PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5‑Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6<i>H</i>-oxazolo­[4,5-<i>e</i>]­indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable β-hairpin “zipper” peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with <i>N</i>-acetyl-cysteine and amyloid β-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 β-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance

Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail <i>Terebra variegata</i>

<div><p>Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from <i>Terebra variegata</i> was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.</p></div

Sequence identification of Tv1 teretoxin.

<p>Shown is the native toxin (black) and the synthesized toxin (green) MS/MS spectrum recorded on a (M +6H)+6 ion after conversion of cysteine residues to dimethyl lysine analogs. Note the overlap of all major fragment ions. The sequence is given above the spectrum and observed c and z-type fragment ions are indicated in the sequence with and , respectively. Doubly charged fragment ions of type c and z are labeled with +2, triply charged ions are of type c and z are indicated with *, z-type fragment ions that resulted from cleavage at cysteine with subsequent loss of the cysteine side chain are denoted in italic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094122#pone.0094122-Chalkley1" target="_blank">[60]</a> and charge reduced species are labeled in the spectrum with #. The spectrum was recorded at a resolution of 7500 at m/z 400 and all fragment ions have a mass accuracy of better than 5 ppm.</p

Disulfide mapping of Tv1.

<p>a) RP-HPLC fractionation of all differentially folded peaks (on HPLC gradient of 20% to 35% buffer B (80% Acetonitrile, 0.1% TFA) in buffer A (water, 0.1% TFA) over 75 min. b) MS-MS spectrum of partially reduced peptide peaks showing representative alkylated b and y ions. c) Schematic representation of Tv1 with predicted disulfide bridges.</p

Comparison of Teretoxin Tv1 and known M superfamily Conotoxins.

<p>Comparison of Teretoxin Tv1 and known M superfamily Conotoxins.</p

Discovery and characterization of disulfide–rich Tv1 teretoxin.

<p>An integrated approach for characterizing sample limited disulfide peptidic natural products was applied: a) RP-HPLC on-line separation of <i>Terebra variegata</i> venom (highlighted is the region where the sequenced peptide eluted). b) ETD and CAD MS/MS analysis recorded on the native peptide to determine the sequence of Tv1 peptide. c) RP-HPLC of linear and folded versions of chemically synthesized Tv1 peptide. d) MS/MS spectrum of partially reduced and alkylated Tv1 used to determine the disulfide connectivity. e) NMR solution structure of folded Tv1 peptide. f) Bioactivity of 20 μM of Tv1 (−) versus normal saline (NS) (−) solution injected into polychaete worms. Y-axis indicates level of activity, where 3 is the normal activity of the NS injected worm, 1 and 2 indicate decreased activity due to partial paralysis.</p

Structural comparison of Tv1 teretoxin and M-superfamily conotoxins.

<p>Comparison of the NMR structure of Tv1 with that of M superfamily conotoxins SmIIIA, mr3e, and KIIIA reveals significant structural differences between Tv1 and these conotoxins despite all having the same cysteine scaffold CC-C-C-CC. All structures are shown in cartoon representation with disulfide bonds highlighted in yellow. All figures were prepared using PyMol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). Conotoxin structural references are as follows: MrIIIe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094122#pone.0094122-Du1" target="_blank">[33]</a> SmIIIA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094122#pone.0094122-Keizer1" target="_blank">[61]</a> and KIIIA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094122#pone.0094122-Khoo1" target="_blank">[62]</a>.</p

Tv1 bioactivity in polychaete worms.

<p>Tv1 bioactivity in polychaete worms.</p