Powerful solvent systems useful for synthesis of sparingly-soluble peptides in solution.

Our maximum protection strategy for the synthesis of human parathyroid hormone(1-84) indicates that fully protected peptide segments in the form of Boc-peptide phenacyl (Pac) ester are relatively soluble in ordinary organic solvents such as DMF, NMP or DMSO, which are suitable for coupling segments. However, about 1% of such segments synthesized were found to be insoluble even in the most polar solvent, DMSO. Thus, a more powerful solvent which can be used for their peptide synthesis was pursued. Among the solvent systems tested, a mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be most powerful for dissolving such sparingly-soluble protected peptides. These solvent systems were confirmed to be useful for the removal reaction of the carboxy-terminal Pac esters from the sparingly-soluble segments. They were then tested for the coupling reactions of fully protected Boc-peptides with other sparingly-soluble peptide esters. The TFE/TCM or TFE/DCM system was extremely useful for coupling segments without danger of racemization and of trifluoroester formation, if WSCI was used as the coupling reagent in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt).     

Solvent effects on coupling yields during rapid solid-phase synthesis of CGRP(8-37) employing in situ neutralization.

The success of solid-phase peptide synthesis is often dependent upon solvation of the resin and the growing resin-bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide-resin in order to obtain satisfactory coupling yields for the rapid solid-phase peptide synthesis, using butyloxycarbonyl-(Boc)-amino acid derivatives, of human-alpha-calcitonin gene-related peptide(8-37) (CGRP(8-37)). Solvation of (p-methylbenzhydrylamine)copoly(styrene-1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin) was correlated to solvent Hildebrand solubility (delta) and hydrogen-bonding (delta(h)) parameters. Contour solvation plots of delta(h) vs. delta revealed maximum solvation regions of resin and peptide-resin. Maximum resin solvation occurred with N-methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide-resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide-resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide-resin solvation shifts to solvents with higher delta(h) values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8-37) was obtained as a single major product in the crude cleaved material.     

Synthesis and conformational investigation of cyclic dipeptides: 7-membered rings containing alpha- and beta-amino acids.

The synthesis of heterocyclic compounds containing the 7-membered ring system [1,4]diazepane-2,5-dione is described. The aim of this study was to elaborate the solid phase and solution synthesis of eight representatives of the cyclic scaffold and to investigate their chemical stability and their conformational properties. The solid phase synthesis was performed on aminomethyl polystyrene resin using 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid as a backbone linker system (BAL-linker). After attachment of the alpha- and beta-amino acid and deprotection of the amino function, the dipeptide ester was obtained. The molecule was cyclized on the solid support by treatment with NaOMe in MeOH/NMP. The product was cleaved from the resin by TFA. For the solution pathway the linear dipeptides were synthesized by coupling of the BOC-protected L-alpha-amino acid with the beta2-amino acid ester (EDC/HOBT). After N- and C-terminal deprotection of the dipeptide, the linear species was cyclized with EDC/HOBT at a concentration of 3 mM in DMF. The products showed high chemical stability after storage in DMSO at room temperature for weeks. The x-ray and two dimensional NMR investigations were performed to investigate the conformation of the molecules. Three types of configuration could be distinguished by NMR, depending on the substitution pattern of the cyclic compounds. The x-ray results confirmed the NMR observations. In general the 7-membered rings showed rigidity, thus they could represent optimal scaffolds for new receptor ligands.     

Synthesis of amphiphilic amylose and starch derivatives

For non-food uses starch generally is modified in order to obtain products with properties suitable for various applications. In the present work, starch and amylose were hydrophobically modified through reactions with long-chain -alkyl epoxides (C₆ and C₁₂) in DMSO solution, in the presence of NaH as a catalyst. The molar substitution (MS) was calculated from NMR spectra. Derivatives with high as well as low MS values were obtained. In order to reach MS values above 1.5, the reaction had to be run for 150–300 h. Viscosity and GPC measurements indicated that the polysaccharides were degraded in DMSO under the influence of methyl sulfinyl anion, which presumably is the active catalyst.

The derivatives were also characterized by FTIR. The ratio between the peak areas for OH stretching and alkyl stretching vibrations, respectively, in the FTIR spectra, was found to be proportional to MS values determined from NMR spectra.

The solubility of the hydrophobically modified polysaccharide in various solvents was tested. Samples having C₁₂-alkyl side chains and MS>1 were soluble in toluene. The C₆ derivatives were water soluble up to a MS value of 0.3.     

Comparative study of lipid extraction from microalgae by organic solvent and supercritical CO2

Pavlova sp. was employed to evaluate the efficiency of different lipid extraction methods. The microalgal crude lipids content determined using the mixed solvent with ultrasonic method was 44.7 wt.%. The triglyceride content obtained by the mixed solvent method was 15.6 wt.%. The extraction yield was the FAME yield divided by the maximum FAME (15.9 wt.%). The extraction yield was improved by cell disruption prior to extraction, and the highest triglyceride extraction yield of 98.7% was observed using the supercritical fluid extraction (SFE) method with bead-beating. The results indicate that the SFE method is effective and provides higher selectivity for triglyceride extraction though the total lipid extracted was less than that using solvent extraction.     

Diffusion and saponification inside porous cellulose triacetate fibers

Cellulose triacetate (CTA) fibers were partially hydrolyzed in 0.054 N solutions of NaOH/H(2)O and NaOMe/MeOH. The surface concentration of acetyl groups was determined using ATR-FTIR. Total acetyl content was determined by the alkaline hydrolysis method. Fiber cross-sections were stained with Congo red in order to examine the interface between reacted and unreacted material; these data were used to estimate the rate constant k and effective diffusivity D(B) for each reagent during the early stages of reaction by means of a volume-based unreacted core model. For NaOH/H(2)O, k = 0.37 L mol(-1) min(-1) and D(B) = 6.2 x 10(-7) cm(2)/sec; for NaOMe/MeOH, k = 4.0 L mol(-1) min(-1) and D(B) = 5.7 x 10(-6) cm(2)/sec. The NaOMe/MeOH reaction has a larger rate constant due to solvent effects and the greater nucleophilicity of MeO(-) as compared to OH(-); the reaction has a larger effective diffusivity because CTA swells more in MeOH than it does in water. Similarities between calculated concentration profiles for each case indicate that the relatively diffuse interface seen in fibers from the NaOMe/MeOH reaction results from factors not considered in the model; shrinkage of stained fiber cross-sections suggests that increased disruption of intermolecular forces may be the cause.     

A combinatorial approach to the synthesis of cystine based organogelators.

A solid-phase approach was used to prepare 20 cystine amide derivatives with disulfide bond formation resulting from an intra-site reaction between neighbouring cysteine residues. Library members were screened as potential organogelators in a range of solvent mixtures and resulted in the identification of a potent gelator able to rigidify water/DMSO mixtures at concentrations as low as 1.3 mM.     

Catalytic activity of Ti-doped NaH nanoclusters towards hydrogenation of terminal alkenes

The reported activity of nanoscale NaH and titanocene towards catalytic reduction of terminal alkenes using molecular hydrogen is surprising considering that both NaH bulk and titanocene are inactive by themselves. In this work, the role of Ti dopants, the importance of NaH nanoclusters and role of the solvent (THF) and cocatalyst (titanocene) are investigated using density functional theory techniques. A plausible mechanism is proposed to explain the origin of the selective catalytic activity. A step-by-step reaction pathway starting with hydrogen chemisorption near the titanium atoms on the NaH nanoparticle surface followed by the reaction of the activated hydrogen with terminal alkenes via a five-membered ring intermediate are discussed.     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

Preparation of C-6 substituted chitin derivatives under homogeneous conditions

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.     

Lipase-catalyzed biodiesel production from waste activated bleaching earth as raw material in a pilot plant

The production of fatty acid methyl esters (FAMEs) from waste activated bleaching earth (ABE) discarded by the crude oil refining industry using lipase from Candida cylindracea was investigated in a 50-L pilot plant. Diesel oil or kerosene was used as an organic solvent for the transesterification of triglycerides embedded in the waste ABE. When 1% (w/w) lipase was added to waste ABE, the FAME content reached 97% (w/w) after reaction for 12 h at 25 degrees C with an agitation rate of 30 rpm. The FAME production rate was strongly dependent upon the amount of enzyme added. Mixtures of FAME and diesel oil at ratios of 45:55 (BDF-45) and 35:65 (BDF-35) were assessed and compared with the European specifications for biodiesel as automotive diesel fuel, as defined by pr EN 14214. The biodiesel quality of BDF-45 met the EN 14214 standard. BDF-45 was used as generator fuel, and the exhaust emissions were compared with those of diesel oil. The CO and SO2 contents were reduced, but nitrogen oxide emission increased by 10%. This is the first report of a pilot plant study of lipase-catalyzed FAME production using waste ABE as a raw material. This result demonstrates a promising reutilization method for the production of FAME from industrial waste resources containing vegetable oils for use as a biodiesel fuel.     

Conformational preference of Leu side chain in melanostatin in DMSO

The solution conformation of melanostatin (Pro-Leu-Gly-NH2) in the neutral and protonated forms of DMSO has been monitored by one and two dimensional NMR techniques at 500 MHz. The temperature coefficients of the amide proton chemical shifts in conjunction with the observed NOESY spectra suggest that melanostatin in neutral form in DMSO adopts a backbone conformation such that leucine amide proton is buried by the proline ring and the side chain of leucine. Similar observation is made for protonated form of melanostatin in DMSO. The results of the present study are at variance with the earlier NMR studies which proposed a beta-turn structure for both the forms of melanostatin. There is, however, no evidence for the presence of beta-turn structure for both the forms of melanostatin in DMSO. In CDCl3 also Leu NH appears to be buried as evident from the solvent titration with DMSO and NOESY spectra.     

Synthesis of N-substituted 1-amino-2,3-dihydro-1H-imidazole-2-thione-N-nucleosides and S-glycosylated derivatives

Fusion of the N-substituted 1-amino-2,3-dihydro-1H-imidazole-2-thiones 1-4 with the peracylated ribose 5 in the presence of iodine afforded the N-nucleosides 6-9 in moderate yields. Deblocking with NaOMe/MeOH gave the free nucleosides 10-13. Alternatively, silylation of 4 followed by ribosylation with 5 in the presence of TMSOTf as catalyst afforded 9 in moderate yield. Ribosylation of 4 with the chlorodeoxyribose derivative 15 as well as 5 in the presence of NaH in DMF afforded the thioglycosides 16 and 18, respectively. Deblocking of 16 and 18 with NaOMe/MeOH gave the free S-thioglycosides 17 and 19, respectively. Thermal rearrangement of 19 at high temperature in the presence of iodine furnished 13 in low yield. The new free nucleosides and thioglycosides were inactive against HIV-1 and HIV-2 induced cytopathicity in human MT-4 lymphocyte cells.     

Zero-waste biorefinery of oleaginous microalgae as promising sources of biofuels and biochemicals through direct transesterification and acid hydrolysis

Oleaginous microalgae are considered as promising sources of biofuels and biochemicals due to their high lipid content and other high-value components such as pigments, carbohydrate and protein. This study aimed to develop an efficient biorefinery process for utilizing all of the components in oleaginous microalgae. Acetone extraction was used to recover microalgal pigments prior to processes for the other products. Microalgal lipids were converted into biodiesel (fatty acid methyl ester, FAME) through a conventional two-step process of lipid extraction followed by transesterification, and alternatively a one-step direct transesterification. The comparable FAME yields from both methods indicate the effectiveness of direct transesterification. The operating parameters for direct transesterification were optimized through response surface methodology (RSM). The maximum FAME yield of 256 g/kg-biomass was achieved when using chloroform:methanol as co-solvents for extracting and reacting reagents at 1.35:1 volumetric ratio, 70 °C reaction temperature, and 120 min reaction time. The carbohydrate content in lipid-free microalgal biomass residues (LMBRs) was subsequently acid hydrolyzed into sugars under optimized conditions from RSM. The maximum sugar yield obtained was 44.8 g/kg-LMBRs and the protein residues were recovered after hydrolysis. This biorefinery process may contribute greatly to zero-waste industrialization of microalgae based biofuels and biochemicals.     

Optimization Studies and Chemical Kinetics of Silica Sulfuric Acid-Catalyzed Biodiesel Synthesis from Waste Cooking Oil

In the present study conversion of waste cooking oil to biodiesel has been carried out via simultaneous esterification and transesterification reaction over silica sulfuric acid as a solid acid catalyst. The process variables that influence the fatty acid methyl ester (FAME) conversion, such as reaction temperature, reaction time, catalyst concentration and methanol to oil molar ratio were investigated and optimized using Taguchi method. Highest FAME production obtained under the optimized condition was 98.66 %. Analysis of variance revealed that temperature was the most significant factor effecting the FAME production among four factors studied. From the kinetic study, the reaction was found to follow pseudo first-order kinetics and rate constant of the reaction under optimum condition was 0.00852 min^?1.     

Refinement of protein structures in explicit solvent

We present a CPU efficient protocol for refinement of protein structures in a thin layer of explicit solvent and energy parameters with completely revised dihedral angle terms. Our approach is suitable for protein structures determined by theoretical (e.g., homology modeling or threading) or experimental methods (e.g., NMR). In contrast to other recently proposed refinement protocols, we put a strong emphasis on consistency with widely accepted covalent parameters and computational efficiency. We illustrate the method for NMR structure calculations of three proteins: interleukin-4, ubiquitin, and crambin. We show a comparison of their structure ensembles before and after refinement in water with and without a force field energy term for the dihedral angles; crambin was also refined in DMSO. Our results demonstrate the significant improvement of structure quality by a short refinement in a thin layer of solvent. Further, they show that a dihedral angle energy term in the force field is beneficial for structure calculation and refinement. We discuss the optimal weight for the energy constant for the backbone angle omega and include an extensive discussion of meaning and relevance of the calculated validation criteria, in particular root mean square Z scores for covalent parameters such as bond lengths.     

几种硫酸化试剂和溶剂对菊糖硫酸化改性的影响

为制取硫酸化菊糖,以硫酸钡比浊法测定硫酸基取代度(DS)、红外光谱测定含硫基团的特征吸收峰、核磁共振碳谱(13C NMR)判断硫酸根取代位置等方法,比较了以N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)和吡啶(Py)三种溶剂,氯磺酸(CA)和三氧化硫(SO3)两种硫酸化试剂对菊糖硫酸酯化的影响.结果表明:以吡啶为溶剂、氯磺酸为硫酸化试剂的方法(CA-Py)与SO3-Py、CA-DMF三种硫酸化方法均获得了硫酸化菊糖,产品均显示不对称S=O键伸缩振动(约1255 cm-1)和对称的C-O-S键伸缩振动(约810 cm-1)特征吸收峰;三种方法的DS分别为:1.24,0.89,1.83;三种产品的13C NMR基本相同,均表明硫酸根连接在C3、C5、C6上.DMSO不适宜用作硫酸化溶剂.三种硫酸化方法是成功的,但以SO3-Py法操作简便,最适于菊糖硫酸化.     

Use of acyl phosphonates for the synthesis of inulin esters and their use as emulsion stabilizing agents

Inulin, the polydisperse polyfructose, extracted from chicory, was modified via esterification with acyl phosphonates. The grafting of an acyl chain onto the inulin backbone under different conditions led to a highly efficient synthesis of a series of inulin esters, with interesting tensioactive properties. The derivatives were evaluated in oil-in-water (O/W) emulsions with isoparaffinic oil, Isopar M. Therefore, a 2% (w/v) aqueous solution of inulin-based surfactant was used in 50/50 O/W emulsions, in nonelectrolyte, and in electrolyte media, using 1 M MgSO4. Longer acyl chains, e.g., dodecanoyl (C12), hexadecanoyl (C16), and octadecanoyl (C18), with degrees of substitution lower than 0.5, gave rise to the highest emulsion stabilities against coalescence.     

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.     

Tosylated and azidated inulins as key substrates for further chemical modifications to access inulin-based advanced materials: an inulin-based glycocluster

We successfully synthesized inulin tosylates by treating commercially available inulin with tosyl chloride and triethylamine in N,N-dimethylacetoamide at the ambient temperature for 24h. The subsequent S(N)2 reactions using sodium azide afford inulin azides that can act as useful substrates for the following Huisgen cycloaddition with alkyne-terminated β-lactoside. The resultant inulin derivative having multiple β-lactosides has excellent affinity towards a β-lactoside binding lectin (RCA(120)). This synthetic strategy has various advantages, such as non-fragmentation of the inulin mainchain and wide applications for various alkyne-terminated functional units. Our strategy can be, therefore, used to develop various inulin derivatives that are applicable for food and medicinal industries.