U.S. patent application number 08/933655 was filed with the patent office on 2002-02-21 for immunological advanced glycation endproduct crosslink.
This patent application is currently assigned to Picower Institute For Medical Research. Invention is credited to AL-ABED, YOUSEF, BUCALA, RICHARD J..
Application Number | 20020022234 08/933655 |
Document ID | / |
Family ID | 25464310 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022234 |
Kind Code |
A1 |
AL-ABED, YOUSEF ; et
al. |
February 21, 2002 |
IMMUNOLOGICAL ADVANCED GLYCATION ENDPRODUCT CROSSLINK
Abstract
There is disclosed a means for standardizing a kit that provides
a means for measuring the formation of advanced glycosylation
endproducts (AGEs). The present invention further provides a novel
isolate AGE that is antigenic and useful for forming antibodies
having utility in diagnostic assays and for standardizing
diagnostic assays.
Inventors: |
AL-ABED, YOUSEF; (NEW YORK,
NY) ; BUCALA, RICHARD J.; (COS COB, CT) |
Correspondence
Address: |
PIPER MARBURY RUDNICK AND WOLFE LLP,
1200 NINETEENTH STREET, N.W.
WASHINGTON
DC
20036-2412
US
|
Assignee: |
Picower Institute For Medical
Research
|
Family ID: |
25464310 |
Appl. No.: |
08/933655 |
Filed: |
September 19, 1997 |
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 33/6842 20130101;
G01N 33/6893 20130101; G01N 33/68 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Claims
We claim:
1. A condensation product advanced glycation endproduct (AGE)
comprising a lysine component, an arginine component and a reducing
sugar component, with the proviso that the AGE is not pentosidine
or cyclic pentosidine.
2. The condensation product AGE of claim 1 wherein the condensation
product is an AGE according to formula I: 5wherein the lysine
component is indicated by the box labeled "K"; the arginine
component is indicated by the box labeled "R"; and the reducing
sugar component is not boxed; and wherein R.sub.1 and R.sub.4 are
independently H or an amide bond to an amino acid residue or a
peptide chain; R.sub.2 and R.sub.3 are, independently, OH or an
amide bond to an amino acid residue or a peptide chain; R.sub.5 is
H, CH.sub.2OH or CHOHCH.sub.2OH; and wherein if more than one of
R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is an amide bond, then the
lysine "K" component and the arginine "R" component may be amino
acid residues of the same or a different peptide chain.
3. The condensation product AGE of claim 2 wherein the condensation
product is an ALI having the structure: 6wherein Z is the remainder
of the polypeptide linked to the Arg and Lys groups.
4. A method for increasing macrophage recognition and elimination
of advanced glycosylation endproducts, comprising administering to
a mammal a therapeutic amount of a condensation product according
to formula I.
5. The method of claim 4, wherein the condensation product is a
compound according to formula II.
6. A method for standardizing an antibody-based diagnostic assay
that measures AGE levels in a sample, comprising adding a defined
amount of an AGE condensation product according to claim 1 to bind
the antibody.
7. The method of claim 6 wherein the condensation product is an AGE
compound according to formula I.
8. The method of claim 6 wherein the condensation product is a
compound according to formula II.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention provides an advanced glycation
endproduct (AGE) crosslink that exhibits immunological
crossreactivity with in vivo AGEs.
BACKGROUND OF THE INVENTION
[0002] The glycation reaction is manifest by the appearance of
brown pigments during the cooking of food, identified by Maillard
in 1912. Maillard observed that glucose or other reducing sugars
react with amino-containing compounds, including amino acids and
peptides, to form adducts that under go a series of dehydrations
and rearrangements to form stable brown pigments. Heat-treated
foods undergo non-enzymatic browning as a result of a reaction
between glucose and a polypeptide chain. Thus, pigments responsible
for the development of brown color that develops as a result of
protein glycosylation possessed characteristic spectra and
fluorescent properties.
[0003] Subsequent reactions (including various dehydrations,
oxidations, eliminations, condensations, cleavages and other
chemical changes) occur to produce a vast array of "early" and
"late" glycation adducts. More advanced glycation adducts are
sometimes described as a class of yellow-brown, fluorescent
pigments with intra- and intermolecular crosslinking activity.
Specific glycation entities are thought to occur at low abundance
within a widely divergent pool of advanced glycation endproducts
(or AGEs). Despite significant research activity, the molecular
structures of only a few of the later glycation adducts and
products have been determined. Moreover, the contribution of these
identified, in vivo-formed advanced glycation structures to
biological processes is poorly understood. Therefore, there is a
need in the art to identify AGEs and determine their biological
properties.
[0004] The process of advanced glycation leads from the reversible
interaction of reducing sugars with amino groups to the formation
of more complex, irreversibly-bound structures with varied spectral
and covalent cross-linking properties. These later products, termed
advanced glycation endproducts or AGEs, form in vivo by chemical
principles first described for the Maillard reaction (Ledl and
Schleicher, Angew. Chem. Int. Ed. Engl. 29:565, 1990; and Maillard,
C. R. Hebd. Seances Acad. Sci. 154:66, 1912). The potential
significance of Maillard-type reactions in living systems however,
has been appreciated only over the last 15 years and the term
advanced glycation has come to refer specifically with those
aspects of Maillard chemistry that involve macromolecules and which
occur under physiological conditions. It is evident that AGEs form
in living tissues under a variety of circumstances, and that they
play an important role in protein turnover, tissue remodeling, and
the pathological sequelae of diabetes and aging (Bucala and Cerami,
Adv. Pharm. 23:1, 1992).
[0005] The initial event in protein glycation is the reaction of a
reducing sugar such as glucose with the N-terminus of a protein or
the .epsilon.-amino group of a lysine to form an aldimine, or
Schiff base. The Schiff base can hydrolyze back to its reactants or
undergo an Amadori rearrangement to form a more stable
N.sup..epsilon.-(1-deoxy-1-fructosyl) lysine (Amadori product, AP).
The reaction pathway leading to reactive crosslinking moieties
(i.e. AGE formation) commences by further rearrangement or
degradation of the AP. Possible routes leading from AP precursors
to glucose-derived, protein crosslinks has been suggested only by
model studies examining the fate of the AP in vitro. One pathway
proceeds by loss of the 4-hydroxyl group of the AP by dehydration
to give a 1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione). An
AP-dione with the structure of an amino-1,4-dideoxyosone has been
isolated by trapping model APs with aminoguanidine, an inhibitor of
the AGE formation (Chen and Cerami, J. Carbohydrate Chem. 12:731,
1993). Subsequent elimination of 5-hydroxy then gives a
1,4,5-trideoxy-1-alkylamino-2,3-hexulos-4-ene (AP-ene-dione), which
has been isolated as a triacetyl derivative of its 1,2-enol form
(Estendorfer et al., Angew. Chem. Ent. Ed. Engl. 29:536, 1990).
Both AP-diones and AP-ene-diones would be expected to be highly
reactive toward protein crosslinking reactions, for example, by
serving as targets for the addition of a guanidine moiety from
arginine or an .epsilon.-amino group from lysine.
[0006] Dicarbonyl containing compounds, such as methylglyoxal,
glyoxal and deoxyglucosones, participate in condensation reactions
with the side chains of arginine and lysine. For example, the
addition of methylglyoxal to the guanidine moiety of arginine leads
to the formation of imidazol-4-one adducts (Lo et al., J. Biol.
Chem. 269:32299, 1994) and pyrimidinium adducts (Al-Abed et al.,
Bioorg. Med. Chem. Lett. 6:1577, 1996). In one study, Sell and
Monnier isolated pentosidine, an AGE fluorescent crosslink which is
a condensation product of lysine, arginine, and a reducing sugar
precursor (Sell and Monnier, J. Biol Chem. 264:21597, 1989) from
human dura collagen. The mechanism of pentosidine formation remains
uncertain but crosslinking requires that the lysine-bound,
glucose-derived intermediate contain a dicarbonyl functionality
that can react irreversibly with the guanidinium group of
arginine.
[0007] Several lines of evidence have established that AGEs exist
in living tissue (Bucala and Cerami, Adv. Pharm. 23:1, 1992), yet
the identity of the major AGE crosslink(s) that forms in vivo
remains uncertain. Recent pharmacologically-based data nevertheless
have affirmed the importance of the AP-dione pathway in stable
crosslink formation (Vasan et al., Nature 382:275, 1996). The lack
of precise data concerning the structure of AGEs has been
attributed to the lability of AGE crosslinks to the standard
hydrolysis methods employed to remove the protein backbone, and to
the possible structural heterogeneity of the crosslinks themselves.
Moreover, there is data to suggest that the pathologically-relevant
crosslinks may not themselves be fluorescent (Dyer et al. J. Clin.
Invest. 91:2463, 1993), a property that has been historically
associated with AGE formation and almost universally used as an
indicator of the Maillard reaction in vivo.
[0008] Hyperimmunization techniques directed against an
AGE-crosslinked antigen produced both polyclonal and monoclonal
antibodies that recognize in vivo formed AGEs (Makita et al., J.
Biol. Chem. 267:1997, 1992). These antibodies made possible the
development of immunohistochemical and ELISA-based technologies
that were free of specificity and other technical problems
associated with prior fluorescence-based assays, and provided the
first sensitive and quantitative assessment of advanced glycation
in living systems. These anti-AGE antibodies were found to
recognize a class of AGEs that was prevalent in vivo but
immunochemically distinct from previously characterized structures
such as FFI, pentosidine, pyrraline, CML, or AFGP (Makita et al.,
J. Biol. Chem. 267:1992, 1992). The specific AGE epitope recognized
by these antibodies increased as a consequence of diabetes or
protein age on various proteins such as collagen, hemoglobin, and
LDL (Makita et al., J. Biol. Chem. 267:1997, 1992; Makita et al.,
Science 258:651, 1992; Wolffenbuttel et al., The Lancet 347, 513,
1996; and Bucala et al. Proc. Natl. Acad. Sci. U.S.A. 91:9441,
1994). One particular polyclonal antibody species, designated "RU",
has been employed in an ELISA assay tested in human clinical
trials. Immunoreactive AGEs were found to be inhibited from forming
by administration of the pharmacological inhibitor, aminoguanidine
(Makita et al., Science 258:651, 1992; and Bucala et al. Proc.
Natl. Acad. Sci. U.S.A. 91:9441, 1994), and to provide important
prognostic information correlated to diabetic renal disease
(Beisswenger et al. Diabetes 44:824, 1995).
[0009] Despite the increasing body of data implicating the advanced
glycation pathway in the etiology of such age- and diabetes-related
conditions as atherosclerosis, renal insufficiency, and amyloid
deposition, elucidation of the structure(s) of the pathologically
important, AGE-crosslinks that form in vivo has been a challenging
problem. Investigations of AGEs that form in vivo have necessarily
relied on chemical methods to purify the crosslinking moieties away
from their macromolecular backbones. These studies have led to a
recognition that the major crosslinks which form in vivo are
largely acid-labile and non-fluorescent (Bucala and Cerami, Adv.
Pharm., 23:1, 1992; Sell and Monnier, J. Biol. Chem.
264:21597,1989; and Dyer et al. J. Clin. Invest. 91:2463, 1993).
However, in view of a predictive antibody-based (ELISA) diagnostic
assay, there is a need in the art to isolate and identify
immunogenic AGEs that can be used to both standardize and improve
such diagnostic assays. The present invention was made in an effort
to achieve the foregoing goals. Further, there is a need in the art
to measure formation of advanced glycosylation endproducts in all
applications where protein aging is a serious detriment. This
includes, for example, the area of food technology (i.e.,
determination of the amount of food spoilage), perishability or
shelf-life determination of proteins and other amino-containing
biomolecules.
SUMMARY OF THE INVENTION
[0010] The present invention provides a means for standardizing a
kit that provides a means for measuring the formation of AGEs as a
diagnostic assay. The present invention further provides a novel
isolate AGE that is antigenic and useful for forming antibodies
having utility in diagnostic assays and for standardizing
diagnostic assays.
[0011] The invention provides a condensation product advanced
glycation endproduct (AGE) comprising a lysine component, an
arginine component and a reducing sugar component. Preferably, the
condensation product is an AGE according to formula I: 1
[0012] wherein the lysine component is indicated by the box labeled
"K"; the arginine component is indicated by the box labeled "R";
and the reducing sugar component is not boxed; and wherein R.sub.1
and R.sub.4 are independently H or an amide bond to an amino acid
residue or a peptide chain; R.sub.2 and R.sub.3 are, independently,
OH or an amide bond to an amino acid residue or a peptide chain;
R.sub.5 is H, CH.sub.2OH or CHOHCH.sub.2OH; and wherein if more
than one of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is an amide bond,
then the lysine "K" component and the arginine "R" component may be
amino acid residues of the same or a different peptide chain. Most
preferably, the condensation product is an ALI having the
structure: 2
[0013] wherein Z is H, carboxybenzoyl, or the remainder of the
polypeptide linked to the Arg and Lys groups; and Y is OH or the
remainder of the polypeptide.
[0014] The present invention further provides a method for
increasing macrophage recognition and elimination of advanced
glycosylation endproducts, comprising administering to a mammal a
therapeutic amount of a compound of formula I.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the dose-dependent activity of synthetic ALI in
an anti-AGE antibody-based competitive ELISA to measure AGE
content. The binding curve is steep and shows 50% inhibition at 500
nmoles. For comparison purposes, the reactivity of FFI, CML, AFGP,
pentosidine and cyclic pentosidine, and ligands with related
epitope structure, such as N.sup..alpha.-Z-arg-lys,
N.sup..alpha.-Z-arg-lys-AP, an imidazolium adduct, a pyrimidinium
adduct, histidine, and lys-his, were studied. ELISA competition
curves for the polyclonal anti-AGE antibody "RU" followed the
methods described by Makita et al. (J. Biol. Chem. 267:1992, 1992).
Assays employed glucose-derived, AGE-BSA as the absorbed antigen.
FFI: 4-furanyl-2-furoyl-1H-imidazole (Ponger et al,. Proc. Natl.
Acad. Sci. U. S. A. 81:2684, 1984), CML: carboxy-methyllysine
(Ahmed et al., J. Biol. Chem. 261:4889, 1986), AFGP:
1-alkyl-2-formyl-3,4-diglycosylpyrrole (Farmar et al., J. Org.
Chem. 53:2346, 1988), PY: pyrraline (Njoroge et al., Carbohydrate
Res. 167:211, 1987), P: pentosidine (Sell and Monnier, J. Biol.
Chem. 264:21597, 1989), CP: cyclic pentosidine (CP) (Al-Abed et
al., Bioorg. Med. Chem. Lett. 5:2929, 1995), AL:
N.sup..alpha.-Z-arg-lys (AL), ALAP: N.sup..alpha.-Z-arg-lys-AP, H:
histidine, LH: lys-his, PA: pyrimidinium adduct (Al-Abed et al.,
Bioorg. Med. Chem. Lett. 6:1577, 1996), and IA: imidazolium adduct
(Brinkmann et al., J. Chem. Soc. Perkin Trans. I:2817, 1995) showed
no detectable crossreactivity with the RU anti-AGE antibody. With
the exception of ALI, none of these compounds showed detectable
crossreactivity with anti-AGE antibodies shown previously to react
with in vivo-formed AGEs.
[0016] FIG. 2 shows a schematic to form the novel 2-amino-4,5
dihydroxyimidazol adduct as an intermediate and then the ALI AGE
product through a dehydration reaction.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention was made by exploiting the specificity
of anti-AGE antibodies reactive with in vivo-formed AGEs to
identify novel crosslinking moieties contained within a synthetic
mixture of AGEs. This selection method found a single,
immunoreactive AGE that formed in 0.6% yield in a synthetic mixture
consisting of glucose and a N.sup..alpha.-blocked dipeptide,
N.sup..alpha.-Z-arg-lys, as reactants. ALI was highly reactive with
the anti-AGE antibody "RU" and showed a steep binding curve at
nmole amounts.
[0018] The present invention found a novel ALI AGE based upon
intramolecular crosslinking of adjacent residues for the formation
of pentosidine-type AGEs, using a lys-arg-type dipeptide. In
particular, the present invention provides a means for
standardizing a kit that provides a means for measuring the
formation of AGEs as a diagnostic assay. The present invention
further provides a novel isolate AGE that is antigenic and useful
for forming antibodies having utility in diagnostic assays and for
standardizing diagnostic assays.
[0019] The invention provides a condensation product advanced
glycation endproduct (AGE) comprising a lysine component, an
arginine component and a reducing sugar component. Preferably. the
condensation product is an AGE according to formula I: 3
[0020] wherein the lysine component is indicated by the box labeled
"K"; the arginine component is indicated by the box labeled "R";
and the reducing sugar component is not boxed; and wherein R.sub.1
and R.sub.4 are independently H or an amide bond to an amino acid
residue or a peptide chain; R.sub.2 and R.sub.3 are, independently,
OH or an amide bond to an amino acid residue or a peptide chain;
R.sub.5 is H, CH.sub.2OH or CHOHCH.sub.2OH; and wherein if more
than one of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is an amide bond,
then the lysine "K" component and the arginine "R" component may be
amino acid residues of the same or a different peptide chain. Most
preferably, the condensation product is an ALI having the
structure: 4
[0021] wherein Z is H, carboxybenzoyl, or the remainder of the
polypeptide linked to the Arg and Lys groups; and Y is OH or the
remainder of the polypeptide. The compounds of formula I were
prepared by incubating one or more polypeptide containing arg and
lys with a reducing sugar, such as ribose, glucose, fructose,
ascorbate or dehydroascorbate, at physiological pH for periods of
10-300 hours and optionally at elevated temperatures for shorter
periods of time. A preferred compound, such as ALI, is prepared by
incubating a N.sup..alpha.-blocked dipeptide (e.g.,
N.sup..alpha.-Z-arg-lys) containing peptide (e.g.,
N.sup..alpha.-CBZ-arg-lys) with a reducing sugar, such as ribose,
glucose, fructose, ascorbate or dehydroascorbate, at physiological
pH for periods of 10-300 hours and optionally at elevated
temperatures for shorter periods of time. A preferred
amine-protecting group is CBZ (carboxybenzyl) group due to its ease
of removal and retention of stereochemistry during manipulations.
The formed AGEs are purified, for example, by HPLC to provide the
AGEs of formula I or formula IIA.
[0022] Among the biological activities of AGEs, the formation of
stable crosslinks may be considered their most important
pathological manifestation. The imidazole-based AGE of formula I,
is a major species of the pathologically-important AGE crosslinks
that form in vivo. The mechanism of formation of ALI points to the
importance of the AP-dione as a critical, reactive intermediate and
further affirms prior, pharmacologically-based studies that have
implicated this intermediate, as well as its dehydration product,
the AP-ene-dione in irreversible, protein-protein crosslinking
(Vasan et al., Nature 382:275, 1996). These intermediates also have
been implicated in the formation of the cyclization product
cypentodine (Zhang and Ulrich, Tetrahedron Lett. 37:4667, 1996),
which may display sufficient redox potential to participate in the
oxidative reactions associated with phospholipid advanced glycation
(Bucala et al., Proc. Natl. Acad. Sci. U. S. A. 90:6434, 1993; and
Bucala, Redox Reports 2, 291, 1996).
[0023] There are several possible in vivo synthetic routes leading
from Amadori product precursors to glucose-derived protein
crosslinks. Applicants have examined models examining the fate of
the Amadori products in vitro. For instance, the Amadori product
can undergo dehydration to give
1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione) (FIG. 2). The
dipeptide N.sup..alpha.-Z-arg-lys was used as the AGE target. The
proximity of arginine and lysine residues to each other promoted
stable intramolecular crosslink formation. A
N.sup..alpha.-Z-arg-lys dipeptide was incubated with 10 equivalents
of glucose in 0.2 M phosphate buffer (pH 7.4) at 37.degree. C. for
five weeks. This reaction mixture produced at least 25 distinct
reaction products after fractionation of this mixture by HPLC. Each
fraction was isolated, concentrated, and analyzed for its
reactivity with a polyclonal anti-AGE antibody (RU) that has been
shown previously to recognize a class of AGEs that increase in vivo
as a consequence of hyperglycemia, and which are inhibited from
forming in human subjects by treatment with the advanced glycation
inhibitor aminoguanidine. The products present within one fraction
(1.5% yield) were found to block antibody binding in a competitive
ELISA assay for AGEs in a dose-dependent fashion. Therefore, the
compound of formula I is useful to standardize AGE-based diagnostic
assays as either a standard target or a standard competitor, or
both. Further purification of this fraction by HPLC revealed the
presence of one major (0.6% yield) immunoreactive compound.
Characterization of this adduct by UV, ESMS and .sup.1H-NMR spectra
revealed the presence of an intramolecular arg-lys-imidazole
crosslink (formula II). This crosslink is non-fluorescent and acid
labile and may represent an important class of immunoreactive
AGE-crosslinks that form in vivo.
EXAMPLE 1
[0024] This example illustrates a synthesis to prepare
Arg-Lys-Imidazole (ALI). To a solution of N.sup..alpha.-Z-arg-lys
(1 g, 0.013 mmol) in 10 ml of aqueous 0.2 M phosphate buffer (pH
7.4) was added D-glucose (0.13 mmol). The reaction mixture was
stirred at 37.degree. C. for five weeks. At intervals, 10 .mu.l of
the reaction mixture was analyzed by HPLC using an analytical
Primesphere column (5C18 MC, 5 micron, 250.times.4.6 mm,
Phenomenex, Torrance, Calif.) and a binary solvent gradient
consisting of 0.05% TFA in H.sub.2O (solvent A), and methanol
(solvent B). Solvent was delivered at a flow rate of 1 ml/min as
follows. From 0-30 min: a linear gradient from A:B (95:5) to A:B
(25:75); from 30-45 min: a linear gradient from A:B (25:75) to
(0:100). Detection was by monitoring UV absorption at .lambda. 214,
254, 280, 320, and 350 nm. At least 25 distinct reaction products
were identified upon fractionation of this mixture by HPLC. Larger
amounts of these products were fractionated using a similar HPLC
method (Primesphere column 5C18 MC, 5 micron, 250.times.21.2 mm,
Phenomenex, Torrance, Calif.). Solvent was delivered at a flow rate
of 10 mL/min using the same gradient as described above. The AGE
crosslink eluted as a mixture of three components at 34.0 min.
Further purification of this subfraction using the same method gave
the desired compound in a high purity (>95%).
EXAMPLE 2
[0025] This example illustrates the discovery and isolation of the
AGE of formula I. The dipeptide N.sup..alpha.-Z-arg-lys was
selected as a target, because a close association of the arginine
and lysine residues provides a significant proximity effect that
promotes crosslink formation. Moreover, a synthetic strategy
employing an arg-lys dipeptide was used successfully in the past to
isolate a cyclic pentosidine in a high yield (Beisswenger et al.,
Diabetes 44:824, 1995). N.sup..alpha.-Z-arg-lys (13 mmoles) was
incubated together with 10 equivalents of glucose in 0.2 M
phosphate buffer (pH 7.4) for five weeks at 37.degree. C. At least
25 distinct reaction products were identified upon fractionation of
this mixture by reverse-phase HPLC. Each fraction was isolated,
concentrated, and analyzed for its reactivity with anti-AGE
antibody by ELISA (Makita et al., J. Biol. Chem. 267:1992, 1992).
Briefly, HPLC fractions and purified compounds were analyzed by an
AGE-specific ELISA following methods described previously (Makita
et al., J. Biol. Chem. 267:1992, 1992). This ELISA employed a
polyclonal anti-AGE antibody raised by hyperimmunization against a
heavily AGE-crosslinked preparation of ribonuclease. Total IgG was
prepared by protein-G affinity chromatography and the ribonuclease
backbone specificities removed by immunoabsorption. For assaying
AGE immunoreactivity, 96-well round bottom microtitre plates
(EIA/RIA plate, Costar, Cambridge, MA) first were coated with
AGE-BSA (3 mg/ml, dissolved in 0.1 M sodium bicarbonate, pH 9.6) by
incubation overnight at 4.degree. C. After washing, the unbound
sites were blocked with SuperBlock.TM. following the manufacturer's
recommendations (Pierce, Rockford, Ill.). After washing, dilutions
of test antigen, together with anti-AGE IgG, were added and the
plates incubated at room temperature for 1 hr. The plates then were
washed again and incubated with a secondary antibody (alkaline
phosphatase-conjugated anti-rabbit IgG) at 37.degree. C. for 1 hr.
The unbound antibodies were removed by extensive washing and bound
antibodies were detected by incubation with p-nitrophenyl phosphate
(PNPP) substrate for 30-60 min, and recording the optical density
at 405 nM by an ELISA reader (EL309, Bio-Tek Instruments Inc.,
Burlington, Vt.). Results were expressed as B/B.sub.0, calculated
as [experimental OD-background OD (i.e. no antibody)]/[total OD
(i.e. no competitor)-background OD].
[0026] The product(s) contained within one distinct fraction,
present in 1.5% yield, were found to block antibody binding in a
dose-dependent fashion. Further purification of this fraction by
HPLC revealed the presence of one major, immunoreactive compound
(0.6% yield), together with two minor ones. The UV and fluorescence
spectrum of the isolated, major product was unremarkable and
similar to that of the starting material. The ESMS spectrum
displayed a molecular ion of m/z 545 [MH].sup.+, an increase of 108
daltons compared to the starting material, N.sup..alpha.-Z-arg-lys
(MW: 436). A .sup.1H-NMR spectrum in D.sub.2O showed, in addition
to the N.sup..alpha.-Z-arg-lys protons, five aliphatic protons that
resonate as a multiplet between 2.35-4.65 ppm (5H), and an olefinic
proton that resonates at 7.3 ppm within the Z-group. Overall, these
data are consistent with the structure of a cyclic, arg-lys
imidazole crosslink (ALI, formula II, FIG. 2).
EXAMPLE 3
[0027] This example illustrates a proposed mechanism of the AGE
formation (FIG. 2). First, dehydration of the lysine-derived AP
(which has been determined to form in 18% yield under these
reaction conditions) gives an obligate, AP-dione reactive
intermediate. Reversible addition of the guanidine moiety to the
dicarbonyl yields the 2-amino-4,5-dihydroxyimidaz- ole adduct,
which then undergoes dehydration to deliver the stable cyclic ALI.
Of importance, this crosslink is non-fluorescent, acid-labile, and
can be inhibited from forming by aminoguanidine.
* * * * *