Low background FRET-substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions

Article abstract of DOI:10.1039/b601151a

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b601151a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Low background FRET-substrates for lipases and esterases suitable
for high-throughput screening under basic (pH 11) conditions†
Yongzheng Yang, Peter Babiak and Jean-Louis Reymond*
Received 24th January 2006, Accepted 16th February 2006
First published as an Advance Article on the web 3rd April 2006
DOI: 10.1039/b601151a
FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from
commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic
1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is
ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme
even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an
unprecedented and practical solution to the long-standing problem of a simple yet efficient
high-throughput screening tool for lipase activities under basic conditions.
are aliphatic esters, and as such they are remarkably resistant
Introduction
towards non-specific degradation. These monoacylated 1,2-diols
proved remarkably reactive towards lipases or esterases, but the
requirement for an oxidative step based on sodium periodate
proved problematic for many applications, in particular for testing
in media containing oxidation sensitive compounds such as
carbohydrates or amino alcohol buffers.
Herein we report fluorogenic substrates 1a–15a for lipases and
esterases having a similar 1,2-diol core, but which are based on
fluorescence resonance energy transfer (FRET) as a signalling
mechanism (Scheme 1). The substrates are highly reactive with
lipases and esterases, yet show essentially no background reaction
in the absence of enzyme. Substrates 1a–15a are prepared in four
steps from commercially available building blocks, which makes
them more readily available than the structurally more complex
FRET-type lipase substrates reported previously.14 In particular
substrate 2a shows a particularly high reactivity across a broad
range of enzymes while being essentially stable even at pH 11 in
the absence of an enzyme.
High-throughput screening (HTS) enzyme assays are essential
tools in biotechnology, in particular for enzyme discovery and
engineering.1 Many enzyme assays are based on fluorogenic
and chromogenic substrates. Although such assays may not be
desirable for directed evolution experiments, they are particularly
useful and popular as reporter assays for routine work and
discovery because they are generally adaptable to a range of
conditions and provide an operationally simple yet reliable test
for enzyme activity. Such assays ideally rely on substrates that
are chemically stable and react only with the target enzyme.
Substrate stability against non-specific degradation is particularly
problematic in assays for lipases and esterases, which is one of the
most important enzyme classes.2 The standard assays use aliphatic
esters of nitrophenol and umbelliferone, which show a high level
of non-specific degradation due to the strong leaving group ability
of the acidic phenol,3,4 although the problem is suppressed when
these substrates are adsorbed onto silicagel prior to the assay.5 The
resistance to non-specific degradation can be improved by using
acyloxymethyl ethers of umbelliferone,6 or acylated cyanohydrins,7
but these fluorogenic esters are still relatively unstable. HTS assays
are also possible with the much less reactive esters of aliphatic
alcohols such as tributyrin using indirect assays based on pH-
indicators,8 pH-titration,9 back-titration of released glycerol,10
or coupled enzymes,11 however the requirement for auxiliary
reagents renders the procedure inherently more complicated and
less flexible.
Results and discussion
Substrate design
FRET-signalling is possible in principle for any bond cleaving
enzyme. The method requires a double labeling of the substrate,
but leaves the reactivity of the enzyme-labile bond unchanged
and therefore allows the use of non-activated functional groups.
FRET-based enzyme activity detection has been used in doubly
labeled peptides for protease assays.15 Several FRET-substrates
have also been reported for lipases and phospholipases, all of
which are close structural analogs of triglycerides.14 However, an
exact structural match to naturally occurring substrates should not
be necessary to obtain lipase/esterase reactive FRET-substrates.
Indeed many highly reactive lipase probes are not close analogues
of natural substrates, with many changes possible on the alcohol
side of the ester. Nevertheless, the presence of an aliphatic acid
such as octanoic acid is generally necessary to obtain high lipase
reactivity, while esterases prefer butyrate esters.
We have recently reported fluorogenic lipase substrates based
on monoesters of 1,2-diols in which the fluorescence signal was
released by a selective oxidation of the 1,2-diol product with
sodium periodate,12 a strategy which is also suitable for releas-
ing volatile perfumes from stable precursors.13 Such substrates
Department of Chemistry & Biochemistry, University of Berne, Freies-
trasse 3, 3012 Berne, Switzerland. E-mail: jean-louis.reymond@ioc.unibe.ch;
Fax: +41 31 631 80 57
† Electronic supplementary information (ESI) available: Rate data for all
enzyme–substrate pairs tested; cocktail assay rate and HPLC traces; NMR
and MS spectra of all compounds. See DOI: 10.1039/b601151a
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Scheme 2 Synthesis of fluorogenic FRET substrates.
were obtained through protocol B in which the aliphatic amine
(methyl-, ethyl-, propyl-, or benzylamine) was first reacted with
0.5 equivalents of the alkenyl bromide in DMF in the presence
of sodium hydride, followed by the addition of DNP–Cl. After
aqueous work-up the crude products were then subjected to cat-
alytic dihydroxylation to give the 1,2-diols, which were separated
from the byproducts by column chromatography and isolated
in low to moderate yields. This procedure was used in various
combinations to give 1,2-diols 3b, 6b, 8b, 9b, 10b, 12b, 13b, 14b,
and 15b.
Scheme 1 FRET-fluorogenic substrates for lipases and esterases.
We set out to prepare FRET-signalling lipase/esterase sub-
strates using pyrenebutyric acid as a fluorescent label. This label
would be attached to a dinitrophenylamino group as a quencher by
mono-esterification to an enzyme reactive 1,2-diol. Variations of
the spacer in terms of branching, hydrophobicity and length were
envisioned to optimize the substrates towards enzyme reactivity.
Additional 1,2-diols incorporating an ethylene glycol unit as
a spacer were prepared starting with N-methylethanolamine.
In this case alkylation with the alkyl bromide involved ether
bond formation at the free hydroxyl group, yielding diols 2b, 4b
and 5b with allyl bromide, 5-bromopentene and 6-bromohexene,
respectively, under procedure B (Scheme 3). Finally, the ster-
ically more demanding secondary alcohol 11b was prepared
starting with 1-bromo-3-methyl-2-butene and propylamine using
protocol B.
The 15 different 1,2-diols 1b–15b were then converted to the
corresponding mono-esters by coupling with pyrenebutyric acid
(Scheme 2 and 3). A previously reported procedure for the
monoesterification of 1,2-diols involving the reaction with the acyl
halide in dichloromethane in the presence of pyridine or collidine
did not work in this case.10 We therefore turned to diimide-
promoted coupling. Esterification to the mono-esters was carried
out by the reaction of pyrenebutyric acid with N-ethyl-Nꢀ-(3-
dimethylaminopropyl)-carbodiimide (EDC) in dichloromethane
Synthesis
Aliphatic 1,2-diols are readily prepared by OsO4-catalyzed di-
hydroxylation of the corresponding alkenes, a reaction which is
compatible with the presence of chromophores and suitable for
the preparation of enzyme substrates.16 The preparation of DNP-
labeled aliphatic alkenes was carried out by the derivatization of
aliphatic primary amines with DNP chloride, followed by a second
alkylation at the nitrogen with allyl bromide. The crude alkenes
were then directly submitted to OsO4-catalyzed dihydroxylation
to yield the dinitrophenylamino-labeled aliphatic 1,2-diols, which
were purified by column chromatography. This procedure A
was used for the preparation of DNP-labeled alkenes 1b (from
methylamine), and 7b (from propylamine) (Scheme 2).
The N-alkylation of the DNP-labeled amine intermediate used
with allyl bromide did not proceed with other less reactive
alkenyl bromides, in particular 4-bromobutene, 5-bromopentene
and 6-bromohexene. In these cases the desired 1,2-diol products
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The initial apparent reaction rates were determined from the
initial slope of the fluorescence–time plots for each of the 540
substrate–enzyme pairs (Table S1†). The fastest reactions were
observed with Mucor miehei lipase (Roche Chiralzyme L8), Ther-
mocyces lanuginose lipase (Roche Chiralzyme L9) and Aspergillus
niger lipase (ANL, Fluka 62294). However seven enzyme samples
did not show any reaction with the substrates. The absence of
a reaction with these seven enzymes is probably caused by the
presence of a pyrenebutyric acid group because the same enzymes
reacted with fluorogenic lipase probes consisting of similar 1,2-
diols esterified to octanoic acid.17
Cocktail assays
We have recently shown that cocktail assays based on HPLC-
analysis of reaction products give highly reproducible activity
fingerprints for enzymes,17,18 which can be used for functional
classification.19 Although substrates 1a–15a indeed reacted as a
cocktail and could be analyzed by HPLC, the reactivity patterns
resulting from this set of substrates were very similar between the
different enzymes, probably because the pyrenebutyric ester group
governs most of the enzyme–substrate interactions. Furthermore,
the same seven enzymes that did not react with individual
substrates also failed to react with the cocktail. Under these
unfavourable circumstances, the use of the cocktail for functional
classification of the enzymes was not investigated further.
Scheme 3 Synthesis of FRET-fluorogenic lipase substrates.
and pyridine as the solvent. The reaction produced the mono-
esters 1a–15a in moderate yields as the only products beside
unreacted 1,2-diols.
Enzyme assays
Substrates 1a–15a were conditioned as 2 mM stock solutions in
DMF. A series of 36 commercially available lipases and esterases
were prepared as 1 mg mL−1 stock solutions. The assays were
conducted at 25 ◦C in aqueous PBS buffer pH 7.4 containing
30% v/v DMSO to ensure complete solubility of the substrates,
using 50 lg mL−1 enzyme and 10 lM of each substrate. The assays
were followed by fluorescence (kex = 300 nm, kem = 376 nm) in a
microtiter plate reader instrument (Fig. 1). While the background
reaction in the absence of any enzyme was not measurable with
any of the substrates, almost all substrates reacted well with the
different enzymes, with the exception of the secondary ester 11b,
which only reacted with three enzymes.
Substrate ranking
The substrates were ranked according to their highest reactivities
to determine the best substrates of the series. For each enzyme, the
relative apparent reactivity of each substrate to the fastest reacting
substrate for the enzyme was determined, and the resulting
percentage values were summed across all enzymes tested. The
data was computed for the initial reaction rates under single
substrate assay conditions, as well as for the relative product
percentages as determined by HPLC analysis of the cocktail
reaction (primary data for all enzymes in Table S1†). Substrate 2a
stood out as the most reactive substrate in both analyses (Fig. 2).
The difference was particularly strong in terms of the initial
reaction rates, for which 2a was 40% more reactive than the second
most reactive substrate 5a. Interestingly, both of these substrates
contain an oxygen atom in the spacer, which renders the substrates
somewhat less hydrophobic. The particularly strong reactivity of
2a might reflect the fact this substrate is directly derived from
glycerol itself, which renders it structurally very similar to natural
triglycerides.
The reactivity of the most reactive substrate 2a against lipases
and esterases was investigated closer in the case of the most reactive
enzymes L8, L9 and ANL. The apparent reaction rate was found
to be independent of the substrate concentration above 10 lM
in all cases, implying that the KM values are much lower than
10 lM. Exact kinetics could not be carried out due to the poor
fluorescence signal at very low substrate concentrations. Tight
substrate binding is probably due to the strongly hydrophobic
nature of the substrates.
Fig. 1 Fluorescence assay of selected lipases and esterases with substrate
12a. Conditions: 10 lM 12a in aq. PBS pH 7.4 with 30% v/v DMSO,
50 lg ml−1 enzyme, 25 ◦C. 100 lL assays were run in 96-well polystyrene
plates. Relative fluorescence units (RFU) are reported with kem = 376 nm
(kex = 300 nm), and are linearly proportional to the concentration of
pyrenebutyric acid in the concentration rage 0–10 lM.
Variation of the pH towards basic conditions showed that
substrate 2a was completely stable towards non-specific hydrolysis,
with no apparent reaction even at pH 11 (Fig. 3). The substrate was
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Table 1 Screening of enzyme activities at pH 11. All enzymes from Table 1
were tested for the reaction with 2a (10 lM) in aq. 20 mM borate buffer
pH 11 containing 30% v/v DMSO, 25 ◦C. The reaction was followed by
fluorescence. Enzymes codes: L8: Roche Thermocyces lanuginose lipase;
CVL: Chromobacterium visc. lipoprotein lipase 2832 U mg−1 (Fluka
62333); MJL: Mucor javanicus lipase 4.9 U mg−1 (Fluka 62304); PSL:
Pseudomonas sp. lipoprotein lipase 1560 U mg−1 (Fluka 62335); E1: Roche
pig liver esterase; MML: Mucor miehei lipase 1.3 U mg−1 (Fluka 62298);
CLL: Candida lipolytica lipase 1.0 U g−1 (Fluka 62303); E2: Roche pig liver
esterase; HrLE: horse liver esterase 250 U mg−1 (Fluka 46058)
Enzyme
Concentration/lg ml−1
% conversion of 2a after 30 min
L8
10
10
1
75
61
41
41
36
34
33
30
29
CVL
MJL
PSL
E1
MML
CLL
E2
1
10
10
1
10
10
HrLE
Fig. 2 Cumulative relative reactivities of fluorogenic lipase substrates
against 29 enzymes. Black bars: sum of the relative initial rate values
for 29 enzyme measurements each under the conditions of Fig. 1. Grey
bars: sum of the relative product abundance values determined by HPLC
after 2 h of reaction (conditions see Fig. S1 and S2†). In each series of
15 measurements with substrates 1a–15a and one enzyme, the substrate
giving the fastest initial rate or the most abundant product receives a value
of 1, and the other substrates receive the ratio of their initial rates or
product abundance to those for the fastest substrate. The rate and product
percentages for each enzyme–substrate pair are given in Table S1†.
an aliphatic 1,2-diol monoacylated with pyrenebutyric acid as
a fluorophore. The substrates showed negligible background
reactivity and a strong and specific reaction with lipases and
esterases. Several enzymes did not show any reaction with these
substrates, preventing the use of this substrate series as a general
functional classification tool. Substrate 2a exhibited the strongest
reactivity towards most enzymes and was also stable at alkaline
pH. Although the assay does not respond with some of the
enzymes and is therefore not completely general, substrate 2a
and its analogs offer an unprecedented and practical solution
to the long-standing problem of an efficient reporter assay for
lipase activity under basic conditions suitable for high-throughput
screening.
Experimental
All reagents were either purchased from Aldrich or Fluka or syn-
thesized following literature procedures. Chromatography (flash)
was performed with Merck silicagel 60 (0.040–0.063 mm). DMF
and THF were dried, solvents were distilled from technical sol-
vents. All reactions were followed by TLC on Alugram SIL UV254
silica gel sheets (Macherey-Nagel) with detection by UV. NMR
spectra were recorded on a BRUKER AC 300 for 1H (300 MHz)
or 13C (75 MHz) measurements. Mass spectra were provided by the
“Service of Mass Spectrometry” of the Department of Chemistry
and Biochemistry.
Fig. 3 Fluorescence assay of lipase L8 (Roche Thermocyces lanuginose
lipase) with pyrene ester 2a at different pH values. Conditions: 10 lM 2a,
10 lg ml−1 lipase in 20 mM PBS buffer pH = 7.24, 20 mM borate buffer
pH = 9.22 and 11.0 with 30% v/v DMSO, 25 ◦C, see also the conditions
in the Experimental section.
Preparation of N-alkyl-2,4-dinitroanilines
2,4-Dinitrophenylchloride (2.3 g, 11.4 mmol) was added to
a solution of the amine in MeOH (2 ml, 1 eq.) and N,N-
diisopropylethylamine (2 ml, 1.2 g, 1 eq.) in CH2Cl2 (20 mL) and
the reaction was stirred for 4 h at 25 ◦C. The precipitate was filtered
off and washed with CH2Cl2, and the filtrate was concentrated to
yield N-methyl-2,4-dinitroaniline (1.58 g, 70%, 8 mmol) or N-
propyl-2,4-dinitroaniline (1.7 g, 66%, 7.5 mmol).
therefore used to screen the activity of the different enzymes under
these strongly alkaline conditions. Nine of the 36 enzymes tested
indeed showed good reactivity with substrate 2a at pH 11 (Table 1).
Enzymes that are inactive are probably denatured at that pH.
3-[N-Methyl-N-(2,4-dinitrophenyl)amino]propane-1,2-diol (1b).
N-Methyl-2,4-dinitroaniline (395 mg, 2 mmol) was dissolved in
7 mL DMF and treated with sodium hydride (55% suspension in
oil, 167 mg, 4 mmol) and allyl bromide (484 mg, 0.35 mL, 4 mmol)
Conclusion
FRET-substrates 1a–15a for lipases and esterases were prepared
featuring a dinitrophenylamino group as a quencher linked to
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◦
and the reaction was stirred at 70 C for 14 h. The reaction was
N-methylethanolamine (300 mg, 4 mmol), 5-bromopent-1-ene
(0.47 mL, 596 mg, 4 mmol), NaH (55% suspension in oil, 335 mg,
8 mmol) and dinitrophenyl chloride (808 mg, 8 mmol), to yield
diol 4b (148 mg, 0.43 mmol, 11%) as a yellow oil, Rf = 0.47
diluted with ethyl acetate (50 mL), washed with water (2 × 30 mL)
and brine (30 mL), dried over MgSO4, filtered and evaporated.
The residue was purified by FC (eluent: hexane–AcOEt 2 : 1). The
main fraction (Rf = 0.48) was concentrated to yield N-methyl-N-
allyl-2,4-dinitroaniline (412 mg, 1.73 mmol, 87%) as a dark yellow
oil. This product was dissolved in acetone (10 mL) and water
(4 mL) and treated with N-methylmorpholine oxide monohydrate
(282 mg, 2.1 mmol) and catalytic OsO4 (0.1 mL of a 2.5% solution
in tert-butanol). After completion of the reaction (12 h at 25 ◦C),
the reaction was diluted with ethyl acetate. Aqueous work-up as
above and purification by FC (CH2Cl2 + 5% MeOH, Rf = 0.44)
gave diol 1b (454 mg, 1.67 mmol, 84%) as a yellow oil. 1H NMR
(300 MHz, CDCl3): d = 8.69 (d, 1H, J = 2.64 Hz), 8.22 (dd, 1H,
J = 9.42, 2.64 Hz), 7.29 (d, 1H, J = 1.14 Hz), 4.08 (m, 1H), 3.79
(dd, 1H, J = 11.1, 3.57 Hz), 3.51 (m, 3H), 3.03 (s, 3H), 2.23 (s,
2H); 13C NMR (75 MHz, CDCl3): d = 149.5, 146.4, 127.7, 123.9,
123.5, 119.0, 69.1, 63.8, 56.2, 41.8; ESI-MS: calc. for C10H13N3O6
[M + H]+: 272.0882, found: 272.0880.
1
(CH2Cl2–MeOH 9 : 1). H NMR (300 MHz, CDCl3): d = 8.64
(d, 1H, J = 2.64 Hz), 8.16 (dd, 1H, J = 9.42, 2.64 Hz), 7.16 (d,
1H, J = 9.42 Hz), 3.56 (m, 6H), 3.37 (m, 3H), 2.99 (s, 3H), 2.20
(s, 2H), 1.53 (m, 2H), 1.34 (m, 2H); 13C NMR (75 MHz, CDCl3):
d = 160.6, 138.7, 127.5, 123.9, 118.3, 71.5 (J = 26.05 Hz), 68.9
(J = 45.26 Hz), 67.7, 66.7, 54.0, 40.5, 30.0, 25.8; ESI-MS: calc. for
C14H21N3O7 [M + H]+: 344.1457, found: 344.1461.
6-[2-(N-Methyl-2,4-dinitrophenyl)amino]ethoxyhexane-1,2-diol
(5b). The procedure for 4b was applied using 6-bromohexene
(0.53 mL, 652 mg, 4 mmol) to yield diol 5b (361 mg, 1.01 mmol,
25%) as a yellow oil, Rf = 0.48 (CH2Cl2–MeOH 9 : 1). 1H NMR
(300 MHz, CDCl3): d = 8.63 (d, 1H, J = 2.82 Hz), 8.15 (dd, 1H,
J = 9.42, 2.64 Hz), 7.17 (d, 1H, J = 9.60 Hz), 3.65 (m, 3H), 3.55
(m, 3H), 3.39 (m, 3H), 2.98 (s, 3H), 2.20 (s, 2H), 1.47 (m, 2H), 1.29
(m, 4H); 13C NMR (75 MHz, CDCl3): d = 149.2, 127.4, 126.2,
123.9, 118.3, 71.2 (J = 57.67 Hz), 66.7 (J = 66.98 Hz), 60.3, 54.0,
40.4, 32.7, 29.5, 22.1, 14.1; ESI-MS: calc. for C15H23N3O7 [M +
H]+: 358.1614, found: 358.1621.
3-[2-(N -Methyl-2,4-dinitrophenyl)amino]ethoxypropane-1,2-
diol (2b). N-Methylethanolamine (188 mg, 2.5 mmol), allyl bro-
mide (302 mg, 2.5 mmol) and 2,4-dinitrophenylchloride (505 mg,
2.5 mmol) were stirred in DMF (5 mL) with◦NaH (55% suspension
in oil, 230 mg, 5 mmol) for 8 hours at 25 C. Aqueous work-up
and FC gave 506 mg of a crude product which was oxidized as
above and purified by FC (CH2CH2–MeOH 9 : 1, Rf = 0.38) to
yield diol 2b (152 mg, 0.55 mmol, 22.4%) as a yellow oil. 1H NMR
(300 MHz, CDCl3): d = 8.64 (d, 1H, J = 2.64 Hz), 8.17 (dd, 1H,
J = 9.60, 2.82 Hz), 7.14 (d, 1H, J = 9.42 Hz), 3.73 (m, 3H), 3.59
(q, 3H, J = 9.99, 5.28 Hz), 3.49 (m, 3H), 2.99 (s, 3H), 2.47 (s, 2H);
13C NMR (75 MHz, CDCl3): d = 149.0, 134.2, 127.5, 123.9, 118.0,
72.6, 70.4, 68.2, 63.6, 53.7, 40.6; ESI-MS: calc. for C12H17N3O7
[M + H]+: 316.1144, found: 316.1056.
6-[2-(N-Methyl-2,4-dinitrophenyl)amino]hexane-1,2-diol (6b).
The procedure for 3b was applied using 6-bromohexene (0.27 mL,
326 mg, 2 mmol) to yield diol 6b (226 mg, 0.72 mmol, 36%) as
1
a yellow waxy solid, Rf = 0.47 (CH2Cl2–MeOH 9 : 1). H NMR
(300 MHz, CDCl3): d = 8.63 (d, 1H, J = 2.82 Hz), 8.14 (dd,
1H, J = 9.42, 2.64 Hz), 7.02 (d, 1H, J = 9.60 Hz), 3.67 (d, 1H,
J = 6.99 Hz), 3.60 (t, 1H, J = 3.0 Hz), 3.37 (q, 3H, J = 11.7,
7.35 Hz), 2.92 (s, 3H), 2.51 (s, 2H), 1.67 (ddd, 2H, J = 19.38,
12.24, 7.71 Hz), 1.37 (m, 4H); 13C NMR (75 MHz, CDCl3): d =
148.8, 136.1, 136.0, 127.6, 124.2, 117.2, 71.8, 66.6, 54.0, 40.5, 32.4,
26.8, 22.6; ESI-MS: calc. for C13H19N3O6 [M + H]+: 314.1352,
found: 314.1345.
5-[N-Methyl-N-(2,4-dinitrophenyl)amino]pentane-1,2-diol (3b).
Methylamine hydrochloride (67.5 mg, 4 mmol) was suspended in
7 mL DMF and treated with 5-bromopentene (298 mg, 0.24 mL,
2 mmol) and sodium hydride (55% suspension in oil, 334 mg,
8 mmol). After 24 h at 25 ◦C, 2,4-dinitrophenylchloride (808 mg,
8 mmol) was added and the reaction was stirred for an additional
3-[N-(2,4-Dinitrophenyl)-N-propylamino]propane-1,2-diol (7b).
The procedure for 3b was applied starting with propylamine
(0.5 mL, 354 mg, 6 mmol) and allyl bromide (0.26 mL, 363 mg,
3 mmol), and using t-BuOK (672 mg, 6 mmol) in 10 mL
dichloromethane, to yield diol 7b (321 mg, 1.07 mmol, 36%) as a
yellow oil, Rf = 0.56 (CH2Cl2–MeOH 9 : 1). 1H NMR (300 MHz,
CDCl3): d = 8.65 (d, 1H, J = 2.64 Hz), 8.21 (dd, 1H, J = 9.21,
2.64 Hz), 7.24 (d, 1H, J = 9.24 Hz), 3.95 (m, 1H), 3.73 (dd, 1H, J =
11.28, 3.57 Hz), 3.44 (m, 3H), 3.08 (m, 2H), 2.33 (s, 2H), 1.56 (ddd,
2H, J = 21.69, 14.55, 7.2 Hz), 0.81 (t, 3H, J = 7.35 Hz); 13C NMR
(75 MHz, CDCl3): d = 149.6, 140.0, 139.1, 127.9, 123.6, 121.0,
68.9, 63.8, 55.5, 53.4, 20.9, 11.1; ESI-MS: calc. for C12H17N3O6
[M + H]+: 300.1184, found: 300.1188.
◦
8 h at 25 C. The reaction was diluted with 50 mL ethyl acetate,
washed with water (2 × 30 mL) and brine (30 mL), dried (Na2SO4),
and concentrated. The residue was dissolved in acetone (10 mL)
and water (4 mL) and treated with N-methylmorpholine oxide
monohydrate (282 mg, 2.1 mmol) and catalytic OsO4 (0.1 mL of
a 2.5% solution in tert-butanol). After completion of the reaction
(12 h at 25 ◦C), the reaction was diluted with ethyl acetate and
worked up as above. Purification of the residue by FC (CH2Cl2–
MeOH 95 : 5) gave diol 3b (263 mg, 0.88 mmol, 44%) as a yellow oil,
Rf = 0.49 (CH2CH2–MeOH 9 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.63 (dd, 1H, J = 2.64, 1.68 Hz), 8.14 (ddd, 1H, J = 9.60,
2.82, 1.32 Hz), 7.04 (d, 1H, J = 9.60 Hz), 3.61 (m, 2H), 3.39 (q,
3H, J = 10.71, 7.32 Hz), 2.92 (s, 3H), 2.42 (bs, 2H), 1.75 (m, 2H),
1.40 (m, 2H); 13C NMR (75 MHz, CDCl3): d = 148.8, 136.4, 127.6,
124.1, 117.3, 71.5, 66.6, 53.9, 40.4, 29.6, 23.1; ESI-MS: calc. for
C12H17N3O6 [M + H]+: 300.1195, found: 300.1206.
4-[N-(2,4-Dinitrophenyl)-N-propylamino)butane-1,2-diol (8b).
The procedure for 3b was applied starting with propylamine
(0.14 mL, 94 mg, 1.6 mmol) and 4-bromobutene (0.08 mL, 108 mg,
0.8 mmol), to yield diol 8b (96 mg, 0.31 mmol, 38%) as a yellow oil,
1
Rf = 0.54 (CH2Cl2–MeOH 9 : 1). H NMR (300 MHz, CDCl3):
d = 8.59 (d, 1H, J = 2.8 Hz), 8.15 (dd, 1H, J = 9.4, 2.8 Hz), 7.12
(d, 1H, J = 9.4 Hz), 3.66 (dddd, 1H, J = 12.2, 10.7, 7.0, 3.4 Hz),
3.57 (dd, 1H, J = 11.1, 3.2 Hz), 3.37 (m, 3H), 3.15 (dddd, 2H,
J = 17.3, 14.1, 9.6, 7.4 Hz), 2.67 (s, 2H), 1.56 (m, 4H), 0.83 (t, 3H,
J = 7.5 Hz); 13C NMR (75 MHz, CDCl3): d = 148.7, 137.9, 137.2,
5-[2-(N -Methyl-2,4-dinitrophenyl)amino]ethoxypentane-1,2-
diol (4b). The procedure for 3b was applied starting with
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127.7, 123.9, 119.2, 69.6, 66.7, 54.6, 48.5, 30.3, 20.7, 11.2; ESI-MS:
d = 8.62 (d, 1H, J = 2.82 Hz), 8.16 (dd, 1H, J = 9.42, 2.64 Hz),
7.05 (d, 1H, J = 9.60 Hz), 3.64 (m, 1H), 3.60 (d, 1H, J = 3.03 Hz),
3.38 (dd, 1H, J = 10.92, 7.35 Hz), 3.20 (ddd, 4H, J = 19.59,
14.52, 7.35 Hz), 2.04 (s, 2H), 1.59 (m, 4H), 1.29 (m, 4H), 0.85 (t,
3H, J = 7.53 Hz); 13C NMR (75 MHz, CDCl3): d = 148.7, 137.0,
127.7, 124.0, 119.0, 71.9, 66.7, 54.1, 52.0, 32.6, 27.3, 22.8, 20.8,
11.2; ESI-MS: calc. for C15H23N3O6 [M + H]+: 342.1665, found:
342.1671.
calc. for C13H19N3O6 [M + H]+: 314.1352, found: 314.1340.
6-[N-Ethyl-N-(2,4-dinitrophenyl)amino]hexane-1,2-diol
(9b).
The procedure for 3b was applied starting with ethylamine
hydrochloride (246 mg, 3 mmol), and 6-bromohexene (489 mg,
3 mmol), to yield diol 9b (199 mg, 0.61 mmol, 20%) as a yellow oil,
1
Rf = 0.38 (CH2Cl2–MeOH 9 : 1). H NMR (300 MHz, CDCl3):
d = 8.61 (d, 1H, J = 2.85 Hz), 8.16 (dd, 1H, J = 9.42, 2.64 Hz),
7.04 (d, 1H, J = 9.60 Hz), 3.61 (m, 2H), 3.26 (m, 5H), 2.13 (s,
2H), 1.60 (m, 2H), 1.33 (m, 4H), 1.19 (t, 3H, J = 7.17 Hz); 13C
NMR (75 MHz, CDCl3): d = 148.2, 134.3, 127.5, 123.8, 118.7,
111.4, 71.8, 66.6, 51.1, 47.2, 32.4, 27.1, 22.7, 12.5; ESI-MS: calc.
for C14H21N3O6 [M + H]+: 328.1508, found: 328.1518.
5-[N-Benzyl-N-(2,4-dinitrophenyl)amino]pentane-1,2-diol (14b).
The procedure for 3b was applied starting with benzylamine
(0.44 mL, 429 mg, 4 mmol), 5-bromopentene (0.24 mL, 298 mg,
2 mmol) to yield diol 14b (111 mg, 0.30 mmol, 15%) as a yellow oil,
Rf = 0.42 (CH2Cl2–MeOH 9 : 1). 1H NMR (300 MHz, CDCl3): d =
8.66 (d, 1H, J = 2.85 Hz), 8.16 (dd, 1H, J = 9.42, 2.82 Hz), 7.28
(m, 3H), 7.18 (q, 2H, J = 3.78, 1.50 Hz), 7.09 (d, 1H, J = 9.42 Hz),
4.47 (s, 2H), 3.59 (m, 2H), 3.36 (dd, 1H, J = 10.74, 7.35 Hz), 3.27
(t, 2H, J = 7.35 Hz), 1.98 (s, 2H), 1.65 (m, 2H), 1.35 (m, 2H);
13C NMR (75 MHz, CDCl3): d = 139.4, 135.4, 128.9, 128.1, 127.7,
127.5, 123.7, 119.6, 71.5, 66.6, 56.2, 52.3, 29.7, 23.4; ESI-MS: calc.
for C18H21N3O6 [M + H]+: 376.1508, found: 376.1523.
5-[N-(2,4-Dinitrophenyl)-N-propylamino]pentane-1,2-diol (10b).
The procedure for 3b was applied starting with propylamine
(0.33 mL, 236 mg, 4 mmol) and 5-bromopentene (0.24 mL, 298 mg,
2 mmol) to yield diol 10b (256 mg, 0.78 mmol, 39%) as a yellow oil,
1
Rf = 0.48 (CH2Cl2–MeOH 9 : 1). H NMR (300 MHz, CDCl3):
d = 8.62 (d, 1H, J = 2.82 Hz), 8.17 (dd, 1H, J = 9.60, 2.82 Hz),
7.07 (d, 1H, J = 9.60 Hz), 3.60 (m, 2H), 3.38 (dd, 1H, J = 10.74,
7.35 Hz), 3.30 (t, 2H, J = 7.35 Hz), 3.20 (t, 2H, J = 7.35 Hz),
2.08 (s, 2H), 1.57 (m, 4H), 1.32 (m, 2H), 0.83 (t, 3H, J = 7.35 Hz);
13C NMR (75 MHz, CDCl3): d = 148.7, 140.4, 127.6, 123.9, 119.0,
71.5, 66.7, 54.0, 51.9, 29.8, 23.5, 20.7, 11.2; ESI-MS: calc. for
C14H21N3O6 [M + H]+: 328.1508, found: 328.1511.
6-[N-Benzyl-N-(2,4-dinitrophenyl)amino]hexane-1,2-diol (15b).
The procedure for 3b was applied starting with benzylamine
(0.19 mL, 184 mg, 1.72 mmol) and 6-bromohexene (0.10 mL,
128 mg, 0.86 mmol) to yield diol 15b (115 mg, 0.30 mmol, 35%) as
a yellow oil, Rf = 0.46 (CH2Cl2–MeOH 9 : 1). 1H NMR (300 MHz,
CDCl3): d = 8.61 (d, 1H, J = 2.82 Hz), 8.12 (dd, 1H, J = 9.39,
2.61 Hz), 7.23 (m, 3H), 7.18 (dd, 2H, J = 6.21, 4.14 Hz), 7.07 (d,
1H, J = 9.60 Hz), 4.46 (s, 2H), 3.60 (q, 1H, J = 5.10, 2.82 Hz), 3.54
(dd, 1H, J = 11.13, 3.03 Hz), 3.32 (dd, 1H, J = 10.92, 7.53 Hz),
3.22 (t, 2H, J = 7.35 Hz), 2.86 (s, 2H), 1.59 (m, 2H), 1.24 (m, 4H);
13C NMR (75 MHz, CDCl3): d = 148.7, 137.8, 137.7, 135.3, 128.8,
127.9, 127.5, 127.4, 123.6, 119.5, 71.7, 66.5, 56.1, 52.4, 32.2, 27.0,
22.5; ESI-MS: calc. for C19H23N3O6 [M + H]+: 390.1665, found:
390.1672.
1-[N-(2,4-Dinitrophenyl)-N-propylamino]-3-methylbutane-2,3-
diol (11b). The procedure for 1b was applied starting with
N-propyl-2,4-dinitroaniline (460 mg, 2 mmol) and 1-bromo-3-
methyl-2-butene (0.4 mL, 596 mg, 4 mmol) to yield diol 11b
(184 mg, 0.56 mmol, 27%) as a yellow liquid, Rf = 0.64 (CH2Cl2–
1
MeOH 9 : 1). H NMR (300 MHz, CDCl3): d = 8.65 (d, 1H,
J = 2.64 Hz), 8.22 (dd, 1H, J = 9.42, 2.82 Hz), 7.23 (d, 1H, J =
9.39 Hz), 3.63 (dddd, 2H, J = 10.74, 6.60, 4.14, 2.46 Hz), 3.34 (dd,
1H, J = 14.49, 10.74 Hz), 3.04 (m, 2H), 1.55 (ddd, 2H, J = 21.63,
14.49, 7.14 Hz), 1.28 (s, 3H), 1.21 (s, 3H), 0.81 (t, 3H, J = 7.35 Hz);
13C NMR (75 MHz, CDCl3): d = 149.7, 138.8, 138.0, 127.7, 123.5,
121.1, 74.1, 71.5, 55.3, 52.5, 26.3, 24.4, 20.8, 11.0; ESI-MS: calc.
for C14H21N3O6 [M + H]+: 328.1508, found: 328.1501.
3-[N -Methyl-N -(2,4-dinitrophenyl)amino]-2-hydroxypropyl-
4-(pyren-1-yl)butyrate (1a). A solution of diol 1b (100 mg,
0.37 mmol) and pyrenebutyric acid (112 mg, 0.39 mmol) in CH2Cl2
(10 mL) and pyridine (1 mL) was treated with N-ethyl-Nꢀ-(3-
dimethylaminopropyl)carbodiimide (EDC) (115 mg, 0.74 mmol)
and 4-dimethylaminopyridine (90 mg, 0.74 mmol) and stirred at
room temperature for 15 h. The reaction was diluted with ethyl
acetate (150 mL), washed with aq. 1 N HCl (2 × 50 mL), sat.
aq. NaHCO3 (50 mL), and brine (50 mL). The organic phase was
concentrated and the residue was purified by FC (AcOEt–hexane
1 : 2) to yield ester 1a (68 mg, 0.13 mmol, 35%) as a yellow oil,
Rf = 0.31 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3): d =
8.54 (d, 1H, J = 3.42 Hz), 8.24 (d, 1H, J = 20.79 Hz), 8.15 (s, 1H),
8.12 (s, 1H), 8.10 (s, 1H), 8.09 (d, 1H, J = 2.85 Hz), 8.06 (d, 1H,
J = 4.14 Hz), 8.00 (s, 2H), 7.94 (q, 2H, J = 7.14, 5.64 Hz), 7.82 (d,
1H, J = 7.89 Hz), 6.92 (d, 1H, J = 9.60 Hz), 3.95 (m, 3H), 3.33
(dd, 2H, J = 14.49, 6.78 Hz), 3.30 (t, 2H, J = 2.28 Hz), 2.84 (s,
3H), 2.46 (t, 2H, J = 6.96 Hz), 2.04 (m, 2H); 13C NMR (75 MHz,
CDCl3): d = 173.4, 149.1, 137.3, 135.2, 131.3, 130.8, 130.0, 128.7,
127.4, 127.4, 127.3, 126.8, 125.9, 125.0, 124.9, 124.8, 123.6, 123.1,
118.6, 67.5, 65.6, 56.3, 41.5, 33.5, 32.5, 26.5; ESI-MS: calc. for
C30H27N3O7 M+: 541.184901, found: 541.1838.
3-[N-Benzyl-N-(2,4-dinitrophenyl)amino]propane-1,2-diol (12b).
The procedure for 3b was applied starting with benzylamine
(0.273 mL, 268 mg, 2.5 mmol) and allyl bromide (0.254 mL,
338 mg, 2.5 mmol) to yield diol 12b (511 mg, 1.47 mmol, 59%) as a
yellow oil, Rf = 0.45 (CH2Cl2–MeOH 9 : 1). 1H NMR (300 MHz,
CDCl3): d = 8.64 (d, 1H, J = 2.82 Hz), 8.18 (dd, 1H, J = 9.42,
2.64 Hz), 7.18 (m, 4H), 7.15 (dd, 2H, J = 5.28, 1.71 Hz), 4.36
(dd, 2H, J = 19.02, 15.24 Hz), 3.66 (m, 1H), 3.61 (dd, 1H, J =
11.28, 3.39 Hz), 3.41 (dd, 1H, J = 11.49, 5.46 Hz), 3.36 (d, 2H,
J = 6.42 Hz), 2.82 (s, 2H); 13C NMR (75 MHz, CDCl3): d =
149.6, 138.8, 138.3, 135.2, 128.9, 128.2, 127.8, 127.8, 123.5, 121.2,
69.2, 63.7, 57.4, 53.7; ESI-MS: calc. for C16H17N3O6Na: 370.1015,
found: 370.1005.
6-[N-(2,4-Dinitrophenyl)-N-propylamino]hexane-1,2-diol (13b).
The procedure with 3b was applied starting with propylamine
(0.33 mL, 236 mg, 4 mmol) and 6-bromohexene (0.3 mL, 362 mg,
2 mmol) to yield diol 13b (345 mg, 1.01 mmol, 51%) as a yellow oil,
1
Rf = 0.47 (CH2Cl2–MeOH 9 : 1). H NMR (300 MHz, CDCl3):
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3-[N-Methyl-N-(2,4-dinitrophenyl)amino]ethoxyl-2-hydroxypropyl-
4-(pyren-1-yl)butyrate (2a). The procedure for 2a was applied
starting with diol 2b (63 mg, 0.2 mmol) and pyrenebutyric acid
(60 mg, 0.21 mmol) to yield ester 2a (65 mg, 0.11 mmol, 55%) as a
yellow oil, Rf = 0.24 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz,
CDCl3): d = 8.59 (d, 1H, J = 2.64 Hz), 8.26 (d, 1H, J = 9.21 Hz),
8.17 (s, 1H), 8.14 (s, 1H), 8.11 (s, 1H), 8.06 (m, 2H), 8.01 (s, 2H),
7.95 (t, 1H, J = 7.74 Hz), 7.83 (d, 1H, J = 7.92 Hz), 6.97 (d, 1H,
J = 9.60 Hz), 3.98 (m, 3H), 3.84 (m, 1H), 3.60 (dd, 2H, J = 9.78,
4.89 Hz), 3.35 (m, 4H), 2.89 (s, 3H), 2.44 (t, 2H, J = 7.14 Hz), 2.04
(m, 2H); 13C NMR (75 MHz, CDCl3): d = 173.4, 148.9, 135.4,
131.3, 130.8, 129.9, 128.7, 127.4, 127.4, 127.3, 126.7, 125.8, 125.0,
124.9, 124.9, 124.8, 123.8, 123.2, 117.9, 72.0, 68.7, 68.2, 65.2,
53.6, 40.5, 33.6, 32.6, 26.6; ESI-MS: calc. for C32H31N3O8 M+:
586.2189, found: 586.2202.
8.17 (t, 1H, J = 1.14 Hz), 8.14 (t, 1H, J = 1.71 Hz), 8.11 (d, 1H, J =
0.75 Hz), 8.08 (dd, 2H, J = 2.82, 1.50 Hz), 8.02 (s, 2H), 7.96 (t, 1H,
J = 7.89 Hz), 7.84 (d, 1H, J = 7.71 Hz), 7.05 (d, 1H, J = 9.42 Hz),
4.18 (m, 1H), 4.08 (dd, 1H, J = 3.96, 0.96 Hz), 3.90 (dd, 1H, J =
11.31, 7.35 Hz), 3.74 (dd, 2H, J = 6.96, 2.82 Hz), 3.56 (q, 2H, J =
6.21, 1.68 Hz), 3.46 (q, 2H, J = 9.42, 4.92 Hz), 3.32 (m, 4H), 2.91
(s, 3H), 2.47 (t, 2H, J = 7.14 Hz), 2.16 (dd, 2H, J = 14.88, 7.35 Hz),
1.94 (bs, 1H), 1.38 (m, 4H); 13C NMR (75 MHz, CDCl3): d = 173.5,
149.0, 135.5, 131.3, 130.8, 129.9, 128.7, 127.4, 127.3, 127.3, 127.3,
126.7, 125.8, 124.8, 124.7, 124.7, 123.8, 123.2, 118.0, 71.1, 69.7,
68.5, 67.5, 53.9, 40.3, 33.6, 32.8, 32.6, 29.4, 26.7, 21.9; ESI-MS:
calc. for C35H37N3O8Na [M + Na]+: 650.2478, found: 650.2492.
6-[N -Methyl-N -(2,4-dinitrophenyl)amino]-2-hydroxyhexyl-4-
(pyren-1-yl)butyrate (6a). The procedure for 1a was applied
starting with 6b (94 mg, 0.3 mmol) and pyrenebutyric acid (91 mg,
0.32 mM) to yield ester 6a (101 mg, 0.17 mmol, 57%) as a yellow
oil, Rf = 0.40 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.57 (d, 1H, J = 2.82 Hz), 8.24 (d, 1H, J = 9.21 Hz), 8.15 (s,
1H), 8.12 (s, 1H), 8.09 (s, 1H), 8.06 (d, 1H, J = 1.32 Hz), 8.00 (s,
2H), 7.94 (dd, 1H, J = 8.10, 7.35 Hz), 7.81 (d, 1H, J = 7.92 Hz),
4.07 (dddd, 1H, J = 12.99, 12.31, 6.15, 3.21 Hz), 3.90 (dd, 1H,
J = 8.49, 7.17 Hz), 3.70 (m, 1H), 3.35 (t, 2H, J = 7.08 Hz), 3.19
(t, 2H, J = 7.35 Hz), 2.77 (s, 3H), 2.46 (t, 2H, J = 7.35 Hz),
2.16 (q, 2H, J = 12.99, 5.73 Hz), 2.05 (bs, 1H), 1.58 (t, 2H, J =
3.18 Hz), 1.38 (t, 2H, J = 5.41 Hz), 1.23 (t, 2H, J = 7.14 Hz);
13C NMR (75 MHz, CDCl3): d = 173.5, 148.5, 136.2, 135.4, 131.3,
130.8, 129.9, 128.6, 127.4, 127.4, 127.3, 126.7, 125.8, 125.0, 124.9,
124.8, 124.7, 124.0, 123.1, 117.0, 69.5, 68.4, 53.8, 40.3, 33.6, 32.6,
32.6, 26.6, 26.6, 22.4; ESI-MS: calc. for C33H33N3O7 [M + H]+:
584.2396, found: 584.2404.
5-[N-Methyl-N-(2,4-dinitrophenyl)amino]-2-hydroxypentyl-4-
(pyren-1-yl)butyrate (3a). The procedure for 1a was applied
starting with 3b (90 mg, 0.3 mmol) and pyrenebutyric acid (91 mg,
0.32 mmol) to yield ester 3a (96 mg, 0.17 mmol, 55%) as a yellow
oil, Rf = 0.18 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.60 (d, 1H, J = 2.82 Hz), 8.26 (d, 1H, J = 9.42 Hz), 8.16 (s,
1H), 8.14 (d, 1H, J = 1.14 Hz), 8.11 (s, 1H), 8.07 (q, 2H, J = 4.32,
2.82 Hz), 8.04 (s, 2H), 8.00 (t, 1H, J = 3.00 Hz), 7.95 (d, 1H, J =
7.53 Hz), 7.83 (d, 1H, J = 8.61 Hz), 6.88 (d, 1H, J = 9.63 Hz), 4.05
(dd, 1H, J = 11.1, 3.21 Hz), 3.89 (dd, 1H, J = 11.46, 7.14 Hz), 3.70
(m, 1H), 3.36 (t, 2H, J = 7.74 Hz), 3.26 (t, 2H, J = 7.32 Hz), 2.82
(s, 3H), 2.46 (t, 2H, J = 7.14 Hz), 2.16 (q, 2H, J = 14.1, 6.96 Hz),
1.63 (m, 2H), 1.35 (dddd, 2H, J = 12.06, 8.67, 3.21 Hz); 13C NMR
(75 MHz, CDCl3): d = 173.5, 148.7, 135.3, 131.3, 130.8, 130.0,
128.7, 127.5, 127.4, 127.4, 127.3, 126.7, 125.9, 125.0, 125.0, 124.9,
124.8, 124.0, 123.2, 117.2, 69.4, 68.4, 53.7, 40.4, 33.6, 32.6, 29.8,
26.6, 22.8; ESI-MS: calc. for C32H31N3O7Na [M + Na]+: 592.2059,
found: 592.2262.
3-[N-(2,4-Dinitrophenyl)-N-propylamino]-2-hydroxypropyl-4-
(pyren-1-yl)butyrate (7a). The procedure for 1a was applied
starting with 7b (90 mg, 0.3 mmol) and pyrenebutyric acid (91 mg,
0.32 mmol) to yield ester 7a (68 mg, 0.12 mmol, 40%) as a yellow
oil, Rf = 0.50 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.56 (d, 1H, J = 2.64 Hz), 8.24 (d, 1H, J = 9.21 Hz), 8.16 (s,
1H), 8.14 (s, 1H), 8.11 (d, 1H, J = 1.32 Hz), 8.05 (ddd, 2H, J =
9.42, 8.49, 2.82 Hz), 8.02 (s, 2H), 7.96 (dd, 1H, J = 8.28, 7.14 Hz),
7.82 (d, 1H, J = 7.71 Hz), 7.02 (d, 1H, J = 9.42 Hz), 4.20 (m, 1H),
4.13 (dd, 1H, J = 7.14, 3.75 Hz), 3.97 (dd, 2H, J = 13.95, 5.46 Hz),
3.24 (m, 4H), 3.02 (m, 2H), 2.68 (m, 1H), 2.46 (t, 2H, J = 7.14 Hz),
2.18 (t, 2H, J = 7.35 Hz), 1.49 (dd, 2H, J = 14.31, 7.14 Hz), 0.75
(t, 3H, J = 7.53 Hz); 13C NMR (75 MHz, CDCl3): d = 173.5,
149.2, 139.0, 138.3, 135.3, 133.6, 131.4, 130.8, 130.0, 128.7, 127.7,
127.5, 127.3, 126.8, 125.9, 125.1, 125.0, 124.9, 124.8, 123.4, 123.2,
120.8, 67.4, 65.6, 55.1, 53.7, 33.6, 32.5, 26.6, 20.8, 11.0; ESI-MS:
calc. for C32H31N3O7 M+: 569.216201, found: 569.2167.
5-[N-Methyl-N-(2,4-dinitrophenyl)amino]ethoxyl-2-hydroxypentyl-
4-(pyren-1-yl)butyrate (4a). The procedure for 1a was applied
starting with 4b (69 mg, 0.2 mmol) and pyrenebutyric acid (60 mg,
0.21 mmol) to yield ester 4a (58 mg, 0.10 mmol, 55%) as a yellow
oil, Rf = 0.30 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz, CDCl3):
d = 8.58 (d, 1H, J = 3.75 Hz), 8.28 (d, 1H, J = 9.21 Hz), 8.17 (t,
1H, J = 1.11 Hz), 8.14 (d, 1H, J = 1.68 Hz), 8.12 (s, 1H), 8.09
(s, 1H), 8.06 (t, 1H, J = 2.64 Hz), 8.02 (d, 2H, J = 1.53 Hz),
7.96 (t, 1H, J = 7.71 Hz), 7.85 (d, 1H, J = 7.74 Hz), 4.11 (m,
2H), 3.92 (dd, 1H, J = 11.28, 6.96 Hz), 3.72 (m, 1H), 3.59 (t, 2H,
J = 5.10 Hz), 3.43 (dd, 2H, J = 12.45, 7.35 Hz), 3.38 (t, 4H, J =
2.61 Hz), 2.90 (s, 3H), 2.45 (dd, 2H, J = 13.38, 6.21 Hz), 2.17
(q, 2H, J = 13.74, 6.39 Hz), 1.40 (m, 4H); 13C NMR (75 MHz,
CDCl3): d = 173.6, 149.0, 136.7, 135.6, 131.4, 130.9, 130.0, 128.7,
127.5, 127.4, 127.4, 127.4, 126.8, 125.9, 125.1, 125.0, 124.9, 124.8,
124.8, 123.8, 123.3, 118.0, 71.2, 69.6, 68.5, 67.6, 53.9, 40.2, 33.7,
32.7, 30.2, 26.8, 25.6; ESI-MS: calc. for C34H35N3O8Na [M +
Na]+: 614.2502, found: 614.2514.
4-[N -(2,4-Dinitrophenyl)-N -propylamino)-2-hydroxybutyl-4-
(pyren-1-yl)butyrate (8a). The procedure for 1a was applied
starting with 8b (63 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mmol) to yield ester 8a (66 mg, 0.11 mmol, 55%) as a yellow
oil, Rf = 0.50 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.56 (d, 1H, J = 2.82 Hz), 8.24 (d, 1H, J = 9.21 Hz), 8.16 (d,
1H, J = 1.29 Hz), 8.14 (d, 1H, J = 0.75 Hz), 8.10 (s, 1H), 8.05 (q,
2H, J = 8.28, 2.82 Hz), 8.01 (s, 2H), 7.96 (t, 1H, J = 7.71 Hz),
7.81 (d, 1H, J = 7.71 Hz), 6.94 (d, 1H, J = 9.30 Hz), 4.03 (q, 1H,
J = 11.31, 3.39 Hz), 3.90 (dd, 1H, J = 11.49, 6.78 Hz), 3.77 (s,
6-[N -Methyl-N -(2,4-dinitrophenyl)amino]ethoxyl-2-hydroxy-
hexyl-4-(pyren-1-yl)butyrate (5a). The procedure for 1a was
applied starting with 5a (71 mg, 0.2 mmol) and pyrenebutyric acid
(60 mg, 0.21 mmol) to yield ester 5a (91 mg, 0.15 mmol, 73%) as a
yellow oil, Rf = 0.38 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz,
CDCl3): d = 8.60 (d, 1H, J = 2.82 Hz), 8.27 (d, 1H, J = 9.21 Hz),
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1H), 3.34 (dddd, 4H, J = 17.34, 14.49, 9.99, 2.28 Hz), 3.06 (ddd,
2H, J = 9.24, 6.78, 1.53 Hz), 2.44 (t, 2H, J = 7.17 Hz), 2.32 (bs,
1H), 2.14 (m, 2H), 1.24 (m, 4H), 0.78 (t, 3H, J = 7.35 Hz); 13C
NMR (75 MHz, CDCl3): d = 173.5, 148.4, 137.9, 137.1, 135.3,
131.3, 130.8, 129.9, 128.7, 127.5, 127.4, 127.4, 127.3, 126.7, 125.9,
124.9, 124.7, 123.7, 123.1, 118.9, 68.3, 67.3, 54.5, 47.8, 33.5, 32.5,
30.5, 26.5, 20.5, 11.0; ESI-MS: calc. for C33H33N3O7 [M + H]+:
584.2396, found: 584.2414.
138.3, 137.6, 135.1, 131.3, 130.7, 129.9, 128.6, 127.4, 127.1, 126.7,
125.8, 125.0, 124.9, 124.8, 124.7, 123.6, 123.0, 119.6, 74.7, 71.4,
54.8, 51.2, 33.5, 32.5, 26.3, 26.1, 25.9, 20.6, 11.0; ESI-MS: calc. for
C34H35N3O7 [M + H]+: 598.2553, found: 598.2545.
3-[N -Benzyl-N -(2,4-dinitrophenyl)amino]-2-hydroxypropyl-4-
(pyren-1-yl)butyrate (12a). The procedure for 1a was applied
starting with 12b (70 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mmol) to yield ester 12a (59 mg, 0.10 mmol, 48%) as a yellow
oil, Rf = 0.32 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.60 (d, 1H, J = 2.82 Hz), 8.24 (d, 1H, J = 9.42 Hz), 8.18
(s, 1H), 8.15 (s, 1H), 8.11 (d, 1H, J = 2.55 Hz), 8.09 (d, 1H, J =
3.03 Hz), 8.06 (t, 1H, J = 2.64 Hz), 8.03 (s, 2H), 7.97 (dd, 1H, J =
8.10, 7.14 Hz), 7.82 (d, 1H, J = 7.92 Hz), 7.24 (dd, 3H, J = 3.57,
1.50 Hz), 7.08 (ddd, 3H, J = 9.24, 6.78, 5.10 Hz), 4.34 (d, 2H, J =
3.03 Hz), 4.04 (m, 2H), 3.94 (q, 1H, J = 12.81, 6.78 Hz), 3.34 (t, 1H,
J = 7.71 Hz), 3.26 (t, 2H, J = 6.96 Hz), 2.42 (t, 2H, J = 7.14 Hz),
2.12 (q, 2H, J = 14.70, 7.35 Hz); 13C NMR (75 MHz, CDCl3): d =
173.4, 149.4, 139.0, 138.6, 135.3, 135.2, 131.3, 130.8, 128.9, 128.2,
127.8, 127.7, 127.4, 127.4, 127.3, 126.7, 125.9, 125.0, 124.8, 123.4,
123.1, 121.2, 67.5, 65.5, 57.3, 53.9, 33.5, 32.5, 26.5; ESI-MS: calc.
for C36H31N3O7 [M + H]+: 618.2240, found: 618.2246.
6-[N -Ethyl-N -(2,4-dinitrophenyl)amino]-2-hydroxyhexyl-4-
(pyren-1-yl)butyrate (9a). The procedure with 1a was applied
starting with 9b (66 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mmol) to yield ester 9a (73 mg, 0.12 mmol, 61%) as a yellow
oil, Rf = 0.50 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz, CDCl3):
d = 8.55 (d, 1H, J = 2.82 Hz), 8.25 (d, 1H, J = 9.21 Hz), 8.15 (s,
1H), 8.12 (s, 1H), 8.09 (s, 1H), 8.07 (s, 1H), 8.02 (dd, 1H, J = 9.60,
2.82 Hz), 8.00 (s, 2H), 7.94 (t, 1H, J = 7.35 Hz), 7.81 (d, 1H, J =
7.71 Hz), 6.83 (d, 1H, J = 9.60 Hz), 4.12 (m, 1H), 3.85 (dd, 1H, J =
11.49, 7.14 Hz), 3.75 (m, 1H), 3.34 (t, 2H, J = 7.92 Hz), 3.12 (m,
4H), 2.45 (t, 2H, J = 7.17), 2.19 (t, 2H, J = 7.71 Hz), 2.11 (bs, 1H),
1.49 (dd, 2H, J = 12.06, 8.67 Hz), 1.40 (m, 3H), 1.21 (m, 1H), 1.07
(t, 3H, J = 7.14 Hz); 13C NMR (75 MHz, CDCl3): d = 173.5, 148.1,
137.5, 136.7, 135.4, 131.3, 130.8, 129.9, 128.6, 127.4, 127.4, 127.3,
126.7, 125.8, 125.0, 124.9, 124.9, 124.7, 124.7, 123.7, 123.2, 118.5,
69.5, 68.4, 51.1, 47.0, 33.6, 32.6, 32.6, 26.9, 26.6, 22.5, 12.4; ESI-
MS: calc. for C34H35N3O7 [M + H]+: 598.2553, found: 598.2543.
6-[N -(2,4-Dinitrophenyl)-N -propylamino)-2-hydroxyhexyl-4-
(pyren-1-yl)butyrate (13a). The procedure for 1a was applied
starting with 13b (102 mg, 0.3 mmol) and pyrenebutyric acid
(91 mg, 0.21 mmol) to yield ester 13a (75 mg, 0.12 mmol, 41%) as
a yellow oil, Rf = 0.15 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz,
CDCl3): d = 8.59 (d, 1H, J = 2.64 Hz), 8.26 (d, 1H, J = 9.21 Hz),
8.17 (s, 1H), 8.14 (d, 1H, J = 0.75 Hz), 8.11 (t, 2H, J = 0.93 Hz),
8.08 (1H, J = 2.61 Hz), 8.02 (s, 2H), 7.96 (t, 1H, J = 7.92 Hz), 7.83
(d, 1H, J = 7.74 Hz), 6.92 (d, 1H, J = 9.40 Hz), 4.06 (dd, 1H, J =
14.61, 3.21 Hz), 3.89 (dd, 1H, J = 11.49, 6.87 Hz), 3.69 (ddd, 1H,
J = 9.81, 6.78, 3.39 Hz), 3.36 (t, 2H, J = 7.92 Hz), 3.12 (q, 4H,
J = 15.06, 7.35 Hz), 2.46 (t, 2H, J = 7.14 Hz), 2.16 (ddd, 2H, J =
14.67, 7.35, 1.47 Hz), 1.51 (ddd, 4H, J = 21.66, 14.58, 4.87 Hz),
1.26 (m, 4H), 0.81 (t, 3H, J = 7.35 Hz); 13C NMR (75 MHz,
CDCl3): d = 173.5, 148.5, 140.0, 136.8, 135.4, 131.3, 130.8, 130.0,
128.9, 127.5, 127.4, 127.4, 127.3, 126.7, 125.8, 125.0, 124.9, 124.9,
124.8, 123.8, 123.2, 118.8, 69.6, 68.5, 53.9, 51.9, 33.6, 32.6, 32.6,
27.1, 26.6, 22.5, 20.6, 11.1; ESI-MS: calc. for C35H37N3O7 [M +
H]+: 612.2709, found: 612.2713.
5-[N -(2,4-Dinitrophenyl)-N -propylamino]-2-hydroxypentyl-4-
(pyren-1-yl)butyrate (10a). The procedure for 1a was applied
starting with 10b (66 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mM) to yield ester 10a (54 mg, 0.09 mmol, 45%) as a yellow
oil, Rf = 0.56 (hexane–AcOEt 1 : 2). 1H NMR (300 MHz, CDCl3):
d = 8.58 (d, 1H, J = 1.74 Hz), 8.26 (d, 1H, J = 9.24 Hz), 8.16 (t,
1H, J = 1.32 Hz), 8.14 (d, 1H, J = 1.32 Hz), 8.11 (s, 1H), 8.06
(dd, 2H, J = 9.42, 2.64 Hz), 8.02 (s, 2H), 7.95 (dd, 1H, J = 7.71,
7.36 Hz), 7.83 (d, 1H, J = 7.71 Hz), 6.91 (d, 1H, J = 9.39 Hz), 4.03
(dd, 1H, J = 8.58, 3.21 Hz), 3.88 (dd, 1H, J = 11.25, 7.14 Hz),
3.67 (dddd, 1H, J = 12.81, 10.47, 7.35, 3.39 Hz), 3.36 (t, 2H, J =
7.71 Hz), 3.19 (t, 2H, J = 7.32 Hz), 3.10 (t, 2H, J = 7.35 Hz), 2.42
(dd, 2H, J = 15.45, 8.10 Hz), 2.15 (m, 2H), 2.02 (bs, 1H), 1.49 (m,
4H), 1.32 (ddd, 2H, J = 11.85, 8.64, 3.00 Hz), 0.80 (t, 3H, J =
7.35 Hz); 13C NMR (75 MHz, CDCl3): d = 173.5, 148.5, 137.7,
136.9, 135.4, 131.3, 130.8, 129.9, 128.7, 127.5, 127.4, 127.4, 127.3,
126.7, 125.8, 125.0, 124.9, 124.9, 124.7, 123.8, 123.1, 118.8, 69.4,
68.4, 53.9, 51.6, 33.6, 32.6, 30.0, 26.6, 23.2, 20.5, 11.1; ESI-MS:
calc. for C34H35N3O7 [M + H]+: 598.2556, found: 598.2552.
5-[N -Benzyl-N -(2,4-dinitrophenyl)amino]-2-hydroxypentyl-4-
(pyren-1-yl)butyrate (14a). The procedure for 1a was applied
starting with 14b (75 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mmol) to yield ester 14a (77 mg, 0.12 mmol, 60%) as a yellow
oil, Rf = 0.44 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.63 (d, 1H, J = 2.82 Hz), 8.27 (d, 1H, J = 9.42 Hz), 8.17 (d,
1H, J = 1.32 Hz), 8.14 (d, 1H, J = 0.75 Hz), 8.12 (s, 1H), 8.07 (m,
2H), 8.02 (s, 2H), 7.96 (dd, 1H, J = 7.92, 7.35 Hz), 7.84 (d, 1H, J =
7.71 Hz), 7.26 (ddd, 3H, J = 8.85, 6.78, 4.89 Hz), 7.13 (q, 2H, J =
8.28, 1.31 Hz), 6.97 (d, 1H, J = 9.39 Hz), 4.38 (s, 2H), 4.03 (dd,
1H, J = 11.49, 3.39 Hz), 3.87 (dd, 1H, J = 11.46, 7.14 Hz), 3.71 (s,
1H), 3.37 (t, 2H, J = 7.71 Hz), 3.19 (t, 2H, J = 7.32 Hz), 2.45 (t,
2H, J = 7.14 Hz), 2.16 (ddd, 2H, J = 14.85, 6.72, 3.57 Hz), 1.61
(m, 2H), 1.34 (dd, 2H, J = 6.99, 3.03 Hz); 13C NMR (75 MHz,
CDCl3): d = 173.6, 148.8, 135.4, 135.4, 131.4, 130.9, 130.0, 128.9,
128.8, 128.1, 127.6, 127.6, 127.5, 127.4, 126.8, 125.9, 125.0, 125.0,
124.8, 123.7, 123.2, 119.6, 69.5, 68.4, 56.2, 52.2, 33.7, 32.7, 30.0,
1-[N-(2,4-Dinitrophenyl)-N-propylamino]-3-hydroxy-3-methyl-
butyl-4-(pyren-1-yl)butyrate (11a). The procedure for 1a was
applied starting with 11b (82 mg, 0.25 mmol) and pyrenebutyric
acid (144 mg, 0.50 mmol) to yield ester 11a (115 mg, 0.19 mmol,
1
76%) as a yellow oil, Rf = 0.77 (hexane–AcOEt 1 : 2). H NMR
(300 MHz, CDCl3): d = 8.60 (d, 1H, J = 2.64 Hz), 8.20 (d, 1H,
J = 6.42 Hz), 8.17 (dd, 1H, J = 4.14, 1.32 Hz), 8.14 (t, 1H, J =
1.11 Hz), 8.10 (d, 1H, J = 4.14 Hz), 8.04 (m, 2H), 8.02 (s, 2H),
7.96 (t, 1H, J = 7.53 Hz), 7.76 (d, 1H, J = 7.71 Hz), 6.97 (d,
1H, J = 9.42 Hz), 5.11 (d, 1H, J = 2.46 Hz), 5.07 (d, 1H, J =
2.43 Hz), 3.12 (m, 5H), 2.00 (m, 4H), 1.82 (bs, 1H), 1.51 (ddd,
2H, J = 21.84, 14.49, 7.17 Hz), 1.22 (s, 3H), 1.18 (s, 3H), 0.82 (t,
3H, J = 7.53 Hz); 13C NMR (75 MHz, CDCl3): d = 172.6, 148.8,
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26.6, 23.2; ESI-MS: calc. for C38H35N3O7 [M + H]+: 646.2553,
Acknowledgements
found: 646.2567.
This work was supported by the University of Berne, the Swiss
National Science Foundation, and Prote´us SA, Nˆımes, France.
6-[N -Benzyl-N -(2,4-dinitrophenyl)amino]-2-hydroxyhexyl-4-
(pyren-1-yl)butyrate (15a). The procedure for 1a was applied
starting with 15b (78 mg, 0.2 mmol) and pyrenebutyric acid (61 mg,
0.21 mmol) to yield ester 15a (80 mg, 0.12 mmol, 60%) as a yellow
oil, Rf = 0.44 (hexane–AcOEt 1 : 1). 1H NMR (300 MHz, CDCl3):
d = 8.62 (d, 1H, J = 2.82 Hz), 8.27 (d, 1H, J = 9.24 Hz), 8.16
(d, 1H, J = 1.32 Hz), 8.14 (d, 1H, J = 1.32 Hz), 8.11 (d, 1H, J =
0.75 Hz), 8.07 (dd, 2H, J = 5.46, 2.82 Hz), 8.02 (s, 2H), 7.96 (t,
1H, J = 7.53 Hz), 7.83 (d, 1H, J = 7.92 Hz), 7.28 (q, 3H, J =
3.84, 2.07 Hz), 7.14 (dd, 2H, J = 7.35, 1.50 Hz), 6.96 (d, 1H, J =
9.39 Hz), 4.40 (s, 2H), 4.04 (dd, 1H, J = 8.49, 6.95 Hz), 3.87 (dd,
1H, J = 11.49, 6.95 Hz), 3.69 (t, 2H, J = 2.82 Hz), 3.36 (t, 2H,
J = 7.89 Hz), 3.15 (t, 2H, J = 7.35 Hz), 2.46 (t, 2H, J = 6.84 Hz),
2.16 (q, 2H, J = 15.06, 7.71 Hz), 1.58 (d, 2H, J = 6.42 Hz), 1.31
(dd, 4H, J = 14.70, 9.24 Hz); 13C NMR (75 MHz, CDCl3): d =
173.5, 148.7, 137.5, 135.4, 131.3, 130.8, 130.0, 128.9, 128.7, 128.0,
127.5, 127.5, 127.4, 127.3, 127.1, 126.7, 125.8, 125.0, 124.9, 124.9,
124.8, 123.6, 123.2, 119.5, 69.5, 68.5, 60.3, 56.1, 52.4, 33.6, 32.6,
32.6, 27.0, 26.6, 22.5; ESI-MS: calc. for C39H37N3O7 [M + H]+:
660.2709, found: 660.2698.
References
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Fluorescence assay. Substrates 1a–15a were conditioned as
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substrate 5 lL of the 2 mM stock solution were diluted in 945 lL
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35 min: A : B = 0 : 100. The column was then washed for 20 min
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1754 | Org. Biomol. Chem., 2006, 4, 1746–1754
This journal is
The Royal Society of Chemistry 2006
©