Tyrosine-derived stimuli responsive, fluorescent amino acids

A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and meta-phenylenevinylene (m-PPV) backbone have been synthesized by palladium-catalyzed Heck couplings.


Introduction
Fluorescence spectroscopy emerged as a powerful tool to investigate complex biological processes such as enzyme activity, protein structure and their interaction with other proteins and nucleic acids. 1 Among the other natural amino acids, tryptophan and tyrosine serve as intrinsic uorescent probes 2 to monitor the above-mentioned processes but suffer from poor optical properties. Therefore continuous efforts have been made to design unnatural amino acids (UAAs) with various uorophores and successfully incorporated into peptides and proteins in a site-specic manner to study their structurefunction properties. 3 Furthermore, relatively smaller size and ease of synthesis of UAAs, as compared to their uorescent protein cohorts, allows chemists ne-tune the structure to obtain desired optical properties. Besides, UAAs bearing functionalities are stimuli-responsive serve as uorescent reporters of micro-environmental changes such as pH, polarity, and redox. 4 Owing to the advancement in synthetic biology methods and imaging techniques, there is always an imperative need for enrichment of UAAs' toolkit encompassing a variety of uorescent scaffolds with diverse spectroscopic properties, shapes and sizes. As the assimilation of synthetic chemistry, biology and imaging furthers, the development of novel uorescent UAAs will continue to be at the forefront to aid the researchers in understanding the fundamental yet complicated biological functions such as protein interactions, recognition, and biosynthesis.
From the synthetic chemistry point of view; two common approaches can be adopted to synthesize the uorescent probes in the form of UAAs, (i) integrating known uorophores into the side-chains of a-amino acids. 5a For example, a polarity-sensitive uorescent UAA, L-Anap was synthesized via covalent attachment of naphthyl uorophore to the hydroxyl group of L-serine using Fukuyama-Mitsunobu reaction, 5b and (ii) constructing a whole new chromophore on a natural amino acid. For example, coumarin-bearing uorescent UAAs were derived from aspartic and glutamic acids; 6 2-(2-furyl)-3-hydroxychromone to probe peptide-nucleic acid complexes was synthesized from L-tyrosine. 7 Latter approach has the advantage of being relatively nonperturbing replacements for the native residues, thereby maintaining the overall native structure of a target peptide or protein. 1a Additionally, a higher chemical stability is expected if the uorophore is linked to the amino acids by a side-chain carbon-carbon bond.
As shown in Fig. 1, augmenting the p-conjugation in tyrosine/phenylalanine to generate structurally novel uorescent UAAs is a unique approach. These UAAs (A-C) showed improved optical properties than the corresponding tyrosine/ phenylalanine amino acids but suffered from multistep synthetic routes and lack of tunability in emission property. 8 Recently, p-conjugated organic systems have received a lot of interests owing to their tunable physical, optical and electronic properties through tailored synthesis of conjugated structures with backbone and/or side chains that renders desired properties. 9 Due to their planar and semi-rigid backbone and p-p stacking potential, these molecules have the propensity to form aggregates that show distinct electronic and optical properties. The physical and optical properties of conjugated molecules can also be ne-tuned by varying the nature of the substitution groups on the terminal phenyl rings. 10 In addition to application in electronic, optical and energy devices, conjugated oligomers are also extensively used in biological and medicinal chemistry. 11 For example, the use of styrene-based compounds as imaging agents and inhibitors of beta amyloid brils is well documented. Recently, Anna and co-workers reported on the synthesis of thiophene-based conjugated oligomers bearing Lamino acid and their use as optical probes for detection of amyloid bril formation in insulin. 12 Encouraged by recent developments in uorescent UAAs, we envisage that the transformation of amino acids into conjugated systems would lead to a whole new class of uorescent UAAs with desired optical properties (Fig. 1). In this regard, herein, we report the design and synthesis of novel a-amino acid analogs constructed via extending the p-conjugation of Ltyrosine. These novel uorescent UAAs consist of stilbene and meta-phenylene vinylene units as uorophores and cover the emission color from blue to near IR. Another unique structural feature is the incorporation of hydroxyl group (phenol) that renders stimuli responsive optical properties. More interestingly, we have observed distinct red, green and blue (RGB) emission spectra simply by controlling the solution pH. We also show the use of these UAAs in solid-phase peptide synthesis (SPPS) to synthesize a cell-penetrating peptide and demonstrate the use of these uorescent peptides for cell imaging.

Results and discussion
Synthesis of p-conjugation extended L-tyrosine amino acids The mono-and bis-styryl-L-tyrosine analogs (compounds 4a-f and 5a-g) were synthesized using strategies shown in Scheme 1. The syntheses of these amino acids have begun with the transformation of commercially available 3-iodo-L-tyrosine (1a) and 3,5-diiodo-L-tyrosine (1b) into their corresponding t-Boc/OMe protected amino acids (2a and 2b, respectively) by following the established literature procedures. Starting from natural amino acid is particularly advantageous because of two reasons: 1 avoiding expensive chiral auxiliaries, which are usually required for the diastereoselective synthesis and thus minimizing the number of steps in synthesis, 13 and 2 the ability to secure the chiral information at the beginning of the synthesis. 14 Although bis-styryl conjugated compounds can be obtained by a variety of synthetic pathways, the use of Heck reactions to synthesize E-congured styryl compounds has been proved to be most promising. 15 Palladium catalyzed mono/double Heck couplings between 2a/2b and appropriate styrene (3a-f) afforded protected tyrosine analogs 4a-f and 5a-g, bearing meta-phenylenevinylene backbone in moderate to good yields. Mindful selection of styrene precursors with different electron-withdrawing and electrondonating groups gave an access to thirteen uorescent UAAs with different end groups, which allowed us to better understand the interplay between dipole interactions and aggregate formation, and fully assess their impact on the electronic and optical properties of p-conjugated UAAs. It is noteworthy that all the Heck couplings proceeded smoothly even without protecting the phenol group, thus minimizing the number of steps in synthesis. Reaction times for the Heck couplings depend on the substituents on the styrene compounds. In general, the presence of electron withdrawing groups at para-position of the styrene analog requires longer reaction times with slightly decreased yields.
We observed that all the Heck couplings progressed with sufficiently high selectivity to generate E-isomers as established by the NMR spectra. The trans-relation of the double bonds was established on the basis of the coupling constant for the vinylic protons in the 1 H NMR spectra (J $ 16 Hz, ESI †).
All of these amino acids are stable as solids at room temperature and can be stored without the need of any special precautions. Absorption and uorescence emission, extinction coefficients, uorescence quantum yields (QY), and optical rotations were measured for each compound and presented in Table 1. The optical properties of L-tyrosine were included for comparison. 16

UV/vis and uorescence spectroscopy
The optical properties of the uorescent amino acids, 4a-f and 5a-g were measured by UV/vis and uorescence spectroscopy in DMSO at room temperature. The results are shown in Fig. 2 and 3.

Mono-styryl-L-tyrosine analogs
We observed two characteristic absorption peaks in the UV-vis spectra of all mono-styryl analogs demonstrating the distinct electronic transitions. For example, all six amino acids have a higher energy absorption band with a l max at roughly 300 nm. For the low energy absorption peak, l max varies from 330 nm to 390 nm depending upon the nature of the para-substitution on the styrene ring.
For all these analogs, photoexcitation at the wavelength that corresponds to both low and high energy peaks show the same emission maxima, but the excitation of the low-energy peak resulted in higher emission intensities. We observed that the DMSO solutions of all amino acids in this series emitted strongly in violet region (400 nm to 435 nm), regardless the nature of substitution group on the styrene ring. The NO 2 compound 4d found to be nonemissive in highly polar (both protic and aprotic) solvents, such as DMSO, methanol, where as it is emissive in non-polar solvents such as chloroform and THF with an emission maxima 400 nm. Compounds 4e-f having electron withdrawing groups (CF 3 and Py) showed a weak shoulder band in emission spectrum at around 590 nm.
Our mono-styrene analogs emit at higher wavelengths (violetblue) due to their short p-conjugation lengths is consistent with literature observation. 17 As illustrated in Fig. 2, these amino acids are derived from tyrosine and composed of a phenol moiety that functions as a latent donor. Deprotonation of phenol leads to the formation of a phenolate active donor that donates a pair of p-electrons into the p-system and thus forming an extended p-conjugated system. As a result of this, a red shi in the emission of these amino acids was observed with increased emission intensities. Moreover, deprotonated phenol also gives rise to a stronger dipole, which facilitates the red shi of emission spectra in a polar solvent. Addition of a base to the DMSO solutions of these amino acids resulted in a red shi in their emissions pushing the emission maxima from 400-435 nm (violet) nm to 510-600 nm (green-orange). This result is in agreement with generation of the phenolate anion, while the spectra of phenol-protected derivatives unaltered by addition of base (ESI †).
Scheme 1 Synthesis of fluorescent unnatural amino acids 4a-f and 5a-g starting from tyrosine.

Bis-styryl-L-tyrosine analogs (Tyr-OMPVs)
Addition of another phenylene vinylene to the meta position of stilbene derivatives leads to a new class of meta-phenylene vinylene bearing amino acids (5a-g). All these amino acids showed absorption wavelengths (l max ) in the UV or visible region ranging from 300-430 nm (Fig. 3). In some cases, multiple absorption bands of lower energies were observed. For example, compounds 5d, 5f and 5g showed the absorption peaks at lower energies (620 nm and 520 nm respectively, Fig. 3). The emergence of the low energy peak is likely due to the donor-acceptor characteristic of the molecules, which causes the HOMO and LUMO to merge, producing smaller band gap energy. 10 As shown in Fig. 3, upon photoexcitation at a wavelength that corresponds to the absorption maxima, all these compounds showed a strong emission in the visible region encompassing the whole visible spectrum (from 400-800 nm), is a key feature of these amino acids. Compounds 5a and 5e, possessing H and SO 3 Na groups, respectively, showed emission  in the blue region. Emission spectra of these compounds also have a small shoulder near 600 nm. As 5a and 5e showed a broad emission starting from 350 nm to 700 nm; these compounds looked more whitish when visualized under UV lamp. Compound 5b and 5c possessing electron-donating groups such as OMe and NH 2 , displayed blue and green emission at 420 nm and 500 nm, respectively. When compared to the other analogs in this series, compound 5d, bearing NO 2 groups on para-positions of the terminal phenyl rings, was not emissive in polar solvents such as DMSO and methanol. However, in apolar solvents such as THF and chloroform, it was emissive with a l max at 512 nm. 5d in polar solvents is nonemissive which is attributed to a complex interplay of single molecule and aggregate emission observed for this particular NO 2 containing compounds. 5d aggregates in DMSO have been detected by dynamic light scattering (DLS) which shows a bimodal distribution with aggregate size of $100 nm. While in THF, compound 5d is mostly monodispersed, non-aggregated species (Fig. S1 in ESI †). The quantum yields of these UAAs in DMSO range from 94% to 4% depending on the solubility and functional groups attached to the end of the styryl part. A red shi of the emission bands was observed when the terminal phenyl rings were replaced with pyridine rings. Compound 5f showed emission in the red region (630 nm) and this emission behavior could be due to the combined effect of the deprotonation of phenol and formation of aggregates. The aggregation behavior of 5f is further validated by concentration dependent uorescence experiments as well as DLS experiments. The aggregate-associated change in PL was conrmed by the concentration experiments, in which we measure PL spectra of 5f in DMSO at concentrations ranging from 1 mM to 100 nM concentrations (Fig. S2, ESI †). At concentrations above 100 nM, the PL emission is dominated by with a l max at 630 nm and a small shoulder band at 430 nm. But, at 100 nM and lower concentrations, we observed that the peak at 630 nm was almost disappeared and the emission peak at 430 nm became dominant. The above results suggest that the blue emission at 430 nm and the red emission at 630 nm are associated with a single molecule and aggregate emission, respectively. It is very important to note that although aggregation induced red shi in uorescence emission with low quantum yield is a wellknown phenomenon in conjugated polymers/oligomers, 18 5f aggregates actually has a fairly high quantum yield comparable to that of its single molecule species. We believe this is probably due to the formation of linear aggregates through Hbonding interaction, rather than p-p interaction between phenyl rings, which typically leads to the quenching of uorescence.
On the other hand, compound 5f showed the blue emission in apolar solvents such as THF (Fig. S3, ESI †) and chloroform, suggesting that these solvents do not facilitate the formation of aggregation as the blue emission is solely coming from the single molecule/non-aggregated species. Compound 5f aggregates have been detected by dynamic light scattering (DLS) with the size of $70 nm in DMSO. While under diluted concentrations (<100 nM) in DMSO and in THF, no noticeable aggregate formation was observed. This result is in agreement with the concentration dependent uorescence spectra, which suggest predominant single molecule species at lower concentrations (<100 nM).

Stimuli response
Red, green, blue (RGB) emission. It has been shown that molecules comprise of nitrogen containing heterocyclic rings such as pyrimidines showed the ability to function as colorimetric and luminescent pH sensors due to the basic character of the nitrogen atoms of the pyrimidine ring. 19 Such character prompts us to study the effect of protonation/deprotonation on the optical properties.
One of the very interesting structural characteristics of 5f is that it contains basic nitrogen atoms that can be protonated and a phenol group that can be deprotonated. Thus, the effect of protonation/deprotonation on the optical properties was found to be particularly interesting. In acetonitrile, 5f underwent a signicant visible color change upon the addition of acid pTSA (p-toluenesulfonic acid) or base (NaOH). Basic, acidic, and neutral solutions of 5f showed red green and blue (RGB) emission respectively, see Fig. 4. Visible/emission color change is fully reversible by neutralization with an acid/base. The emission spectra in acid and basic solutions show a clear red shi when compared to the neutral solution. Emission peak at 535 nm corresponds to the protonated species whereas; the peak at 630 nm represents the phenolate structure.
As expected, most of the compounds exhibit a red shi of their absorption (Fig. S4, ESI †) and emission bands upon addition of pTSA and can be explained by the protonation enhances the accepting effect of the pyridine, thus increasing intramolecular charge transfer from the donor to the pyridinium moiety (acceptor). 20 For the protonated species, the emission is partially quenched (QY ¼ 10%) when compared to its neutral state (QY ¼ 45%). Conversely, addition of NaOH enriched the phenolate population and as a result of extended conjugation, compound 5f exhibited a more intense (QY ¼ 57%) and red shied emission at 630 nm. UAA with pH dependent uorescence emission exhibits distinct red, green and blue color (RGB) with decent quantum yield suggests implications toward sensing, bioimaging and LED devices. 21

Solvatochromism
Compound 5d comprised of terminal NO 2 group showed interesting solvatochromic property. 10 Though the uorescence emission of 5d was quenched by some of the polar solvents, its emission was strong in apolar solvents. Specically, 100 mM solutions of NO 2 -terminated analog 5d in acetonitrile, acetone, THF, dioxane and chlorobenzene exhibits a strong solvatochromism. While the l max of the absorption spectra for the 5d show a relatively small change (<20 nm) among all solvents, the corresponding emission spans the wavelengths from 480 to 570 nm, covering blue, green, yellow and orange region of visible spectrum (Fig. S5, ESI †).
Such strong solvatochromism occurs due to a combination of dipole-solvent interactions, intramolecular charge transfer, and aggregate formation in solution. The above result is also consistent with previously reported observations by our group and others where the conjugated oligomers possessing stronger acceptor end group(s) showing pronounced solvatochromism due to solvent stabilization of intramolecular charge transfer in the excited state. 10 pH and redox response It is well established that perturbations in cellular redox status and pH have important physiological and pathological ramications. 4,22 Investigative tools to monitor these cellular responses are critical to further our understanding of the role of these processes in disease states. One important structural feature of these UAAs is the presence of the phenol group, which is particularly advantageous as it offers stimuli responsive properties in the following ways: (1) interchange between the phenol (acidic) and phenolate (basic) leads to a signicant change in optical properties by varying the pH of the solution; and (2) when treated with an oxidizing agent, such as peroxide, the phenol undergoes oxidation to form the corresponding ketone, which causes perturbation in the conjugation length and thus effecting the optical properties. Since the optical responses of Tyr-OMPVs are reversible, these intrinsic properties were used to explore their potential application as pH and redox stimuli responsive sensors.
A solution (DMSO-water 1 : 4 v/v) of 5b was used to explore the stimuli responsive properties. As shown in (Fig. S6 to S8, ESI †) and Fig. 5, the absorption and emission spectrum of compound 5b was sensitive to pH and redox stimuli. The absorption and emission spectra were recorded for this compound at two pH values (pH ¼ 4.0 and 9.0). Emission of 5b red shied (from 440 nm to 520 nm) from pH 4 to 9, and its uorescence intensity was much higher in the phenolate form than in the phenolic form. To establish the redox sensitivity, compound 5b was subjected to an oxidation-reduction cycle in which ammonium persulfate (APS) was used as an oxidizing agent and hydrazine as a reducing agent. The addition of 2 mM of APS nearly completely quenched the uorescence of 5b, whereas the uorescence was mostly recovered by the addition of 2 mM hydrazine. Note that uorescence recovery was not 100% as the oxidized 5b may have possibly reacted with moisture in the solution.

NIR emission
Recently, Shabat and co-workers have reported on the design of NIR probes based on a D2A p-electron system that can undergo intramolecular charge transfer (ICT) to form a new uorochrome with a longer p-conjugated system. In their design, phenol acts as a latent donor when it is conjugated with two acceptors. Deprotonation of the phenol transforms it into an active phenoxide donor, which donates a pair of p electrons to either one of the conjugated acceptors to exhibit ICT and thus emit at NIR region. 23 As all of the molecules in this study were derived from tyrosine and contain a phenol ring, it is possible to design UAA chromophores with tunable and reversible optical properties wherein the emission extends into NIR by choosing the appropriate end group. Having a water-soluble NIR dye in the form of an amino acid could be useful for various imaging-related applications. Methylation of the pyridine moiety in compound 5f gave a new analog 5g, which comprises a phenol latent donor and two acceptors in the form of a pyridinium moiety.
As described in Fig. 6, upon deprotonation of the phenol, an aqueous solution of compound 5g showed an emission peak in the NIR region at a wavelength of 700 nm. It is known that the presence of the strong acceptor moieties in the dye decreases the pK a of the phenol. Hence, the deprotonation of phenol of 5g occurs under physiological pH and emits NIR uorescence via ICT mode of action (Fig. S10, ESI †). Conversely, the NIR emission was diminished in acidic conditions (pH 2) due to the protonation of the phenolate. Fluorescence emission of 5g in DMSO was further red-shied to NIR region (800 nm), but the quantum yield decreased to 0.002, whereas it showed the moderate in context of NIR emission 24 quantum yield (0.04) in aqueous solution.

Utility in solid phase peptide synthesis (SPPS) and cell imaging
In general, uorescently labeled peptides are oen obtained by the conjugation of the uorescent dye through post-synthesis modications. These typical bioconjugations oen require uorescent dye containing amine-or thiol reactive group which can be problematic in peptides bearing more than one amine or thiol group, presenting the possibility of generating either multiple labeled species or mixtures bearing different labeling patterns. In this regard, the use of uorescent UAA 5b allows for the direct and site-specic introduction of the uorescent moiety as an integrated part of the SPPS process (Scheme 2, ESI †). In order to evaluate the utility of these amino acids in Fmoc-based peptide synthesis, an Fmoc analog of compound 5b was synthesized and incorporated into a cell penetrating Baxinhibiting pentapeptide 24 (sequence ¼ W*VPALK; W* ¼ 5b).
Peptide 1 was further characterized by UV-Vis and uorescence spectroscopy over a range of pH values and the excitation and emission spectra are shown in Fig. S9, ESI. † To assess both the cell permeability and uorescent properties, peptide 1 was incubated with a human epithelial cell line (HeLa) and mouse broblast cells (NIH 3T3) for 3 h and internalization was visualized using laser scanning confocal microscopy (Fig. 7), thus validating the use of these amino acids as intrinsic uorescent labels.

Conclusions
In conclusion, we have developed a direct and facile synthesis of optically pure, uorescent amino acids, using inexpensive natural amino acids as starting material avoiding expensive chiral auxiliaries and lengthy synthetic protocols. The combination of amino acid functionality and stimuli responsive meta-phenylene vinylene moiety in a molecule gives rise to a whole new class of uorescent amino acids that display a broad range of optical properties extending into NIR region. Our synthetic platform utilizes simple Heck coupling reaction and represents a useful tool in the preparation of novel uorescent amino acids, which can be easily incorporated into peptides and used in biological studies. The stimuli responsive nature of these UAAs is resulting from the change in the resonance structure upon variation of solution pH and redox states of the molecules; the UAA has a green color and higher quantum yield in high pH and reduced state, and exhibit blue color and lower quantum yield in low pH and oxidized state. Compound 5f contains basic nitrogen atoms that can be protonated and a phenol group that can be deprotonated exhibits distinct red, green and blue (RGB) emission spectra simply by controlling the solution pH. Given the stimuliresponsive nature and the ability to emit NIR uorescence, our UAAs exhibit a wide range of applications include fabricating optoelectronic devices, probing the cellular processes and other aspects of biological mechanism and function can be expected.