CN114671912A - 一种可光剪切载体辅助均相合成蓝铜三肽的方法 - Google Patents
一种可光剪切载体辅助均相合成蓝铜三肽的方法 Download PDFInfo
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Abstract
本发明提供了一种可光剪切载体辅助均相合成蓝铜三肽的方法,以7‑二苯基膦酰氧基香豆素‑4‑甲醇为保护基兼辅助载体合成了较高纯度的蓝铜肽,最后将蓝铜三肽与醋酸铜反应制备出了蓝铜三肽‑Cu,解决目前蓝铜三肽化学合成方法的均相反应比较复杂,分离步骤多、耗时周期长、纯化规模小、生产成本高,而固相反应的生产规模小,原料价格贵、浪费大,树脂类废弃物多和环境污染严重的问题。
Description
技术领域
本发明涉及生物有机化学领域中多肽的合成方法,具体说是涉及一种基于可光剪切载体辅助均相合成蓝铜三肽的方法。
背景技术
关于蓝铜三肽的活性方面:生物活性肽GHK是一种氨基酸序列为甘氨酰组氨酸赖氨酸的三肽。它自然存在于人的血浆、唾液和尿液中。血浆中GHK水平约为200ng/mL(10-20岁时为7μM),但到60岁时降至80纳克/毫升。GHK水平的下降与生物体再生能力的显著下降相一致。人类肽GHK-Cu于1973年由Pickart分离出来,作为人类白蛋白中的一种活性,可导致老年人肝组织合成像年轻组织一样的蛋白质[1]。随后的研究证实,这种活性是一种氨基酸序列为甘氨酰-L-组氨酸-L-赖氨酸的三肽,对铜具有强烈的亲和力,容易形成复合物GHK-Cu。有人提出,GHK-Cu与Cu2+形成络合物[2]。Pickart等人已经证实GHK-Cu可加速伤口愈合和收缩,改善移植皮肤的吸收,并具有抗炎作用[3-5]。罗伦皮卡特Dr.Loren Pickart发现铜胜肽治疗伤口和皮肤损伤非常有效,不但可减少疤痕组织生成,同时能刺激皮肤自行愈合。在除皱方面,铜胜肽可以把日常的皮肤损伤减到最低程度,延缓老化现象。虽然有些人持怀疑态度,认为研究还不够透彻,但多项大规模研究的结果都认为毫无问题。美国纽奥良州杜兰大学医学院发现,胜肽可改善老化皮肤的说法,有其科学根据。
Borel和Maquart等人指导的后续研究表明,GHK-Cu在非常低的无毒浓度(1-10纳摩尔)下刺激胶原和糖胺聚糖的合成和分解[6]。作为伤口愈合和皮肤重塑过程的主要调节因子,GHK调节金属蛋白酶及其抑制剂(TIMP-1和TIMP-2)的活性[7,8]。GHK调节胶原蛋白、硫酸皮肤素、硫酸软骨素和一种小蛋白聚糖,核心蛋白聚糖[9]。2001年,McCormack等人证实,GHK-Cu在损伤细胞DNA的抗癌放射治疗后恢复了患者成纤维细胞的复制活力[10]。还发现GHK可将免疫细胞和内皮细胞吸引到损伤部位[11]。动物实验证实了GHK-Cu的伤口愈合活性。GHK-Cu可加速伤口愈合,增加血管形成和抗氧化酶水平。该分子还可诱导大鼠、小鼠和猪的全身伤口愈合。它能促进大鼠糖尿病和缺血伤口的愈合,降低TNF-α水平,刺激胶原合成。它还促进了狗的脚垫伤口愈合[12-17]。这种记录良好的皮肤再生活性促使GHK在抗衰老化妆品中广泛使用[18]。GHK-Cu作为一种治疗慢性阻塞性肺疾病(COPD)、皮肤炎症和转移性结肠癌的前瞻性治疗药物而广受关注[19-21]。已经证实它是有能力的人类基因组中至少4000个基因的上调和下调,基本上使DNA恢复到更健康的状态[22]。这些研究为GHK-Cu肽的皮肤重塑活性提供了新的线索。
人体血浆中的三肽Gly-His-Lys(GHK)和二价铜离子有很强的亲和力,能自发形成络合物铜胜肽(copper peptide或GHK-Cu),GHK-Cu不仅在肌肤活化过程中扮演着关键性角色,而且在皮肤正常胶原蛋白、弹力蛋白、蛋白聚糖和葡萄胺聚糖的生成、不同细胞类型的生长速度和迁移、抗炎、抗氧化等生理反应中起到重要作用。最近,王鹏博士通过模拟铜胜肽与铜离子的结合位点,以三肽Gly-His-Lys为探针接受体[22],并将其与荧光团异硫氰酸荧光素巧妙结合构建出可视化多肽生物探针L(Fitc-Ahx-Gly-His-Lys-NH2)。L可以很好地和Cu2+结合,基于铜离子的顺磁淬灭传感机制实现其荧光的快速淬灭。作为一个新颖的可视化荧光探针,L-Cu能够在纯水缓冲溶液中实现对H2S的可视化快速高灵敏度检测,检测极限(LOD)低至14.7nM。另外,L具有较高的准确性和实用性,在两个真实环境样品中特异性高灵敏地检测了Cu2+和H2S。最后,基于良好的生物相容性和较低的生物毒性,将L应用于RKO活细胞中实现了生物成像研究。
综上所述,人们长期以来一直认为人类铜结合肽GHK-Cu可促进皮肤伤口愈合,并刺激皮肤更新,表现出广泛的效果。参与真皮修复和皮肤再生的细胞途径形成了一个复杂而精细的生物化学网络,各种调节分子在其中相互作用。当这种相互作用被破坏时,愈合被延迟,并可能导致过度炎症和疤痕。GHK似乎能够通过将基因模式重置为更健康的状态来恢复皮肤修复中基本细胞途径的健康功能。GHK以低成本大量供应,是一种治疗各种疾病的潜在方法。该分子非常安全,在用作皮肤化妆品或用于人类伤口愈合研究期间,从未出现过任何问题。研究表明,将GHK注射到身体的较远部位,如大腿,以诱导全身愈合,以及每天腹腔注射一次GHK以诱导全身伤口愈合的研究,估计约100-200mg GHK将对人体产生治疗作用。但即使这样也可能高估了分子的必要有效剂量[14]。
关于蓝铜三肽的来源与合成制备方面:1979年,Pickart等人尝试从生物材料中提取GHK,发现从人的血浆中提取和纯化三肽GHK的过程中往往会造成生物活性的损失,而且常伴随着铜离子和铁离子的螯合,这些金属离子对从血浆中分离GHK的多个步骤都会产生干扰[24],这就使得化学合成三肽GHK具有独特的优势。1997年,Liakopoulou等人用Fmoc固相法从C末端合成了GHK,以Fmoc-Lys(Mtt)-OH、Fmoc-His(Mtt)-OH、Fmoc-Gly-OH为原料,以DCCI和HOBt为缩合试剂,经HPLC纯化得到纯的三肽GHK。其中HKZ.肽的产率为67%,GHK三肽的产率仅48%;实验还对其在体外微生物抗性及对DNA,RNA,蛋白合成的影响做了研究,结果表明GHK在一定浓度下能抑制大肠杆菌的生长,迸一步的实验发现GHK抑制大肠杆菌DNAiFfl蛋白质的合成[25]。2001年,Yujun Zheng等人也通过Fmoc固相法,以Fmoc-Lys(Mtt)-OH、Fmoc-His(Trt)-OH、Fmoc-Gly-OH为原料,以DMAP、DIC、HOBt为缩合试剂,10%TFA裂解制得三肽GHK[23]。Conato等人也研究了铜离子,镍离子与其结合的结构特征[26]。目前所见国外合成GHK的报道不多,多属专利。国内的陈钧辉等人在1994年研究了GHK及GHK-Cu2+促使创面愈合的作用[27],1997年报道用液相法成功合成GHK及GHK-Cu2+,并研究了其对于表皮细胞,内皮细胞,成纤维细胞等人源细胞DNA合成的影响[28]。在合成路线研发方面,2003年,彭小三等人选择了先合成苄氧甲酰甘氨酰组氨酸(Z-Gly-His-OH)与ε-氨基和羧基都保护好的赖氨酸(H-Lys(Nε-Z)-OTs,其中Lys的ε-氨基用苄基保护,Lys的羧基用对甲苯磺基保护),再采用DCC法缩合,最后用氢气和钯黑处理,把所有保护基团一并脱去,合成出三肽GHK[29]。GHK与等摩尔数的醋酸铜反应制得GHK-Cu2+。该方法简化了液相合成GHK的工艺路线,但在制备过程中使用钯催化氢化反应后,导致终产物中残留的贵重金属钯含量超标,且不易脱除。
因此,有必要针对上述液相法和固相法合成蓝铜三肽的策略都存在困境和不足,探索一种新的蓝铜三肽合成方法。
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发明内容
本发明的目的在于解决目前蓝铜三肽化学合成方法的均相反应比较复杂,分离步骤多、耗时周期长、纯化规模小、生产成本高,而固相反应的生产规模小,原料价格贵、浪费大,树脂类废弃物多和环境污染严重的问题,而提供了一种可光剪切的基团辅助均相合成蓝铜三肽的方法。
为实现上述目的,本发明中的合成路线设计思路如图1所示,提供的技术解决方案是:
一种7-二苯基膦酰氧基香豆素-4-甲醇类化合物DPCM,其特殊之处在于,分子结构通式为:
本发明提供了上述7-二苯基膦酰氧基香豆素-4-甲醇类化合物DPCM的制备方法,其特殊之处在于,包括以下步骤:
1)以7-羟基香豆素-4-甲基卤素为原料与(R)2POCl反应,在A溶剂中加碱催化进行反应,反应完毕后,旋蒸回收溶剂,残留溶液用乙酸乙酯萃取并与水相分离,合并有机相并用去离子水洗涤多次,分离纯化得到7-二苯基膦酰氧基香豆素-4-甲基卤素类化合物;
2)将7-二苯基膦酰氧基香豆素-4-甲基卤素类化合物经水解反应,转化为相应的7-二苯基膦酰氧基香豆素-4-甲醇类化合物。
所述(R)2POCl与7-羟基香豆素-4-甲基卤素的投料比例为1~4:1,反应温度为-5~50℃,反应时间为1~5小时;
所述A溶剂为四氢呋喃、乙腈、苯、甲苯、氯仿、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮中的一种或几种;
所述碱为叔胺TEA、DIEA、NMM、吡啶及DMAP中的一种或几种。
一种可光剪切载体辅助均相合成蓝铜三肽的方法,其特殊之处在于,包括以下步骤:
1)辅助基团的偶联
辅助基团采用权利要求1所述的7-二苯基膦酰氧基香豆素-4-甲醇类化合物DPCM;氨基酸采用其氨基均被保护的赖氨酸PG1-Lys(Nε-PG2)-OH;
以所述辅助基团替代固相多肽合成中的树脂,在脱水偶联剂的作用下与所述氨基酸进行搅拌反应,使得氨基均被保护的赖氨酸PG1-Lys(Nε-PG2)-OH的C-端与所述辅助基团连接,生成化合物A,PG1-Lys(Nε-PG2)-DPCM;
2)分离纯化
向步骤1)得到的化合物A中加入极性小的烷烃或醚类溶剂,将化合物A与杂质分离,随后对分离后的化合物A进行纯化,纯化方式为过滤和洗涤或者重结晶;
3)脱除N-端PG1
采用脱PG1试剂处理步骤2)纯化后的化合物A,搅拌反应得到化合物B,H-Lys(Nε-PG2)-DPCM;
向化合物B加入极性小的烷烃或醚类溶剂,将化合物B与杂质分离;随后对分离后的化合物B进行纯化,纯化方式为过滤和洗涤或者重结晶;
4)多肽偶联
将步骤3)纯化后的化合物B作为原料,与PG1保护的组氨酸PG1-His(N’-PG3)-OH进行偶联反应,制备得到化合物C,PG1-His(N’-PG3)-Lys(Nε-PG2)-DPCM;对化合物C进行分离纯化和脱除N-端PG1后,得到化合物D,H-His(N’-PG3)-Lys(Nε-PG2)-DPCM;
将化合物D作为原料,与PG1保护的甘氨酸PG1-Gly-OH进行偶联反应,制备得到化合物E,PG1-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM;对化合物E进行分离纯化和脱除N-端PG1后,得到化合物F,H-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM;
5)DPCM辅助基团的光照剪除与侧链脱保护
5.1)采用360nm紫外灯照射步骤4)得到的化合物F的溶液,脱除载体(即辅助基团DPCM),经分离纯化,得到蓝铜三肽的前体化合物G以及载体DPCM;其中,化合物G为H-Gly-His(N’-PG3)-Lys(Nε-PG2)-OH,所述载体DPCM可回收再利用;
5.2)分别以二乙胺的甲醇溶液和三氟乙酸的鸡尾酒溶液为侧链脱保护剂,脱除赖氨酸侧链上的保护基团PG2和组氨酸侧链上的保护基团PG3,经分离纯化得到蓝铜三肽的三氟乙酸盐,TFA*H-Gly-His-Lys-OH;
或者,
5.1)依次采用二乙胺的甲醇溶液和三氟乙酸的鸡尾酒溶液脱除步骤4)得到的化合物F赖氨酸侧链上的保护基团PG2和组氨酸侧链上的保护基团PG3,依次得到化合物G和化合物H;
5.2)采用360nm紫外灯照射得到的化合物H的溶液,脱除载体DPCM,经分离纯化,得到经分离纯化得到蓝铜三肽的三氟乙酸盐,TFA*H-Gly-His-Lys-OH以及载体DPCM;其中,所述载体DPCM可回收再利用;
也就说,即可以先光照剪切掉载体,再进行侧链脱保护,也可以先依次侧链脱保护,最后进行光照剪切;两种方式均不影响蓝铜三肽的合成;
6)蓝铜三肽的分离纯化
通过旋蒸步骤5)得到的蓝铜三肽的三氟乙酸盐回收三氟乙酸,残留溶液采用碳酸氢钠中和,调节pH为8~9,用乙酸乙酯萃取,沉淀析出,经过滤、乙酸乙酯洗涤、干燥得到纯化的蓝铜三肽(GHK),H-Gly-His-Lys-OH。
进一步地,步骤1)中,在0~50℃下搅拌反应1~3小时;所述氨基酸与辅助基团的摩尔比为1~3∶1;所述脱水偶联剂为1︰1摩尔比的脱水偶联活化剂和碱性物质;其中,脱水偶联活化剂如DCC、DIC、EDCI等,碱性物质如DBU、DIEA、DMAP、NMP等;
步骤2)中,烷烃类如正己烷、环己烷和石油醚等,醚类如乙醚,甲基叔丁基醚等;
步骤3)中,在10~50℃下搅拌反应0.5~2小时;
步骤5)中,侧链脱保护的反应条件为5~30℃下,搅拌1~3小时;
所述二乙胺的甲醇溶液中二乙胺的质量分数为20%;
所述三氟乙酸的鸡尾酒溶液中的组分比例为:TFA∶TIPS∶H2O=95∶2.5∶2.5。
上述可光剪切载体辅助均相合成蓝铜三肽过程中辅助基团DPCM的回收再利用的方法,其特殊之处在于:将步骤6)中合并所得的乙酸乙酯萃取溶液旋蒸浓缩至原体积的1/3~1/4,加入极性小的烷烃或醚类溶剂,借助DPCM在不同溶剂系统中易结晶沉淀的特性,可将DPCM与其他杂质分离;对分离后的DPCM进行过滤和洗涤或重结晶操作得到纯化的DPCM,回收后可以直接作为辅助基团再利用。
上述烷烃类如正己烷、环己烷和石油醚等,醚类如乙醚,甲基叔丁基醚等;
除此之外,本发明还提供了上述方法制备过程中得到的N-端PG1保护赖氨酰-DPCM化合物,其特殊之处在于:PG1-Lys(Nε-PG2)-DPCM的分子结构通式为:
上述方法制备过程中得到的脱保护基PG1的赖氨酰-DPCM化合物,其特殊之处在于:H-Lys(Nε-PG2)-DPCM的分子结构通式为:
上述方法制备过程中得到的N-端PG1保护组氨酰-赖氨酰-DPCM化合物,其特殊之处在于:PG1-His(N’-PG3)-Lys(Nε-PG2)-DPCM的分子结构通式为:
上述方法制备过程中得到的脱保护基PG1的组氨酰-赖氨酰-DPCM化合物,其特殊之处在于:H-His(N’-PG3)-Lys(Nε-PG2)-DPCM的分子结构通式为:
上述方法制备过程中得到的N-端PG1保护甘氨酰-组氨酰-赖氨酰-DPCM化合物,其特殊之处在于:PG1-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM的分子结构通式为:
上述方法制备过程中得到的依次脱保护基PG1、PG2和PG3的甘氨酰-组氨酰-赖氨酰-DPCM化合物F、G、H,其特殊之处在于:F、G、H分子结构通式为:
蓝铜三肽,H-Gly-His-Lys-OH的分子结构式为:
本发明的优点是:
与目前已有的合成方法相比,本发明兼备了液相和固相合成法的优点,可以更加简便、快捷、节约、高效地合成制备GHK,而且DPCM辅助基团(也可称之为载体)可以回收并直接再利用,降低原材料浪费,减少废弃物污染,节约成本,利于环保。
附图说明
图1为本发明中合成路线设计的总体思路;
图2为载体分子DPCM的合成路线;
图3为蓝铜三肽的合成路线;
图4为蓝铜三肽的HPLC图谱。
具体实施方式
以下结合附图和具体实施例对本发明的内容作进一步的详细描述,但本发明不限于该具体实例。
本发明可光剪切的基团辅助均相合成蓝铜三肽的方法,包括以下步骤:
(1)辅助基团的偶联:采用我们发展的7-二苯基膦酰氧基香豆素-4-甲醇(DPCM)类化合物及其衍生物为新型保护基兼可光剪切的辅助基团替代固相合成中的树脂载体,在偶联脱水剂的作用下与保护赖氨酸[PG1-Lys(Nε-PG2)-OH]的C-端连接,得到化合物A;
(2)分离纯化:反应完成后,借助DPCM辅助基团在不同溶剂系统中易结晶沉淀的特性,可将化合物A与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的化合物A;
(3)脱除N-端PG1:将化合物A用脱PG1试剂处理后,再借助DPCM辅助基团在不同溶剂系统中易结晶沉淀的特性,可将化合物B与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的化合物B;
(4)多肽偶联:重复以上步骤(1)、(2)、(3),依次与侧链保护的组氨酸[PG1-His(N’-PG2)-OH]和甘氨酸[Fmoc-Gly-OH]进行偶联和脱PG1反应,制备蓝铜三肽的前体F[H-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM](相应的,比如:H-Gly-His(N’-PG3)-Lys(Nε-PG2)-CPPG);
(5)侧链脱保护并剪除DPCM辅助基团:依次采用光照处理、脱Fmoc试剂和脱鸡尾酒型脱保护试剂TFA/TIS/H2O=95:2.5:2.5处理GHK的前体化合物F,脱除侧链上的保护基团Fmoc、tBu、Boc、Pbf以及载体辅助基团DPCM;
(6)反应完毕后,旋蒸回收三氟乙酸(TFA),残留溶液用碳酸氢钠调节pH=8~9,用乙酸乙酯萃取,析出的沉淀,经过滤、洗涤、干燥等操作,即得到GHK,[H-Gly-His-Lys-OH]的固体。
(7)回收DPCM辅助基团:合并(6)中乙酸乙酯萃取的有机相,旋蒸浓缩至原体积的1/3~1/4,再借助DPCM辅助基团在不同溶剂系统中易结晶沉淀的特性,经简单地过滤和洗涤或重结晶操作就可以得到纯化的DPCM,回收再利用DPCM辅助基团。
本发明中一些常用的缩写具有以下含义:
Boc:叔丁氧羰基
DCM:二氯甲烷CH2Cl2
DCC:二环己基碳二亚胺
DEA:二乙胺
DMAP:4-二甲氨基吡啶
DMF:N,N-二甲基甲酰胺
DPCM:7-二苯基膦酰氧基香豆素-4-甲基
DPPC:二苯基膦酰氯
EDC-HCl:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
Fmoc:芴甲氧羰基
GHK-Cu:蓝铜三肽-铜
HATU:2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐
HOBT:1-羟基苯并三唑
HBTU:O-苯并三氮唑-四甲基脲六氟磷酸盐
NMM:N-甲基吗啉
NMP:N-甲基吡咯烷酮
PyBop:六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷
Trt:三苯甲基
TFA:三氟乙酸
THF:四氢呋喃
TIPS:三异丙基硅烷
本实施例以7-二苯基膦酰氧基香豆素-4-甲醇(1,DPCM)为保护基兼辅助载体合成了较高纯度的蓝铜肽(GHK),最后将蓝铜三肽与醋酸铜反应制备出了蓝铜肽-Cu(GHK-Cu),适用于蓝铜肽的合成制备与光解脱除,反应原理与技术路线如下图2和图3所示,具体技术方案如下:
载体分子7-二苯基膦酰氧基香豆素-4-甲醇(1,DPCM)的合成路线见图2。
7-羟基基香豆素-4-甲基氯(a)的合成:量取100mL浓硫酸置于250mL圆底烧瓶中,密封并放置于冰水浴下搅拌10min,是整个体系充分降温;精确称取间苯二酚11.01g(100mmol)和4-氯乙酰乙酸乙酯19.75g(120mmol,约16.2mL)放入50mL的离心管中,轻微加热使间苯二酚尽可能溶解于乙酰乙酸乙酯(不能超声,加热不可太过,间苯二酚无法完全溶解,不影响后续反应);用滴管将间苯二酚和4-氯乙酰乙酸乙酯混合物(溶液混杂着少量固体)缓慢滴加至冷却的浓硫酸中,维持冰水浴,滴加过程中体系逐渐变成棕红色,滴加结束后撤去冰水浴;TLC点板监测反应(展开剂EA:PE=1:1,体积比),12h后间苯二酚反应完全,将反应溶液缓慢倾倒入500mL冰水混合中(大量淡黄色胶状物质析出),倾倒结束后保持搅拌30min,经过滤、洗涤、干燥后将粗产物用50mL 75%的酒精重结晶,最终得到米白色针状晶体15.1g,收率为72%。7-羟基基香豆素-4-甲基氯(a,分子式为C10H7ClO3)的结构表征数据:1H NMR(400MHz,DMSO-d6)δ10.66(s,1H),7.68(d,J=8.7Hz,1H),6.85(dd,J=8.6,2.4Hz,1H),6.76(s,1H),6.42(s,1H),4.96(s,2H);13C NMR(100MHz,DMSO-d6)δ161.92,160.60,155.76,151.45,127.02,113.54,111.53,109.81,102.98,41.83.
7-羟基基香豆素-4-甲醇(b)的合成:准确称取化合物a 10.5g(50mmol),置于250mL的圆底烧瓶中,加入约30mL DMF和45mL去离子水(室温下固体无法完全溶解,体系为乳白色悬浮液),在110℃下加热搅拌,冷凝回流,过夜反应。TLC点板监测反应(展开剂DCM:MeOH=10:1,体积比),反应过夜后化合物5消耗完全,将反应溶液倾倒入200mL饱和食盐水中,用200mL乙酸乙酯萃取水相三次,合并有机相,有机相用饱和食盐水洗涤3次,无水MgSO4干燥,过滤旋蒸除去乙酸乙酯,得到橙黄色粗产物,将粗产物用30mL甲醇重结晶,干燥后得到白色粉末状固体7.392g,收率为77%。7-羟基基香豆素-4-甲基氯(b,分子式为C10H8O4)的结构表征数据:1H NMR(400MHz,DMSO-d6)δ10.52(s,1H),7.52(d,J=8.7Hz,1H),6.77(dd,J=8.7,2.4Hz,1H),6.73(d,J=2.3Hz,1H),6.24(d,J=1.5Hz,1H),5.59(t,J=5.6Hz,1H),4.70(d,J=3.6Hz,2H);13C NMR(100MHz,DMSO-d6)δ161.44,161.09,157.27,155.31,125.93,113.28,110.00,106.98,102.75,59.53;HRMS(ESI)m/z calcd for C10H8O4Na+(M+Na)+215.03148,found 215.03163.
7-二苯基膦酰氧基香豆素-4-甲基氯(c)的合成:精确称取化合物(a)3.5g(16.67mmol)置于100mL的圆底烧瓶中,加入30mL四氢呋喃溶解,密封并放置于冰水浴下搅拌10min,使整个体系充分降温;烧瓶中加入二苯基次膦酰氯4.02g(17mmol,约3.24mL),搅拌使之混合均匀后,缓慢滴加三乙胺1.72g(17mmol,约2.36mL),在滴加三乙胺过程中体系逐渐变浑浊,瓶口有大量白烟生成(按照反应原理应该是先加三乙胺使得化合物5中的酚羟基变为氧负离子,便于和酰氯反应,但由于分子中存在苄氯基团,极易与三乙胺反应生成季铵盐,无法得到目标产物);TLC点板监测反应(展开剂DCM:MeOH=10:1,体积比),半小时后化合物5消耗完全,加入5mL饱和氯化铵水溶液淬灭反应,使用旋蒸除去四氢呋喃,加入50mL二氯甲烷溶解固体,有机相用50mL饱和食盐水洗涤三次,干燥过滤,旋蒸除去二氯甲烷后,通过柱层析(洗脱剂DCM:MeOH=30:1,Vol)获得米黄色固体4.75g,收率为69%。7-二苯基膦酰氧基香豆素-4-甲基氯(c,分子式为C22H16ClO4P)的结构表征数据:1H NMR(400MHz,DMSO-d6)δ8.03–7.94(m,4H),7.82(d,J=8.7Hz,1H),7.62(ddd,J=23.8,7.5,2.7Hz,6H),7.45–7.33(m,2H),6.61(s,1H),4.98(s,2H);13C NMR(100MHz,DMSO-d6)δ159.80,154.56,153.86,150.65,133.58,133.55,132.07,131.97,131.24,129.88,129.61,129.48,127.34,117.58,114.87,114.53,109.28,109.22,41.65;31P NMR(162MHz,DMSO-d6)δ30.95;HRMS(ESI)m/z calcd for C22H17ClO4P+(M+H)+411.05475,found 411.05490.
7-二苯基膦酰氧基香豆素-4-甲醇(DPCM,1)的合成:精确称取化合物b 9.6g(50mmol)置于250mL圆底烧瓶中,加入100mL四氢呋喃(固体无法完全溶解),缓慢滴加三乙胺5.05g(50mmol,约6.9mL),密封并放置于冰水浴下搅拌10min,使整个体系充分降温;在烧瓶中分10批缓慢滴加二苯基次膦酰氯12.4g(52.5mmol,约10mL),冰水浴搅拌,在滴加二苯基次膦酰氯过程中体系逐渐变浑浊,瓶口有大量白烟生成(羟基的酰化可以看作是氧负离子的亲核取代反应,一般来说酚羟基的酸性大于苄醇,在三乙胺存在下酚羟基更易形成氧负离子,比苄醇有更好的亲核性,因此对于化合物7只要控制好酰氯的量,保持低温就可以合成化合物10);TLC点板监测反应(展开剂DCM:MeOH=10:1,体积比),半小时后化合物(b)消耗完全,加入5mL饱和氯化铵水溶液淬灭反应,使用旋蒸除去四氢呋喃,加入30mL甲醇(大量白色固体未溶解),超声使体系分散均匀,将烧瓶放入冰箱冷藏2h,过滤,用冷的50%的甲醇水溶液5mL×3次(除去夹杂的三乙胺盐酸盐),干燥后得到白色粉末状固体15.7g,收率为80.1%。化合物DPCM(1,分子式为C22H17O5P)的结构表征数据:1H NMR(400MHz,DMSO-d6)δ8.05–7.88(m,4H),7.71–7.52(m,7H),7.38(dd,J=2.4,1.1Hz,1H),7.31(ddd,J=8.8,2.4,1.1Hz,1H),6.39(d,J=1.7Hz,1H),5.64(t,J=5.5Hz,1H),4.70(dd,J=5.5,1.7Hz,2H);13C NMR(100MHz,DMSO-d6)δ160.26,156.57,154.18,133.52,133.49,132.06,131.96,131.31,129.95,129.57,129.44,126.36,117.38,117.33,114.75,110.14,109.05,108.99,59.48;31P NMR(162MHz,DMSO-d6)δ30.77;HRMS(ESI)m/z calcd for C22H17O5PNa+(M+Na)+415.07058,found 415.07034.
蓝铜三肽(GHK)的合成路线见图3。
Boc-Lys(Nε-Fmoc)-DPCM(2)的制备:准确称取Nα-Boc-Lys(Nε-Fmoc)-OH(2.57g,5.5mmol,1.1eq)、EDC·HCl(1.44g,7.5mmol,1.5eq)、DMAP(62.5mg,0.5mmol,0.1eq)置于100mL的圆底烧瓶中,加入50mL二氯甲烷,放置于冰水浴下搅拌30min,使体系充分活化(生成活性酯),随后称取CPPG(1.96g,5mmol,1eq)加入反应溶液中,室温下继续搅拌;TLC点板监测反应(展开剂DCM:MeOH=10:1,Vol),30min后反应结束,反应液用饱和NH4Cl水溶液、饱和NaHCO3水溶液和饱和食盐水洗涤后干燥,旋蒸除去二氯甲烷后,固体用乙酸乙酯与石油醚混合溶剂(体积比1:8)洗涤,撇去清液后除去剩余溶剂得到白色泡沫状固体3.9g,收率为93%。(2)Boc-Lys(Nε-Fmoc)-DPCM(C48H47N2O10P)结构表征:1H NMR(400MHz,DMSO-d6)δ7.98(ddd,J=12.4,7.6,1.6Hz,4H),7.87(d,J=7.5Hz,2H),7.72–7.61(m,4H),7.58(td,J=7.3,3.7Hz,4H),7.48–7.27(m,8H),6.44(s,1H),5.36(s,2H),4.31(d,J=6.9Hz,2H),4.21(t,J=6.9Hz,1H),4.10–4.02(m,1H),2.98(q,J=6.3Hz,2H),1.76–1.25(m,15H);31P NMR(162MHz,DMSO-d6)δ30.89;HRMS(ESI)m/z calcd for C48H47N2O10PNa+(M+Na)+865.28605,found 865.28656.
Boc-Lys(Nε-Fmoc)-DPCM脱Boc制备(3):将前文中得到的Boc-Lys(Nε-Fmoc)-DPCM转移到100mL的圆底烧瓶中,加入15mL二氯甲烷溶解,于室温下搅拌并缓慢滴加三氟乙酸(TFA)5mL,(TFA脱Boc基团切忌有水,否则酯键水解),滴加完成后继续在室温下反应,体系中逐渐出现绵密的小气泡;TLC点板监测反应(展开剂DCM:MeOH=10:1,Vol),3h后脱保护完全,通过减压旋转蒸发除去DCM和TFA(注意不能加热在室温下旋蒸即可),旋干后加入20mL二氯甲烷再次溶解样品,有机相分别用饱和NaHCO3和饱和食盐水洗涤2次,经无水MgSO4干燥后旋干溶剂可以得到白色泡沫状固体(3)[H-Lys(Nε-Fmoc)-DPCM]。结构表征:1H NMR(400MHz,Chloroform-d)δ7.99–7.30(m,21H),7.18(dd,J=2.4,1.2Hz,1H),6.39(d,J=1.4Hz,1H),5.24(dd,J=4.3,1.4Hz,2H),4.39(d,J=7.0Hz,2H),4.21(t,J=6.8Hz,1H),3.61(dd,J=7.5,5.2Hz,1H),3.25–3.15(m,2H),1.73–1.35(m,6H);31P NMR(162MHz,Chloroform-d)δ32.65;HRMS(ESI)m/z calcd for C43H39N2O8PNa+(M+Na)+765.23362,found765.23383.
H-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM(4)的制备:偶联Boc-His(N’-Trt)-OH的操作可参照上述步骤,偶联氨基酸所需要的EDC·HCl、HOBt、DIEA和下一个氨基酸的用量都是1.1个当量,可以得到(4)Boc-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM。1H NMR(400MHz,Chloroform-d)δ7.92(dd,J=12.8,7.6Hz,3H),7.74(d,J=7.5Hz,2H),7.6–7.56(m,4H),7.51(td,J=7.6,3.7Hz,4H),7.42–7.29(m,16H),7.18(dd,J=2.4,1.1Hz,1H),7.10(dt,J=7.3,4.1Hz,8H),6.68(s,1H),6.38(s,1H),5.37–5.03(m,2H),4.60(td,J=7.8,5.2Hz,1H),4.49(s,1H),4.45–4.33(m,2H),4.19(t,J=7.0Hz,1H),3.11(d,J=6.4Hz,2H),2.99(d,J=6.3Hz,2H),1.89–1.27(m,16H);31P NMR(162MHz,Chloroform-d)δ32.44;HRMS(ESI)m/z calcd for C73H68N5O11PNa+(M+Na)+1244.45452,found 1244.45520.
参照上述步骤脱除Boc保护基后,经分离纯化得到(5)H-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM。HRMS(ESI)m/z calcd for C68H60N5O9PNa+(M+Na)+1144.40209,found1144.40295.
H-Gly-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM(6)的制备:偶联Boc-Gly-OH的操作可参照上述步骤,偶联氨基酸所需要的EDC·HCl、HOBt、DIEA和下一个氨基酸Boc-Gly-OH的用量都是1.1个当量,反应后可以得到(6)Boc-Gly-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM。HRMS(ESI)m/z calcd for C75H71N6O12PNa+(M+Na)+1301.47598,found 1301.47742。再经脱Boc-保护反应,可以得到(7)H-Gly-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM。
蓝铜三肽(GHK)的制备:将H-Gly-His(N’-Trt)-Lys(Nε-Fmoc)-DPCM产物置于100mL的圆底烧瓶中,加入混合溶剂(TFA:TIS:H2O=95:2.5:2.5,Vol),室温下搅拌反应,TLC点板监测反应(展开剂DCM:MeOH=5:1,Vol),3h后反应结束,通过旋蒸除去TFA和TIS;随后加入30%的二乙胺的乙腈溶液10mL(一锅法完成剪切与脱N’-Fmoc),继续搅拌2h,然后旋干溶剂,然后加入20mL二氯甲烷,有大量白色固体没有溶解,超声振荡后过滤洗涤干燥的得到白色粉末状固体0.29g(0.85mmol),合成该三胜肽的总收率为17%。(8,GHK,C14H24N6O4):1H NMR(400MHz,Deuterium Oxide)δ7.74(d,J=1.2Hz,1H),6.91(d,J=1.2Hz,1H),4.56(dd,J=7.2,6.2Hz,1H),4.02(dd,J=7.9,5.3Hz,1H),3.72–3.58(m,2H),3.08–2.93(m,2H),2.87(t,J=7.6Hz,2H),1.68(dtd,J=13.6,8.1,5.4Hz,1H),1.65–1.47(m,3H),1.24(p,J=7.4,6.8Hz,2H);13C NMR(125MHz,Deuterium Oxide)δ178.36,171.73,168.07,135.71,131.34,117.57,55.05,53.66,40.77,39.18,30.97,28.44,26.29,21.93;HRMS(ESI)m/z calcd for C14H25N6O4 +(M+H)+341.19318,found 341.19266.(注:碳谱在500MHz核磁下测得)
蓝铜三肽-Cu(GHK-Cu)的制备:准确称取蓝铜三肽34mg(0.1mmol),置于25mL的圆底烧瓶中,加入3mL去离子水溶解,量取0.05mol/L的醋酸铜水溶液2mL(含醋酸0.1mmol),室温搅拌下缓慢滴加至蓝铜肽中,溶液由灰蓝色逐渐变为透亮的深蓝色,搅拌至体系颜色稳定后停止反应,通过冷冻干燥除去水得到淡蓝色絮状固体50mg,收率为99%。蓝铜肽的HPLC图谱如图4所示,保留时间为7.598min。
以香豆素衍生物为连接子,在香豆素分子上引入含磷基团作为辅助沉淀基团,设计合成了一系列香豆素基光敏保护基。在与氨基酸偶联时,在氟化钾的存在下,苄氯类的载体虽偶联上了氨基酸但是其含磷基团也被脱除,虽然可以再次引入含磷基团,但是较为繁琐,加上前一步在苄氯香豆素上引入含磷基团的产率不算高(易形成季铵盐),因此苄氯类的香豆素基光敏保护基没有被进一步的研究。
苄醇类的保护基DPCM可以通过传统的酯化方法偶联氨基酸(EDC、DMAP活化酯法),含磷基团不受影响。通过对Boc-Val-DPCM和Boc-Phe-DPCM的脱Boc实验,可以得知DPCM保护基是对酸稳定的,可以沿着Boc-氨基酸途径偶联氨基酸。在对Fmoc-Phe-Val-DPCM进行脱Fmoc保护时没有得到目标产物,而是得到了苯丙氨酸和缬氨酸的环二肽,由此可见DPCM保护基对碱不稳定,其虽然不能沿着Fmoc途径进行偶联,但是此类载体对于合成环二肽有巨大作用。
通过以DPCM为保护基合成出两条直链的多肽:一条六肽(缬氨酸与苯丙氨酸交替偶联而成)和一条三肽(蓝铜三胜肽,甘胺酰-组氨酰-赖氨酸),总体收率分别为12.7%和17%,在偶联过程中很少使用色谱,大多以沉淀的方式进行提纯,大大节省了试剂。由此可以证明DPCM保护基在直链的多肽合成有应用价值。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明公开的技术范围内,可轻易想到各种等效的修改或替换,这些修改或替换都应涵盖在本发明的保护范围之内。
Claims (10)
2.权利要求1所述7-二苯基膦酰氧基香豆素-4-甲醇类化合物DPCM的制备方法,其特征在于,包括以下步骤:
1)以7-羟基香豆素-4-甲基卤素为原料与(R)2POCl反应,在A溶剂中加碱催化进行反应,反应完毕后,旋蒸回收溶剂,残留溶液用乙酸乙酯萃取并与水相分离,合并有机相并用去离子水洗涤多次,分离纯化得到7-二苯基膦酰氧基香豆素-4-甲基卤素类化合物;
2)将7-二苯基膦酰氧基香豆素-4-甲基卤素类化合物经水解反应,转化为相应的7-二苯基膦酰氧基香豆素-4-甲醇类化合物。
3.一种可光剪切载体辅助均相合成蓝铜三肽的方法,其特征在于,包括以下步骤:
1)辅助基团的偶联
辅助基团采用权利要求1所述的7-二苯基膦酰氧基香豆素-4-甲醇类化合物DPCM;氨基酸采用其氨基均被保护的赖氨酸PG1-Lys(Nε-PG2)-OH;
以所述辅助基团替代固相多肽合成中的树脂,在脱水偶联剂的作用下与所述氨基酸进行搅拌反应,使得氨基均被保护的赖氨酸PG1-Lys(Nε-PG2)-OH的C-端与所述辅助基团连接,生成化合物A,PG1-Lys(Nε-PG2)-DPCM;
2)分离纯化
向步骤1)得到的化合物A中加入极性小的烷烃或醚类溶剂,将化合物A与杂质分离,随后对分离后的化合物A进行纯化;
3)脱除N-端PG1
采用脱PG1试剂处理步骤2)纯化后的化合物A,搅拌反应得到化合物B,H-Lys(Nε-PG2)-DPCM;
向化合物B加入极性小的烷烃或醚类溶剂,将化合物B与杂质分离;随后对分离后的化合物B进行纯化;
4)多肽偶联
将步骤3)纯化后的化合物B作为原料,与PG1保护的组氨酸PG1-His(N’-PG3)-OH进行偶联反应,制备得到化合物C,PG1-His(N’-PG3)-Lys(Nε-PG2)-DPCM;对化合物C进行分离纯化和脱除N-端PG1后,得到化合物D,H-His(N’-PG3)-Lys(Nε-PG2)-DPCM;
将化合物D作为原料,与PG1保护的甘氨酸PG1-Gly-OH进行偶联反应,制备得到化合物E,PG1-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM;对化合物E进行分离纯化和脱除N-端PG1后,得到化合物F,H-Gly-His(N’-PG3)-Lys(Nε-PG2)-DPCM;
5)DPCM辅助基团的光照剪除与侧链脱保护
5.1)采用360nm紫外灯照射步骤4)得到的化合物F的溶液,脱除载体DPCM,经分离纯化,得到蓝铜三肽的前体化合物以及载体DPCM;其中,前体化合物为H-Gly-His(N’-PG3)-Lys(Nε-PG2)-OH,所述载体DPCM可回收再利用;
5.2)分别以二乙胺的甲醇溶液和三氟乙酸的鸡尾酒溶液脱除赖氨酸侧链上的保护基团PG2和组氨酸侧链上的保护基团PG3,经分离纯化得到蓝铜三肽的三氟乙酸盐,TFA*H-Gly-His-Lys-OH;
或者,
5.1)依次采用二乙胺的甲醇溶液和三氟乙酸的鸡尾酒溶液脱除步骤4)得到的化合物F赖氨酸侧链上的保护基团PG2和组氨酸侧链上的保护基团PG3,依次得到化合物G和化合物H;
5.2)采用360nm紫外灯照射得到的化合物H的溶液,脱除载体DPCM,经分离纯化,得到经分离纯化得到蓝铜三肽的三氟乙酸盐,TFA*H-Gly-His-Lys-OH以及载体DPCM;其中,所述载体DPCM可回收再利用;
6)蓝铜三肽的分离纯化
通过旋蒸步骤5)得到的蓝铜三肽的三氟乙酸盐回收三氟乙酸,残留溶液采用碳酸氢钠中和,调节pH为8~9,用乙酸乙酯萃取,沉淀析出,经过滤、乙酸乙酯洗涤、干燥得到纯化的蓝铜三肽(GHK),H-Gly-His-Lys-OH。
4.根据权利要求3所述可光剪切载体辅助均相合成蓝铜三肽的方法,其特征在于:
步骤1)中,在0~50℃下搅拌反应1~3小时;所述氨基酸与辅助基团的摩尔比为1~3∶1;所述脱水偶联剂为1︰1摩尔比的脱水偶联活化剂和碱性物质;其中,脱水偶联活化剂如DCC、DIC、EDCI等,碱性物质如DBU、DIEA、DMAP、NMP等;
步骤2)中,烷烃类如正己烷、环己烷和石油醚等,醚类如乙醚,甲基叔丁基醚等;
步骤3)中,在10~50℃下搅拌反应0.5~2小时;
步骤5)中,侧链脱保护的反应条件为5~30℃下,搅拌1~3小时;
所述二乙胺的甲醇溶液中二乙胺的质量分数为20%;
所述三氟乙酸的鸡尾酒溶液中的组分比例为:TFA∶TIPS∶H2O=95∶2.5∶2.5。
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