Chuanzheng Zhou

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

Mechanistic Studies on Histone Catalyzed Cleavage of Apyrimidinic/Apurinic Sites in Nucleosome Core Particles

Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA-protein cross-link (DPC(un)) formation followed by β-elimination to give DPCs containing cleaved DNA (DPC(cl)). Hydrolysis of DPC(cl) produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.

Nucleosome Core Particle-Catalyzed Strand Scission at Abasic Sites

The reactivity of apurinic/apyrimidinic (AP) sites at different locations within nucleosome core particles was examined. AP sites are greatly destabilized in nucleosome core particles compared to free DNA. Their reactivity varied ~5-fold with respect to the location within the nucleosome core particles but followed a common mechanism involving formation of a Schiff base between histone proteins and the lesion. The identity of the histone protein(s) involved in the reaction and the reactivity of the corresponding DNA-protein cross-links varied with the location of the abasic site, indicating that while the relative rate constants for individual steps varied in a complex manner, the overall mechanism remained the same. The source of the accelerated reactivity was probed using nucleosomes containing AP89 and histone H3 and H4 variants. Mutating the five lysine residues in the amino tail region of histone H4 to arginines reduced the rate constant for disappearance almost 15-fold. Replacing histidine 18 with an alanine reduced AP reactivity more than 3-fold. AP89 in a nucleosome core particle composed of the H4 variant containing both sets of mutations reacted only <4-fold faster than it did in naked DNA. These experiments reveal that nucleosome-catalyzed reaction at AP89 is a general phenomenon and that the lysine rich histone tails, whose modification is integrally involved in epigenetics, are primarily responsible for this chemistry.

Probing interactions between lysine residues in histone tails and nucleosomal DNA via product and kinetic analysis.

The histone proteins in nucleosome core particles are known to catalyze DNA cleavage at abasic and oxidized abasic sites, which are produced by antitumor antibiotics and as a consequence of other modalities of DNA damage. The lysine rich histone tails whose post-translational modifications regulate genetic expression in cells are mainly responsible for this chemistry. Cleavage at a C4′-oxidized abasic site (C4-AP) concomitantly results in modification of lysine residues in histone tails. Using LC-MS/MS, we demonstrate here that that Lys8, -12, -16, and -20 of histone H4 were modified when C4-AP was incorporated at a hot spot (superhelical location 1.5) for DNA damage within a nucleosome core particle. A new DNA–protein cross-linking method that provides a more quantitative analysis of individual amino acid reactivity is also described. DNA–protein cross-links were produced by an irreversible reaction between a nucleic acid electrophile that was produced following oxidatively induced rearrangement of a phenyl selenide derivative of thymidine (3) and nucleophilic residues within proteins. In addition to providing high yields of DNA–protein cross-links, kinetic analysis of the cross-linking reaction yielded rate constants that enabled ranking the contributions by individual or groups of amino acids. Cross-linking from 3 at superhelical location 1.5 revealed the following order of reactivity for the nucleophilic amino acids in the histone H4 tail: His18 > Lys16 > Lys20 ≈ Lys8, Lys12 > Lys5. Cross-linking via 3 will be generally useful for investigating DNA–protein interactions.

5-Formylcytosine Yields DNA–Protein Cross-Links in Nucleosome Core Particles

In situ generation of 5-formylcytosine (5fC) in nucleosome core particles (NCPs) reveals that 5fC leads to essential DNA-protein cross-links (DPCs). Mechanistic studies using chemical models and mutated histones demonstrate that DPCs form reversibly between the formyl function of 5fC and primary amines on histones. These results suggest that DPC formation from 5fC in chromatin occurs in addition to its role in DNA demethylation.

Unusual radical 6-endo cyclization to carbocyclic-ENA and elucidation of its solution conformation by 600 MHz NMR and ab initio calculations.

In our previous paper (J. Am. Chem. Soc., 2007, 129, 8362), we reported the synthesis of 7-Me-Carba-LNA and 8-Me-Carba-ENA thymidine through 5-hexenyl or 6-heptenyl radical cyclization. Both 5-hexenyl and 6-heptenyl radical cyclized exclusively in the exo form, giving unwanted exocyclic C7-methyl group. In the present study, we showed that the regioselectivity of the 5-hexenyl radical cyclization could be favorably tuned by introduction of a hydroxyl group to the olefinic double bond, yielding about 9% of the 6-endo cyclization product. Possible pathways to give 6-endo cyclization product 9 compared to the intermediates responsible to give the 5-exo cyclization product 5 has been discussed. Based on this unique 6-endo cyclization strategy, a carbocyclic ENA modified thymidine (carba-ENA) has been successfully synthesized, which also enabled us to perform its full solution conformation analysis by using NMR (1H at 600 MHz) observables for the first time.

DNA damage by histone radicals in nucleosome core particles.

Although DNA binding proteins shield the genetic material from diffusible reactive oxygen species by reacting with them, the resulting protein (peroxyl) radicals can oxidize the bound DNA. To explore this possible DNA damage by protein radicals, histone H4 proteins containing an azoalkane radical precursor at defined sites were prepared. Photolysis of a nucleosome core particle containing the modified protein produces DNA damage that is consistent with selective C4′-oxidation. The nucleotide(s) damaged is highly dependent on proximity to the protein radical. These experiments provide insight into the effects of oxidative stress on protein-bound DNA, revealing an additional layer of complexity concerning nucleic acid damage.

High-quality oligo-RNA synthesis using the new 2′-O-TEM protecting group by selectively quenching the addition of p-tolyl vinyl sulphone to exocyclic amino functions

During the F--promoted deprotection of the oligo-RNA, synthesized using our 2′-O-(4-tolylsulfonyl)ethoxymethyl (2′-O-TEM) group [Org. Biomol. Chem. 5, 333 (2007)], p-tolyl vinyl sulphone (TVS) is f ...

Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones

5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly, and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

Challenges in the Chemistry of Small Interfering RNA as Potential Therapeutics to Inhibit Cellular mRNA Expression

Small interfering RNAs (siRNAs) have been extensively considered as perspective RNA targeting therapeutics since the discovery of cellular RNA interference (RNAi) machinery. However, chemical engineering of siRNA is required for the therapeutic application. In this review, the challenges involved in siRNA therapeutics is briefly discussed and different chemical modifications that have been introduced to siRNAs to circumvent these challenges in the past ten years are summarized. The current development in siRNA delivery and clinical states of siRNA-based drugs are also briefly reviewed.