Biomacromolecules 2000, 1, 519-522
519
Diglycerol Alkenedioates in Suberin: Building Units of a Poly(acylglycerol) Polyester Jose´ Grac¸ a* and Helena Pereira Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade Te´ cnica Lisboa, 1349-017, Lisboa, Portugal Received May 8, 2000; Revised Manuscript Received August 25, 2000
Introduction. Plants are protected from the environment, to avoid uncontrolled water loss and excessive thermal variation, by cells with specialized walls: cutinized and suberized cells. They owe their names to the presence in high quantity of the biopolymers cutin and suberin in their cell walls, believed to be mainly responsible for the confining and insulating properties. Tree barks are rich in suberized cells, and in the case of the outer bark of Quercus suber, a massive, 50% suberin content tissue is produced, allowing the exploitation of commercial cork. Despite their obvious importance, still little is known about the macromolecular structure of these biopolymers. The actual difference between cutin and suberin, though a set of criteria has been proposed,1 remains unclear. Both biopolymers are depolymerized by ester-cleaving reactions, releasing monomers with carboxylic acid and hydroxyl functionalities, predominantly C16-C22 R,ω-diacids and ω-hydroxyacids.2,3 The C18 monomers have at midchain, an unsaturation, an epoxide ring, or a Vic-diol. In suberins, these midchain “modified” C18 monomers constitute, as a rule, the majority of the aliphatic acids. In cutins, the C16 ω-hydroxyacids, substituted with one hydroxyl close to midchain, are frequently the dominant long-chain monomers.4,5 The few studies dedicated to the matter concluded that the carboxylic and hydroxylic groups in R,ω-positions were all involved in ester linkages, but midchain hydroxyls were free in suberins and partially linked in cutins.6-8 In suberins, the ester-depolymerization reactions that release the above aliphatic acids also solubilize significant quantities (roughly a third of the mixture) of ill-characterized phenolics. These phenolics always include small amounts of hydroxycinnamic acids, namely ferulic acid. Together, this knowledge led to the first tentative model for suberin: R,ωdiacids and ω-hydroxyacids interesterified in a linear form and further interesterified to hydroxycinnamic acids and alcohols, and these latter interconnected in a lignin-like manner. In this view, suberin would be an aliphatic-aromatic heteropolymer.8 The picture for the suberin macromolecule has changed in the last few years, with the accumulated evidence that glycerol plays a major role in its structure and that aliphatics and aromatics are distinct polymers occupying different domains in the suberized cell walls. Glycerol, though found in early studies of suberized tissues,9,10 was ignored for a long time. We now know that glycerol is a major monomer released after suberin depolymerization11 and, though few cases have been analyzed, also from cutins.12-14 * To whom correspondence may be addressed: E-mail:
[email protected].
In two suberins, glycerol has been found esterified as monoacylglycerol to most of the aliphatic acids known as monomeric units in these suberins.15,16 These monoacylglycerols resulted from an incomplete depolymerization of suberins through methanolysis using calcium oxide as catalyst.15,16 CaO is a strong base insoluble in alcohols, and we speculate that the methanolysis reaction is due to a superficial catalytic action, yielding reaction products still bearing ester linkages. On the other hand, methanolysis with a soluble strong base, like sodium methoxide, leads to complete depolymerization, even in low concentrations and relative short times, being one of the preferred techniques for the analysis of the monomeric composition of suberins.11 Besides commercial cork, the most studied suberin is the one from the potato tuber periderm. The reasons are the huge importance of the potato as a food stuff, the relatively high proportion of suberin in the potato peel (ca. 20% in extractive-free basis), and the easiness in getting suberized tissue by wound-healing. We applied to potato periderm a methanolysis procedure to achieve partial depolymerization of its suberin, using as catalyst calcium hydroxide instead of calcium oxide, with better results regarding the obtention of oligomeric fragments. With this partial depolymerization of potato suberin we obtained monomers, monoacylglycerols, and diglycerol esters. The former, monomers and monoacylglycerols, are discussed elsewhere.17 Here we report the finding of diglycerol esters in the potato periderm suberin, by the esterification of glycerol at both ends of a R,ω-diacid. These results give support to the hypothesis that glycerol and R,ωdiacids can be continuing pieces of the macromolecular development of the suberin polymer. Materials and Methods. Partial Methanolysis. Potato periderm, 0.25-0.42 mm, dry, extractive-free, was mixed with Ca(OH)2 (2:1, w/w) and refluxed in methanol for 1 h. After filtering, aliquots from the methanolysate filtrates were dried, derivatized with BSTFA, and analyzed by GC-EIMS. Synthesis of the Diglycerol Alkenedioates. Octadec-9en-1,18-dioic acid dimethyl ester was isolated from the NaOCH3-methanolysis products of the potato periderm by preparative silica TLC. R,ω-Diacid dimethyl esters were first separated from the other monomer classes, and the octadec9-en-1,18-dioic acid dimethyl ester was isolated from the saturated counterparts by silver ion impregnated silica TLC. After hydrolysis to obtain the free octadec-9-en-1,18-dioic acid, the position of the double bond was confirmed by the EIMS of the picolinyl and DMOX derivatives. The synthesis of the diglycerol alkenedioates was made by the DCC-activation technique, with DMAP as catalyst.18
10.1021/bm005556t CCC: $19.00 © 2000 American Chemical Society Published on Web 09/19/2000
520
Biomacromolecules, Vol. 1, No. 4, 2000
Communications
Figure 1. High-mass part of the EIMS of 1,1′-diglycerol-octadec-9-en-1,18-dioate, tetrakis(trimethylsilyl) ether, obtained from the partial methanolysis of the potato periderm suberin.
A 6.8 mg (0.022 mmol) sample of octadec-9-en-1,18-dioic acid was dissolved in 1 mL of dimethylformamide, and 250 µL of a dichloromethane solution of 4-(dimethylamino)pyridine (DMAP) (0.5 mmol) and 100 µL of glycerol (1.4 mmol) were added. To the above stirred solution at 0 °C was added 250 µL of a dichloromethane solution of N,N′dicyclohexylcarbodiimide (DCC) (0.11 mmol), and the reaction mixture was allowed to reach ambient temperature. After 24 h of reaction, the solution was concentrated under reduced pressure close to dryness, 1 mL of chloroform was added, and the reaction was successively washed with 2 × 1 mL of 0.5 N HCl/H2O, 2 × 1 mL of a saturated solution of NaHCO3/H2O, and 1 mL of H2O. From the chloroform solution, aliquots were taken to GC-EIMS and ESI-MS/ MS analysis. ESI-MS/MS analysis was carried out in triple quadropole (Micromass Quattro LC) in the negative ion mode. The solution for electrospray ionization was prepared by adding 10 µL of the organic phase-recovered synthesis mixture to 1 mL of 0.2% NH4OH/MeOH. CID MS/MS of the ion of m/z 459, the quasi-molecular ion attributed to the diglyceroloctadec-9-en-1,18-dioates, was made with argon at a pressure of 0.5 bar, with 50 V in the collision cell. Methanolysis of Model Compounds. The Ca(OH)2catalyzed methanolysis above-described was applied under the same conditions separately to 1-monostearoylglycerol and 2-monopalmitoylglycerol and to the following mixtures: 1-monostearoylglycerol, hexadecane-1,16-dioic acid, and eicosane-1,20-dioic acid dimethyl ester; glycerol, hexadecane-1,16-dioic acid, and eicosane-1,20-dioic acid dimethyl ester. After methanolysis, aliquots of the reaction mixtures were prepared for analysis by GC-EIMS as described above. For further details on the experimental procedures see ref 17. Results and Discussion. The Ca(OH)2-catalyzed methanolysis solubilized approximately 2.5% of the potato periderm material, corresponding to 10% of its “suberin” content (materials removed by a NaOCH3 methanolysis). In the identified compounds ca. 50% was glycerol, 35% long-chain monomers, 14% monoacylglycerols and feruloyl esters of the suberin acids,17 and 1% the diglycerol esters we discuss here.
Three peaks in the GC-EIMS runs of the Ca(OH)2 methanolysates of the potato periderm were identified as the diglycerol esters of the octadec-9-en-1,18-dioic acid. This acid is the main monomer in the potato periderm suberin (ca. 40% of monomers excluding glycerol). In the three compounds, glycerol was found linked in its middle and terminal hydroxyl positions: 1,1′-diglycerol-octadec-9-en1,18-dioate (70% of the identified compounds); 1,2′-diglycerol-octadec-9-en-1,18-dioate (29%); 2,2′-diglycerol-octadec9-en-1,18-dioate (1%). Identification of these compounds was made through their electron-impact mass spectra (of their TMS derivatives), and confirmed after their synthesis. The mass spectra of the 1,1′- and 2,2′-diglycerol alkenedioates showed a fragmentation pattern similar to the respectively corresponding 1-monoacylglycerols and 2-monoacylglycerols of R,ω-diacids,15,16 whereas the 1,2′-isomer showed combined characteristics. In the high-mass part of the spectra (Figure 1), the M - 15 ion was used to assign the molecular mass. The M - 73 and M - 90 ions are commonly associated with trimethylsilylated compounds. The M - 103 ion is dominant in the high-mass region of the spectrum of the 1,1′-diglycerol alkenedioate, comparatively smaller in the 1,2′-isomer and practically absent from the 2,2′-isomer. This M - 103 ion, due to the cleavage between C-2 and C-3 in the glycerol moiety, is typical of the mass spectra of TMS derivatives of 1-monoacylglycerols and is absent from the isomeric 2-monoacylglycerols, allowing distinction between them.15,16 The ion at m/z 499 (M - 103-146) follows the same pattern of the ion at M 103, so we presume it originates from its secondary fragmentation. The ion at m/z 513, resulting from the loss of one of the glycerol moieties, is present in the spectra of the three isomeric diglycerol esters. Ions of the low-mass part of the spectra also reflect the substitution in glycerol at C-1, C-2, or both. The relative proportion of the ions at m/z 218 and 219, which come from the glycerol moieties, are different in the three diglycerol esters: in the 1,1′-isomer the m/z 219 dominates; in the 2,2′isomer m/z 218 dominates over the m/z 219; in the 1,2′isomer their abundance is closer. Also, the ion at m/z 191, which is mainly formed by the 2-monoacylglycerols,15,16 is
Communications
Biomacromolecules, Vol. 1, No. 4, 2000 521
Figure 2. CID MS/MS of the ion of m/z 459, obtained by negative-ion electrospray of the synthesized diglycerol-octadec-9-en-1,18-dioates.
present in the 2,2′-isomer, comparatively smaller in the 1,2′isomer, and practically absent in the 1,1′-isomer. To corroborate the interpretation made of the EIMS of the diglycerol alkenedioates, they were synthesized and the mass spectra shown to be identical. The octadec-9-en-1,18dioic acid was obtained from the potato periderm NaOCH3methanolysis products, and the position of the double bond confirmed. The cis/trans isomerism about the double bond was not clarified, but the cis isomer seems to be at least dominant.17 In the synthesis procedure, glycerol was mixed with the octadec-9-en-1,18-dioic acid, intended to obtain esterification in both the 1- and 2-positions of glycerol. A large excess of glycerol was used to avoid esterification in more than one position in each glycerol molecule. After 6 h of reaction all the octadec-9-en-1,18-dioic acid was consumed, and the monoacylglycerol of the acid predominated in the mixture. After overnight reaction all the monoacylglycerol was consumed, and the diglycerol esters were formed. The analysis of the reaction products by GC-MS, after TMS derivatization, showed two peaks of EIMS equal to the ones interpreted as the TMS derivatives of 1,1′-diglycerol-octadec9-en-1,18-dioate (84%) and 1,2′-diglycerol-octadec-9-en1,18-dioate (16%). Only vestigial quantities of the 2,2′diglycerol-octadec-9-en-1,18-dioate were obtained. This synthesis reaction mixture was also analyzed by electrospray mass spectrometry (ESI-MS/MS). In the negative ion-mode, a quasi-molecular ion attributable to the diglycerol octadec-9-en-1,18-dioates was obtained. MS/MS fragmentation of this ion by low-energy CID produced a spectrum where the main parts of the diglycerol octadec-9en-1,18-dioates molecule are recognizable, namely fragment ions associated with the glycerol and acyl moieties (Figure 2). The same Ca(OH)2-catalyzed methanolysis was applied to model monoacylglycerols to confirm the partial methanolysis observed in the potato periderm suberin. About onethird of the initial monacylglycerol esters remained after the reaction in this methanolysis conditions. However, interesterification of the acyl part of the monoacylglycerols was also observed, particularly migration from the 2- to the 1-position of glycerol. Also, the possibility that glycerol esters could arise from a de novo synthesis, based on glycerol
and the free acids or their methyl esters present in the solution, was also checked and shown not to occur. Therefore, this Ca(OH)2 methanolysis was shown to be suited for studies where only partial depolymerization of ester-linked macromolecules is intended. The finding here reported of a R,ω-diacid esterified on both its terminal carboxylic acid positions with glycerol shows that these two types of suberin monomers can be the basis for the growing of the macromolecular structure of suberin, by their successive interlinkages. The glycerol esters of the R,ω-diacids are therefore trimeric building units of the suberin polymer. Glycerol, with its three esterifying positions, can continue the polymer in several planes and to opposite directions. In these trimeric units, glycerol was found to be esterifying the acid both in its terminal hydroxyls, 1-position, and “middle” hydroxyl, 2-position. However, care has to be taken regarding the interpretation of their relative proportions, since migration under analytic conditions from one position to the other cannot be excluded, as discussed above. The availability of hydroxyls from glycerol to esterify the aliphatic carboxylic acid groups seems to be sufficient. Calculations based in the monomeric composition show that in cork suberin the molar proportion of these two groups is close to one, and in the cases of Pseudotsuga bark16 and potato periderm suberin17 the number of glyceridic hydroxyls exceeds the acid groups. For the development of the polymer in the manner discussed above, high proportions of R,ωdiacids will be necessary. In the case of potato periderm suberin R,ω-diacids are the major class of monomers present (55% of the long-chain acids), as is the case in many of the suberins studied, only exceeded sometimes by the proportion of ω-hydroxyacids.2,3 The interlinking of ω-hydroxyacids is also a possibility for the development of a linear polymer. A dimer of the latter case, constituted by the linear interesterification of a C16 ω-hydroxyacid, was actually found in the tomato peel cutin.19 This kind of structure can be more relevant in the case of cutins, where ω-hydroxyacids are frequently dominant, or in the suberins where the same applies. The two types of structures, glycerol-R,ω-diacid-glycerol and ω-hydroxyacidω-hydroxyacid, can coexist in different proportions in suberins and cutins, blurring the frontier between them.
522
Biomacromolecules, Vol. 1, No. 4, 2000
The role of ω-hydroxyacids in suberins can still be another. They can provide the esterifying hydroxyls to the hydroxycinnamic acids, namely ferulic acid, and, through this latter, bridge the glyceryl-aliphatic suberin to the surrounding polymeric aromatics. In three different suberins, including the one here of potato periderm,17 ω-hydroxyacids were found both as glyceryl esters through its carboxylic acid group and as feruloyl esters through its primary hydroxyl.16,20 Several studies have been made by 13C solid-state NMR directly on suberized and cutinized tissue, after enzymatic treatments that remove part of the polysaccharides present.21-24 The analysis of the intact polymers has shown carbons with chemical vicinities consistent with the presence of the glyceryl ester linkages, although that has not been the interpretation of the authors. They have found carbons from primary- and secondary-alcohol ester groups, corresponding with the esterification on the primary and secondary positions of glycerol. Also the NMR spectra evidenced the presence of “short” and “long” alkyl chains with different mobility,21,23 eventually imputable to glycerol and long-chain aliphatics, respectively. Together these results show that suberin can be a polyglyceryl ester structure. The role of glycerol can be, as the finding of the diglycerol alkenedioate trimer supports, the cross-linking monomer of the long-chain aliphatic acids. Because glycerol moieties linked by a R,ω-diacid still have four remaining linking positions, they can be used either to repeat this structure or to esterify ω-hydroxyacids. These latter, in turn, can continue the linear extension of the polymer, through esterification to another ω-hydroxyacid, or end up the aliphatic structure, through esterification to ferulic acid. The fact that suberins and cutins can be acylglycerol-based polyesters will help to understand the protecting properties of the cells and tissues where they are important constituents, since acylglycerols are known to have hydrophobic and insulating properties, namely the triacylglycerols in animal tissues. We also know that acylglycerols and their derivatives, like glycosylacylglycerols, phosphoglycerides or associated with proteins, are the structural elements of both plant and animal biomembranes.25,26 The existence of acylglycerols in the form of a poly(acylglycerol) polyester polymer in the outermost cells of plants, as we propose here, will still widen the vital role of this group of lipids as barrier structures in living bodies. Acknowledgment. Thanks are due to Kevin Thurlow (Laboratory of the Government Chemist, U.K.) for the nomenclature of the diglycerol trimers, and to Isabel Baptista for technical assistance. Work supported by project Praxis 3/3.2/FLOR/2104/95, and Centro de Estudos Florestais, under funding from Fundac¸ a˜o para a Cieˆncia e Tecnologia, Portugal. References and Notes (1) Kolattukudy, P. Cutin, suberin, and waxes. In The biochemistry of plants 4. Lipids; Stumpf, P., Ed.; Academic Press: New York, 1980; Chapter 18.
Communications (2) Holloway, P. Some variation in the composition of suberin from cork layers of higher plants. Phytochemistry 1983, 22, 495-502. (3) Kolattukudy, P.; Espelie, K. Chemistry, biochemistry, and function of suberin and associated waxes. In Natural products of woody plants I; Rowe, J., Ed.; Springer-Verlag: Berlin, 1989; Chapter 6.4. (4) Holloway, P. The chemical constitution of plant cutins. In The plant cuticle, Cutler, D., Alvin, K., Price, Ed.; Academic Press: London, 1982; pp 45-85. (5) Holloway, P. Plant cuticles: physicochemical characteristics and biosynthesis. In Air pollutants and the leaf cuticles; Percy, K., Cape, J., Jagels, R., Simpson, C., Ed.; NATO ASI Series, Series G, Vol. 36; Springer-Verlag: Berlin, 1994; pp 1-13. (6) Rodriguez-M.; B.; Ribas-M.; I. Contribuicion a la estrutura quı´mica de la suberina. An. Quim. 1972, 68B, 1301-1306. (7) Agullo, C.; Seoane, E. Hidrogenolisis de la suberina del corcho con LiBH4; grupos carboxilo libres. An. Quim. 1982, 78C, 389-393. (8) Kolattukudy, P. Lipid polymers and associated phenols, their chemistry, biosynthesis, and role in pathogenesis. Recent AdV. Phytochem. 1977, 77, 185-246. (9) Ku¨gler, K. U ¨ ber den Kork von Quercus suber. Arch. Pharm. (Weinheim, Ger.) 1884, 22, 217-230. (10) Ribas, I.; Blasco, E. Investigaciones sobre el corcho. I. Sobre la existencia de glicerina. An. R. Soc. Esp. Fis. Quim. 1940, 36B, 141147. (11) Grac¸ a, J.; Pereira, H. Methanolysis of bark suberins: analysis of glycerol and acid monomers. Phytochem. Anal. 2000, 11, 45-51. (12) Carvalho, J. Extractos de metano´lise das cortic¸ as. Caracterizac¸ a˜o de alguns componentes em cromatografia em camada fina e espectrofotometria. SilVa Lusit. 1993, 1, 113-122. (13) Moire, L.; Schmutz, A.; Buchala, A.; Yan, B.; Stark, R.; Ryser, U. Glycerol is a suberin monomer. New experimental evidence for an old hypothesis. Plant Physiol. 1999, 119, 1137-1146. (14) Grac¸ a, J.; Pereira, H. Unpublished results. (15) Grac¸ a, J.; Pereira, H. Cork suberin: a glyceryl based polyester. Holzforschung 1997, 51, 225-234. (16) Grac¸ a, J.; Pereira, H. Glyceryl-acyl and aryl-acyl dimers in Pseudotsuga menziesii bark suberin. Holzforschung 1999, 53, 397-402. (17) Grac¸ a, J.; Pereira, H. J. Agric. Food Chem., in press. (18) Neises, B.; Steglich, W. Simple method for the esterification of carboxylic acids. Angew. Chem., Int. Ed. Engl. 1978, 17, 522-524. (19) Osman, S.; Gerard, H.; Fett, W.; Moreau, R.; Dudley, R. Method for the production and characterization of tomato cutin oligomers. J. Agric. Food Chem. 1995, 43, 2134-2137. (20) Grac¸ a, J.; Pereira, H. Feruloyl esters of ω-hydroxyacids in cork suberin. J. Wood. Chem. Technol. 1998, 18, 207-217. (21) Garbow, J.; Ferrantello, L.; Stark, R. 13C Nuclear Magnetic Resonance study of suberized potato cell wall. Plant Physiol. 1989, 90, 783787. (22) Stark, R.; Sohn, W.; Pacchiano Jr., R.; Al-bashir, M.; Garbow, J. Following suberization in potato wound periderm by histochemical and solid-state 13C nuclear magnetic resonance methods. Plant Physiol. 1994, 104, 527-533 and references within. (23) Gil, A.; Lopes, M.; Rocha, J.; Neto, C. A 13C solid-state nuclear magnetic resonance spectroscopic study of cork cell wall structure: the efect of suberin removal. Int. J. Biol. Macromol. 1997, 20, 293305. (24) Yan, B.; Stark, R. A WISE NMR approach to hetereogenous biopolymer mixtures: dynamics and domains in wounded potato tissues. Macromolecules 1998, 31, 2600-2605. (25) Gennis, R. Biomembranes. Molecular structure and function; SpringerVerlag: New York, 1989. (26) Murphy, D. Plant lipids. Their metabolism, function, and utilization. In Plant biochemistry and molecular biology; Lea, P., Leegood, R., Ed.; John Wiley and Sons: Chichester, England, 1993; Chapter 5.
BM005556T
