Synthesis of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Synthesis and activity of the melanocortin Xaa‐d‐Phe‐Arg‐Trp‐NH2 tetrapeptides with amide bond modifications

Abstract: The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His‐Phe‐Arg‐Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure–activity studies using the Ac‐His‐d ‐Phe‐Arg‐Trp‐NH₂ tetrapeptide template have been performed in the context of modifying N‐terminal ‘capping’ groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa‐d ‐Phe‐Arg‐Trp‐NH₂ (Xaa = His or Phe) melanocortin tetrapeptides (N‐site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe‐d ‐Phe‐Arg‐Trp‐NH₂ peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

From β‐N‐tert‐butyloxycarbonyl N‐carboxyanhydrides to β‐aminoacid derivatives

Abstract: β‐N‐tert‐butyloxycarbonyl‐N‐carboxyanhydrides are very reactive β‐amino acid derivatives. They react cleanly and smoothly with different nucleophiles like aminoesters, enolates, N‐methyl‐d ‐glucamine, amidoximes to afford in good to excellent yields peptides, β‐amino ketocompounds, β‐aminosugars and functionalized disubstituted 1,2,4‐oxadiazoles.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Synthesis of N‐methylmorpholine N‐(17O‐oxide) and N‐methylmorpholine 15N‐(17O‐oxide)

N‐Methylmorpholine N‐(¹⁷O‐oxide) and N‐methylmorpholine ¹⁵N‐(¹⁷O‐oxide) were prepared from N‐methylmorpholine and ¹⁵N‐methylmorpholine by oxidation with H₂ ¹⁷O₂. The facile one‐pot procedure provided yields of 82 and 76%, respectively. The labeled hydrogen peroxide was obtained by electrolysis of H₂ ¹⁷O followed by autoxidation of 2‐ethylanthraquinol with the molecular oxygen ¹⁷O₂ generated. The compounds serve for mechanistic studies into gold nanoparticle generation in NMMO solution. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel design and synthesis of a radioiodinated glycolipid analog as an acceptor substrate for N‐acetylglucosaminyltransferase V

Guided by the known molecular recognition interactions between N‐acetylglucosaminyltransferase V (GnT‐V) and certain synthetic substrates, we synthesized a radiolabeled double‐stranded glycolipid composed of a long‐chain alkyl unit and a radioiodinated phenylalkyl unit, [¹²⁵I]‐2‐[N‐(2‐hydroxy‐3‐hexadecyloxy)propyl‐15‐(4‐iodophenyl)pentadecanecarboxamido]ethyl 2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl‐(1→2)‐α‐d ‐mannopyranosyl‐(1→6)‐β‐d ‐glucopyranoside ([¹²⁵I]2), as a novel intravital glycolipid mimic substrate of GnT‐V. The radioactive iodine (¹²⁵I) was incorporated via iododestannylation of the phenyltributyltin derivative, 2‐[N‐(2‐acetoxy‐3‐hexadecyloxy)propyl‐15‐(4‐tributylstannylphenyl)pentadecanecarboxamido]ethyl 3,4,6‐tri‐O‐acetyl‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl‐(1→2)‐3,4,6‐O‐acetyl‐α‐d ‐mannopyranosyl‐(1→6)‐2,3,4‐tri‐O‐acetyl‐β‐d ‐glucopyranoside (26). Subsequent deacetylation at the final step afforded [¹²⁵I]2.     

Caution in the use of 2‐iminothiolane (Traut's reagent) as a cross‐linking agent for peptides. The formation of N‐peptidyl‐2‐iminothiolanes with bombesin (BN) antagonist (d‐Trp6,Leu13‐ψ[CH2NH]‐Phe14)BN6−14 and d‐Trp‐Gln‐Trp‐NH2

Abstract: During a study aimed at generating a bispecific molecule between BN antagonist (d ‐Trp⁶,Leu¹³‐ψ[CH₂NH]‐Phe¹⁴)BN_6‐14 (Antag1) and mAb22 (anti‐FcγRI), we attempted to cross‐link the two molecules by introducing a thiol group into Antag1 via 2‐iminothiolane (2‐IT, Traut's reagent). We found that reaction of Antag1 with 2‐IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, d ‐Trp‐Gln‐Trp‐NH₂ (TP1), the N‐terminal tripeptide of Antag1. Reaction of TP1 with 2‐IT for 5 min gave products 1a and 3a ; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a , the expected Traut product, was identified by collecting it in a vial containing N‐methylmaleimide and then isolating the resultant Michael addition product 2a , which was confirmed by mass spectrometry. Thiol 1a is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH₃ to give N‐TP1‐2‐iminothiolane ( 3a ), ES‐MS (m/z) [602.1 (M+H)⁺], as well as regenerating TP1. Repeat reaction with Antag1 and 2‐IT allowed us to isolate N‐Antag1–2‐iminothiolane ( 3b ), FAB‐MS (m/z) [1212.8 (M+H)⁺] and trap the normal Traut product 1b as its N‐methylmaleimide Michael addition product 2b , ES‐MS (m/z) [1340.8 (M+H)⁺]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.     

Synthesis of deuterium and 15N‐labelled 2,5‐Bis[5‐amidino‐2‐pyridyl]furan and 2,5‐Bis[5‐(methoxyamidino)‐2‐pyridyl]furan

The acetate salt of 2,5‐bis[5‐amidino‐2‐pyridyl]furan‐d₂/¹⁵N₂ ( 4) was synthesized from 2,5‐bis[5‐cyano‐2‐pyridyl]furan‐d₂ ( 2 ), through the bis‐O‐acetoxyamidoxime followed by hydrogenation. Compound 2 was obtained via a Stille coupling reaction of 6‐chloronicotinonitrile with 2,5‐bis[tri‐n‐butyltin]‐furan‐d₂ ( 1 ). 2,5‐bis[5‐amidino‐2‐pyridyl)furan‐d₆ ( 10) was synthesized from 2,5‐bis[5‐cyano‐2‐pyridyl)furan‐d₆ ( 9 ) via a direct reaction with lithium bis(trimethylsilyl)amide, followed by deprotection with ethanolic HCl. ¹⁵N and/or deuterium‐labelled methoxy‐amidines 5a ‐d₂/¹⁵N₂, 5b ‐d₈, 12 , 14 ‐d₆ were prepared in good yield via direct methylation of their respective diamidoximes with either dimethylsulfate‐d₀ or dimethylsulfate‐d₆ in DMF solution and using LiOH as a base. Copyright © 2006 John Wiley & Sons, Ltd.     

Characterization of the dopamine D3 receptor agonist R(+)‐7‐hydroxy‐N,N‐di‐n‐propyl‐2‐aminotetralin‐induced hypo‐ and hypermotility in mice

The present study was designed to characterize the dopamine D₃ receptor agonist R(+)‐7‐hydroxy‐N,N‐di‐n‐propyl‐2‐aminotetralin (R(+)‐7‐OH‐DPAT)‐induced changes in locomotor activity in mice. Although R(+)‐7‐OH‐DPAT (0·01–10 mg/kg) produced a significant decrease in horizontal and vertical motility within 15 min after the start of behavioural measurements, the dopamine D₁ receptor antagonist R(+)‐SCH23390 (0·05 mg/kg) and the dopamine D₃ receptor antagonist (+)‐UH232 (10 mg/kg) had no antagonistic effects on the R(+)‐7‐OH‐DPAT (3 mg/kg)‐induced hypomotility, while the dopamine D₂ receptor antagonist S(−)‐sulpiride (20 mg/kg) augmented it. Although R(+)‐7‐OH‐DPAT (0·01–1 mg/kg) had no marked effects on horizontal or vertical motility, higher doses (3 and 10 mg/kg) of the drug produced a significant increase in horizontal or vertical motility from 30 to 90 min after the start of the behavioural measurements. S(−)‐sulpiride (20 mg/kg) and (+)‐UH232 (10 mg/kg) almost completely inhibited the R(+)‐7‐OH‐DPAT (3 mg/kg)‐induced hypermotility, whereas the antagonistic effects of R(+)‐SCH23390 (0·05 mg/kg) were partial. These results suggest that the R(+)‐7‐OH‐DPAT‐induced hypermotility is mediated principally via dopamine D₂ and D₃ receptors, whereas it is unlikely that the hypomotility results from the activation of presynaptic dopamine D₂ or D₃ receptors. Copyright © 1999 John Wiley & Sons, Ltd.     

Analogues of arginine vasopressin modified in the N‐terminal part of the molecule with enantiomers of N‐methylphenylalanine

Abstract: Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N‐methylphenylalanine were synthesized and studied to assess the influence of N‐methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N‐methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me‐d ‐Phe², Me‐Phe³]AVP and [Me‐d ‐Phe^2,3]AVP, which showed low antiuterotonic activity (pA₂ = 6.6 and pA₂ = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.     

Antagonistic effect of N‐ethylmaleimide on arsenic‐mediated oxidative stress‐induced poly(ADP‐ribosyl)ation and cytotoxicity

Long‐term exposure to arsenic has been known to induce neoplastic initiation and progression in several organs; however, the role of arsenic (As₂O₃) in oxidative stress‐mediated DNA damage remains elusive. One of the immediate cellular responses to DNA damage is poly(ADP‐ribosyl)ation (PARylation), which mediates DNA repair and enhances cell survival. In this study, we found that oxidative stress (H₂O₂)‐induced PARylation was suppressed by As₂O₃ exposure in different human cancer cells. Moreover, As₂O₃ treatment promoted H₂O₂‐induced DNA damage and apoptosis, leading to increased cell death. We found that N‐ethylmaleimide (NEM), an organic compound derived from maleic acid, could reverse As₂O₃‐mediated effects, thus enhancing PARylation with attenuated cell death and increased cell survival. Pharmacologic inhibition of glutathione with l ‐buthionine‐sulfoximine blocked the antagonistic effect of NEM on As₂O₃, thereby continuing As₂O₃‐mediated suppression of PARylation and causing DNA damage. Our findings identify NEM as a potential antidote against As₂O₃‐mediated DNA damage in a glutathione‐dependent manner. Copyright © 2016 John Wiley & Sons, Ltd.     

Cis/trans conformational equilibrium across the N‐methylphenylalanine2‐ N‐methylphenylalanine3 peptide bond of arginine vasopressin analogs

Abstract: Two cyclic analogs of vasopressin, ‐Pro‐Arg‐Gly‐NH₂ ( 1 ) and ‐Pro‐Arg‐Gly‐NH₂ ( 2 ) were synthesized by the solid phase method. Their structure was determined in aqueous solution by two‐dimensional NMR techniques and simulated annealing algorithm from an extended template in X‐PLOR. The total chemical shift correlation spectroscopy and rotating‐frame Overhauser enhancement spectroscopy of the peptides displayed four distinct sets of residual proton resonances. This suggests that both analogs adopt four families of conformations in H₂O/D₂O (9 : 1) (one major and three minor species). In further analysis only signals of major species (M) and of one minor species (m₁) were considered. The major species of both peptides include a trans peptide bond between the first and second residue, and a cis form between the second and third residue. In the minor species, all peptide bonds were found to exist in trans geometry.     

Biological activities of 3,4,5‐trihydroxypiperidines and their N‐ and O‐derivatives

3,4,5‐Trihydroxypiperidines belong to the family of 1,5‐dideoxy‐1,5‐iminosugar natural products and are structural analogues of pentose monosaccharides in the pyranose form. The biological activities of these apparently structurally simple molecules and their N‐ and O‐alkylated and ‐arylated derivatives are no less remarkable than their C‐6 hydroxymethyl counterparts of the hexoses (such as 1‐deoxynojirimycin, DNJ). Their biological profiles indicate that the hydroxymethyl branch is crucial to neither potency nor selectivity, with O‐alkylation demonstrated to produce exquisite selectivity extending beyond glycosidase inhibition, to immunosuppressant and antibacterial activities.     

Development of highly potent and ective dynorphin A analogues as new medicines

Abstract: Dynorphin A (Dyn A), a 17 amino acid peptide H‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln‐OH, is a potent opioid peptide which interacts preferentially with κ‐opioid receptors. Research in the development of selective and potent opioid peptide ligands for the κ‐receptor is important in mediating analgesia. Several cyclic disulphide bridge‐containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A_1?11‐NH₂ cyclic analogues incorporating the sulphydryl‐containing amino acids l ‐ and d ‐Cys and l ‐ and d ‐Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the μ‐ and δ‐receptors) as the parent Dyn A_1?11‐NH₂ peptide in the guinea‐pig brain (GPB), but exhibited a much lower activity in the guinea‐pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure–activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at κ‐opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand‐based drug design.     

Simultaneous quantification of phencynonate and its active metabolite N‐demethyl phencynonate in human plasma using liquid chromatography and isotope‐dilution mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously quantify phencynonate (PCN) and its major metabolite N‐demethyl phencynonate (DM‐PCN) in human plasma. Following one‐step liquid‐liquid extraction, the analytes were separated on a reversed‐phase C₁₈ column. Methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was used as isocratic mobile phase at a flow‐rate of 0.3 mL/min. An API 5000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. Multiple reaction monitoring using the transition of m/z 358.4 → m/z 156.2, m/z 344.4 → m/z 142.2, and m/z 361.3 → m/z 159.2 was performed to quantify PCN, DM‐PCN, and the internal standard (D₃‐PCN), respectively. This approach showed a lower limit of quantification of 10 pg/mL and 25 pg/mL for PCN and DM‐PCN in plasma, respectively. This sensitivity was at least 50‐fold superior to previously reported ones and thus enabled the approach well applicable to low‐dose pharmacokinetic studies. The intra‐ and inter‐day precisions were less than 14.2 % at each QC level for both PCN and DM‐PCN. The inter‐day relative errors ranged from ‐1.9% to ‐4.9% for PCN, and from 0.6% to 6.4% for DM‐PCN. As a proof of principle, the validated method was successfully applied to simultaneous quantification of circulating PCN and DM‐PCN in healthy subjects after a single oral administration of 2 mg phencynonate hydrochloride pellet. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of isotopically labelled glycyl‐L‐prolyl‐L‐glutamic acid (Glypromate®) and derivatives

The related tripeptides glycyl‐L ‐prolyl‐L ‐glutamic acid (GPE) and glycyl‐L ‐2‐methylprolyl‐L ‐glutamic acid (G‐2‐MePE) were labelled with commercially available [1,2,3,4,5‐¹³C₅, 2‐¹⁵N₁]‐L ‐glutamic acid in 3 steps in excellent overall yield with high isotope incorporation. A related cyclic dipeptide was labelled with [2,2‐²H₂, 2‐¹⁵N₁]glycine giving a mixture of compounds resulting from deuterium scrambling. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of the perdeuterated cellulose solvents N‐methylmorpholine N‐oxide (NMMO‐d11) and N,N‐dimethylacetamide (DMAc‐d9)

The synthesis of the perdeuterated cellulose solvents NMMO‐d₁₁ (9) and N,N‐dimethylacetamide‐d₉ (14) is described. NMMO‐d₁₁ was obtained according to a five‐step approach from non‐labeled diglycolic acid (1) via diethylene glycole‐d₈ (4) and its bis‐tosylate (5), which underwent cyclization with benzylamine to N‐benzylmorpholine (6). The removal of the benzyl protecting group, methylation and N‐oxidation completed the synthesis. DMAc‐d₉ (14) was obtained from deuterated acetic acid (10) and dimethylamine–carbon dioxide complex (17) with acidic alumina as the catalyst according to a solvent‐free gas–solid reaction. Copyright © 2008 John Wiley & Sons, Ltd.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.