Interbase Hydrogen Bonds Research Articles

Selective Formation of Pd‐DNA Hybrids Using Tailored Palladium‐Mediated Base Pairs: Towards Heteroleptic Pd‐DNA Systems

AbstractThe formation of highly organized metal‐DNA structures has significant implications in bioinorganic chemistry, molecular biology and material science due to their unique properties and potential applications. In this study, we report on the conversion of single‐stranded polydeoxycytidine (dC15) into a Pd‐DNA supramolecular structure using the [Pd(Aqa)] complex (Aqa=8‐amino‐4‐hydroxyquinoline‐2‐carboxylic acid) through a self‐assembly process. The resulting Pd‐DNA assembly closely resembles a natural double helix, with continuous [Pd(Aqa)(C)] (C=cytosine) units serving as palladium‐mediated base pairs, forming interbase hydrogen bonds and intrastrand stacking interactions. Notably, the design of the [Pd(Aqa)] complex favours the interaction with cytosine, distinguishing it from our previously reported [Pd(Cheld)] complex (Cheld=chelidamic acid). This finding opens possibilities for creating heteroleptic Pd‐DNA hybrids where different complexes specifically bind to nucleobases. We confirmed the Pd‐DNA supramolecular structural assembly and selective binding of the complexes using NMR spectroscopy, circular dichroism, mass spectrometry, isothermal titration calorimetry, and DFT calculations.

Selective Formation of Pd-DNA Hybrids Using Tailored Palladium-Mediated Base Pairs: Towards Heteroleptic Pd-DNA Systems.

The formation of highly organized metal-DNA structures has significant implications in bioinorganic chemistry, molecular biology and material science due to their unique properties and potential applications. In this study, we report on the conversion of single-stranded polydeoxycytidine (dC15 ) into a Pd-DNA supramolecular structure using the [Pd(Aqa)] complex (Aqa=8-amino-4-hydroxyquinoline-2-carboxylic acid) through a self-assembly process. The resulting Pd-DNA assembly closely resembles a natural double helix, with continuous [Pd(Aqa)(C)] (C=cytosine) units serving as palladium-mediated base pairs, forming interbase hydrogen bonds and intrastrand stacking interactions. Notably, the design of the [Pd(Aqa)] complex favours the interaction with cytosine, distinguishing it from our previously reported [Pd(Cheld)] complex (Cheld=chelidamic acid). This finding opens possibilities for creating heteroleptic Pd-DNA hybrids where different complexes specifically bind to nucleobases. We confirmed the Pd-DNA supramolecular structural assembly and selective binding of the complexes using NMR spectroscopy, circular dichroism, mass spectrometry, isothermal titration calorimetry, and DFT calculations.

Mechanism of pH influence on aptamer binding with Cd2+ revealed by molecular dynamics simulation

pH decrease changes the interbase hydrogen bonds and negative charge of the aptamer, destroying its affinity for Cd2+.

Estimating Strengths of Individual Hydrogen Bonds in RNA Base Pairs: Toward a Consensus between Different Computational Approaches.

Noncoding RNA molecules are composed of a large variety of noncanonical base pairs that shape up their functionally competent folded structures. Each base pair is composed of at least two interbase hydrogen bonds (H-bonds). It is expected that the characteristic geometry and stability of different noncanonical base pairs are determined collectively by the properties of these interbase H-bonds. We have studied the ground-state electronic properties [using density functional theory (DFT) and DFT-D3-based methods] of all the 118 normal base pairs and 36 modified base pairs, belonging to 12 different geometric families (cis and trans of WW, WH, HH, WS, HS, and SS) that occur in a nonredundant set of high-resolution RNA crystal structures. Having addressed some of the limitations of the earlier approaches, we provide here a comprehensive compilation of the average energies of different types of interbase H-bonds (EHB). We have also characterized each interbase H-bond using 13 different parameters that describe its geometry, charge distribution at its bond critical point (BCP), and n → σ*-type charge transfer from filled π orbitals of the H-bond acceptor to the empty antibonding orbital of the H-bond donor. On the basis of the extent of their linear correlation with the H-bonding energy, we have shortlisted five parameters to model linear equations for predicting EHB values. They are (i) electron density at the BCP: ρ, (ii) its Laplacian: ∇2ρ, (iii) stabilization energy due to n → σ*-type charge transfer: E(2), (iv) donor–hydrogen distance, and (v) hydrogen–acceptor distance. We have performed single variable and multivariable linear regression analysis over the normal base pairs and have modeled sets of linear relationships between these five parameters and EHB. Performance testing of our model over the set of modified base pairs shows promising results, at least for the moderately strong H-bonds.

How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations.

Computational (DFT and MD simulation) methods are employed to systematically characterize the structural and energetic properties of five hydrophobic nucleobases (FEMO, MMO2, NaM, 5SICS and TPT3) that constitute four unnatural base pairs (FEMO:5SICS, MMO2:5SICS, NaM:5SICS and TPT3:NaM). These hydrophobic bases have been recently shown to be replicated when present between natural bases in DNA duplexes, with the highest replication fidelity and efficiency occuring for the TPT3:NaM pair. Our QM calculations suggest that the preferred (anti) glycosidic orientations of nucleosides containing hydrophobic bases are similar to the natural DNA nucleosides despite differences in their chemical structures. However, due to the inability to form interbase hydrogen bonds, hydrophobic base pairs intrinsically prefer nonplanar, distorted geometries, many of which are stabilized through π-π stacking interactions. Furthermore, the intrinsic stacking potential between a hydrophobic and a natural base is similar to that between two natural bases, indicating that the strength of stacking interactions in DNA duplexes containing hydrophobic bases is likely comparable to natural DNA. However, in contrast to the isolated base-pair geometries, our MD simulations suggest that the hydrophobic base pairs adopt variable geometries within DNA, which range from stacked (5SICS:FEMO) to nearly planar (5SICS:NaM and SICS:MMO2) to planar (TPT3:NaM). As a result, the duplex structural features at the site of modification depend on the identity of the hydrophobic base pair, where the TPT3:NaM pair causes the least structural changes compared to natural DNA. Overall, the structural insight obtained from our calculations on DNA containing hydrophobic base pairs explains the experimentally-observed higher fidelity and efficiency during replication of TPT3:NaM compared to other hydrophobic nucleobase pairs. By providing valuable structural information that explains the intrinsic and duplex properties of this class of unnatural nucleobases, the present work may aid the future design of improved hydrophobic analogues.

Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair.

We investigate the attack of the nitrogen dioxide radical (NO•2) to the guanine—cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO•2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC–NO•2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis.

RB69 DNA Polymerase Structure, Kinetics, and Fidelity

This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n – 1 and n – 2 of the primer strand and position n – 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°–tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.

Urea-Induced Denaturation of PreQ1-Riboswitch

Urea, a polar molecule with a large dipole moment, not only destabilizes folded RNA structures but can also enhance the folding rates of large ribozymes. Unlike the mechanism of urea-induced unfolding of proteins, which is well understood, the action of urea on RNA has barely been explored. We performed extensive all-atom molecular dynamics simulations to determine the molecular underpinnings of urea-induced RNA denaturation. Urea displays its denaturing power in both secondary and tertiary motifs of the riboswitch structure. Our simulations reveal that the denaturation of RNA structures is mainly driven by the hydrogen-bonding and stacking interactions of urea with the bases. Through detailed studies of the simulation trajectories, we found that geminate pairs between urea and bases due to hydrogen bonds and stacks persist only ~0.1-1 ns, which suggests that the urea-base interaction is highly dynamic. Most importantly, the early stage of base-pair disruption is triggered by penetration of water molecules into the hydrophobic domain between the RNA bases. The infiltration of water into the narrow space between base pairs is critical in increasing the accessibility of urea to transiently disrupted bases, thus allowing urea to displace inter-base hydrogen bonds. This mechanism--water-induced disruption of base pairs resulting in the formation of a "wet" destabilized RNA followed by solvation by urea--is the exact opposite of the two-stage denaturation of proteins by urea. In the latter case, initial urea penetration creates a dry globule, which is subsequently solvated by water, leading to global protein unfolding. Our work shows that the ability to interact with both water and polar or nonpolar components of nucleotides makes urea a powerful chemical denaturant for nucleic acids.

Interplay between hydroxyl radical attack and H-bond stability in guanine–cytosine

We combine ONIOM simulations with an elaborated hydrated DNA model to evaluate the effects of the hydroxyl radical, one of the major products in irradiated cells, on the stability of the guanine–cytosine (G:C) base pair. Our results demonstrate that the addition of the hydroxyl radical to the cytosine moiety results in inter-base hydrogen bonds rearrangements, eventually leading to a so-called rare tautomeric isomer. In these processes, the O6(G)–N4(C) hydrogen bond strength plays a crucial role as one of the driving forces for the double proton transfer. We also demonstrate that the surrounding environment affects the stability of the non-canonical mutagenic forms. As in undamaged DNA, the water molecules of the first hydration shell might catalyze the exchange of protons while π-stacking tunes the energetic profile of the reaction. The degenerative effects of the induced rare tautomers are shown to be governed by the transition states along the PT reactions, which in turn determines their lifetimes.

DNA-Based Optomechanical Molecular Motor

An azobenzene-capped DNA hairpin coupled to an AFM is presented as an optically triggered single-molecule motor. The photoinduced trans to cis isomerization of azobenzene affects both the overall length of the molecule and the ability of the DNA bases to hybridize. Using a combination of molecular dynamics simulations and free energy calculations the unfolding of both isomers along the O5'-O3' extension coordinate is monitored. The potentials of mean force (PMFs) along this coordinate indicate that there are two major differences induced by photoisomerization. The first is that the interbase hydrogen bond and stacking interactions are stable for a greater range of extensions in the trans system than in the cis system. The second difference is due to a decreased chain length of the cis isomer with respect to the trans isomer. These differences are exploited to extract work in optomechanical cycles. The disruption of the hairpin structure gives a maximum of 3.4 kcal mol(-1) of extractable work per cycle with an estimated maximum efficiency of 2.4%. Structure-function insights into the operation of this motor are provided, and the effect of the cantilever stiffness on the extractable work is characterized.

Combined effect of stacking and solvation on the spontaneous mutation in DNA

In DNA, base pairs are involved in two reciprocal interactions: interbase hydrogen bonds and stacking. Furthermore, base pairs also undergo the effects of the external entities present in the biological environment, such as water molecules and cations. In this contribution, the double spontaneous mutation has been studied with hybrid theoretical tools in a DNA-embedded guanine-cytosine model accounting for the impact of the first hydration shell. According to our findings, the combination of the neighboring base pairs and surrounding water molecules plays a crucial role in the double proton transfer. Indeed, as a consequence of these interactions, the double proton transfer (DPT) mechanism is altered: on the one hand, stacking and hydration strongly affect the geometry of base pairs, and, on the other hand, vicinal water molecules may play an active role in the tautomeric equilibrium by catalyzing the proton transfer reaction.

Microhydration of 9-methylguanine:1-methylcytosinebase pair and its radical anion: a density functional theory study

In this study, we present density functional theory calculations on the properties of proton transfer and electron binding in isolated, mono-, di-, and pentahydrated 9-methylguanine:1-methylcytosine (mG:mC) base pair radical anions. It was found that the proton transfer in mG:mC(*-) is coupled to the interbase propeller-twisting and stretching motions, which cooperatively shorten the proton-transfer distance. Without the propeller-twisting motion, the interbase stretching will be hindered and the proton-transfer distance will become somewhat longer, which in turn, results in rising of the kinetic barrier for proton transfer. The monohydration can assist or resist the proton-transfer reaction, depending on the hydration sites. Inclusion of five water molecules in the first hydration shell around mG:mC(*-) only moderately lowers the proton-transfer barrier from 3.80 to 3.01 kcal mol(-1) and the reaction energy from -3.16 to -6.40 kcal mol(-1) due to the cancellation between opposite influences of H(2)O molecules. A further consideration of bulk hydration using a polarizable continuum model does not affect the proton-transfer energetics. In contrast, both the first hydration shell and bulk hydration were found to play important roles in stabilizing the excess electron in mG:mC; the adiabatic electron affinity of mG:mC increases from 0.302 to 0.645 eV upon inclusion of five water molecules in the first hydration shell, and further increases to 1.813 eV when the bulk hydration is considered. We noticed that the water molecule can enhance electron binding by direct interaction with the nucleobase that accommodates excess electron or through the indirect effect of tuning interbase hydrogen bonds. In addition, the microhydration effects on proton transfer and electron binding were found to be approximately additive.

Stacking interaction in the middle and at the end of a DNA helix studied with non-natural nucleotides

Base stacking is important for the base pair interaction of a DNA duplex, DNA replication by polymerases, and single-stranded nucleotide overhangs. To study the mechanisms responsible for DNA stacking interactions, we measured the thermal stability of DNA duplexes containing a non-natural nucleotide tethered to a simple aromatic hydrocarbon group devoid of dipole moments and hydrogen bonding sites. The duplexes containing tetrahydrofuran were paired with a deoxyadenosine derivative (A/T base pair analog) or a deoxycytidine derivative (C/G base pair analog) and showed a lower stability than Watson-Crick base pairing, partly due to the loss of interbase hydrogen bonds. Conversely, non-natural nucleotides present at a dangling end yielded an interaction energy as high as that observed with base pairing. Importantly, the non-natural nucleotides yielded an interaction energy with a linear correlation similar to that of the analogous Watson-Crick base pairs both in the middle and at the end of a DNA duplex, although a different stacking mechanism between the middle and the end was suggested. Moreover, a positive cooperativity was observed in dangling end stacking of the nucleotide base moiety and aromatic hydrocarbon group. These observations are useful to understand nucleic acid interactions and to design new non-natural nucleotides.

A Very Stable Cyclic DNA Miniduplex with Just Two Base Pairs

What goes around…︁ Cyclic mini-DNA duplexes, as shown in the scheme, have been synthesized in high yield on the multimicromole scale by using click chemistry. These B-like duplexes are very stable and bind to drugs, such as distamycin A and 7-aminoactinomycin D. Circular dichroism indicates that the bases are stacked, and UV melting and NMR spectroscopy showed that the interbase hydrogen bonds are very stable, even in a dimer.

Probing the role of hydrogen bonds in the stability of base pairs in double‐helical DNA

Aromatic stacking and hydrogen bonding between nucleobases are two of the key interactions responsible for stabilization of DNA double-helical structures. The present work aims at defining the specific contributions of these interactions to the stability of individual base pairs in DNA. The two DNA double helices investigated are formed, respectively, by the palindromic base sequences 5'-dCCAACGTTGG-3' and 5'-dCGCAGATCTGCG-3'. The strength of the N==H...N inter-base hydrogen bond in each base pair is characterized from the measurement of the protium-deuterium fractionation factor of the corresponding imino proton using NMR spectroscopy. The structural stability of each base pair is evaluated from the exchange rate of the imino proton, measured by NMR. The results reveal that the fractionation factors of the imino protons in the two DNA double helices investigated fall within a narrow range of values, between 0.92 and 1.0. In contrast, the free energies of structural stabilization for individual base pairs span 3.5 kcal/mol, from 5.2 to 8.7 kcal/mol (at 15 degrees C). These findings indicate that, in the two DNA double helices investigated, the strength of N==H...N inter-base hydrogen bonds does not change significantly depending on the nature or the sequence context of the base pair. Hence, the variations in structural stability detected by proton exchange do not involve changes in the strength of inter-base hydrogen bonds. Instead, the results suggest that the energetic identity of a base pair is determined by the number of inter-base hydrogen bonds, and by the stacking interactions with neighboring base pairs.

Raman spectroscopic study on structure of human immunodeficiency virus (HIV) and hypericin-induced photosensitive damage of HIV

The first Raman spectra of HIV1-HIV2 in human sera and hypericin-induced photosensitive damage of the virus have been obtained. The prominent Raman lines in the spectra are assigned respectively to the carbohydrates of viral glycoprotein, RNA, protein and lipid. The spectra are dominated by Raman scattering of the carbohydrates. The lines of D-Mannose and N-acetylglucosamine in carbohydrates are obvious and there is a beta-configuration in the anomeric C1 position in D-Mannose. The viral RNA duplexes bound assumes an A-form geometry. The lines of backbone phosphate group, bases (involving interbase hydrogen bonding) and ribose of the RNA are complete and distinct. The secondary structure of the viral protein maintains alpha-helix, beta-sheet, beta-turn and random coil. Its side chains are rich and vary from tryptophan, phenylalanine and "buried" tyrosine; the stable conformation of the S-S bond of gauche-gauche-gauche; the two forms of C-S bonds of gauche and trans; to sulfhydrl group and ionized and unionized carboxyl groups. The viral lipid bilayer molecules are probably in the liquid ordered phase or the gel phase. It was observed that the hypericin-induced photosensitive damage of HIV1-HIV2 in human sera changed various components of HIV1-HIV2 in different degrees: The orderly A-form viral RNA would become a disordered viral RNA. There were a breakage of interbase hydrogen bonds and disruption of vertical base-base stacking interactions. In addition, the groups of ribos and four bases were damaged obviously. A decrease in ordered structure (alpha-helix and beta-sheet) of viral protein is accompanied by an increase in random coil. The Tyr buried in the three-dimensional structure of protein was damaged, but it was still "buried" and the damage of C-S bond of trans form was stronger. The groups of carbohydrates, including D-Mannos and N-acetyl glucosamine, in viral envelope glycoprotein had also been changed. The hydrophilic C-N bond of choline in viral lipid was damaged, which was the possible binding site to hypericin, whereas the viral lipids bilayers were still probably in the liquid ordered phase or the gel phase. So the space structure of HIV1-HIV2 was damaged under the experimental conditions, which might block viral infection and inhibit its growth and breeding. It is apparent that the laser Raman spectra have provided certain direct evidence at the molecular level for photosensitive damage of HIV1-HIV2.

α-Thymidine at 105 K

Two independent molecules in the crystal structure of the title nucleoside, 1-(2-de­oxy-α-d-ribofuranos­yl)-5-methyl­uracil, C10H14N2O5, form a dimer connected by two inter-base hydrogen bonds. The ring puckering modes are envelope C4′-endo and half-chair C3′-exo-C4′-endo, respectively, which are quite uncommon conformations for 2′-deoxy­riboses.

Use of an adjustable soft segment as an effective molecular design for crystal engineering of hydrogen-bonded tape motifs.

From all the 11 alkylsilylated guanosine and adenosine derivatives having different numbers and types of nonpolar and flexible alkylsilyl side chains, 13 crystals were obtained from appropriate solvents in spite of their low molecular symmetry, and their crystal structures were studied by X-ray crystallography and thermal analysis. In these crystals, a clear structural hierarchy was observed, and one-dimensional tape motifs were preferentially formed by multiple inter-base hydrogen bonds. The tape motifs were arranged in lamellar-like (L), herringbone (H) or widened lamella (WL) structures in the crystals. However, the alkylsilylated ribose unit adopted a variety of conformations with notable disorders at the alkylsilyl moiety. These results suggested that role of the adjustable and nonpolar alkylsilyl ribose unit was to provide cushioning or a filling effect as a molecular pad, which assisted the crystal packing of the robust tape motifs. The packing mode of the tape motifs could be understood from the size instead of the shape of the adjustable alkylsilyl ribose moiety, offering a novel approach to their crystal engineering.

Cyclic Adenine-, Cytosine-, Thymine-, and Mixed Guanine−Cytosine-Base Tetrads in Nucleic Acids Viewed from a Quantum-Chemical and Force Field Perspective

We have carried out a systematic study of hydrogen-bonded cyclic A, C, T, and mixed GCGC tetrads resembling conformations occurring in experimental tetraplex structures, using the B3LYP hybrid density functional (DFT) method and the MMFF and AMBER force fields to determine tetrad structures and interaction energies. The results are compared to G and U tetrads analyzed previously, thereby presenting a comprehensive overview of all cyclic tetrads formed from one base type only, in addition to the GCGC data. The DFT calculations indicate that the C tetrad is planar and the GCGC tetrad is nearly planar and correspond to local minima at C4h and Ci symmetry, respectively. For A tetrads with N6−H6···N7 and with N6−H6···N1 H-bonds and for T tetrads, nonplanar structures are more stable than the planar ones, and among the nonplanar structures, S4-symmetric conformations are more stable than C4 structures. Minima confirmed by frequency calculations are found for the planar C tetrad, the Ci-symmetric GCGC tetrad, the C4-symmetric structure of the A tetrad with N6−H6···N7 H-bonds, and the S4-symmetric T tetrad, in addition to the already known local minima of the S4-symmetric structure of the G tetrad and the planar U tetrad with C−H···O hydrogen bonds. The interaction energies (ΔET) corrected for deformation of the bases in the tetrads range from −69.47 kcal/mol for the GCGC tetrad with three pairs of strong Watson−Crick type hydrogen bonds to −11.09 kcal/mol in the planar A tetrad having only a single N6−H62···N1 interbase hydrogen bond. With more than 18%, the largest cooperativity contribution to ΔE is found for the C and G tetrads. The interaction energies derived from the MMFF and AMBER force field are similar to the DFT data for those tetrads having high interaction energies, whereas the relative deviations are much larger for weakly H-bonded tetrads.

Selective Binding of the TATA Box-Binding Protein to the TATA Box-Containing Promoter: Analysis of Structural and Energetic Factors

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding —NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.