A fluorogenic 'click' reaction of azidoanthracene derivatives

Article abstract of DOI:10.1016/j.tet.2008.01.080

Fluorogenic reactions have broad applications in biolabeling, combinatorial synthesis of fluorescent dyes, and materials development. It was recently reported that the highly selective and efficient Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) react

Full text of DOI:10.1016/j.tet.2008.01.080

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 2906e2914
www.elsevier.com/locate/tet
A fluorogenic ‘click’ reaction of azidoanthracene derivatives
Fang Xie, Krishnamoorthy Sivakumar, Qingbing Zeng, Michael A. Bruckman,
*
Blake Hodges, Qian Wang
Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, SC, 29208, USA
Received 4 December 2007; received in revised form 18 January 2008; accepted 18 January 2008
Available online 25 January 2008
Abstract
Fluorogenic reactions have broad applications in biolabeling, combinatorial synthesis of fluorescent dyes, and materials development. It was
recently reported that the highly selective and efficient Cu(I)-catalyzed azideealkyne cycloaddition (CuAAC) reaction can be employed in
designing new types of fluorogenic reactions. In this study, we report a fluorogenic reaction using anthracene azides as starting materials.
The fluorescence of the anthryl core can be greatly inhibited upon introducing electron-donating azido groups in the proximity. Such weakly
fluorescent anthracene azides demonstrate high reactivity with a variety of alkynes under the CuAAC conditions producing a strongly fluorescent
triazole product with high quantum yields. This reaction can be used in the synthesis and screening of fluorescent dyes combinatorially.
Compared with most existing methods, the fluorogenic CuAAC reaction is a much milder and simpler technique to prepare large libraries
of fluorescent dyes without further purification. In order to demonstrate the efficiency of using anthracene azides for biolabeling applications,
both small molecules and biomolecules including the multialkyne-derivatized cowpea mosaic virus and tobacco mosaic virus had been
studied.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
CuAAC reactions have been reported.7,12e15 A representative
fluorogenic dye developed in our laboratory, 3-azido-7-hy-
droxycoumarin, has been successfully used for the labeling
and visualization of synthesized proteins in different cells
and other polymeric systems via the CuAAC reaction.8,16
Compared to the coumarin fluorophore, some anthracene
derivatives have much higher quantum yields, e.g., 9,10-diphe-
nylanthracene is used as a fluorescent standard in quantum
yield determination. In addition, photoinduced electron trans-
fer (PET) process of anthracenes has been widely applied in
ion sensing,17 singlet oxygen detection,18 screening catalysts,19
molecular logic gate construction,20 and cell labeling by rec-
ognition of carbohydrates.21 We therefore exploit the possibil-
ity to design a new type of fluorogenic reaction based on the
PET process of anthracenes. Due to the high electron density
of the a-nitrogen of azido group,13,14 it was envisioned that the
introduction of the azido group close to the anthryl core via a
non-conjugated linker would lead to a favorable electron trans-
fer from the azido to the excited anthryl core and induce the
quenching of fluorescence. After the CuAAC reaction, the
lone pair electrons on nitrogen will become part of the aro-
matic system and become a much weaker electron donor.
Incorporation of exogenous natural or unnatural tags into
proteins and glycans by cellular biosynthetic pathways is an
emerging strategy for investigating their cellular activities.1e3
Since these processes involve multistep enzymatic transfor-
mations that prohibit the incorporation of large signaling
moieties, chemoselective reactions are often employed for post-
labeling.1,3e6 In this case, a bioorthogonal fluorogenic reaction
is invaluable, in which the unreacted reagents show no fluores-
cent background and the purification can be avoided.5,7,8
Being a highly energetic functional group, the organic
azide is stable and unreactive with most biomolecules under
physiological conditions. Recently, it has been employed as
a tag for sequential bioorthogonal labeling via the Bertozzie
Staudinger ligation or the Cu(I)-catalyzed azideealkyne
cycloaddition (CuAAC) reaction.4,6,9e11 Consequently, the
fluorogenic versions of BertozzieStaudinger ligation and
* Corresponding author. Tel.: þ1 803 777 8436; fax: þ1 803 777 9521.
E-mail address: wang@mail.chem.sc.edu (Q. Wang).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.080

F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
2907
Therefore, the fluorescence of the anthryl core will be remedi-
ated resulting in a fluorogenic phenomenon. In this paper, we
report the synthesis of five novel azido derivatized anthracenes
and applied the CuAAC reaction to activate their fluorescence.
This method was applied to label multiple alkyne organic
molecules and large alkyne functionalized biological
macromolecules.
exhibited high reactivity with a variety of alkynes regardless
of the structures under standard CuAAC condition (see Table
1).22,23 A comparison between the maximum emission inten-
sity of the cycloaddition products A1eE1 and that of the cor-
responding azides is shown in Figure 2. The fluorescent
emission of all triazole products is enhanced dramatically
compared to the starting azides except C, which shows a no-
ticeable fluorescence and a bathochromic shift of fluorescence
due to the introduction of 9-cyano group (Fig. S1).
2. Results and discussion
Since the CuAAC reaction can tolerate a great diversity of
the structural features of azides and alkynes, it is possible to
screen the fluorescent properties of the cycloaddition products
in relation to the starting alkynes combinatorially. In our study,
5 different anthracene azides AeE and 34 terminal alkynes 2e
35 (Table 2) were loaded in microtiter plates. Each well thus
represented a unique combination of anthracene and alkyne.
The total volume of each reaction mixture was 200 mL
(DMSOeH2O¼1:1) containing 1 mM azide and alkyne cata-
lyzed with 2 mM CuSO4 and 5 mM NaAsc. Most reactions
were completed in 24 h at room temperature as monitored
by TLC or mass spectrometry.
The formation of the fluorescent triazole products is easily
visualized upon irradiation at 365 nm with a hand-held UV
lamp. Without purification the emission of reaction mixtures
was directly evaluated using a Varian Eclipse fluoriphotome-
ter. As shown in Figure 3 and Table S1 in Supplementary
data, most triazole products of azides A and B had distinct en-
hancement in fluorescent intensity regardless of the structures
of alkynes. Although some factors like reaction yield and co-
ordination with Cu2þ or Cuþ ion would influence the emission
intensity of products,17 we still can get useful information on
the fluorescent properties of the final products.
Compound A was first synthesized and then reacted with
phenylacetylene (1) as shown in Scheme 1.17 The cycloaddi-
tion reaction was performed using catalytic amounts of copper
sulfate and sodium ascorbate (NaAsc) in a solution of DMF
and water (v/v¼1:1).13,22 At the same concentration in pure
DMSO, while the triazolyl product A1 shows almost the
same absorption intensity as A, its fluorescent emission inten-
sity is 75-fold stronger than that of A (Fig. 1). The quantum
yield of A1 is 0.96, much higher than that of A (w0.02).
Moreover, it should be noticed that there is no shift in emis-
sion and excitation wavelength accompanying the change of
fluorescence intensity. All these results indicate a PET process
between azido group and anthryl core that can be adopted for
designing a new type of fluorogenic CuAAC reaction.
In order to examine the scope of the fluorogenic CuAAC
reaction of anthracene derivatives, four more anthracene
azides (BeE) were synthesized via easy transformations
(Scheme 2). Azido groups were attached to the anthracene
core at 1-, 2- or 9-position. In all cases, the fluorescence
was quenched efficiently. All azides are quite stable and no de-
composition has been found after storing on the shelf or re-
fluxing in toluene for a long period of time. Each azide
CuSO4/NaAsc
NaN3
DMF
DMF:H2O = 1:1
N
Cl
N3
N
N
A
1
A1
Scheme 1. Synthesis of azide A and product A1.
Figure 1. Comparison of fluorescent emission (left, excited at 370 nm) and absorption spectra (right) of A and A1 in DMSO (10 mM for emission spectra and
50 mM for absorption spectra).

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F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
OH
N3
OH
N3
1. MsCl, Et3N
CH2Cl2/CH3CN
NaBH4
Ethanol
83%
2. NaN3,DMF
56%
CHO
2b
CHO
1b
B
OAc
N3
1. K2CO3, MeOH/H2O
2. MsCl, Et3N, CH2Cl2
1. H2NOH.HCl
95% EtOH, reflux
2. Ac2O, reflux
74%
3. NaN3, DMF
30%
CN
1c
CN
C
HO
N3
COOH
Et2O
1. MsCl, Et3N, CH2Cl2
2. NaN3, DMF
73%
LiAlH4
85%
D
1d
2d
NH2
N3
NaNO2, H2SO4, NaN3
51%
E
1e
Scheme 2. Synthesis of anthracene azides BeE.
Since CuAAC reaction can tolerate a variety of solvents
(including water), pH ranges, and be compatible to most of
the functionalities, azide and alkyne groups have been broadly
used as biocompatible/chemocompatible tags for biomacro-
molecules and polymers. The fluorogenic CuAAC reaction re-
ported here can therefore be used in the post-labeling of
polyvalent alkyne displayed systems, in particular, in a biolog-
ical environment.
a rod-like particle consisting of 2130 identical protein subunits
arranged helically around a single RNA strand.31 TMV is
initially subjected to an electrophilic substitution reaction at
the ortho-position of the phenol ring of tyrosine-139 residues
with diazonium salts to insert the alkyne functionality.32
MALDI-TOF MS analysis indicated that >95% of the capsid
monomers were converted into alkyne derivatives (Fig. 5A).
The sequential CuAAC reaction is achieved with greatest effi-
ciency using a combination of CuSO4 with NaAsc (Scheme 3).33
The efficiency of the reaction is easily monitored using
MALDI-TOF MS (Fig. 5A) with shift in mass from 17,664
m/z (TMVeAlkyne) to 17,918 m/z (TMVeAnthracene)
and UVevis absorption in the 380e420 nm range (Fig. 5C).
Due to the often destructive nature of organic reactions on
viral particles, the stability of TMV was monitored by TEM
analysis (Fig. 5B) in conjunction with size-exclusion chroma-
tography (Fig. S9) and was found to remain intact and stable
throughout the reaction.33
We chose trispropynyloxybenzene (37), alkyne-derivatized
cowpea mosaic virus (CPMV), and tobacco mosaic virus
(TMV), which have 3, 60, and 2130 terminal alkyne units, re-
spectively, as multivalent model systems to demonstrate the
feasibility of this selective post-labeling strategy. Catalyzed
with in situ generated Cu(I), 37 can be fluorescently derivat-
ized by A in high efficiency (Scheme 3). As previously re-
ported, CPMV has been employed as a nano building block
for many organic reactions.24e28 N-Hydroxysuccinimidal ester
of 34 was synthesized and used to attach terminal alkynes to
the reactive lysines of CPMV.29 The newly synthesized
CPMVeAlkyne was sequentially subjected to the CuAAC re-
action with B catalyzed through the addition of CuBr and
a bathophenanthroline ligand (Scheme 3).30 Gel electrophore-
sis confirmed the covalent attachment of fluorescent anthra-
cene units on CPMV after cycloaddition reaction (Fig. 4A).
The integrity of the CPMV products was confirmed by
TEM and size-exclusion chromatography (Fig. 4B and C).
Due to the strong fluorescence of the cycloaddition product
(the quantum yield of the model compound B34 is 0.51 as
compared to azide B F¼0.02), as low as 0.5 nM of the cyclo-
addition product could be detected without the interference of
starting materials.
3. Conclusions
In summary, we report here a new bioorthogonal fluoro-
genic reaction based on the PET principle, which affords an
efficient way to link two entities together while the conjugat-
ing efficiencies could be reported by the fluorescent emission
assays. It should have broad application in the emerging field
of cell biology and functional proteomics due to the high reac-
tion efficiency of CuAAC reaction at mild reaction conditions
and the distinct fluorescence properties of products. The high
reactivity and selectivity between azides and terminal alkynes
allow further applications in ligating other biomolecules, poly-
mers, nanoparticles and surfaces, which are under exploration.
In addition, since it is generally difficult to construct the
Similarly, the versatility of this fluorogenic reaction is dem-
onstrated via the CuAAC reaction on the surface of TMV,

F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
2909
Table 1
CuAAC reactions of anthracene azides AeE with some typical alkynes
Entry Azide
Alkyne
Product
Yielda Entry Azide
(%)
Alkyne
Product
Yielda
(%)
N
N
N
N
C6H13
N
HO
N
1
2
3
4
5
6
A
A
A
A
A
B
95b
66
71
86
92
69
9
10
11
12
13
14
B
C
D
D
D
D
93c
81
97
93
97
60
A1
B26
N
N
N
N
N
N
Cl
A7
NC
Cl
C1
N
N
N
N
N
N
A17
D1
N
C6H13
O
N
N
N
N
O
N
A26
D11
HO
N
N
N
HO
N
C6H13
D26
N
N
A36
N
N
N
HO
HO
HO
HO
N
N
N
B1
D36
N
N
N
N
N
N
7
8
B
B
63c
15
16
E
E
66
67
E1
B3
N
CF3
N
N
N
OH
N
N
HO
79c
HO
F3C
E27
B14
a
b
c
Isolated yields.
Quantum yields of azide A and the product A1 are 0.02 and 0.97.
Quantum yields: B 0.02, B3 0.95, B14 0.95, and B26 0.97.
highly conjugated fluorophore, only a few reactions have been
applied in the combinatorial synthesis of fluorescent com-
pounds including condensation reactions using aldehydes
and transition metal catalyzed coupling reactions.34e39 The
CuAAC reaction distinguishes from others by its mild reaction
conditions and high transformation efficiency regardless of
the structures of azides and alkynes, which make it an ideal re-
action to synthesize fluorescent dyes in a combinatorial
manner.
4. Experimental section
4.1. General methods
Unless otherwise noted, all chemicals and solvents were
obtained from commercial suppliers and used without purifica-
1
tion. H NMR spectra were recorded on Varian 300 NMR
spectrometer and 13C NMR spectra were recorded either on
Varian 300 NMR or on Varian 400 NMR spectrometer. The

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1000
F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
ZipTip-C18Ò tips to remove the salts before the MS analysis.
900
800
700
600
500
400
300
200
100
0
The quantum yields of anthracene derivatives were deter-
mined using a literature method.40
4.2. Synthesis of azides
The preparation of A,17,41 1b,42 and 1d43 has previously
been described.
A
A1
B
B1
C
C1
D
D1
E E1
4.2.1. 10-Azidomethylanthracene-9-carbaldehyde (2b)
To the stirred suspension of 1b (5.22 g, 22 mmol) in
200 mL dry CH2Cl2 and 200 mL dry acetonitrile under nitro-
gen at 0 ꢀC were added dry triethylamine (3.35 g, 33 mmol)
and methanesulfonyl chloride (3.15 g, 27.5 mmol). The mix-
ture was stirred at room temperature for 4 h under nitrogen
before an aq HCl (1 N, 70 mL) was added to the resulting
solution. The organic layer was washed by water (50 mLꢁ2)
and brine, and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure and the residue was dried
under vacuum. The resulted solid was dissolved in DMF
(250 mL), and NaN3 (2.1 g, 33 mmol) was added. The mixture
was stirred at room temperature overnight. Water (200 mL)
and ethyl acetate (EtOAc, 200 mL) were added. The aqueous
layer was extracted by EtOAc (50 mLꢁ2) and the combined
organic layers were washed with brine and dried over anhy-
drous Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by flash chromatography
on silica gel (EtOAcehexane¼1:9 to 1:4) to give 2b as a yel-
Figure 2. The comparison between the maximum emission intensity of anthra-
cene azides (AeE) and their respective product with alkyne 1. All samples
were measured in DMSO solution ([c]¼10 mM). The original spectra are given
in Supplementary data.
UVevis absorption spectra were measured on Agilent 8453
spectrometer. Emission spectra were measured on Varian
Cary Eclipse fluorescence spectrophotometer. Ultracentrifuga-
tion was performed at the indicated speeds using a Beckman
OptimaÔ L-90K Ultracentrifuge equipped with either SW41
or 50.2 Ti rotors. TEM analyses of viruses were carried out
by depositing 20 mL aliquots of each sample at a concentra-
tion of 0.1e0.3 mg/mL onto 100-mesh carbon-coated copper
grids for 2 min. The grids were then stained with 20 mL of
2% uranyl acetate and viewed with a Hitachi H-8000 TEM
electron microscope. MALDI-TOF MS analyses of viral sub-
unit were carried out using a Bruker Ultra-Flex I TOF/TOF
mass spectrometer with MS grade sinapinic acid in 70% ace-
tonitrile and 0.1% TFA as the matrix. In general, the viruses
were denatured after being treated with guanidinium-HCl
(6.0 M, 4 mL) for 5 min at room temperature. The denatured
protein was spotted onto a MALDI plate using Millipore
1
low solid (3.1 g, 56%). H NMR (300 MHz, CDCl3): d¼11.5
(s, 1H), 8.92e8.88 (m, 2H), 8.41e8.38 (m, 2H), 7.74e7.65
(m, 4H), 5.37 (s, 2H); 13C NMR (75 MHz, CDCl3) d¼194.0,
133.7, 131.2, 130.4, 128.6, 127.7, 127.2, 124.6, 124.5, 46.6.
Table 2
The alkyne building blocks used in this study
F
H2N
F
Cl
F
Br
1
3
2
4
5
9
6
7
8
F
HO
O
N
F3C
F3C
C5H11
O
17
15
13
16
10
11
14
12
N
HO
CF3
S
N
N
H2N
N
N
N
19
21
18
22
20
23
24
OH
OH
OH
HO
HO
25
26
27
29
30
28
O
O
HO
O
O
Cl
HN
OH
HN
OH
HO
36
34
31
32
33
35

F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
2911
Figure 3. Schematic demonstration of the well plate reactions between azidoanthracenes (AeE) and alkynes (2e35). Each cell’s color represents the emission
property of every compound based on the values of Hue, Sat and Lum in an HSL color model. Hue value represents the maximum emission wavelength of
each compound and Lum value represents the maximum emission intensity (more details could be found in Supplementary data).
4.2.2. (10-Azidomethylanthracen-9-yl)methanol (B)
3H); 13C NMR (75 MHz, DMSO) d¼135.6, 130.8, 130.2,
127.5, 126.9, 126.3, 125.2, 56.2, 46.3. HRMS calculated for
C16H13N3O (Mþ): 263.1059, found: 263.1055.
NaBH4 was added in small portions to the suspension of 2b
(360 mg, 1.38 mmol) in 50 mL ethanol at 0 ꢀC. The reaction
mixture was stirred at room temperature for 8 h. Concentrated
HCl solution was added dropwise carefully to destroy the un-
reacted NaBH4 and then water was added until pH was neu-
tral. Dichloromethane (150 mL) was added and the organic
layer was separated, washed with brine, and dried over anhy-
drous Na2SO4. The solvents were removed under reduced
pressure and the residue was purified by flash chromatography
on silica gel (EtOAcehexane¼1:4) to give B as a yellow solid
4.2.3. Acetic acid (10-cyanoanthracen-9-yl)methyl ester (1c)
To a suspension of 1b (0.81 g, 3.43 mmol) in 19 mL of 95%
EtOH (19 mL) was added hydroxylamine hydrochloride
(280 mg, 4.02 mmol, neutralized with Na2CO3) in 4 mL of
water. The mixture was heated on a steam bath for 30 min,
cooled, and diluted with water. The solid was collected, dried
under low pressure, and then acetic anhydride (16 mL) was
added. The resulting solution was refluxed for 8 h and then
added ice water. Yellow precipitate was collected by filtration,
1
(300 mg, 83%). H NMR (300 MHz, DMSO): d¼8.57e8.47
(m, 4H), 7.67e7.60 (m, 4H), 5.52 (s, 2H), 5.49e5.41 (m,
Scheme 3. Reaction conditions: (i) A, CuSO4, NaAsc, DMFeH2O¼1:1, 24 h; (ii) B, CuBr, bathophenanthroline, pH 8.5 buffereDMF¼4:1, 24 h; (iii) B, CuSO4,
NaAsc, pH 8.5 buffereDMF¼4:1, 24 h.

2912
F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
with vigorous stirring. The mixture was then stirred at room
A
B
temperature under nitrogen for 30 min and refluxed for addi-
tional 8 h. The reaction was quenched with water (10 mL)
and concentrated aq HCl (20 mL). The aqueous solution was
separated and extracted by EtOAc (50 mLꢁ2). The combined
organic solution was washed with aq HCl (1 N, 20 mL), satu-
rated aq NaHCO3 (20 mL), and H2O (20 mL), and dried on an-
1
hydrous Na2SO4 to yield crude product 2d (3.2 g, 85%). H
NMR (300 MHz, DMSO): d¼8.69 (s, 1H), 8.58 (s, 1H), 8.13e
8.05 (m, 2H), 8.00 (d, J¼8.4 Hz, 1H), 7.57e7.45 (m, 4H),
5.46 (t, J¼5.1 Hz, 1H), 5.12 (d, J¼5.1 Hz, 2H); 13C NMR
(75 MHz, DMSO): d¼138.5, 132.1, 131.7, 131.5, 129.7,
129.1, 128.5, 128.2, 127.2, 126.3, 126.2, 125.8, 124.1, 122.9,
62.0. HRMS calculated for C15H12O (Mþ): 208.0888, found:
208.0889.
500
400
300
200
100
0
C
Intact Particles
260 nm
280 nm
370 nm
4.2.6. 1-Azidomethylanthracene (D)
The similar procedure of preparation of 2b was followed to
1
prepare D as a yellow solid (yield 73%). H NMR (300 MHz,
CDCl3): d¼8.57 (s, 1H), 8.47 (s, 1H), 8.08e7.99 (m, 3H),
7.54e7.40 (m, 4H), 4.88 (s, 2H); 13C NMR (75 MHz, CDCl3):
d¼132.2, 131.9, 131.2, 130.1, 129.7, 128.8, 128.2, 127.6,
127.1, 126.1, 126.0, 124.7, 122.6, 53.7. HRMS calculated
for C16H15N3 (Mþ): 233.0953, found: 233.0945.
Broken Particles
0
10
Eluting Volume (mL)
20
30
4.2.7. 2-Azidoanthracene (E)
Figure 4. (A) SDS-PAGE of CPMVeAnthracene visualized under UV irradi-
ation (left) and upon staining with Coomassie Blue (right). (B) TEM image of
CPMVeAnthracene. The scale bar is 100 nm. (C) Size-exclusion FPLC
analysis of CPMVeAnthracene.
A solution of sodium nitrite (220 mg, 3.13 mmol) in water
(8 mL) was added dropwise to a solution of 1e (450 mg,
2.45 mmol) in water (15 mL) and concentrated sulfuric acid
(3 mL) at 0 ꢀC over 5 min. The reaction mixture was stirred
at 0 ꢀC for 30 min before a solution of sodium azide (0.3 g,
4.4 mmol) in water (5 mL) was added dropwise over 10 min.
The solution was slowly warmed to room temperature and
kept stirring for 5 h. The precipitate was isolated by filtration,
washed with water, and purified by column chromatography
on a silica column to yield a yellow solid E (260 mg, 51%).
1H NMR (300 MHz, CDCl3): d¼8.40 (s, 1H), 8.32 (s, 1H),
8.02e7.96 (m, 3H), 7.59 (m, 1H), 7.63e7.42 (m, 2H), 7.17e
7.13 (dd, J¼9.0, 2.4 Hz, 1H); 13C NMR (100 MHz, DMSO):
d¼136.4, 131.8, 131.3, 130.8, 128.9, 128.2, 127.8, 126.4,
126.1, 125.5, 124.9, 119.4, 115.0. HRMS calculated for
C14H9N3 (Mþ): 219.0796, found: 219.0799.
washed with water, and finally purified by flash chromatogra-
phy on silica gel (EtOAcehexane¼1:9 to 1:4) to give 1c as
a light yellow solid (0.70 g, 74%). 1H NMR (300 MHz,
CDCl3): d¼8.52e8.43 (dm, J¼7.2 Hz, 4H), 7.78e7.67 (m,
4H), 6.16 (s, 2H), 2.09 (s, 3H); 13C NMR (75 MHz, DMSO)
d¼171.1, 133.4, 133.1, 130.4, 128.8, 127.7, 126.4, 125.1,
117.3, 108.3, 58.4, 21.1.
4.2.4. 10-Azidomethylanthracene-9-carbonitrile (C)
To the stirred suspension of 1c (220 mg, 0.8 mmol) in
methanol (40 mL) and water (10 mL) was added potassium
carbonate (237 mg, 60 mmol). The mixture was stirred at
room temperature overnight before EtOAc (100 mL) was
added into the reaction mixture. The organic layer was washed
by aq HCl (1.0 N, 50 mL) and brine, and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure and
the residue was dried under vacuum. A similar procedure of
preparation of B was followed to yield C as a yellow solid
(60 mg, overall yield 30% from 1c). 1H NMR (300 MHz,
CDCl3): d¼8.53 (dm, J¼7.8 Hz, 2H), 8.39 (dm, J¼7.8 Hz, 2H),
7.80e7.69 (m, 4H), 5.37 (s, 2H); 13C NMR (75 MHz, CDCl3):
d¼133.2, 133.0, 130.1, 128.9, 127.9, 126.7, 124.6, 46.4. HRMS
calculated for C16H10N4 (Mþ): 258.0905, found: 258.0908.
4.3. Typical experimental procedure for preparative scale
CuAAC reactions
Azide A (69 mg, 0.30 mmol) and phenylacetylene (31 mg,
0.30 mmol) were suspended in a 3:1 mixture (v/v) of DMF and
water (24 mL). Copper sulfate (7 mg, 0.045 mmol), sodium
ascorbate (14 mg, 0.09 mmol), and tris[(N-benzyltriazolyl)me-
thyl]amine ligand9 (8 mg, 0.015 mmol) were added. The mix-
ture was then stirred at room temperature for 16e24 h
(monitored by TLC). After the reaction was complete, EtOAc
(20 mLꢁ2) was added to extract the product. The combined
organic layers were washed with brine and dried over anhy-
drous Na2SO4. The solvent was removed under reduced pres-
sure and the residue was purified by flash chromatography on
4.2.5. Anthracen-1-ylmethanol (2d)
To the dispersion of 1d (4.0 g, 18 mmol) in dry ether
(250 mL), LiAlH4 (0.9 g, 26 mmol) was added in portions

F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
2913
B
A
17918 (17925)
TMV-Anthracene
17664 (17662)
TMV-Alkyne
C
1.0
0.8
0.6
0.4
0.2
0.0
TMV-Anthracene
TMV
17534
TMV
16000
17000
18000
19000
250
300
350
400
450
Wavelength (nm)
500
550
600
Figure 5. (A) MALDI-TOF MS data of TMV, TMVeAlkyne, and TMVeAnthracene indicating >95% reactivity per subunit. (Numbers in parenthesis refer to
the expected masses.) (B) TEM image of TMV post-CuAAC functionalization with anthracene. The scale bar is 200 nm. (C) UVevis spectra of TMVand TMVe
Anthracene.
silica gel (EtOAcehexane¼1:9 to 1:4) to yield compound A1
(m, 6H), 8.10 (d, J¼8.7 Hz, 4H), 8.05 (d, J¼8.4 Hz, 2H),
7.99 (s, 2H), 7.84 (s, 1H), 7.60e7.43 (m, 12H), 6.75 (t, J¼
7.8 Hz, 1H), 6.59e6.56 (m, 6H), 6.47 (s, 2H), 4.78 (s, 4H),
4.70 (s, 2H); 13C NMR (100 MHz, DMSO): d¼152.5, 144.1,
143.3, 137.4, 131.7, 131.6, 131.0, 130.9, 129.7, 129.6, 127.8,
127.7, 126.5, 126.4, 126.0, 125.9, 124.8, 124.7, 124.6,
124.2, 108.2, 65.9, 62.4, 46.1, 45.9. HRMS calculated for
C60H45N9O3 (Mþ): 940.3723, found: 940.3707.
as a yellow solid (0.095 g, 95%).
4.4. Typical experimental procedure of combinatorial
CuAAC reactions on a 96-microwell plate
The reaction mixture in each well contained a solution of
anthracene azides (1.0 mM), alkynes (1.0 mM), copper sulfate
(2.0 mM), sodium ascorbate (10.0 mM), and tris[(N-benzyl-
triazolyl)methyl]amine ligand (0.1 mM) in 200 mL DMSOe
water (v/v¼1:1). For each of the five anthracene azides one
control test was performed, which contained all the above
components of the reaction except the alkyne. The reactions
including the control test were allowed to incubate at room
temperature for 24 h. Then the mixture in each well was di-
luted 100 times before the fluorescence intensity was mea-
sured accordingly at proper excitation wavelength. The
excitation wavelength was taken as the maximum of the
band in the UV spectrum.
4.6. Labeling of alkyne-derivatized CPMV with azide B
Multialkyne-derivatized CPMV was synthesized according
to the literature protocol.9 A mixture of CPMVeAlkyne
(4 mg/mL), bathophenanthroline ligand (4 mM), CuBr
(1 mM), and anthracene azide B (100 mM) in HEPES buffer
(pH¼8.5) solution (containing 20% DMF) was incubated at
4 ꢀC for 24 h with nitrogen. CPMVeAnthracene was then
purified by a sucrose gradient ultra-sedimentation. TEM analy-
sis was performed by depositing 20 mL of purified sample onto
100-mesh carbon-coated grids. After waiting for 2 min, thegrids
were stained with uranyl acetate and viewed with Hitachi-8000
electron microscope. FPLC analysis was performed with AKTA
Explorere (Amersham Pharmacia Biotech) equipment, using
SuperoseÔ-6 size-exclusion columns. SDS-PAGE analysis
was carried out in Bio-Rad MiniPROTEANÒ 3 gel electropho-
resis cell. CPMV (1 mg) was denatured by heating at 95 ꢀC for
4.5. Multivalent CuAAC reactions of azidoanthracenesd
reaction of alkyne 37 with azide A
By following the protocol of preparative scale CuAAC re-
1
actions, 38 was obtained as a yellow solid (85%). H NMR
(300 MHz, DMSO): d¼8.68 (s, 2H), 8.61 (s, 1H), 8.57e8.50

2914
F. Xie et al. / Tetrahedron 64 (2008) 2906e2914
11. Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125,
4686e4687.
5 min with TriseHCl buffer containing b-mercaptoethanol, bro-
mophenolblue, and glycerol. The proteins were then resolved on
a 15% polyacrylamide gel at 200 V for 1 h. CPMVeAnthra-
cene was visualized with a UVP Epi Chemi II imager under
UV irradiation before staining. For analysis by coomassie blue
staining, the gel was stained with Bio-Rad BiosafeÒ Coomassie
Blue for 1 h and destained with distilled water.
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4.7. Labeling of TMV with azide B
TMVeAlkyne was prepared using reported procedure.33
Anthracene azide B in a 20% DMSO aqueous solution
(100 mM, 40 mL) and TMVeAlkyne (15 mg/mL, 200 mL)
were mixed in Tris buffer (10 mM, pH 8.0, 660 mL). Then
CuSO4 (100 mM, 10 mL) and sodium ascorbate (200 mM,
10 mL) were added before the mixture was incubated at room
temperature for 18 h. The reaction mixture was then purified
via an ultra-sedimentation on a 10e50% sucrose gradient col-
umn. The collected modified TMV was then pelleted by ultra-
centrifugation at 160,000g for 2.5 h. The pellet was dissolved in
0.01 M PBS buffer and characterized.
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Acknowledgements
24. Lee, L. A.; Wang, Q. Nanomedicine: Nanotechnology, Biology, and
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This work was partially supported by the South Carolina
EPSCoR/CRP program, the W. M. Keck Foundation, and the
NSF-CAREER Program.
27. Wang, Q.; Kaltgrad, E.; Lin, T.; Johnson, J. E.; Finn, M. G. Chem. Biol.
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Supplementary data
28. Wang, Q.; Raja, K. S.; Janda, K. D.; Lin, T.; Finn, M. G. Bioconjugate
Chem. 2003, 14, 38e43.
1H and 13C NMR of all the triazole products, the absorption
and emission spectra of compounds in Table 1; the optical data
of all products from 96-well plate reaction; and the details of
making Figure 3. This material is available free of charge via
the internet. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/j.tet.2008.
01.080.
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