Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: structure activity study

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of l- or d-β-(2-thienyl)-alanine [L- or D-Thi], or l- or d-3-Pyridylalanine [l- or d-3-Pal]) were combined with d-tyrosine(OEthyl) [d-Tyr(Et)] or d-1-naphthylalanine [d-1-Nal] in position 2 and β-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having α-aminoisobutyric acid [Aib] or d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (d-Tic) or diethylglycine (Deg) in position 9 and d-Tyr(Et) or d-1-Nal or d-Tic in position 2 and Mpa or Pen (ββ-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and d-Tyr(Et) or d-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa¹, d-Tyr(Et)², Deg⁹]OT (pA₂ = 8.68 ± 0.26); this analogue was also selective.     

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin

Summary A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by¹H,¹H-¹H,¹³C and¹H-¹³C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.     

Synthesis and biological evaluation of oxytocin analogues containing L-alpha-t-butylglycine [Gly(Bu t)] in positions 8 or 9

We report the solid phase synthesis and some pharmacological properties of seventeen new oxytocin (OT) analogues. Basic modification at positions 8 and/or 9 (introduction of L-alpha-t-butylglycine [Gly(Bu(t))]) was combined with D-Cys(6), D-Tyr(Et)(2), Mpa(1) or Pen(1) modifications and their various combinations. We also present properties of two previously reported re-synthesized analogues ([Gly(Bu(t))(8)]OT and [Mpa(1), Gly(Bu(t))(8)]OT). The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OTR.     

Preparation of polymer-bound trityl-hydrazines and their application in the solid phase synthesis of partially protected peptide hydrazides

Summary Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc- or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl- and tert-butyl protecting groups remain unaffected.     

Synthesis and Biological Evaluation of New GnRH Analogues on Pituitary and Breast Cancer Cells

GnRH analogues have been extensively used in oncology to induce reversible chemical castration due to their hypophysiotropic action. In addition to that, it has recently been shown that many malignant cells, such as breast cancer cells, locally produce GnRH and express the GnRH receptor/s. In order to investigate the structure-activity relationships in both pituitary and extrapituitary biological systems, we synthesized eight new GnRH analogues with modifications in the N-terminal part and/or in position 6 and studied their pituitary binding affinity (in αT3-1 cell membranes) and effect on breast cancer (MCF-7) cell proliferation. 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC) was incorporated instead of pGlu¹-His²- and/or Gly⁶ was substituted by α-aminoisobutyric acid, D-Leu and D-Lys (alone or covalently linked to Gly, Ala, Sar, ATPC). Most GnRH analogues lacked the carboxy-terminal Gly¹⁰-amide of GnRH and an ethylamide residue was added to Pro⁹, a modification common in many potent GnRH agonists, such as leuprolide ([D-Leu⁶, des-Gly¹⁰]-GnRH-NHEt. Results show differential impact of these modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation. ATPC in the N-terminus resulted in analogues with low binding affinity but high antiproliferative effect. Substitutions in position 6 always resulted in high binding affinities. In particular, [D-Lys⁶(Gly), desGly¹⁰]-GnRH-NHEt and [D-Lys⁶(Sar), desGly¹⁰]-GnRH-NHEt have higher pituitary binding affinity than leuprolide, but only the latter had significant antiproliferative effect on both MCF-7 and MDA-MB-231 cells. These results contribute to the on-going research for more potent GnRH analogues. Abbreviations of common amino acids are in accordance with the recommendations of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Arch. Biochem. Biophys. 206, pp.v-xxii (1988), J. Biol. Chem. 264, 668–673 (1989) or J. Peptide Sci. 9, 1–8 (2003).     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

In position 7 l- and d-Tic-substituted oxytocin and deamino oxytocin: NMR study and conformational insights

Incorporation of l- or d-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa¹]OT) resulted in four analogues, [l-Tic⁷]OT (1), [d-Tic⁷]OT (2), [Mpa¹,l-Tic⁷]OT (3) and [Mpa¹,d-Tic⁷]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799–806, 2007). Their NMR study (NOESY, TOCSY, ¹H–¹³C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC₅₀ value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of d analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys⁶-d-Tic⁷ bond is supported by observation of two sets of cross-peaks for ¹H and ¹³C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in ¹H–¹³C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.     

Biological Activities of Analogues of Ser-Val-Gly-Glu (AS-I), a Toxin Pathogenic to Sunflower

The tetrapeptides Ser-Val-Glu-Gly, Hse-Val-Gly-Glu, Thr-Val-Gly-Glu, Val-Ser-Gly-Glu and Ser-Val-Glu-Gly, analogues of the AS-1 toxin which is pathogenic to sunflower, were examined for biological activity on sunflower and eight other plant species. These peptides were synthesized in solution or on a solid phase. Of the species studied by leaf inoculation, sunflower and zucchini were the most susceptible and cotton the most resistant to ail of the toxins tested. The effect of Ser-Val-Glu-Gly was similar to that reported for AS-I, whereas the tetrapeptide Thr-Val-Gly-Glu was the most toxic.     

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50%H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H^N–H^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The -proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H₂ –H^NEth is indicative of proximity between C- and N-termini.     

Thyroxine-binding antibodies inhibit T cell recognition of a pathogenic thyroglobulin epitope

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5' iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A(k)-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.