267
Acta Chim. Slov. 1998, Y5(3), pp. 267-284 (Received 1. 9.1998)
UTILIZATION OF THE CHIROPTICAL SPECTROSCOPIES FOR THE
STRUCTURE ELUCIDATION OF FLAVONOIDS AND RELATED
BENZOPYRAN DERIVATIVEST
Albert Levai
Department of Organic Chemistry, Lajos Kossuth University, P.O.Box 20,
H-4010 Debrecen, Hungary
Abstract
Chiroptical stpectroscopies, mainly the optical rotatory dispersion (ORD) and the circular dichroism (CD) played an important role for the structure elucidation of various naturally occurring organic compounds, with the flavonoids among them, during the last four decades. The aim of the present review article is to provide characteristic examples on the utilization of these techniques in the flavonoid chemistry.
Introduction
Flavonoids are well known natural products of the plant kingdom [1-3]. Several groups  of the naturally occurring flavonoids,  e.g.  flavans,  flavanones,  isoflavans, isoflavanones, etc. are chiral molecules and are isolated in enantiopure form as optically active substances. Numerous flavonoid glycosides are optically active plant products as well. For this reason, the chiroptical techniques are very convenient and efficient tools
268
Dedicated to the late Prof. Dr. Günther Snatzke on the occasion of his 70th birth anniversary for their structure elucidation mainly for the determination of the absolute configuration of their stereogenic centres.
Of the chiroptical techniques, the optical rotatory dispersion (ORD) was utilized first by using the commercially available spectropolarimeter manufactured and marketed by the Rudolph company since 1953. According to Djerassi [4], this instrument "undoubtedly represented the prince who awoke the sleeping beauty of the rotatory dispersion". This prince offered a new opportunity for the flavonoid chemistry by the determination of the absolute configuration of the optically active flavonoids (vide infra). However, because of an easier and more convenient correlation of the structure or stereochemistry and the circular dichrosim (CD) spectra if compared to the ORD properties of a particular molecule, the circular dichroism (CD) spectroscopy came to the front since the middle of the sixties [5]. Owing to the fact that a successful measurement of a CD spectrum requires less than one milligram substance, this technique is especially convenient and helpful method for the structure elucidation of naturally occurring optically active organic compounds. Reference substances with established absolute configuration or the so-called empirical rules like the octant rule [6] and sector rules [7] help to correlate the stereochemistry and the chiroptical properties of optically active flavonoids.
In our present review article, we plan to summarize the most important results of the utilization of the chiroptical spectroscopies in the flavonoid chemistry. One of the aims of this account is to direct the attention of chemists engaged in optically active flavonoids to the utility of the chiroptical techniques.
Flavanones
Since flavanones (2-phenylchromanones) are the first representatives of the known naturally occurring optically active flavonoids, it seems to be expedient to start the discussion of the application of the so-called chiroptical techniques in the flavonoid chemistry with the flavanones. Optically active flavanones, viz. (-)-matteucinol and (-)-demethoxymatteucinol were isolated by Fujise and Kubota [8] already in 1934. Data
269
relating to the determination of the absolute configuration of the stereogenic centre of optically active flavanones were, however, published only in 1960. The absolute configuration of (-)-hesperetin (1), (-)-liquiritigenin (2) and (+)-sakuranetin (3) (Fig. 1)
HO
MeO
1: R1 = OH, R2 = OM e 2: R1 = H, R2 = OH
OH  O
OH
HO^ >H
C"CO2H
,CH„
HO2C
L-malc acid
HO. svH C"
CO2H
.CH„
HO2C
D-malc acid
Figure 1
was established by chemical degradation by Arakawa and Nakazaki [9]. These compounds were subjected to ozonization and subsequent oxidation in order to obtain malic acid of known absolute configuration (Fig. 1, L-malic acid from the (-)-flavanones 1 and 2 and D-malic acid from the (+)-flavanone 3) which gave unequivocally the absolute configuration of the investigated flavanones. Moreover, these results can be used as reference data for the determination of the absolute configuration of other flavanones with the help of chiroptical spectroscopies, e.g. optical rotatory dispersion (ORD) or circular dichroism (CD).
First utilization of a chiroptical spectroscopic method, viz. optical rotatory dispersion (ORD) in the flavonoid chemistry was published by Gaffield and Waiss [10]. They reported that (-)-flavanones 1 and 2 with 2S absolute configuration [8] showed a
270
positive Cotton effect between 320-330 nm due to the n-^%* transition. However, the dextrorotatory (+)-sakuranetin (3) of 2R absolute configuration displayed a negative Cotton effect in the same wavelength region assigned to an n-^%* transition as well. Circular dichroism (CD) spectra of flavanones including compounds 1-3 were measured fist by Arakawa and Masui [11]. Later, the CD spectra of a series of naturally occurring flavanones were studied by Gaffield [12] and the CD spectra of the unsubstituted flavanone enantiomers by Snatzke et al. [13], respectively. Very recently, Kojima et al. [14] applied the CD spectroscopy for the determination of the absolute configuration of optically active flavanones isolated from Iris tenui/olia. All these CD measurements reveal that the sign of the n^7T*-band CD maximum of flavanones are independent on the substitution pattern of the aromatic rings. For this reason, the sign of the n^7T*-band CD maximum can be utilized for the determination of the absolute configuration of the stereogenic centre of flavanones. Thus, the negative n^7T*-band CD maximum proves the absolute configuration to be 2R, while a positive sign of this CD maximum reveals a 2S absolute configuration (Fig. 2). Of course, additional Cotton effects appear at shorter wavelengths, but it is not necessary to correlate their signs with the absolute
O                                                                      O
2R -flavanones                                             2S-flavanones
negative n-^7t*-band CD        positive n-^7t*-band CD
Figure 2
configuration of the molecule. Consideration of the sign of the n^7T*-band CD is enough for this purpose. It is worth mentioning that Gaffield [10] attempted to apply the Snatzke's modified octant rule [15] for the prediction of the sign of the n^7T*-band CD maximum as well.
271
Although it is not the aim of this review article to discuss the utility of the chiroptical techniques for related heterocyclic compounds with heteroatom other than oxygen, we mention that the CD spectroscopy was successfully applied for the determination of the absolute configuration of optically active 1-thioflavanones and the so-called "azaflavanones" by Antus et al. [16].
3-Hydroxyflavanones (dihydroflavonols) are well known and important natural products isolated from numerous plants [1-3]. Owing to their two stereogenic centres,
OH
2R,3S
positive n-^7t*-band CD
2S,3 R
negative n-^7t*-band CD
¦R
O 2R,3R
positive n-^7t*-band CD
O
2S,3S
negative n-^7t*-band CD
Figure 3
they can exist in four optically active stereoisomers (Fig. 3). CD spectroscopy was successfully used for the determination of their absolute configuration [10,12,14,17,18]. A positive n^7T*-band CD maximum in the 330-340 nm wavelength region proves the 2R corfiguration for both the eis and trans isomers. However, a negative CD maximum belonging to a similar wavelength value reveals a 2S absolute configuration for the other two stereoisomers. Configuration at the C-3 atom can then be deduced from these data (Fig. 3). As in the case of flavanones (vide supra), although several CD maxima are generally measured, it is enough to use the sign of the n^7T*-band CD maximum around
272
330-340 nm to determine the absolute configuration of both stereogenic centres of the 3-hydroxyflavanones.
Isoflavanones
Recently, opticaly active isoflavanones (3-phenylchromanones) have also been isolated from various plant sources [19,20]. First utilization of a chiroptical spectroscopy for synthetic isoflavanones was described by Farkas et al. [21] in 1969. They measured the ORD spectra of some isoflavanone glycosides and determined the absolute configuration of the aglycone of the (-)-5-acetoxy-7-methoxy-4'-(tetra-0-acetyl-ß-D-glucosyloxy)isoflavanone by this method. CD spectroscopy was used to determine the absolute configuration of the recently isolated isoflavanones [19,20]. A positive n-^%*-band CD maximum in the 325-352 nm wavelength region reveals a 3R absolute configuration on the basis of the modified octant rule [15] (Fig. 4).
3R -is oDflavanones positive n-^7t*-band CD
1aS,7aR                                                           1aR ,7aS
positive nr^K*-band CD                               negative n-^7T*-band CD
Figure 4
A newest development in the application of the CD spectroscopy for the determination of the absolute configuration of isoflavanone derivatives is our own study
273
on optically active isoflavone epoxides [22] (Fig. 4). It has been found that a negative n-^%* CD maximum of the (-)-compounds uniquivocally proves the laR, 7aS, while a positive sign of the same CD band of their (+)-enantiomers reveals the laS, 7aR absolute configuration by using the Snatzke's "inverse octant rule" [15]. These results are in full harmony with those determined by X-ray diffraction analysis [23]. Thus, it can be concluded that in the case of the optically active isoflavanones or 3,4-epoxyisoflavanones the knowledge of the sign of their n^7T*-band CD maximum is enough to determine the absolute configuration of their stereogenic centres by using the modified octant rule or the inverse octant rule both introcuded by Snatzke [15].
Flavans
In the present review two groups of flavan derivatives, viz. 3-hydroxyflavans and 4-substituted flavans are discussed, the available CD data of which make possible some general considerations.
3-Hydroxyflavans
The stereochemical problems of the 3-hydroxyflavans (catechins and epicatechins) have been summarized in a detailed review article [24]. The absolute configuration of the stereogenic centres of these flavan derivatives were generally determined either by using the Prelog's atrolactic acid method [25] or by azonolysis [26] prior to the availability of the CD spectroscopy. From the early seventies the CD spectroscopy has been successfully used for this purpose as well [27-31].
The absorption bands of these chromophores are found in two regions: around 280 nm (1B2u transition) and at about 240 nm (^Biu transition). In their CD spectra Cotton effects are expected in these two regions. The CD spectra of a series of 3-hydroxyflavans at 25 and -185 °C have been measured by Korver and Wilkins [27]. According to their study, the sign of the Cotton effect due to the 1B2u transition is determined by the chirality of the hetero ring, which in turn is connected with the absolute configuration of the stereogenic centre at position 2 (Fig. 5). Thus, the 2R absolute  configuration  gives  rise  to  a  negative  CD  band,  while  a  2S  absolute
274
configuration to a positive one [27]. The contribution of the C-3 stereogenic centre to this CD band is small. On the basis of low temperature measurements they managed to evaluate the contribution of the phenyl group at the C-2 atom as well. Therefore, it can be concluded that the CD spectroscopy can be advantageously utilized for the determination of the absolute configuration of the 3-hydroxyflavan derivatives.
2R ,3S-3-hydroxyflavan        2S ,3-R-3-hydroxyflavan
2R ,3 R -3-hydroxyflavan        2S,3S-3-hydroxyflavan
Figure 5
4-Substitutedflavans
A series of 4-substituted eis- and trans-Qavan derivatives have been prepared by Bognâr et al. [32-36] by the optical resolution and subsequent chemical transformations of 4-amino- and 4-hydroxyflavans. The CD spectra of these compounds have been studied by Snatzke et al. [13]. The absolute configuration of the two stereogenic centres of these compounds result from their conversion into flavanone enantiomers of known absolute configuration [33]. Moreover, the CD spectroscopy could also be utilized for the determination of the conformation of these 4-substituted flavans. The evaluation of the second- and third-sphere contributions revealed a sofa conformation for both their eis- and trans-isomers [13] (Fig. 6). Sofa conformation has also been suggested by Korver and Wilkins [27]  in the case of    3-hydroxyflavans.  A sector rule for the
275
estimation of the influence of the 4-substituent has also been proposed by Snatzke et al. [13].
R R: OH, NH3+ Cl-, NHAc, NHBz cis-com. pounds
Figure 6
Isoflavans
ORD and CD measurements have been used for the determination of the absolute configuration of naturally occurring isoflavan derivatives [37-41]. Asolute configuration of the (3$-5,7,3',4'-tetramethoxyisoflavan (4) (Fig. 7) was established by its chemical correlation with ,S'-(-)-methylsuccinic acid [42]. This compound served as a reference for the determination of the absolute configuration of other optically active isoflavans by means of chiroptical methods. The ORD spectra of the i^-isoflavans showed a negative Cotton effect in the 260-300 nm region [37]. In the CD spectra of the i^-compounds around 280 nm also a negative maximum, while at about 230-240 nm a positive one were measured. The sign was opposite in both regions for the IR-enantiomers. On all these bases, it can be concluded that the chiroptical techniques can be beneficially used for the determination of the absolute configuration of optically active isoflavans as well (Fig. 7).
3R -isoflavan                                  3S-iso flavan                                                                  4
Figure 7
Flavone and isoflavone glycosides
H
R: OH, OAc trans-com pounds
276
Natural flavones and isoflavones are frequently present in their plant sources attached to mono- or oligosaccharides [1-3]. Both the C-glycosides and O-glycosides of these two types of flavonoids have been isolated from a wide variety of plants [1-3]. Owing to their sugar component, all these natural products are optically active compounds making possible the utilization of the chiroptical techniques for their structure elucidation. Herein we provide some characteristic examples for such application of the ORD and CD spectroscopies.
C-Glycosylflavones
The CD spectra of 6- and 8-glycosylflavones (Fig. 8) have been investigated by Gaffield et al. [43,44]. Their studies prove that the sign of the Cotton effect at 250-275 nm depends upon the position of the C-glycosyl residue on the flavone if the sugar belongs to the D-series and the glycosidic linkage is ß. In this region all the 6-C-glycosylflavones displayed a positive, and the 8-C-glycosylflavones a negative Cotton effect. On this basis the CD spectroscopy appears to be a sensitive method for the differentiation of the 6- and 8-C-glycosylflavones.
O                                                                              O
6-C ^]yosy-]flavones                                           8 -C ^]yosy-]flavones
Figure 8
In view of these findings attempts have been made to use the CD for the differentiation of O-glycosylflavones and O-glycosylisoflavones as well. Prior to the discusssion of the chiroptical properties of the O-glycosylflavones and the O-glycosylisoflavones, CD properties of the simple phenyl glycosides should be mentioned.
277
According to detailed investigations, both 1B2u and ^lu CD maxima are negative for phenyl ß-D-glycosides and positive for the oc-anomers [45,46]. This relationship, however, is not necessarily true for glycosides with a more complicated chromophore, e.g. flavone or isoflavone molecules.
O-Glycosylflavones
CD spectra of few O-glycosylflavones are available in the literature [47,48 ], but the published data do not make possible the establishment of such a relationship between the chiroptical properties and the site of the glycosidic linkage as in the case of C-glycosylflavones [43,44]. To improve these informations, we decided to investigate a systematic series of flavone glycosides, viz. the quercetin glucosides, where the aglycone (3,5,7,3',4'-pentahydroxyflavone), the sugar component (D-glucose) and the glycosidic linkage (ß) are the same [49] (Fig. 9). Compounds under study (5-12) were synthesized by Farkas et al. [50-52].
SrR1 = Gli;R2 = R3 =R4 = R5 = H
ôrR1 = R3 =R4 = R5 = H ;R2 = Gli VrR1 = R2 =R4 = R5 = H ;R3 = Gli SrR1 = R2 =R3 = R4 = H ;R5 = Gli
Gli: ß -D -gllCDSyl
Figure 9
^R1 = R4 = Gli;R2 = R3 = R5 = H 10 rR1 = R5 = Gli;R2 = R3 = R4 = H 11rR1 = R3 = Gli;R2 = R4 = R5 = H 12 rR1 = R2 = R4 = H ;R3 = R5 = Gli
278
A negative CD maximum was observed for all compounds between 340 and 375 nm which seems to belong to the n-^%* transition. The magnitude of this maximum somewhat alters with the position of the glycosidic linkage. Around 250 nm a characteristic intense positive band was measured for the 3-O-glucoside and a negative one for the 5- and 4'-0-glucosides, respectively. 7-Glucosylquercetin, in which the free rotation of the sugar component is not hindered, exhibited only one negative maximum at 375 nm. The CD spectra of the quercetin diglucosides displayed a superposition of the CD spectra of the corresponding two monoglucosides.
O-Glycosylisoflavones
The sugar component of naturally occurring O-glycosylisoflavones known so far are attached to the aglycone through the hydroxyl group either at C-7 or C-4' of isoflavone by a ß-linkage [1-3] (Fig. 10). The position of the glycosidic linkage has been elucidated by means of chemical degradation or by NMR spectroscopy.
negative n-^71*-band CD                                          positive n-^7I*-band CD
Figure 10
The CD spectra [48,53] of a series of synthetic 7-ß-glycosyloxyisoflavones [54-56] and natural 4'-ß-glycosyloxyisoflavones have been measured. These measurements revealed that the sign of the CD maximum due to the n-^%* transition (330-350 nm) is dependent on the site of the glycosidic linkage: it is negative for 7-ß-glycosyloxyisoflavones and positive for 4'-ß-glycosyloxyisoflavones (Fig. 10). These findings refer only to such isoflavone O-glycosides where a D-sugar is attached to the aglycone by a ß-linkage. Therefore, the CD spectroscopy can serve as a simple and
279
convenient method for the elucidation of the site of the glycosidic linkage of newly isolated natural isoflavone glycosides.
Biflavones
First representatives of the optically active biflavones, the (-)-5,5"-dihydroxy-4',4"',7,7"-tetramethoxy-8,8"-biflavone (Fig. 11 (-)-13) was isolated from Araucaria cunninghamii and A cooki in 1968 [57]. Later, several other optically active biflavones, viz. cupressuflavones [57-59], amentoflavones [60,61] and agathisflavones [62] have also been isolated from various plant sources. Their chirality is due to the atropisomerism of the biflavone moiety. Recently, synthesis of optically pure biflavones has also been achieved [63-65]. However, the first data for the determination of their absolute configuration were published only in 1995 by Lin et al. [63]. X-ray diffraction analysis of the (+)-bis-5,5"-camphonate of (+)-13 (Fig. 11) revealed an aR absolute configuration by correlation with the known configuration of the camphorsulfonate group. This compound served as a reference for the determination of the absolute configuration of other optically active biflavones by CD spectroscopy [63-65]. By means of a combined utilization of the X-ray diffraction analysis and the CD spectroscopy Lin et al. [63] assigned an aR configuration to the (+)-biflavones and and aS one to their (-)-enantiomers, whereas Harada et al. [59,65] an aR configuration to compound (-)-13 (Fig. 11) on the basis of the same CD data. This contradiction has not hitherto been solved by the reinvestigation of the absolute configuration of the optically active biflavones.
280
OR  0                                                              OR   O
OR   O                                                                 OR  O
R = H: (+)-13                                                         R = H: (-)-13
R = M e: (+)-14                                                      R = M e: (-)-14
Figure 11
Other benzopyran derivatives
4-Hydroxychromans
Optically active 4-hydroxychromans have been prepared by kinetic resolution of (±)-chroman-4-ols by microbial lipases using vinyl acetate as acyl donor [66,67]. Under these reaction conditions (+)-chroman-4-ols were converted into their acetates and the (-)-chroman-4-ols remained unchaged materials in high enantiomeric excess (Fig. 12). The absolute configuration of these optically active chroman derivatives has been determined by CD spectroscopy [68] either by using the so-called helicity rule [13,16] or by measuring the exciton-coupled CD spectra of the 4-benzoyloxychromans [68]. These measurements revealed a 4R configuration to the (+)-4-acetoxychromans and 4S one to the (-)-4-hydroxychromans (Fig. 12).
281
4S                               4R
Figure 12
2,2-Dimethyl-3,4-epoxychromans
Optically active 2,2-dimethyl-3,4-epoxychromans have been synthesized by several research groups [69-77] by the catalytic enantioselective epoxidation of 2,2-dimethyl-2//-chromenes by using Mn(III)salen catalysts. However, no direct determination of their absolute configuration has been published prior to our paper [77] in 1996. Jacobsen et al. [78] described the measurement of the CD spectra of some optically active 2,2-disubstituted 3,4-epoxychromans, but without mentioning the correlation of the CD data with the configuration of their epoxides. In our own study [77], absolute configuration of one optically active 2,2-dimethyl-3,4-epoxychroman was determined by X-ray diffraction analysis which then could be used as reference substance for the CD study of the related epoxides. Thus, the (+)-compounds proved to be the 3R, Yi?-enantiomers and the (-)-ones are the 3S, •tó'-enantiomers determined by CD measurements (Fig. 13). These CD data [77] may serve as reference for the configurational assignment of related 3,4-epoxychromans by CD spectroscopy or even on the basis of the sign of their specific rotation.
3R,4R                                    3S,4S
Figure 13
282
3,4-Dihydroxy-2,2-dimethylchromans
All four optically active stereoisomers of the 3,4-dihydroxy-2,2-dimethyl-7-methoxychroman (Fig. 14) were synthesized by Jennings [79]. Absolute configuration of these compounds were determined by the CD measurement of their bis-p-N,N-dimethylaminobenzoate by means of the exciton chirality method [80].
MeO
Me
OH
OH
3R,4S
MeO
OH
3S,4R
MeO
Me
OH
3R,4R
MeO
Figure 14
3S,4S
Me
In summary, the present review is aimed to show the utility of the chiroptical spectroscopic methods through the eyes of a synthetic chemist who looks for tools to determine the stereochemistry of optically active flavonoids. Theoretic backgrounds of chiroptical techniques discussed here are, therefore, omitted.
Acknowledgement
The preparation of this review article was sponsored by the Hungarian National Research Foundation (Grant No. OTKA T 014864) for wich our gratitude is experessed.
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Povzetek. Kirooptiène spektroskopske metode, še posebej optièna rotacijska disperzija (ORD) in cirkularni dihroizem (CD), so odigrale pomembno vlogo pri doloèanju strukture razliènih naravnih organskih spojin. Namen èlanka je prikazati uporabo teh metod na na primeru flavonoidov.