Boron-Containing Organic Pigments from a Jurassic Red Alga

Boron-containing organic pigments from a Jurassic red alga

Klaus Wolkensteina,1, Jürgen H. Grossb, and Heinz Falkc

aInstitute of Analytical Chemistry, Johannes Kepler University Linz, 4040 Linz, Austria; bInstitute of Organic Chemistry, University of Heidelberg, 69120 Heidelberg, Germany; and cInstitute of Organic Chemistry, Johannes Kepler University Linz, 4040 Linz, Austria

Edited by Victoria J. Orphan, California Institute of Technology, Pasadena, CA, and accepted by the Editorial Board September 23, 2010 (received for review June 10, 2010) Organic biomolecules that have retained their basic chemical oxide (DMSO). The reddish-colored extracts were purified by structures over geological periods (molecular fossils) occur in a wide solid-phase extraction and characterized by HPLC–diode array range of geological samples and provide valuable paleobiological, detection–electrospray ionization–mass spectrometry (HPLC- paleoenvironmental, and geochemical information not attainable DAD-ESI-MS). From a large Solenopora sample (102.5 g) from from other sources. In rare cases, such compounds are even France, 1.1 mg of crude pigment isolate was obtained as an in- preserved with their specific functional groups and still occur within tensely crimson-colored organic residue (Fig. S1). HPLC analysis the organisms that produced them, providing direct information on of the pigments revealed numerous compounds with similar UV- the biochemical inventory of extinct organisms and their possible visible spectra, with the prominent group at retention time of evolutionary relationships. Here we report the discovery of an 8.0–10.0 min showing a major broad absorption band at 520 nm exceptional group of boron-containing compounds, the borolitho- and a minor one at 420 nm (Fig. 1B), but no Soret band at ~400 chromes, causing the distinct pink coloration of well-preserved nm (which is characteristic of porphyrins). In the negative-ion specimens of the Jurassic red alga Solenopora jurassica. The boroli- mass spectra, corresponding ions at mass-to-charge ratios (m/z) thochromes are characterized as complicated spiroborates (boric of 839, 853, and 867 were detected, indicating that the pigments acid esters) with two phenolic moieties as boron ligands, represent- consist of a homologous series of compounds and accompa- ing a unique class of fossil organic pigments. The chiroptical prop- nying isomers (Fig. 1C). Moreover, all compounds exhibited a erties of the pigments unequivocally demonstrate a biogenic origin, characteristic isotope pattern indicative of the presence of a sin- at least of their ligands. However, although the borolithochromes gle boron atom (Fig. 1D). Based on accurate mass data obtained originated from a fossil red alga, no analogy with hitherto known by HPLC-MS and additional measurements using Fourier present-day red algal pigments was found. The occurrence of the transform ion cyclotron resonance mass spectrometry (FT-ICR- borolithochromes or their possible diagenetic products in the fossil MS) for the ions at m/z 839, 853, and 867, the molecular record may provide additional information on the classification and formulae C50H36O12B, C51H38O12B, and C52H40O12B were de- phylogeny of fossil calcareous algae. termined (Fig. 1D and Fig. S2). The boron could be readily removed from the borolithochromes fossil red algae | molecular preservation | phenolic boric acid esters | optical by reacting the pigments in methanol containing 0.1% tri- activity | liquid chromatography–mass spectrometry fluoroacetic acid. Several series of homologous pigments were isolated by HPLC for further analysis. Solvolysis (methanolysis/ he striking pink coloration of specimens of the fossil calcar- hydrolysis) of a fraction containing various isomers of the pigments Solenopora jurassica − − − − Teous red alga has been a matter of de- [C50H36O12B] ,[C51H38O12B] ,and[C52H40O12B] ([M] ) (Fig. bate for decades and is well known from the Jurassic of Great 2A) resulted in the formation of only two HPLC peaks (Fig. 2B) − Britain (“Beetroot Stone”)(1–3) and France (4). Solenopora with ions at m/z 415 and 429 ([M–H] ), which could be assigned to C25H19O6 and C26H21O6 (Fig. 2C). Accordingly, solvolysis of specimens at the reported localities are well preserved and ex- − a fraction containing isomers of the pigments [C48H32O8B] , hibit preserved tissue structures and regular alternating bands − − – [C49H34O8B] , and [C50H36O8B] resulted in the formation of (2 4) that have been interpreted as seasonal growth structures − (3). The characteristic coloration associated with these bands is two HPLC peaks with ions at m/z 369 and 383 ([M–H] ), which A–C generally more intense in the inner portions of the algal nodules could be assigned to C24H17O4 and C25H19O4 (Fig. S3 ). (2, 3). Given that no traces of the pigments can be found in the Obviously, two equivalent homologous ligands with different surrounding sediment, consisting of white oolitic limestones at substitution patterns give rise to the combinatorial multitude of both locations, there can be no doubt that the pigments are of homologous and isomeric borolithochromes. All solvolysis endogenous origin. Previous reports speculated that the pig- products revealed UV-visible spectra similar to the boron- B Inset ments from the Beetroot Stone likely are porphyrins (2), gen- containing precursors (Fig. 2 , ) with a distinct bath- erally known from bituminous sediments and petroleum (5), ochromic shift of the long-wavelength absorption. Furthermore, whereas the coloration of specimens from France has been at- all borolithochromes and all solvolysis products exhibited deu- tributed to fossil hypericinoid pigments (fringelites) (6), poly- terium-exchangeable protons, indicating the presence of multiple hydroxy groups (Fig. S4). For the homologous borolithochromes cyclic quinones described from purple-colored fossil crinoids − − − (7, 8). Here we provide evidence that the pink coloration of S. [C50H36O12B] to [C52H40O12B] ([M] ), six H/D exchanges jurassica from both occurrences is in fact due to the presence of a unique class of complicated boron-containing organic pig- Author contributions: K.W. designed research; K.W. and J.H.G. performed research; K.W. ments, which we name borolithochromes. collected fossil material; J.H.G. contributed new reagents/analytic tools; K.W., J.H.G., and H.F. analyzed data; and K.W. and H.F. wrote the paper. Results and Discussion The authors declare no conflict of interest. S. jurassica We analyzed distinctly pink-colored specimens (Fig. This article is a PNAS Direct Submission. V.J.O. is a guest editor invited by the Editorial 1A) from two of the localities reported in the literature, in- Board. cluding a part of the neotype from the Beetroot Stone. Following 1To whom correspondence should be addressed. E-mail: [email protected]. dissolution of the carbonate matrix with HCl, crude extracts This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. were obtained by extraction of the residues with dimethyl sulf- 1073/pnas.1007973107/-/DCSupplemental.

19374–19378 | PNAS | November 9, 2010 | vol. 107 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1007973107 Downloaded by guest on September 27, 2021 A C

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ecnabrosbA 3 Wavelength (nm) 2 1 839.23044838.2327 838.2327 841.2356 1 841.2356 842.2382 842.2382 0 6 8 10 12 14 16 18 837 838 839 840 841 842 843 844 Retention time (min) m/z EVOLUTION Fig. 1. Specimen of the Jurassic red alga S. jurassica with exceptional preservation of fossil boron-containing organic pigments (borolithochromes) and analytical data of extracted pigments (DMSO extract). (A) Polished slab of S. jurassica (MNHN 40091), Upper Jurassic, Tannay, France. (B) HPLC chromatogram (detection at 520 nm) of extracted fossil pigments (MNHN 23874) and UV-visible spectrum of the first peak in the chromatogram (Inset). (C) Section from the HPLC chromatogram shown in B (Upper) and extracted ion chromatograms (negative-ion ESI-MS) (Lower) of fossil pigments. (D) Mass spectrum of the main − 11 single isomeric pigment showing the characteristic isotope pattern of boron, observed m/z 839.2306 [M] , calculated for C50H36O12 B: 839.2305). Note the difference of 0.9979 Da (calculated for 11B − 10B: 0.9964) between m/z 838 and 839 and the difference of 1.0028 Da (calculated for 13C − 12C: 1.0034) between m/z 839 and 840. CHEMISTRY − were observed, compared with two H/D exchanges for homologs the borate but the solvolysis products form [M–H] ions, based − − − [C48H32O8B] to [C50H36O8B] ([M] ), suggesting a difference on the number of H/D exchanges, a quinoid structure can be of four hydroxy groups between the two series (Fig. S4 A and B). excluded. Characteristic fragmentation patterns were obtained The distinct hypsochromic shift of 15 nm observed in the UV- by collision-induced dissociation of the ions at m/z 415 and 429 visible spectra of the two series indicates that the hydroxy groups by means of ESI tandem MS (Fig. 2D). Compounds demon- are phenolic (Fig. S5). Accordingly, the corresponding products strated elimination of CH4 and C2H6, indicating the presence of – − C25H19O6 and C24H17O4 ([M H] ) (and their homologs) showed alkyl side chains, followed by elimination of CH2CO and CO2. four and two H/D exchanges, respectively (Fig. S4 C and D), Fragmentation of the ions at m/z 369 and 383 also led to the suggesting a difference of two hydroxy groups between the elimination of CH4 and C2H6 (Fig. S3D), however, followed by products. These data strongly imply that the borolithochromes elimination of CHO, C2H2, and CO, consistent with a phenolic are boric acid esters with two phenolic moieties as boron ligands, structure. Based on the molecular formulae of the solvolysis representing a unique class of spiroborate pigments (Fig. 3). This products, all of which require 16 degrees of unsaturation, as well was also confirmed by 11B NMR spectroscopy of a crude DMSO- as the lack of significant fragments below m/z 200, it can be d6 extract, which displayed a single peak at 2.7 ppm (Fig. S6) concluded that the basic structure of the borolithochrome ligands characteristic of borates (9). Moreover, the tetrahedral co- is a highly condensed aromatic system. The extremely complex ordination of the spiroborates is expressed by their chiroptical composition of organic matter in the samples from Great Britain properties, as revealed by circular dichroism (CD) spectroscopy and France (SI Text, Fig. S7, and Table S1) with very low con- of individual pigments (Fig. 4). Whereas the single isomeric centrations of individual compounds, along with the limited − [C50H36O12B] is chiroptically inactive, the CD spectra of the availability of fossil specimens with distinct coloration, excluded − chromatographically resolvable isomers of [C51H38O12B] and a detailed structural elucidation of the fossil pigments by means of − 1 13 [C52H40O12B] are opposite in sign, indicating that the latter H-, C-, and 2D NMR. compounds are diastereomers (due to a chiral center in addition The borolithochromes represent a previously unknown class of to their inherent C2 symmetry; SI Text) (10). Because MS data organic pigments, fossil as well as recent, and are exceptional in suggest that the borolithochromes are negatively charged from containing the element boron. Because the borolithochromes are

Wolkenstein et al. PNAS | November 9, 2010 | vol. 107 | no. 45 | 19375 Downloaded by guest on September 27, 2021 A C 4 DAD1 - C:Sig=520,4 200110_002.d - ESI Scan (1.664-1.796 min, 9 scans) Frag=200.0V 200110_003.d Subtract

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) U A m A U ) x10 5 399.0870 8 1 c e 2 ( m A U ) Peak 1 Peak 1 399.09 4 -CH22 CH -CO2 2 6 ( m n 0 n m ( -CH 4 3 -CH2 O -CH2 CO 4 n a b r o s b s o r b a n 2 355.10355.0975 327.07 357.08

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s b A b s 0 355.10 327.07 357.0761357.08 429.13 327.0663 340.0722340.07 300.0785 312.0767 372.0642 381.0735381.07 413.0989 429.1345 0 0 2 4 6 8 10 12 250 275 300 325 350 375 400 425 Retention time (min) m/z

Fig. 2. Solvolysis of borolithochromes and formation of free phenolic compounds. (A) HPLC chromatogram (detection at 520 nm) of a HPLC fraction con- − − − − taining only isomers of the pigments [C50H36O12B] ,[C51H38O12B] ,and[C52H40O12B] ([M] at m/z 839, 853, and 867) analyzed immediately after dissolution in methanol containing 0.1% triﬂuoroacetic acid. (B) HPLC chromatogram of the same solution after storage for 30 min at 50 °C with reaction products (peaks − 1 and 2) and UV-visible spectrum of peak 1 (Inset). (C) Mass spectra of the products shown in B (peak 1: observed m/z 415.1191 [M–H] , calculated for − C25H19O6: 415.1187; peak 2: observed m/z 429.1351 [M–H] , calculated for C26H21O6: 429.1344). (D) Collision-induced dissociation mass spectra of m/z 415.12 and 429.13 shown in C (peaks 1 and 2).

found only within pink-colored specimens of Solenopora, and a high-energy depositional environment (Materials and Methods) specific chirality as observed in the pigments is a characteristic and by the occurrence of the pigments within a calcium car- signature of life, the borolithochrome subchromophores are of bonate matrix. The pigments can be extracted only with organic unequivocal biological origin. However, two hypotheses could solvents after dissolution of the carbonate matrix by either the conceivably account for the presence of boron in the pigments: use of a strong acid (HCl or HF) or calcium complexation (i) Although present-day natural compounds containing boron (EDTA). Because salt formation with divalent ions, such as Ca2+, are rare (11, 12), the fossil pigments might represent original bo- is believed to be a key factor in the preservation of fossil ron-containing natural products, and (ii) boron might have been hypericinoid pigments (due to the strong acidity of specific hy- introduced during diagenesis. Because of the thermodynamic sit- droxy groups) (8, 16), the extraordinary stability of the bor- uation of phenolic spiroborates (13, 14), the diagenetic formation olithochromes might be due to salt formation of the negatively of these spiroborates from unboronated precursor compounds charged borates with Ca2+ from the calcitic Solenopora material. would be rather unlikely. This is consistent with the low boron The occurrence of borolithochromes in Middle and Upper content of the surrounding limestone (4.5–15.7 ppm), which is Jurassic specimens of Solenopora from different localities in within the lower range of other marine carbonates (15), as well as Europe suggests that these compounds were common in sol- the lack of intermediate single ligand esters in the fossils. It also enoporacean algae, a family with a fossil record from the early should be noted that no diagenetic complexation process with Paleozoic to the Miocene generally assigned to the Rhodophyta. boron has been reported in the literature. However, although the Even though Solenoporaceae are currently considered a heter- present data point to the preservation of primary boron-containing ogenous group and appear to include chaetetid sponges and pigments, the latter hypothesis cannot be fully excluded, because receptaculids in addition to red algae (17, 18), S. jurassica and diagenetic complexation processes (involving predominately similar fossils from the Jurassic most likely are red algae, given transition metals) are well known for porphyrins (5). the presence of typical features of coralline algae (e.g., filaments Most fossil organic compounds, particularly those of pre- with well-developed cross partitions) (2, 18). Although no indi- Cenozoic age, lost their original functional groups during dia- cations of the presence of phycoerythrin, the present-day red genesis, retaining only their basic chemical structures (5). The algal light-harvesting pigment (19), or its possible degradation exceptional preservation of the highly functionalized bor- products were found in S. jurassica, and no boron-containing olithochromes might be explained by rapid burial of the algae in phenolic pigments are hitherto known from recent organisms,

19376 | www.pnas.org/cgi/doi/10.1073/pnas.1007973107 Wolkenstein et al. Downloaded by guest on September 27, 2021 O O 25

C25H15OOH3 B C25H15OOH3 20 O O 15

) m c l o m L ( L m o l c m ) Fig. 3. Structure of the main single isomeric borolithochrome (C50H36O12B, - 1 10 − [M] at m/z 839). 5

- 0

-5 other phenolic pigments within the red algae are represented by З fl the oridorubin pigments (20, 21). The presence of the bor- Ȉ -10 S. jurassica olithochromes in neither supports nor call into -15 question the status of this species and related forms as red algae, but offers the possibility of clarifying their assignment if similar -20 pigments were to be found in recent organisms. In any case, the -25 fi 250 300 350 400 450 500 550 600 speci c fossil pigments are of potential value as biomarkers for Wavelength (nm) a coherent group of organisms. It would be rather unlikely for such stereochemically complicated and highly unusual pigments Fig. 4. CD spectra of borolithochrome racemate and diastereomers in − fi to have evolved independently in different groups of organisms, DMSO [(M)-C50H36O12B+(P)-C50H36O12B], [M] at m/z 839 (red solid line; rst peak in Fig. 1C), (M,R)-C51H38O12B [with an arbitrarily assigned configuration such as red algae and sponges. Furthermore, horizontal gene − (R) of one of the ligands], [M] at m/z 853 (blue dashed line; second peak in transfers, which might account for the occurrence of similar − Fig. 1C), (P,R)-C51H38O12B, [M] at m/z 853 (blue solid line; third peak in Fig. secondary metabolites in phylogenetically distant organisms, − 1C), (M,R)-C52H40O12B, [M] at m/z 867 (magenta dashed line; fourth peak in have been documented between eukaryotes only rarely. Thus, − Fig. 1C), and (P,R)-C52H40O12B, [M] at m/z 867 (magenta solid line; fifth peak the occurrence of the borolithochromes or their possible diage- in Fig. 1C). netic products in the fossil record may have implications for the phylogenetic status of individual taxa and may provide additional information on the relationship between coralline red algae with matrix compounds (brown-colored fraction) and then with water. The pink- their Mesozoic and Paleozoic ancestors. colored compounds were eluted with acetonitrile, and the solvent was removed under vacuum, leaving an intensely crimson-colored residue (1.1 mg). Materials and Methods Several borolithochrome fractions were obtained by semipreparative HPLC × μ Fossil Material and Geological Setting. Specimens of S. jurassica with distinct on a Phenomenex Gemini C18 column (150 4.6 mm i.d., 5 m) at 30 °C. The pink coloration from two localities reported in the literature were selected HPLC program consisted of a linear gradient of acetonitrile/20 mM aqueous from collection material or were collected at the original localities (2, 4): (i) ammonium acetate (60:40) to 100% acetonitrile in 15 min, followed by fl −1 two pink-colored specimens (MNHN 21556 and MNHN 23874; Muséum Na- isocratic elution at 100% acetonitrile at a ow rate of 1.0 mL min . Finally, tional d’Histoire Naturelle Paris) from Les Petites-Armoises (Département individual borolithochromes were isolated using a Phenomenex Gemini C18 × μ Ardennes, France) and one intensely pink-colored specimen (MNHN 40091) column (250 4.6 mm i.d., 5 m) at 30 °C. The HPLC program consisted of ~ a linear gradient of acetonitrile/20 mM aqueous ammonium acetate (65:35)

from Tannay ( 2 km northeast of the locality near Les Petites-Armoises), EVOLUTION Upper Jurassic, Oxfordian, and (ii) two pink-colored specimens (BMNH to 85% acetonitrile in 40 min, followed by a linear gradient to 100% ace- fl V.60741, representing a part of the neotype, and NHMUK PAL PB V 67825; tonitrile in 2 min and isocratic elution at 100% acetonitrile at a ow rate −1 Natural History Museum London) from Foss Cross Quarry near Chedworth of 1.0 mL min . (Gloucestershire, Great Britain), Middle Jurassic, Bathonian, White Lime- stone Formation, Beetroot Stone. HPLC-MS Analysis. HPLC-MS measurements were carried out using an Agilent The investigated specimens of S. jurassica from both localities came from 1100 Series HPLC system with a diode array detector coupled to an Agilent oolitic limestones deposited in shallow marine environments. At the French 6520 Q-TOF LC/MS mass spectrometer equipped with an ESI source. Sepa- location, Solenopora specimens are found in proximity to coral reefs (4), ration was performed at 30 °C on an Agilent Zorbax Eclipse XDB-C18 column whereas the Solenopora-bearing Beetroot Stone in Great Britain has no (50 × 4.6 mm i.d., 1.8 μm). The HPLC program consisted of a linear gradient CHEMISTRY close reefal association (2). The disturbed orientation of the majority of algal of acetonitrile/20 mM aqueous ammonium acetate (50:50) to 100% aceto- masses suggests that benthic organisms were disrupted and rapidly buried nitrile in 20 min, followed by isocratic elution at 100% acetonitrile at a flow −1 under high-energy conditions (2). rate of 1 mL min . The DAD wavelength was 520 nm, and UV-visible spectra of each peak were recorded in the 200- to 800-nm wavelength range. fi μ fl Extraction and Isolation of Pigments. Fragments of pink-colored Solenopora Extracts were ltered before injection using 0.2- m polytetra uoroethylene material (8.2–31.1 g) were cleaned with acetone. After dissolution of the filters (ReZist; Schleicher & Schuell). Mass spectra were acquired in the −1 carbonate with 10 M HCl, the residues were separated by centrifugation, negative-ion mode (nebulizer gas pressure, 60 psi; drying gas flow, 12 L min ; washed thoroughly with distilled water, and dried overnight at room tem- drying gas temperature, 350 °C; capillary voltage, 4.0 kV) over an m/z range perature under vacuum (~10 Torr). Residues were then sequentially extrac- of 100–1,300. Mass calibration was obtained using purine and the HP-0921 ted by sonication (10 min at 40 °C) and centrifugation in toluene (3×), acetate adduct (C20H21O8N3P3F24) introduced via a reference sprayer. For tetrahydrofuran (3×), and DMSO (1×). Toluene extracts contained no pig- tandem MS experiments, precursor ions measured at defined retention ments and were not analyzed in detail. The reddish-colored tetrahydrofuran times during the HPLC run were mass-selected in the quadrupole and and DMSO extracts were cleaned up by solid-phase extraction. The sorbent fragmented in the collision cell operated at various collision offset voltages. (Bondesil C18, 40 μm) was conditioned by washing with acetonitrile. The Borolithochromes were fragmented at 80, 120, and 160 V, and the solvolysis extracts then were loaded onto the column, and compounds were eluted products were fragmented at 55 V (m/z 415.12 and 429.13) and 70 V (m/z with acetonitrile. Analysis of tetrahydrofuran and DMSO extracts showed 369.11 and 383.13). that both extracts contained the same pigments. Isolation of pigments was done using a 102.5-g sample from of a large FT-ICR-MS. Molecular formulae of the pigments were determined by FT-ICR- specimen (MNHN 23874) from S. jurassica with distinct coloration from Les MS on a Bruker ApexQe instrument equipped with an ESI source and a 9.4-T Petites-Armoises. The material was treated with 10 M HCl and extracted superconducting magnet. All spectra were obtained in the negative-ion sequentially as described above, but using only toluene and successive mode. DMSO extracts were diluted depending on their initial concentration portions of DMSO. The dark, reddish-brown DMSO extract was further pu- from 1:10 to 1:30 in acetonitrile/water 3:1 (vol/vol) plus 0.1 M ammonia and − rified using a modified solid-phase extraction method. The sorbent (Bondesil were delivered to the ESI interface via a syringe pump at 3–6 μLmin 1. The − C18, 40 μm) was conditioned by washing with acetonitrile, followed by solutions were sprayed at 4.5 kV with a nebulizer gas flow of 1.0 L min 1 and − acetonitrile/20 mM aqueous ammonium acetate (50:50). The DMSO extract a desolvation gas flow of 2.0 L min 1 at 200–220 °C. Depending on the then was loaded onto the column, and the sorbent was washed with ace- sample concentration and the type of experiment, the ions were accumu- tonitrile/20 mM aqueous ammonium acetate (50:50) to remove organic lated in the collision hexapole for 1.0–4.0 s and then transferred into the ICR

Wolkenstein et al. PNAS | November 9, 2010 | vol. 107 | no. 45 | 19377 Downloaded by guest on September 27, 2021 cell. The mass spectra were acquired in the broadband mode over an m/z 11B NMR chemical shifts were referenced externally to boron trifluoride range of 250–1,500 with 2 mega data points. Typically, 32 transients were etherate. The broad background signal from any boron-containing glasses accumulated for one magnitude spectrum. External mass calibration was used in the probe or in the sample tube was removed during the processing −1 performed with a solution of arginine [0.2 mg mL in methanol/water 1:1 using Topspin version 2.1 (Bruker). Conversion of the experimental digital- − (vol/vol)] using [arginine–H] cluster ions. The same solution was added to filtered raw data to analog-filtered data was followed by backward linear the analyte solution to establish internal mass calibration. Generally, a mass prediction of the first 64 data points using 128 coefficients. accuracy of 1 ppm was achieved. CD Spectroscopy. CD spectra of individual pigments were recorded in DMSO Solvolysis of Borolithochromes. Isolated pigments were dissolved in methanol at 20 °C on a Jasco J-810 spectropolarimeter using 1-mm quartz cuvettetes. fl (0.02% H2O, according to Karl Fischer titration) containing 0.1% tri uoro- Spectra were obtained by accumulation of 16 scans over the 250- to 600-nm acetic acid (vol/vol). The solution was stored at 50 °C for 30 min, and the wavelength range, and then smoothed using a 25-point Savitzky–Golay fil- reaction was monitored by HPLC-DAD-ESI-MS under the same conditions ter. Concentrations of the sample solutions were in the range of 6.3–11.6 described above. − μmol L 1, as determined on a Varian CARY 100 Bio UV-visible spectropho- tometer based on the long-wavelength absorption maximum of the crude H/D Exchange Experiments and MS Analysis. For H/D exchange experiments, pigment isolate in DMSO. DMSO extracts and isolated fractions dissolved in DMSO were diluted either 3:7 in acetonitrile/H O 6:1 (vol/vol) or 3:7 in acetonitrile/D O 6:1 (vol/vol) 2 2 Boron Elemental Analysis of Carbonate. The boron concentration of the oolitic and delivered to the ESI interface (Agilent 6520 Q-TOF LC/MS mass spec- − limestone matrix of a S. jurassica specimen (NHMUK PAL PB V 67825) from Foss trometer) via a syringe pump at 25 μLmin 1. Mass spectra were acquired Cross was determined by laser ablation inductively coupled plasma MS. Data in the negative-ion mode (nebulizer gas pressure, 20 psi; drying gas flow, 5Lmin−1; drying gas temperature, 325 °C; capillary voltage, 3.5 kV) over an were acquired using a GeoLasC laser ablation system (MicroLas; wavelength m/z range of 100–1,300. Mass calibration was obtained using trifluoroacetic 193 nm) and analyzed with a ELAN 6100 DRC quadrupole mass spectrometer (PerkinElmer). Boron concentrations from single hole measurements of ma- acid and the HP-0921 trifluoroacetic acid adduct (C20H18O8N3P3F27)in- – troduced via a reference sprayer. trix components and cement were in the range of 4.5 15.7 ppm.

11 11B NMR. The 11B NMR measurements were done using a part (13.6 g) from ACKNOWLEDGMENTS. We thank H. Kählig (University of Vienna) for B the large specimen (MNHN 23874) from Les Petites-Armoises. After disso- NMR measurements, D. Günther (ETH Zurich) for LA-ICP-MS, R. Riding (Uni- versity of Tennessee) for helpful information on the status of S. jurassica, lution of the carbonate with 10 M HCl, the organic residue was obtained as and P. Davis (Natural History Museum London), J. Dejax (Muséum National described above. Then the residue was directly extracted with a minimum d’Histoire Naturelle Paris), and A. E. Richter for fossil samples. Discussions 11 volume (0.5 mL) of DMSO-d6. The B NMR spectrum (192.54 MHz) was with W. Buchberger (University of Linz) and J. R. Maxwell (University of recorded at 298 K on a Bruker Avance DRX 600 NMR spectrometer using a 5- Bristol) improved the manuscript. This study was supported by Deutsche mm inverse triple probe (1H, 13C, broadband) with triple-axis gradient coils. Forschungsgemeinschaft Grant WO 1491/1-1 (to K.W.).

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