Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays

D-Alanine (D-Ala) is a ubiquitous constituent of bacterial cell walls. Assays for D-Ala can be used to investigate several aspects of cell wall biosynthesis and the effects of antibiotics on this process. High-sensitivity fluorescent assays for D-Ala were developed in a microtiter plate format based on d-aminoacid oxidase/horseradish peroxidase (DAO/HRP)-coupled reactions. For comparative purposes the classic chromogenic (UV-vis) assay using o-phenylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lower limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commercially available fluorogenic HRP substrates were then tested in this assay. Amplex Red (AR) gave a lower limit of sensitivity of 2 pmol and was linear up to 400 pmol d-Ala. QuantaBlu (QB) based assays exhibited a lag in their response to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calibration, but could be eliminated by addition of 150 pmol D-Ala to all assays. The QB assays were linear up to 3000 pmol D-Ala and gave a lower limit of sensitivity of 10 pmol. These assays are demonstrated for the characterization of the dd-carboxypeptidase activity of a soluble form of Escherichia coli penicillin-binding protein 5 (PBP 5) against the classic PBP substrate diacetyl-L-Lys-D-Ala-D-Ala. AR and QB based assays gave identical v/E(T) profiles, whereas OPD based assays gave slightly (10%) higher activity. This is consistent with the loss of a small amount of E. coli PBP 5 activity during the dilution necessary prior to its use in the highly sensitive fluorescent assays. These assays were then demonstrated for characterization of vancomycin binding to a D-Ala-D-Ala-based substrate.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2,Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala2,Leu5]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH3I-KHCO3 in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me3+Tyr1,D-Ala2,Leu5]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH3I-Ag2O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala2,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).     

Synthesis and biological activity of delta Phe4-enkephalins

The delta Phe4-enkephalins have been synthesized and examined in an in vitro receptor binding assay and an in vivo tail flick analgesia test. The delta Phe4 residue was derived from Boc-Gly-Phe(beta-OH)-OH by spontaneous dehydration and azlactonization. The dipeptide azlactone was coupled directly with H-Leu-OBzl to yield a tripeptide which was converted into the pentapeptides after stepwise coupling with two amino acids using the water soluble EDC-HOBt method. Dehydroenkephalins were liberated with hydrogen fluoride in the presence of anisole. In the radioligand binding assay which did not contain an enzyme inhibitor [D-Ala2, delta Phe4, Leu5] enkephalin was almost twice as active as saturated [D-Ala2, D-Leu5]-enkephalin. The delta Phe4-enkephalins exhibited a considerably diminished activity as compared with the saturated peptide in the in vivo analgesic assay. These results are discussed with regard to the enzyme stability and receptor preference of dehydroenkephalins.     

绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Synthesis and biological activity of eight thymulin analogues

Eight analogues of thymulin, a thymic nonapeptide involved in several aspects of T-cell differentiation, were synthesized by the conventional method in solution. Four were modified in residue 7 (Ala, D-Ala, D-Leu or Sar instead of Gly) and two in residue 8 (D-Ser or Thr instead of Ser); in the others, the Gly6-Gly7 sequence was replaced either by a single glycyl residue or by a triglycyl sequence. The biological activity of the analogues was determined in the rosette assay: five exhibited a prolonged activity in vivo with respect to thymulin. All the analogues inhibited the binding of tritiated thymulin to thymulin receptors on three human lymphoblastoid T-cell lines (HSB2, 1301 and CEM) with the same order of magnitude as non-labelled thymulin.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.     

Synthesis and properties of human beta-endorphin-(1-17) and its analogs

Four analogs of human beta-endorphin (beta h-EP) were synthesized by the solid-phase method: beta h-EP-(1-17) (I), [D-Ala2]beta h-EP-(1-17) (II), [Gln8]-beta h-EP-(1-17) (III) and [D-Ala2, Gln8]-beta h-EP-(1-17) (IV). Measurement in a radio-receptor binding assay with use of tritiated beta h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; beta h-endorphin, 2253.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

The metabolism of neuropeptides. Endopeptidase-24.11 in human synaptic membrane preparations hydrolyses substance P.

Synaptic membrane preparations from human striatum and human diencephalon were shown to contain a phosphoramidon-sensitive metalloendopeptidase that appeared identical with endopeptidase-24.11. The activity of endopeptidase-24.11 was determined with an enzymic assay employing [D-Ala2,Leu5]enkephalin as substrate, and its distribution in human brain was similar to that in pig brain, with the striatum containing the highest levels. The choroid plexus and pons also contained substantial activity. A good correlation (r = 0.97) was obtained for the distribution of the endopeptidase in pig brain and pituitary by the enzymic assay and by an immunoradiometric assay specific for pig endopeptidase-24.11. Synaptic membrane preparations from human striatum and diencephalon hydrolysed substance P at the same sites as did preparations of pig striatal synaptic membranes, and hydrolysis was substantially abolished by phosphoramidon. These results suggest that endopeptidase-24.11 is the principal enzyme hydrolysing substance P in human synaptic membrane preparations.     

Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of intravenous, subcutaneous, and in vitro activity of new agmatine analogs of growth hormone-releasing hormone hGH-RH (1-29)NH2

The effects of new Agmatine (Agm) analogs of human growth hormone-releasing hormone (GH-RH) were compared to GH-RH (1-29)NH2 and to (D-Ala2)GH-RH(1-29)NH2 after intravenous (IV) and subcutaneous (SC) administration to pentobarbital-anesthetised male rats and in vitro using superfused rat pituitary cell system. After IV administration, the analogs: (D-MeAla2,Nle27)GH-RH(1-28)Agm(JG-75), (desamino-Tyr1,D-Ala2,Nle27)GH-RH(1-28)Agm(JG-77), (D-Ala2,Nle27)GH-RH(1-28)Agm(JG-73) and (D-Ala2)GH-RH(1-29)NH2 showed a potency 2.6-3.9 times greater than GH-RH(1-29)NH2 at 5 min and 1.6-2.7 times higher at 15 min. After SC administration these analogs were 30-74 times more potent than GH-RH(1-29)NH2. The ratio between the IV and SC GH-releasing activity of the analogs ranged from 2 to 5, while GH-RH(1-29)NH2 was about 50 times more active IV than SC. This indicates that 20-50% of the analogs can be absorbed from SC tissues, but only 2% of GH-RH(1-29)NH2. The in vitro activity of the agmatine analogs on GH release closely paralleled their IV potency and was 2.8-3.9 times greater than that of GH-RH(1-29)NH2. No significant difference in potency was found between (D-Ala2)GH-RH(1-29)NH2 and JG-75 after IV administration and in vitro, although JG-75 contained only 28 amino acids. We conclude that the reason for the large discrepancies between the previously reported activities of (D-Ala2)GH-RH(1-29)NH2 was simply due to the different ways of administration of this analog, SC vs IV, and not to species specificity. The replacement of Arg29 by Agmatine in (D-Ala2,Nle27)GH-RH(1-29)NH2 causes a 3 fold increase in SC potency, but the replacement of D-Ala2 with D-MeAla2 reduces the SC, but not the IV and in vitro activity in half.     

Modeling of Enterococcus faecalis D-alanine:D-alanine ligase: structure-based study of the active site in the wild-type enzyme and in glycopeptide-dependent mutants

A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was produced using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template. The model passed critical validation criteria with an accuracy similar to that of the template crystallographic structure and showed that ADP and methylphosphinophosphate were positioned in a large empty pocket at the interface between the central and the C-terminal domains, as in E. coli. It evidenced the residues important for substrate binding and catalytic activity in the active site and demonstrated a large body of conserved interactions between the active sites of the E. faecalis and the E. coli D-Ala:D-Ala ligase, the major differences residing in the balance between the hydrophobic and aromatic environment of the adenine. The model also successfully explained the inactivity of four spontaneous mutants (D295 --> V, which impairs interactions with Mg2+ and R293, which are both essential for binding and catalytic activity; S319 --> I, which perturbs recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in the vicinity of the deletion remains unaltered but phosphate transfer from ATP is perturbed because of lack of K253; T316 --> I, which causes the loss of a hydrogen bond affecting the positioning of S319 and therefore the binding of D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this approach, combining molecular modeling and molecular biology, may help in the design of specific ligands which could inhibit the enzyme and serve as novel antibiotics.     

Circadian changes of D-alanine and related compounds in rats and the effect of restricted feeding on their amounts

The circadian changes of D-alanine (D-Ala), an intrinsic D-amino acid found in mammals, were investigated in rats with diurnal and nocturnal habits, and the profiles were compared to those of L-Ala, other D-amino acids and several hormones. Determination of D-Ala in the rat plasma, pancreas and anterior pituitary gland was carried out using a sensitive and selective two-dimensional HPLC system combining a micro-ODS column and an enantioselective column after fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The amount of D-Ala was high during the sleeping period and low during the active period in rats with both diurnal and nocturnal habits, indicating for the first time that the D-Ala is closely related to the activity rhythm of animals. In contrast, L-Ala and other D-amino acids did not show any clear circadian changes. The circadian change of D-Ala inversely correlated with that of the plasma insulin level in rats with both diurnal and nocturnal habits. Considered together with our previous findings that D-Ala is localized in the insulin secreting beta-cells in the rat pancreas, it is strongly suggested that D-Ala has some functional relationships to insulin in mammals.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.     

D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared. According to the fluorescence measurements the distance between the aromatic residues in the deltorphin I peptoid and retropeptoid is similar to that suggested for the delta- and micro-opioids, respectively. Measurements of CD in the presence of beta-cyclodextrin, and some preliminary pharmacological experiments were also performed. No dichroic bands are present in the spectrum of the [Ntyr1,D-Ala2]-deltorphin I, but an increasing dichroic effect appears in the spectra of both the deltorphin I peptoid and retropeptoid. Activity tests on isolated organ preparations showed that the modifications made produced a dramatic decrease in the agonistic activity of the synthetic derivatives.