Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

Photocaged antibody fragments,t ermed photobodies,h ave been developed that are impaired in their antigenbinding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of as ingle tyrosine in an anobody (a single-domain antibody fragment). Tyrosine is af requently occurring residue in central positions of the paratope region. o-Nitrobenzyl-protected tyrosine variants were incorporated into four nanobodies,i ncluding examples directed against EGFR and HER2, and photodeprotection restores the native sequence.Ananti-GFP photobody exhibited an at least 10 000fold impaired binding affinity before photodeprotection compared with the parent nanobody.Abispecific nanobodyphotobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N- S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure-activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the extein acyl group from the α-amino moiety of the intein's first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible extein moiety completely blocked the switch. Furthermore, we investigated the M86 intein, a mutant with faster splicing kinetics previously obtained by laboratory evolution of the Ssp DnaB intein, and the individual impact of its eight mutations. The succinimide formation was decoupled from the first step in the M86 intein, but the acquired H143R mutation acts as a brake to prevent premature C-terminal cleavage and thereby maximizes splicing yields. Together, these results revealed a high degree of plasticity in the kinetic coordination of the splicing pathway. Furthermore, our study led to the rational design of improved M86 mutants with the highest yielding trans-splicing and fastest trans-cleavage activities.

Fluorinated acyl‐Meldrum's acid derivatives were synthesized by electrophilic fluorination of appropriate phenacyl Meldrum's acid substrates using Selectfluor. Reactions with water, ethanol, Grignard, and alkynyllithium reagents gave rise to the corresponding fluoro‐acetophenone, –1,3‐keto ester, –1,3‐diketone and ‐pyran‐4‐one products respectively from the same selectively fluorinated scaffold in one‐step procedures.

No abstract