The oxidative folding pathway of the disulfide containing protein bovine pancreatic trypsin inhibitor (BPTI) was one of the first to be elucidated and has served as a basis for understanding the folding pathways of other proteins. During the oxidative folding of reduced BPTI, two intermediates (N' and N*) accumulate in significant amounts and act as kinetic traps. Both N' and N* bury their two remaining free thiols in their hydrophobic cores, which inhibits further oxidation. Historically, the rate limiting step was considered to be the intramolecular rearrangements of N' and N* to another intermediate with two free thiols, NSH. The two free thiols in NSH are solvent-exposed and easily oxidized to a disulfide, producing native protein (N). Nevertheless, our research using reduced BPTI indicated that the folding rate of N* to N was proportional to the concentration of added glutathione disulfide (GSSG), inconsistent with the slow intramolecular rearrangement of N* to NSH. To confirm our initial results, the intermediate N* was purified and refolded in the presence of GSSG. The conversion of N* to N was dependent upon the disulfide concentration and singly mixed disulfide N*(SG) was observed during folding. These results emphasize that the folding of N* can proceed via a growth type pathway, direct oxidation of the two remaining thiols in N* by an exogenous small molecule disulfide, such as GSSG, to form N. Folding of reduced BPTI via N* was performed under changing concentrations of GSSG and GSH as a function of time. The folding was improved dramatically in terms of rate and yield. Aromatic disulfides and thiols have been demonstrated to improve the folding efficiency of disulfide containing proteins including ribonuclease A (RNase A) and lysozyme. Herein, N* and N' were refolded in the presence of aromatic disulfides. Folding of the two kinetic traps with aromatic disulfides indicated that folding proceed via a growth type pathway. The singly and doubly mixed disulfide intermediates were observed during most folding reactions. The oxidative folding of reduced BPTI with aromatic disulfides and thiols were also investigated. Reduced BPTI can be folded to disulfide intermediates rapidly.

The thesis entitled “Chemistry of Tetrathiomolybdate: Applications in Organic Synthesis” is divided in to six chapters Chapter 1: Synthesis of -amino disulfides, cystines and their direct incorporation into peptides mediated by tetrathiomolybdate In this chapter, we report a simple method for direct access to β-amino disulfides by regioselective ring opening of sulfamidates with benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS4. The versatility of this reaction has been shown by preparing a number of β-amino disulfides having different N-protecting groups and the stability of these protecting groups under the reaction conditions has been evaluated. This methodology is also extended to the synthesis and direct incorporation cystine and 3, 3′-dimethyl cystine derivatives into peptides. Chapter 2: Unusual reactivity of tetrathiomolybdate: A new entry to the synthesis of b-aminothiols In this chapter, we disclose a simple and highly efficient method for the synthesis of β and γ-amino thiols via regioselective ring opening of sulfamidates with tetrathiomolybdate 1. The scope and generality of this methodology has been exemplified by synthesizing a carbohydrate derived β-aminothiol. This methodology has also been extended to the synthesis of isocysteine derivatives in optically pure form. Chapter 3: Part 1: Synthesis of β-aminodiselenides via sequential one-pot, multistep reactions mediated by tetrathiomolybdate In this chapter, we have demonstrated that a variety of N-alkyl-β-aminodiselenides can be synthesized in high yield from appropriate sulfamidates under mild reaction conditions using potassium selenocyanate and tetrathiomolybdate [BnEt3N]2MoS4 via a sequential one-pot multistep process. The compatibility of different protecting groups under the reaction conditions has been discussed. Chapter: 3 Part 2: Synthesis of unnatural seleno amino acids and their direct incorporation into peptides In this chapter, we have demonstrated the first and general method for the synthesis of selenocystine, 3, 3'-dialkylselenocystine, isoselenocystine and their direct incorporation into peptides using a one-pot multistep reaction strategy mediated by tetrathiomolybdate. Chapter 4: Synthesis and functionalization of cysteine, selenocysteine and their derivatives via the formation of unsymmetrical disulfide and sulfur-selenium bond. In this chapter, we present a novel one-pot multi component strategy for the synthesis and functionalization of cysteine, selenocysteine and their derivatives via unsymmetrical disulfides and sulfur-selenium bond formation. Chapter 5: Part 1: A novel method for the synthesis of thioacetates employing benzyltriethylammonium tetrathiomolybdate and acetic anhydride In this chapter, we report a simple and efficient methodology for the synthesis of thioacetates using benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS4 and acetic anhydride as the key reagents, starting from alkyl halides in a multi step, tandem reaction process. The application of this methodology for the synthesis of orthogonally protected cysteine derivatives and anomeric β-thioglycosides has also been demonstrated. Chapter 5: Part 2: One-pot synthesis of β-aminothioacetates using benzyltriethyl-ammonium tetrathiomolybdate and acetic anhydride. In this chapter, we have demonstrated a simple and efficient method for the synthesis of β-amino thioacetates and pseudo thioinositol derivatives, via ring opening of aziridines and aziridino epoxides using tetrathiomolybdate 1 and acetic anhydride as key reagents. Chapter 6: Simple and efficient synthesis of allo and threo-3, 3'-dimethylcystine derivatives in optically pure form In this chapter, we have presented a simple and efficient methodology for the synthesis of allo-3,3'-dimethylcystine and threo-3,3'-dimethylcystine derivatives in optically pure form using L-threonine as the chiral pool and benzyltriethylammonium tetrathiomolybdate 1 as the key reagent. (For structural formula pl see the pdf file)

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

We have described herein a convenient one-pot synthesis of lisulfides/diselenides from a-amino acids mediated by ammonium etrathiomolybdate in water. (Figure 1) (Figure) Figure 1 Transformation of α-amino acids into the corresponding tiiocyanates/selenocyanates/disulfides/diselenides Halo-de-amination of a-amino acids using HBr/NaNCte followed by treatment with ammonium tetrathiomolybdate (NH4)2]VloS4 jLb provided a general route for the the one-pot synthesis of chiral a,a' bis (dithio) carboxylic acids (Figure 1, 2b). The yields were moderate, limited mainly the moderate conversion of a-amino acids into the corresponding chiral a-bromides. It was possible to synthesize the 2-thiocyanto carboxylic acids from the corresponding a-amino acids by a similar strategy. Thus diazotization in the presence of KSCN yielded in the chiral 2-thiocyanto carboxylic acids in moderate yields (Figure 1, 3). Thiocyanato-de-amination thus afforded the thiocyanates which when treated with JJD provided the chiral disulfides (Figure 1, 4a). We could thus synthesize both enantiomers of the disulfide from a single enantiomer of the starting a-amino acid. (Figure 1, 4a,4b) Using a similar strategy we have also demonstrated an efficient method for the synthesis of chiral selenocyanates starting from a-amino acids, using selenocyanate anion as the nucleophile (Figure 1, 5). It is possible to demonstrate a one-pot synthesis of chiral diselenides by reductive coupling of selenocyanates using JJb. (Figure 1, 6) (for figure see the pdf file)

The thesis entitled “Chemistry of Tetrathiomolybdate and Tetraselenotungstate: Studies on Aziridine and Epoxide Ring Opening Reactions” is divided into five chapters. (For Formulas and Equations Refer PDF File) Chapter 1: Part 1: Synthesis of β-Sulfonamidodisulfides and β-Sulfonamidosulfides using Benzyltriethylammonium Tetrathiomolybdate In this chapter, a comprehensive study of general and effective one step procedure for the synthesis of β-sulfonamidodisulfides directly from optically pure N-tosyl aziridines using benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS4 as sulfur transfer reagent in a regio manner under neutral conditions without the use of any Lewis acid or base has been reported. Additionally, we have demonstrated regio- and stereospecific ring opening of di- and trisubstituted aziridines using [BnEt3N]2MoS4 to synthesize substituted β-sulfonamidodisulfides in good yields. This methodology is extended to the synthesis of an optically pure unnatural amino acid with the disulfide bridge and a cyclic seven membered disulfide. Synthesis of a variety of β-sulfonamidosulfides involving cleavage of disulfide bonds assisted by tetrathiomolybdate and the use of masked thiolate for the synthesis of β-sulfonamidosulfides involving multi-step reactions in a one pot is also demonstrated. Chapter 1: Part 2: Synthesis of β-Sulfonamidodiselenides using Tetraethylammonium Tetraselenotungstate In this chapter, we report the results of regio- and stereospecific, nucleophilic ring opening of chirally pure N-tosyl aziridines with tetraethylammonium tetraselenotungstate [Et4N]2WSe4 as selenium transfer reagent to afford a number of β- sulfonamidodiselenides in good yields. Using this methodology, carbohydrate derived β- sulfonamidodiselenides from the corresponding carbohydrate derived aziridines have been synthesized. These enantiopure diselenide derivatives have the potential to be used as chiral ligands in diethyl zinc addition to aldehydes. Chapter 2: Ring Opening of Aziridine/Epoxide, Disulfide Formation, Reduction of Disulfide Bond and Michael Reaction In this chapter, we report a systematic study of tetrathiomolybdate mediated tandem regio- and stereospecific ring opening of aziridines, disulfide formation, in situ reduction of disulfide bond followed by Michael reaction in an one pot operation to give a variety of β-sulfonamidosulfides in good yields. The main advantage of this methodology is that four reactions involving three components take place in a one-pot operation. Chapter 3: Part 1: New Thia-aza Payne type Rearrangement Mediated by Benzyltriethylammonium Tetrathiomolybdate In this chapter, reaction of aziridinemethanol sulfonate esters with tetrathiomolybdate to give thiirane derivatives as the major product and cyclic disulfides as minor product under mild reaction conditions via an unprecedented thia-aza-Payne type rearrangement have been presented. Interestingly, when the reaction of tetrathiomolybdate was carried out with 2-aziridino-cyclohexanol derivatives it resulted in the formation of thia-bicyclo[3.1.1]heptane or dithia-bicyclo[3.2.1]octane derivatives. Chapter 3: Part 2: New selena-aza Payne Type Rearrangement Mediated by Tetraethylammonium Tetraselenotungstate In this chapter, reaction of tetraselenotungstate with simple N-tosyl aziridinemethanol tosylates to give allyl amine derivatives as the only product via an unprecedented selena-aza-Payne type rearrangement is discussed. When the methodology is extended to disubstituted N-tosyl aziridinemethanol tosylates, regio- and stereospecific ring opening of aziridines occurs to afford allyl amine derivatives as the major products and cyclic five membered diselenides as the minor products in good yields. Chapter 3: Part 3: Synthesis of Sulfur and Selenium Heterocycles by Azirdine Ring Opening followed by Cyclization In this chapter, studies on the synthesis of sulfur and selenium-heterocycles by aziridine ring opening followed by cyclization of N-tosyl aziridino-ethanol tosylates using tetrathiomolybdate as a sulfur transfer reagent and tetraselenotungstate as a selenium transfer reagent respectively are presented. Chapter 4: Tetrathiomolybdate Mediated Ring Opening of bis-Aziridines, bis-Epoxides and Aziridino-epoxides In this chapter, studies on the synthesis and ring opening of bis-aziridines, bis-epoxides and aziridino-epoxides with tetrathiomolybdate as the sulfur transfer reagent are presented. This has resulted in the synthesis of optically active sulfur heterocycles ranging from three membered to eight membered ring systems with excellent stereo and regio- control in good yields. Chapter 5: Part 1: Synthesis of Conformationally Locked, Bridged, Bicyclic Mono and Disulfides In this chapter, work related to the synthesis of conformationally locked bridged bicyclic disulfides and sulfides from cis-aziridino-epoxides by ring opening of both aziridines and epoxides in a tandem fashion using tetrathiomolybdate as a sulfur transfer reagent has been discussed. Comparative studies on the behavior of conformationally locked disulfides which has the dihedral angle close to zero (φ = 0) with disulfides having larger dihedral angles (φ>90) have been presented in this chapter. Some correlations have been made on the physicochemical characteristics of the disulfides with change in the dihedral angles. Chapter 5: Part 2: Synthesis of Conformationally Locked, Bridged, Bicyclic Diselenides In this chapter, work related to the development of a general synthetic methodology for the synthesis of conformationally locked, bridged diselena-bicyclo[3.2.1]octane skeleton by regio- and stereospecific, tandem nucleophilic ring opening of cis-1,4-aziridino-epoxides with tetraselenotungstate in one-pot are presented. To compare the behavior of conformationally locked diselenides which has the dihedral angle close to zero (φ = 0) with diselenides having larger dihedral angles (φ > 90), we have synthesized the acyclic diselenide (see chapter 1.2) and cyclic diselenide by regio- and stereospecific ring opening of simple aziridine and bis-aziridine respectively with tetraselenotungstate. Some correlations have been made on the physicochemical characteristics of the diselenides with change in the dihedral angles.

Bisacetylenic cabamates/carbonates are most useful compounds in finger mark development, for the synthesis of polymeric gels and other material applications. Organochalogen derivatives are the organic compounds containing chalcogen (S, Se) atoms. They have been used as chiral ligands for enatioselective catalysis, glycosyl donors and in the synthesis of heterocyclic compounds etc. This thesis describes our efforts towards synthesis of bisacetylenic cabamates/carbonates and development new synthetic strategies using rongalite (Na+HOCH2SO2-) and benzyltriethyl ammonium tetrathiomolybate [BnEt3N]2MoS4 as a reducing agents led to obtain various organochalcogen derivatives. We developed a new reagent, hexa-2,4-diyne-1,6-bisoxycarbonyl chloride [Hbc Cl] for the synthesis of symmetrical diacetylenic biscarbamates/biscarbonates and further studied the solid state structures using X-ray crystallography. Later we described a stereoselective method for the hydrothiolation of buta-1,3-diynes derivatives using diaryldichalcogenides in the presence of rongalite and K2CO3. The buta-1,3-diynes underwent stereoselective addition reaction with in situ generated chalocgenate anion from diaryl dichalcogenides which afforded the corresponding (Z)-chalcogenynes. The reactivity of buta-1,3-diynes with diaryl dichalcogenides was further studied at higher temperature led to a mixture of mono chalcogenated and bischalcogenated products. Then an efficient method was developed for the synthesis of enatiopure β-amino sulfides/selenides via ring opening of sulfamidates using diarylchalcogenides with rongalite as reducing agent. Further we synthesized chalcogeno derivatives of sugars from glycosyl halides and diaryl dichalcogenides in the presence rongalite. In addition, the synthesis of mixed glycosyl dichalcogenides has been demonstrated using [BnEt3N]2MoS4 as sulfur transfer agent as well as reducing agent. Finally the reactivity of [BnEt3N]2MoS4 was studied in detail with various isatioc anhydrides which led to the formation of S-benzyl 2-aminobenzothioate derivatives. Further we synthesized S-alkyl/aryl 2-aminobenzothioate derivatives via ring opening of isatoic anhydrides and diaryl/dialkyl chalcogenides by mean of [BnEt3N]2MoS4 as a reducing agent. We extended this method in a one-pot, tandem fashion with various alkyl halides. In this thesis, details of all of the above studies have been described.

Newcastle disease virus (NDV), an avian paramyxovirus, enters the host cell by fusion of viral and host cell membranes. The fusion of two membranes is mediated by the viral fusion (F) protein. The F protein, like other class I fusion proteins, is thought to undergo major conformational changes during the fusion process. The exact mechanism that leads to major refolding of F protein is not clear. Recently, it has been proposed that disulfide bond reduction in the fusion protein of some viruses may be involved in the conformational changes in fusion proteins. In some viruses, the reduction of disulfide bonds in the fusion protein is mediated by host cell disulfide isomerases belonging to the protein disulfide isomerase (PDI) family. In this study, the role of disulfide bond isomerization in the entry of NDV was analyzed. Using inhibitors of thiol-disulfide isomerases, we found that blocking the reduction of disulfide bonds in the fusion protein inhibited cell-cell fusion as well as virus entry into the host cell. Also, over-expression of isomerases belonging to the PDI family significantly enhanced cell-cell fusion. Taken together, these results suggest that free thiols play an important role in fusion mediated by NDV glycoproteins. Using a thiol specific, membrane impermeable biotin, MPB, we found that free thiols are produced in cell surface-expressed NDV F protein. The production of free thiols was inhibited by inhibitors of thiol-disulfide isomerases. Over-expression of isomerases belonging to the PDI family enhanced detection of free thiols in F protein. In F protein, present in virions or in virus-like particles, free thiols were detected only after the particles were attached to target cells. Taken together, these results suggest that free thiols are produced in F protein and the production of free thiols is mediated by host cell thiol-disulfide isomerases. Using conformation sensitive antibodies, we also studied the conformation of cell surface-expressed F protein in the presence ofthiol-disulfide isomerase inhibitors or in cells over-expressing thiol-disulfide isomerases. In the presence of thiol-disulfide isomerase inhibitors, the cell surface-expressed F protein was in a prefusion conformation while in cells over-expressing thiol-disulfide isomerases the F protein was in a post-fusion conformation. We also correlated the production of free thiols to the conformational changes in F protein. Using temperature-arrested intermediates or F protein with mutations in heptad repeat domains, which are defective in attaining intermediate conformations, we found that free thiols are produced before any of the proposed conformational changes in F protein. Also, the production of free thiols in F protein was found to be independent of its activation by hemagglutinin-neuraminidase (HN) protein. These results suggest that free thiols are probably required for the activation of F protein during membrane fusion.

This thesis describes NMR studies which probe weak interactions between amino acid side chains in folded peptide structures. Aromatic/aromatic interactions between facing phenylalanine residues have been probed in antiparallel β-sheets, while aromatic/proline interactions have been examined using cyclic peptide disulfides that occur in the venom of marine cone snails. Novel intramolecular hydrogen bonded structures in hybrid peptides containing backbone homologated residues, specifically γ-amino acids, are also described. Chapter 1 provides a brief background to the principles involved in the design of antiparallel β-sheet structures and an introduction to previous studies on aromatic/aromatic and aromatic/proline interactions in influencing peptide conformations. A summary of the NMR methods used is also presented. Chapter 2 discusses the structural characterisation of a designed 14 residue, three stranded β-sheet peptide, Boc-LFVDP-PLFVADP-PLFV-OMe (LFV14). The results described in this Chapter support the presence of multiple conformational states about the χ1 (Cα-Cβ) torsional degree of freedom for the interacting aromatic pairs in solution. Chapter 3 presents the structural characterisation of a designed 19 residue three stranded hybrid β-sheet peptide, Boc-LVβFVDPGLβFVVLDPGLVLβFVV-OMe (BBH19). β-amino acid residues (β-phenylalanine, βPhe) were incorporated at facing positions on antiparallel β-sheets. The BBH19 structure provides an example of interaction between the N and C-terminal strands in a three stranded structure with an α/β hybrid backbone. Chapter 4 focuses on studies of the conformations of the contryphan In936 (GCVDLYPWC*) from Conus inscriptus and the related peptide Lo959 (GCPDWDPWC*) from Conus loroissi. Both peptides possess a macrocyclic 23 membered ring, with multiple accessible conformational states. Chapter 5 describes conformational analysis of a novel 20 membered cyclic peptide disulfide, CIWPWC (Vi804), from Conus virgo. NMR structures were calculated for Vi804 and an analog peptide, CIDWPWC, DW3-Vi804. Chapter 6 explores the solution conformation of hybrid sequences containing α and γ residues. Oligopeptides of the type (αγ)n and (αγγ)n have been studied in solution by NMR methods. Chapter 7 provides a summary of the results described in this thesis and highlights the major conclusions.

Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide. Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein. Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.