ChemBioChem

The cover picture shows the envisioned pathways for how the DNA methyltransferase, M.HhaI, can use either S-adenosyl-L-methionine (SAM; left) or an unnatural aziridinium cofactor (right). The aziridinium ion is proposed to arise in situ from the appropriately functionalized 5′ N-mustard precursor. By virtue of its structural similarity to SAM, this highly reactive alkylating agent is bound by a methyltransferase (MTase) and delivered to a specific DNA nucleobase (extrahelical cytosine colored by atom). The stick structures (center) depict the envisioned approach of SAM vs. the proposed aziridinium intermediate to an M.HhaI DNA substrate within the M.HhaI active site. We thank Peter Anderson for assistance in generating the modeling images. The DNA substrate and cofactor structures were adapted from coordinates for the ternary structure of M.HhaI in complex with duplex DNA and S-adenosyl-homocysteine (RCSB PDB). Detailed information regarding this new synthetic DNA alkylating agent and MTase-driven DNA alkylation is reported by S. Rajski and R. Weller on p. 243 ff.

Sea-borne chemistry. Due to their intriguing structures and potent biological activities, marine natural products fascinate chemists and biologists alike. Virtually all marine invertebrates contain associated microorganisms, which are suspected in part to be the biosynthetic source of the extraordinary structural diversity of marine secondary metabolites. The picture shows brunsvicam>ide C, which is produced by a free-living cyanobacterium and structurally closely related to the sponge-derived mozamides.

Special delivery. Amino-functionalized multiwalled carbon nanotubes (NH₂-MWCNTs) were found to interact with negatively charged DNA and the cell membrane (see figure). Through this interaction, NH₂-MWCNTs successfully delivered the GFP (green fluorescent protein) gene into mammalian cells without any cytotoxicity. The results show that NH₂-MWCNT is a novel gene carrier and has potential application in gene delivery.

A broad-spectrum MTase-compatible cofactor has been synthesized. This 5′-N-mustard adenylate is a highly efficient MTase-dependent DNA alkylating agent. Its structural similarity to S-adenosyl-L-methionine and the use of N-mustard chemistry result in a substance that is compatible with a number of different MTases and whose DNA alkylation efficiency far surpasses that of other 5′-aziridine-based cofactors.

A clue towards determining the biosynthetic origin of ansamycin benzoquinone formation by spontaneous oxidation from a corresponding hydroquinone has been provided by ACDL3172 (1), a tricyclic geldanamycin derivative produced by the gel8 gene-inactivated strain of S. hygroscopicus subsp. duamyceticus JCM4427.

Don't stop me now. Anticodon-adjusted tRNA^Ser can be used as unified suppressors of the three types of stop codon. In their presence, translation of pseudonatural mRNAs for DHFR is rendered termination-free and proceeds into the untranslated region (UTR), giving rise to protein–ribosome–mRNA complex with full display of the protein when the translated UTR peptide serving as a spacer arm has a chain-length of ≥50 amino acids (aa). The mRNA template is recovered from the tag-selected complex with a 74-aa UTR in a yield of 2–4 % based on input mRNA.

When DNA duplexes are preformed in water, then DNA-templated synthesis (DTS) can take place efficiently and sequence-specifically in organic solvents with low or minimal water content. This simple method enables reactions that are inaccessible in water, such as pyrrolidine-catalyzed aldol condensation, to be performed. The conditions needed to perform DTS in organic solvents also support DNA-templated reactions that are known to take place in aqueous solution.

The seed of a fold. The conformational state of Alzheimer's peptide Aβ-(1–42) in an aqueous environment has been determined with unprecedented detail by using CD and NMR spectroscopy. Structural data that were integrated by molecular-dynamics simulations highlighted the peptide region that seeds the aggregation process in water (see figure). The nascent β structure that has been identified could represent a target for new pharmaceutical agents against Alzheimer's disease.

Let's stick together. Two-photon fluorescence-correlation spectroscopy allowed the aggregation behavior and relative binding affinities of lectins, such as, soybean agglutinin (SBA) and glycosylated 20 nm fluorescing spheres to be measured (see figure). The assay principle can serve as a model system for studying physical and chemical interactions of unlabeled proteins with carbohydrates, for example, at cell or virus surfaces.

Understanding molecular recognition in cellular immunity. Lipopolysaccharide-binding (LPS-binding) proteins play a major role in responses to bacterial infections, but their ligand-binding domains are poorly defined. Here we have identified an LPS-binding domain of the LPS coreceptor CD14 by screening 20-amino-acid peptides—covering the entire protein—for LPS-neutralizing activity. The importance of three leucine residues facing into the interior of the protein was confirmed by expression of mutant proteins.

Improved precision in solution structures: The structure of hormaomycin in a DMSO-compatible polyacrylamide gel was investigated. In an all-atom superposition, the precision of the ensemble based only on NOE and ³J values, was improved through the incorporation of residual dipolar couplings (RDCs). Without RDC refinement, an overall flexible structure with two major conformations for the macrocyclic ring (in red and blue, top) was derived, whereas after refinement, only one ring conformation remained valid (bottom).

The cytotoxicity of linear polyethylenimine (LPEI22k) can be lowered by using a polypseudorotaxane of LPEI22k and γ-cyclodextrin (LPEI22k/γ-CD), as γ-CD threading decreases the cationic charge of LPEI22k. The high transfection efficiency of LPEI22k was maintained, even though the many secondary amine groups of LPEI22k were included in γ-CDs cavity. Therefore, LPEI22k/γ-CD combines effective transfection and high biocompatibility.

We have synthesized a 140-mer linear polyethylenimine (PEI) ending in a highly nucleophilic hydrazine group. To demonstrate the potential of this PEI as a substrate to prepare synthetic vectors for gene delivery, the building block was regioselectively conjugated to an aldehyde-bearing transferrin. The conjugate (Tf–PEI) was shown to condense plasmid DNA into nanometric particles containing covalently bound transferrin. Moreover, addition of Tf–PEI to PEI/DNA polyplexes increased the transfection efficiency of Tf receptor-expressing K562 cells over that of simple PEI/DNA complexes.

Picking the scope of glycosyltransferases: The synthesis of a series of oligosaccharides, glycoethers and glycoamino acids through the use of a non-Leloir-type glycosyltransferase GTFR with the aid of substrate microarrays has been developed. It provides further understanding of the substrate profiles of glycosyltransferases and simplification of the enzymatic synthesis of a variety of glycoconjugates.

Embracing RNA hairpins: A rapid and modular strategy was developed to construct a series of monomeric and dimeric aminoglycoside–quinacridine conjugates. A dimeric conjugate derived from tobramycin showed remarkable binding selectivity for the P6.1 element of telomerase RNA, which suggested a double wrapping around the stem-loop structure (see schematic representation). A molecular scaffold for small-sized RNA-hairpin recognition, usable for the inhibition of telomerase, was identified.

Add in translation. In vitro deactivation of release factor 1 allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue at a position that was coded by a UAG stop codon. This resulted in the incorporation of puromycin and its analogues into the expressed protein (see figure). This technique can serve as a vehicle for C-terminal conjugation of polypeptides.

Specific recognition by aminoacyl-tRNA synthetases of their cognate amino acid substrate is essential for the translation of the genetic code. For aspartyl-tRNA synthetase, experiments and molecular dynamics simulations are used to perform a triple mutant cycle, revealing long-range electrostatic interactions that determine specificity. The figure shows a model of the enzyme's active site.

Getting a different alcohol. By using error-prone PCR in combination with a novel high-throughput screening specific for primary alcohols, a mutant of the self-sufficient P450 monooxygenase CYP102A3 from Bacillus subtilis with altered regioselectivity was created. While the wild-type enzyme produced a mixture of three secondary alcohols from n-octane and not even a trace of octan-1-ol, a new double mutant formed 48 % octan-1-ol, 52 % of octan-2- and -3-ol and no octan-4-ol.

The enzyme that makes penicillin, isopenicillin N synthase, converts a range of tripeptide substrates to β-lactam products. What happens when the enzyme is challenged with a sterically bulked, depsipeptide analogue and when the reaction is triggered in the crystalline state? X-ray crystallographic analysis reveals an unexpected product (see Figure), apparently formed through oxygen insertion into a primary CH bond in preference to a tertiary CH position.

Bacterial factory. Exogenous galactose and endogenously biosynthesized chitinbiose were used as acceptors for the synthesis of two tetrasaccharides carrying the Lewis x motif (see scheme) by metabolically engineered Escherichia coli strains that overexpress the appropriate glycosyltransferase genes.

The complete nucleotide sequence of the biosynthetic gene cluster for the antitumor depsipeptide thiocoraline (see structure) is reported. Involvement of this cluster in the production of this nonribosomal peptide was demonstrated by insertional inactivation and heterologous expression of the cluster in Streptomyces lividans and S. albus.

Sweeter than sweet. Comparison of the potencies of unsubstituted aspartame-based sweet taste ligands with those of N-arylalkyl-substituted molecules implies that the N-terminal chain above the base of the “L-shaped” structure is responsible for the increased sweetness potency of these molecules. The sweet taste can be amplified in compounds in the L-shaped conformation by an appropriately substituted residue 2 arrayed over the base of the L (see models).