Synthesis of trifunctional bis-azide photoaffinity probe

Article abstract of DOI:10.1080/00397911.2012.697595

Methyl 3-azidomethyl-5-azido-benzoate (1b) and its corresponding hydroxymethyl reduction product (1a) have been utilized for the synthesis of valuable nonradioactive photoaffinity probes. Previous preparations of (1a) depend upon a nonselective monoactiva

Full text of DOI:10.1080/00397911.2012.697595

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Synthesis of Trifunctional Bis-azide
Photoaffinity Probe
Larry L. Klein a & Valentina Petukhova a
a Institute for Tuberculosis Research, University of Illinois at
Chicago , Chicago , Illinois , USA
Accepted author version posted online: 26 Jan 2013.Published
online: 03 Jun 2013.
To cite this article: Larry L. Klein & Valentina Petukhova (2013): Synthesis of Trifunctional Bis-azide
Photoaffinity Probe, Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 43:16, 2242-2245
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Synthetic Communications1, 43: 2242–2245, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2012.697595
SYNTHESIS OF TRIFUNCTIONAL BIS-AZIDE
PHOTOAFFINITY PROBE
Larry L. Klein and Valentina Petukhova
Institute for Tuberculosis Research, University of Illinois at Chicago,
Chicago, Illinois, USA
GRAPHICAL ABSTRACT
Abstract Methyl 3-azidomethyl-5-azido-benzoate (1b) and its corresponding hydroxy-
methyl reduction product (1a) have been utilized for the synthesis of valuable nonradioac-
tive photoaffinity probes. Previous preparations of (1a) depend upon a nonselective
monoactivation of a bis-1,3-hydroxymethylaryl intermediate, leading to the expected stat-
istical range of products=starting material. We report an alternate synthetic approach to
these 1,3,5-trisubstituted molecules using recently described C-H activation and boronate
refunctionalization methods.
Supplemental materials are available for this article. Go to the publisher’s online edition
of Synthetic Communications1 to view the free supplemental file.
Keywords Azide; borylation; photoaffinity
INTRODUCTION
Photoaffinity labeling is a useful tool for investigation of ligand–protein
interaction.[1] This process involves the use of a binding substrate (probe) that has
been modified with a photoactive functional group. Upon irradiation, this group
produces a chemoreactive center (e.g., carbene, nitrene), which can form a covalent
bond to proximal atoms in the protein, thus establishing these proteins as potential
binding targets for this molecule. The actual photoactive groups have included
benzophenones,[2] diazirines,[1,3] and azides,[4] among others, with the former being
less reactive=more selective and the latter being more reactive=less selective. Various
nonradioactive methods have been used to identify these proteins and usually
involve a second chemical connection (‘‘tagging’’) to rhodamines=fluoresceins,[5]
Received May 10, 2012.
Address correspondence to Larry L. Klein, Institute for Tuberculosis Research, University of
Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, USA. E-mail: llk@uic.edu
2242

TRI-FUNCTIONAL PROBE
2243
biotin,[6] or fluorous-related[7] derivatives to isolate the ‘‘tagged’’ protein via visual or
affinity-binding techniques.
DISCUSSION
In recent years, molecules containing a bisazide moiety such as 1a–c (Fig. 1)
have been utilized as a photoaffinity probes[4] to identify or characterize proteins
involved in calcium ion release[8] and HDAC8 inhibitors.[9] In these cases, the ester
or sulfonate group functionality serves as this probe’s point of attachment via amide
or ether linkage, respectively. For this tagging process to be successful, the attach-
ment must occur on the ligand at sites that do not interfere with binding to the pro-
tein. The aryl azide in 1c is selectively photoactivated in the presence of the more
stable benzyl azide[4] and forms the covalent link to the protein. Subsequent Huisgen
reaction of the benzyl azide with a pendant alkyne group of the visualizing agent is
carried out to assist in the detection=isolation of the probe-linked protein. The
1,3,5-substitution pattern was key to isolating the three orthogonal synthetic pro-
cesses for this novel moiety.
The synthesis of probes 1a and 1b have been reported[4] and modified in later
work.[10] In both cases, the key limiting reaction involves a statistical monofunctio-
nalization of the intermediate diol 2, which in turn is prepared from commercially
available diester 3. We were in need of this useful probe and realized that this non-
selective reaction of 2 could be avoided through the use of recently evolving technol-
ogies of C-H activation and boronaterefunctionalization and so report an alternate
synthesis of 1 herein.
Malezcka and Smith[11] and Hartwig[12] reported an efficient regioselective
formation of 1,3,5-trisubstituted aromatic molecules via the use of iridium-based cat-
alyzed borylation reaction. Treatment of methyl 3-methylbenzoate under modified
borylation conditions[13] led to a good yield of the desired regioisomer 6
(Scheme 1). Although consistent reports of the yield of 6 were 91–96%, using non-
Schlenk techniques and microwave irradiation, we were only able to obtain 48%
yield on a multigram scale. With this key intermediate in hand, we found that radical
bromination[14] could be successfully carried out to afford 7, containing a small
amount of the purported a,a-dibromide after recrystallization from cyclohexane.
Because of some instability, this material was used directly for subsequent bromide
displacement with sodium azide to give the corresponding monoazide 8. Recent
Figure 1. Structure of photoaffinity label.

2244
L. L. KLEIN AND V. PETUKHOVA
Scheme 1. Alternate synthesis of 1a. Reagents: (a) B2Pin2, [COD(OMe)Ir]2, dtbpy, hexane; (b) NBS,
Bz2O2, CCl4; (c) 1.5 eq. NaN3, MeOH; (d) 1.5 eq. NaN3, 0.1 eq. CuOAc, MeOH; (e) 3 eq. NaN3, 0.1
eq. CuOAc, MeOH; (f) 4 eq. DiBAH (1 M in hexanes); (g) 2 eq. PhCꢀCH, 0.4 eq. sodium ascorbate,
0.1 eq. Cu2SO4, CH3CN=H2O.
work in the area of copper-catalyzed refunctionalization of boronates with a wide
variety of nucleophiles[15] suggested that the aryl azide could be formed in a subse-
quent step from substrates such as 8.
Treatment of azido-boronate 8 with sodium azide in the presence of catalytic
copper acetate smoothly led to the known bis-azido-ester 1b[4] in good yield. To
further optimize this process, bromide 7 was directly reacted with 3 equivalents of
sodium azide in the presence of copper acetate to afford 1b in 65% yield without
any intervening chromatography. Because the azide products produced no parent
ion under standard mass spectrometry (MS) conditions, further structure proof
for 1b was obtained by derivatizing this compound via ‘‘Click’’ chemistry to give
the bis-adduct 9. A sample of 1b was treated under standard Huisgen conditions
(2 eq. phenyl acetylene, 0.4 eq. sodium ascorbate, 0.1 eq. copper sulfate, in 10%
aqueous acetonitrile stirred at room temperature) for 2 days. The solvents were eva-
porated, and the residue was purified to give an analytical sample of bis-triazole 9.[16]
Although the ester 1b has been coupled via the Staudinger–Bertozzi method-
ology to give amide-linked probes,[17] our work required the corresponding ether
linkage arising from coupling of our protein-binding entity to the benzyl tosylate.
Through the action of diisobutylaluminum hydride, ester 1b was reduced to the
desired alcohol 1a in 74% yield from which the tosylate or mesylate intermediate
can be accessed as per literature.[4,10]

TRI-FUNCTIONAL PROBE
2245
CONCLUSION
This alternate route provides probe 1a–c from easily available starting materi-
als by taking advantage of the continuously evolving C-H activation and boronate-
functionalization protocols.
SUPPORTING INFORMATION
1
Synthetic experimental details and H and 13C NMR characterizations of 1a,
1b, 6, 7, and 8 are available online. HRMS details for bis-‘‘click’’ adduct of 1b
(cpd. 9) are also included.
ACKNOWLEDGMENTS
This work was financially supported by the Potts Foundation (Grant No.
95440). We thank Scott G. Franzblau (director of the Institute for Tuberculosis
Research) for his support, encouragement, and assistance in reviewing this manu-
script. We also thank Raghupathi Neelarapu (Petukhov group, University of Illinois
at Chicago) for an authentic sample of 1a.
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