Photolabile precursors of biological amides: synthesis and characterization of caged o-nitrobenzyl derivatives of glutamine, asparagine, glycinamide, and .gamma.-aminobutyramide

Ramesh, Dharavath; Wieboldt, Raymond; Billington, Andrew P.; Carpenter, Barry K.; Hess, George P.

doi:10.1021/jo00069a021

Cited by 40 publications

(43 citation statements)

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“…Given the established photochemistry of the 2-nitrobenzyl group (8)(9)(10)(11)(12)(13)(14)(15)(16)) and the well characterized Shaker-IR phenotype (26)(27)(28)(29)(30)(31)(32)(33)(34)(35), these results provide convincing evidence that irradiation of Npg leads to peptide backbone cleavage of functional ion channels in vivo. Photolysis of the Npg-containing channels resulted in the liberation of the inactivation ball from the remainder of the channel, thereby slowing inactivation.…”

Section: Resultsmentioning

confidence: 69%

“…The long irradiation times required here indicate that Npg photochemistry may be less efficient than in other related systems (8)(9)(10)(11)(12)(13)(14)(15)(16). Additional work from our labs establishes that more conventional nitrobenzyl caging groups can undergo very efficient photolysis when incorporated into ion channel proteins expressed in Xenopus oocytes (J. Miller, S. K. Silverman, and P.M.E., unpublished work).…”

Section: Resultsmentioning

confidence: 81%

“…Compounds of this type, including Npg itself, have been used as protecting groups in organic synthesis and to produce ''caged'' neurotransmitters, ions, and second messengers that can then be liberated photochemically, but, to our knowledge, Npg has not been incorporated into peptides or proteins (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). As shown in Fig.…”

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confidence: 99%

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Site-specific, photochemical proteolysis applied to ion channels in vivo

England

Lester

Davidson

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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A method for site-specific, nitrobenzylinduced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K ؉ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the ''signature'' Cys128-Cys142 disulfide loop of the nAChR ␣ subunit.A powerful tool for probing protein structure and function is the incorporation of unnatural amino acids by the method of nonsense suppression, a technique developed for in vitro translation systems by Schultz and coworkers (1-3). Recent work has expanded the scope of this methodology by establishing procedures for unnatural amino acid incorporation into proteins expressed in living cells (4-7). This allows the evaluation of neuroreceptors, ion channels, and related proteins that are studied most effectively in vivo using heterologous expression systems such as the Xenopus oocyte. The suppression methodology is especially promising for such integral membrane proteins, which are not yet generally amenable to the methods of high resolution structure determination (e.g., NMR, x-ray crystallography).In this paper we (i) describe the unnatural amino acid 2-(nitrophenyl)glycine (Npg) and show that it can be efficiently incorporated into proteins expressed in vivo; (ii) establish that in vivo irradiation of an Npg-containing protein leads to site-specific, nitrobenzyl-induced photochemical proteolysis (SNIPP) (Fig. 1); and (iii) illustrate the usefulness of this residue by cleavage of the Cys128-Cys142 ''signature'' disulfide loop of the nicotinic acetylcholine receptor, establishing a crucial functional role for this structural feature.The design of the unnatural amino acid Npg was based on the photochemistry of 2-nitrobenzyl derivatives. Compounds of this type, including Npg itself, have been used as protecting groups in organic synthesis and to produce ''caged'' neurotransmitters, ions, and second messengers that can then be liberated photochemically, but, to our knowledge, Npg has not been incorporated into peptides or proteins (8-17). As shown in Fig. 1, incorporation of Npg into a protein, followed by irradiation of the protein, was anticipated to produce peptide backbone cleavage (18) dures (19-20).-(2-nitrophenyl)glycine was protected as the N-pent-4-enoyl (4PO) derivative using standard conditions (21, 22). Na 2 CO 3 (111 mg, 1.05 mmol) was added to a room temperature solution of (2-nitrophenyl)glycine hydrochloride (82 mg, 0.35 mmol) in H 2 O:dioxane (0.75 ml:0.5 ml), followed by a solution of pent-4-enoic anhydride (70.8 mg, ...

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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 81%

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Site-specific, photochemical proteolysis applied to ion channels in vivo

England

Lester

Davidson

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Measurements over the range of 15-55°C gave linear Arrhenius plots and activation energies of 40 + 2 kJ mol -~ for both compounds. These uncaging rates are approximately an order of magnitude faster than those for N-(2-nitrobenzyl) cages of glycine [14], glutamine [15] and 7-aminobutyric acid [16], but slower than for the uncaging of N-(1-(2-nitrophenyl)ethyl)carbamoylcholine iodide [13]. The activation energy for decay of the aci-nitro intermediates of I and II is slightly smaller than the value of 55 kJ mol -] measured for caged ATP [11] under similar conditions.…”

Section: Sa Johnson Et Al/febs Letters 380 (1996) 183-187mentioning

confidence: 86%

“…'Caged' compounds have a functional group modified with a suitable photoremovable protecting or caging group, such as the 2-nitrophenyl substituent [13]. Rates of uncaging of 2-nitrobenzyl-substituted molecules, including amines, via aci-nitro intermediates vary widely [13][14][15][16] but in some cases occur on a sub-millisecond timescale, enabling kinetic measurements to be undertaken on timescales similar to or faster than those using the stopped flow technique. We describe steady-state resonance Raman spectroscopy of the free radical site in pea seedling copper amine oxidase (PSAO).…”

Section: Introductionmentioning

confidence: 99%