Peptide tertiary structure nucleation by side-chain crosslinking with metal complexation and double "click" cycloaddition.

Stabilized helical peptides show potential as therapeutics capable of modulating protein function through enhanced helix-protein binding.[1] Helix turn stabilization may be accomplished though covalent sidechain crosslinking on one face of the helix, in i and i+4 or i+7 positions,[2–4] or replacement of a main chain hydrogen bond with a covalent linker.[3, 5, 6] We report herein a new helix-nucleating[7] i and i+4 crosslinking strategy based on copper catalyzed azide-alkyne [3+2] “click” cycloaddition[8] and demonstrate the ability of this method and metal complexation to restore coiled-coil dimerization in a folding-incompetent sequence crippled by 2 helix-breaking glycine residues. Elegant applications of azide-alkyne cycloaddition chemistry in peptide conjugation[9] and structure stabilization[10] are known in the literature; this design strategy complements known intramolecular methods for structure nucleation such as ring closing metathesis[2, 5, 11] (RCM) and allows convergent installation of functional groups pendant to the bis-alkyne linker that could be used to modulate peptide binding, targeting, and membrane permeability. We studied 21 residue sequences derived from the GCN4 leucine zipper[12] in which the central heptad contains glycine residues in the c and e helix positions and crosslinking residues (X) in the b and f (i and i+4) positions, leaving the hydrophobic core residues in the a and d positions intact as isoleucine and leucine, respectively (Figure 1). Metal complexation with i and i+4 X=His residues is known to induce monomeric helix folding,[4, 13] though it has not been previously demonstrated to restore structure in peptides containing 2 glycine residues. We postulated that the bis-triazole product of a double [3+2] cycloaddition between i and i+4 azidoalanine[14] (Az) residues and a bis alkyne could yield a non-labile covalent linker isosteric with i and i+4 His-His metal complex (Figure 1A). We prepared peptides 1 and 2 using standard Fmoc solid phase synthesis which may be crosslinked in the central heptad by Ni2+ complexation with histidine and bis-alkyne cyclization with Az, respectively (Figure 1B). Indeed, the circular dichroism (CD) spectrum of 1 exhibits a saturable increase in helicity upon treatment with NiCl2, which may be reversed with EDTA treatment (Figure 3). While the free peptide is a completely unfolded monomer, the nickel-complexed peptide melts cooperatively with a Tm of 46 °C, and is found by analytical ultracentrifugation (AUC) to be dimeric (Figure 1D), indicating the ability of sidechain crosslinking to restore tertiary structure even in sequences containing 2 centrally placed glycine residues. Figure 1 (A) Strategies for helix structure nucleation with i and i+4 crosslinking using metal complexation (top) or double click cycloaddition (bottom). aDi-azidoalanine peptide was treated in aqueous buffer with bis alkynes 6–9, sodium ascorbate, CuSO ... Figure 3 (A) Nickel dependent folding of 1, from 0 NiCl2 (ο) to 100 μM NiCl2 (•), with intermediate concentrations shown in black. (B) Binding isotherms of NiCl2 to peptide 1 (•) and peptide 9a (ο), fit to a 1:1 binding ... We sought to use copper catalyzed “click” chemistry to similarly induce secondary[15] and tertiary structure by linking azidoalanines in peptide 2 with bis alkyne linkers 6–9. These intermolecular cyclizations were successful with both resin-bound protected peptide in DMF using CuI/DIEA and in aqueous buffer using a copper (I) catalyst[16] (Figure 1A). Click adducts were subjected to Staudinger reduction conditions with PPh3 (on resin) or TCEP (in aqueous solution) to confirm ring closure; while starting material 2 reacted with phosphine to yield reduction and other products, all adducts remained unchanged, consistent with conversion of azide to triazole. Additionally, MS-MS peptide fragmentation patterns were indicative of cyclic “double-click” products (Figure 2). Fragmentation of the bisalkyne treated diazido peptides yielded all possible peptide bond mass fragments except for those putatively joined by a bis-triazole linker, which should not fragment under these conditions. The identical experiment with the click adduct to a monoazide peptide 3, in which the C-terminal Az residue was replaced with serine yielded all possible peptide mass fragments. Notably, the observed ratio of ion intensities of the 4+ parent ion relative to the common mass fragment y1 is much higher in the spectrum of cycloadduct of 2 (Figure 2B) relative to the cycloadduct of 3. This is consistent with double cycloaddition to 2 that results in the coalescence of ion intensities of 4 possible distinct mass fragments into a single, more intense peak. Figure 2 Representative MS-MS data indicating fragmentation at the peptide bonds of the adduct of linker 6 to (A) peptide 3 and (B) peptide 2 (6a). Arrows indicate diagnostic C-terminal y-fragment ions, N-terminal b-fragment ions and g-parent ions (4+). Interestingly, though linkers were used in large excess on-resin, the bis-adduct was never observed and diazide 2 could be near quantitatively converted on resin to the double cycloadduct (determined as described above), suggesting that the second click reaction to close the ring is much faster than the first, despite the 23 or 24-atom ring closure required. Similarly efficient peptide ring closures have been observed using RCM, suggesting that the peptide backbone may greatly reduce the entropic cost of cyclization through templating effects or hindered amide bond rotations.[2, 11] Cyclization in aqueous buffer also yielded desired product in acceptable (40–50%) isolated yield, though bis-adducts were observed when linker was used in moderate excess; the more rigid unsaturated linkers 7 and 8 gave more bis-adducts relative to linkers 6 and 9, which have single bonds connecting the 2 alkynes. The functional effect of ring-closure via double cycloaddition was obtained by comparing the folding properties of monoazide peptides in which one AzAla residue was replaced by serine. These negative control peptides are also readily coupled with 6–9, but remain unfolded under all conditions, like diazido starting material 2. (Figure 3). Commercially available 1,5-hexadiyne (linker 6) induced the most significant change in helicity upon double cycloaddition, nucleating helical structure in the central heptad with sufficient efficiency to restore dimerization to the folding-crippled 2. Like the nickel complex of 1, the 1,5-hexadiyne adduct peptide (6a) melts cooperatively (Tm=40°C) but dimerizes independently of nickel concentration (Figure 3). Linkers 7–8 were used to probe the steric requirements of side chain constraint. When the ethyl spacer of 1,5-hexadiyne 6 is replaced by the rigid 2 carbon linker of the ortho-diethynylbenzoic acid linker 7, the folded state is not favored. The meta-diethynylbenzoic acid 8 and di-propargylated glycine linker, 9 both extend the chain by 1 atom, with a rigid unsaturated linker and more flexible saturated linker, respectively. The cycloadducts of diazidopeptide 2 with 7 (7a) and 9 (9a) exhibit random coil signatures while 8a is insoluble in aqueous solvent. Interestingly, treatment of 9a with NiCl2 increases the helicity of the system, suggesting the formation of a new nickel binding site, presumably composed of the triazoles, tertiary amine, and carboxylate from the ligated linker. Nickel concentration dependence of ellipticity indicated tighter nickel binding by adduct 9a (Kd=10−6 M) than di-histidine peptide 1 (Kd=10−4 M), consistent with a more organized binding site in cycloadduct relative to the open chain peptide 1. Indeed, the double cycloadduct of propargyl alcohol with 2 installs the two triazole moieties in a non-cyclic product and this peptide remains unfolded even in the presence of nickel chloride, indicating the importance of cyclization (Supporting Information). However, AUC measurements indicated a monomer-dimer equilibrium for 9a under all conditions, suggesting that metal complexation does not completely restore dimerization. This result stands in constrast to the enhanced helicity and dimerization found upon crosslinking with linker 6 to obtain 6a. It is possible that the additional atom in linker 9 actually prevents the formation of a helical turn, and metal binding induces a contraction of the cyclic binding site that allows partial, but incomplete folding and dimerization. Weak dimerization and partially helical signature in 9a suggests that, similar to the parent “uncrippled” coiled-coils, helix folding and oligomerization in these systems is coupled.[17] Peptides 4 and 5 are dimerization-inhibited by charged residues at the dimerization interface or scrambled coiled-coil sequences, respectively. These soluble peptides (X=Az or His) retained random coil CD signatures upon metal complexation with NiCl2 and were insoluble upon cycloaddition, consistent with coupled folding and assembly; this is likely accentuated by the presence of 2 helix-breaking glycines in the sequence. We have thus demonstrated a new methodology for helix structural nucleation using double azide-alkyne [3+2] cycloadditions to form i and i+4 constrained peptide sequences. This chemistry is sufficiently robust to allow intermolecular cyclization with few sideproducts, setting the stage for convergent functionalization and structure nucleation. This technology may be useful as a means of synthesizing new building blocks for peptide-based materials[18] or preparing stabilized helical peptides capable of binding protein interfaces and altering biological function. These studies are currently underway in our lab.