versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.50 n.4 Concepción dic. 2005
J. Chil. Chem. Soc., 50, N° 4 (2005), págs: 719-724
SYNTHESIS AND CHARACTERIZATION OF NEW CHITOSAN-O-ETHYL PHOSPHONATE.
GLORIA PALMA1, PEDRO CASALS2 AND GALO CARDENAS1*
1Departamento de Polímeros, Facultad de Ciencias Químicas, Edmundo Larenas 129, Universidad de Concepción, Concepción (Chile).
The chitin is a natural polymer that is extracted commercially from the shells of crustaceans generated as raw material from the fishing industry. Their chemical structure is constituted by residual units of N-acetyl glucosamine linked by b (1 ® 4) and its derivatives can be obtained from alkaline chitosan.
The chitin, chitosan and their derivatives present very good perspectives to be used in agriculture. These derivatives are biodegradable and they exhibit fungicides, germicides, nemacides properties and natural defensive mechanisms of the plants.
The chlorophosphonic-2-acid (Ethephon®) is a commercial product employed for the early maturation of fruits.
The objective of the present work is the synthesis and characterization of chitosan-O-ethyl phosphonate chitosan to obtain a controlled released system with potential plant growth regulation properties. Alkaline chitosan was obtained; and then reacted with the 2-chlorophosphonic acid.
The synthesis of new chitosan derivatives and its complete characterization by FT-IR, 13C, 1H and 31P NMR is described and gas chromatographic, the effects on field blueberries are also tested. A chitosan 99 % deacetylated and Mv = 89,000 g/mol is prepared. The O-(ethyl phosphonic) chitosan (QOEP) with a degree of substitution of 58% is obtained.
Keywords: chitosan, chitosan-O-derivative, NMR, biopolymer.
Chitosan is chemically built up of 2-amine-2-deoxy-D-glucopyranose units linked b (1®4). This biopolymer can be partially acetylated but the extent of the acetylation which is usually lower than 50% of its amine groups. For industrial applications chitosan may be obtained by chemical deacetylation of chitin isolated from crustacean shells (1).
In recent years much attention had been paid to this polymer regarding its biodegradability and biocompatibility. Furthermore, chitosan possesses reactive -OH and -NH2 which can be derivatized (2).
There are two methods available to prepare biocides chemically bonded. In one methodology the active part of the biocide is bonded to a preformed polymer, natural or synthetic, to prepare a new derivative (3). See scheme 1.
The polymer coating of the pesticide or the binding of the agrochemical to the polymer support slow down the release of the pesticide into the environment, thus increasing its residual effect and reducing contamination.
The advantages of a slow controlled released system (SCR) for agriculture as reported by Kenawy (3,4) are :
The first reports on slow released pesticides using chitin were carried out by Mc Cormick (5). They prepared chitin covalently bonded to the commercial herbicide metribuzin, previously functionalized. This system demonstrated the slow release of the active principle.
The blueberries is a fruit, mainly oriented to exportation, being one of the most profitable fruit crop in Chile. It has grown in the last few years to cover about 5000 hectars harvested as a counter season fruit with respect to the northern hemisphere yielding over 6500 tons of berries per hectar. Ninety percent of the production is exported to the United States with a market value of 180 million dollars. The best price for exports are obtained in October and March (6). The early rippening fruit obtains the higher prices in the import markets therefore the use of a growth regulator such as the chlorophosponic-2-acid is relevant in accelerating the market readiness of the fruit.
Part of the success of a growth regulator is its ability to be absorbed and translocated by the plant and decomposed as in the case of EthephoneR to ethylene (7,8).
The objective of the present study is the synthesis and characterization of new chitosan derivatives bearing moiety in their structure. The new derivative is Chitosan-O-Ethyl Phosphonate with growth regulator properties.
Chitosan was obtained from chitin separated from shrimps shells using conventional methods. The chitin was poured in a round bottomed flask of 1 L with 50% NaOH in a ratio 1:20 (p/v).
The mixture was heated for 4 h at 80ºC, then filtered and washed. The molecular weight of chitosan was determined by viscometer method. From Mark-Houwink equation and values of [h] obtained in static experiment, it is possible to determine the viscosimetric average molecular weight Mv. The Mark-Houwink parameters K = 0.074 and a= 0.76 used were determined by M. Rinaudo (9) et al. for the solvent AcONa 0.2 M/AcOH 0.3 M . The intrinsic viscosity [h] of chitosan samples was measured with an Ostwald viscometer at 25 ± 0.05ºC (10,11). A Mv = 89.000 g/mol was obtained.
Degree of deacetylation
Characterization of the degree of deacetylation was made by potentiometric titration and also by 1H NMR A chitosan 99% deacetylated was prepared.
Synthesis of chitosan-O-ethyl phosphonate.
The obtention of chitosan-O-ethyl phosphonate was prepared using alkaline chitosan. To 5g. of chitosan, 100mL of KOH-methanol (50%) were added. The mixture is agitated during 4 h at reduced pressure then is filtered and dried (12).
To a round bottomed glass of 250mL containing 40mL of methanol and 1g. of dry chitosan the 2-chloro ethyl phosphonic acid is added and then stirred for 12hrs, then filtered and washed with methanol to eliminate the excess of reagent, it is washed again and left it at 25°C. Finally, it is dissolved in distilled water until the pH reaches at 5 and the solution is dialyzed for 72hrs, using Amicon® membranes with porous size of 1000 Da. After filtration the solid was liophylized.
Infrared spectra were measured in a Nicolet Magna 5PC FT-IR spectrophotometer coupled to a PC with OMNIC analysis software. Pellets were prepared by blending the polymer with KBr at 2% concentration. Spectra were recorded at a resolution of 4 cm-1 and 64 scans were accumulated.
1H, 13C, 31P NMR Spectroscopy.
The spectra were recorded on an AC 300 Bruker spectrometer equipped with a process controller, an ASPECT 3000 computer and a variable temperature system. The temperature was 335 K. For NMR measurements, the sample was dissolved in D2O acidified with HCl, freeze dried to displace adsorbed moisture, and then dissolved in the same solvent. The sample concentration was 10 mg/mL in D2O (99.9%). 13C NMR were carried out in a Bruker spectrometer with 400 MHz and the DEPT 135 with a polymer concentration of 100 mg/ml, dissolved in D2O (99,9%) acidulated with HCl. 31P NMR was carried out in a Bruker 250 MHz multinuclear ( 13,14).
Using bottles of 1 mL, 2-chloro ethyl phosphonic acid and chitosan-O-ethyl phosphonate were added, dissolved in 300 µL of acetone (99.9%) distilled. To each bottle 70 µL of KOH 30% were added in deionized water and closing them with a teflon/silicone septum. The bottles were shacked at 150 rpm for 3 h. then cooled at room temperature. The gas samples of 100 µL were obtained from space injection and then injected in a gas chromatography HNU 321 dissembled to an Spectra Physics SP 4292 with a flow of with a FID detector at 150°C. The ethylene content is obtained from the ratio of the standard peak area and the peak are of the sample (15-17).
In the biological evaluation a blueberries field was chosen in Parral, VII Region of Chile.
The experiment was designed in random blocks with five repetitions of two Blueberries plants cv. Elliot which were sprayed with a manual mechanical back pump, harvesting and measuring fruit diameter, fruit weight and fruits per tree at the bluestage phenology level. The soluble solids of the fruits were also recorded. For normalization, the data was transformed to ÷x previous to the Analysis of Variance and means were compared with LSD test at 95% probability level (18). After harvest the soluble solids of the berries were registered with a refractometer. Cytokinin was included in the study as a compound that promotes cytokinesis to be compared with the effect of chitosan and chitosan derivative.
RESULTS AND DISCUSSION
Table 1 summarizes the different chitosan/ethephon ratios and different substitution degree.
When the chitosan/ethephon ratio is 1:1 a substitution degree of 58 % was obtained.
In figure 1 (a,b) the FTIR spectra of chitosan and its derivatives were shown a decrease in the absorption intensity at 3217 cm-1 which corresponds to the v-OH, due to the substitution of the -OH groups. Other absorption appears at 1634 cm-1 and 1542 cm-1 deformation NH3+. The typical bands of phosphonated derivatives are those that appears at 1204cm-1 d (P=O), d asym 1168 cm-1 (P-O-P), v at 914cm-1 (P-O) and d sym at 798 cm-1 (P-O-C) (19). See figure 1.
NMR spectroscopy analysis
The 1H NMR of Q-O-EP shows a -CH3 signal near to a multiplet centered at 2.3 ppm, which corresponds to methylene groups bonded directly to phosphorous. The presence of the multiplet could be due to the coupling of methylene groups with 31P. Other possibility is that the widening of the signals is due to the substitution, in different positions (C-3 or C-6), of the ring. With exception of the anomeric proton (H-1) at 4.75 ppm and H-2 at 3.01 ppm, all the others (H-3,4,5,6) appears from 3.6-4.0 ppm (20). A small amount of CH3- from acetyl at 1.9 ppm is observed. See figure 2.
The glucopyranosic carbons showed more intense band. According to the signal amount and intensities the substitution must occur at C-3. Table 1 shows the chemical shift of the carbon atoms of Q-O-EP obtained from the 13C NMR spectra where C-7 and C-9 appear as a single signal. The signal shown at 39.1 and 41.2 ppm corresponds to carbons bonded to phosphorous. Besides a shift is observed due to the phosphorous presence (21). See figure 3
In the 13C NMR DEPT 135 it is observed in fig. 4 that the inverted signals correspond to the methylene derivative, confirming the five -CH2- in the structure. However, it was not possible to assign which of them is substituted in C-3 or C-6.
To confirm the presence of phosphorous a 31P NMR was carried out. A signal corresponding to the phosphonic groups is shown at 17.9 ppm in figure 5.
The ethephon is a grow plant regulator with systemic properties, penetrating in the tissues decomposing to ethylene and translocate progressively. The 2-chloro ethyl phosphonic is stable in aqueous solution with pH lower than 3.5 and the ethylene is liberated at higher pH.
A new method has been developed for the routine analysis of Ethephon at pH 12-14, avoiding the derivatization (22). Figure 6 shows the amount of ethylene liberated according to the degree of substitution. According to these results there is a progressive ethylene liberation of the derivative that can be used in a SRC.
In figures 7 to 9 it is observed that all treatments were significantly superior(<0.05) to the untreated check when comparing the yield components, fruits per tree, fruit weight, solid soluble and fruit diameter. The addition of ethephon to chitosan increase significantly all yield components with the exception of fruit weight (fig.7) where the effects of 12 L x ha-1 of chitosan and 480 g x ha-1 of chitosan + ethephon were statistically similar (<0.05).
It is interesting to note the similarity of results of chitosan and cytokinin indicating a certain auxinic action of chitosan which is potentiated by the addition of the growth regulator ethephon to elicit not only an accelerated pre-harvest ripening but also a stimulation of the yield components, probably because of the N release of chitosan and the biocidal effect of the product on other harmful organisms to the plant (23).
The chitosan and derivative versus the applied doses in blueberries are summarized in Table 3. Three solutions of 2 % for chitosan, 1 % for OQDEF and 0.1 % for cytokinin were used in the experiment.
The coefficient of variation of each measured parameter is shown in table 4. The lowest source of variability comes from the treatment of chitosan + ethephon when considering the diameter of fruits indicating a good regulatory effect in the cell growth and development of the fruit. The spread of variability distribution is also the smallest when all treatments are compared. The presence of chitin + ethephon counteracts in some way the effect of environmental variability that is shown in other treatments. The soluble solids of blueberries Elliot variety was compared among chitosan, chitosan-OEPA and cytokinin.
The chitosan-OEPA shows the higher value (16º Brix) compared with cytokinin (12ºBrix) being more effective in the mature and sugar content of the fruits.
The substitution of Ethephon occurs at C-3 and C-6 . The chitosan-O-ethyl phosphonate liberates ethylene according to the degree of substitution in the macromolecules.
It is possible to incorporate a growth regulator in a biodegradable polymer such as chitosan to produce a slow release of ethylene. The incorporation of QOEP derivative produces an increase in the blueberries production, diameter and soluble solids, being a potential commercial product.
The new chitosan derivative is effective in the increase of blueberries yields and soluble solids.
The authors would like to thank the financial supports of Innova Bío-Bío FIT B1-050.
We also thank the Laboratories of the Facultad de Ciencias Químicas, Universidad de Concepción and Dirección de Investigación.
1. S. Hirano, Chitin and Chitosan, Ed. G. Skjak-Braek, T. Anthonsen and P.Sandford, Elsevier Applied Science Publishers, London, 1989, 37-43. [ Links ]
2. R. Muzzarelli, Chitin Pergamon Pres Ltda, Great Britain 1197, 1-5.
3. E. Kenawy, M. Sakran, Ind. Eng Chem. Res 1996, 10, 3726-3733. [ Links ]
4. E. Kenawy, D. Sherrington, Eur. Polymer J 1992, 28 (8) 841-862. [ Links ]
5. C. L. Mc Cormick, K. W. Anderson, B. H. Hutchison, J. M. Rev. Macromol. Chem. Phys., 1982-83 c 22 (1), 57-97. [ Links ]
6. Fundación Chile, Diario Pymes, Difusión del cultivo de Arándanos. [ Links ]
7. Royal Society of Chemistry, The Agrochemical Hand book 1987, 502. [ Links ]
8. J. Hurier, M. Mnager, B. Zimmerli, J. Agric. Food Chem. 1978, 25 (2), 475-475. [ Links ]
9. J. Brugnerotto, J. Desbrieres, L. Heux, K. Mazeau, M. Rinaudo, Macromol. Symp. 2001, 168,1-20. [ Links ]
10. D. Le Dung, M. Milas, M. Rinaudo, J. Desbrieres, Carbohydr. Polym., 1994, 24, 209-214. [ Links ]
11. i) G. Cárdenas, C. Bernal, J. Retamal, Bol. Soc. Chil. Quím. 1991, 36, 239, 242. [ Links ]
ii) G. Cárdenas, C. Bernal, J. Retamal, Bol. Soc. Chil. Quím. 1991, 37, 285 - 289. [ Links ]
12. i) E. Taboada, G. Cabrera, G. Cárdenas, J. Chil. Chem. Soc. 2003, 48, 007-012. [ Links ]
ii) G. Cárdenas, S. P. Miranda, J. Chil. Chem. Soc. 2004, 49, 291-295. [ Links ]
13. G. Cárdenas, G. Cabrera, E. Taboada, S. P. Miranda, J. Appl. Polym. Sci. 2004, 93, 1876-1885. [ Links ]
14. P. R. Rege, L. H. Block, Carbohydrate Research 199, 321, 325-245. [ Links ]
15. K. M. Varum, N. W. Anthosen, H. Grasdalen, Carbohydrate Research 1991, 211, 17 - 23. [ Links ]
16. S.H. Tseng, P. C. Chang, S.S. Chou, Food and Drug Analysis 200, 8 (3), 213 -217. [ Links ]
17. S. Tanner, H. Chanzy, M. Vincendon, J. Roux, F. Gail, Macromolecules, 1990, 23, 3576-3583. [ Links ]
18. C. Taiz, E. Zeiger, Plant Physiology 1991, 473 - 482. [ Links ]
19. R. Silverstein, F. Webster, Spectrometric Identification or Organic Compound 1997, 44 - 293. [ Links ]
20. G. Palma, Marine Chemistry thesis, Catholic University Concepción, Chile 2004. [ Links ]
21. R. Zhbankovc, V. Andrianov, M. Marchewka, J. Mol. Struct. 1997, 436/437, 637. [ Links ]
22. J. B. Stuther, Carbon -13 NMR Spectrometric 1978, 24, 390-480. [ Links ]
23. Methods in Enzymology, Academic Press, Inc., 1988, 161, 442-446. [ Links ]
* Correspondence author: e-mail: email@example.com, phone: 56-42-208943, fax: 56-42-203337.