Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation.

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.

Silicon-Containing Neurotensin Analogues as Radiopharmaceuticals for NTS1-Positive Tumors Imaging.

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 68Ga-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, [68Ga]Ga-JMV6659 exhibits high hydrophilicity (log D7.4 = -3.41 ± 0.14), affinity in the low nanomolar range toward NTS1 (Kd = 6.29 ± 1.37 nM), good selectivity (Kd 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 ([68Ga]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, [68Ga]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. [68Ga]Ga-JMV6659 was identified as a promising lead compound suitable for PET imaging of NTS1-expressing tumors.

Phospholyl(borane) Amino Acids and Peptides: Stereoselective Synthesis and Fluorescent Properties with Large Stokes Shift.

The synthesis of phospholyl(borane) amino acids was stereoselectively achieved by reaction of phospholide anion with iodo α-amino ester derived from l-aspartic acid or l-serine, followed by in situ complexation with borane. Phospholyl(borane) amino acids are easy to store and can be subjected to direct transformation into the corresponding free phospholyl, gold complex, oxide or sulfur derivatives as well as phospholinium salts, thus offering a variety of side chains. After selective deprotection of carboxylic function or amine, C- or N- peptide coupling with an alanine moiety proved the possible incorporation into peptides. Such phospholyl amino acid and peptide derivatives exhibit fluorescent properties with a large Stokes shift (160 nm) and fluorescence up to 535 nm, depending on the phosphole aromaticity and the chemical environment. These phospholyl(borane) amino acids constitute a new class of unnatural amino acids useful for structure-activities relationship studies and appear to be promising fluorophores for the development of labeled peptides.

Silicon-Containing Amino Acids: Synthetic Aspects, Conformational Studies, and Applications to Bioactive Peptides.

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.

Exogenous C-S Lyase Enzyme, a Potential Tool To Release Aromas in Wine or Beer?

Varietal thiols have an impact on the overall aroma of many white, rosé, and red wines and beers. They originate from the metabolism of non-odorant aroma precursors by yeast during the fermentation step, via an intrinsic enzyme, the carbon-sulfur ß-lyase (CSL, EC 4.4.1.13). However, this metabolism is directly dependent upon efficient internalization of aroma precursors and intracellular CSL activity. Consequently, the overall CSL activity converts on average only 1% of the total precursors available. To improve the conversion of thiol precursors during winemaking or brewing, we investigated the possibility of using an exogenous CSL enzyme from Lactobacillus delbrueckii subsp. bulgaricus produced in Escherichia coli. We first implemented a reliable spectrophotometric method to monitor its activity on different related aroma precursors and studied its activity in the presence of various competing analogues and at different pH values. This study allowed us to highlight the parameters to define CSL activity and structural insights for the recognition of the substrate, which pave the way for the use of exogenous CSL for the release of aromas in beer and wine.

Rational design of NT-PSMA heterobivalent probes for prostate cancer theranostics.

Targeting the prostate-specific membrane antigen (PSMA) with radiopharmaceuticals for imaging and/or therapy has demonstrated significant advancement in the management of prostate cancer patients. However, PSMA targeting remains unsuccessful in prostate cancers with low expression of PSMA, which account for 15% of cases. The neurotensin receptor-1 (NTS1) has been highlighted as a suitable oncotarget for imaging and therapy of PSMA-negative prostate cancer lesions. Therefore, heterobivalent probes targeting both PSMA and NTS1 could improve the prostate cancer management. Herein, we report the development of a branched hybrid probe (JMV 7489) designed to target PSMA and/or NTS1 bearing relevant pharmacophores and DOTA as the chelating agent. The new ligand was synthesized with a hybrid approach, which includes both syntheses in batch and in the solid phase. Saturation binding experiments were next performed on HT-29 and PC3-PIP cells to derive K d and B max values. On the PC3-PIP cells, [68Ga]Ga-JMV 7489 displayed good affinity towards PSMA (K d = 53 ± 17 nM; B max = 1393 ± 29 fmol/106 cells) in the same range as the corresponding reference monomer. A lower affinity value towards NTS1 was depicted (K d = 157 ± 71 nM; B max = 241 ± 42 fmol/106 cells on PC3-PIP cells; K d = 246 ± 1 nM; B max = 151 ± 44 fmol/106 cells on HT-29 cells) and, surprisingly, it was also the case for the corresponding monomer [68Ga]Ga-JMV 7089. These results indicate that the DOTA macrocycle and the linker are critical elements to design heterobivalent probes targeting PSMA and NTS1 with high affinity towards NTS1.

Design, Synthesis, and Biological Evaluation of the First Radio-Metalated Neurotensin Analogue Targeting Neurotensin Receptor 2.

Neurotensin receptor 2 (NTS2) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS2 overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS2. JMV 7488 (DOTA-(ßAla)2-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with 68Ga and 111In, and investigated in vitro on HT-29 cells and MCF-7 cells, respectively, and in vivo on HT-29 xenografts. [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 were quite hydrophilic (logD7.4 = -3.1 ± 0.2 and -2.7 ± 0.2, respectively, p < 0.0001). Saturation binding studies showed good affinity toward NTS2 (K D = 38 ± 17 nM for [68Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; K D = 36 ± 4 nM for [111In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS1 binding up to 500 nM). On cell-based evaluation, [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 showed high and fast NTS2-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [111In]In-JMV 7488, respectively, along with low NTS2-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [68Ga]Ga-JMV 7488 on HT-29 and increased for [111In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS2 agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS2. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [68Ga]Ga-JMV 7488 uptake although the mechanism was not NTS2-mediated.

Rhodium(I)-catalyzed borylation of nitriles through the cleavage of carbon-cyano bonds.

The reaction of aryl cyanides with diboron in the presence of a rhodium/Xantphos catalyst and DABCO affords arylboronic esters via carbon-cyano bond cleavage. This unprecedented mode of reactivity for a borylrhodium species allows the regioselective introduction of a boryl group in a late stage of synthesis.

Stereoselective synthesis of unsaturated and functionalized L-NHBoc amino acids, using Wittig reaction under mild phase-transfer conditions.

The stereoselective synthesis of a new amino acid phosphonium salt was described by quaternization of melting triphenylphosphine with the γ-iodo NHBoc-amino ester, derived from L-aspartic acid. The deprotection of the carboxylic acid function to afford the phosphonium salt with a free carboxylic acid group was achieved by a palladium-catalyzed desallylation reaction. This phosphonium salt was used in the Wittig reaction with aromatic or aliphatic aldehydes and trifluoroacetophenone, under solid-liquid phase-transfer conditions in chlorobenzene and in the presence of K(3)PO(4) as weak base, to afford the corresponding unsaturated amino acids without racemization. Thus, the reaction with substituted aldehydes allows to graft various functionalized groups on the lateral chain of the amino acid, such as trifluoromethyl, cyano, nitro, ferrocenyl, boronato, or azido. In addition, the reaction of the amino acid Wittig reagent with α,ß-unsaturated aldehydes leads to amino acids bearing a diene on the lateral chain. Finally, this amino acid phosphonium salt appears to be a new powerful tool for the preparation of unsaturated and non-proteinogenic α-amino acids, directly usable for the synthesis of customized peptides.

Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia.

The endogenous tridecapeptide neurotensin (NT) has emerged as an important inhibitory modulator of pain transmission, exerting its analgesic action through the activation of the G protein-coupled receptors, NTS1 and NTS2. Whereas both NT receptors mediate the analgesic effects of NT, NTS1 activation also produces hypotension and hypothermia, which may represent obstacles for the development of new pain medications. In the present study, we implemented various chemical strategies to improve the metabolic stability of the biologically active fragment NT(8-13) and assessed their NTS1/NTS2 relative binding affinities. We then determined their ability to reduce the nociceptive behaviors in acute, tonic, and chronic pain models and to modulate blood pressure and body temperature. To this end, we synthesized a series of NT(8-13) analogs carrying a reduced amide bond at Lys8-Lys9 and harboring site-selective modifications with unnatural amino acids, such as silaproline (Sip) and trimethylsilylalanine (TMSAla). Incorporation of Sip and TMSAla respectively in positions 10 and 13 of NT(8-13) combined with the Lys8-Lys9 reduced amine bond (JMV5296) greatly prolonged the plasma half-life time over 20 h. These modifications also led to a 25-fold peptide selectivity toward NTS2. More importantly, central delivery of JMV5296 was able to induce a strong antinociceptive effect in acute (tail-flick), tonic (formalin), and chronic inflammatory (CFA) pain models without inducing hypothermia. Altogether, these results demonstrate that the chemically-modified NT(8-13) analog JMV5296 exhibits a better therapeutic profile and may thus represent a promising avenue to guide the development of new stable NT agonists and improve pain management.

Insightful Backbone Modifications Preventing Proteolytic Degradation of Neurotensin Analogs Improve NTS1-Induced Protective Hypothermia.

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 <2 min) represents, however, a serious limitation for its use in pharmacological therapy. In light of this, we report here a structure-activity relationship study in which pairs of NT(8-13) analogs have been developed, based on the incorporation of a reduced Lys8-Lys9 bond. To further stabilize the peptide bonds, a panel of backbone modifications was also inserted along the peptide sequence, including Sip10, D-Trp11, Dmt11, Tle12, and TMSAla13. Our results revealed that the combination of appropriate chemical modifications leads to compounds exhibiting improved resistance to proteolytic cleavages (>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.

Revisiting the evaluation strategy of varietal thiol biogenesis.

The varietal thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-mercapto-4-methylpentan-2-one (4MMP) are key aroma compounds in wine due to the tropical notes they impart. They are released by yeast during alcoholic fermentation from different precursors. However, a large part of 3MH origin remains unknown. In this study, we focused on dipeptide forms arising from glutathione S-conjugates to 3MH and 4MMP. Using labelled tracers, we showed in spiked must the release of varietal thiols from 4 different compounds. We highlighted the interconversion between different forms of precursors under defined enological conditions. Cysteinyl-glycine S-conjugates are partially degraded into cysteine S-conjugates, contrary to γ-glutamyl-cysteine S-conjugates. Glutathione S-conjugate to 3MH can be partially degraded to γ-glutamyl-cysteine S-conjugate to 3MH. For the first time, all these labeled forms of precursors were found to release 3MH or 4MMP between 0.17 and 1% molar conversion yield. Two different yeasts were compared without any significant difference.

First identification and quantification of S-3-(hexan-1-ol)-γ-glutamyl-cysteine in grape must as a potential thiol precursor, using UPLC-MS/MS analysis and stable isotope dilution assay.

Varietal thiols are key aroma compounds in wine issued from multiple and complex origins. Several precursor families have been identified in grapes and must and have been widely studied. But a large part of thiol origin still remains unknown. Thus, we only have an incomplete picture of thiol precursors and there is a lack of knowledge on pre-fermentative mechanisms that can impact their levels. Our study focused on the formal identification and the quantification of new varietal thiol precursors in must. First of all, we synthesized natural and labeled standards using an original multi-step strategy, then we developed and validated a UPLC-MS/MS method that allowed us to identify and quantify for the first time a dipeptide S-conjugate to 3MH, the γGluCys-3MH, in Sauvignon B. We observed the S-4-mercapto-4-methylpentan-2-one-l-cysteinyl-glycine (CysGly-4MMP) and S-4-mercapto-4-methylpentan-2-one-N-(l-γ-glutamyl)-l-cysteine (γGluCys-4MMP) but at too low concentration to be quantified.

Efficient synthesis of (P-chirogenic) o-boronated phosphines from sec-phosphine boranes.

An efficient synthesis of boronated phosphines with an o-phenylene-bridge prepared from sec-phosphine boranes and using benzyne chemistry is reported. Successive reactions of sec-phosphine boranes with n-BuLi and 1,2-dibromobenzene, and then with boron reagents, afford the o-boronatophenylphosphine derivatives in 71% yields. The use of P-chirogenic sec-phosphine boranes leads to the free boronated phosphines with retention of configuration at the P-center after decomplexation. The reaction of P-chirogenic o-boronatophenylphosphine with KHF2 affords the corresponding trifluoroborated phosphine with ee >98%.

o-(Hydroxyalkyl)phenyl P-chirogenic phosphines as functional chiral Lewis bases.

The stereoselective synthesis of P-chirogenic phosphines bearing an o-hydroxyalkyl chelating arm is described. The synthesis is based either on the hydroxyalkylation of P-chirogenic o-bromophenylphosphines (borane) or on their carbonatation and then reduction. The hydroxyalkylation with benzaldehyde or pivalaldehyde affords a mixture of epimers which are isolated by chromatography and characterized by their X-ray structures. Preliminary assays of free P-chirogenic o-(hydroxyalkyl)phenyl phosphines, as new functional Lewis bases in catalyzed asymmetric aza-MBH reaction, lead to ß-aminoester derivatives with ee values up to 74%.

P-chirogenic organocatalysts: application to the aza-Morita-Baylis-Hillman (aza-MBH) reaction of ketimines.

The P-chirogenic organocatalysts were found to promote the enantioselective aza-Morita-Baylis-Hillman reaction of ketimines derived from acyclic α-keto esters. In the P-chirogenic organocatalyzed aza-MBH reactions, α,α-disubstituted α-amino acid derivatives were obtained in high yields with high enantioselectivities (up to 97% ee).

Efficient synthesis of quaternary and P-stereogenic phosphonium triflates.

An efficient and general method for the preparation of achiral and chiral phosphonium salts is reported. This synthesis is based on the quaternization of phosphines and their derivatives with arynes generated in situ from 2-(trimethylsilyl)aryl triflates. This methodology is successfully applied to the synthesis of new valuable P-stereogenic phosphonium triflates.