Photoinitiated release of an aziridinium ion precursor for the temporally controlled alkylation of nucleophiles

Article abstract of DOI:10.1016/j.bmcl.2013.02.044

A photo-activatable aziridinium precursor has been developed to investigate the possibility of a photo-initiated traditional nucleophilic reaction. The photolysis of a quaternary amine yields a tertiary amine and has allowed us to temporally control aziridinium formation and subsequent alkylation of a colorimetric nucleophilic reporter molecule. We have also used this photo-initiated reaction to alkylate a sulfhydryl group. This new photo-initiated alkylation strategy is water-soluble and expands the toolkit of photo-activated crosslinkers for protein labeling research.

Full text of DOI:10.1016/j.bmcl.2013.02.044

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2395–2398
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Photoinitiated release of an aziridinium ion precursor for the
temporally controlled alkylation of nucleophiles
Stephen T. McCarron a, Mariel Feliciano a, Jeffreys N. Johnson b, James J. Chambers a,b,
⇑
a Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA
b Neuroscience and Behavior Graduate Program, University of Massachusetts, Amherst, MA 01003, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A photo-activatable aziridinium precursor has been developed to investigate the possibility of a photo-
initiated traditional nucleophilic reaction. The photolysis of a quaternary amine yields a tertiary amine
and has allowed us to temporally control aziridinium formation and subsequent alkylation of a colori-
metric nucleophilic reporter molecule. We have also used this photo-initiated reaction to alkylate a sulf-
hydryl group. This new photo-initiated alkylation strategy is water-soluble and expands the toolkit of
photo-activated crosslinkers for protein labeling research.
Received 28 November 2012
Revised 1 February 2013
Accepted 7 February 2013
Available online 21 February 2013
Keywords:
Photochemistry
Alkylation
Published by Elsevier Ltd.
Ring closure
Nucleophilic addition
Bioconjugation
The development of novel and efficient methods aimed at the
facile alkylation of biological substrates in a spatially- and tempo-
rally-precise manner has been a topic of much effort in the field of
chemical biology.1–4 The ability to covalently deliver a low molec-
ular weight contrast agent to a biological target allows for subcel-
lular imaging and the study of dynamic protein motions on cells. In
fact, new sub-diffraction resolution microscopy methods typically
rely on the delivery and attachment, covalent or non-covalent, of
fluorophores to proteins of interest.5–7 These fluorophores are usu-
ally delivered by one of two methods, genetic tagging of the pro-
tein of interest using a naturally fluorescent reporter protein
such as GFP or by non-covalent labeling using the tried-and-true
antibody method, sometimes in conjunction with a fluorescently-
tagged secondary antibody. These methods, however, rely on
genetic engineering of the target protein or on non-covalent inter-
actions of fairly large antibodies for highlighting the location of the
target biomolecules. More recently, hybrid chemical/genetic strat-
egies such as the tetracysteine/FlAsH and ReAsH or the tetraserine/
RhoBo systems have been developed.8,9 These systems rely on a
genetically engineered protein tag that selectively binds to a com-
plementary fluorophore to label a protein of interest. In addition,
enzyme catalyzed processes have been developed for orthogonal,
selective chemical modification of expressed proteins including
the use of BirA, LplA, and SrtA.10
Recently, the use of small molecular probes for the delivery of
fluorophores to a target of interest has become popular.11–13 These
probes typically consist of a ligand for binding to the target protein,
a fluorophore for visualization, and some form of reactive species,
typically an inherent electrophilic moiety that forms a new cova-
lent bond with the target upon binding to the protein. Quite often,
the native nucleophile is the sulfhydryl of cysteine that is intrinsic
to the target protein and the small molecule-based electrophile
contains a maleimide for selective bioconjugation to this residue.
While these probes offer many advantages in terms of overall size
and their ability to be ‘traceless’, they all suffer from the fact that
an electrophile must be delivered to the biological system and
electrophiles are known to react with off-target biological nucleo-
philes.14 Here, we report the development of a small, photo-
activated, electrophilic moiety that is compatible with aqueous
buffer.
Another method for the delivery of a small molecule to a pro-
tein target is to employ photochemically-activated moieties ap-
pended to small molecules that may target proteins and then
covalently modify the protein of interest for downstream analy-
sis.15–17 There are three classes of commonly used photoreactive
moieties that allow for photo-crosslinking to proteins. These are
aryl azides, diazirines, and substituted benzophenones. Each of
these functional groups is nominally chemically-inert under dark
conditions but can be photo-activated to a reactive species by
the application of high energy, ultraviolet light. Upon photo-activa-
tion, the azides and diazirine groups result in the production of
either a nitrene-like intermediate or a carbene intermediate,
⇑
Corresponding author. Tel.: +1 413 545 3864; fax: +1 413 545 4490.
E-mail address: chambers@chem.umass.edu (J.J. Chambers).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.02.044

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S. T. McCarron et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2395–2398
respectively, while benzophenones depend on radical aryl ketyl
intermediates for reactivity.18,19 However, the reactivity of the
photo-activated products are difficult to tune and often react with
the solubilizing media or buffer in the context of their normal use,
thereby reducing their labeling efficiency. Thus, we focused on a
method to photoinitiate the release of 2-chloro-N,N-dimethyleth-
ylamine, a molecule that is similar in reactivity to bis(2-chloro-
ethyl)ethylamine (HN1), the nitrogen mustard alkylating agent
that is known to form the promiscuous N,N-dialkylaziridinium
electrophile under biologically-relevant conditions.20–23 To date,
there have been a small number of reports of photo-activated, pro-
drugs of electrophilic moieties that are compatible with aqueous
buffer systems.24,25
The N,N-dialkylaziridinium ion formed from HN1 has been
implicated in the alkylation of biological nucleophiles, most
commonly the guanine of DNA after exposure to this nitrogen mus-
tard.20,21 We reasoned that the formation of N,N-dimethylaziridini-
um from 2-chloro-N,N-dimethylethylamine might be initiated with
light if we were to chemically mask the important lone pair of elec-
trons on the tertiary amine of the latter in the form of a photo-
releasable quaternary amine (Scheme 1). Upon investigation of
potential photolabile protecting groups, we decided to employ a
4-methyl-7-methoxycoumarin chromophore for a number of rea-
sons: (1) This chromophore undergoes heterolytic bond cleavage
resulting in the release of a tertiary amine in neutral form with
the lone pair of electrons made available for subsequent ring clo-
sure, (2) this chromophore can be activated by either ultraviolet
light or, potentially important for future biological applications,
two-photon infrared light which could allow for more precise spa-
tial control of labeling and, (3) the quantum yield of release from
this chromophore has been measured and reported to be highly effi-
cient.26 The N,N-dimethylaziridinium that is formed is expected to
be relatively long lived in biological conditions. Reports on similar
aziridinium species suggest that the half-life of the aziridinium in
PBS at 37 °C is about 70 min.27
Scheme 2. Straightforward synthesis of caged N,N-dimethylaziridinium precursor 1.
The pyridinium nitrogen is quarternized by the electrophile and
the alkaline ‘stop solution’, which contains aqueous base, is added
to the mixture. A benzylic proton is removed and the rings then
become conjugated to produce a color that can be rapidly analyzed.
We used this method of analysis to follow the evolution of
N,N-dimethylaziridinium formation of the non-caged 2-chloro-
N,N-dimethylethylamine, of the caged molecule (1), and of the
caged molecule (1) after exposure to a short pulse of 365 nm light
from an LED light source.
Our investigation began with the measurement of the aziridini-
um formation from 2-chloro-N,N-dimethylethylamine (hydrochlo-
ride) in aqueous buffer in the presence of NBP at 37 °C and pH
7.4.33 We performed these reactions at this temperature due to
our future plans to employ this photochemistry on live mammalian
cells which are typically maintained at 37 °C. We followed the time
course of NBP alkylation, presumably via formation of the aziridini-
um ion, by removing aliquots of the reaction mixture and incubating
them with stop solution followed by ethyl acetate extraction of the
colored product (Fig. 1). The OD535 of the ethyl acetate layer was
then quantified. All data points were measured in triplicate and
are from three different reaction runs. In parallel, we prepared an
identical solution of the caged 2-chloro-N,N-dimethylethylamine
(1) and allowed it to react with the NBP in the dark. We expected
to see some background reactivity due to SN2 displacement of the
alkyl chloride on the caged molecule by the nucleophilic pyridine.
To our delight, the caged molecule did not react at a detectable level,
suggesting that the caging group and removal of neighboring group
participation (i.e., quarternization of the tertiary amine) is sufficient
to eliminate much of the electrophilic character of this alkyl chlo-
ride (Fig. 2).
The caged molecule 1 was synthesized by alkylating N,N-
dimethylaminoethanol with 4-chloromethyl-7-methoxycoumarin
(2) in 72% yield28 followed by substitution of the hydroxyl with
chloride using thionyl chloride to afford 1 in 81% yield (Scheme
2).29 To investigate the photoinitiated release of the 2-chloro-
N,N-dimethylethylamine and the subsequent cyclization to form
an aziridinium ion, we employed a colorimetric assay to determine
the rate of putative aziridinium formation by monitoring nucleo-
phile alkylation. 4-Nitrobenzylpyridine (NBP) has been employed
as a detector of alkylation numerous times in past literature be-
cause N-alkylation and facile work-up produces a deep purple col-
or that is easy to quantify using a UV/vis spectrophotometer.30–32
To investigate further this apparent lack of reactivity in the dark
of 1, we added 10 mol % of potassium iodide to encourage Finkel-
stein reaction conditions and facilitate SN2 reactivity. Even after
leaving this reaction for 16 h, only background reactivity similar
Figure 1. Rate of NBP alkylation. Data points represent average of three indepen-
dent experiments (n = 3 for each experiment) of reaction between alkylating
molecule and NBP following work-up and analysis. Lines are calculated as
exponential associations. Green circles are for 2-chloro-N,N-dimethylethylamine,
black boxes are for caged molecule 1 maintained in the dark, and purple triangles
are for caged molecule 1 (at the same concentration and quantity as that used for
the green circles) after exposure to 1 min of 365 nm light as represented by purple
bar at 60 min mark.
Scheme 1. Photo-induced aziridinium formation and subsequent alkylation of the
pyridine-based nucleophile, NBP.

S. T. McCarron et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2395–2398
2397
offer new tools to spatially limit protein modification for the pur-
pose of protein tracking on cells and will add another versatile tool
to the arsenal of spatially- and temporarily-controlled protein
labeling bioorganic chemistry.
General information
All reagents were purchased through Fisher Scientific (Fair
Lawn, NJ, USA). Merck silica gel (35–70 mesh) was used for flash
chromatography. NMR spectra were recorded on a 400 MHz Bruker
NMR spectrometer using the residual proton resonance of the sol-
vent as the standard for proton spectra and the carbon signal of the
deuterated solved as the internal standard for carbon spectra.
Chemical shifts are reported in parts per million (ppm). When peak
multiplicities are given, the following abbreviations are used: s,
singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet. Mass spectra were measured on a Waters ZQ device for
LRMS while HRMS data was collected at the University of Massa-
chusetts Mass Facility which is supported, in part, by the National
Science Foundation. The colorimetric absorption measurements
were made on an Evolution 100 UV/vis spectrometer.
Figure 2. Photographs of alkylation reactions post-workup. (a) 2-chloro-N,N-
dimethylethylamine incubated with NBP at 30 min, (b) caged molecule 1 irradiated
at 365 nm for 1 min and incubated with NBP for 30 min, and (c) caged molecule 1
non-irradiated with NBP at 30 min. The top, color-containing layer is the ethyl
acetate extract that is normally analyzed via UV/vis.
to the solution of 1 that was maintained in the dark was discovered
upon work-up and analysis (data not shown).
Upon photoinitiated release of 2-chloro-N,N-dimethylethyl-
amine from the coumarin cage, NBP alkylation occurred efficiently
and was quite similar in reaction rate to the uncaged 2-chloro-N,
N-dimethylethylamine as determined by sampling over the course
of the incubation. The photoinitiated release of 2-chloro-N,N-dim-
ethylethylamine, we believe, allows the aziridinium formation to
begin at a prescribed time and results in identical alkylation of
the NBP reporter molecule.
Next, we investigated the selectivity of the photo-initiated re-
lease of 2-chloro-N,N-dimethylethylamine towards four amino
acids that represent biologically-relevant potential nucleophiles.34
For all experiments the N-terminus of the amino acids used were
Fmoc-protected to discourage the alkylation of the free amine ver-
Acknowledgments
A portion of this work is related to a Grant from the Human Fron-
tier Science Program (RGY0066/2008). S.T.M. was supported by
start-up funding from the University of Massachusetts, Amherst to
J.J.C. M.F. was supported by a NIH Traineeship administered through
the Chemical–Biology Interface Program at UMA (5T32GM008515).
The authors would like to thank Professors Nathan Schnarr and
Dhandapani Venkataraman for helpful discussion.
sus the side chain functional groups. As
a positive control,
2-chloro-N,N-dimethylethylamine hydrochloride was incubated
with Fmoc-serine, lysine, arginine, or cysteine for 2 h at 37 °C
and pH 7.4 in 2.5 mL of PBS/DMSO (4:1) solution. After 2 h of incu-
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.044.
bation, 50 lL of the reaction mixture was diluted to 1 mL in meth-
anol and analyzed by LC/MS. Under these conditions only cysteine
was found to be alkylated (MW = 415.2). Likewise, caged 1 was
incubated with of each Fmoc-protected amino acid under the same
References and notes
conditions in the dark. After 2 h of incubation, 50 lL of the reaction
mixture was diluted to 1 mL in methanol and analyzed by LC/MS.
The mass corresponding to the alkylated amino acids were not
found under these conditions. In addition, the mass corresponding
to unreacted caged 1 was present, indicating that the cage was not
reactive to the buffer at a detectible level. However, under the
same reaction conditions following exposure to 1 min of 365 nm
light, the mass corresponding to the alkylated amino acids was
only found in the case of cysteine (MW = 415.2). This finding
parallels the results from the positive control experiment with
2-chloro-N,N-dimethylethylamine.
In summary, we have developed a convenient method for the
photoinitiated release of the pro-electrophilic moiety 2-chloro-
N,N-dimethylethylamine that can form N,N-dimethylaziridinium
in situ. While our data does not conclusively confirm the produc-
tion of the aziridinium moiety, we have shown that our caged mol-
ecule is essentially unreactive before the release of light. In future
studies, we plan to synthesize a similar molecule that contains a li-
gand and a propargyl group to allow for the electrophilic attach-
ment to a protein of interest. This alkyne can then be used in a
subsequent 3+2 click reaction for the attachment of an azido-based
fluorophore or biotin. The present molecular system that is the
subject of this manuscript is chemically-inert when kept in the
dark and is stable in phosphate-buffered saline solution for
>30 days at room temperature. The reactive species is only gener-
ated after light is applied to unmask the tertiary amine. The appli-
cation of this technology to protein labeling studies could soon
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S. T. McCarron et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2395–2398
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dimethylethylamine or caged molecule 1 were prepared by dissolving the
hydrochloride salt of 2-chloro-N,N-dimethylethylamine or the chloride salt of 1
in PBS to a final concentration of 25 mM. Nitrobenzylpyridine solution was
prepared in dry acetone to a final concentration of 25 mM.
26. Dore, T. M.; Wilson, H. C. Neuromethods 2011, 57.
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28. Experimental procedure for the synthesis of 2-Hydroxy-N-((7-methoxy-2-oxo-2H-
chromen-4-yl)methyl)-N,N-dimethylethanaminium: To a stirred solution of 4-
chloromethyl-7-methoxycoumarin 2 (500 mg, 2.23 mmol), prepared according
N,N-Dimethylaziridinium formation assays were performed by mixing 1.0 mL
of PBS, 500
dimethylethylamine, and 500
incubate at 37 °C. Aliquots of the reaction mixture (100
predetermined times and were immediately quenched by the addition of
400 ethyl acetate, 500 of water, and 63 L of 1.0 M aqueous KOH.
l
L
of test solution containing either
of NBP solution and allowing them to
L) were removed at
1 or 2-chloro-N,N-
lL
l
to
a
previous report35 in methanol (15 mL) was added N,N-
dimethylethanolamine (0.896 mL, 8.92 mmol). The reaction mixture was
heated to reflux temperature and allowed to stir for 24 h and then cooled to
room temperature and the solvent was removed via rotary evaporation. The
lL
lL
l
Mixtures were vortexed for 1 min and centrifuged to efficiently separate the
layers. The top, organic layer was removed via pipette and was either directly
analyzed by UV absorption or, if highly colored, diluted 10Â or 100Â in order to
produce OD535 measurements between 0.0 and 1.0 AU. Each sample was
measured after zeroing the UV/vis detector to pure ethyl acetate and each data
point was measured in triplicate. Each data point used in the time course plot
is defined by three separate reactions, aliquot removal, and workup. Photolysis
of the protecting group was accomplished by irradiating the sample from the
top of an open 1 dram vial with the light from a LedEngin High Power LED
365 nm source (Mouser Electronics, Mansfield, TX, USA) situated 5 mm from
the top of the vial driven by 600 mA of current.
crude product was dissolved in
a minimal amount of hot methanol and
hexanes were added slowly until a solid precipitated. The solid was collected
by filtration and was extensively rinsed with hexanes and then used without
further purification (503 mg, 72%).
1H NMR (400 MHz, D2O) d ppm = 7.78 (ds, J = 9.09, 2.53 Hz, 1H) 6.99 (dd
J = 9.09, 2.53 Hz, 1H) 6.95 (d, J = 2.53 Hz, 1H) 6.58 (s, 1H) 4.73–4.76 (m, 2H)
4.03–4.09 (m, 2H) 3.83 (s, 3H) 3.59–3.64 (m, 2H) 3.13 (s, 6H).
13C NMR (400 MHz, D2O) d = 163.32, 162.62, 155.31, 142.70, 126.19, 119.26,
113.48, 112.22, 101.64, 67.30, 62.53, 56.03, 55.49, 51.46.
HRMS (FAB+) Calcd for C15H20NO4 [M+]: 278.1387. Found: 278.1389.
29. Experimental procedure for the synthesis of 1: The previously-produced alcohol
(100 mg, 0.319 mmol) was added to thionyl chloride (2.0 mL, 27.6 mmol) and
was stirred at room temperature for 16 h. The volatiles were removed via
rotary evaporation followed by high vacuum to leave a white solid that was
recrystallized from isopropanol to yield a white solid (86 mg, 81%).
1H NMR (400 MHz, D2O) d ppm = 7.79 (d, J = 8.59 Hz, 1H) 6.97–7.05 (m, 2H)
6.58 (s, 1H) 4.76 (s, 2H) 4.00–4.07 (m, 2 H) 3.86–3.92 (m, 2H) 3.84 (s, 3H) 3.17
(s, 6H).
34. Mass spectrometry analysis of amino acid labeling assay: Stock solutions of the
corresponding Fmoc-protected amino acids were dissolved in DMSO to a final
concentration of 14 mM. Control experiments were conducted by dissolving
the hydrochloride salt of 2-chloro-N,N-dimethylethylamine (0.007 mmol) in
200
solution for a final concentration of 28 and 2.8 mM, respectively. Each reaction
was conducted at pH 7.4 and 37 °C for 2 h at which time a 50 L aliquot was
removed, diluted to 1 mL in methanol, and analyzed by mass spectrometry.
Similarly, caged 1 was dissolved in 200 L of PBS followed by the addition of
50 L of each amino acid DMSO solution and allowed to react for 2 h at 37 °C
lL of PBS followed by the addition of 50 lL of the amino acid DMSO
l
l
13C NMR (400 MHz, D2O) d = 163.39, 162.47, 155.31, 142.26, 126.10, 119.36,
113.52, 112.10, 101.68, 66.18, 62.58, 56.05, 51.23, 35.38.
l
and pH 7.4 in the dark. The reaction mixture was then analyzed as previously
described.
HRMS (FAB+) Calcd for C15H19ClNO3 [M+]: 296.1048. Found: 296.1059.
30. Gomez-Bombarelli, R.; Gonzalez-Perez, M.; Calle, E.; Casado, J. Chem. Res.
Toxicol. 2012, 25, 1176.
Lastly, for the photolysis experiment, caged 1 was dissolved in 200
lL of PBS
followed by the addition of 50 L of each amino acid DMSO solution and
l
31. Kim, J. H.; Thomas, J. J. Bull. Environ. Contam. Toxicol. 1992, 49, 879.
32. Kawazoe, Y.; Tamura, N.; Yoshimura, T. Chem. Pharm. Bull. (Tokyo) 1982, 30,
2077.
33. Photolysis and N,N-dimethylaziridinium assay production and detection
conditions: All solutions were kept in the dark (aluminum foil wrapped)
except during UV irradiation conditions. Solutions of 2-chloro-N,N-
warmed to 37 °C. The sample was irradiated for 1 min from a LedEngin High
Power LED 365 nm source and then allowed to react for 2 h at 37 °C and pH 7.4.
The reaction mixture was analyzed as previously described.
35. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-M.; Ahn, B.-Z. Bioorg. Med.
Chem. Lett. 2002, 12, 2345.