Genetically Introducing Biochemically Reactive Amino Acids Dehydroalanine and Dehydrobutyrine in Proteins

Abstract

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-L-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter-and intra-molecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

“…High-resolution MALDI-TOF mass spectrometry study supports the covalent inhibition mechanism of the more potent and selective agents 22 and 24 to be sulfonylation of hNE (maroon peaks, +273 Da for 22, and +238 Da for 24). In both cases, we observed the hNE dehydration product peak (M -18, turquoise) suggesting both agents to effect the covalent modification at the same catalytic serine as 1 did (29,32,(92)(93)(94). To further demonstrate the stringent dependence of SuFEx reactions on protein's tertiary structure, compounds 22 and 24 were incubated, respectively, with inactive denatured hNE and no covalent modification of the enzyme was found (Fig.…”

“…click chemistry | SuFEx | agnostic | covalent inhibitor | elastase S ulfur fluoride exchange (SuFEx)-the new-generation click chemistry, since first introduced in 2014 (1), has quickly found diverse applications across an array of fields (DOI: 10.1039/C8CS00960K), including chemical synthesis (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), material science (13)(14)(15)(16)(17)(18)(19), chemical biology (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32), and drug discovery (33,34). SuFEx creates robust intermolecular links between modules.…”

Systematic Activity Maturation of a Single-Domain Antibody with Non-canonical Amino Acids through Chemical Mutagenesis

“…Here, we report the use of the post-translationally installed synthetically versatile non-canonical amino acid dehydroalanine (Dha) to create a platform for the precise augmentation of the activity of antibodies for inhibiting the aggregation of the 42-residue form of the amyloid-β peptide (Aβ42), a protein fragment closely associated with Alzheimer's disease (AD) ( Hardy and Selkoe, 2002 ; Knowles et al., 2014 ). Dha has proven to be a suitable intermediate for side-chain exploration due to its ease of incorporation through a chemical conversion from cysteine mutant precursors, as well as its ability to react bioorthogonally with a vast number of reagents ( Bernardes et al., 2008 ; Chalker et al., 2011 ; Freedy et al., 2017 ; Tamura and Hamachi, 2018 ; Wright et al., 2016a ; Yang et al., 2019 ). Dha has indeed previously been used in a similar manner to enhance enzyme activity ( Windle et al., 2017 ) and has been used in the complementarity-determining region (CDR) loops of a nanobody to create a Boolean logic gates response ( Gunnoo et al., 2014 ).…”

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion

“…In the present medical context and in the current environment of failing antibiotic protection, an obvious functional expansion is a fortification against post-surgery antibiotic infections, for example by coupling therapeutic proteins to antimicrobial modules. New approaches such as genetically encoded chemical conversion (GECCO) has also been recently introduced (Yang et al, 2019) to overcome limitations of translational machinery. This and other newly emerged approaches such as OTS optimization (Dulic et al, 2018), cell-free protein synthesis (Jin and Hong, 2018), genomically recorded strain development (Amiram et al, 2015), artificial codon box division (Iwane et al, 2016), and quadruplet codon and orthogonal ribosome (d'Aquino et al, 2018) are expected to open new avenues for exploiting chemistry in live systems to bioengineering of lanthipeptides.…”