Capillary electrochromatography of peptides on microfabricated poly(dimethylsiloxane) chips modified by cerium(IV)-catalyzed polymerization

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.     

Synthesis of the C11-C29 fragment of amphidinolide F

[reaction: see text] An efficient synthesis of the C(11)-C(29) fragment 31 of amphidinolide F has been accomplished via a diastereoselective [3 + 2]-annulation reaction of allylsilane 5 and ethyl glyoxylate to prepare the key tetrahydrofuran 15 and a highly stereoselective methyl ketone aldol reaction to generate the C(11)-C(16) segment.     

Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting.     

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.     

Synthesis and anti-tumor activity of β-C-glycoside analogs of the immunostimulant KRN7000

A ring-closing metathesis approach was employed for the synthesis of a β-C-glycoside analog of the immunostimulant KRN7000. The protected C-glycosyl amino acid derivative 18 was converted to amino-olefin 20, and osmylation served to install the diol unit as a mixture of separable syn and anti isomers. Deprotection to the hydroxy-amine 21 was followed by N-acylation and debenzylation to deliver the target compound 5.     

Cyclodimerization versus Polymerization of Methyl Methacrylate Induced by N‐Heterocyclic Carbenes: A Combined Experimental and Theoretical Study

The activation behavior of two N‐heterocyclic carbenes (NHCs), namely, 1,3‐bis(isopropyl)imidazol‐2‐ylidene(NHCiPr) and 1,3‐bis(tert‐butyl) imidazol‐2‐ylidene (NHCtBu), as organic nucleophiles in the reaction with methyl methacrylate (MMA) is described. NHCtBu allows the polymerization of MMA in DMF at room temperature and in toluene at 50 °C, whereas NHCiPr reacts with two molecules of MMA, forming an unprecedented imidazolium–enolate cyclodimer (NHCiPr/MMA=1:2). It is proposed that the reaction mechanism occurs by initial 1,4‐nucleophilic addition of NHCiPr to MMA, generating a zwitterionic enolate 2 , followed by addition of 2 to a second MMA molecule, forming a linear imidazolium–enolate 3 (NHCiPr/MMA=1:2). Proton transfer, generating intermediate 5 , followed by cyclization and release of methanol yielded the aforementioned zwitterionic cyclodimer 1:2 adduct 7 , the molecular structure of which has been established by NMR spectroscopy, X‐ray diffraction, and mass spectrometry. This unexpected difference between NHCtBu and NHCiPr in the reaction with MMA (polymerization and cyclodimerization, respectively) can be rationalized by using DFT calculations. In particular, the nature of the NHC strongly influences the cyclodimerization pathway, the cyclization of 5 and the release of methanol are the discriminating step and limiting step, respectively. In the case of NHCtBu, both steps are strongly disfavoured compared with that of NHCiPr (energetic difference of around 14 and 9 kcal mol^?1, respectively), preventing the cyclization mechanism from a kinetic viewpoint. Moreover, addition of a third molecule of MMA in the polymerization pathway results in a lower activation barrier than that of the limiting step in the cyclodimerization pathway (difference of around 14 kcal mol^?1), in agreement with the formation of polymethyl methacrylate (PMMA) by using NHCtBu as nucleophile.     

From the N‐Heterocyclic Carbene‐Catalyzed Conjugate Addition of Alcohols to the Controlled Polymerization of (Meth)acrylates

Among various N‐heterocyclic carbenes (NHCs) tested, only 1,3‐bis(tert‐butyl)imidazol‐2‐ylidene (NHC^tBu) proved to selectively promote the catalytic conjugate addition of alcohols onto (meth)acrylate substrates. This rather rare example of NHC‐catalyzed 1,4‐addition of alcohols was investigated as a simple means to trigger the polymerization of both methyl methacrylate and methyl acrylate (MMA and MA, respectively). Well‐defined α‐alkoxy poly(methyl (meth)acrylate) (PM(M)A) chains, the molar masses of which could be controlled by the initial [(meth)acrylate]₀/[ROH]₀ molar ratio, were ultimately obtained in N,N‐dimethylformamide at 25 °C. A hydroxyl‐terminated poly(ethylene oxide) (PEO‐OH) macro‐initiator was also employed to directly access PEO‐b‐PMMA amphiphilic block copolymers. Investigations into the reaction mechanism by DFT calculations revealed the occurrence of two competitive concerted pathways, involving either the activation of the alcohol or that of the monomer by NHC^tBu.     

Comparative metabolite profiling of carboxylic acids in rat urine by CE‐ESI MS/MS through positively pre‐charged and 2H‐coded derivatization

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE‐MS and a method for positively pre‐charged and ²H‐coded derivatization. Novel derivatizing reagents, N‐alkyl‐4‐aminomethyl‐pyridinum iodide (alkyl=butyl, butyl‐d9 or hexyl), containing quaternary amine and stable‐isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99–1.32% RSD of migration time) and eliminated problems with capillary coating known in CE‐MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N‐butyl‐4‐aminomethyl‐pyridinum iodide (BAMP) and N‐hexyl‐4‐aminomethyl‐pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE‐MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.     

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.