Juan C. Morales

Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication

The physical and chemical factors that allow DNA to perform its functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of DNA have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving DNA has been the use of DNA base analogues in which functional groups are added, deleted, blocked, or rearranged. Here we describe a different strategy, in which the polar natural DNA bases are replaced by nonpolar aromatic molecules of the same size and shape. This allows the evaluation of polar interactions (such as hydrogen bonding) with little or no interference from steric effects. We have used these nonpolar nucleoside isosteres as probes of noncovalent interactions such as DNA base pairing and protein - DNA recognition. We have found that, while base-pairing selectivity does depend on Watson - Crick hydrogen bonding in the natural pairs, it is possible to design new bases that pair selectively and stably in the absence of hydrogen bonds. In addition, studies have been carried out with DNA polymerase enzymes to investigate the importance of Watson - Crick hydrogen bonding in enzymatic DNA replication. Surprisingly, this hydrogen bonding is not necessary for efficient enzymatic synthesis of a base pair, and significant levels of selectivity can arise from steric effects alone. These results may have significant impact both on the study of basic biomolecular interactions and in the design of new, functionally active biomolecules.

Efficient replication between non-hydrogen-bonded nucleoside shape analogs

DNA polymerase enzymes make an error only once per 104–10 5 initial nucleotide insertions during DNA replication. Most currently held models of this high fidelity cite the hydrogen bonds between complementary pyrimidines and purines as a critical controlling factor. Testing this has been difficult, however, since standard molecular strategies for blocking or removing polar hydrogen-bonding groups cause changes to size and shape as well as hydrogen bonding ability. One answer to this problem is the use of nonpolar molecules that mimic the shape of natural DNA bases. Here we show that a non-hydrogen-bonding shape mimic for adenine is replicated efficiently and selectively against a nonpolar shape mimic for thymine. The results establish that hydrogen bonds in a base pair are not absolutely required for efficient nucleotide insertion. This adds support to the idea that shape complementarity may play as important a role in replication as base–base hydrogen bonds.

Surface-active properties of lipophilic antioxidants tyrosol and hydroxytyrosol fatty acid esters: a potential explanation for the nonlinear hypothesis of the antioxidant activity in oil-in-water emulsions.

Our group has recently observed a nonlinear tendency in antioxidant capacity of different hydroxytyrosol fatty acid esters in fish oil-in-water emulsions, where a maximum of antioxidant efficiency appeared for hydroxytyrosol octanoate. These results appear to disagree with the antioxidant polar paradox. Because the physical location of the antioxidants in an oil-water interface has been postulated as an important factor in explaining this behavior, we have prepared a series of tyrosol and hydroxytyrosol fatty acid esters with different chain length and studied their surface-active properties in water, because these physicochemical parameters could be directly related to the preferential placement at the interface. We have found that tyrosol and hydroxytyrosol fatty acid esters are relevant surfactants when the right hydrophilic-lipophilic balance (HLB) is attained and, in some cases, as efficient as emulsifiers commonly used in industry, such as Brij 30 or Tween 20. Moreover, a nonlinear dependency of surfactant effectiveness is observed with the increase in chain length of the lipophilic antioxidants. This tendency seems to fit quite well with the reported antioxidant activity in emulsions, and the best antioxidant of the series (hydroxytyrosol octanoate) is also a very effective surfactant. This potential explanation of the nonlinear hypothesis will help in the rational design of antioxidants used in oil-in-water emulsions.

Effect of Lipophilization of Hydroxytyrosol on Its Antioxidant Activity in Fish Oils and Fish Oil-in-Water Emulsions

The effect of lipophilization of the antioxidant efficiency of hydroxytyrosol on fish oil enriched systems was studied. Hydroxytyrosol fatty acid esters with increasing size of the alkyl chain and different lipophilicity were tested in bulk fish oils and fish oil-in-water emulsions. Results showed a significant antioxidant activity of hydroxytyrosol esters in both systems especially in emulsions. The introduction of a lipophilic chain decreased the antioxidant effectiveness of hydroxytyrosol in homogeneous systems as fish oils. In emulsion systems, the presence of a short-medium lipophilic chain (acetate, butyrate or octanoate) improved the antioxidant efficiency of hydroxytyrosol favoring the physical location of the antioxidant in the interface, but longer alkyl chain (laurate) maintained or even decreased their antioxidant activity. A maximum of antioxidant efficiency seems to appear when the chain length of the hydroxytyrosol derivative is that of eight carbons which is probably associated with a preferential location of the diorthophenolic moiety in the right geometry. These results are of high importance for the optimum design of effective antioxidants for omega 3 enriched foods, which are very susceptible to suffer oxidation and, then, rancidity.

Metabolites and tissue distribution of resveratrol in the pig

SCOPE trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. METHODS AND RESULTS Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. CONCLUSION This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.

Varied Molecular Interactions at the Active Sites of Several DNA Polymerases: Nonpolar Nucleoside Isosteres as Probes.

We describe a survey of protein-DNA interactions with seven different DNA polymerases and reverse transcriptases, carried out with nonpolar nucleoside isosteres F (a thymidine analog) and Z and Q (deoxyadenosine analogues). Previous results have shown that Z and F can be efficiently replicated opposite each other by the exonuclease-free Klenow fragment of DNA polymerase I from Escherichia coli (KF(-)), although both of them lack Watson-Crick H-bonding ability. We find that exonuclease-inactive T7 DNA polymerase (T7(-)), Thermus aquaticus DNA polymerase (Taq), and HIV-reverse transcriptase (HIV-RT) synthesize the nonnatural base pairs A-F, F-A, F-Z, and Z-F with high efficiency, similarly to KF(-). Steady-state kinetics were also measured for T7(-) and the efficiency of insertion is very similar to that of KF(-); interestingly, the replication selectivity with this pair is higher for T7(-) than KF(-), possibly due to a tighter active site. A second group comprised of calf thymus DNA polymerase α (Pol α) and avian myeloblastosis virus reverse transcriptase (AMV-RT) was able to replicate the A-F and F-A base pairs to some extent but not the F-Z and the Z-F base pairs. Most of the insertion was recovered when Z was replaced by the nucleoside Q (9-methyl-1-H-imidazo[(4,5)-b]pyridine), which is analogous to Z but possesses a minor groove acceptor nitrogen. This strongly supports the existence of an energetically important hydrogen-bonded interaction between the polymerase and the minor groove at the incipient base pair for these enzymes. A third group, formed by human DNA polymerase β (Pol β) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT), failed to replicate the F-Z and Z-F base pairs. No insertion recovery was observed when Z was replaced by Q, possibly indicating that hydrogen bonds are needed at both the template and the triphosphate sites. The results point out the importance of DNA minor groove interactions at the incipient base pair for the activity of some polymerases, and demonstrate the variation in these interactions from enzyme to enzyme.

High-fidelity in vivo replication of DNA base shape mimics without Watson–Crick hydrogen bonds

We report studies testing the importance of Watson–Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson–Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

Carbohydrate-Carbohydrate Interactions in Biological and Model Systems

Carbohydrate-carbohydrate interactions are emerging as a novel and versatile mechanism for cell adhesion and recognition. Although this interaction has not deserved much attention of cell biologists, biochemists, or carbohydrate chemists, new evidence and studies are confirming the importance of this mechanism for specific cell adhesion and communication. The study and evaluation of carbohydrate-carbohydrate interactions is still in its infancy. Their development will go hand in hand with the development of new and more sensitive techniques to study weak interactions such as biosensors, atomic force microscopy, or weak affinity chromatography. In this contribution we will review these new emerging carbohydrate-carbohydrate related interactions including those already established and those where the carbohydrate involvement, at this moment, may be considered possible as in the case of DNA-carbohydrate interaction. The two main research lines on carbohydrate-carbohydrate interaction in biological systems and the efforts, using model systems, to demonstrate and evaluate these interactions are reviewed here in some detail. Considerations about the intermolecular forces and the mechanism that may be involved in carbohydrate-carbohydrate interactions are also presented. The chapter ends with the review of the few examples existing in the literature on quantitative studies of carbohydrate-carbohydrate interaction with model systems.

Highly Polar Carbohydrates Stack onto DNA Duplexes via CH/π Interactions

Carbohydrate-nucleic acid contacts are known to be a fundamental part of some drug-DNA recognition processes. Most of these interactions occur through the minor groove of DNA, such as in the calicheamicin or anthracycline families, or through both minor and major groove binders such as in the pluramycins. Here, we demonstrate that carbohydrate-DNA interactions are also possible through sugar capping of a DNA double helix. Highly polar mono- and disaccharides are capable of CH/π stacking onto the terminal DNA base pair of a duplex as shown by NMR spectroscopy. The energetics of the carbohydrate-DNA interactions vary depending on the stereochemistry, polarity, and contact surface of the sugar involved and also on the terminal base pair. These results reveal carbohydrate-DNA base stacking as a potential recognition motif to be used in drug design, supramolecular chemistry, or biobased nanomaterials.

A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol, and hydroxytyrosol.

A simple and direct strategy to chemically synthesize O-beta-D-glucuronides of urolithin-B 4, resveratrol 5, and the corresponding hydroxytyrosol derivatives 6, 7 (as a regioisomeric mixture), and 8 is described. The critical glycosylation step has been optimized using a structurally simple phenol, urolithin-B, by modification of several reaction parameters (solvent, promoter, and glucuronide donor). Very high yields have been obtained in the first synthesis of the O-beta-D-glucuronide of urolithin-B 4. Extension of these reaction conditions was used for the synthesis of resveratrol-3-O-glucuronide 5 where a higher yield than previously reported was obtained by using the much more common trichloroacetimidate glucuronide donor. Finally, three O-beta-D-glucuronides of hydroxytyrosol 6, 7, and 8 have been synthesized for the first time using chemical synthesis.