Synthesis of immunoreagents for measurement of galactosylhydroxylysine

Article abstract of DOI:10.1016/S0957-4166(99)00331-6

(2S,5R)-(+)-Hydroxylysine (6) was transformed into (-)-succinimidyl ester (13) and conjugated to BSA or KLH to form immunogens (2 and 3) for generation of anti-galactosylhydroxylysine antibodies. Additionally, treatment of (-)-13 with 6-Fln-CH₂

Full text of DOI:10.1016/S0957-4166(99)00331-6

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3157–3165
Synthesis of immunoreagents for measurement of
galactosylhydroxylysine
Maciej Adamczyk ∗ and Rajarathnam E. Reddy
Division Organic Chemistry Research (9NM, AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 17 June 1999; accepted 20 July 1999
Abstract
(2S,5R)-(+)-Hydroxylysine (6) was transformed into (−)-succinimidyl ester (13) and conjugated to BSA or KLH
to form immunogens (2 and 3) for generation of anti-galactosylhydroxylysine antibodies. Additionally, treatment
of (−)-13 with 6-Fln-CH2NH2 (16) or acridinium derivative (17) and subsequent hydrolysis gave the fluorescent (4)
and chemiluminescent (5) tracers, respectively. These immunoreagents (3,4 and 5,6) are essential for development
of assays for diagnosis of osteoporosis. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Galactosylhydroxylysine (GHL 1, Fig. 1) is a structural component of bone collagen, which is formed
by post-translational glycosylation of hydroxylysine.1 The structure of GHL (1) was shown to have a
monosaccharide unit, D-galactopyranose, in which the anomeric center is β-linked at the (5R)-position
of L-lysine.2 During the process of bone resorption, GHL (1) is released into the serum and excreted in
urine.3 Thus, GHL (1) has attracted considerable attention for use as a biochemical marker for diagnosis
of osteoporosis and other metabolic bone diseases.4,5 A number of tedious methods were developed for its
measurement, for example using amino acid analyzer,2b,6 ion-exchange chromatography,7 HPLC,8 and
enzyme-linked immunosorbent assay.9 Development of sensitive and high throughput immunoassays,
such as fluorescent polarization immunoassy (FPIA) and chemiluminescent immunoassay (CLIA), for
GHL (1) is critically important for diagnosis and management of osteoporosis. In this paper, we
describe the synthesis of immunoreagents (immunogens 2,3 and tracers 4,5) starting from (2S,5R)-(+)-
hydroxylysine (6), which are essential for development of assays (FPIA and CLIA) for GHL (1).
∗
Corresponding author. E-mail: maciej.adamczyk@abbott.com
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00331-6

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Figure 1.
2. Results and discussion
In recent years there has been a tremendous surge in the development of new immunoassay te-
chnologies and innovative automated analyzers to meet the numerous challenges faced by the clinical
laboratories.10 The automated immunoassay analyzers developed by Abbott Laboratories, for example
TDx and AxSYM, utilize FPIA,11 whereas the recently launched ARCHITECT instrument is based on
CLIA12 technology. Both FPIA and CLIA methods require two key reagents, antibodies for binding and
a signal generating material (tracer) for detection. Since the low molecular weight analytes (e.g. GHL, 1)
do not, as such, induce the formation of antibodies, they need to be conjugated to a carrier protein, thus
forming an immunogen.13 These immunogens are then used for inoculation of an animal (for example
mouse, hamster, rabbit, sheep) for production of antibodies. Thus, the first phase in the development of
immunoassay is design and synthesis of immunogens and tracers.
There are a number of important issues which need to be considered when preparing the immunogens,
such as: site of the analyte modification, selection of carrier protein, selection of linking arm, method of
conjugation and the hapten density.14 More importantly, the chirality of optically active analytes (e.g.
GHL, 1), need to be preserved in preparing the immunoreagents. In order to allow the host animal
immune system to recognize both chiral centers in the backbone of GHL (1), we envisioned introducing
aminohexanoic acid as a spacer between GHL (1) and carrier protein. This is important, since in the
case of thyroxine (T4),15 we observed that the anti-thyroxine antibodies, which were raised from an
immunogen prepared by direct conjugation of the carboxylic acid of T4 to carrier protein, were blind
to the chiral center. Bovine serum albumin (BSA, molecular weight: 66 430 Da) and keyhole limphet
hemocyanin (KLH, molecular weight: 450–13 000 kDa) are the two commonly used carrier proteins
for the preparation of immunogens. Among these, BSA is well suited as carrier protein because of its
solubility in various aqueous buffers and high content of primary amines (59 lysines and a terminal
amine) available for conjugation with hapten. Although the immunogens prepared using KLH as carrier
protein are difficult to characterize due to the poor solubility and variable mass, they are also frequently
used for production of antibodies. Since the antibodies produced for carrier protein are also present in the
host animal bleeds, an alternative hapten-carrier protein conjugate such as KLH-immunogen, is required
to select the anti-analyte antibodies. Therefore, the key hapten, (−)-acid (12), a modified GHL (1) with
linking arm, was envisioned from (2S,5R)-(+)-hydroxylysine (6).
Thus, (2S,5R)-(+)-6 was treated (Scheme 1) with (Boc)2O in 10% aq. NaHCO3 and THF followed
by refluxing the resulting crude bis-Boc-acid in benzene for 2 h, to produce the (3S,6R)-(−)-lactone
(7) in 94% yield. Opening of the (−)-lactone (7) with linking arm, methyl-6-aminohexanoate (8) in THF
afforded the (2S,5R)-(−)-ester (9) in 78% yield. The next step in the synthesis of hapten (12) was to install
the galactosyl unit from the hydroxyl group of (−)-9. Therefore, (−)-9 was treated with 2.0 equiv. of (+)-
acetobromo-α-D-galactose (10) and 2.2 equiv. of mercury(II) cyanide16 in toluene at 50°C for 2.5 h. The
product was purified by preparative reverse phase HPLC and lyophilized to give the glycosylated product
(−)-11 in >99% purity, but in low yield (13%). Our attempts to improve the yield for a glycosylation step

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3159
by changing the ratios of reagents and reaction conditions were unsuccessful. Finally, alkaline hydrolysis
of (−)-11 using LiOH in THF–H2O followed by HPLC purification afforded the key hapten, (−)-acid
(12), in 63% yield.
Scheme 1.
Since the in situ activation and coupling of acid hapten [for example (−)-12] to the carrier protein
using reagents such as EDAC, can lead to the undesired modification of carrier protein, we found that it
is critical to use pure activated ester [for example (−)-succinimidyl ester (13)] for preparation of the
immunogen. Therefore, the (−)-acid (12) was converted to the corresponding (−)-succinimidyl ester
(13) using N-hydroxysuccinimide and EDAC in DMF, in 65% yield and >98% purity. Conjugation
of the activated ester (−)-13 with carrier protein, BSA, was carried out in DMF and buffer (pH: 8.0)
at room temperature. After 15 h, the crude reaction mixture was purified by dialysis to remove the
hydrolyzed hapten (12) and other low molecular weight impurities. Lyophilization of the product gave an
immunogen precursor (14), in which the Boc protected amino groups of the hapten were unmasked using
trifluoroacetic acid in CH2Cl2. Immunogen (2) was purified by dialysis and isolated as a white powder
after lyophilization. Analysis of the immunogen (2) by gel electrophoresis17 showed a single narrow
band indicating its homogeneity and the absence of free BSA. The resolution of gel electrophoresis is
inadequate to determine the hapten density. Therefore, the molecular weight of the immunogen (2) was
determined by MALDI18 and found to be 77 465, which represents an average of 16 haptens incorporated
per BSA. This substitution rate (26%) was consistent with 16 haptens per BSA, as observed for its
precursor 14. The immunogen (3) was prepared from (−)-13 and KLH by following the procedure
developed for immunogen (2).

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In order to allow the tracer to mimic overall topology of the hapten, which was used to prepare the
immunogen, the fluorescent and chemiluminescent labels were directly attached to hapten (−)-12. Thus,
the active ester (−)-13 was treated (Scheme 2) with 6-aminomethylfluorescein (6-Fln-CH2NH2, 16) in
DMF in the presence of triethylamine at room temperature to afford 18 in 69% yield. Hydrolysis of the
Boc groups in 18 using trifluoroacetic acid in CH2Cl2 at room temperature followed by HPLC purification
afforded the fluorescent tracer (4) in 84% yield and >99% purity as a yellow powder. Similarly, the
active ester (−)-13 was treated with acridinium derivative (Acr-NH2, 17)19 and triethylamine in DMF
at room temperature to afford 19 in 72% yield after purification by preparative reverse phase HPLC.
Finally, hydrolysis of 19 using trifluoroacetic acid in CH2Cl2 followed by HPLC purification afforded
the chemiluminescent tracer (5) in 77% yield and >99% purity as a yellow powder.
Scheme 2.
In summary, two immunogens (2,3) were prepared via conjugation of (−)-succinimidyl ester (13) to
the carrier protein (BSA or KLH) followed by hydrolysis, for generation of anti-galactosylhydroxylysine
antibodies. The key (−)-succinimidyl ester (13) was synthesized from a commercially available (2S,5R)-
(+)-hydroxylysine (6) in five steps. Additionally, treatment of (−)-13 with 6-aminofluorescein (16) or
acridinium derivative (17) followed by hydrolysis gave the fluorescent (4) and chemiluminescent (5)
tracers, respectively, which are essential for development of assays for diagnosis of osteoporosis.
3. Experimental
3.1. General methods and materials
1H and 13C NMR spectra were recorded on a Varian Gemini spectrometer (300 MHz), the chemical
shifts (δ) were reported in ppm relative to TMS and coupling constants (J) were reported in hertz.
Electrospray ionization mass spectrometry (ESI-MS) was carried out on a Perkin–Elmer (Norwalk, CT)
Sciex API 100 Benchtop system employing Turbo Ionspray ion source and HRMS was performed on
Nermang 3010 MS-50, JEOL SX102-A mass spectrometers. Thin layer chromatography was performed
on pre-coated Whatman MK6F silica gel 60 Å plates (layer thickness: 250 µm) and visualized with
UV light and/or using a KMnO4 solution [KMnO4 (1.0 g), NaOH (8.0 g) in water (200 mL)]. Column

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chromatography was performed on silica gel, Merck grade 60 (230–400 mesh). THF and CH2Cl2 were
freshly distilled from sodium benzophenone and CaH2, respectively, under nitrogen. All reagents were
purchased from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and used
without purification, except where noted. All the solvents employed were of HPLC grade purchased from
EM Science (Gibbstown, NJ) and used as received. Analytical reverse phase (RP) HPLC was performed
using a Waters, µBondapak, RCM, C18, 10 µ (8×100 mm) column (solvents ratio v/v reported) unless
otherwise stated. Preparative reverse phase (RP) HPLC was performed using a Waters, µBondapak,
RCM, C18, 10 µ (40×100 mm) column (solvents ratio v/v reported) unless otherwise stated. Optical
rotations were measured on an Autopol III polarimeter, Rudolph Research, Flanders, NJ. Melting points
were recorded in open capillary tubes on an Electrothermal apparatus and were uncorrected.
3.2. (−)-tert-Butyl-(3S,6R)-6-{[(tert-butoxycarbonyl)amino]methyl}-2-oxotetrahydro-2H-pyran-3-
ylcarbamate (7)
(2S,5R)-(+)-Hydroxylysine dihydrochloride monohydrate (6, 0.253 g, 1.0 mmol) was dissolved in 10%
aq. NaHCO3 (10 mL) and THF (5 mL). To this mixture, (Boc)2O (0.872 g, 4.0 mmol, 4.0 equiv.) was
added at room temperature with vigorous stirring in two portions, at 4 h intervals. After stirring the
mixture for 16 h, the pH was adjusted to 3.5 using 6.0N HCl and extracted with CHCl3 (3×40 mL). The
combined organic layers were washed with brine (15 mL), dried (MgSO4) and concentrated on a rotary
evaporator. The residue was dissolved in benzene (20 mL) and refluxed for 2 h while removing the water
azeotropically using a Dean–Stark apparatus. The solvent was removed on a rotary evaporator and the
crude product purified by silica gel column chromatography (50–80% EtOAc in hexanes) to afford 0.324
g of (3S,6R)-(−)-lactone (7) in 94% yield as a colorless glassy material. Rf: 0.58 (80% EtOAc in hexanes);
mp 126–127°C; analytical RP HPLC: MeCN:0.1% aq. trifluoroacetic acid=55:45, 2.0 mL/min at 225 nm,
tR: 3.88 min, >99%; [α]20 −18.1 (c 0.56, CHCl3); 1H NMR (CDCl3): δ 5.28–5.18 (m, 1H), 5.02–4.94
D
(m, 1H), 4.52–4.42 (m, 1H), 4.12–4.02 (m, 1H), 3.54–3.44 (m, 1H), 3.26–3.17 (m, 1H), 2.38–2.46 (m,
1H), 2.03–1.74 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H); 13C NMR (CDCl3): δ 170.6, 155.9, 155.5, 81.9, 80.4,
79.8, 77.2, 51.5, 44.7, 28.3, 28.2, 27.2, 25.7; ESI-MS (m/z): 345 (M+H)+, 362 (M+NH4)+, 367 (M+Na)+,
706 (2×M+NH4)+, 711 (2×M+Na)+.
3.3. (−)-Methyl 6-({(2S,5R)-2,6-bis[(tert-butoxycarbonyl)amino]-5-hydroxyhexanoyl}amino)hexanoate
(9)
Triethylamine (0.106 mL, 6.0 mmol, 4.0 equiv.) was added to a suspension of 6-aminohexanoic acid
hydrochloride (8, 0.545 g, 3.0 mmol, 2.0 equiv.) and THF (8 mL) at room temperature under nitrogen.
To this mixture, a solution of (3S,6R)-(−)-lactone (7, 0.514 g, 1.5 mmol) in THF (8 mL) was added via a
double ended needle and stirred for 4 days. The reaction mixture was concentrated on a rotary evaporator
and the residue dissolved in EtOAc (125 mL) and water (25 mL). The organic layer was separated and
the aqueous layer extracted with EtOAc (50 mL). The combined organic layers were washed with water
(25 mL) and brine (2×25 mL), dried (MgSO4) and the solvent was removed on a rotary evaporator.
The crude compound was purified by silica gel column chromatography (70% EtOAc in hexane to 3%
MeOH in EtOAc) to afford 0.573 g of (−)-ester (9) in 78% yield as a colorless gummy material. Rf: 0.15
(80% EtOAc in hexanes); analytical RP HPLC: MeCN:water=15:85, 2.0 mL/min at 225 nm, tR: 2.34
min, 98.2%; [α]20 −9.6 (c 0.82, CHCl3); 1H NMR (CDCl3): δ 6.43 (dist. t, 1H), 5.42 (d, 1H, J=7.5 Hz),
D
5.06 (dist. t, 1H), 4.20–4.10 (m, 1H), 3.76–3.66 (m, 1H), 3.66 (s, 3H), 3.55–3.48 (m, 1H), 3.32–3.21
(m, 3H), 3.07–2.98 (m, 1H), 2.31 (t, 2H, J=7.5 Hz), 1.91–1.31 (m, 10H), 1.44 (s, 9H), 1.43 (s, 9H); 13
C

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NMR (CDCl3): δ 174.1, 171.9, 81.9, 79.8, 77.2, 71.4, 51.6, 46.6, 39.2, 33.8, 30.1, 29.4, 29.1, 28.4, 28.3,
26.2, 24.4; ESI-MS (m/z): 490 (M+H)+; HRMS (FAB, m/z): calcd for C23H43N3O8: 490.3128 (M+H)+;
observed: 490.3110.
3.4. (−)-Tetraacetate (11)
(+)-Acetobromo-α-D-galactose (10, 3.02 g, 7.362 mmol, 2.0 equiv.) and mercury(II) cyanide (2.08
g, 8.28 mmol, 2.2 equiv.) were added sequentially to a solution of (−)-ester (9, 1.80 g, 3.681 mmol)
dissolved in toluene (55 mL) under nitrogen. The mixture was heated at 50°C for 2.5 h, and concentrated
on a rotary evaporator. The residue was purified by silica gel column chromatography (50% EtOAc in
hexanes to 3% MeOH in EtOAc) to afford 1.077 g of material, which was further purified by preparative
RP HPLC (MeCN:water=50:50, 45.0 mL/min at 225 nm). The product was lyophilized to afford 0.395 g
of (−)-tetraacetate (11) in 13% yield as a white powder. Rf: 0.36 (80% EtOAc in hexanes); analytical RP
HPLC: MeCN:water=55:45, 2.0 mL/min at 225 nm, tR: 7.56 min, >99%; [α]20 −9.1 (c 0.53, CHCl3);
D
1H NMR (CDCl3): δ 6.43 (br. s, 1H), 5.10–5.34 (m, 2H), 5.22–5.15 (m, 2H), 5.00 (dd, 1H, J=10.2,
3.3 Hz), 4.55 (d, 1H, J=7.5 Hz), 4.21–4.30 (m, 2H), 4.02–3.92 (m, 2H), 3.74–3.64 (m, 1H), 3.66 (s,
3H), 3.38–3.08 (m, 4H), 2.31 (t, 2H, J=7.2 Hz), 2.16 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H),
1.66–1.31 (m, 10H), 1.45 (s, 9H), 1.44 (s, 9H); 13C NMR (CDCl3): δ 174.1, 171.7, 170.3, 170.2, 170.1,
169.6, 156.5, 155.8, 101.3, 81.5, 80.1, 79.5, 77.2, 70.8, 70.7, 68.9, 67.0, 61.4, 51.5, 44.0, 39.2, 33.8, 29.1,
29.0, 28.6, 28.5, 28.3, 26.2, 24.2, 20.8, 20.7, 20.6, 20.5; ESI-MS (m/z): 820 (M+H)+, 837 (M+NH4)+,
1639 (2×M+H)+; HRMS (FAB, m/z): calcd for C37H61N3O17: 842.3893 (M+Na)+; observed: 842.3892.
3.5. (−)-Acid (12)
Lithium hydroxide monohydrate (0.199 g, 4.76 mmol, 10.0 equiv.) and water (6.0 mL) were added
sequentially to a solution of (−)-tetraacetate (11, 0.390 g, 0.1 mmol) dissolved in THF (16 mL) at room
temperature. After stirring the mixture for 17 h, the solvent was removed on a rotary evaporator to
dryness. The residue was dissolved in MeCN:0.1% aq. trifluoroacetic acid (10 mL, ratio 35:65) and the
pH of the mixture was adjusted to 5.6 using 1N HCl. The product was purified by preparative RP HPLC
(MeCN:0.1% aq. trifluoroacetic acid=30:70, 45.0 mL/min at 225 nm). The product was lyophilized to
afford 0.191 g of (−)-acid (12) in 63% yield as a white powder. Analytical RP HPLC: MeCN:0.1% aq.
trifluoroacetic acid=35:65, 2.0 mL/min at 225 nm, tR: 3.35 min, >99%; [α]20 −7.9 (c 0.38, MeOH; 1H
D
NMR (CD3OD): δ 4.28 (d, 1H, J=7.2 Hz), 4.00–3.93 (m, 1H), 3.84–3.62 (m, 3H), 3.55–3.41 (m, 3H),
3.26–3.10 (m, 5H), 2.29 (t, 2H, J=7.5 Hz), 1.98–1.86 (m, 1H), 1.70–1.31 (m, 9H), 1.44 (s, 18H); ESI-
MS (m/z): 638 (M+H)+, 655 (M+NH4)+, 660 (M+Na)+; HRMS (FAB, m/z): calcd for C28H51N3O13:
660.3314 (M+Na)+; observed: 660.3316.
3.6. (−)-Succinimidyl ester (13)
N-Hydroxysuccinimide (0.069 g, 0.6 mmol, 2.0 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDAC, 0.086 g, 0.45 mmol, 1.5 equiv.) were added sequentially to a solution
of (−)-acid (12, 0.191 g, 0.3 mmol) dissolved in DMF (3.0 mL) at room temperature under nitrogen.
After stirring the mixture for 16 h, the solvent was removed on a rotary evaporator under vacuum. The
residue was dissolved in MeCN:water (8 mL, ratio 75:25) and purified by RP HPLC (MeCN:0.1% aq.
trifluoroacetic acid=35:65, 45.0 mL/min at 225 nm). The product was lyophilized to afford 0.142 g of
(−)-succinimidyl ester (13) in 65% yield as a white powder. Analytical RP HPLC: MeCN:0.1% aq.

M. Adamczyk, R. E. Reddy / Tetrahedron: Asymmetry 10 (1999) 3157–3165
3163
trifluoroacetic acid=35:65, 2.0 mL/min at 225 nm, tR: 5.53 min, >98%; [α]20 −5.0 (c 0.18, MeOH); 1H
D
NMR (CD3OD): δ 4.28 (d, 1H, J=6.9 Hz), 4.00–3.94 (m, 1H), 3.86–3.62 (m, 3H), 3.56–3.42 (m, 3H),
3.28–3.10 (m, 5H), 2.83 (s, 4H), 2.64 (t, 2H, J=7.2 Hz), 1.98–1.86 (m, 1H), 1.80–1.30 (m, 9H), 1.44
(s, 18H); ESI-MS (m/z): 735 (M+H)+, 752 (M+NH4)+, 757 (M+Na)+; HRMS (FTMS, m/z): calcd for
C32H54N4O15: 757.3477 (M+Na)+; observed: 757.3478.
3.7. BSA-immunogen (2)
A solution of BSA (0.200 g, 0.003006 mmol) dissolved in buffer (pH: 8.00, 10 mL) was added to a
solution of (−)-succinimidyl ester (13, 0.0507 g, 0.06914 mmol, 23.0 equiv.) dissolved in DMF (2 mL)
at room temperature. The mixture was stirred for 15 h and dialyzed in a solution of 0.05N phosphate
buffer (1.8 L) and MeOH (200 mL) twice by changing to fresh buffer–MeOH mixture for a total of 8
h. The mixture was further dialyzed in deionized (DI) water (4.0 L) four times by changing to fresh DI
water every 5 h. Lyophilization of the mixture afforded 0.197 g of immunogen precursor (14) with Boc
protective groups, as a white powder. Analysis of this immunogen precursor (14) by gel electrophoresis
showed a single narrow band and an absence of free BSA. MALDI mass spectrum: 76587 [BSA with 16
haptens (12)]. A mixture of TFA (9.5 mL) and water (0.5 mL) was added to this intermediate immunogen
(0.192 g) at room temperature and stirred for 0.5 h. The solvent was removed on a rotary evaporator to
dryness and the residue was diluted with water (15 mL). The pH of the mixture was adjusted to 8.5 using
1N aq. NaOH. The mixture was dialyzed in 0.05N phosphate buffer (4.0 L) twice by changing to a fresh
buffer solution, for a total of 15 h. The mixture was further dialyzed in DI water (4.0 L) twice by changing
to fresh DI water every 3 h. Lyophilization of the mixture afforded 0.179 g of BSA-immunogen (2) as
a white powder. Analysis of immunogen (2) by gel electrophoresis showed a single narrow band and an
absence of free BSA. MALDI mass spectrum: 73645 [BSA with 16 haptens].
3.8. KLH-immunogen (3)
A solution of KLH (0.200 g), dissolved in buffer (pH: 8.00, 10 mL) was added to a solution of (−)-
succinimidyl ester (13, 0.0507 g, 0.06914 mmol) dissolved in DMF (2 mL) at room temperature. The
mixture was stirred for 15 h and dialyzed in a solution of 0.05 N phosphate buffer (1.8 L) and MeOH
(200 mL) twice by changing to fresh buffer–MeOH mixture every 4 h. The mixture was further dialyzed
in deionized (DI) water (4.0 L) four times by changing to fresh DI water every 5 h. Lyophilization of the
mixture afforded 0.213 g of an immunogen precursor (15) with Boc protective groups. A mixture of TFA
(9.5 mL) and water (0.5 mL) was added to this precursor 15 (0.213 g) at room temperature and stirred for
0.5 h. The solvent was removed on a rotary evaporator to dryness, the resulting residue was diluted with
water (15 mL) and the pH was adjusted to 8.5 using 1N aq. NaOH. The mixture was dialyzed in 0.05 N
phosphate buffer (4.0 L) twice by changing to a fresh buffer solution, for a total of 15 h. The mixture was
further dialyzed in DI water (4.0 L) twice by changing to fresh DI water every 3 h. Lyophilization of the
mixture afforded 0.194 g of KLH-immunogen (3) as a pale gray powder.
3.9. 6-Fluorescenyl-conjugate (18)
6-Fln-CH2NH2 HBr (16, 0.00992 g, 0.0225 mmol, 1.5 equiv.) and triethylamine (0.012 mL, 0.088
mmol, 8.0 equiv.) were added sequentially to a solution of (−)-succinimidyl ester (13, 0.011 g, 0.015
mmol) dissolved in anhydrous DMF (0.3 mL) at room temperature under nitrogen. After stirring the
reaction mixture for 20 h, the solvent was removed on a rotary evaporator under vacuum to dryness.
The residue was dissolved in MeCN:0.1% aq. trifluoroacetic acid (3.0 mL, ratio 1:1) and purified

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by preparative RP HPLC (MeCN:0.1% aq. trifluoroacetic acid=35:65, 45.0 mL/min at 225 nm). The
product was lyophilized to afford 0.0102 g of 18 in 69% yield as a yellow powder. Analytical RP
1
HPLC: MeCN:0.1% aq. trifluoroacetic acid=35:65, 2.0 mL/min at 225 nm, tR: 8.39 min, >99%; H
NMR (CD3OD): δ 8.54–8.48 (m, 45/100H), 7.99 (d, 1H, J=7.8 Hz), 7.92–7.86 (m, 55/100H), 7.64–7.60
(m, 1H), 7.12 (s, 1H), 6.78–6.58 (m, 6H), 4.46–4.42 (m, 2H), 4.27 (d, 1H, J=7.2 Hz), 3.99–3.92 (m, 1H),
3.84–3.40 (m, 6H), 3.28–3.02 (m, 5H), 2.18 (t, 2H, J=7.2 Hz), 1.98–1.18 (m, 10H), 1.43 (s, 9H), 1.42
(s, 9H); ESI-MS (m/z): 982 (M+H)+; HRMS (FAB, m/z): calcd for C49H64N4O17: 1003.4158 (M+Na)+;
observed: 1003.4159.
3.10. Fluorescent tracer (6-Fln-tracer, 4)
Dichloromethane (3.0 mL) and trifluoroacetic acid (3.0 mL) were added sequentially to 6-fluorescenyl-
conjugate (18) (0.010 g, 0.0102 mmol) at room temperature and stirred for 1.5 h. The solvent was
removed on a rotary evaporator to dryness and dissolved in MeCN:0.1% aq. trifluoroacetic acid (5
mL, ratio 30:70). The residue was purified by preparative RP HPLC (MeCN:0.1% aq. trifluoroacetic
acid=25:75, 45.0 mL/min at 225 nm). The product was lyophilized to afford 0.0086 g of fluorescent
tracer-TFA salt (6-Fln-tracer, 4) in 84% yield as a yellow powder. Analytical RP HPLC: MeCN:0.1%
1
aq. trifluoroacetic acid=25:75, 2.0 mL/min at 225 nm, tR: 4.61 min, >99%; H NMR (CD3OD): δ 7.95
(d, 1H, J=7.5 Hz), 7.62–7.58 (m, 1H), 7.09 (s, 1H), 6.67 (d, 2H, J=2.1 Hz), 6.59–6.50 (m, 4H), 4.42 (s,
2H), 4.36 (d, 1H, J=7.5 Hz), 4.02–3.94 (m, 1H), 3.87 (t, 1H, J=6.6 Hz), 3.81–3.41 (m, 5H), 3.22–3.07
(m, 3H), 2.98–2.90 (m, 2H), 2.19 (t, 2H, J=7.5 Hz), 2.14–1.22 (m, 10H), 1.43 (s, 9H), 1.42 (s, 9H); ESI-
MS (m/z): 781 (M+H)+, 803 (M+Na)+; HRMS (FAB, m/z): calcd for C39H48N4O13: 803.3111 (M+Na)+;
observed: 803.3110.
3.11. Acridinium-conjugate (17)
Acr-NH2 TFA (17, 0.0204 g, 0.03 mmol, 1.5 equiv.) and triethylamine (0.022 mL, 0.16 mmol, 8.0
equiv.) were added sequentially to a solution of (−)-succinimidyl ester (13, 0.0146 g, 0.02 mmol)
dissolved in anhydrous DMF (0.4 mL) at room temperature under nitrogen. After stirring the mixture
for 15 h, the solvent was removed on a rotary evaporator under vacuum. The residue was dissolved
in MeCN:0.1% aq. trifluoroacetic acid (5.0 mL, ratio 1:2) and purified by preparative RP HPLC
(MeCN:0.1% aq. trifluoroacetic acid=33:67, 45.0 mL/min at 225 nm). The product was lyophilized
to afford 0.018 g of 19 in 72% yield as a yellow powder. Analytical RP HPLC: MeCN:0.1% aq.
trifluoroacetic acid=35:65, 2.0 mL/min at 225 nm, tR: 4.83 min, >99%; 1H NMR (CD3OD): δ 8.99–8.92
(m, 2H), 8.48–8.43 (m, 2H), 8.19–7.61 (m, 6H), 7.21–7.17 (m, 2H), 5.78–5.74 (m, 2H), 4.32–4.24 (m,
2H), 4.00–3.92 (m, 1H), 3.86–3.04 (m, 15H, merged with CD3OD), 2.86–2.12 (m, 8H), 2.58 and 2.38
(two s, 3H), 2.00–1.28 (m, 12H), 1.43 (s, 9H), 1.42 (s, 9H); ESI-MS (m/z): 1247 (M+H)+, 1269 (M+Na)+;
HRMS (FAB, m/z): calcd for C58H83N7O19S2: 1268.5094 (M+Na)+; observed: 1268.5077.
3.12. Chemiluminescent tracer (Acr-tracer, 5)
CH2Cl2 (4.0 mL) and trifluoroacetic acid (4.0 mL) were added sequentially to 19 (0.018 g, 0.015
mmol) at room temperature and stirred for 1.5 h. The solvent was removed on a rotary evaporator and
the residue was dissolved in MeCN:0.1% trifluoroacetic acid (8.0 mL, ratio 1:1). Purification of the
crude product by preparative RP HPLC (MeCN:0.1% aq. trifluoroacetic acid=25:75, 45.0 mL/min at 225
nm) followed by lyophilization afforded 0.00121 g of Acr-tracer-TFA salt (5) in 77% yield as a yellow

M. Adamczyk, R. E. Reddy / Tetrahedron: Asymmetry 10 (1999) 3157–3165
3165
powder. Analytical RP HPLC: MeCN:0.1% aq. trifluoroacetic acid=25:75, 2.0 mL/min at 225 nm, tR:
1
5.02 min, >99%; H NMR (CD3OD): δ 8.98–8.92 (m, 2H), 8.50–8.42 (m, 2H), 8.22–7.61 (m, 6H),
7.22–7.18 (m, 2H), 5.80–5.70 (m, 2H), 4.44–4.26 (m, 3H), 4.10–3.06 (m, 15H, merged with CD3OD),
3.00–2.10 (m, 8H), 2.58 and 2.38 (two s, 3H), 1.80–1.20 (m, 12H); ESI-MS (m/z): 1047 (M+H)+, 1069
(M+Na)+; HRMS (FAB, m/z): calcd for C48H67N7O15S2: 1046.4215 (M+H)+; observed: 1046.4209.
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