Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.     

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.     

New dipeptide fluorogenic substrates of human tissue kallikrein]

Based on 4-methylcoumarinyl-7-amide (Amc) arginine and a series N-alkyloxycarbonyl derivatives of phenylalanine, eleven Amc-derivatives of the type ROCO-Phe-Arg-Amc (R = alkyl) were synthesized; also were n-C3H7OCO-Leu-Arg-Amc and n-C3H7OCO-D-Phe-Arg-Amc synthesized. The enzymatic hydrolysis of these compounds under the action of tissue and plasma human kallikreins were studied. Tissue kallikrein from human urine hydrolyzed the compounds with R = n-propyl and n-butyl and n-C3H7OCO-Leu-Arg-Amc more readily than the known substrates Z-Phe-Arg-Amc and H-Pro-Phe-Arg-Amc. n-C3H7OCO-D-Phe-Arg-Amc is a weak inhibitor of this enzyme (Ki = 1.5.10(-4) M). Human plasma kallikrein hydrolyzed these novel substrates at a lower rate than Z-Phe-Arg-Amc.     

Biocidal activity of some Mannich base cationic derivatives

A novel series of cationic surfactants was prepared based on Mannich base (produced from the condensation of piperidine and/or morpholine as secondary amine and paraformaldehyde in the presence of 8-hydroxyquinoline). The chemical structures of the synthesized cationic surfactants were confirmed using elemental analyses, FTIR spectroscopy and 1H NMR. Surface activities of the prepared surfactants were measured including: surface tension (gamma), critical micelle concentration (CMC), effectiveness (pi(CMC)), efficiency (Pc20), maximum surface excess (Gamma(max)), minimum surface area (A(min)), interfacial tension (gamma(IT)), emulsification power and foaming power at 25 degrees C. The structural influences on their surface activities and adsorption free energy were discussed. The synthesized cationic surfactants were evaluated for their biocidal activity towards Gram +ve bacteria (Staph. Cocu., Bacillus), Gram -ve bacteria (Salmonella, E. coli), fungi (A. terrus., A. flav.) and yeast (Candida) at 1.0, 2.5 and 5.0mg/mL, respectively. The target compounds showed good inhibition towards Gram +ve bacteria, Gram -ve bacteria and yeast. Meanwhile, excellent fungicidal results were obtained against the various types of fungi under investigation.     

The enthalpy of acyl chain packing and the apparent water-accessible apolar surface area of phospholipids

The energetics of phospholipid aggregation depend on the apparent water-accessible apolar surface area (ASAap), ordering effects of the chains, and headgroup interactions. We quantify the enthalpy and entropy of these interactions separately. For that purpose, the thermodynamics of micelle formation of lysophosphatidylcholines (LPCs, chains C10, C12, C14, and C16) and diacylphosphatidylcholines (DAPCs, chains C5, C6) and C7) are studied using isothermal titration calorimetry. The critical micelle concentration (CMC) values are 90, 15, and 1.9 mM (C5-C7-DAPC) and 6.8, 0.71, 0.045, and 0.005 mM (LPCs). The group contributions per methylene of DeltaDeltaG(0) = -3.1 kJ/mol and DeltaDeltaC(P) = -57 J/(mol. K) for LPCs agree with literature data on hydrocarbons and amphiphiles. An apparent deviation of DAPCs (-2.5 kJ/mol, 45 J/(mol. K)) is due to an intramolecular interaction between the two chains, burying 20% of the surface. The chain/chain interaction enthalpies in a micelle core are by approximately -2 kJ/(mol) per methylene group more favorable than in bulk hydrocarbons. We conclude that the impact of the chain conformation and packing on the interaction enthalpy is very pronounced. It serves to explain a variety of effects reported on membrane binding. Interactions within the water-accessible region show considerable DeltaH, but almost no DeltaG(0). The heat capacity changes suggest about three methylene groups (ASAap approximately 100 A2) per LPC remain exposed to water in a micelle (DAPC: 2 CH2/70 A2).     

Sugar-based anionic surfactants: synthesis and micelle formation of sodium methyl 2-acylamido-2-deoxy-6-O-sulfo-D-glucopyranosides

The following sequence of reactions has been employed to synthesize the title anionic surfactants: [chemical reaction: see text] where R=C7H15; C11H23; and C15H31, respectively, and Py refers to pyridine. Aggregation of the surfactants synthesized (predominantly alpha anomers) in water was studied at 40 degrees C by conductivity measurements. Increasing the chain length of R decreases the critical micelle concentration (CMC) and the degree of counter-ion dissociation. The dependence of the Gibbs free energy of micellization and CMC on the length of R is similar to other ionic surfactants, but the head-group, i.e., the sulfated sugar moiety is less hydrophilic than the structurally related group -(OCH2-CH2)2-OSO3-Na+, most probably because of intermolecular H-bonding in the micellar pseudo-phase     

Time-resolved fluorescence anisotropy of fluorescent-labeled lysophospholipid and taurodeoxycholate aggregates.

Previous work from this laboratory demonstrated that the environment-sensitive lysolipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- monomyristoylphosphatidylethanolamine (N-NBD-MPE), at concentrations below its critical micelle concentration (CMCN-NBD-MPE = 4 microM), reached maximum fluorescence yield upon the addition of taurodeoxycholate (TDC) at concentrations well below its CMC (CMCTDC = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concentrations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to determine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) were measured for N-NBD-MPE at the submicellar concentration of 2 microM, indicating a lack of interaction between the two molecules at this concentration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Based on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi(0.19-0.73 ns) was ascribed to local probe motions, whereas the longer phi was in a time range expected for global rotation of aggregates the size of simple bile salt micelles (3-6.5 ns). From the longer phi, molecular volume and hydrodynamic radii were calculated, ranging from approximately 15 A at 1 mM to approximately 18 A at 5 mM TDC. These data support the conclusion that monomeric lysolipids in solution seed the aggregation of numerous TDC molecules (aggregation number = 16 at 1 mM TDC) to form a TDC micelle with a lysolipid core at concentrations below which they both self-aggregate.     

Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding

Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.     

New chemical constituents from the endophyte Streptomyces species LR4612 cultivated on Maytenus hookeri

From solid cultures of the biologically important endophyte Streptomyces species LR4612, cultivated on Maytenus hookeri, four new and two known compounds were isolated. The new compounds were identified as (2S*,3S*)-5-amino-3-hydroxy-5-oxopentan-2-yl 3-(formylamino)-2-hydroxybenzoate (1), N-[(3R*,4R*)-3-amino-3,4-dihydro-4-methyl-2,6-dioxo-2H,6H-1,5-benzodioxocin-10-yl]formamide (2), (5beta,6alpha)-6,11-dihydroxyeudesmane (3), and 5-(6,7-dihydroxy-6-methyloctyl)furan-2(5H)-one (4); the known compounds were elucidated as sorbicillin (5) and N-acetyltyramine (6). The structures were established by HR-ESI-MS and in-depth NMR analyses.     

Synthesis, characterization and anti-tumour testing of some platinum(II) amine complexes containing 1,1- and 1,2-cyclobutanedicarboxylate ligands

A series of new platinum(II) amine complexes containing 1,1- and 1,2-cyclobutanedicarboxylate ligands, cis-[PtA2(1,1-CBDCA)] (A = RNH2, where R = C2H5, n-C3H7, n-C4H9, n-C5H11, n-C6H13, c-C3H5, c-C5H9, c-C6H11; A2 = ethylenediamine, 1,3-diaminopropane), cis-[PtA2(1,2-CBDCA)] (A = NH3, RNH2 where R = CH3, C2H5, n-C3H7, n-C4H9, c-C3H5) and trans-[Pt(NH3)2(1,1-CBDCAH)2] (CBDCA, CBDCAH = dianion and monoanion of the dicarboxylic acid, respectively) have been synthesized by an improved route. These complexes are stable in aqueous solution and show good aqueous solubility. The [Pt(c-C3H5NH2)2(1,1-CBDCA)] can be isolated in white, grey and blue forms. The grey and blue forms exhibit ESR signals analogous to the so-called platinum blues. The existence of the blue form in aqueous solution is time and temperature dependent. Several of the complexes have been tested against leukaemia L1210 in male BDF mice and activity appears to decrease with the increase in length of the aliphatic chain (or increase in size of the alicyclic ring) of the primary amine. The Yoshida lymphoscarcoma screen, usually insensitive to platinum drugs, was found to respond well to [Pt(n-C4H9NH2)2(1,1-CBDCA)] in 5-day subcutaneously implanted tumours in female Wistar rats.     

Synthesis and antimicrobial activity of symmetrical two-tailed dendritic tricarboxylato amphiphiles

Two series of water-soluble, symmetrical two-tailed homologous dendritic amphiphiles--R(2)NCONHC((CH(2))(2)COOH)(3), 2(n,n), and R(2)CHNHCONHC((CH(2))(2)COOH)(3), 3(n,n), where R=n-C(n)H(2n+1)--were synthesized and compared to R'NHCONHC((CH(2))(2)COOH)(3), 1(n), R'=n-C(n)H(2n+1), to determine whether antimicrobial activity was influenced by total or individual alkyl chain lengths, and whether antimicrobial activity depends on hydrophobicity or tail topology (one or two). In a broad screen of 11 microorganisms, 2(n,n) and 3(n,n) generally displayed higher minimal inhibitory concentrations (MICs) than 1(n) against growth as measured by broth microdilution assays. Chain-length specificity was observed against Candida albicans as 1(16), 2(8,8), and 3(8,8) showed the lowest MIC in their respective series. The one case where two-tailed compounds displayed the lowest MICs-3(10,10), 15 microM; 3(11,11), 7.2microM; and 3(12,12), 6.9 microM-was against Cryptococcus neoformans.     

Mefloquine-oxazolidine derivatives, derived from mefloquine and arenecarbaldehydes: In vitro activity including against the multidrug-resistant tuberculosis strain T113

Ten new mefloquine-oxazolidine derivatives, 4-[(1S,8aR)-3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline (1: aryl=substituted phenyl) and 4-[(1S,8aR)-3-(heteroaryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline [2: heteroaryl=5-nitrothien-2-yl (2a); 5-nitrofuran-2-yl (2b) and 4H-imidazol-2-yl) (2c)], have been synthesized and evaluated against Mycobacterium tuberculosis. Compounds 1f (aryl=3-ethoxyphenyl), 1g (Ar=3,4,5-(MeO)(3)-C(6)H(2)) and 2c were slightly more active than mefloquine (MIC=33μM) with MICs=24.5, 22.5 and 27.4, respectively, whereas compounds 1e (aryl=3,4-(MeO)(2)-C(6)H(3)) and 2a (MICs=11.9 and 12.1μM, respectively) were ca. 2.7 times more active than mefloquine, with a better tuberculostatic activity than the first line tuberculostatic agent ethambutol (MIC=15.9). The compounds were also assayed against the MDR strain T113 and the same MICs were observed. Thus the new derivatives have advantages over such anti-TB drugs as isoniazid, rifampicin, ethambutol and ofloxacin, for which this strain is resistant. The most active compounds were not cytotoxic to Murine Macrophages Cells in a concentration near their MIC values.     

Relationships between structure and molting hormonal activity of tebufenozide, methoxyfenozide, and their analogs in cultured integument system of Chilo suppressalis Walker

The molting hormonal activity of methoxyfenozide (RH-2485), tebufenozide (RH-5992), five analogs with various alkyl groups, and 18 acyl analogs was measured by using cultured integument of rice stem borers, Chilo suppressalis Walker. The hormonal activity of methoxyfenozide was remarkably high (EC(50) = 1.1 x 10(-9) M), being equivalent to that of tebufenozide (RH-5992). The hormonal activity of several tebufenozide analogs with varying alkyl groups such as CH(3), n-C(3)H(7), i-C(3)H(7), n-C(4)H(9) and n-C(5)H(11) at the para-position of the benzene ring furthest from the tert-butyl group was lower than that of tebufenozide (alkyl group is C(2)H(5)). The activity decreased to varying degrees as a result of replacement of the 3,5-dimethylphenyl moiety of tebufenozide with either a phenyl, naphthyl, or cyclohexyl group. Both 1- and 2-naphthyl derivatives were very active (EC(50) = 4.3 x 10(-8) M and 3.2 x 10(-8) M, respectively) without any significant difference between them. The activity of the 1-cyclohexenyl analog (EC(50) = 1.0 x 10(-7) M) was about 40x that of the corresponding 3-cyclohexenyl analog (EC(50) = 4.4 x 10(-6) M), but 1/100 that of tebufenozide. The activity varied parabolically with respect to the molecular hydrophobicity, and decreased with longer acyl moieties.     

Synthesis of acridinyl-thiazolino derivatives and their evaluation for anti-inflammatory, analgesic and kinase inhibition activities

Variety of N-(4-phenyl-3-(2',3',4'(un)substituted phenyl)thiazol-2(3H)-ylidene)-2,4(un)substituted acridin-9-amine (4a-o) and 1-[(2,4-(un)substituted acridin-9-yl)-3-(4-phenyl-3-(2',3',4'(un)substituted phenyl)thiazol-2(3H)-ylidene)]isothiourea (5a-h) derivatives have been synthesized by condensation of 4-phenyl-3-(2',3',4'(un)substituted phenyl)thiazol-2(3H)-imine (3a-g) with 9-chloro-2,4-(un)substituted acridine (1a-c) and 9-isothiocyanato-2,4-(un)substituted acridine (2a-d), respectively. All these compounds were characterized by correct 1H NMR, FT-IR, MS and elemental analyses. These compounds were screened for anti-inflammatory, analgesic and kinase (CDK1, CDK5 and GSK3) inhibition activities. Some compounds exhibited good anti-inflammatory (25-32%) and potent analgesic (50-75%) activities, at 50 mg/kg p.o. A compound, 4o (R1 = H, R2 = OCH3, R3 = CH3, R4 = CH3, R5 = H) exhibited moderate CDK1 (IC50 = 8.5 microM) inhibition activity.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Production of Glycolipid Biosurfactants, cellobiose lipids, by Cryptococcus humicola JCM 1461 and their interfacial properties

Cryptococcus humicola JCM 1461 efficiently produced cellobiose lipids (CLs), bolaform biosurfactants. The main product was identified as 16-O-(2″,3″,4″,6'-tetra-O-acetyl-β-cellobiosyl)-2-hydroxyhexadecanoic acid. The production yield of CLs reached 13.1 g/L under the intermittent feeding of glucose. The critical micelle concentrations (CMC) of the main product at pH 4.0 and 7.0 were 3.3×10(-5) M and 4.1×10(-4) M respectively.     

Enhancement of hydrocarbon wastebiodegradation by addition of a biosurfactantfrom Bacillus subtilis O9

A non-sterile biosurfactant preparation (surfactin)was obtained from a 24-h culture of Bacillussubtilis O9 grown on sucrose and used to study itseffect on the biodegradation of hydrocarbon wastes byan indigenous microbial community at theErlenmeyer-flask scale. Crude biosurfactant was addedto the cultures to obtain concentrations above andbelow the critical micelle concentration (CMC). Lowerconcentration affected neither biodegradation normicrobial growth. Higher concentration gave highercell concentrations. Biodegradation of aliphatichydrocarbons increased from 20.9 to 35.5% and in thecase of aromatic hydrocarbons from nil to 41%,compared to the culture without biosurfactant. Theenhancement effect of biosurfactant addition was morenoticeable in the case of long chain alkanes. Pristaneand phytane isoprenoids were degraded to the sameextent as n-C17 and n-C18 alkanes and, consequently,no decrease in the ratios n-C17/pri and n-C18/phy wasobserved. Rapid production of surfactin crudepreparation could make it practical for bioremediationof ship bilge wastes.     

Cytotoxic steroidal saponins from Polygonatum punctatum

Four new steroidal saponins, polypunctosides A-D (1-4, resp.), were isolated from the rhizomes of Polygonatum punctatum, together with five known steroidal saponins. On the basis of chemical and spectral evidence, the structures of the new saponins were established as (3beta,23S,25R)-23-(alpha-L-arabinopyranosyloxy)spirost-5-en-3-yl 4-O-(6-deoxy-alpha-L-mannopyranosyl)-D-glucopyranoside (1), (3beta,23S,25R)-23-[(2-O-acetyl-alpha-L-arabinopyranosyl)oxy]spirost-5-en-3-yl 4-O-(6-deoxy-alpha-L-mannopyranosyl)-D-glucopyranoside (2), (3beta,23S,25R)-23-[(3-O-acetyl-alpha-L-arabinopyranosyl)oxy]spirost-5-en-3-yl 4-O-(6-deoxy-alpha-L-mannopyranosyl)-D-glucopyranoside (3), and (3beta,23S,25R)-23-[(4-O-acetyl-alpha-L-arabinopyranosyl)oxy]spirost-5-en-3-yl 4-O-(6-deoxy-alpha-L-mannopyranosyl)-D-glucopyranoside (4). The cytotoxic activity of the isolated saponins was evaluated towards HeLa cells.     

Practical stereoselective synthesis of (2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl)acetic acid

Highly enantioselective asymmetric hydrogenation of readily accessible olefins, (E)- and (Z)-[1-(toluene-4-sulfonyl)-1,2,3, 4-tetrahydro-1H-benzo[b]azepin-5-ylidene]acetic acid (4a and 4b, respectively) and [1-(toluene-4-sulfonyl)-2, 3-dihydro-1H-benzo[b]azepin-5-yl]acetic acid (4c), is presented as an efficient and straightforward route to (R)-[1-(toluene-4-sulfonyl)-2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl]acetic acid [(R)-1] which is a key intermediate for the synthesis of non-peptide AVP V2-agonist. Hydrogenation of carboxylic acid 4c gave (R)-1 in quantitative yield and 85% ee using Ru(OAc)2[(S)-H8-BINAP], a Ru(II) complex of a partially hydrogenated BINAP (H8-BINAP), as a catalyst. When (R)-1 of 76% ee was transformed into the corresponding isopropylamide 6, pure enantiomer (R)-6 was obtained in 75% yield by recrystallization from MeOH.     

Conformational studies of aqueous melittin: thermodynamic parameters of the monomer-tetramer self-association reaction

The self-association reaction in which four melittin molecules associate to form an aqueously soluble tetramer was studied by fluorescent spectroscopy. At 23 degrees C, pH 7.15, gamma/2 0.50, the dissociation constant, Kd, is 3.20 x 10(-16) M3. At 23 degrees C, gamma/2 0.60, melittin has an amino acyl group with a proton ionization constant at ca. 10(-6) M, which must be un-ionized for tetramer formation to occur. The change in Kd with temperature indicates the forward reaction (tetramer formation) proceeds primarily by entropic changes, with delta H degrees = -20.3 kJ/mol of monomer and delta S degrees = 211 J/(K . mol of monomer). The observed enthalpic and entropic values for the tetramerization reaction are consistent with the expected contributions of both nascent hydrogen bonds and hydrophobic stabilization to the reaction. The ionic strength dependence of the tetramerization reaction was found to be consistent with an Edsall-Wyman treatment of activity coefficients. Specifically, the calculated charge of melittin varied from 2.5 (pH 10.53, gamma/2 less than 0.08) to ca. 6 (pH 7.15, gamma/2 greater than 0.3) and showed a strong dependence on gamma/2.