Design and synthesis of effective antagonists of substance P

The agonist/antagonist activities of four background analogs of substance P (SP) facilitated design and synthesis of 12 new analogs to achieve effective antagonists. (D-Pro2, D-Phe7, D-Trp9)-SP, (D-Pro2, D-Trp7,9)-SP and (D-Arg1, D-Phe7, D-Trp9)-SP showed no agonist activity; 9 analogs showed weak agonist activity of SP. (D-Pro2, D-Trp7,9)-SP was the most potent antagonist which at a concentration of 10(-5) required a 3-fold increase in SP to allow a 50% response by SP. (D-Pro2, Lys6, D-Phe7)-SP and (D-Pro2, D-pClPhe7, D-Trp9)-SP were also potent, and the antagonism was competitive. For specific pairs of peptides, Lys6 is a promising substituent. D-Trp7,9 was as effective as Lys6, D-Phe7. D-pClPhe7 was three times as effective as D-Phe7. D-Dln6 was 1.33-fold better than D-Gln5. D-Pro2 and D-Pro4 were equally effective. D-Pro2 was 1.5 times as effective as D-Lys3. D-Pro2 may not be important. D-pClPhe9 and D-Trp9 were equally effective.     

Synthesis and cytotoxic activity of Boc-protected pentapeptide amide analogs of somatostatin

Russian Chemical Bulletin - New Boc-protected pentapeptide amides of the general formula Boc—Cys(Boc)—Phe—d-Trp—Lys(Boc)—Thr—NHR, which are analogs of the...     

Engineering strategy to improve peptide analogs: from structure-based computational design to tumor homing

We present a chemical strategy to engineer analogs of the tumor-homing peptide CREKA (Cys-Arg-Glu-Lys-Ala), which binds to fibrin and fibrin-associated clotted plasma proteins in tumor vessels (Simberg et al. in Proc Natl Acad Sci USA 104:932–936, 2007) with improved ability to inhibit tumor growth. Computer modeling using a combination of simulated annealing and molecular dynamics were carried out to design targeted replacements aimed at enhancing the stability of the bioactive conformation of CREKA. Because this conformation presents a pocket-like shape with the charged groups of Arg, Glu and Lys pointing outward, non-proteinogenic amino acids α-methyl and N-methyl derivatives of Arg, Glu and Lys were selected, rationally designed and incorporated into CREKA analogs. The stabilization of the bioactive conformation predicted by the modeling for the different CREKA analogs matched the tumor fluorescence results, with tumor accumulation increasing with stabilization. Here we report the modeling, synthetic procedures, and new biological assays used to test the efficacy and utility of the analogs. Combined, our results show how studies based on multi-disciplinary collaboration can converge and lead to useful biomedical advances.     

Trichogin GA IV: a versatile template for the synthesis of novel peptaibiotics

Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties. This peptaibol is predominantly folded in a mixed 3(10)-/α- helical conformation with a clear, albeit modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial membranes and to induce cell death. In previous papers, we reported on the interesting biological properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5, 6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6 or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments. Interestingly, it turns out that the presence of charged residues induces a transition of the helical conformation adopted by the peptaibols (from 3(10)- to α-helix) as a function of pH in a reversible process. The role played in the analogs by the markedly increased amphiphilicity was further tested by fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some of the least substituted among these analogs are good candidates for the development of new membrane-active antimicrobial agents.     

A general, efficient and stereospecific route to sphingosine, sphinganines, phytosphingosines and their analogs

Sphingosine, sphinganines and phytosphingosines and their analogs were synthesized by an aldol condensation between an iminoglycinate bearing a (+)-(1R,2R,5R)-2-hydroxy-3-pinanone group as chiral auxiliary and an appropriate aldehyde. All condensations proceeded with excellent enantioselectivity to generate the (2S,3R)-D-erythro structures in good yields.     

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.     

Novel cycloundecapeptides related to gramicidin S with both high antibiotic activity and low hemolytic activity

To find candidates with high antimicrobial and low hemolytic activities, many gramicidin S (GS) analogs of various ring sizes have been designed and synthesized. However, syntheses of antimicrobially active analogues of GS having a disordered symmetry structure from C(2) have almost never been reported, because the stable, amphiphilic β-sheet structure of GS with C(2) symmetry is considered essential for its strong antibacterial activity. In the present studies, novel thirteen cycloundecapeptides 1-13 related to GS were synthesized and examined. Among them, cyclo(-Va1(1)-Orn(2)-Leu(3)-D-Phe(4)-X(5)-Pro(6)-Val(7)-Orn(8)-Leu(9)-D-Phe(10)-Pro(11)-) (X=Lys (10), Orn (11), Arg (12) and Lys(Lys) (13)) resulted in high antibiotic activity against both Gram-positive and Gram-negative microorganisms tested. In addition, 11 showed low toxicity against sheep blood cells compared with that of GS. Further, circular dichroism (CD) spectra of 10-13 had a curve similar to each other, suggesting that the conformations of these analogues in methanol are similar to each other. However, CD spectra of 10-13 were different from that of GS in the 190-210 nm region. These results suggest that the presences of one added amino acid residue at position 5 of 10-13 might be partially effective through a structural change in the biological activity of 10-13. In addition, the structural modifications at position 5 lower the undesirable hemolytic activity and enhance the desirable antibiotic activity.     

Novel method for efficient aerobic oxidation of silyl ethers to carbonyl compounds catalyzed with N-hydroxyphthalimide (NHPI) and lipophilic Co(II) complexes

In this work, a new method for highly efficient and selective oxidative deprotection of a variety of structurally diverse trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) ethers using molecular oxygen in the presence of N-hydroxyphthalimide (NHPI) and various types of Co(II) complexes is reported. As a result of the relatively neutral reaction medium, acid-sensitive functional groups such as phenolic TBS ethers survived intact under the presented reaction conditions.     

Oxygen reactivity in flavoenzymes: context matters

Many flavoenzymes--oxidases and monooxygenases--react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused on the importance of a positive charge near N5 of the reduced flavin. Fructosamine oxidase has a lysine near N5 of its flavin. We measured a rate constant of 1.6 × 10(5) M(-1) s(-1) for its reaction with oxygen. The Lys276Met mutant reacted with a rate constant of 291 M(-1) s(-1), suggesting an important role for this lysine in oxygen activation. The dihydroorotate dehydrogenases from E. coli and L. lactis also have a lysine near N5 of the flavin. They react with O(2) with rate constants of 6.2 × 10(4) and 3.0 × 10(3) M(-1) s(-1), respectively. The Lys66Met and Lys43Met mutant enzymes react with rate constants that are nearly the same as those for the wild-type enzymes, demonstrating that simply placing a positive charge near N5 of the flavin does not guarantee increased oxygen reactivity. Our results show that the lysine near N5 does not exert an effect without an appropriate context; evolution did not find only one mechanism for activating the reaction of flavins with O(2).     

Click synthesis of ubiquitin dimer analogs to interrogate linkage-specific UBA domain binding

A new route to the synthesis of triazole-linked ubiquitin dimers (diUbs) as structural analogs of the seven diUbs is reported. Binding studies with the Lys48-specific UBA domain of the Mud1 protein suggest that they represent functionally suitable surrogates of their native counterparts linked by an isopeptide bond.     

Development of the carboxamide protecting group, 4-(tert-butyldimethylsiloxy)-2-methoxybenzyl

The new carboxamide protecting group, 4-(tert-butyldimethylsiloxy)-2-methoxybenzyl (SiMB), has been developed. While this SiMB group can be removed using mild basic desilylation methods, it can also be deprotected under strongly acidic or oxidative conditions. An application of this group to simple carboxamide groups, as well as to more complex and acid-sensitive adenosine derivatives containing a cyclophane scaffold, was also demonstrated.     

Structure-activity relationships of neuromedin U. IV. Absolute requirement of the arginine residue at position 7 of dog neuromedin U-8 for contractile activity

To examine the role of both Arg residues at positions 5 and 7 of dog neuromedin U-8 (d-NMU-8; pGlu1-Phe-Leu-Phe-Arg5-Pro-Arg7-Asn8-NH2) for smooth muscle contractile activity on isolated chicken crop, d-NMU-8 analogs were synthesized where either Arg residue was systematically replaced by various amino acids [X: Ala, Thr, Glu, Gln, Lys, Orn, His, citrulline (Cit) or homoarginine (Har)]. All [X5]-d-NMU-8, except for [Glu5]- and [Des-Arg5]-d-NMU-8, were full agonists, although their affinities to NMU receptors were decreased. No [X7]-d-NMU-8 showed contractile activity even at concentrations of 10(-5) mol/l, except for [Har7]-d-NMU-8, which retained weak biological activity. These analogs had no antagonistic activity against porcine neuromedin U-8 (p-NMU-8). The results revealed that Arg7 of d-NMU-8 is indispensable for receptor binding and activation to induce smooth muscle contraction, and the guanidino group of the side chain at position 7, but not at position 5, is strictly recognized by NMU receptors in the chicken crop.     

Syntheses and biological activities of endothelin-1 analogs.

Endothelin-1 analogs replaced by various amino acids at position 21, namely [X21]-ET-1, were synthesized, and their agonistic vasoconstrictor activity on rat thoracic aortic strips and receptor binding activity on rat brain membrane fraction were examined to elucidate their structure-activity relationship. The vasoconstrictor activities of [Tyr21]- and [Phe21]-ET-1 were one order of magnitude smaller than that of ET-1, and those of [His21]-, [Gly21]-, [Ser21]-, [Ala21]- and [Lys21]-ET-1 were more than two orders of magnitude smaller than that of ET-1. On the other hand, the replacements by Ile, Glu, Gln and Pro resulted in distinguished losses of the vasoconstrictor activities. In addition, preincubation with these analogs did not blunt ET-1-induced vasoconstriction and showed no antagonistic activity. The binding inhibitory activities of these analogs against 125I-ET-1 were approximately conformable to the vasoconstrictor activities with only a slight exception. These findings demonstrate that the phenyl group at position 21 is important for both the vasoconstrictor activity and the receptor binding activity.     

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.     

含环状原酸酯侧基的两亲性嵌段共聚物及其酸敏感胶束状聚集体

合成了含有环状原酸酯基团的单体,甲基丙烯酸5-(5-甲基-2-乙氧基-1,3-二氧六环)甲酯(EDMM),以PEO大分子引发剂引发,通过原子转移自由基聚合方法制备了两个疏水链段(PEDMM)长度不同的酸敏感两亲性嵌段共聚物,PEO-b-PEDMM17和PEO-b-PEDMM34.两个聚合物在水溶液中形成粒径约为60～200nm的球型聚集体,通过动态光散射、透射电镜、荧光探针等手段表征了所得聚集体.结果显示,疏水链段较短的共聚物的临界聚集浓度较高,形成的聚集体尺寸较小.核磁和荧光探针结果表明,胶束状聚集体在中性(pH7.4)水溶液中比较稳定,在酸性水溶液中其疏水核的极性因原酸酯的水解而增加.该类酸敏感胶束状聚集体有望用于肿瘤、细胞内给药等药物传递体系.     

Study of the sensitization of tetradecyl benzyl dimethyl ammonium chloride for the color development reaction between lysine and sodium 1,2-naphthoquinone-4-sulfonate and the determination of lysine in pharmaceutical and biological samples

A rapid, simple and sensitive method for the determination of lysine (Lys) using sodium 1,2-naphthoquinone-4-sulfonate (NQS) and tetradecyl benzyl dimethyl ammonium chloride (Zeph) is presented in this paper. The method is based on the russety product formed from Lys, NQS and Zeph in a buffer solution of pH 9.60, and the stoichiometric ratio of the product is 1:2:2. Beer's law is obeyed in a range of 0.09-18 microg ml(-1) of Lys at the maximum absorption of 474 nm (epsilon(474) is 8.1 x 10(5)l mol(-1)cm(-1)). The equation of linear regression is A=0.40427+0.06112C, with a linearly correlation coefficient of 0.9972. The limit of detection is 0.07 microg ml(-1), R.S.D. 0.8%, and average recovery rate in a range of 98.9-100.1%. This paper further optimizes the determination of Lys compared with the previous methods, and the reaction mechanism is studied intensively. The proposed method has been successfully applied to the determination of Lys in pharmaceutical and biological samples. The common components as nutritional additives in pharmaceuticals and other compounds in biological samples nearly do not interfere with the proposed method.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

Improved Synthesis of 10-(2-Alkylamino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid Derivatives Bearing Acid-Sensitive Linkers

Alkylation of the hydrobromide salts of 1,4,7-tris(methoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane and 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane with appropriate α-bromoacetamides, followed by hydrolysis, provides convenient access to 10-(2-alkylamino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivatives that contain acid-sensitive functional groups. The utility of the method is demonstrated by improved syntheses of two known 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamides bearing acid-sensitive ω-tritylthio alkyl chains in much greater yields based on cyclen as the starting material.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]     

Conformational restriction via cyclization in beta-amyloid peptide Abeta(1-28) leads to an inhibitor of Abeta(1-28) amyloidogenesis and cytotoxicity

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.     

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor,hGRF(1-29)-NH2

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.