TFB-TBOA (CF3-Bza-TBOA)

Excitatory amino acid transporter-1/2/3 (EAAT-1/2/3)

TFB-TBOA (CF3-Bza-TBOA) 在 100 nM 时将突触激活转运电流 (STC) 降低至对照的约 10%。它在大鼠海马切片辐射层的星形胶质细胞中以剂量依赖性方式发挥作用，IC50 为 13 nM[2]。根据 Na+i 响应的幅度，TFB-TBOA 对 200 µM 谷氨酸引起的 Na+i 响应表现出浓度依赖性抑制，IC50 值为 43 nM[1]。

---

谷氨酸转运体的不可运输阻断剂是研究突触传递机制的重要工具。DL-苏-β-苄氧天冬氨酸（DL-TBOA）是兴奋性氨基酸转运体（EAAT）所有亚型的强效阻断剂。我们表征了在各自苯环上具有取代基的新型L-TBOA类似物。类似物显著抑制了标记的谷氨酸摄取，其中最有效的是（2S，3S）-3-{3-[4-（三氟甲基）苯甲酰氨基]苄氧基}天冬氨酸（TFB-TBOA）。在使用瞬时表达EAAT的细胞的摄取试验中，TFB-TBOA对EAAT1、EAAT2和EAAT3的IC50值分别为22、17和300 nM。与L-TBOA相比，TFB-TBOA在抑制EAAT1和EAAT2方面明显更有效（EAAT1-3的IC50值分别为33、6.2和15μM）。电生理分析显示，TBOA类似物阻断了所有五种EAAT亚型中的运输相关电流，也阻断了EAAT5中的漏电流。类似物在抑制底物诱导电流方面的效力排名与摄取试验中观察到的顺序相同。然而，TFBTBOA的动力学与L-TBOA的动力学不同，这可能是因为它具有很强的结合亲和力。值得注意的是，TFB-TBOA不影响其他代表性神经递质转运蛋白或受体，包括离子型和代谢型谷氨酸受体，表明它对EAAT具有高度选择性。此外，侧脑室注射TBOA类似物诱导了小鼠的严重惊厥行为，这可能是由于谷氨酸的积累。综上所述，这些发现表明，新型TBOA类似物，特别是TFB-TBOA，应该成为阐明谷氨酸转运体生理作用的有用工具。[1]

---

在这里，我们描述了最近开发的谷氨酸转运体抑制剂（2S，3S）-3-[3-[4-（三氟甲基）苯甲酰氨基]苄氧基]天冬氨酸（TFB-TBOA）对原代培养的小鼠皮质星形胶质细胞的影响。神经胶质Na（+）-谷氨酸转运系统非常有效，谷氨酸对其的激活会导致细胞内Na（+，i）浓度的快速变化，从而能够实时监测转运蛋白的活性。使用荧光Na（+）敏感探针结合苯并呋喃异戊酸钠，通过荧光显微镜监测单个星形胶质细胞中的Na（+i）。当单独使用时，浓度为1微M的TFB-TBOA会引起Na（+）（i）的微小变化。TFB-TBOA以浓度依赖的方式抑制了200微M谷氨酸盐诱发的Na（+）（i）反应，IC（50）值为43+/-9nM，如Na（+i）反应的振幅所示。TFB-TBOA对谷氨酸诱发的Na（+）（i）增加的最大抑制率>80%，但仅部分可逆。在AMPA/红藻氨酸受体拮抗剂CNQX存在的情况下，残余反应仍然存在。TFB-TBOA还有效地抑制了由d-天冬氨酸（一种不激活非NMDA离子型受体的转运蛋白底物）引起的Na（+）（i）升高。发现TFB-TBOA不影响全细胞膜片钳记录的培养皮层神经元的膜特性。因此，TFB-TBOA因其高效能和明显缺乏神经元效应，似乎是迄今为止研究胶质谷氨酸转运体最有用的药理学工具之一[3]。

谷氨酸转运体迅速吸收突触释放的谷氨酸，并将突触间隙中的谷氨酸浓度维持在较低水平。（2S，3S）-3-[3-[4-（三氟甲基）苯甲酰氨基]苄氧基]天冬氨酸（TFB-TBOA）是一种新型谷氨酸转运体阻断剂，可有效抑制神经胶质转运体的活性TFB-TBOA以剂量依赖的方式抑制大鼠海马切片辐射层星形胶质细胞中的突触激活转运蛋白电流（STC），IC50为13 nM，并在100 nM时将其降低到对照组的约10%。我们研究了TFB-TBOA对CA1锥体细胞谷氨酸能突触传递和细胞兴奋性的影响。TFB-TBOA（100 nM）延长了N-甲基-D-天冬氨酸受体（NMDAR）介导的兴奋性突触后电流（EPSCs）的衰减，而只有当环噻嗪（CTZ）降低AMPAR的脱敏时，它才延长了α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPAR）介导EPSCs的衰减。此外，长期应用TFB-TBOA可诱导自发性癫痫样放电，并伴有膜电位的持续去极化偏移。这些癫痫样活动主要归因于NMDAR的激活。然而，即使在NMDAR的药理学阻断后，TFB-TBOA也会在CTZ存在的情况下通过激活AMPAR诱导类似的变化。因此，神经胶质转运体持续摄取突触释放的谷氨酸对于保护海马神经元免受谷氨酸受体介导的过度兴奋是必不可少的[2]。

神经元全细胞电生理记录[3]
使用电阻为5.5-8MΩ的硼硅酸盐玻璃移液管进行全电池电压钳记录。在电压钳位模式下，钳位电位设置为-70 mV。使用Axopatch 200A放大器进行记录。电流以1kHz进行滤波。数据是用Digidata 1440A以10 kHz的采样率采集的，用Pclamp 10软件控制，用Clampfit软件分析。在建立全细胞配置后，通常允许5分钟的时间。膜片钳细胞内溶液含有（以mM计）葡萄糖酸钾130、NaCl 5、磷酸肌酸钠10、MgCl2 1、EGTA 0.02、HEPES 10、Mg ATP 2和Na3-GTP 0.5，pH 7.3（用KOH调节）。 实验使用开放式灌注室进行。对照细胞外溶液和含有受试药物的溶液在培养细胞上以600μl/min和35°C的速度重力进料。

Hippocampal slices were prepared from 15- to 21-day-old Wistar rats. Rats of either sex were deeply anesthetized with isoflurane and sacrificed by decapitation. The hippocampi were rapidly removed and placed in an ice-cold sucrose Ringer solution containing (in mM): 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10.0 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose, saturated with 95% O2 and 5% CO2. After 5 min incubation in the ice-cold sucrose solution, the hippocampi were glued on the stage of a microslicer with an agar block, and immersed in an ice-cold, oxygenated sucrose Ringer solution. Frontal hippocampal slices 300 μm in thickness were cut with the microslicer and incubated in the control external solution at room temperature for 1 h and maintained for up to 9 h. For recording, the slices were transferred to a 2.5-ml recording chamber mounted on the stage of an upright microscope and perfused at a rate of 2 ml min−1 with external solution maintained at 32 °C with a solution in-line heater.[2]

[1]. Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Mol Pharmacol. 2004;65(4):1008-1015.

[2]. Effects of a novel glutamate transporter blocker, (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), on activities of hippocampal neurons. Neuropharmacology. 2005;48(4):479-491.

[3]. Inhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate. Brain Res. 2010;1316:27-34.

In this study, the TFB-TBOA-induced continuous depolarization with a burst of spike discharges was mimicked by activation of AMPARs when CTZ was co-applied. As described above, CTZ not only increases the affinity of glutamate for AMPARs, but also markedly reduces the desensitization of AMPARs. This indicates that an elevation in the concentration of extracellular glutamate due to a dysfunction of transporters preferentially activates receptors with a high affinity for the agonist and a weak desensitization property. Thus, NMDARs play a more crucial role than AMPARs in epileptogenesis. Shimamoto et al. (2004) have shown that intracerebroventricular injection of TFB-TBOA elicited convulsive seizures within several minutes after the injection in mice. Moreover, we found that intrahippocampal injection of TFB-TBOA elicited similar convulsive seizures in rats (Tsukada et al., unpublished data). These findings are consistent with the present results obtained in hippocampal slices, and indicate directly that the dysfunction of glial glutamate transporters causes convulsive seizures. However, it is presently unknown what extent of blockage of glutamate transporters actually elicits seizures. Obviously, much remains to be done to clarify the involvement of dysfunction of glial glutamate transporters in epileptogenesis in the whole animal. TFB-TBOA will be useful as a pharmacological tool for elucidating the mechanisms for epileptogenesis both in vivo and in vitro experiments. [2]

---

Selective inhibition of the EAAT2 (GLT-1) subtype is classically achieved with dihydrokainate, which, however, possesses a fairly low affinity for the transporter (Bridges and Esslinger, 2005) and causes complex effects on astrocytes in situ (Bernardinelli and Chatton, 2008). A very recent report described the first selective inhibitor of EAAT1, UCPH-101, with an IC50 of ∼1 μM and > 400-fold selectivity over EAAT2 and EAAT3 (Jensen et al., 2009). With TFB-TBOA acting on both glial isoforms, the latter pharmacological tool could represent an interesting complement for the functional studies of glutamate transport. Taken together, the present study showed that TFB-TBOA is able to inhibit Na+-dependent glutamate transport in astrocytes with high potency. Despite a partial reversibility of inhibitory effects that have to be taken into account in experimental designs, TFB-TBOA is to be considered as an extremely valuable tool to study glutamate transport and neuron–glia interactions.[3]

C19H17F3N2O6

426.34

426.104

C, 53.53; H, 4.02; F, 13.37; N, 6.57; O, 22.52

480439-73-4

52941382

White to off-white solid powder

3.112

138.95

4

10

8

30

622

2

C1=CC(=CC(=C1)NC(=O)C2=CC=C(C=C2)C(F)(F)F)CO[C@@H]([C@@H](C(=O)O)N)C(=O)O

LPWONNPEPDHEAI-GJZGRUSLSA-N

InChI=1S/C19H17F3N2O6/c20-19(21,22)12-6-4-11(5-7-12)16(25)24-13-3-1-2-10(8-13)9-30-15(18(28)29)14(23)17(26)27/h1-8,14-15H,9,23H2,(H,24,25)(H,26,27)(H,28,29)/t14-,15-/m0/s1

(2S,3S)-2-amino-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]butanedioic acid

TFB-TBOA; 480439-73-4; (3S)-3-[[3-[[4-(TRIFLUOROMETHYL)BENZOYL]AMINO]PHENYL]METHOXY]-L-ASPARTIC ACID; (2S,3S)-2-amino-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]butanedioic acid; CHEMBL1257519; (2S,3S)-2-amino-3-(3-(4-(trifluoromethyl)benzamido)benzyloxy)succinic acid; CF3-Bza-TBOA; (2~{s},3~{s})-2-Azanyl-3-[[3-[[4-(Trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic Acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (117.28 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.93 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 1.25 mg/mL (2.93 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 1.25 mg/mL (2.93 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。