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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel labeled conjugates for use in specific
binding assays for ligands or their binding partners in a liquid medium.
In particular, the invention relates to flavin adenine dinucleotide
(FAD)-labeled conjugates for use in such assays, particularly for
determining an iodothyronine such as thyroxine in serum. The invention
further relates to intermediate compounds produced in the synthesis of the
novel labeled conjugates.
The iodothyronines have the following general formula:
##STR2##
wherein .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or
iodine. The principal iodothyronines of clinical interest are listed in
Table 1 below.
TABLE 1
______________________________________
Iodothyronine .beta..sup.1 .beta..sup.2
______________________________________
3,5,3'5'-tetraiodothyronine
iodine iodine
(thyroxine; T-4)
3,5,3'-triiodothyronine
iodine hydrogen
(liothyronine; T-3)
3,3',5'-triiiodothyronine
hydrogen iodine
("reverse" T-3)
3,3'-diiodothyronine
hydrogen hydrogen
______________________________________
The quantitative determination of the concentration of the various
iodothyronines, particularly the hormones T-3 and T-4, in serum and of the
degree of saturation of the iodothyronine binding sites on the carrier
protein thyroid binding globulin (TBG) are valuable aids in the diagnosis
of thyroid disorders. Likewise, the determination of other components of
body fluids including serum is useful in assessing the well-being of an
individual. Examples of other substances of clinical interest are evident
from the description below.
2. Brief Description of the Prior Art
Specific binding assay methods have undergone a technological evolution
from the original competitive binding radioimmunoassay (RIA) in which a
radioisotope-labeled antigen is made to compete with antigen from a test
sample for binding to specific antibody. In the RIA technique, sample
antigen is quantitated by measuring the proportion of radioactivity which
becomes associated with the antibody by binding of the radiolabeled
antigen (the bound-species of the labeled antigen) to the radioactivity
that remains unassociated from antibody (the free-species) and then
comparing that proportion to a standard curve. A comprehensive review of
the RIA technique is provided by Skelly et al, Clin. Chem. 19: 146(1973).
While by definition RIA is based on the binding of specific antibody with
an antigen or hapten, radiolabeled binding assays have been developed
based on other specific binding interactions, such as between hormones and
their binding proteins.
From the radiolabeled binding assays have evolved nonradioisotopic binding
assays employing labeling substances such as enzymes as described in U.S.
Pat. Nos. 3,654,090 and 3,817,837. Recently further improved
nonradioisotopic binding assays have been developed as described in German
Offenlegungschriften Nos. 2,618,419 and 2,618,511, based on U.S. Ser. Nos.
667,982 and 667,996, filed on Mar. 18, 1976 and assigned to the present
assignee, employing particularly unique labeling substances, including
coenzymes, cyclic reactants, cleavable fluorescent enzyme substrates, and
chemiluminescent molecules. Flavin adenine dinucleotide is mentioned as
being useful as a coenzyme label since FAD functions as a coenzyme in
useful monitoring reactions. In U.S. Patent application Ser. No. 917,961,
filed June 22, 1978 and assigned to the present assignee, FAD is further
described as useful in improved specific binding assays employing a
prosthetic group as the label because FAD also functions as a prosthetic
group in select biochemical systems.
Various methodologies exist for the determination of iodothyronine
concentrations in serum. A significant advance in iodothyronine assays was
the development of the competitive protein binding assay by Murphy and
Pattee, J. Clin. Endocrinol. Metab. 24:187(1964) in which radiolabeled
iodothyronine competes with serum iodothyronine for binding to TBG. The
development of specific antiserum for the various iodothyronines permitted
radioimmunoassays to be devised in which radiolabeled and serum
iodothyronine compete for binding to antibodies rather than to TBG. In
both the competitive protein binding assay and the radioimmunoassay for an
iodothyronine, the radiolabeled material consists of the native
iodothyronine in which one or more of the iodine atoms are replaced by a
radioactive iodine isotope, usually .sup.125 I. The above-mentioned
nonradioisotopic binding assays have offered even more advantageous
methods for determining iodothyronines, particularly those methods
described in U.S. Pat. Nos. 4,043,872 and 4,040,907 and most especially in
OLS's 2,618,419 and 2,618,511 and U.S. Ser. No. 917,961 mentioned above.
SUMMARY OF THE INVENTION
Novel flavin adenine dinucleotide (FAD)-labeled conjugates have been
devised for use in binding assays for determining ligands, or binding
partners thereof, of analytical interest, such as the iodothyronines, and
particularly for use in the assay referred to hereinbefore employing a
prosthetic group label. The FAD-labeled conjugates have the general
formula:
##STR3##
wherein Riboflavin--Phos).sub.2 Ribose represents the riboflavin-adenine
Pyrophosphate-ribose residue in FAD; n=2 through 6, and preferably is 2 or
6; and --(CO)L is a specifically bindable ligand, or a specific binding
analog thereof, and preferably is an iodothyronine such as thyroxine,
bound through an amide bond.
The specifically bindable ligand or analog thereof in the present labeled
conjugates, in terms of its chemical nature, usually is a protein,
polypeptide, peptide, carbohydrate, glycoprotein, steroid, or other
organic molecule for which a specific binding partner is obtainable. In
functional terms, the ligand will usually be an antigen or an antibody
thereto; a hapten or an antibody thereto; or a hormone, vitamin, or drug,
or a receptor or binding substance therefor. Most commonly, the ligand is
an immunologically-active polypeptide or protein of molecular weight
between 1,000 and 4,000,000 such as an antigenic polypeptide or protein or
an antibody; or is a hapten of molecular weight between 100 and 1,500.
FAD-labeled conjugates wherein the ligand therein is an iodothyronine are
particularly useful in specific binding assays to determine the
iodothyronine in liquid media such as serum and preferably have the
general formula:
##STR4##
wherein Riboflavin--Phos).sub.2 Ribose represents the flavin
mononucleotide-ribose residue in flavin adenine dinucleotide, n=2 through
6, and .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or
iodine.
The FAD-labeled conjugates are used in binding assays for the ligand or a
specific binding partner therefor and are determined, i.e., monitored, for
the purposes of the assay by measuring FAD activity, e.g., the coenzyme or
prosthetic group activity of the labeled conjugate. Preferably the
FAD-labeled conjugates are monitored by measuring holoenzyme activity
generated upon combination of such conjugate with an apoenzyme that
requires FAD to perform its catalytic function as described in detail in
the above-mentioned U.S. Ser. No. 917,961.
The present FAD-labeled conjugates can be prepared by a variety of
synthetic routes. Exemplary of such available synthetic routes is the
following general reaction procedure:
##STR5##
Reaction of 6-chloro-9-(2',3'-O-isopropylidine-.beta.-D-ribofuranosyl)
purine (1) [Hampton et al, J. Am. Chem. Soc. 83:150(1961)] with an
.alpha.,.omega.-diaminoalkane selected from those listed in Table 2
TABLE 2
______________________________________
n .alpha.,.omega.-diaminoalkane
______________________________________
2 1,2-diaminoethane
3 1,3-diaminopropane
4 1,4-diaminobutane
5 1,5-diaminopentane
6 1,6-diaminohexane
______________________________________
yields the intermediate
6-(.omega.-aminoalkyl)-9-(2',3'-O-isopropylidine-.beta.-D-ribofuranosyl)
purine (2).
##STR6##
The amino-purine intermediate (2) is then linked by formation of a peptide
or amide couple with either the ligand, where such contains a carboxylic
acid function, or a binding analog of the ligand (e.g., a derivative of
the ligand) which analog contains the desired carboxylic acid function, to
form the ligand or analog substituted adenosine intermediate (3)
##STR7##
wherein --(CO)L is the ligand or analog thereof bound by an amide bond.
Such condensation reactions can be accomplished by reacting the
amino-purine intermediate (2) directly with the carboxylic acid-containing
ligand or ligand analog using conventional peptide condensation reactions
such as the carbodiimide reaction [Science 144:1344(1964], the mixed
anhydride reaction [Erlanger et al, Methods In Immunology and
Immunochemistry, ed. Williams and Chase, Academic Press (New York 1967) p.
149], and the acid azide and active ester reactions [Kopple, Peptides and
Amino Acids, W. A. Benjamin, Inc. (New York 1966)]. See also for a general
review Clin. Chem. 22:726(1976).
It will be recognized, of course, that other well known methods are
available for coupling the ligand or a derivative thereof to the
amino-purine intermediate (2). In particular, conventional bifunctional
coupling agents can be employed for coupling a ligand, or its derivative,
containing a carboxylic acid or amino group to the amino-purine
intermediate (2). For example, amine-amine coupling agents such as
bis-isocyanates, bis-imidoesters, and glutaraldehyde [Immunochem.
6:53(1969)] can be used to couple a ligand or derivative containing an
amino group to the amino-purine intermediate (2). Also, appropriate
coupling reactions are well known for inserting a bridge group in coupling
an amine (e.g., the amino-purine intermediate) to a carboxylic acid (e.g.,
the ligand or a derivative thereof). Coupling reactions of this type are
thoroughly discussed in the literature, for instance in the
above-mentioned Kopple monograph and in Lowe & Dean, Affinity
Chromatography, John Wiley & Sons (New York 1974).
Such coupling techniques will be considered equivalents to the previously
discussed peptide condensation reactions in preparing useful labeled
conjugates. The choice of coupling technique will depend on the
functionalities available in the ligand or analog thereof for coupling to
the amino-purine intermediate (2) and on the length of bridging group
desired. In all cases, for purposes of this disclosure, the resulting
condensation product will comprise the amino-purine intermediate, which
ultimately is converted to FAD, bound to the remaining portion of the
product, or ultimately to the remaining portion of the FAD-labeled
conjugate, through an amide bond. Such remaining portion of the
condensation product, or conjugate, will be considered as a residue of a
binding analog of the ligand, unless the ligand itself is directly coupled
to the amino-purine intermediate (2). Thus, in this description and in the
claims to follow, the abbreviation --CO)L represents the ligand or a
binding analog thereof coupled through an amide bond, wherein such analog
can be a derivative of the ligand coupled by peptide condensation or can
be the ligand or derivative thereof coupled through a bridging group
inserted by coupling of the ligand or derivative with a bifunctional
coupling agent.
It is evident that in coupling the ligand or derivative thereof to the
amino-purine intermediate (2) it may be desirable to protect certain
reactive groups in such ligand or derivative from participating in side
reactions during coupling. Protection of reactive groups may also be
desirable to prevent interfering reactions during the synthetic steps
described below for completing the preparation of the FAD-labeled
conjugate. Depending upon the specific ligand or derivative involved and
the coupling technique chosen, the addition of protecting groups at the
reactive sites on the ligand or derivative can be accomplished before or
after the coupling to the amino-purine intermediate (2). One skilled in
the art will have a wide variety of conventional blocking reactions from
which to accomplish the desired protection of reactive groups such that
the blocking group added can be readily removed in a subsequent synthetic
step to yield the original ligand or derivative coupled to FAD.
For instance, where the ligand is an iodothyronine, it is preferably
treated to protect the amine group prior to condensation or linkage with
the amino-purine intermediate. The amine-protected iodothyronine
intermediate has the formula:
##STR8##
wherein Y is an amine-protecting group. It will be recognized that
protection of the amine group is a conventional procedure and the
amine-protecting group can be selected from a wide variety of groups,
including trifluoroacetyl, which is preferred, and the like, such as
others of the acyl type (e.g., formyl, benzoyl, phthalyl, p-tosyl, aryl-
and alkylphosphoryl, phenyl- and benzylsulfonyl, tritylsulfenyl,
o-nitrophenylsulfenyl and o-nitrophenoxyacetyl), those of the alkyl type
(e.g., trityl, benzyl and alkylidene) and those of the urethane type
(e.g., carbobenzoxy, p-bromo-, p-chloro- and p-methoxycarbobenzoxy,
tosyloxyalkyloxy-, cyclopentyloxy-, cyclohexyloxy-, t-butyloxy,
1,1-dimethylpropyloxy, 2-(p-biphenyl)-2-propyloxy- and benzylthiocarbonyl.
The substituted adenosine intermediates formed by condensation or linkage
between the amino-purine intermediate (2) and the amine-protected
iodothyronine intermediate (4) are of the formula (3) wherein --(CO)L is:
##STR9##
wherein Y is an amine-protecting group as above.
Treatment of intermediate (3) with phosphorous oxychloride produces the
phosphorylated ligand or analog substituted adenosine intermediate (6)
##STR10##
which upon hydrolysis yields the ligand or analog substituted 5'-adenylic
acid intermediate (7).
##STR11##
Condensation of riboflavin-5'-monophosphate with intermediate (7) activated
to a phosphorimidazolidate by treatment with N,N'-carbonyldiimidazole
yields FAD-labeled conjugates (8).
##STR12##
In the preferred embodiment wherein the ligand is an iodothyronine, and
thus --CO)L is represented by formula (5) above, the resulting
FAD-iodothyronine conjugates are of the formula:
##STR13##
wherein Y is an amine-protecting group or, upon conventional treatment for
removal of such protecting group, Y is hydrogen.
As illustrated above, the novel intermediate compounds (2, 3, 6 and 7)
produced in the course of synthesizing the FAD-labeled conjugates have the
following general formulae [the amino-purine intermediates (2) correspond
to formula A below and the intermediates (3, 6 and 7) correspond to
formula B below]:
formula A
##STR14##
wherein n=2 through 6; and
formula B
##STR15##
wherein --CO)L is a specifically bindable ligand, or a binding analog
thereto, and preferably is of formula (5), bound through an amide bond;
n=2 through 6; .beta..sup.1 and .beta..sup.2 are, independently, hydrogen
or iodine; R.sup.1 is --OH or
##STR16##
when R.sup.2 and R.sup.3 together form the group
##STR17##
or R.sup.1 is
##STR18##
when R.sup.2 and R.sup.3 are --OH.
As stated hereinabove, the ligand which is comprised in the labeled
conjugate or whose binding analog is comprised in the labeled conjugate is
in most circumstances an immunologically-active polypeptide or protein of
molecular weight between 1,000 and 4,000,000, such as an antigenic
polypeptide or protein or an antibody, or is a hapten of molecular weight
between 100 and 1,500. Various methods for coupling such ligands or
analogs thereof to the amino-purine intermediate (2) through an amide bond
in the synthesis of the present FAD-labeled conjugate will now be
presented.
Polypeptides and Proteins
Representative of specifically bindable protein ligands are antibodies in
general, particularly those of the IgG, IgE, IgM and IgA classes, for
example hepatitis antibodies; and antigenic proteins such as insulin,
chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA),
myoglobin, hemoglobin, follicle stimulating hormone, human growth hormone,
thyroid stimulating hormone (TSH), human placental lactogen, thyroxine
binding globulin (TBG), instrinsic factor, transcobalamin, enzymes such as
alkaline phosphatase and lactic dehydrogenase, and hepatitis-associated
antigens such as hepatitis B surface antigen (HB.sub.s Ag), hepatitis B e
antigen (HB.sub.e Ag) and hepatitis B core antigen (HB.sub.c Ag).
Representative of polypeptide ligands are angiotensin I and II, C-peptide,
oxytocin, vasopressin, neurophysin, gastrin, secretin, and glucagon.
Since, as peptides, ligands of this general category possess numerous
available carboxylic acid and amino groups, coupling to the amino-purine
intermediate (2) can proceed according to conventional peptide
condensation reactions such the carbodiimide reaction, the mixed anhydride
reaction, and so forth as described hereinabove, or by the use of
conventional bifunctional reagents capable of coupling carboxylic acid or
amino functions to the amino group in the amino-purine intermediates (2)
as likewise described above. General references concerning the coupling of
proteins to primary amines or carboxylic acids are mentioned in detail
above.
Haptens
Haptens, as a class, offer a wide variety of organic substances which evoke
an immunochemical response in a host animal only when injected in the form
of an immunogen conjugate comprising the hapten coupled to a carrier
molecule, almost always a protein such as albumin. The coupling reactions
for forming the immunogen conjugates are well developed in the art and in
general comprise the coupling of a carboxylic acid ligand or a carboxylic
acid derivative of the ligand to available amino groups on the protein
carrier by formation of an amide bond. Such well known coupling reactions
are directly analogous to the present formation of labeled conjugates by
coupling carboxylic acid ligands or binding analogs to the amino-purine
intermediate (2).
Hapten ligands which themselves contain carboxylic acid functions, and
which thereby can be coupled directly to the amino-purine intermediate
(2), include the iodothyronine hormones such as thyroxine and
liothyronine, as well as other materials such as biotin, valproic acid,
folic acid and certain prostaglandins. Following are representative
synthetic routes for preparing carboxylic acid binding analogs of hapten
ligands which themselves do not contain an available carboxylic acid
function whereby such analogs can be coupled to the amino-purine
intermediate (2) by the aforementioned peptide condensation reactions or
bifunctional coupling agent reactions (in the structural formulae below, n
represents an integer, usually 1 through 6, and Me represents methyl).
Carbamazepine
Dibenz[b,f]azepine is treated sequentially with phosgene, an
.omega.-aminoalkanol, and Jones reagent (chromium trioxide in sulfuric
acid) according to the method of Singh, U.S. Pat. No. 4,058,511 to yield
the following series of carboxylic acids:
##STR19##
Quinidine
Following the method of Cook et al, Pharmacologist 17:219(1975), quinidine
is demethylated and treated with 5-bromovalerate followed by acid
hydrolysis to yield a suitable carboxylic acid derivative.
Digoxin and Digitoxin
The aglycone of the cardiac glycoside is treated with succinic anhydride
and pyridine according to the method of Oliver et al, J. Clin. Invest.
47:1035(1968) to yield the following:
##STR20##
Theophylline
Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.
13:497(1976), 4,5-diamino-1,3-dimethylpyrimidine-2,6-dione is heated with
glutaric anhydride to yield the following:
##STR21##
Phenobarbital and Primidone
Sodium phenobarbital is heated with methyl 5-bromovalerate and the product
hydrolyzed to the corresponding acid derivative of phenobarbital [Cook et
al, Quantitative Analytic Studies in Epilepsy, ed. Kelleway and Peterson,
Raven Press (New York 1976) pp. 39-58]:
##STR22##
To obtain the acid derivative of primidone following the same Cook et al
reference method, 2-thiophenobarbital is alkylated, hydrolyzed, and the
product treated with Raney nickel to yield:
##STR23##
Diphenylhydantoin
Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.
5:767(1973), sodium diphenylhydantoin is reacted with methyl
5-bromovalerate followed by acid hydrolysis to yield the following:
##STR24##
Morphine
Morphine free base is treated with sodium .beta.-chloroacetate according to
the method of Spector et al, Science 168:1347 (1970) to yield a suitable
carboxylic acid derivative.
Nicotine
According to the method of Langone et al, Biochem. 12(24):5025(1973),
trans-hydroxymethylnicotine and succinic anhydride are reacted to yield
the following:
##STR25##
Androgens
Suitable carboxylic acid derivatives of testosterone and androstenedione
linked through either the 1- or 7-position on the steroid nucleus are
prepared according to the method of Bauminger et al, J. Steroid Biochem.
5:739(1974). Following are representative testosterone derivatives:
##STR26##
Estrogens
Suitable carboxylic acid derivatives of estrogens, e.g., estrone, estradiol
and estriol, are prepared according to the method of Bauminger et al,
supra, as represented by the following estrone derivative:
##STR27##
Progesterones
Suitable carboxylic acid derivatives of progesterone and its metabolites
linked through any of the 3-, 6- or 7-positions on the steroid nucleus are
prepared according to the method of Bauminger et al, supra, as represented
by the following progesterone derivatives:
##STR28##
The methods described above are but examples of the many known techniques
for forming suitable carboxylic acid derivatives of haptens of analytical
interest. The principal derivation techniques are discussed in Clin. Chem.
22:726(1976) and include esterification of a primary alcohol with succinic
anhydride [Abraham and Grover, Principles of Competitive Protein-Binding
Assays, ed. Odell and Daughaday, J. B. Lippincott Co. (Philadelphia 1971)
pp. 140-157], formation of an oxime from reaction of a ketone group with
carboxylmethyl hydroxylamine [J. Biol. Chem. 234:1090(1959)], introduction
of a carboxyl group into a phenolic residue using chloroacetate [Science
168:1347(1970)], and coupling to diazotized p-aminobenzoic acid in the
maner described in J. Biol. Chem. 235:1051(1960).
The general reaction scheme described above is exemplified by the following
descriptions of the synthesis of the ethyl (n=2) and hexyl (n=6) analogs
of the FAD-labeled conjugates wherein the ligand is the iodothyronine
thyroxine [i.e., --CO)L is of the formula (5) wherein .beta..sup.1 and
.beta..sup.2 are both iodine]. Also provided are descriptions of assay
methods, and results therefrom, employing the exemplified analogs as
labeled conjugates in a specific binding assay for thyroxine.
1. Ethyl Analog
1-I. PREPARATION OF THE LABELED CONJUGATE
6-(2-Aminoethyl)amino-9-(2',3'-O-isopropylidine-.beta.-D-ribofuranosyl)
purine (2).
13.56 grams (g) [41.5 millimoles (mmol)] of
6-chloro-9-(2',3'-O-isopropylidene-.beta.-D-ribofuranosyl) purine (1)
[Hampton et al, J. Am. Chem. Soc. 83:150(1961)] was added with stirring
over a 15 minute period to a cold excess of 1,2-diaminoethane [75
milliliters (ml)]. The resulting solution was allowed to stand at room
temperature for 24 hours. The solution was evaporated in vacuo and the
resulting yellow oil was stirred with 50 ml of cold, saturated sodium
bicarbonate. The mixture was evaporated in vacuo and the resulting residue
was further repeatedly evaporated in vacuo first from water (3 times from
50 ml) and then from 2-propanol (4 times from 50 ml) to obtain a yellow
glass (15 g). A portion (3 g) of the glass was dissolved in a small volume
of water which was then applied to the top of a 25.times.55 centimeter
(cm) Dowex 50W-X2 cation exchange column in the ammonium form (Bio-Rad
Laboratories, Richmond, Calif. USA).
The column was eluted with a linear gradient generated with 2 liters (L)
each of water and 0.5 molar (M) ammonium bicarbonate. The elution was
completed using a linear gradient generated with 2 L each of 0.5 M and 1 M
ammonium bicarbonate. The effluent from the column was collected in 19 ml
fractions and monitored by elution on silica gel thin layer chromatography
(TLC) plates (E. Merck, Darmstadt, West Germany) with a 9:1 (v:v) mixture
of ethanoland ammonium hydroxide. The developed TLC plates were examined
under ultraviolet light, then sprayed with ninhydrin reagent [Randerath,
Thin Layer Chromatography, Academic Press (1966)]. Fractions numbered 250
through 350 from the column chromatography were combined and evaporated
in vacuo leaving the desired purine (2) as a pale yellow amorphous glass
(1.5 g).
Analysis: Calculated for C.sub.15 H.sub.22 N.sub.6 O.sub.4 : C, 51.42; H,
6.33; N, 23.99; Found: C, 50,92; H, 6.54; N, 23.01
NMR (60 MHz, CDCl.sub.3): .delta.1.37 (s,3H, isopropylidene), 1.63 (s,3H,
isopropylidene), 5.92 (d, 1H, 1'-ribose), 7.90 (s, 1H, purine), 8.26 (s,
1H, purine)
Optical Rotation [.alpha.].sub.D.sup.20 =-74.85.degree. (c 1.0, CH.sub.3
OH)
The remaining crude product (12 g) was purified by chromatography on Dowex
50W-X2 as described above. The overall yield was 8 g (55%).
.alpha.-(N-Trifluoroacetyl)amino-.beta.-[3,5-diiodo-4-(3',5'-diiodo-4'-hydr
oxyphenoxy)phenyl]propanoic acid (4).
This compound was prepared by the method of Blank, J. Pharm. Sci.
53:1333(1964). To a cooled (0.degree. C.), stirred suspension of 5 g (6.4
mmol) of L-thyroxine (Sigma Chemical Co., St. Louis, Mo. USA) in 60 ml of
dry ethyl acetate was added 11.5 ml of trifluoroacetic acid and 1.9 ml of
trifluoroacetic anhydride. After 30 minutes the resulting clear solution
was washed three times with 30 ml of water, once with 30 ml of 5% sodium
bicarbonate, and twice with 50 ml of saturated sodium chloride. The
combined aqueous washings were extracted twice with 20 ml of ethyl
acetate. The ethyl acetate layers were combined and washed with 30 ml of
water, then dried over magnesium sulfate. The dried ethyl acetate solution
was evaporated in vacuo leaving a white solid. Recrystallization from a
mixture of ethyl ether and petroleum ether gave a pinkish-white solid
(3.95 g, 70.5% yield) having a melting point (m.p.) of
228.degree.-230.degree. C. with decomposition.
Analysis: Calculated for C.sub.17 H.sub.10 F.sub.3 I.sub.4 NO.sub.5 : C,
23.39; H, 1.15; N, 1.60; Found: C, 23.00; H, 1.05; N, 1.65
NMR [60 MHz, DCON(CD.sub.3).sub.2 ] .delta.7.28 (s, 2H, aromatic), 8.03 (s,
2H, aromatic), 9.7 (m, 1H, amido)
IR (KCl): 1700 (>C.dbd.O)
Optical Rotation [.alpha.].sub.D.sup.25 =-14.97.degree. (c 1.0
dimethylsulfoxide)
A second recrystallization produced a second precipitate (0.95 g) m.p.
224.degree.-228.degree. C. with decomposition. The overall yield was
87.5%.
N-{2-[N-(trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-i
sopropylidene adenosine (3).
A solution of 8.72 g (10.0 mmol) of .alpha.-(N-trifluoroacetyl)
amino-.beta.-[3,5-diiodo-4-(3',5'-diiodo-4'-hydroxyphenoxy)phenyl]propanoi
c acid (4) and 3.86 g (11.0 mmol) of 6-(2-aminoethyl)
amino-9-(2',3'-O-isopropylidene-.beta.-D-ribofuranosyl) purine (2) in 50
ml of dry dimethylacetamide was prepared under a dry argon atmosphere at
-20.degree. C. To this cold stirred solution was added a solution of 3.04
g (11.0 mmol) of diphenylphosphoryl azide (Aldrich Chemical Co.,
Milwaukee, Wisc. USA) in 10 ml of dry dimethylacetamide followed by the
addition of 1.6 ml (11.0 mmol) of dry triethylamine. The solution was left
at room temperature for 22 hours. The solution was then added dropwise to
300 ml of cold (0.degree. C.) water with stirring. The resulting white
precipitate was collected by filtration and dried in vacuo (56.degree. C.)
to give 13.0 g of a light cream colored solid. The solid was dissolved in
500 ml of acetone and the solution was concentrated by boiling. The white
solid which precipitated from the boiling acetone solution was collected
by filtration while hot. Continued boiling of the filtrate produced two
additional precipitates. The three precipitates were combined to give 8 g
(66.6% yield) of a white solid, m.p. 198.degree.-200.degree. C.
(decomposed).
Analysis: Calculated for C.sub.32 H.sub.30 F.sub.3 I.sub.4 N.sub.7 O.sub.8
: C, 31.89; H, 2.51; N, 8.14; Found: C, 31.95; H, 2.60; N, 7.86
NMR [220 MHz, (CD.sub.3).sub.2 SO] .delta.1.32 (s, 3H, isopropylidene),
1.55 (s, 3H, isopropylidene), 6.14 (d, 1H, 1'-ribose), 7.02 (s, 2H,
thyroxine), 7.82 (s, 2H, thyroxine), 8.25 (s, 1H, purine), 8.36 (s, 1H,
purine), 8.41 (t, 1H, J=6, amido), 9.64 (d, 1H, J=8, trifluoroacetamido)
Optical Rotation [.alpha.].sub.D.sup.25 =-11.82.degree. (c 1.0, pyridine)
N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-i
sopropylidene-5'-adenylic acid monotriethylamine salt monohydrate (6).
A solution of 1.2 g (1.0 mmol) of
N-{2-[N-(trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-
isopropylidene adenosine (3) in 10 ml of dry triethylphosphate was prepared
under a dry argon atmosphere at 0.degree. C. To the cold, stirred solution
was added 0.45 ml (5 mmol) of phosphorous oxychloride. The resulting
solution was kept for 24 hours at 0.degree. C., then added dropwise with
stirring to 1 L of ice water. The resulting precipitate was collected by
filtration and dried in vacuo to give 1.23 g of a white solid. The solid
was dissolved in acetone and 0.32 ml (2.2 mmol) of triethylamine was
added. A precipitate formed. The mixture was evaporated in vacuo and the
resulting residue lixiviated with dry acetone, then recrystalized from a
mixture of dry methyl alcohol and dry ethyl ether to give 390 mg (27.8%
yield) of a white solid, m.p. 173.degree.-183.degree. C. (decomposed).
Analysis: Calculated for C.sub.38 H.sub.48 F.sub.3 I.sub.4 N.sub.8 O.sub.12
P: C, 32.50; H, 3.45; N, 7.98; Found: C, 32.24; H, 3.08; N, 7.58
NMR [60 MHz, (CD.sub.3).sub.2 SO] .delta.1.53 (s, 3H, isopropylidene), 6.2
(d, 1H, 1'H-ribose), 7.1 (s, 2H, thyroxine aromatic), 7.87 (s, 2H,
thyroxine aromatic), 8.27 (s, 1H, purine), 8.52 (s, 1H, purine)
Optical Rotation [.alpha.].sub.D.sup.25 =-17.50.degree. (c 1.0, CH.sub.3
OH)
N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-5'-adenyl
ic acid (7).
200 milligrams (mg) (0.14 mmol) of
N-{2-[N-(trifluoroacetyl-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-i
sopropylidene-5'-adenylic acid monotriethylamine salt monohydrate (6) was
suspended in 1 ml of water (0.degree. C.) and trifluoroacetic acid (9 ml)
was added dropwise with stirring. After 30 minutes a clear solution was
obtained. The solution was kept cold (0.degree. C.) for an additional 15
hours, then evaporated in vacuo (30.degree. C.). The resulting residue was
evaporated four times in vacuo (25.degree. C.) from 20 ml volumes of
anhydrous ethyl alcohol and then dried in vacuo (25.degree. C.) leaving a
white solid.
The solid was stirred for 30 minutes with 10 ml of cold methyl alcohol,
then collected by filtration and dried in vacuo (25.degree. C.) to give a
white solid (135 mg, 76% yield) which slowly melted with decomposition
above 188.degree. C.
Analysis: Calculated for C.sub.29 H.sub.27 F.sub.3 I.sub.4 N.sub.7 O.sub.11
P: C, 27.97; H, 2.19; N, 7.87; Found- C, 28.11; H, 2.31; N, 7.65
NMR [220 MHz, (CD.sub.3).sub.2 SO] .delta.5.95 (d, 1H, 1'-ribose), 7.04 (s,
2H, thyroxine aromatic), 7.84 (s, 2H, thyroxine aromatic), 8.25 (s, 1H,
purine), 8.36 (s, 1H, purine), 8.43 (m, 1H, amido), 9.66 (d, 1H,
trifluoroacetamido)
Optical Rotation [.alpha.].sub.D.sup.25 =-2.72.degree. (c 1.0, pyridine)
Flavin adenine dinucleotide - thyroxine conjugate (8).
498 mg (0.4 mmol) of
N-{2-[N-(trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]aminoethyl}-5'-adeny
lic acid (7) was dissolved in 10 ml of dry dimethylformamide and
tri-n-butylamine [96 microliters (.mu.l), 0.4 mmol] was added followed by
the addition of 1,1'-carbonyldiimidazole (320 mg, 2.0 mmol). After
stirring for 18 hours at room temperature in the absence of moisture,
water (280 .mu.l) was added and then the solvent evaporated in vacuo.
The resulting oil was dried by repeated in vacuo evaporation from dry
dimethylformamide (4 times from 10 ml). The resulting
phosphorimidazolidate was redissolved in 10 ml of dry dimethylformamide
and added dropwise to a 0.4 mmol solution of the tri-n-octylamine salt of
riboflavin-5'-monophosphate in 10 ml of dry dimethylformamide. The salt
was prepared by adding a solution of the ammonium salt of
riboflavin-5'-monophosphate (192 mg, 0.4 mmol) in 10 ml of water to a
stirred solution of tri-n-octylamine (176 .mu.l, 0.4 mmol) in 100 ml of
acetone. After 30 minutes, the resulting mixture was evaporated in vacuo.
The residue was dried by repeated evaporation in vacuo from dry
dimethylformamide leaving the salt as an orange solid.
The above solution containing the phosphorimidazolidate of (7) and the
riboflavin-5'-monophosphate salt was divided into two equal aliquots after
24 hours and one aliquot was evaporated in vacuo. The resulting residue
was chromatographed on a column (2.5.times.78 cm) prepared from 100 g of
Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) which had been
preswollen (18 hours) in a 19:1 (v:v) mixture of dimethylformamide and
triethylammonium bicarbonate (1 M, pH 7.5). The column was eluted with the
above 19:1 (v:v) mixture and 10 ml fractions were collected. The effluent
from the column was monitored by elution on silica gel 60 silanised RP-2
TLC places (E. Merck, Darmstadt, West Germany).
The TLC plates were developed using a 40:40:25:1:1 (v:v) mixture of
acetone, chloroform, methyl alcohol, water, and triethylamine. Fractions
numbered 11 through 17 from the above-mentioned column chromatography were
combined and evaporated in vacuo. The residue was chromatographed on a
column (2.5.times.75 cm) prepared from 125 g of Sephadex LH-20 which had
been preswollen (18 hours) in 0.3 M ammonium bicarbonate. The column was
eluted with 0.3 M ammonium bicarbonate collecting 10 ml fractions. The
effluent was monitored by absorption of ultraviolet light at 254
nanometers (nm). The volume of the fractions was increased to 20 ml
beginning with fraction number 150. The salt concentration of the eluent
was decreased in a stepwise fashion as follows: 0.15 M ammonium
bicarbonate at fraction number 295, 0.075 M ammonium bicarbonate at
fraction number 376, and water at fraction number 430. A total of 480
fractions was collected. Fractions numbered 200 through 235 were combined
and evaporated in vacuo leaving the labeled conjugate (8) as a
yellow-orange residue. An alkaline, aqueous solution of this residue
exhibited ultraviolet absorption maxima at the following wavelengths: 266
nm, 350 nm, 373 nm, and 450 nm. The yield, estimated from the absorption
at 450 was about 5%.
A phosphodiesterase preparation (Worthington Biochemical Corp., Freehold,
N.J. USA) isolated from snake venom (Crotalus Adamanteus) hydrolyzed the
above product to riboflavin-5'-monophosphate and the thyroxine substituted
5'-adenylic acid (7) wherein the trifluoroacetyl blocking group had been
removed.
1-II. BINDING ASSAY FOR THYROXINE
The above-prepared labeled conjugate was used in a prosthetic group-labeled
specific binding assay as follows (further details regarding such an assay
method may be found in the U.S. Patent Application--Ser. No.
917,961--referred to hereinbefore):
A. Preparation of apoglucose oxidase
Purified glucose oxidase with low catalase activity obtained from the
Research Products Division of Miles Laboratories, Inc., Elkhart, Ind., USA
was twice dialyzed for 12 hours each against 0.5% (w:v) mannitol (30
volumes each). Aliquots of the dialyzate containing 100 mg of glucose
oxidase each were lyophilized and stored at -20.degree. C.
Bovine serum albumin (200 mg) was dissolved in 12 ml of water adjusted to
pH 1.6 with concentrated sulfuric acid, mixed with 150 mg charcoal (RIA
grade from Schwarz-Mann, Orangeburg, N.Y., USA), and cooled to 0.degree.
C. Lyophilized glucose oxidase (100 mg) was redissolved in 3.1 ml of water
and 3 ml was added to the stirred albumin-charcoal suspension with
continued stirring for three minutes. The suspension was then filtered
through a 0.8 micron, 25 millimeters (mm) diameter Millipore filter
(Millipore Corp., Bedford, Mass., USA) mounted in a Sweenex filter
apparatus (Millipore Corp.) on a 50 ml disposable plastic syringe. The
filtrate was quickly neutralized to pH 7.0 by addition of 2 ml of 0.4 M
phosphate buffer (pH 7.6) and thereafter 5 N sodium hydroxide. Dry
charcoal (150 mg) was then added and stirred for one hour at 0.degree. C.
The resulting suspension was filtered first through a 0.8 micron Millipore
filter and then through a 0.22 micron Millipore filter. To the filtrate
was added glycerol to 25% (v:v) and the stabilized apoglucose oxidase
preparation was stored at 4.degree. C.
B. Assay Reagents
1. Labeled conjugate--The ethyl analog labeled conjugate prepared as in
section 1-I above was diluted in 0.1 M phosphate buffer (pH 7) to a
concentration of 1 micromolar (.mu.M).
2. Apoenzyme--Apoglucose oxidase was diluted with 0.1 M phosphate buffer
(pH 7) to a concentration of 0.6 .mu.M FAD binding sites. The FAD binding
site concentration of the apoenzyme preparation was determined
experimentally by measuring the minimum amount of FAD required to give
maximum glucose oxidase activity when incubated with the apoenzyme.
3. Insolubilized antibody--A washed, moist cake of Sepharose 4B gel
(Pharmacia Fine Chemicals, Uppsala, Sweden) activated by cyanogen bromide
according to the method of March et al, Anal. Biochem. 60:119 (1974) was
added to a solution of 85 mg of antibody, (isolated from antiserum against
a thyroxine-bovine serum albumin conjugate) in 20 ml of 0.1 M phosphate
buffer (pH 7.0) and agitated slowly for 36 hours at 4.degree. C. Upon
completion of the coupling reaction, 1 ml of 1 M alanine was added and
shaking continued for four more hours to block unreacted sites. The
resulting Sepharose-bound antibody was washed on a scintered funnel with
400 ml each of 50 mM sodium acetate--500 millimolar (mM) sodium chloride
(pH 5) and 50 mM phosphate buffer--500 mM sodium chloride (pH 7), and 800
ml of 100 mM phosphate buffer (pH 7). The moist filter cake was then
suspended in 100 mM phosphate buffer (pH 7) containing 0.01% sodium azide
to give 22 ml of an about 50% suspension.
4. Standard--A 1.15 mM stock solution of thyroxine in 5 mM sodium hydroxide
was diluted to 2 .mu.M in 0.1 M phosphate buffer (pH 7).
5. Monitoring reagent--A glucose oxidase assay reagent was prepared to
contain the following mixture per 130 .mu.l: 25 .mu.l of 1.2 mg/ml
peroxidase (Sigma Chemical Co., St. Louis, Mo., USA) in 0.1 M phosphate
buffer (pH 7), 5 .mu.l of 10 mM 4-aminoantipyrine in water, 20 .mu.l of 25
mM 3,5-dichloro-2-hydroxybenzene sulfonate in 0.1 M phosphate buffer (pH
7), 30 .mu.l of 16.5% bovine serum albumin in 0.1 M phosphate buffer (pH
7), and 50 .mu.l of 1 M glucose in aqueous saturated benzoic acid
solution.
C. Assay Procedure
Binding reaction mixtures were pre | | |
