Transesterification catalysts based on stereochemically defined, modular, functionalized ladder-molecules (named spiroligozymes) were designed, using the "inside-out" design strategy, and mutated synthetically to improve catalysis. A series of stereochemically and regiochemically diverse bifunctional spiroligozymes were first synthesized to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacted with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7 × 10(3)-fold faster than the background reaction with a benzyl alcohol. Two trifunctional spiroligozymes were then synthesized that combined a urea with the pyridine and alcohol to act as an oxyanion hole and activate the bound acyl-spiroligozyme intermediate to enable acyl-transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k(1)/k(uncat) of 2.2 × 10(3) and k(2)/k(uncat) of 1.3 × 10(2). Quantum mechanical calculations identified the four transition states of the catalytic cycle and provided a detailed view of every stage of the transesterification reaction.

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Derivatives of 4-hydroxyproline with a series of hydrophobic groups in well-defined orientations have been tested as catalysts for the aldol reactions. All of the modified proline catalysts carry out the intermolecular aldol reaction in water and provide high diastereoselectivity and enantioselectivity. Modified prolines with aromatic groups syn to the carboxylic acid are better catalysts than those with small hydrophobic groups (1a is 43.5 times faster than 1f). Quantum mechanical calculations provide transition structures, TS-1awater and TS-1fwater that support the hypothesis that a stabilizing hydrophobic interaction occurs with 1a.

Deficiency of the N 6 -methyladenosine (m 6 A) methyltransferase complex results in global reduction of m 6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m 6 A methylation affects cell fate decisions due to the inability to modulate individual m 6 A modification in ESCs with precise temporal control. Here, a targeted RNA m 6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m 6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m 6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m 6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.In mammalian cells, regulatory processes at the posttranscriptional level are often a key determinant of genetic information flow. N 6 -methyladenosine (m 6 A), as the most abundant modification on messenger RNAs (mRNAs), is