Chemical assay-guided natural product isolation via solid-supported chemodosimetric fluorescent probe
As a new model of chemical assay-guided natural product isolation, an effective chemodosimetric assay system was devised. Our chemical assay system features a fluorogenic chemodosimeter immobilized on a solid support, which offers advantages in identifying the desired compounds in complex natural product mixtures. To isolate only compounds with the target functional groups, the click chemistry concept was adopted. The model system presented here was developed for natural products with a terminal alkyne. Using our newly designed alkyne sensing beads with the aforementioned features, we have readily identified, quantified, and isolated compounds with a terminal alkyne group from plant extracts.
Introduction<!>Results and discussion<!>Conclusions
<p>Despite the advent of modern drug discovery technologies, natural products still remain a signicant source of new drugs. 1 In addition, they are valuable tools with which to study biology. 2 At present, bioassay-guided isolation is the favored procedure for identifying new natural substances. 3 Although modern analytical and bioassay techniques have made it considerably competitive, 4 bioassay-guided isolation is a time-consuming process and it still has some limitations caused by false assay signals arising from compounds with unspecic activities. 5 Thus, alternative and complementary approaches are needed to further advance natural product research.</p><p>The isolation of natural products can also be guided by chemical cues associated with functional groups. 6 The chemical reactions of certain functional groups can produce a detectable signal and this has long been used in the natural product isolation process. 7 For example, the phenolic group of tannins was identied by FeCl 3 , and the tertiary amino group of alkaloids was detected by Dragendorff's reagent. However, this approach has become obsolete and is not considered useful, primarily because of the low sensitivity and specicity of the chemical cues produced in natural product extracts. If an effective chemical assay system were to be developed that could easily and reliably identify a compound with a specic functional group in an extract, the systematic exploration of the natural product chemical space would be greatly facilitated.</p><p>The "click reaction" is thermodynamically favorable and reliably leads to a single product under mild reaction conditions. 8 We envisaged that the proper application of click chemistry would guarantee the specicity and reliability of a chemical assay system. Among various click reactions, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is the most popular and well-studied. 9 It produces a 1,4substituted triazole from an azide and a terminal alkyne in generally excellent yield. With this reaction, we designed a chemical assay system to assess the potential of click-based chemical assay-guided natural product isolation. Herein, we present a prototype system designed for terminal alkyne-containing substances, which exhibit a wide array of biological properties. 10</p><!><p>As shown in Fig. 1, the designed alkyne sensing chemical assay system consists of a uorescent chemodosimetric probe, a linker, and a solid support. The uorescent chemodosimetric probe 11 is the key moiety of our system; it captures terminal alkyne groups through the CuAAC reaction and produces a uorescence signal. Immobilization of the uorescent probe on a solid support was adopted for easy handling and tracking. Consequently, the presence of compounds with a terminal alkyne group in the analyte would lead to uorescent responses from the solid bead. In addition, via a cleavable linker, our sensing system allows the captured target component to be released from the bead as a CuAAC product for subsequent spectroscopic analysis.</p><p>The proper choice of solid support and linker is critical to the success of any solid-phase chemistry. TentaGel™ MB amino resin was chosen as a solid support because of its high swelling capacity in various solvents and its relatively large particle size.</p><p>The linker chosen was (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-linker (Fig. 2a), which can be readily cleaved by uoride ion under mild conditions. 12 The prouorophore 3azidocoumarin was chosen as a uorescent chemodosimetric probe because it is transformed into a highly uorescent triazolylcoumarin aer the CuAAC reaction with terminal alkynes. 13 The probe was indirectly attached to the PTMSEL linker by binding through glycolic acid, allowing facile analysis by reverse-phase LC/MS aer cleavage from the linker.</p><p>The alkyne sensing bead 1 was prepared with a loading level of 0.3 mmol g À1 using standard solid-phase synthesis techniques (see ESI †). With the alkyne sensing bead 1 in hand, we explored the optimal conditions for the CuAAC reaction. 1-Pentyne (0.9 mmol) was used as a model alkyne substrate. Aer investigating various sets of conditions, we observed that treatment of bead 1 (0.3 mmol, 1 mg) with CuSO 4 $5H 2 O (1.5 mmol), ascorbic acid (7.5 mmol), N,N-diisopropylethylamine (15.0 mmol), acetic acid (30.0 mmol), and tris[(1-benzyl-1H-1,2,3triazol-4-yl)methyl]amine (3.0 mmol) in DMF (ca. 50 mL) at room temperature generated the desired triazolylcoumarin product in nearly quantitative yield 14 without undesirable side reactions. 15,16 When 1-pentyne was reacted with bead 1 under the aforementioned conditions, uorescence emission was observed from the beads under UV light. However, in a control experiment without the terminal alkyne, no uorescence signal was observed. The uorescence of these "reporter beads" was clearly distinguished by conventional uorescence microscopy (Fig. 2b).</p><p>The resulting on-resin uorescent product was cleaved from the solid support by treatment with TBAF$3H 2 O and subsequently analyzed by LC/MS. Because the detached compound had a UV-active functionality and a hydrophilic carboxylic acid group, as exemplied by compound 3 (Fig. 2e), the analysis was readily achieved using a conventional LC/MS system equipped with a diode-array UV detector and a reverse-phase column. The LC chromatogram of the analyte released from the uorescent beads (analyte B) showed only one peak, which was identied as the molecular ion [M À H] À at m/z 328 in the ESI negative ionization mode. The molecular mass (329 Daltons) of this click product matched well with the theoretical value for   A) also showed only one peak with a molecular ion at m/z 260 ([M À H] À ), which matched well with the theoretical molecular weight of unreacted 3-azidocoumarin 2 (Fig. 2d).</p><p>To further validate the system, a mixture of different terminal alkynes was assayed. A mixture of equimolar amounts of four terminal alkynes 4a-d (0.2 equiv. each with a maximum loading level of beads) was treated with bead 1 (Fig. 3a). The resulting uorescent beads were treated with TBAF$3H 2 O, and the mixture of detached compounds was analyzed by LC/MS. As shown in Fig. 3b, ve peaks were observed in the LC/MS chromatogram. The molecular masses of the peaks exactly matched the theoretical values of triazolylcoumarins 5a-d and 3-azidocoumarin 2. Furthermore, the integration values of the ve peaks were similar, corresponding to the theoretical ratio. The chemical yields of each compound, estimated from the integration of peak areas, were nearly quantitative. 14 This result revealed that the relative quantication of terminal alkynes is possible with this sensing system (see ESI †).</p><p>To demonstrate the utility of alkyne sensing bead 1 in the natural product isolation process, it was rst used in the analysis of the methanol extract of the leaves of Litsea japonica (Lauraceae). A literature search revealed that four terminal alkyne compounds 6a-d have been isolated from the leaves of L. japonica by two independent groups (Fig. 4a). 17 As expected, the leaf methanol extract (3 mg) exhibited uorescence activation aer the click reaction with our alkyne sensing bead 1 (0.5 mg). In the LC/MS analysis of the off-bead mixture, ve peaks were observed in the LC/MS chromatogram in addition to the peak of 2 (Fig. 4b). The deduced molecular mass (521 Daltons) of the major peak (retention time: 18.65 min) matched well with the theoretical molecular weight of 7a, the triazolylcoumarin product resulting from the CuAAC reaction of 6a. The other four peaks exhibited the same m/z value of 538 ([M À H] À ). The  deduced molecular masses (539 Daltons) are in good agreement with those of triazolylcoumarins 7b-d. These results suggested the presence of four terminal alkyne compounds 6a-d and one additional, as yet unidentied terminal alkyne compound with a molecular weight of 278 in the leaves of L. japonica, although we could not exclude the possibility that the stereoisomers of 6a-d are the structures of natural terminal alkynes.</p><p>The methanol extract of the stem heartwood of the same plant (3 mg) also activated the uorescence of the beads. However, the cleaved products showed a somewhat different pattern of peaks in the LC/MS chromatogram compared to the leaf extracts, suggesting that the levels of specic terminal alkyne natural products 6a-d varied among different parts of the plant (Fig. 4c). Because of the characteristic and intense UV absorption of the triazolylcoumarin moiety, the quantication of individual terminal alkyne compounds was possible using the standard curve method. For example, on the basis of the respective integration values, compound 6a is estimated to exist in the methanol extract of the leaves at a concentration of approximately 1.0 mg g À1 , whereas it is present at a concentration of approximately 0.2 mg g À1 in the extract of the stem heartwood (see ESI †).</p><p>Encouraged by the successful results with the crude extract of L. japonica, we performed further investigations to isolate terminal alkyne compounds from the extracts of natural products using the alkyne sensing bead 1. Among the tested crude natural plant extracts of various origins, uorescence activation was observed for the methanol extract of the whole plant of Chrysanthemum morifolium (Compositae) (Fig. 5a and ESI †), from which no terminal alkyne compounds have previously been isolated. Analysis of the LC/MS chromatogram aer resin cleavage indicated the presence of three terminal alkyne compounds. To isolate the terminal alkyne component corresponding to the major peak, 30 g of the methanol extract was suspended in water and successively extracted with hexane, chloroform, ethyl acetate, and n-butanol (Fig. 5b). A small portion of each fraction was subjected to the click reaction with alkyne sensing bead 1. Fluorescence activation was observed only in the hexane fraction. Fractionation of the hexane extract over silica gel produced een different fractions (A-O). The chemical assay with bead 1 and LC/MS analysis indicated that the desired component was present primarily in fraction M. Purication of fraction M by semi-preparative reverse-phase HPLC with the guidance of the extracted ion chromatograms afforded a pure compound with an m/z value of 278 [M + H] + as a white solid. Through analysis of the spectroscopic data, the isolated compound was determined to be terminal diyne 8. 18 The isolated compound is a previously reported natural product. 19 However, this is the rst report of the known natural compound 8 being isolated from the extract of C. morifolium.</p><!><p>In conclusion, we have developed a novel prototype chemical assay-guided natural product isolation method. Click chemistry was used in the chemical assay system to reliably identify target compounds without interference from other components in the extracts. A click reaction-induced uorescence sensing platform was employed to sensitively detect even low concentrations of the target compounds and to permit visualization. Immobilization of this "sensing and reporting" moiety on a solid support was adopted for easy handling and tracking. The presented chemical assay system was designed for the detection of terminal alkyne-containing natural products using CuAAC, the prototypical click chemistry reaction. Our newly designed alkyne sensing bead 1 can successfully and quantitatively identify terminal alkyne compounds on the basis of a uorescence signal. Using this bead, we were able to identify, quantify, and isolate terminal alkyne natural products from plant extracts, demonstrating the prospect of chemical assay-guided natural product isolation. Further studies, including the extension of this chemical assay system to other functional groups through the use of other viable click chemistry reactions, are needed to robustly establish chemical assay-guided natural product isolation. We believe that our "click reaction/uorescent chemodosimetric probe/solid support" chemical assay system could also be applicable to numerous other elds, such as metabolomics and food science, which require the analysis of complex mixtures of compounds.</p>