Note: Descriptions are shown in the official language in which they were submitted.
BACKGROUND OF THE INVENTION
1 . FI ~3LD OF THE I N VI~NTI ON
This invention relates to novel labeled conjugates for
use in specific binding assays for ligands OT their binding
partners in a liquid medium. In particular, the invention
relates to flavin adenine dinucleotide (FAD) - labeled conju-
gates for use in such assays, particularly for determining
an iodothyronine such as thyroxine in serum. The invention
further relates ~o intermediate compounds produced in the
lS synthesis of the novel labeled conjugates.
The iodothyronines ha~e the following general formula:
HOOC-CH-CH2 ~ ~ OH
wherein ~l`and ~2 are, independently, hydrogen or iodine. The
principal iodothyronines of clinical interest are listed in
Table 1 below.
.
- 2 -
~.3~
TABLE 1
Iodothyronine ~1 B2
3,5,3'5'-tetraiodothyronine iodine iodine
(thyroxine; T-4)
3,5,3'-triiodothyronine iodine hydrogen
(liothyronine; T-3)
3,3',5'-triiodothyronine hydrogen iodine
("reverse" T-3)
3,3'-diiodothyronine hydrogen hydrogen
The quantitative determination of the concentration of
the various iodothyronines, particularly ~he hormones T-3 and
T-4, in serum and of the degree of saturation of the iodothy-
ronine 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 DESCRIPT~OD OF THE PRIOR ART ~;
Specific binding assay methods have undergone a techno-
logical evolution from the original competitive binding
radioimmunoassay (RIA) in which a radioisotope-labeled
antigen is made to compete with antigen ~rom a test sample
for binding to specific antibody. In the RIA technique9
sample antigen is quantitated by measuring the proportion of
radioactivity which becomes associated with the antibody by
-- 3
-
~3~
binding of the radiolabeled antigen (the bound-species of
the labeled antigen) to the radioactivi-ty that remains un-
associated from antibody (the free-species) and then com-
paring that proportion to a standard curve. A comprehensive
review of the RIA technique is provided by Skelly, et aZ.,
CZin. Chem. Z9:146(1973). While by definition RIA is based
on the binding of specific antibody with an antigen or hap-
ten, radiolabeled binding assays have been developed based
on other specific binding interactions, such as between hor-
mones and their binding proteins,
From the radiolabeled binding assays have evolvednonradioisotopic binding assays employing labeling substances
such as enzymes as described in U.S. Patents Nos. 3,654,090
and 3,817,837. Recently further improved nonradioisotopic -
binding assays have been developed as described in German
Offenlegungsehriften Nos. 2,618,419 and 2,618,511, assigned
to the present assignee, employing particularly uni~ue la-
beling substanees, including coenzymes, eyclie reaetants,
eleavable fluoreseent enzyme substrates, and ehemilumines-
~0 eent moleeules. Flavin adenine dinucleotide is mentionedas being useful as a coenzyme label since FAD functions as
a eoenzyme in useful monitoring reactions. In Canadian
Patent Application No. 330,233, filed June 21, 1979 and
assigned to the present assignee, FAD is further deseribed
~5 as useful in improved speeifie binding assays employing a
prosthetie group as the label beeause FAD also funetions
as a prosthetie group in seleet bioehemieal systems.
~,
.
.:: .; ,
.
Various methodologies exist for the determination of
iodothyronine concentrations in serum. A significant ad-
vance in iodothyronine assays ~as the development of the
competitive protein binding assay by Murphy and Pattee, J.
CZin. ~ndoe~inoZ . 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 com-
pete for binding to antibodies rather than to TBG. In both
the competitive protein binding assay and the radioimmuno-
assay for an iodothyronine, the radiolabeled material con-
sists of the native iodothyronine in which one or more o~
the iodine atoms are replaced by a radioactive iodine iso-
tope, usually 125I. The above-mentioned nonradioisotopic
binding assays have offered even more advantageous methods
for determining iodothyronines, particularly those methods
described in U.S. Patents Nos. 4,043,872 and 4,040,907 and
most especially in OLS's 2,618,419 and 2,618,511 and
Canadian patent application No. 330,233 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 anal-
ytical interest, such as the iodothyronines, and particu-
larly for use in the assay referred to hereinbefore employ-
ing a prosthetic group label. The FAD-labeled conjugates
have thQ general formula:
~ ~ 5 ~
. .. .. , . ,. . i . ~ ...
NH-~CH2~-nNH-~cO)L
~INXN~
Riboflavin-(Phos)2-Ribose
wherein Riboflavin-(Phos)2-Ribose represents the riboflavin
-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 OT analog thereof in
the present labeled conjugates, in terms of its chemical
nature, usually is a protein, polypeptide, peptide, carbo-
hydrate, glycoprotein, steroid, or other organic molecule
for which a specific binding partner is obtainable. In func- :
tional terms, the ligand will usually be an antigen or an anti- ~.
body 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 poly-
peptide 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 lOO and
1,500.
FAD-labeled conjugates wherein the ligand therein is an
iodothyronine are particularly use~ul in specific binding
assays to determine the iodothyronine in liquid media such as
serum and preferably have the general formula:
- 6 -
NI~ ~CH2~nNH - C - CHCH~
~IN ~N~ Bl 2
Ribofla~in-(Phos)2-Ribose
wherein Ribofla~in-(Phos)2-Ribose represents the riboflavin
-pyrophosphate-ribose residue in flavin adenine dinucleotide,
n = 2 through 6~ and ~1 and ~2 are, independently, hydrogcn or
iodine.
S ` The FAD-labeled conjugates are used in binding assays for
the ligand or a specific binding partner therefor and are deter-
mined, i.e., monitored, for the purposes of the assay by measur-
ing FAD activity, e.g., the coenzyme or prosthetic group activity
~cncrated upon combination of such conjugate with an apoenzyme
that requires FAD to perform its catalytic function as described
in de ail in the abov~-n~ntioned Canadian pat~nt a~plication No. 330,233.
The present FAD-labeled conjugates~ can be prepared by a
~ariety of synthetic routes. Exemplary of such available
synthetic routes is the following general reaction procedure:
Cl
~N~ ' ' ' ' ; ''`
H0 N ~ j
~i I - ' (1,)
0\/0
H3 ~ CH3 :
Reaction of 6-chloro-9-(2',3'-0-isopropylidine~ -ribofuranosyl)
purine (1) ~Hampton et aZ, J. Am. Chem. Soc. 83:150(1961)3 with
an ~,w-diaminoalkan~ selected from those listed in Table 2
~ 7
TA BLE 2
n ~ diaminoalkane
. . . _
2 1,2-diaminoethane
3 1,3-diaminopropane
4 1,4-diaminobutane
1,5-diaminopentane
6 1,6-diaminohexane
yields the intermediate 6-(~-aminoalkyl)-9-(2',3'-0-
isopropylidine-~-D-ribofuranosyl) purine (2).
H-~CH2 ~ NH2
~ ~J
O ~ ( 2 )
n = 2- 6
0~ 0
H3C~CH3 ,
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) ~ .
NH-~CH2 ~ NH-~CO)L
HO N ~ J
O ~ (3)
n = 2-6
0\,0
H C ~ CH
.
~L~ 3~
wherein tCO)L is the ligand or analog thereof bound by an
amide bond. Such condensation reactions can be accomplishe~
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 1~4.1344(1964], the
mixed anhydride reaction [Erlanger et a~, Methods In lmmuno-
~ogy and Immunochemistr~, 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 C~in. Chem. 22: 72~(1976).
It will be recognized, of course, that other well known
methods are available for coupling the ligand or a derivativc
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 car-
bo~ylic acid or amino group to the amino-purine intermediate
(2). For example, amine-amine coupling agents such as
bis-isocyanates, bis-imidoesters, and glutaraldehyde
[Immunoohem. 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 mono-
graph and in Lowe ~ Dean, Affinity ChromatographyJ John
Wiley ~ Sons ~New York 1974).
: . .
Such coupling ~echniques will be considered equivalents
to the previously discussed peptide condensation reactions
in preparing useful labeled conjugates. The choice of cou~-
ling technique will depend on the functionalities a~ailable
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 abbrevia-
tion -~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 pep~ide condensation or
can be the ligand or derivative thereof coupled through a
bridging group inserted by coupling of the ligand or derivative
~ith a bifunctional coupling agent.
It is evident that in coupling the ligand or derivative
thereof to the amino-purine intermediate (2) it may be desir-
able to protect certain reactive groups in such ligand or
derivative from participating in side reactlons during coup-
ling. Protection of reactive groups may also be desirable to
.
, .~ .....
- 10 -
.
prevent interfering reactions during the synthetic ste~s
described below for completing ~he 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 reac~ive sites on the
ligand or derivative can be accomplished before or after the
coupling to the amino-purine intermediate (2). One skille~
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 pro~ect the amine group prior to conden-
sation or linkage with the amino-purine intermedia~e. The
amine-protected iodothyronine intermediate has the formula:
HOOC-CHCH2~ ~O~OH (~)
2 = H or I
. `,:
wherein Y is an amine-protecting group. It will be recogni~ed ~ .
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 pre-
ferred, and the like, such as others of the acyl type (e.g.,
- 11 -
- , , "
.
~ ~ 3'~
formyl, benzoyl 7 phthalyl, p-tosyl, aryl- and alkylphosphoryl,
phenyl- and benzylsulfonyl, tritylsulfenyl, o-nitrophenyl-
sulfenyl and o-nitrophenoxyacetyl), those of the alkyl type
(e.g., trityl, benzyl and alkylidene) and those of the
S urethane type (e.g., carbobenzoxy, p-bromo-, p-chloro- and
p-methoxycarbobenzoxy, tosyloxyalkyloxy-, cyclopentyloxy-,
cyclohexyloxy-, t-butyloxy, l,l-dimethylpropyloxy,
2-(p-biphenyl)-2-propyloxy- and benzylthiocarbonyl.
The substituted adenosine intermediates formed by con-
densation or linkage between the amino-purine intermediate
(2) and the amine-protected iodothyronine intermediate (4)
are of the formula (3) wherein -~CO)L is:
- C-CHCHz ~ O ~ OH (5)
~ 2 = H or I
wherein Y is an amine-protecting group as above.
- 12 -
.
~ ~ 3t~
Treatment of intermediate (3) with phosphorous oxy-
chloride produces the phospho~ylated ligand or analog sub-
stituted adenosine intermediate (6)
NH-~GH2 ~ H-~CO)L
1l <N ~ NJ (6
HO-P-O ~ o ~
~ .
n = 2-6 -
O O :.
H3CXcH3
which upon hydrolysis yields the ligand or analog sub-
stituted 5'-adenylic acid intermediate (7).
NH-~CH2 ~ NH-~CO)L
Il <N ~ N~J (7)
HO-IP-O ~ ~
n = 2-6
H H
Condensation of riboflavin-5'-monophosphate with inter-
mediate (?) activated to a phosphorimidazolidate by treatment i`
with N,N'-carbonyldiimidazole yields FAD-labeled conjugates
~8).
.
NH-~cH2~-n-NH~co)L
<~N ~ N (~)
n = 2-6
Riboflavin- (Phos) 2 ~ Ribose
.~ . .
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:
NH-~CH2~-nNH-ICI-CHCH2 ~ O ~ H
~N ~ N ~1 ~2
\N ~ N ~ n = 2-6
Riboflavin-(Phos)2-Ribose ~1 ~2 = H or I
S 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
PAD-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
NH~CH2 ~NH2
~N
HO N
\1/0
I I
0\~0 ~ :"
3 CH3
wherein n = 2 through 6; and
- 14 -
.. . .... .
formula B
NH-~CH2 ~ NH-~CO)L
~ ~ N
R ~ O
R R3
~herein -~CO)L is a specifically bindable ligand~ or a bind-
ing analog thereto, and preferably is of formula (5), bound
through an amide bond; n = 2 through 6; ~1 and ~2 are,
independently, hydrogen or iodine; Rl is -OH or -O-P-OH
l l OH
when R2 and R3 together form the group X ~ or Rl is
Il 2 3 H3C CH3
-O-~-OH when R and R are -OH.
~H
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 poly- : :
peptide or protein or an antibody, or is a hapten of molecu~
lar weight between 100 and 1,500. Various methods for coupling
such ligands or analogs thereof to the amino-purine inter
mediate (2) through an amide bond in the synthesis of the
present FAD-labeled conjugate will now be presented.
- 15 -
. .
- 1~3~?~
~o7ypeptides and Proteir~
Representative of specifically bindable protein ligands
are antibodies in general, particularly those of the IgG,
IgE, IgM and IgA classes, for example hepa~itis antibodies;
and antigenic proteins such as insulin, chorionic gonadotropin
(e.g., HCG), carcinoembryonic antigen (CEA), myoglob;n, hemo-
globin, follicle stimulating hormone, human growth hormone,
thyroid stimulating hormone (TSH), human placental lactogen,
thyroxine binding globulin ~TBG), instrinsic factor, trans-
cobalamin, enzymes such as alkaline phosphatase and lactic
dehydrogenase, and hepatitis-associated antigens such as
hepatitis B surface antigen (HBsAg), hepatitis B e antigen
(HBeAg) and hepatitis B core antigen (HBCAg). Representative
of polypeptide ligands are angiotensin I and II, C-peptide, -
oxytocin, vasopressin, neurophysin, gastrin, secretin, and
glucagon.
Since, as peptidesg ligands of ~his general category
possess numerous available carboxylic acid and amino groups,
coupling to the amino-purine intermediate (2) can proceed
~0 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 conven-
tional bifunctional reagents capable of coupling carboxylic `
acid or amino functions to ~he amino group in the amino-purine
~5 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 conju-
gate comprising the hapten coupled to a carrier molecule,
almost always a protein such as albumin. The coupling reac-
tions ~or forming the immunogen conjugates are well developed
in the art and in general comprise the coupling of a carboxy-
lic 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 conju-
gates 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 prostag~andins. 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 reac-
tions (in the structural formulae below, n represents an
integer, usually 1 through 6, and Me represents methyl).
- 17 -
.3
Carbamazepine
Dibenz[b,f]azepine is treated sequentially with
phosgene, an ~-aminoalkanol, and Jones re~gent (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:
~ ' .
CONH~C~ ~ COOH
Quinidine
Following the method of Cook et aZ, PharmacoZogist 17:
219tl975), quinidine is demethylated and treated with ~ .
5-bromovalerate followed by acid hydrolysis to yield a
suitable carbcxylic acid derivative.
Digoxin and Di~itoxin
The aglycone of the cardiac glycoside is treated with
succinic anhydride and pyridine according to the method of .
Oliver et aZ, J. C~in. ~nvest. 47 :1035~1968) to yield the
following: ::~
z ~0~0 ~ .
I Me 7 _ .. :
~ ~J ~ ' '
~ H Z c H or OH ,
HOOC~CH2~CO J
- 18 -
Theophylline
Following the method of Cook et aZ, 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:
3 ~N ~ ~ ~ ~ C~ ~ COOH
CH3
Phenobarbital and Primidone
Sodium phenobarbital is heated with methyl 5-bromovalerate .
and the product hydrolyzed to the corresponding acid derivative
of phenobarbital lCook et aZ, Quantitative AnaZytic Studies
in EpiZep~y, ed. Kelleway and Peterson, Raven Press tNew
York 1976~ pp. 39-58]:
HN ~ C2
0~1~0
CC~ ~ COOH
~ 19 r
To obtain the acid derivative of primidone following
the same Cook et aZ reference me~hod, 2-thiophenobarbital
is alkylated, hydrolyzed, and the product treated with
Raney nickel to yield:
., o ~ ".
HN~C2~
~CH2 ~ COOH
Di~henylhydantoin
Following the method of Cook et aZ, Res. Comm. Chem.
Path. Pharm. 5: 767(1973), sodium diphenylhydantoin is reacted
with methyl 5-bromovalerate followed by acid hydrolysis to
yield the following:
~,0
HN~ N ~ CH2 ~ COOH
O .~
h~or~hine -.
: '
Morphine free base is treated with sodium g-chloroacetate
according to the method of Spector et aZ, Soience 168 :1347
~1970) to yield a suitable carboxylic acid derivative.
: ',
- 20 -
. . .
Nicotine
According to the me~hod of Langone et aZ~ Biochem. ~2~24):
5025~1973), tran8-hydroxymethylnicotine and succinic anhydride
are reacted to yield the following:
. ~N ~ fH3
~ N~
HOOC~CH2~C-OCH2J--i
Andro~ens
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 aZ, J. Steroid Biochem. 5: 739(1974). Follow-
ing are representative testosterone derivatives:
l-position
HC~CH2)n
~1
O~V
?-position
o S~CH2 ~ COOH
- 21
-; ; ,.
~3~
Estrogens
Suitable carboxylic acid derivatives of estrogens, e.g.,
estrone, estradiol and es~riol, are prepared according to
the method of Bauminger et aZ~ gUpra ~ as represented by the
following estrone derivative:
N-OCH2-COOH
Progesterones
Suitable carboxylic acid derivatives of progesteron~
and its metabolites linked through any of the 3- J 6- or `
7-positions on the steroid nucleus are prepared according
to the method of Bauminger et a~ ~upra, as represented by
the following progesterone derivatives:
3-posi ~ion :
MeOH
HOOC-CH20-N
.
.
"
- 22 - : :
- .. - , , . ~, , ~,
: ~3'7~
6- posi tion
~3
C=O
Me ¦
0~
S~C~ ~COOH
-
7-position ~H3 ¦
C=O
Me
f~-
O ~ SW~C~ ~ COOH
The methods described abo~e are but examples of the
many known techniques for forming suitable carboxyl.ic
acid derivatives of haptens of analytical interest. The
principal derivation techniques are discussed in CZ~n. Chem.
22:726~1976) and include esterifîcation of a primary alcohol
with succinic anhydride lAbraham and Grover, pr~oip~eB of
Compe~itive Protei~-Binding Assays, ed. Odell and Daughaday,
J.B. Lippincott Co. (Philadelphia 1971) pp. 140-157~, forma-
. tion of an oxime from reaction of a ketone group with carboxyl~
methyl hydroxylamine [J. ~ot. C~em. 234:1090(1959)], intro-
.. .. ..
duction of a carboxyl group into a phenolic residue using
chloroacetate lSaience 168;1347(1970)], and coupling to di~-
zotized p-aminobenzoic acid in the manner described in J. ~io~.
Chem. 235 :1051(1960).
- 23 -
, . . :, . . .. .
The general reaction scheme described above is exempli-
fied by the following descTiptions of the synthesis of the
ethyl ~n=2) and hexyl ~n=6~ analogs of the FAD-labeled conju-
gates wherein the ligand is the iodothyronine thyroxine
[i.e., -~CO)L is of the formula ~s) wherein ~1 and ~2 are
both iodîne]. Also provided are descriptions of assay me~hods,
and results therefrom, employing the exemplified analogs as
labeled conjugates in a specific binding assay for thyroxine.
- 24 -
1. Ethyl Analog
l-I. PREPARATION OF THE LABELED CONJUGATE
6-(2-Aminoethyl)amino- 9-(2',3' O-isopropylidine-~-D
-ribofuranosyl)purine (2).
13.56 grams (g) ~41.5 millimoles (mmol)] of 6-chloro-9-
(2~3l-o-isopropylidene-~-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 dis-
solved in a small volume of water which was then applied to
the top of a 25x55 centimeter (cm) *Dowex 50W-X2 cation exchange
column in the ammonium form (Bio-Rad Laboratories, Richmond,
California 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. Merch, Darmstadt, West Germany) with-a 9:1
(v:v) mixture of ethanol and 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
- 25 -
*Trade Mark
..
- . ,~ .
~-.3~
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 C15H22N6O4: C~ 51.42;
H, 6.33; N, 23.99
Found: C, 50.92; H, 6.54; N, 23.01
NMR (60 MHz, CDC13): ~ 1.37 (s,3H,
isopropylidene), 1.63 (s,3H,
isopropylidene), 5.92 (d, lH, l'-ribose),
7.90 (s, lH, purine), 8.26 ~s, lH, purine)
Optical Rotation [~]D = -74.85 (c 1.0, C~30H)
The remaining crude product (12 g) was purified by chroma-
tography on Dowex 50W-x2 as described above. The overall yield
was 8 g (55~).
~-(N-Trifluoroacet~l)amino-~-[3,5-diiodo-4-(3',5'-diiodo-4'
-hydroxyphenoxy)phenyl]propanoic acid (4).
~ :
This compound was prepared by the method of Blank, J.
Pharm. Sci~ 53:1333(1964). To a cooled (0~C), stirred suspen~
sion of 5 ~ (6.4 mmol) of L-thyroxine (Sigma Chemical Co., St.
~0 Louis, Missouri 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 com-
bined 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 ~-~
- 26 -
3~
and petroleum ether gave a pinkish-white solid (3.~5 ~, 7n.5
yield) having a melting point (m.p.) of 228-2303C with
decomposition.
Analysis: Calculated for C17HloF3I4N05: C, 23.39;
H, 1.15; N, 1.60
Found: C, 23.00; H, 1.05; N, 1.65
NMR [60 MHz, DCON(CD3)2] ~ 7.28 ~s, 2~,
aromatic), 8.03 (S9 2H, aromatic),
9.7 (m, lH, amido)
IR (KCl): 1700 (>C-O)
Optical Rotation E~25 = -14.97 (c 1.0
dimethylsulfoxide)
A second recrystallization produced a second precipitate
(0.95 g) m.p. 224~228C with decomposi~ion. The overall yield
15was 87.5%.
N-{2-[N-~rrifluoroacetyl)-3,3'~5,5'-tetraiodothyronyl]
aminoethyl}-2'~3'-0-isopro~ylidene adenosine (3).
A solution of 8.72 g (10.0 mmol) of a-(N-trifluoroacetyl)
amino-~-[3,5-diiodo-4-(3',5'-diiodo-4'-hydroxyphenoxy)phenyl]
propanoic acid (4) and 3.86 g (11.0 mmol) of 6-(2-aminoethyl)
amino-9-(2',3'-0-isopropylidene-~-D-ribofuranosyl) purine (2)
in 50 ml of dry dimethylacetamide was prepared under a dry
argon atmosphere at -20C. To this cold stirred solution was
added a solution of 3.04 g (11.0 mmol) of diphenylphosphoryl
azide tAldrich Chemical Co., Milwaukee, Wisconsin USA) in 10 ml
of dry dimethylacetamide followed by the addition of l.fi ml
(11.0 mmol) of dry triethylamine. The solution wus ~ert ;It
room temperature for 22 hours. The solution was then a~de~
dropwise to 300 ml of cold (0C) water with stirring. ~he
.
~3 ~
resulting white precipitate was collected by filtration and
dried in vacuo (56C) to give 13.0 g of a light crea~ 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.
198-200C (decomposed).
Analysis: Calculated for C32H30F3I4N7O8: C, 31.89;
H, 2.51; N, 8.14
Found: C, 31.95; H, 2.60; N, 7.86
NMR [220 MHz, (CD3)2SO] ~ 1.32 (s, 3H,
isopropylidene), 1.55 (s, 3H, iso-
propylidene), 6.14 (d, lH, l'-ribose),
7.02 (s, 2H, thyroxine), 7.82 (s, 2H, ~ -
thyroxine), 8.25 (s, lH, purine),
8.36 (s, lH, purine), 8.41 (t. lH.
J=6. amido). 9.64 (d. lH. J=8.
~0 trifluoroacetamido~
~ptical Rotation [~]D = -11.82 (c 1.0,
pyridine)
N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
aminoethyl}-2',3'-O-isopropylidene-5'-adenylic acid
monotriethvlamine salt monohydrate (6).
A solution of 1.2 g (1.0 mmol) of N-{2-[N-(trifluoro- ;;
acetyl)3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-O-isopropyl-
idene adenosine (3) in 10 ml of dry triethy]phosphate was pre-
pared under a dry argon atmosphere at 0C. To the cold, stirred
- 28
`
'.; . ` . `
~3~
solution was added 0.45 ml (5 mmol) of phosphorous oxychloride.
The resulting solution was kept for 24 hours at 0C, 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 re-
crystalized from a mixture of dry methyl alcohol and dry
ethyl ether to give 390 mg (27.8% yield) of a white solid,
m.p. 173-183C (decomposed).
Analysis: Calculated for C38H48F3I4N8 12
H, 3~45; N, 7.98
Found: C, 32.24; H, 3.08; N, 7.58
NMR [60 MHz, (CD3)2SO] ~ 1.53 (s, 3H,
isopropylidene~, 6.2 (d, lH, l'H-ribose), ~ ;
7.1 (s, 2H, thyroxine aromatic), 7.87
(s, 2H, thyroxine aromatic),8.27 (s, lH,
purine). 8.52 (s, lH, Purine)
Optical Rotation [~] D = -17.50 (c 1.0, CH30H)
N-{2-[N-(Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl]
aminoethyl}-5'-adenylic_acid (7)
200 milligrams (mg) (0.14 mmol) of N-{2-[N-(trifluoro-
acetyl-3,3',5,5'-tetraiodothyronyl]aminoethyl}-2',3'-o-isopropyl-
idene-5'-adenylic acid monotriethylamine salt monohydrate (6)
was suspended in 1 ml of water (0C) and trifluoroacetic acid
(9 ml) was added dropwise with stirring. After 30 minutes a clear
- 29 -
solution was obtained. The solution was kept cold ~0C) for
an additional 15 hours, then evaporated in vacuo (30C). The
resulting residue was evaporated four times in vacuo (25C) from
20 ml volumes of anhydrous ethyl alcohol and then dried in
vacuo (25C) leavina 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
(25C) to give a white solid (135 mg, 76% Yield) which slowly
melted with decom~osition above 188C.
Analvsis: Calculated for C29H27F3I4N7OllP: C, 27.97;
H, 2.19; N, 7.87
Found: C, 28.11; H, 2.31; N, 7.65
NMR [220 MHz, (CD3~2SO] ~ 5.95 (d, lH~
l'-ribose), 7.04 (s, 2H, thyroxine
aromatic), 7.84 (s, 2H, thyroxine
aromatic), 8.25 (s, lH, purine),
8.36 (s, lH, purine), 8.43 (m, lH,
amido), 9.66 (d, lH, trifluoroacetamido) `~
Optical Rotation ~]25 = -2.72 (c 1.0,
pyridine)
Flav~n aden n dinucl otide _ thyroxine conjugate (8)
498 mg (0.4 mmol) of N-{2-[N-(trifluoroacetyl)-3,3',-
5,5'-tetraiodothyronyl]aminoethYl}-5'-adenylic acid (7) was
dissolved in 10 ml of dry dimethylformamide and tri-n-butyl-
amine [96 microliters (~1?, 0.4 mmol] was added followed by
the addition of l,l'-carbonyldiimidazole (320 mg, 2.0 mmol).
After stirring for 18 hours at room temperature in the absence
of moisture, water (280 ~1) was added and then the solvent
evaporated in vacuo.
- 30 -
-;
~L~l3~
The resultinq oil was dried by repeated in vacuo evapor-
ation from dry dimethylformamide (4 times from 10 ml). The
resultin~ ~hos~horimidazolidate 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 add-
ing a solution of the ammonium salt of riboflavin-5'-monophos-
phate (192 mg, 0.4 mmol~ in 10 ml of water to a stirred solution
of tri-n-octylamine (176 ~1, 0.4 mmol3 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 oran~e solid.
The above solution containinq 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 (2.5x78 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 dimethyl-
formamide and triethylammonium bicarbonate (1 M, P~I 7.5). Thecolumn 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. Merch, Darmstadt, West Germany).
The TLC plates were developed usinq a 40:40:25:1:1
(v:v) mixture of acetone, chloroform methvl alcohol, water,
and triethylamine. Fractions numbered 11 through 17 from the
- 31 -
*Trade Mark
, . ..
akove-mentioned column chromatography were combined and eva-
porated in va~uo. The resldue was chromatographed on a co-
lumn (2.5 x 75 cm) prepared from 125 g of Sephadex LH-20
which had been preswollen (18 hours) in 0.3 M ammonium bi-
carbonate. The column was eluted with 0.3 M a~monium bicar-
bonate collecting 10 ml fractions. The effluent was moni-
tored by absorption of ultraviolet light at 254 nanometers
(nm). The volume of the fractions was increased to 20 ml
be~inning with fraction number 150. The salt concentration
o~ the eluent was decreased in a stepwise fashion as follows:
0~15 M ammonium bicarbonate at fraction number 295, 0.075
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 evapo-
rated in va~uo leaving the labeled conjugate f8) as a yellow-
orange residue. An alkaline, aqueous solution of this res-
idue exhibited ultraviolet absorption maxima at the follow-
in~ wavelengths: 266 nm, 350 nm, 373 nm, and 450 nm. The
yield, estimated from the absorption at 450 was about 5%.
~0 A phosphodiesterase preparation (Worthington Biochem-
ical Corp., Freehold, New Jersey USA) isolated from snake
venom (~rotaZus A~amanteusJ hydrolyzed the above product to
riboflavin-5'-monophosphate and the thyroxine substituted
5'-adenylic acid (7) wherein the trifluoacetyl blocking
~5 ~roup had been removed. ~-
l-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 Canadian Patent Application No. 330,233 referred to
hereinbefore):
- 32 -
~ .
~ ~ ,.
3~
A. Preparation of apoglucose oxidase
Purified glucose oxidase with low catalase activi ty
obtained from the Research Products Division of Milcs l.lbor~-
tories, Inc., Elkhart, Indiana USA was twice dialyze~ for 12
hours each against 0.5~ (w:v) mannitol (30 volumes e~c~
Aliquots of the dialyzate containing l00 mg o~ glucosc o~ se
each were lyophilized and stored at -20C.
Bovine serum albumin (200 mg) was dissol~ed in 12 ml of
water adjusted to pH l.6 with concentrated sulfuric aci~, nlix~
with 150 mg charcoal (RIA grade from Schwarz-Mann, Or~n~eburg,
New York USA), and cooled to 0C. Lyophilized glucose oxi-la~-
(l00 mg) was redissolved in 3.l ml of water and 3 ml was addc~
to the stirred albumin-charcoal suspension with continued
stirring for three minutes. The suspension was then rilterc-l
through a 0.8 micron, 25 millimeters (mm) diameter Millil)orc
filter ~Millipore Corp., Bedford, Massachusetts USA) moulltc~
in a Sweenex filter apparatus (Millipore CorpO) on a 5n ml
disposable plastic syrings. The filtrate was quickly ne~ltr~-
lized to pH 7.0 by addition of 2 ml of 0.4 M phosphate huffcr
~pH 7.6) and thereafter 5 N sodium hydroxide. Dry charcoal
~l50 mg) was then added and stirred for one hour at 0C. The
resulting suspension was filtered first through a 0.8 miero
Millipore filter and then through a 0.22 micron Millipore
filter. To the filtrate was added glycerol to 25~ (v:v~ ~n~ ;
the stabilized apoglucose oxidase preparation was stored ~t
4C.
~ 33 -
.
~ 3~
B. Assay Reagents
1. Labeled conjugate - The ethyl analog labeled
conjugate prepared as in section l-I above was
diluted in 0.1 M phosphate buffer (pH 7) to a
concentration of 1 micromolar (~M).
2. Apoenzyme - Apoglucose oxidase was diluted with
0.1 M phosphate buffer (pH 7) to a concentration
of 0.6 ~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 oxi-
dase activity when incubated with the apoenzyme.
3. Insolubilized antibody - A washed, moist cake of
Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala,
Sweden) activated bv cvanoaen bromide accordin~ 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
~0 buffer (pH 7.0) and a~itated slowlv for 36 hours at
4C. 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 re-
sulting Sepharose-bound antibody was washed on a
scintered funnel with 400 ml each of 50 mM sodium
acetate - 500 millimolar (mM) sodium chloride IpH 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) containinq 0.01~ sodium azide
to give 22 ml of an about 50% suspension.
- 34 -
:.
r~
4. Standard - A 1.15 mM stock solution of thyroxine in
5 mM sodium hydroxide was diluted to 2 ~M in U. 1 M
phosphate buffer (pH 7).
5. Monitoring rea~ent - A glucose oxidase assay rea~ent
was prepared to contain the following mixture ~er
130 ~1: 25 ~l of 1.2 mg/ml peroxidase (Si~ma
Chemical Co., St. Louis, Missouri USA) in
0.1 M phosphate buffer (pH 7), 5 ~l of lO mM
4-aminoantipyrine in water, 20 ~1 of 25 mM
3J5-dichloro-2-hydroxybenzene sulfonate in 0.l M
phosphate buffer (pH 7), 30 ~1 of 16.5~ bovine serum
albumin in 0.1 M phosphate buffer (pH 7), and 50 ~l
of l M glucose in aqueous saturated benzoic acid
solution.
C. Assay Procedùre
Binding reaction mixtures were prepared by mixin~ 150 ll3
of the insolubilized antibody suspension, 80 ~1 o~ the labeled ~ `
conjugate solution, various amounts of the standard thyroxine
solution to give varying concentrations of thyroxine In the
reaction mixtures, and a sufficient volume of 0.1 M phosphate
`buffer ~pH 7) to make a total volume of 500 ~1. lhe reaction
mixtures were incubated with shaking for two hours a~ 25~C.
Each reaction mixture was then vacuum filtered through a ~lass
wool plugged, dry pasteur pipette previously treated sequcn-
tially with periodate and ethylene glycol solutions to
eliminate possible FAD contamination. To a 300 ~l ali~uot
of each filtrate was adde~ 130 ~l of the monitoring rea~ent
and 5Q ~1 of the apoenzyme solution. After one hour, the
absorbance of each reaction mixturc was measurcd at 520 nm.
- 35
f~
D. Results
Following is Table 3 showing the results of t~le as~y
procedure in measuring thyroxine. The absorbance results ;Ire
expressed as the average of duplicate runs corrected for
residual enzyme activity in the apoenzyme solution (absorbancc~
of 0.522) and for endogenous FAD in the antibody suspension
tabSorbance of 0.142).
TABLE 3
Volùme of Thyroxine Absorbance (520 nm)
10Standard Added ~
0 0.223 `
0 221
0.281 ; ~'
250 0.286
The results demonstrate that the present labeled conjugates
are useful in a specific binding assay method for determining
a ligand in a liquid mediùm.
~ 36
2. HexyZ AnaZo~
2- I. PREPARATION OF THE LABELED CONJUGATE
6-(6-Aminohexyl)amino-9-(2' 93' -O-isopropylidene-~-D-
ribofuranosyl) purine (2).
16.0 g (50 mmol~ of 6-chloro-9-(2',3'-O-isopropylidene-
~-D-ribofuranosyl) purine (1) [Hampton et a~, J. Am. Chem. Soc.
83:1501(1961)] was added with stirring to a molten (70C)
sample of freshly distilled 1,6-diaminohexane (58 g, 500 mmol~.
The resulting mixture was stirred under argon at 40C for
lS hours. The excess diamine was remo~ed by distillation
under reduced pressure (60C, 0.01 mm Hg). The resulting pale
yellow residue was adsorbed onto 150 g of silica gel 60 (E.
~5erck, Darmstadt, West Germany) and used to top a chromato-
graphic 9:1 (v:v) mixture of absolute ethyl alcohol and tri-
ethylammonium bicarbonate (pH 7.5, 1 M). The column was
eluted with the above 9:1 (v:v) solvent mixture and 900 20 ml
fractions were collected. The fractions were examined by thin
layer chromatography (TLC) on silica gel 60 eluting with a 7:3
(v:v) mixture of absolute ethyl alcohol and triethylammonium bi-
carbonate (pH 7.5, 1 M). Fractions numbered 391 through 900 ~`
~rom the column chromatography were combined and e~aporated in
vacuo leaving 15.0 g of a glassy residue (74~ yield). A 1 g
sample of the glass was dissolved in a small volume of methyl
alcohol and applied to the top-of a column prepared from 80 g
of Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden)
preswollen in methyl alcohol. The column was eluted with
methyl alcohol. A total of ninety 8 ml fractions were col-
lected. The fractions were examined by TLC on silica gel 60
eluting with a 7:3 (v:v) mixture of absolute ethyl alcohol
. ,
- 37 -
3r~
and triethylammonium bicarbonate (pH 7.5, 1 M). ~:raction~
numbered 19 through Z7 from the colu~n chromatography were
combined and evaporated in vaCuo leaving 910 mg (91~, rc~ovcry)
of a white glass.
Analysis: Calculated for ClgH30N~O4: C, 56.14;
H, 7.44; N, 20.68.
Found: C, 53.91; H, 7.33; N, 19.18
NMR (60 MHz, CDC13): ~ 1.40 (s, 3H,
isopropylidene), 1.63 (s, 3H, isopro-
pylidene) 5.98 (d, lH, l'-ribose), ;
7.92 (s, lH, purine), 8.36 (s, 1~l,
purine)
Optical Rotation [a]25 = -50.11 (c 1.0,
methyl alcohol)
N-{6-[N-Trifluoroacetyl)-3,3',595'-tetraiodothyronyl]
aminohexyl}-2_',3'-O-isopro~ylidene adenosine (3).
A solution of 4.36 g (5.0 mmol) of ~-tN-trifluoroacetyl)
amino-~-[3,5-diiodo-4-(3',5'-diiodo-4~-hydroxyphenoxy)-phenyl]
propanoic acid t4), prepared as described in section 1 r abovc,
and 2.24 g (5~5 mmol~ of 6-(6-aminohexyl)amino-9-(2',3'-()
isopropylidene-~-D-ribofuranosyl) purine (2) in 100 ml of dry
dimethylformamide was prepared under a dry argon atmosphere at
-20C. To this cold s~irred solution was added a solution of
1.52 g (5.5 mmol) of diphenylphosphoryl azide (Aldrich Chemical
Co., Milwaukee, Wisconsin USA) in 50 ml of dry dimethylforma-
mide followed by the addition of 0.8 ml (5.5 mmol) of dry
triethylamine. l`he solution was left at room temperature for
22 hours. The solution was then added dropwise to 600 ml of
cold (0C) water with stirring. The resulting white precipi-
tate was collected by filtration and dried in v~cuo (60C) to
- 38 -
give 4.90 g (78~ yield) of white solid. A samplc of thi~
solid was recrystallized from a mixture of acetone ~ w.lter
giving a white solid, m.p. 205-207C (decomposed).
Analysis: Calculated for C36H38F3I4N7O~: C~ 34-28;
S H, 3.04; N, 7.77
Found: C, 3q.22; H, 2.99; N, 7.41
Mass Spectrum (20 ma) m/e: 1262 [Mll ],
1164 ~M minus COCF3]
Optical Rotation [~]25 = -21.89 (c 1.(),
pyridine)
N-{6-[N-~Trifluoroacetyl)-3,3',5,5'-tetraiodothyronyl~
aminohexyl}-2',3'-O-isopropylidene-S'-adenylic acid
monotriethylamine salt monohydrate (6~.
A solution of 1.89 g (1,5 mmol) of N-{6-N-
~trifluoroacetyl)-3,3',5,5'-tetraiod~tllyronyl~aminohexyl}-
2',3'-O-isopropylidene adenosine (3) in 15 ml of dry tri-
ethylphosphate was prepared under a dry argon atmospherc at
-10C. To the cold stirred solution was added 0.68 ml
~7.5 mmol) of phosphorous oxychloride. The resultin~ solut iOIl
was kept for 18 hours at -15C then added dropwise with
stirring to 1.5 L o ice water. The resulting precipitate
was colle~ted by filtration and dried in vacuo to give 1. 91 R
t87% yield) of a white solid. The solid was dissolved in
10 ml methyl alcohol and 0.38 ml (2.6 mmol) of tr~ethylaminc
was added. This solution was evaporated in vacuo and the
resulting residue was recrystallized from a mixture of methyl
alcohol and ethyl ether to give 720 mg (33~ yield) of a white
solid, m.p. 151-154C ~decomposed).
- 39 -
Analysis: Calculated for C42H56F3I4N8Ol2
H, 3.86; N, 7.67
Found: C, 35.24; H, 3.88; N, 7.75
Mass Spectrum (20 ma) m/e: 1342 LMH 1,
1244 [M minus COCF3]
Optical Rotation [~]25 = -17.20
(c 1.0, CH30H)
N-{6-[N-(Trifluoroacetyl)-3~3l95~5l-te~raiod4thyr
aminohexyl}-5'-adenylic acid (7).
600 mg (0.41 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,8'
-tetraiodothyronyl]aminohexyl~-2' 3 3'-O-isopropylidene-5'-
adenylic acid monotriethylamine salt monohydrate (6) was
suspended in 0.6 ml of water (0C) and trifluoroacetic aci~
(6 ml) was addéd dropwise with stirring. After 50 minutes
clear solu~ion was obtained. The solution was kept cold
(0C) for an additional 15 hours the~ evaporated ~n v~cu~
(30C). The resulting residue was evaporated in vacuo f ive
times from 20 ml volumes of anhydrous ethyl alcohol then
triturated with 30 ml water and washed with a small volum~ o~
methyl alcohol. The resul~ing white solid (430 mg) was re-
crystallized from methyl alcohol ~o give 290 mg (54.6% yiel~)
of white solid, m.p. 180-183C (decomposed).
Analysis: Calculated for C33H35F3I4N7O
H, 2.71; N, 7.54
Found: C, 30.77; H, 2.55; N, 7.29
Mass Spectrum (20 ma) m/e: 1302 [MH ],
1204 [M minus COCF~]
. - , . . .
.. . .
J~
Flavin adenine dinucleotide - thyrox ne conju~ate (83.
130.13 mg (0.1 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,5'
-tetraiodothyronyl]aminohexyl}-5'-adenylic aci~ (7) was ~lace~l
in an argon atmosphere. To this sample was added a ~oluti~n
of 14 ~1 (0.1 mmol) of triethylamine in 1 ml of dry
dimethylformamide followed by the addition of a solution of
16,2 mg (0.1 mmol) of l,l'-carbonyldiimidazole in 1 n~l of
dry dimethylformamide. After 24 hours, a second equivalent
of l,l'-carbonyldiimidazole (16.2 mg) in 1 ml of dry (~imethyl-
formamide was added. The above reaction was allowed to proccc~
a total of 48 hours at room temperature excluding moisture.
A sample of 47.3 mg (0.1 mmol) of the ammonium salt of ribo-
flavin-5'-monophosphate was converted to the corresponding
tri-n-octylamine salt as described in section l-I above. l`hi~
salt was dissolved in 3 ml of dry dimethylformamide and addcd
to the above solution containing the phosphorimidazolidate of
the adenylic acid intermediate (7).
The resulting solution was allowed to stand in the dark
at room temperature excluding moisture for 24 hours. The
solvent was evaporated in vacuo and the resulting residue w~s
chromatographed on a column (2.5x7B cm) prepared from lO0 ~
of Sephadex LH-20 (Pharmacia Fine Chemicals 9 Uppsala, Swe~en)
which had been preswollen (18 hours) in a 19:1 (v:v) mixture
of dimethylformamide and triethylammonium bicarbonate (1 M,
2S pH 7.5). The column was eluted with the above 19:1 (v:v)
mixture and 5 ml fractions were collected. The effluent
from the column was monitored by elution on silica ~el 6()
silanised RP-2 TLC plates (~. Merck, Darmstadt, West ~.ermany). ~ -
The TLC plates were developed using a 40:40:25:1:1 ~v:v) mix-
~t) ture of acetone, chloroform, methyl alcohol, watcr, ~nd tri-
ethylamine.
- 41 -
.. . . . . .. ... . .
Fractions numbered 24 ~hrough 38 from ~he c~lunln
chromatography were combined and evaporated in v~cuo. 'I'l~e
residue was chromatographed on a column (2.5x85 cm) ~-re~arc~l
from 125 R of Sephadex LH-20 which had been preswollen (18
hours) in 0.1 M ammonium bicarbonate. The column wa~ eluted
with a linear gradient generated from 2 L of 0.1 M ammoniuln
bicarbonate and 2 L o~ water and 23 ml fractions col]~ctc~.
The effluent was monitored by ultraviolet absorption (254 ~ln).
Fractions numbered 170 through 182 were combined and eva~rat~?~l
n ~n vacuo. The residue was chromatographed on a column
(2.5xS5 cm) prepared from 80 g of Sephadex LH-20 which ha~
been preswollen in 0.05 M ammonium bicarbonate. l`he colunln
was eluted with a linear gradient generated from ~ L of
0.05 M ammonium bicarbonate and 2 L of 0.02 M ammonium ~)icar-
bonate. The effluent was monitored by ul~raviole~ abxorptio~
~254 nm). Elutlon was continued with 2 L of 0.2 M ammoniun
bicarbonate, collecting 23 ml fractions. A total of 257
fractions was collected. Fractions numbered 70 throu~h 11l)
were combined and evaporated in vacuo leaving the labeled
~0 conjugate (8) as a yellow-orange residue. An alkalinc,
aqueous solution of this residue exhibited ultraviolet absor~
tion maxima at the following wavelengths: 270 nm, 345 nm, ;Ind
450 nm. The yield, estimated from the absorption at 45() nm,
was about 5%.
A phosphodiesterase preparation (Worthington Biochen~ic~l `
Corp., Freehold, New Jersey USA) isolated from snake venom
(CrotaZus Adamanteus) hydrolyzed the abo~e product to ribo-
flavin-5'-monophosphate and the thyroxine substituted
5'-adenylic acid (?) wherein the trifluoroacetyl hlochi
~() grou~ had been remove~.
42
--II. BINDING ASSAY FOR THYROXINE
The above-prepared labeled conjuga-te was used in a
prosthetic-group labeled specific binding assay as follows
tfurther details regarding such an assay method may be
found in Canadian Patent Application No. 330,233 referred
to hereinbefore):
A. Preparation of apoglucose oxidase
The apoenzyme used was prepared by the method descri-
bed in section l-II, part A above.
B. Assay Reagents
1. Labeled conjugate - The hexyl analog labeled
conjugate prepared as in section 2-I above was
diluted in 0.1 M phosphate buffer (pH 7) to a
concentration of 100 nM.
2. Apoenzyme - This reagent was the same as that
described in section l-II, part B-2 above.
3. Insolubilized antibody - This reagent was the
same as that described in section l-II, part B-3
above.
~0 4. Standard - A 1.15 nM stock solution of thyroxine
in 5 nM sodium hydroxide was diluted to 1 ~M in
0.1 M phosphate buffer (pH 7).
~ _ 43 _
, . .. . ~ ....... . : . ~
.. . ,. , ~ :
3~
5. Monitoring reagent - A glucose oxidase rea~ent W;IS
prepared to contain the following mixture ~cr
117 ~1: 25 ~1 of 1.2 mg/ml peroxidase (Si~ma
Chemical Co ~ St. Louis, Missouri USA) in ~).] M
phosphate buffer (pH 7), 5 ~1 of 10 mM
4-aminoantipyrine in water, 20 ~1 of 25 m~
3,5-dichloro-2-hydroxybenzene sulfona~e in 0.l M
phosphate buffer (pH 7), 17 ~1 of 30% bovine
serum albumin in 0.1 M phosphate buffer (~i 7),
and 50 ~1 of 1 M glucose in aqueous saturated henzoi~
acid solution.
C. Assay Procedure
Binding reaction mixtures were prepared by mixin~ 30 ~1
of the insolubilized antibody suspension, 100 ~1 of the lahclc(l
conjugate solution, either 100 ~1 or none of ~he stand~rd
thyroxine solution, and a sufficient volume of 0.1 M phosl-hate
buffer ~pH 7) to make a to~al volume of 500 ~1. The reuctio
mixtures were incubated with shaking for two hours a~ 25~.
Each reaction mixture was then vacuum filtered through a glass
wool plugged, dry pasteur pipette previously treated sequen-
tially with periodate and ethylene glycol solutions to
eliminate possible FAD contamination. To a 350 ~1 aliquot
o~ each filtrate was added 117 ~1 of the monitoring rcagent
and 50 ~1 of the apoenzyme solution. After one hour, the
absorbance of each reaction mixture was measured at 520 nm.
D. Results
~ `ollowing is Table 4 showing the results of the ilS~y
procedurc in measuring thyroxine. lhe absorbancc reslilts ;lre
expressed as the average of duplicate runs corrected for
- 44 -
- . . . . " ~.-
residual enzyme activity in the apoenzyme solution (al)sorl~an~e
of 0.467) and for endogenous FAD in the antibody susl~ension
(absorbance of 0.041).
~A Bl,E 4
S Volume of Thyroxine Absorbance (520 nm)
Standard Added (~
0.231
100 0.295
The results demonstrate that the present labeled conj Ugiil~CS
are useful in a specific binding assay method for d~tcrmioin~
a ligand in a liquid medium.
- 45 - :
.
. ~
