Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

Lack of specificity of [35S]‐ATPγS and [35S]‐ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio[³⁵S]triphosphate ([³⁵S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X₁, P2X₂, P2X₃, and P2X₄. Similarly, adenosine‐5′‐O‐2‐thio[³⁵S]diphosphate ([³⁵S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [³⁵S]‐ATPγS and [³⁵S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X₄ transfected 1321N1 astrocytoma cells. [³⁵S]‐ATPγS (0.2 nM) and [³⁵S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [³⁵S]‐ATPγS and [³⁵S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y₁ and P2Y₂ receptor subtypes, to 1321N1 cells stably transfected with the human P2X₄ receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X₄ receptor, as measured by cytosolic calcium flux. These data indicate that [³⁵S]‐ATPγS and [³⁵S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999. © 1999 Wiley‐Liss, Inc.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic α‐hydroxymethylamino acids

Abstract: New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic α‐hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to μ and δ opioid receptors. The analogue with (R)‐HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to δ and a significant increase of affinity to μ receptors. In contrast, an analogue with (S)‐HmPhe in the same position, was very potent and more specific to δ receptors than parent DT I. The analogue with (R)‐HmVal at position 5 expressed higher δ affinity and selectivity than parent DT I. The analogue with other possible isomer (S)‐HmVal was less selective for δ opioid receptors, as a result of decreasing affinity to δ and increasing affinity to μ receptors. The analogues with (R)‐ or (S)‐HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

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 tracer labelled [11,12‐3H]‐β‐carotene

The synthesis of tracer labelled [11,12‐³H]‐β‐carotene is described. The procedure uses Wittig condensation of tracer labelled ³H‐retinal (retinal spiked with [11,12‐³H]‐retinal) with retinyl triphenylphosphonium bromide. The preparation of tracer labelled[³H]‐β‐carotene is suitable for studies involving bioavailability and bioconversion of β‐carotene to vitamin A. Copyright © 2003 John Wiley & Sons, Ltd.     

Interaction of morphine but not fentanyl with cerebral α2‐adrenoceptors in α2‐adrenoceptor knockout mice

Objectives α₂‐Adrenergic and μ‐opioid receptors belong to the rhodopsin family of G‐protein coupled receptors and mediate antinociceptive effects via similar signal transduction pathways. Previous studies have revealed direct functional interactions between both receptor systems including synergistic and additive effects. To evaluate underlying mechanisms, we have studied whether morphine and fentanyl interacted with α₂‐adrenoceptor‐subtypes in mice lacking one individual α₂‐adrenoceptor‐subtype (α₂‐adrenoceptor knockout). Methods Opioid interaction with α₂‐adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α_2A‐, α_2B‐ or α_2C‐adrenoceptor deficient mice. Displacement of the radiolabelled α₂‐adrenoceptor agonist [¹²⁵I]paraiodoclonidine from α₂‐adrenoceptors in different brain regions by increasing concentrations of morphine, fentanyl and naloxone was analysed. The binding affinity of both opioids to α₂‐adrenoceptor subtypes in different brain regions was quantified. Key findings Morphine but not fentanyl or naloxone provoked dose‐dependent displacement of [¹²⁵I]paraiodoclonidine from all α₂‐adrenoceptor subtypes in the brain regions analysed. Binding affinity was highest in cortex, medulla oblongata and pons of α_2A‐adrenoceptor knockout mice. Conclusions Our results indicated that morphine interacted with α₂‐adrenoceptors showing higher affinity for the α_2B and α_2C than for the α_2A subtype. In contrast, fentanyl and naloxone did not show any relevant affinity to α₂‐adrenoceptors. This effect may have an impact on the pharmacological actions of morphine.     

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.     

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 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.     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

A new [2H]‐labelled α‐trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates

A new reaction pathway for the synthesis of a [²H]‐labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope‐labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

Synthesis,Biological Activity,and NMR‐Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New α,α‐Disubstituted Glycines

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)‐α‐benzyl‐β‐azidoalanine, α‐benzyl‐β‐(1‐pyrrolidinyl)alanine, α‐benzyl‐β‐(1‐piperidinyl)alanine, and α‐benzyl‐β‐(4‐morpholinyl)‐alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)‐α‐benzyl‐β‐azidoalanine in position 3 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. The conformational behavior of peptides modified with (R) or (S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala, which displays the opposite selectivity, was analyzed by ¹H and ¹³C NMR. The μ‐selective Tyr‐d ‐Ala‐(R)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂ lacks the helical conformation observed in the δ‐selective Tyr‐d ‐Ala‐(S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂. Our results support the proposal that differences between δ‐ and μ‐selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N‐terminal message domain and the C‐terminal address domain.     

Synthesis of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[Methyl‐11C]thymine ([11C]FMAU) via a Stille cross‐coupling reaction with [11C]methyl iodide

1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[methyl‐¹¹C]thymine ([¹¹C]FMAU) [¹¹C]‐ 1 was synthesised via a palladium‐mediated Stille coupling reaction of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐5‐(trimethylstannyl)uracil 2 with [¹¹C]methyl iodide in a one‐pot procedure. The reaction conditions were optimized by screening various catalysts and solvents, and by altering concentrations and reaction temperatures. The highest yield was obtained using Pd₂(dba)₃ and P(o‐tolyl)₃ in DMF at 130°C for 5 min. Under these conditions the title compound [¹¹C]‐ 1 was obtained in 28±5% decay‐corrected radiochemical yield calculated from [¹¹C]methyl iodide (number of experiments=7). The radiochemical purity was >99% and the specific radioactivity was 0.1 GBq/μmol at 25 min after end of bombardment. In a typical experiment 700–800 MBq of [¹¹C]FMAU [¹¹C]‐ 1 was obtained starting from 6–7 GBq of [¹¹C]methyl iodide. A mixed ¹¹C/¹³C synthesis to yield [¹¹C]‐ 1 /(¹³C)‐ 1 followed by ¹³C‐NMR analysis was used to confirm the labelling position. The labelling procedure was found to be suitable for automation. Copyright © 2002 John Wiley & Sons, Ltd.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Efficient O‐ and N‐(β‐fluoroethylation)s with NCA [18F]β‐fluoroethyl tosylate under microwave‐enhanced conditions

Reactions of no‐carrier‐added (NCA) [¹⁸F]β‐fluoroethyl tosylate with amine, phenol or carboxylic acid to form the corresponding [¹⁸F]N‐(β‐fluoroethyl)amine, [¹⁸F]β‐fluoroethyl ether or [¹⁸F]β‐fluoroethyl ester, were found to be rapid (2–10 min) and efficient (51–89% conversion) under microwave‐enhanced conditions. These conditions allow reactants to be heated rapidly to 150°C in a low boiling point solvent, such as acetonitrile, and avoid the need to use high boiling point solvents, such as DMSO and DMF, to promote reaction. The microwave‐enhanced reactions gave about 20% greater radiochemical yields than thermal reactions performed at similar temperatures and over similar reaction times. With a bi‐functional molecule, such as DL‐pipecolinic acid, [¹⁸F]β‐fluoroethyl tosylate reacts exclusively with the amino group. Copyright © 2004 John Wiley & Sons, Ltd.     

Substitution of aromatic and nonaromatic amino acids for the Phe3 residue in the δ-selective opioid peptide deltorphin I: Effects on binding affinity and selectivity

Deltorphins I and II (Tyr-D-Ala-Phe-Asp-Val-Val-Gly NH₂ and Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH₂) display a high degree of 6-opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapep-tides based on Tyr-c[D-Cys-Phe-D-Pen] (JOM-13). With the goal of development of structure-activity relationships for deltorphins and comparison with that of the cyclic tetrapep-tides, ten analogs of deltorphin I were synthesized in which Phe³ was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3-(l-naphthyl)-L-alanine [1-Nal; K_i(μ) = 767 nM, K_i(§) = 7.70 nM], 3-(2-naphthyl)-L-alanine [2-Nal; K_i(μ)= 1910 nM, K_i(§) = 49.2 nM], tryptophan [K_i(μ)= 1250 nM, K_i(§) = 23.9nM], and 3-(3-benzothienyl)-L-alanine [Bth; K_i(μ)= 112nM, K_i(§) = 3.36 nM] were fairly well tolerated at μ- and §-receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both μ (K_i= 4710 nM) and § (K_i= 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced μ binding (K_i= 67.8 nM), leaving § affinity unaffected (K_i= 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [K_i(μ) =≥ 10000 nM for each, K_i(§) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both μ (K_i= 322 and 1240 nM, respectively) and § (K_i= 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr-c[D-Cys-Phe-D-Pen].