Solid-phase Synthesis Conditions Research Articles

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography.

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5'-deoxythymidine-5'-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to -3.1 °C per phosphoroselenolate) when introduced at the 5'-termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

Production of Meta-Iodobenzilguanidine, <sup>123</sup>I Preparation for Medical Diagnostics

This paper demonstrates a method for production of the radiopharmaceutical meta-iodobenzilguanidin (MIBG) labeled with radionuclide iodine-123 (123I). An experimental installation has been developed and the conditions of solid-phase synthesis of the preparation have been described. The influence of the content of ammonium sulfate in the reaction mixture and the temperature and duration of dry synthesis on the process of labeling has been researched. It has been established experimentally that when the ratio of MIBG and ammonium sulfate is 1:4, it is efficient to carry out synthesis at the temperature of 150°C to rapidly obtain a preparation with radiochemical purity of over 97%. Biomedical tests of the synthesized preparation have been carried out in experimental animals. Its functional suitability for scintigraphic studies has been shown.

Protecting‐Group‐Free Solid‐Phase Anchoring of Polyphenolic C‐Glucosidic Ellagitannins and Synthesis of 1‐Alkylamino‐Vescalagin Derivatives (Eur. J. Org. Chem. 23/2014)

The cover picture shows a protecting-group-free approach for the chemo- and stereoselective amination of the C-glucosidic ellagitannin vescalagin. This bioactive plant polyphenol is a characteristic secondary metabolite of fagaceous woody species such as oak (Quercus sp.). Its benzylic secondary alcohol group is first substituted by a thioether unit, which is then activated by the use of a sulfenyl chloride. The resulting thiosulfonium ion reacts with primary amines to afford β-1-alkylamino-vescalagin derivatives. This article by S. Quideau et al. on p. 4963 ff describes this selective amination of such a structurally unique plant polyphenol by a protocol that was adapted to both solution- and solid-phase synthesis conditions. The background picture shows an oak tree hundreds of years old, which is certainly packed with vescalagin, as the amount of this ellagitannin can reach up to 2 %percnt; by weight of dry heartwood.

ChemInform Abstract: Amphiphilic Systems Based on Polysaccharides Produced by Solid‐phase Synthesis ‐ A Review

AbstractReview: 21 refs.

Incorporation Efficiency of 5'-Azido-5'-Deoxyguanosine into 5'-Terminus of RNA for Preparation of Azido-Functionalized RNA

Although the modern biological systems are by large based on DNA genomes and protein enzymes, RNA plays important roles in many fundamental processes in cells, including regulation of protein biosynthesis, RNA splicing, and retroviral replication, with remarkable features. From this point of view, site-specific substitution and derivatization of RNA can provide powerful tools for elucidating RNA structures and functions. The modification of either the 3'and 5'-termini or an internal position of the oligonucleotides with a primary alkylamine group is a widely used method for introducing additional functional groups to the RNA. In particular, several 5'-modifications of RNA molecules such as sulfhydryl modification for functionalizing the 5'-terminus of RNA by a transcription or kinase reaction have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs, since a unique functional group incorporated into the RNA can be subsequently conjugated to the desired molecule by a selective chemical reaction. However, there is still a need to develop coupling chemistry with high stability and yield to modify RNA and other biomolecules. In addition, the coupling functional groups are ideally required to be stable under aqueous reaction conditions, and the coupling reaction should be highly chemoselective. In this regard, Cu-catalyzed azide-alkyne cycloaddition (CuAAC or click chemistry) to form the triazole version of Huisgen’s [2+3] cycloaddition family may be the best choice, because this reaction only occurs between alkynyl and azido functional groups with high yield, and because the resulting 1,2,3-triazoles are stable at aqueous conditions and high temperature. Indeed, the azide group is one of the most utilized bioorthogonal chemical tags for biomolecule-conjugate experiments because of its small size and inertness to most components in a biological environment. We recently reported a two-step synthetic method for 5'azido-5'-deoxyguanosine (azido-G) by adapting literature procedures (Scheme 1) and its efficiency for click chemistry using 6-heptynoyl p-nitroaniline, in consideration that the click chemistry with rapid reaction between the azide and alkyne groups to form a covalent triazole linkage without cross reacting with other functional groups could be used in bioconjugation chemistry. In the present study, we sought enzymatic methods to incorporate azido groups in RNA and measured incorporation efficiency of azido-G into the 5'terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription. This is important in consideration that a terminal azido group is able to be introduced to the 5'termini of RNA molecules, and that azides are unstable under solid-phase synthesis conditions. A 97-mer single-stranded DNA containing a T7 promoter at the 3'-end (5'CAG GAC TGC TCT CAC TCT CAC GCA CCA AGA AGC TGC CAT TGA TCC CGC TGC

Amphiphilic systems based on polysaccharides produced by solid-phase synthesis − A review

The results of research on polymer blends based on polysaccharides (chitin, chitosan, cellulose, starch, etc.) and variously hydrophilic synthetic polymers produced by reaction mixing in solid state under pressure and shear stresses are summarized. The mechanisms of the processes occurring under solidphase synthesis conditions, the properties of the multicomponent systems formed, and the feasibility of their application for creating new biocompatible and biodegradable materials are discussed.

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImNN). Since the ImNN nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImNN unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55°C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImNN were obtained in good yield.

Covalent Tethering of Organic Functionality to the Surface of Glassy Carbon Electrodes by Using Electrochemical and Solid‐Phase Synthesis Methodologies

Organic linkers such as (N-Boc-aminomethyl)phenyl (BocNHCH2C6H4) and N-Boc-ethylenediamine (Boc-EDA) have been covalently tethered onto a glassy carbon surface by employing electrochemical reduction of BocNHCH2C6H4 diazonium salt or oxidation of Boc-EDA. After removal of the Boc group, anthraquinone as a redox model was attached to the linker by a solid-phase coupling reaction. Grafting of anthraquinone to electrodes bearing a second spacer such as 4-(N-Boc-aminomethyl)benzoic acid or N-Boc-beta-alanine was also performed by following this methodology. The surface coverage, stability and electron transfer to/from the tethered anthraquinone redox group through the linkers were investigated by cyclic voltammetry. The effects of pH and scan rate were studied, and the electron-transfer coefficient and rate constant were determined by using Laviron's equation for the different types of linker. The combination of electrochemical attachment of protected linkers and subsequent modifications under the conditions of solid-phase synthesis provides a very versatile methodology for tailoring a wide range of organic functional arrangements on a glassy carbon surface.

Synthesis of a guanosine phosphoramidite derivative containing a photocleavable protecting group at the O6 position via regioselective protection of the 2'-hydroxy group with a TBDMS group

To construct modified RNA that form A-type duplex with photo-irradiation, a photocleavable a-methyl-2-nitropiperonyl (MeNP) group was introduced at O(6) position of guanosine. A guanosine phosphoramidite derivative containing the MeNP group was synthesized via regioselective 2'-O-protection of 3',5'-O-di(t-butyl)silanediylguanosine with TBDMS group. The MeNP group was found to be stable under conditions of solid-phase synthesis of RNA. The MeNP group was also found to be removable by UV irradiation at wavelength of 365 nm for 10 min.

Preparation of fluorinated linkers: use of 19F NMR spectroscopy to establish conditions for solid-phase synthesis of pilicide libraries.

Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared. One of the linkers was used in combination with gel-phase 19F NMR spectroscopy to develop conditions for solid-phase synthesis of two libraries of pilicides, i.e. compounds designed to inhibit assembly of adhesive pili in uropathogenic Escherichia coli. Attachment to and cleavage from the linker could be monitored based on the chemical shift of the fluorine atom of the linker. In addition, use of the linker as internal standard allowed quantification and optimization of reactions occurring further away from the linker when fluorinated building blocks were employed. Importantly, high-quality 19F NMR spectra were obtained for compounds linked to a TentaGel resin in a standard NMR tube using an ordinary NMR instrument.

Improved Conditions for Solid Phase Synthesis of Oligonucleotides on PS-PEG Copolymers

Polystyrene-polyethylene glycol (PS-PEG) tentacle polymers with loadings of up to 60/<μmol/g were used for standard oligonucleotide synthesis. As these resins are easy to handle and stable under reaction and cleavage conditions they may be used alternatively to controlled pore glass (CPG) as the most commonly used solid support for oligonucleotide synthesis. However, structural and chemical properties of the PS-PEG resins require modified conditions to guarantee syntheses with high coupling efficiencies. Oligonucleotides (ODN ) of various sequences and lengths have successfully been synthesized using HPLC and capillary electrophoresis (CE) for purity control. Additionally, electrospray mass spectrometry (ES-MS) was used for product identification.

Solid-phase synthesis of peptides via alpha, beta-unsaturated amino acids: oxytocin, simultaneous incorporation of amide functions in COOH-terminal and endo-positions.

Oxytocin was synthesized via the solid-phase method using dehydroalanine as pseudo-protecting group of the carboxyl-terminal as well as the omega-amide functions of asparagine and glutamine in endo-position. Starting with Boc-Gly-Dha-resin and using Boc-L-Asp(Dha-NHEt)-OH and Boc-L-Glu(Dha-NHEt)-OH as precursors of asparagine and glutamine, respectively, oxytocin was assembled in stepwise manner under solid phase synthesis conditions. Treatment of the protected [Glu(Dha-NHEt)4, Asp(Dha-NHEt)5]-oxytocin-Dha-resin with 1 n HCl in glacial acetic acid in the presence of 3 equiv. water removed the peptide from the support with the simultaneous formation of the asparagine and glutamine residues to give the protected nonapeptide amide: Cbz-Cys(Bzl)-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2, which was deprotected with sodium in liquid ammonia and then oxidized with diiodoethane to give oxytocin. After purification by gel chromatography and countercurrent distribution, the product displayed the chemical and physical properties and oxytocic activity (533 +/- 301U/mg) of a standard oxytocin preparation.

Synthesis of N-tert.-butoxycarbonyl-( alpha-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide alpha-carboxamides.

N-tert.-butoxycarbonyl-aminomethyl( alpha-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide alpha-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-( rho-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH2Cl2 (less than 1% loss after 10h). Upon treatment for 30 min with HF:anisole(9:1) at 0 degrees, 92% cleavage of glycinamide from Gly-handle-resin was obtained. In a test synthesis of a peptide alpha-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminomethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.

Preparation and properties of Nalpha-9-fluorenylmethyloxycarbonylamino acids bearing tert.-butyl side chain protection.

The preparation is described by several Nalpha-9-fluorenylmethyloxycarbonylamino acids and derivatives bearing tert.-butyl type side-chain protection of amine, carboxyl, guanido, hydroxyl, imidazol, and sulfhydryl functionalities. Physicochemical properties of these compounds have been determined. Cleavage of the Fmoc group by various amines appears to depend on the base strength and steric hindrance. Premature deblocking of Fmoc group by amine on solid support is very slow and may be negligible under the conditions of solid-phase synthesis.