Structural Comparison of Enterococcus faecalis and Human Thymidylate Synthase Complexes with the Substrate dUMP and Its Analogue FdUMP Provides Hints about Enzyme Conformational Variabilities.

Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.

Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier.

Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N5-N10-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity.

The intrinsic basicity of the phosphate backbone exceeds that of uracil and thymine residues: protonation of the phosphate moiety is preferred over the nucleobase for pdThd and pUrd.

The gas-phase conformations of the protonated forms of thymidine-5'-monophosphate and uridine-5'-monophosphate, [pdThd+H]+ and [pUrd+H]+, are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and electronic structure calculations. The IRMPD action spectra of [pdThd+H]+ and [pUrd+H]+ are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Low-energy conformations of [pdThd+H]+ and [pUrd+H]+ and their relative stabilities are computed at the MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers indicate that the dominant conformers of [pdThd+H]+ and [pUrd+H]+ populated in the experiments are protonated at the phosphate oxo oxygen atom, with a syn nucleobase orientation that is stabilized by strong P[double bond, length as m-dash]OH+O2 and P-OHO4' hydrogen-bonding interactions, and C2'-endo sugar puckering. Minor abundance of conformers protonated at the O2 carbonyl of the nucleobase residue may also contribute for [pdThd+H]+, but do not appear to be important for [pUrd+H]+. Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of thymidine and uridine, [dThd+H]+ and [Urd+H]+, and the deprotonated forms of pdThd and pUrd, [pdThd-H]- and [pUrd-H]-, provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Most interestingly, the thymine and uracil nucleobases remain in their canonical forms for [pdThd+H]+ and [pUrd+H]+, unlike [dThd+H]+ and [Urd+H]+, where protonation occurs on the nucleobases and induces tautomerization of the thymine and uracil residues.

Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

Kinetico-Mechanistic Studies of Nucleoside and Nucleotide Substitution Reactions of Co(III) Complexes of Fully Alkylated Cyclen.

The solution chemistry of complex [Co{(Me)2(µ-ET)cyclen}(H2O)2](3+) containing a fully substituted tetraammine ligand designed for the avoidance of base-conjugated substitution mechanisms in the 6-8 pH range has been studied. The study should shed some light on the possible involvement of such Co(III) skeleton in inert interactions with biomolecules. The reactivity and speciation of the complex has been found similar to that of the parent cyclen derivative with the presence of mono- and bis-hydroxo-bridged species; at pH < 7.1, all reactivity has been found to be related to the aqua/hydroxo monomeric complexes. Under these pH conditions, the substitution reactions of the aqua/hydroxo ligands by chloride, inorganic phosphate, thymidine, cytidine 5'-monophosphate (5'-CMP), and thymidine-5'-monophosphate (5'-TMP) have been studied at varying conditions; ionic strength has been kept at 1.0 NaClO4 due to the high concentration of 2-(N-morpholino)ethanesulfonic acid (MES) or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) used to ensure buffering. Except for chloride, the process occurs neatly in a one or two step process, showing dissociatively activated substitution mechanisms, having in general large ΔH(⧧), positive ΔS(⧧), and values of ΔV(⧧) close to those corresponding to the liberation of an aqua ligand to the reaction medium. The actuation of noticeable encounter-complex formation equilibrium constants has been found to be the determinant for the reactions with nucleosides and nucleotides, a clear indication of the relevance of hydrogen-bonding interactions in the reactivity of these molecules, even in this highly ionic strength medium. For the substitution of the active aqua/hydroxo ligands with 5'-TMP, the first substitution reaction produces an Nthymine-bound 5'-TMP complex that evolves to a bis-5'-TMP with an Nthymine,Ophosphate-bonding structure. The formation of outer-sphere complexes between the dangling phosphate group of the Nthymine-bound 5'-TMP and the thymine moiety of another entering 5'-TMP has been found to be responsible for this fact, which leaves only the phosphate group for coordination available.

Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >106-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 104-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH3). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²âº bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²âº bimetallo core.

A thymine tetrad assembly templated from thymidylic acid.

A template tetra-coupled with thymidylic acid through a phosphate linkage was characterized in methanol for emergent properties of nucleobase tetrad formation. Intramolecular hydrogen bonded base pairing in the absence of a cation was indicated for the thymidylic acid species supporting a monomeric template-assembled structure. Thus, an initial report of a stabilized individual thymine tetrad assembly is presented here. Consistent with previous investigations, a deoxyguanylic acid variant templated an analogous methanolic monomeric G-tetrad in comparison to the thymine species.

Identification and characterization of second-generation invader locked nucleic acids (LNAs) for mixed-sequence recognition of double-stranded DNA.

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.

Labeling of islet cells with iron oxide nanoparticles through DNA hybridization for highly sensitive detection by MRI.

A labeling method for islet cells with superparamagnetic iron oxide nanoparticles (SPIOs) based on DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified by via Michael reaction by reacting oligo-(deoxyadenylic acid)-bearing a terminal thiol group at the 5'-end ((dA)20-SH) with maleic acid functional groups on the SPIOs. The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)-poly(ethylene glycol)-phospholipid conjugates ((dT)20-PEG-DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes or exfoliated with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2(*)-weighted MR image, whereas non-labeled islet cells could not be detected.

Large scale synthesis of 2'-amino-LNA thymine and 5-methylcytosine nucleosides.

Thymine intermediate 17 has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar 1 in 15 steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate 18 was obtained from 17 in a single step in 96% yield, whereas the key 5-methylcytosine intermediate 20 was obtained from 17 in 2 steps in 58% yield. This highly efficient large scale route necessitates only 2 and 3 novel steps to obtain N2'-functionalized thymine and 5-methylcytosine amino-LNA phosphoramidites from these key intermediates, respectively.

Synthesis, and in vitro enzymatic and antiviral evaluation of d4T polyphosphate derivatives as chain terminators.

A series of d4T di- or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. The steady-state kinetic study of compounds 1-4 in an enzymatic incorporation assay by HIV-1 RT follows Michaelis-Menten profile. In addition, compounds 2-4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells, as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.

Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates.

5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5'-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3'- and 5'-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3'- and 5'-phosphotyrosyl linkage at the 3' and 5' ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5'-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5' activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC(50)=40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate.

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.

A mechanistic approach for the DNA binding of chiral enantiomeric L- and D-tryptophan-derived metal complexes of 1,2-DACH: cleavage and antitumor activity.

A new chiral series of potential antitumor metal-based complexes 1-3(a and b) of L- and D-tryptophan have been synthesized and thoroughly characterized. Both enantiomers of 1-3 bind DNA noncovalently via phosphate interaction with slight preference of metal center for covalent coordination to nucleobases. The K(b) values of L-enantiomer, however, possess higher propensity for DNA binding in comparison with the D-enantiomeric analogs. The relative trend in K(b) values is as follows: 2(a) > 2(b) > 3(a) > 1(a) > 3(b) > 1(b). These observations together with the findings of circular dichoric and fluorescence studies reveal maximal potential of L-enantiomeric form of copper complex to bind DNA, thereby exerting its therapeutic effect. The complex 2a exhibits a remarkable DNA cleavage activity with pBR322DNA in the presence of different activators such as H(2) O(2) , ascorbic acid, 3-mercaptopropionic acid, and glutathione, suggesting the involvement of active oxygen species for the DNA scission. In vitro anticancer activity of complexes 1-3(a) were screened against 14 different human carcinoma cell lines of different histological origin, and the results reveal that 2a shows significant antitumor activity in comparison with both 1a and 3a and is particularly selective for MIAPACA2 (pancreatic cancer cell line).

The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme.

Synthesis and biophysical studies of coronene functionalized 2'-amino-LNA: a novel class of fluorescent nucleic acids.

Incorporation of 2'-N-(coronen-1-yl)methyl-2'-amino-LNA monomer X or 2'-N-4-(coronen-1-yl)-4-oxobutanoyl-2'-amino-LNA monomer Y into short DNA strands induces high binding affinity toward DNA or RNA and a marked red-shift in steady-state fluorescence emission upon hybridization to cDNA or RNA.

Construction of helical J-aggregates self-assembled from a thymidylic acid appended anthracene dye and DNA as a template.

The thymidylic acid appended anthracene dye 2,6-bis[5-(3'-thymidylic acid)pentyloxy]anthracene (1) was synthesized, and the self-assembly of 1 and the binary self-assembly of 1 with a complementary single-stranded 20-meric oligodeoxyadenylic acid (dA(20)) were performed in 0.1 x TE buffer solution (i.e., 1.0 x 10(-3) M Tris-HCl, 1.0 x 10(-4) M ethylenediaminetriacetic acid (EDTA)). The characteristic J-band, small Stokes shift (6 nm), Cotton effect, and helical nanofibers 5.1 nm in diameter are observed in UV/Vis, fluorescence, and circular dichroism (CD) spectroscopies and atomic force microscopy (AFM) measurements for the binary self-assembly of 1 and dA(20) in aqueous solution. These observations revealed that the helical J-aggregates, in which the short-axis transition dipoles of the anthracene moieties are aligned in a head-to-tail fashion, are formed from the binary self-assembly of 1 and dA(20). The UV/Vis absorption and CD band of the anthracene L(a) region were found to be strongly dependent on temperature and showed cooperative changes for the binary self-assembly of 1 and dA(20). The self-assembly of the single-component 1 produced right- and left-handed helical nanofibers with diameters ranging from 4.0 to 10 nm. In contrast, for the binary self-assembly, the UV/Vis and fluorescence spectra showed no J-band and the Stokes shift was at approximately 107 nm for the single-component 1 in aqueous solution. In addition, the binary self-assembly of 1 and noncomplementary single-stranded 20-meric oligothymidylic acid (dT(20)) showed a small J-band and the J-band disappeared at 50 degrees C upon heating. On the basis of these observations, we concluded that thymine-adenine base-pair formation induced supramolecular helical J-aggregates in the binary self-assembly of 1 and dA(20) in aqueous solutions.

Biodegradable protections for nucleoside 5'-monophosphates: comparative study on the removal of O-acetyl and O-acetyloxymethyl protected 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl groups.

The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5'-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5'-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5'-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5'-monophosphate (5'-TMP) is quantitative, while conversion of 1 to 5'-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5'-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.

Functionalized 2'-amino-alpha-L-LNA: directed positioning of intercalators for DNA targeting.

Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting.