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In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114*has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, Cα-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.

Double electron–electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin–spin distance distribution between the two TOAC labels, within a length range of 1.5–8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

The hydrolysis and rearrangement of the dihydroorotic (Hor) residue in the presence of bases, leading to the formation of the hydantoin (Hyd) impurity, represent one of the major problems in manufacturing of the gonadotropin-releasing hormone antagonist Degarelix. In an attempt to find efficient strategies to overcome this problem, we carried out a screening of organic bases in order to select those which afforded both the rapid Fmoc deprotection during the solid-phase synthesis and the absence of this peculiar rearrangement. Among the bases tested, only tert-butylamine did not affect the peptide molecule and was able to perform fast Fmoc removal. The use of tert-butylamine for the synthesis of Degarelix led to a product with excellent purity and yield without a detectable amount of the hydantoin impurity. Thus, we showed that tert-butylamine can be a suitable alternative to piperidine for industrial-scale production of Degarelix or other Hor-containing peptide pharmaceuticals.

The highly potent, long-acting, gonadotropin-releasing hormone antagonist Degarelix is known to be very efficient for prostate cancer treatment. The synthesis of decapeptide Degarelix is complicated because of the presence in its sequence of several unnatural α-amino acids, which are prone to rearrangements and side reactions. In particular, the rearrangement of the dihydroorotic (Hor) moiety with following hydantoin formation in the presence of bases represents one of the major problems. In this study, we describe a novel chemical strategy to overcome this obstacle by the use of the corresponding p-nitrophenylalanine derivative, which is reduced on the solid support to p-aminophenylalanine and acylated with dihydroorotic acid at the end of the solid-phase synthesis. Thus, the contact of Hor with the bases required for Fmoc deprotection is completely avoided. This approach provides a superior purity of Degarelix when the synthesis is carried out in the industrial scale as well.

Water cleaning of paper leaves is a preliminary step in many customary deacidification treatments of ancient books in conservation workshops. Usually, the washing solutions are considered “waste waters” and they are thrown away. In this work, an analytical protocol for the analysis of wash waters for the assessment of the conservation state of ancient books is proposed. Wash waters of leaves under different conservation conditions of a 16th-century-printed book were investigated using UV-Vis, IR, Raman, NMR, EPR, XRF, ESI-MS, ICP-MS, CE-PDA and elemental microanalysis. Analysis of the wash water extracts allowed one to identify a large number of degradation by-products, ranging from low-molecular mass organic acids to cellulose oligomers. Reasons for the very different conservation conditions of the leaves emerged, indicating that wash water analysis provides a deep insight into the conservation state and composition of leaves as well as degradation reactions occurred on them.

Background: The non-receptor spleen tyrosine kinase (Syk; EC 2.7.10.2) is involved in signal transduction in a variety of cell types. In particular, it is a key mediator of immune receptors signaling in host inflammatory cells (B cells, mast cells, macrophages and neutrophils), important for both allergic and antibody-mediated autoimmune diseases. Deregulated Syk kinase activity also allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are able to induce leukemia. Consequently, the development of Syk kinase inhibitors could conceivably treat these disorders and so they have became a major focus in the pharmaceutical and biotech industry. Objective: In this review, we analyze the structure and role of Syk kinase, the use of small molecules, interacting with ATP-binding site, as inhibitors of kinase activity and finally the potential of using inhibitors of Syk kinase expression to attenuate pathological conditions. Conclusion: Syk kinase inhibition is suggested as a powerful tool for the therapy of different pathologies.

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH2] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH2analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr5and/or to Ser10. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe4, Thr5, Ala7and Arg8are crucial residues for OP4receptor activation. Manipulation of Phe1yielded peptides endowed with antagonist activity but [Nphe1] NC(1–13)-NH2acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe1] NC(1–13)-NH2sequence, abolished any residual agonist activity and [Nphe1, βAla7] NC(1–13)-NH2and [Nphe1, βAla11] NC(1–13)-NH2acted as competitive antagonists only. Modification of both Ala7and Ala11abolished the antagonist activity of [Nphe1]NC(1–13)-NH2probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)10] NC(1–13)-NH2displayed an activity comparable with that of NC(1–13)-NH2, [Ser(βGalNAc)10] NC(1–13)-NH2and [βAla11] NC(1–13)-NH2were five and 10 times less active, respectively.Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH2] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH2analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr5and/or to Ser10. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe4, Thr5, Ala7and Arg8are crucial residues for OP4receptor activation. Manipulation of Phe1yielded peptides endowed with antagonist activity but [Nphe1] NC(1–13)-NH2acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe1] NC(1–13)-NH2sequence, abolished any residual agonist activity and [Nphe1, βAla7] NC(1–13)-NH2and [Nphe1, βAla11] NC(1–13)-NH2acted as competitive antagonists only. Modification of both Ala7and Ala11abolished the antagonist activity of [Nphe1]NC(1–13)-NH2probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)10] NC(1–13)-NH2displayed an activity comparable with that of NC(1–13)-NH2, [Ser(βGalNAc)10] NC(1–13)-NH2and [βAla11] NC(1–13)-NH2were five and 10 times less active, respectively.

Syntheses are described of new endomorphin 1and 2peptoid–peptide hybrids in which Tyr1and either one or both Phe3and Phe4have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp2-endomorphin 2analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are β-O-glycosidically linked to the hydroxyproline residue. The Hyp2-endomorphin sequences have also been elongate by adding a C-terminal β-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1and 2are not significantly affected by the Pro/Hyp substitution. Phe4/Nphe substitution in the endomorphin 1reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe3or Phe4, is lower on endomorphin 2. Either modification of both Phe3 and Phe4or glycosylation of the Hyp2-endomorphin 2cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 1]2, [endomorphin 2]2, [Hyp2-endomorphin 1]2, [Hyp2-endomorphin 2]2, [Hyp2-endomorphin 1-Hyp2-endomorphin 2]2 or [Hyp2-endomorphin 2-Hyp2-endomorphin 1]2, are 7–19 times less potent than endomorphin 1on GPI and significantly less active than endomorphins 1and 2on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.Syntheses are described of new endomorphin 1and 2peptoid–peptide hybrids in which Tyr1and either one or both Phe3and Phe4have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp2-endomorphin 2analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are β-O-glycosidically linked to the hydroxyproline residue. The Hyp2-endomorphin sequences have also been elongate by adding a C-terminal β-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1and 2are not significantly affected by the Pro/Hyp substitution. Phe4/Nphe substitution in the endomorphin 1reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe3or Phe4, is lower on endomorphin 2. Either modification of both Phe3 and Phe4or glycosylation of the Hyp2-endomorphin 2cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 1]2, [endomorphin 2]2, [Hyp2-endomorphin 1]2, [Hyp2-endomorphin 2]2, [Hyp2-endomorphin 1-Hyp2-endomorphin 2]2 or [Hyp2-endomorphin 2-Hyp2-endomorphin 1]2, are 7–19 times less potent than endomorphin 1on GPI and significantly less active than endomorphins 1and 2on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.

The side chain orientation of the tyrosine residue included in a peptide, which is an excellent substrate of Syk tyrosine kinase, was fixed in different conformations by either incorporating the tyrosine in cyclic structures (6-OH-Tic, 5-OH-Aic, and Hat derivatives) or adding a sterically bulky substituent in the tyrosine side chain moiety (β-MeTyr). Synthetic peptides containing tyrosine analogues displaying different side chain orientations were analyzed by NMR techniques and tested as potential substrates of the nonreceptor tyrosine kinases Syk, Csk, Lyn, and Fyn. The “rotamer scan” of the phosphorylatable residue generated optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase, while the peptidomimetics were not recognized by the other tyrosine kinases. In particular, l-β-MeTyr and d-Hat containing peptides resulted to be both suitable substrates for the specific monitoring of Syk and consensus sequence scaffolds for the design of potential inhibitors highly selective for this tyrosine kinase.

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149–152), was fixed in the gauche (+) or gauche (−) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the ¹H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This “rotamer scan” of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 478–488, 2003

The side‐chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella‐Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149–152), was fixed in the gauche (+) or gauche (−) conformation by using the 7‐hydroxy‐1,2,3,4‐tetrahydro isoquinoline‐3‐carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine‐type (Hba) structure. The proposed side‐chain orientations were confirmed by the analysis of the 1H‐NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This “rotamer scan” of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src‐related tyrosine kinases Lyn and c‐Fgr. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 478–488, 2003

The heptapeptide EDNEYTA, which reproduces the main autophosphorylation site of Src, has been previously shown to be a good substrate for both Src and Syk tyrosine kinases [Ruzza, P., et al., J. Pept. Sci., 2 (1996) 325]. Four lactam bridge conformationally constrained analogues of this peptide were synthesized by classical solution methods and screened for their suitability as c-Fgr and Syk tyrosine kinase substrates. The kinetic data obtained indicate that the different rings of the lactam peptides influence the capability of the peptides to act as PTK substrates. In general cyclization decreases the peptide phosphorylability, however the sequence containing the greatest lactam ring, ED(EEYTK), resulted in an especially suitable and selective substrate for Syk tyrosine kinase.

The heptapeptide EDNEYTA, which reproduces the main autophosphorylation site of Src, has been previously shown to be a good substrate for both Scc and Syk tyrosine kinases [Ruzza, P., et al., J. Pept. Sci., 2 (1996) 325]. Four lactam bridge conformationally constrained analogues of this peptide were synthesized by classical solution methods and screened for their suitability as c-Fgr and Syk tyrosine kinase substrates. The kinetic data obtained indicate that the different rings of the lactam peptides influence the capability of the peptides to act as PTK substrates. In general cyclization decreases the peptide phosphorylability, however the sequence containing the greatest lactam ring, ED(EEYTK), resulted in an especially suitable and selective substrate for Syk tyrosine kinase.

A growing interest in peptides as active pharmaceutical ingredients (APIs) requires the development of efficient strategies for their preparation. This is particularly challenging in the case of long peptides with a strong tendency for aggregation and folding. Here, we describe the pseudoproline-assisted convergent synthesis of GLP-1 receptor agonist lipopeptides liraglutide and semaglutide, which involves the stepwise condensation of three fragments in the solid phase. The insertion of a pseudoproline residue at the site of fragment coupling prevents aggregation and allows obtaining these peptides with excellent purity and high yield. In addition, for the synthesis of lipidated side chains, we developed a novel approach that involves copper(II) lysinate intermediates and can be particularly suitable for the industrial preparation of both liraglutide and semaglutide and other peptides with a similar branched structure.

We developed a new class of inhibitors of protein–protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2–20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein–protein interactions in the function of SHP2.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT‐IR absorption, CD, 2D‐NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14‐mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in‐depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical‐containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC‐labeled analogs in view of the poorly reactive α‐amino function of this very bulky residue and the specific requirements of its free‐radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α‐helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.