Lactam Bridge Research Articles

Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues.

The N-terminal fragment of PTH(1-34) is critical for PTH1 receptor activation. Various modifications of PTH(1-14) have been shown to result in a considerable increase in signaling potency [Shimizu et al. (2000) J. Biol. Chem. 275, 21836-21843]. Our structural investigations revealed an unusually stable helical structure of the signaling domain (1-14), where residues 6 (Gln) and 10 (Gln or Asn) were located on the same face of the alpha-helix. To test whether a stable N-terminal alpha-helix is required for productive interaction with PTH1 receptor, we designed two conformationally restricted PTH(1-14) analogues, each containing a lactam bridge at positions 6 and 10. Specifically, substitutions Gln(6)-->Glu(6) and Asn(10)-->Lys(10) were introduced into the most potent [Ala(1,3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH2 agonist. Both the Glu(6)-Lys(10) and Lys(6)-Glu(10) lactam-bridged analogues were characterized to examine the importance of orientation of the lactam. According to biological studies [Shimizu et al. (2003) Biochemistry 42, 2282-2290], none of the 6/10 substituted analogues (linear or cyclic) remained as active as the parent peptide. However, relative to their corresponding linear peptides, lactam-bridged analogues either maintained potency or showed 6-fold improvement. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the structural differences between the linear and cyclic PTH(1-14) fragments, supporting the hypothesis that an alpha-helix is the preferred bioactive conformation of the N-terminal fragment of PTH. In addition, our results demonstrate that the structural order of the very first residues (1-4) of the signaling domain plays a significant role in PTH action.

Structure-activity relationships of gamma-MSH analogues at the human melanocortin MC3, MC4, and MC5 receptors. Discovery of highly selective hMC3R, hMC4R, and hMC5R analogues.

It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in alpha-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in gamma-MSH, two different series of gamma-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native gamma-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH(2) with Gly(4)) is more important for selectivity. The 2nd series is made of d-2-naphthylalanine (d-Nal(2')) scan of the gamma-MSH sequence at position 6 and 8 and the replacement of His(5) with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-d-Nal(2')-Arg-Trp-Asp-Arg-Phe-Gly-NH(2), which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.

The Synthesis and Study of Side‐Chain Lactam‐Bridged Peptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues.

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.

Design, Synthesis, Conformational Analysis, and Biological Studies of Urotensin-II Lactam Analogues

Human urotensin II (hU-II; H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) is a disulfide bridged undecapeptide recently identified as the ligand of an orphan G protein-coupled receptor. hU-II has been described as the most potent vasoconstrictor compound identified to date. With the aim of replacing the disulfide bridge by a chemically more stable moiety, we have synthesized and tested a series of lactam analogues of hU-II minimum active fragment, that is hU-II(4–11). The contractile activity of the synthetic analogues on the rat isolated thoracic aorta was found to be dependent upon the dimension of the lactam bridge. The most active peptide, H-Asp-cyclo[Orn-Phe-Trp-Lys-Tyr-Asp]-Val-OH ( 3 ), is approximately 2 logs less potent than hU-II (pD 2=6.3 vs 8.4). A conformational analysis in solution of the active peptide 3 , one of the inactive analogues, and hU-II was performed, using NMR and molecular modelling techniques. A superposition of the calculated structures of hU-II and 3 clearly shows that three out of four key residues (i.e., Phe 6, Lys 8 and Tyr 9) maintain the same side– chain orientation, while the fourth one, Trp 7, cannot be superimposed. This observation could explain the reduced biological activity of the synthetic analogue.

Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600-612 region of the HIV envelope gp41 glycoprotein.

The conformational and immunological properties of different analogues corresponding to the 600-612 disulfide loop of the human immunodeficiency virus (HIV) gp41 glycoprotein envelope were studied. Fourteen analogues were designed and synthesised; namely, a series of seven analogues in which the disulfide bond was replaced by a lactam bridge and a series of seven analogues in which one residue of each analogue at a time, was replaced by its corresponding homologised alpha-amino acid (beta(3)-amino acid). In the case of the lactam analogues, the influence of the two possible CO-NH and NH-CO orientations of the lactam bridge as well as the size of the lactam ring was explored. The analogues were tested in ELISA with monoclonal antibodies raised against the 600-612 cyclic parent peptide as well as with sera from HIV-1 infected patients. A structural analysis of the parent and analogue peptides was carried out in dimethyl sulfoxide (DMSO-d(6)) using two-dimensional NMR techniques and molecular dynamics simulations. Comparison of the own conformation of the cyclic analogues with their either strong or weak reactivity with the antibodies reveals structural features that may be correlated with the antibody reactivity. Thus, a close structural similarity, particularly a characteristic orientation of the side-chains of residues Lys606, Leu607 and Ile608 in the loop, was found in certain beta(3)-analogues that were better recognised than the parent peptide by anti-peptide mouse monoclonal antibodies and patients' antibodies.

Secondary structure of antisauvagine analogues is important for CRF receptor antagonism: development of antagonists with increased potency and receptor selectivity

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF 2) receptor. A structure–activity relationship study was performed to pinpoint residues conferring aSVG’s selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala 22Arg 23Ala 24 (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for α-helicity properties, binding affinities and antagonistic potencies at the rat CRF 1 and mouse CRF 2B receptors. While N-terminal prolongation and replacement of d-Phe 11 by Tyr 11 increased the affinity for the CRF 2 receptor, the introduction of the ARA motif resulted in a loss of CRF 2 receptor selectivity. These data show that aSVG 10–40 analogues are more potent CRF 2 receptor antagonists than aSVG 11–40 peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF 1 receptor.

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.

Stabilization of the helical structure of Y2-selective analogues of neuropeptide Y by lactam bridges.

The importance of helical structure in an analogue of NPY selective for the Y2 receptor, Ac[Leu28,31]NPY24-36, has been investigated by introducing a lactam bridge between positions 28 and 32. The resulting analogue, Ac-cyclo28/32[Ala24,Lys28,Leu31,Glu32]NPY24-36, is a potent Y2-selective agonist. Structural analysis by NMR shows that this analogue forms a helical structure in a 40% trifluoroethanol/water mixture, whereas in water only the region around the lactam bridge (Lys28-Glu32) adopts helical-like structure, with both N- and C-termini being poorly defined. The observation of well-defined helical structure in aqueous TFE contrasts with that reported for a similar analogue, Ac-cyclo28/32[Lys28,Glu32]NPY25-36 (Rist et al. FEBS Lett. 1996, 394, 169-173), which consisted of a hairpin-like structure that brought the N- and C-termini into proximity. We have therefore determined the structures of this analogue, as well as those of Ac-cyclo28/32[Ala24,Lys28,Leu31,Glu32]NPY24-36 and Ac-cyclo28/32[Ala24,Lys28,Glu32]NPY24-36, under identical solution conditions (30% TFE/H2O mixture at 308 K) and find essentially the same helical structure in all three peptides. These findings support the proposal that these Y2-selective analogues adopt a helical structure when bound to the Y2 receptor.

Human somatostatin receptor specificity of backbone-cyclic analogues containing novel sulfur building units.

Somatostatin-14 (somatostatin) and its clinically available analogues octreotide, lanreotide, and vapreotide are potent inhibitors of growth hormone, insulin, and glucagon release. Recently, a novel backbone cyclic somatostatin analogue c(GABA-Phe-Trp-(D)Trp-Lys-Thr-Phe-GlyC3-NH(2)) (analogue 1, PTR 3173) that possesses in vivo endocrine selectivity was described. This long-acting octapeptide exhibits high affinity to human recombinant somatostatin receptors (hsst) hsst2, hsst4, and hsst5. Its novel binding profile resulted in potent in vivo inhibition of growth hormone but not of insulin release. We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to 1. In these analogues, the lactam bridge of 1 was replaced by a backbone disulfide bridge. We present a novel approach for conformational constraint of peptides by utilizing sulfur-containing building units for on-resin backbone cyclization. These disulfide backbone cyclic analogues of 1 showed significant metabolic stability as tested in various enzyme mixtures. Receptor binding assays revealed different receptor selectivity profiles for these analogues in comparison to their prototype. It was found that analogues of 1, bearing a disulfide bridge, had increased selectivity to hsst2 and hsst5; however, they exhibited weaker affinity to hsst4 as compared to 1. These studies imply that ring chemistry, ring size, and ring position of the peptide template may affect the receptor binding selectivity.

Editorial

Our series of invited contributions on “Current Trends in Peptide Science” continues in this issue with a group of articles that encompass a spectrum of topics in peptide structure, pharmacology, and synthesis. In the first article, Mozsolits and Aguilar detail the application of surface plasmon resonance (SPR) spectroscopy to the detailed analysis of peptide affinity for a variety of membrane systems. The authors describe the technique of SPR; the preparation of immobilized lipid surfaces for SPR-based biophysical studies; and provide several examples of information that can be obtained, highlighting the membrane interactions of antimicrobial peptides. Lauer-Fields et al. then review the biological functions—and consequences—of the matrix metalloproteinase (MMP) family of enzymes, describing the capacity of MMPs to catabolize collagen (termed “collagenolysis”), and distinguishing this capacity from triple-helical peptidase activity. The authors point out that the ability to cleave a collagen triple helix is not the limiting factor for collagenolytic activity, which may reside in the enzyme's ability to properly orient and potentially destabilize collagen. The wide-ranging properties of osteogenic growth peptide (OGP) and its C-terminal pentapeptide Tyr–Gly–Phe–Gly–Gly [OGP(10–14)] as bone anabolic agents and hematopoietic stimulators are reviewed by Bab and Chorev. The authors describe, for example, the roles of OGP in the regulation matrix mineralization, in the expression of type I collagen, and as a stimulator of bone marrow regeneration after chemotherapy; and provide a detailed overview of OGP biosynthesis and activity, and of the structure–activity relationships germane to its functions. The final article of this issue features a review by Taylor on the use of side-chain lactam bridges as a valuable method for introducing conformational constraints into a peptide, notably for stabilization of α-helical structures. Through specifically chosen spacing, along with appropriately chosen selectively cleavable protecting groups, desired mono-, di-, and bicyclic lactam-bridged peptides can be prepared by solid-phase methods. The author reviews the key contributions of this methodology to protein biology, including studies of protein folding, ligand–receptor recognition, as well as development of peptide therapeutics with increased potency. The articles in this issue are particularly comprehensive and informative, and form a topical selection that should be of broad interest to peptide chemists and biologists.

The synthesis and study of side-chain lactam-bridged peptides.

Side-chain lactam bridges linking amino acid residues that are spaced several residues apart in the linear sequence offer a convenient and flexible method for introducing conformational constraints into a peptide structure. The availability of a variety of selectively cleavable protecting groups for amines and carboxylic acids allows for several approaches to the synthesis of monocyclic, dicyclic, and bicyclic lactam-bridged peptides by solid-phase methods. Multicyclic structures are also accessible, but segment-condensation syntheses with solution-phase cyclizations are most likely to provide the best synthetic approach to these more complex constrained peptides. Lactam bridges linking (i, i + 3)-, (i, i + 4), and (i, i + 7)-spaced residue pairs have all proven useful for stabilization of alpha helices, and (i, i + 3)-linked residues have also been demonstrated to stabilize beta-turns. These structures are finding an increasing number of applications in protein biology, including studies of protein folding, protein aggregation, peptide ligand-receptor recognition, and the development of more potent peptide therapeutics. Defining the functional roles of the amphiphilic alpha-helices in medium-sized peptide hormones, and studying helix propagation from rigid, alpha-helix initiating bicyclic peptides are among the most exciting developments currently underway in this field.

Designed Peptide Analogues of the Potassium Channel Blocker ShK Toxin

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.

Novel agouti-related-protein-based melanocortin-1 receptor antagonist.

The melanocortin receptors are G-protein coupled receptors (GPCRs) that activate the cAMP signal transduction pathway and are stimulated by the melanocortin agonist alpha-melanocyte stimulating hormone (alpha-MSH). Members of these melanocortin receptors are antagonized by agouti (ASP) and agouti-related protein (AGRP), which are the only known endogenous antagonists of GPCRs identified to date. Structure-function studies of the hAGRP(109-118) decapeptide, Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH(2), by replacing the 26-membered disulfide Cys(2)-Cys(9) ring with lactam bridges resulted in the identification of a novel peripheral skin melanocortin-1 receptor (MC1R) antagonist. This antagonist, Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2), possesses a 27-membered ring with the lactam bridge being formed from the Calpha-carboxyl moiety of Glu (instead of the typical side chain carboxyl moiety) with the amine of the diaminopropionic acid (Dpr) residue. This mouse MC1 receptor antagonist (pA(2) = 5.9) is also an antagonist at the brain melanocortin-4 receptor (pA(2) = 6.9), with no observable pharmacology at the melanocortin-3 or -5 receptors. This MC1R hAGRP(109-118) based decapeptide is novel in that AGRP(83-132) itself does not bind to, agonize, or antagonize the skin MC1R. Structural analysis has been performed using two-dimensional (1)H NMR and computer-assisted molecular modeling (CAMM) techniques in attempts to identify structural features of this Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2) (cyclo Glu alphaCOOH-Dpr betaNH) peptide that can differentially result in antagonist versus agonist properties at the mMC1R.

A new approach to search for the bioactive conformation of glucagon: positional cyclization scanning.

In search for the bioactive conformation of glucagon, "positional cyclization scanning" was used to determine secondary structures of glucagon required for maximal interaction with the glucagon receptor. Because glucagon is flexible in nature, its bioactive conformation is not known except for an amphiphilic helical conformation at the C-terminal region. To understand the conformational requirement for the N-terminal region that appears to be essential for signal transduction, a series of glucagon analogues conformationally constrained by disulfide or lactam bridges have been designed and synthesized. The conformational restrictions via disulfide bridges between cysteine i and cysteine i + 5, or lactam bridges between lysine i and glutamic acid i + 4, were applied to induce and stabilize certain corresponding secondary structures. The results from the binding assays showed that all the cyclic analogues with disulfide bridges bound to the receptor with significantly reduced binding affinities compared to their linear counterparts. On the contrary, glucagon analogues containing lactam bridges, in particular, c[Lys(5), Glu(9)]glucagon amide (10) and c[Lys(17), Glu(21)]glucagon amide (14), demonstrated more than 7-fold increased receptor binding affinities than native glucagon. These results suggest that the bioactive conformation of glucagon may adopt a helical conformation at the N-terminal region as well as the C-terminal region, which was not evident from earlier biophysical studies of glucagon.

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.

Aib-rich peptides containing lactam-bridged side chains as models of the 3(10)-helix.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.

Preparation of 'side-chain-to-side-chain' cyclic peptides by Allyl and Alloc strategy: potential for library synthesis.

Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side-chain-to-side-chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibe method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh3)4/PhSiH3)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium-mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.

Chemical syntheses and biological activities of lactam analogues of alpha-conotoxin SI.

Bicyclization represents an effective method for the introduction of conformational constraints into small, biologically important peptides. Several strategies have been developed for the preparation of bicyclic lactam analogues of alpha-conotoxin SI, a 13-residue peptide neurotoxin found in cone snail venom. Four analogues of the natural regioisomer of alpha-conotoxin SI were designed and synthesized, each with one of the two paired cysteines of the parent peptide being replaced by a side-chain lactam bridged glutamic acid/lysine pair. Solid-phase lactamization was studied to determine rates of formation of the two possible loops and to document the extent of dimerization and higher oligomerization. Radioligand binding assays were carried out on all synthesized peptides, including the naturally occurring two-disulfide form, in order to determine their affinities for nicotinic acetylcholine receptors (nAChRs). Replacement of the Cys(2)-Cys(7) loop of alpha-conotoxin SI with a lactam bridge resulted in complete loss of activity, whereas replacement of the Cys(3)-Cys(13) disulfide loop resulted in a approximately 60-fold reduction in affinity for one orientation and a approximately 70-fold increase in affinity for the other. The two active lactam analogues retain the selectivity exhibited by the naturally occurring peptide for the alpha/delta subunit of nAChRs, as judged by competition experiments with the curariform antagonist metocurine.