A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers.

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2'-isobutyric acid (HPDI). The base acetic acids of thymine 5, Z-cytosine 9, Z-adenine 12, and 6-O-benzyl guanine 17 were prepared and coupled to the immobilized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled.

The retro-inverso form of a homeobox-derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system.

The capacity of a retro-inverso form of a 16-residue peptide from the Antennapedia homeodomain to be taken up by cultured neurones was tested. Like its homologue made of L-amino acids, it was rapidly internalised and distributed throughout the cytoplasm and even the cell nucleus. The amount of retro-inverso peptide in cells after incubation in culture medium was 3.4 times that of the L-peptide form. With a cholesteryl moiety attached to the C-terminus to increase its lipophilicity, the retro-inverso peptide was internalised 8 times better than the L-form. The greater efficiency of the peptidomimetic is probably due to the resistance to proteolytic degradation conferred by the D-amino acids, as the sequence contains two sites sensitive to neuronal endoproteases. The peptide might be the basis for development of system for delivering a variety of molecules into cells.

Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions.

Antipeptide antisera specific for TM-4 and TMBr-3, the two tropomyosin isoforms in neurons, were used to investigate the concentrations and distributions of these F-actin-binding proteins in neurons in vitro and in vivo. TM-4 and TMBr-3 tropomyosins had different developmental profiles. TM-4 was found mainly in immature stages, while the concentration of TMBr-3 increased with maturation. The two isoforms also had different subcellular distributions. TM-4 was concentrated in the growth cones of cultured neurons and, in vivo, in areas where neurites were growing. Later, when development was complete, TM-4 was restricted to postsynaptic sites in the cerebellar cortex, whereas TMBr-3 was found in the presynaptic terminals. These data suggest that the tropomyosin isoforms have different functions, through their interaction with the actin cytoskeleton. TM-4 may be involved in the motile events of neurite growth and synaptic plasticity, while TMBr-3 could play a role in stabilizing neuronal networks and synaptic functioning.

2-Hydroxypropyl-dithio-2'-isobutyric acid (HPDI) as a multipurpose peptide-resin linker for SPPS.

A new type of bifunctional handle for solid-phase peptide synthesis is described that contains both a disulfide and an ester group. Several peptides up to 22 residues long were assembled. Our results demonstrate the stability of this bifunctional handle in the repetitive steps of the Fmoc/tBu protocol and in the corresponding side-chain deprotection conditions. New ways of peptide release were investigated. Cyanolysis in mild conditions led to a C-terminal free-form peptide by a concerted mechanism involving the two groups of the handle. Furthermore, reduction of the disulfide bond, performed with tris-carboxyethyl phosphine, quantitatively releases beta-mercaptoester peptides suitable for biochemical applications (e.g., coupling to protein carriers). Another promising route for peptide release was found: beta-mercaptoester peptides gave, in slightly basic conditions, the C-terminal free-form peptide in good yields and purities. Finally, classical ester cleavage performed with OH or NH3 led to C-terminal free-form or amide peptides, respectively.

Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides.

The use of disulfide bonds for peptide-resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10-20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.

Antibodies cross-reactive with the scorpion-toxin II from Androctonus australis Hector elicited in mice by a synthetic peptide.

With the aim of developing active protection against the noxious effects provoked in humans by scorpion stings, the possibility of eliciting toxin reactive antibodies by immunization with a short peptide was assessed in mice. The amino acid sequence of residues 50 to 59 of Androctonus australis Hector toxin II was chosen, on the basis of previous results indicating that rabbit anti-(50-59) antibodies neutralize the biological effects of the parent toxin. The peptide was prepared by solid-phase synthesis procedures and used in different forms (free, linearly polymerized, coupled to KLH, coupled to a low-molecular weight B-lymphocyte activator) in order to immunize groups of non-congenic NMRI or congenic C57BL/6 mice. The reactivities of each serum with the peptide and with the toxin were assessed in ELISA. Strong reactivities with both the peptide (mean titer over 1:52,600) and the toxin (mean titer 1:800) were observed in all mice from the group that received the KLH-coupled peptide. However, mouse immune sera failed either to recognize the toxin in a liquid-phase radioimmunoassay or to neutralize the lethal effects of the toxin. The requirements, in terms of affinity and recognition of native conformation, for anti-peptide antibodies to display neutralizing properties are discussed.

Disulfide bond as peptide-resin linkage in Boc-Bzl SPPS, for potential biochemical applications.

Use of disulfide bonds for labile linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin). Three bifunctional disulfide handles were synthesized for the introduction of disulfide linkage to the synthesis support. This work showed that only N-Boc aminoethyl 2-propionic acid and N-Boc aminoethyl 2-isobutyric acid were fully compatible with Boc/Bzl peptide synthesis and trifluoromethane sulfonic acid side-chain protection. Qualitative and quantitative synthesis results were comparable to those obtained by conventional peptide synthesis using polyacrylic resins. The resulting peptidyl-resins, which swelled in water or aqueous buffers, may be suitable for various biochemical applications, including use as peptide-resin conjugates for antibody production. Thiolysis by aqueous dithiothreitol released mercapto amide peptides suitable for various uses in solution (e.g., direct coupling with activated protein carrier, specific labeling in the mercapto amide group). Partial thiolysis of the disulfide linkage allowed easy post-synthetic adjustment of the peptide loading of peptidyl-resins.