CN112354519A - 一种含有磷酰胺结构的固定相以及制备蛋白质谱毛细管柱的方法 - Google Patents
一种含有磷酰胺结构的固定相以及制备蛋白质谱毛细管柱的方法 Download PDFInfo
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Abstract
本发明公开了一种含有磷酰胺结构的硅胶固定相，一种制备上述硅胶固定相并进行固定相填充毛细管，制备蛋白质谱色谱柱的方法。二氯磷酸苯酯与长链醇反应得到烷烃氯磷酸苯酯，氨丙基硅胶与烷烃氯磷酸苯酯在干燥的溶剂中反应得到含有磷酰胺结构的固定相，进行石英毛细管装填，制备得到蛋白质谱毛细管柱。相较于碳羰基与硫羰基，磷酰胺结构更接近中等强度酸碱配合物形式，有效对氨基酸、蛋白质类化合物产生的氢键螯合作用，与传统酰胺键结构相比，总体氢键结合能力适中，且完全适用于蛋白质各级结构样本分离。所制备的色谱柱适用于中性氨基酸及所组织的多肽形式分离。
Description
技术领域
本发明涉及色谱固定相，具体涉及一种含有磷酰胺结构的硅胶固定相，以及将所述固定相进行蛋白质谱毛细管制备的方法，本发明属于色谱固定相领域。
背景技术
色谱柱是色谱分离的核心，对蛋白质谱所需要的分离填充毛细管色谱柱来说，色谱填料直接影响其对分析物的选择性和分离效率。近十年来，在蛋白质谱领域软电离方法的迅速发展和成熟不令使质谱技术本身得到长足的发展，而且使它在生命科学领域的应用大为拓展。高效液相-质谱联用通用技术则更是对毛细管蛋白色谱柱的高分辨率、高灵敏度等优点和质谱方法的高精确度，提出了更高的要求。目前，色谱固定相大多以二氧化硅为基质进行表面化学修饰制备而成，根据所键合官能团的不同具有不同的色谱分离机制，进而为分离复杂样品提供多种选择。早期硅胶均预先经过硅烷化试剂处理，而氨丙基硅胶被认为是最适用于制作改性、接枝固定相的载体，因为氨基的极性低于硅羟基，可减少硅胶表面裸露的酸性硅羟基与手性化合物之间的非立体选择性作用。而且由于氨基与纤维衍生物之间易形成氢键，可使衍生物分子有序地排列在担体表面, 这有利于其与手性分子的相互作用。
当前，对蛋白质分离的要求在亲水/油相互作用分离模式下，要求强极性、强亲水性物质均可分离检测，弥补了常规色谱固定相的不足。流动相由一定盐浓度的水溶液和高比例的有机溶剂组成，固定相是强极性吸附剂，分析物通常是极性化合物，因此填料越亲水、溶剂的极性越强，溶质在柱子上的保留越强；随着水系比例的增加，溶质被逐渐洗脱，从而使待分离物质得以分离。近年来，有大量文献报道将极性官能团的有机小分子和亲水碳纳米材料键合到硅胶表面，在模式下用于碱基核苷、氨基酸、天然药物等亲水性物质的分离检测。然而目前，中性氨基酸及其组织的多肽形式的分离，仍然需要借助衍生化反应，难以直接分离。
磷酰胺结构除了质子可以作为亲电试剂活化负电性基团外，在多种耦合反应中，磷酸基团的P═O在实验现象与理论计算方面都被证实可以作为碱性基团接受质子或与活泼氢形成缔合作用。碱性位点是P═O基团，酸性位点为亚胺基团。磷酰胺结构在有机合成领域已有一定数量的报道，其中最为典型的是双官能团磷酸酯催化剂。以磷酸酯催化缩醛转移反应，对映选择性合成O,O-缩醛。缩醛转移反应一般需要强布朗斯特酸催化形成含有羰基碳鎓的中间体。缩醛转移反应的底物与产物结构相似，均为缩醛，其产物也可被强酸催化剂催化形成新的羰基碳鎓，导致最终产物的外消旋现象。而手性的、酸性温和的磷酸被证实可以很好控制体系中的异向产物。磷酸酯的P═O作为氢受体与底物的一个羟基形成氢键，而已经产生缔合作用的羟基将不直接参与缩醛反应，所得产物对映选择性高，er值大于80%。
目前将磷酰胺键引入色谱固定相未有报道，酰胺基团可极大程度的屏蔽硅羟基的作用。与普通反相键合相不同的选择性，尤其是对于极性化合物有不一样的选择性，有潜力在测定极性和碱性等中性氨基酸化合物时，具有极好的峰形，且对流动相选择较宽。
发明内容
本发明的目的是提供一种含有磷酰胺结构的硅胶固定相。
所述的一种含有磷酰胺结构的硅胶固定相具有以下结构：
其中，n为9—17的整数。
本发明的另一目的是提供一种制备磷酰胺结构的硅胶固定相的方法。
包括以下两个步骤：
（1）步骤1：二氯磷酸苯酯与多元醇反应得到烷烃氯磷酸苯酯；
具体措施为：和二氯磷酸苯酯溶于四氢呋喃中，加入氢化钠（分散于矿物油中）。室温下搅拌，向体系加入盐酸水溶液与长链醇再搅拌。分离出有机相，并减压浓缩。粗产物以硅胶色谱柱分离，首先以二氯甲烷/正己烷为洗脱剂，再以乙酸/二氯甲烷为洗脱剂。分离产物溶于二氯甲烷并加入3N盐酸水溶液搅拌。分离有机相，再以3N盐酸水溶液洗涤，以无水MgSO4干燥并过滤，减压旋蒸得到白色固体。
（2）步骤2：氨丙基硅胶与步骤1中所得的烷烃氯磷酸苯酯在干燥的溶剂中反应得到含有磷酰胺结构的固定相。步骤2中所述干燥的溶剂为甲苯、四氢呋喃、二甲苯中的一种。
具体措施为：步骤1所得固体，置入甲苯溶剂，在回流装置上加装分水器，在三口烧瓶中将氨丙基硅胶至于干燥溶剂中，加热至回流，利用恒沸蒸馏的方法，对硅胶进行脱水，加入步骤1中所得白色固体反应后加入咪唑和干燥过甲苯，氮气保护下加热回流。产物过滤后，用适量甲苯洗涤沉淀次，再用甲醇洗涤 3 次，最后加入甲醇/水混合溶液中，搅拌、抽滤，再用适量甲醇洗涤，将产品转移到培养皿中，真空干燥，得到含有磷酰胺结构的硅胶固定相。
步骤1中多元醇为碳原子个数为10~18的直链烷烃醇。
本发明还提供一种以上述固定相为原料制备蛋白质谱毛细管柱的方法，其特征在于以上述固定相为原料，进行石英毛细管装填，制备得到蛋白质谱毛细管柱。
具体措施为：截取石英毛细管，用氢氧化钠/氯化钠混合溶液处理15min，再用超纯水洗涤至流出液呈中性；然后用盐酸溶液处理0.5 h，再用超纯水洗涤至流出液呈中性，将处理后的石英毛细管用于脯氨酸键合硅胶固定相的装填。取脯氨酸键合硅胶加入玻璃小瓶，加入乙腈混匀；将小瓶放入填柱器内，磁力搅拌；安装洗涤过的石英毛细管，打开气阀，填柱压力600—800Bar；调料填充长度15 cm，压柱3h。封口液为硅酸钾、甲酰胺体积比3:1的混合溶液，封口长度2mm，制备得到脯氨酸键合硅胶蛋白质质谱色谱柱。
本发明有益效果：
1. 本发明提供一种具有磷酰胺结构的硅胶固定相，相较于碳羰基与硫羰基，磷酰胺结构更接近中等强度酸碱配合物形式，有效对氨基酸、蛋白质类化合物产生的氢键螯合作用，与传统酰胺键结构相比，总体氢键结合能力适中，且完全适用于蛋白质各级结构样本分离；
2. 本发明还提供了一种制备上述硅胶固定相的过程，及以所述硅胶固定相填充毛细管，制备蛋白质谱色谱柱的方法，所述色谱柱适用于中性氨基酸及所组织的多肽形式分离。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。实施例中所用填柱系统为：PC77-MAG高压装柱系列（尚域发展公司）。
实施例1
烷烃氯磷酸苯酯的制备
手套箱中将二氯磷酸苯酯4.0 mL和癸醇1.6 g溶于THF 20 mL 中，加入氢化钠（60%，分散于矿物油中，480 mg，20 mmol）。室温下搅拌24 h，向体系加入10%的盐酸水溶液与CH2Cl2再搅拌1 h。分离出有机相，并减压浓缩。粗产物以硅胶色谱柱分离，首先以50–100%的CH2Cl2/hexane为洗脱剂，再以3-6% EtOAc/CH2Cl2为洗脱剂。分离产物溶于CH2Cl2 并加入3N盐酸水溶液搅拌4 h。分离有机相，再以3N盐酸水溶液洗涤，以无水MgSO4干燥并过滤，减压旋蒸得到白色固体3.8 g。
实施例2
固定相的制备
在回流装置上加装分水器，在三口烧瓶中加入，再加入4.2 g丙氨基硅胶，加入100 mL干燥的甲苯中，加热至回流，利用恒沸蒸馏的方法，对硅胶进行脱水，将分出的甲苯/水的恒沸混合物冷却后放入专用废液瓶，反应1.5 h 后，将实施例1中所得固体3g加入到反应体系中，再加入4.0 g 咪唑和50 mL干燥甲苯，氮气保护下加热回流10 h。产物过滤后，用适量甲苯洗涤沉淀3次，再用甲醇洗涤3次，最后加入到体积比为9 :1的甲醇/水混合溶液中，50°C下搅拌30 min。抽滤，再用适量甲醇洗涤3次，将产品转移到培养皿中，50 °C 真空干燥8h，置于干燥器中备用。
实施例3
蛋白质谱色谱柱的制备
截取50 cm石英毛细管，用各1 moL/L氢氧化钠/氯化钠混合溶液处理15min，再用超纯水洗涤至流出液呈中性；然后用0.1 moL/L HCl处理0.5 h，再用超纯水洗涤至流出液呈中性，将处理后的石英毛细管用于脯氨酸键合硅胶固定相的装填。取脯实施例3中白色胶装固体2.0 g加入玻璃小瓶，加入5 mL甲苯混匀；将小瓶放入填柱器内，磁力搅拌；安装洗涤过的石英毛细管，打开气阀，填柱压力700Bar；调料填充长度15 cm，压柱3h。封口液为硅酸钾、甲酰胺体积比3:1的混合溶液，封口长度2mm。填柱在烘箱内50℃，制备得到脯氨酸键合硅胶蛋白质质谱色谱柱。
Claims (5)
1.在一种含有磷酰胺结构的固定相，其特征在于，所述固定相具有以下结构：
其中，n为9—17的整数。
2.一种含有磷酰胺结构的固定相，其特征在于，使用以下技术手段制备得到，包括以下两个步骤：
（1）步骤1：二氯磷酸苯酯与长链醇反应得到烷烃氯磷酸苯酯；
（2）步骤2：氨丙基硅胶与步骤1中所得的烷烃氯磷酸苯酯在干燥的溶剂中反应得到含有磷酰胺结构的固定相。
3.根据权利要求2中所述的技术手段，其特征在于，步骤1中长链醇为碳原子个数为10~18的直链烷烃醇。
4.根据权利要求2中所述的技术手段，其特征在于，步骤2中所述干燥的溶剂为甲苯、四氢呋喃、二甲苯中的一种。
5.一种以上述固定相为原料制备蛋白质谱毛细管柱的方法，其特征在于以上述固定相为原料，进行石英毛细管装填，制备得到蛋白质谱毛细管柱。
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