Journal of Molecular Structure 651–653 (2003) 27–37

www.elsevier.com/locate/molstruc

Molecular structural studies of lichen substances II: atranorin,

gyrophoric acid, fumarprotocetraric acid, rhizocarpic acid, calycin,
pulvinic dilactone and usnic acid
Howell G.M. Edwardsa,*, Emma M. Newtona, David D. Wynn-Williamsb,1
a
Department of Chemical and Forensic Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK
b
British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, UK
Received 2 September 2002; accepted 18 September 2002

Abstract
The FT-Raman and infrared vibrational spectra of some important lichen compounds from two metabolic pathways are
characterised. Key biomolecular marker bands have been suggested for the spectroscopic identification of atranorin, gyrophoric
acid, fumarprotocetraric acid rhizocarpic acid, calycin, pulvinic dilactone and usnic acid. A spectroscopic protocol has been
defined for the detection of these molecules in organisms subjected to environmental stresses such as UV-radiation exposure,
desiccation and low temperatures. Use of the protocol will be made for the assessment of survival strategies used by stress-
tolerant lichens in Antarctic cold deserts.
q 2003 Elsevier Science B.V. All rights reserved.
Keywords: Raman; Infrared spectroscopy; Lichens; Antarctica

1. Introduction and carotenoids (from the mevalonic acid pathway,

In an earlier paper of this series [1], the importance
of an unequivocal identification of key Raman
biomarker bands in the spectra of metabolic lichen
products for the evaluation of survival strategies of
lichens and cyanobacteria in stressed habitats was
emphasised. The Raman spectra of the major products
of lichen metabolism show evidence for three groups
of products, namely, polysaccharides and pulvinic
acid (from the shikimic acid pathway, SA) terpenes
* Corresponding author. Tel.: þ 44-1274-233787; fax: þ 44-
1274-235350.
E-mail address: h.g.m.edwards@bradford.ac.uk (H.G.M.
Edwards).
1
Deceased.
0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 2 8 6 0 ( 0 2 ) 0 0 6 2 6 - 9
MVA) and anthraquinones, usnic acid, and orsellinic
acids (from the acetyl-polymalonyl pathway, APM)
(Fig. 1) [2,3]. The latter group is responsible for the
production by lichens of substituted phenolic esters
called depsides. The distribution of depsides among
lichen species is often indicative of the species
concerned and can be used for taxonomic classifi-
cation [2,4]; to this effect, whereas most chemical
studies have been carried out on extracts, this
necessarily involves a destruction of the specimen.
In this paper, we investigate for the first time the
Raman spectra of selected compounds from two of the
three metabolic pathways. These comprise atranorin,
fumarprotocetraric acid and gyrophoric acid (Fig. 2)
which are important members of the depside group of
28 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37

Fig. 1. Probable pathways leading to the major groups of lichen products (after Ref. [3]).

lichen products arising from the APM pathway, growing, the ability to record spectra without
rhizocarpic acid, pulvinic dilactone and calycin detachment of the organism from its substratum
(Fig. 3) from the SA pathway and usnic acid (Fig. 4) will facilitate the monitoring of the response to
from the APM pathway. Carotenoids from the MVA external stress over a period of time which could
pathway have been characterised spectroscopically extend over several years [10].
previously [5]. Although the spectra here have been
obtained from purified extracts of lichen species, the
key biomarker bands indicative of these compounds
will provide the basis of the non-destructive evalu-
ation of their presence in situ in living lichen
communities using Raman spectroscopy. Hence, two
possible broader outcomes of this research will form
the basis of our future work:

† The viability of Raman spectroscopy for the

taxonomic identification of lichen species based
on key spectral characteristic biomarkers;
† The evaluation and assessment of the survival
strategies of lichen communities in stressed
environmental habitats for which the non-destruc-
tive analytical approach will facilitate the moni-
toring of organism response to desiccation,
radiation exposure and cryogenic shock (freeze-
thaw cycling) in cold desert locations based on the
materials produced in the MVA, SA and APM Fig. 2. Structures of (a) atranorin, (b) fumarprotocetraric acid and
metabolic pathways [6 – 9]. Since lichens are slow- (c) gyrophoric acid.
 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37
Fig. 3. Structures of (a) pulvinic dilactone, (b) calycin and (c) rhizocarpic acid.
2. Experimental
2.1. Specimens
For the first set of compounds from the APM
pathway, atranorin was obtained commercially from
Sigma Chemicals; gyrophoric acid and fumarprotoce-
traric acid were extracted from Antarctic lichens. The
molecular structures of these depsides are shown in
Fig. 2; all three are aromatic esters but gyrophoric and
fumarprotocetraric acids also contain carboxylic acid
groups on the terminal ring, whereas atranorin has a
methyl ester group in this position. Atranorin and
fumarprotocetraric acid are both aromatic aldehydes,
but the latter also has an ester side chain with a vinylic
unsaturated carboxylic acid. All three are substituted
toluenes, but fumarprotocetraric acid also has a COC
glycosidic bond between both aromatic rings of the
depside.
For the second set of compounds studied, which
arise from the SA pathway, the characteristic structures
shown in Fig. 3 all contain furan ring systems
conjugated with ketonic CyO and CyC bands; two
specimens, pulvinic dilactone and calycin also contain
fused ring systems and rhizocarpic acid is distinguished
by an amide group.
The third set of compounds studied is exemplified by
usnic acid (Fig. 4) distinguished by its three-ring fused
skeleton and aromatic benzoquinone structure. In this it
differs somewhat from the other members of the APM
pathway shown in Fig. 2, which are all characterised by
29
ester groups bridging highly substituted aromatic rings.
Hence, usnic acid, although also a product of the APM
metabolic pathway, is considered separately here for
the purpose of vibrational spectroscopic analysis.
2.2. Raman and infrared spectroscopy
FT-Raman spectra of all specimens studied were
obtained using a Bruker IFS66 instrument with FRA
106 Raman module attachment and 1064 nm exci-
tation from a Nd3þ/YAG laser. Spectra were recorded
over a 3500– 100 cm21 wavenumber range with up to
2000 spectral accumulations at 4 cm21 resolution to
increase signal-to-noise ratios for spectral quality
enhancement. Wavenumbers are accurate to
^ 1 cm21.
Infrared spectra were recorded using a Nicolet
Omnic IR spectrometer over the wavenumber range
4000– 400 cm21 using potassium bromide disks. The
infrared bands of several of the compounds studied here
have already been reported in the literature but
Fig. 4. Structure of usnic acid.
30 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37

vibrational assignments have not been proposed
hitherto.
3. Results and discussion
3.1. Atranorin, gyrophoric acid
and fumarprotocetraric acid
The FT-Raman and infrared spectra are shown in
Fig. 5(a) – (d) and wavenumbers with possible assign-
ments are listed in Table 1. Whilst a tentative
assignment of the 1666 cm21 band of atranorin to a
n(CHO) aromatic/n(CyO) aldehyde mode and the
1658 cm21 band to the phenyl ester carbonyl stretch
could be made, it should be noted that gyrophoric acid
and fumarprotocetraric acid also have similar phenyl
ester linkages. It is likely that the highly conjugated
carbonyl mode, which could also undergo intra-
molecular hydrogen-bonding in all three cases studied
here, will be significantly lower in frequency than the
literature value of about 1725 cm21 and be present in
all three spectra. It has therefore been assigned here to
the 1666, 1662 and 1679 cm21 bands in the atranorin,

Fig. 5. (a) (i) Infrared and (ii) FT-Raman spectrum of atranorin, wavenumber region 3200–2700 cm21. (b) (i) Infrared and (ii) FT-Raman
spectrum of atranorin, wavenumber region 1800–400 cm21. (c) FT-Raman spectra of (i) atranorin, (ii) gyrophoric acid and (iii)
fumarprotocetraric acid, wavenumber region 3200–1700 cm21. (d) FT-Raman spectra of (i) atranorin, (ii) gyrophoric acid and (iii)
fumarprotocetraric acid, wavenumber region 1800–200 cm21.
 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37 31

Fig. 5 (continued )

gyrophoric acid and fumarprotocetraric acid spectra,
respectively. The aldehyde carbonyl stretch
has been assigned to the 1632 cm 21 band of
atranorin and to the corresponding 1641 cm21 band
of fumarprotocetraric acid. The methyl ester is unique
to atranorin and so the n(CyO) is most likely to be the
1658 cm21 band, which does not have a correspond-
ing band in the other spectra. Gyrophoric acid and
fumarprotocetraric acid both contain an aromatic
carboxylic acid group on the terminal ring; we have
assigned the carbonyl stretch of this group to the
1628 cm21 band of gyrophoric acid, and to a medium
intensity shoulder at 1626 cm21.
3.2. Rhizocarpic acid, pulvinic dilactone and calycin
The FT-Raman and infrared spectra are shown in
Fig. 6 and the wavenumbers listed with proposed
vibrational assignments in Table 2.
As expected, all three compounds have an aromatic
CH stretching band around 3065 cm21, but only
rhizocarpic acid has bands attributable to CH2 and
CH3 stretches. The infrared spectrum of rhizocarpic
acid also has a very strong broad n(N –H) band at
3370 cm21.
Rhizocarpic acid has two very weak FT-Raman
bands (strong in the infrared) at 1769 and 1740 cm21,
32 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37

Table 1
FT-Raman and infrared approximate vibrational assignments of atranorin, gyrophoric acid and fumarprotocetraric acid

n (cm21) Approximate vibrational assignment (present work)

Atranorin Gyrophoric acid Fumarprotocetraric acid

Raman Infrared Raman Infrared Raman Infrared

3094 w 3080 w n(CH) aromatic

3040 vw 3040 vw 3052 w 3050
2986 w 2984 mw 2987 w 2982 w 3000 n(CyCH2)?
2956 mw 2955 m n(CH3) as
2941 m 2945 mw 2940 mw
2932 mw, sh 2932 m n(CH2) as
2876 w 2873 w 2864 w n(CH3) s
1744 m, sh
1727 ms 1720 n(CyO) chain ester
1709 mw, sh n(CyO) chain carboxylic acid, non-dimer form
1666 ms 1667 s, sh 1662 s 1665 1679 w 1690 n(CyO) phenyl ester, conjugated
1658 ms 1653 s n(CyO) methyl ester
1632 m 1630 ms, sh 1641 s 1640 n(CyO) aldehyde
1628 m 1626 m, sh n(CyO) carboxylic acid
1620 mw, sh 1620 m, sh Quadrant ring stretches; n(CyC) aromatic
1612 m 1610 1614 m
1588 mw 1584 m 1587 mw, sh 1574 mw 1568
1508 n(CyC) aromatic
1465
1456 w, sh 1451 m
1450 1446 w 1448
1437 mw 1440 m 1442 w d(CH2), d(CH3)
1411 w 1406 m, br 1408 w 1412
1380 mw 1380 w 1383 mw 1385 1380 mw 1380
1350 w 1360
1334 m 1350
1315 s Ring stretches
1304 s, sh 1310
1303 vs 1303 m, sh 1291 vs
1294 vs 1287 s 1288 ms 1290
1266 ms, sh 1270 s n(C– O) methyl ester
1235 s 1240
1230 w 1230 n(C– O) chain ester
1205 mw 1200 1207 w 1200 n(C– O) carboxylic acid
1167 ms
1150
1138 m 1140 n(CC) ring breathing
1130 w 1125 n(C– O) chain ester
1114 mw 1112 ms
1094 vw 1090
1078 m n(C– O) methyl ester
1068 w 1070
1047 w 1050
1025 vw 1020
1010 vw 1010
997 mw 1000
985 990
920 w
 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37 33

Table 1 (continued)
n (cm21) Approximate vibrational assignment (present work)

Atranorin Gyrophoric acid Fumarprotocetraric acid

Raman Infrared Raman Infrared Raman Infrared

901 mw 899 w 892 w 900

863 w 870 894 vw 880 n(C– O) chain carboxylic acid, H-bonded
861 mw 861 w
844 w 850
830 w 840
821 mw 821 mw
805 w 810
786 w 800
790
781 mw 782 mw
765
750
740
700 708 vw 708
609 mw
588 ms 586 mw 591 mw 592 w
561 m
518 mw

which can be assigned to the CyO stretches of the
lactone and methyl ester groups, respectively. The
amide n(CyO) mode is assigned to the medium –
strong shoulder at 1610 cm21, which is also very
strong in the infrared spectrum. It is possible to assign
the CyO stretch of the ester to the band at 1769 cm21
and the lactone n(CyO) to the band at 1668 cm21.
However, the latter band is more intense in the FT-
Raman spectrum than the infrared, indicating that it is
more likely to be due to a CyC stretch. It has therefore
been assigned to a n(CyC) aromatic mode in the
present work, along with the strong bands at 1653 and
1672 cm21 in calycin and pulvinic dilactone, respect-
ively. A very strong band at 1611 cm21 in calycin is
assigned to the n(CyC) of the conjugated bridging
bond, between the two identical halves of

Fig. 6. FT-Raman spectra of (i) rhizocarpic acid, (ii) calycin and (iii) pulvinic dilactone, wavenumber region 1800–400 cm21.
34 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37

Table 2
FT-Raman (infrared: rhizocarpic acid only) wavenumbers (cm21) and approximate vibrational assignments of rhizocarpic acid, calycin and
pulvinic dilactone.

n (cm21) Approximate vibrational assignment

Rhizocarpic acid Calycin Pulvinic dilactone

Infrared Raman

3370 s n(N–H)
3060 w 3062 mw 3066 vw 3072 vw n(CH) aromatic
3010 w
2975 m, sh 2970 w, sh n(CH3) as
2930 ms 2938 mw n(CH2) as
2899 mw n(CH3) s
2856 mw 2855 mw n(CH2) s
1764 m 1769 w 1798 w n(CyO) lactone
1740 vs 1740 vw n(CyO) ester
1705 mw n(CyO) lactone
1668 m 1665 vs 1653 m, sh 1672 vs n(CyC) arom
1635 s n(CyC) arom
1608 vs 1610 s, sh 1611 vs n(CyO) amide; n(CyC) link
1597 w 1595 vs 1595 s, sh 1603 ms n(CyC) furan
1548 s 1547 w n(C–N) amide
1520 w, sh 1518 ms
1499 ms 1496 ms 1499 w 1499 w
1475 m 1477 ms d(CH3) as
1442 m 1422 w, sh 1461 w 1455 mw n(CyC) furan; d(CH3) s
1405 s d(CH)
1380 w 1380 s
1350 m, sh 1347 mw 1344 mw d(COH) alcohol
1304 s 1303 mw 1304 w 1311 mw n(C–O–C) lactone, conj’d
1285 ms, sh 1280 m Amide III
1250 m 1254 mw, sh 1263 w n(CCO) as alcohol
1240 mw
1190 mw 1189 w 1195 w 1195 w
1164 ms 1163 mw n(C–O) ester
1155 mw
1124 m 1123 w 1118 vw 1117 vw
1075 mw 1074 w n(C–O) ester
1025 mw 1031 w 1034 mw Ring deformation
1003 w 1002 m 999 w 1000 w n(CC) arom
976 mw 973 w 981 m Ring deformation
960 m Ring deformation
950 w 944 mw r(CH3, CH2)
904 mw 902 mw
878 mw Ring deformation
827 w Ring deformation
790 mw 787 mw 782 w n(CCO) s alcohol
740 w
711 w, d
700 m 701 w, d 701 w 706 w
639 w 635 w
620 mw, sh 618 w 616 vw 618 w
602 mw 600 w 595 w d(CCO) furan
527 w
 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37 35

Table 2 (continued)
n (cm21) Approximate vibrational assignment

Rhizocarpic acid Calycin Pulvinic dilactone

Infrared Raman

504 w 498 mw 504 m

484 mw
448 mw
405 w
301 w

Fig. 7. (a) Usnic acid (i) infrared, (ii) FT-Raman spectra, wavenumber region 3200–2700 cm21. (b) Usnic acid (i) infrared, (ii) FT-Raman
spectra, wavenumber region 1800–400 cm21.
36 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37

the molecule. All three FT-Raman spectra have a
strong band at approximately 1600 cm21 and a
weaker feature around 1455 cm21, which are charac-
teristic of the n(CyC) modes of 2-substituted furans.
The methyl ester group of rhizocarpic acid has
additional bands at 1163 and 1074 cm21, which can
be assigned to the C –O stretches.
Rhizocarpic acid and calycin have a tertiary
alcohol function that has characteristic bands at
approximately 1345, 1255 and 785 cm21 which are
assigned to the d(COH), n(CCO) asymmetric and
symmetric modes, respectively.
3.3. Usnic acid

Table 3 The Raman and infrared spectra of usnic acid are

Approximate Raman and infrared band wavenumbers (cm21) and shown in Fig. 7 with the wavenumber listing and
vibrational assignments for usnic acid vibrational assignments in Table 3. Here, it is possible
to assign the conjugated cyclic ketone group to the
n (cm21) Approximate vibrational assignment
1694 cm21 band. Weak bands at 1716 and 1676 cm21
Raman Infrared in the infrared spectrum are assigned to the n(CyO)
non-conjugated cyclic ketone and the non-aromatic
3101 w 3100 vw n(CH) aromatic methyl ketone, respectively. Conjugation, electron
3093 w 3091 w
donating ring substituents and possible intra-molecu-
3008 mw, sh 3009 w, br
2985 m 2983 w n(CH3) asymmetric lar hydrogen-bonding, all contribute to the lower
2930 s 2930 m wavenumber position of the aromatic methyl ketone
2880 w n(CH3) symmetric to 1627 cm21. It is also possible to assign the
2868 w 2869 w, br antisymmetric and symmetric n(COC) aryl alkyl
1716 w n(CyO) cyclic ketone
ether modes to bands at approximately 1288 and
1694 m 1691 ms n(CyO) conjugated cyclic ketone
1676 w, sh n(CyO) non-aromatic methyl ketone 1070 cm21, respectively, with the aid of the infrared
1627 m, sh 1630 vs n(CyO) aromatic ketone spectrum.
1607 ms 1611 s, sh Quadrant ring stretch
1544 vw 1541 s, br n(CyC) aromatic
1484 vw 1483 ms, sh
4. Conclusions
1457 w 1458 s, br d(CH2, CH3)
1444 w 1440 m, sh
1421 m The successful recording of the Raman and
1372 w 1376 m infrared spectra of extracts of important lichen
1358 w, sh 1358 w metabolic products has been accomplished. From
1335 w
this study, it is now possible to identify key molecular
1322 s 1317 w Ring stretch
1289 ms 1290 vs, br n(COC) as aryl alkyl ether biomarkers from which possible protocols for lichen
1221 w 1221 w taxonomy and stress survival strategy could emerge—
1192 m 1191 vs d(OH) phenyl, in plane this will form the subject of a future paper on the
1156 w application of non-destructive Raman spectroscopic
1145 w 1144 ms
analysis for the identification of chemicals in lichens
1119 mw 1118 ms
1071 w 1070 s n(COC) s aryl alkyl ether growing under environmental stresses.
1037 w 1040 s However, here it is appropriate to identify some
992 m 992 mw possible biomarkers for important lichen materials
959 mw 959 mw from the FT-Raman spectra—these are tabulated in
932 w 931 w
Table 4; in a further study, we will describe the
870 w 870 vw, sh
846 mw 841 s selection and observation of these key vibrational
818 s biomarker bands for the monitoring of strategies
731 w, br 733 w adopted by lichens under environmental stress.
699 vw, br 700 mw d(OH) phenyl, out of plane A possible problem is highlighted in the listing of
602 m 601 m
key biomolecular marker bands in Table 4 for the
540 m
identification of lichen substances using FT-Raman
 H.G.M. Edwards et al. / Journal of Molecular Structure 651–653 (2003) 27–37 37

Table 4 band at 1660 –1670 cm21 is not sufficient in itself to

Identification of key molecular biomarkers (indicated in bold) for identify atranorin, since several other compounds in
lichen substances from FT-Raman spectra
the list of those studied here have a feature close to
Lichen substance Key vibrational biomarker in this in wavenumber, the presence of others at 1658,
FTRS (cm21) 1206 and 588 cm21 is unique to atranorin, whereas
Atranorin 166 ms, 1658 ms, pulvinic dilactone has bands at 1405 and 504 cm21,
1632 m, 1437 m,
1303 vs, 1266 ms, 588 ms
and rhizocarpic acid has supporting features at 1499
Gyrophoric acid 1662 s, 1628 m, and 1164 cm21. This procedure will form the basis of
1612 m, 1291 vs, 561 m a future application to living lichen communities and
Fumarprotocetraric acid 1727 ms, 1709 mw, 1614 m, their assessment viability using an FT-Raman spec-
1315 s, 1288 ms, 1235 s, 518 m
troscopy protocol as suggested here [12].
Rhizocarpic acid 3370 s, 1764 m, 1740 vs, 1608 vs,
1548 s, 1499 ms, 1476 m, 1304 s,
1164 ms, 700 m
Calycin 1705 m, 1611 vs, 1595 s, 960 m
Pulvinic dilactone 1672 vs, 1405 s, 504 m References
Usnic acid 1694 m, 1607 ms, 1322 s, 1289 ms,
1192 m, 602 m, 540 m [1] H.G.M. Edwards, E.M. Newton, D.D. Wynn-Williams, S.J.
Coombes, J. Mol. Struct. (2002) in press.
[2] S. Huneck, I. Yoshimura, Identification of Lichen Substances,
spectroscopy in that the presence of more than one Springer, Berlin, 1996.
compound in significant amounts is likely to give rise [3] J.A. Elix, Biochemistry and secondary metabolites, in: T.H.
to a misattribution if only one band is selected as Nash (Ed.), Lichen Biology, Cambridge University Press,
Cambridge, 1996, pp. 154 –180.
characteristic biomarker for each compound con-
[4] V. Ahmadjian, The Lichen Symbiosis, Wiley, Chichester,
cerned [11]. For example, although a band at 1612– 1993.
1614 cm21 is ‘characteristic’ of gyrophoric acid and [5] D.D. Wynn-Williams, H.G.M. Edwards, E.M. Newton, J.M.
fumarprotocetraric acid in the APM pathway, a Holder, Int. J. Astrobiol. 1 (2002) 39.
feature at 1611 cm21 in calycin is characteristic in [6] H.G.M. Edwards, N.C. Russell, D.D. Wynn-Williams,
this compound for the SA pathway compounds J. Raman Spectrosc. 28 (1997) 685.
[7] L. Kappen, Response to extreme environments, in: V.
studied. Any lichen species therefore, that produces
Ahmadjian, M.E. Hale (Eds.), The Lichens, Academic Press,
gyrophoric acid and calycin will negate the individual New York, 1973, pp. 311–380.
spectroscopic identification of these using this band. [8] E.I. Friedmann, Science 215 (1982) 1045.
However, we have already demonstrated that the [9] C.A. Cockell, J. Knowland, Biol. Rev. 74 (1999) 311.
selection of up to three or four ‘key’ features in FT- [10] E.M. Newton, H.G.M. Edwards, D.D. Wynn-Williams, C.A.
Raman spectroscopy will provide a suitable means of Cockell, to be published.
[11] H.G.M. Edwards, F. Garcia-Pichel, E.M. Newton, D.D.
identification of such substances—which is unam-
Wynn-Williams, Spectrochim. Acta Part A 56 (1999) 193.
biguous and recognisable from spectral patterns in [12] D.D. Wynn-Williams, Antarctica as a model for ancient Mars,
living material (namely, Nostoc commune cyanobac- in: J.A. Hiscox (Ed.), The Search for Life on Mars, British
terial mats in the Antarctic). Hence, although a strong Interplanetary Society, London, 1999, p. 49057.