Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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REAGENTS FOR MEASURING LEAD LEVELS VIA THE QUANTIFICATION
OF PORPHOBILINOGEN
Field of the Invention
The present invention relates to new and unique reagents for the direct
qllAnt;fi~ tion of porphobilinogen formed from aminolevulinic acid in the
presence of aminolevulinic acid dehydratase by immunoassay. The amount of
porphobilinogen formed is inversely proportional to the levels of lead in a test15 sample. There is no indirect measurement of porphobilinogen complexes as is
often used in colorirnetric determinations. The present lnvention also relates
to immunogens, antibodies prepared from such immunogens, and labeled
reagents for the quantification of porphobilinogen, preferably for use in a
fluorescence polarization immunoassay.
Background of the Invention
The rapid determination of trace metals in biological and
environmental systems is incre~ingly important in identifying potential
hazards and preserving the public health. The toxicity of certain metals such as2 5 lead is well-known. The absorption of even trace amounts of lead can cause
severe damage to human organs. The numerous and widespread sources of
lead in the environment, including the food supply, compounds the problems
of screening affected groups. It is generally recognized that lead poisoning
occurs in children at blood levels as low as 10-15 mg/dl as noted by S. Cummins
3 0 and L. Goldman in Pediatrics, 1992, 90, 995-996. Lead contamination of
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environmental sources such as water, dust and soil require identification at
even lower levels. In order to measure these amounts, the analytical
techniques must be sensitive, contaminant-specific, and reliable.
S Previous te--hni-lues have relied upon atomic absorption spectroscopy as
described in an article in ain. Chem., 1991, 37, 515-519 by Jacobson et al. or by
measuring biological marlcers for exposure to lead as noted by A. Berlin et al. in
Z. Klin. Chem. Klin. Biochem., 1974, S. 389-390. Atomic absorption spectroscopy
(AAS) is limited in its general availability, high level of technical expertise
1 0 requirements for operation, throughput and expense of investing and
maintaining the necessary instrumentation. The AAS method is generally
carried out in a central laboratory with considerable amount of time between
taking a test sample and obtaining the levels of lead in the test sample. The
biological marker method is based upon an inhibition of the enzyme
1 5 aminolevulinic acid dehydratase (ALAD) where if the activity of ALAD from ablood sample had subnormal activity then that person may have been exposed
to lead. The activity of ALAD after exposure to lead con~ining samples was
further disclosed in US patent 5,354,652 to Silbergeld dated October 11, 1994.
This patent describes a colorimetric method for the quantification of lead. This2 0 method describes the chemical derivatization of porphobilinogen using a
colorimetric agent (Erlich's reagent) where the amount of the resulting colored
porphobilinogen derivative is quantified. The amount of porphobilinogen
produced is inversely proportional to the col~e~E~onding lead levels. The
principles of colorimetric determination of porphobilinogen produced from
2 5 ALAD was originally disclosed by K. D. Gibson et al. in Biochem., 1955,
61:618-629. Silbergeld also disclosed in this patent the use of an antibody to the
ALAD-Lead complex, in this approach the ALAD-Lead specific antibody is used
in an ELISA assay. A sample suspected of containing lead is mixed with ALAD,
the amount of ALAD-Lead complex ~at is formed is detected with a
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horseradish peroxidase-anti-ALAD antibody. In a similar fashion, Jaffe has
disclosed a method for measuring lead exposure by quantifying ALAD activity
using an anti-ALAD antibody in patent application WO 95/04159. This method
also measures the porphobilinogen produced from the ALAD enzyme
5 colorimetrically with Erlich's reagent. H~nkPn.~ et al. disclosed an indirect
enzymatic method for the detection of lead using isocitrate dehydrogenase in
US patent 5,368,707 dated November 29, 1994. This method measured the
inhibition of the enzyme isocitrate dehydrogenase with subsequent
electrochemical detection. A different approach for the detection of lead was
I 0 disdosed by Jing in US patent 5,407,831, it is based on the selective chelation of
lead and detection by atomic absorption spectroscopy.
The above described methods for measuring the levels of lead suffer from
various disadvantages. The colorimetric method which employs Erlich's
l 5 reagent has the disadvantage of the use of strong mineral acid and toxic heavy
metals to give the desired color formation to quantify porphobilinogen. The
method which employs the enzyme isocitrate dehydrogenase coupled with
biosensor detection lacks the desired specificity (lines 28-31, page 3) which had to
be overcome with laborious pretreatment (lines 25-47, page 5). Finally, the
2 0 method employing ALAD-Lead complex employs sandwich assay format where
two antibodies of various specificity are employed in heterogeneous assay
format. It is known that immunoassays based on heterogeneous format are
complex in their manufacturability.
2 5 Therefore, the challenge still exists to accurately and precisely measure levels
of lead in a rapid and selective manner using automated instrumentation that
is currently available in ~lini~l settings in a cost effective manner. This could
be achieved by the direct measurement of porphobilinogen by immunoassay.
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Sltmmary of the Invention
The present invention overcomes the disadvantages of previously
described methods and therefore provides a new, direct method for the
measurement of lead levels by uniquely and directly quantifying the amount of
5 porphobilinogen (PBG) produced from aminolevulinic acid (ALA) catalyzed by
aminolevulinic acid dehydratase (ALAD), where the amount of
porphobilinogen produced is inversely proportional to the amount of lead
present in a test sample. This direct quantification of porphobilinogen is
achieved in a hornogeneous immunoassay forma~ preferably employing
10 fluorescence polarization. This method provides the specificity, speed and
convenience of homogeneous immunoassay to give precise, reliable and low
cost quantification of porphobilinogen, especially well adapted to automated
immunoassay analyzers. There is no indirect quantification of porphobilinogen
via porphobilinogen complexes wllich are formed in processes for colorimetric
I S determination.
The present invention also provides unique antibody reagents and
labeled reagents for the quantification of porphobilinogen in a test sample.
These reagents do not contain toxic heavy metals such as mercury which are
2 0 utilized in the prior methods of the art in the chemical derivatization step.
The present invention further provides synthetic procedures for
preparing unique immunogens which are employed for the production of such
antibody reagents and for preparing such unique labeled reagents. According to
2 5 the present invention, the labeled reagents and the antibody reagents offer an
advance in the art beyond previously known procedures. By the use of these
reagents, the cumbersome and time-consuming method of colorimetric
deterrnination of porphobilinogen is avoided, as is the chemical derivatization
step.
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Brief Description of the Drawings
~IGURE 1 illustrates the synthetic pathway for coupling a
porphobilinogen (~BG) derivative to bovine serum albumin to produce
immunogen (7) according to the method of the present invention.
FIGURE 2 illustrates the synthetic pathway for coupling a PBG derivative
to bovine serum albumin to produce immunogen (14) according to the method
of the present invention.
FIGURE 3 illustrates the synthetic pathway for coupling a PBG derivative
to bovine serum albumin to produce immunogen (19) according to the method
of the present invention.
FIGURE 4 illustrates the synthetic pathway for the preparation of a
fluorescent tracer (21) according to the method of the present invention.
FIGURE 5 illustrates the synthetic pathway for the preparation of a
fluorescent tracer (3(~) according to the method of the present invention.
E;IGURE 6 illustrates the synthetic pathway for the preparation of a
fluorescent tracer (32) according to the method of the present invention.
FIGURE 7 illustrates the synthetic pathway for the preparation of a
fluorescent tracer (34) according to the method of the present invention.
2 0 FIGU3~E 8 illustrates the synthetic pathway for the preparation of a
fluorescent tracer (36) according to the method of the present invention.
~IGIJRE 9 is a graph which illustrates a calibration curve for the
measuring levels of lead via the quantification of porphobilinogen on the
Abbott IMx(g) analyzer.
2 5 FIGURE 10 is a graph which illustrates antibody dilution from animals
inoculated with immunogens (7), (14) and(19).
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Detailed Description of the Invention
According to the present invention, lead levels are measured via the
direct quantiQcation of porphobilinogen formed from aminolevulinic acid in
5 the presence of aminolevulinic acid dehydratase by immunoassay. A test
sample is contacted with a labeled reagent or tracer and an antibody reagent,
either simultaneously or sequentially in either order, and then measuring the
amount of the labeled reagent which either has or has not participated in a
binding reaction with the antibody reagent as a function of the amount of
10 porphobilinogen formed which is inversely proportional to the amount of lead
in the test sample. In particular, the present invention relates to immunogens,
antibodies prepared from such immunogens, and labeled reagents for use in the
fluorescence polarization immunoassay for the quantification of
porphobilinogen thereby measuring the levels of lead.
1 5
Lead is known to inhibit the formation of porphobilinogen (Formula 1)
by ALAD and a measurement of porphobilinogen produced will indirectly
measure the amount of lead present in a test sample. Pretreating a lead
containing test sample frees the lead from within red blood cells and removes
2 0 interfering compounds such as proteins, endogenous ALAD, PBG, ALA and the
like. Acids such as trichloroacetic acid (TCA), nitric acid, 5-sulfosalicylic acid or
perchloric acid are commonly used for sample pretreatment. Such sample is
centrifuged, pellet discarded and resulting supernatant, after neutralization, is
incubated with ALAD to produce an amount of porphobilinogen (PBG)
2 5 inversely proportiona} to the lead concentration present in the test samp}e.This mixture is then assayed by fluorescence polarization immunoassay using
the inventive reagents described herein to detect and quantify the amount of
porphobilinogen present in the mixture and thereby measuring the amount of
lead in the test sample.
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HOOC rCOOH
H~ I ~NH2
Antibodies of the present invention are produced with immunogens
5 which are prepared with derivatives of the Formula II, III and IV:
HOOC~ COOH
I I H)l--N~CNH--X--P
HOOC\ COOH
I I I H ~ I ~ N H2
p
1 0
HOOC~ COOH
I V ) ~C
p_z ~ NH2
wherein for II X is CO-(CH2)n-CO- with n from 0-6, for III Y is SO2-(CH2)n-CO-
with n from Q-6, for IV Z is (CO)m-(CH2)n-CO- with m from 0 to 1 and n from 0-
1 5 6; wherein for II, III and IV P is an immunogenic carrier material.
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Labeled reagents of the presen~ invention are prepared with
porphobilinogen compounds of the Formula V, VI, VII and VIII:
HOOC ~ rCOOH
H)~N~NH--A--Q
H
s
HOOC~ rCOOH
V I H)~NI J~NH--B Q
HOOC
COOH
V I I 1~NJ~NH2
Q--C
~OOC
COOH
V I I I Q -D)~NJ~NH2
H
wherein for V A is ~CO-(CH2)n-CO- with n from 0-6, for VI B is -(CH2)n-CO- with
n from 0-6, for VII C is SO2-(CH2)n-CO- with n from 0-6, for VIII Z is -(CO)m~
(CH2)n-CO- with m from 0 to 1 and n from 0-6; Q is a detectable moiety,
1 5 preferably a fluorescen~ moiety.
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The above immunogens were prepared as described below and as shown in
Figures 1, 2 and 3. An imrnl~nogen of Formula II was prepared by N-acylation
of porphobilinogen with the active ester of adipic acid mono ethyl ester
followed by transformation of the free PBG acids to the corresponding tert-butyl5 esters. Hydrolysis of the ethyl ester to the free acid afforded the desired hapten.
Activation of the free acid and conjugation to bovine serum albumin followed
by treatment with trifluoroacetic acid gave the desired immunogen as shown in
Figure 1. An immunogen of Formula III was prepared from porphobilinogen
by first protecting the side chain nitrogen as a carbamate followed by the
I 0 conversion of the two free acids to tert-butyl esters. The resulting protected
porphobilinogen was sulfonylated on the pyrrole nitrogen with benzyl.4-
chlorosulfonylbutryate followed by hydrogenolysis of the benzyl ester which
afforded the desired hapten. Activation of the free acid and conjugation to
bovine serum albumin followed by treatment with trifluoroacetic acid afforded
15 the desired immunogen as shown in Figure 2. An immunogen of Formula IV
was prepared by the acylation of a PBG di-tert-butyl ester carbamate derivative,subsequent hydrolysis gave the desired hapten as the free acid. Activation of
the free acid and conjugation to bovine serum albumin followed by treatment
with trifluoroacetic acid gave the desired immunogen as shown in Figure 3.
Fluorescent labeled reagents for use in a fluorescence polarization
immunoassay for the measuring lead levels via the quantification of
porphobilinogen were synthesized as shown in Figures 4, 5, 6 and 7. A
porphobilinogen fluorescent derivative of Formula V was prepared by coupling
2 5 the active ester of 6-carboxyfluorescein with porphobilinogen to afford the
desired tracer as shown in Figure 4. A fluorescent derivative of Formula VI was
synfhesi7ed as shown in Figure 5. This derivative was prepared in the
following manner: coupling of the nitroanion of tert-butyl 4-nitrobutryrate and
an aldehyde with subsequent protection as an acetate followed by coupling with
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an isocyanate formed the pyrrole nucleus. Deprotection of the primary alcohol
followed by transformation to the corresponding tert-butyl ester and subsequent
hydrogenolysis of the benzyl ester moiety afforded the desired hapten. The free
acid was activated and coupled to 6-aminomethylfluorescein followed by
5 treatment with trifluoroacetic acid gave the desired porphobilinogen
fluorescent tracer as shown in Figure 5. The preparation of a different
fluorescent tracer of Formula VI was prepared using the same active ester used
in Figure 5. This active ester was coupled with a fluorescent 6-aminocaproic
acid derivative followed by treatment with trifluoroacetic acid afforded the
10 desired fluorescent porphobilinogen derivative shown in Figure 6. A
fluorescent tracer of Formula VII was prepared according to Figure 7 by couplingthe carboxy~lopyl sulfonamide derivative to 5-aminomethylfluorescein with
subsequent treatment with trifluoroacetic acid gave the desired fluorescent
tracer. The fluorescent tracer of Formula VIII was prepared according to Figure
l S 8 coupling 5-aminomethylfluorescein with the 5-carboxypyrrole derivative
followed by trea~nent with trifluoroacetic acid afforded the desired fluorescenttracer.
When following a fluorescence polarization immunoassay (FPIA) format
2 0 employing the reagents according to the present invention, the concentration,
or level, of lead in a test sample can be accurately measured via the
quantification of porphobilinogen. To perform a FPI~ for measuring the levels
of lead, a calibration curve was generated for measuring the levels of lead
(Figure 9).
According to the present invention, it has been found that superior
fluorescence polarization immunoassay results for measuring the levels of lead
via the quantification of porphobilinogen are obtained when employing (i) an
antibody reagent comprising antibodies produced from a porphobilinogen
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(PBG) derived immunogen of Formula III where P is an immunogenic carrier
as described above and (ii) a fluorescent labeled reagent of Formula VII where Qis a fluorescent moiety as described above. For the quantification of
porphobilinogen, the antibody reagent comprises antibodies which are capable
5 of binding to or recognizing porphobilinogen wherein the antibodies are
preferably produced with an immunogen prepared from the porphobilinogen
derivative of Formula III where P is bovine serum albumin and Y is -SO2-
(CH2)3-CO-, the labeled reagent is ~re~erably prepared from the derivative of
Formula VII where Q is a fluorescent moiety and n=3.
1 0
The great advantage of this invention is that the porphobilinogen (PBG)
produced from aminolevulinic acid (ALA) and the enzyme aminolevulinic
acid dehydratase (ALAD) is quantified directly by immunoassay using unique,
specific antibodies of this invention. In the previous art, the porphobilinogen
1 5 produced enzymatically was never measured directly by immunoassay, but
indirectly measured by reacting porphobilinogen with ~rlich's reagent to
produce a derivative of porphobilinogen which was subsequently measured by
colorimetric methods. This latter approach has numerous disadvantages such
as: use of concentrated acid, diminished precision and extended times needed
2 0 for analysis of each sample. The use of corrosive reagents limits the possibilities
for automation and creates environmental problems of disposal.
Derivatization of porphobilinogen adds an extra step in the measuring process
thereby decreasing the precision of an assay that measures porphobilinogen and
makes reliable automation difficult. Extended reaction times necessary for
2 5 derivatization would decrease throughput of the number of assays. Therefore,our inventive reagents offer a vast leap over tl e previous art in (i) direct
measurement of porphobilinogen by immunoassay (ii) use of highly specific
antibodies to porphobilinogen which can operate in the presence of other
porphyrin related compounds (iii) the method for the development of
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porphobilinogen specific antibodies (iv) pairing of porphobilinogen specific
antibodies and labeled reagents for use in the quantification of porphobilinogenand (v) use of inventive reagents in an automated system using existing clinicald~emistry instrumentation, such as Abbott IMx(g) or A~bott AxSYM(~) systems.
When perforrning a fluorescence polarization immunoassay for
measuring the levels of lead via the quantification of porphobilinogen as
described herein, the detectable moiety component of the tracer is a fluorescentmoiety such as fluoresceins, aminofluoresceins, carboxyfluoresceins, and the
1 0 like, preferably aminomethylfluorescein, aminofluorescein, 5-fluoresceinyl, 6-
fluoresceinyl, 6-carboxyfluorescein, 5-carboxyfluorescein, thiourea-
aminofluorescein, and methoxytriazinolyl-aminofluorescein, and the lilce
fluorescent derivatives. The amount of tracer bound to the antibody varies
inversely to the amount of porphobilinogen generated in the test sample.
15 Accordingly, the relative, and therefore characteristic, binding affinities of
porphobilinogen and the tracer to the antibody binding site, are important
parameters of the assay system. Generally, fluorescent polarization techniques
are based on the principle that a fluorescent tracer, when excited by plane
polarized light of a characteristic wavelength, will emit light at another
2 0 characteristic wavelength (i.e., fluorescence~ that retains a degree of the
polarization relative to the incident stimulating light that is inversely related to
the rate of rotation of the tracer in a given medium. As a consequence of this
property, a tracer substance with constrained rotation, such as in a viscous
solution phase or when bound to another solution component with a relatively
2 5 lower rate of rotation, will retain a relatively greater degree of polarization of
emitted light than if in free solution. Therefore, within the time frame in
which the ligand and tracer compete for binding to the antibody, the tracer and
ligand binding rates should yield an appropriate proportion of free and bound
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tracer with the preservation of important performance parameters such as
selectivity, sensitivity, and precision.
When performing a fluorescent polarization immunoassay for
5 measuring the levels of lead via the quantification of porphobilinogen
according to the present invention, a test sample suspected of containing lead is
incubated with ALAD then contacted with antiserum prepared with
immlmogens according to the present invention in the presence of an
appropriately selected fluorescein derivative thereof which is capable of
10 producing a detectable fluorescence polarization response to the presence of
antiserum prepared with immunogens according to the present invention.
Plane polarized light is then passed through the solution to obtain a fluorescent
polarization response and the response is detected as a measure of amount of
porphobilinogen present in the test sample.
1 5
The porphobilinogen derivatives of the present invention are employed
to prepare immunogens by coupling them to conventional carrier materials,
and subsequently used to obtain antibodies. The porphobilinogen derivatives
are also used to prepare labeled reagents which serve as the detection reagents
2 0 in immlmoassays for quantifying the amount of porphobilinogen and thereby
measuring the levels of lead in a test sample.
The porphobilinogen derivatives of the present invention can be coupled
to immunogenic carrier materials by various conventional techniques known
2 5 in the art where P is an immunogenic carrier material in Formula II or III As
would be understood by one skilled in the art, the immunogenic carrier
material can be selected from any of those conventionally known and, in most
instances, will be a protein or polypeptide, although other materials such as
carbohydrates, polysaccharides, lipopolysaccharides, poly(amino) acids, nucleic
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14
acids, and the like, of sufficient size and immunogenicity can also be employed.Preferably, the immunogenic carrier material is a protein such as bovine serum
albumin, lceyhole limpet hemocyanin, thyroglobulin, and the like. The
immunogens according to the present invention are used to prepare antibodies,
S both polyclonal and monodonal, according to methods known in the art for use
in an immunoassay ~y~Lell- according to the present invention. Generally, a
host ~nim~l, such as a rabbit, goat, mouse, guinea pig, or horse is inJected at one
or more of a variety of sites with the immunogen, normally in mixture with an
adjuvant. Further injections are made at the same site or different sites at
1 0 regular or irregular intervals thereafter with bleedings being taken to assess
antibody titer until it is detPrmined that optimal titer has been reached. The
antibodies are obtained by either bleeding the host ~nim~l to yield a volume of
antiserum, or by somatic cell hybridization techniques or other techniques
known in the art to obtain monoclonal antibodies, and can be stored, for
1 S example, at -20~C.
In ~ tiQn to fluorescence polarization immunoassays, various other
immunoassay formats can be followed for the quantification of
porphobilinogen according to the present invention. Such immunoassay
2 0 system formats include, but are not intended to be limited to, competitive,
sandwich and immunometric techniques. Generally, such immunoassay
:jyslellls depend upon the ability of an immunoglobulin, i.e., a whole antibody
or fragment thereof, to bind to a specific analyte from a test sample wherein a
labeled reagent comprising an antibody of the present invention, or fragment
2 S thereof, attached to a label or detectable moiety is employed to determine the
extent of binding. Such detectable labels include, but are not intended to be
limited to, enzymes, radiolabels, biotin, toxins, drugs, haptens, DNA, RNA,
liposomes, chromophores, chemiluminescers, colored particles and colored
microparticles, fluorescent compounds such as aminomethylfluorescein, 5-
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fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein,
aminofluorescein, thioureafluorescein, and methoxytriazinolyl-
aminofluorescein, and the like fluorescent derivatives. As described herein, thetest sample can be a naturally occurring or artificially formed liquid, or an
5 extract thereof, and indudes, but is not intended to be limited to biological test
samples such as whole blood, serum, plasma, urine, feces, saliva, cerebrospinal
fluid, brain tissue, and the like. In addition, the test sample can be an ex~ract of
a test sample, or any derivative thereof.
Typically, the extent of binding in such immunoassay system formats is
determined by the amount of the detectable moiety present in the labeled
reagent which either has or has not participated in a binding reaction with the
analyte, wherein the amount of the detectable moiety detected and measured
can be correlated to the amount of analyte present in the test sample. For
I S example, in a competitive immunoassay system, a substance being measured,
often referred to as a ligand, competes with a substance of close structural
similarity coupled to a detectable moiety, often referred to as a tracer, for a
lirnited number of binding sites on antibodies specific to the portion or portions
of the ligand and tracer with structural similarity, shared with an immunogen
2 0 employed to produce such antibodies.
The present invention will now be illustrated, but is not intended to be
limited by, the following examples. Bold-faced numerals contained in
parenthesis refer to the structural formulae as used in the Figures:
~ 25 Abbreviations/Formulas: acetic acid = HOAc, acetonitrile = CH3CN,
chloroform = CHC13, diethyl ether = Et20, dimethylform~mifle = DMF, ethyl
acetate = EtOAc, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide = EDAC, N-
hydroxysuc~inimi~le = HOSu, methanol = MeOH, methylene chloride = CH2C12,
tetrahydrofuran - THF.
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16
Analytical HPLCs were run using a Waters 8 x 100 mm mBC18R P
cartridge with a flow rate of 2 mL/min and 1 = 225 nm and preparative HPLCs
were run using a Waters 40 x 100 mm mBC~8RP cartridge with a flow rate of 45
mL/min and 1 = 225 nm unless otherwise noted.
1P~ F l
SYNIHESIS OF PORPHOBILINOGEN IMMUNOGEN (7)
To a solution of adipic acid mono ethyl ester (22.8 g, 131 mmol3 in
1 0 dimethylform~mi~le (DMF) (100 mL) was added dicyclohexylcarbodiimide (DCC)
(28.3 g, 137 mmol) and N-hydroxysuc~ inimi~1e (HOSu) (15.7 g, 137 mmol). The
reaction was stirred for 20 hours under N2, filtered and solvents removed in
vacuo. The oil was taken up in EtOAc (100 mL), filtered and dried in vacuo.
The crude material was purified by flash chromatography (50% EtOAc/50%
l 5 Hexane) to afford 30.6 g (85%) of Adipic active ester. lH NMR (d6 DMSO): d 4.05
(q, J=7.14 Hz, 2H), 2.80 ~s, 4H), 2.75-2.65 ~m, 2H), 2.40-2.27 (m, 2H), 1.70-1.55 (m,
4H), 1.16 (t, J=7.14 Hz,3H); mass spec IM+H]+ 272.
Adipic active ester (2.67 g, 9.83 mmol) in DMF (30 mL) was added to a
solution of PBG (1) (800 mg, 3.28 mmol) in 0.1û M NaH2PO4 buffer (30 mL)J1M
2 0 Na2CO3 ~2.4 mL). The reaction was covered with foil, stirred for 6 hours under
N2 and then solvents removed in vacuo. Preparative HPLC purification (25%
CH3CN/75% H2O + 0.1% formic acid) gave 840 mg (67%) of PBG-Adipic ethyl
ester (2). lH NMR (d6 DMSO): d 10.24 (s, lH), 7.93 (t, J=6.41 Hz, lH), 6.39 (s, lH),
4.12 (d, J=4.48 Hz, 2H), 4.05 (q, J----7.12 Hz, 2H), 3.27 (s, 2H), 2.60-2.00 (m, 8H), 1.58-
2 5 1.38 (m, 4H), 1.17 (t, J=7.12 Hz, 3H); mass spec [M+H~+ 383; HPLC (25%
CH3CN/75% H2O + 0.1% formic acid): 5.66 min, >97%.
tert-Butyl isourea (4.7 g, 23.5 mmol) was added to a 5~C solution of PBG-
Adipic ethyl ester (2) (640 mg, 1.67 mmol) in DMF (6 mL) under argon, the
reaction was stirred for 44 hours at room temperature. After adding
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tetrahydrofuran (THF) (15 mL), the reaction was filtered and solvents were
removed in vacuo. Purification by preparative HPLC (60% CH3CN/40% ~2~ +
0.1% formic acid) afforded 300 mg (36%) of PBG-di-tcrt-butyl-Adipic etllyl ester(3). IH NMR (CDC13): d 8.67 (s, lH), 6.62 (t, J=5.38 Hz, lH), 6.41 (s, lH), 4.29 (d,
~=5.56 Hz, 2H), 4.11 (q, J=7.12 Hz, 2H), 3.32 (s, 2H), 2.72-2.10 (m, 8H), 1.70-1.55 (m,
4H), 1.44 (s, 9H), 1.43 (s, 9H), 1.24 (t, J=7.13 Hz, 3H); mass spec [M+H]+ 495; HPLC
(60% CH3CN/40% H2O + 0.1% formic acid): 8.09 min, >93%.
To a 5~C solution of PBG-di-tert-butyl-Adipic ethyl ester (3) (300 mg, 0.607
mmol) in EtOH (7 mL) was added a solution of H2O (7 mL)/KOH (102 mg, 1.82
1 0 mmol); the reaction was warmed to room temperature, covered with foil,
stirred for 3 hours under argon and solvents removed in vacuo. The residue
was dissolved in H2O (150 mL), acidified to pH = 2.8 with 1.5 M H3PO4 and
extracted with EtOAc (4 x 100 mE). The combined extracts were washed with
brine, dried over Na2SO4, filtered and dried in vacuo to give 220 mg (78%) of
1 5 PBG-di-tert-butyl-Adipic hapten (4). IH NMR (CDCl3): d 8.90 (s, lH), 6.89 (t,
~=5.61 Hz, lH), 6.44 (s, lH), 4.30 (d, J=5.73 Hz, 2H), 3.33 (s, 2H), 2.68 (t, J=7.93 Hz,
2H), 2.45 (t, J=7.93 Hz, 2H), 2.33 (t, J= 6.63 Hz, 2H), 2.20 (t, J=6.87 Hz, 2H), 1.70-1.55
(m, 4H), 1.44 (s, 9H), 1.43 (s, 9H); mass spec [M+H]+ 467; HPLC (60% CH3CN/40%
H2O + 0.1% formic acid): 4.07 min, >72%.
2 0 A solution of EDAC (183 mg, 0.955 mmol) in DMF (6 mL) was added to a
solution of PBG-di-tert-butyl-Adipic hapten (4) (110 mg, 0.236 mmol), HOSu (110
mg, 0.956 mmol) in DMF (2 mL); the reaction was covered with foil and stirred
for 20 hours under argon. After removing solvents in vacuo, the residue was
dissolved in a Et2O/0.05 M NaH2PO4 buffer (pH = 5.5) solution and extracted
2 5 with Et2O (4 x 50 mL). The combined extracts were washed with brine, dried
over Na2SO4, filtered and dried in vacuo. Yield was 105 mg (80%) of PBG-di-
tert-butyl-Adipic active ester (5). HPLC (60% CH3CN/40% H2O + 0.1% formic
acid): 5.76 min, >77%.
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PBG-di-tert-butyl-Adipic active ester (5) (37 mg, 0.066 mmol) in DMF (16
mL) was added to a 5~C solution of BSA (210 mg) in 0.1 M NaH2PO4 buffer (pH =
7.7, 20 mL); the reaction was warmed to room temperature, covered with foil
and stirred for 40 hours under N2. Purification by gel filtration [60 g G-25
S Sephadex, 20% MeOH/80% H2O (100 mM ammonium acetate)], fractions
collected and lyophilized. Yield was 280 mg of PBGdi-tert-l:~utyl-Adipic BSA
Immunogen (6).
Trifluoroacetic acid (T~A) (10 mL) was added to a suspension of PBG-di-tert-
butyl-Adipic BSA Tmmllnogen (6) (280 mg) in CH2Cl2 (10 mL), the reaction was
l 0 stirred for 20 minutes under N2 and then solvents removed in vacuo. The
residue was dissolved in H20 (80 mL), neutralized with lM NH40H and
mixture lyophilized. Dissolved solid in H2O (lOO mL), neutralized with lM
NH40H and then lyophili7e~1 The resulting solid was dialyzed in H20 (4 L) for
6 hours and lyophili~etl to give 240 mg of PBG-Adipic-BSA Immunogen (7).
l S
EXA~PT~F ?
SYNTHESIS OF PORPHOBILINOGEN IMMUNOGEN (14)
A solution of di-tert butyl dicarbonate (BOCzO, 2.15 g, 9.4 mmol) in 15 mL
2 0 tetrahydrofuran (THF) was added to a solution of porphobilinogen hydrate (1)(2.3 g, 9.4 mmol) in 30 mL 0.5 M aqueous sodium carbonate (15 mmol) and 15
mL THF. The reaction mixture was stirred under nitrogen for 4 hours, another
10 mL 0.5 M sodiurn carbonate and a 10 mL solution of BOC2O (2.1 g, 9.4 mmol)
in THF were added to the reaction mixture and stirred for another 4 hours.
HPLC indicated <2% starting material and >95~O desired product. The crude
mixture was poured into 200 mL water/5 mL 1 M NaOH after removing
solvents in vacuo and extracted with 50 mL Et2O. The aqueous layer was
acidified with 1 M phosphoric acid, extracted 3 x 10~) mL EtOAc, dried combined
extracts over sodium sulfate and removed solvents in vacuo to afford 2.86 g
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19
(94%) N-BOC PBG diacid (8). IH NMR (DMSO-d6) d 12.02 (br s, lH), 11.98 (br s,
lH), 10.14 (br s, lH), 6.81 (s, lH), 6.36 (s, lH), 4.01 (d, J=5.56 Hz, 2H), 3.45 (s, 2 H),
2.77 (t, J =7.09 Hz, 2~I), 2.56 (t, J=7.09 Hz, 2H), 1.43 (s, 9H); mass spec (M)+ 324.
ter~-Butyl isourea (19.6 g, 98 mmol) was added to a 10~C solution of N-BC:)C
PBG diacid (8) (2.8 g, 8.6 mmol) in 15 mL dimethylformamide (DMF) under
nitrogen and stirred 16 hours at room temperature. Reaction mixture was
poured into 45 mL distilled w~ter and 50 mL phosphate buffer (500 mM,
pH=6.0), extracted 3 x 125 mL Et2O, washed combined extracts with 70 mL brine
and removed solvents in vacuo. Purification by preparative HPI C afforded 1.7
1 0 g (45%) di tert-butyl ester N-BOC PBG (9). lH NMR (CDC13) d 8.58 (br s, 1~), 6.45
(s, lH), 5.26 (br s, lH), 4.15 (d, J=6.07 Hz, 2H), 3.30 (s, 2 H), 2.69 (t, J=8.47 Hz, 2H),
2.45 (t, J=8.47 Hz, 2H), 1.43 (sl br s, 27 H); mass spec (M)~ 438.
Potassium hexamethyldisilylazide (0.50 M, 1.2 mL, 0.60 rnmol) was added to
a 0~C solution of di tert-butyl ester N-~OC PBG (9, 219 mg, 0.50 mmol) in 5 mL
1 5 THF under nitrogen and stirred for 30 minutes at 0~C. A solution of benzyl 4-
chlorosulfonylbutyrate (166 mg, 0.6 mmol) in 1 mL THF was added to the
reaction mixture and stirred overnight. Workup consisted of pouring the
reaction into a mixture of 50 mL water, 5 mL saturated sodium bicarbonate and
extracting with 3 x 75 mL EtOAc, removing solvents in vacuo and purifying the
2 0 residue by preparative HPLC to afford 128 mg (37%) of the desired benzyl N1-sulfonyl compound (10). IH NMR (CDCl3) d 7.39 (br s, 5H), 6.84 (s, lH3, 5.33 (br s,
lH), 5.11 (s, 2H), 4.36 (d, J=6.2 Hz, 2H), 3.52 (s, 2H), 3.31 (t, ~= 7.5 Hz, 2H), 2.63 (t,
J=7.9 Hz, 2H), 2.51-2.39 (m, 4H), 2.03 (p, J=7.3 Hz, 2H), 1.43 (sl br s, 27 H); mass
spec (M+H)+ 679.
A mixture of Nl-sulfonyl compound (10) (110 mg, 0.16 mmol), 25 mL
absolute ethanol and 10% palladiurn on carbon (Pd/C, 60 mg) was stirred under
1 atm hydrogen for 16 hours. Piltered off catalyst and removed solvents in
vacuo to afford 92 mg (84%) desired Nl-sulfonyl compound (11). IH NMR
(CDC13) d 7.90 ~v br s, lH), 6.87 (s, lH), 5.35 (br s, lH), 4.37 (d, J=5.8 Hz, 2H), 3.56
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(br s, 2H3, 3.36 (t, J-7.3 Hz, 2H), 2.63 (t, J=7.6 Hz, 2H), 2.46 (t, J=7.6 Hz, 2H), 2.39 (t,
J=7.2 Hz, 2H), 2.02 (p, J=7.2 Hz, 2H), 1.44 (sl br s, 27 H); mass spec (M)+ 589. EDAC (115 mg, 0.60 mmol) was added to a solution of Nl-sulfonyl
compound (11) (70 mg, 0.12 mmol), HOSu (136 mg, 1.2 mmol) and 3 mL DMF
5 then stirred under nitrogen for 14 hours. The reaction mixture was poured into70 mL Et2O, washed 1 x 30 mL phosphate buffer (500 mM, pH=6.0) and removed
solvents in vacuo to afford 67 mg (82%) of desired active ester (12). lH NMR
(CDCl3) d 6.88 (s, lH), 5.32 (br s, lH), 4.38 (d, J=5.9 Hz, 2H), 3.53 (s, 2 H), 3.40 (t,
J=7.2 Hz, 2~I), 2.83 (s, 4H), 2.74 (t, J=7.2 Hz, 2H), 2.63 (t, J=7.2 Hz, 2H), 2.20-2.08 (m,
l 0 4H), 1.43 (sl br s, 27 H); mass spec (M+H)+ 686.
A solution of active ester (12) (44 mg, 0.064 mmol)/2 mL DMF was added to
a solution of bovine serum albumin (BSA, 220 mg), 22 mL phosphate buffer (50
mM, pH=7.8) and 6 mL DMF, then stirred for 15 hours at room temperature.
Purification of the protected immunogen by G-25 column chromatography and
1 5 lyophilization of fractions containing purified immunogen afforded 278 mg of
desired protected immunogen (13).
Trifluoroacetic acid ~12 mL) was added to a suspension of the protected
immunogen ~13) (277 mg) in methylene chloride (12 mL) and stirred for 30
minutes at room temperature. The reaction mixture solvent were removed
solvents in VRCUO and stirred with 40 mL aqueous ammonium acetate (100
mM) for 15 hours to give a homogeneous solution which was lyophilized. The
material was dissolved in 50 mL water, lyophilized and repeated two times to
give 210 mg desired porphobilinogen immunogen (14).
2 5 ~A~PI.F 3
SYNTHESIS OF PORPHOBILINOGEN IMMUNOGEN (19)
Trichloroacetyl chloride (207 mL, 1.85 mmol) was added to a mixture of di
tert-butyl ester N-BOC PBG ~9, 675 mg, 0.50 mmol), anhydrous potassium
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21
carbonate (2.24 g, 16 mmol) and 15 mL anhydrous Et2O, the reaction mixture
was stirred for 30 minutes under nitrogen at 0~C. Poured mixture into 200 mL
Lt2O/100 mL water, 100 mL saturated sodium bicarbonate, separated, extracted
aqueous layer 1 x 50 mL Et2O and dried combined organic extracts over sodium
5 sulfate. Removal of solvents in vacuo afforded 805 mg of crude C-5 substituted trichloromethyl ketone (15). Mass spec (M+ NH4)~ 602.
Aqueous sodium hydroxide (1.0 M, 3.5 mL, 3.5 mmol) was added to a
solution of crude C-5 substituted trichloromethyl ketone (15) (805 mg, 1.5
mmol), 30 mL acetone and 6 mL water, the reaction mixture was stirred for 25
1 (1 minutes then removed acetone in vacuo. The crude mixture was poured into
100 mL water, extracted with 50 mL Et2O, adjusted pH of aqueous layer to 2.5
with 1.0 M phosphoric acid and extracted 3 x 100 mL EtOAc. The combined
EtOAc extracts were washed 1 x 30 mL brine and removed solvents tn vacuo to
give a dark oil which was purified by preparative HPLC to afford 122 mg (51%
1 5 from 9) desired C-5 substituted carboxylic acid (16). lH NMR ~CDC13) d 9.88 (br s,
2H), 5.5Z (br s, lH), 4.28 (d, J=5.5 Hz, 2H), 3.40 (s, 2 H),3.02 (t, J=7.8 Hz, 2H), 2.49 (t,
J=7.8 Hz, 2H), 1.44 (sl br s, 27 H); mass spec (M+H~+ 483.
EDAC (289 mg, 1.25 mmol) was added to a solution of C-5 substituted
carboxylic acid (16) (122 mg, 0.25 mmol), HOSu (288 mg, 2.5 mmol) and 3.3 mL
2 0 DM~ then stirred under nitrogen for 14 hours. The reaction mixture was
poured into 100 mL Et2O, washed 1 x 50 mL phosphate buffer (500 mM, pH=6.0),
washed 1 x 50 mL water and removed solvents in vacuo to afford 143 mg (98%)
of desired active ester (17). IH NMR (CDCl3) d 10.02 (br s, lH), 5.50 ~br s, lH), 4.24
(d, J=5.5 Hz, 2H), 3.42 (s, 2 H), 3.05 (t, J=7.6 Hz, 2H), 2.82 (s, 4H), 2.51 (t, J=7.6 Hz,
2 5 2H), 1.45 (sl br s, 27 H); mass spec (M+H)+ 597.
A solution of active ester (17) (65.5 mg, 0.113 mmol)/2 mL DMF was added
to a solution of bovine serum albumin (BSA, 150 mg), 50 mL phosphate buffer
(50 mM, pH=8.8) and 10 mL DMF, then stirred for 3 days at room temperature.
Purification of the protected immunogen by G-25 column chromatography and
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W O 97/24621 22 PCT~US96/19544
lyophilization of fractions containing purified immunogen afforded 207 mg of
desired protected immunogen (18).
Trifluoroacetic acid (10 mL) was added to a suspension of the protected
immunogen (18) (206 mg3 in methylene chloride (10 mL) and stirred for 30
5 minutes at room temperature. The reaction solvents were removed in vacuo
and resulting residue wa~ stirred with 40 mL aqueous ammonium acetate (100
mM) for 15 hours to give a homogeneous solution which was lyophilized. The
material was dissolved in 50 mL water, lyophilized and repeated two times to
give 153 mg desired porpho~;linogen immunogen (19).
1 0
1PT .F 4
SYNTHESIS OF PORPHOBILINOGEN TRACER (21)
To a solution of 6-carboxyfluorescein (34 mg, 0.090 mmol) in
1 5 dimethylform~ le (1.7 mL, DMF) was added dicyclohexyl-carbodiimide (18 mg,
0.086 mmol) and N-hydroxysuccinimide (11 mg, 0.087 mmol), the reaction was
covered with foil then stirred for 20 hours under N2. The reaction was filtered to
afforded a solution of 6-carboxyfluorescein active ester (20) which was added to a
solution of porphobilinogen (1) 20 mg, 0.082 mmol) in 0.10 M phosphate (pH=7.7,
2 0 4 mL). The reaction was covered with foil then stirred for 20 hours under N2.
and solvents removed in vaCuo. The crude material was purified twice by HPLC
(30% CH3CN/70% H2O + 0.1% formic acid) to give 8 mg (17%) of PBG-6-AMF (21).
H NMR (CD30D~: d 8.15-8.00 (m, 2H), 7.65 (s, lH), 6.70-6.40 (m, 7H), 4.45 (s, 2H),
3.45 (s, 2H), 2.275-2.40 (m, 4H); mass spec (M+H)+ 585; HPLC (30% CHICN/70%
2 5 H2O + 0.1% ~ormic acid): 8.14 min, >97%.
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23
ExA~lpT~F 5
SYNTHESIS OF PORPHOBIL~NOGEN TRACER (27)
4-Dimethylaminopyridine (DMAP) (1.18 g, 9.7 mmol) was added to a room
temperature solution of t-butyl 4-nitrobutyrate (2.74 g, 14.5 mmol) and 3-
t(tetrahydro-2H-pyran-2-yl)oxy] propanal (1.53 g, 9.7 mmol) in dry CH2Cl2 and
stirred for 96 h. The solvent was removed in vacuo and the crude mixture was
purified by silica gel column chromatography (20-35% EtOAc in n-hexane) to
l 0 afford a-hydroxynitro compound (22) 2.45 g (73%) as a colorless thick gum. 1H
NMR (CDC13): d 4.62-4.54 (m, 2H), 4.3û-3.45 (m, SH), 2.41-2.10 (m, 4H), 1.90-1.70
(m, 2H), 1.57-1.51 (m, 6H), 1.44 (s, 9H); mass spec (M+NH4)+ 365.
Acetic anhydride (4.83 mL, 51.3 mmol) was added dropwise to a 0~C solution
of a-l~ydroxynitro compound (22) (4.45 g, 12.8 mmol), anl-ydrous pyridine (1.15
1 5 mL, 19.2 mmol) and dry CH2Cl2 (50 mL); after 2.0 h, tl~e mixture was allowed to
warm to room temperature and stirred for 14 h. Workup consisted of pouring
the reaction mixture into 10% aqueous NaHCO3 (50 mL), separated layers and
extracted the aqueous layer with CH2Cl2 (2 X 50 m~). The combined organic
layers were washed with 5% HCl (20 mL), water (30 mL), brine (15 mL), dried
(MgSO4) and the solvents were removed in vacuo. Purification by silica gel
column chromatography (20% EtOAc in n-l~exane) afforded 2.63 g of a-
acetoxynitro compound (23) (51%) as a colorless thick liquid. 1H NMR (CDCl3):
d 5.50-5.40 (m, lH), 4.90-4.80 (m, lH), 4.60 (br s, lH), 4.51 (br s, lH), 3.90-3.72 (m,
2H), 3.58-3.32 (m, 2H), 2.45-2.15 (m, 4H), 2.08 (s, 3H), 2.04 (s, 3H), 2.04-1.50 (m, 8H),
2 5 1.44 (s, 9H); mass spec (M+NH4)+ 407.
1,8-Diazabicydol5.4.0]undec-7-ene (DBU) (0.76 mL, 5.08 mmol) was added to a
0~C mixture of a-acetoxynitro compound (23) (1.65 g, 4.24 mmol),
benzylisocyanoacetate (0.890 g, 5.08 mmol) and TH~ (14 mL), stirred for 30 min at
0~C, warmed to room temperature and stirred for 24 h. The resulting orange-red
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W O 97/24621 PCT~US96/19544
24
color solution was quenched with water (20 mL) and extracted with F~OAc (3 X
50 mL). The combined organic layers were washed with water (20 mL), brine (15
mL), dried (MgS04) and the solvents were removed in vacuo. The crude
compound was purified by silica gel column chromatography (20% EtOAc in n-
S hexane) to afford 1.21 g of 24 (63%) as a colorless g~m. lH NMR (CDC13): d 8.86
(br s, lH), 7.45-7.26 (m, 5H), 6.70 (s, lH), 5.30 (m, 2~), 4.51 (br s, lH), 3.88-3.70 (m,
2H), 3.56-3.38 (m, 2H), 3.04 (t, J=7.2 Hz, 2H), 2.75 (t, J=7.8 Hz, 2H), 2.47 (t, J=7.5 Hz,
2H), 1.80-1.43 (m, 6H), 1.42 (s, 9H); mass spec (M+NH4)+ 475.
In a dry single necked round bottom flask equipped with magnetic stir bar,
l 0 nitrogen inlet was placed 2-carbobenzyloxy pyrrole (24) (1.20 g, 2.18 mmol) in
methanol (20 mL) and added pyridinium p-toluenesulfonate (PPTS, 0.58 g, 2.29
mmol) at room temperature and stirred for 48 h. The solvent was removed in
vacuo and the mixture was diluted with water (30 mL)/EtOAc (75 mL). The
aqueous layer was extracted with EtOAc (2 X 50 mL) and the combined organic
1 5 extracts were washed with brine (20 mL) and concentrated in vacuo. Purification
by silica gel column chromatography (30-40% EtOAc in n-hexane) gave 1.66 g
(92%) of pyrrole alcohol (25). 1H NMR (~DC13): d 8.90 (br s, lH), 7.42-7.27 (m,
5H), 6.71 (d, J=2.7 Hz, lH), 5.29 (s, 2H), 3.79-3.74 (m, 2H), 3.03 (t, J=6.3 Hz, 2H), 2.73
(t, J=7.8 Hz, 2H), Z.47 (t, J=7.8 Hz, 2H), 2.15-2.05 (m, lH), 1.41 (s, 9~I); mass spec
2 0 (M+H)~ 374.
Jones reagent (2.67 M, 0.64 mL, 1.69 mmol) was added in one portion to a 0~C
solution of pyrrole alcohol (25) (0.400 g, 1.13 mmol) in acetone (20 mL) and
stirred for 1.5 h then quenched with isopropanol (3.0 mL~ and stirred for 30 minat rt. The mixture was diluted with acetone (22 mL), filtered and washed with
2 5 acetone (30 mL). After concentrating the filtrate in vacuo, the resulting crude
acid was dissolved in DMF (0.5 mL), cooled to 0-5~C with ice bath and added a
solution of O-~-butyl-N,N'-diisopropyl-isourea (0.313 g, 5.0 eq.) in DMF (1.0 mL).
The cooling bath was removed and the mixture was stirred for 43 h at room
temperature before removing solvents in vacuo. Purification by silica gel
.
CA 02239969 1998-06-08
W O97/24621 25 PCTAUS96/19544
column chromatography (20% EtOAc in n-hexane) afforded 0.190 g of tl2%)
pyrrole di t-butyl ester (26). 1H NMR (CDC13): d 8.88 (br s, lH), 7.26-7.41 (m, 5H),
6.73 (d, J=3.0 Hz, lH), 5.29 (s, 2H), 3.77 (s, 2H), 2.70 (t, J=7.2 Hz, 2H), 2.48 (t, J=6.6
Hz, 2H), 1.42 (s, 9H), 1.40 (s, 9H); mass spec calcd for C25H34NO6: 444.2386; found:
444.2383; HPLC: 0.1% formic acid in water:MeCN (75:25), 4.15 min, 97%.
Palladium on carbon (10% Pd/C) (0.029 g) was added to a solution of pyrrole
di-t-butyl ester (26) (0Ø94 g, 0.212 mmol) in absolute ethanol (4 mL) and stirred
under hydrogen (1 atrn) for 2.0 h at room temperature. The mixture was diluted
with ethanol (10 mL), filtered and solvent removed in vacuo to afford 0.065 g
1 0 (89%) of 2-acyl analogue of PBG (27). 1H NMR (acetone-d6): d 10.52 (br s, lH), 6.88
(d, J=3.3 Hz, lH), 3.84 (s, 2H), 2.71 (t, J=8.4 Hz, 2H), 2.49 (t, ~=8.4 Hz, 2H), 1.47 (s,
9H), 1.45 (s, 9H); mass spec calcd for C18H28NO6: 354.1917; found: 354.1914; HPLC:
0.1% formic acid in water:MeCN (25:75), 2.57 min, 96%.
EDAC (0.033 g, 0.175 mmol) was added to a room temperature solution of
l 5 acid (27) (0.017 g, 0.05 mmol) and HOSu (0.015 g, 0.125 mmol) in dry DME (0.5
mL) and stirred for 36 h. The mixture was diluted with potassium phosphate pH
6.0 buffer solution (3 mL) and extracted with Et2O (3 X 20 mL). The combined
ethereal extracts were washed with water (5 mL), dried (MgSO4) and
concentrated in vaCuo to afford 0.021 g of (89%) 2-carboxypyrrole active ester (28).
2 0 1H NMR (CDC13): d 10.32 (br s, lH), 7.12 (s, lH), 3.88 (s, 2H), 2.97 (s, 4H), 2.83 (t,
J=8.1 Hz, 2H), 2.59 (t, J=7.8 Hz, 2H), 1.57 (s, 9H), 1.55 (s, 9H); mass spec (M+NH4)+
468; HPLC: 0.1% formic acid in water:MeCN (25:75), 2.68 min, 89%.
In a dry single-necked round bottom flask equipped with magnetic stir bar
was placed active ester (28) (0.008 g, 0.017 mmol) in dry DMF (0.5 mL); added 6-2 5 (aminomethyl)-fluorescein hydrochloride (6-AMF) (0.010 g, 0.025 mmol)
followed by triethylamine (Et3N) (0.018 mL, 0.13 mmol) to the reaction mixture
and stirred at room temperature for 18 h. The mixture was pllrified by
preparative reversed phase C18 HPLC, eluting with 0.1% formic acid in
water:MeCN/45:55 (v/v), to afford 9.6 mg (77%) of 2-acyl analogue of PBG tracer
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26
di-t-~utyl ester (29~. I H NMR (CD30D): d 7.96 (d, J=7.8 Hz, lH), 7.68 (d, J=7.8 Hz,
lH), 7.21 (s, lH), 6.69-6.48 (m, 7H), 4.59 (s, 2H), 3.52 (s, 2H), 2.67 (t, J=7.5 Hz, 2H),
2.43 (t, J=7.2 Hz, 2H), 1.38 (s, 9H), 1.34 (s, 9H); mass spec (M~H)+ 697; HPLC: 0.1%
formic acid in water:MeCN (45:55), 6.43 min, >99%.
Trifluoroacetic acid (1.0 mL) was added to a mixture of di-t-butyl ester (29)
(0.010 g, 0.14 mmol) in dry CH2Clz (1.0 mL), and stirred at room temperature for1.5 h. The mixture was purified by preparative reversed phase Cl8 HPLC,
eluting witl 0.1% formic acid in water: MeCN (70:30) to afford 2.4 mg (34%) of
PBG 2-acyl analogue tracer (30). 1H NMR (CD30D): d 7.95 (d, J=7.8 Hz, lH), 7.72-1 0 7.68 (m, lH), 7.21 (s, lH), 6.67-6.51 (m, 7H), 4.61 (s, 2H), 3.37 (s, 2H), 2.77 (t, J=7.8
Hz, 2H), 2.50 (t, J=7.2 Hz, 2H); mass spec (M)+ 584; HPLC: 0.1% formic acid in
water:MeCN (70:30), 7.08 min, >99%.
Fx~ yIp~F 6
l 5 SYNTHESIS OF PORPHOBILINOGEN TRACER (32)
In a dry single-necked round bottom flask equipped with magnetic stir bar
was placed ~-t-BOC-6-amino hexanoic acid (1.15 g, 5.0 mmol), HOSu (0.69 g, 6.0
mmol) and dry DMF (15 mL); added EDAC (1.24 g, 6.5 mmol) at room
2 0 temperature and stirred for 24 h. The solvent was removed in vacuo and
diluted with water (25 mL). Extracted the aqueous mixture with Et2O (50 mL, 2 X
30 mL), combined Et2O layers were washed with water (2 X 20 mL), brine (15 mL)
and dried (MgSO4). Removal of the solvent in vacuo afforded 1.52 g of (93%) of
N-t-BOC-6-amino hexanoic acid active ester as a colorless solid. 1 H NMR
2 5 (CDC13): d 4.60 (br s, lH), 3.14-3.05 (m, 2H), 2.97 (s, 4H), 2.83 (s, 4H), 2.61 (t, J=7.2
Hz, 2H), 1.71-1.82 (m, 2H), 1.54-1.40 (m, 4H), 1.43 (s, 9H); HPLC: 0.1% formic acid
in water:MeCN (50:50), 8.98 min.
Triethylamine (0.060 mL, 0.45 mmol) was added to a room temperature
solution of N-t-BOC-6-amino hexanoic acid active ester (û.014 g, 0.045 mmol), 6-
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W O 97/24621 27 PCTrUS96/19544
(aminomethyl)fluorescein hydrochloride (6-AMF) (0.020 g, 0.05 mmol) and dry
DMF (0.4 mL); the mixture was stirred for 24 h at room temperature.
Purification by preparative HPLC, eluting with 0.1% formic acid in water:MeCN
(32:68) afforded 0.026 g (95%) of N-t-BOC-6-aminohexanoic-(6-aminomethyl)-
~ 5 fluorescein. lH NMR (CD30D): d 8.05 (d, J=8.1 Hz, lH), 7.69 (d, J=6.9 Hz, lH), 7.19
(s, lH), 6.87-6.70 (m, 6H), 4.51 (d, J=6.0 Hz, 2H), 3.12-3.05 (m, 2H), 2.30-2.20 (t, J---7.8
Hz, 2H), 1.65-1.46 (m, 4H), 1.~6 (s, 9H), 1.35-1.25 (m, 2Hj; mass spec (M+H)+ 575;
HPLC: 0.1% formic acid in water:MeCN (60:40), 7.98 min, 96%.
In a dry single-necked round bottom flask equipped with magnetic stir bar
1 0 was placed the above prepared N-t-BOC-6-aminohexanoic-(6-aminomethyl)-
fluorescein (0.024 g, 0.069 mmol) in dry CH2C12 (1.0 mL); added trifluoroacetic
acid (0.5 mL~ and stirred at room temperature for 10 min. The mixture was
purified by preparative reversed phase C18 HPLC, eluting with 0.1% formic acid
in water:MeCN (60:40) to give 20 mg (>95%) of aminofluorescein derivative. 1H
1 5 ~MR (CD30D): d 7.96 (d, J=8.1 Hz, IH), 7.55 (d, J=8.7 Hz, lH), 7.10 (s, lH), 6.79-6.53
(m, 6H), 4.86 (s, 2H), 2.83 (t, J=8.1 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H), 1.62-1.50 (m, 4H),
1.38-1.29 (m, 2H); mass spec (M+H)+ 475; HPLC: 0.1% formic acid in H20:MeCN
(60:40), 2.54 min, >99%.
Triethyl~rnine (0.020 mL, 0.15 mmol) was added to a solution of active ester
2 0 (28) (6 mg, 0.013 mmol), aminofluorescein compound (7 mg, 0.014 mmol) and
dry DMF (0.5 mL); the reaction mixture was stirred at room temperature for 20 h.Purification by preparative reversed phase C18 HPLC, eluting with 0.1% formic
acid in water:MeCN (45:55) afforded 4.5 mg (45%) of di-t-butyl ester tracer (31).
1H NMR (CD30D): d 7.97-7.94 (m, lH), 7.80-7.78 (m, lH), 7.08 (s, lH), 6.68-6.53 (m,
2 S 7H), 4.40 (s, 2H), 3.62 (s, 2H), 2.70 (t, J=7.5 Hz, 2H), 2.47 (t, J=8.1 Hz, 2H), 2.20-2.16
(m, 2H3, 1.60-1.20 (m, 8H), 1.42 (s, 9H), 1.40 (s, 9H); mass spec (M+H)+ 810; HPLC:
0.1% formic acid in water:MeCN (45:55), 6.27 min, 98%.
Trifluoroacetic acid (1.0 mL) was added to a 5~C mixture of di-t-butyl ester
(31) (4 mg, 0.005 mmol) in dry CH2Cl2 (1.0 mL) and stirred at room temperature
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28
for 1.5 h. The mixture was purified by preparative reversed phase Clg HPLC,
eluting with 0.1% formic acid in water:MeCN (60:40) to afford 2 mg (59%) of 2-
acyl analogue of PBG tracer (32). 1H NMR (CD30D): d 7.90 (d, J=7.5 Hz, lH), 7.60-
7.55 (m, lH), 7.07 (s, lH), 6.69-6.50 (m, 7H), 4.42 (s, lH), 4.39 (s, lH), 3.53 (s, 2H),
2.78 (t, J=7.5 Hz, 2H), 2.53 (t, J=7.8 Hz, 2H), 2.17 (t, J=6.9 Hz, 2H), 1.56-1.24 (m, 8H);
mass spec (M+H)~ 698; HPLC: 0.1% formic acid in water: MeCN (60:40), 3.46 min,
99%.
FX ~P~.F.7
1 0 SYNTHESIS OF PORPHOBILINOGEN TRACER (33)
EDAC (19.2 mg, 0.10 mmol) was added to a solution of N1-sulfonyl
compound (11) (20 mg, 0.034 mmol), HOSu (19.6 mg, 0.17 mmol) and 1 mL DMF
then stirred under nitrogen for 14 hours. The reaction mixture was poured into
l 5 50 mL Et2O, washed 1 x 30 mL phosphate buffer (500 mM, pH=6.0) and removed
solvents in v~cuo to afford 22.8 mg (99%) of desired active ester (12). lH NMR
(CDCl3) d 6.88 (s, lH), 5.32 (br s, lH), 4.38 (d, J=5.9 Hz, 2H), 3.53 (s, 2 H), 3.40 (t,
J=7.2 Hz, 2H), 2.83 (s, 4H), 2.74 (t, J=7.2 Hz, 2H), 2.63 (t, J=7.2 Hz, 2H), 2.20-2.08 (m,
4H), i.43 (sl br s, 27 H); mass spec (M+H)+ 686.
2 0 5-Aminomethylfluorescein (25 mg, 0.048 mmol) was added to a solution of
active ester ~12) (22.7 mg, 0.033 mmol), diiopropylethylamine (44 mL, 0.25
mmol) and 1 mL DMF, the reaction was stirred for 18 hours and solvents
removed in vaCuo. Preparative HPLC purification gave 14.3 mg (46%) of
desired protected tracer (33). lH NMR (CDCl3) d 7.88-7.80 (m, lH), 7.52-7.40 (m,2 5 lH), 7.05-6.92 (m, lH), 6.87 (s, 2H), 6.70-6.30 (m, 4H), 5.40 (br s, lH), 4.52-4.28 (m,
4H), 4.11~.02 (m, 2H), 3.65-3.52 (m, 2H), 3.35-3.20 (m, 2H), 2.65-2.52 (m, 2H), 2.51-
2.42 (m, 4H), 2.10-1.95 (m, 2H), 1.43 (sl br s, 27~I); mass spec (M)+ 932.
Trifluoroacetic acid (1 mL) was added to a suspension of protected tracer (33)
(14 mg, 0.015 mmol) in 1 mL methylene chloride, stirred 30 minutes at room
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29
temperature and solvents removed in vacuo to afford an orange residue.
Preparative HPLC purification gave 3.1 mg (28%) of desired protected tracer (34).
lH NMR (CD30D + 5 drops CDC13) d 8.03 (s, lH), 7.76 (d, J=8.Q Hz, lH), 7.26 (d,
J=8.0 Hz, 2H), 7.14 (s, lH), 6.97 (s, 2H), 6.95 (d, J=8.9 Hz, 2H), 6.80 (d, J=8.9 Hz, 2H),
4.55 (s, 2H), 4.36 (s, 2H), 3.63 (s, 2H), 3.60-3.46 (m, 2H), 2.71 (t, J=7.1 Hz, 2H), 2.55 (t,
J=7.1 Hz, 2H), 2.01 (t, J=7.2 Hz, 2H); mass spec (M+H)+ 720.
FXAMPLE 8
SYNTHESIS OF PORPHOBILINOGEN TRACER (36)
l O
5-Aminomethylfluorescein (50 mg, 0.085 mmol) was added to a solution of
active ester (17) (24.3 mg, 0.042 mmol), diiopropylethylamine (73 mL, 0.42
mmol) and 0.80 mL DMF, the reaction was stirred for 3.5 days at 50~C and
solvents removed in vacuo. Preparative HPLC purification gave 23 mg (66%~ of
1 5 desired protected tracer (35) 1H NMR (CDCl3 + 2 drops CD30D) d 7.99 (s, lH),7.71 (d, J=6.1 Hz, 2H), 7.12 (d, J=6.1 Hz, 2H), 6.68 (s, 1~), 4.74 (br s, 2H), 4.23 (br s,
2H), 3.35 (s, 2H), 3.00-2.92 (m, 2H), 2.62 (t, J=7.1 Hz, 2H), 1.47 (sl br s, 18 H), 1.38 (s,
9H); mass spec (M)+ 826.
Trifluoroacetic acid (3.5 mL) was added to a suspension of protected tracer
(35) (23 mg, 0.028 mmol) in 3.5 mL methylene chloride, stirred 30 minutes at
room temperature, added 5 mT toluene and solvents removed in ~acuo to
afford an orange residue. Preparative HPLC purification afforded 7.8 mg (45%)
of desired protected tracer (36); mass spec (M)+ 613.
2 5 FXAMPI.F g
A~ll~i~.l~A PRODUCTION
Imrnunogens of Formulas II, III and IV were used for antisera production.
Each of the immunogens was used for immuni2:ation in a separate group of
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rabbits and a separate group of sheep. The rabbits were initially immunized
with 0.5 mg of immunogen and subsequently boosted with 0.25 mg of the
immunogen every 6 weeks while the sheep were initially immunized with 1
mg of immunogen and subsequently boosted with 0.5 mg of the immunogen
5 every 6 weeks. l~nimAl~ were bled at 2 weeks and the bleeds were titrated to
select antisera collections demonstrating adequate binding and displacement at
a reasonable dilution. Each bleed obtained from all ~nimAl~ were tested for
binding of tracer using all tracers synthesized. Testing was performed on the
Abbott IMx(g) analyzer, different dilution's of the antisera in FPIA buffer werel 0 combined with the tracers in a 1 mL final volume for 5-30 minutes. Only
antibodies derived from immunogens (7) and (14) showed significant binding
with selected tracers. A typical pool for measuring the levels of lead via the
quantification of porphobilinogen is diluted 1 to 100, has a binding of about 180
millipolarization units (mP) and a displacement of about 65 mP's with a lead
l 5 solution containing 800 mg lead per milliliter. Figure 10 shows a bar graph of
polarization vs. selected antibodies (derived from immunogens of present
invention)/selected tracers. Each bar represents the polarization obtained at a
1/100 antisera dilution when combined with the matching tracer. A working
calibration curve is demonstrated using tracer (21) and antibodies derived from
2 0 immunogen (7) and using tracer (34) and antibodies derived from immlmogen
(14) as shown in Figure 9.
~AMPLE 10
SAMPLE PRETREATMENT FOR QUA~TIFICATION OF PORPHOBILINOGEN
2 5
A pretreatment solution (200 mL), consisting of 9% trichloroacetic acid, 0.6
N HNO3 and 5 mM periodic acid, was added to 200 mL of test sample in a 1.5
mL microfuge tube which was capped and vortexed for 30 seconds.
Centrifugation at 10,000 rpm for 2 minutes afforded a translucent supernatant
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which was used without further modification in the assay described in Example
11.
F~Al~PT.R 11
S LEAD STANDARD CUl~V~
A gravimetric solution of lead (Pb) traceable to NIST purchased from GFS
Chemicals was diluted to 80, 60, 40 and 20 mg/dl in water that had been adjustedto pH 0.5 with HNO3. This was neutralized to p~I 7.1 with a pH 7.85
l 0 neutralizing buffer consisting of 1.25 M MOPS, 1.25 M HEPES, 30 mM
hydroxyquinoline-5-sulfonic acid and 0.1% Neomycin Sulfate. The enzyme
aminolevulinic acid dehydratase and rabbit antisera solution were diluted into
a buffer consisting of 0.25 M MOPS, 0.9 M ammonium sulfate, 0.5%
polyethylene glycol 8K and 0.1% Neomycin Sulfate pH 7Ø The substrate
l 5 consisted of 50 mM aminolevulinic acid, 25 mM tris(2-carboxyethyl)-phosphinehydrochloride and 250 rnM zinc chloride. The fluorescent tracer was diluted in
Abbott IMx~ FPIA buffer to approximately 13 nM.
The ass~y was run in the Abbott IMx(~ analyzer using the following steps.
A blank read was taken on the empty cuvette. The lead (Pb) standard (150 ml)
2 0 was neutralized with 9Q ml of neutralizing buffer and 50 ml of Abbott IMx~)
FPIA buffer. A portion (140 ml) of this mixture was combined with 90 ml of the
enzyme/antisera solution and 95 ml of Abbott IMx@~ FPIA buffer then incubated
for 6.25 minutes in the cuvette. The substrate (90 ml) and 150 m~ of Abbott
IMx(g) FPIA buffer were added to the cuvette and incubated for 20 minutes
2 5 followed by the addition of fluorescent tracer (220 ml) and 375 ml of FPIA buffer
and a final incubation of 6.25 minutes. ~ final polarization read was measured
on the cuvette solution. Lead standards of 0, 20, 40, 60 and 80 mg/dl were run in
replicates of 2 or 4 (See Figure 9).
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W O 97/24621 32 PCTnUS96/19544
It is expected that the compounds and methods described herein will be
readily adapated to all sorts of automated immunoassay formats, where speed,
cost, convenience and ease of use, as well as accuracy, precision and reliability
are important.
While the invention has been described in each of its various
embodiments, it is expected that certain modifications thereto may be made by
those skilled in the art without departing from the true spirit and scope of theinvention as described in the specification and further set forth in the
10 accompanying claims.
