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 defens that exist in a biochemical system. The Trojan workhor in this
area is the azide, a small but highly energetic functionality that reacts lectively with
veral 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 prent within living
systems.
Our interest in studying host-pathogen interactions associated with dengue virus (DENV), a
flavivirus that infects between 50 and 100 million people per year, led us to investigate
none.6 Herein we report a first generation compound that lectively reacts with azides to install a minimally perturbing diazirine functionality via a traceless Staudinger reaction.The Staudinger ligation, developed by Bertozzi, takes advantage of the exquisite lectivity that phosphines have towards azides.7, 8 The nucleophilic aza-ylide that is formed upon loss of nitrogen gas can be trapped in an intramolecular fashion by a well-positioned reactive carbonyl as oppod to simply hydrolyzing as happens with a traditional Staudinger reaction (Figure 2A).9 The chemistry of Staudinger reactions continued to progress and later in 2000the traceless Staudinger ligation, where the oxidized phosphine is not covalently bound to the resulting amide bond, was independently reported by both Raines and Bertozzi (Figure 2B-C).10 We envisioned that a traceless Staudinger reagent with a small linker to a diazirine would rve our needs and be of u to the growing community of chemists and biochemists who u organic azides. For our application, the major limitation of Staudinger chemistry,namely the slow reaction rate, is greatly outweighed by its remarkable lectively.11 We were somewhat concerned that we did not find examples of a diazirine-containing compound that was subjected to compounds containing phosphorous at our desired oxidation state. Diazirines are somewhat electrophilic and if they readily react with phosphines, that reactivity would terminate our quest at the ont. Certainly triphenylphosphine reacts with highly electron deficient diazo dicarboxylate compounds under mild conditions in the cont
ext of Mitsunobu chemistry, but the combination of a straightforward synthetic route, and the lack of precedent that clearly showed adver phosphinediazirine reactivity prompted us to continue with our strategy.12With veral traceless Staudinger platforms to choo from we weighed the options. Raines’thiol-ester (Figure 2C) has proven most efficient for appending amino acids with chirality alpha 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 rve 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 lf-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
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 n of the utility of PhosDAz we
sought to test it on more complex, congested substrates. Hydrolysis of the aza-ylide
(bracketed in Scheme 2) instead of ligation is always a concern with Staudinger ligations
and if the chemical environment is too congested then the desired amide bond can fail to
form resulting in an amine and oxidized phosphine.
To test more complex, congested azides we focud our effort on the synthesis of two
diazirinyl sugars (Scheme 3). The sugars had been previously synthesized by Kohler and
she has shown to be uful for studying glycosylation in living cells.15 The requisite
peraceylated 2-azido-2-deoxy-gluco, 5, was synthesized in two steps from glucosamine
hydrochloride using diazotransfer 16 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, Ac 4GlcNDAz (6), was
obtained in 76% yield. Ac 4GalNDAz (7) was also synthesized using an analogous route,
with the final coupling step providing the desired diazirine containing sugar in 70% yield.
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We expect that PhosDAz will find general utility amongst tho who wish to add the
delicate diazirine functionality to an azide-containing complex molecule – ubiquitous in this
慨而慷
age of click chemistry – without the need to perform functional group interconversions. For
our rearch purpos, attempts to test PhosDAz in a purely aqueous environment were
thwarted by poor water solubility, something that we are currently addressing with new
上海到普陀山versions of the reagent. The initial approach we are pursuing is analogous to that of Raines’,
who developed a water soluble traceless Staudinger reagent.17 We expect that the 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 saught to explore the utility of
PhosDAz in a more complex reaction environment. In the event we ud PhosDAz to
functionalize an in situ synthesized azide-containing fluorophore and take that forward
without purification to photo-cross-link the fluorophore to a model protein of interest
(Figure 3A). For our fluorophore we cho rhodamine B isothiocyanate becau of its ea
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 rved as a spacer between the
fluorophor and diazirine. The conjugation reactions were performed in a drop of DMSO and
monitored using ReactIR, where the disappearance of initially the isothiocyanate and
subquently the azide were followed. For our model protein of interest we lected bovine
rum albumin (BSA) becau of its ability to bind small organic compounds. 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 employed a UV-LED emitting
at 365 nm as our light source, chon for its narrow peak width in the lower energy region of
wavelengths that cleave diazirines. Light-dependent fluorescent labeling of BSA was哈姆雷特人物形象分析
confirmed by SDS-PAGE analysis. Due to the intrinsic ability of BSA to bind to small
molecules we always obrved a small amount of background labeling – even in the abnce
of photo-cross-linking conditions – but irradiation for 10 minutes provided a BSA sample
with signicantly 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 u to any rearch that eks to
confer photo-cross-linking ability to otherwi photo-unresponsive alkyl azides in complex
双顶径和股骨长samples. The ea of azide incorporation into organic compounds and complex biological
macromolecules makes the azide a powerful functional group and a one-step process to
convert tho azides to amide containing diazirines will prove broadly uful. Finally, the
药苍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
水浒第八回biochemists.Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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, Dr. I. Ghosh (University of Arizona) for u
of his gel scanner.
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18. Significant background photo-cross-linking was obrved when the BSA had not been previously
expod to 365 nm in the prence of a thiol scavenger.
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Figure 1.Extending the bioorthogonal utility of organic azides
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