Ergothioneine in a peptide: Substitution of histidine with 2-thiohistidine in bioactive peptides.

Ergothioneine (EGT) is the betaine of 2-thiohistidine (2-thioHis) and may be the last undiscovered vitamin. EGT cannot be incorporated into a peptide because the α-nitrogen is trimethylated, although this would be advantageous as an EGT-like moiety in a peptide would impart unique antioxidant and metal chelation properties. The amino acid 2-thioHis is an analogue of EGT and can be incorporated into a peptide, although there is only one reported occurrence of this in the literature. A likely reason is the harsh conditions reported for protection of the thione, with similarly harsh conditions used in order to achieve deprotection after synthesis. Here, we report a novel strategy for the incorporation of 2-thioHis into peptides in which we decided to leave the thione unprotected. This decision was based upon the reported low reactivity of EGT with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a very electrophilic disulfide. This strategy was successful, and we report here the synthesis of 2-thioHis analogues of carnosine (ßAH), GHK-tripeptide, and HGPLGPL. Each of these peptides contain a histidine (His) residue and possesses biological activity. Our results show that substitution of His with 2-thioHis imparts strong antioxidant, radical scavenging, and copper binding properties to the peptide. Notably, we found that the 2-thioHis analogue of GHK-tripeptide was able to completely quench the hydroxyl and ABTS radicals in our assays, and its antioxidant capacity was significantly greater than would be expected based on the antioxidant capacity of free 2-thioHis. Our work makes possible greater future use of 2-thioHis in peptides.

Facile removal of 4-methoxybenzyl protecting group from selenocysteine.

Historically, methods to remove the 4-methoxybenzyl (Mob)-protecting group from selenocysteine (Sec) in peptides have used harsh and toxic reagents. The use of 2,2'-dithiobis-5-nitropyridine (DTNP) is an improvement over these methods; however, many wash steps are required to remove the by-product contaminant 5-nitro-2-thiopyridine. Even with many washes, excess DTNP adheres to the peptide. The final product needs excess purification to remove these contaminants. It was recently discovered by our group that hindered hydrosilanes could be used to reduce Cys(Mob). We sought to apply a similar methodology to reduce Sec(Mob), which we expected to be even more labile. Here, we present a gentle and facile method for deprotection of Sec(Mob) using triethylsilane (TES), phenol, and a variety of other scavengers often used in deprotection cocktails. The different cocktails were all incubated at 40 °C for 4 hours. The combination of TFA/TES/thioanisole (96:2:2) appeared to be the most efficient of the cocktails tested, providing complete deprotection and yielded peptide that was mainly in the diselenide form. This cocktail also showed no evidence of side reactions or significant contaminants in the high-performance liquid chromatography (HPLC) and mass spectral (MS) analyses. We envision that our new method will allow for a simple and gentle "one-pot" deprotection of Sec(Mob) following solid-phase peptide synthesis and will minimize the need for extensive purification steps.

Can dimedone be used to study selenoproteins? An investigation into the reactivity of dimedone toward oxidized forms of selenocysteine.

Dimedone is a widely used reagent to assess the redox state of cysteine-containing proteins as it will alkylate sulfenic acid residues, but not sulfinic acid residues. While it has been reported that dimedone can label selenenic acid residues in selenoproteins, we investigated the stability, and reversibility of this label in a model peptide system. We also wondered whether dimedone could be used to detect seleninic acid residues. We used benzenesulfinic acid, benzeneseleninic acid, and model selenocysteine-containing peptides to investigate possible reactions with dimedone. These peptides were incubated with H2 O2 in the presence of dimedone and then the reactions were followed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The native peptide, H-PTVTGCUG-OH (corresponding to the native amino acid sequence of the C-terminus of mammalian thioredoxin reductase), could not be alkylated by dimedone, but could be carboxymethylated with iodoacetic acid. However the "mutant peptide," H-PTVTGAUG-OH, could be labeled with dimedone at low concentrations of H2 O2 , but the reaction was reversible by addition of thiol. Due to the reversible nature of this alkylation, we conclude that dimedone is not a good reagent for detecting selenenic acids in selenoproteins. At high concentrations of H2 O2 , selenium was eliminated from the peptide and a dimeric form of dimedone could be detected using LCMS and 1 H NMR. The dimeric dimedone product forms as a result of a seleno-Pummerer reaction with Sec-seleninic acid. Overall our results show that the reaction of dimedone with oxidized cysteine residues is quite different from the same reaction with oxidized selenocysteine residues.

Comparison of the redox chemistry of sulfur- and selenium-containing analogs of uracil.

Selenium is present in proteins in the form of selenocysteine, where this amino acid serves catalytic oxidoreductase functions. The use of selenocysteine in nature is strongly associated with redox catalysis. However, selenium is also found in a 2-selenouridine moiety at the wobble position of tRNAGlu, tRNAGln and tRNALys. It is thought that the modifications of the wobble position of the tRNA improves the selectivity of the codon-anticodon pair as a result of the physico-chemical changes that result from substitution of sulfur and selenium for oxygen. Both selenocysteine and 2-selenouridine have widespread analogs, cysteine and thiouridine, where sulfur is used instead. To examine the role of selenium in 2-selenouridine, we comparatively analyzed the oxidation reactions of sulfur-containing 2-thiouracil-5-carboxylic acid (s2c5Ura) and its selenium analog 2-selenouracil-5-carboxylic acid (se2c5Ura) using 1H-NMR spectroscopy, 77Se-NMR spectroscopy, and liquid chromatography-mass spectrometry. Treatment of s2c5Ura with hydrogen peroxide led to oxidized intermediates, followed by irreversible desulfurization to form uracil-5-carboxylic acid (c5Ura). In contrast, se2c5Ura oxidation resulted in a diselenide intermediate, followed by conversion to the seleninic acid, both of which could be readily reduced by ascorbate and glutathione. Glutathione and ascorbate only minimally prevented desulfurization of s2c5Ura, whereas very little deselenization of se2c5Ura occurred in the presence of the same antioxidants. In addition, se2c5Ura but not s2c5Ura showed glutathione peroxidase activity, further suggesting that oxidation of se2c5Ura is readily reversible, while oxidation of s2c5Ura is not. The results of the study of these model nucleobases suggest that the use of 2-selenouridine is related to resistance to oxidative inactivation that otherwise characterizes 2-thiouridine. As the use of selenocysteine in proteins also confers resistance to oxidation, our findings suggest a common mechanism for the use of selenium in biology.

Removal of the 5-nitro-2-pyridine-sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis.

We previously reported on a method for the facile removal of 4-methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2'-dithiobis-5-nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2-thio(5-nitropyridyl) (5-Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys-containing or Sec-containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5-Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy.

Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans - 9:91). This was determined by using the 'γ-gauche effect', which makes use of both (1) H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T- conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange.

Compensating for the absence of selenocysteine in high-molecular weight thioredoxin reductases: the electrophilic activation hypothesis.

Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618-12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an "electrophilic activation" mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic activation serves as a compensatory mechanism in the absence of the more electrophilic Sec residue. We present an argument for the importance of S-electrophilicity in Cys orthologs of selenoenzymes.

Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom) or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild-type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between the enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as a donor of an electron to the substrate.

Why is mammalian thioredoxin reductase 1 so dependent upon the use of selenium?

Cytosolic thioredoxin reductase 1 (TR1) is the best characterized of the class of high-molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in a large decrease in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. The Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high-Mr TRs from other organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum, do not require Sec to reduce small molecule substrates, including 5,5'-dithiobis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data show that high-Mr TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum, and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a "guiding bar", amino acids 407-422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and co-workers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. J. Mol. Biol. 370, 116-127], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec → Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes, in contrast, are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater access of the substrate to the N-terminal redox center and because they can make use of alternative mechanistic pathways that are not available to TR1.

A mechanistic investigation of the C-terminal redox motif of thioredoxin reductase from Plasmodium falciparum.

High-molecular mass thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). Trx is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize reduced ß-nicotinamide adenine dinucleotide phosphate to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol while the thiol involved in charge-transfer complexation is denoted as CCT. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer, the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high-molecular mass TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of with a GCUG sequence, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs has its Sec residue replaced with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from Plasmodium falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541, and we classify it as a type II high-molecular mass TR. The oxidized type II redox motif will form a 20-membered disulfide ring, whereas the absence of spacer amino acids in the type I motif results in the formation of a rare eight-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin reductase activity by ∼50-fold, most likely because of disruption of an important hydrogen bond between the amide NH group of Gly541 and the carbonyl of Gly534 that helps to stabilize the ß-turn-ß motif. Alterations of the 20-membered disulfide ring either by amino acid deletion or by substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size caused by using semisynthesis to substitute homocysteine for cysteine also caused significant reductions in catalytic activity, demonstrating the importance of the disulfide ring's geometry in making the C-terminal redox center reactive for thiol-disulfide exchange. The data suggested to us that the transfer of electrons from the N-terminal redox center to the C-terminal redox center may be rate-limiting. We propose that the transfer of electrons from the N-terminal redox center in PfTR to the type II C-terminal disulfide is accelerated by the use of an "electrophilic activation" mechanism. In this mechanism, the type II C-terminal disulfide is polarized, making the sulfur atom of Cys540 electron deficient, highly electrophilic, and activated for thiol-disulfide exchange with the N-terminal redox center. This hypothesis was investigated by constructing chimeric PfTR mutant enzymes containing C-terminal type I sequences GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less thioredoxin reductase activity than the native enzyme but still reduced selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had higher catalytic activity than the native enzyme with Trx, selenocystine, and lipoic acid as substrates. The results suggested to us that (i) Sec in the mutant enzyme accelerated the rate of thiol-disulfide exchange between the N- and C-terminal redox centers, (ii) the type II redox center evolved for efficient catalysis utilizing Cys instead of Sec, and (iii) the type II redox center of PfTR is partly responsible for substrate recognition of the cognate PfTrx substrate relative to noncognate thioredoxins.

Differing views of the role of selenium in thioredoxin reductase.

This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pK (a) and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the "chemico-enzymatic" function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the "chemico-biological" function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO(2)(-), seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO(2)(-) to Cys-SH).

Methaneseleninic acid is a substrate for truncated mammalian thioredoxin reductase: implications for the catalytic mechanism and redox signaling.

Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductase that contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shown that a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail will catalyze the reduction of a number of small molecules. Here we show that the truncated thioredoxin reductase will catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slower than that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Sec residue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO(2)(-)) that it would be quickly reduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be very resistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme with NADPH and subjecting it to high concentrations of H(2)O(2) (up to 50 mM). The results show that the enzyme strongly resisted inactivation by 50 mM H(2)O(2). To determine the redox state of the C-terminal Sec residue, we attempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues and presumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited by dimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H(2)O(2). We also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that it also was resistant to high concentrations of H(2)O(2). One assumption for the use of Sec in enzymes is that it is catalytically superior to the use of cysteine. We and others have previously suggested that there are reasons for the use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented here support this assertion. The results also imply that the redox signaling function of the thioredoxin system can remain active under oxidative stress.

No selenium required: reactions catalyzed by mammalian thioredoxin reductase that are independent of a selenocysteine residue.

Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox-active tetrapeptide of sequence Gly-Cys(1)-Sec(2)-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulysin, HIV Tat protein, H(2)O(2), lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys(2) mutant enzyme or the N-terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild-type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175-550-fold reduction in k(cat). Our data support the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, or (iii) that are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC(50) for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83-142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.

Synthesis, Redox Properties, and Conformational Analysis of Vicinal Disulfide Ring Mimics.

A vicinal disulfide ring (VDR) results from disulfide bond formation between two adjacent cysteine residues. This 8-membered ring is a rare motif in protein structures and is functionally important to those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR mimics, discernment of the preferred conformation of these mimics, and the determination of their respective disulfide redox potentials.

A viable synthesis of N-methyl cysteine.

While a number of methods exist for the production of N-methyl amino acid derivatives, the methods for the production of N-methyl cysteine (MeCys) derivatives are suboptimal as they either have low yields or lead to significant sulfhydryl deprotection during the synthetic protocol. This article focuses on the generation of MeCys and its subsequent use in Fmoc solid-phase peptide synthesis for the generation of N-methyl cystine containing peptides. Various methods for amino methylation of cysteine, in the presence of acid labile or acid stable sulfhydryl protecting groups, are compared and contrasted. Production of MeCys is best attained through formation of an oxazolidinone precursor obtained via cyclization of Fmoc--Cys(StBu)--OH. Following oxazolidinone ring opening, iminium ion reduction generates Fmoc--MeCys(StBu)--OH with an overall yield of 91%. The key to this procedure is using an electronically neutral Cys-derivative, as other polar Cys-derivatives gave poor results using the oxazolidinone procedure. Subsequently, the Fmoc--MeCys(StBu)--OH building block was used to replace a Cys residue with a MeCys residue in two peptide fragments that correspond to the active sites of glutaredoxin and thioredoxin reductase. The examples used here highlight the use of a MeCys(StBu) derivative, which allows for facile on-resin conversion to a MeCys(5-Npys) residue that can be subsequently used for intramolecular disulfide bond formation with concomitant cleavage of the peptide from the solid support. (c) 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 61-68, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Synthesis and Properties of Disulfide-Bond Containing Eight-Membered Rings.

The cyclocystine ring structure (CRS, 3), which results from a disulfide-bond between adjacent cysteine residues, is a rare motif in protein structures and is functionally important to those few proteins that posses it. This communication will focus on the construction of CRS mimics and the determination of their respective redox potentials.

Bleach/acetic acid-promoted chlorinative ring expansion of [2.2.1]- and [2.2.2]-bicycles.

[formula: see text] Treatment of vinyl-substituted [2.2.1]- and [2.2.2]-bicyclocarbinols with NaOCl and AcOH provides [3.2.1]- and [3.2.2]-beta-chloro-bicycloketones, respectively. For [2.2.2]-bicycles, these chlorinative ring expansions are particularly efficient and selective.