Note: Descriptions are shown in the official language in which they were submitted.
1~'76~4~ D ~ 877
This in~ention relates in general to novel
olic acid derivatives and to a process for their pre-
paration. In one aspect, this invention is directed to
folic acid derivatives which can be radioiodinated and
are particularly useful in competitive protein binding
and radioimmuno-assays of folate compounds. In another
aspect, this invention relates to processes for the
preparation of compositions which may be used as ligands
in affinity chromatography or haptens in antigen syn-
theses.
In the 1940's, the structure of the vitamin,
folic acid, was characterized and independently syn-
thesized as reported by R. B. Angier et al.~ Science9 103,
~67 (1946). This compound also known as pteroylglutamic
acid (I) consists of a pterin, p-aminobenzoic~ and
glutamate moieties:
8 i j ¦ COOEI --I
20~2 ~ 7 1 3 ~ 2 ' I I ~ 2
I ~ '~ 9 1 lo ~ 'I cH2
2-amillo-4-hvdroxy-6- p-Amino L- Glutamate
m~thylpteridin~ ben?.oic
(p.erin) acid
~~
Pteroic acicl
D - 11,877
11'~6~
As indicated in the work of R. L. Blakle~ The
Biochemistry of Folic Acid and Related Pteridines, John
Wiley and SOns, New York, 1969, folic acid is a requisite
cofactor in the biological transfer of one carbon units
at varying levels of oxidation. The measurement of folic
acid and other folate cofactors or derivatives is of
significant clinical value for the diagnosis of megaloblastic
anemias, nutritional folate deficiencies such as those
associated with alcoholism, and for monitoring dosage
regimens in leukemia chemotherapy.
The most common, non-microbiological assay
currently practiced utilizes a radioassay procedure based
on competitive protein binding (CPB) between a radiolabeled
folate derivative and a second "unknown" serum folate
cofactor or drug. The technique is based on the abillty
of a specific binding protein and a specific ligand to
form a reversible binder-ligand complex. As is well known,
an assay is performed by adding a fixed quantity of
radiolabeled ligand to a series of samples which contain
the protein binder, and known amounts o~ a "standard"
ligand. During incubation, radiolabeled ligand and un-
labeled ligand compete for a limited number of ~tes on
the binding protein. After incubation, bound ligand is
separated from the free ligand and the ratio of free to
bound can be plotted as a dose response curve. A serum
sample can then be assayed by the same procedure and the
concentration of the unknown determined by referring to
the standard dose response curve.
D - 11 877
~7~2~9
An important, disclosed advance in radioimmuno-
assay has been the replacement in many cases of beta emit-
ting tracers such as tritium and carbon-14 by the more
readily monitored gamma emitters such as iodine (131 and
125) selenium (75) and cobalt (57 and 60). Unfortunately,
in some instances the introduction of a large radiolabel
such as iodine can alter or prevent binding of a radio-
ligand to a binder. Thus, it is extremely important
to design a precursor molecule which will undergo rapid
iodination and will still be competitive under assay
conditions.
This invention describes novel generic deriva-
tives or analogs of folic acid I (and related compo~nds,
such as folate metabolites and folate antagonist drugs),
typified by pteroyltyrosine. These compounds were de-
signed to approximate in size, as closely as possible,
the cofactor, drug, metabolite, etc., but still permit
facile iodination at a remote site. Previous approaches
to folate [125I] radiolabels involved the addition of a
radioiodine accepting moiety to folic acid, whereas this
disclosure describes the novel strategy of replacement of
a significant portion of the folic acid molecule with a
radioiodine acceptor. ~n additional advantage of the
derivatives of this invention is that they can be con-
veniently synthesized and appear to be quite stable in
the radioiodinated form.
~ccordingly, one or more of the following ob-
jects can be achieved by the practice of this invention.
It is an object of this invention to provide folic acid
~ 7~z49 D - 11,877
derivatives, s-lch as radiolabeled pteroyltyrosine, pteroyl-
tyramine, pteroylhistidine and the like Another object of
the invention is to provide derivatives or analogs of folates
which can undergo facile, rapid labeling by radioiodine. A
further object of this invention is to provide a process for
the preparation of the folate compositions. A still further
object is to provide a process for the quantitative detection
of folates by the application of the radioiodinated compounds
of this invention in competitive protein binding and radio-
immuno-assays. Another object is to provide novel antigens,
enzyme conjugates, immunosorbents and affinity ligands pre-
pared from the coupling of the novel folate derivatives to
proteins, enzymes, polypeptides, inorganic materials, poly-
saccharides or plastic articles. These and other objects
will readily become apparent to those skilled in the art in
the light of the teachings herein set forth.
The single drawing is a plot of dose response
curves for folic acid and N5 methyltetrahydrofolic acid
obtained using pteroyltyrosine [l-25I~ prepared by the
process of this invention. Further details on the pro-
cedure used are set forth in Example 7.
In its broad aspect this invention is directed
to a class of novel folate derivatives, a process for their
preparation in both unlabeled and labeled form, their appli-
cation to radioassay and to in vivo diagnostic use, and
the preparation of novel antigens~ immunosorbents, and
polymer bound forms of the genetic compounds.
The generic structure of the unla~eled form
of the compounds prepared by the process of the instant
5 -
11 76~49 D-11,877
invention can be illustrated as follows:
II ~ ~ N ~ N~(7 )m
3 ~ ~ CH2-N- ~ C-X
R' (R)n R"
wherein X represen~s a radiolabel acceptor as hereinafter
defined; R represents hydrogen, lower alkyl, formyl or
iminomethy~ R' and R'r individually represent lower al~yl,
hydroxyl, halo, amino or acetamido; R'il represents R or
nitroso; m has a value of 1, 3 or 4 and n has a value of
zero or 1. Thus, for example, when m equals 4 the ring
containing nitrogen at the 5 and 8 positions will be
saturated and the (R) group can be hydrogen or one of the
substituents indicated aboveO Conversely, if the ring is
unsaturated only one hydrogen will be present at the 7
position and n will be 0.
The structures of the compounds of this inven-
tion in the unlabeled for~s, typified by pteroyltyrosine,
can be viewed to be comprised of three linked components:
a substituted pteridine moiety, a p-aminobenzoyl moiety,
and a radiolabel acceptor. The latter two comprise the
"dipeptide" portion of the generic structure.
It is important to note that the radiolabel
acceptor component of this invention is not formed by the
addition of an accepting moiety to folic acid, but by the
replacement of the L-glut:amate portion of the acid with
such a component. Hence the X component of the above
generic structure will not contain the L-glutama~e moiety
and the aromatic or heterocyclic group containing the
D ~ 77
~76;~49
radiolabel will be separated from the p-aminobenzoic acid
moiety by a linear chain of no more than five atoms. The
chain, of course, can be comprised of atoms other than
carbon and can contain substituents and side chains which
do not adversely affect its reactivity in competitive
protein binding (CPB) and radioimmuno assay applications.
The radiolabel acceptor groups which can be
present in the compositions of this invention include
those wherein X is a single amino acid or des-carboxy
amino acid which contains a readily radioiodinated
aromatic ring and a primary aliphatic amino group for
attachment to the rest of the molecule via an amide bond.
Illustrative X components of the generic structure II are
groups such as the folïowing which are derived from the
indicated compounds:
N
IH2 ~ OH _~-CH2-CH ~ N
-NH-CH-COOZ
tyrosine (Z=H) , histamine ,
20 -~H-cH2-cH2 ~ OH IH2 ~ -OH
-NH-CH-COOZ
tyramine,
5-hydroxytryptophan ~Z=H),
~7~Z~
-NH-CH-COOZ
CH ~ N
-NH-CH-COOZ
histidine (2=H), 2(4'-hydroxyphenyl) glycine (Z=H),
and the like, wherein Z is hydrogen or lower alkyl.
Preferred compositions of this invention are
those wherein the X component or moiety contains up to
24 carbon atoms and more preferably up to 12 carbon atoms.
As illustrated above, X can, of course, contain nitrogen
and oxygen and other substituents which do not adversely
affect the use of the compounds for CPB and radioimmuno-assay
applications.
Variations in the pteridine portion of the molecule
can be used to alter the performance of the subsequent radio-
label in a competitive protein binding or radioimmuno-assay,
in order to gain specificity for primary metabolites of
folic acid, folate antagonists used as drugs, and their
metabolites. For example, radiolabeled species in ~hich
R = CH3 or CHO, R' = OH and R'' = NH2, such as N -methyl-
tetrahydropteroyltyrosine-[l25I] and N5 -formyltetrahydrop-
teroyltyrosine-[125I], are appropriate labels in CPB and
immuno-assays specific for N5-methyl- and N5 -formyltetra-
hydrofolate, respectively. Similarly, species wherein R
and N -H are absent R' = R'' = NH2, R''' = CH3 and X is
any suitable radioiodine acceptor ( such as 4-amino-4-deoxy-
N10 - methylpteroyl- [2-(4'-hydroxyphenyl) glycine]) are
suitable markers for the CPB and immuno-assays of
methotrexate.
--8--
~7~2~3 D - 11,877
In practice, the compounds of this invention
can be prepared by a variety of methods. For example,
pteroyltyrosine can be prepared by condensation of a pro-
tected pteroic acid with L-tyrosine methyl ester (L-TME)
followed by basic hydrolysis to cleave both the ester and
the protecting group. The sequence of reactions can be
illustrated as follows:
f~~COOH (CF3C)~I > ~ ~OOH
OH OX
/_ OEt31~ > ~' 8 CH
Cl OH
+ Nll -l'H C028 __> __~ H ~ H N 8
Hooc-rE~ H2
OH
Because the condensation conducted was between
the carboxylic acid of pteroic acid and the amino group
of L-TME, protection of the reactive Ni0-nitrogen of
pteroic acid was required so that it would not condense
_g_
1~ ~6 Z49 D - 11,877
with itself in the coupling reaction. The protective
group selected was the N-trifluoroacetyl group (N-TFA)
which is readily removed via basic hydrolysis. The
conditions employed for the hydrolysis of the ester
(0.1 N NaOH, steam bath, 45 minutes under nitrogen)
were more than adequate for the cleavage of the N-TFA
groupO N10- trifluoroacetyl pteroic acid was prepared
from the reaction of pteroic acid with neat trifluoro-
acetic anhydride. The 2-amino group of pteroic acid
does not require protection in this scheme because it
is quite unreactive.
~ ondensation of N10-trifluoroacetyl pteroic
acid with L-TME was _ the mixed anhydride procedure.
The acid was treated with iso-butylchloroformate in an
unreactive solvent (dimethylformamide) containing a
tertiary amine base (triethylamine) to yield the mixed
anhydride. This material was then treated with L-
-tyrosine methyl esterO Subsequent hydrolytic work up
with dilute alkali metal hydroxide (0.1 N NaOH) and
careful ion exchange column chromatography gave, after
acidification~ pteroyl-L-tyrosine.
A second approach to the synthesis of pteroyl-
tyrosine was based on the condensation of a ~-
-formylpterin with a ~-aminobenzoic acid ester or
amide and reduction of the Schiff base formed to the
N10-secondary amine.
D - 11,877
6~
The pteroyltyrosine of the instant invention
was prepared by a modification of the previously des-
cribed aldehyde routes as set forth below.
Y~3 1 2 ~ NH-~
OH
~ }--
0i1 C~3t-Oc ,1
(CH3~ 2~ B 3 H N~ N~ N~ ~~ 1 2~=
HOAc N~.~ ~N C~l2~ ~c-N~-cH-coocT~3
011
> --~ ~)` "J CH -~--OH
N~ N \ CH2-NH- ~ -?~H-CH--C(`OH
OH
D - 11,877
11762gL9
The Schiff base between 6-formylpterin and p-aminobenæoyl-
L-tyrosine methyl ester (H-PABA-L-TME) was formed in a
1:1 mixture of trifluoroacetic acid (TFA) and glacial
acetic acid (HOAc) at room temperature, The solvents
were then removed in vacuo and the residue was suspended
in glaci~l acetic acid. Dimethylamine borane was added
to reduce the Schiff base and afford pteroyltyrosine
methyl ester. Basic hydrolysis of the ester moiety
formed the product pteroyl-L-tyrosine,
A novel feature of the prGcedure was the use of
trifluoroacetic acid in the solvent for the initial
condensation. TFA is a powerful solvent for pteridine
derivatives and its inclusion in high concentration allowed
condensation of the generally quite insoluble 6-formylpterin
with ~-aminobenzoyltyrosi.~e methyl ester to be conducted
in homogeneous solution at high solute concentrations.
Imine ~Schiff base~ formation is an acid catalyzed process,
but in the presence of too strong an acid complete
protonation of the amine will prevent condensation with
the aldehyde. Trifluoroacetic acid is a very powerful
acid, and ordinarily it would be a poor choice for imine
formation. However, because of the low basicity of the
amine involved (an aniline), protonation is not complete
and reaction occurs quite rapidly in 50% TFA/50% XOAc
solution for the condensation shown in the above reaction.
-12-
D - 11,877
~'7~2~9
Neat trifluoroacetic acid and other powerful acid/solvent
mixtures such as trichloroacetic acid/methylene chloride,
trichloroacetic acid/acetic acid, methanesulfonic acid/
acetic acid, and others could be used in this condensa-
tion provided the 6-formylpterin is soluble.
Prior to the reduction step the TFA or other
strong acid must be removed from the Schiff base.
Dimethylamine borane reduction of imines is generally
conducted in glacial acetic acid. Strong acids such as 10 TFA react rapidly with amine boranes and must be avoided
in the reduction. Acetic acid on the other hand does
not react with dimethylamine borane, or at least not
c~mpetitively with the rather rapid imine reduction
(half-time of minutes). Formic acid and other amine
boranes can also be used to carry out the reduction stepO
In practice, it has been found that the amine boranes
are preferred for their mildness, simplicity and rapidityO
A virtue in the choice of 6-formylpterin rather
than * -acetyl-6-formylpterin in the synthesis of pteroyl-
tyrosine and folate analogs of this type is that basetreatment to remove an N2-protective group is not required.
Thus, the synthetic scheme above is compatible with pre-
paration directly of a product which bears both a free
N2-nitrogen (amine group) and an ester in the radiolabel
acceptor portion of the molecule. On the other hand, if
an N2-acyl group is desired in the final product one
simply starts with the appropriate N2-acylated 6-formyl-
pterin derivativeO Furthermore, one can prepare the
free acid form of pteroylamino acid derivatives directly
-13-
D - 11,877
1~6249
by starting with the free acid form of the ~-aminobenzoyl
peptide (such as p-aminobenzolyltyrosine or p-amino-
benzoylhistidine~ etc.) Pteroyltyrosine was prepared
in this fashion from 6-formylpterin and ~-aminobenzoyl-
tyrosine, thereby obviating the final basic ester
hydrolysis on the coupled product. In this particular
reaction as shown in Example lf(2), conditions were
not optimized and the product obtained was less pure
than that of coupling the ester followed by hydrolysis.
Nevertheless, the pteroyltyrosine obtained from coupling
p-aminobenzoyltyrosin~ could be radioiodinated to provide
a radiolabel after purification (gel chromatography) which
functioned identically in the folate competitive protein
binding assay to that prepared from the hydrolyzed ester.
As previously indicated, folate [125I3 radiolabels
prepared by prior art methods have consisted of an extension
of folic acid by the covalent coupling of a radioiodine
acceptor to one of the two glutamyl carboxyl groups. In
addition to being conceptually different in design, the
known folate ~125I] radiolabels share unattractive syn-
thetic prob-ems not encountered in the preparation of the
compounds of this invention. Specifically, all of the
known folate [125I] radiolabels bear only one radioiodine
acceptor attached to glutamate. Since the glutamyl residue
contains two reactive carboxyl groups, synthesis of the
desired "folic acid-e~tended" compound necessarily must
D - lL,877
~1762~9
involve either the resolution of a statistical mixture
of one disubstituted plus two possible monosubstituted
compounds, or extensive blocking and deblocking chemistry
in several steps to protect one of the carboxyl groups
and thereby direct the coupling toward the single desired
locus.
In contrast to the known folate [125I] radio-
labels, pteroyltyrosine and its 125I derivative represent
a novel strategy for the design of folate radiolabels
wherein a portion of the folic acid molecule, namely the
glutamate moiety, is replaced by a radioiodinatable group.
This strategy is employed in order to approximate the
species to be assayed in molecular size as closely as
possible. This approach is unique because in modifying
the molecule in this fashion one runs the risk of
~liminating structural ~eatures important or necessary
for protein binding while one is striving to maintain
approximate molecular dimensions in order to insure good
bindingO This strategy has been demonstrated with pteroyl-
tyrosine [125I] as the radiolabel in a folate CPB assaysensitive in the clinically significant concentration
range. In addition, the compounds of this invention are
readily synthesized by straightforward routes which avoid
some of the complexities inherent in the syntheses of
known folate [125I] radiolabels.
That the generic compounds of this invention can
be mildly and rapidly radioiodinated was demonstr~ted by
the radioiodination of pteroyltyrosine to give the compound:
D - 11,877
~17ÇiZ~9
~ ~ ~ CH~- - ~ C-NH-CH C0~ll
Typically, iodine uptake of about 90 percent was observed
in the labeling of 2.5 - 5.0 ~g of pteroyltyrosine with
1 mCi of 125I. The reaction mixtures were fractiona~ed
on short gel filtration columns and the several fractions
at the very maximum of the major radioiodinated peak of
L0 the gel chromatogram were pooled for use in the radioassay.
The generic compounds of this invention in their
radioiodinated form are designed to serve as radiomarkers
in CPB and immuno-assays. To this end the appropriate
choice of substituents allows the synthesis of radiolabels
for the detection and quantification of species such as
folic acid, methotrexate (a cancer chemotherapeutic
folate antagonist), and major metabolites and/or impor-
tant circulating forms of folic acid and methotrexate
and other potential folate antagonistsO It has been
verified that pteroyltyrosine [l25I] is an effective
radiolabel in a sensitive CPB radioassay for foLic acid
in the concen~ration range of 0 - 20 ng/mlO Furthermore,
it has been demonstrated that variation of assay para-
meters .such as buffer, pH, and nature of protein binder
can be exploited to modulate the response of the assay
to the two major circulating forms of folate: folic
acid and N-5-methyltetrahydrofolic acid (N5-methyl THF).
-16-
D - 11,877
~ 76Z~
Thus at high pH the dose response curves for folic acid
and N5-methyl THF generated using pteroyltyrosine [125I~
as label are more nearly coincident than at lower pH.
Radioiodination of biological compounds fre-
uently leads to marked chemical instability and subsequent
degradation o~ the radiolabel. This problem is often
accentuated in the radiolabel when the materials to be
radioiodinated are known to be sensitive to oxygen, light
or extremes in pH, as is the case with folic acid and some
derivatives. An important feature of this invention is
the remarkable stability of the radioiodinated product
pteroyltyrosine [125I] which shows no deterioration in
assay performance over a ten-week period when storPd in a
50% aqueous propylene glycol solution.
Radioiodination of compounds of this invention
can be effected by one or more methods known in the art
and shown in the examples. Alternative procedures for
the radioiodination of the molecules of this invention,
such as the lactoperoxidase, electrolytic, and iodine
monochloride methods can also be employed and may in
certain instances (e.g., sensitivity of precursor to
organic oxidants) be preferred. Variations in the radio-
label which can be introduced to the X moiety include:125I 131I, and 123I.
In another embodiment of this invention it is
known that the inherent reactivity of the group X in the
generic formula toward electrophilic aromatic substitution
D - 11,877
11~76Z~
(e.g., radioiodination) permits simple covalent immobi-
lization to insoluble support materials. For example,
diazotized poly (P-aminOStyrene) can react rapidly with
the compounds of this invention to yield azo-linked
products (see Example 9). In certain cases the group X
of compound II contains additional functionality, such
as carboxylic acids when X is an amino acid, with which
the compounds of this invention may be coupled to macro-
molecules (Example 10). Insolubilized (immobilized)
forms of the novel compounds described herein have
utility as affinity sorbents for the separation and
purification of enzymes, antibodies, binding proteins
and other molecules which form complexes with folic
acid, methotrexate, and related metabolites, analogs,
and derivatives.
-18-
D - 11,877
~176Z~9
The following examples are illustrative:
Example 1
PREPARATION OF PTEROYLT~ROSINE FROM 6-FORMYLPTE~IN
a) N-Carbobenzoxy-p-aminobenzoic acid (Z-PABA-OH)
A 500 ml, round-bottom, three-neck flask was
charged with 300 ml of water, 33.55g (0.40 ~mole) of
sodium bicarbonate, and 19.33g (0.14 mole) of ~-
aminobenzoic acid (PABA). The resultant solution
was stirred mechanically as 25.0 ml (30.0g, 0.18 mole)
of carbobenzoxy chloride was added dropwise over a period
of one hour. Once addition was complete, the thick white
suspension which had formed was allowed to stir overnight.
This suspension was suction filtered, and further work-up
of both the filtrate (A), and the filtered solid (B)
afforded the desired product. The filtrate (A) was acid-
ified with concentrated hydrochloric acid to pH 1, and
the white precipitate which formed was isolated by filtra-
tion and washed with water until the filtrate was neutral.
This precipitate was dissolved in lN NaOH and extracted
with 100 ml of ether. The ether layer was back extracted
twice with 60 ml of lN NaOH and these extracts were pooled
with the first aqueous layer. Acidification of the pooled
basic layer to pH 1 yielded a copious white precipitate
-19-
D - 11,877
~762~g
which was collected on a filter and washed with water
until the filtrate was neutral. The moist solid was freed
of water by dissolving it in ethyl acetate (EtOAc, 200
ml), draining off the several milliliters of water which
rapidly settled out, drying over magnesium sulfate, and
taking the colorless solution to dryness in vacuo. Yield
6.05g of Z-PABA-OH as a white powder.
Additional product was isolated from the original
filter cake (B) in similar fashion by first dissolving it
in lN NaOH, washing with ether, and acidifying the aqueous
layer to pH 1 with concentrated HCl. The resultant white
precipitate was collected on a filter washed with water,
and then freed of water and taken to dryness as above.
Yield:20.16g of white solid. This was further purified
by recrystallization from acetone-cyclohexane. A total
of 16.85g of crystalline Z-PABA-OH was obtained in three
crops, m.p. 217.0-218.0 (dec).
Over-all yield 22.90g (60%).
Anal. Calcd. for ClsH13NO4: C, 66.41; H, 4.83; N, 5.16.
Found: C, 66.26; H, 4.64; N, 5.16.
nmr (DMSO,d6; ~: 5.25 (s, 2H, benzyl CH2); 7.45 (s, 5H,
Cbz aromatic); 7.87 (center of gravity for A2B2
"quartet", 4H, PABA aromatic); 10.17 (s, lH, COOH).
ir ~KBr,~ ) 5.90, 5.99 (doublet, urethane and acid).
W (methanol): ,tmaX258 nm (24,500).
-20-
D - 11,877
11';~6Z49
b) N-Carbobenzoxy-p-aminobenzoyl-L-tyrosine methyl
ester (Z-PABA-T~E)
Z-PABA-OH (9.60g, 0.035 mole) was dissolved in
175 ml of tetrahydrofuran (THF) in a 500 ml, round-
bottom flask containing a magnetic stirring bar. The
solution was cooled in an ice-salt-water bath to -10C.
N-methyl morpholine (4.12 ml, 0.037 mole) was added all
at once, followed shortly thereafter by the addition of
4.8 ml (5.05g, 0.037 mole) of iso-butyl chloroformate.
A milky white suspension was formed which was stirred
magnetically for five minutes. To the suspension at
-10C was added dropwise with stirring, a solution of
L-tyrosine methyl ester (7.60g, 0.03g mole) in 25 ml of
dimethylsulfoxide plus 75 ml of THF over the course of
fifteen minutes. Once addition was complete, the re-
action mixture was stirred at -10C for 2 - 3 hours. It
was thenallowed to warm to room temperature and was stirred
overnight. The mixture was riltered,the precipitate was washed
with THF, and the combined filtrate was concentrated with
gentle warming (30 - 35C) on the rotary evaporator. The
residue, a viscous yellow DMSO solution, was treated with
water (100 ml) and ethyl acetate (200 ml). After being
shaken the aqueous layer was separated and the organic
layer was extracted twice more with 100 ml portions of
water. The EtOAc layer was dried over MgSO4, filtered,
and taken to dr-ytiess on the rotary evaporator. Recry-
stallization of the residue from ethyl acetate -
D - 11,877
~762~9
cyclohexane afforded 5.88 g (37%) of Z-PABA-L-TME,
m.p. 171.5 - 172.0 C, ~a]D25 -67.2 (c=l.0, methanol).
Anal. Calcd. for C2sH24N206: C, 66 95; H, 5.39; N, 6.24.
Found: C, 66.72; H, 5.25; N, 6.26.
nmr (acetone, d6; ~): 3.08 (asym. d, J = 8Hz, 2H, Tyr CH2);
3.67 (s, 3H, CH3); 4.85 (m, lH, Tyr CH); 5.18 (s, 2H,
Cbz CH2); 6,95 (center of gravity for A2B2 "quartet",
4H, Tyr aromatic); 7.38 (s, 3H, Cbz aromatic); 7.73
(center of gravity for A2B2 "quartet", 4H~ PABA aromatic).
ir (KBr pellet~,4 ): 5.8 - 5.9 (broad; amide, ester and
urethane).
UV (methanol); ,~ 265 nm (30,700).
max
c) p-Aminobenzoyl-L-tyrosine methyl ester (H-PABA-L-TME)
Into a 200-ml, round-bottom flask containing a
magnetic stirrer and equipped with a gas inlet tube was
placed 2.38g (5.31 mmole of Z-PABA-L-TME) and 60 ml of
methanol. To the solution was added about 265 mg of 5%
palladium on carbon which had been twice washed with 3 ml
portions of methanol. Hydrogen gas was bubbled through
the stirred suspension for an hour, then an additional
265 mg of washed catalyst was added and the bubbling of
hydrogen was continued for an additional two hours. At
the end of this time, no trace of C02 could be detected
in the effluent gas with saturated barium hydroxide solu-
tion. The catalyst was removed by filtration through a
Standard Super CellL pad (J.M.) and was washed with a
-22-
D - 11,877
4~
copious amount of boiling methanol. The filtrate was
taken to dryness on the rotary evaporator, affording a
tan crystalline residue (1.58g, 95% crude yield). Recry-
~ stallization from 200 ml of chloroform containing a minimumof ethyl acetate (5 - 10 ml) provided 1.44g (86%) of
H-PABA-L-TME as light tan needles,
m.p. 169.5 - 170.5C, [a]D25-76.4(c=l.O, methanol).
17 18 2 4
Found: C, 64.80; H, 5.63; N, 9.08.
nmr (DMSO, d6,): 2.98 (asym. d, J = 8 Hz, 2H, Tyr CH2);
3.56 ~s, 3H, CH3); 4.55 (m, lH, Tyr CH); 5.63 ~s, 2H,
Cbz CH2); 6.3 - 7.8 (eight resonances, 8H, pair of
aromatic A2B2 ~Iquartets~); 8.62 (d, J = 8 Hz, lH, amide
NH).
ir (KBr,~ ): 5.79 (ester); 6.19 (amide)
UV (methanol);l 281 nm ~21,100).
max
d) p-Aminobenzoyl-L-tyrosine (H-PABA-L-Tyr-OH)
A sample of H-PABA-L-TME (1.06g, 3.37 mmole)
was dissolved in 150 ml of O.lN NaOH with swirling. After
1.5 hours the solution was extracted with 50 ml of EtOAc,
and the organic layer was discarded. The aqueous layer
was acidified to pH 5 - 6 with concentrated HCl and was
extracted with EtOAc (4x50 ml). The pooled EtOAc layer
was washed with water (3x50 ml) and was taken to dryness
on the rotary evaporator, affording 0.41g of a tan foam.
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The original aqueous layer was further acidified to pH
3.4 - 3.8 and again was extracted with EtOAc (2x20 ml
and lx30 ml). These EtOAc extracts were pooled and washed
with water (4x30 ml). Concentration of the organic layer
on the rotary evaporator gave 0.32 g of a colorless foam.
The two residues were pooled and recrystallized from EtOAc
to yield 280.8 mg of tan micro-fine prisms:
m.p. 178.5-180C, [a~D 5-59.4 (c=1.0, methanol). The nmr
spectrum of this material revealed the presence of EtOAc
despite having dried the sample at room temperature in
vacuo (0.1 mm Hg) for several hours. Apparently the di-
peptide crystallized as the ethyl acetate solvate; roughly
0.5 mole of EtOAc per mole H-PABA-L-Tyr-OH was present in
the crystalline sample after drying.
nmr (DMSO, d6; ~ ); 1.17 (t, J = 7Hz, ethyl CH3 of EtOAc);
1.98 ~s, CH3CO of EtOAc); 3.0 (asym. d, J = 7 Hz, 2H,
TYr CH2);
- 4.05 (q, J = 7 Hz, CH2 of EtOAc); 4.55 (m, lH, Tyr CH);
6.4 - 7.8 (eight resonances, 8 H, pair of aromatic A2B2
"quartets"); 8.07 (d, J = 8 Hz, lH, amide NH, exchange-
able).
W (methanol): ~ max 278 nm (25,900).
e) Pteroyl-L-tyrosine methyl ester (Pt-L-TME)
A 50 ml round-bottom flask containing a magnetic
stirring bar was charged with 120.5 mg (0.63 mmole) of
6-formyl pterin, prepared by the method of Viscontini,
et al.,(l) (2) 490.2 mg (156 mmole, 2.47 equiv.) of
(1) M. Viscontini, R. Provenzale, S. Ohlgart and
J. Mallevialle, Helv. Chim. Acta., 53, 1202 (1970).
(23 M. Viscontini and JO Bieri, Helv. Chim. Acta., 54,
2291 (1971). -24-
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H-PABA-L-TME and 4 ml of glacial acetic acid (HOAc). With
stirring the H-PABA-L-TME dissolved, but the yellow aldehyde
remained in suspension. The flask was flushed with argon
as 3 - 4 ml of trifluoroacetic acid (TFA) was slowly added
to effect complete dissolution of the suspended solids.
Stirring was continued for an additional 30 minutes from
this point. Most of the solvent then was removed from
the dark brown solution by gentle warming (40C) on the
rotary evaporator (water aspirator). The moist brown re-
sidue was taken to dryness under high vacuum (0.05 mm Hg)
at room temperature overnight with the flask wrapped in
aluminum foil to exclude light. A brown foam or glass
resulted. The vacuum was broken and argon was bled into
the flask. To the brown foam was added 4 ml of HOAc, and
with vigorous swirling and stirring (under argon) the
residue was dislodged from the walls of the flask to form
a light yellow-brown suspension~ Dimethylamine borane,
37.2 mg (0.631 mmole, 1 equiv.) was added to the stirred
suspension; the color rapidly changed to orange-brown and
nearly all of the solid dissolved. After an hour HOAc
(8 - 10 ml) was used to rinse down the neck and walls of
the flask. The reaction mixture was concentrated with
gentle heating(40) on the rotary evaporator to a viscous
residue. Two 25 ml portions of toluene were successively
added to the residue, mixed, then stripped in vacuo with
gentle warming to remove HOAc as its toluene azeotrope.
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~L1762~9
The final toluene-dampresidue was dried to a brown foam
under high vacuum at room temperature overnight. To the
flask was added 30 ml of EtOAc. The foam was scraped
from the walls of the flask with a metal spatula to form
a suspension, which was left standing in the refrigerator
overnight. The insoluble yellow-orange solid was separated
from the yellow supernatant by centrifugation. The pelleted
solid was resuspended in EtOAc and recentrifuged three
times until the final EtOAc wash was colorless. Finally,
the pelleted solid was resuspended in a small volume of
EtOAc, collected on a coarse fitted glass filter under
argon in a pressure filtration apparatus, washed with
several small portions of EtOAc and dried by flowing argon
through the collected filter cake under positive pressure
for an hour.
Yield: 174 mg (56%) buff-colored powder, pteroyl-L-
tyrosine methyl ester,
m.p. 230 - 350 (slowly decomposed).
nmr (DMSO, d6; S ): 3.59 (s; 3H, ester CH3); 4.48 (broad s,
3H, C-9 CH2 and Tyr CH); 6.6 - 7.6 (six line multiplet,
8H, overlapping pair of aromatic A2B2 "quartets");
8.30 (d, J = 8 Hz, amide NH, exchangeable); 8.65
(s, lH, C-7 H).
Mass Spectrum (field desorption): m/e 489 (100%), 314,
207, 192.
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f) Pteroyl-L-Tyrosine (Pt-L-Tyr-OH)
1) Hydrolysis of Pt-L-TME
To a 50 ml round-bottom flask containing 122.0 mg
(0.25 mmole) of pteroyl-L-tyrosine methyl ester under
argon was added 10 ml of 0.1 N NaOH which had been rigor-
ously degassed by sparging with argon. The flask was
stoppered and shaken for ten minutes to dissolve the solid;
a dark yellow-brown slightly turbid solution resulted.
Af~er an additional 35 minutes at room temperature the
turbid reaction mixture was filtered through a mixed
cellulose ester Millipore filter (1.2~ nominal pore size).
The clear,brown filtrate was acidified to pH 2 with 1.0
N HCl and a voluminous, gelatinous brown precipitate was
formed. The suspension was centrifuged at 14,000 rpm in -
the cold (4 - 5C) for 15 minutes, and the supernatant
liquid was decanted. The pelleted solid was washed
successively with 0.1 N HCl (2x 20 ml), distilled water
(2 x 20 ml), absolute ethanol (2 x 20 ml), and ethyl ether
(2 x 20 ml) by resuspension and recentrifugation. The
washed solid was dried under high vacuum at room tempera-
ture to yield 61.1 mg (52%) of a dark-brown solid (Pt-L-
Tyr-OH).
W (0.1 N NaOH): ~ 249 (25,500), 283 (21,700), 362 nm
max
(7,800); acidified to pH 1.8 with conc. HCl: ~max 224
(2~500), 256 (25,200), 280 nm (18,200).
nmr (DMSO, d6; ~ ): 4.46 (m, 3H, C-9 CH2 and Tyr CH);
D - 11,877
1176249
6.6 - 7.6 (six resonances, 8H, overlapping pair of
aromatic A2B2 "quartets"); 8.13 (d, J = 8Hz, lH, amide
NH, exchangeable); 8.64 (s, lH, C-7 H).
Mass Spectrum tField Desorption): m/e 475 (M~), 457,
429, 415, 413, 313, 312, 300, 293, 282, 26~
2) Condensation of 6-Formylpterin with H-PABA-L-Tyr-OH
A 25 ml round-bottom flask containing a magnetic
stirring bar was wrapped in foil to exclude light and
purged with argon. A sample of 6-formylpterin (109.0 mg,
0.57 mmole) was added to the flask and dissolved by the
addition of L.0 ml of TFA. After 15 minutes of stirring
all of the aldehyde had ~issolved, form-ng a bright~ yellow
solution. H-PABA-L-Tyr-OH (216.0 mg, 0.72 mmole, 1.26
equiv.) was added all at once, and to hasten dissolution
an additional 1.0 ml of TFA was added. Fifteen minutes
of continuous swirling and stirring were required to effect
complete dissolution. A dark~brown solution resulted
which was stirred for an additional 40 minutes. The
solvent was stripped in vacuo, affording a thick brown
oil which still contained TFA. HOAc (7 ml) was added to
the oil and a suspension of a fine yellow solid in a dark,
brown supernatant liquid resulted. This was taken to
dryness at 45 - 50C on the rotary evaporator. The yellow-
brown residue was resuspended in HOAc (2 ml), and 26.2 mg
(0.28 mmole) of pyridine borane was added all at once.
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1~76Z~
After 30 minutes the acetic acid was stripped on the rotary
evaporator at 49C, yielding a brownish-yellow solid re-
sidue. The residue was suspended in 10 ml of degassed
EtOAc, filtered under argon, and dried by positive flow
of argon through the filter cake for 30 minutes.
Yield: 224.3 mg of crude Pt-L-Tyr-QH. The nmr spectrum
of this material clearly contained resonances coincident
with those of the more pure sample prepared by hydro-
lysis of Pt-L-TME above. However, unassigned reson-
ances in the spectrum suggested the material was only
30 - 50% pure. A sample of this material (200 mg)
was further purified by dissolution in 10 ml of
0.1 N NaOH and precipitation by acidification to pH 2.5
with 1.0 N HCl. The collected solid (centrifugation)
was washed twice with absolute ethanol and twice with ether
and dried in vacuo to yield 100 mg of brown solid.
Direct radioiodination of this latter material afforded
an isolable fraction which behaved in the folate com-
petitive protein binding assay identically to Pt-L-
Tyr-OH 1 I].
Example 2
PREPARATION OF PTEROYL-L-HISTIDINE (PT-L-HIS-OH)
By the method of Example lb, one prepares
N-carbobenzoxy-p-aminobenzoyl-L-histidine methyl ester
(Z-PABA-L-His-OMe) from one part of histidine methyl
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ester and one part o.f Z-PABA-OH. Catalytic hydrogenolysis
of Z-PAB~-L-His-OMe by the method of Example lc produces
a high yield of ~-aminobenzoyl-L-histidine methyl ester
(H-PABA-L-His-OMe), which is then employed in the reduc-
tive amination of 6-formylpterin by the method of Example
le to yield pteroyl-L-histidine methyl ester (Pt-L-His-OMe).
Subsequent basic hydrolysis of Pt-L-His-OMe by the method
of Example lf(l) affords the final product Pt-L-His-OH.
Example 3
PREPARATION OF PTEROYLTYRAMINE
Pt-L-Tyra is prepared by the method of Example 1
except that tyramine is employed instead of tyrosine methyl
ester, and final basic hydrolysis (Example lf(l)) is
obviated since tyramine does not contain a carboxylic
acid ester.
H2N ~
N CH2 ~ ~r~CH2CH2 ~ -OH
Pteroyltyr~mine
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D - 11,877
~76;~49
Example 4
PREPARATION OF PTEROYLTYROSINE FROM PTEROIC ACID
a~ Synthesis of N10-Trifluoroacetylpteroic Acid
Pteroic acid (130 mg) was refluxed with tri-
fluoroacetic anhydride for 6 hours at which time dis-
solution was complete. The solution was evaporated,
under reduced pressure, and triturated with water (1 ml)
to give a yellow solid. The amorphous material was
washed 3 times with 5 ml portions of water, centrifuged
each time, and dried in vacuo overnight.
b) Pteroyltyrosine
N10-Trifluoroacetylpteroic acid (87.5 mg) and
triethylamine (0.034 ml) were dissolved in N,N-dimethyl-
formamide (2 ml). Iso-butyl chloroformate (0~045 ml) was
added to the mixture and the solution was stirred under
nitrogen at 30, for 45 minO, after which an additional
quantity of triethylamine (~0O9O ml) was added followed
by L-tyrosine methyl ester (124 mg), and stirred at 30
for 24 hours. The reaction mixture was then poured into
Ool N NaOH (36 ml) and heated on a steambath, under a
nitrogen atmosphere for 45 mins. After cooling in an
ice-bath, the solution was adjusted to pH 2 with concen-
trated hydrochloric acid, which gave a gelatinous preci-
pitate. The precipitate was centrifuged and washed 3 times
with small portions of water. The gel was dissolved in
l.OM ammonium bicarbonate (50 ml) diluted to 500 ml with
water and chromatographed on a column of DEAE-cellulose
(105x25 cm). The column was eluted with ammonium bi-
-31-
i) - 11,o, 1
11762~
carbonate (0.5M) and the product was detected in the
fractions appearing after 800 ml of eluant was collected.
The ammonium bicarbonate solution of the deriva-
tive was evaporated under reduced pressure and repeatedly
evaporated with additional quantities of water until the
salts had evaporated. Dissolution of the residue in
water and acidification with hydrochloric acid to pH 2.5
gave a precipitate which was centrifuged, washed with
water, ethanol, ether and then dried in vacuo.
MassSpectrum (Field Desorption):- m/e 475 (M~), 458, 457,
429, 413, 309, 300, 293.
UV(0.5 N NaOH): lmaX252, 281, 364 nm; (0.5 N HCl) ~ max
218, 246 (sh), 282 (sh), 302 nm
Example 5
IODINATION OF PTEROYLTYROSINE
To a mixture of 1.0 millicurie of sodi~m
iodide-125I in 2.5~V 1 of solution, 25 ~ 1 of 0.05M
potassium phosphate buffer, pH 7.5 and 2.5~ g of pteroyl-
tyrosine in 25P 1 buffer in a disposable 1.5 ml micro-
sample tube was added at once 5G~ g ofichloramine T
(N-chloro-p-toluenesulfinamide, sodium salt trihydrate),
in 20~ 1 of 0.05M potassium phosphate buffer, pH 7.5.
After exactly 20 seconds, 100~ g of sodi~ metabisulfite
dissolved in 2~ 1 of 0.05M potassium phosphate buffer
was added, at once, to terminate the reaction.
The reaction mixture was applied to a 1 x 20
cm Sephadex G25, fine~ (Pharmacia Fine Chemicals, Uppsala, -
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D ~ 11,~77
~L7~Z~9
Sweden) column hydrated with distilled water and equil-
ibrated with 0.lM potassium phosphate, pH 7.5. The
column was eluted with ~ potassium phosphate buffer
and 3 ml fractions collected. The product "peak" fractions
32 - 34 ~-7ere collected, diluted 1:1 with propylene ~lycol
and stored helow 0C.
Example 6
IODINATION OF PTEROYL-L-(5-HYDROXYTRYPTOPHAN)
Pteroyl-L-(5-hydroxytryptophan), which is pre-
pared by the method of Example 1 using L-5-hydroxytryptophan
methyl ester instead of L-tyrosine methyl ester is iodin-
ated and purified by the method of Example 5 to yield
pteroyl-L-(5-hydroxytryptophan) [125I].
Example 7
COMPETITIVE PROTEIN BINDING ASSAYS FOR
FOLIC AND N5-METHYLL~:l`~A~Y~('F~!It: ACl~
An application of the novel veneric radioiodin-
ated folate derivatives, typifled by pteroyltyrosine
[125I], is the use of these radiolabels in competitive
protein binding radioassays for folate constituents in
human blood serum. The use of the Centria~ analytical
system, Union Carbide Corporation, in this example, is
meant to be illustrative and other methods~ both automated
and manual, obvious to those s~illed in the clinical diag-
nostic art are also within the scope of this invention.
Briefly, the Centria~ system is a tri-modular
instrument in which reagents are pipetted on to a multi-
well transfer dis~ and mixed centrifugally with standards
-33-
~ 7
1176Z~L9
or patients' samples. After a suitable incubation period.
the components are separated by a centrifu~al elution o~
the bound and rree fractions and the ~ound fractiolls
counted three at a t~me. The data reductior., p~rformed
by a micro-processor is given selectively as either raw
counts. percent bound or in collventional units from a
standard curve derived using one of several trallsforms.
Folic acid and N5-methyltetrahydrofolic acid
standards in the ran~e 0 - 20 r.anograms/milliliter were
prepared in 0.05M sodium borate pH ~.3 conta ning G.1%
human serum albumin. The whole milk binder was prepared
according to the method of Rothenbero et al.,(3~ and
dissolved in sodium borate, pH 9.3 containing 0.1% human
serum albumin
In detail, aliquots of folate standards or un-
knowns (15 microliters) were permitted to compete after
centrifugal transfer and mixing with pteroyltyrosine [125I]
(50 microliters, 20~000 cpm) plus 85 microliters of water
with the limited number of binding sit~s contained in a
200 microliter solution of whole milk folate binder. The
binding reactions may be shown as follows:
pteroyltyrosine 1125I] + serum folate -~ binder -~
(binder-folate)~(binder-pteroyltyrosine L l25I] ) +
folate + pteroyltyrosine [l25I~
After a 10 minute incubation, the reaction mixture was
centrifugally transferred onto DEAE Sephadex A-25 columns
(0.75 x 4 in.) where separation of complexed folate from
free folate was effected with 1.4 ml of elution buffer
(3)S. Rothenberg, M. DaCosta, and Z. Rosenberg, New Eng.
J. Med., 286, 1335 ~1972).
-34-
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~ - 11,877
~176Z49
(0.05M sodium borate, pH 9.3) per sample tube, with the
following results:
In the eluant:
(binder-folate) ~ (binder-pteroyltyrosine-[125I])
On the column:
folate + pteroyltyrosine [125I].
A gamma counter which counts three of
the 36 positions at a time for one minute, so constructed
that only the eluate bottom portion of the test tubes
fits into the counter, was then used to count all samples,
Sequential counting of the tubes thus required about
12 minutes, A small computer with printout capability
then printed out the data after completion of the cycle.
A logit-log plot of dose response curves for folic acid
and N5-methyltetrahydrofolic acid is presented in the
Figure.
Exam~le 8
ENZYMATIC HYDROLYSIS OF PTERO~TYROSINE
Two millilters of a solution of ~teroyltrosine
(0,75 mmoles/ml) in 0.025M Tris-HCl containing 2 m~ zinc
chloride, pH 7,3 were diluted to 10 ml with distilled
water, A portion of this solution (2 ml) was placed in
a 1 cm cuvette and .:n another ~as placed 2 ml of distilled
water. To each of the cuvettes was added 50~ 1 of a
solution of 100~41 carboxypeptidase A suspension (Aldrich
chemical, lot 060637) diluted with 0.9 ml of 10% lithi-lm
chloride.
-35-
i, - 11, ~,i i
76'~4~
The time course of the reaction was ~ollowed
spectrophotometrically (uv) over a period of two hours
at ambient temperature. An increase and shi t in max
from 230 to 277 nm and a marked decrease in absorption
at 220 nm was observed Two isosbestic points at 242
and 282 nm were visible The final spectrum was very
similar to that of pteroic acid.
Example 9
PREPARATION OF AN AFFINITY CHROMATOGRAPHIC MEDIUM
~)K PURIFl~ATION OF FOLATE BINDING PROTEINS CROSS-
5~CTIVE WITH ~TEROY L- L-TY ROSINE
Poly(p-aminostyrene) (2g) is swollen in a mix-
ture of DMSO/3N HCl (1:1, 15 ml) and cooled to 0C in
an ice bath The mixture is gently agitated (stirring,
swirling) as solid sodium nitrite (54 mg, 0 64 mmole)
is added in several small portions over the course of
5 minut~s. After the last portion of sodium nitrite is
added the reaction mixture is filtered in the cold (0-5C)
and ~uickly washed with three 25 ml porti.ons of cold
DMSO/H20(1:1). The moist resin is then quickly added to
a solution of pteroyl-L-tyrosine (100 mg, 0 21 mmole)
in 10 ml of DMSO~0.5 N NaOH (1:1). The pH is readjusted
to 10-ll with cold 4N NaOH and maintained at this pH as
the reaction is allowed to proceed with gentle agitatlon
in the dark at 0-6~C. The mi~ture is then allowed to
warm to room temperature for an hour, acidified to pH 5
with 1 N NaOH and stirred at ambient temperature for an
additional half hours
The mixture is filtered and the resin is washe
with copious quantities of DMSO/water (1:1), then distilled
~ 7/
7~Z9L9
water until no further elution of pteroyl-L-tyrosine can
be detected by radioassay. The water-wet resin is then
washed thoroughly with methanol and methylene chloride,
successively, dried of methylene chloride in vacuo, and
stored dry, in the cold, protected from light.
Example 10
CONJUGATION OF PTEROYL-L-TYROSINE TO BOVINE
SERUM ALBUMIN. A N~VEL FOLATE ANlIGEN
A solution of pteroyl-L-tyrosine (100 mg,
0,21 mmole) and tri-n-butylamine (45 mg, 0.24 mmole) in
DMSO (15 ml) is cooled to 5-6C, and iso-butyl chloro-
formate (33 mg, 0.24 mmole) is added with stirring, The
reaction mixture is stirred for 15 minutes at 5-6C and
then added all at once with stirring to a cold (5C)
solution consisting of bovine serum albumin 51. g,
1.4 x 10-5 mmole) dissolved in 10 ml of distilled water
and adjusted to pH 9.0 with 5% (W/V) potassium carbonate,
The reaction mixture is maintained at pH 9.0 with 5%
potassium carbonate solution while being stirred at 5C
for 4 hours, followed by stirring at ambient temperature
for 1 hour. The mi~ture/solution is filtered if neces-
sary through a Millipore filter (mixed cellulose ester,
0 5~ ) and then dialyzed against 2Q volumes of distilled
water changed once daily for five days The conjugate
solution is lyophilized to a fluffy powder
-37-
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1~76~49
Although the invention has been illustrated
by the preceding examples, it is not to be construed
as being limited to the materials employed herein,
but rather, the invention encompasses the generic area
as hereinbefore disclosed. Various modifications can
be made without departing from the spirit and scope
thereof.
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