ABSTRACT
A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.
Subject(s)
Molecular Biology/methods , Organophosphorus Compounds/pharmacology , Spectrometry, Mass, Electrospray Ionization/methods , Acetonitriles/chemistry , Acetonitriles/pharmacology , Lithium Chloride/chemistry , Lithium Chloride/pharmacology , Macromolecular Substances , Mass Spectrometry/methods , Models, Chemical , Organophosphorus Compounds/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , Reproducibility of ResultsABSTRACT
A method was developed for the synthesis of oligonucleotide-cationic peptide conjugates in solution phase by disulfide bond formation. Precipitation was avoided by the easily removable triethylammonium trifluoroacetate (TEATFAc) salt which served at the same time as a buffer of the reaction mixture. The fast and high yielding disulfide bond formation was due to the Npys thio protecting and activating group of Cys. A solution of the free 5-thiol modified oligonucleotide obtained from Poly-Pak purification was used for conjugation.
Subject(s)
Cations/chemistry , Molecular Biology/methods , Oligonucleotides/chemistry , Peptides/chemistry , Base Sequence , Buffers , Chromatography, High Pressure Liquid , Disulfides/chemistry , Models, Chemical , Molecular Sequence Data , Salts/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time FactorsABSTRACT
The synthesis of free 5'-thiol-modified oligonucleotides using a 4,4',4''-trimethoxytrityl (TMTr)-protected linker and standard Poly-Pak purification has been described.
Subject(s)
Oligodeoxyribonucleotides/chemical synthesis , Sulfhydryl Compounds , Base Sequence , Indicators and Reagents , Oligodeoxyribonucleotides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Sensitivity of ESI-MS analysis of crude PNAs is enhanced using their pyridinium or thiazolium derivatives. Identification of the molecular ion of the product is easier when the label contains bromine, based on the isotope distribution. Study of side reactions, occurred upon the synthesis and/or cleavage, is simple with labelling. Sequencing of non-polar peptides is clear as only a(n) type ions can be observed during their MS/MS analysis.
Subject(s)
Oligopeptides/chemistry , Peptide Nucleic Acids/chemistry , Pyridines , Thiazoles , Indicators and Reagents , Oligopeptides/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
A new, crystalline 5'-thiol modifier phosphoramidite monomer (3), suitable for DNA synthesis, has been prepared. This monomer has been built into an oligonucleotide using the standard protocol. After cleavage, purification and removal of the trityl group with Ag(+), a free 5'-thiol terminal oligonucleotide (15) has been obtained which was subsequently coupled to a cysteine derivative via a disulfide bridge to afford conjugate 16.
Subject(s)
Chromans/chemical synthesis , Chromans/urine , Cysteine/chemistry , Disulfides/chemistry , Oligonucleotides/chemical synthesis , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/chemical synthesis , Pentanoic Acids/chemical synthesis , Pentanoic Acids/urine , Sulfhydryl Compounds/chemistry , Chromatography, High Pressure Liquid , Cysteine/analogs & derivatives , Oligonucleotides/isolation & purification , Spectrometry, Mass, Electrospray IonizationABSTRACT
[reaction: see text]. Ammonium tert-butyl H-phosphonate was used for the phosphorylation of Tyr- and Ser-containing peptides synthesized by an Fmoc strategy. This reaction, leading to a monoprotected peptide phosphate, was found to be highly efficient and generally applicable. Moreover, the method employed avoids undesired side reactions during chain elongation (pyrophosphate formation and beta-elimination catalyzed by piperidine).
Subject(s)
Organophosphonates/chemistry , Phosphopeptides/chemical synthesis , Serine/chemistry , Tyrosine/chemistry , Chromatography, High Pressure Liquid , Phosphopeptides/chemistry , PhosphorylationABSTRACT
In contemporary solid-phase synthesis of oligonucleotides, phosphoramidites containing O-beta-cyanoethyl and N,N -diisopropyl groups are the most widespread monomer units. The N,N -diisopropyl phosphoramidite group can be activated by mild acidic treatment and then it easily reacts with nucleophiles (alcohols, water, etc.) to furnish the required phosphodiester linkage efficiently and cleanly. Owing to these properties, these compounds cannot be investigated using classical electrospray ionization. Their mass spectometric analysis is further hampered by the fact that they are often transiently protected with acid-sensitive groups (4, 4'-dimethoxytrityl, 4-monomethoxytrityl or trityl), which give intense signals in the spectra. Nanoelectrospray measurements from non-aqueous solvents (e.g. acetonitrile, methanol, tetrahydrofuran) were carried out in order to eliminate the nucleophilic water. Different types of alkali metal salts were used to form adduct ions. Among these salts, lithium chloride was found to be the most suitable for the analysis of amidites. Fairly abundant [M+Li](+) and [M+Cl](-) ions are formed in the positive and negative ion mode, respectively. These ions represent the base peaks in most cases whereas the intensities of the peaks corresponding to the protecting group are reduced by approximately 20%. This method is a powerful tool for the mass spectrometric identification of phosphoramidites. Copyright 1999 John Wiley & Sons, Ltd.
