PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5-Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions

Article abstract of DOI:10.1021/acs.jpcb.7b03611

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable β-hairpin zipper peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid β-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 β-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance.

Full text of DOI:10.1021/acs.jpcb.7b03611

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Article
PHOXI: a High Quantum Yield, Solvent-Sensitive Blue
Fluorescent 5-Hydroxytryptophan Derivative Synthesized
within Ten Minutes under Aqueous, Ambient Conditions
Alexandre Grigoryan, Azaria S. Eisenberg, and Laura J. Juszczak
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b03611 • Publication Date (Web): 07 Jul 2017
Downloaded from http://pubs.acs.org on July 18, 2017
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PHOXI: A High Quantum Yield, Solvent-
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Hydroxytryptophan Derivative Synthesized
within Ten Minutes under Aqueous, Ambient
Conditions
ꢀ
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ꢁꢀ
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Alexandre Grigoryan , Azaria S. Eisenberg and Laura J. Juszczak
*
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Department of Chemistry, Brooklyn College of The City University of New York, 2900
Bedford Ave., New York, NY 11210, United States
ꢀ
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Department of Chemistry, The Graduate Center of the City University of New York, New
York, New York 10016, United States
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Department of Biochemistry, The Graduate Center of the City University of New York, New
York, New York 10016, United States
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Abstract
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Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because
individual residue emission is not resolvable. Biosynthetic incorporation of an indole analog
such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of
low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting
framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-
oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 minutes
under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized
using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-
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hydroxytryptophan (5-HTP) and a stable beta hairpin ‘zipper’ peptide with four tryptophan
residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4ꢂ5-HTP. Reaction
optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction
specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid beta
40, a peptide containing all amino acids except tryptophan, proline and cysteine, and lacking
5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents
reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm redshift in
water relative to cyclohexane while high quantum yield is maintained. Thus PHOXI joins the
ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations
reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent
for both the HOMO and LUMO orbitals. Despite the crowded environment of three
additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 β-hairpin structure is
partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI
incorporation can be less disruptive of protein secondary structure, especially at molecular
interfaces and other environments where there is typically less steric hindrance.
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Introduction
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Fluorophores capable of site-specific incorporation into peptides and proteins are
valuable tools for structural studies and imaging applications. While macro fluorophores
such as green fluorescent protein (GFP) can be easily introduced into biological systems
during recombinant expression, their large size can often cause alteration of the structure
or function of the system under study.1,2 Small molecule organic dyes circumvent this issue,
and can be designed to possess favorable photophysical properties that maximize their
utility as biological probes, but they are not readily incorporated in site-specific positions.
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Advances in organic synthesis and calculation methods have allowed for the development
of a plethora of fluorophores with desirable photophysics. Among these, solvatochromic
probes, usually designed to possess an electron donor and acceptor moiety flanking the
aromatic system, have received particular attention because their fluorescence properties
are highly sensitive to the surrounding environment, maximizing their utility in probing
biological systems.3,4 Although many such probes have been developed, their use for
investigation of protein structure, interactions, and dynamics is limited by current methods
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of site-specific incorporation into biomacromolecules. Incorporation of a solvatochromic
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probe into a specific site of a protein using a short and rigid linker maximizes the utility of
any given fluorescence experiment. Current methods of incorporation that meet these
criteria require synthesizing an amino-acid derivative of the fluorophore, and either
introducing it into a peptide using solid phase peptide synthesis for subsequent
introduction into a protein using expressed chemical ligation, or by chemically acylating it
onto an orthogonal tRNA for direct protein incorporation using nonsense suppression.
While the former method limits the placement of the probe to ~40 amino acids from the C-
or N-terminus of the target protein, the latter requires extensive chemical and biological
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manipulation and is not necessarily compatible for every fluorophore amino acid derivative
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investigations, N,N-dimethylamino-6-propionyl-2naphtylamine (PRODAN), and its
derivatives (the DAN fluorophores) have been widely popular in studies of protein
binding,7,8 structure and membrane dynamics. Numerous structural analogs of PRODAN,
which substitute its naphthalene core with other conjugated systems while keeping the
electron donor/acceptor flanking groups, have been synthesized and possess even more
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desirable photophysical properties. For example, synthesis of the amino acid Aladan (also
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known as DANA ), which bears the PRODAN side chain, has allowed for its introduction
into peptides during solid phase peptide synthesis (SPPS) (via the corresponding Fmoc
derivative) and into proteins by means of nonsense suppression. However, in most cases
the challenge of introducing these fluorophores into biological systems in a site-specific
manner remains.
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In our search for a fluorophore that is both a robust probe of microenvironment and is
capable of site-specific incorporation into biological molecules, our attention fell to 2-
phenyl-6H-oxazolo[4,5-e]indole (PHOXI), produced by a fluorogenic oxidative coupling
reaction between 5-hydroxytryptophan (5-HTP) and benzylamine in the presence of mild
oxidative agent in aqueous buffer (Scheme 1). This chemistry, first discovered in the 1980’s
during investigations of the drug 9-hydroxyellipticine, which contains 5-hydroxyindole
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within its structure, has been recently applied to fluorescencebased determination of 5-
hydroxyindole containing analytes (serotonin etc.) in increasingly complex biological
samples14,15 and is touted to be selective for 5-hydroxyindoles within the biological milieu.
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Because there exist straightforward methods for incorporating 5-HTP into peptides via
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Fmoc SPPS, and into proteins through selective pressure incorporation, we envision
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using this chemistry as a means of site-specifically labeling peptides and proteins.
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extinction coefficient (1.54 x 10 cm M ), quantum yield (0.54) and emission in the
visible (461nm), suggest this fluorophore has the essential features of a sensitive
fluorescent tag.
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Recently, this fluorogenic reaction was successfully used to introduce PHOXI into 5-HTP
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containing peptides. Although this report sets an important precedent of the viability of
this reaction to introduce the PHOXI fluorophore into peptides, the authors were employing
this reaction to prepare cyclic peptides, and thus did not investigate its photophysical
properties. Furthermore, because the 5-HTP peptides were prepared using in vitro
translation, their quantities were minute (sub μM concentration in ~10μL), only allowing
for determination of reaction products by mass spectrometry without chromatographic
separation. Although the authors claim, based on mass spectrometric evidence, that only the
desired fluorophore-bearing products are formed, the proposed mechanism for this
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reaction suggests that unwanted products can arise from attack of the intermediate 2 by
nucleophiles other than the intended benzylamine. The susceptibility of 2 to attack even by
weak nucleophiles is further illustrated by the myriad oligomeric products produced upon
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oxidation of 5-hydroxyindole in the absence of benzylamine. Finally, a recent study
exploring the analogous reaction of 5-hydroxyindole-2-acetic acid with benzylamine
reported ~30% yields of the fluorescent adduct.21
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Scheme 1. Fluorogenic oxidative coupling reaction of 5-hydroxyindole (1) and
benzylamine. Initial oxidation forms p-quinoneiminemethide (2) which is highly reactive to
nucleophiles at position 4. Nucleophilic attack by benzylamine at this position, followed by
oxidation to the imine, a 1,5hydride shift, and ring closing yields (3) 2-phenyl-6H-
oxazolo[4,5-e]indole (PHOXI).
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Herein we further probe the utility of the PHOXI derivatization reaction using model
peptides. Using SPPS to produce 5-HTP peptides in greater quantities, we evaluate the
derivatization reaction in greater concentrations ranges, such as those required for
preparing sufficient amounts of labeled biomolecules for structural investigations. Working
with greater quantities of 5-HTP peptides allowed us to perform chromatographic
separation (RP-HPLC) to further interrogate the reaction products and optimize the
derivatization conditions. Consistent with the reaction mechanism, we observed numerous
non-fluorescent byproducts during 5-HTP peptide derivatization. While some of these
byproducts are unavoidable, we show how optimizing the derivatization conditions can
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minimize their formation. Furthermore, we show that PHOXI labeled peptide derivatives
are easily visualized and isolated by RP-HPLC with UV-vis detection.
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We also attempted to create more structurally conservative analogs of PHOXI, guided by the
fact that 6-hydroxyindole-2-acetic acid has been shown to react with amines of the general
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structure R-CH
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were interested in analogs of PHOXI where R is a hydrogen, to minimize the size of this
fluorophore and thus the structural perturbation caused by its introduction into peptides
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and proteins. There are precedents for using methylamine or ethanolamine as
nucleophiles, albeit in organic media, to produce these unsubstituted PHOXI analogs. In
contrast to benzylamine, our attempts to use these reagents to label peptides in a
biologically compatible manner were not met with success (data not shown). While
methylamine derivatization did not produce any fluorescent products (contrary to a patent
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claim ) the reaction with ethanolamine produced 3 fluorescent products, two of which
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were difficult to separate by RP-HPLC. The formation of these three fluorescent adducts is
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consistent with previous reports , but due to the difficulty in resolving them, derivatization
with ethanolamine was not pursued further.
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Finally, we further probe the structure and photophysics of PHOXI, and describe the
solvatochromism of this fluorophore for the first time. Our findings suggest that sensitivity
of PHOXI fluorescence emission to the polarity of the environment is comparable to that of
the DAN fluorophores. While fluorescence emission of PHOXI is only slightly less sensitive
to the polarity of the medium than PRODAN, multiparametric analysis using the Catalan 3P
model23–25 shows that PHOXI’s solvatochromism is more clearly dependent on a single
parameter, general solvent polarity, with half the sensitivity to solvent acidity than
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PRODAN. Additionally, the quantum yields of PHOXI remained above 0.5 in all of the
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solvents studied. This is a considerable improvement in comparison to PRODAN, which
displays very low quantum yields in extremely polar (water) and nonpolar (hexane)
solvents. PHOXI’s solvatochromism and straightforward photophysics along with its
inherent capability to site-specifically incorporate into peptides via derivatization of 5-HTP
make it a very promising fluorescent reporter tag.
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Materials. NH -Trp-5-HTP-OH (98.3% purity) and human Aβ-40 peptide
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(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, purity 98%) were custom
synthesized by Lifetein (Hillsborough, NJ); 5-HTP-TrpZip2 (SWT-5-HTP-ENGKWTWK, 99%
purity) was custom synthesized by Peptide 2.0 (Chantilly, VA). N-acetyl cysteine was
purchased from Sigma-Aldrich, Inc. (St. Louis, MO). The purity of all peptides was confirmed
by HPLC prior to use. Benzylamine (99%) was purchased from Acros Organics (Geel,
Belgium), and distilled before use. 5-hydroxyindole (97%) was purchased from Sigma-
Aldrich, Inc. (St. Louis, MO) and purified by sublimation prior to use.
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Preparation of 2-phenyl-6H-oxazolo[4,5-e] indole (PHOXI)
.
2-phenyl-6H-oxazolo[4,5-
e]indole was prepared from 5-hydroxyindole and benzylamine by adapting a procedure
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described by Boger et al . The procedure is further described in Supporting Information .
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PHOXI H-NMR data confirm the structure (See Supporting Information).
PHOXI derivatization of peptides under biological conditions For optimization of
.
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derivatization conditions, 10-100μM of 5-HTP-containing or control peptide was dissolved
in 0.5M borate buffer, pH 8.0 in the presence or absence of 0.5M benzylamine in an HPLC
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microvial. The reactions were initiated by addition of K
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[Fe(CN) ] to a final concentration of
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2mM. After 10 minutes, the reactions were stopped and the products analyzed by direct
injection onto a Series 200 HPLC system (Perkin Elmer, Waltham, MA) equipped with an
XBridge C18 4.5 X 150mm column (Waters Corporation, Milford, MA). The products were
separated by eluting with a linear gradient of Buffer A (0.1% TFA in water) and buffer B
(0.1% TFA in Acetonitrile) at a flow rate of 1mL/min as follows: 5 min 100% Buffer A, 2
minutes increasing to 20% Buffer B, 20 min increasing to 40% buffer B. The products and
unreacted starting materials were visualized by a UV-Vis flow-cell detector at 220nm
(peptide amide bonds), 280nm (Tryptophan and/or 5-HTP) and 326nm (PHOXI). Product
peaks with appreciable absorption at 326nm were manually collected, lyophilized and
subjected to mass spectrometry to confirm identity of PHOXI derivatized products.
4
5
6
7
8
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1
1
1
1
1
1
1
1
1
1
2
2
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2
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2
2
2
2
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1
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81
Lyophilized NH -Trp-5-HTP-OH derivatization products were dissolved in 50% Acetonitrile
2
containing 0.1% formic acid and analyzed by direct infusion onto an Agilent 6220 ESI-TOF
mass spectrometer. TrpZip2 derivatization products were analyzed using a MALDI-LTQ XL
orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA) using α-Cyano-4-
hydroxycinnamic acid as matrix.
182
1
1
83
84
1
1
85
86
Absorbance measurements. Background corrected absorption spectra were recorded on
a Lambda 650 UV−vis spectrophotometer (Perkin Elmer, Walnut Creek, CA) with a 2 nm
3
-
187
band pass, 1 cm path length, and 1 nm interval. Extinction coefficients of Trp (5.69 × 10 M
1
-1 26
3
-1
-1 27
4
-1
-1 18
1
1
88
89
cm ) 5-HTP (4.8 × 10 M cm ) and PHOXI (1.54 × 10 M cm ) were used to
determine concentrations.
1
1
90
91
Steady-State Fluorescence Emission Measurements. Fluorescence emission spectra of
PHOXI derivatized peptides in water, and of PHOXI in neat solvents were recorded on a
Fluorolog 3 model FL-1000 (Horiba Jobin Yvon, Edison, NJ) fluorimeter. For PHOXI
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derivatized peptides, emission spectra were recorded with an excitation wavelength of 280
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nm (Trp) and 336nm (PHOXI). For quantum yield measurements of PHOXI in neat solvents,
the excitation wavelength used corresponded to the maximum absorbance of PHOXI in each
solvent (332nm for acetonitrile; 336nm for 1,4-dioxane, methanol, toluene and water),
except for cyclohexane, where 322nm had to be used to obtain a complete emission profile.
For all fluorescence measurements, the step size was 1 nm, the integration time was set to
0.1 s. The band pass was 2nm and matched that of absorption spectrophotometer. All
concentrations were < 0.01 mM to avoid inner filter effects.
0
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00
2
8
201
Quantum Yield Determination. Aqueous quinine sulfate (QY = 0.54) and harmane (QY =
29
2
2
2
02
03
04
0.83) in 0.1 M sulfuric acid were used as quantum yield standards. PHOXI quantum yields
(22 ± 2°C) in six neat solvents were determined by the method described by Williams et
3
0
al. Briefly, the quantum yield of the two quantum yield standards are first determined via
reference against each other to ensure confidence in quantum yields measured for the
unknown. The integrated fluorescence intensity of each fluorophore is determined from
emission spectra at several fluorophore concentrations, all less than 0.01 mM, to avoid the
inner filter effect. The integrated intensity is plotted as a function of the measured
absorbance at each concentration. A linear fit is calculated, ensuring that the fit line runs
through the origin. Obviously, the goodness-of-fit should be close to unity. The slope of the
fit is the gradient, G, used in calculation of the quantum yield, QY:
205
2
2
2
2
06
07
08
09
210
2
11
ꢌ
ꢆꢇ
ꢋ
ꢇ
2
12
ꢀꢁ = ꢀꢁ ꢅ ꢊ ꢅ ꢊ
ꢌ
(1)
ꢂ
ꢃꢄ
ꢆꢈꢉ
ꢋ
ꢈꢉ
2
2
13
14
where the subscript, st, signifies the quantities for the quantum yield standard, and x are the
quantities for the fluorophore of unknown quantum yield. n is the refractive index for each
solution: n = 1.333 for water, n = 1.334 for 0.1 M sulfuric acid, n = 1.497 for toluene, n =
1.427 for cyclohexane, n = 1.331 for methanol, n = 1.344 for acetonitrile, n = 1.422 for 1,4-
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19
Trp-PHOXI Förster Distance Calculation.
The Förster distance in Angstroms was calculated using the equation:32
0
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ꢛ
ꢜ
ꢑ
ꢗꢘ
2
2
20
21
ꢍ = 0.2108ꢏꢐ ꢒ ꢖ ꢙꢚ
(2)
ꢎ
ꢓꢔꢕ
Where R is the critical Förster distance, k is the dipole orientation factor and was set to
0
3
3
222
223
2/3, ΦTrp is the quantum yield of Trp in water (0.14), n is the refractive index of the
medium taken as 1.4 for biological macromolecules, and J is the overlap integral, defined as
follows:
2
2
2
24
25
26
ꢟ
ꢎ
ꢘ
ꢙ = ꢝ ꢞ (ꢠ)ꢡꢢꢣꢤꢥꢦ(ꢠ)ꢠ ꢧꢠ
(3)
ꢓꢔꢕ
Where FTrp(λ) is the fluorescence quantum yield of tryptophan, and ꢡꢢꢣꢤꢥꢦ(ꢠ) is the
227
absorption spectrum of PHOXI scaled to its extinction coefficient at 326 nm: ε = 1.54 x 104
-1 -1 18
2
28
cm M .
2
2
29
30
Time-Resolved Fluorescence Lifetime Decay Measurement. Fluorescence lifetime
measurements (22± 2°C) of PHOXI in six neat solvents were carried out on a commercial
instrument that employs a laser diode excitation source with a broad (1.47 ns) instrument
response function (Horiba Jobin Yvon, Edison, NJ). Excitation at 340nm (± 10 nm), 3 nm
excitation and emission band pass, was achieved with a NanoLED source (<1ns pulse). An
experimental instrument response function, necessary for proper deconvolution, was
collected from scattered light from an aqueous casein solution at 340nm. Fluorescence
decay was collected at the varied emission maximum for PHOXI as given in Table 1.
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PHOXI Photodegradation Assays. A 0.33μM solution of PHOXI in either toluene or
water in a 50μL quartz microcuvette was illuminated by a Xenon lamp of a Fluorolog
spectrofluorimeter at 336nm for 5000s. The lamp power at the cuvette was 8.5mW
and the illumination area was 3mm H x 2mmW. Emission and excitation
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0
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monochromator band pass were set to 8 nm. During illumination the fluorescence at
the maximum (459nm for water, 390nm for toluene) was recorded at 1s intervals.
The percentile fraction of residual fluorescence signal after an hour of illumination
243
44
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2
was recorded
Fluorescence Lifetime Decay Analysis
instrument were determined by iterative convolution using the vendor-provided decay
.
.
The lifetimes for decays collected on the diode
2
2
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47
3
4
analysis software, DAS6. Details of this method can be found in reference .
248
Circular Dichroism of PHOXI and 5-HTP containing TrpZip2 peptide. A Chirascan CD
spectrophotometer (Applied Photophysics, Surrey, UK) equipped with a sample
temperature controller unit was used to obtain CD spectra of 25μM aqueous solutions of
TrpZip2 peptides bearing either 5-HTP or PHOXI in place of Trp 4. For each peptide, spectra
were recorded at 25°C, 65°C and 80°C.
2
2
2
2
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50
51
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Structure Calculations. Gaussian 09 software was used for ab initio quantum
2
2
2
2
54
55
56
57
mechanical calculations. The ground-state structure was optimized using the B3LYP density
functional theory method and 6-311G* basis set, with an ultrafine grid. The polarizable
continuum method with implicit solvents was used to allow for the solvent effects, and the
structure was checked for imaginary frequencies to ensure that it was not in a transition
state. Calculations were done on the BOB system at the CUNY High Performance Computing
Center (College of Staten Island of the City University of New York, New York, NY). Chimera
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molecular visualization and analysis software v. 1.10.1 (Resource for Biocomputing,
Visualization, and Informatics, University of California, San Francisco, CA) was used to
calculate the oxazole-phenyl hydrogen bond distances of PHOXI, and to create the image of
the TrpZip2 hairpin shown in Figure 6.
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64 Results
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Incorporation of PHOXI into peptides under biological conditions for fluorescence
enhancement
2
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Two peptides, NH -Trp-5-HTP-OH (WWOH) and a TrpZip2 substituted with 5-HTP in place
2
of Trp in position 4 (SWT-5-HTP-ENGKWTWK or TrpZip2, Trp4ꢂ5-HTP) were used to
269
evaluate the PHOXI derivatization reaction. Both peptides readily reacted with benzylamine
2
2
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in borate buffer pH 8.0, in the presence of 2mM K
3
[Fe(CN) ] to produce fluorescent PHOXI
6
adducts within 10 minutes.
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The HPLC monitoring of reaction products observed during the PHOXI derivatization of
NH -Trp-PHOXI-OH under varying conditions is shown in Figure 1A. The fluorescence
emission spectrum of NH -Trp-PHOXI-OH, showing characteristic PHOXI visible emission
with λmax=451nm and a marked increase in intensity (2.2 x) relative to NH -Trp-5-HTP-OH,
is shown in Figure 2B, right. The PHOXI emission increase is due to the synergistic effects of
2
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2
2
2
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76
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2
3
-1
-1
a high extinction coefficient (4.5 × 10 M cm as measured here in 0.5M Borate pH 8.0) and
quantum yield (0.54). Conducting the oxidation reaction in the absence of benzylamine
(Figure 1A, lower left) produces a number of products. This is consistent with the highly
reactive nature of p-quinoneiminemethide intermediate 2 (Scheme 1) that is formed upon
279
2
2
2
80
81
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0
oxidation of 5-HTP, and with previous studies of oxidation of 5-hydroxyindole showing a
complex mixture of oligomeric products resulting from nucleophilic capture of intermediate
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2 at the 4 position. However, in the presence of large excess of benzylamine, the fluorescent
PHOXI adduct is the major product of the reaction (Figure 1A, upper left). The concentration
of 5-HTP bearing peptide had a profound effect on the number and amount of undesired
byproducts produced during derivatization. Undesired byproducts are produced when the
peptide concentration is increased from 10 to 100μM (Figure 1A, lower right). These
products appear at the same retention time as those formed during the oxidation of WWOH
in the absence of benzylamine. This finding suggests that at higher concentrations
unwanted products result from capture of intermediate 2 by another molecule of peptide.
We found that generally keeping the 5-HTP-bearing peptide at concentration of 20μM or
below eliminates these byproducts.
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These reaction conditions were applied in the derivatization of the peptide, TrpZip,
W4ꢂ5-HTP. HPLC results monitoring this reaction are given in Figure 1B. The left plot in
Fig. 1B shows the elution time of TrpZip2 Trp4ꢂ5-HTP in the absence of reactants. The
right plot in Fig. 1B shows the elution profile for reactants only, grey trace, and the
completed reaction, black trace, with the peak for the PHOXI-derivatized TrpZip2 peptide
denoted as 5. We note the significantly reduced number of byproduct peaks in the HPLC for
the PHOXI TrpZip2. The fluorescence emission spectra for TrpZip2 Trpꢂ5-HTP and the
PHOXI derivatized peptide are shown in Fig. 2C, right panel. Fluorescence spectra of these
products are consistent with free PHOXI emission and excitation (Fig. 2A), further
corroborating their identity as PHOXI peptide adducts.
296
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2
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98
99
00
301
3
02
3
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04
05
After separation of reaction products by HPLC, each peptide produced a major peak with
high absorbance at 326nm, characteristic of PHOXI. These peaks were isolated, and their
identity was confirmed by mass spectrometry as NH -Trp-PHOXI-OH
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07
Figure 1. A. HPLC monitoring of formation of PHOXI. All reactions are carried out in
250mM Borate buffer pH 8.0 for 10min. A. Derivatization of NH2-Trp-5-HTP-OH in the
presence and absence of reactants. Top left: 10μM NH2-Trp-5-HTP-OH only; Top right:
10μM NH2-Trp-5-HTP-OH with 2mM K3[Fe(CN)6] and 0.5M benzylamine; Bottom left:
10μM NH2-Trp-5-HTP-OH with 2mM K3[Fe(CN)6] only; Bottom right: 100μM NH2-Trp-5-
HTP-OH with 2mM K3[Fe(CN)6] and 0.5M benzylamine. Peaks are identified as NH2-Trp-5-
HTP-OH (1), benzylamine (2) and NH2-Trp-PHOXI-OH (3). Grey trace: monitoring
absorbance at 320 nm; black trace: monitoring absorbance at 280 nm B. Derivatization of
10μM Trp-Zip2, Trp4ꢂ5HPT. Left panel: peptide only; right panel: reagents alone, grey
3
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trace; reaction solution, black trace. Peaks are identified as: benzylamine (2), peptide (4),
PHOXI derivatized TrpZip2 (5).
3
18
(calculated [M+H] 508.19, observed [M+H] 508.15) (See Supporting Information, Figure S1)
and SWT-PHOXI-ENGKWTWK, i.e., TrpZip2, Trp4ꢂ5-HTP (calculated [M+H] 1726.79,
observed [M+H] 1726.92) (Supporting Information, Figure S2).
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To ensure that PHOXI derivatization conditions are selective for 5-HTP only, a control
reaction was also conducted using the peptide Aβ-40, which does not contain 5-HTP but has
all canonical amino acid except for cysteine, tryptophan and proline (Figure 3). This peptide
was found to not be affected by reaction conditions used for PHOXI derivatization. As the
peptide sequence for TrpZip2, Trp4ꢂ5-HTP contains three additional Trps, reaction of
benzylamine with Trp is also eliminated as demonstrated by HPLC (Fig. 1B) and mass
spectroscopic analysis of the PHOXI derivatized peptide. Lastly, a similar control reaction
was conducted with N-acetyl cysteine, with similar result, as shown in Figure S3. N-acetyl
Cys remained underivatized in the presence of the potassium ferricynide and benzylamine
reagents.
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3
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1
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Figure 2. Fluorescence excitation (left panels) and emission (right panels) spectra of 5-HTP
and PHOXI, and peptides derivatized with these chromophores. Spectra for 5-HTP-bearing
species given by grey lines. Spectra for PHOXI derivatized species given by black lines.
Emission spectra for PHOXI derivatized species were acquired at two excitation
wavelengths: 280nm (dashed black line) and 336 nm (solid black line). The emission
monochromator was set to the emission maximum as shown in the right panels for
collection of the excitation spectra, given in the left panels. A. Spectra for NH2-5-HTP-OH
(grey lines) and PHOXI (black lines). B. Spectra for NH2-Trp-5-HTP-OH (grey lines) and
NH2-Trp-PHOXI-OH (black lines). The emission intensity for NH2-Trp-5-HTP-OH is scaled
to 2.2x the original intensity. C. Spectra for TrpZip2, Trp4ꢂ5-HTP (grey lines) and
TrpZip2, Trp4ꢂPHOXI (black lines).
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Spectroscopic properties of PHOXI
Fluorescence enhancement of PHOXI derivatized peptides and Tryptophan-PHOXI FRET
Figure 2 shows the fluorescence emission and excitation spectra of 5-HTP (A), WWOH (B)
and TrpZip2, Trp4ꢂ5-HTP (C) before and after PHOXI derivatization. The excitation
maxima of PHOXI analogues of 5-HTP, WWOH and TrpZip2, Trp4ꢂ5-HTP were found to be
336nm for the first two species and 341nm for the last. These wavelengths are outside of
the Trp absorption envelope allowing for selective excitation of PHOXI in the presence of
Trp. The excitation spectrum for 5-HTP in 0.5 M borate buffer, pH 8.0 (Fig. 2A, left panel,
grey line) is narrow, peaking at 316 nm. The normalized (x50) excitation spectrum for
aqueous PHOXI is much broader and almost completely overlaps that of 5-HTP. It peaks at
332 nm. with a small intensity at 280 nm, the wavelength used for Trp excitation. The
emission spectra for 5-HTP and PHOXI are given in Fig. 2A, right. The grey emission
spectrum of 5-HTP (300 nm. excitation) is narrow with an emission maximum at 346 nm.
344
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The breadth of the PHOXI excitation spectrum allows for excitation at 280 nm, a wavelength
typically used to excite Trp emission, and at a wavelength outside of the 5-HTP excitation
band, 336 nm. Emission spectra collected at these two excitation wavelengths prove useful
for demonstrating FRET from Trp to PHOXI in WWOH and TrpZip2, Trp4ꢂ5-HTP, as
discussed below. The emission spectrum for PHOXI excited at 336 nm. (Fig. 2A, right, 17x,
solid black line) consists of a single, broad band with a peak at 459 nm. The emission
spectrum for PHOXI excited at 280 nm. (Fig. 2A, right, 100x, dashed black line) is very weak,
here being magnified 100 times. The emission signal is at the level of noise in the emission
region (300-375 nm) of 5-HTP and Trp.
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Evidence of FRET from Trp residues to the PHOXI residue of WWOH and TrpZip2,
Trp4ꢂ5-HTP can be seen in the excitation and emission spectra of PHOXI bearing peptides
upon excitation at 280nm (Fig. 2B,C, right panels, dashed lines). Compared to the nil 280
nm.-excited emission spectrum of PHOXI alone (Fig. 2A, right panel, dashed line), these
emission spectra show significant intensity at the PHOXI emission maximum of 459 nm,
which can only have resulted from energy transfer from one or more Trp residues. We have
368
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computed the critical Forster distance, R = 23.5Å for the PHOXI-Trp system. This value falls
0
into the useful range for distance measurements in the context of biological
macromolecules.
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Figure 3. PHOXI derivatization conditions do not affect residues other than 5-HTP: HPLC
monitoring of PHOXI derivatization of Aβ-40. All reactions are carried out in 250mM Borate
buffer pH 8.0 for 10min, with the following additions: (A) Derivatization Reagents only:
2mM K3[Fe(CN)6] and 0.5M benzylamine; (B) 20μM Aβ-40 peptide only; (C) 20μM Aβ-40
peptide with 2mM K3[Fe(CN)6] and 0.5M benzylamine; (D) Chromatogram C with A
subtracted.
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Solvatochromism
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To glean the utility of PHOXI as a spectroscopic probe capable of providing structural
information, we synthesized it from 5-hydroxyindole and benzylamine, and studied its
spectroscopic properties in six different neat solvents spanning a range of polarity. The
results are summarized in Table 1, and absorption and emission spectra are shown in
Figure 4A, B.
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Table 1. Spectroscopic properties of PHOXI in neat solvents.
-
1
Solvent
λ
abs(nm)
λ
ex (nm)
λ
em (nm)
γ
Stokes(cm )
φ
F
(±10%) Lifetime (ns)
Cyclohexane 331
331
336
335
332
336
336
372
390
409
427
437
459
3330
5415
5580
7831
7886
9366
0.6 1.69
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Toluene
,4-Dioxane
322
333
320
325
321
0.69
0.78
0.72
0.55
0.54
1.91
2.51
3.29
4.06
5.71
1
Acetonitrile
Methanol
Water
3
89
390
391
PHOXI fluorescence emission maxima were found to be very sensitive to solvent,
exhibiting red-shifted emission with increasing solvent polarity. PHOXI emission redshifts
by 87nm in water relative to cyclohexane, suggesting that PHOXI fluorescence emission is
highly sensitive to the environment. Vibrational structure is present in the emission spectra
of PHOXI in non-polar solvents cyclohexane and toluene, but not in more polar solvents
(Figure 4B).
3
3
3
92
93
94
395
3
96
3
3
3
4
97
98
99
00
Because of the increase in dipole moment that accompanies electronic excitation in a
solvatochromic molecule, considerable solvent reorganization is expected for solvents that
are both hydrogen bond donors and acceptors, i.e., water and methanol. The energy of
reorganization necessary upon excitation is responsible for the observed large Stokes shifts
for PHOXI in these solvents (Table 1), the diminished radiative rate, and the concomitant
lower quantum yield (0.54-0.55) relative to that for other solvents. At the same time, the
excited state is stabilized by the solvent reorganization, resulting in a longer lifetime of 4.06
ns for methanol and 5.71 ns for water. For aprotic solvents like 1,4-dioxane and acetonitrile,
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less reorganizational energy is required upon PHOXI excitation since these are only
hydrogen bond acceptors. Thus, the Stokes shift is not as great, the radiative rate is not as
diminished as for water, and thus quantum yield is higher: 0.72 for acetonitrile and 0.78 for
1,2-dioxane. The excited state is still somewhat stabilized by noncovalent interactions,
yielding fluorescence lifetimes of 3.29 ns for acetonitrile and 2.51 ns for 1,4-dioxane. The
excited state is not stabilized in non-hydrogen bonding solvents like cyclohexane so the
fluorescence lifetime is only 1.69 ns. As little energy is required for solvent reorganization
upon excitation, the Stokes shift is small, and quantum yield is high, certainly within range
for that obtained with acetonitrile given the +10% uncertainty.
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The observation of an inverse relationship between fluorescence quantum yield and
lifetime with changes in solvent hydrogen bonding capability is not without precedent. In a
study of the xanthene dye, rhodamine 6G chloride cation, fluorescence lifetime increased
while quantum yield decreased as the hydrocarbon chain in a series of alcohols decreased.35
The trend culminates in water, where the longest lifetime of 4.08 ns was matched by the
418
, is given by:35
4
4
19
20
lowest quantum yield of 0.09+0.02. Since the radiative rate, k
r
φ
k = ꢩ
(4)
ꢨ
τ
4
4
21
22
Where φ is the quantum yield and τ is the fluorescence lifetime, it follows that the
nonradiative rate, knr, is given by
4
23
kꢪꢨ = (1 − φ)/τ
(5)
424
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Since the distribution of excitation energy between k
r
and knr changes with solvent, it
follows that the ratio, φ/τ, can change with solvent as well. Thus, φ and τ are not
necessarily directly proportional under all solvent conditions.
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Figure 4. Spectroscopic properties of PHOXI in solvents of varying polarity: cyclohexane
(solid black line), toluene (solid grey line), dioxane (dashed black line), acetonitrile (dashed
grey line) methanol (dash-dotted black line) and water (dash-dotted grey line). (A)
absorption, offset; (B) normalized fluorescence emission; (C) Multi-parameter fits of PHOXI
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and PRODAN (Constructed from previously published emission data ) fluorescence
maxima to a global solvent parameter, SP, as defined in the Catalán 3P model23–25
.
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36
37
While PHOXI absorption maxima did not vary significantly (spanning a range of only 10
nm), the quantum yields were found to vary, but remained high (between 0.54 and 0.78,
Table 1) in all solvents studied. Fluorescence lifetimes exhibited single exponential decay
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and varied between 1.69ns (cyclohexane) and 5.71ns (water), increasing with solvent
polarity (See Supporting Information, Figure S5).
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40
To better understand the solvatochromic nature of PHOXI, we fit its solvent-associated
spectral parameters to linear functions of a generalized solvent parameter, SP, using
empirical solvent parameters described by Catalán et al.23–25, and supplying proportionality
constants. A table of solvent parameters used in the analysis is given in Table S1. Figure 4C
shows the dependence of PHOXI emission maxima on SP; an analogous fit of the emission of
PRODAN in the same six solvents is included for comparison (PRODAN emission data used
to construct the graph can be found in ref 32). For PHOXI, a good linear fit of SP to emission
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maximum (R =0.98) was achieved when
SP = 0.79SPP + 0.12SB + 0.09SA
(6)
4
4
49
50
where SPP is solvent polarity-polarizability, SB is solvent basicity, and SA is solvent
2
acidity, while for PRODAN the best fit (R =0.99) is realized when
4
51
SP = 0.69SPP + 0.13SB + 0.18SA.
(7)
4
4
52
53
Notably, almost all of the variance in PHOXI spectral shifts is explained by SPP, with only
minor contributions from acidity and basicity. In fact, a single parameter fit to SPP alone
2
454
gives R =0.94, suggesting that the dipolar nature of the PHOXI excited state is primarily
4
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4
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responsible for the observed solvatochromism. In contrast, PRODAN fluorescence emission
displays roughly twice the sensitivity to solvent acidity. For PHOXI, the minor contributions
of acidity and basicity are not surprising, since it contains both an acidic group (the indolic
NH) and a basic group (the oxazole O or N).
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Variation in fluorescence lifetimes was also found to be primarily affected by SPP
2
(R =0.99), as shown in Figure S4. As ascertained from quantum yield and fluorescence
2
lifetime data, radiative decay (Γ, R =0.97) decreases with increasing solvent polarity. Non-
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radiative decay (knr, R =0.80) and quantum yield (φ , R =0.71) are not as well modeled by
F
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the Catalán et al. generalized solvent parameter, SP (Fig. S4).
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The change between the ground and excited state dipole moments of PHOXI can be
ascertained from its spectral shifts in solvents of differing polarity by means of the Lippert-
Mataga equation:32
ꢑ
ꢱꢲꢳꢴ
ꢑ
467
γꢫꢬꢭꢮꢯꢫ =ꢰ
(Δμ) Δf + constant
(8)
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70
where γꢫꢬꢭꢮꢯꢫ is the Stokes shift expressed as energy, h is Plank’s constant, c is the speed
of light, a is the Onsager radius of the solvent cavity occupied by the fluorophore, Δμ is the
difference in dipole moments in the ground and excited states, and Δf is the orientation
polarizability function defined as
471
ꢵꢗꢶ
ꢪꢌꢗꢶ
4
4
72
73
Δf =ꢰ
−ꢰ
(9)
ꢌ
ꢑꢵꢷꢶ
ꢑꢪ ꢷꢶ
where ε is dielectric constant, and n is the refractive index of the solvent.
474
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Because this equation does not take into account the contributions of specific solvent
interactions, we included the SA term into a multi parameter fit along with Δf, mirroring the
37
4
4
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77
analysis of PRODAN solvatochromism by Catalán . Regression analysis of PHOXI Stokes
shifts affords:
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γStokes = 15035(0.63Δf + 0.37SA) + 4205
(10)
2
479
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Accounting for the variance in Stokes shifts caused by SA in the above equation, and
substituting into the Lippert-Mataga equation gives:
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ꢹꢘꢎꢑꢰꢱꢲꢳꢴ
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82
Δμ =ꢰꢸ
(11)
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ꢑ
483
Assuming an Onsager cavity radius of 4.94 Å, (taken as half of the distance between C4 of
the phenyl substituent and the indolic N, which are the furthest heavy atoms flanking the
PHOXI conjugated system, as ascertained from its geometry optimized ground state
structure), the transition dipole moment of PHOXI is 10.6 Debyes.
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Structural properties of PHOXI
Surprisingly, repeated DFT calculations show that PHOXI adopts a planar conformation,
presumably because greater stabilization is attained if the oxazole oxygen lone pair does
not participate in the aromatic system, while the phenyl substituent does. Figure 5
illustrates the calculated planar geometry of PHOXI showing its ground state dipole vector
(A), as well as its HOMO (B), and LUMO (C) orbitals.
489
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4
4
90
91
92
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Figure 5. DFT calculated planar structure of PHOXI ground state showing the dipole vector
(A), HOMO (B), and LUMO (C) orbitals.
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We probed the structural perturbation caused by PHOXI substitution by obtaining CD
spectra of TrpZip2 bearing either 5-HTP or PHOXI at position 4; these results are shown in
Figure S6. TrpZip2 is engineered to adopt a β-hairpin structure, stabilized by stacking
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interaction of two pairs of tryptophan residues . The NMR structure showing these
interactions is given in Figure 6. The NMR structure reveals that the four Trp residue are
arranged in two pairs with orthogonal molecular planes: Trp 4 aromatic C(4)H points into
the center of the Trp 9 pyrrole ring while Trp 11 C(4)H points into the center of the Trp 2
pyrrole ring. Additionally, the indole molecular planes of Trps 2 and 9 are antiparallel and
at a relative angle of slightly less than 90°. Overall, the Trp indoles in TrpZip2 lie in a closely
packed arch about one face of the beta hairpin. Although Trp 4 is replaced by the bulkier
PHOXI, the PHOXI derivative of TrpZip2 still retains some of its β-hairpin structure. As
shown in Figure S6, the positive ellipticity at 230nm of PHOXI-bearing TrpZip2 at 25°C is
approximately the same as that of 5-HTP-bearing TrpZip2 at 80°C. Thus, despite the fact
that PHOXI is surrounded by three copies of the bulkiest possible amino acid residue, some
native β-hairpin structure is maintained. This provides for some optimism regarding
preservation of secondary structure upon PHOXI incorporation in proteins, especially at
molecular interfaces and other environments where there is typically less steric hindrance.
5
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00
01
02
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508
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12
5
13
Photodegradation
514
Succeptibitily of PHOXI to photodegradation was also tested by exposure to UV light and
monitoring the decrease of its fluorescence intensity at the maximum over time. These
results are shown in Figure S7. The experimental set up was identical to that used to probe
5
5
5
15
16
17
3
6
photostability of PRODAN and its analogue FR0 . After 1 hour, a solution of PHOXI in
518
19
toluene had 72% of its fluorescence intensity remaining, compared to 10% for PRODAN36
and 76% for FR036.
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Figure 6. NMR structure of the beta hairpin peptide, TrpZip2, with tryptophan residues
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specifically displayed (Image created from PDB 1LE1 using Chimera software, University
of California at San Francisco).
524 Discussion
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Incorporation into peptides
We have used two 5-HTP containing model peptides to show that the PHOXI fluorophore
can be introduced by a quick and selective oxidation reaction in aqueous buffer in the
presence of benzylamine. While unintended reaction byproducts may be observed during
derivatization of 5-HTP bearing peptides, the Aβ40 peptide, which is devoid of 5-HTP is
completely unaffected by the reaction conditions, attesting to the selectivity of this reaction.
While N-acetylcysteine in the presence of benzylamine here showed no product formation,
Yamagishi et al. found side-product formation in the cyclization reaction of a fourteen
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amino acid peptide bearing a Phe functionalized with benzylamine and 5-HTP (Fbza-R-T-R-N-
A-WOH-A-G-E-G-F-C-D).18
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Because the byproducts present during the oxidation of 5-HTP peptides in presence of
benzylamine are the same as those formed during oxidation of 5-HTP peptides in its
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absence, we presume that these species are formed by capture of the proposed reaction
intermediate 2 by nucleophiles other than the intended benzylamine.
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Furthermore, we found that lowering the 5-HTP substrate concentration dramatically
reduces the number and amount of these undesired products. This suggests that at higher
substrate concentrations capture of intermediate 2 by nucleophilic moieties on another
molecule of substrate leads to the formation of these products. At substrate concentrations
below 20 μM, the products formed by this intermolecular capture are eliminated.
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Although the presence of these side products is less than desirable in the context of
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peptide labeling, their formation is consistent with the proposed reaction mechanism and
is additionally documented in previous reports.20,21 However, even with this inherent
limitation, we found that PHOXI derivatized products are produced essentially free of
undesired byproducts at low substrate concentrations, and are easily visualized and
isolated by RP-HPLC for every peptide tested. Overall, the oxidative coupling reaction of 5-
HTP to produce PHOXI offers a fast and easy protocol for labeling of peptides: the reaction is
assembled from just three components in aqueous buffer, and is completed in 10 minutes or
less. Furthermore, because PHOXI is assembled in-situ using water-miscible benzylamine, it
avoids the need for inclusion of harsh organic solvents during labeling reactions, an
otherwise necessary step during labeling with many common organic fluorophores, which
suffer from low water solubility.
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Taken together, these above findings suggest that PHOXI labeled peptides are easily
accessed through oxidation of 5-HTP peptides in the presence of benzylamine and
subsequent RP-HPLC purification. The PHOXI fluorophore is introduced site-specifically at
the 5-HTP site, and because it only modifies the indole side chain of 5-HTP, the PHOXI label
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is introduced in a conformationally restricted manner, being linked to the peptide backbone
through a single methylene bridge. For a probe to be useful in characterization of the
microenvironment, this short and rigid tether offers clear advantages over other common
fluorophore attachment strategies (such as through side chain and terminal amine groups,
as well as cysteine thiol groups), which result in longer flexible tethers and greater degrees
of freedom.
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The PHOXI labeling strategy is applicable to wide variety of biomolecules, primarily due
to the ease of introduction of 5-HTP into peptides and proteins. For example, 5-HTP is easily
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incorporated as its Fmoc derivative during SPPS using standard protocols, incorporated
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into proteins in response to a Trp codon during recombinant expression in a Trp
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auxotrophic host, or introduced into a desired site by means of nonsense suppression.
Furthermore, recent work utilizing PHOXI as a fluorescent linker for peptide immobilization
suggests that Fmoc protected tryptophanyl PHOXI derivative can be introduced during SPPS
as well.40
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Applying this chemistry to 5-HTP protein substrates is expected to present challenges
associated with separation of the correctly PHOXI labeled protein from possible undesired
byproducts. While RP-HPLC purification may prove sufficient for some protein substrates,
inadequate resolution and unwanted protein denaturation in HPLC buffers are among the
expected challenges. However, a workaround solution based on substrate flexibility of this
fluorogenic coupling reaction can be envisioned. Several groups14,18 reported that
benzylamines substituted at one or more of the phenyl positions still react with 5-HTP to
form fluorescent adducts analogous to PHOXI. One of these reaction variants included the
introduction of biotynyl benzylamine upon oxidation of a 5-HTP peptide substrate.18
Because unwanted byproducts arise from the capture of the reactive intermediates by
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