CN115043899A - 双胺或胺与硫醇偶联的化合物及其制备方法和应用 - Google Patents
双胺或胺与硫醇偶联的化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了双胺或胺与硫醇偶联的化合物及其制备方法和应用。本发明利用简单的邻苯二甲醛(OPA)试剂通过一锅化学选择性交联两种不同的胺亲核试剂进行生物偶联的新方法及应用。以邻苯二甲醛作为连接子,在温和条件下,快速高效地将两个不同氨基的分子间进行偶联,形成的异吲哚啉亚胺的结构较为稳定。该方法已成功应用于偶联多种偶联天然生物相关分子,包括小分子药物、生物物理探针、多肽、蛋白质、碳水化合物甚至病毒等,并无需任何预功能化。
Description
技术领域
本发明涉及生物化学技术领域,进一步地说,是涉及双胺或胺与硫醇偶联的化合物及其制备方法和应用。
背景技术
生物偶联化学在开发肽或抗体-药物偶联物、疫苗和生物纳米粒子等现代生物制药中发挥着关键作用。除了将相对较小的分子如药物、荧光探针等附着在较大的生物分子上外,越来越需要将蛋白质与肽、蛋白质与核酸等两个复杂的生物分子交联,以期用于更复杂的生物分子工程。这些新的研发需求则需要具有更高效率、选择性和可及性的新型生物偶联反应。到目前为止,通过亲核试剂(Nu)如肽/蛋白上的硫醇和氨基侧链与另一分子的亲电试剂(El)之间的反应为生物偶联提供了一种简单的策略。然而,虽然亲核基团在天然化合物和合成化合物中很常见,但是合适的亲电试剂(如活化酯和卤代烷)却很少存在于天然生物分子上,并且常常需要额外的预功能化修饰。因此,有效、方便且广泛适用的生物偶联方法仍亟待发展。
发明内容
为解决现有技术中出现的问题,本发明提出了双胺或胺与硫醇偶联的化合物及其制备方法和应用。本发明利用简单的邻苯二甲醛(OPA)试剂通过一锅化学选择性交联两种不同的胺亲核试剂进行生物偶联的新方法及应用。以邻苯二甲醛作为连接子,在温和条件下,快速高效地将两个不同氨基的分子间进行偶联,形成的异吲哚啉亚胺的结构较为稳定。该方法已成功应用于偶联多种偶联天然生物相关分子,包括小分子药物、生物物理探针、多肽、蛋白质、碳水化合物甚至病毒等,并无需任何预功能化。
本发明的目的之一是提供一种双胺或胺与硫醇偶联化合物,所述双胺或胺与硫醇偶联的化合物具有以下结构通式或上述结构通式的同分异构体或其盐:
所述R2、R3相同或不同,各自独立地选自C1-C5的烷基;
所述式Ⅰ、式Ⅱ、式Ⅲ、式Ⅳ中的指氨基酸缩合后形成的多肽链,且失去N原子上两个氢原子后的结构,所述多肽链中,至少包括赖氨酸缩合后且失去N原子上两个氢原子后的结构,即为赖氨酸缩合后且赖氨酸的端基胺失去2个氢原子后的结构,所述n为2-25的任意整数;
所述式V、式Ⅵ、式Ⅶ中n值为1-6(在本发明中n大于1时,是因为蛋白质有多个氮的存在);
所述式Ⅷ、式Ⅸ中n值为1;
在本发明所述的赖氨酸缩合后的核苷酸结构,且失去N原子上两个氢原子后的结构中,失去的N原子上两个氢原子是指下式方框中的氢原子。
在本发明所述的双胺或胺与硫醇偶联的化合物,优选地:
更优选地,所述所示的结构中,AA1至AAn中,除去AA外,缩合前对应的天然氨基酸相同或不同,各自独立地选自Na(2-萘甲酸)、Gly (甘氨酸)、Phe(苯丙氨酸)、Arg(精氨酸)、Asp(天冬氨酸)、Ser(丝氨酸)、Asn(天冬酰胺)、Ala(丙氨酸)、lle(异亮氨酸)、leu(亮氨酸)、 Met(甲硫氨酸)、Gln(谷氨酰胺)、Pro(脯氨酸);
在本发明所述的双胺或胺与硫醇偶联的化合物,优选地:
所述R5选自氢、C1-C20的取代或未取代的烷基、取代或未取代的烷基芳基、取代或未取代的杂环基;更优选地,
所述R5选自甲基、叔丁基、C1-C3的烷基苯基、C1-C3的烷基苯酚基、C1-C5 的烷基吲哚基;和/或,
所述R1选自C1-C10的烷基、C1-C5的烷基磺酸基;和/或,
所述R2、R3相同或不同,各自独立地C1-C3的烷基;和/或,所述R4选自烷基取代的苯基,优选C1-C3的烷基取代的苯基。在本发明所述的双胺或胺与硫醇偶联的化合物,优选地:所述双胺或胺与硫醇偶联的化合物包括以下化合物:
在本发明所述的双胺或胺与硫醇偶联的化合物,优选地:所述双胺或胺与硫醇偶联的化合物包括以下化合物:
lysozyme的前体蛋白为溶菌酶;RNaseA的前体蛋白为核糖核酸酶A;
ubiquitin-wT的前体蛋白为泛素;Trastuzumab的前体蛋白为曲妥珠单抗;
ubiquitin-Ko的前体蛋白指将野生型泛素的Lys全部突变为Arg的突变蛋白;
上述与核酸偶联的产物中的n值为1。
本发明的目的之二是提供本发明的目的之一所述的双胺或胺与硫醇偶联的化合物的制备方法,包括以下步骤:
优选地,
所述溶剂选自醇、胺类溶剂、水、磷酸缓冲液和DMSO中的至少一种;优选醇与胺类溶剂的混合溶剂;醇、胺类溶剂和水的混合溶剂;醇、胺类溶剂和磷酸缓冲液的混合溶剂的至少一种、醇、胺类溶剂和磷酸缓冲液和DMSO的混合溶剂的至少一种。
优选地,
所述式(A)与R1-NHX的摩尔比为1:1-10,优选为1:1-6;
所述式(A)与R1-SH摩尔比为1:1-10,优选为1:1-5;
所述式(A)与邻苯二甲醛的摩尔比为1:1-3,优选为1:1-2;
所述式(A)在溶剂中的浓度为0.05mM-10mM,优选为0.1mM-5mM。
优选地,
所述反应的温度为室温;所述反应的时间为10s-1h,优选为5-30min;
所述中醇选自C1-C3的醇,优选为甲醇,所述胺类溶剂选自N,N-二异丙基乙胺;
所述磷酸缓冲液的pH为7.0-11.0。
本发明的目的之三是提供本发明目的之一所述的双胺或胺与硫醇偶联的化合物在药物制备、物理探针、病毒检测中的应用,优选在制备小分子药物中前体中的应用。
在本发明中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。在下文中,各个技术方案之间原则上可以相互组合而得到新的技术方案,这也应被视为在本文中具体公开。
与现有技术相比,本发明至少具有以下优点:
本发明通过对胺的活性进行精准调控,利用双胺之间的反应活性差别,进一步实现产物的选择性生成。在复杂的体系中区分两个独特的氨基并高选择性和高效率的将它们连接起来仍然是比较困难和亟待研究的。本发明报导了一种使用简单的邻苯二甲醛(OPA)试剂通过一锅化学选择性交联两种不同的胺亲核试剂进行生物共轭的新方法。各种α-氨基酸、芳基胺和仲胺可以高效和异源选择性地与肽或蛋白质上赖氨酸的ε-氨基侧链交联,该交联反应具有更广的底物适用范围,可实现产物的选择性生成,并可应用于多种复杂体系。
附图说明
图1为实施例39的反应产物的质谱图;
图2为实施例40的反应产物的质谱图;
图3为实施例41的反应产物的质谱图;
图4为实施例42的反应产物的质谱图;
图5为实施例43的反应产物的质谱图;
图6为实施例44的反应产物的质谱图;
图7为实施例45的反应产物的质谱图;
图8为实施例46的反应产物的质谱图;
图9为实施例47的反应产物的质谱图;
图10为实施例47的反应产物的质谱图;
图11为实施例48的反应产物的质谱图;
图12为实施例50中的甲型H1N1病毒个体包膜表面用H-Lys(Fl)-OH(绿色) 标记的超分辨率图像;
图13为实施例50中的甲型H1N1病毒个体包膜表面用膜染料DID(红色) 标记的超分辨率图像;
图14为实施例50中的甲型H1N1病毒个体包膜表面用H-Lys(Fl)-OH(绿色) 和膜染料DID(红色)双重标记的超分辨率图像;
图15为实施例50中H-Lys(Fl)-OH标记的病毒在两个活MDCK细胞中累积的共聚焦图像。
在本发明图1-图11的质谱图中,横坐标为:解卷积分子量【deconvolutedmass(amu)】。
具体实施方式
下面结合具体附图及实施例对本发明进行具体的描述,有必要在此指出的是以下实施例只用于对本发明的进一步说明,不能理解为对本发明保护范围的限制,本领域技术人员根据本发明内容对本发明做出的一些非本质的改进和调整仍属本发明的保护范围。
另外需要说明的是,在以下具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合。为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,由此而形成的技术方案属于本说明书原始公开内容的一部分,同时也落入本发明的保护范围。
实施例与对比例中采用的原料,如果没有特别限定,那么均是现有技术公开的,例如可直接购买获得或者根据现有技术公开的制备方法制得。
一、前体线性肽(即式I化合物)的制备如下示意:
i)Fmoc保护基脱除;ii)氨基酸缩合;iii)Rink树脂的裂解。
i)Fmoc保护基的脱除:将20%哌啶/DMF加入到固相合成管中振摇反应10 分钟,随后抽掉反应溶剂,以DMF,DCM分别洗两遍;再将上述操作重复一遍完成Fmoc保护基的脱除。
ii)氨基酸缩合:将Fmoc-AA-OH(3.0equiv),2-肟氰乙酸乙酯(3.0equiv)溶解到NMP中并制成澄清溶液,随后向其中加入DIC(3.3equiv)并于冰水浴下反应 5min,随后再将反应溶液加入到固相合成管中,室温下反应1.5小时。再将反应溶剂抽干,分别以DMF,DCM洗两遍,进行下一步脱保护反应。
iii)Rink树脂的裂解:将三氟乙酸、三异丙基硅烷和水按照95:2.5:2.5体积比制得裂解液,随后将其加入到固相合成管中,室温下反应1小时,随后将裂解液收集,除去溶剂,向残留物中加入冷乙醚将多肽沉淀出来,随后通过离心得到C-端为酰胺键的多肽粗品。
本发明中多肽链的合成属于现有常规方法,在此不多更多赘述,本领域技术人员可以根据合成需求,选择其他合适的多肽合成方法。
二、本发明的偶联化合物及其对应的制备方法
实施例1
以多肽Na-Gly-Phe-Lys-NH2(peptide 1)和L-Phe(即下述式子中的N2化合物)为例:
Condition[A]:将多肽底物1(0.05mmol,1.0equiv)和L-Phe(0.15mmol,3.0equiv)在室温下溶于50mL甲醇中,随后加入DIPEA(0.1mmol,2.0equiv)和OPA(0.0525mmol,1.05equiv,140.8μL的0.05mg/μL的甲醇母液)于室温下搅拌反应10分钟得到偶联产物,最后通过HPLC进行纯化分离。
Condition[B]:将多肽底物1(0.05mmol,1.0equiv)和L-Phe(0.15mmol,3.0equiv)在室温下溶于25mL甲醇和25mL水中,随后加入DIPEA(0.1mmol, 2.0equiv)和OPA(1.05equiv,140.8μL的0.05mg/μL的甲醇母液)于室温下搅拌反应10分钟得到偶联产物,最后通过HPLC进行纯化分离。
Condition[C]:将多肽底物1(0.05mmol,1.0equiv)和L-Phe(0.3mmol,6.0 equiv)在室温下溶于25mL甲醇和25mL水中,随后加入DIPEA(0.1mmol,2.0 equiv)和OPA(140.8μL的0.05mg/μL的甲醇母液)于室温下搅拌反应10分钟得到偶联产物,最后通过HPLC进行纯化分离。
Condition[D]:将多肽底物1(0.05mmol,1.0equiv)和L-Phe(0.15mmol,3.0equiv)在室温下溶于25mL甲醇和25mLpH=10PB中,随后加入OPA(1.05 equiv,140.8μL的0.05mg/μL的甲醇母液)于室温下搅拌反应10分钟得到偶联产物,最后通过HPLC进行纯化分离。
Condition[E]:将多肽底物1(0.05mmol,1.0equiv)和L-Phe(0.3mmol,6.0 equiv)在室温下溶于25mL甲醇和25mL pH=10PB中,随后加入OPA(1.05 equiv,140.8μL的0.05mg/μL的甲醇母液)于室温下搅拌反应10分钟得到偶联产物,最后通过HPLC进行纯化分离。
产物的1H-NMR和13C-NMR如下:
1H NMR(400MHz,Methanol-d4)δ8.35(d,J=1.8Hz,1H),7.84(dd,J= 8.6,1.8Hz,1H),7.77(t,J=6.2Hz,2H),7.70(d,J=8.6Hz,1H),7.65–7.58 (m,1H),7.55(t,J=7.6Hz,1H),7.43–7.29(m,6H),7.26(t,J=7.2Hz, 4H),7.20–7.11(m,3H),7.05(t,J=7.2Hz,1H),4.64(dd,J=9.2,5.6Hz, 1H),4.51–4.36(m,2H),4.24–4.08(m,2H),3.98(d,J=16.4Hz,1H),3.44 (dd,J=14.0,4.2Hz,2H),3.33(d,J=12.0Hz,1H),3.24(m,J=27.2,13.6,4.8Hz,3H),3.06(dd,J=13.8,9.2Hz,1H),1.90(m,J=12.4,4.2Hz,1H), 1.66(m,J=13.4,10.8,4.2Hz,1H),1.54–1.35(m,3H),1.27(m,J=14.2, 7.2Hz,1H).
13C NMR(101MHz,Methanol-d4)δ175.1,173.8,172.4,171.2,168.7, 159.5,143.1,137.3,137.1,134.6,132.9,132.2,130.3,129.2,129.1,128.5, 128.4,128.3,128.3,128.3,128.3,127.9,127.8,127.6,127.2,126.9,126.6, 126.5,126.5,125.3,123.5,123.0,60.7,55.6,52.5,45.5,43.2,38.3,36.7, 30.8,25.6,22.6.
实施例2-38
实施例2-38采用与实施例1相同的反应条件制备,区别仅在于原料不同,具体的原料和产物结果如表1和表2所示。
表1
a:LC yield
表2
实施例39
溶菌酶蛋白和氨基葡萄糖的双胺偶联反应:
首先将20μM的溶菌酶蛋白(即lysozyme)和180mM的氨基葡萄糖置于pH=10的磷酸缓冲液(PB)中,然后加入终浓度为0.9mM的OPA(邻苯二甲醛),置于37℃摇床中,反应10min后加甲酸淬灭反应,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图1所示,n=3。
实施例40
首先将20μM的溶菌酶蛋白(即lysozyme)和18mM的生物胞素置于pH=10 的磷酸缓冲液(PB)中,然后加入终浓度为0.2mM的OPA溶液,溶剂为DMSO37℃摇床中,反应10min后加甲酸淬灭反应,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图2所示,n=3。
实施例41
溶菌酶和荧光素的偶联
首先将20μM的溶菌酶蛋白(即lysozyme)和36mM的荧光素(H-Lys(Fl) -OH)置于pH=10的磷酸缓冲液(PB)中,然后加入终浓度为0.6mM的OPA 溶液,溶剂为DMSO,置于37℃摇床中,反应10min后加甲酸淬灭反应,用 ESI-QTOF质谱直接对反应进行监测,质谱结果如图3所示,n=2。
实施例42
首先将140μM的泛素(即Ubiquitin-WT)和98mM的H-Pro-Ala-Phe-NH2短肽置于pH=10的磷酸缓冲液(PB)中,然后加入终浓度为0.56mM的OPA 溶液,溶剂为DMSO37℃摇床中,反应10min后加甲酸淬灭反应,用ESI-QTOF 质谱直接对反应进行监测,质谱结果如图4所示,n=2。
实施例43
首先将10μM的核糖核酸酶A(即RNaseA)和30mM的H-Phe-Gly-Ala-NH2短肽置于pH=10的磷酸缓冲液(PB)中,然后加入终浓度为0.15mM的OPA 溶液,溶剂为DMSO37℃摇床中,反应10min后加甲酸淬灭反应,用ESI-QTOF 质谱直接对反应进行监测,质谱结果如图5所示,n=3。
实施例44
首先将160μM的核苷酸(即5’-NH2-C6-AGTCAGTCAGTC-3’)和80mM 的(R)-2-氨基-3-(2-丙炔基巯基)丙酸置于pH=10的磷酸缓冲液(PB)中,然后加入终浓度为16mM的OPA溶液,溶剂为DMSO37℃摇床中,反应10min后加甲酸淬灭反应,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图6所示, n=1。
实施例45
首先将35μM的溶菌酶蛋白(即lysozyme)和70mM的1-硫葡萄糖置于 pH=8的磷酸缓冲液(PB)中,然后加入终浓度为70μM的OPA溶液,溶剂为 DMSO37℃摇床中,反应10min后,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图7所示,n=2。
实施例46
首先将50μM的溶菌酶蛋白(即lysozyme)和2.5mM的巯基环糊精置于 pH=8的磷酸缓冲液(PB)中,然后加入终浓度为0.5mM的OPA溶液,溶剂为DMSO37℃摇床中,反应10min后,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图8所示,n=4。
实施例47
首先将10μM的曲妥珠单抗(即Trastuzumab)和15mM的卡托普利置于 pH=8的磷酸缓冲液(PB)中,然后加入终浓度为0.15mM的OPA溶液,溶剂为DMSO37℃摇床中,反应10min后,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图9和图10所示,n=2。
实施例48
首先将25μM的泛素-K0(即Ubiquitin-K0,将野生型泛素的所有的Lys均突变为Arg)和10mM的核苷酸(即5’-SH-C6-AGTCAGTC-3’)置于pH=8的磷酸缓冲液(PB)中,然后加入终浓度为0.75mM的OPA(溶剂为DMSO),置于37℃摇床中,反应10min后,用ESI-QTOF质谱直接对反应进行监测,质谱结果如图11所示,n=11。
在本发明中,实施例39-48中,DMSO与PB溶液均为体积比。
实施例49
产物稳定性测试:
不同pH下的稳定性测试:随意抽取偶联产物化合物3,化合物4和化合物 7,分别在pH=5PB(磷酸缓冲溶液),pH=7.3PBS(磷酸缓冲盐溶液)和pH=10 PB(磷酸缓冲溶液)条件下室温处理24h,测试结果如下表所示,此偶联产物具有非常好的稳定性。
化合物 | pH=5PB | pH=7.3PBS | pH=10PB | |
3 | 稳定 | 稳定 | 稳定 | |
4 | 稳定 | 稳定 | 稳定 | |
7 | 稳定 | 稳定 | 稳定 |
该稳定测试结果说明,本发明的双胺或胺与硫醇偶联的化合物非常稳定,可以作为其他反应的底物,进一步参与其余相关反应,且由于本发明是实现多肽链参与的偶联反应,该偶联反应产物在药物制备等领域具有很大的潜力。
实施例50
对于病毒颗粒的标记追踪:
首先将100μL(1mg/mL)的流感病毒、10μM的H-Lys(Fl)-OH(加入100nM OPA)和5μM膜特异性染料双十八烷基四甲基吲哚二羰花青盐(DiD)在室温下共孵育30min。未结合的染料和聚集的病毒分别通过NAP-5凝胶过滤柱(GE Healthcare)和0.2μm孔径过滤器去除。随后将双色标记的病毒置于共聚焦显微镜下进行双通道成像,通过Fiji ImageJ的DeepImageJ功能获取超分辨率图像。
H-Lys(Fl)-OH的结构式为:
甲型H1N1病毒用H-Lys(Fl)-OH和OPA对其进行荧光标记后的结构式为:
将H-Lys(Fl)-OH标记的病毒包膜与Syto82标记的病毒基因组进行共定位分析,以评估H-Lys(Fl)-OH在病毒包膜上的标记效率。将100μL的流感病毒(1 mg/mL)与10μM H-Lys(Fl)-OH(加入100nM OPA)和5μM Syto 82在室温下共孵育30min。用NAP-5凝胶过滤柱(GEHealthcare)和0.2μm孔径过滤器分别去除未结合的染料和聚集的病毒。将双色标记的病毒置于共聚焦显微镜下进行双通道成像,利用Fiji ImageJ软件计算两种荧光信号的共定位效率(Pearson相关系数),即染料对病毒的标记效率。
荧光图像由旋转圆盘共聚焦显微镜(Olympus IXplore SpinSR10)在100 倍物镜下使用在线CO2培养系统(INUB-WELSX-SET)和sCMOS(Prime 95B) 捕获。H-Lys(Fl)-OH和FITC-NHS用488激光和525/50nm发射滤光片成像。Syto 82和DID分别用561和640nm激光以及617/73和685/40nm 发射滤光片成像。来自每个通道的荧光信号在sCMOS上交替成像,以允许同时进行多色成像。
为了跟踪病毒在活细胞内的感染过程,将标记的病毒与MDCK细胞在4℃下孵育10分钟,然后立即放置在配备CO2在线细胞培养系统的转盘共聚焦显微镜上,在37℃下成像。使用Fiji ImageJ软件重建病毒在细胞中移动的时间投影,以连接每一帧中的病毒信号。
通过对MDCK细胞的鼠疫试验检测流感病毒的病毒感染性。将MDCK细胞接种在6孔培养皿中24小时。然后将病毒以1:10-1:10,000的稀释度依次添加到孔中,将处理过的细胞在37℃下孵育1小时并冲洗3次。接下来,将细胞在新鲜感染培养基(含有1.6%琼脂和2%FBS的DMEM)中培养3天,并再次洗涤3次。最后用3.7%甲醛固定细胞,然后用0.6%结晶紫染色,计算病毒滴度。
通过OPA介导的将连在Lys侧链上的荧光素交联到甲型H1N1流感病毒颗粒的两个外壳蛋白血凝素(HA)和神经氨酸酶(NA)上,对其进行标记追踪,如图12所示。得到的荧光素标记病毒进一步用膜特异性染料双十八烷基四甲基吲哚二羰花青盐(DiD)进行染色,得到双标记病毒所示。对病毒个体的超分辨率荧光成像证实,荧光素染料选择性地标记在了包膜蛋白上。同时,用核酸特异性染料Syto82染色的共定位成像分析显示,95%以上的病毒被成功标记,如图 13、图14所示。重要的是,通过OPA标记的病毒在噬斑测定中没有显示出明显的侵染性损失。此外,在与宿主犬肾细胞(MCDK)在37℃下孵育30分钟后, OPA介导标记的病毒成功地穿透细胞膜并在细胞质的特定区域积累,如图15所示。活感染细胞的延时成像共聚焦成像清楚地揭示了单个病毒从细胞膜移动到细胞质的动态过程。这些实验表明,分子间OPA介导的双胺偶联方法可用于标记病毒,并在单病毒颗粒水平上监测活细胞内的病毒感染过程。
以上结合具体实施方式和范例性实例对本发明进行了详细说明,不过这些说明并不能理解为对本发明的限制。本领域技术人员理解,在不偏离本发明精神和范围的情况下,可以对本发明技术方案及其实施方式进行多种等价替换、修饰或改进,这些均落入本发明的范围内。本发明的保护范围以所附权利要求为准。
Claims (10)
1.一种双胺或胺与硫醇偶联化合物,其特征在于,所述双胺或胺与硫醇偶联的化合物具有以下结构通式或上述结构通式的同分异构体或其盐:
所述R2、R3相同或不同,各自独立地选自C1-C5的烷基;
所述式Ⅰ、式Ⅱ、式Ⅲ、式Ⅳ中的指氨基酸缩合后形成的多肽链,且失去N原子上两个氢原子后的结构,所述多肽链中,至少包括赖氨酸缩合后且失去N原子上两个氢原子后的结构,即为赖氨酸缩合后且赖氨酸的端基胺失去2个氢原子后的结构,所述n为2-25的任意整数;
所述式V、式Ⅵ、式Ⅶ中n值为1-6;
所述式Ⅷ、式Ⅸ中n值为1;
2.根据权利要求1所述的双胺或胺与硫醇偶联的化合物,其特征在于:
更优选地,所述所示的结构中,AA1至AAn中,除去AA外,缩合前对应的天然氨基酸相同或不同,各自独立地选自2-萘甲酸、甘氨酸、苯丙氨酸、精氨酸、天冬氨酸、丝氨酸、天冬酰胺、丙氨酸、异亮氨酸、亮氨酸、甲硫氨酸、谷氨酰胺、脯氨酸;
3.根据权利要求1所述的双胺或胺与硫醇偶联的化合物,其特征在于,
所述R5选自氢、C1-C20的取代或未取代的烷基、取代或未取代的烷基芳基、取代或未取代的杂环基;更优选地,
所述R5选自甲基、叔丁基、C1-C3的烷基苯基、C1-C3的烷基苯酚基、C1-C5的烷基吲哚基;和/或,
所述R1选自C1-C10的烷基、C1-C5的烷基磺酸基;和/或,
所述R2、R3相同或不同,各自独立地C1-C3的烷基;和/或,
所述R4选自烷基取代的苯基,优选C1-C3的烷基取代的苯基。
8.根据权利要求6所述的双胺或胺与硫醇偶联的化合物的制备方法,其特征在于:
所述式(A)与R1-NHX的摩尔比为1:1-10,优选为1:1-6;
所述式(A)与R1-SH摩尔比为1:1-10,优选为1:1-5;
所述式(A)与邻苯二甲醛的摩尔比为1:1-3,优选为1:1-2;
所述式(A)在溶剂中的浓度为0.05mM-10mM,优选为0.1mM-5mM。
9.根据权利要求6所述的双胺或胺与硫醇偶联的化合物的制备方法,其特征在于:
所述反应的温度为室温;所述反应的时间为10s-1h,优选为5-30min;
所述中醇选自C1-C3的醇,优选为甲醇,所述胺类溶剂选自N,N-二异丙基乙胺;
所述磷酸缓冲液的pH为7.0-11.0。
10.根据权利要求1-5任一所述的双胺或胺与硫醇偶联的化合物在药物制备、物理探针、病毒检测中的应用,优选在制备小分子药物中前体中的应用。
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