Fullerene-derivatized amino acids: synthesis, characterization, antioxidant properties, and solid-phase peptide synthesis

A series of [60]fullerene-substituted phenylalanine (Baa) and lysine derivatives have been prepared by the condensation of 1,2-(4'-oxocyclohexano)fullerene with the appropriately protected (4-amino)phenylalanine and lysine, respectively. Conversion of the imine to the corresponding amine is achieved by di-acid catalyzed hydroboration. The reduction of the imine is not accompanied by hydroboration of the fullerene cage. The [70]fullerene phenylalanine derivative has also been prepared as have the di-amino acid derivatives. The compounds were characterized by MALDI-TOF mass spectrometry, UV/Vis spectroscopy, and cyclic voltammetry. 1H and 13C NMR spectroscopy allowed the observation of diastereomers. Fullerene-substituted peptides may be synthesized on relatively large scale by solid-phase peptide synthesis. The presence of the C60-substituted amino acid in a peptide has a significant effect on the secondary structures and self-assembly properties of peptides as compared to the native peptide. The antioxidant assay of Baa and a Baa-derived anionic peptide was determined to be significantly more potent than Trolox.     

Electrostatically self-assembled azides on zinc sulfide nanoparticles as multifunctional nanoprobes for peptide and protein analysis in MALDI-TOF MS

A simple method to synthesize electrostatically self-assembled azides on zinc sulfide nanoparticles (ZnS-N₃ NPs) was described and then it was further applied as a multifunctional nanoprobe such as enriching, desalting, accelerating and separation-/washing free nanoprobes for rapid analysis of peptides and proteins and microwave assisted tryptic digested proteins in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ZnS-N₃ NPs were characterized by UV-vis, FT-IR, SEM and TEM spectroscopy. The ZnS-N₃ NPs can effectively enrich signal intensities for 2-10 times for various peptides and proteins including HW6, insulin, ubiquitin, cytochrome c, lysozyme, myoglobin and bovine serum albumin (BSA) in MALDI-TOF MS. Furthermore, we also demonstrated that the ZnS-N₃ NPs can serve as accelerating probes for microwave assisted tryptic digestion of proteins in MALDI-TOF MS. The applicability of the present method on complex sample analysis such as milk proteins from cow milk and ubiquitin and ubiquitin like proteins from oyster mushroom were also demonstrated.     

The peptide toxin of the cyanobacterium Microcystis aeruginosa PCC 7941. Isolation and analysis by nuclear magnetic resonance and fast atom bombardment mass spectroscopy

Toxin was obtained from the cyanobacterium Microcystis aeruginosa PCC7941 by extracting freeze-dried cells with water-saturated, acidified n-butanol, diethyl ether-water distribution, reversed-phase thin-layer chromatography and silica high-performance liquid chromatography (HPLC). Two toxic peptide fractions resulted from HPLC. One of these fractions was analyzed by UV and NMR spectroscopy, amino acid analysis and fast atom bombardment mass spectroscopy. The following amino acid analysis and fast atom bombardment mass spectroscopy. The following amino acids were identified: beta-methyl-Asp, Thr, Glu, Ala, Val, Leu, Phe, Arg, N-methyldehydro-Ala and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. Yet the mass spectroscopic data showed that the fraction was still composed of several, most likely cyclic peptides that did not stain with ninhydrin.     

Simultaneous Analysis of Phosphorylated Peptides by MALDI-TOF-MS

Six peptides with various phosphorylation sensitivities for protein kinase A (PKA) were used for the simultaneous analysis of phosphorylated peptides using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. The mixture of six peptides was reacted with PKA and was analyzed by MALDI-TOF mass spectrometry. The intensity of all peaks except one phosphorylated peptide peak was very low (<20%). Moreover, we examined whether the addition of diammonium citrate to CHCA matrix at concentrations of 1–20 mg mL^?1 can increase the peak intensity of peptides and phosphorylated peptides. The addition of diammonium citrate increased the peak intensity of peptides and phosphorylated peptides, but an increase in the intensity was unsatisfactory. Our study strongly suggests that MALDI-TOF mass spectrometry is not suitable for the simultaneous analysis of phosphorylated peptides.     

Preparation and characterization of 4-amino-2-anilinopyridine and its chlorodiruthenium(III,II) complex

A new bridging agent, 4-amino-2-anilinopyridine (aap), was synthesized and used as an equatorial ligand in the preparation of a diruthenium complex (4,0) Ru₂(aap)₄Cl. Both the ligand and the diruthenium complex were characterized by thermal analysis, MALDI-TOF mass spectrometry, IR and NMR (¹H and ¹³C) spectroscopy, and X-ray crystallography. The structural analysis revealed that the complex existed as a (4,0) isomer, in which the amino group on the pyridyl moiety was not involved in chemical bonding.     

Accurate mass measurement of low molecular weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the accurate measurement of mass of low molecular weight compounds (smaller than 1500 Da), a linear peptide, two types of cyclic depsipeptides, a polyhydroxy-macrocyclic lactone, and two prenylated flavonoids, with delayed extraction in the reflector mode. The performance of the MALDI-TOF instrument was less than those of fast atom bombardment and Fourier-transform ion cyclotron resonance mass spectrometry instruments and insufficient to give acceptable accuracy for literature reporting. Nevertheless, when combined with NMR spectrometry and/or amino acid analysis to give information on the numbers of carbon atoms and index of hydrogen deficiency, MALDI was useful for determination of the elemental composition of the low molecular weight compounds available in small quantities.     

Gel-phase 19F NMR spectral quality for resins commonly used in solid-phase organic synthesis; a study of peptide solid-phase glycosylations

The spectroscopic properties for seven different commercial resins used in solid-phase synthesis were investigated with (19)F NMR spectroscopy. A fluorine-labeled dipeptide was synthesized on each resin, and the resolution of the (19)F resonances in CDCl(3), DMSO-d(6), benzene-d(6) and CD(3)OD were measured with a conventional NMR spectrometer, i.e. without using magic angle spinning. In general, resins containing poly(ethylene glycol) chains (ArgoGel, TentaGel and PEGA) were found to be favorable for the (19)F NMR spectral quality. Three serine containing tri-, penta-, and heptapeptides were then prepared on an ArgoGel resin functionalized with a fluorine-labeled linker. The resin bound peptides were glycosylated utilizing a thiogalactoside glycosyl donor carrying fluorine-labeled protective groups. Monitoring of the glycosylations with gel-phase (19)F NMR spectroscopy allowed each glycopeptide to be formed in similar 80% yield, using a minimal amount of glycosyl donor (3 x 2 equivalents). In addition, it was found that the glycosylation yields were independent of peptide length.     

Identification of<Emphasis Type="Italic"> N</Emphasis>-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.Abbreviations amu atomic mass units - AspN endoproteinase AspN - CAM cell adhesion molecule - ESI electrospray ionization - FTICR Fourier transform–ion cyclotron resonance - IgSF immunoglobulin superfamily - MALDI-TOF matrix-assisted laser desorption ionization–time of flight - MS mass spectrometry - NCAM neural cell adhesion molecule - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase - PSA polysialic acid - TFA trifluoroacetic acid     

Optimized protocol for synthesis of cyclic gramicidin S: starting amino acid is key to high yield

[structures: see text] A simple and highly efficient Fmoc solid-phase protocol for synthesizing the antimicrobial decapeptide gramicidin S and various labeled analogues is presented. When preparing the linear precursor peptides (1a-e), a systematic permutation of the starting amino acid within the cyclic sequence gave different yields between 51% and 93%. Also the subsequent step of cyclization gave widely diverging yields between 26% and 74%, depending again on the starting amino acid. The ease of cyclization was found to correlate with the tendency of the respective linear precursor peptide to assume a preorganized conformation, as observed by circular dichroism. The overall yield is thus critically dependent on the starting amino acid and can be raised from 20% to 70% using (D)Phe. The choice of coupling agent and its counterion was found to play only a marginal role. Irrespective of being able to assume a preorganized conformation, none of the linear precursor peptides exhibited any antimicrobial or hemolytic activity. Using the optimized protocol, which involves only simple Fmoc-couplings and requires no intermittent purification steps, several gramicidin S analogues (3-8) containing 19F-labeled phenylglycine derivatives and/or 15N-labeled amino acids were synthesized for solid-state NMR structure analysis.     

微孔磷酸铝AIPO4-HDA的热分解过程研究

采用差热-热重-质谱(TG-DTA-MS)、粉末X射线衍射(XRD)、红外光谱(FTIR)和固体核磁(Solid-State-MAS-NMR)等技术详细地研究了微孔磷酸铝晶体AlPO4-HDA中模板剂的热分解过程.结果表明,该模板剂的热分解分3步进行:第一步是模板剂和无机骨架之间的部分氢键断裂;第二步为模板剂的Hof-mann降解反应和β-消除反应;第三步是残留积碳的氧化分解反应.固体MASNMR的研究结果表明,随着模板剂的脱出.无机骨架中铝和磷的配位状态发生了变化.     

Oxa[7]superhelicene: A π‐Extended Helical Chromophore Based on Hexa‐peri‐hexabenzocoronenes

A novel π‐extended “superhelicene” based on hexa‐peri‐hexabenzocoronenes (HBCs) has been synthesized by an efficient four‐step synthetic procedure starting from diphenyl ether. Comprehensive structural analysis of the helicene was performed by NMR spectroscopy and mass spectrometry measurements together with X‐ray analysis. Physicochemical analysis of the superhelicene and suitable HBC references revealed it had outstanding fluorescent features with quantum yields of over 80 %.     

Racemic beta-sheets as templates for the generation of homochiral (isotactic) peptides from aqueous solutions of (RS)-valine or -leucine N-carboxy- anhydrides: relevance to biochirogenesis

As part of our program on biochirogenesis of homochiral peptides from racemic precursors, we report the feasibility of obtaining peptides with homochiral sequences composed of up to 25 residues of the same handedness in the polymerization of racemic valine or leucine N-carboxyanhydrides in aqueous solutions, as initiated by amines. The composition of the oligopeptides was determined by MALDI-TOF mass spectrometry, and the sequences of some of the heterochiral diastereoisomers were studied by MALDI-TOF MS/MS performed on samples in which the S enantiomers of the monomer were tagged with deuterium atoms. The process comprises several steps: 1) a Markov mechanism of asymmetric induction in the early stages of the polymerization yields libraries of racemic oligopeptides enriched with isotactic diastereoisomers, together with oligopeptide sequences containing enantiomeric blocks of homochiral residues; 2) the short peptides self-assemble into racemic colloidal architectures that serve as regio-enantioselective templates in the ensuing process of chain elongation; 3) homochiral residues of the amino acids located at the periphery of these colloidal aggregates exert efficient enantioselection, which results in the formation of long isotactic oligopeptides. The final diastereoisomeric distribution of the peptides depends upon the composition of the templates, which is determined by the concentration of the initiator. The racemic mixtures of isotactic peptides can be desymmetrized by using enantiopure methyl esters of alpha-amino acids as initiators.     

Synthesis of a nonavalent mannoside glycodendrimer based on pentaerythritol

A nonavalent glycodendrimer bearing terminal alpha-d-mannopyranoside units has been synthesized with a convergent approach. Terminal trivalent mannoside dendrons bearing p-halophenyl ethers were prepared by glycosylation of pentaerythritol derivatives having three 2-hydroxyethyl ether substituents. Two efficient routes were developed for the synthesis of the pentaerythritol-based core (17), which has three terminal propargyl ethers. Conditions were found under which the triple Sonogashira coupling reaction of the dendron and the tri-O-propargyl ether (17) proceeded efficiently. The product was deprotected and it and precursors were fully characterized by NMR spectroscopy and FT-ICR mass spectrometry.     

An efficient synthesis of a novel analog of octreotide with an unnatural l-lysine-like tetrazolyl amino acid

A new cyclic octreotide-like octapeptide was prepared by incorporation of an unnatural tetrazolyl amino acid, an analog of Fmoc-l-lysine, into the peptide chain. The new tetrazolyl amino acid, (S)-2-{[2-(9H-fluoren-9-yl)acetoxy]amino}-6-(1H-tetrazol-1-yl)hexanoic acid, was obtained by azidation of Fmoc-l-lysine trifluoroacetate with sodium azide in the presence of triethyl orthoformate in glacial acetic acid. The linear peptide sequence was prepared using efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS). Cyclization of the octapeptide was carried out via oxidation with iodine. The structure and purity of the cyclic octapeptide were confirmed by LC–MS, MALDI-TOF MS/MS analysis as well as 1D/2D ¹H and ¹³C NMR spectroscopy.     

An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Au36(SPh)23 nanomolecules

A new core size protected completely by an aromatic thiol, Au(36)(SPh)(23), is synthesized and characterized by MALDI-TOF mass spectrometry and UV-visible spectroscopy. The synthesis involving core size changes is studied by MS, and the complete ligand coverage by aromatic thiol group is shown by NMR.     

Lobocyclamides A-C,lipopeptides from a cryptic cyanobacterial mat containing Lyngbya confervoides

The structures of lipopeptides lobocyclamides A (1), B (2), and C (3) were solved using a combination of mass spectrometry, 2D NMR spectroscopy, and degradative analysis. Lobocyclamides B and C are the first peptides reported with the unusual amino acid 4-hydroxythreonine and also incorporate the rare homologous long-chain beta-amino acids 3-aminooctanoic acid and 3-aminodecanoic acid, respectively. The absolute configurations of the amino acid residues in each compound were assigned, after acid hydrolysis, by either direct chiral HPLC comparison with authentic standards or by prior derivatization by Marfey's method and reversed-phase HPLC. Both compounds exhibited moderate antifungal activity against a panel of Candida spp., including two fluconazole-resistant strains. When tested as a mixture, lobocyclamides A and B displayed synergistic in vitro antifungal activity, a phenomenon noted earlier for the related peptides laxaphycins A and B.     

Study of the interaction of salicyl aldehydes with epichlorohydrin: a simple, convenient, and efficient method for the synthesis of 3,6-epoxy[1,5]dioxocines

An efficient synthetic method for 3,6-epoxy[1,5]dioxocines via the condensation of salicyl aldehydes and epichlorohydrin, using benzyl triethylammonium chloride as catalyst, is described. The use of neutral or electron-deficient salicyl aldehydes in all cases was found to give 3,6-epoxy[1,5]dioxocine derivatives in good yields. The structural features were resolved by IR, NMR spectroscopy, mass spectrometry, and ultimately proved by X-ray crystallographic analysis.     

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).