Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

Antheraea peptide and its analog: Their influence on the maturation of the reproductive system, embryogenesis, and early larval development in Tenebrio molitor L. beetle.

In recent years, many new immunologically active peptides from insects have been identified. Unfortunately, in most cases, their physiological functions are not fully known. One example is yamamarin, a pentapeptide isolated from the caterpillars of the Antheraea yamamai moth. This peptide has strong antiproliferative properties and is probably involved in the regulation of diapause. Additionally, antiviral activity was discovered. The results of the research presented in this paper are, to our knowledge, the first attempt to characterize the biological effects of yamamarin on the functioning of the reproductive processes and embryonic development of insects using a model species, the beetle Tenebrio molitor, a commonly known pest of grain storage. Simultaneously, we tested the possible activity of the molecule in an in vivo system. In this research, we present the multifaceted effects of yamamarin in this beetle. We show that yamamarin influences ovarian growth and development, maturation of terminal oocytes, level of vitellogenin gene transcript, the number of laid eggs, duration of embryonic development, and larval hatching. In experiments with palmitic acid-conjugated yamamarin (C16-yamamarin), we also showed that this peptide is a useful starting molecule for the synthesis of biopharmaceuticals or new peptidomimetics with gonadotropic activity and effects on embryonic development. The data obtained additionally provide new knowledge about the possible function of yamamarin in insect physiology, pointing to the important role of this pentapeptide as a regulator of reproductive processes and embryonic development in a heterologous bioassay with T. molitor.

Disruption of insect immunity using analogs of the pleiotropic insect peptide hormone Neb-colloostatin: a nanotech approach for pest control II.

This work continues our studies on the pleiotropic activity of the insect peptide Neb-colloostatin in insects. In vivo immunological bioassays demonstrated that hemocytotoxic analogs of Neb-colloostatin injected into Tenebrio molitor significantly reduced the number of hemocytes in the hemolymph and impaired phagocytosis, nodulation and phenoloxidase activities in the insects. Among the analogs tested, [Ala1]-,[Val1]-, [Hyp4]- and [Ach4]-colloostatin were particularly potent in disrupting cellular immunity in larvae, pupae and adult insects. This result suggests that the most effective analogs showed increases in the bioactivity period in the hemolymph of insects when compared to Neb-colloostatin. Recently, we demonstrated that it is possible to introduce Neb-colloostatin through the cuticle of an insect into the hemolymph when the peptide is coupled with nanodiamonds. In this study, we showed that [Ala1]-, [Val1]-, [Hyp4]- and [Ach4]-colloostatin, when complexed with nanodiamonds, may also pass through the cuticle into the hemolymph and induce long-term impairments of immunity in T. molitor at all developmental stages. Studies on the tissue selectivity and effectiveness of Neb-colloostatin analogs and efficient methods for their introduction into insects may contribute to the development of eco-friendly pest control methods based on bioactive peptidomimetics.

Copper(ii) complexes with alloferon analogues containing phenylalanine H6F and H12F stability and biological activity lower stabilization of complexes compared to analogues containing tryptophan.

Copper(ii) complex formation processes between alloferon 1 (Allo1) (H1 GVSGH6 GQH9 GVH12G) analogues where the phenylalanine residue is introduced in the place of His residue H6F and H12F have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For the phenylalanine analogues of alloferon 1, complex speciation has been obtained for a 1 : 1, 2 : 1 and 3 : 1 metal-to-ligand molar ratio. At physiological pH and in 1 : 1 metal-to-ligand molar ratio the phenylalanine analogues of alloferon 1 form a CuL complex similar to that of alanine analogues with the 4N{NH2,N1Im,2NIm} coordination mode. The stability of the complexes of the phenylalanine analogues is higher in comparison to those of alanine analogues, but lower in comparison to those containing tryptophan. Injection of Allo12F into insects induced prominent apoptotic changes in all hemocytes. The presence of apoptotic bodies only in the insect hemolymph testifies to the fact that Allo12F is an extremely pro-apoptotic peptide.

The long-term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor.

The subject of this article is a search for the long-term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro-apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p-NH2 )1 ]- and [Phe(p-OMe)1 ]-alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria-challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide-untreated bacteria-challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide-induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half-life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N-terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.

High stability and biological activity of the copper(II) complexes of alloferon 1 analogues containing tryptophan.

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH-1L and/or CuH-2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH-1L, CuH-2L and CuH-3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

Copper(II) complexes of terminally free alloferon mutants containing two histidyl binding sites inside peptide chain structure and stability.

Mononuclear and polynuclear copper(II) complexes of alloferon 1 with point mutations, H1A/H12A H2N-A(1)GVSGH(6)GQH(9)GVA(12)G-COOH, H1A/H9A H2N-A(1)GVSGH(6)GQA(9)GVH(12)G-COOH, and H1A/H6A H2N-A(1)GVSGA(6)GQH(9)GVH(12)G-COOH, have been studied by potentiometric, UV-visible, CD, and EPR spectroscopy, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand molar ratios ranging from 1 : 1 to 3 : 1 was obtained. Over a wide 6-8 pH range, including physiological pH 7.4, and a 1 : 1 metal-to-ligand molar ratio, the peptides studied formed a CuH-1L complex with the 4N{NH2,N(-),2NIm} coordination mode. The presence of the 4N binding site for the CuH-1L complexes prevented the deprotonation and coordination of the second amide nitrogen atom to copper(II) ions (pK-1/-2 7.83-8.07) compared to that of pentaGly (6.81). The amine nitrogen donor and two imidazole nitrogen atoms (H(6)H(9), H(6)H(12) and H(9)H(12)) can be considered to be independent metal-binding sites in the species formed. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2 : 1 and 3 : 1 molar ratios dominate in the solution, respectively. For the Cu(II)-H1A/H9A and Cu(II)-H1A/H12A systems, the Cu3H-9L complexes are likely formed by the coordination of amide nitrogen atoms towards C-termini with ring sizes (7,5,5).

Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H(1) and H(6) or H(9) or H(12)) binding sites Structure Stability and Biological Activity.

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

Effect of alloferon 1 on central nervous system in rats.

Alloferon 1 is an insect-derived peptide with potent antimicrobial and antitumor activity. It was isolated from blood of an experimentally infected insect, the blow fly Callifora vicina. Synthetic alloferon 1 reveals a capacity to stimulate activity of NK cells and synthesis IFN in animal and human models. Moreover, it was demonstrated antiviral and antitumor activity of alloferon 1 in mice. There are no data on influence of alloferon 1 on central nervous system. The aim of present study was to determine an effect of alloferon 1 on rats' central nervous system by some behavioral tests: open field test, hole test, score of rats irritability, and determination of memory consolidation in the water maze test. Moreover, a probable antinociceptive effect of alloferon 1 in rats was determined by a tail immersion test and hot plate test. Experiments were performed on female Wistar rats. Seven days before experiments, rats were anesthetized with ketamine and xylazine and polyethylene cannulas were implanted into the right lateral brain ventricle (i.c.v.). On the day of experiment, alloferon 1 dissolved in a volume of 5 µL of saline was injected directly i.c.v. through implanted cannulas at doses of 5-100 nmol. It was found that alloferon 1 had slight effect on locomotor and exploratory activity, induced some decrease of rat irritability and a weak impairment of rats memory (only at the low dose of 5 nmol). On the other hand, the higher dose of this peptide exerts significant antinociceptive effect. Obtained results indicate that alloferon 1 do not exert any evidently toxic effect on central nervous system in rats. Therefore, alloferon 1 may be good new drug with antitumor and antinociceptive activity.

Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity.

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A(1)GVSGH(6)GQH(9)GVH(12)G (Allo1A) and (H9A) H(1)GVSGH(6)GQA(9)GVH(12)G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H(6),H(12)) leads to the formation of the CuL complex with 4N {NH2,NIm-H(1),NIm-H(6),NIm-H(12)} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH-1L consists of the 3N {NH2,N(-),CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His(1) and His(6) residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

Mono- and polynuclear copper(II) complexes of alloferons 1 with point mutations (H6A) and (H12A): stability structure and cytotoxicity.

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

Novel biological effects of alloferon and its selected analogues: structure-activity study.

The subject of this paper is a search for new biological properties of alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) and a series of its analogues. The studies on structure/activity relationship in alloferon, the synthesis of a series of 28 analogues were performed. The analogues were modified at position 1 or 6, and other were oligopeptides with a shortened peptide sequence. Biological effects of the peptides were evaluated by the pro-apoptotic action in vivo on haemocytes of Tenebrio molitor and in the cardiotropic test in vitro on the heart of T. molitor and Zophobas atratus. In the in vivo bioassays, new biological activities of alloferon and its analogues were discovered. In haemocytotoxic bioassay, alloferon strongly induces T. molitor haemocytes to undergo apoptosis at a dose of 10 nM. Moreover, [Phe(p-NH2)(1)]-, [Tyr(6)]- and [1-10]-alloferon exhibit a two-fold increase of caspases activation in comparison with the alloferon. However, alloferon and its analogues show a weak cardiostimulatory activity in Z. atratus but the heart of T. molitor is not sensitive to these peptides. The results obtained here suggest that alloferon plays pleiotropic functions in insects.

New alloferon analogues: synthesis and antiviral properties.

We have extended our study on structure/activity relationship studies of insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) by evaluating the antiviral effects of new alloferon analogues. We synthesized 18 alloferon analogues: 12 peptides with sequences shortened from N- or C-terminus and 6 N-terminally modified analogues H-X(1)-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH, where X(1) = Phe (13), Tyr (14), Trp (15), Phg (16), Phe(p-Cl) (17), and Phe(p-OMe) (18). We found that most of the evaluated peptides inhibit the replication of Human Herpesviruses or Coxsackievirus B2 in Vero, HEp-2 and LLC-MK(2) cells. Our results indicate that the compound [3-13]-alloferon (1) exhibits the strongest antiviral activity (IC(50) = 38 µM) among the analyzed compound. Moreover, no cytotoxic activity against the investigated cell lines was observed for all studied peptides at concentration 165 µM or higher.

Copper(II) complex formation processes of alloferon I with point mutation H1K; combined spectroscopic and potentiometric studies.

Mononuclear and polynuclear complexes of the alloferon I with point mutation (H1K) Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (AlloK) and its acetylated derivative Ac-Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (Ac-AlloK) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1 for AlloK while to 3:1 for Ac-AlloK. At physiological pH 7.4 and the metal-to-ligand 1:1molar ratio the AlloK peptide forms the CuL complex with the 4N {NH(2), N(-), 2N(Im)} binding mode. In the Cu(II)-AlloK 4:1 system in wide pH 6.5-10 range the Cu(4)H(-7)L complex dominates with the 3N {NH(2),2N(-)} 3×{N(Im),2N(-)} coordination mode. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites of Ac-AlloK. For Ac-AlloK and 1:1 metal-to-ligand molar ratio the CuHL complex with the 3N {3N(Im)} binding sites in pH 4.5-7.5 range is present in solution. The amine nitrogen donor and all of the histidine residues can be considered to be independent metal-binding sites in the species formed in the systems studied. As a consequence, tri- (for the Ac-AlloK) and tetra-nuclear (for the AlloK peptide) complexes for the metal-to-ligand 3:1 and 4:1molar ratios, respectively, are present in the solution.

Further studies on the antiviral activity of alloferon and its analogues.

The subject of our studies was the synthesis, biological evaluation, and conformational studies of insect tridecapeptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) and its analogues such as: [des-His(1) ]-, [Lys(1) ]-, [Arg(1) ]-, and [Ala(1) ]-alloferon. These peptides were synthesized to check the influence of the His residue at position 1 of the alloferon chain on its antiviral activity. Two aspects of the biological effects of these peptides were determined: (i) the cytotoxicity in vitro in the Vero, LLC-MK2, and HEp-2 cell lines, and (ii) the antiviral activity in vitro in respect to DNA and RNA viruses. We found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackie virus replication, whereas [Lys(1) ]-alloferon exhibited a high inhibitory action towards both viruses. Moreover, the peptides did not show any cytotoxic activity against the Vero, LLC-MK2, and HEp-2 cells. The preliminary circular dichroism conformational studies showed that the peptides investigated seem to prefer an unordered conformation.

Mono- and polynuclear copper(II) complexes with fragment of alloferons 1 and 2; combined potentiometric and spectroscopic studies.

Complex formation processes between the SGH(3)GQH(6)GVH(9)G decapeptide fragment of alloferons 1 and 2 and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD) and electron paramagnetic resonance (EPR) methods. This peptide contains two histidines (H(6) and H(9)) and an N-terminal "albumin-like" Xaa-Yaa-His sequence. It was found that the decapeptide is able to bind 3 equiv of metal ions. The SGH(3) sequence is the primary metal binding site and at pH 4 irrespective of the metal-to-ligand molar ratio (1 : 1, 2 : 1, 3 : 1) the CuL species with 4 N {NH(2), 2N(-), N(Im)} binding mode is formed. For the 2 : 1 metal-to-ligand molar ratio the Cu(2)H(-2)L, Cu(2)H(-4)L and Cu(2)H(-5)L complexes, while for the 3 : 1 the Cu(3)H(-2)L, Cu(3)H(-6)L and Cu(2)H(-7)L species dominate in solution. For the metal-to-ligand 2 : 1 and 3 : 1 molar ratios the Cu(3)H(-2)L complex at 4-7 pH range is formed where the {NH(2), 2N(-), N(Im)}{N(Im)}{N(Im)} coordination mode of the decapeptide to copper(II) ions is suggested.

Studies of insect peptides alloferon, Any-GS and their analogues. Synthesis and antiherpes activity.

The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as alloferon, Any-GS and their analogues. The synthesis of 14 peptides was performed by the solid-phase method. Biological effect of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV-1) in vitro using a Vero cell line. It was found that the investigated peptides inhibit the replication of HHV-1 in Vero cells.

Mononuclear copper(II) complexes of alloferons 1 and 2: a combined potentiometric and spectroscopic studies.

Mononuclear copper(II) complexes of the alloferon 1 His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, alloferon 2 Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, Ac-alloferon 1 Ac-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly and Ac-alloferon 2 Ac-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly have been studied by potentiometric, UV-vis, CD and EPR spectroscopic methods. The potentiometric and spectroscopic data shows that acetylation of the amino terminal group induces significant changes in the coordination properties of the Ac-alloferons 1 and 2 compared to the alloferons 1 and 2, respectively. The presence of four (Ac-alloferon 1) or three (Ac-alloferon 2) histidyl residues provides a high possibility for the formation of macrochelates via the exclusive binding of imidazole-N donor atoms. The macrochelation suppresses, but cannot preclude the deprotonation and metal ion coordination of amide functions and the CuH(-3)L species with [N(Im), 3N(-)] bonding mode at pH above 8 are formed. The N-terminal amino group of the alloferons 1 and 2 takes part in the coordination of the metal ion and the 4N complex with [NH(2), 3N(Im)] coordination mode dominates at physiological pH 7.4 for alloferon 1 and the 3N [NH(2), CO, 2N(Im)] binding mode for alloferon 2. However, at higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions.