Biomimetic oxidation of indole by Mn(III)porphyrins

Applied Catalysis A: General 470 (2014) 427–433

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Applied Catalysis A: General journal homepage: www.elsevier.com/locate/apcata

Biomimetic oxidation of indole by Mn(III)porphyrins Margarida Linhares a , Susana L.H. Rebelo a,∗ , Mário M.Q. Simões b , Artur M.S. Silva b , M. Grac¸a P.M.S. Neves b , José A.S. Cavaleiro b , Cristina Freire a a b

REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal QOPNA, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e

i n f o

Article history: Received 25 June 2013 Received in revised form 10 October 2013 Accepted 20 November 2013 Available online 28 November 2013 Keywords: Indole Biomimetic oxidation Mn(III)porphyrins Hydrogen peroxide Indigoid pigments

a b s t r a c t The oxidation of indole under biomimetic conditions in the presence of Mn(III)porphyrins and using hydrogen peroxide as a green oxidant is described. The metalloporphyrins act as chemical models of the enzymes involved in the natural and biocatalytic oxidation of indole to afford indigo dye, but leading to simplified systems, with significantly lower cost requirements, as higher indole conversions and easier product isolation are obtained. The distribution of the products that include 2-oxoindole, isatin, 2,2 bis(3 -indolyl)-3-oxoindole and the indigoid pigments, indigo and indirrubin, was found to be dependent on the reaction time, the amount of oxidant and the electronic characteristics of the metalloporphyrin catalyst. Upon 30 min of reaction time, 85% of indole conversion was achieved. The best conditions for pigment formation and isolation included the separation of the initially formed 3-indoxyl from the oxidizing reaction mixture, followed by heating to obtain the air oxidative dimerization. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The indole nucleus is a structural component of a vast number of natural and unnatural compounds, such as pharmaceuticals, pigments, fragrances and agricultural compounds [1]. Presently, the major focus of interest of the indole-containing molecules are their biological activity and medicinal properties [2,3] and the development of new drugs based on indole or on its oxidized derivatives, oxoindoles, indoxyls and isatins, thus remaining an intense area of research with an increasing number of target applications [4–6]. Nevertheless, the indole functionalization methodologies still require improvements in order to achieve milder conditions and less waste production [7,8]. Indole is historically associated with the natural synthesis of indigoid pigments [9]. Indigo dye or indigotin (I.1, Scheme 1) was the blue color used by almost all the ancient civilizations and marked the twenty century as the color of blue jeans [10]. Until the late 1800 it was obtained from plants of the genus Indigofera and since the industrial revolution until today, almost all the indigo traded worldwide has been produced by synthetic methodologies based on the Pfleger’s method [11]. In these processes, fused mixtures of sodamide with alkali are used to produce 3-indoxyl

∗ Corresponding author. Tel.: +351 961 865 521. E-mail addresses: megui [email protected] (M. Linhares), [email protected] (S.L.H. Rebelo), [email protected] (M.M.Q. Simões), [email protected] (A.M.S. Silva), [email protected] (M.G.P.M.S. Neves), [email protected] (J.A.S. Cavaleiro), [email protected] (C. Freire). 0926-860X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apcata.2013.11.023

that undergoes further dimerization and spontaneous oxidation to indigo by exposure to air. The approach requires high temperatures and involves the production of significant quantities of harsh chemicals that are considered hazardous to the environment and health, and consequently are motivating pertinent environmental concerns. These facts are stimulating the use of natural indigo or its production by biocatalytic methodologies, using microorganisms and mutants [12] or enzymatic systems [13]. Bacteria expressing oxygenase genes have been reported to have the ability to convert indole to indigo, in particular several mutant strains of Pseudomonas putida and Escherichia coli [14–16]. Also, enzymatic systems based on naphthalene-1,2-dioxygenase (NDO) and cytochrome P450 monooxygenases (P450) were able to oxidize indole and the indigo (I.1) and indirubin (I.3) pigments could be identified among the oxidation products, that include mainly 2-oxoindole (I.2) and isatin (I.5) [17–19]. Other indigoid pigments have also shown bioactivity or have been used as imaging dyes and markers [20,21], as well as for cosmetic and alimentary purposes [1]. Indirubin (I.3) is a common impurity of natural indigo, which is also produced during indole oxidation by biocatalytic methods. Recently, significant interest has been directed to this pink pigment, since it has been identified in the formula of traditional medicines from Asia, showing cytotoxic properties against a variety of cell lines by inhibition of cyclin-dependent kinase [22]. Since then, indirubin and derivatives have been under examination as promising antileukemic agents and as treatment for other tumor and viral diseases [23]. Methodologies for indole oxidation and indigo production have been disclosed using m-chloroperoxybenzoic acid as oxidant [24] or

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Scheme 1. Isolated compounds and identified reaction pathways during catalytic oxidation of Indole (I.0).

a catalytic method using an organic hydroperoxide [25]. However, these processes led to the introduction of high amounts of additives in the reaction media, and green and economical conditions could not be achieved. As referred above, mutants of cytochrome P450 monooxygenases have been associated with the oxidation of indole to indigo. This wide family of enzymes is responsible for the activation of molecular oxygen, or hydrogen peroxide, in several oxygenations of organic substrates occurring in vivo, mainly during metabolism. They are characterized by a common active center, the heme group, bonded at the axial position, by a thiolate bond to a cysteine residue. In the last decades, several metalloporphyrins have been tested as chemical models of the prostetic group of P450 monooxygenases [26], and the previous studies showed that manganese(III) complexes of tetraarylporphyrins, carrying electron-withdrawing groups, are among the most efficient and easy to obtain models of P450 enzymes. These metal complexes catalyze the activation of hydrogen peroxide in effective oxidations of organic substrates at room temperature [27–29]. In the present work several Mn(III)porphyrins carrying different substituents (Fig. 1) were tested on the oxidation of indole using hydrogen peroxide as a green

oxidant under mild reaction conditions and ammonium acetate as the co-catalyst. The biomimetic system was optimized in order to obtain the best yields of indigo and indirubin pigments and other relevant products, in eco-compatible conditions, but avoiding the complexity, time consuming and expensive biocatalytic processes. 2. Experimental 2.1. Chemicals All reagents and solvents used throughout the process were of high purity and were used as received. Indole 99%; hydrogen peroxide (aq.) 30% (w/w); urea – hydrogen peroxide adduct (UHP), pyrrole 98% and 2,6-dichlorobenzaldehyde 99% were purchased from Aldrich; ammonium acetate p.a. (NH4 AcO) was purchased from Merck and pentafluorobenzaldehyde was purchased from ACROS organics. Thin layer chromatography (TLC) used for reaction monitoring and preparative separation of the compounds was performed on silica gel 60 UV/254 (Merck). Column chromatographic separations were performed on silica gel 60 (Merck, 0.063–0.200 mm). 2.2. Instrumentation

Fig. 1. Structures of Mn(III)porphyrin catalysts.

Electronic spectra were recorded on a range cell Cary 50 BIO spectrometer. Mass spectra with electrospray ionization in positive mode (ESI-MS) were recorded at Unidad de Masas, Universidade de Santiago de Compostela and with fast atom bombardment in positive mode (FAB+ -MS) were obtained in a VG AutoSpec Q spectrometer, operating at 70 eV. Mass spectra with electronic impact ionization at 70 eV were obtained upon GC/MS analyzes on a Finnigan Trace GC/MS (Thermo Quest instruments) using helium as carrier gas (35 cm/s) and fused silica capillary column DB-5

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429

Table 1 Oxidation of indole by H2 O2 in the presence of different Mn(III)porphyrins and reaction conditions.a

Entry

Catalyst

t (h) (eq.H2 O2 )d

1

[Mn(TDCPP)Cl]

2

[Mn(TDCPP)Cl]

3

[Mn(TDCPP)Cl]

4

[Mn(TDCPP)Cl]

5

[Mn(TDCPP)Cl]

6

[Mn(␤NO2 TDCPP)Cl]

7

[Mn(TF4 NMe2 PP)Cl]

8

–

3 (6) 6 (12) 2 (4) 1 (2) 0.5 (4) 0.5 (4) 0.5 (4) 0.5 (4)

% (S%)b,c I.1 Indigo trace – trace 2 (3) 3 (4) 2 (3) 1 (2) –

C%c I.2 2-oxoindole

I.3 Indirubin

I.4 trimer

I.5 isatin

Otherse

46 (50) 39 (40) 51 (61) 44 (68) 64 (75) 50 (73) 35 (72) –

10 (11) 3 (3) 8 (10) 4 (6) 7 (8) 4 (6) 2 (4) –

11 (12) 13 (13) 7 (8) 4 (6) 5 (6) 6 (9) 7 (14) –

16 (18) 20 (20) 12 (14) 8 (12) 6 (7) 6 (9) 4 (8) –

8 (9) 24 (24) 6 (7) 3 (5) –

85

–

68

–

49

–

0

91 99 84 65

a

Reactions were performed in acetonitrile, using NH4AcO as co-catalyst, at r.t. and the ratio substrate/catalyst was 300. For entries 1–2 are the isolated yields; for entries 3–8 the yields were based on the 1 H NMR of the total reaction mixture and indigo yields confirmed by UV–vis analysis of the total reaction mixture. c The percentages of yield, selectivity and conversion, were calculated as indicated in Section 2.4. d Molar equivalents of H2 O2 relatively to the substrate added. e Include dimerization products and lower molecular weight compounds relatively to indole. b

(30 m, 0.25 mm i.d., 0.25 ␮m film thickness). 1 H and 13 C NMR were recorded on a Brucker Avance III spectrometer at a frequency of 400 and 100 MHz, respectively, and using TMS as reference, at 22 ◦ C. The spectra were recorded in CDCl3 or DMSO-d6 as specified. Connectivities were determined by 1 H–1 H COSY experiments and carbon assignments were obtained from APT, HSQC and HMBC experiments. 2.3. Synthesis of the metalloporphyrins The metalloporphyrin catalysts were prepared in accordance with literature procedures [30–32]. The free-base porphyrins were prepared by condensation of pyrrol and the appropriate benzaldehyde, 2,6-dichlorobenzaldehyde or pentafluorobenzaldehyde, in acidic and oxidizing media, namely in a mixture of acetic acid and nitrobenzene as reaction solvents. The free-base porphyrins were recovered from the reaction media by precipitation and obtained pure after recrystallization [30]. The nitration was performed on the ␤-pyrrolic position by reaction with zinc nitrate in acetic anhydride [31]. Removal of zinc as central atom was obtained by reaction with TFA and the complexation of the porphyrin free-bases was obtained by reaction with manganese chloride in reflux of DMF and in the presence of pyridine [32]. The reaction was controlled by UV-Vis spectroscopy following the bathochromic shift of the Soret band of the porphyrin until completeness of the complexation reaction. The spectroscopic data and yields obtained for pure free-base porphyrin nucleus and corresponding paramagnetic Mn(III) complexes are presented below (an asterisk is assigned to the Soret band on the UV–vis spectra). H2 TDCPP – 5,10,15,20-tetrakis(2,6-diclorophenyl)porphyrin 1 H NMR (CDCl ): ı (ppm): −2.54 (s-broad; 2H; NH); 7.67–7.81 3 (m; 12H; H-Ar); 8.67 (s; 8H; H-␤). MS-FAB+ (m/z): 890 (M+• ). UV–vis (CH2 Cl2 ) max , nm (%): 417* (100); 512 (7); 588 (2); 657 (0.8).  5%. [Mn(TDCPP)Cl] – Chloro[5,10,15,20-tetrakis(2,6diclorophenyl)porphyrinate]manganese(III) UV–vis (CH2 Cl2 ) max , nm (%): 371 (37); 478* (100); 580 (7); 615 (2).  90%. H2 ␤-NO2 TDCPP – 2-nitro-5,10,15,20-tetrakis(2,6-diclorophenyl)porphyrin 1 H NMR (CDCl ): ı (ppm): −2.49 (s-broad; 2H; NH); 7.71–7.83 3 (m; 12H; H-Ar); 8.57 (dd; 2H; H-␤; J 1.0 and J 10.6 Hz); 8.72–8.79

(m; 4H; H-␤); 8.94 (s; 1H; H-␤). MS-FAB+ (m/z): 936 [M+H]+ . UV–vis (CH2 Cl2 ) max , nm (%): 424* (100); 523 (6); 597 (2); 656 (1).  % 76%. [Mn(␤-NO2 TDCPP)Cl] – Chloro[2-nitro-5,10,15,20tetrakis(2,6-diclorophenyl)porphyrinate]manganese(III) UV–vis (CH2 Cl2 ) max , nm (%): 373 (39); 482* (100); 587 (7). % 90%. H2 TF5 PP – 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin 1 H NMR (CDCl ): ı (ppm): −2.93 (s-largo; 2H; NH); 8.92 (s; 8H; 3 H-␤). MS-FAB+ (m/z): 976 [M+2H]+ . UV–vis (CH2 Cl2 ) max , nm (%): 412* (100); 506 (7); 583 (2); 636 (1).  16%. [Mn(TF4 NMe2 PP)Cl] – Chloro[5,10,15,20-tetrakis(4-dimethylamino-2,3,5,6-tetrafluorophenyl)porphyrinate]manganese(III) UV–vis (CH2 Cl2 ) max , nm (%): 365 (42); 474* (100); 572 (10). % 85%. 2.4. Catalytic reactions Initial conditions (Table 1, entries 1–2) were based on previously reported conditions for the oxidation of aromatic compounds by metalloporphyrin catalysis [33]: in a round bottom flask, 35.1 mg of indole (0.3 mmol), 0.3% of the catalyst and 40 mg of ammonium acetate were dissolved in 4 mL of acetonitrile. Hydrogen peroxide was progressively added at a rate of 10 ␮mol min−1 to the mixture under magnetic stirring at room temperature (∼20 ◦ C). The addition was performed during the specified time (a total of 6 or 12 mol equiv. relative to the substrate were added (entries 1 and 2, respectively) and adequate solutions of H2 O2(aq) 30% (w/w) in acetonitrile were prepared to allow the accurate addition). After solvent evaporation, the final reaction mixture was directly applied on preparative TLC plates and fractionated using a mixture of chloroform:ethyl acetate (4:1) as eluent. Spectroscopic data obtained for the characterization of isolated compounds are reported on S.I. As a result of experiments on the optimization of the reaction conditions, the final procedure for the catalytic reaction was as follows (Table 1, entries 5–8): the reaction mixture was prepared as above and the hydrogen peroxide was progressively added at a rate of 60 ␮mol min−1 during 20 min (4 mol equiv. relative to the substrate, adequate solutions of H2 O2 (aq) 30% (w/w) in acetonitrile were prepared to allow the accurate addition). The reaction was left to proceed during 10 min, in a total time of 30 min. To stop the reaction, the mixture was passed through a

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small plug of silica gel in order to retain the excess of catalyst and H2 O2 , and then washed with a (2:1) mixture of chloroform:ethyl acetate. The collected mixture was evaporated to dryness under vacuum. Indigo is insoluble in most of the common solvents, although solutions can be achieved in DMSO or DMF, consequently and in order to achieve convenient quantification of the reaction products, the reactions were performed in duplicate. The residue of the first assay was dissolved in an exact volume of DMSO-d6 for NMR analysis of the total reaction mixture. As a confirmation of the amount of indigo in the reaction mixture, it was also quantified by UV–vis in comparison with standard solutions of indigo in DMSO and the residue of the second assay was dissolved in an exact volume of DMSO for UV–vis analysis. The 1 H NMR analysis of the total reaction mixtures considered the identification of the signals corresponding to individual compounds in DMSO-d6 and relative quantifications based on the integrations of non-superimposed signals. The yields (%), selectivity (S%) and conversion (C%) were based on the mass of obtained products and mass of substrate used: [% = (mass of a product/mass of substrate) × 100]; [S% = (mass of a product/sum of the masses of all isolated products) × 100]; [C% = (sum of the masses of all isolated products/mass of substrate) × 100]. In some experiments, the C% was also calculated based on the mass of recovered substrate and the obtained values were identical. To access the formation of the products as a function of time in the catalytic reaction, an experiment was performed in a quartz UV–vis cell adding H2 O2 progressively in aliquots of 60 ␮mol min−1 during 20 min (total 126 ␮L) and the reaction was left to proceed for 10 min. The UV–vis spectra were acquired at each 3 min.

3. Results and discussion 3.1. Catalytic results and products isolation The catalytic oxidation of indole by aqueous hydrogen peroxide was performed in the presence of the manganese(III) porphyrins shown in Fig. 1. The initial studies were carried out using the metalloporphyrin [Mn(TDCPP)Cl] (Fig. 1) and the results were compared with those obtained with the other two Mn(III)porphyrins carrying different electronic substituents; [Mn(␤-NO2 TDCPP)Cl] with a nitro group in the ␤-pyrrolic position, in addition to the 2,6-dichlorophenyl groups, and the [Mn(TF4 NMe2 PP)Cl] bearing meso-fluorophenyl groups. It is well known that the oxidation reactions catalyzed by metalloporphyrins are strictly dependent on the catalytic conditions used [26]. In previously described protocols, the Mn(III)porphyrins have shown high activity in acetonitrile as solvent and in the presence of an amphoteric co-catalyst such as ammonium acetate (NH4 AcO) [33]. The reactions usually are performed at room temperature, since it was shown that higher temperatures (30–80 ◦ C) led to lower substrate conversions; this behavior has been related with the higher extent of the competing reaction of H2 O2 dismutation, also catalyzed by metalloporphyrins [34,35]. Considering previously reported conditions [28,33], the oxidation of indole was initially performed by progressive addition of H2 O2 to the reaction mixture, at a rate of 10 ␮mol min−1 (2 mol equiv. h−1 ). After 3 h of reaction and a total addition of 6 mol equiv. of oxidant, relatively to the substrate (1.2 mmol), the TLC monitoring showed the appearance of a blue and a red spot, a yellow/orange one and other compounds that were only detected under the UV light. The reaction mixture was then fractionated by preparative TLC and the blue pigment was isolated at trace levels, while the red pigment was isolated in 10% yield. Both pigments were identified by UV–vis spectroscopy in dichloromethane as indigo (I.1) (max = 603 nm) and indirubin (I.3) (max = 550 nm), respectively (Scheme 1, Fig. 2a).

Fig. 2. UV–vis spectra of (a) pure solutions of indigo and indirubin in CH2 Cl2 ; (b) total reaction mixtures in DMSO in the presence of the different Mn(III)porphyrins.

The characterization of the two pigments (S.I. 1 and 2) [18] and of the other reaction products also isolated (Scheme 1 and Table 1) were obtained by 1 H and 13 C NMR, using when necessary 1D and 2D NMR techniques and by MS (S.I). The 2-oxoindole (I.2) was obtained in 46% yield, the trimer 2,2-bis(3 -indolyl)-3-oxoindole (I.4) in 11% yield and the yellow/orange spot identified as isatin (I.5) in 16% yield (Table 1, entry 1). These reaction products have biochemical and biological relevance, as the trimer (I.4) is also produced during indole oxidation in the presence of laccase [36] and an analogous derivative was obtained by biocatalytic trimerization of N-methylindole with marine-derived bacterial cell lines [37]. 2-Oxoindole (I.2) and isatin (I.5) are also commonly observed products during bio-oxidation of indole [19], and these molecules and their derivatives have been described as important heterocyclic building blocks in the synthesis of bioactive compounds [4–6]. In the fraction classified in Table 1 as “others”, obtained in a total yield of 8%, were quantified the non-colored dimer I.6 (Scheme 1) and compounds resulting from opening and decarboxylation of isatin, such as 2-aminobenzaldehyde and 2-aminobenzoic acid, identified by NMR and MS. The percentage of conversion was 91%. A reaction was carried out with longer reaction time (6 h), and the addition of H2 O2 was performed in a higher excess when compared to the previous reaction, 12 mol equiv. relatively to the substrate, although keeping the other above described conditions (Table 1, entry 2). In these conditions, it was observed an increase up to 24% in the amount of the fraction classified as “others” in which, are included the dimer I.6, as it was referred before, and products

M. Linhares et al. / Applied Catalysis A: General 470 (2014) 427–433

resulting from indole ring cleavage. It is worth to refer that during the reaction monitoring by TLC, the blue spot due to indigo was detected but at the end of the reaction the pigment could not be isolated, indicating its degradation in these more severe conditions (longer reaction times and higher excess of oxidant). The yield on indirubin (I.3) decreased to 3% instead of 10%, when compared with the previous conditions. The yield of 2-oxoindole (I.2) also decreased to 39%, while the yield of trimer (I.4) was nearly maintained (13%); however, the yields of isatin (I.5) and of the fraction identified as “others” increased to 20% and 24%, respectively. Probably, isatin and the fraction of “other products” result from further oxidation of the initially formed compounds in the reaction conditions used, and consequently indigo pigment I.1 could not be obtained; therefore, a careful control of reaction time and amount of oxidant added is required in order to avoid over-oxidation of the former derivatives. Some literature results showed that indigo is readily oxidized in solution even in soft oxidizing media, while once precipitated it behaves as a quite stable compound toward oxidation [38]. Thus, the production of pigments should be achieved with shorter reaction times and considering their precipitation or removal from the oxidizing media in the reaction mixture before further oxidation. In this context, in order to have a precise quantification of the indigo produced, the subsequent reactions were performed and stopped, at appropriate time, by passing the reaction mixtures through a small plug of silica gel in order to retain the excess of catalyst and oxidant. The compounds were then eluted using a mixture of chloroform and ethyl acetate and the solvents removed. It should be noted that after elution from the silica, the eluate showed a clear green color, while after solvent evaporation, the formation of pigments could be detected in the residue of the reaction mixture, once the solid residue turned into dark blue or violet color, depending on the relative amounts of indigo and indirubin present. Indigo is highly insoluble in almost all the common solvents, although solutions can be obtained in DMSO or DMF. In order to obtain an accurate quantification of the reaction products, each reaction was performed in duplicate and the residue of the first assay was dissolved in DMSO-d6 for 1 H NMR analysis of the total reaction mixture and quantification of product yields (S.I.3); the residue of the second assay was dissolved in a precise volume of DMSO for UV–vis analysis and quantification of indigo formed, by comparison with standard solutions; this analysis allowed to confirm the yields obtained by 1 H NMR. These procedures were used to obtain the results in all the subsequent studies, shown in Table 1, entries 3–8. Further reactions were performed at shorter times, 2 h and 1 h (Table 1, entries 3 and 4), and keeping the addition rate of H2 O2 at 10 ␮mol min−1 , the total amount of oxidant added was reduced to 4 equiv. and 2 equiv., respectively (Table 1, entries 3 and 4). The observed conversions were 84% and 65%, respectively, lower than in the initially performed reactions, but it was possible to isolate indigo in 2% yield in the last reaction (entry 4). In another experiment, the total addition of 4 mol equiv. of oxidant was considered, but the H2 O2 addition was performed at higher rates than in the previous reactions (60 ␮mol min−1 instead of 10 ␮mol min−1 ) in order to further diminish the reaction time. This reaction was monitored by UV-Vis spectroscopy, at every 3 min intervals and the spectra are shown in Fig. 3. After 20 min, a total amount of 4 equiv. has been added and the monitoring proceeded with no oxidant addition; after 10 min no further spectral changes were observed and a total reaction time of 30 min was considered. Changes in the spectral pattern in Fig. 3 with increasing reaction time can be noticed. The Soret band at  = 478 nm is slightly shifted to lower wavelengths and its intensity decreases as expected from bleaching of the metalloporphyrin [26]. Concomitantly, the band at near  = 375 nm showed a significant and progressive increase in

431

Fig. 3. UV–vis spectra of the reaction mixture in conditions of Table 1, entry 5, recorded at 3 min intervals.

intensity, which cannot be related with the original metal–ligand charge transfer band of the metalloporphyrin, since as mentioned, the concentration of the catalyst decreased as the reaction proceeded. In accordance with reported data, the formed band at near  = 375 nm can be assigned to 2- and 3-oxoindole (Scheme 1) [39], which might be in equilibrium with their enol forms, the indoxyls (hydroxyindoles). However, as can be seen in the spectra of Fig. 3, the pigments are not detected during the reaction time, since no bands are observed for indigo (max = 603 nm) and indirubin (max = 550 nm, Fig. 2a). Only when the reaction mixture was applied on TLC silica plates and after solvent drying, the blue and red spots become visible during elution. This indicates that indigo or indirubin are not significantly formed during the short catalytic reaction of 30 min, but are formed during the work-up. Since indoxyls are the main resulting products of the catalytic reaction and are the key precursors of other reaction products in indole oxidations, specifically 3-indoxyl for indigo, Fig. 3 indicates the kinetic profile of the catalytic reaction of indole oxidation. Furthermore, the possibility of separating the fraction of indoxyls from the reaction media (catalysts and oxidant) can be advantageous since it may allow the isolation of the pigments precursors from the oxidizing media, and performing the coupling reactions in a further stage, thus avoiding their over-oxidation in the reaction mixture. An additional reaction was also performed in the last conditions but the addition of hydrogen peroxide was performed in the form of adduct with urea (UHP), in order to check if this anhydrous oxidant could have a positive effect. No significant differences were observed in the yields of the reaction products relatively to the use of aqueous hydrogen peroxide, showing that this cheapest oxidant, H2 O2 , is appropriate. For comparison, the efficiency of the other two metalloporphyrins, [Mn(␤-NO2 TDCPP)Cl] and [Mn(TF4 NMe2 PP)Cl] were tested in indole oxidation using the conditions described in Table 1, entry 5, for [Mn(TDCPP)Cl]: reaction time of 30 min, with H2 O2 addition at a rate of 60 ␮mol min−1 in a total amount of 4 mol equiv. of H2 O2 relatively to the quantity of substrate (Table 1, entries 6 and 7). The increase of the electron-withdrawing characteristics of the metalloporphyrin nucleus is acceded by the increase of the chemical deviation observed in the 1 H NMR spectra for ␤-protons: ı = 8.67 ppm for H2 TDCPP < ı = 8.72–8.79 ppm for H2 ␤NO2 TDCPP < ı = 8.92 ppm for H2 TF5 PP. A decrease in conversion was observed for the more electron withdrawing metalloporphyrins, namely from 86% with

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[Mn(TDCPP)Cl] to 68% and 49% with [Mn(␤-NO2 TDCPP)Cl] and [Mn(TF4 NMe2 PP)Cl], respectively, and no improvement in pigments yield was observed. With these two catalysts the main product was also 2-oxoindole (I.2). The activation of H2 O2 by the used Mn(III)porphyrins requires the presence of amphoteric co-catalysts, such as NH4 AcO. These co-catalysts mediate the successive activation steps: deprotonation of H2 O2 to form the Mnporphyrin-OOH intermediate; followed by protonation/dehydration of the hydroperoxy group to afford the effective oxidant, a high valent oxo-metalloporphyrin species [Mn(V)=O] [26,40]. The behavior of more electron withdrawing porphyrins has been ascribed to the preferential formation of the oxo-species in a radical form, Mn(IV)-O• . This intermediate showed lower epoxidation capability but improved capability for aromatic hydroxylation [26]. Consequently, the present results showing a decrease on conversion, with the higher electron withdrawing characteristics of the metalloporphyrin, seem to confirm the important role of the initial epoxidation step on indole oxidation. In Fig. 2b, the UV–vis spectra of the reaction mixtures in DMSO and in the presence of the different metalloporphyrins are shown at the same dilution of the reaction residue. The results confirm the indigo yields of 1.4% and 1.7% obtained in the presence of the more electron-withdrawing substituted metalloporphyrins, [Mn(␤-NO2 TDCPP)Cl] and [Mn(TF4 NMe2 )Cl], respectively. When the reaction was performed in the above best conditions (entry 5), but in the absence of catalyst, no indole conversion could be observed (entry 8), while the non-catalytic oxidation by H2 O2 in basic media showed uncontrollable oxidation and the indigo formation was elusive (data not shown). 3.2. Considerations on reaction pathways The present results highlight the catalytic system based on a Mn(III)porphyrin and H2 O2 as an efficient model to reproduce biological pathways of indole oxidation. A general scheme for indole oxidation in these biomimetic conditions can be proposed as illustrated in Scheme 1. The numbered compounds were isolated and characterized, while non-numbered products are probable intermediates, in a conclusion based on the present results and on the biological oxidations of indole reported in literature [18,19]. Correspondence is found between the biomimetic system and the already identified biological pathways, although improved identification of the reaction products was obtained. The reactivity of the initially formed products accounts for the different proportion of reaction products obtained depending on the reaction time [13]. Considering that 3-indoxyl is the key intermediate in all indigo synthesis, including the common industrial synthesis [11], its formation and further dimerization pathways are very relevant reactions in the different synthetic systems. The enzymatic oxidation of indole to indoxyls is proposed to proceed through different mechanisms depending on the enzyme involved. For naphthalene 1,2-dioxygenase (NDO), the formation of a cisdiol derivative was anticipated, followed by spontaneous water elimination to afford 3-indoxyl [17]. In the present conditions, the initial transformation of indole to indoxyls can occur by reaction of the 2,3-bond of indole with the active oxidizing species through epoxidation or aromatic hydroxylation, as both reaction patterns are catalyzed by metalloporphyrins [26]. Attending to the reported capability of the Mn(III)porphyrins to mediate epoxidation reactions of both alkenes [27] and polyaromatic systems [33], and the present results with metalloporphyrin catalysts with different electron withdrawing characteristics, which change the capability for epoxidation [26], it can be rationalized that the oxidation of indole to indoxyls proceed through the initial epoxidation of the 2,3-positions of indole, followed by internal rearrangement of the

oxirane ring to enol (indoxyl) moieties [35]. Instead of the rearrange of epoxide ring, the opening of the epoxide to a diol followed by water elimination can be also considered as an alternative pathway. It is expected that both 2-indoxyl and 3-indoxyl (hydroxyindoles), once formed, can be in equilibrium with the corresponding keto forms. The 2-oxoindole is a particularly stable structure due to the lactam group and this can explain the higher yields found for this compound, which is in accordance with the observed for indole oxidation by cytochrome P450 enzymes [18]. On the other hand, the 3-indoxyl is prone to undergo dimerization followed by spontaneous air oxidation affording indigo, which is facilitated by the stabilization achieved with aromatization and hydrogen bond formation of the final indigo structure or trimerization giving compound I.4. The condensation of two 3-indoxyl units to form indigo is the last step in all indigo dye synthesis, both natural and industrial ones. This reaction was proposed to proceed mainly through a radical mechanism, in which the 3-indoxyl undergoes one electron oxidation in the reaction conditions followed by carbon-carbon coupling [38]. In another pathway, a carbanion coupling is considered, via its zwitterionic form, in which, this carbanion can undergo nucleophilic attack to an epoxide molecule and form a carbon–carbon bounded dimer, followed by air oxidation to indigo [38]. This reactivity could also account for the formation of trimer I.4. In accordance with literature data, indigo over-oxidation was found to afford isatin [41], and probably in this biomimetic conditions isatin is produced by this pathway, since it was observed in higher yields for longer reaction times with the concomitant disappearance of pigments. In the reaction conditions used, isatin formation can be justified by further epoxidation of the intermediate double bond of indigo followed by ring-opening and oxidative cleavage [42]. The chemical synthesis of indirubin can be performed by reaction of isatin and 3-indoxyl [43]; the same pathway can be followed in biosynthesis or alternatively, the coupling of 3-indoxyl and 2indoxyl can occur, in a parallel mechanism to those described for indigo. In the biomimetic conditions described here, both pathways probably can justify indirubin formation, as indirubin was observed in the shorter reactions of 30 min, when the amounts of isatin are minor, but in the 3 h reaction the highest yield (10%) was obtained (Table 1, entry 1) [44]. Compound I.6 is probably the result of a dimerization reaction involving isatin and 3-hydroxy-2-oxoindole [18], which seems to indicate also the formation of this compound during the biomimetic reaction. In the short, 30 min reactions, the formation of pigments was not detected (Fig. 3). After removing the excess of catalyst and H2 O2 , by passing the reaction mixture through a small plug of silica, a fraction enriched in oxoindoles and indoxyls was obtained. When the reaction is being performed at room temperature preserves immediate dimerization to the pigments that were only observed after deposition on silica plates or solvent evaporation; this in turn can facilitate the dimerization and oxidation of indoxyls to the corresponding dimers indigo or indirubin. The enriched fraction in indoxyls has synthetic value since indoxyls also represent important synthons for the synthesis of biologically active indole derivatives and showed their own biological activities [45].

4. Conclusions The oxidation of indole is reported using a biomimetic procedure based on catalysis by manganese(III) porphyrins as models of the enzymes involved in the natural and biocatalytic processes, but with the advantage of avoiding crop production or enzymatic engineering. The pigments indigo and indirubin were obtained by

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controlling the time of the catalytic reaction, the amount of oxidant added and the Mn(III)porphyrin used. The main reaction product was always 2-oxoindole, as observed in the oxidation of indole in the presence of enzymatic strains [18,19]. For longer reaction times, increased amounts of isatin and other products resulting from coupling or cleavage of oxidized indole units were obtained. The best total yields were obtained with the metalloporphyrin carrying less electron-withdrawing substituents. The procedure considered the isolation of a fraction enriched in 2- and 3-indoxyls before pigment formation, allowing the improved synthesis of indigo and indirubin pigments. Furthermore, the present work opens an important field for the development of novel catalytic production of indigoid compounds by metalloporphyrins based catalysis, in particular, leading to the production of indigo dye by milder and cheaper processes. Attending to the relevance of the heterogenation of the present homogeneous process, in order to obtain reusability, the immobilization of appropriate metalloporphyrins on suitable supports, through stable and versatile linkages, is being considered in our labs. Acknowledgements This work was funded by Fundac¸ão para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER and COMPETE through PEst-C/EQB/LA0006/2011 (REQUIMTE) and PEst-C/QUI/UI0062/2011 (QOPNA research unit) and the Portuguese National NMR Network, also supported by funds from FCT. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apcata.2013. 11.023. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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