Click and pick: identification of sialoside analogues for siglec-based cell targeting.
Click and pick: identification of sialoside analogues for siglec-based cell targeting.
复制标题
DOI：
10.1002/anie.201205831
复制
发表时间：
2012-10-29
影响因子：
16.6
通讯作者：
Paulson, James C.
中科院分区：
文献类型：
--
作者：
Rillahan, Cory D.;Schwartz, Erik;McBride, Ryan;Fokin, Valery V.;Paulson, James C.
唾液酸结合蛋白的siglec家族由15个人和9个鼠成员组成，主要由白色血细胞表达，介导先天性和适应性免疫功能。[1]它们作为内吞受体的受限表达模式和活性使得这些蛋白质成为免疫细胞介导的疾病的定向治疗的有吸引力的分子靶标。[2]尽管抗siglec抗体已经在临床上使用，具有唾液酸苷配体的纳米颗粒显示出在体内靶向siglec的前景，从而提供了递送治疗性货物的替代方案。[3]识别具有合适亲和力和选择性的siglec配体的困难限制了带有配体的纳米颗粒的潜力。[1a先前的报道已经证明，在C9-位修饰唾液酸（NeuAc）可以产生对唾液酸粘附素（siglec-1）、CD 22（siglec-2）和髓鞘相关糖蛋白（siglec-4）的增加的亲和力和选择性。[5]还已经提出，在C5-位置处的修饰可以调节对几种单细胞的亲和力和选择性，然而，在该位置处修饰的唾液酸苷类似物尚未充分探索这些性质。[6]因此，尽管在C5和C9处对唾液酸的修饰具有产生有前景的唾液酸苷配体的潜力，并且这些位置使用酶促合成方法进行修饰相对简单，但是缺乏稳健地产生唾液酸苷类似物文库并针对单克隆抗体文库系统地筛选它们的方法阻碍了进展。为了解决这个问题，我们设计了一种简单的“点击和挑选”策略，涉及使用点击化学高通量合成唾液酸苷类似物文库，结合微阵列技术挑选人和鼠单克隆细胞的高亲和力“命中”（图1）。为了产生文库，通过会聚化学酶促方法合成了八个具有乙胺接头的唾液酸苷母体化合物（方案1，以及支持性信息中的方案S1）;这些化合物在唾液酸部分的5-位（A0-D 0）或9-位（E0-H 0）具有叠氮化物或炔取代基，并通过α2-3或α2-6键连接到倒数第二个半乳糖上，这是哺乳动物聚糖中两种最常见的键。然后对这些母体支架进行具有24-30个偶联配偶体的高通量CuI催化的叠氮化物-炔环加成[7]（CuAAC，点击化学）（支持信息，图S1和S2），以生成224种唾液酸苷类似物的文库（支持信息，表S1-S8），几乎所有偶联都有定量转化。由于点击反应与胺酰化化学的正交性，唾液酸苷产物然后可以直接印刷到N-羟基琥珀酰亚胺（NHS）活化的微阵列载玻片上而无需预先纯化，从而允许比先前的努力高得多的筛选通量和文库多样性。[6b为了鉴定单个单克隆抗体的高亲和力配体类似物，将荧光标记的单克隆抗体-Fc嵌合体覆盖在微阵列上（图1）。在Fc嵌合体的最佳浓度下，不存在与天然唾液酸苷对照或亲本支架A0-H 0的结合，从而确保任何命中对应于更高亲和力的配体。[8-9]使用该方法获得的一组人和鼠单克隆细胞的代表性微阵列数据见图2，图3a和支持信息，图3。
The siglec family of sialic-acid binding proteins is comprised of 15 human and 9 murine members that are primarily expressed by white blood cells, which mediate innate and adaptive immune functions.[1] Their restricted expression pattern and activity as endocytic receptors has made these proteins attractive molecular targets for directed therapy for immune-cell-mediated diseases.[2] Although anti-siglec antibodies are already in clinical use, nanoparticles having sialoside ligands show promise for targeting siglecs in vivo, thus providing alternatives for delivery of therapeutic cargo.[3] The difficulty in identifying siglec ligands of suitable affinity and selectivity has limited the potential of ligandbearing nanoparticles.[1a, 2, 4] Previous reports have demonstrated that modification of sialic acid (NeuAc) at the C9-position can produce both increased affinity and selectivity for sialoadhesin (siglec-1), CD22 (siglec-2), and myelinassociated glycoprotein (siglec-4).[5] It has also been suggested that modifications at the C5-position can modulate affinity and selectivity for several siglecs, however, sialoside analogues modified at this position have not been fully explored for these properties.[6] Thus, although modifications to sialic acid at C5 and C9 have potential for yielding promising sialoside ligands, and these positions are relatively straightforward to modify using an enzymatic synthetic approach, the lack of methods to robustly generate sialoside analogue libraries and systematically screen them against a library of siglecs has hampered progress. To address this issue, we devised a facile “click and pick” strategy involving high-throughput synthesis of a sialoside analogue library using click chemistry, coupled with microarray technology to pick high-affinity “hits” for human and murine siglecs (Figure1). To generate the library, eight sialoside parent compounds with ethyl amine linkers were synthesized by a convergent chemoenzymatic approach (Scheme 1, and in the Supporting Information, Scheme S1); these compounds have azide or alkyne substituents at the 5-position (A0–D0) or the 9-position (E0–H0) of the sialic acid moiety, and are attached through an α2-3 or α2-6 linkage to the penultimate galactose, the two most common linkages in mammalian glycans. These parent scaffolds were then subjected to high-throughput CuI-catalyzed azide–alkyne cycloaddition [7](CuAAC, click chemistry) with 24–30 coupling partners (Supporting Information, Figures S1 and S2) to generate a library of 224 sialoside analogues (Supporting Information, Tables S1–S8), with quantitative conversion for nearly all couplings. The sialoside products could then be printed directly onto N-hydroxysuccinimide (NHS) activated microarray slides without prior purification owing to the orthogonality of the click reaction with amine acylation chemistry, thus allowing for far higher screening throughput and library diversity than in previous efforts.[6b, 8] To identify high-affinity ligand analogues of individual siglecs, fluorescently labeled siglec-Fc chimeras were overlayed on the microarrays (Figure 1). At optimal concentrations of the Fc chimeras there was no binding to native sialoside controls or the parent scaffolds A0–H0, thus ensuring that any hits correspond to higher-affinity ligands.[8–9] Representative microarray data obtained using this approach is shown for a panel of human and murine siglecs in Figure 2, Figure3a, and in the Supporting Information, FigureS3.
登录
查看更多内容
影响因子：
15
作者：
O'Reilly, Mary K.;Collins, Brian E.;Paulson, James C.
通讯作者：
Paulson, James C.
影响因子：
16.6
作者：
Rillahan CD;Paulson JC
通讯作者：
Paulson JC
DOI：
10.1046/j.1432-1327.1998.2550663.x
发表时间：
1998-08-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Kelm, S;Brossmer, R;Schauer, R
通讯作者：
Schauer, R
DOI：
10.1084/jem.20091873
发表时间：
2010-01-18
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Duong BH;Tian H;Ota T;Completo G;Han S;Vela JL;Ota M;Kubitz M;Bovin N;Paulson JC;Nemazee D
通讯作者：
Nemazee D
影响因子：
5.7
作者：
Zaccai, NR;Maenaka, K;Jones, EY
通讯作者：
Jones, EY