1. Biomimetic Thioesters as Probes for Enzymatic Assembly Lines: Synthesis, Applications, and Challenges 
Jakob Franke, Christian Hertweck 
Cell Chemical Biology 
Volume 23, Issue 10, Pages (October 2016)
DOI: /j.chembiol
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2. Figure 1 Thioester-Based Activation of Carboxylic Acids in Nature
(A) Activation of carboxylic acids as thioesters and subsequent transformations in biochemistry. Synthetic NAC thioesters can be used as substrate mimics in vivo and in vitro.
(B and C) Chemical structures of thiols used to activate carboxylic acids in Nature (B). Chemical structure of N-acetylcysteamine as a mimic of pantetheine-derived thioesters (C). ACP, acyl carrier protein; PCP, peptidyl carrier protein.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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3. Figure 2
Common Experimental Setups and Problems for In Vitro Assays Using NAC Thioesters as Substrates
(A) NAC thioester substrates can be converted with whole PKS/NRPS (non-ribosomal peptide synthetases) megaenzymes, single modules or similar constructs, or single domains and freestanding enzymes. Selected examples showing possible transformations with NAC substrates.
(B) If a missing interaction with the carrier protein prevents use of an NAC thioester substrate, addition of the corresponding carrier protein can help to convert the substrate in the desired form by the self-acylation activity of the carrier protein.
(C) If substrate degradation is hindering, it can be fruitful to use a photolabile-protecting group to release the substrate only in situ, or to switch the buffer system, but avoiding nucleophilic buffers such as Tris.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 3 Common Feeding Experiments with NAC Thioesters In Vivo
(A) Experimental setups for feeding experiments based on selected examples.
(B) Potentially limiting steps for feeding experiments. The substrate concentration can be monitored to obtain information of whether stability or another factor is limiting.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 4 Common Strategies to Synthesize NAC Thioesters
(A) Reactions to introduce the NAC thioester.
(B) Convenient strategies for common polyketide-like NAC substrates that are often not compatible with standard coupling conditions. Chiral centers are indicated by an asterisk. Aux, chiral auxiliary.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 5 Thioester Mimics Providing Additional Functionality
(A) Generic structure of acyl-coenzyme A substrates.
(B) Sulfonyl mimic for studying dehydratase domains. The probe is coupled chemoenzymatically to ACP and forms a covalent linkage with DH.
(C) Aza mimic as a stable thioester surrogate.
(D) Carba mimic used for off-loading polyketide biosynthesis intermediates by competing with ACP-bound extender units.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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