基於 BODIPY 的螢光探針和生物偶聯物的設計和合成及其在生物系統上的應用

Abstract

神經甾體孕烯醇酮 (P5) 可促進神經微管聚合延伸，減輕與精神分裂症相關的壓力和負面症狀，促進記憶增強，並有助於恢復脊髓損傷。在類固醇生成過程中，孕烯醇酮是第一個從膽固醇中獲得的神經類固醇，它會迅速通過酶代謝轉化為下游類固醇家族成員。因此，很難區分 P5 與大腦中其他下游類固醇的生物活性。為了解決這個問題，在論文第一部分，我們合成並篩選了孕烯醇酮衍生物，通過修飾孕烯醇酮的 C-3、C-5、C-7 和 C-20 位置，希望延緩其代謝機制，同時測試了它們增強微管延伸和神經發育的能力。化合物 JJS-043 被發現是 P5 的有效類似物；它可增強微管聚合，穩定活細胞中的微管動力學，加速神經軸突生長，同時可改變了小鼠小腦顆粒神經元培養物中的生長錐形態。另一方面，它也可促進了斑馬魚小腦軸突中穩定微管軸突的形成。 與孕烯醇酮不同的是，化合物JJS-043不易被代謝，但在體內可維持與 P5 一樣功能活性，可被視為神經發育疾病的治療候選藥物。 結直腸癌 (CRC) 病例在全球範圍內不斷激增，迫切需要新藥來治療 CRC。Signal-transducer and activator of transcription-1 (STAT1) 在腫瘤發生中具有複雜的作用機制，其在 CRC 中的作用仍需深入了解。在論文第二部分，我們發現在 CRC 患者和小鼠模型中，降低 STAT1的磷酸化活性，可抑制腫瘤生長。基於配體的高通量篩選揭示了大豆中富含的大豆異黃酮化合物“金雀異黃酮”（THIF：4',5,7-三羥基異黃酮），是一種新的 STAT1 抑制劑。我們合成了基於 BODIPY 的金雀異黃酮螢光探針用以研究 THIF 和 STAT1 的抑制機制作用。 我們的研究顯示THIF 可阻止 STAT1-STAT1 二聚化，消除了 CRC 中的干性和血管生成，並且小鼠模型中的體內結果減弱了腫瘤生長。除此之外，我們也合成了基於金雀異黃酮的 PROTAC（靶向嵌合體的蛋白水解）降解劑，用以靶向 CRC 中的 STAT1 降解，此研究正持續進行中。 細胞表面的異常醣基化被認為是癌症的標誌之一。追踪此類醣基化途徑有助於了解腫瘤的進展、診斷和治療發展。在論文第三部分，我們採用聚醣代謝工程 (MOE) 來修飾聚醣，其中含有一種含有獨特烯烴標記的生物正交化學報告基團的非天然單醣前體，可用於聚醣代謝工程，並通過生物合成結合到活細胞的多醣中，藉由生物正交化學反應，可與 BODIPY 探針的互補生物正交四嗪官能團發生反應，進行共價螢光標定。我們設計的BODIPY是具有螢光發光性質的探針，發射範圍從綠色到紅色發射 BODIPY 骨架。我們目前的研究表明，這些探針是烯烴功能化單醣的特異性螢光標記試劑，可適用於通過共聚焦顯微鏡觀察細胞中烯烴標記的醣基化共軛物的定位及追蹤。

Neurosteroid pregnenolone (P5) promotes microtubule polymerization, alleviates stress and negative symptoms associated with schizophrenia, promotes memory enhancement, and helps recover spinal-cord injury. During steroidogenesis, pregnenolone is the first neurosteroid obtained from the cholesterol, and it quickly undergoes enzymatic conversion into the downstream steroids. Therefore, it is difficult to distinguish the biological activity of P5 versus other downstream steroids in the brain. To address this issue, we synthesized and screened pregnenolone derivatives to delay its further metabolism via modification at C-3, C-5, C-7, and C-20 positions and tested their ability to enhance microtubule elongation and neural development. Compound JJS-043 was found to be a potent analog of P5; it enhanced microtubule polymerization, stabilized microtubule dynamics in live cells, accelerated neurite growth, and changed the growth cone morphology in mouse cerebellar granule neuronal-culture. It also promoted the formation of stable microtubule tracks in cerebellar-axons of zebrafish. JJS-043, unlike pregnenolone, resist metabolism but recapitulates P5 functions in-vivo and can be considered a therapeutic candidate for neuro-developmental diseases. Colorectal cancer (CRC) cases have a constant surge globally, and there is a urgent need for new medicines to treat CRC. Signal-transducer and activator of transcription-1 (STAT1) have a complex role in tumorigenesis, and its role in CRC still requires in-depth comprehension. As part of this work, an alleviated STAT1 was found in patients with CRC and the mouse model. A ligand-based high-throughput screening revealed a flavonoid abundant in soybeans, “genistein” (THIF: 4′,5,7-trihydroxyisoflavone), a new inhibitor of STAT1. A BODIPY-based genistein fluorescent probe was synthesized to study the mechanistic action of THIF and STAT1. THIF prevented the STAT1-STAT1 dimerization, abolished stemness and angiogenesis in CRC, and the in-vivo results in mice model attenuated tumor growth. In addition, genistein-based PROTAC (Proteolysis targeting chimera) degraders were synthesized to target STAT1 degradation in CRC. Abnormal glycosylation on the cell-surface is considered one of the hallmarks of cancer. Tracking down such glycosylation pathways can help to understand tumor progression, diagnosis and therapeutic development. As part of this work, we employed Metabolic oligosaccharide engineering (MOE) to modify glycans with an unnatural monosaccharide precursor containing a unique alkene tagged bioorthogonal chemical reporter group. The glycans are biosynthetically incorporated to the living cells and allowed to react with, a complementary bioorthogonal tetrazine functional group covalently linked to a set of BODIPY probes. Our designed BODIPY-based probes are fluorogenic with emission ranges from green to red-emitting BODIPY scaffold. Our present study indicates that these probes are specific fluorogenic labeling reagents for alkene-functionalized monosaccharides and is suitable for visualizing the localization of alkene-tagged glycosyl conjugates in cells by confocal microscopy.