Tyrosine-derived stimuli responsive, fluorescent amino acids

Article abstract of DOI:10.1039/c4sc02753a

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 palladi

Full text of DOI:10.1039/c4sc02753a

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TyrosineꢀDerived Stimuli Responsive, Fluorescent Amino Acids
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Pradeep Cheruku, JenꢀHuang Huang, HungꢀJu Yen, Rashi S. Iyer, Kirk D. Rector, Jennifer S. Martinez, and
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HsingꢀLin Wang *
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CꢀPCS, Chemistry Division, Defense System and Analysis Division, Center of Integrated Nanotechnologies (CINT)
Los Alamos National Laboratory, Los Alamos, New Mexico, 87545, United States
*To whom correspondence should be addressed: hwang@lanl.gov
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Abstract: A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and metaꢀ
phenylenevinylene (mꢀPPV) backbone have been synthesized and their optical properties were
studied. These novel UAAs were derived from protected diiodoꢀLꢀtyrosine using palladiumꢀ
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 400ꢀ800 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.
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1. 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
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(1)
nucleic acids. Among the other natural amino acids, tryptophan and tyrosine serve as intrinsic
(
2)
fluorescent probes to monitor the aboveꢀmentioned processes but suffer from poor optical
properties. Therefore continuous efforts have been made to design unnatural amino acids (UAAs)
with various fluorophores and successfully incorporated into peptides and proteins in a siteꢀ
specific manner to study their structureꢀfunction properties. Furthermore, relatively smaller size
and ease of synthesis of UAAs, as compared to their fluorescent protein cohorts, allows chemists
fineꢀtune the structure to obtain desired optical properties. Besides, UAAs bearing functionalities
are stimuliꢀresponsive serve as fluorescent reporters of microꢀenvironmental changes such as pH,
(3)
(4)
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polarity, and redox.
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.
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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
5a
the sideꢀchains of
α
ꢀamino acids. For example, a polarityꢀsensitive fluorescent UAA, ꢀAnap
L
was synthesized via covalent attachment of naphthyl fluorophore to the hydroxyl group of
L
ꢀ
(5bꢀc)
serine using Fukuyama–Mitsunobu reaction,
and (ii) constructing a whole new chromophore
on a natural amino acid. For example, coumarinꢀbearing fluorescent UAAs were derived from
(6)
aspartic and glutamic acids ; 2ꢀ(2ꢀfuryl)ꢀ3ꢀhydroxychromone to probe peptide−nucleic acid
(7)
complexes was synthesized from
L
ꢀtyrosine. Latter approach has the advantage of being
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relatively nonperturbing replacements for the native residues, thereby maintaining the overall
(1a)
native structure of a target peptide or protein. Additionally, a higher chemical stability is
expected if the fluorophore is linked to the amino acids by a sideꢀchain carbon–carbon bond.
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Figure 1. Fluorescent UAAs derived from tyrosine/phenylalanine by extending the πꢀconjugation of
aromatic side chain.
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As shown in Figure 1, augmenting the πꢀconjugation in tyrosine/phenylalanine to generate
structurally novel fluorescent UAAs is a unique approach. These UAAs (AꢀC) showed improved
optical properties than the corresponding tyrosine/phenylalanine amino acids but suffered from
(8)
multistep synthetic routes and lack of tunability in emission property.
Recently, πꢀconjugated organic systems have received a lot of interests owing to their tunable
physical, optical and electronic properties through tailored synthesis of conjugated structures with
(9)
backbone and/or side chains that renders desired properties. Due to their planar and semiꢀrigid
backbone and πꢀπ 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 fineꢀtuned by varying the nature of the substitution groups on the terminal
(10)
phenyl rings. In addition to application in electronic, optical and energy devices, conjugated
(11)
oligomers are also extensively used in biological and medicinal chemistry. For example, the
use of styreneꢀbased compounds as imaging agents and inhibitors of beta amyloid fibrils is well
documented. Recently, Anna and coꢀworkers reported on the synthesis of thiopheneꢀbased
conjugated oligomers bearing
amyloid fibril formation in insulin.
Lꢀamino acid and their use as optical probes for detection of
(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 (Figure 1). In this regard, herein, we report the design and synthesis of

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novel
αꢀamino acid analogs constructed via extending the πꢀconjugation of
Lꢀtyrosine. These
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novel fluorescent UAAs consist of stilbene and metaꢀphenylene 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 solidꢀphase peptide synthesis
(SPPS) to synthesize a cellꢀpenetrating peptide and demonstrate the use of these fluorescent
peptides for cell imaging.
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2. Results and Discussion:
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Synthesis of πꢀconjugation extended
L
ꢀtyrosine amino acids: The monoꢀ and bisꢀ styrylꢀ ꢀ
L
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tyrosine analogs (compounds 4aꢀf and 5aꢀg) were synthesized using strategies shown in Scheme
1. The syntheses of these amino acids have begun with the transformation of commercially
available 3ꢀiodoꢀ
L
ꢀtyrosine (1a) and 3,5ꢀdiiodoꢀ ꢀtyrosine (1b) into their corresponding tꢀ
L
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
(13)
synthesis and thus minimizing the number of steps in synthesis, and (2) the ability to secure the
(14)
chiral information at the beginning of the synthesis.
Although bisꢀstyryl conjugated
compounds can be obtained by a variety of synthetic pathways, the use of Heck reactions to
(15)
synthesize Eꢀconfigured styryl compounds has been proved to be most promising.
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Scheme 1. Synthesis of fluorescent unnatural amino acids 4aꢀf and 5aꢀg starting from tyrosine.
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Palladium catalyzed mono/double Heck couplings between 2a/2b and appropriate styrene (3aꢀf)
afforded protected tyrosine analogs 4aꢀf and 5aꢀg, bearing metaꢀphenylenevinylene backbone in
moderate to good yields. Mindful selection of styrene precursors with different electronꢀ
withdrawing and electronꢀdonating 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 πꢀconjugated UAAs. It is noteworthy that all the Heck couplings proceeded

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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 paraꢀposition 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
Eꢀisomers as established by the NMR spectra. The transꢀrelation of the double bonds was
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established on the basis of the coupling constant for the vinylic protons in the H NMR spectra (J
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∼ 16 Hz, supplemental information).
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[
a]
Table1. Physicochemical properties of tyrosine derived fluorescent amino acids
ꢀ
1
ꢀ1 [b]
[c]
25 [d]
Analog
λAbs (nm) λEm (nm)
ε [cm M ]
11000
16000
14000
18000
25000
19000
26000
29000
30000
39400
28000
34000
28000
5300
QY
[α]D
4
4
a
300, 345
292, 335
320, 360
298, 390
300, 340
300, 340
300, 355
300, 360
320, 375
400, 660
300
400
400
0.41
0.87
0.13
ꢀ
+15
b
+16
+11
+10
+15
+12
+ 8
4
c
440
4
d
ꢀ
4
e
440
0.12
0.13
0.51
0.94
0.47
4
f
440
5
5
a
430, 512,
5
80
b
420, 438
+10
+ 8
5
c
490
ꢀ
5
d
ꢀ
+12
+15
+12
+10
[e]
[e]
(
0.11)
(0.08)
5
e
410, 595
630
0.32
5
f
300, 370,
0.24
5
20
5
g
370, 520
800
0.002[
0.04)
f]
(
Tyr
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352
0.12
ꢀ
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[a] Determined in DMSO (c =10 uM) [b] Extinction coefficient [c] Quantum yield using 9,10ꢀdiphenyl anthracene as a
standard reference [d] See supporting information [e] QY in THF [f] QY in HEPES buffer (pH = 7.3); Italics:
excitation wavelengths.
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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
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presented in Table 1. The optical properties of ꢀtyrosine were included for comparison.
L
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UV/vis and fluorescence spectroscopy: The optical properties of the fluorescent amino
acids, 4aꢀf and 5aꢀg were measured by UV/vis and fluorescence spectroscopy in DMSO at room
temperature. The results are shown in Figures 2 and 3.
Monoꢀstyrylꢀ ꢀtyrosine analogs: We observed two characteristic absorption peaks in the
L
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UV−vis spectra of all monoꢀstyryl analogs demonstrating the distinct electronic transitions. For
example, all six amino acids have a higher energy absorption band with a λmax at roughly 300 nm.
For the low energy absorption peak, λmax varies from 330 nm to 390 nm depending upon the
nature of the paraꢀsubstitution on the styrene ring. In general, strong electron withdrawing groups
such as NO (4d), CF (4e) and Py (4f) caused a red shift in λ for the low energy absorption
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max
peak when compared to their unsubstituted (4a) or electronꢀdonating group substituted analogs
(4bꢀc).
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Figure 2. Absorption and emission spectra of monoꢀstyryl tyrosine analogs 4aꢀf in DMSO: UVꢀvis spectra
(A) before the addition (B) after the addition of NaOH; (C) a clear redꢀshift in emission spectra is due to
the extended conjugation of the phenolate ion; (D) PL spectra; before (solid line) and after (dotted lines) the
addition of NaOH.
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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 lowꢀenergy 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
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group on the styrene ring. The NO compound 4d found to be nonemissive in highly polar (both
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protic and aprotic) solvents, such as DMSO, methanol, where as it is emissive in nonꢀpolar
solvents such as chloroform and THF with an emission maxima 400 nm. Compounds 4eꢀf having
electron withdrawing groups (CF and Py) showed a weak shoulder band in emission spectrum at
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Our monoꢀstyrene analogs emit at higher wavelengths (violet–blue) due to their short πꢀ
(17)
conjugation lengths is consistent with literature observation. As illustrated in Figure 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 πꢀelectrons into the πꢀsystem and thus forming an extended πꢀconjugated 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 400ꢀ435 nm (violet) nm to 510ꢀ600 nm (greenꢀorange). This result is in agreement
with generation of the phenolate anion, while the spectra of phenolꢀprotected derivatives
unaltered by addition of base (supplemental information).
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Bisꢀstyrylꢀ ꢀtyrosine analogs (TyrꢀOMPVs): Addition of another phenylene vinylene to the
L
meta position of stilbene derivatives leads to a new class of metaꢀphenylene vinylene bearing
amino acids (5aꢀg). All these amino acids showed absorption wavelengths (λmax) in the UV or
visible region ranging from 300ꢀ430 nm (Figure 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, Figure 3). The emergence of the low
energy peak is likely due to the donorꢀacceptor characteristic of the molecules, which causes the
(10)
HOMO and LUMO to merge, producing smaller band gap energy.
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Figure 3. Absorption (left) and emission (right) properties of compounds 5aꢀf in DMSO; left
inset: overlapped absorption spectra of 5a, 5b, 5e; right inset: emission colors of fluorescent amino acids in
DMSO that covers the visible spectrum from violet to red
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As shown in Figure 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 400ꢀ800 nm), is a key feature of these amino acids. Compounds 5a
and 5e, possessing H and SO Na groups, respectively, showed emission in the blue region.
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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

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when visualized under UV lamp. Compound 5b and 5c possessing electronꢀdonating groups such
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as OMe and NH , displayed blue and green emission at 420 nm and 500 nm, respectively. When
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compared to the other analogs in this series, compound 5d, bearing NO groups on paraꢀpositions
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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 NO containing compounds. 5d aggregates in
2
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, nonꢀ
aggregated species. (Figure S1 in supporting information).
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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 aggregateꢀassociated 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 (Figure S2, supplemental information). 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 wellꢀknown phenomenon
(18)
in conjugated polymers/oligomers,
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 Hꢀbonding interaction, rather than πꢀπ interaction between phenyl
rings, which typically leads to the quenching of fluorescence.
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On the other hand, compound 5f showed the blue emission in apolar solvents such as THF
(Figure S3, supplemental information) and chloroform, suggesting that these solvents do not
facilitate the formation of aggregation as the blue emission is solely coming from the single
molecule/nonꢀaggregated 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).
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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
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ring. 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 significant visible color change upon the addition of acid pTSA (pꢀ
toluenesulfonic acid) or base (NaOH). Basic, acidic, and neutral solutions of 5f showed red green
and blue (RGB) emission respectively, see Figure 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.
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As expected, most of the compounds exhibit a red shift of their absorption (Figure S4,
supplemental information) 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). 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
(20)
(21)
devices.
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Figure 4. Emission spectra of 5f in neutral (blue), acidic (green) and basic (red) environment showing the
RGB emission
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Solvatochromism: Compound 5d comprised of terminal NO group showed interesting
solvatochromic property. Though the fluorescence emission of 5d was quenched by some of
the polar solvents, its emission was strong in apolar solvents. Specifically, 100 uM solutions of
2
(10)
NO ꢀterminated analog 5d in acetonitrile, acetone, THF, dioxane and chlorobenzene exhibits a
2
strong solvatochromism. While the λmax 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 (Figure
S5, supplemental information).

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Such strong solvatochromism occurs due to a combination of dipoleꢀsolvent 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
(10)
to solvent stabilization of intramolecular charge transfer in the excited state.
pH and redox response: It is well established that perturbations in cellular redox status and pH
(4,22)
have important physiological and pathological ramifications.
Investigative tools to monitor
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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
significant 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 TyrꢀOMPVs are reversible, these intrinsic
properties were used to explore their potential application as pH and redox stimuli responsive
sensors.
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Figure 5. pH (left) and redox (right) sensitivity of compound 5b. Emission spectra were recorded using 50
µM and 10 µM solutions, respectively
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A solution (DMSO: water 1:4 v/v) of 5b was used to explore the stimuli responsive properties. As
shown in (Figures S6 to S8, supplemental information) and Figure 5, 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 oxidationꢀreduction 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

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5b may have possibly reacted with moisture in the solution.
NIR emission: Recently, Shabat and coꢀworkers have reported on the design of NIR probes
based on a D2A πꢀelectron system that can undergo intramolecular charge transfer (ICT) to form
a new fluorochrome with a longer π ꢀ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 π electrons to either one of the conjugated
(23)
acceptors to exhibit ICT and thus emit at NIR region.
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Figure 6. Absorption and emission spectra (left) of NIR emission of deprotonated 5g; c = 100 uM
dissolved in HEPES buffer (pH 7.3). Shown in inset is quenching of NIR emission upon acidification (pH
2) due to the protonation of phenolate; a schematic representation (right) showing the origin of NIR
emission through ICT mechanism
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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 waterꢀsoluble NIR
dye in the form of an amino acid could be useful for various imagingꢀrelated 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 Figure 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 (Figure S10, supporting information). 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 redꢀshifted to NIR region (800 nm), but the quantum yield
(24)
decreased to 0.002, whereas it showed the moderate in context of NIR emission quantum yield
(0.04) in aqueous solution. Despite the low quantum yield, the research on the development of

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NIR emitting water-soluble amino acids is yet to be explored and our efforts to develop more
analogs in this series with improved quantum yields are currently on going and will reported in
due course.
Utility in solid phase peptide synthesis (SPPS) and cell imaging: In general, fluorescently
labeled peptides are often obtained by the conjugation of the fluorescent dye through postꢀ
synthesis modifications. These typical bioconjugations often require fluorescent dye containing
amineꢀ 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 fluorescent UAA 5b allows for the
direct and siteꢀspecific introduction of the fluorescent moiety as an integrated part of the SPPS
process (Scheme 2, supplemental information). In order to evaluate the utility of these amino
acids in Fmocꢀbased peptide synthesis, an Fmoc analog of compound 5b was synthesized and
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incorporated into a cell penetrating Baxꢀinhibiting pentapeptide. (sequence=W*VPALK; W* =
5b).
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Scheme 2: Solid phase synthesis of peptide 1
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Peptide 1 was further characterized by UVꢀVis and fluorescence spectroscopy over a range of pH
values and the excitation and emission spectra are shown in Figure S9, supplemental information.
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 (Figure 7), thus validating the use of
these amino acids as intrinsic fluorescent labels.
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Figure 7. Internalization of peptide 1 by HeLa (left) and NIH 3T3 (right) cells visualized by confocal
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3. Conclusions: In conclusion, we have developed a direct and facile synthesis of optically pure,
fluorescent 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 metaꢀphenylene vinylene moiety in a molecule gives rise to a
whole new class of fluorescent 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 fluorescent amino acids, which can be easily
incorporated into peptides and used in biological studies. 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 stimuliꢀresponsive nature and the ability to emit NIR fluorescence, our
UAAs exhibit strong implications in a wide range of applications include fabricating
optoelectronic devices, probing the cellular processes and other aspects of biological mechanism
and function can be expected.
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Supporting Information
Full experimental details, characterization data of all new compounds is presented. This material
is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgements: This research is supported by the Laboratory Directed Research and
Development (LDRD) program, under the auspices of Department of Energy (DOE). Basic
Energy Science (BES), Biomolecular Materials Program, Division of Materials Science and
Engineering support the synthesis of UAA.
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