Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.     

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.     

Conformationally Constrained Histidines in the Design of Peptidomimetics: Strategies for the χ-Space Control

A successful design of peptidomimetics must come to terms with χ-space control. The incorporation of χ-space constrained amino acids into bioactive peptides renders the χ(1) and χ(2) torsional angles of pharmacophore amino acids critical for activity and selectivity as with other relevant structural features of the template. This review describes histidine analogues characterized by replacement of native α and/or β-hydrogen atoms with alkyl substituents as well as analogues with α, β-didehydro unsaturation or C(α)-C(β) cyclopropane insertion (ACC derivatives). Attention is also dedicated to the relevant field of β-aminoacid chemistry by describing the synthesis of β(2)- and β(3)-models (β-hHis). Structural modifications leading to cyclic imino derivatives such as spinacine, aza-histidine and analogues with shortening or elongation of the native side chain (nor-histidine and homo-histidine, respectively) are also described. Examples of the use of the described analogues to replace native histidine in bioactive peptides are also given.     

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.     

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.     

差示光度法测定高含量茶氨酸

提出了一种测定高含量茶氨酸的新方法.将茶氨酸用浓盐酸水解为乙胺,以8.0 mg· L-1茶氨酸标准溶液为参比,在570 nm波长处进行差示光度测定.结果表明,茶氨酸浓度在8.0～10.0 mg·L-1范围内△C与△A呈线性关系;9.2 mg· L-1茶氨酸溶液的相对标准偏差≤0.3％.该法操作简单、费用低、准确度高,结...     

Samarium‐Promoted Asymmetric Aldol–Tishchenko Reaction: Synthesis of Amino Acid‐Derived 4‐Amino‐1,3‐diols

A samarium‐mediated novel synthesis of enantiopure 4‐amino‐1,3‐diols is carried out through a samarium‐promoted aldol–Tishchenko reaction starting from chiral α′‐amino‐α‐chloro ketones (derived from natural α‐amino acids) and aldehydes. The process takes place with moderate levels of stereoselectivity and in high yields. A mechanism is proposed to explain these results while the absolute configuration and structure of the aldol–Tishchenko adducts were established by X‐ray analysis. This method has also been utilized for the synthesis of enigmols, 1‐deoxysphingoid base analogues.     

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.     

异养小球藻产总脂肪酸的培养基优化

研究了异养小球藻(Chlorella protothecoides)分批培养基中葡萄糖和硝酸盐浓度对生物量、总脂和总脂肪酸产量的影响.结果表明,当细胞在含有40 g·L-1葡萄糖和0.1 mol·L-1 NaNO3的异养培养基中生长时,总脂和总脂肪酸的产量可分别达到最高值为3.83 g·L-1和1.64 g·L-1,同时C18脂肪酸占总脂肪酸的比例超过69%,总脂肪酸占总脂的比例也高达42.63%,总脂含量约为24.3%.通过优化异养小球藻培养基中的葡萄糖和硝酸盐浓度,能够获得高产总脂肪酸,进而生产生物柴油.     

Oxetanes from the ring contraction of alpha-triflates of gamma-lactones: oxetane nucleosides and oxetane amino acids

Alpha-Triflates of gamma-lactones with potassium carbonate in methanol give efficient contraction of the ring to oxetane-1-carboxylates in which the oxygen substituent at C(3) of the oxetane is predominantly trans to the carboxylate at C(2), regardless of the stereochemistry of the starting triflate. The limitations of the procedure are discussed and compared with analogous reactions for the preparation of THF carboxylates. The potential of the contraction in the preparation of oxetane nucleosides (such as oxetanocin) and oxetane sugar amino acids (analogues of oxetin) as peptidomimetics with predisposition to form secondary structural motifs is illustrated.     

Development of antiviral fusion inhibitors: short modified peptides derived from the transmembrane glycoprotein of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) is a naturally occurring pathogen that causes an AIDS-like syndrome in domestic cats and is a valuable model system by which criteria for antiviral vaccines and drugs development can be tested. The cell-entry step of the lentivirus life cycle is regarded as a promising target for the development of new generation inhibitors. We have previously described potent in vitro anti-FIV activity associated with a synthetic octapeptide, termed C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), containing the Trp-rich motif of FIV transmembrane glycoprotein, which shares a common structural framework with the corresponding molecule of HIV and appears to play a similar role in cell entry. In this report, in an attempt to develop simpler potential fusion inhibitors to be tested in vivo, we describe further studies focused on synthetic peptide analogues of C8. Since C8 inhibitory activity is dependent upon the Trp motif, we systematically replaced these residues with bulky and/or aromatic natural and unnatural amino acids, in order to develop a rational structure-activity relationship. Furthermore, the amino acids located between the Trp residues, which are not crucial for inhibitory activity, were replaced by simple alkyl spacers of appropriate length. Design, NMR structural analysis, in vitro anti-FIV activity in lymphoid cell cultures, and serum stability of these new analogues are reported. The final results indicate that a simpler hexapeptide (Ac-Nal2-Ape-Nal2-Ape-Nal2-Ile-NH2; Nal2 = 3-naphthalen-2-yl-L-alanine, Ape = 5-aminopentanoic acid), almost entirely made up of unnatural amino acid residues, has markedly increased enzymatic stability, while maintaining strong antiviral potency in vitro.     

The effect of amino acids and amino acid analogues on growth of an obligate methanotroph,Methylosinus trichosporium OB3b

The sensitivity of the obligate methanotroph, Methylosinus trichosporium OB3b to a number of amino acid analogues and amino acids has been examined. Sulphaguanidine (5 μg ml^?1) norleucine (100 μg ml^?1) m-fluorophenylalanine (50 μg ml^?1), p-fluorophenylalanine (50 μg ml^?1) and S2-aminoethyl-C-cysteine (1000 μg ml^?1) inhibited growth. Proline, threonine, methionine and lysine (2–4 μg ml^?1) also inhibited growth, but arginine and leucine at similar concentrations had no effect. Attempts were made to isolate amino acid analogue resistant mutants, which would be expected to overproduce amino acids. Two approaches were made, selection of spontaneous and induced mutants in plate culture, and selection of a spontaneous mutant during growth in continuous culture under selective conditions, but no mutants were obtained. Possible reasons for failure to obtain such mutants are discussed.     

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.     

Elucidation of the structure-activity relationships of apelin: influence of unnatural amino acids on binding, signaling, and plasma stability

Apelin is the endogenous ligand of the APJ receptor, a member of the G-protein-coupled receptor family. The apelin-APJ complex has been detected in many tissues and is emerging as a promising target for several pathophysiological conditions. There is currently little information on the structure-activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogues of apelin-13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C-terminal portion. Analogues were then tested in binding and functional assays by evaluating Gi/o-mediated decreases in cAMP levels and by assessing β-arrestin2 recruitment to the APJ receptor. The plasma stability of new compounds was also assessed. Several analogues were found to possess increased binding and higher stability than the parent peptide.     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

Novel cell-penetrating peptides based on α-aminoxy acids

The remarkable ability of cell-penetrating peptides (CPPs) to deliver cell-impermeable compounds into living cells makes them attractive transporters for use in biology and medicine. Despite their highly efficient cellular uptake, CPPs consisting of natural amino acids always suffer from degradation and endosomal entrapment, thereby greatly limiting their application in vivo. Here, we describe the preparation of novel CPPs incorporating α-aminoxy acid residues and their cellular uptake behavior. We demonstrate that introducing α-aminoxy acids into the backbones of CPPs enhances their diffuse cytosolic distribution after direct membrane translocation. We also reveal a hybrid peptide, consisting of D-α-aminoxy acids and L-α-amino acids, that achieves efficient diffuse distribution in the cytosol, is stable toward serum, and possesses low cytotoxicity, thus making it a possible vector candidate for in vivo applications. Our results confirm that α-aminoxy acids are useful building blocks when designing novel CPPs possessing favorable properties.     

The Effects of Prodrug Size and a Carbonyl Linker on l-Type Amino Acid Transporter 1-Targeted Cellular and Brain Uptake

The l -type amino acid transporter 1 (LAT1, SLC7A5) imports dietary amino acids and amino acid drugs (e. g., l -DOPA) into the brain, and plays a role in cancer metabolism. Though there have been numerous reports of LAT1-targeted amino acid-drug conjugates (prodrugs), identifying the structural determinants to enhance substrate activity has been challenging. In this work, we investigated the position and orientation of a carbonyl group in linking hydrophobic moieties including the anti-inflammatory drug ketoprofen to l -tyrosine and l -phenylalanine. We found that esters of meta-carboxyl l -phenylalanine had better LAT1 transport rates than the corresponding acylated l -tyrosine analogues. However, as the size of the hydrophobic moiety increased, we observed a decrease in LAT1 transport rate with a concomitant increase in potency of inhibition. Our results have important implications for designing amino acid prodrugs that target LAT1 at the blood-brain barrier or on cancer cells.     

酸性氨基酸二甲酯盐酸盐的合成工艺研究

采用氯化亚砜法,以两种酸性氨基酸、无水甲醇、氯化亚砜为原料酯化合成L-天冬氨酸二甲酯盐酸盐和L-谷氨酸二甲酯盐酸盐,讨论了加样顺序,反应时间tA,反应温度TA,反应时间tB和氨基酸与氯化亚砜的摩尔比对反应产率的影响,优化了两种氨基酸二甲酯盐酸盐的重结晶条件,确定了两种氨基酸二甲酯盐酸盐的最佳合成条件及最优重结晶方案。     

Der „Nornialisierungsfaktor”︁ für die Toinatenmutante chloronerva. XV. Peptidanaloga des Phytosiderophors Nicotianamin

The “Normalizing Factor” for the Tomato Mutant chloronerva. XV. Peptide Analogues of the Phytosiderophore Nicotianamine Some peptide analogues of the phytosiderophore nicotianamine, (2 S , 3′ S , 3″ S )- N -[3-(3-amino-3-carboxypropylamino)-3-carboxypropyl]-azetidine-2-carboxylic acid, have been synthesized from protected amino acids by the EEDQ or the DCC method. They exhibited no biological effect with regard to the phenotypical normalization of the tomato mutant chloronerva.     

Cotranslational incorporation of a structurally diverse series of proline analogues in an Escherichia coli expression system

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline-rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant-protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.