Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

Article abstract of DOI:10.1002/chem.201503908

An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54 % overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 ? resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.

Full text of DOI:10.1002/chem.201503908

DOI: 10.1002/chem.201503908
Full Paper
&
Structural Biology
Azidoethoxyphenylalanine as a Vibrational Reporter and Click
Chemistry Partner in Proteins
Elise M. Tookmanian, Christine M. Phillips-Piro,* Edward E. Fenlon,* and Scott H. Brewer*[a]
Abstract: An unnatural amino acid, 4-(2-azidoethoxy)-l-phe-
nylalanine (AePhe, 1), was designed and synthesized in three
steps from known compounds in 54% overall yield. The sen-
sitivity of the IR absorption of the azide of AePhe was estab-
lished by comparison of the frequency of the azide asym-
metric stretch vibration in water and dimethyl sulfoxide.
AePhe was successfully incorporated into superfolder green
fluorescent protein (sfGFP) at the 133 and 149 sites by using
the amber codon suppression method. The IR spectra of
these sfGFP constructs indicated that the azide group at the
149 site was not fully solvated despite the location in sfGFP
and the three-atom linker between the azido group and the
aromatic ring of AePhe. An X-ray crystal structure of sfGFP-
149-AePhe was solved at 1.45 Š resolution and provides an
explanation for the IR data as the flexible linker adopts a con-
formation which partially buries the azide on the protein
surface. Both sfGFP-AePhe constructs efficiently undergo a bi-
oorthogonal strain-promoted click cycloaddition with a di-
benzocyclooctyne derivative.
tion and the electrostatic environments of proteins by IR[18–26]
and Raman spectroscopy.[26,27] Nitriles and azides are arguably
the two most important functional groups for vibrational UAA
because of their small size, relative stability, sensitivity, and ab-
sorptions that occur in an open window in the IR.[18,28] We re-
cently reported a direct comparison of these two vibrational
reporters and demonstrated that for many applications azides
are superior because they have a significantly larger extinction
coefficient while maintaining excellent sensitivity to their local
environment in terms of frequency shifts.[29] Azides also have
the advantage of being a conduit to other functionalities
through bioorthogonal click cycloaddition reactions with al-
kynes.[30,31] In fact, it is the bioconjugation applications[32–36] of
azido-UAA that are most commonly employed, whereas their
ability to also act as an IR reporter has been underutilized,
with some notable exceptions.[20,23,37–40] Azido-UAA might be
more widely employed as both IR reporters and bioconju-
gation conduits if an efficient and scalable synthesis was devel-
oped from readily available and relatively inexpensive starting
materials. Here we report such a synthesis of an azido-UAA, 4-
(2-azidoethoxy)-l-phenylalanine (AePhe, 1; Figure 1A), and
demonstrate that it can be site-selectively incorporated into
a protein to act as an IR reporter of local hydration and as
a click partner for a bioorthogonal conjugation reaction.
Introduction
The 20 canonical amino acids are genetically encoded and pri-
marily responsible for the amazing diversity found in the hun-
dreds of proteins that enable life. In addition to these, there
are numerous uncommon amino acids that are typically
formed by post-translational modifications (PTM), such as oxi-
dization, methylation, acylation, or phosphorylation, of the can-
onical amino acids. In the past 25 years the ability to incorpo-
rate unnatural amino acids (UAA) into proteins has been devel-
oped and refined.[1–6] To date over 150 UAA have been incorpo-
rated into proteins genetically by codon reassignment and
suppression—only one of several methods of incorporation.[1–6]
UAA offer several advantages for the study of protein structure
and function in that their steric and electronic properties can
be rationally designed for the task at hand. UAA have been
employed for many diverse purposes such as bioconjugation,
reporters, and/or photoreactive groups.[6]
In the case of reporters, a unique spectroscopic signature
that can be observed by fluorescence,[7–10] EPR,[11,12] NMR,[13,14]
or vibrational spectroscopy[15–17] is required. In recent years vi-
brational UAA have been employed to study the local hydra-
[a] E. M. Tookmanian, Prof. Dr. C. M. Phillips-Piro, Prof. Dr. E. E. Fenlon,
Prof. Dr. S. H. Brewer
Department of Chemistry Franklin & Marshall College
P.O. Box 3003, Lancaster, PA 17604 (USA)
E-mail: cpiro@fandm.edu
Results and Discussion
efenlon@fandm.edu
sbrewer@fandm.edu
Design and synthesis of AePhe
Supporting information and ORCID(S) for this article is available on the
WWW under http://dx.doi.org/10.1002/chem.201503908. It contains addi-
tional synthetic procedures, sequence of wt-sfGFP pre- and post-trypsin
digest, additional protein mass spectral data, and sfGFP-149-AePhe X-ray
crystal structure refinement progression.
AePhe (1) was designed with an efficient synthesis in mind as
protected tyrosine 2 is commercially available and relatively in-
expensive. It was also rationalized that the flexible three-atom
linker between the azido group and the aromatic ring would
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Incorporation of AePhe at two sites in sfGFP
Using the amber codon suppression technology, AePhe was
site-specifically incorporated at two unique sites in our model
protein system, superfolder green fluorescent protein (sfGFP),
a 247-residue b-barrel protein (Figure 1B). This system is an
ideal model as it produces robust protein yields, has a readily
visible confirmation of protein expression, and allows the in-
46, 47,49]
corporation a number of UAA at various sites.[23,40,
AePhe was incorporated at the 133 site (sfGFP-133-AePhe) on
a flexible loop and the 149 site (sfGFP-149-AePhe) on the side
of the b-barrel with the side chain facing solvent. A protein gel
indicates protein expression only when the unnatural amino
acid AePhe is added to the media (Figure 2), which indicates
Figure 1. A) Structure of 4-(2-azidoethoxy)-l-phenylalanine. B) Structure of
wild-type sfGFP (PDB ID 2B3P) with the 133 and 149 sites labeled and
shown in dark sticks. The chromophore is shown in stick for reference.
increase the azide’s availability for bioorthogonal click cycload-
dition reactions relative to recently published azido-UAA,
which had a rigid alkynyl linker.[40] The synthesis of AePhe was
accomplished in a three-step process from known materials.
Thus, tyrosine 2 was alkylated with 2-azidoethyl mesylate by
using potassium carbonate as a base to provide azide 3 in
66% yield (Scheme 1). Deprotection of 3 was accomplished by
saponification with lithium hydroxide followed by acid treat-
ment to remove the tert-butoxycarbonyl group and provide
1·HCl in 82% yield for the two steps. The streamlined synthesis
requires only one chromatography purification and readily pro-
vided gram-quantities of AePhe in three steps and 54% overall
yield. This is in contrast to the literature syntheses of other
azido-UAA such as 4-azidomethyl-l-Phe (six steps, 33%
yield),[23] 4-(3-azido-1-propynyl)-l-Phe (six steps, 42% yield),[40]
and 4-(4-azido-1-butynyl)-l-Phe (six steps, 34% yield).[40] The
commercially available azido-UAA 4-azido-l-Phe is unstable
and undergoes photochemical decomposition.[23] This reactivity
is useful for cross-linking experiments[41–44] but the utility of 4-
azido-l-Phe as both a click partner[35,36,44] and a vibrational re-
porter[20,37] requires some precautions because of this unwant-
ed photoreactivity. Numerous additional azido-UAA have been
prepared[45] but few of these have been incorporated into pro-
teins and fewer still have been incorporated for use as vibra-
tional probes.[20,23,37–40] After submission of this manuscript
recent articles were found that describe the syntheses of
AePhe[46–48] and its subsequent incorporation into GFP for bio-
orthogonal reactions at sites other than those studied
here.[46,47] However, these studies did not use the azide as a vi-
brational reporter nor provide X-ray crystallographic analysis.
Figure 2. Coomassie stained 12% tris-glycine SDS-PAGE illustrating efficient,
site-specific incorporation of AePhe with high fidelity at either the 133 or
149 sites. The protein constructs were expressed from pBAD-sfGFP (WT in
lane 2); pBAD-sfGFP-133TAG and pDULE-AzidoPhe in the presence (lane 3)
or absence (lane 4) of 1; pBAD-sfGFP-149TAG and pDULE-AzidoPhe in the
presence (lane 5) or absence (lane 6) of 1. The proteins were purified as de-
scribed in the Experimental Section prior to running the gel.
the selective nature of the synthetase/tRNA system to incorpo-
rate only the UAA. Furthermore, we have confirmed the incor-
poration of AePhe into sfGFP by mass spectral analysis. Com-
paring the observed mass of wt-sfGFP with the observed mass
of sfGFP-133-AePhe or sfGFP-149-AePhe produced the expect-
ed mass differences for the replacement of the 133-aspartic
acid with AePhe (D= +117 Da) and the 149-asparagine with
AePhe (D= +118 Da), see Table 1.
Sensitivity of the azide asymmetric stretch of AePhe to the
local environment
The dependence of the azide asymmetric stretching frequency
of AePhe to solvent was measured to examine the potential of
Scheme 1.
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a hydrogen-bond-donating proton. The direction and magni-
tude of this shift are similar to other azido-modified UAA[40]
and indicates the potential of AePhe to serve as a reporter of
local hydration in proteins in spite of the complicated IR ab-
sorbance profile in the azide region.
Table 1. sfGFP mass spectrometry data.
Protein construct
Exptl.[a]
Exptl.
D wt [Da]
Calcd
D wt [Da]
MW (Da)
wt sfGFP
26,863
26,979
26,981
27,370
27,369
–
116
118
507
507
–
117
118
507
508
sfGFP-133-AePhe
sfGFP-149-AePhe
sfGFP-133-AePhe-DBCO
sfGFP-149-AePhe-DBCO
Figure 3B shows the equilibrium FTIR spectra of AePhe ge-
netically incorporated at either site 149 or 133 in sfGFP dis-
solved in an aqueous pH 7.3 buffer composed of 50 mm
sodium phosphate and 150 mm sodium chloride in the region
2000–2250 cmÀ1 to probe the local protein environment at
these two sites. The buffer was selected for comparison with
previous FTIR studies of sfGFP constructs containing vibrational
reporter UAA.[23,40,49] The absorption profile in the region of the
azide asymmetric stretch vibration in each of these protein
constructs appears less complicated compared to the free
AePhe dissolved in DMSO or water (Figure 3A). This modula-
tion of the absorption profile suggests that the incorporation
of AePhe into the protein alters the anharmonic coupling and/
or the frequency of the potential sub-components of the ob-
served absorption band relative to the free AePhe.
[a] Experimental mass spectrometry data error Æ1 Da.
The azide asymmetric stretching frequency of sfGFP-149-
AePhe and sfGFP-133-AePhe were observed to be 2115.5 and
2118.0 cmÀ1, respectively, as shown in Figure 3B. The position
of the azide asymmetric stretch of sfGFP-133-AePhe indicates
that the azido group is hydrated at this site because of the
similarity of this frequency with that of AePhe dissolved in
water (2120.4 cmÀ1). This hydration state is not surprising since
sfGFP crystal structures[44,53–55] show that this site is a fully sol-
vated loop region of the protein and the side chain does not
appear able to fold back onto the protein surface.
Site 149 is located on one of the b-strands of the b-barrel of
sfGFP in which the side chain of the amino acid is directed
away from the protein into the solvent in numerous crystal
structures.[44,53–55] The azido group of 4-(4-azido-1-butynyl)-l-
Phe, which has a rigid spacer group, at this site was recently
found to be fully solvated.[40] Thus, the azido group of AePhe
has the potential to be fully hydrated depending on the con-
formation of the linker between the azido and the aromatic
ring of AePhe. However, the position of the azide asymmetric
stretch vibration of sfGFP-149-AePhe suggests that the azido
group at this site is partially buried and not fully solvated. This
assignment is based on the red shift of the azide asymmetric
stretching frequency compared to sfGFP-133-AePhe, which
was fully hydrated. On the other hand, the magnitude of the
red shift between the two sites (2.5 cmÀ1) compared to the
magnitude of the red shift of AePhe dissolved in DMSO com-
pared to water (9.7 cmÀ1) suggests that the azido group of
sfGFP-149-AePhe is only partially solvated. To further explore
the conformation of the side chain of AePhe incorporated at
site 149, the crystal structure of this construct was determined.
Figure 3. Transmission FTIR absorbance spectra of AePhe dissolved in either
DMSO or a basic aqueous solution to a concentration of ~50 mm (A) or
sfGFP constructs containing AePhe at either site 149 (solid curve) or 133
(dashed curve) dissolved in a pH 7.3 aqueous buffer composed of 50 mm
sodium phosphate and 150 mm sodium chloride to a concentration of ~1–
3 mm (B). The spectra were recorded at 258C, baseline corrected, and inten-
sity normalized.
AePhe to serve as a vibrational reporter of local environments.
Figure 3A shows the equilibrium FTIR spectra of AePhe in the
2000 to 2250 cmÀ1 region dissolved in either DMSO or water,
which were selected as mimics of the hydrophobic and hydro-
philic environments present in proteins, respectively. These
spectra show that the IR absorbance profile of AePhe in the
azide region in both solvents is complicated and not symmetri-
cal—likely the result of the presence of multiple sub-compo-
nents and/or anharmonic effects.[50,51] The highest intensity fre-
quency component of the profile was assigned to the azide
asymmetric stretch frequency of AePhe similar to previous
studies of azides.[50] This vibrational frequency shifts from
2110.7 cmÀ1 in DMSO to 2120.4 cmÀ1 in water. This 9.7 cmÀ1
blue shift is principally the result of hydrogen bonding be-
tween water molecules and the azido group of AePhe,[23,40,52]
which is absent in the DMSO solution because DMSO lacks
X-ray crystal structure of sfGFP-149-AePhe
The X-ray crystal structure of sfGFP-149-AePhe was solved (Fig-
ure 4A) to confirm the fidelity of the UAA incorporation and
explore the difference in the IR spectra of the asymmetric
stretch vibration of AePhe at the 149 site versus the 133 site.
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Figure 5. Alignment of sfGFP-149-AePhe (magenta) with wt-sfGFP (green) (A)
overall and (B) in the vicinity of the 149 site. AePhe is shown in ball and
stick and side chains of residues within 10 Š of the site are shown in sticks
and labeled. All explicitly shown side chains are colored by atom type with
carbon green (wt) or magenta (AePhe mutant), oxygen red, and nitrogen
blue.
Figure 4. X-ray crystal structure of sfGFP-149-AePhe. A) Overall structure
with chromophore shown in sticks for reference and AePhe shown in ball
and stick and colored by atom type. B) 2FoÀFc electron density at 1.0 s illus-
trating incorporation of AePhe at the 149 site. C) Surface representation sur-
rounding the 149 site emphasizing the orientation of the azido group to-
wards the protein surface and partially buried from solvent by neighboring
residues. The structure of sfGFP-149-AePhe is deposited in the Protein Data-
bank and assigned the PDB ID 5EHU.
side chain movements upon incorporation of AePhe, most no-
tably the approximately 1808 rotation of the 149 and 151 side
chains when compared to wt-sfGFP (Figure 5B), accounting for
the larger all-atom RMSD for the region around the 149 site
compared to the overall structure. However, the side chain
movements do not result in a large cascade of movements
and the backbone of the protein remains mostly unchanged,
as indicated by the low RMSD of Ca atoms in the area. Thus
the crystal structure provides important structural insights into
the nature of the hydration state, which was assigned based
upon the observed azide asymmetric stretching frequency of
AePhe in sfGFP-149-AePhe and sfGFP-133-AePhe thereby pro-
viding further support for the ability of AePhe to serve as a sen-
sitive vibrational reporter UAA.
The 1.45 Š crystal structure illustrated electron density for the
full AePhe amino acid at the 149 site (Figure 4B and Support-
ing Information). The azido group is orientated towards the
protein surface (Figure 4C), supporting the interpretation that
the IR shift described above was caused by a less solvated en-
vironment compared with the 133 site and the fully solvated
4-(4-azido-1-butynyl)-l-Phe previously studied.[40]
The X-ray crystal structure of sfGFP-149-AePhe also permits
the observation of structural changes upon the incorporation
of AePhe in sfGFP. As the 149 site is on the side of the b-barrel
facing towards the solvent it is expected that there would be
little disruption of the protein structure as indicated by the
overall root-mean squared deviation (RMSD) between the wt-
sfGFP and sfGFP-149-AePhe structures. Indeed, the RMSD was
1.25 Š for all-atom and 0.84 Š for Ca atoms only for the overall
structural alignment shown in Figure 5A. An alignment of the
residues within 10 Š of the 149 site indicates RMSDs of 1.45
and 0.25 Š for all-atoms and Ca atoms, respectively. A view of
the environment within 10 Š of the 149 site illustrates a few
Click chemistry in sfGFP at incorporated AePhe residues
The ability of azido-UAA such as AePhe to act as vibrational re-
porters is valuable but they also have the ability to act as
a conduit for PTM of proteins through a bioorthogonal click
chemistry reaction with alkynes. To demonstrate the ability of
AePhe to undergo PTM we explored click reactions under the
two standard conditions, strain-promoted[31] and copper(I)-cat-
Scheme 2.
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alyzed[30] azide–alkyne cycloadditions. The strain-promoted
click reaction proceeded smoothly in duplicate experiments at
both incorporation sites. Thus, when sfGFP-149-AePhe or
sfGFP-133-AePhe were treated with dibenzocyclooctyne
Reactions were stirred with a magnetic stir bar and completed
under a dry argon atmosphere. Reactions carried out above ambi-
ent temperature were conducted in an oil bath. Analytical TLC was
conducted using 0.2 mm silica plastic coated sheets (Selecto Scien-
tific) with F254 indicator and 230–400 mesh silica gel was used for
flash column chromatography.
(DBCO) acid derivative
4 the click reaction cycloadduct
(Scheme 2) was confirmed by mass spectral analysis (see
Table 1). In each case the mass increased by ~390 Da (the
mass of 4) over the AePhe construct which corresponds to
a gain of ~507 Da over wtGFP. As shown in Scheme 2, this re-
action likely produced two isomeric cycloadducts.
All NMR characterization was completed using a Varian INOVA 500
multinuclear Fourier transform NMR spectrometer (1H NMR at
499.7 MHz and 13C NMR at 125 MHz). Chemical shifts are reported
in parts per million (ppm) and coupling constants are reported in
Hertz (Hz). NMR spectra in CDCl3 were referenced to the residual
1
solvent peak (CHCl3 =7.26 ppm) for H NMR and the solvent peak
On the other hand, repeated attempts at the copper(I)-cata-
lyzed click reaction of sfGFP-149-AePhe or sfGFP-133-AePhe
with propargyl alcohol using Hong et al.’s optimized condi-
tions[30] for the catalyst, ligand, and additional reagents failed
to produce the expected cycloadduct according to mass spec-
tral analysis (see Supporting Information). The reasons for this
failure are unclear. When the reaction was attempted with the
polyhistidine-tag (His-tag) additional copper(I) was added, as
recommended, and the reaction was also attempted after the
His-tag had been cleaved by a trypsin digest. Propargyl alcohol
is fully miscible with water, so the solubility of the click partner
was not an issue.
(CDCl3 =77.0 ppm) for 13C NMR. NMR spectra in [D6]DMSO were ref-
erenced to the residual solvent peak ([D5]DMSO=2.49 ppm) for
1H NMR and the solvent peak ([D6]DMSO=39.5 ppm) for 13C NMR.
All IR characterization for synthetic purposes were carried out as
ATR thin films with frequencies reported in cmÀ1. MS analyses of
the free UAA for synthetic purposes were carried out on an Agilent
1100 series LC/MSD SL ion trap mass spectrometer with electro-
spray ionization and MS/MS capabilities.
The ESI-Q-TOF mass analysis was performed on the same purified
protein samples used for the linear IR measurements. This analysis
was performed at the Mass Spectrometry Facility at the University
of Illinois Urbana-Champaign under the direction of Dr. Furong
Sun. The protein samples were desalted into a 20 mm ammonium
acetate solution using PD10 gel filtration columns, lyophilized, and
re-suspended in 1:1 H2O/CH3CN with 0.2% formic acid prior to
analysis.
Conclusion
An efficient and scalable synthesis of AePhe was accomplished
in three steps from known compounds in 54% overall yield.
The IR asymmetric stretch vibration of the azido group of
AePhe was shown to be sensitive to its local environment
through a solvent study. AePhe was successfully incorporated
into sfGFP at the 133 and 149 sites. IR spectroscopy indicated
that, as expected, the azido group at the 133 site was fully sol-
vated, whereas, somewhat unexpectedly, the azido group at
the 149 site was not. An X-ray crystal structure of sfGFP-149-
AePhe provided an explanation for the IR data as the flexible
linker adopts a conformation, which partially buries the azide
on the protein surface. Both sfGFP-AePhe constructs readily
underwent a bioorthogonal strain-promoted click cycloaddi-
tion with DBCO 4 demonstrating that AePhe is valuable both
as a vibrational reporter and as a conduit for bioconjugation.
N-(tert-Butoxycarbonyl)-4-(2-azidoethoxy)-l-phenylalanine
methyl ester (3)
To a solution of 2 (3.01 g, 10.1 mmol, 1 equiv) in dry DMF (100 mL)
were added K2CO3 (4.26 g, 30.4 mmol, 3 equiv) and 2-azidoethyl
methanesulfonate (4.80 g, 29.1 mmol, 2.9 equiv). The heterogene-
ous mixture stirred for 28 h at 708C. The reaction mixture was
cooled and diluted with diethyl ether and water. The organic layer
was washed with brine, dried over MgSO4, filtered through a glass
frit, and concentrated under reduced pressure. The crude product
was purified by column chromatography (hexanes/ethyl acetate
3:1) to yield 3 (2.38 g, 6.55 mmol, 66%) as a light yellow oil:
1H NMR (CDCl3): d=7.05 (d, J=8.6, 2H), 6.85 (d, J=8.6, 2H), 4.97
(d, J=7.8, 1H), 4.54 (m, 1H), 4.13 (t, J=5.1, 2H), 3.71 (s, 3H), 3.58
(t, J=5.1, 2H), 3.04 (m, 1H), 1.42 ppm (s, 9H); 13C NMR (CDCl3): d=
172.37, 157.31, 155.07, 130.39, 128.74, 114.66, 79.91, 66.95, 54.50,
52.20, 50.16, 37.48, 28.29 ppm; IR: n˜ =3357.6 (br, w), 2977.7 (w),
2109.5 (m), 1742.3 (m), 1709.3 (s), 1611.9 (w), 1511.1 (s), 1438.8 (m),
1365.4 (m), 1243.1 (s), 1163.4 (s), 1058.2 (m), 1017.4 (m), 838.9 cmÀ1
(m); MS (ESI) 387 [M+Na+, 100], 265 (15), 422 (10).
Experimental Section
General information
4-(2-Azidoethoxy)-l-phenylalanine hydrochloride (1·HCl)
All chemical reagents were purchased from either Sigma–Aldrich
or TCI America and used without further purification. Deuterated
solvents (CDCl3, 98.8% D enrichment and [D6]DMSO, 99.5% D en-
richment) were purchased from Cambridge Isotope Laboratories.
DH10B cells and pBAD were purchased from Invitrogen. 2-Azi-
doethyl methanesulfonate was synthesized by the literature
method of Demko and Sharpless,[56] except that methanesulfonyl
chloride was utilized in place of toluenesulfonyl chloride (see the
Supporting Information). Protected tyrosine 2 was purchased from
TCI America. Dibenzocyclooctyne (DBCO) C6-Acid derivative 4 was
purchased from Click Chemistry Tools. All aqueous solutions were
prepared with 18 MWcm water.
To a solution of 3 (2.35 g, 6.44 mmol) in THF/H2O (3:1, 20 mL) was
added LiOH monohydrate (0.415 g, 9.66 mmol, 1.5 equiv). The solu-
tion stirred at room temperature for 4 h and then the pH was ad-
justed to ~2.5 by using aq. NaHSO4 (0.5m). The reaction mixture
was diluted with ethyl acetate and the organic layer was washed
with water and brine, dried over MgSO4, filtered through a glass
frit, and concentrated under reduced pressure. The residue was
dissolved in dry HCl in dioxane (2.5m, 17 mL). After stirring for 4 h
at room temperature the solution was concentrated under reduced
pressure to about one-third of its initial volume and pentane was
added. The white precipitate was collected by vacuum filtration to
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1
provide 1·HCl (1.51 g, 5.25 mmol, 82%) as a white powder: H NMR
Equilibrium FTIR measurements
([D6]DMSO): d= 7.20 (d, J=8.8, 2H), 6.90 (d, J=8.8, 2H), 4.13 (t,
J=4.9, 2H), 4.05 (t, J=6.3, 1H), 3.62 (t, J=4.9, 2H), 3.09 ppm (d,
J=6.3, 2H); 13C NMR ([D6]DMSO): d=170.74, 157.65, 131.20,
127.73, 115.00, 67.26, 53.77, 50.02, 35.21 ppm; IR: n˜ =3435.5 (br,
w), 2970.6 (m), 2874.5 (m), 2109.0 (s), 2068.0 (w), 1737.2 (s), 1612.0
(w), 1513.8 (s), 1487.7 (s), 1445.2 (w), 1289.9 (m), 1247.2 (s), 1230.3
(s), 1204.0 (m), 1114.6 (m), 1061.8 (w), 837.7 (m), 803.3 cmÀ1 (m);
MS (ESI) 251.0 [M+, 100], 233.9 (25), 205 (5).
The equilibrium FTIR absorbance spectra were recorded using
a Bruker Vertex 70 FTIR spectrometer, equipped with a globar
source, KBr beamsplitter, and a liquid nitrogen cooled mercury cad-
mium telluride (MCT) detector. The spectra were measured using
a
dual-compartment, temperature-controlled transmission cell
composed of two calcium fluoride windows with a path length of
~100 mm. The spectra were recorded at 258C with a resolution of
1.0 cmÀ1 and were the result of 1024 or 2048 scans. Analysis of the
spectra was performed in Igor Pro (Wavemetrics, Inc).
Expression and purification of sfGFP constructs
Structure determination of sfGFP-149-AePhe
The following plasmids were obtained from Dr. Ryan A. Mehl
(Oregon State University) in which the numbering scheme em-
ployed here corresponds to the published wild-type sfGFP (wt-
sfGFP) crystal structure (PDB ID 2B3P).[53] The plasmid pBAD-sfGFP
was generated by inserting a codon-optimized gene coded with
wt-sfGFP[53] containing a C-terminal 6-His affinity tag into a pBAD
plasmid, generating pBAD-sfGFP.[57,58] Site-directed mutagenesis
was used to individually replace the D133 or N149 codons with
the amber stop codon (TAG) to generate the pBAD-sfGFP-133TAG
and pBAD-sfGFP-149TAG plasmids, respectively. The engineered
aminoacyl-tRNA synthetase for the incorporation of para azido-
modified phenylalanine UAA was inserted into a pDULE plasmid,
generating pDULE-AzidoPhe.[23,57] DH10B E. coli cells were trans-
formed with pBAD-sfGFP for wt-sfGFP, while DH10B E. coli cells
were co-transformed with pBAD-sfGFP-133TAG or pBAD-sfGFP-
149TAG and pDULE-AzidoPhe for the sfGFP-133-UAA and sfGFP-
149-UAA constructs, respectively. Transformed cells were used to
inoculate 6 mL of noninducing media, which grew to saturation
while shaking (250 rpm) at 378C. Autoinduction media (250 mL)
was then inoculated with 2.5 mL of the cultured cells. A 1–2 mL so-
lution of AePhe was added 1 h post-inoculation for a final concen-
tration of 1 mm. Negative control experiments did not include
AePhe in the autoinduction media (Figure 2). The autoinduction
media cultures shook at 378C for 24–30 h when the cells were har-
vested by centrifugation and the expressed protein was purified
using TALON cobalt ion-exchange chromatography (Clontech) simi-
lar to a previous literature procedure.[23] To verify site-specific incor-
poration of the UAA into site 133 and 149 with high efficiency and
fidelity, sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE, Figure 2) and electrospray ionization quadrupole time-
of-flight (ESI-Q-TOF) mass analysis (Table 1) at the University of Illi-
nois at Urbana-Champaign were carried out. The sfGFP constructs
containing AePhe (1) generally yielded 5–10 mg of purified protein
per liter of autoinduction media. Protein yields were calculated
using the extinction coefficient of sfGFP at 488 nm.[53] The follow-
ing protein constructs, sfGFP-133-AePhe and sfGFP-149-AePhe,
were the result of incorporation of AePhe into sfGFP site 133 or
site 149, respectively. PD10 gel filtration columns were used to
desalt the purified protein constructs into an aqueous buffer solu-
tion (50 mm sodium phosphate, 300 mm sodium chloride) with
a pH of 8. The C-terminal 6-His affinity tag was cleaved utilizing
a catalytic amount of trypsin (1%). After 2 h of incubation at 378C,
the 6-His affinity tag was fully cleaved, resulting in a 239 residue
protein. To deactivate the trypsin, a ten-fold molar excess of phe-
nylmethanesulfonyl fluoride (PMSF) compared to trypsin was
added to the solution. Removal of sfGFP still containing a 6-His af-
finity tag sfGFP was accomplished by TALON cobalt ion-exchange
chromatography. The resulting purified UAA containing sfGFP con-
structs were desalted into the appropriate buffer for further steps
utilizing PD10 gel filtration columns or buffer exchange and con-
centration using 10 K MWCO Centricons (Millipore).
A 40 mgmLÀ1 solution of sfGFP-149-AePhe in a 20 mm Hepes
buffer pH 7.5 was combined with a precipitation solution (20%
PEG 8000, 100 mm Hepes pH 7.5) in a 1:1 ratio to form crystals in
a sitting drop well at room temperature. Single green crystals were
mounted on loops, cryoprotected by consecutive soaks of 8, 13,
and 25% ethylene glycol-supplemented precipitant solution and
frozen in liquid nitrogen. Diffraction data were collected at the NE-
CAT 24-ID-E beamline at the Advanced Photon Source (APS) at Ar-
gonne National Lab (ANL) and processed in space group C2 to
1.45 Š using HKL2000.[59] A molecular replacement solution was de-
termined using Phaser[60] (TFZ=27.5, LLG=7651) with the search
model of wild-type sfGFP (PDB ID 2B3P)[53] in which residue 149
was replaced with an alanine. After a single round of refinement
a phenylalanine was modeled into the 2FoÀFc density at position
149; and following a few more rounds of refinement the azido-
ethoxy group at the para position of the phenylalanine was mod-
eled using the FoÀFc difference density (see Supporting Informa-
tion). Rounds of manual refinement in COOT[61] and automated re-
finement in Phenix[62] were continued to produce the reported
structure with R/Rfree of 17.5/20.8% (Table 2).
Table 2. X-ray data collection and processing statistics for the sfGFP-149-
AePhe structure.
Space group
C2
Cell dimensions
a [Š]
129.917
b [Š]
37.492
c [Š]
91.848
a, b, g [8]
l [Š]
90, 106.307, 90
0.97914
T [K]
100
unique reflections
resolution range [Š][a]
average Redundancy[a]
completeness [%][a]
I/s[I][a]
# sfGFP molecules per asu
Rcryst (Rfree) [%][b]
rms deviations
bond lengths [Š]
bond angles [8]
Ramachandran plot [%][c]
preferred
73332
50–1.45 (1.48–1.45)
3.8 (3.9)
96.4 (94.3)
16.5 (1.9)
2
17.5 (20.8)
0.009
1.357
97.6 (404)
1.9 (8)
allowed
outliers
0.5 (2)
[a] The number in parentheses is for the highest resolution shell.
[b] Rcryst =Shkl j jFo(hkl)jÀjFc(hkl)j j/Shkl jFo(hkl)j. Rfree =Rcryst for a test
set of reflections (5%) not included in refinement. [c] Numbers in paren-
theses are the number of residues in each category.
Chem. Eur. J. 2015, 21, 19096 – 19103
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Strain-promoted click cycloaddition reactions
The sfGFP construct (either sfGFP-133-AePhe or sfGFP-149-AePhe)
was initially desalted into a buffer (pH 7.3; 50 mm sodium phos-
phate; 150 mm sodium chloride) using PD10 columns and concen-
trated if necessary utilizing 10 K MWCO Centricons (Millipore). The
strained-promoted cycloaddition reaction was carried out with
a DBCO acid derivative (4, MW=390 Da), which was initially dis-
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Acknowledgements
We thank R. Keyes for the synthesis of 2-azidoethyl methane-
sulfonate, K. Hess for mass spectral (ESI) analyses of small mol-
ecules, L. Mertzman for ordering supplies, and B. Buckwalter
for acquiring NMR spectra. This work was supported by F&M
Hackman funds, F&M Eyler Funds, Leser Scholars Grant, and
Snavely Awards to E.M.T. NSF (CHE-1053946) to S.H.B. and NIH
(R15M093330-02) to E.E.F. and S.H.B. X-ray diffraction data were
collected at the Advanced Photon Source (APS) on the North-
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which is supported by an NIH NIGMS grant P41 GM103403.
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Keywords: azides
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Received: September 29, 2015
Published online on November 26, 2015
Chem. Eur. J. 2015, 21, 19096 – 19103
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