The computer-assisted carboxypeptidase method for C-terminal sequencing of proteins and polypeptides

On further study of the computer-assisted carboxypeptidase method for sequencing the C-terminals of proteins or peptides, two computer programs—DPS and CPA were successfully developed on VAX-11/780 computer and tested with some synthetic peptides and the degradation fragment of the natural protein as model substrates. The C-terminal sequence of CB-3, one of the CNBr degradation fragments of trichosanthin, had first been determined by this method as: -Ser-Ala-Ser-Ala-Ala-Leu-Hse-OH, which was later confirmed by other ways of sequencing. This method is not only able to extend the C-terminal sequencing up to 7 amino acid residues, but also useful for determining the C-terminal sequence with repeating amino acid residues.     

A linear free-energy correlation in the low-energy tandem mass spectra of protonated tripeptides Gly–Gly–Xxx

The backbone cleavages of protonated tripeptide ions of the series Gly—Gly—Xxx, where Xxx ? Gly, Ala, Val, d-Leu, l-Leu, Ile, Phe, Tyr, Trp, Pro, Met and Glu, were studied in a hybrid tandem mass spectrometer. C-Terminal y-type ions and N-terminal a- and b-type ions were noted. A linear relationship between log (y₁/b₂) and the proton affinity of the C-terminal amino acid substituents was found: as the proton affinity of the C-terminal residue increases, the fraction of y₁ ion formation increases. When the C-terminal substituent was more basic than Trp, the b₂ ion was not observed. It is likely that the site of protonation changes from peptide bond to side-chain for just these residues, Lys, His and Arg.     

C-Terminal sequencing of trichosanthin

Preliminary identification of C-terminus of trichosanthin by chemical and enzymic methods, such as hydrazinolysis, thiohydantoin reaction and carboxypeptidase hydrolysis, showed that there may be the possible presence of more than one terminus, i.e., Met and Ala but complicated by side reactions. A computer-assisted carboxypeptidase method was first introduced by the authors to determine the C-terminal sequence of trichosanthin, and showed that trichosanthin is heterogeneous at its C-terminus and has two C-terminal sequences determined as -Arg-Asn-Asn-Met-OH and -Arg-Asn-Asn-Met-Ala-OH respectively. These results have been later unambiguously confirmed by the results from other experiments through the identification of the free alanine always present in the CNBr degradation products of trichosanthin, and the actual separation of two fragments from the finger prints as well as from the HPLC fractions of the trypsin digest of this protein. All shows that their amino acid sequences, determined by manual DABITC/PITC technique, agree well with those of the two C-terminal sequences determined by the computer-carboxy peptidase method.     

Facile Peptide-Bond Formation Using Polystyrene-Bound o-Nitrophenol as an Active Ester

Using a polystyrene-bound o-nitrophenol as the active ester, peptide bond was formed in very high yield (>90%) and optical purity. Peptide fragments with a free C -terminal were synthesized in moderate yields (70-85%) by using an amino acid nucleophile, of which the C-terminal being protected as a salt of phasetransfer reagent.     

Spiro‐Bicyclic Bisborane Catalysts for Metal‐Free Chemoselective and Enantioselective Hydrogenation of Quinolines

A new series of spiro‐bicyclic bisborane catalysts has been prepared by means of hydroboration reactions of C₂‐symmetric spiro‐bicyclic dienes with HB(C₆F₅)₂ and HB(p‐C₆F₄H)₂. When used for hydrogenation of quinolines, these catalysts give excellent yields and enantiomeric excesses, and show turnover numbers of up to 460. The most attractive feature of these metal‐free hydrogenation reactions was the broad functional‐group tolerance, making this method complementary to existing methods for quinoline hydrogenation.     

Novel fragmentation of N-diisopropyloxyphosphoryl dipeptides and tripeptides by fast atom bombardment mass spectrometry

Positive ion fast atom bombardment mass spectrometry of N-diisopropyloxyphosphoryl dipeptides and tripeptides showed a novel cleavage pattern in that only the N-phosphoryl fragment ions gave intense peaks while the C-terminal series of ions was suppressed. The base peak was the N-phosphoryl imino ion responding to the N-terminal residue. These advantages are superior to those of other types of N-protecting groups.     

Association of Radical Chemistry with LanD Flavoprotein Activity for C-Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S-[2-aminovinyl](3-methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C-terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two-stage bioinformatics mining of posttranslational modifications (PTMs) related to C-terminal Cys processing, we report herein that LanD activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.     

Application of parahydrogen for mechanistic investigations of heterogeneous catalytic processes

Parahydrogen-induced polarization technique (PHIP), based on the pairwise addition of molecular hydrogen to a substrate, was successfully applied to obtain novel information on the mechanisms of heterogeneous catalytic hydrogenation, hydrodesulfurization, and oligomerization processes. In particular, the PHIP effects were observed upon hydrogenation with parahydrogen catalyzed by the immobilized neutral complexes of rhodium and iridium, which confirms the similarity in the mechanisms of homogeneous and heterogeneous hydrogenation for such systems. In the study of acetylene oligomerization, a significant NMR signal enhancement was revealed for a number of C₄ oligomers, with the enhancement levels by far exceeding that observed in hydrogenation of carbon-carbon triple bonds. The mechanistic features of heterogeneous hydrogenation of a number of six-membered cyclic hydrocarbons over supported metal catalysts were investigated, and their hydrogenation scheme based on the pairwise addition of molecular hydrogen was proposed. Furthermore, the PHIP technique revealed that heterogeneous hydrodesulfurization of thiophene mainly proceeds via hydrogenation followed by a C—S bond cleavage. A significant enhancement of sensitivity in combination with characteristic line shapes of NMR signals make the PHIP method a unique and highly informative tool for the investigation of heterogeneous catalytic processes.     

Synthesis of Unique Ceramides and Cerebrosides Occurring in Human Epidermis

The sphingolipids 1a , b and 2a , b which play important roles in epidermal barrier function, were synthesized by N-acylation of C₁₈-sphingosine 3 and 1-O-glucosylated C₁₈-sphingosine 6 , respectively, with ω-acyloxy-substituted fatty acids 4 and 5 (Scheme 1). These fatty acids were obtained from ω-hydroxy-substituted fatty acids 8 and 9 by esterification with linoleic acid ( 7 ). The C₃₄-fatty acid 8 was prepared as follows: C₂₅-Compound 18 was obtained by means of a Wittig reaction of C₁₃-aldehyde 13 with C₁₂-phosphonium salt 15 or of C₁₂-aldehyde 24 with C₁₃-phosphonium salt 21 , respectively, and subseqent hydrogenation and O-deprotection (Scheme 2). Alternatively, 8 was prepared via 30 by copper-catalyzed coupling of C₁₃-alkyl halide 19 with the Grignard reagent derived from C₁₂-alkyl bromide 14 (Scheme 2). Oxidation of 18 to aldehyde 39 and Wittig reaction with C₉-phosphonium salt 41 furnished the desired ω-hydroxy-substituted fatty acid 8 , after O-deprotection (Scheme 3). Similarly, Wittig reaction of C₁₁-phosphonium salt 22 with C₁₂-aldehyde 24 furnished C₂₃-aldehyde 40 , after hydrogenation, O-deprotection, and oxidation; Wittig reaction with compound 41 and subsequent deprotection afforded the desired C₃₂-fatty and 9 (Scheme 3). an alternative strategy furnished compound 8 by a coupling reaction of alkyne 53 with ω-bromo-substitued fatty acid 52 , obtained from compounds 24 and 47 by Wittig reaction, hydrogenation, and introduction of bromide (Scheme 4). Hydrogenation (Lindlar's catalyst) of the resulting C₃₄-alkyne 54 and deprotection furnished 8 .     

Metal‐Free Hydrogenation Catalyzed by an Air‐Stable Borane: Use of Solvent as a Frustrated Lewis Base

In recent years ‘frustrated Lewis pairs’ (FLPs) have been shown to be effective metal‐free catalysts for the hydrogenation of many unsaturated substrates. Even so, limited functional‐group tolerance restricts the range of solvents in which FLP‐mediated reactions can be performed, with all FLP‐mediated hydrogenations reported to date carried out in non‐donor hydrocarbon or chlorinated solvents. Herein we report that the bulky Lewis acids B(C₆Cl₅)_x(C₆F₅)_3?x (x=0–3) are capable of heterolytic H₂ activation in the strong‐donor solvent THF, in the absence of any additional Lewis base. This allows metal‐free catalytic hydrogenations to be performed in donor solvent media under mild conditions; these systems are particularly effective for the hydrogenation of weakly basic substrates, including the first examples of metal‐free catalytic hydrogenation of furan heterocycles. The air‐stability of the most effective borane, B(C₆Cl₅)(C₆F₅)₂, makes this a practically simple reaction method.     

Theoretical Study of the Structures,Properties and Spectroscopies on Fullerene Hydrides C<Subscript>26</Subscript>H<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> (<Emphasis Type="Italic">n</Emphasis> = 2, 4, 6, 8)

The structures, stability patterns of C₂₆H _n (n = 2) formed from the initial D _3h C₂₆ fullerene were investigated by use of second-order-Moller–Plesset perturbation theory. The study of the stability patterns of hydrogenation reaction on C₂₆ cage revealed that type (β) carbons were the active site and the analyses of π-orbital axis vector indicated that the reactivity of C₂₆ was the result of the high strain and the hydrogenation reaction on C₂₆ cage was highly exothermic. The calculated ¹³C NMR spectra of C₂₆H _n (n = 2) predicted that the two sp ³ hybridization carbons in C₂₆H _n (n = 2) obviously moved to high field compare with that in D _3h C₂₆. Hence, the C₂₆H₂ should be obtained and detected experimentally. Similarly, the structures and reaction energies of C₂₆H _n (n = 4, 6, 8) were further studied at HF/6-31G*, B3LPY/6-31G* and MP2/6-31G* level. The results suggested the hydrogenation products of C₂₆, C₂₆H _n (n = 4, 6, 8), were more stable than the C₂₆ cage.     

Oxopiperazine capping: Formation of oxopiperazine-containing peptoids via C-terminal cyclization

An unexpected side reaction was observed in peptoids containing a C-terminal carboxamide with a 2-aminoethyl side chain. The reaction proceeded via cyclization and release of NH₃, resulting in C-terminal oxopiperazine formation, analogous to pyroglutamate formation from N-terminal glutamine in peptides. Reaction conditions that promote oxopiperazine formation were developed. In particular, the addition of organic bases accelerated the cyclization, thus providing a simple strategy for the post-synthetic C-terminal capping of peptoids.     

Efficient Solution Phase Synthesis of PPII Helix Mimicking Ena/VASP EVH1 Inhibitors from Proline-Derived Modules (ProMs)

In the search for efficient inhibitors for the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain to reduce cell motility in metastatic cancer, we previously developed a toolkit of proline-derived modules (ProMs), which mimic the PPII helix found in the natural −FPPPP− binding motif of EVH1. In this work, we describe the modular assembly of these ProM-based pentapeptidic EVH1 ligands through liquid phase peptide synthesis. We initially used pentafluorophenyl (Pfp) active esters for amide bond formation and built up the growing peptide chain from the C- to the N-terminus. Switching to HATU/DIPEA coupling conditions and changing the directionality of the synthesis from the N- to the C-terminus afforded the target ligands with improved overall yields and purity. Employing a Fmoc-protected (instead of the N-acetylated) phenylalanine derivative as N-terminal building block significantly reduced epimerization. In contrast to the originally used solid phase peptide synthesis (SPPS), the developed solution phase method allowed for a facile alteration of the C-terminal ProM unit and the production of various pentapeptidic ligands in an efficient fashion even on a multigram scale.     

Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N-terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it providesvaluable information about the sequence of an unknown peptide. The FAB mass spectra containa number of characteristic ions at low-mass region in addition to the sequence ions at high-massregion. It was found that the ions below m/z 200 are characteristic of the amino acid composition ofthe peptide, from which the amino acid composition of the peptide could be estimated. Additionally,mixture analysis is also discussed.     

Factors affecting immonium ion intensities in the high-energy collision-induced decomposition spectra of peptides

Each amino acid in a peptide has a characteristic immonium ion (H₂N⁺?CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.     

Preparation and study of hydrides of fullerenes C60 and C70

Fullerene hydrides were prepared by hydrogenation of fullerences C₆₀ and C₇₀ using proton transfer from 9,10-dihydroanthracene to fullerene and were studied by mass spectrometry (electron impact, field desorption), IR, UV, and¹H and¹³C NMR spectroscopy. The main product of the hydrogenation of C₆₀ is C₆₀H₃₆, which is sufficiently stable. Hydrogenation of fullerene C₇₀ gives a series of polyhydrides C₇₀H _n (n=36–46), and the main product is C₇₀H₃₆. The dehydrogenation of C₆₀H₃₆ by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is not quantitative and results in the formation of fullerene derivatives along with C₆₀. The comparison of the IR and¹H and¹³C NMR spectral data for solid C₆₀H₃₆ with the theoretical calculations suggests that the fullerene hydride has aT-symmetric structure and contains four isolated benzenoid rings located at tetrahedral positions on the surface of the closed skeleton of the molecule. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya. No. 4, pp. 671–678, April, 1997.     

Tunable Anticancer Activity of Furoylthiourea-Based RuII–Arene Complexes and Their Mechanism of Action

Fourteen new Ru^II–arene (p-cymene/benzene) complexes ( C1 – C14 ) have been synthesized by varying the N-terminal substituent in the furoylthiourea ligand and satisfactorily characterized by using analytical and spectroscopic techniques. Electrostatic potential maps predicted that the electronic effect of the substituents was mostly localized, with some influence seen on the labile chloride ligands. The structure–activity relationships of the Ru–p-cymene and Ru–benzene complexes showed opposite trends. All the complexes were found to be highly toxic towards IMR-32 cancer cells, with C5 (Ru–p-cymene complex containing C₆H₂(CH₃)₃ as N-terminal substituent) and C13 (Ru–benzene complex containing C₆H₄(CF₃) as N-terminal substituent) showing the highest activity among each set of complexes, and hence they were chosen for further study. These complexes showed different behavior in aqueous solutions, and were also found to catalytically oxidize glutathione. They also promoted cell death by apoptosis and cell cycle arrest. Furthermore, the complexes showed good binding ability with the receptors Pim-1 kinase and vascular endothelial growth factor receptor 2, commonly overexpressed in cancer cells.     

Asymmetric epoxidation of electron-poor olefins-V : Influence on stereoselectivity of the structure of poly-α-aminoacids used as catalysts

New poly-α-aminoacids modified at the C orN-terminal groups are synthetised and employed in the asymmetric epoxidation of chalcone. Their influence on the stereoselectivity of the reaction is studied.     

In situ N-phosphorylation of oligopeptides for fast atom bombardment mass spectrometry

Positive ion fast atom bombardment mass spectrometry (FABMS) of in situ N-phosphorylated oligopeptides showed intense quasi-molecular ions together with the successive alkene loss fragment ions, which afford multiple checks of the unequivocal reality of the relative molecular mass of the tested samples. More interesting, in a novel cleavage pattern only the N-phosphoryl fragment ions gave intense peaks, the C-terminal series ions being suppressed. For each of the N-terminal ions, losses of alkenes also occur to provide multiple checks for the existence of these ions. The FABMS of the in situ N- phosphorylated oligopeptides might provide an easily accessible routine method for peptide sequencing.     

An Alkene‐Promoted Borane‐Catalyzed Highly Stereoselective Hydrogenation of Alkynes to Give Z‐ and E‐Alkenes

The stereoselective hydrogenation of alkynes to alkenes is an extremely useful transformation in synthetic chemistry. Despite numerous reports for the synthesis of Z‐alkenes, the hydrogenation of alkynes to give E‐alkenes is still not well resolved. In particular, selective preparation of both Z‐ and E‐alkenes by the same catalytic hydrogenation system using molecular H₂ has rarely been reported. In this paper, a novel strategy of using simple alkenes as promoters for the HB(C₆F₅)₂‐catalyzed metal‐free hydrogenation of alkynes was adopted. Significantly, both Z‐ and E‐alkenes can be furnished by hydrogenation with molecular H₂ in high yields with excellent stereoselectivities. Further experimental and theoretical mechanistic studies suggest that interactions between H and F atoms of the alkene promoter, borane intermediate, and H₂ play an essential role in promoting the hydrogenolysis reaction.