Metal-free sequential [3 + 2]-dipolar cycloadditions using cyclooctynes and 1,3-dipoles of different reactivity

Article abstract of DOI:10.1021/ja1081519

Although metal-free cycloadditions of cyclooctynes and azides to give stable 1,2,3-triazoles have found wide utility in chemical biology and material sciences, there is an urgent need for faster and more versatile bioorthogonal reactions. We have found th

Full text of DOI:10.1021/ja1081519

ARTICLE
pubs.acs.org/JACS
Metal-Free Sequential [3 þ 2]-Dipolar Cycloadditions using
Cyclooctynes and 1,3-Dipoles of Different Reactivity
Brian C. Sanders,†,‡ Frꢀedꢀeric Friscourt,† Petr A. Ledin,†,‡ Ngalle Eric Mbua,†,‡ Selvanathan Arumugam,‡
Jun Guo,† Thomas J. Boltje,†,‡ Vladimir V. Popik,‡ and Geert-Jan Boons*,†,‡
†Complex Carbohydrate Research Center, and ‡Department of Chemistry, University of Georgia, 315 Riverbend Road, Athens,
Georgia 30602, United States
S Supporting Information
b
ABSTRACT: Although metal-free cycloadditions of cyclooctynes
and azides to give stable 1,2,3-triazoles have found wide utility in
chemical biology and material sciences, there is an urgent need for
faster and more versatile bioorthogonal reactions. We have found
that nitrile oxides and diazocarbonyl derivatives undergo facile 1,
3-dipolar cycloadditions with cyclooctynes. Cycloadditions with
diazocarbonyl derivatives exhibited similar kinetics as compared to
azides, whereas the reaction rates of cycloadditions with nitrile
oxides were much faster. Nitrile oxides could conveniently be pre-
pared by direct oxidation of the corresponding oximes with BAIB,
and these conditions made it possible to perform oxime formation,
oxidation, and cycloaddition as a one-pot procedure. The methodology was employed to functionalize the anomeric center of
carbohydrates with various tags. Furthermore, oximes and azides provide an orthogonal pair of functional groups for sequential metal-
free click reactions, and this feature makes it possible to multifunctionalize biomolecules and materials by a simple synthetic procedure
that does not require toxic metal catalysts.
’ INTRODUCTION
saccharides and amino acids and can be employed for visualizing
metabolically labeled glycans of living cells.15 Attractive features of
DIBO include easy access to the compound by a simple synthetic
approach, nontoxicity, and the possibility of straightforward attach-
ment of a variety of probes. Furthermore, the structure of DIBO is
amenable to analogue synthesis, and derivatives (3 and 4) have
been introduced that exhibit even higher rates of reaction than the
parent compound.16
Our finding that cyclooctynes can undergo fast cycloadditions
with nitrones has further expanded the scope of metal-free click
reactions,17 and the usefulness of this approach has been demon-
strated by site-specific protein modification by a three-step pro-
tocol entailing periodate oxidation of an N-terminal serine to give
an aldehyde, which could easily be converted into a nitrone and
then reacted with probe-modified dibenzocyclooctynes.
As part of a program to develop metal-free click reactions, we
report here that, in addition to azides and nitrones, nitrile oxides
and diazocarbonyl derivatives readily undergo cycloadditions
with dibenzocyclooctyne to give stable isoxazoles and pyrazoles,
respectively. It has been found that the various 1,3-dipoles exhibit
distinct levels of reactivity, making it possible to perform
sequential cycloadditions. In addition, we have shown, for the first
time, that an oxime can function as a latent 1,3-dipole for a nitrile
Metal-free click cycloadditions of cyclooctynes with azides1 to
give stable 1,2,3-triazoles have found wide utility in labeling gly-
cans,2 proteins,3 and lipids4 of living cells, glycoprotein enrich-
ment for proteomics,5 protein,6 and oligonucleotide modifica-
tion,7 and tissue reengineering.8 These reactions, which have been
coined “strain-promoted alkyne-azide cycloadditions (SPAAC)”,
have also made entry in material sciences and have for example
been employed for the assembly, cross-linking,9 and surface
modification of dendrimers,10 derivatization of polymeric nano-
structures,11 and patterning of surfaces.12
Density functional theory (B3LYP) calculations of the transi-
tion states of cycloadditions of phenyl azide with acetylene and
cyclooctyne indicate that the fast rate of the “strain promoted”
cycloaddition is actually due to a lower energy required for dis-
torting the 1,3-dipole and alkyne into the transition-state geom-
etry.13 The first generation of cyclooctynes proceeded with
relatively slow rates of reaction; however, it has been found that
significant increases in the rate of strain-promoted cycloaddition
can be accomplished by appending electron-withdrawing groups
to the propargylic position of cyclooctyne.2 For example, di-
fluorinated cyclooctyne (DIFO, 1, Figure 1)14 reacts with azides
approximately 60 times faster than similar cycloadditions with an
unsubsituted cyclooctyne. We have reported that derivatives of
4-dibenzocyclooctynol (DIBO, 2) react fast with azido-containing
Received: September 15, 2010
Published: December 23, 2010
r
2010 American Chemical Society
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Table 1. Rate Constants and Yields for the Cycloadditions of
DIBO (2) with Various Nitrile Oxides
entry
R
k (M-1 s-1
)
yield (%)a
1
2
3
4
5
6
7
C6H5b,c,d (5a)
C6H5b,c,d (5a)
3.38 ( 0.03g
2.46 ( 0.03h
2.15 ( 0.02g
8.47 ( 0.03g
3.99 ( 0.05g
3.42 ( 0.03g
3.31 ( 0.06g
93
ND
89
Figure 1. Cyclooctynes for metal-free click reactions.
4-MeO-C6H4b,e (5b)
4-O2N-C6H4b,f (5c)
4-F-C6H4b,c,d (5d)
4-Cl-C6H4b,c,d (5e)
4-Br-C6H4b,c,d (5f)
93
Scheme 1. Rate Constants of Cycloadditions of DIBO (2)
with Various 1,3-Dipoles: Nitrile Oxide, Azide, Nitrone, and
Diazocarbonyl Derivativesa
90
90
93
a Isolated yields of combined isomers. b Second-order rate constants
were determined from pseudo first-order rate constants at various
concentrations of in situ formed nitrile oxides at 25 ( 0.1 °C. c Pseudo
first-order kinetics were determined using UV-vis spectroscopy
by following the decay of the absorbance of compound 2 at 305 nm.
d [2] = 6.0 ꢀ 10-5 M; for details on the concentrations of nitrile oxides,
see the Supporting Information. e [2]=3.0ꢀ10-5 M; [5b] = (2.5-5.0)ꢀ
10-4 M. f Pseudo first-order kinetics were determined by UV-vis
spectroscopy following the decay of the absorbance of 5c at 325 nm;
[5c] = 6.0 ꢀ 10-5 M, [2] = (7.0-17.5) ꢀ 10-4 M. g Reaction was per-
formed in methanol. h Reaction was performed in acetonitrile.
alkynes and benzynes to give pyrazoles and indazoles, respec-
tively.23 These findings inspired us to explore strain-promoted
alkyne-nitrile oxide cycloadditions (SPANOC) and alkyne-
diazocarbonyl (SPADC) with DIBO (2) and compare the rates
of reactions with similar cycloadditions with azides (SPAAC) and
nitrones (SPANC) (Scheme 1).
A range of imidoyl chlorides (5a-f), which can easily be
converted into nitrile oxides by treatment with a mild base, was
prepared by reactions of the corresponding aldehydes with
hydroxylamine24 followed by chlorination of the resulting oximes
with N-chlorosuccinimide.25 Addition of the imidoyl chlorides
5a-f to a solution of DIBO in methanol in the presence of
triethylamine led, within several minutes, to the quantitative
formation of isoxazoles 6a-f (Table 1).
a k 0 is the relative rate with benzyl azide set at 1. The second-order rate
constant for nitrone 9 was determined by using equimolar mixture of
reagents due to a strong absorbance at 305 nm.
Accurate rate measurements of the cycloaddition reactions
were conducted by UV spectroscopy following the growth of the
decay of the characteristic absorbance of the acetylene of DIBO
(2) at 305 nm. The rates were measured in methanol or acetoni-
trile solutions at 25 ( 0.1 °C. The kinetics of the cycloadditions
was studied under pseudo first-order conditions by maintaining a
fixed concentration of DIBO (2), while the concentration of the
dipoles was varied. Consumption of starting material followed a
first-order equation, and the pseudo first-order rate constants
were obtained by least-squares fitting of the data to a single
exponential equation. The observed rate constants were linearly
dependent on the concentration of dipoles,26 and second-order
cycloaddition rate constants calculated from the concentration
dependencies of observed rates are listed in Table 1. As can be
seen, the cycloadditions with the nitrile oxides are exceptionally
fast, and the substituent exerts only small influence. It appears
that strongly electron-withdrawing substituents, such as a nitro
group (entry 4), somewhat increase the rate of reaction.
oxide, which is fully orthogonal with cycloadditions of azides.
These findings make it possible to employ strain-promoted cyclo-
additions for the assembly of complex multifunctional and bioin-
spired materials without the need of employing a toxic metal
catalyst.
’ RESULTS AND DISCUSSION
Nitrile oxides can undergo cycloadditions with terminal alkynes
to give 3,5-isoxazoles;18 however, the success of these reactions is
often compromised by a slow rate of reaction and competing
dimerization of nitrile oxides.19 3,5-Disubstituted isoxazoles have
been prepared in high yield by intramolecular cycloadditions,20 the
use of activated dipolarophiles21 such as benzyne and norbornenes,
or by employing a Cu(I) catalyst.22 Furthermore, diazocarbonyl
reagents, which are sufficiently stable for use in chemical synthesis,
have been employed in 1,3-dipolar cycloadditions with substituted
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Table 2. One-Pot Oxime Formation and SPANOC with
DIBO (2)
a,b,c
entry
R
k (M-1 s-1
)
yield (%)d
1
2
3
C6H5 (6a)
3.44 ( 0.03
3.20 ( 0.03
1.38 ( 0.01
55
51
90
2-Me-C6H4 (6g)
C6H5-CH2CH2 (6h)
a Rate constant was determined from isolated oximes. b Second-order
rate constants were determined from pseudo first-order rate constants at
various concentrations of nitrile oxides at 25 ( 0.1 °C. c Pseudo first-
order kinetics were determined using UV-vis spectroscopy following
the decay of the absorbance of 2 at 305 nm; [2] = 6.0 ꢀ 10-5 M; for
details on the concentrations of nitrile oxides, see the Supporting
Information. d Isolated yields of combined isomers.
Figure 2. Consumption of DIBO 2 (6.0 ꢀ 10-5 M in methanol at
25 °C) in the presence of various dipoles (3 mM). The lines shown were
drawn using parameters obtained by least-squares fitting of single
exponential equation. The inset shows reaction of DIBO with 5a at a
different time scale.
Furthermore, the use of methanol or acetonitrile had only a
marginal influence on the reaction rate (entries 1 and 2).
remaining excess of hydroxylamine and resulted in smooth
formation of the desired isoxazoles 6a,g-h. Furthermore, this
experimental approach made it possible to prepare isoxazoles in
high yield, which have unstable corresponding imidoyl chlorides,
notably in the aliphatic series (Table 2, entry 3).
Next, we compared the reaction rates of 1,3-dipolar cycloaddi-
tions of DIBO (2) with a nitrile oxide derived from imidoyl
chlorides 5a, benzyl derived azide 7, nitrone 9, and diazocarbonyl
derivative 11 to give isoxazole 6a, triazole 8, N-methyl isoxazoline
10, and pyrazole 12, respectively (Scheme 1 and Figure 2). It was
found that the azide, nitrone, and diazocarbonyl exhibit similar
rates of reaction. However, the rate of cycloaddition of the nitrile
oxide was 57 times faster than a similar reaction with benzyl
azide.
Rate constants were measured for the tandem sequence of
oxidation of oximes to nitrile oxides followed by 1,3-dipolar
cycloaddition with 2 establishing that the cycloaddition is the rate-
limiting step and highlighting that oxidation with BAIB is excep-
tionally fast. For example, when benzaldehyde oxime was employed,
the rate constant of the reaction was 3.44 M-1 s-1, which is almost
the same as the value obtained when benzaldehyde imidoyl chloride
was employed (3.38 M-1 s-1). Furthermore, the kinetic data for
compounds 6g and 6h demonstrate further that the nature of the
substituent has only a small effect on the rate of the reactions.
Convenient bioorthogonal reactions require that transforma-
tions are modular, have a high tolerance for the presence of
functional groups, and proceed at ambient temperature using
benign solvents and reagents. To determine whether SPANOC
complies with these requirements, we examined the tagging of a
carbohydrate with a biotin probe. Complex carbohydrates are
involved in a wide variety of biological processes,28 and fluor-
escent, biotin, multivalent, and immobilized saccharide deriva-
tives are important tools to study the intriguing properties of this
class of biomolecules.29 It was expected that such derivatives can
easily be prepared by reaction of sugar oximes by a sequential
reaction of an aldose with hydroxylamine to give an oxime, which
can then be functionalized by reaction with DIBO derivatives in
the presence of BAIB. The attraction of such an approach is that
it allows functionalization of the reducing end of complex
carbohydrates with various probes using low equivalents of
expensive reagents. Thus, reaction of the readily available oxime
1430 with 2 or biotin-modified DIBO 15 (equimolar amounts,
Scheme 2) in the presence of BAIB for 10 min gave the sugar
derivatives 16 and 17, respectively. It is interesting to note that
the use of BAIB did not oxidize primary hydroxyls of lactose or
sulfur of biotin.31 Compounds such as 16 that are modified with a
biotin tag can, for example, be employed for immobilization to a
surface coated with Streptavidin.
Having established that nitrile oxides react exceptionally fast
with DIBO (2), attention was focused on streamlining the
process of nitrile oxide formation and cycloaddition. It was
expected that the number of reaction steps could be reduced
by a direct oxidation of oximes to nitrile oxides by using a mild
oxidant such as [bis(acetoxy)iodo]benzene (BAIB).27 Further-
more, a one-pot multistep sequence in which oxime formation,
oxidation, and cycloaddition are performed by sequential addi-
tion of reagents was expected to reduce the number of workup
and purification steps, thereby increasing the efficiency and
overall yield of the transformation. Thus, a reaction of benzalde-
hyde (13a) with hydroxylamine in methanol gave, after a reaction
time of 2 h, an intermediate benzaldehyde oxime, which was
treated with DIBO and BAIB, and after an additional reaction
time of 10 min, TLC and MS analysis indicated complete con-
version of the oxime into isoxazole 6a, highlighting that the
oxidation and cycloaddition steps proceed with exceptionally
high reaction rates (Table 2, entry 1). Additional experiments
demonstrated that DIBO (2) is stable when exposed to BAIB
alone, and thus the oxidation and cycloaddition could be per-
formed as a tandem reaction sequence. However, hydroxylamine
decomposes DIBO (2) probably by a nucleophilic attack at the
strained alkyne. Thus, the success of the transformation required
the use of either an equimolar quantity of aldehyde and hydro-
xylamine or more conveniently the addition of acetone prior to
cycloaddition to convert the excess hydroxylamine into ketox-
ime, which can react with BAIB but does not provide a 1,3-dipole.
Alternatively, the addition of an excess of BAIB before admin-
istering DIBO (2) also led to complete consumption of the
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Scheme 2. Modification of the Reducing End of Lactose by SPANOC Employing Oxime 14
We envisaged SPANOC can also be used for the installation of
tags into sialic acid containing glycoproteins by mild treatment
with NaIO4 to form a C-7 aldehyde, which upon treatment with
hydroxylamine will give an oxime that can be oxidized to a nitrile
oxide for reaction with derivatives of DIBO. The attraction of
such a strategy is that tags can be installed into glycoproteins by
stable isoxazoles linkages.32 To examine the usefulness of such a
strategy, the glycoprotein fetuin was treated with a 1 mM solu-
tion of NaIO4 for 5 min, after which the excess of oxidizing
reagent was removed by spin filtration. The resulting aldehyde
containing glycoprotein was treated with hydroxyl amine to
install an oxime, which was immediately oxidized to a nitrile
oxide by short treatment with BAIB and then reacted with 15 for
15 min to give a biotin containing sialic acid. As a control, BSA,
which does not contain sugar moieties, was subjected to the same
sequence of reactions. The presence of biotin was examined by
Western blotting using antibiotin antibody conjugated to HRP.
As can be seen in Figure 3, fetuin showed strong reaction when
subjected to the sequential three-step procedure, whereas BSA
was not detected. Furthermore, exclusion of one of the reaction
steps abolished detection, confirming the selectivity of the pro-
cedure. Quantitative protein and biotin determination indicated
that two biotin moieties were installed in each fetuin molecule.
The large difference in reactivity of the cycloaddition of DIBO
with the various 1,3-dipoles should make it possible to perform
sequential click reactions, which may provide opportunities to
prepare multifunctional compounds or materials by a simple syn-
thetic procedure. In particular, it was expected that a highly
reactive nitrile oxide can selectively undergo a cycloaddition in
the presence of an azide. Furthermore, we envisaged that oximes
can function as latent 1,3-dipoles, and therefore, a cyclooctyne
should react with an azide without affecting an oxime. However,
in the presence of BAIB, an oxime is rapidly converted into a nitrile
oxide, which can then be reacted with another functionalized
cyclooctyne. Thus, by careful selection of appropriate reagents, it
should be possible to selectively modify a bifunctional linker (or
complex compound) containing an azide and oxime moiety.
As expected, the addition of monosaccharide-modified DIBO
19 to bifunctional azido-oxime linker 18 in methanol resulted in
selective cycloaddition at the azide moiety to provide the triazole
20 in high yield (Scheme 3). However, when linker 18 was
treated with DIBO derivative 19 in the presence of BAIB, the
oxime moiety was rapidly oxidized to a highly reactive nitrile
Figure 3. Labeling and detection of sialic acids on the glycoprotein
fetuin using SPANOC. Fetuin (samples in lanes 1-4) and BSA (samples
in lanes 5-8) were subjected to periodate oxidation using NaIO4 (samples
in lanes 3, 4, 7, and 8). Next, the generated C-7 aldehyde (on sialic acid)
was reacted with HONH2 HCl to form an oxime, which was oxidized by
3
reacting with BAIB to produce nitrile oxide that was reacted with DIBO
derivative 15 (samples in lanes 2, 4, 6, and 8). Incorporated biotin was then
detected by Western blot using an antibiotin antibody conjugated to HRP.
Total protein loading was confirmed by Coomassie staining.
oxide, which underwent a fast SPANOC resulting in the selective
formation of isoxazole 21.
Having established the orthogonality of azides and oximes/
nitrile oxides, we examined sequential SPAAC-SPANOC click
reactions of bifunctional linker 18 with a biotin (15) or a fluo-
rescent probe (22) and a cluster of glycosides (25)10b modified
with DIBO (Scheme 4). Thus, treatment of azido-oxime linker
18 with DIBO-modified biotin (15) or DIBO-modified coumarin
(22) in methanol or THF, respectively, at ambient temperature for
2 h led to clean formation of monofuntionalized triazoles 23 and
24, respectively. Next, triazoles 23 and 24 were exposed to a
mixture of BAIB to convert the oxime moiety into a highly reactive
nitrile oxide, and reaction with DIBO-modified saccharide cluster
25 lead to a fast SPANOC to give bifunctional compounds 26 and
27, displaying a cluster of galactoses conjugated to biotin or a
fluorescent tag, respectively. It is of interest to note that neither
oxidation of biotin moiety by BAIB nor cycloaddition of the in situ
generated nitrile oxide at the carbon double bond of coumarin33
was observed, highlighting that SPANOC is perfectly suitable for
the conjugation of sensitive compounds.
’ CONCLUSION
Although metal-free cycloadditions between cyclooctynes and
azides to give stable 1,2,3-triazoles have found wide utility in
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Scheme 3. Selective Cycloadditions between Galactoside-Modified DIBO 19 with Either the Azide or the Oxime Moiety of
Linker 18
Scheme 4. Preparation of a Bifunctional Compound by a Sequential SPAAC and SPANOC
chemical biology1 and material sciences,8 there is an urgent need
for faster and more versatile bioorthogonal reactions. We have
found that 1,3-dipolar cycloadditions of cyclooctynes with nitrile
oxides exhibit much faster kinetics than similar reactions with
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azides. The nitrile oxides could easily be prepared by direct
oxidation of the corresponding oximes with BAIB, and these
reaction conditions made it possible for oxime formation, oxida-
tion, and cycloaddition to be performed as a one-pot procedure.
The transformations have a high tolerance for the presence of
functional groups, proceed at ambient temperature using benign
solvents and reagents, and make it possible to modify com-
pounds by a modular approach. Furthermore, the results pre-
sented here demonstrate that oximes and azides provide an
orthogonal pair of functional groups for sequential metal-free
click reactions. In this respect, sequential click reactions have
been reported by Cu(I)-catalyzed alkyne azide cycloaddition34
(CuAAC) using terminal- and silyl-protected alkynes35 and by
exploiting the differential reactivity of CuAAC with SPAAC and
thiol-ene click reactions.36 The usefulness of these approaches
has been demonstrated by the controlled modification of oligo-
nucleotides,37 proteins,38 and fullerenes39 with two or more tags.
The results reported here demonstrate, for the first time, that
strain-promoted click reactions can be performed in a sequential
manner by tuning the reactivity of 1,3-dipoles or by using a latent
1,3-dipole. The attractiveness of the new approach is that it offers
chemical flexibility, avoids toxic metal catalysts, and makes it
possible to multifunctionalize compounds by simple chemical
manipulations.
A variety of methods have been reported for convenient
installment of aldehydes in biomolecules,40 which can easily be
converted into oximes. Thus, it is to be expected that a variety
of biomolecules can be modified by SPANOC. Metal-free
click reactions have found entry into materials science,8 and it
is to be expected that SPANOC will provide an additional tool for
the preparation of increasingly complex materials by simple
and flexible chemical manipulations. Finally, we anticipate that
SPANOC will offer an attractive alternative to the well-estab-
lished oxime ligation41 because the synthesis of oximes is simple,
the isoxazole products are stable, and a combined use with
SPAAC will make it possible to introduce two different func-
tional groups.
equimolar mixture of reagents. Second-order rate constants were deter-
mined by fitting the curves with the following equation:
2
ꢁ
ꢁ ꢁ ꢁ
ꢁ ꢁ
y ¼ ððAo ESMÞ þ ðEP k t ½A0ꢂ ÞÞ=ð1 þ ðk t AoÞÞ
where y is the observed absorbance at given time “t”; A0 is the initial
concentration of the starting materials in molarity; ESM is the sum of
extinction coefficients of starting materials; Ep is the extinction coeffi-
cient of the product; and k is the second-order rate constant in M-1 s-1
.
The obtained second-order rate constants were summarized in Table S15.
1-Benzyl-8,9-dihydro-1H-dibenzocycloocta[1,2,3]triazol-
8-ol (8). Benzyl azide (7) (13 μL, 0.1 mmol) was added dropwise to a
solution of 4-dibenzocyclooctynol (2) (22 mg, 0.1 mmol) in methanol
(5 mL). The reaction mixture was stirred at room temperature for
30 min. The solution was concentrated in vacuo, and the residue was
purified by flash column chromatography on silica gel using a mixture of
hexane and ethyl acetate to give pure triazole 8 (34 mg, 97%). 1H NMR
(300 MHz, CDCl3): δ 2.90-3.65 (m, 2H, CH2CH), 4.55-5.10 (m, 1H,
CHOH), 5.40-5.85 (m, 2H, CH2N), 6.90-7.70 (m, 13H, aromH).
HRMS (MALDI-ToF): 354.1295 (C23H20N3O (M þ Hþ) requires
354.1601).
2-Methyl-3-phenyl-2,3,8,9-tetrahydrodibenzo[3,4:7,8]-
cycloocta-isoxazol-9-ol (10). Phenyl nitrone 9 (14 mg, 0.1 mmol)
was added to a solution of 4-dibenzocyclooctynol (2) (22 mg, 0.1 mmol)
in methanol (5 mL). The reaction mixture was stirred at room tempera-
ture for 30 min. The solution was concentrated in vacuo, and the residue
was purified by flash column chromatography on silica gel using a mixture
of hexane and ethyl acetate to give pure N-methyl dihydroisoxazole 10
(33 mg, 92%). 1H NMR (300 MHz, CDCl3): δ 1.50-1.95 (m, 1H, OH),
2.95-3.65 (m, 5H, CH3, CH2CH), 4.90-5.26 (m, 2H, CHN, CHOH),
6.75-7.65 (m, 13H, aromH). HRMS (MALDI-ToF): 356.1299
(C24H22NO2 (M þ Hþ) requires 356.1645).
N-Benzyl-9-hydroxy-8,9-dihydro-3H-dibenzocycloocta-
pyrazole-3-carboxamide (12). Diazo benzylamide 11 (18 mg,
0.1 mmol) was added to a solution of 4-dibenzocyclooctynol (2)
(22 mg, 0.1 mmol) in methanol (5 mL). The reaction mixture was
stirred at room temperature overnight. The solution was concentrated
in vacuo, and the residue was purified by flash column chromatogra-
phy on silica gel using a mixture of hexane and ethyl acetate to give
pure pyrazole 12 (36 mg, 92%). 1H NMR (300 MHz, CDCl3): δ
2.75-3.60 (m, 2H, CH2CH), 4.00-4.60 (m, 2H, CH2NH), 4.70-5.15
(m, 1H, CHOH), 6.65-7.90 (m, 15H, aromH, CHN, NH). HRMS
(MALDI-ToF): 396.1426 (C25H22N3O2 (M þ Hþ) requires 396.1707).
General Procedure for the Formation of Dibenzocyclooc-
tyl-isoxazoles 6a-f from Imidoyl Chlorides 5a-f. Imidoyl
chloride 5a-f (0.11 mmol) was added to a solution of 4-dibenzocy-
clooctynol (22 mg, 0.1 mmol) and triethylamine (16 μL, 0.11 mmol) in
methanol (10 mL). The reaction mixture was stirred at room tempera-
ture for 10 min. The solution was concentrated in vacuo, and the residue
was purified by flash column chromatography on silica gel using an
appropriate mixture of hexane and ethyl acetate to give pure dibenzo-
cyclooctyl-isoxazoles 6a-f.
’ EXPERIMENTAL PROCEDURES
Kinetic Measurements. The rate measurements of cycloaddi-
tions of dibenzocyclooctynol 2 with various dipoles were conducted
by using Cary 50 and Cary 100 UV-vis spectrophotometers at 25.0 (
0.1 °C. A calculated amount of 0.1 M solutions of a dipole (5a,b,d-f, 7,
9, 11, 13a-h, 14) required to achieve the desired dipole concentration
(2.5 ꢀ 10-4 to 2.7 ꢀ 10-2 M) was added to a thermally equilibrated
solution of dibenzocyclooctynol 2 (3.0 ꢀ 10-5 to 6.0 ꢀ 10-5 M) in
MeOH. In the case of nitrile oxide derivatives of 5a,b,d-f, the imidoyl
chlorides 5a-f in methanol (6.0 ꢀ 10-4 to 1.5 ꢀ 10-2 M) were treated
with triethylamine and then added to a thermally equilibrated solution
of 2, whereas nitrile oxide derivatives of 13a-h were generated by the
oxidation of oximes 13a-h using [bis(acetoxy)iodo]benzene. Reactions
were monitored by following the decay of the characteristic absorbance
of dibenzocyclooctynol 2 at 305 nm. Observed rate constants of the
cycloaddition reactions at various concentrations of dipoles are sum-
marized in Tables S1-S14.26
In the case of the cycloaddition of dibenzocyclooctynol 2 with the
nitrile oxide 5c, 0.1 M solutions of 2 were required to achieve the desired
concentration of 2 (7.0 ꢀ 10-4 to 1.75 ꢀ 10-3 M), and triethylamine
(concentration of triethylamine in the reaction mixture was 1.2 ꢀ 10-4
M) was added to a thermally equilibrated solution of 5c (6.0 ꢀ 10-5 M)
in methanol. Reaction kinetics of nitrone 9 were monitored by following
the second-order growth of the product at 330 nm decay in the
3-Phenyl-8,9-dihydro-dibenzocyclooctanyl-isoxazol-9-ol
(6a). 1H NMR (300 MHz, CDCl3): δ 2.09 (s, 1H, OH), 2.75-3.25
(m, 2H, CH2), 4.66-5.05 (m, 1H, CHOH), 6.55-7.55 (m, 13H,
aromH). HRMS (MALDI-ToF): 340.1075 (C23H18NO2 (M þ Hþ)
requires 340.1332).
3-(40-Methoxyphenyl)-8,9-dihydro-dibenzocyclooctanyl-
isoxazol-9-ol (6b). 1H NMR (300 MHz, CDCl3): δ 208 (s, 1H,
OH), 3.10-3.50 (m, 2H, CH2), 3.70-3.76 (m, 3H, OMe), 5.00-5.65
(m, 1H, CHOH), 6.75-7.55 (m, 12H, aromH). HRMS (MALDI-
ToF): 370.1027 (C24H20NO3 (M þ Hþ) requires 370.1438).
3-(40-Nitrophenyl)-8,9-dihydro-dibenzocyclooctanyl-iso-
xazol-9-ol (6c). 1H NMR (300 MHz, CDCl3): δ 2.08 (brs, 1H, OH),
3.15-3.75 (m, 2H, CH2), 5.00-5.75 (m, 1H, CHOH), 6.70-7.80
954
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ARTICLE
(m, 10H, aromH), 8.00-8.20 (m, 2H, aromH). HRMS (MALDI-
ToF): 385.0961 (C23H17N2O4 (M þ Hþ) requires 385.1183).
3-(40-Fluorophenyl)-8,9-dihydro-dibenzocyclooctanyl-
isoxazol-9-ol (6d). 1H NMR (300 MHz, CDCl3): δ 1.97 (brs,
1H, OH), 3.15-3.75 (m, 2H, CH2), 5.10-5.70 (m, 1H, CHOH),
6.75-7.55 (m, 12H, aromH). HRMS (MALDI-ToF): 358.1016
(C23H17FNO2 (M þ Hþ) requires 358.1238).
5 ꢀ CHgal, 4 ꢀ CHglu), 5.90-6.00 (m, 1H, ArCHOCO), 7.00-7.48
(m, 8H, aromH). HRMS (MALDI-ToF): 998.2939 (C45H61N5O17SNa
(M þ Naþ) requires 998.3681).
Labeling of Sialic Acid Residues on Glycoproteins. Fetuin
(sialylated) and BSA (nonsialylated) as a control were subjected to
periodate oxidation (1 mM NaIO4) for 5 min at 4 °C. The protein
solution was spin filtered at 14 000g for 15 min to remove excess reagent.
Next, the generated C-7 aldehyde (on sialic acid) was reacted with
3-(40-Chlorophenyl)-8,9-dihydro-dibenzocyclooctanyl-
isoxazol-9-ol (6e). 1H NMR (300 MHz, CDCl3): δ 2.08 (brs, 1H,
OH), 3.15-3.75 (m, 2H, CH2), 5.00-5.70 (m, 1H, CHOH), 6.75-
7.65 (m, 12H, aromH). HRMS (MALDI-ToF): 374.0579 (C23H17-
35ClNO2 (M þ Hþ) requires 374.0942).
HONH2 HCl (100 μM in DPBS, pH 6.7) for 1 h at room temperature.
3
The generated oxime was oxidized by reacting with BAIB for 5 min at
room temperature to produce nitrile oxide. After removal of excess
reagent by centrifugation at 14 000g for 15 min, the nitrile oxide was
reacted with DIBO 15 by a copper-free cycloaddition reaction for 30 min
at room temperature. The samples (25 μg of protein per lane) were
resolved on a 4-20% SDS-PAGE gel (Bio-Rad) and transferred to a
nitrocellulose membrane. Next, the membrane was blocked in blocking
buffer (nonfat dry milk (5%; Bio-Rad) in PBST (PBS containing 0.1%
Tween-20 and 0.1% Triton X-100)) for 2 h at room temperature. The
blocked membrane was then incubated for 1 h at room temperature with
an antibiotin antibody conjugated to horseradish peroxidase (HRP)
(1:100 000; Jackson ImmunoResearch Lab, Inc.) in blocking buffer and
washed with PBST (4 ꢀ 10 min). Final detection of HRP activity was
performed using ECL Plus chemiluminescent substrate (Amersham),
exposure to film (Kodak), and development using a digital X-ray imaging
machine (Kodak). Coomassie Brilliant blue staining was used to confirm
total protein loading.
3-(40-Bromophenyl)-8,9-dihydro-dibenzocyclooctanyl-iso-
xazol-9-ol (6f). 1H NMR (300 MHz, CDCl3): δ 2.07 (brs, 1H, OH),
3.15-3.75 (m, 2H, CH2), 5.00-5.60 (m, 1H, CHOH), 6.70-7.85 (m,
12H, aromH). HRMS (MALDI-ToF): 417.9794 (C23H1779BrNO2
(M þ Hþ) requires 418.0437).
General Procedure for the One-Pot Formation of Diben-
zocyclooctyl-isoxazoles 6a,g,h from the Corresponding Al-
dehydes 13a,g,h. Hydroxylamine hydrochloride (10.4 mg, 0.15
mmol) was added to a solution of aldehyde 13a,g,h (1.0 mmol) and
sodium hydroxide (6 mg, 0.15 mmol) in methanol (5 mL). The reaction
mixture was stirred at room temperature for 2 h (monitored by TLC).
[Bis(acetoxy)iodo]benzene (BAIB) (64 mg, 0.20 mmol) was then
added. and the reaction mixture was stirred for 5 min at room tem-
perature. 4-Dibenzocyclooctynol (22 mg, 0.1 mmol) was then added,
and the reaction mixture was stirred for an additional 10 min at room
temperature. The solution was concentrated in vacuo, and the residue
was purified by flash column chromatography on silica gel using an
appropriate mixture of hexane and ethyl acetate to give pure dibenzo-
cyclooctyl-isoxazole 6a,g,h.
Biotin Quantitation. Incorporation of biotin into the protein was
quantified using the Fluorescence Biotin Quantitation Kit (Thermo
Scientific) according to the manufacturer's protocol. Briefly, the bioti-
nylated protein was dissolved in PBS, and DyLight Reporter (a premix of
fluorescent avidin and 40-hydroxyazobenzene-2-carboxylic acid (HABA))
was added to the biotinylated samples and a range of biocytin standards.
The avidin in this reporter fluoresces when the weakly interacting HABA is
displaced by the biotin. A calibration curve of the biocytin standards was
used for calculations. Theextent of biotinylationisexpressedas mol biotin/
mol protein.
3-(20-Toluyl)-8,9-dihydro-dibenzocyclooctanyl-isoxazol-9-ol
(6g). 1H NMR (300 MHz, CDCl3): δ 1.88-2.20 (m, 4H, CH3, OH),
3.20-3.65 (m, 2H, CH2), 5.12-5.48 (m, 1H, CHOH), 6.60-7.60 (m,
12H, aromH). HRMS (MALDI-ToF): 354.1031 (C24H20NO2 (M þ Hþ)
requires 354.1489).
Triazole 20. Azide 18 (10 mg, 0.03 mmol) was added to a solution
of galactose-DIBO derivative 19 (14.3 mg, 0.03 mmol) in methanol
(2 mL). The reaction mixture was stirred at room temperature for 2 h.
The solution was concentrated in vacuo, and the residue was purified by
flash column chromatography on silica gel using a mixture of 10%
methanol in CH2Cl2 to give pure triazole 20 (23 mg, 93%). 1H NMR
(500 MHz, CD3OD): δ 1.74 (m, 2H, CH2), 2.90-3.28 (m, 4H, 2 ꢀ
CH2), 3.35-4.24 (m, 23H, 8 ꢀ CH2, CHCH2, CH2 gal, 3 ꢀ CHgal),
4.50-4.62 (m, 2H, 2 ꢀ CHgal), 5.85-6.20 (m, 2H, CH2CHO, NH),
6.80-7.70 (m, 12H, aromH), 8.01 (s, 1H, CHdN). HRMS (MALDI-
ToF): 844.3492 (C41H51N5O13Na (M þ Naþ) requires 844.3376).
Isoxazole 21. A methanolic solution (1 mL) of galactose-DIBO
derivative 19 (14.3 mg, 0.03 mmol) was added dropwise to a solution of
oxime 18 (12.2 mg, 0.036 mmol) and BAIB (11.6 mg, 0.036 mmol) in
methanol (1 mL). The reaction mixture was stirred at room temperature
for 10 min. The solution was concentrated in vacuo, and the residue was
purified by flash column chromatography on silica gel using a mixture of
8% methanol in CH2Cl2 to give pure isozaxole 21 (14.6 mg, 61%). 1H
NMR (500 MHz, CD3OD): δ 1.70-1.84 (n, 2H, CH2), 3.30-4.30 (m,
29H, 10 ꢀ CH2, CH2CHOH, CH2gal, 5 ꢀ CHgal), 6.10-6.40 (m, 1H,
CH2CHOH), 6.70-7.70 (m, 13H, aromH, NH). HRMS (MALDI-
ToF): 842.2192 (C41H49N5O13Na (M þ Naþ) requires 842.3219).
General Procedure for SPAAC with Bifunctional Linker
18. Bifunctional linker 18 (0.03 mmol, 10.1 mg) and corresponding
DIBO derivative 15 or 22 (0.03 mmol) were dissolved in MeOH or
THF (in case of coumarin-DIBO derivative 22) (2 mL). The reaction
mixture was stirred for 3 h, and the solution was concentrated in vacuo.
The residue was purified by column chromatography on silica gel.
3-(2-Phenylethyl)-8,9-dihydrodibenzocyclooctanyl-isoxazol-
9-ol (6h). 1H NMR (300 MHz, CDCl3): δ 1.50-1.90 (m, 1H, OH),
2.65-3.65 (m, 6H, 3 ꢀ CH2), 4.90-5.10 (m, 1H, CHOH), 6.90-7.70 (m,
13H, aromH). HRMS (MALDI-ToF): 368.1210 (C25H22NO2 (M þ Hþ)
requires 368.1645).
General Procedure for the Formation of Lactose Deriva-
tives. 4-Dibenzocyclooctyl-derivative 2 or 15 (0.1 mmol) was added
to a solution of [bis(acetoxy)iodo]benzene (35 mg, 0.11 mmol) and
lactose oxime 14 (40 mg, 0.11 mmol) in methanol (4 mL), premixed for
1 min. The reaction mixture was then stirred at room temperature for 10
min (TLC monitoring). The solution was concentrated in vacuo, and
the residue was purified either by Iatrobeads using a mixture of 10% of
water in acetonitrile (for 16) or by RP-HPLC (0-2 min 0.1% TFA/
H2O, v/v; 2-5 min gradient of 0-20% 0.1% TFA/CH3CN, v/v; 5-
30 min gradient of 20-60% 0.1% TFA/CH3CN v/v; 30-35 min
gradient of 60-100% 0.1% TFA/CH3CN, v/v; 35-45 min gradient
of 100-0% 0.1% TFA/CH3CN, v/v; t = 21.8 and 23.9 min). Appro-
priate fractions were combined and lyophilized to give pure dibenzocy-
clooctyl-isoxazole 16 and 17, respectively.
3-(Lactose)-8,9-dihydro-dibenzocyclooctanyl-isoxazol-9-ol
(16). 1HNMR(600 MHz, CDCl3):δ3.00-3.85 (m, 11H, 2 ꢀ CH2OH,
4 ꢀ CHgal, 3 ꢀ CHglu), 3.95-4.30 (m, 2H, CH2Ar), 4.35-5.45 (m, 3H,
ArCHOH, OCHO, CHCdN), 7.20-7.80 (m, 8H, aromH). HRMS
(MALDI-ToF): 598.1693 (C28H33NO12Na (M þ Naþ) requires
598.1895).
Lactose-biotin Isoxazole 17. 1H NMR (600 MHz, D2O): δ
1.00-1.45 (m, 6H, 3 ꢀ CH2biotin), 2.00-2.10 (m, 2H, CH2biotin), 2.40-
4.30 (m, 32H, 6 ꢀ CH2PEG, CH2biotin, 3 ꢀ CHbiotin, CH2Ar, 2 ꢀ CH2OH,
955
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ARTICLE
Triazole 23. Purification by silica gel column chromatography
(5 then 10% MeOH in CH2Cl2) gave 23 as a colorless oil (25.1 mg,
87%). 1H NMR (300 MHz, CD3OD): δ 1.34-1.45 (m, 2H, CHCH2-
CH2), 1.52-1.76 (m, 4H, CHCH2CH2CH2), 2.15-2.21 (m, 2H,
CH2CdO), 2.64-2.69 (m, 1H, CHHS), 2.85-3.74 (m, 26H, CHHS,
9 ꢀ CH2O, 2 ꢀ CH2NH, CH2CHO, CHS), 3.83-4.06 (m, 4H, 2 ꢀ
CH2O), 4.21-4.28 (m, 1H, CHNH), 4.41-4.47 (m, 1H, CHNH),
4.55-4.61 (m, 2H, CH2-triazole), 5.89-6.17 (m, 1H, CH2CHO),
6.83-6.88 (m, 2H, aromH), 7.15-7.65 (m, 10H, aromH), 8.01 (s, 1H,
CHdN). MS (MALDI-ToF): 981.4092 (C46H62N8O11SNa (M þ
Naþ) requires 981.4157).
Triazole 24. Purification by silica gel column chromatography (3%
MeOH in CH2Cl2) gave 24 as a yellow amorphous solid (22 mg, 73%).
1H NMR (300 MHz, CDCl3): δ 1.80-2.02 (m, 4H, 2 ꢀ NCH2-
CH2CH2), 2.66-2.86 (m, 4H, 2 ꢀ NCH2CH2CH2), 3.01-4.12 (m,
32H, 2 ꢀ NCH2CH2CH2, 11 ꢀ CH2O, 2 ꢀ CH2NH, CH2CHO),
4.37-4.61 (m, 2H, CH2-triazole), 5.34-6.49 (m, 2H, CH2CHO,
NH), 6.73-6.82 (m, 2H, aromH), 6.93-7.60 (m, 11H, aromH), 7.92-
8.10 (m, 1H, NH), 8.56-8.68 (m, 1H, CHdN), 9.01-9.25 (m, 1H,
CH-vinyl). MS (MALDI-ToF): 1022.4133 (C54H61N7O12Na (M þ
Naþ) requires 1022.4270).
kinetic studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
gjboons@ccrc.uga.edu
’ ACKNOWLEDGMENT
This research was supported by the National Cancer Institute
of the U.S. National Institutes of Health (R01 CA88986, G.-J.B.),
the National Science Foundation Plant Genome Program (IOS-
0923992, G.-J.B.), the National Science Foundation (CHE-
0449478, V.V.P.), and the Georgia Cancer Coalition (V.V.P.).
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General Procedure for SPANOC between Triazoles 23 or
24 and Glycodendrimer 25. To a stirred solution of DIBO-
glycodendrimer 25 (20.5 mg, 5.2 μmol) and oxime 23 or 24 (5.2 μmol)
in MeOH/CH2Cl2 (4/1, v/v, 1.2 mL) was added a solution of BAIB (1.8
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1
55%). H NMR (500 MHz, D2O): δ 0.88-1.22 (m, 23H, 7 ꢀ CH3,
CHCH2CH2), 1.32-1.63 (m, 4H, CHCH2CH2CH2), 1.95-2.22 (m,
18H, 8 ꢀ CH2CH2CH2-triazole, CH2CdO), 2.48-2.80 (m, 17H,
CHHS, 8 ꢀ CH2CH2-triazole), 2.80-3.02 (m, 17H, CHHS, 8 ꢀ
CH2CH2-triazole), 3.08-3.95 (m, 107H, 2ꢀCH2CHO, 4 ꢀ CH2NH,
15 ꢀ CH2O, 8 ꢀ CH-2gal, 8 ꢀ CH-3gal, 8 ꢀ CH-5gal, 8 ꢀ CH2-6gal, 8 ꢀ
CH-4gal, 8 ꢀ CH2CH2CH2-triazole, CHS), 3.99-4.55 (m, 56H, 9 ꢀ
CH2-triazole, 14 ꢀ OCH2, 2 ꢀ CHNH, 8 ꢀ CH-1gal), 5.55-6.15 (m,
2H, 2 ꢀ CH2CHO), 6.33-7.60 (m, 20H, aromH), 7.87 (s, 8H, 8 ꢀ
CHtriazole). MS (MALDI-ToF): 4933.4 (C218H310N34O92SNa (M þ
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2 ꢀ NCH2CH2CH2), 1.99-2.08 (m, 16H, 8 ꢀ CH2CH2CH2-triazole),
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis of oximes 14a-f,
b
imidoyl chlorides 5a-f, azido-oxime linker 18, DIBO-galactose
19, amino-PEG-coumarin 33, and DIBO-glycodendron 25.
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