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Identification of novel specialized metabolites or bioactive compounds represents the main objective in the research field of natural product leads and drug discovery. Mass spectrometry (MS) provides a central tool to expedite and make more efficient the discovery and isolation phases, while minimizing the waste of resources on rediscovery of known compounds. MS contributes acutely to elucidation and identification of numerous species because it allows molecular mass and structural features determination. In particular, identification of the elemental composition of a precursor ion of interest by accurate mass measurement and investigation of dissociative processes undergone by the molecule, represent a worthy methodology to access the structure assignment. The aim of this study was to discover and identify novel antibacterial drugs from microbial source in a jungle of already known compounds. The focus of this paper is on the analytical strategy that permitted the disclosure of a new compound, otherwise confused with other substances. Emphasis is placed on the interpretation of the ESI-MS/MS fragmentation pattern that combined with high resolution mass determination, allowed step by step to properly deduce the exact molecular formula of an unknown component with a molecular weight higher than 1500 Daltons.

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

Natural products (NPs) continue to play an important role for the discovery of new therapeutic candidates. Over the past 30 years, NPs or their derivatives have accounted for 60% of new anticancer agents and almost 75% of all new antibacterial molecules [1-3]. One hundred NP and NP-derived substances were being evaluated in clinical trials or were being registered at the end of 2013 [4]. NPs have been isolated from many terrestrial and marine organisms, including plants, marine invertebrates, and microorganisms, the latter being the source selected more often for pharmaceutical drug discovery programs. Microorganisms (traditionally actinobacteria and fungi, but more recently cyanobacteria and myxobacteria as well) are one of the most prolific sources among living organisms for the production of bioactive molecules. Exploitation of their specialized (commonly termed secondary) metabolism has guaranteed for decades already the discovery of novel antibiotics and other compounds with unprecedented chemical characteristics and biological properties not existing in screening libraries of synthetic compounds [1,5]. Querying the literature, we previously reported [6,7] that among more than 31600 microbial products discovered from 1900 onwards, ca. 20200 possess some biological activity. Among them 35% were produced from filamentous fungi, 48% from actinomycetes, and 17% from other bacteria. According to Berdy [2] ca. 20000 and 22000 bioactive microbial secondary metabolites had been described in the scientific and patent literature by the end of 2000 and 2002, respectively. About 38% of these molecules are produced by filamentous fungi, whereas the largest group (45%) derives from actinomycetes (7600 metabolites from Streptomyces and 2500 from the so-called rare filamentous actinomycetes). The remaining 17% is produced by other bacteria such as Bacillus, Pseudomonas, myxobacteria, and cyanobacteria. During the past 15 years, we have registered the proJOURNAL OF APPLIED BIOANALYSIS, Apr. 2015, p. 55-67. http://dx.doi.org/10.17145/jab.15.010 Vol. 1, No. 2

Extraction and analysis by LC-MS of peptidoglycan precursors represent a valuable method to study antibiotic mode of action and resistance in bacteria. Here, we describe how to apply this method for: (1) testing the action of different classes of antibiotics inhibiting cell wall biosynthesis in Bacillus megaterium; (2) studying the mechanism of self-resistance in mycelial actinomycetes producing glycopeptide antibiotics.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d -alanyl- d -alanine of lipid II with d -alanyl- d -lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 μg/ml, but this increased to ca. 20 μg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d -Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc- l -Ala- d -Glu- meso -Dap- d -Ala and only traces of the pentapeptide UDP-MurNAc- l -Ala- d -Glu- meso -Dap- d -Ala- d -Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d , d -carboxypeptidase activity of vanY . Consistent with this, a vanY -null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae , resulted in higher levels of glycopeptide resistance.

Summary Antibiotics blocking bacterial cell wall assembly (β-lactams and glycopeptides) are facing a challenge from the progressive spread of resistant pathogens. Lantibiotics are promising candidates to alleviate this problem. Microbisporicin, the most potent antibacterial among known comparable lantibiotics, was discovered during a screening applied to uncommon actinomycetes. It is produced by Microbispora sp. as two similarly active and structurally related polypeptides (A1, 2246-Da and A2, 2230-Da) of 24 amino acids linked by 5 intramolecular thioether bridges. Microbisporicin contains two posttranslational modifications that have never been reported previously in lantibiotics: 5-chloro-trypthopan and mono- (in A2) or bis-hydroxylated (in A1) proline. Consistent with screening criteria, microbisporicin selectively blocks peptidoglycan biosynthesis, causing cytoplasmic UDP-linked precursor accumulation. Considering its spectrum of activity and its efficacy in vivo, microbisporicin represents a promising antibiotic to treat emerging infections.

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant toβ-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, namedActinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay againstStaphylococcus aureusand its L-form (also in the presence of aβ-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by anActinoallomurussp. and on a microbisporicin producer not belonging to theMicrobisporagenus.

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the β-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the β-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free β-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.

The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent release of the apoptotic cascade of events. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerization could be used to modulate cell death reinstating apoptosis in cancer cells. An affinity displacement assay based on Bcl-XL/BH3 interaction has been developed. This assay makes use of soluble His-tagged Bcl-XL and fluorescein tagged BH3. Binding is measured as fluorescence associated with magnetic beads. The assay was miniaturized to 96-well microtiter plates and can be employed in high throughput screening (HTS), in addition it is robust enough to be applied to microbial fermentation extracts.

Chitin is a structural polysaccharide present in most fungal cell walls, whose synthesis depends on a family of enzymic activities named chitin synthases (CSs). The specific role of each of them, as well as of their regulatory proteins, in cell morphogenesis and virulence is not well understood. Here, it is shown that most chitin synthesis inCandida albicans, one of the fungi most commonly isolated from opportunistic mycoses and infections, depends onCHS7. Thus,C. albicans chs7Δ null mutants showed reduced levels of chitin and CS activity, and were resistant to Calcofluor. Despite the sequence similarity and functional relationship with ScChs7p, CaChs7p was unable to restore CSIII activity in aSaccharomyces cerevisiae chs7Δ null mutant, because it was unable to direct ScChs3p export from the endoplasmic reticulum.C. albicans chs7Δ null mutants did not show any defect in growth rate, but yeast cells displayed minor morphogenetic defects affecting septum formation, and showed an increased tendency to form filaments. CaChs7p was not required for germ-tube emission, and null mutant strains underwent the dimorphic transition correctly. However, colony morphology appeared distinctively affected.chs7Δ hyphae were very curved and had irregular lateral walls, resulting in very compact colonies that seemed unable to spread out radially on the surface, unlike the wild-type. This growth pattern may be associated with the reduced virulence and high clearance rate observed when thechs7Δ strain was used in a murine model of infection. Therefore, CaChs7p is required for normal hyphal morphogenesis, suggesting that inC. albicansCSIII plays an important role in maintaining cell wall integrity, being essential when invading surrounding tissues.

In this study we describe BI-K0058, a new inhibitor of the transcription-termination factor Rho belonging to a different chemical class from bicyclomycin, the only known antibiotic acting on Rho. BI-K0058 inhibits the poly(C)-dependent ATPase activity of Rho with an IC(50) of 25 microM as well as in vitro transcription-termination of two natural substrates, the Salmonella enterica hisG cistron and the f1 phage intergenic region. BI-K0058 does not affect photolabeling of Rho by ATP. The results of gel mobility shift experiments with a natural RNA substrate demonstrate that BI-K0058 inhibits the formation of the ATP-independent high affinity Rho-RNA complex.

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

A detailed analysis of the secondary liposoluble metabolites of the Verongida sponge Aplysina archery has been performed. Ten bromotyrosine derivatives have been identified of which one, 1 , is a novel compound. Structure of 1 has been assigned on the basis of spectroscopic evidence including 2D-NMR experiments. Absolute configuration has been suggested by helicity rule.

The enzyme properties and location of essential functional groups of solubilized oxidosqualene cyclase of Candida albicans have been studied. We show that the C. albicans enzyme is much more heat-labile compared with Saccharomyces cerevisiae and rat liver cyclases, requires a histidyl residue for enzyme activity, contains an essential thiol residue either close to or in the active site and exhibits a carbocationic mechanism for catalysis, as the enzyme-bound substrate protects the enzyme from inactivation by a site-directed inactivator.

Substituted 2,5-(structure 1) and 2,4-(structure 2) cyclohexadiene-1,3-dicarboxylates were prepared via Birch reductive alkylation of substituted isophthalates with alkylating agents such as alkyl and allyl halides, allkyl dihalides, α-halocarboxylates and epoxides. A brief discussion about their reactivity and stability is presented.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.

The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal d -alanyl- d -alanine of the nascent peptidoglycan with d -alanyl- d -lactate or d -alanyl- d -serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 μg/ml and a MIC of vancomycin of 90 μg/ml during vegetative growth. The vanH , vanA , and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in d -alanyl- d -lactate. No trace of precursors ending in d -alanyl- d -alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 μg/ml), the MIC of teicoplanin remained in the range of 25 to 35 μg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 μg/ml.

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

Compound A40926, produced byActinomadura ATCC 39727, is a lipoglycopeptide antibiotic complex which inhibits Gram-positive bacteria andNeisseria species. Individual components of the complex have an identical glycopeptide core but differ in the acid chains attached to the amino group of the glucuronic moiety. Suspension cultures and resting cells ofActinoplanes teichomyceticus ATCC 31121 were able to deacylate compound A40926 factors to yield the glycopeptide nucleus, which can be then synthetically reacylated to form new analogs. In an optimized fedbatch deacylation process, 0.5 g L−1 of compound A40926 was almost completely converted into the deacyl derivative. Under the same conditions, deacylation was also accomplished withtert-butoxycarbonyl (tert-BOC) A40926, in which the amino group at C15 was blocked to prevent formation of diacyl analogs during reacylation. The deacylase is an endoenzyme whose preliminary characterization is presented.

The abilities of 22,23-epoxy-2-aza-2,3-dihydrosqualene and the corresponding N-oxide, 22,23-epoxy-2-aza-2,3-dihydrosqualene-N-oxide, to inhibit sterol biosynthesis were studied in microsomes and cells of Saccharomyces cerevisiae and Candida albicans. 22,23-Epoxy-2-aza-2,3-dihydrosqualene, which differs from the other inhibitor only in lacking oxygen at position 2, exhibited higher inhibitory properties in all preparations tested. The different levels of effectiveness of the two azasqualene derivatives were evident mostly in microsomes from S. cerevisiae (the 50 inhibitory concentrations of the 2-aza derivative and the corresponding N-oxide on oxidosqualene cyclase were 30 and 120 microM respectively) and in cell cultures of the same strain (1 order of magnitude separated the inhibitory activities of the two compounds on sterol biosynthesis). A possible explanation for the differences between 22,23-epoxy-2-aza-2,3-dihydrosqualene and the corresponding N-oxide arose from the study of their metabolic fates in vivo and in vitro. While the 2-aza derivative did not undergo any transformation, the N-oxide compound was actively reduced to the corresponding amine in microsomes and in cells of both yeast strains. 22,23-Epoxy-2-aza-2,3-dihydrosqualene-N-oxide seems to behave as a proinhibitor of sterol biosynthesis, becoming active only after transformation into the active form 22,23-epoxy-2-aza-2,3-dihydrosqualene.

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion with Staphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.

A series of 2,4- and 2,5-cyclohexadiene-1,3-dicarboxylates were functionalized at the allylic position via oxidation (SeO2, PDC/t-BuOOH) and halogenation (NBS). The regiochemical outcome for different substrates and reactions was studied and the importance of factors such as reaction mechanism, steric hindrance and reaction intermediates stability was discussed. §Present address: GlaxoWellcomc Medicines Research Centre, Via Fleming 4, Verona, Italy $Present Address: Hoechst Marion Roussel, Paris, France

A protein inhibitor which is active against both exogenous and endogenous trypsin has been purified from wheat endosperm. The inhibitor is a single-chain protein of M r 8500 and isoelectric point 8.7. It contains 10 half-cystine residues per mol, all of them linked in disulphide bridges. The N -terminal sequence, 25 residues long, has been determined. The inhibitor is very active against bovine, porcine, cod and wheat trypsin, and inactive against animal chymotrypsin and bacterial subtilisin. The inhibitor binds bovine trypsin with a 1:1 molar stoichiometry.

Two trypsin inhibitors and a number of amylase inhibitors have been purified from wheat flour by a chromatographic procedure. A new putative member of the family of the amylase monomeric isoinhibitors has been isolated. This protein, coded as 0.14, has M r 13 000, 3 disulphide bridges, an amino acid composition comparable to those of other monomeric inhibitors, but it is inactive against animal amylases. Protein 0.14 sequencing by classical procedures did not succeed, thus suggesting that the terminal α-amino group is modified; such modification could be responsible of the inability of protein 0.14 to act as an amylase inhibitor.

Substituted 2,5-(structure 1) and 2,4-(structure 2) cyclohexadiene-1,3-dicarboxylates were prepared via Birch reductive alkylation of substituted isophthalates with alkylating agents such as alkyl and allyl halides, allkyl dihalides, α-halocarboxylates and epoxides. A brief discussion about their reactivity and stability is presented.