A direct comparison of azide and nitrile vibrational probes

Article abstract of DOI:10.1039/c0cp02774j

The synthesis of 2′-azido-5-cyano-2′-deoxyuridine, N ₃CNdU (1), from trityl-protected 2′-amino-2′-deoxyuridine was accomplished in four steps with a 12.5% overall yield. The IR absorption positions and profiles of the azide and nitrile group of

Full text of DOI:10.1039/c0cp02774j

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PAPER
A direct comparison of azide and nitrile vibrational probesw
Xin Sonia Gai, Basil A. Coutifaris, Scott H. Brewer* and Edward E. Fenlon*
Received 3rd December 2010, Accepted 26th January 2011
DOI: 10.1039/c0cp02774j
The synthesis of 20-azido-5-cyano-20-deoxyuridine, N3CNdU (1), from trityl-protected
20-amino-20-deoxyuridine was accomplished in four steps with a 12.5% overall yield. The IR
absorption positions and profiles of the azide and nitrile group of N3CNdU were investigated in
14 different solvents and water/DMSO solvent mixtures. The azide probe was superior to the
nitrile probe in terms of its extinction coefficient, which is 2–4 times larger. However, the nitrile
IR absorbance profile is generally less complicated by accidental Fermi resonance. The IR
frequencies of both probes undergo a substantial red shift upon going from water to aprotic
solvents such as THF or DMSO. DFT calculations supported the hypothesis that the molecular
origin of the higher observed frequency in water is primarily due to hydrogen bonds between the
probes and water molecules.
the mastoparan-X peptide,5,9 myoglobin,13 and ribonuclease
Introduction
S;12 the latter was also studied with the UAAs m-cyanophenyl-
ananine and S-cyanohomocysteine. The chemical conversion of
cysteine thiols into thiocyanates or aryl nitriles has been utilized
to introduce a vibrational probe into numerous peptides and
proteins.16–18 The vibrational Stark effect of a nitrile-containing
enzyme inhibitor was effectively used to measure the electric
field in the active site of human aldose reductase.19 The UAA
5-cyanotryptophan has also been employed as an infrared probe
of protein hydration status.20
The use of vibrational probes to study biomolecules has a long
and rich history. In most of the early work the probe was a
ligand bound to hemoproteins.1 For example, four decades
ago McCoy and Caughey used infrared spectroscopy to
investigate the binding of ligands such as azide, cyanide, and
thiocyanate to metmyoglobin and methemoglobin.2 More
recently, Suydam and Boxer3 examined a series of vibrational
probes for proteins and concluded that the most useful ones
have the following properties: (1) an IR absorption band that
is narrow, intense, and in a clear region of the spectrum; (2)
sensitive to changes in local environment; (3) small size to
minimize structural perturbations; and (4) chemical stability.
It is therefore no surprise that nitriles and azides have emerged
as the most useful vibrational probes, since both meet these
criteria. Within the past decade the use of nitriles to study non-
hemoproteins has become prevalent, and more recently azides
have also been used for this purpose.
In terms of azides, the UAAs azidoalanine and azido-
homoalanine have been synthetically incorporated into peptide
fragments of the Ab peptide and the N-terminal domain of
the ribosomal protein L9 (NTL9), respectively, to serve as
vibrational probes of protein structure.21,22 An engineered
aminoacyl-tRNA synthetase/tRNA pair has been employed
to incorporate the UAA p-azidophenylalanine (pN3Phe) into
the G-protein coupled receptor rhodopsin to study the helix
movements during light activation via infrared spectroscopy.23,24
Aromatic azides such as pN3Phe have an additional property
of photoreactivity. The instability of this probe is a disadvantage
for vibrational experiments but it can be utilized for photo-
crosslinking experiments.25,26 Azido-nicotinamide adenine
dinucleotide was recently synthesized and shown to be a 2D
IR probe of the active sites of NAD-dependent enzymes.27
These same workers showed that the structurally related
3-azidopyridine was less suitable as a vibrational probe because
of a complex absorption profile resulting from accidental Fermi
resonance.28
In terms of nitriles, the unnatural amino acid (UAA)
p-cyanophenylalanine (pCNPhe) has been inserted into proteins
both synthetically4–12 and by in vivo nonsense suppression.13–15
The nitrile of pCNPhe served as a vibrational probe to study the
hydration and electrostatic environments of several protein
systems, including the MLCK peptide–calmodulin complex,4
Franklin & Marshall College, Department of Chemistry, Lancaster,
PA 17604-3003, USA. E-mail: scott.brewer@fandm.edu,
edward.fenlon@fandm.edu; Fax: (717) 291-4343;
The use of nitrile and azide vibrational probes in nucleic
acids has lagged behind the protein work. These functional
groups were first incorporated into nucleic acids decades ago,
but the idea to employ them as vibrational probes was first
proposed in 2007 when the Stark tuning rates of a number of
Tel: (717) 358-4766, (717) 291-4201
w Electronic supplementary information (ESI) available: Full
synthetic procedures; FTIR absorbance spectra of N3CNdU in 14
solvents; line shape analysis of the FTIR absorbance spectrum of
N3CNdU in THF and methanol; variable temperature spectra of
N3CNdU in water; DFT geometry-optimized structures.
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nitrile- and azido-nucleosides were published.29 Krummel and
Zanni30 have demonstrated that the vibrational coupling of
and nitrile groups of N3CNdU to compare and contrast the
utility of these groups as vibrational probes. Fig. 1 shows the
normalized FTIR absorbance spectra of N3CNdU dissolved in
water, tetrahydrofuran (THF), or methanol. The IR absorbance
spectrum of N3CNdU in water shows two well-resolved bands
at 2124.1 cmꢀ1 and 2242.7 cmꢀ1 corresponding to the azide
asymmetric and the nitrile symmetric stretching frequency,
respectively. The fwhm of the azide IR absorbance band is
B26 cmꢀ1 compared to a fwhm of B10 cmꢀ1 for the nitrile IR
absorbance band. These experimental results are consistent
with previous studies of the single-modified nucleosides:
N3dU32 and CNdU.31 A third distinct difference between the
bands is that the azide IR absorbance band is a factor of two
larger than the nitrile IR absorbance band based upon the
peak absorbance of each band. This difference increases to a
factor of five when considering the relative integrated intensity
of each band due to the significant difference in the fwhm.
15
14N- and N-labeled 5-cyano-20-deoxyuridines (CNdU) can
be used to measure distances and angles in a DNA oligomer,
while the effects of solvent, hydrogen bonded heterodimer
formation, and temperature on the nitrile and azido IR
absorbance bands of CNdU and 20-azido-20-deoxyuridine
(N3dU), respectively, have also been reported.31,32 Here, we
believe for the first time, both nitrile and azido functional
groups have been incorporated into the same biomolecular
monomer to facilitate a direct comparison of the vibrational
characteristics of these important probes. 20-Azido-5-cyano-
20-deoxyuridine (N3CNdU, 1) was synthesized and the IR
absorption profiles of the azide and nitrile groups in multiple
solvents and solvent mixtures are compared. This work builds
upon Cho’s comparison of amino acids containing azide,
nitrile, or thiocyante groups in three different solvents.21
Future incorporation of N3CNdU into nucleic acid oligomers
will allow simultaneous monitoring of both the sugar backbone
region and the major groove at a specific nucleotide site
through analysis of the azide and nitrile IR absorbance bands,
respectively.
N3CNdU in tetrahydrofuran (THF). The nitrile IR absor-
bance band of N3CNdU dissolved in the aprotic solvent THF is
centered at 2233.5 cmꢀ1. This position represents a 9.2 cmꢀ1 red
shift of the nitrile stretching frequency in THF relative to water.
The higher frequency in water is the result of H-bonding between
the nitrile group and water, which is absent in THF. This red
shift is consistent with previous experimental studies of the
solvent induced frequency shifts of nitrile-modified molecules
such as acetonitrile, pCNPhe, thiocyanates, and CNdU.4,31,34,35
The impact of H-bonding with water on the nitrile stretching
frequency of N3CNdU was further explored using DFT
calculations at the B3PW91/6-31++G(d,p) level. The calcula-
tions resulted in two distinct geometry-optimized H-bonding
configurations between the water molecule and the nitrile
group of the model 5-cyanouracil (see Supporting Information
for the geometry optimized structures). The first geometry was
characterized by a CRNꢁ ꢁ ꢁH angle of 1531 between the nitrile
group of 5-cyanouracil and the water molecule where a
hydrogen atom of the water molecule interacted with the lone
pair on the nitrogen atom of the nitrile group. This s-H-bond
resulted in a 8.6 cmꢀ1 blue shift in the nitrile stretching
frequency relative to isolated 5-cyanouracil. The CRNꢁ ꢁ ꢁH
Results and discussion
Synthetic chemistry
The synthesis of N3CNdU (1) was accomplished in four
steps with a 12.5% overall yield, Scheme 1. Trityl-protected
20-amino-20-deoxyuridine 233 was subjected to a copper-
catalyzed diazotransfer reaction using triflic azide to afford
azide 3 in 70% yield. Aromatic bromination of 3 with NBS
provided bromide 4 in moderate yield (54%). Cyanation with
potassium cyanide followed by detritylation using dichloroacetic
acid afforded a 33% yield of 1 for the two steps.
Infrared spectroscopy
N3CNdU in water. The dependence of the position, shape,
and complexity of the IR absorbance bands corresponding to
the azide asymmetric or the nitrile symmetric stretch of
N3CNdU was explored in a number of solvents and solvent
mixtures. The solvents and solvent mixtures were selected to
present a wide range of local environments around the azide
Fig. 1 FTIR absorbance spectra of N3CNdU dissolved in water,
tetrahydrofuran (THF), or methanol recorded at 293 K with a
concentration of 50 mM. The maximum absorbance of each spectrum
has been normalized to unity and the spectra have been offset for
comparison.
Scheme 1
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angle in the second configuration was 901, where a hydrogen
atom of the water molecule interacted with the p-orbital of the
nitrile group. The p-H-bond geometry resulted in a 8.4 cmꢀ1
red shift in the nitrile group. Consequently, the nitrile stretching
frequency is dependent on both the presence and geometry of
H-bonding interactions with water. A balance of these
H-bonding interactions results in the experimentally observed
blue shift between the aqueous solutions. The calculations
were not in quantitative agreement with the experimental
values since the calculations were performed in the gas phase,
they do not include effects of anharmonicity, and limitations in
the basis set.36–45 However, these computational results are in
agreement with previous theoretical studies exploring the
impact of H-bonding interactions between the nitrile group of
acetonitrile and water on the nitrile stretching frequency.34,36
The azide IR absorbance band of N3CNdU in THF displays
a complex absorption profile with a maximum absorbance at
2111.5 cmꢀ1. This band position represents a 12.6 cmꢀ1 red
shift relative to its position in water. The azide IR absorbance
band for N3CNdU in THF, however, contains a pronounced
high frequency shoulder resulting in an absorbance band
comprised of at least two spectral components. The central
frequency of the primary component of the azide IR absorbance
band of N3CNdU in THF is 2109.8 cmꢀ1 and the secondary
component (the high frequency shoulder) is centered at
2135.2 cmꢀ1. These positions are based upon a line shape
analysis of the azide IR absorbance band utilizing two line
shape functions described by eqn (1) (see ESIw). This added
complexity is not surprising based upon previous experimental
studies of the azide IR absorbance band of aryl azides such as
3-azidopyridine and pN3Phe.23,28,46,47
molecule characterized by a NQNꢁ ꢁ ꢁH angle of 1501. This
s-H-bond formed by an interaction between the hydrogen
atom of the water molecule and the terminal nitrogen atom of
the azide group resulted in a 7.4 cmꢀ1 blue shift in the azide
asymmetric stretching frequency relative to isolated 2-azido-
1,2-dideoxyribose. The second H-bond geometry involved a
p-H-bond between the water molecule and the azide group
characterized by a NQNꢁ ꢁ ꢁH angle of 1011. This p-H-bond
resulted in a 0.4 cmꢀ1 red shift in the azide asymmetric
stretching frequency relative to isolated 2-azido-1,2-dideoxyribose.
These calculations are in agreement with previous theoretical
studies probing the impact of H-bonding between the azide
group of methyl azide and water.54 The calculations here
demonstrate the sensitivity of the azide asymmetric stretch
to H-bonding with water, although the calculations are not in
quantitative agreement with the experimental results due, in
part, to limitations in the basis set.41–45
N3CNdU in methanol. The azide and nitrile IR absorbance
bands of N3CNdU dissolved in methanol both display complex
absorption profiles. The azide IR absorbance band has an
absorbance maximum at 2113.5 cmꢀ1 with a high frequency
shoulder, which is between the band position observed in
water and THF due to the potential of H-bonding between
methanol and the azide group. Similar to the azide IR
absorbance band of N3CNdU in THF, the azide IR absorbance
band in methanol is comprised of at least two spectral
components. The central frequency of the major component
is 2111.9 cmꢀ1 and the frequency of the minor component is
2135.6 cmꢀ1 based upon line shape analysis of the experi-
mental band to line shape functions described by eqn (1)
(see ESIw). Once again, this complex absorption profile could
be the result of different conformations of N3CNdU but is
more likely due to accidental Fermi resonance, although 2D IR
experiments are needed for a definitive determination.27,28,48–53
The nitrile IR absorbance band of N3CNdU in methanol has
an absorbance maximum at 2237.4 cmꢀ1 and is comprised of
two components positioned at 2236.3 cmꢀ1 and 2241.1 cmꢀ1
as determined by line shape analysis (see ESIw). The complex
absorbance band is likely due to two distinct solvation
states,35,49,55 although Fermi resonance has been observed
previously for nitrile absorbance bands.34,56,57 Based upon
literature precedence, we attribute the high frequency component
to a solvation state where the nitrogen lone pair of the nitrile
group is involved in a s-H-bond with the hydroxyl hydrogen
atom of methanol.49,55 This assignment is supported by the
similarity of the frequency with the observed position of the
nitrile IR absorbance band in water. The low frequency
component is due to a solvation state devoid of a s-H-bond
between the nitrile group and water. It is hypothesized that
these two distinct solvation states give rise to the complex
nitrile absorption profile in methanol.49
The observed complexity of the azide IR absorbance band
of N3CNdU in THF is likely the result of either multiple
conformations of N3CNdU in THF or accidental Fermi
resonance involving the azide asymmetric stretch and a normally
forbidden combination or overtone band of N3CNdU. The
presence of two distinct conformations giving rise to the
complex absorption profile is unlikely since the azide IR
absorbance band for N3dU in THF does not show a
pronounced high frequency shoulder (the band is only slightly
asymmetric).32,48 Consequently, we interpret the 2109.8 cmꢀ1
component to be the azide asymmetric stretching frequency
and the secondary component at 2135.2 cmꢀ1 to be the
normally forbidden combination or overtone band participating
in the Fermi resonance. The assignment of Fermi resonance is
in agreement with a previous literature study,28 although 2D
IR studies of N3CNdU are needed for confirmation.27,28,48–53
Similar to the nitrile IR absorbance band, the observed red
shift in the azide IR absorbance band of N3CNdU in THF
relative to water is due to specific solute–solvent interactions
(H-bonding) between the azide group and water. The sensitivity
of the azide asymmetric stretch to H-bonding with water
was investigated by DFT calculations at the B3PW91/
6-31++G(d,p) level. The calculations resulted in two geome-
try-optimized H-bonding configurations. The calculations
were performed with the model 2-azido-1,2-dideoxyribose
and one explicit water molecule (see Supporting Information
for the geometry-optimized structures). The first configuration
showed a s-H-bond between the azide group and a water
N3CNdU in water/DMSO mixtures. Fig. 2A shows the
dependence of the azide and nitrile IR absorbance bands on
the systematic variation of the solvent system from water to
DMSO in 12.5% increments. These solvent mixtures represent
a wide range of local environments for the azide and nitrile
groups of N3CNdU both in terms of solvent dielectric constants
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percent water is decreased in the water/DMSO solvent mix-
ture. The position of the azide IR absorbance band decreases
from 2124.4 to 2108.7 cmꢀ1, while the nitrile IR absorbance
band decreases from 2242.7 to 2230.2 cmꢀ1 upon going from
water to DMSO. The 15.7 cmꢀ1 and 12.5 cmꢀ1 red shifts are
due to changes in H-bond interactions between the azide and
nitrile groups with water, respectively. The azide IR absor-
bance band displays a larger peak extinction for each solvent
mixture relative to the nitrile IR absorbance band while
exhibiting a larger solvent induced frequency shift. The mag-
nitude of the shift in response to solvent composition high-
lights the potential utility of azide and nitrile vibrational
probes of local environments in nucleic acids.
Additional solvent and temperature studies on N3CNdU. The
IR absorption profiles of N3CNdU in ten additional solvents
(2-propanol, 1-butanol, tert-butanol, 2,2,2-trifluoroethanol,
acetone, nitromethane, formamide, N-methylformamide,
N,N-dimethylformamide, and propylene carbonate) were
investigated (see ESIw). The azide IR absorbance band is
asymmetric in each of these solvents, but is less pronounced
in 2,2,2-trifluoroethanol, nitromethane, and formamide. This
complexity is presumably due to accidental Fermi resonance.
The nitrile IR absorbance band also showed a complex
absorption profile in 2-propanol, 1-butanol, tert-butanol,
formamide, and N-methylformamide (see ESIw). In the alcoholic
solvents the nitrile complexity is likely due to different solvation
states corresponding to H-bonding and non-H-bonding con-
figurations between the hydroxyl group of the solvent and
the nitrile group of N3CNdU. The solvent induced frequency
shifts of the vibrational probes did not correlate with solvent
parameters such as solvent dielectric constant or solvent dipole
moment, similar to previous studies of other nitrile or azide
modified molecules.21,34 Additionally, both the azide and
nitrile IR bands of N3CNdU showed a modest dependence
on temperature in aqueous solution (see ESIw).
Conclusions
Fig. 2 (A) FTIR absorbance spectra of N3CNdU in water/DMSO
mixtures ranging from 0 to 100% water in 12.5% increments recorded
at 293 K with a concentration of 50 mM. The maximum absorbance of
each spectrum has been normalized to unity and the spectra have been
offset for comparison. (B) Dependence of the position of maximum
absorbance of the azide IR absorbance band on solvent composition.
(C) Dependence of the nitrile IR absorbance band position on solvent
composition.
The three-atom azide and two-atom nitrile groups are effective
vibrational probes of local environments as demonstrated by
the solvent dependent positions of the azide asymmetric and
nitrile symmetric stretching frequencies in N3CNdU. On the
whole, the azide probe is superior to the nitrile probe because
its extinction coefficient is 2–4 times larger. However, the
nitrile IR absorbance profile is generally less complicated by
accidental Fermi resonance although both the azide and nitrile
IR absorbance profiles are effectively a single band in the
biologically relevant aqueous solution. The IR frequencies of
both probes exhibit a pronounced red shift upon going from
water to DMSO (or THF). The frequencies of both observables
were higher in water due to H-bonding with water, as confirmed
by DFT calculations. As noted previously for 3-azidopyridine,28
the potential complex absorption profiles for the azide and
nitrile probes must be considered when using them to study
biomolecular structure and dynamics. These complexities28,57,58
could result in significant difficulties in the interpretation of the
infrared signature of azide and nitrile groups in biological
and the potential for specific solute–solvent interactions
(H-bonding). The FTIR spectrum of N3CNdU in DMSO is
similar to the spectrum in THF in that the azide IR absorbance
band shows a complex profile consisting of at least two
components while the nitrile IR absorbance profile is a single
band. Again, the complexity of the azide IR absorbance band
is likely due to accidental Fermi resonance.28
The frequency of maximum absorbance for the azide and
nitrile IR absorbance bands of N3CNdU as a function of the
solvent composition is plotted in Fig. 2B and C, respectively.
The positions of both bands decrease monotonically as the
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systems. A general experimental method to address this issue is
currently under investigation.
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The azide and nitrile groups of N3CNdU have the potential
to simultaneously probe both the sugar backbone region and
the major groove, respectively, of nucleic acids. The vibrational
signatures of both groups are well resolved from each other
permitting the solvent-dependent azide asymmetric and nitrile
symmetric stretching frequencies to yield site-specific information
about nucleic acid structure and dynamics. For instance, this
minimally invasive modified nucleoside has the potential to aid
in the investigation of conformation changes associated with
DNA-protein interactions and RNA folding.
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Acknowledgements
We are grateful to Carol Strausser for assistance in the editing
of the manuscript, Lisa Mertzman for obtaining materials and
supplies, and Beth Buckwalter for acquiring the NMR spectra.
This work was supported by an F&M Snavely Summer
Research stipend and a Snavely Research Award to XSG,
the William M. and Lucille M. Hackman Scholars Program to
BAC, a Mellon/CPC New Tasks/New Tools grant to EEF,
and the NIH (R15GM093330) to SHB/EEF.
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2007, 111, 10020–10025.
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