Synthesis and Base Pairing Properties of 1′,5′‐Anhydro‐L‐Hexitol Nucleic Acids (L‐HNA)

Oligonucleotides composed of 1′,5′‐anhydro‐arabino‐hexitol nucleosides belonging to the L series (L ‐HNA) were prepared and preliminarily studied as a novel potential base‐pairing system. Synthesis of enantiopure L ‐hexitol nucleotide monomers equipped with a 2′‐(N⁶‐benzoyladenin‐9‐yl) or a 2′‐(thymin‐1‐yl) moiety was carried out by a de novo approach based on a domino reaction as key step. The L oligonucleotide analogues were evaluated in duplex formation with natural complements as well as with unnatural sugar‐modified oligonucleotides. In many cases stable homo‐ and heterochiral associations were found. Besides T_m measurements, detection of heterochiral complexes was unambiguously confirmed by LC‐MS studies. Interestingly, circular dichroism measurements of the most stable duplexes suggested that L ‐HNA form left‐handed helices with both D and L oligonucleotides.     

Effects of Six‐Membered Carbohydrate Rings on Structure,Stability, and Kinetics of G‐Quadruplexes

We have evaluated the conformational, thermal, and kinetic properties of d(TGGGGT) analogues with one or five of the ribose nucleotides replaced with the carbohydrate residues hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), or altritol nucleic acid (ANA). All of the modified oligonucleotides formed G‐quadruplexes, but substitution with the six‐membered rings resulted in a mixture of G‐quadruplex structures. UV and CD melting analyses showed that the structure formed by d(TGGGGT) modified with HNA was stabilized whereas that modified with CeNA was destabilized, relative to the structure formed by the unmodified oligonucleotide. Substitution at the fourth base of the G‐tract with ANA resulted in a greater stabilization effect than substitution at the first G residue; substitution with five ANA residues resulted in significant stabilization of the G‐quadruplex. A single substitution with CeNA at the first base of the G‐tract or five substitutions with HNA resulted in striking deceleration or acceleration of G‐quadruplex formation, respectively. Our results shed light on the effect of the sugar moiety on the properties of G‐quadruplex structures.     

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Functionalization of RNA at the 5′‐terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5′‐nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5′‐phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5′‐end of RNA.     

Study on the CID Fragmentation Pathways of Deprotonated 4’-Monophosphoryl Lipid A

Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MSⁿ), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4’-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3’ primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2’ secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2’ primary fatty acids are eliminated as an amide and ketene, respectively; (v) the ^0,2A₂ cross-ring fragment contains a four-membered ring (oxetanose); (vi) the ^0,4A₂ ion is consecutively formed from the ^0,2A₂ ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H₂PO₄⁻ and PO₃⁻ are associated with the formation of sugar epoxide. An understanding of the relation between ^0,2A₂ and ^0,4A₂-type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3’). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.     

3′:5′‐Cyclic nucleotides: two sodium salts of cdTMP

3′:5′‐Cyclic nucleotides play an outstanding role in signal transduction at the cellular level but, in spite of comprehensive knowledge of the biological role of cyclic nucleotides, their structures are not established fully. Two hydrated sodium salts of thymidine 3′:5′‐cyclic phosphate (cdTMP, C₁₀H₁₂N₂O₇P), namely sodium thymidine 3′:5′‐cyclic phosphate heptahydrate, Na⁺·C₁₀H₁₂N₂O₇P⁻·7H₂O or Na(cdTMP)·7H₂O, (I), and sodium thymidine 3′:5′‐cyclic phosphate 3.7‐hydrate, Na⁺·C₁₀H₁₂N₂O₇P⁻·3.7H₂O or Na(cdTMP)·3.7H₂O, (II), have been obtained in crystalline form and structurally characterized, revealing one nucleotide in the asymmetric unit of (I) and eight different nucleotides in (II). All the cyclic nucleotide anions adopt a similar conformation with regard to nucleobase orientation, sugar conformation and 1,3,2‐dioxaphosphorinane ring puckering. In (I), no direct inter‐nucleotide hydrogen bonds are present, and adjacent nucleotide anions interact via water‐mediated and Na⁺‐mediated contacts. In contrast, in (II), direct thymine–phosphate N—H...O inter‐nucleotide hydrogen bonds occur and these are assisted by numerous inter‐nucleotide C—H...O contacts, giving rise to the self‐assembly of cdTMP⁻ anions into three different ribbons. Two of these three ribbons run in the same direction, while the third is antiparallel.     

Hemp Seeds of the Polish ‘Bialobrzeskie’ and ‘Henola’ Varieties (Cannabis sativa L. var. sativa) as Prospective Plant Sources for Food Production

This publication characterizes the nutritional value of the Polish hemp seeds of the ‘Bialobrzeskie’ and ‘Henola’ varieties, including the profile/content of fatty acids and amino acids. Hemp seeds were found to be rich in protein, fat, and dietary fiber. Polyunsaturated fatty acids (PUFA) dominated the unsaturated fatty acids (UFA) profile. Their average share within the total fatty acids (FA) was as high as 75%. Linoleic acid belonging to this group accounted for 55% of the total FA. Lipid profile indices (Σ n − 6/Σ n − 3, Σ PUFA/Σ SFA, the thrombogenicity index, the atherogenicity index and the hypocholesterolemic/hypercholesterolemic ratio) proved the high nutritional value of hemp oil. Considering the tyrosine + phenylalanine and histidine contents, hemp protein exhibited a great degree of similarity to egg protein, which is known and valued for its high biological value.     

Adapting (4,4) Networks through Substituent Effects and Conformationally Flexible 3,2’:6’,3”-Terpyridines

Coordination networks formed between Co(NCS)₂ and 4’-substituted-[1,1’-biphenyl]-4-yl-3,2’:6’,3”-terpyridines in which the 4’-group is Me (1), H (2), F (3), Cl (4) or Br (5) are reported. [Co(1)₂(NCS)₂]_n·4.5nCHCl₃, [Co(2)₂(NCS)₂]_n·4.3nCHCl₃, [Co(3)₂(NCS)₂]_n·4nCHCl₃, [Co(4)₂(NCS)₂]_n, and [Co(5)₂(NCS)₂]_n·nCHCl₃ are 2D-networks directed by 4-connecting cobalt nodes. Changes in the conformation of the 3,2’:6’,3”-tpy unit coupled with the different peripheral substituents lead to three structure types. In [Co(1)₂(NCS)₂]_n·4.5nCHCl₃, [Co(2)₂(NCS)₂]_n·4.3nCHCl₃, [Co(3)₂(NCS)₂]_n·4nCHCl₃, cone-like arrangements of [1,1’-biphenyl]-4-yl units pack through pyridine…arene π-stacking, whereas Cl…π interactions are dominant in the packing in [Co(4)₂(NCS)₂]_n. The introduction of the Br substituent in ligand 5 switches off both face-to-face π-stacking and halogen…π-interactions, and the packing interactions are more subtly controlled. Assemblies with organic linkers 1–3 are structurally similar and the lattice accommodates CHCl₃ molecules in distinct cavities; thermogravimetric analysis confirmed that half the solvent in [Co(3)₂(NCS)₂]_n·4nCHCl₃ can be reversibly removed.     

Chemical Etiology of Nucleic Acid Structure: The Pentulofuranosyl Oligonucleotide Systems: The (1′→3′)‐β‐L‐Ribulo, (4′→3′)‐α‐L‐Xylulo,and (1′→3′)‐α‐L‐Xylulo Nucleic Acids

Under potentially prebiotic scenarios, ribose (pentose), the component of RNA is formed in meager amounts, as opposed to ribulose and xylulose (pentuloses). Consequently, replacement of ribose in RNA, with pentulose sugars, gives rise to prospective oligonucleotide candidates that are potentially prebiotic structural variants of RNA that could be formed by the same type of chemical pathways that gave rise to RNA from ribose. The potentially natural alternative (1′→3′)‐ribulo oligonucleotides and (4′→3′)‐ and (1′→3′)‐xylulo oligonucleotides consisting of adenine and thymine were synthesized and found to exhibit no self‐pairing or cross‐pairing with RNA. This signifies that even though pentulose sugars may have been abundant in a prebiotic scenario, the pentulose nucleic acids (NAs), if and when formed, would not have been competitors of RNA, or interfered with the emergence of RNA as a functional informational system. The reason for the lack of base pairing in pentulose NA highlights the contrasting and central role played by the furanosyl ring in RNA and pentulose NA, enabling and optimizing the base pairing in RNA, while impeding it in pentulose NA.     

Functional Xeno Nucleic Acids for Biomedical Application

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.     

核酸光化学生物学

光敏感基团作为光化学开关被广泛应用于各种生物过程的光调控中。特别是过去十几年内,核苷酸、寡聚核苷酸和DNA/RNA的光敏修饰策略得到了长足的发展,并在细胞信号传导和靶基因的功能调控等诸多生物学研究中发挥重要的作用。本文主要针对常用的光敏感基团、光敏感核酸及其化学生物学研究进展进行简要综述,并对未来核酸光化学生物学的研究进行了展望。     

Quality of Oil Pressed from Hemp Seed Varieties: ‘Earlina 8FC’, ‘Secuieni Jubileu’ and ‘Finola’

In the last decade, the demand for edible niche oils has increased. Therefore, the aim of this study was to characterize the seeds hemp (Cannabis sativa L.) varieties: ‘Finola’ (FIN-314)’, ‘Earlina 8FC’, and ‘Secuieni Jubileu’, and cold and hot pressed oils were prepared from each seed. The seeds were examined for moisture content, granulometric distribution, bulk density, and fat content. Seeds were pressed without and with preconditioning (60 °C), and oil yield and pressing time were recorded. The oil was filtered through cellulose membranes. Oil–water content, oil color, fatty acid profile, and sterol content were studied. From the study conducted, there are significant differences in the parameters of oil recovery and its quality compared to ‘Finola’ seed oil, which is widely reported in the literature. ‘Finola’ oil yield was the lowest, with an average of 79% compared to ‘Earlina’ (82%) and ‘S. Jubileu’ (84%). All oil samples contained a comparable amount of sterols, with campesterol (0.32 mg/g), β-sitosterol (1.3 mg/g) and Δ5-avenasterol (0.15 mg/g) predominating. From the organoleptic evaluation, it was evident that both varieties hemp oils and marc (‘Earlina’ and ‘S. Jubileu’) were not bitter like the “Finola” oil and marc. More detailed studies in this direction have to be undertaken.     

Increased Affinity of 2′‐O‐(2‐Methoxyethyl)‐Modified Oligonucleotides to RNA through Conjugation of Spermine at Cytidines

Structural modification at the 2′‐O‐position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotides that have been tested in clinical trials. Yet, the search for new improved modifications continues in a quest for increased oligonucleotide potency, improved transport in vivo and favorable metabolism. Recently, we described how the conjugation of spermine groups to pyrimidines in oligonucleotides vastly increases their affinity for complementary RNAs through accelerated binding kinetics. Here we describe how spermines can be linked to the exocyclic amino groups of cytidines in MOE‐oligonucleotides employing a straightforward ‘convertible nucleoside approach’ during solid phase synthesis. Singly‐ or doubly‐modified oligonucleotides show greatly enhanced affinity for complementary RNA, with potential for a new generation of MOE‐based oligonucleotide drugs.     

Recognition and detection of oligonucleotides in the presence of chromosomal DNA based on entrapment within conducting-polymer networks

Overlapping voltammetric signals, accrued from redox processes of nucleobases, do not permit discrimination between short oligonucleotides and chromosomal DNA molecules when conventional electrochemical techniques are used. This article describes a new genoelectronic route for discriminating between short oligonucleotides and chromosomal DNA, based on the polypyrrole (PPy) doping process. Such a route relies on the profound effect of short nucleic acid dopants upon the redox activity of PPy, and hence on the square-wave voltammetric signal of the polymer-modified electrode in a blank electrolyte solution. The electropolymeric growth of PPy thus serves for preferential accumulation (by doping) of short oligonucleotides. High selectivity is demonstrated for voltammetric measurements of oligo(dG)₂₀ and oligo(dT)₂₀ in the presence of otherwise interfering ss- and ds-DNA. The signals for the oligonucleotides are also not affected by a large excess of chloride or phosphate ions. The response of the new preconcentration (doping)–medium-exchange–voltammetric protocol is proportional to the concentration of the oligonucleotide dopant. Such a new recognition process, based on the doping of conducting-polymer networks, enhances the scope of electroanalysis of nucleic acids.     

Warum Pentose- und nicht Hexose-Nucleinsäuren?? Teil I. Einleitung und Problemstellung,Konformationsanalyse für Oligonucleotid-Ketten aus 2′,3′-Dideoxyglucopyranosyl-Bausteinen (‘Homo-DNS’) sowie Betrachtungen zur Konformation von A- und B-DNS

Why Pentose and Not Hexose Nucleic Acids? Part I . Introduction to the Problem, Conformational Analysis of Oligonucleotide Single Strands Containing 2′,3′-Dideoxyglucopyranosyl Building Blocks (‘Homo-DNA’), and Reflections on the Conformation of A- and B-DNA Summary in collaboration with Prof. Dr. C.E. Wintner, Haverford College, Haverford, PA 19041-1392; academic guest, ETH, March and June/July, 1991. Chemical rationalization of the structure of a biomolecule can be sought through consideration of two criteria: first, the relationship between the structure and its biological function; and second, the structure's potential for constitutional self assembly. The latter criterion convers the judgment, by chemical reasoning, of the chance of its preformation, that is, a synthetic event which must have been undergone by any molecular structure in order to have been selected (or to have selected itself) to become a biomolecule. One way to further the task of rationalizing a biomolecule's structure by experimental means is the design, synthesis, and study of structural alternatives which might have become biomolecules on the basis of either criterion, but which do not, in fact, appear in Nature today. In the formation of sugar phosphates from glycolaldehyde phosphate under basic conditions, straightforward and selective formation of rac-hexose 2,4,6-triphosphates is observed in the absence of formaldehyde, while rac-pentose 2,4-diphosphates are dominant, when (0.5 equiv.) formaldehyde is present [1]. This and other observations indicate that hexose sugars should be regarded to have had a chance of preformation comparable with that of pentose sugars. Why, then, did Nature choose pentoses and not hexoses as the sugar building blocks of nucleic acids? The reason must be functional; it must be the case that pentose nucleic acids are biologically superior to potential hexose alternatives. To the extent that biological function is a consequence of molecular structure and reactivity, the origin of this superiority should be decipherable through chemical experiment, that is, through synthesis of hexose nucleic acids, systematic study of their chemical properties, and comparison of these properties with those of their natural counterparts. This has been the object of the present investigation, initiated in 1986. The paper introduces a series of papers which will describe the results of a model study, namely, the synthesis, pairing properties and structure of homo-DNA oligonucleotides.     

Nucleic acid and SNP detection via template‐directed native chemical ligation and inductively coupled plasma mass spectrometry

Detection of nucleic acids and single nucleotide polymorphisms (SNPs) is of pivotal importance in biology and medicine. Given that the biological effect of SNPs often is enhanced in combination with other SNPs, multiplexed SNP detection is desirable. We show proof of concept of the multiplexed detection of SNPs based on the template‐directed native chemical ligation (NCL) of PNA‐probes carrying a metal tag allowing detection using ICP‐MS. For the detection of ssDNA oligonucleotides (30 bases), two probes, one carrying the metal tag and a second one carrying biotin for purification, are covalently ligated. The methodological limit of detection is of 29 pM with RSD of 6.7% at 50 pM (n = 5). Detection of SNPs is performed with the combination of two sets of reporter probes. The first probe set targets the SNP, and its yield is compared with a second set of probes targeting a neighboring sequence. The assay was used to simultaneously differentiate between alleles of three SNPs at 5‐nM concentration.     

Mapping the Landscape of Potentially Primordial Informational Oligomers: (3′→2′)‐D‐Phosphoglyceric Acid Linked Acyclic Oligonucleotides Tagged with 2,4‐Disubstituted 5‐Aminopyrimidines as Recognition Elements

The (3′→2′)‐phosphodiester glyceric acid backbone containing an acyclic oligomer tagged with 2,4‐disubstituted pyrimidines as alternative recognition elements have been synthesized. Strong cross‐pairing of a 2,4‐dioxo‐5‐aminopyrimidine hexamer, rivaling locked nucleic acid (LNA) and peptide nucleic acid (PNA), with complementary adenine‐containing DNA and RNA sequences was observed. The corresponding 2,4‐diamino‐ and 2‐amino‐4‐oxo‐5‐aminopyrimidine‐tagged oligomers were synthesized, but difficulties in deprotection, purification, and isolation thwarted further investigations. The acyclic phosphate backbone structure of the protected oligomer seems to be prone to an eliminative degradation owing to the acidic hydrogen at the 2′‐position—an arrangement that renders the oligomer vulnerable to the conditions used for the removal of the protecting groups on the heterocyclic recognition element. However, the free oligomers seem to be stable under the conditions investigated.     

Chemotaxonomic Identification of Key Taste and Nutritional Components in ‘Shushanggan Apricot’ Fruits by Widely Targeted Metabolomics

The chemotypic and the content variation in taste substances and nutrients in ‘Shushanggan apricot’ fruits were detected by UPLC-MS/MS. A total of 592 compounds were identified, of which sucrose contributed mainly to the sweet taste and malic acid and citric acid were important organic acids affecting sweet–sour taste. γ-linolenic acid, α-linolenic acid and linoleic acid were the dominant free fatty acids, and neochlorogenic acid and chlorogenic acid were the predominant phenolic acids. Fruit taste was positively correlated with sucrose and negatively correlated with malic acid and citric acid. The differential metabolites were significantly enriched in the biosynthesis of amino acids and 2-oxocarboxylic acid metabolism pathways, regulating the sugar and organic acid biosynthesis. Taste and nutrient differences could be revealed by variations in composition and abundance of carbohydrates, organic acids and amino acids. The purpose of this study was to provide a comprehensive chemical characterization of taste and nutrient compounds in ‘Shushanggan apricot’ fruits.     

Chemical Morphing of DNA Containing Four Noncanonical Bases

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5‐chloro‐2′‐deoxyuridine, 7‐deaza‐2′‐deoxyadenosine, 5‐fluoro‐2′‐deoxycytidine, and 7‐deaza‐2′deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo‐) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.