A homologation approach to the synthesis of difluorinated cycloalkynes

Article abstract of DOI:10.1021/ol500260d

Difluorinated cyclooctynes are important reagents for labeling azido-biomolecules through copper-free click chemistry. Here, a safe, scalable synthesis of a difluorinated cyclooctyne is reported, which involves a key homologation/ring-expansion reaction. Sequential ring expansions were also employed to synthesize and study a novel difluorinated cyclononyne.

Full text of DOI:10.1021/ol500260d

Letter
pubs.acs.org/OrgLett
A Homologation Approach to the Synthesis of Difluorinated
Cycloalkynes
Ellen M. Sletten,†,# Gabriela de Almeida,† and Carolyn R. Bertozzi*,†,‡,§
†Department of Chemistry, University of California, Berkeley, California 94720, United States
‡Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States
§Howard Hughes Medical Institute, United States
S
* Supporting Information
ABSTRACT: Difluorinated cyclooctynes are important re-
agents for labeling azido-biomolecules through copper-free
click chemistry. Here, a safe, scalable synthesis of a
difluorinated cyclooctyne is reported, which involves a key
homologation/ring-expansion reaction. Sequential ring ex-
pansions were also employed to synthesize and study a
novel difluorinated cyclononyne.
opper-free click chemistry, the reaction between an azide
and a strained cycloalkyne (Figure 1A), has become a
popular ligation strategy for applications that are sensitive to
metals, precluding the use of the copper-catalyzed azide−alkyne
cycloaddition (CuAAC, click chemistry, Figure 1B).1 To date,
the study of live cells and organisms has been the premier
application of cyclooctyne reagents;2 however, the simple
reaction conditions and purification procedures of copper-free
C
click chemistry have resulted in its expansion to a variety of
disciplines including surface immobilization,3 gel synthesis,4
oligonucleotide functionalization,5 dendrimer modification,6
and the decoration of nanostructures.7
The initial report of copper-free click chemistry involved the
use of cyclooctyne 1 (OCT, Figure 1C) for the labeling of
glycan-associated azides.8 This reaction was selective and mild,
but its relatively slow rate remained a liability for many
applications. Since that time, our group and others have focused
on enhancing the rate of the azide-cyclooctyne cycloaddition by
applying classical physical organic chemistry principles to
further activate the strained alkynes for reaction with azides.
Through these experimental efforts, two rate-enhancement
strategies have emerged: the addition of electron-withdrawing
groups at the propargylic position (MOFO 2, DIFO 3)2a,9 and
the augmentation of strain energy through fused aryl (DIBO 4,
DIBAC 5, BARAC 6)10 or cyclopropyl (BCN 7) rings.11 The
second-order rate constant for the [3 + 2] cycloaddition is an
important consideration in the choice of a cycloalkyne reagent,
as rapid reaction kinetics allow for low concentrations of
reagents and offer opportunities for real-time labeling.
However, superior reaction kinetics do not come without
liabilities. For example, BARAC has a limited half-life (24 h) in
phosphate-buffered saline due to hydrolysis of the strained
central amide10c and undergoes rearrangement to indole
products under acidic conditions.12 The balance between
reactivity and stability is a significant challenge, as efforts to
synthesize new cyclooctyne reagents are pursued.13
Despite slower reaction kinetics than BARAC, difluorinated
cyclooctynes have remained useful reagents for copper-free
click chemistry. DIFO is an exceptional cyclooctyne for labeling
glycan-associated azides on the enveloping layer of zebra-
Figure 1. (A) Copper-free click chemistry. (B) Copper-catalyzed
azide−alkyne cycloaddition (CuAAC). (C) Selected cyclooctyne
reagents for copper-free click chemistry.
Received: January 23, 2014
Published: March 3, 2014
© 2014 American Chemical Society
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Letter
fish.2b,14 Also, propargylic difluorination is a much less sterically
encumbering rate-enhancement strategy than aryl-ring fusion.
Smaller reagents are advantageous for reaching hindered azides
as well as for the modification of biomolecules with
cycloalkynes. Recent work exemplifies this as Lemke and co-
workers have discovered that cyclooctyne, but not the larger
dibenzocyclooctyne, can be site-specifically incorporated into
proteins through amber stop codon techniques.15
Scheme 2. Homologation Approach to DIFO2
The major limitation of DIFO surrounds its synthetic
intractability, particularly when compared to other biaryl-
based cyclooctynes with similar reaction kinetics (DIBO,
DIBAC). Previous work from our group addressed this
problem through the synthesis of difluorinated cyclooctyne 8
(DIFO2, Scheme 1), where difluorinated cyclooctanone 9 was
Scheme 1. Retrosynthetic Analysis of Difluorinated
Cycloalkynes from Cyclic 1,3-Diketones
Wittig olefination.20 Despite this limitation, the ring expansion
approach is the preferred method of DIFO2 synthesis within
our group, as it precludes the need for potentially explosive or
highly pyrophoric reagents such as periodic acid and diethyl
zinc.
The homologation strategy also provided a facile opportunity
to access a difluorinated cyclononyne analog through
consecutive ring expansions. Cyclononynes have significantly
less strain energy than their eight-membered ring congeners;
however, they offer valuable insight into the fundamental
physical organic chemistry of copper-free click chemistry,
particularly when all other aspects of the cycloalkynes are
identical. Additionally, slower azide-reactive reagents may prove
useful for applications where proximity-induced reactivity is
desired, such as the study of protein−protein interactions or
binding-promoted ligations.21
a key intermediate.16 The synthesis of DIFO2 was much
improved; however, its reported starting material 1,3-cyclo-
octadione (10) is not readily available in large quantities.
Efforts to improve the safety and scalability of 1,3-cyclo-
octadione have been pursued but remain low yielding.17
To synthesize the target cyclononyne, compound 9 was
treated with trimethylsilyl diazomethane in the presence of
trimethylaluminum followed by acidic conditions to remove the
resulting trimethylsilyl group (Scheme 3). Ketone 13 was
Here, an alternative approach to DIFO2 that begins from the
much more synthetically accessible 1,3-cycloheptadione (12)
and relies upon a key homologation with trimethylsilyl
diazomethane is described. Additionally, two sequential ring
expansions were employed to synthesize cyclononanone 13,
which could be readily transformed into difluorinated cyclo-
nonyne 14 (DIFN). Cycloheptadione 12 was prepared in three
steps from cyclopentanone and dichloroacetyl chloride as
previously reported.18 This procedure has been used to safely
produce up to 50 g of 1,3-cycloheptadione.18b The gem-difluoro
group was installed by treatment of 12 with cesium carbonate
and Selectfluor to give 2,2-difluoro-1,3-cycloheptadione (15)
(Scheme 2). Desymmetrization of 15 was achieved through a
mono-Wittig reaction with phosphonium salt 16 to yield
compound 17. Hydrogenation of the resulting olefin
quantitatively gave ketone 11, which was treated with
trimethylsilyl diazomethane, a safe diazo species,19 in the
presence of trimethylaluminum to yield the eight-membered
ring scaffold. The trimethylsilyl group was hydrolyzed by
treatment with acid to produce difluorinated ketone 9, which
can be taken on to difluorinated cyclooctyne 8 as previously
reported.16 Comparison of the two DIFO2 syntheses reveals
that the homologation approach has a lower overall yield. This
is primarily due to the desymmetrization reaction between 15
and 16 where it is difficult to suppress the competing double
Scheme 3. Synthesis of Difluorinated Cyclononyne 14
readily isolated and converted to the desired alkyne through in
situ vinyl triflate formation followed by syn-elimination of triflic
acid to yield the target cyclononyne (DIFN, 14).
With DIFN in hand, the second-order rate constant for the
reaction of 14 with benzyl azide was measured using an NMR
assay (Scheme S1). The increased ring size led to a reaction
rate that is 3 orders of magnitude less than that of DIFO2 (5.92
× 10−5 2 × 10−7 M−1 s−1 vs 4.2 × 10−2 M−1 s−1 for 14 and 8,
respectively).16 This second-order rate constant is similar to
previously reported monobenzocyclononynes (10−4 to 10−5
M−1 s−1).22 Interestingly, our previous work has shown that
difluorinated cyclooctynes are over 10 times more reactive with
benzyl azide than monobenzocyclooctynes13 suggesting that
electronic modulation, bond polarization, and/or transition
state stabilization23 due to propargylic difluorination have a
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Scheme 4. Synthesis and Reactivity of Tri- and Tetrafluorinated Cycloheptanone Homologation Substrates
large effect only when an alkyne is highly strained. Recent
computational work by Alabugin and co-workers23c has
indicated that minimal reactant destabilization (i.e., low strain
energies) can be completely compensated for by transition-
state stabilization in copper-free click chemistry. DIFN and
other published cyclononynes with experimentally character-
ized azide reactivity22 currently do not support Alabugin’s
conclusion as DIFN’s transition-state stabilizing gem-difluoro
group does not result in increased reactivity with azides.
However, these cyclononynes have not been explicitly designed
to take advantage of transition-state stabilization. Alabugin’s
work exemplifies that although the array of copper-free click
chemistry reagents is rapidly growing, a larger set is still
necessary to understand the importance of each chemical
interaction during the cycloaddition reaction.
ketone. Thus, the homologation methodology is restricted to
the synthesis of gem-difluorocycloalkynes. Using this strategy,
novel difluorinated cyclononyne 14 was synthesized and found
to react with benzyl azide 3 orders of magnitude more slowly
than difluorinated cyclooctyne 8. These data indicate that the
primary importance in the design and synthesis of reactive
cycloalkynes for copper-free click chemistry is ring strain,
followed by electronic activation and steric effects. The
synthesis and analysis of difluorinated cyclononyne 14
represents another important addition to the collection of
copper-free click chemistry reagents, as it will further guide the
design of new more reactive reagents, such as transition-state
stabilized or additionally fluorinated cycloalkynes. New
synthetic strategies toward the putatively highly reactive tri-
and tetrafluorinated cyclooctynes are underway.
Toward this end, and in hopes of achieving highly azide-
reactive reagents, the homologation approach was chosen as a
key retrosynthetic step in the synthesis of tri- and
tetrafluorinated cyclooctynes. Properly substituted ketone
intermediates 18 and 19 became our target ring-expansion
products (Scheme 4). These derivatized cyclooctanones could
be converted to tetra- and trifluorinated cyclooctynes,
respectively, by established methods.1a,2a,9 Tetrafluorinated
ketone 20 and trifluorinated ketone 22 were prepared through
established α-fluorination methods of imine (for 20)24 or silyl
enol ether (for 22 via 21)2a formation followed by treatment
with Selectfluor. However, upon treatment of 20 and 22 with
an array of diazo species in the presence of a variety of Lewis
acids, the desired ring-expansion products were not obtained.25
Instead, primarily oxirane formation (23, 24) was observed,
indicating that 1,2 migration of an electron-deficient C−CFn
bond is disfavored. The high regioselectivity observed in the
homologation of 11 also supports this hypothesis. Thus, it
appears that the homologation strategy is not viable for the
preparation of highly fluorine-substituted (F atoms >2)
cycloalkynes.
A safely scalable synthesis of the second-generation
difluorinated cyclooctyne 8, a reagent that has seen success in
the labeling of azides in living organisms as well as use in
materials science, was devised. The central step in this new
synthesis is a one-carbon ring expansion employing trimethyl-
silyl diazomethane, which proceeds readily on substituted
difluorocycloheptanone and the larger difluorocyclooctanone
substrates; however, the homologation reaction was suppressed
when fluorine atoms were added to both α positions of the
ASSOCIATED CONTENT
* Supporting Information
Procedures and characterization for all new compounds, kinetic
analysis of DIFN, and further discussion of the ring expansion
of tri- and tetrafluorinated ketones. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: crb@berkeley.edu.
Present Address
#Massachusetts Institute of Technology, Cambridge, MA
02139.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant to C.R.B. from the NIH
(GM058867), an ACS Organic Division Fellowship to E.M.S.,
and a predoctoral Ruth L. Kirschstein National Research
Service Award to G.d.A. We thank Jason Kingsbury and Victor
Rendina for helpful discussions regarding the homologation of
20 and 22.
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