Synthesis and backbone conformations of cyclic hexapeptides cyclo-(Xxx-Pro-D-Gln)2

The solution syntheses of cyclo-(Xxx-Pro-D-Gln)₂, where Xxx=Gly, Ala, Leu, Phe and Val are described. Several routes were examined, the most successful involving the intermediate Z-Xxx-Pro-D-Gln-O-tBu and proceeding to cyclization of H-Xxx-Pro-D-Gln-Xxx-Pro-D-Gln-OH using diphenylphosphoryl azide. The N-H regions of the proton magnetic resonance spectra of aqueous solutions of these peptides were examined, and in the Xxx=Leu and Val peptides an unsymmetrical backbone, presumably with one cis Xxx-Pro peptide bond, was found to be important. Previous reports of cyclo-(Xxx-Pro-D-Yyy)₂ peptides have shown only C₂-symmetric forms.     

Circular dichroism investigations on the conformation of linear,cyclic and polymeric peptides containing His,Ser and Asp residues

Conformational studies on the linear, cyclic and polymeric peptides with the sequences -Asp-ßAla-Gly-Ser-ßAla-Gly-His-ßAla-Gly-(nonapeptide series) and -Asp-eAhx-Ser-eAhx-His-eAhx (hexapeptide series) were carried out by means of circular dichroism measurements to clarify the relationship between their conformation and ester hydrolytic activity. It was suggested that all of the present peptides take a certain ordered conformation at acidic pH region and have a tendency to transform to a disordered conformation with a rise of pH. In the course of the conformational change, the linear and polymeric peptides undergo a drastic conformational change at around pH 7.0, while the cyclic peptides maintain the ordered conformation to some extent even at the basic pH region. Although this ordered conformation maintained in the cyclic peptides was estimated not to be a tight one, it was more stable in the cyclic peptide of the latter sequence (Cyclic-6) than in the former sequence (Cyclic-9). As reported previously, Cyclic-6 showed greater activity than the others in the hydrolysis of various ester substrates. Higher activity and more stable ordered conformation of Cyclic-6 seem to be closely related. The effects of urea, organic solvent, salt and change of temperature on their conformation are also reported.     

Solution stability of linear vs. cyclic RGD peptides

Abstract: Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1 ) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH_2; 2 ) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2–12 at 50°C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear ( 1 ) and cyclic ( 2 ) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1 , especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.     

Ring conformation of cyclic octapeptide cyclo (L -Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(l -Pro-Sar)₄. The material [C₃₂H₄₈O₈N₈ · (21/4) H₂O° (1/2) CH₃OH, M_r = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and β = 122.26 (2)° (Λ = 1.54178 Å, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis -or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the γ-carbon atom deviates from the plane of the remaining four atoms in the ring.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides

Abstract: The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1 ) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH₂; 2 ); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1 . Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2 , which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1 , especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2 . This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

The aqueous conformation of cyclo(1,6)Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2

RGD peptides are known as important ligands for integrin receptors in the cell adhesion process. The selectivity of RGD peptides for a certain integrin receptor is partly dependent on the RGD conformation and the residues surrounding the RGD sequence. This paper investigates the effect of the addition of a phenyl-alanine residue on the RGD conformation in cyclo(1,6)Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH₂ (1) as compared to the previously studied cyclo(1,5)Ac-Pen-Arg-Gly-Asp-Cys-NH₂ (2). The conformational study of peptide I was done in aqueous solution using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. This work will increase the understanding of the flanking residue's effect in RGD peptides.     

Characterisation of the solution conformation of a cyclic RGD peptide analogue by NMR spectroscopy allied with a genetic algorithm approach and constrained molecular dynamics

The solution conformation of a cyclic RGD peptide analogue, cyclo-(S,S)-2-merrcaptobenzoate-arginine-glycine-aspartate -2-mercaptoanilide, has been determined via two independent approaches for the searching of conformational space and identification of conformations consistent with NMR and CD spectroscopic data: (i) the use of a binary genetic algorithm and (ii) a molecular dynamics simulation. Inter-proton distances were obtained via analysis of cross-peak volumes from a two-dimensional ROESY NMR spectroscopy experiment at 600 MHz and were used as constraints for the computational calculations. The mercaptoanilide amide proton resonance chemical shift had a very small temperature coefficient, indicating that this proton was hydrogen-bonded. Circular diehroism data showed that, in solution, the torsion angle about the disulfide bond was negative, consistent with one of the distinct conformations around this bond in the 200 ps molecular dynamics simulation. The backbone conformations of the structures resulting from the two different approaches were very similar.     

Synthesis and conformational study of a cyclic hexapeptide analogue of somatostatin: cyclo(Phe-D-Trp-Lys-Thr-o-AMPA)

The active sequence Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰ of somatostatin has been cyclized through o-(aminomethyl)phenylacetic acid, a spacer molecule, designed to mimic a Gly-Gly dipeptide containing a cis-constrained peptide bond. The resulting analogue shows no GH-inhibition. A 2D n.m.r. study reveals conformations different from the proposed bio-active one and still sensitive to the medium (solvent).     

Chemical Degradation Kinetics of Recombinant Hirudin (HVI) in Aqueous Solution: Effect of pH

Purpose. To gain information on the chemical stability pattern and the kinetics of the degradation of recombinant hirudin variant HVI (rHir), a thrombin-specific inhibitor protein of 65 amino acids, in aqueous solution as a function of pH. Methods. Stability of rHir was monitored at 50°C in the framework of a classical pH-stability study in aqueous buffers pH 1–9.5. Two capillary electrophoresis (CE) protocols were used; one for the kinetics of succinimide formation at Asp⁵³-Gly⁵⁴ (C-terminal tail) and Asp³³-Gly³⁴ (loop section), the other for the kinetics of rHir degradation. To check for potential effects of conformational changes by thermal denaturation, circular dichroism (CD) measurements were performed between 25 and 80°C. Results. Throughout the pH range studied no effect of thermal denaturation on rHir confirmation at 50°C was observed. rHir was most stable at a neutral pH whereas, at slightly acidic pH, an intermediate stability plateau was found. Both, strongly acidic and alkaline conditions led to fast rHir degradation. Depending on the pH of degradation, rHir was found to degrade in various combinations of multiple parallel and sequential degradation patterns. Special focus was on succinimide formation at Asp⁵³-Gly⁵⁴ (C-terminal tail) and Asp³³-Gly³⁴ (loop) and on the potential of isoAsp formation in position 53 and 33. Conclusions. Chemical rHir stability in the intermediate pH range depends strongly on succinimide formation. At slightly acidic conditions succinimides represent the major degradation product (up to 40%). Around neutral pH succinimides react further, presumably by isoAsp formation, and concentrations remain low. Relative preference of succinimide formation in the C-terminal tail domain versus the loop domain is explained by higher backbone flexibility in the tail.     

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides

Abstract: In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based ( 3 and 4 ) and the phenylpropionic acid-based ( 5 and 6 ) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu⁵]-enkephalin ( 1 , H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE ( 2 , H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B ) in solution. Conformer A exhibited a type I β-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II β-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4 . The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3 – 6 , but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.     

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability

Abstract: In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu⁵]-enkephalin( 1 , H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE( 2 , H-Tyr-d -Ala-Gly-Phe-d -Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3 : 2 where both conformers exhibit γ and β-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4 . It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4 , with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.     

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro)

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P6₅, Z = 6], shows the presence of one intramolecular N-H?O=C hydrogen bond with the formation of a γ-turn (C₇). The Aib³ residue, at the center of the γ-turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= -73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu⁴ and Pro⁵; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular γ-turn on the opposite side results in an equatorial topology of the side-chains of the Phe¹, Phe² and Leu⁴ residues. Indeed, the C^α-C^βand C^β-C^γ bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu⁴-Pro⁵ peptide bond.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs

Abstract: Ascidiacyclamide (ASC), cyclo(‐Ile¹‐Oxz²‐d ‐Val³‐Thz⁴‐)₂ (Oxz=oxazoline and Thz=thiazole) has a C₂‐symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers 49 , 459–469]. In this study, the modifications were extended. The Ile¹ residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d ‐Ile, and the d ‐Val³ residue was replaced by Val. The structures of these analogs were analyzed by X‐ray diffraction, ¹H NMR and CD techniques. X‐Ray diffraction analyses revealed that the [Ala¹], [Aib¹] and [Phe¹]ASC analogs are folded, whereas [Val¹]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly¹] and [Phe¹]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib¹], [d ‐Ile¹] and [Val³]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d ‐Ile¹] and [Val³]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly¹]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val³]ASC. In the cytotoxicity test using P388 cells, [Val¹]ASC exhibited the strongest activity, whereas the epimers of ASC ([d ‐Ile¹] and [Val³]ASCs), showed fairly moderate activities.     

Novel linear antagonists of the antidiuretic (V2) and vasopressor (V1) responses to vasopressin

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V₂) and the vasopressor (V₁) responses to arginine vasopressin (AVP), d(CH₂)₅[d -Tyr(Et)²,Val⁴]AVP (A). Peptide 1, the linear precursor of (A), (CH₂)₅(SH)-CH₂-CO-d -Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH₂ was modified at position six with α-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu⁶ analogue (No. 2) at position one by substituting cyclohexylacetic acid (Caa), cyclohexylpropionic acid (Cpa), 1-adamantaneacetic acid (Aaa), phenylacetic acid (Phaa), tert.-butylacetic acid (t-Baa), isovaleric acid (Iva), propionic acid (Pa), l -penicillamine (P), tert.-butoxycarbonyl (Boc) or omitting any substituent at this position, and/or in combination with Arg-NH₂⁹, Ala-NH₂, ⁹d -Arg⁸-Arg-NH₂⁹, and desGly⁹ modifications yielded the remaining 14 peptides. All 16 peptides were examined for agonistic and antagonistic potencies in AVP V₂ and V₁ assays in rats. Apart from the Cpa¹ analogue and the analogue lacking any substituent in the 1-position, all exhibit substantial V₂ and V₁ antagonism. A number are as potent as (A) as V₂ antagonists. With an anti-V₂ pA₂= 8.11 ± 0.07, Aaa-d -Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH₂ (No. 6) is as potent as any cyclic AVP V₂ antagonist reported to date. The Pa¹ analogue of No. 6 exhibits promising anti-V₂/anti-V₁ selectivity. These findings prove conclusively that a ring structure is not a requirement for recognition of or for binding to AVP V₂ or V₁ receptors. This discovery thus offers a promising new approach to the design of peptide and non-peptide antagonists of AVP and perhaps also to other cyclic peptides such as somatostatin, atrial-natriuretic factor, insulin, and the recently discovered endothelin. Some of these linear antagonists may be of value as pharmacological tools and as therapeutic agents.     

环二鸟苷酸 (c-di-GMP) 在微生物体内的作用及其类似物的研究

环二鸟苷酸 (cyclic diguanylate, c-di-GMP) 是在细菌中普遍存在的第二信使分子, 参与调节多种生理功能, 包括细胞分化、生物被膜形成、致病因子产生等。细菌细胞内c-di-GMP合成与降解代谢分别受二鸟苷酸环化酶 (diguanylate cyclase, DGC) 和磷酸二酯酶 (phosphodiesterase, PDE) 调控, DGC和PDE共处于同一个蛋白中, 是一个双功能蛋白酶的两个区域, 分别负责菌体内c-di-GMP的合成和降解。c-di-GMP作用菌体内下游靶点包括PilZ结构域和GEMM核开关两种类型。目前发现c-di-GMP核开关是唯一不参与代谢活动而参与信号传导的一类核开关。本文综述了c-di-GMP的代谢途径、调控机制、生物学功能, 以及c-di-GMP结构类似物合成及生物学评价等方面的最新研究进展。     

Effect of alkylation with different sized substituents on the conformation of ovomucoid,lysozyme and ovotransferrin

Conformational changes induced in ovomucoid, lysozyme and ovotransferrin on reductive addition of different sized substituents have been studied employing differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD). The thermograms obtained by DSC revealed that extensive introduction of methyl, isopropyl, cyclopentyl, cyclohexyl, benzyl or n-butyl groups has a detrimental effect on thermal stability (enthalpy of denaturation); the effect generally increases with the size of the substituent. Circular dichroic spectra were affected only to a very limited extent by the modifications, near-u.v. spectra remaining much the same while far-u.v. spectra displayed minor changes. The general conclusion drawn is that the modifications had only limited effects on the conformation of the proteins while, nonetheless, perturbing (or breaking) long-range intramolecular interactions so as to destabilize the structure. Derivatization of lysozyme and ovotransferrin with some of the larger groups has been reported to result in spontaneous precipitation of the proteins [Fretheim, K., Iwai, S. & Feeney, R.E. (1979) Int. J. Peptide Protein Res. 14, 451–456]. The present investigation indicates that precipitation was caused by (partial) denaturation (and ensuing aggregation) as a consequence of modification.     

Crystal structure and conformation of the cyclic tetramer of a repeat tripeptide of elastin,cyclo(l-valyl-l-prolylglycyl)4

X-ray diffraction data were used to determine the crystal structure of cyclo-(l -Val-l -Pro-Gly)₄, the cyclic tetramer of a repeat tripeptide of elastin. The crystals are monoclinic, space group C2, with a = 29.639(3), b = 7.099(1), c = 20.325 (2) Å, and β = 130.4(4)°. The structure was solved by direct methods and refined by least squares to R = 0.082 for 2603 observed reflections. The cyclic dodecapeptide contains two β(II) turns. Hydrophilic and hydrophobic channels that run parallel to the b axis are formed by the stacking of cyclic peptides on twofold axes.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker

Abstract: The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu⁵]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes–Einstein molecular radii similar to those of [Leu⁵]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu⁵]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.     

Effect of calcium (II) and magnesium (II) ions on the 18–23 γ-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin An AMBER molecular mechanics study

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18–23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al, Int. J. Peptide Protein Res. 27, 1986, 530–553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.     

Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67–94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)^73,87] 67–94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.