Mono-amine functionalized phthalocyanines: Microwave-assisted solid-phase synthesis and bioconjugation strategies

Article abstract of DOI:10.1021/jo901424v

(Chemical Equation Presented) Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having two different t

Full text of DOI:10.1021/jo901424v

pubs.acs.org/joc
Mono-amine Functionalized Phthalocyanines: Microwave-Assisted
Solid-Phase Synthesis and Bioconjugation Strategies
S. Sibel Erdem, Irina V. Nesterova, Steven A. Soper, and Robert P. Hammer*,†
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803. †Current address:
New England Peptide, Gardner, MA 01440.
robert.hammer@newenglandpeptide.com
Received July 11, 2009
Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent
tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having
two different types of peripheral substitutents, one for covalent conjugation of the Pc to biomolecules and
others to improve the solubility of the macrocycle, are ideally suited for the desired applications. To date,
difficulties faced during the purification of monofunctionalized Pcs limited their usage in various types of
applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and
bioconjugation techniques for labeling of the oligonucleotides with the near-IR fluors. A novel synthetic
route was developed utilizing a hydrophilic, poly(ethylene glycol) (PEG)-based support with an acid-
labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation
purposes and were decorated with short PEG chains, serving as solubilizing groups. Microwave-assisted
solid-phase synthetic method was successfully applied to obtain pure asymmetrically substituted
monoamine functionalized Pcs in a short period of time. Three different bioconjugation techniques,
reductive amination, amidation, and Huisgen cycloaddition, were employed for covalent conjugation of
Pcs to oligonucleotides. The described microwave-assisted bioconjugation methods give an opportunity
to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours.
Introduction
clinical chemistry applications.1,2 Increasing requirements
for sensitivity and decreased scale as in capillary and
Fluorescence-based detection is the mainstay of many
bioassays including immunoassays, flow cytometry, DNA
sequencing, nucleic acid diagnostics, proteomics, and other
(1) Epstein, J. R.; Biran, I.; Walt, D. R. Fluorescence-based nucleic acid
detection and microarrays. Anal. Chim. Acta 2002, 469, 3–36.
9280 J. Org. Chem. 2009, 74, 9280–9286
Published on Web 11/13/2009
DOI: 10.1021/jo901424v
r
2009 American Chemical Society

Erdem et al.
JOCArticle
microchip devices has put increasing demands on the efficiency
of these assays. One method to improve fluorescence-based
detection is the use of near-IR dyes, which have decreased
background due to scattering and native fluorescence in this
region of the spectrum (650-850 nm).3 Our laboratory and a
number of others have been developing phthalocyanines (Pcs)
as near-IR fluors for bioanalytical applications. Pcs have a
number of improved properties such as narrow absorbance
and emission spectra, resistance to photobleaching, and tun-
ability of spectral and photophysical properties by variation of
substituents around the Pc aromatic core or the central metal.4
Applications of Pcs in bioassays included analysis of PCR
products,5 single gene mutation detection6 and resonance
energy transfer-based assays.7 Structural requirements of
Pcs for most bioanalytical applications include the need for
water-solubility and a unique functional group for attachment
to the biomolecule. Asymmetrically substituted Pcs with two
different types of peripheral substituents, one reactive group
for conjugation and others for solubility, are ideally suited for
these applications, but difficulties in the isolation of these Pcs
have limited their use in biological and bioanalytical applica-
tions. Recently, we have reported solid-phase synthesis of
mono-hydroxylated Pcs.8 Herein we present a time-efficient,
microwave-assisted method for synthesis of pure “AB3-type”
asymmetrically substituted, monoamine-functionalized Pcs on
solid support along with methods for their ready conjugation
to oligonucleotides.
SCHEME 1. Generalized Retrosynthetic Analysis of Mono-func-
tionalized Pc-Biomolecular Conjugates
SCHEME 2. Immobilization of the Amino Acid and Phthaloni-
trile to the Polymer Support
Results and Discussion
Generalized retrosynthetic analysis of a functionalized Pc-
biomolecular conjugate is shown in Scheme 1. The stepwise
synthesis starts with immobilization of one phthalonitrile to
the polymer support via a cleavable linker. Cyclotetrameriza-
tion of the polymer-bound phthalonitrile with the second type
ofphthalonitrile in solutionyields the “AB3-type” target Pcon
the polymer support. Following the cleavage of the desired
mono-functionalized Pcfrom the polymer support, inthe final
step, the AB3-type Pc is covalently conjugated to a biomole-
cule by a variety of conjugation strategies.
Scheme 2 shows the synthesis of an amine-functionalized,
solid-supported phthalonitrile as the starting point for Pc syn-
thesis. Poly(ethylene glycol) (PEG)-based Rink Amide Chem-
Matrix resin with initial loading of 0.52 mmol/g was employed
(2) Smolina, I. V.; Kuhn, H.; Lee, C.; Frank-Kamenetskii, M. D.
Fluorescence-based detection of short DNA sequences under non-denatur-
ing condition. Bioorg. Med. Chem. 2008, 16, 84–93.
(3) Amiot, C. L.; Xu, S. P.; Liang, S.; Pan, L. Y.; Zhao, J. X. Near-
infrared fluorescent materials for sensing of biological targets. Sensors 2008,
8, 3082–3105.
(4) Verdree, V. T.; Pakhomov, S.; Su, G.; Allen, M. W.; Countryman,
A. C.; Hammer, R. P.; Soper, S. A. Water soluble metallo-phthalocyanines:
the role of the functional groups on the spectral and photophysical proper-
ties. J. Fluoresc. 2007, 17, 547–563.
(5) Nesterova, I. V.; Verdree, V. T.; Pakhomov, S.; Strickler, K. L.; Allen,
M. W.; Hammer, R. P.; Soper, S. A. Metallo-phthalocyanine near-IR
fluorophores: Oligonucleotide conjugates and their applications in PCR
assays. Bioconjugate Chem. 2007, 18, 2159–2168.
(6) Zhao, X. J.; Tapec-Dytioco, R.; Wang, K. M.; Tan, W. H. Collection
of trace amounts of DNA/mRNA molecules using genomagnetic nanocap-
turers. Anal. Chem. 2003, 75, 3476–3483.
(7) Nesterova, I. V.; Erdem, S. S.; Pakhomov, S.; Hammer, R. P.; Soper,
S. A. Phthalocyanine dimerization-based molecular beacons using near-IR
fluorescence. J. Am. Chem. Soc. 2009, 131, 2432–2433.
as the polymer support. Synthesis of the monoamine functional
Pcs 7b and 8b started with incorporation of the amine function-
ality to the solid support via coupling of 0.1 mmol Fmoc-
Lys(Mtt)-OH according tostandardprotocols.9 The ε-amine of
the lysine will provide the functional handle for bioconjugation
(8) Erdem, S. S.; Nesterova, I. V.; Soper, S. A.; Hammer, R. P. Solid-
phase synthesis of asymmetrically substituted “AB3-type” phthalocyanines.
J. Org. Chem. 2008, 73, 5003–5007.
(9) Chan, W. C., White, P. D. Fmoc Solid Phase Peptide Synthesis: A
Practical Approach; Oxford University: New York, 2000.
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TABLE 1. Optimization of the Cyclotetramerization Reaction
phthalonitrile
loading on resin
(mmol/g)
reaction concn (M)/equiv of
phthalonitrile in solution
relative to solid support
outcome of the
reaction/crude
mixture yield (%)
sequence on
the resin
reaction conditions
solvent
BuOH
1
2
3
4
5
6
7
Minipeg-Lys(Boc)-phthalonitrile
Minipeg-Lys(Boc)-phthalonitrile
Minipeg-Lys(Mtt)-phthalonitrile
Lys(Mtt)-phthalonitrile
0.46
0.23
0.10
0.30
0.10
0.10
0.10
0.16/9
0.16/9
0.2/12
0.2/12
0.3/15
0.8/24
0.8/24
microwave,
30 min, 200 °C
microwave,
mixture of all Pc
congeners/19
BuOH
mixture of AB3, A2B2
and (AB3þ100)/21
30 min, 180 °C
microwave, 40 min, dodecanol AB3:A2B2 (2:1)/22
150 °C
microwave,
40 min, 150 °C
microwave,
40 min, 150 °C
microwave, 40 min,
150 °C
thermal heat, 24 h,
120 °C
dodecanol AB3:A2B2 (1:1)/18
dodecanol AB3:A2B2 (97:3)/22
Lys(Mtt)-phthalonitrile
Lys(Mtt)-phthalonitrile
BuOH
AB3 > 99%/15-27
Lys(Mtt)-phthalonitrile
BuOH
AB3 > 99%/7
SCHEME 3. Structures of Most Common Pc Congeners
Formed during the Cyclotetramerization Reaction
SCHEME 4. Solid-Phase Synthesis of AB3-Type Pcs
As a result of the dynamic nature of the polymer support,
the outcomes of the solid-phase reactions are affected by
various factors such as the reaction time and temperature,
loading of the resin, linker length, solvent, and the con-
centration of the reaction.10 Base-promoted phthalonitrile
after removal of the acid-labile 4-methyltrityl (Mtt) protecting
group during the cleavage of the Pc from the polymer support.
The remaining amine sites of the resin 1 were capped with an
excess of Ac2O. Following the removal of the 9-fluorenyl-
methoxycarbonyl (Fmoc) protecting group, carboxylate phtha-
lonitrile 2 was anchored to the amino terminus of the polymer-
bound lysine to give a polymer-supported phthalonitrile 3 with
0.1 mmol/g loading (Scheme 2).
(10) Shi, R. R.; Wang, F.; Yan, B. Site-site isolation and site-site inter-
action-two sides of the same coin. Int. J. Pept. Res. Ther. 2007, 13, 213–219.
and references therein.
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FIGURE 1. Absorbance-emission spectra of ZnPc 7b and bioconju-
gate 9: black, ZnPc 7b λmax(abs) = 681 nm (DMSO); green, ZnPc 7b
λmax(em) = 691 nm (DMSO); blue, bioconjugate 9 λmax(abs) = 640,
690 nm (H2O); red, bioconjugate 9λmax(em) =699nm(pH8.5buffer).
FIGURE 2. Labeling of the oligonucleotide with ZnPc 7b by
reductive amination. The chromatograms absorbance data were
extracted at λ = 680 nm.
tetramerization reaction was optimized on the basis of these
criteria as summarized in Table 1. Experiments were per-
formed in CEM MARS extraction system using fiber optic
temperature and pressure probes. Early experiments showed
that higher phthalonitrile loadings (0.46 mmol/g), deter-
mined on the basis of the absorption of the Fmoc adduct,
and longer linker lengths lead to the formation of other Pc
congeners (A2B2, A3B, etc.) on the resin via site-site inter-
actions (Table 1) (Scheme 3). The affect of the linker length
on the outcome of the cyclotetramerization reaction was
examined by incorporating commercially available Fmoc-8-
amino-3,6-dioxaoctanoic acid (Fmoc-miniPEG-(OH)) as a
spacer (Table 1, entries1-3) (Scheme 3). As a result of an
increased mobility of the reactive sites, it was not possible
to have complete site isolation even at lower loadings
(0.1 mmol/g). Thus, the spacer was excluded from the
sequence. Solvent effect is another factor regulating site
interactions on the polymer support.10 Solvents in which
the resin swelling is minimal promote site isolation by limit-
ing the mobility of the reactive sites on the resin.10 Thus, the
cyclotetramerization reaction was performed in dodecanol.
However, the selected solvent did not have a significant effect
on the outcome of the reaction. Although neither the reac-
tion time nor the temperature affected the outcome of the
cyclotetramerization reaction, at elevated reaction tempera-
tures (g180 °C) we saw the decomposition of the PEG resin.
Thus, the following experiments were performed at 150 °C.
The protecting group on the ε-amine of the lysine has a
critical effect on the outcome of the cyclotetramerization
reaction. Initially, we employed lysine with a tert-butylox-
ycarbonyl (Boc) protecting group on the ε-amine, and
MALDI-MS mass spectrometry analysis of the cleaved
product showed a major ion at m/z Mþ100 (Table 1, entry 2)
(Scheme 3), corresponding to displacement of the tert-butyl
group of the Boc protecting group with a n-butyl group of
the solvent, butanol (Scheme 3). Further proof for this
transition was obtained by performing the reaction in dif-
ferent alcohols, such as n-pentanol and 2-butanol (data not
shown). Because of the inseparable mixtures obtained with
Boc-protected amine, Fmoc-Lys(Mtt)-OH was employed
SCHEME 5. Conjugation of ZnPc 7b to an Oligonucleotide by
Reductive Amination or Amidation
for the later experiments. The last parameter that we eval-
uated to optimize the base-promoted cyclotetramerization
reaction was the reaction concentration. Since at high reac-
tion concentrations molecules in solution occupy most of the
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SCHEME 6. Synthesis of Pc-Oligonucleotide Conjugate 14 via Huisgen Cycloaddition
reactive sites,10 high degree of site isolation is achieved. We
successfully suppressed the formation of A2B2-type Pc by in-
creasing the phthalonitrile concentration in solution from 0.3 to
0.8 M (Table 1, entries 5 and 6). On the basis of these results,
optimized conditions for the base-promoted cyclotetrameriza-
tion reaction were determined as shown in Table 1, entry 6.
A suspension of polymer-bound phthalonitrile 3, tri-
(ethylene glycol)-substituted phthalonitrile 4,8 Zn(OAc)2,
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in BuOH
were irradiated for 40 min in a microwave to give polymer-
bound AB3-type ZnPc 6 and the corresponding symmetri-
cally substituted B4-type Pc 7a in solution (Scheme 4). ZnPc
7a8 was removed from the mixture by washing the resin with
hot BuOH. Following the cleavage of the target Pc from the
polymer support, asymmetrically substituted ZnPc 7b was
simply purified by filtration through LH-20 column and was
obtained in 27% yield without any contamination with other
Pc congeners. Water-soluble oligo(ethylene glycol)-subsi-
tuted ZnPc 7b exhibits sharp fluorescence emission in the
near-IR region of the electromagnetic spectrum, which is
highly favorable for the desired bioanalytical applications
(Figure 1).
17-mer oligonucleotides, one of which was functionalized
with a carboxylic acid via a C10-linker at the 50-end and the
other of which was modified with a 4-formyl N-hexylbenza-
mide linker at the 50-end (Scheme 5). While bioconjugate 9
was synthesized via reductive amination by incubating the
reaction in a microwave at 70 °C for 30 min. (79% labeling
efficiency calculated based on the absorbance of the un-
reacted oligonuclotide), as well as at room temperature
overnight (Scheme 5, Figure 2), synthesis of bioconjugate
10 was achieved by amidation in a microwave at 75 °C for
30 min (35%) (Scheme 5). As a result of the competition
between the hydrolysis of the ester, formed in situ, and amide
bond formation, the bioconjugate 10 was obtained in low
yield relative to bioconjugate 9. Comparison of the two
conjugation techniques revealed that the synthesis of Pc-
oligonucleotide conjugates could be achieved in relatively
high yields by employing a reductive amination route. From
the results above it has been concluded that while microwave
irradiation did not significantly affect the labeling efficiency
of the oligonucleotide (Figure 2), it reduced the reaction time
from hours to minutes. Thus, the following experiments were
performed in a microwave.
The same method was employed to synthesize ZnPc 8b,
decorated with six tri(ethylene glycol) chains. Synthesis of
ZnPc 8b endorsed the applicability of the method to different
types of phthalonitriles (Scheme 4).
The successful synthesis of the monoamine-functionalized
oligo(ethylene glycol)-substituted AB3-type Pcs was fol-
lowed by the covalent labeling of oligonucleotides with ZnPc
7b utilizing three different bioconjugation techniques. All
Pc-oligonucleotide conjugates were successfully isolated by
reverse-phase ion-pair HPLC. If desired, excess of the Pcs
can be recycled via simple purification to be used for the next
reaction. Initially, ZnPc 7b was conjugated to two different
Figure 1 shows the absorbance-emission spectra of the
bioconjugate 9 in aqueous medium. Bioconjugate 9 was
aggregated in water as judged by the split Q-band in the
absorbance spectrum. However, it had a sharp emission
band at 699 nm, with 9 nm of Stokes shift, in pH 8.5 buffer.
The scope of the Pc-oligonucleotide bioconjugation tech-
niques was extended with a Huisgen cycloaddition, so-called
“click chemistry”.11 For the ease of the purification as well as
(11) Vsevolod, V. R.; Green, L. G.; Fokin, V. V.; Sharpless, K.B. A A
stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective
ligation of azides and terminal alkynes. Angew. Chem., Int. Ed. 2002, 41,
2596–2599.
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to reduce the reaction time, the reactions were performed on
the solid support under microwave irradiation (Scheme 6). In
order to remove the Mtt protecting group without cleaving
the Pc from the solid support, polymer-supported ZnPc 6
was treated with 2% trifluoroacetic acid (TFA) solution to
yield resin-bound ZnPc 11 (Scheme 6). Bromoacetylation of
the ε-amine of the polymer-bound ZnPc 11 to yield ZnPc 12
was followed by treating the resin with NaN3 in a microwave
for 45 min to give the azide-functionalized resin-bound ZnPc
13 (Scheme 6).12 Azide Pc 13 was cleaved from the solid
support, purified by running through a LH-20 column, and
obtained in 19% overall yield. Conjugation of ZnPc 13 to
an 18-mer oligonucleotide, bearing a hexynyl linker at the
50-end, was carried in a microwave at 60 °C for 1 h
(Scheme 6).13 Pc-oligonucleotide bioconjugate 14 was ob-
tained with 80% labeling efficiency, which was calculated on
the basis of the absorbance of the unreacted oligonucleotide.
(10 mL). The reaction was placed in a shaker and kept at room
temperature for 10 min. Following the filtration of the solution,
fresh cleavage cocktail (10 mL) was added to the resin, and the
reaction was shaken 10 more min at room temperature. The
resin was washed with DMF (5 ꢀ 15 mL), CH2Cl2 (3 ꢀ 15 mL)
and next was submitted to amino acid (phthalonitrile) coupling.
Determination of Loading Capacity of the Phthalonitrile-
Bound Resin. Before the acetylation step, ∼50 mg of the poly-
mer-supported Fmoc-Lys-(Mtt)-OH (1) was placed in 5 mL
syringe equipped with coarse frit and dried in the desiccator
for 4-5 h; 6-7 mg of the dry resin was placed in a 3 mL sy-
ringe equipped with coarse frit and swelled in CH2Cl2 (1 mL)
for 30 min. Following the filtration of the CH2Cl2, the resin was
placed in 20% (v/v) 4-methyl piperidine solution in DMF (1 mL)
and kept in the cleavage solution for 20 min. The solution
was collected by filtration, and the resin was washed with
CH3OH (3 ꢀ 2 mL). Combined filtrate solution (7 mL) was
detected with electronic absorption spectrometry. The loading
capacity (mmol/g) of the resin was calculated using the follow-
ing equation:
Conclusion
ðUV300 ꢀ 106 ꢀ 0:007Þ=ð7800 ꢀ mg of resinÞ
The presented solid-phase synthetic method is an impera-
tive approach to synthesize monoamine-functionalized
water-soluble Pcs in a short period of time. Coupling of the
effective method with microwave irradiation not only im-
proved the yield of the cyclotetramerization reaction but also
reduced the reaction time from hours to minutes.8 Cova-
lent conjugation of Pcs to oligonucleotides, modified with
various types of functional groups, was successfully achieved
by employing different types of bioconjugation techniques.
The oligonucleotides were efficiently labeled and isolated in
high yields. The discussed synthetic methods are being
applied to synthesis of a wide variety of Pcs and Pc-oligo-
nucleotide conjugates for bioanalytical applications.
Polymer-Supported 4-(3,4-Dicyanophenoxy)Benzoic Acid 3.
The resin 1 (0.4 g) with 0.1 mmol/g Lys-(Mtt)-OH loading was
placed in CH2Cl2 (3 mL). HOBt (0.44 mmol, 59 mg), HBTU
(0.44 mmol, 167 mg) and the carboxylated phthalonitrile 2
(0.44 mmol, 110 mg) were dissolved in DMF (1 mL). The
solution and DIEA (0.88 mmol, 0.15 mL) were added to the
resin, and the reaction flask was placed in a shaker and kept at
room temperature for 2 h. (In order to have an efficient coupling
yield, amino acid and/or carboxylated phthalonitrile concentra-
tion in the reaction was adjusted to 0.2 M.) The resin was washed
with CH2Cl2 (4 ꢀ 15 mL), DMF (3 ꢀ 15 mL), and CH2Cl2 (3 ꢀ
15 mL). FT-IR (KBr, cm-1): 3515, 2863, 2232, 1751, 1738, 1656,
1610, 1588, 1542, 1508, 1452, 1348, 1300, 1177, 1146, 1088, 1031,
991, 951, 937, 920, 864, 840, 801, 773, 751, 709, 660, 639.
General Procedure for Synthesis of AB3-Type Pcs 7b, 8b, and
13. The resin 3 (0.4 g) was swelled in BuOH (2 mL) for 30 min in
the microwave vessel. Corresponding phthalonitrile (5 mmol)
and Zn(OAc)2 (1.4 mmol, 0.25 g) were placed in a 20 mL glass
vial in BuOH (4 mL) and heated up to 90 °C in water bath. DBU
(2.8 mmol, 0.38 mL) was added to the suspension, and the
mixture was vigorously stirred until all of the metal salt was
dissolved. The solution was quickly transferred to the micro-
wave vessel, having the resin 3 with BuOH (2 mL). The reaction
was irradiated in the microwave at 850 W. The temperature was
ramped to 150 °C in 5 min and was held at the temperature for
40 min. The resin was washed, until a colorless filtrate was
obtained, first with hot BuOH (12 ꢀ 25 mL) and then hot
CH2Cl2 (3 ꢀ 25 mL).
Experimental Section
Polymer-Supported Fmoc-Lys-(Mtt)-OH 1. Prior to the
synthesis, the Rink Amide resin (0.4 g) with 0.52 mmol/g loading
capacity was swelled in CH2Cl2 (20 mL) for 30 min. The solvent
was filtered off, and CH2Cl2 (7 mL) was added to the
well-swelled resin. Fmoc-Lys-(Mtt)-OH (0.05 mmol, 31 mg),
1-hydroxybenzotriazole (HOBt) (0.05 mmol, 6.7 mg), and O-(1-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoropho-
sphate (HBTU) (0.05 mmol, 19 mg) were dissolved in N,N-
dimethylformamide (DMF) (1 mL). The solution was added to
the resin in CH2Cl2. Diisopropylethyl amine (DIEA) (0.1 mmol,
17.4 μL) was added to the suspension, and the reaction flask was
placed in a shaker and kept at room temperature for 1 h to give
the resin 1 with 0.1 mmol/g of loading. The resin was washed
with CH2Cl2 (4 ꢀ 15 mL), DMF (2 ꢀ 15 mL), and CH2Cl2 (4 ꢀ
15 mL).
Acetylation of the Polymer Support. The resin 1 was suspended
in 0.28 M solution of DIEA in DMF (25 mL). Ac2O (8.4 mmol,
0.8 mL) was added to the suspension, and the reaction was placed
in a shaker and kept at room temperature for 3 h. The resin was
washed with DMF (5 ꢀ 15 mL) and CH2Cl2 (5 ꢀ 15 mL).
General Procedure for Fmoc Cleavage. The resin was sus-
pended in 20% (v/v) solution of 4-methyl piperidine in DMF
General Procedure for Cleavage of AB3-Type Pc 7b, 8b, and
13. The resin was suspended into a solution of TFA/CH2Cl2/
TIPS (triisopropylsilane) (20:78:2; 25 mL) (TFA concentration
was increased to 30% for the cleavage of the ZnPc 13) and
agitated in the shaker for 3 h at room temperature. Filtrate was
evaporated to dryness, and the crude mixture was purified by
filtration through a LH-20 column using CH3OH as eluent (see
Supporting Information for ZnPc 8b).
ZnPc 7b. Obtained as a blue solid (14 mg, 27% yield).
Condition B was employed to perform HPLC. tR = 29.45
min. MALDI-MS: calcd C66H75N11O15Znþ 1325.47; found
1324.98. λmax(abs) (DMSO): 681 ( 1 nm (log ε = 5.2). λmax
(em) (DMSO): 691 nm. φf: 0.10.
(12) Quemener, D.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H.
RAFT and click chemistry: A versatile approach to well-defined block
copolymers. Chem. Commun. 2006, 48, 5051–5053.
(13) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.;
Cloarec, J. P.; Praly, J. P.; Vasseur, J. J.; Morvan, F. Microwave assisted
“Click” Chemistry for the synthesis of multiple labeled-carbohydrate oligo-
nucleotides on solid support. J. Org. Chem. 2006, 71, 4700–4702.
General Procedure for Purification of Symmetrically Substi-
tuted Pcs 7a and 8a. Filtrate solutions were combined and
evaporated to dryness. The crude mixture was dissolved in a
minimum amount of CH3OH, and a large excess of diethyl ether
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Erdem et al.
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(30X) was added to solution. The solution was kept at -20 °C
overnight, and the suspension was centrifuged to give Pcs 7a and
8a as a blue/green solid. If desired, the Pcs can further be purified
by running thorugh LH-20 column (see Supporting Information
for ZnPc 7a).
ZnPc 8a. Obtained as a green solid (1.9 g, 20% yield). (Yields
of the asymmetrically substituted Pcs were calculated on the
basis of the phthalonitrile loading, 0.1 mmol/g, of the resin 3.)
Condition A was employed to perform HPLC. tR = 37.90 min.
ESI calcd C88H128N8O24S8Znþ 2003.93; found 2003.59.
mixtures, and the suspensions were placed in -20 °C and kept
for 1 h. The suspensions were centrifuged at 4 °C at 12,000 rpm
for 30 min. The precipitates were reconstituted in 0.05 M
triethylammonium acetate (TEAA) solution in water and iso-
lated by HPLC in 79% labeling efficiency (76% labeling effi-
ciency was obtained when the reaction was incubated at room
temperature). Condition B was employed to perform HPLC.
tR = 18.95 min. ESI-TOF calcd (M - 3) 2255.14; found 2255.14.
λ
max(abs) (H2O): 640, 690 nm. λmax (em) (pH 8.5 buffer; 4 mM
MgCl2, 15 mM KCl 10 mM TRIS) 699 nm. Unreacted oligo-
nucleotide tR = 1.38 min.
λmax(abs) (DMSO): 709 nm (log ε = 5.5). λmax (em) (DMSO):
716 nm. φf: 0.13
Preparation of Oligonucleotide-Pc Conjugate 10. The se-
quence of the 17mer oligonucleotide is 50(carboxylic acid
C10)-GTA AAA CGA CGG CCA GT 30. To a 3.6 mM
oligonucleotide solution in water (4.4 μL) in an Eppendorf tube
were added NaHCO3 buffer (pH = 7.6; 60 μL), 10 mM ZnPc 7b
solution in CH3OH (30 μL), 150 mM 1-ethyl-3-(3-dimethyl-
aminopropyl)-carbodiimide (EDC) solution in DMSO (5 μL),
and 350 mM N-hydroxysuccinimide (NHS) solution in DMSO
(5 μL) in the given order. The Eppendorf tube was placed in a
screw-capped microwave vessel containing enough water to
surround the Eppendorf tube (∼1.5 mL), and the vessel was
tightened. The microwave vessel was equipped with temperature
and pressure probes, and the reaction was irradiated at 400 W.
The temperature was ramped to 75 °C in 1 min and held at the set
temperature for 30 min. Following the completion of the reaction,
cold ethanol (300 μL) and 3 M NaCl solution in water (10 μL)
were added to reaction mixture, and the suspension kept at
-20 °C for 1 h. The suspension was centrifuged at 4 °C at 12,000
rpm for 30 min. The precipitate was reconstituted in 0.05 M
TEAA solution in water and isolated by HPLC in 35% labeling
Selective Cleavage of Mtt Protecting Group (11). The Pc-
bound polymer support was suspended in a solution of TFA/
CH2Cl2/TIPS (2:96:2; 10 mL) and agitated for 2 min. The
yellow-colored solution was filtered off. This step was repeated
for 7 times using 10 mL of cleavage cocktail at a time. The resin
10 was washed with CH2Cl2 (10 ꢀ 10 mL) and submitted to the
next step.
Bromoacetylation of Polymer-Bound ZnPc 11 (12). Polymer-
bound ZnPc 11 (0.5 g) with 0.1 mmol/g Pc loading, was swelled
in DMF for 30 min prior the synthesis. Following the filtration,
the resin was suspended in 1 mL of DMF. Bromoacetic acid (1.1
mmol, 153 mg) and N,N0-diisopropylcarbodiimide (DIC) (1.32
mmol, 126.2 mg) were dissolved in 2 mL of CH2Cl2/DMF (1:1)
mixture, and the solution was added to resin. The reaction was
agitated for 45 min. The reaction was repeated twice. The resin
was washed with DMF (6 ꢀ 15 mL), CH2Cl2 (3 ꢀ 15 mL), and
DMF (3 ꢀ 15 mL) and used in the next reaction. Approximately
50 mg of the resin was suspended into a solution of TFA/
CH2Cl2/TIPS (20:78:2; 1 mL) for 3 h at room temperature.
The collected filtrate was evaporated to dryness, and the crude
mixture was purified by filtration through a LH-20 column
using CH3OH as eluent. MALDI-MS: calcd C68H76N11-
O16BrZnþH 1449.72; found 1449.80.
Polymer-Bound Azide Pc 13. Polymer-bound bromoacety-
lated ZnPc 12 (0.5 g) was suspended in 4 mL of DMF/H2O (5:1
v/v) solution. To the suspension was added NaN3 (5.5 mmol,
357 mg). The microwave vessel was placed in the microwave,
and the reaction was irradiated at 400 W. The temperature was
ramped to 120 °C in 3 min and was held at that temperature for
45 min. Following the reaction, the resin was washed with DMF
(5 ꢀ 30 mL), MeOH/H2O (6 ꢀ 30 mL), DMF (2 ꢀ 30 mL), and
CH2Cl2 (4 ꢀ 30 mL). ZnPc 13 was cleaved from the polymer
support and purifed by employing general methods described
above.
ZnPc 13. Obtained as a blue solid (11 mg, 19% overall yield).
Condition B was employed to perform HPLC. tR = 27.48 min.
MALDI-MS: calcd C66H76N14O16ZnþH 1409.50; found
1409.50. λmax(abs) (DMSO): 681 ( 1 nm (log ε = 5.6). λmax
(em) (DMSO): 691 nm. φf: 0.09
Preparation of Oligonucleotide-Pc Conjugate 9. The sequence
of the 17mer oligonucleotide is 50(aldehyde C6)-GTA AAA
CGA CGG CCA GT 30. To a 4.3 mM oligonucleotide solution
in water (4.4 μL) in an Eppendorf tube were added NaOAc
buffer (pH = 5.5; 50 μL), 10 mM ZnPc 7b solution in CH3OH
(51.6 μL), and 10 mM NaCNBH3 solution in CH3OH (10 μL) in
the given order. The reaction was incubated either at room
temperature for 16 h or in the microwave for 30 min. The
Eppendorf tube was placed in a screw-capped microwave vessel
containing enough water to surround the Eppendorf tube
(∼1.5 mL), and the vessel was tightened. The microwave vessel
was equipped with temperature and pressure probes, and the
reaction was irradiated at 400 W. The temperature was ramped
to 70 °C in 1 min and held at the set temperature for 30 min.
Following the completion of the reactions, cold ethanol (300 μL)
and 3 M NaCl solution in water (10 μL) were added to reaction
efficiency. Condition B was employed to perform HPLC. tR
19.17 min. Unreacted oligonucleotide tR = 1.43 min.
=
Preparation of Oligonucleotide-Pc Conjugate 14. The se-
quence of the 18mer oligonucleotide is 50(Hexynyl C6)-GTA-
AAA-CGA-CGG-CCA-AGT. To a 5 mM oligonucleotide
solution in water (8 μL) in an Eppendorf tube were added
10 mM ZnPc 7b solution in CH3OH (120 μL), 20 mM solution
of CuSO4 5H2O in water (24 μL), freshly prepared 100 mM
3
solution of Na ascorbate in water (24 μL) and water (4 μL) in the
given order (water/CH3OH, 1:2). The Eppendorf tube was
placed in a screw-capped microwave vessel containing enough
water to surround the Eppendorf tube (∼1.5 mL), and the vessel
was tightened. The microwave vessel was equipped with tem-
perature and pressure probes, and the reaction was irradiated at
400 W. The temperature was ramped to 60 °C in 1 min and held
at the set temperature for 60 min. Following the completion of
the reaction, cold ethanol (300 μL) and 3 M NaCl solution in
water (10 μL) were added to reaction mixture, and the suspen-
sion kept at -20 °C for 1 h. The suspension was centrifuged at
4 °C at 12,000 rpm for 30 min. The precipitate was reconstituted
in 0.1 M TEAA solution in water and isolated by HPLC in 80%
labeling efficiency. Condition B (using 0.1 M TEAA solution)
was employed to perform HPLC. tR = 17.04 min. Unreacted
oligonucleotide tR = 1.56 min.
Acknowledgment. We thank the National Science Foun-
dation (CHE-0304833, EPS-0346411) and the National In-
stitutes of Health (EB006639 and HG01499) for financial
support.
Supporting Information Available: Experimental proce-
dures for the synthesis of phthalonitriles, spectroscopic data,
and HPLC chromatograms for 7-10, 13 and 14. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
9286 J. Org. Chem. Vol. 74, No. 24, 2009