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SELECTIVE OXIDATION AND N-COUPLING BY PURIFIED LACCASE OF XYLARIA POLYMORPHA MTCC-1100
© 2014 Pankaj Kumar Chaurasia#, Sudha Yadava, Shashi Lata Bharati, and Sunil Kumar Singh
Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur, Uttar-Pradesh, 273009 India Received November 13, 2013; in final form, January 29, 2014
The chemical route of oxidation of methyl group to its aldehyde is inconvenient because once a methyl group is attacked, it is likely to be oxidized to the carboxylic acid and it is very difficult to stop the reaction at the aldehyde stage. Fungal laccases can be used for such oxidation reaction and the reaction can be completed sharply within 1—2 hrs. Coupling of amines are another important reactions known for fungal laccases; coupling reactions generally take 3—7 hrs. We have used the purified laccase of molecular weight 63 kDa obtained from the fungal strain Xylaria polymorpha MTCC-1100 with activity of 1.95 IU/mL for selective oxidation of 2-fluorotoluene, 4-fluorotoluene, and 2-chlorotoluene to 2-fluorobenzaldehyde, 4-fluorobenzaldehyde, and 2-chlorobenzaldehyde, respectively, and syntheses of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines. In each oxidation reactions, ABTS was used as mediator molecule. All the syntheses are ecofriendly and were performed at room temperature.
Keywords: laccase, 2-fluorotoluene, 4-fluorotoluene, 2-chlorotoluene, 3-(3,4-dihydroxyphenyl)-propionic acid derivatives
DOI: 10.7868/S0132342314040022
INTRODUCTION
Laccase [benzenediol: oxygen oxidoreductase; E.C. 1.10.3.2] is a polyphenol oxidase, which belongs to the superfamily of multicopper oxidases [1, 2] and catalyzes [3—5] the four electron reduction of molecular oxygen to water. Laccases are dimeric or tetrameric glycoproteins. To perform their catalytic functions, laccases depend on Cu atoms that are distributed at three different copper centers viz. type-1, or blue copper center, type-2, or normal copper center and type-3, or coupled binuclear copper center. The center types differ in their characteristic electronic paramagnetic resonance (EPR) signals [6, 7]. Organic substrate is oxidized by one electron at the active site of the laccase generating a reaction radical which further reacts non-enzymatically. The electron is received at type-1 Cu and is shuttled to the trinuclear cluster where oxygen is reduced to water.
Ortho and para diphenols, aminophenols, polyphenols, polyamines, lignins, and arylamines and some of the inorganic ions are the substrates for laccases. The ability of laccases to catalyze the oxidation of various phenolic, as well as non-phenolic compounds, coupled to the reduction of molecular oxygen to water makes it valuable from the point of view of their com-
# Author for correspondence (phone: +91 9648082044, +91 8896728759; e-mail: pankaj.chaurasia31@gmail.com).
mercial applications [4, 8—10]. The biotechnological importance of laccases have increased after the discovery that oxidizable reaction substrate range could be further extended in the presence of small readily oxi-dizable molecules called mediators [11, 12]. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses [9, 10] having applications in food, pulps, paper, textile, and cosmetics industries and in synthetic organic chemistry [13-16].
Fungal laccases have been used for the selective oxidation reactions [17-22] and N-coupling reactions [16]. N-coupling reactions have been done by using crude fungal laccases, previously. The objective of this communication was to do the selective oxidations of substituted toluenes, such as 2-fluorotoluene, 4-fluorotol-uene, and 2-chlorotoluene to corresponding 2-fluo-robenzaldehyde, 4-fluorobenzaldehyde, and 2-chlorobenzaldehyde in the presence of ABTS as mediator molecule as well as synthesis of3-(3,4-dihydroxyphe-nyl)-propionic acid derivatives by N-coupling reactions using purified laccase which has not been reported so far for this fungal strain. For this purpose, we used purified laccase from Xylaria polymorpha MTCC-1100 of molecular weight of 63 kDa having activity 1.95 IU/mL, directly as reported by Pankaj et al. [23].
RESULTS AND DISCUSSION
One of the best applications of the laccases in organic synthesis is the selective oxidation of the aromatic methyl group to the corresponding aldehyde. The chemical routes of this conversion are inconvenient because methyl groups are preferably converted into carboxylic acids and it becomes very difficult to stop the reaction at aldehyde stage. Moreover, they require drastic reaction conditions which pollutes the environment. The conversion done with pure laccase occurs under milder conditions, the yield is >90% and the process is ecofriendly. The use of purified laccases for this purpose has been studied [17, 18] in the presence of mediator molecules like 2,2'-azino-è/s-(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) [17]. The potentiality of the pure laccase as a biocatalyst for the conversion of aromatic methyl group to the corresponding aldehyde group in the presence of mediator molecule was tested using 2-fluorotoluene, 4-fluorotoluene, and 2-chlorotoluene as substrates which have not reported so far for this fungal strain (Scheme 1).
CH3
CHO
X
Purified laccase pH 4.5, 1-2 hrs, RT ' (Yield >90%)
X
Y
Y
(i) X = F/Cl, Y = H
(ii) X = H, Y = F
Scheme 1. Syntheses of substituted benzaldehydes from substituted toluenes.
Other important reactions of laccases are the coupling reactions [16]. These coupling reactions have been done by using crude laccase previously. In this communication, synthesis of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives have been done by N-coupling reactions with purified laccase of molecular weight 63 kDa having activity 1.95 IU/mL purified by Pankaj et al. from the liquid culture growth medium ofXylaria polymorpha MTCC-1100 [23] (Scheme 2). All the above synthesized products were characterized and identified by HPLC, IR, and NMR spectroscopy.
COOH
COOH
X—NH
+ X—NH
OH
Purified laccase 2 pH 4.5, 3-6 hrs, RT '
75-90%
OH
X—NH
2
HOOC
nh2
H3COOC
nh2
OH
OH ,NH2
f^T
H3COC^^ and H,C'
nh2
Scheme 2. Syntheses of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives by coupling reactions with purified laccase of X polymorpha MTCC-1100.
UV-Visible Spectroscopy
Completions of the reactions were confirmed by spectrophotometry measurements. In all the cases, reaction solution was firstly measured by UV-visible spectrophotometry at zero time of laccase addition and then measured after constant time interval and changes in UV-spectrum were observed. Fig. 1 presents an example of a UV-spectrum of an oxidation reaction of 2-fluorotoluene to 2-fluorobenzaldehyde by laccase in which time taken for reaction completion was 115 minutes.
High performance liquid chromatography (HPLC)
In the above mentioned selective enzyme catalyzed biotransformations, all products formed are easily
available and simple. So, authors have used HPLC technique to confirm the actual product formation by comparing the HPLC profiles ofstandard aldehyde compounds with the enzymatically transformed compounds. Retention times of the standard samples of 2-fluorotolu-ene, 4-fluorotoluene, 2-chlorotoluene, 2-fluorobenzal-dehyde, 4-fluorobenzaldehyde, and 2-chlorobenzalde-hyde were 7.17, 6.97, 7.33, 6.14, 6.11, and 6.25 min, respectively. Retention times of the products of the enzyme catalyzed reaction (6.12, 6.1, and 6.24 min) revealed that they were 2-fluorobenzaldehyde, 4-fluo-robenzaldehyde, and 2-chlorobenzaldehyde. Yields of the extracted 2-fluorobenzaldehyde, 4-fluorobenzal-dehyde, and 2-chlorobenzaldehyde were 94, 94, and 96%, respectively.
Retention time of major peak obtained for N-cou-pling products of 3-(3,4-dihydroxyphenyl)-propionic acid with 4-aminobenzoic acid, methyl 4-aminoben-zoate, 4-aminoacetophenone, and 1-hexylamine were 5.21(yield 89%), 5.16, (82%), 5.10 (86%) and 4.82 min (76%), respectively, while retention times for
3-(3,4-dihydroxyphenyl)-propionic acid, 4-aminoben-zoic acid, methyl 4-aminobenzoate, and 4-aminoace-tophenone were 5.85, 6.21, 5.97, and 6.15, respectively, which demonstrates the formation of coupling products that have been identified and characterized by IR and NMR spectroscopy. HPLC chromatograms of all the coupling products are presented in Fig. 2.
In IR spectra of biotransformed products, a band near 815 cm-1 was due to C—Cl stretching. A band at ~1705 cm-1 was due to aldehydic carbonyl stretching confirming formation of the products. In IR spectra of coupling products, a single stretching band at ~3441 cm-1 was due to the N—H (2-amino group) and showed that these two reactants have been coupled and desired products have been formed.
In 1H NMR spectra of different biotransformed products, a peak at 5 > 9.00 was due to the aldehydic proton, confirming formation of expected benzalde-hydes. In spectra of different coupling products, peak between 3.73—3.85 ppm (s, 1H) was due to NH hydrogen, which shows that reactants have been coupled to form the product.
The results of HPLC, IR and 1H NMR-spectros-copy demonstrate that oxidation products were 2-flu-orobenzaldehyde, 4-fluorobenzaldehyde, and 2-chlo-robenzaldehyde (Scheme 1), while coupling products were 3-(3,4-dihydroxyphenyl)-propionic acid derivatives of 4-aminobenzoic acid, methyl 4-aminobenzoate,
4-aminoacetophenone, and 1-hexylamine (Scheme 2). Possibilities of any side reactions ignored since the rate of side reactions is low; also, this manuscript do not include a study on side reactions of dihydroxyphenyl-propionic acid in the presence of laccase.
Conclusions
Thus, this communication reports the successful involvement of a purified laccase in selective oxidation of aromatic methyl group of substituted toluenes to their respective aldehyde group in the presence of ABTS as mediator molecule and synthesis of 3-(3
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