Synthesis and Evaluation of Novel Iminosugars Prepared from Natural Amino Acids.

Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,ß-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.

Targeted proteomic response to coffee consumption.

PURPOSE: Coffee is widely consumed and implicated in numerous health outcomes but the mechanisms by which coffee contributes to health is unclear. The purpose of this study was to test the effect of coffee drinking on candidate proteins involved in cardiovascular, immuno-oncological and neurological pathways. METHODS: We examined fasting serum samples collected from a previously reported single blinded, three-stage clinical trial. Forty-seven habitual coffee consumers refrained from drinking coffee for 1 month, consumed 4 cups of coffee/day in the second month and 8 cups/day in the third month. Samples collected after each coffee stage were analyzed using three multiplex proximity extension assays that, after quality control, measured a total of 247 proteins implicated in cardiovascular, immuno-oncological and neurological pathways and of which 59 were previously linked to coffee exposure. Repeated measures ANOVA was used to test the relationship between coffee treatment and each protein. RESULTS: Two neurology-related proteins including carboxypeptidase M (CPM) and neutral ceramidase (N-CDase or ASAH2), significantly increased after coffee intake (P < 0.05 and Q < 0.05). An additional 46 proteins were nominally associated with coffee intake (P < 0.05 and Q > 0.05); 9, 8 and 29 of these proteins related to cardiovascular, immuno-oncological and neurological pathways, respectively, and the levels of 41 increased with coffee intake. CONCLUSIONS: CPM and N-CDase levels increased in response to coffee intake. These proteins have not previously been linked to coffee and are thus novel markers of coffee response worthy of further study. CLINICAL TRIAL REGISTRY: http://www.isrctn.com/ISRCTN12547806.

Synthesis and glycosidase inhibition of conformationally locked DNJ and DMJ derivatives exploiting a 2-oxo-C-allyl iminosugar.

A series of analogs of the iminosugars 1-deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (DMJ), in which an extra five or six-membered ring has been fused to the C1-C2 bond have been prepared. The synthetic strategy exploits a key 2-keto-C-allyl iminosugar, easily accessible from gluconolactam, which upon Grignard addition and RCM furnishes a bicyclic scaffold that can be further hydroxylated at the C[double bond, length as m-dash]C bond. This strategy furnished DNJ mimics with the piperidine ring locked in a 1C4 conformation with all substituents in axial orientation when fused to a six-membered ring. Addition of an extra ring to DNJ and DMJ motif proved to strongly modify the glycosidase inhibition profile of the parent iminosugars leading to modest inhibitors. The 2-keto-C-allyl iminosugar scaffold was further used to access N-acetylglycosamine analogs via oxime formation.

Synthesis of tricyclic quinazolinone-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction.

A series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were synthesized from the benzyl protected sugars through three steps. Firstly, the benzyl protected sugar (aldehyde) 5 reacted with o-aminobenzamide by the iodine-induced oxidative condensation to afford the corresponding aldo-quizanolinone 6. Secondly, through the intramolecular cyclization of the unprotected OH and the amide NH in 6, the tricyclic compounds 7 and 8 were constructed by the key Mitsunobu reaction. Finally, removal of the benzyl group gave the target tricyclic quinazolinone-iminosugars 1. The protocol was effective for the preparation of the tricyclic iminosugars in satisfactory yield. Interestingly, an unusual C-2 epimerization was observed with d-mannose and d-ribose compounds under the conditions of the Mitsunobu reaction that generated the products having the trans configuration at the C-2 and C-3 positions. Unfortunately, such tricyclic quinazolinone-iminosugars showed no inhibitory effects on the tested five glycosidases.

Screening and Molecular Identification of Pectinase Producing Microbes from Coffee Pulp.

Application of enzymes in biotechnological process has expanded considerably in recent years. In food and related industry, major importance was being attached to the use of enzymes in upgrading quality, increasing yields of extractive processes, product stabilization, and improvement of flavor and byproduct utilization. Pectinases or pectinolytic enzymes are today one of the upcoming enzymes of the commercial sector. It has been reported that microbial pectinases account for 25% of the global food enzymes sales. For this reason, this study was undertaken with aims of screening microorganisms for the pectinase activity from coffee pulp samples and molecular identification of the potential pectinolytic isolates. In the present investigation, in total, ninety-five (95) isolates were identified from thirty coffee pulp samples. Based on characterization on the selective growth media, the isolates were grouped as actinomycete (21.06%), bacteria (65.26%), and fungi (13.68%). Among these, 31.58% showed colonies surrounded by clear zones which indicate the presence of pectinase activity. After rigorous screening steps, the isolates with high potential pectinase activity were identified molecularly by sequencing 16S rDNA region of the isolates. Based on the molecular identifications, about 70% of the isolates are under genus Bacillus.

Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination.

BACKGROUND: Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. RESULTS: Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. CONCLUSIONS: Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination.

BACKGROUND: Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. RESULTS: A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (ß/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 µg/µL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. CONCLUSIONS: Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Fluorous iminoalditols: a new family of glycosidase inhibitors and pharmacological chaperones.

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D-galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D-galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.

Photosynthesis and photoprotection in coffee leaves is affected by nitrogen and light availabilities in winter conditions.

Coffee is native to shady environments but often grows better and produces higher yields without shade, though at the expense of high fertilization inputs, particularly nitrogen (N). Potted plants were grown under full sunlight and shade (50%) conditions and were fertilized with nutrient solutions containing either 0 or 23 mM N. Measurements were made in southeastern Brazil during winter conditions, when relatively low night temperatures and high diurnal insolation are common. Overall, the net carbon assimilation rate was quite low, which was associated with diffusive, rather than biochemical, constraints. N deficiency led to decreases in the concentrations of chlorophylls (Chl) and total carotenoids as well as in the Chl/N ratio. These conditions also led to qualitative changes in the carotenoid composition, e.g., increased antheraxanthin (A) and zeaxanthin (Z) pools on a Chl basis, particularly at high light, which was linked to increased thermal dissipation of absorbed light. The variable-to-maximum fluorescence ratio at predawn decreased with increasing A+Z pools and decreased linearly with decreasing N. We showed that this ratio was inadequate for assessing photoinhibition under N limitation. Expressed per unit mass, the activities of superoxide dismutase and glutathione reductase were not altered with the treatments. In contrast, ascorbate peroxidase activity was lower in low N plants, particularly under shade, whereas catalase activity was lower in shaded plants than in sun-grown plants, regardless of the N level. Glutamine synthetase activity was greater in sun-grown plants than in shaded individuals at a given N level and decreased with decreasing N application. Our results suggest that the photoprotective and antioxidant capacity per amount of photons absorbed was up-regulated by a low N supply; nevertheless, this capacity, regardless of the light conditions, was not enough to prevent oxidative damage, as judged from the increases in the H(2)O(2) and malondialdehyde concentrations and electrolyte leakage. We demonstrated that N fertilization could adequately protect the coffee plants against photodamage independently of the anticipated positive effects of N on the photosynthetic capacity.

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves.

A cDNA clone (designated CaIRL) encoding an isoflavone reductase-like protein from coffee (Coffea arabica) was retrieved during a search for genes showing organ/tissue-specific expression among the expressed sequence tags (EST) of the Brazilian coffee EST database. The CaIRL cDNA contains a single open reading frame of 946 nucleotides (nt) encoding 314 amino acids (predicted molecular weight of 34 kDa). Several features identified the predicted CaIRL protein as a new member of the PIP family of NADPH-dependent reductases. Expression studies demonstrated that CaIRL is expressed exclusively in coffee leaves and its transcript level is markedly increased in response to fungal infection and mechanical injury. Analysis of transgenic tobacco plants harboring a CaIRL 5'-flanking region (862 nt) fused to uidA reporter gene (GUS) confirmed the responsiveness of the putative promoter to abiotic stress in wounded leaves. In turn, a 5' deletion to -404 completely abolished promoter activation by abiotic stimulus in transgenic plants. The lack of GUS expression in non-wounded leaf tissues in transgenic tobacco was in contrast to the basal level of CaIRL expression observed in non-stressed healthy coffee leaves.

Characterization and expression analysis of genes directing galactomannan synthesis in coffee.

BACKGROUND AND AIMS: Galactomannans act as storage reserves for the seeds in some plants, such as guar (Cyamopsis tetragonoloba) and coffee (Coffea arabica and Coffea canephora). In coffee, the galactomannans can represent up to 25 % of the mass of the mature green coffee grain, and they exert a significant influence on the production of different types of coffee products. The objective of the current work was to isolate and characterize cDNA encoding proteins responsible for galactomannan synthesis in coffee and to study the expression of the corresponding transcripts in the developing coffee grain from C. arabica and C. canephora, which potentially exhibit slight galactomannan variations. Comparative gene expression analysis was also carried out for several other tissues of C. arabica and C. canephora. METHODS: cDNA banks, RACE-PCR and genome walking were used to generate full-length cDNA for two putative coffee mannan synthases (ManS) and two galactomannan galactosyl transferases (GMGT). Gene-specific probe-primer sets were then generated and used to carry out comparative expression analysis of the corresponding genes in different coffee tissues using quantitative RT-PCR. KEY RESULTS: Two of the putative galactomannan biosynthetic genes, ManS1 and GMGT1, were demonstrated to have very high expression in the developing coffee grain of both Coffea species during endosperm development, consistent with our proposal that these two genes are responsible for the production of the majority of the galactomannans found in the grain. In contrast, the expression data presented indicates that the ManS2 gene product is probably involved in the synthesis of the galactomannans found in green tissue. CONCLUSIONS: The identification of genes implicated in galactomannan synthesis in coffee are presented. The data obtained will enable more detailed studies on the biosynthesis of this important component of coffee grain and contribute to a better understanding of some functional differences between grain from C. arabica and C. canephora.

B to O erythrocyte conversion by the recombinant alpha-galactosidase.

BACKGROUND: Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. METHODS: alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O. RESULTS: The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. CONCLUSION: ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

A potent bicyclic inhibitor of a family 27 alpha-galactosidase.

Two isomeric bicyclo[4.1.0]heptane analogues of the glycosidase inhibitor galacto-validamine, (1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptane-2,3,4-triol, have been synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-D-galactose. The inhibitory activities of the two conformationally restricted amines, and their corresponding acetamides, were measured against commercial alpha-galactosidase enzymes from coffee bean and E. coli. The activity of the glycosyl hydrolase family GH27 enzyme (coffee bean) was competitively inhibited by the 1R,6S-amine (7), a binding interaction that was characterized by a K(i) value of 0.541 microM. The GH36 E. coli alpha-galactosidase exhibited a much weaker binding interaction with the 1R,6S-amine (IC(50)= 80 microM). The diastereomeric 1S,6R-amine (9) bound weakly to both galactosidases, (coffee bean, IC(50)= 286 microM) and (E. coli, IC(50)= 2.46 mM).

A simple method to measure effective catalase activities: optimization, validation, and application in green coffee.

Oxidative metabolism in coffee cherries during maturation appears to be regulated by the timely expression of redox enzymes such as catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO). Among these enzymes, CAT is suspected to contribute significantly in setting the redox status of the healthy cherry and the processed bean. The initial redox status of the green bean might further control the nature and dynamics of reactions induced by roasting and eventually quality aspects of the end product. In this respect, Arabica (Coffea arabica) and Robusta (Coffea canephora) typically differ by their cup coffee flavor profiles. We developed an assay that allowed us to screen numerous green coffee samples for effective CAT activities. The proposed assay, which monitors CAT activities by online oxygen sensing in green coffee crude suspensions incubated with H2O2, seeks to integrate potential effects of endogenous inhibitors and activators. After optimization and validation of the assay, 23 Arabicas, 23 Robustas, and 8 Arabustas were analyzed. Nearly all Arabicas (22 of 23) harbored high CAT activity levels, whereas all Robustas harbored low ones. Arabustas performed like Arabicas of the lower CAT activity range. The traditional spectrophotometric assay did not reveal these specificities. Because of its simplicity, our assay might be valuable for assessing effective CAT activities in various plant tissues.

Phenol contents, oxidase activities, and the resistance of coffee to the leaf miner Leucoptera coffeella.

We examined the role of phenolic compounds, and the enzymes peroxidase and polyphenol oxidase, in the expression of resistance of coffee plants to Leucoptera coffeella (Lepidoptera: Lyonetiidae). The concentrations of total soluble phenols and chlorogenic acid (5-caffeoylquinic acid), and the activities of the oxidative enzymes peroxidase (POD) and polyphenol oxidase (PPO), were estimated in leaves of Coffea arabica, C. racemosa, and progenies of crosses between these species, which have different levels of resistance, before and after attack by this insect. The results indicate that phenols do not play a central role in resistance to the coffee leaf miner. Differences were detected between the parental species in terms of total soluble phenol concentrations and activities of the oxidative enzymes. However, resistant and susceptible hybrid plants did not differ in any of these characteristics. Significant induction of chlorogenic acid and PPO was only found in C. racemosa, the parental donator of the resistance genes against L. coffeella. High-performance liquid chromatography (HPLC) analysis also showed qualitative similarity between hybrids and the susceptible C. arabica. These results suggest that the phenolic content and activities of POD and PPO in response to the attack by the leaf miner may not be a strong evidence of their participation in direct defensive mechanisms.

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust.

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.

Biochemical and molecular characterization of alpha-D-galactosidase from coffee beans.

Alpha-D-Galactosidase (alpha-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wall galactomannans. In the present paper it is shown that alpha-galactosidase activities in field-grown coffee beans are variable amongst cultivars of the two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans from greenhouse-cultivated C. arabica as a model, we showed that alpha-Gal activity was undetectable in the bean perispem tissue, but increased gradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the hardening of the endosperm. Alpha-Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of alpha-Gal activity, but were reduced to be undetectable in mature beans at 30 WAF, while alpha-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms accumulated in a linear fashion throughout grain maturation. Alpha-Gal activity was also observed to increase to high levels during in vitro germination of coffee beans suggesting an important function of this enzyme in this process. Alpha-Gal cDNA sequences from Arabica and Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis suggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in 2-DE profiles.

Design and synthesis of glycosidase inhibitor 5-amino-1,2,3,4-cyclohexanetetrol derivatives from (-)-vibo-quercitol.

In continuation of development of bioactive inositol derivatives, a 1-O-methyl derivative of 5-amino-5-deoxy-L-talo-quercitol was designed and synthesized as an analogue of the strong alpha-fucosidase inhibitor, 5a-carba-alpha-L-fucopyranosylamine, the methyl branch being replaced with methoxyl, and demonstrated to be a moderate alpha-fucosidase inhibitor. The present approach provides a possible route to apply alkyl ethers of aminodeoxyinositols as hexopyranose mimics of biological interest.

[Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase].

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.

Plant biochemistry: a naturally decaffeinated arabica coffee.

The adverse side effects of caffeine have increased the market for decaffeinated coffee to about 10% of coffee consumption worldwide (http://www.ncausa.org), despite the loss of key flavour compounds in the industrial decaffeinating process. We have discovered a naturally decaffeinated Coffea arabica plant from Ethiopia, a species normally recognized for the high quality of its beans. It should be possible to transfer this trait to commercial varieties of arabica coffee plants by intraspecific hybridization--a process likely to be simpler than an interspecific hybridization strategy, which could require more than 30 years of breeding to fix the decaffeinated trait and would probably result in an inferior cup of coffee.