Efficient conjugation of peptides to oligonucleotides by "native ligation"
Efficient conjugation of peptides to oligonucleotides by "native ligation"
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DOI：
10.1021/jo000214z
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发表时间：
2000-08-11
影响因子：
3.6
通讯作者：
Gait, MJ
中科院分区：
文献类型：
--
作者：
Stetsenko, DA;Gait, MJ
已经开发出一种将肽与寡核苷酸缀合的新策略。该方法基于N-末端硫酯官能化肽与5'-半胱氨酰寡核苷酸的“天然连接”。合成了两种新试剂，分别用于固相肽和寡核苷酸合成。 S-苄基硫代琥珀酸五氟苯酯用于标准 Fmoc 固相肽组装的最终偶联步骤。用三氟乙酸脱保护，在溶液中生成被 N 末端 S-苄基硫代琥珀酰基部分取代的肽。 O-trans-4-(N-α-Fmoc-S-叔丁基硫基-L-半胱氨基)氨基环己基O-2-氰乙基-N,N-二异丙基亚磷酰胺用于标准亚磷酰胺固相寡核苷酸组装的最终偶联步骤。用氨水溶液脱保护，在溶液中生成5'-S-叔丁基硫基-L-半胱氨酰基功能化寡核苷酸。功能化肽和寡核苷酸无需纯化即可用于水/有机溶液中的天然连接缀合反应，使用三-(2-羧乙基)膦原位去除叔丁基硫基，并使用苯硫酚作为缀合增强剂。通过该途径制备了一系列肽-寡核苷酸缀合物，并通过反相 HPLC 进行纯化。
A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminocyclohexyl O-2-cyanoethyl-N,N-diisopropylphos-phoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.