Acidolytic cleavage of tris(alkoxy)benzylamide (PAL) “internal reference” amino acyl (IRAA) anchoring linkages: validation of accepted procedures in solid-phase peptide synthesis (SPPS)

Under the normal conditions of acidolytic cleavage/deprotection of tris(a1koxy)benzylamide (PAL) anchoring linkages in Fmoc solid-phase peptide synthesis (SPPS), product release occurs by a straightforward single-step pathway. A recently reported cleavage of the NH-VH bond of an amino acyl residue adjacent to PAL [see Int. J. Peptide Protein Res. 38 , 146–153 (1991)] could not be confirmed in novel experiments incorporating a double “internal reference” amino acid (IRAA) design. The results of the present work revalidate the widely accepted application of IRAAs to monitor yields in SPPS, and confirm the reliability of PAL methodology for the preparation of C-terminal peptide amides.     

Affinity purification of a difficult-sequence protein

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41 , 385–393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimen- sional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.     

Comparison of methods for the Fmoc solid-phase synthesis and cleavage of a peptide containing both tryptophan and arginine

A major side reaction which can occur during the synthesis of Trp-containing peptides is modification of the Trp indole by reactive carbonium ion species released during acidolytic cleavage. [Asn²,Trp⁴]Dynorphin A-(1–13), a sequence which is very susceptible to Trp modification, was chosen as a model peptide to compare the effectiveness of various methods proposed to minimize Trp modification during Fmoc solid-phase synthesis. The peptide was synthesized with the side chain of Trp unprotected and cleaved by Reagent K [82.5% trifluoroacetic acid (TFA)/5% phenol/5% water/5% thioanisole/2.5 % ethanedithiol (EDT)] [King, D.S. et al. (1990) Int. J. Peptide Protein Res. 36 , 255–2661, Reagent R [90% TFA/5 % thioanisole/3% EDT/2% anisole] [Albericio, F. et al. (1990) J. Org. Chem. 55 , 3730–3743], TFA containing 20% EDT and 4% water [Riniker, B. & Hartmann, A. (1990) in Peptides: Chemistry, Structure, and Biology (Rivier, J.E. & Marshall, G.R., eds.), pp. 950–952, Escom, Leiden], and TFA containing trialkylsilane, MeOH, and ethylmethyl sulfide [Chan, W.C. & Bycroft, B.W. (1992) in Peptides: Chemistry, Structure, and Biology, Op. cit., pp. 613–614]. Cleavage with Reagent K, Reagent R and TFA containing 20% EDT and 4% water yielded similar results; in addition to the desired peptide, the crude product contained 22–30% of a side product which appeared to result from Trp modification by a Pmc group. Cleavage with the triakylsilane-containing mixture gave the lowest recovery of the desired peptide and the highest levels of Pmc-containing peptides. In contrast, synthesis of the peptide by Fmoc solid-phase synthesis utilizing Fmoc-Trp(Boc) and subsequent cleavage with TFA containing 20% EDT and 5% water yielded the desired peptide in essentially pure form with < 5% of the Pmc-containing side product. Thus, in the Fmoc solid-phase synthesis of [Asn²,Trp⁴]dynorphin A-(1–13) protection of the indole nitrogen by Boc was the most effective method for suppressing the modification of Trp by Pmc. This demonstrates the potential for improving the yield and purity of peptides containing both Trp and Arg by utilizing Fmoc-Trp(Boc) during the Fmoc solid-phase synthesis of these peptides.     

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.     

Divalent metal ion mediated interaction of proteins with negatively charged membranes A model study employing molecular mechanics

Previous molecular mechanics calculations on the effect of Ca(II) and Mg(II) ions on the conformation of the 18-23 loop of bovine prothrombin [Maynard et al. 1988, Int. J. Peptide Protein Res. 31, 137–1491 are extended to include the effect of a model phospholipid head group methyl[l -seryl] phosphate. Whereas the conformation of the Gla-21 Pro-22 amide bond remains decidedly trans in the absence of the model head group, in its presence, the cis Ca(II) ion induced (but not Mg(II)) form is significantly lowered in relative energy. The low energy Ca(II) structures establish a coordination sphere with more ligands than do the low energy Mg(II) ion structures.     

Dynamics of amino acid residues in globular proteins

The dynamic differential equation model developed and tested for bovine pancreatic trypsin inhibitor and tuna ferrocytochrome c in Ponnuswamy, P.K. & Bhaskaran, R. (Int. J. Peptide Protein Res. 24 , 168–179, 1984) is extended for 17 more protein crystals in this work. Average displacements are computed for 20 amino acid residues observed in 19 proteins. Detailed information on the dynamic behaviour of the individual proteins and individual residues is presented. The effect of atomic packing on the fluctuations of the amino acid residues in α-chymotrypsin is illustrated. A number of general points on the dynamic characteristics of globular protein molecules are presented.     

Atrial natriuretic peptide modulators: Dissociation of receptor binding and particulate guanylate cyclase activity

Prazosin stimulated ¹²⁵l-rANP(102–126) binding to bovine adrenal zona glomerulosa membranes (BAZGM) allosterically by converting the lower-affinity state binding sites into the higher-affinity state. The modulatory effect could be mimicked by guanabenz and LY193422, and was concentration-dependent. SC₅₀ (concentration required to stimulate binding by 50%) values for guanabenz, prazosin, and LY193422 were 13, 25, and 1.8 μM, respectively. Prazosin (30 μM) enhanced ¹²⁵l-ANP(102–126) binding by decreasing the K_D from 251 ± 24 to 97.4 ± 8.3 pM without affecting the Bmax. Competitive inhibition of ¹²⁵l-ANP binding by ANP analogs was potentiated by prazosin (30 μM) and LY193422 (50 μM). The modulator-stimulated ¹²⁵l-rANP(102–126) binding was reversible by the addition of unlabelled ANP analogs during incubation. Previously, we also demonstrated that these modulators potentiated the ANP-mediated inhibition of ACTH-induced aldosterone synthesis in rat adrenal zona glomerulosa cells [Horng, J. S., Steinberg, M. I., Wyss, V. L., Wiest, S. A., Schirmer, E. W., McCowan, J. R., and Yu, M. J.: Atrial natriuretic peptide (ANP) receptor modulators: Effects of prazosin and guanabenz analogs on ANP receptor binding and in vitro biological activity. FASEB Journal 3:A733, 1989; Yu, M. J., McCowan, J. R., Steinberg, M. I., Wiest, S. A. Wyss, V. L., and Horng, J. S.: Atrial natriuretic peptide receptor modulators: Effect of disubstituted quinazolines on receptor binding and in vitro biological activity. Journal of Medicinal Chemistry 33:348–353, 1990]. However, neither prazosin nor LY193422 affected basal or ANP-induced particulate guanylate cyclase activity in rabbit glomeruli. This is the first study to our knowledge in which ANP modulators were demonstrated to potentiate ANP-mediated response independent of cGMP. Our studies suggest that potentiation of ANP-mediated inhibition of aldosterone synthesis by these analogs does not involve particulate guanylate cyclase and that these modulators induce a cyclase-uncoupled, high affinity ANP receptor in the adrenal gland, which might be different from cyclase-uncoupled ANP clearance receptor.     

Conformation and hydrogen bonding of N-formylmethionyl peptides

Crystals of N-formyl-L-methionyl-L-phenylalanine (C₁₅H₂₀N₂O₄S). grown from aqueous methanol solution are orthorhombic, space group, P2₁2₁2₁, with cell parameters at 294K of a = 4.900(2), b = 17.947(4), c = 18.726(4)Å, V = 1646.8ų, M.W. = 324.4, Z = 4 and D_m= 1.308 g/cc, and as expected, all nearly identical to that of N-f-D-Met-D-Phe studied by Jeffs, Heald, Chodosh & Eggleston (Int. J. Peptide Protein Res. 24 , 442–446, 1984). The crystal structure was solved and refined using CAD-4 data (1095 reflections ≥ 3σ) to a final R value of 0.042. Molecules related by the a-translation form a parallel β-sheet rather than anti-parallel sheet as stated in the earlier study of Jeffs et al. The formation of the parallel rather than the anti-parallel p-sheet structure, the use of the C-H…O hydrogen bonds to stabilize the b-sheet and the very short O-H…O hydrogen bond between the carboxyl OH and the N-acyl oxygen atom emerge as the main structural features of the chemotactic N-formyl methionyl peptides.     

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.     

Cardiovascular failure produced by a peptide from the venom of the southern Pacific rattlesnake, Crotalus viridis helleri.

R. C. Schaeffer, Jr., T. R. Pattabhiraman, R. W. Carlson, F. E. Russell and M. H. Weil. Cardiovascular failure produced by a peptide from the venom of the Southern Pacific rattlesnake, Crotalus viridis helleri. Toxicon17, 447–453, 1979.—Hemodynamic, metabolic and respiratory effects of a 30 min i.v. infusion of crude venom (1·4 mg/kg) and three venom components (Peptide I, 0·5 mg/kg; Protein I, 1·2 mg/kg, Protein II, 3·4 mg/kg) were studied in 24 sedated rats (270–309 g). Venom shock, characterized by hypotension, lactacidemia, hemoconcentration, hypoproteinemia and death was observed in animals given the crude venom or Peptide I. Just prior to death, respiratory distress was observed in most animals that died. Hemolysis and hematuria were observed in the animals given Protein I or Protein II. These data suggest that the increase in vascular permeability to protein and red blood cells induced by the crude venom can, for the most part, be attributed to the peptide. In addition, Protein I and Protein II appear to account for the hemolytic activity. The toxic effects of the venom components appear to be synergistic.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.     

Ribonuclease inhibitors from the livers of five mammalian species

Inhibitors of neutral ribonuclease have been purified to homogeneity from beef, pig, sheep, mouse, and rat liver by affinity chromatography on Sepharose-RNase A with overall yields ranging from 60–80%. Each of the purified inhibitors presents a single band by SDS-gel electrophoresis; molecular weight estimates by SDS-gel electrophoresis and by gel filtration are ca. 50000. Each of the inhibitors forms a complex with beef pancreatic RNase A with a molecular weight of ca. 64000, suggestive of 1:1 binding on a molar basis. The inhibitors from liver are very similar in properties and amino acid composition to the previously isolated inhibitor from human placenta (Blackburn et al. (1977) J. Biol Chem. 252 , 5904) and beef brain (Burton et al. (1980) Int. J. Peptide Protein Res. 16 , 359). Pig liver offers an alternative to human placenta as a source for an RNase inhibitor of this type (yield, ca. 8 mg/kg of tissue). Immunological similarities were examined using antiserum directed against human placental RNase inhibitor. Cross reactivity of the liver RNase inhibitors with the antiserum raised against placental RNase inhibitor ranged from 15% for mouse RNase inhibitor to as low as 2% for pig and sheep RNase inhibitor.     

Encouraging results for herbal treatment of acute bronchitis in children and adolescents

Kamin W, Maydannik V, Malek FA, Kieser M. Efficacy and tolerability of EPs 7630 in children and adolescents with acute bronchitis: a randomized, double‐blind, placebo‐controlled multicenter trial with a herbal drug preparation from Pelargonium sidoides roots. Int J Clin Pharmacol Ther 2010; 48: 184–91.     

Efficient Fmoc/solid-phase synthesis of Abu(P)-containing peptides using Fmoc-Abu(PO3Me2)-OH

The synthesis of the two 4-phosphono-2-aminobutanoyl-containing peptides, Leu-Arg-Arg-Val-Abu(P)-Leu-Gly-OH.CF₃CO₂H and Ile-Val-Pro-Asn-Abu(P)-Val-Glu-Glu-OH.CF₃CO₂H was accomplished by the use of Fmoc-Abu(PO₃Me₂)-OH in Fmoc solid-phase peptide synthesis. The protected phosphoamino acid, Fmoc-Abu(PO₃Me₂)-OH, was prepared from Boc-Asp-O'Bu in seven steps, the formation of the C—P linkage being effected by the treatment of Boc-Asa-O'Bu with dimethyl trimethylsilyl phosphite. Peptide synthesis was performed using Wang Resin as the polymer support with both peptides assembled by the use of PyBOP® for the coupling of Fmoc amino acids and 20%, piperidine for cleavage of the Fmoc group from the Fmoc-peptide after each coupling cycle. Cleavage of the peptide from the resin and peptide deprotection was accomplished by the treatment of the peptide-resin with 5%, thioanisole/TFA followed by cleavage of the methyl phosphonate group by 1 M bromotrimethylsilane/l M thioanisole in TFA.     

Identification and characterization of a novel synthetic peptide substrate specific for Src-family protein tyrosine kinases

Using a random combinatorial peptide library method [Wu, J., Ma, Q. N. & Lam, K. S. (1994) Biochemistry 33 , 14825–14833] a novel peptide, YIYGSFK, was identified as a substrate for p60^c-src protein tyrosine kinase. Mass spectrometric analysis showed that tyrosine-3 from the N-terminus was the phosphorylation site. Kinetic studies showed that the K_m of YIYGSFK for p60^c-src was 55 μM, about 6.4-fold lower than a peptide derived from p34^cdc2 [cdc2(6–20), KVEKIGEGTYGVVYK], which had been reported to be a specific and efficient substrate for the Src-family protein tyrosine kinases. Comparison of the specificity of YIYGSFK and cdc2(6–20) as a substrate for various Src-family and non-Src-family protein tyrosine kinases suggests that YIYGSFK is a much more specific and efficient substrate for the Src-family protein tyrosine kinases. © Munksgaard 1995.     

Poly(l-lactic acid) microspheres for pulmonary drug delivery: release kinetics and aerosolization studies

We have previously developed poly(l-lactic acid) (PLA) microspheres containing nedocromil sodium and beclomethasone dipropionate (BDP) for aerosolisation to the respiratory tract (El-Baseir, M.M., Phipps, M.A., Kellaway, I.W., Preparation and subsequent degradation of poly(l-lactic acid) microspheres suitable for aerosolisation: a physico-chemical study. Int. J. Pharm. 151 (1997) 145–153). In this study we have investigated the in vitro release kinetics of these two drugs from PLA microspheres and the deposition of the microspheres in an in vitro lung model (Andersen cascade impactor) following aerosolisation from a dry powder inhaler (Spinhaler^®). The in vitro kinetics of drug release revealed a controlled release of nedocromil sodium over 8 days with a burst effect (27–60%, w/w) which varied with the particle size of the microspheres. For BDP entrapped in PLA microspheres, controlled release of BDP occurred over 6 days. BDP release was determined by measuring the shift in the phase transition temperature of dimyristoylphosphatidylcholine (DMPC) liposomes induced by partitioning of the steroid into the lipid bilayers. The residual poly(vinyl alcohol) used as an emulsifier in the production of the microspheres was < 7% (w/w). The in vitro deposition of the microspheres (1.00 ± 0.21 μm) containing BDP from a Spinhaler^® to a cascade impactor at a flow rate of 60 l/min, resulted in 20% of the emitted dose deposited on stages corresponding to particles < 3 μm and approximately 42% < 5 μm.     

N-Methylated Cyclic Enkephalin Analogues Retain High Opioid Receptor Binding Affinity

In an effort to improve the bioavailability of the non-selective, cyclic enkephalin analogues H-Dmt-c[d -Cys-Gly-Phe-d (or L )-Cys]NH₂ (Dmt = 2′,6′-dimethyltyrosine), analogues N-methylated at the Phe⁴ and/or Cys⁵ residue were synthesized. In comparison with the non-methylated parent peptides, all mono- and N-di-methylated analogues in general retained high binding affinities at all three opioid receptors and high opioid agonist potencies in functional opioid activity assays. The results indicate that the progressive conformational restriction in these compounds upon mono- and di-N-methylation did not significantly affect the in vitro opioid activity profile. A low-energy conformer identified for the conformationally most restricted analogue of the series, H-Dmt-c[D -Cys-Gly-Phe(NMe)-L -Cys(NMe)]NH₂ (6), showed good spatial overlap of the essential pharmacophoric moieties with those in the proposed μ receptor-bound conformation of the μ-selective opioid peptide JOM-6 [H-Tyr-c(S-Et-S)[D -Cys-Phe-D -Pen]NH₂] (Pen = penicillamine) [Mosberg M.I. and Fowler C.B. (2002) J Peptide Res; 60:329–335], in agreement with the moderate μ selectivity determined for this compound. An analogue of 6 containing (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Dmt¹ was an opioid antagonist with quite high opioid receptor binding affinities and can be expected to show improved bioavailability because of its further increased lipophilicity and reduced hydrogen-bonding capacity.     

Phosphono Analogues of Glutathione as New Inhibitors of Glutathione S-Transferases

Phosphono-analogues of glutathione containing the O=P(OR)₂ moiety in place of the cysteinyl residue CH₂SH 1a–1d were prepared by solution phase peptide synthesis. Benzyl, benzyloxycarbonyl, and tert-butyl protecting groups were used to mask the individual amino acid functional groups. The formation of peptide bonds was achieved by the usual peptide synthesis via activation of carboxylic functions with cyclohexylcarbodiimide and subsequent reaction with free amino groups. The thus obtained, fully-protected peptides were each purified by normal phase column chromatography. Deprotection was accomplished by hydrogenolysis and by treatment with HBr/acetic acid yielding the desired phosphonic acid diester 1a–1d . The inhibition of the glutathione conjugation of 1-chloro-2,4-dinitrobenzene by human placental glutathione S-transferase was studied by determining the IC₅₀ values of the new glutathione analogues. The IC₅₀ values were 291 μM, 139 μM, 64 μM, and 21 μM for the dimethyl, diethyl, diisopropyl, and di-n-butyl esters, respectively. The results clearly show that the formal substitution of the glutathione thiol function by phosphonic acid esters leads to a new class of glutathione S-transferase inhibitors. Further investigations directed at the question of whether or not these glutathione analogues are suitable for a modulation in chemotherapy are in progress.     

Schisandra extract elevates concentration of tacrolimus in blood of liver transplant patients

Jiang W, Wang X, Xu X, Kong L. Effect of Schisandra sphenanthera extract on the concentration of tacrolimus in the blood of liver transplant patients. Int J Clin Pharmacol Ther 2010; 48: 224–9.     

Synthetic peptide epitope-based polymers: controlling size and determining the efficiency of epitope incorporation

Abstract: The assembly of synthetic peptide-based vaccines that incorporate multiple epitopes is a major goal of vaccine development, because such vaccines will potentially allow the immunization of outbred populations against a number of different pathogens. We have shown that free radical-induced polymerization of individual peptide epitopes results in the incorporation of multiple copies of the same or different epitopes into high molecular weight immunogens (O'Brien-Simpson, N.M., Ede, N.J., Brown, L.E., Swan, J. & Jackson, D.C. (1997) Polymerization of unprotected synthetic peptides: a view toward synthetic peptide vaccines. J. Am. Chem. Soc. 119 , 1183–1188; Jackson, D.C., O'Brien-Simpson, N., Ede, N.J. & Brown, L.E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15 , 1697–1705). The ability to control the size of these polymers, to determine the physical and chemical properties of the backbone material and also to know the extent to which individual peptide epitopes are incorporated are important manufacturing considerations and form the subject of this study. We show here that the polymerization process is highly efficient with at least 70% of peptides incorporated into the resulting polymer, that acrylamide and acryloylated amino acids can be used as comonomers with peptide epitopes in the polymerization reaction and that the choice of the comonomer can influence the properties of the resulting polymer. We also show that the size of chain growth polymers is restricted in the presence of chain transfer agents, that the resulting polymer size can be predicted and that there is little or no difference in the immunogenicity of polymers that range in apparent molecular size between 18 kDa and 335 kDa. The successful polymerization of peptide epitopes with an acryloyl-amino acid creates the potential for introducing different physical and chemical properties into artificial protein immunogens.