The synthesis of two long-chain N-hydroxy amino coumarin compounds and their applications in the analysis of aldehydes

Article abstract of DOI:10.1039/c7ra02177a

Herein, two N-substituted coumaryl hydroxylamines (compounds 1a and 1b) with long aliphatic chains were prepared in 8 steps with a novel synthetic protocol. They served as derivatization agents for aldehydes by the formation of nitrones under mild conditions, which can be readily analysed by LC-MS with good chromatographic performance, excellent fluorescent response, and high ionization strength. We successfully used compounds 1a and 1b in the analysis of furfuraldehydes in raisins, based on the derivatization reactions of standard furfural (F), 5-methylfurfural (5-MF) and 5-(hydroxymethyl)furfural (5-HMF) samples. It proved that they are excellent agents for the analysis of aldehydes in foodstuffs. The derivatization reaction between compound 1a and hexanal suggested that the applications of our designed derivatization agents can be further extended to the analysis of aliphatic aldehydes. This study demonstrated that the designed N-substituted coumaryl hydroxylamines are excellent probes for the analysis of various aldehydes which are important in multiple research areas.

Full text of DOI:10.1039/c7ra02177a

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The synthesis of two long-chain N-hydroxy amino
coumarin compounds and their applications in the
analysis of aldehydes†
Cite this: RSC Adv., 2017, 7, 19707
Zhaobing Guan,‡ Manman Ding,‡ Yao Sun, Sisi Yu, Ao Zhang, Shuguang Xia,
Xiaosong Hu * and Yawei Lin*
Herein, two N-substituted coumaryl hydroxylamines (compounds 1a and 1b) with long aliphatic chains were
prepared in 8 steps with a novel synthetic protocol. They served as derivatization agents for aldehydes by
the formation of nitrones under mild conditions, which can be readily analysed by LC-MS with good
chromatographic performance, excellent fluorescent response, and high ionization strength. We
successfully used compounds 1a and 1b in the analysis of furfuraldehydes in raisins, based on the
derivatization reactions of standard furfural (F), 5-methylfurfural (5-MF) and 5-(hydroxymethyl)furfural (5-
HMF) samples. It proved that they are excellent agents for the analysis of aldehydes in foodstuffs. The
derivatization reaction between compound 1a and hexanal suggested that the applications of our
designed derivatization agents can be further extended to the analysis of aliphatic aldehydes. This study
demonstrated that the designed N-substituted coumaryl hydroxylamines are excellent probes for the
analysis of various aldehydes which are important in multiple research areas.
Received 21st February 2017
Accepted 28th March 2017
DOI: 10.1039/c7ra02177a
rsc.li/rsc-advances
consumer protection and quality control. Besides the points
mentioned above, certain aliphatic aldehydes are key
Introduction
1
–6
7–12
Aldehydes are widely found in foods,
beverages,
the biomarkers for lipid peroxidation in biological organisms,
and biological which has a major effect on a variety of diseases including heart
13–16
17,18
atmosphere,
systems.
environmental water
Concentrations of them have major impact on disease, diabetes mellitus, et al.
human health and quality of life. In the household building thus very helpful for the evaluation of these diseases.
materials industry, the concentrations of aldehydes in the
The combined liquid chromatography-mass spectrometry
materials are very important indexes of quality. Certain alde- (LC-MS) is one of the most widely used technique for the
1
9–24
22–24
The analysis of aldehydes is
13,22–24
hydes have been classied by the World Health Organization analysis of aldehydes.
WHO) as carcinogenic and teratogenic compounds. The to enhance ionization characteristics in LC-MS analysis of
concentration of aldehydes in the materials must be low enough aldehydes based on so ionization techniques such as electro-
Derivatization is primarily applied
(
16
for them to be qualied for home building. In the food spray (ESI) and atmospheric pressure chemical ionization
22
industry, concentrations of aldehydes play important roles in (APCI). This is because aldehydes and other analytes such as
the quality evaluation system. A low abundance of aldehydes sugars and steroids are poorly ionized by ESI or APCI. The
may provide the food with pleasant avour, while a high derivatization process usually involves an organic reaction of an
abundance of them will accelerate the corruption of food and analyte molecule with a suitable derivatization reagent. 2,4-
1
thus affect the freshness. Though aldehydes are minor Dinitrophenylhydrazine (DNPH) is the most widely used deriv-
7
,8,13,22,24
compounds in foodstuffs, they have drawn more and more atization agent for aldehydes.
It contains a reactive
1–5
attention in the detection and analysis of them. The existence functional group (hydrazine) which selectively reacts with
of certain aldehydes can serve as indicators of quality deterio- a complementary functional group (carbonyl) in the analytes.
2,5,6
ration, microbacterial fermentation and so on.
This makes DNPH derivatives of aldehydes are subjected to LC-MS analysis
the determination of aldehydes in foodstuffs critical for using an ultraviolet absorption detector, which can response to
the DNPH moiety of the derivatives.
Derivatization agents containing other reactive function
Department of Chemistry, School of Chemistry, Chemical Engineering and Life
groups, such as hydroxylamines, have also been used in the
Sciences, Wuhan University of Technology, Wuhan, 430070, P. R. China. E-mail:
xhu@whut.edu.cn; linyawei2012@whut.edu.cn
8,17,18,20
analysis of aldehydes.
They are used in various applica-
tions including chemical synthesis, drug discovery and chem-
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra02177a
ical biology, due to their high nucleophilicity and other specic
25
properties.
Both O- and N-substituted hydroxylamine
These authors contributed equally to this work.
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derivatives are widely used in these applications (Scheme 1). O-
Substituted hydroxylamine derivatives realize their applications
through functionalization with O-amino or aminooxy (–ONH2)
group, forming oximes in most situations. Oximes usually need
26
to be further reduced for derivatization study. The applica-
tions of N-substituted hydroxylamine derivatives are carried out
through functionalization with N-hydroxy amino (–NHOH)
group, usually forming nitrones as products. Though nitrones
can be further reduced, the analysis of aldehydes based on N-
substituted hydroxylamine can get satisfactory results without
reduction step. Furthermore, the reactivity of hydroxylamine
with aldehyde is high and the reaction only requires mild
condition. It thus has been chosen in this study as the reactive
function group in the derivatization agents for the aldehyde
analysis.
Scheme 2 Two novel designed long-chain coumaryl derivatives of
hydroxylamine.
26
Scheme 2), and used them in the structural analysis of alde-
hydes based on LC-MS analysis. The hydroxylamine group is
attached to the coumarin ring at 4-position and separated from
the coumarin ring by four carbon atoms. The coumarin ring is
Fluorescence spectroscopy is one of the most sensitive
optical detection technique used with high-performance liquid
chromatography (HPLC). In uorescence, the intensity of the
emission of the sample is usually measured above a low back-
ground level, which explains why uorescence techniques have
high sensitivity. Fluorescence of a molecule can occur naturally,
or it can be introduced by labelling the molecule with a uo-
30,31,35
assembled via Pechmann condensation,
which allows
derivatization at other positions on the coumarin ring, and
provides applicable method to further increase the chain length
between the coumarin and hydroxylamine moieties. Thus more
hydroxyamino coumarin derivatization agents with different
uorescent properties can be prepared and applied in aldehyde
analysis. These coumaryl hydroxylamine was N-substituted,
since it was more convenient for the derivatization as discussed
above.
27–29
rescent tag through derivatization reaction.
We therefore
incorporated uorophore into the designed derivative agents
with hydroxylamine moiety to facilitate the detection during
HPLC-MS analysis. The uorophore we chose is coumarin
group, which has been widely used as uorescent probe in
biological studies due to its minimal toxicity, high uorescence
quantum yield, relatively large Stokes shi, small size, ease to
Furfural (F), 5-methylfurfural (5-MF) and 5-(hydroxymethyl)
furfural (5-HMF) occur in many foodstuffs and avorants at
2,5,10,12
certain level.
The existences and concentrations of these
30–32
furfuraldehydes are relevant to food safety and avor, which
makes the detection and analysis of them quite important. In
this study, we focused on the analysis of them via derivatization
reaction with 1a and 1b. The LC-MS results proved they are
excellent derivatization agents for furfuraldehydes. Derivatiza-
tion reaction of hexanal with 1a indicated the application of
them can be extended to other aldehydes important in envi-
ronmental or biological studies.
synthesize, sensitivity to pH and solvent polarity.
An
unnatural amino acid bearing coumarin group was widely used
30,31
in a variety of biological applications.
Coumarin derivatives
containing hydrodxylamine functional group were used in the
0
33
selective detection of 5-formyl-2 -deoxycytidine in DNA, and
26
the structural analysis of glycans. In these coumaryl deriva-
tives of O- and N-substituted hydroxylamines, hydroxylamine
group is attached to the coumarin ring with a short aliphatic
carbon chain. Muddiman et al. found that increasing the length
of carbon chain in the alkyl tags made them more hydrophobic,
thus improved the chromatographic and electrospray response
Results and discussion
34
of the peptide labeled by the tags. Encouraged by this found- The synthesis of compound 1a and 1b is outlined in Scheme 3.
ing, we synthesized two novel designed long-chain coumaryl The coumarin ring with a 4-bromobutyl group at the 4-position
derivatives of hydroxylamine (compound 1a and 1b in (compound 7) was constructed via Pechmann condensa-
30,31,35,36
tion
between ethyl 7-bromo-3-oxoheptanoate (compound
5) and resorcinol or 3-methoxyphenol, using sulphuric acid as the
condensing agent (7a, 43.3% yield; 7b, 23.5% yield). Compound 7
was then hydrolyzed under reux in water to afford correspond-
ing alcohol 8, which was further oxidized to aldehyde 9 with
37
ꢀ
pyridinium chlorochromate (PCC) in acetone at 65 C. It then
reacted with hydroxylamine hydrochloride to form oxime 10,
which was then treated with sodium cyanoborohydride to afford
N-substituted coumaryl hydroxylamine 1 as the target compound.
Ethyl 7-bromo-3-oxoheptanoate 5 was a major intermediate
compound for the synthesis of the target compound. It was
synthesized in three steps, which started with the Reformatsky
38
reaction between cyclopentanone 2 and ethyl 2-bromoacetate
to form a tertiary alcohol (compound 3). The Reformatsky
Scheme 1 Derivatization of aldehyde using O- and N-substituted
hydroxylamine.
19708 | RSC Adv., 2017, 7, 19707–19716
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Scheme 3 The synthetic protocol for the derivatization agents 1a and 1b.
reaction was chosen to convert 2 into an alcohol, because the resorcinol and ethyl acetoacetate at similar condition. This
enolates formed are less reactive compared with lithium eno- clearly demonstrates that the substituent attached to the b-keto
lates or Grignard reagents, which prevents the occurrence of may have a great effect on the condensation result. The
nucleophilic addition to the ester group. Then under alkaline substitution of hydroxy group at 3-position in resorcinol with
39
bromination condition via retro-Reformatsky fragmentation,
methoxy group drastically decreased the yield of the conden-
the tertiary alcohol was converted efficiently into a,a,u-tri- sation reaction, suggesting that the reactivity of 3-methox-
bromo-b-ketoester (4, 95.3% yield). Compound 4 was reduced yphenol is greatly reduced compared with resorcinol.
using Cu–Zn alloy in saturated ammonium chloride methanol
solution at 10–15 C to afford compound 5 (64.4% yield).
The hydrolysis of compound 7 was straight forward, which
could be achieved smoothly in a sealed tube in a 115 C oil bath
ꢀ
ꢀ
Pechmann condensation was chosen to assemble the to afford the corresponding alcohols (8a, 93.0% yield; 8b, 56.4%
coumarin ring. It is an efficient and practical procedure for the yield). Compound 7b is less soluble in water compared with 7a,
synthesis of 4-substituted coumarins. The length of carbon since it has a methoxy group at 7-postion whereas 7a has a more
chain attached to the 4-the corresponding carbon chain which hydrophilic 7-hydroxy group on the coumarin ring. The less
is attached to u-bromo-b-ketoester. This make it relatively easy solubility of 7b might lead to the lower yield of hydrolysis
37
to adjust the length of the sidechain in the target compounds, product. PCC oxidation was then carried out to convert
ꢀ
which is important for the chromatographic performance and compound 8 to 9 in acetone at 65 C with sodium acetate and
electrospray response of the derived products. Furthermore, diatomite added. It was chosen because it can oxidize the
derivatives with substituents at the 5-, 6- or 8-position can also terminal primary alcohol to aldehyde with high selectivity and
be prepared with Pechmann condensation, some of which can no over-oxidation occurs to form carboxylic acid. Using
serve as potential useful probes with different functionalities or manganese dioxide as oxidant didn't provide comparable yields

uorescent properties for a variety of applications. The Pech- and were less efficient based on our test.
mann condensation used in this study were performed under Oxime 10 was prepared by reacting compound 9 with
mild condition, using sulphuric acid as the condensing agent at hydroxylamine hydrochloride in a mixture of tetrahydrofuran
26
13
ambient temperature. The yield is not very high (7a, 43.3% and water at r.t., while the pH value was adjusted to 4. The
C
yield; 7b, 23.5% yield) compared with condensation between NMR of 10 indicated that both Z- and E-isomer of oxime existed
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and one of them was predominating. To determine which
isomer is the major product needs further investigation. Finally,
oxime 10 was reduced to N-substituted hydroxylamine 1 using
sodium cyanoborohydride in a methanol solution of hydrogen
26
chloride at a pH value of 2–3. Aer TLC indicated the complete
consumption of the reactant, the solution was directly applied
to a RP-HPLC employing a semi-preparative C18 column to
afford 1 as a white solid (1a, 63.9% yield; 1b, 70.6% yield). The
1
13
purity was very high according to H and C NMR spectra.
Sodium cyanoborohydride was chosen as the reductant because
it can selectively reduce the oxime to N-substituted hydroxyl-
amine with a high efficiency at relatively low cost.
This is a relatively efficient protocol to synthesize the N-
substituted hydroxylamine as designed, which consists of 8
steps of reactions in total. Most steps are relatively straight
forward while some steps need more attention. The synthetic
protocol provided a practical way to synthesize N-substituted
coumaryl hydroxylamine with long aliphatic chain, and the
length of it can be varied when proper cyclic ketone with
different ring sizes are used as starting reagents. The Pechmann
condensation method used to install the coumarin moiety,
which makes it possible to introduce substituent groups at the
5-, 6- and 8-position of the coumarin ring. This provides the
possibility to introduce more functional groups at each position
for further derivatization and may improve the uorescent
properties of the designed probes.
The uorescent spectra of derivatization agent 1a and 1b
were shown in Fig. 1. The excitation and emission wavelengths
of compounds 1a are 322 nm and 447 nm, and those of 1b are
Fig. 1 Fluorescence spectra of compounds 1a (a) and 1b (b).
3
20 nm and 390 nm respectively. Stokes shi of 1b is 70 nm,
which is much smaller compared with that of 1a (125 nm). The
quantum yields of 1a and 1b were 0.61 and 0.72 respectively, corresponding nitrone derivative listed below. Mass spectrum
40
which were measured according to the literature using quin- in Fig. 3 is obtained from the peak showing up at 31 min in the
oline sulfate as standard. This result indicates that the modi- HPLC chromatogram in Fig. 2. It indicates that the major peak

cation of the substituents on the coumarin ring will affect the has an m/z value of 328.1180, which corresponds to the singly
uorescent property, which might be important in certain charged [M + H] . This conrms the successful derivatization of
+

investigations based on the derivatization reaction with these furfural with compound 1a to form the nitrone. The intensity of
compounds. The structure modication of the derivatization peak is pretty high, suggesting that the long aliphatic chain in
reagents can be realized via Pechmann condensation discussed the derivative can enhance the ionization strength in mass
previously.
spectroscopy. It also validates that no further reduction reaction
The derivatization reaction was initially tried between is needed in the aldehyde analysis. The HPLC detection limit,
compound 1a with furfural to test the feasibility of the aldehyde
analysis using the hydroxyamino coumarin compounds. Aer
ꢀ
compound 1a reacted with furfural at 70 C in an acetic acid/
sodium acetate buffer for 1 h, the mixture was analysis by
HPLC with a uorescence detector, the excitation and emission
wavelengths of which are set at 322 nm and 447 nm. The result
was compared with that of the sample blank of compound 1a in
an acetic acid/sodium acetate buffer, as shown in Fig. 2. A new
peak with a high intensity showed up around 31 min in the
chromatogram of the derivatization mixture. It suggested that
compound 1a might have reacted with furfural, forming the
nitrone derivative. This was conrmed by collecting the frac-
tions corresponding to the peak showing up at 31 min, and
analyzing them by LC-MS.
The chemical equation for the derivatization reaction is
shown in Scheme 4 with m/z values of compound 1a and the blank of compound 1a.
Fig. 2 HPLC analysis of the furfural derivatization mixture and sample
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compared with that of compound 1a as a sample blank, which is
shown in Fig. 4. It clearly indicated that all of these three
aldehydes can be successfully derived with compound 1a. These
three derivatives have distinct retention times and strong uo-
rescent responses at certain chromatographic condition, which
makes compound 1a an excellent derivatization agent for the
analysis of them. The analysis of a mixture of F, 5-MF and 5-
HMF at 1 : 1 : 1 molar ratio based on derivatization with 1a
further conrmed this. Aer the three aldehydes in the mixture
were derived with compound 1a, the mixture was analyzed with
HPLC, three derivatives were well separated, which is illustrated
in Fig. 5. Each derivative has same retention time as that
derived separately and shown in Fig. 4.
Scheme 4 Derivatization reaction of furfural with compound 1a.
With these results in hand, we further applied compound 1a
in the analysis of F, 5-MF, 5-HMF in common foods. Using
commercially purchased raisin as an example, aer it was
extracted by a mixture of methanol and ammonium acetate and
puried, the supernate was analyzed by compound 1a using
previously discussed method. HPLC result indicated the exis-
tence of F, 5-MF, 5-HMF (Fig. 6). The peak areas of each corre-
sponding signal suggest that the concentration of 5-HMF is
much higher compared with that of F and 5-MF. There is only
trace amount of F and 5-MF existing in the raisin since the
signals of these two derivatives are very weak. It strongly vali-
dates that the designed long-chain hydroxyamino coumarin
compounds are excellent agents for the analysis of aldehydes in
foodstuff, and it should also work well in the analysis of alde-
hydes from other sources.
Besides the derivatization with furfural related aldehydes,
hydroxyamino coumarin compounds can also react with aliphatic
aldehydes. We studied the derivatization reaction between
Fig. 3 Mass spectrum of the nitrone derivative which was generated
from the reaction between furfural and compound 1a.
+
compound 1a and hexanal. A singly charged [M + H] peak with an
m/z value of 332.1858 indicated the successful derivatization of
linear calibration range, regression equation and correlation hexanal, since the corresponding nitrone derivative of hexanal has
coefficient of the 1a-furfural nitrone derivative under our an m/z value of 331.1784 for the most abundant form (Fig. S36 in
experimental condition was measured and summarized (Table ESI†). Because aliphatic aldehydes widely exist in nature, the
S1 in ESI†). The calibration curves showed good linearity usage of these two long-chain hydroxyamino coumarin agents can
between concentration and peak area with correlation coeffi- be extend to more research areas.
cient as 0.9965. The reproducibility of the proposed method,
expressed by relative standard deviation (RSD), is 7.23%. The
detection limit was 1.6 nM at a signal-to-noise ratio (S/N) of 3.
Compared with other analytical methods, the limit of detection
LOD of our method is 1.6 nM, which is more sensitive than
those of GC-MS method (49 nM) and HPLC with pulsed
amperometric detection method (400 nM) according to the ref.
41 and 42 The strong peak at 31 min in Fig. 2 indicates the
excellent uorescent property of the coumarin uorophore.
Aer the successful derivatization of furfural with
compound 1a, we also tested analogous derivatization with
+
compound 1b. A singly charged [M + H] peak with an m/z value
of 342.1339 proved that compound 1b is also a good derivati-
zation agent for aldehydes (Fig. S35 in ESI†). The peak comes
from the corresponding nitrone derivative of furfural which has
an m/z value of 341.13 for the most abundant form.
We then carried out more derivatization reactions of alde-
hydes to include 5-MF and 5-HMF as well. Respective HPLC
Fig. 4 Respective HPLC analysis of F, 5-MF, 5-HMF derivatization
mixtures, compared with corresponding HPLC analysis of compound
analysis of F, 5-MF, 5-HMF derivatization mixtures are 1a as a sample blank.
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followed by LC-MS analysis. Nitrone can be easily formed by the
derivatization reaction between aldehydes and 1a or 1b under
mild condition, and it can be readily analyzed without difficulty,
which proves the effectiveness of N-substituted hydroxylamine
in aldehyde labeling. The excellent uorescent properties of 1a,
1b and their derived products from reacting with aldehydes
validate the correctness of choosing coumarin as the uo-
rophore in the derivatization agents. Aldehydes at mmol level
can be detected and analysed with our agents. Aldehydes
chosen in the study are focused on furfuraldehydes including F,
5-MF, and 5-HMF, the existences and concentrations of which
are closely relevant to food safety and avor. We successfully
used these N-substituted coumaryl hydroxylamines in the
detection of these furfuraldehydes in raisin, based on the
Fig. 5 HPLC analysis of a mixture of F, 5-MF, 5-HMF (1 : 1 : 1 molar derivatization reactions of standard F, 5-MF, and 5-HMF
ratio) derived by compound 1a, which is compared with the corre-
sponding HPLC analysis of compound 1a as a sample blank.
samples. This result proved that they can serve as excellent
agents for the analysis of aldehydes in foodstuffs. The deriva-
tization reaction between 1a and hexanal indicated that the
usage of them can be further extended to the analysis of
aliphatic aldehydes. In the future, we will broaden the appli-
cation of these two agents to the research areas of environ-
mental and biological sciences, and synthesize more
derivatization agents with different sidechain length and
substituents on the coumarin ring. It should be able to diversify
the applications of N-substituted coumaryl hydroxylamine and
further enhance the effectiveness and efficiency of them in the
aldehyde analysis.
Experimental
Materials and instrumentation
All chemicals were purchased from commercial suppliers and
Fig. 6 Analysis of aldehydes in raisin based on derivatization reaction
used without further purication. Flash chromatography was
using compound 1a, compared with the analysis of compound 1a as
carried out with silica gel (200–300 mesh). Analytical TLC was
a sample blank, as well as that of the derivatives of F, 5-MF and 5-HMF
performed with silica gel GF254 plates. Melting points were
as standard.
determined on a SGW X-4 melting point apparatus with
microscope (Shanghai Precision & Scientic Instrument Co.,
Ltd). The H-NMR spectra and C-NMR spectra were recorded at
ꢀ
Conclusion
25 C on 500 MHz or 125 MHz NMR spectrometer (Bruker
Avance III). Chemical shis (d) are reported in PPM using Me Si
4
Through a well-designed synthetic protocol, N-substituted
coumaryl hydroxylamines with long aliphatic chain (1a and 1b)
were prepared and served as derivatization agents for alde-
hydes. The coumarin rings in the derivatization agents were
assembled via Pechmann condensation, which provides
a powerful means to change the length of the sidechain.
Different substituents at 5-, 6-, 7- or 8-position of the coumarin
ring in the derivatization agents can also be introduced via
Pechmann condensation to provide more potential useful
probes with various functionalities or uorescent properties.
This has been veried by the successful preparation of
compound 1a and 1b. These two compounds were prepared
with good yield and high purity as indicated by the NMR
spectra. Long sidechain in the derivatization agents can
improve the chromatographic performance and enhance ioni-
as an internal standard (d ¼ 0 ppm), or using the deuterated
solvent signal as reference. Spin–spin coupling constants (J) are
given in Hz. Mass spectra were measured with an Agilent
Technologies 2100 mass spectrometer, or with a TripleTOF 5600
System (AB SCIEX) equipped with a nanospray source. Fluo-
rescence spectra were recorded on a RF-5301 uorescence
spectrophotometer (Shimadzu, Tokyo, Japan). Chromato-
graphic analyses were performed on a LC-20AT HPLC system
with a RF-20 uorescence detector and a chromatography data
acquisition system (Shimadzu, Tokyo, Japan). Separations were
performed on an Agilent Eclipse 5 mm, XDB-C18 column (USA).
Synthetic procedures
Ethyl 2-(1-hydroxycyclopentyl)acetate (3). Trimethyl chlor-
zation strength in mass spectrometry, as shown in the analysis osilane (TMSCl) (0.9 mL, 72 mmol) and zinc powder (6.3 g, 96
of aldehydes through derivatization reaction with 1a and 1b mmol) were added to anhydrous ether (150 mL). The mixture
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ꢀ
was stirred at r.t. for 15 min and heated to reux at 40 C. A yellow solid formed and was ltered. The ltrate was extracted
mixture of cyclopentanone (5.0 g, 60 mmol) and ethyl 2-bro- with ethyl acetate (3 ꢁ 50 mL) and the organic layered was
moacetate (12 g, 72 mmol) was added in 20 min while reuxing. evaporated to dryness. It was combined with the solid and
The reaction mixture was then stirred for 1 h. TLC analysis puried by ash chromatography (petroleum ether : ethyl
conrmed the complete consumption of ethyl 2-bromoacetate. acetate, 3 : 1) to afford 4-(4-bromobutyl)-7-hydroxy-2H-
Aer the mixture was cool down to r.t., HCl (300 mL, 2 M) was chromen-2-one as a white solid (1.03 g, 3.47 mmol, 43.3% yield).
ꢀ
1
added and it was stirred for 15 min. The organic layer was mp: 139–141 C. H NMR (500 MHz, DMSO-d
separated and the aqueous layer was extracted with ether (2 ꢁ 7.66 (d, 2H, J ¼ 8.9 Hz), 6.80 (dd, 1H, J ¼ 8.9 Hz, J
0 mL). The combined ethereal layer was washed with 5 wt% 6.71 (d, 1H, J ¼ 2.4 Hz), 6.10 (s, 1H), 3.59 (t, 2H, J ¼ 6.7 Hz), 2.77
6
): d 10.05 (s, 1H),
1
2
¼ 2.4 Hz),
5
1
3
aqueous solution of NaHCO , dried with K CO and concen- (t, 2H, J ¼ 7.6 Hz), 1.88–1.94 (m, 2H), 1.70–1.76 (m, 2H);
C
3
2
3
trated. It was puried by ash chromatography (petroleum NMR (125 MHz, DMSO-d ): d 161.56, 160.84, 155.62, 126.83,
6
ether : ethyl acetate, 3 : 1) to afford compound ethyl 2-(1- 113.39, 111.60, 109.85, 102.89, 35.12, 32.27, 30.38, 27.03.
hydroxycyclopentyl)acetate as a colourless liquid (9.66 g,
4-(4-Bromobutyl)-7-methoxy-2H-chromen-2-one (7b). 3-
1
5
2
4
6.1 mmol, 93.5% yield). H NMR (500 MHz, CDCl
3
): d 4.18 (q, methoxyphenol (3.12 g, 25.0 mmol) was carefully dissolved in
ꢀ
H, J ¼ 7.1 Hz), 2.60 (s, 1H), 1.90–1.78 (m, 4H), 1.66–1.50 (m,
H
2
SO
4
solution (95%, 20 mL) at 0 C with stirring. Ethyl 7-
1
3
H), 1.28 (t, 3H, J ¼ 7.1 Hz). C NMR (125 MHz, CDCl
3
): bromo-3-oxoheptanoate (6.20 g, 24.8 mmol) was slowly added to
the solution in 5 min. The mixture was then stirred at r.t. for 5 h.
d 173.27, 79.48, 60.60, 44.58, 39.67, 23.80, 14.19.
Ethyl 2,2,7-tribromo-3-oxoheptanoate (4). Aer ethyl 2-(1- TLC indicated that 3-methoxyphenol was completely consumed
hydroxycyclopentyl)acetate (5.16 g, 30.0 mmol) and K CO
and a uorescent compound formed. The solution was poured
2
3
ꢀ
(24.8 g, 180 mmol) in chloroform (100 mL) was stirred at 0 C for slowly into an ice/water mixture (150 mL) with stirring and then
1
0 min, bromine (24 g, 150 mmol) was added. The reaction extracted with ethyl acetate (3 ꢁ 50 mL) The combined organic
ꢀ
mixture was allowed to continuously stir at 0 C for 10 h. The layer was dried with sodium sulfate and evaporated to dryness,
TLC conrmed the completion of the reaction. Saturated which was further puried by ash chromatography (petroleum
sodium thiosulfate solution was added to the mixture until the ether : ethyl acetate, 3 : 1) to afford 4-(4-bromobutyl)-7-methoxy-
colour of it turned from red brown to pale yellow. The organic 2H-chromen-2-one as a white solid (1.83 g, 5.88 mmol, 23.5%).
ꢀ
1
layer was separated and further washed with saturated sodium mp: 71–72 C. H NMR (500 MHz, DMSO-d ): d 7.75 (d, 1H, J ¼
6
thiosulfate twice and concentrated under vacuum. Purication 8.9 Hz), 7.66 (d, 2H, J ¼ 8.9 Hz, J ¼ 1.5 Hz), 6.95–6.99 (m, 2H),
1
2
by ash chromatography (petroleum ether : ethyl acetate, 30 : 1) 6.18 (s, 1H), 3.60 (t, 3H, J ¼ 6.4 Hz), 3.59 (t, 2H, J ¼ 6.7 Hz),2.80
1
3
gave pure ethyl 2,2,7-tribromo-3-oxoheptanoate as a yellow (t, 2H, J ¼ 6.7 Hz), 1.89–1.94 (m, 2H), 1.71–1.76 (m, 2H);
C
1
liquid (11.7 g, 28.6 mmol, 95.3% yield). H NMR (500 MHz, NMR (125 MHz, DMSO-d
6
): d 162.78, 160.70, 157.02, 155.57,
CDCl
3
): d 4.38 (q, 2H, J ¼ 7.1 Hz), 3.43 (t, 2H, J ¼ 6.4 Hz), 2.98 (t, 126.68, 112.64, 110.74, 101.45, 56.39, 35.14, 32.24, 30.36, 26.98.
1
3
2
H, J ¼ 7.0 Hz), 1.97–1.85 (m, 4H), 1.36 (t, 3H, J ¼ 7.1 Hz).
C
7-Hydroxy-4-(4-hydroxybutyl)-2H-chromen-2-one (8a).
A
NMR (125 MHz, CDCl
3
): d 193.26, 163.73, 64.83, 59.76, 34.96, suspension of 4-(4-bromobutyl)-7-hydroxy-2H-chromen-2-one
ꢀ
3
2
2.89, 31.55, 23.45, 13.77.
(300 mg, 1.01 mmol) in water (40 mL) was heated at 115 C in
Ethyl 7-bromo-3-oxoheptanoate (5). To a solution of ethyl a sealed tube overnight. Aer TLC indicated the completion of the
,2,7-tribromo-3-oxoheptanoate (4.8 g, 12 mmol) in saturated reaction, the reaction mixture was allowed to cool to r.t. and
ammonium chloride/methanol solution was added the cupper- product in the form of white needle-like crystals formed. It was
zinc alloy powder (60 : 40, W/W, 12.3 g, 300 mesh). The mixture collected by ltration and dried in a vacuum oven to afford pure 7-
ꢀ
was then stirred at 10–15 C for 1.5 h. The TLC indicated the hydroxy-4-(4-hydroxybutyl)-2H-chromen-2-one (0.22 g, 0.94 mmol,
ꢀ
1
complete consumption of ethyl 2,2,7-tribromo-3-oxoheptanoate. 93.0% yield). mp: 114–115 C. H NMR (500 MHz, DMSO-d
6
):
Aer the reaction mixture was ltered, the organic layer was d 10.49 (s, 1H), 7.64 (d, 2H, J ¼ 8.9 Hz), 6.80 (dd, 1H, J
1
¼ 8.9 Hz, J
2
separated, dried with sodium sulfate and concentrated. Puri- ¼ 2.4 Hz), 6.71 (d, 1H, J ¼ 2.4 Hz), 6.08 (s, 1H), 4.40 (t, 2H, J ¼ 5.2
cation by ash chromatography (petroleum ether : ethyl acetate, Hz), 3.43 (q, 2H, J ¼ 6.4 Hz, J ¼ 11.6 Hz), 1.61–1.67 (m, 2H), 1.49–
1
2
1
3
3
0 : 1) afforded ethyl 7-bromo-3-oxoheptanoate as a red brown 1.54 (m, 2H); C NMR (125 MHz, DMSO-d ): d 161.56, 160.90,
6
1
liquid (1.94 g, 7.73 mmol, 64.4% yield). H NMR (500 MHz, 157.68, 155.60, 126.84, 113.39, 111.69, 109.74, 102.87, 60.77,
CDCl
3
): d 4.20 (q, 2H, J ¼ 7.1 Hz), 3.44 (s, 2H), 3.41 (t, 2H, J ¼ 6.6 32.50, 31.21, 25.7.
Hz), 2.60 (t, 2H, J ¼ 7.1 Hz), 1.95–1.85 (m, 2H), 1.81–1.72 (m, 2H),
4-(4-hydroxybutyl)-7-methoxy-2H-chromen-2-one (8b).
A
1
3
1
.29 (t, 3H, J ¼ 7.1 Hz). C NMR (125 MHz, CDCl
3
): d 202.17, suspension of 4-(4-bromobutyl)-7-methoxy-2H-chromen-2-one
ꢀ
167.15, 61.43, 49.26, 41.81, 33.14, 31.79, 21.94, 14.11.
(1.87 g, 6.01 mmol) in water (100 mL) was heated at 115 C in
4-(4-Bromobutyl)-7-hydroxy-2H-chromen-2-one (7a). Resor- a sealed tube overnight. Aer TLC indicated the completion of
cinol (0.88 g, 8.0 mmol) was carefully dissolved in H SO solu- the reaction, the reaction mixture was allowed to cool to r.t. and
2
4
ꢀ
tion (95%, 7.2 mL) at 0 C with stirring. Ethyl 7-bromo-3- white needle-like crystals formed. It was ltered and the ltrate
oxoheptanoate (2.20 g, 8.80 mmol) was slowly added to the was extracted with ethyl acetate. The organic layer was evaporate
solution in 5 min. The mixture was then stirred at r.t. for 5 h. to dryness and puried by ash chromatography (petroleum
TLC indicated that resorcinol was completely consumed and ether : ethyl acetate, 5 : 1) to afford another portion of the
a uorescent compound formed. The solution was poured product. In total, 0.842 g of 4-(4-hydroxybutyl)-7-methoxy-2H-
slowly into an ice/water mixture (50 mL) with stirring. A pale chromen-2-one as white needle-like crystals was produced
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ꢀ
1
(
3.39 mmol, 56.4% yield). mp: 97–98 C. H NMR (500 MHz, 1H, J ¼ 2.4 Hz), 6.10 (d, 1H, J ¼ 8.9 Hz), 2.75 (t, 2H, J ¼ 6.7 Hz),
DMSO-d ): d 7.74 (d, 1H, J ¼ 8.9 Hz), 6.95–6.98 (m, 2H), 6.16 (s, 2.77 (t, 2H, J ¼ 7.6 Hz), 1.88–1.94 (m, 2H), 2.18–2.36 (m, 2H),
6
1
3
1
2
H), 4.41 (m, 1H), 3.86 (s, 3H), 3.42–3.46 (m, 2H), 2.75–2.78 (m, 1.70–1.82 (m, 2H); C NMR (125 MHz, DMSO-d ): d 161.55,
6
1
3
H), 1.62–1.68 (m, 2H), 1.51–1.55 (m, 2H); C NMR (125 MHz, 160.85, 157.10, 155.62, 149.50, 126.84, 113.44, 111.61, 109.98,
+
DMSO-d
1
6
): d 162.73, 160.75, 157.54, 155.55, 126.67, 112.63, 102.90, 30.85, 29.12, 25.65. ESI-MS, m/z: 248 [M + H] , 270 [M +
10.63, 101.41, 60.76, 56.37, 32.47, 31.19, 25.12.
+
Na] .
4-(7-Methoxy-2-oxo-2H-chromen-4-yl)butanal oxime (10b).
4
-(7-Hydroxy-2-oxo-2H-chromen-4-yl)butanal (9a). A suspen-
sion
300 mg, 1.28 mmol), pyridinium chlorochromate (417 mg, (292 mg, 1.19 mmol) in THF/H
.93 mmol), sodium acetate (317 mg, 3.86 mmol), and diatomite added hydroxylamine hydrochloride (306 mg, 4.40 mmol), and
of
7-hydroxy-4-(4-hydroxybutyl)-2H-chromen-2-one To a solution of 4-(7-methoxy-2-oxo-2H-chromen-4-yl)butanal
(
1
2
O (6 mL, V : V ¼ 1 : 1) was
ꢀ
(600 mg) in acetone (30 mL) was reuxed at 65 C for 2 h. the pH value of the solution was adjusted to 4 with NaOH (2 M).
Another portion of pyridinium chlorochromate (417 mg, 1.93 It was then stirred at r.t. until TLC indicated the completion of
ꢀ
mmol) was then added, and the mixture was reuxed at 65 C the reaction. The solvent was then removed and the residue was
for 2 h. TLC indicated most of the reactant was consumed. The puried by ash chromatography (petroleum ether : ethyl
reaction mixture was then ltered by silica gel column to acetate, 1 : 1) to afford 4-(7-methoxy-2-oxo-2H-chromen-4-yl)
remove pyridinium chlorochromate and other solid precipitate. butanal oxime as a white powder (249 mg, 0.953 mmol,
1
The ltrate was concentrated and puried by ash chromatog- 80.1%). H NMR (500 MHz, DMSO-d
6
): d 10.44 (s, 1H), 7.75 (d,
raphy (petroleum ether : ethyl acetate, 1 : 1) to afford 4-(7- 1H, J ¼ 8.9 Hz), 7.36–7.38 (m, 1H), 6.95–6.99 (m, 2H), 6.17 (s,
hydroxy-2-oxo-2H-chromen-4-yl)butanal as white powder 1H), 3.32 (s, 3H), 2.78–2.81 (m, 2H), 2.21–2.25 (m, 2H), 1.75–1.81
a
1
13
(
141 mg, 0.607 mmol, 47.4% yield). H NMR (500 MHz, DMSO- (m, 2H); C NMR (125 MHz, DMSO-d ): d 162.77, 160.70,
6
d
6
): d 10.50 (s, 1H), 9.69 (s, 1H), 7.67 (d, 1H, J ¼ 8.9 Hz), 6.80 (d, 156.94, 155.57, 149.48, 126.67, 112.67, 111.87, 101.44, 56.38,
¼ 8.6 Hz, J ¼ 2.1 Hz), 6.71 (d, 1H, J ¼ 2.1 Hz), 6.09 (s, 1H), 30.83, 29.10, 25.60.
.74 (m, 2H), 2.58 (m, 2H), 1.83–1.88 (m, 2H); C NMR (125
): d 203.52, 161.57, 160.83, 156.93, 155.64, To a solution of 4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanal
26.80, 113.42, 111.57, 110.02, 102.91, 42.83, 30.72, 21.18. ESI- oxime (950 mg, 3.84 mmol) in methanol (10 mL) was added
1
2
H, J
1
2
1
3
7-Hydroxy-4-(4-(hydroxyamino)butyl)-2H-chromen-2-one (1a).
MHz, DMSO-d
1
MS, m/z: 233 [M + H] .
-(7-Methoxy-2-oxo-2H-chromen-4-yl)butanal
6
+
sodium cyanoborohydride (1.28 g, 20.4 mmol) batchwise, and the
4
(9b).
A
pH value of the solution was adjust to 2–3 by a methanol solution
suspension of 4-(4-hydroxybutyl)-7-methoxy-2H-chromen-2-one of hydrogen chloride. The reaction mixture was allowed to react
470 mg, 1.89 mmol), pyridinium chlorochromate (612 mg, at r.t. until TLC indicated the complete consumption of the
(
2
.84 mmol), sodium acetate (468 mg, 3.42 mmol), and diatomite reactant. Small amount of water was added to dissolve the solid,
ꢀ
(882 mg) in acetone (40 mL) was reuxed at 65 C for 2 h. and the solution was then applied to a RP-HPLC employing
˚
Another portion of pyridinium chlorochromate (612 mg, 2.84 a semi-preparative C18 column (250 ꢁ 30 mm, 120 A, 10 mm)
ꢀ
mmol) was then added, and the mixture was reuxed at 65 C with a gradient of 40–85% methanol in 35 min at a ow rate of 10
ꢂ1
for 2 h. TLC indicated most of the reactant was consumed. The mL min , which provided 7-hydroxy-4-(4-(hydroxyamino)butyl)-
reaction mixture was then ltered by silica gel column to 2H-chromen-2-one (612 mg, 2.46 mmol, 63.9% yield) as a white
ꢀ
1
remove pyridinium chlorochromate and other solid precipitate. solid. mp: 189–191 C. H NMR (500 MHz, DMSO-d ): d 11.39 (s,
6
The ltrate was concentrated and puried by ash chromatog- 1H), 10.83 (s, 1H), 10.65 (s, 1H), 7.67 (d, 1H, J ¼ 8.9 Hz), 6.83 (d,
raphy (petroleum ether : ethyl acetate, 1 : 1) to afford 4-(7- 1H, J ¼ 8.9 Hz), 6.75 (s, 1H), 6.12 (m, 1H), 3.14 (m, 2H), 2.77 (t,
1
3
6
methoxy-2-oxo-2H-chromen-4-yl)butanal as a white powder 2H), 1.62–1.79 (m, 4H); C NMR (125 MHz, DMSO-d ): d 161.68,
ꢀ
1
(
286 mg, 1.16 mmol, 61.4% yield). mp: 73–75 C. H NMR (500 160.90, 157.05, 155.57, 126.91, 113.46, 109.85, 102.89, 50.22,
+
+
MHz, DMSO-d
(
(
6
): d 9.70 (s, 1H), 7.70 (d, 1H, J ¼ 8.9 Hz), 6.96–6.98 30.77, 25.28, 23.25. ESI-MS, m/z: 250 [M + H] , 272 [M + Na] .
4-(4-(Hydroxyamino)butyl)-7-methoxy-2H-chromen-2-one (1b).
m, 2H), 1.83–1.89 (m, 2H); C NMR (125 MHz, DMSO-d6): To a solution of 4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanal
m, 2H), 6.17 (s, 1H), 3.86 (s, 3H), 2.75–2.78 (m, 2H), 2.57–2.60
1
3
d 203.53, 162.78, 160.69, 156.80, 155.58, 126.63, 112.66, 110.90, oxime (231 mg, 0.877 mmol) in methanol (2 mL) was added
1
01.44, 56.38, 42.80, 30.71, 21.13.
-(7-Hydroxy-2-oxo-2H-chromen-4-yl)butanal oxime (10a). To the pH value of the solution was adjust to 2–3 by a methanol
solution of 4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanal solution of hydrogen chloride. The reaction mixture was allowed
sodium cyanoborohydride (332 mg, 5.28 mmol) batchwise, and
4
a
(
100 mg, 0.431 mmol) in THF/H
2
O (4 mL, V : V ¼ 1 : 1) was to react at r.t. until TLC indicated the complete consumption of
added hydroxylamine hydrochloride (45 mg, 0.65 mmol), and the reactant. Small amount of water was added to dissolve the
the pH value of the solution was adjusted to 4 with NaOH (2 M). solid, and the solution was then applied to a RP-HPLC employing
˚
It was then stirred at r.t. until TLC indicated the completion of a semi-preparative C18 column (250 ꢁ 30 mm, 120 A, 10 mm) with
the reaction. The solvent was then removed and the residue was a gradient of 40–85% methanol in 35 min at a ow rate of 10 mL
ꢂ1
puried by ash chromatography (petroleum ether : ethyl min , which provided 4-(4-(hydroxyamino)butyl)-7-methoxy-2H-
acetate, 1 : 1) to afford 4-(7-hydroxy-2-oxo-2H-chromen-4-yl) chromen-2-one (163 mg, 0.619 mmol, 70.6% yield) as a white
ꢀ
1
6
butanal oxime as a white powder (101 mg, 0.408 mmol, 94.8% solid. mp: 139–141 C. H NMR (500 MHz, DMSO-d ): d 11.40 (s,
ꢀ
1
yield). mp: 136–138 C. H NMR (500 MHz, DMSO-d
6
): d 10.49 (s, 1H), 10.84 (s, 1H), 7.76 (d, 1H, J ¼ 8.9 Hz), 6.95–6.99 (m, 2H), 6.20
1H), 7.64 (dd, 2H, J
1
¼ 8.9 Hz, J
2
¼ 2.2 Hz), 6.80 (m, 1H), 6.71 (d, (s, 1H), 3.86 (s, 3H), 3.12–3.17 (m, 2H), 2.79–2.82 (m, 2H), 1.64–
19714 | RSC Adv., 2017, 7, 19707–19716
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1
3
1
1
2
.78 (m, 4H); C NMR (125 MHz, DMSO-d
6
): d 162.78, 160.72, analysis, the C18 column was pre-equilibrated with the mobile
56.89, 155.52, 126.78, 112.64, 110.77, 101.41, 56.40, 50.17, 30.75, phase for 30 min.
5.21, 23.21. ESI-MS, m/z: 264 [M + H] , 286 [M + Na] .
+
+
Acknowledgements
Fluorescent analysis
We gratefully acknowledge the National Natural Science Foun-
dation of China (21472144 to X. H. and 21405117 to Y. L.), the
Fundamental Research Funds for the Central Universities
The uorescent spectra of compound 1a and 1b were collected
on a Shimadzu RF-5301 uorescence spectrophotometer.
Compound 1a was dissolved in methanol, diluted by 10%
aqueous acetonitrile solution to a concentration of 0.5 mM. It
was further diluted by acetic acid/sodium acetate buffer (0.1 M,
pH ¼ 4) to a nal concentration of 0.5 mM and used in the
(WUT: 2016-IB-005, 2016-IB-007, 2017-IB-007), and Chutian
Scholar Program of the Hubei provincial government (to X. H.)
for nancial support.

uorescent analysis. The procedure for compound 1b was
similar. The only difference was that the nal concentration of
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¼ 0.55). The uorescent
quantum yield was calculated with the expression 4
¼ 4
are the quantum yields of
the samples and the standard, F , F are the peak areas under
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ꢂ1
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RSC Adv., 2017, 7, 19707–19716 | 19715

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