Fr Francium

We describe the synthesis and binding properties of oligonucleotides that contain one or more 2′-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2′-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2′-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2′-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson–Crick rules. The 2′-fluoro-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2′-fluoro monomer adopts a C3′-endo/C2′-exo sugar pucker. Molecular modeling of modified duplexes show that the 2′-fluoro-α-L-RNA nucleotides partake in Watson–Crick base pairing and nucleobase stacking when incorporated in duplexes while the unnatural α-L-ribo configured geometry of the sugar is absorbed by changes in the sugar–phosphate backbone torsion angles. The duplex behavior of our new nucleotide follows that of α-L-LNA, by and large.

Conformational tuning: We have studied the novel 2′-F-α-L-RNA monomer as a potential monocyclic mimic of the bicyclic α-L-LNA (see figure). The two nucleotide analogues are conformationally similar but with respect to duplex stability the α-L-LNA monomer outcompetes 2′-F-α-L-RNA.

Ion-channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage-gated ion channels are affected by pore block or modification of voltage dependence of activation/inactivation. Recent experiments performed on various ion channels show that allosteric modulation is an important mechanism for affecting channel function. For instance, in K_Ca2 (formerly SK) channels, the prototypic “blocker” apamin prevents conduction by an allosteric mechanism, while TRPV1 channels are prevented from closing by a tarantula toxin, DkTx, through an interaction with residues located away from the selectivity filter. The recent evidence, therefore, suggests that in several ion channels, the region around the outer mouth of the pore is rich in binding sites and could be exploited therapeutically. These discoveries also suggest that the pharmacological vocabulary should be adapted to define these various actions.

Channel news: Recent studies on various ion channels have shown that allosteric modulation is an important mechanism for affecting channel function; this indicates that the action of channel modulators is more diverse than initially thought. For example, in SK channels, the “allosteric modulator” apamin was proposed to prevent conduction by binding to the rim of the outer pore (see figure).

A single enzyme (NasB), isolated from a soil metagenomic library, can convert branched-chain α-keto acids into branched-chain acyl-ACPs. These intermediates then enter the type-II fatty acid synthase system, yielding branched long-chain acyl-ACP substrates that are used in the biosynthesis of branched long-chain N-acyl amino acids.

Previous studies have demonstrated that some selected amino monoacids and amino diacids can function as leaving groups in the polymerase-catalyzed incorporation of deoxynucleotides into DNA. Among these, the iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) represents an interesting example, as it could overcome some of the problems observed when using L-aspartic acid as the leaving group, that is, poor chain elongation. We have now synthesized and evaluated a series of IDA-dAMP analogues that bear either an extended aliphatic chain in the amino acid function, or a phosphonic acid moiety (substituting for the carboxylic acid function). Among these compounds, the nucleotide with an iminodipropionic acid leaving group (IDP-dAMP) was identified as the best substrate; the excellent single incorporation (91 % conversion to a P+1 strand at 50 μM) was at a substrate concentration ten times lower than that used for IDA-dAMP). This nucleotide also presented improved kinetics and elongation capability compared to IDA-dAMP. The analogues with T, G, and C base moieties were also investigated for their incorporation ability with HIV-1 RT. The incorporation efficiency was found to decrease in the order A>T>G>C. The properties of the iminodipropionic acid as the leaving group surpass those of previously evaluated leaving groups; this acid will be a prime candidate for in vivo testing.

Pyrophosphate mimics: Iminodiacetic acid phosphoramidates of deoxyadenosine monophosphate analogues, either bearing an extended aliphatic chain in the amino acid function or a phosphonic acid moiety, serve as potential substrates for HIV-1 reverse transcriptase. Iminodipropionic acid was found to be an excellent mimic of the pyrophosphate.

Teflon-like globular proteins: Global incorporation of p-fluorophenylalanine (pFF) in the S phosphotriesterase dimer (see figure) was found to stabilize the protein and led to refoldability and improved activity for substrates at elevated temperatures.

Unite in times of deprivation: We report on the use of a chemostat to elicit cryptic biosynthetic pathways in a fungus. Cutlivation of Aspergillus nidulans under N-limiting conditions in a chemostat led to the specific induction of polyketide biosynthesis genes that were otherwise silent. The merger of an anthraquinone with an orsellinic acid-derived oxanthrene yielded two spiroanthrones, sanghaspirodins A and B (see figure).

Synbody building: We demonstrate how off-the-shelf peptides can be transformed into high affinity protein capture reagents that mimic the recognition properties of natural antibodies (see figure). The designer synthetic antibody amplifies the binding affinity of the individual peptides by ∼1000-fold to bind Grb2 with a K_d of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates.

CouAA-lescence: A fluorescent amino acid (CouAA) was site-specifically incorporated into FtsZ, the bacterial tubulin homologue, in living cells. FtsZ_CouAA is seen at the cleavage furrow during cell division to form the Z-ring. These studies provide the first example of a fully functional protein to be visualized in vivo by using a genetically incorporated fluorescent amino acid.

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe^II:ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

Polar opposites: IPNS converts a wide range of substrate analogues into modified β-lactams, including nurmerous tripeptides in which the third amino acid (normally valine) is substituted. However IPNS does not like polar residues in this position. The crystal structure of the protein with a substrate analogue that incorporates D-thio-allo-isoleucine in place of valine sheds light on this predilection.

Pain relief in a trunc: A truncated peptide was shown to retain the structure of the TLR4-binding hot-spot region of MD2 and to disrupt TLR4–MD2 interactions. The peptide not only demonstrated strong binding affinity in a fluorescence-polarization assay, but also showed high specificity in macrophages. Furthermore, MD2-I was able to suppress neuropathic pain in animal models.