CN111196838A - 一种精氨酸衍生物Cbz-Phe-Arg-AMC的合成方法及应用 - Google Patents
一种精氨酸衍生物Cbz-Phe-Arg-AMC的合成方法及应用 Download PDFInfo
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- CN111196838A CN111196838A CN202010107384.2A CN202010107384A CN111196838A CN 111196838 A CN111196838 A CN 111196838A CN 202010107384 A CN202010107384 A CN 202010107384A CN 111196838 A CN111196838 A CN 111196838A
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- Prior art keywords
- amc
- arg
- phe
- cbz
- pbf
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
本发明申请提供一种精氨酸衍生物Cbz‑Phe‑Arg‑AMC的合成方法及其应用。该方法以7‑氨基‑4‑甲基香豆素(简称AMC)为起始原料，经Fmoc‑Arg(pbf)‑OH缩合、脱Fmoc保护、Cbz‑Phe‑OH偶联获得目标物，用于降纤酶含量测定，比肉眼观察的纤维蛋白原凝固法准确、特异、简单、快速，因此发色底物荧光法比现有降纤酶国家药品标准(WS1‑XG‑031‑2000)中采用的纤维蛋白原凝固法重现性更好，更适合本品低含量效价测定。
Description
技术领域
本发明为多肽合成制备及应用领域，特别是Cbz-Phe-Arg-AMC的合成制备方法及应用。
背景技术
食品安全问题受到人们的日益关注，已经在全世界范围内引起高度重视。而食源性致病菌是引起食源性疾病的重要原因，是食品安全的重大隐患。近年来由食源性致病菌引发的疾病呈上升趋势。如大肠杆菌O157：H7引起了近万人中毒。因此对致病菌进行快速有效的检测就显得尤为重要。传统的检测方法(如分离培养、生化鉴定等)对致病菌的检测特异性不高、灵敏度低、操作繁琐耗时，不能实现快速检测。近年来，特异性酶底物合成和应用发展迅速，已经广泛应用于微生物检测。该方法是将荧光底物或显色底物加入到培养基中，微生物代谢过程中产生的特异性酶将底物分解，释放出色原(直接显色)或荧光团(需在紫外灯下照射显色)，从而实现对微生物的检测和鉴定。尽管特异性显色酶底物法具有操作简便、灵敏性高、特异性好、检测时间短等优点，但是要将该方法作为日常检测方法成本较高，主要是因为现有荧光底物和显色底物的合成工艺复杂、产率低、价格昂贵。因此迫切需要开发出简单易行的合成工艺，降低酶底物的生产成本。近年来通过对常见食源性致病菌显色培养基及其显色底物进行深入研究，目前已成功制备出显色底物磷脂酰基醇(PI)用于检测单核细胞增生性李斯特菌，与进口产品效果相当，明显降低了检测成本。目前国内外应用的荧光底物主要是4-甲基伞形酮和7-氨基-4-甲基香豆素的各类衍生物，包括糖苷类、酯(脂)类、肽类等。4-甲基伞形酮主要用于检测酯(脂)水解酶、糖苷酶等，7-氨基-4-甲基香豆素用与检测肽酶或蛋白酶。
7-氨基-4-甲基香豆素(简称AMC)是重要的荧光物质。因为7位的氨基存在，易与肽链末端羧基缩合形成多肽-香豆素类荧光底物，已成功地应用于微生物检测、免疫检测、生化酶学和多肽合成等研究领域。
发色底物又称发色性多肽底物,显色多肽、产色基质,是一类带有发色基团的小肽,系人工合成的可以代替天然大分子蛋白的底物。1961年,Erlanger等首先用发色性底物苯甲酰-精氨酸-对硝基苯胺(BAPNA)检测丝氨酸蛋白酶。但因该底物未全面照顾到酶与底物相互作用的切点,故专一性和灵敏度欠佳。文献报道采用精氨酸酯酶裂解甲基苯磺酰胺-L-精氨酸甲酯(TAME)，其裂解产物甲基苯磺酰胺-L-精氨酸在247nm波长下有吸收,通过UV法可测得降纤酶的活性。但底物灵敏度不高，在降纤酶浓度为5u/mL时已无法测出。
1972年svednsen等首次合成的发色性三肽Bz-Phe-val-Arg-PNA(S2160)，其模拟了天然纤维蛋白原中凝血酶切点的构型,灵敏度高于BAPNA的100倍,适用于测定凝血酶。雷丹青等采用Bz-Phe-Val-Arg-PNA测定注射用降纤酶的含量，该发色肽释放出对硝基苯胺(PNA)在405nm处有强吸收峰,其显色程度与类凝血酶含量成正比,该试验不受降纤酶制剂中赋形剂右旋糖酐干扰,也不受钙离子影响,因此比肉眼观察的凝固法准确、特异、简单、快速,适于降纤酶原料、半成品、成品的含量测定与常规质量控制。
降纤酶是一种典型的类凝血酶,是以丝氨酸为活性中心的蛋白水解酶,能高选择性的作用于血纤蛋白原,但其对血纤蛋白原的作用比凝血酶更为专一。
现行质量标准采用纤维蛋白原凝固法测定注射用降纤酶含量，该方法的灵敏度虽然高，但存在缓冲液离子浓度、辅料严重影响注射用降纤酶的效价测定，不利于中间体和成品质量控制。如将缓冲溶液稀释一倍，效价结果偏高约一倍左右。辅料右旋糖酐对成品测定也产生干扰，导致结果严重偏离。其次肉眼观察终点误差大，需要探索新的检测方法进行质量控制。
发明内容
本发明针对现有技术的不足，提出一种精氨酸衍生物Cbz-Phe-Arg-AMC的合成方法及应用，具体技术方案如下：
本发明Cbz-Phe-Arg-AMC的结构式如下：
本发明所要解决的技术问题通过以下技术方案实现。本发明是一种Cbz-Phe-Arg-AMC的合成方法，其特点是：包括如下步骤：
步骤A:由AMC、Fmoc-Arg(pbf)-OH缩合反应得到Fmoc-Arg(pbf)-AMC；
步骤B：脱除Fmoc-Arg(pbf)-AMC中胍基侧链保护基得NH2-Arg(pbf)-AMC，偶联Cbz-Phe-OH，获得Cbz-Phe-Arg-AMC。
本发明采用多肽液相合成法，过程为：首先将一个氨基酸被封闭的基团保护氨基酸与AMC缩合，脱掉精氨酸Fmoc保护基，然后再与Cbz-Phe-OH偶联，与氯化氢成盐后得到Cbz-Phe-Arg-AMC。
作为优选，步骤A中偶联包括缩合剂和反应溶剂，所述的缩合剂为DIC、HOBT、HBTU、HATU、HCTU，反应溶剂为DMF、DCM、NMP或DMSO中的一种或两种以上混合液。
步骤B所述的裂解采用的试剂为TFA、苯甲硫醚和EDT的混合液，所述的TFA：苯甲硫醚：水：EDT体积比为92:3:3:2。
7-氨基-4甲基香豆(AMC)的氨基亲核性大于对硝基苯胺(PNA)的氨基，文献报道采用三氯氧磷酰化或DCC混酐法与氨基酸接肽，在反应过程中存在手性氨基酸部分消旋的可能，本发明中，提供的缩合剂如HOBT等可有防止肽类化合物合成过程中手性氨基酸消旋，有利于产品质量控制。本发明工艺具有操作简单，无高温高压，对环境友好的特点。
本发明还提供了一种Cbz-Phe-Arg-AMC用于生化研究，包括用于精氨酸酯酶、凝血酶的发色肽底物。
作为优选，所述的Cbz-Phe-Arg-AMC化合物用于降纤酶的含量测定。
苯甲酰-精氨酸-对硝基苯胺(BAPNA)及Bz-Phe-Val-Arg-PNA发色肽底物释放对硝基苯胺(PNA)可采用紫外吸收(UV)检测，本发明制备的Cbz-Phe-Arg-AMC发色肽底物释放7-氨基-4-甲基香豆素(AMC)具有荧光吸收特性，灵敏度比BAPNA及Bz-Phe-Val-Arg-PNA高(2～3个数量级)，更适合降纤酶低浓度(5U/mL)含量测定。
本发明惊奇的发现，本发明采用的Cbz-Phe-Arg-AMC发色肽底物不受降纤酶制剂中赋形剂右旋糖酐的干扰,因此比肉眼观察的纤维蛋白原凝固法准确、特异、简单、快速,适于降纤酶原料、半成品、成品的含量测定与常规质量控制。因此发色底物荧光法比现有降纤酶国家药品标准(WS1-XG-031-2000)中采用的纤维蛋白原凝固法重现性更好，更适合本品低含量效价测定。
具体实施方式
本发明公开了一种精氨酸衍生物Cbz-Phe-Arg-AMC的合成方法及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中，相关的名词缩写：
Fmoc：9-芴甲氧羰基
HBTU：O-苯并三唑-N-四甲基脲鎓六氟磷酸盐
HATU：O-(7-偶氮苯并三氮唑-1-氧)-N-四甲基脲鎓六氟磷酸盐
DIC二异丙基碳二亚胺
HOBt 1-羟基苯并三唑
HOAt 1-羟基-7-偶氮苯并三唑
Pbf 2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基
DMF N,N-二甲基甲酰胺
DCM二氯甲烷
Bz：苯甲酰
TFA三氟乙酸
NMP：1-甲基-吡咯烷-2-酮
Tris：三羟甲基氨基甲烷
AMC：7-氨基-甲基-香豆素
Cbz：苄氧羰基
BAPNA：苯甲酰-精氨酸-对硝基苯胺
TAME：甲基苯磺酰胺-L-精氨酸甲酯
Bz-Phe-Val-Arg-PNA：苯甲酰-苯丙氨酸-缬氨酸-对硝基苯胺
本发明所用试剂均可由市场购得。
实施例1Fmoc-Arg(pbf)-AMC的合成
溶液1、称取12mmol的AMC、10mmol的Fmoc-Arg(pbf)-OH、12mmol的HOBT，置于100ml的圆底烧瓶中，用20ml的DMF溶解，冷却至0℃备用。
溶液2：称取120mmol的DIC，置于小烧杯中，用10ml的DMF溶解，冷却至0-5℃备用
在0-5℃下，将DIC溶液(溶液2)慢慢滴加到搅拌的溶液1中，反应15分钟，25℃下反应2小时，点板确定反应结束。过滤除去白色沉淀，用30mlDMF洗涤沉淀并过滤，合并滤液，加入大量纯水，出现大量白色固体，过滤得到白色固体。白色固体用1L乙酸乙酯溶解，装入分液漏斗中，分别用稀柠檬酸150ml洗涤3次，用饱和碳酸氢钠150ml洗涤3次，用饱和食盐水200ml洗涤2次，干燥2小时，旋蒸除去溶剂获得产品。纯度为98.4％。
实施例2Cbz-Phe-Arg-AMC的合成
溶液1：称取10mmol的Fmoc-Arg(pbf)-AMC置于100ml的圆底烧瓶中，磁力搅拌下加入哌啶：二氯甲烷＝1：4的溶液溶解，室温下脱保护30分钟，加入大量石油醚，析出大量沉淀，抽滤，用石油醚洗涤数次，获得白色固体产品(NH2-Arg(pbf)-AMC)，在室温下加入脱保护试剂(TFA：苯甲硫醚：水：EDT＝92:3:3:2)30mL，室温下裂解2h，将裂解液倒入预先冷却好的(0℃)无水乙醚中，搅拌出现大量白色固体(NH2-Arg-AMC),固体用10ml的DMF溶解，冷却至0-5℃备用
溶液2：称取10mmol的Cbz-Phe-OH、12mmol的HOBT，置于100ml的圆底烧瓶中，用20ml的DMF溶解，冷却至0℃备用。
溶液3：称取12mmol的DIC，置于小烧杯中，用10ml的DMF溶解，冷却至0-5℃备用
在0-5℃下，将DIC溶液(溶液3)慢慢滴加到搅拌的溶液2中，反应15分钟，将溶液1慢慢滴加到反应液中，在0-5℃下反应15分钟，25℃下反应2小时，点板确定反应结束(具体反应时间以点板时间为准)。过滤除去白色沉淀，用5mlDMF洗涤沉淀并过滤，合并滤液，加入大量纯水，出现大量白色固体，加入大量纯水溶解，加入100mL乙酸乙酯，用饱和碳酸氢钠150ml洗涤3次，无水硫酸钠干燥2小时，过滤后通入氯化氢气体，得到白色固体。HPLC纯度为99.5％。MS[M+H+]:613.3。
实施例3降纤酶含量测定(发色底物荧光法)
在试管中，制备0.3ml 0.2M Tris-HCl缓冲溶液(pH 7.4)和0.1ml样品。向其中加入0.1ml 0.5mM Cbz-Phe-Arg-AMC水溶液，在37℃下加入反应约20分钟后，加入0.5ml 2％乙酸终止反应。在380nm的激发波长和460nm的测量波长下测定游离的7-氨基-甲基-香豆素荧光强度。在相同的条件下，测定对照品溶液的荧光强度，求出相应的供试品标示含量。
降纤酶国家药品标准含量测定(纤维蛋白原凝固法)
标准品溶液的制备取降纤酶标准品，加三羟甲基氨基甲烷缓冲液(pH7.4)制成每1ml中含20、10、5、2.5单位的溶液。
供试品溶液的制备取本品适量，用三羟甲基氨基甲烷缓冲液(pH7.4)(取三羟甲基氨基甲烷2.42g与氯化钠0.585g，加水适量溶解，用1mol/L盐酸溶液调pH值至7.4，加水稀释至500ml)制成制成标准曲线范围内浓度的溶液取试管4支，各精密加入0.4％纤维蛋白原溶液(取纤维蛋白原，用三羟甲基氨基甲烷缓冲液(pH7.4)制成0.4％可凝蛋白溶液)0.2ml，置(37±0.5)5℃水浴中保温2分钟，依次精密量取4种浓度的标准品溶液各0.2ml，迅速加入上述各试管中，立即摇匀，同时计时，于(37±0.5)℃水浴中观察纤维蛋白原的初凝时间，每种浓度测5次，求平均值(5次测定最大与最小值之差不得超过平均值的10％，否则重测)，在双对数坐标纸上，以标准品单位数(U)为横坐标，初凝时间为纵坐标计算回归方程(相关系数应大于0.99)。
表1纤维蛋白原凝固法与发色底物荧光法测定降纤酶含量结果比较
Claims (6)
1.一种精氨酸衍生物Cbz-Phe-Arg-AMC的合成方法，其特征在于：由如下步骤组成：
步骤A:由AMC、Fmoc-Arg(pbf)-OH缩合反应得到Fmoc-Arg(pbf)-AMC；
步骤B：脱除Fmoc-Arg(pbf)-AMC中胍基侧链保护基得NH2-Arg(pbf)-AMC，偶联Cbz-Phe-OH，获得Cbz-Phe-Arg-AMC。
2.根据权利要求1所述的方法，其特征在于：步骤A中包括缩合剂和反应溶剂，所述的缩合剂为DIC、HOBT、HBTU、HATU、HCTU，反应溶剂为DMF、DCM、NMP或DMSO中的一种或两种以上混合液。
3.根据权利要求1所述的方法，其特征在于：步骤B中偶联包括缩合剂和反应溶剂，所述的缩合剂为DIC、HOBT、HBTU、HATU、HCTU，反应溶剂为DMF、DCM、NMP或DMSO中的一种或两种以上混合液。
4.根据权利要求1所述的方法，其特征在于：步骤B所述的裂解采用的试剂为TFA、苯甲硫醚和EDT的混合液，所述TFA：苯甲硫醚：水：EDT体积比为92:3:3:2。
5.精氨酸衍生物Cbz-Phe-Arg-AMC作为精氨酸酯酶、凝血酶的发色肽底物的应用。
6.精氨酸衍生物Cbz-Phe-Arg-AMC用于测定降纤酶含量的应用。
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