Probing the Mechanism of Photoaffinity Labeling by Dialkyldiazirines through Bioorthogonal Capture of Diazoalkanes

Article abstract of DOI:10.1021/acs.orglett.0c02714

Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for biological applications where instantaneous labeling is desirable, as carbene insertions display different chemoselectivity and are much faster than competing mechanisms involving diazo or ylide intermediates. Here, deuterium labeling and diazo compound trapping experiments are employed to demonstrate that both carbene and diazo mechanisms operate in the reactions of a dialkyldiazirine motif that is commonly utilized for biological applications. For the fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling on a model protein under aqueous conditions.

Full text of DOI:10.1021/acs.orglett.0c02714

pubs.acs.org/OrgLett
Letter
Probing the Mechanism of Photoaffinity Labeling by
Dialkyldiazirines through Bioorthogonal Capture of Diazoalkanes
Jessica G. K. O’Brien, Andrew Jemas, Papa Nii Asare-Okai, Christopher W. am Ende,*
and Joseph M. Fox*
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ABSTRACT: Dialkyldiazirines have emerged as reagents of
choice for biological photoaffinity labeling studies. The mechanism
of crosslinking has dramatic consequences for biological
applications where instantaneous labeling is desirable, as carbene
insertions display different chemoselectivity and are much faster
than competing mechanisms involving diazo or ylide intermedi-
ates. Here, deuterium labeling and diazo compound trapping
experiments are employed to demonstrate that both carbene and
diazo mechanisms operate in the reactions of a dialkyldiazirine
motif that is commonly utilized for biological applications. For the
fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products
derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound
intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling
on a model protein under aqueous conditions.
hotoaffinity labeling has become a ubiquitous tool in
activation (>350 nm) and displays high stability in the dark
under neutral or acidic conditions.16 Photochemical reactions
of dialkyldiazirines are relatively challenging to study because
of the susceptibility of both alkylcarbenes and their photo-
excited nitrogenous precursors to undergo intramolecular
rearrangements with α-hydrogens.18 However, several dia-
lkylcarbenes have been characterized and both singlet and
triplet ground states have been observed.19
When diazirines are applied in photoaffinity labeling, it is
frequently assumed that carbene insertion reactions are
responsible for the bimolecular capture of biological targets,
but further consideration is required with dialkyldiazirine
precursors (1, Figure 1A). Like aromatic diazirines, aliphatic
diazirines partially rearrange to diazo compounds upon
irradiation.18 However, aliphatic diazo compounds (2) are
readily protonated at neutral pH to give diazonium compounds
(4) which are reactive alkylating agents.20,21 While both
carbene (3) and diazonium mechanisms can lead to
intermolecular labeling, the chemoselectivity of alkyldiazonium
labeling is likely to differ from that of a carbene mechanism.
Labeling by alkyldiazonium 4 is known to favor esterification
P
chemical biology and drug discovery.1 Aromatic diazirines,
arylazides, and benzophenone derivatives have long served in
photoaffinity labeling applications,1−3 and recent probes based
on nitrile imines4 and photoredox catalysis5 offer new
possibilities. A consideration for the design of any photoaffinity
label (PAL) is the minimization of size and hydrophobicity in
order to ameliorate any detrimental impact of the PAL on the
function, solubility, permeability, or subcellular localization of
the biological molecules being studied.6 Accordingly, dia-
lkyldiazirines have emerged as favored probes for photoaffinity
labeling in cellular experiments7−9 with applications that
include probing small molecule−protein interactions,6,7
protein−protein interactions,10 nucleic acid−protein interac-
tions,11 metabolic oligosaccharide engineering,12 lipid−protein
interactions,13,14 and the study of biological membranes.15
While their physiochemical properties are advantageous for
chemical biology, there are important photochemical differ-
ences between dialkyldiazirines and more traditional aromatic
diazirines. Analogs of α-trifluoromethyl-α-phenyldiazirine are
well established biological probes that upon irradiation give
high intermolecular labeling yields via formal carbene addition
even with aliphatic CH bonds,16 and because α-trifluorometh-
yl-α-phenylcarbene lacks α-CH bonds, it cannot participate in
intramolecular α-C−H insertion processes. α-Trifluoromethyl-
α-phenyldiazirine also leads to ∼35% of α-trifluoromethyl-α-
phenyldiazomethane.16 While this side product undergoes
photochemistry with shorter wavelength UV (∼300 nm),17 it
does not absorb at the wavelengths typically used for diazirine
Received: August 14, 2020
https://dx.doi.org/10.1021/acs.orglett.0c02714
© XXXX American Chemical Society
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Figure 1. Potential mechanisms for photoaffinity labeling with
dialkyldiazirine probes. (A) Both carbene and diazonium intermedi-
ates can potentially lead to crosslinking. (B) For diazirines with
pendant carbonyls, carbene mechanisms do not necessarily involve
direct insertion, but may instead involve formation of ylide and
oxocarbenium intermediates.
Figure 2. (A) Studies of spirobicyclic diazirines by Kirmse and Platz.
(B) Structures of “linear” alkyl diazirines more commonly used in
biological photoaffinity labeling.
reactions,22−25 whereas the reactions of free carbenes are less
selective and include aliphatic C−H insertion as a possibility.26
Moreover, crosslinking reactions are extremely rapid for the
carbene pathway,26 whereas the alkylation reactions of
alkyldiazonium ions are much slower; for example, the reaction
of a methanediazonium ion with water has a relatively long
half-life of ∼0.4 s in water/THF solution.27,28 Thus, in
biological target identification experiments, alkyldiazonium
ions are likely to display a much larger labeling radius than
more reactive carbene intermediates. Additionally, when
alkyldiazonium ions do modify proteins they are likely to
modify carboxylic acid residues,22−25 producing esters which
may be subsequently hydrolyzed by esterases22−24 in live cell
experiments and potentially in the proteomic workflow.
Understanding the relative contributions of carbene and
diazonium intermediates is therefore critical to the interpre-
tation of biological data from photoaffinity experiments.
In seminal studies, Kirmse, Platz and coworkers studied the
photolyses of spirobicyclic diazirines including norbornane-
derived 7, which upon irradiation in MeOH gives a mixture of
products that could derive from either carbene 8 or diazo
Figure 3. (A) Products from photolysis of diazirine 12. (B)
Mechanistic pathways of intramolecular product formation for
photolyses in MeOD leading to deuterated and non-deuterated
alkenes 13−15. (C) D/H ratio serves as a reporter of the carbene 1,2
H-migration pathway. (D) The ratio of alkene:ether products from
amide 12a is similar to that from ester 12.
compound 9 (Figure 2A).18 By studying the changes in
product distribution in the presence of dipolarophile traps for
diazocompounds and by analyzing levels of deuterium
incorporation for photolyses in MeOD, it was possible to
deduce that diazirine 7 produces both carbene 8 and diazo
compound 9 in significant amounts. These studies with
bicyclic diazirines not only demonstrated the importance of
diazonium ions to intermolecular ether formation but also
revealed a structural dependence on the contribution of
carbenes to bimolecular chemistry.
While “linear” dialkyldiazirines have emerged as reagents of
choice for photoaffinity labeling (e.g., 1012 and 11,7 Figure
B
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Figure 4. (A) Preparative reaction of diazirine 12 with the cycloalkyne BCN to produce cycloadduct 23 is proposed to occur by [3 + 2]
cycloaddition with intermediate diazo compound 21, which (B) influence formation of intramolecular products by decreasing the yield and level of
D-incorporation for alkenes 13−15, and (C) on intermolecular chemistry by decreasing the yield of product 17. (D) Effect of product formation by
dipolarophiles BCN and fumaronitrile.
2B), studies on the mechanism of intermolecular labeling are
lacking for the substrate types that are most often used for
biological experiments. Another mechanistic consideration for
such systems is that carbonyl groups are generally used to
attach the probe to a molecule of interest, which opens a
pathway for carbenes to react via carbonyl ylides (5) and
subsequently formed oxocarbenium ions (6) (Figure 1).
Recent LC-MS/MS studies on diazirine photo crosslinking
of proteins have uncovered a preference for labeling carboxylic
acid residues, suggesting an important role of diazo
intermediates.25,29,30 Here, we present direct evidence that
both carbene and diazonium mechanisms operate in the
reactions of a dialkyldiazirine motif commonly utilized for
photoaffinity labeling. We demonstrate that a strained
cycloalkyne can intercept diazocompound intermediates20
and serve as a bioorthogonal probe for studying the
contribution of the diazonium mechanism of photoaffinity
labeling on a model protein under aqueous conditions.
Our study began by observing the products formed when
methanol solutions of diazirine 12 (60 mM) were irradiated at
350 nm in the air to produce three alkenes 13−15, lactone 16,
and the methanol adduct 1731 (Figure 3A). Based on corrected
GC yields there was a 64% yield of the intramolecular products
13−16 and a 32% yield of the intermolecular product 17.
Alkene products 13−15 may arise via carbene 18 or via diazo
21/diazonium 22 (Figure 3B,C). For carbene 18, the alkenes
can form either by 1,2 hydride (1,2 H−) shift or via the
intermediacy of ylide 19 and oxocarbenium 20. Among these
mechanisms, only the carbene concerted 1,2 H-shift does not
involve protonation. Thus, for irradiations carried out in
methanol-d, the D/H ratio reports on the contribution of the
carbene 1,2 H-shift mechanism for the formation of alkenes
13−15,18 as alkenes 13-d, 14-d, and 15-d arise from
C
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toward biological nucleophiles. We therefore sought to identify
a bioorthogonal dipolarophile that could intercept intermedi-
ate diazo compounds under biologically relevant conditions.
Cycloalkynes including the cyclooctyne BCN participate in a
number of bioorthogonal reactions35 including 3 + 2
cycloadditions with diazo compounds.20 As shown in Figure
4A, the photolysis of diazirine 12 in MeOH in the presence of
BCN (1.1 equiv) produces cycloadduct 23 as a mixture of
diastereomers in 22% isolated yield, thus demonstrating the
ability of BCN to intercept diazo intermediate 21. As depicted
in Figure 4B,D, the inclusion of dipolarophile in photolyses of
12 (60 mM) in MeOD also has influence on D-incorporation
for alkene products 13−15. Interception of diazo 21 by BCN is
expected to lead to a decrease in the yield and the D/H ratio
for alkenes 13−15. In the absence of BCN, alkene products
were formed in combined 52% yield and with 31−50% D
incorporation. However, the photolysis of 60 mM 12 in the
presence of 70 mM BCN gave alkenes 13−15 with a yield
decrease to 33% and with only 6−9% D incorporation.36 This
decrease in alkene yield and D-incorporation is consistent with
the capture by BCN of diazo 21, which is partly responsible for
the formation of alkenes 13-d, 14-d, and 15-d. Similar
decreases in yield and D-incorporation were noted for the
photolysis in the presence of fumaronitrile.18
As depicted in Fig 4C,D, the inclusion of dipolarophiles in
photolyses of 12 also decreases the yield of bimolecular
product 17. The yield of ether 17 without added dipolarophile
is 29%, whereas irradiation of 12 (60 mM) with either BCN
(70 mM) or fumaronitrile (200 mM) gives 17 in 14% yield.
Thus, intercepting diazo intermediate 21 leads to a major
reduction in bimolecular product formation. These results
illustrate the importance of diazo compound protonation/
alkylation for intermolecular product formation.
Persistently formed in the photolyses is lactone 16, which is
proposed to arise via protonation of ylide 19 and subsequent
hydrolysis of oxocarbenium 20 by adventitious water.
Intermediates 19 and 20 may arise from either the carbene
pathway (via cyclization of 18) or the diazo pathway (via
cyclization of either 21 to 19, or 22 to 20 (Figure 3B)).
Consistent with all of these mechanisms, 16 shows >99% D-
incorporation when photolyses are carried out in MeOD. The
formation of ylide 19 from carbene 18 provides a possible
explanation for the incomplete ability of dipolarophiles to
suppress D-incorporation in alkenes 13−15, as would be
expected if the carbene 1,2 H-shift was the sole pathway
(Figure 2).37 The formation of 19 from 18 also explains the
incomplete suppression of bimolecular adduct 17 (Figure
4B,C). Thus, while carbene 18 may lead to the formation of an
intermolecular product 17, this is most likely preceded by an
intramolecular reaction to produce ylide 19 based on earlier
conclusions that intramolecular H-shifts are generally too rapid
for intermolecular reactions to compete.18,38−40 Also con-
sistent with the formation of ylide 19 from carbene 18 is the
incomplete ability of dipolarophiles to suppress the formation
of lactone 16, suggesting that 16 arises from a combination of
the carbene and diazo pathways.
Figure 5. (A) Experiment involving BSA, TAMRA-DAz, BCN-
PEG12, and 24. (B) Lane assignments in SDS-PAGE. (C) Relative
fluorescence with 0 to 10 mM BCN-PEG12.
protonation/elimination of diazo 21 or ylide 19. As shown in
Figure 3C, the level of deuterium incorporation for 13, 14, and
15 was 48%, 31%, and 50%, respectively. Hence, the carbene
concerted 1,2 H-shift mechanism is responsible for at least 50−
69% of the alkene products 13−15.
Additionally, the diazirinyl amide 12a was prepared and
irradiated in MeOH (350 nm, 2 h) and by NMR analysis was
observed to give alkene (13a−15a) and ether (17a) products
in 61% yield and ratios that were similar to what was observed
with diazirinyl ester 12. Lactone 16 was not observed, nor was
the cyclic butyrolactam, 5-methyl-1-propylpyrrolidin-2-one.32
Dipolarophiles such as fumaronitrile and diethyl fumarate
participate in cycloaddition reactions with diazo compounds
formed photochemically from diazirines.33 These dipolaro-
philes have been used as mechanistic probes in the reactions of
spirocyclic diazirines,18,34 but are also potent electrophiles
We next sought to use BCN to probe the mechanism of
protein photoaffinity labeling. Rhodamines are known to non-
selectively bind to bovine serum albumin (BSA), and Jewett
had previously demonstrated fluorescent labeling of BSA with
a rhodamine−diazirine conjugate.41 Accordingly, we prepared
a fluorescent diazirine, TAMRA-DAz, and the water-soluble
BCN-PEG12 (Figure 5A). BSA was reduced by DTT and
D
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alkylated with iodacetamide before usage to minimize cysteine
labeling by the BCN reagent.42,43 As shown in Figure 5B,
irradiating a solution of BSA (22 μM) with TAMRA-DAz (24
μM) in PBS/tris buffer for 15 min at 365 nm led to fluorescent
labeling as judged by SDS-PAGE (lane 3), whereas controls
without light gave no labeling (lanes 2, 9, 10). The inclusion of
BCN-PEG12 (2−10 mM) in photolysis experiments with BSA
led to decreased labeling by TAMRA-DAz, with up to a 69%
decrease at the highest BCN-PEG12 concentration (lanes 4−
7). However, no suppression of fluorescence was observed
upon irradiation in the presence of 24, an analog of BCN-
PEG12 that lacks the reactive alkyne (lane 8). As further
evidence that the diazonium mechanism is partly responsible
for protein labeling, an additional control was carried out in the
presence of sodium acetate, which would be expected to
decrease labeling by competing for the diazonium intermedi-
ate.22−25 As shown in Figure S1, inclusion of NaOAc (1 M)
during the irradiation of TAMRA-DAz with BSA led to a 45%
decrease in labeling.
Christopher W. am Ende − Pfizer Worldwide Research and
Development, Groton, Connecticut 06340, United States;
orcid.org/0000-0001-8832-9641;
Email: Christopher.amende@pfizer.com
Authors
Jessica G. K. O’Brien − Department of Chemistry and
Biochemistry, University of Delaware, Newark, Delaware
19716, United States
Andrew Jemas − Department of Chemistry and Biochemistry,
University of Delaware, Newark, Delaware 19716, United
States
Papa Nii Asare-Okai − Department of Chemistry and
Biochemistry, University of Delaware, Newark, Delaware
19716, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02714
Notes
These protein labeling results are consistent with the results
in methanol that show that intermolecular labeling can be
suppressed by capturing diazoalkane intermediates, and
support the hypothesis that the diazonium alkylation pathway
is a major pathway in photoaffinity labeling. The inability to
completely quench labeling by addition of BCN-PEG12 does
suggest a role for the a carbene intermediates in diazirine
photoaffinity labeling. However, the mechanism by which the
carbenes studied here label proteins is unclear and may not
involve commonly assumed carbene insertion reactions, but
could instead involve the intermediacy of ylide and
oxocarbenium intermediates. These mechanistic details have
consequences for chemical biology applications, as alkylations
of diazonium or oxocarbenium ions are much slower than
carbene insertion reactions which may in turn negatively
impact the radius of labeling in protein identification
experiments. Additionally, reactions of diazo compounds
with proteins are known to selectively react with carboxylic
acid residues to give esters,22−25 which may not be stable in
live cell applications due to native esterases and/or the
proteomic workflow employed.22−24
The authors declare the following competing financial
interest(s): C.W.A. is an employee of Pfizer Inc.
ACKNOWLEDGMENTS
■
This work was supported by NIH GM132460 and Pfizer Inc.
J.G.K.O. and A.J. were supported as CBI fellows through T32-
GM133395, and J.G.K.O. thanks the University of Delaware
for fellowship support. We thank R. Knowles and his group for
sending us analytical samples of 13a and 5-methyl-1-
propylpyrrolidin-2-one. Instrumentation was supported by
NIH Awards P20GM104316, P30GM110758,
S10OD025185, S10RR026962, and S10OD016267 and NSF
Awards CHE-0840401, CHE-1229234, and CHE-1048367.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02714.
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(PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Joseph M. Fox − Department of Chemistry and Biochemistry,
University of Delaware, Newark, Delaware 19716, United
States; orcid.org/0000-0002-8258-1640; Email: jmfox@
udel.edu
E
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