ESCA spectra and molecular charge distributions for ...

ESCA spectra and molecular charge distributions for some pyrimidine and purine bases

AND

N . STEWART MCINTYRE Can. J. Chem. Downloaded from www.nrcresearchpress.com by 179.61.180.125 on 03/06/18 For personal use only.

Atrrrlytir (11Scrc~t~c~e Brrrlrch, Wlrrte.cl~~II Nrcclcrrr Rr.rerrrc 11E.ctcrI~lr.tlrtt~et~t, Pitlrrn*rr,Mrrtl., Ctrt~ntlrrHOE /LO Received November 29, 1977 JAMES PEELING, FRANK E. HRUSKA, and N. STEWART MCINTYRE. Can. J. Chcni. 56, 1555 (1978). A series of pyrimidine and purine bases has been studied experinientally by electron spectroscopy for chemical analysis. Individual component peaks in the spectra were assigned on the basis of known substituent effects on core electron binding energies. Binding energies for all the core levcls in the niolecules provide sufficient data to determine the niolec~llarcharge ts favourably with atoniic charges determined theoretically i n distributions. The r e s ~ ~ lconipare the CNDOI2 niolecular orbital formalism. JAMES PELLING, FRANK E. HRUSKA et N. STEWART MCINTYRE. Can. J. Cheni. 56. 1555 (1978). On a etudik exp6rinientalenient, en faisant appel B la spectroscopic d'emission photoelectronique induite par rayons-X (ESCA), une sirie de bases purine et pyrimidine. On a a t t r i b ~ ~les e coniposantes individuelles des spectres en se basant sur dcs etTets connus des substit~~ants sur les energies de liaison des electrons des couches internes. Les energies d c pour liaison P O L I ~tolls Ies nivea~rxinternes des rnolCcules forlrnissent des donnbes s~~ffisantes determiner les distributions des charges ~iioleculaires.Les res~~ltats se comparcnt favorablcnient avec les charges atomiques determinces d'une faqon thboriq~ie par un formalisn~e d'orbitale niolec~~laire du type CNDO/2. [Tradu~tpar Ic journal] I

Introduction

has also recently published the spectraof 5- and (19). 6-azauracil Electron spectroscopy for chen~ical analysis In this paper we describe the core electron spectra (ESCA) or X-ray photoelectron spectroscopy (XPS) has proven to be a valuable tool in stud~esof struc- of common purine and pyrimidine bases, sonie of . I). . ture and chenlical bonding. The binding energies and their aza analogs, and one fluorinated analog (Fig. These analogs have cheniotherapeutic value (20), relative ~ntensities of core photoelectron peaks ly be due to the electronic provide a direct elemental analysis of a sample. At a which ~ ~ l t i ~ n a t eniust finer level of observat~on, chemical shifts in core perturbations elicited by the chemical ~nodifications electron binding energies relate directly to the charge to the base. The measured binding energies are used distributions in ~nolecular solids, in cases where to derive experimental charge distributions in the lattice effects on the binding energies are of secondary molecules. These are conipared with the results o f Importance. In the realm of biologically important molecules, although a number of studies of amino acids (1-4) and protelns (3, 5-8) have been reported, relatively few investigations have been directed towards the area of nucleic acids and their components. The ESCA spectra of tRNA and a few of its colnponents were described briefly (9), and studies on metal complexes of D N A (lo), A M P (1 I), and s o ~ n ethiouracils (12) have appeared. Detailed studies of the ESCA spectra of the purine and pyrimidine bases themselves appear to be limited to the early investigations of Barber and Clark on adenine (13), cytosine and thymine (14), in spite of the interest w h ~ c hthis work created (15-18). Clark 'Post-doctoral fellow: present address: University of Petroleum and Minerals, Dhahran, Saudi Arabia.

FIG. 1. Chemical f o r ~ i i ~ ~ for l a e several of the purine and pyriniidine bases studied here: 5-flnoro~~racil ( I ) , G-azathyniine (2), hypoxanthine (3), and xanthine (4).

1556

C A N . J . CHEM.

VOL.

56, 1978

CNDO (21) ~nolecular orbital theory calculations. Although extensive theoretical studies have been directed towards the elucidation of charge distributions in nucleic acid bases (22-25), a limited quantity of independent experimental data are available.

Can. J. Chem. Downloaded from www.nrcresearchpress.com by 179.61.180.125 on 03/06/18 For personal use only.

Experimental The bases were all commercial samples (Sigma Chemical Company) and were examined, without further purification, as powders mounted on the spectrometer probe either using double-sided tape or by pressing into finely grooved copper flats fixed to the probe. Spectra o f t h e c , , , N,,, O,,,and F,,core levels were obtained on a McPherson ESCA-36, calibrated as described previously (26), using MgK.,,, radiation (1253.6 eV). Several of the samples were run over extended periods of time, and all were examined visually, to check for radiation damage. The constancy of the ratios of the core level peak intensities and their positions were taken as evidence that no substantial radiation damage occurred. Peak positions in incompletely resolved bands were determined by computer deconvolution using the peak fitting routine described by Bancroft e l rrl. (27). The bands used in fitting a peak envelope are defined according to their centroid position, half-width, shape (an equal combination of Gaussian and Lorentzian distributions), and intensity. These parameters are then varied to obtain a minimum in the error sum of squares between the values for the experimental curve and the trial combination of bands. An important feature of this program is its ability to constrain certain parameters of selected bands within set limits, while allowing the others to vary freely. Peak parameters for the intensity of a given core line were constrained to give peak areas proportional to those required by the molecular formula. C I S peak widths were constrained to half-widths (FWHM) between 1.4 and 1.G eV, since preliminary studies showed that peak widths for all carbon sites investigated fell in this range. N,, half-widths were constrained between 1.3 and 1.4 eV. F i g ~ ~2r eshows some examples of envelopes deconvoluted in this manner. Binding energies were determined relative to the C,, peak ascribed to hydrocarbon contamination, which was assigned a value of 285.0 eV (28). This peak gradually increased in intensity with time as residual hydrocarbons deposited on the sample surface.

Results and Discussion ESCA Spectrcr Table 1 gives the experimental binding energies for the bases examined, with corresponding sites in the purines and pyrimidines listed in the same column. In Fig. 2 are some representative examples of deconvoluted C,, bands. The reproducibility of the deconvolution procedure, and of the spectra themselves, was checked for several of the compounds by processing spectra obtained in repeated measurements on a number of different sampl&. For the pyrimidines the absolute binding energies determined in this fashion generally agreed t o better than f 0.15 eV (20), while the relative binding energies for peaks under any one envelope were reproducible to better than f0.10 eV. In the case of the purine bases, the generally greater number of peaks under a given spectral envelope, combined with a smaller spread in binding energies, resulted in increased uncertainties

BINDING ENERGY, eV FIG. 2. Computer deconvoluted C,, bonds for

((1)

uracil,

(6) 5-H~1orouracil,(c) 5-azauracil, and ((1) 6-azauracil. T h e

hydrocarbon contamination peak used for reference is t h e peak at the lowest binding energy. For clarity, the full extent of each Gaussian-Lorentzian band has been truncated in t h e fitted spectra.

in peak positions, the standard deviations being about f0.2 eV for the C I S bands. The poorly resolved N , , bands of the purines presented some difficulties. For xanthine, visual inspection of the N spectral region showed that it consisted of two poorly resolved peaks of relative intensities 3: 1. Computer deconvolution based on this assu~nptiongave a good fit to the experimental spectrum, the peaks being o f equal width. The linewidth was about the same a s that for N , , peaks in the pyrimidines, so the reported binding energies are probably accurate to better than f 0 . 3 eV. A similar approach t o the hypoxanthine N,, band showed that it could be fitted well by two peaks of equal intensity. However, for 8-azaxanthine, guanine, and 8-azaguanine, the N,, envelopes consisted of featureless bands, and the deconvolution procedure consisted of a random fitting under t h e broad spectral envelopes of the number of peaks o f equal intensity required by the molecular formulae.

,,

PEELING ET AL.

The N , , binding energies listed in Table 1 for these latter compounds give a satisfactory but not necessarily unique fit to the experimental spectra. It is difficult to assess quantitatively the accuracy of these data but a reasonable uncertaintv in the bindine energy of any one component peak is 2 0.4 eV. The N , , peaks for uracil, 5-fluorouracil, and thymine consisted of single peaks with linewidths indistinguishable from those found for chemically unique N,, peaks. The same is true in the case of the O,, bands; the peaks observed for the single oxygen in hypoxanthine, guanine, and 8-azaguanine were not sharper than those of the other bases which contain two chemically distinct oxygen species. The lack of resolution in the ESCA technique which becomes apparent in the above discussion is likely to be the limiting factor in carrying out detailed structural studies of the type reported here. The number of incompletely resolved peaks under a given core level band that can be determined uniquely is generally quite small, so even moderately complex molecules like the purine bases are often not amenable to a thorough analysis. Although improved resolution, for instance using monochromatic X-ray sources (29), undoubtedly helps t o some extent, the limited resolution is inherent in the method and the complexity of the molecules that can be studied is thus restricted.

0

m t-

w V l V l V 1 0 o V l

"9N.????


-

Assignment of C,, Sj)ecti.a The assignments of the deconvoluted C,, spectral peaks can be carried out using known substituent effects on core electron binding energies (29). The factors giving rise to ESCA chemical shifts in molecular solids are generally short range in nature. Although in the case of the nucleic acid bases, intermolecular effects, such as hydrogen bonding, may not be insignificant, they are likely to be of secondary importance in comparing similar molecular sites. That they are of secondary importance becomes clear on considering the binding energy data in Table 1. Where the immediate environment of the C(2) carbon is unchanged through the series of compounds, the binding energy remains fairly constant. Replacing the 2-keto oxygen of xanthine and 8-azaxanthine with the less electronegative -NH, group results in a decrease in the binding energy of this carbon. A further decrease in binding energy group t o form occurs on removing the -NH, hypoxanthine. Si~nilarly C(4) shows a significant change in binding energy only for 5-azauracil and 5-fluorouracil, where the substitution of the nearby electronegative group induces an increase in binding energy at this site. As expected, the fluorine substitution also gives rise to an increase in the binding energy of C(6), and to a much more substantial

-NCI-Nm m m m m m mmmvlmm

C0.mm.m': . . m m m m m m m m m m m w m m

momma

d-;ddi

0 b w 0 +0d0U0 d

d

d

Y? u

m w m m ~ !

m

13

o ;o;oo;o m m o m o

-

.2L

m m d m d

3

a a

VI

0

.d

0

0 d

.--

d

c)

:

0

-

13

.5 2 a al

5

m m O O O V l V I O

"9:-.???

"9901"9? o-oo-

L

%S%S$

z

z-M

.a P

a, M

.v .-

O O O V l V l O

m. CI. PI .

-1 m CI 0-330-

%$SF?

%$l l l l l l d t-

2

N

4 4

aJ

m o o m y w 0 o ; o m m o e m m co f m mm t-0

v,

wI oPoI C

N

dG I$ I

V I O

9 i

-o

. . .

w

o

o

Ill0

Cl

VI

. -. ~. .C . I

N

-

U -I W

m

G

m m w NNCI V I O

. .

W -

mtm w N N

N

m w m m m N P I Cl N N

V I V I O

-COW?

mv-,\Dmm "9?9'=?=?

NNCI

w w m w w CI P I N CI CI

I d Gd w m w

V I O O O V I V I

?77?r-': w m m w w m m w m m m w NNNCICIN

m. o .o m .y mooww wooww CI N C I N

m. m m N

V I V I O O O O

t -. m. m.w .w ". . m m m m m m w m w w w w N N N ~ I N N

-.-

2 E.=.= .-E =ssa,x g 2 2.55 0

~

t-P-t-t-t-

T"???':

mO M P -OoW \m Mm W Cl PI C I CI CI

an,

aJ

55

'2

.S .5

c

xzoboob

1558

C A N . J . CHEM. VOL.

56. 1978

TABLE 2. Expel.imental and theorctical C(J)

C(5)

C(6)

CtI,

0.44(045)* [0.33]t 0.40(0.44) 0.46(0.46) 0.39(0.34) 0.42(0.45) 0.40(0.45)

0.33(0.37) [0.271 0.32(0.33) 0.45(0.42) 0.32(0.35) 0.32(0.37) 0.32(0.33)

-0.05(-0.16) [0.011 0.10(0.08)

O.ll(O.18) [0.261 0.13(0.10) 0.21(0.26)

-

0.01(-0.03) - O.OJ(-0.09) 0.03(0.05)

O.OS(0.15)

0.4X0.45) 0.17(0.24) 0.42(0.44)

0.33(0.35) 0.33(0.35) 0.33(0.36) 0.34(0.36) 0.36(0.36)

-0.02(-0.08) -0.04(-0.06) -0.03(-0.06) -0.02(-0.11) -0.05(-0.12)

O.l-l(O.21) O.lJ(0.20) O.lJ(0.19) 0.16(0.22) 0.17(0.23)

C(2)

Compound

Uracil


5-Fluoro~~racil 5-Azauracil 6-Azauracil Thymine 6-Azathymine

Xanthinc 1-lyposanlhine I-Azasruithine Guanine 6-Azaguanine

0.35(0.39) 0.37(0.39)

-

-

N(I)

N(3)

-

-

0.02(-0.00) 0.03(-0.02)

'Values in parentheses represent theorelical charges calculated by CNDOI2 molecular orbital method. t Squ;~rcbrackets contain e\perimental charges for uracil obtained from I-shell S T 0 scattering faclors (ref. 36)

increase in that of C(5). Similarly, C(5) and C(6) in the 6- and 5-azapyriiiiidines, respectively, show higher binding energies, a consequence of replacing a ring carbon with a Inore electronegative nitrogen. However, in the purine bases, the influence of the imidazole nitrogens 011 the binding energies of the analogous carbons (C(5) and C(4)) is considerably smaller.

Assigilme17t of N , , Spectra In spite of the difficulties discussed above in determining the N,, binding energies, a qualitative analysis o f substituent effects can be used in assigning the peaks. The data in Table 1 show that the binding energies of N(3) in the pyrimidine bases and N(1) of the purines, being isolated from major structural changes, are constant t o within i O . 1 5 eV. An increase to 401.20 eV for 5-fluorouracil is the largest change. F o r the 6-azapyrimidines, N(I) is closer to the structural change and an increase in binding energy is apparent here as well as for 5-fluorouracil. In the purines hypoxanthine, guanine, and 8-azaguanine, the binding energy of N(3) decreases by 1.2 eV relative to xanthine. The lower binding

-

\

\

N co~iiparedto the ,N H structural 4 feature has been noted before (1) and is also evident in the case of N(5) and N(6) in the azapyrimidines. Similarly, N(7) in xanthine, hypoxanthine, and guanine has a binding energy of -399.6 eV, while N(9), whose immediate environment is similar t o that of N(l), is assigned t o a component a t -400.9 eV. (An X-ray crystallographic study of guanine (30) has shown that only the tautomeric form having N(9) protonated is f o r ~ n e din the crystals; we assulne that this is also the case for xanthine and hypoxanthine.) In 8-azaxanthine N(8) is protonated and N(9) is unprotonated (31). T h e immediate environments of N(7) and N(9) are thus similar and they are assigned

energy for the

identical binding energies, which, because of t h e influence of N(8), are greater t h a n those of other \

N structural features. The lU(8) nitrogen, being

4

adjacent t o two other nitrogen atoms, is assigned t o the N ,, peak at highest binding energy. The structure of 8-azaguanine (32) on the other hand shows t h a t N(9) is proto~iatedinstead of N(8), so N(7) reniains lower in binding energy than N(9). The limited resolution does not permit a distinction in the binding energies of N(8) and N(9). The --NH, nitrogen in guanine and 8-azaguanine is assigned to the peaks a t 399.5 and 399.6 eV, respectively. Barber and Clark (13) previously have assigned a peak a t 399.5 eV t o an -NH, group in the structurally similar adenine molecule. I n 5-azauracil the assignment o f the N,, peaks a t 401.20 and 400.60 eV to N(1) and N(3), respectively, is not obvious from substituent effects. Therefore, C N D 0 / 2 calculations were carried out for this molecule, and the calculated atomic charges were used to determine the relative binding energies through application of the charge potential model (see below). The order of the N,, binding energies in Table I was confirmed. Further support for this ordering is found in the results o f detailed nb initio ~nolecularorbital theory studies (25, 33), which also show the Is binding energy of N(1) t o be greater t h a n t h a t of N(3).

Tautomeric For/,is T h e binding energy data in Table 1 also provide information on the tautomeric forms of the bases. I n general, the diketo forms of uracil and its derivatives predominate in t h e crystal (as well as solution) state (24, 34), and the N , , binding energies and the C,,y binding energies f o r C(2) and C(4) are in accord w i t h this structure for each of our pyrimidines. Thus, experimental and theoretical studies on model com-

PEELING ET A L

atomic charges in the pyrimidine a n d purine bases N(6)

-

-

-0.35(-0.37)

-0.29(-0.36)

-

-

[-0.301 -0.32(-0.36) -0.40(-0.37) -0.35(-0.3)

-0.34(-0.19)

1-0.281 -0.26(-0.31) -0.30(-0.36) -0.31(-0.33) -0.30(-0.33)

-0.35(-0.19)

-0.30(-0.33)

-0.23(-0.28)

-

-


00)

N(5)

0.00 (0.00)

-0.02(-0.04) N(7)

N(8)

N(9)

NH2

-

-0. IS(-0.13) -0.15(-0.11)

-

-O.lO(-0.16)

-0.06(-0.16) -0.01(-0.04) -O.lO(-0.16) -0.04(-0.05)

I

0.04(0.07)

-O.14(-0.16)

-O.IS(-0.12) O.OS(-0.07) -0.14(-0.17)

pounds have shown that the C,, binding energy of an alcoholic carbon is about 1 eV lower than that of a ketonic carbon (35). Furthermore, in the en01 form \

I

I

,

either N(1) o r N(3) (or both) assumes the / N structure, the N,, binding energy of which is sub\

stantially lower (- 1.5 eV) than in the ,NH

unit.

N o crystal structure of 5-azauracil has appeared as yet. Our C,, and N,, binding energies show n o evidence of the en01 form, the only significant change compared to uracil being an increase in the binding energy of C(4) owing to the influence of N(5). Therefore, it is apparent that this base also exists in the diketo form. Similar arguments utilizing the binding energy data for the purine bases indicate that xanthine and 8-azaxanthine exist in the diketo structure, while in hypoxanthine, guanine, and 8-azaguanine the keto form is assumed at C(6). This is consistent with crystallographic studies on 8-azaxanthine (31), guanine (30), and 8-azaguanine (32). A study of selectively-methylated bases is planned in order t o examine more thoroughly the influence of tautomeric forms on the ESCA spectra. Cliarge Distributions In the electrostatic potential model (36) core electron binding energies E, for a ~nolecularsolid are related to the molecular charge distribution by

where E: is a reference level, q, is the charge on atom i, qj is the charge o n atom j situated a t a distance rij from i, and lci is a constant. The determination of molecular charge distributions from experimental binding energies using eq. [ l ] requires knowledge of the parameters lci and E: for the

-0.37(-0.24) -0.40(-0.23)

O(4)

o(2)

F

-0.25(-0.18)

O(6)

-0.36(-0.38)

-0.30(-0.36) -0.33(-0.38) -0.32(-0.33) -0.32(-0.39) -0.35(-0.38)

0.37(-0.39)

-

different core levels. Clark and T l ~ o i n a s(36) used the C N D 0 / 2 ~nolecularorbital formalism to determine charge distributions in a number of model carbon and oxygen compounds. These charges were used with the experimental binding energies and rnolecular geometries to yield values of lci and E y for the C , , and O,, levels via eq. [I]. We used a similar approach with published N binding energies for a number of simple compounds examined in the solid phase and values of k, = 18.5 f 2.5 and E,' = 399.7 f 0.5 eV were found. In a system composed of C, N , a n d 0, for which the geometry and all the core level binding energies are known, the potential model eq. [ I ] are sufficient to determine all the atomic charges q,, and the neutrality requirement gives one additional equation. However, the nucleic acid bases all contain several hydrogen atoms in addition to C, N , and 0 , so it is necessary t o make some approximations. Experience with C N D 0 / 2 calculations on a variety of molecular systems has shown that in neutral molecules the charge on a hydrogen atom bonded to a carbon is small, whereas hydrogen in an N-H bond has a deficiency of about 0.10 t o 0.15 electrons. For all the bases reported here we have therefore set qk,= 0.0 for C-H and q, = 0.13 for N-H hydrogens. With these approximations there are enough e q u a t i o ~ ~for s 5-fluorouracil to determine the charges on the C, N, 0 , and F atoms, while for the other bases there is one equation more than required to find the charge distributions. The internuclear distances, rij, required in eq. [I 1, were determined from geometries obtained in X-ray crystallographic studies of uracil (37), 5-fluorouracil (38), 6-azauracil (39), thymine (40), 6-azathymine (39), guanine (31), 8-azaguai~ine(32), and 8-azaxanthine (31). The geometries of 5-azauracil, xanthine, and hypoxanthine were estimated by comparison with the experimental geometries of the other bases.

,,

1560

C A N . J . CHE M. VOL. 56. 1978

The experimentally determined charges are given in Table 2. Uncertainties in the values originate to some extent from uncertainties in the binding energies but mainly from uncertainties in the values for ki and E:. The experimental N and O,, binding energies used to determine the constants appropriate to these core levels spanned rather limited ranges, so these constants are less certain than those for the C I S levels. Based on the precision of the experimental binding energies and the constants, uncertainties in the experimental charges are estimated t o be: N I X , 0.08; O,,, 0.06; and C I S ,0.03 electrons. The values of k iin eq. [I] depend on the definition of atomic charge and, when determined using a molecular orbital theory treatment, on the basis set used. The values used in this work were derived from C N D 0 / 2 molecular orbital calculations, so it is appropriate to compare our experimental charges to charges calculated within this theoretical framework. Using the molecular geometries described above, atomic charges were calculated by the C N D 0 / 2 molecular orbital method, and the results are included in Table 2 (in parentheses), Using X-ray scattering factors, Stewart (41) has also obtained an experimental estimate of the charge distribution in uracil, and his data, obtained from L shell Slater type orbitals (STO's) are included in Table 2. The CNDOI2 charges refer to free molecules while the ESCA data refer to the solid phase, where such effects as intermolecular hydrogen bonding may be expected to affect the molecular charge distributions. However, the agreement evident in Table 2 between the experimental and theoretical charges is very good and lends support to the original ESCA peak assignments. For some of the bases, different assignments were assumed and the charges recalculated. In all instances this yielded charges further removed from the theoretical results and in most cases the calculated changes were strongly changed by the alternative assignment. For uracil, and the pyrimidines in general, some consistent trends in the experimental and theoretical results for the relative atomic charges are evident. The larger uncertainties in the experimental data for the purines possibly mask some of the more subtle differences but the major trends in variations in the charge distributions are reproduced. The chemical and biochemical i~nplications of differences in the electron distributions in the nucleic acid bases have been discussed extensively (22-25) and comments here are restricted to a brief mention of inconsistencies between the experimental and theoretical numbers. In the pyrimidines the most notable difference is at the 5 and 6 positions of uracil, 5-azauracil, and thymine. The theoretically


,,

calculated electron excess at the 5 position is greater than that indicated by the ESCA results, and the corresponding electron deficiency at C(6) is also greater. It has been noted that use of [ I ] with C N D 0 / 2 charges predicts the wrong ordering of the C , , levels in 5-azauracil (25, 33), a consequence of the apparent over-polarization o f the 5-6 bond si~ggested by the CNDO/2 calculations. In the purines the magnitudes of the calculated charges a t C(5) and C(4) also exceed those determined by the ESCA procedure. At N(7) the ~nolecular orbital theory calculations show the negative charge t o be greater than the ESCA-derived value. Finally, major disagreement is apparent between the theoretical and experimental charge at the -NH, nitrogen of guanine and 8-azaguanine. Some of these discrepancies could result from effects such as hydrogen bonding o r from the hydrogen charge approximations made in the CNDO/2 calculations. However, a more likely reason for the discrepancies resides in the C N D 0 / 2 method itself which, using Koopmans' theorem, does not take account of core hole relaxation energy differences in differing sites in the molecule. Clark and co-worlters (25) have made rrb itiitio calculations of the neutral and core hole states in 5-azauracil and have shown that the C I S and N , , relaxation energies in this molecule differ with the chemical environment. They have suggested that specific group relaxation energy shifts tin be used t o refine charge calculations which are based on the charge potential model. We intend to apply such procedures to determine if the discrepancies observed in this work c a n be completely accounted for by relaxation effects.

Conclusions This study has shown that in favourable cases application of the charge potential model (eq. [I]) with experimental core electron binding energies can yield atomic charges in the pyrimidine and purine bases. The accuracy of the results f o r small molecules is sufficient to study differences in electron distributions originating in structural modifications. Difficulties in assigning the ESCA spectra, and in the subsequent derivation of charges, arise from the somewhat low resolution of the technique, and apparently limit the complexity of the systems amenable t o this sort of analysis. The pyrimidine bases are readily analyzed, while t h e purines appear to be close to the limit of complexity for thorough analysis. Acknowledgements This research was supported by operating grants from the National Research Council of Canada and

PEELING ET AI..

21. J . A. POPLEilnd G. A. SEGAL.J . Chem. Phys. 43. S 136 (1965). N A. PULLMAN Prog. . Nucleic Acitl Res. 22. B. P U L L M Aand Mol. Biol. 9, 327(1969). 23. B. P U L L M A and N A. P U L L M A N Atlv. . Hete~.ocycl.Cheni. 13.77 (1971). 24. J . S. KWIATKOWSKI ilnd B. P U L L M A N Adv. . Heterocycl. Chem. 18, 199 (1975). , W. S C A N L A Nilnd , J . MULLER.Theor. D. T. C L A R KJ, . PEELING.and L. COLLING.Biochini. 25. D. T. C L A R K1. Chim. Acta. 35,341 (1974). Biophys. Acta, 453.533 (1976). and M. G. COOK.Anal. Chem. 47. 2208 H. RUPPand U. WESER.Biochim. Biophys. Acta, 446. 151 26. N. S . McIN-VYRE (1975). (1976). I. ADAMS.L. L. COA-I.SWOR.I.H, C. D. 27. G. M. BANCROFT, M. M. M I L L A R D Adv. . Exp. Med. Biol. 48.589 (1974). BENNEWI-I.Z, J . D. B R O W Ni~nd , W. D. WES-rwoor~. Anill. H. RUPPand U. WESER.Bioinorg. Chem. 5.21 (1975). Chem. 47,586(1975). J . PEELING. D. T. CLARK.I. M. EVANS.and D. BOULTER. 28. U. GELIUS.E. B A S I L I E R S ., SVENSSON. T . BERGMARK, ant1 J . Sci. Food Agric. 27, 331 (1976). K. S I E G B A HJN. .Electron Spectrosc. Relat. Phenoni. 2,405 J . PEELING, B. G . HASLETT,I. M. EVANS,D. T. C L A R K , (1973). and D. BOULTER. J . Am. Cheni. Soc. 99, 1025 (1977). R. M. M. M I L L A R and D M. S. MASRI.Anal. Chem. 46. 1820 29. U. G E L I U SP., F. H E D E NJ, . H E D M A NB., J . LINDBERG. M A N N ER. , NORDBERG. C . NORDLING, (1974). ilnd K. S I E G B A H N . P. T. ANDREWS, C . E. JOHNSON. B. W A L L B A NR. K,CAMPhys. Scr. 2,70(1970). M A C KD. , 0 . HALL.and K. K. RAO. Biochem. J . 149, 471 30. U. THEWALI-, C. E. BUGG,and R. E. MARSH.Acti~Crystilllogr. B. 27,2358 (1971). (1975). L. D. HULETTand T. A. CARLSON. Clin. Chem.677 (1970). 31. H. C . MEZilnd J . DONOHUE. 2. Krist. 130, 376(1969). M. M. M I L L A R D J ., P. MACQUET, ilnd T. T I ~ E O P H A N I D E32. S . J . SLET.I.EN, E. SLETTEN, and L. H. J E N S E NActs . CrystillBiochim. Biophys. Acta, 402, 166 (1975). logr. B, 24, 1692 (1968). J ., MULLER, and D. B. ADAMS. U. WESER,G. J . STROBEL, and W. VOELTER. FEBS Lett. 33. D. T. C L A R KI., S C A N L A N 111 Proc. SRC Atlas Symp. No. 4, Oxford. April 1974. 41,243 (1974). ilnd U . WESER.Hoppe-Seyler's Z. Physiol. W. H. THOMAS Eciireci by V. R. Snunders and J . Brown. 1974. p. 235. Chem. 358.47 (1977). 34. D. VOETilnd A. RICH.Prog. Niicleic Acid Res. Mol. Biol. M. BARBERilnd D. T. C L A R KChem. . Conimiin. 23 (1970). 10. 83 (1970). 35. D. T . C L A R K111 . Electron emission spectroscopy. Etlirrti M. BARBER and D. T. CLARK. Chem. Comniun. 24 (1970). by W. Dekeyser. D. Reidel Publ. Co., DOI-drecht.Hollancl. A. V A N D E R AVOIRD. Chem. Comniun. 727 (1970). R. R E I N A. , HARTMAN, and S . NIR. Israel J . Chem. 10, 93 1973. p. 373. 36. D. T . CLARKand T . R. THOMAS.J . Polvm. Sci. Polvm. (1972). A. ISHIDA,H. KATO,H. NAKATSUJI, and T. Y O N E Z A W A . Chem. Ed. 14, 1671 (1976). Bull. Chem. Soc. Jpn. 45, 1574 (1972). 37. R. F . STEWAKI'and L. H. JENSEN.A ~ t i lC1.yst21ll0gl.. 23, A. IMAMURA, H. FUJITA,ilnd C . NACATA.J . Am. Chem. 1102 (1967). Soc. 94,6287 (1970). . B, 29,2549 (1973). 38. L . F A L L O NActaCrystnllogr. c polymeric 39. P. S I N G Hand D. J. HODCSON. J . Cheni. S o c . Chem. ComD. T . C L A R K ESCA . applied to o ~ g i ~ n iand systems. 111 Handbook of X-lxy and iilt~.aviolet photomiin. 439 (1973). electron spectroscopy. Edirrd by D. Briggs. Heyden and 40. T . S A K U R A:111d I. Crystallogr. B, 27. I M. O K U N U KActi~ Son Ltd.. London. 1977. 1445 (1971). J . Chem. Phys. 53,205 (1970). 41. R. F . STEWART. J . SKODA.Prog. Niicleic Acid Res. 2, 197 (1963).

the R h Institute of Manitoba. Thanks are due to D. G. Zetaruk, Atomic Energy of Canada Limited, and K. Marat, University of Manitoba, for computing assistance.


I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I.

12. 13. 14. 15. 16. 17. 18. 19.

20.

1561