A catalytic antibody against a tocopherol cyclase inhibitor

Article abstract of DOI:10.1002/chem.200305629

The cyclic ammonium cation 5 and its guanidinium analogue 4 areinhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of

Full text of DOI:10.1002/chem.200305629

FULL PAPER
A Catalytic Antibody against a Tocopherol Cyclase Inhibitor
Roman Manetsch,[a, c] Lei Zheng,[b] Martine T. Reymond,[b] Wolf-Dietrich Woggon,*[a] and
Jean-Louis Reymond*[b]
Abstract: The cyclic ammonium cation
5 and its guanidinium analogue 4 are
inhibitors of tocopherol cyclase. Mono-
clonal antibodies were raised against
protein conjugates of the haptens 1 3
and screened for catalytic reactions
with alkene 8, a short chain analogue
of the natural substrate phytyl-hydro-
quinone 6, and its enol ether analogues
10a,b. Antibody 16E7 raised against
hapten 3 was found to catalyze the hy-
drolysis of Z enol ether 10a to form
hemiacetal 12 with an apparent rate ac-
celeration of kcat/kuncat =1400. Antibody
16E7 also catalyzed the elimination of
Kemp×s benzisoxazole 59. The absence
of cyclization in the reaction of enol
ether 10a was attributed to the compe-
tition of water molecules for the oxo-
carbonium cation intermediate within
the antibody binding pocket. Hapten
and reaction design features contribu-
ting to this outcome are discussed. An-
tibody 16E7 provides the first example
of a carboxyl group acting both as an
acid in an intrinsically acid-catalyzed
process and as a base in an intrinsically
base-catalyzed process, as expected
from first principles. In contrast to the
many examples of general-acid-cata-
lyzed processes known to be catalyzed
by catalytic antibodies, the specific-
acid-catalyzed cyclization of phytyl-hy-
droquinone 6 or its analogue 8 still
eludes antibody catalysis.
Keywords: acid catalysis ¥ base cat-
alysis ¥ catalytic antibodies ¥ cycliza-
tion ¥ transition-state theory
Introduction
gation towards a tocopherol cyclase catalytic antibody. We
describe the preparation of catalytic antibody 16E7, ob-
tained from immunizations against transition-state ana-
logues 1 3 of the tocopherol cyclase reaction, and its catalyt-
ic reactivity (Scheme 1).
The concept of transition-state analogy has provided an in-
credibly productive guiding principle for making enzyme in-
hibitors[1] and has also led to the discovery of catalytic anti-
bodies as tailor-made catalysts.[2] The majority of catalytic
antibodies reported to date accelerate either ester/amide-
bond hydrolysis or pericyclic reactions.[3] By contrast, only a
very few examples of catalytic antibodies for acid-catalyzed
processes have been reported. Herein we report our investi-
[a] Dr. R. Manetsch, Prof. W.-D. Woggon
Department of Chemistry
University of Basel
St. Johanns-Ring 19, 4056 Basel (Switzerland)
Fax : (+41) 61-267-11-02
E-mail: wolf-d.woggon@unibas.ch
[b] Dr. L. Zheng, M. T. Reymond, Prof. J.-L. Reymond
Department of Chemistry and Biochemistry
University of Berne
Scheme 1. Transition state analogues 1 3 of the tocopherol cyclase reac-
tion and inhibitors 4 and 5 of the enzyme tocopherol cyclase.
Freiestrasse 3, 3012 Berne (Switzerland)
Fax : (+41) 31-631-80-57
E-mail: jean-louis.reymond@ioc.unibe.ch
[c] Dr. R. Manetsch
Results
Current address:
The Scripps Research Institute
10550 North Torrey Pines Road, BCC-315
La Jolla, CA 92037 (USA)
The acid-promoted cyclization of phytyl-hydroquinone 6 to
g-tocopherol (7) is the key step in the biosynthesis of the
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DOI: 10.1002/chem.200305629
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
chromanol substructure of the vitamin E family
(Scheme 2).[4] This reaction is of potential industrial interest
for the preparation of this important food additive. This
enzyme-catalyzed ring closure proceeds by Si protonation of
the double bond of 6 and concomittant Re attack of the phe-
nolic oxygen atom.[5] Although the exact nature of the cata-
lytic machinery of this enzyme is not known, the reaction
mechanism clearly requires an acidic residue for catalysis.
strate 8 to form product 9. By contrast, antbodies have been
almost exclusively described for general-acid-catalyzed proc-
esses, which are promoted by weak acids, usually a carboxy-
late side chain within the antibody binding pocket.[8] We
therefore also prepared Z and E enol ethers 10a and 10b.
These substrates undergo a cyclization similar to that of sub-
strate 8 to form acetal 11 or hemiacetal 12, with the advant-
age that the reaction can be promoted by weak acids such
as acetic acid.[9] Additional test substrates were designed to
back up the search for catalysis. These included methylene
derivative 35, enol ethers 44a,b and 45a,b, vinyl ether 50,
and unsaturated esters 55 and 56, all of which might poten-
tially undergo an intramolecular cyclization reaction with
the phenolic hydroxy group.
The design of haptens possibly leading to antibodies pro-
moting specific-acid catalysis of the tocopherol cyclase reac-
tion followed the same principles as those used in previous
systems for acid catalysis. The design should emphasize elec-
trostatic complementarity to the transition state, that is, pos-
itively charged functional groups in the hapten, since specif-
ic-acid catalysis should be triggered not by the presence of a
carboxyl group but by a local electrostatic effect stabilizing
positively charged intermediates more strongly than the sol-
vent water.[10] The quaternary tetrahydroquinolinium cations
1 and 2 and the guanidinium analogue 3 were selected as
transition-state analogues for the reaction. The guanidinium
cation 4 and the ammonium cation 5 are nanomolar inhibi-
tors of the enzyme tocopherol cyclase[11] and related ammo-
nium cations have been used frequently to induce catalytic
antibodies for related acid-catalyzed processes.[8a,c,12] The
guanidinium cation 3 was designed along the lines of related
compounds used previously as haptens to raise catalytic an-
tibodies.[13] A carboxylate was expected on the antibody as a
charge-neutralizing group against the guanidinium group.
Scheme 2. Cyclization of phytyl-hydroquinone 6 to g-tocopherol (7) cata-
lyzed by the enzyme tocopherol cyclase and corresponding model reac-
tions with olefin 8, enol ether 10a and enol ether 10b that were used for
the selection of catalytic antibodies.
Synthesis: Tetrahydroquinolinium hapten 1 was obtained by
quaternization of 2-methyl-6-methoxytetrahydroquinoline
(13) with ethyl 8-bromo-octanoate[14] (Scheme 3). It was
conjugated to carrier proteins KLH (keyhole limpet hemo-
cyanin) and BSA (bovine serum albumin) through its N-hy-
droxysuccinimide ester. Hapten 2 was synthesized by reduc-
tive amination of 6-methoxytetrahydroquinoline (16) with
aldehyde 15. Alkylation of the resulting amine 17 with di-
methylsulfate gave ammonium compound 18. Acidic hydrol-
ysis of the ester gave hapten 2. Conjugation to carrier pro-
teins was carried out as for hapten 1. Guanidinium hapten 3
was prepared from 5-hydroxy-2-nitrobenzaldehyde (19),
which was first protected as the corresponding methyl ether
20 and then transformed into the benzonitrile 21[15] (see
Scheme 4). Reduction of the nitro group with stannous chlo-
ride gave 22, and reduction of the cyano group with LiAlH4/
AlCl3 yielded o-aminobenzylamine 23 in moderate yields.
The thione 24 was prepared by condensation between ben-
zylamine 23 and thiophosgene and was alkylated to give 25.
Substitution with ethyl 8-amino-octanoate[16] gave 26, which
was hydrolyzed under acidic conditions to give hapten 3.
Conjugation to carrier proteins was carried out as above.
Reaction and hapten design: Due to its long isoprene side
chain, phytyl-hydroquinone 6 is strongly hydrophobic and
only poorly soluble in water. We initially decided to delete
this sesquiterpene side chain, which is only important for
recognition by the enzyme,[6] in order to enhance water solu-
bility and therefore enable a reaction setup in the homoge-
neous phase, where most catalytic antibodies have been de-
scribed. The second problematic element in the natural sub-
strate 6 is the dimethylhydroquinone nucleus, which is both
synthetically demanding and highly sensitive to air oxida-
tion. Substitution by a simple ortho-substituted p-methoxy-
phenol would allow a shorter synthesis and would essentially
suppress oxidation sensitivity, while preserving the chemical
reactivity of the system towards cyclization. Modification of
the g-tocopherol precursor 6 along these lines led to the al-
lylic p-methoxyphenol 8 as a model substrate (Scheme 2).
The cyclization of phytyl-hydroquinone 6 is specific-acid
catalyzed, which means that in solution only strong acids
can promote the reaction.[7] Preliminary investigations
showed that this was also the case for the cyclization of sub-
Substrate
8 was prepared according to a reported
method[17] and its cyclization product 9 was obtained by
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Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
fluoride reagents failed in this case due to the reactivity of
the enol ether function. Nevertheless, desilylation proceeded
smoothly by treatment with ethanolamine, to give the de-
sired hydroxy enol ethers. Purification of the isomeric mix-
tures 10a,b and 44a,b by preparative RP-HPLC provided
the isomerically pure enol ethers. In the case of the enol
ether 45a,b, the separation of the E,Z isomers was per-
formed easily by simple column chromatography. Treatment
of acetal 36 with aqueous hydrochloric acid afforded the hy-
drolysis product 12, which in turn was transformed into the
corresponding cyclization acetals 11, 42, and 43 according to
a known procedure.[20]
Scheme 3. Synthesis of haptens 1 and 2. a) Br(CH2)7CO2Et, DMF, 608C,
3h, 84%; b) 1 n HCl/DMF 1:1, RT, 24 h, RP18 HPLC, 71%; c) NaBH-
(OAc)3, DCE, RT, 2 h, 74%; d) dimethylsulfate, CH3CN, RT, 16 h, RP18
HPLC, 55%; e) 4m HCl in dioxane, H2O, RT, 6 h, RP18 HPLC 71%.
DCE=1,2-dichloroethane.
Scheme 4. Synthesis of hapten 3. a) Cs2CO3, DMF, 90 min; MeI, RT, 15 h,
96%; b) H2NOH¥HCl, MgSO4, p-TSA, toluene, D, 15 h, 88%;
c) SnCl2¥2H2O, MeOH, D, 3h, 56%; d) LiAlH 4, AlCl3, Et2O, RT, 5 h,
78%; e) thiophosgene, Et3N, THF, ꢀ788C!RT, 8 h, 84%; f) EtBr,
EtOH, D, 14 h, 98%; g) H2N(CH2)7CO2Et, EtOH, D, 24 h, 65%; h) 1n
HCl/THF, RT, 24 h, RP18 HPLC, 87%. p-TSA=para-toluenesulfonic
acid.
Scheme 5. Synthesis of substrates 35, 10, 44, and 45 as well as the corre-
sponding reference compounds 12, 11, 42, and 43. a) NaH, THF, allyl
bromide, D, 4 h, 95%; b) neat, 2408C, 45 min, 89%; c) TBSCl, imidazole,
DMF, RT, 1 h, 96%; d) BH3¥THF, THF, RT, 2 h; NaOH, H2O2, 08C!RT,
89%; e) DMSO, Et3N, CH2Cl2, oxalyl chloride, ꢀ788C!RT, 90 min,
94%; f) CH3MgCl, THF, RT, 1 h, 89%; g) DMSO, Et3N, CH2Cl2, oxalyl
chloride, ꢀ788C!RT, 1 h, 95%; h) methyltriphenylphosphonium bro-
mide, nBuLi, THF, 08C, 60 min, 75%; i) TBAF, THF, 20 min, 93%; j) for
39: (iPr)2NEt, TMSTf, CH2Cl2 ꢀ78!ꢀ208C for 4 h, 59%; for 40:
(iPr)2NEt, TMSTf, CH2Cl2 ꢀ78!ꢀ208C for 4 h, 63%; for 41: HMDS,
TMSI, CCl4, ꢀ158C for 30 min, 758C for 5 h, 29%; k) for 10: ethanola-
mine, 908C, 4 h, prep. RP18 HPLC, 46% 10b and 44% 10a; for 44: etha-
nolamine, 908C, 3h, prep. RP18 HPLC, 46% 44b and 37% 44a; for 45:
ethanolamine, 908C, 3h, 47% 45b and 45% 45a; l) dioxane/18% HCl
25:1, 508C, 18 h, 94%; m) for 11: thionyl chloride, CH2Cl2, 08C, 15 min;
MeOH, 08C, 15 min, 72%; for 42: thionyl chloride, CH2Cl2, 08C, 15 min;
EtOH, 08C, 15 min, 86%; for 43: thionyl chloride, CH2Cl2, 08C, 15 min;
iPrOH, 08C, 15 min, 76%. TBS=tert-butyldimethylsilyl, TBAF=tetrabu-
tylammonium fluoride, Tf=triflate=trifluoromethanesulfonyl, HMDS=
1,1,1,3,3,3-hexamethyldisilazane.
treatment of 8 with boron trifluoride etherate. Its methylene
analogue 35, poised for exo-trig cyclization, was prepared
from anisole 27 (Scheme 5). O-allylation, Claisen rearrange-
ment, and silylation gave ether 30, which was transformed in
four steps into methyl ketone 34. Wittig reaction with meth-
yltriphenylphosphonium bromide and deprotection gave
substrate 35. Enol ethers 10a,b, 44a,b, and 45a,b were ob-
tained from the corresponding acetals 36 38 by reaction
with trimethylsilyl triflate[18] or trimethylsilyl iodide[19] fol-
lowed by desilylation. Classical desilylation procedures with
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W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
Vinyl ether 50 was synthesized from 2-hydroxy-4-methox-
ybenzaldehyde 46 by silylation to 47, reduction with sodium
borohydride to 48, transetherification catalyzed by
[21]
Hg(OAc)2 to form 49, and mild desilylation with ethanol-
amine (Scheme 6). Reduction of 46 with lithium aluminium
hydride gave benzylalcohol 51, which was transformed into
acetal 52 by acidic treatment with acetaldehyde.[22] Wit-
tig Horner olefination of aldehyde 32 gave the E-configured
a,b-unsaturated ester 53, which was desilylated with TBAF
to yield 55 together with its cyclization product 57 in 56 and
27% yield, respectively. Application of the Still Gennari
variant of the Wittig Horner olefination to aldehyde 32
gave the Z-configured a,b-unsaturated ester 54. Deprotec-
tion with TBAF provide the pure Z isomer 56 together with
its cyclization product 58 in 38 and 39% yield, respectively.
Immunizations and hybridoma selection: The KLH conju-
gates of the haptens were used for immunization following
standard procedures by using either single-hapten or mixed
immunizations (Table 1). Hybridoma generation and initial
screening by ELISA against hapten BSA conjugates gave a
total of 131 positives. Further cell culture up to a volume of
5 mL gave 27 hybridoma cell lines, which were assayed for
catalysis of the cyclization of olefin 8 and enol ethers 10a
and 10b by HPLC. Hybridoma 16E7 (anti-3) and 16G7
(anti-2) catalyzed the hydrolysis of enol ether 10a and their
activity was quantitatively inhibited by addition of the re-
spective haptens. Both hybridomas were subcloned twice to
give stable and homogeneous cell lines. The catalytic activity
and the hapten binding activities were preserved during the
cloning process. Antibodies
Scheme 6. Synthesis of substrates 50, 55, and 56 as well as the corre-
sponding reference compounds 51, 52, 57, and 58. a) (iPr)2NEt, DMF,
TBSCl, 45 min, RT, 97%; b) NaBH4, EtOH, RT, 45 min, 91%; c) ethyl
vinyl ether, Hg(OAc)2 (cat), D, 72%; d) ethanolamine, RT, 16 h, 94%;
e) LiAlH4, THF, RT, 15 min, 84%; f) acetaldehyde, H2SO4, 08C, 4 h,
91%; g) phosphonoacetic acid triethyl ester, NaH, THF, 08C!RT, 14 h,
77%; h) TBAF, THF, RT, 5 min, 56% 55 and 27% 57; i) phosphonoacetic
acid P,P-bis(2,2,2-trifluoroethyl)methyl ester, [18]crown-6, KHDMS,
THF, ꢀ788C, 90 min, 96%; j) TBAF, THF, RT, 5 min, 38% 56 and 39%
58. KHDMS=potassium hexamethyldisilazanide.
were produced from the cell
lines by in vitro cell culture up
Table 1. Data for immunization with haptens 1 3 and screening for catalysis with substrates 8, 10a, and 10b.
to a volume of 1 L and were
purified by ammonium sulfate
precipitation followed by prote-
in G affinity-column purifica-
tion.
Hapten[a] Number
Serum titers Number
of
Number of
screened
Number of
catalytic hybrido-
ma[f]
Clone
name
and sub-
type
of
mice[b]
(conjugate)[c] binders[d]
cell lines[e]
1/1/1
2/2/2
2
4
1 mouse 6400 and
22
5
4
1
0
1
1 mouse 3200 (1 BSA)
3mice 25600 and
1 mouse 12800 (2
BSA)
16G7
kg1
Kinetic characterization: Anti-
body 16E7 was studied in detail
since assays with purified anti-
bodies showed it to be the
more active protein. The anti-
body catalyzed the hydrolysis
of the Z enol ether 10a to form
hemiacetal 12 with multiple
turnover. The reaction was
3/3/3
1/3/3
2
2
2 mice 12800 (3 BSA)
40
3
9
4
1
16E7
kg2a
0
1 mouse 12800 25600
and
5
1 mouse 6400 (1 BSA)
1 mouse 12800 and
1 mouse 0 (3 BSA)
3/1/1
2
30
8
1
[a] First, second, and final boost of the corresponding hapten conjugates used for the immunization. [b] In
total, twelve 129GIX+ mice were immunized according to standard procedures. [c] Dilution factor of blood
serum (after immunization with the hapten KLH conjugates) for 50% reduction of ELISA signal against the
hapten BSA conjugates. A high number indicates a strong immune response. [d] Number of cell lines that test
positive for binding by ELISA against the hapten BSA conjugate after fusion. [e] Number of cell lines that
were grown up to a volume of 5 mL for catalysis testing. [f] Number of fully subcloned and stabilized hybrido-
ma producing antibodies with catalytic properties.
quantitatively
inhibited
by
hapten 3, with the indication of
two active sites per antibody
molecule. Isomeric E enol ether
10b was not accepted as a sub-
strate. The antibody showed a
pronounced hysteresis effect, in
that it required up to eight hours to reach its full catalytic
activity (Figure 1). Similar kinetic retardation effects have
been described in other catalytic antibodies.[23] The kinetics
of antibody 16E7 followed Michaelis Menten kinetics in
both the initial phase and the steady-state phase of conver-
sion, with a result indicating a rate acceleration in the range
of kcat/kuncat =1400 (Table 2), which is a typical value for anti-
body catalysis.
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Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
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Substrate variation studies: Investigations with the pure an-
tibody 16E7 showed that the tocopherol cyclase like cycliza-
tion of the olefinic phenol 8 to form 9 was not catalyzed by
the antibody, even under very low pH conditions and in con-
centrated antibody solution. Even the noncatalyzed reaction
could not be detected under these conditions. The formation
of hemiacetal 12 instead of acetal 11 in the reaction of enol
ether 10a might be caused by a competing water molecule
in the active site. Therefore, the ethyl enol ether 44a,b and
its isopropyl analogue 45a,b were also tested with 16E7,
since these more hydrophobic substrates might reduce the
water content of the active site and suppress the water reac-
tion, thereby leading to hydrolysis. Although both 44a and
45a were accepted as substrates, their reaction led exclusive-
ly to the formation of hemiacetal 12. Acetal 11 was not hy-
drolyzed by the antibody and also did not show any compet-
itive inhibition of the enol ether hydrolysis, a result implying
that it was very poorly recognized by the antibody.
A series of alternative substrates that would potentially
lead to cyclized products under acid base catalysis was
tested for catalysis by antibody 16E7. The methylene ana-
logue 35 might be protonated to form the same carbocation-
ic intermediate as that expected with the original substrate
8, yet starting with an energetically higher point (a disubsti-
tuted versus a trisubstituted olefin) and following a more fa-
vorable overall exo-trig cyclization pathway to the desired
cyclized product as opposed to a relatively disfavored endo-
trig process for the tocopherol cyclase like cyclization of 8.
Similarly, vinyl ether 50 might be as reactive as enol ether
10a and also lead to a cyclized product following a favored
overall exo-trig process. Finally, the unsaturated hydroxy
esters 55 and 56 were expected to undergo a facile acid
base-catalyzed 1,4-addition of the hydroxy group to form 57
and 58, respectively. While all of these substrates indeed un-
derwent the expected processes in organic solvent upon
treatment with strong acid, only vinyl ether 50 was accepted
as a substrate by antibody 16E7 to give alcohol 51 without
cyclization.
Figure 1. Kinetic measurements of the hydrolysis of enol ether 10a by an-
tibody 16E7. All data shown have already been corrected for the uncata-
lyzed reaction. All assays were performed at an antibody concentration
of 0.2mgmLꢀ1 (3.33 mm active-site concentration) in 104 mm NaCl and
29 mm BisTris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) in
water with 10% acetonitrile at 378C and pH 6.24. a) The time-course
plot observed for hydrolysis of enol ether 10a (300 mm) catalyzed by anti-
body 16E7. In general, three parameters can be graphically estimated
from a time-course plot.[42] The initial velocity vi and the steady-state ve-
locity vss can each be obtained by simple linear regression and the appa-
rent rate constant tꢀ1 can be calculated from the intercept of the vss slope
on the time axis (see Equation (1) in the Experimental Section). b) Mi-
chaelis Menten plot for the hydrolysis of the enol ether 10a in the ad-
vanced stages of the catalysis, with constant vss velocities.
Table 2. Kinetic parameters for reactions catalyzed by antibody 16E7.[a]
Substrates
Enol ethers[b]
Benzisoxazoles[c]
10a
44a
45a
50
59
67
[d]
vi [mmsꢀ1
vss [mmsꢀ1
t [h][h]
]
3.22î10ꢀ4[e]
1.75î10ꢀ3[e]
7.9[e]
3.19î10ꢀ4[e]
1.68î10ꢀ3[e]
8.1[e]
1.56î10ꢀ4[e]
1.45î10ꢀ3[e]
8.2[e]
n.d.
n.d.
n.d.
1.23î10ꢀ2[f]
2.34î10ꢀ2[f]
1.2[f]
3.08î10ꢀ3[f]
1.23î10ꢀ2[f]
9.1[f]
[g]
]
kcat [sꢀ1
]
1.11î10ꢀ3
7.99î10ꢀ7
1392
6.93î10ꢀ4
1.66î10ꢀ6
417
5.56î10ꢀ4
4.78î10ꢀ6
116
7.42î10ꢀ4
8.02î10ꢀ6
92
n.d.
n.d.
kuncat [sꢀ1
kcat/kuncat
KM [mm]
]
3.62î10ꢀ6
n.d.
5.96î10ꢀ7
n.d.
171
40
107
132
n.d.
n.d.
kcat/KM [mmꢀ1 sꢀ1
KTS [mm][j]
]
6.47î10ꢀ6
0.124
1.73î10ꢀ5
0.096
5.20î10ꢀ6
0.919
5.64î10ꢀ6
1.432.79î10
1.30î10ꢀ4[i]
7.19î10ꢀ5[i]
8.29î10ꢀ3
ꢀ2
[a] All assays were measured in 104 mm NaCl and 29 mm BisTris in water with 10% acetonitrile at 378C and pH 6.24. [b] All assays were performed at
an active-site concentration of 3.33 mm. [c] All assays were performed at an active-site concentration of 0.32 mm. [d] vi is the initial velocity. [e] For the de-
termination of the values vi, vss, and t an initial substrate concentration of 300 mm was used. [f] For the determination of the values vi, vss, and t an initial
substrate concentration of 671 mm was used. [g] vss is the steady-state velocity. [h] tꢀ1 is the observed rate of the transition between the initial state and
the steady state. [i] The low solubility of 59 and 67 prevented measurements at substrate concentrations above 2500 mm and hence only exact values of
kcat/KM could be obtained. [j] KTS is defined as the dissociation constant of the complex between the catalyst and the transition state of the reaction and
can be calculated by following equation: KTS =kuncat/(kcat/KM).
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W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
Ring-opening reactions: Since our antibody 16E7 did not
induce any acid-catalyzed cyclization, we set out to test its
ability to promote a reverse ring-opening base-catalyzed
process. The deprotonation of Kemp×s benzisoxazole 59,
which undergoes precisely such a base-catalyzed ring-open-
ing process,[24] was therefore investigated (Scheme 7). Two
previous accounts of catalysis of this reaction by antibodies
raised against guanidinium and amidinium haptens similar
to guanidinium 3 were taken into account.[13]
&
Figure 2. The time-course for the decomposition of benzisoxazole 59 ( )
and its deuterated analogue 67 (&). All assays were performed at an anti-
body active-site concentration of 0.32mm and at an initial substrate con-
centration of 671mm in 104 mm NaCl and 29 mm BisTris in water with
10% acetonitrile at 378C and pH 6.24.
form 62 and THP deprotection afforded the desired benzal-
dehyde 63 in good yields. Aldehyde 63 was then trans-
formed by the known procedures[27] to the nitrobenzisoxa-
zole 67.
The comparison of reaction rates between the benzisoxa-
zole 59 and its deuterated analogue 67 was complicated by
the hysteresis effect. Indeed, 59 was converted by more than
30% before the antibody had reached full activity, so the re-
action rate was not approaching its true maximum at any
point during the reaction. With the slower-reacting deuterat-
ed substrate 67, the fully active state of the antibody was
reached at a lower conversion, thereby allowing the rate to
approach closer to a true maximum. Considering these limi-
tations, we estimated that the kinetic isotope effect on the
deprotonation reaction is approximately 2 4, which is con-
sistent with rate-limiting proton transfer and the critical role
of acid base catalysis in the antibody-catalyzed process.
Similar isotope effects have been observed with other cata-
lytic antibodies for the same reaction.[13]
Scheme 7. Deprotonation of Kemp×s benzisoxazole 59 by antibody 16E7
and synthesis of deuterated benzisoxazole 67. a) LiAlD4, THF, 12 h, D,
86%; b) different oxidation methods, low yields; c) nBuLi, Et2O, ꢀ108C,
2 h; [D7]DMF, RT, 8 h, 97%; d) aqueous 1m HCl/THF 1:1; e) hydroxyla-
mine-O-sulfonic acid, Na2SO4, H2O, CH2Cl2; NaHCO3, RT, 1 h, 82%;
f) H2NO3/H2SO4, 08C, 30 min, 53%. Ab=antibody, THP=tetrahydropyr-
an.
Antibody 16E7 efficiently catalyzed the ring-opening re-
action of benzisoxazole 59 to form the yellow cyanonitro-
phenolate 60. As for the hydrolysis of enol ether 10a, the re-
action was quantitatively inhibited by hapten 3, a result
demonstrating that both reactions were taking place within
the antibody binding pocket. A retardation effect similar to
that detected with enol ether 10a was observed (Figure 2).
While there was no detectable substrate binding under the
accessible substrate concentration range (Michaelis Menten
constant of KM >2.5 mm), the catalysis was efficient with a
transition-state dissociation constant of KTS =27.9 nm, which
corresponds to a rate enhancement of 8î106 over the corre-
sponding acetate-catalyzed reaction, if a carboxylate catalyt-
ic group is assumed. Rate-limiting proton transfer was con-
firmed by comparing the kinetics of benzisoxazole 59 with
its deuterated analogue 67.
Initially, compound 67 was prepared by a reported reac-
tion sequence,[25] which caused unexpected difficulties. De-
spite repeated attempts, the oxidation of the deuterated
benzylalcohol 65 provided the corresponding benzaldehyde
63 only in very poor yields, presumably by a primary isotope
effect. We therefore developed an alternative synthesis.
Lithiation of the THP-protected bromophenol 61[26] fol-
lowed by treatment with [D7]-N,N-dimethylformamide to
Discussion
The cyclization of phytyl-hydroquinone 6 to g-tocopherol
(7) is, in principle, a two-step process requiring protonation
of the double bond followed by trapping of the intermediate
carbocation by the hydroxy group. It is believed that preio-
nization of the phenol to a phenolate occurs in the enzyme
binding pocket to favor double-bond protonation, thereby
resulting de facto in a single-step process without an actual
carbocation intermediate. In any case, the enantioselectivity
of the enzyme-catalyzed cyclization implies that the enzyme
induces and immobilizes a single enantiomeric conformation
of the substrate during the reaction.
Tetrahydroquinolinium haptens 1 and 2 provide almost
perfect transition-state analogues: the molecules perfectly
mimic the product-like geometry of a late transition state
while displaying the positive charge of the carbocationic in-
termediate. The cyclic nature of these transition-state ana-
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Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
logues should ensure that the complementary binding pock-
ets of the induced antibodies are able to preorganize the
acyclic precursors for cyclization. The same reasoning ap-
plies, although less perfectly, to the corresponding guanidini-
um hapten 3. Within the framework of an immunization ex-
periment, the positive charge in the haptens 1 3 naturally
induces a charge-neutralizing carboxylate, thereby providing
the necessary catalytic machinery for the reaction. The full
positive charge available in the haptens at neutral pH values
also ensures electrostatic complementarity in the binding
pocket.
As far as can be judged from binding studies by ELISA,
the isolated catalytic antibody 16E7 displays the expected
binding properties, since it binds with both its original
hapten 3 and the quaternary ammonium cations 1 and 2.
This suggests that the antibody binding pocket is indeed
complementary to the geometry of the cyclic product. The
efficient ring-opening reaction with benzisoxazole 59 proba-
bly takes place in the corresponding arrangement, as for the
closely related antibody 34E4 raised against a very similar
hapten.[12a] The question then arises as to why ring closure
with participation of the hydroxy group is never observed
with any of the cyclization precursors tested, despite of the
ability of the antibody binding pocket to react with the
cyclic benzisoxazole 59 in a ring-opening process.
In the case of enol ether 10a, which was used for screen-
ing and could be considered as the ™natural∫ substrate of
antibody 16E7, ring closure would result from intramolecu-
lar trapping of the oxocarbonium cation intermediate, which
occurs in competition with intermolecular trapping by a
water molecule from the solvent. We have described a simi-
lar process in catalytic antibody 14D9,[8,28] in which protona-
tion of hydroxyethyl enol ether 68 led to the formation of
the optically pure cyclic acetal 70 in a 1:10 ratio to ketone
69, which would result from the water reaction
(Scheme 8).[29] In the absence of any directing effect for cyc-
olefin 8, and benzisoxazole 59, but without success. Striking-
ly, antibody 14D9 was obtained from an immunization
against the hydrophobic quaternary ammonium hapten 71,
which is very similar to our tetrahydroquinolinium hapten 1.
In the present series of experiments the fact that hapten 1
did not induce any catalytic antibody could be due to pure
chance.[30] The guanidinium hapten 3 that induced antibody
16E7 probably induces a more hydrophilic binding site.
The present series of experiments to prepare antibody
16E7 was designed in close analogy to our own experience
with the series leading to the hydrolytic antibody 14D9,
which could be interpreted as a bias towards hydrolytic
processes. Janda and co-workers have reported several cata-
lytic antibodies towards hydroxy epoxide and olefinic tosy-
late cyclizations.[31] The functional groups and overall struc-
ture of their haptens is very similar to haptens 1 3 used
here and they incorporate quaternary ammonium and guani-
dinium functional groups next to an immunogenic aromatic
group. However, in contrast to our design, Janda and co-
workers systematically placed the aromatic group of their
haptens between the cationic groups and the linker to the
carrier proteins, which would tend to induce binding pockets
that would become readily sealed off from the solvent upon
substrate binding. Yet another factor affecting the hydrolytic
tendency in the system of Janda and co-workers lies in the
experimental conditions used. Most of their systems, particu-
larly those leading to olefin tosylate cyclizations, operated in
a biphasic hexane/water system,[32] whereby the substrate is
dissolved in the organic phase and comes into contact with a
concentrated buffered antibody solution upon shaking.
These reaction conditions may seem artificial but they may
indeed be quite similar to the membrane-bound conditions
under which many of the natural olefin cyclization enzymes
operate, including various terpene cyclases and tocopherol
6,31f]
cyclase itself.[4
Unfortunately, we did not observe any
conversion of enol ether substrates 10a,b and olefin 8 under
these biphasic conditions. This lack of reactivity under the
biphasic conditions might be related to the particular retar-
dation effect in the onset of activity of 16E7 in aqueous
buffer. Indeed modeling investigations suggest that the ki-
netic retardation effect is induced by a large conformational
movement of the CDR3loop; such a movement could well
be inhibited under phase-transfer conditions.[33]
One of the striking aspects of the reactivity of antibody
16E7 is its dual reactivity consisting in general-acid catalysis
of enol ether hydrolysis on one side and general-base cataly-
sis of the benzisoxazole deprotonation on the other side. In-
hibition of both reactions by hapten 3 implies that they take
place within the same binding pocket. Mutagenesis of
active-site residues on the recombinant chimeric Fab-16E7,
expressed in Escherichia coli, shows that catalysis can be
traced back to glutamate GluL39 in the Fv region of the light
protein chain. (Mutation of GluL39 to either alanine or gluta-
mine completely abolished activity and GluL39 is in direct
contact with the hapten as shown by molecular modeling.)[33]
Antibody 16E7 therefore provides a clear experimental ex-
ample that a general acid base carboxyl group can indeed
function both as an acid in an intrinsically acid-catalyzed
process (enol ether hydrolysis) and as a base in an intrinsi-
Scheme 8. 14D9-catalyzed hydrolysis and ring closure of hydroxyethyl
enol ether 68.
lization in the hapten design, the acetal versus ketone ratio
was interpreted in terms of a low (approximately 3%) water
content of the antibody binding site. Considering the known
versatility of antibody 14D9, we tested whether this anti-
body also catalyzed reactions with enol ethers 10a and 10b,
2493
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
cally base-catalyzed process (benzisoxazole deprotonation).
Both reactions involve rate-limiting proton transfer between
the substrate and the carboxyl group, as proved directly by
the isotope-effect studies discussed above.
Conclusion
Cationic tight-binding inhibitors of tocopherol cyclase were
used as transition-state analogues to search for catalytic an-
tibodies promoting the cyclization of the o-allylphenol sub-
strate 8 and its more reactive enol ether analogues 10a,b as
short-chain models for the natural substrate phytyl-hydro-
quinone 6. Antibody 16E7 was isolated against hapten 3 and
catalyzed the reaction of enol ether 10a to deliver the hy-
drolysis product hemiacetal 12, without participation of the
phenolic function. The antibody also catalyzed the ring-
opening reaction of benzisoxazole 59, thereby demonstrating
its ability to handle cyclic substrates. The more difficult to-
copherol cyclase like cyclization of substrate 8 remained out
of reach. This might be attributed in part to the low reactivi-
ty of the system, as seen by the fact that there is no observa-
ble spontaneous reaction in aqueous buffered systems down
to pH 2.0. Molecular modeling and mutational investigations
with antibody 16E7 are in progress to explain the reaction
mechanism and the unusual hysteresis effect in more detail.
While this dual reactivity can be interpreted as a trivial
manifestation of microscopic reversibility in catalysis, the
particular conditions of each of the two reactions still de-
serves comment. Indeed both reactions show opposite be-
haviors towards solvent effects. Enol ether hydrolysis is
strongly accelerated in polar protic solvents and the reaction
essentially stops as soon as the water content of the medium
decreases, as was studied in detail for the case of hydroxy
enol ether 68 discussed above.[29] In contrast, the deprotona-
tion of benzisoxazole 59 has been extensively discussed in
the literature due to its unusual inverse solvent dependence,
by which the reaction is strongly accelerated in aprotic
media.[27b,34] The effect has been invoked as a key triggering
factor in the benzisoxazole deprotonation by antibody 34E4
of Hilvert and co-workers, as well as in the related decar-
boxylation reaction of antibody 21D8[35] raised against a sul-
fonate hapten. The solvent effect on the benzisoxazole de-
composition by carboxylates can be interpreted in a desolva-
tion of the carboxylate base in aprotic media, which enhan-
ces its basicity.[36]
In contrast, enhancement of basicity of the catalytic gluta-
mate GluL49 side chain within the binding pocket of antibody
16E7 leading to benzisoxazole deprotonation catalysis
cannot be interpreted in terms of medium effects. Indeed,
any decrease in polarity or proticity would be fatal to the
rate of enol ether hydrolysis. This favors an interpretation in
terms of specific interactions within the antibody binding
pocket, such as electrostatic stabilization of the carboxylate
or a particular arrangement preventing the formation of
strong hydrogen-bonding interactions except with the
hapten. Such an arrangement should indeed be selected
during the immune response selecting for the strongest and
most selective hapten binding interactions.
Any effect resulting in an enhanced basicity of the car-
boxylate in antibody 16E7 should indeed enhance its reac-
tivity for both the acid- and the base-catalyzed process.
While it is clear that a higher pKa value of the carboxylic
acid increases the reactivity of the conjugate carboxylate as
a base, it must be stressed that the effect also improves the
acid-catalyzed process at pH values above the pKa value of
the acid, under which the present experiments were done.
The rate enhancement of acid catalysis occurs because a
higher pKa value of the acid increases the effective concen-
tration of the catalytically active carboxyl group at pH
values higher than the pKa. More precisely, this effect is
more pronounced than the corresponding decrease in the re-
activity of the acid for protonation.[14] The ratio of pKa var-
iation to the effect on catalysis is the value a, which can be
interpreted as the extent of proton transfer occurring at the
transition state. The a value is generally around 0.7 for enol
ether protonation by carboxylic acids.[37]
Experimental Section
General: Reactions requiring anhydrous conditions were run under
argon in vacuum- and flame-dried glassware. Diethyl ether and tetrahy-
drofuran were distilled from sodium/benzophenone prior to use. Di-
chloromethane was distilled from calcium hydride. Reagents were pur-
chased from Fluka or Aldrich and were used as received. Reaction prog-
ress was monitored by TLC on Merck silica gel 60 F-254 with detection
by UV light or by immersion in an acidic staining solution (ceric ammo-
nium molybdate or phosphomolybdic acid). Merck silica gel 60 (40
63 mm) was used for column chromatography and flash chromatography.
The following compounds were prepared as previously described: 6-me-
thoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline,[38] ethyl 8-bromo-octa-
noate,[14] 6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride,[38] ethyl
8-amino-octanoate,[16] 4-methoxy-2-(3-methyl-2-butenyl)phenol,[17] 5-
nitro-benzisoxazole,[27b] 2-hydroxy-5-nitrobenzonitrile,[39] 2-(2-bromo-phe-
noxy)tetrahydropyran.[26]
Instrumentation: Melting points were determined on a Kofler apparatus
or with
a
B¸chi 510 apparatus and are uncorrected. 1H NMR and
13C NMR spectra were recorded with Bruker DRX-600, Bruker DRX-
500, Varian VXR-400, or Varian Gemini 300 spectrometers. Proton mag-
netic resonance (1H NMR) spectra were recorded at 600, 500, 400, or
300 MHz. Chemical shifts are expressed in parts per million (d scale)
downfield from tetramethylsilane and are referenced to the residual pro-
tium signals in the solvent (CDCl3, d=7.26 ppm; [D4]MeOH, d=
3.31 ppm; [D6]DMSO, d=2.49 ppm). Data are presented as follows:
chemical shift, multiplicity (s=singlet, d=dublet, t=triplet, q=quartet,
quin=quintet, m=multiplet), integration, and coupling constant (in Hz).
Carbon magnetic resonance (13C NMR) spectra were recorded at 150,
125, 100, or 75 MHz. Chemical shifts are referenced to the carbon signal
for the solvent (CDCl3, d=77.00 ppm; [D4]MeOH, d=49.00 ppm;
[D6]DMSO, d=39.5 ppm). IR spectroscopy was performed on a Perkin
Elmer 1600 FTIR apparatus. Data are presented as follows: frequency of
transmission (cmꢀ1), intensity (s=strong, m=medium, w=weak). The
mass spectra were determined on Varian VG-70 250 (EI), Varian MAT-
312 (EI), or Finnigan Mat LCQ (ESI) spectrometers. HPLC was done on
a Waters 600 controller with a Waters 996 photodiode array detector and
using three different solvents: A (0.1% trifluoroacetic acid (TFA) in
H2O), B (H2O/CH3CN 1:1), and C (H2O). HPLC conditions for selected
compounds are given in Table 3. Preparative HPLC was performed on a
Waters Prep LC and Delta Prep 4000 apparatus with a Waters 486 tuna-
ble absorbance detector.
2494
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
Table 3. Analytical reversed-phase HPLC conditions for selected com-
pounds.[a]
(d, 2J(1,1)=15.2 Hz, 1H; H-C(1)), 4.44 (d, 1H; H-C(1)), 3.75 (s, 3H;
OCH3), 3.68 3.58 (m, 2H; H-C(3)), 3.31 (m, 2H; H-C(1’)), 3.11 (m, 2H;
H-C(4)), 3.02 (s, 3H; NCH3), 2.19 (t, 3J(7’,6’)=7.4 Hz, 2H; H-C(7’)),
1.80 1.70 (m, 2H; H-C(2’)), 1.52 1.46 (m, 2H; H-C(6’)), 1.37 1.21 (m,
6H; H-C(5’), H-C(4’), H-C(3’)) ppm; 13C NMR (125 MHz, [D6]DMSO):
d=174.92 (C(8’)), 159.40 (C(6)), 131.71 (C(4a)), 128.73 (C(8)), 119.36
(C(8a)), 114.08 (C(7)), 113.64 (C(5)), 62.77 (C(1’)), 61.14 (C(1)), 57.14
(C(3)), 55.66 (OCH3), 47.13(NCH 3), 34.01 (C(7’)), 28.66, 28.62 (C(5’),
C(3’)), 26.07 (C(4’)), 24.75 (C(6’)), 23.88 (C(4)), 21.67 (C(2’)) ppm; IR
(NaCl, CH3Cl): n˜ =3180 (m), 2950 (s), 2860 (w), 1730 (s), 1610 (m), 1500
(m), 1460 (m), 1440 (m), 1370 (w), 1100 (s), 1010 (s), 910 (w), 860
(w) cmꢀ1; UV/Vis (EtOH): l=282 nm; MS (FAB): m/z (%): 320 [M +
ꢀCF3COO] (100), 200 (19), 162 (26), 58 (19); HRMS (ESI): m/z: calcd
for C19H30NO3: 320.2226; found: 320.2224 [M +ꢀCF3COO].
Compound
% A
% B
% C
tR [min]
1[b]
50
50
50
50
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
50
50
50
0
0
0
0
0
4.3
7.6
6.5
13.1
7.3
13.2
4.7
4.2
7.1
2.9
7.1
6.9
6.5
7.7
7.0
14.0
15.3
2[b]
3[b]
18[b]
8[c]
50
100
100
64
64
64
64
100
64
64
72
72
64
72
4357
4357
4357
30
30
30
9[c]
0
10a[c]
10b[c]
11[c]
12[c]
35[c]
44a[c]
44b[c]
45a[c]
45a[c]
42[c]
43[c]
50[b]
51[b]
52[b]
55[c]
56[c]
57[c]
58[c]
36
36
36
36
0
36
36
28
28
36
28
Carrier protein conjugates with hapten 1: The KLH and BSA conjugates
of hapten
1 were prepared according to the previously described
method.[40]
5-{[4-(Ethyl-2-one)phenyl]amino}-5-oxo-pentanoic acid ethyl ester (15):
A solution of Dess Martin periodinane (961 mg, 2.26 mmol, 2 equiv) in
CH2Cl2 (30 mL) was added to a stirred suspension of 5-{[4-(ethyl-2-ol)-
phenyl]amino}-5-oxo-pentanoic acid ethyl ester (300 mg, 1.13 mmol,
1 equiv) in CH2Cl2 (10 mL) at ꢀ788C and the resulting suspension was
allowed to warm to room temperature over a period of 1 h with vigorous
stirring. After 16 h, additional Dess Martin periodinane (471.2 mg,
1.1 equiv) was added and the reaction mixture was stirred for an addi-
tional 24 h at room temperature. The mixture was then poured into a vig-
orously stirred mixture of a saturated solution of NaHCO3 (100 mL) and
a saturated solution of Na2S2O3 (100 mL). The organic layer was separat-
ed and washed with brine (100 mL), dried (Na2SO4), and concentrated.
The residue was purified by chromatography (EtOAc/hexane 3:1) to give
pure product 15 (248.2 mg, 0.94 mmol, 83%). Rf =0.40 (EtOAc/hexane
3:1); 1H NMR (400 MHz, CDCl3): d=9.71 (t, 3J(2’’,1’’)=2.4 Hz, 1H; H-
18.2
3
27.4
.1
70
70
70
70
5.5
6.1
10.6
10.3
30
[a] Isocratic elution at 1.0 mLminꢀ1. A=0.1% TFA in H2O, B =CH3CN/
H2O (50:50), C=H2O. [b] Analytical column: Vydac 218TP-54 (C18,
pore size 300 ä), 0.45î22 cm, at 1.5 mLminꢀ1. [c] Analytical column:
Bischoff LiChrospher 100 (C18, pore size 300 ä), 0.46î12.5 cm, at
1.0 mLminꢀ1
.
3
C(6)), 7.52 (d, J(2’,3’)=8.0 Hz, 2H; H-C(2’)), 7.17 (d, 2H; H-C(3’)), 4.14
(q, 3J(CH2CH3,CH2CH3)=7.2 Hz, 2H; (OCH2CH3)), 3.65 (d, 2H; H-
C(1’’)), 2.42 (m, 4H; H-C(4),H-C(2)), 2.04 (m, 2H; H-C(3)), 1.26 (t, 3H;
(OCH2CH3)) ppm; 13C NMR (300 MHz, CDCl3): d=199.66 (C(2™)),
173.73 (C(1)), 170.92 (C(2’)), 137.59 (C(1’)), 130.49 (C(4’)), 127.77
(C(3’)), 120.60 (C(2’)), 60.88 (OCH2CH3), 50.26 (C(1∫)), 36.73 (C(4)),
33.50 (C(2)), 21.12 (C(3)), 14.53 (OCH2CH3) ppm; IR (KBr): n˜ =3290
(s), 3130 (w), 3060 (w), 2940 (w), 2820 (w), 1740 (s), 1670 (s), 1600 (m),
1540 (s), 1410 (m), 1320 (m), 1260 (w), 1180 (m), 1170 (m), 1120 (m),
1070 (m), 1000 (s), 820 (m) cmꢀ1; UV/Vis (MeOH): l (%)=208 (72), 248
(100) nm; MS (EI): m/z (%): 278 (5), 277 [M +] (26), 248 (14), 232 (32),
231 (7), 202 (12), 143 (44), 135 (31), 115 (25), 106 (100), 87 (22), 55 (16);
HRMS (EI): m/z: calcd for C15H19NO4: 277.1314; found: 277.1319 [M +].
Synthesis of the haptens
2-(7-Ethoxycarbonylheptyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoqui-
nolinium bromide (14): 6-Methoxy-2-methyl-1,2,3,4-tetrahydroisoquino-
line (13; 530 mg, 2.99 mmol, 1.0 equiv) and ethyl 8-bromo-octanoate
(750 mg, 3.00 mmol, 1.0 equiv) were dissolved in DMF (1 mL) and the re-
sulting solution was heated for 3h at 60 8C. After evaporation to dryness,
the crude solid was purified by flash chromatography (CH2Cl2/CH3OH
85:15) to give 14 (1.08 g, 2.51 mmol, 84%) as an oil. Rf =0.35 (CH2Cl2/
CH3OH 9:1); 1H NMR (300 MHz, CDCl3): d=7.05 (d, 3J(8,7)=8.5 Hz,
1H; H-C(8)), 6.79 (dd, 4J(7,5)=2.5 Hz, 1H; H-C(7)), 6.72 (d, 1H; H-
C(5)), 4.80 (d, 2J(1,1)=15.1 Hz, 1H; H-C(1)), 4.68 (d, 1H; H-C(1)),
4.22 4.11 (m, 1H; H-C(3)), 4.09 (q, 3J(CO2CH2CH3,CO2CH2CH3)=
7.1 Hz, 2H; CO2CH2CH3), 3.88 3.83 (m, 1H; H-C(3)), 3.79 (s, 3H;
OCH3), 3.71 3.60 (m, 2H; H-C(1’)), 3.42 (s, 3H; NCH3), 3.31 3.05 (m,
2H; H-C(4)), 2.26 (t, 3J(7’,6’)=7.4 Hz, 2H; H-C(7’)), 1.86 1.70 (m, 2H;
2-{5-[(4-Ethyl)phenylamino]-5-oxo-pentanoic acid ethyl ester}-6-me-
thoxy-1,2,3,4-tetrahydroisoquinolium acetate (17): 6-Methoxy-1,2,3,4-tet-
rahydroisoquinoline hydrochloride (16; (803mg, 3.17 mmol, 4 equiv) was
dissolved in a 50% aqueous K2CO3 solution (35 mL) and extracted with
tert-butyl methyl ether (MTBE) (3î50 mL). The combined organic
layers were dried (Na2SO4), and evaporated under reduced pressure. The
residue was mixed with aldehyde 15 (220 mg, 0.793mmol, 1 equiv), dis-
solved in 1,2-dichloroethane (20 mL) and then treated with sodium tria-
cetoxyborohydride (252 mg, 1.18 mmol, 1.5 equiv). The mixture was stir-
red at room temperature for 30 min, then the reaction was quenched by
adding aqueous saturated NaHCO3 (100 mL) and the product was ex-
tracted with EtOAc (4î100 mL). The combined organic layers were
dried (Na2SO4) and concentrated. The residue was purified by chroma-
tography (MeOH/CH2Cl2 1:9) to give pure product 17 (285.0 mg,
0.588 mmol, 74%). Rf =0.50 (MeOH/CH2Cl2 1:9); m.p. 88 908C;
1H NMR (400 MHz, CDCl3): d=7.48 (s, 1H; NH), 7.45 (d, J(2’’,3’’)=
8.5 Hz, 2H; H-C(2’’)), 7.18 (d, 2H; H-C(3’’)), 6.96 (d, 3J(8,7)=8.7 Hz,
1H; H-C(8)), 6.74 (dd, 4J(7,5)=2.5 Hz, 1H; H-C(7)), 6.66 (d, 1H; H-
C(5)), 4.15 (q, 3J(OCH2CH3,OCH2CH3)=7.6 Hz, 2H; (OCH2CH3)), 3.78
(s, 3H; (OCH3)), 3.65 (s, 2H; H-C(1)), 3.05 (m, 4H; H-C(3), H-C(4)),
2.64 (m, 4H; (NCH2CH2-C(4’’)), 2.42 (m, 4H; H-C(4’), H-C(2’)), 2.09 (s,
3H; CH3COO), 2.05 (m, 2H, H-C(3’)), 1.24 (t, 3H; (OCH2CH3)) ppm;
13C NMR (400 MHz, CDCl3): d=175.96 (CH3COO), 173.79 (C(1’)),
170.92 (C(5’)), 158.72 (C(6)), 136.72 (C(1’’)), 135.59 (C(3’’)), 134.89
(C(4a)), 129.59 (C(2’’)), 128.10 (C(8)), 125.21 (C(2’’)), 120.45 (C(8a)),
113.61 (C(7)), 112.96 (C(5)), 60.91 (NCH2CH2-C(4’’)), 59.11 (OCH2CH3),
3
H-C(2’)), 1.56 (quin, J(6’,7’) = 3J(6’,5’)=7.4 Hz, 2H, H-C(6’)), 1.41 1.25
(m, 6H; H-C(5’), H-C(4’), H-C(3’)), 1.22 (t, 3H; CO2CH2CH3) ppm;
13C NMR (75 MHz, CDCl3): d=174.34 (C(8’)), 160.60 (C(6)), 130.89
(C(4a)), 129.35 (C(8)), 118.59 (C(8a)), 114.91 (C(7)), 114.08 (C(5)), 62.91
(C(1’)), 62.68 (C(1)), 60.87 (CO2CH2CH3), 58.42 (C(3)), 56.09 (OCH3),
48.90 (NCH3), 34.76 (C(7’)), 29.39, 29.32, 26.71 (C(5’), C(4’), C(3’)), 25.26
(C(6’)), 24.88 (C(4)), 23.04 (C(2’)), 14.89 (CO2CH2CH3) ppm; IR (NaCl):
n˜ =3180 (m), 2950 (s), 2860 (w), 1730 (s), 1610 (m), 1510 (s), 1460 (m),
1440 (m), 1380 (w), 1320 (m), 1250 (m), 1180 (m), 1160 (m), 1100 (s),
1030 (s), 910 (w), 860 (w) cmꢀ1; UV/Vis (CHCl3): l=280 nm; MS (FAB):
m/z (%): 348 [M +ꢀBr] (100), 214 (12), 176 (19), 147 (5), 58 (11); HRMS
(ESI): m/z: calcd for C21H34NO3: 348.2538; found: 348.2554 [M +ꢀBr].
2-(7-Carboxyheptyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolini-
um trifluoroacetate (1): A solution of compound 14 (45.5 mg, 107 mmol)
in DMF (10 mL) was treated with 1n aqueous HCl (10 mL) and the reac-
tion mixture was stirred at 258C for 24 h. The solution was neutralized
by adding solid KOH and the resulting mixture was lyophilized. Purifica-
tion by preparative HPLC gave product 1 (32.9 mg, 76.0 mmol, 71%).
Rf =0.24 (CH2Cl2/CH3OH/CH3COOH 8:2:0.1); 1H NMR (500 MHz,
[D6]DMSO): d=11.92 (s, 1H; COOH), 7.10 (d, 3J(8,7)=8.1 Hz, 1H; H-
4
C(8)), 6.88 (dd, J(7,5)=2.2 Hz, 1H; H-C(7)), 6.85 (d, 1H; H-C(5)); 4.50
2495
Chem. Eur. J. 2004, 10, 2487 2506
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
55.65 (C(1)), 54.64 (CH3O-C(6)), 50.38 (C(3)), 36.77 (C(4’)), 33.64
(NCH2CH2-C(4’’)), 28.25 (C(4)), 22.21 (C(3’)), 21.25 (CH3COO), 14.62
(OCH2CH3) ppm; IR (KBr): n˜ =3450 (m), 3310 (m), 3020 (w), 2940 (m),
1730 (s), 1690 (s), 1660 (s), 1610 (s), 1540 (m), 1510 (s), 1410 (m), 1380
(w), 1310 (m), 1240 (s), 1160 (w), 1030 (m), 830 (w) cmꢀ1; UV/Vis
(MeOH): l (%)=208 (100), 246 (86) nm; MS (FAB): m/z (%): 426 (20),
425 [M +] (72), 424 (8), 423(19), 192 (10), 177 (12), 176 (100), 175 (6),
162 (14), 160 (5), 146 (5), 143(11), 134 (4), 120 (17), 115 (8), 106 (5), 55
(10); elemental analysis calcd (%) for C27H36N2O6: C 66.92, H 7.49, N
5.78, O 19.81; found: C 66.68, H 7.68.
zaldehyde (19; 10.0 g, 59.8 mmol, 1.0 equiv) and Cs2CO3 (27.3g,
83.7 mmol, 1.4 equiv) in DMF (60 mL). After stirring for 15 h at room
temperature, the reaction was quenched by adding H2O (300 mL) and
the aqueous solution was extracted with EtOAc (7î60 mL). The organic
layers were dried (Na2SO4) and evaporated, then the crude residue was
purified by column chromatography (hexane/MTBE 1:1) to give 20
(10.3g, 56.8 mmol, 96%) as a yellow solid. Rf =0.34 (hexane/MTBE 1:1);
1
m.p. 858C; H NMR (500 MHz, CDCl3): d=10.50 (s, 1H; CHO), 8.17 (d,
4
3J(3,4)=9.1 Hz, 1H; H-C(3)), 7.34 (d, J(6,4)=2.8 Hz, 1H; H-C(6)), 7.16
(dd, 1H; H-C(4)), 3.97 (s, 3H; OCH3) ppm; 13C NMR (125 MHz,
CDCl3): d=188.53 (CHO), 164.00 (C(5)), 142.31 (C(2)), 134.34 (C(1)),
127.26 (C(3)), 118.68 (C(4)), 113.19 (C(6)), 56.35 (OCH3) ppm; IR
(KBr): n˜ =3100 (w), 3030 (m), 2980 (m), 2940 (w), 1740 (s), 1580 (s),
1520 (s), 1500 (m), 1480 (m), 1440 (m), 1420(m), 1250 (m), 1160 (m),
1080 (m), 1030 (m), 930 (s), 900 (m) cmꢀ1; UV/Vis (CHCl3): l (%)=248
(100), 322 (74) nm; MS (EI): m/z (%): 181 [M +] (56), 151 (60), 123(39),
108 (82), 106 (58), 95 (54), 63(100); HRMS (EI): m/z: calcd for
C8H7NO4: 181.0375; found: 181.0377; elemental analysis calcd (%) for
C8H7NO4: C 53.04, H 3.90, N 7.73, O 35.33; found: C 52.86, H 4.05, N
7.61, O 35.59.
2-Methyl-2-{5-[(4-ethyl)phenylamino]-5-oxo-pentanoic acid ethyl ester}-
6-methoxy-1,2,3,4-tetrahydroisoquinolinium trifluoroacetate (18): Com-
pound 17 (50 mg, 0.117 mmol) was dissolved in a 50% aqueous K2CO3
solution (35 mL) and extracted with TBME (3î50 mL). The combined
organic layers were dried (Na2SO4) and evaporated under reduced pres-
sure. The residue was redissolved in CH3CN (5 mL) and then dimethyl-
sulfate (14 mL, 0.106 mmol, 1.1 equiv) was added to this solution. The re-
action mixture was stirred for 15 h at room temperature and then the sol-
vent was evaporated under reduced pressure. The residue was purified by
preparative HPLC to yield 18 (29.1 mg, 0.053mmol, 55%). Rf =0.35
CH2Cl2/CH3OH/CH3COOH 8:2:0.1); 1H NMR (400 MHz, CDCl3): d=
7.45 (d, 3J(2’’,3’’)=8.6 Hz, 2H; H-C(2’’)), 7.01 (d, 2H; H-C(3’’)); 6.98 (d,
3J(8,7)=7.8 Hz, 1H; H-C(8)), 6.81 (dd, 4J(7,5)=2.4 Hz, 1H; H-C(7)),
6.71 (d, 1H; H-C(5)), 4.56 (d, 2J(1,1)=15.2 Hz, 1H; H-C(1)), 4.39 (d,
1H; H-C(1)), 4.08 (q, 3J(OCH2CH3,OCH2CH3)=7.4 Hz, 2H;
(OCH2CH3)), 3.78 (s, 3H; (CH3O)), 3.72 (m, 2H; H-C(3)), 3.60 (s, 3H;
(NCH3)), 3.50 (m, 2H; (NCH2CH2-C(4’’))), 3.13 (m, 2H; H-C(4)), 2.96
(m, 2H; (NCH2CH2-C(4’’))), 2.39 (t, 3J(2’,3’)=7.5 Hz, 2H; H-C(2’)), 2.34
(t, 3J(4’,3’)=7.4 Hz, 2H; H-C(4’)), 1.92 (m, 2H; H-C(3’)), 1.21 (t, 3H;
(OCH2CH3)) ppm; 13C NMR (400 MHz, CDCl3): d=173.74 (C(1’)),
172.06 (C(5’)), 160.45 (C(6)), 138.35 (C(1’’)), 130.34 (C(4’’)), 129.60
(C(4a)), 129.05 (C(2’’)), 121.20 (C(8)), 117.95 (C(8a)), 114.82 (C(7)),
113.78 (C(5)), 64.91 (NCH2CH2-C(4’’)), 62.55 (C(1)), 60.78 (C(3)), 58.45
(OCH2CH3), 55.78 (CH3O)), 47.26 (NCH3)), 36.32 (C(4’)), 33.84 (C(2’)),
5-Methoxy-2-nitrobenzonitrile (21):
A suspension of compound 20
(9.50 g, 52.4 mmol, 1.0 equiv), H2NOH¥HCl (4.00 g, 57.7 mmol,
1.1 equiv), MgSO4 (25.2 g, 210 mmol, 4.0 equiv), and p-toluenesulfonic
acid monohydrate (2.00 g, 10.5 mmol, 0.2 equiv) in toluene (200 mL) was
stirred under reflux conditions. After stirring for 15 h, CHCl3 (100 mL)
was added to the warm suspension. The warm suspension was filtered
and the filtrate was washed with H2O (3î100 mL), dried (Na2SO4), and
evaporated. The crude residue was purified by flash chromatography
(hexane/EtOAc 1:1) to give 21 (8.21 g, 46.1 mmol, 88%) as a yellow
solid. Rf =0.34 (hexane/EtOAc 1:1); m.p. 968C; 1H NMR (300 MHz,
CDCl3): d=8.32 (d, 3J(3,4)=9.2 Hz, 1H; H-C(3)), 7.33 (d, 4J(6,4)=
2.8 Hz, 1H; H-C(6)), 7.22 (dd, 1H; H-C(4)), 3.98 (s, 3H; OCH3) ppm;
13C NMR (75 MHz, CDCl3): d=163.67 (C(5)), 141.39 (C(2)), 127.84
(C(3)), 120.60 (C(4)), 118.14 (C(6)), 114.98 (CN), 109.87 (C(1)), 56.62
(OCH3) ppm; IR (NaCl, CHCl3): n˜ =3120 (w), 3030 (w), 2950 (w), 2850
(w), 2210 (m), 1580 (s), 1520 (s), 1490 (m), 1460 (m), 1440 (w), 1340 (s),
28.41
(NCH2CH2-C(4’’)),
24.41
(C(4)),
1.19
(C(3’)),
14.56
(OCH2CH3)) ppm; IR (CHCl3, NaCl): n˜ =3570 (w), 3160 (w), 2990 (w),
2960 (w), 2840 (w), 2250 (s), 1790 (m), 1730 (m), 1690 (s), 1610 (m), 1560
(w), 1510 (w), 1460 (s), 1380 (s), 1320 (w), 1280 (w), 1100 (s), 1010 (m),
910 (s), 820 (w) cmꢀ1; UV/Vis (MeOH): l (%)=210 (82), 246 (100) nm;
MS (ESI): m/z (%): 440 (28), 439 [M +] (100), 305 (29); HRMS (EI): m/
z: calcd for C26H35N2O4: 279.1471; found: 279.1470 [M +ꢀCF3COO].
1280 (s), 1130 (m), 1100 (m), 1090 (s), 1080 (m), 1030 (m), 890 (w) cmꢀ1
;
UV/Vis (CH2Cl2): l=314 nm; MS (EI): m/z (%): 178 [M +] (100), 148
(68), 117 (65), 89 (31); elemental analysis calcd (%) for C8H6N2O3: C
53.94, H 3.39, N 15.72, O 26.94; found C 53.78, H 3.53, N 15.53, O 26.95.
2-Amino-5-methoxybenzonitrile (22): A mixture of compound 21 (5.73g,
32.2 mmol, 1.0 equiv) and SnCl2¥H2O (32.0 g, 141.9 mmol, 4.4 equiv) in
MeOH (30 mL) was heated at 708C under argon. After 3h the solution
was allowed to cool and then poured into ice (400 g). The pH value was
made slightly basic (pH 8) by addition of saturated aqueous NaHCO3
before being extracted with ethyl acetate (3î60 mL). The organic phase
was thoroughly washed with brine, dried (Na2SO4), and evaporated. The
crude residue was purified by column chromatography (hexane/MTBE
1:1) to give 22 (2.67 g, 18 mmol, 56%) as a white solid. Rf =0.30 (hexane/
MTBE 1:1); m.p. 468C; 1H NMR (300 MHz, CDCl3): d=6.95 (dd,
3J(4,3)=8.9, 4J(4,6)=2.9 Hz, 1H; H-C(4)), 6.86 (d, 1H; H-C(6), 6.66 (d,
1H; H-C(3)), 4.15 (s, 2H; NH2), 3.72 (s, 3H; OCH3) ppm; 13C NMR
(75 MHz, CDCl3): d=151.69 (C(5)), 144.32 (C(2)), 122.77 (C(4)), 117.67
(CN), 117.20 (C(6)), 114.73(C(3)), 96.20 (C(1)), 55.94 (OCH 3) ppm; IR
(KBr): n˜ =3460 (s), 3440 (s), 3370 (s), 3240 (m), 3060 (w), 3010 (w), 2920
(w), 2840 (w), 2220 (s), 1640 (m), 1620 (w), 1510 (s), 1470 (m), 1430 (m),
1310 (m), 1280 (s), 1250 (m), 1180 (m), 1160 (m), 1130 (m), 1040 (s), 930
(w), 890 (m), 810 (m) cmꢀ1; UV/Vis (CH2Cl2): l (%)=244 (100), 308
(40) nm; MS (EI): m/z (%): 148 [M +] (78), 134 (15), 133 (100), 105 (32);
elemental analysis calcd (%) for C8H8N2O: C 64.85, H 5.44, N 18.91, O
10.80; found: C 64.64, H 5.44, N 18.71, O 10.91.
2-Methyl-2-{5-[(4-ethyl)phenylamino]-5-oxo-pentanoic acid}-6-methoxy-
1,2,3,4-tetrahydroisoquinolinium trifluoroacetate (2): Compound 18 was
dissolved in a mixture of 4m HCl in dioxane (2 mL) and water (250 mL)
and the resulting reaction mixture was stirred for 6 h. With aqueous
K2CO3 solution the pH value was adjusted to 7. The mixture was then
purified by preparative HPLC to yield 2 (19.6 mg, 0.037 mmol, 71%).
Rf =0.22 (CH2Cl2/CH3OH/CH3COOH 8:2:0.1); 1H NMR (500 MHz,
[D4]MeOH): d=7.54 (d, 3J(2’’,3’’)=8.5 Hz, 2H; H-C(2’’)), 7.25 (d, 2H;
H-C(3’’)), 7.11 (d, 3J(8,7)=7.7 Hz, 1H; H-C(8)), 6.91 (m, 1H; H-C(7)),
6.89 (m, 1H; H-C(5)), 4.62 (d, 2J(1,1)=15.2 Hz, 1H; H-C(1)), 4.54 (d,
1H; H-C(1)), 3.81 (s, 3H; (CH3O)), 3.78 (m, 2H; H-C(3)), 3.60 (m, 2H;
NCH2CH2-C(4’’)), 3.26 (m, 2H; H-C(4)), 3.22 (s, 3H; (NCH3)), 3.19 (m,
2H; NCH2CH2-C(4’’)), 2.43(t, 3J(2’,3’)=7.5 Hz, 2H; H-C(2’)), 2.38 (t,
3J(4’,3’)=7.4 Hz, 2H; H-C(4’)), 1.96 (m, 2H; H-C(3’)) ppm; 13C NMR
(500 MHz, [D4]MeOH): d=176.79 (C(1’)), 173.79 (C(5’)), 161.55 (C(6)),
139.21 (C(1’’), 132.45 (C(4’’)), 132.06 (C(4a)), 130.38 (C(3’’)), 129.57
(C(8)), 121.74 (C(2’’)), 119.42 (C(8a)), 115.28 (C(7)), 114.37 (C(5)), 65.52
(NCH2CH2-C(4’’)), 63.04 (C(1)), 59.26 (C(3)), 55.85 (CH3O), 47.80
(NCH3)), 36.84 (C(4’)), 34.06 (C(2’)), 28.96 (NCH2CH2-C(4’’)), 24.99
(C(4)), 2.05 (C(3’)) ppm; UV/Vis (MeOH):
l (%)=208 (100), 248
(87) nm; IR (CHCl3, NaCl): n˜ =3570 (w), 3160 (w), 2990 (w), 2960 (w),
2840 (w), 2250 (s), 1790 (m), 1730 (m), 1680 (s), 1610 (m), 1560 (w), 1510
(w), 1470 (s), 1380 (s), 1320 (w), 1280 (w), 1100 (s), 1010 (m), 910 (s), 820
(w) cmꢀ1; MS (ESI): m/z (%): 412 (25), 411 [M +ꢀCF3COO] (100), 277
(21); HRMS (ESI): m/z: calcd for C24H31N2O4: 411.2284; found: 411.2286
[M +ꢀCF3COO].
2-Amino-5-methoxybenzylamine (23): LiAlH4 (133 mg, 3.50 mmol,
12.0 equiv) and AlCl3 (467 mg, 3.50 mmol, 3.0 equiv) were suspended in
dry Et2O (15 mL) at 08C. After 15 min, a solution of compound 22
(173mg, 1.17 mmol, 1.0 equiv) in Et 2O (15 mL) was added dropwise.
After 5 h at room temperature, the reaction was quenched with water
(60 mL) until no further evolution of hydrogen was observed. 6n aque-
ous H2SO4 (120 mL) was then added and the mixture was stirred for a
further 30 min. The grey suspension was then washed with Et2O (3 î
50 mL). The grey aqueous layer was basicified by addition of solid KOH,
which resulted in the formation of a white suspension. This suspension
Carrier protein conjugates of hapten 2: The KLH and BSA conjugates of
hapten 2 were prepared according to the previously described method.[40]
5-Methoxy-2-nitrobenzaldehyde (20): Methyl iodide (80.0 mL, 182.4 g,
1.285 mol, 21 equiv) was added to a suspension of 5-hydroxy-2-nitroben-
2496
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
was filtered over celite and the filtrate was extracted with Et2O (4î
50 mL). The combined organic layers were dried (Na2SO4) and filtered,
then the solvent was removed by evaporation to yield product 23
24.79 (C(7’)), 14.23(CO CH2CH3) ppm; IR (NaCl, CHCl3): n˜ =3190 (m),
2
2960 (s), 2940 (s), 2860 (m), 1720 (s), 1670 (s), 1630 (s), 1600 (w), 1510
(s), 1460 (m), 1380 (w), 1350 (w), 1170 (w), 1100 (m), 1040 (m), 870
(w) cmꢀ1; UV/Vis (CHCl3): l=262 nm; MS (FAB): m/z (%): 348 [M +
ꢀCH3COO] (100), 347 (14); HRMS (ESI): m/z: calcd for C19H30N3O3:
348.2287; found: 348.2302 [M +ꢀCH3COO].
1
(138 mg, 0.91 mmol, 78%). Rf =0.51 (EtOH/16% aq NH3 17:3); H NMR
(300 MHz, CDCl3): d=6.68 6.59 (m, 3H; H-C(6), H-C(4), H-C(3)), 3.89
(s, 2H; CH2NH2), 3.84 (s, 3H; OCH3), 1.80 (s, 4H; NH2, CH2NH2) ppm;
13C NMR (75 MHz, CDCl3): d=152.36 (C(5)), 139.57 (C(2)), 127.82
(C(1)), 116.81 (C(3)), 115.12 (C(6)), 113.16 (C(4)), 55.75 (OCH3), 44.86
(CH2NH2) ppm; IR (NaCl, CHCl3): n˜ =3320 (w), 3000 (m), 2960 (s), 2860
(m), 2830 (m), 1600 (s), 1500 (s), 1470 (m), 1440 (m), 1380 (w), 1160 (w),
1100 (s), 1010 (m), 860 (w) cmꢀ1; UV/Vis (MeOH): l (%)=246 (100),
308 (41) nm; MS (EI): m/z (%): 152 [M +] (55), 136 (75), 135 (100), 134
(41), 120 (52), 93(18); HRMS (EI): m/z: calcd for C8H12N2O: 152.0950;
found: 152.0948 [M +].
2-(N-Aminocapryl acid)-6-methoxy-3,4-dihydroquinazolinium trifluoro-
acetate (3): A solution of compound 26 (45.5 mg, 111 mmol) in THF
(5 mL) was treated with 1n aqueous HCl (15 mL) and the reaction mix-
ture was stirred at 258C for 24 h. The solution was neutralized by adding
solid KOH and the resulting mixture was lyophilized. Purification by
preparative HPLC gave 3 (41.8 mg, 96.6 mmol, 87%). Rf =0.72 (CH2Cl2/
MeOH/CH3COOH 24:16:0.5); 1H NMR (600 MHz, [D4]MeOH): d=7.1
3
(d, J(8,7)=8.6 Hz, 1H; H-C(8)), 6.85 (dd, 4J(7,5)=2.7 Hz, 1H; H-C(7)),
6-Methoxy-3,4-dihydro-(1H)-quinazoline-2-thione (24):
A solution of
6.77 (d, 1H; H-C(5)), 4.52 (s, 2H; H-C(4)), 3.81 (s, 3H; OCH3), 3.28 (t,
3J(2’,3’)=7.2 Hz, 2H; H-C(2’)), 2.29 (t, 3J(8’,7’)=7.4 Hz, 2H; H-C(8’)),
1.66 1.60 (m, 4H; H-C(7’), H-C(3’)), 1.42 1.28 (m, 6H; H-C(6’), H-C(5’),
H-C(4’)) ppm; 13C NMR (125 MHz, [D4]MeOH): d=177.84 (C(9’)),
158.50 (C(6)), 154.87 (C(2)), 127.69 (C(8a)), 121.02 (C(4a)), 117.71
(C(8)), 115.03(C(7)), 112.13 (C(5)), 56.06 (OCH 3), 42.77 (C4), 42.59
(C(2’)), 35.21 (C(8’)), 30.98, 30.68, 30.56 (C(6’), C(5’), C(4’)), 27.48
(C(3’)), 25.79 (C(7’)) ppm; IR (KBr): n˜ =3440 (s), 3080 (m), 2940 (s),
2860 (m), 2800 (m), 1600 (s), 1510 (w), 1470 (m), 1380 (m), 1350 (m),
1280 (w), 1250 (w), 1120 (m), 1020 (m) cmꢀ1; UV/Vis (MeOH): l=
258 nm; MS (FAB): m/z (%): 320 [M +ꢀCF3COO] (100); HRMS (ESI):
m/z: calcd for C17H26N3O3: 320.1974; found: 320.1961 [M +ꢀCF3COO].
benzylamine 23 (117 mg, 0.77 mmol, 1.0 equiv) and Et3N (250 mL,
181 mg, 1.79 mmol, 2.3equiv) in dry Et 2O (3.5 mL) was cooled to ꢀ788C.
A solution of thiophosgene (70 mL, 103mg, 0.93mmol, 1.2 equiv) dis-
solved in dry Et2O (1.2 mL) was added to this mixture. After 30 min, the
heterogeneous mixture was allowed to warm to 258C and stirred at this
temperature for an additional 9 h. The mixture was concentrated to half
the original volume and the remaining solution was purified by column
chromatography (CH2Cl2/MeOH 37:3) to give 24 (126 mg, 0.65 mmol,
84%) as a brown solid. Rf =0.54 (CH2Cl2/MeOH (37:3)); m.p. 1788C;
1H NMR (400 MHz, [D6]DMSO): d=10.24 (s, 1H; H-N(1)), 8.40 (s, 1H;
H-N(3)), 6.85 (d, 3J(8,7)=8.6 Hz, 1H; H-C(8)), 6.73(dd, 4J(7,5)=2.9 Hz,
1H; H-C(7)), 6.70 (dd, 1H; H-C(5)), 4.31 (s, 2H; H-C(4)), 3.67 (s, 3H;
OCH3) ppm; 13C NMR (100 MHz, [D6]DMSO): d=175.00 (C(2)), 155.16
(C(6)), 128.86 (C(4a)), 118.63(C(8a)), 114.98 (C(8)), 11.354 (C(5)),
111.12 (C(7)), 55.28 (OCH3), 42.94 (C(4)) ppm; IR (KBr): n˜ =3600 (m),
3240 (s), 3020 (s), 2830 (m), 1570 (s), 1540 (s), 1380 (m), 1270 (s), 1240
(m), 1200 (s), 960 (m), 900 (m), 880 (w), 840 (w) cmꢀ1; UV/Vis (MeOH):
l (%)=218 (49), 288 (100) nm; MS (EI): m/z (%): 194 [M +] (100), 193
(31), 136 (59), 120 (24); HRMS (EI): m/z: calcd for C9H10N2OS:
194.0514; found: 194.0515 [M +].
Carrier protein conjugates of hapten 3: The KLH and BSA conjugates of
hapten 3 were prepared according to the previously described method.[40]
Synthesis of the substrates and reference compounds
4-Methoxy-2-(3-methyl-2-butenyl)phenol (8): Olefin 8 was prepared ac-
cording to a known procedure.[17]
2,2-Dimethyl-6-methoxychroman (9): Olefin
8
(1.39 g, 7.23 mmol,
1.0 equiv) and BF3¥Et2O (7.23mmol, 1.0 equiv) in CH 2Cl2 (5 mL) were
stirred for 24 h at RT. The reaction mixture was partitioned between
water (50 mL) and MTBE (50 mL). The aqueous layer was separated
and extracted further with MTBE (2î50 mL). The combined organic
layers were washed with 2m aqueous HCl, water, saturated aqueous
Na2CO3 solution, and finally water. The organic layers were dried over
Na2SO4 and the solvent was removed by evaporation. The residue was
purified by flash chromatography (hexane/EtOAc 9:1) to give 9 (1.19 g,
2-(Ethylsulfanyl)-6-methoxy-3,4-dihydroquinazolinium bromide (25):
Ethylbromide (7.0 mL) was added to a solution of compound 24 (120 mg,
0.62 mmol) in dry EtOH (5.0 mL). The mixture was stirred under reflux
conditions for 14 h. The resulting solution was concentrated on the rotary
evaporator, whereupon product 25 crystallized (185 mg, 0.64 mmol,
98%). Rf =0.27 (CH2Cl2/MeOH 19:1); m.p. 1948C; 1H NMR (500 MHz,
[D6]DMSO): d=12.08 (s, 1H; H-N(1)), 10.24 (s, 1H; H-N(3)), 7.03 (d,
3J(8,7)=8.5 Hz, 1H; H-C(8)), 6.90 (dd, 4J(7,5)=2.8 Hz, 1H; H-C(7)),
6.85 (dd, 1H; H-C(5)), 4.72 (s, 2H; H-C(4)), 3.67 (3îs, 3H; OCH3), 3.29
(q, 3J(SCH2CH3,SCH2CH3)=7.3Hz, 2H; SC H2CH3), 1.31 (t, 3H;
SCH2CH3) ppm; 13C NMR (125 MHz, [D6]DMSO): d=160.40 (C(2)),
157.87 (C(6)), 125.44 (C(8a)), 119.78 (C(4a)), 117.53(C(8)), 114.19
(C(7)), 111.69 (C(5)), 55.51 (OCH3), 43.12 (C(4)), 25.92 (SCH2CH3),
14.29 (SCH2CH3) ppm; IR (NaCl, CHCl3): n˜ =3410 (s), 2960 (s), 2840
(w), 1600 (s), 1500 (m), 1460 (w), 1400 (w), 1260 (s), 1100 (s), 1010 (s),
865 (m), 640 (w) cmꢀ1; UV/Vis (EtOH): l=296 nm; MS (FAB): m/z
(%): 223[ M +] (100), 102 (51), 57 (30), 55 (29); HRMS (ESI): m/z: calcd
for C11H15N2OS: 223.0910; found: 223.0917 [M +ꢀBr].
6.22 mmol, 86%) as
a colorless oil. Rf =0.32 (hexane/EtOAc 9:1);
1H NMR (300 MHz, CDCl3): d=6.80 6.56 (m, 3H; H-C(8), H-C(7), H-
C(5)), 3.75 (s, 3H; OCH3), 2.78 (t, 3J(3,4)=7.2 Hz, 2H; H-C(3)), 1.77 (t,
2H; H-C(4)), 1.25 (s, 6H; (CH3)2-C(2)) ppm; 13C NMR (75 MHz,
CDCl3): d=152.88 (C(6)), 147.95 (C(8a), 121.45 (C(4a)), 117.73(C(8)),
113.96 (C(5)), 113.39 (C(7)), 73.78 (C(2)), 55.72 (OCH3), 32.83 (C(3)),
26.76 ((CH3)2-C(2)), 22.83(C(4)) ppm; IR (NaCl, CHCl 3): n˜ =2980 (m),
2940 (m), 2900 (m), 2880 (m), 1500 (s), 1450 (m), 1440 (w), 1410 (w),
1310 (w), 1300 (m), 1280 (s), 1240 (m), 1200 (s), 1150 (m), 1100 (s), 1040
(s), 910 (m), 890 (s) cmꢀ1; UV/Vis (CHCl3): l=292 nm; MS (EI): m/z
(%): 193(16), 192 [ M +] (100), 177 (20), 163 (6), 138 (11), 137 (98), 136
(74), 108 (21), 79 (6), 78 (9), 77 (11), 65 (11), 55 (5); HRMS (EI): m/z:
calcd for C12H16O2: 192.1150, found: 192.1150 [M +]; elemental analysis
calcd (%) for C12H16O2: C 74.97, H 8.39, O 16.64; found: C 74.99, H 8.41,
O 16.54.
2-(N-Aminocapryl acid ethyl ester)-6-methoxy-3,4-dihydroquinazolinium
acetate (26): A solution of compound 25 (130 mg, 0.43 mmol, 1.0 equiv)
and ethyl 8-amino-octanoate (160 mg, 0.86 mmol, 2.0 equiv) in dry EtOH
(10.0 mL) was heated under reflux conditions for 24 h. After evaporation
of the solvent, the residue was purified by flash chromatography
(CH2Cl2/MeOH/CH3COOH 19:1:0.2) to give 26 (115 mg, 0.28 mmol,
65%) as an oil. Rf =0.34 (CH2Cl2/MeOH/CH3COOH 19:1:0.2); 1H NMR
(600 MHz, CDCl3): d=7.02 (d, 3J(8,7)=8.6 Hz, 1H; H-C(8)), 6.75 (dd,
4J(7,5)=2.7 Hz, 1H; H-C(7)), 6.52 (d, 1H; H-C(5)), 4.52 (s, 2H; H-
C(4)), 4.13(q, 3J(CO2CH2CH3,CO2CH2CH3)=7.2 Hz, 2H; CO2CH2CH3),
1-Allyloxy-4-methoxybenzene (28): A solution of hydroquinone mono-
methyl ether (27; 87.0 g, 701 mmol, 1 equiv) in THF (200 mL) was added
to a suspension of sodium hydride (25.2 g, 1.05 mol, 1.5 equiv) in THF
(1.5 L) at 08C. The mixture was stirred for 30 min prior to the addition
of allyl bromide (90 mL, 128.7 g, 1.06 mol, 1.5 equiv). The mixture was
heated under reflux conditions for 4 h and then the reaction was
quenched by addition of saturated aqueous NH4Cl solution. The volume
of the reaction was reduced to 400 mL and the remaining mixture was
extracted with MTBE (5î80 mL). The organic phases were combined,
washed with brine, dried (Na2SO4), and evaporated. The crude residue
was purified by distillation at reduced pressure (1148C/10 mbar) to
afford pure 28 (109 g, 664 mmol, 95%) as a colorless oil. Rf =0.46
(hexane/MTBE 9:1); b.p. 1148C/10 mbar; 1H NMR (400 MHz, CDCl3):
d=6.88 6.81 (m, 4H; H-C(3), H-C(2)), 6.05 (ddt, 3J(2’,3’a)=17.3,
3J(2’,3’b)=10.6, 3J(2’,1’)=5.1 Hz, 1H; H-C(2’)), 5.40 (dq, 2J(3’,3’) and
3
3.76 (s, 3H; OCH3), 3.17 (m, 2H; H-C(2’)), 2.29 (t, J(8’,7’)=7.4 Hz, 2H;
H-C(8’)), 2.07 (s, 3H; CH3COO), 1.73 1.64 (m, 4H; H-C(7’), H-C(3’)),
1.62 1.58 (m, 2H; H-C(4’)), 1.38 1.24 (m, 4H; H-C(6’), H-C(5’)), 1.22 (t,
3H; CO2CH2CH3) ppm; 13C NMR (125 MHz, CDCl3): d=181.53
(CH3COO), 173.88 (C(9’)), 156.66 (C(6)), 151.78 (C(2)), 126.58 (C(8a)),
118.60 (C(4a)), 116.94 (C(8)), 114.02 (C(7)), 111.22 (C(5)), 60.23
(CO2CH2CH3), 55.58 (OCH3), 42.42 (C(4)), 41.89 (C(2’)), 34.21 (C(8’)),
28.85, 28.79, 28.68 (C(6’), C(5’), C(4’)), 26.60 (CH3COO), 26.48 (C(3’)),
2497
Chem. Eur. J. 2004, 10, 2487 2506
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
4J(3’,1’)=1.5 Hz, 1H; H-C(3’)), 5.27 (dq, 1H; H-C(3’)), 4.49 (dt, 2H; H-
C(1’)), 3.93 (s, 3H; OCH3) ppm; 13C NMR (100 MHz, CDCl3): d=154.34,
153.18 (C(4), C(1)), 134.04 (C(2’)), 117.86 (C(3’)), 116.16, 115.03(C(3),
C(2)), 69.95 (C(1’)), 56.12 (OCH3) ppm; IR (NaCl, CHCl3): n˜ =3010 (m),
2960 (m), 2940 (m), 2910 (m), 2870 (w), 2840 (m), 1650 (w), 1590 (w),
1510 (s), 1470 (m), 1440 (m), 1430 (w), 1410 (w), 1360 (w), 1290 (m),
1110 (m), 1040 (s), 1000 (m), 930 (m), 830 (s) cmꢀ1; UV/Vis (EtOH): l
(%)=228 (100), 290 (37) nm; MS (EI): m/z (%): 164 [M +] (29), 124
(11), 123 (100), 95 (23), 41 (17), 39 (10); HRMS (EI): m/z: calcd for
C10H12O2: 164.0837; found: 164.0836 [M +].
(s, 9H; SiC(CH3)3), 0.21 (s, 6H; Si(CH3)2) ppm; 13C NMR (100 MHz,
CDCl3): d=154.33 (C(5)), 147.74 (C(2)), 133.49 (C(1)), 119.51 (C(3)),
116.20 (C(6)), 111.94 (C(4)), 62.51 (C(1’)), 55.97 (OCH3), 33.38 (C(2’)),
27.07 (C(3’)), 26.24 (SiC(CH3)3), 18.63(Si C(CH3)3), ꢀ3.79
(Si(CH3)2) ppm; IR (NaCl): n˜ =3360 (s), 2990 (s), 2900 (m), 2860 (s),
1490 (s), 1470 (s), 1420 (m), 1390 (m), 1360 (m), 1250 (s), 1220 (s), 1150
(m), 1120 (w), 1060 (s), 1030 (s), 950 (s), 900 (s), 840 (s), 800 (m) cmꢀ1
;
UV/Vis (MTBE): l=289 nm; MS (EI): m/z (%): 296 [M +] (15), 223(6),
222 (22), 221 (100), 211 (13); HRMS (EI): m/z: calcd for C16H28O3Si:
296.1808; found: 296.1808 [M +]; elemental analysis calcd (%) for
C16H28O3Si: C 64.82, H 9.52, O 16.19, Si 9.47; found: C 64.60, H 9.75.
2-Allyl-4-methoxyphenol (29): Allyl phenyl ether 28 was heated at 2408C
until the starting material had been consumed. The crude residue was pu-
rified by distillation at reduced pressure (1028C/10ꢀ1 mbar) to yield 29
(85.9 g, 523mmol, 89%) as an oil. Rf =0.27 (hexane/EtOAc 9:1); b.p
1028C/10ꢀ1 mbar; 1H NMR (400 MHz, CDCl3): d=6.78 6.66 (m, 3H; H-
C(6), H-C(5), H-C(3)), 6.01 (ddt, 3J(2’,3’a)=17.6, 3J(2’,3’b)=9.8,
3J(2’,1’)=6.3Hz, 1H; H-C(2 ’)), 5.19 5.12 (m, 2H; H-C(3’)), 4.56 (s, 1H;
OH), 3.76 (s, 3H; OCH3), 3.38 (d, 2H; H-1’) ppm; 13C NMR (100 MHz,
CDCl3): d=154.24 (C(4)), 148.38 (C(1)), 136.55 (C(2’)), 126.81 (C(2)),
116.97 (C(6)), 116.92 (C(3)), 116.36 (C(3’)), 113.06 (C(5)), 56.12 (OCH3),
35.75 (C(1’)) ppm; IR (NaCl, CHCl3): n˜ =3530 (m), 3360 (s), 3080 (m),
3000 (s), 2980 (s), 2940 (s), 2910 (m), 2840 (s), 1850 (w), 1640 (m), 1610
(m), 1500 (s), 1470 (s), 1450 (s), 1430 (s), 1330 (s), 1280 (s), 1110 (m),
1100 (m), 1040 (s), 1000 (s), 920 (s), 870 (m), 850 (m) cmꢀ1; UV/Vis
3-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-propionaldehyde
(32): Oxalyl chloride (1.2 mL, 1.78 g, 13.6 mmol, 1.3 equiv) was added
dropwise to a solution of DMSO (2.0 mL, 2.18 g, 27.3mmol, 2.6 equiv) in
CH2Cl2 (100 mL) at ꢀ788C. After 10 min, a solution of alcohol 31
(3.09 g, 10.5 mmol, 1.0 equiv) in CH2Cl2 (5 mL) was added dropwise.
Triethylamine (8.6 mL, 6.28 g, 62 mmol, 5.0 equiv) was added after stir-
ring at ꢀ788C for 15 min and the reaction mixture was allowed to warm
to room temperature. The reaction mixture was partitioned between
water (300 mL) and MTBE (300 mL). The aqueous layer was separated
and extracted further with MTBE (2î150 mL). The combined organic
layers were washed with 2m aqueous HCl, water, saturated aqueous
Na2CO3 solution, and finally water. The organic layers were dried over
Na2SO4 and the solvent was removed by evaporation to give 32 (2.90 g,
9.85 mmol, 94%) as a colorless oil. An analytical sample was purified by
column chromatography over basic aluminium oxide (hexane/EtOAc
12:1). Rf =0.22 (hexane/MTBE 9:1); 1H NMR (400 MHz, CDCl3): d=
9.81 (t, 3J(1’,2’)=1.5 Hz, 1H; H-C(1’)), 6.71 (d, 3J(3,4)=8.8 Hz, 1H; H-
C(3)), 6.69 (d, 4J(6,4)=2.8 Hz, 1H; H-C(6), 6.62 (dd, 1H; H-C(4)), 3.88
(s, 3H; OCH3), 2.88 (t, 3J(3’,2’)=7.9 Hz, 2H; H-3’), 2.72 (td, 2H; H-
C(2’)), 1.03(s, 9H; SiC(CH 3)3), 0.21 (s, 6H; Si(CH3)2) ppm; 13C NMR
(100 MHz, CDCl3): d=202.35 (C(1’)), 154.21 (C(5)), 147.78 (C(2)),
132.07 (C(1)), 119.35 (C(3)), 116.20 (C(6)), 112.38 (C(4)), 56.01 (OCH3),
44.36 (C(2’)), 26.20 (SiC(CH3)3), 24.23(C(3 ’)), 18.59 (SiC(CH3)3), ꢀ3.78
(Si(CH3)2) ppm; IR (NaCl): n˜ =2960 (s), 2930 (s), 2860 (s), 1730 (s), 1610
(w), 1580 (w), 1500 (s), 1470 (m), 1430 (m), 1390 (w), 1360 (w), 1260 (m),
1230 (s), 1160 (m), 1120 (w), 1040 (m), 940 (m), 900 (s), 840 (m), 780
(EtOH): l (%)=228 (100), 294 (87) nm; MS (EI): m/z (%): 164 [M +
]
(100), 149 (66), 91 (20), 77 (27); HRMS (EI): m/z: calcd for C10H12O2:
164.0837; found: 164.0834 [M +].
(2-Allyl-4-methoxyphenoxy)-tert-butyldimethylsilane (30): Imidazole
(68.9 g, 1.01 mol, 2.0 equiv) and tert-butyldimethylsilyl chloride (100 g,
658 mmol, 1.3equiv) were added sequentially to a solution of compound
29 (83.1 g, 506 mmol, 1.0 equiv) in DMF (350 mL). After being stirred
for 1 h at room temperature, the reaction mixture was partitioned be-
tween water (600 mL) and MTBE (200 mL). The aqueous layer was sepa-
rated and extracted further with MTBE (3î200 mL). The combined or-
ganic layers were dried (Na2SO4) and concentrated. The residue was pu-
rified by distillation at reduced pressure (1178C/10ꢀ1 mbar) to yield 30
(135.4 g, 486 mmol, 96%) as a colorless oil. Rf =0.64 (hexane/EtOAc
5:1); b.p. 1178C/10ꢀ1 mbar; 1H NMR (400 MHz, CDCl3): d=6.78 6.69
(m, 2H; H-C(6), H-C(3)), 6.64 (dd, 3J(5,6)=8.6, 4J(5,3)=3.3 Hz, 1H; H-
C(5)), 5.97 (ddt, 3J(2’,3’a)=17.7, 3J(2’,3’b)=9.6, 3J(2’,1’)=6.6 Hz, 1H; H-
C(2’)), 5.11 5.04 (m, 2H; H-C(3’)), 3.90 (s, 3H; OCH3), 3.35 (dt,
4J(1’,3’)=1.5 Hz, 2H; H-C(1’)), 1.03(s, 9H; SiC(CH 3)3), 0.21 (s, 6H;
Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3); d=154.24 (C(4)), 147.58
(C(1)), 137.21 (C(2’)), 131.94 (C(2)), 119.33 (C(6)), 116.12 (C(3’)), 116.07
(C(3)), 112.16 (C(5)), 55.99 (OCH3), 35.01 (C(1’)), 26.25 (SiC(CH3)3),
18.64 (SiC(CH3)3), ꢀ3.78 (Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =3080
(w), 3010 (s), 2960 (s), 2930 (s), 2900 (m), 2860 (s), 2840 (w), 1640 (m),
1610 (w), 1590 (w), 1500 (s), 1470 (s), 1430 (m), 1390 (w), 1360 (w), 1150
(m) cmꢀ1; UV/Vis (MTBE): l=290 nm; MS (EI): m/z (%): 294 [M +
]
(12), 238 (20), 237 (89), 209 (23), 208 (20), 207 (100), 181 (31); HRMS
(EI): m/z: calcd for C16H26O3Si: 294.1651; found: 294.1655 [M +].
4-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-butan-2-ol
(33):
Methyl magnesium chloride (15 mL of a 3m solution in THF, 45 mmol,
4.5 equiv) was added dropwise to a solution of aldehyde 32 (2.91 g,
9.87 mmol, 1.0 equiv) in anhydrous THF (50 mL). After stirring for 1 h at
room temperature, saturated aqueous NH4Cl solution (200 mL) was
added. The aqueous mixture was extracted with MTBE (3î150 mL) and
the combined organic layers were dried over Na2SO4. The solvent was re-
moved by evaporation and the residue was purified by flash chromatog-
raphy (hexane/EtOAc 4:1) to give 33 (2.73g, 8.81 mmol, 89%) as a color-
less oil. Rf =0.19 (hexane/EtOAc 4:1); 1H NMR (400 MHz, CDCl3): d=
6.72 (d, 3J(3,4)=8.8 Hz, 1H; H-C(3)), 6.70 (d, 3J(6,4)=3.1 Hz, 1H; H-
C(6)), 6.61 (dd, 1H; H-C(4)), 3.76 (s, 3H; OCH3), 3.72 (tq, 3J(2’,3’) and
3J(2’,1’)=6.3Hz, 1H; H-C(2 ’)), 2.76 2.59 (m, 2H; H-C(4’)), 2.66 (t,
3J(1’,2’)=7.4 Hz, 2H; H-C(1’)), 1.94 (s, 1H; HO-C(2’)), 1.75 1.68 (m,
2H; H-C(3’)), 1.19 (d, 3H; H-C(1’)), 1.02 (s, 9H; SiC(CH3)3), 0.21 (s,
6H; Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=154.38 (C(5)),
147.65 (C(2)), 133.69 (C(1)), 119.58 (C(3)), 116.10 (C(6)), 111.90 (C(5)),
67.61 (C(2’)), 55.98 (OCH3), 40.05 (C(3’)), 27.13(C(4 ’)), 26.26
(SiC(CH3)3), 23.71 (C(1’)), 18.66 (SiC(CH3)3), ꢀ3.79 (Si(CH3)2) ppm; IR
(NaCl, CHCl3): n˜ =3600 (s), 3490 (s), 3010 (s), 2960 (s), 2940 (s), 2860
(s), 2840 (s), 1600 (m), 1580 (m), 1490 (s), 1460 (s), 1420 (m), 1390 (w),
1370 (w), 1150 (m), 1120 (w), 1060 (s), 1040 (s), 940 (s), 900 (s), 860 (s),
(m), 1100 (w), 1040 (s), 1000 (m), 950 (m), 920 (s), 880 (s), 840 (s) cmꢀ1
UV/Vis (MTBE): l=290 nm; MS (EI): m/z (%): 278 [M +] (48), 222,
;
(26), 221 (100), 193(13), 181 (42); HRMS (EI):
m/z: calcd for
C16H26O2Si: 278.1702; found: 278.1703[ M +]; elemental analysis calcd
(%) for C16H26O2Si: C 69.01, H 9.41, O 11.49, Si 10.09; found: C 68.87, H
9.63.
3-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]propan-1-ol
(31):
BH3¥THF (56.4 mL of a 1m solution in THF, 56.4 mmol, 1.1 equiv) was
added dropwise to a stirred solution of alkene 30 (14.5 g, 52.1 mmol,
1.0 equiv) in THF (200 mL) at 08C over a period of 15 min. After stirring
for 10 min, the ice bath was removed and stirring was continued for an
additional 2 h at room temperature. The reaction mixture was cooled to
08C and then aqueous NaOH (20 mL of a 3m solution) and H2O2 (20 mL
of a 30% solution) were slowly added. After stirring for 15 min, the cool-
ing bath was removed and stirring was continued for additional 1 h. Then
the aqueous solution was extracted with MTBE (3î200 mL). The organic
layers were dried and evaporated, then the crude residue was purified by
column chromatography (hexane/EtOAc 3:1) to give 31 (13.8 g,
840 (m) cmꢀ1; UV/Vis (MTBE): l=290 nm; MS (EI): m/z (%): 310 [M +
]
(21), 235 (15), 211 (100), 193 (33); HRMS (EI): m/z: calcd for
C17H30O3Si: 310.1964; found: 310.1968 [M +].
4-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-butan-2-one (34):
Oxalyl chloride (570 mL, 864 mg, 6.8 mmol, 1.4 equiv) was added drop-
wise to a solution of DMSO (1.0 mL, 1.09 g, 13.6 mmol, 2.8 equiv) in
CH2Cl2 (50 mL) at ꢀ788C. After 10 min, a solution of alcohol 33 (1.51 g,
4.86 mmol, 1.0 equiv) in CH2Cl2 (5 mL) was added dropwise. After stir-
ring at ꢀ788C for 15 min, triethylamine (4.0 mL, 2.95 g, 29.1 mmol,
46.4 mmol, 89%) as
a colorless oil. Rf =0.16 (hexane/EtOAc 4:1);
1H NMR (400 MHz, CDCl3): d=6.72 (d, 3J(3,4)=8.7 Hz, 1H; H-C(3)),
6.70 (d, 3J(6,4)=3.3 Hz, 1H; H-C(6)), 6.61 (dd, 1H; H-C(4)), 3.92 (s,
3H; OCH3), 3.60 (t, 3J(3’,2’)=6.3Hz, 2H; H-C(3 ’)), 2.66 (t, 3J(1’,2’)=
7.4 Hz, 2H; H-C(1’)), 1.90 1.85 (m, 2H; H-C(2’)), 1.71 (s, 1H; OH), 1.02
2498
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
6.0 equiv) was added and the reaction mixture was allowed to warm to
room temperature. The reaction mixture was partitioned between water
(300 mL) and MTBE (300 mL). The aqueous layer was separated and ex-
tracted further with MTBE (2î150 mL). The combined organic layers
were washed with 2m aqueous HCl, water, saturated aqueous Na2CO3
solution, and finally water. The organic layers were dried over Na2SO4,
and the solvent was removed by evaporation. The residue was purified
by flash chromatography (hexane/MTBE 4:1) to give 34 (1.42 g,
MS (EI): m/z (%): 192 [M +] (60), 137 (100), 109 (10); HRMS (EI): m/z:
calcd for C12H16O2: 192.1150; found: 192.1146 [M +].
3-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-1,1-dimethoxypro-
pane (36): Aldehyde 32 (2.78 g, 9.49 mmol, 1.0 equiv) was treated with
methanol (30 mL), trimethyl orthoformate (15 mL), and (Æ)-camphorsul-
fonic acid (175 mg, 0.76 mmol, 8 mol%) at 458C for 30 min. The mixture
was evaporated to dryness and the crude solid was purified by flash chro-
matography (hexane/MTBE 85:15) to yield 36 (2.75 g, 8.09 mmol, 85%)
1
4.59 mmol, 95%) as
a colorless oil. Rf =0.22 (hexane/MTBE 4:1);
as an oil. Rf =0.26 (hexane/MTBE 9:1); H NMR (300 MHz, CDCl3): d=
1H NMR (400 MHz, CDCl3): d=6.70 (d, 3J(3,4)=8.8 Hz, 1H; H-C(3)),
6.69 (d, 4J(6,4)=3.0 Hz, 1H; H-C(6)), 6.61 (dd, 1H; H-C(4)), 3.86 (s,
3H; OCH3), 2.81 (m, 2H; H-C(4’)), 2.72 (m, 2H; H-C(3’)), 2.02 (s, 3H;
H-C(1’)), 1.04 (s, 9H; SiC(CH3)3), 0.21 (s, 6H; Si(CH3)2) ppm; 13C NMR
(100 MHz, CDCl3): d=208.59 (C(2’)), 154.19 (C(5)), 147.78 (C(2)),
132.74 (C(1)), 119.31 (C(3)), 116.18 (C(6)), 112.23 (C(4)), 56.00 (OCH3),
44.08 (C(3’)), 30.33 (C(1’)), 26.18 (SiC(CH3)3), 25.84 (C(4’)), 18.56
(SiC(CH3)3), ꢀ3.79 (Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =3010 (m),
2960 (s), 2930 (s), 2860 (s), 2840 (w), 1710 (s), 1610 (m), 1580 (m), 1490
(s), 1470 (m), 1440 (w), 1430 (w), 1360 (m), 1160 (m), 1120 (w), 1060
(m), 1030 (s), 940 (s), 890 (s), 840 (m) cmꢀ1; UV/Vis (CHCl3): l=
288 nm; MS (EI): m/z (%): 308 [M +] (8), 251 (100), 235 (15), 207 (36),
181 (28); HRMS (EI): m/z: calcd for C17H28O3Si: 308.1808; found:
308.1812 [M +]; elemental analysis calcd (%) for C17H28O3Si: C 66.19, H
9.15, O 15.56, Si 9.10; found: C 66.14, H 9.36.
6.71 (d, 4J(6,4)=3.2 Hz, 1H; H-C(6)), 6.69 (d, 3J(3,4)=8.8 Hz, 1H; H-
C(3)), 6.61 (dd, 1H; H-C(4)), 4.37 (t, 3J(1’,2’)=5.8 Hz, 1H; H-(C1’)),
3.88 (s, 3H; OCH3), 3.33 (s, 6H; (CH3O)2C(1’)), 2.65 2.55 (m, 2H; H-
C(3’)), 1.88 (td, 3J(2’,3’)=5.8 Hz, 2H; H-C(2’)), 1.01 (s, 9H; SiC(CH3)3),
0.20 (s, 6H; Si(CH3)2) ppm; 13C NMR (75 MHz, CDCl3): d=153.71
(C(5)), 147.41 (C(2)), 133.04 (C(1)), 118.93 (C(3)), 115.67 (C(6)), 111.53
(C(4)), 104.22 (C(1’)), 55.60 (OCH3), 52.79 (CH3O)2C(1’)), 32.55 (C(2’)),
26.05 (C(3’)), 25.84 (SiC(CH3)3), 18.24 (SiC(CH3)3), ꢀ4.10
(Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =3010 (s), 2960 (s), 2930 (s), 2900
(s), 2860 (s), 2830 (m), 1610 (w), 1580 (w), 1500 (s), 1470 (s), 1450 (m),
1430 (w), 1390 (w), 1360 (w), 1160 (m), 1130 (s), 1080 (s), 1050 (s), 940
(m), 900 (s), 840 (s) cmꢀ1; UV/Vis (CH3OH): l (%)=222 (100), 288
(68) nm; MS (EI): m/z (%): 340 [M +] (39), 251 (28), 225 (69), 196 (22),
195 (100), 89 (24), 75 (20); elemental analysis calcd (%) for C18H32O4Si:
C 63.49, H 9.47, O 18.79, Si 8.25; found: C 63.53, H 9.41.
4-Methoxy-2-(3-methylbut-3-enyl)phenol (35): nBuLi (1.7 mL of a 1.6m
nBuLi solution in hexane, 2.75 mmol, 2.2 equiv) was added to a suspen-
sion of methyltriphenylphosphonium bromide (893mg, 2.5 mmol,
2 equiv) in THF (50 mL) at 08C. The mixture was stirred for 15 min at
room temperature and then ketone 34 (385 mg, 1.25 mmol, 1.0 equiv) dis-
solved in THF (5 mL) was added dropwise. After stirring for 60 min, the
reaction was quenched with water (100 mL) and extracted with MTBE
(3î50 mL). The combined organic layers were dried over Na2SO4 and
and the solvent was removed by evaporation. The residue was purified
by flash chromatography (hexane/MTBE 9:1) to yield 4-[2-(tert-butyldi-
3-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-1,1-diethoxypro-
pane (37): Aldehyde 32 (2.49 g, 8.46 mmol, 1.0 equiv) was treated with
ethanol (30 mL), triethyl orthoformate (15 mL), and (Æ)-camphorsulfon-
ic acid (181 mg, 0.78 mmol, 9 mol%) at 458C for 30 min. The mixture
was evaporated to dryness and the crude solid was purified by flash chro-
matography (hexane/MTBE 9:1) to yield 37 (2.34 g, 6.36 mmol, 76%) as
an oil. Rf =0.21 (hexane/MTBE 9:1); 1H NMR (400 MHz, CDCl3): d=
6.73(d, 4J(6,4)=3.1 Hz, 1H; H-C(6)), 6.70 (d, 3J(3,4)=8.5 Hz, 1H; H-
C()3), 6.63(dd, 1H; H-C(4)), 4.73(t,
3J(1’,2’)=5.7 Hz, 1H; H-C(1’)),
3.78 (s, 3H; OCH3), 3.74 3.46 (m, 4H; C(OCH2CH3)2), 2.70 2.60 (m,
2H; H-C(3’)), 1.98 1.86 (m, 2H; H-C(2’)), 1.24 (t, 3J(C(OCH2CH3)2,-
C(OCH2CH3)2)=5.7 Hz, 6H; C(OCH2CH3)2), 1.04 (s, 9H; SiC(CH3)3),
0.24 (s, 6H; Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=154.10
(C(5)), 147.79 (C(2)), 133.59 (C(1)), 119.29 (C(3)), 115.93 (C(6)), 111.83
(C(4)), 103.08 (C(1’)), 61.46 (C(OCH2CH3)2), 55.98 (OCH3), 33.93
(C(2’)), 26.54 (C(3’)), 26.29 (SiC(CH3)3), 18.62 (SiC(CH3)3), 15.79
(C(OCH2CH3)2), ꢀ4.16 (Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =3010 (s),
2960 (s), 2930 (s), 2900 (s), 2880 (m), 1610 (m), 1580 (m), 1490 (s), 1470
(s), 1440 (m), 1420 (w), 1390 (w), 1370 (m), 1340 (w), 1130 (s), 1060 (s),
1040 (s), 1000 (m), 940 (m), 900 (s), 840 (s) cmꢀ1; UV/Vis (MeOH): l
(%)=222 (100), 288 (62) nm; MS (EI): m/z (%): 368 [M +] (36), 252
(17), 251 (26), 239 (21), 195 (100), 73 (29); elemental analysis calcd (%)
for C20H36O4Si: C 65.17, H 9.84, O 17.36, Si 7.62; found: C 64.91, H 9.61.
methylsilanyloxy)-5-methoxyphenyl]-2-methyl-but-1-enyl
(287 mg,
937 mmol, 75%) as an oil. Rf =0.45 (hexane/MTBE 4:1); 1H NMR
(400 MHz, CDCl3): d=6.73(d, 4J(6,4)=3.0 Hz, 1H; H-C(6)), 6.70 (d,
3J(3,4)=8.8 Hz, 1H; H-C(3)), 6.61 (dd, 1H; H-C(4)), 4.71 (s, 2H; H-
3
C(1’)), 3.86 (s, 3H; OCH3), 2.71 (t, J(4’,3’)=8.3Hz, 2H; H-(4 ’)), 2.72 (t,
2H; H-C(3’)), 1.83(s, 3H; CH 3C(2’)), 1.02 (s, 9H; SiC(CH3)3), 0.23(s,
6H; Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=154.12 (C(5)),
147.74 (C(2)), 146.15 (C(2’)), 132.07 (C(1)), 119.20 (C(3)), 116.06 (C(6)),
111.67 (C(4)), 110.25 (C(1’)), 55.99 (OCH3), 38.34 (C(3’)), 29.64 (C(4’)),
26.22 (SiC(CH3)3), 23.09 (CH3C(2’)), 18.61 (SiC(CH3)3), ꢀ3.79
(Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =3090 (w), 3000 (w), 2940 (m),
2830 (m), 1660 (m), 1610 (w), 1500 (s), 1460 (m), 1440 (m), 1430 (m),
1380 (w), 1340 (w), 1260 (m), 1150 (m), 1100 (w), 1040 (s), 890 (s) cmꢀ1
;
UV/Vis (MeOH): l=288 nm; MS (EI): m/z (%): 306 [M +] (100), 251
(33), 249 (31), 207 (67), 193 (83); HRMS (EI): m/z: calcd for C18H30O2Si:
306.2015; found: 306.2012 [M +]. A solution of tetrabutylammonium fluo-
ride (250 mL of a 1m TBAF in THF, 250 mmol, 1.3equiv) was added to a
3-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-1,1-diisopropoxy-
propane (38): Aldehyde 32 (895 mg, 3.03 mmol, 1.0 equiv) was treated
with isopropanol (10 mL), triisopropyl orthoformate (5 mL), and (Æ)-
camphorsulfonic acid (60.0 mg, 0.26 mmol, 10 mol%) at 458C for 50 min.
The mixture was evaporated to dryness and the crude solid was purified
by flash chromatography (hexane/MTBE 19:1) to yield 38 (985 mg,
2.49 mmol, 83%) as an oil. Rf =0.52 (hexane/MTBE 9:1); 1H NMR
(400 MHz, CDCl3): d=6.71 (d, 4J(6,4)=3.1 Hz, 1H; H-C(6)), 6.68 (d,
3J(3,4)=8.6 Hz, 1H; H-C(3)), 6.60 (dd, 1H; H-C(4)), 4.58 (t, 3J(1’,2’)=
5.6 Hz, 1H; H-C(1’)), 3.87 (septet, 3J(OCH(CH3)2,OCH(CH3)2)=6.2 Hz,
2H; OCH(CH3)2), 3.78 (s, 3H; OCH3), 2.64 2.58 (m, 2H; H-C(3’)), 1.90
1.83(m, 2H; H-C(2 ’)), 1.19 (d, 3H; OCH(CH3)2), 1.13(d, H3 ;
OCH(CH3)2), 1.02 (s, 9H; SiC(CH3)3), 0.20 (s, 6H; Si(CH3)2) ppm;
13C NMR (100 MHz, CDCl3): d=154.10 (C(5)), 147.81 (C(2)), 133.83
(C(1)), 119.23(C(3)), 115.94 (C(6)), 111.72 (C(4)), 100.51 (C(1 ’)), 67.99
(OCH(CH3)2), 55.98 (OCH3), 35.65 (C(2’)), 26.74 (C(3’)), 26.27
(SiC(CH3)3), 23.85, 23.03 (OCH(CH3)2), 18.62 (SiC(CH3)3), ꢀ3.78
(Si(CH3)2) ppm; IR (NaCl): n˜ =2970 (s), 2930 (s), 2900 (m), 2860 (m),
2840 (w), 1610 (w), 1580 (w), 1490 (s), 1460 (m), 1380 (m), 1370 (m),
1330 (w), 1250 (m), 1220 (s), 1180 (w), 1160 (m), 1130 (m), 1030 (s), 990
(w), 950 (m), 890 (m), 840 (s), 800 (m) cmꢀ1; UV/Vis (MeOH): l (%)=
225 (100), 286 (68) nm; MS (EI): m/z (%): 396 [M +] (58), 336 (25), 293
(63), 251 (44), 237 (100), 211 (34), 193 (20), 75 (38); elemental analysis
solution
of
4-[2-(tert-butyldimethylsilanyloxy)-5-methoxyphenyl]-2-
methyl-but-1-enyl (59.1 mg, 192 mmol, 1 equiv) in THF (2 mL) at 08C.
After being stirred at 08C for 20 min, the reaction mixture was parti-
tioned between saturated aqueous NaHCO3 solution (20 mL) and MTBE
(20 mL). The aqueous layer was separated and extracted further with
MTBE (2î20 mL). The combined organic layers were dried over Na2SO4
and concentrated. The oily residue was purified by flash chromatography
(hexane/MTBE 2:1) to give product 35 (34.4 mg, 179 mmol, 93%) as a
colorless oil. Rf =0.33 (hexane/MTBE 2:1); 1H NMR (400 MHz, CDCl3):
d=6.75 (d, 4J(3,5)=3.0 Hz, 1H; H-C(3)), 6.71 (d, 3J(6,5)=8.8 Hz, 1H;
H-C(6)), 6.59 (dd, 1H; H-C(5)), 4.81 (s, 2H; H-C(4’)), 4.40 (s, 1H; OH),
3.77 (s, 3H; OCH3), 2.73(t, 3J(1’,2’)=8.2 Hz, 2H; H-(1’)), 2.42 (t, 2H; H-
C(2’)), 1.83(s, 3H; CH 3C(3’)) ppm; 13C NMR (100 MHz, CDCl3): d=
154.13(C(4)), 147.83(C(1)), 146.10 (C(3 ’)), 129.84 (C(2)), 116.34 (C(6)),
116.22 (C(3)), 112.21 (C(5)), 110.69 (C(4’)), 56.14 (OCH3), 38.14 (C(2’)),
29.24 (C(1’)), 23.06 (CH3C(3’)) ppm; IR (NaCl, CHCl3): n˜ =3600 (m),
3350 (m), 3080 (w), 3010 (w), 2940 (m), 2840 (m), 1650 (m), 1610 (w),
1510 (s), 1470 (m), 1450 (m), 1430 (m), 1380 (w), 1330 (w), 1260 (m),
1150 (m), 1100 (w), 1040 (s), 890 (s) cmꢀ1; UV/Vis (CHCl3): l=292 nm;
2499
Chem. Eur. J. 2004, 10, 2487 2506
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
calcd (%) for C22H40O4Si: C 66.62, H 10.16, O 16.14, Si 7.08; found: C
66.41, H 10.09.
(Si(CH3)2)) ppm; IR (NaCl): n˜ =3030 (w), 2960 (s), 2930 (s), 2900 (w),
2860 (m), 2830 (w), 1660 (m), 1610 (w), 1590 (w), 1500 (s), 1470 (m),
1440 (m), 1390 (w), 1370 (w), 1360 (w), 1250 (s), 1220 (s), 1150 (m), 1110
(s), 1040 (m), 950 (m), 900 (s), 840 (s), 800 (m), 780 (s) cmꢀ1; UV/Vis
(MeOH): l=288 nm; MS (EI): m/z (%): 322 [M +] (73), 237 (66), 236
(41), 209 (72), 207 (46), 181 (100), 161 (19); HRMS (EI): m/z: calcd for
C18H30O3Si: 322.1964, found: 322.1959 [M +]; elemental analysis calcd
(%) for C18H30O3Si: C 67.03, H 9.38, O 14.88, Si 8.71; found: C 67.29, H
9.52.
(E,Z)-[2-(3-Methoxy-2-buten-1-yl)-4-methoxyphenoxy]-tert-butyldime-
thylsilane (39): Compound 36 (2.58 g, 7.59 mmol, 1 equiv) and N-ethyldii-
sopropylamine (1.8 mL, 1.38 g, 10.63 mmol, 1.4 equiv) were dissolved in
CH2Cl2 (50 mL). After the solution was cooled to ꢀ788C, TMSOTf
(1.6 mL, 2.03g, 9.11 mmol, 1.2 equiv) was added dropwise through a sy-
ringe with stirring. The pale yellow mixture was stirred at ꢀ208C for 4 h.
The reaction was quenched by the addition of a saturated aqueous
NaHCO3 solution (200 mL) and the aqueous layer was separated and ex-
tracted further with MTBE (2î50 mL). The combined organic layers
were dried over Na2SO4 and concentrated. The crude solid was purified
by flash chromatography (hexane/MTBE 47:3) to yield 39 (1.37 g,
(E,Z)-[2-(3-Propoxy-2-buten-1-yl)-4-methoxyphenoxy]-tert-butyldime-
thylsilane (41): Compound 38 (165 mg, 416 mmol, 1 equiv) was dissolved
in CCl4 (5 mL) and treated at ꢀ108C with anhydrous hexamethyldisila-
zane (140 mL, 86.7 mg, 666 mmol, 1.6 equiv) and iodotrimethylsilane
(74 mL, 108 mg, 540 mmol, 1.3equiv). After stirring for 1 h at ꢀ108C, the
mixture was heated at 758C for an additional 15 h. The reaction was
quenched by the addition of a saturated aqueous NaHCO3 solution
(15 mL) and the aqueous layer was separated and extracted further with
CH2Cl2 (2î20 mL). The combined organic layers were dried over
Na2SO4 and concentrated. The crude solid was purified by flash chroma-
tography (hexane/MTBE 48:2) to yield 41 (40.5 mg, 120 mmol, 29%) as a
yellow oil. Rf =0.25 and 0.22 (hexane/MTBE 96:4) for Z-41 and E-41, re-
4.48 mmol, 59%) as
a yellow oil. Rf =0.30 (hexane/MTBE 47:3);
1H NMR (400 MHz, CDCl3): d=6.77 (d, 4J(Z-3,Z-5)=3.3 Hz, 1H; H-
C(Z-3)), 6.73 (d, 4J(E-3,E-5)=3.1 Hz, 1H; H-C(E-3)), 6.69 (d, 3J(E-6,E-
3
5)=8.6 Hz, 1H; H-C(E-6)), 6.68 (d, J(Z-6,Z-5)=8.6 Hz, 1H; H-C(Z-6)),
6.62 6.56 (m, 2H; H-C(E-5), H-C(Z-5)), 6.37 (d, 3J(E-3’,E-2’)=12.6 Hz,
1H; H-C(E-3’)), 6.02 (d, 3J(Z-3’,Z-2’)=6.3Hz, 1H; H-C( Z-3’)), 4.88 (td,
3J(E-2’,E-1’)=7.3Hz, 1H; H-C( E-2’)), 4.52 (td, 3J(Z-2’,Z-1’)=7.3Hz,
1H; H-C(Z-2’)), 3.75 (s, 3H; E-(C(4)-CH3)), 3.74 (s, 3H; Z-(C(4)-
OCH3)), 3.62 (s, 3H; E-(C(3’)-OCH3)), 3.52 (s, 3H; Z-(C(3’)-OCH3)),
3.38 (d, 2H; H-C(Z-1’)), 3.22 (d, 2H; H-C(E-1’)), 1.01 (s; 9H; E-
(SiC(CH3)3), Z-(SiC(CH3)3)), 0.19 (s, 9H, E-(SiC(CH3)3), Z-
(SiC(CH3)3)) ppm; 13C NMR (100 MHz, CDCl3): d=154.303 (C(E-4),
C(Z-4)), 147.52 (C(E-1), C(Z-1)), 147.07 (C(E-3’), C(Z-3’)), 133.52 (C(E-
2), C(Z-2)), 119.25 (C(Z-6)), 119.24 (C(E-6)), 115.86 (C(E-3), C(Z-3)),
111.77 (C(E-5)), 111.66 (C(Z-5)), 105.24 (C(Z-2’)), 102.10 (C(E-2’)), 59.9
(Z-(C(3’)-OCH3)), 56.28 (E-(C(3’)-OCH3)), 55.98 (E-(C(4)-OCH3)),
55.96 (Z-(C(4)-OCH3)), 28.81 (C(E-1’)), 26.26 (E-(SiC(CH3)3), Z-
(SiC(CH3)3)), 25.15 (C(Z-1’)), 18.99 (E-SiC(CH3)3), Z-(SiC(CH3)3)),
ꢀ3.80 (E-(Si(CH3)2), Z-(Si(CH3)2)) ppm; IR (NaCl): n˜ =2960 (s), 2930
(s), 2910 (w), 2860 (m), 2830 (w), 1650 (w), 1600 (w), 1560 (w), 1500 (s),
1470 (m), 1460 (m), 1430 (w), 1390 (w), 1370 (w), 1150 (m), 1110 (s),
1040 (w), 1010 (w), 940 (m), 900 (m), 840 (s) cmꢀ1; UV/Vis (CHCl3): l=
290 nm; MS (EI): m/z (%): 308 [M +] (63), 251 (51), 236 (16), 219 (45),
195 (22), 145 (25), 89 (100), 59 (22); HRMS (EI): m/z: calcd for
C17H28O3Si: 308.1808; found: 308.1811 [M +]; elemental analysis calcd
(%) for C17H28O3Si: C 66.19, H 9.15, O 15.56, Si 9.10; found: C 66.34, H
9.25.
1
spectively; H NMR (400 MHz, CDCl3): d=6.80 6.56 (m, 6H; H-C(E-6),
3
H-C(Z-6), H-C(E-5), H-C(Z-5), H-C(E-3), H-C(Z-3)), 6.15 (d, J(E-3’,E-
3
2’)=12.4 Hz, 1H; H-C(E-3’)), 6.09 (d, J(Z-3’,Z-2’)=6.1 Hz, 1H; H-C(Z-
3’)), 5.00 (dt, 3J(E-2’,E-1’)=7.3Hz, 1H; H-C( E-2’)), 4.54 (td, 3J(Z-2’,Z-
1’)=7.3Hz, 1H; H-C( Z-2’)), 3.97 (septet, 3J(OCH(CH3)2,OCH(CH3)2)=
6.2 Hz,
1H;
Z-(OCH(CH3)2)),
3.92
(septet,
3J(OCH(-
CH3)2,OCH(CH3)2)=6.2 Hz, 1H; E-(OCH(CH3)2)), 3.74 (s, 3H; E-
(OCH3)), 3.73 (s, 3H; Z-(OCH3)), 3.37 (d, 2H; H-C(Z-1’)), 3.19 (d, 2H;
H-C(E-1’)), 1.24 (d, 6H; Z-(OCH(CH3)2)), 1.22 (d, 6H; E-
(OCH(CH3)2)), 1.01 (s, 9H; Z-(SiC(CH3)3)), 0.99 (s, 9H; E-(SiC(CH3)3)),
0.19 (s, 12H; E-(SiC(CH3)3), Z-(SiC(CH3)3)) ppm; 13C NMR (100 MHz,
CDCl3): d=154.22 (C(E-4), C(Z-4)), 147.55 (C(E-1’), C(Z-1’)), 146.42
(C(E-3’)), 144.72 (C(Z-3’)), 133.75 (C(E-2)), 133.57 (C(Z-2)), 119.27
(C(Z-6)), 119.20 (C(E-6)), 115.73(C( Z-3)), 115.67 (C(E-3)), 111.76 (C(E-
5)), 111.60 (C(Z-5)), 105.31 (C(Z-2’)), 104.18 (C(E-2’)), 74.25 (Z-
(OCH(CH3)2)), 72.76 (E-(OCH(CH3)2)), 56.00 (E-(OCH3)), 55.94 (Z-
(OCH3)), 28.73(C( E-1’)), 26.27 (Z-(SiC(CH3)3)), 26.25 (E-(SiC(CH3)3)),
25.17 (C(Z-1’)), 22.88 (Z-(OCH(CH3)2)), 22.55 (E-(OCH(CH3)2)), 18.66
(E-(SiC(CH3)3), Z-(SiC(CH3)3)), ꢀ3.77 (E-(Si(CH3)2)), ꢀ3.80 (Z-
(Si(CH3)2)) ppm; IR (NaCl): n˜ =3040 (w), 2960 (s), 2930 (s), 2910 (w),
2900 (w), 2860 (m), 2830 (w), 1660 (m), 1600 (w), 1500 (s), 1470 (m),
1460 (m), 1430 (m), 1370 (w), 1340 (m), 1250 (s), 1220 (s), 1150 (m), 1120
(m), 1100 (m), 1050 (s), 950 (m), 900 (s), 840 (s), 800 (m) cmꢀ1; UV/Vis
(MeOH): l=288 nm; MS (EI): m/z (%): 336 [M +] (39), 237 (53), 181
(100); HRMS (EI): m/z: calcd for C19H32O3Si: 336.2121; found: 336.2125
[M +].
(E,Z)-[2-(3-Ethoxy-2-buten-1-yl)-4-methoxyphenoxy]-tert-butyldimethyl-
silane (40): Compound 37 (2.10 g, 5.70 mmol, 1 equiv) and N-ethyldiiso-
propylamine (1.4 mL, 1.03g, 7.98 mmol, 1.4 equiv) were disolved in
CH2Cl2 (50 mL). After the solution was cooled to ꢀ788C, TMSOTf
(1.2 mL, 1.52 g, 6.84 mmol, 1.2 equiv) was added dropwise through a sy-
ringe with stirring. The pale yellow solution was stirred at ꢀ208C for 4 h.
The reaction was quenched by the addition of a saturated aqueous
NaHCO3 solution (200 mL) and the aqueous layer was separated and ex-
tracted further with MTBE (2î50 mL). The combined organic layers
were dried over Na2SO4 and concentrated. The crude solid was purified
by flash chromatography (hexane/MTBE 19:1) to yield 40 (1.158 g,
(E and Z)-2-(3-Methoxy-2-buten-1-yl)-4-methoxyphenol (10a and 10b):
Enol ether 39 (34.6 mg, 112 mmol) was dissolved in ethanolamine (4 mL)
and heated at 908C for 3h. Evaporation of the solvent at reduced pres-
sure (398C/4î10ꢀ1 mbar) and column chromatography (hexane/MTBE/
NEt3 16:4:0.2) of the crude residue gave a mixture of the two isomers.
Purification of the isomeric mixture by preparative RP18 HPLC provided
isomerically pure E enol ether 10b (10.2 mg, 51.5 mmol; 46%) and Z
enol ether 10a (9.5 mg, 49.2 mmol, 44%). Analytical data for (E)-2-(3-
methoxy-2-buten-1-yl)-4-methoxyphenol (10b). Rf =0.14 (hexane/MTBE
8:2); 1H NMR (400 MHz, CDCl3): d=6.84 (d, 3J(6,5)=8.4 Hz, 1H; H-
C(6)), 6.69 (d, 4J(3,5)=3.0 Hz, 1H; H-C(3)), 6.56 (dd, 1H; H-(5)), 6.41
(d, 3J(3’,2’)=12.8 Hz, 1H; H-C(3’)), 4.79 (dt, 3J(2’,1’)=7.2 Hz, 1H; H-
C(2’)), 4.36 (s, 1H; OH), 3.78 (s, 3H; C(4)-OCH3), 3.58 (s, 3H; C(3’)-
OCH3), 3.26 (d, 2H; H-C(1’)) ppm; 13C NMR (100 MHz, CDCl3): d=
154.10 (C(4)), 149.12 (C(1)), 148.88 (C(3’)), 128.45 (C(2)), 116.72 (C(6)),
116.06 (C(3)), 112.70 (C(5)), 100.74 (C(2’)), 56.44 (C(3’)-OCH3), 56.13
(C(4)-OCH3), 29.54 (C(1’)) ppm; IR (NaCl): n˜ =3410 (s), 3060 (w), 3040
(w), 3000 (w), 2940 (m), 2920 (m), 2840 (m), 1660 (s), 1620 (w), 1510 (s),
1470 (m), 1440 (m), 1350 (w), 1270 (m), 1210 (s), 1150 (m), 1110 (m),
1040 (m), 940 (m), 870 (w), 810 (m), 790 (w), 720 (m) cmꢀ1; UV/Vis
(EtOH): l (%)=224 (100), 292 (73) nm; MS (EI): m/z (%): 195 (11), 194
[M +] (84), 163 (5), 162 (13), 161 (35), 147 (9), 137 (13), 136 (100), 135
(5), 108 (33), 107 (4), 91 (10), 79 (7), 78 (10), 77 (9), 65 (11), 63 (3), 55
1
3.59 mmol, 63%) as a yellow oil. Rf =0.40 (hexane/MTBE 9:1); H NMR
4
(400 MHz, CDCl3): d=6.79 (d, J(Z-3,Z-5)=3.0 Hz, 1H; H-C(Z-3)), 6.74
(d, 4J(E-3,E-5)=3.0 Hz, 1H; H-C(E-3)), 6.69 (d, 3J(E-6,E-5)=8.6 Hz,
1H; H-C(E-6)), 6.68 (d, 3J(Z-6,Z-5)=8.6 Hz, 1H; H-C(Z-6)), 6.62 6.56
(m, 2H; H-C(E-5), H-C(Z-5)), 6.30 (d, 3J(E-3’,E-2’)=12.7 Hz, 1H; H-
C(E-3’)), 6.05 (d, 3J(Z-3’,Z-2’)=6.1 Hz, 1H; H-C(Z-3’)), 4.92 (td, 3J(E-
2’,E-1’)=7.3Hz, 1H; H-C( E-2’)), 4.53(td, 3J(Z-2’,Z-1’)=7.3Hz, 1H; H-
3
C(Z-2’)), 3.81 (q, J(OCH2CH3,OCH2CH3)=7.1 Hz, 2H; Z-(OCH2CH3)),
3.75 (s, 3H; E-(OCH3)), 3.74 (s, 3H; Z-(OCH3)), 3.72 (q, 3J(OCH2-
CH3,OCH2CH3)=7.1 Hz, 2H; E-(OCH2CH3)), 3.38 (d, 2H; H-C(Z-1’)),
3.19 (d, 2H; H-C(E-1’)), 1.27 (t, 6H; E-(OCH2CH3), Z-(OCH2CH3)),
1.01 (s, 6H; E-(SiC(CH3)3), Z-(SiC(CH3)3)), 0.19 (s, 12H; E-(SiC(CH3)3),
Z-(SiC(CH3)3)) ppm; 13C NMR (100 MHz, CDCl3): d=154.25 (C(E-4),
C(Z-4)), 147.59 (C(E-1), C(Z-1)), 145.77 (C(E-3’), C(Z-3’)), 133.58 (C(E-
2), C(Z-2)), 119.25 (C(Z-6)), 119.21 (C(E-6)), 115.79 (C(E-3), C(Z-3)),
111.76 (C(E-5)), 111.62 (C(Z-5)), 105.22 (C(Z-2’)), 102.41 (C(E-2’)),
67.98 (Z-(OCH2CH3)), 65.00 (E-(OCH2CH3)), 56.02 (E-(OCH3)), 55.97
(Z-(OCH3)), 28.84 (C(E-1’)), 26.25 (E-(SiC(CH3)3), Z-(SiC(CH3)3)),
25.17 (C(Z-1’)), 18.64 (E-(SiC(CH3)3), Z-(SiC(CH3)3)), 15.71 (Z-
(OCH2CH3)), 15.17 (E-(OCH2CH3)), ꢀ3.80 (E-(Si(CH3)2), Z-
2500
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
(5), 51 (6); HRMS (EI): m/z: calcd for C11H14O3: 194.0943; found:
194.0947 [M +].
148.58 (C(1)), 147.27 (C(3’)), 128.09 (C(2)), 116.77 (C(6)), 115.98 (C(3)),
112.79 (C(5)), 103.23 (C(2’)), 73.17 (OCH(CH3)2), 56.13(OCH 3), 29.83
(C(1’)), 22.51 (OCH(CH3)2) ppm; IR (NaCl): n˜ =3410 (s), 3080 (w), 2970
(s), 2920 (s), 2850 (m), 2830 (m), 1670 (s), 1650 (s), 1500 (s), 1460 (m),
1430 (m), 1390 (w), 1370 (m), 1340 (m), 1260 (m), 1200 (s), 1160 (s), 1120
(s), 1040 (s), 930 (m) cmꢀ1; UV/Vis (MeOH): l=292 nm; MS (EI): m/z
(%): 222 [M +] (49), 180 (100), 137 (61), 136 (96); HRMS (EI): m/z:
calcd for C13H18O3: 222.1256; found: 222.1258 [M +].
(Z)-2-(3-Methoxy-2-buten-1-yl)-4-methoxyphenol
(10a):
Rf =0.17
(hexane/MTBE 8:2); 1H NMR (400 MHz, CDCl3): d=6.82 (d, 3J(6,5)=
8.7 Hz, 1H; H-C(6)), 6.62 (dd, 4J(5,3)=3.2 Hz, 1H; H-C(5)), 6.64 (d,
1H; H-C(3)), 6.31 (s, 1H; OH), 6.19 (d, 3J(3’,2’)=6.2 Hz, 1H; H-C(3’)),
4.46 (td, 3J(2’,1’)=8.6 Hz, 1H; H-C(2’)), 3.78 (s, 3H; C(4)-OCH3), 3.52
(s, 3H; C(3’)-OCH3), 3.30 (d, 2H; H-C(1’)) ppm; 13C NMR (100 MHz,
CDCl3): d=153.66 (C(4)), 148.89 (C(1)), 146.45 (C(3’)), 127.55 (C(2)),
117.08 (C(6)), 116.01 (C(3)), 113.12 (C(5)), 105.52 (C(2’)), 60.31 (C(3’)-
OCH3), 56.43(C(4)-O CH3), 26.29 (C(1’)) ppm; IR (NaCl): n˜ =3420 (s),
3060 (w), 3040 (w), 3010 (m), 2950 (m), 2930 (m), 2830 (m), 1660 (s),
1620 (w), 1520 (s), 1480 (m), 1440 (m), 1370 (w), 1260 (m), 1210 (s), 1150
(m), 1110 (m), 1040 (m), 940 (m), 870 (w), 840 (m), 810 (m), 770 (m), 720
(m) cmꢀ1; UV/Vis (EtOH): l (%)=224 (100), 292 (43) nm; MS (EI): m/z
(%): 195 (12), 194 [M +] (87), 163 (5), 162 (12), 161 (35), 147 (9), 137
(11), 136 (100), 119 (6), 108 (33), 91 (10), 79 (9), 78 (11), 77 (16), 65 (18),
55 (7), 45 (12), 41 (8), 39 (10); HRMS (EI): m/z: calcd for C11H14O3:
194.0943; found: 194.0948 [M +].
(Z)-2-(3-Isopropoxy-2-buten-1-yl)-4-methoxyphenol
(45a):
Rf =0.39
(hexane/MTBE 3:1); 1H NMR (400 MHz, CDCl3): d=6.80 (d, 3J(6,5)=
8.6 Hz, 1H; H-C(6)), 6.67 (dd, 4J(5,3)=3.0 Hz, 1H; H-C(5)), 6.64 (d,
1H; H-C(3)), 6.40 (s, 1H; OH), 6.09 (d, 3J(3’,2’)=6.1 Hz, 1H; H-C(3’)),
4.66 (td, 3J(2’,1’)=8.6 Hz, 1H; H-C(2’)), 4.07 (septet, 3J(OCH(-
CH3)2,OCH(CH3)2)=6.2 Hz, 1H; C(OCH(CH3)2)), 3.75 (s, 3H; OCH3),
3.27 (d, 2H; H-C(1’)), 1.33 (d, 6H; C(OCH(CH3)2)) ppm; 13C NMR
(100 MHz, CDCl3): d=153.55 (C(4)), 149.15 (C(1)), 143.61 (C(3’)),
127.68 (C(2)), 117.13 (C(6)), 115.98 (C(3)), 113.08 (C(5)), 105.55 (C(2’)),
75.51
(OCH(CH3)2),
56.14
(OCH3),
26.71
(C(1’)),
22.61
(OCH(CH3)2) ppm; IR (NaCl): n˜ =3380 (s), 3080 (w), 2970 (s), 2930 (s),
2870 (w), 2840 (w), 1660 (s), 1640 (w), 1610 (w), 1500 (s), 1470 (m), 1450
(m), 1430 (m), 1380 (m), 1370 (m), 1340 (w), 1230 (s), 1200 (m), 1180
(m), 1150 (s), 1100 (s), 1050 (s), 910 (w) cmꢀ1; UV/Vis (MeOH): l=
292 nm; MS (EI): m/z (%): 222 [M +] (55), 180 (100), 161 (28), 136 (89);
HRMS (EI): m/z: calcd for C13H18O3: 222.1256; found: 222.1259 [M +].
(E and Z)-2-(3-Ethoxy-2-buten-1-yl)-4-methoxyphenol (44a and 44b):
Enol ether 40 (19.4 mg, 60.1 mmol) was dissolved in ethanolamine (4 mL)
and heated at 908C for 3h. Evaporation of the solvent at reduced pres-
sure (398C/4î10ꢀ1 mbar) and column chromatography (hexane/MTBE/
NEt3 15:5:0.2) of the crude residue gave a mixture of the two isomers.
Purification of the isomeric mixture by preparative RP18 HPLC provided
isomerically pure E enol ether 44b (5.8 mg, 27.8 mmol, 46%) and Z enol
ether 44a (4.6 mg, 22.1 mmol, 37%). 44b: Rf =0.18 (hexane/MTBE 3:1);
1H NMR (400 MHz, CDCl3): d=6.73(d, 3J(6,5)=8.6 Hz, 1H; H-C(6)),
6.70 (d, 4J(3,5)=2.9 Hz, 1H; H-C(3)), 6.66 (dd, 1H; H-(5)), 6.41 (d,
3J(3’,2’)=12.6 Hz, 1H; H-C(3’)), 4.93(dt, 3J(2’,1’)=7.1 Hz, 1H; H-C(2’)),
4.81 (s, 1H; OH), 3.75 (s, 3H; OCH3), 3.73 (q, 3J(OCH2-
CH3,OCH2CH3)=6.9 Hz, 2H; OCH2CH3), 3.26 (d, 2H; H-C(1’)), 1.27 (t,
1H; OCH2CH3) ppm; 13C NMR (100 MHz, CDCl3): d=154.10 (C(4)),
148.52 (C(1)), 148.37 (C(3’)), 128.24 (C(2)), 116.76 (C(6)), 116.00 (C(3)),
112.74 (C(5)), 101.50 (C(2’)), 65.24 (OCH2CH3), 56.13(OCH 3), 27.37
(C(1’)), 15.12 (OCH2CH3) ppm; IR (NaCl): n˜ =3420 (s), 3060 (w), 3030
(w), 2980 (s), 2930 (s), 2900 (w), 2830 (m), 1650 (m), 1610 (w), 1500 (s),
1460 (m), 1450 (m), 1430 (m), 1390 (w), 1350 (w), 1260 (m), 1200 (s),
1150 (s), 1110 (m), 1040 (s), 930 (m), 870 (m), 850 (s), 800 (m), 720
(m) cmꢀ1; UV/Vis (EtOH): l (%)=224 (100), 292 (73) nm; MS (EI): m/z
(%): 208 [M +] (82), 161 (31), 137 (26), 136 (100), 108 (29); HRMS (EI):
m/z: calcd for C12H16O3: 208.1099; found: 208.1098 [M +].
6-Methoxychroman-2-ol (12): An 18% aqueous HCl solution (1.5 mL)
was added to a solution of compound 36 (321 mg, 943 mmol) in dioxane
(7 mL). The resulting mixture was heated to 558C for 10 h. The mixture
was diluted with water (300 mL) and the aqueous layer was extracted
with MTBE (3î75 mL). The organic layers were combined, dried over
Na2SO4, and evaporated to dryness. The crude residue was purified by
flash chromatography (hexane/MTBE 3:1) to yield product 12 (159 mg,
886 mmol, 94%) as an oil. Rf =0.17 (hexane/MTBE 3:1); 1H NMR
(500 MHz, CDCl3): d=6.75 (d, 3J(8,7)=8.8 Hz, 1H; H-C(8)), 6.69 (dd,
4J(7,5)=3.1 Hz, 1H; H-C(7)), 6.62 (d, 1H; H-C(5)), 5.64 5.58 (m, 1H;
H-C(2)), 3.71 (s, 3H; OCH3), 3.05 2.95 (m, 1H; H-C(3)), 2.91 (s, 1H;
OH), 2.78 (dt, 2J(3,3)=16.4, 3J(3,4)=5.3Hz, 1H; H-C(3)), 2.11 1.90 (m,
2H; H-C(4)) ppm; 13C NMR (100 MHz, CDCl3): d=154.12 (C(6)),
146.30 (C(8a)), 122.97 (C(4a)), 117.89 (C(8)), 114.27 (C(5)), 113.88
(C(7)), 92.38 (C(2)), 56.13 (OCH3), 27.40 (C(3)), 20.97 (C(4)) ppm; IR
(NaCl): n˜ =3420 (s), 2940 (m), 2900 (m), 2830 (m), 1720 (w), 1610 (m),
1590 (w), 1500 (s), 1470 (m), 1430 (m), 1350 (m), 1320 (m), 1300 (m),
1270 (s), 1180 (m), 1150 (m), 1100 (m), 1060 (s), 1000 (m), 950 (s), 910
(m), 870 (m) cmꢀ1; UV/Vis (EtOH): l (%)=227 (100), 292 (54) nm; MS
(EI): m/z (%): 180 [M +] (100), 161 (20), 136 (59), 108 (32), 77 (20);
HRMS (EI): m/z: calcd for C10H12O3: 180.0786; found: 180.0787 [M +].
(Z)-2-(3-Ethoxy-2-buten-1-yl)-4-methoxyphenol (44a): Rf =0.23(hexane/
MTBE 3:1); 1H NMR (400 MHz, CDCl3): d=6.80 (d, 3J(6,5)=8.8 Hz,
1H; H-C(6)), 6.67 (dd, 4J(5,3)=3.0 Hz, 1H; H-C(5)), 6.64 (d, 1H; H-
C(3)), 6.30 (s, 1H; (OH)), 6.06 (d, 3J(3’,2’)=6.1 Hz, 1H; H-C(3’)), 4.66
(td, 3J(2’,1’)=8.6 Hz, 1H; H-C(2’)), 3.93 (q, 3J(OCH2CH3,OCH2CH3)=
7.1 Hz, 2H; OCH2CH3), 3.75 (s, 3H; OCH3), 3.30 (d, 2H; H-C(1’)), 1.35
(t, 1H; OCH2CH3) ppm; 13C NMR (100 MHz, CDCl3): d=153.59 (C(4)),
149.10 (C(1)), 144.90 (C(3’)), 127.57 (C(2)), 117.10 (C(6)), 115.97 (C(3)),
113.09 (C(5)), 105.48 (C(2’)), 68.74 (OCH2CH3), 56.13(OCH 3), 27.38
(C(1’)), 15.46 (OCH2CH3) ppm; IR (NaCl): n˜ =3400 (s), 3040 (w), 2980
(s), 2930 (s), 2900 (w), 2840 (m), 1670 (w), 1650 (m), 1620 (w), 1610 (w),
1510 (s), 1470 (m), 1430 (m), 1390 (w), 1350 (w), 1260 (s), 1200 (s), 1150
2,6-Dimethoxychroman (11):
A solution of compound 12 (36.63 mg,
203 mmol, 1 equiv) in CH2Cl2 (1.5 mL) was stirred with ice-bath cooling
while a solution of thionyl chloride (1.3mL of a 267 m m thionyl chloride
solution in CH2Cl2, 3 74mmol, 1.9 equiv) was added dropwise. After being
stirred in the cold for 30 min, the green solution was treated with dry
methanol (3mL). The resulting bluish solution was stirred in the cold for
an additional 90 min and then poured into saturated aqueous NaHCO3
(75 mL). The organic layer was separated and the aqueous layer was ex-
tracted with MTBE (3î30 mL). The combined organic extracts were
dried over Na2SO4 and evaporated under reduced pressure. The crude
residue was purified by flash chromatography (hexane/MTBE 7:1) to
yield product 11 (28.11 mg, 145 mmol, 72%) as an oil. Rf =0.5 (hexane/
MTBE 6:1); 1H NMR (500 MHz, CDCl3): d=6.77 (d, 3J(8,7)=8.8 Hz,
1H; H-C(8)), 6.68 (dd, 4J(7,5)=3.0 Hz, 1H; H-C(7)), 6.60 (d, 1H; H-
C(5)), 5.14 5.11 (m, 1H; H-C(2)), 3.71 (s, 3H; (CH3O)-C(6)), 3.47 (s,
3H; (CH3O)-C(2)), 2.97 2.90 (m, 1H; H-C(3)), 2.61 (ddd, 2J(3,3)=16.2,
3J(3,4)=5.9, 3J(3,4)=3.1 Hz, 1H; H-C(3)), 2.06 1.90 (m, 2H; H-
C(4)) ppm; 13C NMR (125 MHz, CDCl3): d=153.60 (C(6)), 145.75
(C(8a)), 123.14 (C(4a)), 117.53 (C(8)), 113.87 (C(5)), 113.29 (C(7)), 97.94
(C(2)), 55.68 (C(6)-OCH3), 55.51 (C(2)-OCH3), 26.23(C()3), 20.59
(C(4)) ppm; IR (NaCl): n˜ =2990 (m), 2940 (m), 2830 (m), 1610 (w), 1500
(s), 1470 (m), 1450 (m), 1430 (m), 1370 (m), 1320 (w), 1270 (m), 1250
(m), 1200 (s), 1150 (m), 1110 (m), 1060 (s), 1050 (s), 1000 (s), 910 (s), 870
(m), 850 (w), 810 (m) cmꢀ1; UV/Vis (EtOH): l (%)=228 (100), 290
(54) nm; MS (EI): m/z (%): 194 [M +] (96), 163(64), 161 (70), 136 (100),
(s), 1110 (m), 1040 (s), 930 (m), 870 (m), 850 (s), 800 (m), 720 (m) cmꢀ1
;
UV/Vis (EtOH): l (%)=224 (100), 292 (43) nm; MS (EI): m/z (%): 208
[M +] (87), 161 (30), 137 (23), 136 (100); HRMS (EI): m/z: calcd for
C12H16O3: 208.1099; found: 208.1098 [M +].
(E and Z)-2-(3-Isopropoxy-2-buten-1-yl)-4-methoxyphenol (45a and
45b): Enol ether 41 (17.4 mg, 51.7 mmol) was dissolved in ethanolamine
(3mL) and heated at 90 8C for 4 h. Evaporation of the solvent at reduced
pressure (398C/4î10ꢀ1 mbar) and column chromatography (hexane/
MTBE/NEt3 15:5:0.2) of the crude residue gave isomerically pure E enol
ether 45b (5.4 mg, 24.3 mmol, 47%) and Z enol ether 45a (5.2 mg,
23.4 mmol, 45%). 45b: Rf =0.27 (hexane/MTBE 3:1); 1H NMR
(400 MHz, CDCl3): d=6.74 (d, 3J(6,5)=8.6 Hz, 1H; H-C(6)), 6.69 (d,
4J(3,5)=2.8 Hz, 1H; H-C(3)), 6.66 (dd, 1H; H-C(5)), 6.26 (d, 3J(3’,2’)=
12.4 Hz, 1H; H-C(3’)), 5.02 (dt, 3J(2’,1’)=7.1 Hz, 1H; H-C(2’)), 4.83(s,
1H; OH), 3.98 (septet, 3J(OCH(CH3)2,OCH(CH3)2)=6.2 Hz, 1H;
OCH(CH3)2), 3.75 (s, 3H; OCH3), 3.25 (d, 2H; H-C(1’)), 1.22 (d, 6H;
OCH(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=154.10 (C(4)),
2501
Chem. Eur. J. 2004, 10, 2487 2506
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
108 (72), 91 (30), 65 (38); HRMS (EI): m/z: calcd for C11H14O3:
194.0943; found: 194.0944 [M +].
tracted further with Et2O (2î200 mL). The combined organic layers
were dried over Na2SO4 and then concentrated at reduced pressure. The
residue was purified by column chromatography (hexane/MTBE 2:1) to
2-Ethoxy-6-methoxychroman (42): Following the procedure for com-
pound 11 with the use of ethanol instead of methanol afforded product
42 (23.2 mg, 111 mmol, 86%). Rf =0.42 (hexane/MTBE 4:6); 1H NMR
(400 MHz, CDCl3): d=6.75 (d, 3J(8,7)=8.8 Hz, 1H; H-C(8)), 6.68 (dd,
4J(7,5)=3.0 Hz, 1H; H-C(7)), 6.60 (d, 1H; H-C(5)), 5.23 5.19 (m, 1H;
H-C(2)), 3.87 (q, 3J(OCH2CH3,OCH2CH3)=7.08 Hz, 2H; (CH3CH2O)-
C(2)), 3.74 (s, 3H; (CH3O)-C(6)), 2.97 2.90 (m, 1H; H-C(3)), 2.61 (ddd,
2J(3,3)=16.2, 3J(3,4)=5.8, 3J(3,4)=3.2 Hz, 1H; H-C(3)), 2.06 1.88 (m,
2H; H-C(4)), 1.18 (t, 3H; (CH3CH2O)-C(2)) ppm; 13C NMR (100 MHz,
CDCl3): d=153.92 (C(6)), 146.50 (C(8a)), 123.61 (C(4a)), 117.84 (C(8)),
114.29 (C(5)), 113.61 (C(7)), 97.16 (C(2)), 63.93 (C(2)-OCH2CH3), 56.08
(C(6)-OCH3), 26.95 (C(3)), 21.31 (C(4)), 15.55 (C(2)-OCH2CH3) ppm;
IR (NaCl): n˜ =2970 (m), 2940 (m), 2910 (m), 2830 (m), 1730 (w), 1610
(m), 1590 (w), 1500 (s), 1470 (m), 1450 (m), 1430 (m), 1370 (m), 1350
(m), 1320 (m), 1300 (m), 1270 (m), 1200 (s), 1150 (m), 1120 (s), 1110 (s),
1060 (s), 1000 (s), 970 (s), 930 (m), 910 (m), 880 (m), 840 (m), 810
(m) cmꢀ1; UV/Vis (EtOH): l (%)=228 (100), 290 (54) nm; MS (EI): m/z
(%): 208 [M +] (72), 194 (23), 162 (41), 136 (100), 108 (37); HRMS (EI):
m/z: calcd for C12H16O3: 208.1100; found: 208.1103[ M +].
provide 48 (961 mg, 3.58 mmol, 91%) as
a colorless oil. Rf =0.26
(hexane/MTBE 2:1); 1H NMR (400 MHz, CDCl3): d=6.89 (d, 4J(6,4)=
2.8 Hz, 1H; H-C(6), 6.73(d, 3J(3,4)=8.6 Hz, 1H; H-C(3)), 6.70 (dd, 1H;
H-C(4)), 4.65 (d, 3J(CH2,OH)=8.6 Hz, 2H; CH2OH), 3.78 (s, 3H;
CH3O), 2.10 (t, 1H; HOCH2), 1.01 (s, 9H; SiC(CH3)2), 0.23(s, 6H;
Si(CH3)) ppm; 13C NMR (100 MHz, CDCl3): d=154.43(C(5)), 147.44
(C(2)), 132.62 (C(1)), 119.47 (C(3)), 114.37 (C(4)), 113.87 (C(6)), 62.32
(CH2OH), 56.07 (CH3O), 26.16 (SiC(CH3)3), 18.55 (SiC(CH3)3), ꢀ3.82
(Si(CH3)2) ppm; IR (NaCl): n˜ =3420 (m), 3000 (w), 2960 (s), 2930 (s),
2890 (w), 2860 (m), 1500 (s), 1460 (m), 1430 (m), 1390 (w), 1360 (w),
1270 (s), 1220 (s), 1150 (m), 1110 (w), 1040 (m), 1010 (w), 940 (m), 900
(s), 840 (s), 820 (w), 800 (w), 780 (m), 690 (w) cmꢀ1; UV/Vis (MeOH): l
(%): 228 (100), 288 (60) nm; MS (EI): m/z (%): 268 [M +] (7), 212 (22),
211 (100), 193(42), 75 (85); HRMS (EI): m/z: calcd for C14H24O3Si:
268.1495; found: 268.1494 [M +].
2-(tert-Butyldimethylsilanyloxy)-5-methoxybenzyl vinyl ether (49): A stir-
red solution of benzyl alcohol 48 (441 mg, 1.65 mmol), ethyl vinyl ether
(50 mL), and Hg(OAc)2 (60.0 mg, 188 mmol, 11 mol%) was heated at
908C for 16 h. Hg(OAc)2 (52.0 mg, 163 mmol, 10 mol%) was added and
the mixture was heated to 908C for an additional 16 h. The solvent was
removed under reduced pressure and the crude product was purified by
flash chromatography (hexane/MTBE 9:1) to yield 49 (348 mg,
2-Isopropoxy-6-methoxychroman (43): Following the procedure for com-
pound 11 with the use of isopropanol instead of methanol afforded prod-
uct 43 (26.84 mg, 121 mmol, 76%). Rf =0.58 (hexane/MTBE 6:1);
1H NMR (300 MHz, CDCl3): d=6.73(d, 3J(8,7)=8.8 Hz, 1H; H-C(8)),
6.67 (dd, 4J(7,5)=3.0 Hz, 1H; H-C(7)), 6.60 (d, 1H; H-C(5)), 5.28 5.21
(m, 1H; H-C(2)), 4.43(septet, 3J(OCH(CH3)2,OCH(CH3)2)=6.2 Hz, 2H;
C(2)-(OCH(CH3)2)), 3.74 (s, 3H; (CH3O)-C(6)), 3.02 2.84 (m, 1H; H-
C(3)), 2.61 (m, 1H; H-C(3)), 2.04 1.76 (m, 2H; H-C(4)), 1.18 (d, 3H;
C(2)-(OCH(CH3)2)), 1.13(d, 3H; C(2)-(OCH(C H3)2)) ppm; 13C NMR
(75 MHz, CDCl3): d=153.51 (C(6)), 146.38 (C(8a)), 123.35 (C(4a)),
117.53 (C(8)), 113.96 (C(5)), 113.22 (C(7)), 95.15 (C(2)), 69.52 (C(2)-
OCH(CH3)2), 55.78 (C(6)-OCH3), 27.04 (C(3)), 23.55 (C(4)), 21.95, 21.12
(C(2)-OCH(CH3)2) ppm; IR (NaCl): n˜ =2970 (m), 2940 (m), 2830 (w),
1610 (m), 1500 (s), 1470 (m), 1430 (m), 1380 (m), 1340 (m), 1320 (m),
1270 (m), 1250 (m), 1200 (s), 1150 (m), 1120 (m), 1100 (m), 1060 (s),
1040 (s), 1000 (s), 910 (m), 870 (m), 810 (m) cmꢀ1; UV/Vis (EtOH): l
(%)=230 (100), 290 (53) nm; MS (EI): m/z (%): 222 [M +] (57), 180
(100), 161 (32), 136 (67); HRMS (EI): m/z: calcd for C13H18O3: 222.1256;
found: 222.1256 [M +].
1.18 mmol, 72%) as
a colorless oil. Rf =0.51 (hexane/MTBE 9:1);
1H NMR (400 MHz, CDCl3): d=6.92 (m, 1H; H-C(3)), 6.72 (m, 2H; H-
3
3
C(6), H-C(4)), 6.56 (dd, 1H; J(3’,4’a)=14.2, J(3’,4’b)=6.8 Hz; H-C(3’)),
4.76 (s, 2H; H-C(1’)), 4.28 (dd, 1H; 2J(4’a,4’b)=2.2 Hz; H-C(4’a)), 4.07
(dd, 1H; H-C(4’b)), 3.77 (s, 3H; OCH3), 1.03(s, 9H; SiC(CH )3), 0.18 (s,
3
6H; Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=154.39 (C(5)),
152.11 (C(3’)), 147.24 (C(2)), 128.68 (C(1)), 119.62 (C(3)), 114.48 (C(4)),
114.47 (C(6)), 87.47 (C(4’)), 65.89 (C(1’)), 56.07 (OCH3), 26.17
(SiC(CH3)3), 18.60 (SiC(CH3)3), ꢀ3.89 (Si(CH3)2) ppm; IR (NaCl,
CHCl3): n˜ =3000 (w), 2960 (m), 2930 (m), 2900 (w), 2860 (m), 1640 (w),
1610 (m), 1500 (s), 1460 (m), 1430 (w), 1320 (m), 1150 (m), 1050 (m),
1010 (w), 940 (m), 900 (s), 840 (s) cmꢀ1; UV/Vis (MeOH): l (%)=228
(100), 290 (50) nm; MS (EI): m/z (%): 294 [M +] (11), 251 (60), 237 (29),
207 (40), 195 (100), 181 (15), 75 (21); HRMS (EI): m/z: calcd for
C16H26O3Si: 294.1651; found: 294.1650 [M +].
2-Hydroxy-5-methoxybenzyl vinyl ether (50): Compound 49 (25.1 mg,
85.2 mmol) was dissolved in ethanolamine (10 mL) and stirred at room
temperature for 16 h. The reaction mixture was diluted with saturated
aqueous NH4Cl solution and the resulting mixture was extracted with
MTBE (4î80 mL). The combined organic layers were dried over Na2SO4
and then concentrated at reduced pressure. The residue was purified by
column chromatography (hexane/MTBE 3:1) to provide product 50
(14.4 mg, 80.0 mmol, 94%) as a colorless oil. Rf =0.22 (hexane/MTBE
3:1); 1H NMR (400 MHz, CDCl3): d=6.82 (d, 3J(3,4)=8.6 Hz, 1H; H-
C(3)), 6.78 (dd, 4J(4,6)=2.8 Hz, 1H; H-C(4)), 6.72 (d, 1H; H-C(6)), 6.53
(dd, 3J(3’,4’a)=14.5, 3J(3’,4’b)=6.8 Hz, 1H; H-C(3’)), 5.83(s, 1H; HO-
2-(tert-Butyldimethylsilanyloxy)-5-methoxybenzaldehyde (47): A stirred
solution of 2-hydroxy-5-methoxybenzaldehyde (46; 2.0 mL, 2.44 g,
16.0 mmol, 1.0 equiv) and tert-butyldimethylsilyl chloride (3.80 g,
25.6 mmol, 1.6 equiv) in DMF (40 mL) was treated with N,N-diisopropyl-
ethylamine (6.0 mL, 4.45 g, 29.0 mmol, 1.8 equiv). After 30 min at room
temperature, the reaction solution was poured into saturated aqueous
NaHCO3 (500 mL) and this mixture was extracted with MTBE (4î
150 mL). The organic layers were combined, dried over Na2SO4, and con-
centrated at reduced pressure. The oily residue was purified by column
chromatography (hexane/MTBE 9:1) yielding silyl ether 47 (4.96 g,
15.6 mmol, 97%) as
a yellowish oil. Rf =0.39 (hexane/MTBE 9:1);
2
1H NMR (400 MHz, CDCl3): d=10.40 (s, 1H; CHO), 7.27 (d, 4J(6,4)=
3.3 Hz, 1H; H-C(6)), 7.04 (dd, 3J(4,3)=9.1 Hz, 1H; H-C(4)), 6.82 (d,
1H; H-C(3)), 3.78 (s, 3H; OCH3), 1.01 (s, 9H; SiC(CH3)3), 0.23(s, 6H;
Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3): d=190.28 (CHO), 154.46
(C(5)), 153.72 (C(2)), 127.53 (C(1)), 124.29 (C(3)), 122.00 (C(4)), 109.90
(C(6)), 56.11 (OCH3), 26.08 (SiC(CH3)3), 18.71 (SiC(CH3)3), ꢀ3.97
(Si(CH3)2) ppm; IR (NaCl): n˜ =3070 (w), 3000 (w), 2960 (s), 2930 (s),
2890 (m), 2860 (s), 1680 (m), 1610 (w), 1580 (w), 1490 (s), 1470 (s), 1420
(s), 1390 (s), 1360 (w), 1280 (s), 1220 (m), 1190 (w), 1150 (s), 1040 (s),
1010 (w), 960 (w), 940 (m), 900 (s), 840 (s), 820 (m), 800 (m), 780 (m),
750 (m), 690 (m) cmꢀ1; UV/Vis (MeOH): l (%)=224 (100), 256 (50), 346
(25) nm; MS (FAB): m/z (%): 267 [M ++H] (49), 209 (100), 73(46); ele-
mental analysis calcd (%) for C14H22O3Si: C 63.12, H 8.32, Si 10.54, O
18.02; found: C 63.15, H 8.22.
C(2)), 4.86 (s, 2H; H-C(1’)), 4.42 (dd, J(4’a,4’b)=2.5 Hz, 1H; H-C(4’a)),
4.18 (dd, 1H; H-C(4’b)), 3.76 (s, 3H; OCH3) ppm; 13C NMR (100 MHz,
CDCl3): d=153.80 (C(5)), 150.95 (C(3’)), 149.27 (C(2)), 123.12 (C(1)),
117.61 (C(3)), 115.23 (C(4)), 114.60 (C(6)), 89.54 (C(4’)), 68.89 (C(1’)),
56.20 (OCH3) ppm; IR (NaCl, CHCl3): n˜ =3420 (s), 3000 (w), 2920 (s),
2900 (w), 2850 (m), 1620 (s), 1500 (s), 1460 (s), 1450 (s), 1430 (s), 1370
(m), 1320 (s), 1110 (w), 1040 (m), 1010 (w), 990 (w), 960 (w), 900
(w) cmꢀ1; UV/Vis (EtOH): l (%)=220 (100), 294 (40) nm; MS (EI): m/z
(%): 180 [M +] (26), 138 (14), 137 (100), 108 (17); HRMS (EI): m/z:
calcd for C10H12O3: 180.0786; found: 180.0786 [M +].
2-Hydroxy-5-methoxybenzylalcohol (51): A solution of 2-hydroxy-5-me-
thoxybenzaldehyde (46; 1.0 mL, 1.22 g, 8.02 mmol, 1.0 equiv) in THF
(5 mL) was added to a stirred ice-cold suspension of LiAlH4 (114 mg,
3.00 mmol, 1.5 equiv) in THF (35 mL). This mixture was stirred for
15 min and the reaction was quenched by addition of H2O (50 mL). The
suspension was slowly poured into ice-cold aqueous 1m HCl solution
(150 mL) to dissolve the precipitated aluminium hydroxide. The resulting
solution was extracted with MTBE (3î150 mL). The organic layers were
combined, dried over Na2SO4, and filtered through a celite pad. The re-
moved solids were washed thoroughly with Et2O and the filtrate was con-
2-(tert-Butyldimethylsilyanyloxy)-5-methoxybenzylalcohol (48): Sodium
borohydride (195 mg, 5.15 mmol, 1.3equiv) was added to an ice-cold sol-
ution of aldehyde 47 (1.05 g, 3.94 mmol, 1.0 equiv) in absolute ethanol
(40 mL). The reaction mixture was stirred at 08C for 45 min and then
was carefully partitioned between saturated aqueous NH4Cl solution
(350 mL) and Et2O (200 mL). The aqueous layer was separated and ex-
2502
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
centrated. The residue was purified by flash chromatography (hexane/
MTBE 4:6) to yield 51 (1.03g, 6.65 mmol, 84%) as a colorless oil. Rf =
0.22 (hexane/MTBE 4:6); 1H NMR (400 MHz, CDCl3): d=6.95 (s, 1H;
HO-(C(2)); 6.78 (d, 3J(3,4)=8.7 Hz, 1H; H-C(3)); 6.73 (dd, 4J(4,6)=
2.5 Hz, 1H; H-C(4)); 6.56 (d, 1H; H-C(6)); 4.82 (s, 2H; CH2OH); 3.78
(s, 3H; OCH3); 2.68 (s, 1H; CH2OH) ppm; 13C NMR (100 MHz, CDCl3):
d=153.50 (C(5)), 150.06 (C(2)), 126.05 (C(1)), 117.46 (C(3)), 114.74
(C(4)), 113.97 (C(6)), 64.74 (CH2OH), 56.25 (OCH3) ppm; IR (KBr): n˜ =
3440 (s), 3180 (s), 3010 (s), 2980 (m), 2900 (m), 2840 (m), 1620 (w), 1520
(s), 1480 (s), 1460 (m), 1440 (s), 1400 (w), 1380 (w), 1300 (m), 1280 (m),
1260 (m), 1230 (m), 1200 (s), 1160 (s), 1110 (w), 1040 (s), 1010 (s), 980
(s), 930 (m), 860 (s), 820 (s), 780 (m), 760(m), 700 (m) cmꢀ1; UV/Vis
and treated with KN(TMS)2 (820 mL of a 0.5m solution in toluene,
0.41 mmol, 1.2 equiv). The aldehyde 32 (100 mg, 0.34 mmol, 1 equiv) was
then added and the resulting mixture was stirred at ꢀ788C for 90 min.
Saturated aqueous NH4Cl solution (150 mL) was added and the product
was extracted into MTBE (3î75 mL). The ether extracts were dried over
Na2SO4 and evaporated. The product was purified by flash chromatogra-
phy (hexane/MTBE 9:1) to give pure Z isomer 54 (116 mg, 0.33 mmol,
96%) as
a
colorless oil. Rf =0.35 (hexane/MTBE 9:1); 1H NMR
(400 MHz, CDCl3): d=6.73 6.68 (m, 2H; H-C(3’), H-C(4’)), 6.63 6.59
(m, 1H; H-C(6’)), 6.24 (dt, 3J(3,2)=12.2, 3J(3,4)=7.4 Hz, 1H; H-C(3)),
5.77 (dt, 4J(2,4)=2.6 Hz, 1H; H-C(2)), 3.74 (s, 3H; OCH3), 3.69 (s, 3H;
CO2CH3), 2.90 (td, 3J(4,5)=7.0 Hz, 2H; H-C(4)), 2.70 (t, 2H; H-C(5)),
0.99 (s, 9H; SiC(CH3)3), 0.20 (s, 6H; Si(CH3)2) ppm; 13C NMR
(100 MHz, CDCl3): d=167.13(C(1)), 154.13(C(5 ’)), 150,12 (C(3)),
147.82 (C(2’)), 132.90 (C(1’)), 120.03(C(2)), 119.24 (C(3 ’)), 116.13
(C(6’)), 112.11 (C(4’)), 56.01 (OCH3), 51.37 (CO2CH3), 30.24 (C(4)),
29.51 (C(5)), 26.21 (SiC(CH3)3), 18.60 (SiC(CH3)3), ꢀ3.79
(Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =2950 (s), 2857 (m), 1723(s), 1646
(w), 1498 (s), 1471 (m), 1436 (w), 1405 (w), 1254 (w), 1224 (s), 1177 (m),
1044 (w), 897 (m), 838 (m), 779 (m) cmꢀ1; UV/Vis (MeOH): l=289 nm;
MS (EI): m/z (%): 350 [M +] (12), 294 (13), 293 (56), 225 (52), 196 (18),
195 (100), 89 (25), 75 (12), 73 (33), 59 (12); HRMS (EI): m/z: calcd for
C19H30O4Si: 350.1913 [M +]; found: 350.1902; elemental analysis calcd
(%) for C19H30O4Si: C 65.10, H 8.63, O 18.26, Si 8.01; found: C 65.26, H
8.67.
(MeOH): l (%)=230 (100), 294 (86) nm; MS (EI): m/z (%): 154 [M +
]
(22), 136 (100), 108 (47), 78 (36); HRMS (EI): m/z: calcd for C8H10O3:
154.0630; found: 154.0632 [M +]; elemental analysis calcd (%) for
C8H10O3: C 62.33, H 6.54, O 31.13; found: 62.31, H 6.54, O 30.92.
6-Methoxy-2-methyl-4H-benzo[1,3]dioxane (52): Two drops of concen-
trated H2SO4 were added to
a solution of compound 51 (110 mg,
710 mmol) in acetaldehyde (10 mL). The reaction mixture was stirred at
48C for 3h and then diluted with H 2O (200 mL). After extraction with
MTBE (3î150 mL), the combined organic layers were washed sequen-
tially with a 3m aqueous NaOH solution (100 mL) and H2O (100 mL)
and dried over Na2SO4, then the solvent was removed. The residue was
purified by flash chromatography (hexane/MTBE 9:1) to yield 52
(116 mg, 644 mmol, 91%) as a colorless solid. Rf =0.24 (hexane/MTBE
9:1); m.p. 988C; 1H NMR (400 MHz, CDCl3): d=6.79 (d, 3J(8,7)=
9.1 Hz, 1H; H-C(8)), 6.72 (dd, 4J(7,5)=3.0 Hz, 1H; H-C(7)), 6.49 (d,
1H; H-C(5)), 5.12 (q, 3J(2,CH3)=5.1 Hz, 3H; H-C(2)), 4.98 (d, 2J(4,4)=
14.7 Hz, 1H; H-C(4)), 4.80 (d, 1H; H-C(4)), 3.80 (s, 3H; CH3O), 1.52 (d,
3H; H3C-C(2)) ppm; 13C NMR (100 MHz, CDCl3): d=154.33 (C(6)),
147.34 (C(8a)), 121.67 (C(4a)), 117.74 (C(8)), 114.37 (C(7)), 109.91
(C(5)), 97.38 (C(2), 66.78 (C(4), 56.12 (CH3O), 21.04 (H3C-C(2)) ppm;
IR (KBr): n˜ =2990 (m), 2940 (m), 2910 (m), 2890 (m), 1500 (s), 1460 (m),
1450 (w), 1400 (w), 1320 (w), 1300 (m), 1270 (s), 1250 (m), 1220 (s), 1150
(E)-5-[2-Hydroxy-5-methoxyphenyl]pent-2-enoic acid ethyl ester (55)
and (rac)-(6-methoxychroman-2-yl)acetic acid ethyl ester (57): TBAF
(0.55 mL of 1m solution in THF, 0.55 mmol, 1.0 equiv) was added to a
stirred solution of compound 53 (202 mg, 0.55 mmol, 1.0 equiv) in THF
(5 mL) and the resulting red reaction mixture was directly quenched by
addition of saturated aqueous NH4Cl solution (40 mL). The mixture was
extracted with MTBE (3î25 mL), the combined organic layers were
dried over Na2SO4, and the solvent was removed by evaporation. The
crude mixture was purified by flash chromatography (hexane/EtOAc 3:1)
to give the pure E isomer of the a,b-unsaturated ester 55 (77.4 mg,
0.31 mmol, 56%) and chroman 57 (37.3 mg, 0.15 mmol, 27%). 55: Rf =
0.17 (hexane/MTBE 3:1); 1H NMR (400 MHz, CDCl3): d=7.05 (dt,
(m), 1100 (s), 1030 (s), 930 (m), 900 (s), 860 (m), 820 (m), 790 (m) cmꢀ1
;
UV/Vis (MeOH): l (%)=223(100), 292 (23) nm; MS (EI): m/z (%): 180
[M +] (20), 136 (100), 108 (40); HRMS (EI): m/z: calcd for C10H12O3:
180.0786; found: 180.0786 [M +].
3
3J(3,2)=16, J(3,4)=7.2 Hz, 1H; H-C(3)), 6.70 6.60 (m, 3H; H-C(3’), H-
C(4’), H-C(6’)), 5.85 (d, 1H; H-C(2)), 4.18 (q, 3J(OCH2-
CH3,OCH2CH3)=7.1 Hz, 2H; OCH2CH3), 3.74 (s, 3H; OCH3), 2.74 (t,
3J(5,4)=7.3Hz, 2H; H-C(5)), 2.51 (dt, 2H; H-C(4)), 1.28 (t, 3H;
OCH2CH3) ppm; 13C NMR (100 MHz, CDCl3): d=167.21 (C(1)), 154.12
(C(5’)), 148,89 (C(3)), 147.84 (C(2’)), 128.71 (C(1’)), 122.07 (C(2)), 116.30
(C(6’), C(3’)), 112.19 (C(4’)), 60.63(O CH2CH3), 56.14 (OCH3), 32.69
(C(4)), 29.39 (C(5)), 14.66 (OCH2CH3) ppm; IR (NaCl, CHCl3): n˜ =3400
(s), 2980 (m), 2940 (m), 2830 (w), 1690 (s), 1650 (m), 1510 (s), 1470 (w),
1450 (w), 1430 (m), 1370 (w), 1310 (m), 1270 (m), 1200 (s), 1150 (m),
1070 (w), 1040 (s), 970 (w), 800 (m), 710 (w) cmꢀ1; UV/Vis (CH3OH):
l=293nm; MS (EI): m/z (%): 250 [M +] (38), 204 (60), 176 (36), 161
(46), 137 (100); HRMS (EI): m/z: calcd for C14H18O4: 250.1205; found:
250.1197 [M +]; elemental analysis calcd (%) for C14H22O3Si: C 67.18, H
7.25, O 25.57; found: C 66.98, H 7.16, O 25.58.
(E)-5-[2-(tert-Butyldimetylsilanyloxy)-5-methoxyphenyl]-pent-2-enoic
acid ethyl ester (53):
A suspension of triethyl phosphonoacetate
(0.73 mL, 3.65 mmol, 2 equiv) and NaH (79 mg, 3.29 mmol, 1.8 equiv) in
anhydrous THF (15 mL) was cooled to 08C. Aldehyde 32 (537 mg,
1.83mmol, 1 equiv) dissolved in THF (5 mL) was added dropwise to the
cold suspension. The mixture was then stirred for 12 h at room tempera-
ture. Saturated aqueous NH4Cl solution (150 mL) was added and the
product was extracted into MTBE (3î75 mL). The organic extracts were
dried over Na2SO4 and evaporated. The crude product was purified by
flash chromatography (hexane/EtOAc 9:1) to give pure E isomer 53
(534 mg, 1.46 mmol, 77%) as a colorless oil. Rf =0.37 (hexane/MTBE
9:1); 1H NMR (400 MHz, CDCl3): d=7.00 (dt, 3J(3,2)=16.2, 3J(3,4)=
7.2 Hz, 1H; H-C(3)), 6.71 6.65 (m, 2H; H-C(3’), H-C(4’)), 6.64 6.60 (m,
1H; H-C(6’)), 5.83(d, 1H; H-C(2)), 4.18 (q, 3J(OCH2CH3,OCH2CH3)=
3
7.1 Hz, 2H; OCH2CH3), 3.74 (s, 3H; OCH3), 2.70 (t, J(5,4)=7.3Hz, 2H;
(rac)-(6-Methoxychroman-2-yl)acetic acid ethyl ester (57): Rf =0.40
(hexane/MTBE 5:1); 1H NMR (500 MHz, CDCl3): d=6.71 (d, 3J(8,7)=
8.8 Hz, 1H; H-C(8)), 6.65 (dd, 4J(7,5)=2.9 Hz, 1H; H-C(7)), 6.58 (d,
1H; H-C(5)), 4.45 4.37 (m, 1H; H-C(2)), 4.18 (q, 3J(OCH2-
CH3,OCH2CH3)=7.2 Hz, 2H; (OCH2CH3)), 3.74 (s, 3H; (CH3O)-C(6)),
H-C(5)), 2.47 (dt, 2H; H-C(4)), 1.28 (t, 3H; OCH2CH3), 0.99 (s, 9H;
SiC(CH3)3), 0.20 (s, 6H; Si(CH3)2) ppm; 13C NMR (100 MHz, CDCl3):
d=167.05 (C(1)), 154.15 (C(5’)), 148,85 (C(3)), 147.77 (C(2’)), 132.65
(C(1’)), 121.98 (C(2)), 119.28 (C(3’)), 116.13(C(6 ’)), 112.19 (C(4’)), 60.53
(OCH2CH3), 56.01 (OCH3), 32.99 (C(4)), 29.89 (C(5)), 26.21
2.92 2.83(m, 1H; H-C()3), 2.78 (dd,
2J(CH2CO2Et,CH2CO2Et)=15.4,
3J(CH2CO2Et,2)=6.1 Hz, 1H; (CH2-CO2Et)), 2.61 (ddd, 2J(3,3)=16.3,
3J(3,4)=5.9, 3J(3,4)=3.0 Hz, 1H; H-C(3)), 2.57 (dd, 1H; (CH2-CO2Et)),
2.10 2.02 (m, 1H; H-C(4)), 1.82 1.72 (m, 1H; H-C(4)), 1.28 (t, 3H;
(OCH2CH3)) ppm; 13C NMR (125 MHz, CDCl3): d=171.23(CO 2Et),
153.37 (C(6)), 148.47 (C(8a)), 122.09 (C(4a)), 117.41 (C(8)), 113.97
(C(5)), 113.32 (C(7)), 72.14 (C(2)), 60.52 (OCH2CH3), 55.71 (C(6)-
OCH3), 40.64 (CH2CO2Et), 27.46 (C(3)), 24.72 (C(4)), 14.76
(OCH2CH3) ppm; IR (NaCl, CHCl3): n˜ =2980 (m), 2950 (m), 2830 (w),
1740 (s), 1620 (m), 1500 (s), 1430 (m), 1380 (w), 1360 (m), 1320 (w), 1290
(m), 1260 (s), 1200 (m), 1150 (m), 1050 (s), 1030 (m), 1010 (m), 920 (m),
890 (w) cmꢀ1; UV/Vis (CH3OH): l=293nm; MS (EI): m/z (%): 250
[M +] (100), 176 (16), 161 (40), 136 (41); HRMS (EI): m/z: calcd for
C14H18O4: 250.1205; found: 250.1203[ M +].
(SiC(CH3)3),
18.59
(SiC(CH3)3),
14.66
(OCH2CH3),
ꢀ3.78
(Si(CH3)2) ppm; IR (NaCl, CHCl3): n˜ =2955 (s), 2950 (s), 2857 (m), 1721
(s), 1653 (w), 1499 (s), 1471 (m), 1426 (w), 1390 (w), 1367 (w), 1317 (w),
1268 (s), 1225 (s), 1193(m), 1151 (m), 1044 (s), 895 (m), 839 (m), 779
(m) cmꢀ1; UV/Vis (MeOH): l=289 nm; MS (EI): m/z (%): 364 [M +
]
(12), 307 (85), 251 (16), 211 (59), 195 (100), 193 (40), 75 (54); HRMS
(EI): m/z: calcd for C20H32O4Si: 364.2070; found: 364.2072 [M +]; elemen-
tal analysis calcd (%) for C20H32O4Si: C 65.89, H 8.85, O 17.56, Si 7.70;
found: C 65.72, H 8.82.
(Z)-5-[2-(tert-Butyldimethylsilanyloxy)-5-methoxyphenyl]-pent-2-enoic
acid methyl ester (54): A solution of phosphonoacetate-P,P-bis(2,2,2-tri-
fluoroethyl)methyl ester (95 mL, 0.44 mmol, 1.3equiv) and 18-crown-6
(448 mg, 1.69 mmol, 5 equiv) in anhydrous THF was cooled to ꢀ788C
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W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
(Z)-5-[2-Hydroxy-5-methoxyphenyl]pent-2-enoic acid methyl ester (56)
and (rac)-(6-methoxychroman-2-yl)acetic acid methyl ester (58): TBAF
(1.1 mL of 1m solution in THF, 1.08 mmol, 1.1 equiv) was added to a stir-
red solution of compound 54 (331 mg, 0.94 mmol, 1.0 equiv) in THF
(10 mL) and the resulting orange reaction mixture was directly quenched
by addition of saturated aqueous NH4Cl solution (40 mL). The mixture
was extracted with MTBE (3î25 mL), the combined organic layers were
dried over Na2SO4, and the solvent was removed by evaporation. The
crude mixture was purified by flash chromatography (hexane/MTBE 2:1)
to give the pure Z isomer of the a,b-unsaturated ester 56 (161 mg,
0.36 mmol, 38%) and chroman 58 (165 mg, 0.37 mmol, 39%). 56: Rf =
0.18 (hexane/MTBE 2:1); 1H NMR (400 MHz, CDCl3): d=6.80 (d,
3J(3’,4’)=7.6 Hz, 1H; H-C(3’)), 6.69 6.65 (m, 2H; H-C(4’), H-C(6’)), 6.49
(dt, 3J(3,2)=12.4, 3J(3,4)=7.6 Hz, 1H; H-C(3)), 5.86 (d, 1H; H-C(2)),
3.78 (s, 3H; OCH3), 3.74 (s, 3H; CO2CH3), 2.82 2.69 (m, 4H; H-C(5), H-
C(4)) ppm; 13C NMR (100 MHz, CDCl3): d=167.70 (C(1)), 153.72
(C(5’)), 150,11 (C(3)), 148.94 (C(2’)), 127.76 (C(1’)), 120.71 (C(2)), 117.10
(C(3’)), 115.93(C(6 ’)), 113.09 (C(4’)), 56.13(OCH 3), 51.94 (CO2CH3),
31.04 (C(4)), 30.14 (C(5)) ppm; IR (NaCl, CHCl3): n˜ =3430 (s), 2980 (w),
2950 (m), 2830 (w), 1700 (s), 1640 (m), 1510 (s), 1440 (s), 1340 (w), 1200
(s), 1040 (m), 820 (m) cmꢀ1; UV/Vis (MeOH): l=293nm; MS (EI): m/z
(%): 236 [M +] (37), 204 (49), 176 (26), 161 (29), 137 (100), 77 (11);
HRMS (EI): m/z: calcd for C13H16O4: 236.1049; found: 236.1049 [M +].
concentrated at reduced pressure to provide compound 63 (2.55 g,
20.7 mmol; 97%) as
a colorless oil. Rf =0.26 (hexane/MTBE 2:1);
1H NMR (400 MHz, CDCl3): d=7.56 7.48 (m, 2H; H-C(6), H-C(4)),
7.03 6.95 (m, 2H; H-C(5), H-C(3)) ppm; 13C NMR (100 MHz, CDCl3):
d=196.66 (t, 2J(CDO,CDO)=27.22 Hz; CDO), 162.10 (C(2)), 137.38
(C(4)), 134.08 (C(6)), 120.99 (C(1)), 120.23 (C(5)), 118.01 (C(3)) ppm; IR
(NaCl): n˜ =3180 (w), 3060 (w), 2940 (m), 2360 (w), 2130 (m), 2060 (w),
1650 (s), 1640 (s), 1620 (m), 1580 (m), 1490 (m), 1460 (m), 1350 (w), 1280
(s), 1250 (m), 1230 (m), 1210 (m), 1150 (m), 1110 (w), 1040 (m), 1020
(m), 870 (m), 760 (m), 750 (m), 710 (m) cmꢀ1; UV/Vis (MeOH): l (%)=
254 (100), 325 (32) nm; MS (EI): m/z (%): 123[ M +] (100), 121 (71), 93
(20), 76 (26); HRMS (EI): m/z: calcd for C7H5DO2: 123.0431; found:
123.0430 [M +].
a-Deuterio-benzo[d]isoxazole (66): Following the reported procedure[27a]
,
benzaldehyde 63 (2.47 g, 20.1 mmol, 1.0 equiv) afforded benzisoxazole 66
(1.98 g, 16.5 mmol, 82%) as an oil. Rf =0.48 (hexane/MTBE 8:1);
1H NMR (100 MHz, CDCl3): d=7.83(d, 3J(6,5)=7.7 Hz, 1H; H-C(4)),
7.68 7.35 (m, 2H; H-C(6), H-C(5)), 7.22 (d, 3J(7,6)=7.4, 1H; H-
C(7)) ppm; 13C NMR (100 MHz, CDCl3): d=162.66 (C(7a)), 146.35 (t,
2J(C(3),C(3)-D)=28.71 Hz; C(3)), 130.44 (C(6)), 124.14 (C(4)), 122.39
(C(5)), 121.61 (C(3a)), 110.11 (C(7)) ppm; IR (NaCl): n˜ =3080 (m), 2940
(m), 2870 (w), 1940 (w), 1640(s), 1620 (s), 1500 (s), 1470 (s), 1430 (s),
1340 (w), 1300 (m), 1250 (m), 1220 (s), 1200 (w), 1150 (m), 1120 (m),
1010 (m), 940 (w), 890 (m), 860 (s), 810 (s), 770 (m), 750 (s) cmꢀ1; UV/
Vis (MeOH): l (%)=235 (100), 281 (32) nm; MS (EI): m/z (%): 120
[M +] (89), 92 (100), 64 (51), 38 (24); HRMS (EI): m/z: calcd for
C7H5DO2: 120.0434; found: 120.0433 [M +].
(rac)-(6-Methoxychroman-2-yl)acetic acid methyl ester (58): Rf =0.340
(hexane/MTBE 2:1); 1H NMR (500 MHz, CDCl3): d=6.74 (d, 3J(8,7)=
8.9 Hz, 1H; H-C(8)), 6.65 (dd, 4J(7,5)=3.0 Hz, 1H; H-C(7)), 6.58 (d,
1H; H-C(5)), 4.46 4.37 (m, 1H; H-C(2)), 3.74 (s, 3H; (CH3O)-C(6)),
3.72 (s, 3H; CO2CH3), 2.92 2.67 (m, 3H; H-C(3), (CH 2-CO2Me)), 2.62
(dd, 2J(CH2CO2Me,CH2CO2Me)=15.5, 3J(CH2CO2Et,2)=6.0 Hz, 1H;
(CH2-CO2Me)), 2.10 2.02 (m, 1H; H-C(4)), 1.81 1.72 (m, 1H; H-
C(4)) ppm; 13C NMR (125 MHz, CDCl3): d=171.63(CO 2Me), 153.38
(C(6)), 148.47 (C(8a)), 122.49 (C(4a)), 117.56 (C(8)), 113.91 (C(5)),
113.33 (C(7)), 72.09 (C(2)), 55.76 (C(6)-OCH3), 51.79 (CO2CH3), 40.44
(CH2CO2Me), 27.21 (C(3)), 24.71 (C(4)) ppm; IR (NaCl, CHCl3): n˜ =
2980 (m), 2950 (m), 2830 (w), 1740 (s), 1620 (m), 1500 (s), 1430 (m), 1380
(w), 1360 (m), 1320 (w), 1290 (m), 1260 (s), 1200 (m), 1150 (m), 1050 (s),
1030 (m), 1010 (m), 920 (m), 890 (w) cmꢀ1; UV/Vis (CH3OH): l=
293nm; MS (EI): m/z (%): 236 [M +] (100), 176 (18), 162 (42), 136 (40);
HRMS (EI): m/z: calcd for C13H16O4: 236.1049; found: 236.1051 [M +].
3-Deuterio-5-nitrobenzo[d]isoxazole (67): Following the reported proce-
dure[27b]
,
benzisoxazole 66 (1.64 g, 13.7 mmol) afforded product 67
(1.19 g, 7.26 mmol, 53%) as
a
white solid: m.p. 76 8C; 1H NMR
(400 MHz, CDCl3): d=8.73(d, 3J(4,6)=2.4 Hz, 1H; H-C(4)), 8.49 (dd,
3J(6,7)=9.1 Hz, 1H; H-C(6)), 7.76 (d, 1H; H-C(7)) ppm; 13C NMR
(100 MHz, CDCl3): d=164.81 (C(7a)), 147.20 (t, 2J(C(3),C(3)-D) =
29.91 Hz; C(3)), 145.21 (C(5)), 125.99 (C(6)), 122.19 (C(3a)), 119.60
(C(4)), 110.88 (C(7)) ppm, IR (NaCl): n˜ =3100 (m), 2920 (m), 2860 (w),
1920 (w), 1790 (w), 1620 (m), 1530 (s), 1520 (s), 1490 (s), 1450 (m), 1430
(m), 1350 (w), 1300 (m), 1260 (m), 1250 (m), 1230 (s), 1130 (m), 1080
(m), 1000 (m), 960 (w), 930 (m), 900 (s), 850 (s), 830 (s), 810 (m), 770
(m), 740 (s) cmꢀ1; UV/Vis (EtOH): l (%)=224 (100), 277 (32) nm; MS
(EI): m/z (%): 165 [M +] (100), 135 (30), 119 (30), 91 (61), 63 (92);
HRMS (EI): m/z: calcd for C7H3DN2O2: 165.0285; found: 165.0285
[M +].
2-a-Deuterio-(tetrahydropyran-2-yloxy)benzaldehyde (62): A solution of
2-(2-bromophenoxy)tetrahydropyran (61; 5.91 g, 22.7 mmol, 1 equiv) in
Et2O (30 mL) was cooled in an ice bath. nBuli (14.2 mL of a 1.6m solu-
tion in hexane, 22.7 mmol, 1 equiv) was added dropwise. After stirring at
08C for 2 h, a solution of [D7]DMF (2.0 g, 24.95 mmol, 1.1 equiv) in Et2O
(5 mL) was added dropwise and the resulting reaction mixture was stir-
red for 8 h at room temperature. The reaction was quenched by addition
of H2O (60 mL) and then the aqueous layer was separated and extracted
further with Et2O (3î30 mL). The combined organic layers were dried
over Na2SO4 and concentrated. The oily residue was purified by flash
chromatography (hexane/MTBE 3:1) to give benzaldehyde 62 (4.56 g,
22.0 mmol, 97%) as an oil. Rf =0.47 (hexane/MTBE 2:1); 1H NMR
(400 MHz, CDCl3): d=7.83(d, 3J(6,5)=7.8 Hz, 1H; H-C(6)), 7.48 (t,
3J(4,5) and 3J(4,3)=7.3Hz, 1H; H-C(4)), 7.22 (d, 1H; H-C(3)), 7.03 (t,
1H; H-C(5)), 5.61 5.57 (m, 1H; H-C(2’)), 3.90 3.83 (m, 1H; H-C(3’)),
3.64 3.59 (m, 1H; H-C(3’)), 2.04 1.57 (m, 6H; H-C(4’), H-C(5’), H-
C(6’)) ppm; 13C NMR (100 MHz, CDCl3): d=189.80 (t, 2J(CDO,CDO)=
27.22 Hz; CDO), 159.92 (C(2)), 136.03 (C(4)), 128.40 (C(6)), 125.74
(C(1)), 121.91 (C(5)), 115.86 (C(3)), 96.98 (C(2’)), 62.49 (C(6’)), 30.49
(C(3’)), 25.41 (C(5’)), 18.89 (C(4’)) ppm; IR (NaCl): n˜ =3060 (w), 2940
(m), 2870 (w), 2120 (m), 1670 (m), 1650 (s), 1640 (s), 1600 (m), 1580 (m),
1480 (m), 1460 (m), 1390 (w), 1350 (w), 1280 (s), 1240 (m), 1230 (m),
1200 (m), 1180 (m), 1150 (m), 1120 (m), 1070 (m), 1040 (m), 1020 (m),
960 (m), 920 (m), 870 (m), 820 (w), 760 (m), 750 (m), 720 (m), 660
(m) cmꢀ1; UV/Vis (MeOH): l (%)=252 (100), 320 (38) nm; MS (EI): m/
z (%): 207 [M +] (15), 123(6)3, 121 (51), 85 (100), 84 (41), 67 (22);
HRMS (EI): m/z: calcd for C12H13DO3: 207.1006; found: 207.1006 [M +].
Preparation of antibodies and kinetic measurements
Immunization, fusion, and cell culture: Eight mice (129GIX/boy+) were
immunized against the KLH conjugates of either 1, 3, or a succession of
1 and 3 or 3 and 1 (two mice each) following standard procedures.[41]
Four mice (129GIX/boy+) were immunized against the KLH conjugate
of 2. Each mouse received two intraperitoneal injections of hapten KLH
conjugate (100 mg in 200 mL phosphate-buffered saline (PBS; aqueous
10 mm phosphate, 160 mm NaCl, pH 7.4) emulsified with monophosphor-
yl-lipid A (MPL) and trehalose dicorynomycolate (TDM) adjuvants
(Sigma M6536)), one injection on day 1 and one on day 14. Samples of
serum were taken by means of a tail bleed on day 21 to estimate the
titer. Fusion process and cell cultures were done according to the previ-
ously described procedure.[40]
Screening for catalysis: Cell-culture supernatant (5 mL) was passed onto
Protein G gel (100 mg; Gammabind Plus sepharose, Pharmacia Biotech)
in a cotton-plugged Pasteur pipette. The gel was washed with PBS (2î
1 mL) and 0.1m aqueous NaCl (2î1 mL) and finally eluted with 50 mm
citrate (2î150 mL, pH 2.7). BisTris solution (30 mL of a 1m aqueous solu-
tion) was added to the eluted acidic buffer containing the antibody in
order to adjust the pH value to 6.2. The antibody solution was transfer-
red into three different vials (90 mL in each vial) and a different substrate
(10 mL of a 5 mm substrate solution in CH3CN, substrates 8, 10a, or 10b
were used for the screening) was added to each vial. A 50-mL aliquot of
each sample was immediately transferred to another vial containing a sol-
ution of the corresponding hapten 1 3 (1 mL, 1mm hapten in PBS). The
capped vials were kept at 378C for at least 48 h. The substrate and prod-
uct concentrations of each vial were determined by RP18 HPLC on a
Bischoff LiChrospher 100 (4.6î125 mm) column with isocratic elution at
a-Deuterio-2-hydroxybenzaldehyde (63): Benzaldehyde 62 (4.42 g,
21.3mmol) was dissolved in THF (10 mL) and 1 n aqueous HCl solution
(10 mL) was added. After stirring for 10 h at room temperature, the mix-
ture was diluted with water (200 mL) and extracted with Et2O (5î
50 mL). The combined organic layers were dried over Na2SO4 and then
2504
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2004, 10, 2487 2506

Catalytic Antibody against Tocopherol Cyclase Inhibitor
2487 2506
a rate of 1 mLminꢀ1 with a premixed acetonitrile/water solution of the
desired proportions. Substrate and product concentrations were deter-
mined after 24 and 48 h incubation time, respectively. The difference in
apparent rate between the antibody and the inhibited antibody (with
hapten) samples was used as the criterion for specific catalysis. The
screening assay was performed repeatedly with each individual hybrido-
ma at all stages of cell culture.
d) J. Hasserodt, Synlett 1999, 12, 2007 2022; e) J.-L. Reymond, Top.
Curr. Chem. 1999, 200, 59 93.
[3] a) N. R. Thomas, Nat. Prod. Rep. 1996, 13, 479 511; b) J. D. Steven-
son, N. R. Thomas, Nat. Prod. Rep. 2000, 17, 535 577.
[4] A. Stocker, A. R¸ttimann, W.-D. Woggon, Helv. Chim. Acta 1993,
76, 1729 1738.
[5] A. Stocker, T. Netscher, A. R¸ttimann, R. K. M¸ller, H. Schneider,
L. J. Todaro, G. Derungs, W.-D. Woggon, Helv. Chim. Acta 1994, 77,
1721 1737.
[6] A. Stocker, H. Fretz, H. Frick, A. R¸ttimann, W.-D. Woggon,
Bioorg. Med. Chem. 1996, 4, 1129 1134.
Kinetic measurements: A solution of antibody in BisTris buffer (pH 6.24)
at 378C was mixed with a substrate solution (acetonitrile/buffer 1:1) to
provide a final solution containing 29mm BisTris, 104mm NaCl, and 10%
acetonitrile. The final antibody concentration was 250 mgmLꢀ1 for the hy-
drolysis of the enol ethers and 24 mgmLꢀ1 for the Kemp elimination.
Product formation was monitored by RP18 HPLC on a Bischoff LiChros-
pher 100 (4.6î125 mm) column with isocratic elution at rate of
1 mLminꢀ1 with a premixed acetonitrile/water solution of the desired
proportions. The 16E7-catalyzed elimination was monitored by measur-
ing the absorbance increase at 380 nm with a Molecular Devices microtit-
er plate reader. Measurements were taken at intervals of 60, 90, or 120 s.
With all substrates, the antibody-catalyzed reactions were quantitatively
inhibited by the hapten.
[7] Specific-acid or specific-base catalysis means that only H+ or OHꢀ,
and not weak acids (that is, general acids), can catalyze the reaction.
The definition applies to chemical catalysts in solution. Many en-
zymes accelerate specific-acid- or specific-base-catalyzed reactions
by using general acid base catalysis. Achieving this mechanistic
change requires the contributions of the enzyme binding pocket,
such as substrate immobilization. See also: W. P. Jencks, Catalysis in
Chemistry and Enzymology, Dower Publications, New York, 1987,
p. 165.
[8] a) J.-L. Reymond, K. D. Janda, R. A. Lerner, Angew. Chem. 1991,
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8528 8529; c) J.-L. Reymond, K. D. Janda, R. A. Lerner, J. Am.
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2630.
Data treatment: For the hydrolysis of the enol ethers, the values for the
initial velocity, vi, and for the final velocity, vss, were graphically estimat-
ed from the slopes of the progress curve. From Equation (1), the value,
T, of the intercept on the time axis was used to determine the apparent
rate constant, t, for the transition between the two velocities vi and vss.[42]
ðvssꢀviÞ
t
ð1Þ
T
¼
vss
[11] R. Manetsch, A. Chougnet, W.-D. Woggon, unpublished results.
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For the Kemp elimination the data were analyzed with the Kaleidagraph
program (Abelbeck Software). The three parameters vi, vss, and t were
obtained from Equation (2).[42]
½Pꢁ ¼ vsstꢀðvssꢀviÞð1ꢀeꢀt=tÞt
ð2Þ
Michaelis Menten kinetics for the hydrolysis of the enol ethers: The hy-
drolysis of the enol ethers was initiated by adding the substrate to the an-
tibody in a capped microtiter plate. The microtiter plate was kept in a
closed plastic box with a wet paper towel inside for humidity saturation
at 378C for at least 12 h and then the substrate and product concentra-
tions were measured by RP18 HPLC. The determination of the substrate
and product concentrations was repeated four and eight hours later, re-
spectively. During this period the catalyzed reaction is in the stable phase
with constant velocity, which allows the determination of the final veloci-
ty, vss, for the different substrate concentrations used in the measure-
ments. After correction of the rates for the uncatalyzed reaction rates in
BisTris buffer, the net vss rates were obtained. These rates were used to
derive the Michelis Menten constants, KM, and the maximum velocity,
Vmax, from the Lineweaver Burk plot of 1/Vmax versus 1/[S]. The catalytic
constant, kcat, was obtained by dividing Vmax by the antibody concentra-
tion.
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Michaelis Menten kinetics for the Kemp elimination: For the Kemp
elimination the obtained vss rates from Equation (2) were used to deter-
mine the value of KM/Vmax from the Lineweaver Burk plot of 1/Vmax
versus 1/[S].
[24] a) M. L. Casey, D. S. Kemp, K. G. Paul, D. D. Cox, J. Org. Chem.
1973, 38, 2294 2301; b) D. S. Kemp, M. L. Casey, J. Am. Chem. Soc.
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Acknowledgement
This work was supported by the Swiss National Science Foundation, the
University of Basel, and the University of Berne.
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2505
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

W.-D. Woggon, J.-L. Reymond et al.
FULL PAPER
[30] Even when considering that approximately 25% of the 45 monoclo-
nal antibodies obtained in the immunization series against hapten 71
leading to antibody 14D9 showed catalytic activity, an overview of
other immunization series shows that the occurrence of catalysis is
often not statistical by numbers of monoclonal antibodies but corre-
lates with individual mice, a fact implying that success may be tied
to obtaining viable monoclonal antibodies from a ™catalytic∫ mouse.
[31] a) T. Li, K. D. Janda, J. A. Ashley, R. A. Lerner, Science 1994, 264,
1289 1293; b) J. Hasserodt, K. D. Janda, R. A. Lerner, J. Am.
Chem. Soc. 1996, 118, 11654 11655; c) J. Hasserodt, K. D. Janda,
R. A. Lerner, J. Am. Chem. Soc. 1997, 119, 5993 5998; d) T. Li,
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Received: October 16, 2003[F5629]
Published online: March 22, 2004
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