Synthesis and mass spectrometry studies of branched oxime ether libraries. Mapping the substitution motif via linker stability and fragmentation pattern

Article abstract of DOI:10.1021/jo015765j

The oxime ether chemistry has recently been used as a convenient approach to preparing potentially highly diverse combinatorial libraries. The synthetically easiest way to form the libraries is convergent, i.e., via reaction of a branched scaffold containing two or more aminooxy linker groups, with a variety of carbonyl substituents. We show here that such reactions between aldehydes and ketones of different structure with the scaffolds containing different types of aminooxy groups can lead to the formation of virtually all expected components in the model mixtures 1-3 formed from three scaffolds (7-9) and eight substituents (R₁-R₈). One important problem with the branched libraries is that the libraries formed from the more complex scaffolds, such as 11, contain multiple regioisomers. The results of extensive analysis of a variety of library components by mass spectrometry presented here show that the differences in the MS-MS fragmentation energies for different linkers yield regiochemical information essential for identification of individual library components.

Full text of DOI:10.1021/jo015765j

J . Org. Chem. 2002, 67, 59-65
59
Syn th esis a n d Ma ss Sp ectr om etr y Stu d ies of Br a n ch ed Oxim e
Eth er Libr a r ies. Ma p p in g th e Su bstitu tion Motif via Lin k er
Sta bility a n d F r a gm en ta tion P a tter n
Noureddin Nazarpack-Kandlousy,† Marina I. Nelen,† Vasiliy Goral,† and Alexey V. Eliseev*,†,‡
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, and
Therascope AG, Hans-Bunte-Str. 20, 69123 Heidelberg, Germany
eliseev@buffalo.edu
Received May 17, 2001
The oxime ether chemistry has recently been used as a convenient approach to preparing potentially
highly diverse combinatorial libraries. The synthetically easiest way to form the libraries is
convergent, i.e., via reaction of a branched scaffold containing two or more aminooxy linker groups,
with a variety of carbonyl substituents. We show here that such reactions between aldehydes and
ketones of different structure with the scaffolds containing different types of aminooxy groups can
lead to the formation of virtually all expected components in the model mixtures 1-3 formed from
three scaffolds (7-9) and eight substituents (R1-R8). One important problem with the branched
libraries is that the libraries formed from the more complex scaffolds, such as 11, contain multiple
regioisomers. The results of extensive analysis of a variety of library components by mass
spectrometry presented here show that the differences in the MS-MS fragmentation energies for
different linkers yield regiochemical information essential for identification of individual library
components.
In tr od u ction
throughput synthesis/screening strategy, it has to be
complemented by appropriate methods for screening and
structure identification of individual components.
The oxime ether chemistry and the relevant chemistry
of aminooxy compounds12-15 has recently attracted at-
tention as a convenient approach to the rapid formation
of combinatorial libraries, both in a divergent16,17 and in
a convergent18 mode. In the divergent case, the libraries
are formed by the reaction of a scaffold containing two
or more aminooxy groups with a mixture of carbonyl
compounds (Scheme 1). The reaction proceeds under mild
conditions and is rapid and selective in that the carbonyl
groups are preferred over the majority of other function-
alities capable of reacting with the aminooxy nucleophile.
The resulting libraries have been shown to contain an
excellent representation of the expected components that
are formed from aldehydes with varying steric and
electronic properties.16 Among other advantages of the
oxime ethers is their potential suitability19 as components
for the dynamic libraries.20-24
Combinatorial chemistry and related techniques of
organic synthesis have been rapidly developing over the
recent years as tools for efficient generation of large
arrays of compounds for biological screening.1-7 It is now
clear that, even with the most efficient combinatorial
synthesis methods, it is not possible to cover the full
desired diversity space even for small drug-like mol-
ecules. Still, there is a big demand for new synthetic and
analytical approaches that can quickly generate and
assess large number of diverse chemical structures. Most
of the organic compound libraries, e.g., in the pharma-
ceutical industry, are synthesized by parallel techniques
as arrays of individual compounds. A potentially much
faster approach to molecular diversity is the formation
of mixture libraries8-11 via a convergent or divergent
combination of building blocks. The mixture formation
is attractive in its simplicity, but to fit in an overall high-
† State University of New York at Buffalo.
‡ Therascope AG.
(1) Lowe, G. Chem. Soc. Rev. 1995, 24, 309 ff.
(2) Thompson, L. A.; Ellman, J . A. Chem. Rev. 1996, 96, 555-600.
(3) Balkenhohl, F.; Von dem Bussche-Hunnenfeld, C.; Lansky, A.;
Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2289-2337.
(4) Patel, D. V.; Gordon, E. M. Drug Discov. Today 1996, 1, 134-
144.
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Melnyk, P.; Deprez, B.; Tartar, A. Eur. J . Med. Chem. 1996, 31, 87-
98.
(12) Khomutov, A. R.; Vepsalainen, J . J .; Shvetsov, A. S.; Hyvonen,
T.; Keinanen, T. A.; Pustobaev, V. N.; Eloranta, T. O.; Khomutov, R.
M. Tetrahedron 1996, 52, 13751-13766.
(13) Choong, I. C.; Ellman, J . A. J . Org. Chem. 1999, 64, 6528-
6529.
(14) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis 2000, 1189-
1207.
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3, 139-142.
(6) Maehr, H. Bioorg. Med. Chem. 1997, 5, 473-491.
(7) Weber, L. Curr. Opin. Chem. Biol. 2000, 4, 295-302.
(8) Blondelle, S. E.; Crooks, E.; Ostresh, J . M.; Houghten, R. A.
Antimicrob. Agents Chemother. 1999, 43, 106-114.
(9) Houghten, R. A.; Pinilla, C.; Appel, J . R.; Blondelle, S. E.; Dooley,
C. T.; Eichler, J .; Nefzi, A.; Ostresh, J . M. J . Med. Chem. 1999, 42,
3743-3778.
(16) Nazarpack-Kandlousy, N.; Zweigenbaum, J .; Henion, J .; Eli-
seev, A. V. J . Comb. Chem. 1999, 1, 199-206.
(17) Nazarpack-Kandlousy, N.; Chernushevich, I. V.; Meng, L. J .;
Yang, Y.; Eliseev, A. V. J . Am. Chem. Soc. 2000, 122, 3358-3366.
(18) Maly, D. J .; Choong, I. C.; Ellman, J . A. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 2419-2424.
(19) Polyakov, V. A.; Nelen, M. I.; Nazarpack-Kandlousy, N.; Ryabov,
A. D.; Eliseev, A. V. J . Phys. Org. Chem. 1999, 12, 357-363.
(20) Ganesan, A. Angew. Chem., Int. Ed. Engl. 1998, 37, 2828-2831.
(21) Eliseev, A. V.; Lehn, J . M. Comb. Chem. Biol. 1999, 243, 159-
172.
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10.1021/jo015765j CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/07/2001

60 J . Org. Chem., Vol. 67, No. 1, 2002
Nazarpack-Kandlousy et al.
Sch em e 1
One of the inherent problems with the structures
shown in Scheme 1, and with the branched libraries
overall, is the simultaneous formation of multiple regioi-
somers, which are difficult to characterize structurally,
particularly when in a mixture with similar compounds.
We have addressed this problem previously via a com-
bined synthetic/analytical approach (regiochemical tag-
ging)17 that involved synthesis of isotopically labeled
aminooxy scaffolds. In the ensuing paper, we show that
the intrinsic gas-phase properties of the oxime ether
library components make it possible to assign their
structure via straightforward analysis using tandem
mass spectrometry, and in many cases without invoking
special tagging procedures. It is also shown that the
synthesis and analysis of the oxime ether libraries can
incorporate a broad range of building blocks, such as
variety of aromatic and aliphatic aldehydes and ketones,
with different steric and electronic properties.
F igu r e 1. ESI MS of the mixture libraries 2 (a) and 3 (b)
formed by mixing scaffolds 8 and 9, respectively, with four
carbonyl compounds R1dO, R2dO, R3dO, R6dO.
Resu lts a n d Discu ssion
cluded all of the expected components, even those result-
ing from carbonyls with different electronic and steric
properties. While the exact ratio of products could not
be precisely determined by mass spectrometry, due to the
lack of individual standard compounds, all predicted
oxime ether components appeared as molecular peaks in
the single MS spectra (Figure 1). This fact simplified the
subsequent structural studies and supported previously
made conclusion about low selectivity of the oximation
reaction.16,17
We have previously shown that the stability of oxime
ether linker groups in the collision-induced dissociation
MS-MS analysis is sensitive to the chemical environ-
ment of the linker.17 This fact played a useful role in the
structural identification of the components, because
substituents linked to different attachment points were
cleaved off at different energies, and the regiochemistry
of the compound could thus be determined.
To explore the chemistry of the oxime ethers in a
combinatorial context, it was important to test diverse
types of both scaffolds and substituents in the formation
of library components, as well as to assess their suit-
ability for structural analysis by mass spectrometry.
Initially, we synthesized three types of libraries 1-3 on
the basis of disubstituted scaffolds 7-9, respectively, each
with two identical aminoxy groups. The synthetic routes
for the aminooxy scaffolds are depicted in Scheme 2. To
compare the properties of different substituents, we
tested different types of aldehydes and ketones, both
aliphatic and aromatic (R1-R8), with all three scaffolds.
The compounds for mass-spectrometry studies were
prepared in the form of small mixture libraries resulting
from the condensations of the scaffolds with two to four
carbonyl substituents. The condensation products in-
The molecular peaks of the oxime ether library com-
ponents in the single MS were then subjected to collision-
induced dissociation25 in a triple quadrupole mass spec-
trometer. We were particularly interested in observing
the “informative” fragments that correspond to the cleav-
age of substituents from the scaffolds, allowing the
identification of the substitution motif. The collision
energies were optimized for maximum intensity of each
fragment, and the optimal values were used to character-
ize the stability of the fragmented bonds in the gas phase.
The three sets of disubstituted compounds (1-3) showed
differences both in the fragmentation energies and the
fragment patterns, as summarized in Table 1.
Among the major fragments from libraries 1 and 2
were those resulting from the homolytic cleavage of the
N-O bonds. One of the reasons for testing these libraries
was to reinforce our earlier finding that the presence of
(22) Lehn, J . M. Chem. Eur. J . 1999, 5, 2455-2463.
(23) Cousins, G. R. L.; Poulsen, S. A.; Sanders, J . K. M. Curr. Opin.
Chem. Biol. 2000, 4, 270-279.
(25) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/
Mass Spectrometry; VCH Publishers: Weinheim, Germany, 1988.
(24) Lehn, J . M.; Eliseev, A. V. Science 2001, 291, 2331-2332.

Studies of Branched Oxime Ether Libraries
J . Org. Chem., Vol. 67, No. 1, 2002 61
Sch em e 2a
a
Legend: (a) CuCN, DMF, 150 °C, 17 h, 37%; (b) DIBAL-H, toluene, 0 °C, 1 h, 67%; (c) HONX/K2CO3, DMF, rt, 1 h, 97%; (d) NaBH3CN/
Ti(O-i-Pr)4, CHCl3/MeOH, rt, 1 h, 45%; (e) PBr3, CH2Cl2/ether, rt, 3 h, 75%; (f) MeBnNH/K2CO3, DMSO, rt, 2 h, 95%; (g) N2H4‚H2O,
CD3OH/CDCl3, rt, 12 h, 50%; (h) HONX/K2CO3, DMF, 75 °C, 3 h, 56%; (i) MeNH2/NaBH3CN/Ti(O-i-Pr)4, CHCl3/MeOH, rt, 1 h, 45%; (j)
N2H4‚H2O, CD3OH/CDCl3, rt, 2 h, 55%; (k) HONX/K2CO3, DMSO, rt, 1 h, 96%; (l) MeNH2/NaBH3CN/Ti(O-i-Pr)4, CHCl3/MeOH, rt, 1 h,
50%; (m) N2H4‚H2O, CD3OH/CDCl3, rt, 3 h, 60%; (n) NaBH3CN/Ti(O-i-Pr)4, CHCl3/MeOH, rt, 1 h, 64%; (o) CH2Cl2, PPh3, DEAD, rt, 10
h, 82%; (p) MeNH2.HCl, NaBH3CN/Ti(O-i-Pr)4, CHCl3/MeOH, rt, 1 h, 52%; (q) N2H4‚H2O, CD3OH/CDCl3, rt, 15 h, 50%; (r) 22, Ti (O-i-
Pr)4, NaBH3CN, CHCl3/MeOH, 50%;(s) N2H4‚H2O, CD3OH/CDCl3, rt, 2.5 h, 60%.
substituents in the position ortho to the O-NdR group
facilitates the fragment formation. Indeed, the results
showed that the cleavage of substituents from 1 was
typically observed in the energy range between 10 and
17 eV, while similar cleavage in 2 required 17-20 eV.
Similar positional effects had been observed previously
in the compounds with both ortho and para substitu-
ents;17 however, only here were we able to quantify the
energy difference. Based on our studies of libraries 4, we
proposed previously that the low energy of the ortho
substituent cleavage might be due to assistance of the
neighboring protonated amino group. In the current
study, we tested this hypothesis with library 5 formed
from scaffold 10 in which the protonated amino group
was separated from the fragmentation center by a rigid
substituent. The MS-MS spectra of 5 showed a differ-
ence between the ortho and para substituent cleavage
energies similar to 4, which indicates that difference is
most likely due to steric effects. (MS-MS of 4 and 5 was
performed on a QqTOF mass spectrometer (Sciex), while
the spectra of other libraries were recorded on the triple
quadrupole instrument. Therefore, the fragmentation
energies could not be directly compared between these
series of compounds.)
The MS-MS of libraries 3 showed a somewhat differ-
ent fragmentation pattern. The presence of hydrogen in
the beta-position to the oxime nitrogen led to the loss of
the substituent fragment of one mass unit higher, ap-
parently resulting from the hydrogen abstraction from
the CH2 group (Scheme 3). However, the optimum energy
needed for cleaving the substituent increased dramati-
cally, to 30-35 eV. This observation points to the much
higher stability of the aliphatic O-NdR group, as
compared to aromatic.
Nearly all of the fragments observed at less than 35
eV contained some part of the central scaffold. This fact
is not surprising because the electrospray ionization is
known to preserve the solution chemistry of the ions,26
where the ionic charge is concentrated on the nitrogen
of the amino groups. However, for structural analysis it
is particularly attractive to be able to directly observe
fragments of the substituents, which became possible in
(26) Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. J . Chem. Educ.
1996, 73, A 82 ff.

62 J . Org. Chem., Vol. 67, No. 1, 2002
Nazarpack-Kandlousy et al.
Ch a r t 1
the case of aliphatic oxime ethers 3. At the fragmentation
energies above 40 eV, peaks of the RN+ type were
detected in the positive ionization mode MS-MS for a
variety of substituents (Table 1). The relative intensity
of the peaks was dependent on the electronic properties
of the aldehyde and varied from barely visible (R4) to the
most intense in the spectrum (R5). However, the corre-
sponding peaks from all substituents could always be
detected, even in the compound containing both R4 and
R5. These types of fragments were never observed in the
aromatic oxime ethers 1 and 2, nor in the previously
studied scaffolds.17 We believe that these fragments
result from direct heterolytic cleavage, as shown in
Scheme 3. The scaffold part of the cleavage (M + H -
RN) was not observed in the spectrum, being neutral
overall.
The reaction of 11 with more than one carbonyl
compound leads to the formation of multiple isomers that
show up as one peak and make the MS-MS energy
analysis difficult. Hence, we restricted the MS studies
to the corresponding oxime ethers with three identical
substituents (Table 2). These individual compounds bore
the combination of linkers of the three previously studied
types, i.e., ortho- and para-substituted aromatic and
aliphatic oxime ethers. Even though the fragmentation
of 6 was expected to cleave off chemically similar sub-
stituents, the difference in the linkers would result in
different energy optima and the fragment pattern.
The fragments resulting from the first substituent
cleavage (MH - RxN) had optimum energies in the range
somewhat higher than that observed for 1. Because in
this case we could not distinguish between the frag-
ments with the remaining ortho and para substituents,
the optimization graph shown in Figure 2 most likely
reflects the combination of both fragments and results
in the average energy. However, as shown above with
disubstituted scaffolds, similar fragmentation performed
on the compound with different substituents should show
a distinct difference in the energy optima and clearly
identify the position of each of the substituents. Unlike
libraries 1-3, structures 6 generated a new type of
informative fragments, MH - (RxN)2, with the optimum
energy between 25 and 32 eV. In the disubstituted 1-5,
similar fragments were always observed, but represented
the bare central moiety and therefore did not provide any
The experiments with the disubstituted scaffolds dem-
onstrate (i) that different types of the oxime ether
linkages exhibit different fragmentation energies and
patterns and (ii) that these differences are by and large
determined by the variation of the scaffold structure, and
to a much lesser degree by the types of substituents.
Therefore, by combining different types of linkages in
more complex scaffolds, one should be able to determine
the regiochemistry of a variety of library components, i.e.,
to obtain the fragment of each substituent with a unique
energy and pattern, which is characteristic of its attach-
ment position. This hypothesis was tested with libraries
6 based on trisubstituted scaffold 11.

Studies of Branched Oxime Ether Libraries
J . Org. Chem., Vol. 67, No. 1, 2002 63
Ta ble 1. Op tim a l F r a gm en ta tion En er gies for th e Ser ies
of Com p ou n d s 1-3
characteristic
fragments
(positive mode) energy plots, (0.5 eV
maxima of fragment
intensity vs collision
substituents
(Rx,Ry)
scaffold
1
1
R1, R1
R1, R2
MH-R1N
MH-R1N
MH-R2N
MH-R1N
MH-R3N
MH-R1N
MH-R6N
MH-R3N
MH-R4N
MH-R3N
MH-R5N
MH-R3N
MH-R6N
MH-R4N
MH-R5N
MH-R7N
MH-R7N
MH-R8N
MH-R8N
MH-R1N
MH-R1N
MH-R2N
MH-R1N
MH-R3N
MH-R2N
MH-R2N
MH-R3N
MH-R3N
MH-R3N
MH-R4N
MH-R3N
MH-R5N
MH-R4N
MH-R5N
MH-R1NH
MH-R3NH
R3N
12.5
11.0
13.0
11.5
13.5
10.1
15.2
16.0
12.5
11.7
13.4
11.5
15.4
12.0
17.0
13.2
13.3
13.0
13.0
16.8
16.7
17.0
17.0
17.5
17.3
17.0
17.0
17.8
20.0
17.0
17.5
18.0
20.0
17.5
29.0
32.0
37.5
35.0
47.0
54.0
35.0
29.0
40.5
43.0
37.5
54.0
35.0
27.5
40.0
35.0
50.0
1
1
1
1
1
1
R1, R3
R1, R6
R3, R4
R3, R5
R3, R6
R4, R5
1
1
R7, R7
R7, R8
1
2
2
R8, R8
R1, R1
R1, R2
2
R1, R3
2
2
R2, R2
R2, R3
2
2
R3, R3
R3, R4
F igu r e 2. ESI MS-MS of the parent peak of compound 6(R3)3
at 13 eV (a) and 50 eV (b) and energy optimization curves (c)
for different fragments.
2
R3, R5
2
2
3
3
R4, R4
R5, R5
R1, R1
R3, R3
Sch em e 3
3
R3, R4
MH-R3NH
R3N
R4N
3
R3, R5
MH-R3NH
MH-R5NH
R3N
R5N
3
3
R4, R4
R4, R5
MH-R4NH
R4N
MH-R5NH
R4N
R5N
3
R5, R5
MH-R5NH
R5N
regiochemical information. In contrast, this type of
fragmentation in 6 is important because it shows what
substituent remains on the more stable aliphatic linker.
The latter piece of information can be confirmed by
detecting the RxN fragments coming off at yet higher
energy from the aliphatic linker (Table 2).
Ta ble 2. Ch a r a cter istic F r a gm en t En er gies for th e
Ser ies of Com p ou n d s 6
optimum of collision
Rx
R1
fragment
energy, (0.5 eV
MH-R1N
MH-(R1N)2
R1N
MH-R2N
MH-(R2N)2
R2N
MH-R3N
MH-(R3N)2
R3N
MH-R6N
MH-(R6N)2
R6N
15.0
25.0
65.5
11.5
29.0
48.0
20.0
25.0
55.0
20.0
31.5
40.0
Although the above studies of 6 have been performed
with individual compounds, they have clear implications
for the analysis of libraries based on 11 or similar
scaffolds. The observation of the substituent cleavage at
different energies from different positions bears impor-
tant regiochemical information about the parent com-
pounds. Thus, if an individual library component based
on type 6 or similar scaffolds were isolated, e.g., after
affinity selection with a biological target, then its sub-
stitution motif could be mapped by the MS-MS analysis
at varying energies. The new fragmentation patterns
discovered in the aliphatic oxime ethers allow one to
proofread the structural identification based on different
types of fragments.
R2
R3
R6
Con clu sion
This study has shown that minor structural variations
in the oxime ether linkers utilized with branched polysub-
stituted structures can allow one to determine the

64 J . Org. Chem., Vol. 67, No. 1, 2002
Nazarpack-Kandlousy et al.
stirred for 1 h at 0 °C. A 15 mL portion of chloroform was then
added followed by 25 mL of 10% HCl, and the solution was
stirred at room temperature for 1 h. The organic layer was
separated followed by extraction with additional 2 × 25 mL
of CHCl3. The combined organic layers were washed with 2 ×
25 mL of water and dried over anhydrous MgSO4. Purification
by silica chromatography (hexanes/CH2Cl2, 1:1) gave 285 mg
(67%) of 14. 1H NMR (δ ppm, 500 MHz, CDCl3): 10.29 (s, 2H),
8.46 (t, J ) 8.0 Hz, 1H), 7.09 (t, J ) 9.8 Hz, 1H). 13C NMR (δ
regiochemistry of substitution by mass spectrometry. In
combination with the previously developed regiochemical
tagging method,17 this approach leverages the practical
application of mixture-based combinatorial libraries, and
specifically those with labile linkers such as oxime ethers.
It is theoretically possible to synthesize complex branched
scaffolds or even dendrimers with a variety of attachment
points and rapidly generate vast diversity of compounds
in one pot (specific oxime ether chemistry is particularly
advantageous, because it gives access to the large array
of commercially available aldehydes and ketones). The
attachment points in the scaffolds can be differentiated
either by fragmentation energies and patterns, or with
the isotopic labeling motif. The MS-MS properties of the
scaffolds can be studied with a few symmetrically sub-
stituted compounds and then the trends identified can
potentially be applied to the structural analysis of
complex library components.
ppm, 125 MHz, CDCl3): 184.63 (t, J ) 2 Hz),167.85 (dd, J 1
)
14.6 Hz, J 2 ) 269.8 Hz), 131.42 (t, J ) 4.8 Hz), 121.74 (dd, J 1
) 4.5 Hz, J 2 ) 7.9 Hz), 106.01 (t, J ) 24.4 Hz). FAB MS: 170.0
([M + H]+).
P r otected 4,6-Dia m in ooxyisop h th a la ld eh yd e (15). To
a solution of 14 (289 mg, 1.7 mmol) and endo-N-hydroxy-5-
norbornene-2,3-dicarboximide (942 mg, 5.1 mmol) in anhy-
drous DMF (5 mL) was added anhydrous potassium carbonate
(705 mg, 5.1 mmol). After the solution was stirred for 1 h, the
residue was partitioned between saturated aqueous NaCl (30
mL) and CH2Cl2 (30 mL). The water layer was extracted with
CH2Cl2 (2 × 20 mL). Combined organic layers were washed
with brine (4 × 30 mL) and water (2 × 50 mL) and dried over
anhydrous MgSO4. After removal of the solvent in vacuo,
purification by silica chromatography (10% EtOAc in CH2Cl2)
yielded 806 mg (97%) of 15. 1H NMR (δ ppm, 500 MHz, DMSO-
d6): 10.25 (s, 2H), 8.29 (s, 1H), 6.67 (s, 1H), 6.24 (t, J ) 2.0
Exp er im en ta l Section
Mass spectrometry analysis was performed using a model
API-3000 triple quadrupole mass spectrometer (Applied Bio-
system/Sciex, USA) coupled to LC and equipped with a turbo
ionspray interface. The mass spectrometer was operated at a
unit resolution. Samples were introduced into the ionization
source at a flow rate of 5 µL/min with a syringe pump. The
ionspray needle was operated at +5500 V, and the orifice
voltage was set at 35-40 eV. Full-scan MS spectra were
acquired over the mass range m/z 50-1000 by scanning the
first quadrupole (Q1) with 0.1 step size at a scan rate of 2 s.
The product-ion spectra were obtained with collision energy
ranging from 5 to 60 eV.
The optimum of collision energy for each certain fragmenta-
tion was determined as collision energy corresponding to the
maximum of a characteristic fragment-ion peak. The optimiza-
tion was performed with collision energy varying from 5 to
100 eV at a step size of 3-5 eV.
Hz, 4H), 3.59 (m, 4H), 3.39(s, 4H), 1.63 (dd, J 1 ) 9 Hz, J 2
)
28 Hz, 4H). 13C NMR (δ ppm, 125 MHz, DMSO-d6): 186.16,
171.10, 163.38, 135.04, 131.78, 120.93, 100.94, 51.06, 44.37,
42.87. FAB MS: 489.3 ([M + H]+).
P r otected 4,6-Dia m in ooxyisop h th a lyl Alcoh ol (16). To
a solution of 15 (489 mg, 1.0 mmol) in CHCl3 (48 mL) was
added titanium(IV) isopropoxide (0.767 mL, 2.5 mmoL), and
the mixture was refluxed for 30 min. After the mixture was
cooled to room temperature, methanol (16 mL) and NaBH3-
CN (165 mg, 2.5 mmol, in two portions, within 1 h) were added.
The residue was partitioned between 100 mL of CH2Cl2 and
50 mL of brine and stirred for 1 h. The aqueous layer was
extracted twice more with CH2Cl2, and the combined organic
layers were washed with brine and water, and dried over
anhydrous MgSO4. Removal of the solvent followed by silica
chromatography (step gradient from 10% EtOAc in CH2Cl2 to
50% EtOAc in CH2Cl2) resulted in 222 mg (45%) of 16. 1H NMR
(δ ppm, 500 MHz, CDCl3): 7.41 (s, 1H), 6.64 (s, 1H), 6.25 (t, J
) 2.0 Hz, 4H), 4.69 (s, 4H), 3.75 (br s, 2H), 3.45 (br s, 4H),
3.31(m, 4H), 1.80 (dt, J 1 ) 1.8 Hz, J 2 ) 12.5 Hz, 2H), 1.54 (d,
J ) 9.0 Hz, 2H). 13C NMR (δ ppm, 125 MHz, CDCl3): 172.06,
156.19, 134.84, 131.72, 128.80, 106.77, 59.30, 51.46, 44.85,
42.77.
Solutions were prepared in 25% (v/v) aqueous acetonitrile
with sample concentration ranging from 5 to 30 µM.
Syn th esis. 1,5-Dibr om o-2,4-d iflu or oben zen e (12) was
synthesized on the basis of the procedure used for the synthesis
of 5-bromo-2,4-difluorotoluene.27 The pure product was ob-
tained in 75% yield after distillation (35-38 °C, 2 mmHg). 1H
NMR (δ ppm, 500 MHz, CDCl3): 7.76 (t, J ) 7.3 Hz, 1H), 7.00
(t, J ) 8.3 Hz, 1H). 13C NMR (δ ppm, 125 MHz, CDCl3): 158.36
(dd, J 1 ) 11.3 Hz, J 2 ) 250 Hz), 136.33 (t, J ) 1.1 Hz), 105.78
(t, J ) 26.9 Hz), 104.55 (dd, J 1 ) 8.9 Hz, J 2 ) 17.4 Hz). FAB
MS: 269.8, 271.8, 273.8 ([M + H]+, Br isotopes).
P r otected 4,6-Dia m in ooxy-r,r′-d ibr om o-m -xylen e (17).
To a solution of 16 (123 mg, 0.25 mmol) in 10 mL of CH2Cl2-
ether (1:1) was added PBr3 (1 M in CH2Cl2, 0.30 mL, 0.3 mmol).
After being stirred at room temperature for 3 h, the mixture
was partitioned between 25 mL of CH2Cl2 and 10 mL of water.
The organic layer was washed with water (3 × 20 mL), dried
over MgSO4, and purified by silica chromatography (1% EtOAc
4,6-Diflu or oisop h th a lon itr ile (13). To 8.16 g (30 mmol)
of 12 in 100 mL of DMF was added 6.18 g (69 mmol) of cuprous
cyanide. The mixture was refluxed for 17 h and then cooled to
room temperature. After the mixture was passed through a
filter and solvent evaporated, it was stirred in a mixture of
200 mL of CH2Cl2 and 50 mL of ice-cold water. The mixture
was extracted with additional 2 × 50 mL of CH2Cl2, and the
combined organic layers were washed with 3 × 100 mL of
water, filtered, and dried over anhydrous MgSO4. The solvent
was then evaporated, and the crude mixture was purified by
silica chromatography (10-40% of hexanes in CH2Cl2) to yield
1
in CH2Cl2). Yield: 116 mg (75%). H NMR (δ ppm, 300 MHz,
DMSO-d6): 7.68 (s, 1H), 6.41 (s, 1H), 6.19 (s, 4H), 4.70 (s, 4H),
3.50 (br s, 4H), 3.33 (br s, 4H), 1.59 (dd, J 1 ) 8.4 Hz, J 2 ) 20.7
Hz, 4H). 13C NMR (δ ppm, 75 MHz, DMSO-d6): 171.32, 156.29,
134.92, 134.04, 122.92, 102.13, 51.10, 44.22, 42.69, 26.61.
P r otected 4,6-Diam in ooxy-r,r′-di(ben zylm eth ylam in e)-
m -xylen e (18). To a mixture of 6 (155 mg, 0.25 mmol) and
K2CO3 (86.4 mg, 0.625 mmol) in 5 mL of DMSO was added
N-methylbenzylamine (0.081 mL, 0.625 mmol). After being
stirred at room temperature for 2 h, the mixture was parti-
tioned between 25 mL of CH2 Cl2 and 10 mL of brine. The
organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (2 × 10 mL). The combined organic
layers were washed with water (4 × 25 mL) and dried over
MgSO4. Evaporation of the solvent resulted in the analytically
pure product (162.4 mg, 95%). 1H NMR (δ ppm, 500 MHz,
CDCl3 ): 7.79 (s, 1H), 7.39 (d, J ) 7.3 Hz, 4H), 7.30 (d, J ) 7.3
Hz, 4H), 7.24 (t, J ) 7.3 Hz, 2H), 6.60 (s, 1H), 6.26 (s, 4H),
3.79 (s, 4H), 3.57 (s, 4H), 3.47 (s, 4H), 3.29 (m, J ) 4.5 Hz,
1
1.82 g (37%) of 13. H NMR (δ ppm, 500 MHz, CDCl3): 8.04
(t, J ) 6.8 Hz, 1H), 7.24 (t, J ) 8.5 Hz, 1H). 13C NMR (δ ppm,
125 MHz, CDCl3): 166.04 (dd, J 1 ) 13.5 Hz, J 2 ) 270.9 Hz),
138.70 (t, J ) 2.5 Hz), 110.95, 107.07 (t, J ) 24.1 Hz), 100.15
(dd, J 1 ) 7.3 Hz, J 2 ) 13.4 Hz). FAB MS: 164.0 ([M + H]+).
4,6-Diflu or oisop h th a la ld eh yd e (14). Compound 13 (410
mg, 2.5 mmol) was dissolved in 10 mL of anhydrous toluene
and cooled to 0 °C. DIBAL-H (5 mL of 1.5 M solution in
toluene, 7.5 mmol) was added dropwise. The solution was
(27) Schweitzer, B. A.; Kool, E. T. J . Org. Chem. 1994, 59, 7238-
7242.

Studies of Branched Oxime Ether Libraries
J . Org. Chem., Vol. 67, No. 1, 2002 65
4H), 2.21 (s, 6H), 1.81 (d, J ) 9.0 Hz, 2H), 1.54 (d, J ) 9.0 Hz,
2H). 13C NMR (δ ppm, 125 MHz, CDCl3): 171.34, 155.59,
139.52, 134.87, 132.00, 128.85, 128.10, 126.74, 125.87, 104.33,
61.92, 54.34, 51.37, 44.80, 42.84, 42.20. ESI MS (CH3CN/water
1/3 v/v) 699.6 ([M + H]+).
(s, 1H), 7.22-7.35 (m, 11H), 6.85 (br s, 4H), 3.48 (s, 4H), 3.41
(s, 4H), 2.08 (s, 6H). ESI MS 407.6 ([M + H]+).
Sca ffold 8: 0.40 mmol H2NNH2‚H2O, 2 h, solvent not
evaporated upon completion, silica purification in 1-2% MeOH
1
in CH2Cl2. Yield: 55%. H NMR (δ ppm, 500 MHz, CD3CN):
Syn th esis and analytical data of compounds 19, 20, 22, 23,
25, 26, and 28 are described in the Supporting Information.
4-(P r otected 2,4-15N-d ia m in ooxyben zyloxy)-N,N d im -
eth ylben zyla m in e. To a solution of aldehyde 26 (237.2 mg,
0.42 mmol) in anhydrous CHCl3 (10 mL) was added titanium-
(IV) isopropoxide (0.84 mmol, 0.250 mL). Dimethylamine
hydrochloride (51.4 mg, 0.63 mmol) was then added, and the
mixture was refluxed for 30 min. After the mixture was cooled
to room temperature, methanol (3.3 mL) and NaBH3CN (79.2
mg, 1.26 mmol, in two portions, within 1 h) were added. The
residue was partitioned between 50 mL of CH2Cl2 and 25 mL
of brine and stirred for 1 h. The aqueous layer was extracted
twice more with CH2Cl2, and the combined organic layers were
washed with brine and water and dried over anhydrous
MgSO4. Removal of the solvent followed by silica chromatog-
raphy (step gradient from 1 to 8% MeOH in CH2Cl2) yielded
130.3 mg (52%) of the product. 1H NMR (δ ppm, 500 MHz,
CDCl3): 7.41 (d, J ) 9.0 Hz, 1H), 7.29 (d, J ) 8.5 Hz, 2H),
7.02 (d, J ) 8.5 Hz, 2H), 6.81 (dd, J ) 2.5 Hz, J ) 8.5 Hz,
7.21 (d, J ) 8.5 Hz, 4H), 7.03 (d, J ) 8.5 Hz, 4H), 6.14 (s, 4H),
3.38 (s, 4H), 2.04 (s, 3H). ESI MS 274.5 ([M + H]+).
Sca ffold 9: 0.60 mmol H2NNH2‚H2O, 3 h, solvent evapo-
rated upon completion, silica purification in 1-3% MeOH in
CH2Cl2. Yield: 60%. 1H NMR (δ ppm, 400 MHz, DMSO-d6):
7.26 (dd, J ) 8.4 Hz, J ) 16.0 Hz, 8H), 6.00 (br s, 4H), 4.51
1
2
(s, 4H), 3.44 (s, 4H), 2.03 (s, 3H). ESI MS 302.5 ([M + H]+).
Sca ffold 10: 0.10 mmol H2NNH2‚H2O, 15 h, solvent evapo-
rated upon completion, silica purification in 2.5% Et3N in CH2-
1
Cl2. Yield: 50%. H NMR (δ ppm, 500 MHz, DMSO-d6): 7.26
(d, J ) 2.5 Hz, 1H), 7.18 (d, J ) 8.5 Hz, 2H), 7.17 (d, J ) 8.0
Hz, 1H), 6.96 (s, 2H), 6.95 (s, 1H), 6.91 (d, J ) 8.5 Hz, 2H),
6.82 (s, 1H), 6.57 (dd, J ) 2.0 Hz, J ) 9.0 Hz, 1H), 4.86 (s,
1
2
1H), 3.39 (s, 2H), 2.12 (s, 6H). ESI MS 305.2 ([M + H]+).
Sca ffold 11: 0.60 mmol H2NNH2‚H2O, 2.5 h, solvent not
evaporated upon completion, silica purification in 2.5% Et3N
in CH2Cl2. Yield: 60%. 1H NMR (δ ppm, 400 MHz, DMSO-
d6): 7.29 (dd, J ) 8.8 Hz, J ) 14.0 Hz, 4H), 7.21 (d, J ) 2.8
1
2
Hz,, 1H), 7.14 (d, J ) 8.4 Hz, 1H), 6.86 (s, 2H), 6.82 (s, 2H),
6.56 (dd, J ) 2.4 Hz, J ) 8.4 Hz, 1H), 6.04 (br s, 2H), 4.55
1
2
1H), 6.76 (d, J ) 2.5 Hz, 1H), 6.34 (t, J ) 1.8 Hz, 2H), 6.25 (t,
1
2
J ) 1.8 Hz, 2H), 5.26 (s, 2H), 3.79 (s, 2H), 3.51 (dm, J ) 3.5
(s, 2H), 3.47 (s, 2H), 3.37 (s, 2H), 2.06 (s, 3H). ESI MS 319.4
1
Hz, J ) 6.5 Hz, 4H), 3.37 (dm, J ) 3.5 Hz, J ) 10.5 Hz,
([M + H]+).
2
1
2
4H), 2.53 (s, 6H), 1.84 (t, J ) 9.0 Hz, 2H), 1.58 (t, J ) 9.0 Hz,
2H). 13C NMR (δ ppm, 75 MHz, CDCl3): 171.12 (d, J ) 9.1
Hz), 171.03, 158.77, 158.19, 155.56, 135.12, 134.93, 131.58,
129.60, 124.27, 121.73, 115.16, 110.62, 102.41, 63.55, 61.67,
51.47, 51.40, 44.79, 44.76, 43.00, 42.90. ESI MS (CH3CN/water
1/3 v/v) 597.3 ([M + H]+).
Libr a r y F or m a tion was performed as described in refs 16
and 17.
Ack n ow led gm en t. We are grateful to the NIH for
the financial support of this work (Grant No. 6035201
from NIGMS) and Dr. I. Chernushevich (Sciex) for
recording the spectrum of compound 5. The LCMS was
obtained with Shared Instrumentation Grant No.
S10RR14572 from the National Center for Research
Resources, NIH. We thank the Pharmaceutical Sciences
instrumentation facility at the University at Buffalo for
data acquisition.
Dep r otection of P r otected Am in ooxy Sca ffold s. In a
typical procedure, varying amount of hydrazine monohydrate
was added to a solution of 0.025 mmol of the protected
aminooxy scaffold in 1 mL of 10-15% CD3OD in CDCl3 in an
NMR tube, and the deprotection progress was monitored by
NMR at room temperature. The pure products were isolated
by silica chromatography. Specific conditions used for different
scaffolds are given below. ESI MS spectra were recorded in
acetonitrile/water 1/3 v/v.
Su p p or tin g In for m a tion Ava ila ble: Synthesis and ana-
lytical data of intermediates, Scheme 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Sca ffold 7: 0.063 mmol H2NNH2‚H2O, 12 h, solvent evapo-
rated upon completion, silica purification in 2.0% Et3N in CH2-
1
Cl2. Yield: 50%. H NMR (δ ppm, 400 MHz, DMSO-d6): 7.59
J O015765J