Design and synthesis of water-soluble bioconjugatable trans-AB-porphyrins

Article abstract of DOI:10.1016/j.tet.2008.08.096

Three free base porphyrins have been prepared that bear a polar and facially encumbering 2,4,6-tris-(carboxymethoxy)phenyl motif at one meso (5-) position. The only other substituent (15-position) comprises phenyl, formyl, or p-aminophenyl. The porphyrins exhibit solubility in water (or aqueous buffer solutions) at pH ≥7 and concentrations >1 mM at room temperature. The concise syntheses, water solubility, and bioconjugatable handle make these porphyrin constructs suitable for biological applications.

Full text of DOI:10.1016/j.tet.2008.08.096

Tetrahedron 64 (2008) 11440–11448
Contents lists available at ScienceDirect
Tetrahedron
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Design and synthesis of water-soluble bioconjugatable trans-AB-porphyrins
*
Ana Z. Muresan, Jonathan S. Lindsey
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2008
Received in revised form 19 July 2008
Accepted 20 August 2008
Available online 4 September 2008
Three free base porphyrins have been prepared that bear a polar and facially encumbering 2,4,6-tris-
(carboxymethoxy)phenyl motif at one meso (5-) position. The only other substituent (15-position)
comprises phenyl, formyl, or p-aminophenyl. The porphyrins exhibit solubility in water (or aqueous
buffer solutions) at pH ꢀ7 and concentrations >1 mM at room temperature. The concise syntheses, water
solubility, and bioconjugatable handle make these porphyrin constructs suitable for biological
applications.
Keywords:
Porphyrin
Ó 2008 Elsevier Ltd. All rights reserved.
Water
Hydrocarbon
Water-soluble
Facial encumbrance
Bioconjugation
1. Introduction
alkylcarboxylic acids,22–26 or by attachment of small aryl motifs
such as N-alkyl pyridinium or phenylsulfonic acid units to the meso
Tetrapyrrolic molecules have distinct chemical and photo-
physical properties that are attractive for a wide range of diagnostic
and therapeutic applications in the field of photomedicine. The
applications encompass optical imaging of diseased tissue,1–3
fluorescent labeling in flow cytometry,4–7 photodynamic in-
activation of microbial infections,8–10 and photodynamic therapy of
solid tumors.11–15 A key challenge to the implementation of tetra-
pyrrolic molecules in photomedicine entails tailoring the molecules
to exhibit appropriate solubility. In particular, synthetic motifs that
impart water solubility are important for a number of biological
studies. Tetrapyrrolic macrocycles (porphyrins, chlorins, and bac-
teriochlorins) present a particular challenge in this regard owing to
the large size of the planar hydrocarbon macrocycle. In recent
years, approaches for solubilizing related carbon-rich molecules
such as fullerenes and nanotubes have relied on grafting polar
substituents to create a polar layer or sheath that encompasses the
hydrophobic molecule.16–20 For biological applications of tetra-
pyrrolic macrocycles, a typical requirement is to introduce sub-
stituents to achieve both water solubility and facility for
conjugation to biomolecules.21
sites of synthetic porphyrins27 (Chart 1). In the latter case, four
polar substituents (one at each of the meso sites) traditionally were
introduced to impart water solubility. The use of four substituents
stemmed from the synthetic methods of the day,28 which only
readily supported the synthesis of such A4-porphyrins. Regardless,
the most easily synthesized membersdthe pyridinium- or sul-
foaryl-derivatized porphyrinsdexhibit only limited solubility in
aqueous media. Moreover, the sense that four such groups were
essential also presented an impediment to further functionalization
of the macrocycle, as required for applications where bio-
conjugation is required. The advent of versatile and rational syn-
thetic routes to compact porphyrins that bear less than four meso
substituents29,30 (e.g., trans-AB-porphyrins) has opened the door to
consideration of new molecular designs for water solubilization
that are both potent and compatible with features required for
bioconjugation.
In parallel with the development of new synthetic methods,
new water-solubilization motifs have been developed to serve
a variety of objectives. Representative examples of such solubili-
zation motifs include oligoethylene glycol monomers31 and den-
drimers,32 alkyl polyamines,33 and polycarboxy chains.34 While
each such group has merit, most are quite large and are not well
suited for our goals in developing compact bioconjugatable mole-
cules for use in photomedical applications.
Water solubilization of porphyrins has traditionally been achieved
by use of uroporphyrin, which bears eight
b-substituted
We initiated a program to develop water-solubilization motifs
that ideally adhere to the following design guidelines: (1) ionic
* Corresponding author. Tel.: þ1 919 5156406; fax: þ1 919 5132830.
E-mail address: jlindsey@ncsu.edu (J.S. Lindsey).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.096

A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
11441
HO2C
sulfhydryls).44 To perform bioconjugation in aqueous solution, and
therefore avoid the solubility problems that can arise with mixed
aqueous–organic media, the carboxy moieties must be deprotected.
Hence, the conjugatable groups should be compatible with the
carboxylic motif to allow for selective bioconjugation. We have
elected to focus on aldehyde and amine groups, which are suitable
for reductive amination in aqueous media. Reductive amination
has been used successfully for derivatization of tetrapyrrolic
macrocycles.45–48 This design complements prior examples of
trans-AB-porphyrins that bear a bioconjugatable handle including
isothiocyanate,21 carboxylic acid/ester,7,38 and iodoacetamide.37
Taken together, the work described herein provides an additional
approach for the design and synthesis of compact, water-soluble,
bioconjugatable porphyrins.
HO2C
CO2H
CO2H
HO2C
HO2C
NH
N
N
HN
CO2H
HO2C
Uroporphyrin III
Ar
Ar
N
N
N
N
N
Ar
Ar
M
2. Results and discussion
2.1. Molecular design and synthesis strategy
SO3
Ar
meso-tetraarylporphyrin
Each target molecule (H2P-1–3) is a free base trans-AB-por-
phyrin (Chart 3). The trans-AB-porphyrin architecture, wherein the
porphyrin contains only two meso substituents, was chosen to
maintain a compact size. Each of the porphyrins is tailored with
a water-solubilizing 2,4,6-tris(carboxymethoxy)phenyl substituent
at one meso position. The 2,4,6-tris(carboxymethoxy)phenyl unit is
relatively compact. The mass of this water-solubilizing moiety
(C12H11O9, 299 Da) is comparable to that of the core porphine unit
(C20H12N4, 308 Da) in the trans-AB-porphyrin.
The 2,4,6-tris(carboxymethoxy)phenyl unit is an o,o0-di-
substituted meso-aryl motif, whereby the polar groups are (i) in
close proximity to the porphyrin macrocycle and (ii) are thrust in an
orientation that encumbers the faces of porphyrin, thereby im-
peding aggregation. The introduction of 2,6-disubstituted aryl units
Chart 1.
groups are more potent than non-ionic groups for solubilization, (2)
anionic groups are generally preferred over cationic groups to
minimize non-specific binding to cellular structures, (3) the pro-
jection of polar substituents above and below the plane of the
macrocycle is expected to suppress cofacial aggregation of the
porphyrins, and (4) solubilization is desired with a single group so
as to achieve a compact molecular architecture. The emphasis on
groups that project over the porphyrin macrocycle stemmed
from our finding that the mesityl group and homologues (e.g.,
2,4,6-triethylphenyl) impart increased organic solubility (versus
phenyl) to porphyrins.35,36 The results from this program include
HOOC
a
1,5-diphosphonopent-3-yl group (swallowtail moiety, I),7,37
a N,N-diethylimidazolium-2-yl unit (II),38 and a 2,6-bis(phospho-
nomethoxy)phenyl moiety (III)39 (Chart 2). The first two solubili-
zation moieties (I, II) have been incorporated as one substituent in
trans-AB-porphyrins wherein the distal substituent constituted
a bioconjugatable handle (carboxylic acid, iodoacetamide).7,37,39
The diphosphonate solubilization moieties (I, III) have been
incorporated in a synthetic chlorin.39 Jux also has developed fa-
cially encumbering motifs for porphyrins by reaction of a 2,6-
bis(bromomethyl)aryl group located at the porphyrin meso position
with 4-tert-butylpyridine or with diethyl malonate.40–43
Here we describe an additional water-solubilization motif that
provides facial encumbrance, the 2,4,6-tris(carboxymethoxy)-
phenyl unit. This motif is incorporated as one substituent in
a trans-AB-porphyrin. The other substituent is a handle suitable for
bioconjugation. Typical groups for bioconjugation include iso-
thiocyanate and imidate esters (for amines) and iodoacetamide (for
O
COOH
N
HN
N
O
NH
O
HOOC
H2P-1
HOOC
O
COOH
N
NH
HN
N
OHC
O
O
HOOC
H2P-2
HO
O
HO
P
O
HOOC
O
OH
O
P
OH
N
N
COOH
N
HN
N
OH
OH
H2N
O
I
O
P
P
NH
O
O
HO
O
HO
HOOC
H2P-3
I
II
III
Chart 2.
Chart 3.

11442
A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
to porphyrins has presented challenges over the years; however,
the o-benzyloxy motif is compatible with the steric constraints
encountered upon formation of the pyrromethane intermediate
(e.g., dipyrromethane, porphyrinogen) on the path to the porphy-
rin.49 Indeed, we previously synthesized porphyrins that bear 2,6-
bis(benzyloxy) and 2,4,6-tris(benzyloxy) substituents at the meso
positions wherein a number of substituents were introduced on the
arene moiety of the benzyloxy group.36,50,51
OH
HO
OH
CHO
Br
O
82%
O
K2CO3, DMF, 80 °C
The conjugatable groups should be compatible with carboxylic
acids to allow for selectivity during the bioconjugation step. Hence,
conjugatable groups include a formyl group for porphyrin H2P-2
and a p-aminophenyl group for H2P-3. Porphyrin H2P-1, which has
a meso-phenyl substituent in lieu of a bioconjugatable handle, was
prepared as a benchmark for the solubility studies.
The synthesis relied on two methods for the preparation of trans-
AB-porphyrins. One method employs condensation of a 1,9-bis(N,N-
dimethylaminomethyl)dipyrromethane with a dipyrromethane in
refluxing ethanol containing zinc acetate followed by DDQ oxida-
tion.29 The second method requires the 1,9-diformylation of
a dipyrromethane and subsequent imination of the 1,9-diformyl-
dipyrromethane by treatment with n-propylamine. Reaction of the
resulting 1,9-bis(imino)dipyrromethane with a dipyrromethane in
the presence of zinc acetate and air leads to the corresponding trans-
AB-porphyrin.30 In both routes the zinc porphyrin is obtained. Both
routes typically afford a single porphyrin product, albeit in rather
low yield (5–30%).
O
O
O
1
O
O
O
O
O
CHO
O
pyrrole (excess), InCl3
35%
O
O
O
2a
O
O
O
O
O
O
The tert-butyl group was chosen as the protective moiety for
each of the three carboxylic acids of the water-solubilization motif.
The water-solubilization motif to be introduced thus contains three
phenolic ethers and three esters. Accordingly, the 1,9-functionali-
zation (imination or aminoalkylation) was performed on the
complementary dipyrromethane.
NH HN
N
HN
HN
3a
18%
N
2.2. Synthesis
Zn(OAc)2, EtOH, refux,16 h
2.2.1. Porphyrin H2P-1
The O-alkylation51,52 of 2,4,6-trihydroxybenzaldehyde with
tert-butyl bromoacetate in the presence of K2CO3 gave the alky-
lated product 1 in 82% yield. Condensation53 of aldehyde 1 with
pyrrole (40 equiv) in the presence of InCl3 followed by crystalli-
zation gave dipyrromethane 2a in 35% yield (Scheme 1).
Condensation of dipyrromethane 2a with 1,9-bis(imino)dipyrro-
methane 3a30 in refluxing ethanol containing excess Zn(OAc)2
with exposure to air gave the corresponding protected zinc ester-
porphyrin ZnP-10 in 18% yield. Treatment of ZnP-10 with aqueous
KOH (2 M) in THF at 60 ꢁC for 8 h afforded cleavage of the esters.
The porphyrin was extracted in the aqueous layer and concen-
trated to give the zinc porphyrin with free carboxylic acids. Upon
adjusting an aqueous solution of the latter compound to pH¼3 (by
addition of 2 M aqueous HCl), the zinc porphyrin was demetalated
and the resulting free base porphyrin H2P-1 was isolated as
a precipitate in 80% yield (overall from ZnP-10). The porphyrins
were characterized by 1H NMR spectroscopy, ESI-MS or LD-MS
(laser desorption mass spectrometry in the absence of a matrix),54
absorption spectroscopy, and fluorescence spectroscopy.
OR
O
O
OR
O
N
N
N
N
O
Zn
O
O
OR
ZnP-1′ : R = tert-butyl
(1) aqueous KOH (2 M), THF, 60 °C
80% (2) H2O (gives R = H)
(3) HCl (2 M)
H2P-1
Scheme 1.
H2P-2 was confirmed by LD-MS, ESI-MS, and 1H NMR analyses.
The purity of the porphyrin H2P-2 was estimated to be 90%
according to the 1H NMR analysis. Attempts to purify porphyrin
H2P-2 employing reversed-phase (RP) chromatography were not
successful.
To achieve greater purity, stepwise deprotection conditions
were employed. The carboxylic acid units were unveiled by
treatment of porphyrin ZnP-20 with aqueous KOH (2 M) in THF at
60 ꢁC for 12 h. Aqueous–organic workup and subsequent treat-
ment of the crude product with neat TFA at 50 ꢁC for 24 h
resulted in deprotection of the formyl group. The crude reaction
2.2.2. Porphyrin H2P-2
Condensation of the 1,9-bis(N,N-dimethylaminomethyl)-5-
acetaldipyrromethane 429 and dipyrromethane 2a in refluxing
ethanol containing Zn(OAc)2 followed by oxidation with DDQ gave
the corresponding zinc porphyrin ZnP-20 in 10% yield (Scheme 2).
Attempts to simultaneously cleave both the tert-butyl and the
acetal protective groups with a biphasic solution55 of TFA/H2O/
CH2Cl2 gave a mixture of porphyrins. Treatment of ZnP-20 with
neat TFA at room temperature for 3 days unveiled the carboxylic
and formyl groups, giving H2P-2 in 77% yield. The structure of

A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
11443
2a
O
(1) Zn(OAc)2, EtOH, reflux
NH
NH
O
HN
N
2b
HN
HN
O
O
10%
68%
DMF, POCl3, 1 h
CHO
N
4
(2) DDQ
O
NH
O
5
HN
O
NH
O
CHO
O
O
O
N
N
N
N
O
O
NH2
O
quantitative
Zn
THF, rt, 1 h
O
O
N
O
O
NH
O
HN
NH
N
ZnP-2′
(1) THF, aqueous KOH (2 M), heat
(2) TFA, heat
3b
65%
2a, Zn(OAc)2, toluene
28%
reflux, 12 h
H2P-2
Scheme 2.
O
O
mixture was diluted with H2O to pH 3 to induce precipitation of
the porphyrin. Sonication, centrifugation, decanting, and drying
of the resulting solid pellet gave porphyrin H2P-2 (overall 65%
yield) in pure form without chromatography. Porphyrin H2P-2
was characterized by 1H NMR and 13C NMR spectroscopy, ab-
sorption and fluorescence spectroscopy, and LD-MS and ESI-MS
analyses.
O
O
O
O
N
N
O
HN
O
Zn
N
N
O
O
O
2.2.3. Porphyrin H2P-3
ZnP-3′
Dipyrromethane 2b56 was subjected to Vilsmeier formylation
to give the corresponding 1,9-diformyldipyrromethane 5 in 68%
yield. Condensation of 5 with excess n-propylamine gave the
corresponding 1,9-bis(imino)dipyrromethane 3b quantitatively.
The resulting 1,9-bis(imino)dipyrromethane 3b was employed
promptly for porphyrin formation without further purification.
The condensation of 1,9-bis(imino)dipyrromethane 3b and
dipyrromethane 2a in refluxing toluene for 12 h gave porphyrin
ZnP-30 in 28% yield (Scheme 3). No other porphyrin products
(e.g., derived from scrambling) were detectable by LD-MS
analysis.
(1) aqueous KOH, THF, 60 °C, 11 h
(2) neat TFA, rt, 12 h
83%
H2P-3
Scheme 3.
2.3. Characterization
2.3.1. Molecular modeling
The deprotection of ZnP-30 was examined under various
conditions, including TFA in CH2Cl2 (6 days), neat TFA (2 days),
concentrated aqueous HCl/CH2Cl2 (5 days), TMSBr/triethylamine
in CHCl3 (2 days), and LiOH in aqueous THF (1 day), all of which
gave incomplete reaction or mixtures of products. Treatment
with KOH (2 M) and THF for 28 h at 60 ꢁC gave a porphyrin that
lacked the tert-butyl esters but retained the tert-BOC protective
group on the amino moiety, as indicated by LD-MS and ESI-MS
data. Treatment with TFA at room temperature for 11 h gave the
target porphyrin H2P-3 in 83% overall yield. Porphyrin H2P-3
was characterized by 1H NMR and 13C NMR spectroscopy, ab-
sorption and fluorescence spectroscopy, and LD-MS and ESI-MS
analyses.
A molecular model of porphyrin H2P-1 is shown in Figure 1. The
figure shows the projection of the carboxymethoxy substituents
above and below the plane of the porphyrin macrocycle. A con-
siderable range of motion is available to the 2,6-carboxymethoxy
units owing to rotation about the single bonds of the carboxy-
methoxy moiety and the torsion of the aryl unit with respect to the
plane of the porphyrin (by rotation about the single bond between
the aryl carbon and the porphyrin meso carbon). On account of the
combined motions, the hydroxy moiety of the carboxylic acid can
extend as far as the pyrrolic NH and pyrrolenyl units (and contact
the p-chromophore of the porphyrin), project almost normal to the
plane of the porphyrin macrocycle, or project back over the aryl
unit.

11444
A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
Figure 1. Molecular model of porphyrin H2P-1 showing different orientations (not
energy-minimized) of the 2,6-carboxymethoxy units attached to the aryl moiety.
2.3.2. Chemical characterization
All porphyrins were characterized by 1H NMR and 13C NMR
spectroscopy, mass spectrometry (LD-MS, ESI-MS and/or FABMS),
and absorption and fluorescence spectroscopy. Analytical RP-HPLC
was also employed to confirm the purity of the hydrophilic por-
phyrins (H2P-1–3). The hydrophobic porphyrins (ZnP-10–30) were
examined by 1H NMR spectroscopy in CDCl3, and by absorption and
emission spectroscopy in toluene. The hydrophilic porphyrins
(H2P-1–3) were examined by 1H NMR spectroscopy in DMSO, and
by absorption and emission spectroscopy in water.
The hydrophilic porphyrins did not give observable NH and
COOH signals upon 1H NMR spectroscopic analysis in DMSO-d6 due
to broadening/exchange with solvent. The 1H NMR spectra were
typically characteristic of meso-substituted porphyrins, with the
exception of a splitting in the peak attributed to the inner NH of the
free base porphyrins; this phenomenon occurred with each of H2P-
1–3. A variable temperature experiment with H2P-2 over the range
20–80 ꢁC showed that the peaks coalesced upon heating to 80 ꢁC,
with a coalescence temperature estimated to be w60 ꢁC (Fig. 2).
The origin of the splitting at room temperature is not known but
likely stems from different conformations of the 2,6-bis(carboxy-
methoxy)phenyl unit (vide supra).
The hydrophilic porphyrins were examined by absorption
spectroscopy in a range of solvents, including THF, DMSO, water,
and aqueous buffer solutions. In each case, the Soret band
(w410 nm) was broadened in all solvents examined versus that of
a benchmark trans-A2-porphyrin (5,15-diphenylporphyrin,57 H2P-
4) in toluene or DMSO. The absorption spectrum of H2P-3 is dis-
played in three solvents (water, THF, DMSO) in Figure 3.
Figure 2. Variable temperature 1H NMR spectra for porphyrin H2P-2 in DMSO-d6.
these features are characteristic of the meso-formyl porphyrin in
aqueous media rather than due to aggregation. Finally, upon in-
creasing the pH to 10, the Soret band broadened to a fwhm of
63 nm (entry 19, Table 1).
The porphyrins also were characterized by fluorescence emis-
sion spectroscopy with excitation into the Soret band. The hydro-
phobic, zinc ester-porphyrins were analyzed in toluene and
exhibited emission characteristic of zinc trans-AB-porphyrins,
The breadth of the Soret band was assessed by the full width at
half maximum (fwhm) for the hydrophilic porphyrins in a range of
solvents (THF, MeOH, DMSO, water, aqueous buffer solutions of pH
7 and 10) and for the hydrophobic porphyrins in toluene and
DMSO. Regardless of solvent, the hydrophilic porphyrins (H2P-1–3)
exhibited broader Soret bands (w20–30 nm or greater vs w11–
14 nm) than those of the hydrophobic porphyrins (ZnP-10–30). This
difference does not stem from the different metalation states (the
former are free base porphyrins whereas the latter are zinc por-
phyrins) but must be due to the different conformations of the
polar, ionizable carboxylic acid moieties (at the 2,6-positions of the
meso-aryl unit) and resulting range of electronic interaction with
the p-chromophore of the porphyrin. Indeed, the benchmark por-
phyrin (H2P-4) lacks the solubilizing feature and exhibits a sharp
Soret band (fwhm¼13 nm). The results from an extensive study are
summarized in Table 1.
The only notable absorption spectral distinction observed in
water versus other solvents among the hydrophilic porphyrins
occurred for H2P-2, the porphyrin bearing a meso-formyl group. In
DMSO, examination of the visible region showed peaks (declining
order of intensity) at 561, 641, 584 (shoulder), and 518 nm. In water
the peaks in the visible region were substantially broader, which
remained unchanged upon dilution (vide infra), suggesting that
Figure 3. Absorption spectra of porphyrin H2P-3 at room temperature in water (solid
line), THF (dashed line), and DMSO (dotted line).

A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
11445
Table 1
Absorption spectra of porphyrins in various solvents
Entry Compd
M
Solvent
lSoret (nm) fwhm (nm) lQ (nm)
1
2
3
4
5
6
7
8
ZnP-10
ZnP-10
ZnP-10
ZnP-20
ZnP-20
ZnP-20
ZnP-30
ZnP-30
ZnP-30
H2P-1
H2P-1
H2P-1
H2P-1
H2P-1
H2P-2
H2P-2
H2P-2
H2P-2
H2P-2
H2P-3
H2P-3
H2P-3
H2P-3
H2P-3
H2P-4
H2P-4
Zn
Zn
Zn
Zn
Zn
Zn
Zn
Zn
Zn
Toluene 413
13
10
10
14
10
11
13
11
11
27
21
30
20
20
31
33
33
41
63
34
22
24
23
24
13
13
539, 573
544, 579
548, 583
502, 539, 574
542, 577
502, 545, 580
539, 575
545, 580
MeOH
DMSO
Toluene
MeOH
DMSO
410
417
411
408
414
Toluene 414
THF
DMSO
414
418
416
406
402
402
403
415
411
407
408
395
414
409
404
405
405
9
548, 583
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
H, H DMSO
H, H MeOH
H, H H2O
H, H pH 7
H, H pH 10
H, H DMSO
H, H MeOH
H, H H2O
H, H pH 7
H, H pH 10
H, H DMSO
H, H THF
H, H H2O
H, H pH 7
H, H pH 10
H, H Toluene 408
H, H DMSO 407
506, 547, 577, 632
501, 542, 574, 629
505, 564, 620
504, 541, 617
504, 541, 617
518, 560, 641
516, 559, 642
519, 561, 637
518, 562, 633
519, 565, 637
507, 548, 579, 647
504, 541, 579, 636
505, 543, 568, 623
506, 544, 567, 622
506, 544, 567, 622
503, 535, 576, 633
502, 537, 574, 629
which consisted of two bands at w578 and 632 nm. The hydro-
philic, deprotected free base porphyrin analogues were analyzed in
water and also exhibited two emission bands. The emission max-
ima were typical for free base trans-AB-porphyrins bearing meso-
aryl substituents for H2P-1 (625 and 686 nm) and H2P-3 (629 and
688 nm), but were shifted bathochromically for H2P-2 (641 and
707 nm). The shift in the emission spectrum of H2P-2 stems from
the presence of the meso-formyl substituent, which is known to be
a potent auxochrome.58,59
2.3.3. Water solubility
The hydrophilic porphyrins (H2P-1–3) were examined for sol-
ubility at room temperature in aqueous media of physiologic pH 7.4
and higher. Solubility was assessed by visual inspection using pure
water or aqueous buffer solutions (pH 7, 7.4, 8, and 10) at porphyrin
concentrations of 10–14 mM (using short-pathlength cells). Each of
the porphyrins H2P-1–3 gave an optically clear solution in an
aqueous buffer solution at pH 7 or higher for porphyrin concen-
trations in this regime.
One method to detect the aggregation of porphyrins entails the
comparison of absorption spectra of various concentrations. In the
case of no aggregation, the shape of the absorption spectra remains
constant upon change of concentration of the sample. For this de-
termination, stock solutions of porphyrins H2P-1 (14 mM), H2P-2
(10 mM), and H2P-3 (14 mM) were prepared in an aqueous buffer at
pH 7. The absorption spectra were collected in the same buffer
solution at concentrations obtained by 1-, 5- and 10-fold dilution
using cuvettes of different path lengths (l¼1, 5, and 10 mm),
thereby in principle affording equal absorbancies. The spectra of
porphyrins H2P-1–3 are shown in Figure 4. A very slight change was
observed in the spectra of H2P-1 (over the concentration of 2.8,
0.56, and 0.28 mM) and H2P-2 (2.5, 0.5, and 0.25 mM), whereas the
three spectra of porphyrin H2P-3 (3, 0.6, and 0.3 mM) were su-
perimposable with each other. The spectra suggest that any ag-
gregation across this concentration regime is at most very slight.
Figure 4. Absorption spectra of porphyrins in aqueous buffer solution (pH¼7) at room
temperature as
a function of concentration. (A) Porphyrin H2P-1: (solid line)
c¼2.8 mM, l¼1 mm; (dotted line) c¼0.56 mM, l¼5 mm; (dashed line) c¼0.28 mM,
l¼10 mm. (B) Porphyrin H2P-2: (solid line) c¼2.5 mM, l¼1 mm; (dotted line)
c¼0.5 mM, l¼5 mm; (dashed line) c¼0.25 mM, l¼10 mm. (C) Porphyrin H2P-3: (solid
line) c¼3 mM, l¼1 mm; (dotted line) c¼0.6 mM, l¼5 mm; (dashed line) c¼0.3 mM,
l¼10 mm.
must be functionalized to achieve water solubility (and bio-
conjugatability). The use of tetrapyrrolic molecules in aqueous
media might seem unnecessarily complex, given the availability of
numerous organic dyes that are soluble in water. However, such
dyes typically bear an intrinsic charge; consequently, the synthetic
chemistry and purification of elaborate analogues of such dyes can
be quite cumbersome.60 The tetrapyrrole family presents the
opportunity to carry out all synthetic manipulations on neutral,
3. Outlook
The molecular design of water-soluble tetrapyrrolic molecules
generally begins with a non-polar, carbon-rich macrocycle that

11446
A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
non-ionic compounds in organic solution, and then unveil the
groups for water solubilization and bioconjugation in the last step
of the synthetic process. In addition, few organic dyes present the
opportunities for molecular tuning that are available with the tet-
rapyrrolic macrocycles by virtue of chelation of different metals,
reduction of the macrocycle (porphyrin, chlorin, bacteriochlorin),
and introduction of diverse substituents.
95% B; 40–50 min 100% A; A¼water (0.1% TFA), B¼methanol (0.1%
TFA); detection at 405 nm. In all cases, the void volume corre-
sponded to t¼2 min.
4.2. Noncommercial compounds
Dipyrromethanes 2b,56 3a,30 and 429 were prepared following
The trans-AB-porphyrins described herein provide a compact
architecture with controlled substitution pattern. The presence of
a single conjugatable group enables formation of a single product
upon bioconjugation, as opposed to a mixture that results with
porphyrins that have multiple reactive functional groups. It is
noteworthy that chromatography was not employed in the purifi-
cation of the water-soluble porphyrins upon unveiling the water-
solubilizing carboxylic acid moieties. As anticipated, introduction of
the polar and facially encumbering 2,4,6-tris(carboxymethoxy)-
phenyl motif on the macrocyclic perimeter increased the hydro-
philicity of the porphyrins. The hydrophilic porphyrins proved to be
soluble in aqueous buffer solutions at pH ꢀ7 and concentrations of
at least 1 mM at room temperature. The target molecules are
amenable to conjugation with biologically active molecules.
We now have described four motifs for imparting water solu-
bility to tetrapyrrolic macrocycles,7,37–39 all of which rely on facial
encumbrance of polar groups. The potency of water solubilization
appears to decline in the order of diphosphonate-bearing groups
(swallowtail, 2,6-aryl)>2,4,6-tris(carboxymethoxy)phenyl>N,N-
dialkylimidazolium-2-yl. One distinction between the three types
of groups is that the diphosphonate-derivatized group is expected
to be ionized (and hence to impart solubility) at essentially all
physiological pH ranges, the carboxy-derivatized group would be
expected to be ionized only at neutral to basic pH ranges, and the
imidazolium group is permanently charged. Accordingly, the use of
the carboxy-containing motif described herein may be expected to
enable ready penetration of lipid membranes and also selective
precipitation upon exposure to acidic environments. The former
feature may be essential for bioavailability whereas the latter may
be attractive for localization in sub-cellular domains, as required for
staining or for targeted molecular brachytherapy.61
reported procedures.
4.3. Synthetic procedures
4.3.1. 2,4,6-Tris(tert-butoxycarbonylmethoxy)benzaldehyde (1)
Following the K2CO3 alkylation method,51,52 a solution of 2,4,6-
trihydroxybenzaldehyde (2.0 g, 13 mmol) in anhydrous DMF
(43 mL, 0.3 M) was treated with K2CO3 (17.8 g, 129 mmol, dried in
vacuo at 140 ꢁC overnight), and the mixture was heated at 60 ꢁC.
After 30 min, tert-butyl bromoacetate (7.70 mL, 51.6 mmol) was
added dropwise to the reaction mixture and the temperature was
set at 80 ꢁC. After stirring for 1 h at 80 ꢁC, the reaction mixture
was allowed to reach ambient temperature. The reaction mixture
was treated with water (20 mL) and extracted with ethyl acetate.
The organic layer was washed (brine and water), separated, dried
(Na2SO4), and concentrated to a red oil. Purification by column
chromatography [silica, hexanes/ethyl acetate (1:1)] gave a red-
brown viscous oil that solidified upon standing for 1 day at room
temperature (5.2 g, 82%): mp 55–60 ꢁC. 1H NMR
1.49 (s, 9H), 4.49 (s, 2H), 4.57 (s, 4H), 6.01 (s, 2H), 10.43 (s, 1H); 13C
NMR 28.2, 65.8, 66.8, 82.9, 83.2, 93.5, 110.6, 162.3, 163.7, 166.9,
d 1.48 (s, 18H),
d
167.1, 187.5; FABMS obsd 497.2396, calcd 497.2387 (C25H36O10).
Anal. Calcd for C25H36O10: C, 60.47; H, 7.31. Found: C, 60.03; H,
7.33.
4.3.2. 5-[2,4,6-Tris(tert-butoxycarbonylmethoxy)-
phenyl]dipyrromethane (2a)
Following a procedure for dipyrromethane formation,53 a sam-
ple of aldehyde
1 (1.53 g, 3.08 mmol) and pyrrole (8.4 mL,
0.12 mol) was purged with argon for 10 min. Then InCl3 (66 mg,
0.30 mmol) was added and the mixture was stirred at room
temperature under argon. After 1 h, the reaction mixture was
treated with powdered NaOH (360 mg, 6.00 mmol) and stirring
was continued for 30 min. The reaction mixture was filtered
through a Buchner funnel and the filtrate was concentrated. The
resulting solid was treated with hexanes and the suspension was
sonicated. The volatile components were evaporated. This se-
quence of operations was repeated thrice in order to remove
traces of pyrrole. Crystallization from ethanol/water afforded an
4. Experimental section
4.1. General methods
All 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz)
were collected in CDCl3 unless noted otherwise. The DMSO-d6
employed was 99.9% pure. Absorption spectra were collected in
toluene at room temperature unless noted otherwise. Mass spectra
of porphyrins were obtained via laser desorption mass spectrom-
etry (LD-MS) without a matrix,54 by high-resolution fast atom
bombardment mass spectrometry (FABMS) using a matrix of
nitrobenzyl alcohol and polyethylene glycol, and by electrospray
ionization mass spectrometry (ESI-MS). Positive ions were detected
unless noted otherwise. The target carboxy-porphyrins gave posi-
tive ions upon analysis in a water/methanol mixture (25:75 v/v)
ash-gray solid (659 mg, 35%): mp 107–112 ꢁC. 1H NMR
d 1.45 (s,
18H), 1.50 (s, 9H), 4.39 (s, 6H), 5.94–5.97 (m, 2H), 6.01–6.06 (m,
2H), 6.09 (s, 2H), 6.23 (s, 1H), 6.64–6.68 (m, 2H), 9.50 (br, 2H); 13C
NMR
d 28.26, 28.29, 32.8, 66.1, 66.8, 82.8, 106.3, 107.4, 116.2, 116.6,
132.7, 158.1, 167.8, 168.3; FABMS obsd 612.3082, calcd 612.3047
(C33H44N2O9).
4.3.3. Zn(II)-5-Phenyl-15-[2,4,6-tris(tert-butoxycarbonyl-
methoxy)phenyl]porphyrin (ZnP-10)
containing 0.1% ammonium hydroxide. Silica gel (40 mm average
particle size) was used for column chromatography. Neutral alu-
mina was used as received. Molecular modeling was carried out
using PCMODEL version 7.50.00 (Serena Software).
The aqueous buffer solutions employed were the following: (i)
potassium phosphate (0.05 M, pH 7); (ii) potassium phosphate
monobasic (0.05 M, pH 7) was titrated with sodium hydroxide to
give pH 7.4 or pH 8; and (iii) potassium carbonate/potassium bo-
rate/potassium hydroxide (0.05 M, pH 10).
Following a reported procedure,30 a solution of 2a (150 mg,
0.245 mmol) and 3a (88.0 mg, 0.245 mmol) in EtOH (25 mL,
0.01 M) was treated with Zn(OAc)2 (450 mg, 2.45 mmol) and
heated to reflux for 16 h. The heat source was removed and the
reaction mixture was concentrated. Purification by column chro-
matography [alumina, hexanes/ethyl acetate (1:3)] gave a pink
solid (40 mg, 18%). 1H NMR
d 1.24 (s, 18H), 1.25 (s, 9H), 4.11 (s, 4H),
4.75 (s, 2H), 6.51 (s, 2H), 7.77–7.80 (m, 3H), 8.25–8.28 (m, 2H), 9.11
Analytical RP-HPLC in all cases was carried out using a reversed-
(d, J¼4.4 Hz, 2H), 9.21 (d, J¼4.7 Hz, 2H), 9.40 (d, J¼4.4 Hz, 4H), 10.26
phase, C-18 column (5
mm, 125 mmꢂ4 mm) with the following
(s, 2H); 13C NMR
d 28.1, 28.4, 66.4, 66.7, 82.2, 82.9, 93.7, 105.9, 126.8,
elution program: flow rate¼1.5 mL/min; 0–2 min, 0% B; 2–40 min
127.6, 131.5, 132.26, 132.39, 132.54, 134.9, 143.0, 149.5, 149.8, 151.4,

A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
11447
159.83, 159.99, 167.9; LD-MS obsd 914.5; ESI-MS obsd 915.2927,
mixture was diluted with H2O to adjust the pH to 3. The resulting
suspension was sonicated and centrifuged. Decanting afforded
a solid pellet. Water was added followed by sonication, centrifu-
gation, and decanting; this treatment was performed three times,
whereupon the supernatant was colorless. The pellet was dried in
calcd 915.2942 [(MþH)þ; M¼C50H51N4O9Zn]; labs 412, 538 nm; lem
(lex¼412 nm) 578, 632 nm.
4.3.4. 5-Phenyl-15-[2,4,6-tris(carboxymethoxy)phenyl]-
porphyrin (H2P-1)
vacuo to a green solid (6.5 mg, 65%). 1H NMR (DMSO-d6)
d
ꢃ2.58 (s,
A solution of ZnP-10 (20 mg, 0.022 mmol) in THF (4 mL, 5 mM)
was treated with 1 mL of aqueous KOH (2 M) and heated at 60 ꢁC.
After 8 h, CH2Cl2 (15 mL) and H2O (15 mL) were added to the re-
action mixture. The aqueous layer, which contained the porphyrin,
was separated and concentrated to a pink solid. The solid was
dissolved in H2O (3 mL) and treated with aqueous HCl (2 M) to
obtain pH¼3, whereupon the porphyrin precipitated. The mixture
was sonicated, centrifuged, and decanted to afford a solid pellet.
Water was added followed by sonication, centrifugation, and
decanting; this treatment was performed four times, whereupon
the supernatant was colorless. The pellet was dried in vacuo to
afford a red-brown solid (12 mg, 80% yield). 1H NMR (DMSO-d6)
1H), ꢃ2.52 (s, 1H), 4.44 (s, 4H), 4.91 (s, 2H), 6.65 (s, 2H), 9.09 (d,
J¼4.7 Hz, 2H), 9.46 (d, J¼4.3 Hz, 2H), 9.78 (d, J¼4.7 Hz, 2H), 10.32 (d,
J¼4.7 Hz, 2H), 10.59 (s, 2H), 12.52 (s, 1H); 13C NMR
d 64.6, 65.1, 92.7,
104.9, 116.6, 128.8, 131.87, 132.04, 134.9, 143.7, 145.6, 147.4, 148.9,
158.8, 160.1, 170.0; LD-MS obsd 637.0; ESI-MS obsd 636.1514, calcd
636.1492 (C33H24N4O10); labs (H2O) 409, 562, 633 nm; lem (H2O,
lex¼409 nm) 641, 707 nm; RP-HPLC tR¼28.94 min.
4.3.7. 1,9-Diformyl-5-{4-[N-(tert-butoxycarbonyl)-
amino]phenyl}dipyrromethane (5)
Following a reported procedure,30 a solution of 2b (500 mg,
1.48 mmol) in anhydrous DMF (1.50 mL) at 0 ꢁC under argon was
treated dropwise with POCl3 (0.28 mL, 3.1 mmol). The mixture
was stirred at room temperature. After 1 h the reaction mixture was
poured into a saturated aqueous solution of NaOAc (50 mL). The
milky reaction mixture was extracted with CH2Cl2. The organic
extract was washed with water, dried (Na2SO4), and filtered. The
filtrate was concentrated to dryness. The crude residue was chro-
matographed [silica, CH2Cl2/ethyl acetate (3:1)] to afford a beige
d
ꢃ3.32 (s, 1H), ꢃ3.24 (s, 1H), 4.46 (s, 4H), 4.95 (s, 2H), 6.66 (s, 2H),
7.87–7.90 (m, 3H), 8.28–8.31 (m, 2H), 9.01 (d, J¼4.9 Hz, 2H), 9.10 (d,
J¼4.4 Hz, 2H), 9.53 (d, J¼4.4 Hz, 2H), 9.63 (d, J¼4.9 Hz, 2H), 10.53 (s,
2H); 13C NMR
d 64.7, 65.2, 92.8, 105.1, 110.9, 127.3, 130.5, 131.3, 132.1,
134.6, 159.1, 169.7, 170.1; ESI-MS obsd 684.18505, calcd 684.1856
(C38H28N4O9); labs (H2O) 401, 504, 563 nm; lem
lex¼401 nm) 625,
(
686 nm; RP-HPLC tR¼30.05 min. In one case, the deprotection was
carried out with isolation of the zinc ester-porphyrin. A solution of
ZnP-10 (9 mg, 0.009 mmol) in THF (2.0 mL, 4.5 mM) was treated
with 0.5 mL of aqueous KOH (2 M) and heated at 60 ꢁC. After 8 h,
CH2Cl2 (10 mL) and H2O (10 mL) were added to the reaction mix-
ture. The aqueous layer, which contained the porphyrin, was sep-
arated and concentrated to give Zn(II)-5-phenyl-15-[2,4,6-
tris(carboxymethoxy)phenyl]porphyrin as a pink solid. 1H NMR
solid (400 mg, 68%): mp 196–202 ꢁC. 1H NMR
1H), 6.05–6.08 (m, 2H), 6.84–6.90 (m, 2H), 7.15–7.18 (m, 2H), 7.34–
7.32 (m, 2H), 9.24 (s, 2H), 10.94 (br, 2H); 13C NMR
28.5, 43.9, 111.7,
d 1.51 (s, 9H), 5.51 (s,
d
119.2, 122.7, 129.2, 132.8, 133.8, 138.1, 142.2, 179.3; FABMS obsd
394.1773, calcd 394.1767 [(MþH)þ; M¼C22H23N3O4].
4.3.8. 1,9-Bis(propylimino)-5-{4-[N-(tert-butoxy-
(methanol-d4)
d
4.09 (s, 4H), 4.69 (s, 2H), 6.78 (s, 2H), 7.80–7.82 (m,
carbonyl)amino]phenyl}dipyrromethane (3b)
3H), 8.22–8.25 (m, 2H), 8.98 (d, J¼4.4 Hz, 2H), 9.17 (d, J¼4.4 Hz, 2H),
9.36–9.40 (m, 4H), 10.23 (s, 2H); ESI-MS obsd 745.0917, calcd
745.0918 [(MꢃH)þ; M¼C38H26N4O9Zn]; labs (H2O) 409, 543 nm;
Following a reported procedure,30 a solution of 5 (250 mg,
0.630 mmol) and n-propylamine (1.00 mL, 12.7 mmol) in THF
(2 mL) was stirred at room temperature for 1 h. Removal of the
solvent and excess n-propylamine gave a beige solid (300 mg,
lem
(
lex¼409 nm) 587, 638 nm.
quantitative): mp 65–68 ꢁC. 1H NMR
d
0.88 (t, J¼7.2 Hz, 6H), 1.49 (s,
4.3.5. Zn(II)-5-(5,5-Dimethyl-1,3-dioxan-2-yl)-15-[2,4,6-tris-
(tert-butoxycarbonylmethoxy)phenyl]porphyrin (ZnP-20)
9H), 1.57–1.63 (m, 4H), 1.99 (s, 2H), 3.38 (t, J¼7.2 Hz, 4H), 5.40 (s,
1H), 5.92 (d, J¼3.6 Hz, 2H), 6.41 (d, J¼3.6 Hz, 2H), 7.07–7.10 (m, 2H),
Following a reported procedure,29 a solution of 2a (0.163 g,
0.267 mmol) and 4 (0.100 g, 0.267 mmol) in ethanol (26 mL,
10 mM) was treated with anhydrous Zn(OAc)2 (0.489 mg,
2.67 mmol) and heated to reflux. After 16 h, DDQ (0.181 g,
0.801 mmol) was added and the mixture was stirred for 15 min.
7.23–7.25 (m, 2H), 7.82 (s, 2H); 13C NMR
d 11.9, 24.3, 28.5, 43.9, 61.5,
109.8, 118.81, 118.84, 129.14, 129.20, 135.2,137.7, 139.0, 151.6; FABMS
obsd 476.3026, calcd 476.3056 [(MþH)þ; M¼C28H37N5O2].
4.3.9. Zn(II)-5-{4-[N-(tert-butyloxycarbonyl)amino]phenyl}-15-
[2,4,6-tris(tert-butoxycarbonylmethoxy)phenyl]porphyrin (ZnP-30)
Following a reported procedure,30 a solution of 2a (123 mg,
0.200 mmol) and 3b (95 mg, 0.20 mmol) in toluene (20 mL, 0.01 M)
was treated with anhydrous Zn(OAc)2 (367 mg, 2.00 mmol) and
refluxed for 12 h with exposure to air. The reaction mixture was
concentrated to dryness. Column chromatography [alumina, hex-
anes/ethyl acetate (1:1)] afforded a purple solid (58 mg, 28%). 1H
Triethylamine (187 mL, 1.33 mmol) was added and the reaction
mixture was concentrated to dryness. Column chromatography
[alumina, hexanes/ethyl acetate, (1:3)] afforded a purple solid
(26 mg, 10%). 1H NMR
d 1.23 (s, 27H), 1.64 (s, 6H), 4.11 (s, 4H), 4.38–
4.39 (m, 4H), 4.74 (s, 2H), 6.49 (s, 2H), 8.10 (s, 1H), 9.19 (d, J¼4.3 Hz,
2H), 9.36 (d, J¼4.3 Hz, 2H), 9.50 (d, J¼4.3 Hz, 2H), 10.16 (d, J¼4.7 Hz,
2H), 10.24 (s, 2H); 13C NMR
d 22.9, 25.4, 28.0, 28.2, 29.9, 31.2, 66.4,
66.6, 67.6, 80.5, 82.2, 82.9, 93.5, 106.0, 107.0, 130.4, 131.8, 132.43,
132.58, 149.3, 149.5, 149.7, 150.7, 159.7, 159.9, 167.8; LD-MS obsd
952.3; FABMS obsd 952.3222, calcd 952.3237 (C50H56N4O11Zn); labs
NMR d 1.15 (s, 9H), 1.62 (s, 27H), 4.15 (s, 4H), 4.79 (s, 2H), 6.49 (s,
2H), 7.76–7.78 (m, 2H), 8.17 (d, J¼8.0 Hz, 2H), 9.18 (d, J¼4.0 Hz, 2H),
9.10 (d, J¼4.0 Hz, 2H), 9.16 (d, J¼4.0 Hz, 2H), 9.37 (dd, J¼4.3, 4.3 Hz,
411, 543, 578 nm; lem
(lex¼411 nm) 578, 632 nm.
2H), 10.20 (s, 2H); 13C NMR
d 28.0, 28.4, 28.7, 66.4, 66.6, 82.2, 82.9,
90.7, 105.9, 110.3, 114.9, 116.8, 119.7, 131.4, 132.21, 132.34, 135.4,
137.84, 137.96, 149.4, 149.7, 149.9, 151.3, 153.3, 159.7, 159.9, 163.2,
167.8, 168.0, 204.21, 204.25, 211.1; LD-MS obsd 1029.2; FABMS obsd
1029.3558, calcd 1029.3503 (C55H59N5O11Zn); labs 414, 545,
4.3.6. 5-Formyl-15-[2,4,6-tris(carboxymethoxy)phenyl]-
porphyrin (H2P-2)
A sample of porphyrin ZnP-20 (15 mg, 0.016 mmol) in THF
(1.6 mL, 0.01 M) was treated with aqueous KOH (0.75 mL, 2 M) at
60 ꢁC for 10 h. The reaction mixture was allowed to reach ambient
temperature. The reaction mixture was treated with H2O (10 mL)
and CH2Cl2 (10 mL). The aqueous layer, which contained the por-
phyrin, was separated and concentrated to a pink solid. The solid
was treated with neat TFA (1 mL) and stirred at 50 ꢁC for 24 h. The
580 nm; lem
(
lex¼414 nm) 579, 634 nm.
4.3.10. 5-(4-Aminophenyl)-15-[2,4,6-tris(carboxy-
methoxy)phenyl]porphyrin (H2P-3)
A solution of ZnP-30 (20 mg, 0.019 mmol) in THF (1.9 mL,
0.01 M) was treated with aqueous KOH (0.5 mL, 2 M) at 60 ꢁC for

11448
A.Z. Muresan, J.S. Lindsey / Tetrahedron 64 (2008) 11440–11448
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28 h. The reaction mixture was treated with water and CH2Cl2. The
aqueous layer, which contained the porphyrin, was separated and
concentrated to a pink solid. The solid was diluted with water. The
solution was acidified with aqueous HCl (2 M) to pH 3, whereupon
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fuged, and decanted to afford a solid pellet; this procedure was
repeated four times, whereupon the supernatant was colorless. The
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treatment was performed three times, whereupon the supernatant
was colorless. The resulting solid was dried in vacuo to afford
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a dark-purple solid (11 mg, 83%). 1H NMR (DMSO-d6)
d
ꢃ3.21 (s,
1H), ꢃ3.11 (s, 1H), 4.45 (s, 4H), 4.94 (s, 2H), 6.65 (s, 2H), 7.07 (d,
J¼8.0 Hz, 2H), 7.95 (d, J¼8.4 Hz, 2H), 9.06 (d, J¼4.4 Hz, 2H), 9.13 (d,
J¼4.4 Hz, 2H), 9.49 (d, J¼4.8 Hz, 2H), 9.58 (d, J¼4.4 Hz, 2H), 10.46 (s,
2H); 13C NMR
d 64.8, 65.2, 92.7, 104.8, 110.1, 110.8, 112.9, 120.4,
127.66, 127.68, 130.8, 131.0, 131.6, 132.0, 135.9, 144.4, 146.5, 148.1,
148.7, 159.1, 159.8, 169.8, 170.1; LD-MS obsd 700.3; ESI-MS obsd
699.19663, calcd 699.19653 (C38H29N5O9); labs (H2O) 404, 505, 544,
567, 622 nm; lem (H2O, lex¼407 nm) 629, 688 nm; RP-HPLC
tR¼22.25 min.
36. Loewe, R. S.; Tomizaki, K.-Y.; Youngblood, W. J.; Bo, Z.; Lindsey, J. S. J. Mater.
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Acknowledgements
This research was supported by
a grant from the NIH
(GM36238). Mass spectra were obtained at the Mass Spectrometry
Laboratory for Biotechnology at North Carolina State University.
Partial funding for the Facility was obtained from the North Caro-
lina Biotechnology Center and the National Science Foundation. We
thank NingNing Xu, Ibrahim Bori, and Dhanalekshmi Savithri for
early work on this project.
39. Borbas, K. E.; Chandrashaker, V.; Muthiah, C.; Kee, H. L.; Holten, D.; Lindsey, J. S.
J. Org. Chem. 2008, 73, 3145–3158.
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