Design and synthesis of protoporphyrin IX/vitamin B12 molecular hybrids via CuAAC reaction

Article abstract of DOI:10.1142/S1088424612501350

The design and synthesis of new molecular hybrids composed of protoporphyrin IX (PPIX) and vitamin B₁₂ via copper catalyzed alkyne azide cycloaddition reaction is described. New, clickable aminoazide and aminoalkyne linkers were prepared and subsequently attached to PPIX (via vinyl group) and to vitamin B₁₂ giving desired building blocks. Preliminary results showed that respective water soluble hybrids were formed under CuAAC reaction. Gratifyingly, Cu incorporation into the PPIX core was avoided, which was important for further biological studies. Copyright

Full text of DOI:10.1142/S1088424612501350

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2013; 17: 104–117
DOI: 10.1142/S1088424612501350
Published at http://www.worldscinet.com/jpp/
Design and synthesis of protoporphyrin IX/vitamin B12
molecular hybrids via CuAAC reaction
Rafał Loska, Anita Janiga and Dorota Gryko*◊
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
Received 14 August 2012
Accepted 15 October 2012
ABSTRACT: The design and synthesis of new molecular hybrids composed of protoporphyrin IX
(
PPIX) and vitamin B via copper catalyzed alkyne azide cycloaddition reaction is described. New,
12
clickable aminoazide and aminoalkyne linkers were prepared and subsequently attached to PPIX
via vinyl group) and to vitamin B12 giving desired building blocks. Preliminary results showed that
(
respective water soluble hybrids were formed under CuAAC reaction. Gratifyingly, Cu incorporation
into the PPIX core was avoided, which was important for further biological studies.
KEYWORDS: CuAAC, click chemistry, azides, alkynes, vitamin B12, protoporphyrin IX, molecular
hybrids.
decreases due to the growing tolerance [1]. Some new
INTRODUCTION
NO-independent activators of sGC have already been
introduced and their action depends on the heme in the
regulatory domain of the enzyme, similarly to NO [3].
Ignarro and coworkers showed that some corrin and
tetrapyrrole compounds, in particular protoporphyrin
IX (1, PPIX), strongly regulate sGC in in vitro studies,
however not in vivo [4]. It was proposed that porphyrin
For many years, nitroglycerine and other organic
nitrates have been used for treatment of cardiovascular
disorders, particularly coronary heart diseases, heart
failure and hypertension. Such drugs release nitric oxide
(
NO) by spontaneous decomposition or bioconversion.
This key signaling molecule is involved in the regulation
of a variety of biological and physiological processes
in mammals including blood pressure control and
neurotransmission via activating soluble guanylate
cyclase (sGC) [1]. The enzyme is activated by NO which
binds to the heme moiety in the regulatory domain.
Formation of nitrosyl heme disrupts the axial bond with
histidine 105, which leads to conformational changes in
the structure of the enzyme causing a dramatic increase
of its activity. The NO-activated enzyme catalyses the
transformation of guanosine triphosphate (GTP) into
cyclic guanosine monophosphate (cGMP), an increased
level of cGMP results in vasodilatation and inhibition
of platelet aggregation [2]. However, prolonged
administration of NO-releasing drugs causes many
unwanted side effects, and effectiveness of the therapy
1
interacts with the regulatory domain of sGC causing
its activation by replacing heme and inducing structural
changes similar to those occurring after complexation to
NO. It was also reported that the propionic acid groups
of porphyrin 1 are crucial as they interact with tyrosine
1
35 and arginine 139 of sGC [5]. Dicyanocobinamide,
another corrin activator discovered by Martin et al.,
unlike other sGC regulators interacts directly with the
catalytic domain of sGC by a mechanism of activation
which is still unknown [6].
Thanks to the existence of highly efficient dietary
uptake system in mammals [7], vitamin B12 has been
utilized as a delivery vehicle for several bioactive and
imaging molecules [8]. In particular, bioconjugates of
vitamin B12 have been recently used in efficient oral
delivery of insulin [8(h),8(j)] as well as anti-cancer
drugs [8(r)] and in cancer diagnostics [8(l),8(p)]. Since
the uptake system involves a series of transport proteins,
conjugates of vitamin B12 must be recognized by them.

SPP full member in good standing
*
Correspondence to: Dorota Gryko, email: dorota.gryko@icho.
edu.pl, tel: +48 22-343-2051, fax: +48 22-632-6681
Copyright © 2013 World Scientific Publishing Company

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
105
It has been shown that recognition of vitamin B by the
On the other hand, the role of vitamin B12 fragment
would be to deliver the whole hybrid from the intestine
to endothelium cells. Thus, it should be linked to PPIX
in such a way that neither molecule obstructs each other
and that does not interfere with vitamin B12 binding to
proteins. We chose the 5′-OH group as it can be readily
functionalized in a selective manner [8(b),8(p)]. The
other option would involve the synthesis of e-acid via
mild statistical hydrolysis [7].
1
2
proteins, involved in its uptake (haptocorrin, intrinsic
factor and transcobalamin II (TC-II)), is not impaired
only when it is modified at specific sites, such as 1)
5
′-hydroxy group on the ribose unit, 2) the phosphate
unit, and 3) the e peripheral propionamide unit. In the
B -TC-II complex the “tail” of vitamin B , containing
1
2
12
ribose ring and phosphate group is the only part of the B12
molecule accessible for the solvent [7(b)].
We envisaged that vitamin B12 could also facilitate
uptake of PPIX and as such increase its bioavailability.
Therefore, a hybrid molecule [9] composed of PPIX and
vitamin B moieties was designed. Herein, we describe
The final step that is connection of PPIX (1) to
vitamin B12 derivative could be achieved using the
copper-catalyzed azide alkyne cycloaddition reaction
(CuAAC) [11], which has been already used in the
preparation of various tetrapyrroles derivatives [12]. The
designed approach required the synthesis of linkers with
either terminal azide or alkyne moieties and on its other
terminus the amino group allowing its attachment either
to PPIX or vitamin B at positions specified above. The
1
2
the synthesis of linker molecules suitable for linking
vitamin B12 to PPIX (1), the synthesis of “clickable”
vitamin B and PPIX derivatives, and finally our attempts
1
2
to prepare their water-soluble bioconjugates.
1
2
strategy is outlined in Fig. 1.
RESULTS AND DISCUSSION
One potential difficulty with this method lay with
the incorporation of copper into porphyrins which was
very likely to occur under typical CuAAC conditions.
Protection of the PPIX core by metalation with, for
example, Zn would increase the number of synthetic
steps and subsequent Zn removal, performed on the
final hybrid, might be problematic. Therefore, we
concentrated on finding such conditions for CuAAC
under which Cu incorporation into the porphyrin core
would be avoided.
Design of the molecular hybrids
PPIX and its analogs can regulate sGC activity via
binding to the regulatory domain. It was postulated that
PPIX binds to the NO-dependent heme binding site.
Moreover, the propionic acid groups at positions 2 and
1
8 form electrostatic bonds with amino acids’ basic
groups (for example arginine) and such interactions are
critical for the enzyme activation [5]. Therefore, it would
be optimal to incorporate PPIX into the hybrid molecule
by linking it to vitamin B via meso-position or one of
Synthesis of aminoazide linkers
1
2
its vinyl groups. We chose the latter possibility as we
thought that it would not affect binding of PPIX to the
enzyme, and because methods for functionalization of
PPIX at vinyl groups are known [10].
Various aminoazide linkers were synthesized
starting either from terminal dibromides or ethylene
glycols. Selective monosubstitution of terminal
leaving groups in symmetrical 1,6-dibromohexane
CONH2
CONH2
H NOC
2
CN
Co
N
N
CONH2
N
N
H NOC
2
CONH2
O
N
HO2C
N
NH
N
NH
O
HO
N
vitamin B12 (2)
NH
O
HO2C
P
O
O O
OH
PPIX (1)
CuAAC
N3
L I N K E R
NH2
H N
2
L I N K E R
Fig. 1. The general strategy of preparation of molecular hybrids containing PPIX (1) and vitamin B12 (2)
Copyright © 2013 World Scientific Publishing Company
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106
R. LOSKA ET AL.
(i)
(ii)
(iii)
Br
PthN
PthN
N3
Br
Br
N3
H2N
3
a
4a (87%)
5a (92%)
6a (70%)
(iv)
(i)
(ii)
O
O
n
O
HO
OH
TsO
OTs
PthN
OTs
n
n
n = 1 3b (81%)
n = 3 3d (90%)
n = 1 4b (75%)
n = 3 4d (61%)
(v)
O
O
PthN
N3
H2N
N3
n
n
n = 1 5b (93%)
n = 3 5d (82%)
n = 1 6b (63%)
n = 3 6d (92%)
Scheme 1. (i) PthNH, K CO , DMF, rt; (ii) NaN , DMF, 60 °C; (iii) H NNH , EtOH/H O, 60 °C; (iv) TsCl, NaOH, THF/H O, 0 °C
2
3
3
2
2
2
2
to rt; (v) MeNH , EtOH, reflux
2
(i)
(ii)
to desired amines 6 possessing a terminal
azide group (Scheme 2) [13].
O
O
HO
OH
TsO
OTs
n
n
Synthesis of aminoalkyne linkers
n = 1, 2, 3
n = 1 3b (81%)
n = 2 3c (19%; + 30% mono)
n = 3 3d (90%)
The simplest alkyne linker used in this
work was propargylamine. A longer chain
analog could be obtained by the selective
alkylation reaction of 1,6-dibromohexane
(3a) with propargyl alcohol. Subsequent
substitution of -Br with -N group, using
3
(
iii)
standard conditions, gave azide 9a. The
labile azidoalkyne was directly reduced with
triphenylphosphine in the presence of water
to afford aminoalkyne 10a in 46% yield
O
O
N3
N3
H2N
N3
n
n
n = 2 7c (63%)
n = 3 7d (92%)
n = 1 6b (56% after 2 stages)
n = 2 6c (32%)
n = 3 6d (85%)
(Scheme 3).
A more efficient approach to aminoalkyne
linkers, which avoided the low-yielding
desymmetrization step and allowed to
prepare the unstable alkyneazides 9 under
milder conditions, involved introduction of
Scheme 2. (i) TsCl, NaOH, THF/H O, 0 °C to rt (for n = 1, 3) or TsCl, Py,
2
CH Cl , 0 °C to rt (for n = 2); (ii) NaN , DMF, 60 °C; (iii) Ph P, CH Cl /
2
2
3
3
2
2
THF/2M HCl(aq)
Br
(i)
(ii)
Br
(
3a) or glycol derivatives 3b and 3d with
Br(CH2)6O
phthalimide followed by the introduction of
the azide group at the other terminus gave
protected amines 5 in good yield. Treatment
of phtalimide derivatives 5a, 5b, and 5d with
methylamine afforded desired aminoazide
linkers 6 (Scheme 1).
+
OH
8
(54%)
(
iii)
N3(CH2)6O
H N(CH ) O
2
2 6
The simplified synthesis involved the
Staudinger reaction as a crucial step. Bis(tosyl)
derivatives 3b–3d were reacted with sodium
azide furnishing (bis)azides 7. Subsequent,
selective reduction of only one azide group led
9
a (95%)
10a (46%)
Scheme 3. (i) NaH, DMF, rt; (ii) NaN , DMF, rt; (iii) Ph P, THF/CH Cl /
3
3
2
2
H O, rt
2
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 106–117

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
107
the azido group as the first step, followed by the alkylation
allow to attach two molecules of vitamin B12 to one
porphyrin.
of azidoalcohols 12 with propargyl bromide, preferably
under PTC conditions with tetra-n-butylammonium
bromide (TBAB) as a catalyst. Reduction of azide 9 to a
primary amine was again accomplished under Staudinger
conditions. Starting monotosylates 11 of 1,w-diols were
The reaction of protoporphyrin dimethyl ester (13)
with Tl(NO ) ·3H O first gave dimethyl acetal derivatives
3
3
2
which after hydrolysis with formic acid afforded a
mixture of two isomeric, inseparable monoaldehydes 14
and bis(aldehyde) 15. Under optimal conditions desired
monoaldehydes 14 were obtained in 40% combined
yield and bis(aldehyde) 15 in 44% yield (Scheme 5). All
attempts to increase the yield of monoaldehydes 14 by
decreasing the amount of Tl(NO ) ·3H O or lowering
available through standard tosylation in NaOH/H O/THF
2
system with one equivalent of TsCl (Scheme 4).
Synthesis of PPIX derivatives with terminal azide and
alkyne linkers
3
3
2
the reaction temperature led to a very low conversion of
the substrate while an increase in the amount of oxidant
resulted in the predominant formation of bis(aldehyde)
Linkers 6 and 10 were attached to PPIX (1) by
oxidizing one or two vinyl groups of PPIX to the aldehyde
and subsequent reductive amination reaction.
Attempts to oxidize free PPIX (1) resulted in its
decomposition therefore dimethyl ester 13 was used as
a starting material. Oxidation of protoporphyrin IX ester
1
5.
A mixture of monoaldehydes 14, after separation
from bis(aldehyde) 15, was subjected to reductive
amination with linkers 6. First attempts were performed
as follows: aldehydes 14 were stirred with an amine and
1
3 with thallium(III) nitrate leads to PPIX bis(aldehyde).
a drying agent (Na SO ) in CH Cl to form two isomeric
This approach is one of the most convenient methods for
the synthesis of this molecule [14], but it has been also
reported to provide some quantities of monoaldehydes.
From our point of view such monoaldehydes would be
more desirable, though bis(aldehyde) would, in theory,
2
4
2
2
imines; after filtration and evaporation, crude imines
were reduced with NaBH in MeOH. This procedure
4
provided porphyrin derivative 16a in a reasonable yield
(
Scheme 6).
(
(
ii) (n = 1)
iii) (n = 2)
(
i)
(iv)
O
O
O
TsO
OH
N3
OH
N3
O
n
n
n
Br
n = 1 11a
n = 2 11b
n = 1 12a (85%)
n = 2 12b (62%)
n = 1 9b (46%)
n = 2 9c (84%)
O
H2N
O
n
n = 1 10b (68%)
n = 2 10c (83%)
(
i)
(iii)
(iv)
TsO
OH
N3
OH
N3
O
2
H N
O
11c
12c (83%)
9d (69%)
10d (78%)
Scheme 4. (i) NaN , DMF, rt; (ii) NaH, DMF, rt (iii) 50% NaOH , TBAB, toluene; (iv) Ph P, THF/CH Cl /H O, rt
3
(aq)
3
2
2
2
CHO
CHO
NH
N
N
NH
N
N
CHO
NH
N
N
NH
N
N
CHO
(
i)
+
+
NH
NH
NH
NH
MeO2C
CO2Me
MeO2C
CO2Me
MeO2C
CO2Me
MeO2C
CO2Me
13
14 (40%)
15 (44%)
Scheme 5. Oxidation of porphyrin 13. (i) (1) 2.2 equiv. Tl(NO ) ·3H O, (2) conc. HCl , (3) HCO H
3
3
2
(aq)
2
Copyright © 2013 World Scientific Publishing Company
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108
R. LOSKA ET AL.
H
N
NH
N
CHO
NH
N
N
N
(
i)
NH
N
N
(ii)
N
HN
HN
HN
N3
N3
MeO C
CO2Me
MeO C
CO2Me
6a (41%)
2
2
MeO2C
CO2Me
1
4 (+ the other regioisomer)
1
Scheme 6. Reductive amination of PPIX monoaldehydes 14. (i) 6a, Na SO , CH Cl ; (ii) NaBH , CH Cl /MeOH, 0 °C to rt
2
4
2
2
4
2
2
NHR
observed. Its yield could be increased by using
.3 equivalents of Tl(NO ) ·3H O instead of 2.2.
3
3
3
2
Other methods of attaching linkers to PPIX
NHR
+
NH
N
N
NH
N
N
(
i)
(1) through its vinyl groups were considered as
well, but they proved unsuccessful or inefficient.
Alkene metathesis reaction of ester 13 or its zinc
complex with Cbz-protected 8-amino-1-octene
gave only traces of unidentified product while the
Wittig reaction of a mixture of aldehydes 14 and
14
NH
NH
MeO C
CO2Me
MeO C
CO2Me
2
2
15 with an ylide stabilized with an amide group
led to the decomposition of the starting material.
N3
H2NR =
H2N
16a (80%)
16b (25%)
Preparation of vitamin B12 derivatives with
azide and alkyne linkers at 5’ position
H2N
O
N3
Linker molecules were attached to the 5′
hydroxyl group of the ribose ring of vitamin
B using the well-established methodology of
Scheme 7. Reductive amination of PPIX monoaldehydes 14. (i) RNH2,
NaBH CN, AcOH, MeCN
3
1
2
carbamate formation mediated by carbonyldiimidazole
CDI) or carbonylditriazole (CDT) [8(b),(p)]. In the
However, the above procedure did not work well
with linkers other than 6a and even in that case, the
yields were not reproducible. A much more reliable
(
first reaction propargylamine and aminoalkyne 10c gave
derivatives 18a and b in 35 and 38% yield respectively.
A similar reaction of 10c but using CDT instead of
CDI gave 82% of 18b. Therefore, the former coupling
reagent was used in other examples of azide- and alkyne-
functionalized vitamins 18 (Scheme 9).
protocol employed NaBH CN as a reducing agent
3
(
Scheme 7) [15].
PPIX aldehydes 14 and 15 are moderately stable and
their separation was troublesome. For these reasons,
in terms of yield, as well as the number of laboratory
operations needed and solvents and materials used, the
overallefficiencyofthesynthesisof“clickable”porphyrin
building blocks was further improved. When PPIX
aldehydes 14 and 15 were not separated and purified, but
instead the crude reaction mixture after oxidation and
hydrolysis was used directly in the reductive amination
reaction the overall yield increased. Using this protocol
several PPIX derivatives of type 16 and 17 were obtained,
containing both azide and acetylene linkers (Scheme 8).
Overall yields, based on ester 13, were moderate, but
they include 3 reaction steps: oxidation, cleavage of the
acetal and reductive amination. Unlike aldehydes 14
and 15, products 16 and 17 were reasonably stable and
could be fairly easily separated from each other and
characterized. Regioisomers of porphyrins 16 containing
one linker and one vinyl group could not be separated.
In some cases product 17 with two linkers was not even
These new derivatives 18 were easily separated from
small-molecule organic compounds (mainly DMSO after
the reaction) by precipitation from a mixture of Et O with
2
CHCl or CH Cl [8(p)] and washing with Et O. Further
3
2
2
2
purification involved reverse-phase chromatography
and then another precipitation and washing with Et O.
2
Purity of amines 18 was confirmed using reverse-phase
1
HPLC. H NMR and HPLC proved that the reaction was
selective at the 5’ primary hydroxyl group (products of
the reaction with 2′ hydroxyl were not observed).
Attempted synthesis of hybrid molecules
The first attempt of the CuAAC reaction of porphyrin
azide16bwithvitaminB derivedalkyne18c(Scheme10)
1
2
was performed under standard conditions: CuSO ·5H O,
4
2
sodium ascorbate, TBTA (tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine), t-BuOH/H O [11(a)]. The
2
Copyright © 2013 World Scientific Publishing Company
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DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
109
NHR
NHR
NHR
NHR
(
i), (ii)
NH
N
N
NH
N
N
NH
N
N
1
3
+
+
NH
NH
NH
MeO2C
CO2Me
MeO2C
CO2Me
MeO C
2
CO Me
2
1
6 (mono; mixture of isomers)
17a (15%*)
17 (bis)
N3
H2NR =
H2N
H2N
16d (21-27%)
7d (6-11%; 20%*)
1
H2N
O
O
N3
O
16b (27%)
7b (-)
H2N
O
16e (24%)
17e (0-4%)
1
O
O
H2N
O
N3
16c (21%)
7c (5%)
H2N
O
O
O
16f (25 %)
17f (0-5%)
1
H2N
*
3.3 equiv. of Tl(NO3)3
16g (25-30%)
17g (9%)
Scheme 8. Preparation of PPIX derivatives with linkers (i) (1) 2.2 equiv. Tl(NO ) ·3H O, (2) conc. HCl , (3) HCO H; (ii) linkers
3
3
2
(aq)
2
6
or 10, NaBH CN, AcOH, MeCN
3
CONH2
CONH2
CONH2
CONH2
CONH2
H2NOC
H2NOC
CN
CN
N
N
N
CONH2
N
N
N
N
Co
Co
(
i), (ii)
N
H2NOC
O
H2NOC
O
CONH2
CONH2
N
N
NH
O
NH
O
HO
HO
N
N
O
O
P
O
P
O
O O
O O
O
OH
O
R
N
2
18
H
O
O
RNH2 =
NH2
18a (35%*)
N
N
N
N
N
N
N
N
N
N
O
NH2
NH2
18b (82%)
18c (69%*)
O
O
CDI
CDT
O
NH2
N3
18d (63%)
18e (38%)
18f (63%)
O
N3
O
NH2
O
*
obtained using CDI
N3
O
NH2
Scheme 9. Preparation of vitamin B12 derivatives with azide and alkyne linkers: (i) 1.5 equiv. CDT, DMSO, 40 °C, 30 min. (ii) 3.3
equiv. RNH , rt, 24 h
2
Copyright © 2013 World Scientific Publishing Company
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110
R. LOSKA ET AL.
CONH2
CONH2
CONH2
N
H2NOC
H
CN
Co
O
N
N
NH
N
N
N3
N
N
NH
H2NOC
(i)
+
CONH2
O
N
NH
O
HO
N
MeO2C
CO2Me
O
P
O
16b
O O
O
(
+ the other regioisomer)
O
O
N
H
CONH2
18c
CONH2
CONH2
H2NOC
H2NOC
CN
N
N
N
N
Co
N
CONH2
H
O
O
N
N
N
N
N
N
NH
N
HO
N
N
Cu
O
O
O
P
O
O
O
O
O
N
H
MeO2C
CO2Me
1
9a
very low solubility in any solvent
Scheme 10. Cycloaddition reaction of porphyrin derivative 16b with vitamin alkyne derivative 18c: (i) CuSO ·5H O, sodium
4
2
ascorbate, TBTA, tBuOH/H O (5:1), rt, 20 h
2
formation of the expected product 19a was confirmed by
MS (as a copper complex), but unfortunately hybrid 19a
was insoluble in any solvents, most importantly in water.
It was probably due to the peculiar character of this
molecule — it contains a strongly hydrophilic vitamin
part and a hydrophobic porphyrin part.
Water-soluble porphyrin lithium salts 20 and 21
were then employed in CuAAC reactions. In order
to avoid copper insertion into the porphyrin core,
we tried conditions different from those shown in
Scheme 10, which was CuI/TBTA/sodium ascorbate
in EtOH/H O 1:1 or in DMF. The reaction of azide
2
Considering the potential application of the PPIX-
vitamin B12 hybrids, their water solubility was crucial.
Therefore, in the next reactions we decided to use PPIX
derivatives with hydrolyzed ester groups. Hydrolysis
of selected PPIX derivatives 16 and 17 was performed
using NaOH in MeOH/H O or LiOH·H O in THF/H O/
18f with porphyrin alkyne 20c in EtOH/H O led only
2
to the recovery of the starting materials, while the
reaction in DMF gave respective hybrids 19b and 19c
(Scheme 12). Under the developed conditions hybrid
19c was obtained in good yield. These conjugates were
fairly soluble in water and polar solvents like DMF
or DMSO allowing their purification by precipitation
and washing, similarly to vitamin derivatives 18.
However, chromatographic purification of these
compounds was troublesome therefore their structure
could only be, so far, confirmed by mass spectrometry.
Importantly, in the case of compound 19b ESI-MS
spectrum demonstrated no incorporation of copper into
the porphyrin core of the hybrid during the CuAAC
reaction carried out with CuI/TBTA/sodium ascorbate
catalytic system. Disappointingly, the main signal
in the ESI-MS spectrum of hybrid 19c was assigned
as a copper complex. The isotopic distributions for
the ESI-MS peaks of hybrids 19a–19c matched the
theoretical ones.
2
2
2
MeOH or dioxane/H O/MeOH (Scheme 11). When using
2
NaOH, some side products of PPIX decomposition were
formed and were very difficult to remove. Gratifyingly,
hydrolysis with LiOH proceeded smoothly though
purification and characterization of lithium salts 20 and
2
1 also presented difficulties. Reverse-phase C-18 silica
gel could not be used because of too strong adsorption on
stationary phase while cationites caused decomposition.
A fairly good method for their purification turned out to
be chromatography on lipophilic Sephadex with water
and MeOH as eluents. Signals in NMR spectra of such
amphiphilic porphyrins were broad, probably due to
formation of micelle-like aggregates. The best results
were obtained in DMSO-d or sometimes in CD OD.
6
3
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 110–117

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
111
RNH2 =
H2N
H2N
O
20c (56%)
NH
R
NH
R
NH
N
N
NH
N
N
(
i)
O
O
NH
NH
O
N3
20e (76%)
20f (51%)
O
H2N
O
MeO2C
CO2Me
LiO2C
CO2Li
1
6c, e
N
N
H
H
NH
NH
NH
N
N
NH
N
N
(
ii)
NH
NH
MeO2C
CO2Me
LiO2C
CO2Li
1
7d
21d (82%)
.
.
Scheme 11. Hydrolysis of the ester groups in PPIX derivatives 16, 17. (i) LiOH H O, dioxane/H O/MeOH; (ii) LiOH H O,
2
2
2
THF/H O/MeOH
2
CONH2
CONH2
CONH2
H2NOC
CN
N
N
N
N
Co
H
N
NH
N
N
(i)
H2NOC
O
1
8f
+ 20c
NH
CONH2
O
N
N
NH
O
HO
N
N
N
LiO2C
CO2Li
O
P
O
O O
O
O
CONH2
N
O
19b
O
N
H
O
CONH2
CONH2
H2NOC
CN
N
N
Co
N
H2NOC
O
CONH2
N
NH
O
HO
N
(
i)
H
O
18e + 20f
N
P
O
NH
N
N
O O
O
O
NH
O
O
N
N
N
H
O
N
LiO2C
CO2Li
19c
Scheme 12. Cycloaddition reactions. (i) CuI, TBTA, sodium ascorbate, DMF, rt
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 111–117

112
R. LOSKA ET AL.
m), 3.57 (3H, s), 3.59 (6H, s), 3.61 (3H, s), 3.65 (6H, s),
EXPERIMENTAL
3
4
.39 (4H, t, J = 7.8 Hz), 5.02 (4H, s), 9.85 (2H, s), 10.0
HH
(
1H, s), 10.1 (1H, s), 10.2 (2H, s).
Reductive amination of PPIX aldehydes. A mixture
General information
of 14 and 15 (crude, obtained from 0.542 mmol of
3) was dissolved in acetonitrile (75 mL) followed
Analytical grade solvents were used as received. All
reagents were purchased from Sigma Aldrich or POCH.
H and C NMR spectra were recorded at rt on Bruker
or Varian 500 MHz with TMS as an internal standard.
DCVC (dry column vacuum chromatography) was
performed using Merck Silica Gel (200–300 mesh).
Analytical thin layer chromatography (TLC) was
performed using Merck Silica Gel GF254, 0.20 mm
thickness and preparative TLC on Merck Silica Gel 60,
1
1
13
by the addition of acetic acid (250 ml) under an argon
atmosphere. Then, over a 15–20 min time period a
solution of primary amine (2.71 mmol) in MeCN (5 mL)
andAcOH (250 mL) was added dropwise, simultaneously
with portionwise addition of NaBH CN (8.24 mmol,
3
5
11 mg). After 1 h of stirring, the reaction mixture was
diluted with chloroform (c.a. 100 mL) and washed with
water (3 × 100 mL) and saturated Na CO (50 mL),
1
mm thickness. Chromatographic purification of water-
2
3(aq)
dried over Na SO and evaporated. The products were
soluble compounds was performed of LiChroprep RP-18
gel or Lipophilic Sephadex LH-20. High resolution ESI
mass spectra were recorded on a Mariner spectrometer.
UV-vis absorption spectra were recorded in DCM on a
Perkin Elmer λ-25.
2
4
separated using chromatography on silica gel starting
with 1-2% MeOH in CH Cl up to 33% MeOH. Each
2
2
product was then further purified on a new column or by
using a preparative SiO TLC plate.
2
-1
Porphyrin 16a. Yield 80%. IR (KBr): nmax, cm
3
1
311, 2924, 2857, 2688, 2091, 1733, 1434, 1361, 1194,
Synthesis of PPIX derivatives with terminal azide and
alkyne linkers
162, 1105, 906, 833, 726, 676. HRMS (ESI): calcd.
+
for C H N O ([M + H] ) 733.4184, found 733.4190.
4
2
53
8
4
Oxidation of porphyrin (13) — preparation of
PPIX adehydes 14 and 15 [14]. To a 250 mL round-
bottom flask porphyrin 13 (0.542 mmol, 320 mg) and
CH Cl (120 mL) were added under an argon atmosphere.
st
1
1
-
(
isomer: H NMR (500 MHz; CDCl ; Me Si): d , ppm
4.62 (2H, bs), 0.86 (2H, m), 0.95–1.32 (6H, m), 2.91
3
4
H
4H, m), 3.16 (4H, m), 3.40 (8H, m), 3.63 (3H, s), 3.64
3
2
2
(9H, s), 4.16–4.33 (6H, m), 6.02 (1H, dm, J = 10.5 Hz),
HH
The mixture was heated to 43 °C and a solution of
Tl(NO ) ·3H O (1.19 mmol, 530 mg) in MeOH (25 mL)
3
3
6
.16 (1H, dm, J = 18 Hz), 7.96 (1H, dd, J = 17.2
HH HH
13
3
3
2
Hz, 11.5 Hz), 9.33–9.80 (4H, m). C NMR (125 MHz,
was added in one portion with vigorous stirring of the
reaction mixture. After 15 min (monitoring by TLC – 1%
CDCl ; Me Si): d , ppm 11.7, 11.4, 11.5, 12.5, 21.6,
3
4
C
2
1.7, 26.2, 26.4, 28.4, 29.6, 31.4, 31.9, 36.8, 36.8, 48.3,
MeOH in CH Cl ) the reaction mixture was cooled (cold
2
2
48.4, 51.0, 51.7, 95.8, 96.1, 96.3, 96.7, 97.0, 97.1, 120.1,
water bath) and filtered through a pad of cotton. Conc.
HCl(aq) (2 mL) was added to the filtrate and it was stirred
nd
1
1
20.2, 130.1, 136.5 (bm), 173.5, 173.5. 2 isomer: H
NMR (500 MHz, CDCl ): d , ppm -4.62 (2H, bs), 0.86
3
H
vigorously. After about five minutes solid Na CO and
2
3
(2H, m), 0.95–1.32 (6H, m), 2.91 (4H, m), 3.16 (4H, m),
3.40 (8H, m), 3.63 (3H, s), 3.64 (9H, s), 4.16–4.33 (6H,
Na SO were added and then the solution was filtered and
2
4
evaporated. The residue (a mixture of dimethyl acetals
of porphyrin) was dissolved in 85% formic acid (about
3
3
m), 6.04 (1H, dm, J = 9.4 Hz), 6.20 (1H, dm, J
=
HH
HH
3
1
9
8.4 Hz), 7.96 (1H, dd, J = 18.1 Hz, 12.0 Hz), 9.33–
.80 (4H, m). C NMR (125 MHz; CDCl ; Me Si): d ,
HH
1
2
00 mL) and stored overnight at 0 °C or stirred at rt for
h. The acidic solution was then diluted with an equal
13
3
4
C
ppm 11.7, 11.4, 11.6, 12.6, 21.6, 21.7, 26.2, 26.4, 28.5,
29.7, 31.4, 31.9, 36.8, 36.8, 48.3, 48.4, 51.0, 51.7, 95.8,
amount of brine and extracted with CH Cl (3 × 100
2
2
mL). The combined organic layers were washed with
water (2 × 150 mL) and brine (150 mL), dried over a
mixture of Na CO (or K CO ) and Na SO , filtrated and
9
6.1, 96.3, 96.7, 97.0, 97.1, 120.1, 120.2, 130.1, 136.5
(bm), 173.5, 173.5.
-1
2
3
2
3
2
4
Porphyrin 17a.Yield 15%. IR (KBr): nmax, cm 3445,
concentrated.
3
1
311, 2934, 2858, 2326, 2092, 1732, 1435, 1362, 1259,
A mixture of PPIX monoaldehydes 14 was separated
from bis(aldehyde) 15 by column chromatography on
SiO using 2–5% MeOH/CH Cl .
195, 1164, 1105, 835, 726, 676. HRMS (ESI): calcd.
+
for C H N O ([M + H] ) 875.5403, found 875.5394.
4
8
67 12
4
1
2
2
2
H NMR (600 MHz; CDCl ; Me Si): d , ppm -3.98 (2H,
3
4
H
1
Monoaldehydes 14 [14]. Yield 40%. H NMR (200
bs), 1.28–1.38 (8H, m), 1.48 (4H, m), 1.59 (4H, m), 2.60
MHz; CDCl ; Me Si): d , ppm -3.84 (2H, bs), 3.27 (4H,
3
3
4
H
(8H, m), 3.10 (8H, m), 3.25 (4H, m, J = 8.4 Hz), 3.46
(3H, s), 3.48 (3H, s), 3.54 (3H, s), 3.55 (3H, s), 3.64 (3H,
HH
3
t, J = 7.8 Hz), 3.45–3.65 (9H, m), 3.65–3.80 (9H, m),
.38 (4H, m), 4.98 (2H, s), 6.18 (1H, d, J = 10.9 Hz),
.36 (1H, d, J = 17.8 Hz), 8.25 (1H, dd, J = 17.1
HH
3
4
6
3
HH
s), 3.66 (3H, s), 3.98 (2H, t, J = 7.8 Hz), 4.05 (2H, t,
HH
3
3
3
HH
HH
JHH = 8.0 Hz), 4.34 (4H, m), 9.84 (1H, s), 9.90 (1H, s),
Hz, 11.8 Hz), 9.80 (1H, s), 10.0 (2H, s), 10.1 (1H, s),
0.2 (1H, s).
Bis(aldehyde) 15 [14]. Yield 44%. H NMR (200
MHz; CDCl ; Me Si): d , ppm -3.82 (2H, bs), 3.27 (4H,
13
9
.91 (1H, s), 9.99 (1H, s). C NMR (150 MHz; CDCl ;
3
1
Me Si): d , ppm 11.6, 11.6, 11.6, 21.8, 24.1, 24.2, 26.6,
2
51.3, 51.7, 51.7, 57.5, 57.6, 59.8, 59.9.
4
C
1
6.7, 27.1, 27.1, 27.2, 27.2, 28.7, 28.8, 36.9, 42.4, 42.4,
3
4
H
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 112–117

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
113
-
1
st
Porphyrin 16b. Yield 27%. IR (film): nmax, cm 3686,
= 7.6 Hz), 3.53–3.64 (20H, m), 3.67 (2H, s, 1 isomer),
nd
3
6
7
(
(
600, 3474, 2953, 2110, 1734, 1606, 1438, 1171, 841,
80. HRMS (ESI): calcd. for C H N O ([M + H] )
3.70 (2H, s, 2 isomer), 4.21 (2H, m), 4.28–4.42 (4H,
m), 6.17 (1H, d, J = 11.5 Hz, 1 isomer), 6.18 (1H, d,
= 11.6 Hz, 2 isomer), 6.39 (1H, d, J = 17.7 Hz,
1 isomer), 6.41 (1H, d, J = 18.1 Hz, 2 isomer), 8.42
(1H, dd, J = 17.1 Hz, 11.6 Hz, 1 isomer), 8.46 (1H,
dd, J = 17.2 Hz, 11.6 Hz, 2 isomer), 10.11–10.17
HH
(2H, m), 10.17–10.24 (2H, m). 1 isomer: C NMR (125
MHz; CDCl ; Me Si): d , ppm 11.6, 11.6, 11.7, 12.7,
21.7, 36.9, 37.9, 50.4, 51.7, 71.5, 81.8, 96.1, 96.4, 96.6,
96.9, 97.0, 97.5, 120.5, 130.4, 137 (m), 173.5, 173.6. 2
isomer: C NMR (125 MHz; CDCl ; Me Si): d , ppm
11.6, 11.6, 11.8, 12.8, 21.8, 37.0, 37.9, 50.4, 51.7, 71.5,
81.8, 96.1, 96.4, 96.6, 96.9, 97.0, 97.5, 120.5, 130.4, 137
(m), 173.5, 173.6.
Porphyrin 17d. Yield 20%. IR (KBr): nmax, cm
3288, 2912, 2857, ~2130, 1732, 1637, 1435, 1363, 1226,
1195, 1163, 1108, 833, 737, 677. HRMS (ESI): calcd.
for C H N O ([M + H] ) 701.3810, found 701.3831.
H NMR (400 MHz; CDCl ; Me Si): d , ppm -3.86
(2H, bs), 2.16 (2H, m), 3.28 (4H, t, J = 7.7 Hz), 3.56
(8H, m), 3.60 (3H, s), 3.63 (3H, s), 3.65 (6H, s), 3.66
(6H, s), 4.21 (4H, m), 4.41 (4H, t, J = 7.6 Hz), 10.03
(1H, s), 10.05 (2H, s), 10.07 (1H, s). C NMR (100
MHz; CDCl ; Me Si): d , ppm 11.7, 11.8, 11.9, 21.8,
+
3
st
4
0
49
8
5
HH
nd
3
3
21.3820, found 721.3856. UV-vis (CH Cl ): lmax, nm
J
2
2
HH
st
HH
3
nd
log e) 404 (5.16), 502 (3.98), 536 (3.95), 570 (3.87), 625
HH
st
1
3
st
3.49). 1 isomer: H NMR (500 MHz; CDCl ; Me Si):
3
4
HH
3
nd
dH, ppm -3.84 (2H, bs), 2.99 (2H, m), 3.11 (2H, m), 3.27
4H, m), 3.45–3.54 (4H, m), 3.54–3.60 (6H, m), 3.61–
.64 (6H, m), 3.65–3.70 (8H, m), 4.21 (2H, m), 4.39 (4H,
m), 6.16 (1H, dm, J = 11.6 Hz), 6.34 (1H, dm, J
7.7 Hz), 8.26 (1H, dd, JHH = 17.8 Hz, 11.4 Hz), 9.99
1H, s), 10.02 (1H, s), 10.06 (1H, s), 10.16 (1H, s). 2
isomer: H NMR (500 MHz, CDCl ): d , ppm -3.84 (2H,
bs), 2.99 (2H, m), 3.11 (2H, m), 3.27 (4H, m), 3.45–3.54
4H, m), 3.54–3.60 (6H, m), 3.61–3.64 (6H, m), 3.65–
.70 (8H, m), 4.21 (2H, m), 4.39 (4H, m), 6.16 (1H, dm,
st
13
(
3
3
4
C
3
3
=
HH
HH
3
nd
1
(
nd
13
3
4
C
1
3
H
(
-
1
3
3
3
JHH = 11.6 Hz), 6.34 (1H, dm, J = 17.7 Hz), 8.25 (1H,
dd, J = 17.7 Hz, 11.7 Hz), 10.00 (1H, m), 10.03 (2H,
HH
3
HH
+
s), 10.06 (1H, s), 10.12 (1H, s).
4
2
49
6
4
1
Porphyrin 16c. Yield 21%. HRMS (ESI): m/z calcd.
for C H N O ([M + H] ) 809.4345, found 809.4326.
H NMR (500 MHz; CDCl ;Me Si): d , ppm -4.16 (2H,
bs), 2.88 (2H, m), 2.94 (4H, m), 3.04 (2H, m), 3.10
2H, m), 3.24 (4H, t, J = 7.6 Hz), 3.36 (4H, m), 3.42
2H, m), 3.52 (3H, s), 3.55 (5H, m), 3.59 (3H, s), 3.65
3H, s), 3.66 (3H, s), 3.67 (3H, s), 4.03 (2H, m), 4.34
4H, m), 6.13 (1H, m), 6.28 (1H, d, J = 17.7 Hz, 1
isomer), 6.31 (1H, d, J = 2 isomer), 8.15 (1H, dd,
3
4
H
+
3
4
4
57
8
7
HH
1
3
4
H
3
HH
3
13
(
(
(
(
HH
3
4
C
26.7, 36.9, 38.1, 50.6, 51.7, 51.7, 71.8, 81.6, 96.3, 96.7,
96.7, 96.8, 173.6. Anal. calcd. for C H N O ·H O: C,
3
st
HH
42 48
6
4
2
3
nd
70.17; H, 7.01; N, 11.69%. Found: C, 70.25; H, 6.78;
N, 12.18%.
HH
3
st
3
JHH = 18.0 Hz, 11.5 Hz, 1 isomer), 8.19 (1H, dd, J
17.8 Hz, 11.5 Hz, 2 isomer), 9.74–9.83 (2H, m),
.87–10.05 (2H, m). 1 isomer: C NMR (125 MHz,
HH
nd
=
9
Porphyrin 16e. HRMS (ESI): calcd. for C H N O
4
4
54
5
5
st
13
+
1
([M + H] ) 732.4119, found 732.4123. H NMR (600
CDCl ; Me Si): d , ppm 11.5, 11.5, 11.6, 11.6, 21.8,
MHz; CDCl ; Me Si): d , ppm -5.07 (2H, bs), 1.38 (2H,
3
4
C
3
4
H
4
3
9
6.9, 50.2, 51.5, 51.7, 69.4, 69.7, 69.9, 69.9, 70.0, 70.2,
6.0, 96.3, 97.0, 97.4, 120.4, 130.4, 136.2 (m), 137.4
m), 1.50 (2H, m), 1.86 (2H, m), 2.29 (1H, t, J = 2.2
HH
Hz), 2.89 (2H, m), 3.05 (2H, m), 3.11 (2H, m), 3.27 (2H,
m), 3.33 (2H, m), 3.49 (3H, s), 3.52 (3H, s), 3.59 (3H,
s), 3.63 (3H, s), 3.63 (3H, s), 3.64 (3H, s), 3.93 (2H, m),
4.11 (2H, m), 4.15 (4H, m), 5.94 (1H, m), 6.06 (1H, d,
nd
13
(
m), 173.5, 173.6. 2 isomer: C NMR (125 MHz;
CDCl ; Me Si): d , ppm 11.5, 11.6, 11.6, 11.8, 21.7,
3
4
C
3
9
6.8, 50.2, 51.4, 51.7, 69.4, 69.7, 69.9, 69.9, 70.1, 70.2,
6.0, 96.4, 96.9, 97.4, 120.4, 130.4, 136.2 (m), 137.4
3
st
3
nd
JHH = 18 Hz, 1 isomer), 6.11 (1H, d, J = 18 Hz, 2
HH
3
st
(
m), 173.5, 173.6. Anal. calcd. for C H N O ·1.5H O:
isomer), 7.77 (1H, dd, J = 18 Hz, 12 Hz, 1 isomer),
7.86 (1H, dd, J = 18 Hz, 12 Hz, 2 isomer), 9.23 (1H,
4
4
56
8
7
2
HH
3
nd
C, 63.22; H, 7.11; N, 13.40%. Found: C, 63.15; H,
HH
7
6
.11; N, 13.20%. Anal. calcd. for C H N O ·2H O: C,
2.54; H, 7.16; N, 13.26%. Found: C, 62.63; H, 7.24;
s), 9.39–9.64 (3H, m).
4
4
56
8
7
2
Porphyrin 17e. Yield 4%. HRMS (ESI): calcd. for
+
N, 13.42%.
C H N O ([M + H] ) 873.5273, found 873.5301.
5
2
69
6
6
Porphyrin 17c. Yield 5%. MS (ESI): m/z 514.4 ([M +
Porphyrin 16f. Yield 25%. HRMS (ESI): calcd. for
2
+
+
1
+
1
2
H] ), 1027.7 ([M + H] ). H NMR (400 MHz; CDCl ;
C H N O ([M + H] ) 734.3912, found 734.3909. H
3
43 52 5 6
Me Si): d , ppm -3.78 (2H, bs), 3.02 (4H, m), 3.07, (8H,
NMR (500 MHz; CDCl ; Me Si): d , ppm -4.16 (2H,
3
4
H
3
4
H
m), 3.14 (4H, m), 3.20 (4H, m), 3.29 (8H, m), 3.35–3.56
12H, m), 3.67 (18H, m), 4.28 (4H, m), 4.43 (4H, m),
bs), 2.16 (1H, m), 2.96 (2H, m), 3.23 (6H, m, J = 7.4
HH
(
Hz), 3.41 (6H, m), 3.42 (5H, m), 3.55–3.63 (7H, m), 3.66
(6H, m), 3.73 (2H, m), 4.02–4.17 (2H, m), 4.32 (4H, m),
1
0.10 (2H, m), 10.12 (2H, m).
-
1
3
3
Porphyrin 16d.Yield 27%. IR (KBr): nmax, cm 3309,
946, 2910, 1732, 1614, 1434, 1361, 1226, 1195, 1162,
107, 905, 832, 725, 677. UV-vis (CH Cl ): lmax, nm
6.13 (1H, dm, J = 11.3 Hz), 6.29 (1H, dm, J = 17.7
HH HH
3
2
1
Hz), 8.16 (1H, m, J = 17.2 Hz, 11.5 Hz), 9.80 (1H, s),
HH
9.90 (2H, s), 10.00 (1H, s). C NMR (125 MHz; CDCl ;
1
3
2
2
3
(
5
log e) 331 (3.68), 404 (4.65), 502 (3.41), 537 (3.41),
70 (3.39), 602 (2.92), 626 (2.92). HRMS (ESI): calcd.
Me Si): d , ppm 11.5, 11.6, 11.6, 11.8, 12.7, 12.8, 21.7,
4
C
21.8, 36.8, 36.9, 36.9, 48.5, 48.6, 51.1, 51.2, 51.7, 58.0,
58.0, 68.6, 68.6, 69.1 (m), 70.0, 70.1, 74.4, 74.4, 79.2,
96.0, 96.3, 96.5, 96.9, 97.0, 97.4, 120.4, 130.4, 136.0
(m), 137.4 (m), 173.5, 173.6.
+
for C H N O ([M + H] ) 646.3388, found 646.3383.
3
9
44
5
4
1
H NMR (500 MHz, DMSO-d , Me Si): d , ppm -4.08
6
4
H
3
(
2H, s), 3.11 (1H, m), ~3.30 (4H, m), 3.45 (2H, t, JHH
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 113–117

114
R. LOSKA ET AL.
Porphyrin 17f. Yield 5%. HRMS (ESI): calcd. for
Preparation of vitamin B derivatives with azide and
12
alkyne linkers at 5’ position
+
C H N O ([M + H] ) 877.4858, found 877.4860.
Porphyrin 16g. Yield 30%. IR (KBr): nmax, cm
309, 2912, 2862, 2446, 2111, 1732, 1610, 1435, 1349,
5
0
65
6
8
-1
Vitamin B12 (0.15 mmol, 200 mg) was dissolved in
dry, degassed DMSO (5 mL, ultrasonic bath, bubbling
argon for 15 min) at 35 °C under an argon atmosphere.
With stirring, solid CDT (0.225 mmol, 37 mg) was added.
After 30 min, amine (0.5 mmol) was added in dry DMSO
3
1
(
7
-
3
3
(
228, 1195, 1164, 1104, 912, 832, 726, 676. HRMS
+
ESI): calcd. for C H N O ([M + H] ) 778.4174, found
4
5
56
5
7
1
78.4166. H NMR (500 MHz; CDCl ; Me Si): d , ppm
4
3
4
H
4.37 (2H, s), 2.26 (1H, m, J = 2.2 Hz), 2.90 (2H, m),
HH
(0.5 mL). After 30 min the heating bath was removed and
.00 3.09 (4H, m), 3.12 (2H, m), 3.19 (4H, m), 3.340–
.42 (6H, m), 3.44 (3H, s), 3.45 (3H, s), 3.52 (3H, s), 3.63
the reaction mixture was stirred overnight. The reaction
mixture was then added dropwise to a vigorously stirred
4
3H, s), 3.64 (3H, s), 3.66 (3H, s), 3.84 (1H, d, J = 2.1
HH
1
:1 mixture of Et O and CH Cl (50 mL). After 30 min
st
4
nd
2 2 2
Hz, 1 isomer), 3.86 (1H, d, J = 2.1 Hz, 2 isomer),
HH
the clear solution above the red precipitate was removed
by decantation, the precipitate was separated using a
centrifuge, washed twice with CH Cl /Et O 2:1 and three
3
st
3
3
7
.98 (2H, t, J = 7.2 Hz, 1 isomer), 4.05 (2H, t, J
=
HH
HH
nd
3
.3 Hz, 2 isomer), 4.23 (4H, m), 6.06 (1H, d, J = 11.8
HH
nd
st
3
2
2
2
Hz, 1 isomer), 6.09 (1H, d, J = 11.7 Hz, 2 isomer),
HH
times with Et O. After drying in air, the precipitate was
3
st
3
2
6
=
1
.21 (1H, d, J = 17.8 Hz, 1 isomer), 6.26 (1H, d, J
17.9 Hz, 2 isomer), 8.03 (1H, dd, J = 17.9 Hz,
1.6 Hz, 1 isomer), 8.11 (1H, dd, J = 17.7 Hz, 11.5
Hz, 2 isomer), 9.64–9.90 (4H, m). C NMR (125 MHz;
CDCl ;Me Si): d , ppm 11.1, 11.1, 11.2, 11.5, 12.5, 12.9,
HH
nd
HH
dissolved in a small amount of water and separated using
3
HH
chromatography on RP gel using 0–30% EtOH in H O.
st
3
2
HH
After evaporation, the product was dissolved in DMF (2
mL) and further purified using the same precipitation,
centrifugation and washing procedure as described above.
Vitamin 18a. Yield 35%. HRMS (ESI): m/z calcd.
nd
13
3
4
C
3
6
9
(
(
6.3, 36.3, 46.4, 48.6, 51.3, 57.3, 57.4, 65.9, 68.4, 69.4,
9.6, 69.7, 69.8, 77.1, 80.2, 96.6, 96.9, 97.0, 97.1, 97.2,
7.2, 120.4. 130.0, 137 (m), 172.9, 172.9, 172.9. UV-vis
2
+
for C H N O PCoNa ([M + 2Na] ) 740.7837, found
6
7
91 15 15
1
2
7
40.7808. H NMR (400 MHz, DMSO-d ): d , ppm 0.34
6
H
CH Cl ): lmax, nm (log e) 403 (5.03), 502 (3.91), 536
2
2
(
(
(
3H, s), 0.85 (1H, m), 1.01 (3H, s), 1.03 (3H, s), 1.20
3H, s), 1.26 (3H, s), 1.35 (3H, s), ~1.4 (1H, m), 1.55
1H, m), 1.71 (3H, s), 1.73–1.80 (5H, m), 2.05 (3H, m),
3.83), 571 (3.74), 625 (3.41).
Porphyrin 17g. Yield 9%. IR (KBr): nmax, cm
444, 3261, 2945, 2866, 2447, 2111, 1733, 1438, 1351,
-1
3
1
2
.19 (3H, s), 2.21 (3H, s), 2.15–2.35 (5H, m), 2.42 (3H,
s), 2.35–2.51 (5H, m), 2.50 (3H, s), 2.70 (1H, m), 2.80
1H, m), 2.92 (1H, m), 3.11 (1H, m), 3.17–3.25 (2H, m),
251, 1196, 1104, 836, 732. HRMS (ESI): calcd. for
+
C H N O ([M + H] ) 965.5383, found 965.5382.
5
4
73
6
10
(
UV-vis (CH Cl ): lmax, nm (log e) 402 (5.03), 498 (3.77),
2
2
3
4
.66 (1H, m), 3.81 (2H, m), 3.96 (1H, m), 4.08 (1H, m),
5
33 (3.77), 567 (3.77, 621 (3.27).
3
.10–4.20 (3H, m), 4.33 (1H, d, J = 10.2 Hz), 4.65
HH
Hydrolysis of PPIX derivatives. Dimethyl ester of
a porphyrin derivative (0.130 mmol) was dissolved in
3
(
(
1H, m), 4.69 (1H, d, J = 8.3 Hz), 5.92 (1H, s), 6.24
1H, s), 6.43 (1H, s), 6.46 (1H, s), 6.57 (1H, s), 6.80 (1H,
HH
dioxane or THF (9 mL) and MeOH (1.5 mL). LiOH·H O
2
s), 6.83 (1H, s), 7.03 (1H, s), 7.07 (1H, s), 7.10 (1H, s),
.19 (1H, s), 7.24 (1H, s), 7.37 (1H, s), 7.41 (1H, d, JHH
7.2 Hz), 7.58 (1H, s), 7.62 (1H, s), 7.73 (1H, s), 7.75
(
3.25 mmol, 136 mg) and water (3 mL) were added and the
7
=
reaction mixture was stirred under an argon atmosphere
for 24 h. The reaction mixture was diluted with water (15
mL) and extracted with CH Cl (3 × 10 mL). Water phase
(
1H, s), 7.80 (1H, s).
Vitamin 18b. Yield 82%. IR (KBr): nmax, cm
346, 3200, 2936, 2134, 1668, 1573, 1498, 1402,
2
2
-1
was diluted with a few volumes of EtOH and evaporated.
The residue was dissolved in water (2-3 mL), separated
on a lipophilic sephadex column using first water and
then 30% MeOH in water and evaporated.
3
1
222, 1144, 1083, 559. HRMS (ESI): m/z calcd. for
2
+
C H N O PCoNa ([M + 2Na] ) 806.8230, found
8
s), 0.88 (1H, m), 0.95 (1H, m), 1.05 (3H, s), 1.11 (3H,
d, J = 6.5 Hz), 1.24 (3H, s), 1.26 (3H, s), 1.30 (3H,
7
3
103 15 18
1
2
06.8231. H NMR (600 MHz, D O): d , ppm 0.30 (3H,
2
H
Porphyrin 20c. Yield 56%. HRMS (ESI): m/z calcd.
+
for C H N O ([M + 3H] ) 704.3806, found 704.3802.
4
2
50
5
5
3
HH
Porphyrin 20e. Yield 76%. HRMS (ESI): m/z calcd.
s), 1.66–1.72 (2H, m), 1.72 (3H, s), 1.75–1.85 (4H, m),
+
for C H N O ([M + 3H] ) 781.4032, found 781.4040.
4
2
53
8
7
3
1
(
.85 (2H, m), 1.98 (1H, td, J = 14.1 Hz, 3.2 Hz), 2.04
1
HH
H NMR (500 MHz, DMSO-d ): d , ppm -3.92 (2H, bs),
6
H
3
2
1H, d, J = 13.7 Hz), 2.12 (6H, s), 2.26 (2H, AB, J
H
H
H
H
2
(
8
.88 (4H, m), 3.00–3.90 (34H, m), 4.28 (6H, m), 6.18
=
13.3 Hz), 2.30–2.57 (7H, m), 2.40 (3H, s), 2.42 (3H,
3
3
1H, d, J = 11.2 Hz), 6.42 (1H, d, J = 17.6 Hz),
HH HH
s), 2.61 (2H, m), 2.86 (1H, m), 3.15–3.24 (3H, m), 3.29
.49 (1H, m), 10.19 (1H, s), 10.28 (2H, s), 10.85 (1H, s).
3
(
3
1H, dd, J = 10.9, 5.2 Hz), 3.44–3.49 (5H, m), 3.49–
HH
Porphyrin 21d. Yield 82%. HRMS (ESI): m/z calcd.
3
.56 (6H, m), 3.94 (1H, d, J = 10.1 Hz), 4.01 (2H,
s), 4.04 (1H, dd, J = 8.9 Hz, 2.0 Hz), 4.07 (1H, m),
.15 (1H, m), 4.18 (1H, m), 4.46 (1H, d, J = 11.3 Hz),
.60–4.71 (2H, m), 5.93 (1H, s), 6.18 (1H, d, J = 2.4
Hz), 6.36 (1H, s), 6.96 (1H, s), 7.13 (1H, s). C NMR
150 MHz, D O): d , ppm 15.0, 15.2, 15.6, 16.6, 18.8,
+
HH
for C H N O ([M + 3H] ) 673.3497, found 673.3509.
4
0
45
6
4
3
1
HH
H NMR (500 MHz, CD OD): d , ppm 3.14 (2H, m),
3
H
3
4
4
4
HH
3
3
4
1
.16 (2H, m, J = 1.6 Hz), 3.30 (6H, m), 3.39 (2H, m),
HH
3
HH
.53 (5H, m), 3.59 (3H, s), 3.67 (m, 6H), 4.04 (2H, m),
.16 (2H, m), 4.44 (4H, m), 9.93 (1H, s), 10.07 (1H, s),
0.09 (1H, s), 10.34 (1H, s).
1
3
(
2
C
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 114–117

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
115
1
3
4
6
7
1
1
1
3
9
8.8, 19.1, 19.1, 19.2, 19.7, 25.8, 27.7, 31.1, 31.2, 31.5,
7.05 (1H, s), 7.09 (1H, s), 7.14 (1H, s), 7.32 (1H, s), 7.44
(1H, s), 7.50 (2H, s), 7.67 (2H, s), 7.76 (1H, s), 7.80 (1H,
1.7, 31.9, 34.2, 34.7, 36.8, 38.8, 40.1, 42.5, 42.7, 44.9,
7.0, 47.9, 51.2, 53.4, 55.5, 56.1, 57.7, 58.9, 62.7, 68.5,
8.5, 69.2, 69.4. 69.4, 69.5, 72.8, 73.1, 74.7, 78.8, 78.9,
9.7, 84.9, 86.8, 94.6, 103.9, 107.4, 111.2, 116.3, 129.7,
33.0, 135.0, 136.4, 141.6, 158.0, 165.1, 165.8, 173.4,
74.4, 174.9, 175.4, 175.6, 176.7, 176.8, 177.5, 177.6,
1
3
s). C NMR (150 MHz, DMSO-d ): d , ppm 15.4, 16.1,
6
C
16.8, 16.9, 19.1, 20.2, 20.3, 20.4, 20.5, 20.5, 26.0, 26.1,
26.3, 27.6, 28.6, 29.7, 30.3, 31.5, 31.9, 32.1, 32.1, 34.3,
35.6, 38.5, 40.6, 42.1, 42.6, 45.0, 47.0, 47.8, 50.7, 51.0,
53.4, 54.4, 55.4, 59.0, 63.4, 69.3, 70.7, 73.3, 75.2, 79.9,
84.8, 86.5, 94.0, 103.5, 106.3, 112.2, 116.7, 130.2, 131.6,
132.9, 136.7, 142.6, 156.6, 165.0, 165.8, 171.4, 171.5,
172.9, 173.0, 173.2, 173.7, 174.0, 174.3, 175.5, 178.7,
78.8, 179.9. UV-vis (H O): lmax, nm (log e) 278 (4.13),
2
05 (3.91), 322 (3.83), 361 (4.39), 408 (3.49), 548 (3.88),
68 (1.92).
Vitamin 18c. Yield 69%. HRMS (ESI): m/z calcd. for
179.9. UV-vis (H O): lmax, nm (log e) 278 (4.15), 305
2
2
+
C H N O Na PCo ([M + 2Na] ) 790.8280, found
(3.92), 322 (3.84), 361 (4.41), 408 (3.49), 519 (3.83), 549
(3.89), 962 (0.48).
7
3
103 15 16
1
2
7
(
(
90.8242. H NMR (600 MHz, DMSO-d ): d , ppm 0.32
6
H
1
3H, s), 0.86 (1H, m), 0.99 (3H, s), 1.02 (3H, s), 1.06
3H, s), 1.18 (3H, s), 1.21–1.32 (4H, m), 1.33 (3H, s),
Vitamin 18e. Yield 38%. H NMR (600 MHz,
DMSO-d ): d , ppm 0.29 (3H, s), 0.84 (1H, m), 1.02
6
H
1
.35–1.41 (2H, m), 1.45–1.52 (3H, m), 1.60–1.74 (2H,
m), 1.69 (3H, s), 1.75–1.84 (4H, m), 1.90–2.10 (3H, m),
.12–2.26 (4H, m), 2.16 (3H, s), 2.18 (3H, s), 2.27–2.45
(3H, s), 1.02 (3H, s), 1.14 (3H, s), 1.20 (3H, s), 1.29 (3H,
s), 1.46 (1H, m), 1.61 (1H, m), 1.66 (3H, s), 1.67–1.80
(5H, m), 2.00 (3H, m), 2.13 (3H, s), 2.14 (3H, s), 2.15–
2.21 (3H, m), 2.25 (1H, m), 2.32 (1H, m), 2.35 (3H, s),
2
3
(
(
(
7H, m), 2.30 (3H, s), 2.39 (2H, t, JHH = 5.4 Hz), 2.48
3H, s), 2.57 (1H, m), 2.77 (1H, m), 2.89 (1H, m), 2.95
2.38–2.51 (5H, m), 2.44 (3H, s), 2.57 (1H, m), 2.75 (1H,
3
3
3
2H, m), 3.09 (1H, d, JHH = 10.7 Hz), 3.15 (1H, dd, J
m), 2.86 (1H, m), 3.05 (1H, d, J = 10.4 Hz), 3.11 (2H,
HH
HH
3
=
10.5 Hz, 5.6 Hz), 3.25 (1H, dd, J = 10.5 Hz, 6.0 Hz),
.41 (2H, m), 3.69 (1H, m), 3.91 (1H, d, J = 10.8 Hz),
.01 (1H, m), 4.06 (1H, m), 4.09 (1H, dm, J = 2.4 Hz),
.15 (1H, m), 4.29 (1H, d, J = 9.8 Hz), 4.61 (1H, m),
m), 3.30–3.42 (5H, m), 3.54 (2H, m), 3.65 (1H, m), 3.91
HH
3
3
3
3
4
4
4
6
(1H, d, J = 10.5 Hz), 3.98 (1H, d, J = 9.9 Hz), 4.03
HH
HH HH
4
3
(2H, m), 4.13 (1H, m), 4.26 (1H, d, J = 10.0 Hz), 4.66
HH
HH
3
(2H, m), 5.84 (1H, s), 6.27 (1H, s), 6.30 (1H, s), 6.40
(1H, s), 6.50 (1H, s), 6.76 (2H, s), 6.97 (1H, s), 7.00 (1H,
s), 7.05 (1H, s), 7.09 (1H, s), 7.13 (1H, s), 7.33 (1H, s),
7.42 (1H, s), 7.50 (1H, s), 7.65 (1H, s), 7.72 (2H, s), 7.83
HH
3
.69 (1H, d, JHH = 7.0 Hz), 5.90 (1H, s), 6.28 (1H, s),
.36 (1H, s), 6.44 (1H, s), 6.53 (1H, s), 6.74 (1H, s), 6.78
1H, s), 7.03 (1H, s), 7.07 (1H, s), 7.08 (1H, s), 7.13 (1H,
(
1
3
s), 7.18 (1H, s), 7.36 (2H, s), 7.54 (1H, s), 7.68 (1H, s),
(1H, s). C NMR (150 MHz, DMSO-d ): d , ppm 15.5,
6
C
1
3
7
.71 (1H, s), 7.78 (1H, s), 7.81 (1H, s). C NMR (150
16.1, 16.8, 16.9, 19.1, 20.2, 20.3, 20.3, 20.5, 20.5, 26.0,
26.1, 27.5, 30.3, 31.5, 31.9, 32.1, 32.1, 34.3, 35.6, 38.5,
40.5, 42.1, 42.5, 45.0, 47.0, 47.8, 50.4, 50.7, 53.4, 54.4,
55.4, 59.0, 63.6, 69.3, 69.4, 69.4, 70.8, 73.2, 75.2, 79.9,
84.8, 86.5, 94.0, 103.5, 106.3, 112.1, 116.7, 130.2, 131.6,
133.0, 136.7, 142.6, 156.7, 165.0, 165.8, 171.4, 171.6,
173.0, 173.0, 173.2, 173.7, 174.0, 174.4, 175.5, 178.7,
MHz, DMSO-d ): d , ppm 15.0, 15.2, 16.4, 16.5, 18.7,
6
C
1
2
4
5
8
1
1
1
9.8, 19.9, 20.0, 20.1, 20.1, 25.3, 25.4, 26.0, 26.1, 27.1,
8.7, 28.8, 29.3, 29.9, 31.0, 31.6, 31.7, 34.0, 35.1, 38.0,
0.2, 42.0, 42.1, 44.6, 46.5, 47.3, 50.3, 52.9, 54.0, 54.9,
7.2, 58.6, 62.8, 67.2, 68.8, 69.1, 70.3, 74.8, 76.8, 76.9,
0.6, 84.4, 85.9, 93.5, 103.1, 105.8, 111.8, 116.4, 129.7,
31.3, 132.6, 136.2, 142.2, 156.1, 164.6, 165.4, 170.9,
71.1, 172.5, 172.8, 173.2, 173.3, 173.5, 173.6, 174.0,
78.2, 179.5.
179.9. UV-vis (H O): lmax, nm (log e) 279 (4.17), 306
2
(3.94), 323 (3.89), 361 (4.41), 518 (3.86), 548 (3.92).
Vitamin 18f. Yield 63%. IR (KBr): nmax, cm
3347, 3197, 2934, 2109, 1669, 1573, 1498, 1402,
-1
-
1
Vitamin 18d. Yield 63%. IR (KBr): nmax, cm
3
1
334, 3195, 2934, 2097, 1668, 1573, 1498, 1401,
1224, 1144, 1082, 559. HRMS (ESI): m/z calcd. for
2
+
238, 1145, 1071, 559. HRMS (ESI): m/z calcd. for
C H N O PCoNa ([M + 2Na] ) 822.3315, found
72 2
104 18 18
1
+
C H N O PCoNa ([M + Na] ) 1545.6577, found
822.3296. H NMR (600 MHz, DMSO-d ): d , ppm 0.29
7
0
100 18 15
1
6
H
3
1
0
1
1
545.6519. H NMR (600 MHz, DMSO-d ): d , ppm
(3H, s), 0.84 (1H, m), 1.01 (3H, d, J = 6.2 Hz), 1.02
6
H
HH
3
.29 (3H, s), 0.84 (1H, bs), 1.01 (3H, d, JHH = 6.6 Hz),
.02 (3H, s), 1.14 (3H, s), 1.20 (3H, s), 1.25 (4H, m),
.29 (3H, s), 1.37 (2H, m), 1.47 (2H, m), 1.56–1.70 (2H,
(3H, s), 1.14 (3H, s), 1.20 (3H, s), 1.30 (3H, s), 1.46 (1H,
m), 1.58–1.71 (2H, m), 1.66 (3H, s), 1.76 (4H, m), 2.01
(3H, m), 2.13 (3H, s), 2.15 (3H, s), 2.14–2.27 (3H, m),
2.33 (2H, m), 2.35 (3H, s), 2.38–2.48 (3H, m), 2.44 (3H,
s), 2.51 (2H, s), 2.55 (1H, m), 2.75 (1H, m), 2.86 (1H, m),
m), 1.66 (3H, s), 1.70–1.80 (5H, m), 1.95–2.40 (3H, m),
.13 (3H, s), 2.14 (3H, s), 2.15–2.28 (3H, m), 2.33 (1H,
m), 2.36 (3H, s), 2.38–2.50 (6H, m), 2.44 (3H, s), 2.55
2
3
3
3.06 (1H, d, J = 11.0 Hz), 3.09 (2H, m, J = 5.7 Hz),
HH
HH
3
3
(
(
1H, m), 2.74 (1H, m), 2.84 (1H, m), 2.92 (2H, m), 3.06
3.35 (2H, t, J = 4.8 Hz), 3.38 (2H, t, J = 6.2 Hz),
HH HH
3
3
1H, d, J = 10.7 Hz), 3.27 (2H, t, J = 7.1 Hz), 3.42
3.42 (1H, m), 3.45–3.51 (6H, m), 3.51–3.53 (2H, m),
HH
HH
3
3
3
(
1H, m), 3.65 (1H, dd, JHH = 9.3, 5.4 Hz), 3.91 (1H, d,
JHH = 10.5 Hz), 3.96 (1H, d, J = 10.1 Hz), 4.03 (2H,
m), 4.11 (1H, m), 4.25 (1H, d, J = 10.1 Hz), 4.66 (2H,
m), 5.84 (1H, s), 6.31 (1H, d, JHH = 9.1 Hz), 6.40 (1H,
3.56 (2H, t, J = 5.0 Hz), 3.65 (1H, dd, J = 9.5, 5.5
HH HH
3
3
3
Hz), 3.91 (1H, d, J = 10.7 Hz), 3.98 (1H, m), 4.01 (1H,
m), 4.05 (1H, m), 4.12 (1H, m), 4.23 (1H, d, J = 10.0
HH
HH
3
3
HH
HH
3
Hz), 4.66 (2H, m), 5.84 (1H, s), 6.31 (2H, bs), 6.40 (1H,
s), 6.49 (1H, s), 6.75 (2H, s), 6.95 (1H, s), 6.99 (1H, s),
s), 6.50 (1H, s), 6.75 (2H, s), 6.97 (1H, s), 6.99 (1H, s),
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 115–117

116
R. LOSKA ET AL.
7
.06 (1H, s), 7.09 (1H, s), 7.14 (1H, s), 7.32 (1H, s), 7.42
Hybrid 19c. MS (ESI): m/z 1140.5 ([C H N O
109 143 23 22
PCoCu] ).
2
+
(
1H, s), 7.50 (1H, s), 7.65 (1H, s), 7.68 (1H, s), 7.76 (1H,
1
3
s), 7.81 (1H, s). C NMR (150 MHz, DMSO-d ): d ,
ppm 15.5, 16.1, 16.8, 16.9, 19.1, 20.2, 20.3, 20.4, 20.5,
6
C
CONCLUSION
2
3
5
7
1
1
1
0.5, 26.0, 26.1, 27.5, 30.3, 31.5, 31.9, 32.1, 32.1, 34.4,
5.6, 38.5, 40.9, 42.1, 42.5, 45.0, 47.0, 47.8, 50.4, 50.7,
3.4, 54.4, 55.4, 59.0, 63.6, 69.3, 69.6, 69.7, 70.0, 70.1,
0.2, 70.2, 70.7, 73.2, 75.2, 79.9, 84.8, 86.5, 94.0, 103.5,
06.3, 112.2, 116.7, 130.2, 131.6, 133.0, 136.7, 142.7,
56.7, 165.0, 165.8, 171.4, 171.5, 172.9, 173.0, 173.2,
A series of PPIX derivatives containing one or two
linkers, attached through the PPIX vinyl group making them
suitable for CuAAC reaction, was synthesized. Considering
the versatility of CuAAC, these derivatives could be used
for bioconjugation with a broad variety of substrates. We
have also showed that propionyl methyl esters could be
easily hydrolyzed with lithium hydroxide in good yield.
CuAAC reactions of porphyrin derivatives with suitably
functionalized vitamin B derivatives gave desired, water
73.7, 174.0, 174.3, 175.5, 178.7, 179.9. UV-vis (H O):
2
lmax, nm (log e) 278 (4.15), 305 (3.93), 322 (3.85), 361
4.40), 408 (3.52), 519 (3.84), 549 (3.89), 962 (1.59).
(
12
CuAAC reaction of alkine 18c with azide 16b in
soluble hybrids. The presence of free carboxylate groups in
PPIX part are the prerequisite for hybrid’s water solubility.
Interestingly, under the optimal conditions for CuAAC
copper insertion into the PPIX part of the hybrid was
avoided, which is a rare case in the chemistry of porphyrins.
Further effort is needed to develop methods of processing
and purification of such hybrids to enable their appropriate
characterization and pharmacological evaluation.
t-BuOH/H O – hybrid 19a
2
PPIX azide derivative 16b (12 mmol, 8.9 mg), TBTA
16 mmol, 8.3 mg), sodium ascorbate (78 mmol, 15.5 mg)
(
and vitamin B12 alkyne derivative 18c (13 mmol, 20.0
mg) were added to degassed (ultrasonic bath, bubbling
Ar for 15 min) mixture of water and tert-BuOH (1:3.7,
2
4
.5 mL). After stirring for 10 min CuSO ·5H O (16 mmol,
4
2
.0 mg) was added and the reaction mixture was stirred at
Acknowledgements
rt for 20 h. By TLC analysis, the starting PPIX derivative
and most of vitamin B12 derivative was consumed. A
dark red precipitate was formed. It was filtered, washed
with water (3 × 2 mL), tert-BuOH (2 mL) and CH Cl
This work was supported by the European Regional
Development Found with the TEAM program, Grant no.
TEAM/2009-3/4.
2
2
(
3 × 2 mL). The solid obtained after drying (6 mg) was
insoluble in any common solvents (DMF, DMSO, etc.).
It was analyzed using MS ESI spectroscopy.
Supporting information
Experimental procedures for the synthesis of linkers
Hybrid
19a.
MS
(ESI):
m/z
2319.8
6
and 10 are given in the supplementary material. This
+
2+
(
[C H N O PCoCu] ),1159.9([C H N O PCoCu] ).
113 150 23 21
113 151 23 21
material is available free of charge via the Internet at
http://www.worldscinet.com/jpp/jpp.shtml.
CuAAC reaction in DMF
CuI (80 mmol, 15 mg) and TBTA (201 mmol, 107 mg)
were dissolved to dry, degassed (ultrasonic bath, bubbling
Ar for 15 min) DMF (5 mL). After 15 min, when the
solution was completely clear, vitamin derivative (18f or
REFERENCES
1. a) Chen Z, Zhang J and Stamler JS. Proc. Nat. Acad.
Sci. USA 2002; 99: 8306–8311. b) Nitric Oxide:
Biochemistry, Molecular Biology, and Therapeutic
Implications, Ignarro LJ and Murad F. (Eds.) Aca-
demic Press: 1995. c) Carpenter AW and Schoen-
fisch MH. Chem. Soc. Rev. 2012; 41: 3742–3752. d)
Maxwell AJ. Nitric oxide 2002; 6: 101–124.
2. a) Murad F. N. Engl. J. Med. 2006; 355: 2003–2011.
b) Martin E, Sharina I, Kots A and Murad F. Proc.
Nat. Acad. Sci. USA 2003; 100: 9208–9213. c)
Friebe A and Koesling D. Nitric Oxide 2009; 21:
149–156.
1
8e, 30.7 mmol), PPIX derivatived (20c or 20f, 28 mmol)
and sodium ascorbate (160 mmol, 32 mg) were added. The
reaction mixture was stirred overnight at rt and then it was
added dropwise to a vigorously stirred mixture of Et O
2
and CH Cl (1:1, 25 mL). After 30 min the clear solution
2
2
above the red precipitate was removed by decantation,
the precipitate was separated using a centrifuge, washed
twice with CH Cl /Et O 2:1 and three times with Et O.
2
2
2
2
After drying, the precipitate was dissolved in water and
separated by chromatography on RP gel. Unreacted
vitamin B12 was first removed with 10% EtOH in H O,
and the new product was then eluted with 50% EtOH
3. Boerrigter G and Burnett JC. Cardiovasc. Drug Rev.
2007; 25: 30–45.
2
in H O or 50% i-PrOH in H O, in some difficult cases
4. a) Wolin MS, Wood KS and Ignarro LJ. J. Biol.
Chem. 1982; 257: 13312–13320. b) Ignarro LJ,
Wood KS and Wolin MS. Proc. Nat. Acad. Sci. USA
1982; 79: 2870–2873. c) Waldman SA, Sinacore
MS, Lewicki JA, Chang LY and Murad F. J. Biol.
Chem. 1984; 259: 4038–4042. d) Dierks EA, Hu S,
2
2
basified with 2% LiOH. After evaporation it was dissolved
in DMF (1.5–2 mL), precipitated and washed in the same
way as described for the crude reaction mixture.
Hybrid 19b. MS (ESI): m/z 1147.9 ([C H N O
1
13 151 22 23
2
+
PCoLi ] ).
3
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 116–117

DESIGN AND SYNTHESIS OF PROTOPORPHYRIN IX/VITAMIN B12 MOLECULAR
117
Vogel KM,Yu AE, Spiro TG and Burstyn JN. J. Am.
j) Chalasani KB, Russell-Jones GJ, Yandrapu SK,
Diwan PV and Jain SK. J. Controlled Release 2007;
117: 421–429. k) Russell-Jones GJ, McTavish K,
McEwan J, Rice J and Nowotnik D. J. Inorg. Bio-
chem. 2004; 98: 1625–1633. l) Viola-Villegas N,
Rabideau AE, Bartholoma M, Zubieta J and Doyle
RP. J. Med. Chem. 2009; 52: 5253–5261. m) Bag-
nato JD, Eilers AL, Horton RA and Grissom CB. J.
Org. Chem. 2004; 69: 8987–8996. n) Hogenkamp
HPC, Collins DA, Live D, Benson LM and Naylor
S. Nucl. Med. Biol. 2000; 27: 89–92. o) Smeltzer
CC, Cannon MJ, Pinson PR, Munger Jr. JD, West
FG and Grissom CB. Org. Lett. 2001; 3: 799–801. p)
Lee M and Grissom CB. Org. Lett. 2009; 11: 2499–
2502. q) Alsenz J, Russell-Jones GJ, Westwood S,
Levet-Trafit B and de Smidt PC. Pharm. Res. 2000;
17: 825–832. r) Wilson S, Reinhard KS and Gao X.
US Patent 2007; 0066561 A1. s) Wang X, Wei L and
Kotra LP. Bioorg. Med. Chem. 2007; 15: 1780–1787.
9. a) Meunier B. Acc. Chem. Res. 2008; 41: 69–77. b)
Ojima I. Acc. Chem. Res. 2008; 41: 108–119.
10. Pavlov VY. Russ. J. Org. Chem. 2007; 43: 1–34.
11. a) Kolb HC, Finn MG and Sharpless KB. Angew.
Chem. Int. Ed. 2001; 40: 2004–2021. a) Meldal M
and Tornøe CW. Chem. Rev. 2008; 108: 2952–3015.
b) Iha RK, Wooley KL, Nystrom AM, Burke DJ,
Kade MJ and Hawker CJ. Chem. Rev. 2009; 109:
5620–5686.
Chem. Soc. 1997; 119: 7316–7323. e) Ignarro LJ.
J. Physiol. Pharm. 2002; 53: 503–514. f) Carr HS,
Tran D, Reynolds MF, Burstyn JN and Spiro TG.
Biochemistry 2002; 41: 10149–10157.
5
6
. Ignarro LJ, Ballot B and Wood KS. J. Biol. Chem.
1
984; 259: 6201–6207.
. a) o Proinsias K, Giedyk M, Sharina IG, Martin E
and Gryko D. ACS Med. Chem. Lett. 2012; 3: 476–
4
79. b) Sharina IG, Sobolevsky M, Doursout M-F,
Gryko D and Martin E. J. Pharmacol. Exp. Ther.
012; 340: 723–732.
2
7
. a) Gruber K, Puffer B and Kraütler B. Chem. Soc.
Rev. 2011; 40: 4346–4363. b) Petrus AK, Fairchild
TJ and Doyle RP. Angew. Chem. Int. Ed. 2009; 48:
1
022–1028. c) Wuerges J, Garau J, Geremia S,
Fedosov SN, Petersen TE and Randaccio L. Proc.
Nat. Acad. Sci. USA 2003; 100: 4386–4391. d) Wil-
bur DS, Pathare PM, Hamlin DK, Rothenberg SP
and Quadros EV. Bioconjugate Chem. 1999; 10:
9
12–920.
8
. a) Clardy SM, Allis DG, Fairchild TJ and Doyle RP.
Expert Opin. Drug Deliv. 2011; 8: 127–140. b) JF
McEwan, HS Veitch and GJ Russell-Jones. Biocon-
jugate Chem. 1999; 10: 1131–1136. c) Pathare PM,
Wilbur DS, Heusser S, Quadros EV, McLoughlin P
and Morgan AC. Bioconjugate Chem. 1996; 7: 217–
232. d) Russell-Jones GJ. Adv. Drug Del. Rev. 1996;
2
0: 83–97. e) Howard, Jr. WA, Bayomi A, Natara-
12. Dumoulin F and Ahsen V. J. Porphyrins Phthalo-
cyanines 2011; 15: 481–504.
jan E, Aziza MA, El-Ahmady O, Grissom CB and
West FG. Bioconjugate Chem. 1997; 8: 498–502.
f) Mundwiler S, Spingler B, Kurz P, Kunze S and
Alberto R. Chem. Eur. J. 2005; 11: 4089–4095. g)
Siega P, Wuerges J, Arena F, Gianolio E, Fedosov
SN, Dreos R, Geremia S, Aime S and Randaccio
L. Chem. Eur. J. 2009; 15: 7980–7989. h) Petrus
AK, Allis DG, Smith RP, Fairchild TJ and Doyle
RP. ChemMedChem 2009; 4: 421–426. i) van Sta-
veren DR, Benny PD, Waibel R, Kurtz P, Pak J-K
and Alberto R. Helv. Chim. Acta 2005; 88: 447–460.
13. a) Schwabacher AW, Lane JW, Schiesher MW,
Leigh KM and Johnson CW. J. Org. Chem. 1998;
63: 1727–1729. b) Klein E, DeBonis S, Thiede B,
Skoufias DA, Kozielski F and Lebeau L. Bioorg.
Med. Chem. 2007; 15: 6474–6488.
14. Kenner GW, McCombie SW and Smith KM.
Liebigs Ann. Chem. 1973; 1329–1338.
15. a) Borch RF, Bernstein MD and Durst HD. J. Am.
Chem. Soc. 1971; 93: 2897–2904. b) Lane CF. Syn-
thesis 1975; 135–146.
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