Di-cysteine S,S-tetrazine: A potential ultra-fast photochemical trigger to explore the early events of peptide/protein folding

Abstract

The tetrazine chromophore holds promise as an effective photochemical trigger to achieve structural release, directed at the determination of peptide/protein motions that occur early in the folding processes. The photochemistry of 3,6-di-cysteine-S,S-tetrazines was examined by femtosecond IR transient absorption spectroscopy. Excitation of the tetrazine chromophore by visible and near UV light in the end yields chemically inert, structurally unobtrusive photoproducts that are not expected to interfere with the conformational dynamics of peptides and proteins. Dicysteine S,S-tetrazine is suggested to undergo photocleavage via a photochemical pathway different than the parent molecule s-tetrazine, based on kinetic measurements that reveal a stepwise reaction pathway of photofragmentation, whereby the initial ring cleavage event occurs prior to the formation of the SCN groups.

Applications of two-dimensional infrared spectroscopy

“…10, 74–76, 81–83 For example, 2D IR spectroscopy has been extensively applied toward the study of amyloid fibril formation. 77, 78 Figure 7 shows 2D IR spectra from one recent study of human γD-crystallin protein that employed a combination of 2D IR spectroscopy and segmental 13 C labeling to elucidate the involvement of the N- and C-terminal domains in the formation of amyloid fibrils.…”

The Design and Synthesis of Alanine-Rich α-Helical Peptides Constrained by an S,S-Tetrazine Photochemical Trigger: A Fragment Union Approach

“…Upon photolysis, the yield of 21, measured by nanosecond transient absorption, was 28%, slightly larger than the 20% previously observed for the non-cyclic S,S -tetrazine peptide 15 . 28 The increase in photochemical yield is likely related to the conformational strain exerted on the S , S -tetrazine system when the Cys residues are in an i, i +2 relationship within cyclic peptide 21 . The timescale of SCN formation upon flash photolysis of 21 was determined by femtosecond transient spectroscopy 27,28 by measuring the time dependence of the increase of the SCN absorption band intensity of photoproduct peptide 22 , centered at 2163 cm −1 (Figure 4).…”

“…28 The increase in photochemical yield is likely related to the conformational strain exerted on the S , S -tetrazine system when the Cys residues are in an i, i +2 relationship within cyclic peptide 21 . The timescale of SCN formation upon flash photolysis of 21 was determined by femtosecond transient spectroscopy 27,28 by measuring the time dependence of the increase of the SCN absorption band intensity of photoproduct peptide 22 , centered at 2163 cm −1 (Figure 4). The rate of SCN formation was determined to be ~56 ps, again slightly faster than previously reported for the acyclic system 15 .…”

“…Based on these experiments, formation of the final photoproduct 22 is expected to be on the order of tens of picoseconds, while the initial timescale of S,S- tetrazine ring cleavage was determined by prior experiments with acyclic 15 to be ~10 ps. 28 …”

“…27–29 Importantly, the photoproducts comprise inert and unobtrusive thiocyanates and nitrogen; moreover, the S,S -tetrazine phototrigger can be inscribed readily between two cysteine residues to constrain a peptide within a narrow distribution of conformers. 28 We have also described UV-pump transient 2D-IR-probe experiments to monitor the structural relaxation of a 24 residue alanine-rich peptide containing a kink induced by the S,S -tetrazine phototrigger (Figure 1B). 30 Upon release of the constraint, initiated by a sub-picosecond pulse of UV irradiation (λ = 355 nm), formation of a single α-helix turn was directly observed by 2D-IR spectroscopy without ‘unzipping’ of nearby helix turns ( vide infra ).…”