lnfrared spectra and molecular association of lumiflavin and riboflavin derivatives

Spectrochimica Acta, Vol. 42A, No. 9, pp. 1059-1068, 1986. Printed in Great Britain.

0584-8539/86 $3.00+0.00 Pergamon Journals Ltd.

Infrared spectra and molecular association of lumiflavin and riboflavin derivatives M. ABE,* Y. K Y O G O K U , IIT. KITAGAWA,t Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan K. KAWANO,~ N. OHISHI,§ A. TAKAI-SUzUKI and K. YAGI § Institute of Biochemistry, Faculty of Medicine, University of Nagoya, Nagoya 466, Japan (Received 9 January 1986; in final form 25 March 1986; accepted 25 March 1986)

Abstract--Infrared spectra of lumiflavin (Lf), [3-ND]Lf, [3-NMc]Lf, [ 2 : SC]Lf, [4a-t aC]Lf, [1,3-1SN]Lf, [1,3,5-tSN]Lf, riboflavin (Rf), [3-NMe]Rf, riboflavin-2',3',4',5'-tetraacctate (RfT) and [3-NMe]RfT were measured in their solid and solution states. The vibrational assignments of lumifiavin have been made on the basis of the observed isotope frequency shifts in the i.r. spectra. The differences between the skeletal vibrations of Lf and those of Rf are discussed. The modes of molecular association of Lf and Rf are also proposed based on the i.r. spectra of the carbonyl stretching region.

have not been observed in the resonance Raman spectra, despite that the carbonyl and imino groups of ring III (Fig. 1) are chemically very active and important in characterizing the isoailoxazine ring. Flavin molecules have the possibility to associate with each other or with other kinds of molecules by forming hydrogen bonds at the N 1, Na, Ns and O atoms of the flavin nucleus. For instance, specific association of riboflavin and adenine derivatives in chloroform was reported by KYOGOKU and YU [ 17], and the presence of flavin-protein hydrogen bonding in riboflavinbinding protein was suggested by KITAGAWA et al. [10] and SCHMIDTet al. [15]. The effect of association through hydrogen bonding is reflected in the vibrational spectra. Especially when the hydrogen bonds are formed at the oxygen or N a - H atom, the effect should be reflected by the carbonyl stretching or N a - H bending vibrations in the i.r. spectra. For successful application of vibrational spectra to the study of flavoproteins, it is necessary to analyze both i.r. and Raman spectra and to establish their vibrational assignments.

INTRODUCTION

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are coenzymes which contain an isoalloxazine ring. In the process of flavoprotein catalysis, the isoalloxazine ring plays an essential role. For this reason, many workers have an interest in the structure of flavin derivatives and have studied them by various techniques such as absorption spectra [1], circular dichroism [2], nuclear magnetic resonance [3], fluorescence [1] and recently by resonance Raman spectra [4-15]. As the vibrational frequencies depend sensitively on the molecular structure, Raman spectroscopy becomes noteworthy in the study of the isoalloxazine ring. Especially, in the resonance Raman spectra of flavoprotein, the vibrations of the isoalloxazine ring can be selectively observed without interference by the Raman lines of apoproteins. The resonance Raman spectra of lumiflavin (Lf) [12-15] and riboflavin (Rf) [4-11], including their isotopically substituted compounds, have been studied, and the vibrational assignments have been made experimentally and recently by normal mode analysis [16]. On the other hand, the i.r. spectra of such a complicated molecule as flavin, in which almost all the vibrations of the molecule appear, cannot be analyzed in detail. However, i.r. data provide information about the modes which are not observed by Raman spectra. In fact the carbonyl stretching and the N a - H bending vibrations of the isoalioxazine ring

H

R

9

1 C

H,c/' *Present address: Shoin Women's University, Nadaku, Kobe 657, Japan. tPresent address: Institute for Molecular Science, Myodaiji, Okazaki 444, Japan. :~Presentaddress: Faculty of Dentistry, Kyushu University, Fukuoka 812, Japan. §Present address: Institute of Applied Biochemistry, Mitake, Gifu 505-01, Japan. IPAuthor to whom correspondence should be addressed.

10

I

% if H

1

I] C

~N°~"4'~C°il/ N~ H 0

Lf:R=CH3 f 2" 3' 4' 5" Rf:R=CH2-CH(OH)-CH(OH)-CH(OH)-CH20H Fig. 1. Structure and numbering of the isoalloxazine ring.

1059

M. AsE et al.

1060

The main purpose of this work is to obtain quantitative information about the vibrations of the isoalloxazine ring. Lf has a simpler structure than Rf, and the i.r. spectra of carbon-13 and nitrogen-15 labeled lumiflavins, [2-13C]Lf, [4a-13C]Lf, [5-15N]Lf, [1,3~SN]Lf and [1,3,5-15N]Lf, and the N 3 substituted lumiflavins, [3-ND]Lf and [3-NMe]Lf, have been measured. In order to study the effect of molecular association on the isoalloxazine ring, the i.r. spectra of Rf, [3-NMe]Rf, riboflavin-2',3',4',5'-tetraacetate(RtT) and [3-NMe]RfT were examined. Comparing the i.r. spectra of Lf and Rf derivatives, the association of the flavin molecule and vibrational assignments of the isoalloxazine ring on the basis of the isotope frequency shifts are discussed.

,.,

..r ¢,~

..r to

tx~

vYU;,,

EXPERIMENTAL

1BOO

Materials Lf and ~SN or 13C-labeled Lf were prepared by the photolysis [18] of Rf and labeled Rf [19] or FMN [3]. [3-ND]Lf was obtained by dissolving Lf in alkaline D20 solution, then neutralizing it with DCI followed by freezedrying. Syntheses of RfT and [3-NMe]RIT have been described previously [17].

Measurement of infrared spectra The i.r. spectra of the above compounds were measured using a Hitachi 225 i.r. spectrophotometer. Lf, Rf and their isotopicallysubstituted compounds and N3-methylatedcompounds were measured in KBr discs because of their low solubility in any solvent. By the acetylation of the ribityl group, riboflavinbecomes soluble in chloroform. In order to study the structure of the associatedmolecules,the i.r. spectra of RtT and [3-NMe]RtT in chloroform were measured and compared with those in the solid state. Infrared spectra of Rfl" in chloroform at different concentrations (0.02-0.13 M) were also measured. In order to ascertain the differences in the molecular structures arising from the different sample conditions for i.r. and Raman measurements, the i.r. spectra of FMN sodium salt in D20 were measured in the region of 1800-1200cm-1 and were compared with its resonance Raman spectra obtained in D20. RESULTS AND DISCUSSION The i.r. spectra in the 1800-1200cm -1 region of F M N sodium salt in a KBr disc and in D 2 0 are shown in Fig. 2. The observed frequencies agree well with those of the resonance Raman spectra obtained in the same solution [6]. In Fig. 3, the i.r. spectra of Rf in a KBr disc and [3-ND]Rf in the same disc are compared. It can be said that the molecular structure of the skeletal part of the isoalloxazine ring in solution is basically not very different from that in the solid state, because the observed frequencies of corresponding bands are almost the same in both spectra, though a few wavenumbers are higher in solution than in the solid state. The i.r. spectra ofLf, [3-ND]Lfand [3-NMe]Lfare shown in Fig. 4, and observed frequencies of these compounds and isotope frequency shifts of labeled lumiitavins are given in Table 1. The i.r. spectra of Rf, [3-NMe]Rf, RfT and [3-NMe]RfT in the solid state

'

16bo ' 14bo ' l e b o WAVE NUMBER (cm-')

Fig. 2. Infrared spectra of FMN sodium salt in KBr disc and in D20: - - - , solid in KBr disc; - - - , 0.085 M in D20 (pD 6.1).

are shown in Fig. 5. The spectra of RfT and [3NMe]RfT in solution are given in Fig. 6 and the concentration dependence of RfT in chloroform solution are shown in Fig. 7. The observed frequencies of Rf derivatives are listed in Table 2.

Assignments of the carbonyl stretchino vibrations The amide I vibration of the cis CONH group, in which the C--O stretching mode mainly contributes, is usually observed in the 1650-1720 cm- 1 region [20]. In the i.r. spectra of Lf and Rf in the solid state, two strong bands were observed above 1630 cm- 1, which were sensitive to 13C substitution at C2 and N3deuteration or methylation and also to substitution of the groups at Nto. These two bands can be assigned to the two carbonyl stretching vibrations, which could not be observed in the resonance Raman spectra of free riboflavins, although the C4=O stretching band was observed in the spectra of Ru(II)-flavincomplexes [21]. In Lf, the lower frequency band at 1663 cm- 1 shows a large low frequency shift on 13C2 substitution, which suggests that the C2=O stretching mode mainly contributes to this band. According to X-ray analysis [22], the bond lengths of N s - C ~ and C10~-Nt are markedly shorter than those of the other skeletal bonds, and the bond length of C2=O is a little longer than that of (24=0. The C2---O bond may be slightly conjugated with the two C-N bonds mentioned above, and this conjugation may be one of the reasons why the C2=O carbonyl bond has a lower vibrational frequency than the C4=0. The conjugation should not be so strong as to increase the intensity of the carbonyl stretching vibration in the resonance Raman spectra. This lower frequency band also shows a considerable low frequency shift on N3-deuteration, which indicates

Infrared spectra of iumiflavin and riboflavin i

i i

u ii

,

r ~l

i r

i , ~ n n i'~,~

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u

l

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0

Z

:

',

v

,

I

i

1061

~

~

~

I

~

(.O

Rf

~oro

~~~V~~~~~~[3_D N fR ]

o3

i... tD --LO

3000

2000 1500 WAVE NUMBER (cm "1)

1000

500

Fig. 3. Infrared spectra of Rf and [3-ND]Rf in the solid state (KBr disc).

I

1800

L

I

1600

~

I

i

,

I

1400 12JO0 I000 WAVE NUMBER (cm-I)

,

i

~

600

4()0

Fig. 4. Infrared spectra of Lf, [3-ND]Lf and [3-NMe]Lf in the solid state (KBr disc).

that this band is not a pure C2=O stretching mode but is coupled with the vibration of the N3-H group. The higher frequency carbonyl band near 1710 crn-t was observed as a doublet for most of the isotopically substituted compounds of Lf, but was observed as only one peak in N 3 substituted c o m SA(A) 42:9-H

pounds, [3-ND]Lf and [3-NMe]Lf. This band, which showed a slight frequency shift on t 3C2 substitution, but no remarkable isotope frequency shift on N 3deuteration, is regarded as arising mainly from the C4carbonyl stretching vibration, slightly coupled with the C2-carbonyl stretching vibration. The doublet for

1062

M . ABE et al.

Table 1. Observed infrared frequencies and isotopic frequency shifts of lumiflavin derivatives (in cm -1)

Lf

Observed frequencies [3-ND]Lf [3-NMe]Lf [2-'sC]

t 1721m* 1708 s 1663 s

t

1621 w 1583 s 1552 sb 1513w 1493 vw 1461 w 1447 sh 1425 w 1413 vw 1396m 1388 w 1359 w 1346m 1301m 1283m 1272 s 1262m 1238 s 1206m 1169m 1081 w 1031m 1006m 980 w

1710 1649 1580 1546 1506

1700 1670 1625 1586 1552 1512

1461 1450 1425

1424

1392 1379 1359 1349 1301 1292

1381 1362 1347 1306 1288

1208 1190 1074 1030 1012

1463

1273 1214 1187 1159 1088 1028 1010

-3 -2 -32 0 0 0 -2 -2 -2 0 -13 -4 - 1 -6 - 1 -1 -3 -1 -11 -4 -3

Isotopic frequency shifts [4a-13C] [5-'5N] [1,3-'SN] 0 0 2 0 -12 -4 -2 - 1

0 0 -1 0 -3 -10 -6 -2 0

-4 -1 0 0 -2 -2 -12

0 -11 0 -10 -16

- 1

-4

-2 0

-7

-2 -3 -4 -2 -1 -4 -5

-1 - 1 0 -2 -3

-3

-2

- 1

-4 -3 -2 - 1 - 1

- 1 0 -8 - 1 -2

-1 -2 -1 -4 - 10

[l,3,5-'SN]

-3

0

- 12 -3 -2 - 1

-3

- 10 -10 -2 2 -5 -4 -13 - 1 -2

0 0 0 -1

-8 -1 -2

-2

- 1 2 -2 0 - 1

- 1 -4 -2 -1 - 1 - 1 0

0 -3 -5 -1 -2 0 1

0 -4 -7 -1 -2 -2 0

-4 -14 -3 -4 0 0

-2

-4

-2

-4

-4

- 1 -2 -1 - 1 0 -1 0

-2 - 1 -2 -3 -3 -1

-3 -2 -4 -4 -2 -1

-3 -2 -6 -2 -2 -4

-3 2 -3 -3

0

-2

-1

0

-2

-1

-12

0

-1 -13

-3 -3

0 2

967 937

t

881 sh 876 m 855 m 824 m 810m 785 w 773 w 735 m 676 m 627 vw 604 m 538 w 522 w 513w 503 w 462m

881 878 853 823 810 779 770 736 693 675 660 635 600 536

818 808 769 729 692 684 625 588 536

501

511 504

456

454

413

434 412

449 m 414 w

0

-4

0 -2

*s: strong, m: medium, w: weak, sh: shoulder, b: broad, v: very.

m o s t o f L f derivatives m a y arise f r o m t h e F e r m i resonance of the C4-carbonyl stretching vibration with the combination tone of the vibrations including the N 3 - H d e f o r m a t i o n m o d e , b e c a u s e it d i s a p p e a r s o n d e u t e r a t i o n a n d m e t h y l a t i o n at t h e N 3 p o s i t i o n . A s f o r R f t h e l o w e r f r e q u e n c y c a r b o n y l b a n d at 1647 c m - 1 also s h o w s a large low f r e q u e n c y shift o n ~3C 2 s u b s t i t u t i o n [23"] a n d is a s s i g n e d m a i n l y to t h e C2=O stretching mode. However, the pattern of the

f r e q u e n c y shift o f t w o c a r b o n y l s t r e t c h i n g b a n d s o n N 3 d e u t e r a t i o n is different f r o m t h a t o f L f (Fig. 3). T h e extent of the coupling between the carbonyl stretching a n d t h e N a - H b e n d i n g v i b r a t i o n s m u s t b e different in t h e s e t w o c o m p o u n d s . T h e h i g h e r f r e q u e n c y b a n d at 1731 c m - 1 o f R f w a s a l w a y s o b s e r v e d as o n e p e a k , a n d t h e i s o t o p e f r e q u e n c y shift (6 c m - 1) o f this b a n d o n 13C 2 s u b s t i t u t i o n 1-23] is larger t h a n t h a t o f L f (3crn-l), which suggests greater coupling with the

Infrared spectra of lumiflavin and riboflavin I

I

.

.

.

.

I

.

.

.

.

I

.

.

1063 .

.

I

Rf

RfT

3000

2000

1500 I000 WAVENUMBER(era"l)

500

Fig. 5. Infrared spectra of Rf, [-3-NMelRf, RIT and [3-NMe]RtT in the solid state (KBr disc).

RfT

~

[3-NMe]RfT

~

solution

_ 1800

~

1600

1400

I

I

cm'l

1800

i

I 1600

i

I 1400

Fig. 6. Infrared spectra of the carbonyl stretching region of RtT and [3-NMe]Rfr in the solid state (KBr disc) and in solution (0.02 M in chloroform).

C2--O stretching mode. It is interesting that the two carbonyl stretching vibrational frequencies and their behavior on isotope substitution are different between Lfand Rfwhose molecular structures differ only in the groups attached to the N10 atom. The origin of the difference will be discussed later. The strong band near 1745 crn -1 observed in RtT and [3-NMe]RIT (Fig. 5) can be assigned to the carbonyl stretching vibration of acetyl groups, since the carbonyl stretching vibration in an ester is usually observed at 1735-1750cm - t . The lower frequency side shoulder of the 1746 crn -1 band of RfT and the 1710 c m - 1 band of [3-NMe]RtT in the solid state are assignable to the C4--O stretching, and the 1664 crn- 1 band of Rfl" and the 1665 c m - 1 band of [3-NMe]RtT are assigned to the C 2 ~ stretching vibrations of the isoalloxazine ring from the similarity to those of Rf.

The effect of association on the carbonyl bands The lower carbonyl stretching band, to which the stretching mode of the C2=O bond mainly contributes,

1064

M. ABE et al.

RfT

--0.02M ....... 0 . 0 5 M ...... 0.07M ............. 0 . 1 3 M

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i

¢.D

i

:

..

'Jl

",j I

1800

1700

16'00cm_,

Fig. 7. Concentration dependence of the i.r. spectra of RfT in chloroform solution.

shows a 17 c m - 1 higher frequency shift on acetylation at the ribityl group of Rf (see Table 2 and Fig. 5). This suggests that a specific interaction exists between the C2=O group and the OH groups of the ribityl moiety, for instance, intra- and/or intermolecular hydrogen bonding between the OH groups and the C2 carbonyl group. Moreover, this C2=O stretching band of RfT showed a 30cm -1 higher frequency shift when RtT was dissolved in chloroform to be 0.02 M as shown in Fig. 6. Instead of the two strong bands at 1746 and 1664 cm- 1 in the solid state, three bands at 1745, 1717 and 1694 cm -1 appeared in solution. On the other hand, in the spectra of [3-NMe]RtT compared in the same figure, three bands were observed in both solid and solution states, and large frequency shifts could not be observed between them. The three bands observed in solutions of RtT and [3-NMe]RfI" may be assigned, from higher frequency to lower, to the C=O stretching vibration of the acetyl group, the C4=O stretching and the C2=O stretching vibrations of the isoalloxazine ring, respectively. As shown in Fig. 7, the peak intensity of the 1694 c m - ~band of Rf'l" decreased as the concentration increased, and the intensity at a position about 10cm -~ lower than the maximum increased. These lines of evidence indicate that in the solid state of Rf'I" there exist intermolecular hydrogen bonds between the N3-H group and the C2 carbonyl group, similar to the association of Rf with adenine derivatives as shown by KYOGOKU and Yu [17]. In dilute solution the molecular association disappeared, and the C2=O stretching frequency (1694 c m - 1) aris-

ing from the hydrogen bond-free molecule was observed. In the case of [3-NMe] R tT, the methylation of the N 3 position removes the possibility of hydrogen bond formation with other molecules through the N3-H group, and, therefore, observed spectra obtained in solid and solution states are almost the same. The i.r. spectra of Rf in dilute solution could not be obtained because of its low solubility in any organic solvent. There is a possibility of forming hydrogen bonds with other molecules through the N3-H group as in the case of RfT. Accordingly, in the solid state of Rf, the C2 carbonyl group may form hydrogen bonds with both the N 3-H group of another molecule and the OH group in the ribityl group of the same or another molecule. The frequency difference, 47 c m - 1, between Rf (1647cm -1, Fig. 5) in the solid state and RIT (1694cm -1, Fig. 6) in solution is a result of both effects, since the C2=O stretching frequency of hydrogen bond-free Rf is estimated to be the same as that of RtT. After subtraction of 17 c m - 1, which is thought to be due to the hydrogen bonding through the ribityl group, the resultant 30 c m - 1 may reflect the effect of intermolecular N3-H hydrogen bonding and it coincides with the shift value found for the intermolecular N 3 H hydrogen bonding of RfT. In the case of [3-NMe] R f, there is no intermolecular hydrogen bond formation through the N3-H group. However, there still remains the possibility of hydrogen bond formation by the ribityl group within the same and/or with another molecule. The higher frequency shift by 44cm 1 arising from the acetylation of the ribityl group of [3-NMe]Rf is thought to be due to the loss of interaction between the OH groups of the ribityl group and the C2-carbonyl group. The 44 cm-1 shift is distinctly larger than 17 cm-1, which is thought to be the magnitude of the shift due to hydrogen bonding between a ribityl OH group and the C2-carbonyl group in Rf. However it is comparable with the value 47 c m - 1, which is estimated as the total effect of the hydrogen bonding by the ribityl OH and N3-H. Therefore, in [3-NMe]Rf, another ribityl OH group, in place of the N s - H group, seems to form a hydrogen bond to the C2-carboyl group and the shift value of 44 c m - t would be the total effect of two kinds of hydrogen bonding by the ribityl OH groups. We could not determine whether these are due to intra- or intermolecular hydrogen bonding, because of the lack of spectral data of Rf and [3-NMe]Rf in dilute solution. As mentioned above and shown in Table 3, the C2=O stretching frequency of hydrogen bond-free molecules of Rfand RfI" is estimated to be 1694 c m - 1; and those of [3-NMe]Rf and [3-NMe]RtT, 1663cm -1. In the methylated compounds, the C2 carbonyl stretching frequency is lowered by the loss of vibrational coupling with the N a - H bending mode. The difference, 31 cm -1, is similar to the case of Nmethylacetamide in the cis and t r a n s conformations of the C O N H group; in cis CONH, amide I is observed at 1690 c m - 1, and in t r a n s CONH at 1657 cm- 1. Some

1065

Infrared spectra of lumiflavin and riboflavin Table 2. Observed infrared frequencies of riboflavin derivatives (in c m - 1 ) Rf

RfT

[3-NMe]Rf

[3-NMe]Rfr

1746 s 1731 s 1647 s 1621 w 1578 s 1540sb

1664 s 1624 w 1576 s ~1535s

1504s 1465 sh 1457m 1436m 1396 s

1742 s 1710 s 1665 s 1615 vw 1582 s 1538s

1712 s 1621 s 1581 s ~1535sb

1505m 1462m

Rf

RtT

896w 883m 867 w 850m (816sh

[3-NMe]Rf

900vw

1806m ~786 sh [775m 756 vw 742m 719w

808w ~784 vw (776w

1349m 1307w ~1272 s (1258 s

~679m (673m 626w

676 w

1296w

1345 s 1305 s 1275 s

1246s 1222w

1245 s 1215 s

1230 s 1202 s

(1240s )1212 s

596m

1178m 1154m

1183 sh 1158m

l181m 1154m

k 1 1 7 4 sh l150m 1132m

572m 532m 519m

1076 s 1069 s 1058m 1032m 1014 s

1086w

1084 s 1067 s 1052 s 1037 s 1026 s

ll12w 1082m

502m 486w 474 w 449m 410m 382w

1436m 1399m ( 1384 sh ~1372s L1350 sh

1368m 1344s 1303m 1274m

~1445 s [1432 s 1394m

1052m lO12w

)

(1044sh ~1033 s klOllm

909w 899 vw 878w

892m 876w 860m 831m

853w 834w

1493m 1464m ~1440 sh (1431m ~1399 sh |1374 s

[3-NMe]Rfr

849m ~826w [817 w 807m

807 s

762m 751 w 733 w 716 w 693m

774 s 754 vw 731w 710w 696w 685 w

~741 w [725 sh

667w 642w 632w 626 w 608m 588w 574 w 547 w 538 w 517 vw ~505 w (500w 485w 451m 436w 414m 388w 372 w

~628w (621 sh ~606sh [596m 588 w 561 vw 530 w

~600w t590sh 571 w

501w

506w 487w

~460sh (450m 414w

530 w 520 vw

449m 421w 375w

335 w 989 vw

956 vw

983m

922 w

935 w

926 w

972m 952 w 938 w

294m 337 w

Table 3. Carbonyl stretching frequencies [v(C2=O)] and the effect of hydrogen bonding (HB) in riboflavin and lumiflavin derivatives Rf v(C2=O) in the solid state

Rfl"

O=C2) (~) - ( ~ ) -17 Effect of HB (NH...O---C2) ( ~ - (~) + 17 (~) - ( ~ ) -30 -30 1694"

(~) 1694

®-® -44 --

1578

1576

--

1663"

(~) 1663

-31"

(~) - (~) -31

Effect of N a methylation v(C4a-Ns, CI0a-N1)

[3-NMe]RfF

(~) 1647 c m - t (~) 1664 cm - t (~)1621 cm -~ ( ~ 1 6 6 5 c m -~

Effect of H B ( O H . . '

v(C2--O) of HB free molecule

[3-NMe]Rf

1581

1582

Lf

[3-NMe]Lf

v(C2--O) in the solid state Effect of HB ( N H - . . O--C2) v(C2--O ) of HB free molecule Effect o f N 3 methylation

1663 -30* 1693"

1670

v(C4a-Ns, Cloa-N~)

1583

*Estimated values.

1670" -23* 1586

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M. ABE et al.

skeletal structural change is also induced by methylation. For instance, the strong band near 1580cm-1, which shows considerable isotope frequency shift on 15NI,a, ~, ISN 5 and laC4~ substitutions and in which a large contribution of the C4~N ~ and C t ~ - N 1 stretching vibrations is expected, shows a slightly higher frequency shift on N3-methylation in Rf derivatives. On methylation of the Na position, the double bond nature of C4~-N s and Cl0~-N 1 bonds increases and the C2----O bond is slightly weakened, and, therefore, the C2---O stretching force constant diminishes. Besides, small alterations in force constants of skeletal stretching and deformation vibrations are induced, which also result in the low frequency shift of the C4=O stretching vibration. In the case of Lf, where the ribityl group of Rf is replaced by the CH 3 group, there is only the possibility of forming intermolecular hydrogen bonds through the N a - H group; that is, the spectrum of Lf in the carbonyl stretching region should rather resemble that of Rfr. In fact, the frequency of the C2=O stretching vibration of Lf is close to that of RtT; and that of [3-NMe]Lf to that of [3-NMe]RtT (see Table 3). Assuming the effect of an intermolecular hydrogen bond is almost the same as that in Rf, the C2=O stretching frequency of a free molecule of Lf is estimated to be 1693 c m - x, and the effect of methylation is about 23 c m - 1. The 1583 c m - t band shows about a 3cm -~ higher frequency shift on N 3methylation, and small structural change is also expected in the methylated Lf. Assignments of other vibrations o f lumiflavin

In the structure of ¢is CONH, the NH bending mode usually couples with the C=O stretching vibration. Accordingly, by the formation of hydrogen bonds between the C2=O of one molecule and the N a - H of another molecule, the frequency of the C2=O stretching and the N3-H bending mode affect each other. MIYAZAWA[20] assigned the 1443 c m - 1 band to the NH bending vibration in the cis CONH structure. NISHIMURA et al. [24] assigned the band at 1423 c m - I (in the solid state) and MILES et al. [25] assigned the band at 1417 cm -1 (in solution) to the NH bending vibration of 1-methyluracil. The weak i.r. band at 1413 cm-1 of Lf cannot be seen in [3-ND]Lf and [3-NMe]Lfand is assignable to the N a - H bending vibration, which has not been reported by resonance Raman study. The new band at 937 cm- 1 of [3-ND]Lf must be due to the N3-D bending vibration. The b a n d a t 1396 cm-1 shows a considerable low frequency shift on 13C2, 15N1, 3 and 15N1,3, 5 substitutions. This band is assignable to the N1-C2-N3 vibration, probably the antisymmetric stretching mode, which may correspond to the Raman band at 1406 c m - 1 [14]. The band at 1238 c m - ~ of Lf disappeared on deuteration and methylation at the N 3 position and seemed to shift to a higher frequency region, ~ 1270cm -1 in [3-ND]Lf and 1273 cm -1 in [3-NMe]Lf, and showed a considerable low frequency

shift on 13C2 ' t SNl, 3 and 15Nt. a, s substitutions. These characteristic frequency shifts indicate that this band corresponds to the resonance Raman band at 1243 c m - 1 of Lfin solution reported by NISH1NAet al. [14] and the 1252cm -1 band of Rf reported by KITAGAWA et al. [10]. This band was empirically assigned by KITAGAWA et al. [10] to the C2-N a stretching vibration which corresponds to the amide III vibration of the cis amide group, that is, the CN stretching mode coupled with the NH bending mode. The amide III band usually shows a higher frequency shift on N-deuteration [20]. Since we do not have Casubstituted compounds, reliable information about the displacement of the C4 atom could not be obtained in this work. However, this band may be mainly assigned to the C2-N3-C 4 stretching vibration coupled with the N a - H bending mode, because two cis amide groups exist in the isoalloxazine ring as in the case of 1-methyluracil. The band at 1272 cm-1 which also shows a higher frequency shift on N3-deuteration and methylation and a lower frequency shift on 13C2 and 1SN5 substitutions may be due to the ring III and ring II skeletal stretching vibrations coupled with the N a - H bending mode. The band at 103l c m - 1 shows a large isotope frequency shift on 15N1, 3 and 15N1,3. 5 substitutions and could be assigned to the N1-C2-N3 symmetric stretching vibration. As shown in this paragraph, ring III skeletal vibrations couple more or less with the N3-H bending mode, which makes the correspondence of the spectra between Lf and its N 3substituted ones very complicated. The bands at 1583, 1552, 1301 and 1169 cm -1 may be mainly due to ring II stretching vibrations. The 1583 and 1552 cm -1 bands show isotope frequency shifts o n 13C4a , 15N5, 15N1, a and 1SN1,3,5 substitutions. The 1583 cm-1 band may correspond to the Raman bands at 1585 c m - ~ for Lfand 1584 c m - 1 for Rfin solution, which have been assigned to the C4a-N 5 stretching mode [10]. Although no information about the displacement of the 10a atom could be obtained from the present experiment, both 1583 and 1552 cm -~ bands can be assigned to the ring II and ring III skeletal stretching vibrations, in which two C=N bonds (C4~=N5 and Cl0~=N~) stretch with interaction on each other. The band at 1301 cm- t also shows an isotope frequency shift on lSN 5 and taC4a substitutions, but shows almost no shift on 15N1. 3 substitution, and may be mainly due to ring II vibration including the C4~-N 5 stretching mode, though the exact vibrational mode could not be assumed empirically. The 1169cm -~ band shows considerable down shift on ~3C4~ substitution and up shift on N3 deuteration and methylation, but almost no isotope frequency shift on 13C 2 and 15N~. 3 substitutions, and is assigned mainly to the ring II vibration in which the C4a atom is displaced considerably. The higher frequency shift of this band on N 3 deuteration and methylation may arise from the alteration in the vibrational coupling with the N3-D bending or Na-Me stretching vibrations, respectively,

Infrared spectra of lumiflavin and riboflavin Several bands which do not change much or change only a little in frequency on any isotopic substitutions in ring II and ring III are associated with ring I or partly ring II vibrations. These are the bands at 1621, 1461, 1425, 1206 and 1081 cm -1. To the bands observed in the region below 1000 c m - 1, the skeletal deformation vibrations mainly contribute. In most deformation modes, vibration is not restricted to one ring but spreads over two or three rings because of the fused ring nature of the molecule. For instance, the band at 824 cm -1 shows a considerable isotope frequency shift on 15N5, lSN1, 3 and 15N1.3. 5 substitutions and is assumed to be due to the C - N - C deformation mode of ring II and ring III. However, it is difficult to estimate the exact vibrational mode only by experimental isotope frequency shifts. In the spectrum of [3-ND]Lf or [3-NMe]Lf, a N a - D bending or N a - M e stretching vibration should appear in the frequency region of the skeletal stretching and sometimes in that of the skeletal deformation vibrations of the isoalloxazine ring. They may bring a change in the vibrational coupling and accordingly in the skeletal vibrational modes in this region. As a result, some vibrations of Lf, for instance the vibrations at 1272, 1238, 1169, 1081 and 1031cm -1, apparently show a higher frequency shift as shown in Table 1 and Fig. 4. The N a - D bending mode has been already assigned to the band at 937 cm-1. The new bands at 1424, 1159, 967 and 692 crn- 1 observed in [3N M e ] L f may be assigned to N3-Me stretching or bending, or to methyl group vibrations. The N3-Me stretching vibration is expected to appear in the range 1200-900 cm-1. The bands at 1159 and/or 967 c m - t may be assigned to this mode, which couples with other skeletal stretching vibrations of the isoalloxazine ring and makes the spectrum of this region different from that of Lf.

Comparison of the infrared spectra of lumiflavin and riboflavin The difference in the spectra of Lf and Rf in the carbonyl stretching region has already been discussed, which may arise mostly from the difference in intra- and/or intermolecular interactions. In the 1630-1100 c m - 1 frequency region in Rf, the vibrations of skeletal modes, the N a - H bending mode, the methyl group deformation modes and the methylene and OH deformation modes of the ribityl group are expected to appear. If the effect of the ribityl group on the isoalloxazine skeleton is nil or very little, the observed frequencies for ring skeletal vibrations of Lf and Rf should be almost the same. In fact, the bands observed for the two compounds correspond well with each other. However, no deuteration-sensitive band could be observed for Rf at around 1410 era-1 where Lf gives the N a - H bending mode. As shown in Figs 3 and 5, the 1368cm -1 band of Rf disappears on N adeuteration and methylation. Accordingly it might be possible to assign the band to the N3-H bending mode

1067

of Rf, though the N3-D bending mode could not be detected in the region of 800-1100 cm - 1. Even if the effect of intermolecular hydrogen bond formation between C2=O and N3-H groups is almost the same in both compounds, the alteration in the observed carbonyl stretching frequencies due to the intramolecular hydrogen bonding in Rf suggests that the vibrational coupling of the C2---O stretching vibration with the N3-H bending mode is not the same as that of Lf. Therefore, the N3-H bending frequency of Rf may not be the same as that of Lf. For molecules with a ring structure, especially those with a conjugated ring system, skeletal vibrations are not localized at specific atoms. As for the isoalloxazine ring the skeletal vibrations may spread over at least one ring and sometimes over two or three rings as mentioned in the preceding paragraph. A small change in coupling between several vibrations results in the alteration of the observed frequencies of several bands and sometimes even in the alteration of vibrational modes. The frequency difference between Lf and Rf may be explained by such a reason. The spectrum of Rf below 1000cm -1 is considerably different from that of Lf. Instead of the N 10- M e group vibrations of Lf (N 1o-CHa stretching, N l o - C H s bending and CH3 rocking vibrations), the vibrations of the ribityl group (C-C stretching, C - O stretching, C - H deformation, C - O H deformation, C - C - C deformation vibrations etc.) are expected to appear in Rf and to overlap with the ring vibrations in this region, which makes the spectrum of Rf more complicated. However, the i.r. bands of Rf whose corresponding bands are observed in the resonance Raman spectrum, where the skeletal vibrations of the isoalloxazine ring are enhanced, can also be found as corresponding bands in the i.r. spectrum of Lf. This means that the skeletal vibrational frequencies of the isoalloxazine ring are not very different in Lf and Rf. The broad band at 1436cm- 1 of Rf may include the OH in-plane bending mode of the ribityl group, and the strong bands near 1070 and 920cm -1 may be assigned to the C - O and C-C stretching vibrations of the ribityi group, respectively, The band at 719cm-1 disappeared in RfT and may be assigned to the OH out-of-plane bending vibration. As mentioned in the discussion of the earbonyl stretching vibrations, the C2=O stretching vibrational frequency of [3-NMe]RfT resembles that of [3NMe]Lf. The bands at 1431, 1150, 972 and 693 c m - 1 observed in [3-NMe]RfT, but not in Rf and RfT, correspond well to the bands at 1424, 1159, 967 and 692 c m - 1 observed in [3-NMe]Lf, respectively. In conclusion, Lf and Rf in the solid state form intermolecular hydrogen bonds through the oxygen atom of the C 2 carbonyl and the hydrogen atom attached to the N s atom; and, moreover, in Rf there exists hydrogen bond between C2=O and OH of the ribityl group. The effect of the formation of hydrogen bonds appears more directly in the carbonyl stretching and the N3-H bending vibrations which were observed

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only in i.r. spectra. They have a small effect on the skeletal vibrations which were selectively observed in the resonance Raman spectra. The assignments of i.r. bands of L f were made on the basis of isotope frequency shifts observed in i.r. spectra. The details of the vibrational modes o f Lf and its derivatives are discussed elsewhere [26] including resonance Raman data on the basis of vibrational analysis by normal coordinate treatment. The skeletal vibrations o f the isoalloxazine ring in R f seem to correspond well to those in Lf, and the results of vibrational analysis for skeletal vibrations of Lf may be reasonably applied to those of Rf. REFERENCES

[1] K. YAGI and T. YAMANO (eds), Flavins and Flavoproteins. Japan Scientific Societies Press, Tokyo (1980). [2] K. SHmA, K. HORItKE, Y. NISmNA, A. ISOMOTOand T, YAMANO, J. Biochem. Tokyo 81, 1465 (1977) and references cited therein. [3] K. YAGI, N. OHlsm, A. TAKAI, K. KAWANOand Y. KYOGOKU,Biochemistry, 15, 2877 (1976) and references cited therein. [4] J. NESTOR, T. G. SPIRO and G. KLAUMINZER,Proc. HatH. Acad, Sci. U.S.A. 73, 3329 (1976). [5] P.K. DUTTA,J. R. NESTORand T. G. SPIRO,Proc. HatH. Acad. Sci. U.S.A. 74, 4146 (1977). [6] Y. NISH1MURAand M. TSUBOl,Chem. Phys. Lett. 59, 210 (1978). [7] Y. NISHINA,T. KITAGAWA,K. SHIGA, K. HORnKE, Y. MATSUMURA,O. WATARIand T. YAMANO,J. Biochem., Tokyo 84, 925 (1978). [8] P. K. DUTrA, J. NESTOR and T. G. SPIRO, Biochem. Biophys. Res. Commun. 83, 209 (1978).

[9] P. K. DUTrA and T. G. SPIRO, J. chem. Phys. 69, 3119 (1978). [10] T. KITAGAWA,Y. NISHINA,Y. KYOGOKU,T. YAMANO, N. OHISHI,A. T. SUZUKIand K. YAC31,Biochemistry 18, 1804 (1979). [I 1] P. K. DUTrA, R. SPENCER,C. WALSHand T. G. SPIRO, Biochim. biophys. Acta 623, 77 (1980). [12] L. M. SCHOPFERand M. D. MORRIS, Biochemistry 19, 4932 (1980). [13] L. M. SCHOPFER, J. P. HANSHALTER,M. SMITH, M. MILAD and M. D. MORRIS, Biochemistry 20, 6734 (1981). [14] Y. NISHINA,K. SHIGA,K. HORIIKE,H. ToJo, S. KASAI, K. YANASE,K. MATSUI,H. WATARIand T. YAMANO,J. Biochem. Tokyo 88, 403 (1980). [15] J. SCHM1DT, P. COUDRON, A. W. THOMPSON, K. L. WATrERSand J. T. MACFARLAND,Biochemistry 22, 76 (1983). [16] W. D. BOWMANand T. G. SPIRO, Biochemistry 20, 3313 (1981). [17] Y. KYOGOKUand B. S. Yu, Bull. Chem. Soc. Japan 42, 1387 (1969). [18] A. T. SUZUKI,N. OHISHIand K. YAGI, J. Chromatogr. 169, 459 (1979). [19] K. YAGI, N. OHiSHI, A. TAKAI, K. KAWANOand Y. KYOGOKU,Flavins and Flavoproteins, p. 775 (edited by T. P. SINGER).Elsevier, Amsterdam (1976). [20] T. MIYAZAWA,J. molec. Spectrosc. 4, 155 (1960). [21] M. J. BENECKY,M. G. DOWLING, M. J. CLARKEand T.G. SPIRO, lnor#. Chem. 23, 865 (1984). [221 N. TANAKA,Kagakuno Ryoiki 26, 326 (1972). [23] Y. NISHINA,personal communication. [24] Y. NISHIMURA,H. HARUYAMA,K. NOMURA, A. Y. HIRAKAWAand M. TSUBOl,Bull. Chem. Soc. Japan 52, 1340 (1979). [25] H.T. MILES,T. P. LEWIS,E. D. BECKERand J. FRAZIER, J. biol. Chem. 248, 1115 (1973). [26] M. ABE and Y. KYOGOKU, Spectrochim. Acta, submitted.