Microwave assisted solid-phase synthesis of substituted tetraazaporphyrins and a phthalocyanine-peptide conjugate

Article abstract of DOI:10.1142/S1088424610002744

Various asymmetrically substituted phthalocyanines (Pcs) and porphyrazines (Pzs) have been synthesized in good yields using a solid-phase synthesis method with a poly(ethylene glycol) (PEG) resin attached to an indole linker as the solid support. These co

Full text of DOI:10.1142/S1088424610002744

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2010; 14: 891–903
DOI: 10.1142/S1088424610002744
Published at http://www.worldscinet.com/jpp/
Microwave assisted solid-phase synthesis of substituted
tetraazaporphyrins and a phthalocyanine-peptide conjugate
Mercy Mudyiwa, Margaret W. Ndinguri, Steven A. Soper and Robert P. Hammer*†
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1803, USA
Received 28 March 2010
Accepted 30 September 2010
ABSTRACT: Various asymmetrically substituted phthalocyanines (Pcs) and porphyrazines (Pzs) have
been synthesized in good yields using a solid-phase synthesis method with a poly(ethylene glycol)
(PEG) resin attached to an indole linker as the solid support. These compounds are formed by cross
condensation of maleonitrile or phthalonitrile with another phthalonitrile covalently bonded to the solid
support with an amino linking group. The polymer bound Pc or Pz is separated by filtration, and washing
the symmetrical Pc or Pz by-product. The amine Pc-appended to polyethylene glycol resin is further
reacted to yield azide whilst still on the solid support. Cleavage of the Pc or Pz off the solid support results
in 3:1 asymmetric Pc or Pz with high degree of purity, requiring minimal further purification. The use of
hydrophilic PEG-based resin allows the symmetrical compound to be removed completely by washing
whereas the acid labile indole linker makes it easier to cleave the product under mild conditions. The
conjugation abilities of these compounds have been demonstrated by the successful conjugation of one
of the azide Pcs with a peptide elaborated with an alkyne function. Use of microwave for the synthesis of
these compounds results in shorter reaction times, higher yields and higher degree of purity.
KEYWORDS: solid-phase synthesis, microwave assisted, tetraazaporphyrins, phthalocyanine-peptide
conjugate, macrocyclization.
applications derives from the increased ability to tune
INTRODUCTION
the exact structure and physical properties of the por-
Phthalocyanines (Pcs) and porphyrazines (Pzs) and
phyrinoids to suit the application. The ability to specifi-
their congeners are members of the tetraazaporphy-
cally design properties of Tazps is due in large part to
rin (Tazp) family of aromatic macrocycles that have
advances in the synthetic approaches that allow control
uniquely tunable physical, photophysical, and chemical
of structural elements such as the type and position
properties, which make them suitable for a wide range of
of peripheral functionalization [14] variation of the
applications that range from their original use as dyes and
central metal core [5] and symmetry/asymmetry of the
pigments [1], to cancer therapy [2], to designer supra-
porphyrinoid.
molecular assemblies [3] and nanotechnology [3, 4].
Unsymmetrically substituted Tazps (A3B,A2B2, (AB)2,
The synthesis and applications of Tazps have been sum-
etc.) are particularly useful in biological and materials
marized in a recent monograph [4] as well as in a num-
applications as they allow orientation of functionality
ber of review articles [1, 4–13]. The continued growth
around the Tazp in a defined manner [10, 12]. A number a
of interest in Tazp derivatives for so many different
synthetic approaches to unsymmetrical Tazps have been
developed including statistical tetramerization of two or
SPP full member in good standing
more monomer species (e.g. malonitrile, phthalocyanine)
*Correspondence to: Robert P. Hammer, email: robert.
hammer@newenglandpeptide.com, tel: +1 978-630-0020
†Current address: New England Peptide, 65 Zub Lane, Gardner,
MA 01440, USA
[11], ring expansion of subphthalocyanines (and related
structures) [13] and polymer-supported approaches in
solution [7] or in the solid-phase [9]. There are a num-
ber of recent examples of the use of improved synthetic
Copyright © 2010 World Scientific Publishing Company

892
M. MuDyIWa et al.
approaches to unsymmetrical Pcs, Pzs and related deriv-
atives to provide compounds with improved biological
or materials properties. For example, Barrett, Hoffman
and coworkers reported a multigram synthesis of an A3B
porphyrazine derivative using a statistical condensation
approach [15]. The same groups have made extensive
use of polymerization-based capture-release synthesis of
porphyrinoids [6, 7, 16] by statistical condensation of two
different monomers, one containing a norbornene group
that is polymerizable by ROM, which allows selective
capture and subsequent release of the A3B compounds.
Torres and collaborators have utilized a statistical con-
densation approach to prepare an asymmetrically-sub-
stituted, monoazide-functionalized phthalocyanine that
is utilized to decorate single-wall carbon nanotubes via
Click chemistry [17]. The resulting Pc-nanotube conju-
gates function as a highly efficient light capturing photo-
voltaic cell. A number of groups are developing near-IR
absorbing asymmetrically substituted Tazps as photody-
namic therapy agents including recent work by Zimick
and coworkers on aza-substituted porphyrazines [18],
as well as developments by the groups of Barrett and
Hoffman on napthyl-substitued [19] and chiral porphy-
razines [20].
of tetrazaporphyrinoids from mixtures of maleonitrile
and phthalonitrile monomers. Additionally, one of the
resulting asymmetrically-substituted tetraazaporphyrin
containing an azide functional group is utilized in a solid-
phase “Click” conjugation protocol to provide a peptide-
Tazp conjugate.
RESULTS AND DISCUSSION
The retrosynthetic scheme for the tetraazaporphyrins
is as shown in Fig. 1. In this case the amine phthalonitrile
group is attached to a hydrophilic, crosslinked polyeth-
ylene glycol (PEG) support and reacted with an excess
of a different phthalonitrile or maleonitrile in solution
providing a 3+1 condensation product attached to the
resin which upon cleavage provides the 3+1 asymmetric
Tazp product. Conjugation can be carried out whilst the
product is still on the solid support. Aminomethyl-Chem-
Matrix PEG resin was chosen because of the hydrophilic
nature of the polymer core, which allows for complete
removal of the symmetrical Tazp by washing. We also
chose an aldehyde-containing linker, based on formyl
indole that allows the attachment of the phthalonitrile
through an amine function by reductive amination. This
amine serves as diversity point for attachment of a wide
range of functionalities, making the synthesis of asym-
metric Pcs or Pzs with different functional groups pos-
sible. Using the amine whilst still attached to the resin,
we were able to link the Pcs to the azide functionality and
not much purification was required on cleavage of the Pc
Our laboratory has been developing porphyrinoids as
fluorescent tags for biological molecules and bioanalyti-
cal applications [21–26]. This has required addition of
water-solubilizing groups as well as a site for covalent
attachment of the porphyrinoids. For example, we have
utilized carboxylated or pegylated Pcs for labeling of
DNA oligonucleotides for both PCR-based
assays and in mutation detection schemes as
molecular beacons [21, 25]. Building on the
precedent of Leznoff and coworkers [27, 28],
we have developed a generalized solid-phase
method for asymmetrically substituted Pcs,
using modified Linstead cyclotetramerization
approach [22, 23]. In this method one phtha-
lonitrile group is attached to a hydrophilic,
FG'
FG
SGn
SGn
N
M
N
N
M
N
N
N
N
N
N
N
N
N
N
N
N
N
SGn
SGn
SGn
SGn
crosslinked polyethylene glycol (PEG) sup-
port and reacted with an excess of a differ-
ent phthalonitrile in solution providing a 3+1
condensation product attached to the resin.
This approach allows the incorporation of
functional group handles into the solid-sup-
ported monomer and solubilizing groups into
the three phthalonitrile subunits that add from
the solution phase. The use of the hydrophilic
PEG-based resin is critical to the reproduc-
ibility and high yield of the approach. Also,
the utilization of microwave heating greatly
TAP-bioconjugate
"AB3-type" TAP
X FG
SGn
CN
N
X
FG
CN
+
N
N
N
N
M
N
N
CN
N
SGn
CN
SGn
SGn
reduces the time required to prepare the resin-
bound tetraazaporphyrins and provides higher
= Polymer support
SGn = Solubilizing group(s)
FG, FG' = Functional group
X
yields [22]. The current paper presents an
expansion of this method wherein alterative
linker chemistries are applied and solid-sup-
ported synthesis is applied to the synthesis
with a cleavable linker
M = Metal
Fig. 1. Retrosynthetic scheme of the tetraazaporphyrins
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MICrOWave aSSISteD SOlID-PhaSe SyntheSIS Of SubStItuteD tetraazaPOrPhyrInS
893
NH2
SR
RS
RS
CN
CN
NC
NC
SR
SR
CN
CN
O
CN
CN
i: 10
ii: 11
12
i: 6
ii: 7
1
6, 10, 12: R = (C2H4O)2CH3
7: R = p-C6H4COOBu
11: R = C3H6COOBu
Fig. 2. Phthalonitriles and maleonitriles used in the Pcs and Pzs synthesis
off the resin. The azide Pc on solid support was also suc-
cessfully conjugated to a peptide, also making purifica-
tion of the product on cleavage off the resin much easier.
The phthalonitriles and maleonitriles used for the syn-
thesis of the Pcs and Pzs in this study are shown in Fig. 2.
These were prepared using base-catalyzed nucleophilic
displacement of nitro, chloride or bromide on commer-
cially available phthalonitriles and disodium maleonitrile
(Na2MNT). See the Supporting information section for
the details of their synthesis.
As depicted in Scheme 1, the polymer was attached
to the indole linker, after which the amine-phthalonitrile
1, prepared using commercially available 4-nitro-
phthalonitrile and p-hydroxyaniline in 72% yield, was
covalently attached by the reductive amination method to
the so-formed indole PEG resin 2 to give polymer bound
phthalonitrile 3.
of the commercially available 4,5-dichlorophthalonitrile
with monomethyl diethylene glycol thiol 4 [29–31]. Con-
densation of resin-bound phthalonitrile 3 with the phthalo-
nitrile 6 in the presence of zinc acetate dihydrate, catalytic
amount of DBU and 1-BuOH was achieved by micro-
wave heating. The temperature was ramped to 180 °C in
15 min and maintained at that temperature for 1 h to give
compound 13a in solution and 13b on solid support. The
symmetrical Pc 13a was drained, and the solid supported
compound 13b was further washed with 1-BuOH and
CH2Cl2 until the washings were clear.
Amines bound on indole resin can easily be cleaved by
5–10% TFA/CH2Cl2 (4 h) with no need for a cation scav-
enger [32, 33]. However, we were able to increase the
acid concentration in the cocktail mixture to 20–25% in
the presence of a scavenger such as, triisopropyl silane,
and reduced the Pc or Pz exposure to acid by 1–2 h. The
crude products showed a high degree of purity with no
evidence of the symmetrical compound but some of the
The synthesis of phthalonitriles 6 was accomplished by
base-catalyzed nucleophilic aromatic chloride displacement
NH2
O
O
CN
CN
O
1) DCM, DMF, rt
DIC, HOBt
N
NH2
1
O
O
+
O
2)
N
O
O
DCE, TMOf, rt
NaBH(OAc)3
24 h
HN
DIPEA, DCM, rt , 2 h
2
OH
CN
CN
O
NH
N
O
HN
3
Scheme 1. Attachment of phthalonitrile 1 to PEG-resin bound indole linker
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 893–903

894
M. MuDyIWa et al.
2:2 compound was also present as determined by MALDI
MS characterization. This problem was attributed to the
reaction between the polymer-bound phthalonitriles if
the active sites were too close to each other as a result of
low concentration of the phthalonitrile in solution or high
concentration of the active sites on the starting polymer.
This problem was solved by either significantly increas-
ing the concentration of the starting phthalonitrile (30
times) or reducing the available active sites. Although
both methods worked well, we settled for the reduction
of the loading capacity and capping the remaining amine
sites because the other method consumed a lot of the
starting phthalonitrile, making it synthetically non-via-
ble. The starting resin loading capacity of 0.6 mmol.g-1
was reduced to 0.19 mmol.g-1 and the remaining free
amines capped with acetic anhydride in the presence of
diisopropyl amine to give resin-bound phthalonitrile 3
with a reduced loading capacity. Condensation of 3 with
phthalonitrile 6 in the presence of zinc(II) acetate dihy-
drate and 1-BuOH by microwave irradiation gave Pc 13a
in solution and the solid supported, compound 13b. The
asymmetric Pc 13c was freed from the polymer support
after washing off 13a from the solid supported 13b and
passed through a plug of Sephadex LH-20 to remove low
molecular weight impurities. The yield of this compound
was 10% based on the loading of the phthalonitrile 1 on
the resin. The symmetrical Pc 13a was purified by silica
column, eluting with 10% MeOH in CH2Cl2 to give 13a
in 17% yield. Compounds 13a and 13c show Q-bands
above 700 nm, which is typical of 4,5-dithio-substituted
compounds [34]. The fluorescent quantum yield of 13a
was found to be 0.15 and is within the expected range of
similar phthalocyanines with zinc as the metal at the cen-
ter [35]. The normalized absorbance and emission spec-
tra of 13a are shown in Fig. 3. Compound 13a exhibits
highly pronounced Q-band at 710 nm and emission band
centered at 719 nm. The Q-band of 13c is slightly blue
shifted to 705 nm and has a maximum emission band at
714 nm.
N,N-diisopropylethylamine (DIPEA) for 16 h resulted in
chloro-acetamide Pc bound to the resin which was dried
in vacuo before the nucleophilic displacement of the
chloride by NaN3 in DMSO. The azide-functionalized
resin bound Pc was then subjected to the 20% TFA in
CH2Cl2 cocktail to free the asymmetrical azide Pc 13d.
Following purification of the crude azide Pc 13d by pass-
ing through a plug of Sephadex LH-20 to remove low
molecular weight impurities, the asymmetric azide Pc
13d was obtained in 14% yield based on the loading of
the starting phthalonitrile 1 on the resin. The azide Pc
13d has maximum absorption centered at 700 nm and
maximum emission band at 712 nm.
The synthesis of compounds 14a and 14c started by
Fischer esterification of p-benzoic thiol with 1-butanol
and H2SO4 as catalyst to give butyl 4-mercaptobenzoate
5, which was used to synthesize the ester phthalonitrile 7
by base-catalyzed nucleophilic aromatic displacement of
4,5-dichlorophthalonitrile in 81% yield. As depicted in
Scheme 2, macrocyclization of phthalonitrile 7 with the
resin-bound phthalonitrile 3 using zinc acetate dihydrate,
catalytic amount of DBU and 1-BuOH heating at 180 °C
by microwave irradiation gave Pc 14a in solution and
the solid supported, compound 14b. Following wash-
ing of 14a and cleavage off the solid support of Pc 14c,
the compounds were further purified on a silica column
in the case of 14a and on a Sephadex LH20 column for
asymmetric Pc 14c. Pc 14a, obtained in 19% yield, has
its Q-band centered at 706 nm whereas that of 14c with a
yield of 11% is blue shifted to 697 nm.
Asymmetric Pz were also obtained by the solid sup-
port method. Disodium maleonitrile (Na2MNT) [36]
was used to synthesize maleonitrile 10 by the reaction
similar to literature procedures in which 2-(2-methoxy-
ethoxy)ethyl bromide, catalytic amount of NaI and
Na2MNT were reacted by refluxing in dry acetone for
24 h [37, 38]. Pure maleonitrile 10 was obtained as yel-
low viscous oil upon chromatography of the crude dark
brown oil obtained after rotary evaporation of the reac-
tion mixture on silica, eluting with diethyl ether/CH2Cl2
in the ratio of 2:1. Unfortunately, attempts to directly
form an asymmetric zinc inserted Pz were not success-
ful as the cleavage resulted in mixture of the correct
product together with the free-base version. This was
attributed to the basic nature of the Pz core which made
it possible for the zinc to dissociate from the core of the
Pz under acid conditions. As shown in Scheme 3, cross-
over macrocyclization of maleonitrile 10 with the resin
bound phthalonitrile 3, refluxing with 1-BuOH for 24 h
in the presence of Mg(OBu)2 resulted in Pzs 15a and
15b on solid support. Following washing, cleavage, and
purification, 15a was obtained in 23% yield whereas
15b was obtained in 11% yield. The Zn metal insertion
of Pz 15b was carried out in DMF and Zn(II) acetate at
100 °C for 24 h to obtain 15d in yield of 9%. Treatment
of 15a with 10% acetic acid gave the free-base Pz 15c
in 17% yield.
Azide Pc 13d was synthesized as depicted in
Scheme 2. Compound 13b was dried under vacuum
for 24 h. Reaction of the swollen 13b in anhydrous
CH2Cl2 with chloroacetyl chloride in the presence of
Fig. 3. Normalized absorbance and emission spectra of 13a in
DMSO
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MICrOWave aSSISteD SOlID-PhaSe SyntheSIS Of SubStItuteD tetraazaPOrPhyrInS
895
H
N
CN
R1
R4
BuOH, DBU
Zn(OAc)2·2H2O
R2
R3
CN
CN
CN
+
N
O
O
microwave 180 °C, 1 h
HN
3
H
N
R3
R2
R3
O
R1
R4
R4
R2
R1
R2
R1
R3
R4
N
N
N
R3
R2
N
N
N
N
N
N
N
R1
R4
O
NH
R1
R2
Zn
N
N
Zn
N
N
R2
R1
N
+
R3
N
N
R4
R4
R3
R1
R2
R4
N
N
N
N
N
1) wash off symmetric Pc
R
Zn
N
N
3
R1
R4
R1
R4
2) 15% TFA in DCM
rt, 2 h
R2
R2
R3
R3
R1
R2
i: 13b
ii: 14b
i: 13a
ii: 14a
N
1) wash off symmetrical Pc
2) 2-chloroacetyl chloride
DIPEA, CH2Cl2
R2
O
R2
i: 13c
ii: 14c
NH2
3) NaN3, DMSO
4) 20% TFA in CH2Cl2
R3
R2
R1
R4
R2
R1
R3
N
R3
N
N
N
Zn
N
N
N
N
13a, 13b, 13c, 13d: R2 & R3 = S(C2H4O)2CH3; R1 & R4 = H
14a, 14b, 14c: R2 & R3 = p-SC6H4COOBu; R1 & R4 = H
R1
R2
R4
R3
O
13d
O
NH
–N
N+
N
Scheme 2. Solid-phase synthesis of symmetrical Pcs 13a and 14a, amine Pcs 13c and 14c, azide Pc 13d
Pzs 16a and 16b were obtained by first converting
disodium maleonitrile (Na2MNT) [36] to maleonitrile
11. The coupling reaction of 1-butanol and 4-bromo-
butyric acid using diisopropylcarbodiimide (DIC) and
diisopropylcarbodiimide (DMAP) in solvents, dichlo-
romethane and dimethyl formamide, gave an oily sus-
pension containing butyl 4-bromobutanoate 9 [39]. The
oily suspension was filtered to remove the precipitate
before further purification on a silica column, eluting with
dichloromethane to give the desired product, butyl 4-bro-
mobutanoate 9, as yellow oil in 91% yield. Maleonitrile
11 was then obtained by the reaction of 4-bromo-butyl
butanoate 9 in a reaction similar to literature procedures
whereby the two reactants Na2MNT and 4-bromo-butyl
butanoate 9 were heated to reflux in dry acetone in the
presence of catalytic amount of NaI for 24 h [38]. Pure
maleonitrile 11 was obtained in 72% yield following
flash chromatography, eluting with dichloromethane.
The Pzs 16a and 16b were obtained by macrocyclization
of resin bound phthalonitrile 3 with maleonitrile 11 in
the presence of Mg(OBu)2, and 1-butanol at 110 °C for
18 h to give a deep blue color of 16a in solution and 16b
attached to the solid support. After washing of 16a, 16b
was cleaved from the solid support using 15% TFA in
CH2Cl2. The compounds were purified by passing each
through a column of silica, eluting with 10% MeOH in
ethyl acetate in both cases to give 16a in 22% yield and
16b in 9% yield. The Zn metal insertion of Pz 16b was
carried out using Zn(II) acetate dihydrate in the DMF at
100 °C for 24 h to obtain 16d in yield of 8%. Treatment
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896
M. MuDyIWa et al.
Mg(OBu)2
BuOH
NC
NC
SR
SR
110 °C, 24 h
i: 10
ii:11
SR
N
SR
RS
N
O
N
N
SR
SR
SR
SR
RS
RS
N
N
N
N
N
N
N
N
HN
M
M
+
N
N
RS
N
N
N
SR
SR
RS
RS
O
15a: M = Mg
16a: M = Mg
NH
10% AcOH
CH2Cl2
1) wash off symmetric Pz
2) 15% TFA in DCM
15c: M = 2H
16c: M = 2H
SR
RS
SR
N
O
N
N
SR
SR
RS
N
N
N
N
NH
N
Zn(OAc)2·2H2O
DMF 100 ˚C
H2N
N
Zn
N
N
N
HN
N
RS
SR
RS
N
RS
SR
RS
i: 15b
ii: 16b
i: 15d
ii: 16d
O
NH2
i) 10, 15a, 15b, 15c, 15d: R = (C2H4O)2CH3
ii) 11, 16a, 16b, 16c, 16d: R = C3H6COOBu
Scheme 3. Solid-phase synthesis of symmetrical Pzs 15a, 15c, 16a and 16c and amine Pzs 15b, 15d, 16b and 16d
of 16a with 10% acetic acid gave the free-base Pz 16c in
18% yield. The free-base compound 16c showed a split
Q-band with one centered at 670 nm and the other one at
710 nm. The compounds with the metal at the core have
their Q-bands, slightly broadened around 675 nm.
The water soluble peptide 17 used for conjugation
was synthesized by standard solid phase chemistry using
an Fmoc-PAL-PEG-PS support on a Pioneer Peptide
synthesizer (see the Supporting information section for
details of synthesis).
The solid-supported azide Pc 18 was synthesized in a
similar manner to the one depicted in Scheme 2 except
that phthalonitrile 12, synthesized by base-catalyzed
nucleophilic aromatic nitro displacement of the commer-
cially available 3-nitrophthalonitrile with monomethyl
diethylene glycol thiol 4 [29–31] was used in place of
phthalonitrile 6. The conjugation of peptide 17 to solid
supported azide Pc 18 was then carried out using ‘Click
chemistry’ whereby the solid supported azide functional-
ized Pc 18 was reacted with the water soluble alkyne-
functionalized peptide using a method similar to the
one described by François Morvan by use of microwave
irradiation [40]. The alkyne functionalized water soluble
peptide 17, the solid-supported azide Pc 18, copper(II)
sulfate, sodium ascorbate, methanol and water were all
transferred into a microwave vial. The vial was sealed
and irradiated with microwave at 60 °C for 45 min. Treat-
ment of the resin beads with a cocktail of TFA/CH2Cl2
(1:4) for 2 h at room temperature afforded the triazole
conjugate in 31% yield. The conjugate was characterized
by ESI mass spectrometry and purity verified by HPLC,
carried out using the gradient elution with TEAA, MeOH
and THF. Compound 19 is readily soluble in MeOH and
is partially soluble in a MeOH/H2O solution, an indica-
tion of great improvement on the solubility properties of
the Pc.
All Pcs and Pzs were characterized by HPLC,
MALDI, and absorbance and fluorescence spectroscopy.
The yields of all asymmetrical compounds were between
8–14% based on the loading of the phthalonitrile bound
to the resin, whilst those of the symmetrical compounds
were between 17–23% as shown in Table 1. Except for
compounds 15b, 15d, 16b and 16d, which were purified
on a silica column, all asymmetric compounds were puri-
fied on Sephadex LH20 column, whereas all symmetric
ones were purified on a silica column. These compounds
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MICrOWave aSSISteD SOlID-PhaSe SyntheSIS Of SubStItuteD tetraazaPOrPhyrInS
897
O
MeO
O
N
H
OMe NH
Boc-Glu(OtBu)-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-DLys(Aloc)-Leu-Arg(Pbf)-Pro-Gly
a) Pd(0), 4 methylmorpholine
acetic acid, CHCl3, rt, 2 h
b) PyAOP/ 4-pentynoic acid
c) cocktail (TFA-Tips-H2O-Phenol)
RS
N
RS
N
N
N
N
N
Zn
N
NH
O
SR
N
N
O
18
O
HN
N
pGlu-His-Trp-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
O
17
O
N3
R =
O
a) CuSO4, sodium ascorbate, H2O, MeOH
60 °C, 45 min
b) 20% TFA, DCM
RS
N
H
O
N
N
N
N
N
N
SR
O
N
O
N
N
N
HN
N
pGlu-His-Trp-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
SR
19
Scheme 4. Conjugation of PEG resin-bound azide Pc 18 to alkyne peptide 17
are highly soluble in most common organic solvents
such as EtOAc, CH2Cl2, CHCl3, DMSO, DMF, THF, and
MeOH. The increased solubility is due to the periphery
substitution of the macrocycles with solubilizing groups
that is ethylene glycol, ester, amine and azide groups.
Except for the octa-thio butyl ester substituted compound
14a, which was taken in THF; all electronic spectra were
taken in DMSO. The spectra for Pcs are typical of metal-
lated Pcs, characterized by narrow Q-bands as an indica-
tion of monomeric behavior in DMSO [41]. The HOMO
and LUMO energy gap is reduced by introducing sulfur
as well as free nitrogen at the periphery of macrocycles
resulting in compounds absorbing well in the red (703–
710 nm). Compound 13a shows a Q-band above 700 nm,
which is typical of 4,5-dithio-substituted compounds
[34]. Porphyrazines exhibit pronounced B-band between
370 and 400 nm and a split Q-band at 643 and 710 nm for
free-base ester Pz 16c. The splitting is caused by loss of
degeneracy of the lowest unoccupied molecular orbitals.
The band between 400–550 nm can be ascribed to S(n)
to π* transitions [37]. Free-base as well as metallated
asymmetrical porphyrazines showed broadened Q-bands
typical of Pzs of reduced symmetry [42, 43]. Fluores-
cence quantum yields were determined by a comparative
Copyright © 2010 World Scientific Publishing Company
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898
M. MuDyIWa et al.
Table 1. Compound properties
-1
-1
·
Compound
Yield, %
Solvent
Q-band λmax, nm
Log ε, cm M
Emission λmax, nm
Stokes shift, nm
ΦF
13a
13c
13d
14a
14c
15a
15b
15c
15d
16a
16b
16c
16d
17
10
14
19
11
23
11
17
9
DMSO
DMSO
DMSO
THF
710
705
700
705
703
675
675
674
675
675
674
670
675
5.2
5.1
5.5
5.5
5.1
5.1
4.6
4.8
4.7
4.9
4.7
4.7
4.7
719
714
712
716
713
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
9
9
0.15
0.051
0.12
0.15
0.049
n.d.
12
11
10
—
—
—
—
—
—
—
—
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
n.d.
n.d.
n.d.
22
9
n.d.
n.d.
18
8
n.d.
n.d.
the resin in CH2Cl2 (20 mL) for 15 min and then adding
dry diisopropyl ethyl amine DIPEA (12 mL) and acetic
anhydride (8 mL) to the swollen resin and shaking the
mixture at room temperature for 2 h. The solvents were
then drained and the resin further washed with DMF
(2 × 30 mL), MeOH (2 × 30 mL) and CH2Cl2 (2 ×
30 mL), respectively. The resin was then dried under vacuum
overnight to give the indole functionalized PEG resin 2.
Reductive amination of polymer-bound N-ethyl-2-(3-
formyl-indol-1-yl)acetamide(3).Polymer-boundN-ethyl-
2-(3-formyl-indol-1-yl)acetamide (4.027 g, 0.886 mmol)
was swelled in dry DCE (40 mL) for 15 min in a 100 mL
two neck round bottom flask, with one neck sintered.
Trimethylorthoformate (20 mL), the amine phthalonitrile 1
(2.08 g, 8.84 mmol) and NaBH(OAc)3 (1.88 g, 8.87 mmol)
were added to the swollen resin. The mixture was shaken
at room temperature for 16 h, after which the solvents were
drained, and resin was washed with DMF (2 × 20 mL),
MeOH (2 × 20 mL) and CH2Cl2 (2 × 20 mL) respectively.
The resin was then dried under vacuum for 24 h.
method [44] using methylene blue (ΦF = 0.03) [45] in
methanol as the standard. All fluorescence measurements
were performed in solutions with absorbances of 0.047–
0.054 for both Pc and standard with excitation ranging
from 630 nm to 665 nm depending on the absorbances
of the Pcs. Emission recorded in all cases refers to the
Q-band emission. Fluorescence quantum yields of the
compounds are within the normal range for those com-
pounds [46]. The Pzs did not show any fluorescence
when excited near the Q-band region. Whilst the sulfur
and nitrogen’s n to π* transitions contribute to the absor-
bance in the red, the atoms appear to cause rapid decay
of the excited states through radiationless conversion to
sulfur and nitrogen (n, π*) states as well as the ground
states [37], hence reducing the quantum yields. This
is particularly pronounced in asymmetrical molecules
bearing the sulfur as well as the free amine group at the
periphery. The properties of the compounds are summa-
rized in Table 1.
Determination of loading capacity of resin 3. Poly-
mer bound N-ethyl-2-(3-formyl-indol-1-yl)acetamide 2
(0.2 g) was suspended in dry dichloroethane (DCE)
(4 mL) for 15 min in a 50 mL two neck round bottom flask,
with one neck sintered. Trimethyl orthoformate (2.0 mL),
mono-Fmoc 1,3-diaminopropane hydrochloride (0.2 g,
0.60 mmol) and NaBH(OAc)3 (0.127 g, 0.60 mmol) were
added to the swollen resin. The mixture was shaken at
room temperature for 16 h, after which the solvents were
drained, and resin was washed with DMF (2 × 10 mL),
MeOH (2 × 10 mL) and CH2Cl2 (2 × 10 mL) respectively
to give the Fmoc-amino acid resin. The resin was then
dried under vacuum and then 5.1 mg of the dried resin
was shaken in piperidine:DMF (3:7) (0.5 mL) for 30 min,
following which MeOH (6.5 mL) was added and the
resin filtered off. For reference, piperidine:DMF:MeOH
(0.3:0.7:39) solution was prepared. Spectrophotometric
EXPERIMENTAL
See the Supporting information section for the general
experimental procedures and the synthesis of phthalo-
nitriles and maleonitriles.
Polymer-bound N-ethyl-2-(3-formyl-indol-1-yl)ace-
tamide (2). Aminomethyl PEG resin (3.0 g, 0.66 mmol.g-1)
was swelled in dry CH2Cl2 (36 mL) for 30 min in a 100 mL
two neck round bottom flask, with one neck sintered. To
the swollen resin was added dry DMF (4 mL), 4-formyl
indoyl acetic acid (0.1341 g, 60 mmol), HOBt (0.26 g,
1.98 mmol) and DIC (0.2329 g, 1.85 mmol). The mixture
was shaken under argon at room temperature for 24 h.
The resin was drained, washed with DMF (2 × 30 mL),
MeOH (2 × 30 mL), and CH2Cl2 (2 × 30 mL) respectively.
The resin was then dried under vacuum after which the
remaining amine functions were capped by first swelling
Copyright © 2010 World Scientific Publishing Company
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MICrOWave aSSISteD SOlID-PhaSe SyntheSIS Of SubStItuteD tetraazaPOrPhyrInS
899
analysis was carried out at 301 nm, with comparison to
chloride bound resin. The acetyl chloride bound resin
(0.8 g, 0.18 mmol) was swelled in DMSO for 30 min. To
this swelled resin was added sodium azide (1.5 g, 2.31
mmol). The mixture was stirred on a shaker at room tem-
perature for 48 h after which it was washed with distilled
water (2 × 50 mL), DMF (2 × 20 mL), and CH2Cl2 (2 ×
30 mL) respectively. The resin was dried in vacuo. The
dried azide bound resin (0.8 g, 0.18 mmol) was swelled
in CH2Cl2 (5 mL). To the swelled resin was added a
cocktail of TFA/CH2Cl2 (1 mL:4 mL). The mixture was
shaken for 2 h. The resin was drained and washed with
CH2Cl2 (5 × 10 mL). The washings were combined and
evaporated at room temperature to give a dark green
solid. The solid was purified by chromatography on Sep-
hadex LH 20 column, eluting with 10% MeOH in CH2Cl2
to give the azide Pc 13d as a dark green solid. Pc 13d.
Yield 40 mg (14%). UV-vis-NIR (DMSO): λmax, nm 700.
MS (MALDI-TOF): m/z 1570.41 (calcd. for [M + H]+
C70H82N12O14S6Zn 1570.37). ΦF: 0.12. HPLC retention
time (tR = 43.43 min).
Octa- and hexa-substituted thio-butyl benzoate Pcs
(14a and 14c). A mixture of 4,5-(4-mercaptobenzoic
butyl ester)phthalonitrile 7 (1.25 g, 2.31 mmol), dried
resin bound amine phthalonitrile 3 (0.7 g, 0.154 mmol),
zinc acetate dihydrate (0.32 g, 1.36 mmol) in dry 1-bu-
tanol (15 mL) was heated in a two neck round bottom
flask connected to a condenser under argon to 90 °C
before catalytic amount of DBU (0.1 g) was added to
the mixture. Heating continued for 24 h, maintaining the
temperature at 110 °C. During this period the mixture
changed from pale yellow to green and finally dark green.
The solution containing 14a was drained, leaving the
resin bound, compound 14b in the flask. Compound 14b
was then washed to completely remove the symmetrical,
first with hot 1-1-BuOH, mixture of 1-BuOH/CH2Cl2,
and CH2Cl2 until the washings were clear. The washings
were combined, rotary evaporated, and further purified
on silica column eluting with 10% MeOH in CH2Cl2 to
give the symmetrical Pc 14a in (0.197 g, 19%) yield.
Compound 14b was treated with a cocktail of 5 mL TFA,
0.2 mL triisopropylsilane and 19.8 mL CH2Cl2 for 2 h to
cleave the Pc 14c. The solution containing the cleaved Pc
14c was drained; the resin washed and drained three more
times to remove all the cleaved Pc, 14c from the resin.
The combined washings were rotary evaporated at room
temperature to give a dark green solid which was purified
by chromatography on LH 20 column, eluting with 10%
MeOH in CH2Cl2 to give the asymmetrical Pc 14c. Pc
14a. Yield 0.197 g (19%). UV-vis-NIR (THF): λmax, nm
705. MS (MALDI-TOF): m/z 2240.3 (calcd. for [M + H]+
C120H112N8O16S8Zn 2240.53). HPLC retention time (tR =
47.3 min). ΦF: 0.15. Pc 14c. 29.7 mg (11%). UV-vis-NIR
(DMSO): λmax, nm 703. MS (MALDI-TOF): m/z 1931.5
(calcd. for [M + H]+ C104H93N9O13S6Zn 1931.45). HPLC
retention time (tR = 45.83 min). ΦF: 0.049.
a free Fmoc Ala of known concentration treated under
similar conditions. The loading capacity of the resin 3
was found to be 0.19 mmol.g-1.
Octa- and hexa-substituted-[2-(2-methoxy-ethoxy)
ethylsulfanyl]Pcs (13a and 13c). The dried resin bound
amine phthalonitrile 3 (0.5622 g, 0.123 mmol), 4,5-[2-(2-
methoxy-ethoxy)ethyl sulfanyl]-phthalonitrile 6 (0.8 g,
3.053 mmol) and zinc acetate dihydrate (0.2 g, 0.91 mmol)
were added into a dry microwave reaction vessel purged
with argon. 1-butanol (15 mL) was added to the mix. The
mixture was heated in a water bath to swell the resin and
dissolve the phthalonitrile as well as the zinc acetate.
To this reaction mixture was added catalytic amount of
DBU (0.1 g) before the reaction was purged again with
argon and microwave vessel sealed. The microwave
temperature was ramped to 180 °C in 15 min. The reac-
tion temperature was held at 180 °C for 1 h to complete
the reaction. After cooling to 50 °C the contents in the
microwave were poured into a three neck round bottom
flask, with one neck sintered and the symmetrical Pc 13a
drained leaving the resin bound, compound 13b in the
flask. Compound 13b was then washed, first with hot
1-butanol, a solution of 1-butanol/CH2Cl2, and CH2Cl2
until washings were clear and the symmetrical Pc 13a
completely removed. The washings containing the sym-
metrical Pc 13a were combined, rotary evaporated at
room temperature to give a green solid which was further
purified by column chromatography on a silica column,
eluting with 10% MeOH in CH2Cl2. The symmetrical Pc
13a was obtained in (0.19 g, 17%) yield. The resin sup-
ported, compound 13b was then treated with a cocktail
of CH2Cl2 (12 mL, TFA (3.5 mL) and triisopropylsilane
(0.3 mL). This mixture was shaken at room temperature
for 2 h to cleave the polymer bound Pc 13c. The resin
was then drained and washed 3 more times with CH2Cl2
(5 mL) to completely remove the cleaved Pc 13c. The
filtrate was evaporated using rotary evaporator at room
temperature to give a green solid. Chromatography of the
crude Pc 13c on a Sephadex LH 20 column, eluting with
10% MeOH in CH2Cl2 gave the desired product Pc 13c.
Pc 13a. Yield 0.19 g (17%). UV-vis-NIR (DMSO): λmax
,
nm 710. MS (MALDI-TOF): m/z 1648.43 (calcd. for [M +
H]+ C72H96N8O16S8Zn 1648.4). HPLC retention time (tR =
43.19 min). ΦF: 0.15. Pc 13c. Yield 18 mg (10%). UV-
vis-NIR (DMSO): λmax, nm 705. MS (MALDI-TOF): m/z
1487.38 (calcd. for [M + H]+ C68H81N9O13S6Zn 1487.36).
HPLC retention time (tR = 42.93 min). ΦF: 0.051.
Asymmetric azide Pc (13d). Compound 13b (0.8 g,
0.18 mmol) was swelled in dry CH2Cl2 (15 mL) for
15 min. To this swelled resin was added dry DIPEA
(0.37 mL, 2.12 mmol) followed by dropwise addition
of chloroacetyl chloride (0.123 mL, 1.54 mmol). The
mixture was shaken under argon for 14 h after which the
resin was drained, washed with dry DMF (2 × 20 mL),
MeOH (2 × 30 mL) and CH2Cl2 (2 × 30 mL) respectively.
The resin was then dried under vacuum to give acetyl
Octa- and hexa-substituted-[2-(2-methoxy-ethoxy)
ethylsulfanyl]Pzs (15a and 15b). Magnesium turnings
Copyright © 2010 World Scientific Publishing Company
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900
M. MuDyIWa et al.
(0.1, 4.1 mmol) and 0.01 g of I2 were suspended in 1-bu-
tanol (20 mL) in microwave vessel. The microwave tem-
perature was ramped to 180 °C in 5 min. The reaction
temperature was held at 180 °C for 1 h to form Mg(OBu)2.
After cooling to 50 °C the contents in the microwave
were poured into a round bottom flask, with one of
the necks sintered. The flask was connected to a con-
denser and purged with argon. Maleonitrile 10 (2.17 g,
6.26 mmol), resin bound amine phthalonitrile 3 (0.7 g,
0.15 mmol) were added and the suspension heated at
110 °C for 24 h. During the heating period, the reaction
mixture turned yellow, and then gradually turned deep
blue. After 24 h, the symmetrical Pz 15a in solution was
removed by draining. The resin remaining in the flask
was washed first with hot 1-butanol (10 × 10 mL), 1-bu-
tanol/CH2Cl2 mix (10 × 10 mL), MeOH (5 × 10 mL) and
CH2Cl2 (10 × 10 mL) until the washings became clear.
The washings were combined, rotary evaporated to give
a dark blue solid which was further purified by column
chromatography on silica eluting with 10% MeOH/
CH2Cl2 to give the symmetrical Pz 15a. The Pz bound
resin was dried in vacuo, swelled in CH2Cl2 (10 mL)
for 10 min. To the swollen resin triisopropylsilane (2–3
drops) and a cocktail of TFA/CH2Cl2 (1 mL:4 mL) were
added. The treated resin was shaken at room temperature
for 30–45 min after which the free unsymmetrical Pz 15b
was drained. The resin was washed 3 more times with
CH2Cl2 (3 × 10 mL) to completely remove the cleaved
Pz. The washings were combined, rotary evaporated at
room temperature to give a dark blue solid which was
further purified by column chromatography on silica
eluting with 10% MeOH/CH2Cl2 to give the unsymmetri-
cal Pz 15b. Pz 15a.Yield 0.47 g (23%). UV-vis (DMSO):
blue solution was then rotary evaporated to give crude
Pz 15d. The crude 15d was purified on silica column
using 10% MeOH in CH2Cl2 to give 15d in (0.99 mg,
9%) yield based on the starting phthalonitrile on the resin.
Pz 15d. Yield 0.99 mg (9%). UV-vis (DMSO): λmax, nm
675. MS (MALDI-TOF): m/z 1337.28 (calcd. for [M +
H]+ C56H75N9O13S6Zn 1337.31). HPLC retention time
(tR = 42.54 min).
Octa- and hexa-substituted thio-butyl butanoate Pzs
(16a and 16b). Magnesium turnings (0.10 g, 4.1 mmol)
and 0.01 g of I2 were suspended in 1-butanol (20 mL)
in microwave vessel. The microwave temperature was
ramped to 180 °C in 15 min. The reaction temperature
was maintained at 180 °C for 1 h to form Mg(OBu)2.
After cooling to 50 °C the contents in the microwave
were poured into a round bottom flask with one neck sin-
tered. The flask was connected to a condenser and purged
with argon. Maleonitrile 11 (2.82 g, 6.26 mmol), resin
bound amine phthalonitrile 3 (0.7 g, 0.15 mmol) were
added into the flask and the suspension heated at 110 °C
for 24 h. During the heating period, the reaction mixture
turned yellow, and then gradually turned deep blue. After
24 h, the symmetrical Pz 16a in solution was removed by
draining. The resin attached to the asymmetric Pz 16b
was washed first with hot 1-butanol, 1-butanol/CH2Cl2,
MeOH and CH2Cl2 until the washings became clear. The
washings were combined, rotary evaporated to give a dark
blue solid which was further purified by column chroma-
tography on silica, eluting with 5% MeOH in CH2Cl2 to
give the symmetrical Pz16a.The Pz bound resinwas dried
in vacuo, swelled in CH2Cl2 (10 mL) for 10 min. To the
swollen resin triisopropyl silane (2–3 drops) and a cock-
tail of TFA:DCM (1 mL:4 mL) were added. The treated
resin was shaken at room temperature for 30–45 min
after which the free unsymmetrical Pz 16b was drained.
The resin was washed 3 more times with (3 × 10 mL)
CH2Cl2 to completely remove the cleaved Pz. The wash-
ings were combined, rotary evaporated at room tempera-
ture to give a dark blue solid which was further purified
by column chromatography on silica eluting with 5%
MeOH in CH2Cl2 to give the unsymmetrical Pz 16b. Pz
16a. Yield 0.55 g (22%). UV-vis (DMSO): λmax, nm 675.
MS (MALDI-TOF): m/z 1728.56 (calcd. for [M + H]+
C80H120MgN8O16S8: 1728.64). HPLC retention time (tR =
45.37 min). Pz 16b. Yield 20 mg (9%). UV-vis (DMSO):
λ
max, nm 675. MS (MALDI-TOF): m/z 1408.29 (calcd.
for [M + H]+ C56H88MgN8O16S8 1408.39). HPLC reten-
tion time (tR = 44.35 min). Pz 15b. Yield 21 mg (11%).
UV-vis (DMSO): λmax, nm 675. MS (MALDI-TOF): m/z
1275.15 (calcd. for [M + H]+ C56H77N9O13S6 1275.4).
HPLC retention time (tR = 42.62 min).
Octa-substituted-[2-(2-methoxy-ethoxy)ethylsulfa-
nyl]Pz (15c). A mixture of Mg Pz 15a (1.2 g, 0.85 mmol),
and 10% AcOH (6 mL) in dry CH2Cl2 (5 mL) was stirred
under argon at room temperature for 2 h. To this mixture
was added ammonium hydroxide dropwise until the solu-
tion was basic. The mixture was poured into a separating
funnel and washed with water. The organic layer was col-
lected, dried with MgSO4 after which it was rotary evap-
orated to give crude 15c which was further purified on
silica column to give Pz 15c. Pz 15c. Yield 0.82 g (17%).
UV-vis (DMSO): λmax, nm 674. MS (MALDI-TOF): m/z
1386.4 (calcd. for [M + H]+ C56H90N8O16S8 1386.42).
HPLC retention time (tR = 42.48 min).
λ
max, nm 674. MS (MALDI-TOF): m/z 1515.7 (calcd. for
[M + H]+ C74H101N9O13S6: 1515.58). HPLC retention time
(tR = 42.36 min).
Octa-substituted thio-butyl butanoate Pzs (16c).
A mixture of Mg Pz 16a (1.5 g, 0.87 mmol), and 10%
AcOH in dry CH2Cl2 (12 mL) was stirred under argon
at room temperature for 2 h. To this mixture ammonium
hydroxide was added dropwise until the solution was
basic. The mixture was poured into a separating funnel
and washed with water. The organic layer was collected,
dried with MgSO4 after which it was rotary evaporated
to give crude Pz 16c which was further purified on silica
Hexa-substituted-[2-(2-methoxy-ethoxy)ethylsulfa-
nyl]Pz (15d). A mixture of asymmetric Pz 15b (1.2 mg,
0.000093 mmol) and Zn(II) acetate dihydrate (0.013 g,
0.00093 mmol) in dry DMF (5 mL) was heated in a 25 mL
round bottom flask at 90 °C for 24 h under argon. The
Copyright © 2010 World Scientific Publishing Company
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MICrOWave aSSISteD SOlID-PhaSe SyntheSIS Of SubStItuteD tetraazaPOrPhyrInS
901
column, eluting with 10% MeOH in CH2Cl2 to give the
The contents of the microwave were then placed in a two
neck flask, with one of the necks sintered. The solvents
were drained and the remaining contents washed with
MeOH (2 × 20 mL) and CH2Cl2 (2 × 30 mL) to remove
the unreacted peptide. Treatment of the resin beads with a
cocktail of TFA:CH2Cl2 (1:4) for 2 h at room temperature
enabled the triazole conjugate which was further purified
on a Sephadex LH20 column, eluting with 100% MeOH
to give the azide-peptide conjugate 19 in (3 mg, 31%).
The compound was characterized by ESI mass spectrom-
etry and purity verified by HPLC. ESI-MS: m/z 2519.89
(calcd. for [M + H]+ C119H143N30O23S3Zn 2519.94). HPLC
retention time (tR = 41.1 min).
title compound. Pz 16c. Yield 1.21 g (18%). UV-vis
(DMSO): λmax, nm 670. MS (MALDI-TOF): m/z 1706.68
(calcd. for [M + H]+ C80H122N8O16S8: 1706.67). HPLC
retention time (tR = 42.9 min).
Hexa-substituted thio-butyl butanoate Pz (16d). A
mixture of asymmetric Pz 16b (1.1 mg, 0.000073 mmol)
and Zn(OAc)2·2H2O (0.013 g, 0.00093 mmol) in dry
DMF (5 mL) was heated in a 25 mL round bottom flask
at 90 °C for 24 h under argon. The blue solution was then
rotary evaporated to give crude Pz 16d. The crude 16d
was purified on silica column using 5% MeOH in EtOAc
to give 16d in (0.95 mg, 8%) yield based on the starting
phthalonitrile on the resin. Pz 16d. Yield 0.95 mg (8%).
UV-vis (DMSO): λmax, nm 675. MS (MALDI-TOF): m/z
1577.47 (calcd. for [M + H]+ C74H99N9O13S6Zn: 1577.5).
HPLC retention time (tR = 44.3 min).
CONCLUSION
We have successfully synthesized several asymmetri-
cal Pcs and Pzs which contain not only solubilizing groups
but also labile functional groups namely the amine and
azide groups appropriate for tagging compounds with
carbonyl, acid, active ester and alkyne functionalized
groups, DNA or peptides for example. The conjugation
abilities of these compounds have been demonstrated by
the successful conjugation of one of the azide Pcs with an
alkyne peptide. The conjugate is highly soluble in MeOH,
an improvement of solubility properties of the Pc.
Because these compounds fluoresce in the near-IR
region of the spectrum, have high molar absorption
properties and high fluorescence quantum yields, their
bio-conjugates can potentially be used in fluorescence
detection for analysis of nucleic acids, proteins and other
biological targets such as tumors [21, 25, 47–49]. These
compounds could also find application as photosensitiz-
ers for the photodynamic therapy of tumors and as anti-
viral agents for blood sterilization. Use of microwave for
the synthesis of these compounds allowed us to shorten
the reaction times from 24 h to 1 h and the compounds
so obtained had a high degree of purity. The use of this
mild solid support synthesis method and ready-post
TAP assembly functionalization will make it possible to
synthesize many other important functional groups for
tagging.
Polymer-bound asymmetric azide Pc (18). The dried
resin bound amine phthalonitrile 3 (0.6 g, 0.123 mmol),
phthalonitrile 12 (1.5 mmol, 0.4 g), 1-butanol and zinc(II)
acetate dihydrate (0.3 g, 1.37 mmol) were added to the
microwave reaction vessel purged with argon. The mix-
ture was heated in a water bath to swell the resin and
dissolve the phthalonitrile as well as the zinc acetate
dihydrate. The reaction mixture was purged again with
argon and the reaction vessel was closed. The microwave
temperature was ramped to 180 °C in 15 min. The reac-
tion temperature was held at 180 °C for 1 h to complete
the reaction. After cooling to 50 °C the contents in the
microwave were poured into a round bottom flask, with
one neck sintered and the symmetrical Pc drained leav-
ing the resin bound Pc in the flask. The resin bound Pc
was washed first with hot 1-BuOH, mixture of CH2Cl2/
1-BuOH and CH2Cl2 until the washings were clear. The
resin was then dried under vacuum for 24 h. The resin
bound to the amine Pc was swelled in dry CH2Cl2 (15 mL)
for 15 min. To this swelled resin dry DIPEA (0.37 mL,
2.12 mmol) was added, followed by dropwise addition of
chloroacetyl chloride (0.123 mL, 1.54 mmol). The mix-
ture was shaken under argon for 14 h after which the resin
was drained, washed with dry DMF (2 × 20 mL), MeOH
(2 × 30 mL) and CH2Cl2 (2 × 30 mL) respectively. The
resin was then dried under vacuum to give acetyl chloride
bound resin. The acetyl chloride bound resin was swollen
in DMSO before adding sodium azide (1.5 g, 2.31 mmol).
The mixture was stirred on a shaker at room temperature
for 48 h after which it was washed with distilled water
(2 × 50 mL), DMF (2 × 20 mL), and CH2Cl2 (2 × 30 mL)
respectively to give the resin bound to the azide Pc,
compound 18.
Supporting information
A full list of general experimental procedures, synthe-
sis of phthalonitriles and maleonitriles, 1D proton and
carbon NMR of phthalonitriles and maleonitriles and
HPLC chromatograms of Pcs, Pzs, peptide and conjugate
are given in the supplementary material. This material is
available free of charge via the Internet at http://www.
worldscinet.com/jpp/jpp.shtml.
Azide Pc-peptide conjugate (19). Solid-supported
azide Pc 18 (0.2 g, 0.05 mmol), alkyne functionalized
water soluble peptide 17 (0.25 g, 0.23 mmol), copper(II)
sulfate (9 mg, 0.009 mmol), sodium ascorbate (0.036 g,
0.045 mmol) methanol (6.0 mL), and water (6.0 mL)
were transferred into a microwave vial. The vial was
sealed and irradiated with microwave at 60 °C for 45 min.
Acknowledgement
We thank the National Science Foundation (CHE-
0304833, EPS-0346411) and the National Institutes of
Health (EB006639 and HG01499) for financial support.
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M. MuDyIWa et al.
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