Sequential Photochemistry of Dibenzo[a,e]dicyclopropa[c,g][8]annulene-1,6-dione: Selective Formation of Didehydrodibenzo[a,e][8]annulenes with Ultrafast SPAAC Reactivity

Article abstract of DOI:10.1021/acs.joc.6b01545

An order of magnitude difference in photoreactivity between bis- (photo-DIBOD, 1) and mono-cyclopropenone-caged dibenzocyclooctadiynes (MC-DIBOD, 5) allows for selective monodecarbonylation of 1. Alternatively, 5 is prepared by selective mono-cyclopropanation of dibenzo[a,e]cyclooctadiyne (DIBOD). MC-DIBOD (5) permits efficient sequential SPAAC cross-linking of azide-derivatized substrates. Cycloaddition to 5 converts an azide moiety into a photocaged form of triazole-fused dibenzo[a,e]cyclooctyne (3). While the azide reactivity of MC-DIBOD (5) and DIBOD is similar to that of other dibenzocyclooctynes, fusion of triazole to the dibenzocyclooctyne system in 3 results in a 3 orders of magnitude enhancement in SPAAC rates. In methanol, 3 reacts with butyl azide at an astonishing rate of 34 M^-1 s^-1, thus representing the most reactive cyclooctyne analogue reported so far. MC-DIBOD (5) was utilized in the preparation of mixed bis-triazoles and derivatization of the protein BSA with fluorescent dye and polyethylene glycol.

Full text of DOI:10.1021/acs.joc.6b01545

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Article
Sequential photochemistry of dibenzo[a,e]dicyclopropa[c,
g][8]annulene-1,6-dione: selective formation of
didehydrodibenzo[a,e][8]annulenes with ultrafast SPAAC reactivity
Dewey A Sutton, and Vladimir V. Popik
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01545 • Publication Date (Web): 16 Sep 2016
Downloaded from http://pubs.acs.org on September 20, 2016
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Sequential photochemistry of dibenzo[a,e]dicyclopropa[c,g][8]annulene-1,6-dione:
selective formation of didehydrodibenzo[a,e][8]annulenes with ultrafast SPAAC
reactivity
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Dewey A. Sutton and Vladimir V. Popik*
Department of Chemistry, University of Georgia, Athens, GA 30602
*vpopik@uga.edu
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Abstract: An order of magnitude difference in photo-reactivity between bis- (photo-
DIBOD, 1) and mono-cyclopropenone - caged dibenzocyclooctadiynes (MC-DIBOD, 5)
allows for selective mono-decarbonylation of 1. Alternatively, 5 is prepared by selective
mono-cyclopropanation of dibenzo[a,e]cyclooctadiyne. MC-DIBOD (5) permits efficient
sequential SPAAC cross-linking of azide-derivatized substrates. Cycloaddition to 5
converts an azide moiety into
a
photo-caged form of triazole-fused
dibenzo[a,e]cyclooctyne (3). While azide reactivity MC-DIBOD (5) and dibenzo[a,e]
cyclooctadiyne (DIBOD, 2) is similar to other cyclooctynes, fusion of triazole to
dibenzocyclooctyne system in 3 results in three orders of magnitude enhancement in
SPAAC rates. In methanol, 3 reacts with butyl azide at an astonishing rate of 34 M-1s-1,
thus representing the most reactive cyclooctyne analog reported so far. MC-DIBOD (5)
was utilized in the preparation of mixed bis-triazoles and derivatization of the protein
(BSA) with fluorescent dye and polyethylene glycol.
TOC Graphics:
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Introduction
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Catalyst-free cycloaddition of organic azides across the triple bond in cyclooctynes
(SPAAC)1 has become one of the most popular “click-chemistries” employed for the
functionalization, cross-linking, and immobilization of various substrates.2 The utility of
this technique is hampered by the need of the differential functionaliztion of the substrates
involved, one with an azide group, and the second with cyclooctyne moiety. The azide
functionalization is well developed for a large variety of substrates, but incorporation of
the cyclooctyne fragment is often synthetically challenging and expensive. Cross-linking
of two azide-derivatized units by a bis-cyclooctyne linker, therefore, provides an attractive
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alternative
to
cyclooctyne-azide
coupling.3,4,5
Sondheimer
diyne
(DIBenzo[a,e]cycloOctaDiyne or DIBOD 2) is the most atom economical double-SPAAC
cross-linker, as both strained triple bonds belong to the same eight-membered ring.4 The
parent dibenzocyclooctadiyne (2a) has found applications in the macrocyclization of bis-
azide functionalized peptides,6 post-synthetic modification of MOF thin films,7 protein
derivatization,4a the development of fluorescent dyes,8 and the preparation of tetramers
of HIV-related peptides.9 The utility of the DIBOD cross-linkers (2), however, is limited by
its short shelf lifetime and rapid decomposition in aqueous solutions ( ~ 10 min at pH =
½
7.4).10 To alleviate this shortcoming, we have recently synthesized photochemical
precursors to diynes 2a-c, dibenzo[a,e]dicyclopropa[c,g][8]annulene-1,6-dione (photo-
DIBOD, 1a-c).10 Both triple bonds in photo-DIBOD (2) are masked as cyclopropenone
moieties, which are known to undergo quantitative decarbonylation upon UVA11 or NIR12
photolysis. Photo-DIBODs (1) is a very stable compound, despite the presence of an
antiaromatic cyclooctatetraene core, substantial angle strain, and high unsaturation (DU=
15). Irradiation of photo-DIBOD (1) with 350 or 420 nm fluorescent lamps in the presence
of two azide-functionalized substrates results in the efficient formation of bis-triazole 4,
via the apparent formation of Sondheimer diyne (2) and mono-adduct (3, Scheme 1).10 In
addition to enhanced stability of the precursor 1, photochemical generation of reactive
diyne 2 allows for the control of the ligation of two azides in space and in time.13
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Scheme 1. Azide cross-linking using bis-caged dibenzo[a,e]cyclooctyne
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While DIBOD (2) is an efficient cross-linking reagent, selective conjugation of two
different azides is hard to achieve, because the addition of the second equivalent of azide
to mono-triazole 3 proceeds much faster than the first “click” step. The actual rates of the
addition of organic azides across the triple bond of the didehydro[8]annulene 3 were
unknown before the present work (vide infra). To achieve a high degree of selectivity in
the sequential conjugation of different azide-tagged substrates, we envisaged a strategy
utilizing a mono-cyclopropenone protected analog of Sondheimer diyne (6,7-didehydro-
1H-dibenzo[a,e]cyclopropa[c][8]annulene-1-one, MC-DIBOD, 5). The addition of the first
azide to MC-DIBOD (5) is followed by the photo-release of second triple bond, making it
available for the reaction with the second azide (Scheme 2, isomers of 3 and 4 are omitted
for clarity).
Scheme 2. Selective cross-linking of two azide-derivatized substrates using MC-DIBOD (5)
Our interest in this system was further piqued by the recent report of the
preparation of 4,9-dimethoxy-substituted analog of 5 (R1, R3= OCH3, R2= H)14 This
compound reacts with azide to give a fluorescent analog of triazole 6 (R1, R3= OCH3, R2=
H, Scheme 2), which was found to be photochemically inert. Since it was the first example
of a photo-stable and fluorescent cyclopropenone, known to the authors, we have decided
to conduct a detailed investigation of the photochemistry and photophysics of this system.
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Results and Discussion
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To synthesize MC-DIBODs 5a,b we have employed our recently developed
methodology of selective mono-cyclopropanation the symmetric diynes. Thus, the
treatment of the Sondheimer diyne 2a or 2,9-dibutoxy-substituted DIBOD (2b) with 1
equivalent of trifluoromethyltrimethylsilane15 and sodium iodide in dilute THF, followed by
silica gel - promoted hydrolysis afforded the mono-caged diyne in 70% yield (Scheme 3).
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Scheme 3. Synthesis of MC-DIBOD (5)
Interestingly, MC-DIBOD (5a) can be generated photochemically by selective
decarbonylation of one of the cyclopropenone moieties in photo-DIBOD (1a). The
controlled irradiation of methanol-DCM solution of bis-cyclopropenone 1a with 350 nm
fluorescent lamps results in the formation of MC-DIBOD (5a) in 90% preparative yield.
The main byproduct of this reaction is DIBOD (2a). MC-DIBOD (5a) can be also converted
into diyne 2, but that requires much longer irradiation. This selective sequential
decarbonylation of cyclopropenone moieties in photo-DIBOD (1a) is possible due to the
significant difference in quantum yields of the photoreaction between cyclopropenones
1a (350 = 0.05) and 5a (350 = 0.006). The loss of one carbonyl group results in the blue
shift of characteristic diaryl-substituted cyclopropenone bands from 342 (log ε=4.0) and
361 nm (log ε= 4.1) in 1a to 340 (log ε=3.8) and 353 nm (log ε=3.9) in 5a, accompanied
by a weak hypochromic effect (Fig. 1). The position and intensity of two stronger bands
at shorter wavelengths (260 and 268 nm, log ε~ 4.8) are not significantly affected by the
mono-decarbonylation of 1a.
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Wavelength (nm)
Figure 1. UV Spectra of ca. 0.22 mmol solution of photo-DIBOD (1a, solid line) and MC-DIBOD
(5a, dashed line) in MeOH-DCM (9:1).
MC-DIBODs 5a,b, as well as their parent photo-DIBODs 1a,b, show no detectable
fluorescence. The reduced quantum yields of photo-decarbonylation of 1 and 5 compared
to their saturated analogue 4,9-dialkoxy-6,7-dihydro-1H-dibenzo[a,e]cyclopropa[c]
[8]annulene-1-one (photo-DIBO)16 and acyclic diaryl-cyclopropenones,11a as well as their
lack of fluorescence, suggest that these rather rigid compounds undergo surprisingly
efficient vibrational relaxation.
The reactivity of MC-DIBOD 5 towards organic azides is similar to other carbocyclic
dibenzocyclooctynes and this reaction produces a nearly quantitative yield of
corresponding triazoles. Thus, 1-(butyl)-dibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-d]-
1,2,3-triazol-8-one (6a-Bu) and two isomers 5,10-dibutoxy-1-(butyl)-dibenzo[3,4:7,8]
cyclopropa [5,6]cycloocta[1,2-d]-1,2,3-triazol-8-one (6b-Bu) were isolated in 90-95%
yield upon reaction of 5a,b with butyl azide (Scheme 4).
Scheme 4. SPAAC of butyl azide across the triple bond of MC-DIBOD (5)
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The conversion of MC-DIBOD 5 into triazole 6 is accompanied by the
disappearance of 353 nm and 340 nm bands and the formation of the broad band
centered at 310 nm (log ε=3.86, Fig. 2). In a sharp contrast to 5,11-dimethoxy-substituted
analog of 6a, which was reported to be moderately fluorescent (F= 0.12),14 both the
parent triazole 6a-Bu and its 5,10-dibutoxy-substituted analog 6b-Bu show very weak
fluorescence. In DCM:MeOH solution fluorescent quantum yields are F= 0.0039 ±0.0006
and F= 0.0076 ±0.0006 correspondingly. The aqueous medium (1:4 MeOH:H2O)
somewhat enhances fluorescence of 6a-Bu (F= 0.0119 ±0.0009), but its efficiency still
remains very low. Furthermore, irradiation of the triazole 6a-Bu with UVB fluorescent lamp
for 5 min results in the smooth loss of carbon monoxide and the formation of triazole-
fused didehydro[8]annulene 3a-Bu, which can be followed by the bleaching of 310 nm
band (Scheme 5, Fig. 2). Alkyne 3a-Bu could be detected by ESI-MS in dilute solution,
but upon concentration, even at low temperature, forms a mixture of dimers and
oligomers.
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1.2
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Wavelength (nm)
Figure 2. UV Spectra of ca. 0.18 mmol solution of 1-(butyl)-dibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-
d]-1,2,3-triazol-8(1H)-one (6a-Bu, solid line) and mono-triazole (3a-Bu, dashed line) in MeOH-DCM (9:1).
Photolysis of 1-(butyl)-dibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-d]-1,2,3-
triazol-8(1H)-one 6a-Bu in the presence of benzyl azide results in the formation of two
isomeric of bis-triazole 7 and 8 in 78% isolated yield (Scheme 5).
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Scheme 5. Photo-decarbonylation of MC-DIBOD (5) followed by SPAAC of benzyl azide.
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It is interesting to note that the photo-decarbonylation of triazole 6 is over an order
of magnitude more efficient (= 0.09 ± 0.01) than the MC-DIBOD 5. This fortuitous
difference in photo-sensitivity between photo-DIBOD 1 (= 0.05), MC-DIBOD 5 (=
0.006), and triazole 6 (= 0.09) allows for selective cross-linking of different azide-
derivatized substrates. Thus, one-pot synthesis of mixed bis-triazoles 4 (Scheme 1) was
demonstrated using sequential photo-decarbonylation of photo-DIBOD 1a. A solution
containing an equimolar mixture of bis-cyclopropenone 1a and butyl azide was irradiated
with 350 nm fluorescent lamps for 6 min and incubated for 3 hours. The addition of one
equivalent of benzyl azide, followed by 5 min 300 nm photolysis allowed us to isolate bis-
triazoles in a good yield 7 and 8 (Scheme 6).
Scheme 6. Selective one-pot formation of mixed triazoles using photo-DIBOD 1a
Kinetics of SPAAC. Successful preparation of MC-DIBOD 5 gave us access to
mono-triazole 3 and allowed for the direct experimental comparison of the azide reactivity
of Sondheimer diyne (2), its cyclopropenone- (5) and triazole-fused (3) derivatives. The
rate of this 1,3-dipolar cycloaddition (SPAAC) depends on the distortion of the bond
angles in the acetylenic fragment,17 and the electronic effects of substituents.18 Since
compounds 2, 3, and 5 possess the same dibenzo[a,e]cyclooctatetraene core, the
reactivity in the series should follow the apparent strain increase from 3 to 5 to 2. Some
experimental evidence, as well as the density functional computation analysis, suggests,
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however, that the addition of a second equivalent of azide to the DIBOD (2a) proceeds
much faster than the first.19 To assess the relative reactivity of strained cycloalkynes 2, 3,
and 5 in SPAAC, we have determined the second-order rate constants of the addition of
butyl azide. The accurate rate measurements were conducted under pseudo-first order
conditions using 20-fold or higher concentration of azide at 25.0 ± 0.10 C in methanol-
DCM mixture (1:1).20 The second order rate constants were calculated by the least
square analysis of the dependence of the observed rates on the azide concentration
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(Figure 3). The SPAAC reactivity of the DIBOD (k(BuN ) = (6.65 ± 0.03) x10-2 M-1s-1 for 2a
3
and k(BuN ) = (6.5 ± 0.02) x10-2 M-1s-1 for 2b) is surprisingly similar to that of significantly
3
less strained dibenzo[a,e]cyclooctyne (DIBO),16 and agrees well with previous estimates.4
MC-DIBOD (5a), as expected, is 3-4 fold less reactive than 2a (k(BuN ) = (1.66 ± 0.02) x10-
3
2 M-1s-1, Figure 3). To our great surprise, mono-triazole 3a (R= Bu), generated by photo-
decarbonylation of 6a-Bu, is more than 500-fold more reactive than DIBOD 2a (k(BuN )
=
3
(34 ± 1 M-1s-1). This rate represents the fastest ever measured cyclooctyne-azide click
reaction in organic solution reported to date, beating the previous record set by ODIBO
(1.7 M-1s-1)21 by more than an order of magnitude.
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Azide Concentration (M)
Figure 3. Observed pseudo-first order rate constants of reaction of acetylenes 2a (), 3a-Bu (),
and 5a () at different concentration of butyl azide in MeOH-DCM (1:1).
To test the suitability of MC-DIBOD (5) for the ligation of biologically relevant
azides, we have employed it for the functionalization a model azide-tagged protein,
bovine serum albumin (BSA-N3).10 BSA-N3 was treated cyclopropenone 5a to produce
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MC-DIBOD derivatized protein 9 (Scheme 7). The BSA derivative 9 was irradiated for 8
min with 300 nm fluorescent lamps and incubated with Rhodamine B azide.22 The in-gel
fluorescent image of the SDS-PAGE of the resulting protein demonstrates the efficient
labeling of azido-BSA with the dye (Lane 1, Fig. 4). An overnight incubation of the triazole
9 with Rhodamine B azide in the dark does not produce labelled BSA 11 (Lane 2, Fig. 4).
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To illustrate the selectivity of the sequential azide cross-linking, the BSA-
Rhodamine B conjugate 11 has been prepared by an alternative procedure. MC-DIBOD
– equipped Rhodamine B 10 was prepared in situ by the reaction of Rhodamine B azide
with 5a (Scheme 7). Triazole 10 has been added to the solution of BSA-N3 in PBS and
irradiated for 8 min. The resulting conjugate 11 shows as a brightly fluorescent band on
the gel (Lane 3, Fig. 4). The dark control of the BSA-N3 mixture with 10 produces no
fluorescent bands on SDS-PAGE (Lane 4, Fig. 4).
Scheme 7. Cross-coupling of azido-BSA with Rhodamine B azide using MC-DIBOD 5a
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75 kDA marker
In-gel fluorescence
Coomassie Brilliant Blue stain
Figure 4. SDS-PAGE analysis of BSA-N3 photo-conjugation with Rhodamine B azide. Lane 1:
BSA-N3 treated with MC-DIBOD 5a and irradiated in the presence of Rhodamine B – azide; Lane
2: BSA-N3 treated with 5a and then Rhodamine B – azide in the dark; Lane 3: Rhodamine B –
azide reacted with 5a and irradiated in the presence of BSA-N3; Lane 4: Rhodamine B – azide
reacted with 5a and incubated with BSA-N3 in the dark.
The preparation of mixed triazoles (7, 8, Scheme 5) and cross-conjugation of BSA-
N3 with Rhodamine B azide demonstrated the utility of MC-DIBOD 5 for cross-linking of
small azides and for the protein functionalization. The applicability of this platform for the
cross-linking of larger substrates was demonstrated on the example of BSA-N3 and
PEG5000-azide23 cross-linking (Scheme 8). First, PEG-azide was incubated with an
equimolar amount of MC-DIBOD 5a for 12 hours at room temperature. The IR spectrum
of the product showed disappearance of azide stretching vibrations at 2100 cm-1. Three
fold excess of the crude triazole 12 was added to a PBS solution of BSA-N3, irradiated
with 300 nm lamps for 8 min, and incubated overnight. The BSA-PEG5000 conjugate was
isolated by spin filtration (MWCO 10,000) and purified on a Sephadex column. According
to the MALDI-TOF spectrum, the product contains a mixture of the native BSA with
proteins bearing one, two, three, and even four PEG fragments (Fig. S3).20
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Scheme 8. PEGylation of BSA
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As we reported previously,10 this inhomogeneity of the protein functionalization is
due non-selective derivatization of native BSA. The treatment of the BSA-N3 sample used
in this experiment with PEG5000-acetylene24 under conventional copper-catalyzed click
conditions produced the same mixture of BSA-PEG5000 conjugates (Fig. S5).20 Thus, the
photo-derivatization of azido-protein with MC-DIBOD derivative 12 occurs only at azide
groups and is at least as efficient as CuAAC coupling. The MALDI-TOF spectrum of the
protein isolated after the incubation of BSA-N3, with triazole 12 in the dark overnight
contains only the single mass peak of a native protein (Fig S4). 20
Conclusions
An order of magnitude difference in the photo-sensitivity between bis-
cyclopropenone derivative of dibenzo[a,e]cyclooctatetraene 1 and mono-cyclopropenone
5 allows for the selective mono-decarbonylation of photo-DIBOD (1, Scheme 9). MC-
DIBOD (5) was also synthesized by selective mono-cyclopropanation of
dibenzocyclooctadiyne (2). The addition of an azide across the triple bond of MC-DIBOD
(5) to give triazole 6 restores the photo-reactivity and permits the efficient generation of
triazole-fused didehydrodibenzo[a,e][8]annulene 3 (the mono-adduct of azide to DIBOD
2, Scheme 9). The alkyne 3 displays extraordinary reactivity towards azides: the rate of
addition of butyl azide in methanol (34 M-1s-1) represents the fastest ever reported SPAAC
reaction of cyclooctynes in organic solvents.
This fortuitous combination of photo-sensitivity and azide reactivity of
dibenzo[a,e]cyclooctatetraene derivatives 1 - 6, makes MC-DIBOD 5 a very convenient
platform for selective sequential cross-linking of azide-tagged substrates. We have
illustrated the utility of the cross-linker 5 by one-pot preparation of mixed bis-triazoles,
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cross-conjugation of azido-derivatized protein and azido-terminated polymer, and by the
efficient labelling of protein with fluorescent dye.
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Scheme 9.
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Experimental
General Methods. Tetrahydrofuran was freshly distilled from sodium benzophenone ketyl prior
to use. Dichloromethane was freshly distilled from CaH2 prior to use. Solutions were prepared
using HPLC grade water and methanol. Flash chromatography was performed using 40-63 m
silica gel. Photolyses were conducted using a photoreactor equipped with sixteen 4W fluorescent
lamps with emission at 300 or 350 nm. The quantum yields of photolysis were measured against
the 4-nitroveratrole actinometer.25 NMR spectra were recorded using 400 MHz spectrometer in
deuterochloroform and referenced to TMS unless otherwise noted. High resolution mass spectra
were obtained using electron spray ionization and orbitrap mass analyzer.
Materials: All reagents were purchased from commercial sources and used as received, unless
otherwise noted. Rhodamine-B azide,22 BSA-N3,10 PEG5000 azide,10 DIBOD (2a,b),10 and photo-
DIBOD (1a,b)10 were prepared following the procedures reported previously.
6,7-Dehydrodibenzo[a,e]cyclopropa[c][8]annulen-1-one (5a). TMSCF3 (0.191 mL, 1.29
mmol) was added to a solution of Sondheimer diyne (0.258 g, 1.29 mmol) and NaI (0.232 g, 1.55
mmol) in THF (24.3 mL) in a pressure vessel. The reaction mixture was heated at 110 ℃ for 2 h,
cooled to r.t., and quenched by adding a saturated bicarbonate solution (20 mL), diluted in DCM
(200 mL) and extracted with deionized water and brine. The organic phase was dried over
anhydrous K2CO3, concentrated, and loaded onto a silica column (10% DCM/hexanes to 1%
MeOH/ DCM). The difluorocyclopropene immediately hydrolyzed upon chromatography to give
0.195 g (67%) of the title compound as a bright yellow solid (m.p. 204°C decomp.). 1H NMR: 7.55
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(d, J=7.4 Hz, 2H), 7.23 (m, 4H), 6.91 (d, J=8.3Hz, 2H)13C NMR: 154.3, 150.7, 135.2, 133.7, 132.1,
130.1, 127.2, 125.2, 106.8. IR: 1845cm-1 (C=O). HRMS m/z: (M+H+) Calcd. for C17H8O:229.0648,
found: 229.0649.
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Preparation of 5a from photo-DIBOD 1a. A solution of 1a (25 mg, 0.098 mmol) in 440 mL of
DCM : MeOH mixture (1 : 9) was irradiated using 350 nm lamps. The consumption of starting
material was followed by the disappearance of the band at 363 nm (ca. 4 min of exposure). The
solvent was evaporated in vacuum and the crude mixture was purified via flash chromatography
(0.5 - 2% DCM/MeOH) to give 20 mg (90%) of the desired product as a yellow solid.
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Preparation of 5b from photo-DIBOD 1b. A solution of bis-butoxy photo-DIBOD 1b (39 mg,
0.098 mmol) in 0.5 L of DCM: MeOH mixture (1:9) was irradiated using 350 nm lamps following
the disappearance of λmax at 370 nm (ca. 5 min of irradiation). The solution was evaporated in
vacuum and the crude mixture was purified via flash chromatography (0.5-2% DCM/MeOH) to
give 34 mg (73%) of the desired product as a yellow solid. 1H-NMR: 7.45 (d, J = 8.5 Hz, 1H), 7.08
(d, J = 2.6 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.68 (dd, J = 8.5, 2.6 Hz, 1H), 6.58 (dd, J = 8.5, 2.6
Hz, 1H), 6.40 (s, 1H), 3.95 (q, J = 6.6Hz, 4H), 1.75 (m, 4H), 1.48 (m, 4H), .99 (t, J = 7.4 Hz, 6H).
13C-NMR: 163.5, 160.6, 154.1, 151.2, 145.8, 137.1, 133.8, 128.4, 127.9, 124.2, 121.2, 118.5,
116.0, 114.5, 113.4, 107.9, 104.7, 68.4, 68.2, 31.1, 31.0, 19.1, 13,8, 13.8. HRMS m/z: (M+H+)
Calcd. for C25H25O3: 373.1798, found: 373.1795.
1-butyldibenzo[3,4;7,8]cycloocta[1,2-d][1,2,3]triazol-8-one (6a). Butyl azide (43 mg, 0.44
mmol) was added to a solution of cyclopropenone 5a (50 mg, 0.22 mmol), in 20 mL of DCM/MeOH
mixture (1:4). The mixture was stirred at r.t. overnight, concentrated, and purified via flash
chromatography (0.5% MeOH/DCM) to give 66 mg (92%) of the title compound as an off-white
solid (m.p. 153-156°C). 1H NMR: 7.90 (d, J=7.7Hz, 1H), 7.73 (d, J=7.6Hz, 1H), 7.65-7.53 (m, 4H),
7.46 (t, J=7.6 Hz, 1H) 7.39 (d, J=7.6Hz, 1H), 4.35 (m 1H), 4.18 (m, 1H), 1.69 (m, 2H), 1.21 (m,
2H), .81 (m, 3H). 13C NMR: 157.7, 154.2, 151.9, 142.9, 133.8, 133.1, 132.7, 132.2, 132.1, 131.7,
131.5, 131.3, 130.5, 129.3, 128.9, 126.9, 124.4, 49.2, 32.0, 19.6, 13.3. IR: 1852 cm-1 (C=O).
HRMS m/z: (M+H+) Calcd. for C21H17N3O: 328.1444, found: 328.1444.
6,11-dibutoxy-1-butyldibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-8-one
(6b)
and
5,10-dibutoxy-1-butyldibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-
d][1,2,3]triazol-8-one (6b’). Butyl azide (11 mg, 0.11 mmol) was added to a solution of the mono-
cyclopropenone 5b (34 mg, 0.091 mmol) in 1mL of MeOH/DCM (1:1). The mixture was stirred for
12 h, evaporated, purified via flash chromatography (1% MeOH/DCM), evaporated, and
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recrystallized from DCM/hexanes to give 0.025 g (58%) of the desired product as a yellow solid
1
13
(mixture of isomers). H and C NMR spectra are provide in supporting information file(S12-S-
13).20 HRMS m/z: (M+H+) Calcd. for C29H34N3O3: 472.2595, found: 472.2593.
7
8
9
1-benzyl-8-butyldibenzo[3,4;7,8]cycloocta[1,2-d:5,6-d’]bis([1,2,3]triazole) (7) and 1-benzyl-
10-butyldibenzo[3,4;7,8]cycloocta[1,2-d:5,6-d’]bis([1,2,3]triazole) (8). A solution of the
cyclopropenone 6a-Bu (30 mg, 0.092 mmol) in 0.5 L of DCM/MeOH mixture (1:1), was irradiated
for 8 min at 300 nm. After irradiation, benzyl azide (610 mg, 4.6 mmol) was added. The mixture
was stirred at r.t. overnight, concentrated, and purified via flash chromatography (0.3%
MeOH/DCM) to give 31 mg (78%) of a mixture of the bis-triazoles as viscous oil. Triazoles could
be partially separated for NMR characterization: 1H NMR: 7.75 (d, J=6.7Hz, 1H), 7.68 (d,
J=7.7Hz, 1H), 7.51 (m, 4H), 7.39 (t, J=8.2Hz, 1H) 7.26 (m, 3H), 7.10 (d, J=7.2Hz, 2H), 7.01 (m,
2H), 5.50-5.33 (dd, J=90.8, 15.3Hz, 1H) 4.32 (m 1H), 4.18 (m, 1H), 1.67 (m, 2H), 1.04 (m, 2H),
.74 (m, 3H).13C NMR: 145.2, 144.8, 135.1, 135.0, 134.4, 132.9, 132.7, 131.4, 131.3, 130.2, 130.0,
129.9, 129.4, 128.9, 128.8, 128.6, 128.3, 127.1, 127.0, 126.4, 52.2, 48.4, 31.8, 19.4, 13.2. HRMS
m/z: (M+H+) Calcd. for C27H25N6: 433.2135, found: 433.2130.
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Preparation of bis-triazoles 7 and 8 from 1a. A solution of DIBOD 1a (25 mg, 0.098 mmol) in
0.5 L of DCM/MeOH (1:10) was irradiated with 350 nm lamps, following the disappearance of
absorption peak at 363 nm (ca. 6 minutes). Butyl azide (19 mg, 0.195 mmol) was added to the
photolysate and the reaction mixture was stirred for 3 h at r.t. Benzyl azide (1.8 mL, 0.98 mmol)
was added to the mixture, irradiated with 300 nm lamps for 5 minutes, stirred for 1 h, and solvents
were removed in vacuum. The crude material was purified via flash chromatography (DCM to
3%MeOH/DCM) to give 0.020 g (47%) the mixture of 7 and 8 as a tan oil.
N-(6-(diethylamino)-9-(2-((3-(8-oxodibenzo[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-
d][1,2,3]triazol1(8H)-yl)propoxy)carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-
ethylethaneaminium chloride 10. MC-DIBOD 5a (0.028 g, 0.123 mmol) was added to a solution
of rhodamine azide (50 mg, 0.094 mmol) in 5mL of MeOH/DCM (9:1). The mixture was stirred
overnight, evaporated and purified via flash chromatography (1% MeOH/DCM) to give 65 mg
1
(91%) of the desired compound as a violet, amorphous solid. H-NMR 8.03-8.02 (d, J = 7.7 Hz,
1H), 7.88 (d, J = 7.7Hz, 1H), 7.82-7.76 (m, 2H), 7.69-7.57 (m, 6H), 7.52-7.49 (t, J = 8.4 Hz, 2H),
7.10-7.05 (m, 2H), 7.02-6.99 (dd, J = 9.6, 2.3 Hz, 1H), 6.93-6.91 (dd, J = 9.6, 2.3 Hz, 1H), 6.81-
6.79 (m, 2H), 4.46-4.41 (m, 1H), 4.31-4.28 (m, 1H), 4.05-3.94 (m, 2H), 3.64 (m, 8H), 1.95 (m, 2H),
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1.33 (m, 12H). C-NMR; 12.6, 28.7, 46.1, 46.1, 46.4, 62.1, 96.2, 113.38, 113.41, 113.6, 114.0,
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114.3, 114.5, 124.3, 126.4, 128.2, 129.3, 129.4, 130.2, 130.4, 130.8, 131.2, 131.3, 131.6, 132.1,
131.2, 132.9, 133.1, 133.2, 133.2, 133.6, 133.7, 142.4, 142.9, 151.8, 152.1, 154.3, 155.5, 155.6,
157.4, 157.6, 157.7, 158.6, 164.5. HRMS m/z: (M+) Calcd. for C48H44N5O4754.3388 Found:
754.3391.
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Kinetics. The accurate rate measurements of butyl azide addition to alkynes 2a, 3a, and 5a were
performed in MeOH-DCM (1:1) solution at 25.0±0.1°C under pseudo-first order conditions, using
20 fold or higher excess of azide. The consumption of alkynes was followed by the decay of the
absorbance band of the starting material: 287 nm for 2a, 266 nm for 3a, and 350 nm for 5a. The
experimental data fits the single exponential equation well (Figure S1 and S2).20 Linear
dependence of the observed pseudo-first order rate constants on azide concentration (Table S1-
S3) was analyzed by the least squares method to obtain the bimolecular rate constant (Inserts in
Figures S1 and S2).20
Photo-conjugation of BSA-N3 with Rhodamine B azide.
Procedure 1. Rhodamine B azide (95 mg, 0.180 mmol) was added to a solution of MC-DIBOD
5a (41 mg, 0.18 mmol) in 8 mL of DCM/MeOH (1:3) mixture, stirred overnight at r.t., and solvents
evaporated in a vacuum. The crude solid was used in the subsequent step without further
purification. A solution of the triazole 10 (2 mg, 3.90 μmol) in MeOH (241 µL) was added to a
solution of BSA-N3 (17.5 mg, 0.265 μmol), in PBS (10 mL). The solution was irradiated for 8 min
at 300 nm and stirred overnight in the dark. The resulting protein was concentrated by spin
filtration (MWCO 10,000), purified on a PD-10 Sephadex column, and lyophilized. The BSA-
Rhodamine conjugate, as well as a dark control, were resolved on 12% SDS-PAGE gel and
visualized through in-gel fluorescence using a GE Typhoon scanner with excitation wavelength
fixed at 532 nm and emission wavelength fixed at 580 nm.
Procedure 2. A solution of MC-DIBOD 5a (5 mg, 0.022 mmol) in MeOH (2 mL) was added to a
solution of BSA-N3 (145 mg, 2.19 µmol) in PBS (10 mL), the reaction mixture was stirred overnight
at r.t., concentrated by spin filtration (MWCO 10,000), purified on a PD-10 Sephadex column, and
lyophilized. A portion of the resulting triazole 9 (25 mg, 0.379 μmol) was reconstituted in PBS (2.5
mL), and Rhodamine B azide (2 mg, 3.79 μmol) was added to it. The solution was irradiated for
8 min at 300 nm and stirred overnight in the dark. The resulting protein was concentrated by spin
filtration (MWCO 10,000), purified on a PD-10 Sephadex column, and lyophilized. The BSA-
Rhodamine conjugate, as well as a dark control, was resolved on 12% SDS-PAGE gel and
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visualized through in-gel fluorescence using a GE Typhoon scanner with excitation wavelength
fixed at 532 nm and emission wavelength fixed at 580 nm.
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Photo-crosslinking of BSA-N3 and PEG5000-N3. PEG5000-N3 (0.500g, 0.100 mmol) was added to
a solution of 5a (23 mg, 0.10 mmol) in MeOH, the mixture was stirred at r.t. for 12 h, and
concentrated to give 519 mg (99%) of the crude product as a white solid. Evaluation of the IR
spectrum of the product revealed the disappearance of (N=N=N) stretch at 2100 cm-1. The crude
solid was used in the subsequent step without further purification. A solution of the PEG-triazole
12 (5 mg, 0.956 µmol) in MeOH (1 mL) was added slowly to a solution of BSA-N3 (21 mg, 0.319
µmol) in PBS buffer (2 mL). The mixture was irradiated at 300 nm for 8 min and incubated
overnight. The resulting protein was concentrated by spin filtration (MWCO 10,000), purified on a
PD-10 Sephadex column, and lyophilized. The product was characterized by MALDI-TOF
analysis and compared with a dark control (same treatment without irradiation). The MALDI-TOF
analysis of the BSA-PEG5000 conjugate revealed multi-functionalization of BSA (Figure S3).
Incubation of the mixture containing the triazole 12, BSA-N3, and PEG5000-azide in PBS overnight,
didn’t produce any BSA-PEG5000 conjugate (Figure S4).20
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Copper-catalyzed click (CuAAC) conjugation of BSA-N3 and propargyl-PEG5000. A solution
BSA-N3 (13.2 mg, 0.20 μmol), propargyl-PEG5000 (10 mg, 2.0 μmol), ascorbic acid (7.0 μg, 0.040
μmol) and CuSO4 (3.2 μg, 0.020 μmol) in PBS (7 mM, 6 mL) was stirred for 12 h, concentrated to
1.5 mL by spin filtration (MWCO 10,000), purified over a PD-10 column, and lyophilized. The
MALDI-TOF analysis of the protein shows that it contains a mixture of unmodified BSA and BSA
molecules derivatized with one, two, three, and four PEG5000 fragments (Fig. S5).20 The ratio of
these proteins (60 : 23 : 11) was almost identical to MC-DIBOD 5a pegylation experiment.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr. Y. G. Zheng and Mr. Z. Han’s help in the analysis
of dye-labeled BSA. This project was supported by grants from the National Science
Foundation (CHE-1565646 and CHE-1213789).
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Supporting Information Available: Kinetic and MALDI-TOF data and NMR spectra of
newly synthesized compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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