evidence (16) that the Karplus curve is subject to vertical displacements of at least 2 c.p.s., only values less than 1 c.p.s. should be taken as indicative of the trans configuration (17)). These considerations would of course not apply should it be possible to predict with reasonable certainty the conformations of anomeric furanosides. Viscontini and Hiirzeler-Jucker (3) oxidized the compound now known to be I11 with periodic acid to form a dialdehyde product with a specific rotation of +67". We have confirmed this result. The periodic acid oxidation of a N-(D-ribofuranosy1)-3-carboxamidopyridinium chloride preparation with rotation of +4g0 provided (3) a dialdehyde with a specific rotation of -70". Since both dialdehydes were derived from D-sugars, the diastereoisomeric compounds differ only in the configuration of the anomeric center. Since

5 LEMIEUX AND LOWN: CONFIGURATION FIG. 3. Nuclear magnetic resonance spectra measured in 1:l deuterium oxide - pyridine mixture and the chemical shifts expressed in p.p.m. from the signal for tetramethylsilane as external reference. A. Diphosphopyridine nucleotide (DPN). B. Reduced diphosphopyridine nucleotide (DPNH). compound I11 is now known definitely to have the P-D-configuration, the dextrorotatory ribonucleoside which was oxidized must have had the a-d-configuration. Therefore, the levorotatory anomer used by Hughes, Kenner, and Todd (1) to synthesize DPN must have had the P-D-configuration and this must be the configuration of the anomeric center of the ribosyl nicotinamide portion of DPN.

6 894 CANADIAN JOURNAL OF CHEMISTRY. VOL The optical rotatory dispersion curves shown in Fig. 4 show that the rotations at all wavelengths for the 0-D-glucopyranosides are less dextrorotatory than those for the Wavelength. mu,- FIG. 4. Optical rotatory dispersion curves for the D - ~ ~ U C Onicotinamides S ~ ~ I, 11, 111, and IV described in Figs. 1 and 2. a-d-anomers. The marked effect of acetylation on the shape of the curve in the case of the p-anomers is noteworthy. Also, the resemblance of the curve for the unacetylated 0-anomer to those for the a-anomers clearly demonstrates the hazard attending any attempt to establish the configuration of such a glycoside by optical rotation. The optical rotatory dispersion curves reproduced in Fig. 5 confirm the p-d-configuration for the ribosyl

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8 896 C4NADIAN JOURNAL OF CHEMISTRY. VOL. 41, 1963 EXPERIMENTAL All n.m.r. spectra were recorded on a Varian A-60 analytical spectrometer operating at a fixed radio frequency of 60 Mc/sec. The spectra were measured on approximately 20% (w/v) solutions in an appropriate solvent using tetramethylsilane as a standard. The optical rotatory dispersion curves were measured on a Rudolph automatic recording spectropolarimeter, Model 260/655/ , using about M aqueous solutions at room temperature. The nucleotides reported in Figs. 3 and 5 were purchased from Pabst Laboratories, Milwaukee, Wisconsin. Tetra-0-acetyl-p-~-glucopyranosyl-S-carboxamidopyridinzum Bromide (I) and its a-anomer (IT) A solution of 15 g of freshly recrystallized tetra-0-acetyl-a-d-glucopyranosyl bromide and 5 g of nicotinamide in 70 ml of dry acetonitrile was heated on the steam bath for 4 hours under conditions similar to those employed by Haynes and Todd (10). The crystalline precipitate, 12 g (60% yield), was purified by recrystallization from methanol. The pure substance (I) melted with decomposition at ' and possessed a [aid24 = -20.5' (c, 2.09 in water). The values reported (10) for these physical constants are m.p ' (decamp.) and [a]nl9 = -18.3". The n.m.r. spectrum is reported in Fig. 1A and the optical rotatory dispersion in Fig. 4. The filtrate was evaporated to dryness under reduced pressure and the residual dark oil taken up in 30 ml of chloroform. After filtration, 150 ml of anhydrous ether was added. The resulting white precipitate was washed with ether and dried in vacuo. The n.m.r. spectrum showed two doublets, of approximately equal intensity, at 2.94 and 3.54 s assignable to anomeric protons. The material was therefore subjected to partition chromatography (18) on Celite with n-butanol- diethyl ether (3:2) saturated with water as eluant. The fraction of the eluant which contained the a-nucleoside was identified by n.m.r. and was purified by several recrystallizations from a mixture of isopropanol and acetonitrile. The white crystalline powder (11), m.p ' and [ a ] = ~ $22.1 ~ ~ (c, 7 in water), possessed the n.m.r. spectrum reported in Fig. IB, which showed it to be free of the p-anomer. The optical rotatory dispersion is shown in Fig. 4. Haynes and Todd (10) have reported an amorphous preparation of I1 with a specific rotation of $20.9" in water. N-(6-D-Glucopyranosy1)-3-carboxamidopyridiim Bromide (111) The acetylated glucoside (I), 2.03 g, was dissolved in 200 ml of a 3% solution of hydrobromic acid and the solution was kept at 45O for 20 hours. The solution was evaporated to dryness and the residual oil was treated with 1 ml each of isopropanol and acetonitrile and set aside at 0'. After 3 weeks, crystallization occurred and the solid was purified by recrystallization from a 1:1 mixture of isopropanol and ethanol. The yield was g (39y0) of material (111) with the physical constants, m.p ', [@ID2' $29.4 (c, 3.1 in water), in good agreement with the literature values (19). The n.m.r. spectrum is reported in Fig. 2A and the optical rotatory dispersion in Fig. 4. A solution of 98.2 mg of the glucoside (111) in 5 ml of water was kept at 0' with 192 mg of sodium metaperiodate. After 4 hours, the rotation of the solution reached a steady value, [ol]~~' = $63.8' (c, 2.5 in water). N-(a-D-G1ucopyranosyl)-3-carboxamidopyridinum Bromide (I V) The pure crystalline acetylated a-d-glucoside (141, 164 mg, was dissolved in 10 ml of a 3Y0 solution of hydrobromic acid and kept at 45' for 24 hours. The solution was lyophilized to leave 100 mg of a dark gum which could not be induced to crystallize. The substance was dissolved in chloroform and reprecipitated as a yellow gum by the addition of anhydrous ether. The n.m.r. spectrum reproduced in Fig. 2B establishes beyond doubt the identity of the substance (IV) which gave the optical rotatory dispersion shown in Fig. 4. ACKNOWLEDGMENTS This research was supported in part by a National Research Council of Canada grant (T172) to R. U. L. The n.m.r. spectra and the optical rotatory dispersions were measured by R. N. Swindlehurst of this laboratory. REFERENCES 1. N. A. HUGHES, G. W. KENNER, and A. R. TODD. J. Chem. Soc (1957). 2. L. J. HAYNES, N. A. HUGHES, G. \Y. KENNER, and A. R. TODD. J. Chem. Soc (1957). 3. h1. VISCONTINI and E. HURZELER-JUCKER. Helv. Chim. Acta, 39, 1620 (1956). 4. C. S. HLDSON. J. Am. Chem. Soc. 31, 66 (1909) KJ~LBERG. Acta Chem. Scand. 14, 1118 (1960). E. BERNER and 0. KJ~LBERG. Acta Chem. Scand. 14, 909 (1960). 6. R. U. LEMIEUX and M. HOFFER. Can. J. Chem. 39, 111 (1961). 7. R. U. LEMIEUX, R. K. KULL~IG, H. J. BERNSTEIN, and W. G. SCHNEIDER. J. Am. Chem. Soc. 80, 6098 (1958). 8. R. U. LEMIEUX. Can. J. Chem. 39, 116 (1961).

9 LEMIEUX AND LOWN: CONFIGURATION 9. M. KARPLUS. J. Chem. Phys. 30, 11 (1959). 10. L. J. HAYNES and A. R. TODD. J. Chem. Soc. 303 (1950). 11. F. A. L. ANET. Can. J. Chem. 39, 2262 (1961). 12. J. I. MUSHER and E. J. COREY. Tetrahedron, 18, 791 (1962). 13. J. D. ROBERTS. An introduction to spin-spin splitting in high resolution nuclear magnetic resonance spectra. W. A. Benjamin, Inc., New York L. M. JACKMAN. Applications of nuclear magnetic resonance spectroscopy in organic chemistry. Pergamon Press, New York K. S. PITZER and W. E. DONATH. J. Am. Chem. Soc. 81, 3213 (1959). 16. R. U. LEMIEUX, J. D. STEVENS, and R. R. FRASER. Can. J. Chem. 40, 1955 (1962). 17. K. L. RINEHART, JR., M. HICKENS, A. D. ARGOUDELIS, W. S. CHILTON, H. E. CARTER, M. P. GEORGIADIS, C. P. SCHAFFNER, and R. T. SCHILLINGS. J. Am. Chem. Soc. 84, 3218 (1962). 18. R. U. LEMIEUX, C. T. BISHOP, and G. E. PELLETIER. Can. J. Chem. 34, 1365 (1956). 19. M. VISCONTINI, R. HOCHREUTER, and P. KARRER. Helv. Chim. Acta, 36, 1777 (1953).