Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

Syntheses,Receptor Bindings,in vitro and in vivo Stabilities and Biodistributions of DOTA‐Neurotensin(8–13) Derivatives Containing β‐Amino Acid Residues – A Lesson about the Importance of Animal Experiments

Neurotensin(8–13) (NTS(8–13)) analogs with C‐ and/or N‐terminal β‐amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1 – 6 ). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivatives, 6a , into a crystallographically identified receptor NTSR1 (Fig. 1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell‐membrane homogenates, while, with NTSR1‐exhibiting cancer tissues, affinities in the single‐digit nanomolar range can be observed (Table 2). Most of the β‐amino acid‐containing NTS(8–13) analogs (Table 1 and Fig. 2), including the ⁶⁸Ga complexes of the DOTA‐substituted ones ( 6 ; Figs. 2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two ⁶⁸Ga complexes (of 6a and 6b ) in HT29 tumor‐bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET‐imaging experiments with the tumor‐bearing mice (Fig. 6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10–15 min) of the two ⁶⁸Ga complexes shows that they are rapidly cleaved in the animals (Fig. 5).     

Synthesis and Immunostimulating Properties of Novel Adamant‐1‐yl Tripeptides

The aim of this work was to prepare L ‐ and D ‐(adamant‐1‐yl)‐Gly‐L ‐Ala‐D ‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala) and structurally related adamant‐2‐yl tripeptides.     

Effect of one D‐Leu residue on right‐handed helical ‐L‐Leu‐Aib‐ peptides in the crystal state

Four diastereomeric‐Leu‐Leu‐Aib‐Leu‐Leu‐Aib‐peptides, Boc‐D ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (1), Boc‐L ‐Leu‐D ‐Leu‐Aib‐L ‐Leu‐L ‐Leu‐Aib‐OMe (2), Boc‐L ‐Leu‐L ‐Leu‐Aib‐D ‐Leu‐L ‐Leu‐Aib‐OMe (3), and Boc‐L ‐Leu‐L ‐Leu‐Aib‐L ‐Leu‐D ‐Leu‐Aib‐OMe (4), were synthesized. The crystals of the four hexapeptides were characterized by X‐ray crystallographic analysis. Two diastereomeric hexapeptides 1 and 2 having D ‐Leu(1) or D ‐Leu(2) were folded into right‐handed (P) 3 ₁₀ ‐helical structures, while peptide 3 having D ‐Leu(4) was folded into a turn structure nucleated by type III′ and I$' \bf{\beta}$ ‐turns, and peptide 4 having D ‐Leu(5) was folded into a left‐handed (M) 3 ₁₀ ‐helical structure. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Dodecyl α‐L‐Rhamnopyranosyl‐(1→2)‐β‐D‐fucopyranoside Derivatives from the Glandular Trichome Exudate of Erodium pelargoniflorum

Chemical investigation of the glandular trichome exudate of Erodium pelargoniflorum (Geraniaceae) led to the isolation of two dodecyl disaccharide derivatives, named pelargoside A1 and pelargoside B1 ( 1 and 2 , resp.). The structures of 1 and 2 were determined as dodecyl 4‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside and dodecyl 3,4‐di‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside, respectively, by spectroscopic studies, including 2D‐NMR, and chemical transformations. In addition, undecyl, tridecyl, and tetradecyl homologs of 1 and 2 , named pelargosides A2–A4 and pelargosides B2–B4, were also characterized as minor constituents of the exudate.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel analogues of arginine vasopressin containing α‐2‐indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D ‐enantiomer (Igl or D ‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl² substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA₂ values ranging from 7.19 to 7.98) and practically did not interact with V_1a and V₂ receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D ‐Igl² substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D ‐Igl peptides showed a strong antioxytocic potency (pA₂ values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Three New Polyacetylene Glycosides from the Roots of Heracleum dissectum and Their Triglyceride Accumulating Activities in 3T3‐L1 Cells

Continually phytochemical study of the roots of Heracleum dissectum had led to the isolation of three previously undescribed polyacetylene glycosides ( 1 – 3 ), together with seven known compounds, including one polyacetylene ( 8 ) and six coumarins ( 4 – 7 and 9 – 10 ) using diverse chromatographic methods. The structures of these three new compounds were characterized and identified as deca‐4,6‐diyn‐1‐yl β‐d ‐glucopyranosyl‐(1→6)‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranoside ( 1 ), (8Z)‐dec‐8‐ene‐4,6‐diyn‐1‐yl β‐d ‐glucopyranosyl‐(1→6)‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranoside ( 2 ), and (8E)‐dec‐8‐ene‐4,6‐diyn‐1‐yl β‐d ‐glucopyranosyl‐(1→6)‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranoside ( 3 ) based on their physicochemical properties and extensive analyses of various spectroscopic data. Their triglycerides accumulating activities were assayed and the results showed that the three new polyacetylene glycosides ( 1 – 3 ) exhibited triglyceride accumulating activities in 3T3‐L1 adipocytes.     

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH₂ insertion into the peptides mentioned in the title is described. This involves replacement of the N‐terminal amino acid residue by a β²‐ and of the C‐terminal amino acid residue by a β³‐homo‐amino acid moiety (β²hXaa and β³hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N‐terminal to the C‐terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1 ) and its C‐terminal fragment NT(8–13) are ligands of the G‐protein‐coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b – 2e , for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5–1.3 vs. 0.6 nM ). At the same time, one of the homologated NT analogs, 2c , survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and Fig. 8) reveals that this N‐terminal NT fragment folds to a turn in CD₃OH. – In the case of the human analgesic opiorphin ( 3a ), a pentapeptide, and of the HIV‐derived B27‐KK10 ( 4a ), a decapeptide, terminal homologation (→ 3b and 4b , resp.) led to a 7‐ and 70‐fold half‐life increase in plasma (Fig. 9). With N‐terminally homologated NPY, 5c , we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c , and 5c , were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability‐increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.     

Simenoside A,a New Triterpenoid Saponin from Gypsophila simonii Hub.‐Mor.

Saponins are amphiphilic glycoconjugates which give soap‐like foams in H₂O. A new triterpenoid saponin, simenoside A ( 1 ), based on gypsogenin aglycone, and the known saponin 2 were isolated from Gypsophila simonii Hub.‐Mor. The structure of the new saponin was elucidated as 3‐O‐β‐D ‐galactopyranosyl‐(1→2)‐[β‐D ‐xylopyranosyl‐(1→3)]‐β‐D ‐glucuronopyranosylgypsogenin 28‐O‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐xylopyranosyl‐(1→4)]‐α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐fucopyranosyl ester on the basis of extensive spectral analyses and chemical evidence. Saponins 1 and 2 were isolated from G. simonii for the first time.     

Synthesis,characterization, and evaluation of novel cell‐penetrating peptides based on TD‐34

In this study, six N‐1, N‐2, or N‐11 derivatives of TD‐34 (a cationic cyclic cell‐penetrating peptide [CPP], ACSSKKSKHCG) were designed and synthesized including both linear peptides and cyclic peptides, such as DL‐1 (KWSSKKSKHCG), DLCC‐1 (cyclopeptide, KWSSKKSKHCG), DL‐2 (KWSSKKSKHCG‐NH₂), DLCC‐2 (cyclopeptide, KWSSKKSKHCG‐NH₂), DL‐3 (RWSSKKSKHCG), and DLCC‐3 (cyclopeptide, RWSSKKSKHCG). The cyclic peptides were synthesized by disulfide bound linkages formed by N‐2 and N‐10 cysteine. In vitro penetration experiment was conducted to investigate the transdermal enhancement ability of these derivatives, using triptolide (TP) as model drug. The results display that at the presence of DLCC‐2, the accumulative penetration amount of TP increased 1.71‐fold (P < .05) within 12 hours, displaying better transdermal enhancing ability than TD‐34. Meanwhile, DL‐3 and DLCC‐3 slightly decreased the transdermal delivery of TP, and the presence of DL‐1 and DLCC‐1 shows no obvious effect. In order to clarify the factors on the transdermal ability of peptides, the solubility of TP in phosphate buffer saline (PBS) at the presence of different peptides and the mechanism of transdermal delivery of CPPs was investigated. The result shows that most of these peptides have no significant effect on the solubility of TP except DLCC‐3 (the solubility of TP slightly increased). And in order to investigate transdermal absorption route of DLCC‐2, polyarginine linked to rhodamine b (Rh b) derivative is used. The result proved that the transdermal route of polyarginine is via hair follicle, which may change the transdermal route of its cargo molecule (TP). Our group previously proved that polyarginine and TD‐34 have similar transdermal enhancing mechanism (changing the transdermal route of their cargo molecule); it is reasonably speculated that the transdermal route of DLCC‐2 is the same as polyarginine and then changes the transdermal absorption route of TP. Furthermore, such results have laid a solid foundation for further investigation of CPPs and paved a way for both designing and synthesizing of new drug delivery system for therapy molecules.     

Four New Steroidal Glycosides,Protolinckiosides A – D,from the Starfish Protoreaster lincki

Four new steroidal glycosides, protolinckiosides A – D ( 1 – 4 , resp.), were isolated along with four previously known glycosides, 5 – 8 , from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 – 4 were elucidated by extensive NMR and ESI‐MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)‐4,6,7,8,15,16,26‐heptahydroxycholestan‐3‐yl 2‐O‐methyl‐β‐d ‐xylopyranoside ( 1 ), (3β,5α,6β,15α,24S)‐3,5,6,8,15‐pentahydroxycholestan‐24‐yl α‐l ‐arabinofuranoside ( 2 ), sodium (3β,6β,15α,16β,24R)‐29‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]stigmast‐4‐en‐15‐yl sulfate ( 3 ), and sodium (3β,6β,15α,16β,22E,24R)‐28‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]ergosta‐4,22‐dien‐15‐yl sulfate ( 4 ). The unsubstituted β‐d ‐galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 – 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.     

Identification of a naturally‐occurring 8‐[α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]daidzein from cultivated kudzu root

Introduction – Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one‐year‐old kudzu root cultivated in Vietnam. Objective – To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS‐MS, ¹H‐, ¹³C‐NMR and enzymatic hydrolysis. Methodology – Samples were prepared by extraction of one‐year‐old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV‐light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results – ESI⁺ TOF MS‐MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H‐, ¹³C‐NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α‐1,6‐glycosidic bond. This compound is the first naturally‐occurring 8‐[α‐D ‐glucopyranosyl‐(1→6)‐β‐D ‐glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl‐α‐1,6‐puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion – The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water‐soluble glycosylated puerarin. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,Structure, and Biological Applications of α‐Fluorinated β‐Amino Acids and Derivatives

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α‐fluorinated β‐amino acids and derivatives. General methods are described for the preparation of monosubstituted α‐fluoro‐β‐amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α‐amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt? Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α‐Difluoro‐β‐amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α‐Fluoro‐β‐amino acids and derivatives analyzed by X‐ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2–6). α‐Fluoro amide and β‐fluoro ethylamide/amine effects can influence the secondary structure of α‐fluoro‐β‐amino acid‐containing derivatives including peptides and peptidomimetics (Figs. 7–9). α‐Fluoro‐β‐amino acids are also components of a diverse range of bioactive anticancer (e.g., 5‐fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.     

Antibacterial Activity of Enrofloxacin and Ciprofloxacin Derivatives of β‐Octaarginine

β³‐Octaarginine chains were attached to the functional groups NH and CO₂H of the antibacterial fluoroquinolones ciprofloxacin (→ 1 ) and enrofloxacin (→ 2 ), respectively, in order to find out whether the activity increases by attachment of the polycationic, cell‐penetrating peptide (CPP) moiety. For comparison, simple amides, 3 – 5 , of the two antimicrobial compounds and β³‐octaarginine amide ( βR₈ ) were included in the antibacterial susceptibility tests to clarify the impact of chemical modification on the microbiological activity of either scaffold (Table).     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ‐dependent 4‐amino‐4‐deoxy‐l‐arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare‐associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram‐negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine‐containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two‐component system (TCS) directly activates 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l ‐Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB_Ecl TCS. Instead, PhoP_Ecl binds to the arnB_Ecl promoter to induce l ‐Ara4N biosynthesis and PmrAB‐independent addition to the lipid A disaccharolipid. Therefore, PhoPQ_Ecl contributes to regulation of CAMP heteroresistance in some ECC clusters.