唾液酸是一种含有九个碳的酸性单糖，通常存在于细胞和糖蛋白表面糖聚合物上糖链的末端。这些唾液酸化聚糖具有多种生理生化功能，同时唾液酸也被鉴定为不同类型癌症的标志物用于早期诊断和治疗。鉴于唾液酸在生物学和生理学中的重要作用，对其准确、简单和灵敏地定量在人类健康状况的临床诊断和评估中具有重要意义。目前已经开发了各种技术来定量唾液酸及其衍生物单糖，但大多都存在特异性差和灵敏性低等不足。此外，LC-MS法常用的传统唾液酸衍生剂（例如OPD、DMB和DMBA）在质谱离子化过程中，由于异质离子的加入降低了检测的灵敏度。因此，开发新型唾液酸衍生剂用于高特异性和高灵敏性的LC-MS检测具有重要意义。

离子液体类探针（ITag）在糖化学和糖生物学中有着广泛的应用。咪唑基ITag由于其易于合成并且能够结合其他不同的化合物结构，而作为ITag中常用的一种类别。目前，ITag在糖科学中的最新发展突显了它们作为质谱技术中灵敏探针的巨大潜力。咪唑类离子液体自身带有的永久性正电荷的特性与非离子化合物相比，可以显著增加质谱信号，适用于复杂生物基质中痕量唾液酸的鉴定。

因此，基于上述研究背景和技术现状，本文设计并合成了一种基于芳香胺咪唑类离子液体的新型唾液酸衍生剂（DAPMI），经过结构表征、功能验证及衍生条件优化后，对其在UPLC-ESI-MS上的检测灵敏度进行定量分析，并将其应用于不同物种（脊椎动物和软体动物）唾液酸类型及含量的鉴定。针对不同物种表现出的唾液酸组成差异性，本文围绕唾液酸代谢中的关键酶，唾液酸醛缩酶（NPL），深入探索了不同物种来源重组NPL的底物特异性，以期阐明不同物种唾液酸的体内组成差异与唾液酸的生物合成途径的相关性，为解释唾液酸组成的种属差异提供分子机制支撑。具体研究结果如下：

1. 基于芳香胺咪唑类离子液体的新型唾液酸标记物的设计与合成

本文采取将自身带有正电荷的咪唑基探针（ITag）和具有功能性芳香胺结构的液相荧光衍生物相结合，设计并合成了一种新型的芳香胺咪唑基唾液酸标记物（DAPMI）。该衍生剂可以选择性地与唾液酸中的酸性部分反应，同时将唾液酸本身转化为带正电的物质，以提高MS质谱离子化效率和检测灵敏度，NMR和HRMS表征结果显示DAPMI的结构正确且纯度较高。

然后，本文对DAPMI衍生唾液酸的功能进行了验证，采用HPLC-ESI-MS证明它可以实现对唾液酸（Neu5Ac、Neu5Gc和KDN）及经过基团修饰的唾液酸类似物进行衍生，验证了该法的可行性和普适性。为了提高衍生效率，本文对不同的衍生条件进行优化，以Neu5Ac为标准唾液酸进行衍生，并使用HPLC检测，条件优化结果如下：DAPMI衍生唾液酸的最适温度为80 °С，最佳衍生时间为45 min，最适衍生剂DAPMI浓度为20 mg/mL和还原剂NaHSO₃的浓度为100 mM；最佳衍生溶剂为水溶液。DAPMI衍生后的储藏稳定性显示无论是在4 °С还是20 °С的避光或室内光条件下产物具有较好的稳定性。

2. 基于DAPMI衍生唾液酸的检测性能分析

本文为了进一步分析DAPMI衍生化唾液酸在液相色谱的荧光强度（UPLC-FLD）和电喷雾质谱（ESI-MS）离子化效率方面的性能，以实现定量分析DAPMI的HPLC-ESI-MS检测灵敏度。为了体现DAPMI的优良衍生效率及检测灵敏性，还与传统衍生剂（OPD）进行了比较。经过两种衍生剂的大体系衍生制备得到衍生化唾液酸OPD-Neu5Ac（2.2 mg）和DAPMI-Neu5Ac（8.8 mg），并采用核磁波谱（NMR）和高分辨质谱（HRMS）验证了分子结构正确并且纯度较高。荧光扫描显示，DAPMI-Neu5Ac的最佳激发波长/发射波长为356 nm/412 nm，OPD-Neu5Ac的最佳激发波长/发射波长为354 nm/416 nm。

衍生化唾液酸的检测限和定量限定量分析表明：在UPLC-FLD检测中，OPD-Neu5Ac的检出限和定量限分别为512 fmol和1512 fmol，而DAPMI-Neu5Ac的检出限和定量限分别为380 fmol和1505 fmol。在ESI-MS质谱检测中，OPD-Neu5Ac的LOD和LOQ分别是326.15 fmol和890.67 fmol，而DAPMI-Neu5Ac的LOD和LOQ分别是2.55 fmol和28.78 fmol。综上，DAPMI衍生唾液酸的荧光强度与传统唾液酸衍生剂OPD衍生唾液酸相比荧光强度略高，而质谱结果显示DAPMI衍生唾液酸的质谱响应值即在ESI-MS中的离子化效率和检测灵敏度显著提高，与OPD衍生唾液酸相比提高了130倍，检测限LOD（S/N=3）降低了130倍，定量限LOQ（S/N=10）降低了31倍。此外，当在各自的激发和发射最大值下检测时，DAPMI-Neu5Ac在FLD分析中的荧光检测灵敏度略高于OPD衍生的Neu5Ac。

本文还与传统唾液酸检测方法（酶法、化学法）的检测性能进行对比，结果显示这些方法灵敏度和特异性不高，并且适用于检测唾液酸含量较高的样本，只能测定总唾液酸含量，无法分开鉴定不同种类的唾液酸，这更加突出了DAPMI高效的检测性能。

3. 新型唾液酸标记物DAPMI用于不同物种唾液酸组成差异性研究

本文验证了咪唑类离子液体唾液酸标记物（DAPMI）应用于复杂生物样本中唾液酸检测的可行性，尤其适用于微量唾液酸的检测。DAPMI分别对脊椎动物（人、CMAH小鼠、禽蛋）和软体动物（牡蛎）中的唾液酸进行鉴定。

通过外标法对人血清和CMAH小鼠组织中的唾液酸分析结果表明，在野生型和杂合型CMAH（+/-）小鼠各组织中Neu5Ac/Neu5Gc比例不同。在纯合子CMAH（-/-）小鼠的组织和人血清中检测到了微量的Neu5Gc。为了减少定量误差，本文利用“一釜五酶”法生物合成了唾液酸内标物（Neu5FoGalX），并且MALDI-ToF-MS、NMR和HRMS证实了Neu5FoGalX具有正确的结构和较高的纯度。将此内标物应用于ESI-MS定量分析禽蛋中的唾液酸，结果显示禽蛋中的唾液酸由Neu5Ac及少量的KDN组成。此外，本文在牡蛎的唾液酸中首次发现了游离KDN，ESI-MS/MS表明牡蛎中的唾液酸主要由KDN构成，还存在少量的KDN类似物（5-epi-KDN和5,7-di-epi-KDN）。

4. 不同物种唾液酸生物合成途径的差异比较

针对脊椎动物和软体动物中的唾液酸在组成及含量上的差异性，本文进一步对不同物种生物合成途径进行比较，尤其是唾液酸醛缩酶参与的唾液酸代谢途径。本文选择几种代表性的脊椎动物（鸡）和软体动物（牡蛎、蜗牛）来源的唾液酸醛缩酶（NPL）并探究他们对唾液酸（Neu5Ac、KDN及其类似物）的合成与分解的催化活性。经过基因合成、重组蛋白表达与纯化后，获得家禽鸡NPL（chNPL）、淡水蜗牛NPL（sNPL）和太平洋牡蛎（CgNPL），采用TLC、化学法、酶法及HPLC-ESI-MS法进行底物活性鉴定的结果显示，软体动物源NPL（sNPL和CgNPL）无法催化由ManNAc和丙酮酸生成Neu5Ac的可逆反应，但可催化一系列己醛糖（甘露糖、葡萄糖和半乳糖）与丙酮酸生成相应的KDN及KDN类似物（5-epi-KDN和5,7-di-epi-KDN），这种催化活性的区别与不同动物体内唾液酸组成差异性具有较强的相关性。

Sialic acid is a nine-carbon acidic monosaccharide typically found at the terminal ends of glycan chains on cell surfaces and glycoprotein-associated sugar polymers. These sialylated glycans participate in various physiological and biochemical functions, and sialic acid has also been identified as a biomarker for different types of cancers, aiding in early diagnosis and treatment. Given the significant role of sialic acid in biology and physiology, its accurate, simple, and sensitive quantification is of great importance for clinical diagnosis and assessment of human health. Various techniques have been developed to quantify sialic acid and its derivative monosaccharides, but most suffer from limitations such as poor specificity and low sensitivity. Additionally, traditional sialic acid derivatization reagents (e.g., OPD, DMB, and DMBA) commonly used in LC-MS methods reduce detection sensitivity during mass spectrometry ionization due to the introduction of heterogeneous ions. Therefore, developing novel sialic acid derivatization reagents for highly specific and sensitive LC-MS detection is of significant importance.

Ionic liquid-based probes (ITags) have broad applications in glycochemistry and glycobiology. Among them, imidazole-based ITags are widely used due to their ease of synthesis and ability to incorporate diverse compound structures. Recent advancements in ITags within glycoscience highlight their great potential as highly sensitive probes in mass spectrometry. The inherent permanent positive charge of imidazole-based ionic liquids can significantly enhance mass spectrometry signals compared to non-ionic compounds, making them suitable for identifying trace sialic acids in complex biological matrices.

Based on the above research background and current technological landscape, this study designed and synthesized a novel sialic acid derivatization reagent (DAPMI) based on an aromatic amine-imidazole ionic liquid. After structural characterization, functional validation, and optimization of derivatization conditions, its detection sensitivity on UPLC-ESI-MS was quantitatively analyzed, and it was applied to identify the types and concentrations of sialic acids in different species (vertebrates and mollusks). Given the observed variations in sialic acid composition among species, this study focused on a key enzyme in sialic acid metabolism — sialic acid aldolase (NPL) — to explore the substrate specificity of recombinant NPLs from different species, aiming to elucidate the correlation between in vivo and in vitro sialic acid synthesis. The specific research findings are as follows:

1. Design and Synthesis of a Novel Sialic Acid Tagging Reagent Based on Aromatic Amine-Imidazole Ionic Liquid

This study describes the design and synthesis of DAPMI by combining a positively charged imidazolium tag (ITag) with a functional aromatic amine-based fluorescent derivatization group. This derivatization reagent selectively reacts with the carboxyl group of sialic acids while converting them into positively charged species to enhance MS ionization efficiency and detection sensitivity. NMR and HRMS analyses confirmed the correct structure and high purity of DAPMI.

We then validated DAPMI's functionality for sialic acid derivatization using HPLC-ESI-MS, demonstrating its applicability to sialic acids (Neu5Ac, Neu5Gc, and KDN) and their modified analogs, confirming the method's feasibility and universality. To optimize derivatization efficiency, we systematically evaluated reaction conditions using Neu5Ac as the standard substrate, with HPLC detection. The optimized conditions were: optimal temperature of 80°C, derivatization time of 45 minutes, DAPMI concentration of 20 mg/mL, NaHSO3 concentration of 100 mM, and aqueous reaction medium. Stability studies showed that DAPMI-derivatized products maintained good stability under both 4°C and 20°C storage conditions, whether protected from light or exposed to ambient light.

2. Performance Analysis of DAPMI-Derivatized Sialic Acids

This study further analyzed the performance of DAPMI-derivatized sialic acids in terms of fluorescence intensity (UPLC-FLD) and electrospray ionization efficiency (ESI-MS) to quantitatively evaluate the detection sensitivity of DAPMI in HPLC-ESI-MS. To demonstrate DAPMI's superior derivatization efficiency and detection sensitivity, we conducted comparative studies with the traditional derivatization reagent OPD. Large-scale derivatization using both reagents yielded OPD-Neu5Ac (2.2 mg) and DAPMI-Neu5Ac (8.8 mg), with their molecular structures and high purity confirmed by NMR and HRMS. Fluorescence scanning revealed optimal excitation/emission wavelengths of 356 nm/412 nm for DAPMI-Neu5Ac and 354 nm/416 nm for OPD-Neu5Ac.

Limit of detection (LOD) and quantification (LOQ) analyses showed: (1) in UPLC-FLD: OPD-Neu5Ac (LOD=512 fmol, LOQ=1512 fmol) vs DAPMI-Neu5Ac (LOD=380 fmol, LOQ=1505 fmol); (2) In ESI-MS: OPD-Neu5Ac (LOD=326.15 fmol, LOQ=890.67 fmol) vs DAPMI-Neu5Ac (LOD=2.55 fmol [130-fold improvement], LOQ=28.78 fmol [31-fold improvement])

While DAPMI-derivatized sialic acids showed slightly higher fluorescence intensity than OPD derivatives, their MS response values (reflecting ionization efficiency and detection sensitivity) were dramatically improved - 130-fold higher for LOD (S/N=3) and 31-fold higher for LOQ (S/N=10). Additionally, when detected at their respective maximum excitation/emission wavelengths, DAPMI-Neu5Ac showed slightly higher sensitivity in FLD analysis than OPD-derivatized Neu5Ac.

Comparative studies with traditional sialic acid detection methods (enzymatic and chemical assays) revealed their limitations in sensitivity and specificity, as they could only measure total sialic acid content without distinguishing between different sialic acid types, further highlighting DAPMI's superior detection performance.

3. DAPMI-Based Analysis of Species-Specific Sialic Acid Composition Variations

This study validated the feasibility of applying imidazolium ionic liquid sialic acid tags (DAPMI) to detect sialic acids in complex biological samples, particularly for trace-level analysis. DAPMI was used to profile sialic acids in vertebrates (humans, CMAH mice, avian eggs) and mollusks (oysters).

External standard analysis of human serum and CMAH mouse tissues revealed different Neu5Ac/Neu5Gc ratios across wild-type and heterozygous CMAH (+/-) mouse tissues. Trace Neu5Gc was detected in homozygous CMAH (-/-) mouse tissues and human serum, likely due to dietary intake of Neu5Gc-containing foods.

To minimize quantification errors, we biosynthesized a sialic acid internal standard (Neu5FoGalX) using a "one-pot five-enzyme" method, with its structure and high purity confirmed by MALDI-ToF-MS, NMR, and HRMS. Application of this internal standard in ESI-MS quantification revealed that avian egg sialic acids consisted primarily of Neu5Ac with minor KDN components.

Free KDN was identified for the first time in oysters. ESI-MS/MS analysis showed oyster sialic acids were predominantly KDN, with minor amounts of KDN analogs (5-epi-KDN and 5,7-di-epi-KDN).

4. Comparison of Differences in Salic Acid Biosynthetic Pathways among Different Species

To investigate the interspecies differences in sialic acid composition, we characterized the catalytic activities of representative NPL enzymes from vertebrates (chicken) and mollusks (oyster, snail) toward sialic acid (Neu5Ac, KDN and analogs) synthesis and degradation. Following gene synthesis, recombinant protein expression and purification, we obtained chicken NPL (chNPL), freshwater snail NPL (sNPL), and Pacific oyster NPL (CgNPL). Activity assays using TLC, chemical methods, enzymatic assays, and HPLC-ESI-MS revealed that molluskan NPLs (sNPL and CgNPL) could not catalyze the reversible reaction of Neu5Ac synthesis from ManNAc and pyruvate, but could catalyze the formation of KDN and its analogs (5-epi-KDN and 5,7-di-epi-KDN) from various hexoses (mannose, glucose, galactose) and pyruvate. This catalytic difference likely contributes to the distinct sialic acid profiles observed in different species.

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