Biological activity of 3-(2-benzoxazol-5-yl)alanine derivatives.

Searching for new drugs is still a challenge for science, mainly because of civilization development and globalization which promote the rapid spread of diseases, which is particularly dangerous in the case of infectious ones. Moreover, readily available already known antibiotics are often overused or misused, possibly contributing to the increase in the number of multidrug-resistant microorganisms. A consequence of this is the need for new structures of potential drugs. One of them is a benzoxazole moiety, a basic skeleton of a group of fluorescent heterocyclic compounds already widely used in chemistry, industry, and medicine, which is also present in naturally occurring biologically active compounds. Moreover, synthetic benzoxazoles are also biologically active. Considering all of that, a large group of non-proteinogenic amino acids based on 3-(2-benzoxazol-5-yl)alanine skeleton was studied in search for new antimicrobial and anticancer agents. Screening tests revealed that antibacterial potential of 41 compounds studied is not very high; however, they are selective acting only against Gram-positive bacteria (B. subtilis). Moreover, almost half of the studied compounds have antifungal properties, also against pathogens (C. albicans). Most of studied compounds are toxic to both normal and cancer cells. However, in a few cases, toxicity to normal cells is much lower than for cancer cells indicating these compounds as future anticancer agents. The research carried out on such a large group of compounds allowed to establish a structure-activity relationship which enables to select candidates for further modifications, necessary to improve their biological activity and obtain a new lead structure with potential for therapeutic use.

Fluorescent analogs of trypsin inhibitor SFTI-1 isolated from sunflower seeds--synthesis and applications.

This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-L-alanine-[(4NPh2 )Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-L-alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe(5)]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects.

Unexpected photoproduct generated via the acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine in a water/isopropanol solution: experimental and computational studies.

The acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine (5-BrdU) in a water/isopropanol solution with 300 nm photons leads to the formation of 2'-deoxyuridine (dU) and a comparable amount of another photoproduct that has not been reported in the literature so far. The negative and positive mass spectra recorded for this species indicate that they originate from the molecular mass of 286 Da, which corresponds to an adduct of 2'-deoxyuridine and 2-propanol. Quantum chemical calculations carried out at the DFT and TDDFT levels reveal both the structure and the UV spectrum of that adduct. The latter computational characteristic matches well the experimental UV spectrum of the new photoproduct. Our findings indicate that the acetone-sensitized photolysis of 5-BrdU is more complicated than has hitherto been assumed. Nevertheless, since electron transfer is one of the pathways responsible for 5-BrdU decay, acetone-sensitized photolysis of the halogen derivatives of nucleobases could be a convenient tool for studying their radiosensitivity in aqueous solutions.

Highly specific substrates of proteinase 3 containing 3-(2-benzoxazol-5-yl)-l-alanine and their application for detection of this enzyme in human serum.

A set of benzoxazolyl-l-alanine derivates along with the MCA moiety (donors of fluorescence) were introduced into a proteinase 3 (PR3) substrate with a C-terminal ANB-NH(2) that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-NH(2) were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest k(cat)/K(M) value, 1.5 x 10(6) M(-1) s(-1), was obtained for (Pyr)Box-Ala-Tyr-Tyr-Abu-NH(2) where (Pyr)Box-Ala stands for N-methylpyrrole benzoxazole-l-alanine. Titration of this peptide with proteinase 3 resulted in measurable fluorescence at an enzyme amount equal to 29 pmol. This substrate was selected used to detect quantifiable levels of proteinase 3 in serum samples, including those of normal subjects. For all c-ANCA-positive samples (diagnosed Wegener granulomatosis), a significant increase of PR3 concentration was observed. Wegener granulomatosis is a severe autoimmune disease causing inflammation of the blood vessels. Our results clearly show that this substrate can be used for the construction of a very reliable, inexpensive, and easy to use diagnostic test for PR3 determination.

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region.

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH(2), where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k(cat)/K(M) = 275,000 M(-1) s(-1)), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.

Design of selective substrates of proteinase 3 using combinatorial chemistry methods.

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates' sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X3-X2-X1-ANB-NH2 yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH2 displayed the highest value of specificity constant (k(cat)/K(M)=189 x 10(3) M(-1) s(-1)) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods.

In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.