DL-ジチオトレイトール 化学特性,用途語,生産方法
外観
白色, 結晶〜粉末又は塊
溶解性
水, エタノール, アセトンに易溶。エタノール、アセトン及び水に溶けやすい。
解説
ジチオスレイトール，やや潮解性のある結晶。 DTTと略記する。融点 42～43℃，37℃で昇華。水，エチルアルコール，エーテルに可溶。－SH基の保護剤として酵素反応によく利用される。次の構造をもつ。
用途
DTTの利用法として良くあるのはチオール化したDNAを還元して「脱保護」するものである。チオール化DNAの末端の硫黄原子は（特に酸素存在下の）溶液中で二量体を作りやすい。例えばバイオセンサーとして金表面にDNAを固定化するような場合、二量体が形成されるとカップリング反応の効率が著しく低下する。そこでDNA溶液にDTTを加えて反応を助け、その後濾過やクロマトグラフィーによってDTTを除去（「脱塩」）する。
DTTはタンパク質のジスルフィド結合を還元し、タンパク質のシステイン残基の間で分子内または分子間のジスルフィド結合が形成されないようにするためによく使われる。しかしDTTは埋没した（溶液にさらされていない）ジスルフィド結合を還元することはできないので、ジスルフィド結合の還元は高温または高濃度の変性剤（6M塩酸グアニジン、8M尿素、1% SDSなど）を加えた変性条件で行うことが多い。逆に、個々のジスルフィド結合が溶液にさらされているかどうかをDTTによる還元速度で分析することもできる。
DTTは酸化剤として用いることもできる。グルタチオンなど他の薬剤とは違い、ジスルフィド種が混合しないという利点がある。非常に稀なことだが、DTTの2つの硫黄原子が異なる硫黄原子とジスルフィド結合をつくりDTT付加物が生成することがある。その場合は、自由なチオール基がないためDTTは環状化できない。
用途
タンパク質架橋剤、SH基酸化防止剤。
使用上の注意
不活性ガス封入
化学的特性
Dithiothreitol (DTT), also known as Cleland's reagent, is a small-molecule redox reagent . Its oxidized form is a disulfide-bonded 6-membered ring. DTT has an epimeric ('sister') compound, dithioerythritol (DTE).
DTT is a white crystalline powder. It is highly soluble in water (clear solution, OD<0.05 at 0.02M), but also in ethanol, chloroform, ether and ethyl acetate.
DTT is an unusually strong reducing agent, with a redox potential of -0.33 V at pH 7. The pKa of thiol groups is typically ~8.3.
The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions.
使用
DL-dithiothreitol (DTT) is a sulfhydryl compound that acts both as a reagent reducing disulfide bonds and as a protein denaturant on staphylococcal biofilm (Wu et al. 2011). Thanks to these properties, DTT is routinely used in clinical microbiology to liquefy respiratory specimens, but it has also been proven effective to detach biofilm from orthopaedic prosthesis (Drago et al. 2012, 2013). Since it is crucial to discriminate implant-related infections from aseptic loosening in orthopaedics, and because of the difficulty to diagnose subclinical infections, a prompt diagnosis of indwelling device-related infection is important for successful treatments (Borens et al. 2013). A fast microbiological diagnosis and bacterial identification should be recommended in order to set up a specific antimicrobial therapy. According to this strategy, the bacterial detachment from biofilm on explants should be accelerated by supporting the pathogen viability.
定義
ChEBI: 1,4-dithiothreitol is the threo-diastereomer of 1,4-dimercaptobutane-2,3-diol. It has a role as a reducing agent, a chelator and a human metabolite. It is a dithiol and a 1,4-dimercaptobutane-2,3-diol.
主な応用
Dithiothreitol (DTT) is a water-soluble reducing reagent used for various applications in biotechnology, biology and biochemistry :
reduces quantitatively disulfides, generating sulfhydryls (used typically at 1-10mM for protein SS reduction)
reduction of proteins before SDS-PAGE analysis, studies of protein structure and function (Kaji 1993)
keep sulfhydryl groups of biomolecules in the reduced state - protects biomolecules in various applications (enzymes or receptors, living cells under ionizing radiations)
prevents the fading of fluorescence such as FITC labeled conjugates (Picciolo 1984)
一般的な説明
1,4-Dithiothreitol (DTT) is the threo isomer of 2,3-dihydroxy-1,4-dithiolbutane, and an isomer of 1,4-dithioerythritol. DTT is used in molecular biology to maintain sulfhydryl (-SH) groups in the reduced state and for quantitative reduction of disulfide (-S-S-) groups, as described by Cleland in his pioneering publication from the 1960's. Its usefulness as an reducing agent stems from its water solubility and reduced odor compared to previous thiol compounds.
DTT is oxidized to the cyclic disulfide, and thereby ensures the reduction of other disulfides in solution. The disulfide reduction is complete in minutes at pH 8. DTT is less pungent and less toxic than 2-mercaptoethanol. Typically, a 7-fold lower concentration of DTT (100 mM) is required compared to 2-mercaptoethanol (5% v/v, 700 mM).
合成
The most widely used DL-Dithiothreitol (DTT) synthetic method is to be raw material with Soviet Union's moss sugar alcohol, use potassium permanganate oxidation earlier, obtains the sulfo-intermediate again under the thioacetic acid effect, and this sulfo-intermediate of hydrolysis obtains DTT at last.
Yu discloses a synthesizing method of DL-Dithiothreitol.1,4-butylene glycol is used as an initiating raw material and undergoes an addition reaction with bromine at first to obtain 2,3-dibromo-1,4-butylene glycol; hydrolyzation under the catalyzation of alkali is carried out to obtain dioxirane; addition reaction with thioacetic acid is carried out to obtain dithiothreitol diacetate; and finally hydrolyzation under the catalyzation of alkali is carried out to obtain the DL-Dithiothreitol.
貯蔵
Store lyophilized at 4°C, desiccated. In lyophilized form, the chemical is stable for 12 months. Once in solution, store at -20°C and use within 3 months to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.
DTT solutions should be prepared fresh daily. If improperly stored (including room temperature and solution forms) its reducing ability may be reduced. Exposure to air should be minimized, even though DTT has a lower tendency to be oxidized directly by air than other reducing agents.
Recorded half-life of DTT solutions at various pH and temperatures (all are in M potassium phosphate buffer):
pH 6.5 @ 20°C= 40 hours
pH 7.5 @ 20°C = 10 hours
pH 8.5 @ 20°C = 1.4 hours
pH 8.5 @ 0°C = 11 hours
pH 8.5 @ 40°C = 0.2 hours
pH 8.5 @20°C (+0.1 mM Cu2+) = 0.6 hours
pH 8.5 @20°C (+0.1 mM EDTA) = 4 hours
DL-ジチオトレイトール 上流と下流の製品情報
原材料
準備製品