2,2'-Dithiobis(5-nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups.

Of all the commercially available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of side-chain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during peptide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide connectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide's architecture. It is important to continuously discover new vectors of deprotection for these different blocking protocols in order to achieve the highest degree of orthogonality between the removal of one species in the presence of another. We report here a complete investigation of the scope and limitations of the deprotective potential of 2,2'-dithiobis(5-nitropyridine) (DTNP) on a selection of commercially available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions. Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation of disulfide architecture within a more complex cysteine-containing peptide template.

Binding and toxicity of apamin. Characterization of the active site.

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.

[Basic peptides in been venom, V. Synthesis of four fragments from the sequence of apamin (author's transl)]. / Basische Peptide des Bienengifts, V. Synthese von vier Peptidfragmenten aus der Sequenz des Apamins.

The synthesis of the following four fragments by conventional methods is described: Pos. 15-17 Boc-Cys(SiPr)-Gln(Mbh)-Gln(Mbh) (I) Pos. 10-14 Boc-Leu-Cys(Trt)-Ala-Arg(Tos)-Arg(Tos) (II) Pos. 5-9 Boc-Ala-Pro-Glu(gammaBzl)-Thr-Ala (III) Pos. 1-4 Boc-Cys(SiPr)-Asn-Cys(SiPr)-Lys(Z) (IV) These peptides are fragments of apamin, a basic, neurotoxic peptide from bee venom. The purity of the products was examined by thin-layer chromatography, amino acid and elemental analysis. It is possible to synthesize apamin by liquid-phase fragment condensation coupling these peptides stepwise on a polyethylene-histidine support.

Use of synthetic analogs for a study on the structure-activity relationship of apamin.

[Lys13,Lys14]Apamin, [Lys13]apamin and [Lys14]apamin, three structural analogs of the bee venom neurotoxin, have been obtained by solid-phase peptide synthesis while an attempt to obtain [Cit13]apamin failed, probably at the step of reoxidation of cysteines. After the chemical purity of these three derivatives had been assessed, further chemical modifications led to three new peptides: [Ac-Cys1,Lys(Ac)4,Lys(Ac)13]apamin, [Ac-Cys1,Lys(Ac)4,Lys(Ac)14]apamin and [Har4,Har13,Har14]apamin. These six analogs have been tested for their neurotoxicity, i.e. determination of LD50 for mouse by subcutaneous injection. A lethal potency is observed only when the region 13-14 of the sequence contains a double positive charge. One arginyl residue is necessary for a high biological activity, while its location in position 13 or 14 is of minor importance. When homoarginine (Har) replaces arginyl residues the neurotoxicity is lowered.

Synthesis and conformational analysis of apamin analogues with natural and non-natural cystine/selenocystine connectivities.

By replacing two cysteine residues in apamin with selenocysteine, the three possible isomers related to the side-chain connectivities of a bis-cystinyl-peptide were synthesized in regioselective manner exploiting the low redox potential of the diselenide bond. Nuclear magnetic resonance conformational analysis of monoselenocystine analogue apamin with the natural diselenide/disulfide network confirmed the highly isomorphous character of the sulfur replacement with selenium despite its slightly larger atomic radius and increased bond lengths. The comparative conformational analysis of the apamin analogues containing the non-natural side-chain links with wild type apamin clearly revealed retention of the main structural fold and thus the high propensity of these small molecules to adopt the secondary structure elements present in natural apamin. These findings offered interesting hints for a better understanding of the oxidative refolding pathway of the bis-cystinyl peptide that leads exclusively to the correct natural isomer.

Solid phase synthesis of 13-lysine-apamin, 14-lysine-apamin and the corresponding guanidinated derivatives.

In order to study the importance of arginine residues 13 and 14 in apamin, the bee venom neurotoxin, four analogues, [Lys13]-apamin, [Lys14]-apamin, [Har4, Har13]-apamin and [Har4, har14]-apamin were synthesized and tested with respect to their neurotoxicity. The two lysine-apamins were prepared by the solid phase method on benzhydrylamine resins. Before oxidation to disulphides, the (S-Acm)4-peptides were isolated and characterized. Portions of the purified lysin peptides were converted to homoarginine analogues by guanidination. The four apamin analogues were lethal, but the lethal doses differed significantly. The results demonstrate that the arginine residue at position 14 is more important for the high toxicity than is the one at position 13. The circular dichroism (CD) spectrum of [Lys13]-apamin was identical with that of apamin itself, whereas the spectrum of [Lys14]-apamin showed certain deviations.

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin.

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.

Investigation of the dimethylsulfoxide-trifluoroacetic acid oxidation system for the synthesis of cystine-containing peptides.

Disulfide bonds of peptides were effectively established between S-protected cysteine residues as well as free cysteine residues by the action of dimethylsulfoxide in trifluoroacetic acid. Oxytocin and alpha-human calcitonin gene-related peptide were synthesized using this oxidation system. The feasibility of this method for the formation of two disulfide bridges of apamin was also examined.

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide.

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.

Preparation of a pure monoiodo derivative of the bee venom neurotoxin apamin and its binding properties to rat brain synaptosomes.

The preparation and purification of an active monoiodo derivative of apamin is described. Radiolabeled monoiodoapamin (2000 Ci/mmol) binds specifically to rat brain synaptosomes at 0 degrees C and pH 7.5 with a second order rate constant of association (ka = 2.6 x 10(7) M-1 s-1) and a first order rate constant of dissociation (kd = 3.8 x 10(-4) s-1). The maximal binding capacity is 12.5 fmol/mg of protein and the dissociation constant is 15-25 pM for the monoiodo derivative and 10 pM for the native toxin. The apamin receptor is destroyed by proteases suggesting that it is of a proteic nature. Neurotensin and its COOH-terminal partial sequences are the only molecules unrelated to apamin that are able to displace monoiodoapamin from its receptor at low concentrations. Half-displacement occurs at 170 nM neurotensin. This property is due to the presence in the COOH-terminal sequence of neurotensin of two contiguous arginine residues, a structure analogous to that of the apamin active site. The binding of monoiodoapamin to its receptor is sensitive to cations. Increasing K+ or Rb+ concentrations from 10 microM to 5 mM selectively enhances the binding by a factor of 1.8. Increasing the concentration of any cation from 1 to 100 mM completely inhibits iodoapamin binding. Both effects are due to a cation-induced modulation of the affinity of monoidoapamin for its receptor without any change of the maximal toxin binding capacity of synaptosomes. Guanidinium and molecules containing a guanidinium group are better inhibitors of iodoapamin binding than other inorganic cations or positively charged organic molecules.