Sapphyrin derivatives and conjugates

The present invention provides various covalently modified sapphyrin derivatives and conjugates, and also, polymers including sapphyrin or derivatives thereof. Disclosed are water soluble sapphyrins, including polyhydroxysapphyrins and sapphyrin-sugar derivatives; sapphyrin-metal chelating conjugates; sapphyrin nucleobase conjugates; and polymer supported sapphyrins. Novel sapphyrin dimers, trimers, oligomers and polymers are also described, which polymers may include repeating units of sapphyrin or sapphyrin derivatives alone, or may further incorporate other units such as nucleobases.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to macrocyclic expanded porphyrin 
compounds, and particularly, to novel sapphyrin derivatives and 
conjugates, and polymers thereof. The covalently modified sapphyrin 
monomer derivatives of this invention include water soluble sapphyrins, 
such as polyhydroxysapphyrins and sapphyrin-sugar derivatives, 
sapphyrinmetal chelating derivatives, and sapphyrin nucleobase conjugates. 
The present invention also concerns polymer supported sapphyrins, and 
oligomers and polymers comprising sapphyrin or novel sapphyrin 
derivatives, either alone, or in combination with other units such as 
nucleobases. 
2. Description of the Related Art 
Expanded porphyrins are large pyrrole-containing macrocyclic analogues of 
the porphyrins (e.g. porphine, structure I, FIG. 1A). A number of expanded 
porphyrin systems are now known. However, only a few fully conjugated 
examples have been reported that contain more that four pyrrolic subunits, 
namely the smaragdyrins, sapphyrins, pentaphyrins, hexaphyrins, and 
superphthalocyanines.sup.1 (Sessler & Burrel, 1991). Sapphyrin, in its 
generalized substituent-free form, is represented by structure II (FIG. 
1B). Structure III (FIG. 1C) provides a generalized representation of 
.beta.-substituted sapphyrins. 
Sapphyrin, first discovered serendipitously by Woodward.sup.2 is one of the 
more intriguing products to emerge from initial studies directed towards 
the synthesis of Vitamin B.sub.12..sup.2,3 It is a 22 pi-electron 
pentapyrrolic macrocycle which exhibits an intense Soret-like band at 
about 450 nm (CHCl.sub.3) along with weaker Q-type transitions in the 620 
to 690 nm region. These optical properties, along with the presence of a 
large central cavity which serves for metal binding, renders sapphyrin 
useful for certain biomedical applications, including photodynamic therapy 
(PDT) and magnetic resonance imaging enhancement (MRI). 
In addition to the above, certain expanded porphyrins, including especially 
those of the sapphyrin series, have been found to act as halide anion 
chelating agents in both solution and the solid state.sup.4. This finding, 
along with an appreciation, that the diprotonated form of 
3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin acts as an 
efficient carrier for the through-dichloromethane-membrane transport of 
nucleotide monophosphates, such as e.g. guanosine-5' monophosphate, and 
related entities at acidic pH.sup.5, led the inventors to consider that 
the basic sapphyrin structure and related compounds such as the rubyrins, 
if suitably modified, could be used to bind, recognize, and transport 
phosphorylated entities at or near neutral pH. 
Unfortunately, all sapphyrins known at the time of this invention were 
known both to be essentially insoluble in water and also known to be 
ineffective as through membrane carriers for phosphate monoesters 
including those specifically that define the class of compounds known as 
nucleotides and nucleotide analogues.sup.5. These two deficiencies limited 
the potential utility of sapphyrins for any applications associated with 
their use at or near neutral pH and, more generally, any conditions 
involving partial or complete association with an aqueous environment. 
In addition, the sapphyrins known prior to the present invention were all 
of such simple character in terms of peripheral substituents, such that 
only hydrogen, alkyl and carboxy alkyl were known.sup.1,6 that said 
systems, even if they were to demonstrate binding to nucleotides or 
nucleotides, would be expected to do so without any degree of specificity 
with regards the nature of the nucleic acid base ("nucleobase") attached 
to the phosphate core. Thus, at the time of this invention, it was 
considered that the development of a sapphyrin-derived species capable of 
binding, recognizing, and/or transporting a mononucleotide (or related 
entity) at or near neutral pH would represent a significant advance, 
especially if such system or systems could be made to achieve this 
binding, transport, or recognition in a nucleobase specific manner. 
Furthermore, it was recognized that the synthesis of one or more water 
soluble sapphyrins would represent a considerable advantage, not only in 
terms of phosphate entity recognition and transport, but also because it 
would allow for a detailed study of the basic binding phenomena in aqueous 
media. This latter would be particular true if said water soluble 
sapphyrin were neutral in character. 
A considerable number of ionic (e.g. phosphorylated) nucleotide analogues 
are known that exhibit antiviral activity in cell-free extracts and yet 
are inactive in vivo due to their inability to cross lipophilic cell 
membranes.sup.7,8. The anti-herpetic agent, acyclovir, is typical in that 
it enters the cell only in its uncharged nucleoside-like form. However, 
this compound is phosphorylated in the cytoplasm resulting in the active, 
ionic triphosphate species. In contrast, many other potential antivirals, 
including the anti-HIV agent, Xylo-G, are not phosphorylated 
intracellularly and are therefore largely or completely inactive.sup.9. 
If, however, the active monophosphorylated forms of these putative drugs 
could be transported into cells, it would be possible to fight viral 
infections with a large battery of otherwise inactive materials. 
At present, no general set of nucleotide transport agents exists.sup.10. In 
early work, Tabushi was able to effect adenosine nucleotide transport 
using a lipophilic, diazabicyclooctane-derived, quaternary amine 
system.sup.10a. However, this same system failed to mediate the transport 
of guanosine 5'-monophosphate (GMP) or other guanosine-derived 
nucleotides. Since then, considerable effort has been devoted to the 
generalized problem of nucleic acid base ("nucleobase") recognition, and 
various binding systems have been reported. 
Currently known nucleotide binding systems include various acyclic, 
macrocyclic, and macrobicyclic polyaza systems.sup.10a-10n ; 
nucleotide-binding bis-intercalands.sup.10k ; guanidinium-based 
receptors.sup.10f,10n ; and various rationally designed H-bonding 
receptors.sup.10o-10u. These latter H-bonding receptors have been shown to 
be effective for the chelation of neutral nucleobase and/or nucleoside 
derived substrates but, without exception, have all proved unsatisfactory 
for the important task of charged nucleotide recognition. Thus, despite 
intensive efforts in this field, there is currently no synthetic system 
capable of effecting the recognition, or through-membrane transport, of 
phosphate-bearing species such as anti-viral compounds. Furthermore, there 
are presently no rationally designed receptors which are "tunable" for the 
selective complexation of a given nucleobase-derived system. 
Not surprisingly, the transport of larger polyphosphorylated compounds 
across cellular membranes also poses significant problems. The 
difficulties in transporting oligonucleotides across the plasma membrane 
and into mammalian cells is one of the factors currently limiting the 
successful application of antisense technology to human therapy. Further 
limitations may also result from the dynamics of oligonucleotide 
recognition, binding and functional inhibition which occurs 
intracellularly, subsequent to any import that does occur. 
There is clearly, therefore, a major need for novel drug delivery systems 
to be developed. Compounds which would allow negatively-charged (anionic) 
structures, particularly phosphate-bearing compounds, including 
nucleotides and nucleotide derivatives such as anti-viral compounds and 
anti-sense oligonucleotides, to be transported across naturally lipophilic 
cellular membranes would represent an important scientific and medical 
advance. 
In addition to the delivery of compounds to cells, there still remains, 
obviously, considerable scope for the design of improved chemotherapeutic 
compounds which act upon DNA once inside a target cell. Since currently 
available chemotherapeutic agents have complex structures, or complicated 
modes of interaction with their targets that preclude systematic 
improvement, the development of a novel class of DNA binding compounds 
would open up new avenues for the design of improved therapeutics. In this 
regard, a class of compounds that can be modified in a number of different 
ways whilst maintaining their overall monomeric, or preferably polymeric, 
structure would be particularly advantageous. The same is true for 
compounds that can be activated by light, or other means, to produce 
singlet oxygen or hydroxyl radicals, once bound to DNA. These 
considerations provided the present inventors with further impetus for the 
design and synthesis of improved sapphyrins such as those embodied by the 
present invention. 
SUMMARY OF THE INVENTION 
The present invention addresses these and other shortcomings in the prior 
art through the synthesis of several novel monomeric and polymeric 
sapphyrin derivatives and other monomeric and polymeric compounds, based 
generally upon the sapphyrin molecule. 
This invention encompasses new sapphyrin derivatives and conjugates and 
polymers thereof. In a general and overall sense, included within the 
novel compounds of the invention are covalently modified sapphyrin 
monomers of the following general types: water soluble sapphyrins, 
sapphyrin-metal chelating derivatives, sapphyrin nucleobase conjugates, 
polymer supported sapphyrins and sapphyrin polymers and oligomers. 
Oligomers and polymers will typically comprise sapphyrin or sapphyrin 
derivatives alone, or sapphyrin in combination with other units such as 
nucleobases, as well as complexes of sapphyrin-nucleobase polymers with 
oligonucleotides. 
In general terms, sapphyrin derivatives of the present invention can be 
defined by the following general structure: 
##STR1## 
wherein each of R.sup.1 -R.sup.10 is separately or collectively an H, 
alkyl, alkene, alkyne, halide, alkyl halide, hydroxyalkyl, glycol, 
polyglycol, thiol, alkyl thiol, aminoalkyl, carboxyalkyl, alkoxyalkyl, 
aryloxyalkyl, alkyloxycarbonyl, aryloxycarbonyl, aldehyde, ether, ketone, 
carboxylic acid, phosphate, phosphonate, sulfate, phosphate substituted 
alkyl, phosphonate substituted alkyl, or sulfate substituted alkyl, such 
that the total number of carbon atoms in each substituent R is less than 
or equal to 10. 
The novel aspect of the foregoing structure is the fact that in the context 
of the present invention, at least one R group substituent of the 
foregoing general structure will be of the general formula X-B, wherein X 
is any sapphyrin compound and B is a substituent that 1) confers water 
solubility, 2) is a metal chelating compound, 3) is a nucleobase compound, 
4) is a polymeric matrix or solid support, or 5) is a polymer or oligomer 
of sapphyrin or one of the foregoing sapphyrin derivatives. 
The novel aspects of the invention may most readily be denoted through the 
use of the structure X--(CH.sub.2).sub.n --A--(CH.sub.2).sub.m --B, 
wherein X is any sapphyrin macrocycle, and A is CH.sub.2, O, S, NH or 
NR.sup.11, wherein R.sup.11 is alkyl, alkene, alkyne, halide, alkyl 
halide, hydroxyalkyl, glycol, polyglycol, thiol, alkyl thiol, substituted 
alkyl, phosphate, phosphonate, sulfate, phosphate substituted alkyl, 
phosphonate substituted alkyl, sulfate substituted alkyl, COO, CONH, CSNH, 
or CONR.sup.11. 
The B substituent can include any alkene, alkyne, halide, alkyl halide, 
hydroxyalkyl, glycol, polyglycol, thiol, alkyl thiol, substituted alkyl, 
phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate 
substituted alkyl, sulfate substituted alkyl, hydroxyalkyl, sugar, sugar 
derivative, polysaccharide, metal chelating group, nucleobase, modified 
nucleobase, oligonucleotide, sapphyrin, sapphyrin derivative, polymeric 
sapphyrin, alkylating agent, steroid, steroid derivative, amino acid, 
peptide or polypeptide, polymeric matrix or solid support, and n is 0 to 
10 and m is 0 to 10. 
Certain particular embodiments of the invention relate to sapphyrin 
derivatives that are polyhydroxylated and therefore water-soluble. Water 
soluble sapphyrins are particularly desirable where one would like to 
exploit the various surprising properties of the sapphyrin macrocycle in 
connection with human or animal applications. The nature of the 
polyhydroxylation is not particularly critical to achieving water 
solubility of the sapphyrin derivative, so long as at least three or four 
hydroxyl groups per sapphyrin macrocycle are incorporated into the 
structure. The inventors have found that the introduction of at least 3 
hydroxyl groups per macrocycle will be sufficient to achieve some degree 
of water solubility. 
One means for introducing hydroxyl groups into the sapphyrin macrocycle 
structure is simply through the addition of alkyl substituents to the 
basic sapphyrin macrocycle unit, wherein the added substituents include 
one or more hydroxyl groups within their structures. Thus, exemplary 
polyhydroxylated sapphyrins will be those that are modified to include 
structures such as hydroxymethyl, hydroxyethyl, hydroxypropyl, 
hydroxybutyl, dihydroxyalkyl, trihydroxyalkyl, or the like, at one or more 
R positions of the basic sapphyrin structure shown above. Exemplary 
polyhydroxylated, water soluble sapphyrins are set forth in FIG. 2B, and 
are denoted as structures 1 and 3. 
An alternative means of achieving polyhydroxylation is through the addition 
of sugar moieties such as a saccharide, polysaccharide, saccharide 
derivative or aminosaccharide, to the sapphyrin macrocycle structure. In 
such cases, it has been found that the addition of a single saccharide 
molecule to a sapphyrin macrocycle will achieve a degree of water 
solubility. These structures are referred to broadly herein as simply 
sapphyrin-sugar compounds, conjugates or derivatives. The nature of the 
sugar is not particular critical to the achievement of water solubility, 
and a non-exhaustive, exemplary list of useful sugars in this regard are 
set forth in Table I. Of course, any sugar or modified sugar may be 
employed including sugars having additional phosphate, methyl or amino 
groups and the like. Moreover, the use of both D- and L- forms, as well as 
the .alpha. and .beta. forms are also contemplated. Particularly preferred 
are sugars such as glucose, galactose, galactosamine, glucosamine and 
mannose. Exemplary structures in this category are denoted as structures 
4, 4a, 4b, 4c, 6, 6a and 6b of FIG. 2B. 
In other aspects, the invention concerns sapphyrin derivatives which 
incorporate a metal chelator moiety at the B position. It has been found 
that the addition of a chelator moiety confers exciting new properties 
onto the sapphyrin macrocycle, including most notably an ability to cleave 
DNA through an as yet unknown mechanism. This is exciting because it 
allows one to prepare sapphyrin macrocycles that have the ability to both 
bind to and cleave DNA. Thus, not only will such molecules have clear in 
vitro uses, such as for DNA shearing or cleaving, but it opens the door 
for in vivo uses. For example, it is quite possible that 
sapphyrin-chelator complexes will have the ability to bind to, and cleave, 
the DNA of blood-borne viruses. Alternatively, it is possible that these 
structures will be useful in disrupting enzymatic action, by competing for 
requisite metal cofactors or by cleaving proteins. It is posited that due 
to their strong attraction for charged phosphate groups, the sapphyrins of 
the present invention will be particularly useful in selectively cleaving 
phosphorylated proteins, which are known to play a role in expression and 
activation of gene products including oncogene products. These structures 
may also be useful in an in vivo context through their introduction into 
cells, where they would be expected by the present inventors to effect 
cleavage of intracellular DNA or RNA. It may even be possible to effect a 
base or sequence specific cleavage through modification of the sapphyrin 
macrocycle structure, such as by substituent modification. 
In particular embodiments relating to chelator conjugates, the 
sapphyrin-chelator derivative will include a metal chelating group such as 
1,10-phenanthralene, EDTA, EGTA, DTPA, DOTA, crown ether, azacrown, 
catecholate or ethylene diamine. An exemplary structure is set forth as 
structure 8 of FIG. 3B, wherein the conjugated chelating group is EDTA. 
This particular molecule has been found to cleave DNA in a fashion that 
results in a "ladder" effect upon gel electrophoresis of the fragments 
that are generated. 
In still other embodiments, the present invention relates to what are 
referred to as sapphyrin-nucleobase conjugates. As used herein, the term 
"sapphyrin-nucleobase conjugate" is intended to refer broadly to any 
conjugate formed by the covalent conjugation of any sapphyrin macrocycle 
to any nucleobase. Moreover, as used herein the term "nucleobase"is 
intended to refer broadly to any moiety that includes within its structure 
a purine or pyrimidine, a nucleic acid, nucleoside, nucleotide, or any 
derivative of any of these. Thus, the term nucleobase includes adenine, 
cytosine, guanine, thymidine, uridine, inosine, or the like, bases, 
nucleotides or nucleosides, as well as any base, nucleotide or nucleoside 
derivative based upon these or related structures. A particular example of 
a useful nucleobase are the so-called antimetabolites that are based upon 
the purine or pyrimidine structure. These structures typically exert their 
biological activity as antimetabolites through competing for enzyme sites 
and thereby inhibiting vital metabolic pathways. However, in the context 
of the present invention, the inventors are employing the term 
"antimetabolite nucleobase" quite broadly to refer to any purine or 
pyrimidine-based molecule that will effect an anticellular, antiviral, 
antitumor or antienzymatic effect, regardless of the underlying mechanism. 
Exemplary structures are shown in Table 2, including preferred conjugates 
such as purine or pyrimidine antimetabolites such as FU, AraC, AZT, ddI, 
xylo-GMP, Ara-AMP, PFA or LOMDP. 
In still further embodiments, the nucleobase component of 
sapphyrin-nucleobase conjugates will include a protected nucleobase having 
attached substituents that protect the nucleobase from inappropriate or 
undesirable chemical reaction. Examples include substituents such as 
9-fluorenylmethyl carbonyl, benzyloxycarbonyl, 
4-methoxyphenacyloxycarbonyl, t-butyl oxycarbonyl, 1-adamantyloxycarbonyl, 
benzoyl, N-triphenylmethyl, N-di-(4-methoxyphenyl)phenylmethyl, and the 
like. 
It is contemplated that sapphyrin-nucleobase conjugates will have a wide 
variety of applications, ranging from their use as agents for selectively 
delivering an associated, biologically active nucleobase to a particular 
body or even subcellular locale. For example, in the case of 
antimetabolite nucleobases, it is contemplated that the 
sapphyrin-nucleobase conjugates will act to deliver the antimetabolite to 
subcellular sites through the DNA binding activity of the sapphyrin 
portion of the conjugate. Perhaps more importantly, it is recognized that 
many, many nucleobase antimetabolites can not be readily employed in 
therapy due to the fact that their charged nature inhibits their uptake by 
target cells, or otherwise inhibits or suppresses their unencumbered 
movement across biological membranes. Typically, this shortcoming is due 
to the presence of charged structures such as phosphates, phosphonates, 
sulfates or sulfonates on the nucleobase, which due to their charged 
nature prevents or inhibits their crossing of a biological membrane. It is 
proposed that sapphyrins of the present inventions can be employed as 
transport agents for carrying such nucleobases across membranes, (whether 
the nucleobase is directly conjugated to the macrocycle or simply 
complexed with it). 
Generally speaking, in the context of sapphyrin-nucleobase constructs 
designed for drug delivery it will usually be the case that one will 
employ only one nucleobase-containing substituent for each sapphyrin 
macrocycle, however this is in no way a limitation upon the invention. For 
example, sapphyrin-nucleobase conjugates of the present invention may have 
any number of nucleobases or nucleobase oligomers or polymers attached. 
The foregoing can be most readily appreciated through consideration of 
other embodiments and utilities that are contemplated by the inventors. 
For example, it has been surprisingly discovered by the inventors that 
sapphyrin-nucleobases which include a selected nucleotide nucleobase 
conjugate will serve to selectively bind, through hydrogen bonding 
interactions, the complementary nucleotide. Thus, a sapphyrin-nucleobase 
conjugate such as sapphyrin-adenine will selectively bind thymidine, 
presumably through a hydrogen bonding of the two nucleotides that is 
stabilized through the interaction of the charged phosphate groups of the 
hydrogen bonded nucleotide with the macrocycle structure. Such structures 
will likely have a wide variety of applications, such as intracellular 
carriers for nucleobases that are hydrogen bonded rather than being 
covalently attached. Furthermore, as discussed in more detail below, it is 
contemplated that polymers of sapphyrin-nucleobase conjugates can be 
employed to carry hydrogen bonded poly- or oligo-nucleotides into target 
cells through complementarity with the sequence of bases "encoded" on the 
sapphyrin-nucleobase polymer. 
The foregoing general structure could be exemplified by the formulas: 
##STR2## 
wherein X is the sapphyrin macrocycle, N is the conjugated nucleobase 
structure, and Y is the hydrogen bonded poly- or oligonucleotide. 
Alternatively, it is contemplated that sapphyrins of the present invention 
may serve as a carrier for polymers of nucleobases, wherein the nucleobase 
polymers are attached covalently to the sapphyrin macrocycle, such as 
might be exemplified through the structural designation: 
##STR3## 
wherein X is a single sapphyrin macrocycle, and N is a selected oligomeric 
or polymeric nucleotide or other nucleobase, and Y is a hydrogen bonded 
poly- or oligonucleotide. Such a structure would be useful in a number of 
contexts, such as a specific carrier for complementary nucleotides such as 
antisense molecules. 
Chemically speaking, any number of nucleobase structures can be attached to 
the sapphyrin macrocycle. The ultimate number of such residues that are 
attached will, of course, depend upon the application. Where one intends 
to employ such a structure to carry complementary nucleotides, one may 
well desire to employ a structure having a polymer of at least 10 or so 
bases attached. However, for other applications, such as for intracellular 
delivery of the nucleobase or other charged compounds of non-polymeric 
size, it may be convenient to design and employ sapphyrin-nucleobase 
constructs employing from one to three nucleobases per single macrocycle. 
Moreover, the nature of the intended use will dictate the number of 
attachment points there are on the sapphyrin macrocycle for attaching 
nucleobase moieties. Thus, it may typically be the case that a single 
attachment site will suffice for most applications, for certain particular 
applications those of skill may find it particularly advantageous to 
attach various nucleobases at various of the subcomponents of the 
macrocycle. 
A number of the simpler sapphyrin-nucleobase conjugates contemplated by the 
inventors are set forth in FIG. 3, and serve as simple examples of the 
overall concept. Thus, for example, one may wish to refer to structures 9, 
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 of FIG. 3A and 3B. 
As mentioned above, a particular aspect of the present invention involves 
the realization that novel sapphyrin structures may be prepared through 
the construction of sapphyrin polymers or oligomers. As used herein, the 
terms "sapphyrin polymer" or "polysapphyrin" are intended to refer to any 
compound which includes at least two sapphyrin macrocycles joined 
covalently. Moreover, the term "sapphyrin oligomer" or "oligosapphyrin" is 
intended to refer to sapphyrin-containing structures having a more defined 
length, such as from 2 or 3 up to 20, 30 or, at most, 40, sapphyrin 
units/molecule. 
In still further embodiments, the invention concerns compositions which are 
composed of a sapphyrin derivative in accordance with any one of the 
embodiments discussed above complexed to a second compound, wherein the 
second compound includes within its structure a negatively charged 
phosphate, phosphonate, sulfate, or sulfonate moiety. More particularly, 
the second compound will be one that will bind to the sapphyrin by means 
of its negative charge, afforded by a sulfate, sulfonate, an ester of 
sulfate or sulfonate, a phosphate, a phosphate mono or diester, a 
phosphonate or phosphonate ester moiety. 
In particular embodiments, the second compound will include a purine or 
pyrimidine, or an analog of either, within its structure. Exemplary 
structures include purine or pyrimidine antimetabolites such as FU, AraC, 
AZT, ddI, xylo-GMP, Ara-AMP, PFA or LOMDP. In other embodiments, the 
second compound of the composition will simply be DNA or RNA. 
In still further embodiments, the invention concerns a method for forming a 
complex between a sapphyrin derivative and a second compound which 
includes within its structure a negatively charged phosphate, phosphonate, 
sulfate or sulfonate moiety, wherein the method involves preparing a 
sapphyrin derivative as described above; obtaining the second compound; 
and contacting the sapphyrin derivative with the second compound under 
conditions effective to allow the formation of a complex between the 
sapphyrin derivative and the second compound. 
It will be appreciated by those of skill in the art that the invention is 
also generally applicable to the introduction of a sapphyrin molecule, 
alone or complexed with a second molecule, into an organism or, more 
generally, a cell contained within an organism. This may be employed as a 
means, for example, of successfully introducing the second compound 
(typically a charged compound) into the cell. An example might be 
introduction of a complex which includes an antimetabolic or antienzymatic 
compound such as an antiviral antimetabolic or antienzymatic compound, 
which one desires to introduce into a virally infected target cell. 
Another example would be the introduction of an antimetabolic or 
antienzymatic antitumor or antiproliferative compound that is introduced 
into a targeted tumor or proliferating cell. Of course, it is contemplated 
that the target cell may be located within an animal or human patient, in 
which case the complex is administered in effective amounts in an 
effective manner to the patient. 
Generally speaking, it is contemplated by the inventors that useful 
pharmaceutical compositions of the present invention will include the 
selected sapphyrin derivative (which preferably incorporates a water 
soluble sapphyrin macrocycle) in a convenient amount that is diluted in a 
physiological buffer, such as phosphate buffered saline. The route of 
administration and ultimate amount of material that is administered to the 
patient or animal under such circumstances will depend upon the intended 
application and will be apparent to those of skill in the art in light of 
the examples which follow. Preferred routes of administration will 
typically include parenteral or topical routes. 
In still further embodiments, the invention concerns a method of cleaving 
DNA comprising preparing a sapphyrin-chelator derivative and contacting 
DNA with said sapphyrin under conditions effective to promote cleavage of 
the DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Sapphyrins are large pyrrole-containing macrocyclic analogous to porphyrins 
(e.g. porphine, FIG. 1A, structure I,). The synthesis of various 
sapphyrins has been previously reported.sup.2-6,11,12, see also, U.S. Pat. 
No. 5,159,065, incorporated herein by reference. Structural information is 
available for a limited number of sapphyrin analogues, for 
example.sup.4,6,11. The present invention concerns a variety of new 
sapphyrin-based systems, in which the sapphyrin molecule has been 
derivatized in a number of novel ways. In particular, this invention 
encompasses, but is not limited to, four broad new groups of 
sapphyrin-based molecular structures. These may be generally defined as: 
(I) water soluble sapphyrins; (II) sapphyrin-metal chelating conjugates; 
(III) sapphyrin-nucleobase conjugates; and (IV) oligomeric and polymeric 
sapphyrin derivatives, of which a sub-group is the polymer-supported 
sapphyrins. 
Individually and collectively these new sapphyrin species overcome known 
deficiencies associated with extant sapphyrins. This is because all 
sapphyrins known at the time of this invention were exclusively monomeric 
in nature and insoluble in aqueous media at or near neutral pH. Thus, the 
sapphyrins known prior to the present invention were incapable of forming 
well-characterized, water soluble complexes with phosphorylated entities, 
including DNA, RNA, nucleotides, nucleotide analogues, and simple 
phosphate and phosphorate monoesters, at or near neutral pH. 
In addition, all sapphyrins known at the time of this invention were 
recognized to be quite limited in terms of their substitution patterns, 
bearing either hydrogens, alkyl groups, or carboxy alkyl groups in the 
so-called .beta.-positions. Thus, these systems could not and did not 
display any kind of binding selectivity as far as phosphate chelation was 
concerned; no specificity, for instance, for or against a particular 
nucleotide (i.e. guanosine-5'-monophosphate vs. cytosine 5'-monophosphate) 
was observed in such cases where such binding was inferred.sup.5. 
Furthermore, this same lack of substituent versatility meant that sapphyrin 
systems carrying potentially reactive side chains were completely unknown 
and this too was recognized as limiting the utility of those few 
sapphyrins known to be extant at the time of this invention. Thus, the 
inventors felt it worthwhile to prepare 1) water soluble sapphyrins, 2) 
sapphyrins bearing specific recognition units such as nucleobases, 3) 
sapphyrins bearing reactive sites, such as the metal chelating derivatives 
embodied in modified EDTA side chains, and 4) polymer supported sapphyrins 
and 5) oligomeric and polymeric sapphyrin systems, wherein the binding and 
recognition affects achieved in the monomeric sapphyrins might be expected 
to be greatly amplified. 
Water soluble porphyrin and porphyrin-like derivatives, such as sapphyrins, 
are known to be of interest in biomedical applications including 
photodynamic therapy (PDT).sup.1. The present inventors also recognized 
their potential for use in DNA recognition and modification. They reasoned 
that water soluble sapphyrin-based compounds without ionizable groups may 
be particularly advantageous for use in a number of ways, such as in PDT, 
cellular recognition and targeting and in the transport of biologically 
important molecules. 
Anionic phosphorylated entities are ubiquitous in biology. They play a 
critical role in a variety of fundamental processes ranging from gene 
replication to energy transduction..sup.13 In addition, certain 
phosphate-bearing nucleotide analogues, such as, e.g., 
9-(.beta.-D-xylofuranosyl)guanine-5'-monophosphate (Xylo-GMP), are known 
to display antiviral activity in vitro..sup.9 However, Xylo-GMP, like a 
considerable number phosphorylated nucleotide analogues which exhibit 
antiviral activity in cell-free extracts, is inactive in vivo.sup.9 due to 
its inability to cross lipophilic cell membranes.sup.7,8. 
The anti-herpetic agent, acyclovir 
(9-[(2-hydroxyethoxy)methyl]-9H-guanine), is active in vivo. Acyclovir can 
enter the cell only in its uncharged nucleoside-like form. Once in the 
cytoplasm, it is phosphorylated, first by a viral-encoded enzyme, 
thymidine kinase, and then by relatively nonspecific cellular enzymes to 
produce an active, ionic triphosphate nucleotide-like species. There it 
functions both as an inhibitor of the viral DNA polymerase and as a chain 
terminator for newly synthesized herpes simplex DNA. 
The biological limitations of many other potential antiviral agents, 
including Xylo-G, arise from the fact that they are not phosphorylated 
once inside the cell and are therefore largely or completely inactive. If, 
however, the active monophosphorylated forms of these putative drugs could 
be transported into cells, it would be possible to fight viral infections 
with a large battery of otherwise inactive materials. If such specific 
into-cell transport were to be achieved, it would therefore greatly 
augment the treatment of such debilitating diseases as, for example, AIDS, 
herpes, hepatitis and measles. Given the fact that AIDS is currently a 
major national health problem of frightening proportions, and that 
something so nominally benign as measles still claims over 100,000 lives 
per year world-wide, treatment of these diseases would be particularly 
timely and worthwhile. 
Not surprisingly, in recent years, increasing effort has been devoted to 
the problem of phosphate recognition and a number of phosphate-binding 
receptors are now known..sup.10 In spite of this, there are currently no 
artificial entities capable of effecting the selective through-membrane 
transport of mononucleotides and oligomeric polynucleotides at neutral or 
near-neutral pH, i.e., at a biological pH. A major aim of the inventors 
studies has been therefore to provide a means of transporting active mono- 
and polyphosphorylated forms of these and other agents into cells. This 
would allow a wide range of otherwise inactive compounds, such as 
antivirals, to be employed therapeutically, and would also create new 
possibilities for gene therapy. 
In preliminary work concerning nucleoside transport, the present inventors 
employed triisopropylsilyl (TIPS) substituted (phosphate-free) 
nucleosides.sup.14. It was found that efficient and selective 
through-membrane transport of non-charged nucleoside analogues could be 
achieved by using the complementary TIPS derivatives as carriers.sup.14. 
Not surprisingly, however, these same TIPS derivatives proved completely 
ineffective as transport agents for the analogous phosphate-containing 
nucleotide derivatives. Thus, whilst confirming the viability of a 
base-pairing approach to selective nucleotide recognition, this work 
served to highlight further the need for an organic soluble, neutralizing, 
phosphate binding group. 
The inventors reasoned that if sapphyrin-based systems were to be made 
effective as neutral-regime carriers, say, e.g. for GMP, it would require 
the construction of polytopic receptor systems in which a nucleobase 
recognition unit, in this case, a cytosine-like group, were "appended" 
directly onto the phosphate-chelating expanded porphyrin core. Naturally, 
they also contemplated the use of nucleobases recognition units other than 
cytosine for use in the specific binding and transport of the 
complementary nucleobases and nucleobase-containing compounds. 
To synthesize multitopic receptors, the inventors developed strategies to 
address the following objectives: (i) the independent development of 
molecular recognition strategies for the complexation of two very 
different kinds of substrates (charged anionic and neutral nucleobase); 
(ii) their subsequent co-combination so as to provide receptors bearing 
both kinds of binding subunits; and (iii) various alternative methods of 
receptor oligomerization so as to provide oligomeric species bearing 
numerous combinations of multitopic receptors. 
Pursuing these strategies led to the development of the sapphyrin-based 
ditopic receptor systems of the present invention, capable of recognizing 
both the anionic phosphate and the neutral portions of the nucleotide 
derivatives, such as the purine or pyrimidine moieties. Molecules of this 
type are, indeed, capable of the binding and transport of nucleotides and 
their derivatives. This theme was extended to the preparation of 
oligomeric, multitopic, receptors capable of recognizing multiple 
phosphate anions and nucleobase portions of nucleotide derivatives 
arranged in specific sequences. 
The ditopic receptor systems are ideal vehicles for the intracellular 
transport of nucleotides and their derivatives, including anti-viral 
agents. The multitopic receptors, likewise, are contemplated to be of use 
in binding to oligonucleotides and specific sections of DNA or RNA and in 
transporting such nucleic acid segments into cells. The phosphate and 
nucleic acid base ("nucleobase") recognition, through-membrane transport 
and cell delivery properties of the present invention are thus applicable 
to the recognition and delivery of a large variety of monomeric and 
oligomeric species, including DNA, RNA and antisense constructs. 
As outlined above, the present invention therefore encompasses, but is not 
limited to, the following four groups of novel sapphyrin-based molecular 
structures: (I) water soluble sapphyrins; (II) sapphyrin-metal chelating 
conjugates; (III) sapphyrin-nucleobase conjugates; and (IV) polymer 
supported sapphyrins and oligomeric and polymeric sapphyrin derivatives. 
The water soluble sapphyrins include sapphyrin hydroxyalkylamide and sugar 
derivatives. The sapphyrin-nucleobase conjugates include an extensive 
group of structures in which nucleobase-like recognition units are 
appended directly into the phosphate-chelating sapphyrin core. The last 
broad group comprises both polymer supported sapphyrins, and also 
oligomers and polymers of sapphyrin or sapphyrin derivatives and 
sapphyrin-nucleotide oligomers and polymers. The oligomeric linkage in 
such molecules may take place at various places in the sapphyrin or 
sapphyrin-nucleobase monomers. 
In addition, it will be understood that the synthetic strategies developed 
by the inventors, wherein a functionalized sapphyrin is appended to a 
moiety of desirable chemical function, can be used to prepare an extremely 
wide variety of sapphyrin-containing conjugates. Sapphyrins may thus be 
conjugated to, not only metal chelating agents, sugars, nucleobases, and 
other sapphyrins, sapphyrin derivatives, or polysapphyrins, but also to a 
variety of other substances. These include, for example, phosphates, 
phosphonates, sulfates, sulfonates, amino acids, peptides, polypeptides, 
steroids, steroid derivatives, alkylating agents, and polymers glasses or 
solids, such as agarose, polyacrylamide, controlled pore glass, silica 
gel, polystyrene or sepharose. It is contemplated that one of skill in the 
art will be able to prepare sapphyrin conjugates including those listed 
above, without undue experimentation, given the extensive synthetic 
methodology disclosed throughout the present application 
I. WATER SOLUBLE SAPPHYRIN DERIVATIVES 
Water soluble porphyrin and porphyrin-like derivatives, especially 
sapphyrin derivatives, are of potential interest in a variety of 
applications ranging from photodynamic therapy (PDT) to DNA recognition 
and modification to cellular recognition and transport. The present 
inventors considered that the development of water soluble sapphyrin-based 
compounds without ionizable groups would likely be advantageous in a 
number of these applications. 
In regard to PDT, water soluble sapphyrins may be used as 
photosensitization agents for the photodynamic inactivation of infectious 
agents having membranous envelopes. As such they may be employed in the 
photo-eradication of cell-free viruses from blood samples, such as, for 
example, the hepatitis viruses HBV and NANB, and especially HIV-1. In this 
process, sapphyrin localizes selectively at or near the morphologically 
characteristic viral envelope. Upon photoirradiation, it catalyzes the 
formation of highly reactive singlet oxygen which, in turn, destroys the 
essential membrane envelope, thus killing the virus and eliminating its 
infectivity, see U.S. Pat. No. 5,041,078, incorporated herein by 
reference. 
The search for compounds for use in in vivo cellular transport and uptake, 
where diffusion across a membrane is involved, led the inventors to 
synthesize and characterize a range of novel water soluble sapphyrin 
derivatives. Generally speaking, the water soluble sapphyrin derivatives 
of this invention will include at least four OH groups, such as can be 
supplied by a variety of different polyhydroxy groups, or a single sugar 
residue. This broad class of water soluble sapphyrins can be further 
divided into water soluble polyhydroxysapphyrins and water soluble 
sapphyrin sugar derivatives. These groups include a variety of distinct 
molecules, such as, for example, the compounds represented by structures 1 
and 3-6 (FIG. 2B), and substituted derivatives thereof. 
Naturally, those of skill in the art will understand that a wide range of 
water soluble sapphyrins are encompassed by the present invention. Both a 
variety of polyhydroxysapphyrins and sapphyrin sugar derivatives may be 
synthesized according to the methodology disclosed herein. For example, 
any one, or more, of the many sugar and modified sugar units depicted in 
Table 1 may be linked to a sapphyrin core to create a water-soluble 
sapphyrin in accordance herewith. 
A. Water Soluble Polyhydroxysapphyrins 
These are water soluble sapphyrin derivatives based on two 
(poly)hydroxyalkylamido units attached to the macrocyclic periphery. 
Examples of compounds of this type include those represented by structures 
1 and 3 (FIG. 2B). Although, naturally, it will be understood that a wide 
variety of different, and yet analogous, substituted derivatives may be 
prepared in accordance herewith. Polyhydroxysapphyrins may be prepared 
from an activated form of a sapphyrin acid (acid chloride, mixed 
anhydride, O-acylurea derivative, N-acylimidazole) and polyhydroxyamines. 
B. Water Soluble Sapphyrin-Sugar Derivatives 
The second general group of water soluble sapphyrin derivatives are the 
sapphyrin-sugar derivatives, where any one of a number of various sugar 
subunits are connected to the macrocycle periphery. Specific examples of 
this type of compound are represented by structures 4 & 6 (FIG. 2B). 
Again, in light of the present disclosure, those of skill in the art will 
be able to prepare a wide variety of distinct sapphyrin-sugar derivatives 
without undue experimentation. Examples of sugars and sugar-derivatives 
that may be employed in accordance herewith are listed in Table 1. The 
sugars employed may be either D or L forms and may also be either .alpha. 
or .beta. forms. The use of modified sugars is also envisioned, such as 
those including, for example, phosphate, methyl or amino groups. It is 
contemplated that preferred sugars for use in accordance herewith will 
include, for example, glucose, glucosamine, galactose, galactosamine and 
mannose. 
TABLE 1 
______________________________________ 
Examples of Sugars and Sugar Derivatives 
______________________________________ 
Ribose Fructose 
Arabinose Sorbose 
Xylose Tagatose 
Lyxose Fucose 
Allose 
Altrose Methylglucoside 
Glucose Glucose 6-phosphate 
Mannose 
Gulose N-Acetylgalactosamine 
Idose N-Acetylglucosamine 
Galactose Sialic Acid 
Talose 
Ribulose 
Xylulose 
Psicose 
______________________________________ 
For example, the efficiency of those compounds shown in FIGS. 4c and 6b for 
singlet oxygen generation has already been tested, when it was found to be 
11% (in comparison with ZnTPPS.sub.4). These water soluble sapphyrins 4c 
and 6b thus have apparent utility as a potential cellular targeting agent. 
As is generally known, glycoconjugates have important roles in the control 
of cell division and intercellular association. Changes in the biochemical 
and organizational structures occur during malignant 
transformation.sup.15. Therefore it may be therapeutically advantageous to 
alter or inhibit the biosynthesis of these tumor cell surface 
constituents. This might result in tumor cell death caused by the 
inhibition of the biosynthesis of vital membrane components. In this 
regard, D-glucosamine derivatives have been proven to be efficient 
inhibitors of tumor growth.sup.16. 
It is envisioned that differential tumor toxicity and specific organ 
targeting can be achieved with different sugar-sapphyrin derivatives. For 
instance, modified sugars such as e.g., Glc-NAc can be included within the 
image of substituents that can be appended to the sapphyrin core. All that 
would be needed is to start with an activated sapphyrin and 
2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-.beta.-D-glucopyranosylamine. The 
synthesis and use of this and other related systems are thus considered to 
fall within the scope of the present invention. 
The synthesis of representative compounds 4 and 6 involve compounds that 
are connected via glycoside bonds and obtained starting from 
dihydroxysapphyrin and .alpha.-D-acetobromoglucose, as precursors, and 
using silver triflate in dichloromethane to effect coupling. The second 
set of systems is connected via amide bonds. These later materials are 
obtained starting from sapphyrin(bis)acid chloride and 
1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-.alpha.-D-glucopyranose 
(tetraacetyl-D-glucosamine). In both series, after removing the protecting 
groups from the sugar moieties, the desired water soluble sapphyrin 
derivatives may be obtained. 
It is contemplated that compounds such as those represented by structures 4 
and 6 will have utility in both photophysical and biological embodiments. 
For example, it is envisioned that these new sapphyrins will have improved 
solubility and/or phosphate anion chelation properties, rendering them of 
use in protocols such as anti-viral transport and RNA/DNA recognition and 
binding. 
II. SAPPHYRIN-CHELATING CONJUGATES 
The second general group of novel monomeric sapphyrin derivatives of the 
present invention are the sapphyrin-metal chelating derivatives. Suitable 
chelating groups contemplated for use in such conjugates include, but are 
not limited to, EDTA, EGTA, DTPA, DOTA, ethylene diamine, bipyridine, 
1,10-phenanthralene, crown ether, aza crown and catechols. 
A specific example of this type of sapphyrin derivative is the 
sapphyrin-EDTA conjugate represented by structure 8 (FIG. 3B). The 
inventors have demonstrated that, in the presence of Fe.sup.2+ and 
dithiothreitol, this molecule, in micromolar concentrations, can cleave 
DNA. Sapphyrin-EDTA conjugates will therefore be useful in effecting 
affinity cleavage of double stranded DNA. It is particularly contemplated 
that they will be of use in cleaving double stranded DNA of a defined 
sequence, or more importantly, perhaps will cleave double stranded DNA 
with a particular structural motif. 
The sapphyrin-EDTA conjugate is believed to bind to DNA in a novel manner, 
based generally upon binding to the phosphate residues of the 
sugar-phosphate backbone. As such, it is contemplated that this sapphyrin 
conjugate will be capable of sensing conformational changes about the DNA 
back-bone. Sapphyrin-EDTA will thus be particularly useful as a structural 
probe of DNA, and the sapphyrin-metal chelating derivatives in general 
will be of use in a variety of photophysical embodiments. 
III. SAPPHYRIN NUCLEOBASE DERIVATIVES 
The inventors initially found that organic-solubilized, 2', 3', 
5'-tris(triisopropylsilyl)-substituted nucleosides would enhance the 
through-CH.sub.2 Cl.sub.2 transport of the corresponding Watson-Crick 
complementary phosphate-free nucleoside in a standard 3-phase Aq 
I--CH.sub.2 Cl.sub.2 --Aq II liquid membrane cell..sup.14 They also 
reported that the deproteinated form of sapphyrin, a pentapyrrolic 
"expanded porphyrin",.sup.2,3,4a,6 acts as an efficient but non-selective 
carrier for nucleotide monophosphates at pH&lt;4..sup.5 Rubyrin.sup.17, and a 
large excess of C-Tips (ca. 100-fold) was also found capable of effecting 
the selective through-transport of GMP at neutral pH (C-Tips is 
2',3',5'-tris(triisopropylsilyl)-cytosine. 
However, sapphyrin, which remains monoprotonated in the ca. 
3.5.ltoreq.pH.ltoreq.10 .sup.5,18 was itself found to be ineffective as a 
GMP carrier at pH 7, even in the presence of a large excess of 
C-Tips..sup.18 Thus, it was thought that if sapphyrin-based systems were 
to be made effective as neutral-regime carriers, it would require the 
construction of sapphyrin systems in which nucleotide recognition units 
are "appended" directly onto the phosphate-chelating expanded porphyrin 
core. 
Precisely these types of sapphyrin derivatives have now been synthesized 
and form an important part of the present invention. These sapphyrin 
nucleobase conjugates are molecules which have been derivatized by the 
addition of one or more nucleobase compounds, i.e., one or more purines, 
pyrimidines, or derivatives thereof. Sapphyrin derivatives with one 
nucleobase per sapphyrin molecule may be referred to as ditopic receptors, 
whereas those with 2 nucleobases per molecules are termed tritopic 
receptors. 
Sapphyrin mononucleobase derivatives may include any of the 
naturally-occurring purine or pyrimidine nucleobases, namely, cytosine, 
guanine, thymidine, adenine or uridine. Equally, they may include modified 
versions of any of these, such as the heterocyclic components of those 
nucleoside/nucleotide analogues listed in Table 2. 
TABLE 2 
______________________________________ 
MODIFIED NUCLEOSIDE/NUCLEOTIDE 
ANALOGUE ANTI-METABOLITES 
______________________________________ 
AraC 
AraAMP 
Azaribine 
Azathioprine 
Azauridine 
AZT 
Bromodeoxyuridine 
Chlorodeoxyuridine 
Cytarabine 
Deoxyuridine 
DideoxyInosine DDI 
Erythrohydroxynonyladenine 
Floxuridine 
Fluorouracil (5-FU) 
Idoxuridine 
LOMPD 
Mercaptopurine 
PFA 
Thioguanine 
Trifluoromethylde-oxyuridine 
Xylo-GMP 
______________________________________ 
Also included within the invention are the sapphyrin mononucleobase 
derivatives including chemically modified nucleobase such as "protected" 
bases. Protecting groups are used to protect reactive groups, such as 
amino and carboxyl groups, from inappropriate chemical reactions. 
Sapphyrin-nucleobase conjugates with protected bases include, for example, 
conjugates wherein one or more base has a protecting group, such as 
9-fluorenylmethylcarbonyl, benzyloxycarbonyl, 
4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl, 1-adamantyloxycarbonyl, 
benzoyl, N-triphenylmethyl or N-di-(4-methoxyphenyl)phenylmethyl on the 
amino group of the nucleobase. 
As clearly detailed in the description of the figures, any of the groups 
R.sup.1 -R.sup.10 may be H, alkyl, alkene, alkyne, halide, alkyl halide, 
hydroxyalkyl, glycol, polyglycol, thiol, alkyl thiol, aminoalkyl, 
carboxyalkyl, alkoxyalkyl, aryloxyalkyl, alkyloxycarbonyl, 
aryloxycarbonyl, aldehyde, ether, ketone, carboxylic acid, phosphate, 
phosphonate, sulfate, phosphate substituted alkyl, phosphonate substituted 
alkyl, or sulfate substituted alkyl; with at least one of these R groups 
being: (CH.sub.2).sub.n --A--(CH.sub.2).sub.m --B, wherein A may include 
any of the groups listed above and B will be one or more nucleobases, 
nucleobase derivatives or protected nucleobases. 
Conjugation of a nucleobase to a sapphyrin derivative to form a 
mononucleobase sapphyrin conjugate may be via any of the R groups R.sup.1 
-.sup.10. Conjugation of the two separate nucleobases to a sapphyrin 
derivative to form a dinucleobase sapphyrin conjugate may also be via any 
two of the R groups R.sup.1 -.sup.10. However, it is contemplated that the 
creation of a symmetrical molecule, such as by substitution on R.sup.4 and 
R.sup.7, or R.sup.5 and R.sup.6, will generally be preferred. 
Specific examples of sapphyrin mononucleobase derivatives are represented 
by structures 9-15 (FIG. 4A-2 through 4A-7). These include, but are 
clearly not limited to, the cytosine-containing sapphyrin derivative 
represented by structure 10; and the guanine-containing sapphyrin 
compounds 12, 14 & 15. The doubly substituted sapphyrin analogues, or 
tritopic sapphyrin receptors, of this invention may contain any 
combination of nucleobases. These sapphyrin derivatives may be 
homonucleobase conjugates, containing two of the same bases, or 
heteronucleobase constructs containing two distinct nucleobases in any 
combination. Thus, for example, in the nomenclature used herein, a 
sapphyrin nucleobase derivative with a single cytosine residue is 
represented by structure 10; a sapphyrin nucleobase derivative with two 
guanine residues is represented by structure 18, and a sapphyrin 
nucleobase derivative with one guanine residue and one cytosine residue is 
represented by structure 16. 
These sapphyrin nucleobase monomers will act as selective carriers for the 
through-membrane transport of nucleotide monophosphates at biological, 
i.e. near-neutral pH. Thus, a sapphyrin nucleobase derivative bearing a 
cytosine residue will function in the binding and transport of GMP, and 
likewise, each nucleobase conjugates will be able to effect the transport 
of its complementary base, i.e., the base with which it naturally 
base-pairs. The nucleobase conjugates will be particularly useful for the 
transport of nucleotide derivatives, such as acyclovir monophosphate, 
Xylo-GMP and Ara-AMP, phosphonate derivatives and simple species such as 
the pyrophosphate derivatives PFA and COMDP, each of which function as 
antiviral agents. 
For initial studies, ditopic and tritopic sapphyrin receptors bearing one 
and two cytosine molecules were first synthesized. The ditopic receptor is 
represented by structure 10 (FIG. 4A-3), and the tritopic receptor by 
structure 20 (FIG. 4B-5). These sapphyrin nucleobase derivatives were 
prepared by trifluoroacetic acid (TFA) induced detritylation of protected 
conjugates. The protected conjugates in question are those wherein the 
nucleobase derivative contains, instead of a single hydrogen, a C(C.sub.6 
H.sub.5).sub.3 group. These, in turn, were prepared by coupling 
1-(2-aminoethyl)-4-[(triphenylmethyl)amino]-pyrimidin-2-one.sup.19 with 
the appropriate sapphyrin mono- or diacid chlorides. 
The inventors have also prepared a variety of sapphyrin nucleobase 
conjugates by the condensation of sapphyrin mono and bis acids with 
conveniently modified nucleobases. Various spacers may be used for the 
connection, such as, for example, oligomethylene bridges with terminal 
amino, or hydroxy function, which allow formation of amide and ester bond 
for the connection of the sapphyrin and nucleobase units. This bridge may 
also be modified, e.g., by the reduction of the amide bond to give the 
amine function..sup.19 Satisfactory spectroscopic and analytic data have 
been obtained for all such new compounds. The present invention thus 
encompasses many possibilities for the connection of the same or different 
nucleobases to one sapphyrin macrocycle. 
Transport studies, using a standard.sup.20 Aq I--CH.sub.2 Cl.sub.2 --Aq II 
liquid membrane cell, were carried out using the sapphyrin cytosine 
conjugates represented by structures 10 and 20. It was found that both 10 
and 20 were able to effect the selective through-membrane transport of GMP 
at, or near, neutral pH (Table 3). In all cases, compound 20 displayed a 
higher selectivity for GMP, by a factor of 8-100, relative to either AMP 
or CMP, than its congener 10. 
These results clearly demonstrate that the transport of a normally 
organic-insoluble species, namely guanosine-5'-monophosphate (GMP), can be 
effected by preparing and using an appropriate sapphyrin-nucleobase 
conjugate. A similarly designed sapphyrin receptor approach may be used to 
achieve the into-cell in vivo delivery of other nucleotides and nucleotide 
derivatives, such as, for example, Xylo-GMP and other antiviral 
nucleotide-based drugs. 
IV. OLIGOMERIC AND POLYMERIC SAPPHYRIN DERIVATIVES 
Further groups of novel sapphyrin-based compounds embodied by the present 
invention are the sapphyrin oligomers and polymers. These include 
relatively low-number conjugates, such as dimers and trimers, and also 
larger oligomers or polymers. The oligomers will generally include between 
about 4 and about 8 residues, or even up to 12 residues, whereas the 
polymers may generally comprise from about 13 to about 100 residues, or 
even up to about 200. 
The monomeric units employed in the synthesis of sapphyrin-sapphyrin 
conjugates may be known sapphyrin molecules, such as those described in 
U.S. Pat. No. 5,159,065, incorporated herein by reference. Equally, any of 
the novel sapphyrin derivatives disclosed herein may be employed, in any 
combination, to create further novel sapphyrin dimers, trimers, oligomers 
or polymers. Encompassed within the terms oligomers or polymers are those 
sapphyrin conjugates synthesized by the controlled addition of particular 
monomeric units and those produced by more uncontrolled polymerization 
methods. 
Due to the unique mode of DNA interaction, sapphyrin polymeric molecule 
will possess an unrivaled ability to act as a general DNA binding 
platform. This has the distinct advantage that it can be modified so as to 
adjust both target cell specificity and degree of interaction with the 
DNA. For sapphyrins and sapphyrin polymers, the basic site of interaction 
with the DNA involves the interior of the sapphyrin macrocycle, so that 
the exterior positions R.sub.1 -R.sub.10 can be substantially modified 
without significantly disrupting the DNA binding interaction. These 
exterior positions can be used to systematically adjust features such as 
solubility, membrane permeability and cell selectivity. Furthermore, 
groups designed to modulate interaction with DNA can be attached to the 
exterior of the sapphyrin polymers including alkylating functions 
(bromoacetamido groups, epoxides etc.) to provide covalent attachment to 
DNA or ene-diyne moieties to allow for double stranded cleavage. 
A further and important advantage of the sapphyrin system is that the 
simple DNA binding motif has been extended to several multimeric 
structures, in which multiple sapphyrins covalently linked together will 
be able to bind simultaneously and thus strengthen the entire interaction. 
This feature will allow a modular approach in which the appropriate number 
(2-10) of sapphyrin molecules is attached in a single molecule, perhaps 
with different sapphyrin units containing sapphyrin derivatives with 
different groups attached that control such important properties such as 
solubility, target cell specificity and DNA modification ability. 
A. Sapphyrin Oligomers 
Specific examples of sapphyrin oligomers include, but are not limited to, 
those compounds represented by structures 21-24 (FIG. 5B-2 through 5B-5). 
Sapphyrin oligomers were been prepared by the condensation of sapphyrin 
mono and bis acids with amino groups as spacers units. As di- and 
tri-amino spacers, ethylenediamine, 1,3-diaminopropane, 1,3- and 
1,4-phenylenediamine, diaminonaphtalenes and anthracenes, for example, 
have used in the synthetic approach. Sapphyrin trimers were built in one 
step reaction, in which 3 molar equivalents of sapphyrin mono acid were 
combined with a trisamino component, e.g. tris(aminoethyl) amine, in very 
high yield. 
For the coupling reaction, a variety of different methods have been used. 
These include, for example, acid chloride, O-acylurea, mixed anhydride and 
N-acylimidazole, which were found to be particularly successful. The 
synthetic methodology employed was essentially the same as that developed 
to prepare sapphyrin-nucleobase conjugates. It is important to emphasize 
that the synthesis of both the sapphyrin nucleobase conjugates and the 
sapphyrin oligomers and polymers may be readily performed using a standard 
automated oligonucleotide synthesizer. 
The present inventors have prepared mono- di- and tri-sapphyrins and have 
proven them to be very efficient recognition species for nucleotides, 
monophospates, diphosphates and triphosphates. The formation of 
noncovalently bonded complexes between sapphyrin-like oligomers and a 
nucleotide allows the transport of nucleotide mono, di and triphosphates 
across cell membranes to occur at physiological pH. This will likely be of 
direct use in the transport of antiviral triphosphates to mammalian cells, 
especially for the treatment of AIDS. 
As discussed above, any of the novel sapphyrins of the present invention 
may be employed, in any combination, in the synthesis of novel sapphyrin 
oligomers or polymers. The synthetic approach developed is equally 
suitable to the use of one or more novel sapphyrin derivatives as starting 
materials as it is to the use of known sapphyrins. Importantly, methods 
are disclosed herein for the generation of covalently bonded 
sapphyrin-nucleotide complexes. Importantly, the sapphyrin-sapphyrin 
nucleobase and sapphyrin-DNA linkage chemistry of the present invention is 
compatible with automated oligonucleotide synthesis, and phosphoramidate 
chemistry. 
The present invention encompasses two categories of sapphyrin polymers; 
these may be described generally as polymeric sapphyrins and polymer 
supported sapphyrins. The polymeric sapphyrins may be employed in a 
variety of different embodiments, for example, relating to oligonucleotide 
binding and transport, as will be discussed more fully below. The polymer 
supported sapphyrin group includes resin-synthesized sapphyrins. These 
sapphyrin polymers may ultimately be cleaved, resulting in the generation 
of a free polymer. However, following resin-bound synthesis, the polymers 
may be maintained covalently bound to the parent resin, thus opening 
further possibilities for their use. 
Sapphyrin polymers which are maintained bound to the parent resin may be 
advantageously employed as a "column material" for use in chromatography, 
for example, in the separation of nucleotides or in photoactivation. With 
regards to the latter, it is to be appreciated that polymer-supported 
sapphyrins could prove particularly advantageous for the in vitro 
inactivation of viruses and other blood borne pathogens: the fact that no 
sapphyrin would be left in the blood (or other substance) being purged 
would militate against any toxicity problems. 
B. Polymeric Sapphyrins 
One class of sapphyrin polymers contemplated by the present invention are 
those compounds resulting from the polymerization of monomeric units, by 
various types of processes other than those using a resin. For example, 
radical polymerization of olefin substituted sapphyrin may be employed to 
give a polyethylene type of polymer. Alternatively, polycondensation of 
sapphyrin bis acid with sapphyrin diamine could be used to give a 
polyamide type of polymer, or polycondensation of sapphyrin bis acid with 
sapphyrin bis alcohol may be employed resulting in a polyester type of 
polymer. In all of these cases, sapphyrin-based polymers may be prepared 
both with and without covalently bonded nucleobases. Examples of the 
latter are 26A and 26B. 
C. Polymer Supported Sapphyrin 
One class of sapphyrin polymers are those based on the covalent connection 
of sapphyrin derivatives to different types of polymeric resins. To 
achieve this, amide, ester, ether and amino bonds have been used. The 
great advantage of this procedure is that different numbers of sapphyrin 
molecules may be introduced per polymer unit simply by varying the molar 
ratio of sapphyrin derivative to the number of the groups bonded on 
polymer surface, illustrated both schematically, and with specific 
examples, throughout FIG. 5D-1 through 5D-4. 
Nucleobase-sapphyrin conjugates of the present invention which posses 
another functional group, namely a carboxy group, could also be used 
advantageously for attaching sapphyrins to polymeric matrices resulting in 
novel polymer-bonded sapphyrins with unique properties, for example, for 
use in the specific binding of oligonucleotides and nucleic acids. 
Sapphyrin polymers contemplated within this group include those where the 
sapphyrin units are bonded to natural occurring polymers, for example, to 
polysaccharides or nucleic acids, via sugar or nucleobase units. 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventor to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. For example, other macrocyclic, positively-charged entities can 
be envisioned as binding to phosphate-containing species such as 
nucleotides, oligonucleotides and DNA by means of the same or similar 
oriented electrostatic interactions described herein. 
EXAMPLE 1 
SYNTHESIS OF POLYHYDROXYSAPPHYRINS 
A. Preparation of 
3,12,13,22-tetraethyl-8,17-bis[di(hydroxyethyl)aminocarbonylethyl]-2,7,18, 
23-tetramethylsapphryin, structure 1 
Sapphyrin bis acid structure 2a (66 mg, 0.1 mmol) was dissolved/suspended 
in dry dichloromethane (30 ml) and 0.5 ml oxalylchloride and 1 drop of DMF 
was added. The reaction mixture was stirred at room temperature for 3 
hours, then evaporated to dryness. Sapphyrin bis acid chloride was 
dissolved in dry dichloromethane (20 ml) and slowly added under argon to 
the solution of diethanoamine (52.5 mg, 0.5 mmol) in dry dichloromethane 
(30 ml), which contained also 5 mg of 4-dimethylaminopyridine and 0.2 ml 
pyridine. The reaction mixture was stirred at room temperature for 24 
hours and then washed with brine, which contained 5% hydrochloric acid. 
The water phase was washed 3 times with dichloromethane containing 20% of 
methanol. The combined organic extracts were dried over sodium sulfate and 
evaporated. Crystalization from ethanol-hexane (1:3) gave 75 mg (86.9%) of 
product 1. 
.sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.:-5.13 (2H, s, NH), -4.95 (1H, s, 
NH), -4.78 (2H, s, NH), 2.18 (4H, t, CH.sub.2 CH.sub.3), 2.20 (4H, t, 
CH.sub.2 CH.sub.3) 2.24 (4H, t, CH.sub.2 CH.sub.3), 2.88 (8H, t, NCH.sub.2 
CH.sub.2 OH), 2.94 (8H, t, NCH.sub.2 CH.sub.2 OH), 3.56 (4H, t, CH.sub.2 
CH.sub.2 CON), 4.07 (6H, s, CH.sub.3), 4.25 (6H, s, CH.sub.3), 4.49 (2H, 
q, CH.sub.2 CH.sub.3), 4.52 (2H, q, CH.sub.2 CH.sub.3), 4.79 (2H, q, 
CH.sub.2 CH.sub.3), 5.15 (2H, q, CH.sub.2 CH.sub.3), 5.30 (4H, t, CH.sub.2 
CH.sub.2 CON), 6.05 (4H, br s, OH), 11.66 (2H, s, meso-H), 11.78 (2H, s, 
meso-H). FAB MS m/e (rel. intensity) 863 (98, [MH].sup.+), 864 (78, 
[MH.sub.2 ].sup.+), 862 (56, [M].sup.+). HRMS Calcd for C.sub.50 H.sub.68 
N.sub.7 O.sub.6 :862.520676. Found 862.523102 UV/VIS (H.sub.2 O): 
.lambda..sub.max 410.5, 621.0, 672.0. 
B. Preparation of 
3,12,13,22-tetraethyl-8,17-bis{[tris(hydroxymethyl)methylamino]-carbonylet 
hyl}-2,7,18,23-tetramethylsapphryin, structure 3. 
Sapphyrin bis acid structure 2a (66 mg, 0.1 mmol) was dissolved in dry 
tetrahydrofuran (20 ml) and 1,1'-carbonyldiimidazole (33 mg, 0.2 mmol) was 
added and solution was stirred at room temperature for 1 hour. A solution 
of tris(hydroxymethyl)aminomethane (24.2 mg, 0.2 mmol) in 3 ml of water 
was added. The reaction mixture was stirred for 12 hours then imidazole 
was filtered off, the solvent was evaporated in vacuo and the product 
crystalized from mixture methanoldichloromethane (1:10), the yield of 
product 3 was 73 mg (81.5%). 
FAB MS m/e (rel. intensity) 896 (100,[M].sup.+), 897 (60, [MH].sup.+). 
HRMS: Calcd. C.sub.50 H.sub.69 N.sub.7 O.sub.8 895.52072. Found 895.52099. 
UV/VIS (H.sub.2 O): .lambda..sub.max 412,622,673. 
EXAMPLE 2 
SYNTHESIS OF SAPPHYRIN DIGLYCOSIDES 
Sapphyrin mono and diglycosides were prepared by the glycosylation of 
sapphyrin alcohols with .alpha.-D-acetobromoglucose and 
.alpha.-D-acetobromogalactose with a silver catalyst. The most 
advantageous catalyst was found to be silver triflate, although silver 
tetrafluoroborate and silver carbonate also gave very good results. With 
polyalcohols it is possible to determine the conversion to glycosides by 
the molar ratio alcohol-halogenose/silver catalyst. The inventors were 
able to introduce 1 or 2 sugar units as a function of the molar ratio of 
hydroxy groups/halogenose/silver catalyst. 
A. Preparation of 8,17-di(tetraacetate-.alpha., 
.beta.-D-glucopyranoxypropyl)-3,12,13,22-tetraethyl-2,7,18,23-tetramethyls 
apphyrin, structure 4A. 
3,12,13,22-Tetraethyl-2,7,18,23-tetramethyl-8,17-di(hydroxypropyl) 
sapphyrin structure 5 (132 mg, 0.2 mmol) was dried with silver triflate 
(0.2569 g, 1 mmol) and barium carbonate (0.5 g) for 2 hours at 20.degree. 
C./1.32 mm Hg in apparatus equipped with septum. The apparatus was flushed 
with argon (2.times.) and dry dichloromethane (50 ml) was added through 
the septum. After dissolution, the mixture was cooled to -45.degree. C. 
and solution of .alpha.-D-glucopyranosylbromide tetraacetate (0.411 g 1 
mmol) in dichloromethane (20 ml) was gradually added through septum under 
stirring. The reaction mixture was stirred at -45.degree. C. for 1 hour, 
then allowed to warm to room temperature with exclusion if light and 
stirred for 8 hours. The reaction mixture was diluted with 50 ml of 
dichloromethane, filtered with celite, the filtrate was washed with 
saturated solution of sodium hydrogencarbonate and water, dried over 
sodium sulfate and solvent was evaporated. Pure product was obtained by 
column chromatography on silica gel with dichloromethane with 4% of 
methanol as a eluent. The yield of product 4a was 250 mg (94.7%). 
.sup.1 H NMR spectrum (300 MHz, CDCl.sub.3): .delta.-6.21-6.07, -5.81, 
-5.75, 2.03, 2.05, 2.11, 2.13, 2.16, 2.28, 2.31, 3.09, 4.11, 4.17, 4.24, 
4.33, 4.51, 4.53, 4.72, 4.74, 5.29, 11.59, 11.66. FAB MS, m/e (rel. 
intensity): 1321 (90, [MH].sup.+), 1322 (56, [MH.sub.2 ].sup.+), 1320 (45, 
[M].sup.+). HRMS Calcd. for C.sub.70 H.sub.89 N.sub.5 O.sub.20 1319.6100. 
Found 1320.617916 ([MH].sup.+). 
The same experimental procedure was used for the preparation of 
tetraacetylgalactose and tetraacetylmannose substituted sapphyrins. In 
these cases, the sugar unit was varied using the same protecting group. 
B. Preparation of 
8,17-di(tetrabenzoate-.alpha.,.beta.-D-glucopyranoxypropyl)-3,12,13,22-tet 
raethyl-2,7,18,23-tetramethylsapphyrin, structure 4B. 
The same procedure as for tetraacetylderivative with 
.alpha.-D-glucopyranosylbromide tetrabenzoate (0.660 g, 1 mmol) gave 
product 4b in 97.6% yield. 
FAB MS m/e (rel. intensity):1817 (95, [MH].sup.+), 1818 (67, 
[MH.sub.2].sup.+), 1816 (62, [M].sup.+). HRMS Calcd. for C.sub.110 
H.sub.105 N.sub.5 O.sub.20 1815.7346. Found 1816.743117 ([MH].sup.+). 
C. Preparation of 
8,17-di(.alpha.,.beta.-D-glucopyranoxypropyl)-3,12,13,22-tetraethyl-2,7,18 
,23-tetramethylsapphyrin, structure 4c. 
The product was prepared from protected derivatives (acetyl, benzoyl) by 
splitting of protecting group in methanol with a catalytic amount of 
sodium methoxide, or potassium hydroxide, or potassium cyanide. Pure 
product was obtained by crystalization, or reverse phase chromatography 
(C.sub.18 -modified silicagel) with methanol as a eluent. The yield of 
product 4c was 67%. 
FAB MS m/e (rel. intensity):986 (70, [M].sup.+), 987 (56, [MH].sup.+). HRMS 
Calcd. for C.sub.54 H.sub.75 N.sub.5 O.sub.12 985.54108. Found 985.5417. 
Elemental analysis: calc. 65.77% C, 7.67% H, 7.10% N; found 65.65% C, 
7.69%H, 7.04% N. UV/VIS (H.sub.2 O): .lambda..sub.max 416,597.5, 642,712; 
(MeOH): .lambda..sub.max 445. 
EXAMPLE 3 
SYNTHESIS OF SAPPHYRIN BIS(GLYCOSAMIDES) 
Sapphyrin bis(glycosamides) were prepared by condensation of an activated 
form of the above-described sapphyrin acid (acid chloride, mixed 
anhydride, O-acylurea, N-acylimidazole derivative) with free, or 
O-acetylated glycoamines (2-amino-2-deoxy-glucopyranose, mannopyranose, 
galactopyranose). 
A. Preparation of 
3,12,13,22-Tetraethyl-8,17-bis[1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-.alp 
ha.,.beta.-D-glucopyranose)-carbonylethyl]2,7,18,23-tetramethylsapphyrin, 
structure 6A 
Sapphyrin bis acid structure 2a (69 mg, 0.1 mmol) was converted to bis acid 
chloride as previously described. Bis acid chloride was dissolved in dry 
dichloromethane under argon and slowly added to the solution of 
1,3,4,6-tetra-O-acetyl-2-amino-2-desoxy-.alpha.-D-glucopyranose (0.1736, 
0.5 mmol) in dichloromethane, which also contained 10 mg of 
4-dimethylaminopyridine and 0.5 ml of dry pyridine at room temperature. 
The reaction mixture was stirred for 14 hours, then washed with water, the 
organic layer was evaporated and the product obtained by column 
chromatography on silica gel with dichloromethane containing 2-10% 
methanol as an eluent. The yield of product 6a was 114.6 mg (85.0%). 
.sup.1 H NMR spectrum (300 MHz, CDCl.sub.3): .delta.=-4.91, -4.59, 1.94, 
2.06, 2.12, 2.27, 3.41, 3.689, 3.84, 3.91, 3.93, 4.05, 4.12, 4.22, 4.54, 
4.63, 4.72, 5.04, 5.19, 5.22, 11.59, 11.69. 
FAB MS, m/e (rel. intensity):1347 (96, [M].sup.+), 1348 (84, [MH].sup.+). 
HRMS: Calcd. C.sub.70 H.sub.89 N.sub.7 O.sub.20 1347.61428. Found 
1347.61624. 
B. Preparation of 
3,12,13,22-Tetraethyl-8,17-bis[(2-amino-2-deoxy-.alpha.,.beta.-D-glucopyra 
nose)-carbonylethyl]-2,7,18,23-tetramethylsapphyrin, structure 6b. 
The above-described acetylated sapphyrin derivative 6a (13.4 mg, 0.01 mmol) 
was dissolved in methanol (10 ml) and a solution of 3 mg KOH in methanol 
was added. The reaction mixture was stirred for 4 hours and then the pH 
adjusted to 6 by adding hydrochloric acid. After evaporating to dryness, 
the product was crystallized from methanol-dichloromethane (1:1), or 
obtained by reverse phase chromatography with methanol as eluent. The 
yield of product 6b was 10.38 mg (88.0%). 
FAB MS, m/e (rel. intensity): 1012 (76, [MH].sup.+), 1011 (54, [M].sup.+). 
UV/VIS (H.sub.2 O): .lambda..sub.max 413,621,671. 
Deacetylation could also be achieved under basic conditions by using, e.g., 
NH.sub.3 in methanol, sodium methoxide in methanol, DABCO in methanol, or 
KCN in methanol, each with good yields. 
EXAMPLE 4 
SYNTHESIS OF SAPPHYRIN-EDTA CONJUGATE (STRUCTURE 8) 
One example of the sapphyrin-metal chelating conjugates of the present 
invention is sapphyrin-EDTA. To a solution of diethylenetriamine (22.0 ml, 
210 mmol) in 200 ml of dry dichloromethane at 0.degree. C., was added 
triphenylmethylchoride (1.8 g, 6.5 mmol) and the solution was warmed to 
room temperature with stirring overnight. The organic layer was washed 1M 
NaOH (200 ml.times.3), dried with Na.sub.2 SO.sub.4, and the solvent was 
removed by rotary evaporation. Purification on silica gel using 
methanol/dichloromethane yielded 2.1 g (94%) of a viscous oil that gave a 
positive test using ninhydrin. 
.sup.1 H NMR .delta. (CD.sub.2 Cl.sub.2) 2.27 (2H, t, .phi..sub.3 
CNHCH.sub.2 CH.sub.2), 2.55 (2H, t, .phi..sub.3 CNHCH.sub.2 CH.sub.2), 
2.70 (4H, m, RNHCH.sub.2 CH.sub.2 NH.sub.2), 7.23-7.65 (15H, m, 
aromatic-H's), .sup.13 CNMR .delta. (CDCl.sub.3) 41.5, 43.1, 49.9, 42.1, 
70.7, 126.1, 127.7, 128.4, 146.1; MS FAB, [MH].sup.+ :m/z 346; HRMS, 
[MH].sup.+ : 346.2278 (calcd for C.sub.23 H.sub.28 N.sub.3 :346.2283). 
Tert-butyl bromoacetate (1.3 ml, 8.5 mmol), tritylated diethylenetriamine 
(4, 0.89 g, 2.6 mmol), K.sub.2 CO.sub.3 (1.18 g, 8.5 mmol), and Cs.sub.2 
CO.sub.3 (1.69 g, 5.2 mmol) in 15 ml of acetonitrile were stirred at 
0.degree. C. overnight. The inorganic salts were removed by vacuum 
filtration, and the solvent was removed by rotary evaporation. 
Purification on silica gel using dichloromethane/hexanes yielded 1.2 g 
(68%) of a light yellow oil: 
.sup.1 H NMR .delta. (CD.sub.2 Cl.sub.2) 1.41 (9H, s, .phi..sub.3 
CNRCH.sub.2 CO.sub.2 C(CH.sub.3).sub.3), 1.44 (18H, s, N(CH.sub.2 CO.sub.2 
C(CH.sub.3).sub.3).sub.2) 2.21 (2H, t, .phi..sub.3 CNRCH.sub.2), 2.62 (2H, 
t, .phi..sub.3 CNRCH.sub.2 CH.sub.2), 2.72 (4H, m, NRCH.sub.2 CH.sub.2 
NR.sub.2), 3.11 (2H, s, CH.sub.2 CH.sub.2 NRCH.sub.2 CO.sub.2 t-butyl) , 
3.36 (4H, s, N(CH.sub.2 CO.sub.2 t-butyl).sub.2) ,7.17-7.51 (15H, m, 
aromatic H's); .sup.13 CNMR: .delta. (CDCl.sub.3) 27.9, 28.1, 41.1, 52.6, 
52.8, 54.5, 55.0, 55.6, 56.1, 70.6, 80.8, 126.1, 127.7, 128.7, 146.3, 
170.6, 170.9 (one quaternary carbon missing); MS FAB, [MH].sup.+ : 688.5; 
HRMS, [MH].sup.+ :688.4323 (calcd for C.sub.41 H.sub.58 N.sub.3 O.sub.6 : 
688.4326). 
The tritylated amine (120 mg, 0.2 mmol) and palladium black (150 mg) were 
stirred in 5.0 ml of methanol for 16 hours under an atmosphere of hydrogen 
gas. The palladium metal was removed by vacuum filtration and the solvent 
removed by rotary evaporation. Purification on silica gel using 
methanol/chloroform yielded 50 mg (64%) of a yellow oil: 
.sup.1 H NMR: .delta. (CDCl.sub.3) 1.42 (27H, s, CO.sub.2 
C(CH.sub.3).sub.3), 2.14 (2H, b, NH.sub.2), 2.69 (4H, m, H.sub.2 NCH.sub.2 
CH.sub.2), 2.77 (4H, m, RHNCH.sub.2 CH.sub.2 NR.sub.2), 3.27 (2H, s, 
CH.sub.2 CH.sub.2 NRCH.sub.2 CO.sub.2 t-butyl) , 3.42 (4H, s, N(CH.sub.2 
CO.sub.2 t-butyl) .sup.13 CNMR .delta. (CDCl.sub.3) 28.1, 39.9, 52.3, 
52.8, 56.1, 57.1, 80.8, 80.9, 170.6, 171.1; MS FAB, [MH].sup.+ :m/z 446; 
HRMS, [MH].sup.+ : 446.3242 (calcd for C.sub.22 H.sub.44 N.sub.3 O.sub.6 : 
446.3230). 
To the sapphyrin mono-carboxylic acid structure 7a (37 mg, 50 .mu.mol) in 
20 ml of dry dichloromethane was added one drop of dimethylformamide 
followed by the cautious dropwise addition of 2.5 ml (5.0 mmol) of a 2.0M 
solution of oxalyl chloride in dichloromethane. The resulting solution was 
stirred at room temperature for 4 hours and the solvent was removed under 
vacuum. The resulting solids were redissolved in 20 ml of dry 
dichloromethane and 0.1 ml of dry pyridine and the amine (68 mg, 150 
.mu.mol) in 20 ml of dry dichloromethane was added dropwise over 1 hour. 
The reaction was allowed to stir overnight and the solvent was removed 
under vacuum. Purification on silica gel using methanol/dichloromethane 
yielded 32 mg of structure 8 as a blue solid (57%). The macrocycle can be 
further purified by recrystallization from dichloromethane/pentane: 
.sup.1 H NMR: .delta. (CDCl.sub.3) -5.03 (2H, m, NH), -4.57 (1H, s, NH), 
-4.38 (2H, s, NH), 1.32 (18H, s, CO.sub.2 C(CH.sub.3).sub.3), 1.38 (9H, s, 
CO.sub.2 C(CH.sub.3).sub.3), 3.29 (2H, s, R.sub.2 NCH.sub.2 CO), 3.42 (4H, 
s, N(CH.sub.2 CO).sub.2), 3.49 (4H, m, NCH.sub.2 CH.sub.2 N), 4.12 (6H, s, 
CH.sub.3), 4.23 (6H, s, CH.sub.3), 4.28 (3H, s, CH.sub.3), 4.53 (4H, q, 
CH.sub.2 CH.sub.3), 4.70 (4H, q, CH.sub.2 CH.sub.3), 5.09 (2H, t, CH.sub.2 
CH.sub.2 CONH), 7.58 (1H, b, CONH), 11.62 (s, 1H, meso-H), 11.70 (s, 2H, 
meso-H), 11.80 (s, 1H, meso-H); MS FAB, M.sup.+ : 1057; HRMS, M.sup.+ : 
1056.6790 (calcd for C.sub.62 H.sub.88 N.sub.8 O.sub.7 : 1056.6776). 
EXAMPLE 5 
SYNTHESIS OF SAPPHYRIN DERIVATIVES 7a; 7b, FOR USE AS PRECURSORS 
The synthesis of the precursor 
3,8,17,22-Tetraethyl-12-(carboxyethyl)-2,7,13,18,23-pentamethyl-sapphyrin, 
structure 7a, is a two part procedure requiring the preparation of the 
ester 
3,8,17,22-tetraethyl-12-(methoxycarbonylethyl)-2,7,13,18,23-pentamethylsap 
phyrin, structure 7b and subsequent hydrolysis to the sapphyrin acid of 
general structure 7a. Ester 7b was prepared in accord with the general 
optimized procedure for the production of substituted sapphyrins.sup.4a, 
incorporated herein by reference. 
4,4'-diethyl-5,5'-diformyl-3,3'-dimethyl-2,2'-bipyrrole (272 mg, 1.0 mmol) 
and 
2,5-bis(5-carboxy-3-ethyl-4-methyl-pyrrol-2-ylmethyl)-3-methoxycarbonyleth 
yl-4-methylpyrrole (523 mg, 1.0 mmol) were condensed to give this desired 
sapphyrin product in 75.4% yield (0.490 g). 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-4.78 (1H, s, NH), -4.76 (1H, 
s, NH), -4.32 (1H, s, NH), -4.13 (2H, s, NH), 2.35-2.43 (12H, m, CH.sub.2 
CH.sub.3), 3.85 (2H, t, CH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3), 3.99 (3H, s, 
CH.sub.3), 4.29 (6H, s, CH.sub.3), 4.38 (3H, s, CH.sub.3), 4.44 (3H, s, 
CH.sub.3), 4.67-4.74 (8H, m, CH.sub.2 CH.sub.3), 5.22 (2H, t, CH.sub.2 
CH.sub.2 CO.sub.2 CH.sub.3), 11.82 (1H, s, meso-H), 11.85 (1H, s, meso-H), 
11.88 (2H, s, meso-H). .sup.13 C NMR (75 MHz, CDCl.sub.3): .delta.=12.7, 
13.1, 15.9, 17.8, 17.9, 21.0, 23.0, 37.1, 52.1, 91.5, 92.0, 98.3, 98.4, 
126.9, 127.0, 129.5, 129.6, 130.2, 132.7, 132.8, 134.7, 135.3, 135.5, 
136.6, 136.7, 137.7, 139.1, 141.5, 141.7, 173.3. HRMS: Calcd. for C.sub.41 
H.sub.49 N.sub.5 O.sub.2 : 643.3886. Found 643.3887. 
The second part of the procedure involves the synthesis of sapphyrin acid 
7a which was prepared as follows: a ca. 1:1 v.v. mixture of 
trifluoroacetic acid and conc. hydrochloric acid (10 ml for 100 mg of 
starting sapphyrin 7b) was used to hydrolyze the ester. The reaction was 
run at 50.degree. C. for 2 days after which time the desired sapphyrin 
acid product was obtained as its bis HCl adduct. After drying in vacuo, 
this protonated product was purified by column chromatography on silica 
gel (methanol 5% in dichloromethane, eluent). The yield was ca. 95%. 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-5.84 (2H, bs, NH), -5.35 (3H, 
bs, NH), 2.20 (12H, t, CH.sub.3 CH.sub.2), 3.23 (2H, t, CH.sub.2 CH.sub.2 
CO.sub.2 H), 4.03 (3H, s, CH.sub.3), 4.15 (6H, s, CH.sub.3), 4.23 (3H, s, 
CH.sub.3), 4.41 (3H, s, CH.sub.3), 4.65 (4H, q, CH.sub.2 CH.sub.3), 4.74 
(4H, q, CH.sub.2 CH.sub.3), 4.79 (2H, m, CH.sub.2 CH.sub.2 CO.sub.2 H), 
11.42 (2H, s, meso-H), 11.55 (1H, s, meso-H), 11.58 (1H, s, meso-H). 
.sup.13 C NMR (75 MHz, CDCl.sub.3): .delta. 12.7, 12.9, 14.3, 15.9, 17.7, 
17.9, 20.6, 20.9, 22.8, 36.5, 36.7, 61.9, 91.6, 98.1, 120.7, 120.9, 125.4, 
125.4, 127.3, 129.1, 129.2, 130.0, 132.7 132.8, 134.8, 134.9, 135.2, 
135.4, 135.6, 135.7, 136.1, 136.8, 136.9 137.0, 137.7, 139.31, 141.4, 
141.8, 141.8, 174.4. FAB MS, m/e (rel intensity): 631 (48, 
[MH.sub.2].sup.+), 630 (100,[MH].sup.+), 629 (52, M.sup.+); Calcd. for 
C.sub.40 H.sub.47 N.sub.5 O.sub.2 : Found 630.3798 ([MH].sup.+); for 
C.sub.40 H.sub.48 N.sub.5 O.sub.2 [MH].sup.+ : Calcd. 63808. 
EXAMPLE 6 
SYNTHESIS OF SAPPHYRIN MONONUCLEOBASE DERIVATIVES 
3,8,17,22-Tetraethyl-12-[2-[1-[2-oxo-4-[(triphenylmethyl)amino]pyrimidyl]et 
hyl]aminocarbonylethyl]-2,7,13,18,23-pentamethylsapphyrin, structure 9. 
Method A: The sapphyrin acid 7a, as prepared above (63 mg, 0.1 mmol), was 
dissolved in 10 ml of dry dichloromethane under argon. Oxalyl chloride 
(0.2 ml) was added followed by 0.03 ml of DMF. The reaction mixture was 
stirred at room temperature for 3 hours under argon and then evaporated to 
dryness in vacuo. The sapphyrin acid chloride so obtained was then 
redissolved in dry dichloromethane (20 ml) and added slowly under argon 
and at room temperature to a solution of 59.4 mg (0.15 mmol) of 
1-(2-aminoethyl)-4-[(triphenylmethyl)-amino]-pyrimidin-2-one.sup.19 
containing 5 mg of 4-dimethylaminopyridine and 0.4 ml of dry pyridine in 
20 ml of dry dichloromethane. After the addition was complete (ca. 1 
hour), the reaction mixture was stirred overnight. The reaction mixture 
was then washed in succession with first dilute hydrochloric acid (3%, 20 
ml), then water (20 ml) followed by saturated sodium bicarbonate (20 ml), 
and then finally water (20 ml) once again. The organic phase was then 
dried over sodium sulfate and the solvent removed in vacuo. The desired 
product 9 was isolated by column chromatography on silica gel using 
methanol, 2-5% in dichloromethane, as the eluent. The yield obtained this 
way was 91.0 mg (ca. 90%). 
Method B. The sapphyrin acid 7a, described above (31.5 mg, 0.05 mmol), was 
dissolved in dry dichloromethane (20 ml). The resulting solution was then 
cooled to 0.degree. C. and dicyclohexylcarbodiimide (41.27 mg, 0.2 mmol) 
and 1-hydroxybenzotriazole (5 mg) were added. The resulting solution was 
then stirred in an ice bath for 30 min. and the amino-functionalized 
cytosine, 1-(2-aminoethyl)-4-[(triphenylmethyl)amino]pyrimidin-2-one (29.7 
mg, 0.075 mmol) was then added followed by 0.1 ml of dry pyridine. The 
reaction mixture was then stirred, first for 0.5 hours at 0.degree. C. and 
then for 48 hours at room temperature. Acetic acid (0.2 ml) was then added 
and the solution stirred a further 1 hour at room temperature. 
Dicyclohexylurea was then filtered off and the reaction worked up as per 
method A. The yield of 9 obtained using this method was 42 mg (83.3 %). 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-5.43 (2H, bs, NH), -4.67 (2H, 
bs, NH), 2.21 (12H, t, CH.sub.2 CH.sub.3), 2.73 (2H, t, CONHCH.sub.2 
CH.sub.2), 3.11 (2H, t, CH.sub.2 CH.sub.2 CONH), 4.07 (9H, s, CH.sub.3), 
4.18 (3H, s, CH.sub.3), 4.30 (3H, s, CH.sub.3), 4.50 (4H, m, CH.sub.2 
CH.sub.3), 4.60 (4H, m, CH.sub.2 CH.sub.3), 4.67 (2H, m, CONHCH.sub.2 
CH.sub.2), 4.90 (2H, m, CH.sub.2 CH.sub.2 CONH), 6.20 (1H, d, C.sup.5 H ), 
6.80, (1H, bs, NH), 7.06-7.26 (15H, m, Tr), 7.54 (1H, s, CONH), 7.73 (1H, 
d, C.sup.6 H), 11.55 (2H, s, meso-H), 11.56 (2H, s, meso-H). FAB MS, m/e 
(rel. intensity): 1008 (25, [MH].sup.+), 1007 (58, M.sup.+), 1006 (22, 
[M-H].sup.+), 765 (22, [M-Tr].sup.+). HRMS: Calcd. for C.sub.65 H.sub.69 
N.sub.9 O.sub.2 (M.sup.+): 1007.5574. Found 1008.5654 ([MH].sup.+); for 
C.sub.65 H.sub.70 N.sub.9 O.sub.2 ([MH].sup.+): calcd. 1008.5652. 
EXAMPLE 7 
SYNTHESIS OF SAPPHYRIN MONONUCLEOBASE STRUCTURES 
3,8,17,22-Tetraethyl-12-[2-[1-(4-amino-2-oxopyrimidinyl)-ethyl]-aminocarbon 
ylethyl]-2,7,13,18,23-pentamethylsapphyrin, structure 10. 
Compound 9 (50.4 mg, 0.05 mmol) was dissolved in trifluoroacetic acid (5 
ml) and the solution was heated at reflux for 1 hour. After allowing the 
solution to cool, the solvent was removed in vacuo. The residue was then 
redissolved in dichloromethane, filtered, and taken to dryness on a rotary 
evaporator. The crude product so obtained was purified by 
recrystallization from a dichloromethane-hexane (1:3, v.v.) mixture, or by 
column chromatography on silica gel using dichloromethane-methanol 9:1 
v.v. as the eluent. Such purifications afforded compound 10 as its bis 
trifluoroacetic salt in ca. 75% yield (28.5 mg). Prior to use in transport 
studies, this trifluoroacetate salt was dissolved in dichloromethane and 
washed with either a 1M solution of NaOH in H.sub.2 O or with a saturated 
aqueous solution of sodium bicarbonate. 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-6.66 (1H, s, NH), -6.57 (1H, 
s, NH), -6.50 (1H, s, NH ), -6.45 (1H, s, NH), -5.79 (1H, s, NH), 2.17 
(12H, m, CH.sub.3 CH.sub.2), 2.37 (2H, t, CONHCH.sub.2 CH.sub.2), 3.08 
(2H, t, CH.sub.2 CH.sub.2 CONH), 3.35 (2H, t, CONHCH.sub.2 CH.sub.2), 4.11 
(6H, s, CH.sub.3), 4.20 (3H, s, CH.sub.3), 4.23 (3H, s, CH.sub.3), 4.25 
(3H, s, CH.sub.3), 4.54 (4H, q, CH.sub.2 CH.sub.3), 4.68 (4H, q, CH.sub.2 
CH.sub.3), 5.12 (2H, t, CH.sub.2 CH.sub.2 CONH), 5.50 (1H, d, J= 7.2, 
C.sup.5 H), 6.26 (2H, s, NH.sub.2), 6.84 (1H, d, J=7.2, C.sup.6 H) 7.47 
(1H, s, CONH), 11.52 (1H, s, meso-H), 11.65 (1H, s, meso-H), 11.69 (1H, s 
meso H), 11.70 (1H, s, meso-H)..sup.13 C NMR, (125 MHz, CDCl.sub.3): 
.delta.=12.52, 12.67, 12.86, 15.47, 15.83, 17.33, 17.51, 17.55, 17.57, 
20.57, 20.68, 20.75, 22.55, 36.99, 37.74, 48.46, 91.50, 91.81, 91.87, 
98.08, 98.22, 121.47, 126.37, 127.08, 127.82, 129.08, 129.92, 130.15, 
130.20, 132.95, 132.98, 135.02, 135.25, 135.35, 135.61, 137.41, 138.51, 
138.59, 140.49, 142.03, 142.10, 142.48, 147.08, 158.57, 173.65. FAB MS, 
m/e (rel. intensity): 768 (65, [MH.sub.2 ].sup.+), 767 (78,[MH].sup.+), 
766 (100, M.sup.+), 766 (45, [M-H].sup.+). HRMS: Calcd. for C.sub.46 
H.sub.55 N.sub.9 O.sub.2 (M.sup.+): 765.4478. Found 766.4535 ([MH].sup.+); 
for C.sub.46 H.sub.56 N.sub.9 O.sub.2 ([MH].sup.+): calcd. 766.4556. 
B. preparation of 
3,8,17,22-tetraethyl-12-{2-[7-(2-benzamide-6-oxopurinyl)ethyl]-aminocarbon 
ylethyl}-2,7,13,18,23-pentamethylsapphyrin, structure 11. 
Sapphyrin monoacid 7a (63 mg, 0.1 mmol) was dissolved in dry 
tetrahydrofurane (30 ml) and cooled to 0.degree. C., 
dicyclohexylcarbodiimide (103.2 mg, 0.5) was added with 10 mg of 
hydroxybenzotriazole and solution was kept at 0.degree. C. for 30 minutes. 
7-(2-Aminoethyl)-2-benzamidopurin-6-one was added with stirring. Reaction 
mixture was kept at 0.degree. C. for 1 hour, then allowed to room 
temperature, and stirred for 48 hours. Acetic acid (0.1 ml) was added and 
stirred 1 hour, dicyclohexylurea was filtered off, solvent evaporated in 
vacuo. Redissolved in dichloromethane with 5% methanol, washed with 
diluted hydrochloric acid (10 ml, 3%), saturated solution of sodium 
hydrogencarbonate (10 ml) and with water (10 ml). Organic phase was dried 
over sodium sulfate and evaporated to dryness. Product 11 was purified by 
column chromatography on silica gel with 2-5% methanol. The yield was 81 
mg (89.0%). 
FAB MS, m/e (rel. intensity): 911 (96,[MH].sup.+), 910 (86, [M].sup.+). 
HRMS: Calcd. for C.sub.54 H.sub.60 N.sub.11 O.sub.3 910.48739. Found 
910.48806. 
C. Preparation of 
3,8,17,22-tetraethyl-12-{2-[7-(2-amino-6-oxopurinyl)ethyl]amino-carbonylet 
hyl}-2,7,13,18,23-pentamethylsapphyrin, structure 12. 
This was prepared by splitting of benzoyl protecting group from derivative 
11 described above using NH.sub.3 in methanol at room temperature for 24 
hours in 85% yield. 
FAB MS, m/e (rel. intensity: 806(96,[M].sup.+), 807 (68,[MH].sup.+). HRMS: 
Calcd. for C.sub.47 H.sub.56 N.sub.11 O.sub.2 806.46131. Found 806.46185. 
D. 
3,12,13,22-Tetraethyl-8-(methoxycarbonylethyl)-17-{2-[7(2-benzamido-6-oxop 
urinyl)-ethyl]aminocarbonylethyl}-2,7,18,23-tetramethylsapphyrin, structure 
13. 
3,12,13,22-Tetraethyl-8-(carboxymethyl)-17-(methoxycarbonylethyl)-2,7,18,23 
-tetramethylsapphyrin 2c (23.5 mg, 0.033 mmol) was dissolved in dry 
dichloromethane (20 ml), solution was cooled by ice, 
dicyclohexylcarbodiimide (51.2 mg, 0.2 mmol) was added together with 5 mg 
of hydroxybenzotriazol, reaction mixture was kept at 0.degree. C. for 30 
minutes and then 7-(2-aminoethyl)-2-benzamidopurin-6-one (30 mg, 0.1 mmol) 
was added. The solution was kept cooled for 1 hour, then allowed to warm 
to room temperature and stirred for 30 hours. Acetic acid (0.1 ml) was 
added and stirred for 1 hour, dicyclohexylurea was filtered off, organic 
phase washed with water (10 ml), product was purified by crystallization 
from dichloromethane-hexane (1:1), final purification was made by column 
chromatography on silica gel with dichloromethane with 10% methanol and 
0.3% of trifluoroacetic acid as a eluent. The yield of product 13 28 mg 
(79.5%). 
.sup.1 H NMR spectrum (300 MHz, CDCl.sub.3): .delta.=-5.50 (1H, s, NH), 
-5.31 (1H, s, NH), -5.04 (1H, s, NH), -4.97 (1H, s, NH), -4.95 (1H, s, 
NH), 2.05 (6H, t, CH.sub.2 CH.sub.3), 2.20 (6H, t, CH.sub.2 CH.sub.3), 
2.91 (2H, t, CH.sub.2 CH.sub.2 NH), 3.46 (2H, t, CH.sub.2 CH.sub.2 N), 
3.51 (3H, s, CO.sub.2 CH.sub.3), 3.63 (2H, t, CH.sub.2 CH.sub.2 CO), 4.08 
(6H, s, CH.sub.3), 4.23 (6H, s, CH.sub.3), 4.50 (2H, t, CH.sub.2 CH.sub.2 
NH), 4.52 (4H, q, CH.sub.2 CH.sub.3), 4.67 (4H, q, CH.sub.2 CH.sub.3) 4.96 
(2H, t, CH.sub.2 CH.sub.2 CONH), 7.40 (2H, m, BzH), 7.58 (1H, m, BzH), 
7.65 (2H, m, BzH), 7.98 (1Hm br s, C.sup.8 H), 8.01 (1H, s, CONH), 11.62 
(2H, s, meso-H), 11.63 (2H, s, meso-H). 
.sup.13 C NMR (125 MHz, CDCl.sub.3 with 20% CD.sub.3 OD): .delta.=12.68, 
12.82, 16.52, 16.61, 17.76, 17.86, 18.47, 18.57, 20.56, 20.61, 20.65, 
20.76, 22.76, 23.50, 29.58, 36.97, 51.98, 91.71, 96.25, 126.88, 127.41, 
127.46, 128.27, 129.44, 129.59, 129.85, 129.96, 132.23, 133.96, 134.45, 
138.12, 139.23, 143.45, 144.79, 145.10, 173.23, 173.35, 173.45. 
FAB MS m/e (rel. intensity): 982 (95,[M-H].sup.+), 983 (87, [M].sup.+). 
HRMS: Calcd. for C.sub.57 H.sub.64 N.sub.11 O.sub.5 982.51087. Found 
982.50919. 
E. Preparation 
3,12,13,22-Tetraethyl-8-(methoxycarbonylethyl)-17-{2-[17-(2-amino-6-oxopur 
inyl)ethyl]-aminocarbonylethyl}-2,7,18,23-tetramethylsapphyrin, structure 
14. 
This compound was prepared in 88% yield from the benzoyl derivative 13 
previously described using a saturated solution of NH.sub.3 in methanol at 
room temperature for 30 hours. 
FAB MS, m/e (re. intensity): 879 (98,[M].sup.+), 880 (68,[MH].sup.+). HRMS: 
Calcd. for C.sub.50 H.sub.61 N.sub.11 O.sub.4 879.49291. Found 879.080. 
F. Preparation of 
3,12,13,22-Tetraethyl-8-(carboxymethyl)-17-{2-[7-(2-amino-6-oxopurinyl)eth 
yl]-aminocarbonylethyl}-2,7,18,23-tetramethylsapphyrin, structure 15. 
This compound was prepared in 75% yield from the benzoyl derivative 13 by 
hydrolysis with KOH solution in methanol at room temperature for 48 hours. 
This is a very convenient building block for construction of sapphyrin 
conjugates with different nucleobases. 
FAB MS, m/e (re intensity): 866 (68,[M].sup.+), 867 (56,[MH].sup.+, 865 
(54, [M-H].sup.+). HRMS: Calcd. for C.sub.49 H.sub.59 N.sub.11 O.sub.4 
865.47511. Found 865.47451. 
EXAMPLE 8 
SYNTHESIS OF SAPPHYRIN DINUCLEOBASE STRUCTURES 
A. Preparation of 
3,12,13,22-Tetraethyl-8-{2-[1-[2-oxo-4-[triphenylmethyl)-amino]pyrimidyl]e 
thyl]aminocarbonylethyl}-17-{2-[7-(2-amino-6-oxopurinyl)ethyl]-aminocarbony 
lethyl}-2,7,18,23-tetramethylsapphyrin, structure 16. 
This compound was prepared in 69% yield from sapphyrin 15 as described 
above by condensation (carbodiimide method) with 
1-(2-aminoethyl-4-[(triphenylmethyl)amino]-pyrimidin-2-one. 
FAB MS, m/e (rel. intensity): 1244 (78,[M].sup.+), 1245 (65,[MH].sup.+). 
HRMS: Calcd. for C.sub.74 H.sub.81 N.sub.15 O.sub.4 1243. 659547. Found 
1243.65940. 
B. Preparation of 
3,12,13,22-Tetraethyl-8,17-bis{2-[7-(2-benzamido-6-oxopurinyl)ethyl]-amino 
carbonylethyl}-2,7,18,23-tetramethylsapphyrin, structure 17. 
Sapphyrin bis acid 2a (34.5 mg, 0.05 mmol) was converted to its 
corresponding bis acid chloride as previously described herein. A solution 
of this acid chloride in dry dichloromethane (20 ml) was then slowly added 
to a solution of 7-(2-aminoethyl)-2-benzamidopurin-6-one (60 mg, 0.2 
mmol), which contained 0.3 ml of dry pyridine and 10 mg of 
4-dimethylaminopyridine. The reaction mixture was stirred at room 
temperature for 36 hours. Dichloromethane-methanol (5:1) 20 ml was added 
and the resulting solution then washed first with diluted hydrochloric 
acid (10 ml, 3%), then with saturated solution of sodium hydrogencarbonate 
(10 ml) and then finally with water (10 ml). The organic phase was dried 
over sodium sulfate and evaporated to dryness. The product 17 was 
crystalized from methanol-dichloromethane (1:15). The yield was 55 mg 
(88.0%). 
.sup.13 C NMR spectrum (125 MHz, CDCl.sub.3 with 25% CD.sub.3 OD): 
.delta.=13.90, 15.63, 17.63, 18.41, 20.72, 20.83, 22.20, 22.81, 23.26, 
33.99, 46.32, 46.36, 63.07, 92.00, 98.80, 110.10, 111.59, 123.02, 127.16, 
127.73, 127.89, 128.34, 128.65, 128.95, 129.97, 131.09, 133.21, 133.41, 
138.57, 138.91, 142.66, 144.00, 148.28, 151.44, 168.59, 168.89, 172.91, 
173.30. FAB m/e (rel. intensity): 1249 (88, [M].sup.+), 1250 (56, 
[MH].sup.+), 1251 (45, [M+2H].sup.+). HRMS: Calcd. for C.sub.70 H.sub.77 
N.sub.17 O.sub.6 1251. 62667. Found 1251.62427. [MH.sub.2 ].sup.+. 
C. Preparation of 
3,12,13,22-tetraethyl-8,17-bis{2-[7-(2-amino-6-oxopurinyl)ethyl]-amino-car 
bonylethyl}-2,7,18,23-tetramethylsapphyrin, structure 18. 
This compound was prepared in 78% yield from the above-described bis 
benzoyl derivative 17 by stirring with NH.sub.3 in methanol at room 
temperature for 30 hours. 
FAM MS, m/e (rel. intensity): 1041 (95, [M].sup.+), 1040 (78, [M-H].sup.+), 
1042 (56, [MH].sup.+). HRMS: Calcd. for C.sub.56 H.sub.76 N.sub.17 O.sub.4 
1041.55787. Found 1041.55619. 
D. 
3,12,13,22-tetraethyl-8,17-bis[2-[1-[2-oxo-4-[(triphenylmethyl)amino]pyrim 
idyl]-ethyl]aminocarbonylethyl]-2,7,13,18,23-tetramethylsapphyrin, 
structure 19. 
3,12,13,22-Tetraethyl-8,17-bis(methoxycarbonylethyl)-2,7,18,23-tetramethyl- 
sapphyrin 2b and 
3,12,13,22-tetraethyl-8,17-bis(carboxyethyl)-2,7,18,23-tetramethylsapphyri 
n 2a were prepared as described by Sessler et al. (1990; reference 4a), 
incorporated herein by reference. This latter diacid 2a (34.5 mg, 0.05 
mmol) was then suspended in dry dichloromethane (20 ml), and, under argon, 
treated with oxalyl chloride (0.3 ml) and DMF (0.03 ml). The reaction 
mixture was stirred at room temperature for 3 hours and then taken to 
dryness in vacuo. The resulting sapphyrin bis acid chloride was 
redissolved in dry dichloromethane (15 ml) and added slowly to a solution 
of 1-(2-aminoethyl-4-[(triphenylmethyl)amino]-pyrimidin-2-one (51.48 mg, 
0.13 mmol) in dry dichloromethane (20 ml) containing 
4-dimethylaminopyridine (5 mg) and dry pyridine (0.3 ml). The reaction 
mixture was stirred under argon at room temperature for 12 additional 
hours, then washed with first dilute hydrochloric acid (3%, 30 ml), then 
water followed by saturated aqueous sodium bicarbonate, and then, finally, 
with water once again. After drying over anhydrous sodium sulfate, the 
product was isolated by column chromatography on silica gel using 
dichloromethane-methanol (1.fwdarw.10%, gradient) as the eluent. The yield 
of compound 19 obtained in this manner was 65.0 mg (89.9%). 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-5.25 (2H, s, NH), -5.05 (2H, 
s, NH), 2.13 (12H, t, CH.sub.3 CH.sub.2), 2.93 (4H, m, CONHCH.sub.2 
CH.sub.2), 3.23 (4H, m, CH.sub.2 CH.sub.2 CONH), 3.83 (4H, t, CONHCH.sub.2 
CH.sub.2), 4.00 (6H, s, CH.sub.3), 4.15 (3H, s, CH.sub.3), 4.25 (3H, s, 
CH.sub.3), 4.57 (4H, q, CH.sub.2 CH.sub.3), 4.75 (4H, q, CH.sub.2 
CH.sub.3), 4.99 (4H, m, CH.sub.2 CH.sub.2 CONH), 5.50 (2H, d, C.sup.5 H), 
6.73 (2H, s, NH), 7.01-7.29 (30H, m, Tr), 7.28 (2H, d, C.sup.6 H), 7.67 
(2H, s, CONH), 11.43 (4H, bs, meso-H). FAB MS m/e (rel intensity): 1446 
(58, M.sup.30 ), 1447 (38, [MH].sup.+). HRMS Calcd for C.sub.92 H.sub.95 
N.sub.13 O.sub.4 : 1445.7645. Found 1445.7658. 
E. 
3,12,13,22-Tetraethyl-8,17-bis[2-[1-(4-amino-2-oxopyrimidinyl)-ethyl]-amin 
ocarbonylethyl]-2,7,13,18,23-pentamethylsapphyrin, structure 20. 
The bis(trityl) sapphyrin-cytosine derivative 19 (72.3 mg, 0.05 mmol) was 
dissolved in trifluoroacetic acid (5 ml) and heated to reflux for 0.5 
hour. After cooling, the trifluoroacetic acid was removed by evaporation 
and the product purified (as its trifluoracetate salt) by column 
chromatography on silica gel using methanol (15% by volume) in 
dichloromethane as the eluent. Alternatively, this salt could be purified 
by recrystallization from dichloromethane-hexane-methanol (1:1:0.1 v.v.v.) 
to give 38.0 mg (79.0%) of deprotected product 20, (CF.sup.3 CO.sub.2 
H).sub.2. Prior to use in transport studies, this trifluoroacetate salt 
was dissolved in dichloromethane and washed with either a 1M solution of 
NaOH in H.sub.2 O or with a saturated aqueous solution of sodium 
bicarbonate. 
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.=-5.91 (1H, s, NH), -5.70 (2H, 
s, NH), -5.47 (2H, s, NH), 2.08 (6H, m, CH.sub.3 CH.sub.2), 2.13 (6H, m, 
CH.sub.3 CH.sub.2), 2.71 (4H, t, CONHCH.sub.2 CH.sub.2), 3.08 (4H, t, 
CH.sub.2 CH.sub.2 CONH), 3.50 (4H, t, CONHCH.sub.2 CH.sub.2), 4.07 (3H, 
s, CH.sub.3), 4.13 (3H, s, CH.sub.3), 4.20 (3H, s, CH.sub.3), 4.23 (3H, s, 
CH.sub.3), 4.51 (4H, q, CH.sub.2 CH.sub.3), 4.68 (4H, q, CH.sub.2 
CH.sub.3), 5.29 (4H, m, CH.sub.2 CH.sub.2 CONH), 5.69 (2H, d, J=7.20, 
C.sup.5 H), 6.35 (4H, bs, NH.sub.2), 6.89 (2H, d, J=7.20, C.sup.6 H), 7.50 
(2H, s, CONH) , 11.52 (.sup.1 H, s, meso-H) , 11.57 (.sup.1 H, s, meso-H) 
, 11.61 (1H, s, meso-H), 11.63 (1H, s, meso-H). .sup.13 C NMR (125 2 MHz, 
CDCl.sub.3 with 10% CD.sub.3 OD): .delta.=12.88, 12.90, 12.98, 16.03, 
17.55, 17.63, 17.70, 18.23, 18.29, 18.37, 18.43, 20.23, 20.55, 20.75, 
20.89, 20.91, 20.96, 22.58, 29.59, 35.58, 37.69, 37.84, 37.91, 48.80, 
48.98, 49.15, 49.32, 49.48, 49.66, 49.83, 97.79, 97.91, 97.94, 97.97, 
122.99, 128.27, 129.62, 129.74, 129.84, 129.86, 129.91, 129.95, 130.06, 
130.10, 130.15, 130.24, 130.32, 135.38, 135.43, 138.93, 139.53, 143.23, 
144.23, 144.59, 172.66. FAB MS m/e (rel intensity): 962 (45, M.sup.+) , 
963 (38, [MH].sup.+). HRMS: Calcd for C.sub.54 H.sub.67 N.sub.13 O.sub.4 : 
961.5439. Found 961.5448. 
EXAMPLE 9 
PREATION OF OLIGOMERIC SAPPHYRINS 
PREATION OF SAPPHYRIN DIMERS 
Sapphyrin monoacid 7a (126 mg, 0.2 mmol) was converted to acid chloride as 
previously described hereinabove. A solution of acid chloride in dry 
dichloromethane (20 ml) was slowly added to the solution of 0.1 mmol 
aromatic bis(amino) compound in dry dichloromethane (20 ml), which 
contained 5 mg 4-dimethylaminopyridine and 0.3 ml of dry pyridine. The 
reaction mixture was stirred 48 hours at room temperature, then washed 
with water, organic phase was dried with magnesium sulfate and evaporated. 
The product was isolated by column chromatography on silicagel in 
dichloromethane with 2-10% of methanol as a eluent. Reaction with 
1,8-diaminonapthalene (0.1 mmol, 15.8 mg) gave 80 mg (57.89%) of product 
21. 
FAB MS m/e (rel. intensity) 1382 (67,[MH].sup.+), 1381 (56,[M].sup.+). HRMS 
Calcd. for C.sub.90 H.sub.100 N.sub.12 O.sub.2 : 1380.80916. Found 
1380.8093. 
Reaction with m-phenylenediamine (0.1 mmol, 10.8 mg) gave 69 mg (51.80%) of 
product 
FAB MS m/e (rel. intensity) 1333 (78,[MH].sup.+), 1332 (65,[M].sup.+). HRMS 
Calcd. for C.sub.86 H.sub.98 N.sub.12 O.sub.2 : 1330.79352. Found 
1330.79349. 
Reaction with aliphatic diaminocompounds was carried with DCC as a coupling 
reagent. Sapphyrin acid 7a (126 mg, 0.2 mmol) was dissolved in dry 
dichloromethane under argon and cooled by ice. Dicyclohexylcarbodiimide 
(0.5 g) was added with 5 mg of hydroxybenzotriazole. Reaction mixture was 
stirred at 0.degree. C. for 30 minutes and then 1,3-diaminopropane (7.4 
mg, 0.1 mmol) was added. Reaction mixture was stirred 30 minutes at 
0.degree. C. and 48 hours at room temperature. Acetic acid (0.2 ml) was 
added, stirred 1 hour, dicyclohexylurea was filtered off, product was 
isolated by column chromatography on silica gell with dichloromethane 
contains 5-10% of methanol. Yield of compound 23 is 96 mg (73.97%) . 
B. PREATION OF SAPPHYRIN TRIMERS 
The inventors used the same coupling procedures as described above for the 
sapphyrin dimers. Thus sapphyrin monoacid 7a (189 mg, 0.3 mmol) was 
coupled (DCC method, 0.75 g) with tris(2-aminoethyl)amine (14.6 mg, 0.1 
mmol) giving 160 mg (80.73%) of compound 24. 
FAB MS m/e (rel. intensity) 1983 (36, [MH].sup.+), 1982 (32, [M].sup.+). 
HRMS Calcd. for C.sub.126 H.sub.153 N.sub.19 O.sub.3 : 1980.240296. Found 
1980.240289. 
EXAMPLE 10 
PREATION OF SAPPHYRIN POLYMERS (