Page 345 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

15.2 Cascade Reactions for Assaying Transketolase Activity In Vitro 321

the result of the stability of the cis-enediolate intermediate generated under base-
catalyzed hydrogen abstraction α to the α-hydroxylated aldehyde (Scheme 15.8)
[24c]. An analytical approach by LC/UV/MS [24a] was developed to monitor
the product distribution from the fluorogenic probe 1 upon TK catalysis. Thus
β-elimination reactions were found to be rate limiting, and the reprotonation
reaction was preferred. Nevertheless, TK activity was detectable from the release of
umbelliferone in Hepes buffer down to 0.7 mIU, with a significant 340-fold S/N
ratio.

B OH
HO H H-B
O O
O O Umb
Umb
H

O
H O
H-B β-Elimination
H O O O Umbelliferone
Umb Umb
B
OH
Scheme 15.8 Base-catalyzed hydrogen abstraction pathways for hydroxyaldehyde 4.

In conclusion, fluorogenic substrate 1 can be considered as a reliable probe
to monitor low wild-type yeast TK activity [24c]. For assaying TK with a reverse
stereoselectivity or nonnatural ability to transfer a formyl group, suitable probes
2 and 3 have been synthesized respectively with l-erythro (3S,4S) configuration
2 or bearing a formyl instead of the hydroxyacetyl group 3 (Scheme 15.9) [24b].
They meet the conditions required to screen libraries of TK mutants, including an
excellent Z-factor, lending them a strong advantage for HTS.

O R 2 R 3
O O O Mutant TK
R
1
BSA HO O O
OH
Umbelliferone
2 R = H, R = OH, and R = H
1 2 3

3 R 1 = CH 2 OH, R 2 = H, and R 3 = OH
Scheme 15.9 Stereochemical probes 2 and 3 for the screening of mutant TK libraries.
15.2.1.3 Coupling with BSA and Polyphenol Oxidase
In a fashion similar to the coumarol release, probe 7 was studied for release
of protected l-tyrosine upon cleavage by yeast TK, followed by BSA catalyzed β-
elimination from intermediate 8 under weakly alkaline conditions (Scheme 15.10)
[25]. Generation of 9 was of interest for its potential use in a selection assay with E.
coli auxotrophs or by a redox detection system.