A traceless staudinger reagent to deliver diazirines

Article abstract of DOI:10.1021/ol402404n

A triarylphosphine reagent that reacts with organic azides to install amide-linked diazirines is reported. This traceless Staudinger reagent reacts with complex organic azides to yield amide-linked diazirines, thus expanding the scope of the utility of bo

Full text of DOI:10.1021/ol402404n

ORGANIC
LETTERS
2013
Vol. 15, No. 19
5060–5063
A Traceless Staudinger Reagent
To Deliver Diazirines
Ali M. Ahad, Stephanie M. Jensen, and John C. Jewett*
University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721,
United States
jjewett@email.arizona.edu
Received August 20, 2013
ABSTRACT
A triarylphosphine reagent that reacts with organic azides to install amide-linked diazirines is reported. This traceless Staudinger reagent reacts
with complex organic azides to yield amide-linked diazirines, thus expanding the scope of the utility of both azide and diazirine chemistry.
The area of bioorthogonal chemistry relies on the ability
of chemists to sneak unnatural reactivity past the defenses
that exist in a biochemical system. The Trojan workhorse
in this area is the azide, a small but highly energetic
functionality that reacts selectively with several soft, non-
natural nucleophiles.1 The diminutive size of the azide, in
particular, has allowed it to be incorporated into myriad
biological macromolecules, providing an azide labeled
biomolecular metabolic building block to the machinery
present within living systems.
Our interest in studying hostꢀpathogen interactions
associated with dengue virus (DENV), a flavivirus that
infects between 50 and 100 million people per year, led us
to investigate compounds that can photo-cross-link patho-
genic proteins to those found within the host.2 We have
recently found that various alkyl azides can be incorpo-
rated into DENV. Regrettably, unlike their aryl counterparts,
alkyl azides are not easily degraded photochemically.3
In lieu of re-engineering our incorporation strategy, we
sought to convert our existing azides to something more
susceptible to photochemical cross-linking. Fortunately,
the azide’s less stable chemical cousin, the diazirine, is
readily degraded using conditions that have been shown
to be relatively benign to living systems.4 The resulting
carbenes are highly reactive and cross-link with whatever
is in close proximity. There are several unnatural diazirine-
containing metabolites that have proven useful for
biochemical photo-cross-linking studies, but these are
often complicated by questions of levels and locations
of incorporation.5 These complications stem from the
fact that probes that selectively react with diazirines to
report on their presence have yet to be described. All of
these issues are summarized in Figure 1. Upon searching
for reagents that would selectively transform our azides
into diazirines, either through direct functional group
(4) For a recent review on biologically relevant photo-cross-linking,
see: Pham, N. D.; Parker, R. B.; Kohler, J. J. Curr. Opin. Biol. 2013, 17, 90.
(5) (a) Suchanek, M.; Radzikowska, A.; Thiele, C. Nat. Methods
2005, 2, 261. (b) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.;
Taunton, J. J. Am. Chem. Soc. 2007, 129, 14560. (c) Tanaka, Y.; Kohler,
J. J. J. Am. Chem. Soc. 2008, 130, 3278. (d) Zhang, M.; Lin, S.; Song, X.;
Liu, J.; Fu, Y.; Ge, X.; Fu, X.; Chang, Z.; Chen, P. R. Nat. Chem. Biol.
2011, 7, 671. (e) Li, Z.; Hao, P.; Li, L.; Tan, C. Y. J.; Chang, X.; Chen,
G. Y. J.; Sze, S. K.; Shen, H.-M.; Yao, S. Q. Angew. Chem., Int. Ed. 2013,
52, 8551.
(1) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666.
(2) WHO, Dengue: Guidelines for Diagnosis, Treatment, Prevention
and Control; World Health Organization: Geneva, 2009.
(3) (a) Krieg, U. C.; Walter, P.; Johnson, A. E. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 8604. (b) Schnapp, K. A.; Poe, R.; Leyva, E.; Soundararajan,
N.; Platz, M. S. Bioconjugate Chem. 1993, 4, 172.
(6) The closest transformation is found in the conversion of organic
azides to diazo compounds; see: Meyers, E. L.; Raines, R. T. Angew.
Chem., Int. Ed. 2009, 48, 2359.
r
10.1021/ol402404n
Published on Web 09/23/2013
2013 American Chemical Society

interconversion or using the azide’s reactivity to install a
diazirine, we were surprised to find that there were none.6
Herein we report a first generation compound that selec-
tively reacts with azides to install a minimally perturbing
diazirine functionality via a traceless Staudinger reaction.
(Figure 2BꢀC).10 Weenvisioned thatatraceless Staudinger
reagent with a small linker to a diazirine would serve our
needs and be of use to the growing community of chemists
and biochemists who use organic azides. For our applica-
tion, the major limitation of Staudinger chemistry, namely
the slow reaction rate, is greatly outweighed by its remark-
able selectivity.11 We were somewhat concerned that we
did not find examples of a diazirine-containing compound
that was subjected to compounds containing phosphorus
at our desired oxidation state. Diazirines are somewhat
electrophilic, and if they readily react with phosphines,
that reactivity would terminate our quest at the onset.
Certainly triphenylphosphine reacts with highly electron
deficient diazo dicarboxylate compounds under mild con-
ditions in the context of Mitsunobu chemistry, but the
combination of a straightforward synthetic route, and the
lack of precedent that clearly showed adverse phosphine-
diazirine reactivity prompted us to continue with our
strategy.12
Figure 1. Extending the bioorthogonal utility of organic azides.
Scheme 1. Synthesis of PhosDAz
With several traceless Staudinger platforms to choose from
we weighed the options. Raines’ thiol-ester (Figure 2C)
has proven most efficient for appending amino acids with
chirality R to the amide bond being formed,13 but the
requisite diazirine-containing portion to be ligated was
sterically unencumbered. As such we reasoned that any
traceless Staudinger scaffold that had been shown to
efficiently transfer simple acyl groups to form amides
from azides would serve well as proof of concept for
further optimization studies. Starting with commercially
available 2-hydroxydiphenylphosphinylbenzene (1, Scheme 1),
we coupled diazirine acid 2 using standard coupling
conditions to give PhosDAz (3) in excellent yield. Upon
establishing that PhosDAz was not self-destructing or
overly prone to oxidation it was treated with benzyl azide
to cleanly provide amide 4 in 96% yield (Scheme 2).
Indeed, PhosDAz is stable for months when stored in a
freezer, and also stable at room temperature in a DMSO/
water solution.14
Figure 2. Staudinger ligations. (A) A generic example of
Bertozzi’s Staudinger ligation.7 An acetate functionality can
be delivered by traceless Staudinger ligations reported by either
Bertozzi (B)10b or Raines (C).10a
The Staudinger ligation, developed by Bertozzi, takes
advantage of the exquisite selectivity that phosphines have
toward azides.7,8 The nucleophilic aza-ylide that is formed
upon loss of nitrogen gas can be trapped in an intramole-
cular fashion by a well-positioned reactive carbonyl as
opposed to simply hydrolyzing as happens with a tradi-
tional Staudinger reaction (Figure 2A).9 The chemistry of
Staudinger reactions continued to progress, and later in
2000 the traceless Staudinger ligation, where the oxidized
phosphine is not covalently bound to the resulting amide,
was independently reported by both Raines and Bertozzi
Benzyl azide does not suffer from steric congestion, and
its simplicity makes it an ideal model azide for testing
general reactivity. To get a better sense of the utility of
PhosDAz we sought to test it on more complex, congested
(11) (a) Lin, F.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686. (b) Soellner, M. B.;
Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820.
(12) (a) Swamy, K. C. K.; Kumar, N. N. B.; Balaraman, E.; Kumar,
K. V. P. P. Chem. Rev. 2009, 109, 2551. (b) During the course of this
work, a paper wherein diazirines were submitted to conditions that
contained triphenyl phosphine demonstrated that the diazirine func-
tionality remained intact; see ref 5e.
(7) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
(8) Schilling, C. I.; Jung, N.; Biskup, M.; Schepers, U.; Brase, S.
Chem. Soc. Rev. 2011, 40, 4840.
(9) (a) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635. (b)
Gololobov, Y. G. Tetrahedron 1981, 37, 437.
(10) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,
2, 1939. (b) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000,
2, 2141.
€
(13) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3, 9.
Org. Lett., Vol. 15, No. 19, 2013
5061

Scheme 2. Reaction of PhosDAz with Benzyl Azide
substrates. Hydrolysis ofthe aza-ylide (bracketed in Scheme 2)
instead of ligation is always a concern with Staudinger
ligations, and if the chemical environment is too con-
gested then the desired amide bond can fail to form
resulting in an amine and oxidized phosphine.
To test more complex, congested azides we focused our
effort on the synthesis of two diazirinyl sugars (Scheme 3).
These sugars had been previously synthesized by Kohler,
and she has shown them to be useful for the study of
glycosylation in living cells.15 The requisite peraceylated
2-azido-2-deoxy-glucose, 5, was synthesized in two steps
from glucosamine hydrochloride using diazotransfer16
chemistry followed by peracetylation of the remaining
alcohols. We noted that the reaction of 5 with PhosDAz
proceeded more slowly than that with benzyl azide, but
upon consumption of 5, the resulting sugar, Ac4GlcNDAz
(6), was obtained in 76% yield. Ac4GalNDAz (7) was also
synthesized using an analogous route, with the final cou-
pling step providing the desired diazirine-containing sugar
in 70% yield.
Figure 3. (A) One-drop synthesis of Rhodamine B conjugated
to a diazirine (Rhod-DAz) and photo-cross-linking to bovine
serum albumin (BSA). (B) SDS-PAGE gel showing passivized/
photobleached BSA18 treated with Rhod-DAz and either
not-exposed (ꢀ) or exposed (þ) to 365 nm light for 10 min
(Lanes 4 and 5 respectively). A control rhodamine conjugated to
ethanolamine (rhod-OH) showed minimal light-dependent la-
beling (Lanes 2 and 3). The fluorescent signal in the BSA sample
exposed to photo-cross-linking conditions is significantly more
intense (bottom gel scan). The coomassie stained gel (top gel
scan) shows that lanes were loaded with similar amounts
of protein.
versions of the reagent. The initial approach we are pursu-
ing is analogous to that of Raines’, who developed a water-
soluble traceless Staudinger reagent.17 We expect that
these next generation probes will further expand the utility
of this transformation and facilitate our work with DENV.
In the meantime, in spite of its poor water solubility, we
sought to explore the utility of PhosDAz in a more com-
plex reaction environment. In the event, we used PhosDAz
to functionalize an in situ synthesized azide-containing
fluorophore and took that compound forward without
purification to photo-cross-link the fluorophore to a
model protein of interest (Figure 3A). For our fluorophore
we chose rhodamine B isothiocyanate because of its ease of
conjugation and absorption at a wavelength of light that is
red-shifted compared with the optimal diazirine photo-
cross-linking wavelength. A primary amine linked to an
azide via a short PEG chain to confer additional water
solubility served as a spacer between the fluorophore and
diazirine. The conjugation reactions were performed in
a drop of DMSO and monitored using ReactIR, where
the disappearance of initially the isothiocyanate and sub-
sequently the azide were followed. For our model protein
of interest we selected bovine serum albumin (BSA)
because of its ability to bind small organic compounds.
Scheme 3. Syntheses of Ac4GlcNDAz (6) and Ac4GalNDAz (7)
We expect that PhosDAz will find general utility among
those who wish to add the delicate diazirine functionality
to an azide-containing complex molecule, ubiquitous in
this age of click chemistry, without the need to perform
functional group interconversions. For our research pur-
poses, attempts to test PhosDAz in a purely aqueous
environment were thwarted by poor water solubility,
something that we are currently addressing with new
(14) A solution of PhosDAz in degassed DMSO-d6/D2O (80:20) was
monitored for degradation by NMR. After 24 h at room temperature no
degradation was observed by proton or phosphorus NMR. This is
longer than a typical traceless Staudinger reaction is allowed to proceed.
(15) (a) Bond, M. R.; Zhang, H.; Vu, P. D.; Kohler, J. J. Nat. Protoc.
2009, 4, 1044. (b) Yu, S.-H.; Boyce, M.; Wands, A. M.; Bond, M. R.;
Bertozzi, C. R.; Kohler, J. J. Proc. Natl. Acad. Sci. U.S.A. 2012,
109, 4834. (c) Bond, M. R. Ph.D. Thesis, Stanford University, 2010.
(16) Ye, H.; Lui, R.; Li, D.; Liu, Y.; Yuan, H.; Guo, W.; Zhou, L.;
Cao, X.; Tian, H.; Shen, J.; Wang, P. G. Org. Lett. 2013, 15, 18.
(17) Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2007,
129, 11421.
(18) Significant background photo-cross-linking was observed when
the BSA had not been previously exposed to 365 nm in the presence of a
thiol scavenger.
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Org. Lett., Vol. 15, No. 19, 2013

This binding was expected to provide a sufficiently tight
interaction to trap the highly reactive carbene generated
upon liberation of nitrogen from the diazirine. We em-
ployed a UV-LED emitting at 365 nm as our light source,
chosen for its narrow peak width in the lower energyregion
of wavelengths that cleave diazirines. Light-dependent
fluorescent labeling of BSA was confirmed by SDS-PAGE
analysis. Due to the intrinsic ability of BSA tobind tosmall
molecules we always observed a small amount of back-
ground labeling, eveninthe absence ofphoto-cross-linking
conditions, but irradiation for 10 min provided a BSA
sample with significantly enhanced fluorescence compared
with the no-light control (Figure 3B).
In conclusion, we reported a reagent, PhosDAz, that
bridges the gap in reactivity between azides and diazirines.
We expect that it will be of general use to any research that
seeks to confer photo-cross-linking ability to otherwise
photo-unresponsive alkyl azides in complex samples. The
ease of azide incorporation into organic compounds and
complex biological macromolecules makes the azide a
powerful functional group, and a one-step process to
convert those azides to amide-containing diazirines will
prove broadly useful. Finally, the remarkable simplicity by
which this reagent is made, from a commercially available
phosphine and diazirine, makes it accessible to a wide
spectrum of chemists and biochemists.
Acknowledgment. This work was supported by funding
provided to J.C.J. by the University of Arizona COS, Bio5,
and CBC. S.M.J. was partially supported by an NIH
predoctoral training grant (T32 GM008804). We would
like to thank Dr. J. Njardarson (University of Arizona) for
feedback regarding this work and Dr. I. Ghosh (University
of Arizona) for use of his gel scanner.
Supporting Information Available. The preparation and
characterization of the compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 19, 2013
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