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The 13C nuclear magnetic resonance spectra of some 2-, 6-, and 2,6-substituted purines
JOURNAL
OF MAGNETIC
RESONANCE
15,98-112
(1974)
The 13CNuclear Magnetic ResonanceSpectra of Some 2-, 6-, and 2,643ubstitutedPurines MARTHA Kettering-Meyer
C. THORPE, W. C. COBURN, JR., AND JOHN A. MONTGOMERY Labora:ory, Birmingham,
Southern Research Alabama 35205
Institrrte,
Received February 13, 1974 The 13C NMR spectra of purine and 26 substituted purines are presented. In most cases, proton-coupled spectra as well as the decoupled spectra were obtained. Chemical shift assignments are reported, and one-bond and long-range coupling constants are given where measurable. Substituent effects on chemical shifts were investigated and shielding constants derived and found to be approximately additive. Correlations of &-a with o,+ and of ace5 with aR were found. Tautomerism of the labile proton between N-7 and N-9 is proposed as the cause of line broadening, particularly of the lines due to C-4 and C-5, in nearly half of the substituted purines studied. INTRODUCTION
We report in this paper a study of the 13C NMR spectra of some simply substituted purines-three 2-substituted, twelve 6-substituted, and eleven 2,6-disubstituted purines. Most of these are the same compounds for which lH NMR spectra were reported earlier (174. Reviews of 13C spectra of aromatic nitrogen heterocycles, including purine, 1, have recently been given by Stothers (3) and Levy and Nelson (4). The earliest work on
purine itself was reported by Pugmire et al. (5). By means of deuterated compounds they were able to assign the chemical shifts of carbons 2, 6, and 8. The peak at highest field was assigned to C-5 on the basis that it is theoretically predicted to have the highest rc-electron density. The remaining peak at lowest field was assigned to C-4. These same workers later compared the spectra of the purine anion, the neutral molecule, and the cation (6). They concluded that the cation is protonated at N-l and that tautomeric averaging occurs such that there is little distinction for either N-7 or N-9 as the preferred site of protonation of the anion. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
98
CARBON-1
3
SPECTRA
OF SUBSTITUTED
PURINES
99
Jones et al. (7) give a suggested assignment for adenine, based on their assignment for adenosine. In the direction of increasing field, the ordering of peaks they suggest is C-6. C-2, C-4, C-8, and C-5. This assignment is confirmed in the present work. We have been able to make unambiguous assignments of the chemical shifts for almost all of the compounds studied here and have investigated substituent additivity effects as well as relations between chemical shifts and substituent reactivity parameters. 7-H, 9-H tautomerism is observed to have a marked effect on the appearance of the spectra of substituted purines. EXPERIMENTAL
Materials. Dimethyl sulfoxide-d, (DMSO-d,) was obtained either from Merck and Co., Inc., or from Columbia Organic Chemicals Co., Inc., and was stored over molecular sieve after opening. The preparations of the substituted purines have been previously reported. Purine, 2-aminopurine, and 2-amino-6-methylpurine were purchased from Cycle Chemical. Purin-6-yltrimethylammonium chloride, 6-cyanopurine, and 6-bromopurine were purchased from Aldrich Chemical Co., Inc. 6-Iodopurine was purchased from Sigma Chemical Co. NMR measurements. All spectra were measured at 25.16 MHz in the pulsed, Fouriertransform mode on a Varian XL-loo-15 spectrometer equipped with a Digilab Model 400-2 pulser and NMR-3 data system. Solutions in DMSO-d, were as concentrated as possible and, except for a few cases in which sample was limited, were near saturation. Probe temperature was estimated to be about 35°C. Tetramethylsilane was used as internal reference. Proton-coupled spectra were obtained using gated, noise decoupling in order to achieve greater sensitivity due to the NOE. In most cases, the proton-coupled spectra were done under conditions resulting in transformed spectra of 16K data points. while decoupled spectra were done under conditions resulting in transformed spectra of either 8K or 16K data points. RESULTS
AND
DISCUSSION
The proton-decoupled spectra, in general, consist of four peaks assignable to C-2, C-4, C-6, and C-8 in the region of approximately 140-160 ppm downfield from internal TMS and a fifth peak assignable to C-5 well-separated from the other four and located near 120 ppm. This affords a ready assignment of C-5, but the ranges of the peaks for the other four carbons overlap and their assignment is thus not immediately apparent. Peaks for carbons bearing hydrogens are normally most pronounced because of the nuclear Overhauser enhancement, but carbons bearing amino groups also give rise to strong, sharp lines. Peaks corresponding to the quaternary carbons C-4 and C-5 are weaker and frequently broad. Their appearance is of help in picking out C-4 from the remaining three low-field peaks. In most cases the proton-coupled spectrum was also obtained. Hydrogen-bearing carbons can then definitely be picked out. In some cases C-8 can be assigned by comparison of the magnitude of its lJCH coupling constant with that measured from the 13C satellites in the ‘H NMR spectrum (8), since these spectra were previously assigned (1, 2). This method can also be employed for C-2 in the 6-
100
THORPE,
COBURN
AND
MONTGOMERY
substituted compounds. C-4 and C-6 can frequently be assigned on the basis of the longrange splittings observed in the coupled spectrum. In compounds having methyl-bearing functional groups, it is often possible to assign the carbon to which the group is attached from the long-range splittings from the methyl hydrogens observable in the coupled spectrum. In cases where other long-range splittings obscure these patterns, selective irradiation of the methyl protons gives a clue to the identity of the attached carbon by simplifying its appearance and causing an Overhauser enhancement. Of additional assistance in making assignments is the additive effect of substituents in the 2- and 6-positions on the chemical shifts of the ring carbons. As will be shown below, shielding constants for the different substituents were derived from the chemical shifts of the monosubstituted compounds, relative to the chemical shifts of purine, and were used to predict the chemical shifts of the 2,6-disubstituted purines. Table I shows the assignments and 13C chemical shifts of the purines studied. Purine itself is included since its chemical shifts in DMSO-d,, the solvent used for all of the compounds in Table 1, have not been previously reported. Chemical shift assignments. The decoupled spectrum of purine, 1, shows five sharp lines, the lowest and highest field ones being of about one-half the intensity of the others. These two are assigned, respectively, to C-4 and C-5, following Pugmire et al. (5). The ‘JcH values, obtained from the coupled spectrum, agree well with those measured from the 13C satellites in the lH NMR spectrum (8) and, therefore, support these latter workers’ assignment of C-2, C-6, and C-S. 2-Aminopurine, 2, also shows five sharp lines in its decoupled spectrum. C-6 and C-S are assigned on the basis of the magnitudes of their lJcH coupling constants (see the following section). C-4 and C-5 are assigned on the basis of their relatively weaker intensities and their multiple splitting by H-6 and H-8 in the coupled spectrum. The line assigned to C-2 is the strongest in the decoupled spectrum and appears as a doublet in the coupled spectrum, being split by H-6. 2-Fluoropurine, 3, shows splitting by the fluorine of all five carbons in the protondecoupled spectrum. C-2 is readily assigned on the basis of its large ‘JczF, value of -207.5 Hz. C-4 and C-5 are much less intense than the peaks of the other three carbons. They are distinguished not only by the normal upfield position of C-5 but also by their C-F coupling constants : / 3J,-,F,1= 17. I Hz and 14JCsF21 = 3.7 Hz. C-6 and C-8 are assigned from the magnitudes of their lJCH values and from the small value of /‘JcsF2/ = I .8 Hz (13JCgF21= 15.9 Hz). Both C-4 and C-5 are very weak and broad in the spectrum of 2-chloropurine, 4, and are assigned on this basis. C-6 and C-S are assigned from the magnitudes of their ‘JCH values. C, is the sharpest peak in the decoupled spectrum and gives rise to a sharp doublet in the coupled spectrum with 13J,2H6/= 14.0 Hz. All of the peaks in the decoupled spectrum of 6-methylpurine, 5, are sharp. C-4 and C-6 are readily assigned from the coupled spectrum, where C-4 is a doublet of doublets from three-bond couplings with H-2 and H-S, and C-6 is a doublet of quartets from couplings with H-2 and the methyl hydrogens. C-5 is an unresolved multiplet in the coupled spectrum from couplings with H-S and the methyl group, but selective irradiation of the methyl hydrogens gives sharp doublets for both C-5 and C-6, showing residual splitting, respectively, from H-8 and H-2. C-2 and C-8 were distinguished in
-H -NH2 -F -Cl -H -H -H -H -H -H -H -H -H -H -H -H -SMe -NH2 -Cl -Me -NH* -SMe -NH2 -Cl -F -Cl -Et
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25f 26 27
Substituent
-H -H -H -H -Me -0Me -SMe -NH2 -NHMe -NMe* -NEt, -Cl -Br -I -CN -NMe: -SMe -NH, -Cl -NH, -Me -NH, -SMe -0Me -NH, -NH, -Cl
6 152.10 + 0.02 160.59 + 0.02 158.31 + 0.02 152.69 f 0.02 151.76 + 0.01 151.30 f 0.01 151.57 f 0.02 152.37 + 0.01 152.42 + 0.01 151.76 k 0.02 151.91 f 0.01 151.50 f 0.01 151.52 + 0.02 151.72 5 0.02 152.20 + 0.01 150.33 +_ 0.01” 163.75 + 0.01 160.20 + 0.01 150.98 f 0.01 160.72 + 0.01 160.11 + 0.02 163.89 + 0.02 159.65 + 0.01 151.13 + 0.01 158.77 + 0.01 152.78 + 0.02 165.01 & 0.02
k-2
AND
“C
TABLE
154.77 155.11 158.22 157.68 153.87 155.06 150.21 151.30 150.0 151.23 151.08 154.16 152.98 150.02 154.97 151.62 151.83 152.77 156.23 151.8 154.34 152.20 151.65 156.95 153.36 152.78 155.11
+ 0.02 + 0.02 * 0.02 f 0.08 + 0.01 + 0.01 +_ 0.02 + 0.01 +0.1 + 0.02 * 0.01 + 0.01 + 0.02 f. 0.02’ * 0.01 f 0.01’ f 0.08 f 0.01 f 0.01 kO.1 + 0.02 + 0.02 + 0.01 _+ 0.01 +_ 0.01 + 0.02 + 0.02
h‘c-4
CHEMICAL
1 PURINES
L-5 145.50 147.69 147.25 146.91 155.67 159.26 158.70 155.30 154.72 154.29 153.07 147.78 140.12 136.53 127.80 156.59 159.78 155.78 148.10 154.94 157.25 154.92 159.20 159.57 156.81 155.94 147.59
TMS) * 0.02 + 0.02 + 0.02 f 0.02 ?c 0.01 If: 0.01 +_ 0.02 + 0.01 ? 0.02 f 0.02 + 0.01 : 0.01 + 0.02 + 0.02’ f 0.01’ f 0.01’ * 0.08 + 0.01 f 0.01 + 0.05 f 0.02 + 0.02 & 0.05 + 0.01 z!z 0.01 + 0.02 Y!z0.02
SC-6
Chemical shift” downfield from internal
OF SUBSTITUTED
130.46 zb 0.02 125.52 + 0.02 128.79 + 0.02 129.06 f 0.08 129.59 + 0.01 118.14 f 0.01 129.40 +_ 0.02 117.61 +r 0.01 118.2 ; 0.1 118.97 * 0.02 118.46 + 0.01 129.23 _+ 0.01 131.97 If: 0.02 120.18 f 0.02’ 133.47 !z 0.01” 137.74 * 0.01 127.86 ? 0.08 112.50 + 0.01 128.51 f 0.01 115.8 kO.1 124.40 C 0.02 115.42 + 0.02 123.97 + 0.01 116.83 + 0.01 115.53 +_ 0.01 116.20 5 0.02 127.70 rf: 0.02
(ppm
SHIFTS
146.09 141.62 147.98 147.79 144.51 142.59 143.13 139.29 138.76 137.74 137.86 146.18 145.94 145.02 149.29 147.32 141.99 135.91 147.44 138.62 140.02 138.47 138.74 143.83 140.06 140.17 145.99
+ 0.02 + 0.02 f 0.02 + 0.02 f 0.01 + 0.01 f 0.02 f 0.01 + 0.01 f 0.02 + 0.01 f 0.01 + 0.02 + 0.02 f 0.01 + 0.01’ * 0.01 Ifr 0.01 f 0.01 rt 0.05 + 0.02 + 0.02 k 0.01 + 0.01 +_ 0.01 * 0.02 + 0.02
SC-S
27.22 + 0.02 37.84 + 0.02 13.49 * O.Olb
19.48 + 0.01 53.66 & 0.01 11.30 + 0.02
6cm
+ O.OZg
12.61
0.01 0.02 0.02 0.01 0.01
+ + + f +
25.33 19.02 13.63 10.80 54.70
114.43 * O.Old 54.27 f 0.01 e
-
L116K transforms give a statistical accuracy of +O.Ol ppm while 8K give +0.02 ppm. Larger uncertainties resulted in some cases because of weak, broad peaks with a low S/N ratio. * The CH, absorption appears at 42.19 + 0.01 ppm. c These assignments are uncertain; see text. d Assigned to the cyano group carbon. “ The 2-SMe absorbs at 14.02 +_ 0.01 ppm and the 6-SMe at 11.23 + 0.01 ppm, by comparison with 6-methylthiopurine. I Run at a concn of 12 mg/0.15 ml in a 5-mm semimicro cell. Referenced approximately to external TMS; thus, although the internal shift differences are accurate to +O.Ol ppm, the chemical shifts from TMS are probably only good to +O.l ppm. g The CH, absorption appears at 31.44 & 0.02 ppm.
2
Compound No.
ASSIGNMENTS
102
THORPE,
COBURN
AND
MONTGOMERY
this case by a variety of methods. In an off-resonance decoupling experiment, the proton region was irradiated coherently 50 Hz downfield from the position of H-2 [6,,-, = X.76 ppm and 6,-, = 8.53 ppm (2)J. One of the two unassigned carbon resonances shows proportionately more residual splitting than the other and is accordingly assigned as C-X, since H-8 was farther away from the frequency of proton irradiation. This assignment of C-2 and C-8 was later confirmed by running the r3C spectrum of 6-methylpurine-2-d. In addition, the lJ,-- values agree with those measured from 13C satellites in the proton spectrum (8). The sample designated above as 6-methylpurine-2-d was an old sample which had been stored in a desiccator for several years. Although originally substituted at the 2-position, deuterium exchange had evidently taken place during this period. Its r3C spectrum shows the presence of some deuterium at C-8 and in the methyl group. C-2 of the 2-D compound is 0.34 ppm upfield from C-2 of the 2-H compound, C-8 of the 8-D compound is 0.24 ppm upfield from the 8-H compound, and the CH,D carbon resonance is 0.29 ppm upfield from the CH, carbon resonance. The decoupled spectrum of 6-methoxypurine, 6, consists, in the low-field region, of two strong, sharp lines and three weak lines, two of which are broad. The coupled spectrum proves the strong lines to correspond to C-2 and C-X, and they are assigned on the basis of the magnitudes of their ‘Jcu values. The other three resonances are weak, broad. and unresolved. The most upfield of these is, as usual, assigned to C-5. In a separate experiment the methyl hydrogens were selectively decoupled, resulting in the lowest-field resonance appearing as a sharp doublet. This is assigned to C-6, with a residual coupling to H-2. The remaining resonance is assigned to C-4. Both C-4 and C-5 are sharpened somewhat, but they remain weak and unresolved. All five purine carbon resonances of 6-methylthiopurine, 7, are comparatively sharp in its decoupled spectrum. The coupled spectrum allows assignment of C-2 and C-X from the lJ,-- values. C-5 appears as a slightly broadened doublet, split by H-8 and probably by a small coupling with the methyl protons. C-4 is a doublet of doublets, split by H-2 and H-8, while C-6 is an incompletely resolved multiplet, split by H-2 and by the methyl protons. The decoupled spectrum of adenine, 8, shows three sharp lines and two weak, moderately broad ones. These latter two are assigned accordingly to C-4 and C-5. The coupled spectrum allows the assignment of C-2 and C-X on the basis of their ‘JcH values. C-6 appears as a doublet with an 11.6-Hz coupling constant, j3JCgH2/.This assignment confirms the proposed assignment for adenine made by Jones et al. (7). The appearance of the decoupled spectrum of 6-methylaminopurine, 9, is almost identical with that of adenine in the ring-carbon region, and the chemical shifts are very similar. The assignment is thus based on that made for adenine. No coupled spectrum was obtained in this case. 6-Dimethylaminopurine, 10, also has a similar appearance to adenine in its decoupled spectrum, although the peaks assigned to C-4 and C-5 are somewhat sharper. The coupled spectrum yields lJcH values for C-2 and C-8 which agree well with those obtained from their “C satellites in the lH NMR spectrum (8). C-4 appears as a doublet of doublets from coupling to H-2 and H-8, C-5 as a doublet from coupling to H-X, and C-6 is broad and unresolved from coupling to H-2 and the methyl protons. 6-Diethylaminopurine, 11, again has a similar appearance to adenine in its decoupled spectrum, except that C-4 and C-5 are now sharp. C-2 and C-8 are again assigned
CARBON-
13
SPECTRA
OF SUBSTITUTED
PURINES
103
on the basis of the magnitudes of their ‘JCHvalues in the coupled spectrum. There. also, C-5 is a doublet, C-4 is a doublet of doublets, and C-6 an incompletely resolved multiplet, and they are assigned as in 6-dimethylaminopurine. In contrast to 2-chloropurine, all five lines in the decoupled spectrum of 6-chloropurine, 12, are sharp. The coupled spectrum allows assignment of C-2 and C-8 from their typical ‘JcH values. The lowest-field resonance is a doublet of doublets and hence must be C-4; C-5 and C-6 are doublets, coupling, respectively, with H-8 and H-2. The appearance of the decoupled spectrum of 6-bromopurine, 13, is very much like that of 6-chloropurine, except that C-6, being more shielded by the bromine, is shifted upfield by almost 8 ppm. It shows the same multiplicities in the coupled spectrum and is assigned accordingly. The decoupled spectrum of 6-iodopurine, 14, shows five moderately sharp peaks, two strong and three weak. The strong peaks are assigned to C-2 and C-8 on the basis of their lJCH values in the coupled spectrum. However, the other three resonances appear as weak, broad, unresolved peaks in the coupled spectrum, so no coupling information is available to aid in their assignment. The assignment given in Table I is, therefore. only a suggested one. The assignment given in Table 1 for 6-cyanopurine, 15, is again an uncertain one. The decoupled spectrum gives six sharp lines. The most upfield of these is assigned to the cyano-group carbon since it remains a sharp singlet in the coupled spectrum. C-2 and C-8 are assigned from the magnitudes of their lJCH values. The lowest-field resonance is a doublet of doublets and thus assigned as C-4. The remaining two absorptions in the coupled spectrum are doublets, as expected for C-5 and C-6, but since they are both upfield in the normal range for C-5, it is not certain which is which. We assign the lower field resonance of this pair to C-5, since it gives a better fit to a correlation of the chemical shift of C-5 with Taft’s cR (9), to be discussed below. Purin-6-yltrimethylammonium chloride, 16, decomposed in DMSO-d, at probe temperature to 6-dimethylaminopurine. The chemical shifts of its five ring carbons are measurable from the decoupled spectrum, but only C-2 and C-8 can be measured in the coupled spectrum. These two cannot be distinguished because their lJCHvalues are too close in magnitude. A tentative assignment is given in Table 1. A correlation of the chemical shift of C-8 with Brown and Okamoto’s a,+ (ZO),to bediscussedbelow,predicts a value of 148.1 ppm, in good agreement with the assignment proposed here. The decoupled spectrum of 2,6-bis(methylthio)purine, 17, shows two sharp lines and three weak, broad peaks in the ring-carbon region. The coupled spectrum shows the upfield, sharp line to be C-8, since it shows a ‘J CHsplitting. The other sharp line, at lowest field, is split into a quartet with 13J,--I = 4.6 Hz; therefore, this resonance must be associated with either C-2 or C-6, splitting with the protons of a methyl group. C-5 is assigned to the highest field resonance on the basis of its chemical shift. C-2, C-4, and C-6 are assigned on the basis of substituent additivity relations, to be discussed below. 2,6-Diaminopurine, 18, gives five sharp lines in its decoupled spectrum. C-4, C-5. and C-8 are readily assigned as they are doublets in the coupled spectrum. C-2 and C-6 remain singlets and are assigned on the basis of substituent addivitity relations. 2,6-Dichloropurine, 19, also gives five moderately sharp lines in its decoupled spectrum. Its multiplicities in the coupled spectrum are the same as those of 2,6-diaminopurine, and it is assigned in the same manner.
104
THORPE.
COBURN
AND
MONTGOMERY
6-Amino-2-methylpurine, 20, was not very soluble in DMSO-d,. It was run as a saturated solution at a concentration somewhat below 2 wt %. Consequently, only the methyl absorption is observable in the coupled spectrum. C-8, however, can be assigned on the basis of its position and relative intensity in the decoupled spectrum. C-4 and C-5 are also assigned on the basis of their positions and the fact that they appear as very weak, broad absorptions. In a separate experiment, the methyl group protons were selectively irradiated. This spectrum allows assignment of C-2 and C-6 from the relative enhancement of C-2. In the decoupled spectrum of 2-amino-6-methylpurine, 21, only C-5 is weak and broad. In the coupled spectrum, C-4, C-5, and C-6 are unresolved multiplets, presumably because of splitting with the methyl-group protons. However, C-6 appears as an enhanced singlet when the methyl-group protons are selectively irradiated, allowing its certain assignment and distinguishing it from C-4. The decoupled spectrum of 6-amino-2-methylthiopurine, 22, shows three strong, sharp ring-carbon lines and two that are weak and broadened. That these latter two are C-4 and C-5 is borne out by the coupled spectrum in which they remain weak, broad, and unresolved; C-2 is resolved into a quartet from coupling to the methyl protons, C-6 remains a sharp singlet, and C-8 shows its normal lJcH splitting. The decoupled spectrum of 2-amino-6-methylthiopurine, 23, consists of two strong, sharp peaks and three that are weak and broad. The coupled spectrum allows assignment of C-4, C-5, and C-8. Selective irradiation of the methyl protons results in enhancement of C-6 with respect to the other peaks, distinguishing it from C-2 and C-4. All of the peaks are sharp in the decoupled spectrum of 2-chloro-6-methoxypurine, 24. It appears from the coupled spectrum that H-8 is coupling with all five ring carbons: C-2, C-4, C-5, and C-8 are doublets, while C-6 is probably a doublet of quartets from splitting with H-8 and the methyl protons, although its exact mutliplicity is obscured by noise in this spectrum. C-2 can be distinguished from C-4 by the small size of C-2’s coupling constant, 1“JcZH81= 0.6 Hz and j3JcqHsj= 8.5 Hz. As the amount of 6-amino-2-fluoropurine, 25, available was limited to 12 mg, no coupled spectrum was obtained, and the decoupled one has a low signal-to-noise ratio. C-8 and C-5 show no measurable splitting by the fluorine, and C-5 is so weak and broad as to obscure any. C-2 is readily assigned on the basis of its ‘JcZF, value of -202.0 Hz. C-4 and C-6 both show three-bond splittings by the fluorine, respectively, of magnitudes 9.2 and 21.4 Hz. They are assigned on the basis of substituent additivity relations. Only four moderately sharp peaks are observed in the decoupled spectrum of 6amino-2-chloropurine, 26. C-8 is assigned from the coupled spectrum and C-2 and C-6 from substituent additivity relations. The coincidence of C-4 and C-2 was demonstrated by the addition of a few drops of trifluoroacetic acid to the solution. In the subsequent spectrum all the peaks are shifted slightly, and C-2 and C-4 appear as two distinct peaks. All lines are sharp in the decoupled spectrum of 6-chloro-2-ethylpurine, 27. However, it is readily assigned on the basis of the coupled spectrum. C-4, C-5, and C-8 are doublets from coupling with H-8, C-6 remains a singlet, and C-2 is a complex multiplet from coupling with the protons of the ethyl group. The multiplicity of C-2 is not clear because some components are lost in noise in the baseline. Coupling constanrs. All of the measurable one-bond r3C-lH coupling constants are
CARBON- 13 SPECTRA OF SUBSTITUTED PURINES
105
given in Table 2. Although the ranges for C-2 and C-S overlap slightly, the values are characteristic enough to be useful in making assignments. For 11 compounds, ‘Jc,n, TABLE
2
ONE-BOND 13C-1H COUPLING Compound No. .--__ 1 2 3 4
5 6 7
8 10 11 12 13 14 15 16 17
18 19 20 21 22 23 24 26 27
Substituent 2
-H -NH2 -F -Cl -H -H -H -H -H -H -H -H -H -H -H -SMe -NH2 -Cl -Me -NH, -SMe -NH2 -Cl -Cl -Et
CONSTANTS
Coupling 6
-H -H -H -H -Me -0Me -SMe -NH, -NMe* -NEtz -Cl -Br -1 -CN -NMe,+ -SMe -NH2 -Cl -NH2 -Me -NH* -SMe -0Me -NH2 -Cl
c-2 203.2
C-6
5 0.3
184.0 179.4 188.05
202.0 203.9 204.5 197.8 197.8
197.8 208.7 209.4 208.1 209.4
212.4 ’
& 0.3 + 0.3 f 0.3 zk 0.3 a 0.3 c 0.3 + 0.3 & 0.3 f 0.3 c 0.3 k 0.3’
Other
C-8
f 0.3 * 0.3
189 f2b
constant (Hz)
0.3
210.6 5 0.3 210.0 + 0.3 211.8 kO.3 211.8 kO.3 210.0 L- 0.3 210.6 5 0.3 211.2 * 0.3 209.4 + 0.3 210.0+ 0.3 210.0 + 0.3 212.4 k 0.3 212.4 k 0.3 212.4 + 0.3 213.6& 0.3 213.6 5 0.3" 212.4 + 0.3 208.1 rl: 0.3 214.4 + 0.3 208.7 210.0 210.6 211.8 211.8 211.2
i 0.3 f 0.3 fc0.3 kO.3
+ 0.3 + 0.3
128.0 k 0.3c 147.4 + 0.3' 142.2 +0.3"
137.9 t 0.3’ d
I
126.3 127.6 140.4 141.4 148.3
& + 5 * +
0.39 0.3" 0.3' 0.3' 0.3'
h
n Although we have not determined the signs of these coupling constants, we take them a11 to be coupling (see, for example, Ref. (3)). positive since this has been established for one-bond %-iH * The uncertainty in this value of ‘JcgHg p robably arises from a small splitting of C-6 by H-8. c This value is for the methyl group. d The value for the methyl group is 126.3 + 0.3 Hz; that for the methylene group is 138.0 & 0.5 Hz (there may be a small splitting of the methylene carbon by H-2 or H-S). e Assignments are uncertain for these two resonances. S The value for the 2-SMe group is 141.0 ? 0.3 Hz; the value for the 6-SMe group is 142.2 F 0.3 Hz (this latter value also agrees with that for 6-methylthiopurine). g This value is for the methyl group. The concentration was too low to allow measurement of ‘JQ”S. h The value for the methyl group is 127.3 + 0.3 Hz; the value for the methylene group is 127.9 f 0.3 Hz. has a mean value of 203.9 + 3.8 Hz with a total range of 11.6 Hz. For four compounds ‘JC& has a mean value of 185.1 k 3.4 Hz with a total range of 10 Hz. For 23 compounds,
‘JC,H,has a mean value of 211.1 k 1.2 Hz with a total range of 6.3 Hz. That there is the
106
THORPE,
COBURN
AND
MONTGOMERY
smallest variation in ‘JcsH8is undoubtedly owing to the fact that C-8 is farthest from the two sites of substitution studied here. Table 3 gives the long-range 13C-lH coupling constants measured for the few 2substituted purines available and for purine itself. Figure 1 shows the coupled spectrum of purine and illustrates the complexity of the resonances of C-4 and C-5 owing to the TABLE LONG-RANGE
Compound Lo.
1 2 3 -l
COUPLING
3
CONSTANISOF~-SUBSTITUTED
PURINES
Coupling constant (Hz)
3JC2Hg 11.0 f 0.3 11.6+0.3 13.4 + 0.3 14.0 rt O.?
‘Jc2us
-cl?
3J~q~g 4.8 + 0.3? -5.8 b
3JC4HS 11.6 + 0.3? -5.8 b
‘Jc,,,, 6.4 + 0.3? 5.8 f 0.3? c
3JCgHS
‘+JC&
9.2 + 0.3 1 0.6 IO.3 10.7 + 0.3? c
” Since signs of coupling constants were not determined, all values in the table are absolute values. * In coupled spectrum dvI,z z 40 Hz; 13JCqH6 + 3Jc4H81 2 23 Hz. ’ 11: coupled spectrum dv l,z z 22 Hz; I’Jc,~,,+ 3JcsHsl z 18 Hz. -C-*7
FIG I. The proton-coupled
13C spectrum of purine at 25.16 MHz; concn = 120 mg/ml in DMSO-d,.
long-range coupling to the three protons. The question marks in the table indicate uncertainty as to which proton is coupling to the indicated carbon. In the case of C-5, we have assumed that the larger value should be associated with H-8, since this is a three-bond coupling while that to H-6 is a two-bond coupling; in benzene, 3J,-His the largest of the long-range couplings (4). However, in the case of C-4, we are unable to determine whether the larger coupling is with H-6 or H-8 since these are both three-bond couplings. No coupling is observable between C-8 and H-6, a four-bond coupling, although a small splitting is observed between CL6 and H-S in the case of purine, I.
CARBON-
13
SPECTRA
OF SUBSTITUTED
107
PURINES
The long-range coupling constants of the 6-substituted purines and purine are given in Table 4. Again the question marks indicate assignment ambiguities. C-4 appears as a doublet of doublets in the coupled spectra, and it is not clear whether the larger coupling is with H-2 or H-8. C-5 of purine shows splittings with all three protons; the smallest TABLE LONG-RANGE
COUPLING
1
8.1 k 0.3?
11.6+0.3?
5 7 8
7.9 + 0.3? 7.0 f 0.3?
11.6 2 0.3‘~ 11.0+0.6?
4.6 5.5 7.3 7.3
11.6+0.4? 12.2 + 0.3? 12.2 + 0.3? 12.2 + 0.3?
10 11’
12 13 15’
CONSTANTS
1.5 + 0.3‘~
4 OF 6-~~JBSTITUTED
9.2 & 0.3? >4
PURINES
10.7 + 0.3 10.2 +- 0.3
11 -t-l 12
+ 0.4? t 0.3? + 0.3? + 0.3'!
0.6 i. 0.3
rfl
11.4 + 0.4
10 +1 10.4 & 0.3 10.4 * 0.3 10.4* 0.3
14.0 + 0.3 13.4 IO.3 12.2 + 0.3
a Sincesignsof coupling constantswere not determined, all valuesin the table are absolutevalues. ,aI‘Jc61,ye= 6.7 + 0.3 Hz. c l-?I.c6,,Mej = 3.7 + 0.3 Hz. ’ For the methyl carbon splitting aith the rnethylene protons, IzJc,,/ = 2.9 i 0.3; for the methylene carbon splitting with the methyl protons, 12JcHI = 4.3 + 0.3; there also appearsto be a small splitting of the methylene carbon by H-2 or H-8 of about 1 Hz. C-6 is unresolvedwith dvliz r 25 Hz. u ‘“J<4,q2 + “Ji4,,& = 18.3 + 0.3 Hz. of these is probably with H-2 as this is a four-bond coupling, whereas that with H-6 is a two-bond coupling and that with H-8 is a three-bond coupling. No five-bond couplings of C-2 with H-8 or C-8 with H-2 are observable. Table 5 shows the observable long-range coupling constants of 2,6-disubstituted purines. No assignment ambiguities exist here, except those mentioned above for purine, since the only ring proton available is now H-8. A definite five-bond coupling constant is measurable for C-2 of 2-chloro-6-methoxypurine, 24. The measurable 13C-19F coupling constants for the two compounds having fluorine as a substituent are shown in Table 6. The values of ‘JCFin these two cases are smaller, i.e., more positive, than those for substituted fluorobenzenes (about -240 Hz), probably indicating greater s character in the C-F bond of the fluoropurines (3). In the decoupled spectrum of 2-fluoropurine, every resonance is a doublet, showing measurable splitting by the fluorine of each carbon in the molecule, including a five-bond coupling to C-8 of 1.8 Hz magnitude. Substituent effects. Shielding constants for the various substituents at C-2 and C-6 were derived from spectra of the monosubstituted purines by means of the relation : AC,, = GC,(purine) - K,(derivative), where AC, is in ppm. In three cases, 2-SMe, 2-Me, and 2-Et, shielding constants were
108
THORPE,
COBURN
AND
MONTGOMERY
TABLE 5 LONG-RANGE COUPLING CONSTANW OF 2,6-DISUBSTITUTED PURINES
Compound
no. ---.
1 17b 18 19 22' 24 27'
5JczHs
Coupling constant (Hz) 3JCq”* 3Jcsll* 11.6+0.3?
9.2 + 0.3?
6.9 + 0.4
10.4 * 0.3 9.9 * 0.4
8.5 + 0.3 6.7 + 0.3
9.2 IL-0.3 ll.OkO.3
6.1 + 0.3
0.6 k 0.3
4JC&
0.6 rt 0.3
R Since signs of coupling constants were not determined, all values in the table are absolute values. b The only long-range coupling observed was that between C-2 and the methyl protons, 13JcHl= 4.6 + 0.3 Hz; all other peaks were too broadened by tautomerism (see discussion below). c See footnote b; here j3JcHI = 4.9 + 0.3 Hz. d j4Jc6~Jz 13J~6~OMel ~55.5H z; exact multiplicity uncertain because of noise in baseline. e For the methyl carbon coupling with the methylene protons, /2JcHI = 4.9 of:0.3 Hz; for the methylene carbon coupling with the methyl protons, /*JcHl = 4.6 & 0.3. TABLE
6
13C-19F COUPLING CONSTANTS Compound No.
3 25
Substituent 2 6 -F
-H
-F
-NH2
Coupling constant (Hz)
1Jc& -207.5 5 0.3 -202.0 * 0.3
3Jc41z2
4JCgF2
17.1 + 0.6 9.2 + 0.3
3.7 + 0.6
3JC& 15.9 + 0.3 21.4 + 0.3
SJCsFZ 1.8 + 0.3
(1Since signs of coupling constants were not determined, the long-range coupling constants are absolute values. It has been established that ‘JcV values are negative (see, for example, Ref. (3)).
derived from disubstituted purines (because the appropriate were not available) by means of the relation: AC, = X,(monosubstituted
monosubstituted
purine) - E,,(disubstituted
purine).
purines PI
These shielding constants are listed in Table 7. They were found to be very useful in predicting carbon chemical shifts of disubstituted purines, as is shown by the comparison of predicted and experimental values in Table 8. We also investigated the possibility that 13C chemical shifts of substituted purines might be predictable by means of some of the reactivity parameters, as we had found to be the case earlier (I, 2) for a,-, and S,-, of 6- and 2,6-substituted purines. For bcm8and
CARBON-
3 SPECTRA
]
OF SUBSTITUTED TABLE
‘T Substituent
CONSTANTS
AC.,
2 2 2 2 2 2 6 6 6 6 6 6 6 6 6
F Cl SMe Me Et Me OMe SMe NH2 NHMe NMez NEtz Cl Br values
I FOR SUBSTITUTED
Position
NH2
n AC,
SHIELDING
-8.49 -6.21 -0.59 -11.52 -8.35 -13.51 0.34 0.80 0.53 -0.21 -0.32 0.34 0.19 0.60 0.58
P~RINES”
AC,
-0.34 -3.45 -2.91 -0.90 -0.51 -0.95 0.90 -0.29 4.56 3.41 4.8 3.54 3.69 0.61 1.79
AC,
4.94 1.67 1.40 2.19 1.83 1.53 0.87 12.32 1.06 12.85 12.3 11.49 12.00 1.23 -1.51
-2.19 -1.75 -1.41 0.38 0.36 0.19 -10.17 -13.76 -13.20 -9.80 -9.22 -8.19 -7.57 -2.28 5.38
4.47 -1.89 -1.69 0.82 0.67 0.19 1.58 3.50 2.96 6.80 7.33 8.35 8.23 -0.09 0.15
are in ppm. TABLE
COMPARISON
109
PURINES
OF OBSERVED
AND
PREDICTED
VALUES
8 OF THE Y
CHEMICAL
SHIFTS
OF DISIJBSTITUTED
PURIN&
Chemical Substituent 2 -Cl -Cl -Cl -NH2 -NH2 -NH, --F -SMe
not
fit-2 6 -NH, -Cl -0Me -Me -NH2 -SMe -NH, -SMe
&-‘l
Exp.
Calc.
Exp.
Calc.
152.78 150.98 151.13 160.11 160.20 159.65 158.77 163.75
152.96 152.09 151.89 160.25 160.86 160.06 158.58 163.09
152.78 156.23 156.95 154.34 152.77 151.65 153.36 151.83
154.21 157.07 157.97 154.21 151.64 150.55 154.75 151.11
n Data from 6-amino-2-methylthiopurine, included because they were used
shift (ppm) k-5 Exp. Calc.
116.20 128.51 116.83 124.40 112.50 123.97 115.53 127.86
116.21 127.83 116.74 124.64 112.67 124.46 115.94 127.21
6-amino-2-methylpurine, to derive shielding constants
&-Ii
L-8
Exp.
Calc.
Exp.
Calc.
155.94 148.10 159.57 157.25 155.78 159.20 156.81 159.78
156.71 149.19 160.67 157.86 157.49 160.89 157.05 158.32
140.17 147.44 143.83 140.02 135.91 138.74 140.06 141.99
140.98 147.87 144.28 140.04 134.82 138.66 141.18 142.31
and 6-chloro-2-ethylpurine from Eq. [2].
were
a,-, such relations were found, but for the carbons at other positions no simple relations are apparent. The chemical shift of C-8 is predictable from 0,’ values (10) of the substituents in a relation similar to that reported earlier (1, 2) for H-8. The equation, representing 24 points, is : &,
= (146.20 + 0.34) + (4.74 f 0.25)a& + (3.38 + 0.33)0&, Y = 0.98.
[31
110
THORPE,
COBURN
AND
MONTGOMERY
Since both a,-, and an8 correlate well with cr,+ of the substituents, a correlation also exists between them. This relation is primarily interesting because the coefficient relating Bcm8and a,-, is about half as large as those reported for some aryl and heteroaromatic systems (3). 6,-, = (50.5 + 5.0) + (11.03 k 0.60)6,-,, r = 0.97. (41 A satisfactory correlation was found for C-5 with cR (9). This equation, derived from data for 22 compounds, is: d,-, = (132.37 ) 0.50) + (19.1 f 1.4&r,; + (6.7 k l.S)a,,, r = 0.96. I51 Tuutomerism. In the proton-decoupled spectra of more than half of the substituted purines, all of the lines are sharp. In the others, varying degrees of line broadening are observed, particularly in the lines due to C-4 and C-5. This suggested to us the probability of tautomerism of the labile proton between N-7 and N-9. Krugh (II) reported the observation of 13C line broadening caused by tautomerism in the spectra of formycin A (II) and formycin B (III). These compounds each have two
HO
OH
HO
II
OH III
possible tautomeric sites, but it has been demonstrated by Chenon et al. (12) that the broadening is caused by tautomeric averaging of the proton between N-l and N-2.
C-6
c-4
c-2
C-6
2. The proton-decoupled 13C spectrum of 6-methoxypurine; Chemical shifts are in ppm downfield from internal TMS. FIG.
C-5
concn = 50 mg/ml in DMSO-d,.
CARBON-
13 SPECTRA
OF SUBSTITUTED
PURINES
111
We chose to investigate two compounds which have only one possible site of tautomerism. In these two cases, we have demonstrated that exchange of the proton between N-9 and N-7 is indeed the source of the line broadening. In the proton-decoupled spectrum of 2,6-bis(methylthio)purine at about 35”C, the lines due to C-4, C-5, and C-6 are all very broad. With the sample temperature raised to lOO’C, all the lines in the spectrum are sharp. The proton-decoupled spectrum of 6-methoxypurine, shown in Fig. 2, has broad peaks for both C, and C,. This broadening is absent in the spectrum of 6-methoxy-9-
FIG. 3. The proton-decoupled 13C spectrum of 6-methoxy-9-(B-D-ribofuranosyl)purine; concn = 150 mg/ml in DMSO-rl,. Peak assignments were made by comparison with the spectrum of 6-methoxypurine, and confirmed by the splittings observed in the proton-coupled spectrum. Chemical shifts are in ppm downfie!d from internal TMS.
(P-D-ribofuranosyl)purine, shown in Fig. 3, pulsed under the same conditions. In this case, the possibility of tautomerism was removed by the replacement of the labile proton with the sugar moiety. Thus we conclude that the degree of line broadening, particularly of the lines due to C-4 and C-5, depends on the rate of tautomerism of the labile proton between N-7 and N-9. In those cases where the lines are sharp, the rate of tautomerism is either very fast on the NMR time scale or else the proton is essentially fixed at N-7 or N-9, although the latter explanation seems less likely. When the rate is intermediate, broadening occurs. REFERENCES 1. W. C. COBURN, JR., M. C. THORPE, J. A. MONTGOMERY,AND K. HEWSON.J. Org. Chem. 30,111O (1965).
2. W. C. COBURN, JR., M. C. THORPE, J. A. MONTGOMERY,AND K. HEWSON,J. Org. Chem. 30, 111-i (1965). 3. J. B. &OTHERS, “Carbon-13 NMR Spectroscopy,” Vol. 24 of “Organic Chemistry, A Series of Monographs” (A. T. Blomquist and H. Wasserman, Eds.), Academic Press, New York, 1972. 4. G. C. LEVY AND G. L. NELSON, “Carbon-13 Nuclear Magnetic Resonance for OrganicChemists,” Wiley-Interscience, New York, 1972. 5. R. J. PUGMIRE, D. M. GRANT, R. K. ROBINS, AND G. W. RHODES,J. Amer. Chem, Sot. 87,225 (1965).
112 6. 7. 8. Y. 10. 11. 12.
THORPE,COBURN
AND MONTGOMERY
R. J. PUGMIRE AND D. M. GRANT, J. ,4mer. Chem. Sot. 93, 1880 (1971). A. J. JONES, D. M. GRANT, M. W. WINKLEY, AND R. K. ROBINS, J. Amer. Chem. Sot. 92,4079 ( 1970). Unpublished data from this laboratory. R. W. TAFT, JR., S. EHRENSON, I. C. LEWIS, AND R. E. GLICK,J. Amer. Chem. Sot. 81,532 ( 1959). H. C. BROWN AND Y. OKAMOTO, J. Amer. Chem. Sot. 80,4979 (1958). T. R. KRUGH, J. Amer. Chem. Sot. 95, 4761 (1973). M.-T. CHENON, R. J. PUGMIRE, D. M. GRANT, R. P. PANZICA, AND L. B. TOWNSEND, J. Heteroc.vcl. Chem. 10,431 (1973).
OF MAGNETIC
RESONANCE
15,98-112
(1974)
The 13CNuclear Magnetic ResonanceSpectra of Some 2-, 6-, and 2,643ubstitutedPurines MARTHA Kettering-Meyer
C. THORPE, W. C. COBURN, JR., AND JOHN A. MONTGOMERY Labora:ory, Birmingham,
Southern Research Alabama 35205
Institrrte,
Received February 13, 1974 The 13C NMR spectra of purine and 26 substituted purines are presented. In most cases, proton-coupled spectra as well as the decoupled spectra were obtained. Chemical shift assignments are reported, and one-bond and long-range coupling constants are given where measurable. Substituent effects on chemical shifts were investigated and shielding constants derived and found to be approximately additive. Correlations of &-a with o,+ and of ace5 with aR were found. Tautomerism of the labile proton between N-7 and N-9 is proposed as the cause of line broadening, particularly of the lines due to C-4 and C-5, in nearly half of the substituted purines studied. INTRODUCTION
We report in this paper a study of the 13C NMR spectra of some simply substituted purines-three 2-substituted, twelve 6-substituted, and eleven 2,6-disubstituted purines. Most of these are the same compounds for which lH NMR spectra were reported earlier (174. Reviews of 13C spectra of aromatic nitrogen heterocycles, including purine, 1, have recently been given by Stothers (3) and Levy and Nelson (4). The earliest work on
purine itself was reported by Pugmire et al. (5). By means of deuterated compounds they were able to assign the chemical shifts of carbons 2, 6, and 8. The peak at highest field was assigned to C-5 on the basis that it is theoretically predicted to have the highest rc-electron density. The remaining peak at lowest field was assigned to C-4. These same workers later compared the spectra of the purine anion, the neutral molecule, and the cation (6). They concluded that the cation is protonated at N-l and that tautomeric averaging occurs such that there is little distinction for either N-7 or N-9 as the preferred site of protonation of the anion. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
98
CARBON-1
3
SPECTRA
OF SUBSTITUTED
PURINES
99
Jones et al. (7) give a suggested assignment for adenine, based on their assignment for adenosine. In the direction of increasing field, the ordering of peaks they suggest is C-6. C-2, C-4, C-8, and C-5. This assignment is confirmed in the present work. We have been able to make unambiguous assignments of the chemical shifts for almost all of the compounds studied here and have investigated substituent additivity effects as well as relations between chemical shifts and substituent reactivity parameters. 7-H, 9-H tautomerism is observed to have a marked effect on the appearance of the spectra of substituted purines. EXPERIMENTAL
Materials. Dimethyl sulfoxide-d, (DMSO-d,) was obtained either from Merck and Co., Inc., or from Columbia Organic Chemicals Co., Inc., and was stored over molecular sieve after opening. The preparations of the substituted purines have been previously reported. Purine, 2-aminopurine, and 2-amino-6-methylpurine were purchased from Cycle Chemical. Purin-6-yltrimethylammonium chloride, 6-cyanopurine, and 6-bromopurine were purchased from Aldrich Chemical Co., Inc. 6-Iodopurine was purchased from Sigma Chemical Co. NMR measurements. All spectra were measured at 25.16 MHz in the pulsed, Fouriertransform mode on a Varian XL-loo-15 spectrometer equipped with a Digilab Model 400-2 pulser and NMR-3 data system. Solutions in DMSO-d, were as concentrated as possible and, except for a few cases in which sample was limited, were near saturation. Probe temperature was estimated to be about 35°C. Tetramethylsilane was used as internal reference. Proton-coupled spectra were obtained using gated, noise decoupling in order to achieve greater sensitivity due to the NOE. In most cases, the proton-coupled spectra were done under conditions resulting in transformed spectra of 16K data points. while decoupled spectra were done under conditions resulting in transformed spectra of either 8K or 16K data points. RESULTS
AND
DISCUSSION
The proton-decoupled spectra, in general, consist of four peaks assignable to C-2, C-4, C-6, and C-8 in the region of approximately 140-160 ppm downfield from internal TMS and a fifth peak assignable to C-5 well-separated from the other four and located near 120 ppm. This affords a ready assignment of C-5, but the ranges of the peaks for the other four carbons overlap and their assignment is thus not immediately apparent. Peaks for carbons bearing hydrogens are normally most pronounced because of the nuclear Overhauser enhancement, but carbons bearing amino groups also give rise to strong, sharp lines. Peaks corresponding to the quaternary carbons C-4 and C-5 are weaker and frequently broad. Their appearance is of help in picking out C-4 from the remaining three low-field peaks. In most cases the proton-coupled spectrum was also obtained. Hydrogen-bearing carbons can then definitely be picked out. In some cases C-8 can be assigned by comparison of the magnitude of its lJCH coupling constant with that measured from the 13C satellites in the ‘H NMR spectrum (8), since these spectra were previously assigned (1, 2). This method can also be employed for C-2 in the 6-
100
THORPE,
COBURN
AND
MONTGOMERY
substituted compounds. C-4 and C-6 can frequently be assigned on the basis of the longrange splittings observed in the coupled spectrum. In compounds having methyl-bearing functional groups, it is often possible to assign the carbon to which the group is attached from the long-range splittings from the methyl hydrogens observable in the coupled spectrum. In cases where other long-range splittings obscure these patterns, selective irradiation of the methyl protons gives a clue to the identity of the attached carbon by simplifying its appearance and causing an Overhauser enhancement. Of additional assistance in making assignments is the additive effect of substituents in the 2- and 6-positions on the chemical shifts of the ring carbons. As will be shown below, shielding constants for the different substituents were derived from the chemical shifts of the monosubstituted compounds, relative to the chemical shifts of purine, and were used to predict the chemical shifts of the 2,6-disubstituted purines. Table I shows the assignments and 13C chemical shifts of the purines studied. Purine itself is included since its chemical shifts in DMSO-d,, the solvent used for all of the compounds in Table 1, have not been previously reported. Chemical shift assignments. The decoupled spectrum of purine, 1, shows five sharp lines, the lowest and highest field ones being of about one-half the intensity of the others. These two are assigned, respectively, to C-4 and C-5, following Pugmire et al. (5). The ‘JcH values, obtained from the coupled spectrum, agree well with those measured from the 13C satellites in the lH NMR spectrum (8) and, therefore, support these latter workers’ assignment of C-2, C-6, and C-S. 2-Aminopurine, 2, also shows five sharp lines in its decoupled spectrum. C-6 and C-S are assigned on the basis of the magnitudes of their lJcH coupling constants (see the following section). C-4 and C-5 are assigned on the basis of their relatively weaker intensities and their multiple splitting by H-6 and H-8 in the coupled spectrum. The line assigned to C-2 is the strongest in the decoupled spectrum and appears as a doublet in the coupled spectrum, being split by H-6. 2-Fluoropurine, 3, shows splitting by the fluorine of all five carbons in the protondecoupled spectrum. C-2 is readily assigned on the basis of its large ‘JczF, value of -207.5 Hz. C-4 and C-5 are much less intense than the peaks of the other three carbons. They are distinguished not only by the normal upfield position of C-5 but also by their C-F coupling constants : / 3J,-,F,1= 17. I Hz and 14JCsF21 = 3.7 Hz. C-6 and C-8 are assigned from the magnitudes of their lJCH values and from the small value of /‘JcsF2/ = I .8 Hz (13JCgF21= 15.9 Hz). Both C-4 and C-5 are very weak and broad in the spectrum of 2-chloropurine, 4, and are assigned on this basis. C-6 and C-S are assigned from the magnitudes of their ‘JCH values. C, is the sharpest peak in the decoupled spectrum and gives rise to a sharp doublet in the coupled spectrum with 13J,2H6/= 14.0 Hz. All of the peaks in the decoupled spectrum of 6-methylpurine, 5, are sharp. C-4 and C-6 are readily assigned from the coupled spectrum, where C-4 is a doublet of doublets from three-bond couplings with H-2 and H-S, and C-6 is a doublet of quartets from couplings with H-2 and the methyl hydrogens. C-5 is an unresolved multiplet in the coupled spectrum from couplings with H-S and the methyl group, but selective irradiation of the methyl hydrogens gives sharp doublets for both C-5 and C-6, showing residual splitting, respectively, from H-8 and H-2. C-2 and C-8 were distinguished in
-H -NH2 -F -Cl -H -H -H -H -H -H -H -H -H -H -H -H -SMe -NH2 -Cl -Me -NH* -SMe -NH2 -Cl -F -Cl -Et
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25f 26 27
Substituent
-H -H -H -H -Me -0Me -SMe -NH2 -NHMe -NMe* -NEt, -Cl -Br -I -CN -NMe: -SMe -NH, -Cl -NH, -Me -NH, -SMe -0Me -NH, -NH, -Cl
6 152.10 + 0.02 160.59 + 0.02 158.31 + 0.02 152.69 f 0.02 151.76 + 0.01 151.30 f 0.01 151.57 f 0.02 152.37 + 0.01 152.42 + 0.01 151.76 k 0.02 151.91 f 0.01 151.50 f 0.01 151.52 + 0.02 151.72 5 0.02 152.20 + 0.01 150.33 +_ 0.01” 163.75 + 0.01 160.20 + 0.01 150.98 f 0.01 160.72 + 0.01 160.11 + 0.02 163.89 + 0.02 159.65 + 0.01 151.13 + 0.01 158.77 + 0.01 152.78 + 0.02 165.01 & 0.02
k-2
AND
“C
TABLE
154.77 155.11 158.22 157.68 153.87 155.06 150.21 151.30 150.0 151.23 151.08 154.16 152.98 150.02 154.97 151.62 151.83 152.77 156.23 151.8 154.34 152.20 151.65 156.95 153.36 152.78 155.11
+ 0.02 + 0.02 * 0.02 f 0.08 + 0.01 + 0.01 +_ 0.02 + 0.01 +0.1 + 0.02 * 0.01 + 0.01 + 0.02 f. 0.02’ * 0.01 f 0.01’ f 0.08 f 0.01 f 0.01 kO.1 + 0.02 + 0.02 + 0.01 _+ 0.01 +_ 0.01 + 0.02 + 0.02
h‘c-4
CHEMICAL
1 PURINES
L-5 145.50 147.69 147.25 146.91 155.67 159.26 158.70 155.30 154.72 154.29 153.07 147.78 140.12 136.53 127.80 156.59 159.78 155.78 148.10 154.94 157.25 154.92 159.20 159.57 156.81 155.94 147.59
TMS) * 0.02 + 0.02 + 0.02 f 0.02 ?c 0.01 If: 0.01 +_ 0.02 + 0.01 ? 0.02 f 0.02 + 0.01 : 0.01 + 0.02 + 0.02’ f 0.01’ f 0.01’ * 0.08 + 0.01 f 0.01 + 0.05 f 0.02 + 0.02 & 0.05 + 0.01 z!z 0.01 + 0.02 Y!z0.02
SC-6
Chemical shift” downfield from internal
OF SUBSTITUTED
130.46 zb 0.02 125.52 + 0.02 128.79 + 0.02 129.06 f 0.08 129.59 + 0.01 118.14 f 0.01 129.40 +_ 0.02 117.61 +r 0.01 118.2 ; 0.1 118.97 * 0.02 118.46 + 0.01 129.23 _+ 0.01 131.97 If: 0.02 120.18 f 0.02’ 133.47 !z 0.01” 137.74 * 0.01 127.86 ? 0.08 112.50 + 0.01 128.51 f 0.01 115.8 kO.1 124.40 C 0.02 115.42 + 0.02 123.97 + 0.01 116.83 + 0.01 115.53 +_ 0.01 116.20 5 0.02 127.70 rf: 0.02
(ppm
SHIFTS
146.09 141.62 147.98 147.79 144.51 142.59 143.13 139.29 138.76 137.74 137.86 146.18 145.94 145.02 149.29 147.32 141.99 135.91 147.44 138.62 140.02 138.47 138.74 143.83 140.06 140.17 145.99
+ 0.02 + 0.02 f 0.02 + 0.02 f 0.01 + 0.01 f 0.02 f 0.01 + 0.01 f 0.02 + 0.01 f 0.01 + 0.02 + 0.02 f 0.01 + 0.01’ * 0.01 Ifr 0.01 f 0.01 rt 0.05 + 0.02 + 0.02 k 0.01 + 0.01 +_ 0.01 * 0.02 + 0.02
SC-S
27.22 + 0.02 37.84 + 0.02 13.49 * O.Olb
19.48 + 0.01 53.66 & 0.01 11.30 + 0.02
6cm
+ O.OZg
12.61
0.01 0.02 0.02 0.01 0.01
+ + + f +
25.33 19.02 13.63 10.80 54.70
114.43 * O.Old 54.27 f 0.01 e
-
L116K transforms give a statistical accuracy of +O.Ol ppm while 8K give +0.02 ppm. Larger uncertainties resulted in some cases because of weak, broad peaks with a low S/N ratio. * The CH, absorption appears at 42.19 + 0.01 ppm. c These assignments are uncertain; see text. d Assigned to the cyano group carbon. “ The 2-SMe absorbs at 14.02 +_ 0.01 ppm and the 6-SMe at 11.23 + 0.01 ppm, by comparison with 6-methylthiopurine. I Run at a concn of 12 mg/0.15 ml in a 5-mm semimicro cell. Referenced approximately to external TMS; thus, although the internal shift differences are accurate to +O.Ol ppm, the chemical shifts from TMS are probably only good to +O.l ppm. g The CH, absorption appears at 31.44 & 0.02 ppm.
2
Compound No.
ASSIGNMENTS
102
THORPE,
COBURN
AND
MONTGOMERY
this case by a variety of methods. In an off-resonance decoupling experiment, the proton region was irradiated coherently 50 Hz downfield from the position of H-2 [6,,-, = X.76 ppm and 6,-, = 8.53 ppm (2)J. One of the two unassigned carbon resonances shows proportionately more residual splitting than the other and is accordingly assigned as C-X, since H-8 was farther away from the frequency of proton irradiation. This assignment of C-2 and C-8 was later confirmed by running the r3C spectrum of 6-methylpurine-2-d. In addition, the lJ,-- values agree with those measured from 13C satellites in the proton spectrum (8). The sample designated above as 6-methylpurine-2-d was an old sample which had been stored in a desiccator for several years. Although originally substituted at the 2-position, deuterium exchange had evidently taken place during this period. Its r3C spectrum shows the presence of some deuterium at C-8 and in the methyl group. C-2 of the 2-D compound is 0.34 ppm upfield from C-2 of the 2-H compound, C-8 of the 8-D compound is 0.24 ppm upfield from the 8-H compound, and the CH,D carbon resonance is 0.29 ppm upfield from the CH, carbon resonance. The decoupled spectrum of 6-methoxypurine, 6, consists, in the low-field region, of two strong, sharp lines and three weak lines, two of which are broad. The coupled spectrum proves the strong lines to correspond to C-2 and C-X, and they are assigned on the basis of the magnitudes of their ‘Jcu values. The other three resonances are weak, broad. and unresolved. The most upfield of these is, as usual, assigned to C-5. In a separate experiment the methyl hydrogens were selectively decoupled, resulting in the lowest-field resonance appearing as a sharp doublet. This is assigned to C-6, with a residual coupling to H-2. The remaining resonance is assigned to C-4. Both C-4 and C-5 are sharpened somewhat, but they remain weak and unresolved. All five purine carbon resonances of 6-methylthiopurine, 7, are comparatively sharp in its decoupled spectrum. The coupled spectrum allows assignment of C-2 and C-X from the lJ,-- values. C-5 appears as a slightly broadened doublet, split by H-8 and probably by a small coupling with the methyl protons. C-4 is a doublet of doublets, split by H-2 and H-8, while C-6 is an incompletely resolved multiplet, split by H-2 and by the methyl protons. The decoupled spectrum of adenine, 8, shows three sharp lines and two weak, moderately broad ones. These latter two are assigned accordingly to C-4 and C-5. The coupled spectrum allows the assignment of C-2 and C-X on the basis of their ‘JcH values. C-6 appears as a doublet with an 11.6-Hz coupling constant, j3JCgH2/.This assignment confirms the proposed assignment for adenine made by Jones et al. (7). The appearance of the decoupled spectrum of 6-methylaminopurine, 9, is almost identical with that of adenine in the ring-carbon region, and the chemical shifts are very similar. The assignment is thus based on that made for adenine. No coupled spectrum was obtained in this case. 6-Dimethylaminopurine, 10, also has a similar appearance to adenine in its decoupled spectrum, although the peaks assigned to C-4 and C-5 are somewhat sharper. The coupled spectrum yields lJcH values for C-2 and C-8 which agree well with those obtained from their “C satellites in the lH NMR spectrum (8). C-4 appears as a doublet of doublets from coupling to H-2 and H-8, C-5 as a doublet from coupling to H-X, and C-6 is broad and unresolved from coupling to H-2 and the methyl protons. 6-Diethylaminopurine, 11, again has a similar appearance to adenine in its decoupled spectrum, except that C-4 and C-5 are now sharp. C-2 and C-8 are again assigned
CARBON-
13
SPECTRA
OF SUBSTITUTED
PURINES
103
on the basis of the magnitudes of their ‘JCHvalues in the coupled spectrum. There. also, C-5 is a doublet, C-4 is a doublet of doublets, and C-6 an incompletely resolved multiplet, and they are assigned as in 6-dimethylaminopurine. In contrast to 2-chloropurine, all five lines in the decoupled spectrum of 6-chloropurine, 12, are sharp. The coupled spectrum allows assignment of C-2 and C-8 from their typical ‘JcH values. The lowest-field resonance is a doublet of doublets and hence must be C-4; C-5 and C-6 are doublets, coupling, respectively, with H-8 and H-2. The appearance of the decoupled spectrum of 6-bromopurine, 13, is very much like that of 6-chloropurine, except that C-6, being more shielded by the bromine, is shifted upfield by almost 8 ppm. It shows the same multiplicities in the coupled spectrum and is assigned accordingly. The decoupled spectrum of 6-iodopurine, 14, shows five moderately sharp peaks, two strong and three weak. The strong peaks are assigned to C-2 and C-8 on the basis of their lJCH values in the coupled spectrum. However, the other three resonances appear as weak, broad, unresolved peaks in the coupled spectrum, so no coupling information is available to aid in their assignment. The assignment given in Table I is, therefore. only a suggested one. The assignment given in Table 1 for 6-cyanopurine, 15, is again an uncertain one. The decoupled spectrum gives six sharp lines. The most upfield of these is assigned to the cyano-group carbon since it remains a sharp singlet in the coupled spectrum. C-2 and C-8 are assigned from the magnitudes of their lJCH values. The lowest-field resonance is a doublet of doublets and thus assigned as C-4. The remaining two absorptions in the coupled spectrum are doublets, as expected for C-5 and C-6, but since they are both upfield in the normal range for C-5, it is not certain which is which. We assign the lower field resonance of this pair to C-5, since it gives a better fit to a correlation of the chemical shift of C-5 with Taft’s cR (9), to be discussed below. Purin-6-yltrimethylammonium chloride, 16, decomposed in DMSO-d, at probe temperature to 6-dimethylaminopurine. The chemical shifts of its five ring carbons are measurable from the decoupled spectrum, but only C-2 and C-8 can be measured in the coupled spectrum. These two cannot be distinguished because their lJCHvalues are too close in magnitude. A tentative assignment is given in Table 1. A correlation of the chemical shift of C-8 with Brown and Okamoto’s a,+ (ZO),to bediscussedbelow,predicts a value of 148.1 ppm, in good agreement with the assignment proposed here. The decoupled spectrum of 2,6-bis(methylthio)purine, 17, shows two sharp lines and three weak, broad peaks in the ring-carbon region. The coupled spectrum shows the upfield, sharp line to be C-8, since it shows a ‘J CHsplitting. The other sharp line, at lowest field, is split into a quartet with 13J,--I = 4.6 Hz; therefore, this resonance must be associated with either C-2 or C-6, splitting with the protons of a methyl group. C-5 is assigned to the highest field resonance on the basis of its chemical shift. C-2, C-4, and C-6 are assigned on the basis of substituent additivity relations, to be discussed below. 2,6-Diaminopurine, 18, gives five sharp lines in its decoupled spectrum. C-4, C-5. and C-8 are readily assigned as they are doublets in the coupled spectrum. C-2 and C-6 remain singlets and are assigned on the basis of substituent addivitity relations. 2,6-Dichloropurine, 19, also gives five moderately sharp lines in its decoupled spectrum. Its multiplicities in the coupled spectrum are the same as those of 2,6-diaminopurine, and it is assigned in the same manner.
104
THORPE.
COBURN
AND
MONTGOMERY
6-Amino-2-methylpurine, 20, was not very soluble in DMSO-d,. It was run as a saturated solution at a concentration somewhat below 2 wt %. Consequently, only the methyl absorption is observable in the coupled spectrum. C-8, however, can be assigned on the basis of its position and relative intensity in the decoupled spectrum. C-4 and C-5 are also assigned on the basis of their positions and the fact that they appear as very weak, broad absorptions. In a separate experiment, the methyl group protons were selectively irradiated. This spectrum allows assignment of C-2 and C-6 from the relative enhancement of C-2. In the decoupled spectrum of 2-amino-6-methylpurine, 21, only C-5 is weak and broad. In the coupled spectrum, C-4, C-5, and C-6 are unresolved multiplets, presumably because of splitting with the methyl-group protons. However, C-6 appears as an enhanced singlet when the methyl-group protons are selectively irradiated, allowing its certain assignment and distinguishing it from C-4. The decoupled spectrum of 6-amino-2-methylthiopurine, 22, shows three strong, sharp ring-carbon lines and two that are weak and broadened. That these latter two are C-4 and C-5 is borne out by the coupled spectrum in which they remain weak, broad, and unresolved; C-2 is resolved into a quartet from coupling to the methyl protons, C-6 remains a sharp singlet, and C-8 shows its normal lJcH splitting. The decoupled spectrum of 2-amino-6-methylthiopurine, 23, consists of two strong, sharp peaks and three that are weak and broad. The coupled spectrum allows assignment of C-4, C-5, and C-8. Selective irradiation of the methyl protons results in enhancement of C-6 with respect to the other peaks, distinguishing it from C-2 and C-4. All of the peaks are sharp in the decoupled spectrum of 2-chloro-6-methoxypurine, 24. It appears from the coupled spectrum that H-8 is coupling with all five ring carbons: C-2, C-4, C-5, and C-8 are doublets, while C-6 is probably a doublet of quartets from splitting with H-8 and the methyl protons, although its exact mutliplicity is obscured by noise in this spectrum. C-2 can be distinguished from C-4 by the small size of C-2’s coupling constant, 1“JcZH81= 0.6 Hz and j3JcqHsj= 8.5 Hz. As the amount of 6-amino-2-fluoropurine, 25, available was limited to 12 mg, no coupled spectrum was obtained, and the decoupled one has a low signal-to-noise ratio. C-8 and C-5 show no measurable splitting by the fluorine, and C-5 is so weak and broad as to obscure any. C-2 is readily assigned on the basis of its ‘JcZF, value of -202.0 Hz. C-4 and C-6 both show three-bond splittings by the fluorine, respectively, of magnitudes 9.2 and 21.4 Hz. They are assigned on the basis of substituent additivity relations. Only four moderately sharp peaks are observed in the decoupled spectrum of 6amino-2-chloropurine, 26. C-8 is assigned from the coupled spectrum and C-2 and C-6 from substituent additivity relations. The coincidence of C-4 and C-2 was demonstrated by the addition of a few drops of trifluoroacetic acid to the solution. In the subsequent spectrum all the peaks are shifted slightly, and C-2 and C-4 appear as two distinct peaks. All lines are sharp in the decoupled spectrum of 6-chloro-2-ethylpurine, 27. However, it is readily assigned on the basis of the coupled spectrum. C-4, C-5, and C-8 are doublets from coupling with H-8, C-6 remains a singlet, and C-2 is a complex multiplet from coupling with the protons of the ethyl group. The multiplicity of C-2 is not clear because some components are lost in noise in the baseline. Coupling constanrs. All of the measurable one-bond r3C-lH coupling constants are
CARBON- 13 SPECTRA OF SUBSTITUTED PURINES
105
given in Table 2. Although the ranges for C-2 and C-S overlap slightly, the values are characteristic enough to be useful in making assignments. For 11 compounds, ‘Jc,n, TABLE
2
ONE-BOND 13C-1H COUPLING Compound No. .--__ 1 2 3 4
5 6 7
8 10 11 12 13 14 15 16 17
18 19 20 21 22 23 24 26 27
Substituent 2
-H -NH2 -F -Cl -H -H -H -H -H -H -H -H -H -H -H -SMe -NH2 -Cl -Me -NH, -SMe -NH2 -Cl -Cl -Et
CONSTANTS
Coupling 6
-H -H -H -H -Me -0Me -SMe -NH, -NMe* -NEtz -Cl -Br -1 -CN -NMe,+ -SMe -NH2 -Cl -NH2 -Me -NH* -SMe -0Me -NH2 -Cl
c-2 203.2
C-6
5 0.3
184.0 179.4 188.05
202.0 203.9 204.5 197.8 197.8
197.8 208.7 209.4 208.1 209.4
212.4 ’
& 0.3 + 0.3 f 0.3 zk 0.3 a 0.3 c 0.3 + 0.3 & 0.3 f 0.3 c 0.3 k 0.3’
Other
C-8
f 0.3 * 0.3
189 f2b
constant (Hz)
0.3
210.6 5 0.3 210.0 + 0.3 211.8 kO.3 211.8 kO.3 210.0 L- 0.3 210.6 5 0.3 211.2 * 0.3 209.4 + 0.3 210.0+ 0.3 210.0 + 0.3 212.4 k 0.3 212.4 k 0.3 212.4 + 0.3 213.6& 0.3 213.6 5 0.3" 212.4 + 0.3 208.1 rl: 0.3 214.4 + 0.3 208.7 210.0 210.6 211.8 211.8 211.2
i 0.3 f 0.3 fc0.3 kO.3
+ 0.3 + 0.3
128.0 k 0.3c 147.4 + 0.3' 142.2 +0.3"
137.9 t 0.3’ d
I
126.3 127.6 140.4 141.4 148.3
& + 5 * +
0.39 0.3" 0.3' 0.3' 0.3'
h
n Although we have not determined the signs of these coupling constants, we take them a11 to be coupling (see, for example, Ref. (3)). positive since this has been established for one-bond %-iH * The uncertainty in this value of ‘JcgHg p robably arises from a small splitting of C-6 by H-8. c This value is for the methyl group. d The value for the methyl group is 126.3 + 0.3 Hz; that for the methylene group is 138.0 & 0.5 Hz (there may be a small splitting of the methylene carbon by H-2 or H-S). e Assignments are uncertain for these two resonances. S The value for the 2-SMe group is 141.0 ? 0.3 Hz; the value for the 6-SMe group is 142.2 F 0.3 Hz (this latter value also agrees with that for 6-methylthiopurine). g This value is for the methyl group. The concentration was too low to allow measurement of ‘JQ”S. h The value for the methyl group is 127.3 + 0.3 Hz; the value for the methylene group is 127.9 f 0.3 Hz. has a mean value of 203.9 + 3.8 Hz with a total range of 11.6 Hz. For four compounds ‘JC& has a mean value of 185.1 k 3.4 Hz with a total range of 10 Hz. For 23 compounds,
‘JC,H,has a mean value of 211.1 k 1.2 Hz with a total range of 6.3 Hz. That there is the
106
THORPE,
COBURN
AND
MONTGOMERY
smallest variation in ‘JcsH8is undoubtedly owing to the fact that C-8 is farthest from the two sites of substitution studied here. Table 3 gives the long-range 13C-lH coupling constants measured for the few 2substituted purines available and for purine itself. Figure 1 shows the coupled spectrum of purine and illustrates the complexity of the resonances of C-4 and C-5 owing to the TABLE LONG-RANGE
Compound Lo.
1 2 3 -l
COUPLING
3
CONSTANISOF~-SUBSTITUTED
PURINES
Coupling constant (Hz)
3JC2Hg 11.0 f 0.3 11.6+0.3 13.4 + 0.3 14.0 rt O.?
‘Jc2us
-cl?
3J~q~g 4.8 + 0.3? -5.8 b
3JC4HS 11.6 + 0.3? -5.8 b
‘Jc,,,, 6.4 + 0.3? 5.8 f 0.3? c
3JCgHS
‘+JC&
9.2 + 0.3 1 0.6 IO.3 10.7 + 0.3? c
” Since signs of coupling constants were not determined, all values in the table are absolute values. * In coupled spectrum dvI,z z 40 Hz; 13JCqH6 + 3Jc4H81 2 23 Hz. ’ 11: coupled spectrum dv l,z z 22 Hz; I’Jc,~,,+ 3JcsHsl z 18 Hz. -C-*7
FIG I. The proton-coupled
13C spectrum of purine at 25.16 MHz; concn = 120 mg/ml in DMSO-d,.
long-range coupling to the three protons. The question marks in the table indicate uncertainty as to which proton is coupling to the indicated carbon. In the case of C-5, we have assumed that the larger value should be associated with H-8, since this is a three-bond coupling while that to H-6 is a two-bond coupling; in benzene, 3J,-His the largest of the long-range couplings (4). However, in the case of C-4, we are unable to determine whether the larger coupling is with H-6 or H-8 since these are both three-bond couplings. No coupling is observable between C-8 and H-6, a four-bond coupling, although a small splitting is observed between CL6 and H-S in the case of purine, I.
CARBON-
13
SPECTRA
OF SUBSTITUTED
107
PURINES
The long-range coupling constants of the 6-substituted purines and purine are given in Table 4. Again the question marks indicate assignment ambiguities. C-4 appears as a doublet of doublets in the coupled spectra, and it is not clear whether the larger coupling is with H-2 or H-8. C-5 of purine shows splittings with all three protons; the smallest TABLE LONG-RANGE
COUPLING
1
8.1 k 0.3?
11.6+0.3?
5 7 8
7.9 + 0.3? 7.0 f 0.3?
11.6 2 0.3‘~ 11.0+0.6?
4.6 5.5 7.3 7.3
11.6+0.4? 12.2 + 0.3? 12.2 + 0.3? 12.2 + 0.3?
10 11’
12 13 15’
CONSTANTS
1.5 + 0.3‘~
4 OF 6-~~JBSTITUTED
9.2 & 0.3? >4
PURINES
10.7 + 0.3 10.2 +- 0.3
11 -t-l 12
+ 0.4? t 0.3? + 0.3? + 0.3'!
0.6 i. 0.3
rfl
11.4 + 0.4
10 +1 10.4 & 0.3 10.4 * 0.3 10.4* 0.3
14.0 + 0.3 13.4 IO.3 12.2 + 0.3
a Sincesignsof coupling constantswere not determined, all valuesin the table are absolutevalues. ,aI‘Jc61,ye= 6.7 + 0.3 Hz. c l-?I.c6,,Mej = 3.7 + 0.3 Hz. ’ For the methyl carbon splitting aith the rnethylene protons, IzJc,,/ = 2.9 i 0.3; for the methylene carbon splitting with the methyl protons, 12JcHI = 4.3 + 0.3; there also appearsto be a small splitting of the methylene carbon by H-2 or H-8 of about 1 Hz. C-6 is unresolvedwith dvliz r 25 Hz. u ‘“J<4,q2 + “Ji4,,& = 18.3 + 0.3 Hz. of these is probably with H-2 as this is a four-bond coupling, whereas that with H-6 is a two-bond coupling and that with H-8 is a three-bond coupling. No five-bond couplings of C-2 with H-8 or C-8 with H-2 are observable. Table 5 shows the observable long-range coupling constants of 2,6-disubstituted purines. No assignment ambiguities exist here, except those mentioned above for purine, since the only ring proton available is now H-8. A definite five-bond coupling constant is measurable for C-2 of 2-chloro-6-methoxypurine, 24. The measurable 13C-19F coupling constants for the two compounds having fluorine as a substituent are shown in Table 6. The values of ‘JCFin these two cases are smaller, i.e., more positive, than those for substituted fluorobenzenes (about -240 Hz), probably indicating greater s character in the C-F bond of the fluoropurines (3). In the decoupled spectrum of 2-fluoropurine, every resonance is a doublet, showing measurable splitting by the fluorine of each carbon in the molecule, including a five-bond coupling to C-8 of 1.8 Hz magnitude. Substituent effects. Shielding constants for the various substituents at C-2 and C-6 were derived from spectra of the monosubstituted purines by means of the relation : AC,, = GC,(purine) - K,(derivative), where AC, is in ppm. In three cases, 2-SMe, 2-Me, and 2-Et, shielding constants were
108
THORPE,
COBURN
AND
MONTGOMERY
TABLE 5 LONG-RANGE COUPLING CONSTANW OF 2,6-DISUBSTITUTED PURINES
Compound
no. ---.
1 17b 18 19 22' 24 27'
5JczHs
Coupling constant (Hz) 3JCq”* 3Jcsll* 11.6+0.3?
9.2 + 0.3?
6.9 + 0.4
10.4 * 0.3 9.9 * 0.4
8.5 + 0.3 6.7 + 0.3
9.2 IL-0.3 ll.OkO.3
6.1 + 0.3
0.6 k 0.3
4JC&
0.6 rt 0.3
R Since signs of coupling constants were not determined, all values in the table are absolute values. b The only long-range coupling observed was that between C-2 and the methyl protons, 13JcHl= 4.6 + 0.3 Hz; all other peaks were too broadened by tautomerism (see discussion below). c See footnote b; here j3JcHI = 4.9 + 0.3 Hz. d j4Jc6~Jz 13J~6~OMel ~55.5H z; exact multiplicity uncertain because of noise in baseline. e For the methyl carbon coupling with the methylene protons, /2JcHI = 4.9 of:0.3 Hz; for the methylene carbon coupling with the methyl protons, /*JcHl = 4.6 & 0.3. TABLE
6
13C-19F COUPLING CONSTANTS Compound No.
3 25
Substituent 2 6 -F
-H
-F
-NH2
Coupling constant (Hz)
1Jc& -207.5 5 0.3 -202.0 * 0.3
3Jc41z2
4JCgF2
17.1 + 0.6 9.2 + 0.3
3.7 + 0.6
3JC& 15.9 + 0.3 21.4 + 0.3
SJCsFZ 1.8 + 0.3
(1Since signs of coupling constants were not determined, the long-range coupling constants are absolute values. It has been established that ‘JcV values are negative (see, for example, Ref. (3)).
derived from disubstituted purines (because the appropriate were not available) by means of the relation: AC, = X,(monosubstituted
monosubstituted
purine) - E,,(disubstituted
purine).
purines PI
These shielding constants are listed in Table 7. They were found to be very useful in predicting carbon chemical shifts of disubstituted purines, as is shown by the comparison of predicted and experimental values in Table 8. We also investigated the possibility that 13C chemical shifts of substituted purines might be predictable by means of some of the reactivity parameters, as we had found to be the case earlier (I, 2) for a,-, and S,-, of 6- and 2,6-substituted purines. For bcm8and
CARBON-
3 SPECTRA
]
OF SUBSTITUTED TABLE
‘T Substituent
CONSTANTS
AC.,
2 2 2 2 2 2 6 6 6 6 6 6 6 6 6
F Cl SMe Me Et Me OMe SMe NH2 NHMe NMez NEtz Cl Br values
I FOR SUBSTITUTED
Position
NH2
n AC,
SHIELDING
-8.49 -6.21 -0.59 -11.52 -8.35 -13.51 0.34 0.80 0.53 -0.21 -0.32 0.34 0.19 0.60 0.58
P~RINES”
AC,
-0.34 -3.45 -2.91 -0.90 -0.51 -0.95 0.90 -0.29 4.56 3.41 4.8 3.54 3.69 0.61 1.79
AC,
4.94 1.67 1.40 2.19 1.83 1.53 0.87 12.32 1.06 12.85 12.3 11.49 12.00 1.23 -1.51
-2.19 -1.75 -1.41 0.38 0.36 0.19 -10.17 -13.76 -13.20 -9.80 -9.22 -8.19 -7.57 -2.28 5.38
4.47 -1.89 -1.69 0.82 0.67 0.19 1.58 3.50 2.96 6.80 7.33 8.35 8.23 -0.09 0.15
are in ppm. TABLE
COMPARISON
109
PURINES
OF OBSERVED
AND
PREDICTED
VALUES
8 OF THE Y
CHEMICAL
SHIFTS
OF DISIJBSTITUTED
PURIN&
Chemical Substituent 2 -Cl -Cl -Cl -NH2 -NH2 -NH, --F -SMe
not
fit-2 6 -NH, -Cl -0Me -Me -NH2 -SMe -NH, -SMe
&-‘l
Exp.
Calc.
Exp.
Calc.
152.78 150.98 151.13 160.11 160.20 159.65 158.77 163.75
152.96 152.09 151.89 160.25 160.86 160.06 158.58 163.09
152.78 156.23 156.95 154.34 152.77 151.65 153.36 151.83
154.21 157.07 157.97 154.21 151.64 150.55 154.75 151.11
n Data from 6-amino-2-methylthiopurine, included because they were used
shift (ppm) k-5 Exp. Calc.
116.20 128.51 116.83 124.40 112.50 123.97 115.53 127.86
116.21 127.83 116.74 124.64 112.67 124.46 115.94 127.21
6-amino-2-methylpurine, to derive shielding constants
&-Ii
L-8
Exp.
Calc.
Exp.
Calc.
155.94 148.10 159.57 157.25 155.78 159.20 156.81 159.78
156.71 149.19 160.67 157.86 157.49 160.89 157.05 158.32
140.17 147.44 143.83 140.02 135.91 138.74 140.06 141.99
140.98 147.87 144.28 140.04 134.82 138.66 141.18 142.31
and 6-chloro-2-ethylpurine from Eq. [2].
were
a,-, such relations were found, but for the carbons at other positions no simple relations are apparent. The chemical shift of C-8 is predictable from 0,’ values (10) of the substituents in a relation similar to that reported earlier (1, 2) for H-8. The equation, representing 24 points, is : &,
= (146.20 + 0.34) + (4.74 f 0.25)a& + (3.38 + 0.33)0&, Y = 0.98.
[31
110
THORPE,
COBURN
AND
MONTGOMERY
Since both a,-, and an8 correlate well with cr,+ of the substituents, a correlation also exists between them. This relation is primarily interesting because the coefficient relating Bcm8and a,-, is about half as large as those reported for some aryl and heteroaromatic systems (3). 6,-, = (50.5 + 5.0) + (11.03 k 0.60)6,-,, r = 0.97. (41 A satisfactory correlation was found for C-5 with cR (9). This equation, derived from data for 22 compounds, is: d,-, = (132.37 ) 0.50) + (19.1 f 1.4&r,; + (6.7 k l.S)a,,, r = 0.96. I51 Tuutomerism. In the proton-decoupled spectra of more than half of the substituted purines, all of the lines are sharp. In the others, varying degrees of line broadening are observed, particularly in the lines due to C-4 and C-5. This suggested to us the probability of tautomerism of the labile proton between N-7 and N-9. Krugh (II) reported the observation of 13C line broadening caused by tautomerism in the spectra of formycin A (II) and formycin B (III). These compounds each have two
HO
OH
HO
II
OH III
possible tautomeric sites, but it has been demonstrated by Chenon et al. (12) that the broadening is caused by tautomeric averaging of the proton between N-l and N-2.
C-6
c-4
c-2
C-6
2. The proton-decoupled 13C spectrum of 6-methoxypurine; Chemical shifts are in ppm downfield from internal TMS. FIG.
C-5
concn = 50 mg/ml in DMSO-d,.
CARBON-
13 SPECTRA
OF SUBSTITUTED
PURINES
111
We chose to investigate two compounds which have only one possible site of tautomerism. In these two cases, we have demonstrated that exchange of the proton between N-9 and N-7 is indeed the source of the line broadening. In the proton-decoupled spectrum of 2,6-bis(methylthio)purine at about 35”C, the lines due to C-4, C-5, and C-6 are all very broad. With the sample temperature raised to lOO’C, all the lines in the spectrum are sharp. The proton-decoupled spectrum of 6-methoxypurine, shown in Fig. 2, has broad peaks for both C, and C,. This broadening is absent in the spectrum of 6-methoxy-9-
FIG. 3. The proton-decoupled 13C spectrum of 6-methoxy-9-(B-D-ribofuranosyl)purine; concn = 150 mg/ml in DMSO-rl,. Peak assignments were made by comparison with the spectrum of 6-methoxypurine, and confirmed by the splittings observed in the proton-coupled spectrum. Chemical shifts are in ppm downfie!d from internal TMS.
(P-D-ribofuranosyl)purine, shown in Fig. 3, pulsed under the same conditions. In this case, the possibility of tautomerism was removed by the replacement of the labile proton with the sugar moiety. Thus we conclude that the degree of line broadening, particularly of the lines due to C-4 and C-5, depends on the rate of tautomerism of the labile proton between N-7 and N-9. In those cases where the lines are sharp, the rate of tautomerism is either very fast on the NMR time scale or else the proton is essentially fixed at N-7 or N-9, although the latter explanation seems less likely. When the rate is intermediate, broadening occurs. REFERENCES 1. W. C. COBURN, JR., M. C. THORPE, J. A. MONTGOMERY,AND K. HEWSON.J. Org. Chem. 30,111O (1965).
2. W. C. COBURN, JR., M. C. THORPE, J. A. MONTGOMERY,AND K. HEWSON,J. Org. Chem. 30, 111-i (1965). 3. J. B. &OTHERS, “Carbon-13 NMR Spectroscopy,” Vol. 24 of “Organic Chemistry, A Series of Monographs” (A. T. Blomquist and H. Wasserman, Eds.), Academic Press, New York, 1972. 4. G. C. LEVY AND G. L. NELSON, “Carbon-13 Nuclear Magnetic Resonance for OrganicChemists,” Wiley-Interscience, New York, 1972. 5. R. J. PUGMIRE, D. M. GRANT, R. K. ROBINS, AND G. W. RHODES,J. Amer. Chem, Sot. 87,225 (1965).
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