A hydrophilic azacyclooctyne for cu-free click chemistry

Article abstract of DOI:10.1021/ol801141k

(Chemical Equation Presented) Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4- yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

Full text of DOI:10.1021/ol801141k

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2008
Vol. 10, No. 14
3097-3099
A Hydrophilic Azacyclooctyne for
Cu-Free Click Chemistry
Ellen M. Sletten and Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes
Medical Institute, UniVersity of California, The Molecular Foundry, Lawrence
Berkeley National Laboratory, Berkeley, California 94720
crb@berkeley.edu
Received May 19, 2008
ABSTRACT
Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity
can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne
(DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes.
However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.
A growing area of reaction methodology focuses on the
development of chemical transformations with the selectivity,
kinetics, and biocompatibility required to proceed in living
systems.1 Several efforts in this area have converged on the
azide as a reagent. Its small size, diverse modes of reactivity,
and bioorthogonality make the azide an ideal chemical
reporter group for biomolecules.2 Azides can be incorporated
into glycans, lipids, and proteins using metabolic machin-
eries,2a–d
covalent
inhibitors,2e
or
enzymatic
transfers.2fStaudinger ligation with phosphines,2a Cu-cata-
lyzed cycloaddition with terminal alkynes (often referred to
as click chemistry),3 or strain-promoted cycloaddition with
Figure 1. Cu-free click chemistry with (A) cyclooctyne reagents
or (B) 6,7-dimethoxyazacyclooct-4-yne (DIMAC) reagents.
(1) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13–21.
(2) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010. (b)
Dieterich, D. C.; Link, A. J.; Graumann, J.; Tirrell, D. A.; Schuman, E. M.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9482–9487. (c) Hang, H. C.;
Geutjes, E. J.; Grotenbreg, G.; Pollington, A. M.; Bijlmakers, M. J.; Ploegh,
H. L. J. Am. Chem. Soc. 2007, 129, 2744–2755. (d) Sawa, M.; Hsu, T-L.;
Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt, P. K.; Wong, C-H. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 12371–12376. (e) Speers, A. E.; Adam,
G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686–4687. (f)
Fernandez-Suarez, M.; Baruah, H.; Martinez-Hernandez, L.; Xie, K. T.;
Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Nat. Biotechnol. 2007, 25, 1483–
1487.
cyclooctynes4 allow for modification of the azido biomol-
ecules with diverse probes. The latter reaction, also termed
Cu-free click chemistry (Figure 1A), was designed to
eliminate the cytotoxic metal catalyst required for conven-
tional click chemistry. Incorporating the alkyne into a strained
eight-membered ring system promoted the cycloaddition with
(3) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599. (b) Breinbauer, R.; Kohn,
M. ChemBioChem 2003, 4, 1147–1149.
(4) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046–15047.
10.1021/ol801141k CCC: $40.75
Published on Web 06/13/2008
2008 American Chemical Society

Scheme 1. Synthesis of DIMAC
azides without compromising selectivity in biological sys-
tems.4 Since the initial report, fluorination5 and fused phenyl
rings6 have improved the reaction kinetics, and a difluori-
nated analogue has been employed to image glycans in
developing zebrafish.7
zinc reduction/reductive amination reaction followed by
amide formation with methyl succinyl chloride.10 The eight-
membered ring was generated by a Grubbs ring-closing
metathesis reaction to yield 3.11 Allylic alcohol 3 was
converted to ketone 4 via oxidation to the enone followed
by hydrogenation.
We explored two routes for conversion of the ketone to
the corresponding alkyne: vinyl triflate formation followed
by syn-elimination of triflic acid5 and formation of a
selenadiazole followed by fragmentation to the alkyne.12
Although successful in our previous cyclooctyne syntheses,5
the vinyl triflate method proved too harsh for the target
compound. Thus, we condensed compound 4 with semicar-
bazide and oxidized the resulting intermediate to yield
selenadiazole 5. Subsequent thermal decomposition of the
selenadiazole followed by saponification of the methyl ester
produced DIMAC (6).
The reactivity of DIMAC was tested in a model 1,3-dipolar
cycloaddition reaction with benzyl azide (Scheme S2,
Supporting Information). The reaction proceeded cleanly with
a second-order rate constant of 3.0 × 10-3 M-1 s-1 (Figure
S1, Supporting Information), slightly higher than that for the
parent cyclooctynes (1-2 × 10-3 M-1s-1).4,5a This slight
enhancement in rate might reflect added ring strain due to
the shorter C-N bond length or the sp2 character of the
amide nitrogen.13
These achievements reflect optimization of the reaction
for use with live cells and simple model organisms. However,
we envision applications of Cu-free click chemistry in
mammalian disease models where the bioavailability and
pharmacokinetic properties of the reagents become important.
The cyclooctynes currently employed for Cu-free click
chemistry comprise hydrocarbon scaffolds that limit their
solubility in aqueous solutions. The hydrophobicity of these
cyclooctyne scaffolds could also promote sequestration by
membranes or nonspecific binding to serum proteins, thereby
reducing their bioavailable concentrations. Consequently, we
have focused on designing strained cycloalkynes with
enhanced water solubility.
Here, we report the synthesis and biological evaluation
of a novel heterocyclic and heteroatom-substituted cyclooc-
tyne. The compound, 6,7-dimethoxyazacyclooct-4-yne
(DIMAC, Figure 1B), has a nitrogen atom within the strained
ring system that interrupts the hydrophobic surface area and
provides a convenient site for probe conjugation. We
reasoned that two methoxy groups would enhance the
compound’s polarity, and placement of one at the propargylic
position would impart reaction kinetics similar to existing
simple cyclooctynes.4,5a LogS calculations for DIMAC
methyl amide gave a value of ∼-2.7, while that of a parent
cyclooctyne methyl amide was ∼-3.1.8
Next, we tested the ability of DIMAC to label glycan-
associated azides within cell lysates and on the surface of
live cells. DIMAC was first conjugated to biotin (Figure 2A;
(8) Calculated for compounds 7 and 8 where R ) CH3 using logS
software developed by Tetko et al., www.vcclab.org/lab/alogps/. More
negative values indicate poorer water solubility. (a) Tetko, I. V.; Tanchuk,
V. Y.; Kasheva, T. N.; Villa, A. E. J. Chem. Inf. Comput. Sci. 2001, 41,
246–252. (b) Tetko, I. V.; Tanchuk, V. Y.; Kasheva, T. N.; Villa, A. E. P.
J. Chem. Inf. Comput. Sci. 2001, 41, 1488–1493.
As shown in Scheme 1, DIMAC was synthesized in nine
steps beginning from methyl 6-bromoglucopyranoside (1).9
First, compound 1 was transformed to acyclic diene 2 via a
(5) (a) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi,
C. R. ACS Chem. Biol. 2006, 1, 644–648. (b) Baskin, J. M.; Prescher, J. A.;
Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli,
J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793–
16797.
(9) (a) Jones, K.; Wood, W. W. Carbohydr. Res. 1986, 155, 217–222.
(b) Scheme S1, Supporting Information.
(10) Sletten, E. M.; Liotta, L. J. J. Org. Chem. 2006, 71, 1335–1343.
(11) Miller, S. J.; Kim, S. H.; Chen, Z. R.; Grubbs, R. H. J. Am. Chem.
Soc. 1995, 117, 2108–2109.
(6) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G-J. Angew. Chem., Int.
Ed. 2008, 47, 2253–2255.
(12) (a) Meier, H.; Voigt, E. Tetrahedron 1972, 28, 187–198. (b) Meier,
H.; Stavridou, E.; Storek, C. Angew. Chem., Int. Ed. 1986, 9, 809–810. (c)
Petersen, H.; Kolshorn, H.; Meier, H. Angew. Chem., Int. Ed. 1978, 17,
461–462.
(7) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R.
Science 2008, 320, 664–667.
3098
Org. Lett., Vol. 10, No. 14, 2008

Scheme S3, Supporting Information), providing the means
to detect its cycloadducts. For comparative purposes, we
performed parallel experiments with previously reported
cyclooctyne-biotin conjugate 8.5a The difference in water
solubility of these two reagents was apparent during the
preparation of 2.5 mM stock solutions. DIMAC-biotin 7
was readily soluble in aqueous buffer, whereas cyclooctyne-
biotin 8 required an organic cosolvent (30% DMF).
peroxidase (HRP)-conjugated R-biotin. Both DIMAC-biotin
(7) and the control cyclooctyne 8 labeled glycoproteins from
the lysate in an azide-dependent manner (Figure 2B).
However, compound 8 produced a greater extent of non-
specific protein labeling.
To test DIMAC’s reactivity with azides on live cells, Jurkat
cells were again incubated with 25 µM Ac4ManNAz for 3
days and then reacted with 0 or 250 µM 7 or 8. The cells
were stained with fluorescein isothiocyanate (FITC)-conju-
gated avidin and analyzed by flow cytometry. DIMAC-biotin
(7) and cyclooctyne-biotin 8 selectively labeled cell surfaces
in an azide-dependent manner with comparable efficiencies
(Figure 3C).14 Compound 8, however, showed significantly
higher nonspecific background labeling. The DIMAC reagent
labeled cells with a signal/background ratio of 26:1, whereas
the ratio for cyclooctyne 8 was 14:1. Indeed, the fluorescence
of cells lacking azides but treated with DIMAC-biotin
followed by FITC–avidin was indistinguishable from those
treated with FITC–avidin alone (Figure S4, Supporting
Information). Importantly, DIMAC did not show any cyto-
toxicity during cell labeling (Figures S5-7, Supporting
Information). DIMAC also exhibited time- and dose-de-
pendent labeling (Figure S8, Supporting Information).
In summary, DIMAC represents a new class of heterocy-
clic substrates for Cu-free click chemistry. Its superior
polarity and water solubility reduce nonspecific protein and
cell binding and improve the sensitivity of azide detection.
The synthetic route can be adapted to various carbohydrate
starting materials and is amenable to functional group
manipulations directed at tuning reactivity, solubility, and
perhaps biodistribution. DIMAC and its congeners are
therefore promising reagents for Cu-free click chemistry in
mammalian systems.
Figure 2. (A) Biotin conjugates of DIMAC (7) and a parent
cyclooctyne (8).5a (B) Western blot of Jurkat cell lysates. Cells
were treated with (+Az) or without (-Az) 25 µM Ac4ManNAz
for 3 d, and lysates (35 µg total protein) were reacted with 250
µM 7 or 8 overnight at 25 °C. Equal protein loading was confirmed
by Ponceau S staining (Figure S2, Supporting Information). The
blot was probed with HRP-conjugated R-biotin. (C) Flow cytometry
analysis of Jurkat cells. Cells metabolically labeled as in (B) were
reacted with 250 µM 7 or 8 for 1 h at 25 °C and then stained with
FITC-avidin. Similar data were obtained in three replicate experi-
ments.
Acknowledgment. We thank J. M. Baskin, S. T. Laughlin,
and J. A. Codelli at UC Berkeley for helpful discussions.
This work was supported by a grant to C.R.B. from the
National Institutes of Health (GM058867).
Supporting Information Available: Experimental pro-
cedures, protocols, and spectral data for all new compounds
as well as Figures S1-S8 and Schemes S1-S3. This material
is available free of charge via the Internet at http://pubs.acs.org.
Jurkat cells were grown in the presence (+Az) or absence
(-Az) of 25 µM N-azidoacetylmannosamine (Ac4ManNAz)
for 3 days, resulting in the metabolic labeling of cell-surface
glycans with N-azidoacetyl sialic acid (SiaNAz) residues.2a
Cell lysates were incubated overnight with 250 µM 7 or 8
and then analyzed by Western blot probing with horseradish
OL801141K
(13) Meier, H.; Hanold, N.; Molz, T.; Bissinger, H. J.; Kolshorn, H.;
Zountsas, J. Tetrahedron 1986, 42, 1711–1719.
(14) For a comparison of DIMAC to a difluorinated cyclooctyne as well
as a triarylphosphine for reaction via the Staudinger ligation, see Figure
S3, Supporting Information.
Org. Lett., Vol. 10, No. 14, 2008
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