222
CHRISTINE 11. JARDETZKY
[CONTRIBUTIOS FROM THE RETINA
AND
OLEGJ A R D E T Z K Y
Vol. 82
FOIJSDATIOX AND THE
DEPARTMENTS O F CHEMISrRY A V D PIIARMACOLOGT, IIARVARD UNIVERSITY]
Investigation of the Structure of Purines, Pyrimidines, Ribose Nucleosides and Nucleotides by Proton Magnetic Resonance. 11’ BY CHRISTINE D.
JARDETZKY~AND
OLEG JARDETZKY
RECEIVED MARCH6, 1959 Several purine and pyrimidine proton resonances, as well as those of nucleosides and nucleotides, were observed in SOlUtions of DzO a t different hydrogen ion concentrations. Tentative assignments for the heterocyclic ring proton peaks were made by comparing the shifts for these protons in the dissociated pnrines with those in the corresponding ribose and ribose5’-mOnO- or tri-phosphate derivatives. The electron shielding effect of amino and oxy substituents of the purine ring, and a concentration dependence of the proton shifts which was found to be different for the various protons of a given Purine were also observed. Titration of the dissociated oxy groups caused very little change in the chemical shifts of the heterocyclic ring protons, while titration of the amino groups had pronounced eiiects on the shifts of the base protons 111 both purine and pyrimidine nucleosides and nucleotides. Definite tautomeric structures for the cations of cytidine and adenosine and for the guanosine triphosphate ion were considered to be in agreement with the nuclear magnetic resonance results an! with existing evidence from X-ray crystallography. On the basis of the coupling constant for the CI’ proton of ribose, it was suggested t h a t the ribose ring configuration in the pyrimidine nucleosides may be different from t h a t present in either the purine suggested or the purine nucleotides.
Introduction Ultraviolet and infrared spectroscopy have greatly contributed to the understanding of the structure of the heterocyclic bases and, in particular, of the nucleic acid derivative^.^-^ The dissociation constants for many compounds are known from titrimetric studies4and X-ray crystallography has established the fact that the ribose is in the Ns- or NI-fl-D-furanosyl form with the furanose ring almost perpendicular to that of the planar purine or pyrimidine base, r e s p e c t i ~ e l y , a~t, ~least in the solid state. However, very little is known about the electron distribution in the ring structure of the bases and, thus, about the most important tautomeric and resonance forms in aqueous solution. On the other hand, the specificity with which these compounds are active in various enzymatic reactions and with which derivatives of similar structure may act, either as normal metabolites or as metabolic inhibitors, indicates that the reactivity or electron shielding a t the various ring atoms may be directly linked with the role of the compound in a biological reaction. Correlations of calculated electron densities with the antitumor activity of purine and pyrimidine derivatives already have been made.8t9 Recently, important contributions to the understanding of structure and mechanism of interaction in solution have been made by the use of nuclear magnetic resonance spectroscopy. LVith the (1) This investigation was supported in p a r t b y a research grant
(B-904)from t h e National Institute of Neurological Diseases and Blindness. Public Health Service, a n d in p a r t by a research grant (H2297(c)) from t h e National Heart Institute, Public Health Service. Paper 77, Retina Foundation. (2) Biological Laboratories, Harvnrd University. Cambridge, hf assachusetts. (3) G. E. W. Wulstenholmc and C. R.1 O’Connor, “Ciba Foundation Symposium on t h e Chemistry and Biology of Purines,” Little, Brown & Company, Boston, hlass., 1967. (1) E. Chargaff and J. N. Davidson. “Nucleic Acids,” Vol. I , Academic Press, Inc., New York, N.Y., 1968. ( 5 ) H . T. Milcs, Biochim. B i o p h y s . A c f a , 27, 40 (1955). ( 6 ) S. Furberg, Acto Chem. Scnnd., 4, 753. (1350). (7) L. Paulin:: and R . B. Curey, Arch. Biochrm. B i o p h y s . , 66, 164 ( 19%).
( 8 ) B. Pullman and A. Pullman, B d . SOC. chim. France, 973 (1955). (9) A. Pullman and B. Pullman, Compt. rend., 246, 611 (19573. (10) “Chemical Society Symposia,” T h e Chemical Society, B u r l i n e ton House, W I , London, Specixl Publication KO. 12, p. 211. 1958. (11) 0. V. S t . Whitelwk, A n i i . N . Y . A c a d . Sei., 7 0 , 763 (1958).
purpose of elucidating some of the problems mentioned, a study has been made of the proton resonance spectra of purines, pyrimidixles and their ribose derivatives in solutions of D20 a t different hydrogen ion concentration^.'^ Experimental All spectra mere obtained with the 40 megacycle high resolution n.m.r. spectrometer from Varian Associates in a field of about 9,400 gauss, T h e purines and pyrimidines were dissolved in sulfuric acid or sodium hydroxide of different normalities, and the final concentration was 30 g. in 100 ml. However, effects on the proton chemical shifts due t o high concentration soon became apparent, and the solute concentration in all later studies was kept a t 0.2 M: The nucleosides and nucleotides studied were dissolved in DtO and the pH was adjusted to the desired value by addition of small amounts of anhydrous sodium peroxide crystals or concentrated hydrochloric acid. For exact 911 measurements, 3 to 5 ml. samples in DzO and 0.2 Af in solute concentration were prepared and 0.2 t o 0.3 ml. aliquots were removed after titrating and determining the pH wlth the Beckman Model G pH meter. The compounds used were obtained from the Catlfornia Foundation for Biochemical Research, from the Sigma Chemical Company and from the Mann Research Laboratories. The 0.2 t o 0.5 ml. samples were pipetted into Pyres glass tubes of 5 mm. outside diameter and were used for obtaining the spectra within a few hours after preparation or were stored a t -20” for a few days before the recording of the spectra. All spectra were obtained with the Sanborn Recorder in both the presence and absence of an external standard. ( I n many instances the reversibility of the spectrum of the substance in D.,O was assured by increasing or decreasing the pH with sodium peroxide or hydrochloric acid and then titrating back t o the original pH.) The reference compound, toluene, was introduced m the sample by inserting a toluene containing capillary of about 1 mm. outside diameter. From the distance between the aromatic and the methyl peaks of toluene which was measured bp the side band modulation methodl4 and found t o be equal to 196.6 0.2 C.P.S. at a room temperature of Z O O , the frequency shift for the other peaks was calculated. At least two spectra with the field increasing and two with the field decreasing were analyzed separately, and the average value vsrird within f0.8 C.P.S. I n some instances, additional checks on the frequency separation were obtained with the side band modulation method and the means from both de(12) J . D. Roberts, ”Nuclear Magnetic Kesonance, Applicatims t o Organic Chemistry,” McGraw-Hill Book Co., Xew York, N. Y., l93. (12a) J. A. Puple, W. G. Scbneider and €1. J Bernstein, “HighResolution Nuclear Magnetic Resnnance,” McGraw-Hill Book Co., Kew York, N . Y.,1959. (13) C. D. Jardetzky and 0. Jardetzky, FedsralioM PronH nomc
I
I%.~CPS
OCPS N h
b
I
I
1
196.6 CPS
0 CPS
196 ~
OCPS
C P S
r14
C
1966~~s
OCPS
d 1 I
n
0
A i 5
6
CPS
Fig. 1.-Pyrimidine nucleosides, a , uridine, PH 7.5. b, c and d, cytidine hemisulfate, p H 2.95, 6.0 and 10.1, respectively. e, Nl-methylcytosine, pH 3.2. Peak (1) H g doublet, Ha', Hd', (2) Ha diiublet, (3) HI' doublet, (4)H20, ( 5 ) HZ', (6) ~ H s ' (71, , (8) aromatic and methyl protons of toluene, the external standard and (9) side band (due t o the spinning of the sample). In this and subsequent figures the concencentration is 0.2 M i n D20 and the magnetic field is increasing from left to right except in case le.
(21) J. (1958).
N. Shoolery and M. T. Rogers, THISJOURNAL, 80, 5121
1966CPS
OCPS
0 CPS
H4') due to the methylenic protons being very close to one another, but not exactly superimposed. This situation has been shown to exist in the 21acetoxy-20-one steroids for the Cpl-methylene resonance.21 Further work is planned to resolve this point in the nucleosides and nucleotides. The effect of protonation of the purine ribosides may be seen in Tables VI1 and VI11 for adenosine and inosine. Only a limited @Hrange was studied because of the low solubility of the compounds. In the case of adenosine a t high fiH, the peaks due to the Czand C8 protons were assigned on the basis of arguments discussed in the previous section. However, a t low pH, where the difference in shift between these protons becomes much smaller, the assignment is purely intuitive. At any rate, there is a greater shift to lower field of the C:,than of the Cs proton. Furthermore, a shift to lower field is noted for the C1' proton of ribose by about 0.30 p.p.m. between the extremes of the @H range
6
I
I
I
1%
5
196.6 CPS
I OCPS
7 n4
I OCPS
1966CP5
nucleosides, a , b, adenosine, p H 1 8 2 aiid c, d and e, inosine pH 2.2, 9 4 alid 14, respectively. f, g, Xanthosine pH 10.7 and 14, respectively. (2) H8. The rest of the peak identification and Peak (1) Ho, remarks as in Fig. 1. Fig 2.-Puritie 14, respectively.
studied. These results can be understood in terms of a definite tautomeric structure of adenosine in which the hydrogen is attached to N1 and the positive charge is distributed among the nitrogen atoms of the adenine ring. Further evidence for such a structure is provided by the X-ray crystallographic data for adenine hydrochloride according to which the proton is located on N1 rather than on N U of the amino g r o ~ p . ~ ~ J ~ For inosine, on the other hand, one observes that protonation of the (NC0)- group with a (22) W. Cochran, Acfa Crysf., 4 , 81 (1951). (23) J. M. Broomhead, i b i d , , 4, 92 (1951).
Jan. 5, 1960
PURINE AND PYRIMIDINE PROTON JfAGNETIC
RESO~-A~;CES
337
il
TABLE VI1 PROTON CHEMICAL SHIFTS I N ADENOSINE~ OH
1.82
12.5
3.42
14.0
-2.09 -1.96 -1.75 -1.78 -2.01 -1.83 -1.57 -1.53 $0.32 $0.38 $0.60 $0.62 f1.57 $1.54 $1.37 f 1.84 $1.85 $2.00 f1.99 $2.22 f2.12 $2.22 f2.21 $2.22 $2.26 2Hb‘ +2.57 +2.56 $2.60 +2.57 a ~ K A= , 3.45 due t o ionization of a hydrogen from the adenine ring. pKa, = 12.5 due t o ionization of a ribose hydrogen. TABLE VI11 P R O T O N CIiEMICAL SHIFTS I N I N O S I N E Hs Hz HI’ Silvent H2‘, Hs’, H,’
3H
H?,& HI’ 2H5‘
7.4
2.2
-1.87 -1.85 -1.69 -1.72 f0.40 f0.40 f 1 ’ 5 R +1’59 +*’“ f2’58
8.3
9.4
14.0
-1.79 -1.66 $0.45 f1’59 f2’61
-1.59 -1.48 +0.50 f1’55 f2’60
-1.75 -1.75 +0.5R 3.1’38 +2’61
PKA of 8.P4results in little change in the shielding
I
136.6~~1
OCPS
2 1
I
I 196 6 c p 1
OCPS
I
I
0 CPS
136.6c ps
Fig. 3.-hlononucleotides, a, 2‘, 3‘-cytidilic acid, pH 5.36. b, and c, 5’-adenylic acid, pH 3.7 and 9.5, respectively. Peaks (1) and ( 2 ) in a are due to H6 and Ha respectively of the cytidine 2’- and 3’-phosphates. The rest of the peaks and remarks as in Figs. 1 and 2.
of the purine protons as seen by comparing the data a t pH between 2.2 to 8.3 and 14. Existing evidence from the infrared spectra of hypoxanthine points to the fact that the protonated group is
finip
0
It
2 1
probably in the form of -N-C-.26
I
I
0 LDS
1 196 6cps
H TABLE IX CHEMICAL SHIFTS OF SOME NON-EXCHANGEABLE PROTONS IN NUCLEOSIDES AND NUCLEOTIDES Compound
Adenosine pH 12.5-14.0 2’3’-AMP pH 5 . 2 - 9 . 8 5’-AMP pH 6.0-11.0 ATP pH 6.5-10.0 Inosine pH 14.0 5’-IMP PH 1 1 . 6 Guanosine pH 14.0 2’3’-GMP pH 1 0 . 5 GTP pH about 12 Cytidine pH 6.2-14.0 2’3 ’-CMP pH 6.0-8.0 5’-CMP
pH 6 . 5
H2
Ha
HI’
2Hr’
-1.55
-1.79
$0.61
$2.58
-1.49
-1.79
$ .45
$2.53
-1.51
-1.98
+
.43 $2.42
-1.56
-1.99
4-
.38 $2.20
-1.75
-1.75
4- .59 $ 2 . 6 1
-1.63
-1.95
$ .39
-1.42
+
-1.50
+ , 5 1 $2.57
-1.69
+
,52 $2.20
+
’55
Ha
2
1
I OCPS
OCPS
1966~~s
1966~~s
$2.48
.71 $2.60
+2’55
-1.42
+2,56
-1.63
+2,37
(24) The ~ K values A were obtained from “Properties of Nucleic Acid Derivatives,” California Corporation for Biochemical Research and those for guanosine triphosphate from the Pabst Circular ORIO. (25) s. F. Mason, “Chemistry and Biology of Purines,” G. E. w. Wolstenholme and C. M. O’Connor. 1957, p. 60.
OCPS
136.6~~~
Fig. 4.-Nuclenside triphosphates, a, b, adenosine triphosphate pH 4.0 and 6.5, respectively. c, d, guanosine triphosphate pH 2.9 and 6.5, respectively. Peaks and remarks as in Fig. 2.
It is noted that a t PH of about 2, where both adenosine and inosine exist in the protonated forms, the base protons of inosine are more shielded than those of adenosine. This is interesting in view of the fact that inosine has an additional ionization with a P K Aof 1.2 which refers to the ionization of one of the ring nitrogens. In the completely protonated form, therefore, the Cz and Cs protonsof inosine mustbe Seen at a lower field. Nucleotides.-The presence of the phosphate group in the nucleotides may cause a further
CHRISTINE D. J.ZRDETZKY A N D Or,m
228
From the data on the titration of the adenine iiucleotides, it is seen that the Cs protori undergoes very little change in shielding, while the Cz proton moves to lower field by about 0.40 p.p.m. In the case of 3’-adenylic acid (AMP), it was not possible to obtain a 0.2 Msolution a t pH less than 3.7 so that the titration data, although consistent with those of adenosine triphosphate (ATP), are not complete. The proton shifts due to titration of these nucleotides point to the fact that the proton is added to the pyrimidine portion of the ring since both the CP,
TAI~L XE P R O I O N C\IJEMICAL
HI’ Solvent H?‘,I f ; ’ , l ~ i , ’ 211;‘
SHIFTS I N ;’-&\DENYLICA%C11)
3.70
4.50
-2.01
-1.90
$H
Hu H.
-1.75 f0.37 +l.61 $2.04 $2.20 $2.33
Xcetcine
9.50
11 00
-2.00
-2 01 -1 52 +o 42 $1 55 rl 91 $2 04 +2 42 +4.24
5.95
-1.94 -1.56 -1.51 f0.44 $ 0 . 4 4 -1 56 $ 1 . 5 5 $ 2 . 0 6 +2.06 +2.21 + 2 . 2 5 $ 2 . 3 3 $2.38 +4.25
-1.49 $0.42
$1.56 $1.92 +2.03 $2.4.5
V(11. s:!
JARDETZKI’
T.4BLB X I PROWSCHEMICAL SHIFTSIS ADENOSINETRIPHOSPHATB~
fiH
Hs H? HI
’
Solvent H*’,H$’,Hd‘
2.00
3.00
4.00
4.75
-2.10 -1.95 +0.33 $1.54
-2.09 -1.93 $0.34 +1.55 f1.89 $2.00 +2.12
-2.07 -1.83 +0.35 $1.55
-2.00 -1.70 $0.36 $1.57 $1.82 +2.02 $2.17
+1.87 S2.02 +2.14
$1.85
+2.00 +2.13
5.90
7.3.5
6.50
9.95
-1.99 -1.98 -157 -1.54 $0.37 $0.38 +1.55 $1.56 +1.76 +1.75 +2.04 +2.06 $2.20 $2.21 +4.24 pK,, = 6.5 due to ionization of the phosphate groups. -2.00 -1.58 $0.38 $1.56 $1.78 $2.04 +2.19
-1.94 -1.56 +0.36 +1.54 +1.78 +2.00 $2.16
A r e t ( 111e
/iK\!= 1 I refcrq t o ionization of a hydrogen from the adenine ring. TABLE XI1 PROTON CHEMICAL OH
H8
I .2
1.9
SHIFTS I S C;UASOSISE
TRIPHOSPIIATIY 6.n
5.3
2.9
8.5
12b
-1.65 -1.69 Hi’ +0.55 $0.52 Solvent $1.50 $l,XS -1.83 H?’, HI’, Hq’ 2H,‘ + 2 1
