Solid-phase synthesis of azaphthalocyanine-oligonucleotide conjugates and their evaluation as new dark quenchers of fluorescence

Article abstract of DOI:10.1021/bc100226x

Hydrophobic nonaggregating metal-free azaphthalocyanines (AzaPc) of the tetrapyrazinoporphyrazine type were synthesized, characterized, and used for oligonucleotide labeling. Both 3′-end and 5′-end labeling methods using solid phase synthesis suitable for automatic processes in the DNA/RNA synthesizer were developed. The hydrophobic character of AzaPc enabled the anchoring of the conjugates on reverse phase of the oligonucleotide purification cartridge, thus enabling their simple purification. AzaPc did not show any fluorescence and extremely low singlet oxygen quantum yields (Φ_Δ = 0.015-0.018 in DMF) in a monomeric state due to ultrafast intramolecular charge transfer. That is why they were investigated as a new dark quencher structural type. They profit particularly from absorption in a wide range of wavelengths (300-740 nm) that covers all fluorophores used in hybridization assays nowadays. As an example, quenching efficiency was evaluated in a simple hybridization assay using monolabeled probes. AzaPc-based probes efficiently quenched both fluorescein and Cy5 fluorescence by both resonance energy transfer and contact quenching. The results were compared with three established dark quenchers, and the AzaPc exerted better (BHQ-1 and BHQ-2) or comparable (BBQ-650) quenching efficiencies for both fluorophores.

Full text of DOI:10.1021/bc100226x

1872
Bioconjugate Chem. 2010, 21, 1872–1879
Solid-Phase Synthesis of Azaphthalocyanine-Oligonucleotide Conjugates
and Their Evaluation As New Dark Quenchers of Fluorescence
Kamil Kopecky, Veronika Novakova, Miroslav Miletin, Radim Kucˇera, and Petr Zimcik*
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in
Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic. Received May 14, 2010; Revised Manuscript Received
August 30, 2010
Hydrophobic nonaggregating metal-free azaphthalocyanines (AzaPc) of the tetrapyrazinoporphyrazine type were
synthesized, characterized, and used for oligonucleotide labeling. Both 3′-end and 5′-end labeling methods using
solid phase synthesis suitable for automatic processes in the DNA/RNA synthesizer were developed. The
hydrophobic character of AzaPc enabled the anchoring of the conjugates on reverse phase of the oligonucleotide
purification cartridge, thus enabling their simple purification. AzaPc did not show any fluorescence and extremely
low singlet oxygen quantum yields (Φ∆ ) 0.015-0.018 in DMF) in a monomeric state due to ultrafast
intramolecular charge transfer. That is why they were investigated as a new dark quencher structural type. They
profit particularly from absorption in a wide range of wavelengths (300-740 nm) that covers all fluorophores
used in hybridization assays nowadays. As an example, quenching efficiency was evaluated in a simple hybridization
assay using monolabeled probes. AzaPc-based probes efficiently quenched both fluorescein and Cy5 fluorescence
by both resonance energy transfer and contact quenching. The results were compared with three established dark
quenchers, and the AzaPc exerted better (BHQ-1 and BHQ-2) or comparable (BBQ-650) quenching efficiencies
for both fluorophores.
otide conjugates, and several new Pc-based near-infrared
emitting fluorophores for DNA-hybridizations assays were
developed (6, 7). The use of Pc-oligonucleotide conjugates for
a specific DNA catalytic or photosensitized modification was
also described (8, 9). The conjugation of Pc with the oligo-
nucleotide is usually performed in a solution that requires the
water solubility of the Pc moiety and subsequent extensive
purification involving HPLC. The Pc-oligonucleotide labeling
approaches tested until this time cover isothiocyanate (5) or
succinimidyl methods (6, 10, 11), Huisgen cycloaddition (12),
and reductive amination (12).
During our studies of AzaPc, we systematically observed that
alkylamino substituted derivatives even in the strictly monomeric
form are lacking any fluorescent or photosensitizing properties
typical for other AzaPc derivatives (13). Recently, we discovered
and fully explained that ultrafast intramolecular charge transfer
(ICT) is responsible for the quenching of the S1 excited state
of such AzaPc (14). This unique mechanism of S1 state
deactivation, not yet observed at Pc derivatives, is responsible
for the lack of fluorescence. These properties, together with
strong absorption over a wide range of wavelengths, make
alkylamino AzaPc ideal candidates for the modern dark quench-
ers. In order to confirm the hypothesis, suitable AzaPc were
synthesized, simple solid-phase labeling of oligonucleotide probe
was developed, and the conjugates were subjected to hybridiza-
tion assays with two commonly used fluorophores.
INTRODUCTION
Modification of oligonucleotides with dyes is a commonly
used procedure in genomic assays, e.g., in the monitoring of
real-time polymerase chain reaction (PCR1). Most of the
hybridization assays are based on the combination of a fluoro-
phore with a quencher leading to the decrease of the fluorophore
emission after detection of the target (1). The dark quenchers
are a modern type of quenchers with no intrinsic fluorescence,
which substantially simplifies the hybridization assays and
increases their sensitivity. However, the currently used dark
quenchers are based on two or three structural types, and they
are often suitable only for a restricted number of fluorescent
labels due to a limited spectral overlap. Therefore, the develop-
ment of new highly efficient and versatile dark quenchers is
desirable.
Phthalocyanines (Pc) and their aza-analogues azaphthalocya-
nines (AzaPc) are long known organic dyes with application in
various areas (2). Despite deep investigations in Pc and AzaPc
chemistry and applications, Pc conjugates with oligonucleotides
were described only recently (3-5); furthermore, those using
AzaPc have not been reported at all. For example, strong
fluorescent properties of Pc were appreciated in Pc-oligonucle-
* To whom correspondence should be addressed. Tel: +420
495067257. Fax: +420 495067167. E-mail: petr.zimcik@faf.cuni.cz.
1Abbreviations: AzaPc, azaphthalocyanine; BBQ, BlackBerry Quench-
er; BHQ, Black Hole Quencher; CPG, controlled pore glass, DCC, N,N′-
dicyclohexylcarbodiimide; DMF, dimethylformamide; DMSO, dime-
thylsulfoxide, DMTr, 4,4′-dimethoxytrityl; DMTrCl, 4,4′-dimethoxytrityl
chloride; FAM, fluorescein; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; ICT, intramolecular charge
transfer; lcaa, long chain aminoalkyl; MALDI-TOF, matrix-assisted
laser desorption/ionization time-of-flight; MeOH, methanol; NHS,
N-hydroxysuccinimide; OPC, oligonucleotide purification cartridge; Pc,
phthalocyanine; PCR, polymerase chain reaction; RET, resonance
energy transfer; TFA, trifluoroacetic acid; THF, tetrahydrofuran; ZnPc,
zinc phthalocyanine.
EXPERIMENTAL PROCEDURES
All organic solvents were of analytical grade. Anhydrous
butanol was stored over magnesium and distilled prior to use.
Anhydrous pyridine was distilled from potassium hydroxide and
stored over molecular sieves. Anhydrous tetrahydrofuran (THF)
was stored over sodium and distilled prior to use. All chemicals
for synthesis were obtained from established suppliers (Aldrich,
Acros, Merck) and used as received. TLC was performed on
Merck aluminum sheets with silica gel 60 F254. Merck
10.1021/bc100226x  2010 American Chemical Society
Published on Web 09/20/2010

Azaphthalocyanines as Dark Quenchers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1873
Kieselgel 60 (0.040-0.063 mm) was used for column chroma-
tography. Long chain amino alkyl (lcaa) solid phase (500 Å
controlled pore glass (CPG)) was obtained from ChemGenes
Corporation (Wilmington, MA, USA). All HPLC chromato-
graphic separations were performed on a Shimadzu chroma-
tography system consisting of a communication bus module
CBM 20A, a diode array detector SPD-M20A, two pumps LC-
20AD, an autoinjector SIL-20AC, a column compartment CTO-
20AC, a degasser DGU-20A3, and a computer-based chromato-
graphic software LC solution (Shimadzu, Tokyo, Japan). The
infrared spectra were measured on IR-Spectrometer Nicolet
Impact 400 in KBr pellets. The 1H and 13C NMR spectra were
recorded on Varian Mercury-Vx BB 300 (299.95 MHz 1H, and
75.43 MHz 13C). The reported chemical shifts are relative to
tetramethylsilane. The UV/vis spectra were recorded using a
Shimadzu UV-2401PC spectrophotometer. The fluorescence
spectra were obtained by an AMINCO-Bowman Series 2
luminescence spectrometer. The matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF) mass spectra of com-
pounds 1, 2, and 7-10 were collected by a Voyager-DE STR
mass spectrometer (Applied Biosystems) in R-cyano-4-hydroxy-
cinnamic acid or trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-
propenylidene]-malononitrile as the matrix. Mass spectra of
oligonucleotide probes were obtained using a MALDI-TOF
Bruker Autoflex II mass spectrometer. Compound 3 was
prepared according to Mørkved et al. (15), and compounds 1
(16) and 6 (17) have been published by our group earlier.
Synthesis. Azaphthalocyanine (2). AzaPc 1 (100 mg, 0.09
mmol) in anhydrous dichloromethane (3 mL) was added to
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (102 mg,
0.36 mmol) and diisopropylethylamine (126 µL, 0.72 mmol)
in anhydrous dichloromethane (5 mL) and stirred for 2 h at rt
under an argon atmosphere. Progress of the reaction was
monitored on TLC (dichloromethane/acetone/methanol 50:1:1;
product Rf ) 0.56; compound 1 Rf ) 0.12). Thereafter, the
solution was washed with saturated NaHCO3, dried by Na2SO4,
and evaporated to dryness. Any possible purification on a silica
column failed due to the decomposition of 2 on silica. That is
why the product was used directly for oligonucleotide modifica-
tion without any further purification. The similar instability of
phosphoramidites on silica or alumina has been described also
by other researchers (18). The MALDI-TOF spectra of 2 showed
the mass at expected m/z ) 1322.6, approximately 10% of the
oxidized product at m/z ) 1338.6, and a fragment at m/z )
1105.6 caused by laser ionization. Yield, 105 mg; 89% as a
deep purple solid. MS (MALDI-TOF) m/z ) 1105.6 (55)
[M-OPN(iPr)2OCH2CH2CN]+, 1322.6 (100) [M]+, 1338.6 (10)
[M + O]+.
mobile phase of chloroform/methanol 10:1 and finally recrystal-
lized from ethanol/water. Yield, 324 mg; 62% of yellow powder;
mp 263.3-266.2 °C (decomp.). 1H NMR (300 MHz, [D6]
3
DMSO-): δ ) 7.79 (d, J(H,H) ) 8.8 Hz, 2H, arom. CH2),
7.09 (d, 3J(H,H) ) 8.8 Hz, 2H,arom. CH), 3.48 (s, 3H, N-CH3),
3
3
3.25 (q, J(H,H) ) 7 Hz, 4H, N-CH2) and 0.78 (t, J(H,H) )
7 Hz, 6H, CH2-CH3) ppm. 13C NMR (75 MHz, [D6] DMSO):
δ ) 167.0, 148.5, 147.6, 143.9, 130.9, 126.0, 124.1, 120.4,
118.3, 115.4, 115.2, 42.8, 39.2, and 12.5 ppm. IR (KBr): ν )
2976, 2936, 2876, 2360, 2229 (CN), 1686, 1607, 1533, 1501,
1404, and 1292 cm-1
.
2-[(4-Carboxyphenyl)methylamino]-3,9,10,16,17,23,24-heptak-
is(diethylamino)-1,4,8,11,15,18,22,25-(octaaza)phthalocyanine (7).
Metal lithium (168 mg, 24 mmol) was added to the refluxing
mixture of compounds 5 (300 mg, 0.85 mmol) and 6 (700 mg,
2.57 mmol) in anhydrous butanol (20 mL) and the reflux
continued for the next 3 h. The solvent was removed under
reduced pressure, dilute acetic acid (50%, 50 mL) was added,
and the suspension was stirred for 30 min at rt. The crude
product was filtered and washed thoroughly with water. The
resulting mixture of congeners was separated on silica with
dichloromethane/acetone/methanol 20:1:1 as the mobile phase.
The second intense purple fraction (product) was purified again
on silica with dichloromethane/acetone/methanol 30:1:1. Finally,
pure compound 7 was dissolved in 5 mL of dichloromethane,
dropped into 200 mL of hexane, and cooled to -30 °C
overnight. The precipitated fine suspension was filtered and
washed with hexane. Yield, 150 mg; 15% of deep purple solid.
1H NMR (300 MHz, [D5] pyridine): δ ) 13.83 (br s, 2H; central
NH), 8.48 (d, 3J(H,H) ) 9 Hz, 2H; arom. CH), 7.20 (d, overlap
with solvent, 2H; arom. CH), 3.96-3.86 (m, 24H, N-CH2),
3
3.85 (s, 3H; N-CH3), 3.72 (q, J(H,H) ) 7 Hz, 4H; N-CH2)
and 1.27-1.06 ppm (m, 42H; CH3). 13C NMR (75 MHz, [D5]
pyridine): δ ) 169.0, 153.2, 151.0, 150.9, 150.5, 146.7, 146.6,
143.0, 142.9, 141.6, 140.0, 139.9, 139.7, 139.7, 131.4, 117.3,
43.8, 43.1, 43.1, 39.1, 13.2, 13.1, and 13.1 ppm (some signals
were overlapped with signals of solvent). IR (KBr): ν ) 2969,
2931, 2873, 1710 (CO), 1639, 1605, and 1426 cm-1. UV/vis
(THF): λmax (ε) ) 680 (80000), 654 (66500), 622sh, 597sh, 518
(54300) and 369 nm (93200 M-1cm-1). MS (MALDI-TOF):
m/z ) 1168.6 [M]+. Anal. (C60H80N24O2 + 2 H2O) calcd. C
59.78, H 7.02, N 27.89; found, C 60.18, H 7.30, N 27.53.
Azaphthalocyanine (8). AzaPc 7 (290 mg, 248 µmol) was
dissolved in anhydrous tetrahydrofuran (40 mL). N,N′-dicyclo-
hexylcarbodiimide (DCC) (56.2 mg, 273 µmol) and subse-
quently N-hydroxysuccinimide (NHS) (31.4 mg, 273 µmol) were
added. The reaction was stirred for 72 h at rt. The precipitated
solid (N,N′-dicyclohexylurea) was filtered off and washed with
THF. 3-Amino-1,2-propandiol (113 mg, 1.24 mmol) in methanol
was added into the THF solution, and the reaction was stirred
at rt. The reaction was protected from light by aluminum foil.
After 36 h, the solvent was evaporated at reduced pressure, and
the rest was suspended in water, filtered, and washed thoroughly
with water. Purification was performed on silica with a
dichloromethane/acetone/methanol gradient from 30:1:1 to 10:
4-[(3-Chloro-5,6-dicyanopyrazin-2-yl)methylamino]benzoic
Acid (4). A mixture of compound 3 (400 mg, 2 mmol) and
4-(methylamino)benzoic acid (604 mg, 4 mmol) in THF (75
mL) was refluxed for 12 h. The reaction was cooled down and
filtered, and the filtrate was purified on silica with chloroform/
acetone/methanol 10:1:1 as the mobile phase. The product was
recrystallized from ethanol/water. Yield, 520 mg; 83% as a
yellow powder; mp 208.1-211.6 °C. 1H NMR (300 MHz, [D6]
acetone): δ ) 8.09 (d, 3J(H,H) ) 8.5 Hz, 2H, arom. CH); 7.48
1
1:1. Yield, 150 mg; 49% as deep purple solid. H NMR (300
MHz, [D5] pyridine): δ ) 13.81 (br s, 2H; central NH), 9.32 (t,
3
3
3J(H,H) ) 6 Hz, 1H; NHCO), 8.32 (d, J(H,H) ) 9 Hz, 2H;
(d, J(H,H) ) 8.5 Hz, 2H, arom. CH2) and 3.66 (s, N-CH3,
3H,) ppm. 13C NMR (75 MHz, [D6] acetone): δ ) 166.7, 153.5,
149.6, 141.1, 131.9, 130.7, 130.0, 125.8, 121.9, 114.3, 114.2,
and 42.6 ppm. IR (KBr): ν ) 3066, 2957, 2663, 2546, 2360,
2343, 2234 (CN), 1694, 1607, 1537, 1494, 1406, and 1375 cm-1.
arom. CH), 7.17 (d, overlap with solvent, 2H; arom. CH),
4.53-4.42 (m, 1H; CH-OH), 4.32-4.00 (m, 4H; NH-CH2,
O-CH2), 3.97-3.84 (m, 24H; N-CH2), 3.83 (s, 3H, N-CH3),
3
3
3.69 (q, J(H,H) ) 7 Hz; 4H, N-CH2) 1.21 (t, J(H,H) ) 7
3
4-[(5,6-Dicyano-3-diethylaminopyrazin-2-yl)methylamino]ben-
zoic Acid (5). Compound 4 (312 mg, 0.89 mmol) was refluxed
in THF (50 mL) with diethylamine (1.3 g 18 mmol) for 2 h.
The reaction was cooled down, filtered, and the solvent
evaporated. The crude product was purified on silica with a
Hz, 30H; CH3), 1.14 (t, J(H,H) ) 7 Hz, 6H; CH3) and 1.06
3
ppm (t, J(H,H) ) 7 Hz, 6H; CH3). 13C NMR (75 MHz, [D5]
pyridine): δ ) 168.4, 153.1, 151.0, 150.9, 150.9, 150.6, 150.5,
150.0, 147.0, 146.1, 142.8, 142.7, 141.5, 140.1, 139.9, 139.8,
139.7, 139.6, 129.4, 128.9, 128.3, 126.1, 122.9, 117.9, 116.7,

1874 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kopecky et al.
114.4, 72.2, 65.2, 43.7, 43.1, 43.0, 39.2, 30.2, 13.3, 13.1, and
13.1 ppm (some signals were overlapped with signals of
solvent). IR (KBr): ν ) 2969, 2931, 2873, 1636 (CONH), 1607,
1506, and 1427 cm-1. UV/vis (THF): λmax (ε) ) 681 (80000),
655 (65600), 622sh, 597sh, 518 (53500) and 370 nm (91100
M-1cm-1). MS (MALDI-TOF) m/z ) 1241.3 [M]+. Anal.
(C63H87N25O3 + 3 H2O) calcd. C 58.36, H 7.23, N 27.01; found,
C 58.50, H 7.33, N 26.97.
the test tube was sealed and shaken. The optical absorbance of
the sample, diluted to fit an interval of 0.2-1.0 AU was
measured at 498 nm in a 1 cm cuvette. The loading was
calculated as given bellow:
loading (µmol/g) ) (absorbance at 498) × dilution ×
143/(weight of support (mg))
The loading of DMTr on solid phase 11 was determined to be
30.8 µmol/g.
Azaphthalocyanine (9). AzaPc 8 (124 mg, 0.1 mmol), 4,4′-
dimethoxytrityl chloride (DMTrCl) (44 mg, 0.13 mmol), and a
catalytic amount of 4-N,N-dimethylaminopyridine were dis-
solved in anhydrous pyridine (20 mL) and stirred for 14 h at rt.
After that time, more DMTrCl (169 mg, 0.5 mmol) was added,
and the reaction was stirred further. The reaction vessel was
protected from light by aluminum foil. After 24 h, TLC
(dichloromethane/pyridine 40:1) indicated full conversion. The
product was used directly for the subsequent reaction without
any isolation. A sample was taken from the solution and
analyzed using mass spectrometry. MS (MALDI-TOF) m/z
1543.7 [M]+, after the addition of trifluoroacetic acid (TFA)
into the matrix (deprotection of 4,4′-dimethoxytrityl (DMTr)):
m/z 1241.6 [M-DMTr]+.
Synthesis of Oligonucleotides. Oligonucleotides were syn-
thesized on a Perkin-Elmer Applied Biosystems 394 DNA/RNA
synthesizer. 3′-Labeling of the oligonucleotides was achieved
by synthesis on modified solid phases (solid phase 11 and
commercially obtained supports of Black Hole Quencher (BHQ)
or BlackBerry Quencher (BBQ)). 5′-Labeling of the oligonucle-
otides was performed on the same synthesizer employing
standard phosphoramidite chemistry and corresponding dye
2-cyanoethyl-(N,N-diisopropyl)-phosphoramidites (compound 2
and commercially obtained dyes). 3′-BHQ-1 CPG, 3′-BHQ-2
CPG, 5′-BHQ-1 phosphoramidite, 5′-BHQ-2 phosphoramidite,
Cy5 phosphoramidite, and 5′-fluorescein phosphoramidite were
supplied by Glen Research, Sterling, VA, USA. 3′-BBQ-650
CPG and 5′-BBQ-650 phosphoramidite were supplied by Berry
& Associates, Inc., Dexter, MI, USA. The synthesis and
purification of oligonucleotides labeled with commercially
obtained dyes were performed according to the supplier’s
recommendations.
OPC Purification of Oligonucleotides S1-S6 Modified
with AzaPc. Prepared oligonucleotides S1-S6 with AzaPc were
purified on oligonucleotide purification cartridges (OPCs), Puri-
Pak cartridges, (ChemGenes Corp., Wilmington, MA, USA)
directly after deprotection from the solid phase. The OPC was
washed with acetonitrile (4 mL) and 2 M triethylammonium
acetate (2 mL). A solution of oligonucleotide (2-3 mL) in
deprotecting solution was loaded on the OPC repeatedly until
all of the color was absorbed to the top of OPC. Thereafter, the
OPC was washed with a diluted solution of ammonia (2.5%, 4
mL), then with 8% acetonitrile (4 mL) and 30% acetonitrile (2
mL). The desired sequence was obtained by freeze-drying the
30% acetonitrile fraction. Purified oligonucleotides were stored
at -20 °C.
S1 (AzaPc): MS (MALDI-TOF), a cluster peaking at m/z
8805 [M + H]+ and adducts with sodium and potassium. S2
(AzaPc): MS (MALDI-TOF), a cluster peaking at m/z 10325
[M + H]+ and adducts with sodium and potassium. S3 (AzaPc):
MS (MALDI-TOF), a cluster peaking at m/z 11846 [M + H]+
and adducts with sodium and potassium. S4 (AzaPc): MS
(MALDI-TOF), a cluster peaking at m/z 8685 [M + H]+ and
adducts with sodium and potassium. S5 (AzaPc): MS (MALDI-
TOF), a cluster peaking at m/z 7400 [M + H]+ and adducts
with sodium and potassium. S6 (AzaPc): MS (MALDI-TOF),
a cluster peaking at m/z 5819 [M + H]+ and adducts with
sodium and potassium.
HPLC Analysis. A gradient elution was used to analyze
S1-S6 modified with AzaPc. The separation was performed
on a Hypersil BDS C18 column (100 × 4.6 mm, 3 µm particle
size) using a mobile phase consisting of acetonitrile and 50 mM
triethylammonium acetate. Mobile phase A was 12% acetonitrile
in 50 mM triethylammonium acetate, and mobile phase B was
acetonitrile. The gradient time program was set as follows: 0-2
min 0%B; 2-18 min 0-85% B; 18-25 min 85% B; 25-25.5
min 85-0% B. The column was equilibrated for 4.5 min. The
column temperature was maintained at 40 °C, and the flow rate
was set at 0.7 mL/min. The compounds were analyzed by a
diode array detector. The corresponding controls were analyzed
separately to allow the determination of their elution patterns.
Azaphthalocyanine (10). Succinic anhydride (100 mg, 1
mmol) was added into the previous reaction, and the mixture
was heated for 12 h at 60 °C. Thereafter, the solvent was
removed under reduced pressure, and the crude product was
purified on silica with a step gradient: dichloromethane/pyridine
40:1, dichloromethane/pyridine 20:1, and dichloromethane/
methanol/pyridine 20:1:1. Yield, 40 mg; 25% (after two steps)
1
as a deep purple solid. H NMR (300 MHz, [D5] pyridine): δ
3
) 13.86 (br s, 2H; central NH), 9.11 (t, J(H,H) ) 5 Hz, 1H;
NHCO), 6.88-8.46 (m, 17H; arom. CH), 4.33-4.20 (m, 1H;
CH-O), 3.98-3.79 (m, 26H; 1 × NH-CH2, 12 × N-CH2),
3.75 (s, 3H; N-CH3), 3.73-3.56 (m, 12H; 2 × O-CH3, 1 ×
O-CH2, 2 × N-CH2), 2.91-2.87 (m, 2H; CH2-CO), 2.85-2.81
3
(m, 2H; CH2-CO), 1.20 (t, J(H,H) ) 7 Hz, 30H; CH3), 1.14
3
3
(t, J(H,H) ) 7 Hz, 6H; CH3) and 1.06 ppm (t, J(H,H) ) 7
Hz, 6H; CH3). 13C NMR (75 MHz, [D5] pyridine): δ ) 174.4,
172.2, 167.1, 158.3, 152.4, 150.3, 150.2, 150.1, 148.5, 146.3,
145.1, 144.7, 135.7, 135.3, 134.1, 131.0, 129.9, 128.3, 127.9,
127.6, 126.4, 116.9, 113.0, 85.8, 72.4, 62.2, 54.5, 42.9, 42.4,
42.3, 38.5, 29.5, 29.3, 29.1, 12.4 ppm (some signals were
overlapped with the signals of the solvent). IR (KBr): ν ) 3428,
3305, 3050, 2966, 2930, 2871, 1734 (CO-O), 1640(CO-NH),
1607, 1508, 1425, and 1250 (O-CH3) cm-1. MS (MALDI-
TOF): m/z ) 1643.7 [M]+; after the addition of TFA into the
matrix, m/z ) 1341.7 [M-DMTr]+.
Solid Phase Modified with Azaphthalocyanine (11). AzaPc
10 (8 mg, 5 µmol), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethy-
luronium hexafluorophosphate (HBTU) (3 mg, 10 µmol), and
anhydrous triethylamine (10 µL) were dissolved in anhydrous
dimethylformamide (DMF) (2 mL), and 100 mg of lcaa CPG
was added. The reaction vial was sealed, and the mixture was
shaken for 24 h at rt on an orbital shaker protected from light.
Thereafter, the solid phase was washed with DMF, acetonitrile,
and diethylether and dried over the silica under reduced pressure.
Unreacted amino groups were subsequently capped. A mixture
of anhydrous THF, pyridine, and acetic anhydride (8:1:1 (v/v),
1 mL) was mixed with a 10% (m/m) solution of 1-methylimi-
dazole in anhydrous THF (1 mL) and shaken with the solid
phase 11 for 30 min at rt. Thereafter, the solid phase was washed
with DMF, acetonitrile, and diethylether and dried over silica
under reduced pressure. 4,4′-Dimethoxytrityl loading was
determined as described in the following procedure. Ap-
proximately 2-3 mg of the support was accurately weighed
out directly to the test tube, perchloric acid solution (10 mL,
51.4 mL of 70% HClO4 + 46 mL of methanol) was added, and

Azaphthalocyanines as Dark Quenchers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1875
Unlabeled oligonucleotides were eluted with tR of approximately
2 min and free dye (1 or 8) with tR of approximately 23 min.
Hybridization Assays. Absorption Spectra of Duplexes.
Spectra of duplexes were measured in hybridization buffer.
Stock solutions of oligonucleotides S1, S3, and S4 labeled with
AzaPc and SF and SFX labeled with Cy5 were prepared to a
concentration 100 µM. Hybridization buffer (1 mL) was
transferred to a cuvette, the stock solution of oligonucleotide
probe with the quencher (10 µL) was added, and the absorption
spectrum was measured. Thereafter, the stock solution of the
oligonucleotide labeled with fluorophore (10 µL) was added,
and the solution was heated to 50 °C for 1 min. The absorption
spectrum of the duplex was measured after 30 min when the
sample cooled down to laboratory temperature. The final
concentration of both oligonucleotides was 1 µM. Absorption
spectra of SF and SFX were measured also separately after the
addition of the stock solution (10 µL) to hybridization buffer
(1 mL).
Determination of Quenching Efficiency. The method was
adopted from Marras et al. (1). Oligonucleotides S1-S6
modified with AzaPc, BHQ-1, BHQ-2, and BBQ-650 or without
any quencher were dissolved in hybridization buffer to a stock
concentration 10 µM. Oligonucleotide SF was dissolved in
hybridization buffer to a concentration 0.05 µM and transferred
to a cuvette (800 µL), and the fluorescence was measured at
663 and 517 nm for Cy5 and fluorescein (FAM), respectively
(excitation wavelengths 651 and 492 nm for Cy5 and FAM,
respectively). Subsequently, the oligonucleotide with the quench-
er (20 µL of stock solution; final concentration 0.25 µM) was
added, and the solution was heated to 70 °C for 5 min and
allowed to cool to laboratory temperature for 30 min (S6 was
cooled to 4 °C). Thereafter, the fluorescence of the duplex was
measured. A similar procedure was used for oligonucleotide
SFX. The quenching efficiency (QE) was calculated according
to the following equation (1, 19):
Figure 1. Structures of compounds 1 and 7.
HPLC separation. The design of AzaPc labels was based on
the following considerations: (1) the macrocycle must carry
mostly hydrophobic substituents and one modifiable group; (2)
the macrocycle must be substituted with alkylamino substituents
in order to allow ICT to quench efficiently excited states; and
(3) the hydrophobic substituents must be sufficiently bulky in
order to reduce aggregation typical for planar AzaPc and Pc
macrocycles. On the basis of these requirements, compounds 1
and 7 (Figure 1 and Schemes 1 and 2) were synthesized and
further modified for both 3′- and 5′-end solid-phase labeling.
Postsynthetic modification of AzaPc was easily performed
because bulky N,N-diethylamino substituents reduced the ag-
gregation to a minimum, and the typical problems with low
solubility, silica binding (22), or tailing of AzaPc on TLC (14)
were not observed (see Supporting Information, Figure S1).
5′-End modified oligonucleotides S4-S6 were prepared from
1 (16) via phosphoramidite 2 (Scheme 1). Phosphoramidites
are highly reactive intermediates (23, 24) finding their place in
automatic DNA synthesis for both DNA elongation and labels
attachment. Compound 2 was therefore introduced as the last
step into the DNA/RNA synthesizer during solid-phase syn-
thesis. Contrary to the in-solution conjugation methods, this
approach simplifies the labeling and enables the general
automatic processes to be used.
For the 3′-end labeling of oligonucleotides, the long chain
aminoalkyl (lcaa) solid phase was modified by AzaPc 7 (Scheme
2) before the synthesis of the desired oligonucleotide chain
(S1-S3). First, unsymmetrical AzaPc 7 was synthesized by
statistical condensation of 5 and 6 and isolated from the mixture
of six congeners (see Supporting Information, Figure S2) with
a yield of 15%. Compound 5 was prepared by two step
nucleophilic substitution of chlorine atoms in 3. Although
several selective strategies were developed for the synthesis of
unsymmetrical Pc (25) including recent efforts in the improve-
ment of solid-phase synthesis (26), the statistical condensation
gave reasonable yields of 7 with very simple isolation of the
desired congener. To verify the stability of 7 during the 3′-end
labeling of oligonucleotides, the compound was subjected to
the conditions occurring in the course of automated chemical
oligonucleotide synthesis. It was shown to be stable in all the
range of chemical conditions tested (see Supporting Informa-
tion). Compound 7 was converted to dihydroxyamide 8 via an
active succinimidyl ester. Protection of the primary hydroxy
group with DMTrCl (9) allowed for selective modification of
the secondary hydroxyl with succinate (10) and subsequent
attachment of the AzaPc to lcaa solid phase (11). Unreacted
amino groups in solid phase were capped with acetic anhydride.
Solid phase 11 allows for the start of the synthesis of the DNA
probe on hydroxyl after DMTr deprotection and simple cleavage
of the synthesized conjugate from the solid phase by basic
hydrolysis of the ester bond. Such a modified solid phase was
QE ) (1 - FX/F0) × 100
where F0 is the fluorescence of fluorophore SF alone, and FX
is the fluorescence of the duplex.
Singlet Oxygen Quantum Yields and Fluorescence. Quan-
tum yields of a singlet oxygen (Φ∆) of 1 and 7 were determined
according to a previously published procedure (20) using the
decomposition of 1,3-diphenylisobenzofuran in DMF. Zinc
phthalocyanine (ZnPc) was used as a reference (Φ∆ ) 0.56 in
DMF 21, 22). The absorption of the dyes in the Q-band area
was set approximately to 0.1. The presented data represent the
mean of three measurements.
An attempt to detect any fluorescence signals from quenchers
was also made. Compounds 1 and 7 in DMF, dimethylsulfoxide
(DMSO), and THF or oligonucleotides S1-S6 with AzaPc in
hybridization buffer were excited at the B-band (365 nm) or
Q-band (650 nm). Absorption at the excitation wavelength was
set to approximately 0.1. Fluorescence was monitored in a range
of 550-900 nm.
RESULTS AND DISCUSSION
Synthesis. All conjugation procedures between Pc and
oligonucleotides until now were performed in water solution
with an excess of Pc reagent. Isolation of the desired conjugate
therefore always included several steps including the final HPLC
separation. Solid-phase synthesis of the conjugates allows for
the reduction of the side products, and the excess of the reagent
can be easily washed out. In order to further simplify the
purification steps, the AzaPc labels were designed to be highly
hydrophobic, and that is why the conjugates may be anchored
in a short OPC allowing easy purification without the need for

1876 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kopecky et al.
Scheme 1. Synthesis of 5′-End Modified Oligonucleotides Labeled with AzaPca
a (i) anhydr. DCM, diisopropylethylamine, rt; (ii) 5-ethylthiotetrazole, anhydr. acetonitrile, DNA/RNA synthesizer; (iii) ammonia/methylamine,
rt.
Scheme 2. Synthesis of 3′-End Modified Oligonucleotides Labeled with AzaPca
a (i) THF, reflux; (ii) diethylamine, THF, reflux; (iii) anhydr. butanol, Li, reflux; (iv) DCC, NHS, anhydr. THF/methanol, rt; (v) DMTrCl, 4-(N,N-
dimethylamino)pyridine, anhydr. pyridine, rt; (vi) anhydr. pyridine, 60°C; (vii) HBTU, triethylamine, anhydr. DMF, rt; (viii) acetic anhydride,
1-methylimidazole, THF/pyridine, rt; (ix) DNA synthesis in DNA/RNA synthesizer.
Figure 2. HPLC chromatogram of purified oligonucleotide S3 labeled
with AzaPc. Red line, 254 nm; blue line, 673 nm.
Figure 3. MALDI-TOF spectrum of oligonucleotide S3 labeled with
AzaPc.
used in a standard oligonucleotide synthesis in a DNA/RNA
synthesizer. Also, this approach allows the general automatic
processes in a DNA/RNA synthesizer to be used for both
synthesis and cleavage of the conjugate from solid phase.
In all of the cases (S1-S6), the AzaPc conjugates cleaved
from the solid phase were purified by passing through short
OPC. The hydrophobic character of AzaPc labels allowed for
the anchoring of the conjugates to reverse phase in OPC, and
the unlabeled oligonucleotides were washed out with 8%
acetonitrile. Subsequently, the increase of acetonitrile concentra-
tion to 30% released the AzaPc-oligonucleotide conjugates from
OPC. The purified conjugates were analyzed by HPLC (see
Figure 2), and two important peaks were detected in the
chromatograms (typical retention times tR ) 2 min and tR ) 17
min). On the basis of an analysis of absorption spectra (see
Supporting Information, Figure S5) and retention times of the
controls, the base peak (tR ) 17 min) belongs to the oligo-
nucleotide labeled with AzaPc. Unlabeled oligonucleotides were
eluted in the second minute and did not exceed 4% in all cases
(based on an area under the curve calculation at 254 nm).
Nonconjugated AzaPc control eluted with tR ) 23 min and was
not detected in chromatograms of AzaPc-oligonucleotide
conjugates. In order to verify the composition of conjugates,
MALDI-TOF spectra were recorded, and the expected mass to
charge ratio was confirmed (Figure 3).
UV-Vis Spectral, Photophysical, and Photochemical
Properties. Absorption spectra of AzaPc 1 and 7 were analyzed
in different solvents (DMSO, THF, methanol (MeOH), DMF,
and acetone). The spectra in all solvents showed the shape
characteristic for monomeric species with two absorption bands
typical for AzaPc or Pc: a high energy B-band at approximately
360 nm and a low energy Q-band at approximately 660 nm.
Both compounds also showed another broad band at ap-
proximately 550 nm due to the conjugation of lone electron
pairs of N,N-diethylamino substituents with a macrocyclic
system (n-π* transitions) (14, 27). The Q-bands of 1 and 7 were
split due to the unsymmetrical composition of the whole
macrocycle because of the presence of the central hydrogens
(16). The splitting was sharp in THF but became broad and

Azaphthalocyanines as Dark Quenchers
Bioconjugate Chem., Vol. 21, No. 10, 2010 1877
Figure 4. Absorption spectra of 7 in THF (blue), DMSO (green), and
oligonucleotide S1 with AzaPc in buffer (red) at a concentration of
1 µM.
Figure 5. Absorption spectra of oligonucleotide S1 labeled with AzaPc
(red line, filled area), BHQ-1 (yellow), BHQ-2 (black), and BBQ-650
(green). The spectra were normalized to the same absorption at 254
nm (which is due to the oligonucleotides). The arrows indicate emission
maxima of FAM and Cy5 fluorophores.
Table 1. Spectral and Photochemical Properties of AzaPc 1 and 7
λ (ε)
λ (ε)
λ (ε) in
in DMSO
in THF
hybridization buffer
compound (nm (M-1 cm-1)) (nm (M-1 cm-1)) (nm (M-1 cm-1))a Φ∆ (DMF)
1
685 (57400)
661 (50900)
530 (51900)
372 (84800)
686 (68000)
542 (60000)
378 (91500)
677 (95300)
647 (69500)
508 (58400)
365 (108000)
680 (86300)
654 (72300)
516 (59600)
370 (102700)
664 (45200)
536 (66300)
373 (105900)
0.015
7
668 (43000)
537 (60300)
376 (97900)
0.018
a Conjugate S1 (for 7) or S5 (for 1).
featureless in solvents with increased polarity (e.g., DMSO),
and completely disappeared when the conjugates where dis-
solved in hybridization buffer (Figure 4), indicating considerable
effect of the solvent polarity on the Q-band shape. Such spectral
changes in DMSO or buffer could also be attributed to
aggregation, but neither the shape of the spectra nor the
extinction coefficient changed in a very wide range of concen-
tration (in DMSO 2.5 × 10-4 M-1.0 × 10-7 M). Also, no new
bands at shorter or longer wavelengths corresponding to
H- (7, 28) or J-dimer (29) formation were observed. Another
support for the statement that the aggregation is not responsible
for the broad Q-band is that the spectra of the conjugates did
not change after hybridization with complementary oligonucle-
otides (see, e.g., the duplex S4-SF in Supporting Information,
Figure S6-c).
Photophysical and photochemical parameters of AzaPc 1
and 7, which are important for their prospective use as dark
quenchers, were determined. No fluorescence of either AzaPc
1 and 7 in THF, DMF, DMSO, or the AzaPc labeled probes
S1-S6 in hybridization buffer was detected. Singlet oxygen,
usually strongly produced by AzaPc after illumination (30),
is a highly reactive species that may destroy oligonucleotide
probes or the target sequence (8, 31), and thus, its formation
is undesirable in hybridization assays. Importantly, AzaPc 1
and 7 reached very low singlet oxygen quantum yields (Φ∆),
0.015 and 0.018, respectively. For comparison, the Φ∆ value
for FAM, a routinely used fluorophore in hybridization
assays, ranges from 0.03 to 0.1 (32). It must be noted that
no aggregates were detected during measurements, and
therefore, the lack of any detectable fluorescence can be
attributed solely to the deactivation of the S1 excited state
by ICT (14). Spectral and photochemical data of compounds
1 and 7 are summarized in Table 1.
Figure 6. Sequences of modified oligonucleotides. F ) fluorophore
(FAM or Cy5), and Q ) quencher (AzaPc, BHQ-1, BHQ-2, or BBQ-
650).
Figure 7. Fluorescence emission spectra of oligonucleotide SF with
FAM (a) and Cy5 (b) before (red) and after (blue) the addition of the
complementary oligonucleotide S1 with AzaPc.
ment. In order to prove the quenching ability of AzaPc,
oligonucleotide SF labeled with two commonly used fluoro-
phores was chosen for hybridization assays. For example, FAM
(λem ) 517 nm) emits at short wavelengths, while Cy5 (λem
)
663 nm) emits at long wavelengths. For comparative purposes,
the oligonucleotides S1-S6 were labeled with the currently used
dark quenchers BHQ-1, BHQ-2, and BBQ-650, which are highly
efficient at the quenching of FAM or Cy5 (33).
Oligonucleotides S1-S6 (Figure 6) were designed to inves-
tigate both mechanisms of quenching utilized in hybridization
assays. Resonance energy transfer (RET) requires the emission
spectrum of a fluorophore and the absorption spectrum of a
quencher to overlap with the effective distance between labeling
dyes being 20-100 Å (19). During contact quenching, the
fluorophore and the quencher must be very close to each other
in order to form a ground-state complex that does not emit any
light. Overlap of the spectra is not required in this mechanism (19,
34). According to Marras et al. (1), the oligonucleotide S1 can
be used for the detection of the contact quenching after
hybridization with SF because the fluorophore and the quencher
may form the nonemitting heterodimer. S3 is designed to allow
purely RET quenching, and S2 may involve both mechanisms.
However, the absorption spectra of the duplex of AzaPc-labeled
S3 with Cy5-labeled SF revealed the presence of a certain level
DNA Hybridization Assays and Quenching Efficiency. The
absorption spectra of AzaPc-oligonucleotide conjugates S1-S6
in hybridization buffer show a wide range of absorption from
300 to 740 nm due to the AzaPc moiety (Figure 5). Thus, it
efficiently overlaps the emission spectra of all fluorophores
currently used in hybridization assays. Such versatility cannot
be found in any other structural type of dark quenchers and
makes these compounds highly promising for future develop-

1878 Bioconjugate Chem., Vol. 21, No. 10, 2010
Kopecky et al.
Table 2. Quenching Efficiency (%)a
fluorophore (SF)
quencher
S1
S2
S3
S4
S5
S6
S1 + SFX
FAM
AzaPc
BHQ-1
BHQ-2
BBQ-650
no quencher
AzaPc
BHQ-1
BHQ-2
97.0 ( 0.12
98.5 ( 0.05
98.6 ( 0.02
97.6 ( 0.02
15.2 ( 0.71
93.8 ( 0.15
96.9 ( 0.05
92.5 ( 0.28
98.2 ( 0.04
3.0 ( 1.42
92.3 ( 0.23
94.9 ( 0.08
95.7 ( 0.03
90.3 ( 0.11
5.5 ( 1.31
95.1 ( 0.11
95.7 ( 0.08
96.3 ( 0.07
96.3 ( 0.14
-35.0 ( 1.07
84.5 ( 0.74
88.4 ( 0.45
89.3 ( 0.10
78.9 ( 0.13
9.6 ( 2.15
96.9 ( 0.04
86.9 ( 0.51
96.7 ( 0.08
82.2 ( 0.06
-38.1 ( 1.76
35.8 ( 1.19
21.4 ( 3.73
9.9 ( 1.30
30.7 ( 0.32
34.9 ( 2.11
28.0 ( 2.38
18.5 ( 1.61
36.6 ( 0.52
54.9 ( 0.44
48.5 ( 0.69
48.5 ( 0.35
49.9 ( 0.03
Cy5
27.5 ( 1.55
-0.5 ( 3.65
5.5 ( 1.49
25.1 ( 0.87
3.5 ( 5.36
11.7 ( 0.95
26.1 ( 0.19
45.1 ( 0.42
4.1 ( 1.00
33.1 ( 0.57
46.5 ( 0.38
-9.5 ( 10.2
-4.5 ( 8.45
-9.1 ( 10.8
6.05 ( 1.01
4.6 ( 15.5
BBQ-650
no quencher
18.6 ( 0.76
a The mean of three measurements ( SD. The mathematical symbol minus means that fluorescence increased after hybridization.
of the heterodimer suggesting a looping of freely rotating 10T
overhang (see Supporting Information, Figure S6). The het-
erodimer has a different absorption spectrum than a simple sum
of the fluorophore and the quencher absorption spectra. Thus,
S3 cannot be used in this case for the detection of pure RET-
based quenching.
In order to overcome this problem, oligonucleotides S4-S6
were investigated in hybridization assays as well. The rigid
double strand of their duplexes with SF prevents looping and
thus permits the determination of pure RET quenching. At the
same time, it enables the investigation of the quenching
efficiency at a longer distance. The absorption spectra of the
duplexes S4-SF corresponded well with a simple sum of
spectra of S4 and SF indicating the absence of the heterodimer
(Supporting Information, Figure S6).
routinely used fluorophore and any hybridization assay. Finally,
AzaPc represents flexible macrocycles with the possibility of
shifting their absorption maximum over 750 nm (17). Therefore,
they appear to have a great potential in genetic analysis,
especially for the imaging of gene expression in living cells,
where excitation and emission at long wavelengths is highly
desired.
ACKNOWLEDGMENT
The work has been supported by grant KJB401100801 (The
Grant Agency of the Academy of Sciences of the CR), by
Research Project MSM0021620822, and by grant SVV-2010-
261-001. We gratefully thank Jirˇ´ı Kunesˇ for the measurements
of NMR spectra.
After hybridization, all of the prepared AzaPc-labeled oli-
gonucleotides S1-S6 efficiently decreased the fluorescence
signal of SF, either Cy5 or FAM-containing (Figure 7). The
quenching efficiency (1, 19) is shown in Table 2. While all tested
quenchers were highly efficient and comparable at a shorter
distance (S1-S3), marked differences appeared at a longer
distance between the fluorophore and the quencher (S4-S6).
BHQ-1 quenched only FAM emission, BHQ-2 quenched both
FAM and Cy5 emission, but its efficiency considerably de-
creased with distance. The AzaPc quencher was able to quench
the fluorescence of both Cy5 and FAM with higher efficiency
than the above-mentioned BHQ dyes and with efficiency
comparable to that of the BBQ-650 quencher. This is most likely
a consequence of better overlap of the AzaPc quencher
absorption and fluorophore emission spectra (Figure 5).
In order to exclude nonselective quenching, two kinds of
controls were involved. The decrease of SF fluorescence
emission by the S1-S3 sequence without any attached quencher
was negligible (Table 2). Nonspecific quenching of fluorescence
was excluded using Cy5-labeled oligonucleotide SFX that was
not complementary to S1. No quenching occurred for any of
the quenchers tested (Table 2).
Supporting Information Available: TLC of the AzaPc 7
in a mixture before isolation, structures of the AzaPc congeners
from statistical condensation, stability tests of compound 7,
absorption spectra from HPLC analysis, and absorption spectra
of duplexes. This material is available free of charge via the
Internet at http://pubs.acs.org.
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