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International audience; Several independent studies have demonstrated the overexpression of NTS1 in various malignancies, which make this receptor of interest for imaging and therapy. To date, radiolabeled neurotensin analogues suffer from low plasmatic stability and thus insufficient availability for high uptake in tumors. We report the development of Ga-68-radiolabeled neurotensin analogues with improved radiopharmaceutical properties through the introduction of the silicon-containing amino acid trimethylsilylalanine (TMSAla). Among the series of novel radiolabeled neurotensin analogues, [Ga-68]Ga-JMV6659 exhibits high hydrophilicity (log D-7.4 = -3.41 +/- 0.14), affinity in the low nanomolar range toward NTS1 (K-d = 6.29 +/- 1.37 nM), good selectivity (K-d NTS1/Kd NTS2 = 35.9), and high NTS1-mediated internalization. It has lower efflux and prolonged plasmatic half-life in human plasma as compared to the reference compound ([Ga-68]Ga-JMV6661 bearing the minimum active fragment of neurotensin and the same linker and chelate as other analogues). In nude mice bearing HT-29 xenograft, [Ga-68]Ga-JMV6659 uptake reached 7.8 +/- 0.54 %ID/g 2 h post injection. Uptake was decreased to 1.38 +/- 0.71 %ID/g with injection of excess of non-radioactive neurotensin. Radiation dose as extrapolated to human was estimated as 2.35 +/- 0.6 mSv for a standard injected activity of 100MBq. [Ga-68]Ga-JMV6659 was identified as a promising lead compound suitable for PET imaging of NTS1-expressing tumors.

The synthesis of silole amino acids was achieved through hydrosilylation of alkene or alkyne‐containing amino acids with 1‐methyl‐2,3,4,5‐tetraphenyl‐1H‐silole, using Karstedt's catalyst with yield up to 95 % and without epimerization. After selective deprotection of carboxylic acid or amine functions respectively, C‐ or N‐peptide coupling with an alanine moiety proved their possible incorporation into peptides. A model tripeptide was synthesized by solid phase synthesis with the N‐Fmoc protected silole amino acid version. The silole moiety can be also grafted on a precursor peptide directly on the solid support. These amino acids and peptides exhibit AIE properties with λem ca. 500 nm and Δλ ca. 100 nm. This approach constitutes an alternative and promising strategy for incorporation of such AIE fluorogens to peptides.

International audience; Fusion of nonopioid pharmacophores, such as neurotensin, with opioid ligands represents an attractive approach for pain treatment. Herein, the μ-/δ-opioid agonist tetrapeptide H-Dmt-D-Arg-Aba-β-Ala-NH 2 (KGOP01) was fused to NT(8-13) analogues. Since the NTS1 receptor has been linked to adverse effects, selective MOR-NTS2 ligands are preferred. Modifications were introduced within the native NT sequence, particularly a β 3-homo amino acid in position 8 and Tyr 11 substitutions. Combination of β 3 hArg and Dmt led to peptide 7, a MOR agonist, showing the highest NTS2 affinity described to date (K i = 3 pM) and good NTS1 affinity (K i = 4 nM), providing a >1300-fold NTS2 selectivity. The (6-OH)Tic-containing analogue 9 also exhibited high NTS2 affinity (K i = 1.7 nM), with low NTS1 affinity (K i = 4.7 μM), resulting in an excellent NTS2 selectivity (>2700). In mice, hybrid 7 produced significant and prolonged antinociception (up to 8 h), as compared to the KGOP01 opioid parent compound.

Therapeutic hypothermia represents a brain-protective strategy for multiple emergency situations, such as stroke or traumatic injury. Neurotensin (NT), which exerts its effects through activation of two G protein-coupled receptors, namely NTS1 and NTS2, induces a strong and long-lasting decrease in core body temperature after its central administration. Growing evidence demonstrates that NTS1 is the receptor subtype mediating the hypothermic action of NT. As such, potent NTS1 agonists designed on the basis of the minimal C-terminal NT(8-13) bioactive fragment have been shown to produce mild hypothermia and exert neuroprotective effects under various clinically relevant conditions. The high susceptibility of NT(8-13) to protease degradation (half-life 24 h; 16). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla13 (4) and Sip10 (6) or the di-substitution Lys11 - TMSAla13 (12), D-Trp11-TMSAla13 (14), and Dmt11-Tle12 (16) produced sustained hypothermic effects (−3°C for at least 1 h). Importantly, we observed that hypothermia was mainly driven by the increased stability of the NT(8-13) derivatives, instead of the high binding-affinity at NTS1. Altogether, these results reveal the importance of the reduced amine bond in optimizing the metabolic properties of the NT(8-13) peptide and support the development of stable NTS1 agonists as first drug candidate in neuroprotective hypothermia.

Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8–13) (NT(8–13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8–13). We first examined the binding affinities of this novel NT(8–13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article “Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog” published in European Journal of Pharmacology [1] . The reader is directed to the associated article for results interpretation, comments, and discussion.

International audience; Since the discovery of (L)-phosphinothricin in the year 1970, the development of α-amino acids bearing a phosphorus group has been of renewed interest due to their diverse applications, including their use in [18F]-fluorolabeling, as fluorescent probes, as protecting groups and in the reversible immobilization of amino acids or peptide derivatives on carbon nanomaterials. Considerable progress has also been achieved in the field of antiviral agents, through the development of phosphoramidate prodrugs, which increase significantly the intracellular delivery of nucleoside monophosphate and monophosphonate analogues. This review aims to summarize the strategies reported in the literature for the synthesis of P(III), P(IV) and P(V) phosphorus-containing amino acids with P–C, P–O, P–S or P–N bonds in the side chains and their related applications, including their use in natural products, ligands for asymmetric catalysis, peptidomimetics, therapeutic agents, chemical reagents, markers and nanomaterials. The discussion is organized according to the position of the phosphorus atom linkage to the amino acid side chain, either in an α-, β-, γ- or δ-position or to a hydroxyl, thiol or amino group.

Neurotensin (NT) exerts its analgesic effects through activation of the G protein-coupled receptors NTS1 and NTS2. This opioid-independent antinociception represents a potential alternative for pain management. While activation of NTS1 also induces a drop in blood pressure and body temperature, NTS2 appears to be an analgesic target free of these adverse effects. Here, we report modifications of NT at Tyr11 to increase selectivity toward NTS2, complemented by modifications at the N-terminus to impair proteolytic degradation of the biologically active NT(8-13) sequence. Replacement of Tyr11 by either 6-OH-Tic or 7-OH-Tic resulted in a significant loss of binding affinity to NTS1 and subsequent NTS2 selectivity. Incorporation of the unnatural amino acid β3hLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both β3hLys8 and 6-OH-Tic11 into NT(8-13) produced a potent and NTS2-selective analogue with strong analgesic action after intrathecal delivery in the rat formalin-induced pain model with an ED50 of 1.4 nmol. Additionally, intravenous administration of this NT analogue did not produce persistent hypotension or hypothermia. These results demonstrate that NT analogues harboring unnatural amino acids at positions 8 and 11 can enhance crucial pharmacokinetic and pharmacodynamic features for NT(8-13) analogues, i.e., proteolytic stability, NTS2 selectivity, and improved analgesic/adverse effect ratio.

A concise synthesis of ( S )-γ-fluoroleucine is described in five steps from commercially available compounds, with an overall yield of 57%. The Markovnikov hydrofluorination reaction of the unsaturated amino acid precursor as last step of the synthesis proved to be effective. This fluorination can also be performed directly on a short peptide model with the same efficiency. This reaction could be in principle applicable for the preparation of radiolabeled amino acids and peptides.

N-Substituted glycines (NSG) constitute mimics of natural amino acids, and bring stability to peptide analogues. We developed a method to synthesize NSG bearing reactive secondary heterofunctionality. It was shown that persilylation could be used for temporary protection of reactive groups present on the substrate, with the aim of selectively alkylating the amine groups. NSG ready for peptide coupling were obtained by C-terminal deprotection and N-terminal protection of the resulting amino acid derivatives. This method was applied to prepare N-alkylated glycines as analogues of serine, cysteine, tyrosine and tryptophan. The last two derivatives were obtained in good yields and N-homotyrosine (N-hTyr) has been introduced into a peptide sequence of interest using automated solid-phase peptide synthesis (SPPS).

Unnatural α-amino acids form a family of essential molecules used for, among other applications, the synthesis of modified peptides, to improve resistance to proteolytic enzyme degradation, and to modulate physico- and biochemical properties of bioactive peptides as well as chiral inducers in asymmetric synthesis. Among them, silicon-containing unnatural amino acids are becoming an interesting new class of building blocks. The replacement of carbon atoms in bioactive substances with silicon is becoming increasingly popular. Peptides containing silyl amino acids hold great promise for maintaining or reinforcing the biological activity of active compounds, while they simultaneously enhance their resistance to enzyme degradation. In addition, the lipophilicity of the silicon atom facilitates their membrane crossing and their bioavailability. Nowadays, the interest of the pharmaceutical industry in peptide- and protein-based therapies is increasing. In this respect, silicon-containing amino acids and peptides...

International audience; The determination of pKa values for molecules containing multiple acidic groups in solution is challenging both experimentally and theoretically. We propose a general method to obtain these values by combining a graphical analysis based on a predominance diagram, for amino acids and nicotianamine polyacid, with first principle DFT calculations. Implicit and semiexplicit water solvent models were included to account for solvation. This strategy enables the investigation of the protonation states of compounds containing acidic moieties in solution depending on the pH domain. The method was first validated on a set of amino acids with pKa values calculated with an accuracy within 0.5–1.0 pKa unit and then on the chalenging nicotianamine polyacid with six pKa values. This approach is particularly well suited for such a complex system including both zwitterionic structures and unknown experimental pKa values.

Unnatural α-amino acids form a family of essential molecules used for, among other applications, the synthesis of modified peptides, to improve resistance to proteolytic enzyme degradation, and to modulate physico- and biochemical properties of bioactive peptides as well as chiral inducers in asymmetric synthesis. Among them, silicon-containing unnatural amino acids are becoming an interesting new class of building blocks. The replacement of carbon atoms in bioactive substances with silicon is becoming increasingly popular. Peptides containing silyl amino acids hold great promise for maintaining or reinforcing the biological activity of active compounds, while they simultaneously enhance their resistance to enzyme degradation. In addition, the lipophilicity of the silicon atom facilitates their membrane crossing and their bioavailability. Nowadays, the interest of the pharmaceutical industry in peptide- and protein-based therapies is increasing. In this respect, silicon-containing amino acids and peptides are likely to be a significant part of future innovations in this area, and more generally in the area of biomolecules. In this process, commercial availability of silicon-containing amino acids is necessary: new syntheses have been developed, and work in this area is ongoing. This review aims to be a comprehensive and general summary of the different methods used to prepare silicon-containing amino acids and their implications on conformational structures and biological applications when they are incorporated into bioactive molecules.

A method using hydrosilylation of unsaturated amino acids was developed and optimised to obtain silylated amino acids. The platinum (Bu4N)2PtCl6 complex was identified as the best catalyst, and the procedure tolerated different unsaturated substrates, silane reagents and protecting groups. Seven silicon-containing α-amino acids were prepared in an enantiomerically pure form under mild conditions in good yields with orthogonal protections for versatile use in peptide synthesis.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

The silylated amino acid (l)-(trimethylsilyl)alanine (TMSAla) was incorporated at the C-terminal end of the minimal biologically active neurotensin (NT) fragment, leading to the synthesis of new hexapeptide NT[8–13] analogues. Here, we assessed the ability of these new silylated NT compounds to bind to NTS1 and NTS2 receptors, promote regulation of multiple signaling pathways, induce inhibition of the ileal smooth muscle contractions, and affect distinct physiological variables, including blood pressure and pain sensation. Among the C-terminal modified analogues, compound 6 (JMV2007) carrying a TMSAla residue in position 13 exhibits a higher affinity toward NT receptors than the NT native peptide. We also found that compound 6 is effective in reversing carbachol-induced contraction in the isolated strip preparation assay and at inducing a drop in blood pressure. Finally, compound 6 produces potent analgesia in experimental models of acute and persistent pain.

International audience; A concise synthetic pathway yielding to hydrophobic α,α-disubstituted disilylated amino acids based on a hydrosilylation reaction is described. As a first example of utilization in solid-phase peptide synthesis, TESDpg was introduced in replacement of Aib in an alamethicin sequence, leading to analogues with modified physicochemical properties and conserved helical structures. This study highlights the potential of these new amino acids as tools for peptide modulation.

International audience; Stereoselective synthesis of unsaturated α-amino acids was performed by asymmetric alkylation. Two methods were investigated and their enantiomeric excess measured and compared. The first route consisted of an enantioselective approach induced by the Corey–Lygo catalyst under chiral phase transfer conditions while the second one involved the hydroxypinanone chiral auxiliary, both implicating Schiff bases as substrate. In all cases, the use of a prochiral Schiff base gave higher enantiomeric excess and yield in the final desired amino acid.

A new polymer-supported BOP (P-BOP) has been prepared starting from the commercially available polystyrene-bound 1-hydroxybenzotriazole (P-HOBt) and successfully used as a solid-supported reagent for peptide-coupling reactions. Compared to BOP, less epimerization was observed. P-BOP is also a suitable activating reagent for difficult peptide coupling reactions involving α,α-dialkyl amino acids. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The need to replace natural amino acids in peptides with nonproteinogenic counterparts to obtain new medicinal agents has stimulated a great deal of innovation on synthetic methods. Here, we report the incorporation of non-natural silylated amino acids in substance P (SP), the binding affinity for the two hNK-1 binding sites and, the potency to stimulate phospholipase C (PLC) and adenylate cyclase of the resulting peptide. We also assess the improvement of their stability towards enzyme degradation. Altogether, we found that replacing glycine with silaproline (Sip) in position 9 of SP leads to a potent analogue exhibiting an increased resistance to angiotensin-converting enzyme hydrolysis.

Silaproline (Sip) is a proline analogue that exhibits similar conformational properties as the natural amino acid in peptides. Moreover, the presence of a dimethylsilyl group confers to silaproline higher lipophilicity as well as improved resistance to biodegradation. The stereoselective synthesis of protected silaproline and two routes to obtain Fmoc-(L)Sip-OH on gram scale using chiral HPLC resolution are reported. Silaproline was introduced into the sequences of various natural peptides, and the influence of the silylated proline analogues on bioactivity was studied. In particular, considering the importance of polyproline II helices (PPII) in protein–protein molecular interactions and biology, a series of silaproline oligomers from dimer to pentamer were studied and shown to preferentially populate the polyproline type II secondary structure in both chloroform-d and methanol-d4 as shown by circular dichroism (CD), NMR spectroscopy, and molecular modeling.

Tentoxin is a naturally occurring phytotoxic cyclic tetrapeptide excreted by fungi of the Alternaria alternata family. The four total syntheses of tentoxin published to date give poor total yields, mainly owing to two difficulties, the introduction of the dehydro amino acid and more especially the cyclization step. Here we describe a method that stereospecifically introduces Z-dehydrophenylalanine (deltaZPhe) by a modified Erlenmeyer aldolization reaction. The linear tetrapeptide, Boc-R1Ala-Leu-R2deltaZPhe-G1y-OMe (R1, R2: CH3, 14CH3), the precursor of tentoxin, was obtained in a 72% yield from Boc-Leu-Gly-OH. This linear tetrapeptide, labelled with carbon-14, was used for a comparative study of four cyclization reagents DPPA, DCC-PfpOH, HBTU and HATU. This last was the most effective and gave tentoxin in a 81% cyclization yield. The activated ester formed with this reagent displayed an enhanced capacity for cyclization, permitting cyclization in concentrated medium (10 mM). This new synthetic route gave tentoxin in a 60% yield from Boc-Leu-Gly-OH and offers a means of achieving the synthesis of hitherto elusive analogues.

Small cyclopeptides of four to six residues are very interesting for their biological properties. Unfortunately, the synthesis of the linear precursor is generally fastidious and the cyclization often occurs in low yields. Molecular modeling used through the genmol program is a powerful tool for predicting the best precursor, as was shown in a previous paper about five tetrapeptides. However, sometimes all the linear precursors of a cyclopeptide can be unfavorable for cyclization when no structural feature (N-Me amino acid. Pro, D-amino acid) is present in the peptide. This led us to develop a method using a reversible chemical modification of the peptide main chain in order to favor the cisoid conformation able to cyclize easily. Tetraphenylalanine was used as a model, with the tert-butyloxycarbonyl (Boc) group as substituent on the main-chain nitrogen atoms. The cyclization yield increases from less than 1 % to 27% after this chemical modification and cleavage of the Boc groups. Molecular modeling on such molecules shows that this yield increase is due to a preferred conformation having the terminal functions close together induced by the Boc substituents.

N-Bis-trimethylsilylation of α-amino acids using the powerful trimethylsilyl triflate reagent is difficult, and is rendered impossible in the case of bulky side-chains (valine). However, favorable entropy changes resulting from a cyclization reaction allow the formation of “benzostabase” N-diprotections regardless of the side-chain bulk.

A synthesis of Meso-lanthionine with discrimination of the chiral centers is reported. Two cysteine residues of opposite configuration and with orthogonal protections are temporarily and reversibly linked in order to promote the formation of an intramolecular disulfide bridge thus avoiding symmetrization reactions during the sulfur extrusion step.

An improved preparation of the cyclic tetrapeptide Tentoxin is reported employing an azlactone as key intermediate. This new synthetic route offers the advantage over existing methodologies that the dehydro amino acid would easily be varied, thus allowing the simple preparation of analogues.

N-Bis-trimethylsilylation of α-amino acids using the powerful trimethylsilyl triflate reagent is difficult, and is rendered impossible in the case of bulky side-chains (valine). However, favorable entropy changes resulting from a cyclization reaction allow the formation of “benzostabase” N-diprotections regardless of the side-chain bulk.

New alpha,alpha'-disubstituted amino acids with silylated side chains have been synthesized in racemic form. Starting from a Schiff base of glycine tert-butyl ester, a large variety of alpha,alpha'-dialkylated amino acids has been obtained, depending on the alkylating reagents. The application of a hydrosilylation methodology enabled the synthesis of the same unnatural amino acids in an enantiomerically pure form. The ability of these bulky amino acids to be incorporated into peptides by solution-phase methodology has also been demonstrated, since constrained silylated dipeptides have been synthesized. These new lipophilic building blocks could be useful and innovative in the design of peptaibol analogues.

A method using hydrosilylation of unsaturated amino acids was developed and optimised to obtain silylated amino acids. The platinum (Bu4N)2PtCl6 complex was identified as the best catalyst, and the procedure tolerated different unsaturated substrates, silane reagents and protecting groups. Seven silicon-containing α-amino acids were prepared in an enantiomerically pure form under mild conditions in good yields with orthogonal protections for versatile use in peptide synthesis.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

The asymmetric synthesis of a new proline surrogate, incorporating the dimethylsilyl group at position 4 of proline using Schollkopf's bis-lactim ether method, is described.

An improved preparation of the cyclic tetrapeptide Tentoxin is reported employing an azlactone as key intermediate. This new synthetic route offers the advantage over existing methodologies that the dehydro amino acid would easily be varied, thus allowing the simple preparation of analogues.