Soluble precipitable porphyrins for use in targeted molecular brachytherapy

Article abstract of DOI:10.1039/b714127k

In a new therapy that aims to concentrate and immobilize therapeutic radionuclides in nanoscale assemblies within solid tumors, a soluble precipitable reagent (SPR) is administered as the radionuclide carrier and is converted to non-diffusible precipitate

Full text of DOI:10.1039/b714127k

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Soluble precipitable porphyrins for use in targeted molecular
brachytherapyw
Zhen Yao, K. Eszter Borbas and Jonathan S. Lindsey*
Received (in Gainesville, FL, USA) 13th September 2007, Accepted 23rd October 2007
First published as an Advance Article on the web 2nd November 2007
DOI: 10.1039/b714127k
In a new therapy that aims to concentrate and immobilize therapeutic radionuclides in nanoscale
assemblies within solid tumors, a soluble precipitable reagent (SPR) is administered as the
radionuclide carrier and is converted to non-diffusible precipitate by an enzyme located in tumor
tissues. To meet the objective of such an SPR, we have prepared and examined a class of
porphyrin–alkyldiphosphates that are soluble in aqueous solution and that are rendered insoluble
upon removal of the two phosphate groups. The porphyrins examined herein are of the trans-AB
architecture wherein the substituents are a bis(dihydroxyphosphoryloxy)alkyl group and a phenyl
(or p-bromophenyl) group. Provisions for later incorporation of radionuclides have been
established by preparation of the analogous copper chelate or the meso-iodo free-base porphyrin.
Altogether, four porphyrins bearing a bis(dihydroxyphosphoryloxy)alkyl group were examined
and found to exhibit satisfactory solubility in water (41 mM). Dephosphorylation reactions have
been carried out in vitro using the enzyme shrimp alkaline phosphatase. In each case, enzyme-
induced precipitation was observed. The soluble-to-insoluble conversion has been examined by
visual inspection, absorption spectroscopy, electrospray ionization mass spectrometry, and
nephelometry using non-radiolabeled porphyrins.
obstacles to further progress of targeted cancer therapies: (1)
Introduction
the current therapeutic agents only target a subset of cancer
cells in the intrinsically heterogeneous tumor, whereupon the
remaining cell types give rise to resistant colonies;3 (2) the
agents also kill normal cells because of non-exclusive selectiv-
ity toward cancer cells; and (3) the agents are not potent
enough to kill resistant cancer cells or to overcome the ability
of cancer cells to adapt to the agents and become resistant.4
Note also that a combination of multiple chemotherapeutic
drugs, each having distinct chemical structure and each block-
ing a different biochemical pathway, has not significantly
prolonged the survival of cancer patients.13
Fundamentally new approaches are urgently needed for ther-
apeutic treatment of cancer, which has surpassed heart disease
as the leading cause of death in the United States for persons
younger than 85.1 Of all types of cancers, solid tumors are now
estimated to account for more than 80% of all incidents of
cancer diagnosis and death.2 Surgery and radiation therapy
may be successful for treating localized solid tumors, but not
for metastases. Chemotherapy of metastasized tumors is lim-
ited due to the heterogeneity of tumors, the plasticity of cancer
cells, and the imperfect differentiation between healthy and
tumor tissue.3,4 Although systemic agents are essential for the
treatment of metastatic cancer, the occurrence of adverse side
effects typically limits the maximum dose of the anti-cancer
agent that can be administered.
A decade ago, a fundamentally new strategy to treat cancer
was proposed by Rose.14–16 The proposed approach mimics
the treatment of thyroid cancer with radioiodide. Compared to
other types of cancer, thyroid cancer often can be successfully
treated.17 The thyroid naturally concentrates iodide from body
fluids; therefore, the administration of radioiodide results in
localization of most of the radiation damage inside the thyroid
with little systemic damage. Similarly, the goal of Rose’s
method is to concentrate radionuclides inside tumors (both
primary and metastatic tumors) by systemic administration of
a set of therapeutic agents, including both non-radiolabeled
agents and radiolabeled agents.
Diverse targeted cancer therapies (e.g., antibodies, macro-
molecules, nanoparticles, viral vectors)5–12 have been exam-
ined in an effort to increase the therapeutic index (i.e.,
selectively deliver the therapeutic agents to the tumor location
and thereby diminish the systemic cytotoxicity). Despite tre-
mendous effort, however, the various targeted methods devel-
oped to date have generally not been successful for treating
disseminated cancers. There appear to be three universal
To selectively deliver the radiolabeled materials to tumors
and maintain the radiation within a well-defined area, Rose’s
method employs a multi-step regimen that employs the follow-
ing four reagents in successive steps:
Department of Chemistry, North Carolina State University, Raleigh,
NC 27695-8204, USA. E-mail: jlindsey@ncsu.edu; Fax: 1 919 513
2830; Tel: 1 919 515 6406
w Electronic supplementary information (ESI) available: ESI-MS data
for enzymatically treated samples of water-soluble porphyrins. See
DOI: 10.1039/b714127k
(1) A soluble reagent bearing a cancer-targeting agent and
an enzyme-binding moiety is administered (Step-1 reagent).
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Such a reagent is endocytosed preferentially but not exclu-
sively in cancer cells owing to the presence of the cancer-
targeting agent. Upon endocytosis, a chemical conversion
occurs that results in the formation of a precipitate. The
precipitate, termed ‘platform’, constitutes a nanoscale archi-
tecture that is non-diffusible and that contains the enzyme-
binding moiety in an accessible state (vide infra). Due to the
capacity for soluble-to-insoluble conversion, this reagent
is a type of soluble precipitable reagent (SPR), in particular
a Step-1 SPR.
ing groups, such as phosphates,21 sulfates,22 or sugars23 (e.g.,
glucosides or galactosides). One SPR presently under devel-
opment24 (at Oncologic, Inc., a company founded by Rose) is
designed around derivatives of an indoxyl glycoside. Such a
chemical structure was chosen because mammalian lysosomes
contain enzymes that will cleave the sugar to form the
corresponding indoxyl. The indoxyl undergoes oxidative
(aerobic) dimerization to give insoluble indigo (Scheme 1).
With appropriate prior chemical modification, the indigo
precipitate provides the platform for subsequent localization
of radioactive material. In vitro studies showed the accumula-
tion of indigo (blue) inside the tumor cells, and the presence of
a binding handle (e.g., a Loracarbef moiety) on the precipitate
was demonstrated by an immunoassay.19 These preliminary
results prove the viability of the strategy on which the STAR
method is established.
(2) A low dose of a cytotoxic drug (e.g., doxorubicin) is
administered (Step-2 reagent), which kills a small fraction of
the super-sensitive cancer cells. The subsequent cell lysis
releases some of the platform to the extracellular medium.
The normal cells are unaffected by the low dose of the
cytotoxic drug, hence the platform is retained inside such cells.
(3) An enzyme conjugate, which is not naturally present or
accessible in the extracellular area, is administered (Step-3
reagent). The enzyme attaches covalently to the platform
(released into the extracellular medium in step 2) via the
enzyme-binding moiety, thereby localizing and immobilizing
the enzyme in the tumor. The Step-3 reagent is cell-imperme-
able to preclude binding to the platform located inside cells in
normal tissues (as well as un-lysed cancer cells). It is known
that enzymes in aggregated or precipitated forms can maintain
a high level of activity.18
Although the full STAR method has not yet been demon-
strated, in recent years, other approaches have been developed
that embody selected steps of the STAR method. Pretargeted
radioimmunotherapy employs an antibody bearing avidin to
target the cancer, followed by administration of a radiolabeled
biotin derivative.6,7,9,10 This approach suffers from lack of
immobilization of the antibody in the tumor, and rapid
clearance of the antibodies in the kidney.25 Enzyme-mediated
cancer therapy employs a water-soluble radiolabeled prodrug
that is hydrolyzed enzymatically in vivo to give a water-
insoluble compound; the group to be cleaved is the substrate
of an enzyme that is overexpressed extracellularly by tumor
cells.26 Thus, the water-soluble radiolabeled prodrug is in
essence a Step-4 SPR. The one example that has been demon-
strated to date is a radiolabeled iodo-quinazolinone that bears
a phosphoryloxy group; dephosphorylation results in forma-
tion of a radiolabeled precipitate.27 The design of a Step-4
SPR also has some commonality with certain reagents for
enzyme histochemical staining, wherein a soluble chromogen
(e.g., diaminobenzidine, or even indoxyl phosphate) is
converted to an insoluble form.28
(4) A radiolabeled SPR, which precipitates upon action of
the enzyme attached to the platform in step 3, is administered
(Step-4 reagent). The radiolabeled (and immobile) precipitate
generated by the Step-4 SPR forms a radiation field inside the
tumors, destroying potentially every cancer cell within the
effective radius of the radiation.
One term for this four-step procedure is ‘‘selectively targeted
amplified radiotherapy’’ (STAR),19 which denotes the follow-
ing features: (1) a combination of two ‘‘imperfect’’ targeting
steps that causes localization of the therapeutic agent near the
most sensitive cancer cells; (2) generation of a strong radiation
field to destroy every cell in a given micro-region; and (3)
amplified localization and immobilization of radiotherapy
sources in the tumor. Although the concept of the STAR
method was inspired by the treatment for thyroid cancer, the
overall therapeutic effect has great similarity to that of
brachytherapy,20 wherein radioactive seeds, typically small
capsules enclosing radioactive materials, are surgically im-
planted in or near tumor tissues. The radiolabeled precipitate
formed in the STAR method is comparable in principle to the
radioactive seed in traditional brachytherapy, but has the
advantages of (1) nanoscale assembly in situ, (2) targeting of
both primary and metastatic tumor tissues; (3) separation of
the targeting and therapeutic steps, and (4) administration
without surgery. Therefore, Rose’s method constitutes a ‘‘tar-
geted molecular brachytherapy’’, given the ability to selec-
tively deposit radiation sources inside the tumor and cause
micro-regional destruction of surrounding cancer cells.
The cornerstone of the STAR method is a novel class of
soluble chemical compounds particularly the Step-1 SPR, the
Step-4 SPR, and the enzyme conjugate (Step-3 reagent). The
core of a SPR is an inherently insoluble entity that is rendered
soluble by the attachment of enzymatically cleavable solubiliz-
In this paper, we describe our work concerning the devel-
opment of porphyrin-based SPRs. There are multiple reasons
for choosing porphyrins as the backbone of an SPR: (1) the
extensive
knowledge-base
concerning
water-soluble
Scheme 1
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porphyrins29 and aggregation of porphyrinic compounds;30 (2)
rational synthetic methods for preparing porphyrins enable
introduction of up to four functional groups in distinct
patterns;31–35 (3) the robustness of the synthetic porphyrin
macrocycle in biological systems; (4) low molecular weight
(o1000 m); and (5) the ability to use porphyrins as carriers for
diverse radionuclides encompassing radioiodine and radio-
active metals.
Our initial focus concerns the design and synthesis of a
water-soluble porphyrin that serves as a Step-4 SPR. Solu-
bilization is achieved by the attachment of a symmetrically
branched alkyl chain equipped with phosphate groups at the
termini (‘‘swallowtail’’ motif).36 The essential soluble-to-insol-
uble conversion has been investigated via various spectro-
scopic methods. Synthetic routes to a porphyrin-based Step-
4 SPR bearing iodo groups or a copper chelate were investi-
gated, thereby providing the foundation for later preparation
of radiolabeled analogues (e.g., containing 131I or 64Cu).
Taken together, this work provides the foundation for the
development of a new class of molecules for use in targeted
molecular brachytherapy.
Chart 1
terminus of each alkyl group. The projection of polar groups
over both faces of the porphyrin macrocycle was expected to
render the molecules highly water-soluble.
Diphosphate-derivatized swallowtail porphyrins in a trans-
AB design were prepared in a rational synthesis.38 A trans-
AB-porphyrin bears substituents only at the 5- and
15-positions, resulting in a compact design that is well suited
for biological applications. Indeed, the free-base porphyrin
P1-(PO4)2 (5-(4-bromophenyl)-15-[1,5-bis(dihydroxyphos-
phoryloxy)pent-3-yl]porphyrin), which bears one swallowtail
substituent, one derivatizable group (4-bromophenyl), and
no substituents at the flanking meso positions, displays
excellent solubility in water (Chart 1).
Results and discussion
1
Molecular design
The structural motifs for a Step-4 reagent include (1) the
ability to undergo conversion from soluble-to-insoluble forms,
(2) ability to carry radionuclides, and (3) formation of a non-
diffusible precipitate.
Low aqueous solubility is a common problem with porphy-
rins because such planar aromatic compounds tend to aggre-
gate upon p–p interactions among the aromatic rings.29 Low
solubility in aqueous media is an excellent property for the
product of the soluble-to-insoluble conversion of an SPR, but
first the porphyrin must be rendered soluble in aqueous media.
Substituted phenyl groups such as 3,5-di-tert-butylphenyl and
mesityl groups impart higher organic solubility to the por-
phyrin than the phenyl group.36 A likely explanation is that
the alkyl moieties on the meso-phenyl groups shield the space
above and below the macrocycle, and thus suppress p–p
interactions. Another type of meso-substituent that solubilizes
porphyrins by the same shielding strategy is a symmetrically
branched alkyl group (i.e., swallowtail, Fig. 1).36,37 Taking
advantage of the facial encumbrance of the swallowtail group,
the swallowtail design was modified to accommodate polar
moieties such as phosphate or phosphonate groups at the
During the development of the water-solubilizing swallow-
tail motifs, it was observed that the presence of a central zinc
atom in the porphyrin–alkyldiphosphates yielded a precipitate
in protic solvents within a short time. The precipitate was
found to be the poorly aqueous-soluble dihydroxyporphyrin
ZnP1-(OH)2. The facile ZnP1-(PO4)2 - ZnP1-(OH)2 conver-
sion was ascribed to coordination of one of the phosphate
oxygens to the zinc, followed by nucleophilic attack by a
solvent molecule (Scheme 2). We hypothesized that a similar
soluble-to-insoluble transformation could be achieved upon
treatment of the free-base porphyrin with a phosphatase
enzyme. Similarly, a metalloporphyrin wherein the metal does
not support apical binding (e.g., copper) could be used.
A further advantage of using a trans-AB porphyrin as the
SPR is the availability of two meso positions for radioiodina-
tion, and the possibility of metalation with a radioactive metal
to give the metalloporphyrin. In this regard, a number of
radiolabeled porphyrins have been employed both in diagnosis
and in therapy.39 Representative radiometals for inclusion as
the centrally coordinated metal in a metalloporphyrin include
the following: 51Mn (ref. 40), 54Mn (ref. 41), 57Co (ref. 42),
58Co (ref. 43), 64Cu (refs. 44 and 45), 65Zn (ref. 43), 67Cu (refs.
44 and 46), 67Ga (ref. 47), 90Y (refs. 48 and 49), 99mTc (ref 50),
109Pd (refs. 45, 51 and 52), 111In (refs. 47 and 53), 166Ho (ref.
48), and 169Yb (ref. 49). The radiolabel 123I (ref. 54) or 18F (ref.
55) has been substituted on the aryl ring of a meso-tetra-
arylporphyrin (but not the meso-position itself). While already
quite diverse, other radiolabeled (metallo)porphyrins also can
be envisaged.
Fig. 1 A porphyrin bearing a meso-swallowtail substituent. (a) Side
view; (b) Newman projection. Adapted from ref. 36.
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Scheme 2
2
Synthesis
A. Porphyrin synthesis. The target porphyrins are of the
trans-AB type, bearing the swallowtail motif and an aromatic
substituent at the 5- and 15- (meso) positions. This design
exploits synthetic accessibility and employs the minimum
number of substituents. Trans-AB-porphyrins are available
via a number of routes, of which the following were attractive
for the structures herein: (1) reaction of a 1,9-bis(N,N-
dimethylaminomethyl)dipyrromethane + a dipyrromethane
in the presence of zinc acetate in ethanol followed by oxidation
with DDQ,32 and (2) reaction of a 1,9-bis[(propylimino)-
methyl]dipyrromethane + a dipyrromethane in the presence
of zinc acetate in ethanol.33 The latter was utilized to gain
access to the desired Step-4 reagents.
Scheme 3
was treated with N-chlorosuccinimide, yielding dibenzyl chlo-
rophosphate.56 The product was sufficiently pure for use in the
subsequent step after removal of the hydroxysuccinimide side-
product by filtration. Treatment of dipyrromethane–dicarbi-
nol 138 with the crude dibenzyl chlorophosphate in the pre-
sence of a catalytic amount of 4-dimethylaminopyridine
The synthesis of P1-(PO4)2 and P1-(OH)2 has been reported
previously (Chart 1).38 Porphyrin P1-(PO4)2 was subjected to
enzymatic dephosphorylation in the precipitation experiments
(vide infra). Porphyrin P1-(OH)2 was used as an authentic
sample for the fully dephosphorylated product. The synthesis
of P1-(PO4)2 employed methyl groups to protect the phos-
phate moieties during the porphyrin synthesis. The cleavage of
the methyl esters in the last step of the synthesis required
extensive optimization to avoid cleavage of the phosphate
groups.38 To avoid such complications, we pursued the
synthesis of benzyl-protected porphyrin–alkyldiphosphates
(Scheme 3).
(DMAP)
yielded
dipyrromethane–alkyldiphosphate
1-(PO4Bn)2 in 43% yield. A lesser amount (29%) of the
monophosphate derivative was also isolated. The condensa-
tion of 1-(PO4Bn)2 and bis[(propylimino)methyl]dipyrro-
methane 233 in refluxing toluene in the presence of zinc
acetate
furnished
porphyrin–alkyldiphosphate
ZnP2-
(PO4Bn)2 in 10% yield (a typical yield for trans-AB-porphyr-
ins with these types of substituents). Scaling up the synthesis
(1.35 mmol scale) resulted in slightly decreased yields (5–8%).
B. Iodination. The porphyrins to be employed in the
enzymatic study, P1-(PO4)2 and P2-(PO4)2, each have two
free meso positions for the later introduction of 131I. There-
fore, we investigated the synthesis of a swallowtail-diphos-
phate diiodoporphyrin with the following objectives: (1) to
The synthesis of swallowtail porphyrin–alkyldiphosphate
ZnP2-(PO4Bn)2, which contains a phenyl moiety rather than
a p-bromophenyl group, was accomplished using standard
procedures.32,33,38 Commercially available dibenzyl phosphite
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establish the synthetic pathway to such iodoporphyrins, (2) to
examine the aqueous solubility of such iodoporphyrins as well
as the soluble-to-insoluble conversion, and (3) to potentially
employ the cold iodoporphyrin in in vivo biodistribution
studies. The corresponding hot agent bearing radioiodine
may be synthesized in the same manner.
conditions, such as TFA60 and NaI,61 failed to yield the
desired water-soluble iodoporphyrin.
Diiodination of the zinc chelate ZnP2-(PO4Bn)2 was sig-
nificantly faster than that of the corresponding free-base
porphyrin, and proceeded in 88% yield under identical con-
ditions. Debenzylation (and demetalation) of ZnP2I2-
(PO4Bn)2 was carried out by treatment with TFA containing
5% thiophenol. The diiodo-substituted free-base porphyrin
was isolated after neutralization of the reaction mixture with
aqueous NaOH, followed by preparative reversed-phase chro-
matography. The product had poor solubility in water (even
upon the addition of base) or in methanol. The Soret band
appeared at lmax = 423 nm (MeOH); such a red-shifted
spectrum was consistent with the presence of the two iodines
attached to the macrocycle, given that unsubstituted trans-AB-
porphyrins (A = aryl, B = swallowtail) typically absorb near
412 nm.33,38 Analytical HPLC showed the sample to be pure,
however, attempts at more rigorous characterization were
hampered by the low solubility of the product, despite the
presence of the two phosphates.
Demetalation of ZnP2-(PO4Bn)2 with TFA afforded free-
base porphyrin–alkyldiphosphate P2-(PO4Bn)2 upon column
chromatography in 43% yield. The low yield likely stems from
partial cleavage of the benzyl protecting groups.57 Superior
conditions entailed use of p-TsOHÁH2O in CH2Cl2 for 20 min,
which afforded pure P2-(PO4Bn)2 by simple aqueous–organic
extraction in 96% yield (Scheme 4). Porphyrin P2-(PO4Bn)2 is
the common precursor of two candidate SPRs, free-base
model porphyrin P2-(PO4)2 and diiodinated P2I2-(PO4)2.
Iodination of P2-(PO4Bn)2 with a mixture of bis(trifluoro-
acetoxy)iodobenzene and iodine in CHCl3–pyridine (2 : 1)58
yielded meso diiodoporphyrin P2I2-(PO4Bn)2. However, the
debenzylation of diiodoporphyrin P2I2-(PO4Bn)2 was not
successful. Treatment with Pd/C and H259 removed the benzyl
protection as well as the iodo groups. Other deprotection
An alternative approach was to remove the benzyl groups
first and then perform the iodination in aqueous solution.
Such aqueous iodination reactions are known.62 Thus, treat-
ment of ZnP2-(PO4Bn)2 with a mixture of thiophenol in TFA
(5 : 95) for 2 h at room temperature,63 followed by neutraliza-
tion of the sample (aqueous NaOH) and preparative reversed-
phase chromatography afforded water-soluble porphyrin P2-
(PO4)2 in 99% yield as a dark red solid (Scheme 5). It is worth
mentioning that this deprotection is significantly milder and
Scheme 4
Scheme 5
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easier to execute than the method employed previously
(TMS–Br, CH2Cl2, Ar).38 None of the dephosphorylated
side-products was observed either by ESI-MS or HPLC
analysis of the sample.
troscopic analysis of either the reaction mixture or the pure
sample.
Attempts to cleave the phosphoester moieties in CuP2-
(PO4Bn)2 were problematic: (i) thiophenol/TFA gave the
free-base porphyrin; (ii) TMS–Br in the presence of pyridine64
gave a complex mixture that contained both partially depro-
tected and dephosphorylated porphyrins; and (iii) hydrogena-
tion (formic acid, H2, 10% Pd/C)65 yielded a mixture of water-
soluble copper porphyrins that rapidly decomposed during
chromatographic purification. Cleavage of the benzyl protect-
ing groups was eventually accomplished in near-quantitative
yield by treatment of CuP2-(PO4Bn)2 with excess TMS–Br in
CHCl3 under an argon atmosphere. The strong acid removed
a small fraction of the central copper atoms (as shown by UV-
Vis absorption and emission spectroscopy), but the product
consisted predominantly of the desired copper porphyrin
CuP2-(PO4)2.
Iodination of P2-(PO4)2 in aqueous solution was accom-
plished with in situ-generated NaICl2.62 Upon addition of the
reagent, precipitation occurred, presumably due to the low
solubility of the porphyrin–alkyldiphosphate at low pH (conc.
HCl solution). Nevertheless the iodination reaction proceeded
with high yield (87%), albeit only monosubstitution occurred.
Neither uniodinated nor diiodinated products were isolated.
1
The H NMR spectrum of pure P2I1-(PO4)2 clearly showed
the disappearance of the broad singlet at 10.32 ppm corres-
ponding to one of the meso-proton signals. Thus, the strategy
of phosphoester deprotection followed by aqueous-solution
iodination proved superior to the route of organic-solution
iodination followed by deprotection.
D. Benchmark compounds. A series of model compounds
was prepared as benchmarks for use in the enzymatic reac-
tions. Enzymatic dephosphorylation of P2-(PO4)2 or CuP2-
(PO4)2 is expected to produce dihydroxyporphyrin P2-(OH)2
or CuP2-(OH)2, respectively. Free-base dihydroxyporphyrin
P2-(OH)2 was synthesized as a reference compound (Scheme
7). Thus, reaction of bis[(propylimino)methyl]dipyrromethane
C. Copper insertion. Copper metalation of P2-(PO4)2 was
not possible in water, as the porphyrin–alkyldiphosphates
(both protected and unprotected) are sensitive to metal salts
in protic solvents.38 Thus, metalation was carried out at the
benzyl phosphate stage in a mixture of CHCl3 and THF (3 : 1)
by treatment of P2-(PO4Bn)2 with Cu(OAc)2 (Scheme 6). The
copper chelate CuP2-(PO4Bn)2 was isolated in 79% yield as a
dark orange solid after silica column chromatography. Resi-
dual free-base porphyrin was not observed by emission spec-
2
and TBDMS-protected swallowtail dipyrromethane
1-(OTBDMS)2 in the presence of zinc acetate in refluxing
toluene gave the porphyrin ZnP2-(OTBDMS)2 in 32% iso-
lated yield. Cleavage of the TBDMS protecting groups with
TBAF followed by demetalation with TFA/CH2Cl2 gave
P2-(OH)2 in 93% yield. Treatment of P2-(OH)2 with
Cu(OAc)2 in a mixture of CHCl3 and MeOH (3 : 1) yielded
copper chelate CuP2-(OH)2 in 98% yield after chromato-
graphy.
The synthesis of CuP2-(PO4)2 shown in Scheme 6 relied on
copper insertion in organic solution followed by phosphoester
deprotection. An alternative synthesis reverses the order of the
two steps (Scheme 7). The synthesis of methyl phosphate-
substituted porphyrin CuP2-(PO4Me)2, of which analogues
have been already been reported,38 was attempted from CuP2-
(OH)2 by treatment with excess dimethyl chlorophosphate in
the presence of DMAP. The analogous reaction with zinc or
free-base porphyrins results in at most trace amounts of the
desired diphosphates, presumably due to zinc-mediated phos-
phate loss, or interference from the porphyrinic nitrogen
atoms, respectively. By contrast, the reaction with CuP2-
(OH)2 proceeded well, and CuP2-(PO4Me)2 was isolated as
a bright orange solid in (unoptimized) 35% yield after silica
column chromatography. LD-MS analysis showed the
presence of several major peaks in addition to the molecule
ion (m/z 768.8), which were assigned to demetalated P2-
(PO4Me)2 (m/z 708.5), and copper or free-base dihydroxy-
porphyrin CuP2-(OH)2 (m/z 552.3) or P2-(OH)2 (m/z 488.4),
respectively. We attribute these peaks to demetalation and
phosphate cleavage under the analysis conditions, as free-base
porphyrin was not observed upon UV-Vis absorption spectro-
scopic analysis of the sample, and the product was pure by
TLC and HPLC analysis.
Scheme 6
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soluble porphyrin (as well as the dihydroxyporphyrins) was
examined by HPLC. NMR spectra were obtained of the
porphyrin–alkyldiphosphates at B1 mM concentration in
D2O with no noticeable signs of aggregation. At much more
dilute concentrations, the absorption spectra of the porphyrin–
alkyldiphosphates in water exhibited characteristic absorption
bands with no noticeable signs of aggregation. The aqueous
solutions of P2-(PO4)2, P2I1-(PO4)2 and CuP2-(PO4)2 were
stable at room temperature for extended periods of time
(several days), when shielded from the light.
3
Enzymatic studies
We performed a series of experiments with the water-soluble
porphyrins P1-(PO4)2, P2-(PO4)2, P2I1-(PO4)2 and CuP2-
(PO4)2 to assess conversion to a precipitate upon enzymatic
treatment as required for a porphyrin-based SPR. Extensive
experiments were carried out with P1-(PO4)2, and representa-
tive experiments were then performed with the other three
porphyrins.
Porphyrin–alkyldiphosphates were treated in aqueous solu-
tion with shrimp alkaline phosphatase (SAP), an enzyme
known to rapidly remove phosphate groups. The product of
the reaction is expected to be a mono-hydroxyporphyrin or a
dihydroxyporphyrin (Scheme 8). Each dihydroxyporphyrin
examined herein is expected to be insoluble in water, and
hence expected to precipitate. The insolubility of P1-(OH)2
and P2-(OH)2 was confirmed by testing with authentic sam-
ples. The reaction course was monitored by several methods
Scheme 7
E. Chemical characterization. All porphyrins were fully
1
characterized by H NMR spectroscopy (except copper che-
lates), LD-MS, high resolution ESI-MS, and UV-Vis absorp-
tion and emission spectroscopy. The copper chelates were non-
emissive, as expected. LD-MS analysis of each benzyl-pro-
tected porphyrin–alkyldiphosphate showed the presence of
both the molecule ion and peaks corresponding to the loss
or gain of one and two benzylic groups. In two cases
[P2I1-(PO4)2, CuP2-(PO4)2], ESI-MS data were not obtained.
The identity of the mono-iodo diphosphate free-base porphyr-
in P2I1-(PO4)2 (the non-iodinated precursor of which was fully
1
characterized) was confirmed by H NMR spectroscopy (loss
of meso proton), absorption and emission spectroscopy (red-
shifted spectra), and HPLC. The identity of the diphosphate
copper porphyrin CuP2-(PO4)2 (the benzyl-protected precur-
sor of which was fully characterized) was confirmed by water
solubilization, absorption spectroscopy, emission spectro-
scopy, and HPLC.
All but one of the porphyrins bearing free phosphate groups
were soluble in pure water. The water-soluble porphyrins
include P1-(PO4)2, P2-(PO4)2, P2I1-(PO4)2 and CuP2-(PO4)2;
the water-insoluble porphyrin was P2I2-(PO4)2. Each water-
Scheme 8
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including visual inspection, absorption spectroscopy, ESI-MS,
and nephelometry.
A. Qualitative composition upon enzymatic treatment. The
enzymatic treatment was carried out with an aqueous solution
of P1-(PO4)2 at 68 mM in a Tris-HCl buffer (50 mM, pH 9.0)
as described in the Experimental section. One enzymatic
reaction (E) and three negative control reactions were set up.
The controls included use of heat-inactivated enzyme and
enzyme buffer (dE), enzyme buffer but no enzyme (MZ), and
neither enzyme buffer nor enzyme (NE). All four tubes were
incubated at 37 1C for 2 h followed by centrifugation. The
results are shown in Fig. 2. Treatment with enzyme afforded
rapid discoloration of the reaction mixture (o1 min). After
2 h, no precipitate was observable by visual inspection, but
was readily observed upon centrifugation. No significant
changes were observed in the absence of enzyme, or with the
other two controls (not shown). We note that centrifugation is
not essential for eventual visual observation of the precipitate
in the case of enzymatic treatment; indeed, a red–brown
precipitate was observed at the bottom of the tube after
standing for several days.
Absorption spectra and ESI-MS data were obtained for
aqueous aliquots removed from the tubes. Any precipitate was
isolated, washed with water, dissolved in DMF and analyzed
by ESI-MS. The origin of the discoloration of the reaction
mixture upon treatment with enzyme is displayed in the
absorption spectrum. Prior to the addition of the enzyme
SAP, the P1-(PO4)2 exhibited a strong absorption in aqueous
solution at 401 nm (Soret band). Following treatment (prior to
or following centrifugation), a sample from the aqueous phase
shows extensive broadening (and diminished intensity) of the
porphyrin absorption bands. By contrast, the spectrum of the
sample without enzyme remains characteristic of a monomeric
porphyrin in solution (Fig. 2). The reaction mixtures in tube
MZ and dE showed slight decreases (22–25%) in the absorp-
tion intensity at 401 nm, which may be attributed to the
presence of divalent salts (e.g., Mg2+, Zn2+).
ESI-MS analysis of the supernatant upon enzyme treatment
showed peaks corresponding to the dephosphorylated por-
phyrin P1-(OH)2 and a small amount of P1-OH/PO4. The
molecule ions were typically cationized with H+ and/or Na+
(see ESIw). It was surprising to observe a peak attributed to
P1-(OH)2, given that an authentic sample of P1-(OH)2 does
not dissolve appreciably in water, and attempts to obtain an
ESI-MS spectrum from an aqueous sample did not give the
corresponding peak. Analysis of the precipitate (following
isolation and dissolution in DMF) showed that P1-(OH)2
was the major component. ESI-MS analysis showed the sole
presence of starting P1-(PO4)2 in the control reactions with no
enzyme or with enzyme buffer, whereas the mass spectrum of
the reaction mixture treated with deactivated enzyme dis-
played peaks attributed to P1-(PO4)2, P1-(OH)2 and
P1-OH/PO4. Apparently, the condition that we employed
for deactivation (75 1C for 20 min) did not completely
deactivate the enzyme. However, the heat treatment did sig-
nificantly lower the enzyme activity given that P1-(PO4)2
remained present after incubation for 2 h.
Fig. 2 Time course of P1-(PO4)2 in aqueous solution with (a) no
enzyme (NE) or (b) active SAP (E). Samples with a buffer solution or
heat-inactivated SAP gave the same results as with no enzyme (not
shown). A brown precipitate is clearly visible following centrifugation
of the tube containing enzyme. The absorption spectrum of the
aqueous phase following incubation for 2 h shows no aggregation
(control reaction lacking enzyme) or extensive aggregation (reaction
mixture with enzyme).
B. Kinetic study upon enzymatic treatment. The porphyrin
P1-(PO4)2 at 60 mM in water containing a Tris-HCl buffer (50
mM, pH 9.0) was treated with the enzyme SAP at 37 1C.
Aliquots were periodically removed from the reaction tube
and heated to deactivate the enzyme (70 1C at 10 min),
whereupon absorption spectra were obtained in water at 402
and 450 nm as shown in Fig. 3. The data at 402 nm pre-
dominantly measure the concentration of the monomeric
intact porphyrin, whereas 450 nm provides a gauge of the
amount of aggregated porphyrin (of unknown molar
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intensity of scattered light over the period examined (2 h). On
the other hand, the porphyrin solution treated with active SAP
enzyme gave a clear increase in scattered light, steadily in-
creasing above background over the course of B6 h (Fig. 4A).
A slight further increase was observed after 18 to 20 h. Mass
analysis showed the presence of P1-OH/PO4 even when the
intensity of the scattered light leveled off. A second batch of
SAP was added, but after 6 h the mass peak attributed to
P1-OH/PO4 still remained. The P1-OH/PO4 mass peak final-
ly disappeared after several days. The slow dephosphorylation
may stem from the low temperature (B25 1C instead of 37 1C)
inside the nephelometer.
D. Further studies. Water-soluble porphyrin–alkyldiphos-
phates P2-(PO4)2, P2I1-(PO4)2 and CuP2-(PO4)2 were treated
in the same manner as for P1-(PO4)2 and monitored by visual
inspection, absorption spectroscopy, and nephelometry. The
final reaction mixtures were analyzed by HPLC. In each case,
the results generally mirrored those described above for P1-
(PO4)2. Some slight differences were observed by nephelome-
try, where precipitate formation was more rapid than that
observed with P1-(PO4)2. In particular, the amount of pre-
cipitate increased until approximately 2 h and leveled off
thereafter. An example is shown for P2I1-(PO4)2 (Fig. 4B).
When more concentrated porphyrin solutions were employed,
precipitate formation could be seen initially, followed by a
period of gradual decrease. As a small amount of orange
Fig. 3 Absorption spectral monitoring of the reaction course upon
treatment of P1-(PO4)2 with enzyme. Legend: monomeric porphyrin
in water (402 nm: solid line, circles); aggregated porphyrin in water
(450 nm; dotted line, squares); total porphyrin in DMF (406 nm:
dashed line, triangles). The tf datapoints were collected after overnight
reaction.
absorption coefficient). To measure the total porphyrin pre-
sent, and ensure there was no porphyrin decomposition during
the course of the reaction, samples were also removed and
dissolved in DMF, which dissolves all of the porphyrin species
[substrate porphyrin P1-(PO4)2 and the hydrolyzed product
porphyrins].
The sharp drop in absorption intensity at 402 nm (by more
than half in the first 10 min after the addition of enzyme) was
accompanied by the intensification of absorption at 450 nm.
The peak at 450 nm likely represents aggregated complexes of
P1-(OH)2 and P1-OH/PO4 [and possible entrainment of some
P1-(PO4)2]. On the other hand, the absorption intensity at 406
nm determined in DMF changed little during the reaction
course. Finally, P1-(PO4)2 was incubated with enzyme for 5 h,
whereupon the entire reaction mixture was dissolved in DMF,
thereby eliminating any sampling error caused by taking
aliquots from heterogeneous media. The absorption intensity
at 406 nm changed little after the incubation (0.61 to 0.65; not
shown). Thus, the intensity decrease of the 402 nm peak must
stem from aggregation and precipitation of P1-(OH)2 and/or
P1-OH/PO4 formed via enzymatic dephosphorylation.
C. Kinetics of precipitation. We assessed the extent of
precipitation during the course of the reaction by nephelome-
try, a technique that measures scattered light. A nephelometer
is readily available by use of a fluorimeter, where the light
deflected by the precipitate in the sample in a cuvette is
detected at a right angle from the incident beam.66 For use
with porphyrins, the wavelength of the incident beam was
chosen as 800 nm to avoid any absorption by the porphyrin.
The dephosphorylation process was carried out with a
gently stirred sample of P1-(PO4)2 at 35 mM in a 3-mL
fluorescence cuvette, and the scattered light was measured
continuously as a function of time. The porphyrin solution
treated with deactivated SAP enzyme gave no increase in the
Fig. 4 Light scattering (nephelometry) over time upon treatment of a
bis(dihydroxyphosphoryloxy)alkylporphyrin in water with the enzyme
SAP. (a) The sample of porphyrin P1-(PO4)2 was disturbed upon
removal of aliquots (for ESI-MS analysis) at 2, 4 and 6 h. (b) The
porphyrin P2I1-(PO4)2.
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coating was observed on the stir bar, the decrease may be due
to the formation of large aggregates of porphyrin, which settle
out of the suspension. Regardless, in each case, the porphyrins
underwent the soluble-to-insoluble conversion upon treatment
with the enzyme SAP.
Experimental
General
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded in CDCl3 unless noted otherwise. Absorption spectra
and fluorescence spectra were collected at room temperature in
CH2Cl2 unless noted otherwise. Hydrophobic porphyrins were
analyzed in neat form by laser desorption mass spectrometry
(LD-MS).70 The water-soluble porphyrins were analyzed by
direct infusion of water–acetonitrile (40:60) or water–metha-
nol (40 : 60) solutions by atmospheric pressure electrospray
mass spectrometry (ESI-MS). Both in LD-MS and ESI-MS
analyses, positive ions were detected unless noted otherwise.
Benzyl-protected porphyrin–alkyldiphosphates gave clusters
of peaks in the molecule ion region that were attributed to
the addition or loss of benzyl groups; in each case, the
molecule ion only is listed. Solvents were dried according to
standard procedures. The solvent CHCl3 contained the inhi-
bitor amylenes or ethanol; the former is distinguished by
referral as ‘‘CHCl3–amylenes’’ whereas ‘‘CHCl3’’ refers to
the solvent containing ethanol.
Outlook
A porphyrin-based model compound for use as a Step-4
reagent in the STAR method has been synthesized.
The soluble form carries phosphate groups at the termini of
a branched alkyl chain. The phosphates can be efficiently
cleaved upon treatment with a phosphatase enzyme, resulting
in the formation of a precipitate. The potential for radiolabel-
ing has been investigated through iodination and metalation
experiments. Each of four porphyrins examined underwent
the desired soluble-to-insoluble conversion. Accordingly, it
should be possible to employ water-soluble porphyrin–alkyl-
phosphates to deliver radioactive loads to the target,
and thereby form an insoluble precipitate as a result of
enzymatic action. The syntheses developed herein incorporate
iodine or copper (surrogates for radionuclides) near the
end of the preparative manipulations, which is highly desirable
for medical applications. The ability to form an immobile
precipitate in situ rather than deliver intact nanoparticles
may afford greater control and versatility. It should be noted
that while the present porphyrins contain phosphates,
other enzymatically cleavable groups such as sulfates or
glucuronides also could be employed. In addition, the presence
of phosphates may make the radiolabeled analogues
of the porphyrins compounds viable alone for some thera-
pies given the overexpression of phosphatases in many can-
cers.67
Non-commercial compounds
Compounds 1-(OTBDMS)2,38 138 and 233 were synthesized as
described in the literature.
Chromatography
Preparative chromatography was performed using silica or
alumina (80–200 mesh). Thin layer chromatography was
performed on silica or alumina. Transparent samples were
visualized by UV-light (254 nm and 365 nm), Br2-vapor, or
KMnO4/K2CO3. Reversed-phase preparative column chroma-
tography was carried out using C-18-coated silica and eluants
containing water admixed with methanol. Analytical HPLC in
all cases was carried out using a reversed-phase C-18 column
(5 mm, 4.6 mm  150 mm) with the following elution program
with solvents A (water containing 0.1% TFA) and B (metha-
It warrants emphasis that for the successful development of
the STAR method, the design, synthesis, and validation of a
Step-4 SPR is a necessary prelude to the design of a Step-1
SPR. Step-1 and Step-4 SPRs have different chemical struc-
tures, yet both types of SPRs share the requirement for
soluble-to-insoluble conversion, and thus will share some
structural features. A Step-1 SPR presents a greater challenge
given the requirements to (1) bear a cancer-targeting agent, (2)
bear a ligand for binding of the enzyme conjugate (i.e.,
bispecific or Step-3 reagent), (3) undergo the soluble-to-
insoluble conversion, and (4) display the ligand for binding
of the enzyme conjugate upon precipitation to give the plat-
form. Molecular designs centered around porphyrins that
meet these criteria have been described.68 The work described
herein may provide the basis for refined designs of Step-1
SPRs and ultimately prove useful in new therapeutic ap-
proaches for treating solid tumors. By all accounts, the devel-
opment of radiotherapeutics is in its infancy.8 Synthetic
porphyrins may prove useful for a variety of radiotherapeutic
applications given their inherent tailorability and versatility as
carriers, either by covalent linkage (e.g., I) or by chelation
(e.g., Cu, Ga, Y, Rh, In, Lu, Re, Bi) for essentially all of the
radionuclides8,25,69 that have been identified or proposed in
recent years for radiotherapy.
nol containing 0.1% TFA): flow rate = 1.5 mL minÀ1
;
0–2 min, 0% B; 5–35 min, 0 - 95% B; 35–40 min, 95%
B; detection at 254, 400, and 417 nm; void volume typically
1.1 min.
Enzymatic experiments
HPLC-grade water was used to prepare all aqueous solutions
and absorption spectral measurements. The concentration of
the enzyme SAP in the stock solution was 2.55 mM, calculated
from the unit concentration (unit mLÀ1), specific activity (unit
mgÀ1) and molecular weight (g molÀ1; all data provided by
Promega). Porphyrin concentrations were calculated from
absorption spectra assuming eSoret = 500 000 MÀ1 cmÀ1
The enzymatic reaction mixture was analyzed by direct infu-
sion of water–methanol (40 : 60) solutions by ESI-MS in the
positive-ion mode.
.
(i) Reaction composition. The free-base porphyrin P1-
(PO4)2 shows good solubility in water; however, the porphyrin
needs to be soluble in a buffer suitable for the dephosphoryla-
tion reaction catalyzed by SAP. Mixing the aqueous solution
of the porphyrin and a buffer solution from the enzyme
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provider (Promega) yielded an immediate red precipitate.
Aqueous solutions of P1-(PO4)2 of a range of concentrations
(up to 0.61 mM) were prepared and mixed with the SAP
buffer. With high concentrations of porphyrin, a precipitate
was observed (by visual inspection) to form very rapidly. With
low concentrations, a precipitate was observed only upon
centrifugation of the sample. In each case, the porphyrin
concentration in the supernatant (calculated from absorption
spectra) was 1–2 mM. To avoid such precipitation, a Tris-HCl
buffer (50 mM, pH 9.0) was used (lacking SAP and metal salt)
to prepare the porphyrin solution. Neither precipitation nor
change of color in the solution was observed. Although the
lack of Mg2+, which is a cofactor of SAP, may lower the
enzyme activity, a higher starting substrate concentration is
preferable to better distinguish the reaction composition be-
fore and after dephosphorylation. On the other hand, the
lower enzyme activity can be compensated for by increasing
the enzyme/substrate ratio. After several preliminary experi-
ments, we found that a suitable molar ratio of phosphoester
[each P1-(PO4)2 molecule contains two phosphoesters] to SAP
is approximately 5000 : 1.
tion spectra in both water and DMF were recorded for each
aliquot and the remaining solution.
A solution of P1-(PO4)2 in 300 mL of a Tris-HCl buffer
(50 mM, pH 9.0) was prepared in an Eppendorf tube at a
concentration of 38 mM. A 100-mL aliquot of the solution was
transferred to a cuvette filled with 3 mL of DMF, and the
absorption spectrum was recorded. A 1.7-mL aliquot of the
SAP stock solution was added to the Eppendorf tube, and the
reaction was incubated at 37 1C with shaking for 5 h. The
entire reaction mixture was transferred to a cuvette filled with
3 mL of DMF, and a second absorption spectrum was
recorded. The absorption intensity at 406 nm in the second
spectrum was divided by 2 and compared to the absorption
intensity at 406 nm in the first spectrum.
(iv) Protocol for precipitate detection. A simple light-scat-
tering instrument (nephelometer)66 was constructed using a
fluorimeter (Photon Technology International). Excitation
and emission monochromators were both set at the same
wavelength to allow scattered incident light to enter the
photomultiplier. The wavelength 800 nm was chosen to mini-
mize direct absorption by the porphyrin. The instrument
parameters were as follows: photomultiplier voltage, 800 V;
slit widths, 1 mm; integration time, 10 s. The enzymatic
reaction was performed in a 1-cm pathlength fluorescence
cuvette (3 mL) at room temperature. The cuvette was
equipped with a stirring bar, and the reaction mixture was
stirred at a modest rate (no vortex formation). The data are
obtained as counts/sec over time.
(ii) Representative protocol for qualitative analysis. A solu-
tion of P1-(PO4)2 in 1600 mL of a Tris-HCl buffer (50 mM, pH
9.0) was prepared. A 60-mL aliquot of this solution was diluted
in 3 mL of water, and an absorption spectrum was obtained.
The concentration calculated from the intensity at 401 nm was
68 mM. The remaining porphyrin solution was distributed
evenly into four 1.5-mL Eppendorf tubes, of which one
received the enzyme and enzyme buffer (tube E), and three
were used for controls (enzyme buffer and heat-deactivated
enzyme, dE; enzyme buffer but no enzyme, MZ; and neither
enzyme buffer nor enzyme, NE). A 3.8-mL aliquot of the SAP
stock solution [containing 1 mM MgCl2, 0.1 mM ZnCl2,
25 mM Tris-HCl (pH 7.6) and 50% (v/v) glycerol] was added
to tube E. Another 3.8 mL of the SAP stock solution was
heated at 75 1C for 20 min and then added to tube dE. A buffer
solution was prepared, which contained 1 mM MgCl2, 0.1 mM
ZnCl2, 20 mM Tris-HCl (pH 7.2) and 40% (v/v) glycerol. A
3.8-mL aliquot of the buffer solution was added to tube MZ.
All four tubes were incubated at 37 1C for 2 h and then
centrifuged at 7,000 rpm for 8 min. From each tube, 60 mL of
the solution (tube dE, NE and MZ) or supernatant (tube E)
was transferred into 3 mL of water for absorption analysis.
The remaining solutions and supernatant were used for mass
analysis. The precipitate in tube E was washed with water,
dissolved in DMF, and analyzed by ESI-MS.
A fluorescence cuvette was filled with 3 mL of an aqueous
solution of P1-(PO4)2 in a Tris-HCl buffer (50 mM, pH 9.0).
The concentration of the porphyrin solution was 35 mM. An
aliquot (16.8 mL) of the SAP stock solution was added to the
cuvette, and the light scattering measurement was started
immediately. A 500-mL aliquot of the reaction mixture was
taken out of the cuvette for mass analysis at 2, 4, 6 and 18 h.
After the mass spectrum was obtained, the aliquot was trans-
ferred back to the cuvette to keep the total volume constant. A
second batch of SAP (16.8 mL) was added at 20 h, and the
cuvette was stirred at room temperature. ESI-MS spectra were
obtained every 2 h by the same procedure described above.
For P2-(PO4)2 and CuP2-(PO4)2, the reactions were
monitored over 3 h by nephelometry. At the end of the
reaction, an aliquot of each sample was diluted with methanol,
and the sample was analyzed by HPLC with P2-(OH)2 or
CuP2-(OH)2 as reference. For P2I1-(PO4)2, the reaction was
monitored over 18 h by nephelometry.
5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]dipyrromethane
(1-(PO4Bn)2)
(iii) Protocol for kinetics experiments. A solution of P1-
(PO4)2 in 500 mL of a Tris-HCl buffer (50 mM, pH 9.0) was
prepared in an Eppendorf tube at a concentration of 60 mM. A
5.3-mL aliquot of the SAP stock solution was added, and the
tube was incubated at 37 1C with shaking. A 50-mL aliquot of
the reaction mixture was removed from tube 1 every 10 min
from 0 min (before the addition of SAP) to 70 min. The
remaining solution was incubated at 37 1C for 16 h, and then
heated to 70 1C for 10 min. Each aliquot was heated to 70 1C
in a water bath for 10 min to deactivate the enzyme. Absorp-
Following a literature procedure,56 a solution of N-chlorosuc-
cinimide (612 mg, 4.57 mmol) in dry toluene (25 mL) was
treated with dibenzyl phosphite (1.01 mL, 4.57 mmol) under
argon at room temperature for 2 h. The reaction mixture was
filtered through a filter paper. The filtrate was concentrated
and dried under vacuum. The resulting colorless liquid was
dissolved in dry CH2Cl2 (10 mL) to afford the ‘‘dibenzyl
chlorophosphate solution.’’ Following a literature procedure38
with modifications, the dibenzyl chlorophosphate solution was
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slowly added to a solution of 1 (260 mg, 0.935 mmol) and
DMAP (557 mg, 4.57 mmol) in dry CH2Cl2 (10 mL). The
mixture was stirred under argon at room temperature for 20 h.
The reaction mixture was diluted in CH2Cl2 and washed with
water. The organic layer was dried (Na2SO4). Removal of
solvent gave the crude product as a dark oil. Chromatography
[silica, CH2Cl2–MeOH (49 : 1)] of the crude product yielded a
light-yellow, viscous oil (309 mg, 43%): 1H NMR d 1.40–1.50
(m, 2H), 1.69–1.79 (m, 2H), 2.20–2.30 (m, 1H), 3.87–3.94 (m,
4H), 4.19 (d, J = 4.8 Hz, 1H), 4.94–5.04 (m, 8H), 5.91–5.95
(m, 2H), 6.07–6.11 (m, 2H), 6.60–6.64 (m, 2H), 7.29–7.36 (m,
20H), 8.57 (br, 2H); 13C NMR d 32.26, 32.32, 35.35, 40.59,
66.27, 66.33, 69.46, 69.51, 106.72, 108.21, 117.04, 128.09,
128.13, 128.73, 128.76, 130.83, 135.86, 135.92; FAB-MS obs.
769.2841, calc. 769.2808 [(M + H)+, M = C42H46N2O8P2].
Anal. Calc. for C42H46N2O8P2: C, 65.62; H, 6.03; N, 3.64.
Found: C, 65.67; H, 6.03; N, 3.61%.
5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (P2-(PO4Bn)2). Method A
Following a literature procedure,38 a solution of ZnP2-
(PO4Bn)2 (25 mg, 23 mmol) in CH2Cl2 (4 mL) was treated
with TFA (500 mL) at room temperature under argon for 2 h.
The reaction mixture was concentrated to dryness and dis-
solved in CH2Cl2. The resulting solution was diluted in
CH2Cl2 and washed with saturated aqueous NaHCO3. The
organic layer was separated and dried (Na2SO4). Removal of
the solvent yielded the crude product as a dark solid, which
upon chromatography (silica, ethyl acetate) gave a purple
solid (10 mg, 43%): 1H NMR d À2.92 (br, 1H), –2.86 (br,
1H), 2.97–3.07 (m, 2H), 3.28–3.39 (m, 2H), 3.76–3.84 (m, 2H),
3.93–4.01 (m, 2H), 4.39–4.50 (m, 6H), 4.58–4.65 (m, 2H),
5.59–5.69 (m, 1H), 6.71–6.76 (m, 2H), 6.78–6.82 (m, 2H),
6.83–6.94 (m, 8H), 6.94–7.00 (m, 4H), 7.79–7.84 (m, 3H),
8.24–8.29 (m, 2H), 9.05–9.09 (m, 2H), 9.30 (d, J = 4.7 Hz,
1H), 9.34 (d, J = 4.7 Hz, 1H), 9.39 (d, J = 4.4 Hz, 1H), 9.42
(d, J = 4.7 Hz, 1H), 9.56 (d, J = 4.7 Hz, 1H), 9.67 (d, J = 4.7
Hz, 1H), 10.19 (s, 1H), 10.27 (s, 1H); LD-MS: obs. 1009.5;
ESI-MS: obs. 1009.3491, calc. 1009.3489 [(M + H)+,
M = C59H54N4O8P2]; labs 406, 503, 536, 576, 631 nm.
Data for 5-[1-(dibenzyloxyphosphoryloxy)-5-hydroxypent-3-
yl]dipyrromethane (1-OH/PO4Bn)
5-[1-(Dibenzyloxyphosphoryloxy)-5-hydroxypent-3-yl]dipyr-
romethane, which is more polar than 5-[1,5-bis(dibenzyl-
oxyphosphoryloxy)pent-3-yl]dipyrromethane by TLC analy-
sis, was also isolated as a light-purple, viscous oil (207 mg,
29%): 1H NMR d 1.34–1.44 (m, 1H), 1.46–1.56 (m, 1H),
1.61–1.70 (m, 1H), 1.72–1.81 (m, 2H), 2.32–2.41 (m, 1H),
3.51–3.60 (m, 2H), 3.86–4.02 (m, 2H), 4.17 (d, J = 5.7 Hz,
1H), 4.95–5.05 (m, 4H), 5.95–6.00 (m, 2H), 6.08–6.12 (m,
2H), 6.59–6.62 (m, 2H), 7.29–7.38 (m, 10H), 8.66 (br, 1H),
8.69 (br, 1H); 13C NMR d 29.76, 32.84, 32.89, 34.71, 35.23,
41.23, 60.85, 66.66, 66.72, 69.58, 69.64, 106.61, 106.85,
108.14, 108.21, 116.96, 117.03, 128.14, 128.16, 128.81,
131.34, 131.62, 135.80, 135.87; FAB-MS: obs. 508.2143, calc.
508.2127 (C28H33N2O5P).
5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (P2-(PO4Bn)2). Method B
A solution of ZnP2-(PO4Bn)2 (50.2 mg, 0.0469 mmol) in
CH2Cl2 (14.5 mL) was treated with p-TsOHÁH2O (522 mg,
2.73 mmol) at room temperature for 20 min. The solution was
diluted in CH2Cl2 and washed with saturated aqueous NaH-
CO3 solution. The organic layer was dried (Na2SO4). Removal
of the solvent yielded the crude product as a dark solid that
was sufficiently pure (495%) without further purification
(45.4 mg, 96%). Characterization data (1H NMR, LD-MS,
labs) were identical to those reported with Method A.
lem (lexc 408 nm) 634, 700 nm.
Zn(II)-5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (ZnP2-(PO4Bn)2)
5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]-10,20-
diiodo-15-phenylporphyrin (P2I2-(PO4Bn)2)
Following a literature procedure,33,38 a solution of 1-(PO4Bn)2
(175 mg, 228 mmol) and 2 (103 mg, 286 mmol) in toluene (25
mL) was treated with Zn(OAc)2 (417 mg, 2.28 mmol). The
mixture was refluxed open to air for 4.5 h. The reaction
mixture was cooled to room temperature and diluted in
CH2Cl2. The resulting dark solution was washed with water
and brine. The organic layer was separated, dried (Na2SO4)
and concentrated to dryness. Chromatography [silica,
CH2Cl2–ethyl acetate (4 : 1)] yielded a red solid. The resulting
crude product was chromatographed [silica, hexanes–ethyl
Following a literature procedure58 with modification, a solu-
tion of bis(trifluoroacetoxy)iodobenzene (57 mg, 134 mmol)
and iodine (34 mg, 134 mmol) in CHCl3–amylenes (1 mL) was
treated with anhydrous pyridine (500 mL) at room temperature
for 30 min. The color of the reaction mixture changed from
purple to light yellow. A white solid was observed in the
reaction mixture. The resulting yellowish mixture was slowly
added to a solution of P2-(PO4Bn)2 (9.0 mg, 8.9 mmol) in
CHCl3–amylenes (1 mL). The mixture was stirred at room
temperature for 1 h. The reaction mixture was diluted in
CH2Cl2 and washed with saturated aqueous Na2S2O3. The
organic layer was separated and dried (Na2SO4). Removal of
the solvent yielded the crude product as a dark purple solid.
Chromatography [silica, CH2Cl2–ethyl acetate (1 : 1)] yielded a
1
acetate (2 : 3)] to yield a red solid (25 mg, 10%): H NMR d
2.36–2.48 (m, 2H), 2.78–2.95 (m, 6H), 3.32–3.43 (m, 2H),
3.48–3.72 (m, 6H), 5.13–5.23 (m, 1H), 6.30 (d, J = 7.4 Hz,
2H), 6.37 (d, J = 7.7 Hz, 2H), 6.70–6.82 (m, 8H), 6.85–6.91
(m, 4H), 7.69–7.80 (m, 3H), 8.13 (d, J = 6.6 Hz, 2H),
9.02–9.08 (m, 3H), 9.29 (d, J = 4.4 Hz, 1H), 9.33 (d, J =
4.7 Hz, 1H), 9.36 (d, J = 4.4 Hz, 1H), 9.45 (d, J = 4.7 Hz,
1H), 9.52 (d, J = 4.4 Hz, 1H), 10.01 (s, 1H), 10.20 (s, 1H); LD-
MS: obs. 1071.0; FAB-MS: obs. 1070.2572, calc. 1070.2552
(C59H52N4O8P2Zn); labs 408, 538, 635 nm.
1
purple solid (2.5 mg, 22%): H NMR d À2.71, 2.89–3.03 (m,
2H), 3.15–3.28 (m, 2H), 3.72–3.84 (m, 2H), 3.92–4.04 (m, 2H),
4.41–4.57 (m, 6H), 4.60–4.70 (m, 2H), 5.46–5.58 (m, 1H),
6.75–6.81 (m, 4H), 6.85–7.03 (m, 16H), 7.72–7.84 (m, 3H),
8.12 (d, J = 7.4 Hz, 1H), 8.70–8.79 (m, 2H), 9.36 (d, J = 4.4
Hz, 1H), 9.51 (d, J = 4.7 Hz, 1H), 9.56 (d, J = 4.7 Hz, 1H),
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New J. Chem., 2008, 32, 436–451 | 447

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9.60 (d, J = 4.4 Hz, 2H), 9.72 (d, J = 5.0 Hz, 1H); LD-MS:
obs.; FAB-MS: obs. 1261.1421, calc. 1261.1422 [(M + H)+,
M = C59H52I2N4O8P2]; labs 426, 524, 563, 604, 664 nm.
5-[1,5-Bis(dihydroxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (P2-(PO4)2)
A solution of 1 (22.7 mg, 22.5 mmol) in TFA (4.25 mL) was
treated with thiophenol (0.25 mL) at room temperature for 2
h. The solution was neutralized with concentrated aqueous
NaOH, and applied onto a reversed-phase silica column
equilibrated with H2O. Elution with H2O–MeOH (0 -
50%) yielded a dark purple solution that was concentrated
in vacuo. The resulting dark purple residue was dissolved in
distilled water, and the resulting solution was filtered through
a plug of cotton wool. The filtrate was freeze-dried to yield a
dark red film (quantitative): 1H NMR d 3.53–3.60 (m, 4H),
3.91 (m, 2H), 4.30 (m, 2H), 5.85 (m, 1H), 7.58 (d, J = 6.0 Hz,
2H), 7.73–7.76 (m, 2H), 7.87–7.91 (m, 1H), 8.39 (br 1H), 8.49
(br 1H), 8.86 (br 1H), 9.05 (br 1H), 9.65 (br, 1H), 9.76 (br 1H),
9.96 (br 1H), 10.16 (br 1H), 10.42 (br, 1H), 10.32 (br, 1H);
ESI-MS: obs. 649.1629; calc. 649.1611 [(M + H)+; M =
C31H30N4O8P2]; labs (H2O) 401, 505, 540, 568, 619;
lem (lexc 401 nm) 627, 690 nm; HPLC tR = 16.62 min.
Zn(II)-5-[1,5-Bis(tert-butyldimethylsilyloxy)pent-3-yl]-15-
phenylporphyrin (ZnP2-(OTBDMS)2)
Following a standard procedure,33 a solution of 2 (258 mg,
0.716 mmol) and 1-(OTBDMS)2 (341 mg, 0.716 mmol) in
toluene (78 mL) was treated with Zn(OAc)2 (1.35 g, 7.16
mmol). The mixture was refluxed for 18 h open to the air.
The toluene was evaporated, and the resulting residue was
chromatographed (silica, CH2Cl2) to give a purple solid (176
mg, 32%): 1H NMR d À0.14 (s, 12H), 0.89 (s, 18H), 3.07–3.14
(m, 2H), 3.32-3.39 (m, 2H), 3.61–3.72 (m, 4H), 5.98 (m, 1H),
7.80 (app s, 3H), 8.18–8.20 (m, 2H), 9.05 (s, 2H), 9.31–9.34 (m,
2H), 9.47–9.50 (m, 2H), 9.92–9.93 (m, 1H), 10.08–10.09 (m,
1H), 10.18 (s, 1H), 10.22 (s, 1H); LD-MS: obs. 778.9; ESI-MS:
obs. 779.31444, calc. 779.31495 [(M
+ M =
H)+,
C43H54N4O2Si2Zn]; labs 407, 538 nm; lem (lexc 407 nm) 578,
631 nm.
5-(1,5-Dihydroxypent-3-yl)-15-phenylporphyrin (P2-(OH)2)
5-[1,5-Bis(dihydroxyphosphoryloxy)pent-3-yl]-10-iodo-15-
phenylporphyrin (P2I1-(PO4)2)
A solution of ZnP2-(TBDMS)2 (140 mg, 0.179 mmol) was
dissolved in THF containing TBAF (3.6 mL of 1.0 M solution,
water content B5%). The reaction was allowed to proceed for
1 h. The sample was concentrated. The residue was dissolved
in CH2Cl2 (2 mL). The solution was treated with TFA (2 mL).
The mixture was stirred at room temperature for 30 min. The
mixture was concentrated at reduced pressure. The residue was
dissolved in ethyl acetate, and the resulting solution was
washed with water. The aqueous layer was extracted with
ethyl acetate. The combined organic extract was washed with
water, dried (Na2SO4), and concentrated. Column chromato-
graphy [neutral alumina, CH2Cl2–MeOH (98 : 2 - 90 : 10]
afforded a bright purple solid (85 mg, 97%): 1H NMR
(CDCl3–CD3OD) d 3.03–3.08 (m, 2H), 3.25–3.31 (m, 2H),
3.58–3.60 (m, 4H), 5.73 (m, 1H), 7.78 (app s, 3H), 8.21 (app s,
2H), 9.03–9.04 (m, 2H), 9.37–9.40 (m, 2H), 9.46–9.48 (m, 2H),
9.76–9.78 (m, 2H), 9.88–9.89 (m, 1H), 10.29–10.30 (s, 1H);
LD-MS: obs. 489.2; FAB-MS: obs. 488.2207, calc. 488.2212
(C31H28N4O2); labs 404, 502, 534, 576 nm; lem (lexc 404 nm)
634, 701 nm; HPLC tR = 32.20 min.
Following a literature procedure62 with modification, a solu-
tion of NaICl2 [prepared from aqueous NaOCl (10–13%,
49 mL), NaI (22 mg, 147 mmol) and conc. HCl (28 mL)] was
added to a solution of P2-(PO4Bn)2 (15 mg, 0.023 mmol) in
water (600 mL). A solid precipitate formed immediately.
The mixture was stirred at room temperature for 12 h. The
reaction mixture was diluted by dropwise addition of aqueous
NaOH (2.5 M) until all solid dissolved. The resulting
solution was applied onto a reversed-phase silica column
equilibrated with water. Chromatography [H2O–MeOH
(0 - 50%)] yielded a purple solution. The sample was
concentrated in vacuo, and the resulting solid residue was
dissolved in water. The solution was filtered through a plug
of cotton wool. The filtrate was freeze-dried to yield a dark
1
red, voluminous solid (15.4 mg, 87%): H NMR d 3.93 (m,
2H), 4.30 (m, 2H), 5.81 (m, 1H), 7.31–7.62 (m, 5H), 8.10 (m,
1H), 8.22 (m, 1H), 8.48 (m, 1H), 8.71 (m, 1H), 9.47 (br, 1H),
9.9.65 (br, 1H), 9.91 (br, 1H), 10.15 (br, 1H), 10.26 (br, 1H);
labs (H2O) 412, 563 nm; lem (H2O, lexc 412 nm) 621, 677 nm;
HPLC tR = 26.02 min.
Cu(II)-5-(1,5-Dihydroxypent-3-yl)-15-phenylporphyrin
(CuP2-(OH)2)
Cu(II)-5-[1,5-Bis(dibenzyloxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (CuP2-(PO4Bn)2)
A
sample of P2-(OH)2 (11.9 mg, 0.0246 mmol) in
CHCl3–MeOH (3 : 1, 2 mL) was treated with Cu(OAc)2
(44.5 mg, 0.246 mmol, 10 equiv). Stirring was continued for
12 h at room temperature. The sample was diluted with ethyl
acetate and water. The layers were separated, and the aqueous
layer was extracted with ethyl acetate. The organic layer was
dried (Na2SO4). Chromatography [neutral alumina,
CH2Cl2–MeOH–THF (100 : 0 : 0 - 5 : 1 : 1)] yielded an
orange solid (13.2 mg, 98%): LD-MS: obs. 487.4 (M À Cu)+,
548.8 (M+); FAB-MS: obs. 489.2277, calc. 489.2285 [(M À
A solution of P2-(PO4Bn)2 (23.9 mg, 0.0237 mmol) in
CHCl3–THF (3 mL, 2 : 1) was treated with Cu(OAc)2 (42.9
mg, 0.237 mmol, 10 equiv). The mixture was stirred at room
temperature for 12 h. The reaction mixture was diluted with
CH2Cl2 and water. The phases were separated, and the aqu-
eous layer was extracted with CH2Cl2. The organic layer was
dried (Na2SO4) and concentrated. Chromatography [silica,
CH2Cl2–ethyl acetate (4 : 1)] yielded an orange solid (20.0
mg, 79%): LD-MS: obs. 1069.7; ESI-MS: obs. 1070.26165,
calc. 1070.26252 [(M + H)+, M = C59H52CuN4O8P2]; labs
404, 529 nm.
Cu)+ M = C31H28CuN4O2]; labs 403, 530 nm; HPLC tR
=
30.53 (P2-(OH)2), 35.76 (CuP2-(OH)2), 32.56 (minor), 36.75
(minor) min.
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Cu(II)-5-[1,5-Bis(dimethoxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (CuP2-(PO4Me)2)
5-[1,5-Bis(dihydroxyphosphoryloxy)pent-3-yl]-15-phenyl-10,20-
diiodoporphyrin (P2I2-(PO4)2)
A solution of P2-(OH)2 (9.80 mg, 0.0182 mmol) in dry CH2Cl2
(500 mL) under argon was treated with DMAP (44.4 mg, 0.364
mmol, 10 equiv). The mixture was stirred at room temperature
for 10 min, after which dimethyl chlorophosphate (40 mL, 0.37
mmol) was added dropwise. The mixture was stirred at room
temperature for 12 h. The mixture was loaded onto a silica
column. Chromatography [CH2Cl2–MeOH (0 - 3%)] yielded
a bright orange solid (5.4 mg, 35%): LD-MS: obs. 768.8
(CuP2-(PO4Me)2+), 708.5 [P2-(PO4Me)2+], 552.3 [CuP2-
(OH)2+], 525.2 (unassigned), 497.0 (unassigned), 488.4
[P2-(OH)2+]; FAB-MS: obs. 765.1310, calc. 765.1304
(C35H36CuN4O8P2); labs 404, 529 nm.
A solution of ZnP2I2-(PO4Bn)2 (30.4 mg, 0.0230 mmol) in
TFA (1.90 mL) was treated with thiophenol (0.10 mL) at room
temperature for 2 h. The solution was neutralized with con-
centrated aqueous NaOH, and applied onto a reversed-phase
silica column equilibrated with H2O. Elution with
H2O–MeOH (0 - 50%) yielded a dark green solution that
was concentrated in vacuo. The dark purple residue was
dissolved in distilled water. The resulting solution was filtered
through a plug of cotton wool. The filtrate was freeze-dried to
yield a dark green film that proved too insoluble in either
aqueous or organic solvents for sufficient characterization
(13.4 mg, 65%): labs (MeOH) 423, 525, 564, nm; tR = 34.59
min, tcol = 1.09 min.
Cu(II)-5-[1,5-Bis(dihydroxyphosphoryloxy)pent-3-yl]-15-
phenylporphyrin (CuP2-(PO4)2)
Acknowledgements
A solution of CuP2-(PO4Bn)2 (9.6 mg, 0.0090 mmol) in
CHCl3–amylenes (500 mL) was treated with TMS-Br (30 mL)
at room temperature for 30 min. MeOH (1 mL) was added,
and stirring was continued for 20 min. The sample was
concentrated. The solid residue was dissolved in dilute
aqueous NaHCO3, and the resulting solution was applied
onto a reversed-phase silica column equilibrated with H2O.
Elution with H2O–MeOH (0 - 50%) yielded a dark orange
solution that was concentrated in vacuo. The resulting dark
orange residue was dissolved in distilled water, and the result-
ing solution was filtered through a plug of cotton wool. The
filtrate was freeze-dried to yield a dark red voluminous solid
(quantitative): labs (H2O) 400, 530; HPLC tR = 26.56 min.
This work was supported by the NIH (GM36238) and by
Oncologic, Inc. Mass spectra were obtained at the Mass
Spectrometry Laboratory for Biotechnology at North Caroli-
na State University. Partial funding for the facility was
obtained from the North Carolina Biotechnology Center and
the NSF. We also thank the Oncologic team of Dr Sam Rose
(deceased), Dr Henry Rapaport (deceased), Dr Winston
Salser, Dr George Mayers, and Dr Chris Knudsen for
stimulating discussions.
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