Glucose oxidase (GOx) was covalently modified at pH 7.2 with ferrocenecarboxylic acid (FCA), ferrocenedicarboxylic acid (FDA), and ferroceneacetic acid (FAA) using 1-ethyl-3-( 3-(dimethy1amino)propyl)-carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide to promote selective coupling to surface lysines.Reagent ratios were varied to obtain derivatives with 5-12 ferrocene groups per GOx dimer and 260% activity. For comparison, GOx was derivatized with FCA in the presence of 3 M urea using only EDC as a promoter. Varying reagent ratios yielded derivatives with 4-39 FCA groups per GOx and 530% activity; linear sweep voltammetry results showed a slow but readily detectable release of FCA upon storage of these derivatives. Tryptophan fluorescence quenching in two media, 0.1 M phosphate buffer (pH 7.0) and 8 M urea, confirmed that GOx was covalently modified and not merely associated with ferrocene. In all cases, the GOx derivatives exhibited significantly greater quenching than controls containing native GOx with free ferrocenes. The results of voltammetric dilution experiments (performed in oxygen-free solutions in the presence of excess glucose) were consistent with rate-limiting intramolecular electron transfer from the reduced flavin centers to bound ferricinium. Using an expression derived here, values between 0.16 and 0.90 s-1 were obtained for intramolecular electron transfer in the FCA derivatives, suggesting that the location (rather than the number) of bound ferrocene groups is rate-determining. Approximately lo3-fold slower intramolecular electron transfer was measured in an FDA derivative, consistent with fluorescence quenching data which indicated that bound FDA is more solvent-exposed than bound FCA. The results of lysine-targeted modification of GOx are interpreted in light of the recently published 3-D structure of GOx; since the critical flavin-lysine separations are all >23 A, an alternative approach is necessary to obtain GOx derivatives for use in a practical, reagentless glucose sensor.
Poly(ester amide)s (PEAs) composed
of variable diols, dicarboxylic
acids, and α-amino acids were synthesized and characterized
to elucidate structure–property relationships. The PEAs were
synthesized using both solution and interfacial approaches to obtain
varying molecular weights (MWs) and polydispersities. Studies using
differential scanning calorimetry and powder X-ray diffraction revealed
a range of amorphous and semicrystalline behavior that was dependent
on structure. The viscosities of the PEAs as a function of temperature
were studied by rheology, and phase transitions were confirmed for
some polymers. Mechanical properties were also investigated, and it
was found that the PEAs could be tuned to exhibit elastomeric or brittle
behavior by varying both the length of the diol and the amino acid
moiety. This information provides new insights into the effects of
PEA structure, MW, and MW distribution on PEA properties and will
aid in the design and selection of PEAs with optimal properties for
targeted applications.
ortho-Benzoquinones substituted with an electron-withdrawing group (EWG = CO2Me, COSMe, COCH3, CHO) at C-3 or C-4 react as dienophiles at the C3—C4 double bond in Diels–Alder reactions with several dienes, with predictable regiochemistry. The adducts undergo migration of the "angular" EWG substituent with concomitant aromatization to produce substituted catechols. The bicyclic products can be oxidized in situ and annelated by a further Diels–Alder reaction to yield 9,10-phenanthraquinone systems. When (2E)-2,4-pentadienol is employed as the diene in the second cycloaddition, reaction of the alcoholic hydroxyl group with one of the quinone carbonyl groups results in the production of tetracyclic lactols 22a, 22b, and 23. Keywords: benzoquinone, Diels–Alder, rearrangement, phenanthraquinone, phenanthrofuran.
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