Design and syntheses of three haptens to generate catalytic antibodies that cleave amide bonds with nucleophilic catalysis

Article abstract of DOI:10.1016/S0968-0896(98)00203-X

Design principles and syntheses of three haptens that were recently reported to generate amide bond cleaving catalytic antibodies are described. The hapten designs sought to induce acidic and/or basic residues in antibody binding sites via charge compleme

Full text of DOI:10.1016/S0968-0896(98)00203-X

Bioorganic & Medicinal Chemistry 7 (1999) 279±286
Design and Syntheses of Three Haptens to Generate Catalytic
Antibodies that Cleave Amide Bonds with Nucleophilic Catalysis
Oguz Ersoyy, Roman Fleck, Marõa-Jesus Blanco and Satoru Masamune*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 26 May 1998; accepted 14 September 1998
AbstractÐDesign principles and syntheses of three haptens that were recently reported to generate amide bond cleaving catalytic
antibodies are described. The hapten designs sought to induce acidic and/or basic residues in antibody binding sites via charge
complementarity, and also to generate a hydrophobic binding pocket for an external phenol nucleophile. The charged yet aromatic
nature of these haptens presented some unique synthetic challenges and solutions to which are described below. # 1999 Elsevier
Science Ltd. All rights reserved.
Introduction
A unimolecular version of this reaction was also
designed where the phenolic group is tethered to the
amide as shown in substrate 1 (Fig. 1). This substrate
technically simpli®ed the initial screening where 74 dif-
ferent clones were tested. Three catalysts were found to
accelerate the intramolecular reaction.3a When these
clones were tested in the presence of phenol and pro-
pionyl p-nitroanilide, all three were found to accelerate
this target amide cleavage reaction, although, of course,
with kinetic constants di€erent from those obtained in
the unimolecular case.3b
In our recent work, we have focused on the generation
of antibody catalysts for the cleavage of amide bonds,1
a highly challenging task.2 To this end, we devised a
heterologous immunization protocol where two struc-
turally similar but di€erently functionalized haptens are
used to immunize animals in sequence. The aim of this
approach is to generate multiple charged residues in
antibody binding sites while avoiding complicated
syntheses of zwitterionic haptens. This method yielded
highly ecient esterolytic antibodies, although amide
hydrolysis met with little success.
In both reaction systems, antibody 14±10 generated by
heterologous immunization was found to be a ®vefold
to tenfold more ecient catalyst (in terms of kcat/Km)
than the antibodies 6±17 and 3±49 raised by homo-
logous immunization with only one of the haptens.
Furthermore, the percentage of catalysts among all
hapten binding antibodies was signi®cantly higher with
the heterologous immunization protocol (ca. 20%) than
with either one of the homologous immunization pro-
tocols (ca. 5%).
A novel approach to amide hydrolysis was adopted in
the present work with the use of an externally supplied
nucleophilic cofactor.3 This approach would pattern
after the mechanism of serine proteases,4 which use an
active-site serine residue to initiate the nucleophilic
attack on the peptide bond. We chose phenol as the
externally supplied nucleophile for antibody catalyzed
cleavage of the amide bond which would generate a
water labile phenyl ester. In addition to the high water
solubility and good nucleophilicity of phenol, its aro-
matic ring provides a convenient recognition element
for antibody binding pockets via hydrophobic interac-
tions. Propionyl p-nitroanilide was chosen as a simple
chromogenic amide substrate. This mechanism is shown
in Scheme 1.
The three haptens (O1, O2, O3) were designed to gen-
erate antibody binding pockets with the following
important features: (1) a hydrophobic binding pocket
for the phenyl ring, (2) an acidic residue complementary
to the oxyanionic transition state (O1, O3), and (3) a
basic residue to aid the deprotonation of the phenol
nucleophile and hopefully protonation of the departing
amine afterwards (O2). In addition to homologous
immunization using each of the haptens separately,
hapten O2 would be paired with either O1 or O3 in a
heterologous immunization protocol. Upon initial
immunizations, N-oxide hapten O3 failed to generate a
strong immune response and therefore, all further studies
Key words: Antibodies; catalysts; phosphonamidates; quinolizinium
ions.
*Corresponding author. Fax: +1-617-253-1340; e-mail: masamune@-
mit.edu
Present address: Laboratory for Molecular Immunology, Kar-
olinska Institute, S-17176 Stockholm, Sweden.
y
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00203-X

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O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
Scheme 1.
Figure 1.
were carried out with haptens O1 and O2. Since kinetic
studies on the antibody catalysis were discussed exten-
sively,3 this article describes the syntheses of haptens
O1, O2 and O3 to complete a full documentation of this
subject.
ment of 5 with anhydrous p-toluenesulfonic acid (in
re¯uxing xylene to azeotropically remove generated
ethanol), a€orded the cyclic phosphonate 6, achieving
two steps in a one-pot reaction in good yield (90%).
The formation of the phosphonamidate bond was not
straightforward. The phosphonyl chloride of 6 was
found to be very unreactive and failed to react with 4-
nitroaniline, aniline or even with a carbamate protected
phenylenediamine under various reaction conditions.9
Only unprotected phenylenediamine (in excess) showed
the required nucleophilicity to react with this phos-
phonyl chloride. The problem of handling excess of
phenylenediamine was solved by a careful and exhaus-
tive puri®cation which involved removal of the excess of
phenylenediamine via sublimation, followed by two
¯ash column chromatography steps to a€ord the
desired phosphonamidate 7. This compound was trea-
ted with glutaric anhydride to give hapten O1 (5 steps,
26% overall yield).
Results and discussion
The synthesis of hapten O1 (Scheme 2) started with 2-
benzyloxybenzaldehyde 2, easily prepared by benzyla-
tion of commercially available salicylaldehyde. The
Horner±Emmons ole®nation of 2 with bisphosphonate
3 under biphasic conditions5 provided trans-stilbene-
phosphonate 4 in good yield. Catalytic hydrogenation
of 4 reduced the ole®n and removed the benzyl protecting
group in one step, which yielded diethyl phosphonate 5.
Since the phosphonamidate functionality present in
hapten O1 is known to be rather labile,6 it was decided
to ®rst construct a new P±O bond (to cyclize 5) rather
than a P±N bond. Thus phosphonate 5 was treated with
PCl5 in order to carry out the cyclization step. Although
one of the ethoxy groups of the phosphonate 5 was
easily exchanged for chloride, various attempts failed to
yield the desired phosphonic acid 6. Other strategies
such as generating the monophosphonic acid and treat-
ing it with various acyl-coupling reagents (e.g. BOP7 or
carbodiimides) were also unsuccessful.8 Finally, treat-
The synthesis of hapten O2 was accomplished in 13
steps (10% overall yield) as shown in Scheme 3. Alky-
lation of the phosphonate 8 with 4-nitrobenzyl bro-
mide10 followed by a Horner±Emmons reaction with
formaldehyde11 a€orded the unsaturated ester 9.
The DIBAL-H reduction of ester 9 at 78 ꢀC, followed
by triethylsilyl protection of the resulting primary alcohol

O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
281
Scheme 2.
Scheme 3.
10 and subsequent epoxidation of the double bond with
m-CPBA provided 12 in good yield (86%, 3 steps). The
nitro group of 12 impeded the nucleophilic opening of
the epoxide with 2-picolyllithium even when high order
cyano cuprates12 were used, so we decided to reduce it
to an amine and protect it. Hydrogenation of the nitro
group with Pd/C followed by protection of the resulting
amino moiety with trityl chloride proceeded almost
quantitatively. The opening of the epoxide 13 with 2-
picolyllithium13 proceeded then smoothly to give 14 in
69% yield. Deprotection of the TES group with TBAF
gave the diol 15. Activation of the primary hydroxyl
group with methanesulfonyl chloride (MsCl) and sub-
sequent cyclization in re¯uxing acetone14 a€orded the
quinolizinium salt, 16. Finally, the trityl-group of 16
was removed under biphasic conditions by re¯uxing it
in a mixture of water/ethyl acetate, followed by con-
densation of obtained 17 with glutaric anhydride to
yield hapten O2 as an amorphous solid.
The synthesis of hapten O3 was straightforward as
shown in Scheme 4. Thus, 1,2,3,4 tetrahydro-isoquino-
line 18 was alkylated with 4-nitrobenzylbromide to give
19 followed by the catalytic reduction of the nitro

282
O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
Scheme 4.
group. The thus obtained aniline 20 was coupled with
glutaric anhydride to a€ord 21. The tertiary amine 21
was relatively unreactive towards oxidation to the N-
oxide, and after several attempts (including di€erent
peroxides and dioxirane) m-CPBA was found to be the
most ecient reagent providing hapten O3 in 77%
overall yield (4 steps).
follows: s (singlet), d (doublet), t (triplet), c (quartet), q
(quintuplet), m (multiplet), br (broad). Coupling con-
stants (J) are reported in Hz. Proton-decoupled-carbon
(13C NMR) and phosphorus (31P NMR) magnetic
resonance spectra were recorded at 75.44 MHz and
121.44 MHz, respectively, on a Varian XL-300 instru-
ment. The chemical shifts of 13C NMR spectra were
referenced to CDCl3 (77.0 ppm), DMSO (39.5 ppm),
CD2Cl2 (53.8 ppm), or CD3OD (49.0 ppm). 31P NMR
spectra were referenced to 85% H3PO2 as an external
standard (capillary tube within NMR tube). Infrared
(IR) spectra were recorded on a Perkin±Elmer 283B
spectrometer. Melting points were not corrected. High-
resolution mass spectra (HRMS) were recorded on a
Finnigan MAT 8200 spectrometer.
In summary, we have disclosed here the full experi-
mental details of the syntheses of three novel haptens
that were used to generate amide cleaving antibodies.
Considerable synthetic challenges were encountered
especially in the synthesis of hapten O1 both in the
cyclization of phosphonate 5, and also in the formation
of the P±N bond of phosphonamidate 7. A novel acid
catalyzed cyclization protocol followed by a careful
matching of phosphonyl and amine reactivities helped
to successfully overcome the respective challenges.
2-Benzyloxybenzaldehyde (2). A solution of anhydrous
K2CO3 (29.9 g, 216 mmol) in MeOH (300 mL) and
CHCl3 (600 mL) was re¯uxed for 15 min under a stream
of argon. Salicylaldehyde (5.25 mL, 49.1 mmol) and
benzyl bromide (6.45 mL, 54.0 mmol) were added, and
the resulting mixture was re¯uxed for 1 day. The reac-
tion mixture was ®ltered, the ®ltrate evaporated and the
resulting residue was partitioned between 1N HCl
(200 mL) and CH2Cl2 (2Â300 mL). The combined
organic layers were washed with brine, dried, ®ltered
and concentrated to a€ord 10.3 g (99%) of 2 as a
Experimental
Air sensitive reactions were carried out under an argon
atmosphere in oven-dried glassware. Flash chroma-
tography was performed using Merck silica gel 60 (230±
400 mesh). Solvents used for elution are noted for the
speci®c examples. Solvents extracts were routinely dried
over MgSO4 and concentrated. Anhydrous solvents,
starting materials, and reagents were purchased from
Aldrich Chemical Co. and used as obtained. THF and
Et2O were distilled from Na/benzophenone. CH2Cl2
and pyridine were distilled from CaH2. Proton (1H
NMR) magnetic resonance were recorded at 300 MHz
on a Varian XL-300 instrument. The chemical shifts of
1H NMR spectra were referenced to CDCl3 (7.24 ppm),
DMSO (2.49 ppm), CD2Cl2 (5.32 ppm), or CD3OD
(3.30 ppm). 1H NMR peak multiplicities are reported as
1
slightly yellow oil. H NMR of the crude showed it to
be very pure and it was used in the next step without
any further puri®cation. IR (®lm) 3053, 3020, 2751,
1
1687, 1598 cm
.
1H NMR (CDCl3) d 10.57 (s, 1H),
7.86 (d, J=8.0 Hz, 1H), 7.57±7.49 (m, 1H), 7.47±7.32
(m, 5H), 7.08±7.00 (m, 2H), 5.19 (s, 2H).
(E) Diethyl [(2-benzyloxy)phenyl]-ethenyl-phosphonate
(4). NaOH (50% aq, 6 mL) was added to a solution of
2 (0.74 g, 3.48 mmol) and bisphosphonate 3 (0.86 mL,

O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
283
3.48 mmol) in CH2Cl2 (6 mL). The biphasic reaction was
stirred vigorously (open to ambient athmosphere) for
4 h. The reaction mixture was diluted with H2O (20 mL)
and extracted with CH2Cl2 (2Â20 mL). The combined
organic layers were washed with brine, dried, ®ltered and
concentrated to give 1.1 g (92%) of 4 as a colorless oil. IR
(®lm) 3209, 2977, 2745, 2612, 1594, 1500, 1456 cm 1; 1H
amine (0.87 g, 8.0 mmol), pyridine (0.67 mL, 8.0 mmol)
and DMAP (cat.) in DMF (2 mL). The resulting mix-
ture was stirred for 6 h at rt. The reaction mixture was
diluted with CH2Cl2 (40 mL), ®ltered (in order to
remove the pyridinium chloride) and the ®ltrate was
concentrated. The excess of unreacted phenylene dia-
mine was removed by sublimation in vacuo (1 mm Hg,
120 ꢀC) and the remaining residue was puri®ed by ¯ash
chromatography (gradient 2 to 5% MeOH in CH2Cl2).
The fractions containing the product 7 with the con-
taminating phenylene diamine were then pooled, con-
centrated in vacuo, and puri®ed again by ¯ash
chromatography (gradient 75±100% EtOAc±hexanes
with 0.1% NH4OH) to yield 364 mg (35%) of 7 as a
white solid. This compound is stable inde®nitely under
NMR (CDCl3) d 7.88 (dd, JP±H=23.4 Hz, JH±H
=
17.7 Hz, 1H), 7.46 (dd, J=4.8, 1.5 Hz, 1H), 7.41±7.21
(m, 6H), 6.92 (t, J=9.0 Hz, 2H), 6.32 (dd, JP±H=19.5 Hz,
JH±H=17.7 Hz, 1H), 5.19 (s, 2H), 4.07 (q, J=7.2 Hz,
4H), 1.28 (t, J=7.2 Hz, 6H); 13C NMR (CDCl3) d 154.3,
143.9 (d, JC±P=117.2 Hz), 141.2 (d, JC±P=11.1 Hz),
133.5, 131.7, 128.7, 128.5, 128.3, 127.6, 127.0, 126.6,
122.8, 63.3, 62.1 (d, JC±P=4.9 Hz), 31.8. HRMS (M+)
for C19H23O4P: calcd. 346.3630, found 346.3629.
1
ambient atmosphere. H NMR (CD3OD) d 7.26±7.12
(m, 2H), 7.08±6.94 (m, 4H), 6.80 (d, J=8.4 Hz, 2H),
3.18±3.02 (m, 2 H), 2.27±2.11 (m, 2 H). 13C NMR
(DMSO-d6) d 150.9 (d, JC±P=6.6 Hz), 136.5, 132.6,
128.6, 127.7, 124.2 (d, JC±P=10.4 Hz), 123.3, 121.0 (d,
JC±P=5.5 Hz), 118.3 (d, JC±P=6.9 Hz), 118.2, 23.9 (d,
JC±P=9.3 Hz), 20.5 (d, JC±P=115.9 Hz). 31P NMR
(DMSO-d6) d 27.16. HRMS (M+) for C14H15O2PN2:
calcd. 274.2591, found 274.2592.
Diethyl 2-(2-hydroxyphenyl)-ethyl-phosphonate (5). Pd/C
(10%, catalytic amount) was added to a solution of 4
(1.99 g, 5.72 mmol) in deoxygenated EtOH (30 mL). The
¯ask was purged with argon and then evacuated
(vacuum) and pressurized (H2) three times. The reaction
mixture was mechanically shaken under 52 psi of H2 for
3 days. The catalyst was removed by ®ltration, and the
®ltrate was evaporated to a€ord 1.45 g (98%) of 5 as a
colorless oil. The crude product was used in the next
5-({4-[2-(3,4-Dihydro-1,2-benzoxaphosphorin-2-oxide)-
amino]phenyl} amino)-5-oxo-pentanoic acid (hapten O1).
A mixture of phosphonamidate 7 (215 mg, 0.785 mmol)
and glutaric anhydride (990 mg, 0.86 mmol) in CH2Cl2
(20 mL) were stirred at rt for 3 h. The precipitated white
solid was ®ltered and dried under vacuum to a€ord
275 mg (91%) of hapten O1, which can be stored inde-
1
step without further puri®cation: H NMR (CDCl3) d
8.45±8.10 (br, 1H), 7.10±7.02 (m, 2H), 6.88 (d,
J=8.2 Hz, 2H), 6.78 (t, J=7.8 Hz, 2H), 3.99 (q,
J=7.2 Hz, 4H), 2.89 (dt, JP±H=22.8 Hz, JH±H=7.2 Hz,
1H), 2.12 (dt, JP±H=17.1 Hz, JH±H=7.2 Hz, 1H), 1.33
(t, J=7.2 Hz, 6H). 13C NMR (CDCl3) d 131.7, 130.8,
128.6, 128.2, 126.6, 121.5, 62.1 (d, JC±P=4.8 Hz), 31.8,
25.6 (d, JC±P=11.5 Hz), 20.5 (d, JC±P=125.6 Hz). 31P
NMR (CDCl3) d 45.58. HRMS (M+) for C12H19O4P:
calcd. 258.2542, Found 258.2537.
1
®nitely. H NMR (CD3OD) d 7.36 (d, J=9.6 Hz, 2H),
7.21±7.13 (m, 2H), 7.04±6.95 (m, 4H), 3.20±3.02 (m,
2H), 2.35 (t, J=7.5 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H),
2.24±2.11 (m, 2H), 1.94 (q, J=7.2 Hz, 2H). 13C NMR
(CD3OD) d 176.4, 172.8, 152.1 (d, JC±P=8.0 Hz), 136.1,
134.0, 129.9, 129.0, 125.3 (d, JC±P=12.7 Hz), 124.6,
122.1, 120.3 (d, JC±P=5.7 Hz), 119.7 (d, JC±P=6.9 Hz),
2-Hydroxy-3,4-dihydro-1,2-benzoxaphosphorin-2-oxide (6).
A solution of 5 (1.8 g, 6.97 mmol) and anhydrous p-
toluenesulfonic acid (1.57 g, 9.07 mmol) in xylene
(40 mL) were re¯uxed open to the air for 24 h. The
reaction mixture was cooled to rt, concentrated in vacuo
to a volume of 3±4 mL and stored at 5 ꢀC overnight. The
resulting white solid was ®ltrated and recrystallized
from acetone to give 1.15 g (90%) of 6. 1H NMR
(DMSO-d6) d 7.29±7.16 (m, 2H), 7.12±6.98 (m, 2H),
3.04 (dt, JP±H=23.6 Hz, JH±H=7.4 Hz, 2H), 2.12 (dt,
JP±H=17.9 Hz, JH±H=7.4 Hz, 2H). 13C NMR (DMSO-
36.5, 33.8, 25.2 (d, JC±P=8.1 Hz), 21.8 (d, JC±P
=
114.3 Hz), 21.6. 31P NMR (CD3OD) d 27.49. HRMS
(M+) for C19H21O5PN2: Calcd. 388.3600, found
388.3598.
Ethyl 2-[(4-nitrophenyl)methyl]-acrylate (9). A solution
of triethyl phosphono-acetate 8 (900 mL, 4.5 mmol) in
THF (5 mL) was added dropwise to a suspension of
sodium hydride (200 mg, 5 mmol, 80%) in THF (5 mL)
at 0 ꢀC. The resulting mixture was allowed to stir at rt
for 15 min (potassium salt precipitated as white solid)
followed by the addition of a solution of 4-nitro-ben-
zylbromide (970 mg, 4.5 mmol) in THF (5 mL). The
resulting solution was stirred for 1 h at rt (the pre-
cipitate was completely dissolved). The mixture
obtained was partitioned between hydrochloric acid
(10 mL, 5% aq) and CH2Cl2 (3Â10 mL). The combined
organic layers were dried, ®ltered and evaporated.
Without further puri®cation, the alkylated phosphono-
acetate was suspended in formaldehyde solution
(1.8 mL, 18 mmol, 35% aq) and a solution of potassium
carbonate (1.4 g, 10 mmol) in H2O (2 mL) was added
dropwise at rt. The reaction mixture was stirred at 80 ꢀC
for 7 h. After cooling, the reaction mixture was partitioned
d6) d 129.8, 129.5, 128.7, 127.7, 124.9, 119.3 (d, JC±P
7.5 Hz), 32.5 (d, JC±P=9.8 Hz), 28.0 (d, JC±P
107.9 Hz). 31P NMR (DMSO): d 25.76. HRMS (M+)
=
=
for C8H9O3P: calcd. 184.1314, Found 184.1311.
2-[(4-Aminophenyl)amino]-3,4-dihydro-1,2-benzoxaphos-
phorin-2-oxide (7). A suspension of 6 (1.15 g, 6.3 mmol)
and PCl5 (2.0 g, 9.6 mmol) in CH2Cl2 (10 mL) was
re¯uxed overnight. Xylene was added and the mixture
was concentrated in vacuo. Azeotropic removal of
xylene was repeated several times to remove the POCl3
by-product of the reaction. The phosphonyl chloride
obtained (0.77 g, 3.8 mmol) was redissolved in CH2Cl2
(10 mL) and added dropwise (over 1 h) via an addition-
funnel to a solution of freshly-sublimed phenylene di-

284
O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
between ammonium chloride (sat, 5 mL) and Et2O
(2Â6 mL). The combined organic layers were washed
with brine, dried and concentrated. Puri®cation by ¯ash
chromatography (gradient 50±90% EtOAc±hexanes)
a€orded 443 mg (42%) of 9. IR (®lm): 3105, 3095, 2978,
(1 atm) at rt. After hydrogen consumption stopped (4 h)
the solution was ®ltered through Celite and evaporated.
The residue was immediately dissolved in CH2Cl2
(135 mL), cooled at 0 ꢀC and Et3N (1.86 mL,
13.25 mmol) and tritylchloride (3.72 g, 13.25 mmol) were
added. After 1 h, the solution was washed with
NaHCO3 (sat), brine, dried, ®ltered and concentrated.
The residue was puri®ed by ¯ash chromatography (gra-
dient 5±20% EtOAC in hexanes) to a€ord 6.9 g (96%)
of 13: IR (®lm) 3413, 3060, 3013, 2954, 2910, 2874,
1614, 1513 cm 1. 1H NMR (CD2Cl2) d 7.67±7.45 (m, 15
H), 7.14 (d, J=8.1 Hz, 2H), 6.58 (d, J=8.0 Hz, 2H),
3.82 (d, J=11.7 Hz, 1H), 3.23 (d, J=11.9 Hz, 1H), 3.09
(d, J=13.8 Hz, 1H), 2.88 (d, J=13.9 Hz, 1H), 2.87 (d,
J=5.0 Hz, 1H), 2.73 (d, J=4.8 Hz, 1H), 1.19 (t,
J=8.4 Hz, 9H), 0.85 (c, J=8.4 Hz, 6H). 13C NMR
(CD2Cl2) d 146.1, 145.5, 130.0, 129.5, 128.5, 127.1,
126.1, 116.5, 72.0, 60.6, 50.3, 37.3, 7.2, 4.9. HRMS
(M+) for C35H41NSiO2: Calcd. 535.29066, found
535.29110.
1
1713, 1631, 1601, 1519 cm 1. H NMR (CDCl3) d 8.13
(d, J=7.8 Hz, 2H), 7.35 (d, J=7.8 Hz, 2H), 6.29 (s, 1H),
5.55 (s, 1H), 4.15 (c, J=6.8 Hz, 2H), 3.71 (s, 1H), 1.23
(t, J=7.7 Hz, 3H). 13C NMR (CDCl3) d 166.4, 147.0,
139.2, 129.9, 127.2, 123.8, 61.1, 38.3, 14.3. HRMS (M+)
for C12H13O4N: Calcd. 235.08446, found 235.08449.
2-Triethylsilyloxymethyl-2-[(4-nitrophenyl)methyl]-oxirane
(12). DIBAL-H (8.5 mL, 8.5 mmol, 1.0 M in hexane)
was added dropwise to a cooled ( 78 ꢀC) solution of
acrylester 9 (940 mg, 4 mmol) in THF (35 mL). After
1 h, the solution was allowed to warm up to rt. The
reaction mixture was partitioned between hydrochloric
acid (30 mL, 10% aq) and Et2O (2Â40 mL). The com-
bined organic layers were washed with NaHCO3 (satd),
brine, dried, ®ltered and concentrated. The residue was
®ltered through silica gel (hexanes:EtOAc 1:3) to yield
2-Hydroxy-2-[(N-trityl-4-aminophenyl)methyl]-4-(2-pyridyl)-
1-butanol (15). To a solution of phenyllithium (21.5 mL,
38.6 mmol, 1.8 M in cyclohexane/ether) in Et2O
(100 mL) was added dropwise (20 min) a solution of 2-
picoline (3.81 mL, 38.6 mmol) in Et2O (40 mL) The
solution turned dark red. After 1 h of stirring at rt the
solution was cooled to 0 ꢀC and a solution of epoxide 13
(6.9 g, 12.85 mmol) in Et2O (40 mL) was added over a
period of 30 min (with a syringe pump). After 3 h, the
reaction mixture was partitioned between ammonium
chloride bu€er (pH 8, sat, 150 mL) and Et2O
(2Â200 mL). The combined organic layers were dried
over potassium carbonate, ®ltered and concentrated.
The residue was puri®ed by ¯ash chromatography (gra-
dient 30±50% EtOAc±hexanes) to a€ord 5.64 g (69%)
of 14: IR (®lm) 3386, 3061, 3030, 2988, 1625, 1601,
760 mg (90%) of the allylic alcohol 10: IR (®lm) 3366,
1
3084, 2919, 2848, 1648, 1596, 1614, 1437 cm
.
1H
NMR (CDCl3) d 8.13 (d, J=8.4 Hz, 2H), 7.35 (d,
J=8.4 Hz, 2H), 5.17 (s, 1H), 4.87 (s, 1H), 4.02 (s, 2H),
3.49 (s, 2H), 1.68 (s, 1H). 13C NMR (CDCl3) d 147.2,
147.0, 130.0, 123.8, 113.1, 65.4, 39.6. HRMS (M+) for
C10H11NO3: calcd. 193.07389, found 193.07394.
Triethylamine (2.78 mL, 20 mmol), chlorotriethylsilane
(20 mL, 20 mmol, 1.0 M in THF) and DMAP (15 mg,
0.12 mmol) were added to a stirred solution of alcohol
10 (4.22 g, 20 mmol) in THF (150 mL) at rt. After 3 h the
solution was ®ltered and evaporated. The residue was
puri®ed by ¯ash chromatography (hexanes:EtOAc 4:1)
to give 6.5 g (100%) of 11: IR (®lm) 3072, 2954, 2873,
1649, 1595, 1519, 1461, 1413 cm 1. 1H NMR (CDCl3) d
8.13 (d, J=8.7 Hz, 2H), 7.34 (d, J=8.7 Hz, 2H), 5.17 (s,
1H), 4.81 (s, 1H), 4.02 (s, 2H), 3.45 (s, 2H), 0.91 (t,
J=8.1 Hz, 9H), 0.57 (c, J=8.1 Hz, 6H). 13C NMR
(CDCl3) d 147.4, 146.7, 129.8, 123.6, 112.8, 65.1, 39.4,
6.8, 4.5. HRMS (M+) for C16H25O3NSi: calcd.
307.16037, found 307.16046.
1
1559 cm 1. H NMR (CD2Cl2): d 8.44 (d, J=4.2 Hz,
1H), 7.58 (dt, J=7.8 Hz, 1H), 7.18±7.39 (m, 15H), 7.09±
7.13 (m, 2H), 6.80 (d, J=8.6 Hz, 2H), 6.30 (d,
J=8.5 Hz, 2H), 3.34 (d, J=9.6 Hz, 1H), 3.28 (d,
J=9.6 Hz, 1H), 2.87 (m, 2H), 2.64 (d, J=13.2 Hz, 1H),
2.56 (d, J=13.4 Hz, 1H), 1.82 (m, 2H), 0.94 (t,
J=7.8 Hz, 9H), 0.59 (c, 7.9 Hz, 6H); 13C NMR
(CD2Cl2): d 161.1, 149.0, 146.1, 145.4, 137.2, 130.6,
129.7, 128.4, 127.1, 126.8, 123.7, 121.6, 116.7, 72.0, 67.3,
43.0, 35.6, 32.4, 7.0, 4.6.
To a solution of silylether 11 (6.5 g, 20 mmol) in CH2Cl2
(200 mL) was added m-CPBA (9.65 g, 40 mmol, 80%).
The solution was re¯uxed for 3 h and then stirred at rt
for another 3 h. The solution was washed with NaHCO3
(satd), brine, dried, ®ltered and concentrated. The resi-
due was puri®ed by ¯ash chromatography (gradient
TBAF (2.22 mL, 2.22 mmol, 1.0 M in THF) was added
at 0 ꢀC to a solution of 14 (1.4 g, 2.22 mmol) in THF
(20 mL). After 10 min, the solvent was evaporated and
the residue was puri®ed by ¯ash chromatography (gra-
dient 0±10% acetone±EtOAc) to yield 1.03 g (90%) of
5±20% EtOAc±hexanes) to yield 6.5 g (95%) of 12: IR
1
(®lm) 3048, 2955, 2872, 1601, 1519 cm
.
1H NMR
(CDCl3) d 8.14 (d, J=8.5 Hz, 2H), 7.40 (d, J=8.5 Hz,
2H), 3.54 (s, 2H), 3.09 (s, 2H), 2.69 (d, J=4.6 Hz, 1H),
2.53 (d, J=4.6 Hz, 1H), 0.93 (t, J=8.0 Hz, 9H), 0.57 (c,
J=8.0 Hz, 6H). 13C NMR (CDCl3) d 147.3, 144.7,
130.9, 123.6, 65.1, 59.6, 50.4, 37.7, 6.9, 4.6.
15: IR (CHCl3) 3405, 3057, 3021, 2932, 1612,
1
1596 cm
.
1H NMR (CD2Cl2) d 8.43 (d, J=4.8 Hz,
1H), 7.62 (dt, J=7.7 Hz, 1H), 7.13±7.40 (m, 17H), 6.78
(d, J=8.1 Hz, 2H), 6.32 (d, J=8.7 Hz, 2H), 3.32 (d,
J=9.5 Hz, 1H), 3.28 (d, J=9.5 Hz, 1H), 2.92 (m, 2H),
2.60 (s, 2H), 1.82 (m, 2H). 13C NMR (CD2Cl2) d 161.4,
149.1, 146.3, 145.5, 137.4, 130.9, 129.8, 128.5, 127.4,
126.9, 123.8, 121.8, 116.8, 72.2, 67.7, 43.1, 35.4, 32.2.
HRMS (M+) for C35H34N2O2: calcd. 514.26203, found
514.26215.
2-Triethylsilyloxymethyl-2-[(N-trityl-4-aminophenyl)methyl]-
oxirane (13). Pd/C (cat. 10%) was added to a solution
of 12 (4.5 g, 13.25 mmol) in deoxygenated EtOAc
(200 mL). The resulting solution was stirred under H2

O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
285
1,2,3,4-Tetrahydro-3-hydroxy-3-[(N-trityl-4-aminophenyl)-
methyl]-quinolizinium methanesulphonate (16). Triethyl-
amine (279 mL, 2.0 mmol) and methanesulfonylchloride
(155 mL, 2.0 mmol) were added to a precooled (0 ꢀC)
solution of diol 15 in THF (20 mL). After 2 h, the solu-
tion was ®ltered, evaporated and the residue was puri-
®ed by ¯ash chromatography (5% MeOH in CH2Cl2).
The resulting mesylate turned out to be not very stable
and was therefore immediately submitted to the next
step. The crude product was dissolved in acetone
(100 mL) and the solution was re¯uxed overnight. The
suspension obtained was cooled to 0 ꢀC, the product
was ®ltered o€ and dried under vacuum to yield 820 mg
(70%) of 16: IR (CH3OH) 3402, 3055, 2934, 2516, 1632,
1,2,3,4-Tetrahydro-2-[(4-nitrophenyl)methyl]-isoquinoline
(19). To a stirred suspension of NaH (0.5 g, 16.5 mmol,
80%) in THF (20 mL) was added dropwise 1,2,3,4 tet-
rahydro-isoquinoline 18 (1.9 mL, 15 mmol). The result-
ing mixture was stirred at rt for 3 h. A solution of 4-
nitrobenzyl bromide (3.24 g, 15 mmol) in THF (20 mL)
was added dropwise and the reaction mixture was stir-
red overnight. The reaction mixture was ®ltered, con-
centrated and puri®ed by ¯ash chromatography to
1
a€ord 3.76 g (94%) of 19 as a yellow solid. H NMR
(CDCl3) d 8.83, (d, J=8.7 Hz, 2H), 7.58 (d, J=8.7 Hz,
2H), 7.20±7.05 (m, 3H), 7.00±6.95 (m, 1H), 3.77 (s, 2H),
3.65 (s, 2H), 2.92 (t, J=6.0 Hz, 2H), 2.67 (t, J=6.0 Hz,
2H); 13C NMR (CDCl3) dd 146.5, 134.3, 134.1, 129.4,
128.7, 126.5, 126.3, 125.7, 123.6, 77.4, 77.0, 76.6, 61.8,
56.1, 50.8, 29.1.
1
1613 cm 1. H NMR (CD3OD) d 8.53 (d, J=6.3 Hz,
1H), 8.28 (t, J=4.4 Hz, 1H), 7.83 (d, J=8.4 Hz, 1H),
7.75 (t, J=6.8 Hz, 1H), 7.08±7.32 (m, 15H), 6.74 (d,
J=8.8 Hz, 2H), 6.37 (d, J=8.3 Hz, 2H), 4.40 (d,
J=13.5 Hz, 1H), 4.27 (d, J=13.5 Hz, 1H), 3.30 (m, 2H),
2.72 (s, 2H), 2.62 (s, 3H), 1.96 (m, 1H), 1.82 (m, 1H).
13C NMR (CD3OD) d 158.0, 147.2, 146.5, 145.8, 132.5,
131.4, 130.6, 130.0, 128.9, 128.2, 127.8, 126.3, 117.8,
72.7, 70.4, 65.6, 46.6, 39.7, 27.4. HRMS (M+) for
C35H33N2O: Calcd. 497.25929, found 497.25952.
1,2,3,4-Tetrahydro-2-[(4-aminophenyl)methyl]isoquinoline
(20). A mixture of 19 (1.67 g, 6.23 mmol), Pd/C (cat.
10%) and hydrazine hydrate (1.0 g, 20 mmol) in iso-
propyl alcohol (20 mL) was re¯uxed for 24 h. After
cooling, the solution was decanted, concentrated and
puri®ed by ¯ash chromatography. Recrystallization
from acetonitrile a€orded 1.27 g (86%) of 20 as a white
1
solid. IR (®lm) 2902, 2799, 2352, 1600, 1496, 1462 cm
;
5-{4-[3-(1,2,3,4-Tetrahydro-3-hydroxy-quinolizinium meth-
anesulphonate) methyl]phenyl}-amino-5-oxopentanoic acid
(Hapten O2). A suspension of 16 (670 mg, 1.13 mmol)
in H2O:EtOAc (2:1, 35 mL), was vigorously stirred and
re¯uxed until both phases clear up (2 h). The aqueous
phase was washed with EtOAc (2Â20 mL) and the water
was azeotropically removed (coevaporation with 1:1
benzene:acetonitrile) to yield 396 mg (100%) of 17: IR
(CH3OH) 3331, 3082, 3018, 2940, 2474, 2232, 2065, 1632,
1H NMR (DMSO-d6) d 8.27 (s, 1H), 8.19 (s, 1H), 7.13
(d, J=8.4 Hz, 2H), 7.10±7.02 (m, 3H), 3.52 (s, 2H), 3.32
(s, 2H), 2.78 (t, J=6.1 Hz, 2H), 2.64 (t, J=6.1 Hz, 2H);
13C NMR (DMSO-d6) d 151.1, 135.0, 134.2, 129.7,
129.1, 128.9, 128.4, 126.3, 125.9, 125.8, 125.4, 112.6,
61.8, 55.4, 50.1, 28.8.
5-{4-[2-(1,2,3,4-Tetrahydroisoquinolyl)methyl]phenyl}amino-
5-oxo-pentanoic acid (21). A solution of 20 (1.0 g,
4.2 mmol) and glutaric anhydride (0.57 g, 5.0 mmol) in
EtOAc (40 mL) was stirred for 1 h (open to air atmo-
sphere). The reaction mixture was concentrated and
puri®ed by ¯ash chromatography (gradient 15±50%
MeOH±CH2Cl2) to yield 1.45 g (98%) of 21 as a white
1
1614 cm 1. H NMR (CD3OD) d 8.54 (d, J=5.7 Hz,
1H), 8.29 (t, J=6.9 Hz, 1H), 7.82 (d, J=7.5 Hz, 1H),
7.74 (t, J=6.3 Hz, 1H), 6.99 (d, J=8.4 Hz, 2H), 6.63 (d,
J=8.4 Hz, 2H), 4.47 (d, J=14.1 Hz, 1H), 4.33 (d,
J=13.2 Hz, 1H), 3.31 (m, 2H), 2.83 (s, 2H), 2.62 (s, 3H),
2.01 (m, 1H), 1.87 (m, 1H); 13C NMR (CD3OD) d
157.8, 147.9, 146.3, 145.7, 132.4, 129.4, 126.4, 125.8,
116.6, 70.3, 65.4, 65.3, 46.4, 39.6, 27.3. HRMS (M+) for
C16H19N2O: calcd. 255.14974, found 255.14975.
1
solid which was recrystallized from EtOAc/hexanes. H
NMR (DMSO-d6) d 9.88 (s, 1H), 7.41 (d, J=8.4 Hz),
7.25 (d, J=8.4 Hz), 7.12±7.02 (m, 3 H), 7.02±6.95 (m, 1
H), 3.55 (s, 2H), 3.16 (s, 2H), 2.78 (t, J=6.1 Hz, 2H),
2.64 (t, J=6.1 Hz, 2H), 2.33 (t, J=7.8 Hz, 2H), 2.26 (t,
J=7.2 Hz, 2H), 1.79 (q, J=7.4 Hz, 2H). 13C NMR
(DMSO-d6) d 173.6, 170.1, 137.6, 134.2, 133.6, 132.2,
128.5, 127.8, 125.8, 125.4, 124.9, 118.4, 60.9, 54.8, 49.6,
34.9, 32.5, 28.1, 20.0. HRMS (M+) for C21H24N2O3:
calcd 354.4332, found 354.4335.
To a solution of the resulting aniline quinolizinium salt
17 (396 mg, 1.13 mmol) in acetonitrile (15 mL) was
added glutaric anhydride (193 mg, 1.64 mmol) at rt.
After 36 h, the solvent was evaporated and the residue
redissolved in water. The aqueous solution was washed
with EtOAc and then azeotropically evaporated (coevap-
oration with 1:1 benzene:acetonitrile) to yield 460 mg
(88%) of hapten O2: IR (CH3OH) 3412, 3107, 3048,
5-{4-[2-(1,2,3,4-Tetrahydroisoquinolyl-N-oxide)methyl]-
phenyl}amino-5-oxo-pentanoic acid (hapten O3). To a
stirred solution of 21 (0.446 g, 1.27 mmol) in THF
(10 mL) was added m-CPBA (386 mg, 1.9 mmol, 85%)
over 2 min. After 10 min, the reaction was complete and
the precipitated product was ®ltered and the residue
washed with THF and CH2Cl2, and dried under
1
2931, 2647, 1719, 1666, 1628 cm 1. H NMR (CD3OD)
d 8.54 (d, J=6.3 Hz, 1H), 8.29 (t, J=7.7 Hz, 1H), 7.83
(d, J=8.1 Hz, 1H), 7.73 (t, J=6.5 Hz, 1H), 7.43 (d,
J=8.4 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 4.48 (d,
J=14.1 Hz, 1H), 4.29 (d, J=14.1 Hz, 1H), 3.31 (m, 2H),
2.90 (s, 2H), 2.58 (s, 3H), 2.28 (m, 4H), 2.02 (m, 1H),
1.86 (m, 2H), 1.78 (m, 1H); 13C NMR (D2O) d 180.7,
177.5, 158.5, 147.4, 147.2, 138.5, 134.8, 134.0, 131.2,
127.8, 124.7, 72.1, 67.0, 66.0, 47.2, 41.1, 38.1, 35.7, 31.5,
28.3. HRMS (M+) for C21H25N2O4: calcd. 369.18143,
found 369.18092.
1
vacuum to give 400 mg (97%) of hapten O2: H NMR
(DMSO-d6) d 10.13 (s, 1H), 7.63 (d, J=8.4 Hz, 2H),
7.49 (d, J=8.4 Hz, 2H), 7.23±7.10 (m, 3H), 6.98 (d,
J=8.2 Hz, 2H), 4.64 (s, 3H), 4.17 (d, J=15.1 Hz, 2H),
3.70±3.54 (m, 3H), 3.34±3.18 (m, 3H), 2.32 (t,
J=7.4 Hz, 2H), 2.23 (t, J=7.4 Hz, 2H), 1.83±1.69 (m,

286
O. Ersoy et al. / Bioorg. Med. Chem. 7 (1999) 279±286
2H). 13C NMR (DMSO-d6) d 175.5, 171.3, 140.4, 133.4,
131.5, 129.1, 128.1, 127.1, 126.8, 126.2, 123.8, 118.5,
71.0, 67.0, 64.0, 35.8, 34.5, 25.1, 21.0; HRMS (M+) for
C21H24N2O4: calcd. 370.4326, found 368.4323.
Gibbs, R. A.; Taylor, S.; Benkovic, S. J. Science 1992, 258,
803. (i) Liotta, L. J.; Benkovic, P. A.; Miller, G. P.; Benkovic,
S. J. J. Am. Chem. Soc. 1993, 115, 350.
3. (a) Ersoy, O.; Fleck, R.; Sinskey, A. J.; Masamune, S. J.
Am. Chem. Soc. 1996, 118, 13077. (b) Ersoy, O.; Fleck, R.;
Sinskey, A. J.; Masamune, S. J. Am. Chem. Soc. 1998, 120,
817.
Acknowledgements
4. (a) Carter, P.; Wells, J. A. Nature 1988, 332, 564. (b) Fink,
A. L. In Enzyme Mechanisms; Page, M. I.; Williams, A., Eds.;
The Royal Society of Chemistry: London, 1987; pp 229±239.
5. Mikolajczyk, M.; Grzejszczak, G.; Midura, W.; Zatorski,
A. Synthesis 1976, 396.
S.M. acknowledges generous support from the National
Science Foundation (CHE-9424283) and Kao Corpora-
tion, Japan. M.J.B. thanks the Spanish Ministry of
Education for a postdoctoral fellowship.
6. Jacobsen, N. E.; Bartlett, P. A. J. Am. Chem. Soc. 1981,
103, 654.
7. Campagne, J.-M.; Coste, J.; Jouin, P. Tetrahedron Lett.
1993, 34, 6743.
References and Notes
1. (a) Suga, H.; Ersoy, O.; Tsumuraya, T.; Lee, J.; Sinskey, A.
J.; Masamune, S. J. Am. Chem. Soc. 1994, 116, 487. (b) Suga,
H.; Ersoy, O.; Williams, S. F.; Tsumuraya, T.; Margolies, M.
N.; Sinskey, A. J.; Masamune, S. J. Am. Chem. Soc. 1994, 116,
6025. (c) Tsumuraya, T.; Suga, H.; Meguro, S.; Tsukanawa,
A.; Masamune, S. J. Am. Chem. Soc. 1995, 117, 11390.
8. Sampson, N. S.; Bartlett, P. A. J. Org. Chem. 1988, 53,
4500.
9. Musiol, H.-J.; Grams, F.; Rudolph-Bohner, S.; Moroder,
L. J. Org. Chem. 1994, 59, 6144.
10. Clark, R. D.; Kozar, L. G.; Heathcock, C. H. Synthesis
1975, 635.
2. (a) Lerner, R. A.; Benkovic, S. J.; Schultz, P. G. Science
1991, 252, 659. (b) Schultz, P. G.; Lerner, R. A. Acc. Chem.
Res. 1993, 26, 391. (c) Schultz, P. G.; Lerner, R. A. Science
1995, 269, 1835. (d) Macbeath, G.; Hilvert, D. Chem. Biol.
1996, 3, 433. (e) Janda, K. D.; Schloeder, D.; Benkovic, S. J.;
Lerner, R. A. Science 1988, 241, 1188. (f) Pollack, S. J.; Hsiun,
P.; Schultz, P. G. J. Am. Chem. Soc. 1989, 111, 5961. (g)
Iverson, B. L.; Lerner, R. A. Science 1989, 243, 1184. (h)
11. Ravikumar, V. T.; Swaminathan, S.; Rajagopalan, K.
Tetrahedron Lett. 1984, 25, 6045.
12. Lipshutz, B. H.; Wilhelm, R. S.; Kozlowski, J. A.; Parker,
D. J. Org. Chem. 1984, 49, 3928.
13. Begley, M. J.; Crabb, T. A.; Roch, O. J. Magn. Reson.
Chem. 1986, 24, 292.
14. Undheim, K.; Nordal, V.; Tjùnneland, K. Acta Chem.
Scan. 1969, 23, 1704.