The permeation peptide sequence used was based on the wild-type HIV-1 Tat sequence (Tat
47–60). D-isomer amino acid peptides were used to prevent in vivo proteolysis. The D-isomer has also been shown to have enhanced cellular uptake.
4 Substitution of ornithine for glutamine was also found to increase cellular uptake.
4 A second peptide sequence from a viral protein (HSV-1 VP-22) served as a control. Both peptides were conjugated at the C terminus to a fluorescent marker for direct visualization of cellular uptake within retinal sections.
The peptides Ac-rkkrrorrrgc-NH
2 (modified Tat) and Ac-plssifsrigdp-AHA-εkgc-NH
2 (control) were prepared by solid-phase peptide synthesis (Tufts University Peptide Synthesis Core Facility, Boston, MA) using standard BOP/HOBt coupling chemistry and all D N-α-Fmoc-protected amino acids.
18 Peptides were obtained pure and were conjugated to Alexa Fluor 594 maleamide (AF; 1.2 equiv; Molecular Probes, Eugene, OR) by thiol conjugation at ambient temperature in 0.5 × PBS for 2 hours. Quantitative yields were observed for all reactions, as analyzed by C
18 reverse-phase HPLC (RP-HPLC). Peptides were purified by RP-HPLC at a flow rate of 1 mL/min using, as eluent, Solvent A (0.1% trifluoroacetic acid in 5% acetonitrile/95% water [0.1% TFA/(5% CH
3CN/H
2O)]) modified with Solvent B (0.1% trifluoroacetic acid in 90% acetonitrile/10% water [0.1% TFA/(90% CH
3CN/H
2O)]) by a linear gradient of 100% A to 40% A over 40 minutes (modified Tat) or 100% A to 65% A over 15 minutes to 50% A at 30 minutes (control) before washing with Solvent B to obtain the following pure peptides: Ac-rkkrrorrrgc(AF)-NH
2 (modified Tat,
t R = 21.6 minutes;
m/z: 2414.0; calc: 2412.7) and Ac-plssifsrigdp-AHA-εkgc(AF)-NH
2 (control,
t R = 25.9 minutes;
m/z: 2618.0; calc: 2617). According to previous studies, isolated doublets from the modified Tat peptides were determined by electrospray mass spectrometry (ESMS) to have identical mass, indicating that the doublet represents two independent conformers of the desired product.
19
As a control for uptake mediated by the fluorophore, nonreactive Alexa Fluor 594 was obtained through succinimidyl ester hydrolysis by incubating Alexa Fluor 594 succinimide (Invitrogen, Carlsbad, CA) in water at pH 9 for 9 hours at ambient temperature. RP-HPLC analysis using the above gradient for purification of the modified Tat peptides revealed an increase over time of a doublet peak at earlier retention times (t R = 21.1/22.2) than the parental doublet peak (t R = 23.4/24.1). Near completion of hydrolysis (80%) was observed as determined by integration of the resultant doublet peak. The hydrolyzed doublet peaks were isolated, analyzed by ESMS, and determined to have identical mass (m/z: 723.3; calc. 722.9), indicating that the doublet represented two independent conformers.