Conformationally rigid pyrazoloquinazoline α-amino acids: One- And two-photon induced fluorescence

Article abstract of DOI:10.1039/c9cc09064a

The synthesis and photophysical properties of a new class of α-amino acid bearing a rigid pyrazoloquinazoline chromophore are described. Confromational constraint of the amino acid side-chains resulted in high emission quantum yields, while the demonstration of two-photon-induced fluorescence via near-IR excitation signifies their potential for sensitive bioimaging applications.

Full text of DOI:10.1039/c9cc09064a

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Conformationally rigid pyrazoloquinazoline -amino acids: one-
and two-photon induced fluorescence
Jonathan D. Bell,a Alexander H. Harkiss,a David Nobis,a Eilidh Malcolm,a Astrid Knuhtsen,a
Received 00th January 20xx,
Accepted 00th January 20xx
Christopher R. Wellaway,b Andrew G. Jamieson,a Steven W. Magennisa and Andrew Sutherland*a
DOI: 10.1039/x0xx00000x
for excitation. Prolonged exposure to UV light causes cell and tissue
damage through photo-bleaching. In recent years, this limitation has
been overcome by the use of two-photon fluorescence microscopy,
which permits excitation of the chromophore at longer wavelength,
allowing long-term imaging of biological specimens, deeper tissue
penetration, with minimization of photo-bleaching and photo-
toxicity.7 While this technique has been utilized with a wide range of
fluorophores and applied to the imaging of various biological
processes and diseases,8 there are relatively few reports with
unnatural -amino acids. Examples include the seminal work of Mély
and co-workers who used two-photon excitation of peptides
containing 3-hydroxyflavone derived -amino acids (e.g. 1, Fig. 1) for
monitoring interactions with cell membranes and intracellular RNA,9
while the Vendrell group used two-photon excitation of a cyclic
The synthesis and photophysical properties of a new class of -
amino acid bearing a rigid pyrazoloquinazoline chromophore are
described. Confromational constraint of the amino acid side-chains
resulted in high emission quantum yields, while the demonstration
of two-photon-induced fluorescence via near-IR excitation signifies
their potential for sensitive bioimaging applications.
Fluorescence spectroscopy is a powerful technique for studying
biological structure and function and for visualizing molecular
interactions and intracellular processes.1 This is due to its high
sensitivity and information content, its multiplexing capability, the
ability to probe processes over a wide time range, and the availability
of an array of versatile fluorescent probes. The investigation of
cellular functions such as enzyme mechanisms and protein-protein
interactions have often utilized extrinsic fluorescent labels such as
green fluorescent protein (GFP) and its derivatives2 or intrinsic labels
such as the proteinogenic fluorescent -amino acids, tyrosine or
tryptophan.3 However, the attachment of a fluorescent protein can
alter the stability and functionality of the fusion partner, while
tyrosine and tryptophan have poor optical properties and the
occurrence of multiple residues in different environments can
complicate their analysis.3
peptide bearing
a tryptophan-BODIPY conjugate (2) for the
visualization of fungal infections.10
In recent years these limitations have been overcome by the
development of unnatural fluorescent -amino acids.4 The
photophysical properties of such amino acids can be tuned for a
particular application and they can be incorporated into proteins and
peptides by solid phase peptide synthesis (SPPS) or by unnatural
amino acid mutagenesis.5 Of particular interest are amino acids with
side-chain chromophores that can be easily incorporated into
peptides and proteins with minimal disruption to structure and
function.4,6 While some of these fluorescent -amino acids have
found application in biological and medicinal imaging, an inherent
issue of small chromophores is that ultraviolet (UV) light is required
Fig. 1 Selected Unnatural Fluorescent -Amino Acids.
We previously reported the synthesis of 5-arylpyrazole-derived
-amino acids such as 3 (Fig. 1).11 These compounds possessed some
interesting photophysical properties however, the rotational
flexibility between the aryl and pyrazole rings resulted in low
quantum yields and brightness. As conformationally rigid, molecular
structures permit more efficient -overlap and subsequently brighter
a. WestCHEM, School of Chemistry, The Joseph Black Building, University of
Glasgow, Glasgow, G12 8QQ, UK. Email: Andrew.Sutherland@glasgow.ac.uk
b. Medicinal Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood
Road, Stevenage, SG1 2NY, UK.
†Electronic Supplementary Information (ESI) available: Experimental procedures,
characterisation data for compounds, photophysical data for amino acids and, NMR
spectra for all novel compounds. See DOI: 10.1039/x0xx00000x
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chromophores,12 we proposed that conformational restriction of
The UV/Vis absorption and emission spectra of the amino acids
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–5
DOI: 10.1039/C9CC09064A
these 5-arylpyrazole motifs would lead to -amino acids with were recorded in methanol at a concentration of 0.5 × 10 M (Fig. 2
enhanced photophysical properties. Here we describe a new class of and Table 1).17 As expected for chromophores that are planar in the
-amino acid (4), bearing planar pyrazoloquinazoline chromophores. ground state, the amino acids, particularly 4a–d, showed structured
We also report the photophysical properties of these compounds, absorption spectra with well-resolved vibrational bands (Fig. 2a).18
demonstrating how conformational restraint leads to fluorophores The emission spectra of amino acids 4a–e, following one-photon
with high quantum yields and spectroscopic properties that are excitation at the longest wavelength absorption band, showed
sensitive to environment polarity. We also show the application of strong fluorescence ranging from 342–414 nm, with a vibrational
these -amino acids to two-photon excitation, for potential use in progression which mirrored that of the absorption spectra (Fig. 2b).19
imaging UV sensitive, biological systems.
Amino acid 4e bearing the electron rich dimethyl-amino substituent
A synthetic route for the preparation of pyrazoloquinazoline- gave a red-shifted, structureless emission maximum at 414 nm,
containing -amino acids 4 was developed from L-aspartic acid. which is indicative of a combination of locally excited and internal
Initially, L-aspartic acid was converted to phosphononorvaline charge transfer states.20 More importantly, the quantum yields of
derivative 5 in five steps and 84% overall yield using previously 4a–e were characteristic of conformational restriction and showed a
described methods.13,14 A range of 2-nitrobenzaldehydes was ten-fold increase in comparison to the non-rigid pyrazole-derived
reacted with phosphononorvaline 5 via a Horner-Wadsworth- amino acid 3. As a result, these -amino acids are substantially
Emmons reaction, which gave the E-isomer of enones 6a–e in 42– brighter than the corresponding non-rigid systems.
82% yields (Scheme 1).14,15 The enone moiety was used to construct
the pyrazole ring by reaction of 6a–e with hydrazine via a one-pot
(a)
condensation/aza-Michael process. Low temperature oxidation of
the resulting 2-pyrazolines with DDQ gave 5-(2-nitroaryl)pyrazoles
7a–e.11 Carbonylation was then achieved by reduction of the
nitroaryl moiety, followed by reaction with triphosgene under basic
conditions.16 This gave pyrazoloquinazolines 8a–e in 30–75% yields
over the two steps. Deprotection to the parent amino acids was
either conducted in one step under acidic conditions (4b, 37%; 4c,
40%) or more efficiently by a two-step strategy involving ester
hydrolysis, followed by removal of the Boc-protecting group under
milder acidic conditions (4a, 80%; 4d, 82%; 4e, 78%).
0.15
0.1
0.05
0
4a
4b
4c
4d
4e
230
280
330
380
Wavelength (nm)
(b)
1
4a
4b
4c
4d
4e
0.75
0.5
0.25
0
300
350
400
450
500
550
Wavelength (nm)
Fig. 2 (a) Absorption spectra of amino acids 4a–4e recorded at 0.5 × 10–5 M in
MeOH (b) Emission spectra of 4a–4e at 0.5 × 10–5 M in MeOH.
Table 1: Photophysical data for 5’-(naphthylen-2”-yl)-1’-phenylpyrazole amino acid 3 and
pyrazoloquinazoline-derived -amino acids 4a–e.a
Amino
acid
3
Abs
(nm)
249
321
350
(279)
328
337
349
 (cm–1 M–1)
Em
(nm)
356
342
372
(373)
353
363
414
Fb
Brightness
(cm–1 M–1)c
950
23700
8300
0.04
0.42
0.47
(0.27)
0.47
0.56
0.48
4a
3500
7700
3600
4b
(24,800)
10000
11200
12200
(6700)
4700
4c
4d
4e
6300
5900
a All spectra were recorded at 0.5 × 10–5 M in MeOH. b Quantum yields (F) were
determined in MeOH using anthracene and L-tryptophan as standards.
c
Brightness was calculated as the product of the QY and the molar absorptivity at
the wavelength maximum specified.
Scheme 1 Synthesis of pyrazoloquinazoline -amino acid 4a–e.
2 | J. Name., 2012, 00, 1-3
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As amino acid 4e showed the most interesting photophysical photophysical properties of -amino acid 4e were retained, including
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–1 –1
DOI: 10.1039/C9CC09064A
properties, this compound was further investigated. The a high quantum yield (0.45) and brightness value (6900 cm M ).
fluorescence decay was measured at an emission wavelength of 430
(a)
424
374
1
0.8
0.6
0.4
0.2
0
nm in methanol using time-correlated single-photon counting,
following excitation at 390 nm (see SI). Three decay components
were fitted with lifetimes of 0.79, 2.17 and 7.90 ns, and A-factors of
0.60, 0.36 and 0.04, respectively. This suggests the molecule exists
predominantly in two states, as has been observed previously for
dimethylamino-substituted fluorophores.21 The amplitude-weighted
average lifetime is 1.57 ns, which is comparable to fluorophores
typically used in fluorescence lifetime imaging microscopy (FLIM).22
To assess the environmental sensitivity of amino acid 4e, its
photophysical properties were examined in a range of solvents. The
absorption maxima (342–348 nm) were found to be independent of
polarity (see SI), indicating negligible intramolecular interaction
between the electron-rich dimethylamino and electron-deficient N-
acylpyrazole moieties in the ground state. In contrast, the emission
spectra were found to be highly sensitive to the solvent used, with
increasing polarity leading to concomitantly broadened,
structureless emission spectra at longer wavelengths (Fig. 3a).23,24
For example, in THF, an emission maximum at 374 nm was observed,
while in phosphate-buffered saline (PBS), the emission maximum
was found at 424 nm. These spectral features and strong
solvatochromism are likely due to increasing solvent stabilization of
both the locally excited and intramolecular charge transfer states.
Application of amino acid 4e for two-photon spectroscopy was
next investigated. Two- and three-photon excitation has recently
been shown to be a promising approach for the ultrasensitive
analysis of fluorescent nucleobases, suggesting a similar approach
may be applicable to -amino acids.25 Two-photon absorption was
confirmed with a quadratic dependence of the emitted intensity on
the excitation intensity (see SI). Furthermore, the molecule has no
one-photon absorption in the near IR, precluding linear excitation.
The emission spectra of amino acid 4e in methanol after one- and
two-photon excitation have a similar profile (Fig. 3b). The two-
photon cross section was measured to be 0.38±0.06 GM using
rhodamine B as a reference compound (see SI). This value is
comparable to other molecules of similar size.8d,e Thus, 4e can
undergo two-photon absorption using near-IR excitation, thereby
avoiding the use of UV excitation and the issues of photobleaching.
The potential of these amino acids for incorporation into
peptides via SPPS methodology was also investigated. A model
pentapeptide (Val-Pro-Thr-Leu-Lys), based on the Bax-binding
domain of Ku70, a multifunctional protein involved in DNA repair and
cell-death regulation was chosen.26 This pentapeptide and similar
analogues have been shown to have low cytotoxicity and are
effective at penetrating living cells.26b The pentapeptide was
THF
MeCN
DMSO
MeOH
PBS
355
405
455
Wavelength (nm)
505
555
(b)
Fig. 3 (a) Solvatochromic study of 4e. Spectra were recorded using a concentration of
0.5 × 10–5 M. (b) Emission of 4e after two-photon absorption (red) and one-photon (blue)
absorption in MeOH. For the two-photon measurement, a solution of 200 M in MeOH
and an excitation centred around 700 nm with FWHM of 10 nm was used.
Scheme 2 SPPS and photophysical data of hexapeptide 10.
prepared in an automated, peptide synthesizer using TentaGelTM
S
0.2
0.15
0.1
1000
750
500
250
0
Rink Amide (RAM) resin as the polymer support27 and routine SPPS
methodology (Scheme 2). Coupling of each Fmoc-protected amino
acid was performed by HCTU28 activation, followed by piperidine-
mediated N-deprotection, which gave the N-terminal unprotected
pentapeptide. Boc-Protected pyrazoloquinazoline-derived amino
acid 9 (see SI for preparation) was then coupled manually onto the
polymer-supported pentapeptide. A TFA cleavage cocktail was used
to remove the protecting groups and release the hexapeptide from
the polymer support. Purification by reverse phase-HPLC gave
hexapeptide 10 in 72% yield with >95% purity. Analysis of the
photophysical properties of hexapeptide 10 showed that apart from
a small hypsochromic shift in the emission spectra (Fig. 4), the key
0.05
0
200
300
400
500
Wavelength (nm)
Fig. 4 Absorption (dotted lines) and emission (solid lines) spectra of amino acid 4e
(blue) and hexapeptide 10 (red), recorded at 0.5 × 10–5 M in MeOH.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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In summary, a new class of conformationally rigid, unnatural -
amino acid has been synthesized using a highly regioselective
olefination reaction to introduce side-chain diversity, followed by a
one-pot condensation/aza-Michael reaction and carbonylation
process to form the key pyrazoloquinazoline ring system. Although
containing relatively small side-chains, these compounds were found
to be strongly fluorescent with high quantum yields (42–56%). The
potential of amino acid 4e as a sensitive bio-imaging tool was
demonstrated with successful excitation of this compound at 700 nm
using two-photon spectroscopy. Furthermore, amino acid 4e was
efficiently incorporated into a biologically relevant peptide as part of
a SPPS process. Based on the exciting optical properties of this new
class of -amino acid, current work is investigating the introduction
of these minimally invasive compounds into other biologically
relevant peptides for in vitro and in vivo spectroscopic and
microscopic applications.
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Conflicts of interest
There are no conflicts to declare.
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4b. Excitation of the peak at 279 nm gave the most intense
fluorescence and highest brightness value (Table 1).
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Acknowledgements
Financial support from EPSRC (studentships to J.D.B., EP/N509176/1
and A.H.H., EP/K503058/1), University of Glasgow (studentship to
D.N.) and GlaxoSmithKline is gratefully acknowledged.
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