Cysteine-linked aromatic nitriles as UV resonance Raman probes of protein structure

Article abstract of DOI:10.1002/jrs.3167

Nitriles introduced into peptides and proteins can serve as useful vibrational spectroscopic probes, because the nitrile C≡N stretch is well isolated from backbone and sidechain vibrational bands. Aromatic nitriles offer large nC≡N absorption intensity in infrared spectra and resonance enhancement in Raman spectra with ultraviolet excitation. We report the ultraviolet resonance Raman spectra of cyanophenylalanine attached to cysteine, through linkage reactions that are applicable to cysteine residues in proteins. Excitation profiles are reported, and the nC≡N detection limit is estimated to be 5μM. The band position is sensitive to solvent polarity and especially to strong H-bonding. The derivatization of mastoparan X peptide at introduced cysteine residues demonstrated the effectiveness of a cyanophenylcysteine probe in reporting the lowered environmental polarity when the peptide was incorporated into liposomes. For an asymmetrical cyanophenyl derivative, 2-CBCys, the intensity ratio of asymmetric and symmetric ring modes, n8_b and n8_a, was found to respond to solvent polarity and not to H-bonding. 2012 John Wiley & Sons, Ltd.

Full text of DOI:10.1002/jrs.3167

Research Article
Received: 3 November 2011
Revised: 15 December 2011
Accepted: 21 December 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jrs.3167
Cysteine-linked aromatic nitriles as UV
resonance Raman probes of protein structure
Colin L. Weeks,a Hyunil Jo,b Brandon Kier,c William F. DeGradob and
Thomas G. Spiroc*
Nitriles introduced into peptides and proteins can serve as useful vibrational spectroscopic probes, because the nitrile C ꢀ N
stretch is well isolated from backbone and sidechain vibrational bands. Aromatic nitriles offer large nC ꢀ N absorption inten-
sity in infrared spectra and resonance enhancement in Raman spectra with ultraviolet excitation. We report the ultraviolet res-
onance Raman spectra of cyanophenylalanine attached to cysteine, through linkage reactions that are applicable to cysteine
residues in proteins. Excitation profiles are reported, and the nC ꢀ N detection limit is estimated to be 5 mM. The band position
is sensitive to solvent polarity and especially to strong H-bonding. The derivatization of mastoparan X peptide at introduced
cysteine residues demonstrated the effectiveness of a cyanophenylcysteine probe in reporting the lowered environmental
polarity when the peptide was incorporated into liposomes. For an asymmetrical cyanophenyl derivative, 2-CBCys, the intensity
ratio of asymmetric and symmetric ring modes, n8b and n8a, was found to respond to solvent polarity and not to H-bonding.
Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: peptides; unnatural amino acids; proteins; UV resonance Raman; cyanophenylalanine
possible.[9,10] Labeling of proteins by a more facile method is desir-
able, and the use of arylation or alkylation reactions to attach phenyl
Introduction
Vibrational spectroscopy provides a powerful approach to moni-
tor structure and dynamics in proteins because vibrational modes
are sensitive to local conformation and molecular environment.
However, the method is often limited by spectral crowding and
the overlap of bands arising from multiple copies of the same
structural elements, the peptide links[1,2] and the amino acid
sidechains.[3] Introduction of additional probes, with unique
vibrational signatures, can provide a valuable adjunct to protein
studies. Nitrile derivatives are particularly attractive, because the
nC ꢀ N band, at ~2200 cmÀ1, is well removed from other bands
in the protein spectrum.
or benzyl nitriles to cysteine residues for IR studies was recently
reported in a preliminary communication.[11] These methods are
described more fully in the present paper, and UV resonance Raman
(UVRR) characteristics of the aromatic nitrile adducts are reported.
Boxer and coworkers have incorporated the thiocyanate (ÀSCN)
label into proteins, for IR[12] and NMR studies. To determine whether
UVRR spectroscopy could be applied to these labels, we investi-
gated the UVRR spectra of ethylthiocyanate (EtSCN). Unfortunately,
resonance enhancement was found to be weak, not greater than
that of cyanide ion itself, even with deep UV excitation. Thus, UVRR
spectroscopy is useful only for aromatic nitriles (Fig. 1).
Because of the polarizability of the C–N bond, the nC ꢀ N
wavenumber responds to the polarity of the nitrile environ-
ment[4–7] and to the proximity of H-bond donors. Boxer and
coworkers have shown that the dipolar effect is less pronounced
than that of specific H-bonding and that the two effects can be
distinguished by correlating nC ꢀ N with 13C nuclear magnetic
resonance (NMR) chemical shifts.[8]
The nC ꢀ N band can be measured using infrared (IR) or Raman
spectroscopy. The C ꢀ N stretch produces a strong induced IR
dipole, particularly if the C ꢀ N group is attached to an aromatic
ring. The environmental sensitivity of nC ꢀ N (Stark tuning rate)
is also larger for aromatic than for aliphatic nitriles.[4,5] Neverthe-
less, millimolar concentrations of protein are required for IR spec-
troscopy. For Raman spectroscopy, however, detection limits are
lowered into the micromolar range with ultraviolet (UV) excita-
tion of the cyanophenylalanine (PheCN) Raman spectrum, thanks
to resonance enhancement.
Methods
Raman spectra were obtained on samples sealed in 5-mm quartz
NMR tubes, which were spun to ensure mixing of the solution, using
5 Â 2 min of acquisition times to check for sample degradation
(decrease in peak intensity vs NaClO4 or NaCN standard and/or peak
deformation) and averaged. The scattered light was collected at
*
Correspondence to: Thomas G. Spiro, Department of Chemistry, University of
Washington, Box 351700, Seattle, WA, 98195, USA. E-mail: spiro@chem.
washington.edu
a Department of Chemistry and Biochemistry, University of Northern Iowa, Cedar
Falls, IA, 50614, USA
b Department of Biochemistry and Biophysics, University of Pennsylvania,
Philadelphia, PA, 19104-6059, USA
Cyanophenylalanine can be incorporated into chemically synthe-
sized polypetides. Incorporation into recombinant proteins via
the difficult method of orthogonal tRNA synthetase pairs is also
c
Department of Chemistry, University of Washington, Seattle, WA, 98195, USA
J. Raman Spectrosc. (2012)
Copyright © 2012 John Wiley & Sons, Ltd.

C. L. Weeks et al.
Scheme 1. S-arylation of N-acetylcysteine 6b.
Figure 1. Aromatic nitrile derivatives of cysteine: S-4-nitro-2-cyanophenyl-
N-acetylcysteine (4-N-2-CPCys, 1), S-4-cyanophenyl-N-acetylcysteine
(4-CPCys, 2), S-4-nitro-2-cyanobenzyl-N-acetylcysteine (4-N-2-CBCys, 3),
S-4-cyanobenzyl-N-acetylcysteine (4-CBCys, 4), S-2-cyanobenzyl-N-acetylcysteine
(2-CBCys, 5).
Both 2,4-dinitro benzonitrile and 4-nitro benzonitrile reacted
smoothly to provide the corresponding S-arylated product 5-N-
2-CPCys 1 (5-nitro-2-cyanophenyl cysteine) and 4-CPCys 2 (4-
cyanophenyl cysteine), respectively, using Cs2CO3 as a base in
DMSO.[17] The second nitro moiety in 2,4-dinitro benzonitrile
was included to increase the electrophilic character of the aro-
matic ring but did not prove essential for the coupling reaction.
135ꢁ geometry and focused onto a 1.26-m spectrograph (Spex
1269, 3600 grooves/mm grating) equipped with a liquid nitrogen-
cooled CCD camera (Roper Scientific). The laser power at the sample
was 0.5 mW. Variable wavelength excitation was provided by an
intracavity frequency-doubled Ar+ laser at 257 and 244 nm (Coher-
ent Innova 300 FRED) and by a Q-switched frequency quadrupled
Ti:sapphire laser (GM30, Photonics International, Inc.) at 197 nm
(20 ns, 1 kHz) and at 212, 220 and 229 nm (0.5 mJ/pulse).[13–15]
Raman excitation profiles were determined for the nC ꢀ N
bands of the cysteine derivatives in 0.1-mM aqueous solution con-
taining 100-mM NaCN as internal standard. Raman cross-sections
were calculated as previously described.[16] Aqueous stock solu-
tions were made slightly basic (1–2 drops of 1-M NaOH added
per 10 mL) to improve sample solubility and reduce the hazard
of HCN production. The solvent dependence of the nC ꢀ N band
was determined in H2O, tetrahydrofuran (THF), 1,1,1,3,3,3-hexa-
fluoroisopropanol (HFIP) and cyclopentanol, using 229-nm excita-
tion. Calibration and analysis of the spectra were carried out
using GRAMS AI software (Thermo Electron Corp.). Acetone and
DMSO-d6 were used as the calibration standards.
Aqueous preparation of 4-N-2-CBCys 3, 4-CBCys 4 and 2-CBCys 5
Because post-translational modification of peptides and proteins
is best carried out in aqueous solution, aromatic nitrile incorpora-
tion under aqueous condition was investigated. The conventional
bioconjugation techniques unique to cysteine thiols, including
a-iodoacetamide or maleimide derivatives, were also considered.
However, less sterically demanding benzyl bromide derivatives
were selected, and the use of benzyl halide for S-alkylation of
Cys residues[17–21] suggested that selective alkylation on the
cysteine sulfur would prove viable under aqueous conditions.
Indeed, the coupling of cyanobenzyl moieties to N-acetyl cysteine
1 was successfully achieved using either buffered NH4HCO3
system[20] or aqueous NH4OH,[21] yielding 4-N-2-CBCys 3, (4-nitro-
2-cyanobenzylcysteine), 4-CBCys 4 (4-cyanobenzylcysteine) and
2-CBCys 5 (2-cyanobenzylcysteine; Scheme 2)
Mastoparan X peptides were investigated at concentrations
of~40 mM. DOPC (1,2-dioleoylphosphatidylcholine) vesicles (0.05 mm)
were prepared by extrusion using an Avanti Polar Lipids extruder.
Ascorbate was added (20x peptide concentration) to mitigate
potential degradation of the CBCys or Trp residues, as had been
observed for Trp and PheCN in previously investigated mastoparan
X peptides.[16]
Ultraviolet–visible absorption spectra were recorded in a 0.10-cm
quartz cell using an Agilent 8453 diode array spectrophotometer.
Fluorescence spectra were recorded with an LS-50B Perkin Elmer
Luminescence Spectrophotometer. NMR spectra were recorded
using a Bruker 500-MHz instrument.
Results and discussion
Linkage chemistry
Non-aqueous preparation of 5-N-2-CPCys 1 and 4-CPCys 2
The introduction of cyanophenyl groups to N-acetyl cysteine 6
was achieved via nucleophilic aromatic substitution reactions
using nitrobenzonitriles as electrophiles (Scheme 1).
Scheme 2. Preparation of 4-N-2-CBCys 3, 4-CBCys 4 and 2-CBCys 5.
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Cysteine-linked aromatic nitriles as UV resonance Raman probes of protein structure
Attachment of probes to mastoparan X
residue (Scheme 4). It is noteworthy that the reaction was very
selective and high yielding as shown in the high-performance
liquid chromatography profile of the crude alkylation reaction
mixture. (See supporting information, Part 1).
Because our goal was an accessible route to a minimally disrup-
tive post-translational addition of probe moieties, we verified that
our linkage chemistry could be used to attach the probes to
whole peptides. We chose mastoparan X as our model system,
as it associates with lipid membranes, allowing for hydrophobic
burial of the probe, in the presence of lipids.[22] Mastoparan X
was previously used in our UVRR study of the PheCN probe,
which was incorporated by peptide synthesis.[16] Four consecu-
tive residues (positions 2–5) were chosen for mutation to cysteine
and subsequent alkylation with 4-cyanobenzyl bromide.
We first attempted direct solid-phase installation of this
probe by alkylation, which could facilitate purification and
allow for other modification. Two cysteine variants of masto-
paran X (K4C and G5C) were prepared using conventional
fluorenylmethyloxycarbonyl-based solid-phase peptide synthe-
sis. Selective 4-methoxytrityl (para-Monomethoxytrityl) depro-
tection on cysteine (structure 7 – Scheme 3) was achieved
by 1% trifluoroacetic acid/triisopropylsilane/dichloromethane
treatment,[23] and the subsequent alkylation was smoothly accom-
plished via identical conditions to yield the desired peptides 8 and
9 (Scheme 3). Polyethylene glycol-based resin was used because of
its convenient swelling in aqueous buffer, but polyethylene glycol-
based resins are not strictly required for probe incorporation.[24]
Next, we turned our attention to the solution-phase method
that could be useful for probe installation in native peptides
and proteins. The other two cysteine variants of mastoparan X
(N2C and W3C) were prepared via standard fluorenylmethyloxy-
carbonyl solid-phase peptide synthesis. After cleavage and
deprotection, the purified peptides were subject to conditions
identical to standard N-acetyl cysteine alkylation, again using
4-cyano benzyl bromide to introduce the probe to the cysteine
Characterization
Stability
The stability of the cysteine derivatives was monitored, as photo-
degradation can be a problem when UV excitation is used to
acquire Raman spectra. Considerable photodegradation was
evident for an aqueous solution of 5-N-2-CPCys after acquisition
of consecutive Raman spectra. The position of the nC ꢀ N band
shifted from 2230 to 2242 cmÀ1, and the peak broadened and
weakened. (Figs. S3–S5, supporting information). 4-N-2-CBCys
also showed poor stability in aqueous solution, even in the
absence of laser exposure. After a sample was stored overnight
(in the dark at 4 ꢁC), the intensity of the nC ꢀ N band had decreased
by ~15%, and there was a 4-cmÀ1 shift in its position. (Fig. S6,
supporting information). Degradation of both derivatives was
evidenced by a color change (from orange to pale yellow) and
the appearance of new species in the NMR spectrum (Fig. S5 right
panel, supporting information). The similar chemical shifts and
coupling constants for all but the sidechain methylene (beta) pro-
tons suggests that the initial degradation event is cleavage at the
S–C bond. However, after >3 h of laser exposure, a complex mix
of products is evident (Fig. S5 last panel, supporting information).
The instability is attributable to the electronic effects of the nitro
substituents, because none of the compounds lacking the nitro
groups, 2-CBCys, 4-CBCys or CPCys, showed any change in the nC
N position or other signs of photodegradation during Raman spec-
tral acquisition. A significant decrease in the band intensity (>10%)
was only observed after storage of the solutions for extended
periods (10+ days; Figs. S7–S9, supporting information).
Scheme 3. On resin probe incorporation.
Scheme 4. Solution-phase probe incorporation.
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C. L. Weeks et al.
Fluorescence
PheCN derivatives showed any appreciable fluorescence. It is likely
that the fluorescence is quenched by the cysteine sulfur atom.
Although 4-PheCN fluoresces strongly,[25,26] and has been used in
fluorescence energy transfer (FRET) measurements with trypto-
phan residues as acceptors,[25,27] none of the cysteine-linked
UV resonance Raman enhancement
The nC ꢀ N band was strongly enhanced with UV excitation for all
cysteine derivatives, as it is for PheCN itself.[16] Excitation profiles
(Fig. 2) show maxima near 235 nm, coincident with the absorption
bands in this region, indicating resonance with the benzene-like La
electronic transitions.[28] Significantly larger cross-sections were seen
with 197 nm excitation, in resonance with the stronger benzene-like
Ba,b electronic transitions. However, the noise level at 197 nm was
greater than at 229 nm, due (at least in part) to the superior efficiency
of our detector at longer wavelengths. Signal/noise was optimal at
229 nm, as was found for PheCN.[16] Consequently, 229 nm excitation
was used for studying detection limits and solvent effects.
Figure 3 shows the intensity variation of the 2-CBCys nC ꢀ N
band as the concentration is decreased from 50 to 2 mM, in
100 mM NaCN, present as an internal standard. Although the
band is detectable at 2 mM, we judge 5 mM to be the practical de-
tection limit. A similar result was obtained for 4-CBCys.
Figure 2. Raman excitation profiles (symbols) and UV–visible absorption
spectra (lines) for NCPCys, NCBCys, CPCys, 2-CBCys and 4-CBCys.
Figure 4. Solvent-dependent shifts in the nC ꢀ N bands of 4-CBCys in
H2O (200 mM), THF (500 mM) and HFIP (360 mM) and CPCys in H2O (100 mM),
THF (100 mM), HFIP (90mM) and cyclopentanol (saturated solution). nC ꢀ N
intensities normalized.
Figure 3. UV Raman spectra of 4-CBCys at the indicated concentrations,
showing its nC ꢀ N band along with that of the CNÀ from the 100-mM
NaCN intensity standard.
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J. Raman Spectrosc. (2012)

Cysteine-linked aromatic nitriles as UV resonance Raman probes of protein structure
For comparison with the aromatic Cys derivatives, EtSCN (ethyl
thiocyanate) was investigated with 197 nm excitation, because its
absorbance peak near 197 nm suggested potential resonance
enhancement for UVRR. Thiocyanate adducts of proteins are readily
prepared by reacting cysteine sidechains with cyanide and have
been exploited by Boxer and coworkers as IR probes. Unfortunately,
the nC ꢀ N intensity of EtSCN was similar to that of free cyanide
(Fig. S10, supporting information). Ala-CN was tested and also failed
to display resonance enhancement beyond that of free cyanide.
Interestingly, we found a direct correlation with solvent polarity
for the intensity ratio of the n8a-type and n8b-type aromatic ring
modes of 2-CBCys (Fig. 5 and Table 1). These are stretching modes
of the phenyl ring, with orientations parallel (n8a) and perpendicular
(n8b) to the pseudo twofold axis, defined by the CN substituent. If
this axis is maintained, as in 4-CBCys, the perpendicular mode is
weak; but it gains strength when the symmetry is perturbed, as in
2-CBCys. The perturbation is seen to depend on the solvent, with
the n8b relative intensity increasing in the order THF < HFIP < water.
The fact that HFIP is not an outlier in this series indicates that the
perturbation results from a dipole–dipole interaction, rather than
from H-bonding. Thus, the n8b/n8a intensity ratio could be used
in conjunction with the nC ꢀ N shift to distinguish H-bonding and
polarity effects when 2-CBCys is used as a UVRR probe.
Solvent and H-bonding dependence
To assess the environmental sensitivity of the nC ꢀ N band, we
recorded Raman spectra of 4-CBCys, CPCys (Fig. 4) and 2-CBCys
(Fig. S1, supporting information) in a variety of solvents. As seen
previously for PheCN, the wavenumber depends on solvent po-
larity, shifting up in aqueous solution (~2236 cmÀ1) from its value
in THF or cyclopentanol (~2230 cmÀ1). However, a much higher
wavenumber, ~2250 cmÀ1, was produced in HFIP. HFIP has a
lower dielectric constant than cyclopentanol (Table 1), but it is
known to form unusually strong H-bonds, an effect clearly seen
in the anomalously high nC ꢀ N. Boxer and coworkers have
reported specific H-bond effects on nC ꢀ N of EtSCN and have
shown how to distinguish them from polarity shifts, using com-
bined IR and 13C NMR measurements.[8]
Mastoparan X peptides
The four 4-CBCys-containing mastoparan X peptides revealed
5–6 cmÀ1 downshifts between aqueous solution and DOPC lipo-
somes (Fig. 6), consistent with transfer of the probes to a less
polar environment. When bound to liposomes, the peptides form
amphiphilic helices,[29] perpendicular to the lipid axis.[30] How-
ever, the observed shifts do not distinguish which side of the
helix is labeled, indicating that sidechains on the solvent-facing
side of the helix are shielded from solvent by the lipid head
groups. A similar result was reported from a previous IR study of
mastoparan X having synthetically incorporated PheCN probes.[22]
We note that the degradation observed in our previous UVRR
study of mastoparan X-bearing liposomes[16] was greatly reduced
by the inclusion of ascorbate, in 20 molar excess, in the buffer. In
addition to its anti-oxidant properties, ascorbate acts as a UV
shield for molecules in the laser path, including those at the rear
of the sample tube, outside of the focal volume from which
Raman scattering was collected by the optics. Interference from
the ascorbate Raman spectrum was negligible.
Table 1. Comparison of dielectric constants for different solvents
Solvent
nC ꢀ N (cmÀ1
CPCys 4-CBCys 2-CBCys
)
n8a/b ratio Dielectric
constant[31]
2-CBCys
H2O
2238 2235 2231
2.50
80.10a
18.5c
Cyclopentanol 2230 (No data) (No data) (No data)
HFIP
THF
2253
2229
2250
2228
2244
2225
2.05
1.35
16.70a
7.52b
HFIP, hexafluoroisopropanol; THF, tetrahydrofuran.
aat 293 K;
bat 295 K;
cat 288 K.
Conclusions
Cyanophenyl groups can be linked to the thiolate group of cyste-
ine using electrophilic aromatic substitution with nitrocyanophenyl
derivatives or via alkylation of the thiolate with cyanobenzyl
Figure 6. UV Raman spectra of mastoparan X W3CBCys derivative in aque-
ous buffer with (dashed line) and without (solid black line) DOPC liposomes
(Concentration: ~50 mM). See figure S12 for all mastoparan X peptide data.
Figure 5. Solvent-dependence of the n8a-type and n8b-type bands of 2-
CBCys in H2O (200 mM), THF (saturated solution) and HFIP (450 mM).
J. Raman Spectrosc. (2012)
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C. L. Weeks et al.
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The resulting cysteine-linked cyanophenyl or cyanobenzyl labels
afford intense UVRR bands for the isolated nC ꢀ N mode, which
serves as a monitor of the probe environment. Its wavenumber
increases with increasing polarity and is particularly sensitive to
strong H-bond donation. The 4-CBCys probe attached to cysteine
residues introduced into mastoparan X reveals a less polar environ-
ment when the peptide inserts into liposomes. The nC ꢀ N shift is
the same for probes attached to either side of the amphiphilic helix,
indicating that sidechains facing the surface are also protected
from solvent. PheCN and its derivatives greatly outperform their
tyrosine and, especially, phenylalanine analogs as Raman probes
because of both superior enhancement over a broad range of wave-
lengths and also the isolated, environment-sensitive nC ꢀ N band.
For the 2-CBCys molecule, the n8b/n8a intensity ratio increases
with solvent dielectric constant, without anomalous values asso-
ciated with strong H-bonding. This probe could therefore be
used to disentangle specific H-bond and general polarity effects,
analogous to the combination of IR and 13C NMR spectroscopy
used by Boxer and coworkers for thiocyanate probes.[8]
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Supporting information
Supporting information can be found in the online version of this
article.
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