Quantitative 1H NMR spectroscopy (qNMR) in the early process development of a new quorum sensing inhibitor

Article abstract of DOI:10.1002/mrc.4906

2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one (called 6-oxo) is presented as a new AI-1 quorum sensing inhibitor for Vibrio harveyi. The development of a chemical process to afford traceable materials for new biological assays demands the development of analytical methods to ensure their purity and quality. This work describes the use of quantitative ¹H nuclear magnetic resonance (NMR) spectroscopy (qNMR) to assess the purity of a sample of 6-oxo (99.88%) and a sample of its major process impurity (E)-1-(2-hydroxycyclohex-2-en-1-yl)but-2-en-1-one (called HCB; 98.28%). To explore the scope of the use of qNMR to quantify the amount of low-content components in samples related to the chemical process for 6-oxo synthesis, this work also determined the amount of 6-oxo in two HCB samples: (a) the high-purity HCB sample described above and (b) a crude HCB sample collected during the chemical process. Despite the complexity of the crude sample, the amount of 6-oxo was readily assessed and could help to estimate the extent to which 6-oxo was already formed during the HCB synthesis. This information can help the understanding of how the process parameters can be modified to improve the performance of the whole process, by controlling the reaction mechanisms working at each step of this chemical process. In this context, our results reinforce qNMR as a complementary analytical tool for the quantification of the main component found in a sample, contributing to the standardization of reference materials and thus allowing the development of analytical methods for process control and traceability of the samples used for biological assays.

Full text of DOI:10.1002/mrc.4906

Machado Angelo H. L. (Orcid ID: 0000-0003-4284-9929)
Full title: Quantitative 1H NMR spectroscopy (qNMR) in the early process development of a
new quorum sensing inhibitor.
Short title: 1H qNMR to ensure quality of a new quorum sensing inhibitor.
Robson A. F. Cavalcante,a Felipe L. Silva,b Fernada Favero,a,b Inês S. Resck,a
Alex L. Pereira,b* and Angelo H. L. Machadoa*
a. Instituto de Química, Universidade de Brasília, Campus Universitário Darcy
Ribeiro, CEP 70910-900, Asa Norte, Brasília-DF, Brazil.
b. Campus de Ceilândia, Universidade de Brasília, Centro Metropolitano, CEP:
72220-275, Ceilândia Sul, Ceilândia-DF, Brazil.
*Corresponding authors. E-mail: nagelo@unb.br and alexpereira@unb.br;
Tel +55-61-31073858 and +55-61-31073045
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/mrc.4906
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Abstract
2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one (called 6-oxo) is presented as a new AI-
1 quorum sensing inhibitor for Vibrio harveyi. The development of a chemical process to
afford traceable materials for new biological assays demands the development of analytical
1
methods to ensure their purity and quality. This work describes the use of quantitative H
NMR spectroscopy (qNMR) to assess the purity of a sample of 6-oxo (99.88%) and a sample
of its major process impurity (E)-1-(2-hydroxycyclohex-2-en-1-yl)but-2-en-1-one (called
HCB; 98.28%). To explore the scope of the use of qNMR to quantify the amount of low-
content components in samples related to the chemical process for 6-oxo synthesis, this work
also determined the amount of 6-oxo in two HCB samples: (1) the high-purity HCB sample
described above and (2) a crude HCB sample collected during the chemical process. Despite
the complexity of the crude sample, the amount of 6-oxo was readily assessed and could help
to estimate the extent to which 6-oxo was already formed during the HCB synthesis. This
information can help the understanding of how the process parameters can be modified to
improve the performance of the whole process, by controlling the reaction mechanisms
working at each step of this chemical process. In this context, our results reinforce qNMR as
a complementary analytical tool for the quantification of the main component found in a
sample, contributing to the standardization of reference materials, and thus allowing the
development of analytical methods for process control and traceability of the samples used
for biological assays.
Keywords: purity; quantitative nuclear magnetic resonance; process development;
related compound; process control.
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1.
Introduction
In recent decades, health authorities have reported an increase in bacterial antibiotic
resistance and the emergence of multi-resistant pathogenic strains. These problems have
driven the pharmaceutical industry to discover new therapeutic alternatives for conventional
drugs [1][2][3]. Recent studies have showed the relationship between bacterial quorum
sensing (QS), a chemical signaling system, and virulence expression factors [4][5]. Greater
knowledge concerning QS mechanisms and the role of their signaling molecules, called
autoinducers (AIs), can lead to the discovery of new strategies to help control bacterial
infections [3]. In this context, the prospection of molecules to work as quorum sensing
inhibitors (QSIs) has been introduced as promising anti-virulence drugs for treating bacterial
infections. [1][5][6][7]
Preliminary studies in our workgroup have shown 2-methyl-5,6,7,8-tetrahydro-2H-
chromen-4(3H)-one, called 6-oxo (1), as a new bacterial QS inhibitor. In order to determine
the effect of 6-oxo on luminescence in Vibrio harveyi and to dismiss artifacts possibly
generated by inhibitory effects on bacterial growth, the luminescence expression was
measured simultaneously at different points of the bacterial growth curve (Fig. 1). The assays
showed that 6-oxo reduced the V. harveyi luminescence at 60 %, without interfering with
bacterial population growth (luminometry assay and statistical analyses are described in the
supplementary information). Moreover, the inhibitory effect produced by 6-oxo occurred in a
dose-dependent manner and was seen to be more effective in the AI-1 QS system (Fig. 2) [8].
Driven by this biological response, the synthesis of batches of this chromenone with known
quality is indispensable, as good as that required for active pharmaceutical ingredients
(APIs), aiming to obtain this product with high purity for further biological assays [9].
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To assess the purity requirements of organic molecules to ensure a desired quality, the
development of analytical methods addressing quality is mandatory. However, during the
early development of a new drug substance, no commercially available certified reference
material is expected to be found on the market. For this reason, most common
chromatographic methods applied to purity determination cannot be used for this proposal
(e.g. HPLC and GC)[10].
Quantitative Nuclear Magnetic Resonance (qNMR) is an attractive analytical tool to
assess the purity of organic molecules, due to its easy procedure for sample preparation with
a simple method development [9][10][11][12]. The qNMR analysis can be considered as a
primary ratio method of measurement, especially because it allows the direct comparative
measurement of the signal area ratio between an analyte and a standard of the same quantity.
This method can be described completely by mathematical equations, from which a full
uncertainty budget may be derived. For this reason, calibrations or reference standards
analogous to analytes are not needed [13][14]. Other positive features include: the assignment
of the chemical structure of the analyte and its impurities, the absence of intensity calibration
requirements for determination of ratios (the signal area is proportional to the number of
resonant nuclei), relatively short measuring times (this parameter depends on the relaxation
times, of the analyte concentration and the spectrometer used), easy handling and sample
preparation and the simultaneous analysis of different analytes in the same sample,
particularly important for the analysis of impurities [11][13][14]. This technique has already
been used in the analysis of drugs and made official by some pharmacopoeias, such as in the
U.S. Pharmacopoeia (USP) monographs on orphenadrine citrate and isoamyl nitrite [13][15].
Recently, the methodology (based on an internal standard approach) was extended and
validated for quantification of MDMA in ecstasy tablets.[16]
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Given that 6-oxo is a potential pharmaceutical candidate during early stages of
development (scheme 1), it is indispensable to define an assay method for its purity
determination in terms of mass fractions [17][18]. However, current regulatory requirements
for the pharmaceutical industry demands not only the synthesis of 6-oxo samples as a highly
pure active pharmacological substance, but also knowledge about the potential impurities
present in these samples, and the definition of process parameters that are directly responsible
for impurities formation [19]. Therefore, our contribution describes the characterization of 6-
oxo and its main impurity (E)-1-(2-hydroxycyclohex-1-en-1-yl)but-2-en-1-one, called HCB
(2) by 1H NMR. It also describes the application of 1H qNMR using two different certificated
internal reference standards, for the determination of the purity of 6-oxo and HCB samples.
In addition, qNMR methodology was performed in two different approaches to quantify the
amount of 6-oxo as minor chemical component. The first one was focused on assessing the
individual amount of 6-oxo in a high-purity HCB sample prepared to be used as a reference
material in our analytical developments. The other application of the qNMR method was
focused on measuring the amount of 6-oxo already produced after the enamine acylation step
of the 6-oxo synthesis, highlighting the potential of the use of qNMR for the optimization of
this chemical process.
2.
Experimental section
2.1. Chemicals and reagents
Chemical compounds and reagents used were purchased and supplied by Sigma-
Aldrich Chemicals. All chemicals were of analytical grade and used without additional
purification procedures. Benzoic acid (Sigma-Aldrich, Buchs, CH, TraceCERT quality -
99.97 %) and maleic acid (Sigma-Aldrich, Buchs, CH, TraceCERT quality - 99.94 %) were
used as certified reference material (internal standard).
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2.2. Synthesis of 6-oxo (2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one)
To a three-neck round-bottomed flask (250 mL) equipped with condenser, addition
funnel and anhydrous CaCl2 tube, 1-morpholinocylohexene (4.8 mL, 30 mmol), triethylamine
(5.4 mL, 38 mmol) and anhydrous dichloromethane (30 mL) were added. To this solution,
magnetically stirred and kept in an ice bath (10-12 °C), a solution of crotonoyl chloride (3.2
mL) in anhydrous dichloromethane (25 mL) was added over 30 minutes. The reddish-brown
mixture was kept in a warm water bath (38-40 °C) for 18 h. A solution prepared with 30 mL
of HCl (10 % w/w) and 15 mL of ethanol (95 %) was sequentially added, and the resulting
mixture was refluxed for a further 10 h. After cooling, the organic layer was separated, and
the aqueous portion was extracted with chloroform (3 x 20 mL). The combined organic
extract was washed successively with water (3 x 50 mL), saturated aqueous solution of
sodium bicarbonate (30 mL) and saturated aqueous solution of sodium chloride (30 mL).
Drying over anhydrous Na2SO4 and evaporation of solvent gave a reddish-brown liquid.
Without further purification, a solution prepared with Acetic acid (99.85%): HCl (37%): H2O
(1:1:1, 1 mL of each component for 1 g of reddish-brown liquid) was added to the round-
bottomed flask containing the reddish-brown liquid. A condenser was connected to the
round-bottomed flask and the reaction mixture was refluxed (72-75 ºC), under stirring for 4h.
The cooled reaction mixture was diluted with water (20 mL) and extracted with
dichloromethane (3 x 20 mL). The combined extract was washed with water (3 x 20 mL),
saturated aqueous solution of sodium bicarbonate (30 mL) and brine (30 mL). Drying over
anhydrous Na2SO4 and rotoevaporation of the solvent gave the crude product (brown solid).
The crude was dissolved in hot hexane/ethyl acetate solution (95:5 v/v, 20 mL, 38-40 °C) and
kept under stirring over activated charcoal for 6 h. This solution was vacuum filtered through
a diatomaceous earth layer, and the solvent was removed by rotoevaporation to give a light
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yellow solid. This product was solubilized in hot hexane (25 mL). After cooling, the
crystallization yielded the pure 6-oxo as white needles (3.3 g, 65 %).
2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one 6-oxo (1)
White needles. mp 47- 48 °C (literature 47-48 °C [20]).
1H NMR (600 MHz, DMSO-d6) δ (ppm) 4.45 (dqd, J = 12.7, 6.3, 3.8 Hz, 1H), 2.41 (dd, J =
16.7, 13.3 Hz, 1H), 2.34 (dd, J = 16.7, 3.8 Hz, 1H), 2.25 – 1.98 (m, 4H), 1.75 – 1.37 (m, 4H),
1.34 (d, J = 6.3 Hz, 3H).
13C NMR (151 MHz, CDCl3) δ (ppm) 192.5, 171.4, 112.0, 74.6, 43.3, 28.7 22.2, 22.0, 20.8
20.7.
IR (KBr, υmax/ cm-1) 2973, 2929, 2865, 1664, 1612.
EI/MS (70 eV) m/z 166 (M+), 151, 138, 124, 110, 96, 83, 68 (100), 55.
2.3. Synthesis of HCB ((E)-1-(2-hydroxycyclohex-1-en-1-yl)but-2-en-1-one)
The pure sample of HCB was obtained by the chromatographic purification (silica-gel
and dichloromethane) of the crude HCB (reddish-brown liquid collected just after the first
extraction step).
Yellow solid. mp 47-48 ºC (literature 46-48 °C [21]).
1H NMR (600 MHz, DMSO-d6) δ (ppm) 6.88 (dq, J = 15.2, 6.9 Hz, 1H), 6.46 (dq, J = 15.2,
1.7 Hz, 1H), 2.38 (t, J = 6.2 Hz, 2H), 2.34 (t, J = 6.2 Hz, 2H), 1.92 (dd, J = 6.9, 1.7 Hz, 3H),
1.68 – 1.60 (m, 4H).
13C NMR (151 MHz, CDCl3) δ (ppm) 191.7, 182.2, 141.6, 124.5, 106.1, 33.7, 23.7, 23.0,
21.8, 18.7.
EI/MS (70 eV) m/z 166 (M+), 151(100), 138, 123, 109, 95, 79, 69, 55.
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2.4. 1H NMR and qNMR analysis.
The NMR solutions were prepared with Sigma-Aldrich deuterated chloroform (CDCl3)
for 13C NMR analyses (Figs. S3 and S4, supplementary information) and deuterated dimethyl
1
sulfoxide (DMSO-d6) for H qNMR analyses. Five qNMR samples were prepared weighing
12 to 16 mg of analyte and internal standard, both dissolved in 600 µL of deuterated solvent.
The qNMR experiments were carried out on a Bruker Avance III HD 600 MHz
spectrometer equipped with a 5 mm broadband observe (BBFO) probe. Locking and
shimming adjustments were performed automatically. Tuning and matching adjustment were
done manually for each sample. The 1H NMR spectra were acquired without sample spinning
and with 13C decoupling during acquisition (zgig30 pulse sequence). T1 measures of all
analytes were previously performed in qNMR experiments. To ensure complete relaxation,
all acquisitions were made with a relaxation delay of 7 times the largest T1 value. Acquisition
parameters (Table 1) were adjusted to ensure a signal-to-noise ratio at least 300 for the
relevant peaks. The integration of the internal standard signal and sample signal were done in
the same way. Aiming to integrate completely each quantified signal, the integration range
was extended from the centre of the signal at least eighty times of the full width at half height
(FWHM) on either side of the measured signal. The limits of the integration range to
multiplets were based on the outermost signals. Spectrum data were processed with the
TopSpin 3.2 software. Manual adjustments in phase and baseline were performed for each
sample, and all spectra were referenced using TMS signal (0 ppm). The spectra showed were
edited with MestReNova software (Mestrelab Research S.L., Santiago de Compostela, Spain,
version: 11.0.4, 2017).
Equation S1 was used to calculate the purity (% w/w) of each analyte in the samples
(supplementary information) [12][13][14].
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The uncertainty budget was based on main uncertainty sources to our qNMR
measurements and these are illustrated in a cause-effect diagram in the supplementary
information (fig. S1). The combined uncertainty
was calculated by equation S2,
was calculated to a confidence
(supplementary information). The expanded uncertainty
level of 95%, by multiplying of the combined uncertainty to a confidence level factor of k = 2
[14][22][23].
3.
Results and discussion
3.1. Chemical synthesis of the samples
The synthesis of 6-oxo (1) was based on the acylation of 1-morpholinocyclohexene (3)
by crotonoyl chloride (4) in the presence of triethylamine, followed by acid hydrolysis to
afford the crude HCB (2). After that, the final tandem isomerization / cyclization step was
performed under acidic conditions to yield the pure 6-oxo after crystallization in hexane
(Scheme 1). The whole procedure is based on one previously described by Mahajan and
Resck [20].
The intermediate HCB (2) is the last chemical intermediate of the 6-oxo synthesis and
is its main process impurity. A high-purity sample of HCB was prepared by chromatographic
purification (silica gel and dichloromethane) of the crude HCB, a process sample collected
just after the work-up of the enamine acylation step.
The measurement of the mass fraction of a chemical substance present in a complex
sample is a recognized qNMR limitation [24]. To address this issue, we also proposed the
measurement of the 6-oxo amount present in a crude sample of HCB collected just before
quenching the first reaction step, the acylation of the enamine.
3.2.
NMR analyses
1
The 6-oxo H NMR spectrum (Fig. 3a) showed chemical shifts and multiplicities that
endorse its molecular structure. A double quartet of doublets (dqd), observed at 4.45 ppm,
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was assigned to H13, due to its coupling with H12, H9´ and H9´´. Two doublets doubles of
doublets (dd), observed at 2.41 and 2.34 ppm, were assigned to the diastereotopic hydrogens
H9´ and H9´´, due to their couplings with H13. A multiplet, 2.25 – 1.98 ppm, integrating for
4 hydrogens, was assigned to the allylic methylenes H3 and H6. From 1.75 to 1.37, a
multiplet could be observed corresponding to the methylenes H1 and H2. Finally, at 1.42
ppm, a doublet could be observed, assigned to the methyl H12, due to its coupling with H13.
1
The HCB H NMR spectrum also matched the proposed chemical structure (Fig. 4a).
At 6.88 ppm, a double of quartets (dq) was attributed to H9, due to its coupling with H8 and
H10. At 6.46 ppm another dq was attributed to H8, due to its coupling with H9 and H10. At
2.38 and 2.34 ppm, two triplets, which were respectively assigned to the methylene
hydrogens H1 and H4, due to their couplings with H2 and H3, could also be detected. At 1.92
ppm a double doublet (dd) assigned to the methyl H10, due to its coupling with H8 and H9. A
multiplet corresponding to the resonance of the methylenes H2 and H3 was detected, ranging
from 1.68 to 1.60 ppm. A doublet observed at 1.34 ppm was assigned to the methyl group of
6-oxo, which was detected as an impurity in this HCB sample, confirming the information
previously obtained by the GC-MS and HPLC analysis.
3.3. Purity determination and 6-oxo quantification in samples of HCB
The 6-oxo 1H NMR spectrum has a signal at 4.45 ppm (dqd) that was resolved enough
to allow its integration free of the contribution of another hydrogen nuclei (T1: 4 s CDCl3 and
3 s to DMSO4-d). For this reason, this signal was chosen to perform the purity assay of this 6-
oxo sample based on the integral value of the maleic acid signal (6.27 ppm, Fig. 3a) and
benzoic acid signal (8.13 ppm, Fig. 3b), chosen to be separately used as the internal
1
standards. The results of the H qNMR analysis are presented in Table 2 for both internal
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standards, where the purity calculation was performed by means of eqn. 1. The analyzed
samples showed a high purity for both standards, (PMA= 99.88%, UMA=0.33%) and (PBA=
99.87%, UBA=0.31%), having the acceptable purity required for its use in our future
biological assay related to QS.
1
The purity analysis of the pure HCB sample by H NMR spectroscopy was also
performed with maleic acid (Fig. 4a) and benzoic acid (Fig. 4b) as internal standards. The
HCB characteristic resonance signals for all its hydrogen nuclei could be readily assessed. In
addition, a doublet at 1.34 ppm (T1: 2 s CDCl3 and 1 s to DMSO4-d6), assigned to 6-oxo
methyl group, was detected (Fig. 4a). The presence of this impurity had already been
predicted by GC-MS and HPLC analysis.
For maleic acid as the internal standard, the purity analysis of HCB was performed with
the same parameters used to assess 6-oxo purity (table 1). The signal at 6.88 ppm (T1: 6 s
CDCl3 and 4 s to DMSO4-d6) had enough resolution to be used as the reference resonance
signal to acquire the qNMR data of this sample of HCB (Fig. 4a) and yielded a purity of
98,28 % (UMA=0.33%) (Table 3). The percentage of 6-oxo in this sample could also be
calculated through its doublet in 1.34 ppm (Fig. 4a) and showed an amount of 1.58 % .
Benzoic acid was also used as an internal standard to assess the purity of the HCB by
1H qNMR analysis. In addition to the expected resonance signals for HCB and 6-oxo, some
extra signals were detected (highlighted by squares in Fig. 4b). These signals were credited to
unexpected chemical interactions between the analyte and internal standard. Not necessarily
due to the acid promoted decomposition of the analyte, but most probably due to the
acid/base equilibrium between HCB and benzoic acid. The afforded salt could have its ionic
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pair close enough to allow differential anisotropic shield/deshield promoted by the benzoic
acid aromatic ring for some nuclei. This hypothesis was supported by the lower HCB purity
(97.56%, UBA=0.40%) assessed when benzoic acid was the internal standard in contrast to the
previous purity (98,28 %, UMA=0.33%) observed when maleic acid was the internal standard.
1
These findings highlight the need to scan different internal standards to assess purity by H
qNMR analysis.
The possibility to measure the amount of 6-oxo when it is an impurity in a sample of
HCB prompted us to answer an opened question in respect to our process development. How
much 6-oxo is formed just after the hydrolysis step that affords HCB? This hydrolysis is
performed under acidic conditions (aqueous HCl solution) and, in the same way as that
promoted by AcOH/HCl/H2O for the cyclization of HCB, it could promote 6-oxo formation
(Scheme 1). The confirmation of this hypothesis could open the opportunity to redesign the
process to a one pot hydrolysis/cyclization step to yields 6-oxo, reducing the number of the
unit operations and, consequently, lowering the process timeline and the exposure of the
desired product to acidic conditions responsible to degrade it.
1
1
To evaluate the H qNMR methodology with this different approach, H NMR
spectrum of the crude HCB was acquired (Figure 5a) and revealed new resonance signals
ranging especially from 6.0 to 6.5 ppm, when compared to the pure HCB sample (Figure 4).
For this reason, only benzoic acid was used as an internal standard. Despite the high
complexity of the acquired spectrum, the 6-oxo signal at 4.45 ppm showed enough resolution,
even after the addition of the internal standard, and was used to perform qNMR analysis (Fig.
5b). The results showed a 6.58 % mass fraction of 6-oxo on average and a minor data
variation (Table S1, supplementary information). Despite the success on the detection and
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quantification of 6-oxo at this process step, the low amount of the final product discouraged
us from attempting the redesign 6-oxo chemical process.
4.
Conclusion.
The measurement of the mass fraction of the components of a sample, especially the
assay of its main compound, is important to properly evaluate the data obtained by the
1
biological activity studies. This scientific contribution detailed the use of quantitative H
NMR spectroscopy to determine the purity of a new quorum sensing inhibitor called 6-oxo.
1
The H qNMR results, assessed by two different internal standards, provided high accuracy
and demonstrated the achievement of a high-purity product.
1
The same H qNMR methodology was used to assess the purity of an HCB sample, a
synthetic intermediate of the 6-oxo chemical process, usually observed as the main impurity
in final samples of 6-oxo. The data showed good results when maleic acid was used as the
internal standard. Attempts to perform the analysis when benzoic acid was the internal
1
standard led to additional resonance signals in the H NMR spectrum, yielding a lower
percentage of HCB when compared to the result returned using maleic acid as internal
standard. This was credited to unwanted chemical interaction between the analyte and the
internal standard, highlighting the need for the screening of the proper internal standard to the
desired purity determination.
1
The H qNMR methodology was also used successfully to assess the mass fraction of
the 6-oxo in a crude sample of HCB, despite the presence of other impurities. This can allow
6-oxo formation to be followed at each step of the chemical process and potentially helps its
optimization toward the improvement of 6-oxo yield. This technique can be incorporated into
the daily workflow of 6-oxo quantification by NMR, even if it is applied to the analysis of a
complex matrix, as usually observed for in-process control steps.
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Acknowledgements
The authors are grateful to the Brazilian Health Regulatory Agency (Anvisa). This
study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brazil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento
Científico e Tecnológico – Brazil (CNPq) - 477418/2013-9, Fundação de Apoio à Pesquisa
do Distrito Federal – Brazil (FAPDF) 193.001.049/2015 and 0193.001.560/2017, Decanto de
Pesquisa e Pós-graduação (DPG-UnB). We also acknowledge Dr. Luiz E. C. Benedito
(CAIQ-UnB) and Profª. Aline Lima de Oliveira (IQ-UnB).
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Table 1 - qNMR acquisition parameters
Parameter (Bruker code)
Digitization mode (DIGMOD)
Filter correction (FILCOR)
Number of dummy scans
Pre-scan delay (DE)
Value
Baseopt
1.5 µs
4
10 µs
automatic
15 s
Receiver gain (RG)
Relaxation delay (D1)
Sweep width (SW)
24 ppm
64k
Time domain size (TD)
Total number of scans (NS)
32
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1
Table 2 - Data of 6-oxo H qNMR analysis with maleic acid (MA) or benzoic acid (BA)
as internal standard.
Mx
MStd
mStd
m
PStd
Px
Sample Std Ix /10
IStd /10 NStd Nx
(g/mol) (g/mol) (mg) (mg) (% ) (% )
1
2
3
4
5
1
2
3
4
5
3.19
3.86
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
166.22 116.07 14.0 12.8 99.94 99.90
166.22 116.07 12.4 13.7 99.94 99.87
166.22 116.07 12.0 13.7 99.94 99.94
166.22 116.07 12.5 13.9 99.94 99.95
166.22 116.07 12.5 14.0 99.94 99.73
166.22 122.12 15.9 12.4 99.97 99.99
166.22 122.12 15.3 12.2 99.97 99.89
166.22 122.12 15.6 12.1 99.97 99.84
166.22 122.12 14.9 12.5 99.97 99.91
166.22 122.12 15.0 12.6 99.97 99.72
MA 3.99
3.88
3.90
2.87
2.93
BA 2.85
3.08
3.08
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Table 3 - Data from HCB 1H qNMR analysis with maleic acid as internal standard
Mx MStd mStd PStd
m
Px
Sample Analyte Ix /10 IStd /10 NStd Nx
(g/mol) (g/mol) (mg) (mg) (% ) (% )
1
2
3
4
5
1
2
3
4
5
3.72
3.82
3.69
3.81
3.79
0.18
0.17
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
10.00
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
3
3
3
3
3
166.22 116.07 14.2 15.4 99.94 98.25
166.22 116.07 13.4 14.9 99.94 98.37
166.22 116.07 13.0 14.0 99.94 98.17
166.22 116.07 12.6 14.0 99.94 98.21
166.22 116.07 12.7 14.0 99.94 98.40
166.22 116.07 14.2 15.4 99.94 1.54
166.22 116.07 13.4 14.9 99.94 1.45
166.22 116.07 13.0 14.0 99.94 1.67
166.22 116.07 12.6 14.0 99.94 1.65
166.22 116.07 12.7 14.0 99.94 1.60
HCB
6-oxo 0.19
0.19
0.19
Table 4 - Data from HCB 1H qNMR analysis with benzoic acid as internal standard.
Mx MStd mStd PStd
m
Px
Sample Ix/10
IStd/10 NStd Nx
(g/mol) (g/mol) (mg) (mg) (%) (%)
166.22 122.12 15.1 16.0 99.97 97.37
166.22 122.12 15.7 15.3 99.97 97.77
166.22 122.12 13.4 14.0 99.97 97.41
166.22 122.12 13.4 13.9 99.97 97.69
166.22 122.12 12.4 13.5 99.97 97.54
1
2
3
4
5
3.79
3.50
3.74
3.72
3.90
10.00
10.00
10.00
10.00
10.00
2
2
2
2
2
1
1
1
1
1
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Lines represent spline interpolations
Error bars indicate standard errors of the means
“a” indicates p < 0.001 for the comparison between two indicated groups
Figure 1 - Effect of 6-oxo at concentration of 752 μmol/L on QS-induced luminescence
produced by V. harveyi strain BB886. a) Representative image showing the inhibitory
effect of 6-oxo on the luminescence produced by V. harveyi strain BB886. b) 6-oxo reduced
the luminescence produced by V. harveyi luminescence at 60.7% without interfering with
bacterial population growth.
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Figure 2 - 6-oxo inhibits the QS system AI-1 more efficiently in V. harveyi. 6-oxo at
concentration of 752 μmol/L reduced the luminescence of the strains BB120 (wild-type
strain: sensor AI-1+, sensor AI-2+), BB170 (mutant strain: sensor AI-1 mutant, sensor AI-2
wild-type) and BB886 (mutant strain: sensor AI-1 wild-type, sensor AI-2 mutant),
respectively at 89.7% ± 6.6; 63.1% ± 5.6; and 97.0% ± 2.5. When compared to the controls
the reductions were statistically significant (P < 0.05). 1
1
Further comments about the quorum sensing system in the Vibrio harveyi bacteria can be found in the
supplementary information
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Figure 3 - 1H NMR spectrum (600 MHz) of the pure 6-oxo with maleic acid (a) or
benzoic acid (b) as internal standard.
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Figure 4 - 1H NMR spectra (600 MHz, DMSO-d6) of the pure HCB with maleic acid (a)
or benzoic acid (b) as internal standard (Resonance signals highlighted by the squares
were attributed to chemical interactions between HCB and benzoic acid).
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Figure 5 - 1H NMR spectra (600 MHz, DMSO-d6) of the crude HCB (a) and the crude
HCB with benzoic acid (b)
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Scheme 1 - Chemical steps for the synthesis of 6-oxo (1)
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Graphical Abstract
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