Neuroprotective and Anti-inflammatory Effects of Rubiscolin-6 Analogs with Proline Surrogates in Position 2.

Naturally occurring peptides, such as rubiscolins derived from spinach leaves, have been shown to possess some interesting activities. They exerted central effects, such as antinociception, memory consolidation and anxiolytic-like activity. The fact that rubiscolins are potent even when given orally makes them very promising drug candidates. The present work tested whether rubiscolin-6 (R-6, Tyr-Pro-Leu-Asp-Leu-Phe) analogs have neuroprotective and anti-inflammatory effects. These hypotheses were tested in the 6-hydroxydopamine (6-OHDA) injury model of human neuroblastoma SH-SY5Y and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The determination of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), Caspase-3 activity, lipid peroxidation and nitric oxide (NO) production allowed us to determine the effects of peptides on hallmarks related to Parkinson's Disease (PD) and inflammation. Additionally, we investigated the impact of R-6 analogs on serine-threonine kinase (also known as protein kinase B, AKT) and mammalian target of rapamycin (mTOR) activation. The treatment with analogs 3 (Tyr-Inp-Leu-Asp-Leu-Phe-OH), 5 (Dmt-Inp-Leu-Asp-Leu-Phe-OH) and 7 (Tyr-Inp-Leu-Asp-Leu-Phe-NH2) most effectively prevented neuronal death via attenuation of ROS, mitochondrial dysfunction and Caspase-3 activity. Peptides 5 and 7 significantly increased the protein expression of the phosphorylated-AKT (p-AKT) and phosphorylated-mTOR (p-mTOR). Additionally, selected analogs could also ameliorate LPS-mediated inflammation in macrophages via inhibition of intracellular generation of ROS and NO production. Our findings suggest that R-6 analogs exert protective effects, possibly related to an anti-oxidation mechanism in in vitro model of PD. The data shows that the most potent peptides can inhibit 6-OHDA injury by activating the PI3-K/AKT/mTOR pathway, thus playing a neuroprotective role and may provide a rational and robust approach in the design of new therapeutics or even functional foods.

The Therapeutic Potential of Naturally Occurring Peptides in Counteracting SH-SY5Y Cells Injury.

Peptides have revealed a large range of biological activities with high selectivity and efficiency for the development of new drugs, including neuroprotective agents. Therefore, this work investigates the neuroprotective properties of naturally occurring peptides, endomorphin-1 (EM-1), endomorphin-2 (EM-2), rubiscolin-5 (R-5), and rubiscolin-6 (R-6). We aimed at answering the question of whether well-known opioid peptides can counteract cell injury in a common in vitro model of Parkinson's disease (PD). Antioxidant activity of these four peptides was evaluated by the 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity, oxygen radical absorbance capacity (ORAC), and ferric-reducing antioxidant power (FRAP) assays, while neuroprotective effects were assessed in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y). The mechanisms associated with neuroprotection were investigated by the determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and Caspase-3 activity. Among the tested peptides, endomorphins significantly prevented neuronal death induced by 6-OHDA treatment, decreasing MMP (EM-1) or Caspase-3 activity (EM-2). Meanwhile, R-6 showed antioxidant potential by FRAP assay and exhibited the highest capacity to recover the neurotoxicity induced by 6-OHDA via attenuation of ROS levels and mitochondrial dysfunction. Generally, we hypothesize that peptides' ability to suppress the toxic effect induced by 6-OHDA may be mediated by different cellular mechanisms. The protective effect caused by endomorphins results in an antiapoptotic effect (mitochondrial protection and decrease in Caspase-3 activity), while R-6 potency to increase a cell's viability seems to be mediated by reducing oxidative stress. Our results may provide new insight into neurodegeneration and support the short peptides as a potent drug candidate to treat PD. However, further studies should be conducted on the detailed mechanisms of how tested peptides could suppress neuronal injuries.

Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-D-alanine.

Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2). Cyclization was achieved through an amide bond between side-chains of D-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-D-alanine (D-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2',6'-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-D-ACAla showed high affinity for both, µ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-D-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.

Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2.

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-ß-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-ß-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-ß-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-ß-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the µ-opioid receptor (MOR) antagonist, ß-funaltrexamine (ß-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-ß-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.

Novel glycosylated endomorphin-2 analog produces potent centrally-mediated antinociception in mice after peripheral administration.

We report the synthesis and pharmacological characterization of a novel glycosylated analog of a potent and selective endogenous µ-opioid receptor (MOP) agonist, endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2), obtained by the introduction in position 3 of the tyrosine residue possessing the glucose moiety attached to the phenolic function via a ß-glycosidic bond. The improved blood-brain barrier permeability and enhanced antinociceptive effect of the novel glycosylated analog suggest that it may be a promising template for design of potent analgesics. Furthermore, the described methodology may be useful for increasing the bioavailability and delivery of opioid peptides to the CNS.

Natural plant materials as a source of neuroprotective peptides.

In many circumstances, some crucial elements of the neuronal defense system fail, slowly leading to neurodegenerative diseases. Activating this natural process by administering exogenous agents to counteract unfavourable changes seems promising. Therefore, looking for neuroprotective therapeutics, we have to focus on compounds that inhibit the primary mechanisms leading to neuronal injuries, e.g., apoptosis, excitotoxicity, oxidative stress, and inflammation. Among many compounds considered neuroprotective agents, protein hydrolysates and peptides derived from natural materials or their synthetic analogues are good candidates. They have several advantages, such as high selectivity and biological activity, a broad range of targets, and high safety profile. This review aims to provide biological activities, the mechanism of action and the functional properties of plant-derived protein hydrolysates and peptides. We focused on their significant role in human health by affecting the nervous system and having neuroprotective and brain-boosting properties, leading to memory and cognitive improving activities. We hope our observation may guide the evaluation of novel peptides with potential neuroprotective effects. Research into neuroprotective peptides may find application in different sectors as ingredients in functional foods or pharmaceuticals to improve human health and prevent diseases.

New Uracil Analog with Exocyclic Methylidene Group Can Reverse Resistance to Taxol in MCF-7 Cancer Cells.

Introduction: Taxol (Tx), a microtubule-stabilizing drug, has been widely used as a chemotherapeutic in several types of cancer. However, the development of resistance limited its application. One of the strategies used to prevent the emergence of drug resistance is combination treatment, involving at least two drugs. The aim of the current study was to assess if a new uracil analog, 3-p-bromophenyl-1-ethyl-5-methylidenedihydrouracil (U-359) can prevent the development of Tx resistance in breast cancer cells. Methods: The cytotoxicity of the new drug was tested in MCF-7 (hormone receptor (ER, PR) positive cell-line) and MCF-10A cell lines using MTT method. For the detection of apoptosis and necrosis, the Wright and Giemsa staining was used. Gene expression was measured by real-time PCR, and changes in the protein levels were evaluated by ELISA and bioluminescent method. Results: We investigated the effect of Tx and U-359 on cancer MCF-7 and normal MCF-10A cells alone and in combination. Tx co-administered with U-359 inhibited proliferation of MCF-7 cells to 7% while the level of ATPase drastically decreased to 14%, compared with effects produced by Tx alone. The apoptosis process was induced through the mitochondrial pathway. These effects were not seen in MCF-10A cells, showing the wide safety margin. The obtained results have shown that U-359 produced a synergistic effect with Tx probably by reducing Tx resistance in MCF-7 cells. To elucidate the possible mechanism of resistance, expression of tubulin III (TUBIII), responsible for microtubule stabilization and tau and Nlp proteins, responsible for microtubule dynamics, was assessed. Conclusion: Combination of Tx with U-359 reduced overexpression of TUBIII and Nlp. Thus, U-359 may stand for a potential reversal agent for the treatment of MDR in cancer cells.

Kinetic studies of novel inhibitors of endomorphin degrading enzymes.

Endomorphins (EMs), two endogenous µ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3), which were designed earlier in our laboratory. The presented data give evidence that EMDB-2 and EMDB-3 are potent inhibitors of enzymes responsible for endomorphin cleavage. These compounds are stable and easily synthesized. EMDB-2 and EMDB-3 are competitive inhibitors of both, DPP IV and APM, with K(i) values in micromolar range. They are less potent than diprotin A in protecting EMs against DPP IV but more potent than actinonin in protecting these peptides against APM.

Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs.

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH(2) or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH(2), respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure. Dmt(1)-substituted analogs displayed high affinity at the µ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting µ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration. Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood-brain barrier permeability.

Whether short peptides are good candidates for future neuroprotective therapeutics?

Neurodegenerative diseases are a broad group of largely debilitating, and ultimately terminal conditions resulting in progressive degeneration of different brain regions. The observed damages are associated with cell death, structural and functional deficits of neurons, or demyelination. The concept of neuroprotection concerns the administration of the agent, which should reverse some of the damage or prevent further adverse changes. A growing body of evidence suggested that among many classes of compounds considered as neuroprotective agents, peptides derived from natural materials or their synthetic analogs are good candidates. They presented a broad spectrum of activities and abilities to act through diverse mechanisms of action. Biologically active peptides have many properties, including antioxidant, antimicrobial, antiinflammatory, and immunomodulatory effects. Peptides with pro-survival and neuroprotective activities, associated with inhibition of oxidative stress, apoptosis, inflammation and are able to improve cell viability or mitochondrial functions, are also promising molecules of particular interest to the pharmaceutical industries. Peptide multiple activities open the way for broad application potential as therapeutic agents or ingredients of health-promoting functional foods. Significantly, synthetic peptides can be remodeled in numerous ways to have desired features, such as increased solubility or biological stability, as well as selectivity towards a specific receptor, and finally better membrane penetration. This review summarized the most common features of major neurodegenerative disorders, their causes, consequences, and reported new neuroprotective drug development approaches. The author focused on the unique perspectives in neuroprotection and provided a concise survey of short peptides proposed as novel therapeutic agents against various neurodegenerative diseases.

Signal Peptides - Promising Ingredients in Cosmetics.

Naturally occurring peptides found in the human skin can serve particular biological activities and play roles as signaling molecules of various physiological processes such as homeostasis, growth, defense or immunity. Their great biological activity resulted in a growing interest in the pharmaceutical industry. Researchers consider peptides either promising compounds with potential application for human diagnosis, therapy or cosmetics. Peptides are becoming interesting cosmetic ingredients with the functions to reduce premature skin aging, improve the barrier function of skin, moisturize the skin, protect it from UV damage, and anti-inflammatory properties that alleviate acne and irritation. Till now, peptides of different origins were investigated in formulation developed to enhance collagen or elastin production, increase fibroblast proliferation, improve wound healing or skin condition. Most of them are obtained by chemical synthesis or by partial digestion of animal proteins. Short and easily synthesized peptides with alternative amino acid sequence, and combinations have created a new field of molecules inspired by nature and implemented in the cosmetic industry. Nowadays, peptides are cheaper and easier to produce in large quantities. The efficient process development methods allow obtaining nearly unlimited sequences, which makes them functionally preferred. Generally, cosmetic peptides are categorized as carrier peptides, neurotransmitter- affecting peptides, enzyme inhibitor peptides and signal peptides. The use of signal peptides in cosmetics increased over a few years. These molecules trigger a signaling cascade and stimulate fibroblast collagen production, the proliferation of elastin, fibronectin, laminin, etc. Thus, a literature search on a topical application of the most common signal peptides; and their current status in the cosmetic industry was carried out.

Biological activity of endomorphin and [Dmt1]endomorphin analogs with six-membered proline surrogates in position 2.

Endogenous mu-opioid receptor (MOR) selective peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), unlike so called 'typical opioids', are characterized by the presence of Pro(2) residue, which is a spacer connecting aromatic pharmacophoric residues. In order to investigate structural requirements for position 2, we synthesized endomorphin analogs incorporating, instead of Pro, unnatural amino acids with six-membered heterocyclic rings, such as piperidine 2-, 3- or 4-carboxylic acids (Pip, Nip and Inp, respectively). (R)-Nip residue turned out to be favourable for improving MOR affinity. Introduction of 2',6'-dimethyltyrosine (Dmt) instead of Tyr(1) led to obtaining [Dmt(1), (R)-Nip(2)]EM-2 which showed exceptional MOR affinity and high stability against enzymatic degradation in rat brain homogenate. In in vivo hot-plate test in mice, this analog given intracerebroventicularly (i.c.v.), produced profound supraspinal analgesia, being much more potent than EM-2. The antinociceptive effect of this analog lasted about 170 min and was almost completely reversed by beta-funaltrexamine (beta-FNA), a selective MOR antagonist.

[Endomorphins--endogenous ligands of the mu-opioid receptor]. / Endomorfiny--endogenne ligandy receptora opioidowego mu.

Two endogenous opioid peptides with extremely high mu-opioid receptor affinity and selectivity, endomorphin-1 and endomorphin-2, were: discovered and isolated from the mammalian brain in 1997. Endomorphins are amidated tetrapeptides, structurally different from so called typical opioids: enkephalins, dynorphins and endorphins. A protein precursor of endomorphins and a gene encoding their sequence remain unknown. Endomorphins are unable to cross the blood-brain barrier because of their low hydrophobicity. In animal models, these peptides turned out to be very potent in relieving neuropathic and inflammatory pain. In comparison with morphine, a prototype opioid receptor ligand, endomorphins produces less undesired side effects. In this article we describe the discovery of endomorphins, their cellular localization and functions in the organism, as well as their structure-activity relationships and biodegradation pathways.

Rubiscolins - Highly Potent Peptides Derived from Plant Proteins.

Rubiscolins are two naturally occurring, linear peptides, isolated from the pepsin digests of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from spinach leaves. Their sequences are as follows: Tyr-Pro-Leu-Asp-Leu (YPLDL, rubiscolin-5) and Tyr-Pro-Leu-Asp-Leu-Phe (YPLDLF, rubiscolin-6, also known as rubixyl). Though their structure does not resemble typical opioid peptides, rubiscolins were found to exhibit high affinity and selectivity for the delta opioid receptor and an antinociceptive effect in mice after oral administration. Moreover, orally administered rubiscolin-6 was shown to possess orexigenic, anxiolytic-like, and memory-enhancing activities in mice. This review summarizes various biological activities of rubiscolins in mammalian organism and recent developments on the structure-activity relationship of rubiscolin analogs, aimed at improving their pharmacological properties. Naturally occurring substances, such as rubiscolins, may provide a rational and powerful approach in the design of new therapeutics or functional foods.

Synthesis and activity of opioid peptidomimetics with ß2- and ß3-amino acids.

Morphiceptin (Tyr-Pro-Phe-Pro-NH2) is a selective ligand of the mu opioid receptor, an important target in pain regulation. In this study, morphiceptin was modified at positions 2 or 3 by introduction of ß2- or ß3-amino acids and additionally in position 1 by replacing Tyr by Dmt (2',6'-dimethyltyrosine), which resulted in obtaining enzymatically stable analogs with mixed opioid receptor affinity profiles. An analog of the sequence Dmt-d-Ala-(R)-ß2-1-Nal-Pro-NH2 [Nal=3-(1-naphthyl)-alanine] showed very high activity at the mu and delta receptors in the calcium mobilization functional test but did not cross the artificial membrane imitating the blood-brain barrier. In the in vivo test this analog induced strong antinociceptive effect in the writhing test in mice after intraperitioneal but also oral administration and inhibited diarrhea similarly to loperamide. Therefore, it may become an interesting lead compound in the development of peripherally restricted drugs for the treatment of gastrointestinal disorders.

Correction for "Synthesis of Mixed Opioid Affinity Cyclic Endomorphin-2 Analogues with Fluorinated Phenylalanines".

[This corrects the article DOI: 10.1021/acsmedchemlett.5b00056.].

Synthesis of mixed MOR/KOR efficacy cyclic opioid peptide analogs with antinociceptive activity after systemic administration.

Cyclic pentapeptide Tyr-c[D-Lys-Phe-Phe-Asp]NH2, based on the structure of endomorphin-2 (EM-2), which shows high affinity to the µ-opioid receptor (MOR) and a very strong antinociceptive activity in mice was used as a parent compound for the structure-activity relationship studies. In this report we synthesized analogs of a general sequence Dmt-c[D-Lys-Xaa-Yaa-Asp]NH2, with D-1- or D-2-naphthyl-3-alanine (D-1-Nal or D-2-Nal) in positions 3 or 4. In our earlier papers we have indicated that replacing a phenylalanine residue by the more extended aromatic system of naphthylalanines may result in increased bioactivities of linear analogs. The data obtained here showed that only cyclopeptides modified in position 4 retained the sub-nanomolar MOR and nanomolar κ-opioid receptor (KOR) affinity, similar but not better than that of a parent cyclopeptide. In the in vivo mouse hot-plate test, the most potent analog, Dmt-c[D-Lys-Phe-D-1-Nal-Asp]NH2, exhibited higher than EM-2 but slightly lower than the cyclic parent peptide antinociceptive activity after peripheral (ip) and also central administration (icv). Conformational analyses in a biomimetic environment and molecular docking studies disclosed the structural determinants responsible for the different pharmacological profiles of position 3- versus position 4-modified analogs.

Synthesis of mixed opioid affinity cyclic endomorphin-2 analogues with fluorinated phenylalanines.

As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.

Bioavailability of endomorphins and the blood-brain barrier--a review.

Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain because they modulate nociceptive pathways at supraspinal, spinal and peripheral levels. Unfortunately, peptides are generally hydrophilic compounds and therefore unable to cross the blood-brain barrier (BBB) by passive diffusion to reach the central nervous system (CNS) in an amount sufficient to activate appropriate receptors. Endomorphins (EMs) belong to the class of endogenous opioids eliciting the strongest analgesic effect, but only after direct administration to the CNS. Extensive research is in progress to better understand the relationships between EM structure and bioavailability. This article deals with the recent investigations that allow the design of stable and neuroactive EM analogs with enhanced brain passage and uptake.

Cyclic pentapeptide analogs based on endomorphin-2 structure: cyclization studies using liquid chromatography combined with on-line mass spectrometry and tandem mass spectrometry.

The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt=2',6'-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.