Synthesis and properties of chemiluminescent acridinium ester labels with fluorous tags

Article abstract of DOI:10.1039/c4ob00456f

Acridinium dimethylphenyl esters are highly sensitive chemiluminescent labels that are used in clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of the cationic surfactant cetyltrimethylammonium chl

Full text of DOI:10.1039/c4ob00456f

Organic &
Biomolecular Chemistry
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Synthesis and properties of chemiluminescent
acridinium ester labels with fluorous tags†
Cite this: Org. Biomol. Chem., 2014,
12, 3887
Anand Natrajan,* David Wen and David Sharpe
Acridinium dimethylphenyl esters are highly sensitive chemiluminescent labels that are used in clinical
diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of the cat-
ionic surfactant cetyltrimethylammonium chloride (CTAC). CTAC compresses emission times of these
labels to <5 seconds and also increases overall light yield 3–4 fold. The observed enhancement in acridi-
nium ester chemiluminescence (light yield) is quite sensitive to the polarity of the micellar interface. In the
current study, we report the synthesis of new acridinium ester labels with fluorous tags of varying fluorine
content and their chemiluminescence in the presence of cationic micelles of CTAC, anionic micelles of
sodium perfluorooctanoate (SPFO) as well as mixed micelles of CTAC and SPFO. These studies indicate
that in the presence of the mixed micelle system of CTAC and SPFO and at low mole fractions of SPFO,
polarity of the mixed micelle interface is lower than that of CTAC leading to a greater enhancement of
chemiluminescence for both fluorinated acridinium esters as well as a structurally analogous but non-
fluorinated acridinium ester. Chemiluminescence stability of the fluorinated acridinium esters was either
comparable to or better than the stability of the non-fluorinated acridinium ester. Non-specific binding to
paramagnetic microparticles was higher for fluorinated acridinium esters requiring a surfactant wash to
reduce their non-specific binding to the same extent as that observed for the non-fluorinated acridinium
ester.
Received 27th February 2014,
Accepted 28th April 2014
DOI: 10.1039/c4ob00456f
www.rsc.org/obc
excited state acridone VI.2 At physiological pH, most acridi-
nium ester labels and their conjugates exist as water-adducts
Introduction
Acridinium dimethylphenyl esters1 (Fig. 1) are highly sensitive that are commonly referred to pseudobases (I in Fig. 2).1f,g,3
and stable chemiluminescent labels that are used in clinical Consequently, an acid pre-treatment step is needed to convert
diagnostics in Siemens Healthcare Diagnostics’ ADVIA the pseudobase I to the acridinium form II before the latter
Centaur® systems. These chemiluminescent compounds are can react with peroxide. In practice, at the end of each immuno-
used to label both proteins such as antibodies as well as small assay on the ADVIA Centaur® system, light emission is trig-
molecules and the resulting conjugates are used in conjunc- gered by the sequential addition of 0.3 mL of 0.1 M nitric acid
tion with magnetic microparticles in automated immuno- with 0.5% hydrogen peroxide followed by 0.3 mL of 0.25 M
assays. A variety of structural modifications can be introduced NaOH containing 7 mM cetyltrimethylammonium chloride
in the acridinium ring or the phenolic ester leaving group to (CTAC).1d,f CTAC compresses emission kinetics of the labels
increase light yield, manipulate emission kinetics, increase from approximately 60 seconds to <5 seconds and increases
aqueous solubility and lower the non-specific binding of the overall light yield 3–4 fold.1d,f The increase in emission kine-
labels.1
tics can be attributed to strong hydroperoxide ion binding to
Light emission from acridinium esters and their conjugates the surface of cationic CTAC micelles which increases their
is triggered with alkaline peroxide which adds to C-9 of the local concentration and facilitates formation of the peroxide
acridinium ring (Fig. 2) thereby initiating a series of reactions adduct III. CTAC also provides a low polarity environment
that ultimately result in the formation of the primary emitter, (alcohol-like) which is conducive to formation of both dioxe-
tane IV and dioxetanone V precursors to excited state acridone
VI.1d,f Obviously, both these effects also require strong parti-
tioning of the acridinium esters and their conjugates to CTAC
Siemens Healthcare Diagnostics, Advanced Technology and Pre-Development, 333
Coney Street, East Walpole, MA 02032, USA. E-mail: anand.natrajan@siemens.com;
micelles which occurs mainly through a combination of hydro-
Fax: +1-508-660-4591; Tel: +1-508-660-4582
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
phobic and charge interactions.1f In our detailed studies
we provided evidence that minimally, two acridinium ester
c4ob00456f
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Fig. 1 Structures of chemiluminescent acridinium dimethylphenyl ester labels1 that are used in automated immunoassays in Siemens Healthcare
Diagnostics’ ADVIA Centaur® systems.
Fig. 2 Simplified reaction pathway of chemiluminescence from N-sulfopropyl acridinium esters.1d,f Cationic surfactants such as CTAC accelerate
conversion of II to III by increasing the local concentration of hydroperoxide ions. They also facilitate formation of dioxetane IV and/or dioxetanone
V by offering a lower polarity medium for these reactions which involve charge dispersal in their transition states.
intermediates bind with different strengths to CTAC micelles signal while minimizing non-specific binding. Acridinium
and that micelles affect light emission by two discrete mechan- esters tend to be hydrophobic and in recent publications,1 we
isms.1d,f
have outlined strategies to mitigate non-specific binding of
In immunoassays that use acridinium ester labels (or any these labels to microparticles, without disrupting their
other luminescent tag) along with microparticles, optimal binding to CTAC micelles, by incorporating poly(ethylene)
assay sensitivity is dependent upon maximizing the specific glycol (PEG) or zwitterions in these labels. These PEG and
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zwitterion containing acridinium esters display excellent
aqueous solubility, low non-specific binding and consequently
improved sensitivity in immunoassays.1
PEG or zwitterion modification of surfaces is also a very
useful and widely described strategy to reduce the ‘stickiness’
of various surfaces to proteins and other macromolecules.4
More recently, an interesting approach to create protein-resist-
ant surfaces has been described in the literature using fluori-
nation rather than PEG or zwitterion modification.5
Fluorinated surfaces show low non-specific binding and
appear very useful for the construction of protein arrays as well
as for the immobilization of molecules containing fluorous
tags via fluorous (F–F) interactions.5,6 Fluorous interactions
are very specific and have been used in a wide range of appli-
cations including fluorous immobilization and extraction,6,7
fluorous chromatography8 and numerous other applications.
Fluorinated molecules are also selectively solubilized in aggre-
gates of fluorinated surfactants.9 A recent book edited by
Horváth describes some of the more recent applications of
fluorous chemistry.10
Another vast body of research pertaining to fluorinated
materials is in the field of fluorinated surfactants and a com-
prehensive compilation of research in this area can be found
in a book edited by Kissa.11 We were particularly interested to
learn from published reports that the commercially available,
perfluorinated anionic surfactant sodium perfluorooctanoate
(SPFO) forms mixed micelles with cationic surfactants such as
cetyltrimethylammonium bromide (CTAB) and dodecyltri-
methylammonium chloride.12 SPFO and the two cationic sur-
factants were reported to interact synergistically where the
cationic surfactant serves as the solvent for SPFO at low mole
fractions of SPFO.12 Moreover, an investigation of the micellar
properties of the mixed micelle system formed by CTAB and
SPFO using a fluorescent and an ESR probe indicated a micro-
environment with a low dielectric constant, high microvisco-
sity and significant penetration of the SPFO chains in the
interior of these mixed micelles.12 Although these mixed
micelle systems have been investigated using these types of
spectroscopic techniques, there are few studies on how such
mixed micellar systems would impact reaction pathways.
In our previous studies, we observed that acridinium ester
chemiluminescence is quite sensitive to the polarity of the
micellar interface.1d,13 Less polar micelles derived from cat-
ionic surfactants with large head groups increase the lumines-
cence (light yield) of acridinium esters by facilitating the
formation of dioxetane IV and dioxetanone V (Fig. 2). Based on
the above reports on the interaction of SPFO with cationic
micelles, we became interested in determining whether fluo-
Fig. 3 Panel A: Model for interaction of acridinium esters (AEs) contain-
ing fluorous tags with mixed micelles of cetytrimethylammonium chlor-
ide (CTAC) and sodium perfluorooctanoate (SPFO). Binding of
fluorinated acridinium esters to mixed micelles is through a combination
of hydrophobic, charge and fluorous interactions. Only fluorous inter-
actions are shown. Panel B: Model for interaction of acridinium esters
(AEs) with fluorous tags with anionic micelles of SPFO. Fluorinated acri-
dinium esters bound to SPFO micelles exhibited a greater inhibition of
emission kinetics and loss of light yield with increasing fluorous content.
son to CTAC and SPFO micelles individually, and what could
be learnt about the micellar environment of these mixed
micelles in relation to the proposed mechanism outlined in
Fig. 2? Other specific questions we hoped to address were,
how does fluorous content in acridinium esters with, or
without charge augmentation (to SPFO) affect chemilumine-
scence (emission kinetics and enhancement) from mixed
micelles of CTAC + SPFO? Finally, we also wanted to evaluate
the non-specific binding of the fluorinated acridinium esters
to microparticles to determine their relative lipophilicity when
compared to a non-fluorinated acridinium ester.
To answer all these questions, in the present study we
report the syntheses and properties of new acridinium ester
labels with fluorous tags and varying fluorine content with or
without positive charges appended near the fluorous chains
(Fig. 4).
Results and discussion
Synthesis of fluorinated acridinium esters and bovine serum
albumin (BSA) conjugates
rous tags incorporated in acridinium esters could be used as a The structures of fluorinated acridinium esters that we elected
handle to enhance solubilization in both CTAC as well as to synthesize in the present study are illustrated in Fig. 4. Both
mixed micelles of CTAC + SPFO (Fig. 3, panel A). Would fluo- for ease of synthesis and to ensure strong partitioning of the
rous chains in acridinium esters increase partitioning into less acridinium ring in these compounds into surfactant aggre-
polar regions of CTAC micelles which would then be reflected gates, we decided to incorporate the fluorous chains at C-2 of
by an increase in light yield? We also wanted to investigate the acridinium ring. In structures 5a, 5b and 5c, the length of
how mixed micelles of CTAC + SPFO would affect chemilumi- the fluorous ‘ponytail’ was increased from 9 fluorines in 5a, to
nescence from fluorinated acridinium ester labels in compari- 13 fluorines in 5b and 17 fluorines in 5c. For convenience we
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Fig. 4 Structures of fluorinated acridinium esters (FnAEs) 5a–5c and (Fn+AEs) 10a–10c synthesized in the current study. AEs 5a, 5b and 5c contain
fluorous chains of increasing length and are designated by the number of fluorine atoms (F9AE, F13AE and F17AE). Compounds 10a, 10b and 10c
contain a positive charge in addition to fluorous chains of increasing length and are designated by the number of fluorine atoms with a positive
charge (F9+AE, F13+AE, F17+AE). Compound 11,1f is an acridinium ester without any fluorines but with the same C-2 alkoxy substitution pattern.
shall henceforth refer to these compounds as F9AE for 5a pleted by condensation with glutaric anhydride followed by
(AE = acridinium ester), F13AE for 5b and F17AE for 5c. In activation of the resulting carboxylic acids.
structures 10a (9 fluorines), 10b (13 fluorines) and 10c (17
In a similar vein, the fluorinated acridinium esters F9+AE,
fluorines), a positive charge in the form of a quaternary nitro- F13+AE and F17+AE with a quaternary nitrogen adjacent to the
gen was introduced between the acridinium ring and adjacent fluorous chains were synthesized using an analogous synthetic
to the fluorous chains. Using a similar nomenclature, as scheme as illustrated in Fig. 6. In this case, the starting acri-
described for 5a–5c, compounds 10a, 10b and 10c shall be dine ester was compound 6 which is accessible by the Mitsu-
referred to as F9+AE, F13+AE and F17+AE respectively.
nobu reaction between compound 1 and 3-dimethylamino-1-
The common synthetic scheme for F9AE, F13AE and F17AE propanol.1f N-alkylation of the exocyclic dimethylamino group
is shown in Fig. 5. Compound 1, whose synthesis we described in compound 6 with the triflates i–iii led to exclusive reaction
previously1f was O-alkylated at the phenolic group using at this nitrogen to give the fluorinated acridine esters 7a–7c
1H,1H,2H,2H-perfluorohexyl, -perfluorooctyl and -perfluoro- which in turn were converted to the N-sulfopropyl acridinium
decyl triflates to introduce the various fluorous chains in the carboxylic acids 8a–8c by N-alkylation of the acridine nitrogen
acridine ring. The triflates were synthesized from the corres- with 1,3-propane sultone in [BMIM][BF4] followed by acid
ponding fluorinated alcohols using a literature procedure.14 hydrolysis of the methyl ester. Completion of the rest of the
The fluorinated O-alkylated products 2a–2c were then con- synthesis was accomplished as described earlier. HPLC traces
verted to the final targets using chemistry we have described of all the intermediates and final compounds of Fig. 5 and 6
previously.1f,g Thus, N-alkylation of 2a–2c with 1,3-propane are shown in the ESI (Fig. S1–S24†). Yields of intermediates
sultone in the ionic liquid 1-butyl-3-methylimidazolium hexa- and the final products are indicated in Fig. 5 and 6 and were
fluorophosphate [BMIM][PF6] was followed by acid hydrolysis generally good except for some of the F17 compounds.
of the methyl ester to give the acridinium carboxylic acids
The fluorous phase is often thought of as a third phase that
3a–3c. The water soluble zwitterionic linker1e iv was then intro- is distinct from aqueous and hydrocarbon phases.10 Based on
duced in 3a–3c to give compounds 4a–4c, which are the amine this classification, the fluorinated acridinium esters of the
derivatives of the final targets. Conversion of 4a–4c to the present study (Fig. 4) can be considered to be comprised of
N-hydroxysuccinimide esters (5a–5c, FnAEs, Fig. 5) was com- three distinct regions: (a) a relatively hydrophobic acridinium
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Fig. 5 Synthetic scheme for FnAEs 5a–5c. Reagents: (a) potassium carbonate, MeCN; (b) 1,3-propane sultone, 2,6-di-tert-butylpyridine, 1-butyl-3-
methylimidazolium hexafluorophosphate [BMIM][PF6]; (c) 1–2 M HCl–MeCN; (d) N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoro-
borate (TSTU), diisopropylethylamine (DIPEA), DMF; (e) 0.25 M NaHCO3–DMF; (f) glutaric anhydride, DMF–MeOH; (g) TSTU, DIPEA, DMF.
ring and phenolic ester, (b) a highly hydrophilic region con- all gave a similar level of label incorporation of 2–3 labels
taining a sulfobetaine linker that is attached para to the phe- (Table 1) as determined by mass spectroscopy.
nolic ester and, (c) a fluorous region that is attached to the C-2
Chemiluminescence measurements
of the acridinium ring. In contrast, compound 11 (Fig. 4, refer-
ence compound1f in the current study) has an electronically
Emission spectra of the fluorinated acridinium esters as well
similar C-2 alkoxy functional group but only contains hydro-
phobic and hydrophilic regions.
BSA conjugates of the various acridinium esters of Fig. 4
were prepared using five equivalents input of the N-hydroxy-
succinimide esters of the labels as described in the ESI† and
compound 11 were measured using a spectral camera as
described in the ESI.† Emission maxima are listed in Table 2
and the complete emission spectra for each compound are
shown in the ESI (Fig. S25–S31†). The emission spectra were
recorded in a mostly (∼90%) aqueous medium and the amine
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Fig. 6 Synthetic scheme for Fn+AEs 10a–10c. Reagents: (a) potassium carbonate, MeCN; (b) 1,3-propane sultone, 2,6-di-tert-butylpyridine,
1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]; (c) 1–2 M HCl–MeCN; (d) N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetra-
fluoroborate (TSTU), diisopropylethylamine (DIPEA), DMF; (e) 0.25 M NaHCO3–DMF; (f) glutaric anhydride, DMF–MeOH; (g) TSTU, DIPEA, DMF.
derivatives of the compounds were used because of their listed in Table 1. For all the acridinium esters, we used BSA
greater aqueous solubility compared to the N-hydroxysuccini- conjugates instead of the free labels to ensure complete solubi-
mide esters. The emission spectra of all the compounds were lity of the labels in aqueous buffer especially at high dilution
quite similar and the emission maxima spanned a range of that were needed for measurements in the linear range of the
445–457 nm. The presence of the electron withdrawing fluo- photomultiplier tube in the luminometer. (In our previous
rous chains closer to the acridinium ring in the FnAE series study we observed that micellar effects on the chemilumine-
caused a small hypsochromic shift in their emission maxima scence of acridinium ester labels are very similar for the free
compared to the Fn+AE compounds. The reference compound labels as well as the conjugates of the labels.)1d The conjugates
11, with a similar C-2 alkoxy group substituent displayed an ∼2 mg mL−1 were serially diluted into buffer to final concen-
emission maximum at 454 nm.
trations of approximately 0.3 nanomolar (nM) and chemilumi-
Light yield (specific activity) from BSA conjugates of the nescence was measured using a luminometer from Berthold
fluorinated acridinium esters as well as compound 11 are Technologies. Light emission from a 0.01 mL sample was
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in excess, the overall charge of the mixed micelle is expected to
be positive. Both surfactants were dissolved in 0.25 M sodium
hydroxide (reagent 2) and reagent 1 was unchanged in these
experiments. No surfactant in 0.25 M sodium hydroxide was
used for the control experiment. Besides the CTAC–SPFO
mixed micellar system, the effect of aggregates of the individ-
ual surfactants CTAC (described above) and SPFO were also
investigated. SPFO was used at twice its reported critical
micelle concentration.15
The effect of the different micellar systems on emission
kinetics of the BSA conjugates of the various fluorinated acridi-
nium esters and compound 11 are listed in Table 3 and the
complete emission profiles are shown in Fig. S32–S39 (ESI†).
The values in Table 3 reflect the time needed (in seconds) for
≥95% emission. For the cationic micellar systems (CTAC and
Table 1 Acridinium ester incorporation in BSA conjugates measured
my mass spectroscopy and specific chemiluminescent activity (SCA) of
the labels. Light emission was triggered by the sequential addition of
0.3 mL reagent 1 (0.1 M nitric acid + 0.5% peroxide) followed by 0.3 mL
reagent 2 (7 mM CTAC in 0.25 M NaOH). (RLU = relative light unit)
No. of labels by
mass spectroscopy
SCA × 10−19
RLUs mol−1
Label
11
3
3
2
2
2
2
2
7.0
8.2
11.3
10.5
10.9
10.8
10.4
5a, F9AE
10a, F9+AE
5b, F13AE
10b, F13+AE
5c, F17AE
10c, F17+AE
CTAC
+ SPFO mixtures) where fast light emission was
Table 2 Emission spectra of acridinium esters. Spectra were recorded
in >90% aqueous media using a PR-740 spectroradiometer (camera)
from Photo Research Inc. Bandwidth slit: 2 nm; aperture: 2 degrees;
exposure time: 5000 ms. The amine intermediates were used for these
measurements
observed, light emission was measured for a total period of
20 seconds integrated at 0.1 second intervals. Light emission
was significantly slower in the absence of surfactant and in the
presence of anionic micelles of SPFO and in these two cases,
light emission was measured for a total period of 120 seconds
integrated at 0.5 second intervals.
Emission maximum
Compound (amine intermediate)
(nm)
From the data in Table 3, in the absence of any surfactant,
light emission from the conjugates of the different labels was
observed to be quite slow with some intrinsic differences
between the different labels. In particular, increasing the bulk
of the C-2 substituent had an accelerative effect on emission
kinetics. For example whereas the time for ≥95% emission was
71 seconds for 11 with a C-2 isopropoxy group, these emission
times were compressed to 48.5, 32.5 and 29 seconds for F9AE,
F13AE and F17AE respectively. Similarly, emission times
decreased from 72, 53.5 and 38 seconds with increasing chain
length for F9+AE, F13+AE and F17+AE respectively. The latter
compounds with a positive charge near the fluorous chains
were observed to be slower emitters. Light emission from acri-
dinium esters is initiated by the addition of hydroperoxide
ions at C-9 (Fig. 2) and a remote C-2 functional group is un-
likely to influence this reaction. It is possible that an increase
in size of the C-2 substituent facilitates faster decomposition
of the dioxetane IV or dioxetanone V precursors presumably
because of relief of steric congestion in these reactive inter-
mediates. As to what role a remote positive charge may play in
this process is unclear at present.
In mixtures of CTAC and SPFO, at the ratios we examined,
overall micellar charge is positive and consequently, light
emission from all acridinium ester conjugates was very rapid
with emission times of ∼2 seconds. Emission times increased
marginally as overall positive charge in the mixed micellar
system was attenuated by increasing SPFO concentration.
Nevertheless this effect was quite small as indicated by the
data in Table 3. Clear evidence for fluorous interactions
between SPFO and the fluorinated acridinium esters were
noticed when emission times were measured in pure anionic
micelles of SPFO. For example, whereas the conjugate of non-
fluorinated acridinium ester 11 showed similar emission times
in the absence of surfactant and SPFO (71 seconds versus
Amine intermediate1f of 11
4a, F9AE
9a, F9+AE
4b, F13AE
9b, F13+AE
454
446
457
445
453
445
453
4c, F17AE
9c, F17+AE
initiated by the sequential addition of 0.3 mL of 0.1 M nitric
acid with 0.5% hydrogen peroxide (reagent 1) followed by the
addition of 0.25 M sodium hydroxide (reagent 2) containing
7 mM of the cationic surfactant CTAC. Light was measured for
20 seconds integrated at 0.1 s intervals (additional details can
be found in the ESI†). The output of the instrument was
expressed as Relative Light Units (RLUs). As can be noted from
Table 1, the specific chemiluminescent activities, expressed as
RLUs per mole, of the fluorinated labels were either compar-
able or slightly higher compared to that of the non-fluorinated
reference compound 11. The observed small increases in
overall light yield increased with increasing fluorous content
of the labels. Because all labels have the same C-2 alkoxy
substitution pattern which is the main determinant of light
yield,1c,f the results in Table 1 are expected. Moreover, these
results also indicate that the fluorinated acridinium esters can
partition strongly into micelles of a non-fluorinated cationic
surfactant through a combination of hydrophobic and charge
interactions1f in a similar manner to 11.
We then investigated the properties of the mixed micelle
system of CTAC and SPFO on acridinium ester chemilumine-
scence (both light yield and emission kinetics), at different
mole fractions of CTAC and SPFO as indicated in Tables 3 and
4. Specifically, CTAC : SPFO ratios were varied from 10 : 1, 5 : 1,
3.3 : 1 and 2.5 : 1. At all ratios of CTAC : SPFO, because CTAC is
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Table 3 Time (seconds) for ≥95% total light emission for BSA conjugates of acridinium esters (average of triplicate measurements). Light emission
was triggered by the sequential addition of 0.3 mL reagent 1 (0.1 M nitric acid + 0.5% peroxide) followed by 0.3 mL of 0.25 M NaOH containing
CTAC (7 mM) or CTAC–SPFO mixtures as indicated. No surfactant in 0.25 M NaOH was the control experiment
Reagent 2
11
5a, F9AE
10a, F9+AE
5b, F13AE
10b, F13+AE
5c, F17AE
10c, F17+AE
No CTAC
75 mM SPFO
CTAC
CTAC : SPFO, 10 : 1
CTAC : SPFO, 5 : 1
CTAC : SPFO, 3.3 : 1
CTAC : SPFO, 2.5 : 1
71.0
79.5
1.5
2.0
2.3
2.0
2.1
48.5
79.0
1.2
1.7
1.8
1.9
1.9
72
78
32.5
70
53.5
57
29
74
38.0
51.5
0.8
1.2
1.2
1.1
1.2
1.0
1.5
1.8
1.8
2.1
1.4
1.6
1.9
1.8
2.0
0.8
1.0
1.1
1.1
1.4
1.3
1.6
2.0
1.7
1.9
Table 4 Observed relative change in light output of BSA conjugates of acridinium esters (average of triplicate measurements). Light emission was
triggered by the sequential addition of 0.3 mL reagent 1 (0.1 M nitric acid + 0.5% peroxide) followed by 0.3 mL of 0.25 M NaOH containing CTAC
(7 mM) or CTAC–SPFO mixtures as indicated. No surfactant in 0.25 M NaOH was the control experiment. Observed RLUs in the absence of any
surfactant were normalized to one for each label. Maximum observed enhancement values are indicated in bold
Reagent
11
5a, F9AE
10a, F9+AE
5b, F13AE
10b, F13+AE
5c, F17AE
10c, F17+AE
No CTAC
75 mM SPFO
CTAC
CTAC : SPFO, 10 : 1
CTAC : SPFO, 5 : 1
CTAC : SPFO, 3.3 : 1
CTAC : SPFO, 2.5 : 1
1.0
0.8
5.1
5.3
5.5
5.1
5.1
1.0
0.6
5.3
5.7
5.7
5.4
5.3
1.0
1.0
4.1
4.4
4.3
4.1
3.7
1.0
0.6
4.5
5.1
5.1
5.0
5.0
1.0
0.9
3.4
3.7
3.8
3.6
3.6
1.0
0.4
3.9
4.3
4.3
4.1
3.9
1.0
0.7
2.9
3.3
3.3
3.1
3.1
79.5 seconds respectively), the fluorinated acridinium esters tants CTAC and SPFO. To enable easy comparisons, we have
showed strong inhibition of emission kinetics which increased normalized the light yield in the absence of surfactant to one
with increasing fluorous content. Emission times for F9AE, for all acridinium ester conjugates. Inspection of the data in
F13AE and F17AE increased from 48.5, 32.5 and 29 seconds Table 4 indicates that in the mixed micellar system of CTAC
respectively in the absence of surfactant to 79, 70 and 74 and SPFO, light yield is slightly enhanced, compared to CTAC
seconds respectively in the presence of SPFO. Increasing fluo- alone, for all acridinium ester conjugates especially at CTAC :
rous content in these acridinium esters leads to greater parti- SPFO ratios of 10 : 1 and 5 : 1. The conjugate of compound 11
tioning from bulk medium into fluorinated micelles of SPFO which contains no fluorines also showed enhanced light yield
which repel hydroperoxide ions and inhibit emission kinetics. in mixed micelles of CTAC : SPFO at low mole fractions of
The inhibition in emission kinetics is also clearly evident from SPFO. These results indicate that at CTAC : SPFO ratios of 10 : 1
a comparison of the emission profiles for the various conju- and 5 : 1, the polarity of the mixed micelles is lower than that
gates in the absence of surfactant and in the presence of SPFO of CTAC. The observed enhancements in the mixed micellar
micelles as illustrated in Fig. S39 (ESI†). Interestingly, the com- system at these ratios of CTAC and SPFO are comparable to
pounds F9+AE, F17+AE and F17+AE which were designed to what we observed previously in less polar aggregates of cat-
interact with SPFO micelles both by fluorous and charge inter- ionic surfactants with large head groups such as cetyltripropyl-
actions, showed a more attenuated inhibition of emission ammonium chloride.1d The conjugates of the fluorinated
kinetics with emission times of 72, 53.5 and 38 seconds acridinium esters showed a similar trend and the magnitude
respectively in the absence of surfactant, to 78, 57 and 51.5 of the observed enhancement actually decreased slightly with
seconds respectively in the presence of SPFO for ≥95% emis- increasing fluorous content for both series of acridinium
sion. We conclude from these observations that partitioning of esters. These observations coupled with the data in Table 1
these fluorinated acridinium ester substrates into SPFO suggest that the fluorous chains in the fluorinated acridinium
micelles is poorer which suggests an absence of any attractive esters do not enable partitioning into less polar regions of
charge interaction between the anionic head group of SPFO micelles of either CTAC or CTAC + SPFO.
and the quaternary nitrogen in these compounds. Increased
aqueous solubility of these charged labels may also play a role to decrease back to the levels of enhancement seen with
in poorer partitioning into SPFO micelles. micelles of pure CTAC. At higher mole fractions of SPFO,
As SPFO concentration increased, light output was observed
Micelles also strongly influence light yield from acridinium partial separation of the CTAC and SPFO phases may occur
esters and in Table 4 we have summarized the effect the mixed similar to what has been reported in other mixed micelle
micellar system of CTAC and SPFO on light yields and have systems16 which could explain the drop in light enhancement
compared it to light yields observed in the absence of surfac- although the emission kinetics data (Table 3) indicate that
tant and in the presence of micelles of the individual surfac- phase separation may not be occurring. A more reasonable
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explanation is that at higher concentrations of SPFO, polarity systems. Assay sensitivity is a function of both light output of
of the mixed micelle is higher and similar to that of CTAC. the label and the background signal caused by non-specific
Table 4 also indicates that in anionic micelles of SPFO, light binding of the label. We evaluated the non-specific binding of
output is suppressed and the magnitude of this inhibition the BSA conjugates of the various fluorinated acridinium ester
increased with increasing fluorous content of the acridinium labels to paramagnetic particles (PMPs) using the non-fluori-
esters F9AE, F13AE and F17AE where observed light yields nated acridinium ester 11 as the reference compound
were 0.6, 0.6 and 0.4 respectively compared to the no surfac- (Table 5). The PMPs used for measurement of non-specific
tant control experiment. These results parallel the strong inhi- binding were, 1–10 micron-sized iron(III) oxide particles,
bition in emission kinetics that was observed with these coated with an anti-fluorescein antibody on the amino-sila-
compounds in SPFO micelles (Table 3). Inhibition of light nized particle surface using commonly used glutaraldehyde
output presumably results from unfavorable electrostatic inter- coupling chemistry. The particles were mixed with solutions of
actions between the anionic head group of SPFO and nega- the acridinium ester-labeled conjugates and were then magne-
tively charged dioxetane intermediates (IV in Fig. 2) that are tically separated. They were then washed twice using three
precursors to excited state acridone. The Fn+AE series of fluori- different wash solutions: (a) phosphate buffer, pH 7.2, (b) the
nated acridinium esters exhibited less severe inhibition of non-ionic surfactant Tween-20 at a concentration of 0.8 mM in
emission kinetics and their light yields were also less affected phosphate buffer, and (c) a combination of 0.8 mM Tween-20
by anionic micelles of SPFO.
with 50 mM SPFO in phosphate buffer. (The cationic surfac-
tant CTAC resulted in particle loss during the magnetic separ-
ation step so could not be evaluated as a wash detergent.) The
chemiluminescence associated with the particles was then
measured. The ratio of this chemiluminescence value in com-
parison to the total chemiluminescence input is referred to
fractional non-specific binding (FNSB) and is a reflection of
the resistance of the conjugate towards non-specific adsorp-
tion to the microparticles. The results of these measurements
are tabulated in Table 5 and details are described in the ESI.†
Compared to the non-fluorinated reference compound 11,
all conjugates of the fluorinated acridinium esters showed sig-
nificantly higher FNSB when the PMPs were washed only with
buffer strongly suggesting that the acridinium rings in these
compounds are much more hydrophobic than that of 11
(Table 5). This non-specific binding was exacerbated with
increasing fluorous content and the addition of a positive
Non-specific binding and chemiluminescence stability
Finally, we examined chemiluminescence stability and non-
specific binding of the BSA conjugates of the fluorinated
acridinium esters (Fig. 7 and Table 5). Stability is essential for
long term storage of reagents and in this regard, the fluori-
nated acridinium ester labels exhibited stability which was
comparable to the reference compound 11. Under refrigeration
at 4 °C, no loss of chemiluminescence occurred whereas at
37 °C, chemiluminescence stability increased with increasing
fluorous content which was more clearly evident for the FnAE
series. The FnAE series of compounds exhibited better stability
than the Fn+AE series as can be noted from Fig. 7.
Acridinium dimethylphenyl ester labels are used in con-
junction with magnetic microparticles in automated immuno-
assays in Siemens Healthcare Diagnostics’ ADVIA Centaur®
Fig. 7 Chemiluminescence stability of BSA conjugates of acridinium esters at 4 °C (blue lines) and 37 °C (red lines). BSA conjugates of the various
acridinium esters were stored in buffer at pH 7.4 either at 4 °C or 37 °C and residual chemiluminescence was measured as a function of time. There
was no loss of chemiluminescence at 4 °C. At 37 °C, stability of the fluorinated acridinium ester labels was comparable to the reference compound
11 with slightly enhanced stability with increasing fluorous content which was more evident for the FnAE series.
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Table 5 Fractional non-specific binding (FNSB) of BSA conjugates of fluorinated acridinium esters and the non-fluorinated acridinium ester 11 to
paramagnetic microparticles (PMPs) using three different wash protocols. The fluorinated acridinium esters were observed to be more lipophilic and
exhibited higher non-specific binding when the PMPs were washed with only buffer. The non-ionic detergent Tween-20 was effective in reducing
the non-specific binding of the fluorinated acridinium esters with lower fluorous content. The addition of SPFO in the wash solution did not reduce
non-specific binding any further
FNSB after wash
Relative FNSB
Phosphate buffer +
0.8 mM Tween-20 +
50 mM SPFO
Phosphate buffer +
0.8 mM Tween-20 +
50 mM SPFO
Phosphate
buffer, pH 7.2
Phosphate buffer +
0.8 mM Tween-20
Phosphate
buffer, pH 7.2
Phosphate buffer +
0.8 mM Tween-20
Conjugate
11
3.3 × 10−5
2.1 × 10−4
2.2 × 10−4
2.1 × 10−4
6.6 × 10−4
3.6 × 10−4
5.4 × 10−4
4.7 × 10−5
3.4 × 10−5
6.8 × 10−5
4.6 × 10−5
3.9 × 10−4
9.4 × 10−5
1.4 × 10−4
3.8 × 10−5
3.5 × 10−5
7.8 × 10−5
4.0 × 10−5
4.3 × 10−4
1.1 × 10−4
1.5 × 10−4
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.4
0.2
0.3
0.2
0.6
0.3
0.3
1.1
0.2
0.4
0.2
0.7
0.3
0.3
5a, F9AE
10a, F9+AE
5b, F13AE
10b, F13+AE
5c, F17AE
10c, F17+AE
charge in the Fn+AE series of compounds. However, when the a small impact on their chemiluminescence emission spectra
non-ionic detergent Tween-20 was used in buffer to wash the when compared to an analogously C-2 alkoxy substituted, non-
particles, the FNSB values of the fluorinated acridinium esters, fluorinated acridinium ester.
especially those with lower fluorous content and no positive
2. Fluorinated acridinium esters partition equally well into
charge (F9AE and F13AE), were effectively reduced down to the micelles of the cationic surfactant CTAC as a non-fluorinated
same FNSB value observed for non-fluorinated 11. Interest- acridinium ester. Fast light emission and similar quantum
ingly, Tween-20 was no more effective than plain buffer as a yields (comparable to a non-fluorinated acridinium ester) are
wash solution for 11 which indicates that overall, 11 is a rela- observed for these new labels. Thus, fluorous tags in acridi-
tively hydrophilic compound. Thus from Table 5, when the nium esters do not lead to solubilization of the labels into a
observed FNSB values of the various acridinium esters using a less polar region of CTAC micelles as reported for SPFO in
buffer wash are normalized to one, a significantly greater drop CTAC micelles. Because charged reactive ions and charged
in the relative FNSB was observed for the fluorinated labels reaction intermediates are involved in the chemiluminescence
when compared to 11 (which was essentially unchanged) when reaction pathway of acridinium esters, these results are not
Tween-20 was used as the wash detergent. To determine entirely unexpected. The chemiluminescence reaction most
whether specific fluorous interactions between SPFO and the likely proceeds at the Stern layer of the micelle.
fluorinated acridinium esters could be used to augment the
3. Mixed micelles of CTAC : SPFO at low mole fractions of
wash efficacy of Tween-20, a third wash protocol using a SPFO behave like less polar micelles of cationic surfactants
mixture of SPFO and Tween-20 was evaluated (Table 5) but we with large head groups such as cetyltripropylammonium
observed no further reduction in the FNSB values of the fluori- chloride based on the observed enhancement in light
nated acridinium esters. The dominant interaction between yields when compared to CTAC micelles.1d Fluorous inter-
the conjugates of the fluorinated acridinium esters and the actions between fluorinated acridinium esters and SPFO in the
PMPs appears to be largely hydrophobic in nature and specific mixed micelles do not lead to greater enhancement of light
fluorous interactions between these labels and SPFO are too yields for the same reason noted in 2. Evidence for fluorous
weak to overcome this interaction.
interactions between the fluorinated acridinium esters and
SPFO was noted in pure anionic SPFO micelles where
inhibition of emission kinetics and depressed light yield was
observed.
4. Chemiluminescence stability of fluorinated acridinium
esters at 37 °C and neutral pH are either comparable to or
better than the stability of a non-fluorinated acridinium ester.
5. Fluorinated acridinium esters are lipophilic and require
a detergent wash to reduce their non-specific binding to para-
magnetic microparticles.
From the current study, we can identify two fluorinated
acridinium esters, compound 5a (F9AE) and 5b (F13AE) as
improvements over the non-fluorinated acridinium ester 11
owing to their higher light yield (Table 1), better chemilumi-
nescence stability (Fig. 7) and equivalent non-specific binding
(Table 5).
Conclusions
In the current study we have described the synthesis of new
acridinium esters with fluorous tags and have investigated
their light emission in the presence of cationic micelles of
CTAC, anionic micelles of the perfluorinated surfactant SPFO
as well as mixed micelles these two surfactants. We have also
compared the chemiluminescence stability and non-specific
binding of these labels to that of a structurally analogous but
non-fluorinated acridinium ester. Our main findings and
observations can be summarized in the following points:
1. Fluorous tags in acridinium esters, installed at C-2 ether
linkages of the acridinium ring, have either a minimal or only
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Synthesis of acridinium esters (Fig. 4–6, and Fig. S1–S24 ESI†)
Experimental
General
Compound 2a. Compound 1 (100 mg, 0.249 mmol) was dis-
solved in acetonitrile (15 mL) and potassium carbonate
(38 mg, 0.274 mmol) was added followed by 1H,1H,2H,2H-per-
fluorohexyl triflate (197 mg, 0.498 mmol) and the reaction was
stirred at room temperature for 16 hours. The reaction mixture
was concentrated under reduced pressure. The residue was
extracted with dichloromethane (70 mL) and subsequently
washed with water (30 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was purified by flash chromato-
graphy on silica (40 g column) using a 30 minute gradient of
0 → 100% B (solvent A: hexanes; solvent B: hexanes–ethyl
acetate, 1 : 1). Yield = 145 mg (90%). δH (500 MHz, CDCl3) 2.47
(s, 6H), 2.75 (m, 2H), 3.95 (s, 3H), 4.42 (t, 2H, J = 6.7 Hz), 7.54
(dd, 1H, J = 9.4 Hz, 2.6 Hz), 7.61 (d, 1H, J = 2.6 Hz), 7.63–7.72
(m, 1H), 7.76–7.86 (m, 1H), 7.92 (s, 2H), 8.24 (d, 1H, J =
9.4 Hz), 8.31 (dd, 1 H, J = 8.7 Hz, 1.1 Hz), 8.43 (dd, 1H, J =
8.8 Hz, 1.1 Hz). δF (470 MHz, CDCl3) −80.99 (t, 3F, J = 9.6 Hz),
−113.46 (m, 2F), −124.44 (m, 2F), −125.93 (m, 2F). MALDI-TOF
MS m/z 648.2 (M + H)+; HRMS m/z 648.1423 (M + H)+ (648.1433
calculated).
Chemicals were purchased from Sigma-Aldrich (Milwaukee,
Wisconsin, USA) unless indicated otherwise. The ionic liquids
[BMIM][PF6] and [BMIM][BF4] were dried under high vacuum
over P2O5 prior to use. Sodium perfluorooctanoate (SPFO) was
purchased from Alfa-Aesar.
All final acridinium esters, intermediates and reaction
mixtures were analyzed and/or purified by HPLC using a
Beckman-Coulter HPLC system. For analytical HPLC, a Pheno-
menex, Kinetex, XB-C18, 2.6 micron, 4.6 × 50 mm column
was used with MeCN–water (each with 0.05% trifluoroacetic
acid, TFA) as the solvents at a flow rate of 1 mL min−1 and UV
detection at 260 nm. Preparative HPLC was performed using a
Phenomenex, Luna C18, 5 micron, 250 × 30 mm column at a
flow rate of 16–20 mL min−1 and UV detection at 260 nm.
Flash chromatography was performed using an ‘Autoflash’
system from TELEDYNE ISCO. MALDI-TOF (Matrix-Assisted
Laser Desorption Ionization-Time of Flight) mass spectroscopy
was performed using a VOYAGER DE Biospectrometry Work-
station from ABI. This is a bench top instrument operating in
the linear mode with a 1.2 meter ion path length, flight tube.
Spectra were acquired in positive ion mode. For small mole-
cules, α-cyano-4-hydroxycinnamic acid was used as the matrix
and spectra were acquired with an accelerating voltage of
20 000 volts and a delay time of 100 ns. For protein conjugates,
sinapinic acid was used as the matrix and spectra were
acquired with an accelerating voltage of 25 000 volts and a
delay time of 85 ns.
For HRMS (High Resolution Mass Spectra), samples were
dissolved in HPLC-grade methanol and analyzed by direct-flow
injection (injection volume = 5 μL) ElectroSpray Ionization
(ESI) on a Waters Qtof API US instrument in the positive ion
mode. Optimized conditions were as follows: capillary =
3000 kV, cone = 35, source temperature = 120 °C, desolvation
temperature = 350 °C.
NMR spectra (proton and fluorine) were recorded on a
Varian 500 MHz spectrometer. To suppress or minimize pseudo-
base formation (Fig. 2, II to 1 conversion), spectra of the
fluorinated acridinium esters and intermediates were acquired
in an acidic solvent, namely deuterated acetic acid so that two
forms of the compounds were not present during spectral ana-
lysis. Significant line broadening was observed in deuterated
trifluoroacetic acid and therefore this acidic solvent was unsui-
table for proton NMR analysis of the fluorinated acridinium
esters. Because of limited solubility of the fluorinated acridi-
nium compounds in deuterated acetic acid, carbon NMR
spectra could not be acquired.
Compounds 2b and 2c were synthesized similarly.
Compound 2b. Yield = 80 mg (86%). δH (500 MHz, CDCl3)
2.47 (s, 6H), 2.75 (m 2H), 3.95 (s, 3H), 4.42 (t, 2H, J = 6.7 Hz),
7.53 (dd, 1H, J = 9.5 Hz, 2.6 Hz), 7.61 (d, 1H, J = 2.6 Hz), 7.68
(m, 1H), 7.82 (m, 1H), 7.92 (s, 2H), 8.24 (d, 1H, J = 9.5 Hz), 8.31
(m, 1H), 8.43 (m, 1H). δF (470 MHz, CDCl3) −80.76 (t, 3F, J =
11.1 Hz), −113.26 (m, 2F), −121.82 (m, 2F), −122.84 (m, 2F),
−123.51 (m, 2F), −126.12 (m, 2F). MALDI-TOF MS m/z 748.4
(M + H)+; HRMS m/z 748.1363 (M + H)+ (748.1369 calculated).
Compound 2c. Yield = 95 mg (90%). δH (500 MHz, CDCl3)
2.47 (s, 6H), 2.75 (m, 2H), 3.94 (s, 3H), 4.42 (t, 2H, J = 6.7 Hz),
7.53 (dd, 1H, J = 9.4 Hz, 2.7 Hz), 7.61 (d, 1H, J = 2.6 Hz), 7.68
(m, 1H), 7.81 (m, 1H), 7.92 (s, 2H), 8.24 (d, 1H, J = 9.5 Hz), 8.30
(br d, 1H, J = 8.5 Hz), 8.43 (br d, 1H, J = 8.4 Hz). δF (470 MHz,
CDCl3) −80.73 (t, 3F, J = 9.9 Hz), −113.24 (m, 2F), −121.60 (m,
2F), −121.88 (m, 4F), −122.69 (m, 2F), −123.46 (m, 2F),
−126.09 (m, 2F). MALDI-TOF MS m/z 848.1 (M + H)+; HRMS
m/z 848.1290 (M + H)+ (848.1305 calculated).
Compound 3a. Compound 2a (100 mg, 0.154 mmol) in
[BMIM][PF6] (1 mL) and 2,6-di-tert-butylpyridine (0.256 mL,
1.158 mmol) was treated with 1,3-propane sultone (283 mg,
2.317 mmol). The mixture was heated at 155 °C in a sealed vial
for 16 hours and then cooled to room temperature. The reac-
tion mixture was diluted with ethyl acetate (1 mL) and purified
by flash chromatography on silica (40 g column) using pure
ethyl acetate as eluent to remove unreacted starting material
and base followed by 40% ethyl acetate in methanol to elute
product. Yield = 99 mg (83% yield). This N-sulfopropyl acridi-
nium methyl ester was carried as such to the next step. Thus,
89 mg of the acridinium methyl ester was refluxed in a mixture
of 2 N HCl–acetonitrile (1 : 1) for 16 hours. The reaction
mixture was cooled to room temperature and product 3a was
purified by preparative HPLC using a 30 minute gradient of
Chemiluminescence measurements were carried out using
a Berthold Technologies’ AutoLumat Plus LB 953 lumino-
meter. Emission spectra were recorded in >90% aqueous
media using a PR-740 spectroradiometer (camera) on loan
from Photo Research Inc. Bandwidth slit: 2 nm; aperture:
2 degrees; exposure time: 5000 ms.
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10 → 90% MeCN–water (each with 0.05% TFA). The HPLC frac-
tions were concentrated under reduced pressure. Yield =
Compounds 4b and 4c were synthesized similarly.
Compound 4b. Yield 23 mg (59%). δH (500 MHz,
=
84 mg (80%). δH (500 MHz, CD3COOD) 2.69 (s, 6H), 2.86–2.96 CD3COOD) 2.32–2.42 (m, 2H), 2.42–2.52 (m, 4H), 2.53 (s, 6H),
(m, 2 H), 2.99–3.13 (m, 2H), 3.63 (m, 2H), 4.80 (t, 2H, J = 2.73–2.88 (m, 2H), 2.97–3.15 (m, 2H), 3.23 (t, 2H, J = 6.6 Hz),
6.2 Hz), 6.02 (m, 2H), 7.98 (d, 1H, J = 2.7 Hz), 8.15 (m, 2H), 3.29 (s, 3H), 3.42 (t, 2H, J = 7.1 Hz), 3.56 (m, 2H), 3.61–3.74 (m,
8.30 (dd, 1H, J = 8.8 Hz, 6.7 Hz), 8.45 (dd, 1H, J = 10.0 Hz, 4H), 3.74–3.86 (m, 4H), 4.69 (t, 2H, J = 6.0 Hz), 5.92 (m, 2H),
2.8 Hz), 8.66 (m, 1H), 8.78 (dd, 1H, J = 8.8 Hz, 1.3 Hz), 9.15 7.71 (m, 1H), 7.91 (s, 2H), 8.23 (m, 1H), 8.41 (dd, 1H, J =
(d, 1H, J = 9.4 Hz), 9.22 (d, 1H, J = 10.1 Hz). δF (470 MHz, 10.0 Hz, 2.6 Hz), 8.52 (d, 1H, J = 8.7 Hz), 8.62 (m, 1H), 9.10 (d,
CD3COOD) −81.95 (t, 3F, J = 9.8 Hz), −113.88 (m, 2F), −124.93 1H, J = 9.5), 9.16 (d, 1H, J = 10.1 Hz). δF (470 MHz, CD3COOD)
(m, 2F), −126.57 (m, 2F). MALDI-TOF MS m/z 756.2 (M + H)+; −81.71 (t, 3F, J = 10.1 Hz), −113.78 (m, 2F), −122.36 (m, 2F),
HRMS m/z (M + H)+ 756.1304 (756.1314 calculated).
Compounds 3b and 3c were synthesized similarly.
−123.39 (m, 2F), −124.02 (m, 2F), −126.75 (m, 2F). MALDI-TOF
MS m/z 1105.3 (M + H)+; HRMS m/z 1105.2769 (M + H)+
Compound 3b. Yield
CD3COOD) 2.67 (s, 6H), 2.80–2.93 (m, 2H), 2.98–3.10 (m, 2H),
=
38 mg (67%). δH (500 MHz, (1105.2761 calculated).
Compound 4c. Yield
=
8 mg (52%). δH (500 MHz,
3.57–3.64 (m, 2H), 4.78 (t, 2H, J = 6.2 Hz), 6.00 (t, 2H, J = CD3COOD) 2.31–2.39 (m, 2H), 2.39–2.53 (m, 4H), 2.63 (s, 6H),
9.5 Hz), 7.97 (d, 1H, J = 2.8 Hz), 8.13 (s, 2 H), 8.28 (dd, 1H, J = 2.78–2.95 (m, 2H), 2.97–3.15 (m, 2H), 3.24 (m, 2H), 3.26 (s,
8.8 Hz, 6.7 Hz), 8.44 (m, 1H), 8.64 (m, 1H), 8.76 (dd, 1H, J = 3H), 3.38 (t, 2H, J = 7.1 Hz), 3.55–3.71 (m, 6H), 3.71–3.85 (m,
8.8 Hz, 1.3 Hz), 9.13 (d, 1H, J = 9.4 Hz), 9.21 (d, 1H, J = 10.1 4H), 4.75 (t, 2H, J = 6.1 Hz), 5.98 (m, 2H), 7.89 (d, 1H, J =
Hz). δF (470 MHz, CD3COOD) −81.70 (t, 3F, J = 10.1 Hz), 2.8 Hz), 7.94 (s, 2H), 8.27 (m, 1H), 8.42 (dd, 1H, J = 9.9 Hz,
−113.60 (m, 2F), −122.33 (m, 2F), −123.37 (m, 2F), −123.92 2.7 Hz), 8.63 (m, 1H), 8.70 (d, 1H, J = 8.7 Hz), 9.12 (d, 1H, J =
(m, 2F), −126.70 (m, 2F). MALDI-TOF MS m/z 856.2 (M + H)+; 9.5 Hz), 9.19 (d, 1H, J = 10.1 Hz). δF (470 MHz, CD3COOD)
HRMS m/z 856.1249 (M + H)+ (856.1250 calculated).
Compound 3c. Yield
−81.67 (t, 3F, J = 10.1 Hz), −113.75 (m, 2F), −122.09 (m, 2F),
=
20 mg (36%). δH (500 MHz, −122.38 (m, 4F), −123.24 (m, 2F), −123.93 (m, 2F), −126.67
CD3COOD) 2.67 (s, 6H), 2.83–2.94 (m, 2H), 2.98–3.13 (m, 2H), (m, 2F). MALDI-TOF MS m/z 1205.4 (M + H)+; HRMS m/z
3.60 (m, 2H), 4.78 (t, 2H, J = 6.2 Hz), 6.00 (m, 2H), 7.96 (d, 1H, 1205.2703 (M + H)+ (1205.2697 calculated).
J = 2.7 Hz), 8.13 (s, 2H), 8.27 (dd, 1H, J = 8.8 Hz, 6.7 Hz), 8.43
Compound 5a. Compound 4a (30 mg, 0.030 mmol) was dis-
(dd, 1H, J = 10.0 Hz, 2.7 Hz), 8.63 (m, 1H), 8.76 (dd, 1H, J = solved in methanol (2 mL) and diisopropylethylamine (DIPEA,
8.8 Hz, 1.3 Hz), 9.13 (d, 1H, J = 9.4 Hz), 9.20 (d, 1H, J = 10.1 0.026 mL, 0.149 mmol) and glutaric anhydride (17.0 mg,
Hz). δF (470 MHz, CD3COOD) −81.67 (t, 3F, J = 10.0 Hz), 0.149 mmol) were added. The reaction was stirred at room
−113.57 (m, 2F), −121.11 (m, 2F), −122.36 (m, 4F), −123.22 temperature and monitored by analytical HPLC which showed
(m, 2F), −123.85 (m, 2F), −126.68 (m, 2F). MALDI-TOF MS m/z 98% conversion. The reaction mixture was concentrated under
956.1 (M + H)+; HRMS m/z 956.1197 (M + H)+ (956.1186 reduced pressure. The residue was dissolved in DMF (2 mL)
calculated).
and DIPEA (0.104 mL, 0.597 mmol) and TSTU (180 mg,
Compound 4a. Compound 3a (60 mg, 0.079 mmol) was sus- 0.597 mmol) were added. The reaction was stirred at room
pended in DMF (3 mL) and diisopropylethylamine (0.021 mL, temperature and monitored by analytical HPLC which showed
0.119 mmol) and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)- complete conversion to the N-hydroxysuccinimide ester. The
uronium tetrafluoroborate (TSTU, 29 mg, 0.095 mmol) were product 5a was purified by preparative HPLC as described
added. The reaction was stirred at room temperature for earlier using a 40 minute gradient of 10 → 60% MeCN–water
1 hour and was then added drop wise to a stirred solution of iv (each with 0.05% TFA). The HPLC fractions containing
(170 mg, 0.397 mmol, HBr salt) dissolved in 0.5 M sodium product 5a were frozen at −80 °C and lyophilized to dryness.
bicarbonate (3 mL) cooled in an ice bath. After 16 hours, the Yield = 28 mg (76%). δH (500 MHz, CD3COOD) 2.28–2.36 (m,
reaction mixture was purified by preparative HPLC as 2H), 2.36–2.47 (m, 2H), 2.51–2.64 (m, 8H), 2.81 (m, 6H),
described earlier using a 40 minute gradient 10 → 40% 2.88–3.12 (m, 8H), 3.17–3.28 (m, 5H), 3.44–3.65 (m, 8H),
MeCN–water (each with 0.05% TFA). The HPLC fractions con- 3.67–3.77 (m, 4H), 4.72 (t, 2H, J = 6.0 Hz), 5.95 (m, 2H), 7.81
taining product 4a were concentrated under reduced pressure. (d, 1H, J = 2.8 Hz), 7.93 (s, 2H), 8.25 (dd, 1H, J = 8.7 Hz,
Yield = 60 mg (75%). δH (500 MHz, CD3COOD) 2.28–2.39 (m, 6.8 Hz), 8.41 (dd, 1H, J = 10.0 Hz, 2.7 Hz), 8.62 (m, 2H), 9.12
2H), 2.39–2.52 (m, 4H), 2.55 (s, 6H), 2.78–2.86 (m, 2H), (d, 1H, J = 9.6 Hz), 9.18 (d, 1H, J = 10.1 Hz). δF (470 MHz,
2.96–3.10 (m, 2H), 3.21 (m, 2H), 3.27 (s, 3H), 3.40 (t, 2H, J = CD3COOD) −81.90 (m, 3F), −113.88 (m, 2F), −124.92 (m, 2F),
7.2 Hz), 3.56 (m, 2H), 3.59–3.72 (m, 4H), 3.72–3.83 (m, 4H), −126.53 (m, 2F). MALDI-TOF MS m/z 1216.4 (M + H)+; HRMS
4.70 (t, 2H, J = 6.1 Hz), 5.92 (m, 2H), 7.76 (d, 1H, J = 2.7 Hz), m/z 1216.3448 (M + H)+ (1216.3305 calculated).
7.91 (s, 2H), 8.23 (dd, 1H, J = 8.8 Hz, 6.7 Hz), 8.39 (dd, 1H, J =
10.0 Hz, 2.7 Hz), 8.59 (m, 2H), 9.09 (d, 1H, J = 9.8 Hz), 9.15 (d,
Compounds 5b and 5c were synthesized similarly.
Compound 5b. Yield mg (45%). δH (500 MHz,
=
8
1H, J = 10.0 Hz); δF (470 MHz, CD3COOD) −81.92 (t, 3F, J = CD3COOD) 2.28–2.37 (m, 2H), 2.37–2.47 (m, 2H), 2.47–2.64 (m,
9.9 Hz), −113.91 (m, 2F), −124.94 (m, 2F), −126.54 (m, 2F). 8H), 2.76–2.87 (m, 6H), 2.95–3.12 (m, 8H), 3.15–3.25 (m, 5H),
MALDI-TOF MS m/z 1005.4 (M + H)+; HRMS m/z 1005.2835 3.46–3.65 (m, 8H), 3.68–3.78 (m, 4H), 4.71 (t, 2H, J = 6.1 Hz),
(M + H)+ (1005.2825 calculated).
5.93 (m, 2H), 7.78 (d, 1H, J = 2.8 Hz), 7.92 (s, 2H), 8.24 (dd, 1H,
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J = 8.8 Hz, 6.7 Hz), 8.41 (dd, 1H, J = 9.9 Hz, 2.7 Hz), 8.55–8.67 (1 mL). The reaction was heated at 155 °C in a sealed vial for
(m, 2H), 9.11 (d, 1H, J = 9.5 Hz), 9.17 (d, 1H, J = 10.1 Hz). δF 16 hours and then cooled to room temperature. The reaction
(470 MHz, CD3COOD) −81.69 (t, 3F, J = 10.0 Hz), −113.69 (m, mixture was then transferred into a flask with acetonitrile
2F), −122.30 (m, 2F), −123.36 (m, 2 F), −123.96 (m, 2F), (5 mL) and 2 N HCl (20 mL) was added. The reaction was
−126.69 (m, 2F). MALDI-TOF MS m/z 1316.8 (M + H)+; HRMS refluxed for 16 h and then cooled to room temperature. The
m/z 1316.3292 (M + H)+ (1316.3242 calculated).
Compound 5c. Yield
crude reaction mixture was purified by preparative HPLC as
2 mg (17%). δH (500 MHz, described earlier using a 40 minute gradient of 10 → 70%
=
CD3COOD) 2.27–2.36 (m, 2H), 2.36–2.46 (m, 2H), 2.46–2.57 (m, MeCN–water (each with 0.05% TFA). The HPLC fractions
2H), 2.62 (s, 6H), 2.71–2.90 (m, 6H), 2.93–3.13 (m, 8H), contain product 8a were concentrated under reduced pressure.
3.17–3.27 (m, 5H), 3.46–3.66 (m, 8H), 3.67–3.77 (m, 4H), 4.74 Yield = 119 mg (quantitative). δH (500 MHz, CD3COOD) 2.62 (s,
(t, 2H, J = 6.1 Hz), 5.96 (m, 2H), 7.86 (d, 1H, J = 2.8 Hz), 7.93 (s, 6H), 2.64–2.73 (m, 2H), 2.77–2.88 (m, 2H), 2.99–3.15 (m, 2H),
2H), 8.25 (m, 1 H), 8.41 (dd, 1H, J = 10.0 Hz, 2.7), 8.62 (m, 1H), 3.47 (s, 6H), 3.55 (t, 2H, J = 6.2 Hz), 3.88–4.06 (m, 4H), 4.56 (t,
8.67 (m, 1H), 9.11 (d, 1H, J = 9.4 Hz), 9.18 (d, 1H, J = 10.1 Hz). 2H, J = 5.6 Hz), 5.93 (m, 2H), 7.84 (d, 1H, J = 2.6 Hz), 8.08 (s,
δF (470 MHz, CD3COOD) −81.66 (t, 3F, J = 10.0 Hz), −113.69 2H), 8.23 (dd, 1H, J = 8.8 Hz, 6.7 Hz), 8.35 (m, 1H), 8.57 (m,
(m, 2F), −122.06 (m, 2F), −122.36 (m, 4F), −123.21 (m, 2F), 1H), 8.70 (d, 1H, J = 8.7 Hz), 9.04 (d, 1H, J = 9.4 Hz), 9.13 (d,
−123.90 (m, 2F), −126.67 (m, 2F). MALDI-TOF MS m/z 1416.5 1H, J = 9.9 Hz); δF (470 MHz, CD3COOD) −81.95 (t, 3F, J =
(M
+
H)+; HRMS m/z 1416.3582 (M
+
H)+ (1416.3178 9.8 Hz), −114.10 (m, 2F), −124.33 (m, 2F), −126.64 (m, 2F).
MALDI-TOF MS m/z 841.4 (M)+; HRMS m/z 841.2216 (M)+
calculated).
Compound 7a. Compound 6 (100 mg, 0.206 mmol) was dis- (841.2205 calculated).
solved in acetonitrile (15 mL) and 1H,1H,2H,2H-perfluorohexyl
triflate (163 mg, 0.411 mmol) was added. The reaction was
Compounds 8b and 8c were synthesized similarly.
Compound 8b. Yield 35 mg (63%). δH (500 MHz,
=
stirred at room temperature for 1 hour and monitored by CD3COOD) 2.66 (s, 6H), 2.67–2.76 (m, 2H), 2.81–2.92 (m, 2H),
analytical HPLC which showed 89% conversion. The crude 3.00–3.17 (m, 2H), 3.48 (s, 6H), 3.59 (m, 2H), 3.93–4.05 (m,
mixture was purified by preparative HPLC as described earlier 4H), 4.58 (t, 2H, J = 5.7 Hz), 5.98 (m, 2H), 7.88 (d, 1H, J =
using a 30 minute gradient of 10 → 90% MeCN–water (each 2.7 Hz), 8.12 (s, 2H), 8.26 (dd, 1H, J = 8.8 Hz, 6.8 Hz), 8.38 (dd,
with 0.05% TFA). The HPLC fractions containing product 7a 1H, J = 10.0 Hz, 2.7 Hz), 8.61 (m, 1H), 8.75 (dd, 1H, J = 8.7 Hz,
were concentrated under reduced pressure. Yield = 163 mg 1.3 Hz), 9.08 (d, 1H, J = 9.5 Hz), 9.19 (d, 1H, J = 10.1 Hz); δF
(94%). δH (500 MHz, CD3CN) 2.31 (m, 2H), 2.49 (s, 6H), (470 MHz, CD3COOD) −81.95 (m, 3F), −113.96 (m, 2F),
2.72–2.86 (m, 2H), 3.11 (s, 6H), 3.55 (m, 2H), 3.65 (m, 2H), 3.90 −122.43 (m, 2F), −123.44 (m, 4F), −126.75 (m, 2F). MALDI-TOF
(s, 3H), 4.26 (t, 2 H, J = 5.6 Hz), 7.63 (m, 2H), 7.76–7.86 (m, MS m/z 941.4 (M)+; HRMS m/z 941.2151 (M)+ (941.2141
1H), 7.88–7.96 (m, 3H), 8.31 (d, 1H, J = 9.4 Hz), 8.34 (d, 1H, J = calculated).
8.7 Hz), 8.45 (d, 1H, J = 8.7 Hz); δF (470 MHz, CD3CN) −81.80
Compound 8c. Yield = 40 mg (72%). δH (500 MHz,
(t, 3F, J = 9.7 Hz), −114.10 (m, 2F), −124.35 (m, 2F), −126.52 CD3COOD) 2.67 (s, 6H), 2.69–2.78 (m, 2H), 2.80–2.92 (m, 2H),
(m, 2F). MALDI-TOF MS m/z 733.3 (M)+; HRMS m/z 733.2347 3.00–3.20 (m, 2H), 3.43–3.70 (m, 8H), 3.92–4.20 (m, 4 H), 4.62
(M)+ (733.2324 calculated).
(m, 2H), 5.98 (m, 2H), 7.88 (m, 1H), 8.12 (s, 2H), 8.26 (m, 1H),
Compounds 7b and 7c were synthesized similarly.
8.40 (m, 1H), 8.61 (m, 1H), 8.75 (d, 1H, J = 8.6 Hz), 9.09 (d, 1H,
Compound 7b. Yield = 25 mg (63%). δH (500 MHz, CD3CN) J = 9.3 Hz), 9.19 (m, 1H); δF (470 MHz, CD3COOD) −81.67 (t,
2.31 (m, 2H), 2.49 (s, 6H), 2.72–2.86 (m, 2H), 3.11 (s, 6H), 3.55 3F, J = 10.1 Hz), −113.79 (m, 2F), −122.12 (m, 2F), −122.36 (m,
(m, 2H), 3.65 (m, 2H), 3.90 (s, 3H), 4.26 (t, 2H, J = 5.6 Hz), 4 F), −123.22 (m, 4F), −126.69 (m, 2F). MALDI-TOF MS m/z
7.59–7.64 (m, 2H), 7.79 (m, 1H), 7.90 (m, 1H), 7.92 (s, 2H), 8.28 1041.2 (M)+; HRMS m/z 1041.2089 (M)+ (1041.2077 calculated).
(m, 1H), 8.31 (m, 1H), 8.45 (m, 1H); δF (470 MHz, CD3CN)
Compound 9a. Compound 8a (54 mg, 0.062 mmol) dis-
−81.54 (t, 3F, J = 9.8 Hz), −113.84 (m, 2 F), −122.31 (m, 2F), solved in DMF (2 mL) was treated with DIPEA (0.016 mL,
−123.37 (m, 4F), −126.62 (m, 2F). MALDI-TOF MS m/z 833.3 0.092 mmol) and TSTU (20 mg, 0.068 mmol). The reaction was
(M)+; HRMS m/z 833.2259 (M)+ (833.2260 calculated).
stirred at room temperature for 30 minutes and then this solu-
Compound 7c. Yield = 81 mg (73%). δH (500 MHz, CD3CN) tion was added drop wise to an ice cold, stirred solution of iv
2.31 (m, 2H), 2.48 (s, 6H), 2.70–2.86 (m, 2H), 3.11 (s, 6H), 3.55 (133 mg, 0.31 mmol, HBr salt) dissolved in 0.5 M sodium
(m, 2H), 3.65 (m, 2H), 3.90 (s, 3H), 4.25 (t, 2H, J = 5.7 Hz), bicarbonate (2 mL). The reaction was stirred at 0 °C for
7.57–7.63 (m, 2H), 7.77 (m, 1H), 7.88 (m, 1H), 7.92 (m, 2H), 2 hours. The crude product was purified by a preparative
8.25 (m, 1H), 8.28 (m, 1H), 8.44 (m, 1H); δF (470 MHz, CD3CN) HPLC as described earlier using a 40 minute gradient of 10 →
−81.52 (m, 3F), −113.86 (m, 2F), −122.12 (m, 2F), −122.32 (m, 60 MeCN–water (each with 0.05% TFA). The HPLC fractions
4F), −123.15 (m, 2F), −123.38 (m, 2F), −126.58 (m, 2F). MAL- containing product 9a were concentrated under reduced
DI-TOF MS m/z 933.4 (M)+; HRMS m/z 933.2164 (M)+ (933.2196 pressure. Yield = 38 mg (55%). δH (500 MHz, CD3COOD)
calculated).
2.27–2.38 (m, 2H), 2.47 (m, 4H), 2.58 (s, 6H), 2.70 (m, 2H), 2.82
Compound 8a. Compound 7a (100 mg, 0.118 mmol), 2,6-di- (m, 2H), 2.99–3.24 (m, 4H), 3.26 (s, 3H), 3.38 (m, 2H), 3.48 (s,
tert-butylpyridine (0.196 mL, 0.886 mmol) and 1,3-propane 6H), 3.52–3.85 (m, 10H), 3.98 (m, 4H), 4.49 (m, 2H), 5.93 (m,
sultone (216 mg, 1.772 mmol) were added to [BMIM][BF4] 2H), 7.73 (d, 1H, J = 2.7 Hz), 7.88 (s, 2H), 8.25 (m, 1H), 8.33
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(dd, 1H, J = 10.0 Hz, 2.7 Hz), 8.58 (m, 1H), 8.70 (m, 1H), 9.04 (m, 2H), 3.23 (s, 3H), 3.49 (s, 6H), 3.51–3.90 (m, 8H), 3.95–4.08
(d, 1H, J = 9.4 Hz), 9.14 (d, 1H, J = 10.1 Hz); δF (470 MHz, (m, 4H), 4.51 (t, 2H, J = 5.6 Hz), 5.97 (m, 2H), 7.77 (d, 1H, J =
CD3COOD) −81.93 (m, 3F), −114.06 (m, 2F), −124.31 (m, 2F), 2.7 Hz), 7.91 (s, 2H), 8.28 (dd, 1H, J = 8.8 Hz, 6.7 Hz), 8.35 (dd,
−126.61 (m, 2F). MALDI-TOF MS m/z 1090.5 (M)+; HRMS m/z 1H, J = 10.0 Hz, 2.7 Hz), 8.61 (m, 1H), 8.76 (dd, 1H, J = 8.7 Hz,
1090.3828 (M)+ (1090.3716 calculated).
1.4 Hz), 9.07 (d, 1H, J = 9.5 Hz), 9.18 (d, 1H, J = 10.1 Hz);
Compounds 9b and 9c were synthesized similarly.
δF (470 MHz, CD3COOD) −81.67 (m, 3F), −113.84 (m, 2F),
Compound 9b. Yield
= 20 mg (65%). δH (500 MHz, −122.37 (m, 2F), −123.36 (m, 4F), −126.69 (m, 2F). MALDI-TOF
CD3COOD) 2.27–2.38 (m, 2H), 2.49 (m, 4H), 2.63 (s, 6H), 2.73 MS m/z 1402.9 (M + H)+; HRMS m/z 1401.4303 (M)+ (1401.4133
(m, 2H), 2.85 (m, 2H), 3.04–3.13 (m, 2H), 3.17 (m, 2H), 3.27 (s, calculated).
3H), 3.37 (m, 2H), 3.49 (s, 6H), 3.55–3.90 (m, 10H), 3.96–4.05
Compound 10c. Yield = 3 mg (50%). δH (500 MHz,
(m, 4H), 4.52 (m, 2 H), 5.97 (m, 2H), 7.78 (d, 1H, J = 2.7 Hz), CD3COOD) 2.26–2.34 (m, 2H), 2.42–2.61 (m, 6H), 2.63 (s, 6H),
7.92 (s, 2H), 8.28 (dd, 1H, J = 8.7 Hz, 6.7 Hz), 8.35 (dd, 1H, J = 2.67–2.74 (m, 2H), 2.78–2.90 (m, 6H), 2.95–3.12 (m, 10 H), 3.19
10.0 Hz, 2.7 Hz), 8.61 (m, 1H), 8.78 (d, 1H, J = 8.7 Hz), 9.08 (d, (m, 2H), 3.22 (s, 3H), 3.50 (s, 6H), 3.51–3.90 (m, 8H), 3.95–4.08
1H, J = 9.4 Hz), 9.19 (d, 1H, J = 10.1 Hz); δF (470 MHz, (m, 4H), 4.52 (m, 2H), 5.98 (m, 2H), 7.79 (d, 1H, J = 2.7 Hz),
CD3COOD) −81.69 (m, 3F), −113.90 (m, 2F), −122.39 (m, 2F), 7.91 (s, 2H), 8.28 (dd, 1H, J = 8.7 Hz, 6.7 Hz), 8.35 (dd, 1H, J =
−123.39 (m, 4F), −126.73 (m, 2F). MALDI-TOF MS m/z 10.0 Hz, 2.7 Hz), 8.61 (m, 1H), 8.79 (d, 1H, J = 8.5 Hz), 9.07 (d,
1191.5 (M + H)+; HRMS m/z 1190.3691 (M)+ (1190.3652 1H, J = 9.4 Hz), 9.19 (d, 1H, J = 10.1 Hz); δF (470 MHz,
calculated).
CD3COOD) −81.66 (t, 3F, J = 10.1 Hz), −113.83 (m, 2F),
Compound 9c. Yield
=
4
mg (14%). δH (500 MHz, −122.13 (m, 2F), −122.37 (m, 4F), −123.25 (m, 4F), −126.69
CD3COOD) 2.27–2.38 (m, 2H), 2.48 (m, 4H), 2.62 (s, 6H), 2.72 (m, 2F). MALDI-TOF MS m/z 1501.9 (M)+; HRMS m/z 1501.4374
(m, 2H), 2.85 (m, 2H), 3.02–3.21 (m, 4H), 3.27 (s, 3H), 3.38 (m, (M)+ (1501.4069 calculated).
2H), 3.49 (s, 6H), 3.55–3.92 (m, 10H), 3.96–4.08 (m, 4H), 4.51
(m, 2H), 5.97 (m, 2H), 7.77 (d, 1H, J = 2.7 Hz), 7.91 (s, 2H),
8.28 (m, 1H), 8.35 (m, 1H), 8.61 (m, 1H), 8.77 (d, 1H, J =
8.6 Hz), 9.07 (d, 1H, J = 9.4 Hz), 9.19 (d, 1H, J = 10.1 Hz);
δF (470 MHz, CD3COOD) −81.67 (t, 3F, J = 10.1 Hz), −113.83
Acknowledgements
(m, 2F), −122.13 (m, 2F), −122.35 (m, 4F), −123.25 (m, 4F), We thank Dr Shenliang Wang for assistance in emission
−126.68 (m, 2F). MALDI-TOF MS m/z 1290.5 (M)+; HRMS m/z spectra measurements.
1290.3627 (M)+ (1290.3588 calculated).
Compound 10a. Compound 9a (20 mg, 0.018 mmol) was
dissolved in methanol (2 mL) and DIPEA (0.031 mL,
0.178 mmol) and glutaric anhydride (10 mg, 0.089 mmol) were
added. The reaction was stirred at room temperature for
References
1 hour and was then concentrated under reduced pressure.
The residue was dissolved in DMF (2 mL) and DIPEA
(0.062 mL, 0.355 mmol) and TSTU (107 mg, 0.355 mmol) were
added. The reaction was stirred at room temperature for
1 hour. The crude reaction mixture was purified by preparative
HPLC as described earlier using a 40 minute gradient of 10 →
60% MeCN–water (each with 0.05% TFA). The HPLC fractions
containing product 10a were frozen at −80 °C and lyophilized
to dryness. Yield = 22 mg (93%). δH (500 MHz, CD3COOD)
2.27–2.38 (m, 2H), 2.42–2.57 (m, 6H), 2.63 (s, 6H), 2.67–2.78
(m, 2H), 2.80 (m, 2H), 2.84 (m, 4H), 2.96–3.12 (m, 10H), 3.17
(m, 2H), 3.23 (s, 3H), 3.50 (s, 6H), 3.51–3.90 (m, 8H), 3.95–4.08
(m, 4H), 4.52 (m, 2H), 5.98 (m, 2H), 7.78 (d, 1H, J = 2.7 Hz),
7.93 (s, 2H), 8.29 (m, 1H), 8.36 (dd, 1H, J = 9.9 Hz, 2.7 Hz),
8.61 (m, 1H), 8.77 (m, 1H), 9.08 (d, 1H, J = 9.4 Hz), 9.19 (d, 1H,
J = 10.1 Hz); δF (470 MHz, CD3COOD) −81.92 (t, 3F, J = 9.9 Hz),
−114.07 (m, 2F), −124.29 (m, 2F), −126.60 (m, 2F). MALDI-TOF
MS m/z 1301.7 (M)+; HRMS m/z 1301.4341 (M)+ (1301.4191
calculated).
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Compound 10b. Yield = 12 mg (68%). δH (500 MHz,
CD3COOD) 2.27–2.35 (m, 2H), 2.42–2.57 (m, 6H), 2.60 (s, 6H),
2.67–2.78 (m, 2H), 2.78–2.94 (m, 6H), 2.95–3.12 (m, 10H), 3.17
3900 | Org. Biomol. Chem., 2014, 12, 3887–3901
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