A biomimetic approach for enhancing the in vivo half-life of peptides.

The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes.

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches ("flipids") that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.

A convenient method for the relative and absolute quantification of lipid components in liposomes by 1H- and 31P NMR-spectroscopy.

OBJECTIVE: Liposomes are promising delivery systems for pharmaceutical applications and have been used in medicine in the recent past. Preparation of liposomes requires reliable characterization and quantification of the phospholipid components for which the traditional cumbersome molybdate method is used frequently. The objective was to improve relative and absolute quantification of lipid components from liposomes. METHODS: A reliable method for quantification of lipid composition in liposome formulations in the 1-10 µmol range with 1H- and 31P NMR spectroscopy at 600 MHz has been developed. The method is based on three crystalline small-molecule standards (Ph3PO4, (Tol)3PO4, and Ph3PO) in CDCl3. RESULTS: Excellent calibration linearity and chemical stability of the standards was observed. The method was tested in blind fashion on liposomes containing POPC, PEG-ceramide and a pH-sensitive trans-aminocyclohexanol-based amphiphile (TACH).1 Relative quantification (percentage of components) as well as determination of absolute lipid amount was possible with excellent reproducibility with an average error of 5%. Quantification (triplicate) was accomplished in 15 min based on 1H NMR and in 1 h based on 31P NMR. Very little change in mixture composition was observed over multiple preparative steps. CONCLUSION: Liposome preparations containing POPC, POPE, DOPC, DPPC, TACH, and PEG-ceramide can be reliably characterized and quantified by 1H NMR and 31P NMR spectroscopy at 600 MHz in the µmol range.

Fliposomes: pH-triggered conformational flip of new trans-2-aminocyclohexanol-based amphiphiles causes instant cargo release in liposomes.

A new type of pH-sensitive liposomes (fliposomes) was designed based on the amphiphiles that are able to perform a pH-triggered conformational flip (flipids). This flip disrupts the liposome membrane and causes rapid release of the liposome cargo, specifically in response to lowered pH. The flipids (1) and (2) are equipped with a trans-2-aminocyclohexanol conformational switch. pH-sensitive fliposomes containing one or both of these flipids, as well as POPC and PEG ceramide, were constructed and characterized. These compositions were stable at 4°C and pH 7.4 for several months. Fliposomes loaded with ANTS/DPX performed an unusually quick content release within a few seconds at pH below 8.5 (in case of 2) and 6.0 (in case of 1). This difference in pH sensitivity demonstrates a potential for the custom design of flipids by variation of the amino group to target areas with specific pH values. The pH titration curves for the fliposome leakage parallel the curves for the acid-induced conformational flip of 1 and 2 studied by ¹H NMR. A plausible mechanism of pH sensitivity starts with an acid-triggered conformational flip of 1 or 2, which changes the molecular size and shape, shortens the lipid tails, and perturbs the liposome membrane, resulting in the content leakage.

trans-2-Aminocyclohexanol as a pH-sensitive conformational switch in lipid amphiphiles.

Protonation-induced conformational change of lipid tails is reported as a novel strategy to render pH-sensitive lipid amphiphiles and lipid colloids.

Fliposomes: trans-2-aminocyclohexanol-based amphiphiles as pH-sensitive conformational switches of liposome membrane - a structure-activity relationship study.

Recently developed lipids with the trans-2-aminocyclohexanol (TACH) moiety represent unique pH-sensitive conformational switches ("flipids") that can trigger the membrane of liposome-based drug delivery systems at lowered pH as seen in many pathological scenarios. A library of flipids with various TACH-based headgroups and hydrocarbon tails were designed, prepared, and characterized to systematically elucidate the relationship between their chemical structures and their ability to form and to trigger liposomes. Liposomes (fliposomes) consisting of a flipid, POPC and PEG-ceramide were stable at 4°C, pH 7.4 for up to several months and yet released the encapsulated fluorophore in seconds upon acidification. The colloidal properties and encapsulation efficiencies of the fliposomes depended on the structure features of the flipids such as the polarity of the headgroups and the shape and fluidity of the lipid tails. The pH-triggered release also depended on the flipid structure, where shorter linear tails yielded more efficient release. The release of fliposomes was enhanced at different narrow pH ranges, depending on the basicity of the flipid headgroup, which can be estimated either by calculated pKa or by acid/base titration of the flipids while its conformation is monitored by 1H NMR. The structure-activity relationship of the flipids supports "lipid tail conformational shortening" as the mechanism to disrupt lipid membranes and would provide great flexibility in the design of pH-sensitive drug delivery systems.

Arylsulfanyl groups - Suitable side chains for 5-substituted 1,10-phenanthroline and nickel complexes as G4 ligands and telomerase inhibitors.

Guanine-rich DNA sequences can undergo self-assembly into unique G-quadruplex structures that interfere with the binding of proteins to the same DNA region. The formation of DNA G-quadruplexes requires monovalent cations (Na+ and K+) or small molecules known as G-quadruplex (G4) ligands. Phenanthroline is a type of G4 ligand scaffold known for its coordination with metal ions to form complexes with a large aromatic surface area, which aptly stack with G-quartets. In this report, we have investigated the side chain effect on G-quadruplex recognition by evaluating a series of 5-substituted phenanthroline-based metal complexes (Phen-Ni) binding to telomeric G-quadruplex DNA. Results from biophysical methods including fluorescence and circular dichroism (CD) thermal denaturation, CD titration, and the fluorescent intercalator displacement (FID) assay suggest that several Phen-Ni complexes bind to G-quadruplex DNA with submicromolar G4DC50 values. Arylsulfanyl groups at the 5 position of 1,10-phenanthroline are the best side chains regarding binding affinity and selectivity towards G-quadruplex DNA. Most of the G-quadruplex binding Phen-Ni complexes can inhibit telomerase activity in vitro as indicated by the telomeric repeat amplification protocol (TRAP) assay and such inhibition is clearly concentration dependent. Our results here provide a guidance of utilizing 5-substituted phenanthroline derivatives as a viable and facile approach to design novel G4 ligands.

Capacious and programmable multi-liposomal carriers.

Spherical polycationic brushes (SPBs) were synthesized by grafting polycationic chains onto 100 nm polystyrene particles. These particles were exposed to unilamellar egg-lecithin (EL) liposomes with a mean diameter of 40 nm that had been rendered anionic via the presence of 10 molar% of phosphatidylserine (PS(1-)). The liposomes also contained 30 mole% of a morpholinocyclohexanol-based lipid (MOCH) that undergoes a conformational flip when the pH is decreased from 7.0 to 5.0. Mixtures of SPBs and liposomes at pH 7 gave an electrostatically-driven complex possessing, on average, about 40 liposomes for each SPB particle. It was found that the bound liposomes rapidly release much of their contents when the pH is reduced from 7.0 to 5.0 owing mostly to a MOCH conformational change that creates defects in the bilayer membrane. The drop in pH does not, however, induce a separation of the liposomes from the SPBs. Around 50-60% of the liposome contents escape before, it is reasoned, lateral and transmembrane motion of the membrane components heals the defects and prevents further release. Remarkably, the liposomes complexed with SPB release their cargo much faster than the identical but non-complexed liposomes.

Palladium-charcoal-catalyzed reduction of tri-O-acetyl-beta-L-fucopyranosyl cyanide: a route to small cluster oligosaccharide mimetics (SCOMs).

[reaction: see text] Synthesis of glycosyl cyanides was optimized with a new catalyst system. Reduction of tri-O-acetyl-beta-L-fucopyranosyl cyanide with Pd-hydrogen, in the presence of Ac(2)O and Boc(2)O, gave N-protected-mono- and -di-(2,3,4-tri-O-acetyl-beta-L-fucopyranosylmethyl)-amines, which allow for the syntheses of small cluster oligosaccharide mimetics of fucopyranosylomethyl-substituted ureas. From di-(2,3,4-tri-O-acetyl-beta-L-fucopyranosylmethyl) amine was also prepared a carbamoyl chloride as a potentially useful synthon for preparation of more complex C-glycosidic conjugates.

C-H-deprotonation mediated by a remote syn-axial acetoxy group--an unprecedented double bond formation upon cyanation of the dimer from L-fucal.

Replacement of the anomeric acetate by a cyanide group in the dimer of di-O-acetyl-L-fucal by the action of mild Lewis acid [Hg(CN)(2)-HgBr(2)-Me(3)SiCN], resulted not only in the desired transformation but also in the introduction of an additional double bond between C-2A and C-3A. Due to its configuration, the remote C-4A acetoxy group may facilitate the deprotonation by functioning as an internal base. 1H NMR spectroscopy and X-ray crystallography indicate that the conformations of both rings A and B and their relative orientation in the resulting C-linked disaccharidic glycosyl cyanide, 4-O-acetyl-2-C-(4-O-acetyl-2,3-dideoxy-alpha-L-threo-hex-2-enopyranosyl)-2,3-dideoxy-2-eno-alpha-L-fucopyranosyl cyanide, in solution are virtually identical to the crystal structure.

Fliposomes: stimuli-triggered conformational flip of novel amphiphiles causes an instant cargo release from liposomes.

This review presents a new strategy for the design of stimuli-responsive liposomes for targeted delivery - the construction of a liposome membrane (lipid bilayer) using amphiphiles able to perform a stimuli-triggered conformational flip ('flipids'). When done simultaneously by a major or significant part of the bilayer molecules, this massive flip disrupts the liposome membrane and induces a rapid release of the liposome load specifically in response to the initial stimulus. The conformational switches incorporated into the amphiphilic molecules could potentially be controlled by various internal or external factors (pH, metal complexation, light, electric field, etc.). Using this concept, we designed a series of pH-triggerable flipids, and prepared and tested 'fliposomes' with extraordinary characteristics: high stability in storage and in serum combined with an instant release of their cargo in response to a weakly acidic medium.

Fliposomes: pH-controlled release from liposomes containing new trans-2-morpholinocyclohexanol-based amphiphiles that perform a conformational flip and trigger an instant cargo release upon acidification.

A new type of pH-sensitive liposome (fliposomes) was designed based on the amphiphiles that are able to perform a pH-triggered conformational flip (flipids). This flip disrupts the liposome membrane and causes rapid release of the liposome cargo, specifically in the areas of increased acidity. The flipids (1-3) are equipped with a trans-2-morpholinocyclohexanol conformational switch, pH-Sensitive fliposomes containing one of these flipids, POPC and PEG-ceramide (molar ratio 50/45/5) were constructed and characterized. These compositions were stable at 4 degrees C and pH 7.4 for several months. Fliposomes loaded with ANTS/DPX demonstrated an unusually quick content release (in a few seconds) at pH below 5.5, which was more efficient in the case of flipid 1 with the shorter linear C12-tails. The pH-titration curve for the fliposome leakage paralleled the curve for the acid-induced conformational flip of 1-3 studied by 1H NMR. A plausible mechanism of the pH-sensitivity starts with an acid-triggered conformational flip of 1, 2 or 3, which changes the molecular size and shape, shortens the lipid tails, and perturbs the liposome membrane resulting in the content leakage.

Fliposomes: pH-Sensitive Liposomes Containing a trans-2-morpholinocyclohexanol-Based Lipid That Performs a Conformational Flip and Triggers an Instant Cargo Release in Acidic Medium.

Incorporation of a pH-sensitive conformational switch into a lipid structure enables a drastic conformational flip upon protonation that disrupts the liposome membrane and causes rapid release of cargo specifically in areas of increased acidity. pH-sensitive liposomes containing the amphiphile (1) with trans-2-morpholinocyclohexanol conformational switch, a phospholipid, and a PEG-lipid conjugate were constructed and characterized. The optimized composition-1/POPC/PEG-ceramide (50/45/5)-could be stored at 4 °C and pH 7.4 for up to 1.5 years, and was stable in blood serum in vitro after 48 h at 37 °C. Liposomes loaded with ANTS/DPX or methotrexate demonstrated an unusually quick content release (in a few seconds) at pH below 5.5, which was independent of inter-liposome contact. The pH-titration curve for the liposome leakage paralleled the curve for the acid-induced conformational flip of 1 studied by 1H-NMR. Freeze-fracture electron microscopy images showed budding and division of the bilayer at pH 5.5. A plausible mechanism of pH-sensitivity involves an acid-triggered conformational flip of 1, shortening of lipid tails, and membrane perturbations, which cause the content leakage. The methotrexate-loaded liposomes demonstrated much higher cytotoxicity in HeLa cells than the free drug indicating that they can serve as viable drug delivery systems.

Novel easily accessible glucosidase inhibitors: 4-hydroxy-5-alkoxy-1,2-cyclohexanedicarboxylic acids.

Glycosidases are very important enzymes involved in a variety of biochemical processes with a special importance to biotechnology, food industry, and pharmacology. Novel structurally simple inhibitors derived from cyclohexane-1,2-dicarboxylic acids were synthesized and tested against several fungal glycosidases from Aspergillus oryzae and Penicilliumcanescens. The presence of at least two carboxylic groups and one hydroxy group was essential for efficient inhibition. Significant selective inhibition was observed for alpha- and beta-glucosidases, the magnitude of which depended on the configuration of substituents; inhibition increased for beta-glucosidase by lengthening the alkoxy group of the inhibitor.

Mild synthesis of disaccharidic 2,3-enopyranosyl cyanides and 2-C-2-deoxy pyranosyl cyanides with Hg(CN)(2)/HgBr(2)/TMSCN.

Lewis acid-catalyzed dimerization of mono- and disaccharidic per-O-acetylated glycals gave di- and tetrasaccharidic O-acetylated C-glycosides, respectively. 2,3-Enopyranosyl cyanides were obtained from per-O-acetylated glycals by a new, mild anomeric S(N)'-acetoxy displacement with Hg(CN)(2)/HgBr(2)/TMSCN. Per-O-acetylated 2-C-2-deoxy-pyranoses were converted into pyranosyl cyanides by the same reagent. An unprecedented acetic acid elimination from dimers with D-galacto- and L-fuco-configurations accompanied the S(N)-displacement under those conditions. A new set of (1)H NMR coupling constants for 2,3-enopyranosyl systems was used for configurational assignment of complicated tetrasaccharide mimics.