Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates  and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.In  Mycobacterium chelonae and capped with phosphoinositol (PI) to form PILAM in Mycobacterium smegmatis , the termini of the arabinan are further substituted with short mannan caps (di- or tri-saccharide) (M. chelonae does not induce cytokine secretion or apoptosis of macrophages (4: (β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)- α-D-Araf) (A) and a branched terminus Ara6:(-α-D-Araf-(1→5)-α-D-Araf) (B) have been shown to be the epitopes recognized by anti-LAM monoclonal antibodies (mAb) (fs: β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf- (C) and a lack of the branched Ara6 (B). In addition, covalent acyl modifications such as succinyl monoester at the 3-hydroxyl (C3) of the 2-linked arabinose (the penultimate Araf at the nonreducing end) were also reported  is a heterogeneous lipoglycan, a major component of the cell wall of mycobacteria , 2. It imegmatis . In the charide) , 5, resucharide)  and dendcharide) , 8. On trophages . The lin-D-Araf) A and a b-D-Araf) B have bees (mAb) , 12, 13 es (mAb) , 15, 16 es (mAb) , 18. We es (mAb) , 19, 20,-D-Araf- C and a lreported . To thisocations .Figure 1Mycobacterium tuberculosis complex (MTBC) exhibits a strong phylogeographical population structure, with some lineages occurring globally and others showing a strong geographical restriction  wherein1H-13C correlation spectra (HSQC full spectrum presented in A–D) revealed that all LAM has similar glycosidic linkages as reported in the literature  at δ 5.02, 5.09 (H-1), δ 107.5, 107.2 (C-1) ppm; 3,5-linked arabinofuranoses, i.e., α-D-Araf -(1→5)- α-D-Araf –[α-D-Araf -(1→3)]-(1→5) at δ 5.07 (H-1), δ 107.2 (C-1) ppm 2-linked Araf; β-D-Araf (1→2)- α-D-Araf -(1→5) at δ 5.10 (H-1), δ 105.7 (C-1) ppm, 2-linked Araf (branch); β-D-Araf (1→2)- α-D-Araf -(1→3) at δ 5.18 (H-1), δ 105.5 (C-1) ppm and terminal nonmannose-capped and mannose-capped arabinofuranoses; t- β-D-Araf- (1→2) and α-D-Man-(1→5)- β-D-Araf-(1→2) at δ 5.07 (H-1), δ 100.6, 100.8 (C-1) ppm, respectively. The anomeric protons of Manps are assigned to: 2,6-linked Manp; & α-D-Manp-(1→6)-Manp-[α-D-Manp-(1→2)]-(1→6) at δ 5.07 (H-1), δ 98.4 (C-1) ppm, 6-linked Manp; Manp-α(1→6) at δ 4.85 (H-1), δ 99.8 (C-1) ppm, and terminal as well as 2-linked Manp (mannan core and mannose caps (p- (1→2) at δ 4.95, 4.97 (H-1), δ 102.4 (C-1) ppm respectively. Additionally, anomeric proton/carbon for MTX (5-SMe-α-D-Xylf) was also visible at δ 5.32 (H-1), δ 102.5 (C-1) ppm as indicated in 1H-1H correlation (TOCSY) spectroscopy. TOCSY NMR spectrum of LAM from HN878, IO and H37Rv is presented in the A–C.1D-proton NMR showed no marked difference among LAM isolated from HN878, EAI, IO compared with laboratory strain H37Rv . Among tterature . The repterature  revealedA). The cross peak at δ 4.85 ppm (1H) and δ 79.2 ppm (13C) was attributed to the H3/C3 of an arabinose ring. In fact, all the acyl functionalities should be linked through this proton unless they occupy the primary –OH groups. However, the absence of any downfield proton that could be attributed to H5 of Araf or H6 of Manp supports this finding. In addition, the absence of any anomeric proton in 1,3-bond correlation (in TOCSY) with the acylated ring proton ruled out acylation at the 2-position of Araf (B). RvLAM on the other hand was found to have two sets of acylated ring protons (A) with different intensities. The major cross peak was the H3/C3 of the Araf ring as found in all four LAM, and the minor cross peak was found at δ 4.83 ppm (1H) and δ 81.1 ppm (13C). Unfortunately, the weak peak could not affirm 1,3-bond correlation (TOCSY) with any anomeric proton, in the event the succinate was at the 2-position. The presence of a succinyl residue at the 2-position of 3,5-linked Araf was reported earlier in arabinogalactan isolated from Mtb H37Rv  showed two sets of protons corroborating to a -C(O)-CH2-CH2-C(O)- structure unequivocally establishing a succinyl monoester residue. For confirmation, additional support was sought from enzymatic digestion of LAM. As a representative structure, the extracted ion chromatogram (LC/MS of enzyme digested LAM arabinan) showed same retention time for the ion m/z 1101.34 [M-H]¯ corresponding to Suc1Man2Ara5 for all the 3× LAM species . This indicated that the monosuccinylated Man2Ara5 has a similar structure in all LAM. MS-MS was performed on this ion to reveal the sequence . Possibly, a charge-based association of succinate with EDTA  is responsible for this shift of peaks.The TOCSY of HN878 LAM B showed sequence . This shr in LAM . The ana 2.6 ppm , A–C. PoO-acetyl butyrate-like structure. The downfield shift of the α-proton of α-hydroxy butyrate from δ 4.1 to δ 5.2 ppm was attributed to the electron withdrawing effect of the O-acetyl group. These variations were less prominent in IO-LAM and undetectable in HN878 LAM and RvLAM.The TOCSY NMR of EAI-LAM  clearly in vivo LAM, at that time, because of lack of material, MS-MS analysis on enzyme-released fragments was not possible. Herein, analysis was achieved on m/z 911.29  by LC/MS-MS on EAI-LAM. The ion at m/z 911.29 [M-H]¯can also arise from monoacetyl Man2Ara4. However, after sequential digestion with α-mannosidase followed by endoarabinanase, no acetyl was found associated with Ara4 residues in case of the EAI LAM. Three consecutive losses of 132 Da (each) were accounted for three unsubstituted pentoses (Araf) at the reducing end. The corresponding ions were found at m/z 779.2 [M-H]¯ (C3)¯(B), m/z 647.2 [M-H]¯ (C2)¯ , and m/z 515.1 [M-H]¯ (C1)¯ respectively. The ion (C1)¯ underwent a specific fragmentation that is associated to a loss of 46 Da-(formic acid), at m/z 469.2 [M-H]¯ (C1-46)¯. This fragmentation clearly suggested that a free carboxylic group was present, thereby indicative of the lactyl group attached to LAM via ether linkage. The presence of m/z 307.0 [M-H]¯ (Y2α)¯  indicated that (the loss of) a hexose (Manp) is not associated to the lactyl. So, the lactyl group is attached to one of the two Araf units at the nonreducing end. At this point, we also noticed that the ion m/z 647.2 [M-H]¯ (C2)¯  undergoes some specific cleavages. The loss of 90 Da (72 Da from lactyl group and 18 Da for loss of water), which accounts for the ion at m/z 557.1 [M-H]¯ (C2-90)¯ (F), is suggestive of the lactyl group associated to a secondary ring hydroxy-group. Sequential losses of 132 Da (m/z 425.1 [M-H]¯ (C1-90)¯  followed by 162 Da (m/z 263.1 [M-H]¯ (Y2α)¯(C)) conclude again that the lactyl group is attached to one of the two Araf units at the nonreducing end. The presence of a specific ion at m/z 291.1 [M-H]¯ (C2-Z1α-44)¯  is indicative of (i) the carboxyl unit lost as carbon dioxide (44 Da) and (ii) neither the mannose cap nor the β-Araf bear lactyl ether. However, the presence of two specific D-type ions at m/z 275.0 [M-H]¯ ¯  and m/z 229.0 1 [M-H]¯ ¯  not only suggests the structural assignment of 2b but also establishes that the lactyl-ether is attached to the 2-linked Araf, which has a 3-position, available for the lactyl substitution.Lactyl group was identified in all the LAM samples from four different Mtb species by LC-MS. Although, we had reported earlier  that alkH]¯ (C3)¯B, m/z 64(C2)¯ 2; C; , and (C1)¯2a; C respect1-46)¯1; D. This fY2α)¯ 1; E indicat(C2)¯ 2; C undergo(C2-90)¯ F, is sug90)¯ 2a; G followe]¯ (Y2α)¯C) conclu44)¯ 2b; H is indi1α)¯ 2c; I and m/z46)¯ 2d; J not onl5 and the branched Ara6 termini of EAI LAM. This was established by the LC/MS-MS analysis in the negative ion mode. Representative ion at m/z 763.2 [M-H]¯ , obtained after sequential enzymatic digestion, revealed a specific neutral loss of 58 Da to produce m/z 705.1 [M-H]¯ . This loss was attributed to the loss of propanal (58 Da) produced by the α-cleavage of α-hydroxy butyrate  via a neutral loss of 104 Da (86 Da from α-hydroxy butyryl group +18 Da from water) is indicative of the substitution associated to a secondary ring hydroxy-group. The formate-Ara5 (m/z 705.1 [M-H]¯) (3a) underwent further fragmentation with a loss of 150 Da (Araf). This ion at m/z 555.1 [M-H]¯ (Z1α-58)¯ , accounts for a loss of 18 Da (water) along with cleavage of Araf from the nonreducing end. This assignment was made since C-Z-type fragmentations were found to be dominant during fragmentation pathway. Further loss of 3xAraf from 3b the reducing end gave ions at m/z 423.0 [M-H]¯ (C3- Z1α -58)¯ , m/z 291.1 [M-H]¯ (C2- Z1α -58)¯ , and m/z 159.0 [M-H]¯ (C1- Z1α -58)¯; . Formation of these ions also suggests that the 2-linked Araf is also linked to the formyl or α-hydroxy butyryl group. Also, the characteristic cross-ring cleavage , a major fragment ion of 2-linked Araf, accounts for a loss of 176 Da (Araf ring contributes 90 Da and α-hydroxy butyryl group contributes 86). Sequential losses of 3× Araf (m/z 455.1 (C3-176)¯ , 323.1 (C2-176)¯ , and 191.0 (C1-176)¯ [M-H]¯;  support the presence of the α-hydroxy butyryl group at the 2-linked Araf. The formylated counter ion was also found at m/z 117.0 [M-H]¯ ¯ . Collectively, these specific ions suggest that the α-hydroxy butyrate group is attached to the 2-linked Araf, which has only 3-position, available as a secondary –OH group, for substitution.The α-hydroxy butyryl group mainly associated with the linear Ara-H]¯ 3a; B. This lbutyrate . The prem/z 673.2 [M-H]¯  by LC/MS-MS experiment. Two consecutive losses of 132 Da each giving ions at m/z 541.1 [M-H]¯ ¯) and m/z 409.1 [M-H]¯ (C1)¯ were observed. This indicated that two Araf units are not acylated. Further loss of 150 Da for a terminal Araf unit produced an ion at m/z 259.1 [M-H]¯ (Z1α)¯signifying that the substitution could be on the second Araf unit on either terminal. An ion at m/z 145.0 [M-H]¯ . This was suggestive of a substitution associated to secondary -OH of the Araf unit, i.e., 2- or 3-OH. Subsequently, sequential losses of 2× Araf units (132 Da each) from 4a resulted in ions at m/z 395.1 [M-H]¯ (C2)¯) and m/z 263.0 [M-H]¯ (C1)¯ suggesting that the two successive Araf units at the reducing end are not substituted. The ion at m/z 217.0 [M-H]¯ ¯ suggests a loss of 218 Da (90 from the Araf ring and 128 from the acetoxybutyrate). The corresponding ¯ ion at m/z 455 [M-H]¯ (4b) was also present. These ions signify that the acetoxybutyrate is linked at 3-position of an Araf ring. It also allows limiting the two arabinose units of the nonreducing end where these 0,2A-type fragments can occur because the remaining two arabinose units are α-(1→5) linked. Further, sequential losses of 132 Da from 4b gave ions m/z 323.0 (C2)¯ [M-H]¯ and m/z 191.0 (C1)¯ [M-H]¯. These ions clearly indicate that the reducing end arabinose units do not carry the butyrate functionality. The cross-ring cleavage giving m/z 191.0 [M-H]¯ ¯ can be produced from the reducing end, and this is crucial for further structural assignments. A series of ¯ ions such as m/z 481.1 [M-H]¯ (5), m/z 439.1 [M-H]¯ (5a), m/z 421.0 [M-H]¯ (5b), m/z 381.0 [M-H]¯ (5c), and m/z 353.0 [M-H]¯ (5d) account for the losses of acetate , propanal , and carbon monoxide . Significantly, further loss of 150 Da, the terminal Araf, from each of these 0,2A1 ions, produced (Z1α)¯ ions at m/z 331.0 [M-H]¯, m/z 289.0 [M-H]¯, m/z 271.0 [M-H]¯, m/z 231.0 [M-H]¯, and m/z 203.0 [M-H]¯ respectively. These ions decisively establish that the terminal β-Araf is not substituted. Furthermore, it is conclusive that the α-acetoxybutyryl ester is at the 3-position of the 2-linked Araf. Chromatograms of all the ions are presented in the A and B.We were able to investigate M-H]¯ 4;   was also found to be present. The presence of MTX/MSX in relatively high NMR peak intensity signifies that there could be multiple sites that bear MTX and/or MSX. So far, it has been shown that MTX in LAM is present one per molecule of LAM (The presence of methylthioxylose (MTX) residue is evident in the NMR spectra of all LAM preperations-HN878, EAI, IO, and H37Rv. The correlation (TOCSY) of H5a and H5b, the diastereotopic protons, (δ 2.6 and 2.7 ppm) with δ 4.12 (H2), δ 4.18 (H3), δ 4.31 (H4) ppm respectively confirm , 29 the e of LAM . Our curendoarabinanase and LC/MS of released ararbinan fragments of LAM, low-intensity ions corresponding to MTXMan1Ara5 (m/z 1001.30 [M-H]¯) were obtained overlapping with Man2Ara5 (m/z 1001.32 [M-H]¯) in LAM  and 1149.34 [M-H]¯  supports the presence of sulfoxide functionality.We had reported earlier that following digestion with ) in LAM . Such a 4 [M-H]¯  as abund1Ara5  can exist in one of the resonating structures 7′ . Subsequently, sequential loss of 4× Araf (132 Da each) confirms the presence of methylsulfoxyxylose unit at the nonreducing end. The ion at m/z 195.03 [M-H]¯  confirms that the sulfoxide unit is attached to a pentose (Xylf). However, the formation of m/z 143.03 [M-H]¯  can be attributed to a D-type cleavage to a Manp unit. This indicates that the MTX/MSX unit is linked to Manp either of the 2-, 3-, or 4- hydroxyl group. Previous work showed that it is linked to (1→4) α-D-Manp  and its mannose capped variations that are not succinylated (A). Two most prominent motifs are Ara6 and Man2Ara6 (H37Rv > HN878 > IO > EAI). Among the monosuccinylated versions, it seemed that the linear Ara4 and Ara5 with and without mannose capping dominated, although one needs to be cautious that this relative quantification is based on ion intensity only and is not absolute. It appears that four termini  bear the succinates (B). In addition, relative succinylation (C) estimated from LC/MS ion abundance analysis reveals that HN878, EAI- and IO-LAM have ∼45% succinylated termini (green/orange segments). Based on the LC/MS ion abundance, Figure 8D showed that EAI-LAM has approximately 10–12% less Man capping (green/yellow segment) compared with the LAMs from three other strains in this study.The LC/MS profile of inylated A. Two moccinates B. In addnylation C estimatp or not, LAM from each strain was first treated with α-mannosidase (Jack beans), and the residual glycan was passed through a Biogel P4 column. The 1H-NMR of digested LAM revealed that the acyl groups and MTX/MSX residues were still present in all 4× LAM molecules (A and B). TOCSY experiment on the digested RvLAM sample, chosen as the pilot because of its available amount, confirmed the presence of succinates along with MTX/MSX. However, MTX/MSX cross peaks were of relatively low intensity. The possibility of slower nonspecific digestion of MTX-Manp linkages cannot be ruled out. Major changes were noticeable in the anomeric region of HSQC NMR spectrum (C). The two sets of overlapping protons for β-D-Araf-(1→2)-Araf arising from Man capped and uncapped arabinan termini merged into one peak, thereby confirming almost complete removal of mannose caps, as expected. A weak residual signal for t-α-D-Manp-(1→2)-Manp supports this observation. Unlike the parent RvLAM molecule, the peak for Manp-α-(1→6) appears more intense than 2,6-linked α-Manp (A). Although there is a collateral loss in structure, it does not deter any alteration in antibody recognition activity.In order to unequivocally establish if the acyl substituents were on the terminal end Manolecules , A and Bspectrum C. The twd α-Manp A. Althouendoarabinanase. This allowed us to characterize the released arabinan termini  bearing acyl groups and MTX, without heterogeneity arising from mannose capping. We found that monosuccinylated Ara4 and Ara5 were major linear species with lesser amounts of branched Ara6 (B). Abundant nonacylated Ara5 and Ara6 were found in the RvLAM digest compared with the other LAM species in this study.α-Mannosidase-treated LAM was subsequently digested with hed Ara6 B. AbundaA) indicating its larger size. As expected, CS35 mAb recognized LAM from each isolate equally well (A).LAM extracted and purified from the Mtb strains were run on a 10–20% Tricine gel, followed by periodic acid-silver staining and transferred to nitrocellulose membrane for a Western Blot using mouse mAb CS35. The smear for RvLAM appeared to be running slower than the clinical isolates A indicatlly well A.Figure endoarabinanase-treated enzyme resistant cores were analyzed by SDS/PAGE (B) followed by Western Blot using mAb CS35 (B1). As evident from the SDS/PAGE analyses, (B) there was only a shift of ∼2 kDa after mannosidase treatment and selective removal of Man capping did not abrogate any antibody binding activity. On the other hand, following the endoarabinanase treatment, which afforded only partial removal of the arabinan termini, the resistant core shifted down to 10–15 kDa (C) and lost all binding capacity to the antibody (C1). Compositional analyses of this material showed Ara:Man ratio of 0.9:1 indicating 57% of Ara still remaining and linkage analyses of this core by GC/MS showed some 2 Manp, 2 Araf are present along with large amounts of 5-linked Araf (A–C).LAM, α-mannosidase-treated LAM, and SDS/PAGE B followemAb CS35 B1. As evnalyses, B there w0–15 kDa C and losantibody C1. Compoked Araf , A–C.4/Ara6)-CS35Fab recognition has been reported (4/Ara6) with or without a succinyl residue at the 3-position of the 2-linked-Araf and CS35Fab. The lowest docking score  clearly showed that the binding of the succinylated Ara4/Ara6 to CS35-Fab is unfavorable in comparison to nonsuccinylated Ara4/Ara6 (I and Ia). It is apparent  that the succinyl residue tends to pose outward the (II and IIa) binding groove of CS35Fab indicating conformational mismatch  appeared to be more abundant, whereas 2-linked Manp in lower abundance was observed by linkage analysis suggesting relatively lower mannose capping in EAI-LAM (C). Although succinylation is the most pronounced covalent modification of the LAM-arabinan, the TOCSY NMR of EAI-LAM . Only recently lactate and other short chain acids have been implicated to be present only as a contaminant in a LAM preparation 2Ara6. Therefore, it is conceivable that the abundance of Ara6 should preferentially dictate the response to CS35 as LAM-binding epitopes. If, however, Ara6 is less abundant than (Man2)2Ara6, total mAb response may equate the sum of the responses from binding to all available Ara6 and (Man2)2Ara6. The LC/MS profile of all the four LAMs after digestion with endoarabinanase (A) revealed that the relative abundance of Ara6 in HN878, EAI, IO, and H37Rv is 6.7%, 16.1%, 11.3%, and 5.3%, respectively and that of (Man2)2Ara6 is 12.6%, 8.4%, 10.9%, and 16.3%, respectively. It is understandable that for EAI and IO LAM, the response from CS35 will have approximately 2-fold more contribution from Ara6 than for HN878 and H37Rv. This is what is supported by the ELISA  as removal of Man caps does not influence the abundance of original Ara6. EAI and IO LAM did not show any drastic change in OD values after digestion with mannosidase . This justifies the reduced OD values for the mannosidase-digested HN878 and H37Rv LAM from their natives (blue line) by approximately 15–30%. These results indicate that the use of α-mannosidase as pretreatment reported for the detection of urinary LAM may be inconsistent -α-D-Araf-(1→5)-α-D-Araf-(1→5)-. According to our findings, all the acyl groups as well as lactyl ether are at the 3-position of the 2-linked penultimate Araf. Steric hindrance, offered by these acyls, may not allow the acylated epitopes to bind to CS35. The docking studies indicate that the binding of succinylated Ara4/Ara6 is less favored than their nonsuccinylated versions. Conformational mismatching may be responsible, thereby supporting our hypothesis. It is expected that deacylation, achieved by mild alkaline hydrolysis, should enable better availability of antigenic epitopes and consequently, enhancement of mAb-response and better sensitivity of immunoassay may be achieved . This was not unexpected because immobilization of LAM on polystyrene-ELISA plates relies on the PI anchor of LAM, and deacylation should remove these. Therefore, inefficient immobilization should lead to loss of antigen during washing and consequently, the mAb response should drop. To circumvent the antigen immobilization issue, the TB capture-ELISA . These results demonstrate that the acyl groups impart some inhibitory effect towards intact LAM detection by capture ELISA, and deacylation is perhaps a rapid, cheap, and efficient way to achieve better sensitivity with the existing mAbs. However the caveat is, there is no evidence whether acyl functionalities such as mono-succinyl ester are present in LAM in biofluids.We monitored the effect of Man cap removal by indirect ELISA whereby LAM/LAM derivatives were immobilized and subsequently detected by CS35, a well-known anti LAM murine mAb having strong binding affinity to LAM termini motifs. Earlier we reported  that CS3binanase A revealensistent . Ara6, Are-ELISA  was emplA, I–IV) due to the loss of termini motifs.Not unexpected, a drastic (∼70–80%) loss in CS35 response was observed with arabinanase resistant LAM-core compared with intact LAM -α-D-Araf-(1→5)-α-D-Araf-(1→5)- arrangement is lost from the nonreducing terminus, antigenic response to CS35 was compromised pointing that overall, abundance of specific arrangement of arabinan termini is crucial for the LAM-mAb recognition.It is evident that the structure of LAM, from different demographic origin of The biological significance of the structural features and their variations among strains remains to be determined. That said, the finding of covalent substituents such as succinates, methylthioxylose (MTX), and other acyl functions modifying the glycan in all M.tb strains suggests that M.tb has evolved strategies as other prokaryotes to promote their survival in its host environments. From the diagnostic perspective, the importance of specific arabinan nonreducing-end motifs, irrespective of its modifications cannot be understated. These could be the ultimate feature requirement for TB specific Ab binding.M. tuberculosis (M.tb) were originally received from the Trudeau mycobacterial culture collection (H37Rv), the laboratory of Dr James Musser (HN878), or the laboratory of Dr Sebastian Gagneux (T17 – “IO” and 91-0079 – “EAI”). All growth conditions were performed by incubation at 37 °C. Seed stocks were made by colony isolation, subculture to 7H9-OADC (100 ml), and freezing 0.8 ml samples in 20% glycerol. Genotyping was confirmed for the seed stocks by spoligotyping. Working stocks (3 × 1 ml) were used to inoculate 7H10-OADC plates with passage of bacterial lawns to 1 L Fernbach flasks containing GAS media . Quantitation was based on 3-O-methyl -glucose, used as an internal standard . The mass spectrum was scanned from standard .2O (0.56 ml) at 25 °C. All PRESAT 1H NMR was recorded on a Bruker Neo 400 MHz NMR instrument. 1H-13 C correlation (HSQC) NMR spectra were recorded with 128 scans and relaxation delay 1.2 s on Bruker Neo 400 MHz NMR instrument. The default Bruker parameters were used for recording spectra. TOCSY (1H-1H correlation) NMR spectra were recorded with 256 scans on Varian Innova 500 MHz instrument. All chemical shifts are based on the reference to the HOD peak at 4.64 ppm. The default Varian parameters were used for recording spectra. Spectra were processed using MestReNova x64.All NMR spectra were recorded with ∼5 mg LAM in DLAM (1 mg/0.2 ml water) was treated with 0.085 ml (5 units) of α-Mannosidase  and incubated at 37 °C (water bath) for 20 h. The reaction mixture was then frozen at –80 °C for 2 h. After inactivation of the enzyme at 80 °C for 15 min, the reaction mixture was purified over Biogel-P4 column .endoarabinanase as previously described  from each strain was digested with escribed  and the Indirect ELISA was carried out as previously described . LAM samA 96-well polystyrene high binding microplate  was coated with CS35 . The plates were blocked . LAM and derivatives  were serially diluted 2-fold and applied in duplicate to the plate/s. After incubation  and wash, detection antibody  was applied. Streptavidin-Horseradish Peroxidase (R&D Systems) (25 min) followed by Ultra TMB ELISA chromogenic substrate (Thermo Scientific) were added to develop color. The reaction was stopped by adding 2 M sulfuric acid, and the optical density was measured at 450 nm.m/z) range of 110−4000 at 1 Hz scan rate. For MS-MS experiments, the MS was in multiple reaction monitoring (MRM) scan mode with collision-induced dissociation (CID) energies of 40 eV and 60 eV on target masses with m/z width of 10 Da. Internal instrument mass-scale calibration was performed by infusing the Agilent ESI-L low-concentration tuning mix. Instrument controls were performed via the Bruker HyStar v4.1 software package. Data were processed using Bruker Compass 2.0 Data Analysis 4.4 software.Enzyme-released oligosaccharides were subjected to ultraperformance liquid chromatography (UPLC) separation on a Waters Acquity UPLC H-Class system in line with a Bruker MaXis Plus quadrupole time-of-flight (QTOF) mass spectrometer (MS). Separation was performed in gradient mode with a Waters Atlantis T3 3.0 μm column (2.1 × 150 mm) at 40 °C. Mobile-phase components were 10 mM ammonium acetate in water (A) and 10 mM ammonium acetate in acetonitrile (B). The flow rate was 0.3 ml/min. The data were acquired in the negative electrospray ion (ESI) mode with a mass-to-charge ratio (6/SucAra6 and 3HNT with Ara4/SucAra4) on PyRx-Vina platform. The.pdbqt files were further analyzed using Discovery-studio-visualizer.The methyl oligoarabinofuranosides with proper stereochemistry were drawn using Chemdraw 20.0 and 3D mm2 energy minimization was done. The protein structures of CS35Fab (3HNS and 3HNT) were obtained from Protein Data Bank. Docking studies were performed (3HNS with AraAll data are contained within the manuscript, either in the main body or in the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Scientific Reports 10.1038/s41598-020-79201-5, published online 13 January 2021Correction to: The original version of this Article contained an error in Figure 6B where were the y-axis numbering “0, 10, 20, 30” was incorrectly given as “0, 1, 2, 3”.The original Figure The original Article has been corrected.


Background: There are limited reports available on investigations into the molecular spectrum of thalassemia and hemoglobinopathy in Fujian province, Southeast China. Here, we aim to reveal the spectrum of the thalassemia mutation and hemoglobinopathy in Quanzhou prefecture, Fujian province.Methods: We collected data from a total of 17,407 subjects with the thalassemia trait in Quanzhou prefecture. Gap-PCR, DNA reverse dot blot hybridization, and DNA sequencing were utilized for common and rare thalassemia gene testing.Results: In our study, we identified 7,085 subjects who were carrying thalassemia mutations, representing a detection rate of 40.70% . Among them, 13 different α-thalassemia gene mutations were detected, with the most common mutation being –SEA (69.01%), followed by –α3.7 (21.34%) and –α4.2 (3.96%). We also discovered 26 β-thalassemia gene mutations, with the mutations of IVS-II-654 (C > T) (36.28%) and CD41/42(–TCTT) (29.16%) being the most prevalent. Besides, a variety of rare thalassemia variants were identified. Among them, the –FIL, βMalay, βIVS–I–130, and βIVS–II–672 mutations were identified in Fujian province for the first time. Additionally, we detected 78 cases of hemoglobinopathies, of which Hb Owari was the first reported case in Fujian province and Hb Miyashiro was the first case identified in the Chinese population.Conclusion: Our study indicates that there is a diverse range of thalassemia mutations, and it also reveals the mutation spectrum of rare thalassemia and hemoglobinopathies in Quanzhou, Fujian province. It provides valuable data for the prevention and control of thalassemia in Southeast China. Thalassemia is a hereditary blood disorder caused by human globin gene synthesis disorders, of which α- and β-thalassemia are the most common genotypes . It mostin utero or shortly after birth, and this condition also often leads to the mortality of the pregnant mother  routine hematology examination showed abnormal mean corpuscular volume (MCV) <82 fl/or mean corpuscular hemoglobin (MCH) <27 pg; (2) abnormal hemoglobin electrophoresis; (3) parents or siblings carried the thalassemia gene mutation; (4) at least one of the couple was identified as a thalassemia carrier.2 for routine blood analysis and hemoglobin electrophoresis analysis. We performed routine blood detection on all of the subjects using an automated cell counter  and analyzed the hemoglobin components by hemoglobin electrophoresis . Positive hematological screening was defined as an MCV of less than 82 fl and/or an MCH concentration of less than 27 pg and/or hemoglobin A2 (HbA2) levels greater than 3.4% or less than 2.6% or an Hb F value of more than 2.0%. All patients with positive hematological analysis results underwent thalassemia gene testing.Approximately 4 ml of peripheral blood was collected from each subject and anticoagulated with EDTA-KFor the serum ferritin test, approximately 3 ml of peripheral blood was collected from the patients. Then, it was centrifuged at 3,500 rpm for 10 min to separate the serum. The serum ferritin test was performed with Siemens Healthcare Diagnostics equipment and kit  and using the ADVIA Centaur XP Immunoassay System .For each subject with positive hematological analysis results for the molecular analysis of common α- and β-thalassemia, we collected a further 2 ml of peripheral blood. An automatic nucleic acid extractor  was used to extract the genomic DNA of the subjects. We also used Gap-PCR to detect the three common deletional α-thalassemia mutations . The PCR reverse dot hybridization technique (PCR-RDB) was utilized to detect the three common non-deletional α-thalassemia mutations and 17 common β-thalassemia mutations . The β-thalassemia mutations we detected were as follows: CD41-42(-TCTT), IVS-II-654(C > T), –28 (A > G), CD71/72(+ A), CD17(AAG > TAG), CD26(GAG > AAG), CD43(GAG > TAG), –29(A > G), CD31(-C), –32(C > A), IVS-I-1(G > T), CD27/28(+ C), –30(T > C), CD14-15(+ G), Cap + 40-43(–AAAC), initiation codon(ATG > AGG), and IVS-I-5(G > C). The experimental operations were performed strictly according to the protocols of the manufacturers.THAI, –α27.6, −α21.9) and rare β-thalassemia genotype screening kits  were utilized for suspected carriers of rare α- or β-thalassemia deletion. Indicators included low levels of Hb A2 without common α-thalassemia variants, as well as high or low levels of Hb A2 without common β-thalassemia variants.Rare α-thalassemia genotype screening kits (–FIL. We designed specific primers according to the known DNA sequences around the breakpoints. These primer sequences were P1: TCTAAAAATTCATCCTTAAGAGAATAA and P2: GATCTAATGTAGTAGAGATAATAACCTTTA. All primers were synthesized at Sangon Biotech (Shanghai). The amplification conditions were 96°C for 5 min, then 35 cycles of 98°C for 30 s, 60°C for 1 min, 72°C for 2 min, and finally 72°C for 10 min. Subsequently, we performed an electrophoresis analysis.Gap-PCR was performed to identify the deletion of –We employed a multiplex ligation-dependent probe amplification (MLPA) assay using the SALSA MLPA Probemix P140-C1HBA  to detect the known or unknown globin gene deletions. DNA sequencing was performed when we observed suspected carriers of rare thalassemia mutations.t-test to compare the means of the two groups. We also applied the chi-square test to compare the detection rates between the groups. A value of p < 0.05 was considered statistically significant.The statistical analysis was conducted using SPSS19.0 software. The measurement data were expressed as SEA/αα (70.17%) mutation was the most common deletional mutation, followed by –α3.7/αα (18.37%) and –α4.2/αα (3.34%). The three most common non-deletional mutations were ααQS/αα, ααCS/αα, and ααWS/αα, with frequencies of 2.05%, 1.31%, and 0.94%, respectively. Besides, nine genotypes of rare α-thalassemia were detected, of which –THAI/αα, Hkαα/–SEA, and ααIVS–II–55(T > G)/αα were the most common. Of these nine genotypes, our study was the first to detect a –FIL/αα case in Fujian province, while –α27.6/–SEA and ααIVS–II–55(T > G)/αα had never before been identified in Quanzhou prefecture /αα, we performed a comparison of the hematological parameters among the different genotypes of silent α-thalassemia. As QS/αα-positive cases exhibited lower levels of Hb, MCV, and MCH than those of other groups, and the hematological phenotype was similar to that of α-thalassemia minor. Conversely, the rare genotype ααIVS–II–55(T > G)/αα displayed a milder hematological phenotype similar to ααWS/αα.To further analyze the hematological phenotype ααIVS–II–654/βN (36.19%) and βCD41–42/βN (29.82%), followed by βCD17/βN (16.78%), βCD26/βN (5.35%), and β–28/βN (4.57%). These five common mutations accounted for 92.71% of β-thalassemia gene mutations. In this study, 17 cases of rare β-thalassemia variants were identified, of which βCD54–58/βN and βIVS–II–806/βN mutations were the first to be reported in Quanzhou, while βMalay/βN and βIVS–II–672/βN had not previously been identified in Fujian province. Moreover, one subject carried two rare β-thalassemia mutations (βIVS–I–130/βIVS–II–81), which has never been encountered before ] and one case of the novel δ-globin gene mutation [CD44(TCC > TGC) (HBD:c.134C > G)] were identified .SEA (69.01%), followed by –α3.7 (21.34), –α4.2 (3.96%), ααQS (2.19%), ααCS (1.59%), and ααWS (1.48%)  thalassemia mutation chromosomes, including 13 α-thalassemia gene mutations and 26 β-thalassemia mutations. Of the α-thalassemia mutant chromosomes, 5,205 chromosomes carried α-thalassemia gene mutations, of which the most frequent mutation was – (1.48%) . Regardi (1.48%) .To further investigate the hemoglobin variants, we performed a DNA sequencing analysis. Altogether, we detected 24 cases of Hb Q-Thailand [CD74(GAC > CAC)], two cases of Hb G-Honolulu [CD30(GAG > CAG)], and one case of Hb Owari [CD121(GTG > ATG)], all of which were induced by the α-globin gene mutation. Similarly, we identified 37 cases of Hb New York [CD113(GTG > GAG)], 12 cases of Hb J-Bangkok [CD56(GGC > GAC)], one case of Hb Miyashiro [CD23(GTT > GGT)], and one case of Hb G-Coushatta [CD22(GAA > GCA)]. These cases were caused by the β-globin gene mutation .QS was the most common non-deletional α-thalassemia variant in Fujian and Jiangxi province, Guangdong and Hunan province mainly exhibited the ααCS mutation, and ααWS was the most widespread non-deletional α-thalassemia variant in Guangxi province. The IVS-II-654(C > T) and CD41-42(-TCTT) mutations were the most common β-thalassemia mutations in Fujian and its neighboring provinces, except in Guangxi province, which mainly carried CD41-42(-TCTT) and CD17(A > T) mutations.By analyzing the spectrum of thalassemia genotypes in Fujian and the neighboring provinces, we discovered that the highest frequency genotypes of deletional α-thalassemia were similar. However, the mutations in α-thalassemia and β-thalassemia showed distinct regional differences. As In China, there is a high prevalence of thalassemia in the regions south of the Yangtze River, particularly in Guangdong and Guangxi. In 2019, a study showed that the prevalence of thalassemia in Fujian was 6.8% . Few stuSEA, –α3.7, and –α4.2, which is consistent with previous studies in Fujian province and neighboring provinces /αα mutation in this study, a mutation that was first identified in Fuzhou city and subsequently reported in the Nanping region of Fujian province (IVS–II–55(T > G) may be an increasingly common mutation that will possibly become more prevalent in Fujian province. Further analysis of the hematological phenotype of ααIVS–II–55(T > G)/αα provided similar results as tests for ααWS/αα.We discovered 7,085 subjects who harbored thalassemia mutations; therefore, the detection rate was 40.70%. As rovinces . Howeverrovinces . Among tprovince . Before province . The –FIprovince . This inIVS–II–806/βN mutation, which was first identified in the Nanping region north of Fujian, had been identified in humans  and higher levels of Hb A2 (4.5%) were observed. Thus, more research should be conducted to reveal whether the IVS-II-672(A > C) mutation causes β+-thalassemia. Additionally, we identified a rare compound mutation, which is a combination of the IVS-I-130(G > C)  and IVS-II-81(C > T) mutations. This causes minor thalassemia, which suggests that the IVS-II-81(C > T) mutation may be a silent mutation.In this study, we detected 2,223 chromosomes carrying β-thalassemia gene mutations. The most frequent mutation was IVS-II-654(C > T), which was consistent with several regions in South China . Howevern humans . Additioded case . This muded case . The IVSded case . However0- or δ+-thalassemia. In our case, the -77(T > C) mutation was combined with the CD17(A > T) mutation, resulting in normal levels of Hb A2 (2.7%). This indicates that the diagnosis of β-thalassemia could be hindered when it is combined with δ-thalassemia. Moreover, we made the first discovery in the Chinese population of the novel δ-globin gene mutation CD44(TCC > TGC) (HBD:c.134C > G). The mutation at codon 44, which results in Ser→Cys acid substitution, is believed to cause δ+-thalassemia.Besides, we identified two δ-globin gene mutations, including -77(T > C) and CD44 (TCC > TGC) (HBD:c.134C > G). Previous studies have shown that the -77(T > C) mutation is the most common δ-globin gene mutation in China  and may 4.2) and causes Hb Q-H disease when associated with α-thalassemia (mainly –SEA) (12 g/L), low levels of MCV (56 fl) and MCH (18.3 pg), and increased Hb A2 (6.0%). Our results were consistent with those of a previous study, which suggested that the coexistence of Hb J-Bangkok and β-thalassemia may not aggravate the phenotype  has previously been reported in Chinese and other Southeast Asian populations. It is invariably linked to a leftward single α-globin gene deletion (–αly –SEA) . Moreovely –SEA) . Additioly –SEA) . In thishenotype . Moreovehenotype . TherefoA diverse range of rare thalassemia mutations was identified in this study, which is consistent with the location and the increasing population in Quanzhou prefecture. Notably, with thalassemia detection kits, we identified some rare thalassemia mutations that were more pervasive than the so-called common thalassemia mutations, such as CD31(-C), –32(C > A), and –30(T > C), which have never been detected in this region before. Nowadays, next-generation sequencing is being increasingly used to improve thalassemia detection . Thus, DIn this study, we conducted a comprehensive large-scale study on the thalassemia mutation in Quanzhou prefecture, which provides valuable data for the prevention and control of thalassemia. Our study is the first to reveal the spectrum of rare thalassemia mutations and hemoglobinopathies in the Quanzhou region. From this study, a diversity of rare thalassemia mutations and hemoglobinopathies was identified. This research, combined with the use of DNA sequencing and gap-PCR technology, shows great value in the investigation of rare and novel thalassemia gene mutations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of The Women’s and Children’s Hospital of Quanzhou (2020No.8). The patients/participants provided their written informed consent to participate in this study.JZ designed the study and wrote the article. NZ and JZ performed conventional thalassemia analysis. YZ performed specific gap-PCR amplification and DNA sequencing. NZ and HZ analyzed the data. YX, YJ, DC, and YW revised and polished the manuscript. All authors have approved the final article.YZ was employed by the company Yaneng BIOscience (Shenzhen) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the crystal structure, infinite supramolecular layers in the  15H21NO3, the urethane function and the benzoyl group are almost perpendicular to each other [dihedral angle 88.97 (5)°]. In the crystal structure, infinite supramolecular layers in the bc plane are formed by weak C—H⋯O hydrogen bonds.In the molecular structure of the title compound, C The same is true for the benzoyl group . These two planes subtend a dihedral angle of 88.97 (5)° and therefore an almost perpendicular arrangement Fig. 1. OtherwiB⋯O3 links molecules of the title compound into infinite chains parallel to the c-axis direction. Additional C2—H2B⋯O1 and C9—H9B⋯O2 interactions link these infinite chains to a supramolecular sheet parallel to the bc plane -Ph group attached to the carbamate oxygen atom is reported  dissolved in a small amount of DMSO was added in one portion. The consumption of 2-bromo-1-phenylethan-1-one was monitored by TLC and after 30 min the reaction mixture was poured on ice to quench the reaction. After extraction with dichloromethane (3×), the combined organic phases were washed with brine, separated and dried over Na2SO4. The solvent was removed in vacuo and the crude product was recrystallized from n-hexane/ethanol (4:1) to afford the title compound  as a colorless solid, m.p. 347.5°C. 1H NMR  [ppm]: δ = 7.90 , 7.55 , 7.45 , 5.33 , 3.97 , 1.25 ; 13C NMR  [ppm]: δ = 193.91 (C=O), 154.80 (NC=O), 134.69, 133.65, 128.84, 127.83 (Car), 66.36 (O=C—O), 46.32 [(H3C)2CH–], 20.99 [(H3C)2CH–].Diisopropylamine  and 1 equiv. of cesium carbonate  were placed in a Schlenk tube and dissolved in anhydrous DMSO (150 mL). The tube was sealed with a septum and two balloons filled with COUiso(H) = 1.2×Ueq(C) for methylene and hydrogen atoms of the phenyl group or 1.5×Ueq(C) for methyl groups.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021006927/zl5014sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021006927/zl5014Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021006927/zl5014Isup3.cmlSupporting information file. DOI: 2094771CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Lipoxygenases are one of the critical signaling mediators which can be targeted for human prostate cancer (PC) therapy. In this study, 4-methyl-2-(4-methylpiperazinyl)pyrimidobenzothiazine (4-MMPB) and its two analogs, 4-propyl-2-(4-methylpiperazinyl)pyrimidobenzothiazine (4-PMPB) and 4-ethyl-2-(4-methylpiperazinyl)pyrimidobenzothiazine (4-EMPB), were proposed to have anti-tumor properties in prostate cancer.After synthesizing the compounds, cytotoxic effects of 4-MMPB and its two analogs against PC-3 cancerous and HDF normal cells were investigated by 3--2,5-diphenyltetrazolium bromide (MTT) assay and then mechanism of cell death was assessed by flow cytometry. Finally, the anti-tumor effects of the mentioned compounds were investigated in an immunocompromised C57BL/6 mouse model.4-PMPB and 4-EMPB had similar anti-cancer effects on PC-3 cells as compared with 4-MMPB, while they were not effective on normal cells. Moreover, apoptosis and ferroptosis were the main mechanisms of induced cell death in these cancerous cells. Furthermore, in vivo results indicated that both analogs had similar anti-cancer effects as 4-MMPB, leading to delayed tumor growth without any noticeable side effects in weight loss and histological investigations.Thus, our results suggest that specific targeting of lipoxygenases via 4-MMPB analogs can be considered as a treatment of choice for PC therapy, although it requires further investigations. Cancer is considered as a serious concern affecting the health of all human societies. Prostate cancer (PC) is the most common malignancy in males worldwide, representing the second leading cause of mortality . VariousS-hydroperoxyoctadecadienoic acid (13S-HpODE), but also metabolizes AA to 12S- and 15S-hydroperoxyeicosatetraenoic acid (15S-HpETE) benzothiazine (4-MMPB) as a heterocyclic compound, functions as an effective and specific 15-lipoxygenase inhibitor with an ICof 18 µM . Moreove of 9 µM . In thisAll chemical reagents and solvents were purchased from Merck  and used as received without further purification. 3--2,5 diphenyltetrazolium bromide (MTT) and trypsin–EDTA were purchased from Tinab Shimi . Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium and Dulbecco’s modified Eagle’s medium (DMEM) were bought from Gibco . Fetal bovine serum , and penicillin–streptomycin were obtained from Gibco . Soybean LOX-1  and linoleic acid (L1376) were bought from Sigma‐Aldrich . Itraconazole and co-amoxiclav were purchased from Tehran Darou Pharmaceutical Co. and AFA Chemi, Pharmaceutical Co. , respectively.4-MMPB and 4-PMPB were synthesized as previously described , 47. The−1, 1H NMR (CDCl3): 1.27 , 2.87 , 4.30 , 6.77–7.43 , 13C NMR (CDCl3): 14.1, 29.8, 114.4, 117.5, 118.5, 120.4, 128.0, 128.7, 147.3, 157.4, 161.5, 162.8).To a solution of 5-bromo-2,4-dichloro-6-ethylpyrimidine 1 (CAS No. 1373331–48-6)  and triethylamine (1.2 g) in chloroform (30 ml), 2-aminothiophenol  was added dropwise with vigorous stirring over a minute. Solvent was removed and the residue was washed with warm water and then crystallized from ethanol . IR: 3300, 3432 cm−1, 1H NMR: (CDCl3) 1.26 , 2.34 2), 2.86 , 3.73 ), 6.92 , 7.35 , 8.20–8.42 ; 13C NMR: (CDCl3) 14.1, 23.9, 39.5, 44.1, 46.7, 56.4, 94.7, 120.5, 122.7, 124.1, 131.1, 136.7, 141, 155.6, 159.6, 167.0; Elemental analysis of C17H21N5S: Calculated: C, 62.36; H, 6.46; N, 21.39; S, 9.79. Found: C, 62.41; H, 6.49; N, 21.27; S, 9.72.A mixture of 2-[(5-bromo-2-chloro-6-ethylpyrimidin-4-yl)sulfanyl]aniline 2  and 4-methylpiperazin (30 mmol) in ethanol (20 ml) was heated under reflux for 5 h. Water (40 ml) was added to the solution and separated sticky liquid (intermediate 3) was dried at 80 °C for an hour and immediately utilized without purification. Sodium amide  was added to stirred solution of the intermediate 3 in acetonitrile (30 ml), and then heated under reflux condition (3 h). The solvent was removed and a solution of 2% acetic acid (20 ml) was added to the residue and filtered. Then the residue was crystallized from benzene to give 4-ethyl-2-(4-methylpiperazinyl)pyrimidobenzothiazine (4-EMPB) (4) as a yellow powder (55% yield). Melting Point: 171–173 °C; IR: 3348 cm−1, 3 ml) in 50 mM phosphate buffer at pH 5.0 (25 ml). A linoleic acid solution was prepared by mixing 5.6 mg linoleic acid with 0.5 ml methanol and then diluted with 100 mM KOH to a final volume of 5 ml (4 mM). In the assay, a solution of inhibitors in dimethyl sulfoxide (DMSO) (12.5 μl), soybean LOX-1 , and phosphate buffer, pH 7.0  were mixed in 48-well plates, and pre incubation was carried out for 10 min at 30 °C. A control test was performed with the same volume of DMSO. After the pre incubation, linoleic acid solution (25 μl) was added to start the peroxidation reaction at 30 °C, and 10 min later, solution A (135 μl) and then solution B (65 μl) were added to start the color formation. After 3 min, 100 μl of a 2% sodium dodecyl sulphate (SDS) solution was added to terminate the reaction. The absorbance at 598 nm was compared with the control. These experiments were performed in triplicate and the data were analyzed using GraphPad Prism 6.0 .Linoleic acid and two assay solutions (A and B) were prepared in advance. Solution A was 50 mM 3-(dimethylamino)benzoic acid (DMAB) in l00 mM phosphate buffer (pH 7.0). Solution B was a mixture of l0 mM 3-methyl-2-benzothiazolinonehydrazone hydrochloride , and hemoglobin (5 mg ml−1) were then prepared by serial dilution from the primary stock solution and further diluted in the culture medium. Moreover, culture medium containing 0.25% HCl 0.3 N was used as a negative control in all experiments for a better evaluation.Two mg of each compound was dissolved in 100 μl hydrochloric acid (HCl 0.3 N) to make a primary stock solution. Different concentrations  and routinely cultured in RPMI 1640 supplemented with 10% FBS. In order to investigate the toxicity of the compounds on normal cells, in vitro experiments were also performed on HDF cells. These cells were kindly provided by Academic Center for Education, Culture and Research  and grown in DMEM supplemented with 10% FBS. Both cell lines were kept at 37 °C containing 5% COThe MTT reduction assay is a standard quantitative method to evaluate cytotoxicity. This assay measures cellular dehydrogenases, and is considered as an easy, safe and highly reproducible method. Viable cells reduce yellow tetrazolium salt to purple formazan crystals and thus the color change serves as a marker reflecting cell viability .2. Next day, the media were changed and both cell lines were treated with various concentrations (1.56–50 μg ml−1) of the compounds and cisplatin. Experiments were carried out in triplicate for 4-PMPB, 4-EMPB and 4-MMPB as well as cisplatin as a positive control. After 24, 48 and 72 h of treatments, 20 μl MTT solution  was added to each well, and cells were incubated in the dark for 3–4 h. The media were then removed and purple crystals were dissolved in 160 μl DMSO. The absorbance in each well was measured at 540 nm as reference wavelength in an enzyme-linked immunosorbent assay (ELISA) reader  and relative optical density (OD) values were recorded. The percentage of viable cells was calculated using the following equation [PC-3 and HDF cells were seeded in 96-well plates  at a density of 6000 and 8000 cells per well, respectively and incubated at 37 °C with 5% COequation :\documen50 values of 4-PMPB + , 4-EMPB + , and 4-MMPB + deferoxamine (80 µM)) on PC-3 cells, to distinguish between ferroptosis and apoptosis mechanisms [50 and IC50 values of 4-PMPB, 4-EMPB, 4-MMPB, and cisplatin for 24 h. Then the cells were collected by trypsinization (0.25% trypsin-1 mM EDTA), washed twice with PBS, followed by centrifugation, and resuspended in a binding buffer. Samples were stained with Annexin V-FITC/ PI followed by 15 min incubation at room temperature. Finally, cells were subjected to flow cytometry  and the data were analyzed by FlowJo 7.6.1 software . Moreover, PC-3 cells treated with 0.3 N HCl were used as a control to eliminate the effects of solvent.To determine the mechanism of cell death induced by 4-MMPB analogs, flow cytometry was employed using the Annexin V-FITC kit with PI  according to the manufacturer’s instructions. We performed co-treatment experiments with the compounds and a ferroptosis inhibitor  (IR.UM.REC.1399.001). C57BL/6 mice were inbred in an animal house at FUM and maintained under pathogen-free conditions. Immunosuppression of male C57BL/6 mice (4–6 weeks old) was carried out according to the protocol described by Jivrajani et al. . However6 cells in 100 μl RPMI 1640 into the right flank of the immunocompromised mice. When the tumor volume reached 100–200 mm3, the animals were randomly divided into 9 groups (n = 8 per group) and treated with: 1) PBS as a negative control; 2) HCl (0.3 N) as solvent control; 3) 4-PMPB (10 mg kg−1); 4) 4-PMPB (50 mg kg−1); 5) 4-EMPB (10 mg kg−1); 6) 4-EMPB (50 mg kg−1); 7) 4-MMPB (10 mg kg−1); 8) 4-MMPB (50 mg kg−1) and 9) cisplatin as a positive control (2 mg kg−1). The changes in tumor size and body weight were monitored every other day. The tumor volumes were calculated using the following formula [The PC-3 tumor model was generated by subcutaneous injection of 3 × 10 formula :\documenTo validate the anti-tumor activity in different experimental groups, mice were sacrificed 15 days post treatment. Meanwhile, blood samples were collected and histological analyses were also performed to investigate the possible side effects. Hematological analyses were performed in an automated hematology analyzer . The tissue slices were stained with hematoxylin and eosin (H&E) and observed by an optical microscope .t-test (group pairs) or one-way ANOVA (multiple groups) followed by Tukey multiple comparison tests. Experiments were performed in triplicate and data are presented as mean ± standard error or standard deviation as required. p-values < 0.05 were considered as significant.All statistical analyses were carried out using GraphPad Prism software 6.0 . First the Kolmogorov–Smirnov test was performed to evaluate the normal distribution of the data. Then, differences between groups were assessed with Student’s 50 values) of the mentioned compounds were calculated from their sigmoidal dose–response curves  and 4-MMPB (IC50 = 17.1 μM).Lipoxygenase inhibitory activity of 4-MMPB and its analogs was assessed on soybean LOX-1 and the inhibitory potencies  increased from 0.133% in control to 52.38%, 60.9%, 41.5% and 67.8% in 4-PMPB-, 4-EMPB-, 4-MMPB-, and cisplatin- treated PC-3 cells, respectively, when treated with IC50 values of the compounds.To evaluate the mechanism of cell death, PC-3 cells co-treated with 4-PMPB, 4-EMPB, or 4-MMPB in combination with deferoxamine, a ferroptosis inhibitor, were assessed using Annexin V-FITC/ PI staining and compared with those treated with the compounds only. The results revealed that the population in Q2 (Annexin V positive/PI positive) was markedly decreased after co-treatment with deferoxamine as compared to those evaluated in the absence of this ferroptosis inhibitor. These observations suggest that 4-MMPB and its two analogs have probably induced both apoptosis and ferroptosis in PC-3 cells  was confirmed in immunocompromised mice , 4-EMPB (10 and 50 mg kg−1), 4-MMPB (10 and 50 mg kg−1), and cisplatin, and then their body weights and tumor volumes were measured every other day. As shown in Fig. After administration of immunosuppression protocol, blood samples were collected and their parameters were compared with normal C57BL/6 mice. Significant reduction in WBC, lymphocyte, and neutrophil counts (p < 0.01). However, no significant changes in body weights were observed between 4-PMPB- (10 and 50 mg kg−1), 4-EMPB- (10 and 50 mg kg−1) and 4-MMPB- (10 and 50 mg kg−1) treated groups.The side effects of the compounds were evaluated by measuring the body weights, hematological analyses, and H & E staining of different organs. Body weights of various groups were compared and as shown in Fig. −1 had significantly elevated AST and ALT levels compared with immunosuppressed control group. Similarly, the group treated with cisplatin had significantly increased serum liver enzymes compared to control group (p < 0.0001). Histological analysis showed no obvious tissue damages in major organs including liver, kidney, and spleen, following 4-PMPB, 4-EMPB, and 4-MMPB treatments. However, mild and local accumulation of inflammatory cells was noticed in cisplatin treated group  and alanine aminotransferase (ALT) were evaluated in different experimental groups Fig. . Urea leoup Fig. . Meanwhioup Fig. . ApoptosALOX15 expression is regulated by a mutant form of P53 and results in promoting PC [S-HODE, the major metabolite of 15-LOX-1 can interact with anti-apoptotic mediators such as B-cell lymphoma 2 (BCL2) and insulin-like growth factor receptor-1 (IGF-1R) which leads to inhibition of apoptotic signaling pathways in PC [. [. [−1) in combination with ketoconazole (10 mg kg−1) after 18 days. In present study, human PC xenograft was developed according to the Jivrajani’s protocol in C57BL/6 mice [−1) possessed similar anti-cancer effects as compared with 4-MMPB, leading to delayed tumor growth without noticeable side effects in weight loss and histological assessments.In recent years, cancer incidence rate has increased significantly especially in developing countries and discovering more effective treatments has emerged as a major concern. Chemotherapy is one of the conventional treatment modalities, which can increase patient’s survival. However, therapeutic index can be decreased due to serious limitations of chemotherapy including drug resistance, poor specificity, and undesirable, long term side effects , 54. Disoting PC . Moreoveys in PC –61. Addiys in PC , 62, andys in PC . In ordeys in PC , 65. Nudys in PC ; therefoys in PC . Cunha ein PC [.  reportedPC [. [.  reportedPC [. [.  reportedL/6 mice . Blood cTargeted therapeutic strategies such as lipoxygenase inhibition in PC can reduce tumor growth and help in improving clinical outcomes. Data generated from our study showed that, use of 4-PMPB and 4-EMPB can be considered as a treatment of choice to trigger both apoptosis and ferroptosis pathways and inhibit PC cell survival in vitro and in vivo probably via specific targeting of lipoxygenases. For this purpose, different approaches can be recommended, and use of nanotechnology-based drug delivery systems (DDSs) is one of the most attractive strategies. In this regard, mentioned compound inhibitors of lipoxygenases can be encapsulated into specific nanocarriers such as liposomes, which would enable us to use them in clinical trials. Moreover, surface of nanocarriers can be decorated with specific ligands referred as active targeting, to reduce possible side effects on normal tissues and facilitate their availability to tumor microenvironment.


Racemic 1–3 were further separated by chiral HPLC. Their absolute configurations were assigned by spectroscopic and computational methods. It is interesting that all of these isolates are natural product hybrids. Antiviral, immunosuppressive, antitumor and anti-inflammatory properties of all the isolates were evaluated.Five new compounds including three pairs of enantiomeric xanthine analogues, parvaxanthines D–F ( Cyclopelta parva is mainly distributed in southern China, such as in Hunan, Hubei, Guangdong and Yunnan provinces. Previous pharmacological studies revealed that C. parva possesses significant anticancer, antibacterial, antifatigue activities 20D − 11.8 ; CD (MeOH) Δε209 + 1.15, Δε233 − 1.42; 1a}; {[α]20D + 6.3 ; CD (MeOH) Δε209 − 0.86, Δε233 + 1.03; 1b}; HRESIMS ; 1H and 13C-NMR data, see Parvaxanthine D (2): white solid; UV (MeOH) λmax (logε) 258 (3.99), 241 (3.93), 200 (4.34) nm; {[α]20D − 3.2 ; CD (MeOH) Δε208 + 1.10, Δε233 − 1.23; 2a}; {[α]20D + 6.7 , CD (MeOH) Δε208 − 1.26, Δε233 + 0.45; 2b}, HRESIMS ; 1H and 13C-NMR data, see Parvaxanthine E (3): white solid; UV (MeOH) λmax (logε) 269 (3.70), 201 (4.22) nm; {[α]20D + 7.7 ; CD (MeOH) Δε214 + 0.42; 3a}; {[α]D20 − 10.0 ; CD (MeOH) Δε214 − 0.20; 3b}; HRESIMS ; 1H and 13C-NMR data, see Parvaxanthine F (6): yellow gum; UV (MeOH) λmax (logε) 255 (3.80), 200 (4.16) nm; {[α]20D + 108.3 ; CD (MeOH) Δε201 − 2.33, Δε216 + 1.48, Δε249 − 1.41, Δε281 + 0.58; 6}; HRESIMS ; 1H and 13C-NMR data, see Asponguanosine C (7): yellow gum; UV (MeOH) λmax (logε) 254 (3.94), 200 (4.25) nm; {[α]20D − 17.4 ; CD (MeOH) Δε200 − 1.90, Δε214 + 1.54, Δε276 − 0.38; 7}; HRESIMS ; 1H and 13C-NMR data, see Asponguanosine D (6 or 7 (each 0.5 mg) was submitted to hydrolysis with 0.8 mL of 6 N HCl at 60 °C for 1.5 h. The reaction mixtures were concentrated and l-cysteine methyl ester in pyridine (0.8 mL) solution was added to it and reflected at 60 °C for 1 h. Then 2-methylphenyl isothiocyanate was added to the reaction mixtures and further heated for one more hour. In the same way, the standards ribose d and l were also derivatized and the reaction mixtures were directly analyzed by RP-18 HPLC . The retention times (tR) of the derivatives of standard d-ribose and l-ribose were determined at 16.8 min and 13.3 min, respectively. By the comparison of retention times (tR) of compounds 6 and 7 derivatives with the standards (d/l-ribose derivatives), the ribose in compounds 6 and 7 were determined to be d-form  and DFT/TDDFT were calculated with Spartan’14 software package and Gaussian 09 program package. Electronic circular dichroism (ECD) calculations were conducted at the B3LYP SCRF (PCM)/6-311G level and the CD spectra were produced by the program SpecDis 1.62 .4 cells/well. After 24-h incubation, the growth medium ) was discarded, and the cells incubated with compounds were diluted to 3 series concentrations  in maintenance solution (RPMI-1640 medium supplemented with 2% FBS). The experiment was repeated three times with 7 replicates for each concentration. The negative control wells represented Vero cells with maintenance medium without virus. All cells were cultured at 37 °C in a 5% CO2 atmosphere for 72 h and the cytopathic condition was observed under an inverted microscope. When the cytopathic effect in the virus control group (positive control) reached more than 75%, we observed and recorded the cytopathic effect (CPE) of each well  were incubated for 24 h in the presence of various concentrations of compounds. Cell viability was measured at 570 nm by MTT uptake. T lymphocytes obtained from C57/BL6 mice were cultured with compounds (20 μM) for 4 h, then treated with murine IFN-γ (5 ng/mL) for 30 min. After the incubation, proteins were extracted and assessed by Western blot analysis. Mouse T lymphocytes (2 × 105/well) obtained from splenocytes of C57/BL6 mice were stimulated by Con A (5 μg/mL) for 48 h, in the presence of compounds (20 μM) and their proliferation was evaluated using MTT assay (see The cytotoxicity of the compounds (20 μM) on resting lymphocytes from C57/BL6 mice was determined. Spleen cells  supplemented with 100 U/ml penicillin-streptomycin and 10% FBS. Cell viability was evaluated with Cell Count Kit-8 (CCK-8) assay kit and exponentially growing cells were seeded at 3–8 × 103 cells per well in 96-well culture plates for 24 h. Cells were treated with increasing concentrations (up to 40 μM) of these compounds for 48 h and an equal volume of DMSO was used as a control. CCK-8 solution (10 μL) was added to each well and the light absorbance was measured at 450 nm after incubation for another 0.5–4 h. The cell survival values were determined by the PrismPad program [All the cell lines were purchased from the Cell Bank of China Science Academy and the cell lines were incubated at 37 °C under 5% CO program ,32. Cyto2. RAW264.7 (2 × 104 cells/mL) cells were cultivated overnight into 96-well plates with completed DMEM and treated with DMSO or various concentrations of compounds for 24 h. Then, 10 μL CCK-8 solution was added into each well for 1 h at 37 °C and the absorbance of each well was recorded at 450 nm using a microplate reader. The cells (RAW264.7) were pre-treated with different concentrations of compounds for 2 h and then stimulated with LPS (1 μg/mL) for an additional 4 h. The culture supernatant from each well was collected and assayed with IL-6 ELISA Kit.RAW264.7, a mouse macrophage cell line, was cultured in high-glucose DMEM supplemented with 10% FBS, 100 μg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified environment containing 5% COC. parva. In addition, antiviral, immunosuppressive, anti-tumor and anti-inflammatory properties of all the isolates were determined. We concluded that only compound 5a could suppress the production of IL-6 in LPS-stimulated RAW264.7 cells in a concentration dependent manner.Herein we report the isolation and structural elucidation of five new and two known metabolites from the insect


R)-limonene led to the formation of bisdichlorodinickel as a new dinuclear nickel complex.The reaction of a nickel precursor with an enantiomerically pure amino-oxime issued from ( S)-limonene based aminooxime ligand has been isolated and its crystal structure determined. The resolved structure of dichloridobis{-2-methyl-5-(prop-1-en-2-yl)-2-[(pyridin-2-yl)methylamino]cyclohexan-1-one oxime}dinickel(II), [Ni2Cl2(C16H23ClN3O)2], at 100 K has monoclinic (P21) symmetry. The two NiII ions in the dinuclear complex are each coordinated in a distorted octahedral environment by three nitrogen atoms, a terminal chloride and two μ bridging chlorides. Each oxime ligand is coordinated to nickel(II) by the three nitrogen atoms, leading to two five-membered chelate rings, each displaying an envelope conformation. In the crystal, numerous intermolecular and intramolecular hydrogen bonds lead to the formation of a three-dimensional network structure.A dinuclear nickel complex with ( The ligand was first synthesized from (R)-limonene through the addition of nitrosyl chloride, NOCl, to a picolylamine moiety, allowing the formation of the oxime moiety.Asymmetric synthesis allows the preparation of enantiomerically enriched compounds either by using a chiral auxiliary, which will be temporarily introduced, or by using catalytic procedures  and 91.99 (4)°, respectively, together with Cl1—Ni1—N2 and Cl2—Ni1—N1 angles of 165.04 (11) and 88.69 (10)°, respectively. A similar arrangement can be found around the Ni2 atom .The title compound Fig. 1 crystalltrans-position at distances of 2.4408 (12) and 2.4077 (14) Å from the metal centers Ni1 and Ni2, respectively. The two metal centers are linked by two bridging Cl atoms with an average Ni—Cl distance of 2.42 Å, which is normal for these bond lengths. All these values compare well with literature values. The two nickel ions are separated by a distance of 3.5198 (7) Å, which is similar to average values  by the three nitrogen atoms, leading to two five-membered chelate rings, each displaying an envelope conformation (with N2 as the flap for Ni1/N1/C5/C6/N2 and N5 for Ni2/N4/C21/C22/N5). The six-membered carbocycles of the limonene units adopt a chair conformation. The lengths of the Ni1—N1, Ni1—N2 and Ni1—N3 bonds are 2.077 (3), 2.126 (4) and 2.041 (3) Å, respectively, while Ni2—N4, Ni2—N5 and Ni2—N6 are 2.095 (4), 2.103 (4) and 2.027 (3) Å. Atoms Cl1 and Cl4 are in a A⋯Cl4 hydrogen bonds and in intermolecular C3—H3⋯O1 and C26—H26⋯O2 interactions.The crystal structure is stabilized by numerous intermolecular and intramolecular hydrogen bonds Table 1, which lN-coordinated cationic complexes as enantiopure compounds  in MeOH (5 mL) was added -1-picolylamino-p-menth-8-en-2-one-oxime  dissolved in MeOH (3 mL). The solution turned green. The mixture was stirred overnight at room temperature during which time the mixture changed color to blue–green. The solvent was then evaporated to produce a crude solid that was washed with diethyl ether before crystallization. Single crystals were grown by slow diffusion at room temperature of diethyl ether into a dichloromethane solution. Elemental analysis calculated for C32H46Cl4N6Ni2O2: C, 46.33; H, 5.54; N, 9.65. Found: C, 46.35; H, 5.672; N, 9.77.To a solution of NiUiso(H) = 1.2Ueq(N) or 1.5Ueq(O). H atoms were positioned geometrically(C—H = 0.95–1.00 Å) and refined as riding with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl)Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021010537/ex2048sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021010537/ex2048Isup2.hklStructure factors: contains datablock(s) I. DOI: 2115017CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information: