Pradeep
Cheruku
a,
Jen-Huang
Huang
b,
Hung-Ju
Yen
a,
Rashi S.
Iyer
b,
Kirk D.
Rector
a,
Jennifer S.
Martinez
c and
Hsing-Lin
Wang
*a
aC-PCS, Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. E-mail: hwang@lanl.gov
bDefense System and Analysis Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA
cCenter of Integrated Nanotechnologies (CINT), Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA
First published on 31st October 2014
A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and meta-phenylenevinylene (m-PPV) backbone have been synthesized and their optical properties were studied. These novel UAAs were derived from protected diiodo-L-tyrosine using palladium-catalyzed Heck couplings with a series of styrene analogs. Unlike the other fluorescent UAAs, whose emissions are restricted to a narrow range of wavelengths, these new amino acids display the emission peaks at broad range wavelengths (from 400–800 nm); including NIR with QY of 4% in HEPES buffer. The incorporation of both pyridine and phenol functional groups leads to distinct red, green, and blue (RGB) emission, in its basic, acidic and neutral states, respectively. More importantly, these amino acids showed reversible pH and redox response showing their promise as stimuli responsive fluorescent probes. To further demonstrate the utility of these UAAs in peptide synthesis, one of the amino acids was incorporated into a cell penetrating peptide (CPP) sequence through standard solid phase peptide synthesis. Resultant CPP was treated with two different cell lines and the internalization was monitored by confocal fluorescence microscopy.
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 fluorescent scaffolds with diverse spectroscopic properties, shapes and sizes. As the assimilation of synthetic chemistry, biology and imaging furthers, the development of novel fluorescent 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 fluorescent probes in the form of UAAs, (i) integrating known fluorophores into the side-chains of α-amino acids.5a For example, a polarity-sensitive fluorescent UAA, L-Anap was synthesized via covalent attachment of naphthyl fluorophore 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 fluorescent 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 fluorophore is linked to the amino acids by a side-chain carbon–carbon bond.
As shown in Fig. 1, augmenting the π-conjugation in tyrosine/phenylalanine to generate structurally novel fluorescent 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
Fig. 1 Fluorescent UAAs derived from tyrosine/phenylalanine by extending the π-conjugation of aromatic side chain. |
Recently, π-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 π–π 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 fine-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 fibrils is well documented. Recently, Anna and co-workers reported on the synthesis of thiophene-based conjugated oligomers bearing L-amino acid and their use as optical probes for detection of amyloid fibril formation in insulin.12
Encouraged by recent developments in fluorescent UAAs, we envisage that the transformation of amino acids into conjugated systems would lead to a whole new class of fluorescent UAAs with desired optical properties (Fig. 1). In this regard, herein, we report the design and synthesis of novel α-amino acid analogs constructed via extending the π-conjugation of L-tyrosine. These novel fluorescent UAAs consist of stilbene and meta-phenylene vinylene units as fluorophores 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 fluorescent peptides for cell imaging.
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 electron-donating groups gave an access to thirteen fluorescent 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 π-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 1H 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 fluorescence emission, extinction coefficients, fluorescence 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
Analog | λ Abs (nm) | λ Em (nm) | ε [cm−1 M−1] | QYc | [α]25Dd |
---|---|---|---|---|---|
a Determined in DMSO (c = 10 uM). b Extinction coefficient. c Quantum yield using 9,10-diphenyl anthracene as a standard reference. d See ESI. e QY in THF. f QY in HEPES buffer (pH = 7.3); italics: excitation wavelengths. | |||||
4a | 300, 345 | 400 | 11000 | 0.41 | +15 |
4b | 292, 335 | 400 | 16000 | 0.87 | +16 |
4c | 320, 360 | 440 | 14000 | 0.13 | +11 |
4d | 298, 390 | — | 18000 | — | +10 |
4e | 300, 340 | 440 | 25000 | 0.12 | +15 |
4f | 300, 340 | 440 | 19000 | 0.13 | +12 |
5a | 300, 355 | 430, 512, 580 | 26000 | 0.51 | + 8 |
5b | 300, 360 | 420, 438 | 29000 | 0.94 | +10 |
5c | 320, 375 | 490 | 30000 | 0.47 | + 8 |
5d | 400, 660 | −(0.11)e | 39400 | −(0.08)e | +12 |
5e | 300 | 410, 595 | 28000 | 0.32 | +15 |
5f | 300, 370, 520 | 630 | 34000 | 0.24 | +12 |
5g | 370, 520 | 800 | 28000 | 0.002 (0.04)f | +10 |
Tyr | 278 | 352 | 5300 | 0.12 | — |
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 NO2 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 (CF3 and Py) showed a weak shoulder band in emission spectrum at around 590 nm.
Our mono-styrene analogs emit at higher wavelengths (violet-blue) due to their short π-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 π-electrons into the π-system and thus forming an extended π-conjugated system. As a result of this, a red shift 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 shift of emission spectra in a polar solvent. Addition of a base to the DMSO solutions of these amino acids resulted in a red shift 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†).
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 SO3Na 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 NH2, displayed blue and green emission at 420 nm and 500 nm, respectively. When compared to the other analogs in this series, compound 5d, bearing NO2 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 λmax 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 NO2 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 shift 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 fluorescence experiments as well as DLS experiments. The aggregate-associated change in PL was confirmed 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 λmax 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 shift in fluorescence emission with low quantum yield is a well-known phenomenon in conjugated polymers/oligomers,185f 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 H-bonding interaction, rather than π–π interaction between phenyl rings, which typically leads to the quenching of fluorescence.
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 fluorescence spectra, which suggest predominant single molecule species at lower concentrations (<100 nM).
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 significant 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 shift 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.
Fig. 4 Emission spectra of 5f in neutral (blue), acidic (green) and basic (red) environment showing the RGB emission. |
As expected, most of the compounds exhibit a red shift 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 = 10%) when compared to its neutral state (QY = 45%). Conversely, addition of NaOH enriched the phenolate population and as a result of extended conjugation, compound 5f exhibited a more intense (QY = 57%) and red shifted emission at 630 nm. UAA with pH dependent fluorescence emission exhibits distinct red, green and blue color (RGB) with decent quantum yield suggests implications toward sensing, bioimaging and LED devices.21
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
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 = 4.0 and 9.0). Emission of 5b red shifted (from 440 nm to 520 nm) from pH 4 to 9, and its fluorescence 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 fluorescence of 5b, whereas the fluorescence was mostly recovered by the addition of 2 mM hydrazine. Note that fluorescence recovery was not 100% as the oxidized 5b may have possibly reacted with moisture in the solution.
Fig. 5 pH (left) and redox (right) sensitivity of compound 5b. Emission spectra were recorded using 50 μM and 10 μM solutions, respectively. |
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 pKa of the phenol. Hence, the deprotonation of phenol of 5g occurs under physiological pH and emits NIR fluorescence 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-shifted to NIR region (800 nm), but the quantum yield decreased to 0.002, whereas it showed the moderate in context of NIR emission24 quantum yield (0.04) in aqueous solution.
Peptide 1 was further characterized by UV-Vis and fluorescence 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 fluorescent properties, peptide 1 was incubated with a human epithelial cell line (HeLa) and mouse fibroblast 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 fluorescent labels.
Fig. 7 Internalization of peptide 1 by HeLa (left) and NIH 3T3 (right) cells visualized by confocal scanning microscope. |
Footnote |
† Electronic supplementary information (ESI) available: Full experimental details and characterization data of all the new compounds. See DOI: 10.1039/c4sc02753a |
This journal is © The Royal Society of Chemistry 2015 |