Two-photon uncaging of bioactive thiols in live cells at wavelengths above 800 nm.

Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from biology to materials science. In chemical biology, induction of biological processes via photoactivation is a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable protecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethylamine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has a bathochromic shift in the one-photon absorption maximum from λmax = 315 nm with the unfunctionalized NDBF scaffold to λmax = 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm. To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting, caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live cells using 850-900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experiments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm. Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments in chemical biology and materials science.

Methoxy-Substituted Nitrodibenzofuran-Based Protecting Group with an Improved Two-Photon Action Cross-Section for Thiol Protection in Solid Phase Peptide Synthesis.

Photoremovable caging groups are useful for biological applications because the deprotection process can be initiated by illumination with light without the necessity of adding additional reagents such as acids or bases that can perturb biological activity. In solid phase peptide synthesis (SPPS), the most common photoremovable group used for thiol protection is the o-nitrobenzyl group and related analogues. In earlier work, we explored the use of the nitrodibenzofuran (NDBF) group for thiol protection and found it to exhibit a faster rate toward UV photolysis relative to simple nitroveratryl-based protecting groups and a useful two-photon cross-section. Here, we describe the synthesis of a new NDBF-based protecting group bearing a methoxy substituent and use it to prepare a protected form of cysteine suitable for SPPS. This reagent was then used to assemble two biologically relevant peptides and characterize their photolysis kinetics in both UV- and two-photon-mediated reactions; a two-photon action cross-section of 0.71-1.4 GM for the new protecting group was particularly notable. Finally, uncaging of these protected peptides by either UV or two-photon activation was used to initiate their subsequent enzymatic processing by the enzyme farnesyltransferase. These experiments highlight the utility of this new protecting group for SPPS and biological experiments.

Synthesis and NMR Characterization of the Prenylated Peptide, a-Factor.

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins.