FRET Pairs with Fixed Relative Orientation of Chromophores

Article abstract of DOI:10.1002/ejoc.201600489

Synthetic routes to different oligospirothioketal (OSTK) F?rster resonance energy transfer (FRET) constructs are described and the photophysics of these constructs were explored in different solvents. The FRET efficiencies were determined from the experim

Full text of DOI:10.1002/ejoc.201600489

DOI: 10.1002/ejoc.201600489
Full Paper
FRET Rods
FRET Pairs with Fixed Relative Orientation of Chromophores
Pablo Wessig,*[a] Nicole Behrends,[a] Michael U. Kumke,*[a] and Ursula Eisold[a]
Dedicated to Professor Jochen Mattay on the occasion of his 70th birthday
Abstract: Synthetic routes to different oligospirothioketal mined from the experimental data and compared with theoreti-
(OSTK) Förster resonance energy transfer (FRET) constructs are
described and the photophysics of these constructs were ex-
plored in different solvents. The FRET efficiencies were deter-
cal values. The influence of the outstanding rigidity of the novel
OSTK compounds on the FRET is discussed.
Introduction
surface and an extracellular matrix or between two cells. The
force is also measured on the basis of the alteration of the
FRET efficiency. The linker connecting the D–A pair is here a
key element for the dynamic range of accessible forces.
Mechanical forces represent a major factor in the modulation
of cellular processes at different levels, such as adhesion to sur-
faces, the stiffness of cell membranes, or in the proliferation
and differentiation of cells.[1] In general, two kinds of forces can
be distinguished based on their origin: Exogenous and endoge-
nous. Exogenous forces are caused, for example, by gravity or
fluid shear, whereas endogenous forces originate from osmotic
pressure or from proteins (e.g., motor proteins).[2] The measure-
ment of the impact of such diverse forces on cells in vivo is a
major challenge. Progress is strongly coupled to innovations in
experimental methods (instruments but also suitable molecular
probes).
The development/progress of force-based microscopic tech-
niques, such as atomic force microscopy (AFM), and the availa-
bility of genetically encoded (intrinsic) fluorescence probes
(GFP-based probes) have advanced in vivo detection at a single-
cell/molecule level.[3] Based on the tremendous progress made
in genetic engineering, the labeling of specific proteins inside
the cell or in the membrane, which react to different external
force stimuli initiating a downstream signaling cascade or act
as an origin of force themselves (e.g., motor proteins), is now
being developed.[2] The latter approach is based on the concept
of Förster resonance energy transfer (FRET) and transduces the
force-related alteration in the relative positions of fluorescent
molecules into an optical signal [in the case of intrinsic probes
two GFPs act as a donor (D)–acceptor (A) pair]. In addition, ex-
trinsic sensors utilizing the FRET concept are also applied. Here,
D and A are separated by a linker and one side of the FRET pair
is attached to a surface (anchor side) and the other side to
the cell surface (e.g., by specific binding through an antibody–
antigen interaction)[3c] to monitor the forces between the cell
Intrinsic as well as extrinsic FRET sensors are further defined
by the specific spectroscopic parameters of the D–A pair, which
defines the distance range (and therefore also the force range)
accessible by a specific pair. Here, the so-called Förster radius
R0 is commonly used as a reference parameter. It describes the
distance between D and A at which the efficiency of the FRET
is 50 %. As a rule of thumb, a specific FRET pair can be used to
monitor distance alterations in the range 0.5R0 < r < 2R0. The
combination of different intrinsic (spectroscopic) properties of
D and A determine the value of R0 [see Equation (1)]. The over-
lap integral J is defined by the spectral overlap between the
donor emission and acceptor absorption spectra and is a meas-
ure of the overall number of resonant transitions for D and A.
Achieving an improvement in J is a frequently used approach
to increase R0. Also, the fluorescence quantum yield of D has
been addressed to pushinfluence R0. On the other hand, the
influence of the orientation factor κ2 [see Equation (2)] has also
been recognized but attempts to tailor this parameter are
sparse. The parameter κ2 relates the relative orientation of the
transition dipole moments of D and A in space and is defined
ˆ
by the dot product of the respective unit vectors d, â, and rˆ
[see Equation (2a)] or, more handily, by the respective angles
[see Equation (2b)].[4]
[a] Institut für Chemie, Universität Potsdam,
Karl-Liebknecht-Str. 24–25, 14476 Potsdam, Germany
E-mail: wessig@uni-potsdam.de
http://ag-wessig.chem.uni-potsdam.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600489.
The influence of κ2 on the calculated donor (D) acceptor (A)
distance can be large. For a D–A couple in solution, both free
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to rotate, κ2 will be 2/3. In cases in which D and/or A are linked at the same time, the distance between D and A is decreased,
to a (macro)molecule, the rotational freedom can be distinctly which should lead to an increase in the FRET efficiency. A sim-
altered. Depending on the nature of the linker and the chemical ple estimate shows that the diminishing value of κ2 dominates
microenvironment created by the (macro)molecule, limitations for a small amount of bending (see the Supporting Informa-
in the accessible space due to intramolecular interactions or tion). The second case occurs when the transition moments
due to conformational hindrance can lead to distinct alterations of D and A are arranged perpendicularly to each other and,
in the value of κ2. The miscalculation of κ2 can cause an error additionally, the transition moment of the acceptor (or the do-
of up to 40 % in the calculation of R0.[4a] The situation may nor) is also perpendicular to the line connecting D and A in the
become even more complex in cases in which the κ2 value and “idle state” (Figure 1, b). According to Equation (2b), κ2 is zero
the D–A distance are no longer independent.[5]
Measurement of the forces should be possible if the two
fluorescent dyes of a FRET pair are rigidly connected (and sub-
sequently fully controlled) by a molecular rod (R). In view of the
and no FRET should take place. Upon bending, the orthogonal-
ity between the transition moments is cancelled resulting in
increasing FRET.
To realize this concept, a FRET pair needs fluorescent dyes
functional principles one can distinguish between two border- that can be rigidly connected with a molecular rod. Further-
line cases, which are depicted in Figure 1. The first case exists more, the angle between the transition moments of the dyes
if, in the “idle state”, the transition moments (green arrows) of and the direction axis of the rod should ideally be 0 or 90°.
the donor and acceptor are collinear with respect to each other Rigidity is of the utmost importance because it has been shown
and to the line connecting D and A (Figure 1, a). Under these that due to molecular vibrations very efficient FRET is observed
circumstances, the orientation factor κ2 reaches the maximum even for orthogonally aligned transition dipole moments of D
possible value of 4. If the FRET rod is bent by an external stimu- and A.[6] Based on our long-term experience with oligospiro-
lus (blue arrows) the FRET efficiency should decrease as a result ketal (OSK) rods[7] and the recently developed oligospirothio-
of the decreasing κ2 [cf. Equation (2)]. It should be noted that, ketal (OSTK) rods,[8] we decided to use these rods in the current
project. Very recently we reported on a new FRET pair consist-
ing of a coumarin dye as donor and a dioxolobenzodioxole
(DBD) dye[9] as acceptor.[10] The structures of these dyes meet
the requirements stated above perfectly. Thus, in both cases
the transition moments and the direction axes are nearly collin-
ear and a point of connection to the OS(T)K rods is available
(Figure 2). Herein we report on the synthetic routes to FRET
pairs with rigid connection of the dyes in line with principle a
in Figure 1.
Figure 2. Coumarin (Cou) and [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) dyes
with transition moments (green) and direction axis of the rods (blue).
Results and Discussion
To compare the properties of the rigidly joined FRET pairs with
more flexible counterparts we developed for each dye both a
rigid and flexible combinable building block, hereinafter re-
ferred to as RC and FC dyes, respectively. The synthesis of the
RC coumarin commenced with the commercially available 6,7-
dihydroxycoumarin 1. After silylation of the hydroxy groups, ac-
etalization with 4-pivaloyloxycyclohexanone under Noyori con-
ditions[11] gave the spirane 3. After removal of the pivaloyl pro-
tecting group and oxidation we obtained the RC coumarin 5
(Scheme 1).
Figure 1. Functional principles of force-responsive FRET rods (D = donor, A =
The FC coumarin building block was synthesized in two
acceptor, R = molecular rod, green arrows = transition dipole moments, blue
arrows = external stimuli).
steps starting with the previously described carboxylic acid 6.[1]
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Scheme 1. Synthesis of ketone 5. Reagents and conditions: i) TMSCl, Et3N,
toluene, quant.; ii) TMSOTf (cat.), DCM, 49 %; iii) LiOH·H2O, MeOH, 94 %;
iv) Dess–Martin periodinane, DCM, 87 %.
Coupling with 4-hydroxypiperidine followed by oxidation of the
hydroxy group afforded the FC coumarin 8 (Scheme 2).
Scheme 3. Synthesis of ketone 17 (R = TBDMS). Reagents and conditions:
i) PPTSA, toluene, 95 %; ii) K2[ON(SO3)2], KH2PO4 (pH 5–6), MeOH/acetone
(1:3), 89 %; iii) 1. PtO2, H2, THF, 2 h; 2. NaH, MOMBr, –10 °C, 91 %; iv) 1. BuLi,
CuI, THF; 2. butyryl chloride, 90 %; v) pTSA, MeOH, quant.; vi) benzyl propio-
late, DMAP, DCM, 91 %; vii) Dess–Martin periodinane, DCM, 60 %.
Scheme 2. Synthesis of ketone 8. Reagents and conditions: i) 4-hydroxypiper-
idine, DCC, HOBt, DMF, 91 %; ii) Dess–Martin periodinane, DCM, 90 %.
The synthesis of the RC DBD building block proved to be
considerably more sophisticated, because an acetalization in
analogy to that used for coumarin 1 fails with DBD dyes. There-
fore the spirane moiety has to be installed at an early stage of
the synthesis. First, we prepared spirane 11 from the silyl-pro-
tected acetal of 4-hydroxycyclohexanone 9[12] and hydroxy-
hydroquinone 10. Oxidation with Fremy's salt {K2[ON(SO3)2]}
gave the quinone 12 in very good yield.[13] Reduction of 12
and subsequent protection of the resulting catechol with me-
thoxymethyl (MOM) groups gave compound 13. It should be
noted that the MOM groups are necessary for complete two-
fold lithiation in the next step. After Li–Cu exchange, reaction
with butyryl chloride afforded the MOM-catechol 14. Deprotec-
tion with p-toluenesulfonic acid (pTSA) gave the nonfluorescent
catechol 15 quantitatively. In the next step, the DBD skeleton
was completed by 4-(dimethylamino)pyridine (DMAP)-catalyzed
cyclization[14] with benzyl propiolate to give DBD dye 16. The
last step consists in the oxidation of the secondary hydroxy
group to give the RC DBD building block 17 (Scheme 3).
Scheme 4. Synthesis of ketone 20. Reagents and conditions: i) 4-hydroxy-
piperidine, DCC, HOBt, DCM, quant.; ii) (COCl)2, DMSO, DCM, 58 %.
With the RC (5, 17) and FC (8, 20) building blocks in hand
we next investigated their combination with OSK rods. For this
purpose we performed model reactions with diol 21[15] by us-
ing the double-activation method (here cyclohexanone was the
placeholder for the other dye).[7a] Unfortunately, the reaction
The FC DBD building block 20 was prepared in a similar way between ketone 5 and diol 21 provided only the symmetric
to FC coumarin 8 starting from the previously described DBD OSK rod 22 bearing two coumarin chromophores instead of the
acid 18 (Scheme 4).[9c]
desired unsymmetrical rod (Scheme 5).
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Scheme 5. Formation of rod 22. Reagents and conditions: i) 1. NaH, TMSCl;
2. TMSOTf, 83 %.
The same result was obtained when the FC building block 8
was used instead of 5 (Scheme 6). This behavior, which is the
consequence of several transacetalization steps, has frequently
been observed in the past and is the principal disadvantage of
OSK rods. The considerably lower solubility of symmetrical OSK
rods is the main driving force for this outcome.
Scheme 7. Synthesis of compounds 26–29. Reagents and conditions:
i) TMSCl, Et3N, toluene, quant.; ii) 1. NaH, TMSCl; 2. TMSOTf, Et2O, 74 % (26),
55 % (27); iii) TMSOTf, DCM, 78 % (28), 93 % (29).
After these preliminary studies we turned our attention to
the synthesis of Cou-DBD FRET pairs. Starting with the previ-
ously developed building block 30,[8] the coumarin diol 31 was
obtained by the iodine-catalyzed formation of the thioketal fol-
lowed by removal of the TBDMS groups by HF. It should be
noted that silyl groups are ideally suited for the protection of
hydroxy groups in the presence of thiol groups due to the low
S–Si affinity compared with the high O–Si affinity. Finally, 31
was converted into the bis-silyl ether 32, which smoothly re-
acted with 17 to yield the rigid FRET rod 33 and with 20 to
form the semi-flexible FRET rod 34 (Scheme 8 and Scheme 9).
Scheme 6. Formation of compound 23. Reagents and conditions: i) 1. NaH,
TMSCl; 2. TMSOTf, 39 %.
Very recently we reported on oligospirothioketal (OSTK) rods,
which differ from OSK rods in that one or more ketals are re-
placed by thioketals.[5] In contrast to ketals, the formation of
thioketals from dithiols and ketones is not an equilibrium reac-
tion and thioketals are significantly more stable towards hydro-
lytic conditions than ketals. For this reason we hypothesized
that OSTK rods could circumvent the problems outlined in
Scheme 5 and Scheme 6. To this end, we used diol 24[8,16] bear-
ing a 1,3-dithiane instead of a 1,3-dioxane and smoothly ob-
tained coumarins 26 and 27 in satisfactory yields by using the
double-activation method (Scheme 7).[7a] For the preparation
of the analogous products with DBD dyes (28 and 29), it proved
beneficial to apply Noyori conditions[11] with compound 25,
prepared from 24 by silylation in quantitative yield (Scheme 7).
Scheme 8. Synthesis of FRET pair 33. Reagents and conditions: i) 1. I2; 2. HF,
DCM, 87 %; ii) TMSCl, Et3N, toluene, quant.; iii) TMSOTf, DCM, 64 %.
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Table 1. Spectroscopic properties of single and double labeled rods 26, 29, 33, and 34.
Solvent
ΦD
ΦA
εD (340 nm) [L/mol cm]
εA (450 nm) [L/mol cm]
τD [ns]
τDA [ns]
τA [ns]
E (Φ)
E (τ)
26 acetone
CHCl3
29 acetone
CHCl3
0.12 0.006
0.27 0.01
–
–
–
–
9240 460
12300 620
–
–
–
–
0.8 0.1
1.7 0.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.54 0.03
0.65 0.03
1950 100
2320 120
2840 140
22.5 0.1
24.1 0.1
33 acetone
0.01 0.001 0.58 0.03 9330 470
0.1 0.03 (68 %) 18.9 0.1 18.9 0.1 0.92 0.01 0.88 0.05
1.2 0.1 (32 %)
CHCl3
0.01 0.001 0.71 0.04 13600 680
3200 160
0.1 0.03 (93 %) 22.3 0.1 22.3 0.1 0.96 0.01 0.95 0.02
1.8 0.1 (7 %)
34 acetone
0.02 0.001 0.60 0.03 9700 490
0.05 0.003 0.74 0.04 13900 700
1455 70
1855 90
0.5 0.1
1.2 0.1
18.1 0.1 18.1 0.1 0.83 0.02 0.38 0.2
22.5 0.1 22.5 0.1 0.81 0.02 0.30 0.1
CHCl3
chromic shift in the absorption and fluorescence bands of the
Cou-OSTK rod upon changing the solvent polarity. More distinct
are the alterations in the extinction coefficients and the fluores-
cence quantum yields Φ as well as in the fluorescence decay
times τ. Changing the solvent from acetone to chloroform leads
to an increase in these photophysical parameters. The fluores-
cence decay curves (λex = 340 nm, λem = 420 nm) of the Cou-
OSTK rod 26 show monoexponential behavior in polar and non-
polar solvents. The fluorescence decay time for the Cou-OSTK
rod in acetone (τ = 0.8 ns) is shorter than that in chloroform
(τ = 1.7 ns) and correlates with the results of the steady-state
fluorescence measurements and quantum yields (Table 1).
The spectroscopic properties of the DBD dyes have previ-
ously been reported to be sensitive to the local environment
and this is especially reflected in the huge alteration of the
spectral intensity distribution.[9a] The Stokes shift (Δλ) of more
than 100 nm for the DBD-OSTK rod 29 is very large. In acetone
it is about 117 nm (λabs = 419 nm, λem = 536 nm) and in chloro-
form it is about 124 nm (λabs = 427 nm, λem = 551 nm). The
solvent dependence of the spectroscopic properties is also evi-
denced by the fluorescence parameters Φ and τ. The fluores-
cence decay time (λex = 450 nm, λem = 650 nm) for the DBD-
OSTK rod 29 in acetone (τ = 22.5 ns) is slightly shorter than
that in chloroform (τ = 24.1 ns). This is complementary to the
results for the fluorescence quantum yields Φ, which were de-
termined to be 0.54 and 0.65 in acetone and chloroform, re-
spectively.
Scheme 9. Synthesis of FRET pair 34. Reagents and conditions: i) TMSOTf,
DCM, 50 %.
The mono- and double-labeled OSTK rods were character-
ized by absorption and fluorescence spectroscopy. With respect
to the latter, steady-state as well as time-resolved data were
acquired. In general, the dyes show a distinct solvent depend-
ence of their photophysical properties independent of the at-
tachment to an OSTK rod unit (see Table 1 and Figure 3).
Double-Labeled OSTK Rods 33 and 34
The fluorescence emission of the Cou-OSTK rod (donor) shows
a very good spectral overlap with the absorption of the DBD-
OSTK rod (acceptor, see the Supporting Information). From the
spectral overlap integral the theoretical Förster distance of 33
was calculated by Equation (1) with κ2 = 3.9 to be R0 = 4.8 nm.
The distance R between the donor and acceptor is 1.9 nm.
Based on these data, a very efficient FRET with E ≥ 99 % was
expected for compound 33 and confirmed by the experimental
results (see below).
Figure 3. Excitation and emission spectra of 33 in CHCl3.
Single-Labeled OSTK Rods 26 and 29
The spectroscopic properties of the coumarin do not change
upon linking to the OSTK rod. No change in the spectral posi-
tions of the absorption or fluorescence emission compared with
the coumarin parent compound (6) is found, because the “di-
The fluorescence decay curve of the rigid FRET-OSTK rod 33
rect” substituents at the 6- and 7-positions are not altered. in chloroform shows a biexponential fluorescence decay. The
Upon excitation an intramolecular charge transfer (“push–pull” strong quenching of the donor fluorescence yields a very short
system) is initiated, which is, in general, the origin of the ob- fluorescence decay time of τ1 = 0.1 ns (93 %). The small contri-
served photophysics of the 6,7-dialkoxycoumarins. The ex- bution of the second decay time τ2 = 1.8 ns (7 %) was attrib-
pected solvent dependence can be observed as a slight batho- uted to a minor amount of unbound coumarin precursor still in
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the sample. The experimental FRET efficiency for the rigid FRET- rods show some distinct features that make them highly attract-
OSTK rod 33 was calculated by Equation (3) using the fluores- ive as building blocks for further development in the field of
cence decay times as well as the fluorescence quantum yields. optical force detection. The relative orientation of D and A is
Both sets of data are in excellent agreement and for both sol- highly fixed especially in 33 and an averaging out of errors
vents FRET efficiencies of >90 % were determined, which fit well should not be operative, which also makes the good agreement
with the theoretical value calculated with Equation (3).
between theoretical and experimental FRET efficiencies inter-
esting from a fundamental point of view. Compared with re-
cently reported FRET systems with orthogonally fixed align-
ment of D and A, no distinct influence of vibrations or “environ-
mental noise” was observed.[6,18] The excellent agreement be-
tween theoretically expected and experimentally found FRET
efficiencies underlines the outstanding rigidity of the OS(T)K
rods. The minimization of vibrational influences on FRET effi-
ciency is a prerequisite for application in force measurements
based on alterations in the fluorescence parameters of a D–A
pair because it will significantly improve the precision of the
measurement by limiting uncertainties due to force-unrelated
motions.[17]
OSTK rod 34 was investigated as a reference compound in
which the DBD dye is connected to a flexible linker yielding a
distinctly higher degree of freedom with respect to the rotation
of the DBD dye relative to the Cou dye. The overall maximum
distance between donor and acceptor was approximately the
same (ca. 2.2 nm), but because of the greater flexibility the κ2
value was reduced to 2/3, which yielded a smaller R0 value of
2.7 nm. Consequently a reduced FRET efficiency was expected.
From Table 1 it can be seen that in the case of compound
34 the calculated FRET efficiencies based on the fluorescence
quantum yields and the fluorescence decay times are distinctly
different, and the solvent seems to have no influence on the
determined values. This observed difference in E may be an
With the successful proof-of-concept the next steps will in-
indication of the formation of an equilibrium between two (lim- clude a systematic increase in the OS(T)K rod length to monitor
iting) forms of 34 (“limiting” conformers). One “limiting” con- the contour length of the rods to determine the critical length
former is represented by the DBD dye unit rotated in such a at which significant alterations, for example, due to vibrational
way that it becomes closer towards the rod and the coumarin motions, become effective. Moreover, complementary con-
unit. Here, the distance between the two dyes (D–A distance) structs carrying D and A with an orthogonal orientation of their
is highly reduced, but at the same time the relative orientation respective transition dipole moments as well as OS(T)K rods
of the transition dipole moments (κ2 influence) could be less with a link adding flexibility (again) are currently being synthe-
efficient for FRET.
sized to create a tool box of linkers. We are also exploring the
incorporation of OS(T)K rods into vesicles as biomimetic sys-
tems for cells, which will be combined with AFM, serving as an
artificial force stimulus, to further develop the concept of opti-
cal force measurements based on our novel probes.
In the second “limiting” conformer an elongated form is
present in which the distance is longer but due to an almost
collinear orientation of the transition dipole moments (κ2 be-
comes close to four) a more efficient FRET may be operative.
Although for very efficient energy transfer the corresponding
fluorescence decay time becomes extremely short and is conse-
quently not resolvable, in the case of less efficient energy trans-
fer a quenched fluorescence decay time can be measured. For
such a system only in the quantum yield measurements will
both (limiting) conformers contribute to the calculation of the
FRET efficiency, whereas in the case of decay time measure-
ments the “dark form” does not contribute and a smaller FRET
efficiency is determined. The spectroscopic properties of 34,
Experimental Section
General Information: See the Supporting Information.
4-Methyl-6,7-bis[(trimethylsilyl)oxy]-2H-chromen-2-one (2): An-
hydrous NEt3 (9.48 mg, 93.67 mmol, 6.0 equiv.) and TMSCl (10.18 g,
93.67 mmol, 6.0 equiv.) were added to a suspension of 6,7-di-
hydroxy-4-methylcoumarin (1; 3.00 g, 15.61 mmol) in anhydrous tol-
uene (100 mL). The reaction mixture was stirred overnight at room
compared with 33, underline the strong influence of κ2 on the temperature, filtered through Celite®, washed with petroleum ether
(PE), and the solvent was evaporated. The residue was suspended
in PE and filtered through Celite® once more. Evaporation of the
solvent yielded 2 (15.20 g, 15.45 mmol, 99 %) as a white solid, m.p.
FRET efficiency. It should be noted that in 33 as well as in 34
the distance between D and A is small (<5 nm) and the dipole
approximation of the FRET theory may not be fully applica-
ble.[16] The relative orientation of D and A is highly fixed, espe-
cially in 33, and an averaging out of errors should not be opera-
tive, which makes the good agreement between the theoretical
and experimental FRET efficiency for 33 especially interesting
from a fundamental point of view. Work is in progress to investi-
gate this topic in greater detail by systematically altering the
1
57–60 °C. H NMR (300 MHz, CDCl3): δ = 6.99 (s, 1 H), 6.82 (s, 1 H),
6.15 (s, 1 H), 2.35 (s, 3 H), 0.31 (s, 9 H), 0.27 (s, 9 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 161.4, 152.0, 150.5, 149.2, 143.7, 115.1, 114.0,
112.5, 108.5, 18.6, 0.2 ppm. IR (ATR): ν = 3155, 2960, 2787, 2769,
˜
1722, 1666, 1609, 1552, 1503, 1418, 1387, 1368, 1292, 1249, 1227,
1214, 1170, 1151, 1061, 1033, 1005, 941, 904, 884, 865, 843, 758,
695, 670, 605 cm–1. HRMS: calcd. for C16H25O4Si2 337.1291 [M + H]+;
distance between D and A by using the unique rigidity of the found 337.1263.
OSTK rods.
8′-Methyl-6′-oxo-6′H-spiro[cyclohexane-1,2′-[1,3]dioxolo[4,5-
g]chromen]-4-yl Pivalate (3): A solution of 2 (4.00 g, 11.98 mmol)
and 4-oxocyclohexyl pivalate (2.47 g, 12.48 mmol, 1.05 equiv.) in
anhydrous DCM (150 mL) was treated with 2 drops of TMSOTf. After
stirring overnight the solvent was removed under reduced pressure
and the residue was purified by flash silica gel column chromatog-
Conclusions
The FRET system based on OS(T)K rods has been successfully
developed and compared with other rigid spacer concepts the raphy [Hex/ethyl acetate (EE), 5:1] to yield 3 (2.15 g, 5.77 mmol,
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1
49 %) as a white solid, m.p. 215–218 °C. H NMR (300 MHz, CDCl3):
3422, 3057, 2927, 2861, 2694, 2669, 1582, 1493, 1449, 1402, 1386,
1347, 1264, 1204, 1141, 1116, 1075, 1047, 1029, 1010, 961, 920,
856, 806, 779, 745, 711, 676, 640 cm–1. HRMS: calcd. for C18H19NO6
345.1244 [M]+; found 345.1221.
4
δ = 6.89 (s, 1 H), 6.76 (s, 1 H), 6.14 (d, J = 0.9 Hz, 1 H), 5.04–4.95
(m, 1 H), 2.38–2.32 (m, 3 H), 2.19–1.90 (m, 8 H), 1.26–1.17 (m, 9
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 177.7, 161.3, 152.4, 150.6,
150.4, 144.6, 119.7, 113.5, 111.9, 101.9, 98.3, 67.9, 38.9, 31.0, 27.2,
1-[2-(8-Methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-2-yl)acet-
yl]piperidin-4-one (8): DMP (848 mg, 2.00 mmol, 1.1 equiv.) was
added to a solution of 7 (628 mg, 1.82 mmol) in anhydrous DCM
(80 mL). The mixture was stirred at room temperature overnight
and then washed with an aqueous solution of NaHCO3/Na2S2O3
(250 g/L, 3×) and brine, and extracted several times with DCM. The
combined organic layers were dried with MgSO4, evaporated, and
the resulting residue was purified by flash silica gel column chroma-
tography (DCM/MeOH, 100:1) to yield 8 (561 mg, 1.63 mmol, 90 %)
as a white solid, m.p. 174 °C. 1H NMR (300 MHz, CDCl3): δ = 6.92 (s,
1 H), 6.76 (s, 1 H), 6.74 (t, 3J = 4.99 Hz, 2 H), 6.11 (s, 1 H), 3.93 (t,
27.1, 19.1 ppm. IR (ATR): ν = 3056, 2968, 2940, 2872, 1719, 1623,
˜
1583, 1493, 1452, 1399, 1376, 1344, 1266, 1249, 1228, 1206, 1165,
1150, 1138, 1118, 1065, 1043, 1000, 972, 921, 896, 846, 808, 769,
741 cm–1. HRMS: calcd. for C21H24O6 372.1573 [M]+; found 372.1565.
4-Hydroxy-8′-methyl-6′H-spiro[cyclohexane-1,2′-[1,3]dioxolo-
[4,5-g]chromen]-6′-one (4): A solution of 3 (100 mg, 0.27 mmol)
and LiOH·H2O (33 mg, 0.81 mmol, 3.0 equiv.) in MeOH (10 mL)
was stirred at room temperature until complete conversion of 3,
monitored by TLC. The organic layer was washed with 1
N HCl and
brine and extracted with DCM (3×). The combined organic layers
were dried with MgSO4 and concentrated under reduced pressure.
The resulting residue was purified by flash silica gel column chro-
matography (DCM/MeOH, 100:1) to yield 4 (73 mg, 0.25 mmol,
3
3
3J = 5.93 Hz, 2 H), 3.77 (t, J = 6.12 Hz, 2 H), 3.14 (d, J = 4.90 Hz, 2
H), 2.51 (q, J = 5.65 Hz, 4 H), 2.33 (s, 3 H) ppm. 13C NMR (75 MHz,
3
CDCl3): δ = 205.8, 166.0, 161.0, 152.2, 150.5, 150.4, 144.5, 113.9,
112.3, 110.7, 102.2, 98.4, 44.1, 40.9, 40.7, 40.5, 38.5, 19.0 ppm. IR
1
94 %) as a white solid, m.p. 145 °C. H NMR (300 MHz, CDCl3): δ =
6.92–6.87 (m, 1 H), 6.81–6.71 (m, 1 H), 6.14 (d, 4J = 0.9 Hz, 1 H),
4.05–3.94 (m, 1 H), 2.39–2.33 (m, 3 H), 2.26–2.14 (m, 2 H), 2.05–1.78
(m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 161.5, 152.6, 150.9,
150.4, 144.8, 120.1, 113.4, 111.8, 101.9, 98.2, 66.4, 31.2, 30.7,
˜
(ATR): ν = 3126, 2933, 2858, 1730, 1644, 1583, 1493, 1446, 1402,
1381, 1366, 1346, 1263, 1221, 1201, 1165, 1142, 1092, 1037, 963,
918, 888, 847, 824, 806, 778, 745, 728, 709, 648 cm–1. HRMS: calcd.
for C18H17NO6 343.1056 [M]+; found 343.1064.
19.1 ppm. IR (ATR): ν = 3879, 3078, 2771, 2596, 2535, 2297, 2233,
˜
tert-Butyl[(4,4-dimethoxycyclohexyl)oxy]dimethylsilane (9): A
solution of tert-butyl[(4,4-dimethoxycyclohexyl)oxy]dimethylsilane
(1.5 g, 6.57 mmol) and pTSA (12.5 mg, 0.07 mmol, 0.01 equiv.) in
trimethyl orthoformate/MeOH (5:1, 24 mL) was stirred until com-
plete conversion (1 h), controlled by TLC. NEt3 (100 μL) was added
and the solvent was removed in vacuo. The residue was purified by
flash silica gel column chromatography (Hex/EE, 20:1) to yield 9
(1.8 g, 6.56 mmol, quant.) as a colorless oil. 1H NMR (300 MHz,
CDCl3): δ = 3.81–3.65 (m, 1 H), 3.11–3.00 (m, 6 H), 2.04–1.83 (m, 2
H), 1.71–1.46 (m, 6 H), 1.05–0.92 (m, 9 H), 0.11 to –0.01 (m, 6 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 100.0, 69.3, 47.9, 47.5, 32.0, 29.4, 26.4,
1707, 1624, 1581, 1494, 1453, 1401, 1368, 1346, 1270, 1249, 1207,
1142, 1119, 1071, 1045, 979, 951, 924, 896, 854, 809, 770, 743 cm–
1. HRMS: calcd. for C16H16O5 288.0998 [M]+; found 288.0990.
8′-Methyl-6′H-spiro[cyclohexane-1,2′-[1,3]dioxolo[4,5-g]chrom-
ene]-4,6′-dione (5): Dess–Martin periodinane (DMP; 668 mg,
1.58 mmol, 1.1 equiv.) was added to a solution of 4 (413 mg,
1.43 mmol) in anhydrous DCM (50 mL). The mixture was stirred at
room temperature overnight and then washed with an aqueous
solution of NaHCO3/Na2S2O3 (250 g/L, 3×) and brine, and extracted
several times with DCM. The combined organic layers were dried
with MgSO4, evaporated, and the resulting residue was purified by
flash silica gel column chromatography (PE/EE, 3:1) to yield 5
(358 mg, 1.25 mmol, 87 %) as a white solid, m.p. >180 °C (decomp.).
1H NMR (300 MHz, CDCl3): δ = 6.97 (s, 1 H), 6.83 (s, 1 H), 6.18–6.15
(m, 1 H), 2.73–2.60 (m, 5 H), 2.44–2.33 (m, 8 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 207.3, 161.1, 152.3, 150.5, 150.2, 144.3, 118.1,
–4.2 ppm. IR (ATR): ν = 2951, 2930, 2856, 1472, 1462, 1435, 1375,
˜
1249, 1236, 1132, 1102, 1051, 1019, 935 914, 905, 865, 850, 833,
809, 771, 675 cm–1
.
4′-[(tert-Butyldimethylsilyl)oxy]spiro[benzo[d][1,3]dioxole-2,1′-
cyclohexan]-5-ol (11): Anhydrous toluene (150 mL) and pyridinium
p-toluenesulfonate (PPTSA; 17.0 mg, 0.07 mmol, 0.01 equiv.) were
added to a two-necked flask equipped with a distillation apparatus
and the mixture was stirred and heated to 80 °C. Benzene-1,2,4-
triol (10; 835.4 mg, 6.62 mmol) was added and the mixture was
heated at reflux. Compound 9 (2.0 g, 7.26 mmol, 1.10 equiv.) was
added in three portions every 15 min. The solution was then heated
at reflux for an additional hour. After cooling the solvent was evapo-
rated and the residue purified by flash silica gel column chromatog-
raphy (PE/EE, 10:1) to yield 11 (2.1 mg, 6.30 mmol, 95 %) as a beige
113.8, 112.2, 102.4, 98.5, 36.9, 33.8, 19.0 ppm. IR (ATR): ν = 3436,
˜
3120, 3043, 2934, 2903, 1781, 1708, 1621, 1579, 1493, 1450, 1417,
1400, 1364, 1348, 1326, 1273, 1238, 1149, 1115, 1059, 1043, 1008,
959, 930, 809, 776, 761, 745, 707 cm–1. HRMS: calcd. for C16H14O5
286.0841 [M]+; found 286.0842.
2-[2-(4-Hydroxypiperidin-1-yl)-2-oxoethyl]-8-methyl-6H-[1,3]di-
oxolo[4,5-g]chromen-6-one (7): 4-Hydroxypiperidine (20 mg,
0.20 mmol, 1.05 equiv.), N,N′-dicyclohexylcarbodiimide (DCC; 28 mg,
0.21 mmol, 1.1 equiv.), and 1-hydroxybenzotriazole (HOBt; 43 mg,
0.21 mmol, 1.1 equiv.) were added to a solution of 6 (50 mg,
0.91 mmol) in anhydrous DMF (10 mL). After stirring overnight at
room temperature the precipitate was filtered through Celite®. The
1
solid, m.p. 80–82 °C. H NMR (300 MHz, CDCl3): δ = 6.60–6.54 (m, 1
3
H), 6.39–6.33 (m, 1 H), 6.21 (dd, J = 2.4, 8.3 Hz, 1 H), 3.99–3.91 (m,
1 H), 2.24–2.11 (m, 2 H), 1.94–1.70 (m, 7 H), 0.92 (s, 10 H), 0.09 (s, 7
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 150.2, 148.1, 141.5, 118.6,
107.8, 105.8, 98.1, 66.4, 31.1, 30.5, 25.8, 18.1, –4.8 ppm. IR (ATR): ν =
˜
organic layer was washed with 0.1
N HCl, aq. NaHCO3, and brine
3319, 3262, 3088, 2953, 2931, 2855, 2709, 1636, 1618, 1478, 1437,
1374, 1322, 1249, 1198, 1144, 1112, 1062, 1043, 1014, 964, 947, 896,
853, 830, 776, 712, 677, 641, 608 cm–1. HRMS: calcd. for C18H28O4Si
336.1757 [M]+; found 336.1763.
and dried with MgSO4. The solvent was evaporated and the residue
purified by flash silica gel column chromatography (DCM/MeOH,
100:1) to yield 7 (60 mg, 0.17 mmol, 91 %) as a white solid, m.p.
1
98–100 °C. H NMR (300 MHz, CDCl3): δ = 6.91 (s, 1 H), 6.76 (s, 1 H),
6.71 (t, 3J = 5.09 Hz, 1 H), 6.12 (s, 1 H), 3.90–4.09 (m, 2 H), 3.62–3.75
(m, 1 H), 3.17–3.38 (m, 2 H), 3.04 (d, 3J = 5.09 Hz, 2 H), 2.23 (m, 3
4′-[(tert-Butyldimethylsilyl)oxy]spiro[benzo[d][1,3]dioxole-2,1′-
cyclohexane]-5,6-dione (12): Fremy's salt (K2NO7S2, 12.54 g,
H), 1.87 (m, 2 H), 1.56 (m, 2 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 37.40 mmol, 2.50 equiv.) was added to a solution of KH2PO4 (1.4 g,
165.4, 161.2, 152.5, 150.6, 150.4, 144.6, 113.8, 112.0, 111.0, 102.1, 10.29 mmol, 0.55 equiv.) in H2O (90 mL) at 0 °C. Compound 11
98.2, 66.4, 42.8, 38.8, 38.4, 34.2, 33.5, 19.0 ppm. IR (ATR): ν = 3830,
(6.29 g, 18.70 mmol, 1.0 equiv.) dissolved in MeOH/acetone (1:3,
˜
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Full Paper
20 mL) was added to the solution within 15 min. After stirring at
residue washed with H2O/DCM. The solvent was dried with MgSO4
0 °C for 4 h, the suspension was stirred overnight at room tempera- and evaporated to yield 15 (190 mg, 502 μmol, quant.) as a red
ture. The resulting yellow precipitate was washed with H2O and
extracted with DCM several times. The combined organic layers
were dried with MgSO4 and evaporated to yield 12 (2.1 mg,
6.30 mmol, 95 %) as an orange solid, m.p. 197–199 °C. 1H NMR
(300 MHz, CDCl3): δ = 5.94 (m, 2 H), 4.10–3.99 (m, 1 H), 2.44–2.27
(m, 2 H), 1.90–1.76 (m, 6 H), 0.91 (s, 9 H), 0.17–0.01 (m, 6 H) ppm.
solid, m.p. 190–192 °C. 1H NMR (300 MHz, CDCl3): δ = 4.14–3.93 (m,
1 H), 3.10–2.85 (m, 4 H), 2.34–2.18 (m, 2 H), 2.06–1.79 (m, 6 H), 1.79–
1.64 (m, 4 H), 1.12–0.92 (m, 6 H) ppm. 13C NMR (75 MHz, CDCl3):
δ = 205.2, 143.6, 143.6, 138.4, 119.6, 109.7, 109.6, 66.3, 45.5, 45.5,
30.9, 17.4, 17.4, 13.8 ppm. IR (ATR): ν = 3249, 3041, 2963, 2938,
˜
2874, 1631, 1450, 1377, 1339, 1288, 1272, 1233, 1177, 1147, 1117,
13C NMR (75 MHz, CDCl3): δ = 177.9, 161.0, 125.0, 101.1, 101.0, 64.7, 1065, 1056, 1034, 1006, 979, 953, 938, 909, 878, 759 cm–1. HRMS:
30.8, 30.6, 25.7, 18.0, –4.9 ppm. IR (ATR): ν = 3088, 3070, 2953, 2929, calcd. for C20H26O7 378.1679 [M]+; found 378.1686.
˜
2883, 2856, 1722, 1654, 1469, 1438, 1399, 1360, 1334, 1292, 1233,
Benzyl 2-{4,8-Dibutyryl-4′-hydroxyspiro[benzo(1,2-d:4,5-d′)-
1200, 1155, 1134, 1119, 1064, 1047, 1018, 962, 898, 866, 844, 816,
bis([1,3]dioxole)-2,1′-cyclohexan]-6-yl}acetate (16): DMAP
772, 631, 619 cm–1. HRMS: calcd. for C18H26O5Si 350.1550 [M]+;
(125 mg, 1.02 mmol, 1.5 equiv.) was added to a solution of 15
found 350.1569.
(336 mg, 681 μmol) and benzyl prop-2-ynoate (120 mg, 750 μmol,
({5,6-Bis(methoxymethoxy)spiro[benzo[d][1,3]dioxole-2,1′-
1.1 equiv.) in anhydrous DCM (8 mL) under N2 at room temperature.
cyclohexan]-4′-yl}oxy)(tert-butyl)dimethylsilane (13): A suspen- After stirring overnight, the solvent was removed under reduced
sion of 12 (1.16 g, 3.32 mmol) and PtO2 (2 mg, 9.13 μmol,
0.001 equiv.) in dry THF (150 mL) was stirred under hydrogen
[p(H2) = 1 atm] at room temperature. After the complete conversion
of 12, monitored by TLC (2 h), the solution was cooled to –10 °C
under N2. NaH (451.36 mg, 11.29 mmol, 3.4 equiv.) was added and
the reaction mixture was stirred for 30 min. MOMBr (903 μL,
9.96 mmol, 3.0 equiv.) dissolved in dry THF (10 mL) was added
pressure. Purification of the residue by flash silica gel column chro-
matography (DCM/MeOH, 100:1) afforded 16 (406 mg, 621 μmol,
91 %) as an orange, vitreous solid. 1H NMR (300 MHz, CDCl3): δ =
3
7.36 (s, 5 H), 6.66 (t, J = 5.0 Hz, 1 H), 5.20 (s, 2 H), 4.04–3.93 (m, 1
3
H), 3.11 (d, J = 5.1 Hz, 2 H), 2.91–2.78 (m, 4 H), 2.30–2.14 (m, 2 H),
2.04–1.89 (m, 4 H), 1.88–1.80 (m, 2 H), 1.75–1.65 (m, 4 H), 1.04–0.92
(m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 196.3, 167.5, 135.1,
dropwise within 15 min. The solution was stirred overnight at room 128.6, 128.4, 128.2, 120.4, 109.5, 66.9, 66.5, 45.6, 40.0, 31.1, 30.9,
temperature. The resulting white suspension was washed with H2O
and brine and extracted several times with Et2O. The solvent was
dried with MgSO4, evaporated, and the residue was purified by flash
silica gel column chromatography (PE/EE, 10:1) to yield 13 (1.34 g,
17.2, 13.8 ppm. IR (ATR): ν = 3525, 2960, 2938, 2874, 1737, 1684,
˜
1439, 1370, 1282, 1174, 1101, 1051, 993, 750, 698, 603 cm–1. HRMS:
calcd. for C30H34O9 538.2203 [M]+; found 538.2211.
Benzyl 2-{4,8-Dibutyryl-4′-oxospiro[benzo(1,2-d:4,5-d′)-
bis([1,3]dioxole)-2,1′-cyclohexan]-6-yl}acetate (17): DMP
(175 mg, 0.41 mmol, 1.5 equiv.) was added to a solution of 16
(148 mg, 0.27 mmol) in anhydrous DCM (10 mL) and the mixture
was stirred overnight at room temperature. It was then washed with
an aqueous solution of NaHCO3/Na2S2O3 (250 g/L, 2×) and brine.
The organic layers were dried with MgSO4, evaporated, and the
resulting residue purified by flash silica gel column chromatography
(DCM/MeOH, 100:1) to yield 17 (89 mg, 0.17 mmol, 60 %) as an
1
3.03 mmol, 91 %) as a colorless oil. H NMR (300 MHz, CDCl3): δ =
6.68 (s, 2 H), 5.10 (s, 4 H), 3.99–3.88 (m, 1 H), 3.53 (s, 6 H), 2.27–2.10
(m, 2 H), 1.95–1.69 (m, 6 H), 0.95–0.88 (m, 9 H), 0.08 (s, 6 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 142.4, 142.4, 141.4, 141.3, 118.8, 100.8,
100.7, 96.9, 66.4, 56.1, 31.1, 30.5, 25.8, 18.1, –4.8 ppm. IR (ATR): ν =
˜
2951, 2930, 2894, 2855, 1491, 1377, 1251, 1208, 1148, 1071, 1043,
962, 922, 874, 835, 772, 684, 623, 477 cm–1. HRMS: calcd. for
C22H36O7Si 440.2230 [M]+; found 440.2209.
1
1,1′-{4′-[(tert-Butyldimethylsilyl)oxy]-5,6-bis(methoxymethoxy)-
spiro[benzo[d][1,3]dioxole-2,1′-cyclohexane]-4,7-diyl}bis-
(butan-1-one) (14): A solution of 13 (1.52 g, 3.45 mmol) in anhy-
orange, vitreous solid. H NMR (300 MHz, CDCl3): δ = 7.36 (s, 5 H),
3
3
6.69 (t, J = 5.0 Hz, 1 H), 5.20 (s, 2 H), 3.13 (d, J = 5.1 Hz, 2 H), 2.85
(t, 3J = 7.2 Hz, 4 H), 2.70–2.57 (m, 4 H), 2.48–2.34 (m, 4 H), 1.78–1.61
3
drous THF (100 mL) was cooled to –60 °C. nBuLi (1.6
M, 5.61 mL,
(m, 4 H), 0.96 (t, J = 7.4 Hz, 6 H) ppm. 13C NMR (75 MHz, CDCl3):
8.98 mmol, 2.6 equiv.) was added over 10 min and the mixture
stirred for 1.5 h at 0 °C. Anhydrous CuI (1.64 g, 8.64 mmol, 2.5 equiv.)
was then added and the suspension stirred for 1.5 h. Then butyryl
δ = 207.5, 196.1, 167.4, 140.5, 140.3, 135.1, 128.6, 128.5, 128.3, 118.5,
110.2, 109.7, 67.0, 45.6, 40.0, 37.1, 33.7, 17.1, 13.8 ppm. IR (ATR): ν =
˜
3033, 2963, 2936, 2875, 1739, 1722, 1687, 1472, 1455, 1439, 1416,
chloride (1.43 mL, 13.82 mmol, 4.0 equiv.) was added and the reac- 1401, 1369, 1285, 1217, 1177, 1118, 1104, 1065, 1046, 989, 889, 752,
tion mixture stirred for a further 2 h. An aqueous solution of NH4Cl
was added and the mixture extracted with Et2O. The organic layer
was dried with MgSO4, evaporated, and the residue purified by flash
silica gel column chromatography (PE/EE, 30:1) to yield 14 (1.80 g,
3.10 mmol, 90 %) as a pale-yellow oil. 1H NMR (300 MHz, CDCl3):
δ = 5.01 (s, 4 H), 3.95 (br. s, 1 H), 3.47 (s, 6 H), 2.93–2.77 (m, 4 H),
2.27–2.09 (m, 2 H), 1.99–1.47 (m, 10 H), 1.03–0.93 (m, 6 H), 0.91 (s,
9 H), 0.07 (s, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 162.3, 140.9,
121.2, 100.5, 100.5, 66.0, 57.7, 57.7, 46.2, 46.2, 31.0, 30.5, 25.8, 18.1,
699 cm–1. HRMS: calcd. for C30H32O9 536.2046 [M]+; found 536.2066.
1,1′-{2-[2-(4-Hydroxypiperidin-1-yl)-2-oxoethyl]benzo-
(1,2-d:4,5-d′)bis([1,3]dioxole)-4,8-diyl}bis(butan-1-one) (19): 4-
Hydroxypiperidine (46 mg, 0.45 mmol, 1.1 equiv.), DCC (102 mg,
0.49 mmol, 1.2 equiv.), and HOBt (67 mg, 0.49 mmol, 1.2 equiv.)
were added to a solution of 18 (150 mg, 0.41 mmol) in anhydrous
DCM (40 mL). After stirring overnight at room temperature the or-
ganic layer was washed with 0.1
N HCl, aq. NaHCO3, and brine and
dried with MgSO4. The solvent was evaporated and the residue was
purified by flash silica gel column chromatography (DCM/MeOH,
100:1) to yield 19 (182 mg, 0.41 mmol, quant.) as an orange solid,
17.2, 17.2, 13.8, 13.7, 13.6, –4.9 ppm. IR (ATR): ν = 3095, 2959, 2934,
˜
2879, 2858, 1738, 1708, 1462, 1433, 1377, 1359, 1280, 1252, 1242,
1213, 1158, 1138, 1112, 1094, 1079, 1054, 1003, 939, 859, 837, 813,
774, 676 cm–1. HRMS: calcd. for C30H48O9Si 580.3068 [M]+; found
580.3088.
1
3
m.p. 90–91 °C. H NMR (300 MHz, CDCl3): δ = 6.71 (t, J = 5.0 Hz, 1
H), 6.08 (s, 2 H), 4.14–3.82 (m, 2 H), 3.82–3.61 (m, 1 H), 3.37–3.17
3
(m, 2 H), 3.10 (d, J = 4.9 Hz, 2 H), 3.03–2.70 (m, 4 H), 1.97–1.81 (m,
1,1′-(4′,5,6-Trihydroxyspiro[benzo[d][1,3]dioxole-2,1′-cyclohex-
ane]-4,7-diyl)bis(butan-1-one) (15): A solution of 14 (300 mg,
516 μmol) and pTSA·H2O (1.28 mg, 6 μmol, 0.01 equiv.) in MeOH
2 H), 1.77–1.61 (m, 4 H), 1.61–1.44 (m, 2 H), 0.95 (t, 3J = 7.4 Hz, 6
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 196.4, 165.3, 140.9, 140.7,
111.4, 110.0, 102.5, 66.7, 45.6, 43.1, 39.1, 38.3, 34.3, 33.7, 17.1,
(7 mL) was stirred overnight. The solvent was evaporated and the 13.7 ppm. IR (ATR): ν = 3419, 2960, 2930, 2934, 2875, 1684, 1630,
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1473, 1435, 1283, 1242, 1114, 1063, 951, 751, 579 cm–1. HRMS: calcd.
for C23H29NO8 447.1893 [M]+; found 447.1896.
1221, 1203, 1141, 1090, 1045, 962, 940, 920, 888, 863, 806, 744, 729,
711, 650 cm–1. HRMS: calcd. for C41H42N2O14 786.2636 [M]+; found
786.2648.
1,1′-{2-[2-Oxo-2-(4-oxopiperidin-1-yl)ethyl]benzo(1,2-d:4,5-d′)-
bis([1,3]dioxole)-4,8-diyl}bis(butan-1-one) (20): Oxalyl chloride
(1.4 mL, 0.34 mmol, 1.5 equiv.) was added dropwise to a solution
(1,5-Dithiaspiro[5.5]undecane-3,3-diyl)dimethylenebis(oxytri-
methylsilane) (25): NEt3 (586 μL, 4.23 mmol, 5.0 equiv.) and TMSCl
of DMSO (33 μL, 0.46 mmol, 2.0 equiv.) in anhydrous DCM (10 mL) (536 μL, 4.23 mmol, 5.0 equiv.) were added to a suspension of 24
at –78 °C and the mixture stirred for 30 min. Compound 19 (102 mg, (210 mg, 0.85 mmol) in anhydrous toluene (30 mL). The mixture was
0.28 mmol) dissolved in anhydrous DCM (3 mL) was added drop- stirred overnight at room temperature. The precipitate was filtered
wise. After stirring for 30 min the reaction mixture was treated with through Celite®, washed with petroleum ether (PE) and the solvent
NEt3 (158 μL, 1.1 mmol, 5 equiv.) and stirred for 30 min while warm- evaporated. The residue was treated with PE and filtered through
ing up to room temperature. DCM was added and the organic layer
was washed with 1 HCl and brine, dried with MgSO4, evaporated,
and the resulting residue purified by flash silica gel column chroma-
tography (DCM/MeOH, 100:1) to yield 20 (59 mg, 0.13 mmol, 58 %)
as an orange solid, m.p. 128–129 °C. 1H NMR (300 MHz, CDCl3): δ =
Celite® once again. Removing of the solvent yielded 25 (333 mg,
0.85 mmol, 100 %) as a white solid. 1H NMR (300 MHz, C6D6): δ =
3.72 (s, 4 H), 2.67 (s, 4 H), 2.06–1.89 (m, 4 H), 1.63–1.47 (m, 4 H),
1.32–1.19 (m, 2 H), 0.12 (s, 18 H) ppm. 13C NMR (75 MHz, C6D6): δ =
64.0, 52.3, 38.9, 37.1, 29.9, 26.5, 23.1, 0.0 ppm, m.p. 70–72 °C. IR
N
3
3
6.74 (t, J = 4.9 Hz, 1 H), 6.10 (s, 2 H), 3.94 (t, J = 6.3 Hz, 2 H), 3.81
(ATR): ν = 2956, 2935, 2925, 2911, 2901, 2861, 2851, 1463, 1440,
˜
3
3
3
(t, J = 6.1 Hz, 2 H), 3.20 (d, J = 4.9 Hz, 2 H), 2.88 (t, J = 7.2 Hz, 4
1408, 1322, 1250, 1128, 1111, 1065, 1011, 876, 862, 833, 754, 746,
H), 2.60–2.48 (m, 4 H), 1.70 (sext, 3J = 7.3 Hz, 5 H), 0.96 (t, 3J = 733, 687 cm–1. HRMS: calcd. for C17H36O2S2Si2: 392.1695 [M]+; found
7.4 Hz, 7 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 206.1, 196.2, 165.8,
392.1689.
141.0, 140.6, 111.1, 110.1, 102.5, 45.6, 44.4, 41.0, 40.9, 40.6, 38.6,
8′′′′-Methyl-6′′′′H-tetraspiro[cyclohexane-1,2′-[1,3]dithiane-
5′,5′′-[1,3]dioxane-2′′,1′′′-cyclohexane-4′′′,2′′′′-[1,3]dioxolo[4,5-
g]chromen]-6′′′′-one (26): NaH (9 mg, 0.22 mmol, 1.2 equiv.) and
TMSCl (28 μL, 0.22 mmol, 1.2 equiv.) were added to a solution of 5
(53 mg, 0.18 mmol) in anhydrous Et2O (30 mL) and DCM (5 mL) at
0 °C. After stirring for 1 h, 24 (50 mg, 0.20 mmol, 1.1 equiv.) and
1 drop of TMSOTf were added and the suspension was stirred at
room temperature until complete conversion of 5, monitored by
TLC. The solvent was evaporated and the residue purified by flash
silica gel column chromatography (DCM) to yield 26 (70 mg,
0.14 mmol, 74 %) as a white solid, m.p. >230 °C (decomp.). 1H NMR
(300 MHz, CDCl3): δ = 6.89 (s, 1 H), 6.76 (s, 1 H), 6.14 (s, 1 H), 3.98–
17.1, 13.7 ppm. IR (ATR): ν = 3623, 3421, 3188, 2960, 2930, 2874,
˜
2754, 2601, 2547, 2292, 1706, 1681, 1468, 1435, 1416, 1399, 1367,
1347,1276,1242,1218,1126,1081,1038,986,960,931,737,663,620cm–1
HRMS: calcd. for C23H27NO8 445.1737 [M]+; found 445.1732.
.
8,8′′′′′-Dimethyl-6H,6′′′′′H-pentaspiro[[1,3]dioxolo[4,5-g]chrom-
ene-2,1′-cyclohexane-4′,2′′-[1,3]dioxane-5′′,5′′′-[1,3]diox-
ane-2′′′,1′′′′-cyclohexane-4′′′′,2′′′′′-[1,3]dioxolo[4,5-g]chrom-
ene]-6,6′′′′′-dione (22): NaH (9 mg, 0.22 mmol, 1.3 equiv.) and
TMSCl (28 μL, 0.22 mmol, 1.3 equiv.) was added to a suspension of
5 (48 mg, 0.17 mmol) in anhydrous Et2O (8 mL) at 0 °C. After stirring
for 1 h, compound 21 (36 mg, 0.17 mmol, 1.0 equiv.) and 1 drop of
TMSOTf were added and the suspension was stirred for 24 h at 3.78 (m, 4 H), 2.74 (s, 4 H), 2.36 (s, 3 H), 2.15–2.02 (m, 8 H), 2.02–
room temperature. The solvent was evaporated and the residue was
purified by flash silica gel column chromatography (DCM/MeOH,
1.88 (m, 4 H), 1.72–1.55 (m, 4 H), 1.55–1.38 (m, 2 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 161.4, 152.5, 150.7, 150.4, 144.7, 120.2, 113.4,
100:1) to yield 22 (47 mg, 0.07 mmol, 83 %) as a white solid, m.p. 111.8, 102.0, 98.3, 97.1, 66.6, 66.5, 51.7, 37.6, 37.5, 31.2, 31.0, 29.1,
1
>220 °C (decomp.). H NMR (300 MHz, [D6]DMSO): δ = 7.22 (s, 2 H),
28.6, 25.8, 22.2, 19.1 ppm. IR (ATR): ν = 3525, 3505, 3485, 3415,
2931, 1723, 1624, 1580, 1499, 1449, 1401, 1376, 1344, 1301, 1272,
˜
7.02 (s, 2 H), 6.20 (s, 2 H), 3.80–3.64 (m, 8 H), 2.34 (s, 6 H), 1.74–1.56
(m, 8 H), 1.51–1.28 (m, 8 H) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 1235, 1220, 1204, 1139, 1123, 1109, 1085, 996, 954, 942, 921, 907,
160.4, 153.9, 150.3, 150.0, 144.2, 120.2, 113.4, 111.2, 103.1, 98.0, 96.5,
62.9, 62.1, 39.5, 32.2, 31.0, 28.6, 27.1, 18.8 ppm. IR (ATR): ν = 3057,
2962, 2925, 2858, 2800, 2364, 1621, 1584, 1493, 1441, 1398, 1377,
1342, 1270, 1223, 1207, 1159, 1139, 1088, 1043, 993, 970, 944, 909,
865, 807, 774, 735, 705, 690 cm–1. HRMS: calcd. for C37H36O12
672.2207 [M]+; found 672.2211.
850, 834, 768 cm–1. HRMS: calcd. for C27H33O6S2 517.1713 [M + H]+;
found 517.1703.
˜
8-Methyl-2-[2-oxo-2-(7,21-dioxa-11,18-dithia-3-azatrispiro-
[5.2.2.512.29.26]henicosan-3-yl)ethyl]-6H-[1,3]dioxolo[4,5-
g]chromen-6-one (27): NaH (13 mg, 0.32 mmol, 1.2 equiv.) and
TMSCl (41 μL, 0.32 mmol, 1.2 equiv.) was added to a solution of 8
(92 mg, 0.27 mmol) in anhydrous Et2O (20 mL) and DCM (2 mL) at
0 °C. After stirring for 1 h, 24 (70 mg, 0.28 mmol, 1.1 equiv.) and
1 drop of TMSOTf were added and the suspension was stirred at
room temperature until complete conversion of 8, monitored by
TLC. The solvent was evaporated and the residue purified by flash
silica gel column chromatography (DCM) to yield 27 (85 mg,
0.15 mmol, 55 %) as a white solid, m.p. >230 °C (decomp.). 1H NMR
(300 MHz, CDCl3): δ = 6.93 (s, 1 H), 6.80 (s, 1 H), 6.73 (t, 3J = 5.0 Hz,
1 H), 6.15 (s, 1 H), 3.94–3.78 (m, 4 H), 3.67 (t, 3J = 5.4 Hz, 2 H), 3.52–
3.40 (m, 2 H), 3.05 (d, 3J = 5.1 Hz, 2 H), 2.77 (s, 2 H), 2.66 (s, 2 H),
2.35 (s, 3 H), 1.99–1.76 (m, 8 H), 1.68–1.54 (m, 4 H), 1.51–1.38 (m, 2
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 165.4, 161.1, 152.4, 150.6,
150.5, 144.6, 113.8, 112.2, 110.9, 102.1, 98.4, 96.7, 66.3, 66.2, 51.6,
42.5, 38.5, 38.4, 37.5, 37.5, 33.7, 30.9, 30.9, 29.1, 25.7, 22.2, 19.1 ppm.
2,2′-[(7,11,18,21-Tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]-
henicosane-3,15-diyl)bis(2-oxoethane-2,1-diyl)]bis(8-methyl-
6H-[1,3]dioxolo[4,5-g]chromen-6-one) (23): NaH (13 mg,
320.39 μmol, 1.1 equiv.) and TMSCl (41 μL, 320.39 μmol, 1.1 equiv.)
was added to a suspension of 8 (100 mg, 291.26 μmol) in anhydrous
Et2O (40 mL) at 0 °C. After stirring for 1 h, compound 21 (63 mg,
291.26 μmol, 1.0 equiv.) and 1 drop of TMSOTf were added and the
suspension was stirred for 24 h at room temperature. The solvent
was evaporated and the residue purified by flash silica gel column
chromatography (DCM/MeOH, 100:1) to yield 23 (45 mg,
57.19 μmol, 39 %) as a white, vitreous solid. 1H NMR (300 MHz,
3
CDCl3): δ = 6.91 (s, 2 H), 6.77 (s, 2 H), 6.71 (t, J = 4.7 Hz, 2 H), 6.13
(s, 2 H), 3.86–3.76 (m, 4 H), 3.76–3.58 (m, 8 H), 3.53–3.36 (m, 4 H),
3.04 (d, J = 4.7 Hz, 4 H), 2.34 (s, 6 H), 1.96–1.74 (m, 8 H) ppm. 13C
3
NMR (75 MHz, CDCl3): δ = 165.4, 161.1, 152.3, 150.6, 150.5, 144.6, IR (ATR): ν = 3518, 3484, 2930, 2855, 2777, 2751, 1726, 1635, 1583,
˜
113.8, 112.2, 110.9, 102.1, 98.3, 96.8, 63.4, 63.3, 38.5, 38.4, 33.8, 33.6,
1493, 1449, 1405, 1386, 1345, 1298, 1264, 1220, 1204, 1141, 1108,
33.2, 31.0, 30.9, 19.0 ppm. IR (ATR): ν = 3484, 3469, 2955, 2932, 1081, 1035, 1012, 954, 918, 890, 862, 806, 778, 745 cm–1. HRMS:
˜
2867, 2747, 2659, 1729, 1637, 1582, 1492, 1448, 1384, 1345, 1263,
calcd. for C29H35NO7S2 573.1855 [M]+; found 573.1851.
Eur. J. Org. Chem. 0000, 0–0
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Benzyl {4,8-Dibutanoyltetraspiro[benzo(1,2-d:4,5-d′)bis[1,3]di-
oxole-2,1′-cyclohexane-4′,2′′-[1,3]dioxane-5′′,5′′′-[1,3]dithiane-
2′′′,1′′′′-cyclohexan]-6-yl}acetate (28): A drop of TMSOTf was
added to a solution of 17 (20 mg, 37.27 μmol) and 25 (22 mg,
55.91 μmol, 1.5 equiv.) in anhydrous DCM (5 mL) and the reaction
dithian]-6-one (32): Anhydrous NEt3 (635 μL, 4.58 mmol,
10.0 equiv.) and TMSCl (582 μL, 4.58 mmol, 10.0 equiv.) were added
to a suspension of 31 (200 mg, 0.46 mmol) in anhydrous toluene
(10 mL). The reaction mixture was stirred overnight at room temper-
ature, filtered through Celite®, washed with PE, and the solvent
mixture was stirred overnight at room temperature. After evapora- evaporated. The residue was suspended in PE and filtered through
tion of the solvent the residue was purified by flash silica gel col-
umn chromatography (DCM DCM/MeOH, 100:1) to yield 28 (22 mg,
28.68 μmol, 78 %) as an orange solid, m.p. 161–163 °C. 1H NMR
Celite® once more. Evaporation of the solvent yielded 32 (266 g,
0.46 mmol, quant.) as a white solid, m.p. 142–144 °C. 1H NMR
(300 MHz, CDCl3): δ = 6.55 (s, 1 H), 6.40 (s, 1 H), 5.84 (d, 4J = 1.1 Hz,
1 H), 3.71 (s, 4 H), 2.63 (s, 4 H), 2.25–2.14 (m, 4 H), 2.05–1.94 (m, 4
H), 1.50 (d, 4J = 1.1 Hz, 3 H), 0.16–0.10 (m, 19 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 160.5, 151.5, 151.5, 151.0, 144.8, 120.0, 114.0,
3
(300 MHz, CDCl3): δ = 7.45–7.30 (m, 5 H), 6.65 (t, J = 5.0 Hz, 1 H),
5.19 (s, 2 H), 3.95–3.79 (m, 4 H), 3.11 (d, 3J = 5.1 Hz, 2 H), 2.83 (t,
3J = 7.2 Hz, 4 H), 2.77–2.68 (m, 4 H), 2.15–2.03 (m, 8 H), 2.03–1.92
3
(m, 4 H), 1.76–1.53 (m, 8 H), 1.53–1.37 (m, 2 H), 0.96 (t, J = 7.3 Hz, 113.1, 102.7, 98.8, 63.9, 49.7, 44.5, 36.8, 35.3, 31.9, 30.2, 18.7,
6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 196.4, 168.5, 151.8, 141.4,
–0.08 ppm. IR (ATR): ν = 3055, 2956, 2930, 2914, 2858, 1726, 1633,
˜
128.6, 128.2, 110.9, 109.6, 99.6, 66.9, 66.5, 45.6, 41.1, 37.6, 32.2, 31.9
1620, 1580, 1489, 1439, 1360, 1344, 1225, 1208, 1158, 1135, 1116,
31.0, 30.9, 29.1, 22.2, 17.2, 13.8 ppm. IR (ATR): ν = 2960, 2933, 2870,
1091, 1065, 1042, 967, 947, 921, 906, 872, 838, 809, 748, 690 cm–1
HRMS: calcd. for C27H40O6S2Si2 580.1805 [M]+; found 580.1812.
.
˜
1739, 1687, 1465, 1442, 1398, 1377, 1299, 1281, 1271, 1246, 1188,
1122, 1110, 1038, 1005, 982, 958, 919, 903, 736, 696 cm–1. HRMS:
calcd. for C41H51O10S2 767.2924 [M + H]+; found 767.2925.
Benzyl 2-{4,8-Dibutyryl-8′′′′′-methyl-6′′′′′-oxo-6′′′′′H-penta-
spiro[benzo(1,2-d:4,5-d′)bis([1,3]dioxole)-2,1′-cyclohexane-
4′,2′′-[1,3]dioxane-5′′,5′′′-[1,3]dithiane-2′′′,1′′′′-cyclohexane-
4′′′′,2′′′′′-[1,3]dioxolo[4,5-g]chromen]-6-yl}acetate (33): A drop of
TMSOTf was added to a solution of 17 (20 mg, 37.27 μmol) and 32
(24 mg, 41.00 μmol, 1.1 equiv.) in anhydrous DCM (5 mL) and the
reaction mixture was stirred overnight at room temperature. After
evaporation of the solvent the residue was purified by flash silica
gel column chromatography (DCM DCM/MeOH, 50:1) to yield 33
1,1′-{2-[2-Oxo-2-(7,21-dioxa-11,18-dithia-3-azatrispiro-
[5.2.2.512.29.26]henicosan-3-yl)ethyl]benzo(1,2-d:4,5-d′)-
bis([1,3]dioxole)-4,8-diyl}bis(butan-1-one) (29): A drop of
TMSOTf was added to a solution of 20 (27 mg, 60.61 μmol) and 25
(36 mg, 90.92 μmol, 1.5 equiv.) in anhydrous DCM (10 mL) and the
reaction mixture was stirred overnight at room temperature. After
evaporation of the solvent the residue was purified by flash silica
gel column chromatography (DCM DCM/MeOH, 50:1) to yield 29
(38 mg, 56.23 μmol, 93 %) as an orange solid, m.p. 94–96 °C. 1H
NMR (300 MHz, CDCl3): δ = 6.74 (t, 3J = 4.3 Hz, 1 H), 6.10 (s, 2 H),
3.86 (br. s, 4 H), 3.72–3.61 (m, 2 H), 3.53–3.44 (m, 2 H), 3.16–3.06 (m,
2 H), 2.88 (t, 3J = 7.2 Hz, 4 H), 2.79–2.61 (m, 4 H), 2.02–1.80 (m, 8
H), 1.76–1.56 (m, 8 H), 1.50–1.37 (m, 2 H), 0.97 (t, 3J = 7.4 Hz, 6
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 196.3, 165.2, 140.9, 140.6,
111.3, 110.0, 102.5, 96.7, 66.2, 51.6, 45.6, 42.6, 38.5, 38.4, 37.5, 33.5,
1
(23 mg, 24.08 μmol, 64 %) as an orange solid, m.p. 115 °C. H NMR
(300 MHz, CDCl3): δ = 7.42–7.29 (m, 5 H), 6.90 (s, 1 H), 6.77 (s, 1 H),
6.66 (t, 3J = 4.9 Hz, 1 H), 6.15 (s, 1 H), 5.19 (s, 2 H), 3.90 (s, 4 H), 3.11
3
(d, J = 4.9 Hz, 2 H), 2.90–2.72 (m, 8 H), 2.36 (s, 3 H), 2.32–2.21 (m,
3
4 H), 2.21–1.97 (m, 12 H), 1.78–1.59 (m, 4 H), 0.96 (t, J = 7.3 Hz, 6
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 196.4, 167.5, 161.4, 152.5,
150.6, 150.4, 144.5, 140.8, 139.9, 135.2, 128.6, 128.4, 128.2, 120.4,
119.6, 113.5, 111.9, 110.1, 109.6, 102.0, 98.3, 97.2, 66.9, 66.4, 49.3,
45.6, 40.0, 36.9, 34.2, 31.3, 31.2, 31.0, 29.0, 19.1, 17.2, 13.8 ppm. IR
31.2, 30.9, 29.1, 25.7, 22.2, 17.1, 13.7 ppm. IR (ATR): ν = 2960, 2931,
˜
2873, 2856, 1684, 1642, 1601, 1474, 1434, 1409, 1363, 1348, 1282,
1241, 1224, 1205, 1188, 1107, 1061, 1039, 1013, 951, 942, 934, 891,
864, 800, 732, 701, 597 cm–1. HRMS: calcd. for C34H45NO9S2
675.2536 [M]+; found 675.2535.
(ATR): ν = 2961, 2931, 2872, 2851, 1725, 1687, 1637, 1627, 1585,
˜
1495, 1440, 1403, 1377, 1348, 1283, 1273, 1259, 1225, 1207, 1140,
1107, 1067, 1043, 1012, 969, 922, 905, 863, 802, 744 cm–1. HRMS:
calcd. for C51H54O14S2 955.3033 [M]+; found 955.3015.
5′′,5′′-Bis(hydroxymethyl)-8-methyl-6H-dispiro[[1,3]dioxolo-
[4,5-g]chromene-2,1′-cyclohexane-4′,2′′-[1,3]dithian]-6-one
(31): A solution of 5 (249 mg, 869.77 μmol), 30 (380 mg,
956.75 μmol, 1.10 equiv.), and iodine (29 mg, 113.07 μmol,
0.13 equiv.) in DCM (20 mL) was stirred at room temperature until
complete conversion of 5, monitored by TLC. HF (48 %, 63 μL,
1.74 mmol, 2.00 equiv.) was added and the mixture stirred for a
further 2 h. The organic layer was washed with aq. NaHCO3 and aq.
Na2S2O3 (20 %), dried with MgSO4, and the solvents evaporated.
The residue was purified by flash silica gel column chromatography
(DCM/MeOH, 100:1) to yield 31 (330 mg, 755.95 μmol, 87 %) as a
white solid, m.p. >205 °C (decomp.). 1H NMR (300 MHz, [D6]-
acetone): δ = 7.13 (s, 1 H), 6.83 (s, 1 H), 6.13 (s, 1 H), 3.89 (s, 4 H),
2.93–2.88 (m, 4 H), 2.41 (s, 3 H), 2.35–2.22 (m, 4 H), 2.21–2.10 (m, 4
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 163.3, 154.9, 151.8, 151.0,
145.7, 120.8, 114.4, 111.8, 103.0, 98.8, 65.0, 64.9, 49.7, 44.6, 36.4,
1,1′-[2-(2-{8′′′′-Methyl-6′′′′-oxo-6′′′′H-tetraspiro[piperidine-4,2′-
[1,3]dioxane-5′,5′′-[1,3]dithiane-2′′,1′′′-cyclohexane-4′′′,2′′′′-
[1,3]dioxolo[4,5-g]chromen]-1-yl}-2-oxoethyl)benzo(1,2-d:4,5-
d′)bis([1,3]dioxole)-4,8-diyl]bis(butan-1-one) (34): A drop of
TMSOTf was added to a solution of 32 (40 mg, 68.86 μmol) and 20
(34 mg, 75.74 μmol, 1.1 equiv.) in anhydrous DCM (10 mL) and the
reaction mixture was stirred overnight at room temperature. After
evaporation of the solvent the residue was purified by flash silica
gel column chromatography (DCM DCM/MeOH, 50:1) to yield 34
1
(30 mg, 34.72 μmol, 50 %) as an orange solid, m.p. 113 °C. H NMR
(300 MHz, CDCl3): δ = 6.89 (s, 1 H), 6.82–6.65 (m, 2 H), 6.25–6.02 (m,
3 H), 3.97–3.80 (m, 4 H), 3.72–3.62 (m, 2 H), 3.54–3.41 (m, 2 H), 3.11
(d, 3J = 4.1 Hz, 2 H), 2.88 (t, 3J = 7.2 Hz, 4 H), 2.84–2.65 (m, 4 H),
2.35 (s, 3 H), 2.30–2.19 (m, 4 H), 2.18–2.09 (m, 4 H), 1.94–1.81 (m, 4
H), 1.77–1.62 (m, 4 H), 0.97 (t, 3J = 7.4 Hz, 6 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 196.4, 165.3, 161.3, 152.4, 150.5, 150.4, 144.5,
141.0, 140.6, 119.6, 113.5, 111.9, 111.3, 110.0, 102.5, 102.0, 98.3, 96.9,
66.1, 49.2, 45.6, 42.6, 38.5, 38.4, 34.1, 33.6, 31.3, 31.2, 29.1, 19.1, 17.1,
35.2, 31.9, 30.1, 19.5 ppm. IR (ATR): ν = 3710, 2923, 2650, 2585,
˜
2370, 2355, 1715, 1696, 1624, 1583, 1493, 1450, 1401, 1365, 1345,
1304, 1270, 1256, 1208, 1142, 1109, 1086, 1067, 968, 923, 838, 811,
745, 727 cm–1. HRMS: calcd. for C21H25O6S2 437.1093 [M + H]+;
found 437.1064.
13.7 ppm. IR (ATR): ν = 3423, 3364, 3064, 2961, 2933, 2874, 2251,
˜
1718, 1686, 1639, 1584, 1495, 1474, 1451, 1436, 1403, 1368, 1347,
1282, 1273, 1259, 1242, 1224, 1141, 1108, 1067, 1045, 925, 906, 863,
812, 773, 731, 647, 596, 496 cm–1. HRMS: calcd. for C44H49NO13S2
863.2645 [M]+; found 863.2668.
8-Methyl-5′′,5′′-bis{[(trimethylsilyl)oxy]methyl}-6H-di-
spiro[[1,3]dioxolo[4,5-g]chromene-2,1′-cyclohexane-4′,2′′-[1,3]-
Eur. J. Org. Chem. 0000, 0–0
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Full Paper
J. Org. Chem. 2012, 77, 3907; f) C. Grimm, T. Meyer, S. Czapla, J. Nikolaus,
H. A. Scheidt, A. Vogel, A. Herrmann, P. Wessig, D. Huster, P. Müller, Chem.
Eur. J. 2013, 19, 2703; g) A. Techen, S. Czapla, K. Möllnitz, D. Budach, P.
Wessig, M. U. Kumke, Helv. Chim. Acta 2013, 96, 2046; h) P. Wessig, M.
Gerngroß, S. Pape, P. Bruhns, J. Weber, RSC Adv. 2014, 4, 31123; i) P.
Wessig, D. Budach, A. Thünemann, Chem. Eur. J. 2015, 21, 10466; j) P.
Wessig, R. Merkel, P. Müller, Beilstein J. Org. Chem. 2015, 11, 74.
[8] P. Wessig, M. Gerngroß, D. Freyse, P. Bruhns, M. Przezdziak, U. Schilde, A.
Kelling, J. Org. Chem. 2016, 81, 1125.
[9] a) P. Wessig, R. Wawrzinek, K. Möllnitz, E. Feldbusch, U. Schilde, Tetrahe-
dron Lett. 2011, 52, 6192; b) R. Wawrzinek, P. Wessig, K. Möllnitz, J. Niko-
laus, R. Schwarzer, P. Müller, A. Herrmann, Bioorg. Med. Chem. Lett. 2012,
22, 5367; c) R. Wawrzinek, J. Ziomkowska, J. Heuveling, M. Mertens, A.
Herrmann, E. Schneider, P. Wessig, Chem. Eur. J. 2013, 19, 17349; d) J.
Heuveling, V. Frochaux, J. Ziomkowska, R. Wawrzinek, P. Wessig, A. Herr-
mann, E. Schneider, Biochim. Biophys. Acta (BBA) - Biomembranes 2014,
1838, 106; e) C. Meyners, R. Wawrzinek, A. Krämer, S. Hinz, P. Wessig, F.-
J. Meyer-Almes, Anal. Bioanal. Chem. 2014, 4889; f) R. Wawrzinek, P. Wes-
sig, Dyes Pigm. 2015, 123, 39; g) D. Bader, D. T. Klier, C. Hettrich, F. F. Bier,
P. Wessig, Anal. Methods 2016, 8, 1235.
Acknowledgments
The financial support of this work by the Deutsche Forschungs-
gemeinschaft (DFG) (WE 1850/10-1 and KU 1162/12-1) is grate-
fully acknowledged.
Keywords: Fluorescence · Energy transfer · FRET ·
Chromophores · Spiro compounds
[1] a) M. D. Brenner, R. Zhou, T. Ha, Biopolymers 2011, 95, 332; b) D. M.
Morris, G. J. Jensen, Ann. Rev. Biochem. 2008, 77, 583; c) A. L. Plant, K.
Bhadriraju, T. A. Spurlin, J. T. Elliott, Biochim. Biophys. Acta – Mol. Cell Res.
2009, 1793, 893.
[2] C. Yang, X. Zhang, Y. Guo, F. Meng, F. Sachs, J. Guo, Biochim. Biophys. Acta
– Mol. Cell Res. 2015, 1853, 1889.
[3] a) B. Liu, W. Chen, C. Zhu, Ann. Rev. Phys. Chem. 2014, 66, 427; b) B.
Hochreiter, A. P. Garcia, J. A. Schmid, Sensors 2015, 15, 26281; c) C. Jurch-
enko, K. S. Salaita, Mol. Cell. Biol. 2015, 35, 2570; d) B. Kainz, E. A.
Oprzeska-Zingrebe, J. L. Herrera, Biotechnol. J. 2014, 9, 51; e) D. Shrestha,
A. Jenei, P. Nagy, G. Vereb, J. Szöllösi, Int. J. Mol. Sci. 2015, 16, 6718.
[4] a) B. W. Van Der Meer, Rev. Mol. Biotechnol. 2002, 82, 181; b) B. W.
Van der Meer, Forster Theory, in: FRET Förster Resonance Energy Transfer:
From Theory to Applications (Eds.: I. Medintz, N. Hildebrandt), Wiley-VCH,
Weinheim, Germany, 2014, p. 23–62.
[10] P. Wessig, N. Behrends, M. U. Kumke, U. Eisold, T. Meiling, C. Hille, RSC
Adv. 2016, 6, 33510.
[11] T. Tsunoda, M. Suzuki, R. Noyori, Tetrahedron Lett. 1980, 21, 1357.
[12] K. Kumamoto, Y. Ichikawa, H. Kotsuki, Synlett 2005, 2254.
[13] H. H. Hussain, G. Babic, T. Durst, J. S. Wright, M. Flueraru, A. Chichirau,
L. L. Chepelev, J. Org. Chem. 2003, 68, 7023.
[14] X. Ariza, O. Pineda, J. Vilarrasa, G. W. Shipps Jr., Y. Ma, X. Dai, Org. Lett.
[5] D. B. VanBeek, M. C. Zwier, J. M. Shorb, B. P. Krueger, Biophys. J. 2007, 92,
4168.
[6] H. Langhals, A. J. Esterbauer, A. Walter, E. Riedle, E. I. Pugliesi, J. Am.
Chem. Soc. 2010, 132, 16777.
[7] a) P. Wessig, K. Möllnitz, C. Eiserbeck, Chem. Eur. J. 2007, 13, 4859; b) P.
Wessig, K. Möllnitz, J. Org. Chem. 2008, 73, 4452; c) P. Müller, J. Nikolaus,
S. Schiller, A. Herrmann, K. Möllnitz, S. Czapla, P. Wessig, Angew. Chem.
Int. Ed. 2009, 48, 4433; Angew. Chem. 2009, 121, 4497; d) J. Nikolaus, S.
Czapla, K. Möllnitz, C. T. Höfer, A. Herrmann, P. Wessig, P. Müller, Biochim.
Biophys. Acta (BBA) - Biomembranes 2011, 2781; e) P. Wessig, K. Möllnitz,
2001, 3, 1399.
[15] M. C. Murguia, S. E. Vaillard, R. J. Grau, Synthesis 2001, 1093.
[16] H. J. Backer, A. F. Tamsma, Rec. Trav. Chim. 1938, 57, 1183.
[17] A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, B. Mennucci,
Biophys. J. 2009, 96, 4779.
[18] P. Nalbach, I. Pugliesi, H. Langhals, M. Thorwart, Phys. Rev. Lett. 2012, 108,
1.
Received: April 19, 2016
Published Online: ■
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
FRET Rods
P. Wessig,* N. Behrends,
M. U. Kumke,* U. Eisold ..................... 1–12
FRET Pairs with Fixed Relative Orienta-
tion of Chromophores
FRET pairs with a fixed relative orientation spirothioketal (OSTK) rods. Their photo-
of chromophores have been synthesized physical properties have been investi-
by combining fluorophores with oligo- gated and are described herein.
DOI: 10.1002/ejoc.201600489
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim