Structural Characterization of Rubber from Jackfruit and Euphorbia as

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Biomacromolecules 2004, 5, 2013-2019

2013

Structural Characterization of Rubber from Jackfruit and Euphorbia as a Model of Natural Rubber Dararat Mekkriengkrai,† Koiichi Ute,‡ Ewa Swiezewska,§ Tadeusz Chojnacki,§ Yasuyuki Tanaka,† and Jitladda T. Sakdapipanich†,* Department of Chemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400 Thailand, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Putthamonthon, Nakornpathom 73170, Thailand, Division of Chemistry, Department of Material Engineering Science, Faculty of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan, and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warszawa, Poland Received April 29, 2004; Revised Manuscript Received June 18, 2004

A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from HeVea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from HeVea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The R-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal dCH-CH2OP group. Introduction HeVea brasiliensis has been established as practically the only important natural source of rubber termed natural rubber (NR) owing to high yields with desirable physical properties. It is well-known that NR is composed of cis-1,4 polyisoprene. However, the detailed structure of both chain ends has not been clarified. This may be due to the limitation derived from extremely high molecular weight of NR as well as the difficulty of purification to analyze remarkably small amounts of functional groups in NR. Young nascent HeVea leaves and HeVea seedlings were reported to contain low molecular weight rubber enough to analyze the detailed structure.1 However, the 13C NMR analysis of these low molecular weight rubbers showed no dimethylallyl group at the initiating ω end. Naturally occurring low molecular weight rubbers, originated from a mushroom (Lactarius Volemus), sunflower (Helianthus annuus) and goldenrod (Solidago altissima), have been declared as the good models of NR.2,3 However, it was difficult to analyze the original structure of in vivo rubber from mature trees, i.e., HeVea trees due to the modification in lactiferous tubes of virgin mature HeVea trees and also storage latex for several days.4,5 Jackfruit of the family Moraceae (Artocarpus heterophyllus Lam. or Arto* To whom correspondence should be addressed. Phone: 66-28893116. Fax: 66-28893116. E-mail: [email protected]. † Mahidol University and Institute of Science and Technology for Research and Development. ‡ Osaka University. § Institute of Biochemistry and Biophysics.

carpus integrifolia) is a kind of rubber-producing plants widely planted in Thailand. All parts of the tree contain a sticky white latex.6 The rubber from Jackfruit is a good model for structural analysis of NR because of facility to get both low and high molecular weight rubbers from the fruit and trunk. Painted spurge is one species of the family Euphorbiaceae (E. heterophylla), which is widely distributed in Thailand. It has been revealed that Artocarpus integrifolia, Euphorbia hirta, E. splendens, and Ficus religiosa contain cispolyisoprene as in the case of NR.7 It has been reported that the chemical constituents of the rubber from Artocarpus integra latex are composed of 72% resins soluble in acetone and rubber-like residue which is solubilized in petroleum ether and benzene.8 The low molecular weight rubber isolated from Euphorbia lactiflua latex was chlorinated and found to show comparable properties with chlorinated rubber obtained from liquid NR.9 The present work focuses on the structural characterization of low molecular weight rubbers from Jackfruit and a wild grass grown in Thailand, Euphorbia heterophylla as model compounds of NR. Materials and Method Plant Materials. The rubbers used for structural characterization in this work were obtained from HeVea brasiliensis (natural rubber), Artocarpus heterophyllus Lam (Jackfruit), and Euphorbia heterophylla shown as follows: Extraction of Jackfruit Rubber. Jackfruit latex was collected directly into a beaker containing hexane, followed by

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Table 1. Rubber Yield and Molecular Weight of Rubbers from Various Sources GPC rubber source

rubber part

yield (%)

Artocarpus heterophyllus

trunk ripe fruit mature tree young tree mature leaves whole tree

0.65 0.45 30 a 0.72 a

Hevea brasiliensis

Euphorbia heterophylla a

M hw

(×105)

1.77 2.25 8.52 3.00 1.20 2.60

M h n (×105)

M h w/M hn

0.87 1.18 2.43 0.83 0.36 0.73

2.04 1.90 3.50 3.63 3.38 3.57

These values could not estimated.

coagulation and washing with ethanol and acetone to remove impurities soluble in ethanol and acetone. The resulting rubber was purified by precipitation from toluene solution into methanol. The purified rubber was dried in vacuo at room temperature. Extraction of Rubber from HeVea Tree of RRIM 600 Clone. Fresh leaves collected from a mature tree were washed with water, crushed into small pieces by a blender, and extracted with ethanol to remove the ethanol-soluble fraction. Pale leaves were further extracted by acetone in Soxhlet apparatus. The rubber was extracted by toluene and purified by reprecipitation from toluene solution with ethanol for several times. The purified rubber was dried in vacuo. HeVea latex from one-year-old HeVea trees was collected directly into a beaker containing acetone. The precipitated rubber was dissolved in toluene and centrifuged. The toluene-soluble fraction was purified several times by reprecipitation with methanol and dried in vacuo. Fresh latex from a mature HeVea tree was collected in an ice-cooled cup and centrifuged to separate C-serum (CS) and the bottom fraction (BF). The rubber cream fraction was collected and dissolved in a surfactant solution. Rubber solution was cast to make a thin film and dried. Extraction of Euphorbia Rubber. Latex from a wild grass, Euphorbia heterophylla, was collected by cutting the stem and dipping it directly into 1% Triton® X-100 aqueous solution. The solid fraction was dissolved in hexane and centrifuged to remove the hexane-insoluble fraction. The rubber fraction was recovered by precipitation of the hexane solution into ethanol. The resulting rubber was purified by reprecipitation from hexane solution with ethanol and dried in vacuo. Polymer Fractionation. The purified Jackfruit rubber was dissolved in toluene (1% w/v) containing 0.1% w/v 2,6-ditert-butyl-p-cresol as an antioxidant and fractionated into several fractions by successive addition of methanol in the usual way.10 Molecular Weight and Molecular Weight Distribution. Molecular weight (MW) and MWD were determined by GPC using two columns in series, packed with polystyrenedivinylbenzene copolymer gels having exclusion limits of 2.0 × 108 and 4.0 × 105, with THF as an eluent. The chromatogram was recorded at 35 °C, monitoring with a refractive index detector (RI). Samples were prepared at a concentration of 0.01-0.1% g dL-1 in THF and then filtered through a Millipore LS pre-filter and 0.45 µm membrane filter. Standard polyisoprenes purchased from PSS Polymer Standards Service GmbH (Mainz, Germany), were used for preparing a calibration curve.

Particle Size Distribution. Particle size distribution was determined using a Malvern 2000 laser particle size analyzer with Hydro 2000MU machine. The latex was dispersed in distilled water as a dispersant in the range of 10-20% dilution prior to measurement. Structural Analysis. Purified rubbers were subjected to FT-IR and NMR analyses. FT-IR Spectroscopy. The FT-IR spectra were obtained by a JASCO-460 FT-IR spectrometer at a resolution of 4 cm-1 with 100 scans. The rubbers were dissolved in chloroform solution and cast on KBr disc to make a thin film. NMR Spectroscopy. The 1H NMR spectra of rubbers were recorded using a BRUKER NMR spectrometer at 300 MHz with CDCl3 at 50 °C, with pulse repetition time of 1 s. The 1H NMR measurements at 750 MHz were performed with a Varian UNITY INOVA-750 MHz NMR spectrometer using C6D6 as a solvent with TMS as an internal standard at 50 °C, with pulse repetition time of 2 s for 45° pulse, pulse width 6 µs and 2000 scans. The 13C NMR measurements at 188 MHz were carried out at 50 °C, with a pulse repetition time of 10 s, pulse width 5.1 µs and 65 536 scans. The chemical shifts were measured by taking the central signal in C6D6 as an internal reference. The 1H NMR spectra of polyprenol phosphates were obtained by using a JEOL Alpha-500 spectrometer at 500 MHz in CDCl3 solution using TMS as internal standard at 27 °C with a pulse repetition time of 7 s. Results and Discussion Rubber Yield and Molecular Weight. Table 1 shows the content and molecular weight of rubber in Jackfruit latex eluted from the trunk and ripe fruit, compared with those of rubber from HeVea latex obtained from a mature tree and mature leaves, as well as the rubber extracted from E. heterophylla. The rubber content in Jackfruit latex was 0.40.7%, which was very low compared with that of 30-35% in the latex from the HeVea tree. Likewise, it has been reported that the latex of Artocarpus integra Merr. contains 0.94% rubber, whereas no rubber was presented in the latex of Artocarpus elastica Reinw.11 It is interesting to note that the Jackfruit rubber from ripe fruit gave a higher molecular weight than that from the Jackfruit tree. On the other hand, the rubber in HeVea leaves showed a molecular weight lower than that in the latex eluted from the HeVea trunk. The same phenomenon was also observed for trans-polyisoprene obtained from Eucommia ulmoides.12 The relationship between the molecular weight and the part of rubber occurrence

Structural Characterization of Jackfruit Rubber

Figure 1. Molecular weight distribution of rubbers from (A) ripe fruit of Jackfruit, (B) Jackfruit trunk, (C) cream fraction of Hevea latex, (D) cream fraction of Hevea latex of one year-old Hevea tree, (E) leaves of mature Hevea tree, and (F) E. heterophylla.

in the tree may be concerned with the role of rubber in the tree as well as the biosynthesis mechanism of rubber formation. Low molecular weight rubber was also obtained from E. heterophylla with low rubber content. Figure 1 shows the MWD of rubber from different sources. Both Jackfruit rubbers showed a unimodal distribution with h n. This suggests that a narrow polydispersity index, i.e., M h w/M the biosynthesis of rubber proceeds on a limited site of chain elongation using an initiating species. The molecular weight of the resulting rubbers may reflect the supply of isopentenyl diphosphate as a monomer or the activity of rubber transferase as a polymerizing enzyme for chain elongation. It has been revealed that the overexpressed rubber transferase in Escherichia coli catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 × 103 - 1 × 104 Da.13 Both of the rubbers from latex collected from mature and young HeVea trees showed a bimodal MWD with a shoulder peak at low and high molecular weight fractions, respectively. The tendency of increasing the high molecular weight fraction with increasing the rubber tree-age is in accordance with that in the previous report.14 The rubber from leaves of a mature HeVea tree provided low molecular weight rubber with a broad unimodal MWD. The rubber from all parts of the HeVea tree showed a high polydispersity index as compared to that from Jackfruit rubber. The rubber from E. heterophylla showed a unimodal MWD with a wide polydispersity index, which differs from the typically bimodal distribution of HeVea rubber.15 The peak-top of Jackfruit and Euphorbia rubbers fell between the values of the high and low molecular weight peaks in the MWD of cream rubber at ca. 4.0 × 105. This MWD is similar to that of the rubber recovered from small rubber particles in skim after centrifugation of fresh latex. 16 Rubber Particle Size Distribution. Figure 2 shows a typical particle size distribution of latex from E. heterophylla and Jackfruit. The rubber particle in latex from E. heterophylla shows a unimodal distribution ranging from 0.03 to 0.7 µm, with a mean diameter of 0.12 µm. It is noteworthy that the mean particle size of rubber from E. heterophylla is

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Figure 2. Rubber particle size distributions of rubber particles from latex of (A) E. heterophylla, (B) original Jackfruit, and (C) Jackfruit latex after sonication.

Figure 3. 1H NMR spectra at 300 MHz in CDCl3 of rubber extracted from (A) ripe fruit of Jackfruit, (B) Hevea leaves, and (C) E. heterophylla.

equal to that of small rubber particles, 0.13 µm.16 On the other hand, the rubber particles in Jackfruit latex shows two size distributions ranging from 0.5 to 5.8 µm and from 17.4 to 417 µm. The latter is presumed to be formed by aggregation of rubber particles after cutting. As a proof, the particle size distribution changed clearly after sonication of the Jackfruit latex (Figure 2C). Infrared Analysis. The rubbers from Jackfruit, E. heterophylla and HeVea leaves showed the FT-IR bands characteristic of cis-1,4 polyisoprene at 1664 and 835 cm-1, which are due to CdC stretching and C-H bending, respectively. The rubber extracted from Jackfruit and E. heterophylla contained some non-rubber constituents, i.e., nitrogenous and ester compounds as in the case of HeVea rubber. This indicates that rubbers from Jackfruit and E. heterophylla can be applied as model compounds for the analysis of both terminal groups of high molecular weight HeVea rubber.

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Figure 4. 1H NMR spectra at 750 MHz in C6D6 of fractionated Jackfruit rubber. (A) High molecular weight fraction 6th, M h n ) 1.4 × 105 and (B) Low molecular weight fraction 7th, M h n ) 0.7 × 105. Here, the signals a, b, c, d, and e correspond to ω(t), ω-t-t, t-t-c, R, and ω(c), respectively. 1 H NMR Analysis. Figure 3 shows the 300 MHz 1H NMR spectra of rubber obtained from different sources using CDCl3 as a solvent. The rubbers extracted from Jackfruit and E. heterophylla showed three major signals of cispolyisoprene at 5.13, 2.06 and 1.68 ppm, which correspond to the olefinic proton (H-CdC), methylene proton (-CH2CdC) and methyl proton (cis-CH3-CdC), respectively, as compared with the rubber from HeVea leaves. It is remarkable that Jackfruit rubber showed a small triplet signal centered at 4.05 ppm (J ) 6.5 Hz), which was also observed in the 1H NMR spectrum of HeVea rubber and presumed to be derived from the methylene protons linked to a phosphate group (dCH-CH2OP).17 On the other hand, the characteristic methylene proton signal of CH2-OH may show the doublet signal at 4.055 ppm. The Jackfruit rubber was fractionated to obtain low molecular weight fractions of 1.7 × 105 and 9.8 × 104 with narrow MWD and subjected to the analysis of both terminal groups by high-resolution 1H NMR at 750 MHz in d6benzene solution as shown in Figure 4. It was reported that well resolved signal of the methyl protons from cis-1,4 and trans-1,4 units was obtained in the benzene solution, resonating at 1.79 and 1.65 ppm, respectively.18 According to the signal assignment of model compounds,19 the signal at 1.579 ppm (a) is assigned to the methyl proton in the trans configuration of a dimethylallyl group at the ω-terminal. The signal of methyl protons in the cis configuration of the dimethylallyl group was overlapped with the 13C-satellite signal at 1.684 ppm (e) (Figure 4B). By comparison with the 1H NMR spectrum of the rubber from Lactarius mushroom20 and the relative intensities of signals, the signals resonating at 1.621 (b) and 1.640 (c) ppm can be correlated to the methyl-protons of a trans-isoprene unit in ω-transtrans-, trans-trans-cis sequences, respectively. Here, the

Figure 5. 1H NMR spectra at 500 MHz in CDCl3 of (A) Prenol-11 monophosphate and (B) Prenol-18 diphosphate. Here, a and b correspond to dCH-CH2-OP and dCH-CH2-OP, respectively.

trans-isoprene unit in bold face indicates the unit of interest in the sequence of isoprene units. This indicates that Jackfruit rubber contains the dimethylallyl group and two transisoprene units as an initiating species. The methyl-proton signal of a cis-isoprene unit in the R-terminal was presumed to resonate at 1.645 ppm, although there was no information on the structure of the functional group at the R-terminal.

Structural Characterization of Jackfruit Rubber

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Figure 6. 13C NMR spectra at 188 MHz in C6D6 of fractionated Jackfruit rubber from ripe fruit. (A) High molecular weight fraction 6th and (B) Low molecular weight fraction 7th.

Furthermore, both low molecular weight fractions of Jackfruit rubbers showed a triplet signal at 4.0-4.1 ppm. The higher molecular weight fraction showed an asymmetric triplet signal centered at 4.083 ppm, which correspond to two doublets with J ) 7.02 Hz and J ) 6.72 as shown by decoupling from the R-C-2 olefinic proton (dCH) at 5.293 ppm (Figure 4A). This triplet signal was assigned to the R-C-4 methylene proton linked to a phosphate group based on the information from model compounds. This asymmetric triplet signal was also observed in the spectra of prenol-11 monophosphate, prenol-18 monophosphate, and prenol-11 diphosphate resonating at 4.388 (J ) 6∼6.5 Hz), 4.390 (J ) 6.5 Hz), and 4.362 ppm, respectively, observed in CDCl3 solution, as shown in Figure 5. A similar splitting of the R-C-4 methylene proton was observed for geranylgeranyl diphosphate (GGDP), solanesol diphosphate (SDP), trans1,4 polyisoprene from Eucommia ulmoides12 and prenols14, 15, 18 and 19 monophosphates as well as phosphorylated rubber from L. Volemus,16 although the chemical shifts of the latter compounds were slightly shifted downfield due to

the effect of the protecting group for phosphate. This is supporting evidence showing the presence of a phosphate group linked with the Jackfruit rubber at the R-terminal, which provides a triplet signal centered around 4.08 ppm. Otherwise, the lower molecular weight fraction of Jackfruit rubber showed two triplet signals centered at 4.079 (J ) 6.9 Hz) and 3.510 ppm (J ) 6.87 Hz and J ) 6.3 Hz) (Figure 4 B). The former triplet signal is assigned to the same proton as that mentioned above, while the latter is attributed to the methylene proton linked to some functional group in the R-terminal end. At present, there is no direct evidence to verify this functional group. The difference of terminal group between the high and low molecular weight fractions suggests that the termination process of Jackfruit rubber would differ by molecular weight. A similar tendency was observed for the rubbers from young and mature sporophores of Lactarius mushroom.21 13C NMR Analysis. The presence of a dimethylallyl group at the ω-terminal and an R-terminal link-up with a phosphate group in Jackfruit rubber was further confirmed by 13C NMR

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Table 2. Assignment of Characteristic

13C

Mekkriengkrai et al.

NMR Signals

chemical shift (ppm) in C6D6

in CDCl3

carbon atoma

Jackfruit

Jackfruit

prenol-18

R-C-2 ω-C-2 in ω-trans R-C-3 trans-C-1 cis-C-1 cis-C-4 cis-C-5 ω-C-5 trans-C-5 ester-CH3

135.47 125.80 40.32 32.82 27.09 23.75 17.75 16.58 14.43

135.25 125.18 39.81 32.34 26.52 23.40 17.46 16.03 14.05

139.81 131.15 124.35 39.81 32.11 26.95-26.50 23.41 17.66 16.03 -

ω, dimethylallyl group; R, cis-1,4 isoprene unit at the chain end. Carbon atoms in isoprene unit including dimethylallyl group are numbered as follows: a

measurement in C6D6 as shown in Figure 6. The signals due to the C-1 and C-5 methylene carbon atoms of trans-isoprene units in the trans-trans and/or dimethylallyl-trans linkages were detected at 40.24 and 16.58 ppm, respectively. The C-1 methylene carbon atom in the trans-trans linkage is expected to resonate at 39.8 ppm, while that resonating at 40.0 ppm is assignable to the trans C-1 carbon in the cis-trans linkage as reported in the case of a synthetic cis-1,4 polyisoprene.22 However, the chemical shift values of signals in Jackfruit rubber shifted due to the effect of the solvent used. Table 2 shows the assignment of 13C NMR signals observed for Jackfruit rubber and for Prenol-18 in CDCl3. Both signals observed for the lowest molecular weight fraction showed clear splitting, which was assigned to the trans-isoprene unit in dimethylallyl-trans at the downfield region and to that in the trans-trans linkage at the upfield region, for both C-1 methylene and C-5 methyl carbon signals23 (Figure 6). By the intensity ratio of the cis-C-1 and trans-C-1 methylene carbon signals, the number of cis-isoprene units per chain was estimated to be 1810 and 880 for fraction 6 and fraction 7, respectively, which is in agreement with the degrees of polymerization of 2000 and 1070 estimated from GPC measurements by using polyisoprene standards. A small signal was detected at 17.75 ppm, which is assignable to the methyl carbon atom in dimethylallyl group. This clearly indicates that the Jackfruit rubber contains a dimethylallyl group and two trans-isoprene units at the ω-terminal end. However, the C-2 olefinic carbon signal of a dimethylallyl group linked to the trans-isoprene unit, which is expected to resonate at 131.14 ppm, was not detected. The R-terminal of Jackfruit rubber was presumed to be a phosphate group based on the observation of the signal resonating at 64.54 ppm. It is remarkable that the rubber from Jackfruit showed no signal due to the terminal isoprene units having a hydroxyl group and fatty acid group, which are expected to resonate at 59.06 and 60.84 ppm in the case of rubbers from sunflower, goldenrod and Lactarius mushroom.2,23,24 On the other hand, in the 13C NMR spectrum, the lower molecular

weight fraction of Jackfruit rubber showed no signal due to an R-terminal around 64 ppm and also no signal due to longchain fatty acid ester around 14 ppm. This may be explained by assuming that the low molecular weight rubber contains a diphosphate chain end for chain elongation as in the case of small rubber particles in natural rubber,25 which is difficult to determine at the terminal end. Acknowledgment. The present study was supported by a grant (PHD/0160/2543) from Thailand Research Fund (TRF), government of Thailand. References and Notes (1) Tangpakdee, J.; Tanaka, Y. Long-Chain polyprenol and rubber in young leaves of HeVea brasiliensis. Phytochemistry 1998, 48, 447450. (2) Tanaka, Y.; Kawahara, S.; Eng, A. H.; Shiba, K.; Ohya, N. Initiation of biosynthesis in cis-polyisoprene. Phytochemistry 1995, 39, 779784. (3) Tanaka, Y. Structural characterization of naturally occurring cis- and trans-polyisoprenes by 13C NMR spectroscopy. J. Appl. Polym. Sci. Appl. Polym. Symp. 1989, 44, 1-9. (4) Tangpakdee, J.; Tanaka, Y. Why rubber trees produce polyisoprene?A possible role of natural rubber in the HeVea tree. J. Rubber Res. 1998, 1, 77-83. (5) Tangpakdee, J.; Tanaka, Y.; Jacob, J. L.; d’ Auzac, J. Characterization of HeVea brasiliensis rubber from virgin trees: A possible role of cis-polyisoprene in unexploited tree. Rubber Chem. Technol. 1999, 72, 299-307. (6) Morton, J. F. Fruits of Warm Climates. Distributed by Creative Resources Systems, Inc., 1987; pp 58-63. (7) Augustus, G. D. P. S.; Jayabalan, M.; Seiler, G. J. Alternative energy sources from plants of Western Ghats (Tamil Nadu, India). Biomass Bioenergy 2003, 24 (6), 437-444. (8) Tanchico, S. S.; Magpantay, C. R. Analysis and composition of Artocarpus integra latex. Philipp. J. Sci. 1958, 87, 149-157. (9) Gnecco, S.; Pooley A.; Lefimil, C.; Pino, C.; Valenzuela, L. Chlorination of low-molecular weight Euphorbia lactiflua natural rubber. Polymer Bulletin (Berlin) 1997, 39 (5), 605-612. (10) Francuskiewicz, F. Polymer Fractionation; Springer-Verlag: Berlin Heidelberg, Germany, 1994; Chapter 5, pp 39-63. (11) Ultee, A. J. Latexs in which rubber is absent. Bull. Jardin Botan. Buitenzorg 1924, 6, 264-8. (12) Tangpakdee, J.; Tanaka, Y.; Shiba, K.-I.; Kawahara, S.; Sakurai, K.; Suzuki, Y. Structure and biosynthesis of trans-polyisoprene from Eucommia ulmoides. Phytochemistry 1997, 45 (1), 75-80. (13) Asawatreratanakul, K.; Zhang, Y. W.; Wititsuwannakul, D.; Wititsuwannakul, R.; Takahashi, S.; Rattanapittayaporn, A.; Koyama, T. Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from HeVea brasiliensis: A key factor participating in natural rubber biosynthesis. Eur. J. Biochem. 2003, 270, 4671-4680. (14) Tangpakdee, J.; Tanaka, Y.; Wititsuwannakul, R.; Chareonthiphakorn, N. Possible mechanisms controlling molecular weight of rubbers in HeVea brasiliensis. Phytochemistry 1996, 42, 353-355. (15) Subramaniam A. Molecular weight and molecular weight distribution of natural rubber. Rubber Res. Inst. Malaysia Technol. Bull. No. 4, 1980, p 1-25. (16) Sakdapipanich, J. T.; Suksujartporn, S.; Tanaka, Y. Structural characterization of the small rubber particles in fresh latex. J. Rubber Res. 1999, 2 (2), 160-168. (17) Sakdapipanich, J. T.; Mekkriengkrai, D.; Gou, L.; Tanaka, Y. Structural characterization of the terminating groups of some polyprenyl phosphates and of rubber from the Lactarius mushroom J. Rubber Res. 2001, 4 (2), 118-130. (18) Chen, H. Y. Determination of cis-1, 4 and trans-1, 4 contents of polyisoprene by high-resolution nuclear magnetic resonance. Anal. Chem. 1962, 34, 1793-1795. (19) Tanaka, Y.; Hirasawa H. Sequence analysis of polyprenols by 500 MHz 1H NMR spectroscopy. Chem. Phys. Lipid. 1989, 51, 183189. (20) Ohya, N.; Takizawa, J.; Kawahara, S.; Tanaka, Y. Molecular weight distribution of polyisoprene from Lactarius Volemus. Phytochemistry 1998, 48, 781-786.

Structural Characterization of Jackfruit Rubber (21) Ohya, N.; Tanaka, Y. Structural characterization of rubber from Lactarius mushrooms; 1H NMR analysis of terminal groups in the rubber chain. J. Rubber Res. 1998, 1 (2), 84-94. (22) Eng, A. H.; Kawahara, T.; Tanaka, Y. Trans-Isoprene units in natural rubber. Rubber Chem. Technol. 1993, 67, 159-168. (23) Tanaka, Y.; Mori, M.; Ute, K.; Hatada, K. Structure and biosynthesis mechanism of rubber from fungi. Rubber Chem. Technol. 1990, 63, 1-7.

Biomacromolecules, Vol. 5, No. 5, 2004 2019 (24) Tanaka, Y. Structure and biosynthesis mechanism of natural polyisoprene. Prog. Polym. Sci. 1989, 14, 339-371. (25) Ohya, N.; Tanaka, Y.; Wititsuwannakul, R.; Koyama, T. Activity of rubber transferase and rubber particle size in HeVea latex. J. Rubber Res. 2000, 3 (4), 214-221.

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