A Dynamic Membrane-Active Glycopeptide for Enhanced Protection of Human Red Blood Cells against Freeze-Stress.

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical α-helical/ß-sheet structure-driven regulations of ice crystals during freeze-thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Genetic and transcriptomic evidences suggest ARO10 genes are involved in benzenoid biosynthesis by yeast.

Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.

Biosynthesis of Benzylic Derivatives in the Fermentation Broth of the Edible Mushroom, Ischnoderma resinosum.

Employing isotope incubation studies, the biosynthetic pathway leading to a series of benzylic derivatives was elucidated in the fermentation broth of the edible mushroom Ischnoderma resinosum (P. Karst). Twenty-six hydroxy- and methoxy- benzylic derivatives were screened by gas chromatography-mass spectrometry (GC-MS) of which 13 were detected in the culture media. Results from the isotope incubation studies showed the transformation of both benzyl alcohol and benzoic acid into benzaldehyde. Benzaldehyde was then converted into 4-methoxybenzaldehyde via hydroxylation and subsequent methylation of the 4-C position. The resulting 4-methoxybenzaldehyde was then hydroxylated in the 3-C position followed by methylation into 3,4-dimethoxybenzaldehyde. Based on these findings, a novel metabolic scheme for the biosynthesis of benzylic derivatives in I. resinosum was proposed. The knowledge of the biosynthetic pathway was utilized to produce 4-hydroxy-3-methoxybenzaldehyde (vanillin) from 4-hydroxy-3-methoxybenzoic acid (vanillic acid). This is the first report to elucidate the biosynthetic pathway of benzyl derivatives and production of vanillin from I. resinosum.

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation.

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86-99 % ee and 130-1020 total turnover numbers.

Haloquadratum walsbyi Yields a Versatile, NAD+/NADP+ Dual Affinity, Thermostable, Alcohol Dehydrogenase (HwADH).

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.

Functional Synthetic Model for the Lanthanide-Dependent Quinoid Alcohol Dehydrogenase Active Site.

The oxidation of methanol by dehydrogenase enzymes is an essential part of the bacterial methane metabolism cycle. The recent discovery of a lanthanide (Ln) cation in the active site of the XoxF dehydrogenase represents the only example of a rare-earth element in a physiological role. Herein, we report the first synthetic, functional model of Ln-dependent dehydrogenase and its stoichiometric and catalytic dehydrogenation of a benzyl alcohol. Density functional theory calculations implicate a hydride transfer mechanism for these reactions.

Last-Step Pd-Mediated [11C]CO Labeling of a Moxestrol-Conjugated o-Iodobenzyl Alcohol: From Model Experiments to in Vivo Positron Emission Tomography Studies.

The fast, efficient, and functional group tolerant last-step radiolabeling of bioconjugates is crucial for positron emission tomography (PET) applications. In this context, o-iodobenzyl alcohol based structures were identified as ideal tags for an easy Pd-catalyzed carbonylation after bioconjugation, and a moxestrol-conjugated precursor was chosen as the model compound for the further studies. Despite scale and time constraints, conditions developed with [12C]CO and [13C]CO were easily transferred to the 11C isotope, and the desired radioactive product was obtained in amounts up to 740 MBq with radiochemical purities higher than 99%. Radio-high-performance liquid chromatography analyses of rat blood samples demonstrated excellent in vivo stability within the time of the acquisition. MicroPET-magnetic resonance imaging showed excretion pathways similar to moxestrol, and molecular modeling was also performed to evaluate the potential ability of this conjugate to bind estrogen receptors α. Thus, being both synthetically and biologically suitable, this strategy clears the path to potential novel biotracers for preclinical PET imaging.

A new approach to synthesis of benzyl cinnamate: Optimization by response surface methodology.

In this work, the new approach to synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid with benzyl alcohol is optimized by response surface methodology. The effects of various reaction conditions, including temperature, enzyme loading, substrate molar ratio of benzyl alcohol to cinnamic acid, and reaction time, are investigated. A 5-level-4-factor central composite design is employed to search for the optimal yield of benzyl cinnamate. A quadratic polynomial regression model is used to analyze the experimental data at a 95% confidence level (P<0.05). The coefficient of determination of this model is found to be 0.9851. Three sets of optimum reaction conditions are established, and the verified experimental trials are performed for validating the optimum points. Under the optimum conditions (40°C, 31mg/mL enzyme loading, 2.6:1 molar ratio, 27h), the yield reaches 97.7%, which provides an efficient processes for industrial production of benzyl cinnamate.

Pharmacokinetics in Elderly Women of Benzyl Alcohol From an Oil Depot.

Pharmaceutical oil depots are meant to release active substances at a sustained rate. Most of these depots contain benzyl alcohol (BOH) to facilitate the production and administration. Because BOH changes the solubility of components in both the body fluid and the oil formulation, it is relevant to know the change in the BOH concentration in the oil over time. In this study, volunteers were subcutaneously injected with an oil depot that contained 10% BOH, nandrolone decanoate, and cholecalciferol. The aim of this study was to determine the pharmacokinetic profiles of BOH and its metabolites benzoic acid and hippuric acid simultaneously in serum to estimate the BOH release out of the depot. For this, an HPLC bioassay was developed and adequately validated. Hereafter, the bioassay was applied to serum samples obtained at several time points between 0 and 35 days. BOH appeared immediately in serum after injection. The pharmacokinetic profile revealed that all BOH was depleted from the depot within 52 h after injection. Thus, the partition coefficient of active substances between the oil formulation and the body tissue changes rapidly in the first days after injection but will remain constant hereafter.

Effect of yeast assimilable nitrogen on the synthesis of phenolic aroma compounds by Hanseniaspora vineae strains.

In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid-related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non-Saccharomyces yeast strains have a well-recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape-derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate and 2-phenylethyl acetate are significantly synthesized by H. vineae strains. Levels of these compounds found in fermentations with 11 H. vineae different strains were one or two orders of magnitude higher than those measured in fermentations with a known S. cerevisiae wine strain. The implications for winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate and 2-phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of sucrose and benzyl alcohol on GCSF conformational dynamics revealed by hydrogen deuterium exchange mass spectrometry.

Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations. In this study, we examined the roles of the excipients, sucrose and benzyl alcohol, on the conformational dynamics of recombinant human granulocyte colony stimulating factor using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS). Under physiological pH and temperature, sucrose globally protects the whole molecule from deuterium uptake, whereas benzyl alcohol induces increased deuterium uptake of the regions within the α-helical bundle, with even larger extent. The HDX experiments described were incorporated a set of internal peptides (Zhang et al., 2012. Anal Chem 84:4942-4949) to monitor the differences in intrinsic exchange rates in different formulations. In addition, we discussed the feasibility of implementing HDX-MS with these peptide probes in protein formulation development.

Pumping iron to keep fit: modulation of siderophore secretion helps efficient aromatic utilization in Pseudomonas putida KT2440.

Studies of biotechnology applications of Pseudomonas putida KT2440 have been predominantly focused on regulation and expression of the toluene degradation (TOL) pathway. Unfortunately, there is limited information on the role of other physiological factors influencing aromatic utilization. In this report, we demonstrate that P. putida KT2440 increases its siderophore secretion in response to the availability of benzyl alcohol, a model aromatic substrate. It is argued that accelerated siderophore secretion in response to aromatic substrates provides an iron 'boost' which is required for the effective functioning of the iron-dependent oxygenases responsible for ring opening. Direct evidence for the cardinal role of siderophores in aromatic utilization is provided by evaluation of per capita siderophore secretion and comparative growth assessments of wild-type and siderophore-negative mutant strains grown on an alternative carbon source. Accelerated siderophore secretion can be viewed as a compensatory mechanism in P. putida in the context of its inability to secrete more than one type of siderophore (pyoverdine) or to utilize heterologous siderophores. Stimulated siderophore secretion might be a key factor in successful integration and proliferation of this organism as a bio-augmentation agent for aromatic degradation. It not only facilitates efficient aromatic utilization, but also provides better opportunities for iron assimilation amongst diverse microbial communities, thereby ensuring better survival and proliferation.

Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,ß-unsaturated alcohols from allylic aldehydes and ketones.

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,ß-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

Stereospecific multiple isotopic labeling of benzyl alcohol.

Isotopically labeled enzymatic substrates and biological metabolites are useful for many mechanistic analyses, particularly the study of kinetic and equilibrium isotope effects, determining the stereospecificity of enzymes, and resolving metabolic pathways. Here, we present the one-pot synthesis, purification, and kinetic analysis of 7R-[(2) H]-phenyl-[(14) C]-benzyl alcohol. The procedure involves a chemoenzymatic synthesis that couples formate dehydrogenase to alcohol dehydrogenase with a catalytic amount of nicotinamide cofactor. The reaction goes to completion overnight, and the measurement of a competitive kinetic isotope effect on the enzymatic oxidation of the purified product identified no (1) H contamination. This measurement is very sensitive to such isotopic contamination and verified the high level of isotopic and enantiomeric purity yielded by the new synthetic procedure.

Dehydration and clearing of adult Drosophila for ultramicroscopy.

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Dehydration and clearing of whole mouse brains and dissected hippocampi for ultramicroscopy.

This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, mouse brains and hippocampi are carefully dissected and dehydrated, and then cleared in a solution of benzyl benzoate and benzyl alcohol.

Janus PEG-based dendrimers for use in combination therapy: controlled multi-drug loading and sequential release.

The increasing use of drug combinations to treat disease states, such as cancer, calls for improved delivery systems that are able to deliver multiple agents. Herein, we report a series of novel Janus dendrimers with potential for use in combination therapy. Different generations (first and second) of PEG-based dendrons containing two different "model drugs", benzyl alcohol (BA) and 3-phenylpropionic acid (PPA), were synthesized. BA and PPA were attached via two different linkers (carbonate and ester, respectively) to promote differential drug release. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of BA and PPA, namely, (BA)(2)-G1-G1-(PPA)(2), (BA)(4)-G2-G1-(PPA)(2), (BA)(2)-G1-G2-(PPA)(4), and (BA)(4)-G2-G2-(PPA)(4). Release studies in plasma showed that the dendrimers provided sequential release of the two model drugs, with BA being released faster than PPA from all of the dendrons. The different dendrimers allowed delivery of increasing amounts (0.15-0.30 mM) and in exact molecular ratios (1:2; 2:1; 1:2; 2:2) of the two model drug compounds. The dendrimers were noncytotoxic (100% viability at 1 mg/mL) toward human umbilical vein endothelial cells (HUVEC) and nontoxic toward red blood cells, as confirmed by hemolysis studies. These studies demonstrate that these Janus PEG-based dendrimers offer great potential for the delivery of drugs via combination therapy.

Effect of benzyl alcohol on recombinant human interleukin-1 receptor antagonist structure and hydrogen-deuterium exchange.

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90-97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol.

Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86.

Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD(+) dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD(+), while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K (m) value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K (m) and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.

Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit.

The volatile compounds that constitute the fruit aroma of ripe tomato (Solanum lycopersicum) are often sequestered in glycosylated form. A homology-based screen was used to identify the gene SlUGT5, which is a member of UDP-glycosyltransferase 72 family and shows specificity towards a range of substrates, including flavonoid, flavanols, hydroquinone, xenobiotics and chlorinated pollutants. SlUGT5 was shown to be expressed primarily in ripening fruit and flowers, and mapped to chromosome I in a region containing a QTL that affected the content of guaiacol and eugenol in tomato crosses. Recombinant SlUGT5 protein demonstrated significant activity towards guaiacol and eugenol, as well as benzyl alcohol and methyl salicylate; however, the highest in vitro activity and affinity was shown for hydroquinone and salicyl alcohol. NMR analysis identified isosalicin as the only product of salicyl alcohol glycosylation. Protein modelling and substrate docking analysis were used to assess the basis for the substrate specificity of SlUGT5. The analysis correctly predicted the interactions with SlUGT5 substrates, and also indicated that increased hydrogen bonding, due to the presence of a second hydrophilic group in methyl salicylate, guaiacol and hydroquinone, appeared to more favourably anchor these acceptors within the glycosylation site, leading to increased stability, higher activities and higher substrate affinities.