Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae

ABSTRACT: An extract of the chloromonad Heterosigmacarterae (Raphidophyceae), cultivated in natural seawater, con-tained a complex mixture of sulfoquinovosyl diacylglycerols.Palmitoyl (16:0), three isomers of hexadecenoyl (16:1 cis ∆9,∆11, ∆13), and eicosapentenoyl (20:5) were found to be themain fatty acyl substituents. Exact double-bond sites were de-termined by mass spectrometry analysis of the correspondingnicotinyl derivatives. Four major sulfoquinovosyl diacylglycerolcomponents were partially purified and identified as 1–4 by in-terpretation of their nuclear magnetic resonance and mass spec-tral data. In addition, complete analysis of the H. carterae sulfo-quinovosyl diacylglycerols was performed using high-perfor-mance liquid chromatography combined with electrospraytandem mass spectrometry.Lipids 32, 1101–1112 (1997)

Blooms of the microalga Heterosigma carterae (formerly H.akashiwo) (1) have been implicated in massive fish mortali-ties at aquaculture sites along the North American PacificCoast (2) and also in some other regions of the world, partic-ularly Chile (3), Japan (4), New Zealand (5), and Spain (6).Biochemical and biological studies indicated that H. carteraeproduced a toxic substance responsible for the fish deaths (1),and this prompted us to investigate the molecular structuresof the sulfoquinovosyl diaclglycerol (SQDG) componentsproduced at high levels by this organism (7,8). SQDG com-ponents isolated from other organisms have been shown toexhibit potent antiviral activity, particularly against thehuman immunodeficiency virus (HIV-1) (9), and also antitu-mor-promoting activities (10). Furthermore, glycolipids have

been implicated in fish mortalities caused by Chrysochro-mulina polylepis (11).

In practice, the isolation of pure SQDG from the complexmixtures usually encountered in nature, which differ only inthe length and/or degree of unsaturation of their fatty acylsubstituents, is extremely difficult (12). Furthermore, a prac-tical and rapid method for the structural analysis of these sub-stances has been elusive. The goal of the present study wastherefore to develop a new method for the structural elucida-tion of the SQDG produced by H. carterae, utilizing liquidchromatography combined with electrospray tandem massspectrometry (LC/MS/MS). As the structure of the sulfo-glycoglycerol moiety is essentially the same for all SQDGisolated and identified to date [i.e., 3-0-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol] (13), the objective was tofocus on determination of the structures and positional assign-ments (sn-1 or sn-2) of the fatty acyl substituents. Nuclearmagnetic resonance (NMR) spectroscopy was used to con-firm the structure of the sulfoquinovosyl glycerol moiety, andalso to firmly establish the structures of purified H. carteraeSQDG identified using the LC/MS/MS technique.

RESULTS AND DISCUSSION

The SQDG fraction was obtained from the chloroform/meth-anol extract of H. carterae cells using silica gel column chro-matography. In order to minimize air oxidation of any polyun-saturated fatty acyl moieties present in the SQDG fraction,the extraction and lipid fractionation were completed within1 d, and fractions were stored under nitrogen, at −80°C, untilfurther use. Analysis by 1H and 13C NMR spectroscopy andby thin-layer chromatography showed the SQDG fraction tobe free of other glycolipids.

Identification of the fatty acyl substituents of H. carteraeSQDG. A portion of the H. carterae SQDG fraction was re-acted with boron trifluoride in methanol to liberate the fattyacyl substituents as their methyl ester derivatives, which wereanalyzed by gas chromatography/mass spectrometry (GC/MS).The major fatty acid methyl esters (FAME) were identified as14:0, 16:0, 20:5, and three isomers of 16:1 (see Table 1). Minorcomponents were determined to be 15:0, 18:3, and 18:4 (Table1). By comparison of the GC/MS data with those obtained forFAME standards, the structure of one of the 16:1 isomers was

Copyright © 1997 by AOCS Press 1101 Lipids, Vol. 32, no. 10 (1997)

1Current address: Institut für Pharmazeutische Biologie, Universität Bonn,Nußallee 6, D-53115 Bonn, Germany.*To whom correspondence should be addressed at the Institute for MarineBiosciences, National Research Council of Canada, 1411 Oxford St., Hali-fax, Nova Scotia B3H 3Z1 Canada. E-mail: [email protected]: AQ, acquisition time; COSY, correlated spectroscopy; EI,electron impact; FA, fatty acid; FAME, fatty acid methyl ester; GC, gas chro-matography; GC/MS, gas chromatography/mass spectrometry; HMBC, het-eronuclear multiple bond correlation; HMQC, heteronuclear multiple quan-tum correlation; LC, liquid chromatography; LC/MS/MS, combined liquidchromatography/tandem mass spectrometry; LSIMS, liquid secondary ionmass spectrometry; MS/MS, tandem mass spectrometry; NMR, nuclear mag-netic resonance; SQDG, sulfoquinovosyl diacylglycerol; SQMG, sulfo-quinovosyl monoacylglycerol; SW, spectral width; TOCSY, total correlatedspectroscopy.

Sulfoquinovosyl Diacylglycerolsfrom the Alga Heterosigma carterae

Michael Keusgen1, Jonathan M. Curtis*, Pierre Thibault,John A. Walter, Anthony Windust, and Stephen W. Ayer

Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia B3H 3Z1, Canada

determined to be the methyl ester of palmitoleic acid (16:1cis-∆9), and the 14:0, 16:0, 18:3, and 20:5 FAME were iden-tified as the methyl esters of myristic, palmitic, linolenic (cis-∆9, ∆12, ∆15), and eicosapentaenoic (cis-∆5, ∆8, ∆11, ∆14,∆17) acids, respectively. The position and stereochemistry ofthe carbon-carbon double bonds present in the remaining two16:1, and the 18:4 FAME could not be determined by com-parison of the GC/MS data with available standards. [Afterthis work was completed, it came to our attention that the 18:4(all-cis ∆6, ∆9, ∆12, ∆15) FAME is now available fromSigma Chemical Company (St. Louis, MO).]

To determine the position of the double bonds in theunidentified 16:1 and 18:4 SQDG fatty acyl groups and toconfirm the assignments previously made using FAME stan-dards analyzed by GC/MS, a portion of the H. carteraeSQDG fraction was reacted with lithium aluminum hydrideto liberate the fatty acyl moieties as the corresponding alco-hols. The alcohols were then converted to their nicotinate de-rivatives, and the resulting mixture was analyzed by GC/MS.Interpretation of the mass spectra of the nicotinate derivatives(discussed shortly) allowed assignment and confirmation ofall the carbon-carbon double-bond sites (Table 1). The twounknown SQDG hexadecenoyl moieties were identified as11-hexadecenoyl (16:1 ∆11) and as 13-hexadecenoyl (16:1∆13) (7), and the 18:4 SQDG fatty acyl group was determinedto be 18:4 (∆6, ∆9, ∆12, ∆15). Additionally, the 15:0 fattyacyl substituent was found only as the saturated species.

Previous investigations have reported the use of picolinylester derivatives of fatty acids (FA) in order to establish thecarbon-carbon double-bond positions (14–18). However, theuse of picolinyl ester or nicotinate derivatives for determina-tion of double-bond positions in SQDG fatty acyl moietieshas not yet been described. For SQDG, three steps would berequired for preparation of the picolinyl esters: (i) cleavageof the SQDG to liberate the FA, (ii) formation of the FA chlo-ride using thionyl chloride, and (iii) esterification withpyridylcarbinol. Steps i and ii involve harsh conditions forpolyunsaturated FA prone to air oxidation, although a modifi-cation has recently been described for step ii (17,18). Theoverall reaction procedure described here for the formation

of the nicotinate derivatives from the SQDG fatty acyl sub-stituents proceeds in good yield with minimal side reactions,and only two steps are required. The preparation of nicotinatederivatives of long-chain alcohols and their analysis by elec-tron impact mass spectrometry (EI/MS) was previously de-scribed by Vetter and Meister (19) and by Harvey (16). How-ever, in contrast to a previous report (16), we have found thatthe formation of the nicotinate derivatives from long-chainalcohols is highly reproducible when using nicotinyl chloridedirectly as the reactant in anhydrous pyridine.

To determine the extent of decomposition of the polyun-saturated fatty acyl moieties, the synthesis of the nicotinatederivatives from the SQDG fatty acyl groups was monitoredby 1H NMR spectroscopy. A comparison was made betweenthe NMR spectra of the initial SQDG mixture and those ofboth the long-chain alcohol intermediates and nicotinateproducts. It was found that there is a stable ratio between theintegrals for the protons resonating at ca. δ 5.3 ppm (olefinicprotons), ca. δ 2.8 ppm (doubly allylic methylene protonscharacteristic of polyunsaturated FA), and the signals in therange between δ 0.7 and δ 2.0 ppm (protons of the aliphaticmethylene and terminal methyl groups). This result indicatedthat the polyunsaturated SQDG fatty acyl moieties were notdegraded to a detectable amount more than were the saturatedfatty acyl groups, an important objective of this study.

The EI mass spectra of the nicotinate derivatives of the sat-urated 14:0, 15:0, and 16:0 SQDG fatty acyl moieties show aregular series of fragment ions down to m/z 178, resultingfrom charge-site remote radical-induced cleavage along thehydrocarbon chain. Figure 1 (A and B) shows the EI massspectra of the nicotinate derivatives of two of the three iso-meric SQDG hexadecenoyl (16:1) acyl groups. These twobackground-subtracted mass spectra (as well as that for the16:1 ∆9 isomer, not shown) were taken from a single GC/MSexperiment, at the crests of GC peaks eluting within a 30-speriod. Each 16:1 nicotinate derivative exhibited a molecularion at m/z 345, but the series of fragment ions between m/z345 and m/z 178 showed marked differences, in terms of boththe m/z values and the relative intensities of the fragment ionsobserved. As described by Vetter and Meister (19), a mini-mum in the relative intensities of the fragment ions for thenicotinate derivatives of monounsaturated fatty alcohols de-notes the position of the double bond. Straightforward inter-pretation of the mass spectra of the nicotinate derivatives al-lowed assignment of two of the 16:1 SQDG fatty acyl groupsas being 11-hexadecenoyl (Fig. 1A) and 9-hexadecenoyl (notshown). The latter assignment is also consistent with a com-parison of the GC retention time of the corresponding FAME(determined in a GC/MS experiment) to that of a 16:1 ∆9standard. The assignment of the final 16:1 SQDG fatty acylmoiety as 13-hexadecenoyl was not possible by simple inter-pretation of the mass spectrum of its nicotinate derivative(Fig. 1B). In fact, the mass spectral data could easily be mis-interpreted as arising from the nicotinate derivative of a 12-hexadecenoyl moiety. However, we have studied the H.carterae SQDG 13-hexadecenoyl moiety in detail (7) and

1102 M. KEUSGEN ET AL.

Lipids, Vol. 32, no. 10 (1997)

TABLE 1Fatty Acid Composition of the SQDG Isolatedfrom Heterosigma carteraea

Fatty acid Rt (min) Amount (%)b

14:0 n 7.0 6.015:0 n 7.5 1.416:0 n 8.2 24.016:1 ∆9 cis 8.4 7.716:1 ∆11 cis 8.5 11.816:1 ∆13 cis 8.6 31.218:3 ∆9, ∆12, ∆15 all-cis 11.3 2.618:4 ∆6, ∆9, ∆12, ∆15 11.6 2.720:5 ∆5, ∆8, ∆11, ∆14, ∆17 all-cis 14.6 12.7aAbbreviations: SQDG, sulfoquinovosyl diacylglycerol; Rt, retention time.bAmounts were estimated from peak areas in the gas chromatography/massspectrometry total ion current chromatograms for the fatty acid methyl esters.

have shown that its nicotinate derivative gives unexpectedlyhigh relative intensities for the fragment ions observed at m/z316 and 302, as is seen in Figure 1B, which is consistent withthe observed fragmentation of the picolinyl ester of the ho-mologous 15-octadecenoyl compound (18). These resultswere in contrast to previous investigations of the total FA-fraction of H. carterae, where FAME were used exclusivelyfor structural elucidation of the FA residues (20).

Figure 1 (C and D) shows the EI mass spectra of the nicoti-nate derivatives of the polyunsaturated H. carterae SQDGfatty acyl groups. The signal-to-noise ratio evident in thesespectra is much lower than that achievable with more abun-dant components or with standards, although such a resultmay be realistic for many minor polyunsaturated componentsof interest. Furthermore, fragmentations arising from thecharge-site remote radical-induced cleavage are seen to bemuch less intense and increasingly ambiguous with increas-

ing degree of unsaturation. Additionally, the polyunsaturatednicotinate derivatives eluted at higher column temperaturesthan the saturated or monounsaturated derivatives so that ahigher level of background ions was observed. This back-ground noise may not be completely eliminated by back-ground subtraction (e.g., m/z 384 in Fig. 1C). The assign-ments of the 18:3 and 20:5 polyunsaturated SQDG fatty acylmoieties obtained by GC/MS of the FAME were confirmedby the EI mass spectra of their nicotinate derivatives (Fig. 1Cand 1D, respectively). The spectrum of the 18:3 nicotinateshows fragmentation similar to that observed by Harvey (15)for the picolinyl ester derivative of cis ∆9, ∆12, ∆15-octade-catrienoic acid. In the present case, the dominant signal at m/z300 (Fig. 1C) can be explained by the loss of an allylic radi-cal. The corresponding ion for 18:4 is seen at m/z 298 whilethat for 20:5 is at m/z 324 (Fig. 1D), consistent with the as-signed structures. However, for 18:4 the signal-to-noise ratio

SULFOQUINOVOSYL DIACYLGLYCEROLS FROM HETEROSIGMA CARTERAE 1103

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FIG. 1. The gas chromatography/electron impact mass spectra of the nicotinate derivatives of the polyunsaturated sulfoquinovosyl diacylglycerolfatty acyl moieties. A) 16:1 ∆11, B) 16:1 ∆13, C) 18:3 ∆9, ∆12, ∆15, D) 20:5 ∆5, ∆8, ∆11, ∆14, ∆17.

is too low to allow confirmation by the presence of the ex-pected fragment ions at m/z 178 and 192 that the additionalunsaturation is indeed located at C-6. It can be seen from Fig-ure 1 that, for the nicotinate derivatives of saturated and mo-nounsaturated fatty alcohols, an ion containing a C-4 chain atm/z 178 is generally observed. It was found that for deriva-tives of polyunsaturated SQDG fatty acyl moieties an ion atm/z 178 is seen in the case of 18:3 but is weak for 18:4 and20:5, consistent with the presence of the proximal doublebond at C-5 or C-6.

To assign the configuration of the carbon-carbon doublebonds in the 11- and 13-hexadecenoyl SQDG fatty acyl sub-stituents, the purified (7) methyl ester derivatives were exam-ined by 1H NMR spectroscopy. For the methyl ester deriva-tive of the 13-hexadecenoyl substituent, decoupling by irradi-ation of the allylic methylene protons at δ 2.05 ppm collapsedthe multiplet resonating at δ 5.32 ppm to a doublet of dou-blets with a coupling constant of 10.8 Hz, allowing assign-ment of the cis (Z) configuration (21) for the carbon-carbondouble bond (Table 2). In the same experiment, the triplet res-onance at 0.95 ppm, assigned to the terminal methyl group,collapsed to a singlet. This result proved that the carbon-car-bon double bond was in the n-3 (∆13) position (7). By usingthe same decoupling technique, the methyl ester derivative ofthe 11-hexadecenoyl substituent also gave a doublet of dou-blets for the olefinic protons, with a coupling constant of 10.9Hz. Thus, the configuration of the double bond in the 11-hexadecenoyl substituent was also assigned as cis (Z).

The purified methyl ester derivative of the 9-hexadecenoylsubstituent was found to be in the cis (Z) configuration basedon GC/MS comparison with an authentic standard. This de-rivative gave, upon irradiation at 2.05 ppm, a singlet reso-nance at 5.34 ppm for the two olefinic protons. NMR exami-nation of cis and trans isomers of the methyl esters of four ∆9monounsaturated FA standards performed under the same de-coupling conditions as described above showed that the cisisomers gave a singlet resonance at 5.34 ppm for the olefinicprotons, whereas for the trans isomers the singlet was ob-served at 5.38 ppm (Table 2). This result confirmed that the9-hexadecenoyl fatty acyl substituent was the cis (Z) isomer.

13C NMR analysis of the methyl ester derivatives of the

isolated 9-, 11-, and 13-hexadecenoyl moieties also supportedthe stereochemical assignments (Table 3). For the cis andtrans standards of 16:1 ∆9, significant differences were ob-served for both the chemical shifts of the olefinic carbons (cis:130.8 and 130.9 ppm; trans: 131.5 and 131.6 ppm) and forthe corresponding signals of the adjacent methylene carbons(cis: 28.1 and 28.2 ppm; trans 33.7 and 33.8 ppm). The 13CNMR chemical shifts assigned to the allylic methylene car-bons in both the 16:1 ∆11 and 16:1 ∆13 fatty acyl substituents(Table 3) supported the cis stereochemical assignment for thedouble bonds and are in good agreement with published data(22–24).

Investigation of the intact SQDG from H. carterae byLC/MS and LC/MS/MS. With the analysis of the fatty acylsubstituents completed, the next step was to examine the in-tact H. carterae SQDG in order to locate the acyl substituentson the sulfoquinovosyl glycerol backbone. Figure 2 shows theflow injection electrospray mass spectrum of the H. carteraeSQDG in the negative-ion mode. The peaks at m/z 837, 813,791, and 763, which represent singly deprotonated molecules[M − H]−, are the most prominent. On the basis of tandemmass spectrometry (MS/MS) experiments, these ions couldbe tentatively assigned as SQDG with the following fatty acylpairings (as described in the following discussion); 20:5/16:1and/or 18:3/18:3 (m/z 837); 18:3/16:1 (m/z 813); 16:0/16:1(m/z 791); and 14:0/16:1 (m/z 763). On the basis of the frag-mentation patterns in their MS/MS spectra, the peaks at m/z823 and m/z 869 in Figure 2 were determined to represent per-oxidized forms of the two most abundant SQDG (see Table4) having the 16:0/16:1 and 20:5/16:1 fatty acyl pairings, re-spectively.

Although it was possible to analyze the major H. carteraeSQDG by flow-injection MS/MS without chromatographicseparation, it was necessary to use LC/MS and LC/MS/MS


Lipids, Vol. 32, no. 10 (1997)

TABLE 2Chemical Shifts of Olefinic Protons of Different Unsaturated FattyAcid Methyl Esters (FAME) in CD3OD and Olefinic H-H CouplingConstants Measured After Irradiation at δ 2.05

FAME

13-Hexadecenoyl methyl ester 16:1 n-3 ∆13 5.30/5.34J = 10.8 Hz

11-Hexadecenoyl methyl ester 16:1 n-5 ∆11 5.33/5.36J = 10.9 Hz

9-Hexadecenoyl methyl ester 16:1 n-7 ∆9 5.34a

Palmitoleic acid (cis) 16:1 n-7 ∆9 5.34 sPalmitelaidic acid (trans) 16:1 n-7 ∆9 5.38 sOleic acid (cis) 18:1 n-9 ∆9 5.34 sElaidic acid (trans) 18:1 n-9 ∆9 5.38 saSignals for both olefinic protons are identical. Abbreviation: s, singlet.

TABLE 3Carbon Nuclear Magnetic Resonance Shifts of Fatty Acid MethylEsters in CD3OD

Synthetic IsolatedPosition 16:1 ∆9 cis 16:1 ∆9 trans 16:1 ∆13 16:1 ∆11 16:1 ∆9

Methyl 52.0 52.0 52.0 52.0 52.01 175.9 175.6 175.9 175.9 175.92 34.8 34.9 34.8 34.8 34.83 26.1 26.1 26.0 26.0 26.04 30 30 30 30 305 30 30 30 30 306 30 30 30 30 307 30 30 30 30 308 28.1a 33.7 30 30 28.09 130.8 131.5 30 30 130.8

10 130.9 131.6 30 28.1 130.811 28.2 33.8 30 130.6 28.112 31 31 28.0 131.0 3013 31 31 130.2 27.9 3114 33.0 33.0 132.5 30 33.215 23.8 23.9 21.4 24.0 23.816 14.5 14.5 14.8 14.1 14.3aCharacteristic chemical shifts for cis and trans isomers are in boldface type.

methods to resolve isomeric and closely related componentsas well as some minor SQDG components found in this frac-tion. A separation method employing a basic mobile phaseand an alkali-resistant octadecylsilyl stationary phase pro-vided the best separation of the H. carterae SQDG (Fig. 3).As demonstrated by the extracted ion chromatograms (Fig. 4)taken from the LC/MS total ion chromatogram shown in Fig-ure 3, closely related SQDG could be adequately resolved.

Analysis of the MS data showed that the two major peaksin Figure 4B corresponded to two SQDG differing by twomass units. The peak at 22.9 min corresponds to an SQDG ofmass 766 ([M − H]− m/z 765, 16:0/14:0 fatty acyl pair),whereas the peak at 20.4 min corresponds to the second iso-tope peak (i.e., two mass units higher than the nominal mass)of the SQDG of nominal mass 764 ([M − H]− m/z 763,14:0/16:1 fatty acyl pair, see Fig. 4A). This second isotopepeak arises owing to the natural abundance of 13C and 34S re-sulting in an ion at m/z 765 for the 14:0/16:1 fatty acyl pair at17% abundance relative to the all 12C and 32S ion at m/z 763.Similarly, the peaks at 25.1 and 28.3 min in Figure 4F corre-spond to SQDG of mass 792 ([M − H]− m/z 791, 16:0/16:1fatty acyl pair) and 794 ([M − H]− m/z 793, 16:0/16:0 fattyacyl pair), and the peaks at 20.2 and 20.9 min in Figure 4I cor-

respond to SQDG of mass 838 ([M − H]− m/z 837, 20:5/16:1fatty acyl pair) and 840 ([M − H]− m/z 839, 20:5/16:0 fattyacyl pair). In each of these cases, the SQDG components dif-fering by two mass units were nearly baseline resolved, andMS/MS experiments could be performed without interfer-ences due to 13C and 34S isotopic variants of a related SQDGof similar mass (see Table 4 for a complete summary of theLC/MS/MS results).

The MS/MS spectrum of the major SQDG (16:0/16:1 fattyacyl pairing) found in the H. carterae cell extract, taken froman LC/MS/MS run, is shown in Figure 5. The observed frag-mentation pattern is significantly different from that previ-ously observed by fast atom bombardment ionization-MS/MSof SQDG (25). Of particular note is the absence of fragmentions resulting from cleavages along the fatty acyl chainswhich only arise at higher collision energies than are accessi-ble on the triple quadrupole mass spectrometer used for thisexperiment. Also, in contrast to the earlier fast atom bom-bardment ionization-MS/MS results, the most abundant high


Lipids, Vol. 32, no. 10 (1997)

FIG. 2. Flow injection negative ion electrospray mass spectrum of theHeterosigma carterae sulfoquinovosyl diacylglycerols.

TABLE 4SQDG Composition of Heterosigma carterae

FA composition LC/MS LC/MS/MSsn-1 sn-2 Rt (min) %a Mr [M − H − Rsn-1CO2H]− [M − H − Rsn-2CO2H]−

14:0 16:1 20.4 8.1 764 535 50916:0 14:0 22.9 3.0 766 509 53715:0 16:1 21.8 2.8 778 523 53516:1 16:1 20.7 2.4 790 535 53516:0 16:1 25.1 53.0 792 535 53716:0 16:0 28.3 0.6 794 537 53718:3 16:1 20.3 3.7 814 535 55918:3 18:3 19.4 0.2 838 559 55920:5 16:1 20.2 21.8 838 535 58320:5 16:0 20.9 0.8 840 537 583

Othersb 3.6aAmounts estimated from LC/MS peak areas.bSQDG in smaller amounts (e.g., pairings with the 18:4 fatty acid). Abbreviations: FA, fatty acid; LC/MS, liquid chro-matography/mass spectrometry; LC/MS/MS, combined liquid chromatography/tandem mass spectrometry. For other ab-breviations see Table 1.

FIG. 3. Total ion current liquid chromatography/mass spectrometrychromatogram for the Heterosigma carterae sulfoquinovosyl diacylglyc-erols.

mass fragment ions (at m/z 537 and m/z 535) can be rational-ized as arising from a fragmentation involving loss of oneacyl substituent from the molecular ion ([M − H − C16H3002]−

and [M − H − C16H3202]−) and corresponding to fragmentions b and e shown in Scheme 1. In this Scheme, the frag-mentation of SQDG under MS/MS conditions is shown. Thefragments b–e may be used for determination of the FA sub-stitution. Fragment ions at m/z 553 and m/z 555 arising fromthe loss of a ketene analog ([M − H − R1C=0]− and [M − H −R2C=0]− (25), ions c and d in Scheme 1) are of very low in-tensity in the LC/MS/MS spectrum. Peaks assigned as the

fragment ions corresponding to the free FA ([C16H3102]− and[C16H2902]− are clearly visible at m/z 255 and m/z 253. Otherpeaks appearing in Figure 5 could be readily assigned to frag-ment ions f–o which can be considered characteristic for thesubstance class of SQDG under MS/MS conditions, indepen-dent of their FA substitution pattern (Scheme 2) (25).

Extensive interpretation of the LC/MS/MS data allowedcomplete assignment of the SQDG produced by H. carterae(Table 4). The positional assignments of the fatty acyl moietieslisted in Table 4 were made on the basis of the relative intensi-ties of the [M − H − Rsn-1CO2H]− and [M − H − Rsn-2CO2H]−

fragment ions in the MS/MS spectrum of each SQDG. Theseassignments were supported by the positional assignments forthe SQDG components with the 16:0/16:1 and 20:5/16:1 fattyacyl pairings as determined by interpretation of the NMR spectraand regioselective enzymatic hydrolysis (see below). It was foundthat, in general, the relative intensity of the fragment ion result-ing from loss of the sn-1 fatty acyl moiety ([M − H − Rsn-1CO2H]−,m/z 535 in Figure 5) is significantly higher than the relative in-tensity of the fragment ion resulting from loss of the sn-2 fattyacyl group ([M − H −Rsn-2CO2H]−, m/z 537 in Figure 5). Theonly exception to this rule appears to be when a highly polyun-saturated fatty acyl group is attached to the sn-2 position. Inthis case, only a small difference in the relative intensity of thetwo fragment ions occurs.

NMR analyses of SQDG fractions. To obtain SQDG com-ponents for structural analysis by NMR spectroscopy and forregioselective enzymatic cleavage of the sn-1 fatty acyl moi-ety, the SQDG mixture from H. carterae was separated bypreparative LC. The fractions containing a mixture of threeSQDG components having the 16:0/16:1 fatty acyl pairing(compounds 1, 2, and 3), and the fraction containing mainlythe SQDG with the 20:5/16:1 fatty acyl pair (substance 4),were subjected to NMR analysis. The 1H and 13C NMR spec-tra of both fractions exhibited nearly identical chemical shiftsfor resonances assigned to the sulfoquinovosyl-glycerol moi-eties. The NMR assignments for the fraction containing themixture of SQDG with the 16:0/16:1 fatty acyl pairing arelisted in Tables 5 and 6, and were made on the basis of inter-pretation of one- and two-dimensional [(including 1H-1Hcorrelated spectroscopy (COSY), total correlated spec-troscopy (TOCSY), nuclear Overhauser enhancement spec-troscopy, heteronuclear multiple quantum correlation (HMQC),and heteronuclear multiple bond correlation (HMBC)] NMRspectra. Where comparison was possible, the resonances ob-served for the sulfoquinovosyl-glycerol moieties are in excel-lent agreement with literature values (26).

The positional distribution of the fatty acyl groups in themixture of SQDG having the 16:0/16:1 fatty acyl pairing wasdetermined by two different methods, viz., NMR spec-troscopy and regioselective cleavage of the acyl group at thesn-1 position. By using 3:1 (vol/vol) CD3OD/C6D6 as the sol-vent for NMR examination of the mixture of compounds 1, 2,and 3, the resonance at 5.44 ppm, assigned to the proton atthe sn-2 position of the glycerol moiety, could be resolvedfrom the resonances attributed to the olefinic protons in the


Lipids, Vol. 32, no. 10 (1997)

FIG. 4. Extracted ion chromatograms reconstructed from the full-scanliquid chromatography/mass spectrometry data shown in Figure 3 forthe Heterosigma carterae sulfoquinovosyl diacylglycerols.

FIG. 5. Tandem mass spectrum of the [M − H]− ion of the major sulfo-quinovosyl diacylglycerol (16:0/16:1 fatty acyl pairing) found in theHeterosigma carterae cell extract, taken from a combined chromatogra-phy/tandem mass spectrometry run.

16:1 acyl substituents. In the HMBC spectrum of the mixture,a correlation was observed between the proton resonance at5.44 ppm and the carbon resonating at δ 174.96 ppm, thus al-lowing assignment of the latter resonance to the carbonyl car-bon of the fatty acyl group attached to the sn-2 position. Themethylene protons resonating at 2.32−2.35 ppm showed acorrelation in the HMBC spectrum to the same carbonyl car-bon (δ 174.96), thereby allowing assignment of these protonsas the methylene protons on the carbon adjacent to the car-

bonyl carbon in the fatty acyl group attached at the sn-2 posi-tion. Similarly, correlations from the resonances at 4.28 ppmand at 2.26–2.27 ppm to the carbonyl carbon at 175.11 ppmproved the assignment of these resonances to the sn-1 posi-tion and its associated FA. The TOCSY spectrum of the mix-ture revealed that the protons resonating at 2.02 ppm (as-signed to the allylic protons in the 16:1 fatty acyl groups; seeTable 6) belonged to the same spin systems as those resonat-ing at 2.32–2.35 ppm, a result which allowed assignment of


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SCHEME 1

SCHEME 2

all three 16:1 fatty acyl groups exclusively to the sn-2 posi-tion.

Enzymatic cleavage of SQDG and analyses of the result-ing sulfoquinovosyl monoacylglycerol (SQMG). To confirmthis assignment, the 16:0 fatty acyl moiety at the sn-1 posi-tion of the mixture was regioselectively hydrolyzed using li-pase from the fungus Rhizopus arrhizus (7,26). Negative ionliquid secondary ion mass spectrometry (LSIMS) analysis ofthe resulting SQMG gave an [M − H]− ion at m/z 553, clearly

indicating that the remaining fatty acyl group was 16:1. How-ever, NMR analysis of this SQMG (including 1H-1H COSY,HMBC, and HMQC spectra, see Table 7) revealed a mixtureof compounds 5, 6, and 7 which have the remaining 16:1 fattyacyl moiety attached to the sn-1 position. This is clearly seenfrom the 4.16 ppm chemical shift of the methine proton at-tached to the sn-2 position, and the chemical shifts of 4.16and 4.27 ppm observed for the methylene protons attached tothe sn-1 position. The location of the 16:1 fatty acyl moiety


Lipids, Vol. 32, no. 10 (1997)

TABLE 5Nuclear Magnetic Resonance Assignments of Substances 1–3

Solvent

CD3OD CD3OD/C6D6

Positions 1H Sys. J (Hz) 13C 1H 13C

1 4.77 d 3.8 100.1 4.85 100.32 3.38 dd 3.8/9.8 73.5 3.53 73.63 3.62 dd 8.8/9.8 74.88 3.79 75.064 3.06 dd 8.8/9.9 74.90 3.22 75.135 4.05 ddd 2.1/9.2/9.9 69.8 4.21 69.96 2.90 dd 9.2/14.3 54.2 3.08 54.6

3.37 dd 2.1/14.3 3.501′ 3.55 dd 6.4/10.8 67.1 3.64 67.4

4.08 dd 5.3/10.8 4.192′ 5.30 m na 71.7 5.44 71.83′ 4.18 dd 6.9/12.0 64.3 4.28 64.5

4.49 dd 3.0/12.0 4.59

FA attached to C2′ (sn-2)

1 174.9 174.962 2.33 m 7.5/15.7 35.1 2.32 35.4

2.353 1.59 m na 26.1 1.58 26.1

FA attached to C3′ (sn−1)

1 175.1 175.112 2.31 m 7.5/15.7 35.0 2.26 35.2

2.273 1.59 m naa 26.1 1.58 26.1ana, not available. For other abbreviations, see Table 4.

TABLE 6Nuclear Magnetic Resonance Assignments of Substances 1–3 (solvent CD3OD)

16:0 (1–3) 16:1 n-3/∆13 (in 3) 16:1 n-5/∆11 (in 2)

Position 1H 13C 1H 13C 1H 13C

4 1.3 30 1.3 30 1.3 305 1.3 30 1.3 30 1.3 306 1.3 30 1.3 30 1.3 307 1.3 30 1.3 30 1.3 308 1.3 30 1.3 30 1.3 309 1.3 30 1.3 30 1.3 30

10 1.3 30 1.3 30 2.02 28.211 1.3 30 1.3 30 5.33 130.6

(J = 11.7 Hz)12 1.3 30 2.02 28.0 5.35 131.013 1.3 30 5.31 130.2 2.02 28.2

(J = 10.6 Hz)14 1.29 33.1 5.34 132.4 1.28 30.115 1.29 23.8 2.04 21.5 1.27 24.016 0.88 14.5 0.93 14.8 0.89 14.2

(J = 7.0 Hz) (J = 7.6 Hz) (J = 7.2 Hz)

at the sn-1 rather than the expected sn-2 position can be ex-plained by an acyl migration favored by proton-donating sol-vents (28,29) since in this experiment, the SQMG had beenpurified using a C18 derivatized silica gel column followed bypreparative LC, involving the use of triethylamine and water.

The enzymatic hydrolysis experiment was then repeatedusing an alternatve purification procedure in which, follow-ing hydrolysis, proteins and salts were precipitated by alco-hols. The NMR spectra of the SQMG products were then rundirectly after filtration of the alcoholic solution. Using this al-ternative purification procedure, the product was determinedto be an SQMG mixture composed of compounds 8, 9, and10 (selected NMR assignments are listed in Table 7). In con-trast to the NMR data for the SQMG containing 5, 6 and 7,the methine proton attached to the sn-2 position now res-onated at 5.08 ppm and the signals assigned to the two sn-1methylene protons had identical chemical shifts at 3.72 ppm.The isolation of either the sn-1 or sn-2 acylated product, de-pending on purification conditions, demonstrates the impor-tance of conducting regioselective enzymatic cleavage andsubsequent workup under carefully controlled conditions inorder to obtain the expected sn-2 monoacylated product.

The sn-1 selective enzymatic cleavage followed by nega-tive-ion LSIMS analysis was also applied to different SQDGfractions containing 14:0/16:1, 16:0/14:0, 18:3/16:1, and20:5/16:0 FA pairings to yield an [M − H]− for the corre-sponding SQMG at m/z 553 (loss of 14:0 FA), m/z 527 (lossof 16:0 FA), m/z 553 (loss of 18:3 FA), and m/z 555 (loss of20:5 FA), respectively. These results are in full accordancewith the positional assignments obtained by the LC/MS/MSexperiments described earlier (see Table 4).

To confirm the structure of the sulfoglycoglycerol moiety,the mixture of 5, 6, and 7 was hydrolyzed under acid condi-tions to give the deacylated product 11. The 1H and 13C NMRdata for 11 (Table 7) are in excellent agreement with those re-ported for 3-0-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol(26,30). These data, together with comparison of the NMRdata of the intact SQDG discussed earlier, support the rela-

tive stereochemistry of the sulfoglycoglycerol moiety asshown in Scheme 3. Although the data presented here do notexclude the possibility that the H. carterae SQDG sulfoglyco-glycerol moiety actually has the opposite absolute configura-tion to that presented in structure 11, it is unlikely that H.carterae would be the only organism to produce such a sulfo-quinovosyl glycerol.

In conclusion, it has been shown that H. carterae containsnumerous SQDG, all with the same sulfoquinovosyl-glycerolbackbone substituted by various saturated, monounsaturated,and polyunsaturated FA. The most abundant SQDG containthe fatty acids 16:0, 16:1, and 20:5. Isolation of the individ-ual SQDG was deemed impractical, so an LC/MS/MS systemfor their separation and identification was developed to eluci-date their profile in extracts of H. carterae. The positional as-signments of the fatty acyl moieties on the sulfoquinovosylglycerol backbone could be made on the basis of the relativeintensity of the [M − H − Rsn-1CO2H]− and [M − H − Rsn-2CO2H]− fragment ions in the LC/MS/MS spectra. The loca-tions of carbon-carbon double bonds on the fatty acyl chainswas determined from the GC/EI mass spectra of theirnicotinyl derivatives. For monounsaturated fatty acyl chains,the configurations of these double bonds were determined byNMR analyses of the corresponding FAME. The combinedresults obtained from the different analytical techniques al-lowed an almost complete determination of the SQDG in H.carterae.

MATERIALS AND METHODS

All solvents were analytical grade, and purchased either fromBaker (Phillipsburg, NJ), BDH (Toronto, Canada), or Sigma(St. Louis, MO). Distilled or Milli-Q-grade water was usedfor chromatography.

Extraction and isolation of SQDG. Hetersigma carterae(isolate 102R) was obtained from the Notheast Pacific Cul-ture Collection, University of British Columbia, Vancouver,British Columbia, Canada. The cultures were grown in f/2


Lipids, Vol. 32, no. 10 (1997)

TABLE 7Nuclear Magnetic Resonance Assignments of Substances 5–10

5–7a 8–10b 11a

Position 1H 13C 1H 13C 1H 13C

1 4.84 100.3 4.75 100.1 4.85 100.02 3.49 73.9 3.40 73.7 3.50 73.53 3.77 75.3 3.62 75.1 3.74 75.14 3.12 75.2 3.07 74.8 3.16 74.75 4.22 70.0 4.07 69.8 4.21 69.76 3.04 54.6 2.92 54.2 3.02 54.2

3.47 3.33 3.441′ 3.47 70.0 3.52 67.3 3.44 70.5

4.16 4.08 4.122′ 4.16 70.8 5.08 74.8 3.98 72.73′ 4.16 66.6 3.72 62.2 3.68 64.0

4.27aSolvent: 3:1 CD3OD/C6D6.bSolvent: CD3OD.

seawater medium (31) at 22°C on a 16:8 h light/dark cycleunder an irradiance of 80 mmol-photons m−2s−1 provided by40-W cool-white fluorescent lamps. Wet cell material (160 g)was extracted into MeOH/CHCl3 (7:3, vol/vol), yielding atotal of 850 mg of SQDG after silica gel column chromatog-raphy, as described in Reference 7. A portion of this crudeSQDG fraction was further fractionated on a Nova-Pak C18LC column (7.8 mm i.d. × 300 mm; Waters Ltd., Milford,MA) using a linear gradient (7).

sn-1 Regioselective enzymatic cleavage of the SQDG. Theenzymatic digestion is based on a procedure described in Ref-erence 27 and is described in detail in Reference 7. Lipasetype XI from Rhizopus arrhizus (Sigma) was used. Approxi-mately 5 mg of each SQDG-fraction was incubated in Trisbuffer (pH 7.4) at 37°C for 15 min. The reaction was stoppedby adding isopropanol (15 mL), and the solvent was evapo-rated under reduced pressure. The residue was loaded onto aflash column (8 mm i.d.) packed with 2.7 g of C18 derivatizedsilica gel (Baker) and eluted with 20 mL water, 5 mL of amixture of triethylamine, acetonitrile and water (1:1:8, to con-vert acidic compounds into their corresponding salts), 20 mLwater, and finally 40 mL MeOH. The MeOH fraction was an-alyzed by mass spectrometry (LSIMS). For NMR experi-ments, this fraction was further purified by LC using a Nova-Pak C18 column (4.5 × 300 mm) with an isocratic solvent sys-tem containing MeOH/water (9:1), a Waters 510 pumpcombined with a Waters refractive index detector RI 410, anda flow rate of 1.2 mL/min. In this way 1 mg of SQMG wasobtained (Rt, 3.8 min), liberated FA eluted at Rt, 12.8 min anda small amount of undigested SQDG eluted at Rt 20.9 min.Note that this procedure resulted in a migration of the sn-2fatty acyl moiety onto the sn-1 position after enzymatic di-gestion.

The alternative procedure, adopted to yield 8–10, was to

stop the enzymatic reaction with isopropanol (20 mL), thenevaporate the solvent under reduced pressure. The residuewas dissolved in 2 mL MeOH and transferred to a clean vial,and the solvent evaporated by a stream of dry nitrogen. Theresidue was dissolved in 1 mL MeOH and filtered (0.5 µm fil-ter cartridge). NMR analysis of the protein-free filtrate wasthen performed on the same day.

Formation of FAME. Twenty mg of the purified SQDGfraction was dissolved in 2 mL 0.5 M NaOH/MeOH and equi-librated for 30 min at room temperature. A 2-mL aliquot ofBF3 in MeOH (20%) was added, and after 30 min the volumewas reduced to 0.5 mL by a stream of nitrogen. Saturatedaqueous NaCl solution (20 mL) was added and the mixtureextracted three times with 20 mL n-hexane each. The hexanelayers were combined and volume reduced to 25 mL underreduced pressure. The hexane fraction was washed with 20mL H2O, dried over Na2SO4 for about 10 min, and solventwas evaporated under reduced pressure to yield 2.6 mg of acolorless oil which was analyzed by GC/MS.

MS. LSIMS and GC/MS experiments were performedusing a VG ZAB-EQ mass spectrometer (Micromass Ltd.,Manchester, United Kingdom) coupled to a Hewlett-PackardSeries II gas chromatograph with on-column injector (PaloAlto, CA). For LSIMS experiments, a 20 keV beam of Cs+

ions and a triethanolamine matrix were used. MS/MS experi-ments were performed in the first field-free region by a linkedscan at constant B/E. Ar was used as collision gas at a pres-sure which attenuated the ion beam by 50% and at a collisionenergy of 8 keV. EI experiments were performed at an ionsource temperature of 200°C and an ionizing electron energyof 70 eV. For further details see Reference 7.

LC/MS/MS experiments were performed using a Hewlett-Packard 1090 HPLC system combined with a PE-SCIEX APIIII+ triple quadrupole mass spectrometer (Concord, ON,


Lipids, Vol. 32, no. 10 (1997)

Substance no. R1 (FA)a R2 (FA)

1 16:0 16:1 ∆9 cis2 16:0 16:1 ∆11 cis3 16:0 16:1 ∆13 cis4 20:5 ∆5, ∆8, ∆11, ∆14, ∆17 all-cis 16:1 ∆13 cis5 16:1 ∆9 cis H6 16:1 ∆11 cis H7 16:1 ∆13 cis H8 H 16:1 ∆9 cis9 H 16:1 ∆11 cis

10 H 16:1 ∆13 cis11 H HaAbbreviation: FA, fatty acid.

SCHEME 3

Canada) operated in negative ion ionspray mode. A ZorbaxSB C18 LC column (0.21 × 15 cm) was used at a flow rate of0.2 mL min−1. The optimal LC separation was achieved using0.03% NH4OH in H2O and CH3CN as solvents in linear gra-dients such that their respective percentages were 70:30 ini-tially; 50:50 at 10 min; 20:80 at 30 min; 0:100 at 35 min; and70:30 at 45 min.

NMR spectroscopy. NMR spectra at 20°C were recordedon a Bruker AMX-500 spectrometer (Bruker Analytik, Karls-ruhe, Germany) at 500.13 MHz (1H), 125.7 MHz (13C), using5-mm sample tubes (Wilmad Glass, Buena, NJ; model 535pp)and standard Bruker pulse sequences. Solvents CD3OD or 3:1CD3OD/C6D6 were used as noted in the text or tables. Chem-ical shifts were referred to CHD2OD at 3.30 ppm (1H), or13CD3OD at 49.0 ppm (13C).

Acquisition conditions, one-dimensional spectra: (1H)spectral width (SW) 4098 Hz, acquisition time (AQ) 4.0 s,66° pulse, delay after acquisition (D1) 2 s, decoupling fieldwhere used ca. 250 Hz; (13C) SW 33,333 Hz, AQ 0.98 s, 40°pulses, Waltz decoupling of 1H, D1 0.1 s. Spectra wereprocessed with zero filling after exponential weighting (linebroadening 0.2 Hz for 1H, 0.5 Hz for 13C) or resolution en-hancement by Lorentz-Gauss multiplication. Couplings ofsome protons were obtained by simulation.

Two-dimensional spectra (1H TOCSY and double-quan-tum filtered COSY): SW 3676 Hz, AQ 0.07 s, D1 2.0 s, 512× 512 increments, TOCSY spin lock for 160 ms at fieldstrength 8 kHz, processed with zero filling and apodizationby 90° shifted sine-bell squared in both dimensions; (1H/13CHMQC and HMBC) 1H SW 3676 Hz, 13C SW 16,600 or22,726 Hz, AQ 0.07 s, D1 1.5 s, 512 × 1024 increments, zerofilling both dimensions, apodization as above for HMQC, un-shifted sine-bell squared in both dimensions for HMBC.Delay for HMBC 60 ms and 90 ms.

Isolated compounds. 1′-O-(6-desoxy-6-sulfo-α-D-glu-copyranosyl)-2′-O-(hexadeca-9- enoyl)-3′-O-hexadecanoyl-sn-glycerol, 1; 1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-2′-O-(hexadeca-11-enoyl)-3′-O-hexadecanoyl-sn-glycerol, 2;1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-2′-O-(hexa-deca-13-enoyl)-3′-O-hexadecanoyl-sn-glycerol, 3; total yield100 mg; NMR data, see Tables 5 and 6; MS see Table 4 andReferences 7, 25.

1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-2′-O-(hexa-deca-13-enoyl)-3′-O-(eicosapenta-5,8,11,14,17-enoyl)-sn-glycerol, 4; 69.2 mg. 1H NMR data of the eicosapentaenoicacid moiety: δ 2.25 (2H, t; H at C2), δ 1.60 (2H, m; H at C3),δ 2.02 (4H, m; H at C4, C19), δ 5.36 (10H, m; olefinic pro-tons), δ 2.80 (8H, m; H at C7, C10, C13, C16), δ 0.96 (3H, t, J= 7.6 Hz; H at C20). All other shifts are identical with thosegiven in Table 5. The signal at δ 4.49 (1H at C3′) is slightlydownfield-shifted (δ 4.50). 13C NMR data: methyl groups: δ15.0, δ 15.1; methylene groups: δ 21.7−δ 35.4; olefinic car-bons: δ 128.3–δ 133.0 (CDCl3/C6D6, 3:1); all other values areidentical with those given in Table 5. MS data: see Table 4.

1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-3′-O-hexa-decaenoyl-sn-glycerols, 5–7; yield after high-performance

liquid chromatography separation ca. 1 mg. Enzymatic cleav-age and purification are described in Reference 7. NMR data:see Table 7. MS data are given in Reference 7.

1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-2′-O-hexa-decaenoyl-sn-glycerol, 8–10; yield ca. 5 mg. NMR data, seeTable 7; [M − H]− at m/z 553.4; fragmentation series identicalwith 4–7.

1′-O-(6-desoxy-6-sulfo-α-D-glucopyranosyl)-sn-glycerol,11; yield 3.6 mg. Acid-catalyzed hydrolysis: 15 mg of theSQDG (16:1/16:0) were dissolved in 5 mL absolute MeOHand a few drops of trifluoroacetic acid were added. Com-pound 11 was purified by LC as described in Reference 7.NMR data are given in Table 7; further analytical data: seeReferences 26 and 30.

ACKNOWLEDGMENTS

We gratefully acknowledge Ping Seto for obtaining the NMR spec-tra and Dr. Robert K. Boyd and Dr. Archie McCulloch for review-ing the manuscript. This is NRCC publication number 39767.

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[Received May 27, 1997, and in final revised form August 25, 1997;revision accepted August 26, 1997]


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Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae

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