A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ
Graphical abstract
Introduction
Branched peptides have been found to be useful in several research fields including the development of vaccines, as antimicrobial agents, for tumour targeting, etc.1, 2, 3, 4 These molecules are characterized by a central branched scaffold (core) to which a variable number of peptide sequences are linked. Branched peptides can be prepared by using either divergent or convergent approaches. With the former approach, a variable number of peptide arms are assembled by stepwise solid phase peptide synthesis methods on the branched core (in most cases polylysine). Purification of compounds prepared with this procedure is complicated, due to the contamination of the desired product by tens of deletion compounds (lacking one or more amino acid) with chemical-physical properties (molecular weight, charge, polarity, hydrophilicity, etc.) very similar to the desired product. Even using the most efficient purification techniques available, the overall yield of this process is very low and high purity levels cannot be always obtained.1, 5 With the convergent approach the desired product is produced by using a two-step procedure: first, the peptide sequences and the core are separately synthesized and purified; second, peptides and the core are stapled together through mutually reactive functional groups. The crucial issue of the convergent approach is the chemoselectivity of the reaction used to link together the peptide sequences and the core. In fact the presence of several reactive functionalities of the amino acid side-chains impose the use of an extremely chemoselective reaction between the core and the attachment point of the peptide sequence.6, 7, 8, 9, 10 The thiol-Michael reaction perfectly meets this requirement. The reaction occurs between a thiol (the side chain of the Cys residue inserted into the peptide sequence) and an α,β-unsaturated carbonyl group (not present as peptide side chain) to yield a thioether addition product.
Multibranched (in particular tetra branched) peptides have been also synthesised and tested as ligands for peptidergic receptors. Available examples were obtained using the sequences of different peptides including enkephalins, neurotensin, nociceptin/orphanin FQ (N/OFQ),11 and melanocyte stimulating hormone.6 These multibranched peptides displayed high affinity for their receptors associated to a dramatic reduction of susceptibility to peptidase action.11, 12 These features make multibranched peptides interesting candidates as novel ligands for peptidergic receptors.
N/OFQ has been identified as the endogenous ligand13, 14 of a previously orphan receptor now named N/OFQ peptide receptor (NOP). The N/OFQ–NOP receptor system displays structural, functional and transductional similarities with classical opioid systems being however pharmacologically distinct.15 In fact N/OFQ does not bind classical opioid receptors and the NOP receptor does not recognize opioid ligands. Via selective activation of the NOP receptor N/OFQ modulates several important functions both in the central nervous system (pain transmission, anxiety, mood, memory, locomotion, food intake, drug abuse) and in the periphery (cardiovascular, respiratory, gastrointestinal, genitourinary systems).15 Thus NOP selective ligands may be developed as innovative drugs for the treatment of a variety of conditions and pathological states.16 In particular, based on available evidence, NOP receptor agonists can be proposed as analgesics, anxiolytics, as orexigenic agents, as treatment for drug abuse, and for the control of the cough and the micturition reflex. On the other hand, NOP receptor antagonists are worthy of testing as antidepressant and as innovative drugs for the control of Parkinson disease.15
Here we present a novel and facile synthesis of tetra branched derivatives of N/OFQ (PWT1-N/OFQ, PWT2-N/OFQ, and PWT3-N/OFQ) characterized by high yield and purity of the desired final product. In addition the N/OFQ tetra branched derivatives were pharmacologically characterized as NOP ligands in vitro using as N/OFQ sensitive preparation the electrically stimulated mouse vas deferens. Finally, PWT2-N/OFQ was assayed in vivo in mice for its ability to stimulate food intake after supraspinal administration.
Section snippets
Results and discussion
In Scheme 1 the synthesis of the maleimido cores PWT1, PWT2 and PWT3 is reported. The commercially available Boc-Lys(Boc)-OH (1) and H-Lys-NH2 (2) were reacted by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) in dimethylformamide (DMF) in the presence of 1-hydroxybenzotriazole (HOBt) to afford the coupling product [Boc-Lys(Boc)]2-Lys-NH2 which was then treated with trifluoroacetic acid (TFA) to give 3. Coupling of 3 with 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic
Conclusions
Altogether the findings summarized in the present study demonstrated that the PWT chemical strategy can be successfully applied to the N/OFQ peptide sequence to generate with unprecedented high purity and yield tetra branched compounds. These compounds demonstrated an in vitro pharmacological profile superimposable to that of the natural sequence in terms of full agonism and NOP selectivity of action associated to increased potency. In addition the in vivo investigation of PWT2-N/OFQ in food
General
Amino acid derivatives, reagents and solvents were purchased from Sigma Aldrich (Steinheim, Germany) or Bachem (Bubendorf, Switzerland). The purity of the tested compounds PWT1-N/OFQ, PWT2-N/OFQ, and PWT3-N/OFQ has been assessed by RP-HPLC. All compounds showed >95% purity. One-dimensional and two dimensional NMR spectra were recorded on a VARIAN 400 MHz instrument. Chemical shifts are given in ppm (δ) relative to solvent signal and coupling constants are in Hz. MS analyses were performed on a
Acknowledgment
This work was supported by funds from UFPeptides s.r.l.
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