Genetically encoded unnatural amino acids (UAAs) enable novel chemical and physical properties to be selectively introduced into proteins directly in live cells, which provides great potential for addressing biological questions at the molecular and cellular level in native settings.[1-3] UAAs have been genetically incorporated into proteins in mammalian cells using orthogonal tRNA-codon-synthetase sets,[4-6] yet the current low incorporation efficiency hinders their effective application. Efforts to improve efficiency have focused on optimizing the expression and activity of the orthogonal tRNA and synthetase,[5-7] whereas the bioavailability of the UAA inside mammalian cells, a prerequisite for incorporation, has not been addressed. In addition, there are many UAAs that have not been genetically incorporated into proteins successfully, such as glycosylated and phosphorylated amino acids. These amino acids can be invaluable for studying the contribution of posttranslational modifications to protein function and the role of a target protein in cellular signal transduction. Among many reasons, the inability of the UAA to enter cells prevents evolving a mutant synthetase specific for the UAA using cell-based selections or screens. Here we show that an UAA structurally deviating from the canonical amino acids in side chain could not be efficiently transported into mammalian cells, but masking the carboxyl group of the UAA as an ester greatly increased the rate of cellular uptake and intracellular concentration of the UAA. This resulted in a significant increase in the incorporation of this UAA into proteins in mammalian cells. Among three esters tested, acetoxymethyl ester (AME) yielded the highest UAA incorporation efficiency with a concomitant reduction of the UAA required in the growth media.
UAAs with side chains similar to canonical amino acids can be transported into cells by endogenous amino acid transporters,[3, 8] which are relatively nonspecific for substrates. However, UAAs significantly deviating from canonical amino acids in side chain structure may not be recognized by these transporters. Cell membranes are more permeable to neutral than charged molecules. As a zwitterion, UAA has only a very small proportion present in the neutral form at the physiological pH, making it difficult to cross the cell membrane. Esters have been widely used in prodrugs to derivatize carboxyl, hydroxyl and thio functionalities to promote membrane permeability of the parent drugs. In particular, Tsien et al. pioneered the use of AME to enhance cellular uptake of various charged molecules such as carboxylate-containing Ca2+ chelators and phosphate-containing second messengers.[11-12] Inspired by these examples, we reasoned that masking the carboxyl group of the UAA with an ester would convert the UAA into a protonated weak base, which has a higher percentage of neutral form and increased lipophilicity to translocate the membrane. Once inside the cell, intracellular esterases can cleave the ester to regenerate the original UAA for incorporation.
We demonstrated this strategy with UAA 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (DanAla, 1), which does not resemble and is bulkier in size than any canonical amino acid. Its fluorescence property also makes it an attractive candidate to develop optical reporters for imaging protein activities in live cells. We synthesized the methyl, ethyl and acetoxymethyl ester of DanAla using methods shown in Scheme 1b. Compound 5 was synthesized from Boc-Dap-OH and dansylchloride according to the known procedure. Alkylation of 5 with iodomethane and iodoethane gave intermediates 6 and 7, which afforded DanAla-OMe (2) and DanAla-OEt (3) after deprotection, respectively. However, in the presence of bromomethyl acetate and DIPEA, compound 5 was transformed to the undesired cyclization product 8, instead of Boc-DanAla-OAM. Therefore, the carboxyl group and the sulfonamide group were protected with the benzyl group and the Boc group, respectively. After debenzylation, carboxylic acid 10 was transformed to acetoxymethyl ester 11, which furnished DanAla-OAM (4) after deprotection of the Boc groups.
To determine if the DanAla esters could enhance the incorporation of DanAla into proteins in mammalian cells, we used an in cellulo fluorescence assay to evaluate the incorporation efficiency of DanAla.[6-7] The incorporation of DanAla into the green fluorescent protein (GFP) was measured using a stable clonal HeLa cell line, in which the GFP gene containing a premature UAG stop codon at a permissive site (Tyr182) was integrated into the genome. The cells were transfected with the orthogonal tRNA-synthetase pair specific for DanAla, and DanAla or DanAla ester was added in the growth media. The incorporation of DanAla at the UAG182 position results in full-length, fluorescent GFP, whereas no incorporation results in truncated, nonfluorescent GFP. The total fluorescence intensity of cells was measured by flow cytometry. As shown in Figure 1a, the cell fluorescence intensity increased when more DanAla was added to the growth media, but reached a plateau from 0.25 mM to 1.00 mM. The plateau of DanAla incorporation suggests that either the concentration of DanAla inside cells has saturated the synthetase or the cellular availability of DanAla is limiting. When DanAla-OMe was used, the cell fluorescence intensity doubled in comparison to cells incubated with same concentrations of DanAla, indicating that the cellular availability of DanAla was the limiting factor. Similar results were also obtained for DanAla-OEt. Nonetheless, there was no significant increase in cell fluorescence intensity when the DanAla-OMe or DanAla-OEt was increased from 0.10 mM to 0.25 mM. In contrast, DanAla-OAM doubled the fluorescence intensity at 0.10 mM and quadrupled it at 0.25 mM. In comparison to 1.00 mM of DanAla, the amount often used in UAA incorporation, the DanAla-OAM increased the cell fluorescence intensity 4 fold in addition to requiring 75% less compound in the growth media (0.25 mM). Western blot analysis of the cell lysates confirmed the increase in the amount of GFP produced by the AME modification (Figure 1b). These results suggest that all three ester modifications were able to increase DanAla incorporation into proteins with a concomitant reduction of the extracellular supply of UAA. Furthermore we identified DanAla-OAM as the most effective out of the three esters tested.
To understand how DanAla-OAM increases DanAla incorporation, we first monitored the uptake of DanAla and DanAla-OAM into cells using fluorescence microscopy. We added 0.10 mM of DanAla or DanAla-OAM to the media of HEK293T cells and measured the cytosolic fluorescence intensity of cells at sequential time points using confocal microscopy (Figure 2a). For cells incubated with DanAla, the cytosolic fluorescence intensity was almost flat in the range of 620-750 AU. A striking difference was observed in the DanAla-OAM incubated sample. Within 5 mins, the intracellular fluorescence intensity rapidly rose to 1100 AU, a 48% increase from the DanAla sample. The intensity increased for 3 hrs peaking at 1613 AU, which was 2-fold of the DanAla peak intensity. The intensity of DanAla-OAM treated cells then gradually dropped, possibly due to the depletion of the extracellular DanAla-OAM in combination with the equilibration of converted DanAla in and outside of cells. The AME modification thus significantly accelerates the cellular uptake rate of the compound.
Next we quantified the intracellular concentrations of DanAla and DanAla-OAM using HPLC. HEK293T cells were incubated with 0.10 mM of the compounds for 1 hr, and cell contents were extracted. Small molecules in the cell extracts were separated by HPLC, and peaks corresponding to DanAla and DanAla-OAM were verified using pure compounds and mass spectrometry. Surprisingly, after only 1 hr incubation, cells incubated with DanAla-OAM showed no peak for DanAla-OAM but a large peak for DanAla (Figure 2b), indicating that all intracellular DanAla-OAM had been hydrolyzed to DanAla. The peak area for DanAla was used to determine its intracellular concentration. Cells incubated with DanAla and DanAla-OAM had 0.28 mM and 8.9 mM intracellular concentration of DanAla, respectively. The AME modification dramatically increased the intracellular concentration of DanAla by 31-fold.
When the concentration of DanAla-OAM was further increased to 0.50 mM, the cell fluorescence intensity decreased unexpectedly (Figure 1a). We used propidium iodide (PI) staining to assess the health of cells incubated with DanAla or DanAla-OAM (Figure 2c). For cells treated with DanAla from 0.10 mM to 0.50 mM, the percentage of PI positive cells was similar to that of cells without UAA. A slight increase of PI positive cells was observed at 1.00 mM of DanAla. In contrast, cells treated with DanAla-OAM showed significantly higher percentage of PI positive cells, especially at the concentration of 0.50 mM. DanAla-OAM hydrolysis releases formaldehyde, which will negatively affect cell health at high concentration. High intracellular concentration of DanAla may be toxic to cells as well. Therefore, an optimal concentration of the AME ester should be used to achieve highest UAA incorporation efficiency with minimal negative effect to cell health.
In summary, we demonstrated that masking the carboxyl group of DanAla with AME increased the uptake rate and intracellular concentration of the UAA in mammalian cells. This resulted in a higher DanAla incorporation efficiency accompanied by the added benefit of reducing the amount of UAA in the growth media. Efficient UAA incorporation would prove valuable for the effective application of genetically encoded UAAs in studying various biological processes in mammalian cells. Modification of UAAs with AME may be generally applicable to other UAAs that are difficult to enter mammalian cells. To test this hypothesis, more unnatural amino acids need to be modified and their incorporation efficiency determined. However, for these candidate unnatural amino acids there are currently no orthogonal tRNA-synthetase available for their incorporation, because a synthetase cannot be evolved for an UAA that cannot readily enter the cell. We are applying the esterification strategy to highly polar amino acids such as glycosylated and phosphorylated amino acids to overcome this problem with the final goal to genetically incorporate these biologically important amino acids into proteins.