Lead-contaminated drinking waters in the public schools of Philadelphia.

UNLABELLED: Lead exposure is a preventable environmental health concern. Young children between the ages of 1 to 6 are most susceptible to its clinical effects. This article reports the results of lead level determinations in the drinking water of Philadelphia's public school buildings and remediation efforts aimed at dealing with this public health concern. METHODS: Water samples were collected from drinking sources in 292 school buildings in Philadelphia from May 2000 through January 2001. These samples were collected and sent to reference laboratories for determination of lead levels. RESULTS: A total of 42.5% (124) of schools had water lead levels not exceeding the action level of 20 ppb, of which 3.1% had nondetectable levels or levels less than 5 ppb. A total of 28.7% of buildings had water lead levels ranging from 20 to 50 ppb, 11.6% had levels between 50-100 ppb, and 17.1% had water lead levels of 100 ppb or more. CONCLUSION: A total of 57.4% of Philadelphia's public school buildings had water lead levels exceeding the Environmental Protection Agencies (EPA) action level of 20 ppb, and 28.7% of school buildings had water with mean lead levels in excess of 50 ppb. Depending on the volume of water consumed, drinking water from school buildings may be a significant source of lead exposure for children in their formative years of development. Although Philadelphia's public school buildings were evaluated, lead-contaminated drinking water in schools is not only an urban concern. School buildings in suburban and rural areas may have similar water lead levels, and testing programs are desirable.

Inhibition of human multidrug resistance P-glycoprotein 1 by analogues of a potent delta-opioid antagonist.

Analogues Dmt-Tic (2',6'-dimethyl-L-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) pharmacophore, a potent delta-opioid receptor antagonist, inhibited hMDR1 P-GP expressed in a G-185 fibroblast cell line in a manner similar to verapamil. N,N(Me)2-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-Ala-NH-1-adamantane and N,N(Me)2-Dmt-Tic-NH-tBut were highly effective inhibitors. Weaker inhibition was observed with N,N(Et)2-Dmt-Tic-OH, H-Dmt-Tic-Ala-NH-tert-butyl amide and cyclo(Dmt-Tic). Results demonstrate that N- and C-terminal hydrophobic/lipophilic analogues of the Dmt-Tic pharmacophore inhibit hMDR1 and point to a potential role as chemosensitizing agents in chemotherapy for cancers containing hMDR1.

Opioid pseudopeptides containing heteroaromatic or heteroaliphatic nuclei.

In lieu of H-Dmt-Tic-OH, H-Dmt-analogues included 2-amino-3(1H-benzoimidazol-2-yl)-propionic acid, N(Bzl)Gly, L-octahydroindole-2-carboxylic acid, [3S-(3alpha,4abeta, 8abeta)]-decahydro-3-isoquinoline carboxylic acid, benzimidazole-, pyridoindole- or spiroinden-derivatives, or C-terminally modified. L- or D-Ala, Sar, or Pro were spacers between aromatic nuclei. Only H-Dmt-(Xaa-)-pyridoindole exhibited high affinities with delta and mu antagonism. The peptides competed equally against [3H]DPDPE (delta agonist) or [3H]N,N(CH3)2-Dmt-Tic-OH (delta antagonist) signaling a single delta binding site. The data confirm the importance of Tic for delta affinity and antagonism, while heterocyclic or heteroaliphatic nuclei, or spacer exert effects on mu- and delta-receptor properties.

Synthesis of stereoisomeric analogues of endomorphin-2, H-Tyr-Pro-Phe-Phe-NH(2), and examination of their opioid receptor binding activities and solution conformation.

All sixteen stereoisomeric analogues of endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)) were synthesized by Fmoc-strategy using solid phase methods. Although synthetic endomorphin-2 exhibited similar mu- and delta-opioid receptor-binding activity to the natural compound, endomorphin-2 analogues containing d-amino acid isomers exhibited lower interaction with mu-receptors depending on the particular combination. The data clearly indicated that the three dimensional structure of endomorphin-2 with the natural l-configuration was the most suitable for binding within the mu receptor, but specific residues are important for activity. Circular dichroism studies verified that changes in chirality of amino acids in the endomorphin-2 sequence resulted in structural conformation. These alterations significantly reduced the specificity for mu-receptor-binding sites.

Inverse agonism by Dmt-Tic analogues and HS 378, a naltrindole analogue.

The potent delta-opioid receptor antagonist H-2',6-L-tyrosine(Dmt)-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic-OH) exhibited partial inverse agonism (EC(50)=6.35 nM, E(max)=-18.87%) for [35S]GTPgammaS binding and H-Dmt-Tic-NH(2) was a neutral antagonist (no effect up to 30 microM). In contrast N,N(CH(3))(2)-Dmt-Tic-NH(2) was a full inverse agonist (EC(50)=2.66 nM, E(max)=-35.95%) similar to ICI 174864 ([N,N-diallyl-Tyr(1),Aib(2,3),Leu(5)]enkephaline) but with a 3.5-fold higher EC(50). In comparison, naltrindole was a neutral antagonist while its analogue HS 378 was a partial inverse agonist (E(max)=-12.99%).

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist.

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective delta opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3',5'-I2)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the delta opioid receptor whereas its binding at mu and kappa opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue ¿35SGTPgammaS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study delta opioid receptors in vitro and in vivo.

Assessment of substitution in the second pharmacophore of Dmt-Tic analogues.

The Dmt-Tic pharmacophore exhibits potent delta-opioid receptor antagonism. Analogues with substitutions in the second pharmacophore with (1, 1') or without a COOH function (2-9) were synthesized: several had high delta affinity (1', 2, 7, and 9), but exhibited low to non-selectivity toward mu receptors similar to H-Dmt-Tic-amide and H-Dmt-Tic-ol. Functional bioactivity indicated high delta antagonism (pA2 7.4-7.9) (1', 2, and 9) and modest mu agonism, pEC50 (6.1-6.3) (1', 2, 8, and 9), but with Emax values analogous to dermorphin. These Dmt-Tic analogues with mixed delta antagonist/mu agonist properties would appear to be better candidates as analgesics than pure mu agonists.

Further studies on the Dmt-Tic pharmacophore: hydrophobic substituents at the C-terminus endow delta antagonists to manifest mu agonism or mu antagonism.

Twenty N- and/or C-modified Dmt-Tic analogues yielded similar K(i) values with either [(3)H]DPDPE (delta(1) agonist) or [(3)H]N, N(Me)(2)-Dmt-Tic-OH (delta antagonist). N-Methylation enhanced delta antagonism while N-piperidine-1-yl, N-pyrrolidine-1-yl, and N-pyrrole-1-yl were detrimental. Dmt-Tic-X (X = -NHNH(2), -NHCH(3), -NH-1-adamantyl, -NH-tBu, -NH-5-tetrazolyl) had high delta affinities (K(i) = 0.16 to 1 nM) with variable mu affinities to yield nonselective or weakly mu-selective analogues. N, N-(Me)(2)Dmt-Tic-NH-1-adamantane exhibited dual delta and mu receptor affinities (K(i)delta = 0.16 nM and K(i)mu = 1.12 nM) and potent delta antagonism (pA(2) = 9.06) with mu agonism (IC(50) = 16 nM). H-Dmt-betaHTic-OH (methylene bridge between C(alpha) of Tic and carboxylate function) yielded a biostable peptide with high delta affinity (K(i) = 0.85 nM) and delta antagonism (pA(2) = 8.85) without mu bioactivity. Dmt-Tic-Ala-X (X = -NHCH(3), -OCH(3), -NH-1-adamantyl, -NHtBu) exhibited high delta affinities (K(i) = 0.06 to 0.2 nM) and elevated mu affinities (K(i) = 2.5 to 11 nM), but only H-Dmt-Tic-Ala-NH-1-adamantane and H-Dmt-Tic-Ala-NHtBu yielded delta receptor antagonism (pA(2) = 9.29 and 9.16, respectively). Thus, Dmt-Tic with hydrophobic C-terminal substituents enhanced mu affinity to provide delta antagonists with dual receptor affinities and bifunctional activity.

Acylcarnitine analogues as topical, microbicidal spermicides.

Acylcarnitine analogues, (+)-6-Carboxylatomethyl-2-alkyl-4,4-dimethylmorpholinium (Z-n, where n = the number of carbons in the alkyl chain), synthesized in multi-gram quantities show in vitro activities as spermicides, anti-HIV agents, and inhibitors of the growth of Candida albicans. Activity improves with increasing chain length. Compound Z-15 is a candidate for further study as a topical, microbicidal spermicide.

Synthesis of pyrazinone ring-containing opioid mimetics and examination of their opioid receptor-binding activity.

Cyclization of dipeptidyl chloromethyl ketones gave 6-(4-aminobutyl)-3-carboxyethyl-5-methyl-2(1H)-pyrazinone, 3-(4-aminobutyl)-6-carboxyethyl-5-methyl-2(1H)-pyrazinone, and 3,6-bis(4-aminobutyl)-5-methyl-2(1H)-pyrazinone, which were inserted into the enkephalin sequence to give opioid mimetics. Thus, it was confirmed that a pyrazinone ring can be easily inserted into a peptide sequence in order to evaluate structural components required for biologically active peptides.

What peptides these deltorphins be.

The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.

Amino acids and peptides. LII. Design and synthesis of opioid mimetics containing a pyrazinone ring and examination of their opioid receptor binding activity.

An amino group was introduced to the 3 or 6 position of a pyrazinone ring by cyclization of dipeptidyl chloromethyl ketones. Boc-Tyr-OH was coupled with the amino function, followed by removal of the Boc group to give pyrazinone ring-containing tyrosine derivatives. Of the various tyrosine derivatives prepared, 5-methyl-6-beta-phenethyl-3-tyrosylaminobutyl-2(1H)-pyrazinone exhibited strong binding to the mu-opioid receptor with a Ki value of 55.8 nM and to the delta-opioid receptor with a Ki value of 2165 nM and with a Ki mu/Ki delta value of 0.026.

Rational design of dynorphin A analogues with delta-receptor selectivity and antagonism for delta- and kappa-receptors.

Substitution of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in place of Gly2 in dynorphin A-(1-13)-NH2 and -(1-11)-NH2 (DYN) analogues (1 and 2) decreased the affinity to the kappa, delta, and mu receptors, and kappa selectivity. The analogue [D-Ala2, des-Gly3]DYN (4), a chimera between deltorphin/dermorphin N-terminal tripeptide and DYN, was virtually inactive for kappa-sites while the affinities for delta- and mu-receptors remained essentially unchanged. The doubly substituted analogue [2',6'-dimethyl-L-tyrosine (Dmt1)-Tic2]DYN (3) exhibited high delta-affinity (Ki=0.39 nM) while mu- and kappa-affinities were only an order of magnitude less (4-5 nM). Bioactivity of [Tic2]DYN peptides (1-3) on guinea-pig ileum and rabbit jejunum revealed potent delta- and kappa-antagonism, while the delta agonist potency of 4 was comparable to DYN. Thus, conversion from a kappa-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with delta- and mu-opioid agonists containing a Tic2 residue.

Evolution of the Dmt-Tic pharmacophore: N-terminal methylated derivatives with extraordinary delta opioid antagonist activity.

The delta opioid antagonist H-Dmt-Tic-OH (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) exhibits extraordinary delta receptor binding characteristics [Ki delta = 0.022 nM; Ki mu/Ki delta = 150,000] and delta antagonism (pA2 = 8.2; Ke = 5.7 nM). A change in chirality of Dmt at C alpha (1, 2, 6, 8, 10, 13) curtailed delta receptor parameters, while replacement of its alpha-amino function by a methyl group (3) led to inactivity; Tyr-Tic analogues 4 and 11 weakly interacted with delta receptors. N-Alkylation of H-Dmt-Tic-OH and H-Dmt-Tic-Ala-OH with methyl groups produced potent delta-opioid ligands with high delta receptor binding capabilities and enhanced delta antagonism: (i) N-Me-Dmt-Tic-OH 5 had high delta opioid binding (Ki delta = 0.2 nM), elevated delta antagonism on mouse vas deferens (MVD) (pA2 = 8.5; Ke = 2.8 nM), and nondetectable mu activity with guinea pig ileum (GPI). (ii) N,N-Me2-Dmt-Tic-OH (12) was equally efficacious in delta receptor binding (Ki delta = 0.12 nM; Ki mu/Ki delta = 20000), but delta antagonism rose considerably (pA2 = 9.4; Ke = 0.28 nM) with weak mu antagonism (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:5640). N-Me-(9) and N,N-Me2-Dmt-Tic-Ala-OH (15) also augmented delta opioid receptor binding, such that 15 demonstrated high affinity (Ki delta = 0.0755 nM) and selectivity (Ki mu/Ki delta = 20132) with exceptional antagonist activity on MVD (pA2 = 9.6; Ke = 0.22 nM) and weak antagonism on GPI (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:7180). Although the amidated dimethylated dipeptide analogue 14 had high Ki delta (0.31 nM) and excellent antagonist activity (pA2 = 9.9; Ke = 0.12 nM), the increased activity toward mu receptors in the absence of a free acid function at the C-terminus revealed modest delta selectivity (Ki mu/Ki delta = 1655) and somewhat comparable bioactivity (GPI/MVD = 4500). Thus, the data demonstrate that N,N-(Me)2-Dmt-Tic-OH (12) and N,N-Me2-Dmt-Tic-Ala-OH (15) retained high delta receptor affinities and delta selectivities and acquired enhanced potency in pharmacological bioassays on MVD greater than that of other peptide or non-peptide delta antagonists.

Helix-inducing alpha-aminoisobutyric acid in opioid mimetic deltorphin C analogues.

The achiral symmetric alpha-aminoisobutyric acid (Aib) replaced the critical N-terminal residues of the amphibian skin opioid deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) without detriment to the physicochemical requirements for delta opioid receptor recognition. Substitutions by the alpha, alpha-dialkyl amino acid in place of D-Ala2 or Phe3, or both, exhibited high delta receptor affinity (Ki delta = 0.12-3.6 nM) and 5-9-fold greater selectivity (Ki mu/Ki delta = 5000-8500) than the parent compound. This is the first definitive demonstration that the D-chirality of alanine and the aromaticity of phenylalanine are replaceable by an achiral alpha, alpha-dialkylated residue without detrimental effects on ligand binding. Incorporation of the mono-alpha-alkyl amino acid L- or D-Ala at the third position also produced highly selective delta ligands (Ki mu/Ki delta = 2000-3500), albeit with reduced delta affinities (Ki delta = 6-15 nM). Replacement of the anionic residue Asp4 by Aib yielded an opioid peptide that fit two-site binding models for the delta receptor (eta = 0.763; P < 0.0001) and displayed dual high affinity for both delta and mu receptors, emphasizing the repulsive effect by a negative charge at mu receptor sites and the insignificance of Asp for delta affinity. Molecular dynamics conformation analyses suggested that Aib residues caused distinct changes in deltorphin C secondary structure when substituted for D-Ala2, Asp4, and simultaneously D-Ala2 and Phe3 but not when substituted for Phe3. These conformational changes might be critical factors for the proper orientation of reactive constituents of residues in the N-terminal region of deltorphin C. Disparities between binding data and functional bioassays of [Aib3] indicated that Phe3 was required for bioactivity in mouse vas deferens but not for interaction with delta opioid receptors in rat brain membranes.

Opioid diketopiperazines: refinement of the delta opioid antagonist pharmacophore.

Bioactive models for a delta opioid receptor antagonist are proposed based on the structurally rigid, diketopiperazine containing cyclo 2',6'-dimethyl-L-tyrosyl (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic). Monte Carlo conformational analysis of c(Dmt-Tic) generated three low energy clusters (I-III) of conformers. The lowest energy conformer representing cluster I superimposed best with the X-ray crystal structures of c(Tyr-Tic), an inactive diketopiperazine with similar framework as c(Dmt-Tic), with H-Tyr-Tic-NH2, a dipeptide of moderate delta opioid affinity and lacking bioactivity, and with H-Tyr-Tic-Phe-Phe-OH (TIPP), a selective and potent delta opioid receptor antagonist. Clusters I and II superimposed best with three different overlays of naltrindole, a potent delta opiate antagonist, and with two other H-Tyr-Tic-NH delta opioid antagonist pharmacophores proposed by Temussi et al. (1994) and Wilkes and Schiller (1995). The 3-dimensional topography of these two clusters of c(Dmt-Tic) conformations may represent bioactive models for interaction of an antagonist at delta opioid receptors. Cluster I conformers exhibited gauche- (- 64 degrees) and gauche+ (53 degrees) orientations of the side chains Dmt and Tic, respectively, while cluster II contained trans (179 degrees) and gauche+ (62 degrees) orientations of those side-chains. Aromatic ring distances were 5.4 A for cluster I conformations and 8.2 A for cluster II structures. Orientation about the peptide bond N-C' was cis (- 5 degrees and 3 degrees) for both clusters, respectively. These structural features may provide optimal alignment of the physicochemical moieties important for delta opioid receptor interaction, such as the hydrophobic methyl groups of Dmt, hydrogen bonding of the dimethyltyrosine hydroxyl group within the receptor pocket and cation-pi interactions involving the aromatic rings of Dmt and Tic, as profiled by the three point attachment hypothesis.

Opioid diketopiperazines: synthesis and activity of a prototypic class of opioid antagonists.

Discovery of high affinity and ultraselective delta opioid dipeptide antagonists composed of 2',6'-dimethyl-L-tyrosyl (Dmt) and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic (Tic) served as the basis for the conformationally restricted diketopiperazine cyclo(Dmt-Tic) and related open chain analogues. These peptides primarily bind to delta opioid receptors: c(Dmt-Tic) displayed 30- to 50-fold higher delta affinity (Ki delta) than its diastereo-isomeric analogues and more than 4000-fold greater than its Tyr cognate; all of the c(Tyr-Tic) analogues were essentially inactive; c[(N-methyl)Dmt-Tic] lost 5-fold in Ki delta, while Ki mu increased 10-fold to yield a nonselective peptide; and the c(Dmt-Phe) series exhibited considerably reduced binding which indicated a synergism between Dmt and Tic in the binding mechanism. Whereas acetyl-Dmt-Tic linear peptides weakly interacted with opioid receptors, Ac-Dmt-Tic-NH2, exhibited better delta antagonist activity than c(Dmt-Tic) and greater delta receptor selectivity (Ki mu/Ki delta = 570). A three point attachment hypothesis for the interaction between c(Dmt-Tic) and the delta receptor was proposed: hydrophobicity imparted by the aromatic rings and the methyl groups of Dmt, hydrogen bonding through the tyramine hydroxyl group, and cation-pi interactions were suggested as contributing factors in binding the diketopiperazine in the receptor pocket. Although c(Dmt-Tic) exhibited a weak antagonist activity with mouse vas deferens, this diketopiperazine may provide a scaffolding for the formation of more potent antagonists for potential therapeutic applications.

Opioid receptor selectivity alteration by single residue replacement: synthesis and activity profile of [Dmt1]deltorphin B.

The single amino acid replacement of 2',6'-dimethyl-L-tyrosine in deltorphin B (H-Dmt-D-Ala-Phe-Glu-Val-Val-Gly-NH2) yielded high affinity for mu- and delta-binding sites. [Dmt1]Deltorphin B lacks activity at kappa-opioid binding sites. Bioactivity in vitro with guinea-pig ileum confirmed that [Dmt1]deltorphin B interacted with mu-opioid receptors by reducing electrically induced contractions in a naloxone-reversible manner and was 150-fold more potent than morphine and comparable to [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO). The inhibition of spontaneous contractions of rabbit jejunum provided evidence for delta-opioid receptor interaction. Analgesia (hot plate and tail flick tests) revealed that [Dmt1]deltorphin B was 180- to 200-fold more potent than morphine. Pretreatment with naloxone, naltrindole or H-Dmt-Tic-Ala-OH (a highly selective delta-opioid receptor antagonist) prevented [Dmt1]deltorphin B antinociception. Thus, [Dmt1]deltorphin B exhibited remarkably high dual affinity and bioactivity toward delta- and mu-opioid receptors.

Design and synthesis of 1-aminocycloalkane-1-carboxylic acid-substituted deltorphin analogues: unique delta and mu opioid activity in modified peptides.

Deltorphin analogues were substituted by a series of achiral C alpha,alpha-dialkyl cyclic alpha-amino acids (1-aminocycloalkane-1-carboxylic acids, Ac chi c, where chi = a hexane, pentane, or propane cycloalkane ring) in position 2, 3, 4, or 2 and 3 in deltorphin C, and in position 2 in [Ac6c2,-des-Phe3]deltorphin C hexapeptide. Receptor assays indicated that even though Ac6c2 and Ac6c3 exhibited a diminished Ki delta by ca. 20-fold (2.5-3.3 nM) relative to deltorphin C (Ki delta = 0.15 nM), selectivity was marginally elevated (Ki mu/Ki delta = 1250) or enhanced by about 70%, and both peptides fitted stringent iterative calculations for a two-site binding model (eta = 0.625 and 0.766, respectively, P < 0.0001). The disubstituted [Ac6c2,3]- or [Ac6c2,des-Phe3]deltorphin analogues yielded peptides with decreased Ki delta, such that the latter peptide was essentially inactive. The presence of Ac5c or Ac3c in place of Phe3 further diminished Ki delta (15.4 to 19.0 nM), yet delta selectivity only fell about one-half (Ki mu/Ki delta = 440 and 535, respectively), and only the former peptide fitted a two-site binding model (eta = 0.799). The replacement of Asp4 by Ac6c, Ac5c, or Ac3c produced essentially nonselective analogues through the acquisition of high mu affinities (2.5, 0.58 and 0.27 nM, respectively) while maintaining high delta affinities (Ki delta = 0.045-0.054 nM) which were about 3-fold greater than that of deltorphin C. Using pharmacological assays in vitro (mouse vas deferens and guinea pig ileum), position 3-substituted analogues all indicated substantial losses in bioactivity, whereas substitution by 1-aminocycloalkanes at the fourth position retained high delta activity. In fact, the bioactivity of [Ac3c4]deltorphin C indicated a peptide with relatively weak delta selectivity, which was comparable to the observations with the receptor binding data. In summary, the data confirmed that (i) delta selectivity occurs in the absence of D-chirality at position 2, (ii) the aromaticity of Phe3 is replaceable by an achiral residue with a hydrophobic ring-saturated side chain, and (iii) the acquisition of dual high-affinity analogues occurs through the elimination of the anionic function at position 4 and replacement by an amino acid with a hydrophobic side chain.