Synthesis and protein incorporation of azido-modified unnatural amino acids

Article abstract of DOI:10.1039/c4ra14244f

Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-l-phenylalanine (1) and 4-azidomethyl-l-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

Full text of DOI:10.1039/c4ra14244f

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PAPER
Synthesis and protein incorporation of
azido-modified unnatural amino acids†
Cite this: RSC Adv., 2015, 5, 1274
Elise M. Tookmanian, Edward E. Fenlon* and Scott H. Brewer*
Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-
phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs)
containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine.
These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large
extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The
sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein
environments. Three of the four azido-modified phenylalanine residues were successfully genetically
incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered,
orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity.
SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The
observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP
indicated that the azide groups were hydrated in the protein.
Received 10th November 2014
Accepted 24th November 2014
DOI: 10.1039/c4ra14244f
www.rsc.org/advances
in a relatively clear region of the infrared spectrum and is
sensitive to local environment. Additionally, the relative small
Introduction
Amino acids covalently modied with spectroscopic reporters size of most of these reporters helps to minimize any pertur-
offer the potential to probe local protein hydration with high bation to the native protein structure.
spatial and temporal resolution when coupled with the appro-
Recent work in our laboratory has focused on unnatural
priate spectroscopic technique. The development of effective amino acids containing the azide vibrational reporter to take
unnatural amino acids (UAAs) to serve as sensitive, site-specic advantage of the relatively large oscillator strength of the azide
probes rests on several key principles. First, the UAAs must have asymmetric stretch vibration and the high sensitivity of this
a readily measurable spectroscopic observable that is sensitive vibration to local environment. This work has included the
to local environment. Secondly, the synthesis of the UAA should development of 4-azidomethyl-L-phenylalanine (2), which is a
be efficient, cost-effective, and amenable to a large scale. stable analogue of the photo-reactive UAA, 4-azido-L-phenylal-
21
Thirdly, the UAA must be able to be incorporated in either anine (1). As expected, the azide asymmetric stretch of 2 was
peptides or proteins in a site-specic manner with high effi- sensitive to local protein environment as demonstrated through
ciency and delity. Lastly, the UAA needs to be stable and the incorporation of this UAA at multiple sites in the 247
minimally invasive to the native protein structure.
residue, b-barrel superfolder green uorescent protein (sfGFP,
A number of unnatural amino acids containing a variety of Fig. 2) utilizing an engineered, orthogonal aminoacyl-tRNA
vibrational probes have been utilized to study protein structure synthetase.
34
1–12
13–21
and dynamics. For instance, the nitrile (–CN), azide (–N
3
),
and metal acids to serve as sensitive, site-specic vibrational probes of
vibrational reporters have been utilized for this local protein hydration. The series of azido-modied UAAs
Here, we have extended the toolkit of azido-modied amino
22–26
27–29
thiocyanate (–SCN),
carbonyl
selenocyanate (–SeCN)
30–32
purpose. These reporter moieties have been incorporated into (shown in Fig. 1) were designed to systematically alter the
peptides and/or proteins by standard solid-phase peptide
1
22,31
synthesis, post-translational modications,
and/or genetic
33
techniques. Each probe has a vibrational signature that occurs
Franklin & Marshall College, Department of Chemistry, Lancaster, PA 17604-3003,
USA. E-mail: edward.fenlon@fandm.edu; scott.brewer@fandm.edu; Fax: +717 291
4
343; +717 291 4343; Tel: +717 291 4201; +717 358 4766
†
Electronic supplementary information (ESI) available: Sequence of wt-sfGFP and
ESI-Q-TOF mass analysis of the sfGFP protein constructs. See DOI:
Fig. 1 Structure of the series of the azido-modified unnatural amino
1
0.1039/c4ra14244f
acids.
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1
13
following frequencies: H (499.7 MHz) and C (125 MHz).
Chemical shis are reported in parts per million (ppm) while all
1
coupling constants are reported in hertz (Hz). H NMR spectra
in CDCl were referenced to the residual solvent peak at 7.26
3
1
3
ppm while C NMR spectra in CDCl
solvent peak at 77.0 ppm. H NMR spectra in DMSO were
3
were referenced to the
1
1
3
referenced to the residual solvent peak at 2.49 ppm while
NMR spectra in DMSO were referenced to the solvent peak at
9.5 ppm. All IR characterization for synthetic purposes were
carried out as ATR thin lm analyses with all peaks reported in
C
3
ꢀ1
wavenumbers (cm ). Mass spectral (MS) analyses of the free
UAAs (3, 4) for synthetic purposes were carried out on an Agilent
1100 series LC/MSD SL ion trap mass spectrometer with elec-
trospray ionization and MS/MS capabilities. Protein constructs
were analyzed by electrospray ionization quadrupole time-of-

ight (ESI-Q-TOF) mass analysis at the University of Illinois at
Urbana-Champaign.
Fig. 2 Structure of wt-sfGFP (PDB ID 2B3P) with site 150 (magenta)
highlighted.
Boc-4-(3-hydroxy-1-propynyl)-L-Phe methyl ester (7a)
3 2 2
Pd(PPh ) Cl (693 mg, 0.0987 mmol, 1 mol%) and CuI (376
mg, 0.19 mmol, 2 mol%) were suspended in triethylamine (40
mL) and 6 (4.00 g, 9.87 mmol) was added, followed by prop-
argyl alcohol (632 mL, 610 mg, 10.9 mmol, 1.1 equiv.), which
caused immediate darkening of the solution to a brown
color. The reaction was stirred at room temperature for 5 h.
The crude mixture was ltered through a glass frit and the
distance between the azide group and the phenyl ring of the
modied phenylalanine residues. The alkyne group in 3 and 4
was designed as a rigid spacer that limits the conformational

exibility between the azide group and aromatic ring. In
concert, this series has the potential to probe protein hydration
systematically with high spatial resolution thus creating a
vibrational hydration ruler. The synthesis of 3 and 4, the genetic
incorporation of these UAAs into sfGFP at site 150 (Fig. 2), and
the subsequent infrared analysis of the resulting sfGFP
constructs is discussed in addition to future applications of
these UAAs.
3
solid residue was washed with NEt . The ltrate was
concentrated under reduced pressure and the residue was
puried by ash column chromatography (hexanes/ethyl
acetate, 3 : 2) to give 7a as a yellow-orange oil (2.73 g, 8.18
mmol, 83% yield). IR 3379.5 (br, w), 2977.5 (w), 1741.2 (s),
1
1
693.3 (s), 1509.2 (s), 1437.6 (m), 1366.4 (s), 1252.0 (m),
218.6 (m), 1164.2 (s), 1027.1 (s), 951.9 (w), 823.6 (w), 781.1
1
(
w). H NMR (CDCl
3
) 7.34 (d, J ¼ 8.5, 2H), 7.06 (d, J ¼ 8.5, 2H),
Experimental
General information
4
3
1
5
.96 (d, J ¼ 7.5, 1H), 4.56 (m, 1H), 4.47 (d, J ¼ 6.0, 2H), 3.68 (s,
13
3
H), 3.05 (m, 2H), 1.39 (s, 9H). C NMR (CDCl ) 172.09,
55.01, 136.45, 131.65, 129.12, 121.26, 87.60, 84.87, 79.97,
4.17, 52.13, 51.18, 38.05, 28.12.
Chemical reagents were purchased from Sigma-Aldrich, Strem,
Gelest, Alfa Aesar, Peptech, and Tokyo Chemical Industry and
used without further purication. Deuterated chloroform
(
(
98.8% D enrichment) and deuterated dimethyl sulfoxide
99.5% D enrichment) were purchased from Cambridge Isotope
Boc-4-(4-hydroxy-1-butynyl)-L-Phe methyl ester (7b)
Laboratories. DH10B cells and pBadA were purchased from 7b was synthesized in the same manner as 7a using the
Invitrogen. 4-Azido-L-phenylalanine (1) was purchased from following reagents: 3-butyn-1-ol (626 mL, 583 mg, 8.22 mmol, 1.1
Bachem. The synthesis of Boc-4-iodo-L-Phe methyl ester (6) was equiv.), Pd(PPh ) Cl (524 mg, 0.075 mmol, 1 mol%), CuI (285
3
2
2
35
carried out following a literature procedure. All aqueous mg, 0.149 mmol, 2 mol%) and 6 (3.03 g, 7.47 mmol) in NEt (30
3
solutions were prepared using 18 MU cm water.
mL). Purication by ash column chromatography (hexanes/
Reactions were carried out under a dry argon atmosphere ethyl acetate, 3 : 2) yielded 7b as a light orange oil (2.02 g,
and stirred with a magnetic stir bar. An ice bath was utilized for 5.79 mmol, 78% yield). IR 3374.3 (br, w), 2977.3 (w), 1694.0 (s),
reactions performed below ambient temperature. Analytical 1509.9 (s), 1436.1 (m), 1366.0 (s), 1249.9 (m), 1216.3 (m), 1163.9
1
thin layer chromatography (TLC) was carried out using 0.2 mm (s), 1053.6 (s), 1021.4.4 (m), 729.8 (m). H NMR (CDCl
3
) 7.28 (d, J
silica plastic coated sheets (Selecto Scientic) with F254 ¼ 7.9, 2H), 7.01 (d, J ¼ 7.9, 2H), 5.03 (m, 1H), 4.52 (m, 2H), 3.75
indicator. Flash column chromatography was carried out using (t, J ¼ 6.4, 2H), 3.65 (s, 3H), 3.05 (m, 2H), 2.62 (t, J ¼ 6.4, 2H),
1
3
230–400 mesh silica gel.
1.37 (s, 9H). C NMR (CDCl ) 172.11, 154.98, 135.88, 131.68,
3
NMR spectra were obtained with a Varian INOVA 500 129.11, 122.01, 86.55, 81.90, 79.92, 61.00, 54.20, 52.15, 38.07,
multinuclear Fourier transform NMR spectrometer at the 28.16, 23.70.
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Boc-4-(3-azido-1-propynyl)-L-Phe methyl ester (8a)
temperature for 4 h. 0.5 M NaHSO
4
was added to adjust the pH
of the solution to ꢃ2.5. The mixture was extracted with ethyl
acetate (2 ꢂ 25 mL) and the combined organic layers were
washed with water (30 mL) and brine (30 mL), dried over
A solution of 7a (2.1813 g, 6.81 mmol), dimethyl formamide
(
5.4 mL) and triethylamine (1.41 mL, 10.21 mmol, 1.5 equiv.)
ꢁ
was cooled to 0 C under argon. Methanesulfonyl chloride (686
4
MgSO , ltered through a glass frit, and concentrated under
mL, 8.85 mmol, 1.3 equiv.) was added over approximately 6 min
reduced pressure. The crude product was immediately taken to
the next step. The residue was dissolved in a solution of dry HCl
in dioxane (2.5 M, 11 mL) and stirred for 4 h at room temper-
ature. The solution was concentrated under reduced pressure to
about a third of its initial volume, and pentane was added to
precipitate the solid product. The product was isolated by
causing the solution to become cloudy. Aer an additional
ꢁ
6
min at 0 C, the solution was allowed to stir at room
temperature for 30 min. The reaction was then quenched with
water, extracted with diethyl ether (2 ꢂ 30 mL), and the
combined organic layers were washed with H O (50 mL) and
2
4
brine (50 mL) before drying over MgSO , ltering through a

ltration to obtain 3 as an offwhite to yellow powder (817 mg,
glass frit, and concentrating under reduced pressure. This
crude product was immediately moved forward to the next step.
The residue was dissolved in dimethyl formamide (5.4 mL) and
sodium azide (664 mg, 10.21 mmol, 1.5 equiv.) was added using
appropriate precautions. The solution was allowed to stir at
temperature for 72–96 h, or until TLC conrmed that the reac-
tion was complete. Water (20 mL) was added and the product
was extracted with ethyl acetate (3 ꢂ 25 mL). The combined
1
3
.34 mmol, 86%). H NMR (DMSO) 7.43 (d, J ¼ 8.5, 2H), 7.30
(d, J ¼ 8.5, 2H), 4.36 (s, 2H), 4.13 (t, J ¼ 6.2, 1H), 3.13 (d, J ¼ 6.5,
1
3
2H). C-NMR (DMSO) 170.24, 136.24, 131.78, 129.99, 120.18,
+
86.16, 82.60, 52.89, 39.00, 35.51. MS(ESI) 245.0 (M , 100), 171.0
(40), 145.1 (20), 488.8 (15).
4-(4-Azido-1-butynyl)-L-Phe (4)
organic layers were washed with water (1 ꢂ 25 mL), saturated 4 was synthesized by the same method as 3 using the following
aqueous NH
Cl (1 ꢂ 25 mL), water (1 ꢂ 25 mL), and brine (1 ꢂ reagents: 8b (1.961 g, 5.27 mmol), THF : H O (3 : 1, 21.1 mL),
5 mL). The organic layer was dried over MgSO
4
2
2
4
, ltered and LiOH monohydrate (331 mg, 7.90 mmol). Following reac-
through a glass frit, and concentrated under reduced pressure. tion completion and isolation, 2.5 M HCl in dioxane (13.9 mL).
Purication by ash column chromatography (hexanes/ethyl Filtration yielded 4 as a pale pink powder (1.353 g, 4.59 mmol,
1
acetate 3 : 1) yielded 8a as a pale yellow oil (1.523 g, 4.42 87%). H NMR (DMSO) 7.34 (d, J ¼ 8.3, 2H), 7.27 (d, J ¼ 8.3, 2H),
mmol, 65% yield). IR 3405.5 (br, w), 2976.8 (w), 2118.8 (m), 4.13 (t, J ¼ 5.9, 1H), 3.49 (t, J ¼ 6.4, 2H), 3.14 (d, J ¼ 6.4, 2H), 2.73
1
3
1
(
743.3 (m), 1701.1 (s), 1508.0 (s), 1437.1 (m), 1366.2 (s), 1247.8 (t, J ¼ 6.4, 2H). C NMR (DMSO) 170.18, 135.30, 131.79, 129.86,
m), 1164.5 (s), 1108.5 (w), 1058.0 (m), 1020.8 (m), 860.5 (w). 121.59, 87.57, 81.67, 52.99, 49.24, 35.43, 19.89. MS(ESI) 259.1
1
+
H NMR (CDCl
3
) 7.34 (d, J ¼ 8.1, 2H) 7.05 (d, J ¼ 8.1, 2H), 5.03 (M , 100), 213.1 (5), 517.1 (2 M, 25).
(
(
1
4
d, J ¼ 8.3, 1H), 4.53 (m, 1H), 4.08 (s, 2H), 3.66 (s, 3H), 3.03
13
m, 1H), 1.36 (s, 9H). C NMR (CDCl
3
) 171.97, 154.90, 137.05,
31.88, 129.25, 120.50, 86.99, 81.03, 79.86, 54.14, 52.15, 42.65,
0.42, 38.13, 28.13.
Expression and purication of sfGFP constructs
A codon-optimized gene coded with a C-terminal 6-His affinity
tag for wild-type sfGFP (wt-sfGFP)
10,21,34,36,37
was inserted into
pBadA, generating pBad–sfGFP. The N150 codon was replaced
through site-directed mutagenesis with the amber stop codon
Boc-4-(4-azido-1-butynyl)-L-Phe methyl ester (8b)
b was synthesized in the same manner as 8a using the
following reagents: 7b (1.1788 g, 3.39 mmol), dimethyl form-
amide (2.7 mL), triethylamine (704 mL, 5.09 mmol, 1.5 equiv.),
and methanesulfonyl chloride (342 mL, 4.41 mmol, 1.3 equiv.).
Following work up and concentration, the product was
(TAG) generating pBad–sfGFP–150TAG. The engineered,
8
orthogonal aminoacyl-tRNA synthetase for the incorporation
of 1, 2, 3, and 4, was inserted into pDule, generating
10,21,35
pDule–pN
3
Phe.
These plasmids were obtained from
Dr Ryan A. Mehl (Oregon State University).
pBad–sfGFP was transformed into DH10B E. coli cells and
3
pBad–sfGFP–150TAG was co-transformed with pDule–pN Phe
combined with dimethyl formamide (2.7 mL) and NaN
331 mg, 5.09 mmol, 1.5 equiv.). Purication yielded 8b as a
clear oil (706 mg, 1.90 mmol, 56% yield) IR 3354.0 (br, w),
978.2 (w), 2108.6 (m), 1743.7 (m), 1712.1 (s), 1508.7 (m), 1438.0
m), 1364.9 (s), 1249.6 (m) 1165.6 (s), 1056.7 (m), 1016.5 (m),
3
(
into DH10B E. coli cells. Five milliliters of noninducing media
were inoculated with the transformed cells, which grew to
2
ꢁ
saturation while shaking (250 rpm) at 37 C. A portion of the
(
1
cultured cells (2.5 mL aliquot) was used to inoculate 250 mL of
autoinduction media containing the appropriate UAA at 1 mM.
The negative control experiment excluded the UAAs from the
7
2
6
78.3 (m). H NMR (CDCl
3
) 7.30 (d, J ¼ 8.1, 2H), 7.02 (d, J ¼ 8.1,
H), 4.97 (d, J ¼ 7.3, 2H), 4.53 (m, 1H). 3.66 (s, 3H), 3.42 (t, J ¼
1
3
.9, 2H), 3.03 (m, 2H), 2.67 (t, J ¼ 6.9, 2H), 1.38 (s, 9H). C NMR
ꢁ
autoinduction media. Aer shaking at 37 C for 24–30 h, the
(CDCl
3
) 172.05, 154.95, 136.11, 131.65, 129.17, 121.81, 85.85,
cells from the autoinduction media were collected by centrifu-
gation and the expressed protein was puried using TALON
cobalt ion-exchange chromatography (Clontech) similar to
8
2.17, 79.89, 54.21, 52.14, 49.85, 38.15, 28.19, 20.53.
4
8
-(3-Azido-1-propynyl)-L-Phe (3)
10,21
previous procedures.
a (1.34 g, 3.87 mmol) was dissolved in a mixture of THF : H O
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
2
(3 : 1, 15.5 mL) and LiOH monohydrate (244 mg, 5.80 mmol, (SDS-PAGE) and electrospray ionization quadrupole time-of-
1.5 equiv.) was added and the mixture was stirred at room ight (ESI-Q-TOF) mass analysis were used to verify the
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incorporation of the UAAs into sfGFP at site 150 with high Solvent dependence of the azide asymmetric stretch of the
efficiency and delity. The sfGFP constructs containing 1, 2, or 4 UAAs
yielded between 5–150 mg of puried protein per liter of auto-
Fig. 3 illustrates the sensitivity of the azide asymmetric stretch
of 3 and 4 to local environment. Fig. 3A shows the FTIR spectra
induction media. The yields for protein expression were calcu-
34
lated using the extinction coefficient of sfGFP at 488 nm.
of 3 dissolved in either DMSO or water to mimic a buried or a
solvated position in a protein, respectively. Each spectrum
contains at least three components. The most intense high
frequency component is assigned as resulting from the azide
asymmetric stretch vibration based upon literature prece-
Equilibrium FTIR measurements
A Bruker Vertex 70 FTIR spectrometer, equipped with a globar
source, KBr beamsplitter, and a liquid nitrogen cooled mercury
cadmium telluride (MCT) detector, was used to record all
equilibrium FTIR absorbance spectra. The spectra were recor-
ded using a temperature-controlled transmission cell with an
embedded thermocouple to measure the temperature of the
cell. The dual-compartment transmission cell consisted of two
calcium uoride windows with a path length of ꢃ100 mm. The
spectra were the result of 1024 scans recorded at a resolution of
14,16,18,21,38
dent.
The other components of this absorption band
are likely the result of anharmonic coupling such as accidental
39,40
Fermi resonance.
Alternatively, this complexity could be the
result of the azide group sampling multiple distinct environ-
14
ments. Based upon this assignment, the azide asymmetric
ꢀ
1
stretch vibration of 3 shis from 2115.1 cm in DMSO to
ꢀ1
ꢀ1
2
126.2 cm in water. This 11.1 cm blue shi is mainly the
ꢀ
1
ꢁ
result of hydrogen bonding interactions between water and the
1
.0 cm . The spectra were recorded at 25 C, baseline cor-
13,28,41
azide group of 3 in the aqueous solution.
rected, and intensity normalized. Igor Pro was used for the
analysis of all spectra (Wavemetrics).
Similarly the FTIR spectra of 4 in either DMSO or water
Fig. 3B) show a complex absorption prole in the region of the
azide asymmetric stretch likely due to anharmonic effects.
(
39,40
Results and discussion
Synthesis of azido-modied UAAs
The spectrum in DMSO contains at least two components
although the two most prominent components have nearly the
same intensity in this spectrum. The high frequency component
The syntheses of 3 and 4 were accomplished in gram quantities
in just four steps from the commercially available Boc-4-iodo-L-
Phe (5) in overall yields of 42% and 34%, respectively
ꢀ
1
(
2110.2 cm ) is the most intense component and is assigned as
arising from the azide asymmetric stretch vibration of 4. The
spectrum in water also contains at least two components where
(Scheme 1). The rst step for both syntheses was the protection
ꢀ
1
the more intense high frequency component (2123.3 cm ) is
assigned as resulting from the azide asymmetric stretch. Thus,
of the carboxylic acid of 5 as its methyl ester (6), which was
accomplished in 90% yield by the alkylation of the carboxylate
ꢀ1
35
this vibration blue shis 13.1 cm upon moving from DMSO to
water. This shi is slightly larger than the solvent-induced shi
in this vibration of 3, although the difference could be the result
of variations in the strength of the anharmonic-coupling
with methyl iodide following a known procedure. The
syntheses diverged in the Sonogashira coupling step; coupling
with propargyl alcohol led to alcohol 7a (83%), whereas
coupling with 3-butyn-1-ol provided alcohol 7b (78%). Azides 8a
and 8b were then prepared in 65% and 56% yields, respectively,
by a two-step procedure of mesylate formation followed
by displacement by sodium azide. Finally, the well-established
28,30,42–44
present in these spectra.
The solvent-dependence of the azide asymmetric stretch of
3
and 4 is similar to the solvent dependence of this vibration
35
in 1 and 2. We have previously shown that the azide asym-
deprotection method
of saponication with lithium
ꢀ
1
metric stretch vibration of 1 shis from 2115.5 cm to 2128.6
hydroxide, Boc-removal with HCl-dioxane, and pentane
precipitation provided UAA 3 (86%) and UAA 4 (87%) in excel-
lent yields.
ꢀ
1
cm
upon moving from DMSO to water, respectively,
ꢀ
1
21
resulting in a 13.1 cm blue shi. The azide asymmetric
Scheme 1
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Fig. 3 FTIR absorbance spectra of 3 (Panel A) and 4 (Panel B) dissolved in either DMSO or an aqueous basic solution (200 mM NaOH) at a
concentration of ꢃ50 mM.
stretch vibration of 2 was also shown to blue shi upon going
The azido-modied phenylalanine residues 1, 2, and 4 were
genetically incorporated into site 150 of sfGFP in response to an
ꢀ1
from DMSO to water. This vibration shied from 2097.7 cm
ꢀ1
to 2111.2 cm upon moving from DMSO to water, respec- amber codon in an efficient, site-specic manner with high
ꢀ
1
21
tively, resulting in a 13.5 cm
blue shi. These results delity utilizing an engineered, orthogonal aminoacyl-tRNA
illustrate that the azide groups of 1–4 are effective vibrational synthetase. The incorporation was veried by SDS-PAGE (see
reporters of local environment where the azide asymmetric Fig. 4) and ESI-Q-TOF mass analysis (see ESI†). The
stretch of each of these UAAs has a similar sensitivity to local
environment.
Genetic incorporation of azido-modied UAAs into sfGFP
Site 150 in sfGFP was selected since it is located on the edge of
the b-barrel where the side chain is directed away from the
34
protein and into the solvent. Thus, the solvation of the protein
can be studied at varying distances from the protein surface and
the incorporated UAAs should be minimally perturbative to the
native protein structure compared with the UAA side chains
directed into the interior of the protein.
Fig. 4 Coomassie blue stained tris-glycine SDS-PAGE illustrating UAA
incorporation into sfGFP at site 150 with high fidelity. The protein
constructs were expressed from pBad–sfGFP–150TAG and pDule–
3
pN Phe (lanes 2–6) in the presence (lanes 2–5) or the absence (lane 6)
of 1, 2, 3, or 4, respectively.
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Fig. 5 FTIR absorbance spectra of sfGFP constructs containing 1, 2, or 4 incorporated at site 150 dissolved in a pH 7.3 aqueous buffer containing
0 mM sodium phosphate and 150 mM sodium chloride at a concentration of ꢃ1 mM.
5
incorporation of 1, 2, or 4 into site 150 resulted in the produc- than one spectral component in this region of the spectrum is,
39,40
tion of the protein constructs sfGFP-150-1, sfGFP-150-2 and once again, likely the result of anharmonic effects.
sfGFP-150-4, respectively. The high delity of the UAA incor- Based upon these assignments, the azide asymmetric stretch
poration was demonstrated by the lack of protein expression for sfGFP-150-1, sfGFP-150-2 and sfGFP-150-4 was observed at
ꢀ
1
ꢀ1
ꢀ1
when the UAAs were not included in the expression media (see 2126.6 cm , 2107.3 cm , and 2119.1 cm , respectively. These
lane 6 of Fig. 4). frequencies suggest that the azide group in each of these
Although a band corresponding to a sfGFP construct con- protein constructs is solvated since these frequencies are
taining an UAA was observed in the SDS-PAGE (Fig. 4, lane 4) similar to the frequencies of the respective free unnatural
when 3 was present in the expression media, further analysis by amino acids dissolved in water (see above). The exact nature of
ESI-Q-TOF mass spectroscopy and FTIR spectroscopy revealed the hydration state is partially complicated due to the anhar-
this sfGFP construct did not contain an azide group. The monic effects present in these spectra. However, the position
expected mass was not observed in the ESI-Q-TOF mass spec- and prole of the absorption band of sfGFP-150-2 suggests that
trum and the FTIR spectrum of this construct did not contain the hydration state of the azide group at site 150 is slightly
an azide asymmetric stretch vibration. This was surprising different than the corresponding hydration state of this reporter
45
because azides in biomolecules are known to be quite stable.
incorporated at site 134 of sfGFP, which is in a fully solvated site
The loss of the azide group from 3 is likely due to the reactivity in a loop region of the protein. Specically, the absorption band
propargyl linker as the methylene protons are somewhat acidic resulting primarily from the azide asymmetric stretch vibration
0
ꢀ1
and the benzylic position can act as an electrophile in a SN2
for 2 incorporated at site 134 occurs at 2109.8 cm and is fairly
21
ꢀ1
mechanism. These mechanistic pathways are not likely for the symmetrical. This 2.5 cm blue shi compared to sfGFP-150-
other three UAAs, consistent with the loss of the azide group 2 and different line shape are indicators that the azide vibra-
only with 3. It is unclear whether 3 was incorporated into the tional reporter can probe different hydration states and
protein before or aer the azide group was lost.
dynamics, although complementary techniques, such as 2D IR
spectroscopy, would be needed to extract out the ner details of
28,30,42–44
this difference.
IR characterization of sfGFP constructs containing UAAs
UAAs 1, 2, and 4 place the azide in different locations relative
to the protein backbone in sfGFP. In a more general sense,
when coupled with 2D IR spectroscopy, these probes hold great
potential to investigate protein hydration dynamics with high
spatial and temporal resolution near the protein–water
interface.
ꢀ1
The FTIR spectra, in the region 2000–2200 cm , of sfGFP-150-1,
sfGFP-150-2 and sfGFP-150-4 dissolved in an aqueous buffer are
shown in Fig. 5. The IR spectrum of sfGFP-150-1 shows a fairly
symmetrical absorption band resulting from the azide asym-
metric stretch vibration, while the absorption band for sfGFP-
150-2 in this region is asymmetric towards higher frequency.
The IR spectrum of sfGFP-150-4 contains at least two spectral
components in this region with the high frequency component
Conclusion
having the largest intensity, which is assigned as resulting from In this study, we have successfully synthesized two novel azi-
the azide asymmetric stretch vibration. The presence of more dophenylalanine residues,
3
and 4, through multi-step
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syntheses. Similar to 4-azido-L-phenylalnine (1) and 4-azido-
methyl-L-phenylalanine (2), the azide asymmetric stretch
vibration for these new UAAs was found to be sensitive to local
environment. The position of this vibration for 3 and 4 blue
shied 11.1 cm and 13.1 cm , respectively, upon going from
DMSO to water, which is similar to the previously observed shi
for 1 and 2 in these solvents. 1, 2, and 4 were genetically
incorporated with high efficiency and delity into site 150 of
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Beth Buckwalter for acquiring NMR spectra; and Kenneth Hess
for technical assistance with the mass spectral analysis of 3 and
4
. This work was supported by F&M Hackman, Leser, and
Snavely funds to EMT; NSF (CHE-1053946) to SHB; and NIH
R15GM093330-02) to SHB/EEF.
(
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