1.4- Dithiols dithiothreitol

Turk and Hollocher (1992) have attempted to trap nitroxyl using the dithiol, dithiothreitol, during the reduction of NO by purified nitric oxide reductase from Pa. denitrificans. Because the dithiol served in this system as both electron donor to the enzyme and trap for nitroxyl, the ratio of thiol groups oxidized to NO consumed would have been 1 if nitroxyl trapping did not occur and 3 if nitroxyl... 

However, hybrid bilayers are not suitable for protein incorporation because water is needed at the inner part of the bilayer to avoid protein denaturalization. In order to avoid this problem, Au surfaces were functionalized with a short hydroxylated dithiol (dithiothreitol, DTT) which adopts a lying down configuration with the OH groups exposed to the environment. Vesicle fusion on these DTT surfaces allows phospholipid bilayer formation with a water layer between the DTT SAM and the inner phospholipid monolayer. The phospholipid bilayer exhibits good fluidity as has been shown by in situ AFM (atomic force microscopy) imaging. These bilayers have been formed on both planar and nanostructured [SERS (surface enhanced Raman spectroscopy) active] gold surfaces. ... 

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

I. 1.4.1] catalyzes the reaction of 2-methyl-3-phytyl-l,4-naphthoquinone with oxidized dithiothreitol and water to produce 2,3-epoxy-2,3-dihydro-2-methyl-3-phytyl-l,4-naphthoquinone and 1,4-dithiothreitol. In the reverse reaction, vitamin K 2,3-epoxide is reduced to vitamin K and possibly to vitamin K hydroquinone by 1,4-dithioer-ythritol (which is oxidized to the disulfide). Some other dithiols and butane-4-thiol can also act as substrates. This enzyme is strongly inhibited by warfarin. 

A third enzyme is required to reduce the epoxide to vitamin K (Eq. 15-57). The biological reductant is uncertain but dithiols such as dithiothreitol serve in the laboratory.518 See also Eq. 18-47. Protonation of an intermediate enolate anion would give 3-hydroxy-2, 3-dihydrovitamin K, an observed side reaction product. 

While azides were far more susceptible to reduction by dithiols, the rates of reduction of a diazotrifluoropropionyl derivative by dithiothreitol, (3-mercaptoethanol, cysteine, and reduced glutathione did not differ widely. Thioglycolic acid was however a poor reductant and it was suggested that it should be used to replace (3-mercaptoethanol or DTT when diazo reagents are used. The reduction may be monitored by TLC or by a 500-fold increase in the absorbance at 260 nm. 

Although the reduction of ribonucleotides to the corresponding 2 -deoxyribonucleotides is catalyzed by enzyme systems differing in their cofactor requirements and/or in the level of phosphorylation of the substrates, the overall reduction process is very similar in all systems. For all the systems NADPH is the ultimate reductant, the hydrogen is transferred by thioredoxin reductase to thioredoxin, which in turn is oxidized by ribonucleotide reductase with the concomitant production of a 2 -deoxyribonucleotide. In these systems the thioredoxin-thiore-doxin reductase reducing system can be replaced by dithiols such as dihydrolipoate or dithiothreitol. 

An extensive study revealed that the A-dithiasuccinyl-protected azide 224 offers a major advantage in the synthesis of A-glycans. Efficient reduction of the A -dithiasuccinyl- and azido-functionality in 224 could be achieved, either in solution by utilizing simultaneous in situ reduction with Zn in THE AcOH AC2O, or on solid phase upon treatment with ethyldiisopropylamine and an excess of dithiothreitol, propane-1,3-dithiol, or 2-mercapto-A-methylacetamide leading to the known 1 or 225. 

Although this mechanism has not been established for an in vivo process, in vitro studies show that the reactivity of excess thiol with RSNOs leads to disulfide and HNO formation (26). Moreover, HNO is also generated from the nitrosation of dithiol compounds such as dithiothreitol and lipoic acid (22). 

No unimolecular cyclization routes to the 1,4-dithiocins have been reported. Addition of 1,4-dihalides to vicinal dithiols and of 1,4-dithiols to vicinal dihalides both represent common approaches to the ring system. Thus, 2,2 -dithiolbiphenyl reacts with c75-l,2-dichloroethene to afford a low yield of dibenzo[e, ][l,4]dithiocin (181) (Scheme 62) <69ZCI84>. Similarly, cis-1,2-dichloroethene reacts with the acetone ketal of dithiothreitol to afford tetrahydrodithiodn (182 R2 = CMe2) in 40% yield, with the low yield in part due to losses upon purification <77T2i5i>. 

Disulfides (134) (oil) and (135) (mp 155°C) were prepared (in the dithiol forms) as alternative reagents to dithiothreitol for the reduction of disulfide linkages. The dithiol of (135) was found to be an acceptable replacement to dithiothreitol <93JOC633>. 

Studies show that microsomal thiol S-methyltransferase in rat salivary glands is highly specific to aliphatic thiols. Relative activity of 4 mmol butane-1,4-dithiol/l was 95.6% (compared with 100% for dithiothreitol). The authors concluded that microsomal thiol S-methyltransferase activity in rat salivary glands detoxifies extracellular thiols and intracellular hydrogen sulfide to protect normal secretory functions (Yashiro Takatsu, 2001). 

As shown in Fig. 1, the enzyme catalyzes the reduction of ribonucleoside diphosphates (Fig. lA) by dithiothreitol (Fig. IB). K values for CDP and DTT are 70 x Af and 20 mAf, respectively. The requirement for a dithiol suggests that, as for other class II RNRs, such as the extensively studied enzyme from Lactobacillus leichmannii, the hydrogen donor is very likely to be a dithiol protein such as thioredoxin or glutaredoxin. However, there is still no experimental evidence that an archaeal thioredoxin operates as an electron source for RNRs. The enzyme also requires AdoCbl for which a value of 1 pAf has been obtained (Fig. 1C). Finally, the reaction has an optimal temperature of 80° (Fig. ID), with very little activity at 30°. How AdoCbl resists such a high temperature and how the enzyme controls Co-C bond homolysis required for catalysis in thermophilic AdoCbl-dependent enzymes is an intriguing question. These properties are shared by other isolated thermophilic class II RNRs (Table II). 

In Lactobacillus leischmanii, reduced thioredoxin is the natural reductant, but dithiols (dihydrolipoate, dithiothreitol) and monothiols (2-mercaptoethanol and glutathione) have been found to be active reduc-tants. In this reaction, coenzyme B12 is believed to act as a hydrogen carrier. 

The emphasis here is on the properties of glutathione (or cysteine if data for cysteine, but not GSH are available). Dithiols which form cyclic or stabilized disulphide radical anions, such as dithiothreitol (//ireo-l,4-dimercapto-2,3-butanediol) [29-33], or the reduced form of lipoic acid (6,8-dithiooctanoic acid) [15, 34-37], in effect have uncharacteristically high values of to be good models for glutathione. In addition, biological effects may be complicated by thiol/disulphide exchange since these thiols will reduce many disulphides [38-40]. Arenethiyl radicals (e.g. phenylthiyl or derived from 2-mercaptoimidi-azole) are closer to phenoxyl radicals in nature than aliphatic thiyl radicals [41-44]. 

Of all the synapses which have cholinergic transmission, that at the motor end-plate (at the volimtary neuromuscular junction) has been most studied. Its structure, visible even in the light microscope and sketched in Fig. 7.1, has revealed yet further complexities to the electron microscope, to electro-physiological measurements, to assays of acetylcholine vesicles and of acetylcholinesterase, and to autoradiography. The chemical nature of the receptors in the end-plate is imperfectly known, but a disulphide (S-S) group is essential for its functioning. This follows from the inhibition of the receptor by dithiothreitol (a disulphide reducer) and restoration of sensitivity by 5,5 -dithio-fe 5-2-nitrobenzoic acid (which restores a dithiol to the disulphide state) (Karlin and Bartels, 1966). 

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