Jens Peter Jacobsen

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2′‐O, 4′‐C‐methylene bridge. We have investigated a modified 9‐mer single stranded oligonucleotide as well as three 9‐ and 10‐mer modified oligonucleotides hybridized to unmodified DNA. The single‐stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson–Crick base‐pairing with all the nucleotides in anti‐conformation. By use of selective DQF‐COSY spectra we determined the ratio between the N‐type (C3′‐endo) and S‐type (C2′‐endo) sugar conformations of the nucleotides. In contrast to the corresponding single‐stranded DNA (ssDNA), we found that the sugar conformations of the single‐stranded LNA oligonucleotide (ssLNA) cannot be described by a major S‐type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N‐type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N‐type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S‐type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N‐type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases. Copyright

Lna (Locked Nucleic Acid)

Abstract LNA (Locked Nucleic Acid) forms duplexes with complementary DNA, RNA or LNA with unprecedented thermal affinities. CD spectra show that duplexes involving fully modified LNA (especially LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMR examination of an LNA:DNA duplex confirm the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA is demonstrated suggesting strand invasion by LNA. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has been accomplished.

LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets.

The remarkable binding properties of LNA (Locked Nucleic Acid) and α-L-LNA (the α-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2′-O-Me-RNA chimera and LNAs with a “dangling” nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and α-L-LNA sequences in relation to the unprecedented binding properties of LNA and α-L-LNA.

α‐L‐LNA (α‐L‐ribo Configured Locked Nucleic Acid) Recognition of DNA: An NMR Spectroscopic Study

We have used NMR and CD spectroscopy to study and characterise two α-L-LNA:DNA duplexes, a nonamer that incorporates three α-L-LNA nucleotides and a decamer that incorporates four α-L-LNA nucleotides, in which α-L-LNA is α-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson–Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H–1H NOE measurements. In general, the deoxyriboses in both of the α-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified α-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H–1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the α-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson–Crick base pairing.

Synthesis and NMR-studies of dinucleotides with conformationally restricted cyclic phosphotriester linkages

Abstract Four diastereomeric dinucleotides in which the phosphodiester linkages are conformationally restricted in cyclic phosphotriester structures are synthesised. From the epimeric 5′- C -vinyl thymidine derivatives, dinucleotides containing two terminal alkene moieties are constructed via standard phosphoramidite chemistry, and applied as substrates in ring-closing metathesis (RCM) reactions. Hereby, four diastereomeric dinucleotides with seven membered phosphepine rings in the inter-nucleoside linkages are obtained and separated, and their configurations elucidated by advanced NMR-studies in combination with restrained molecular dynamics (rMD) simulations. The seven membered rings are found to give some degree of conformational restriction in the natural nucleic acid backbone, and one of the four dinucleotides is found to favour stacking between the two adjacent thymine moieties.

Phenoloxidase catalyzed coupling of catechols. Identification of novel coupling products.

Phenoloxidases from insect cuticle as well as from other sources oxidize catechols resulting in the formation of various coupling products. The two dominating products from 4-methylcatechol and the main product from N-acetyldopamine were purified and identified by means of plasma desorption and electron impact mass spectrometry and by 1H- and 13C-NMR spectroscopy. The main product from both catechols has a quinoid trihydroxybiphenyl structure, indicating oxidative coupling between a catechol and the corresponding trihydroxy derivative. The second product from 4-methylcatechol is a biphenyltetrol derivative, indicating oxidative coupling between two catechols.

Spin-lattice relaxation time measurements of D2O in a lyotropic phase

Abstract The transformation between the Hoffman relaxation matrix, Λ, and the Redfield relaxation matrix, R , has been derived. The relation is exemplified by the theoretical descriptions of spin-lattice relaxation time measurements on the two lines in the quadrupole doublet of the 2 H NMR spectrum of deuterium oxide in a lyotropic phase. Two relaxation times were determined, resulting in the conclusion that at least two correlation times must be introduced in order to describe the motions of the water molecules.

A comparison of the solution structures of an LNA:DNA duplex and the unmodified DNA:DNA duplex

Modified oligonucleotides, containing restricted nucleotides with a 2′-O,4′-C-methylene bridge (LNA), hybridized toward either DNA or RNA display an unprecedented increase in melting temperatures. In order to understand the structural basis for this high stability we have used 1H NMR spectroscopy to determine the high resolution solution structures of an LNA-modified oligonucleotide, as well as the structure of the corresponding unmodified duplex. The modified duplex is an LNA:DNA duplex containing three thymidine LNA modifications (TLL), d(C1TLL2G3A4TLL5A6TLL7G8C9):d(G10C11A12T13A14T15C16A17G18). A full relaxation matrix approach by the program RANDMARDI was used to obtain interproton distance bounds from NOESY cross peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Both duplexes have right-handed helix conformations with all bases in the anti conformation forming normal Watson–Crick base pairs. The LNA strand in the modified duplex has predominantly N-type sugar conformations compared to the S-type conformations of the complementary strand. The unmodified DNA:DNA strand has almost exclusively S-type sugar conformations. The structural strain introduced by the conformational changes of the ribose rings in the LNA:DNA duplex is released by unwinding the helix and widening the minor groove, but as a whole the structure of the duplex is surprisingly unaffected by introducing the modified LNA nucleotides.

Hydrogen NMR spectra of pyridazine and pyrazole containing 15N

Abstract Preparation and 1H NMR spectra are described for [15N]pyridazine and [15N]-pyrazole. CNDO/2 and INDO calculations of spin-spin coupling constants are reported for pyridazine, pyrazole, pyridine, pyrrole, and thiazole. For 2JNH and 3JHH correlations between experimental and calculated data are established.

Catecholamine-protein conjugates: Isolation of an adduct of N-acetylhistidine to the side chain of N-acetyldopamine from an insect-enzyme catalyzed reaction

Nα-Acetyl-Nτ-[1-(3,4,-dihydroxyphenyl)-2-N-acetylamino-ethyl]-histidine (4) was isolated from a biomimetic reaction containing N-acetyl-dopamine 1, Nα-acetyl-L-histidine and cuticle of silkmoth larvae as an enzyme source. The formation of 4 suggests an 1,6-addition of the Nτ imidazole nitrogen to the p-quinone methide 3.