合成Exendin-4的体外人血浆稳定性研究

为了解决Ⅱ型糖尿病患者注射用药频繁的问题, 需要比Exendin-4更加稳定的GLP-1的激动剂, 本文的研究目的是找到Exendin-4较易水解的氨基酸位点, 为设计新的长效的Exendin-4模拟肽提供相关信息.     

Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation

Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.     

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.     

化学裂解结合生物质谱对多肽二硫键的定位

二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键. 当多肽中存在多个半胱氨酸时, 形成的二硫键可能会存在多种配对方式. 快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要. 本文开发了一种基于化学裂解和生物质谱的新方法, 对利那洛肽中3对二硫键进行了精准定位. 通过解析裂解后特异肽段的二级质谱图, 确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6, Cys2-Cys10和Cys5-Cys13. 该方法为二硫键的定位研究提供了新思路.     

Effects of disulfide linkages on gas-phase reactions of small multiply charged peptide ions

Multiply protonated ions of disulfide-intact and -reduced peptides were generated by electrospray ionization and studied by Fourier transform ion cyclotron resonance mass spectrometry. The effects of disulfide bonds on gas-phase deprotonation reactions and hydrogen/deuterium (H/D) exchange were investigated. Insight into conformations was gained from molecular dynamics calculations. For ions from three small peptides containing 9–14 amino acid residues, H/D exchange is more sensitive to changes in conformation than deprotonation. However, with both gas-phase reactions the more diffuse forms of the peptides (as determined by molecular modeling) react more readily. The effects of disulfide cleavage on the conformations and on the reactions were found to depend upon the sequence of the peptide. For [M + 3H]³⁺ of TGF-α (34–43), reduction of the disulfide linkage leads to a greatly extended structure and a dramatic increase in the rate and extent of H/D exchange. In contrast, [M + 2H]²⁺ of Arg⁸ -vasopressin becomes slightly more compact upon cleavage of the disulfide bond; these reduced ions are slower to react. For [M + 3H]³⁺ of somatostatin-14, reduction of the disulfide bond has little effect on conformation or gas-phase reactivity. Overall, these results indicate that there is no general rule on how cleavage of a disulfide bond will effect a peptide ion’s gas-phase reactivity.     

An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry

Desfuroylceftiofur (DFC) is a bioactive beta-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg(8)]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H](2+) from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H](4+) shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ∼9–10 kcal mol^–1 than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.     

Determination of disulfide bond patterns in laminin beta1 chain N-terminal domains by nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.     

Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method

We have utilized sequence information from an antiheme monoclonal antibody to develop novel porphyrin-binding peptides. Several peptides which have an intramolecular disulfide bond in different positions and different chain lengths were prepared. The affinities of peptides for meso-tetrakis(4-carboxyphenyl)porphyrin were increased by an appropriate conformational restraint using a disulfide bond. Detailed studies with a representative 12-peptide, 12C4, whose length was reduced from 20 residues of the complementarity-determining region (CDR), indicated that both the hydrophobic and electrostatic interactions were essential factors in the peptide-porphyrin binding. Moreover, two-dimensional 1H NMR spectroscopy revealed the conformation of the peptide and the critical residues for the porphyrin-binding. According to the obtained results, a further minimized 9-peptide, 9L, was successfully redesigned with a sequence capable of forming a beta-turn instead of a disulfide bond. Furthermore, affinity maturation studies of 9L were performed by using a combinatorial approach such as the spot-synthesis method. Peptides with an improved affinity for porphyrins were prepared by systematic amino acid replacement. Thus, the design of peptides targeted to porphyrins was demonstrated by the combination of antibody information and the rationally designed combinatorial method.     

Electron Transfer Dissociation (ETD) of Peptides Containing Intrachain Disulfide Bonds

The fragmentation chemistry of peptides containing intrachain disulfide bonds was investigated under electron transfer dissociation (ETD) conditions. Fragments within the cyclic region of the peptide backbone due to intrachain disulfide bond formation were observed, including: c (odd electron), z (even electron), c-33 Da, z + 33 Da, c + 32 Da, and z–32 Da types of ions. The presence of these ions indicated cleavages both at the disulfide bond and the N–Cα backbone from a single electron transfer event. Mechanistic studies supported a mechanism whereby the N–Cα bond was cleaved first, and radical-driven reactions caused cleavage at either an S–S bond or an S–C bond within cysteinyl residues. Direct ETD at the disulfide linkage was also observed, correlating with signature loss of 33 Da (SH) from the charge-reduced peptide ions. Initial ETD cleavage at the disulfide bond was found to be promoted amongst peptides ions of lower charge states, while backbone fragmentation was more abundant for higher charge states. The capability of inducing both backbone and disulfide bond cleavages from ETD could be particularly useful for sequencing peptides containing intact intrachain disulfide bonds. ETD of the 13 peptides studied herein all showed substantial sequence coverage, accounting for 75%–100% of possible backbone fragmentation.     

Can metal ions be used as gas‐phase disulfide bond cleavage reagents? A survey of coinage metal complexes of model peptides containing an intermolecular disulfide bond

The role that a metal ion can have in promoting disulfide bond cleavage has been assessed by surveying the tandem mass spectra of the following metal complexes of model peptides containing an intermolecular disulfide bond: [M--H+Cu(II)](+); [M--H+Cu(II)(bipy)](+); [M+Ag(I)](+); and [M+Au(I)(PMe(3))](+). In comparison to previously studied protonated peptides, these binary and ternary metal complexes generally yield more abundant S--S and/or C--S bond cleavage. In general, [M--H+Cu(II)](+) ions cleave the adjacent C--S bond more readily, while the [M+Au(I)(PMe(3))](+) ion cleaves the S--S bond more readily. The ternary metal complex [M--H+Cu(II)(bipy)](+), on the other hand, fragments by exclusive loss of the bipyridyl ligand for the larger model peptides studied. Of all coinage metal systems studied, Me(3)PAu(+) is superior in promoting disulfide bond cleavage.     

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.     

Induction and identification of disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

The disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 (β-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis using software developed in-house. E. coli O157:H7 was induced to express Stx2 by culturing on solid agar media supplemented with 10-50 ng mL(-1) of ciprofloxacin (CP). Bacterial cell lysates at each CP concentration were analyzed by MALDI-TOF-MS. A prominent ion at mass-to-charge (m/z) ~7820 was observed for the CP concentration range: 10-50 ng mL(-1), reaching a maximum signal intensity at 20 ng mL(-1). Complex MS/MS data were obtained of the ion at m/z ~7820 by post-source decay resulting in top-down proteomic identification as the mature, signal peptide-removed, disulfide-intact β-Stx2. Eight fragment ion triplets (each spaced Δm/z ~33 apart) were also observed resulting from backbone cleavage between the two cysteine residues (that form the intra-molecular disulfide bond) and symmetric and asymmetric cleavage of the disulfide bond. The middle fragment ion of each triplet, from symmetric disulfide bond cleavage, was matched to an in silico fragment ion formed from cleavage of the backbone at a site adjacent to an aspartic acid or glutamic acid residue. The flanking fragment ions of each triplet, from asymmetric disulfide bond cleavage, were not matched because their corresponding in silico fragment ions are not represented in the database. Easier to interpret MS/MS data were obtained for the disulfide-reduced β-Stx2 which resulted in an improved top-down identification.     

Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry

The gas‐phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o‐TEMPO‐Bz‐conjugated peptides with an intra‐ and intermolecular disulfide bond was investigated using MSⁿ tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLC RIVVIRVC R), TGF‐α (C HSGYVGVRC ), MCH (DFDMLRC MLGRVFRPC WQY) and Adrenomedullin (16–31) (C RFGTC TVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o‐TEMPO‐Bz‐conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S? S or C? S bond was readily cleaved. The observed peptide backbone fragments included a‐, c‐, x‐ or z‐types, which indicates that the radical‐driven peptide fragmentation mechanism plays an important role in TEMPO‐FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S? S or C? S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of ?SH or ?SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S? S bond, the abstraction of a hydrogen atom at C_β by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H‐abstraction at C_α is suggested to lead to C? S bond cleavage, which yields [ion ± S] fragments or the loss of ?SH or ?SSH. Copyright © 2011 John Wiley & Sons, Ltd.     

Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation

We explored matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry for the analysis of N-glycosylated peptides, using horseradish peroxidase (HRP) as a test case. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing information on peptide sequence and glycan attachment site. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a (0,2)X-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Loss of the complete N-glycan moiety occurred by cleavage of both the N-glycosidic bond and the side-chain amide group of the N-glycosylated asparagine, yielding a characteristic peak doublet with a mass difference of 17 Da, which revealed the individual masses of the N-glycan and the peptide moiety. Analysis of a HRP tryptic digest at the sub-picomole level allowed the characterization of various N-glycosylated peptides including those with internal disulfide linkages, a glycopeptide linked via a disulfide bond to another peptide, and a 5 kDa glycopeptide carrying two N-glycans. The potential of our approach was illustrated by the detection of the following novel features of HRP glycosylation: (i) The conjugation of a xylosylated trimannosyl N-glycan without core-fucosylation to site Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, which might represent a Fuc(alpha1-3)GlcNAc- moiety arising from the processing of N-glycans by a horse-radish endoglycosidase during biosynthesis of HRP.     

A disulfide bond replacement strategy enables the efficient design of artificial therapeutic peptides

We demonstrate that disulfide bond replacement is an efficient strategy for engineering therapeutic peptides. In previous work, short peptide fragments, known as WP9QY, with sequence homology with the predicted ligand contact surface of the receptor activator of NF-κB (RANK) were crosslinked through intramolecular disulfide bonds to inhibit RANK ligand (RANKL)-induced signaling, osteoclastogenesis, bone resorption in vitro, and bone loss in vivo. We report that replacement of the disulfide bond of WP9QY with an amine cross-linkage results in a significant improvement in enzymatic stability, with only a slight loss of bone resorption-blocking activity in vitro. Furthermore, the WP9QY derivative inhibits bone loss significantly in vivo, whereas the native form of WP9QY was not effective under the same conditions.     

Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes

Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method. This is accomplished by collisional activation of singly and doubly charged cationic Na⁺ and Ca²⁺ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by 66 mass units resulting from selective S-C bond cleavages. Highly selective elimination of 66 mass units, which corresponds to the molecular weight of hydrogen disulfide (H₂S₂), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the S-C bond. Further activation of the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest fragments. For example, the doubly charged Ca²⁺ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting from selective S-C bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation. Further activation of the product comprising the A chain reveals the presence of a second disulfide bridge, an intramolecular linkage. Experimental and theoretical studies of the disulfide linked model peptides provide mechanistic details for the selective cleavage of the S-C bond.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric observation of a peptide triplet induced by thermal cleavage of cystine.

Heat-induced (90 degrees C, 30 min) beta-elimination of a cystine residue leads to cleavage of a disulfide bond and produces a set of three peptides with a cysteine residue, a thiocysteine residue (+32Da), and a dehydroalanine residue (-34Da). This characteristic feature was observed from somatostatin and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Mass spectrometric observation of this triplet is useful in identifying the presence of a cystine residue in a peptide, and could assist mass spectrometric identification of the peptide from a database.     

Photocycloaddition of Benzothiazole-2-thiones to Alkenes

The photocycloaddition of benzothiazole-2-thiones to electron-rich and aryl-substituted alkenes are described. Irradiation of N-unsubstituted benzothiazole-2-thione ( 1 ) in the presence of alkenes 3 gave 2-(2′-mercaptoalkyl)benzothiazoles 4 , and 2-substituted benzothiazoles 5 and 6 (in the case of 3a and 3h , resp.) through the ring cleavage of an intermediate 2-aminothietane (Schemes 1 and 3 ). The latter was formed by [2+2] cycloaddition of the C?S bond of 1 and the C?C bond of 3 . Irradiation of N-methylbenzothiazole-2-thione ( 2 ) and 2-methylpropene ( 3a ) gave the spiro-1,3-dithiane 8 , 1,2,6-benzodithiazocin-5-one 9 , and disulfide 10 . The structure of 9 was established by X-ray crystal-structure analysis.