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Consequently, the yield of acetal-PEG-COOH is high and the intermediate is obtained in good yield.
Under these conditions, the phosphoenzyme intermediate is obtained in the reverse direction of the hydrolytic reaction at the end of the catalytic cycle.
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At each step, the behavior of intermediate medium is obtained by any analytical homogenization method and is used as matrix of the following step.
First one constitutes a Suzuki coupling reaction between aromatic dibromine functional polymer and 3-aminophenylboronic acid, when a diamino-containing intermediate was obtained.
After the protection of both the 1α- and 3β-hydroxyl in 1α-hydroxydehydroepiandrosterone with TBS groups, followed by bromination with NBS and debromination in the presence of γ-collidine, the diene intermediate was obtained.
After recrystallizing with alcohol and drying, the white crystal of 3-chloro-2-hydroxyl propanesulfonate intermediate was obtained.
This last key intermediate was obtained with 54% overall yield following the synthetic methodology developed in this work.
Further insight into the nature of the cross-linked intermediate was obtained from Mössbauer spectroscopy.
The F intermediate was obtained by the addition of peroxide at pH 8.75 (a small aliquot of catalase added during the pulsing procedure was not removed).
The P intermediate was obtained by gently blowing CO gas onto the enzyme for 10 s at pH 8.75, with subsequent incubation leading to complete formation after a few minutes.
The 2.0 Å structure of the 2-deoxy-2-fluoroglucopyranosyl HoBGLA intermediate was obtained by soaking crystals with only 2FGlc and not with an activated compound such as 2,4-dinitrophenyl 2-deoxy-2-fluoro β- d-glucoside (DNP- 2FGlc) or 2-deoxy-2-fluoro β- d-glucosyl fluoride.
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