3.1.1 6-Nitroveratryl-based caged amino acids
The nitroveratryl (NV) group is a derivative of the o-nitrobenzyl group first introduced as a PPG in 1970.[178] Methoxy groups in 4- and 5- positions induce a bathochromic shift of the absorption maximum enabling cleavage at ~360 nm. However, shorter wavelengths are more effective.[179] Photolysis occurs under the mechanism shown in Scheme 3.1Scheme 2.1.[180]
Scheme 3.1: Photorelease mechanism of NV protected groups.[180]
Irradiation of a NV-caged compound (a) elevates the nitroveratryl group to an exited state (b). Through intramolecular proton shift the aci-intermediate (c) is formed which converts to the isoxazolidin-1-ol intermediate (d) by irreversible cyclization. Ring opening product hemiacetal (e) is hydrolyzed in the rate-limiting step releasing the leaving group.
Byproduct of the photolysis is o-nitroso veratraldehyde (f), a potential toxin to
31 surrounding bioprocesses. Despite poor quantum yield (Φ = 0.006, 365 nm),[179] NVOC is one of the most popular PPGs both for applications in solution and on solid support.
Due to its straightforward and cost-effective synthesis it was tested in this project.
Fmoc-L-Lys(NVOC)-OH (4) was synthesized as part of a related master thesis in a two-step procedure (Scheme 3.2).[181] 6-nitroveratryl alcohol (5) was reacted with 4-nitrophenyl chloroformate (6) to produce activated anhydride (7). Nucleophilic attack from the ε-amino group of Fmoc-L-Lys-OH at the carbonate gave Fmoc-L -Lys(NVOC)-OH (4) with an overall yield of 42%.
Scheme 3.2: Synthesis overview of Fmoc-L-Lys(NVOC)-OH.[181]
Deprotection rate was evaluated with the available cleavage setup by method a) (see section 7.1.5) in methanol (3.4 mM) and followed by analytical HPLC (see the appendix, Figure-A 1). Solvent and concentration were chosen diverging from the final application conditions (HEPES buffer, ~0.7 µM) to effectively dissolve the amino acid and clearly observe it in analytical HPLC. After 10 min of irradiation only a trace of the caged amino acid could be detected.
Despite being a common protecting group for carboxylic acids, NV was disqualified from being used on glutamic acid for not withstanding reaction conditions of solid phase peptide synthesis.[182] Alternatively, the closely related 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl (DMNPB) group was chosen to protect the glutamic acid residues.[183,184] It has been developed to overcome some of the drawbacks of NV by having a higher quantum yield (Φ = 0.26, 365 nm) and a less toxic photolysis byproduct.[184] In literature, DMNPB protected carboxylic acids (g) are reported to traverse aci-nitro intermediate (h) and finally release free carboxylic acid and nitrostyrene derivative (i) (Scheme 3.3).[184]
However, uncaging tests in methanol (3.4 mM) revealed multiple product peaks in analytical HPLC analysis (see the appendix, Figure-A 2), suggesting a more diverse byproduct composition. On the other hand, the caged amino acid was completely consumed after 5 min of irradiation, confirming a more effective photorelease.
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Scheme 3.3: Photorelease of a DMNPB protected carboxylic acid.[184]
Synthesis of the SPPS-ready Fmoc-L-Glu(DMNPB)-OH (8) was adapted from WIRKNER
et al.[183] with minor changes and performed by HOA NAM NGUYEN for his bachelor thesis (Scheme 3.4).[185]
Scheme 3.4: Synthesis overview of Fmoc-L-Glu(DMNPB)-OH.[185]
First, commercially available 3,4-dimethoxy-phenylacetone (9) was methylated with NaH and methyl iodide to give ketone (10) as a racemic mixture. Postponing reduction of the carbonyl until after nitration allowed the use of aggressive nitrating acid to give compound 11 in high yields. Reduction to 12 could then be chemoselectively achieved with NaBH4. STEGLICH esterification connected the protecting group to the glutamate side chain to produce 13 and acidic hydrolysis of tBu and Boc followed by Fmoc protection of the amine yielded Fmoc-L-Glu(DMNPB)-OH (8) as a threo/erethro mixture with a total yield of 19%. It is important to note, that loss of more than 50% in yield in the final exchange of protecting groups could have been avoided by use of commercially available Fmoc-L-Glu-OtBu in the STEGLICH esterification. This has been considered in following synthesis strategies.
33 3.1.2 7-Diethylamino-4-methylcoumarin (DEACM) protected amino acids After NVOC and DMNPB protected SNARE-analogs were tested in fusion experiments and were found to be released too slowly (see section 3.3.3), a different type of caging group had to be chosen. DEACM was predicted be a good choice, especially to substitute NVOC, for its high quantum yield, high excitation wavelength (DEACM-cAMP, Φ = 0.24, 395 nm) and ultrafast release.[186,187] Uncaging can be efficient at wavelengths as high as 420 nm making it suitable for future microscopic applications as many confocal microscopes are equipped with a 405 nm laser. Furthermore, DEACM had already been established for caging both amines and carboxylic acids.[188,189]
In the mechanism of photolysis which is accepted to be common for (coumarin-4-yl)methyl esters (Scheme 3.5)[190,187], water plays a critical role. The heterolytically cleaved methylene-O-bond (l) will recombine if not scavenged by a nucleophile, for example water.
Scheme 3.5: A) Proposed mechanism of photocleavage for (coumarin-4-yl)methyl esters. In photoexcited k heterolytic bond cleavage can occur (k1) or deactivation by fluorescence or nonradiave procecces (kfl+knr). Recombination of l (krec) competes with solvent separation (kesc) and subsequent reaction with water (khyd). Published in: J. Phys. Chem.
A 2007, 111, 5768-5774. Copyright © 2007 American Chemical Society.[190] B) Translation of the proposed mechanism to DEACM caged amine. Final decarboxylation reveals the free amine.
As the utilization of DEACM caged glutamic acid in SPPS had previously been reported[188] and DEACM caged lysine was not yet literature known, first, Fmoc-L -Lys(DEACM)-OH (14) was synthesized (Scheme 3.6) and tested with regard to SPPS and photocleavage. Following a synthesis procedure by ZHANG et al,[191] 7-Diethylamino-4-methylcoumarin (15) was oxidized with SeO2 in xylene by heating to reflux for two days and the intermediate aldehyde was reduced to alcohol 16 with sodium borohydride.
The use of these specific experimental conditions is emphasized, as other procedures with lower boiling point solvents and longer reaction times have been published,[192,193] but this procedure gave the best effort to yield ratio. Also, upscaling to more than 5 g is not recommended as selenium side products accumulate at the glass walls and stirring rod, interfering with stirring, and yields are reduced. Activation of the alcohol and conjugation to Fmoc-L-Lys-OH was achieved analogous to compound 4.
34
Scheme 3.6: Synthesis route to produce Fmoc-L-Lys(DEACM)-OH.
Short test peptide 18 (Scheme 3.7) was synthesized by manual SPPS and no major side reactions were observed. The caged peptide was used to determine photocleavage efficiency under the available experimental setup (method a, section 7.1.5). Owing to the high extinction coefficient of DEACM (16000 mol-1 cm-1)[194] a lower concentration was traceable by UPLC. Furthermore, considering the photorelease mechanism, the uncaging was expected to proceed best in aqueous medium so a 3.6 µM solution in HEPES buffer pH 7.4 was tested (see the appendix, Figure-A 3). Within 1 min, the caged peptide was consumed and the formation of photocleavage byproduct DEACM-OH was completed.
Additionally, peptides 19-21 (Scheme 3.7) were synthesized, to determine whether the cage would be available for photocleavage if the coiled coil was formed. The coiled coils were also chosen to test cleavage with a hand-held 405 nm 100 W laser pointer (method c, section 7.1.5), that would allow to condense the cleavage procedure in the fusion experiments. Uncaging proved to be complete within one minute (see the appendix Figure-A 4).
35
Scheme 3.7: Overview of test peptides used to assess photocleavage efficiency of DEACM caged lysine.
DEACM caged glutamic acid was synthesized by STEGLICH esterification of DEACM-OH (16) and Fmoc-L-Glu-OtBu as depicted in scheme 5.8. Acidic deprotection of the C-terminus produced Fmoc-L-Glu(DEACM)-OH (23) in an overall yield of 50%.
Scheme 3.8: Synthesis of Fmoc-L-Glu(DEACM)-OH.