Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

Synthesis of N‐(3‐[18F]Fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane ([18F]FP‐β‐CIT)

N‐(3‐[¹⁸F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane ([¹⁸F]FP‐β‐CIT) was synthesized in a two‐step reaction sequence. In the first reaction, 1‐bromo‐3‐(nitrobenzene‐4‐sulfonyloxy)‐propane was fluorinated with no‐carrier‐added fluorine‐18. The resulting product, 1‐bromo‐3‐[¹⁸F]‐fluoropropane, was distilled into a cooled reaction vessel containing 2β‐carbomethoxy‐3β‐(4‐iodophenyl)‐nortropane, diisopropylethylamine and potassium iodide. After 30 min, the reaction mixture was subjected to a preparative HPLC purification. The product, [¹⁸F]FP‐β‐CIT, was isolated from the HPLC eluent with solid‐phase extraction and formulated to yield an isotonic, pyrogen‐free and sterile solution of [¹⁸F]FP‐β‐CIT. The overall decay‐corrected radiochemical yield was 25 ± 5%. Radiochemical purity was > 98% and the specific activity was 94 ± 50 GBq/µmol at the end of synthesis. Copyright © 2006 John Wiley & Sons, Ltd.     

Deleterious effects of non‐synonymous single nucleotide variants of human IL‐1β gene

The IL‐1β gene is currently topic of interest for its important role in the pathogenesis of intervertebral disk degeneration. The new sequencing technology makes it crucial to study the effects of variants in IL‐1β. Thus, 714 IL‐1β variants with evidence supporting were collected from the EMBL database. Among them, 62 were non‐synonymous single nucleotide variants (nsSNVs). Furthermore, six common nsSNVs were predicted to have damaging effects by SIFT, PolyPhen, PROVEAN and SNPs&GO. Based on the constructed three‐dimensional structure of pro‐IL‐1β, rs375479974 with a mutation of Phe to Ser was proposed to reduce the stability of the pro‐IL‐1β protein. The rs375479974 variant was found to cause least common stabilizing amino acid residues, decrease hydrophilic and increase hydrophobic surface areas in the greatest degree, and have the lowest free energy alterations in I‐Mutant 2.0 sequence analysis. When analyzing the interaction between the experimental 3D structure of mature IL‐1β and its neutralizing McAb canakinumab complex, the rs775174784 substitution of Leu with Phe was found to attenuate this interaction by reducing binding energy, while rs375479974 not. Molecular dynamics simulation results in intervertebral disk environment supported rs775174784's effects. These results suggest that both rs375479974 and rs775174784 may have potential clinical and drug target implications.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

Radiosynthesis of 1‐(4‐(2‐[18F]fluoroethoxy)benzenesulfonyl)‐3‐butyl urea: a potential β‐cell imaging agent

Tolbutamide ( 1 ) is a sulfonurea agent used to stimulate insulin secretion in type 2 diabetic patients. Its analogue 1‐(4‐(2‐[¹⁸F]fluoroethoxy)benzenesulfonyl)‐3‐butyl urea ( 3 ) was synthesized in overall radiochemical yields of 45% as a potential β‐cell imaging agent. Compound 3 was synthesized by ¹⁸F‐fluoroalkylation of the corresponding hydroxy precursor ( 2 ) with 2‐[¹⁸F]fluoroethyltosylate in DMF at 120°C for 10 min followed by purification with HPLC in a synthesis time of 50 min. Insulin secretion experiments of the authentic ¹⁹F‐standard compound on rat islets showed that the compound has a similar stimulating effect on insulin secretion as that of tolbutamide ( 1 ). The partition coefficient of compound 3 between octanol/water was determined to be 1.3±0.3 (n=5). Copyright © 2002 John Wiley & Sons, Ltd.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N‐terminal part of the molecule with l‐β‐homophenylalanine

Abstract: In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l ‐β‐homophenylalanine (β‐Hph). Two of them had just this single modification, the next two peptides are analogues of the V₂ agonist, namely [3‐mercaptopropionic acid (Mpa)¹]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V_1a antagonist, [1‐mercaptocyclohexaneacetic acid (Cpa)¹]AVP, with β‐Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph³]AVP, turned out to be uterotonic antagonists with pA₂ = 6.3 ± 0.2, 6.3 ± 0.1, 6.0 ± 0.3 respectively. The last one exhibited antipressor properties also (pA₂ = 6.4 ± 0.1).     

Synthesis and Evaluation of 3‐(furo[2,3‐b]pyridin‐3‐yl)‐4‐(1H‐indol‐3‐yl)‐maleimides as Novel GSK‐3β Inhibitors and Anti‐Ischemic Agents

A series of novel 3‐(furo[2,3‐b]pyridin‐3‐yl)‐4‐(1H‐indol‐3‐yl)‐maleimides were designed, synthesized, and biologically evaluated for their GSK‐3β inhibitory activities. Most compounds showed favorable inhibitory activities against GSK‐3β protein. Among them, compounds 5n , 5o , and 5p significantly reduced GSK‐3β substrate tau phosphorylation at Ser396 in primary neurons, indicating inhibition of cellular GSK‐3β activity. In the in vitro neuronal injury models, compounds 5n , 5o , and 5p prevented neuronal death against glutamate, oxygen–glucose deprivation, and nutrient serum deprivation which are closely associated with cerebral ischemic stroke. In the in vivo cerebral ischemia animal model, compound 5o reduced infarct size by 10% and improved the neurological deficit. The results may provide new insights into the development of novel GSK‐3β inhibitors with potential neuroprotective activity against brain ischemic stroke.     

Synthesis of (R)‐ and (S)‐[O‐methyl‐11C]N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐methoxy‐phenyl)‐urea as candidate high affinity β1‐adrenoceptor PET radioligands

Molecular imaging and quantification of myocardial β₁‐adrenoceptor (AR) rather than total β‐AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β₁‐AR density is reduced in patients with chronic heart failure whereas cardiac β₂‐AR density may vary. Positron emission tomography (PET), with appropriate radioligands, offers the possibility to assess β‐AR density non‐invasively in humans. However, no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Here some derivatives of the well characterized β₁‐AR selective antagonist, ICI 89,406, namely the enantiomers of N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐hydroxy‐phenyl)‐urea ( 5a and 5b ) were synthesized and evaluated in vitro. The (R)‐isomer 5a was more β₁‐selective but has lower affinity than its (S)‐enantiomer 5b (β₁‐AR selectivity: 6100 vs 1240; β₁‐affinity: K₁ = 0.288 nM vs K₁ = 0.067 nM). Etherification of the analogous desmethyl precursors, 5e and 5f , respectively, with [¹¹C]iodomethane gave ¹¹C‐labelled versions of 5a and 5b , namely 5g and 5h , in 44 ± 5% radiochemical yield (decay‐corrected) and 97.4 ± 1.3% radiochemical purity with specific radioactivities of 26.4 ± 9.4 GBq/µmol within 41.2 ± 3.4 min from the end of bombardment (n = 14). 5g and 5h are now being evaluated as candidate radioligands for myocardial β₁‐ARs. Copyright © 2005 John Wiley & Sons, Ltd.     

Simplified synthesis of N‐(3‐[18F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([18F]β‐CFT‐FP) using [18F]fluoropropyl tosylate as the labelling reagent

A synthesis method has been developed for the labelling of N‐(3‐[¹⁸F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([¹⁸F]β‐CFT‐FP), a potential radioligand for visualization of the dopamine transporters by positron emission tomography. The two‐step synthesis includes preparation of [¹⁸F]fluoropropyl tosylate and its use without purification in the fluoroalkylation of 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane (nor‐β‐CFT). The final product is purified by HPLC. Optimization of the two synthesis steps resulted in a greater than 30% radiochemical yield of [¹⁸F]β‐CFT‐FP (decay corrected to end of bombardment). The synthesis time including HPLC‐purification was approximately 90 min. The radiochemical purity of the final product was higher than 99% and the specific radioactivity at the end of synthesis was typically 20 GBq/µmol. In comparison to alkylation by [¹⁸F]fluoropropyl bromide, the procedure described here results in an improved overall radiochemical yield of [¹⁸F]β‐CFT‐FP in a shorter time. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of [13C2, 15N]‐1,3‐2H‐1‐benzyl‐(Z)‐3‐(benzylidene)indolin‐2‐one

Parkinson disease (PD) is a neurodegenerative disorder characterized by the accumulation of α‐synuclein into Lewy bodies. 3‐Benzylidine‐indolin‐2‐one represents a class of compounds, which are known to inhibit the accumulation of α‐synuclein. In this paper, we report the synthesis of [¹³C] and [¹⁵N] labelled 1‐benzyl‐(Z)‐3‐(benzylidene)indolin‐2‐one from commercially available [¹³C₂]‐chloroacetic acid and [¹⁵N]‐aniline in five steps. The product will be used to study its metabolites in human liver microsomes by liquid chromatography‐tandem mass spectrometry.     

Synthesis and preliminary characterization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase‐3β (GSK‐3β) in the brain

We report on the synthesis and preliminary characterization of two radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimides, 3‐(benzofuran‐3‐yl)‐4‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]5), and 3‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐4‐(6‐methoxybenzofuran‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase‐3β (GSK‐3β). In this study, we used ¹²⁵I as a surrogate of ¹²³I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10–30%. In vitro binding experiments suggested that both compounds show high affinity for GSK‐3β at a level similar to a known GSK‐3β inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK‐3β.     

Synthesis of (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐ 3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate‐1‐14C

γ‐Cyhalothrin ( 1a ), (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate, is a single‐isomer, synthetic pyrethroid insecticide marketed by Pytech Chemicals GmbH, a joint venture between Dow AgroSciences and Cheminova A/S. As a part of the registration process there was a need to incorporate a carbon‐14 label into the cyclopropyl ring of this molecule. A high yielding radiochemical synthesis of γ‐cyhalothrin was developed from readily available carbon‐14 labeled N‐t‐Boc protected glycine. This seven step synthesis, followed by a preparative normal phase HPLC separation of diastereomers, provided 21.8 mCi of γ‐cyhalothrin‐1‐¹⁴C ( 1b ) with >98% radiochemical purity. Copyright © 2007 John Wiley & Sons, Ltd.     

Radiosynthesis of the α4β2 nicotinic acetylcholine receptor ligand: 5‐((1‐[11C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)vinyl)pyridine

5‐((1‐[¹¹C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)‐vinyl)pyridine ([¹¹C]‐FPVC) was synthesized from [¹¹C]‐methyl iodide and the corresponding normethyl precursor. The average time of synthesis, purification, and formulation was 42 min with an average non‐decay‐corrected radiochemical yield of 19%. The average specific radioactivity was 359 GBq/µmol (9691 mCi/µmole) at end of synthesis (EOS). Copyright © 2006 John Wiley & Sons, Ltd.     

Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution‐phase synthesis and circular dichroism study of protected 17‐residue partial peptides in the first putative transmembrane domain*

Abstract: Charcot‐Marie‐Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc‐IVLH(Bom)VAVLVLLFVSTIV‐OMe ( 1 ) and its Pro¹⁶ mutant Boc‐IVLH(Bom)VAVPVLLFVSTIV‐OMe ( 2 ) were synthesized. Circular dichorism (CD)‐spectral analysis suggested that peptide 1 adopts a stable α‐helical conformation in membrane‐mimetic solvent,1‐BuOH/1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) system. On the contrary, the mutant 2 favors β‐sheet conformation in the same solvent system. Interestingly, α‐helix to β‐sheet transition of 2 was observed at higher contents of 1‐BuOH than 70%.     

Synthesis of [guanido‐13C]‐γ‐hydroxyarginine

This report describes an efficient method of synthesizing [guanido‐¹³C]‐γ‐hydroxyarginine HCl salt. Iodolactonization of N‐Boc‐protected allylglycine mainly provided the cis iodo compound 2. This was converted to an amine through azide 4. The amine 5 was reacted with N‐Boc‐protected [¹³C]thiourea to afford N‐Boc‐protected [¹³C]guanidine 6, which underwent base catalyzed ring opening. Removal of the N‐Boc group afforded [guanido‐¹³C]‐γ‐hydroxyarginine HCl salt 7 giving a 30% overall yield of the final product from N‐Boc protected allylglycine 1 in five steps. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.