Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids.

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined.

Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.

Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

Nonpolar nucleoside mimics as active substrates for human thymidine kinases.

We describe the use of nonpolar nucleoside analogues of systematically varied size and shape to probe the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. Comparison of polar thymidine with a nonpolar isostere, 2,4-difluorotoluene deoxyriboside, as substrates for the two enzymes establishes that TK1 requires electrostatic complementarity to recognize the thymine base with high efficiency. Conversely, TK2 does not and phosphorylates the hydrophobic shape mimic with efficiency nearly the same as the natural substrate. To test the response to nucleobase size, thymidine-like analogues were systematically varied by replacing the 2,4 substituents on toluene with hydrogen and the halogen series (H, F, Cl, Br, I). Both enzymes showed a distinct preference for substrates having the natural size. To examine the shape preference, we prepared four mono- and difluorotoluene deoxyribosides with varying positions of substitutions. While TK1 did not accept these nonpolar analogues as substrates, TK2 did show varying levels of phosphorylation of the shape-varied set. This latter enzyme preferred toluene nucleoside analogues having steric projections at the 2 and 4 positions, as is found in thymine, and strongly disfavored substitution at the 3-position. Steady-state kinetics measurements showed that the 4-fluoro compound (7) had an apparent V(max)/K(m) value within 14-fold of the natural substrate, and the 2,4-difluoro compound (1), which is the closest isostere of thymidine, had a value within 2.5-fold. The results establish that nucleoside recognition mechanisms for the two classes of enzymes are very different. On the basis of these data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for future bioactive molecules.