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1. Chemical Modification of Silk Proteins via Palladium‐Mediated Suzuki−Miyaura Reactions

   https://doi.org/10.1002/macp.202300307  

   Santen, Racine M.; Owens, Kayla M.; Echague, Keith C.; Murphy, Amanda R. (October 2023, Macromolecular Chemistry and Physics)

   Abstract Suzuki−Miyaura cross‐coupling reactions are used to modify the tyrosine residues onBombyx morisilkworm silk proteins using a water‐soluble palladium catalyst. First, model reactions using tyrosine derivatives are screened to determine optimal reaction conditions. For these reactions, a variety of aryl boronic acids, solvents, buffers, and temperature ranges are explored. Qualitative information on the reaction progress is collected via high‐performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). Optimized reactions are then applied to silk proteins. It is demonstrated the ability to modify silk fibroin in solution by first iodinating the tyrosine residues on the protein, and then carrying out Suzuki‐Miyaura reactions with a variety of boronic acid derivatives. Modification of silk is confirmed with NMR, ion‐exchange chromatography (IEC), UV‐vis, and infrared spectroscopy (IR). 

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2. Quantitative Functionalization of the Tyrosine Residues in Silk Fibroin through an Amino‐Tyrosine Intermediate

   https://doi.org/10.1002/macp.202200119  

   Hausken, Kian G.; Frevol, Romane L.; Dowdle, Kimberly P.; Young, Aleena N.; Talusig, Jeremy M.; Holbrook, Carolynne C.; Rubin, Benjamin K.; Murphy, Amanda R. (June 2022, Macromolecular Chemistry and Physics)

   Abstract Here, a reaction sequence that can be used to quantitatively modify the tyrosine residues in silk protein fromB. morisilkworms is demonstrated. A primary amine is installed ortho to the hydroxyl group on the tyrosine ring using a diazonium coupling reaction followed by reduction of the azo bond. The resulting amine is then acylated using carboxylic acid or NHS‐ester derivatives at room temperature and neutral pH conditions. The silk derivatives are characterized using¹H NMR, UV–vis spectroscopy, ATR‐FTIR, and a unique method to follow this reaction sequence using isotopically labeled reagents and 2D NMR spectroscopy is also used. This study further demonstrates that this sequence can be used to install alkyne or azide functional groups which can undergo further bio‐orthogonal cycloaddition reactions under mild conditions. Finally, methods to carry out these modifications on solid silk microparticles and electrospun mats are also described. 

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3. Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

   https://doi.org/10.1002/anie.201803907  

   Wang, Yifan; Griffith, Wendell P.; Li, Jiasong; Koto, Teruaki; Wherritt, Daniel J.; Fritz, Elizabeth; Liu, Aimin (June 2018, Angewandte Chemie International Edition)

   Abstract Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C−S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink. 

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4. Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

   https://doi.org/10.1002/ange.201803907  

   Wang, Yifan; Griffith, Wendell P.; Li, Jiasong; Koto, Teruaki; Wherritt, Daniel J.; Fritz, Elizabeth; Liu, Aimin (June 2018, Angewandte Chemie)

   Abstract Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C−S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink. 

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5. Occupied and abandoned structures from ecosystem engineering differentially facilitate stream community colonization

   https://doi.org/10.1002/ecs2.2734  

   Tumolo, Benjamin B.; Albertson, Lindsey K.; Cross, Wyatt F.; Daniels, Melinda D.; Sklar, Leonard S. (May 2019, Ecosphere)

   Abstract Ecosystem engineers transform habitats in ways that facilitate a diversity of species; however, few investigations have isolated short‐term effects of engineers from the longer‐term legacy effects of their engineered structures. We investigated how initial presence of net‐spinning caddisflies (Hydropsychidae) and their structures that provide and modify habitat differentially influence benthic community colonization in a headwater stream by conducting an in situ experiment that included three treatments: (1) initial engineering organism with its habitat modification structure occupied (hereafter caddisfly); (2) initial habitat modification structure alone (hereafter silk); and (3) a control with the initial absence of both engineer and habitat modification structure (hereafter control). Total invertebrate colonization density and biomass was higher in caddisfly and silk treatments compared to controls (~25% and 35%, respectively). However, finer‐scale patterns of taxonomy revealed that density for one of the taxa, Chironomidae, was ~19% higher in caddisfly compared to silk treatments. Additionally, conspecific biomass was higher by an average of 50% in silk treatments compared to controls; however, no differences inHydropsychesp. biomass were detected between caddisfly treatments and controls, indicating initially abandoned silk structures elevated conspecific biomass. These findings suggest that the positive effects of the habitat modification structures that were occupied for the entirety of the experiment may outweigh any potential negative impacts from the engineer, which is known to be territorial. Importantly, these results reveal that the initial presence of the engineer itself may be important in maintaining the ecological significance of habitat modifications. Furthermore, the habitat modifications that were initially abandoned (silk) had similar positive effects on conspecific biomass compared to caddisfly treatments, suggesting legacy effects of these engineering structures may have pertinent intraspecific feedbacks of the same magnitude to that of occupied habitat modifications. Elucidating how engineers and their habitat modifications differentially facilitate organisms will allow for a clearer mechanistic understanding of the extent to which animal engineers and their actions influence aspects of community organization such as colonization. 

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