The design and synthesis of alanine-rich α-helical peptides constrained by an S,S-tetrazine photochemical trigger: a fragment union approach

Abstract

The design and synthesis of alanine-rich α-helical peptides constrained in a partially unfolded state by incorporation of the S,S-tetrazine phototrigger has been achieved, permitting, upon photochemical release, observation by 2D-IR spectroscopy of the subnanosecond conformational dynamics that govern the early steps associated with α-helix formation. Solid-phase peptide synthesis was employed to elaborate the requisite fragments, with full peptide construction via solution-phase fragment condensation. The fragment union tactic was also employed to construct (13)C═(18)O isotopically edited amides to permit direct observation of conformational motion at or near specific peptide bonds.

Figure 1

A. Photolysis of the S,S-tetrazine phototrigger can be achieved with laser pulses at various wavelengths that afford two thiocyanates and molecular nitrogen in less than 50 ps. B. Schematic representation of the S,S-tetrazine phototriggering applied to a S,S-Tet-AKA. Photolysis of the S,S-tetrazine (shown in space filling representation) removes the structural constraint to furnish the bis-SCN photoproduct peptide. The 2D-IR measurements at various time delays following the photolysis pulse (T_opt) directly monitor the conformational relaxation within the central CAC motif (the main chain of the peptide in the CAC region is shown in sticks).

Figure 2

A. The AKA peptide was employed for the design of an S,S-tetrazine constrained synthetic target: S,S-Tet-AKA.

Figure 3

The far-UV CD spectra of the AKA_A11P in phosphate buffer (dark blue) and TFE (magenta); the AKA_A10C/A12C peptide in TFE (red); and final synthetic target S,S-Tet-AKA in TFE (green).

Figure 4

The time-dependent spectrum of the SCN transient absorption observed upon photolysis (355 nm excitation) of S,S-tetrazine tripeptide 21 to furnish bis-SCN tripeptide 22, measured at 2163 cm⁻¹ from 0–100 ps. The raw data points are pictured in black with blue outline, while the exponential fit to data, with a time constant τ=56 ps, is shown by the green curve.

Figure 5

Isotopically edited peptides S,S-Tet-AKA_C10*/A11*, S,S-Tet-AKA_A5*/A6* and S,S-Tet-AKA_A17*/A18* were synthesized to resolve the amide-I stretching mode from the remaining backbone stretching modes. The position of the ¹³C=¹⁸O labeled amides in the synthetic peptides is shown in blue circles.

Scheme 1

The Synthesis of AKA variants AKA_A11P and AKA_A10C/A12C.

Scheme 2

The SPPS of tetrapeptide 3 and steady state photolysis to afford bis-thiocyante tetrapeptide 4.

Scheme 3

Attempted synthesis of the S,S-Tet-AKA peptide on the N-methyl indole solid support.

Scheme 4

A. Preparation of Lys-protected S,S-tetrazine hexapeptide 9. B. Union of S,S-tetrazine hexapeptide 9 to the C-terminal peptide fragment 10 of the S,S-Tet-AKA target peptide, with the corresponding residue numbers shown, to furnish 18-mer 11.

Scheme 5

A. Evaluation of the benzyloxycarbonyl (Cbz) as an alternative protecting group for the lysine side chain and validation of deprotection conditions. B. The S,S-tetrazine ring of 15 is partially reduced to the dihydro-S,S-tetrazine system 16 upon exposure to hydrogenolysis conditions.

Scheme 6

The successful three-fragment union approach to synthesize S,S-Tet-AKA.