Neelakandan Prakash P, Hariharan Mahesh, Ramaiah Danaboyina
Photosciences and Photonics, Regional Research Laboratory (CSIR), Trivandrum 695019, India.
J Am Chem Soc. 2006 Sep 6;128(35):11334-5. doi: 10.1021/ja062651m.
With the objective of developing small molecule based receptors for nucleosides and nucleotides, interactions of a cyclic donor-acceptor conjugate 1 with adenosine, AMP, ADP, CTP, UTP, ITP, ATP, and GTP have been investigated by absorption, steady-state, and time-resolved fluorescence, cyclic voltammetry (CV), NMR, and fluorescence indicator displacement techniques. Titration of 1 with the fluorescent indicator, 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS), resulted in nearly complete fluorescence quenching of HPTS, along with 25% hypochromicity in its absorption spectrum. Benesi-Hildebrand analysis gave a 1:1 stoichiometry for the complex between the receptor 1 and HPTS with an association constant (Kass) of 4.66 x 104 M-1 in buffer. The driving force for such a complexation was evaluated to be the synergistic effects of pi-stacking and electrostatic interactions inside the cavity as confirmed by the effect of ionic strength, temperature, and the negative results obtained with the model compound 2. Titration of the nonfluorescent complex [1.HPTS] with various nucleosides and nucleotides resulted in revival of fluorescence of the indicator, HPTS. It was observed that GTP induces maximum displacement of HPTS from the complex [1.HPTS] with an overall fluorescence enhancement of ca. 150-fold. The addition of adenosine, AMP, ADP, CTP, and UTP showed negligible changes, whereas ca. 45- and 50-fold enhancement was observed with ATP and ITP, respectively. The competitive displacement of the indicator by various analytes is found to be in the order GTP (buffer) approximately GTP (biofluid) > ITP approximately ATP > UTP > CTP approximately ADP approximately AMP approximately Ade. By virtue of having a better pi-electron cloud, GTP undergoes effective electronic, pi-stacking, and electrostatic interactions inside the cavity and forms a stable complex with the receptor 1. The uniqueness of this assay is that it differentiates GTP from ATP and other nucleotides and signals the event through a visual "turn on" fluorescence mechanism in buffer as well as in biological fluids.
为了开发基于小分子的核苷和核苷酸受体，通过吸收光谱、稳态荧光和时间分辨荧光、循环伏安法（CV）、核磁共振（NMR）以及荧光指示剂置换技术，研究了环状供体-受体共轭物1与腺苷、AMP、ADP、CTP、UTP、ITP、ATP和GTP的相互作用。用荧光指示剂8-羟基-1,3,6-芘三磺酸盐（HPTS）滴定1，导致HPTS的荧光几乎完全猝灭，同时其吸收光谱出现25%的减色效应。Benesi-Hildebrand分析得出受体1与HPTS之间的复合物化学计量比为1:1，在缓冲液中的缔合常数（Kass）为4.66×104 M-1。通过离子强度、温度的影响以及模型化合物2得到的阴性结果证实，这种络合作用的驱动力是腔内π-堆积和静电相互作用的协同效应。用各种核苷和核苷酸滴定非荧光复合物[1·HPTS]导致指示剂HPTS的荧光恢复。观察到GTP诱导HPTS从复合物[1·HPTS]中最大程度地置换出来，总体荧光增强约150倍。加入腺苷、AMP、ADP、CTP和UTP显示变化可忽略不计，而ATP和ITP分别观察到约45倍和50倍的增强。发现各种分析物对指示剂的竞争性置换顺序为GTP（缓冲液）≈GTP（生物流体）>ITP≈ATP>UTP>CTP≈ADP≈AMP≈腺嘌呤核苷。由于具有更好的π电子云，GTP在腔内发生有效的电子、π-堆积和静电相互作用，并与受体1形成稳定的复合物。该检测方法的独特之处在于它能够区分GTP与ATP和其他核苷酸，并通过缓冲液以及生物流体中的视觉“开启”荧光机制来指示该事件。
