Investigation of Strain-Promoted Azide-Alkyne Cycloadditions in Aqueous Solutions by Capillary Electrophoresis

Article abstract of DOI:10.1021/acs.joc.7b02092

The Cu-free 1,3-dipolar cycloaddition of cyclooctynes and azides is an up-and-coming method in bioorganic chemistry and other disciplines. However, broad application is still hampered by major drawbacks such as poor solubility of the reactants in aqueous media and low reaction rates. It is thus of high demand to devise a fast and user-friendly strategy for the optimization of reaction conditions and reagent design. We describe a capillary electrophoresis (CE) study of reaction kinetics in strain-promoted azide-alkyne cycloadditions (SPAAC) using substrates with acidic or basic functionalities. This study reveals that the pH value has a significant effect on reaction rates as a result of changes in the reactants' charge state via protonation or deprotonation, and the concomitant changes of electronic properties. This novel experimental setup also enables the study of even more challenging conditions such as reactions in micelles and we did indeed observe much faster SPAAC reactions in the presence of surfactants. Careful combination of the above-mentioned parameters resulted in the identification of conditions enabling remarkable rate enhancement by a factor of 80. This electrophoretic method may thus serve as a versatile, fast and reliable tool for screening purposes in all research areas applying SPAAC reactions.

Full text of DOI:10.1021/acs.joc.7b02092

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Article
Investigation of Strain-Promoted Azide-Alkyne Cycloadditions
in Aqueous Solutions by Capillary Electrophoresis
Jana Steflova, Golo Storch, Sarah Wiesner, Skrollan Stockinger, Regina Berg, and Oliver Trapp
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02092 • Publication Date (Web): 26 Dec 2017
Downloaded from http://pubs.acs.org on December 29, 2017
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Investigation of Strain-Promoted Azide-Alkyne
Cycloadditions in Aqueous Solutions by Capillary
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Electrophoresis
Jana Steflova,*,†,‡ Golo Storch,† Sarah Wiesner,† Skrollan Stockinger,† Regina Berg† and Oliver Trapp*,†
†Department Chemie, LudwigꢀMaximiliansꢀUniversität München,
Butenandtstr. 5–13, 81377 München, Germany
oliver.trapp@cup.uniꢀmuenchen.de
‡Charles University, Faculty of Science, Department of Physical and Macromolecular Chemistry, Hlavova
8, 128 40 Prague 2, Czech Republic
svobod.j@natur.cuni.cz
Abstract
The Cuꢀfree 1,3ꢀdipolar cycloaddition of cyclooctynes and azides is an upꢀandꢀcoming method in
bioorganic chemistry and other disciplines. However, broad application is still hampered by
major drawbacks such as poor solubility of the reactants in aqueous media and low reaction rates.
It is thus of high demand to devise a fast and userꢀfriendly strategy for the optimization of
reaction conditions and reagent design. We describe a capillary electrophoresis (CE) study of
reaction kinetics in strainꢀpromoted azideꢀalkyne cycloadditions (SPAAC) using substrates with
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acidic or basic functionalities. This study reveals that the pH value has a significant effect on
reaction rates as a result of changes in the reactants’ charge state via protonation or
deprotonation, and the concomitant changes of electronic properties.
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This novel experimental setup also enables the study of even more challenging conditions like
reactions in micelles and we did indeed observe much faster SPAAC reactions in the presence of
surfactants. Careful combination of the aboveꢀmentioned parameters resulted in the identification
of conditions enabling remarkable rate enhancement by a factor of 80. This electrophoretic
method may thus serve as a versatile, fast and reliable tool for screening purposes in all research
areas applying SPAAC reactions.
Introduction
Click chemistry is a concept that describes highꢀyielding and extremely chemoꢀ, regioꢀ and
stereoselective chemical transformations that do neither require the exclusion of water and air nor
chromatographic purification procedures.1 Despite this rather broad definition, the term Click
reaction is often used synonymously for the Cu(I)ꢀcatalyzed Huisgen azideꢀalkyne cycloaddition
(CuAAC).2,3 1,3ꢀDipolar cycloadditions of azides and alkynes resulting in the formation of 1,2,3ꢀ
triazoles had been developed by Huisgen in the 1960s.4ꢀ8 However, in the absence of any catalyst
this reaction only proceeds under harsh conditions with low reaction rates giving a mixture of
1,4ꢀ and 1,5ꢀdisubstituted 1,2,3ꢀtriazole regioisomers. In 2002, the group of Meldal presented a
copper(I)ꢀcatalyzed solidꢀphase synthesis of 1,2,3ꢀtriazoles.9 At the same time, the group of
Sharpless independently reported on a copper(I)ꢀcatalyzed azideꢀalkyne cycloaddition under
solution phase conditions, most interestingly also in aqueous media.2 Based on the development
of more and more advanced ligand systems (NHC, iminophosphoranes, tris(triazolyl)methanol),
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there have also been several examples of CuAAC reactions being performed in water and under
aerobic conditions.10ꢀ12 Experimental and DFTꢀbased mechanistic investigations have shown the
copper(I)ꢀcatalyzed variant of the Huisgen cycloaddition to proceed by a stepwise mechanism
with activation of the alkyne by coordination to two copper(I) ions.13ꢀ17 Since 2002, the CuAAC
reaction has rapidly found a plethora of applications in chemistry and related disciplines, among
them many in biological sciences.18ꢀ24 In particular, the CuAAC reaction is widely used for
coupling a biomolecule to another (bio)molecule via a covalent molecular linker
(bioconjugation), as its characteristics and reaction conditions are compatible with some of the
requirements and conditions in this field (bioorthogonality),25ꢀ27 i.e. tolerance towards air and
aqueous media of physiological pH, ambient temperature, biological and chemical inertness of
the starting materials and the cycloadduct, rapid and full conversion, and high specificity.
However, the reaction’s major limitation in biological contexts lies in the use of a transition metal
catalyst, as Cu(I) ions have been shown to be toxic for isolated cells as well as for living
organisms due to oxidative damage.28,29 For this reason there is a strong interest in developing
suitable alternatives for CuAAC compatible with this additional requirement of bioorganic
applications (nonꢀtoxicity).
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In 2004, the group of Bertozzi developed a StrainꢀPromoted [3+2] AzideꢀAlkyne Cycloaddition
(SPAAC) applying cyclooctyne dipolarophiles in order to increase the rate of reaction by taking
advantage of the lower activation energy as a result of the strained substrate geometry in
connection with an early – and thus substrateꢀlike – transition state (according to the Hammond
postulate).27,30,31 This idea has its foundations in reports by Blomquist and Liu (1953) and by
Wittig and Krebs (1961), both describing the explosionꢀlike 1,3ꢀcycloaddition of cyclooctyne
with phenyl azide.32,33 The high reactivity of cyclooctynes in comparison to acyclic or unstrained
cyclic alkynes is explained by destabilization of the ground state due to substantial bond angle
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distortion, which leads to a lower activation energy for the cycloaddition reaction.34ꢀ36 The
SPAAC has been accelerated by introduction of electronꢀwithdrawing fluoride substituents
(monoꢀ and difluorinated cyclooctyne substrates, MOFOs and DIFOs).37ꢀ39 Substantial rate
enhancement in SPAAC reactions was also achieved by using dibenzocyclooctyne (DIBO or
DBCO),40 difluorobenzocyclooctyne (DIFBO),41 azadibenzocyclooctyne (DIBAC)42 or
biarylazacyclooctynone (BARAC) substrates.43,44
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However, there is still demand for improvement of the SPAAC, as even with stateꢀofꢀtheꢀart
cyclooctynes these reactions proceed slower than common CuAAC reactions. While we were
working on this project, the groups of Huber45 and Heemstra46 independently presented strategies
to increase the rate of SPAAC reactions by adding surfactants for the formation of micelles as
confined compartments for the reaction to take place at high concentration. This strategy has
recently been greatly advanced by the group of Lipshutz by applying novel designer surfactants
for the acceleration of diverse transformations in water, such as Heck reactions, metalꢀcatalyzed
cross couplings, olefin metathesis and 1,4ꢀaddition of cuprates to enones.47ꢀ49 Huber and
coworkers had noted that azide residues of unnatural amino acids pointing into a hydrophobic
pocket of a protein were more readily labeled by Alexa488ꢀDIBO than those exposed to the
aqueous environment. The authors thus deduced that the high hydrophobicity of the cyclooctyne
substrates prevents them to react efficiently in SPAAC reactions in aqueous media and that the
local environment at the site of reaction within the protein has a major effect on the rate of the
SPAAC. By solubilizing the GPC receptors in their study with detergent (nꢀdodecyl
βꢀDꢀ
maltoside), they observed a rate enhancement of up to a factor of 1000 compared to SPAAC
reactions with azide groups on waterꢀexposed surfaces. Heemstra and coworkers used the
strongly hydrophobic DIBAC derivative to investigate its SPAAC reaction with a a) hydrophobic
and b) hydrophilic (PEGylated) azide employing i) neutral (Tween, Triton Xꢀ100), ii) cationic
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(DTAB, CTAB) and iii) anionic (SDS) surfactant additives using absorbance UV spectroscopy
for the determination of reaction rate constants. In conclusion, they found that rate enhancement
with the hydrophobic azide is much more pronounced and especially strong with cationic
surfactants (by a factor of 179). Recently, the Heemstra group has also systematically
investigated the effect of buffer identity (NaPi, Tris, TBE, MES, MOPS, HEPES, acetate), ionic
strength (by addition of NaCl) and organic cosolvents (DMSO, MeCN, NMP, MeOH, EtOH)
showing that moderate quantities of organic cosolvent lead to rate enhancement, whereas choice
of buffer and ionic strength has no substantial effect on the rate of reaction, as long as conditions
remain neutral.50 Our goal was now to investigate SPAAC kinetics under a variety of reaction
conditions and buffer additives with special emphasis on pH. Such screening requires selection of
a suitable analytical method, which would allow for continuous monitoring of reaction progress
in combination with a wide range of conditions. This method needs to be applicable with
biological samples as well. Capillary zone electrophoresis does ideally fulfill these requirements.
It is a broadly applicable analytic method allowing for efficient separation of analytes with a wide
variety of compound classes ranging from small inorganic salts to complex biological samples.
Quantitative determination of physical chemical parameters of various compounds as well as
investigation of reaction kinetics by capillary electrophoresis has been shown to proceed with
high reliability.51ꢀ58 This method does also offer deep insights into the separation process, as
electromigration is very well described with a solid theoretical foundation. The complete
mathematical model was implemented in several simulation programs, thus enabling optimization
of separation conditions and detailed planning of experiments.59ꢀ62 Another advantage is the
method’s versatility, as separation/reaction conditions are widely variable.
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In this study, we use capillary electrophoresis as the method of choice for monitoring the
progress of SPAAC reactions under various reaction conditions to optimize the reaction by
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significant reaction rate enhancement. In a series of test reactions, cycloadditions of
dibenzocyclooctyneꢀamine (3ꢀaminoꢀ1ꢀ(11,12ꢀdidehydrodibenzo[b,f]azocinꢀ5(6H)ꢀyl)propanꢀ1ꢀ
one, DBCOꢀamine) with several azides were investigated. Systematically screening the reaction
medium’s pH, we observed clear trends regarding the dependence of the reaction rate on the
charge state of the various azide substrates with acidic/basic substituents as well as on the charge
state of DBCOꢀamine. Plausible structural scenarios were identified by DFT calculations. Next,
the knowledge gained by our study varying the pH of the reaction medium was applied in
reactions with surfactant additives. Due to the large rate enhancement, we propose the addition of
surfactants to achieve efficient and fast SPAAC reactions in aqueous solution.
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Results and Discussion
Capillary electrophoresis was utilized for precise and fast determination of reaction kinetics. This
novel electrophoretic method allows for continuous monitoring of the reaction mixture’s
composition. A suitable separation buffer was selected in order to provide baselineꢀseparated
symmetrical peaks of reactants and products, which do not coincide with any system peaks. The
buffer composition was optimized under the premise that all reactants were soluble within a wide
range of concentrations. PeakMaster 5.3 was utilized for optimization of separation
conditions.61,62 After careful optimization of the buffer composition for the different reaction
mixtures, 7 mM βꢀalanine/10 mM LiOH buffer with 20 mM SDS (pH 11.45) and 28 vol% of
acetonitrile was chosen for reactions with 4ꢀazidophenol (PhOHꢀN3), 4ꢀazidobenzene sulfonic
acid (PhSO3HꢀN3), azidobenzoic acid (PhCOOHꢀN3), or (S)ꢀ2ꢀazidoꢀ3ꢀphenylpropionic acid
(PheꢀN3) as substrate, and a buffer composition of 15 mM citric acid/10 mM LiOH buffer with 15
mM SDS (pH 3.34) was used in reactions with azidobenzene (PhꢀN3). With these buffers all
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reactants were soluble up to a concentration of 10 mM. The cycloaddition products of DBCO
with a variety of azides have already been investigated and resulting triazoles have been
characterized.63,64
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Our CE method was optimized for fast and effective separation of reactants and products
covering a wide range of reaction rates (10ꢀ3–102 dm3molꢀ1sꢀ1). Advantageously, neither internal
standard nor calibration was necessary for data evaluation, as the ratio of the peak areas (and not
the absolute values) were needed for kinetic evaluation (see Supporting Information, Section 1,
equation 4). The CE method employed was tested by using simulation tool Simul 5 Complexꢀ
Kinetic,59,60 which allowed us to computationally check the applicability of our method over a
wide range of reaction rates (Supporting Information, Section S1). An illustrative sequence of
electropherograms for one reaction and the resulting evaluation of the observed peak area ratios
is depicted in Figure 1.
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Figure 1 A) Reaction scheme for SPAAC of PheꢀN3 with DBCOꢀamine. B) Electropherograms
of the reaction mixture of PheꢀN3 and DBCOꢀamine at different reaction times with 20 mM SDS
(for pH 6.59 at the start of the reaction). C) Evaluation of the electropherograms, plot of
APheꢀN3/ADBCOꢀamine over time, black solid line: exponential fit. For residual plot of the exponential
fit see Figure SIꢀ3.
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We focused mainly on the selection of a suitable reaction buffer regarding pH and choice of
additives, as changing reaction conditions is the most efficient and timeꢀsaving way of optimizing
reaction rates, for example in comparison to structural modifications on the substrate
cyclooctynes. The reaction was optimized for commercially available DBCOꢀamine. The azides
were chosen in a way to include functional groups with electron donating and withdrawing
effects featuring acidic and basic functionalities that can be protonated/deprotonated depending
on the pH of the reaction mixture. The substrates are shown in Figure 2b.
First, we put our focus on the influence of buffer pH on the rate of SPAAC reaction. The pKa
values of individual reactants were determined by capillary zone electrophoresis (for details see
Supporting Information, Section S3), the resulting values are depicted in Figure 2b (azides) and
2c (DBCOꢀamine). Figure 2a shows the plot of the reactants’ effective charge (ordinate) in
ꢀ
dependence on the reaction mixture’s pH (abscissa) (PhSO3 ꢀN3 is fully negatively charged and
PhꢀN3 is neutral along the complete pH range). Based on these data, we were able to select
suitable buffers covering the pH regions of various charge states of individual reactants (pH 2.29,
3.77, 7.09, 11.55). The exact composition of buffers is given in the Supporting Information
(Table SIꢀ2).
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Figure 2 (a) Effective charge (|z|) of the reactants in dependence on the buffer’s pH; black solid
line: DBCOꢀamine; black dashed line: PhOHꢀN3; red dotted line: PhCOOHꢀN3; blue dotꢀdashed
line: PheꢀN3; the vertical lines represent individual buffer pH values, the composition of the
corresponding buffer is given above each line. (b) Rate constants obtained for different azide
substrates at various reaction buffer pH values, experimentally determined pKa values for
individual molecules are given in blue next to the respective acidic or basic functional group. (c)
Rate constant k (left axis) of the SPAAC in dependence on the pH of the reaction buffer; black
ꢀ
squares: PheꢀN3; red dots: PhSO3 ꢀN3; dashed black line: effective charge (|z|) of DBCOꢀamine
(right axis). pKa of DBCOꢀamine is depicted as a vertical line.
ꢀ
In the case of neutral PhꢀN3 and negatively charged sulfonate PhSO3 ꢀN3, the reaction was
performed at pH 11.55 and pH 3.77, where the DBCOꢀamine is neutral and positively charged,
respectively. The same conditions also applied for the reaction of PhOHꢀN3 as the pKa values of
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PhOHꢀN3 and DBCOꢀamine are very close (10.40 and 9.07, respectively). In the case of
PhCOOHꢀN3 and PheꢀN3, measurements were additionally performed at pH 7.09, where both
DBCOꢀamine and the azides’ carboxylic acid groups in the side chain are charged.
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As PheꢀN3 is a moderately strong acid (pKa 3.54), it is mainly dissociated (α = 61 %) in the citric
buffer solution (pH 3.77). For this reason, the rate of the SPAAC reaction with PheꢀN3 as
substrate was also measured in maleic acid buffer at pH 2.29, where the degree of dissociation is
about 5 % (Figure 2a).
The determined reaction rates are summarized in Figure 2b. The highest reaction rates were
observed for PhOHꢀN3, which can be explained by the highly electron donating hydroxyl group.
This effect is even more pronounced for the deprotonated azidophenol (PhOꢀꢀN3). With
increasing pH value and the resulting deprotonation to the respective phenolate, the reaction rate
of the SPAAC with DBCOꢀamine increases by a factor of 3.4. In comparison, the reaction rates
ꢀ
of PhCOOHꢀN3 and PhSO3 ꢀN3 are significantly lower, which correlates well with the electron
withdrawing effect of the substituent. The unsubstituted azidobenzene (PhꢀN3) is highly volatile
and its concentration decreases in the course of the reaction. Figure SIꢀ4 shows the dependence of
the relative area of the PhꢀN3 peak on time, which proves that the concentration of azide
decreases in the same extent independently on the composition of the buffer used. No effect of
pH on the reaction rate of PhꢀN3 was observed.
Obviously, the reaction rates observed for SPAAC reactions with PheꢀN3 increase with pH in
acidic pH regions, which can be explained by deprotonation of the carboxyl group as the medium
becomes more alkaline. In contrast to COOH, the carboxylate COOꢀ is characterized by a much
attenuated electronꢀwithdrawing effect. Surprisingly, the reaction rate is lower at basic
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conditions. The same trend is observed for all azides (except for PhOHꢀN3, where the effect of
OH deprotonation to the phenolate PhOꢀꢀN3 predominates). Figure 2c shows in detail the reaction
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rate’s dependence on pH for these azide substrates. Clearly, for both PheꢀN3 and PhSO3 ꢀN3
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substrates, we observe the same drop in SPAAC reaction rate in the pH range 8–10. This is
ꢀ
highly surprising as the charge state of neither PhSO3 ꢀN3 nor PheꢀN3 changes in this pH region.
This leads to the conclusion that deprotonation of the DBCOꢀammonium ion in this pH regime
has significant impact on the SPAAC reaction rate, independent on the structural characteristics
of the azide reaction partner, which is further supported by the fact that the inflection point of the
resulting curve corresponds very well with the pKa of DBCOꢀamine (Figure 2c). This result was
surprising because of the large distance of the amino group as the site of protonation from the
reactive triple bond of DBCO.
Thermodynamic parameters of the SPAAC reaction of PheꢀN3 and DBCOꢀamine were
determined experimentally in order to characterize the influence of the effective charge of
DBCOꢀamine on the rate of reaction. The rate constants were determined at various temperatures
(15, 20, 25, 30 and 40°C) in acidic and basic media. The corresponding Eyring plots and
thermodynamic parameters are shown in Figure 3. The Gibbs energy was calculated at 20 °C,
which gave values of Δꢀꢁ‡ = 71.5 ± 0.1 kJ molꢀ1 and Δꢀꢂ‡ = 70.0 ± 0.4 kJ molꢀ1 for neutral DBCOꢀ
amine and DBCOꢀammonium ion, respectively. Thus, the difference in activation energies of
SPAAC reactions with (unprotonated) DBCOꢀamine and (protonated) DBCOꢀammonium ion is
1.6 ±0.5 kJ molꢀ1 with the ammonium ion featuring lower activation energy and higher rate of
reaction.
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Figure 3 Eyring analysis of the experimental rate constants of the SPAAC reaction with Phe-N3
as determined by CE offline reaction. Solid squares: neutral DBCOꢀamine, transparent circles:
protonated DBCOꢀamine (= DBCO ammonium ion). Errors are expressed as standard deviation;
gray lines show the confidence interval on the 95 % level.
Inspired by the pioneering work of Bertozzi and Houk,34,35,44 we subsequently investigated the
SPAAC reaction by DFT calculations65,66 with the aim of identifying different plausible reaction
pathways that would rationalize the rate constant’s pH dependence. We were aware of the small
energy differences of the corresponding transition states and, therefore, we did not attempt to
pinpoint one single favored mechanism, but rather screen possible trajectories thoroughly. All
calculations were performed with Gaussian09 Rev.ꢀD.0167 on a B3LYP68ꢀ70/ccꢀpVDZ71ꢀ73 level of
theory including the D3 version of Grimme’s dispersion correction.74 Augmented basis sets
(augꢀccꢀpVDZ) were used for all atoms involved in bond formation and hydrogen bonding
interactions and a CPCM solvent model (acetonitrile unless otherwise specified) was applied.75,76
The focus of our theoretical investigations was the reaction of DBCOꢀamine with PheꢀN3 in
acetonitrile. We initially performed a detailed geometry analysis of all possible conformers of
DBCOꢀamine that arise due to the flexible βꢀalanine amide side chain in order to determine the
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ground states of the reaction participants. Both in the protonated and in the nonꢀprotonated case,
the amide bond Eꢀconfiguration was found to be energetically favored. In the protonated state,
there was a strong preference for conformers that allowed intramolecular hydrogen bonding
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interactions between the NH3 group and the amide carbonyl (see Supporting Information,
Section 4 for details).
We investigated the transition states considering protonated as well as nonꢀprotonated forms of
the βꢀalanine side chain as well as the formation of both triazole regioisomers (DBCOꢀamineꢀ11
and DBCOꢀamineꢀ12). We realized that the approach of the phenyl azide (PhꢀN3) might either be
of syn or anti orientation with respect to the βꢀalanine group. In the nonꢀprotonated state, both syn
approaches also featured weak hydrogen bonding interactions of the NH2 group and two azide
nitrogen atoms. Additionally, we calculated one trajectory of an anti approach. The energy levels
ꢁ{E+ZPE} and ꢁG‡(298 K) of the three transition states were found to be quite similar
(Figure 4a). We also identified three syn and one anti transition state for the protonated βꢀalanine
side chain, some of them featuring hydrogen bonding interactions with the approaching azide.
These transition states were found to be energetically comparable to those of the nonꢀprotonated
analogues (Figure 4b).
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Figure 4 DFT study of the reaction of strained DBCOꢀamine with PhꢀN3. The transition state
structures of the nonꢀprotonated (a) and protonated βꢀalanine side chain (b) are depicted together
with the respective energy levels. Energy levels given in parentheses are results from calculations
applying the CPCM solvent model for water (instead of MeCN). DFT calculations were
performed at the B3LYP/ccꢀpVDZ level of theory including the D3 version of Grimme’s
dispersion correction. Augmented basis sets (augꢀccꢀpVDZ) were used for all atoms involved in
bond formation and hydrogen bonding interactions and a CPCM solvent model was applied.
Detailed inspection of the transition state structures revealed that the protonation state of the βꢀ
alanine side chain might indeed affect the transition state energy. This might either be due to nonꢀ
covalent interactions with the incoming azide molecule or due to subtle changes in electronic
properties, which both can easily cause changes in the range of 1–2 kJ molꢀ1, which would match
the differences in the experimentally observed rate constants of protonated and unprotonated
DBCOꢀamine in SPAAC reactions.
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In the next step, we extended our study to the SPAAC reaction with methyl azide in order to
identify potential differences when an aliphatic azide takes part in the reaction. We identified two
transition states (protonated and nonꢀprotonated βꢀalanine side chain) for the formation of each of
the two possible regioisomers (Figure SIꢀ6). Interestingly, the resulting transition state energies
of ꢁ{E+ZPE} = 26.0–28.5 kJ molꢀ1 and ꢁG‡(298 K) = 80.7–81.7 kJ molꢀ1 are very similar to
those with phenyl azide (PhꢀN3) as reactant.
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All calculated transition state Gibbs energies seem to be systematically overestimated when
compared to the experimental results. This is in agreement with the studies by Bertozzi and Houk
et al., who reported the B3LYP functional to result in overestimations of about 27 kJ molꢀ1 for
calculations of SPAAC reactions.44,77
Summarizing this part of the study, we have determined the rate constants of SPAAC reactions at
various pH values of different MeCN/water reaction media by continuous reaction monitoring
with capillary electrophoresis. We have shown that the composition of the buffered reaction
medium can significantly influence the reaction rate of the SPAAC reaction. Our study points to
the necessity to always optimize SPAAC reaction conditions for a specific set of reactants with
regard to the choice of buffer and pH, and introduces a versatile and powerful method to do so
(e.g. in comparison to the much more tedious tuning of the reactants’ structural characteristics). A
strong influence on the rate of the SPAAC reaction was observed for different protonation states
of the functionalized acidic or basic azides as well as for our test cycloalkyne substrate DBCOꢀ
amine. DFT calculations illustrate the manifold transition state geometries similar in energy that
can be identified depending on the substrate’s protonation state, and support our experimental
findings.
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Sodium dodecyl sulfate (SDS) micelles can have an advantageous effect on the rate of reactions
featuring highly hydrophobic compounds in polar reaction media, as has recently been reported
for SPAAC reactions by the groups of Heemstra and Huber45,46. This effect is based on the
incorporation of the hydrophobic reactants into the SDS micelles, which results in an increase in
the reactants’ local concentration and thus an enhancement of the rate of reaction.
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At first, the interaction of all reactants with SDS micelles was characterized by capillary zone
electrophoresis (CZE; Figure SIꢀ7). Recently, we showed that SDS micelles can even be formed
in media containing high concentrations of MeCN, with the critical micelle concentration (CMC)
being only slightly shifted to higher concentrations of SDS.78 To characterize the micellization in
our particular solvent mixture (28 vol% MeCN in water), we determined the CMC of SDS by
fluorescence correlation spectroscopy (FCS) as well as mobility measurements (see Figure SIꢀ8A
and SIꢀ8B for details). The resulting value of CMC in the water/MeCN mixture (28 vol% MeCN)
is 13 mM. Simultaneously, the mobility of all reactants was measured in dependence on the SDS
concentration (Figure 5 and SIꢀ7). Based on these data two concentrations above the CMC, 20
mM and 50 mM SDS, were chosen for investigations of the SPAAC reaction kinetics.
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Figure 5 Effective mobility of the reactants in dependence on the concentration of SDS in the
background electrolyte (BGE). a) DBCOꢀamine, b) PheꢀN3. Solid squares pH 11.55 (MeCN/H2O,
pH at SDS 0 mM), transparent squares pH 11.49 (H2O, pH at SDS 0 mM); solid circles pH 3.77
(MeCN/H2O, pH at SDS 0 mM), transparent circles 3.37 (H2O, pH SDS 0 mM), solid triangles
pH 2.29 (MeCN/H2O, SDS 0 mM), transparent triangles pH 2.39 (H2O, pH SDS 0 mM),
transparent symbols: measurements in aqueous buffers, solid symbols: in H2O/MeCN buffer. For
pH values at different SDS concentration see Table SIꢀ1. Errors are expressed as standard
deviations.
Figure 6 (a) Rate constants of SPAAC reactions with various azides and DBCOꢀamine in
dependence on pH and concentration of SDS. (b) Rate constant of reaction of PheꢀN3 with
DBCOꢀamine depending on concentrations of SDS, pH and concentration of NaCl. Buffer
solutions containing 28 vol% MeCN labeled MeCN/H2O and aqueous buffers labeled H2O.
Errors are given as standard deviations. For pH values at different SDS concentration see Table
SIꢀ1.
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Based on these findings, the reaction kinetics was investigated at 0 mM, 20 mM and 50 mM
SDS. The reaction rate of PhꢀN3 increases with addition of SDS independently on the pH value of
the reaction medium. We attribute this effect to the strong interaction of both PhꢀN3 and DBCOꢀ
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amine with SDS micelles. On the other hand, PhSO3 ꢀN3 is very hydrophilic, which disfavors its
incorporation in micelles, which results in a decrease in the reaction rate after addition of SDS at
both acidic and basic pH (Figure 6a), because the azide concentration is lowered in the micelle
under these conditions.
PhOHꢀN3’s pKa is in a similar range as the pKa of protonated DBCOꢀamine, which means that in
acidic pH the phenol moiety of PhOHꢀN3 is not deprotonated, while DBCOꢀamine is fully
protonated (ammonium ion). From the observed rate enhancement of the SPAAC reaction, we
derive that both compounds interact strongly with SDS, which results in significant increase of
rate constants. The opposite effect can be observed at basic pH, where PhOHꢀN3 is negatively
charged upon deprotonation of the phenol and DBCOꢀamine is not protonated and neutral.
Higher concentrations of SDS lead to an increase in the rate constants of SPAAC reactions with
PhCOOHꢀN3 at acidic pH, where the azide’s carboxylic acid group is not deprotonated (neutral)
and DBCOꢀamine protonated (cationic) both interacting strongly with micelles. On the other
hand, at pH 11.55 PhCOOHꢀN3 is fully dissociated and avoids the micelle environment, which
results in a decrease of reaction rate. This effect is even more pronounced at pH 7.09, where
DBCOꢀamine is cationic and interacts with SDS significantly (see Figure 6a). Very similar
behavior was observed for PheꢀN3. The largest rate enhancement was observed at pH 2.29, where
the azide reactant PheꢀN3 interacts strongest with SDS. At pH 3.77 almost no effect of SDS
addition was observed, as PheꢀN3 is mainly dissociated (
α = 60 % at pH 3.77). Similarly to
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PhCOOHꢀN3, the decrease of the reaction rate is evident at pH 11.55 and most pronounced at pH
7.09.
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We have to point out that addition of SDS results in simultaneous increase of ionic strength,
which can result in changes of reaction kinetics. By increasing the ionic strength, the reaction rate
can either increase or decrease depending on the effective charges of reactants according to the
following equation
ꢃꢄꢅꢆ = ꢃꢄꢅꢆꢇ + 1.02ꢈꢉꢈꢊ√ꢋ,
(1)
where k0 is the rate constant at infinite dilution, zA and zB are the effective charges of reactants, I
is ionic strength. It means that for reactants of opposite charge the rate constant decreases with
increasing ionic strength and vice versa. If one of the reactants is neutral, no effect of the ionic
strength on rate constants can be observed.
Our reactants are always of opposite charge or at least one of them is neutral, so the rate constants
should either decrease with ionic strength or be independent of ionic strength , respectively.
These theoretical predictions were experimentally confirmed for SPAAC reactions with PheꢀN3.
The rate constants were determined at higher ionic strength by addition of 50 mM NaCl to the
SPAAC reaction mixture. The experimental results are summarized in Figure 6b. At highly acidic
or basic pH, where either the functionalized azide or DBCOꢀamine is neutral, the effect of ionic
strength on the reaction kinetics is negligible. A very slight decrease of rate constants was
observed at pH 3.77, where PheꢀN3 is partially dissociated. At pH 7.09, where both reactants are
charged, the decrease of SPAAC reaction rate is the most remarkable. At this pH, the rate
constants were determined also at 20 mM concentration of NaCl to be able to compare the effect
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of ionic strength with the effect of SDS at the same concentration. Only a slight decrease of the
reaction rate was observed. Thus, we conclude that the effect of ionic strength on the rate
constants is negligible in comparison to the influence of adding SDS.
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The major drawback of the SPAAC reaction is the poor solubility of cyclooctyne reactants in
water, which makes the use of MeCN/water mixtures for the reaction necessary. Acetonitrile is
toxic, which limits the applicability of SPAAC in biological applications. We have shown that
DBCOꢀamine interacts with SDS (vide supra), which indicates that addition of SDS should
improve the solubility of DBCOꢀamine in aqueous buffers. We were able to reach a solubility of
DBCOꢀamine of up to 5mM in 20 mM SDS solution. Similar improvement of solubility by
addition of SDS was observed also for neutral azides.
Thus, we were able to perform SPAAC reactions in purely aqueous reaction media. The reaction
was monitored for commercially obtained PheꢀN3. Similarly to the previously described reactions
carried out in MeCN/H2O, the reactants’ interactions with SDS were characterized by
electrophoretic measurements (Figure 5). DBCOꢀamine and the nonꢀdissociated carboxylic acid
PheꢀN3 interact much stronger with micelles in aqueous environment than in MeCN/H2O buffer
solutions. Although the CMC of SDS determined at the same ionic strength is 5 mM, the
intensive interaction with SDS was observed already at much lower concentration (2 mM), which
is evident from its highly negative mobility (Figure 5a). Based on these results, we expect
significant improvement of the rate of the SPAAC reaction in acidic pH regions, while the
reaction at basic pH should be slowed down.
The rate constants were determined at 2 mM, 20 mM and 50 mM concentrations of SDS, as
shown in Figure 6b. The rate constant observed at an SDS concentration of 2 mM (pH 2.39) is
11.4 dm3molꢀ1sꢀ1, which is about ten times higher than in the MeCN/H2O mixture
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(1.3 dm3molꢀ1sꢀ1) under comparable conditions. The rate constants slightly decrease with
increasing concentration of SDS, as the maximum interaction between reactants and micelles is
already achieved at a concentration of 2 mM and addition of further micelles causes dilution of
the reactants in the micellar space. Also, we observed a very slight increase of pH after addition
of SDS under these conditions, possibly due to partial protonation of SDS (pKa (sodium dodecyl
sulfonic acid) = 1) (Table SIꢀ1).
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At pH 3.37 the rate constants are already lower (2 mM SDS, 4.4 dm3molꢀ1sꢀ1), but still
significantly higher than in MeCN/H2O buffers in the absence of any surfactant. The rate
constants change with the SDS concentration only in the range of experimental error.
At neutral and basic pH, the rate constants are much lower than in the acidic pH region or in
MeCN/H2O buffers without any surfactant. The rate of the reaction is highest at 2 mM SDS
concentration and decreases with increasing concentrations of SDS, as higher fractions of DBCOꢀ
amine are incorporated in the micelles, while the azide is mostly found in the aqueous phase.
Optimal results were achieved by combining the rateꢀenhancing effects of an adequate choice of
pH for the respective reactants and addition of a surfactant for micelle formation. Thus, the
reaction rate of the SPAAC reaction between PheꢀN3 and DBCOꢀamine can be accelerated by a
factor of up to 80 in the presence of 2 mM SDS in an aqueous buffer at pH 2.39. Thus, we
conclude that all reaction parameters need to be carefully optimized. The reaction rate constants
determined in our study range from three times lower up to ten times higher compared to rate
constants reported in literature for SPAAC reactions with similar azide substrates and under
comparable reaction conditions (see Table SIꢀ6).50,79
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Our novel and userꢀfriendly electrophoretic method allows for continuous, fast and reliable
determination of the rate of reaction in SPAAC. The method is suitable for a wide range of
reaction conditions. Thus, screening of various reaction conditions becomes feasible in a timeꢀ
efficient manner. Screening parameters include the choice of buffer, buffer additives and the pH
of the reaction medium. Having established this analytical method, we gained valuable insights
for the optimization of SPAAC reaction conditions: we found that the charge state of
functionalized azides containing either acidic or basic functional side chains has a major impact
on the rate of the SPAAC reaction with test cyclooctyne DBCOꢀamine. These results were in
good correlation with theoretical trends, which proved the high accuracy of our methods. As we
found the charge state of cyclooctyne substrate DBCOꢀamine to also have an impact on the rate
of reaction, we computationally calculated reaction trajectories featuring this cyclooctyne in
different protonation states by DFT methods. Our results suggest that subtle changes in transition
states, like hydrogen bonding interactions or minor changes in the electronic structure, account
for the observed changes of rate constants. We also investigated the effect of SDS micelle
formation on the rate of reaction, thus proving the power of the newly developed CE method. We
observed substantial impact of SDS addition on the rate of SPAAC reaction with highly
hydrophobic compounds. Carrying out the SPAAC reaction in micellar environment also allowed
for running the reaction in a purely aqueous environment. Based on the proper selection of
reaction conditions, the reaction rate was improved up to a factor of 80.
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Experimental Section
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All electrophoretic experiments were performed on/with Agilent 3DCE capillary electrophoresis
equipment operated under/by ChemStation software (Agilent Technologies, Waldbronn,
Germany). Fused silica capillaries (50 µm i.d., 375 µm o.d.) were provided by MicroQuartz
(Munich, Germany). The experiments were performed in bare fused silica capillaries with a total
length and effective length to the diode array detector (DAD) of approximately 35.9 cm and
27.4 cm, respectively. New capillaries were flushed with H2O for 5 min, then for 5 min with 1M
NaOH and for 10 min with H2O again. UVꢀdetection was performed at wavelengths λ = 214 nm
and 230 nm, i.e. at the reactants’ absorbance maxima.
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The pH values were determined by a LAB 850 pH meter (SI Analytics, Lyon, France) calibrated
with standard IUPAC buffers at pH 1.679, pH 7.000, pH 10.012 and pH 12.450.
Reaction kinetics was determined by means of CZE, the reaction was performed directly in
sample vial. Reaction mixture was regularly injected to the system and analyzed. In addition to a
20 kV separation voltage, a hydrodynamic pressure of 50 mbar was applied during the
measurements in order to increase the separation speed and allow for a short injection interval of
about 3 min. The temperature of the cassette as well as the reaction vials was kept constant at
20°C.
The capillary was flushed with the separation buffer for 4 min before each sequence of
measurements. The capillary was not flushed in between individual runs in order to shorten the
time between injections. We did not observe any baseline deviations even after 15 injections.
The samples were injected hydrodynamically at 30 mbar×5 s. Sample preparation was performed
according to the following procedure: the stock solution of DBCOꢀamine was prepared in a
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concentration of 10 mg/ml (36.2 mM) in acetonitrile (MeCN), due to its insolubility in water. The
same stock solution was used for all measurements. The stock solutions of azides at a mass
concentration of 10 mg/ml were prepared directly in the respective buffer.
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The stock solutions of DBCOꢀamine and azides were further diluted by the reaction buffer
directly in the reaction vial. The concentration of DBCOꢀamine was 0.5 mM and the
concentration of azide varied between 0.6 mM to 1.5 mM depending on the reaction rate under
particular conditions and with the type of azide derivative used. Reactions were performed
directly in the electrophoretic vial with a total volume of 300 µl.
Reaction kinetics was determined at various pH values in order to elucidate the effect of
dissociation of the reactants. The composition and pH of the buffers used in this study are
summarized in Table SIꢀ1. Buffers contained 28 vol% of acetonitrile to ensure sufficient
solubility of the reactants.
The rate constants were also measured at 2mM, 20 mM and 50 mM concentrations of SDS and
after addition of 20 mM and 50 mM NaCl in the case of PheꢀN3.
Kinetics of the cycloaddition of PheꢀN3 with DBCOꢀamine was also investigated in purely
aqueous reaction media without addition of MECN in the presence of SDS. The rate of reaction
was measured at three concentrations of SDS: 2 mM, 20 mM and 50 mM. The composition of the
buffers was the same as in the case of MeCN/H2O buffers, see Table SIꢀ1.
All measurements were performed in triplicates.
Synthetic Procedures
Phenyl azide,80 pꢀazidophenol81 and pꢀazidobenzoic acid82 were prepared according to literature
procedures. All spectroscopic data was in accordance with reported characterization.
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Synthesis of sodium p-azidobenzenesulfonate.83 This compound was prepared using a modified
literature procedure.82 An aqueous solution of NaNO2 (497 mg, 7.22 mmol) was added dropwise
to a solution of sulfanilic acid (1.00 g, 5.77 mmol) in 2 N hydrochloric acid (20 mL) under ice
cooling. After an hour, an aqueous solution (3 mL) of sodium azide (2.00 equiv.) was added and
stirred at room temperature for at least 15 hours. The solid sodium salt was formed by adding
sodium chloride, separated by filtration and dried under reduced pressure. Sodium pꢀ
azidobenzenesulfonate was obtained in 56 % yield (711 mg, 3.21 mmol), including
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approximately 10 % sodium chloride according to elemental analysis. H NMR (400 MHz, d6ꢀ
DMSO) δ 7.64 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H) ppm; 13C{1H} NMR (101 MHz, d6ꢀ
DMSO) δ 145.0, 139.5, 127.5, 118.4 ppm; elemental analysis: calculated C – 32.58 %, H – 1.82
%, N – 19.00 %, S – 14.50 %, Na – 10.39 %, O – 21.70 %; found: C – 29.40 %, H – 1.91 %, N –
17.31 %, Cl – 7.82 %.
DFT calculations
All computational results of this work were obtained by applying density functional theory
methods (DFT)65,66 using the Gaussian09 RevisionꢀD.01 program package.67 The B3LYP
functional68,69 has been used together with the ccꢀpVDZ basis set71,72 for all structures. All
geometries were optimized without symmetry restrictions and with the D3 version of Grimme’s
dispersion correction (gd3).74 Solvation energy corrections were calculated using the CPCM
model.75,76 Transitions states were confirmed by occurrence of one, and only one, imaginary
frequency in the Hesse matrix.
Associated Content
The supporting information is available free of charge on the ACS Publication website.
Experimental Procedures – Electrophoretic Methods – Synthetic Procedures – Calculational Data
with Optimized Ground State Geometries – Determination of Effective Mobilities and Critical
Micelle Concentrations – NMR Spectra
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Notes
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The authors declare no competing financial interest.
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Acknowledgements
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J.S. thanks the Alexander von Humboldt Foundation for a postdoctoral fellowship and G.S.
acknowledges the Fonds der Chemischen Industrie for a Ph.D. fellowship.
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