Bioconjugatable, PEGylated hydroporphyrins for photochemistry and photomedicine. Narrow-band, red-emitting chlorins

Article abstract of DOI:10.1039/c6nj01154c

Chromophores that absorb and emit in the red spectral region (600-700 nm), are water soluble, and bear a bioconjugatable tether are relatively rare yet would fulfill many applications in photochemistry and photomedicine. Here, three molecular designs have been developed wherein stable synthetic chlorins-analogues of chlorophylls-have been tailored with PEG groups for use in aqueous solution. The designs differ with regard to order of the installation (pre/post-formation of the chlorin macrocycle) and position of the PEG groups. Six PEGylated synthetic chlorins (three free bases, three zinc chelates) have been prepared, of which four are equipped with a bioconjugatable (carboxylic acid) tether. The most effective design for aqueous solubilization entails facial encumbrance where PEG groups project above and below the plane of the hydrophobic disk-like chlorin macrocycle. The chlorins possess strong absorption at ～400 nm (B band) and in the red region (Q_y band); regardless of wavelength of excitation, emission occurs in the red region. Excitation in the ～400 nm region thus provides an effective Stokes shift of >200 nm. The four bioconjugatable water-soluble chlorins exhibit Q_y absorption/emission in water at 613/614, 636/638, 698/700 and 706/710 nm. The spectral properties are essentially unchanged in DMF and water for the facially encumbered chlorins, which also exhibit narrow Q_y absorption and emission bands (full-width-at-half maximum of each <25 nm). The water-solubility was assessed by absorption spectroscopy over the concentration range ～0.4 μM-0.4 mM. One chlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The conjugate displayed a sharp signal when excited by a violet laser (405 nm) with emission in the 620-660 nm range. Taken together, the designs described herein augur well for development of a set of spectrally distinct chlorins with relatively sharp bands in the red region.

Full text of DOI:10.1039/c6nj01154c

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Bioconjugatable, PEGylated hydroporphyrins
for photochemistry and photomedicine.
Cite this:DOI: 10.1039/c6nj01154c
Narrow-band, red-emitting chlorins†
Mengran Liu,a Chih-Yuan Chen,a Amit Kumar Mandal,b Vanampally Chandrashaker,a
Rosemary B. Evans-Storms,c J. Bruce Pitner,*c David F. Bocian,*d Dewey Holten*b
and Jonathan S. Lindsey*a
Chromophores that absorb and emit in the red spectral region (600–700 nm), are water soluble, and bear
a bioconjugatable tether are relatively rare yet would fulfill many applications in photochemistry and
photomedicine. Here, three molecular designs have been developed wherein stable synthetic chlorins –
analogues of chlorophylls – have been tailored with PEG groups for use in aqueous solution. The designs
differ with regard to order of the installation (pre/post-formation of the chlorin macrocycle) and position
of the PEG groups. Six PEGylated synthetic chlorins (three free bases, three zinc chelates) have been
prepared, of which four are equipped with a bioconjugatable (carboxylic acid) tether. The most effective
design for aqueous solubilization entails facial encumbrance where PEG groups project above and below
the plane of the hydrophobic disk-like chlorin macrocycle. The chlorins possess strong absorption at
B400 nm (B band) and in the red region (Qy band); regardless of wavelength of excitation, emission
occurs in the red region. Excitation in the B400 nm region thus provides an effective Stokes shift of
4200 nm. The four bioconjugatable water-soluble chlorins exhibit Qy absorption/emission in water at
613/614, 636/638, 698/700 and 706/710 nm. The spectral properties are essentially unchanged in DMF
and water for the facially encumbered chlorins, which also exhibit narrow Qy absorption and emission
bands (full-width-at-half maximum of each o25 nm). The water-solubility was assessed by absorption
spectroscopy over the concentration range B0.4 mM–0.4 mM. One chlorin was conjugated to a mouse
polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle.
The conjugate displayed a sharp signal when excited by a violet laser (405 nm) with emission in the
620–660 nm range. Taken together, the designs described herein augur well for development of a set of
spectrally distinct chlorins with relatively sharp bands in the red region.
Received (in Montpellier, France)
13th April 2016,
Accepted 5th July 2016
DOI: 10.1039/c6nj01154c
www.rsc.org/njc
half of the solar photons are at wavelengths 4600 nm.1 For
Introduction
optical imaging in medicine,2 the deepest penetration in soft
Photochemistry in the red (600–700 nm) and near infrared tissue occurs in the NIR spectral region wherein light exhibits
(NIR, 700–1400 nm) spectral regions has been less investigated significantly less scattering than at shorter wavelengths and
than in the shorter wavelength visible or ultraviolet regions, yet minimal absorption by natural pigments and the vibrational
holds powerful attractions. For solar photoconversion, nearly overtones of water.3 For flow cytometry and related analytical
methods in clinical diagnostics, an objective is to squeeze as many
spectrally distinct fluorophores into the ultraviolet-visible-NIR
a Department of Chemistry, North Carolina State University, Raleigh,
region as possible thereby enabling multiplex analyses (i.e.,
NC 27695-8204, USA. E-mail: jlindsey@ncsu.edu
b Department of Chemistry, Washington University, St. Louis, MO 63130-4889, USA.
polychromatic flow cytometry4–6). Nonetheless, far fewer chromo-
phores are available for photochemical studies in the red and NIR
spectral regions than at wavelengths of 200–600 nm, and most NIR
absorbers that are available have quite broad spectral features.7,8
Nature’s chosen chromophores for the red and NIR spectral
regions are chlorophylls and bacteriochlorophylls, respectively.9
The fundamental chromophore of a chlorophyll or bacterio-
chlorophyll is a chlorin or bacteriochlorin, respectively, wherein
E-mail: holten@wustl.edu
c NIRvana Sciences, Research Triangle Park, NC 27709-3169, USA.
E-mail: bruce@nirvanasciences.com
d Department of Chemistry, University of California, Riverside, CA 92521-0403,
USA. E-mail: david.bocian@ucr.edu
† Electronic supplementary information (ESI) available: Studies and results
concerning exploratory syntheses of target chlorins, and spectral data for new
compounds. See DOI: 10.1039/c6nj01154c
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Chart 2 Synthetic porphyrins designed for aqueous solubility.
A variety of porphyrins bearing PEG groups similarly positioned
in the plane of the macrocycle have been prepared.29–32
One strategy implemented to mitigate aggregation has been
to place polar groups above and below the plane of the macro-
cycle, thereby shielding the intrinsically hydrophobic disk-like
macrocycle by facial encumbrance. This approach has been
employed with tetrapyrrole macrocycles as illustrated in Chart 3.
The representative examples displayed include porphyrins
bearing 3,5-disubstituted meso-aryl groups (I),33 porphyrins bearing
2,6-disubstituted meso-aryl groups (II),34–36 and the phthalocyanine
La Jolla Blue.37,38 In La Jolla Blue the apical bonding sites of the
centrally coordinated silicon bear bulky polar groups, and the
perimeter of the macrocycle is substituted with two carboxylic
acid groups for bioconjugation. The chlorin mTHPC,39 prepared
by hydrogenation of the corresponding porphyrin, contains only
one polar site on each meso-aryl group. The expected statistical
Chart 1 Natural pigments and core chromophores.
one or two b,b0-double bonds has been reduced relative to the distribution of conformers (due to rotation about the carbon–
p-system of a porphyrin (Chart 1). Synthetic methodology for carbon single bond between the tetrapyrrole and each aryl unit)
preparing such tetrapyrrole macrocycles has advanced enormously would afford positioning of groups on both (a,b) sides of the
over the years, with the ability now in hand to create reasonably macrocycle for three conformers (aaab,abab,aabb) but not the
diverse macrocycles and tailor the structure of the immediate fourth conformer (aaaa), likely limiting solubility (not shown).40
environment.10–21 Yet, suitable molecular designs for the afore- Regardless, the chlorins mTHPC and PEGylated derivative PEG-
mentioned photochemical applications typically must satisfy mTHPC,41 while widely used in photomedicine, lack a stabilizing
multiple criteria. The criteria encompass wavelength tunability, structural motif in the pyrroline ring and are susceptible to aerobic
features for bioconjugation, strong absorption, good fluores- dehydrogenation leading to the corresponding porphyrin.
cence quantum yield (a proxy for a reasonably long-lived singlet
Our own efforts to thwart aggregation in water have given rise to
excited state), and tailorable polarity for solubility in diverse chlorins42 that contain facially encumbering phosphonates (Chart 3,
media.22 Meeting all such criteria simultaneously often poses lower right), as well as a broader selection of ionic groups with
design challenges and can stretch the limits of synthetic capabilities. porphyrins43–46 and to some extent with bacteriochlorins.47,48
Moreover, many of the applications of red- and NIR-functional Because the ionic groups impart solubility in water but not
chromophores require solubilization in water. In this regard, (typically) in non-aqueous solvents, the polar group typically
the development of general strategies for aqueous solubilization must be installed or unveiled in the last synthetic step, where-
of tetrapyrrole macrocycles has remained a stubborn unsolved upon purification often entails merely washing the product
challenge.
with solvents. Moreover, the preparation of an activated ester
A classic design for aqueous solubilization of porphyrins relies (for bioconjugation) in the presence of ionic groups such as
on appending polar groups at the perimeter of the macrocycle, phosphates, phosphonates, and carboxylates can result in
exemplified by pyridyl and sulfonato porphyrins.23,24 A more recent deleterious cross-reaction.47 The PEG group is an attractive
example entails similar appendages of oligoethyleneoxy (i.e., PEG) alternative to these ionic species.49 The PEG group is nonionic,
groups, which have found extensive application and success is commercially available in various lengths27 (monodisperse or
across organic chemistry for aqueous solubilization.25–27 The polydisperse) with a single derivatizable handle,25,27,50,51 and is
mere introduction of PEG groups is not necessarily a panacea, soluble in water as well as a variety of organic solvents.49
however, as illustrated by the meso-tetrakis[( p-PEG10)phenyl]- Accordingly, workup of PEGylated compounds can be achieved
porphyrin, T(p-PEG10)PP (Chart 2), which exhibited significant by partitioning crude reaction mixtures between an aqueous
aggregation in water at a concentration as low as 0.1 mM.28 phase and dichloromethane.
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Chart 3 Tetrapyrroles with facially encumbering motifs.
In this paper we report the design and synthesis of a While diverse porphyrins have been used in bioconjugation
handful of chlorins bearing PEG groups and a bioconjuga- studies,24,54 few PEGylated synthetic chlorins have hereto-
table handle. A companion paper reports the development of fore been reported,41,55–58 and only one was equipped (but
corresponding bacteriochlorins.52 Our specific motivations not tested) with a bioconjugatable tether.58 The chlorins
for preparing bioconjugatable, PEGylated (bacterio)chlorins described herein have been characterized for solubility in
stemmed from a desire to pursue two research areas. One dilute aqueous solution. The photophysical properties,
area entails utilization of the PEGylated molecules as light- including rate constants and yields for fluorescence emission,
harvesting constituents in biohybrid antenna complexes via have been measured. One chlorin was attached to an anti-
attachment at hydrophilic terminal regions of membrane- body and examined via flow cytometry. Taken together, the
spanning photosynthetic peptides.53 A second research area advances reported herein broaden the scope of synthetically
focuses on the utilization of the PEGylated (bacterio)chlorins accessible chromophores for use in the red spectral region,
as fluorophores in clinical diagnostics, particularly flow particularly where sharp absorption and emission bands are
cytometry, where multiple antibodies are to be labeled. advantageous.
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Results and discussion
Molecular design and synthesis strategy
other designs were investigated and abandoned owing to synthetic
limitations (see the ESI†).
The de novo synthesis of chlorins developed in our laboratory
provides the foundation for the creation of stable chlorin macro-
cycles that contain diverse substituents about the perimeter.15 Each
synthetic chlorin bears a geminal dimethyl group in the reduced,
pyrroline ring to block adventitious dehydrogenation (Chart 4).
Tuning the position of the long-wavelength (Qy) absorption band
can be accomplished chiefly by (1) introduction of auxochromes
at positions along the y-axis (i.e., the b-pyrrole positions 2, 3, 12
and 13 in rings A and C), and (2) metalation, and to lesser
extent by (3) introduction of auxochromes at positions along
the x-axis (i.e., the b-pyrrole positions 7 and 8, and the b-pyrroline
position 17) in rings B and D. Given the desire to exploit the
b-pyrrole positions for wavelength tuning, a water-solubilization
motif and a bioconjugatable tether are ideally installed at positions
5, 10, and/or 15. Chlorin nomenclature is described in detail in
ref. 15.
Exploration of a number of designs has led to the synthesis
of the target compounds shown in Chart 5. The synthesis relies
on conversion of an aldehyde (compound 1 series) to a dipyrro-
methane (2 series), a 1-acyldipyrromethane (3 series), and a 1-acyl-
9-bromodipyrromethane (3-Br series) to give the Eastern half,
which is reacted with a tetrahydrodipyrrin Western half (4 or 5)
to form the chlorin. The terminology for Eastern and Western
halves dates to the work of Battersby some 40 years ago.15 In the
first design, the PEG groups are installed via click chemistry with
a PEG-azide following construction of a trialkynyl-substituted
chlorin macrocycle. The chlorin is prepared from a 2,4,6-tris-
(propargyloxy)phenyl-substituted Eastern half and an unsubstituted
Western half. The resulting zinc chlorin ZnC1 and free base chlorin
FbC1 were examined for water solubility but lack a bioconjugatable
tether. Click chemistry has found growing use in tetrapyrrole
chemistry but only occasionally applied with chlorins.59 In the
second design, the PEG groups and a bioconjugatable tether were
pre-installed in the Eastern half to give chlorins ZnC2 and FbC2.
The first two designs rely on a 2,6-disubstituted meso-aryl group
to impart facial encumbrance and achieve water solubility. In the
third design, a 3,13-diacetylchlorin bearing a bioconjugatable
group was converted to the corresponding chalcone thereby
affording chlorins ZnC3 and FbC3. This third design lacks the
facial encumbrance of the first two designs but, as in the first
design, is attractive in the installation of the PEG groups in the
final step of the synthesis. As part of this work, a number of
Synthesis
Dipyrromethanes. The synthesis of an Eastern half begins with
an aldehyde and pyrrole. Two aldehydes (1a and 1b) required herein
have been made from 2,4,6-trihydroxybenzaldehyde (phloroglucinol
carboxaldehyde), whereas the other aldehyde 1c60 is a known
compound. The reaction of 2,4,6-trihydroxybenzaldehyde and
propargyl bromide in the presence of K2CO3 at 80 1C afforded
2,4,6-tris(propargyloxy)benzaldehyde 1a in 87% yield without
chromatographic purification (Scheme 1). The O-alkylation of
2,4,6-trihydroxybenzaldehyde with 1-bromo-3,6,9,12-tetraoxatridecane
[CH3(OC2H4)4Br] in the presence of K2CO3 for 1.5 h46 gave
PEGylated benzaldehyde 1b in a yield of 36%. Partially PEGylated
product 1b0 was also isolated in 20% yield.
Dipyrromethanes were readily synthesized in a solventless
process from the corresponding aldehyde in a solution of excess
pyrrole containing InCl3 or TFA as catalyst60 (Scheme 2). Thus,
reaction of aldehyde 1a or 1b with pyrrole in the presence of InCl3
gave dipyrromethane 2a or 2b in yield of 52% or 74%, respectively.
Alternatively, a streamlined route to dipyrromethane 2b entailed
the following: (1) the O-alkylation of 2,4,6-trihydroxybenzaldehyde
was prolonged to 16 h; (2) the crude product 1b (containing a trace
amount of 1b0) was directly used in condensation with pyrrole
and B1 equiv. of InCl3; and (3) flash chromatography afforded
dipyrromethane 2b in a total yield of 54% (see Experimental
section). Dipyrromethane 2c60 was synthesized as described in
the literature.
Design I – chlorins without a bioconjugatable tether. Design I
relies on
a tris(propargyloxy)phenyl motif at the chlorin
10-position, whereupon click chemistry can be employed with
the three alkynes and a PEG-azide. The synthesis of the Eastern
half proceeded by Vilsmeier formylation61 of 2a with POCl3/DMF,
which afforded 1-formyldipyrromethane 3a as the major
product in 51% yield along with 1,9-diformylated byproduct
3a0 (not shown) in 20% yield (Scheme 3). Bromination of 3a
with 1 equiv. of NBS at ꢀ78 1C afforded 3a-Br9 in 68% yield.
Because 3a-Br9 decomposed quickly at room temperature, the
chlorin synthesis was initiated immediately. The chlorin-
forming reaction62 of 3a-Br9 and Western half 463 entailed acid-
catalyzed condensation ( p-TsOHꢁH2O in MeOH/CH2Cl2 under
argon for 30 min) followed by cyclization with accompanying
zinc chelation, oxidation, and HBr elimination [Zn(OAc)2,
2,2,6,6-tetramethylpiperdine (TMPi), and AgOTf in CH3CN at
reflux exposed to air for 22 h]. In this manner, tris(propargyl)
zinc chlorin 6 was obtained in 15% yield.
The click reaction of zinc chlorin 6 with PEG-azide 7 was
examined under several conditions concerning the solvent and
amount of copper (see Table S1, ESI†). Ultimately, treatment of
6 and 7 in DMSO with a stoichiometric amount of Cu(I) catalyst
(freshly prepared from CuSO4ꢁ5H2O and sodium ascorbate) in
H2O at 40 1C gave the zinc triazole-PEG-chlorin ZnC1 in 58%
yield. Demetalation with TFA gave the corresponding free base
chlorin FbC1 in 89% yield (Scheme 3). Performing the click
reaction with the zinc rather than the free base chlorin was
Chart 4 Chlorin nomenclature and spectroscopic axes.
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Chart 5 Molecular design and key precursors of synthetic chlorins.
essential to avoid undesired copper insertion, given that (1)
PEGylated chlorins FbC1 and ZnC1 were examined by UV-Vis
copper(II) chlorins are unsuitable for many photochemical spectroscopy in water and exhibited good water solubility (see
applications due to the short excited-state lifetime,64 and (2) the ESI,† Fig. S1). Successful installation of the PEG moiety by
demetalation of a copper chlorin requires strong acid and often click reaction and resulting water solubility prompted examination
proceeds in low yield.65 Still, a zinc chlorin can potentially of routes to analogous chlorins equipped with a bioconjugatable
transmetalate with copper. Two tests proved the absence of any tether. Several synthetic problems were encountered, however, that
copper chlorin in the target chlorins: (1) upon demetalation of limited the viability of this approach (see ESI†).
zinc chlorin ZnC1 with TFA, the expected bathochromic shift of
the Qy band was observed for the neutralized sample; and (2) no 5-position. Incorporation of the bioconjugatable tether at
copper chlorin peak was observed upon MALDI-MS analysis. the 5-position of chlorin can be achieved via acylation of the
Design II – chlorins with a bioconjugatable tether at the
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Scheme 1 Synthesis of aldehydes.
Scheme 3 Synthesis of PEGylated chlorins FbC1 and ZnC1 via click
chemistry.
Scheme 2 Synthesis of dipyrromethanes from the corresponding aldehydes. with n-Bu4POH in THF at 0 1C under argon, followed by the
addition of tert-butyl bromoacetate68 to form the phenolic ether 8
in 87% yield (Scheme 4). Benzoic acid 8 was prepared previously via
corresponding dipyrromethane.62,66 The acylation reagent a 3-step procedure.69 Reaction of 8 with oxalyl chloride under argon
(an S-2-pyridyl benzothioate)67 was prepared by selective alkylation in anhydrous CH2Cl2 afforded the acid chloride, which was treated
of 4-hydroxybenzoic acid. Thus, 4-hydroxybenzoic acid was treated with 2-mercaptopyridine in anhydrous THF to give pyridyl
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Scheme 4 Synthesis of Mukaiyama acylation reagent.
thioester 9 in 45% yield. Alternatively, streamlined procedures
beginning with 4-hydroxybenzoic acid and use of recrystalliza-
tion in lieu of chromatography afforded 9 in 32% yield.
Treatment of PEGylated dipyrromethane 2b with 9 afforded
acyldipyrromethane 3b in 41% yield (Scheme 5). Bromination70
of acyldipyrromethane 3b with 1 equivalent of NBS at ꢀ78 1C
followed by reduction with NaBH4 in THF/MeOH at room
temperature afforded the 9-bromodipyrromethane-1-carbinol.
Condensation of the latter with Western half 4 in the presence
of TFA followed by zinc-mediated oxidative cyclization gave zinc
chlorin 10 in 11% yield. Treatment of 10 with 20% TFA in
CH2Cl2 to hydrolyze the tert-butyl ester afforded PEGylated free
base chlorin FbC2. Subsequently, FbC2 was treated with
Zn(OAc)2 in CH2Cl2 to give zinc chlorin ZnC2 in 95% yield.
Chlorins FbC2 and ZnC2 constitute water-soluble, bioconjugatable,
spectrally distinct chromophores with absorption and emission in
the red spectral region. Examination of 10 by 1H NMR spectroscopy
validated the geometric design, showing the projection of the
2,6-substituted PEG groups in the vicinity of the p-electron cloud
of the chlorin macrocycle, but the 4-substituted PEG group not
in the ring current (see ESI,† Fig. S3). Attempts to extend this
synthetic approach to a collection of chlorins encompassing a wider
spectral region, which required incorporation of auxochromes in
rings A and C, were not successful (see ESI†); hence, we turned to
an alternative design.
Chlorin FbC2 was converted to the N-hydroxysuccinimide (NHS)
active ester by reaction with N-hydroxysuccinimide (HOSu) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
as shown in Scheme 6. The resulting active ester FbC2-NHS could
Scheme 5 Synthesis of a PEGylated chlorin.
be used for protein derivatization, but also was amidated with a chlorin prior to use in biological applications. The chlorin NHS
longer PEG-azide (11) followed by NHS activation with EDC to give esters were characterized (absorption spectroscopy, 1H NMR
chlorin FbC2-R-NHS, which contains 24 PEG (i.e., –OCH2CH2O–) spectroscopy and MALDI mass spectrometry) and were estimated
units. The two chlorin NHS esters were prepared for use in flow to be B80% pure.
cytometry studies (vide infra). As is customary in bioconjugate
Design III – chlorins with a bioconjugatable tether at the
chemistry, the active esters were not purified to homogeneity given 10-position. Chlorins with chalcones at the 3,13-positions are
that the chlorin–protein conjugate can be readily freed of unbound known to have a bathochromically shifted Qy band, but this
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Scheme 6 Synthesis of a PEGylated, bioconjugatable chlorin.
design had not previously been adapted for water solubility and
bioconjugation. While the PEGylated porphyrin shown in Chart 2
had very poor water solubility, we sought to examine whether the
presence of multiple PEG groups on each arene as well as the
nonplanar configuration of the chlorin–chalcone architecture would
impart aqueous solubility. Thus, vicinal dibromination of known
1-formyldipyrromethane 3c gave Eastern half 3c-Br8,9 (Chart 5),18
which was used directly without further purification (Scheme 7).
Condensation of 3c-Br8,9 with bromo-Western half71 5 gave
Scheme 7 Synthesis of PEGylated chlorins ZnC3 and FbC3.
Handling of PEGylated compounds. The PEG groups are
3,13-dibromochlorin 12 in 10% yield from 3c. Stille coupling72 with distinct from many other groups used to impart water solubility
tributyl(1-ethoxyvinyl)tin73 and a catalytic amount of (Ph3P)2PdCl2 in two regards: (1) PEG groups are non-ionic, and (2) PEGylated
in CH3CN/DMF (3 : 2) followed by acidic hydrolysis gave compounds can be partitioned preferentially into organic solvents
3,13-diacetylchlorin 13 in 90% yield. The aldol condensation74 from an aqueous medium.49 Thus, the PEGylated chlorins were
of 13 with excess aldehyde 1b in ethanolic NaOH under micro- quite soluble in CH2Cl2 or ethyl acetate, and could be extracted from
wave (MW) irradiation afforded chlorin–chalcone FbC3. Metala- aqueous solution to CH2Cl2 or ethyl acetate without significant loss.
tion of FbC3 with Zn(OAc)2 in CHCl3/CH3OH gave ZnC3 in 84% The purification of PEGylated compounds entailed gradient elution
yield. This chlorin–chalcone strategy provides a concise and chromatography. For some of the PEGylated tetrapyrrole macro-
efficient route, particularly given the ease of purification afforded cycles, the improved purification involved a sequence of three
by installation of the PEG moieties in the last step of the process. chromatography procedures:75 (1) removal of common impurities
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by silica chromatography; (2) separation of PEG-containing
For chlorins, ZnC1, FbC1, ZnC2 and FbC2, the Qy absorption
impurities by gravity-flow size-exclusion chromatography (SEC); bands have a full-width-at-half-maximum (FWHM) in the range
and (3) final silica chromatography to remove residual impurities 12–18 nm (14 nm average) in DMF and 14–19 nm (17 nm
(see the ESI†).
average) in water (Table 1). The greater FWHM in water versus
Characterization. The PEGylated, bioconjugatable chlorins DMF is paralleled by a decrease in Qy-band peak intensity
and the corresponding precursors typically were characterized by (relative to the Soret maximum) in water versus DMF. The
absorption and fluorescence spectroscopy (not for precursors), compensating effects of bandwidth with peak height indicate
1H NMR spectroscopy, 13C NMR spectroscopy (where quantity that the integrated intensity (oscillator strength) of the Qy band
and solubility allowed), MALDI-MS and ESI-MS. Several other target generally does not change appreciably with solvent. For the two
molecules were also designed with expectation to tune the wave- chlorin–chalcones (ZnC3 and FbC3), the peak intensity of the
length and were the subject of exploratory syntheses (see the ESI†). Qy band (relative to the Soret maximum) is much higher than
that of the other four chlorins, and the FWHM again increases
Photophysical characterization
in water versus DMF.
Static spectral properties. Fig. 1A shows the absorption
The spectra in Fig. 2 and Qy band data in Table 1 reveal that
spectra of chlorins ZnC2, FbC2, ZnC3, and FbC3 in DMF at room the addition of a bioconjugatable tether at the 5-position in
temperature normalized to the B band maximum (416–455 nm). chlorins ZnC2 and FbC2 gives a small (B2 nm) bathochromic
Fig. 1B focuses on the NIR region with spectra normalized to the shift in the Qy bands with respect to ZnC1 and FbC1 in DMF.
characteristic sharp Qy band (610–687 nm). The corresponding A small diminution of intensity of the Qy band is also observed
fluorescence emission bands are in the range 613–692 nm.
in the case of FbC1 with respect to that of FbC2 in water for
The absorption and fluorescence spectra of the same four incorporation of the bioconjugatable tether at the 5-position
chlorins in DMF are shown in Fig. 2A along with those for FbC1 (Table 1). For chlorin–chalcone FbC3, the Qy band shows a
and ZnC1. The spectra of all six chlorins in water are shown in B15 nm bathochromic shift with respect to the corresponding
Fig. 2B. The absorption spectra in Fig. 2 are normalized to the zinc chlorin ZnC3 in DMF.
total (300–900 nm) absorption intensity obtained upon integration of
The fluorescence spectra of all PEGylated chlorins are domi-
the spectra plotted against wavenumbers (cmꢀ1). This approach is nated by the Qy(0,0) emission band, with a weaker (0,1) band to
useful for comparing relative peak-intensity changes for related longer wavelength (Fig. 2A and B). For chlorins ZnC1, FbC1,
tetrapyrroles, and overcomes some of the drawbacks with other ZnC2 and FbC2, the fluorescence maximum lies within 3 nm of
normalization methods or using (uncertain) extinction coefficients.76 the Qy(0,0) absorption maximum in both DMF and water,
Each absorption spectrum contains three main features: the strong indicating a very small Stokes shift. On the other hand, in
near-ultraviolet (NUV) Soret bands (Bx and By, 410–455 nm), a very DMF the Stokes shift for chlorin–chalcones ZnC3 and FbC3 are
weak green-orange Qx band (520–560 nm) and a red or NIR Qy band 10 nm and 5 nm, respectively. In water, the shift is smaller,
(609–706 nm). The Soret and Qy band positions are listed in Table 1. 2 nm for ZnC3 or 4 nm for FbC3. For chlorins ZnC1, FbC1 and
Several general spectral characteristics are as follows: (1) the NUV By FbC2, the solvent effect causes a hypsochromic shift of 1–2 nm
and Bx bands overlap completely with each other for the zinc in the Qy fluorescence maximum in water versus DMF. Contrarily,
chlorins but only partially overlap for the free base chlorins. (2) The the Qy fluorescence maximum shifts bathochromically for ZnC2
weak Qx bands of free base chlorins are further weakened in the case (1 nm), ZnC3 (18 nm) and FbC3 (8 nm).
of zinc chlorins. (3) The Qy bands for the two chlorin–chalcones
Excited-state properties. The measured photophysical properties
(ZnC3 and FbC3) are moderately strong whereas the Qy bands for the of the chlorins are the lifetime (tS) of the lowest singlet excited state
other four chlorins (ZnC1, FbC1, ZnC2 and FbC2) are relatively weak. (S1), the fluorescence quantum yield (Ff), i.e. the yield of S1 to S0,
(4) All the origin bands are typically accompanied by one weaker and the triplet yield (Fisc), i.e., the yield of S1 to T1 intersystem
vibronic satellite band to higher energy by roughly 1000 cmꢀ1
.
crossing. The yield of S1 to S0 internal conversion is obtained by the
Fig. 1 (A) Absorption spectra of PEGylated chlorins in DMF; (B) normalized Qy absorption and emission spectra of chlorins in DMF.
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Fig. 2 Absorption (blue solid) and fluorescence (red dashed) spectra of PEGylated chlorins in (A) DMF and (B) water. The emission intensities are
normalized to the Qy absorption for ease of presentation.
Table 1 Spectral properties of PEGylated chlorins in DMF and in water
Table 2 Photophysical properties of PEGylated chlorins in DMF and
watera
Bmax Qy abs Qy
abs FWHM abs
Chlorin Solventa (nm) (nm)
Qy
Qy/ em
By (nm) (nm)
Qy em
FWHM
P
P
ꢀ1
ꢀ1
IQ
/
Qy em tS
Chlorin Solventb (nm)
(ns)
kfꢀ1 kisc
kic
y
(nm) IB
Ff
Fisc Fic (ns) (ns)
(ns)
y
ZnC1
FbC1
ZnC2
FbC2
ZnC3
FbC3
DMF
Water
DMF
Water
DMF
Water
DMF
Water
DMF
Water
DMF
410
408
406
404
416
414
416
412
455
445
443
445
609
610
637
636
610
613
640
636
672
698
687
706
18 0.16 0.20
19 0.14 0.15
15 0.25 0.12
17 0.20 0.12
14 0.15 0.16
16 0.15 0.15
12 0.25 0.15
14 0.14 0.06
28 0.68 0.38
71 0.57 0.38
25 0.52 0.19
42 0.52 0.22
612
611
639
637
613
614
640
638
682
700
692
710
20
22
16
18
17
24
15
17
30
60
26
58
ZnC1
FbC1
ZnC2
FbC2
ZnC3
FbC3
DMF
Water
DMF
Water
DMF
Water
DMF
Water
DMF
Water
DMF
612
611
639
637
613
614
640
638
682
700
692
710
2.0 0.066 0.92 0.01 30
1.8 0.030 0.88 0.09 61
8.4 0.20 0.73 0.07 42 11
8.2 0.14 0.76 0.10 58 11
2.1 0.080 0.91 0.01 26
1.8 0.013 0.93 0.06 138
2
2
140
20
120
82
210
31
85
2
2
10.2 0.25 0.63 0.12 41 16
9.7 0.17 0.68 0.15 57 14
5.1 0.34 0.48 0.18 15 11
65
28
c
c
c
c
c
c
c
—
—
—
—
—
—
—
6.0 0.33 0.43 0.24 18 14
25
c
c
c
c
c
c
c
Water
Water
—
—
—
—
—
—
—
a
a
The samples in water contained 5% DMF as a cosolvent.
The typical errors (percent of value) of the photophysical properties
are as follows: tS (ꢂ7%), Ff (ꢂ5%), Fisc (ꢂ15%), Fic (ꢂ20%), kf (ꢂ10%),
kisc (ꢂ20%), kic (ꢂ25%). The error bars for tS, Ff, and Fisc were
determined from select repeat measurements, and those for the Fic, kf, kisc
and kic were obtained from propagation of errors. b The samples in water
difference: Fic = 1 ꢀ Ff ꢀ Fisc. The fluorescence (kf), internal
conversion (kic), and intersystem crossing (kisc) decay rate
constants of the lowest singlet excited state are related via the
c
contained 5% DMF as a cosolvent. Not examined due to low solubility.
expressions tS = (kf + kic + kisc
)
ꢀ1 and Fx = kxꢁtS, where x = f, isc or
ic. Thus, all three excited-state rate constants can be calculated ZnC3 and FbC3, the Ff values are relatively higher (0.34 and 0.33,
from the lifetime and yields (Table 2).
respectively) than for the other chlorins in DMF (Table 2).
Some important characteristic features gleaned from Table 2
The Ff values range from 0.013 to 0.080 and the tS values
range from 1.8 ns to 2.1 ns for the zinc chlorins ZnC1 and ZnC2 are as follows: (1) the Ff values of the free base chlorins
(Table 2) in both DMF and water. For the corresponding free (FbC1, FbC2) are greater than the zinc chlorins (ZnC1, ZnC2),
base analogues, the Ff values are higher (0.14–0.25) and the and are similar for the two chlorin–chalcones (FbC3, ZnC3). (2)
tS values are much longer (8.2–10.2 ns). For the chlorin–chalcones, The ts values of the zinc chlorins are much shorter than the free
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base chlorins. These changes can be traced largely to greater
kisc values for the zinc chelates relative to the free base forms, as
expected due to more facile intersystem crossing associated with
the heavy-atom effect on spin–orbit coupling in the metallo-
chlorins (Table 2). Metalation only increases kisc modestly for
ꢀ1
chlorin–chalcone ZnC3 relative to FbC3. The kic
values
decrease (kic increases) for chlorins ZnC1, FbC1, ZnC2 and
FbC2 in water versus DMF. As noted above, the low solubility
of the two chlorin–chalcones (ZnC3 and FbC3) precludes the
photophysical studies in water. In each case, the calculated
radiative rate constant (kf) in water is reduced considerably
from the values in DMF. Similarly, the intensity of the Qy band
relative to the Soret is either reduced or similar in water versus
DMF (Table 1). The effects are in the same direction and in
good agreement considering experimental error. The connec-
tion between these two observables is the direct proportionality
of the Einstein coefficients for absorption and spontaneous
emission. This connection explains the slight diminution in
relative Qy absorption intensity (Table 1) and the moderate
decrease of Ff (Table 2) in water compared to DMF. Some small
change in radiative probability for the compounds in the two
media is expected due to the difference in refractive index
values. However, other effects may also come into play, including
the influence of the media on the relative energies of the frontier
molecular orbitals of the complexes. The latter issue has not
been explored in detail for tetrapyrroles.
Effect of concentration on spectral properties. Absorption
versus concentration studies were conducted to assess the
aqueous solution properties of the PEGylated chlorins over a
1000-fold range of concentration (B450 mM to B0.45 mM). The
methodology for this type of study has been previously reported
in detail;77 the same approach was utilized herein. The spectral
properties of the PEGylated chlorins ZnC2 and FbC2 in neat
Fig. 3 Absorption versus concentration of ZnC2 (top) and FbC2 (bottom)
over a 1000-fold range. The spectra are normalized at the Qy band; the
path length of the sample cell and FWHM of the Qy band are also listed in
the inset. The concentrations are based on the absorbance of the B5 mM
solution measured at the B band (assuming64 e = 160 000 Mꢀ1 cmꢀ1).
deionized water are shown in Fig. 3; data for ZnC1 and FbC1 are antibody-capture compensation beads, a tool frequently used for
shown in ESI† (Fig. S1). ZnC2 exhibits almost unchanged setting up multicolor flow cytometry experiments to determine
spectral properties over a concentration range of 1000-fold, proper corrections due to spectral overlap between fluorophore-
indicating exceptionally high solubility of this chlorin in water. labeled reagents.78
On the other hand, FbC2 exhibits changes in the shape of the
The chlorin FbC2-R-NHS was used to label mouse IgG antibody
B bands at higher concentrations (between 4.8 and 48 mM) for detection using mouse IgG antibody-specific compensation
indicating some degree of aggregation. The absorption spectra beads. The antibody was labeled at room temperature for 3 h
of chlorin–chalcones, ZnC3 and FbC3 exhibit clear broadening with a 35-fold molar excess of FbC2-R-NHS. The conjugate was
in neat water (see ESI,† Fig. S2) at B5 mM, which indicates dialyzed against a 20 kD molecular weight cutoff membrane
limited water solubility of these compounds.
to remove byproducts, and the labeled antibody was further
purified by affinity binding to Protein A agarose beads. The
resulting chlorin–antibody conjugate FbC2–Ab had a fluorophore/
Flow cytometry
The development of fluorophores with spectrally distinct emission protein ratio of 2.2 on the basis of absorption spectroscopy. This
bands (i.e., distinct ‘‘colors’’) as well as supporting technology ratio is relatively low but it ensures minimal dye–dye quenching on
that enables polychromatic flow cytometry is critically impor- the antibody. Flow cytometry experiments used a 405 nm laser for
tant for achieving increased accuracy and efficacy in clinical excitation with 600 nm longpass and 620/60 nm bandpass filters
diagnostics.4–6 In this regard, the 405 nm (violet) diode laser is for emission. Fig. 4 shows a histogram of the flow cytometry signals
becoming one of the most commonly used excitation sources in for the FbC2–Ab-bound compensation beads, for unbound beads
flow cytometers.5 This excitation wavelength is ideal for chlorins (negative population), and for beads bound to a monoclonal anti-
because these molecules typically absorb strongly near 405 nm; body labeled with Brilliant Violet 650 (BV650–Ab, the positive
the resulting emission in the red region affords an effective Stokes control). Brilliant Violet 650 is from a recently introduced dye family
shift of 4200 nm (Fig. 1). To demonstrate the use of chlorins in (based on conjugated polymers) that are among the brightest
flow cytometry, chlorin-labeled antibodies were detected using fluorophores available for flow cytometry.79 The FbC2–Ab exhibits
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multiplexing; (2) collect most of the emission from the chlorin but
only a fraction from current dyes with broader emission; and
(3) effectively collapse any apparent greater (integrated) brightness
of another dye compared to a chlorin.
Finally, a key issue for flow cytometry and many other
photochemical applications concerns photostability. The PEGylated
chlorins were stable for routine handling and spectroscopic
measurements. For photophysical measurements the solutions
were purged with argon to remove O2. Such solutions typically
showed less than 3% change in the spectrum during the course
of spectroscopic studies carried out here, as evidenced by the
diminution in the overall spectral amplitude generally without
appearance of prominent new features.
Fig. 4 Histogram from a flow cytometry experiment using compensation
beads stained with either 0.5 mg of chlorin–antibody FbC2–Ab (solid line) or
0.12 mg of Brilliant Violet 650–antibody BV650–Ab (dashed line), plus unstained
beads (dotted line). The coefficient of variation (CV) was 36 (FbC2–Ab),
30 (BV650–Ab), and 155 (unstained beads). The analysis used the 405 nm
violet laser with 600 nm longpass and 620/60 nm bandpass filters.
Conclusions
The installation of PEG groups in a facially encumbering arrange-
ment enables aqueous solubilization of the hydrophobic, disk-like
chlorin macrocycle. The resulting PEGylated chlorins are water-
soluble but neutral and nonionic. A single PEG-substituted
2,4,6-trialkoxy arene (for ZnC1, FbC1, ZnC2, FbC2) suffices to
impart aqueous solubility, whereas PEG groups at the terminus
of chalcones (for FbC3, ZnC3) were only partially effective in
this regard. Chlorins ZnC1, FbC1 or ZnC2, FbC2 are substituted
with fewer PEG units (12 units or 18 units in total, respectively)
than T(p-PEG10)PP (40 units in total; Chart 2), yet the former
designs impart significant solubility of the macrocycle in water.
A distinct feature of the synthetic chlorins is the relatively sharp
long-wavelength absorption and companion fluorescence emission
band. Such sharp bands are very attractive for use in polychromatic
flow cytometry, light-harvesting, and energy-cascade processes.
roughly the same fluorescence intensity as a four-fold dilution of the
BV650–Ab for the same relative protein concentrations. Fig. 5 shows
the relative emission spectral overlap of both dyes with the bandpass
filter. The BV650–Ab is a commercial product with an undisclosed
fluorophore/protein ratio, so a more precise comparison of relative
dye spectral properties was not possible.
The flow cytometry data suggest that chlorins should prove
valuable as labels for polychromatic experiments, especially ones
requiring a large number of fluorophores excited from a violet
laser. Although not as bright as Brilliant Violet 650, FbC2-R-NHS
has far narrower emission (FWHM o20 nm) than the commercial
label (FWHM B50 nm; Fig. 5). Thus, within the red spectral
range, it should be possible to discriminate more chlorin-labeled
biomolecules with less spectral overlap (‘‘spillover’’)78 than with
other fluorophore families currently available for flow cytometry.
Fig. 5 suggests that the overall advantage of the narrow-emitting
chlorins would be further enhanced if the filter bandpass were
reduced (e.g., from 60 nm to 30 nm). This would (1) fit more
discrete emission channels in a given wavelength span to enhance
Experimental section
General methods
1H NMR (400 MHz) spectra and 13C NMR spectra (100 MHz)
were collected at room temperature in CDCl3 unless noted
otherwise. Silica gel (40 mm average particle size) was used for
adsorption column chromatography. Size-exclusion chromato-
graphy (SEC) was carried out at the preparative level using Bio
Beads S-X3 (200–400 mesh) and elution with toluene (HPLC
grade). All solvents were reagent grade and were used as
received unless noted otherwise. THF was freshly distilled from
sodium/benzophenone ketyl. Electrospray ionization mass
spectrometry (ESI-MS) data are reported for the molecular ion
or cationized molecular ion. Matrix-assisted laser-desorption
mass spectrometry (MALDI-MS) was performed with the matrix
1,4-bis(5-phenyl-2-oxaxol-2-yl)benzene.80 Sonication was carried
out using a benchtop sonication bath. Compounds 2c,60 3c,18 4,63
and 571 were prepared as described in the literature.
Synthesis
2,4,6-Tris(propargyloxy)benzaldehyde (1a). Following an
alkylation procedure,46 a solution of 2,4,6-trihydroxybenzaldehyde
(5.05 g, 32.7 mmol) in anhydrous DMF (150 mL) was treated with
Fig. 5 Fluorescence spectra of chlorin FbC2-R-NHS (red line) and Bril-
liant Violet 650 (dotted line) overlaid with the Chroma bandpass filter
ET620/60 (shadowed gray).
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K2CO3 (27.6 g, 200 mmol), and the resulting suspension was heated (7.4 mL, 0.11 mol) and 1b (0.77 g, 1.1 mmol) was degassed with
to 60 1C. After 30 min, propargyl bromide (80% in toluene, 11.5 mL, a stream of argon for 10 min. InCl3 (2.4 mg, 11 mmol) was
105 mmol) was added, and the temperature was increased to 80 1C. added, and the mixture was stirred under argon at room
After stirring for 1.5 h, the reaction mixture was allowed to cool to temperature for 1.5 h. The mixture turned yellow during the
room temperature, and then diluted with ethyl acetate (500 mL). course of the reaction. Powdered NaOH (0.13 g, 3.3 mmol) was
The organic fraction was washed [water (3 ꢃ 200 mL) and brine added to quench the reaction. After stirring for 45 min, the
(2 ꢃ 200 mL)], dried (Na2SO4), and concentrated to obtain a brown mixture was filtered by gravity flow. The filtrate was concen-
1
solid (7.65 g, 87%): mp 137–139 1C; H NMR (300 MHz) d 2.56 trated and chromatographed (silica, CH2Cl2 with 2% MeOH) to
(t, J = 2.4 Hz, 2H), 2.60 (t, J = 2.4 Hz, 1H), 4.76 (d, J = 2.4 Hz, 2H), afford a pale yellow liquid (0.66 g, 74%): 1H NMR d 3.36 (s, 6H),
4.78 (d, J = 2.4 Hz, 4H), 6.39 (s, 2H), 10.38 (s, 1H); 13C NMR d 32.9, 3.37 (s, 3H), 3.50–3.57 (m, 15H), 3.62–3.70 (m, 24H), 3.80–3.82
56.0, 56.6, 76.5, 77.4, 93.8, 95.0, 110.3, 161.5, 163.2, 187.2; ESI-MS (m, 3H), 4.00–4.07 (m, 6H), 5.86 (s, 2H), 5.85–5.87 (m, 2H), 6.10
obsd 269.08107, calcd 269.08084 [(M + H)+, M = C16H12O4].
(s, 3H), 6.60–6.61 (m, 2H), 9.20 (s, 2H); 13C NMR d 32.6, 59.0,
2,4,6-Tris(3,6,9,12-tetraoxatridecyloxy)benzaldehyde (1b). Follow- 67.5, 69.6, 70.4, 70.6, 70.75, 70.79, 71.9, 93.2, 105.5, 107.2,
ing an alkylation procedure,46 a solution of 2,4,6-trihydroxybenz- 113.5, 115.9; ESI-MS obsd 841.4682, calcd 841.4698 [(M + H)+,
aldehyde (0.45 g, 2.9 mmol) in anhydrous DMF (5.0 mL) was M = C42H68N2O15].
treated with K2CO3 (1.5 g, 12 mmol), and the resulting suspen-
Streamlined synthesis of 2b. A solution of 2,4,6-trihydroxy-
sion was heated to 60 1C. After 30 min, 1-bromo-3,6,9,12- benzaldehyde (0.82 g, 5.3 mmol) in anhydrous DMF (10.6 mL)
tetraoxatridecane (3.0 g, 11 mmol) was added. The mixture was treated with K2CO3 (2.4 g, 18 mmol), and the resulting
was heated at 80 1C and stirred for 1.5 h. The reaction mixture suspension was heated to 60 1C. After 30 min, 1-bromo-3,6,9,12-
was allowed to cool to room temperature, and then diluted with tetraoxatridecane (5.0 g, 18 mmol) was added, and the tem-
CH2Cl2. The mixture was washed with brine. The organic layer perature was raised to 80 1C. After 16 h, the reaction mixture
was dried (Na2SO4) and concentrated. Chromatography (silica, was concentrated under vacuum (40 mmHg) at 60 1C. The
CH2Cl2 with 5% MeOH) afforded the title compound (0.77 g, resulting liquid was diluted with CH2Cl2, dried (Na2SO4) and
36%) and 2,4-bis(3,6,9,12-tetraoxatridecyloxy)-6-hydroxybenz- filtered. The filtrate was concentrated in a 100 mL flask to
aldehyde (1b0, 0.30 g, 20%), each as a pale yellow liquid. Data afford a pale yellow liquid. Pyrrole (37 mL, 0.54 mol) was added,
for 1b: 1H NMR d 3.38 (s, 9H), 3.53–3.56 (m, 6H), 3.63–3.72 and the mixture was degassed with a stream of argon for 10 min.
(m, 30H), 3.85–3.91 (m, 6H), 4.14–4.17 (m, 6H), 6.11 (s, 2H), InCl3 (0.60 g, 3.0 mmol) was added, and the mixture was stirred
1
10.36 (s, 1H); 13C NMR d 59.0, 69.6, 68.7, 69.3, 70.6, 70.8, 71.0, under argon at room temperature. After 1 h, H NMR analysis
71.9, 92.2, 109.4 163.0, 165.0, 187.4; ESI-MS obsd 725.3942, showed the reaction was not complete. Additional InCl3 (0.60 g,
calcd 725.3954 [(M + H)+, M = C34H60O16]. Data for 1b0: 1H NMR 3.0 mmol) was added, and the mixture was stirred for 2.5 h.
d 3.38 (s, 6H), 3.54–3.56 (m, 4H), 3.63–3.73 (m, 20H), 3.84–3.89 NaOH (0.60 g, 0.020 mol) was added to quench the reaction.
(m, 4H), 4.13–4.16 (m, 4H), 5.97 (dd, J = 21.7 Hz, J = 2.01 Hz, After stirring for 45 min, the mixture was filtered by gravity flow.
2H), 10.12 (s, 1H), 12.48 (s, 1H); 13C NMR d 59.0, 67.8, 68.1, The filtrate was concentrated and chromatographed (silica,
69.2, 70.6, 70.8, 70.9, 71.9, 91.84, 91.90, 93.5, 93.6, 106.1, 162.6, CH2Cl2 with 2% MeOH) to afford a pale yellow liquid (2.4 g,
166.2 167.1, 192.0; ESI-MS obsd 535.2725, calcd 535.2749 54%). The characterization data (1H NMR) were consistent with
[(M + H)+, M = C25H42O12].
those reported above.
5-[2,4,6-Tris(propargyloxy)phenyl]dipyrromethane (2a). Following
1-Formyl-5-[2,4,6-tris(propargyloxy)phenyl]dipyrromethane (3a).
a reported procedure,60 a solution of aldehyde 1a (2.68 g, 10.0 mmol) Following a standard procedure,61 the Vilsmeier reagent was
in pyrrole (69.3 mL, 1.00 mol) was degassed with a stream of argon prepared by treatment of dry DMF (2 mL) with POCl3 (341 mL,
for 10 min. InCl3 (221 mg, 1.00 mmol) was added, and the mixture 3.65 mmol) at 0 1C and stirring of the resulting mixture for
was stirred for 1.5 h under argon flow. Powdered NaOH (1.2 g) was 10 min under argon. In a separate flask, a solution of 2a (1.30 g,
added, and the mixture was stirred for 45 min. The mixture was 3.38 mmol) in DMF (15 mL) was treated with the freshly
filtered. The filtrate was concentrated under high vacuum (to remove prepared Vilsmeier reagent at 0 1C. The resulting mixture was
excess pyrrole) and then diluted with ethyl acetate (100 mL). The stirred at 0 1C for 1.5 h. The reaction mixture was treated with
mixture was washed with water, dried (Na2SO4), concentrated and saturated aqueous NaOAc (B15 mL) for 2 h. CH2Cl2 was added,
chromatographed [silica, hexanes/ethyl acetate (4 : 1)] to give a then the organic phase was washed (water, brine), dried (Na2SO4),
1
viscous oil (2.03 g, 52%): H NMR (300 MHz) d 2.50–2.57 (m, 3H), concentrated, and chromatographed [silica, hexanes/ethyl acetate
1
4.52 (b, 4H), 4.66 (d, J = 2.7 Hz, 2H), 5.90–5.94 (m, 2H), 6.07–6.12 (3 : 2)] to afford an orange oil (713 mg, 51%): H NMR d 2.53
(m, 3H), 6.44 (s, 2H), 6.62–6.66 (m, 2H), 7.56–7.64 (b, 2H); 13C NMR (t, J = 2.4 Hz, 2H), 2.56 (t, J = 2.4 Hz, 1H), 4.56 (d, J = 2.4 Hz, 4H),
d 14.0, 32.8, 56.2, 57.6, 76.1, 76.2, 78.4, 78.6, 96.2, 106.3, 108.0, 4.66 (d, J = 2.4 Hz, 2H), 5.92–5.94 (m, 1H), 6.06 (s, 1H), 6.11–6.12
115.2, 116.5, 116.8, 132.8, 157.2, 157.8; ESI-MS obsd 383.13838, (m, 2H), 6.40 (s, 2H), 6.68–6.70 (m, 1H), 6.83–6.84 (m, 1H),
calcd 383.13902 [(M–H)+, M = C24H20N2O3; this likely is the 8.90 (s, 1H), 9.26 (s, 1H), 9.33 (s, 1H); 13C NMR d 32.7, 32.8,
protonated dipyrrin, which is far more easily protonated than 56.8, 76.1, 76.2, 76.4, 77.9, 78.07, 95.44, 95.49, 107.7, 107.9,
the dipyrromethane and can be detected in minute quantities].
109.4, 112.7, 117.6, 122.2, 129.8, 131.5, 144.1, 156.7, 158.2,
5-[2,4,6-Tris(3,6,9,12-tetraoxatridecyloxy)phenyl]dipyrromethane 177.9; ESI-MS obsd 413.14984, calcd 413.14958 [(M + H)+,
(2b). Following a standard procedure,60 a solution of pyrrole M = C25H20N2O4].
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1-{4-[2-(tert-Butoxy)-2-oxoethoxy]benzoyl}-5-[2,4,6-tris(3,6,9,12- (THF-d8) d 29.6, 30.8, 45.1 50.9, 57.4, 75.9, 79.0, 93.6, 94.7, 96.0,
tetraoxatridecyloxy)phenyl]dipyrromethane (3b). Following 108.6, 114.2, 116.0, 126.4, 126.5, 127.7, 127.8, 131.8, 132.2, 145.7,
a
standard procedure62 with slight modification of reaction time 146.1, 147.2, 148.3, 153.0, 153.4, 158.1, 159.2, 159.6, 168.4;
and reagent equivalents, a solution of 2b (840. mg, 1.00 mmol) in MALDI-MS obsd 641.4263, calcd 641.1526 [(M + H)+, M =
THF (2.0 mL) was treated with EtMgBr (2.8 mL, 0.9 M, 2.5 mmol) at C37H28N4O3Zn]; ESI-MS obsd 640.14376, calcd 640.14474
room temperature for 30 min. The solution was cooled to ꢀ78 1C, (M+, M = C37H28N4O3Zn); labs (toluene) 407, 609 nm.
whereupon a solution of 9 (202 mg, 1.20 mmol) in THF (2.4 mL)
4-[2-(tert-Butoxy)-2-oxoethoxy]benzoic acid (8). Following a
was added. After 1 h, the reaction mixture was quenched by procedure for phenol alkylation,68 a solution of 4-hydroxy-
addition of saturated aqueous NH4Cl solution (10 mL). The mixture benzoic acid (3.00 g, 21.7 mmol) in THF (43.4 mL) at 0 1C
was extracted with CH2Cl2 (3 ꢃ 20 mL). The organic layer was dried under argon was treated with 40% aqueous n-Bu4POH (30.3 mL,
(Na2SO4), concentrated, and chromatographed (silica, CH2Cl2 with 43.4 mmol), whereupon tert-butyl bromoacetate (3.20 mL,
2–5% MeOH) to afford a pale yellow liquid (439 mg, 41%): 1H NMR 21.7 mmol) was added. The resulting reaction mixture was
d 1.50 (s, 9H), 3.36 (s, 6H), 3.37 (s, 3H), 3.51–3.55 (m, 6H), 3.60–3.73 allowed to warm to room temperature for 1 h. The organic
(m, 33H), 3.82–3.84 (m, 3H), 3.97–4.16 (m, 6H), 4.58 (s, 2H), 5.90– solvent was removed under vacuum, and the aqueous residue
5.92 (m, 1H), 6.05–6.07 (m, 1H), 6.08–6.10 (m, 1H), 6.11–6.15 (m, was acidified with 2 N aqueous HCl. No product precipitated
3H), 6.71–6.74 (m, 2H), 6.91–6.96 (m, 2H), 7.81–7.85 (m, 2H), 9.41 after acidification, so ethyl acetate and brine were added. The
(s, 1H), 9.90 (s, 1H); 13C NMR d 28.0, 32.9, 59.0, 65.5, 67.5, 69.53, organic phase was washed (water), dried (Na2SO4), and concentrated
70.46, 70.71, 70.77, 71.83, 82.6, 93.1, 107.0–107.3 (m), 109.0, to give a colorless oil. The resulting oil was filtered through a silica
111.3, 114.0, 117.8, 119.7, 129.2, 130.5, 130.7, 132.2, 143.5, 157.2, pad with ethyl acetate. The filtrate was chromatographed [silica,
159.3, 160.5, 167.5, 182.3; ESI-MS obsd 1075.5566, calcd hexanes/ethyl acetate (3 : 1)] to afford a white solid (4.76 g, 87%): mp
1075.5585 [(M + H)+, M = C55H82N2O19].
114–116 1C; 1H NMR d 1.46 (s, 9H), 4.57 (s, 2H), 6.91 (d, J = 9.0 Hz,
Zn(II)-10-[2,4,6-tris(propargyloxy)phenyl]-18,18-dimethylchlorin 2H), 8.04 (d, J = 9.0 Hz, 2H), 11.23 (br s, 1H); 13C NMR d 28.0, 65.5,
(6). Following a standard procedure,70 a solution of 3a (540 mg, 82.9, 114.3, 122.6, 132.4, 162.3, 167.5, 171.8; ESI-MS obsd 275.08850,
1.31 mmol) in anhydrous THF (13 mL) was treated with NBS calcd 275.08899 [(M + Na)+, M = C13H16O5].
(233 mg, 1.31 mmol) under argon at ꢀ78 1C. The reaction mixture
S-2-Pyridyl 4-[2-(tert-butoxy)-2-oxoethoxy]benzothioate (9).
was stirred for 1 h at ꢀ78 1C, after which the cooling bath was Following a reported procedure,67 a sample of 8 (4.76 g,
removed. Upon reaching 0 1C, hexanes and water were added. The 18.8 mmol) was dissolved in anhydrous CH2Cl2 (188 mL) under
mixture was extracted with ethyl acetate. The organic layer was argon and several drops of DMF were added. The mixture
dried (Na2SO4), concentrated, and chromatographed [silica, was cooled to 0 1C and treated dropwise with oxalyl chloride
hexanes/ethyl acetate (2 : 1)] to afford 9-bromo-1-formyl-5- (2.40 mL, 28.3 mmol). The resulting reaction mixture was
[2,4,6-tris(propargyloxy)phenyl]dipyrromethane (3a-Br9) as an allowed to warm to room temperature for 40 min, and then
1
orange oil (440 mg, 68%): H NMR d 2.56 (t, J = 2.4 Hz, 2H), the solvent was removed under vacuum to afford a colorless oil.
2.57 (t, J = 2.4 Hz, 1H), 4.62 (d, J = 2.4 Hz, 4H), 4.70 (d, J = 2.4 Hz, The resulting oil was dissolved in THF (37.6 mL) and treated
2H), 5.96–5.97 (m, 1H), 6.00 (s, 1H), 6.04–6.05 (m, 2H), 6.42 with 2-mercaptopyridine (2.10 g, 18.8 mmol) under argon. After
(s, 2H), 6.85–6.87 (m, 1H), 8.76 (s, 1H), 9.05 (s, 1H), 9.20 (s, 1H). stirring for 30 min at room temperature, the reaction mixture
Bromodipyrromethane 3a-Br9 in CDCl3 solution darkened and was treated with diethyl ether/aqueous NaHCO3. The organic
decomposed, and hence was used directly in the chlorin- phase was washed (water), dried (Na2SO4), concentrated, and
forming process.
chromatographed [silica, hexanes/ethyl acetate (3 : 1)] to afford
Following a general procedure,62 a solution of 4 (170 mg, a white solid (2.8 g, 45%): mp 105–106 1C; 1H NMR d 1.49
0.896 mmol) and 3a-Br9 (440 mg, 0.896 mmol) in CH2Cl2 (24 mL) (s, 9H), 4.60 (s, 2H), 6.96 (d, J = 9.0 Hz, 2H), 7.31–7.34 (m, 1H),
was treated with a solution of p-TsOHꢁH2O (852 mg, 4.48 mmol) 7.71–7.73 (m, 1H), 7.76–7.80 (m, 1H), 8.00 (d, J = 9.0 Hz, 2H),
in methanol (6 mL) under argon. The reaction mixture immediately 8.66–8.68 (m, 1H); 13C NMR d 28.2, 65.7, 83.0, 114.7, 123.6,
turned red. The mixture was stirred for 30 min under argon, then 129.9, 130.1, 131.1, 137.2, 150.56, 150.64, 162.5, 167.3; ESI-MS
treated with 2,2,6,6-tetramethylpiperidine (1.10 mL, 6.72 mmol) obsd 346.11081, calcd 346.11076 [(M + H)+, M = C18H19NO4S].
and concentrated to dryness. The resulting brown solid was
Streamlined synthesis of 9. A solution of 4-hydroxybenzoic
suspended in acetonitrile (90 mL) followed by the successive acid (6.0 g, 43 mmol) in THF (86 mL) at 0 1C under argon was
addition of 2,2,6,6-tetramethylpiperidine (3.00 mL, 17.9 mmol), treated with 40% aqueous n-Bu4POH (60. mL, 87 mmol) followed
Zn(OAc)2 (2.46 g, 13.4 mmol), and AgOTf (691 mg, 2.69 mmol). by tert-butyl bromoacetate (6.4 mL, 43 mmol). The resulting
The resulting suspension was refluxed for 22 h exposed to air. reaction mixture was allowed to warm to room temperature
The crude mixture was filtered through a silica pad with CH2Cl2. for 1 h. Ethyl acetate (300 mL) and aqueous HCl solution
The filtrate was chromatographed [silica, hexanes/CH2Cl2 (1: 1)] (300 mL, pH = 2 by pH test paper) were added to the reaction
1
to afford a green solid (86 mg, 15%): H NMR (THF-d8) d 2.06 mixture. The organic phase was separated and washed with
(s, 6H), 2.65–2.67 (m, 3H), 4.38 (d, J = 2.8 Hz, 4H), 4.55 (s, 2H), aqueous HCl solution (150 mL ꢃ 3, pH = 2 by pH test paper),
5.03 (d, J = 2.8 Hz, 2H), 6.93 (s, 2H), 8.46 (d, J = 4.4 Hz, 1H), 8.54 dried (Na2SO4), and then concentrated to give a white solid.
(d, J = 4.4 Hz, 1H), 8.60–8.66 (m, 3H), 8.70 (d, J = 4.4 Hz, 1H), 8.74 Recrystallization (hot water) afforded 9 (8.2 g, 30 mmol, 69%),
(d, J = 4.4 Hz, 1H), 9.04 (d, J = 4.4 Hz, 1H), 9.55 (s, 1H); 13C NMR which was dissolved in its entirety in anhydrous CH2Cl2 (380 mL)
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containing 1 mL of DMF under argon. The mixture was cooled to
3,13-Dibromo-10-[4-(methoxycarbonyl)phenyl]-18,18-dimethyl-
0 1C and treated dropwise with oxalyl chloride (4.8 mL, 56 mmol). chlorin (12). Following a reported procedure,18 a solution of 3c
The resulting reaction mixture was allowed to warm to room (1.70 g, 5.52 mmol) in anhydrous THF (55 mL) was treated with
temperature for 40 min, and then the solvent was removed under NBS (1.96 g, 11.0 mmol) under argon at ꢀ78 1C. The reaction
vacuum to afford a colorless oil. The oil was dissolved in THF mixture was stirred for 1 h at ꢀ78 1C, after which the cooling bath
(75 mL) and treated with 2-mercaptopyridine (4.7 g, 45 mmol) under was removed. Upon reaching ꢀ20 1C, hexanes and water were
argon. After stirring for 30 min at room temperature, the reaction added. The mixture was extracted with ethyl acetate. The
mixture was treated with diethyl ether/aqueous NaHCO3. The organic layer was dried (Na2SO4) and concentrated to afford
organic phase was washed (water), dried (Na2SO4), and concentrated. 8,9-dibromo-5-[(4-methoxycarbonyl)phenyl]dipyrromethane
The resulting solid was recrystallized (hot ethanol) to afford a yellow (3c-Br8,9 18
as a yellow oil, which was used in the next step
)
solid (4.7 g, 32% from 4-hydroxybenzoic acid). The characterization without further purification.
data (1H NMR) were consistent with those reported above.
Following a general procedure,62 a solution of 5 (5.5 mmol)
Zn(II)-18,18-dimethyl-5-[4-(2-(tert-butoxy)-2-oxoethoxy)phenyl]- and 3c-Br8,9 (1.48 g, 5.50 mmol) in CH2Cl2 (140 mL) was treated
10-[2,4,6-tris(3,6,9,12-tetraoxatridecyloxy)phenyl]chlorin (10). Follow- with a solution of p-TsOHꢁH2O (5.20 g, 27.5 mmol) in methanol
ing a standard procedure,62 a solution of 3b (482 mg, 449 mmol) in (37 mL) under argon. The reaction mixture immediately turned
THF (12 mL) was treated with NBS (83.0 mg, 446 mmol) at ꢀ78 1C for red. The mixture was stirred for 30 min under argon, then
1 h. A mixture of hexanes (26 mL) and water (26 mL) was added, and treated with 2,2,6,6-tetramethylpiperidine (7.00 mL, 41.3 mmol)
the reaction mixture was allowed to warm to room temperature for and concentrated to dryness. The resulting brown solid was
20 min. The organic phase was separated. The aqueous phase was suspended in acetonitrile (550 mL) followed by the successive
extracted with CH2Cl2 (26 mL). The combined organic extract was addition of 2,2,6,6-tetramethylpiperidine (23.60 mL, 137.5 mmol),
dried (Na2SO4) and concentrated, whereupon THF (7.3 mL) and Zn(OAc)2 (25.00 g, 137.5 mmol), and AgOTf (4.20 g, 16.5 mmol).
anhydrous methanol (1.8 mL) were added. The solution was treated The resulting suspension was refluxed for 22 h exposed to air. The
with NaBH4 (300. mg, 7.93 mmol) at room temperature for 45 min. crude mixture was filtered through a silica pad with CH2Cl2. The
The reaction mixture was quenched by the addition of saturated filtrate was concentrated. The resulting crude product was dis-
aqueous NH4Cl (26 mL), and then extracted with CH2Cl2 (52 mL). solved in anhydrous CH2Cl2 (100 mL). TFA (500 mL) was added
The organic extract was dried (Na2SO4) and concentrated under dropwise to the resulting mixture. After 10 min, saturated aqueous
reduced pressure without heating to B4 mL, whereupon anhydrous NaHCO3 was added slowly. The organic layer was dried (Na2SO4),
CH3CN (10 mL) was added, and again the solution was concentrated concentrated and chromatographed [silica, hexanes/CH2Cl2 (2 : 1)]
by blowing with argon to B4 mL. The resulting solution (which to afford a green solid (332 mg, 10%): 1H NMR d ꢀ2.19 (br s, 1H),
contained the Eastern half 3b-Br9) was treated with anhydrous ꢀ1.79 (br s, 1H), 2.00 (s, 6H), 4.10 (s, 3H), 4.60 (s, 2H), 8.15
CH3CN (9 mL, total volume of B13 mL of CH3CN), Western half (d, J = 8.0 Hz, 2H), 8.40 (d, J = 8.0 Hz, 2H), 8.47 (d, J = 4.4 Hz, 1H),
4 (86.6 mg, 456 mmol) and TFA (34 mL, 447 mmol). The reaction 8.73 (s, 1H), 8.75 (s, 1H), 8.91 (d, J = 4.4 Hz, 1H), 8.93 (s, 1H), 9.11
mixture was stirred at room temperature for 30 min and then (s, 1H), 9.82 (s, 1H); 13C NMR d 31.3, 46.6, 52.1, 52.7, 94.9, 95.7,
diluted with CH3CN (42 mL). Samples of triethylamine (1.8 mL, 106.0, 113.9, 118.8, 120.3, 124.7, 128.4, 128.8, 129.9, 132.3, 132.5,
14 mmol), Zn(OAc)2 (1.2 g, 12 mmol), and AgOTf (0.34 mg, 1.3 mmol) 133.4, 134.0, 134.2, 137.3, 140.2, 145.9, 151.3, 152.5, 159.6, 163.8,
were added. The resulting mixture was refluxed in the presence of air 167.5, 176.1; MALDI-MS obsd 631.0874; ESI-MS obsd 631.03316,
for 24 h. The reaction mixture was quenched by the addition of calcd 631.03388 [(M + H)+, M = C30H24Br2N4O2]; labs (toluene)
water, and extracted with CH2Cl2 (50 mL ꢃ 4). The organic extract 411, 652 nm.
was dried (Na2SO4) and concentrated. The mixture was purified by
3,13-Diacetyl-10-[4-(methoxycarbonyl)phenyl]-18,18-dimethyl-
(vide supra): (1) flash chromatography (silica, CH2Cl2 with 5% chlorin (13). Following a procedure for Stille coupling of
MeOH), (2) preparative SEC, and (3) flash chromatography (silica, chlorins,72 a mixture of 12 (100 mg, 0.158 mmol), tributyl(1-
CH2Cl2 with 2–5% MeOH). The purification procedure afforded a ethoxyvinyl)tin (267 mL, 0.790 mmol), and (Ph3P)2PdCl2 (22.2 mg,
blue solid (62 mg, 11%): 1H NMR d 1.60 (s, 9H), 1.77–1.80 (m, 4H), 0.0316 mmol) was stirred in CH3CN/DMF [8 mL (3 : 2)] under
1.89–2.18 (m, 12H), 2.02 (s, 6H), 2.23–2.41 (m, 8H), 2.46 (s, 6H), 2.99– argon for 4 h at 83 1C in a Schlenk line. The reaction mixture was
3.02 (m, 4H), 3.40 (s, 3H), 3.57–3.60 (m, 2H), 3.68–3.80 (m, 8H), 3.84– treated with 10% aqueous HCl (5 mL) at room temperature for
3.87 (m, 2H), 3.89–3.92 (m, 4H), 4.01–4.04 (m, 2H), 4.35–4.37 20 min. CH2Cl2 was added. The organic layer was separated,
(m, 2H), 4.49 (s, 2H), 4.77 (s, 2H), 6.54 (s, 2H), 7.19 (d, J = 8.8 Hz, washed (saturated aqueous NaHCO3, water, and brine), dried
2H), 7.69 (d, J = 8.8 Hz, 2H), 8.30, 8.33 (AB, J = 4.4 Hz, 2H), 8.47 (Na2SO4), concentrated, and chromatographed [silica, CH2Cl2/
(s, 1H), 8.51 (d, J = 4.4 Hz, 1H), 8.53 (s, 1H), 8.58 (d, J = 4.4 Hz, 1H), CH3OH (99 : 1)]. The resulting solid product was washed [hexanes/
8.610 (d, J = 4.4 Hz, 1H), 8.614 (d, J = 4.4 Hz, 1H); 13C NMR d 28.2, ethyl ether (20 : 1)] five times (to remove impurities derived from
1
31.1, 45.0, 50.5, 58.2, 59.1, 66.0, 67.7, 68.64, 68.71, 68.89, 68.95, 69.14, the tin reagent) to afford a brown solid (80 mg, 90%): H NMR d
69.17, 69.5, 69.9, 70.7, 70.9, 71.9, 82.5, 93.4, 93.9, 95.8, 112.6, 115.1, ꢀ1.78 (br s, 1H), ꢀ1.58 (br s, 1H), 1.99 (s, 6H), 3.04 (s, 3H), 3.12
115.7, 123.2, 126.1, 126.9, 127.5, 128.6, 132.4, 132.6, 134.6, 136.4, (s, 3H), 4.14 (s, 3H), 4.57 (s, 2H), 8.06 (d, J = 8.0 Hz, 2H), 8.38–8.40
145.4, 146.8, 147.3, 148.6, 153.1, 153.2, 157.3, 158.7, 159.9, 160.4, (m, 3H), 8.77 (s, 1H), 8.84 (d, J = 4.0 Hz, 1H), 8.94 (s, 1H), 9.17
168.3, 169.7; ESI-MS obsd 1303.5576, calcd 1303.5614 [(M + H)+, (s, 1H), 10.14 (s, 1H), 10.53 (s, 1H); 13C NMR d 29.8, 29.9, 31.1, 46.4,
M = C67H90N4O18Zn].
52.3, 52.7, 66.1, 96.6, 99.3, 109.0, 122.1, 127.8, 128.3, 130.07, 130.12,
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131.3, 132.3, 133.0, 133.2, 134.0, 134.1, 135.0, 137.4, 138.1, 145.8, 114.3, 122.5, 122.8, 123.0, 123.6, 124.6, 127.5, 128.3, 131.9,
153.6, 154.7, 165.9, 167.4, 176.8, 196.7, 196.9; MALDI-MS obsd 132.4, 132.6, 133.8, 136.6, 139.7, 140.5, 143.5, 150.8, 154.8,
559.2522; ESI-MS obsd 559.23347, calcd 559.23398 [(M + H)+, 159.9, 161.2, 162.5, 174.2; MALDI-MS obsd 1566.0934, calcd
M = C34H30N4O4]; labs (toluene) 433, 686 nm.
1564.7910 [(M + Na)+, M = C76H111N13O21]; ESI-MS obsd
Zn(II)-10-[2,4,6-tris(2,5,8,11,14,17-hexaoxanonadecyl-1H-1,2,3- 771.90605, calcd 771.90815 [(M + 2H)2+, M = C76H111N13O21];
triazol-4-ylmethoxy)phenyl]-18,18-dimethylchlorin (ZnC1). The labs (toluene) 408, 640 nm.
Cu(I) catalyst was prepared by treatment of CuSO4ꢁ5H2O (15.7 mg,
5-[4-(Carboxymethoxy)phenyl]-18,18-dimethyl-10-[2,4,6-tris-
0.0630 mmol) and sodium ascorbate (25.0 mg, 0.126 mmol) with (3,6,9,12-tetraoxatridecyloxy)phenyl]chlorin (FbC2). Following a
600 mL deionized H2O under argon. The reaction mixture turned standard procedure,77 a solution of 10 (15 mg, 12 mmol) in
brown immediately and was stirred until homogeneous. In a CH2Cl2 (4.2 mL) was treated with TFA (1.1 mL). The reaction
separate vial, a solution of chlorin 6 (20.0 mg, 0.0315 mmol) and mixture was stirred at room temperature for 3 h. Water (B6 mL)
PEG-azide 7 (150 mg, 0.468 mmol) in DMSO (1200 mL) was was added. The organic phase was separated, washed with water
treated with freshly prepared Cu(I) catalyst (300 mL) under argon. (6 mL ꢃ 3), dried (Na2SO4) and concentrated. Hexane (HPLC-
The reaction mixture was stirred at 40 1C for 16 h. H2O was grade) was added to the residue, and the suspension was
added, and the organic phase was extracted with CH2Cl2. The sonicated for 3 min followed by centrifugation. The supernatant
extract was washed (brine), dried (Na2SO4), and concentrated. was discarded to afford a brown solid (11 mg, 80%): 1H NMR (the
The resulting mixture was purified as follows: (1) chromato- CO2H proton peaks were not observed) d ꢀ1.91 (br s, 2H), 2.06
graphy [silica, CH2Cl2/CH3OH (93 : 7 - 19: 1)], (2) preparative (s, 6H), 2.14–2.28 (m, 6H), 2.61–2.63 (m, 4H), 2.91–2.93 (m, 6H),
SEC, and (3) chromatography (silica, CH2Cl2 with 8% CH3OH). 3.02–3.08 (m, 2H), 3.18–3.21 (m, 4H), 3.25 (s, 6H), 3.28–3.31
The purification procedure afforded a blue oil (29 mg, 58%): (m, 4H), 3.41 (s, 3H), 3.59–3.61 (m, 2H), 3.69–4.05 (m, 18H),
1H NMR (THF-d8) d 1.98 (s, 6H), 2.78 (s, 6H), 2.92–2.94 (m, 4H), 4.37–4.40 (m, 2H), 4.60 (s, 2H), 4.88 (s, 2H), 6.55 (s, 2H), 7.24
3.04–3.06 (m, 4H), 3.12–3.17 (m, 6H), 3.22–3.25 (m, 4H), 3.27 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 8.35, 8.50 (AB, J =
(s, 3H), 3.30–3.36 (m, 12H), 3.38–3.41 (m, 4H), 3.44–3.45 (m, 2H), 4.2 Hz, 2H), 8.72–8.75 (m, 3H), 8.80 (d, J = 4.8 Hz, 1H), 8.83
3.53–3.68 (m, 28H), 3.80 (t, J = 5.2 Hz, 4H), 3.97 (t, J = 5.2 Hz, 2H), (s, 1H), 8.90 (s, 1H); 13C NMR d 31.2, 46.3, 51.8, 58.70, 58.79, 59.0,
4.52 (s, 2H), 4.62 (t, J = 5.2 Hz, 2H), 4.96 (s, 4H), 5.44 (s, 2H), 6.47 65.2, 65.8, 67.7, 68.5, 69.1, 69.3, 69.41, 69.53, 69.62, 69.77, 69.83,
(s, 2H), 6.97 (s, 2H), 8.24 (s, 1H), 8.42 (d, J = 4.4 Hz, 1H), 8.52 70.53, 70.65, 70.70, 70.9, 71.5, 71.9, 92.72, 92.80, 94.6, 96.1,
(d, J = 4.0 Hz, 1H), 8.58–8.60 (m, 3H), 8.69 (d, J = 4.0 Hz, 1H), 8.76 112.80, 112.93, 113.2, 114.1, 121.2, 122.6, 123.2, 128.0, 128.4,
(d, J = 4.4 Hz, 1H), 9.01 (d, J = 4.0 Hz, 1H), 9.54 (s, 1H); 13C NMR 131.2, 131.7, 134.4, 134.9, 135.6, 135.92, 140.04, 140.26, 151.4,
(THF-d8) d 32.4, 47.2, 51.2, 51.9, 52.4, 60.0, 64.2, 65.0, 70.8, 71.6, 153.5, 157.4, 159.9, 160.8, 163.2, 170.8, 174.2; MALDI-MS obsd
71.8, 72.12, 72.13, 72.18, 72.22, 72.31, 72.35, 72.40, 72.44, 72.5, 1186.7898; ESI-MS obsd 1185.5815, calcd 1185.5853 [(M + H)+,
72.6, 72.7, 73.9, 74.0, 95.1, 96.0, 97.6, 110.5, 116.8, 117.4, 125.0, M = C63H84N4O18].
126.4, 128.3, 129.6, 130.0, 133.7, 134.4, 145.4, 147.5, 147.8, 149.2,
Zn(II)-5-[4-(carboxymethoxy)phenyl]-18,18-dimethyl-10-[2,4,6-
150.4, 154.8, 155.4, 160.0, 161.8, 162.6, 171.5; MALDI-MS obsd tris(3,6,9,12-tetraoxatridecyloxy)phenyl]chlorin (ZnC2). A solution
1604.8876, calcd 1604.7225 [(M + H)+, M = C76H109N13O21Zn]; of FbC2 (11 mg, 9.3 mmol) in CH2Cl2 (0.5 mL) was treated with
ESI-MS obsd 1625.70380, calcd 1625.69664 [(M + Na)+, M = a solution of Zn(OAc)2ꢁ2H2O (30.0 mg, 136 mmol) in methanol
C76H109N13O21Zn]; labs (toluene) 409, 609 nm.
(0.5 mL). The reaction mixture was stirred at room temperature
10-[2,4,6-Tris(2,5,8,11,14,17-hexaoxanonadecyl-1H-1,2,3-triazol- for 16 h, and then treated with water (B4 mL). The organic
4-ylmethoxy)phenyl]-18,18-dimethylchlorin (FbC1). A solution phase was separated, washed with water (4 mL ꢃ 3), dried
of ZnC1 (8.0 mg, 0.0050 mmol) in CH2Cl2 (16 mL) was treated (Na2SO4) and concentrated. HPLC-grade hexanes was added to
dropwise with TFA (40 mL). After 5 min, saturated aqueous the residue, and the suspension was sonicated for 3 min
NaHCO3 was added slowly. The organic layer was separated, dried followed by centrifugation. The supernatant was discarded to
(NaSO4), and concentrated. The crude product was chromato- afford a blue solid (11 mg, 95%): 1H NMR (CDCl3, 300 MHz, the
graphed [silica, CH2Cl2/CH3OH (19 : 1)] to afford a green solid CO2H proton peak was not observed) d 2.02 (s, 6H), 2.08–2.69
(7.0 mg, 89%): 1H NMR (THF-d8) d ꢀ2.22 (s, 1H), ꢀ1.94 (s, 1H), (m, 24H), 2.71 (s, 6H), 2.93–3.05 (m, 4H), 3.40 (s, 3H), 3.57–3.60
2.06 (s, 6H), 2.55 (s, 6H), 2.92–2.94 (m, 4H), 3.04–3.06 (m, 4H), (m, 4H), 3.68–3.94 (m, 10H), 4.01–4.04 (m, 4H), 4.35–4.38
3.12–3.17 (m, 6H), 3.22–3.25 (m, 4H), 3.27 (s, 3H), 3.30–3.36 (m, 2H), 4.48 (s, 2H), 4.77 (br s, 2H), 6.53 (s, 2H), 7.17 (d, J =
(m, 12H), 3.38–3.41 (m, 4H), 3.44–3.45 (m, 2H), 3.53–3.68 8.3 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 8.26, 8.34 (AB, J = 4.3 Hz,
(m, 28H), 3.80 (t, J = 5.2 Hz, 4H), 3.97 (t, J = 5.2 Hz, 2H), 4.52 2H), 8.47 (s, 1H), 8.50 (d, J = 4.4 Hz, 1H), 8.52 (s, 1H), 8.57–8.61
(s, 2H), 4.63 (t, J = 5.2 Hz, 2H), 5.02 (s, 4H), 5.47 (s, 2H), 6.26 (m, 3H); 13C NMR d 31.0, 45.0, 58.2, 59.0, 68.6, 68.8, 69.0, 69.2,
(s, 2H), 7.10 (s, 2H), 8.27 (s, 1H), 8.54 (d, J = 4.4 Hz, 1H), 8.75 69.8, 70.50, 70.62, 71.9, 114.9, 125.3, 128.2, 129.0, 137.8, 159.8,
(d, J = 4.4 Hz, 1H), 8.82 (d, J = 4.4 Hz, 1H), 8.86 (d, J = 4.4 Hz, 169.8; MALDI-MS obsd 1247.6318; ESI-MS obsd 1247.4956,
1H), 8.98–9.04 (m, 3H), 9.28 (d, J = 4.4 Hz, 1H), 9.87 (s, 1H); calcd 1247.4988 [(M + H)+, M = C63H82N4O18Zn].
13C NMR (THF-d8) d 30.5, 30.6, 46.2, 49.3, 50.1, 52.0, 58.1, 58.2,
18,18-Dimethyl-5-[4-(succinimidooxycarbonylmethoxy)phenyl]-
62.4, 63.2, 68.85, 68.95, 69.6, 69.7, 69.8, 69.94, 69.95, 70.01, 10-[2,4,6-tris(3,6,9,12-tetraoxatridecyloxy)phenyl]chlorin (FbC2-NHS).
70.06, 70.09, 70.23, 70.38, 70.40, 70.44, 70.51, 70.56, 70.58, Following a standard procedure,52 a mixture of FbC2 (4.0 mg,
70.74, 70.76, 70.80, 72.0, 72.1, 94.0, 94.2, 96.3, 107.0, 113.1, 3.4 mmol), N-hydroxysuccinimide (0.47 mg, 4.1 mmol) and
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EDC (0.78 mg, 4.1 mmol) in CH2Cl2 (5.0 mL) was stirred in the 137.1, 138.4, 138.6, 146.4, 153.3, 154.2, 161.1, 161.2, 162.38, 162.48,
dark at room temperature. After 6 h, the reaction mixture was 165.4, 168.8, 176.5, 189.6, 190.2; MALDI-MS obsd 1980.6716, calcd
diluted with CH2Cl2 (3 mL) and washed with water. 1979.9554 [(M + Na)+, M = C101H144N4O34]; ESI-MS obsd 979.49094,
The organic layer was separated, dried (Na2SO4) and concen- calcd 979.49094 [(M + 2H)2+, M = C101H144N4O34]; labs (toluene) 442,
trated to a red-brown solid (4.6 mg, B80% purity estimated 686 nm.
by 1H NMR spectroscopy and MALDI-MS): MALDI-MS obsd
1283.73, calcd 1282.60 [(M + H)+, M = C67H87N5O20]; labs tris(3,6,9,12-tetraoxatridecyloxy)phenyl]prop-2-en-1-onyl}chlorin
(CH2Cl2) 414, 508, 641 nm. (ZnC3). A solution of FbC3 (15 mg, 7.7 mmol) in CHCl3 (0.5 mL)
Zn(II)-5-(4-carboxyphenyl)-18,18-dimethyl-3,13-bis{(E)-3-[2,4,6-
18,18-Dimethyl-5-(4-((42-(succinimidooxy)-2,42-dioxo-6,9,12,15,- was treated with a solution of Zn(OAc)2 (27.0 mg, 150 mmol) in
18,21,24,27,30,33,36,39-dodecaoxa-3-azadotetracontyl)oxy)phenyl)- methanol (0.5 mL). The reaction mixture was stirred at room
10-(2,4,6-tris(3,6,9,12-tetraoxatridecyloxy)phenyl)chlorin (FbC2-R- temperature for 16 h, and then treated with water (B4 mL). The
NHS). A mixture of FbC2-NHS (5.5 mg, 80% purity, 3.4 mmol), organic phase was separated, washed with water (4 mL ꢃ 3),
PEG-amine 11 (2.5 mg, 4.0 mmol) in dry CH2Cl2 (1.7 mL) was dried (Na2SO4) and concentrated. HPLC-grade hexanes was
stirred in the dark at room temperature. After 6 h, the reaction added to the residue, and the suspension was sonicated
mixture was diluted with CH2Cl2 (3 mL) and washed with water. followed by centrifugation. The supernatant was discarded
1
The organic layer was separated, dried (Na2SO4) and concentrated. to afford a green solid (13 mg, 84%): H NMR (THF-d8) d 2.10
The resulting material was treated with N-hydroxysuccinimide (s, 6H), 3.24 (s, 6H), 3.31 (s, 6H), 3.36–3.37 (m, 4H), 3.39
(0.29 mg, 2.5 mmol) and EDC (0.48 mg, 2.5 mmol) in CH2Cl2 (s, 3H), 3.40 (s, 3H), 3.44–3.50 (m, 8H), 3.51–3.52 (m, 16H),
(2.1 mL) in the dark at room temperature. After 20 h, the reaction 3.55–3.58 (m, 8H), 3.61–3.63 (m, 4H), 3.65–3.76 (m, 28H),
mixture was diluted with CH2Cl2 (3 mL) and washed with aqueous 3.79–3.82 (m, 4H), 3.86–3.90 (m, 2H), 3.90–3.95 (m, 6H),
HCl solution (pH = 5). The organic layer was separated, dried 4.04–4.09 (m, 4H), 4.15–4.20 (m, 2H), 4.20–4.22 (m, 2H),
(Na2SO4) and concentrated. The resulting material was treated 4.25–4.29 (m, 4H), 4.30–4.34 (m, 4H), 4.60 (s, 2H), 6.35
with hexanes/ethyl acetate (1 : 2), sonicated, and centrifuged. (s, 2H), 6.41 (s, 2H), 8.24 (d, J = 8.0 Hz, 2H), 8.41–8.45
The supernatant was discarded, leaving a red-brown solid (m, 3H), 8.47–8.48 (m, 3H), 8.61 (d, J = 15.6 Hz, 1H), 8.70
(3.8 mg, B80% purity estimated by 1H NMR spectroscopy (s, 1H), 8.80 (d, J = 15.6 Hz, 1H), 8.91 (d, J = 4.4 Hz, 1H), 9.18
and MALDI-MS): MALDI-MS obsd 1882.75, calcd 1881.95 (s, 1H), 9.47 (s, 1H), 10.06 (s, 1H), the –CO2H proton was not
[(M + H)+, M = C94H140N6O33]; labs (CH2Cl2) 414, 508, 641 nm. detected; ESI-MS obsd 1032.42903, calcd 1032.42907 [(M + 2Na)2+,
5-(4-Carboxyphenyl)-18,18-dimethyl-3,13-bis{(E)-3-[2,4,6-tris- M = C101H142N4O34Zn]; labs (toluene) 447, 666 nm.
(3,6,9,12-tetraoxatridecyloxy)phenyl]prop-2-en-1-onyl}chlorin (FbC3).
Photophysical measurements
A mixture of 13 (50.0 mg, 0.0895 mmol), benzaldehyde 1b (649 mg,
0.895 mmol), and NaOH (143 mg, 3.58 mmol) in absolute ethanol All studies were performed at room temperature for compounds
(45 mL) was refluxed in the open air upon microwave irradiation at in DMF, or in HPLC-grade water (Sigma Aldrich) with 5% DMF.
60 W. The microwave irradiation protocol was as follows: (1) heat The small amount of DMF was employed to pre-solubilize any
from room temperature to 100 1C (irradiate for 2 min), (2) hold at solid compound with limited solubility in pure water, and this
100 1C (irradiate for 18 min), (3) allow to cool to room temperature. method was used in all cases for consistency. To simplify the
The reaction mixture was concentrated. The resulting crude product presentation, the solvent consisting of water plus 5% DMF is
was dissolved in CH2Cl2 and washed with saturated aqueous NH4Cl. referred to as ‘‘water’’. Dilute (mM) argon-purged samples were
The organic layer was separated, dried (Na2SO4), and concentrated. used for static emission spectral, fluorescence quantum yield
The mixture was purified as follows: (1) flash chromatography (Ff) and singlet excited-state lifetime (tS) studies. Static emission
(silica, CH2Cl2 with 5% MeOH), (2) preparative SEC (toluene), and spectra were acquired using 2–4 nm excitation and detection
(3) flash chromatography (silica, CH2Cl2 with 10% MeOH). The bandwidths and corrected for instrument spectral response. The
purification procedure afforded a blue solid (98 mg, 56%): 1H NMR Ff values were determined for samples having A r 0.1 at lexc
d ꢀ1.52 (br s, 1H), ꢀ1.36 (br s, 1H), 2.07 (s, 6H), 3.24 (s, 6H), 3.31 (s, (typically in the Soret region) using replicate measurements with
6H), 3.36–3.37 (m, 4H), 3.39 (s, 3H), 3.40 (s, 3H), 3.44–3.50 (m, 8H), an integrating sphere (Horiba, Quanti-Phi).
3.51–3.52 (m, 16H), 3.55–3.58 (m, 8H), 3.61–3.63 (m, 4H), 3.65–3.76
Singlet excited-state lifetimes (tS) are the average result of
(m, 28H), 3.79–3.82 (m, 4H), 3.86–3.90 (m, 2H), 3.90–3.95 (m, 6H), two measurements. The first method used a stroboscopic
4.04–4.09 (m, 4H), 4.15–4.20 (m, 2H), 4.20–4.22 (m, 2H), 4.25–4.29 fluorescence decay apparatus with an B1 ns Gaussian instru-
(m, 4H), 4.30–4.34 (m, 4H), 4.67 (s, 2H), 6.26 (s, 2H), 6.27 (s, 2H), ment response function (Laser Strobe TM-3; Photon Technol-
8.29 (d, J = 8.0 Hz, 2H), 8.46 (d, J = 16.0 Hz, 1H), 8.52–8.54 (m, 3H), ogy International) and samples excited in the blue to green
8.62 (d, J = 16.0 Hz, 1H), 8.67 (d, J = 15.6 Hz, 1H), 8.72 (d, J = 15.6 Hz, spectral regions by a dye laser pumped by a nitrogen laser. The
1H), 8.93 (s, 1H), 8.00 (d, J = 4.4 Hz, 1H), 9.22 (s, 1H), 9.49 (s, 1H), second method utilized transient absorption spectroscopy
10.34 (s, 1H), 10.81 (s, 1H), the –CO2H proton was not detected; employing B100 fs excitation flashes (typically in the Qx region)
13C NMR d 31.2, 46.4, 52.4, 59.0, 59.1, 67.7, 68.2, 68.3, 69.4, 69.6, from an ultrafast laser system (Spectra Physics) and acquisition of
69.7, 70.3, 70.40, 70.44, 70.52, 70.56, 70.61, 70.67, 70.70, 70.9, 71.8, difference spectra (360–900 nm) using a white-light pulsed laser
72.0, 92.6, 92.8, 96.2, 99.2, 107.4, 109.1, 121.7, 124.6, 125.5, 126.5, (B1 ns rise time) in 100 ps time bins with variable pump–probe
128.6, 129.9, 132.4, 132.5, 132.9, 133.9, 134.2, 134.7, 135.5, 136.2, spacing up to 0.5 ms (Ultrafast Systems, EOS). The latter
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apparatus was also used to determine the yield of S1 - T1 to buffer and adjusted to a final volume of 250 mL. Blanks were
intersystem crossing (Fisc) by comparing the extent of bleaching treated by the following sequence of washes and centrifugation
of the ground-state Bx, Qx or Qy absorption bands (relative to the but without antibody addition, then resuspended in 1.0 mL of
featureless excited-state absorption) for the T1 state at long times buffer. For labeled antibodies, 1.0 mL aliquots of bead solution
compared to that due to S1 right after the flash. The contribution were prepared from 4 drops of beads, then 50 mL was aliquoted
of stimulated emission (to S1 spectra) was taken into account for per sample. For the experiment shown in Fig. 4, 0.5 mg of FbC2–
studies in the Qy region.
Ab, and 0.12 mg of the positive control Brilliant Violet 650-labeled
antibody (BV650–Ab) were added to each aliquot, and each
sample was mixed gently for 30 min at ambient temperature
Flow cytometry measurements
Instrumentation and software. Samples were analyzed at the in the dark. Solutions were washed twice by addition of buffer
University of North Carolina Core Flow Cytometry Facility on a (1 mL) to each tube, centrifugation at 3000 ꢃ g for 3 min, and
19-parameter LSR-II SORP flow cytometer (BD Biosciences, San Jose, removal of supernatant. Samples were resuspended in 1.0 mL of
CA) equipped with seven lasers (355, 405, 488, 532, 561, 594, and buffer for analysis.
633 nm) using FACSDiva 8.0 acquisition software. The longpass filter
in the ‘‘A’’ detector channel of the 355 nm laser was replaced
Experiment and data analysis
with a 600 nm longpass filter provided with the instrument and
the standard bandpass filter in this channel was replaced with a
620/60 nm bandpass filter (ET620/60, Chroma Technology Corp.,
Bellows Falls, VT). Data were collected using the 100 mW 405 nm
laser. Post-experimental analysis was performed with FlowJo
software (version 10.0.8, FlowJo, LLC, Ashland, OR).
The FbC2–Ab and the positive control BV650–Ab were excited
with the 405 nm laser and read in channel A with a 600 nm
longpass filter and a 620/60 nm bandpass filter in place.
Compensation beads were identified on the basis of forward
and side light scatter. Gating was applied to exclude events with
both lower and higher scatter than single beads, and all further
data were analyzed using this gating. For all experiments, 5000
gated events were collected.
Materials
Simply Cellulart anti-Mouse for Violet Laser compensation standard
beads (Bangs Laboratories, Fishers, IN) were used for all flow
cytometry experiments. Antibodies used included BD Horizon
Brilliant Violet 650-labeled CD8 clone RPA-T8 (BV650–Ab, positive
control) and Protein A-purified mouse IgG (MU-003-C, Immuno-
Reagents, Raleigh, NC).
Conflict of interest statement
D. F. B., D. H. and J. S. L. are cofounders of NIRvana Sciences,
which develops chlorins and bacteriochlorins for use in clinical
diagnostics.
Bioconjugation to form FbC2–Ab
Prior to bioconjugation, dialysis was used to exchange anti-
bodies into 50 mM borate buffer (pH 8.5). The chlorin FbC2-R-
NHS (0.1 mg) was dissolved in 20 mL of the borate buffer and
added to 0.25 mg of mouse IgG solution to achieve a 35-fold
ratio of fluorophore to protein in a final reaction volume of 86 mL.
This reaction solution was gently rotated in a microcentrifuge
tube protected from light for 3 h at ambient temperature and
then dialyzed against phosphate buffered saline (PBS) with a
20 kD MWCO dialysis membrane for at least 4 h at ambient
temperature to remove unreacted materials. The chlorin–
antibody bioconjugate was further purified by affinity purifica-
tion using Pierce Protein A Agarose beads (Thermo Scientific,
Rockford, IL) and the manufacturer’s protocol for immuno-
precipitation. The resulting chlorin–antibody bioconjugate,
FbC2–Ab, exhibited a fluorophore/protein ratio of 2.2 as deter-
mined by absorption spectroscopy using a molar absorption coeffi-
Acknowledgements
Synthesis and photophysical characterization were supported by
the Photosynthetic Antenna Research Center (PARC), an Energy
Frontier Research Center funded by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Award No.
DE-SC0001035. Flow cytometry studies were supported by a grant to
NIRvana Sciences (1R41EB020470) from the National Institutes of
Health Small Business Technology Transfer Program. Cytometry
was performed at the UNC Flow Cytometry Core Facility, which is
supported in part by P30 CA016086 Cancer Center Core Support
Grant to the UNC Lineberger Comprehensive Cancer Center. 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 Carolina Biotechnology
Center and the National Science Foundation.
cient for the chlorin (e414nm = 160 000 Mꢀ1 cmꢀ1; e280nm
=
=
22 000 Mꢀ1 cmꢀ1
)
and for the antibody81 (e280nm
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