Synechoxanthin, an aromatic C40 xanthophyll that is a major carotenoid in the cyanobacterium Synechococcus sp. PCC 7002

Article abstract of DOI:10.1021/np800310b

A major aromatic, dicarboxylate carotenoid (> 15% of total) was isolated from the euryhaline cyanobacterium Synechococcus sp. PCC 7002. This compound, which was given the common name synechoxanthin (1), has been assigned the structure (all-E) χ,χ-caroten-18,18′-dioic acid by a combination of spectroscopic (UV-vis, FT-IR, ¹H and ¹³C NMR, LC-MS) and chemical methods. This discovery conclusively establishes that some cyanobacteria are capable of synthesizing aromatic carotenoids.

Full text of DOI:10.1021/np800310b

J. Nat. Prod. 2008, 71, 1647–1650
1647
Synechoxanthin, an Aromatic C40 Xanthophyll that Is a Major Carotenoid in the
Cyanobacterium Synechococcus sp. PCC 7002
Joel E. Graham,†,‡ Juliette T. J. Lecomte,§, and Donald A. Bryant*,†
Department of Biochemistry and Molecular Biology and Department of Chemistry, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802
ReceiVed May 22, 2008
A major aromatic, dicarboxylate carotenoid (>15% of total) was isolated from the euryhaline cyanobacterium
Synechococcus sp. PCC 7002. This compound, which was given the common name synechoxanthin (1), has been assigned
the structure (all-E) ꢀ,ꢀ-caroten-18,18′-dioic acid by a combination of spectroscopic (UV-vis, FT-IR, 1H and 13C NMR,
LC-MS) and chemical methods. This discovery conclusively establishes that some cyanobacteria are capable of
synthesizing aromatic carotenoids.
Aryl carotenoids were first discovered in the marine sponge
Reniera japonica,1 a brightly colored, orange-red sponge that grows
in abundance in the Sea of Japan. Three bicyclic aromatic
compounds, renieratene (ꢀ,φ-carotene), isorenieratene (φ,φ-caro-
tene), and renierapurpurin (ꢀ,ꢀ-carotene), were all initially isolated
from this organism. The discovery of monocyclic φ,ψ-chlorobactene
in a variety of green sulfur bacteria followed,2 and monocyclic ꢀ,ψ-
okenone was subsequently identified as a common pigment in purple
sulfur bacteria.3 The bicyclic carotenoid φ,φ-isorenieratene was later
proven to be identical to leprotene from mycobacteria;4 it was first
isolated from Streptomyces mediolani and is also produced by
various other Streptomyces species.5,6 Isorenieratene is also pro-
duced as a major carotenoid in brown-colored, green sulfur bacteria
that synthesize bacteriochlorophyll e.7,8 Because of their association
with photosynthetic green and purple sulfur bacteria, aromatic
carotenoids have become important biomarkers.9,10 The diagenetic
products of aromatic C40 carotenoids have been used as biomarkers
to predict the occurrence of anaerobic environments in the ancient
oceans.11 Aromatic carotenoids have further been studied as models
to understand the preservation pathways of sedimentary carbon.12
The organisms previously known to synthesize aromatic carotenoids
therefore include green sulfur bacteria, purple sulfur bacteria,
and actinomycetes.6,13 In contrast, cyanobacteria have long been
Figure 1. Chromatogram showing HPLC separation of pigments
extracted from Synechococcus sp. PCC 7002 grown to early
stationary phase: 1. synechoxanthin (compound 1, Figure 2); 2.
myxoxanthophyll; 3. zeaxanthin; 4. cryptoxanthin; 5. echinenone/
chlorophyll a; 6. renierapurpurin (compound 4, Figure 2); 7.
ꢁ-carotene. Structures of all other compounds may be found in
ref 16.
assumed to be devoid of aromatic carotenoids.14,15
Here we describe the isolation and structural characterization of
a previously unreported, aromatic carotenoid diacid from the
cyanobacterium Synechococcus sp. PCC 7002. In recognition of
its origin, we have given this compound the common name
synechoxanthin. Synechoxanthin occurs as one of the major
pigments (>15% of total carotenoids) in the wild-type strain of
this organism; it is found in several other cyanobacteria, including
the freshwater and soil organisms Synechocystis sp. PCC 6803 and
Nostoc sp. PCC 7120.16 Additionally, renierapurpurin was isolated
in smaller quantities from Synechococcus sp. PCC 7002 cells grown
into stationary phase.
Whole Synechococcus sp. PCC 7002 cells were extracted with
acetone/methanol (7:2, v/v) with sonication. Examination of the
crude extract by reversed-phase HPLC revealed a highly polar
carotenoid compound that eluted much earlier than myxoxantho-
phyll from this organism (Figure 1). This compound, synechox-
anthin (compound 1 in Figure 2), was isolated by extraction from
whole cells in 70% (v/v) MeOH/H2O, followed by extraction into
CH2Cl2. The free acid was then purified by preparative HPLC. The
free acid was treated with trimethylsilyldiazomethane in MeOH to
form the dimethyl ester (2), which was repurified by preparative
HPLC. Synechoxanthin was not reduced by NaBH4, had no
chemically modifiable hydroxyl groups, and was unaffected by
saponification. Visible spectra, including λmax and spectral fine
structure (%III/II),17 of the reduction (LiAlH4) product (3) were
consistent with that of renierapurpurin (4). The visible spectrum
of 1 was similar to that of 3 and 4 but had a 4-nm red shift consistent
with carbonyl functions at the 18,18′ positions (Figure 3).13 LC-
MS analysis of 1 gave a molecular mass of 588 Da, whereas
compound 2 had a mass of 616 Da. FT-IR analysis of 2 showed
that synechoxanthin dimethyl ester had bands at 1710, 1290, 1130,
and 970 cm-1. Similar bands are detected in methylbenzoate (1730,
1280, 1110, and 970 cm-1) and are assigned to the CdO stretch,
C-O stretches, and OdC-O bending vibrations of an aryl ester.18
* To whom correspondence should be addressed. Tel: 814-865-1992.
Fax: 814-863-7024. E-mail: dab14@psu.edu.
† Department of Biochemistry and Molecular Biology.
‡ Current address: Center for Marine Biotechnology, University of
Maryland Biotechnology Institute, Baltimore, MD 21202.
§ Department of Chemistry.
Current address: Department of Biophysics, 110 Jenkins Hall, Johns
Hopkins University, Baltimore, MD 21218.
10.1021/np800310b CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/21/2008

1648 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
Table 1. 1H and 13C NMR Spectroscopic Data for Dimethyl
ꢀ,ꢀ-Carotene-18,18′-dioate (2)
position
δ (ppm)
multiplicity and J (Hz)
H-4
H-5
7.57
7.40
d, J(4,5) ) 8.2
d
H-7
H-8
6.89
6.83
d, J(7,8) ) 15.7
d
H-10
H-11
H-12
H-14
H-15
H-16
H-17
H-18
H-19
H-20
H-18e
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-18ea
6.39
6.73
6.47
6.33
6.69
2.33
2.47
na
m, J(10,11) ) 11.5, J(10,19) ≈ 0.9
dd, J(11,12) ) 14.8
m, J(12,20) < 0.9
m, J(14,15) ∼ 11.9
m, J(15,15′) ∼ 14.3
s
s
2.08
2.01
3.86
d
d
s
135.7
138.2
130.1
127.6
123.0
140.7
125.7
137.2
136.4
134.3
125.4
139.2
137.2
133.7
130.9
16.0
Figure 2. Structures of synechoxanthin [(all-E) ꢀ,ꢀ-caroten-18,18′-
dioic] (1), synechoxanthin dimethyl ester [(all-E) dimethyl ꢀ,ꢀ-
caroten-18,18′-dioate] (2), ꢀ,ꢀ-caroten-18,18′-diol (3), and renier-
apurpurin [(all-E) ꢀ,ꢀ-carotene] (4).
17.5
169.3
13.2
13.1
52.2
a 18e refers to the ester methyl at C-18.
Figure 3. In-line UV-vis absorption spectra of synechoxanthin
(solid line) and its LiAlH4 reduction product, ꢀ,ꢀ-caroten-18,18′-
diol (dashed line).
Figure 4. Vinylic-aromatic region of the 600 MHz proton spectrum
of synechoxanthin dimethyl ester in CD2Cl2, 25 °C. The numbering
corresponds to 2 in Figure 2; chemical shifts and J values are listed
in Table 1. Additional NMR data are presented in the Supporting
Information.
To elucidate the structure, 1H NMR data were collected on the free
acid (1), and 1H and 13C NMR data were collected on the dimethyl
ester (2).
The one-dimensional 1H NMR spectrum of 1 collected in DMSO-
d6 contained a broad peak at ∼12.75 ppm (Supporting Information
Figure S1), a shift that is characteristic of carboxylic acid protons.
Saturation of the water line by rf irradiation at 3.55 ppm resulted
in the disappearance of the peak at 12.75 ppm, consistent with its
assignment to labile protons. The 1H NMR spectrum of 2 in CD2Cl2
(Supporting Information Figure S2) displayed a total of five resolved
methyl signals and nine signals downfield of 6 ppm and was
therefore indicative of 2-fold symmetry. The covalent structure of
2 was established by homodecoupling experiments, rotating-frame
1
nuclear Overhauser effect (ROE) difference spectra, and H-13C
correlation experiments (Supporting Information Figures S3-S18).
Chemical shifts and coupling constants for 2 are listed in Table 1.
Figure 4 shows the aromatic and vinylic region of the 1H
spectrum. The downfield-shifted protons at 7.57 and 7.40 ppm are
J-coupled to each other (∼8 Hz) and to no other protons. The
HMBC data (Supporting Information Figure S7) contained a
correlation between 7.57 and 169 ppm, a shift consistent with a

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1649
Biosystems Sciex 3200 Q-Trap LC/MS/MS system was used in either
positive atmospheric pressure chemical ionization (APCI) mode or
electrospray ionization (ESI) mode.
carbonyl carbon. Long-range correlations to four additional qua-
ternary carbons were observed from 7.57 and 7.40 ppm (Supporting
Information Figure S8); these described an aromatic ring. A long-
range correlation between the proton at 7.40 ppm and the carbon
bearing the proton at 6.89 ppm placed the 7.40 ppm proton ortho
to the polyene chain. Additional correlations placed methyl groups
ortho and meta to the chain. Thus, the data indicated a ꢀ substitution
pattern of the benzene ring. The chemical shifts calculated for C-4
and C-5 with CH3 at C-1, CH3 at C-2, COOCH3 at C-3, and polyene
at C-6 of the benzene ring agreed with the observed values
(Supporting Information Table S1 and Figure S20). The location
of the methyl groups on the polyene chain (at C-9 and C-13) was
determined with the HMBC data. Stronger coupling was detected
through the double bond than the s-trans bond (Supporting
Information Figure 9). These results unambiguously established the
covalent structure of 2 as dimethyl ꢀ,ꢀ-carotene-18,18′-dioate
(Figure 2). The all-E configuration was apparent through the trans
3JH-H coupling values and ROE effects (Supporting Information
Figures S11-18). The trans geometry at the 15-15′ bond was
supported by the 14.3 Hz vicinal coupling constant required in the
simulation of the AA′BB′ spin system (Supporting Information
For NMR analyses, the sample of synechoxanthin was ∼1.0 mM in
DMSO-d6, and the sample of synechoxanthin dimethyl ester was ∼1.6
mM in CD2Cl2. 1D spectra for integration and J measurement were
collected with a resolution of 0.18 Hz (32 k data points, spectral width
of 6 kHz). The relaxation delay was 5 s. 1D-ROE (CAMELSPIN)
difference spectra26 were collected by inverting the signal of interest
with a 32 ms sinc pulse. Locking was achieved with a succession of
180° pulses (10 kHz) sustained for 150 to 600 ms. A total of 32
transients were acquired for each frequency, and the relaxation delay
1
was 4 s. Spectra were collected in pairs (on/off resonance). H-13C
HMQC data27 were collected in the phase-sensitive mode (TPPI28).
Acquisition parameters were 4 k points and a spectral width of 5.3
1
kHz in the H dimension and 278 real points and a spectral width of
23 kHz in the indirect dimension. The delay τ was 3.57 ms (short-
range) or 50 ms (long-range). GARP decoupling29 (80 µs) was applied
to 13C nuclei during acquisition. Relaxation time was 2 s. 1H-13C
HMBC data30 were collected with gradient selection, magnitude mode,
with a τ delay of 3.57 ms. 1H-13C H2BC data31 were collected in the
phase-sensitive mode (echo-antiecho) with a third-order low-pass J
filter. Acquisition parameters were 4 k points and a spectral width of
3
1
Figure S19). JH-H coupling across the single 14-15 bond was
5.3 kHz in the H dimension and 180 complex points and a spectral
11.9 Hz, in agreement with trends observed in other carotenoids.19
The UV-vis absorption spectrum and HPLC retention time were
consistent with the all-E configuration. Furthermore, we have
established that all-trans ꢁ-carotene is the biochemical precursor
for the synthesis of synechoxanthin.16,20
width of 23 kHz in the indirect dimension with GARP decoupling (80
µs) during acquisition. Relaxation time was 2 s. Data were processed
with XWIN-NMR (Bruker, Biospin) and analyzed with Sparky.32
Chemical shifts were referenced indirectly to CHDCl2 at 5.32 ppm (1H)
and 53.8 ppm (13C). Spinworks33 was used to simulate the proton
spectrum shown in Figure 4, in particular the 14-15-15′-14′ system.
Bacterial Material. Synechococcus sp. PCC 7002 strains were grown
axenically at 38 °C in 30 mL tubes, or 12 L carboys with constant
Renierapurpurin (4) was identified as a minor compound that
accumulated in cells grown to stationary phase. Its elution time by
HPLC was similar to that of isorenieratene, although its visible
absorption spectrum was nearly identical to that of the reduction
product of synechoxanthin (3) and to the reported absorption
spectrum of renierapurpurin.21 The molecular mass of 4 was
illumination by standard fluorescent lamps (250 µmol photons m-2 s-1
)
and constant bubbling with 1% (v/v) CO2 balanced with air. A crtR
mutant,20 deficient in the synthesis of 3,3′-hydroxy carotenoids, was
used to produce samples for NMR experiments, because this mutant
strain synthesizes fewer xanthophylls but produces more synechoxanthin
than the wild type. Unless otherwise specified, cells were harvested in
midexponential phase by centrifugation (20 min, 5500g, 4 °C) and
frozen until required.
1
determined to be 528 Da. The H NMR spectrum confirmed the
structure of 4 as renierapurpurin (ꢀ,ꢀ-carotene). Renierapurpurin
is presumed to be a precursor to synechoxanthin.
The demonstration that Synechococcus sp. PCC 7002 synthesizes
the aromatic carotenoid synechoxanthin establishes that this cy-
anobacterium is the first organism proven to be capable of de noVo
production of a ꢀ,ꢀ-carotenoid. Although the presence of aromatic
carotenoids has been reported in some marine sponges, the source
of these compounds has usually been attributed to bacterial
symbionts.22 Recent work has identified an astounding array of
sponge-associated cyanobacteria, including members of the genera
Synechoccoccus, Prochlorococcus, and Oscillatoria among others.23
Therefore, it is possible that cyanobacterial symbionts are the source
of the ꢀ,ꢀ-carotene found in marine sponges.
Perhydro-renierapurpurin (renierapurpurane), a diagenetic product
of ꢀ,ꢀ-carotenoids, has been used alongside other preserved
carotanes as geochemical biomarkers for photosynthesis.11,24
Because these compounds have generally been believed to be
synthesized by photosynthetic sulfur bacteria, it has been suggested
that these compounds could be used to identify ancient anaerobic
environments.11 Although monocyclic ꢀ,ψ-carotenes have been
isolated from purple sulfur bacteria,13 no photosynthetic sulfur
bacterium has yet been described that produces ꢀ,ꢀ-carotenes. The
production of synechoxanthin by Synechococcus sp. PCC 7002
establishes that cyanobacteria can synthesize aromatic carotenoids,
and this brings into question the origin of diagenetic products of
renierapurpurin in the fossil record.11,25
Isolation of Carotenoids. General precautions for work with
carotenoids were taken.34 For isolation of synechoxanthin, cells were
extracted with 70% (v/v) MeOH/H2O via sonication, then pelleted by
centrifugation. The supernatant contained large amounts of synechox-
anthin, but was largely devoid of other pigments. Synechoxanthin was
then extracted into CH2Cl2, and fractions were pooled and dried under
N2 in glass. The pigment was resuspended and purified by preparative
HPLC or treated with trimethysilyldiazomethane in MeOH to generate
the diester (2) and then purified by preparative HPLC.
HPLC Separation. Pigments were separated by HPLC (Agilent
model 1100) equipped with a diode array detector (model G1315B)
and controlled with Agilent ChemStation software (Agilent Technolo-
gies, Palo Alto, CA) on a 25 cm by 4.6 mm, 5 µm Discovery C18
analytical column or a 25 cm by 10 mm semipreparative column
(Supelco, Bellefonte, PA). A gradient elution method was used. Solvent
A was H2O/MeOH/CH3CN (62.5:21:16.5) containing 10 mM NH4OAc.
Solvent B was 50% MeOH, 30% EtOAc, and 20% CH3CN. The
gradient was [time, %B][0, 20][10, 70][40, 100][50, 100]. Flow rates
were 1 mL min-1 for the analytical column and 3.5 mL min-1 for the
semipreparative column.
Synechoxanthin ((all-E) ꢀ,ꢀ-caroten-18,18′-dioic) (1): UV-vis
(MeOH) λmax 442 (sh), 470, 497 nm (%III/II ) 4);17 (pyridine) 463
(sh), 494, 522 nm (%III/II ) 2); (DMSO) 463 (sh), 494, 522 nm (%III/
II ) 2); (HPLC system, 13 min) 447 (sh), 475, 503 nm (%III/II ) 9);
CIMS m/z 589 [M+ + 1].
Synechoxanthin dimethyl ester ((all-E) dimethyl ꢀ,ꢀ-caroten-
18,18′-dioate) (2): methyl ester of 1 produced by treatment of 1 with
(trimethylsilyl)diazomethane in MeOH; UV-vis (HPLC system, 34
min) λmax 451 (sh), 477, 503 nm (%III/II ) 0); 1H NMR (CD2Cl2, 600
MHz) see Table 1; HMQC correlations, H-4/C-4; H-5/C-5; H-7/C-7;
H-8/C-8; H-10/C-10; H-11/C-11; H-12/C-12; H-14/C-14; H-15/C-15;
H3-16/C-16; H3-17/C-17; H3-18e/C-18e; H3-19/C-19; H3-20/C-20;
H2BC correlations, H-4/C-5; H-5/C-4; H-7/C-8; H-8/C-7; H-10/C-11;
H-11/C-10; H-11/C-12; H-12/C-11; H-14/C-15; H-15/C-14; HMBC
Experimental Section
General Experimental Procedures. UV/vis spectra were recorded
on a Genesis 10 spectrophotometer or were recorded in-line during
HPLC by diode array detector (Agilent model G1315B). 1H and inverse-
detected 13C data were collected on a Bruker DRX-600 spectrometer
operating at a 1H frequency of 600.18 MHz and equipped with a triple
axis gradient TXI shielded probe. For mass analyses an Applied

1650 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
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correlations, H3-16 to C-1, C-2, C-6; H3-17 to C-1, C-2, C-3; H-4 to
C-2, C-6, C-18; H-5 to C-1, C-3, C-7; H-7 to C-5, C-9; H-8 to C-6,
C-19, C-10; H3-19 to C-8, C-9, C-10; H-10 to C-8, C-12, C-19; H-11
to C-13; H-12 to C-14, C-20; H3-20 to C-12, C-13, C-14; H-14 to C-12,
C-15′; H-15 to C-13, C-14′; 1D-ROE connectivities, H-4/H-5; H-5/H-
8; H-7/H3-16; H-7/H3-19; H-8/H-10; H-10/H-12; H-11/H3-19; H-11/
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617 [M+ + 1] (79), 585 (100), 571 (4), 553 (28), 439 (5).
(all-E) 18,18′-Dihydroxy-ꢀ,ꢀ-carotene (3): produced either from
1 or 2 by reduction with lithium aluminum hydride; UV-vis (HPLC
system, 20 min) λmax 446 (sh), 472, 500 nm (%III/II ) 13); CIMS m/z
561 [M+ + 1].
Renierapurpurin [(all-E) ꢀ,ꢀ-carotene] (4): minor product from
Synechococcus sp. PCC 7002; UV-vis (benzene) λmax 457 (sh), 484,
516 nm (%III/II ) 20), (HPLC system, 42 min) λmax 446 (sh), 472,
1
500 nm (%III/II ) 12); H NMR (CD2Cl2, 500 MHz) δ 7.25 (1H, d,
J ) 7.75 Hz, H-5), 6.96 (1H, d, J ) 7.75 Hz, H-4), 6.89 (1H, d, J )
15.92, H-7), 2.30 (1H, s, Me, H-16), 2.28 (1H, s, Me, H-18), 2.20 (1H,
s, Me, H-17), 2.07 (1H, s, Me, H-19); CIMS m/z 529 [M+ + 1] (100),
409 (19), 396 (12), 277 (36).
Acknowledgment. The authors thank C. Falzone for assistance in
interpreting the NMR data. The authors thank Dr. L. Zhang of the
Proteomics and Mass Spectrometry Core Facility at Penn State for his
assistance with mass spectrometry. This work was supported by
National Science Foundation grants MCB-0349409 to J.T.J.L. and
MCB-0077586 and MCB-0519743 to D.A.B.
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