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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the preparation and use of molecules
carrying attached thereon metal complexing agents or biotin-containing
detectable groups, as well as the products themselves.
2. Description of the Prior Art
The use of radioactively labelled diagnostic and therapeutic agents
obtained by labeling such agents with metal ions has recently received
renewed interest. In this technique, a chelating moiety is covalently
attached to the molecule of interest, and a radioactive ion is chelated by
the sequestering groups of the chelator. The radioactively labelled agents
can then be used both in vitro (for example in radioimmunoassay systems)
and in vivo (for example, both in diagnostic imaging techniques and in
radiation therapy techniques). The use of metal labelling of the
nonradioactive type is also of interest, as for example, in the
utilization of nuclear magnetic resonance, electron spin resonance,
catalytic techniques, and the like.
Different metal chelating groups have been attached to biopolymers in the
prior art. Activated analogues of ethylenediaminetetraacetic acid (EDTA)
derived from 1-(p-benzenediazonium)EDTA (I) have been used on proteins:
##STR3##
(See, for example, Meares et al. U.S. Pat. No. 4,043,998, Sundberg et al.
Journal of Medicinal Chemistry 17:1304-1307 (1974); or Sundberg et al.,
Nature 50:587-588 (1974).) The p-benzenediazonium EDTA of formula I is
coupled via an azo linkage to selected tyrosine, histidine or amine
residues of proteins, the latter forming triazines which are acid labile.
Diethylenetriaminepentaacetic acid (DTPA) is a metal chelator which has
also been attached to polypeptides (see, for example, Krejcarek et al
Biochemical Biophysical Research Communications 77:582-585 (1977),
Hnatovich Science 220: 613-615 (1983), or Khaw, ibid, 209:295-297 (1980).)
The chelator is attached through one of its carboxyl groups via an amide
linkage to a protein-derived amino group, as shown in formula II:
##STR4##
This DTPA conjugate is achieved by first preparing the di-anhydride and
reacting the same with a protein. (See for example, Scheinberg, Science
215:1511-1513 (1982).) Involvement of the di-anhydride, however, may cause
potential crosslinking problems which are either intramolecular or
intermolecular. Also, attachment of the chelator through one of its
carboxy groups may remove this carboxy group from consideration as a
complexing moiety, thus decreasing the chelating efficiency, by a
modification of the binding affinity constant and geometry.
Wieder et al U.S. Pat. No. 4,352,751 also suggest the attachement of metal
chelating groups to proteins, utilizing
trans-diaminocyclohexanetetraaacetic acid (DCTA), attached through one of
its carboxy groups to the amino group of a protein. As a model, Wieder et
al show the reaction with ethylamine to form compound (III):
##STR5##
This compound may suffer from the same problems as the DTPA complex, in
that conjugation occurs through one of the carboxy groups, thus
potentially decreasing the binding affinity, and modifying the geometry of
the resulting metal complexes.
Other metal chelating groups have also been attached to biopolymers, e.g.,
methylpicolinimidate on lysozyme (Benisek et al, J. Biol. Chem.,
243:4267-4271 (1968)), ferritin on monoclonal antibodies (Block et al,
Nature 301:342-344 (1983)), and the like. A possible means of overcoming
the aforementioned problems of loss of affinity, limitation on protein
reactive residues, and change in geometry or crosslinking is disclosed in
commonly assigned copending patent application Ser. No. 391,440 filed on
June 23, 1982 for "Modified Nucleotides, Methods of Preparing and
Utilizing, and Compositions Containing the Same" by Engelhardt et al,
which is herein fully incorporated by reference. The Engelhardt et al
application discloses the coupling of a thiocyanate derivative of DCTA to
an allylamine-modified deoxyUTP and its possible incorporation into
polynucleotides. See IV:
##STR6##
The use of the deoxyUTP allylamine and its attachment to other detectable
groups, such as biotin, has also been disclosed (See, for example Langer
et al Proc. Nat. Acad. Sci. 78:6633-06637 (1981)) or copending U.S.
application Ser. No. 255,223 filed Apr. 17, 1981 at the U.S. Patent and
Trademark Office to Ward et al, entitled "Modified Nucleotides and Methods
of Preparing and Using Same," herein incorporated by reference).
There would be an advantage to utilize the DCTA chelating agent or other
chelating agents without having to extensively modify nucleotides a
priori, to utilize physiological chemical process conditions, and to
provide a wide range of alternative methods utilizable in polypeptide,
polynucleotide, polysaccharide and small molecular chemistry.
The development of such methodology would allow the use of high affinity,
versatile metal chelating agents such as DCTA, and might also be extended
and applied to the attachment of other chelators or detectable moieities,
such as biotin.
SUMMARY OF THE INVENTION
The present invention is partly based on the discovery of methods of the
quick, mild and versatile attachment of metal chelating groups and biotin
to polymers, and especially biopolymers such as polynucleotides,
polypeptides or polysaccharides. The attachment methods include both the
use of known intermediate linking agents (which, however, had heretofore
not been used for this purpose), or in some instances, includes the
development of novel linking or bridging groups. The invention also
relates to the products obtained from these methods and extends to
products comprising both polymers linked to chelators and analogues
thereof, to biotin and analogues thereof, and to various intermediates.
In addition, the invention also provides certain low molecular weight (MW
less than about 2,000) molecules linked to a variety of detectable agents
such as various chelating agents, and also to biotin moieties. The low
molecular weight compounds can be linked by direct bonds or by any known
linking arms to any chelating molecule or potentially chelating molecule.
The low molecular weight conjugates between low molecular weight compounds
and chelating molecules thus have the formula (V):
A.sup.1 . . . Det.sup.a (V)
where A.sup.1 is a low molecular weight compound of molecular weight
preferably below 2,000, and Det.sup.a is biotin or a detectable chemical
moiety comprising a substituted or unsubstituted metal chelator or a
compound capable of yielding a metal chelating compound, most preferably
one of the formula (VI):
##STR7##
where M and R.sup.3 are defined below; and the link ".sup. . . . "
indicates a direct covalent bond or an appropriate spacer arm which does
not interfere with the signalling ability of Det.sup.a, with the molecular
recognition properties of A.sup.1 and which assures a stable conjugate
between A.sup.1 and Det.sup.a.
In a preferred embodiment, another aspect of the invention comprises a
detectable molecule of the formula (VII):
A.sup.3 (X--R.sup.1 --E--Det.sup.b) (VII)
where
A.sup.3 is A.sup.2 or a polymer, both A.sup.2 or the polymer having at
least one modifiable reactive group selected from the group consisting of
amino, hydroxy, cis 1,2-di OH, halide, aryl, imidazoyl, carbonyl, carboxy,
thiol or a residue comprising an activated carbon;
A.sup.2 is a chemical entity having a molecular weight of less than about
2,000;
--X-- is selected from the group consisting of --NH--CO--, --NH--CNH--,
--N.dbd.N--, --NH--SO.sub.2 --, --OSO.sub.2 --, --NH--N.dbd.N--,
--NH--CH.sub.2 --, --CH.sub.2 --NH--, --O--CO--, --NH--CO--CH.sub.2 --S--,
--NH--CO--CH.sub.2 --NH--, --O--CO--CH.sub.2 --, --S--CH.sub.2 --;
--O--CO--NH--
##STR8##
or a C.sub.1 -C.sub.10 branched or unbranched alkyl or aralkyl, which may
be substituted by OH;
--Y-- is a direct bond to --S--, or --Y-- is --S--R.sup.2 --, where R.sup.2
is a C.sub.1 -C.sub.10 branched or unbranched alkyl;
Z.sub.a is chlorine, bromine or iodine;
E is O, --N-- or an acyclic divalent sulfur atom;
Det.sup.b is a detectable chemical moiety comprising biotin or a
substituted or unsubstituted metal chelator, or a compound capable of
yielding a metal chelating compound, preferably a compound of the formula:
##STR9##
where R.sup.3 is C.sub.1 -C.sub.4 alkyl or is --CH.sub.2 --COOM, and each
M is a suitable cation;
m is an integer from one to the total number of modified reactive groups on
A.sup.3.
Yet another aspect of the present invention comprises a detectable modecule
of the formula:
##STR10##
wherein A.sup.3 is as defined above, j is an electron withdrawing group, K
is a signal generating entity or a solid matrix, r is an integer from one
to about two and n is as defined above.
Other specific aspects of the invention comprise individual nucleotides,
saccharides or amino acids modified with a group X--R.sup.1 --E--Det as
above. Still other aspects of the invention relate to synthetic methods of
preparing, as well as general methods of using the aforementioned
products.
The resulting covalent conjugates between the biopolymers or small
molecules and metal chelators or biotin moieties are utilizable in a wide
range of applications. For example, the products can be used as detectable
products, by chelating radiometals thereto. They can then be used in a
wide range of in vivo and in vitro therapeutic, diagnostic, imaging and
assay (immunoassay or hybridization assay) techniques. Biotin labelled
biopolymers or small molecules can be used as detectable molecules
wherever biotin/avidin or biotin/streptavidin-based pairs or detection
systems have been used in the prior art. The synthetic polymers of the
invention can be utilized in the same applications as the biopolymers or
small molecules, by attaching the synthetic polymer to biopolymers or
small molecules. Thus, for example, such synthetic polymers can provide
numerous radiometals per biopolymer or small molecule, which results in a
very strong signal being produced.
The ease on introduction, physiological process conditions, versatility and
other such advantages using the DCTA-based chelating agents are
particularly capable of providing a chelator with high affinity, without
loss of its geometry, and avoidance of crosslinking in its introduction.
The methods also have the ability of introducing, at least with certain
linking procedures described hereinbelow, quantitatively more labeling
agent per molecule than the prior art.
BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS
Products
By the small molecular weight entity A.sup.2 is meant to include the so
called ligands generally involved in immunoassays for their determination.
These include drugs which are used for therapeutic purposes, naturally
occurring physiological compounds, metabolites, pesticides, pollutants,
enzyme substrates, the reaction product of an enzyme and its substrate,
and the like. (For a list of useful entities A.sup.2 see, for example
columns 12, 13, 14 and 15 of Rowley et al. U.S. Pat. No. 4,220,722, herein
fully incorporated by reference.) For example, included in A.sup.2 are
alkaloids, steroids, lactams, aminoalkylbenzenes, benzheterocyclics,
purines, vitamins, prostaglandins, antibiotics, amino acids, pesticides,
and the like. The molecular weight of A.sup.2 is less than about 2,000,
especially less than about 1,000.
By the small molecular weight compound A.sup.1, on the other hand are
included all of the aforementioned compounds for A.sup.2 with the proviso
that A.sup.1 is not a monosaccharide, or a mononucleotide. Preferably
A.sup.1 is not an amino acid either. A.sup.1 thus generally comprises such
compounds as pesticides, drugs, pollutants, other physiological compounds,
and the like. For example A.sup.1 includes alkaloids, steroids, lactams,
aminoalkylbenzenes, benzheterocyclics, prostaglandins, antibiotics and the
like.
Certain other products within the present invention are detectable polymers
which comprise synthetic polymers and biopolymers such as polynucleotides,
polypeptides or polysaccharides, or larger fractions containing these.
By "polynucleotide" is meant to include both polyribonucleotides,
polydeoxyribonucleotides, or anypolypurine, poly-pyrimidine or analogue,
or combinations thereof. Examples are DNA, RNA, or fragments thereof.
By "polypeptide" is meant to include any polyamino acid chains, whether
high or low in molecular weight. These include proteins, hormones,
enzymes, immunoglobulins, such as for example, monoclonal antibodies,
protein complexes, and the like.
By "polysaccharide" is meant to include any polysaccharide either naturally
or non-naturally occurring, linear, non-linear or crosslinked,
aqueous-soluble or insoluble, unsubstituted or partly or wholly
substituted. These include cellulose, starch, amylose, amylopectin, and
the like.
By "synthetic" polymer is meant to include any synthetic polymer having at
least one modifiable reactive group selected from the group consisting of
amino, hydroxy, 1,2- cis di OH, halides, aryl, imidazoyl, carbonyl,
carboxy, thiol or a residue comprising an activated carbon. Nonlimiting
examples of suitable polymers that can be modified to have such a
modifiable reactive group include polyethylene, polyacrylamide,
polyurethane, polystyrene, polyethylene glycol, polybutadiene, polyvinyl
alcohols and halides and copolymers thereof. If the polymer does not
contain the modifiable reactive group, then such group can be attached to
the polymers by any of the methods well known to those having ordinary
skill in the art of organic chemistry.
It is necessary that the entity A.sup.3 (which can be either A.sup.2,
supra, or a polymer) prior to reaction have at least one and up to several
modifiable reactive groups selected from the group consisting of amino
groups (such as for example -amino group of lysine, amino groups in
proteins, amino groups in aminopolysaccharides or reactive amino groups on
nucleotide bases), hydroxy groups or cis OH groups (such as for example
those in steroids, saccharides, serine or in sugar moieties of
polynucleotides, such as terminal 3' or 5' hydroxy), carboxyl groups (such
as for example aspartate, glutamate, or derivatives thereof), thiol groups
(such as for example cysteine), carbonyl groups (such as those existing in
certain steroids, alkaloids, on terminal portions of naturally occurring
proteins, or obtainable by modification, as is shown hereinbelow), or
residues comprising activated carbon groups (such as the C-3 or C-5 carbon
site on tyrosine residues, the C-4 site in histidine residues, the
reactive carbon site on guanine, inosine, cytidine or analogues thereof.
For example, guanine has a reactive carbon atom at position C-8.) Also,
A.sup.3 can have modifiable reactive groups such as imidazoyl groups in
proteins as part of a histidine residue or aryl groups as part of tyrosine
residue, or halides as part of a synthetic polymer. The reactive carbon
atoms of these molecules or molecular portions of A.sup.3, should be
capable of covalently reacting with electrophiles such as diazoaryl
functionalities, and undergo coupling (e.g., diazo coupling reactions).
The modifiable reactive group on A.sup.3 may also be present by
modification of A.sup.3, and introduction thereinto of such a group. It
may also be present, for example, in an enzyme cofactor which may be
linked, covalently or noncovalently with a polypeptide.
The number of modifiable reactive groups on A.sup.3 will depend on the
presence or absence of such groups in A.sup.2 or certain reactive amino
acids, bases or saccharides in the polypeptide, polynucleotide or
polysaccharide, respectively. This, in the case of the biopolymer, will
depend on the actual chemical composition of the biopolymer, on the
molecular weight thereof, as well as the three dimensional structure of
the biopolymer, and thus the relative accessibility of reactive groups to
the approach and covalent interaction with reactive partners. It is known,
for example, that in proteins there are certain residues which are more
reactive than others, given the fact that they may be closer to the
surface, present in certain active regions, or the like. When the
biopolymer is modified according to the present invention, with an excess
of modifying reagent, the aforementioned factors will determine the amount
and extent of modification. Thus, one, several and possibly all reactive
residues, bases or sugar moieties may react with an appropriate reactive
partner.
Alternatively, an individual unit of a biopolymer, such as an individual
amino acid or an individual nucleotide or saccharide might be previously
modified, and then incorporated into a final, build-up biopolymer.
In any event, it is a matter of routine to those of ordinary skill in the
art to estimate whether there exist reactive residues in a given entity
A.sup.2 or biopolymer, and how many such residues have reacted, in order
to determine the final stoichiometry of the conjugate between A.sup.2 or
the biopolymer and the modifying group. Such techniques as radiolabeling
can be used to estimate the extent of modification, and to actually count
the number of modified reactive groups. In most instances, the number of
actually modified groups will be less than the number of potentially
available modifiable groups of any particular chemical species.
Among the preferred products of the invention are those of the formula
(VII):
A.sup.3 --(X--R.sup.1 --S--Det.sup.b) (VII)
where A.sup.3 is A.sup.2 or the biopolymer comprising a polynucleotide,
polypeptide or polysaccharide.
--X-- generally comprises a covalent bonding function between one of the
A.sup.3 -modifiable reactive groups and the group R.sup.1. X may be a
single function, such as an amide or ester, or --X-- may be a bridge or
link between a modifiable reactive group on A.sup.3 and the R.sup.1 group.
For example, when X is --NH--CO--, the --NH portion thereof is normally
derived from an amine functionality of A.sup.1 or of the biopolymer, and X
is a standard amide group. When X is --N.dbd.N-- (azo linkage), this
linkage is usually attached to an activated carbon-containing modifiable
group on A.sup.2 or on the biopolymer.
R.sup.1 may be unsubstituted phenyl or phenyl substituted by a halogen such
as chlorine or bromine. R.sup.1 may also be a phenyl substituted by a
group --Y-- where Y may be a direct covalent bond to --S-- or may be
--S--R.sup.2 --. R.sup.2 may be divalent C.sub.1 -C.sub.10 branched or
unbranched alkyl, preferably lower alkyl (C.sub.1 -C.sub.6), most
preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or pentyl.
R.sup.1 may also be a divalent C.sub.1 -C.sub.10 branched or unbranched
alkyl, as described for R.sup.2, supra, and preferably lower alkyl
(C.sub.1 -C.sub.6), most preferably C.sub.2 -C.sub.4. R.sup.1 may also be
a C.sub.1 -C.sub.10 aralkyl, such as phenyl substituted by lower alkyl,
especially benzyl.
Det.sup.b is a detectable chemical moiety which comprises either biotin, or
a modified biotin molecule, or comprises Det.sup.a, which is a metal
chelating compound or a compound capable of yielding a metal chelating
compound. Preferred among these compounds are such molecules as EDTA, DTPA
or DCTA or analogues or homologues thereof. Most preferred is the compound
of the formula (VIII):
##STR11##
This formula depicts a cyclohexane-based metal chelator which may be
attached to sulfur S through positions 4 or 5, and which carries from 1 to
4 metal or nonmetal cations, monovalent cations or the alkaline earth
metals. Thus, with metals of oxidation state +1, each individual
cyclohexane-based molecule may carry up to 4 metal cations (where both
R.sup.3 groups are CH.sub.2 COOM). As is more likely, with higher
oxidation states, the number of metals will decrease to 2 or even 1 per
cyclohexane skeleton. The cyclohexane functionality admits of varying
stereochemistry, and the aforementioned formula is not intended to limit
the molecule to any specific stereochemistry. In particular, both amino
functionalities may be either cis or trans to each other.
The cyclohexane may be unsubstituted (except for the two nitrogen
functionalities and the sulfur substituent) or may be substituted,
especially at the 4-position, with a hydroxy or acylated hydroxy group,
such as with a lower acyl substitution.
For purposes of this invention, other cyclohexanebased analogues such as
alkyl derivatives (e.g., lower alkyl) or substitution products, wherein
the derivatization or substitution do not interfere with the linking of
the cyclohexane skeleton to sulfur, with the chelating ability (affinity,
geometry, etc.) of the individual chelating moieties, or with the overall
biological activity of the modified A.sup.3 are equivalent to those
actually shown. Substitutions which are equivalent for the purposes of
this invention are such as hydroxy, acyl, halogen, amino, and the like.
The A.sup.3 moieties having attached cyclohexane moieties may be in the
acid form (M.dbd.H) or a non-radioactive metal or non-metal form (e.g.,
M.dbd.Mg.sup.+2, Na' K.sup.+, Li.sup.+, NH.sub.4.sup.+, etc.) or in a
radioactive metal form.
Any metal capable of being detected in a diagnostic procedure in vivo or in
vitro, or capable of effecting therapeutic action in vivo or in vitro can
be used. Both nonradioactive and radioactive metals can be utilized for
this purpose. Thus, metals capable of catalyzing chemical reactions,
metals capable of effecting NMR or ESR spectra, or metals capable of
emitting radiation of various types or intensities could be utilized.
Particularly, any radioactive metal ion capable of producing a therapeutic
or diagnostic result in a human or animal body or in an in vitro
diagnostic assay may be used in the practice of the present invention.
Suitable ions include the following: Antimony-124, Antimony-125,
Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-206, Bismuth-207,
Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139, Cerium-141, Cerium-144,
Cesium-137, Chromium-51, Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60,
Erbium-169, Europium-152, Gadolinium-153, Gold-195, Gold-199, Hafnium-175,
Hafnium-175+181, Indium 111, Iridium-192, Iron-55, Iron-59, Krypton-85,
Lead-210, Manganese-54, Mercury- 197, Mercury-203, Molybdenum-99,
Neodymium-147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191,
Palladium-103, Platinum-195m, Praseodynium-143, Promethium-147,
Protactinium-233, Radium-226, Rhenium-186, Rubidium-86, Ruthenium-103,
Ruthenium-106, Scandium-44, Scandium-46, Selenium-75, Silver-110m,
Silver-111, Sodium-22, Strontium-85, Strontium-89, Strontium-90,
Sulfur-35, Tantallum-182, Tecnetium-99m, Tellurium-125, Tellurium-132,
Turbium-160, Thallium-204, Thorium-228, Thorium-232, Thallium-170,
Tin-113, Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49,
Ytterbium-169, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, and
Zirconium-95.
Preferred subgroups within the above formula (VII) are:
--NH--CO-- combined with C.sub.1 -C.sub.10 branched or unbranched alkyl;
activated carbon on tyrosine, histidine or guanine, inosine, or cytidine
combined with --N.dbd.N--Aryl--, where aryl is as defined in formula
(VII).
--NH--CO--CH.sub.2 --S-- combined with C.sub.1 -C.sub.10 branched or
unbranched alkyl or with aryl, where aryl is as defined in formula (VII);
Specific examples of modified A.sup.3 entities according to the present
invention are shown in Table I below:
TABLE 1
__________________________________________________________________________
A.sup.2
(modified reac-
tive group)
X R S Det
__________________________________________________________________________
Dextran (OH)
OCN CH.sub.2CH.sub.2
S DCTA
protein (NH.sub.2)
##STR12## CH.sub.2CH.sub.2
S DCTA
polynucleotide (G,C.sup.8)
NN
##STR13## S CH.sub.2 CH.sub.2 NHCO(CH.sub.2).sub.4
Biotin
protein (NH.sub.2)
##STR14## CH.sub.2CH.sub.2
S DCTA
protein (tyr, his)
NN
##STR15## S DCTA
protein (tyr, his)
NN
##STR16## S DCTA
polynucleotide (G,C.sup.8)
NN
##STR17## S DCTA
polynucleotide (uridineallyl amine)
##STR18## CH.sub.2CH.sub.2
S DCTA
__________________________________________________________________________
Specific examples of A.sup.1 or A.sup.2 low molecular weight entities are
digoxin, morphine, codein, heroin, diterpene alkaloids, estrogens, DES,
barbiturates, amphetamines, catecholamines, chlorpromazine, azepines,
diazepines, caffeine, theophylline, cannabinol, THC, penicillins,
ethambuzol, chloromycetin, nitrofurantoin, methadone, serotonin,
antihistamines, polyhalgenated biphenyls, phosphate esters,
thiophosphates, carbamates, and metabolites, derivatives and analogues
thereof.
Other preferred products of the invention are those of the formula (IX):
##STR19##
wherein a A.sup.4 is A.sup.2 or a polymer, both A.sup.2 or the polymer
having at least one modifiable reactive group selected from the group
consisting of amino, aryl, imidazoyl and a residue comprising an activated
carbon; A.sup.2 is a chemical entity having a molecular weight less than
about 2,000; j is an electron withdrawing group, K is a signal generating
entity or a solid matrix, n is an integer from one to about two,
preferably two, and m is as defined above.
j can be essentially any electron withdrawing group. Preferably, j is
selected from the group consisting of chlorine, fluorine, bromine, sulfone
groups and iodine, with chlorine being most preferred.
K can encompass virtually any of the signal generating entities used in the
prior art, and any system to be developed in the future. It comprises a
moiety which generates a signal itself, e.g. a radio label or a moiety
which upon further reaction or manipulation will give rise to a signal,
e.g. an enzyme linked system. Non limiting examples of suitable signal
generating entities are disclosed in co-pending, co-assigned, U.S. patent
application Ser. No. 391,440, filed on June 23, 1982. K can be attached to
the benzene ring by any method known in the prior art. Also, K can be a
solid matrix such as cellulose. The diazonium product can be fixed to
cellulose by the method disclosed in Seed, U.S. Pat. No. 4,286,964.
The preferred products of formula (IX) are:
##STR20##
The products in formula IX are suprisingly stable and are strong
electrophiles. Such stability and strong electrophilicity permits one to
attach the products of formula (IX) to A.sup.3 when the modifiable
reactive group is very inert, such as the reactive carbon at the C-8
position of guanine. It is believed that such stability and strong
electrophilicity is due to the electron withdrawing group or groups on the
benzene ring.
Other products within the present invention are individual modified
mononucleotides (ribo- and deoxyribo-) according to the formula (X):
##STR21##
where P.sub.z is
##STR22##
or metal or non-metal salts thereof;
Q.sup.1 is H or OH;
BA is a modifiable purine or pyrimidine base, such as guanine, inosine, or
cytidine.
Preferred among these products are those wherein BA has the formula (XI):
##STR23##
Still other products within the present invention are various intermediates
which are described further hereinbelow.
Methods
Reactions involving the preferred cyclohexanebased skeleton can be carried
out on DCTA or analogues, homologues, or substitution derivatives thereof,
which are prepared according to any of the following Schemes:
##STR24##
Scheme I shows the reduction of 3,4 dinitro phenol (I-1) to 3,4-diamino
cyclohexane (I-2); bromination of 3,4-diaminocyclohexane to form
3,4-diaminobromocyclohexane (I-3); and further reaction of this compound
with a halide-substituted carboxymethyl compound to produce the
tetracarboxymethyl derivative thereof yielding the title compound (I-4).
Details of these reactions can be found in Engelhardt et al, copending
Ser. No. 391,440, filed June 23, 1982.
##STR25##
Scheme II shows the use of 4-cyclohexene-1,2-dicarboxylic anhydride (II-1)
as a starting material. Reaction with alcohol followed by hydrazine yields
a dihydrazide (II-3) which, when reacted with nitrate and heated,
undergoes re-arrangement to a diurethane (II-5). Treatment of the
diurethane with base leads to a diamine (II-6) which can than be
carboxyalkylated to yield 1,2-diamino-4-cyclohexenetetraacetic acid
(II-7). This compound can, for example, then be treated with
N-bromosuccinimide (NBS) to yield 4-bromo-5-hydroxy DCTA derivative
(II-8). Details of these reactions can be found in the accompanying
Examples.
##STR26##
Scheme III shows the use of 1,4-cyclohexadiene (III-1) to produce dibromo
derivative III-2, which can further be reacted with N-alkyl substituted
glycine to yield the title compound (III-3).
In the above Schemes I, II or III, it is of course understood that
different halogens, or even pseudohalogens could be used, since the object
is to substitute the cyclohexane with a leaving group capable of being
displaced by a mercapto group, SH. Such a leaving group could be chlorine,
bromine, cyano, tosylate, mesylate, and the like.
The intermediates or starting materials used in these Schemes (such as for
example the diester cyclohexene (II-2)), can be used for the preparation
of further substituted cyclohexane skeletons as will be readily
appreciated by one of skill in organic chemistry. Thus, a wide variety of
modifications and substitutions can be introduced into the cyclohexane
skeleton without affecting the basic chemistry of the chelating groups or
of the displaceable leaving group.
The attachment of the (substituted or unsubstituted) cyclohexane skeleton
to A.sup.3 is carried out via a basic nucleophilic substitution reaction
between the oxygen, nitrogen or preferably, the sulfur atom of a
thiol-containing compound, and the displaceable group or groups on the
cyclohexane. The attachment can take any of three general routes.
First, one can attach the A.sup.3 --X--R--SH moiety to the leaving
group-containing cyclohexane by nucleophilic substitution.
Second, one can attach an A.sup.3 moiety containing a reactive group, to a
previously prepared X'--R--S--Det.sup.b, where X' is a group capable of
reacting with the modifiable reactive group on A.sup.3, to yield X.
Third, one can use a combination of both the first and second approaches,
in that A.sup.3 is first reacted with part of the bridging group, which in
turn is reacted with a previously modified cyclohexane to give the final
conjugate.
In the second approach (Scheme IV, below), one can prepare a diazo aryl
moiety-containing cyclohexane (IV-2, bonded to the cyclohexane via sulfur)
and react the same with a protein or a polynucleotide as follows.
##STR27##
In the third approach, for example, one can previously modify A.sup.3 by
reacting modifiable reactive groups thereon with a haloacyl group, and
then reacting this modified A.sup.3 with a modified cyclohexane containing
a nucleophilic group such as a thiol or amine (Scheme V).
##STR28##
The preparation of haloacyl A.sup.3 's as in Scheme V is shown, for
example, in the book "Chemical Modification of Proteins", by Means and
Feeney, Holden-Day, Inc., 1971, and in Rowley et al, U.S. Pat. No.
4,220,722, both of which are herein incorporated by reference.
The "A.sup.3 -NH" moiety in Scheme V above can also be modified instead by
means of a compound containing a diazo aryl group (such as a 3,4,5
trichlorobenzenediazonium salt) containing a leaving group. Such a
compound is known in the tetrafluoroborate form (Korzeniowsky et al,
Journal of Organic Chemistry, 1981, 46:2153-2159). Attachment of this
compound to a modifiable reactive group A.sup.3 modifies the resulting
A.sup.3 by attaching thereto a displaceable chlorine atom. (Such a scheme
would be a modification of Scheme IV, above, obtained by inverting the
steps). Generally, the attachment of (other) aryl diazonium functions to
biopolymers is known (see Seed, U.S. Pat. No. 4,286,964, and Meares et al,
U.S. Pat. No. 4,043,998).
Other possible A.sup.3 modifications, especially for biopolymers, useful to
prepare the final products of the present invention comprise the reaction
of amino groups with diketene to yield acetoacetyl containing A.sup.3 's,
possibly followed by reduction. (Means and Feeney, supra, page 80-81). The
availability of the ketone group of acetoacetyl is useful in reductive
amination reactions, where the cyclohexane chelator carries a nucleophilic
amine.
Amine-containing biopolymers can be reacted with imido esters in alkaline
solution to form imido amides, so-called amidines (Means and Feeney,
supra, page 90-91). Reaction occurs at moderately alkaline pH, in aqueous
solvent and at room temperature. Appropriately substituted amidines can be
prepared which are then capable of reacting with modified cyclohexane
chelators.
Sulfonyl halides and substituted sulfonyl halides, such as chlorides and
fluorides, are known to react with amino, sulfhydro, imidazole, and
phenolic hydroxy groups of proteins (Means and Feeney, supra, page 97).
Reaction with aliphatic hydroxy groups is somewhat slower. Appropriately
substituted sulfonyl halides can be used to introduce displaceable groups,
such as displaceable chlorines, into a biopolymer.
Individual modified mononucleotides can be prepared by applying any of the
above-described methods to said mononucleotides.
Attachment of Det.sup.b to polysaccharides can be carried out e.g. by
reacting any cis-diol containing polysaccharide with cyanogen biomide and
then reacting the resulting water soluble or insoluble activated
polysaccharide with an appropriately modified, nucleophilic group
containing a cyclohexane chelator a precursor thereof, or biotin. The same
scheme can be applied to the preparation of low molecular weight cis-diol
containing molecules, such as digoxin, for example.
The attachment of a detectable moiety comprising biotin would generally
require modification of the biotin side chain by attachment of a
sulfur-containing nucleophile. An example of such a modification is shown
below in Scheme VI:
##STR29##
Scheme VI exemplifies the use of 1-amino, 2-mercapto ethane. The Scheme
also exemplifies the use of 3,4,5 trichlorobenzenediazonium salt, but
other such coupling agents can be utilized. For example, when A.sup.3 is a
biopolymer, the same can be modified with a suitable halide, and the
mercapto derivative VI-5 can be reacted therewith to yield the final
product.
Generally, the reactions with the cyclohexane chelator or derivatives
thereof can be carried out with the molecule in the neutralized form (COOH
or COONa or COOalkyl form), or in the presence of stoichiometric amounts
of other metals such as magnesium. Preparation of the active, detectable
cyclohexane moiety containing radiolabelled metal or metal capable of
being detected by or imaged by nonradioactive methodologies (NMR, ESR,
etc.) can be carried out after the final step in the organic synthesis.
Of particular interest is the preparation of a radiolabelled product prior
to the utilization of the agent. A method of preparing a radioactively
labelled diagnostic or therapeutic molecule generally comprises contacting
a therapeutic or diagnostic agent comprising a molecularly recognizable
portion and a chelating portion capable of chelating with a radioactive
metal ion, with an ion exchange material having the radioactive metal ion
bound thereto and having a binding affinity for the radioactive metal ion
less than the binding affinity of the chelating portion for the
radioactive metal ion, wherein, prior to the contact, the chelating
portion is unchelated or is chelated with a second metal ion having a
binding affinity with the chelating portion less than the binding affinity
of the radioactive metal ion, whereby a radiolabelled therapeutic or
diagnostic agent is produced by the contacting, and then separating the
radiolabelled therapeutic or diagnostic agent from the ion exchange
material. The so formed radiolabelled material is then immediately used in
an in vitro or in vivo diagnostic procedure. Such a method is disclosed in
commonly assigned co-pending application U.S. Pat. No. 4,703,440 filed on
even date herewith by Y. Stavrianopoulos, for "METHOD OF RADIOACTIVELY
LABELLING DIAGNOSTIC AND THERAPEUTIC AGENTS CONTAINING A CHELATING GROUP,"
herein fully incorporated by reference.
Among other aspects of the invention are various intermediates used in the
aforementioned synthetic procedures. Thus, the invention also includes a
modified compound of the formula (XII):
##STR30##
where A.sup.3 is as defined above, and contains at least one modifiable
reactive group selected from the group consisting of amine and a residue
comprising an activated carbon;
Z.sub.b is chlorine, bromine or iodine; and
m is an integer from 1 to the total number of modified reactive groups on
A.sup.3.
This modified A.sup.3 is useful in preparing the preferred final detectable
products of the invention.
The invention also includes a compound of the formula (XIII):
##STR31##
or the 4-hydroxy or acyloxy derivative thereof, where M is as defined
previously;
--S-- is divalent sulfur atom; and
Q.sup.2 -- is H; branched or unbranched C.sub.1 -C.sub.10 alkyl or aralkyl
which carries a group selected from the group consisting of --OH, --SH,
--NH.sub.2, --CONHNH.sub.2, or --C--Lv, where Lv is a displaceable leaving
group; or Q.sup.2 is
##STR32##
where Z.sub.c is hydrogen, chlorine, bromine or iodine, and J is
--NH.sub.2 or --N.sub.2.sup.+ CA.sup.-, where CA.sup.- is a counteranion.
Examples of Lv are --N.sub.3, --Cl, --Br, tosylate, mesylate, and the like.
Examples of CA.sup.- are fluoroborate, tetrafluoroborate, tosylate,
perchlorate, and the like.
Still other intermediates are modified mononucleotides of the formula XIV:
##STR33##
where P.sub.z, BA and Q.sup.1 are as defined above.
The modified mononucleotides (XIV) can be integrated into a polynucleotide
and then reacted with appropriate SH-group containing cyclohexane chelator
or biotin. Alternatively, the modified mononucleotides (XIV) are reacted
with a cyclohexane chelator or biotin, and the resulting products are
incorporated into a polynucleotide.
Still other intermediates include compounds of the formula XV:
##STR34##
wherein j, n and K are as defined previously, and CA.sup.- is a suitable
counteranion.
The preparation of small molecular weight chelator-containing compounds
(V):
A.sup.1 . . . Det.sup.a (V)
can be carried out by any of the well known methods of linking metal
chelating moieties or potential metal chelating moieties to molecules. For
example such chelators as EDTA, DTPA or DCTA can be attached to amino or
hydroxy groups of A.sup.1 with formation of amides or esters. Diazoaryl
containing chelators or potential chelators can be attached to activated
aromatic groups on A.sup.1.
Applications
The uses and applications of the chelator or biotin-containing compounds of
the invention are unlimited, and extend to all of those uses to which
detectably labelled compounds of this type had been put in the prior art.
For example, any compound desired to be detected and analyzed in a sample
can be modified according to the techniques of the present invention. Of
particular interest are the modification of antibodies for use in
immunoassay procedures, such as sandwich immunoassay procedures. Also of
interest is the modification of drugs for radioimmunoassay procedures or
of proteins associated with or known to be present on microorganism walls
or membranes. Detectably labelled proteins prepared in such manner can
also be used in competitive immunoassay procedures. Labelled
polynucleotides can be used in hybridization assays.
Another use of the detectable compounds, especially biopolymers, of the
invention is in imaging, especially with monoclonal antibodies. These can
be modified according to the techniques of the invention and allowed to
carry a metal onto a given site in a living material, such as an animal
body. Detection can then be carried out by radiological techniques. A
metal carried to such site can also be chosen to be an emitter, thus
producing localized radioth | | |
