Electronic spectra of some pterins and deazapterins

Chemical Physics 179 (1994) 55-69 North-Holland

Electronic spectra of some pterins and deazapterins Paul Wormell School ofScience, University of Western Sydney, Hawkesbury, Richmond, NSW 2753, Australia

and Jill E. Gready * Department of Biochemistry, The University of Sydney, Sydney, NSW 2006, Australia Received 3 1 March 1993; in final form 10 September 1993

The electronic absorption spectra of some derivatives of pterin and NSdeazapkxin are analysed using the CNDO/S-CI method, including allowance for solvent shifts. These calculations give good agreement with the spectra, which may be assigned to a group of ti+x transitions. There are some differences between calculated values for gas-phase and solution models but their general level of conformity with experiment is similar. Band shifts caused by methyl substitution, protonation and replacement of ring nitrogens are investigated, and a number of structuraland spectroscopic problems are addressed. The spectral predictions agree well with experimental assignments for tautomeric forms and protonation sites for known compounds, and predictions are made for the spectra of NBdeazapterin and N5,NSdideazapterin which have not been reported.

1. Introduction The electronic spectra of pterin and its derivatives have been important in establishing the structures and properties of a wide range of naturally-occurring compounds [ 11, and they are now being used similarly to study new pterin-analogue families of potential antitumour agents [ 2,3]. Pterins are the principal naturally-occurring derivatives of pteridine; this group includes many pigments and the enzyme cofactor folic acid. Structures of pterin and some related molecules are shown in fig. 1; the atomic numbering convention is also shown. 8-Substituted pterins and Nkkazapterins are two new families of mechanism-based compounds active as substrates and inhibitors respectively of dihydrofolate reductase (DHFR) which are being investigated as part of a drug design program [ 3,4]. Their electronic spectra are characterised by strong absorption in the ultraviolet and blue/violet regions, and many also fluoresce strongly. Band energies and intensities are sensitive l

Corresponding author.

P@rin

N3(H) tautomer

li

A NB(H)-pterin

Nl (H)-pterin

enol pteltn

b3 8-methyl ptenn

NB-deazapterin

bHS N3*-8-methyl pterin

NB.N8diieazapterin

Fig. 1. Molecular structures of some pterins and deazapterins.

to the tautomeric form of the molecule, the pattern of methylation and state of protonation. Band shifts, together with changes in quantum yield and solvent

0301-0104/94/$07.00 Q 1994 Elsevier Science B.V. All tights reserved. SSDZ 0301-0104(93)E0338-V

56

P, Wormell,J.E. Gready/ ChemicalPhysicsI79 (I 994) 55-69

shifts, are being used to characterise the fluorescence spectra to help in the development of a fluorimetric probe of the binding to DHFB [ 5 1. Two other interesting related molecules are N8deazapterin and NS,N8dideazapterin, neither of which has yet been synthesised but for which folate derivatives have been reported [ 6 1. Although many spectra of pterin derivatives have been tabulated and reviewed [ 7 1, the electronic transitions which produce them have not been studied in any detail. In this paper we use semiempirical MO calculations to characterise the transitions and study the band shifts for the different molecules with their associated tautomeric and protonated forms. The calculations confirm a number of structural assignments, and predict spectra of some other recentlysynthesised or unknown compounds. As the CNDO/SCI method [8] has been used successfully on many other one and two-ring heterocycles [ 9, lo] we have used it to model the electronic transitions, and checked the predictions against known experimental data. As experimental geometries are not available for the pterins and deazapterins, and our initial calculations showed some sensitivity to input geometry, we have tested the stability of the spectral predictions with theoretical geometries at the AM1 and SCF/STG-3G, 3-21G and 63 lG# levels [ 1l-l 31. These compounds are difficult to crystallize, so few X-ray structures are available for comparison [ 141, but where data are available theory at the 3-21 G and 6-3 lG** levels shows good agreement.

2. Methods Pterin was from the Sigma Chemical Company. The 8-substituted pterins and NS-deazapterins were gifts from Mark Keen [ 15 ] and Michael Ivery [ 3,16 1. W-visible absorption spectra were recorded at room temperature using a Cary 3 spectrophotometer. Since p& values are known for all these compounds [ 2,3,16,17], spectra were recorded in aqueous phase at two different pH values, corresponding to the protonated and neutral species. CNDO/S-CI calculations were carried out using the program of Del Bene et al. [ 81 with input ab initio SCF geometries from Gready [ Ill. The CI calcula-

tion included the 100 lowest singly-excited configurations (typically up to an energy of 100000 cm- ’ ) . This did not include all ti*ex configurations, but those which were excluded had higher energies than the lowest doubly-excited configurations, which were also excluded. Solvent shifts in water were approximated using the non-polar&able spherical-cavity model [ 18,19 1. The solvent-cavity radius was taken to be that of a sphere with the same volume as the van der Waals volume of the solute molecule.

3. Results and discussion 3.1. Pterin spectra The electronic spectrum of pterin illustrates some of the general conclusions which can be drawn from this study. A summary of predicted band energies, oscillator strengths and transition types is given in table 1, with solution-phase spectral data for comparison. Several x*+x and x*+-n transitions are predicted to occur between 200 and 400 nm. The titn transitions have not been observed experimentally since pterin and its derivatives are virtually insoluble in non-polar solvents, and x*tn bands are usually blueshifted under stronger x++n absorptions in water or other polar solvents [ 201. The calculated transition energies are sensitive to input geometry: for example, predictions for the lowest-energy x*+-n band range from 24960 (3-21G) to 27270 cm-’ (SIG-3G). The strong lictlc transitions which produce the observed absorption spectra show similar sensitivity. They are predicted to occur at about 33000 and 37000, and above about 45000 cm-‘. Experimentally, strong relatively featureless bands are seen at 29 500,37 000 and above 40000 cm-‘, as shown in fg 2. The energy of the first of these is thus overestimated by about 3500 cm-‘, a typical result for this class of compound, as discussed below. Predictions for the four theoretical geometries give the same number and ordering of rr*cx transitions below 50000 cm-‘. The predicted band intensities (expressed as oscillator strengths, j) are similar for the various geometries, but the predicted band energies show some variation. The li+ex transition energies obtained from AM 1 ga ometries are consistently lower than those from the

P. Wormell,J.E. Gready/Chemical Physics179 (I 994) 55-69

57

Table 1 Comparison of CNDG/SCI predictions for pterin using different theoretical input geometries. Y=wavenumber (gas phase) (in cm-‘); &oscillator strength; Arkcalculated solvent shift (in cm-‘) Band type

6-31G” pv)

rP+n titn

26010(0.007) 32240(0.0004) 34090(0.26) 36900(0.42) 37010(0.0005) 38380(0.0001) 40910(0.009) 47360(0.16) 51790(0.12) 52090(0.002) 52990(0.48)

SIG-3G

3-21G AP -220 320 - 1720 1010 790 - 1070 230 1400 -1770 760 -80

PO

AV

24960(0.006) 31800(0.0004) 34090(0.27) 37220(0.42) 35750(0.0003 ) 37400(0.0008 ) 39870(0.008) 47610(0.17) 51460(0.16) 52870(0.002) 53020(0.44)

- 190 27270(0.008) 180 32230(0.0004) - 1910 32990(0.27) 1140 37470(0.35) 1030 38590(0.0001 ) -670 40720(0.005) 490 42900(0.006) 1730 46550(0.15) - 1600 50440(0.20) 1000 50760(0.0001 ) -40 52150(0.61)

‘) Absorption spectrum in aqueous solution

AP

YY)

Experimental

AM1 vcf)

- 120 26750(0.007) 340 31660(0.0003) - 1480 32000(0.29) 780 34230(0.39) 160 37130(0.001) 130 38760(0.001) -80 40620(0.007) 1110 44280(0.12) -2720 49270(0.23) - 1410 52330(0.003) -780 50840(0.47)

AV

v

-240 500 - 1740 29500 ‘) 1520 37000a’ 790 - 760 -20 1660 42900 ‘) - 1620 1830 150

[ 241.

3-2 1G geometries by 2000-3400 cm- ’ for pterin and the &methyl derivatives. The STO-3G minimal-basis geometry gives energies which are within 1100 cm- ’ of those for 3-21G, and are usually lower. The 3-2 1G and 6-3 1G” results are typically within 350 cm-’ of each other for transitions up to about 50000 cm-‘, but with the occasional larger difference, especially for higher-energy bands. Typical experimental uncertainties for the corresponding solution band maxima are IL120 cm-’ for a band at 25000 cm-’ (+2nmat400nm) and f400cm-‘forabandat 45 000 cm-’ ( + 2 nm at 222 run), so for our purposes the 3-2 1G geometries are reasonable approximations to those obtained with the higher-level 63 1Gee basis set, which might be expected to be more accurate. Since the 3-2 1G calculations require much less computer time, especially for methylated sps ties, we have used these geometries as standard inputs for the spectroscopic calculations. The calculated solvent shifts typically have magnitudes of 1000 up to 1900 cm-’ for the X*+-Xtransitions. The differences in transition energy for the various input geometries are accompanied by differences also in solvent shift, as might be expected, but this does not affect the conclusions which we have drawn above. The overall effects of the calculated solvent shifts on the correlation between theory and experiment are discussed below.

3.2. Spectral comparisons Table 2 summarises the first five predicted rP*crt transitions for 37 molecules and cations, together with experimental data where available. Experimental band energies are those of the band maxima for the reasons set out in ref. [ 211. The species comprise various tautomeric and protonated forms of pterin, NSdeazapterin, NSdeazapterin and NS,NSdideazapterin, and a range of mono-, di- and u-i-methyl derivatives (neutral and protonated) of pterin and N5deazapterin. Typical absorption spectra of neutral and protonated 8-methyl pterins and NSdeazapterins in aqueous solution are shown in figs. 2a and 2b, and spectra of pterin and N 1+-pterin are shown in fig. 2c for comparison. These spectra exhibit three regions of absorption: A, B and C. For some species regions B and C are predicted to contain composite bands, so we refer to transitions Bl, B2, Cl and C2. We are interested only in bands below about 50000 cm-’ since they are accessible experimentally and CNDO predictions become unreliable at higher energies unless multiply-excited configurations are considered 191. Band A is especially interesting since it is well separated from the other major bands, contains only one transition, is sensitive to molecular structure, and correlates with the fluorescence band used in DHFR studies. Theoretical predictions agree well with ex-

P. Wormell,J. E. Gready/Chemical PhysicsI 79 (I 994) 55-69 ENERGY I cm” 4wOO

2OOw

2oOoO

(a)

perimental trends, but consistently overestimate the band energy. In fig. 3 gas-phase theoretical energies are plotted against experimental values for 24 species. A linear least squares fit for the complete data set is satisfactory, and there is even better fit within groups of closely-related molecules. Analysis of the CI coefficients reveals that, for all but one species, band A is mainly attributable to the (LUMO)n* c (HOMO)x one-electron promotion. The excep tion is the neutral form of NSdeazapterin, for which the transition is a combination of (32)x*+ (30)x, (31)ft(28)x and (31)x*+ (30)x, with the first of these predominating.

400

300 WAVELENQTH

I nm

3.3. Solvent dependence of spectra

ENERGY I cd

(:I

r

I f\

I

I I

2(1Iwo ,

2OOOo

4OOw

W ----N3+-y~

I

-mycNwawpM

I I

A

260

400

WAVELENQTH

I nm

ENERGY/ cd

4oooo

2woo

2owo

a00 WAVELENGTH

400 I nm

Fig. 2. Absorption spectra in aqueous solution of (a) 8-methyl pterin and N3+-8-methyl pterin; (b) 8-methyl-NS-deazapterin and N3+-8-methyl-NS-deazapterin; (c) pterin and Nl +-pterin.

The discrepancies between the calculated and experimental band energies could, in part, be caused by solvent shifts in aqueous solution. The typical blue shift of an x’cn transition is well known, but tiex bands may also be shifted, We are investigating the possibility that environmentally-induced shifts may be a useful probe in DHFR binding studies [ 21, and there is considerable current interest in accurately calculating band shifts in various solvents, including water [22,23]. We have calculated a major component of the solvent shift to test whether this approach gives better predictions of band energies for this class of molecules. The ground- and excited-state dipole moments generated by the CNDG program were used to calculate solvent shifts based on the non-polarizable spherical-cavity model [ 18,19 1. Large changes in dipole moment occur during electronic transitions in pterin-like molecules, so these calculated shifts should make a substantial contribution to the total shift. As shown in table 2, shifts of up to 6400 cm-l for the A band were calculated. There are, of course, other contributions to solvent shifts but these are harder to calculate even for smaller molecules. The simple model we have used has several shortcomings: the solventcavity radius is a rather arbitrary parameter, the polarizabilities of the solvent and solute have not been considered, and neither have specific solvent-solute interactions [ 19,22,23]. However, as discussed below, this approach is sufficient to test whether the inclusion of solvent effects is useful at this level of MO theory for these bicyclic species. Gas-phase and solvent-shifted results for the A band

3-21G 3-21G 6-31G” 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 6-31G# 6-31G”

3-21G 3-21G 3-21G 3-21G 6-31G# 6-31G” 3-21G 3-21G 6-3 1G6-31cP

T T+S T E T T+S E T T+S E T T+S T T+S E E T T+S T T+S T T+S E T T+S T T+S E

N8(H )-pterin

Nl(H )-pterin

enol-pterin

N 1+-pterin

N5+-pterin

N8+-pterin

NSdeaxapterin

36100(0.15) 35200 36100(0.14) 35200 31900 f,

29900(0.59) 28400 30000(0.59) 28700 [25900] d,

27500(0.28) 29600

35100(0.26) 41000 35000(0.24) 41000 31800 ‘) 31800

32300(0.29) 32000 [27900] =)

35500(0.28) 32400 [30600] b,

30000(0.43) 25700 30200(0.43) [25100] d,

34100(0.27) 32200 34100(0.26) 32400 [28300] b, 29500 c’ 29400

3-21G 3-21G 6-31G’” 6-31Ga

T T+S T T+S E E E

PteliU (N3(H)-tautomer)

42200(0.02) 40100 [351OO(sh)] b,

37900(0.05) 36400 38200(0.06) [ 309001 d,

Band Bl Jr0

Band A rv)

46900(0.93) 46400 51600(0.25) 56500 52700(0.31) 57000

48000(0.18) 47300 49800(0.03) 48500 50000(0.04) 48700 50700(0.51) 51500 50800(0.56) 51400

44900(0.05) 45000 [43500] =) 44300(0.19) 44700 44200(0.20) 44400 437OO(sh) =r 446OO(sh) 43800(0.31) 45300 49100(0.19) 47400 49200(0.18) 47300 [ 385001 d, 47700(0.45) 48800 48000(0.38) 49000 46900 ‘)

39600(0.20) 39300 [385OO(sh)] =) 37900(0.11) 35700 38000(0.12) 35200 41300 =r 416OO(sh) 35700(0.42) 36300 37800(0.18) 34800 37800(0.18) 34900 [ 365001 d, 37800(0&t) 38600 37700(0.44) 38400 37900 f’

continued on next page

52700(0.27) 50600 48600(0.51) 44300

45600(0.22) 41300 [41700] b,

46500

51500(0.16) 49900 51800(0.12) 50000

51700(0.25) 53000 51500(0.28)

47600(0.17) 49300 47400(0.16) 48800 [41700] b) 42900 f, 43600

37200(0.42) 38400 36900(0.42) 37900 [36500] ‘) 37000 =) 37100

ru,

Band C2

49400(0.18) 45000 49800(0.17)

fro

rv)

40100(0.23) 39000 40100(0.22) [37900] d,

Band C 1

Band B2

and oscillator strengths cr) for some pterins and deaxapterins

Geometry

band energies (F, cm-‘)

Theory/expt solvent a)

and experimental ti+x

Compound

Table 2 Summary of CNDG/S-Cl

3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G 3-21G

T T+S T T+S T T+S T T+S E T T+S T T+S T T+S T T+S T T+S T T+S E T T+S E T T+S E

Nl+-NSdcazapterin

N8+-NSdeazapterin

N8-deazapterin

Nl +-N&deazapterin

NS+-N8deazapterin

NS,N&dideazapterin

Nl+-NS,N8-dideazapterin

&methyl ptcrin

N3+-&methyl pterin

6,8dimethyl pterin

3-21G 3-21G

3-21G 3-21G 6-31G-” 6-31Gn

3-21G 3-21G 6-31G” 6-31G-

3-21G 3-21G 6-31G” 6-31G-

T T+S T T+S E

NI(H)-NS-deazapterin

Geometry

Thwry/expt solvent l)

Compound

Table 2 (continued)

28700(0.44) 24600 24400

29900(0.57) 29200 25900

29600(0.43) 25800 25100

35300(0.17) 40000

34800(0.16) 33900

30100(0.19) 32600

35200(0.18) 39720

34600(0.14) 31900

32400(0.52) 33000 32400(0.52) 33000 29200 ‘)

35800(0.18) 42200 35900(0.18) 42000

31000(0.39) 30900 31000(0.38) 31200 [27900] ‘)

37100(0.01) 36100 31300

37600(0.01) 36000 30900

37200(0.03) 36000

38200(0.07) 32100

39300(0.05) 38500 39400(0.05) 38400

38500(0.01) 39300 38300(0.02) 39300

Band Bl vcf)

BandA FcfI

39200(0.26) 38900 37700

37800(0.19) 35600 365OO(sh)

39800(0.26) 39500 37900

40300(0.02) 45300

38400(0.35) 39400

36000(0.40) 38000

44400(0.15) 42600

37800(0.49) 38400

39OcKqO.17) 38100 39000(0.17) 38200 36600 ‘)

44300(0.10) 47300 44500(0.11) 47000

41200(0.25) 45500 40900(0.25) 45500 [ 388001 I)

flu-I

Band B2

48500(0.16) 43700

49000(0.14) 47600 38500

49100(0.19) 44700

44200(0.32) 42700

45900(0.72) 46200

43800(0.43) 43900

45700(0.17) 48800

46800(0.26) 46600

49500(0.12) 49000 49700(0.11) 49200 47200 ‘)

46100(0.20) 51900 46600(0.18) 52500

48900(0.19) 46200 49200(0.16) 47600 [46100] *)

rcn

BandCl

Band C2

50700(0.12) 50300

49600(0.11) 47300

51 lOO(O.18) 50900

50400(0.88) 48600

48800(0.15) 48900

48000(0.22) 47100

51900(0.98) 45800

49700(0.31) 47000

51600(0.01) 53100 51800(0.01) 53200

51700(0.98) 51800 52000(0.98) 52100

50500(0.03) 54900 50000(0.03) 55300

fro

T T+S E T T+S E T T+S E T T+S E

N3+-6,8dimethyl pterin

7,Mimethyl pterin

7-methylene-S-methyl pterin

N3+-7,8dimethyl

T T+S E

N3+-6,7,8+imethyl

3-21G 3-21G 3-21G 3-21G

T T+S E T T+S E T T+S E

S,&dimethyl-NS-deazapterin

N3+-5,8dimethylNSdeazapterin

6,8-dimethyl-NS-deazapterin

3-21G 3-21G

3-21G 3-21G

T T+S E

N3+-8-methyl-NSdeazapterin

3-21G 3-21G

T T+S E

3-21G 3-21G

3-21G 3-21G

3-21G 3-21G

3-21G 3-21G

3-21G 3-21G

3-21G 3-21G

Geometry

8-methyl-NSdeazapterin

pterin

T T+S E

6,7&trimethyl pterin

pterin

Theory/apt solvent a)

Compound

Table 2 (continued)

30100(0.39) 29700 27400

33300(0.47) 33800 29700

31100(0.37) 34100 28700

32300(0.50) 33100 28700

30700(0.38) 31000 27900

27900(0.58) 27500 25300

28000(0.48) 24000 24600

28900(0.58) 28700 25700

28200(0.29) 28700 [24300] h,

28900(0.48) 25100 24900

28700(0.56) 27800 25300

37800(0.03) 38000 32600

38100(0.03) 41700 33000

38000(0.02) 38700 34000

36800(0.004) 36200 31800

35800(0.46) 33600 [31900] h,

37400(0.01) 36700 31400

BandBl flv)

Band A rcf)

Band B2

40500(0.22) 44800 38900

39200(0.16) 38500 36200

41700(0.23) 49600 39100

38600(0.17) 38000 36100

41100(0.24) 45700 38800

35900(0.24) 34600 35200

39000(0.26) 39300 37700

36900(0.20) 35500 35600

38700(0.19) 40200 [32700] ‘)

39600(0.25) 39800 37700

36400(0.23) 34500 35800

rcn

49600(0.05) 52200

50900(0.60) 50100

49500(0.10) 54400

51300(0.08) 52400

49900(0.06) 53700

48600( 0.08) 46300

50100(0.10) 49200

49100(0.14) 46600

46700(0.04) 44700

50700(0.21) 50300

48600(0.06) 46400

continuedon next page

48100(0.08) 46200 45700

47600(0.19) 48400 43500

47500(0.05) 49500 43100

49000(0.10) 49100 46200

48800(0.12) 46800 46100

46600(0.13) 45500 39500

48200(0.16) 42900

47800(0.12) 46700 39100

41800(0.26) 39500 [ 374001 h)

48800(0.16)

47600(0.13) 46400 38500

Band C2 FffJ

BandCl PO

3-21G 3-21G

3-21G 3-21G

3-21G 3-21G

T T+S E T T+S E T T+S E T T+S E T T+S E T T+S E

7,8dimethyLNSdeaxapterin

N3+-7,SdimethyL NSdeaxapterin

5,6,8-trimethyl-NSdeaxapterin

N3+-5,6,8_trimethylNSdeaxapterin

6,7,8-trimethyl-NSdeaxapterin

N3+-6,7&trimethylNS-deaxapterin 30700(0.51) 31300 28200

29600(0.42) 31200 27500

32400(0.45) 32500 28900

30700(0.37) 32700 28200

31600(0.51) 32500 28800

37700(0.04) 40400

38200(0.05) 40800

37400(0.23) 37000 35700

40500(0.20) 48200 39000

38200(0.19) 37400 36000

41300(0.20) 48500 39100

38300(0.20) 38000 36000

41100(0.22) 46200 39200

S denotes that the calculated

47500(0.10) 47800 45700

48000(0.09) 47500 45600

46000(0.20) 47000 42900

46500(0.08) 47700 42300

48500(0.13) 48300 46500

48800(0.14) 45900 46700

47800(0.08) 48600 46100

IO

37700(0.22) 37000 36100

Band Cl

Band B2 fiu)

‘) All experimental band energies were recorded in aqueous solution, and are from this work unless otherwise indicated; has been included in the results. b, Spectra of 3-methyl and l-methyl pterin reported in ref. [ 241. ‘) Ref. [24]. d, Approximated by the spectra of neutral and protonated 8-methyl pterin. ‘) Spectrum of 2-amino4methoxypteridine (4-metboxy pterin) reported in ref. [ 25 1. ‘) Ref. [26]. *) Approximated by the spectrum of S-methyl-NSdeazapterin. ‘) Approximated by the spectrum of the 7exo-methylene form of 6,7dimethyl-8(2’ -hydroxyethyl)pterin [ 271.

3-21G 3-21G

3-21G 3-21G

38100(0.03) 39200 33000

31400(0.49) 31800 28200

3-21G 3-21G

T T+S E

N3+-6,8dimethylNS-deaxapterin

30200(0.42) 30400 28000

Fu)

3-21G 3-21G

Band Bl

Band A p(f)

Geometry

Theory/expt solvent a)

Compound

Table 2 (continued) Band C2

solvent shift

49500(0.06) 50600

49300(0.03) 54500

49800(0.45) 49100 45900

49600(0.10) 53200

51 lOO(O.03) 52400

49600(0.04) 53700

49900(0.12) 50400

rr(f)

63

P. Wormell,J.E. Gready/ ChemicalPhysicsI79 (1994) 55-69

h(experimental)lnm 300

350

400 38000 I

I

I xl 016 14g*4

23

I

260004 24000

26000

32000

E(~xperlmental)/cti’

Fig. 3. Comparison of experimental and gas-phase theoretical energies for the A bands of some pterins and NSdeaxaptetins. ( x ) Neutral and protonated pterin and NSdeaxapterin. (0 ) Neutral and protonated g-methyl pterins. ( 0 ) Neutral and protonated 8-methyl-NSdeaxapterins. ( 1) pterin; (2) Nl +-pterin; (3) NSdeaxapterin; (4) N8+-NSdeaxapterin; (5) g-methyl pterin; (6) N3+-8-methyl pteriq (7) 6,8dimethyl pterin; (8) N3+-6,8dimethyl pteriq (9) 7,ldimethyl pterin; (10) N3+-7,& dimethyl pterin; (11) 6,7,8-trimethyl pterin; (12) N3+-6,7,& trimethyl pterin; (13) 8-methyl-NSdeaxapterin; (14) N3+-& methyl-N5deaxapteriq (15) 5,8dimethyl-NSdeaxapteriq (16) N3+-5,8dimethyl-NSdeaxapteriq (17) 6,8dimethyl-NSdeaxapteriq ( 18) N3+-6,8dimethyl-NSdeaxapterin; ( 19) 7,8dimethyl-N5deaxapterin; (20) N3+-7,8-dimethyl-NSdeaxapterm; (21) 5,6,8-trimethyl-NSdeaxapterin; (22) N3+-5,6,8trimethyl-N5deaxapterin; (23) 6,7,8-trimethyl-NS-deaxapterm; (24) N3+-6,7,8-trimethyl-N5deazapterin.

are compared in fig. 4. The diagonal line corresponds to an exact correlation between theory and experiment. The solvent-shifted result for N 1+-pterin has been omitted because the energy ordering of the A and B2 transitions has been reversed. Overall, the solvent-shifted results are no better than the gas-phase (unshifted) results. The solvent-shifted results for the neutral 8-substituted pterins near 25 000 cm- ’ appear to be very accurate, but these results could also be regarded as outliers when compared with those for the other molecules. Similar conclusions are drawn when theory and experiment are compared for the B2 band (see fig. 6 below). Some specific aspects of the solvent-shift calculations are highlighted below. 3.4. Spectral dependence on tautomer Two crucial problems in pterin chemistry have been the identification of tautomeric forms, and the sites

25000

3ocoo

35000

E(experimental)/cti’

Fig. 4. Comparison of calculated energiesfor the A bands of some pterins and NSdeaxapterins in gas and solution phase with experimental energies. ( 0 ) Gas-phase calculations. (A ) Solutionphase calculations.

of protonation in acidic solution [ 17 1. The most stable tautomer of pterin is predicted to be the N3 (H) form [ 121, shown in fig. 1. The Nl (H) and N8(H) tautomers and the 4-hydroxy (enol pterin) form have not been isolated (the N5 (H) form requires charge separation) but the spectra of l-methyl, S-methyl and Cmethoxy pterin, together with 3-methyl pterin, have been reported and compared with that of pterin itself [ 24,25,28]. The comparison establishes that pterin exists as the N3 (H) tautomer in aqueous solution. Our CNDO/S-CI predictions support this assignment, and there are good correlations between the predicted spectra of the tautomers and the reported spectra of the methyl derivatives [ 24,25,28]. The A bandsoftheNl(H),N3(H),N8(H) andenolforms are predicted to have energies of 32400, 30900, 26 700 and 29 100 cm-’ respectively (corrected using the line of best fit from fig. 3 ) . The experimental value of 29400 cm-’ for pterin is consistent with either the enol or N3 (H) forms, with the former giving the best fit. The same conclusion may be drawn from the spectra of the methyl derivatives [ 241. However, the assignment to the N3 (H) tautomer is confirmed by the B and C regions of the spectra: pterin has a strong B2 band at 37 100 cm-’ as predicted by theory for the N3 (H) tautomer, whereas 4-methoxy pterin has a shoulder at 38 500 cm-‘, which is consistent with the prediction of a weak band at 39 600 cm-’ for enol pterin (bearing in mind that the CNDO/S-

64

P. Wormell,LE. Gready/ ChemicalPhysicsI79 (1994) H-69

CI calculations slightly overestimate the energies of B2 bands, as discussed below). The A and B bands of l-methyl pterin and 8-methyl pterin also correlate well with the theoretical predictions for the Nl (H) and N8 (H) tautomers. Spectroscopic arguments [ 171 also support the ab initio SCF prediction [ 121 that pterin is protonated at Nl, rather than N5 or N8. The CNDO/S-CI calculations, corrected using the line of best fit from fig. 3, predict that the A bands of the Nl+, N5+ and N8+ cations should occur at about 32 000,24 100 and 26 600 cm-’ respectively. The experimental A band of protonated pterin occurs at 3 1800 cm- l, which is consistent with Nl +-pterin being the preferred protonated form. The pterin A transition is essentially the (3 1)A*+- (30)~ one-electron promotion. It involves both rings and the exocyclic nitrogen and oxygen, with significant changes in MO coefficients on C4a and C8a. In the N8 (H) tautomer, the transition is again mainly (31)7c*+ (30)x, but C4a and the exocyclic atoms are much less important, and the transition mainly involves the pyrazine ring. This tautomer, although unstable relative to the N3(H) form, is the prototype of the stable and isolable I-methyl derivatives. Their spectra are distinctively different from the normal pterins, i.e. the N3 (H) tautomers, owing to different patterns of rr-conjugation [ 12,291. The A band is both predicted and observed to occur at longer wavelengths, resulting in bright yellow compounds and blue/green fluorescence. The calculations also correctly predict that the 8-methyl NSdeazapterin spectra occur at shorter wavelengths than the corresponding pterin derivatives. While the A transitions for pterin and NSdeazapterin are quite different in orbital character, those of the corresponding N8 (H) tautomers and I-methyl derivatives are similar. The transitions for the 8-methyl-NS-deazapterins are again mainly (LUMO)lc*+ (HOMO)n, and principally involve the pyridine ring. For all the 8-methyl pterins and NS-deazapterins the largest changes in MO coefficients during the (LUMO)ti+ (HOMO)a transition occur at N5 (C5 for the deazapterins) and C7, with smaller but still significant changes at C6. This suggests that methylation at these positions, especially C7 (and C5 for the deazapterins), could perceptibly affect the spectra of the various species, and this is indeed the case.

3.5. Effect of methylation Different patterns of methylation produce small but distinct wavelength shifts, which are also seen in the corresponding fluorescence spectra [ 2,3 1. The energy shifts have ranges of 500 cm-’ for the various 8methyl pterins and 1500 cm-’ for the 8-methyl-NSdeazapterins. Theory reproduces the general trends, and indeed exaggerates them. The calculated solvent shifts do not significantly affect these trends. For the neutral pterins the energy ordering is (6, 7, 8) = (6, 8)<(7,8)=(8) (experimental)and(6,7,8)<(6, 8) < (7, 8) < (8) (theoretical). On protonation the orderingis (6, 7, 8)=(6, 8)<(8)<(7, 8) (experimental) and (6, 7,8) < (6,8)< (7, 8) < (8) (theoretical). For the neutral NS-deazapterins the orderingis(6,8)<(6,7,8)<(8)<(7,8)<(5,6,8)<(5, 8) (experimental) and (6, 7, 8)<(6, 8)<(7, 8)<(5, 6, 8)=(8)<(5, 8) (theoretical), and on protonation, (6,7,8)=(6,8)<(8)<(7,8)<(5,6, 8)~ (5, 8) (experimental) and (6, 7, 8)< (6, 8)x(7, 8)<(8)<(5, 6, 8)<(5, 8) (theoretical). There is thus a tendency for 5-substitution to cause a blue-shift relative to the other 8-methyl species, while 6-substitution causes a red-shift. In practice ‘I-substitution causes a slight blue-shift, but the theoretical results make no consistent prediction. Presumably these shifts are, in part, related to the substantial involvement of N5/C5, C6 and C7 in the electronic transitions, as discussed above. However, we find no clear relationships between the MO coefficients for the various atoms and the directions of the band shifts. 3.6. Eflect ofprotonation The spectra of the pterins and NS-deazapterins change on protonation, and the wavelength shift of band A and the corresponding fluorescence have proved useful for determining p& values and establishing the state of protonation of DHFR-pterin complexes [ 2,3,5]. Most of the molecules in this study show a blue-shift on protonation, about 600 cm- ’ for 8-methyl pterins and 800 cm-’ for 8-methyl-NS-deazapterins. The CNDO/S-CI results are not entirely satisfactory, since they show virtually unchanged energies on protonation of the pterins, and blue-shifts of between 1100 and 2200 cm- ’ for the deazapterins.

P. Wormell,J.E. Gready/ ChemicalPhysicsI79 (1994) 55-69

Our solvent-shifted results show blue shifts of about 3400 cm-l for the pterins; some of the corresponding NS-deazapterins are predicted to have blue shifts of up to 2 100 cm- ‘, while others have red shifts of up to 300 cm-‘. Despite the difficulties encountered in modelling the protonation shifts for &methyl derivatives, theory gives good results for the parent compounds, pterin and NSdeazapterin, which have very different behaviour. Pterin has a large blueshift on protonation, while deazapterin shows a large red-shift [ 26 1. This behaviour reflects the different character of the electronic transitions in the two molecules (see above) and different protonation behaviour, i.e. in the pyrimidine and pyridine rings respectively. Ab initio SCF calculations predict that NS-deazapterin should be protonated at N8 in acidic solutions, while pterin is protonated at Nl [ 11,12 1. The CNDO/SCI calculations predict that the A band should occur at 32400 cm-’ (corrected value 29200 cm-’ using the line of best tit from fig. 3) for the NI-protonated NS-deazapterin, and 35 800 cm-’ (32 700 cm-‘) for the Nl-protonated form. The experimental value is 29 200 cm-’ [ 261, which supports the prediction of NS-protonation. 3.7. Spectral B-region While all of the species in table 2 have only one, fairly strong band in the A region of the spectrum, there are characteristic differences in the B region. We have reserved the designation Bl for the weak band cf< 0.07) which is predicted to lie between the A band and the stronger B2 band in the neutral 8methyl pterins and NSdeazapterins. A similar band is also predicted for Nl +-NS,N8dideazapterin, and the non-preferred N 1+-N8deazapterin [ 111 and Nl(H)-pterin [ 121. A strong cf=O.46) band is predicted at a similar energy for 7-methylene-8-methyl pterin; this distinctive band is discussed below. Pterin and NSdeazapterin each have only one B band, designated B2. The main contributor for pterin is the (32)ft (30)~ promotion, while NSdeazapterin is again anomalous, with (3 1)tit (30)x predominating. The N8 (H) tautomer of pterin has both B 1 and of B2 bands, deriving from combinations (32)tit (30)x and (31 )Irt (28)x, with the latter predominating in Bl. (31)lc’t (28)x involves the pyrazine ring, N3 and the exocyclic nitrogen and ox-

65

ygen, while C4a and C8a are relatively unimportant. (32)x*+ (30)x involves both rings and the oxygen, with C7, Nl, N3 and C8a being particularly important. Experimentally the B 1 band is most prominent in the spectrum of 8-methyl pterin [ 301, as shown in fig. 2a. Weaker bands are seen in the spectra of the other 8-methyl pterins and 8-methyl NSdeazapterins (except for the 5,6,8- and 6,7,8-trimethyl N5deazapterins). Bl bands have not been seen in the spectra of the protonated 8-methyl compounds. Although the qualitative predictions are firhilled, the fit between theoretical and experimental band energies is poor. Theory overestimates Bl energies by up to 6700 cm-’ for the 8-methyl pterins, and about 5 100 cm- ’ for the corresponding NSdeazapterins. Since the B 1 bands are so weak in (and indeed sometimes absent from) the spectra of the 8-methyl NSdeazapterms, and since some of the spectra show shoulders on the low-energy edges of the B2 bands, we must consider the possibility that the observed Bl bands have some other source than the transition predicted by theory. We have observed that the Bl bands are more prominent in ethanolic solutions of these compounds, which raises the possibility that the bands are caused by appreciable equilibrium concentrations of hydrated species or other covalent-addition compounds. However, this is unlikely since NMR spectra [ 27 ] show no trace of such species in freshlyprepared basic solutions of S-methyl pterin, which has a prominent Bl band in its spectrum. We conclude that the Bl band may be correlated with the weak transition which is predicted to lie between the A and B2 bands, but it is not always strong enough to be seen and theory significantly overestimates its energy. All of the spectra in this study contained strong B2 bands with maximum absorbances between 35000 and 39 500 cm- *. As with the A bands, the CNDO/ S-C1 calculations predict that these bands should exist, and fig. 5 shows the correlation between theoretical and experimental band energies. The data for N 1+-pterin have been omitted from this graph since it is not clear whether the shoulder observed at 41600 cm- ’ should be correlated with the predicted weak B2 transition at 37900 cm-’ or the slightly stronger Cl transition at 44 300 cm- ‘. The theoretical energies are typically about 1700 cm-’ too high - a smaller discrepancy than shown by the A bands - and some clear trends are apparent. Theory and experiment

P. WormeN,J.E. Gready/Chemical Physics179 (1994) 55-69

66

again overestimates the sizes of the shifts. 6- and 7substitution consistently cause red-shifts for the 842000 methyl pterins, but the behaviour of the I-methyl N5015 _ 240 ‘30 02’ deazapterins is more complicated. Theory predicts 016 that 5substitution should cause a blue-shift, and 6‘7823 I I and 7-substitution should cause red-shifts. However, ‘E 4000005 -250 5 x 4 z experiment supports only the prediction for Wubsti16 x ;; t 4 14k tution, while the results for 6- and 7-methyl derivaz 11 ‘ii $ 20 A -260 p tives show no consistent trends. g 3600022 x3 16A “, Gas-phase and solvent-shifted results for the B2 B 24~ _. I I band are compared in fig. 6, where the diagonal line 10m 270 corresponds to an exact correlation between theory n6 t n 12 and experiment. As in the case of the A band, the sol36000 35000 vent-shifted results are comparable with the un40000 37500 shifted results, except in this case the neutral 8E(experlmental)/cni’ methyLN5deazapterins are calculated to have Fig. 5. Comparison of experimental and gas-phase theoretical anomalously large blue-shifts. energies for the B2 bands of some pterins and N5deazapterin.x Jeong and Gready [ 27 ] propose that in methanol ( x ) Neutral and protonated pterin and NS-deazapterin. (Cl ) solution 6,7dimethyl-8-( 2’-hydroxyethyl)pterin Neutral 8-methyl pterins. ( 0 ) Neutral 8-methyl-NS-deazaptertautomerises mostly to a 7-exe-methylene form which ins. ( n) FVotonated8-methyl pterins. (A ) Protonated 8-methylNSdeazapterins. (1) pteriq (3) NSdeazapterin; (4) N8+-N5has a strong and characteristic absorption at 3 13 nm deazapterin; ( 5) I-methyl pterin; (6) N3+-8-methyl pterin; (7) ( 3 1900 cm- ’ ) . Other workers report similar bands 6,8dimethyl pteti, (8) N3+-6$-dimethyl pterin; (9) 7,8difor 7-methyl-substituted pterins [ 3 1,321. This intermethyl pterin; (10) N3+-7,8dimethyl pterin; (11) 6,7,8-trime pretation is supported by the CNDO/S-CI calculathy1pttin; ( 12) N3+-6,7,8&methyl pterin; ( 13) 8-methyl-NSdeazapterin; ( 14) N3+-8-methyl-NSdeazapterin; ( 15) 5,8ditions in which the weak Bl cf~O.01 at 37400 cm-‘) methyLN5-deazapterin; ( 16) N3+-5,8-dimethyl-NS-deazapand strong B2 cf=O.25 at 39600 cm-‘) bands of teriq ( 17) 6,8dimethyl-NSdeazapterin; ( 18) N3+-6,8& neutral 7,8-dimethyl pterin are replaced by strong B 1 methyl-N5deazapterin; (19) 7,8dimethyl-NSdeazapterindeazapterin; (20) cf= 0.46 at 35 800 cm- ’ ) and weaker B2 (SC 0.19 at N3+-7,8dimethyl-N5-deazapterin; (21) 5,6,8-trimethyl-NSX(experlmental)/nm

260

270

260

250

I

deazapterin; (22) N3+-5,6,8-trimethyl-N5deazapterin; (23) 6,7,8-trimethyl-NS-deazapterin; (24) N3+-6,7,8-trimethyl-N5-

i

show the B2 bands for the neutral 8-methyl NSdeazapterins to be significantly blue-shifted relative to the corresponding pterins. Experimentally, the bands for the pro&mated 8-methyl compounds all lie in the same range, but theory places the protonated 8-methyl NS-deazapterins a mean value of 1250 cm-’ higher in energy. In contrast to the A bands, the B2 bands exhibit strong red-shifts on protonation, as shown in figs. 2a and 2b. The B2 bands show small wavelength shifts for the various methyl derivatives of pterin and NS-deazapterm, just as was observed for the A bands. Again the shifts are usually small, with ranges of 400 and 500 cm-’ for the neutral and protonated I-methyl N5deazapterins respectively, and 200 and 1300 cm-’ for the neutral and protonated 8-methyl pterins. Theory

A A

A

1

35000

37k

39boo

E(experimentel)/cni’

Fig. 6. &qxukon of calculatedenexgiesfor the B2 bands ofsome pterins and NS-deazapterins in gas and solution phase with experimental energies. ( q ) Gas-phase calculations. ( A ) Solutionphase calculations.

P. Wormell,J.E. Gready/ ChemicalPhysics179 (1994) U-69

61

39 700 cm- l) bands in methanol solution. After giving satisfactory correlation between theory and experiment for the A, Bl and B2 bands, the performance of the CNDO/S-CI method falls off at higher energies. This is to be expected if multiply-excited configurations are not used in the CI calculations. As shown in fig. 2a, the B2 band in the spectrum of S-methyl pterin splits into two components on protonation. In the dimethyl and trimethyl derivatives the splitting becomes so large, about 3000 cm-‘, that it is almost certain that two transitions are involved. However, the theoretical results listed in table 2 predict that the next strong band should be about 10 000 cm-’ higher in energy; this constitutes a large discrepancy. As may be seen in fig. 2b, neutral and protonated g-methyl NS-deazapterin have a strong band, Cl, at about 46000 cm-‘. Theory is in reasonable agreement, although the calculations underestimate the band’s intensity relative to the B2 band. We are, however, cautious about making any assignments in this spectral region, since the speo trum of the protonated compound has a shoulder between the B2 and C 1 bands which is not predicted by theory. A definitive study of the C region awaits a more appropriate semiempirical parameterisation with extensive CI, or an extension of CASSCF [ 331 or similar methods to bicyclic systems.

are The spectra of Nl +- and NS+-N8deazapterin predicted to be sufficiently different that the preferred site of protonation may be determined spectroscopically. Ab initio SCF calculations [ 111 suggest that protonation of N8deazapterin should occur at N5, rather than Nl. Using the line of best tit from fig. 3, the N5+ and Nl + forms are calculated to have A bands at 375 and 312 nm. The Nl+ form should have a weak B 1 band and a weak B2 which, if visible, should occur at about 236 nm. In contrast, the N5+ form should have no B 1 band, and a strong B2 band at about 289 nm. It should thus be possible to test the ab initio SCF prediction spectroscopically. For both Ngdeazapterin and NS,NI-dideazapterin the A bands are mainly attributable to the (3 1)Ir+ (30)~ one-electron promotions, although (32)x*+- (29)x is also significant for NIdeazapterin and (32)x*+ (30)x for NS,N&dideazapterin. Overall, the transitions resemble those of pterin, with both rings and the oxygen being important. However C2, C8a and the exocyclic nitrogen are less important than for pterin, while Nl , C6 and C7 are more important. C8 is more important for NI-deazapterin, but less important for the dideazapterin. These differences have implications for the spectra of methylated derivatives of these compounds.

3.8. Predictions for other species

4. Conclusions

Since theory and experiment correlate well for the A and B bands of the pterins and NS-deazapterins, predictions may be made for some species which have not yet been synthesised. Using the line of best fit from fg 3, the A bands of the neutral and Nl-protonated forms of N5,NIdideazapterin are predicted to occur at about 3 16 nm and 3 11 nm respectively. The A band for NSdeazapterin should occur at about 318 nm. These wavelengths are shorter than for pterin, but comparable with those for NS-deazapterm. Using the line of best fit from fa. 5 the B2 band should occur at 276 and 272 nm for the neutral forms of NSdeazapterin and NS,N8dideazapterin respeo tively. Nl +-NS,N8dideazapterin should have a weak B 1 band and an almost equally weak B2 band at about 260 nm. Experimentally, the B2 band may either be swamped by the strong Cl band, or show enhanced intensity as a result of vibronic interactions with it.

CNDO/S-CI calculations successfully model the prominent long-wavelength absorption bands in the spectra of neutral and protonated pterin, NSdeazapterin and various g-methyl derivatives of these biologically-interesting molecules. The correlation between theory and experiment is particularly good for the strong A and B2 bands, but falls off at higher energies. Theory predicts the overall appearance of the spectra, including the weak Bl bands seen in the neutral g-methyl pterins and some of the g-methyl NSdeazapterins. It provides insight into the x*+-x transitions which are responsible for the bands, and successfully models the anomalous spectrum of neutral NSdeazapterin. Theoretical and experimental band energies may be correlated for the prominent A and B2 bands, and the correlation is sufficiently good that predictions may be tested spectroscopically against experimental assignments of tautomeric forms

68

P. Wormell, J.E. Gready / Chemical Physics 179 (1994) 55-69

and sites of protonation. For known compounds these predictions confirm the experimental assignments. Spectroscopic predictions of the spectra of NSdeazapterin and NS,NSdideaxapterin which have not been reported are expected to be reliable, and may allow ab initio SCF predictions of protonation behaviour to be tested. Although theory satisfactorily models the spectra of these compounds, there are still significant discrepancies. Band energies are overestimated by roughly 3200,540O and 1700 cm- ’ for the A, B 1 and B2 bands respectively. Part of the discrepancy may be attributable to solvent effects, but even the large calculated shifts we report did not consistently correct for this. It seems likely that the CNDO/S parameter&&ion and the limited CI calculation are principally responsible for much of the discrepancy, but careful attention to solvent shifts will be important when more accurate calculations are available for these bicyclic systems. As discussed above, the SCF/ 3-2 1G geometries should be satisfactory for the present semiempirical calculations, but experimental geometries could give marginal improvements. However, improved geometries would be desirable if higher-level calculations were to be undertaken, as the results listed in table 1 show that the calculations are sensitive to input geometry. The success of this study suggests that the CNDG/ S-C1 method may be usefully applied to other pterinlike molecules, and even better models of the spectra may be possible as higher-level calculations with solvent corrections become available.

References [ 1] W. Weiderer, J. Heterocyclic Chem. 29 (1992) 583. [ 21 S.-S. Jeong, P. Wormell and J.E. Gready, Pteridin~ 4 ( 1993) 32. [3] J.E. Gready, M.T.G. Ivery, M.J. Koen and H.J. Yang, in: pteridines and related biogenic amines and folates 1992, eds. N. Blau, H.-C. Curtius, R. Levine and J. Yim (Hamim Publishing, Seoul, 1992) p. 265. [4] J.E. Gready, in: Chemistry and biology of pteridines 1989, eds. H.-C. Curtius, S. Ghisla and N. Blau (de Gruyter, Berlin, 1990) p. 23. [ 5 ] S.-S. Jeong and J.E. Gready, in: Chemistry and biology of pteridines and folates, eds. J.E. Ayling, M.G. Nair and C.M. Baugh (Plenum Press, New York, 1993) p. 529. [6] D.C. Palmer, J.S. Skotnicki and E.C. Taylor, in: Progress in medicinal chemistry, Vol. 25, eds. G.P. Ellis and G.B. West (Elsevier, Amsterdam, 1988) p. 85. Brown, Fused pyrimidines, The chemistry of heterocyclic compounds, Vol. 24 part 3 (Wiley, New York, 1988) p. 167, and references therein.

[ 71 D.J.

[S] J.DelBene,H.H. Jaff6,R.L. EllisandG. Kueh&nz,QCPE program No. 174, Department of Chemistry, Indiana University, Bloomington, IN 47405, USA. [Q] RL. Ellis, G. Kuelmlenz and H.H. Jaffk, Theoret. Chim. Acta (1972) 131. [lo] P. Wormell andA.R Lacey, Chem. Phys. 160 (1991) 55.

[ 111 J.E. Gready, to be published. [ 121 J.E. Gready, J. Comput. Chem. 6 (1985) 377. [ 131 J.E. Gready, J. Mol. Struct. THEGCHBM 124 (1985) 1. [ 141 M.J. Koen, T.W. Hambley and J.E. Gready, unpublished results. [15]M.J.KoenandJ.E.Gready,J.Grg.Clxm.58(1993)

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[16]M.T.G.IvexyandJ.E.Gready,tobepublished. [17]W.~eidercs,in:Unconjugatcdpterinsinneurobiology,~ W. Loevenberg and R.A. Levine (Taylor and Francis, London, 1987) p. 29.

[ 181 A.T. Amos and B.L. Burrows, in: Advances in quantum

Acknowledgement

chemistry, Vol. 7, ed. P.-O. L&din York, 1973) p. 296. [ 191 W. Rettig, J. Mol. Struct. 84 (1982)

We wish to thank Dr. Mark Koen and Michael Ivery for gifts of chemicals, Soon-Seog Jeong for the spectra of some g-methyl pterins, and Dr. Jeff Reimers for making a VAX version of the CNDG/S-CI program available to us, and for his advice on solvent shifts. We gratefully acknowledge financial assistance from the National Health and Medical Research Council.

(Academic Press, New 303.

[20] J.M. Hollas, Modern spectroscopy, 2nd Ed. (Wiley, New York, 1992) p. 249. [21] J. Ridley and M. &rner, Theoret. Chim. Acta 32 (1973) 111. [22]M.M.KarelsonandM.C.Zemer,J.Phys.Cbem.96(1992) 6949. [23]J.Zeng,J.S.Craw,N.S.HushandJ.RReimers,Chem.Phys. Letters, in press. [24] W. Ptleiderer, E. Liedek, R. Lohrmann and M. R&vied, Chem. Ber. 93 (1960) 2015. [25]W.PfleidererandR.Lohrmann,Chem.Ber.94(1961) [26] M. Pfleiderer, Dissertation, Germany (1986) p. 19.

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