The role of the maleimide ring system on the structure-activity relationship of showdomycin.

Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. In order to understand structure-activity relationships of showdomycin, we synthesized a series of derivatives with modifications in the maleimide ring at the site of alkylation to moderate its reactivity. The showdomycin congeners were designed to retain the planarity of the base ring system to allow Watson-Crick base pairing and preserve the nucleosidic character of the compounds. Consequently, we synthesized triphosphates of showdomycin derivatives and tested their activity against RNA polymerases. Bromo, methylthio, and ethylthio derivatives of showdomycin were incorporated into RNA by bacterial and mitochondrial RNA polymerases and somewhat less efficiently by the eukaryotic RNA polymerase II. Showdomycin derivatives acted as uridine mimics and delayed further extension of the RNA chain by multi-subunit, but not mitochondrial RNA polymerases. Bioactivity profiling indicated that the mechanism of action of ethylthioshowdomycin was altered, with approximately 4-fold reduction in both cytotoxicity against human embryonic kidney cells and antibacterial activity against Escherichia coli. In addition, the ethylthio derivative was not inactivated by medium components or influenced by addition of uridine in contrast to showdomycin. The results explain how both the maleimide ring and the nucleoside nature contribute to the bioactivity of showdomycin and demonstrates for the first time that the two activities can be separated.

Controlled Monofunctionalization of Molecular Spherical Nucleic Acids on a Buckminster Fullerene Core.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs.

Expanding the Scope of the Cleavable N-(methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates.

Oligonucleotides modified by a 2'-deoxy-2'-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-ß-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a ß-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to ß-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.

Kinetic and NMR spectroscopic study of the chemical stability and reaction pathways of sugar nucleotides.

The alkaline cleavage of two types of sugar nucleotides has been studied by 1H and 31P NMR in order to obtain information on the stability and decomposition pathways in aqueous solutions under alkaline conditions. The reaction of glucose 1-UDP is straightforward, and products are easy to identify. The results obtained with ribose 5-UDP and ribose 5-phosphate reveal, in contrast, a more complex reaction system than expected, and the identification of individual intermediate species was not possible. Even though definite proof for the mechanisms previously proposed could not be obtained, all the spectroscopic evidence is consistent with them. Results also emphasise the significant effect of conditions, pH, ionic strength, and temperature, on the reactivity under chemical conditions.

Immobilized Carbohydrates for Preparation of 3'-Glycoconjugated Oligonucleotides.

A detailed protocol for preparation 3'-glycoconjugated oligonucleotides is described based on one-pot immobilization of 4,4'-dimethoxytrityl-protected carbohydrates to a solid support followed by on-support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post-synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate-modified supports using conventional phosphoramidite chemistry produces 3'-glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 1-O-tert-butyldimethylsilyl-6-O-(4,4'-dimethoxytrityl)-ß-D-glucose Basic Protocol 2: Synthesis of 6-O-dimethoxytrityl-2,3,1',3',4',6'-hexa-O-benzoylsucrose Basic Protocol 3: Synthesis of 6″-O-dimethoxytrityl-N-trifluoroacetyl-protected aminoglycosides Basic Protocol 4: Synthesis of 3-O-dimethoxytrityl-propyl ß-D-galactopyranoside Basic Protocol 5: Synthesis of trivalent N-acetyl galactosamine cluster Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates.

Conjugation of Oligonucleotides to Peptide Aldehydes via a pH-Responsive N-Methoxyoxazolidine Linker.

The formation of N-methoxyoxazolidines in the preparation of oligonucleotide-peptide conjugates was evaluated. The reaction occurred between unprotected 2'-N-(methoxy)amino-modified oligonucleotides and peptide aldehydes in reasonable yields when isolated. The reaction is reversible under slightly acidic conditions, and it is pH-responsive. The rate and the equilibrium constant may be varied with structurally different aldehydes, allowing an optimization of the ligation and cleavage rate of the resultant conjugates. Therefore, this concept can be considered a cleavable linker.

Culturally and linguistically diverse healthcare students' experiences of the clinical learning environment and mentoring: A qualitative study.

Development of educators' and students' global competence in higher education is increasingly important due to internationalization. Internationalization significantly influences healthcare education through an increase in the mobility of students. When conducting clinical practice in healthcare education, culturally and linguistically diverse healthcare students face the challenges of having limited learning opportunities and social isolation. Further investigation is required of students' experiences in this area while asking them to share their experiences. This study aimed to describe students' experiences of clinical learning environment and mentoring. A qualitative research design was used during 2013-2016. Data were collected from 133 culturally and linguistically diverse healthcare students, including exchange students and students studying in English language-taught degree programmes at eight Universities of Applied Sciences in Finland. Data were analysed using inductive content analysis. Students' experiences were related to their mentors' competence in mentoring, culturally diverse pedagogical atmosphere, and aspects of diversity that influence clinical learning. Students reported that they had experienced social isolation, discrimination, bullying, sexual harassment and prejudice during their clinical placements. These issues related to mistreatment of students need to be addressed. Also, structured clinical environments should be developed in which competent mentors assist students in reaching their clinical placement goals.

DNA-Templated N(Me)-Alkoxyamine Glycosylation.

The potential of N(Me)-alkoxyamine glycosylation as a DNA-templated ligation has been studied. On a hairpin stem-template model, a notable rate enhancement and an increased equilibrium yield are observed compared to the corresponding reaction without a DNA catalyst. The N-glycosidic connection is dynamic at pH 5, whereas it becomes irreversible at pH 7. The N(Me)-alkoxyamine glycosylation may hence be an attractive pH controlled reaction for the assembly of DNA-based dynamic products.

Metal ion-promoted cleavage of nucleoside diphosphosugars: a model for reactions of phosphodiester bonds in carbohydrates.

Cleavage of five different nucleoside diphosphosugars has been studied in the presence of Cu(2+) and Zn(2+) complexes. The results show that metal ion catalysts promote the cleavage via intramolecular transesterification whenever a neighbouring HO group can adopt a cis-orientation with respect to the phosphate. The HO group attacks the phosphate and two monophosphate products are formed. If such a nucleophile is not available, Cu(2+) complexes are able to promote a nucleophilic attack of an external nucleophile, e.g. a water molecule or metal ion coordinated HO ligand, on phosphate. With the Zn(2+) complex, this was not observed.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

A procedure for the preparation and isolation of nucleoside-5'-diphosphates.

Tris[bis(triphenylphosphoranylidene)ammonium] pyrophosphate (PPN pyrophosphate) was used in the SN2 displacements of the tosylate ion from 5'-tosylnucleosides to afford nucleoside-5'-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5'-diphosphates from crude reaction mixtures.

Phosphate analogues in the dissection of mechanism.

Phosphoryl group transfer is central to genetic replication, cellular signalling and many metabolic processes. Understanding the mechanisms of phosphorylation and phosphate ester and anhydride cleavage is key to efforts towards biotechnological and biomedical exploitation of phosphate-handling enzymes. Analogues of phosphate esters and anhydrides are indispensable tools, alongside protein mutagenesis and computational methods, for the dissection of phosphoryl transfer mechanisms. Hydrolysable and non-hydrolysable phosphate analogues have provided insight into the nature and sites of phosphoryl transfer processes. Kinetic isotope effects and crystallography using transition state analogues have painted more detailed pictures of transition states and how enzymes work to stabilise them.

There is no universal mechanism for the cleavage of RNA model compounds in the presence of metal ion catalysts.

The transesterification of uridine 3'-phosphodiesters with a wide range of leaving group alcohols has been studied in the presence of monometallic and bimetallic complexes. The catalysis of isomerization of the phosphodiester bond was studied with a nucleoside 3'-phosphonate as a substrate. The results obtained are consistent with a step-wise mechanism, where metal ions are able to enhance both the nucleophilic attack and the departure of the leaving group. The mechanism of the catalysis depends on the acidity of the catalyst and of the leaving group alcohol: a change from general base catalysis to general acid catalysis is proposed. Catalysis of the isomerization requires efficient stabilization of the phosphorane by strong interactions with the catalyst. Catalytic strategies utilised by bimetallic complexes are also briefly discussed.

The mechanism of cleavage and isomerisation of RNA promoted by an efficient dinuclear Zn2+ complex.

The cleavage and isomerisation of uridine 3'-alkylphosphates was studied in the presence of a dinuclear Zn(2+) complex, 3. The rate acceleration of the cleavage by 1 mM 3 is approximately 10(6)-fold under neutral conditions. Most remarkably, the complex also promotes the isomerisation of phosphodiester bonds, although the rate-enhancement is more modest: under neutral conditions complex 3 (1 mM) catalyses isomerisation by about 500-fold. The observation of this reaction shows that the reactions of these substrates catalysed by 3 proceed through a stepwise mechanism involving an intermediate phosphorane. A ß(lg) value of -0.92 was determined for the 3-promoted cleavage reaction, and modest kinetic solvent deuterium isotope effects ranging from 1.5 to 2.8 were observed. Isomerisation was less sensitive to the nature of the esterifying group, with a ß value of -0.5, and the kinetic solvent deuterium isotope effects were less than 1.5. Most of these characteristics of the 3-promoted cleavage are very similar to those for the cleavage of nucleoside 3'-phosphotriesters. These data are explained by a mechanism in which the complex primarily acts as an electrophilic catalyst neutralising the charge on the phosphate and stabilising an intermediate phosphorane, with general acid catalysis promoting the cleavage reaction. In contrast to the behaviour of triesters, isomerisation is significantly slower than cleavage; this suggests that the changes in geometry that occur during isomerisation lead to a much less stable complex between 3 and the phosphorane intermediate.