Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.     

L-3-氟-酪氨酸的合成研究

以L-酪氨酸为原料,经由羧基、氨基保护后,在其苯环上3-位直接硝化,硝化后的产物在铁粉和醋酸的作用下还原成3-位氨基的衍生物,再重氮化、光照下发生氟代反应,最后,氟代完成后的产物盐酸水解除去保护基后,即得到光学纯的L-3-氟-酪氨酸.经熔点、旋光度、1H-NMR及MS检测,结果与文献值一致.本文方法是合成L-3-氟-酪氨酸的理想方法.     

Influence of the Type of Amino Acid on the Permeability and Properties of Ibuprofenates of Isopropyl Amino Acid Esters

Modifications of (RS)-2-[4-(2-methylpropyl)phenyl] propanoic acid with amino acid isopropyl esters were synthesised using different methods via a common intermediate. The main reaction was the esterification of the carboxyl group of amino acids with isopropanol and chlorination of the amino group of the amino acid, followed by an exchange or neutralisation reaction and protonation. All of the proposed methods were very efficient, and the compounds obtained have great potential to be more effective drugs with increased skin permeability compared with ibuprofen. In addition, it was shown how the introduction of a modification in the form of an ion pair affects the properties of the obtained compound.     

Probing the Outstanding Local Hydrophobicity Increases in Peptide Sequences Induced by Incorporation of Trifluoromethylated Amino Acids

In order to achieve accurate determination of the local hydrophobicity increases in peptide sequences produced by incorporation of trifluoromethylated amino acids (TfmAAs), the chromatographic hydrophobicity indexes (?₀) of three series of tripeptides containing three unnatural trifluoromethylated amino acids have been measured and compared with those of their non‐fluorinated analogues. The hydrophobic contribution of each fluorinated amino acid was quantified by varying the position and the protection of (R)‐ and (S)‐α‐trifluoromethylalanine (TfmAla), (R)‐trifluoromethylcysteine (TfmCys), and (S)‐trifluoromethionine (TFM) in a short peptide sequence. As a general trend, strong increases in hydrophobicity were precisely measured, even exceeding the high hydrophobic contribution of the natural amino acid isoleucine. This study validates the incorporation of trifluoromethylated amino acids into peptide sequences as a rational strategy for the fine‐tuning of hydrophobic peptide–protein interactions.     

新型可溶性透明聚芳酰胺的合成与表征

以2-氯-5-硝基三氟甲苯和1,4-环己二醇为起始原料,通过两步有机反应合成了一种新的含氟二胺单体：1,4-双[4-胺基-2-(三氟甲基)苯氧基]环己烷;并由该二胺单体和对苯二甲酸、间苯二甲酸、4,4'-二苯醚二甲酸和2,2-双(4-羧基苯基)六氟丙烷经缩聚反应制备了一系列新型聚芳酰胺,其特性黏度在0.89~1.29 dL/g之间。该类聚芳酰胺表现出了优良的溶解性能和光学性能,室温下不仅可以溶于N-甲基-吡咯烷酮、N,N-二甲基乙酰胺、N,N-二甲基甲酰胺等强极性溶剂中,还能溶于低沸点的四氢呋喃、氯仿、二氯甲烷等溶剂中;由该类聚合物溶液所制的薄膜无色透明,截断波长在335~357 nm,在400 nm后具有高透明性。此外,该聚芳酰胺还表现出了良好的热学性能,玻璃化转变温度在202~223 ℃,氮气中10 %热失重温度在330~364 ℃。     

Synthesis and Cytotoxicity of Octahydroepoxyisoindole-7-carboxylic Acids and Norcantharidin–Amide Hybrids as Norcantharidin Analogues

Octahydroepoxyisoindole analogues of norcantharidin were accessed through a Diels–Alder reaction of an amine-substituted furan with maleic anhydride and subsequent reduction of the bicyclo[2.2.1]heptene olefin. Despite retention of the carboxylate and the ether bridgehead known to impart cytotoxic activity to norcantharidin, none of these analogues displayed notable cytotoxicity against the 11 cell lines examined: HT29 (colon), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), SJ-G2 and U87 (glioblastoma), MIA (pancreatic), and SMA (spontaneous murine astrocytoma). The incorporation of an amino-substituted system post-synthesis of norcantharidin afforded facile access to 14 acid/amide-substituted norcantharidin analogues. Of these, only four displayed sufficient activity at the initial 25 μm compound screening dose to warrant full evaluation of growth inhibition. Common to these analogues was the presence of a 4-biphenyl moiety, and in particular 3-(2-(furan-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid ( 13 c ) and 3-(2-(pyrrole-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid ( 24 ) displayed high levels of cytotoxicity, returning GI₅₀ values of 15 nm (HT29) to 2.9 μm (U87) and 17 nm (SMA) to 2.8 μm (U87), respectively. These are the most cytotoxic norcantharidin analogues reported to date.     

Stabilization of bzip peptides through incorporation of fluorinated aliphatic residues

Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88 %, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27 degrees C in melting temperature when isoleucine was replaced by 5TFI. However, the T(m) of the peptide containing 4TFV was increased by only 4 degrees C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which DeltaDeltaG(unfold) in water was determined to be 2.1 or 0.3 kcal mol(-1) upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.     

Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.     

Synthesis of Analogues of Thyrotropin-Releasing Hormone

The synthesis of ten new thyrotropin-releasing hormone analogues ( 15–24 ) which contain uncoded amino acids (L- and D-homoleucine, L- and D-homophenylalanine, L-homoproline and 6-ketopipecolic acid) is described here. The peptide bond formation was achieved in solution phase using pentafluorophenyl ester activation of the N-protected amino acids. The analogues were tested for their ability to release thyrotropin and for CNS activities and proved to be fully inactive.     

Extraction Equilibria of Trimellitic and [ 1,1′-Biphenyl]-2,2′-dicarboxylic Acid with 1-Octanol, 50%TBP,and 10%TRPO in Kerosene

To explore the feasibility of extracting aromatic acid products from oxidizing coal, two aromatic acids, trimellitic and [1,1′-biphenyl]-2,2′-dicarboxylic acid, were selected as the solutes, and the extraction equilibrium of the acids were studied with 1-octanol, 50% tributyl phosphate （TBP） in kerosene, and 10% trialkylphosphine oxide （TRPO） in kerosene. The results showed that the degree of extraction of [1,1′-biphenyl]-2,2′-dicarboxylic acid was larger than that of trimellitic acid for all of the solvent, and the extraction capacity with TRPO is more effective than the one with TBP. The extraction behavior of aromatic polyacid is different from that of carboxylic acid, and the reactive extraction function of aromatic acids with TBP and TRPO is not as effective as that of carboxylic acid. 1-octanol could be used to remove [1,1′-biphenyl]-2,2′-dicarboxylic acid from the mixture of trimellitic acid and [1,1′-biphenyl]-2,2′-dicarboxylic acid. Because the weak hydrogen bond association exists between -OH in 1-octanol and -COOH in aromatic acid, the extractive selectivity of [ 1, 1′-biphenyl]-2,2′-dicarboxylic to trimellitic acid depends on the stoichiometric ratio.     

SSTR1- and SSTR3-selective somatostatin analogues

We prepared the two enantiomers of 3‐(3′‐quinolyl)‐alanine (Qla, 1 ) in multigram scale by asymmetric hydrogenation. These amino acids, protected as Fmoc derivatives, were then used in the solid‐phase synthesis of two new somatostatin 14 (SRIF‐14) analogues 8 a and 8 b , tetradecapeptides in which the tryptophan residue (Trp8) is replaced by one of the two enantiomers of 3‐(3′‐quinolyl)‐alanine (Qla8) and therefore lack the N? H bond in residue 8. The selectivity of these new analogues for the somatostatin receptors, SSTR1–5, was measured. Substitution with L ‐Qla8 yielded peptide 8 a , which was highly selective for SSTR1 and SSTR3, with an affinity similar to that of SRIF‐14. Substitution by D ‐Qla gave the relatively selective analogue 8 b , which showed high affinity for SSTR3 and significant affinity for SSTR1, SSTR2 and SSTR5. The biological results demonstrate that bulky and electronically poor aromatic amino acids at position 8 are compatible with strong activity with SSTR1 and SSTR3. Remarkably, these high affinity levels were achieved with peptides in which the conformational mobility was increased with respect to that of SRIF‐14. This observation suggests that conformational rigidity is not required, and might be detrimental to the interaction with receptors SSTR1 and SSTR3. The absence of an indole N proton in Qla8 might also contribute to the increased flexibility observed in these analogues.     

Stereoselective Biocatalytic α-Deuteration of L-Amino Acids by a Pyridoxal 5’-Phosphate-Dependent Mannich Cyclase

α-Deuterated amino acids are valuable building blocks for developing deuterated drugs, and are important tools for studying biological systems. Biocatalytic deuteration represents an attractive strategy to directly access enantiopure α-deuterated amino acids. Here, we show that a PLP-dependent Mannich cyclase, LolT, involved in the biosynthesis of loline alkaloids, is capable of deuterating a diverse range of L-amino acids, including basic and acidic, nonpolar and polar, aliphatic and aromatic amino acids. Furthermore, complete deuteration of many amino acids can be achieved within minutes with exquisite control on the site- and stereoselectivity. During the course of this investigation, we also unexpectedly discovered that LolT exhibits β-elimination activity with L-cystine and O-acetyl-L-serine, confirming our previous hypothesis based on structural and phylogenetic analysis that LolT, a Cα−C bond forming enzyme, is evolved from a primordial Cβ−S lyase family. Overall, our study demonstrates that LolT is an extremely versatile biocatalyst, and can be used for not only heterocyclic quaternary amino acid biosynthesis, but also biocatalytic amino acid deuteration.     

Organosoluble and light‐colored fluorinated polyimides derived from 5,5′‐bis[4‐(4‐amino‐2‐trifluoromethylphenoxy) phenyl]‐4,7‐methanohexahydroindan

A novel bis(ether amine) monomer, 5,5′‐bis[4‐(4‐amino‐2‐trifluoromethylphenoxy)phenyl]‐4,7‐methanohexahydroindan ( 2 ), was synthesized through the nucleophilic aromatic substitution reaction of 5,5′‐bis‐(4‐hydroxyphenyl)‐4,7‐methanohexahydroindan with 2‐chloro‐5‐nitrobenzotrifluoride to yield the intermediate dinitro compound, followed by catalytic reduction with hydrazine and Pd/C. A series of polyimides were synthesized from 2 and various aromatic dianhydrides using a standard two‐stage process with chemical or thermal imidization of poly(amic acid). All of these polymer films were soluble in amide‐type solvents above 10% w/v, had tensile strengths of 97–117 MPa, and the 10% weight loss temperature was above 464 °C with their residues exceeding 46% at 800 °C in nitrogen. Compared with the non‐fluorinated polyimides, the fluorinated series were observed to have lower dielectric constants (2.92–3.28 at 1 MHz) and lower moisture absorptions (0.15–0.43 wt%) as well as lower color intensity and better solubility. Copyright © 2006 Society of Chemical Industry     

Elongation and desaturation of dietary fatty acids in turbotScophthalmus maximus L., and rainbow trout,Salmo gairdnerii rich

Turbot and rainbow trout, which had previously recieved diets free of fat, were fed [1-14C] fatty acids. The distribution of radioactivity in the tissue fatty acids was examined 6 days later. In rainbow trout fed [1-14C] 18:3omega3, 70% of the radioactivity was present in 22:6omega3 fatty acid. In contrast, turbot fed [1-14C] 18:1omega9, 18:2omega6, or 18:3omega3 converted only small amounts of labeled fatty acids (3-15%) into fatty acids of longer chain length. The major product of the limited modification found in turbot was the dietary acid elongated by 2 carbon atoms.     

Partition of ketone bodies into cholesterol and fatty acids in vivo in different brain regions of developing rats

The proportions of labeled ketone bodies and glucose incorporated into cholesterol and fatty acids in different regions of the brain in developing rats were compared. In cerebrums of 15- and 18-day-old rats, the ratios of dpm cholesterol/dpm fatty acids incorporated from [3-¹⁴C] acetoacetate and [3-¹⁴C] β-hydroxybutyrate ranged from 0.4 to 0.7, or 50 to 100% higher than values obtained with [U-¹⁴C] glucose. Much higher ratios were obtained with younger animals: from 1 to 12 days of life, the values ranged from 1.0 to 1.3 with [3-¹⁴C] β-hydroxybutyrate as substrate, and, from 1 to 5 days, with [3-¹⁴C] acetoacetate, they were 1.0 or greater. During the first 12 days of life, the ratios resulting from administration of [U-¹⁴C] glucose were 0.4–0.7. Clearly, a greater proportion of acetoacetate and β-hydroxybutyrate was incorporated into cholesterol during the first week of life than the remaining suckling period. Like cerebrum, other brain regions (i.e., cerebellum, midbrain, brain stem and thalamus) yielded higher ratios of dpm cholesterol/dpm fatty acids from [3-¹⁴C] β-hydroxybutyrate during the first 12 days of life than on day 17. Brain stem was the most active region for lipid synthesis, and had the highest dpm cholesterol/dpm fatty acid ratio. Since active synthesis of cholesterol from ketone bodies during the early postnatal period coincides with a period of rapid brain growth, the results indicate that ketone bodies are more important early in the suckling period as sources of cholesterol for brain growth.     

Side Chain Orientation of Tryptophan Analogues Determines Agonism and Inverse Agonism in Short Ghrelin Peptides

We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe⁴ is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL-NH₂, the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid-phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.     

Lithocholic acid in human liver: Identification of ∈-lithocholyl lysine in tissue protein

Human liver had been shown to contain two forms of lithocholic acid. One form is extractable by 95% ethanol-ammonia, 1000∶1, v/v (soluble lithocholate, SL) and the other form is firmly bound to the tissue residue. The latter, tissue-bound lithocholic acid (TBL), can be enzymatically released by means of the specific clostridial peptide bond hydrolase, cholylglycine hydrolase (cholanoyl amino acid hydrolase, EC no. 3.5). Solvolytic procedures for the analysis of hepatic lithocholic acid sulfate revealed that almost all of the TBL was non sulfated, while in the SL fraction there was an apparent preponderance of the sulfated form. Cholylglycine hydrolase liberates free labeled lithocholic acid from synthetic [24-¹⁴C] lithocholyl-bovine serum albumin and from [24-¹⁴C]lithocholyl polylysine. By analogy, the enzyme releases lithocholic acid from tissue protein in which the bile acid is conjugated through amino groups of basic side chains. Hydrolysis of [24-¹⁴C] lithocholyl polylysine with 6n HC1 yielded ∈-[24-¹⁴C] lithocholyl lysine, which was chromatographically similar to the product obtained by acid hydrolysis of TBL. Chromatographic, infrared, and mass spectroscopic studies with synthetic N-α-lithocholyl lysine, N-∈-lithocholyl lysine, and N-α-∈-Bis lithocholyl lysine showed the hydrolytic product from TBL to be N-∈-lithocholyl lysine. Since monohydroxy bile acids, such as lithocholic acid, are known to show unusual cytotoxic properties, the identification of TBL in human liver poses important questions regarding the role of lithocholate in liver injury.     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis: II. Perfused rat liver

Livers from rats with experimental hypoproteinemia induced by aminonucleoside-nephrosis or plasmapheresis were perfused with a [¹⁴C]-labeled amino acid mixture at physiological concentration. Compared to control rats, a significantly increased incorporation of the amino acid label was found in the apolipoproteins of the ultracentrifugally separated very low and high density lipoproteins (VLDL, HDL), and into albumin secreted into the perfusate. However, no increase in the amino acid-derived label was detected in VLDL- or HDL-borne lipids in nephrosis or plasmapheresis. Perfusion with U-[¹⁴C] leucine as a lipogenesis precursor at >10 times higher than physiological concentration resulted in 5-fold increase in the label incorporation into perfusate proteins in nephrosis but only in a slightly significant increase in perfusate lipids. In contrast, the incorporation of a preformed fatty acid, 9,10-[³H] oleate into VLDL and HDL lipids increased 3- to 4-fold in nephrosis. Both with leucine and oleate as precursors, the increments in the label appearing in perfusate proteins or lipids, respectively, were markedly greater than the increases in hepatic tissue proteins or lipids. The results indicate that amino acids are preferentially directed by the liver into the synthesis of circulating apolipoproteins and albumin in hypoproteinemia and do not seem to constitute an important precursor of the lipoprotein lipids. The increased production of apolipoproteins is associated with an increased incorporation of preformed fatty acids into lipoprotein lipids in addition to the previously reported stimulation of hepatic de novo lipid synthesis from precursors other than amino acids.     

Lipid composition and de novo lipid biosynthesis of human palmar fat in Dupuytren's disease

Seventy-two surgically obtained Dupuytren's disease palmar-fat (DDPF) specimens and 18 location-matched specimens from patients not suffering from this disease (controls) were studied for their total lipid composition and de novo lipogenic activity. Incubation of "DDPF" with 1-[14C]acetate in oxygen produced [14C]palmitate and [14C]stearate in approximately equal yields as those obtained from "controls." No [14C]octanate was formed in any of the palmar-fat preparations. The lipids and fatty acid analysis revealed differences: (a) DDPF specimens were richer in free fatty acids, methyl esters of fatty acids and free-cholesterol than specimens of controls. (b) DDPF specimens contained less phospholipids. (c) DDPF specimens showed a significantly higher content of octanoate and other short-chain fatty acids than specimens of controls. The above findings are not incompatible with the results expected if some mild hypoxia occurred in DDPF; this has been suggested in the statistical correlations observed for this disease and alcoholism with liver involvement.     

Transdermal Delivery Systems for Ibuprofen and Ibuprofen Modified with Amino Acids Alkyl Esters Based on Bacterial Cellulose

The potential of bacterial cellulose as a carrier for the transport of ibuprofen (a typical example of non-steroidal anti-inflammatory drugs) through the skin was investigated. Ibuprofen and its amino acid ester salts-loaded BC membranes were prepared through a simple methodology and characterized in terms of structure and morphology. Two salts of amino acid isopropyl esters were used in the research, namely L-valine isopropyl ester ibuprofenate ([ValOiPr][IBU]) and L-leucine isopropyl ester ibuprofenate ([LeuOiPr][IBU]). [LeuOiPr][IBU] is a new compound; therefore, it has been fully characterized and its identity confirmed. For all membranes obtained the surface morphology, tensile mechanical properties, active compound dissolution assays, and permeation and skin accumulation studies of API (active pharmaceutical ingredient) were determined. The obtained membranes were very homogeneous. In vitro diffusion studies with Franz cells were conducted using pig epidermal membranes, and showed that the incorporation of ibuprofen in BC membranes provided lower permeation rates to those obtained with amino acids ester salts of ibuprofen. This release profile together with the ease of application and the simple preparation and assembly of the drug-loaded membranes indicates the enormous potentialities of using BC membranes for transdermal application of ibuprofen in the form of amino acid ester salts.