Synthesis of tetra(2-hydroxyethoxy)-substituted dibenzocyclooctyne derivatives as novel, highly hydrophilic tool compounds for strain-promoted alkyne-azide cycloaddition applications

Article abstract of DOI:10.1055/s-0032-1316875

The synthesis of a novel, hydrophilic dibenzocyclooctyne derivative for strain-promoted alkyne-azide cycloaddition (SPAAC) is described. Starting from 2-(3,4-dimethoxyphenyl)acetaldehyde, the corresponding activated carbonate of 2,3,8,9-tetrakis(2-hydroxyethoxy)-5,6-dihydro-11,12-didehydrodibenzo[a,e][8] annulene, termed THE-DIBO, was prepared in seven steps and 24% overall yield. A water-soluble THE-DIBO analogue showed a k₂ value of (21.9 ± 0.2) × 10^-2 M^-1 s^-1 for SPAAC with 2-azidoethanol in water. The high hydrophilicity and steric bulk of THE-DIBO derivatives should to suppress nonspecific thiol-yne addition of proteins to strained alkynes observed for SPAAC labeling in complex proteomes. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0032-1316875

PAPER
▌1207
paper
Synthesis of Tetra(2-hydroxyethoxy)-Substituted Dibenzocyclooctyne Deriva-
tives as Novel, Highly Hydrophilic Tool Compounds for Strain-Promoted
Alkyne-Azide Cycloaddition Applications
Tetra(2-hydroxyethoxy)-Substituted Dibenzocyclooctynes
Martin Golkowski,* Thomas Ziegler
Institut für Organische Chemie, Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
Fax +49(7071)295244; E-mail: Martin.Golkowski@uni-tuebingen.de
Received: 17.02.2013; Accepted after revision: 05.03.2013
strain-promoted azide-alkyne cycloaddition (SPAAC) has
Abstract: The synthesis of a novel, hydrophilic dibenzocyclooc-
been introduced as a novel strategy for rapid and che-
tyne derivative for strain-promoted alkyne-azide cycloaddition
moselective conjugation of biomolecules and materials.6
(SPAAC) is described. Starting from 2-(3,4-dimethoxyphenyl)acet-
The methodology obviates the use of cytotoxic, environ-
mentally harmful copper and can proceed at significantly
higher reaction rates than classical CuAAC.
aldehyde, the corresponding activated carbonate of 2,3,8,9-tetrakis(2-
hydroxyethoxy)-5,6-dihydro-11,12-didehydrodibenzo[a,e][8]an-
nulene, termed THE-DIBO, was prepared in seven steps and 24%
overall yield. A water-soluble THE-DIBO analogue showed a k2
value of (21.9 ± 0.2) × 10–2 M–1 s–1 for SPAAC with 2-azidoethanol
in water. The high hydrophilicity and steric bulk of THE-DIBO de-
rivatives should to suppress nonspecific thiol-yne addition of pro-
teins to strained alkynes observed for SPAAC labeling in complex
proteomes.
SPAAC has been shown to enable bioorthogonal conjuga-
tion of azide-functionalized biomolecules in various ex-
perimental settings, most prominently the labeling of
azide-functionalized glycans on the surface of living cells.
Nonetheless, in the more recent past reports on the incom-
plete chemoselectivity of SPAAC in a physiological envi-
ronment have become more frequent.6a,7,8 We and others
have observed extensive nonspecific addition of biopoly-
mers, i.e. proteins, to strained alkynes causing significant
nonspecific background labeling alongside specific label-
ing of azide-bearing biomolecules.
Key words: alkynes, azides, conjugation, proteins, SPAAC, click
chemistry, thiol-yne addition, bioorthogonal chemistry
Over the last decade the copper-catalyzed Huisgen-cyclo-
addition reaction between an azide moiety and a terminal
alkyne (CuAAC) has become a versatile chemical tool for
the rapid assembly of complex organic structures.1,2 Click
reactions, such as the archetypical CuAAC, but also the
Staudinger ligation reaction, oxime ligation, and natural
chemical ligation, among others, are defined as processes
that proceed chemoselectively, in high yield, and under
mild reaction conditions.3 The bioorthogonal character of
CuAAC allowed efficient synthetic manipulation of bio-
molecules functionalized with the azide or alkyne moiety
in vitro and in vivo.4,5 More recently, the concept of
Regarding this issue, cyclooctyne derivatives, such as
compounds derived from bicyclo[6.1.0]nonyne (BCN) 1,9
dibenzocyclooctyne (DIBO) 2,6a and azadibenzocyclooc-
tyne (DIBAC) 310 (Scheme 1), were shown to be suscep-
tible to thiol-yne addition of proteins under physiological
conditions. Whether the thiol-yne addition to strained al-
kynes follows a radical or ionic mechanism could not be
elucidated unambiguously.8,11 Thus, it was found that ali-
phatic thiols spontaneously add to cyclooctyne with ex-
clusion of light. It was hypothesized that the conversion
MeO
OMe
OMe
N
MeO
+Na–O3SO
OSO3–Na+
H
H
O
O
O
O
O
O
NH
O
O
S
OpNP
OpNP
O
OpNP
OH
OH
DIBO (2)
DIBAC (3)
TMDIBO (5)
BCN (1)
S-DIBO (6)
O
hydrophilic strained alkyne
HO(H2C)2O
O(CH2)2OH
O(CH2)2OH
O
MeO
7 steps
HO(H2C)2O
O
O
MeO
24%
overall yield
OpNP
THE-DIBO (4)
Scheme 1 Various cyclooctynes for SPAAC applications described in the literature and the novel, hydrophilic THE-DIBO 4
SYNTHESIS 2013, 45, 1207–1214
Advanced online publication: 28.03.2013
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0032-1316875; Art ID: SS-2013-T0888-OP
© Georg Thieme Verlag Stuttgart · New York

1208
M. Golkowski, T. Ziegler
PAPER
was triggered by reactive oxygen species and proceeds via The molecular architecture of 4 was inspired by the tetra-
free radicals, but the exact mechanism could not be clari- methoxy-functionalized alkyne TMDIBO 5 (Scheme 1).13
fied.11
TMDIBO 5 was reported to be significantly more stable
during storage and application in biological experimental
settings than for example DIBO 2, while retaining very
fast reaction kinetics in the SPAAC reaction.13 Due to
these facts and the straightforwardness of the synthetic
route leading to 5, we deemed the tetraalkoxy-DIBO scaf-
fold superior for our purposes as compared to the previ-
ously reported ionic S-DIBO 6 (Scheme 1).12
In a study published by van Geel et al.8 it was found that
thiol-yne addition to the strained alkynes 1–3 is not sup-
pressed in the presence of sodium ascorbate. It was also
found that small, soluble thiols [i.e., 2-mercaptoethanol
(BME), dithiothreitol (DTT), and reduced glutathione
(GSH)] as well as bovine serum albumin (BSA) compete
for the thiol-yne addition by proteins only when present in
large excess (>25 fold). Likewise, our findings and reports To increase the hydrophilicity as well as the steric bulk
from other groups suggest that BME, DTT, and GSH do around the strained alkyne moiety, the methoxy groups
not readily add to strained alkynes derived from the DIBO found in TMDIBO 5 were replaced by 2-hydroxyethoxy
scaffold, but that free thiols found in proteins (e.g., de- moieties (Scheme 1). The synthetic route to 4-nitrophenyl
rived from whole-cell lysates) are capable of doing so (un- (pNP) activated carbonate building block 4 starts from
published results from our group). Thus, it appeared known tetrahydroxy aromatic 8.14 The latter could be ob-
feasible that reactive thiols in the hydrophobic environ- tained in 98% yield over two steps by treatment of 2-(3,4-
ment of proteins preferentially engage in thiol-yne addi- dimethoxyphenyl)acetaldehyde with iodotrimethylsilane
tion and said thiols are primarily responsible for in dichloromethane15 followed by demethylation of di-
nonspecific SPAAC labeling in complex proteomes.
merization product 7 by boron tribromide in dichloro-
methane (Scheme 2).
In order to obtain a strained alkyne warhead exhibiting
minimal interaction with proteins and thus having a lower Contrary to the literature report,14 the direct route via con-
tendency for nonspecific thiol-yne addition, the hydro- densation of 2-(3,4-dimethoxyphenyl)acetaldehyde with
philic SPAAC reagent 4 was envisaged. Activated THE- excess boron tribromide led to low yields of 8 and com-
DIBO 4 was synthesized in seven steps and 24% overall plex product mixtures. In order to establish the tetrakis(2-
yield from commercially available starting materials. Fur- hydroxyethoxy) substitution pattern, 8 was peralkylated
thermore, the reaction rate constants of the click reaction with TBS-protected 2-iodoethanol16 using cesium carbon-
between a 1-methylpiperazine carbamate derivative of ate as the base in refluxing acetonitrile. Hereby, tetraeth-
THE-DIBO 4 and 2-azidoethanol in water and methanol oxy derivative 9 was obtained in 67% yield (Scheme 2).
were determined by UV spectrophotometry12 and com- Attempts to peralkylate 8 with TBS-protected 2-bromo-
pared with the corresponding DIBO 2 analogue. Finally, or iodoethanol in N,N-dimethylformamide or dimethyl
the stability of THE-DIBO 4 toward thiol-yne addition sulfoxide with bases such as potassium carbonate or sodi-
was studied by competitive treatment of our model com- um hydride were unsuccessful. This was most likely due
pounds with 2-azidoethanol and GSH or BME in aqueous to precipitation of the corresponding sodium or potassium
buffer solution using LC-MS.
phenolate salts formed in situ.
TBSO(H2C)2O
OH
O(CH2)2OTBS
O(CH2)2OTBS
MeO
MeO
OMe
HO
HO
a
b
O
O
O
TBSO(H2C)2O
OMe
OH
9
8
7
c, d
OTES
OR
TBSO(H2C)2O
TBSO(H2C)2O
O(CH2)2OTBS
O(CH2)2OTBS
TBSO(H2C)2O
TBSO(H2C)2O
O(CH2)2OTBS
O(CH2)2OTBS
e
12
10: R = H
11: R = TES
f
OH
OpNP
TBSO(H2C)2O
TBSO(H2C)2O
O(CH2)2OTBS
O(CH2)2OTBS
O
O
g
TBSO(H2C)2O
TBSO(H2C)2O
O(CH2)2OTBS
O(CH2)2OTBS
h
4
13
14
Scheme 2 Synthesis of the activated THE-DIBO carbonate 4. Reagents and conditions: (a) BBr3, CH2Cl2, –78 °C to 25 °C, 2 h, 98%; (b)
I(CH2)2OTBS, Cs2CO3, MeCN, reflux, 72 h, 67%; (c) BuLi, THF, –50 °C, 4 h; (d) TESCl, imidazole, DMF, 0 °C to 25 °C, 14 h, 96%; (e) 1.
dioxane–Br2, THF, 0 °C, 30 min, 2. KHDMS, THF, –78 °C to 25 °C, 1 h, 74%; (f) HF·Py, THF, –50 °C, 10 h, 61%; (g) 4-O2NC6H4OCOCl,
Py, CH2Cl2, 0 °C to 25 °C, 14 h, 94%; (h) HF·Py, THF, 0 °C, 2 h, 91%.
Synthesis 2013, 45, 1207–1214
© Georg Thieme Verlag Stuttgart · New York

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Tetra(2-hydroxyethoxy)-Substituted Dibenzocyclooctynes
1209
In the next stage, the epoxy scaffold in 9 was cleaved uti- In order to obtain model alkynes for further tests in aque-
lizing excess butyllithium in tetrahydrofuran–hexane. Un- ous systems and complex proteomes, the water soluble pi-
expectedly, the elimination reaction proceeded smoothly perazine conjugates 15 and 16 and THE-DIBO-biotin 18
at temperatures as low as –50 °C leading to alcohol 10 were prepared. Thus, 4 was reacted with excess 1-methyl-
(compare13). Due to the highly acid labile nature of 10, the piperazine in N,N-dimethylformamide to give 15 in 94%
free alcohol was directly converted into the corresponding yield (Scheme 3). Likewise, pNP-DIBO 2 (Scheme 1) was
triethylsilyl ether with chlorotriethylsilane and imidazole converted into the corresponding piperazine carbamate 16
in N,N-dimethylformamide to yield 11 in 96% yield over in virtually quantitative yield; 16 should serve as a water-
two steps (Scheme 2). Like 10, 11 was acid labile and col- soluble reference compound for the measurement of
umn chromatography was performed on pre-neutralized SPAAC rate constants and the stability of 15 toward thiol
silica gel. Introduction of the alkyne moiety by a sequence reagents. As expected, the THE-DIBO warhead 15 was
of bromination and elimination was found to be more dif- found to be highly soluble in aqueous buffer solutions,
ficult than expected. Treatment with bromine in dichloro- e.g. 50 mM sodium acetate, 200 mM ammonium acetate,
methane at low temperature followed by brief workup and or 50 mM sodium phosphate buffer. To obtain THE-
elimination with excess potassium tert-butoxide/1-meth- DIBO-biotin 18, activated THE-DIBO 4 was reacted with
ylpiperazine in tetrahydrofuran, as utilized in the synthe- biotin derivative 176a (Scheme 3) giving 18 in 89% yield.
sis of TMDIBO 5, led to very low yields of alkyne 12 Biotin conjugate 18 possesses a remarkable water solubil-
(<10%).13 Likewise, treatment of 11 with bromine in di- ity of approx. 6 mM and exhibits the same stability char-
chloromethane at different temperatures and variations of acteristics as the piperazine conjugate 15 (see below).
the workup procedure followed by elimination with potas-
Second-order rate constants of the SPAAC reaction with
sium tert-butoxide in tetrahydrofuran alone were unsuc-
THE-DIBO analogue 15 were determined by UV-spectro-
cessful. Gratifyingly, efficient bromination–elimination
photometric measurement of the disappearance of the al-
could be achieved by treatment of 11 with dioxane–bro-
mine complex in tetrahydrofuran at 0 °C immediately
followed by treatment with excess potassium hexamethyl-
disilazanide at –78 °C and warming to 25 °C. Hereby, the
alkyne 12 was obtained in 74% yield (Scheme 2). Next,
the transiently installed secondary TES ether group was
selectively removed in presence of the primary TBS ether
moieties. Here, treatment of 12 with 2 vol% hydrogen flu-
kyne absorption band at around 310 nm as reported
previously.12 Plotting of the pseudo-first-order rate con-
stants obtained by treatment of 15 and 16 with excess al-
kyl azide against azide concentration yielded the k2
values. To get an impression of the impact of the aryl sub-
stituents in 15, the nature of the azide and the solvent used
on the SPAAC rates, DIBO analogue 16 was included in
the experiments as a control. The alkynes were reacted
with either 2-azidoethanol or benzyl azide in methanol
and/or water, respectively (Table1).
oride–pyridine in tetrahydrofuran at –50 °C led to the best
result and free alkynol 13 was obtained in 61% yield. Re-
acting free alcohol 13 with 4-nitrophenyl chloroformate
Markedly, the second-order rate constants for THE-DIBO
conjugate 15 increased slightly (approx. 1.5-fold) com-
pared to DIBO analogue 16 for the azides and solvents
tested (Table 1). Further, the rates for reaction between 2-
azidoethanol and 15/16 decreased slightly compared to
under standard conditions6a led to carbonate 14 in 94%
yield. Finally, deprotection of the TBS groups in 14 was
achieved with excess hydrogen fluoride–pyridine in tetra-
hydrofuran at 0 °C giving the activated THE-DIBO build-
ing block 4 in 91% yield.
Me
N
Me
N
O
N
N
NH
HN
H
O
O
O
H
O
O
HO(H2C)2O
HO(H2C)2O
O(CH2)2OH
O(CH2)2OH
a
a
O
4
2
S
H2N
N
2
H
15
16
17
Me
N
H
H
N
O(CH2)2OH
O
HO(H2C)2O
N
O
S
NH
OH
O
H
HO(H2C)2O
O(CH2)2OH
O(CH2)2OH
HO(H2C)2O
HO(H2C)2O
O(CH2)2OH
O(CH2)2OH
b
H+, H2O
CO2
O
O
4
O
HO(H2C)2O
NH
N
H
N
O
H
2
N
N
N
N
N
OH
N
N
OH
Me
O(CH2)2OH
20
18
19
HO(H2C)2O
Scheme 3 Synthesis of the water-soluble model alkynes 15, 16, THE-DIBO-biotin 18 and acid-catalyzed fragmentation of carbamate 19. Re-
agents and conditions: (a) 1-methylpiperazine, DMF, r.t., 12 h, 94% (15), 99% (16); (b) 17·TFA, DIPEA, DMF, r.t., 24 h, 89%.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1207–1214

1210
M. Golkowski, T. Ziegler
PAPER
Table 1 Measurement of the SPAAC Rate Constantsa
Alkyne
Solvent
2-Azidoethanol k2 (M–1 s–1)
Solvent
MeOH
Benzyl azide k2 (M–1 s–1)
15
H2O
MeOH
(21.8 ± 0.2) × 10–2
(11.4 ± 0.6) × 10–2
(16.7 ± 0.3) × 10–2
16
H2O
MeOH
(14.6 ± 0.2) × 10–2
(6.9 ± 0.3) × 10–2
MeOH
(9.3 ± 0.4) × 10–2
a T = 295 K.
the reaction with benzyl azide. It should also be men- port the assumption that small, hydrophilic thiols do not
tioned that the k2 value for SPAAC between benzyl azide readily add to DIBO-derived SPAAC reagents.
and THE-DIBO analogue 15 was found to be 1.5-fold
At this point it should be mentioned that the triazole 19
higher than the k2 value for a TMDIBO 5 analogue in the
(Scheme 3) formed by SPAAC reaction between 15 and
same reagent system that was reported in the literature
2-azidoethanol seems to posses only a limited half-life in
1
(evaluated by H NMR at 300 K13). Also, a twofold rate
neutral to slightly acidic buffer media (pH 7 to 6.3). Thus,
enhancement for SPAAC between 2-azidoethanol and
reexamination of these sample mixtures after 48 hours un-
der ambient conditions by LC-MS analyses showed clear
15/16 could be observed when switching solvent from
methanol to more polar water. This is in good accordance
to the observation that polar solvents can accelerate cyclo-
signs of fragmentation of the triazole carbamate 19
(tR = 6.2 min, m/z = 674.5 [M + H]+) to the corresponding
addition reactions, e.g. water in Diels–Alder-type reac-
benzyl alcohol 20 (tR = 9.5 min, m/z = 547.8 [M + H]+; ap-
tions.17 This effect was previously described for SPAAC
prox. 5%; Scheme 3). This effect was neither observed for
reactions.6a,9
the parent alkyne 15 nor for the biotin conjugate 18 (see
To further highlight the utility of our THE-DIBO-based above). Also, the corresponding DIBO-derived 2 triazole
SPAAC reagents, the storability of 4, 15, and 18 as well carbamates did not show any fragmentation under the
as the susceptibility of 15 toward thiol-yne addition was same conditions. Apparently the 4-alkoxybenzyl position
evaluated. Activated carbonate 4 could be stored at 7 °C in 19 experiences significant activation upon transforma-
under ambient atmosphere for nine months without no- tion of 15 to the triazole in regard to acid-catalyzed hydro-
ticeable loss of activity in the synthesis of follow-up com- lysis via an SN1 mechanism. When shifting to more basic
pounds. The corresponding TFA salt of 15 and THE- buffer media (TRIS pH 8.1) carbamate fragmentation in
DIBO 18 did not show any sign of decomposition upon 19 was suppressed and only trace amounts of the corre-
storage under ambient conditions for at least six months as sponding benzyl alcohol could be detected by LC-MS af-
judged by LC-MS analyses. Further, the aqueous stock so- ter 48 hours under ambient conditions. Based on these
lution of 15 used in the UV experiments was found to be findings we strongly recommend that the triazoles formed
stable for at least four weeks at 7 °C (LC-MS). Thiol-yne from THE-DIBO derivatives are kept in basic buffers
addition to the water-soluble strained alkynes was inves- when stored for longer periods of time.
tigated by LC-MS. Thus, 1.4mM solutions of 15 and 16 in
In conclusion, we described a practical synthetic route to
200 mM aqueous ammonium acetate were treated either
the novel, highly hydrophilic and stable tetrakis(2-hy-
with two equivalents of 2-azidoethanol (2.8 mM azide,
droxyethoxy)-substituted dibenzocyclooctyne THE-
control), two equivalents of 2-azidoethanol, and 10 equiv-
DIBO 4, which is a versatile building block for the assem-
alents of GSH or BME (14 mM thiol, competition) or
bly of strained alkyne probes for SPAAC applications.
blank buffer (control), respectively. After stirring for 24
Activated THE-DIBO 4 was obtained in seven steps from
commercially available 2-(3,4-dimethoxyphenyl)acetal-
dehyde in an overall yield of 24%; 4 was further coupled
hours under ambient conditions, the mixtures were ana-
lyzed by LC-MS. With the exception of the sample con-
taining DIBO-conjugate 16, 2-azidoethanol and GSH, no
to 1-methylpiperazine and biotin conjugate 17 to yield the
addition of thiol to the alkynes and only formation of the
water-soluble model alkyne 15 and THE-DIBO-biotin 18.
corresponding triazoles was observed. In the given sam-
The second-order reaction rate constant of the click reac-
ple, the 16-GSH adduct (tR = 15.4 min, m/z = 654.4 [M +
tion between model compound 15 and 2-azidoethanol was
H]+) was present only in trace amounts (approx. 1–2%). In
found to be (21.8 ± 0.2) × 10–2 M–1 s–1 as measured by UV
a further control experiment 15 and 16 were treated with
spectrophotometry in water. Further, alkyne 15 proved to
GSH and BME alone under the aforementioned condi-
be highly resistant toward thiol-yne addition by small hy-
tions. LC-MS analyses of the sample mixtures after 24
drophilic thiols GSH and BME. A possible weakness of
the synthetic route presented here is the low yield in the
alkylation step (step b in Scheme 2) and the TES-deprot-
ection step (step f in Scheme 2) with 67% and 61% yields,
hours indicated that no addition of thiol to 15 had oc-
curred. The adducts 16-BME (tR = 17.0 min, m/z = 448.0
[M + Na]+) and 16-GSH were traceable in the total ion
chromatogram (TIC), but the bands (220 nm) were of too
low intensity to be integrated (<1%). These findings sup-
catalyzed hydrolysis of the triazoles obtained from THE-
respectively. Furthermore, the potential liability to acid-
Synthesis 2013, 45, 1207–1214
© Georg Thieme Verlag Stuttgart · New York

PAPER
Tetra(2-hydroxyethoxy)-Substituted Dibenzocyclooctynes
1211
tone); light beige solid/colorless crystals; yield: 819 mg (98%); mp
285 °C (dec); Rf = 0.45 (toluene–acetone, 1:1).
DIBO conjugates might diminish their usefulness in ap-
plications where long term stability of the triazole conju-
gates is important. A possible solution would be to switch
from the apparently labile carbamate linkage to more sta-
ble ether bonds. Derivatives of THE-DIBO are currently
tested for their performance in our previously reported
capture-release protocol for azide tagged biomolecules.18
We hope that probes derived from THE-DIBO 4 are less
prone to nonspecific thiol-yne addition, which is a major
obstacle for successful application of SPAAC in complex
proteomes like cell lysates, the interior of living cells or in
animals.
IR (KBr): 3346, 2917, 2886, 2830, 1610, 1528, 1459, 1438, 1348,
1287, 1264, 1190, 1160, 1106, 1051, 1018, 933, 866, 827, 800, 515
cm–1.
1H NMR (400 MHz, DMSO-d6): δ = 2.32 (d, J = –15.8 Hz, 1 H,
ArCH2), 3.14 (dd, J = –15.8, 5.9 Hz, 1 H, ArCH2), 4.96 (d, J = 5.9
Hz, 1 H, ArCHO), 6.33 (s, 2 H, ArH), 6.45 (s, 2 H, ArH), 8.63 (s, 4
H, OH).
13C NMR (100 MHz, DMSO-d6): δ = 35.2, 68.2, 112.0, 115.2,
122.0, 128.5, 143.4, 144.2.
MS (FAB): m/z (%) = 152.6 (100), 287.1 (40) [M + H]+.
Anal. Calcd for C16H14O5: C, 67.13; H, 4.93. Found: C, 67.36; H,
4.92.
All commercially available chemicals were used as received unless
noted otherwise. Dry solvents were prepared according to standard
methods, distilled, and stored over molecular sieves 3 Å under an at-
mosphere of N2 until used. NMR spectra were recorded on a Bruker
Avance 400 spectrometer and calibrated for the solvent-signal (1H-
2,3,8,9-Tetrakis[2-(tert-butyldimethylsiloxy)ethoxy]-5,6,11,12-
tetrahydro-5,11-epoxydibenzo[a,e][8]annulene (9)
Tetraol 8 (5.70 g, 19.9 mmol) was dissolved in MeCN (300 mL).
Cs2CO3 (38.9 g, 119 mmol, 6 equiv) and tert-butyl(2-iodoeth-
oxy)dimethylsilane16 (34.1 g, 119 mmol, 6 equiv) were added and
the mixture was heated at reflux temperature for 72 h. After cooling
to 25 °C, the mixture was concentrated under reduced pressure, the
residue taken up in EtOAc (400 mL), and the slurry was transferred
to a separatory funnel. The organic phase was washed with H2O ( 3
× 200 mL) and brine (100 mL) and dried (MgSO4); the solvent was
removed under reduced pressure. The residue was subjected to col-
umn chromatography (silica gel, toluene–EtOAc, 39:1) to afford
pure TBS-protected ether 9 as a colorless viscous oil; yield: 12.2 g
(67%); Rf = 0.31 (toluene–EtOAc, 39:1).
1
MeOH-d4: δ = 3.31; 13C-MeOH-d4: δ = 49.00, H-DMSO-d6: δ =
1
2.50; 13C-DMSO-d6: δ = 39.52; H-acetone-d6: δ = 2.05; 13C-ace-
tone-d6: δ = 28.84). SPAAC reaction kinetics were measured by UV
spectrophotometry following the published protocol12 on a Perkin-
Elmer Lambda 25 UV-Vis spectrophotometer using a 1-cm quartz
cuvette and were measured in duplicate. Absorbance was measured
at 316 nm (15) and 305 nm (16), respectively. FT-ICR-MS spectra
were recorded on a Bruker Apex II FT-ICR-MS (FAB) spectrome-
ter and FAB-spectra on a Finnigan model TSQ 70. Melting points
were determined with a Büchi Melting Point M-560 and are uncor-
rected. Elemental analysis was performed on a HEKAtech Euro EA
Analyzer. IR spectra were recorded on a Bruker Tensor 27. TLC-
analysis was performed with Macherey-Nagel Polygram SIL G/UV
pre-coated polyester sheets. Column chromatography was per-
formed with silica gel bead size 0.04–0.063 mm. Pre-neutralized
silica gel was prepared by adding 5 vol% 7 M NH3 in MeOH to a
suspension of silica gel in CHCl3 followed by brief shaking and re-
moval of the solvent under reduced pressure. All reactions were per-
formed in flame-dried glassware under an atmosphere of N2 using
dry solvents unless stated otherwise. 2-(3,4-Dimethoxyphenyl)acet-
aldehyde and 2,3,8,9-tetramethoxy-5,6,11,12-tetrahydro-5,11-
epoxydibenzo[a,e][8]annulene (7)15 and tert-butyl(2-iodoeth-
oxy)dimethylsilane16 were prepared according to the published pro-
cedures. The SPAAC/thiol-yne addition competition experiments
with 15 an 16 were conducted by treatment of a 1.4 mM soln of the
water-soluble strained alkyne component in 200 mM NH4OAc buf-
fer with 2 mol equiv 2-azidoethanol (2.8 mM) alone (control), with
additional 10 mol equiv (14 mM) of GSH or BME (competition) or
blank buffer (control), respectively. After stirring for 24 h at r.t. un-
der ambient atmosphere the mixtures were analyzed by LC-MS
(ESI; Bruker Daltonics Esquire 3000 plus) on a Macherey-Nagel
EC-Nucleodur 100-5 C18 ec column eluting with a MeOH–H2O +
1 vol% HCO2H gradient of 20 vol% MeOH to 100 vol% MeOH in
30 min.
IR (neat/KBr): 2929, 2857, 2738, 1612, 1516, 1472, 1428, 1361,
1343, 1254, 1197, 1110, 960, 835, 778 cm–1.
1H NMR (400 MHz, acetone-d6): δ = 0.08 (s, 6 H, TBS-CH3), 0.09
(s, 6 H, TBS-CH3), 0.12 (s, 12 H, TBS-CH3), 0.89 (s, 18 H, TBS-t-
Bu), 0.92 (s, 18 H, TBS-t-Bu), 2.69 (d, J = –16.0 Hz, 2 H, ArCH2),
3.33 (dd, J = –16.0, 6.0 Hz, 2 H, ArCH2), 3.90–4.06 (m, 16 H,
OCH2), 5.13 (d, J = 5.7 Hz, 2 H, ArCHO), 6.58 (s, 2 H, ArH), 6.78
(s, 2 H, ArH).
13C NMR (100 MHz, acetone-d6): δ = –5.0, 18.8, 18.9, 26.3, 26.3,
36.2, 62.9, 62.9, 69.7, 71.1, 71.3, 112.0, 115.1, 125.1, 131.4, 148.3,
148.9.
HRMS (FAB): m/z [M + Na]+ calcd for C48H86O9Si4Na: 941.5241;
found: 941.5243.
2,3,8,9-Tetrakis[2-(tert-butyldimethylsiloxy)ethoxy]-5-(trieth-
ylsiloxy)-5,6-dihydrodibenzo[a,e][8]annulene (11)
TBS-protected ether 9 (196 mg, 213 μmol) was dissolved in THF (5
mL) and the soln cooled to –78 °C followed by dropwise addition
of 2.5 M BuLi in hexanes (596 μL, 7 equiv) via a syringe. Then, the
mixture was allowed to warm to –50 °C and stirring was continued
for 4 h. Excess BuLi was quenched by the addition of H2O (1 mL),
the mixture was diluted with Et2O (25 mL) and transferred to a sep-
aratory funnel. The aqueous layer was separated and the organic
layer washed with H2O (2 × 10 mL) and brine (10 mL), and dried
(MgSO4); the solvent removed under reduced pressure. The crude
alcohol 10 was obtained as an amber-colored viscous oil and used
for the next step without further purification. Crude alcohol 10 was
dissolved in DMF (1 mL) and the soln cooled to 0 °C. Imidazole
(31.9 mg, 469 μmol, 2.2 equiv) and TESCl (39.3 μL, 234 μmol, 1.1
equiv) were added and the mixture stirred for 2 h at 0 °C and an ad-
ditional 12 h at 25 °C. Subsequently, the mixture was diluted with
Et2O (25 mL), transferred to a separatory funnel, and washed with
H2O (3 × 10 mL) and brine (10 mL). The organic layer was dried
(MgSO4); the solvent was removed under reduced pressure. Col-
umn chromatography of the residue (pre-neutralized silica gel, light
petroleum ether–EtOAc, 22:1) yielded pure TES ether 11 as a col-
5,6,11,12-Tetrahydro-5,11-epoxydibenzo[a,e][8]annulene-
2,3,8,9-tetraol (8)
2,3,8,9-Tetramethoxy-5,6,11,12-tetrahydro-5,11-epoxydiben-
zo[a,e][8]annulene15 (7, 1 g, 2.92 mmol) was dissolved in CH2Cl2
(50 mL) and the soln cooled to –78 °C. Then, BBr3 (1.13 mL, 11.7
mmol, 4 equiv) was added dropwise via a syringe and the soln al-
lowed to warm to 25 °C over a period of 1 h. The mixture was stirred
for an additional 1 h at 25 °C and then poured onto ice-water (100
mL), stirred for 1 h, transferred to a separatory funnel, and extracted
with EtOAc (3 × 50 mL). The combined organic layers were
washed with brine (3 × 20 mL) and dried (MgSO4); the solvent was
removed under reduced pressure to yield pure tetraol 8. An analyti-
cal sample of 8 was obtained by recrystallization (benzene–ace-
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1207–1214

1212
M. Golkowski, T. Ziegler
PAPER
orless oil; yield: 211 mg (96%); Rf = 0.33 (light petroleum ether–
EtOAc, 22:1).
yield pure alkynol 13. An analytical sample of 13 was obtained by
recrystallization (n-hexane); white solid/colorless needles; yield:
322 mg (61%); mp 135 °C; Rf = 0.33 (light petroleum ether–EtOAc,
17:3).
IR (neat/KBr): 2930, 2737, 1604, 1512, 1462, 1361, 1254, 1188,
1105, 1107, 960, 835, 778, 743 cm–1.
IR (KBr): 3511, 2953, 2930, 2884, 2857, 2857, 1602, 1561, 1502,
1471, 1328, 1255, 1222, 1136, 1109, 1078, 964, 836, 779 cm–1.
1H NMR (400 MHz, acetone-d6): δ = 0.08–0.16 (m, 24 H, TBS-
CH3), 0.55–0.63 (m, 6 H, TES-CH2), 0.87–0.99 (m, 45 H, TBS-t-Bu
+ TES-CH3), 3.13 (dd, J = –15.1, 10.1 Hz, 1 H, ArCH2), 3.36 (dd,
J = –15.1, 5.6 Hz, 1 H, ArCH2), 3.90–4.14 (m, 16 H, OCH2), 5.41
(dd, J = 10.1, 5.6 Hz, 1 H, ArCHO), 6.65 (d, J = 12.3 Hz, 1 H,
ArCH), 6.67 (s, 2 H, ArH), 6.72 (s, 1 H, ArH), 6.74 (d, J = 12.3 Hz,
1 H, ArCH), 7.12 (s, 1 H, ArH).
13C NMR (100 MHz, acetone-d6): δ = –5.0, –5.0, –5.0, 5.6, 7.3,
18.9, 26.3, 47.2, 62.7, 62.8, 62.9, 71.0, 71.0, 71.2, 71.2, 72.9, 112.8,
114.9, 116.7, 116.7, 128.3, 129.6, 129.7, 130.4, 132.8, 137.2, 147.4,
148.0, 148.7, 148.7, 149.0.
1H NMR (400 MHz, acetone-d6): δ = 0.13, 0.13 (2 s, 24 H, TBS-
CH3), 0.92 (s, 36 H, TBS-t-Bu), 2.74 (dd, J = –14.3, 3.5 Hz, 1 H,
ArCH2), 3.07 (dd, J = –14.3, 1.9 Hz, 1 H, ArCH2), 3.98–4.05 (m, 8
H, OCH2), 4.08–4.18 (m, 8 H, OCH2), 4.42–4.47 (m, 1 H, ArCHO),
4.80 (d, J = 4.64 Hz, 1 H, OH), 6.91, 6.92 (2 s, 2 H, ArH), 7.10 (s,
1 H, ArH), 7.45 (s, 1 H, ArH).
13C NMR (100 MHz, acetone-d6): δ = –5.0, 18.9, 26.3, 50.2, 62.9,
62.9, 63.0, 63.0, 71.2, 71.4, 71.6, 75.6, 110.9, 112.1, 112.4, 112.5,
112.8, 113.8, 116.9, 117.2, 146.8, 148.2, 148.5, 149.7, 150.1, 151.7.
HRMS (FAB): m/z [M + Na]+ calcd for C54H100O9Si5Na:
HRMS (FAB): m/z [M + Na]+ calcd for C48H84O9Si4Na: 939.5085;
1055.6106; found: 1055.6102.
found: 939.5084.
Anal. Calcd for C48H84O9Si4: C, 62.83; H, 9.23. Found: C, 62.78; H,
9.28.
2,3,8,9-Tetrakis[2-(tert-butyldimethylsiloxy)ethoxy]-5-(trieth-
ylsiloxy)-11,12-didehydro-5,6-dihydrodibenzo[a,e][8]annulene
(12)
4-Nitrophenyl {2,3,8,9-Tetrakis[2-(tert-butyldimethylsil-
oxy)ethoxy]-5,6-dihydro-11,12-didehydrodibenzo[a,e][8]annu-
len-5-yl} Carbonate (14)
Alkene 11 (870 mg, 842 μmol) was dissolved in THF (20 mL) and
the soln cooled to 0 °C. Dioxane–bromine complex (292 mg, 1.18
mmol, 1.4 equiv) was added in one portion and the mixture stirred
for 30 min at 0 °C in the dark. Subsequently, the mixture was cooled
to –78 °C and 1 M KHDMS in THF (8.42 mL, 8.42 mmol, 10 equiv)
was added via a syringe. The mixture was allowed to warm to 25 °C
over a period of 30 min and stirred for additional 30 min at 25 °C.
The mixture was quenched by addition of sat. NH4Cl soln (5 mL),
transferred to a separatory funnel, and diluted with H2O (10 mL)
and Et2O (25 mL). The organic layer was separated and the aqueous
phase extracted with Et2O (2 × 10 mL). The combined organic lay-
ers were washed with H2O (2 × 10 mL) and brine (10 mL), and dried
(MgSO4); the solvent was removed under reduced pressure. The
residue was subjected to column chromatography (silica gel, light
petroleum ether–EtOAc, 24:1) to give pure, protected alkynol 12 as
a colorless viscous oil; yield: 643 mg (74%); Rf = 0.33 (light petro-
leum ether–EtOAc, 24:1).
Alkynol 13 (260 mg, 282 μmol) was dissolved in CH2Cl2 (2 mL)
and the soln cooled to 0 °C. Then, pyridine (115 μL, 1.42 mmol, 5
equiv) was added followed by 4-nitrophenyl chloroformate (115
mg, 574 μmol, 2 equiv), the mixture stirred for 2 h at 0 °C and for a
further 12 h at 25 °C. Subsequently a few drops of butane-1,4-diol
were added and the mixture stirred for 30 min at 25 °C in order
quench excess 4-nitrophenyl chloroformate. The mixture was dilut-
ed with EtOAc (25 mL) and transferred to a separatory funnel, the
organic layer washed with H2O (3 × 10 mL) and brine (10 mL), and
dried (MgSO4); the solvent was removed under reduced pressure.
The residue was subjected to column chromatography (light petro-
leum ether–EtOAc, 7:1) to give pure activated carbonate 14 as a
light yellow resin; yield: 289 mg (94%); Rf = 0.37 (light petroleum
ether–EtOAc, 7:1).
IR (KBr): 3442, 2930, 2857, 1772, 1604, 1561, 1529, 1506, 1471,
1345, 1256, 1220, 1108, 964, 835, 778 cm–1.
IR (neat/KBr): 2929, 2856, 1601, 1562, 1503, 1471, 1407, 1329,
1252, 1224, 1186, 1134, 1107, 1082, 1006, 969, 835, 776 cm–1.
1H NMR (major isomer, 400 MHz, acetone-d6): δ = 0.09–0.17 (m,
24 H, TBS-CH3), 0.89–0.96 (m, 36 H, TBS-t-Bu), 2.88 (dd, J =
–15.2, 3.8 Hz, 1 H, ArCH2), 3.39 (dd, J = –15.2, 1.8 Hz, 1 H,
ArCH2), 3.97–4.07 (m, 8 H, OCH2), 4.09–4.22 (m, 8 H, OCH2),
5.45–5.49 (m, 1 H, ArCHO), 6.96 (s, 1 H, ArH), 7.02 (s, 1 H, ArH),
7.20 (s, 1 H, ArH), 7.22 (s, 1 H, ArH), 7.59–7.65 (m, 2 H, pNP),
8.32–8.38 (m, 2 H, pNP).
13C NMR (mixture of isomers, 100 MHz, acetone-d6): δ = –5.1,
–5.0, 18.9, 26.3, 26.3, 39.9, 46.5, 62.8, 62.8, 62.9, 62.9, 71.3, 71.5,
71.5, 71.6, 71.6, 79.2, 82.6, 109.6, 110.1, 111.1, 111.4, 111.5,
112.1, 112.4, 112.6, 113.3, 114.1, 116.2, 116.5, 117.6, 119.3, 119.4,
119.5, 122.9, 123.2, 125.9, 126.1, 140.8, 141.8, 144.4, 145.1, 146.2,
146.5, 148.9, 149.0, 149.0, 149.0, 149.9, 150.0, 150.2, 152.5, 152.7,
156.4, 156.7.
1H NMR (400 MHz, acetone-d6): δ = 0.11–0.14 (m, 24 H, TBS-
CH3), 0.55–0.63 (m, 6 H, TES-CH2), 0.89–0.972 (m, 45 H, TBS-t-
Bu + TES-CH3), 2.79 (dd, J = –14.4, 3.5 Hz, 1 H, ArCH2), 3.06 (dd,
J = –14.4, 1.8 Hz, 1 H, ArCH2), 3.98–4.21 (m, 16 H, OCH2), 4.50–
4.54 (m, 1 H, ArCHO), 6.91 (s, 1 H, ArH), 6.93 (s, 1 H, ArH), 7.09
(s, 1 H, ArH), 7.40 (s, 1 H, ArH).
13C NMR (100 MHz, acetone-d6): δ = –5.0, –5.0, 5.4, 7.3, 18.9,
26.3, 26.3, 51.0, 62.8, 62.8, 63.0, 63.0, 71.1, 71.4, 71.5, 71.5, 76.9,
110.6, 112.0, 112.3, 113.0, 113.6, 116.8, 117.2, 146.1, 148.3, 148.6,
149.5, 149.9, 151.0.
HRMS (FAB): m/z [M + Na]+ calcd for C54H98O9Si5Na: 1053.5949;
found: 1053.5945.
2,3,8,9-Tetrakis[2-(tert-butyldimethylsiloxy)ethoxy]-5-hy-
droxy-11,12-didehydro-5,6-dihydrodibenzo[a,e][8]annulene
(13)
HRMS (FAB): m/z [M + Na]+ calcd for C55H87NO13Si4Na:
1104.5147; found: 1104.5147.
In a 15-mL polypropylene tube equipped with a stirring bar and a
rubber septum, TES ether 12 (593 mg, 575 μmol) was dissolved in
THF (9 mL) under an atmosphere of N2. The soln was cooled to
–50 °C and HF·Py (180 μmol, 6.3 mmol, 11 equiv) was added at a
final concentration of 2 vol%. The mixture was stirred for 10 h at
–50 °C, poured into ice-cold sat. NaHCO3 soln, and transferred to a
separatory funnel. The mixture was extracted with Et2O (3 × 15 mL)
and the combined organic layers were washed with H2O (2 × 10
mL) and brine (10 mL), and dried (MgSO4); the solvent was re-
moved under reduced pressure. The residue was purified by column
chromatography (silica gel, light petroleum ether–EtOAc, 17:3) to
4-Nitrophenyl [2,3,8,9-Tetrakis(2-hydroxyethoxy)-5,6-dihydro-
11,12-didehydrodibenzo[a,e][8]annulen-5-yl] Carbonate (4)
In a 10-mL polypropylene tube equipped with a stirring bar and a
rubber septum activated carbonate 14 (52.6 mg, 49 μmol) was dis-
solved in THF (1 mL) under an atmosphere of N2. The soln was
cooled to 0 °C, HF·Py (100 μL, 3.48 mmol, 71 equiv) was added,
and the mixture was stirred for 2 h at 0 °C. Then the soln was diluted
with EtOAc (2 mL) and silica gel (1 g) was added in order to quench
excess HF·Py. The soln was filtered, the solids were washed with
MeOH (3 × 5 mL), and the combined organic layers were concen-
Synthesis 2013, 45, 1207–1214
© Georg Thieme Verlag Stuttgart · New York

PAPER
Tetra(2-hydroxyethoxy)-Substituted Dibenzocyclooctynes
1213
trated under reduced pressure. The residue was subjected to column
chromatography (CHCl3–MeOH, 12:1) to afford pure deprotected
active THE-DIBO 4. An analytical sample of 4 could be obtained
by recrystallization (acetone); white solid/colorless crystals; yield:
27.9 mg (91%); mp 145 °C (dec); Rf = 0.28 (CHCl3–MeOH, 12:1).
3.90 (m, 4 H, pip-CH2), 2.84, 2.93 (2 dd, J = –13.6, 2.0, –14.9, 3.9
Hz, 1 H, ArCH2), 3.16, 3.71 (2 dd, J = –14.9, 2.1, –13.6, 10.3 Hz, 1
H, ArCH2), 5.52–5.57 + 6.28 (m + dd, J = 10.3, 2.0 Hz, 1 H, Ar-
CHO), 7.23–7.51 (m, 8 H, ArH).
13C NMR (mixture of isomers, 400 MHz, CDCl3): δ = 39.8, 43.3,
44.1, 46.1, 46.3, 46.5, 54.1, 54.4, 54.9, 74.3, 77.5, 109.9, 110.3,
110.8, 113.1, 121.4, 123.2, 123.7, 123.8, 125.1, 126.0, 126.1, 126.4,
126.9, 127.1, 127.2, 127.8, 128.0, 128.1, 128.2, 129.0, 130.0, 131.4,
132.2, 148.2, 149.7, 151.1, 152.1, 154.2, 154.3.
IR (KBr): 3406, 2934, 1758, 1602, 1562, 1506, 1334, 1221, 1082,
982, 860 cm–1.
1H NMR (major isomer, 400 MHz, DMSO-d6): δ = 2.75 (dd, J =
–15.2, 3.9 Hz, 1 H, ArCH2), 3.42 (dd, J = –15.2, 1.5 Hz, ArCH2),
3.66–3.79 (m, 8 H, OCH2), 3.98–4.11 (m, 8 H, OCH2), 4.82–4.95
(m, 4 H, OH), 5.29–5.34 (m, 1 H, ArCHO), 6.99 (s, 1 H, ArH), 7.07
(1s, 1 H, ArH), 7.09 (s, 1 H, ArH), 7.24 (s, 1 H, ArH), 7.64–7.69 (m,
2 H, pNP), 8.32–8.38 (m, 2 H, pNP).
HRMS (FAB): m/z [M + H]+ calcd for C22H23N2O2: 347.1754;
found: 347.1756.
LC-MS: tR = 18.9 min; m/z = 369.9 [M + Na]+.
13C NMR (mixture of isomers, 100 MHz, DMSO-d6): δ = 45.1,
59.4, 59.5, 59.6, 70.6, 70.8, 77.8, 81.3, 108.7, 109.3, 110.3, 110.7,
111.1, 111.4, 111.7, 112.5, 112.5, 114.6, 114.8, 116.9, 117.7, 118.3,
118.6, 122.1, 122.8, 125.4, 125.5, 139.7, 140.7, 143.1, 144.1, 145.1,
145.3, 147.4, 147.5, 147.6, 147.8, 148.6, 148.8, 149.0, 151.1, 151.4,
154.9, 155.3.
2,3,8,9-Tetrakis(2-hydroxyethoxy)-5,6-dihydro-11,12-didehy-
drodibenzo[a,e][8]annulen-5-yl {2-[2-(2-{5-[(3aS,4S,6aR)-2-
Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanami-
do}ethoxy)ethoxy]ethyl}carbamate (18)
To a soln of activated carbonate 4 (200 mg, 319 μmol) in DMF (2
mL) was added the TFA salt of biotin conjugate 176a (234 mg, 479
μmol, 1.5 equiv) and DIPEA (124 mg, 957 μmol, 3 equiv) and the
mixture was stirred for 24 h at r.t. After removal of the volatiles un-
der reduced pressure, the residue was subjected to column chroma-
tography (CHCl3–MeOH, 4:1) to furnish pure THE-DIBO-biotin 18
as a colorless foam; yield: 240 mg (89%); Rf = 0.45 (CHCl3–
MeOH, 10:3).
HRMS (ESI): m/z [M + Na]+ calcd for C31H31NO13Na: 648.1687;
found: 648.1685.
2,3,8,9-Tetrakis(2-hydroxyethoxy)-5,6-dihydro-11,12-didehy-
drodibenzo[a,e][8]annulen-5-yl 4-Methylpiperazine-1-carbox-
ylate (15)
IR (KBr): 3389, 2929, 2870, 2482, 2482, 2150, 1694, 1642, 1603,
1560, 1504, 1456, 1408, 1332, 1263, 1222, 1082, 1039, 934, 904
cm–1.
To a soln of activated carbonate 4 (100 mg, 160 μmol) in DMF (2
mL) was added 1-methylpiperazine (80.1 mg, 800 μmol, 5 equiv)
and the mixture was stirred for 12 h at r.t. Then, the solvent was re-
moved under reduced pressure and the residue subjected to column
chromatography (CHCl3–MeOH–7 M NH3 in MeOH, 20:4:1),
which led to pure piperazine conjugate 15. The basic alkyne could
later be converted into its TFA salt by dissolving 15 in MeOH, add-
ing an equimolar amount of TFA, and removal of the solvent under
reduced pressure; colorless foam; yield: 88.2 mg (94%); Rf = 0.55
(CHCl3–MeOH–7 M NH3 in MeOH, 20:4:1).
1H NMR (major isomer, 400 MHz, MeOH-d4): δ = 1.29–1.42 (m, 2
H, biotin-CH2), 1.46–1.72 (m, 4 H, biotin-CH2), 2.15 (t, J = 7.0 Hz,
2 H, biotin-CH2), 2.61–2.76 (m, 2 H, biotin-SCH2 + ArCH2), 2.86
(ddd, J = –12.7, 4.9, 1.9 Hz, 1 H, biotin-SCH2), 3.05–3.16 (m, 2 H,
biotin-SCH2 + ArCH2), 3.24–3.38 (m, 4 H, NCH2), 3.41–3.73 (m, 8
H, OCH2), 3.82–3.95 (m, 8 H, OCH2), 3.99–4.16 (m, 8 H, OCH2),
4.20 (dd, J = 7.7, 4.4 Hz, 1 H, biotin-NCH), 4.42 (dd, J = 7.7, 4.9
Hz, 1 H, biotin-NCH), 5.27–5.36 (m, 1 H, ArCHO), 6.85 (s, 1 H,
ArH), 6.87 (1s, 1 H, ArH), 7.01 (s, 1 H, ArH), 7.19 (s, 1 H, ArH).
IR (KBr): 3419, 2929, 2875, 2742, 1683, 1602, 1561, 1506, 1460,
1432, 1408, 1321, 1285 cm–1.
13C NMR (major isomer, 400 MHz, MeOH-d4): δ = 26.8, 29.4, 29.7,
36.7, 40.2, 41.0, 41.7, 47.2, 56.9, 58.3, 61.4, 61.5, 63.2, 70.5, 70.9,
71.2, 71.3, 71.8, 71.8, 71.9, 78.2, 110.3, 111.4, 112.1, 113.4, 114.8,
117.2, 146.4, 147.5, 148.6, 148.6, 148.9, 149.7, 149.9, 150.0, 157.9,
165.9, 176.0.
1H NMR (mixture of isomers, 400 MHz, MeOH-d4): δ = 1.37–1.50,
1.82–1.96 (2 m, 1 H, pip-CH2), 2.16–2.31, 2.44–3.15 (2 m, 6 H, pip-
CH2 + ArCH2), 2.19, 2.40 (2 s, 3 H, pip-CH3), 3.40–3.65 (m, 2 H,
pip-CH2 + ArCH2), 3.66–3.95 (m, 9 H, pip-CH2 + OCH2), 3.98–
4.19 (m, 8 H, OCH2), 5.33–5.38, 6.07–6.13 (2 m, 1 H, ArCHO),
6.85, 6.88, 6.88, 6.93, 6.96, 7.00, 7.02, 7.15 (8 s, 4 H, ArH).
HRMS (FAB): m/z [M + Na]+ calcd for C41H56N4O14SNa:
13C NMR (mixture of isomers, 400 MHz, MeOH-d4): δ = 40.5, 43.5,
44.5, 45.6, 45.9, 47.1, 54.9, 55.5, 61.5, 61.6, 61.6, 72.0, 72.0, 72.0,
72.1, 72.2, 76.0, 79.4, 79.5, 110.2, 110.9, 111.0, 111.3, 111.7,
112.4, 112.8, 112.8, 113.4, 115.0, 117.1, 117.2, 117.5 119.0, 119.1,
119.8, 142.5, 144.2, 146.2, 146.8, 148.9, 148.9, 149.1, 149.2, 150.0,
150.0, 150.1, 150.3, 155.7, 155.7.
883.3406; found: 883.3410.
LC-MS: tR = 20.2 min; m/z = 883.1 [M + Na]+.
Acknowledgment
We wish to thank Dr. Markus Kramer, Dr. Dorothee Wistuba, and
Paul Schuler for their analytical services.
HRMS (FAB): m/z [M + H]+ calcd for C30H39N2O10: 587.2599;
found: 587.2597.
LC-MS: tR = 14.3 min; m/z = 587.5 [M + H]+.
References
5,6-Dihydro-11,12-didehydrodibenzo[a,e][8]annulen-5-yl
4-Methylpiperazine-1-carboxylate (16)
(1) Huisgen, R. Angew. Chem. 1963, 75, 742.
Analogous to the synthesis of 15 DIBO carbonate 2 (100 mg, 259
μmol) was treated with 1-methylpiperazine (130 mg, 1.30 mmol, 5
equiv). Column chromatography (CHCl3–7 M NH3 in MeOH, 49:1)
yielded pure piperazine conjugate 16. DIBO derivative 16 could lat-
er be converted into the corresponding TFA salt analogous to 15;
colorless foam; yield: 85.4 mg (99%); Rf = 0.25 (CHCl3–7 M NH3
in MeOH, 49:1).
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