CN104837507B - 检测真菌细胞的方法 - Google Patents
检测真菌细胞的方法 Download PDFInfo
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- CN104837507B CN104837507B CN201380036385.0A CN201380036385A CN104837507B CN 104837507 B CN104837507 B CN 104837507B CN 201380036385 A CN201380036385 A CN 201380036385A CN 104837507 B CN104837507 B CN 104837507B
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- ddao
- candida
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- caspofungin
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Abstract
本发明涉及靶向剂和利用所述靶向剂检测受试者的真菌细胞的方法。
Description
相关申请的交叉参考
本申请要求于2012年5月8日提交的美国临时申请第61/644,283号的权益，该申请以其全部公开内容通过引用并入本文。
关于联邦政府资助研究的声明
本项工作由国家卫生研究院(National Institutes of Health)提供资助(AI069397)。美国政府拥有本发明的某些权利。
技术领域
本发明涉及靶向剂和利用所述靶向剂检测受试者的真菌细胞的方法。
背景技术
侵袭性真菌感染(Invasive fungal infections，IFI)由于引起免疫损害疾病和慢性感染而对人类健康构成日益增强的威胁。在大多数考虑IFI诊断的情况中，临床表现常常是不确定的，而且可以由大范围的感染性有机体、潜在的疾患或者治疗并发症引起。成功的IFI诊断由于在疾病定义和选择建立诊断的标准方法方面的不确定性和争议性而更加复杂。真菌细胞壁组分如葡聚糖和半乳甘露聚糖在生长和发育的过程中主动脱落，成为基于生物标记物的商业抗原检测快速诊断测试的基础，但是它们的价值受到由于各种各样的因素而引起的可能的假阳性和假阴性结果的限制。成像是疾病(如侵袭性曲霉病(invasiveaspergillosis，IA))诊断中的一个重要组成部分。来自常规的X-射线和更高级的计算机断层扫描(CT)的特征性图像能够用于鉴别中性白细胞减少症患者的疾病损害以及帮助管理IA。然而，诊断成像本身就是非特异性的，并且依赖于其它临床体征和症状。因此需要一种具有标记物的广谱真菌特异性靶向分子来选择性地检测受试者的侵袭性真菌感染。
发明内容
本发明涉及靶向剂和利用所述靶向剂检测受试者的真菌细胞的方法。本发明利用一种广谱真菌特异性靶向分子满足了对检测患者的真菌的方法的需求。
一方面，本发明提供了一种用于检测受试者的真菌的方法，所述方法包括对所述受试者施用靶向剂，其中所述靶向剂包含与可检测标记物共价结合的抗真菌药物；以及检测所述靶向剂。可检测标记物可以是荧光标记物、放射性同位素或者造影剂。荧光标记物可以是硼-二吡咯甲川(boron-dipyrromethene，BODIPY)、7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)(DDAO)、7-氨基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)(7-氨基DDAO)，或者它们的衍生物。抗真菌药物可以是多烯、唑类化合物(azole)和棘球白素(echinocandin)。抗真菌药物可以是那他霉素(natamycin)、龟裂杀菌素(rimocidin)、非律平(filipin)、制霉菌素(nystatin)、两性霉素B(amphotericin B)、坎底辛(candicin)、咪康唑(miconazole)、酮康唑(ketoconazole)、克霉唑(clotrimazole)、益康唑(econazole)、联苯苄唑(bifonazole)、布康唑(butoconazole)、芬替康唑(fenticonazole)、异康唑(isoconazole)、奥西康唑(oxiconazole)、舍他康唑(sertaconazole)、硫康唑(sulconazole)、噻康唑(tioconazole)、氟康唑(fluconazole)、伊曲康唑(itraconazole)、艾沙康唑(isavuconazole)、雷夫康唑(ravuconazole)、泊沙康唑(posaconazole)、伏立康唑(voriconazole)、特康唑(terconazole)、阿巴芬净(abafungin)、特比萘芬(terbinafine)、阿莫罗芬(amorolfine)、萘替芬(naftifine)、布替萘芬(butenafine)、阿尼芬净(anidulafungin)、卡泊芬净(caspofungin)和米卡芬净(micafungin)。靶向剂可以是卡泊芬净-7-氨基DDAO。
也可以在施用所述靶向剂之前对受试者施用预处理抗真菌药物，其中所述预处理抗真菌药物和所述靶向剂的抗真菌药物为同一抗真菌药物，例如卡泊芬净。
也可以在施用所述靶向剂之前对受试者施用预处理抗真菌药物，其中所述预处理抗真菌药物和所述靶向剂中的抗真菌药物不是同一抗真菌药物，并且其中所述预处理抗真菌药物与所述靶向剂中的抗真菌药物不与相同的靶点结合，例如预处理抗真菌药物是泊沙康唑，而所述靶向剂中的抗真菌药物是卡泊芬净。
可以使用成像装置来检测靶向剂，所述成像装置包括但不限于x-射线成像装置、红外线成像装置、荧光成像装置、核磁共振成像装置、磁共振光谱装置和正电子发射断层装置。可以检测的真菌包括但不限于白色念珠菌(Candida albicans)、光滑念珠菌(Candidaglabrata)、近平滑念珠菌(Candida parapsilosis)、克鲁斯念珠菌(Candida krusei)、烟曲霉菌(Aspergillus fumigatus)、黑曲霉菌(Aspergillus niger)、黄曲霉菌(Aspergillus flavus)、新型隐球菌(Cryptococcus neoformans)、尖端赛多孢子菌(Scedosporium apiospermum)、接合菌(Zygomycetes)、荚膜组织胞浆菌(Histoplasmacapsulatum)、粗球孢子菌(Coccidioides immitis)、巴西副球孢子菌(Paracoccidiioidesbrassiliensis)和皮炎芽生菌(Blastomyces dermatitidis)。
在第二方面，本发明提供一种靶向剂，该靶向剂包含与可检测标记物直接结合的抗真菌剂。可检测标记物可以是荧光标记物，并且抗真菌剂可以是卡泊芬净或者泊沙康唑。可检测标记物可以是硼-二吡咯甲川、7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)、7-氨基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)，或者它们的衍生物。
在第三方面，本发明提供了一种用于检测生物样品或者受试者的真菌的试剂盒，所述试剂盒包含如上所述的靶向剂和使用说明书。
附图说明
图1示出了与硼-二吡咯甲川(BODIPY)共价连接的卡泊芬净(CSF)的衍生物的化学结构，并指出了胺连接部位。
图2描述了7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)(DDAO)、7-氨基DDAO及其衍生物的合成和光发射特性。
图3描述了与7-氨基DDAO共价结合的卡泊芬净(CSF)以及与7-氨基DDAO共价结合的泊沙康唑(POS)。
图4描述了将7-氨基DDAO共价连接到POS的合成步骤。
图5描述了将BODIPY共价连接到POS(POS-BOD)的合成步骤。
图6描述了泊沙康唑(POS)的化学结构，标记物的连接位点以及与BODIPY共价连接的POS(POS-BOD)。
发明详述
本发明涉及靶向剂和靶向剂用于检测受试者的真菌的用途。
靶向剂和使用方法
在一个实施方案中，本发明提供了一种靶向剂，所述靶向剂包含与可检测标记物共价结合的抗真菌药物。抗真菌剂可以是多烯、唑类化合物或棘球白素。多烯的实例包括哈霉素(hamycin)、那他霉素、龟裂杀菌素、非律平、制霉菌素、两性霉素B和坎底辛。唑类化合物的实例包括咪康唑、酮康唑、克霉唑、益康唑、联苯苄唑、布康唑、芬替康唑、异康唑、奥昔康唑、舍他康唑、硫康唑、噻康唑、氟康唑、伊曲康唑、艾沙康唑、雷夫康唑、泊沙康唑、伏立康唑和特康唑。棘球白素的实例包括阿尼芬净，卡泊芬净和米卡芬净。在另一个实施方案中，抗真菌药物特异性地与抗真菌靶点结合，而不与哺乳动物在生物学上产生的靶点结合。真菌靶点可以是真菌来源的而非哺乳动物来源的碳水化合物、肽、脂质，或者它们的组合，例如，β(1,3)葡聚糖合酶。在一个优选实施方案中，靶向剂包括卡泊芬净、泊沙康唑或者本领域中已知的它们的衍生物。
可检测标记物可以是荧光标记物、放射性同位素或者造影剂。在某些实施方案中，抗真菌药物用放射性同位素标记，所述放射性同位素如砹211、14碳、51铬、36氯、57钴、58钴、铜67、152Eu、镓67、氢、碘、碘、碘、铟、铁、磷、铼、铼、硒、35硫、锝(technicium)99m和钇90。125I、锝99"1和铟1。将选择的放射性同位素加入到抗真菌剂中、与抗真菌剂共价结合的方法是本领域中已知的方法。
本文所使用的术语“荧光团”、“荧光部分”、“荧光标记物”和“荧光染料”在本文中可互换使用。它们是指在溶液中且当受到适当波长的光激发后发射光的分子。大量的具有多种多样的结构和特性的荧光标记物适合用于本发明的实践中。类似地，荧光标记感兴趣的分子的方法和材料是已知的(参见，例如，R.P.Haugland，"Molecular Probes:Handbookof Fluorescent Probes and Research Chemicals 1992-1994",5.sup.th Ed.,a 1994,Molecular Probes,Inc.)。在选择荧光标记物时，通常期望荧光标记物高效率地吸收光并发射荧光(即，分别对应于高摩尔吸收系数和荧光量子产率)，并且是光稳定的(即，其在进行分析所必需的时间内在光激发时不经历显著的降解)。此外，在选择标记物时，优选荧光标记物是(1)小分子，例如(FW＝294)；(2)长波长发射；(3)亮度适中；(4)不依赖于pH值；(5)发射最大值高达约680nm，这时体组织是最透明的。在本发明的一个优选实施方案中，荧光标记物是7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)(DDAO)、7-氨基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮)(7-氨基DDAO)，或者它们的衍生物。DDAO衍生物可以用于共价标记感兴趣的生物分子如靶向剂。DDAO衍生物在7位含有胺或氨基基团，而不含羟基基团。在一个优选的实施方案中，羟基基团被下列式NH-(CH2)X-NHY替代，其中X＝1-10且Y＝H、C、烷基、CS，(CH2)X也可以被另一间隔物或者聚合物替代，如聚乙二醇或其它具有相同性质和长度的聚合物。在7位含有氨基基团的DDAO荧光团在本文中被称为7-氨基DDAO。合成中间体7-(4-氨基丁基)氨基DDAO能够容易地转化成可用于生物偶联(bioconjugation)的其它反应形式(例如硫醇基-或点击反应性(click-reactive))，方法是本领域中已知的。DDAO衍生物比原DDAO亮1.4-2.3倍。在另一实施方案中，荧光标记物是硼-二吡咯甲川(BODIPY)或其衍生物。
在某些实施方案中，抗真菌药物用造影剂如用于核磁共振成像(MRI)的顺磁性金属离子标记。这样的顺磁性金属离子的实例包括但不限于钆III(Gd3+)、铬III(Cr3+)、镝III(Dy3+)、铁III(Fe3+)、锰II(Mn2+)、以及镱III(Yb3+)。钆是FDA批准的用于MRI的造影剂，并且已知在身体不同区域的正常组织和异常组织之间提供较大的对比。
通过本领域中已知的技术方法，使抗真菌药物与可检测标记物共价结合，使得所得到的靶向剂保持对抗真菌剂靶点的特异性和敏感性。
在另一实施方案中，本发明的靶向剂可以被配制成药物组合物，并且可以以适于所选的施用途径的各种形式，即口服或者肠道外、通过静脉、肌内、局部、皮下或者其它途径施用于哺乳动物类宿主，如人类患者。因此，本发明的药物组合物可以与药学上可接受的赋形剂如惰性稀释剂组合进行全身性施用，例如，口服。它们可以被直接并入到患者饮食的食物。对于口服治疗施用，本发明的组合物可以以酏剂、糖浆剂等形式使用。在制备任何单位剂型中使用的任何材料应当是药学上可接受的并且在所用剂量下是基本上无毒的。为了将药物组合物施用于受试者，优选将分子配制成包含一种或多种药学上可接受的载体的组合物。
“药学上可接受的载体”包括任何以及全部的临床可用的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。然而，也可利用其它溶剂。在常规的储存和使用条件下，这些制剂可以包含用于防止微生物生长的防腐剂，和本领域中已知的其它制剂成分。
本发明进一步提供了一种通过如下步骤检测受试者的真菌的方法：对受试者施用靶向剂，所述靶向剂包含与可检测标记物共价结合的抗真菌药物，接着用成像装置检测所述靶向剂。术语“真菌”是指与靶向剂结合的真菌细胞和相关真菌结构，例如葡聚糖合酶。本发明的靶向剂能够通过本领域中已知的许多方法中的任意方法施用于受试者。
“受试者”是指人类和非人类动物。非人类动物的实例包括所有的脊椎动物，例如哺乳动物，如非人灵长类动物(特别是高级灵长类动物)、狗、啮齿动物(例如小鼠或大鼠)、豚鼠、猫，和非哺乳类动物，如鸟类、两栖动物、爬行动物等。在一个优选的实施方案中，受试者是人。在另一个实施方案中，受试者是实验动物或者适合作为疾病模型的动物。通常，在本文中术语“受试者”和“患者”在指人类受试者时可互换使用。
本发明的靶向剂可以以适于所选的施用途径的各种形式施用，例如，口服或肠胃外，通过静脉，肌内，局部，皮下，或其它途径等。可以在例如水中和/或与药学上可接受的载体一起制备溶液。
在另一个实施方案中，本发明提供了一种具有其它步骤的方法，其中在施用靶向剂前先将预处理抗真菌药物施用于受试者。在一个优选的实施方案中，预处理抗真菌药物和靶向剂的抗真菌药物是同一抗真菌药物。在一个优选的实施方案中，预处理抗真菌药物是卡泊芬净，而靶向剂的抗真菌药物也是卡泊芬净。在另一实施方案中，预处理抗真菌药物和靶向剂的抗真菌药物不是同一抗真菌药物，并且预处理抗真菌药物与靶向剂的抗真菌药物不与相同的靶点结合。出于举例说明的目的，预处理抗真菌药物可以是泊沙康唑，而靶向剂的抗真菌药物可以是卡泊芬净，并且能由本领域普通技术人员选择。
在某些实施方案中，检测靶向剂的成像装置是可以是磁成像装置、x-射线成像装置、红外成像装置、荧光成像装置、核磁共振成像装置、磁共振光谱装置和正电子发射断层装置。本领域普通技术人员将采用适当的仪器(modality)来检测如前所述的靶向剂。
本发明提供广谱靶向剂来检测多种真菌。可以检测的真菌类型包括但不限于白色念珠菌、光滑念珠菌、近平滑念珠菌、烟曲霉菌、黑曲霉菌、黄曲霉菌、新型隐球菌、尖端赛多孢子菌、接合菌、荚膜组织胞浆菌、粗球孢子菌、巴西副球孢子菌和皮炎芽生菌。更进一步地，本领域普通技术人员可以决定用于检测的受试者的组织、器官或体液类型，例如肺、肾、痰、BAL、血液、血清或尿液。
在另一个实施方案中，本发明提供一种用于检测生物样品或受试者的真菌的试剂盒，所述试剂盒包含如前所述的靶向剂和使用说明书。本文所用的“生物样品”是指生物组织或流体的样品。这样的样品包括但不限于从动物分离出的组织。生物样品也可包括组织切片如活检样品和尸检样品、出于组织学目的采集的冷冻切片、血液、血浆、血清、痰、唾液、粪便、泪液、粘液、毛发和皮肤。生物样品还包括来源于患者组织的外植体(explant)和原代和/或转化细胞培养物。生物样品可以通过从动物移除细胞样品来提供，也可以通过使用以前分离的细胞(如，从另一个人、在另一个时间和/或出于另一目的分离的)来完成。
实施例
现正一般性地描述本发明，通过参考以下实施例会更容易地理解本发明，包括的这些实施例仅用于解释说明本发明中的某些方面和实施方案，并且不用来限制本发明。
方法和材料
BODIPY标记的药物的合成。生产利用BODIPY(BOD)获得的CSF衍生物，并用多种真菌病原体测试CSF-BOD(BODIPY是类似于荧光素但是更小且更疏水的荧光素类似物)。BODIPY-琥珀酰亚胺酸酯(succinimidate)在DMF中在作为质子受体的三乙胺的存在下与纯CSF孵育。粗产物通过TLC硅胶色谱法纯化，并用质谱法、荧光和UV色谱法进行表征。修饰后的试剂保持其对真菌靶点的特异性和敏感性。为了测试这些性质，对修饰的和未修饰的根化合物(root compound)的抗真菌活性进行评价，发现抗真菌活性在标记物的存在下实际上未发生改变(针对白色念珠菌，MIC(未标记的)＝0.06μg/ml，而MIC(标记的)＝0.12μg/ml)，证实了其保持其固有的效力。通过用琥珀酸酐修饰泊沙康唑的一个羟基而形成与BODIPY偶联的泊沙康唑(POS)(图6)。
在DCC的存在下对酰化产物和4-硝基苯酚的孵育生成一个新化合物，该新化合物与形成的活化酯(图5，化合物II)一致。对该化合物与乙二胺的孵育得到在405nm有特征吸收的硝基苯酚盐阴离子，其可作为泊沙康唑活化酯被二胺酰化的指征。用BODIPY荧光团的氨基丁烷衍生物(图5，化合物III)孵育该酯生成有荧光的泊沙康唑-BODIPY加合物IV，其具有光吸收谱。
细胞标记。为了举例说明CSF-BOD和POS-BOD使真菌细胞可视化的能力，使用该试剂来探测在包括固体和液体生长培养基在内的各种基质中的念珠菌属(Candida)和曲霉菌属(Aspergillus)种类的存在。对于所有的实验使用临床烟曲霉菌野生型菌株R21和白色念珠菌ATCC菌株90028。对于曲霉菌属，将一滴酵母菌提取物蛋白胨葡萄糖(YPD)琼脂置于15孔多测试玻片的每孔的右上角落处，接着加入10μl含有105个R21的分生孢子的生理盐水。将玻片放置于用蒸馏水提供湿润环境的无菌陪替式培养皿(petri dish)中，并在37℃孵化器中孵育10-16个小时，以促进菌丝成分(hyphal elements)的萌发和生长。将10μL CSF-BOD(170ng/ml)或POS-BOD(150ng/ml)的等分样品加入到每个孔中，并在37℃孵育6小时，然后用无菌水洗涤3次，并真空干燥。对于念珠菌属，使白色念珠菌的培养物过夜生长，离心洗涤并再悬浮于dH2O中。向RPMI中加入酵母菌细胞，并在37℃、200rpm下孵育1小时以形成萌发管，然后洗涤并再悬浮于1ml CSF-BOD(120ng/ml)或POS-BOD(150ng/ml)溶液中。将细胞和药物在37℃、200rpm下孵育1小时，洗涤并再悬浮于50ml dH2O中。准备15孔玻片，使用聚-L-赖氨酸使细胞紧贴在孔中。将10μL念珠菌细胞的等分样品放置在每个孔中，并孵育10分钟，吸出，并加入1μL Slow Fade Antifade试剂以延长每个玻片上的细胞的荧光寿命和水分含量。采用Nikon Eclipse 90i荧光显微镜的全内反射物镜(TIRF)在100倍放大下观察每个玻片。使用Volocity 3D图像分析软件(PerkinElmer)对玻片的15个孔各自进行单独的检查。在量视场照明(bright field lighting)下对每个孔、菌丝成分和细胞簇进行可视化、分析和捕捉，并在绿色荧光蛋白(GFP)照明装置下重复。
结果
在37℃、MIC(120ng/ml)下孵育白色念球菌1小时，在母细胞膜中普遍形成荧光，并沿着萌发管轴向生长尖端具有略微更清晰的点状荧光，与推定的葡聚糖合成酶从成簇的高尔基小泡复合体的细胞内小泡转运一致。在6小时和37℃的条件下，烟曲霉菌在芽孢中显示出明亮的荧光，但是在菌丝成分朝向生长顶点的表面上显示出更为分散的标记，其与葡聚糖合酶的膜位置一致。
标记是高度温度敏感的，观察到在37℃下6小时内具有最大标记。单独的卡泊芬净和BODIPY未生成任何标记。在一种充分表征的fskl-S645F突变体中，结合水平极大降低，所述fskl-S645F突变体具有降低的葡聚糖合酶对棘球白素的敏感性，这与探针与其预期靶点结合一致。
在相同的条件下使代表性革兰氏阴性和革兰氏阳性菌：铜绿假单胞菌(Pseudomonas aeruginosa)、肺炎克雷伯菌(Klebsiella pneumoniae)、肺炎链球菌(Streptococcus pneumoniae)、粘质沙雷菌(Serratia marcescens)、金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)生长和标记。在任何细胞内均未观察到荧光。
在与卡泊芬净相同的标记条件下，用念珠菌属和曲霉菌属标记的POS-BOD显示母细胞的普遍荧光标记和延长的菌丝成分。用未标记的泊沙康唑或伏立康唑预处理极大地减少或消除荧光信号，而用卡泊芬净预处理对细胞经POS-BOD的标记具有极小的影响。
用四种不同的唑类化合物伏立康唑(Pfizer)、伊曲康唑(Janssen)、泊沙康唑(Merck)和氟康唑(Pfizer)在一个低于最小抑菌浓度稀释度下预处理细胞(白色念珠菌1小时和烟曲霉菌2小时)，接着如上所述进行CSF-BOD标记，对标记没有影响。在有和没有预处理的情况下，所有样品显示相同的荧光强度，这与唑类化合物与单独的细胞内靶点结合一致。然而，在用CSF-BOD探针进行标准标记之前，用棘球白素阿尼芬净和米卡芬净预处理的细胞消除标记。用卡泊芬净预处理使得荧光增强。
DDAO荧光衍生物的合成
DDAO-NH-(CH2)4-NH2(图2)。将10mg DDAO(7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮))(33μmol)溶解在100μl 1M二氨基丁烷二乙酸酯的80％DMSO水溶液中。TLC分析(展开系统为乙腈-水(14:1))检测到移动低于原产物(Rf＝0.9)的强蓝色产物(Rf＝0.45)。在95℃下孵育10小时后，将反应混合物补充2ml水，并用乙酸乙酯(3x5ml)萃取。用10M的KOH将水层的pH值调节至11-11.5，之后用乙酸乙酯(2x5ml)萃取。收集有机层，并减压蒸干，得到4mg化合物I。UVλmax＝(ε＝M-1cm-1)，λmin＝(ε＝M-1cm-1)。MS：DDAO-NH-(CH2)4-NH2(+1)378.0887(实测值)378.288(计算值)。
卡泊芬净-DDAO衍生物(图3)。将卡泊芬净(2.6mg，2μmol)溶解在230μl5mM DDAO-NH-(CH2)4-NCS的DMF溶液中，加入0.5μl TEA，之后在60℃下孵育90分钟。TLC分析(展开系统为乙腈-水(5:1))检测到蓝色反应产物(Rf＝0.65)。卡泊芬净和DDAO-NH-(CH2)4-NCS的Rf值分别是0.48和1.0。产物经制备TLC纯化，展开系统为乙腈-水(7:1)，50％甲醇水溶液洗脱，溶液减压蒸发至最终浓度为0.33mM。UVλmax＝(ε＝M-1cm-1)，λmin＝(ε＝M-1cm-1)。DDAO-NH2-(CH2)4-NCS-卡泊芬净(+H)1515.7242(实测值)1515.673(计算值)。
泊沙康唑-DDAO衍生物的合成(图4)。将2毫克化合物III(图4)溶解在0.1ml 20mM的化合物I溶液中。将混合物补充2ml三乙胺，并在室温下放置20分钟。TLC分析(展开混合物是乙酸乙酯-乙醇(8:1))显示化合物I完全转化为反应产物。将混合物用2ml水稀释，离心收集残留物，将其溶解在DMF中，并在相同的系统中进行制备TLC。得到0.5μmol。
为衍生核心化合物DDAO，使用早先发现的利用更简单的苯酚-或萘酚-衍生物的Hamilton反应(Malmberg,E.,W.,Hamilton,C.,S.,J.Am.Chem.Soc.&0,2415,(1948)；Willenz,J.J.Chem Soc.,1955,2049)。反应包括氨基化合物酸-催化进攻芳香族羟基衍生物的内消旋酮式。以高收率获得与1,4-二氨基丁烷的反应产物，并通过萃取纯化。将得到的DDAO氨基-衍生物通过用硫羰基二咪唑处理，之后用三氟乙酸孵育，转化为相应的异硫氰酸酯(ITC)(图2)。得到的&-氨基DDAO衍生物用于标记抗真菌药物泊沙康唑和卡泊芬净(图3)。卡泊芬净由ITC在单步反应中在药物的两个脂肪族氨基中的一个氨基处发生衍生化。为了在泊沙康唑分子中引入DDAO荧光标记，在DMSO中在亲核催化剂N-甲基咪唑的存在下首先在羟基基团处通过琥珀酸酐对药物进行酰化(图4)。将得到的产物通过用4-硝基苯酚和DCC孵育转化成活化酯。将这个合成中间体引入到与1,4-二氨基丁基-DDAO化合物的反应中，得到最终产物，该产物使用制备TLC纯化。
7-氨基DDAO、卡泊芬净-DDAO和泊沙康唑-DDAO衍生物的光吸收和荧光谱。DDAO的修饰导致光吸收最大值出现可检测的蓝移(对应于653nm和673nm)。7-氨基DDAO的摩尔消光系数(55000M-1cm-1)由连接的具有已知摩尔吸收系数的参考发色团测定。已标记的卡泊芬净和泊沙康唑衍生物的光吸收谱接近于7-(4-氨基丁基)氨基DDAO和相应的药物的光吸收谱的叠加。7-(4-氨基丁基)氨基-DDAO的荧光谱(图6B)与DDAO的离子化形式(图6A)相比显示蓝移。因此，DDAO的激发和发射最大值相应地是653nm和660nm，而对于7-(4-氨基丁基)氨基-DDAO则相应地移动到671nm和679nm。增加有机溶剂(MeOH)的含量导致光发射增强和激发的特征性变化。因而将50％甲醇替换为水不会影响7-氨基DDAP的激发光谱的形状，但使光发射增大约2.5倍。将化合物置于100％甲醇导致激发光谱曲线的急剧变化，最大值从670nm移动至620nm，而发射最大值只有少许移动，从680nm移动至670nm。尤其是光发射强度下降1.7倍。显著地，DDAO的离子化形式的激发光谱曲线的形状在50％甲醇和100％甲醇中是一样的。而且，与7-氨基DDAO对比，观察到发射光在100％甲醇中比在50％甲醇中增强1.3倍。
卡泊芬净-DDAO衍生物在体内用于真菌感染成像。对小鼠静脉接种5*105CFU野生型白色念珠菌使其感染，感染最主要发生在肾脏。感染48小时后，在0、2、4、8小时通过尾静脉注射固定浓度为0.12ug/mL的CSF-DDAO，评估感染可视化的最佳时间。在每个时间点，在非侵入性整体动物成像系统中使小鼠成像来检测荧光能量。感染白色念珠菌的动物在48小时后表现出肾脏中的真菌感染的增殖。通过整体成像测定，加入CSF探针导致在靶器官中细胞随着时间的进行性标记。在8小时时出现最大标记。在无感染的情况下，CSF-DDAO在肾脏不蓄积。
在本说明书中引用的所有出版物都以全文形式通过引用并入本文。在本文中对任何参考文献的引用不是承认这样的参考文献是本发明的现有技术。
说明书中的实施方案提供对本发明的实施方案的举例说明，不应理解为限制本发明的范围。本领域技术人容易认识到本发明涵盖很多其它实施方案。本领域技术人员将认识到，或者仅通过常规实验就能够确定，本发明所述具体实施方案的许多等同实施方案。这些等同实施方案意图被下面的实施方案涵盖。
Claims (8)
1.靶向剂在制备用于检测受试者的真菌的试剂盒中的用途，其中所述靶向剂选自：
并且其中所述真菌选自白色念珠菌(Candida albicans)、光滑念珠菌(Candidaglabrata)、近平滑念珠菌(Candida parapsilosis)、克鲁斯念珠菌(Candida krusei)、烟曲霉菌(Aspergillus fumigatus)、黑曲霉菌(Aspergillus niger)和黄曲霉菌(Aspergillus flavus)。
2.权利要求1所述的用途，其中所述靶向剂是卡泊芬净-7-氨基DDAO。
3.权利要求1所述的用途，其中利用成像装置检测所述靶向剂。
4.权利要求3所述的用途，其中所述成像装置是荧光成像装置。
5.一种靶向剂，其选自：
6.权利要求5所述的靶向剂，其中所述靶向剂是卡泊芬净-7-氨基DDAO。
7.权利要求5所述的靶向剂，其中所述靶向剂是泊沙康唑-7-氨基DDAO。
8.一种用于检测生物样品或受试者的真菌的试剂盒，包括权利要求5所述的靶向剂，和使用说明书，其中所述真菌选自白色念珠菌、光滑念珠菌、近平滑念珠菌、克鲁斯念珠菌、烟曲霉菌、黑曲霉菌和黄曲霉菌。
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