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. Author manuscript; available in PMC: 2025 Dec 12.
Published in final edited form as: Org Lett. 2000 Jun 1;2(11):1545–1547. doi: 10.1021/ol005856n

New Phototriggers:1 Extending the p-Hydroxyphenacyl π–π* Absorption Range

Peter G Conrad II 1, Richard S Givens 1, Jörg F W Weber 1, Karl Kandler 1,†
PMCID: PMC12697336  NIHMSID: NIHMS593992  PMID: 10841475

Abstract

Introducing 3-methoxy or 3,5-dimethoxy substituents on the 4-hydroxyphenacyl (pHP) photoremovable protecting group has been explored with two excitatory γ-amino acids, l-glutamic acid and γ-amino butyric acid (GABA). These substituents significantly extend the absorption range of the pHP chromophore, e.g., the tail of absorption bands of 2a,b extend above 400 nm, well beyond the absorptions of aromatic amino acids and nucleotides. Irradiation releases the amino acids with rate constants of ~107 s−1 and appearance efficiencies (Φapp) of 0.03–0.04. The photoproducts are formed through the pHP excited triplet and are primarily products of photoreduction and photohydrolysis. 1a,b also rearranged to the phenylacetic acid 3.

Graphical Abstract

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We report herein the use of 3-methoxy- and 3,5-dimethoxy-4-hydroxyphenacyl (MeO-pHP and (MeO)2-pHP) as new members of the p-hydroxyphenacyl (pHP) series of carboxylate and phosphate photoremovable protecting groups. Earlier we2 introduced the p-hydroxyphenacyl (pHP) phototrigger as an efficient, rapid photoremovable protecting group for applications in neurobiology, physiology, and biochemistry. Since its introduction, a variety of pHP derivatives have been employed for the release of ATP,2 glutamic acid,3,4 GABA,3 the dipeptide Ala-Ala,1 and the C-terminus of the nonapeptide bradykinin.1 Several in vitro biochemical studies have shown that the attachment of pHP to the γ-carboxyl group of glutamic acid or GABA or the C-terminal carboxylic acid group of peptides completely suppresses the normal biological activity of the natural substrates.1,3

In several studies, we have demonstrated that the photorelease occurs from the triplet state of the phototrigger since it can be quenched with either potassium sorbate or sodium 2-naphthalenesulfonate1–5 with excellent Stern–Volmer-derived kinetic rates of ~108 s−1 or faster for release for these substrates. A concomitant rearrangement of the phototrigger to p-hydroxyphenylacetic acid (Scheme 1) was shown to be the major pathway6 for the pHP chromophore. The rearrangement is preceded by an excited-state deprotonation7 of the phenolic proton yielding the triplet phenolate ion,5 the apparent driving force for the rearrangement.4

Scheme 1.

Scheme 1.

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Mechanism for the Photorelease from pHP

To expand the dynamic range for this phototrigger and to move the effective absorption away from the range of normal peptides and nucleotides, we have explored the effects of added methoxy substituents on the pHP chromophore and the resulting photochemistry. Our results are presented here.

The synthesis of the methoxy pHP esters of l-glutamate (1a, 2a) and of γ-aminobutyric acid (GABA, 1b, 2b) were accomplished by the DBU-catalyzed displacement of bromide from the appropriate α-bromoacetophenone followed by deprotection of the remaining amino acid protecting groups with TFA to give the methoxy phenacyl protected neurotransmitters 1a,b and 2a,b (Scheme 2).

Scheme 2.

Scheme 2.

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Synthesis of Methoxy pHP GABA’s and l-Glutamates

As shown in Figure 1, the absorption maxima for the new 3-methoxy-4-hydroxyphenacyl and the 3,5-dimethoxy-4-hydroxyphenacyl chromophores 1 and 2 are in fact shifted to 350 and 370 nm, respectively.

Figure 1.

Figure 1.

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Ultraviolet spectra of three caged l-glutamates, 3-methoxy-pHP 1a, 3,5-dimethoxy-pHP 2a, and pHP 3, and three caged GABA’s, 3-methoxy-pHP 1b, 3,5-dimethoxy-pHP 2b, and pHP 4.

Photolyses of 1 and 2 were performed in aqueous solutions with either 300 or 350 nm lamps (Scheme 3) and were monitored by HPLC or 1H NMR (D2O). The release of the amino acids occurred with quantum efficiencies8 of 0.03 to 0.04, somewhat below those that we reported for the parent pHP amino acids.3

Scheme 3.

Scheme 3.

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Photorelease of l-glutamic Acid and GABA from 1a,b and 2a,b

Photolysis of 2a in phosphate buffer (pH 7.2) in the presence of 0.001–0.010 M potassium sorbate quenched the photorelease of the amino acids and gave good Stern–Volmer kinetics (KSV = 343 M−1) resulting in a calculated triplet lifetime of 46 ns and rate constant for release of 2.2 × 107 s−1 (assuming kdiff = kq = 7.4 × 109 M−1 s−1).9

Phosphorescence spectra gave triplet energies of 69 kcal/mol for 1a and 71 kcal/mol for 2a. The phosphorescence emission for 1a was also quenched by potassium sorbate. These results clearly indicate a reaction pathway through the triplet excited state of these new phototriggers and are in accord with our recent results for p-hydroxyphenacyl (pHP).4 In contrast to the earlier results, however, rearrangement to the phenylacetic acid has become a minor pathway for 1 and is not observed at all for 2. These alternative photochemical pathways have been reported in a wide array of other phenacyl derivatives.10

The biological efficacy of these new phototriggers has likewise been tested using electrophysiological whole-cell patch clamp recordings for CA1 neurons in acute hippocampal brain slices4 and from cultured neocortical neurons.11 Photolysis12 of a 200 mM solution of 3,5-dimethoxy-4-hydroxyphenacyl GABA (2b) bathing a CA1 hippocampal neuron from a 7-day-old rat elicited a response from the cell as indicated by the detection of outward currents as shown in Figure 2. In all neurons tested (n = 12), photolysis of 2b consistently elicited fast membrane currents. These responses were due to the specific stimulation of GABA A-receptors (a ligand gated chloride channel) because application of Bicuculline abolished all inward currents. Figure 2 depicts the responses stimulated by the release of GABA from 2b.

Figure 2.

Figure 2.

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Peak amplitude of currents elicited by released GABA is plotted against stimulus number (interval between stimuli 20 s) and is the average of 10 consecutive traces. After stimulus #22, 10 M Bicuculline14 was added to the bath solution, which completely blocked the responses elicited by photorelease of GABA. The relatively constant amplitude (114.2 pA ± 1.4 SEM) for the 20 flashes prior to receptor shut down by Bicuculline demonstrates that the cell is not harmed by the transient exposure to the UV flashes or to the appearance of the phototrigger side products during photolysis.

These preliminary results for the 3-methoxy-4-hydroxyphenacyl and 3,5-dimethoxy-4-hydroxyphenacyl phototriggers demonstrate the potential for effective release of certain α-amino acids14 and for C-terminal protected peptides. Furthermore, release of amino acids, oligopeptide, and proteins in biological environments employing laser flash photolysis and other fast kinetic techniques using pHP phototriggers such as 1 or 2 is possible at λ > 300 nm and even beyond λ = 400 nm.

Supporting Information Available:

Details of the synthesis and 1H and 13C NMR, UV, IR, and HRMS data for 1, 2, and 6–14. This material is available free of charge via the Internet at http://pubs.acs.org. Complete experimental details and additional examples of substituted pHP derivatives will be provided in our full paper.

Acknowledgment.

This research was supported by the University of Kansas and by the NSF (OSR-9255223).

References

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