合成類法呢焦磷酸酯之螢光探針用以探討基質與十一異戊二烯焦磷酸酯合成酶之交互作用

十一異戊二烯焦磷酸合成酶 (Undecaprenyl Pyrophosphate Synthase, UPPs) 催化其天然受質法呢焦磷酸酯 (Farnesyl Pyrophosphate, FPP) 與八個異戊二烯焦磷酸酯 (Isopropyl Pyrophosphate, IPP) 進行縮合生成十一異戊二烯焦磷酸酯類似物 (Undecaprenyl Pyrophosphate, UPP)，其在細菌細胞壁的合成中扮演將lipid II由細胞膜內翻轉至細胞膜外的角色。本論文設計四個FPP螢光類似物以追蹤細菌細胞壁的生合成。希望此四個FPP螢光類似物可以被UPPs辨識為天然受質並進行後續的鏈延長生合成反應，且能與已經修飾在UPPs底部的螢光團Alexa Fluoro 488 產生螢光共振能量轉移 (Fluorescence resonance energy transfer, FRET) 的現象，藉此來觀測鏈延長的過程。而另一目的則可以利用螢光性質來追蹤此生合成的產物最後座落的位置。這四個FPP螢光探針分別為BD-GPP、DC-GPP、3HF-GPP以及GPP-3HF。其組成主要由螢光團連接香葉醇基焦磷酸酯 (geranyl pyrophosphate, GPP)，如圖示。我們主要選擇三個放射峰在500 nm的螢光團。其中之一為boron-dipyrromethene (BODIPY)，BODIPY 連接GPP的骨架結構與FPP相當類似，且此螢光團具有量子產率高以及光穩定性高的特性趨使我們利用此螢光團做為此系統的運用。而另一個螢光團為dicyanomethylenedihydrofuran (DCDHF)，其具有分子結構極強的推拉偶極作用易於調控其螢光放射性質以及螢光團結構利於修飾小分子等特色。而第三個螢光團為3-hydroxyflavone (3-HF)。此螢光團在單一激發波長下可以有兩個不同波長的螢光放射峰，且此兩螢光放射峰的比例與環境極性密切相關，此性質也趨使我們將其運用在此系統。將此四個FPP螢光類似物測試其與UPPs的生物活性後卻發現皆無法進行鏈延長的反應。所以無法與已修飾於UPPs內部的Alexa Fluoro 488 產生FRET的現象，但此四個化合物可以當作UPPs良好的抑制劑。

關鍵字

類法呢焦磷酸螢光探針

並列摘要

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C55 undecaprenyl pyrophosphate (UPP), which is a carrier transporting lipid II across the membrane in the biosynthesis of bacteria cell wall. Four FPP fluorescent analogues are designed to aim for probing the biosynthetic pathway of bacterial cell wall. It is hoped that these FPP analgues can bind with UPPs as its natural substrate, and follow the original biosynthetic pathway. It is also hoped that these FPP analogues have fluorescence resonance energy transfer phenomena to Alexa Fluoro 488 that has been modified in the active site of UPPs when the chain elongation reaction occurs. In addition, we can apply the fluorescence property for probing the position of the biosynthetic product. These FPP fluorescent analogues, denoted as BD-GPP、DC-GPP、3HF-GPP and GPP-3HF are composed of fluorophores and geranyl pyrophosphate (GPP) (see figure). Three fluorophores which emit at 500 nm are chosen. One of them is boron-dipyrromethene (BODIPY). The framework of BODIPY connected with geranyl pyrophosphate is similar to UPPs natural substrate, and its high quantun yield and photostability motivate us to apply it to our probe design. The second fluorophore is called dicyanomethylenedihydrofuran (DCDHF). The push-pull structural characteristics renders modification of its photophysical properties possible, and its amino group can be derived easily. The third fluorophore is 3-hydroxyflavone, which has dual emissions and the ratios of the two emissions depending on the polarity of microenvironment. This property that one fluorophore has dual emission stimulates us to apply this fluorophore to this system. After testing these four FPP analogues binding and biological properties activity with UPPs, we find these four FPP analogues can not proceed chain elongation reactions when bound in the active site of UPPs, but all of them can be reasonably good inhibitors.

並列關鍵字

FPP fluorescent analogues

參考文獻

1. Wang, K. C.; Ohnuma, S.-I. Biochim. Biophys. Acta 2000, 1529, 33-48.

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12. Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A.1995, 92, 5008-5011.

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14. Gibbs, R. A.; Krishnan, U.; Dolence, J. M.; Poulter, C. D. J. Org. Chem. 2002, 60, 7821-7829.

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合成類法呢焦磷酸酯之螢光探針用以探討基質與十一異戊二烯焦磷酸酯合成酶之交互作用

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