Hydrophilic bioconjugatable trans-AB-porphyrins and peptide conjugates

Article abstract of DOI:10.1142/S1088424615500121

Porphyrins bearing a single bioconjugatable group and a single water-solubilization motif in a trans-AB-architecture (with no other substituents) provide a compact design of value for studies in diverse disciplines. Established synthetic methods have been

Full text of DOI:10.1142/S1088424615500121

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2015; 19: 1–16
DOI: 10.1142/S1088424615500121
Published at http://www.worldscinet.com/jpp/
Hydrophilic bioconjugatable trans-AB-porphyrins
and peptide conjugates
Tuba Sahin, Pothiappan Vairaprakash, K. Eszter Borbas,
Thiagarajan Balasubramanian and Jonathan S. Lindsey*◊
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA
Received 29 October 2014
Accepted 28 November 2014
ABSTRACT: Porphyrins bearing a single bioconjugatable group and a single water-solubilization
motif in a trans-AB-architecture (with no other substituents) provide a compact design of value for
studies in diverse disciplines. Established synthetic methods have been employed to prepare four new
free base porphyrins and one Mn(III) chelate. The hydrophilic motif includes 4-N-methylpyridinium,
2,4,6-tris(carboxymethoxy)phenyl,2,6-bis(phosphonomethoxy)phenyl,andcarboxy;thebioconjugatable
unit includes carboxy, maleimido, and N-hydroxysuccinimido (NHS) ester. Bioconjugation experiments
with a protected porphyrin-diphosphate or unprotected porphyrin-diphosphonate were examined in
organic solution or water, respectively. Both approaches were employed to conjugate to the e-amino
group of Lys11 inAcKPV-NH2, a tripeptide fragment [Ac-a-MSH(11-13)-NH2] of melanocyte stimulating
hormone, yielding porphyrin-peptide conjugates.
KEYWORDS: water-soluble, manganese, dipyrromethane, molecular design, facial encumbrance.
set of porphyrins [2]. The Gunter and Mander approach
INTRODUCTION
thus represented a significant advance in liberating the
field from the tyranny of statistical reactions. On the other
hand, the advance was beset with two limitations: first,
the presence of two b-methyl groups flanking each of
the meso-aryl groups results in steric deformation of the
porphyrin macrocycle [3]. Second, access to a trans-AB-
porphyrin (with or without flanking b-alkyl groups) was
only achievable by statistical formation of a mixture of
the trans-A2-porphyrin, trans-AB-porphyrin, and trans-
B2-porphyrin (e.g. by reaction of two dipyrromethanes
and trimethyl orthoformate [4]).
Methodology for the rational synthesis of trans-A2-
porphyrins and trans-AB-porphyrins that lack flanking
b-alkyl groups was developed over the course of a
generationfollowingtheworkofGunterandMander[5–9].
One route is shown in Scheme 1. A dipyrromethane [10]
is subjected to Vilsmeier formylation and then imination
with an alkylamine (e.g. propylamine); subsequent
condensation of the bis(imino)dipyrromethane with a
dipyrromethane in the presence of zinc acetate gives the
trans-AB-porphyrin [7]. Other synthetic routes also can
be employed [8, 9].
The synthesis of trans-AB-porphyrins has distant
origins in the seminal work of Gunter and Mander, who
reacted o-nitrobenzaldehyde with 2,3,7,8-tetramethyl-
dipyrromethane in a two-step one-flask procedure of acid
catalysis followed by oxidation [1]. The resulting trans-
A2-porphyrinbearseightb-methylgroupsandtwolinearly
disposed meso-aryl groups, with two meso-positions
unsubstituted (Scheme 1). The only other approach at
that time for gaining access to such trans-disubstituted
porphyrins relied on reaction of two aryl aldehydes with
pyrrole, thereby forming a statistical distribution of
meso-tetraarylporphyrins with six types of substitution
patterns: A4, A3B, cis-A2B2, trans-A2B2, AB3 and B4. The
chromatographic separation of the mixture (particularly
to isolate the cis-A2B2 and trans-A2B2 isomers in pure
form) is often difficult, and the trans-A2B2-porphyrin
constitutes only 12.5% (on a statistical basis) of the total
◊ SPP full member in good standing
*Correspondence to: Jonathan S. Lindsey, email: jlindsey@
ncsu.edu, fax: +1 919-513-2830
Copyright © 2015 World Scientific Publishing Company

2
T. SAHIN ET AL.
Gunter-Mander route:
A
(1) p-TsOH·H2O
MeOH
NH HN
NH
N
rt, 6 h;
4 °C 16 h
N
HN
(2) DDQ, THF, rt
CHO
A
A
imine route:
A
NH HN
A
H
H
N
N
R
R
N
N
N
N
Zn(OAc)2
Zn
EtOH, reflux
R = propyl
NH HN
B
B
Scheme 1. Routes to trans-substituted porphyrins
The imine route has been exploited to gain access to
a variety of trans-AB-porphyrins [11–15]. trans-AB-
porphyrins that bear a hydrophilic substituent and a bio-
conjugatable group are particularly attractive given the
sparsely substituted molecular design, which enables
the porphyrin chromophore to be utilized in diverse
applications wherein a compact architecture is desirable.
The applications range from medicine (e.g. photodynamic
therapy, free radical scavenging) to clinical diagnostics
(e.g. imaging, flow cytometry) to energy sciences (e.g.
light-harvesting, charge separation). A common theme in
these diverse studies is the ability to (1) attach the porphyrin
to a particle, macromolecule or surface, and (2) achieve
a resulting conjugate that does not aggregate in aqueous
solution, either with itself or with other entities in the
sample (e.g. non-specific binding with proteins or cells).
Motifs for bioconjugation can be incorporated with
tetrapyrrolemacrocyclesinafairlystraightforwardmanner
[16]. On the other hand, satisfactory approaches for water-
solubilization of the intrinsically hydrophobic, disk-
like tetrapyrrole macrocycle are less developed despite
extensive work [17, 18]. Most such work concerns meso-
tetrasubstituted porphyrins rather than the more compact
disubstituted porphyrins bearing a single hydrophilic
motif. Motifs that have been employed with trans-AB-
porphyrins are shown in Chart 1. N-pyridinium moieties
(P1a, P1b) and the p-(N,N,N-trimethylammonium)-
phenyl motif (P1c) place the charged moiety at sites
removed from the porphyrin macrocycle (such porphyrins
were prepared via statistical methods [4]). By contrast,
the 2,4,6-tris(carboxymethoxy)phenyl unit (P2) [15]
positions ionizable functional groups above and below
the face (as well as at the perimeter) of the macrocycle, as
Chart 1. Prior relevant trans-AB-porphyrins
do the swallowtail motifs bearing phosphate (P3) [11] or
phosphonate (P4) [11, 14] termini.
The groups distal to the hydrophilic motif include those
often used for bioconjugation (amino, a-iodoacetamido,
isothiocyanato, carboxy) and groups used rarely if at all
(formyl, bromo). Other notable examples of trans-AB-
porphyrins, while not containing both a bioconjugatable
group and a hydrophilic group, include the following
substituent pairs: p-decyloxyphenyl/PEG-dendrimer [19];
p-aminophenyl/formyl [20]; protected imidazole/ester
[12]; hydroxymethyl/ethyne [21]; and hydroxymethyl/
ethoxycarbonyl [21].
In this paper, we present the design and synthesis of
a set of trans-AB-porphyrins that build from the prior
examples yet are more versatile in the nature of both the
bioconjugatable handle and the water-solubilization motif
(Chart 2). The bioconjugatable handles include the NHS
ester (e.g. for amidation with a protein amine) and the
maleimide group (for Michael addition with a cysteinyl
thiol), which are common motifs for bioconjugation
[22]. The hydrophilic substituents include carboxylates,
phosphonates, and quaternized pyridinium moieties.
Three examples of bioconjugation also are reported.
Copyright © 2015 World Scientific Publishing Company
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
3
HO2C
CO2H
TMS
O
O
O
O
O
NH
N
N
CO2H
O
O
O
N
O
O
HN
O
O
DCC, DMAP
86%
CHO
+
A
HO2C
P5
TMS
CHO
HO
O
O
O
NH
N
N
I
Scheme 2. Synthesis of an aryl aldehyde
N
O
N
HN
O
P6
NH
NH
O
acid
O
+
R
N
H
N
N
N
N
R
H
N
M
CO2H
NH
O
Yield, %
R
Cmpd
Acid
O
P7: M = H, H
MnP7: M = ClMn(III)
TMSCH2CH2O2C
O
InCl3
33 (here)
B
OH
HO
P
O
t-BuO2C
O
O
O
O
NH
N
C
35 (ref 15)
InCl3
O
t-BuO2C
HO
O
N
HN
t-BuO2C
N
O
HO
P
O
P8
D
E
heat
TFA
58 (ref 26)
74 (ref 27)
OH
Chart 2. Target trans-AB-porphyrins
O2N
RESULTS AND DISCUSSION
TFA
CH2Cl2
t-BuO2C
18 (ref 21)
F
TFA
CH2Cl2
G
35 (ref 28)
EtO2C
Synthesis of porphyrins
The overall synthesis proceeds along the series
aldehydesàdipyrromethanesàa-disubstituteddipyrro-
methanes à porphyrins à derivatized porphyrins. All
but one of the aldehydes required herein have been made
previously or are commercially available. The reaction
of (4-formylphenoxy)acetic acid [23] and (2-trime-
thylsilyl)ethanol was carried out in the presence of
N,N-dicylohexylcarbodiimide (DCC) [24] and 4-N,N-
dimethylaminopyridine (DMAP) in 86% yield to give
aryl aldehyde A (Scheme 2).
InCl3
O
H
88 (ref 11)
t-BuO2C
OEt
O
P
OEt
O
InCl3
88 (ref 29)
I
O
Meso-substituted dipyrromethanes are readily synthe-
sized in a solventless process from the corresponding
aldehyde in a solution of excess pyrrole containing an acid
catalyst (Scheme 3) [25]. Thus, reaction of aldehyde A
OEt
P
O
OEt
Scheme 3. Synthesis of aryl aldehydes
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 3–16

4
T. SAHIN ET AL.
R
The porphyrins were subjected to functional group
transformations as required to prepare the target
compounds. The transformations are as follows:
NH HN
ZnP5-1 was treated with tetrabutylammonium
fluoride (TBAF) to remove the trimethylsilylethyl
protecting group (giving ZnP5-2), whereupon the
unmasked carboxylic acid was reacted with N,N′-
disuccinimidyl carbonate [30] to install the activated
NHS bioconjugatable handle, giving ZnP5-3. The latter
was treated with trifluoroacetic acid (TFA) to both cleave
the tert-butyl groups and demetalate the porphyrin while
leaving the NHS ester unaffected (Scheme 6).
ZnP6-1 was treated with CH3I at 60°C to quaternize
the pyridine unit. The crude product was treated with
TFA to remove the trimethylsilylethyl group and the
central zinc, affording the porphyrin P6-2 in 94% yield.
The latter was reacted with N-hydroxysuccinimide in
the presence of (3-dimethylaminopropyl)carbodiimide
(EDC) to afford the target porphyrin in 79% yield
(Scheme 7).
POCl3/DMF
R
NH HN
H
H
O
O
Yield, %
91
Cmpd
R
TMSCH2CH2O2C
O
B-(CHO)2
E-(CHO)2
53
O2N
Manganese porphyrins exhibit multiple stable redox
states [31]. The electrochemical properties of manganese
hematoporphyrin IX in water have been characterized for
the 2+, 3+ and 4+ oxidation states [32–34]. Manganese
porphyrins have been examined as superoxide dismutase
mimics [28, 35, 36], catalase mimics [37], oxygen-atom
transfer catalysts [38], magnetic resonance imaging
contrast agents [19], and in studies of electron transfer
across lipid bilayers [39–41].
O
O
O
H-(CHO)2
45
Scheme 4. Diformylation of dipyrromethanes
with pyrrole in the presence of InCl3 gave dipyrromethane
B in 33% yield. Dipyrromethanes C [15], D [26], E [27],
F [21], G [28], H [11] and I [29] were synthesized as
described in the literature. Dipyrromethane D was
prepared without added acid, whereas F was prepared
from an ester precursor.
The imine route was chosen for the synthesis of the
trans-AB-porphyrins given the presence of aryl groups
at both of the meso-positions. To prepare the requisite
To prepare a bioconjugatable manganese porphyrin,
ZnP7-1a or ZnP7-1b was treated with Zn/NH4Cl [42]
to reduce the nitro group (Scheme 8). In each case, the
crude product was treated with 3N HCl to give the free
base porphyrin P7-2a or P7-2b, respectively. Other
reagent systems such as Sn/HCl or Pd/C/ammonium
formate did not result in the amino product but led to
decomposition of the starting material. For the porphyrin
ethyl ester (P7-2b), saponification of the ester with
NaOH in a mixed solvent (H2O/MeOH/THF) afforded
putative P7-3b, but attempts to couple the latter with
4-maleimidobutyric acid failed to give the expected
product. On the other hand, the porphyrin tert-butyl ester
(P7-2a) was coupled with 4-maleimidobutyric acid in the
presence of DCC to install the bioconjugatable handle,
affording P7-3a in 52% yield. The tert-butyl group
was then removed with 40% TFA/CH2Cl2 to unmask
the hydrophilic substituent of the target porphyrin,
affording P7 in 80% yield. For the synthesis of the
manganese porphyrin (MnP7), first metalation [36] was
performed with P7-3a using MnCl2·4H2O and a hindered
pyridine base (here 2,6-di-tert-butylpyridine instead of
2,6-dimethylpyridine) in CHCl3/MeOH at 50°C to give
MnP7-3a in 62% yield. The metalation begins with
the Mn(II) reagent but affords the Mn(III) chelate [43].
The apical counterion is assumed to be chloride. The
protected manganese porphyrin was treated with 20%
bis(imino)dipyrromethanes, dipyrromethanes
B and
E were formylated under Vilsmeier conditions to give
B-(CHO)2 and E-(CHO)2, respectively (Scheme 4).
Diformyldipyrromethane H-(CHO)2 has been prepared
previously [11].
Formation of zinc porphyrins starts with conver-
sion of a dipyrromethane-1,9-dicarboxaldehyde to the
corresponding bis(imine) as shown in Scheme 5. A
dipyrromethane is treated with excess alkylamine (e.g.
propylamine or butylamine) to give the corresponding
bis(imine) [7], which without further purification is
reacted with the appropriate choice of dipyrromethane
in the presence of Zn(OAc)2 and air to give the
zinc(II) trans-AB-porphyrin. Thus, B-(CHO)2 was
treated with propylamine to give the corresponding
bis(imine) B-(imine)2, and the latter was reacted with
dipyrromethane C to give ZnP5-1 (the first porphyrin
precursor to the target P5) in 25% yield. In a similar
manner, four other porphyrins were prepared in yields
ranging from 8–32%.
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 4–16

HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
5
H
H
H
HN
HN
R2
R
R
O
O
N
NH
NH
NH
NH
N
N
N
N
R1
R1
R1
Zn
R2
RNH2
Zn(OAc)2
refluxing ethanol
or toluene
N
H
R1
R2
Reactants
R
Yield, %
Porphyrin
CO2t-Bu
O
O
TMSCH2CH2O2C
TMSCH2CH2O2C
O
O
ZnP5-1
B-(imine)2 + C
Pr
25
CO2t-Bu
CO2t-Bu
ZnP6-1
B-(imine)2 + D
E-(imine)2 + F
Pr
O
N
8
CO2t-Bu
32
Bu
ZnP7-1a
O2N
CO2Et
26
E-(imine)2 + G
Bu
ZnP7-1b
O2N
OEt
EtO P
O
O
O
19
ZnP8-1
H-(imine)2 + I
Pr
t-BuO2C
O
EtO P
EtO
O
Scheme 5. Porphyrin formation
TFA/CH2Cl2 to remove the tert-butyl group affording
MnP7 in 87% yield.
such a band is missing in the spectrum of MnP7.
Fluorescence emission also was absent, as expected
for the Mn(III)porphyrin. The MALDI-MS spectrum
showed the expected molecular ion upon loss of the
anionic apical ligand. The manganese chelates were
not characterized by NMR spectroscopy given their
paramagnetic character.
Treatment of ZnP8-1 with p-TsOH·H2O in CH2Cl2 at
room temperature [13] resulted in the free base porphyrin
P8-1 in 90% yield. The tert-butyl ester remained intact
under these conditions. Porphyrin P8-1 was treated with
excess KOH in aqueous THF at room temperature for 24 h
(Scheme 9). The resulting porphyrin salt was liberated
The manganese porphyrin MnP7 was characterized
by absorption and fluorescence spectroscopy (in aqueous
solution) and by mass spectrometry (MALDI-MS,
ESI-MS). The absorption spectrum of MnP7 in
aqueous phosphate buffer at neutral pH (Fig. 1) shows
the characteristic bands [31] of a Mn(III)porphyrin.
The characteristic weak absorption feature at 765 nm
also was observed (inset in Fig. 1). A nearly identical
spectrum was observed in DMF at room temperature.
The free base porphyrin (P7-3a or P7) exhibits a
characteristically strong Soret band at 407 nm (toluene);
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T. SAHIN ET AL.
ZnP5-1
quantitative
ZnP7-1a or ZnP7-1b
TBAF
(1) Zn/NH4Cl/EtOH/H2O
60% (2) 3N HCl
(3) satd aq NaHCO3
t-BuO2C
O
N
N
N
N
HO2C
O
CO2t-Bu
NH
N
N
Zn
O
CO2R
O
HN
H2N
ZnP5-2
t-BuO2C
P7-2b (R = Et), 61%
THF
MeOH
H2O
P7-2a (R = tert-Bu), 60%
O
O
N
O
P7-2a only
DCC/CH2Cl2
quantitative
N
O
O
NaOH
O
O
O
52% HO
N
P7-3b (R = H)
O
t-BuO2C
O
O
O
O
O
N
N
N
N
O
N
CO2t-Bu
TFA
NH
N
N
CH2Cl2
N
O
O
Zn
CO2R
80%
O
O
HN
NH
O
O
P7
(R = H)
ZnP5-3
t-BuO2C
P7-3a (R = tert-Bu)
quantitative TFA
MnCl2·4H2O
CHCl3/MeOH, 50 °C
P5
62%
Scheme 6. An NHS ester-porphyrin-tricarboxylate
N
ZnP6-1
O
N
Cl
N
N
N
N
O
O
(1) CH3I, DMF, 60 °C
94%
Mn
(2) TFA, CH2Cl2
NH
O
O
NH
N
N
I
HO2C
MnP7-3a
87% TFA, CH2Cl2
O
N
HN
O
N
P6-2
Cl
N
N
N
N
O
Mn
CO2H
79%
EDC,
N
OH
NH
O
O
O
MnP7
P6
Scheme 8. A maleimido-porphyrin-carboxylate
Scheme 7. An NHS ester-porphyrin-pyridinium
Bioconjugation
with a small amount of acetic acid, whereupon the
porphyrin was treated with excess trimethylsilyl bromide
(TMSBr) in CH2Cl2 [29] at room temperature for 4 h.
Quenching with aqueous methanol, concentration and
trituration with CH2Cl2 afforded the desired porphyrin
P8 as a green solid in quantitative yield.
Bioconjugation of porphyrins can be carried out
in aqueous or organic solution [4, 16, 44, 45]. To gain
information about handling of porphyrin-diphosphates
in bioconjugation processes, the tetramethyl-protected
porphyrin-diphosphate P3a-1 [11] was reacted with
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
7
O
O
O
O
O
O
P OMe
NH
N
N
OMe
N
O
OMe
HN
O
P
OMe
O
P3a-2
(1) EDC, DMF, 10 min, rt
(2) N-hydroxysuccinimide, 24 h, rt
O
O
O
O
P OMe
NH
N
N
OMe
OMe
HO
O
HN
P
OMe
O
P3a-1
Fig. 1. Absorption spectrum of MnP7 in aqueous phosphate
buffer (pH 7) at room temperature. The inset shows the weak
absorption at long wavelength
D-PheOEt, EDC/HOBt, DMF, rt
O
OEt
O
O
P OMe
NH
N
N
EtO
P
O
OMe
OMe
O
O
HN
O
O
O
O
P
OMe
N
N
N
N
H
N
O
O
M
O
O
D-Phe-P3a-1
Et
EtO
ZnP8-1: M = Zn
P
O
TMSBr, CHCl3, rt, 3 h
OEt
O
90%
p-TsOH·H2O, CH2Cl2, rt, 2 h
O
O
P
OH
OH
NH
N
N
OH
P8-1: M = H,H
(1) 2 M KOH, THF/H2O, rt, 24 h
O
O
OH
P
HN
O
(2) AcOH
(3) TMSBr, CH2Cl2, rt, 4 h
H
O
N
H
O
P8
D-Phe-P3a
Scheme 9. A carboxylic acid-porphyrin-diphosphonate
Scheme 10. Phe-porphyrin-diphosphate: bioconjugation in DMF
reverse-phase column chromatography yielded D-Phe-
P3a as a purple solid. The sample was homogeneous by
reverse-phase HPLC but ESI-MS showed peaks attributed
to monomethyl and dimethyl/ethyl esters. Increasing
the reaction time or the amount of TMSBr resulted in
decomposition of the porphyrin moiety.
D-PheOEt in DMF containing the coupling reagents
EDC and 1-hydroxybenzotriazole (HOBt) (Scheme 10).
The product D-Phe-P3a-1 was isolated after conventional
aqueous-organic workup and silica column chromato-
graphy. The use of CHCl3 containing TMSBr [46] gave
the best results to afford the target porphyrin D-Phe-P3a.
ESI-MS analysis indicated that the starting porphyrin
(m/z = 953.1) disappeared after ~3 h. The major products
were the fully deprotected D-Phe-P3a (m/z = 868.0) and
a compound with a mass spectral signal at 28 mass units
higher. The latter could arise from either a dimethyl-
protected molecule with the cleaved ethyl moiety, or,
more likely, the free diphosphate with an uncleaved
phenylalanine ethyl ester. Concentration of the sample,
followed by neutralization with dilute aqueous NaOH and
Porphyrin P3a-1 also was converted to the
corresponding NHS ester (P3a-2) by treatment with EDC
and N-hydroxysuccinimide. The NHS ester was isolated
along with the free carboxylic acid in a 4:1 ratio. The
1
mixture was characterized by H NMR spectroscopy
and mass spectrometry and used as such in conjugation
reactions.
Anextobjectivewastoconjugateporphyrin-diphosphate
P3a-1toAc-KPV-NH2,thetripeptidecarboxamide-terminal
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T. SAHIN ET AL.
segment of a-melanocyte stimulating hormone (a-MSH)
[47, 48]. a-MSH is a tridecapeptide that is produced by a
wide variety of mammalian cell types, is widely distributed
in the central nervous system, exhibits enhanced uptake by
melanoma cells, and exhibits immunomodulatory effects,
particularly anti-inflammatory activity [49–51]. Extensive
studies of truncated segments of a-MSH have been carried
out [52]. One finding is that Ac-KPV-NH2 also exhibits
anti-inflammatory activity; moreover, within this peptide,
L-Pro12 is essential for such activity whereas L-Lys11 can
be replaced with D-Lys11 without loss of activity [48]. The
sole site of nucleophilicity is the lysine e-NH2 group (given
the protected C- and N-termini), which was expected to
result in selective reaction with the carboxylic acid in
the porphyrin-diphosphate P3a-1. Derivatives of a-MSH
itself have been prepared; representative examples include
those that contain an N-terminal fatty acid (for receptor
localization studies) [51] or a metal chelating ligand (for
melanoma uptake studies) [49, 53]. In studies of the latter,
amidation at the e-amino group of Lys11 dramatically
reduced kidney uptake and thereby afforded increased
retention for radiolabeling studies of melanoma uptake.
Accordingly, an analogous porphyrin-containing construct
might prove viable for biomedical imaging studies to
identify likely sites of uptake or localization of a-MSH
including for melanoma diagnoses.
Attempts to react the NHS ester-porphyrin P3a-2 with
Ac-KPV-NH2·HCl were unsuccessful, as only starting
materials were observed by ESI-MS even after 24 h under
rigorously anhydrous conditions. Therefore, the conjug-
ation was carried out in the same manner as for D-Phe-
P3a-1, with in situ activation of the carboxy-porphyrin
P3a-1 by use of EDC/HOBt in DMF (Scheme 11).
N,N-Diisopropylethylamine (DIEA) was included to
liberate the peptide from its HCl-salt. The product MSH-
P3a-1 was isolated after silica column chromatography
in good yield as a purple solid. Porphyrin MSH-P3a-1
exhibited the molecular ion peak upon mass spectral
analysis. No unconjugated porphyrin or tripeptide was
observed. Upon examination by H NMR spectroscopy,
1
the peptidyl NH-protons, the characteristic amino acid
CH-resonances, and the porphyrin aromatic protons
were readily identified by gCOSY experiments. The
integrations of the signals were of the expected ratios.
1
On the other hand, as expected, the H NMR spectrum
was rather complicated in the aliphatic region and
unambiguous assignment of the signals in this region was
not possible.
1
The complexity of the H NMR spectrum of MSH-
P3a-1 was expected given our prior studies of swallowtail
porphyrins [11, 13, 14, 54]. The following structural
features are relevant: (1) the C1′H is more or less in the
plane of the porphyrin macrocycle, which results in two
sets of b-protons and two different meso-proton signals,
and (2) the swallowtail alkyl groups (which preferentially
occupy positions out-of-plane with the porphyrin ring)
are hindered in their rotation around the carbon–carbon
single bond between the porphyrin meso-position and the
swallowtail branching site (i.e. C5–C1′H).
Removal of the protecting groups of MSH-P3a-1
was achieved with TMSBr in CHCl3. Purification by
reverse-phase column chromatography yielded a purple
solid, which upon ESI-MS analysis (m/z = 1088, [M +
H]+) proved to be consistent with the desired deprotected
peptide-porphyrin-diphosphate conjugate (MSH-P3a),
yet also containing a trace of monomethyl-MSH-P3a.
Both porphyrin conjugates D-Phe-P3a and MSH-P3a
displayed excellent solubility in water.
The initial porphyrin conjugates D-Phe-P3a-1 and
MSH-P3a-1 were prepared with the phosphate moiety
in protected form, leaving the unveiling of the water-
solubilization motif to thelast step of the overallsynthesis.
For conjugation to water-soluble macromolecules,
however, this approach is not feasible. Therefore, the
water-soluble porphyrin-diphosphonate P4a was reacted
with Ac-KPV-NH2·HCl in the presence of EDC/HOBt
NH2
O
H
N
P3a-1
DIEA
O
EDC
N
HOBt
O
+
O
H
DMF, rt
O
O
P
OX
OX
O
NH
N
N
H
N
OX
O
N
O
O
O
NH2
O
H
N
O
N
HN
P
H
N
OX
O
MSH-P3a-1: X = CH3
TMSBr, CHCl3, rt, 3 h
MSH-P3a: X = H
Scheme 11. Synthesis of a peptide-porphyrin-diphosphate: bioconjugation in DMF
Ac-Lys-Pro-Val-NH2·HCl
NH3Cl
Copyright © 2015 World Scientific Publishing Company
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
9
P-4a
OH
OH
O
NH2
O
P
P
H
NH
N
N
O
N
HN
O
OH
OH
N
OH
DIEA
EDC
HOBt
OH
O
O
O
O
H
O
P
P
NH
N
N
+
H
OH
OH
N
H2O, rt
O
N
O
O
O
HN
O
NH2
OH
H
N
O
N
H
MSH-P4a
N
O
Ac-Lys-Pro-Val-NH2·HCl
NH3Cl
Scheme 12. A peptide-porphyrin-diphosphonate: bioconjugation in water
in water containing DIEA as shown in Scheme 12.
(Porphyrin-diphosphonate P4a is highly water-soluble,
given that 8–10 mg completely dissolved in 500 mL
of distilled water, consistent with a concentration of
≥ 20 mM.) The reaction was monitored by ESI-MS:
the peak corresponding to the protonated porphyrin
(m/z = 691) was very weak after 24 h, while that of the
desired peptide-porphyrin conjugate (m/z = 1056.4) was
observed. The sample was then diluted with a small
volume of water and purified on a short reverse-phase
silica column. The peptide-porphyrin-diphosphonate
conjugate (MSH-P4a) was isolated as a deep purple
solid. The conjugate was found to be homogeneous upon
analysis by RP-HPLC, and upon ESI-MS analysis gave
the protonated molecular ion (m/z = 1056.3) and a doubly
charged cationized molecular ion [m/z = 539.3, (M + H
+ Na)2+] in the positive ion mode, vs. the deprotonated
molecular ion (m/z = 1054.3) in the negative ion mode.
MALDI-MS and electrospray ionization mass spectro-
metry (ESI-MS) data are reported for the molecular
ion or cationized molecular ion unless noted otherwise.
Noncommercial compounds (4-formylphenoxy)acetic
acid [23], C [15], D [26], E [27], F [21], G [28], H [11],
I [29], P3a-1 [11], and P4a [11] were obtained following
literature procedures. The tripeptide Ac-KPV-NH2·HCl
was obtained from Bachem California, Inc. The coupling
reagent(3-dimethylaminopropyl)carbodiimide(EDC)was
employed as the hydrochloride salt. All other compounds
were used as received from commercial sources.
Chromatography
Silica gel (40 mm average particle size) was used
for column chromatography. Preparative size-exclusion
chromatography (SEC) was performed using BioRad
Bio-Beads SX-1 (200–400 mesh) beads. Reverse-phase
preparative column chromatography was carried out
using C-18-coated silica and eluants based on water
admixed with methanol. Analytical RP-HPLC was
carried out using an ODS C-18 column (5 mm, 125 mm ×
4 mm); flow rate = 1 mL/min; detection at 254, 410 and
417 nm; and the following elution program with solvents
A (water containing 0.1% TFA) and B (acetonitrile
containing 0.1% TFA): 0-2 min, 0% B; 2-20 min, 90%
B; 20-23 min, 90% B.
EXPERIMENTAL
General methods
1HNMR(300or400MHz)or13CNMR(75or100MHz)
spectroscopy was performed at room temperature in
CDCl3 unless noted otherwise. All solvents were reagent
grade and were used as received unless noted otherwise.
THF was freshly distilled from sodium/benzophenone
ketyl. Anhydrous CH2Cl2 was used as received. Laser-
desorption mass spectrometry (LD-MS) was performed in
the absence of a matrix. Matrix-assisted laser-desorption
mass spectrometry (MALDI-MS) was performed with the
matrix 1,4-bis(5-phenyl-2-oxaxol-2-yl)benzene (POPOP)
[55] or a-cyano-4-hydroxycinnamic acid. LD-MS,
Synthesis of porphyrin precursors
4-[2-(Trimethylsilyl)ethoxycarbonylmethoxy]
benzaldehyde (A). Following a reported procedure
[24], a solution of (4-formylphenoxy)acetic acid (1.00 g,
5.55 mmol) in DMF/CH2Cl2 (30 mL, 1:1) was treated
with 2-(trimethylsilyl)ethanol (1.19 mL, 8.33 mmol)
and DMAP (0.135 g, 1.11 mmol), and the mixture was
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 9–16

10
T. SAHIN ET AL.
cooled to 0°C. DCC (1.26 g, 6.11 mmol) was added, and
the mixture was stirred and allowed to warm overnight
to room temperature. The precipitate was filtered. The
filtrate was concentrated and chromatographed (silica,
1,9-Diformyl-5-(3-nitrophenyl)dipyrromethane
(E-(CHO)2). The Vilsmeier reagent was prepared by
treatment of dry DMF (20.0 mL) with POCl3 (3.73 mL,
40.0 mmol) at 0°C and stirring of the resulting mixture for
10 min. In a separate flask, a solution of dipyrromethane E
(4.01 g, 15.0 mmol) in DMF (75 mL) was treated with the
freshly prepared Vilsmeier reagent at 0°C. The resulting
mixture was stirred at 0°C for 15 min and then for 1 h at
room temperature. The reaction mixture was diluted with
CH2Cl2 (200 mL) and then treated with saturated aqueous
sodium acetate. After stirring for 1 h, the aqueous phase
was separated and extracted with CH2Cl2 (3 × 50 mL).
The combined organic extract was washed (water), dried
(Na2SO4), concentrated and chromatographed [silica,
CH2Cl2/ethyl acetate (19:1)] to afford a pale yellow solid
1
CH2Cl2) to afford a yellow oil (1.34 g, 86%). H NMR
(300 MHz, CDCl3): d, ppm 0.05 (s, 9H), 0.99–1.06 (m,
2H), 4.29–4.34 (m, 2H), 4.69 (s, 2H), 7.01 (d, J = 8.4 Hz,
2H), 7.85 (d, J = 8.7 Hz, 2H), 9.90 (s, 1H). 13C NMR
(100 MHz, CDCl3): d, ppm -1.4, 17.4, 64.3, 65.5, 115.0,
130.8, 132.1, 162.8, 168.3, 190.9. ESI-MS: m/z obsd.
281.1210; calcd. 281.1204 ([M + H]+, M = C14H20O4Si).
5-[4-(2-(Trimethylsilyl)ethoxycarbonylmethoxy)
phenyl]dipyrromethane (B). Following a reported
procedure [25], a solution of aldehyde A (2.44 g, 8.70
mmol) in pyrrole (60 mL, 870 mmol) was degassed with
a stream of argon for 10 min. InCl3 (192 mg, 0.868 mmol)
was added, and the mixture was stirred under argon
at room temperature for 1.5 h. NaOH (1 g, powdered)
was added to quench the reaction, and the mixture was
stirred for 45 min. The mixture was filtered. The filtrate
was concentrated and chromatographed [silica, hexanes/
CH2Cl2/ethyl acetate (7:2:1)] to afford a yellow viscous
1
(2.57 g, 53%): mp 189–190°C. H NMR (300 MHz,
CDCl3/CD3OD): d, ppm 5.73 (s, 1H), 6.08 (d, J = 3.9 Hz,
2H), 6.96 (d, J = 3.9 Hz, 2H), 7.53–7.62 (m, 2H), 8.11
(brs, 1H), 8.17 (dt, J1 = 3.9 Hz, J2 = 1.8 Hz, 1H), 9.35
(s, 2H). 13C NMR (100 MHz, CDCl3/CD3OD): d, ppm
43.6, 111.2, 122.7, 123.3, 129.9, 132.9, 134.5, 140.1,
141.9, 148.5, 179.6. ESI-MS: m/z obsd. 324.0938, calcd.
324.0979 ([M + H]+, M = C17H13N3O4).
1
oil (1.13 g, 33%). H NMR (400 MHz, CDCl3): d, ppm
0.09 (s, 9H), 1.05–1.09 (m, 2H), 4.31–4.35 (m, 2H), 4.58
(s, 2H), 5.39 (s, 1H), 5.89–5.90 (m, 2H), 6.15–6.17 (m,
2H), 6.65–6.66 (m, 2H), 6.84 (d, J = 8.4 Hz, 2H), 7.12
(d, J = 8.8 Hz, 2H), 7.96 (brs, 2H). 13C NMR (100 MHz,
CDCl3): d, ppm -1.5, 17.4, 43.1, 63.9, 65.5, 107.1, 108.3,
114.7, 117.3, 129.5, 132.8, 135.5, 156.7, 169.2. ESI-MS:
m/z obsd. 397.1940; calcd. 397.1942 ([M + H]+, M =
C22H28N2O3Si).
Formation of porphyrins
Zn(II)-5-[4-(2-(trimethylsilyl)ethoxycarbonyl-
methoxy)phenyl]-15-[2,4,6-tris(tert-butoxycarbonyl-
methoxy)phenyl]porphyrin (ZnP5-1). Following a
reported procedure [7], a mixture of B-(CHO)2 (1.18 g,
2.61 mmol) and propylamine (4.00 mL, 52.2 mmol) in
THF (15 mL) was stirred at room temperature for 1 h.
Removal of the solvent and the excess propylamine
affordedB-(imine)2 asabrownviscousoil,whichwasused
without further purification in the next step. A solution
of B-(imine)2 (60 mg, 0.11 mmol) and dipyrromethane
C (69 mg, 0.11 mmol) in toluene (11 mL, 0.01 M) was
treated with anhydrous Zn(OAc)2 (202 mg, 1.10 mmol)
and refluxed for 15 h with exposure to air. The reaction
mixture was concentrated and chromatographed [basic
alumina, hexanes/ethyl acetate (3:1)] to afford a purple
1 , 9 - D i fo r m y l - 5 - [ 4 - ( 2 - ( t r i m e t h y l s i l y l )
ethoxycarbonylmethoxy)phenyl]dipyrromethane
(B-(CHO)2). Following a reported procedure [7] with
slight modification, the Vilsmeier reagent was prepared
by treatment of dry DMF (3.63 mL) with POCl3 (560 mL,
5.98 mmol) at 0°C and stirring of the resulting mixture for
10 min. In a separate flask, a solution of dipyrromethane
B (1.13 g, 2.85 mmol) was treated with the freshly
prepared Vilsmeier reagent at 0°C. The resulting mixture
was stirred at 0°C for 1 h. Then the reaction mixture
was allowed to warm to room temperature upon which
a mixture of saturated aqueous NaOAc/CH2Cl2 [50 mL
(1:1)] was added, and the reaction mixture was stirred for
1 h. The aqueous phase was separated and extracted with
CH2Cl2. The combined organic extract was washed with
brine, dried (Na2SO4), concentrated and chromatographed
[silica pretreated with 1% triethylamine in hexanes,
CH2Cl2/ethyl acetate (4:1)] to afford a brown viscous
1
solid (30 mg, 25%). H NMR (400 MHz, CDCl3): d,
ppm 0.15 (s, 9H), 1.16 (s, 18H), 1.19–1.25 (m, 2H), 1.67
(s, 9H), 3.95 (s, 4H), 4.43–4.47 (m, 2H), 4.74 (s, 2H),
4.88 (s, 2H), 6.43 (s, 2H), 7.30 (d, J = 8.4 Hz,
2H), 8.14 (d, J = 8.4 Hz, 2H), 9.06 (d, J = 4.4 Hz,
2H), 9.16 (d, J = 4.4 Hz, 2H), 9.34 (d, J = 4.4 Hz, 2H), 9.37
(d, J = 4.4 Hz, 2H), 10.19 (s, 2H). 13C NMR (100 MHz,
CDCl3): d, ppm -1.3, 17.6, 27.9, 28.4, 64.1, 66.0, 66.4,
82.2, 82.9, 93.5, 105.8, 110.3, 112.9, 114.7, 119.5, 131.3,
132.1, 132.2, 132.3, 135.7, 136.5, 149.3, 149.6, 149.8,
151.2, 157.6, 159.6, 159.8, 167.8, 168.0, 169.5. ESI-MS:
m/z obsd. 1089.3652; calcd. 1089.3654 ([M + H]+, M =
C57H64N4O12SiZn). UV-vis (toluene): labs, nm 413, 539,
574; (lexc = 413 nm, toluene): lem, nm 579, 633.
1
oil (1.18 g, 91%). H NMR (400 MHz, CDCl3): d, ppm
0.03 (s, 9H), 0.99–1.03 (m, 2H), 4.26–4.30 (m, 2H), 4.55
(s, 2H), 5.52 (s, 1H), 5.99–6.01 (m, 2H), 6.81–6.83 (m,
4H), 7.17–7.19 (m, 2H), 9.12 (s, 2H), 10.86 (brs, 2H). 13C
NMR (100 MHz, CDCl3): d, ppm -1.5, 17.4, 43.6, 63.9,
65.5, 111.6, 115.0, 122.4, 129.7, 132.6, 132.6, 142.2,
157.2, 169.0, 179.1. ESI-MS: m/z obsd. 453.1845; calcd.
453.1840 ([M + H]+, M = C24H28N2O5Si).
Zn(II)-5-[4-(2-(trimethylsilyl)ethoxycarbonyl-
methoxy)phenyl]-15-(4-pyridyl)porphyrin (ZnP6-1).
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
11
Following a reported procedure [7], a solution of
obtain a dark red solid (145 mg, 26%). 1H NMR (400 MHz,
CDCl3/CD3OD): d, ppm 1.87 (t, J = 7.2 Hz, 3H), 5.15 (q,
J = 7.2 Hz, 3H), 7.34 (s, 2H), 7.97 (t, J = 8.0 Hz, 1H),
8.57 (dt, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H), 8.69 (ddd, J1 =
8.4 Hz, J2 = 2.4 Hz, J3 = 0.8 Hz, 1H), 8.94 (d, J = 4.4 Hz,
2H), 9.10 (t, J = 2.0 Hz, 1H), 9.39 (d, J = 4.4 Hz, 2H),
9.49 (d, J = 4.8 Hz, 2H), 9.62 (d, J = 4.4 Hz, 2H), 10.29
(s, 2H). 13C NMR (100 MHz, CDCl3/CD3OD): d, ppm
14.9, 63.3, 107.0, 108.0, 118.4, 122.7, 127.6, 128.4, 131.1,
131.9, 132.1, 133.3, 140.0, 144.8, 146.9, 148.8, 148.9,
150.0, 150.1, 173.5. ESI-MS: m/z obsd. 565.0727, calcd.
565.0723 ([M]+, M = C29H19N5O4Zn). UV-vis (toluene):
labs, nm 413.
Zn(II)-5-[4-(tert-butoxycarbonylmethoxy)phenyl-
15-[3,5-bis(diethylphosphonomethoxy)phenyl]-
porphyrin (ZnP8-1). Following a reported procedure
[7], a sample of H-(CHO)2 (200 mg, 0.490 mmol) in
propylamine (3.00 mL) was stirred at room temperature.
After 1 h, the excess propylamine was removed under
reduced pressure. A solution of the resulting solid and
dipyrromethane I (271 mg, 0.490 mmol) in ethanol
(45.0 mL) was treated with zinc acetate (917 mg,
5.00 mmol) under reflux for 18 h. The solvent was
removed in vacuo, affording a dark pink residue. The
residue was chromatographed [silica, CH2Cl2/methanol
(0–10%)] to afford a pink solid (90.0 mg, 19%). 1H NMR
(CD3OD): d, ppm -0.22 (t, J = 6.8 Hz, 12H), 1.65 (s, 9H),
2.28–2.5 (m, 8H), 4.28 (s, 2H), 4.31 (s, 2H), 5.05 (s, 2H),
7.30 (d, J = 8.8 Hz, 2H), 7.38 (m, 2H), 7.88 (t, J = 8.4 Hz,
1H), 8.18 (d, J = 8.0 Hz, 2H), 8.92 (d, J = 4.8 Hz,
2H), 9.04 (d, J = 4.0 Hz, 1H), 9.37 (d, J = 4.4 Hz,
2H), 9.40 (d, J = 4.4 Hz, 2H), 10.22 (s, 2H). ESI-MS:
m/z obsd. 986.23973, calcd. 986.23993 ([M]+, M =
C48H52N4O11P2Zn). UV-vis (CH2Cl2): labs, nm 409, 538.
B-(imine)2 (1.92 g. 3.58 mmol) and dipyrromethane D
(0.800 g, 3.58 mmol) in toluene (358 mL, 0.01 M) was
treated with anhydrous Zn(OAc)2 (6.57 g, 35.8 mmol) and
refluxedfor15hwithexposuretoair.Thereactionmixture
was concentrated and chromatographed [silica, ethyl
acetate/CH2Cl2 (1:4)] to afford a purple solid (200 mg,
8%). 1H NMR (400 MHz, THF-d8): d, ppm 0.19 (s, 9H),
1.13–1.17 (m, 2H), 4.39–4.43 (m, 2H), 4.95 (s, 2H), 7.35
(d, J = 8.4 Hz, 2H), 7.98 (d, J = 5.5 Hz, 2H), 8.15 (d, J =
8.8 Hz, 2H), 8.28 (brs, 2H), 8.86 (d, J = 4.4 Hz, 2H), 9.07
(d, J = 4.4 Hz, 2H), 9.39 (d, J = 2.6 Hz, 2H), 9.41 (d, J =
2.6 Hz, 2H), 10.28 (s, 2H). 13C NMR (100 MHz, THF-
d8): d, ppm -1.2, 18.3, 63.9, 66.6, 106.9, 113.8, 120.9,
121.4, 128.9, 130.6, 132.0, 132.5, 132.9, 133.2, 136.6,
137.2, 149.9, 150.7, 150.9, 151.5, 152.3, 159.3, 169.6.
ESI-MS: m/z obsd. 700.17150; calcd. 700.17169 ([M +
H]+, M = C38H33N5O3SiZn). UV-vis (toluene): labs, nm
413, 539; (lexc = 413 nm, toluene): lem, nm 580, 631.
tert-Butyl Zn(II)-15-(3-nitrophenyl)porphyrin-5-
carboxylate (ZnP7-1a). Following a reported procedure
[7] with slight modification, a sample of E-(CHO)2
(360 mg, 1.11 mmol) was treated with butylamine
(2.00 mL) at room temperature for 3 h. The mixture
was filtered through a short pad of Na2SO4, which was
washed with CH2Cl2. The filtrate was concentrated
and dried to obtain a brown residue. The residue was
treated with dipyrromethane F (250 mg, 1.01 mmol) and
Zn(OAc)2·2H2O (2.20 g, 10.0 mmol) in ethanol (100 mL)
under argon. The resulting pink solution was stirred
under reflux for 18 h open to the atmosphere. The
mixture was filtered. The filtrate was concentrated and
chromatographed (silica, CH2Cl2) to obtain a dark red
1
solid (192 mg, 32%). H NMR (400 MHz, THF-d8): d,
ppm 2.10 (s, 9H), 8.03 (t, J = 8.0 Hz, 1H), 8.60 (d, J =
7.2 Hz, 1H), 8.71 (dd, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H), 8.97
(d, J = 4.4 Hz, 2H), 9.08 (t, J = 2.0 Hz, 1H), 9.44 (d, J =
4.8 Hz, 2H), 9.51 (d, J = 4.4 Hz, 2H), 9.67 (d, J = 4.8 Hz,
2H), 10.34 (s, 2H). 13C NMR (100 MHz, THF-d8): d,
ppm 28.9, 83.4, 107.2, 112.0, 118.8, 123.2, 128.4, 129.0,
131.9, 132.3, 132.7, 133.4, 140.8, 145.8, 148.0, 149.6,
149.8, 150.8, 150.9, 171.7. ESI-MS: m/z obsd. 594.1114,
calcd. 594.1114 ([M + H]+, M = C31H23N5O4Zn). UV-vis
(toluene): labs, nm 413.
Ethyl Zn(II)-15-(3-nitrophenyl)porphyrin-5-car-
boxylate (ZnP7-1b). Following a reported procedure [7]
with slight modification, a sample of E-(CHO)2 (323 mg,
1.00 mmol) was treated with butylamine (3.00 mL) at room
temperature for 2 h. The mixture was filtered through a
short pad of Na2SO4 and washed with CH2Cl2. The filtrate
was concentrated and dried to obtain a brown residue.
The residue was treated with dipyrromethane G (218 mg,
1.00 mmol) and Zn(OAc)2·2H2O (2.20 g, 10.0 mmol) in
ethanol (200 mL) under argon. The resulting pink solution
was stirred under reflux for 15 h at open atmosphere. The
mixture was filtered. The filtrate was concentrated and
chromatographed [silica, hexanes/ethyl acetate (9:1)] to
Modification of porphyrins
Zn(II)-5-[4-(carboxymethoxy)phenyl]-15-[2,4,6-
tris(tert-butoxycarbonylmethoxy)phenyl]porphyrin
(ZnP5-2). A solution of ZnP5-1 (29 mg, 0.027 mmol)
in anhydrous THF (2.7 mL) was treated with TBAF
solution (40 mL, 0.04 mmol, 1 M in THF) at 0°C. The
solution was allowed to reach room temperature and was
stirred for 1.5 h. Ethyl acetate, H2O and saturated aqueous
NH4Cl were added, and the mixture was extracted with
ethyl acetate. The organic phase was dried (Na2SO4),
concentrated and chromatographed [silica, ethyl acetate/
MeOH (9:1)] to afford a purple solid (26 mg, quantitative).
1HNMR(400MHz,CDCl3/CD3OD):d,ppm1.26(s,18H),
1.66 (s, 9H), 4.16 (s, 4H), 4.82 (s, 2H), 5.12 (s, 2H), 6.58
(s, 2H), 7.38 (d, J = 8.2 Hz, 2H), 8.18 (d, J = 8.2 Hz, 2H),
9.07 (d, J = 4.3 Hz, 2H), 9.11 (d, J = 4.6 Hz, 2H), 9.34 (d,
J = 4.6 Hz, 2H), 9.36 (d, J = 4.6 Hz, 2H), 10.18 (s, 2H).
13C NMR (100 MHz, CDCl3/CD3OD): d, ppm 29.3, 29.7,
31.5,57.5,68.0,68.3,84.2,84.8,95.8,107.0,108.0,111.2,
114.6, 117.4, 121.1, 132.9, 133.6, 137.5, 138.4, 151.2,
151.4, 151.7, 152.8, 159.6, 161.5, 161.6, 170.1, 170.5.
Copyright © 2015 World Scientific Publishing Company
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T. SAHIN ET AL.
ESI-MS: m/z obsd. 989.2935; calcd. 989.2946 ([M +
H]+, M = C52H52N4O12Zn). UV-vis (toluene): labs, nm 414,
540, 575; (lexc = 414 nm, toluene): lem, nm 579, 632.
Zn(II)-5-[4-(N-succinimidyloxycarbonylmethoxy)
phenyl]-15-[2,4,6-tris(tert-butoxycarbonylmethoxy)-
phenyl]porphyrin (ZnP5-3). A sample of ZnP5-2
(50 mg, 0.050 mmol) was dissolved in a mixture of dry
DMF (770 mL) and dry pyridine (39 mL). Disuccinimidyl
carbonate (19 mg, 0.075 mmol) was added, and the
reaction mixture was stirred at room temperature for 5 h.
Water and ethyl acetate were added to the reaction
mixture. The organic phase was separated and washed
(water and brine), dried (Na2CO3) and concentrated. The
crude product was treated with 2% CH2Cl2 in hexanes,
sonicated, and centrifuged. The supernatant was decanted
to give the title product as a purple solid (38 mg, 70%
yield). MALDI-MS: m/z (POPOP) 1085.6; calcd. 1086.3
([M+H]+, M=C56H55N5O14Zn). UV-vis(toluene):labs, nm
413, 540; (lexc = 413 nm, toluene): lem, nm 579, 633.
5-[4-(N-succinimidyloxycarbonylmethoxy)
phenyl]-15-[2,4,6-tris(carboxymethoxy)phenyl]
porphyrin (P5). A solution of ZnP5-3 (26 mg,
0.024 mmol) in CH2Cl2 (3.8 mL) was treated with TFA
(960 mL), and the mixture was stirred for 4 h at room
temperature. Water was added, and the aqueous layer was
extracted with ethyl acetate. The organic layer was dried
(Na2SO4) and concentrated to give a purple solid (20 mg,
quantitative). MALDI-MS: m/z 856.2; calcd. 856.2 ([M +
H]+, M = C44H33N5O14). UV-vis (0.5 M phosphate buffer,
pH 7.0): labs, nm 404, 504, 542, 573; (lexc = 404 nm, 0.5 M
phosphate buffer, pH 7.0): lem, nm 626, 687.
M = C34H26IN5O3). UV-vis (DMF): labs, nm 414, 504,
548, 576, 637; (lexc = 414 nm, DMF): lem, nm 642, 704.
5-[4-(N-succinimidyloxycarbonylmethoxy)
phenyl]-15-(N-methylpyridinium-4-yl)porphyrin
iodide (P6). A solution of P6-2 (38 mg, 0.057 mmol)
and N-hydroxysuccinimide (66 mg, 0.57 mmol) in DMF
(1 mL) was treated overnight with EDC (109 mg, 0.568
mmol) at room temperature. Diethyl ether was added.
The mixture was sonicated and centrifuged whereupon
the supernatant was decanted. The same procedure was
repeated twice with water then once with THF. Drying
in vacuo afforded the product as a purple solid (35 mg,
1
79%, assuming an iodide counterion): H NMR spectra
were recorded in two different solvents given that the
CD3OD signal overlapped with the methyl and methylene
protons, and the DMF-d7 signal overlapped with the NHS
1
ester protons. H NMR (400 MHz, DMF-d7): d, ppm
-3.03 (brs, 1H), -3.08 (brs, 1H), 5.02 (s, 3H), 5.16 (s, 2H),
7.56 (d, J = 8.8 Hz, 2H), 8.32 (d, J = 8.3 Hz, 2H), 9.21
(d, J = 4.4 Hz, 2H), 9.24 (d, J = 6.8 Hz, 2H), 9.29 (d, J =
4.9 Hz, 2H), 9.75−9.77 (m, 4H), 9.89 (d, J = 4.4 Hz, 2H),
10.81 (s, 2H). 1H NMR (400 MHz, CD3OD): d, ppm 2.68
(s, 4H), 7.47 (d, J = 8.3 Hz, 2H), 8.20 (d, J = 8.8 Hz,
2H), 8.99 (d, J = 6.9 Hz, 2H), 9.15 (d, J = 4.4 Hz, 2H),
9.18 (d, J = 4.4 Hz, 2H), 9.37 (d, J = 6.3 Hz, 2H), 9.55
(d, J = 4.9 Hz, 2H), 9.67 (d, J = 4.4 Hz, 2H), 10.56 (s,
2H). MALDI-MS: m/z obsd. 649.4, ESI-MS: m/z obsd.
649.2178; calcd. 649.2939 ([M – I]+, M = C38H29IN6O5).
UV-vis (DMF): labs, nm 414, 505, 548, 579, 635; (lexc
414 nm, DMF): lem, nm 641, 703.
=
tert-Butyl15-(3-aminophenyl)porphyrin-5-carboxy-
late (P7-2a). Following a reported procedure [42], a
suspension of ZnP7-1a (150 mg, 0.252 mmol), Zn dust
(817 mg, 12.5 mmol) and NH4Cl (1.34 g, 25.0 mmol) in
ethanol (40.0 mL) and water (10.0 mL) was stirred under
reflux for 4 h. The mixture was filtered, and the filtrate
was concentrated. The resulting residue was treated
with 3 N HCl, stirred for 1 h at room temperature, and
then washed with ethyl acetate (colorless). The acidic
aqueous solution (green) was neutralized with saturated
aqueous NaHCO3 solution followed by extraction with
ethyl acetate. The resulting organic solution (purple) was
concentrated and chromatographed [silica, CH2Cl2/ethyl
acetate (9:1)] to obtain a dark purple solid (75.9 mg,
60%). 1H NMR (300 MHz, THF-d8): d, ppm -3.10 (s, 1H)
-2.95 (s, 1H), 2.09 (s, 9H), 4.90 (s, 2H), 7.01–7.05 (m,
1H), 7.44–7.45 (m, 2H), 7.50–7.51 (m, 1H), 9.16 (d, J =
4.8 Hz, 2H), 9.37 (d, J = 4.5 Hz, 2H), 9.49 (d, J = 4.2 Hz,
2H), 9.67 (d, J = 4.8 Hz, 2H), 10.36 (s, 2H). 13C NMR
(100 MHz, THF-d8): d, ppm 28.9, 83.8, 106.6, 109.7,
114.5, 122.3, 123.3, 125.0, 128.2, 130.7, 131.9, 132.3,
133.5, 142.6, 146.1, 146.6, 147.3, 147.8, 148.2, 170.4.
MALDI-MS: m/z (POPOP) obsd. 501.1, calcd. 501.2165
([M]+, M = C31H27N5O2). UV-vis (toluene): labs, nm 408.
tert-Butyl15-[3-(4-(N-maleimido)-1-oxobutylamino)
phenyl]porphyrin-5-carboxylate (P7-3a). A mixure
of P7-2a (25.0 mg, 50.0 mmol), DCC (51.5 mg,
5-[4-(Carboxymethoxy)phenyl]-15-(N-methyl-
pyridinium-4-yl)porphyrin iodide (P6-2). A sample of
ZnP6-1 (38 mg, 0.054 mmol) in DMF (1 mL) containing
iodomethane (300 mL, 4.8 mmol) in a capped vial was
heated overnight at 60°C with stirring. The reaction
mixture was concentrated, and the resulting crude solid
was treated with anhydrous diethyl ether followed by
sonication in a benchtop sonication bath. The resulting
suspensionwascentrifuged.Thesupernatantwasremoved
leaving the desired product as a solid (45 mg, quantitative,
assuming an iodide counterion). MALDI-MS: m/z obsd.
713.8. ESI-MS: m/z obsd. 714.1865, calcd. 714.1873
([M – I]+, M = C39H36N5O3SiIZn]. A solution of this
product (1.5 mL, CH2Cl2) was treated with TFA (0.5 mL)
at room temperature for 5 h. The reaction mixture was
concentrated to dryness. Water was added followed by
sonication, centrifugation and decantation, which was
performed twice (each time retaining the pellet). The
final pellet was dried in vacuum to afford a black-purple
1
solid (34 mg, 94%, assuming an iodide counterion). H
NMR (400 MHz, TFA-d): d, ppm 4.82 (s, 3H), 5.14 (s,
2H), 7.63 (d, J = 8.8 Hz, 2H), 8.49 (d, J = 8.4 Hz, 2H),
9.06–9.08 (m, 4H), 9.17 (d, J = 6.6 Hz, 2H), 9.31 (d, J =
6.2 Hz, 2H), 9.58 (d, J = 4.8 Hz, 2H), 9.69 (d, J = 4.8 Hz,
2H), 11.11 (s, 2H). MALDI-MS: m/z obsd. 552.5,
ESI-MS: m/z obsd. 552.2032; calcd. 552.2030 ([M – I]+,
Copyright © 2015 World Scientific Publishing Company
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
13
0.250 mmol) and 4-maleimidobutyric acid (45.8 g,
and chromatographed [silica, CH2Cl2/ethyl acetate (9:1)]
to obtain a dark purple solid (43 mg, 61%). H NMR
1
0.250 mmol) in CH2Cl2 (1.00 mL) was stirred at room
temperature under argon for 15 h. The mixture was
diluted with CH2Cl2 (20 mL) and washed with water.
The organic phase was separated, washed (brine), dried
(Na2SO4), concentrated and chromatographed [silica,
CH2Cl2/MeOH (99.5:0.5)] to obtain a mixture containing
the title compound and 4-maleimidobutyric acid. Further
chromatography [basic alumina, CH2Cl2/ethyl acetate
(400 MHz, CDCl3/CD3OD): d, ppm -3.10 (s, 1H) -2.95
(s, 1H), 1.88 (t, J = 7.6 Hz, 3H), 5.13 (q, J = 7.6 Hz, 2H),
7.13 (d, J = 1.2 Hz, 1H), 7.57 (brs, 2H), 7.61–7.67 (m,
1H), 9.16 (d, J = 4.4 Hz, 2H), 9.34 (d, J = 4.8 Hz, 2H),
9.46 (d, J = 4.8 Hz, 2H), 9.69 (d, J = 4.8 Hz, 2H), 10.32
(s, 2H). 13C NMR (100 MHz, THF-d8): d, ppm 14.2, 62.5,
106.0, 113.8, 121.6, 123.0, 124.4, 125.2, 127.5, 130.3,
131.2, 131.6, 133.0, 137.6, 141.7, 145.4, 145.8, 147.0,
147.4, 170.3. MALDI-MS: m/z (POPOP) obsd. 473.5,
calcd. 473.2 ([M]+, M = C29H23N5O2). UV-vis (toluene):
labs, nm 408.
ClMn(III) 5-(tert-Butoxycarbonyl)-15-[3-(4-(N-
maleimido)-1-oxobutylamino)phenyl]porphyrin
(MnP7-3a). Following a literature procedure [36] with
slight modification, a sample of P7-3a (29 mg, 0.043
mmol) in CHCl3/MeOH (2:1, 18 mL) was treated with
MnCl2·4H2O (273 mg, 1.38 mmol) and 2,6-di-tert-
butylpyridine (8 drops). The reaction mixture was stirred
at 50°C for 4 days. The reaction mixture was diluted
with CHCl3 and extracted with water. The organic
extract was separated, dried (Na2SO4), concentrated and
chromatographed [silica, CHCl3 à CHCl3/MeOH (9:1)]
to afford a red-brown solid (20.0 mg, 62% assuming
a chloride counterion): MALDI-MS: m/z (a-cyano-
4-hydroxycinnamic acid) obsd. 719.1, ESI-MS: m/z
obsd. 719.1809; calcd. 719.1809 ([M – Cl]+, M =
C39H32ClMnN6O5). UV-vis (DMF): labs, nm 368, 390,
411, 458, 552.
ClMn(III) 5-Carboxy-15-[3-(4-(N-maleimido)-1-
oxobutylamino)phenyl]porphyrin (MnP7). A sample
of MnP7-3a (10 mg, 0.014 mmol) in CH2Cl2 (2.4 mL)
was treated with TFA (0.6 mL), and the mixture was
stirred for 4 h at room temperature. Hexanes was added,
and the resulting mixture was sonicated in a benchtop
sonication bath. After centrifuging the suspension, the
supernatant was carefully decanted and this process
was repeated with diethyl ether to obtain the title
product as a red-brown solid (8.5 mg, 87% assuming
a chloride counterion): MALDI-MS: m/z (a-cyano-
4-hydroxycinnamic acid) obsd. 663.1, ESI-MS: m/z
obsd. 663.1159; calcd. 663.1183 ([M – Cl]+, M =
C35H24ClMnN6O5). UV-vis (0.5 M phosphate buffer, pH
7.0): labs, nm 372, 394, 459, 549.
5-[4-(tert-Butoxycarbonylmethoxy)phenyl-15-[3,5-
bis(diethylphosphonomethoxy)phenyl]porphyrin
(P8-1). Following an established procedure for de-
zincation in the presence of phosphono esters [13], a
solution of ZnP8-1 (30. mg, 0.030 mmol) in CH2Cl2 (10
mL) was treated with p-TsOH·H2O (480 mg, 2.52 mmol)
at room temperature for 1 h. The reaction mixture was
diluted with CH2Cl2 and washed with saturated aqueous
NaHCO3 solution. The organic layer was separated, dried
(Na2SO4) and filtered. The filtrate was concentrated to a
dark pink solid. The solid was chromatographed [silica,
CH2Cl2/methanol(0–10%)]toaffordapinksolid(25.3mg,
1
(2:1)] afforded a dark brown solid (17.3 mg, 52%). H
NMR (400 MHz, CDCl3/CD3OD): d, ppm 1.91–1.97
(m, 2H), 2.10 (s, 9H), 2.32 (t, J = 7.2 Hz, 2H), 3.51 (t, J
= 6.8 Hz, 2H), 6.53 (s, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.96
(d, J = 7.6 Hz, 1H), 8.02 (m, 1H), 8.29 (t, J = 1.6 Hz, 1H),
9.07 (d, J = 4.8 Hz, 2H), 9.32 (d, J = 4.4 Hz, 2H), 9.45
(d, J = 4.8 Hz, 2H), 9.56 (d, J = 4.4 Hz, 2H), 10.22 (s,
2H). 13C NMR (100 MHz, CDCl3/CD3OD): d, ppm 25.2,
29.2, 34.9, 37.7, 84.5, 107.0, 110.1, 119.6, 121.6, 126.7,
127.5, 130.9, 131.3, 132.0, 133.2, 133.3, 134.6, 137.1,
144.1, 148.7, 150.0, 150.1, 150.4, 171.7, 172.1, 173.5.
ESI-MS: m/z obsd. 667.2649, calcd. 667.2663 ([M +
H]+, M = C39H34N6O5). UV-vis (toluene): labs, nm 407.
15-[3-(4-(N-maleimido)-1-oxobutylamino)phenyl]
porphyrin-5-carboxylic acid (P7). A solution of P7-3a
(16.6 mg, 25.0 mmol) in CH2Cl2 (1.20 mL) was treated
with TFA (800 mL) for 4 h at room temperature. The
volatile components (solvent and TFA) were removed
by purging with argon. The resulting residue was washed
with water and chromatographed [silica, CH2Cl2/MeOH
(19:1)]. The resulting solid was dissolved in CH2Cl2/
MeOH (5.0 mL, 19:1) and filtered to remove insoluble
matter. The filtrate was added to hexanes (200 mL). The
resulting precipitate was separated by filtration and dried
1
to obtain a dark brown solid (12.2 mg, 80%). H NMR
(400 MHz, CDCl3/CD3OD): d, ppm 2.03 (qt, J = 7.2 Hz,
2H), 2.44 (t, J = 7.2 Hz, 2H) 3.56 (t, J = 6.8 Hz, 2H), 6.64
(s, 2H), 7.72 (t, J = 8.0 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H),
8.08 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 9.05 (d, J = 4.4 Hz,
2H), 9.28 (d, J = 4.4 Hz, 2H), 9.37 (brs, 2H), 9.76
(brs, 2H), 10.20 (s, 2H). 13C NMR (100 MHz, CDCl3/
CD3OD): d, ppm 25.1, 34.8, 37.8, 106.5, 109.6, 120.2,
121.3, 127.0, 128.0, 131.0, 131.4, 131.9, 132.0, 133.1,
133.3, 134.7, 137.8, 142.0, 146.0, 146.9, 171.9, 172.7.
ESI-MS: m/z obsd. 611.2035, calcd. 611.2037 ([M +
H]+, M = C35H26N6O5). UV-vis (toluene): labs, nm 407.
Ethyl 15-(3-aminophenyl)porphyrin-5-carboxylate
(P7-2b). Following a reported procedure [42], a
suspension of ZnP7-1b (85 mg, 0.15 mmol), Zn dust
(0.13 g, 2.0 mmol) and NH4Cl (0.11 g, 2.0 mmol) in
ethanol (50 mL) and water (5.0 mL) was stirred under
reflux for 4 h. The content was filtered, and the filtrate
was concentrated. The resulting residue was treated with
3N HCl, stirred for 1 h at room temperature, and then
washed with ethyl acetate (colorless). The acidic aqueous
solution (green) was neutralized with saturated aqueous
NaHCO3 solution and then extracted with ethyl acetate.
The resulting organic solution (purple) was concentrated
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 13–16

14
T. SAHIN ET AL.
1
90%). H NMR: d, ppm -3.15 (brs, 2H), -0.38 (t, J =
(m, 4H), 10.28 (s, 2H). LD-MS: m/z obsd. 877.2, calcd.
875.2 (C41H43N5O13P2); also obsd. 778.9 (P3a-1). This
crude material was used in pilot conjugation experiments
without further characterization.
7.2 Hz, 12H), 1.64 (s, 9H), 2.27–2.44 (m, 8H), 4.18 (s,
2H), 4.20 (s, 2H), 4.85 (s, 1H), 4.95 (s, 1H), 7.14 (d, J =
8.8 Hz, 2H), 7.36 (m, 2H), 7.82 (t, J = 8.8 Hz, 1H), 8.21
(m, 2H), 8.95 (d, J = 4.4 Hz, 2H), 9.10 (m, 2H), 9.33 (d,
J = 4.8 Hz, 2H), 9.38 (d, J = 4.8 Hz, 2H), 10.24 (s, 2H).
ESI-MS: m/z obsd. 924.32462, calcd. 924.32643 ([M]+,
M = C48H54N4O11P2). UV-vis (CH2Cl2): labs, nm 406, 501,
534, 575.
(D-Phe-P3a-1). A solution of P3a-1 (5.41 mg,
6.95 mmol) in anhydrous DMF (400 mL) was treated
with EDC (6.25 mg, 32.7 mmol) and HOBt (5.60 mg,
41.5 mmol). The solution was stirred at room temperature
for 10 min. D-Phe-OEt (p-toluenesulfonic acid salt,
7.61 mg, 20.9 mmol) and DIEA (5.6 mL, 31 mmol) were
added. Stirring was continued for 24 h. The mixture
was diluted with CH2Cl2 and washed with water. The
aqueous phase was extracted with CH2Cl2. The combined
organic extract was washed (water), dried (Na2SO4) and
chromatographed [silica, CH2Cl2/MeOH (97:3)] to afford
5-[4-(Carboxymethoxy)phenyl-15-[3,5-bis-
(phosphonomethoxy)phenyl]porphyrin (P8). A solu-
tion of P8-1 (30 mg, 0.032 mmol) in THF/H2O (10 mL,
4:1) was treated with aqueous 2M KOH (200 mL,
0.400 mmol). The mixture was stirred for 24 h at room
temperature. Then, THF was removed under vacuum.
The residue was partioned between water (5 mL) and
CH2Cl2 (5 mL). The aqueous layer was freeze-dried to
obtain the potassium salt of the porphyrin. The solid was
suspended in CH2Cl2 (5 mL), whereupon a few drops of
acetic acid were added. The mixture was filtered. The
filtrate was concentrated. The resulting solid was dried
under high vacuum. The solid was dissolved in CH2Cl2
(5 mL), whereupon TMSBr (300 mL, excess) was added.
The solution was stirred for 4 h at room temperature.
The solvent and excess TMSBr were removed in vacuo,
then methanol/water (5 mL, 4:1) was added. Stirring was
continued for 1 h, whereupon the volatile components
were removed under high vacuum. The resulting green
solid was dried under high vacuum, then triturated with
CH2Cl2 (10 mL × 3) and finally dried to obtain a green
1
a deep purple solid (4.70 mg, 71%). H NMR: d, ppm
-2.89 (s, 1H), -2.84 (s, 1H), 1.32 (t, J = 6.9 Hz, 3H), 3.20–
3.49 (m, 18H), 3.91 (m, 2H), 4.06 (m, 2H), 4.28 (q, J =
6.9 Hz, 2H), 4.82 (s, 2H), 5.09–5.11 (m, 1H), 5.80 (m,
1H), 7.30–7.38 (m, 7H), 8.19 (d, J = 7.5 Hz, 2H), 9.06 (s,
2H), 9.38–9.40 (m, 2H), 9.49 (s, 2H), 9.66 (s, 1H), 9.83
(s, 1H), 10.28 (s, 1H), 10.31 (s, 1H); LD-MS: m/z obsd.
955.6; FAB-MS: m/z obsd. 976.3107, calcd. 976.3064
[(M + Na)+, M = C48H53N5O12P2). UV-vis: labs, nm 406,
504; lem, nm (lexc 406 nm) 633, 700.
(D-Phe-P3a). A solution of D-Phe-P3a-1 (5.15 mg,
5.40 mmol) in anhydrous CHCl3 (0.5 mL) was treated with
TMSBr (5.7 mL, 4.3 mmol). The solution was stirred at
room temperature for 3 h. The solution was concentrated,
and the resulting residue was dissolved in dilute aqueous
NaOH (1 mL, 0.1 M). The mixture was stirred for 1 h.
Chromatography [C-18 silica, water/MeOH (0 à 70%)]
yielded a deep purple solid (6.30 mg): ESI-MS: m/z obsd.
(–) 868.0 (M – H), also obsd. 881.0 (M + methylene –
H)-. ESI-MS: m/z obsd. 870.2 [M + H]+, 891.2 [M + Na]+,
calcd. 869.2 (M = C42H41N5O12P2). UV-vis: labs, nm 402,
505; lem, nm (lexc 402 nm) 627, 686. HPLC tR = 9.60 min.
(MSH-P3a-1). A solution of P3a-1 (4.76 mg, 6.12
mmol) in anhydrous DMF (400 mL) was treated with EDC
(11.7 mg, 61.2 mmol) and HOBt (9.09 mg, 67.3 mmol)
and stirred at room temperature for 10 min. Samples
of Ac-a-MSH(11-13)-NH2·HCl (3.75 mg, 8.94 mmol)
and DIEA (2.4 mL, 13 mmol) were added. Stirring was
continued for 24 h. The solution was diluted with CH2Cl2
and washed with water. The aqueous phase was extracted
with CH2Cl2. The combined organic extract was washed
(water), dried (Na2SO4) and chromatographed [silica,
CH2Cl2/MeOH (0 à 12%)] to afford a deep purple solid
1
solid (25 mg, quantitative). H NMR (CD3OD): d, ppm
4.18 (s, 2H), 4.20 (s, 2H), 5.12 (s, 2H), 7.41 (d, J =
7.6 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 8.02 (t, J = 8.8 Hz,
1H), 8.59 (d, J = 7.6 Hz, 2H), 9.34 (m, 4H), 9.89 (d,
J = 5.2 Hz, 2H), 9.93 (d, J = 4.8 Hz, 2H), 11.34 (s, 2H).
ESI-MS: m/z (–) obsd. 756.14070, calcd. 756.13863
([M]-, M = C36H30N4O11P2). UV-vis (H2O): labs, nm 404,
504, 541, 565.
Bioconjugation of porphyrins
5-[4-(N-succinimidyloxycarbonylmethoxy)
phenyl]-15-[1,5-bis(dimethoxyphosphoryl)pent-3-yl]
porphyrin (P3a-2). A solution of P3a-1 (10.1 mg,
13.0 mmol) in anhydrous DMF (400 mL) was treated with
EDC (4.88 mg, 25.5 mmol). The solution was stirred at
room temperature for 10 min. N-hydroxysuccinimide
(2.94 mg, 25.6 mmol) was added. Stirring was continued
for 24 h. The mixture was diluted with CH2Cl2 and
washed with water. The aqueous phase was extracted
with CH2Cl2. The combined organic extract was washed
with water. The organic layer was dried (Na2SO4).
Concentration of the sample gave a mixture of P3a-1 and
P3a-2 (approximately 1:4 ratio). 1H NMR: d, ppm 1.09 (t,
J = 6.9 Hz, 3H), 2.66–3.68 (m, approximately 22H), 5.30
(s, 2H), 5.82–5.93 (m, 1H), 7.39–7.42 (m, 2H), 8.19–8.21
(m, 2H), 9.07 (app s, 2H), 9.38–9.48 (m, 2H), 9.61–9.76
1
(5.30 mg, 76%). H NMR: d, ppm -2.89 (s, 1H), -2.84
(s, 1H), 0.93–0.98 (m, 6H), 1.42–1.76 (m, 12H), 3.20–
3.22 (m, 2H), 3.40–3.49 (s, 14H), 3.65–4.06 (m, 7H),
4.26–4.31 (m, 1H), 4.55 (m, 1H), 4.81 (s, 2H), 5.78 (br,
2H), 6.17 (s, 1H), 6.72 (d, J = 7.5 Hz, 1H), 6.92 (d, J =
8.4 Hz, 1H), 7.08 (s, 1H), 7.37–7.40 (m, 2H), 8.19–8.22
(m, 2H), 9.06 (s, 2H), 9.37–9.40 (m, 2H), 9.49 (m,
2H), 9.65 (m, 1H), 9.81 (m, 1H), 10.28 (s, 1H), 10.30
(s, 1H). ESI-MS: m/z obsd. (–) 1142.3 [M – H]-, 1179.3
Copyright © 2015 World Scientific Publishing Company
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HYDROPHILIC BIOCONJUGATABLE TRANS-AB-PORPHYRINS AND PEPTIDE CONJUGATES
15
[M + Cl – H]-, 1188.3 [M + Na + Cl – H]-. ESI-MS: m/z
characterized. As the hydrophilicity of the porphyrins
increased upon elaboration, characterization became
more challenging. Excellent characterization of the
protected porphyrin conjugates was generally obtained
whereas the partial characterization of the deprotected
conjugates (D-Phe-P3a, MSH-P3a, and MSH-P4a)
causes their assignments to remain provisional.
obsd. 1144.4 [M + H]+, 572.7 [M + 2H]2+, calcd. 1143.5
(M = C55H71N9O14P2). LD-MS: m/z obsd. 1146.6, 1169.3
[M + Na]+. UV-vis: labs, nm 406, 503, 538, 577; lem, nm
(lexc 406 nm) 634, 699.
(MSH-P3a). A solution of MSH-P3a-1 (3.18 mg,
2.78 mmol) in anhydrous CHCl3 (0.5 mL) was treated
with TMSBr (2.9 mL, 22 mmol) and stirred at room
temperature for 3 h. The mixture was concentrated, and
the resulting residue was dissolved in MeOH (3 mL).
The resulting solution was stirred for 1 h and then
concentrated. The resulting dark green solid was treated
with dilute aqueous NaOH (0.5 mL of 0.05 M solution).
Chromatography [C-18 silica, water/MeOH (0 à 70%)]
yielded a deep purple solid (4.24 mg). ESI-MS: m/z obsd.
(–) 1086.3 [M – H]-, calcd. 1087.4 (M = C51H63N9O14P2).
ESI-MS: m/z obsd. 1100.2 [M + methylene – H]+, 1115.3
[M + 2 methylene – H]+. UV-vis: labs, nm 402, 505; lem,
nm (lexc 402 nm) 626, 687. HPLC tR = 11.76 min.
(MSH-P4a). A solution of P4a (1.04 mg, 1.51 mmol),
EDC (4.36 mg, 22.8 mmol) and HOBt (3.00 mg,
22.2 mmol) in water (180 mL) was treated with Ac-a-
MSH(11-13)-NH2 (1.65 mg, 3.93 mmol) and DIEA
(3.2 mL, 34.5 mmol). Stirring was continued for 24 h. The
solution was diluted with water (1 mL). Chromatography
[C-18 silica, water/MeOH (0 à 70%)] afforded a deep
purple solid (1.68 mg). ESI-MS: m/z obsd. 539.3 [M +
Na + H]2+, 1056.3 [M + H]+. ESI-MS: m/z obsd. (–)
1054.3 [M – H]-, 573.3 [M – H + 2Cl + Na]2-, 609.3 [M –
H + Cl + DIEA]2-, calcd. 1055.4 (M = C51H63N9O12P2),
also obsd. 691.1 (P4a + H)+. UV-vis: labs, nm 403, 507;
lem, nm (lexc 403 nm) 628, 688. HPLC tR = 10.58 min.
Acknowledgements
The various compounds collectively described herein
were prepared over a period of 10 years to realize distinct
research objectives. Energy sciences studies have been
supported since 2009 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, underAward
no. DE-SC0001035 (all except P3a, P4a and P8 series of
compounds). Biomedical sciences studies were supported
earlier by a grant from the NIH (GM36238 through
2007) and a grant from the National Institute of Allergy
and Infectious Diseases (R41AI072854, 2007–2009) to
NIRvana Pharmaceuticals, Inc. The content is solely the
responsibility of the authors and does not necessarily
represent the official views of the NIAID or the NIH. We
thank Prof. Paul A. Loach for suggesting the manganese
porphyrin. 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.
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