Methyl caffeate

In a search for allelopathic agents from common weeds, Amaranthus palmerl S. Wats (Palmer amaranth) and Ambrosia artemisiifolia L. (Louisiana annual ragweed) have been analysed for their organic natural products. From A. palmerl phytol, chondrlllasterol, vanillin, 3-methoxy-4-hydroxynitrobenzene and 2,6-dimethoxy- benzoquinone were isolated. From the roots of Ambrosia artemisiifolia four polyacetylenes, a mixture of sesquiterpene hydrocarbons, methyl caffeate, and a mixture of 8-sitosterol and stlgmasterol were obtained. 

Campanula glomerata L. f. canescens (Maxim.) Kitag. C. glomerata L. var. dahurica Fisch. ex Ker-Gawl. C. punctata Lam. Feng Lin Cao (whole plant) Quercetin, isorhamnetin, kaempferol, hyperoside, isoquercetin, trifolin, chlorogenic acid, methyl caffeate, coumaroylquinic acid.48 For throat infection, headache... 

Five transformants produced a major secreted protein band, with an estimated molecular weight of 40 kDa and no protein was detected in the control. The estimated molecular weight is greater than that predicted or observed from E. coli, which is likely due to post-translational modification. P6 and P10 transformants were retained to perform enzymatic assays. The culture supernatants were assays for activity against methyl caffeate (MCA) and methyl ferulate (MFA). The recombinant proteins were shown to be active as a feruloyl esterase and show the characteristics of a type B ferulic acid esterase.6 Feruloyl esterase activity is reported in Table 1. 

Occurrence in the Solanaceae. p-Coumaric acid, caffeic acid, methyl caffeate, and methyl ferulate as well as certain of their 2,3-dihydro derivatives have been identified as constituents of the leaves of Cestrum parqui L Herit. with good phytotoxic activity against different species (D Abrosca et al. 2004). Family-specific phenylpropanoid acids like tropic acid or 2-hydroxytropic acid as acyl moieties of tropane alkaloids are synthesized via phenylalanine -> phenylpyruvic acid (l )-3-phenyllactic acid (Fig. 3.14 Table 3.1 (T5-T7-B)]. Tropic acid may occur as a metabolite of, e.g., hyoscyamine, but the free acid is not synthesized as such (for details see Sect. 3.4). 

Conversion of caffeoylquinic acids to methyl caffeate with chlorogenate hydrolase... 

The aim of this study was the development of conversion system of caffeoylquinic acids to valuable compounds. When an IL, l-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([bmim][NTf2]) was used as a reaction solvent, we found that immobilized chlorogenate hydrolase (Kikkoman) catalyzed the conversion of 5-caffeoylquinic acid to methyl caffeate with methanol (Fig. 1). The immobilized enzyme was prepared with chlorogenate hydrolase using quaternary ammonium sepabeads (Mitsubishi Chemical Co., Tokyo, Japan) (Kurata et al., 2011). To synthesize valuable compounds from caffeoylquinic adds, we attempted to develop a method for the conversion of caffeoylquinic acids to CAPE analogues via methyl caffeate. In section 2, we describe the properties of immobilised chlorogenate hydrolase in ILs. Using various caffeoyl quinic acid prepared from immature coffee beans, we developed a system to produce methyl caffeate. 

A) Alcoholysis of various caffeoyl quinic aicds (compound 1) was catalysed by chlorogenate hydrolase with methanol and [bmim][NTf2] as the reaction solvent, and methyl caffeate (compound 2) was produced. (B) Structures of substrates for synthesis of methyl caffeate with chlorogenate hydrolase are shown. 

When the concentration of the aqueous solution was 1% or lower, the production of methyl caffeate increased. However, the production of methyl caffeate was decreased in aqueous solutions greater than 2%. The production of caffeic acid was increased with addition of the buffer, indicating that the enzyme probably catalyzed hydrolysis of 5-caffeoylquinic acid to produce caffeic add rather than alcoholysis to produce methyl caffeate. Thus, the result indicated that the addition of 1% aqueous solution was suitable for the production of methyl caffeate. It was suggested that ILs are able to maintain active strudures of the enzymes with a monomolecular layer of water (Feher et al., 2007). Thus, chlorogenate hydrolase would maintain the active structure with the layer of the buffer in [bmim][NTf2]. 

Fig. 2. Effects of water concentration on conversion of 5-caffeoylquinic acid to methyl caffeate by chlorogenate hydrolase (Kurata et al., 2011). The reaction was performed at 40°C using chlorogenate hydrolase and [bmim][NTF2] as the reaction solvent with a 0-5% (v/ v) aqueous solution of 50 mM sodium phosphate (pH 6.5). Each symbol indicates methyl caffeate (closed circle) and caffeic add (open circle).

We examined the production of methyl caffeate using the alcoholysis reaction by chiorogenate hydrolase in [bmim][NTf2] (Table 1). Methyl caffeate was produced using various caffeoylquinic adds (15 pmol) and methanol (2200 pmol). Using 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic add, and 4,5-dicaffeoylquinic add, methyl caffeate was synthesized at concentrations of 9.0 mM (9.0 (imol), 9.2 mM (9.2 pmol), 12.9 mM (129 pmol), 13.9 mM (13.9 pmol), and 17.1 mM (17.1 (imol), respectively (Kurata et al., 2011). 

Because dicaffeoylquinic acid and caffeoylquinic acid have two and one caffeoyl groups, respectively, the volumes of methyl caffeate prepared from 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were greater than those of methyl caffeate prepared from 3-caffeoylquinic acid, 4-caffeoylquinic add, and 5-caffeoylquinic add. In the cases of 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, both caffeoyl groups would be used for synthesis of methyl caffeate. 

Additionally, using a mixture of 3,4-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, which is a crude fraction prepared from coffee beans, HPLC analysis showed that aU peaks of caffeoylquinic acids disappeared and that the peak of methyl caffeate occurred after a 4-h readion with chiorogenate hydrolase in [bmim][NTf2] (Kurata et al., 2011). 

Chiorogenate hydrolase acted on caffeoylquinic acids and dicaffeoylquinic acids. Thus, methyl caffeate was produced from various caffeoylquinic acids prepared from coffee beans using this procedure. 

Conversion of methyl caffeate to CAPE analogues with Novozyme 435... 

In section 2, we described success in converting various caffeoylquinic acids to methyl caffeate with good yields. Next, we tried to convert methyl caffeate to valuable compounds, namely, CAPE analogues, using IL as the reaction solvent (Fig. 5). 

We initially selected methyl caffeate and 3-cyclohexyl-l-propanol as substrates for comparative study of the enzyme s performance in [bmim][NTf2]. Four commercially available lipases, C. antarctica lipase B (Novozyme 435, Novozymes, 30 U mg-i), Rhizotnncor miehei lipase (RMIM, Novozymes, 1370 U mg-i), B. cepacia lipases (PS-CI, Wako Pure Chemical, Osaka, Japan, 2560 U mg-i), and Thermomyces lanuginosus lipase (TLIM, Novozymes, 1850 U mg-i) were tested (Table 2). 

Table 2. Selection of lip>ase for conversion of methyl caffeate (Kurata et al., 2010). The reaction was performed with 1,200,000 U of lipases.

We examined the production of CAPE analogues using transesteiification with [bmim][NTf2] (Kurata et al., 2010). CAPE analogues were produced using 50 mM methyl caffeate (50 pmol) and 300 mM alcohols (300 pmol). We synthesized 48.8 mM 2-cyclohexylethyl caffeate (48.8 pmol), 46.9 mM 3-cyclohexylpropyl caffeate (46.9 pmol), 49.4 mM 4-phenylbutyl caffeate (49.4 pmol), and 42.0 mM 5-phenylpentyl caffeate (42.0 pmol). The conversion yields are shown in Table 3. 

Table 3. Conversion yields of CAPE analogues. The reaction was performed with methyl caffeate, each of alcohol, Novozyme 435, and [bmim][NTf2], which was used as a reaction medium. For production of 2-cyclohexylethyl caffeate, 3-cydohexylpropyl caffeate, 4-phenylbutyl caffeate, and 5-phenylpentyl caffeate, alchols used were shown as follows 2-cyclohexylethanol, 3-cyclohexyl-l-propanol, 4-phenyl-l-butanol, 5-phenyl-l-pentanol, respectively(Kurata et al., 2010).

We described the alcoholysis of various caffeoylquinic acids with methanol to produce methyl caffeate in section 2, and the transesteiification of methyl caffeate to various CAPE analogues in section 3. Both reactions was performed in the same IL, namely, [bmim][NTf2], with good production yields. In section 4, we investigated a one-pot consecutive conversion of 5-caffeoylquinic acid to a CAPE analogue, 3-cyclohexylcaffeate, via methyl caffeate (Fig. 9). In the case of a one-pot two-step reaction, pnirification of the reaction intermediate is not required, so that the intended product was expected to be obtained with high conversion yield. 

Fig. 9. Consecutive enzymatic reactions for synthesis of 3-cyclohexylpropyl caffeate from 5-caffeoylquinic acid. In all reaction steps, [bmim][NTf2] was used as the solvent. Compound 1. 5-caffeoylquinic acid, 2. methyl caffeate, 3 3-cydohexyl-l-propanol, and 4 3-cyclohexylpropyl caffeate.

We initialy examined the production of methyl caffeate or 3-cyclohexylpropyl caffeate using immobilized chlorogenate hydrolase in [bmim][NTf2] (Fig. 10). 

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