Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.
The 6-deoxyerythronolide B hydroxylase (EryF) is a soluble cytochrome P450 responsible for the stereospecific C-6 hydroxylation of the erythromycin precursor, 6-deoxyerythronolide B. Using the expression of the eryF gene in Escherichia coli [Andersen, J. F., & Hutchinson, C. R. (1992) J. Bacteriol. 174, 725-735] as the enzyme source, we examined the catalytic activity of the EryF protein toward several macrolide substrates related to 6-deoxyerythronolide B. The results of these studies were compared with measurements of the apparent dissociation constants for various substrates and with information from molecular modeling studies of the substrates and the enzyme-substrate complex. Only minor changes in the structure of 6-deoxyerythronolide B resulted in substrates with catalytic rates less than 1% of those seen with the natural substrate. Although the 9S epimer of 9-deoxo-9-hydroxy-6-deoxyerythronolide B was hydroxylated at a rate approximately equal to the natural substrate, the 9R epimer was hydroxylated at a 2-fold lower rate. Examination of molecular models revealed that the position of the 9-hydroxyl oxygen in the 9S epimer resembles that of the 9-oxo oxygen in the natural substrate more closely than in the 9R epimer. 8,8a-Deoxyoleandolide, which is identical to 6-deoxyerythronolide B except for the presence of a C-13 methyl group, and its (9S)-9-deoxo-9-hydroxy derivative were C-6 hydroxylated at a 4-fold lower rate than the natural substrate, and the 9-oxo form showed a substantially larger apparent dissociation constant.(ABSTRACT TRUNCATED AT 250 WORDS)
A new, simple, and reproducible method is described for the determination of selenium(IV) based on differential pulse cathodic stripping voltammetry. The optimized experimental conditions are as follows: selenium(IV) ions in an acidic medium (0.06 M HCl-0.07 M HNO(3)) are electrodeposited on a rotating silver disk electrode as silver selenide at -0.4 V vs SCE for 30 min; the deposit is then cathodically stripped in another solution (2 M NaOH) at a scan rate of 50 mV s(-1) to -1.2 V vs SCE. The cathodic stripping results in only a single well-defined peak at about -0.95 V vs SCE. The calibration (peak height vs selenium concentration) graph is linear up to at least 40 ng mL(-1) of selenium(IV) and passes through the origin, with a relative standard deviation of 2.7% for 20 ng mL(-1) (n = 5). The detection limit (3σ) is 0.20 ng mL(-1). The possible interferences have been evaluated. Dissolved oxygen does not affect the peak height of selenium. The electrode can be used repeatedly at least 20 times with excellent reproducibility without further polishing. The proposed method is an improvement over the existing cathodic stripping techniques.
To investigate the role of the 2'-hydroxy group at the C-13 side chain of docetaxel in the antitumor activity, we prepared several 2',2'-difluoro derivatives of docetaxel and evaluated their cytotoxicity against mouse leukemia and human tumor cell lines and their microtubule disassembly-inhibitory activity. These analogues were prepared by esterification of protected 10-deacetylbaccatin III (21) with appropriate alpha, alpha-difluorinated carboxylic acids (Charts 1 and 2). Among these 2',2'-difluorodocetaxel derivatives, 2',2'-difluorodocetaxel (23b) was approximately 3-10 times as active as 2'-fluorodocetaxel (29a) in terms of cytotoxicity. In addition, the 3'-(2-furyl) (23h) and 3'-(2-pyrrolyl) (23p) analogues showed activity comparable or superior to that of docetaxel (2).
The oligomycin B spiroketal portion, [2S,2(2R),3S,6R,8S,8(3R),9S,10R,11S]-2-[2-(t-butyldiphenylsilyloxy)propyl]-8-[3-(hydroxymethyl)pentyl]-3,9,11-trimethyl-1,7-dioxaspiro[5.5]undecane-5,10-diol (2), and polypropionate portion, ethyl (2E,4S,5R,6R,7S,8S,9R,10S,12R,13S,14R,16E)-5-(t-butyldimethylsilyloxy)7,9-(isopropylidenedioxy)-12,13-(4-methoxybenzylidenedioxy)-4,6,8,10,12,14-hexamethyl-11-oxo-18-phenylsulfonyloctadeca-2,16-dienoate (3), have been synthesized. The C19-C21 Wittig salt, [(2S,3R)-2-ethyl-3,4-(isopropylidenedioxy)butyl]triphenylphosphonium iodide (6), prepared from 2-butene-1,4-diol via Sharpless epoxidation, was coupled with the C22-C27 aldehyde, benzyl 2,4-dideoxy-3-O-(4-methoxybenzyl)-2,4-di-C-methyl-α,β-l-galacto-hexodialdopyranoside-(1,5) (7), prepared from (Z)-2-butene-1,4-diol via Sharpless epoxidation and the Brown’s crotylboration. The resulting coupling product was transformed to the C19-C27 lactone, [3S,4R,5R,6S,6(3R,4R)]-6-[3-ethyl-4,5-(isopropylidenedioxy)pentyl]-4-(4-methoxybenzyloxy)-3,5-dimethyl-3,4,5,6-tetrahydro-2H-pyran-2-one (4). The C28-C34 organostannane compound, (2R,4S,5S,7RS)-2-(t-butyldiphenylsilyloxy)-5-methyl-7-(tributylstannyl)-4-(triethylsilyloxy)-7-[(2-trimethylsilylethoxy)methoxy]heptane (5b), was prepared from (R)-methyl 3-hydroxybutyrate via the Brown’s crotylboration and the Still’s stannylation. After lithiation of 5b with butyllithium, the resulting α-alkoxy organolithium compound was coupled with 4 and the product was converted to the C19-C34 spiroketal, [2S,2(2R),3S,6R,8S,8(3R,4R),9S,10R,11S]-2-[2-(t-butyldiphenylsilyloxy)propyl]-8-[3-ethyl-4,5-(isopropylidenedioxy)pentyl]-10-(4-methoxybenzyloxy)-3,9,11-trimethyl-1,7-dioxaspiro[5.5]undecan-5-ol (37). The synthetic 2, derived from 37, was identical to the oligomycins (A, B, C mixture) degradation product in all respects, which elucidates the absolute stereochemistry of oligomycin B (1b). The C3-C9 aldehyde, (2-trimethylsilylethoxy)methyl 2,4,6-trideoxy-3-O-(4-methoxybenzyl)-2,4,6-tri-C-methyl-d-glycero-α-l-ido-heptodialdopyranoside-(1,5) (9), was prepared from (2S)-3-(t-butyldimethylsilyloxy)-2-methylpropanal via Keck’s crotylstannane addition and Brown’s crotylboration. The aldol coupling between the zinc enolate of the C10-C16 ketone, t-butyldimethylsilyl 2,3,7,8-tetradeoxy-4-O-(4-methoxybenzyl)-3,5-di-C-methyl-α-l-xylo-octopyranosid-6-ulose (10), prepared from methyl (R)-(+)-lactate via Brown’s crotylboration and a metallated methoxyallene addition, and aldehyde 9 gave the C8-C9 syn, C9-C10 syn product, which was transformed to the oligomycin B polypropionate portion 3 through elongation of the C1-C2 and C17-C18 carbon units.
Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologicallyrelevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins, whereas the reversible modification of Tyr with 1o switched protein function on and off in an enzyme and in a monoclonal antibody by modification and deconjugation processes. ASSOCIATED CONTENTSupporting Information. Experimental procedures, characterization data, computational procedure, and data. The Supporting Information is available free of charge at https://pubs.acs.org.
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