Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins

Article abstract of DOI:10.1021/acs.orglett.5b03268

A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivatives were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene-alanine derivatives exhibited efficient trans/cis photoswitching upon irradiation with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene-alanine analogues were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.

Full text of DOI:10.1021/acs.orglett.5b03268

Letter
pubs.acs.org/OrgLett
Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene
Amino Acids into Proteins
Alford A. John, Carlo P. Ramil, Yulin Tian, Gang Cheng,† and Qing Lin*
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260-3000, United States
S
* Supporting Information
ABSTRACT: A series of red-shifted azobenzene amino acids
were synthesized in moderate-to-excellent yields via a two-step
procedure in which tyrosine derivatives were first oxidized to the
corresponding quinonoidal spirolactones followed by ceric
ammonium nitrate-catalyzed azo formation with the substituted
phenylhydrazines. The resulting azobenzene−alanine derivatives
exhibited efficient trans/cis photoswitching upon irradiation with
a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying
the azobenzene−alanine analogues were expressed in E. coli in good yields via amber codon suppression with an orthogonal
tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.
ntil recently, the use of azobenzene photoswitches in
biological systems typically required short-wavelength UV
Scheme 1. Approaches for Synthesis of Azobenzene Amino
Acids
U
light in order to induce isomerization of the azo bond, which is
undesirable because of the phototoxicity to cells. To minimize
phototoxicity and reduce light scattering, recent efforts have
focused on the design of highly substituted red-shifted
azobenzenes.1 On the basis of an earlier observation2 that
bulky substituents at the ortho positions of the azo bond caused
a red shift in the n → π* band and a blue shift in the π → π*
́
band, Woolley3 and Bleger4 designed tetra-o-methoxy- and
tetra-o-fluoro-substituted, red-shifted azobenzenes, respectively,
that showed robust photoswitching properties with the use of
visible light. On the other hand, three strategies have been
employed to introduce the azobenzenes into proteins site-
selectively for the photoregulation of protein function in vivo.
These include the design of the cysteine-reactive tethered
ligands,5,6 the genetic encoding of the azobenzene amino acids
in E. coli7 and mammalian cells,8 and the use of bioorthogonal
ligation with genetically encoded bioorthogonal reporters.9
Among them, the genetic encoding of azobenzene amino acids
is particularly attractive as it offers the highest incorporation
efficiency in vivo without interference from intracellular thiols
as well as potential side reactions found in bioorthogonal
reactions. In addition, a genetically encoded azo bridge for
proteins was reported recently, showing potentially improved
photo control of protein function in vivo.10
To expand genetically encodable azobenzene photoswitches
for photochemical control of protein function in vivo, efficient
synthetic methods need to be developed. Previously, the
azobenzene amino acids were prepared by treating anilines with
Oxone to generate the nitrosobenzene intermediates, which
reacted with the p-aminophenylalanine to afford the protected
azobenzene amino acids in moderate yields (Scheme 1a).7,8
Since the nitroso intermediates are unstable and the p-
aminophenylalanine analogues are not widely available,
alternative synthetic methods are highly desirable. Herein, we
report the facile synthesis of the highly substituted azobenzene
amino acids through the reaction of quinonoidal spirolactones,
which can be prepared in one step from the tyrosines, and
phenylhydrazines in the presence of ceric ammonium nitrate
(CAN) catalyst (Scheme 1b). Furthermore, nine new
azobenzene amino acids were incorporated into the superfolder
green fluorescent protein (sfGFP) in E. coli using a reported
orthogonal tRNA/PylRS pair. One of the resulting azo-sfGFP
showed durable photoswitching with the LED light.
In search of efficient methods for the synthesis of azobenzene
amino acids, we were intrigued by a report from Carreno and
̃
co-workers where they showed that the azobenzenes can be
prepared in good-to-excellent yields from quinone bisacetals
and arylhydrazines in the presence of a catalytic amount of
CAN.11 To examine whether this method can be extended to
the quinone analogue derived from tyrosine, we prepared the
quinonoidal spirolactone 1a (structure in Table 1 scheme) in
37% yield by treating N-Boc-L-tyrosine with a slight excess
amount of phenyliodonium diacetate (PIDA).12 Separately, the
Received: November 12, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b03268
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
provide a potential cross-linking site for a proximal cysteine
(e.g., at the i+7 position) through the thiol−ene click reaction
as reported.14 Similarly, the acetylene moiety at the meta
position of 3f offers a bioorthogonal handle for conjugation
with an azide-containing ligand via either copper-catalyzed click
chemistry15 or copper-catalyzed Glaser−Hays bioconjugation
Table 1. Synthesis of N-Boc-Protected Azobenzene−Alanine
Analogues
reaction.16 Since Bleg
́
er et al. showed that o-fluorine
substitution leads to superior photoswitching properties,4 we
investigated the reactivity of a series of fluorine-substituted
phenylhydrazines 2g−k in the CAN-catalyzed azo formation
reaction. To our satisfaction, the fluorinated azobenzene−
alanine analogues were obtained in good to excellent yields
(entries 7−11, Table 1), irrespective of the number of fluorines
as well as additional substituents.
Since the ortho-substituted azobenzenes show red-shifted
absorbance and improved photophysical properties compared
to the azobenzene parent, we chose two commercially available
ortho-halogenated L-tyrosine derivatives and prepared their
corresponding quinonoidal spirolactones, 1b and 1c, using the
PIDA method (structures are shown in the Table 1 scheme).
To maximize potential improvement in photophysical proper-
ties, these two spirolactones were reacted with the ortho-
difluorinated phenylhydrazines 2g, 2h, and 2k. All six
halogenated azobenzene−alanine products 3l−q were obtained
uneventfully in moderate yields (entries 12−17, Table 1),
though longer incubation times were required for the two
chlorinated azo products, presumably due to the bulky chlorine
substituent that slows down the nucleophilic addition.
a
entry
X
R
product
yield (%)
1
H
H
H
H
H
H
H
H
H
H
H
F
4-Me (2a)
3a
3b
3c
3d
3e
3f
73
72
72
85
68
2
2-OMe (2b)
3
4-CN (2c)
4
3-CN (2d)
5
3-CHCH2 (2e)
3-CCH (2f)
2,6-F2 (2g)
b
6
83
7
3g
3h
3i
95
91
8
2,4,6-F3 (2h)
2,6-F2-4-CONH2 (2i)
2,6-F2-4-I (2j)
2,3,4,5,6-F5 (2k)
2,6-F2 (2g)
c
9
94
d
,
e
10
11
12
13
14
15
16
17
3j
82
3k
3l
89
72
72
60
F
2,4,6-F3 (2h)
2,3,4,5,6-F5 (2k)
2,6-F2 (2g)
3m
3n
3o
3p
3q
F
e,f
Cl
Cl
Cl
68
f g
67 ,
2,4,6-F3 (2h)
2,3,4,5,6-F5 (2k)
63
a
Isolated yields were reported. Unless noted otherwise, reactions were
Because the ortho-fluorinated azobenzenes were reported to
show good separation of trans/cis n → π* bands, and as a result
achieve high cis and trans photostationary state (pss),4 we
investigated photoswitching properties of nine fluorinated
azobenzene−alanine analogues we synthesized. The simplest
difluorinated azobenzene analogue, 3g, showed a wavelength
separation of 8 nm and 66% cis and 62% trans pss after 30 min
photoirradiation with green (530 nm) and blue (448 nm) LED
light, respectively (Table 2). Adding additional fluorine on one
side of the azo bond increased the wavelength separation but
led to lower cis-pss (compare 3h and 3k to 3g in Table 2).
When a fluorine was introduced in the ortho position of the
internal benzene ring, all three tri-ortho-fluorinated azoben-
zene−alanine analogues (3l, 3m, and 3n) showed greater
wavelength separation (12−16 nm) than 3g; most strikingly,
3m exhibited 76% cis-pss after irradiation with green light.
However, when a chlorine was placed at the ortho position of
the internal benzene ring, divergent behaviors were observed
carried out by stirring a solution of spirolactone (1a−c) with 1.1 equiv
of the substituted phenylhydrazine in MeCN at room temperature for
b
c
12−17 h. 24 h reaction time. MeOH/MeCN (1:4) was used as
d
e
solvent. 1.5 equiv of the substituted phenylhydrazine was used. 72 h
reaction time. 2.5 equiv of the substituted phenylhydrazine was used.
48 h reaction time.
f
g
phenylhydrazine derivatives are either commercially available or
can be readily prepared from appropriate anilines through the
diazonium salt formation followed by reduction with SnCl2 in
HCl.13
With the substrates in hand, we performed the azo formation
reaction in acetonitrile in the presence of 3 mol % of CAN, and
the results are summarized in Table 1. For monosubstituted
phenylhydrazines 2a−f, the N-Boc-protected trans-dominant
azobenzene−alanines were obtained in 68−85% yield (entries
1−6, Table 1) with no obvious electronic and steric effects. The
vinyl group at the meta position in the azo product 3e might
Table 2. Photophysical Properties of N-Boc-Protected Azobenzene−Alanine Analogues
a
compd
X
R
trans λmax n→π* (nm)
cis λmax n→π* (nm)
n→π* separation (nm)
trans:cis 530 nm
trans:cis 448 nm
b
b
3g
3h
3k
3l
H
H
H
F
2,6-F2
435
439
446
442
436
442
428
425
448
427
429
428
426
424
429
426
425
434
8
10
18
16
12
13
2
34:66
62:38
c
c
2,4,6-F3
38:62
61:39
c
c
2,3,4,5,6-F5
2,6-F2
43:57
64:36
b
b
36:64
60:40
b
b
3m
3n
3o
3p
3q
F
2,4,6-F3
24:76
61:39
c
c
F
2,3,4,5,6-F5
2,6-F2
35:65
62:38
b
b
Cl
Cl
Cl
23:77
70:30
b
b
2,4,6-F3
0
28:72
71:29
b
b
2,3,4,5,6-F5
15
34:66
67:33
a
b
c
Wavelength separation was calculated using the following equation: Δ = λmax,trans − λmax,cis. Trans/cis ratio was determined by 19F NMR. Trans/cis
ratio was determined by 1H NMR. Sample was dissolved in DMSO-d6 in an NMR tube to obtain a concentration of 15−22 mM. The
photoirradiation was performed by placing the NMR tube on top of an LED light in an enclosed compartment for 30 min.
B
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Table 3. Genetic Incorporation of Azobenzene−Alanine Analogues into Superfolder GFP
a
compd
X
R
calcd mass, Da
obsd mass, Da
yield (mg/L)
% Gln incorp
4d
4g
4h
4k
4l
H
H
H
H
F
3-CN
27856.3
27868.1
27886.1
27921.3
27886.1
27903.3
27941.1
27902.1
27920.5
27857.6 1.3
27867.6 1.0
27885.2 0.9
27920.3 1.8
27886.0 1.1
27901.9 1.6
27940.3 1.7
27904.6 1.6
27920.3 1.2
32.0
22.3
31.6
5.4
0
0
2,6-F2
2,4,6-F3
2,3,4,5,6-F5
2,6-F2
0
36
4
10.2
3.9
4m
4n
4o
4p
F
2,4,6-F3
2,3,4,5,6-F5
2,6-F2
21
50
68
27
F
4.8
Cl
Cl
7.8
2,4,6-F3
4.4
a
The Gln incorporation into sfGFP in the purified protein samples was calculated on the basis of ion counts using the following equation: Gln % =
IsfGFP‑S2Q/(IsfGFP‑S2Q + IsfGFP‑S2→Aba), where IsfGFP‑S2Q and IsfGFP‑S2→Aba are the ion counts of sfGFP-S2Q and sfGFP-S2 → Aba, respectively, in the
deconvoluted mass spectra.
for the resulting three azobenzene−alanine analogs (3o, 3p,
and 3q). For di- and trifluorinated analogues (3o and 3p), there
was hardly any separation for the n → π* bands, but 3o showed
the highest cis-pss (77%) detected in the series along with the
second highest trans-pss (70%). For 3q, despite the largest
bathochromic shift in the trans n → π* band (448 nm) and
improved wavelength separation compared to 3g, the cis-pss
remained unchanged while the trans-pss showed a modest
improvement. It is noteworthy that the photostationary states
were reached after only 5 min photoirradiation in a time−
course study with 3h (Table S1, Supporting Information) and
that the cis-pss of 3h did not show measurable reduction after 4
days of dark adaptation at room temperature (Figure S1,
Supporting Information). Taken together, there was no
apparent correlation between the wavelength separation and
the pss, suggesting an alternative mechanism may be needed to
account for this discrepancy. Unexpectedly, the o-chlorine-
substituted di-o-fluoroazobenzene−alanine analogues 3o and
3p exhibited essentially no wavelength separation but highest
cis- and trans-pss values, the parameters most relevant in
photochemical control of protein function in biological system.
Since several substituted azobenzene−alanines have been
site-specifically incorporated into proteins using amber codon
suppression with the orthogonal MmPylRS/tRNACUA pairs,8 we
surmised that our new azobenzene−alanine analogues could be
similarly incorporated into proteins using the same system.
Accordingly, pEvol-PylT-MmPylRS plasmid encoding a Pyl-
tRNACUA and a PylRS variant carrying four mutations (A302T,
L309S, N346V, and C348G) in its active site was prepared and
then cotransformed into BL21(DE3) cells along with the pET-
sfGFP-S2TAG reporter plasmid. The resulting transformants
were grown in 10 mL LB medium supplemented with 1 mM
azobenzene−alanine (Aba) analogue at 37 °C, and the Aba-
encoded sfGFP proteins were isolated through Ni−NTA
affinity chromatography. Among the azobenzene−alanine
analogues tested, small substituents on the distal benzene
ring such as cyano and fluorine are generally well accepted by
the synthetase with expression yields reaching as high as 32.0
mg L−1 (Table 3), along with high fidelity evidenced by mass
spectrometry (see the Supporting Information). One exception
is pentafluoro analogue 4k, which produced a lower yield of 5.4
mg L−1 with 36% near-cognate Gln-suppression product,17
indicating the m-fluoro substituents are not well tolerated by
the synthetase. When the o-fluorine was introduced into the
internal benzene ring, the azobenzene−alanine derivatives (4l,
4m, and 4n) showed lower expression yields, along with the
erosion of fidelity. A similar phenomenon was also observed for
the ortho-substituted azobenzene−alanine analogues 4o and 4p,
presumably due to the repulsion between the internal halogen
and the distal azo-nitrogen, which twists the azo bond out of
coplanarity with the internal benzene ring4 resulting in poorer
substrate properties. With these observations, it appears that
new PylRS variants are warranted in order to efficiently charge
the twisted tri-o-halogenated azobenzene−alanines with high
fidelity in E. coli.
To examine whether the genetically encoded azobenzenes in
proteins can respond to visible-light induced photoswitching,
we subjected the highly expressed sfGFP mutant sfGFP−4h to
alternating 5 min green-blue irradiation cycles and monitored
the π → π* bands (Figure S2, Supporting Information) because
of their relatively high intensity in the UV−vis spectra. Closer
examination revealed rhythmic changes in absorbance at 340
nm over 10 cycles, consistent with reversible photoswitching
between the trans (high absorbance at 340 nm) and cis (low
absorbance at 340 nm) form of the azo bond with no sign of
“fatigue”, the erosion of cis ratio during photoswitching cycles
(Figure 1). The photoswitching durability of the trifluorinated
azobenzene in sfGFP−4h also highlighted an exceptional
photostability for the fluorinated azobenzene system.4
In summary, we have synthesized a series of red-shifted
azobenzene−alanine analogues from the substituted tyrosine
and phenylhydrazine derivatives via a two-step procedure. The
route involves storable spirolactone intermediates and enables
facile unsymmetrical azo formation with the phenylhydrazines
in the presence of the ceric ammonium nitrate catalyst. The
photophysical properties of the fluorinated azobenzene-alanine
analogues were characterized, with the o-chlorodifluoroazoben-
zene 3o reaching the highest photostationary states (77% cis-
pss after 530 nm photoirradiation and 70% trans-pss after 448
nm photoirradiation). In addition, nine azobenzene−alanine
amino acids were genetically incorporated into proteins in E.
coli with good-to-excellent yields and varying degrees of fidelity
via amber codon suppression with an orthogonal tRNA/PylRS
pair; eight of them are reported here for the first time. One
azobenzene-containing protein, sfGFP−4h, showed durable
photoswitching upon photoirradiation with alternating green-
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Organic Letters
Letter
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specific tRNA/PylRS pairs, should greatly enhance our
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b03268.
Supplemental figures, experimental procedure, and
characterization of all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: qinglin@buffalo.edu.
Present Address
†Zhejiang University, College of Pharmaceutical Sciences,
China.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Institutes of Health
(GM 85092) for financial support. We thank Prof. Wenshe Liu
at Texas A&M University for providing the pEvol-MmPylRS
and pET-sfGFPS2TAG plasmids and Tracey Lewandowski in
the Q.L. lab at SUNY Buffalo for assistance with cloning.
REFERENCES
■
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DOI: 10.1021/acs.orglett.5b03268
Org. Lett. XXXX, XXX, XXX−XXX