CN114933629A - 一种基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法 - Google Patents
一种基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法 Download PDFInfo
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- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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Abstract
本发明属于生物技术领域,具体公开了一种基于巯基‑烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,该方法以带有巯基的化合物与带有烯基叠氮基团的化合物为反应物,生成含有β‑羰基硫醚基团的氨基酸、多肽或蛋白质生物偶联体,以实现化学修饰。本发明的方法条件温和,溶剂选择性宽泛,反应温度为37‑40℃,反应时间为10min‑48h。该方法在功能多肽或蛋白质制备、蛋白质标记以及生物医药等方面具有良好的应用前景。
Description
技术领域
本发明属于生物技术领域,具体公开了一种基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法。
背景技术
生物大分子的化学修饰是指通过化学反应的手段将特定功能的基团安装到目标生物大分子上。多肽或蛋白质是化学修饰的主要目标,因为化学修饰可以改善多肽和蛋白质的功能,还可以赋予其新功能,多肽或蛋白质的化学修饰在生命科学、医药、生物材料中发挥了重要的作用。
半胱氨酸(Cys)重要且独特,其含有的巯基也是很多酶的催化位点。与其它亲核基团相比,巯基展现出鲜明的反应性,并且分布于97%的人源蛋白质中,这意味着基于半胱氨酸的化学修饰比较容易达成位点选择性,同时也具有普适性。特异性蛋白质半胱氨酸修饰通常基于以下三个反应(反应式如下):
(I)二硫化合物的交换反应,目标Cys与二硫试剂进行硫醇交换,实现功能基转移,该方法特异性好,条件温和,但新生的二硫键会继续发生硫醇交换,失去功能分子;(II)亲核取代反应,载有亲电位点的功能分子烷基化Cys,但是此类反应易受其它亲核残基的干扰;(III)迈克尔加成反应,负载了功能小分子的迈克尔受体与巯基加成,但是此类偶联产物易因逆迈克尔反应丢失功能分子。
最近也有其它的方法出现在文献和专利中,包括1)半胱氨酸巯基侧链对多氟芳烃、磺酰基氮杂芳烃等芳香亲电试剂的芳香亲核取代反应(SNAr);2)全氟烷基、烯基和炔基高碘试剂对半胱氨酸残基的烷基化、烯基化和炔基化;3)过度金属有机试剂对半胱氨酸残基的芳基化、硼基化等。然而这些方法由于各自的局限性,目前还未得到普遍应用,尤其难以在医药工业界中实施。
另外,巯基对普通烯/炔烃的加成反应,即Thiol-ene/Thiol-yne反应(反应式如下)也被用于蛋白质的修饰,但这类反应需要紫外光、光敏剂或自由基引发剂,这些额外的试剂或条件以及反应本身的效率上的缺陷严重削弱了这些方法的应用前景。
发明内容
针对背景技术指出的不足,本发明公开了一种基于新机理的修饰方法,能够有效解决现有技术存在的不足。本发明提供了一种基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,其中巯基-烯基叠氮反应的化学原理为:含有巯基的化合物中的巯基自由基对含有烯基叠氮基团的化合物的β位点进行自由基Thiol-ene加成,生成的中间体裂解释放出氮气分子,紧跟着水解,释放氨分子,产物为含有β-羰基硫醚的化合物,通过这个方法化学选择性地在巯基上连接酮羰基。
其具体方法为:含有巯基的化合物与含有烯基叠氮基团的化合物在反应介质中反应,使含有巯基的化合物修饰为含有β-羰基硫醚的化合物。
具体反应式如下所示:
其中,含有巯基的化合物为半胱氨酸及其衍生物,带有自由烷基巯基,来自于液相或固相合成;其结构式(I)为其中P1选自氢,烷基,芳基,杂芳基,-(C=O)-烷基,-(C=O)-芳基,-(C=O)-杂芳基,-(C=O)-O-烷基,-(C=O)-O-芳基,-(C=O)-O-杂芳基,-(C=O)-NH-烷基,-(C=O)-NH-芳基,-(C=O)-NH-杂芳基,二肽基,或多肽基,上述二肽和多肽是指由2个以上任意天然氨基酸、常见非天然氨基酸或它们的任意组合通过酰胺键连接而构成分子片段。
P2选自羟基,-O-烷基,-NH-烷基,-NH-芳基,-NH-杂芳基,-NH-二肽基,或-NH-多肽基以及环胺基,如:脯氨酸衍生物。
含有巯基的化合物也可以是天然多肽或天然蛋白、基因工程多肽蛋白以及其它修饰蛋白,下表1列出了本专利涉及的部分氨基酸、肽和蛋白,但不限于此表所收集的氨基酸、肽和蛋白。
表1 含有巯基的氨基酸、肽和蛋白
含有烯基叠氮基团的化合物的结构式(II)为其中,R1为烷基,芳基,R2和R3为氢,R1可选择性地被一个或多个独立的Q1基团取代:Q1为胺基,环烷基,杂环烷基,芳基,杂芳基,杂环芳基,-OR4,-S(O)nR5,-NR6R7,-SO2NR6R7,-NR6SO2R5;
上述R4,R5,R6,R7可选择性地被一个或多个独立的氢,卤素,-CN,-OH,-NH2,-NO2,氧基,-CF3,-OCF3,-CO2H,-S(O)nH,烷基,芳基,杂芳基,环烷基,杂环烷基,杂环芳基或-O-烷基取代,其中,n取自0,1或2。
结构式(II)中的R1、R4、R5和Q1还可以是包含功能分子的有机片段,如:药物分子、酶的抑制剂、受体的拮抗剂或激动剂、荧光发色分子、(多)糖基、多肽、天然产物分子、多齿配体、有机催化基团。
表2列出了本发明涉及的部分含有烯基叠氮基团的化合物,但不限于此表所收集的烯基叠氮化合物。
表2 部分含有烯基叠氮基团的化合物
反应温度在37-40℃,反应时间为10min-48h;
反应的溶剂为四氢呋喃、二氧六环、丙酮、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲亚砜、水、甲醇、乙醇、异丙醇、乙腈、缓冲溶液中的一种或几种混合物;
含有β-羰基硫醚的化合物为含有β-羰基硫醚的半胱氨酸及其衍生物、含有β-羰基硫醚的多肽或者含有β-羰基硫醚的蛋白质;其中,含有β-羰基硫醚的半胱氨酸及其衍生物的结构式(III)为表3列出了部分修饰后的含有β-羰基硫醚的化合物,但不限于此。
表3 部分修饰后的含有β-羰基硫醚的化合物
本发明有益技术效果如下:
1)本反应是巯基自由基引发的自由基链式反应,反应条件温和,受溶剂影响较小,在有机溶剂、水、缓冲溶剂以及混合溶剂中都能顺利进行;
2)采用非亲电的烯基叠氮,不受其它亲核残基,比如胺基、(酚)羟基、羧基、咪唑基、吲哚等的干扰;
3)反应无需添加剂,也不需催化剂,副产物是氮气和氨,容易后处理;
4)制备的硫醚很稳定,酮羰基可以是方便的二次修饰位点;
5)提供了一个全新的化学工具,此工具可以应用于体内和体外生物偶连体的制备及应用,包括蛋白的荧光标记、抗体偶联药物的制备、蛋白组学分析、共价抑制剂药物的开发等。
附图说明
图1为磺酰-乙烯基叠氮化物修饰的BSA的凝胶电泳图,其中,(a)为凝胶的CBB染色,(b)为凝胶的荧光图像。
图2为用YPet-ECFP和STAV AF568标记Ni-NTA树脂制备方法图。
图3为标记的与未标记的Ni-NTA树脂荧光成像图。
具体实施方式
下面结合实施例对本发明做进一步描述,但不限于此。
实施例1
将半胱氨酸衍生物26(0.2mmol,1equiv)溶解在3mL THF中,接着在混合溶液中加入烯基叠氮化物1(2mmol,10equiv),将反应混合物在空气氛围中于25℃搅拌直至多肽消耗完(通过TLC监测),0.5h后反应完成,减压旋干溶剂。以石油醚和乙酸乙酯用作所得粗产物的洗脱液,石油醚和乙酸乙酯的体积比为3:1,目标产物48可通过硅胶柱快速纯化并真空浓缩,产率77%。
实施例2
将半胱氨酸衍生物26(0.2mmol,1equiv)溶解在3mL乙腈(MeCN)中,接着在混合溶液中加入烯基叠氮化物1(2mmol,10equiv),将反应混合物在空气氛围中于40℃搅拌直至多肽消耗完(通过TLC监测),35min后反应完成,减压旋干溶剂。以石油醚和乙酸乙酯用作所得粗产物的洗脱液,石油醚和乙酸乙酯的体积比为3:1,目标产物48通过硅胶柱快速纯化并真空浓缩,产率75%。
实施例3
将半胱氨酸衍生物26(0.4mmol,2equiv)溶解在3mL THF中后,添加乙烯基叠氮化物16(0.2mmol,1equiv),将反应混合物在空气氛围中于0℃冰浴搅拌直至肽被消耗(通过TLC监测),24h后反应完成,旋干溶剂。以石油醚和乙酸乙酯做洗脱剂,石油醚和乙酸乙酯的体积比为3:1,修饰产物79通过硅胶柱快速纯化并真空浓缩,产率99%。
实施例4
将谷胱甘肽44(0.2mmol,1equiv)溶解在2mL PBS(pH7.4)中,然后加入溶解在2mLTHF中的乙烯基叠氮化物1(0.4mmol,2equiv),将反应混合物在空气气氛中于40℃搅拌直至肽被消耗(通过TLC监测,正丁醇,乙酸,水的体积比为3:1:1),4h后反应完成,旋干溶剂。粗产物用3%甲醇的水溶液作为洗脱流动相,经过反相柱层析纯化,并在真空中冷冻干燥浓缩得到修饰产物80,产率99%。
用相同的步骤,更换溶剂在其他条件实施了反应,如表1所示:a40℃下,0.2mmol谷胱甘肽44(GSH)(1.0equiv)和0.4mmol乙烯叠氮化物(2.0equiv)进行反应,使用2.0mL PBS作为助溶剂,其中,cPBS的pH值为7.4;dPBS的pH值为7.2,b产率为73-99%;由此可以看出,该反应可以在上述各种溶剂下进行。
表1 反应实施条件a
实施例5
将谷胱甘肽44(0.2mmol,1equiv)溶解在2mL PBS(pH7.4)中,然后加入溶解在2mLTHF中的乙烯基叠氮化物15(0.4mmol,2equiv),将反应混合物在空气气氛中于30℃搅拌直至肽被消耗(通过TLC监测,正丁醇/乙酸/水=3:1:1),48h后反应完成,旋干溶剂。粗产物用3%甲醇的水溶液作为洗脱流动相,经过反相柱层析纯化,并在真空中冷冻干燥浓缩得到修饰产物85,产率78%。
实施例6
将谷胱甘肽44(0.2mmol,1equiv)溶解在2mL PBS缓冲液(10mmol/L,pH7.4)中,然后加入溶解在THF中的乙烯基叠氮化物18(0.4mmol,2equiv),将所得溶液在空气氛围中60℃搅拌8h,通过HPLC-MS分析反应,得到产物68,产率为63%。
实施例7
将谷胱甘肽44(0.2mmol,1equiv)溶解在2mL PBS缓冲液(10mmol/L,pH7.4)中,然后加入乙烯基叠氮化物22(0.4mmol,2equiv),将所得溶液在空气氛围中40℃搅拌10min,通过HPLC-MS分析反应,得到产物77,产率为81%。
实施例8
丹磺酰-乙烯基叠氮化物与牛血清蛋白结合
对牛血清蛋白(BSA)的修饰采用如下方法:反应在1.5mL离心管中进行,向对应离心管中加入10μL溶解在PBS中的BSA 46(1.5×10-6mmol,1equiv),40μL EtOH和140μL PBS(10mmol/L,pH7.4)(或90μL EtOH和90μL PBS)。向预混溶液中加入溶解在EtOH中的10μL苯基烯基叠氮6(1.5×10-4mmol,100equiv),得到混合物的最终体积为200μL,蛋白含量为1.5×10-6mmol,将离心管封上保鲜膜并用牙签扎孔,然后在40℃下摇晃24h,通过SDS-PAGE分析样品。
将蛋白质溶液与SDS上样缓冲液混合,首先制备10%的分离胶100mL(0.25M Tris-HCl,10%SDS,30%甘油和0.05%溴酚蓝),待分离胶凝固后,添加SDS-PAGE浓缩凝胶100mL(15%丙烯酰胺,0.375M Tris(pH8.8),0.1%SDS,0.1%APS和0.05%TEMED),胶全部凝固后,将样品连同PageRuler Plus预染蛋白Marker上样到凝胶凹槽上,在缓冲液(25mM Tris、0.19M甘氨酸和0.1%SDS)中运行。凝胶中蛋白质的荧光用Gel Doc TM XR+凝胶成像仪和Image lab TM软件拍摄并记录,凝胶用10μL浓度为2.5g/L的考马斯亮蓝染料R250上样染色后,再次用凝胶成像仪记录。
在图1中,M为PageRuler Plus预染蛋白标记;泳道1为在EtOH/PBS(v/v=1:1)条件下反应,BSA丹磺酰偶联物88用5×SDS-PAGE样品缓冲液处理并在100℃下煮沸5min;泳道2为在EtOH/PBS(v/v=1:3)条件下反应,BSA丹磺酰偶联物88用5×SDS-PAGE样品缓冲液处理并在100℃下煮沸5min;泳道3为没有修饰的BSA;泳道4为丹磺酰基-乙烯基叠氮化物6。
图1中(a)为凝胶的CBB染色,(b)为凝胶的荧光图像。(a)、(b)两图对比,泳道1和2中的BSA被丹磺酰叠氮6修饰后,在(b)中显示出了荧光,但仅有BSA的泳道3却没有荧光,说明BSA被丹磺酰叠氮88修饰成功,获得BSA-丹磺酰偶联物,即完成了对蛋白质BSA的荧光标记。
实施例9
用YPet-ECFP和STAV AF568标记Ni-NTA树脂制备的三元荧光蛋白偶联物,制备方法如图2所示。
将Biotin 7溶解在THF中并稀释至8.4×10-3μmol/μL,将链霉亲和素Alexa Fluor568偶联物溶解在PBS(pH7.4)中并稀释至36μM,由BCA蛋白检测试剂盒测定纯化后的YPet-ECFP 47蛋白浓度为0.5808μg/μL。
反应在两个500μL离心管(1)和(2)中进行:向(1)号管添加Biotin 7(10μL,8.4×10-2μmol,100equiv)和YPet-ECFP 47(100μL,8.4×10-4μmol,1equiv),向(2)号管添加YPet-ECFP 47(100μL,8.4×10-4μmol,1equiv),两种混合物均在37℃轻轻摇动约40h,然后向两种反应混合物中分别加入50μL镍NTA琼脂糖珠(Ni-NTA)(购自Thermo Scientific)并在室温下摇动1h以确保YPet-ECFP与Ni-NTA树脂完全结合。Ni-NTA树脂及其吸附物在低速离心机中通过离心沉淀,去除上清液,每管用1mL PBS洗涤3次,以洗去多余的生物素,向两个离心管中均加入链霉亲和素Alexa Fluor 568(12.5μL,4.5×10-4μmol,36μM)和PBS(100μL),然后在室温下振摇5min,将两种反应混合物用1mL PBS洗涤3次,以去除过量的Streptavidin Alexa Fluor 568,最后,加入100μL PBS使Ni-NTA树脂悬浮,并将两种混合物置于共聚焦盘底部进行显微镜成像(CarlZeiss,Germany,primo vert)。根据显微镜成像结果,Ni珠本身不带荧光(图3-IV),可以判定所呈现的荧光为其吸附物的荧光。图3-I、II、III中的青色荧光和黄色荧光则主要来源于YPet-ECFP 47。图3-I、II中的红色荧光来源于偶联物链霉亲和素Alexa Fluor 568中的STAV AF 568染料。当叠氮与蛋白上的半胱氨酸巯基发生反应时,叠氮上的生物素会随着蛋白一起吸附于Ni珠表面。加入染料STAV AF 568时,由于生物素与亲和素的特异性结合,染料会优先通过生物素-亲和素连接在蛋白上,一起吸附在Ni珠表面。从而可以观察到Ni珠表面上一层红色光圈,其位置与青色荧光、黄色荧光一致(图3-I),故此可以得出,红色荧光是通过蛋白吸附Ni珠呈现的。若叠氮修饰反应未发生,加入染料后,染料会均匀的由外向里渗透进Ni珠内部(图3-II)。在图3-I、II的两种情况下,对Ni珠周围的光密度进行处理分析,发现经过修饰的Ni珠周围的光密度大于未经过修饰的Ni珠,具有统计学的差异,由此可以得出链霉亲和素Alexa Fluor568偶联物中的红色荧光是通过生物素-叠氮-巯基连接到Ni珠表面,因此证明了叠氮修饰反应在蛋白质水平上也能快速发生。
Claims (9)
1.一种基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,其特征在于,所述方法为:将含有巯基的化合物与含有烯基叠氮基团的化合物在反应介质中反应,使含有巯基的化合物修饰为含有β-羰基硫醚的化合物;反应温度为37-40℃,反应时间为10min-48h;含有巯基的化合物为半胱氨酸及其衍生物、含有巯基的多肽或者含有巯基的蛋白质。
4.根据权利要求3所述的基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,其特征在于,所述含有烯基叠氮基团的化合物的结构式(II)中R1被一个或多个独立的Q1基团取代:Q1为氢,卤素,羟基,胺基,-CN,-CF3,-OCF3,-NO2,叠氮基,烷基,烯基,炔基,环烷基,杂环烷基,芳基,杂芳基,杂环芳基,-OR4,-S(O)nR5,-NR6R7,-SO2NR6R7,-(C=O)-R5,-(C=O)-NR6R7,-(C=O)-OR5,-O-(C=O)-R5。
5.根据权利要求1所述的基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,其特征在于,所述含有β-羰基硫醚的化合物为含有β-羰基硫醚的半胱氨酸及其衍生物、含有β-羰基硫醚的多肽或含有β-羰基硫醚的蛋白质。
7.根据权利要求1所述的基于巯基-烯基叠氮偶联反应的多肽或蛋白质定向修饰方法,其特征在于,所述反应介质为四氢呋喃、二氧六环、丙酮、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲亚砜、水、甲醇、乙醇、异丙醇、乙腈、缓冲溶液中的一种或几种的混合物。
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CN114349822A (zh) * | 2021-12-31 | 2022-04-15 | 深圳湾实验室坪山生物医药研发转化中心 | 一种基于乙烯基锍盐的生物大分子修饰方法 |
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CN114349822A (zh) * | 2021-12-31 | 2022-04-15 | 深圳湾实验室坪山生物医药研发转化中心 | 一种基于乙烯基锍盐的生物大分子修饰方法 |
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