However, complications may arise when applying this sequence to systems with multiple inequivalent 14N nuclei, especially a single 1H sitting close to multiple 14N atoms. Due to its broadband characteristics, the PM pulse saturates all 14N atoms; hence, the single 1H simultaneously experiences the RESPDOR effect from multiple 1H-14N couplings. Consequently, no reliable H-N distances are obtained. To overcome the problem, selective 14N saturation is desired, but it is difficult because 14N is an integer quadrupolar nucleus. Alternatively, 14N overtone (OT) NMR spectroscopy can be employed owing to its narrow bandwidth for selectivity. Moreover, owing to the sole presence of two energy levels (m = ± 1), the 14N OT spin dynamics behaves similarly to that of spin-1/2. This allows the interchangeability between RESPDOR and rotational-echo double-resonance (REDOR) since their principles are the same except the degree of 14N OT population transfer; saturation for the former whereas inversion for the latter. As the ideal saturation/inversion is impractical due to the slow and orientation-dependent effective nutation of 14N OT, the working condition is usually an intermediate between REDOR and RESPDOR. The degree of 14N OT population transfer can be determined from the results of protons with short distances to 14N and then can be used to obtain long-distance determination of other protons to the same 14N site. Herein, we combine the 14N OT and REDOR/RESPDOR to explore the feasibility of selective 1H-14N distance measurements. Experimental demonstrations on simple biological compounds of L-tyrosine.HCl, N-acetyl-L-alanine, and L-alanyl-L-alanine were performed at 14.1 T and MAS frequency of 62.5 kHz. The former two consist of a single 14N site, whereas the latter consists of two 14N sites. The experimental optimizations and reliable fittings by the universal curves are described. The extracted 1H-14N distances by OT-REDOR are in good agreement with those determined by PM-RESPDOR and diffraction techniques.Accurate distance measurements between proton and nitrogen can provide detailed information on the structures and dynamics of various molecules. The combination of broadband phase-modulated (PM) pulse and rotational-echo saturation-pulse double-resonance (RESPDOR) sequence at fast magic-angle spinning (MAS) has enabled the measurement of multiple  H-N distance is of importance for deeper insights into the structures and dynamics of chemical and biological systems due to the ubiquity of both proton and nitrogen. Such distance can be obtained by solid-state nuclear magnetic resonance (ssNMR) through the determination of H-N dipolar coupling, which is inversely proportional to the cube of the H-N distance. There are a few reasons why ssNMR has advantages for the H-N measurement over traditional diffraction techniques. First, ssNMR spectroscopy is applicable to various systems no matter their states, i.e., lacking long-range order or even being a disorder, which are intractable by diffraction techniques. Second, it enables the precise location of the H-atom positions, which is poorly determined by X-ray diffraction (XRD) or electron diffraction .15N and 14N. The former is preferred in ssNMR because it is a spin-1/2 nucleus; thus, it is easy to manipulate and to obtains high resolution. Many methods have been designed to measure 1H-15N distances . It makes the measurements lengthy for sufficient signal-to-noise ratio (S/N); otherwise, 1) the isotopic labeling is needed, which is not always simple and cost-effective or 2) dynamic nuclear polarization experiments are required , but it suffers from the severe quadrupolar broadening and complicated spin dynamics because 14N is an integer quadrupolar nucleus (spin I = 1). For protons, the intense 1H–1H homonuclear dipolar couplings in the solid state cause 1H line broadening and shorten the 1H coherence time. Consequently, these unfavorable characteristics of both N isotopes and H nucleus make H-N distance measurement by NMR challenging.Despite such advantages, the H-N distance measurement by NMR has difficulties due to some unfavorable nuclear characteristics of these two elements. For nitrogen, it has two stable and NMR active isotopes, istances . Neverthrequired . HoweverνR ≥ 60 kHz) with proton detection has made 14N NMR spectroscopy a routinely used method, overcoming the difficulty associated with quadrupolar interaction  pulse (21 recoupling (1H-14N distances with high accuracy at fast MAS of 70 kHz (14N quadrupolar coupling constant (CQ) and 1H-14N dipolar coupling. This broadband characteristics of PM pulse is useful when we work on systems containing a single 14N site, as shown in the previous study. However, complications may arise for systems where multiple 14N sites are present, as shown below.The development of fast magic-angle spinning  pulse , SR421 coupling , and rotcoupling    . This ap14N overtone (OT) NMR spectroscopy, where the forbidden transitions |Δm| = 2 are weakly allowed (m is the energy level) , the spin dynamics of 14N OT behaves similarly to that of spin-1/2. Hence, for a 1H-14N OT system, the working conditions under RESPDOR can also be described by rotational-echo double-resonance (REDOR) (14N OT and REDOR sequence (1H-14N OT-REDOR) to explore its feasibility for distance measurements. This sequence is firstly demonstrated using two model biological compounds of L-tyrosine.HCl (Tyr) and N-acetyl-L-alanine (AcAla) and then applied to a more complex dipeptide system of L-alanyl-L-alanine (AlaAla) that involves two inequivalent nitrogen sites in a single molecule.An alternative approach is the y level) . Becausen of 14N . The nar (REDOR)  dependin (REDOR) . Since t1H-14N OT-REDOR sequence. It is identical to the conventional S-REDOR sequence (21 recoupling (lasting for τmix) is used to recover the 1H-14N dipolar coupling and CW (lasting for τCW) is used to saturate/invert the 14N OT populations between the two energy levels. We note that since SR421 is not γ-encoded, the interval between the two SR4 blocks should be rotor-synchronized to avoid the spatial modulation of the recoupled 1H-14N dipolar couplings. For distance measurement, we measure two signals, S0 and S’, acquired without and with CW pulse, respectively, for obtaining the fraction curve ΔS/S0 = (S0–S′)/S0 as a function of τmix.sequence , where ST2 relaxation, making it dependent solely on the dipolar coupling constant as for the case of REDOR with complete inversion. However, for 14N OT, complete inversion is difficult to achieve. Subsequently, the distance is extracted by fitting the fraction curve to either the numerically exact or universal curves. For the numerically exact curve, the simulation is extremely difficult as it must work in the laboratory frame without high-field approximation, leading to time-consuming calculations . Thus, for objective fitting, we use the universal curve approach, which is derived by following the original work of Gan or later analysis of Chen and coworkers  is the population of spin state m = i under high-temperature approximation, Wij is the population transfer probability from m = i to m = j spin state, and REDOR(|Δm|) presents the normalized dipolar-dephased signal intensity for classical REDOR. The general expression helps to derive the universal curves mentioned in 14N isotope is 99.6%, very close to 100%; hence, we can safely neglect it.where 14N (I = 1) spin, under the Zeeman interaction with the external magnetic field, there are three energy levels of m = 0 and ±1. We assume the population for each level is 1/3. The population transfers among the energy levels are categorized into zero- (ZQ or Δm = 0), single- (SQ or |Δm| = 1), and double-quantum (DQ or |Δm| = 2) transitions, which determine the RESPDOR effect. Both energy levels m = +1 and m = −1 are involved in all three ZQ, SQ, and DQ transitions, as shown in 14N spin, Wj±1 for these transfers are equal; hence, each transition has Wj±1 of 1/3. On the other hand, the energy level m = 0 is only involved in ZQ and SQ transitions, but there are two SQ transitions of (m = 0 → m = −1) and (m = 0 → m = +1). Hence, for m = 0, Wj0 of ZQ, SQ, and DQ transitions are 1/3, 2/3, and 0/3, respectively. Taken together, under the complete saturation of 14N spin, ΣPiWij for ZQ, SQ, and DQ transitions for m =  are 1/3·3/3, 1/3·4/3, and 1/3·2/3, respectively (see Pi and Wij in 14N is given byFor vely see . ReplaciJ±1/4 denotes the ±1/4-order Bessel functions of the first kind and b1H–14N/(2π) is the 1H-14N dipolar coupling constant while τmix is the total mixing time of SR421 recoupling sequence. 1H-14N RESPDOR in the literature ), 0, and 1/3·2f, respectively. The universal expression for REDOR/RESPDOR on 14N OT is given byNext, we consider the case of m| = 2) is proportional to f, affecting the slope of the fraction curve. However, since f uniformly affects the other elements in the equation, the universal curves derived from τmix no matter f value. It is worth noting that the introduction of f makes the fitting among universal curves and experimental fraction curve better, but it makes the extracted b1H–14N inaccurate. Particularly, when the fraction curve has not reached the maximum ΔS/S0, universal curves generated by different combinations of f and b1H–14N can reproduce the very similar fraction curve, thus giving ambiguous results. To avoid this situation, our fitting strategy consists of two steps. The first is to determine f, which is possible only when the fraction curve of the shortest H-N distance must show the maximum. Under this condition, the fitting parameter f is determined as the ratio of the experimental and theoretical maxima ΔS/S0 (2/3 = 0.67). That precisely known f leads to the unambiguous determination of b1H–14N. For longer H-N distance, the REDOR curve may possibly not show the maximum while the oscillation is damped, making the fitting difficult. Under this situation, accurate distance determinations are still possible by the second step. It is to use this observed f from the first step for measuring longer 1H-14N distances of the same 14N site. This strategy is a disadvantage of OT-REDOR compared to PM-S-RESPDOR. The latter does not require the prior knowledge of the fitting parameter f owing to the complete saturation of all 14N crystallites by the PM pulse, thus enabling the reliable fitting even when the maximum is not observed.From 1H-14N RESPDOR and 1H-14N OT-REDOR. This can be done by comparing the universal curves resulting from b1H–14N. f = 0.5 (complete saturation) and f = 1.0 (complete inversion). These two f values are chosen because they correspond to ideal RESPDOR (f = 0.5) and REDOR (f = 1.0) conditions and our working condition is an intermediate between these two, as mentioned in the Introduction. The two curves from 1H-14N OT and that of 1H-14N is that the dephasing rate of the former curves is about two times faster than that of the latter . This is because, for 1H-14N OT, the REDOR effect is determined by the DQ transitions, whereas, for 1H-14N, the RESPDOR effect is determined by both the SQ and DQ transitions. Such a faster dephasing rate associated with the multiple quantum transitions has been known in the literature  1H-{14N OT} D- or T-HMQC (sequences in n = -2 SSB) for the highest S/N. Such n value depends on the sense of rotation with respect to the magnetic field (14N OT SSB (n = −2). 14N OT frequency and its correlations to both proton sites of NH3 and CH by the D-HMQC experiment. The smaller correlation of N to CH under τmix of 0.51 ms is explained due to the longer 1H-14N distance compared to the directly bonded H-N distance of the NH3 group. This 2D spectrum is in agreement with the 1H-{14N} D-HMQC spectrum in the previous study  under varying τCW values.After the approach . However1H-14N OT-REDOR experiment on Tyr. 1H spin echo (S0), the dephased (S′), and the difference (S0–S’) spectra, in which the former two were extracted from the 1H-14N OT-REDOR experiment with τmix of 0.51 ms, whereas the latter is calculated from the former two. The NH3 and CH sites are evidently assigned in H3 and CH are shown in H3 in S/S0 is reached at τmix of 0.6 ms. In our previous study, the 1H-14N PM-S-RESPDOR fraction curve shows the maximum at τmix of ∼1.4 ms  to the experimental fraction curves (black circles) presented in τmix of ∼1.8 ms, the fitting by the universal curves is only up to τmix of ∼1.0 ms, owing to the poor agreement between the experimental and universal curves for NH3 at τmix > 1.0 ms  in 1H-14N dipolar couplings. It is noted that, for NH3 (2(cos(θ))  is used for the dynamic average of the N-H dipolar coupling due to the threefold rotation. The 1H-14N distances by OT-REDOR are shown in 1H-14N distances. It is worth noting that the longer distances by ssNMR than those from neutron result from the different vibrational averages of the internuclear distances of the two techniques  determination of 14N OT resonance frequency, 2) optimization of τCW, and 3) implementation of OT-REDOR. These experiments were all performed at the second 14N OT SSB for the highest S/N. For step 1, the 1H-{14N OT} T-HMQC was performed  at τmix of 0.19 ms (or three loops of SR421 recoupling blocks) and CH (red circles) at τmix of 0.96 ms (or 15 loops of SR421 recoupling blocks) under identical τCW range is shown in τmix is due to the large difference between 1H-14N distances for these proton sites. The optimum τCW of 0.192 ms for 14NH in AcAla is shorter than τCW of 0.464 ms for 14NH3 in Tyr. This result is expected since the 14N site of NH has a larger quadrupolar interaction, thus resulting in a larger 14N OT nutation field and shorter pulse length for efficient saturation/inversion.To further demonstrate the feasibility of OT-REDOR for a system with a larger erformed . Again, 1H spin echo (S0), the dephased (S′), and the difference (S0–S′) spectra. The former two were extracted from the 1H-14N OT-REDOR experiment with τmix of 0.26 ms, whereas the latter results from the difference of the former two. For readability, the intensity of the difference spectrum is triple, showing the NH and CH sites which experience the REDOR effect. These two sites are also located in the molecular structure of AcAla , AcAla experiences the saturation of overall magnetization (f = 0.50). The difference may arise from the large frequency linewidth  at the top, S0–S’) spectra where 1) NH3 and 2) NH sites are saturated/inverted during 1H-14N OT-REDOR experiments with τmix of 0.51 ms and 0.19 ms, respectively. The NH, NH3, CH(1), and CH(2) sites are unambiguously assigned  for the highest S/N to determine 14N OT frequencies . Then, τCW for 14NH3 and 14NH of AlaAla were optimized, which were 0.384 and 0.128 ms, respectively  (black circles) are shown in H3, the experimental curve shows the oscillation and the maximum ΔS/S0 is 0.63 , similar to the case of NH3 of Tyr. Another similarity to Tyr is that significant deviation is observed, especially at long τmix for the full-scale fitting between the fraction curve of H(1), the fraction curve reaches the plateau ΔS/S0 of 0.35 at τmix of 1.50 ms  and CH(2) sites, in which only CH(1) is close to 14NH3. For a good match with the experimental fraction curve, we need to halve the universal curves in 1H-14N distance by NMR is in poor agreement with that reported from XRD (see 1H-14N OT-REDOR fraction curves gives the 1H-14N distances of 1.05 ± 0.03 Å for NH3 (after a modulation factor of P2(cos(θ)) as described in L-Tyrosine.HCl and Pulse Sequence and the Universal Expression and 2.09 ± 0.05 Å for CH(1) . Both measured distances are in good agreement with those reported from XRD /CH(1) (black circles) are shown in H in S/S0 of 0.25 at τmix of 0.50 ms and then large fluctuations of ΔS/S0 at τmix larger than 1.50 ms. The origin of such fluctuations may be due to t1 noise from spinning frequency fluctuation  to the 1H-14N OT-REDOR fraction curve, the 1H-14N distance is measured to be 1.19 ± 0.20 Å. This value is in excellent agreement with 1H-15N distance by inverse cross-polarization with variable contact (CPVC) /CH(1) fraction curve, its fraction signal ΔS/S0 is larger than 0.25 and continues to grow at long τmix   (Supplemt (CPVC)  and in ct (CPVC) . An advasite see . For CH, N-acetyl-L-alanine (AcAla), and L-alanyl-L-alanine were purchased from Sigma-Aldrich and used as received. The samples were separately packed into 1.0 mm zirconia rotors and then inserted into 1 mm 1H and 14N OT Larmor frequencies are 600.0 and 86.8 MHz, respectively. For the highest S/N, the 14N OT frequency was set at the second SSB (n = −2). The 14N and 14N OT shifts are referenced to CH3NO2, whose 14N and 14N OT shifts are equal to 0 ppm or 0 kHz. The 1H rf-field was 328 kHz for π/2 and π pulses and 140 kHz for the SR421 recoupling sequence. The 14N OT rf-field was 120 kHz.All NMR experiments were recorded at a room temperature of 25 °C on JNM-ECZ600R (JEOL RESONANCE Inc.) at 14.1 T solid-state NMR spectrometers. The 1H-{14N} D-HMQC spectrum in t1 points, and rotor-synchronized t1 increment of 16.0 µs. τp, τmix, and recycling delay (RD) were 200 µs, 512 µs, and 4 s, respectively. The experimental time was about 0.6 h. The States-TPPI method was employed for quadrature detection along the indirect dimension. For τCW was optimized within the range from 16 µs to 656 µs with a step of 32 µs; the 14N OT frequency was −1.73 ppt (parts per thousand), the τmix was fixed at 1152 µs, the number of scans (NS) was 18, and RD was 5.0 s. The experimental time was 1.1 h. For 1H-14N OT-REDOR was performed at τCW of 464 µs, 14N OT frequency of −1.73 ppt, NS of 72, RD of 6.5 s, and τmix from 0 to 3456 µs with a step of 64 µs. The experimental time was 14.3 h.For Tyr, the 2D τCW optimization in 14N OT frequency was −1.267 ppt, NS was 18 and RD was 10.0 s, and τmix was fixed at 192 µs for NH at 7.9 ppm and 960 µs for CH at 3.9 ppm. The experimental times for both experiments were 1.5 h. For 1H-14N OT-REDOR was performed at τCW of 192 µs, 14N OT frequency of −1.267 ppt, NS of 108, RD of 10 s, and τmix from 0 to 2048 µs with a step of 64 µs. The experimental time was 19.8 h.For AcAla, the 1H-14N OT-REDOR experiments in τmix from 0 to 3072 µs with a step of 64 µs, and τCW, 14N OT frequencies were of 384 µs, −1.72 ppt and 128 µs, −1.26 ppt for 14NH3 and 14NH, respectively. The experimental times for both experiments were 9.8 h.For AlaAla, the NMR data are available upon request.


Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT.l-Trp via two enzyme cascades for the first time. First, a chassis cell that can convert l-Trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous tetrahydrobiopterin (BH4) module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 25.9 mol%) at the optimal conditions .Here we propose a one-pot production of 5-HT from l-Trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.This protocol provided an efficient one-pot method for converting. The online version contains supplementary material available at 10.1186/s12934-022-01745-0. Amongecursors .Moringa Oleifera seeds) [l-Trp in the nervous system of human and animals via two enzymatic steps: l-Trp is hydroxylated to 5-HTP by pterin-dependent tryptophan hydroxylase (TPH), then converted to 5-HT by tryptophan decarboxylase (TDC), which is one of the Aromatic amino acid decarboxylase [l-Trp or 5-HTP (prefer to l-Trp), with the result that a one-pot cascade reaction cannot be performed and has therefore never been reported [Currently, the main supply of 5-HT depends on the extraction from mammals  or plants  . Howeverboxylase . Howeverboxylase . Aromatireported , 11 (Alll-dopa to dopamine [l-Trp [Dopa decarboxylase (DDC), another AAAD, is able to convert dopamine . In addie [l-Trp , 14. Howe [l-Trp , 15.l-Trp into 5-HT using a whole-cell factory for the first time. First, artificial endogenous cofactor tetrahydrobiopterin (BH4) and tetrahydromonapterin (MH4) modules were constructed and compared to catalyse l-trp into 5-HTP using a tryptophan hydroxylase (TPH) from Schistosoma mansoni with high stability and catalytic ability. Then, a DDC from Harmonia axyridis that specifically catalyses 5-HTP to produce 5-HT was introduced to the chassis cell to form the cell factory. The optimal conditions of the recombinant cell for 5-HT production were also studied.In this study, we proposed a one-pot enzymatic catalytic approach for converting l-Trp, a chassis cell that could efficiently convert l-Trp to the intermediate product 5-HTP, needed to be constructed first. Previous studies have reported that TPH from S. mansoni (SmTPH) possesses good activity and stability compared that of other resources [SmTPH was chosen for converting l-Trp into 5-HTP in this study. However, SmTPH is a cofactor-dependent enzyme and requires the expensive cofactor biopterin [4) and tetrahydromonapterin (MH4) modules were constructed in the chassis cell containing SmTPH [4 and MH4 increased until 60 h of fermentation, then decreased with the further increase of time. The observed decrease in 5-HTP concentration may have been due to the oxidation and degradation of 5-HTP as the fermentation time increased [4 and MH4, respectively. Zhang et al. reported that the production of 5-HTP from SmTPH using BH4 as a cofactor was higher than that when using MH4 as a cofactor in S. cerevisiae, which correlated with the findings of our study [4 and SmTPH was used in the next experiment.To produce 5-HT from esources ; therefoiopterin , 17. To ng SmTPH . As showur study . TherefodrDDC) has attracted significant attention, due to its activity for both 5-HTP and l-DOPA but with no activity to l-Trp [Harmonia axyridis  for 5-HT synthesis.Dopa decarboxylase from Drosophila melanogaster △tnaA. The recombinant HaDDC was purified by a Ni–NTA resin, with a recovery yield of 80.3%.As shown in Additional file HaDDC, l-Trp and 5-HTP were used as substrates respectively. As shown in Fig. l-Trp within 4 h or longer (data not shown), indicating that the recombinant HaDDC had no activity toward l-Trp. Meanwhile, as shown in the Fig. l-Trp. It was clearly that 5-HT was released within 4 h by HaDDC with 5-HTP as a substrate in the Fig. HaDDC exhibited strict substrate specificity similar to that displayed by drDDC from D. melanogaster [HaDDC exhibited a high efficiency on 5-HTP with a yield of 88.45% at 4 h, which was much higher than that of TDC (16.04%) from Catharanthus roseus which was higher than TDC reported previously [HaDDC from H. axyridis was chosen for converting 5-HTP into 5-HT △tnaA/BH4/HaDDC-SmTPH and E. coli BL21(DE3)△tnaA/BH4/SmTPH-HaDDC, respectively. The production of 5-HT was obtained in the former but not in the latter (data not shown). To explore the reasons, SDS-PAGE was used to analyse the protein expression of E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH and E. coli BL21(DE3)△tnaA/BH4/SmTPH-HaDDC. As shown in Fig. E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH exhibited better protein expression than E. coli BL21(DE3)△tnaA/BH4/SmTPH-HaDDC with HaDDC in the second multiple cloning site. This phenomenon may be attributed to the insert position of the HaDDC gene, which affects the expression of both enzymes. Thus, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH was chosen to produce 5-HT from l-Trp. As shown in Fig. l-Trp concentration decreased continuously from 2.00 to 0.899 g/L within 96 h. Meanwhile, the 5-HT concentration increased as time increased and reached the highest concentration (0.3136 g/L) at 84 h, which was 2.04-fold higher than that obtained from the two-stage fermentation process by CtAAAH and TDC [l-Trp could be also degraded by other tryptophanases in E.coli and caused the broth turned dark [The enzyme cascade of purified ime Fig. . Meanwhi and TDC . It may ned dark –24.Fig. l-Trp concentration on 5-HT production was illustrated in Fig. l-Trp concentration from 0.5 to 2.0 g/L before 48 h, followed by decreasing slightly. However, as the concentration of l-Trp increased to 2.5 g/L, the 5-HT concentration lower than that from 1.0, 1.5, 2.0 g/L l-Trp. Therefore, 2.0 g/L l-Trp was chosen for subsequent experiments.To determine the optimal conditions for 5-HT production, a number of catalysis conditions were investigated. The effect of The effect of induction temperature on the production of 5-HT is shown in Fig. The effects of concentration of IPTG on 5-HT synthesis were investigated △tnaA/BH4/HaDDC-SmTPH, then decreased. Thus, the optimal time for induction was 12 h.As shown in Fig. l-Trp increased as the catalysis temperature increased from 25 to 30 ℃, and the maximum concentration of 5-HT  was reached at 30℃. However, with the temperature increased from 30 to 40℃, the yield of the product decreased rapidly.The relationship between 5-HT concentration and catalysis temperature was presented in Fig. E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH were 12 h for the expression of proteins at 25 ℃ with 0.05 mM IPTG, and the optimal conditions for catalysis were 30 ℃ for 48 h. The highest yield of 5-HT from l-trp was 25.9 mol%.Based on these results, the optimal conditions for 5-HT production of one-pot catalysis using l-Trp into 5-HT was successfully constructed. First, a chassis cell that contained a screened artificial endogenous BH4 module as a cofactor and tryptophan hydroxylase from Schistosoma mansoni (SmTPH) was constructed, which could convert L-Trp into 5-HTP efficiently. The dopa decarboxylase from Harminia axyridis (HaDDC) that can specific catalyse 5-HTP to 5-HT was chosen for 5-HT production. Finally, the two enzymes were cascaded in vivo for converting L-Trp to 5-HT, which lead to a yield of 414.5 ± 1.6 mg/L at the optimal conditions. This study provides a novel and important foundation for artificial cell factory synthesis of 5-HT.In this study, a cell factory capable of efficiently converting l-Tryptophan (l-Trp), 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT) standards were purchased from Aladdin Industrial Corporation . (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4), pyridoxal 5-phosphatemonohydrate (PLP) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . Other chemicals and solvents in this study were purchased from local suppliers and were of analytical grade or higher purity.E.coli Trans1T1 was used for plasmid construction and propagation, and E. coli BL21(DE3) was used for protein expression. As tryptophanase A (tnaA) gene, which encodes tryptophanase, is necessary to accelerate degrade l-Trp and 5-HTP into indole and 5-hydroxy indole in E.coli, respectively. Therefore, tnaA gene should be knocked out in E. coli BL21(DE3) to ensure the biosynthesis of 5-HTP or 5-HT, using the previously established CRISPR/Cas9 protocol [protocol . The expBacillus subtilus were codon-optimized, synthesized and inserted into pCDF-Duet-1 to acquire E. coli BL21(DE3)△tnaA/BH4 [4), whose relative genes were also constructed in pCDF-Duet-1, was used to acquire E. coli BL21(DE3)△tnaA/MH4 according to a previous study [SmTPH from Schistosoma mansoni was codon-optimised for E. coli expression, synthesised, and inserted into pET-28a( +), then transferred into E. coli BL21(DE3)△tnaA/BH4 and E. coli BL21(DE3)△tnaA/MH4 to acquire E. coli BL21(DE3)△tnaA/BH4/SmTPH and E. coli BL21(DE3)△tnaA/MH4/SmTPH. HaDDC from Harminia axyridis was also codon- optimised for E. coli expression, synthesized and inserted into pET-28a ( +), then transferred into E. coli BL21(DE3)△tnaA to acquire E. coli BL21(DE3)△tnaA/HaDDC.Plasmid construction and DNA manipulation were performed following the standard molecular cloning protocols. First, the PTPS, SPR, PCD, DHPR human genes and mtrA from us study . SmTPH fHaDDC was amplified from pET-28a ( +)-HaDDC using the primer pairs, HaDDC-F-BamHI, HaDDC-R-HindIII, HaDDC-F-EcoRI, and HaDDC-R-SalI. First, BamHI- and HindIII-digested polymerase chain reaction (PCR) products of HaDDC were ligated to BamHI- and HindIII- digested pET28a ( +)-SmTPH and EcoRI- and SalI- digested pET28a ( +)-SmTPH to construct the plasmids pET28a ( +)-SmTPH-HaDDC and pET28a ( +)-HaDDC-SmTPH. The plasmids were constructed in E. coli Trans1T1 and transferred into E. coli BL21(DE3)△tnaA/BH4 a to acquire E. coli BL21(DE3)△tnaA/BH4/SmTPH/HaDDC and E. coli BL21(DE3)△tnaA /BH4/HaDDC/SmTPH. The plasmids and strains along with the primers and specific structures of the plasmids, are shown in Table The gene fragment of li BL21DE△tnaA/BH4l-Trp to 5-HTP [600 reached 0.4–0.6, the cells were induced with isopropyl-β-d-thiogalactoside (IPTG) for 12 h at either 25℃ (E. coli BL21(DE3)△tnaA/BH4/SmTPH) or 18℃ (E. coli BL21(DE3)△tnaA/HaDDC).Luria–Bertani (LB) medium was used for cell cultivation and enzyme expression . Modifieto 5-HTP . All theE. coli BL21(DE3)△tnaA/BH4/SmTPH and E. coli BL21(DE3)△tnaA/MH4/SmTPH were centrifuged at 4000 rpm for 10 min, and the cells were respectively transferred to M9Y media containing 2 g/L of l-Trp. Sampling and measuring were conducted at 12-h intervals.The fermentation broth of HaDDC was purified using a fast protein liquid chromatography (FPLC) system  with a Ni-nitrilotriacetic acid affinity chromatography (Ni–NTA) column (His Trap™ FF5 mL) according to the manufacturer’s instructions. Protein concentrations were determined at 595 nm using the Bradford method with bovine serum albumin as the standard [SmTPH was same as that for HaDDC.The cells harvested from cultures were washed, resuspended in 50 mM HEPES (pH 7.0), and lysed at 4 ℃ by JY92-IIN ultrasonication . The lysates were centrifuged at 12,000 rpm at 4 ℃ for 20 min, and the supernatant was used as a crude enzyme solution. The recombinant standard . All proHaDDC activity was assayed at 35 ℃ in a reaction mixture of 2 mL, containing 50 mM HEPES (pH 7.0), 0.4 mM PLP, 50 μL purified HaDDC (protein concentration of 15 g/L) and 200 μM L-Trp or 5-HTP.The recombinant SmTPH and HaDDC was conducted in a reaction system (2 mL) that contained 7 mM DTT, 0.1 M ferrous ammonium sulfate, 0.1 mg/mL catalase, 0.6 mM BH4, 0.1 mM PLP, 0.5 mM L-Trp, 100 µL purified SmTPH (1.0 g/L), 100 µL purified HaDDC (1.0 g/L) and 50 mM HEPES (pH 7.0), and incubated at 30 ℃ [The in vitro cascade of  at 30 ℃ .SmTPH and HaDDC was performed as follows: the E. coli BL21(DE3)△tnaA/BH4/SmTPH/HaDDC and E. coli BL21(DE3)△tnaA /BH4/HaDDC/SmTPH fermentation broths were centrifuged at 4000 rpm for 10 min and the cells  were transferred to 100 mL of M9Y media containing 2 g/L L-Trp [The in vivo cascade of Cells were transferred to 100 mL of M9Y medium in a 500 mL flask and shaken at 200 rpm in various conditions.In this study, single-factor experiments using five predominant factors  were conducted to investigate the effects of these factors on the production of 5-HT . InvestiThe investigation was carried out at varying concentrations of L-Trp , IPTG , induced time , induced temperature , and catalysis temperature .600) was measured using an UV1000D ultraviolet–visible spectrophotometer . High-performance liquid chromatographic (HPLC) analysis of the L-Trp, 5-HTP and 5-HT contents was performed on an Agilent 1260 series LC system  with an ultraviolet detector reading at 276 nm [l-Trp, 5-HTP and 5-HT standards using a calibration curve. Tryptamine standard was eluted via column chromatography  in the following gradients: methanol (solvent A) and 0.1% formic acid in water (solvent B): 0–15 min 10% A; 15–16 min, 55% A; 16–22 min, 100% A; 22–26 min, 10% A; the flow rate was 0.8 mL/min, and the reaction absorption was recorded at 280 nm.The cell growth density at 600 nm .5-HT yield (mol%) = 5-HTP released (mol)/Additional file 1: Fig. S1. SDS-PAGE analysis of different induction temperatures on the protein expression of SmTPH. Fig. S2. SDS-PAGE of cell extracts of E.coli BL21(DE3)/pET28a-DDC. Fig. S3. Effects of different induction temperatures on the catalysis in 5-HTP of HaDDC. Fig. S4. Effects of different pH on the catalysis in 5-HTP of HaDDC. Fig. S5. Effects of different PLP concentration on the catalysis in 5-HTP of HaDDC. Fig. S6. Phylogenetic analysis of HaDDC with other DDCs. Fig. S7. Plasmids construction of SmTPH gene and HaDDC gene at different cloning sites in the pET-28a ( +). Table S1. The synthesized protein sequence applied in this study.


Their structures were elucidated by extensive NMR and HRESIMS spectrometric analyses. All compounds were evaluated for immunosuppressive activity. Fortunately, compound 2 exhibits certain inhibition specifically against the LPS-induced proliferation of B lymphocyte cells with IC50 value 16.16 μM.Three new pyrrole alkaloids albifipyrrols A–C (The online version contains supplementary material available at 10.1007/s13659-022-00327-2. The human immune system is a complex network of defensing against foreign invaders. Autoimmune diseases arise when the immune system fails to distinguish between self and non-self , 2. ImmuTaxomyces andreance , seven aromatic or olefinic methines and four nonprotonated carbons [including one ketone carbonyl at δC 197.4 (C-7)]. Among them, one benzene ring, an acetyl group and four olefinic carbons occupied seven degrees of unsaturation. Hence, the remaining one degree of unsaturation can only be due to the presence of one ring. The key HMBC correlations  on the pyrrole nucleus were also confirmed at C-3, C-4 based on the HMBC correlations from H-8 to C-3 and from H-6 to C-3/C-4/C-5. Compound 1 was, therefore, established as albifipyrrol A, as depicted.Compound 2 was obtained as yellow oil. The molecular ion peak of HR-ESI–MS was at m/z 280.1306 [M + Na]+ , which deduced that the molecular formula of compound 2 was C16H19NO2, with eight degrees of unsaturation. The 1H-NMR and 13C-NMR data , which indicated that the molecular formula of compound 3 is C12H17NO4, with five degrees of unsaturation. The 1H-NMR ; δC 52.2 (C-5′)], one carboxyl group [δC 174.8 (C-4′)], three methylenes . The methyl butyrate unit was established by the 1H-1H COSY correlations between H2-1′, H2-2′ and H2-3′ and the key HMBC correlations  induced T cell proliferation and lipopolysaccharide (LPS) induced B cell proliferation. Compound 18 reversed phase column  .1D and 2D NMR spectra were recorded on Bruker DXR-600 instrument (600 and 150 MHz) and Bruker DXR-500 instrument (500 and 126 MHz). The UV data were detected by Hitachi UH5300 spectrophotometer . IR spectra were conducted on IRT racer-100  with KBr pellets. HR-ESI–MS data were obtained on a UPLC-Q Exactive MS system . The packing for column chromatography (CC) is silica gel  or Sephadex LH-20 . The semi-prepared HPLC was carried out on an Agilent Technologies 1260 Infinity II system with a diode array detector. And the chromatographic column was CC. chinensis collected from Enshi, Hubei province, and was identified as Albifimbria viridis via 18S rDNA sequences and deposited at South-Central University for Nationalities, China. The sequence data for this strain had been submitted to the DDBJ/EMBL/Genbank with accession No. MT110686.1.The strain were isolated from the roots of Albifimbria viridis was fermented on solid rice medium  and was cultured at 30 °C for one month. The fermented material was soaked in absolute methanol (20 L × 4). The combined extracts were evaporated under reduced pressure to afford an crude extract, which was further dissolved in water and extracted three times with EtOAc (10 L × 4) to yield 110 g of the extract. The crude extract was subjected to silica gel column chromatography  to yield six fractions (A‒F). Fraction B (6 g) was separated into eight sub-fractions (B1‒B8) by ODS MPLC. The eluent is composed of methyl alcohol: H2O . Fraction B3 was purified by semi-preparative HPLC  to give compound 1 . Fraction C (5 g) was isolated from Sephadex LH-20 eluting with MeOH and purified by semi-preparative HPLC  to obtain compound 2 . Fraction D (7.5 g) was isolated by Sephadex LH-20 column chromatography (MeOH) to obtain six sub-fractions (D1‒D6). Fraction D5 was purified by semi-preparative HPLC  to yield compound 3 .The fungus max (log ε): 210 (1.97). HR-ESI–MS m/z found 266.1150 [M + Na]+ . IR (KBr) νmax (cm–1): 3401, 2949, 2837, 1655, 1450, 1117, 1024. 1H and 13C-NMR see (Table Yellow oil. UV (MeOH) λm/z found 280.1306 [M + Na]+ . IR (KBr) νmax (cm–1): 3364, 2945, 2833, 1670, 1452, 1119, 1032. 1H and 13C-NMR see (Table Yellow oil. UV (MeOH) λmax (log ε): 210 (1.82). HR-ESI–MS max (log ε): 255 (2.01). HR-ESI–MS m/z found 262.1046 [M + Na]+ . IR (KBr) νmax (cm–1): 3400, 2950, 1734, 1632, 1526, 1157. 1H and 13C-NMR see (Table Yellow oil. UV (MeOH) λ: 255 2.0. HR-ESI–6 cells) were cultured in triplicate on a 96-well plate for 48 h at 37 °C in a humidified incubator containing 5% CO2 (with or without different concentrations of compounds). During the last 8 h of culture, a certain amount of CCK-8 was added to each well. At the end of culture, the OD values at 450 nm was measured by a bio-RAD 650 microplate reader. Cells with viability above 85% were further screened for their inhibitory activity against T and B lymphocytes. The 5 × 105 spleen cells were cultured at the same conditions as those mentioned above. T cell or B cell proliferation was induced with 10 µg ml−1 of LPS or 5 µg ml−1 of ConA, respectively. Proliferation was assessed in terms of uptake of [3H]-thymidine during 8 h of pulsing with 25 µL/well of [3H]-thymidine, and then cells will be harvested onto glass fiber filters. The incorporated radioactivity was counted using a Beta scintillation counter . Cells treated without any stimuli were used as negative control. The immunosuppressive activity of each compound was expressed as the concentration of compound that inhibited ConA induced T cell proliferation or LPS-induced B cell proliferation to 50% (IC50) of the control value. Both the cytotoxicity and proliferation assessment repeated twice. Cyclosporin A (CsA) an immunosuppressive agent, was used as a positive control . Spleen cells  spectrum of compound 1. Figure S2.13C NMR  spectrum of compound 1. Figure S3. HSQC spectrum of compound 1. Figure S4. COSY spectrum of compound 1. Figure S5. HMBC spectrum of compound 1. Figure S6. HRESIMS of compound 1. Figure S7. UV spectrum of compound 1. Figure S8. IR spectrum of compound 1. Figure S9.1H NMR  spectrum of compound 2. Figure S10.13C NMR  spectrum of compound 2. Figure S11. HSQC spectrum of compound 2. Figure S12. COSY spectrum of compound 2. Figure S13. HMBC spectrum of compound 2. Figure S14. HRESIMS of compound 2. Figure S15. UV spectrum of compound 2. Figure S16. IR spectrum of compound 2. Figure S17.1H NMR  spectrum of compound 3. Figure S18.13C NMR  spectrum of compound 3. Figure S19. HSQC spectrum of compound 3. Figure S20. COSY spectrum of compound 3. Figure S21. HMBC spectrum of compound 3. Figure S22. HRESIMS of compound 3. Figure S23. UV spectrum of compound 3. Figure S24. IR spectrum of compound 3.


As Yb2O3-MgO was added into the BT-NBT, the phase changes from tetragonal to pseudo-cubic, with the tetragonality c/a decreases from 1.011 to 1.008 and XRD peaks broadened. The combined study of XRD and TEM image revealed a formation of core–shell structure in grains with core of 400–600 nm and the shell of a thickness 60–200 nm. There is a slowly phase transition against temperature from the variable temperature Raman analysis. The ferroelectric relaxor peak of BT-NBT decreases from ~4000 to ~2000 and a new broad dielectric peak with an equivalent maximum (εr′~2300) appears in the temperature dependent dielectric constant curve (rε′-T), which produces a flat rε′-T curve. Sample 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.005 MgO and 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.01MgO give a εr′ variation within ±14% and ±10% in 20–165 °C. The core–shell microstructure should take account for the flattened εr′–T behavior of these samples.Dielectric properties and structure of 0.015Yb The c/a = 1 for an ideal cubic phase and c/a > 1 for tetragonal BT phase. However, for c/a ≈ 1, it is hard to recognize between tetragonal and cubic ones, which are considered as a pseudo-cubic phase. The undoped BT-NBT ceramics give an obvious tetragonal perovskite structure with separated (200) (020) peak and (002) peak  peak b. The teinements c is 1.01 ≈ 1.008 c. The teg et al.  proposedP4mm, #99) has the eight first-order Raman active modes (3A1 + B1 + 4E). The broad modes locate at ~118, 152, 188, 289, 509, and 737 cm−1 mean a local symmetry fluctuation in the cubic background (pseudo-cubic). Refer to results of Ba(ZrxTix1−)O3 [x1−Srx)TiO3 ceramics [−1 to A1(TO1), E(TO2), A1(TO3), and A1(LO3) modes, respectively.The room temperature depolarized Raman spectra of BT-NBT and BT-NBT-YM ceramics are plotted in Ti1−x)O3  and  ceramics are plotted in E(TO2) mode at ~307 cm−1 is considered to be the key modes associates with the tetragonal-cubic phase transition [The temperature-dependent Raman spectra of BT-NBT and BT-NBT-YM (ansition  in compl3 with a low Curie temperature (shell) and undoped-BaTiO3 with a high Curie temperature (core) is known to show a flat permittivity over a wide temperature. To confirm the “core–shell” structure in the BT-NBT-YM samples, we investigated the TEM image of a selected sample (x = 0.015). A typical TEM image of the ceramic is shown in Generally, a composite of doped-BaTiOObvious “core–shell” grains and grain boundaries can be recognized. There are ferroelectric domain patterns (A) in the core region of grain surrounded by paraelectric shell region (B) of grain and grain boundary (C). For the selected grain, the size of the core (A) is 400–600 nm and the thickness of the shell is 60–200 nm. The crystal structure of the core and shell region should be tetragonal phase and cubic/pseudo-cubic phase, respectively. The crystal structure between them should be quite similar and the small size of core region and shell region causes the broadening of their XRD peaks, which makes the character peaks of them hard to be distinguished in the overlapped XRD patterns.εr′) and the dielectric loss (tanδ) of the BT-NBT-YM samples are shown in εr* = εr′ − jεr″) of samples are shown in εr′–T  but the decreases with the increase of frequency at higher T (>180 °C). The dielectric loss at higher and lower temperature here is named as tanδ′ and tanδ″, respectively. The tanδ″ is almost independent with T but the tanδ′ is greatly influenced by temperature T.The temperature-dependent real permittivity (2O3 was added into the BT-NBT (BT-NBT-Yb2O3), the peak “A” decreases from ~4000 to ~2000 and a new broad dielectric peak (labelled as “B” in εr′~2300) appears in εr′–T curve. The two-peak like behavior is also observed in the εr″–T curve (εr′–T peaks (both A and B) slightly decrease but the imaginary εr″–T peaks slightly increase with the increase of frequency, which means that both are ferroelectric relaxor responses. As MgO was added to the Yb2O3-BT-NBT forming BT-NBT-YM, the dielectric behavior is similar to that of Yb2O3-BT-NBT system. With the increase of x (MgO), the dielectric peak “A” almost keeps unchanged (~2200). However, the peak “B” shifts to lower temperature for x ≤ 0.01 with the position almost unchanged but the maximum increases for x ≥ 0.015, e.g., εr′ = 2300 at 325 K for x = 0.005, εr′ = 1900 at 323 K for x = 0.01, and εr′ = 2500 at 330 K for x = 0.02, at 200 kHz. Thus, YM significantly improves the ferroelectric relaxing performance. The dielectric loss especially tanδ′ decreases at x ≤ 0.01 but keeps almost unchanged at x ≥ 0.015. Sample 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.005MgO and 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.01MgO give the best ferroelectric relaxor behavior, which give an εr′ variation within ± 14% and ± 10% in 20–165 °C at 200 kHz.As 1.5 mol % Yb–T curve . Similarεr′–T behavior of BT-NBT-YM here should be caused by the “core–shell” microstructure. The broad permittivity temperature characteristics should be originated from the compositional inhomogeneity in grains, the dielectric behavior should be a superposition of the characteristics of ferroelectric grain core (peak A), paraelectric grain shell (peak B) and concentration gradient region between them according to ref. [2O3 and MgO in this study) from the liquid matrix containing BT-NBT during sintering, and the core region is essentially large BT-NBT grains. Though it is not distinguished in our XRD analysis for BT-NBT-YM samples due to the sensitivity and peak broadening, the samples should be in a complex subtle inhomogeneous microstructure and the tetragonality of core should be larger than that of the shell. Thus, for the observed εr′–T behaviors, the peak A should be dominated by the ferroelectric core . Both theoretical and experimental details on the mechanism of the ferroelectric relaxation of BT-NBT-YM need to be investigated in our future studies.Refer to the studies reported on BT-based dielectric materials and our XRD, TEM, and Raman results, the observed  to ref. , Jeon et2O3-x mol MgO doped 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3 ceramics with x = 0.0–0.025 are prepared by the solid state reaction method. The ceramics were analyzed by x-ray diffractions, dielectric measurements, TEM and Raman spectra. As Yb2O3-MgO added, the phase changes from tetragonal to pseudo-cubic, with the tetragonality c/a decreases from 1.011 to 1.008 and XRD peaks broadened obviously. There is a slow phase transition with the temperature changed from variable temperature Raman measurements and core–shell grain microstructures with core of 400–600 nm and a shell of 60–200 nm thickness is observed. A new broad peak in temperature dependent dielectric constant curve is also observed as Yb2O3–MgO added into the 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3, which improves the overall relaxor behavior. Sample 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.005MgO and 0.92BaTiO3-0.08(Na0.5Bi0.5)TiO3-0.015Yb2O3-0.01MgO give the best ferroelectric relaxor behavior, which give an εr′ variation within ± 14% and ± 10% in 20–165 °C.1.5% Yb


A pyrimidine analog, 5 fluoro-uracil is a chemotherapeutic drug used for treating multiple solid malignant tumors. But its application is limited, as it has side effects like low bioavailability and high toxicity. Molecular docking is an exemplary tool, helps in identifying target and designing a drug containing high bio-availability and minimum toxicity.1,2,4-oxadiazole derivatives exhibited significant anti-cancer activity when they were evaluated, against human cancer cell lines. They also showed anti-inflammatory, analgesic, diabetic, immunosuppressive, α,β1HNMR, 13CNMR and Mass spectral analysis. Further, these compounds were investigated for their anticancer activity towards a panel of four human cancer cell lines such as , lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Among them, compounds 7a, 7b, 7c, 7d and 7i demonstrated more promising anticancer activity than standard.A set of 1,2,4-oxadiazole linked 5-fluoruracil derivatives (7a–j) were synthesized and their structures were confirmed by Synthesized derivatives (7a–j) of 1,2,4-oxadiazole linked 5-fluorouracil and investigated for their anticancer activity towards a panel of four human cancer cell lines.The online version contains supplementary material available at 10.1186/s13065-021-00757-y. The reaction mixture was stirred at 0 °C for an additional half an hour. To this mixture, (1E)-4--yl)methyl)-Nʹ-hydroxybenzamidine (5)  was added and stirring was continued for another half an hour at 0 °C. The reaction mixture was slowly allowed to attain room temperature with continuous stirring. Then it was heated to 110 °C for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled down to the room temperature, to obtain the solid product, which was then filtered and washed with CH2Cl2. The resultant white solid was heated for 3 h by dissolving it in a mixture of ethanol (20 ml), sodium acetate  and water (3 ml). The reaction mixture was then cooled down to room temperature. The resultant solid was filtered and purified by recrystallization using ethanol to obtain the desired solid compound 7a with 32% (210.4 mg) yield.A solution of substituted benzoic acid : 13C NMR : +.Mp: 316–318 °C, IR (KBr): 3176 (N–H stretching vibration), 2965 (Ar–C–H str.), 2944 (C–H str.), 1642 (NH(C=O)–C=C– stretching vibration of amide), 1598, 1425 (C=N and C=C stretching vibration of pyridine Nucleus), 1065 (C–F stretching vibration), 776 (2-Substituted pyridine), 7b was prepared following the method described for the preparation of the compound 7a, employing 5  with 3,4,5-trimethoxybenzoic acid (6b) , EDC·HCl  and sodium acetate  to afford pure compound 7b, 308.4 mg in 38% yield. Mp: 330–332 °C,IR (KBr): 3181 (N–H stretching vibration), 3062 (Ar–C–H str.), 2923 (C–H str.), 1657 (NH(C=O)–C=C– stretching vibration of amide), 1602, 1403 (C=N and C=C stretching vibration of pyridine Nucleus), 1093 (C–F stretching vibration), 773 (2-Substituted pyridine), 1HNMR : 13CNMR : +.The compound, 7c was prepared similar to the preparation of the compound 7a, in which compound 5  was treated with 3,5-dimethoxybenzoic acid (6c) , EDC·HCl  and sodium acetate (83 mg 0.0017 mol) to afford pure compound 7c, 284.2 mg in 37% yield. Mp: 345–347 °C, IR (KBr): 3181 (N–H stretching vibration), 3065 (Ar–C–H str.), 2912 (C–H str.), 1621 (NH(C=O)–C=C– stretching vibration of amide), 1612, 1423 (C=N and C=C stretching vibration of pyridine Nucleus), 1053 (C–F stretching vibration), 762 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound, 7d was prepared following the method described for the preparation of the compound 7a, employing 5  with 4-methoxybenzoic acid (6d) , EDC·HCl  and sodium acetate  to afford pure compound 7d, 291.3 mg in 41% yield. Mp: 342–344 °C, IR (KBr): 3181 (N–H stretching vibration), 3021 (Ar–C–H str.), 2895 (C–H str.), 1641 (NH(C=O)–C=C– stretching vibration of amide), 1618, 1386 (C=N and C=C stretching vibration of pyridine Nucleus), 1093 (C–F stretching vibration), 784 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound, 7e was prepared following similar method used for the preparation of the compound 7a, employing 5  with 4-chlorobenzoic acid (6e) , EDC·HCl  and sodium acetate  to afford pure compound 7e, 313.5 mg in 44% yield. Mp: 338–340 °C,IR (KBr): 3163 (N–H stretching vibration), 3075 (Ar–C–H str.), 2897 (C–H str.), 1691 (NH(C=O)–C=C– stretching vibration of amide), 1610, 1422 (C=N and C=C stretching vibration of pyridine Nucleus), 1094 (C–F stretching vibration), 794 (2-Substituted pyridine), 1H NMR : 13C NMR : +.This compound, 7f was prepared using the method described for the preparation of the compound 7a, employing 5  with 4-bromobenzoic acid (6f) , EDC·HCl  and sodium acetate  to afford pure compound 7f, 372.4 mg in 47% yield. Mp: 362–364 °C, IR (KBr): 3203 (N–H stretching vibration), 3055 (Ar–C–H str.), 2965 (C–H str.), 1643 (NH(C=O)–C=C– stretching vibration of amide), 1632, 1403 (C=N and C=C stretching vibration of pyridine Nucleus), 1093 (C–F stretching vibration), 769 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound 7g was prepared as described above, employing 5  with 4-nitrobenzoic acid (6g) , EDC·HCl  and sodium acetate  to afford pure compound 7g, 367.3 mg in 50% yield. Mp: 370–372 °C, IR (KBr): 3176 (N–H stretching vibration), 2906 (Ar–C–H str.), 2966 (C–H str.), 1668 (NH(C=O)–C=C– stretching vibration of amide), 1631, 1396 (C=N and C=C stretching vibration of pyridine Nucleus), 1112 (C–F stretching vibration), 791 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound, 7h was prepared as per the method used for the preparation of the compound 7a, in which compound 5  treated with 3,5-dinitrobenzoic acid (6h) , EDC·HCl  and sodium acetate  to afford pure compound 7h, 342.8 mg in 42% yield. Mp: 376–378 °C, IR (KBr): 3206 (N–H stretching vibration), 3112 (Ar–C–H str.), 2887 (C–H str.), 1648 (NH(C=O)–C=C– stretching vibration of amide), 1603, 1403 (C=N and C=C stretching vibration of pyridine Nucleus), 1114 (C–F stretching vibration), 769 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound 7i was prepared as described above. In this, compound 5  was reacted with 4-methylbenzoic acid (6i) , EDC·HCl  and sodium acetate  to afford pure compound 7i, 239.6 mg in 35% yield. Mp: 356–358 °C, IR (KBr): 3224 (N–H stretching vibration), 3102 (Ar–C–H str.), 2986 (C–H str.), 1643 (NH(C=O)–C=C– stretching vibration of amide), 1632, 1405 (C=N and C=C stretching vibration of pyridine Nucleus), 1087 (C–F stretching vibration), 756 (2-Substituted pyridine), 1H NMR : 13C NMR : +.The compound 7j was prepared following the method described for the preparation of the compound 7a, in which compound 5  was made to react with 4-bromo-3,5-dinitrobenzoic acid (6j) , EDC·HCl  and sodium acetate  to afford pure compound 7j, 356.9 mg in 37% yield. Mp: 386–388 °C, IR (KBr): 3125 (N–H stretching vibration), 3098 (Ar–C–H str.), 2995 (C–H str.), 1664 (NH(C=O)–C=C– stretching vibration of amide), 1609, 1409 (C=N and C=C stretching vibration of pyridine Nucleus), 1076 (C–F stretching vibration), 748 (2-Substituted pyridine), 1H NMR : 13C NMR : +.This compound 7a–j) were examined for their anticancer activity against a panel of four human cancer cell lines including breast cancer , lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Etoposide was used as reference standard and the obtained results were summarized in Table The target compounds (7a–j) synthesized 7a, 7b, 7c, 7d and 7i possessed good activity with IC50 values ranging from 0.011 ± 0.009 to 19.4 ± 8.11 µM and standard reference showed IC50 values range from 1.91 ± 0.84 to 3.08 ± 0.135 µM, respectively. The structure–activity relationship (SAR) studies indicated that the compound 7a without substituent on the phenyl ring attached to 1,2,4-oxadiazole moiety has showed good anticancer activity against four cell lines . Up on introduction of electron-donating 3,4,5-trimethoxy group on the phenyl ring resulted compound 7b, showed more significant anticancer activity  than standard against cancer cells presented in Table 7c displayed slightly decreased activity on four cell lines  when compared with 7b. Where, compound 7d with 4-methoxy substituent has showed reduced anticancer activity  than 7c. Instead of 4-methoxy group with weak electron-donating 4-methyl group (7i) exhibited acceptable activity . Further, the compounds 7e, 7f, 7g, 7h and 7j, which possess electron-withdrawing substituents on the phenyl ring, showed moderate activities compared to those compounds 7a, 7b, 7c, 7d and 7i, without electron withdrawing and with electron-donating substituents.Among the compounds (7a–7j) were performed using Molegro Virtual Docker (MVD). The crystal structure of Human VGEFR-2 enzyme (PDB ID: 1YWN) along with the crystal ligand imatinib was downloaded from protein databank [The docking studies of the potent 1,2,4-Oxadiazole linked 5-Fluorouracil derivatives (databank . Human V7a–7j) was assessed by using DataWarrior Software [In silico ADMET screened for 5-fluoro-1-benzyl)pyrimidine-2,4-dione derivatives . The crystal structure of human Vascular Endothelial Growth Factor Receptors (VEGFR-2) enzyme (PDB ID: 1YWN) along with the co-crystal ligand was downloaded from protein databank. Design of VEGFR-2 could be potential target for inhibition of blood vessel formation and leads to the development of anti-angiogenesis agents. All the chemical structures were prepared by using Marvin sketch and minimized. Mol file format structures were converted into Mol2 file format by using Discovery studio. MVD was used to perform computational studies, cavity prediction, assigning bond orders, structure refinement, defining the active binding sites of the VEGFR-2 and structure preparation. The protein preparation was carried out with MVD and the chain was treated to add missing hydrogen, assign proper bond orders and deleted water molecules. The structure output format was set to pose viewer file so as to view the output of resulting docking studies and hydrogen bond interactions of different poses with the protein. The 2D interactions were generated from Protein Plus and 3D interactions were generated from MVD.The docking studies of the potent compounds In the present study, the synthesized compounds were docked into X-ray crystal structures of Human VEGFR-2 enzyme (PDB ID: 1YWN) to understand the possible target mechanism of action. The cavities were detected with MVD and the following are the active residues involved with co-crystal ligand, as it forms hydrogen bond interactions with Glu883, Glu915, Cys917, Asp1044. Arg1049 residues and hydrophobic interactions with Leu838, Val846, Val897, Val914, Cys1043, and Asp1044, residues. The docking has validated and found same interactions with Moldock score − 199.37 and H-bond energy − 6.24 kcal/mol  established a hydrogen bond (NH—ON) with amino group of Asp1044 and oxygen atom of oxadiazole (NH—O) with amino group of Lys866 with a H-bond energy − 7.40 and − 7.34, respectively and with Moldock score values are − 156.20 and − 157.88, respectively. The 7j and 7h also form same hydrophobic interactions with Arg840, Val846, Asp1044, Gly1046, and Arg1049 amino acid residues  with amino hydrogen of Gly1046 with a H-bond energy of − 2.59 kcal/mol  and 7h  showed good inhibitory constant and excellent free energy of binding, which might be the reason for anticancer activity.All the synthesized compounds form hydrogen bond interactions with Asp1044 and have shown same interaction with crystal ligand. The binding affinity of the docked compounds were expressed in negative binding energy kcal/mol (Moldock score). The ligands with more negative value of Moldock score will have more affinity with protein binding. 3,5-dinitro substituted compounds (All the synthesized compounds generated pharmacophore features by using PharmaGist web server . It will7a–7j) was assessed by using DataWarrior Software benzyl)pyrimidine-2,4-dione derivatives  and all these compounds were characterized by 1HNMR, 13CNMR and Mass spectral analysis. Further, these compounds were investigated for their anticancer activity towards a panel of four human cancer cell lines such as , lung cancer (A549) and prostate cancer (DU-145) by using MTT method. Among them, compounds 7a, 7b, 7c, 7d and 7i were demonstrated more promising anticancer activity than standard. Further docking studies of all the compounds were presented, where the designed compounds showed good anticancer activity and excellent free energy of binding interactions with VEGFR-2 kinase domain.In conclusion, we have synthesized a library of 1,2,4-oxadiazole linked 5-fluorouracil derivatives (Additional file 1: Spectra of synthesized compounds.


The pyridazine ring in the molecule of the title compound adopts a screw-boat conformation. The whole molecule is flattened, the dihedral angles subtended by the least-suares plane of the central aromatic ring with those of the terminal benzene and pyridazine rings being 15.18 (19) and 11.23 (19)°, respectively. In the crystal, the molecules are linked by pairs of N—H⋯O bonds into centrosymmetric dimers and by C—H⋯π contacts into columns. 20H17ClN2O3, adopts a screw-boat conformation. The whole molecule is flattened, the dihedral angles subtended by the least-squares plane of the central aromatic ring with those of the terminal benzene and pyridazine rings being 15.18 (19) and 11.23 (19)°, respectively. In the crystal, the molecules are linked by pairs of N—H⋯O bonds into centrosymmetric dimers and by C—H⋯π contacts into columns. The results of the Hirshfeld surface analysis show that the most prominent interactions are H⋯H, accounting for 36.5% of overall crystal packing, and H⋯O/O⋯H (18.6% contribution) contacts.The pyridazine ring in the title compound, C The pale-red spots on the surface represent short N—H⋯O and C—H⋯O interactions -2-(thiophen-2-yl)ethenyl]-4,5-dihydropyridazin-3(2H)-one -6-(4-hydroxy-3-methoxyphenyl)ethenyl-4,5-dihydropyridazin-3(2H)-one -6-(4-hydroxystyryl)-4,5-dihydropyridazin-3(2H)-one , K2CO3  and 2-chloro-1-(4-chlorophenyl)ethan-1-one  in acetone (50 ml) was refluxed overnight. After cooling, the solution was filtered and the solvent removed under reduced pressure. The residue was purified by recrystallization from ethanol to afford single crystals (yield 72%).A mixture of (Uiso(H) = 1.5 Ueq(C)], C—H = 0.93 Å for aromatic [Uiso(H) = 1.2 Ueq(C)] and C—H = 0.98 Å for methine [Uiso (H) = 1.2 Ueq(C)] H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698902101238X/yk2160sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902101238X/yk2160Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902101238X/yk2160Isup3.cmlSupporting information file. DOI: 2123627CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information: