M.-H. Zhao

Diastereoselective Total Synthesis of (±)‐Schindilactone A

Schindilactone A (1) and structures 2–4 (Scheme 1a) are representative members of a novel group of nortriterpenoids isolated by Sun and co-workers from the plants of Schisandraceae, which have been used in China for the treatment of rheumatic lumbago and related diseases. Preliminary biological assays indicated that some of them possess biological activities for inhibiting hepatitis, tumors, and HIV-1. The synthetic challenge posed by 1 stems from the complexity of its molecular structure: a highly oxygenated framework bearing 12 stereogenic centers, eight of which are contiguous chiral centers located in the FGH tricyclic ring system, and an oxa-bridged ketal that lies within an unprecedented 7–8 fused carbocyclic core. The structural complexity together with the attractive biological activities has rendered 1 a target for synthetic studies. Herein we report our efforts on the development of synthetic methods and a strategy centered on the construction of the polycyclic ring system that allowed the first total synthesis of ( )-schindilactone A. This concise strategy opens a pathway for the syntheses of other compounds related to schindilactone A (2–4, Scheme 1a), as well as their derivatives and analogues.

Binding capacity and pathophysiological effects of IgA1 from patients with IgA nephropathy on human glomerular mesangial cells

IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge‐region O‐glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with 125I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal‐regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF‐β mRNA and production of supernatant fibronectin were tested by RT‐PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose‐dependent, saturable manner, and was saturated at approximately 500 pmoles per 0·5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8·89 ± 2·1) × 10−8mversus (4·3 ± 1·2) × 10−7m for aIgA1 from healthy controls (P = 0·026).The binding was specific because it was only inhibited by unlabelled Mono‐IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF‐βmRNA and secretion of fibronectin in HMC in a similar time‐dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0·05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.

Neutrophil extracellular traps can activate alternative complement pathways

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg‐EGTA)‐treated human serum. After incubation of serum with supernatants enriched in ANCA‐induced NETs, levels of complement components in supernatants were measured by enzyme‐linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b‐9 on NETs incubated with heat‐inactivated normal human serum (Hi‐NHS) or EGTA‐treated Hi‐NHS (Mg‐EGTA‐Hi‐NHS) were significantly less than that on NETs incubated with NHS or EGTA‐treated NHS (Mg‐EGTA‐NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b‐9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I‐degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV.

Anti-endothelial cell antibodies (AECA) in patients with propylthiouracil (PTU)-induced ANCA positive vasculitis are associated with disease activity.

Increasing evidence has demonstrated that propylthiouracil (PTU) could induce ANCA positive vasculitis. However, our previous work has suggested that only one‐fifth of the PTU‐induced ANCA positive patients had clinical vasculitis and so the mechanism is not clear. Anti‐endothelial cell antibodies (AECA) have been implicated in the pathogenesis of various vasculitides, including primary ANCA positive systemic vasculitis. The purpose of this study is to investigate the prevalence of AECA and their possible role in the pathogenesis of patients with PTU‐induced ANCA positive vasculitis. Sera from 11 patients with PTU‐induced ANCA positive vasculitis at both active and quiescent phases, and sera from 10 patients with PTU‐induced ANCA but without clinical vasculitis, were studied. Sera from 30 healthy blood donors were collected as normal controls. Soluble proteins from 1% Triton‐100 extracted in vitro cultured human umbilical vein endothelial cells were used as antigens and an immunoblotting technique was performed to determine the presence of AECA, and their specific target antigens were identified. In patients with PTU‐induced ANCA positive vasculitis, 10 of the 11 patients in an active phase of disease were serum IgG‐AECA positive and six protein bands of endothelial antigens could be blotted (61 kD, 69 kD, 77 kD, 85 kD, 91 kD and 97 kD). However, in the quiescent phase, seven of the 10 positive sera turned negative. None of the ANCA positive but vasculitis negative patients or normal controls were AECA positive. In conclusion, AECA could be found in sera from patients with PTU‐induced ANCA positive vasculitis and were associated more closely with vasculitic disease activity.

Diastereoselective Total Synthesis of (±)‐Schindilactone A, Part 3: The Final Phase and Completion

The final phase for the total synthesis of (±)-schindilactone A (1) is described herein. Two independent synthetic approaches were developed that featured Pd-thiourea-catalyzed cascade carbonylative annulation reactions to construct intermediate 3 and a RCM reaction to make intermediate 4. Other important steps that enabled the completion of the synthesis included: 1) A Ag-mediated ring-expansion reaction to form vinyl bromide 17 from dibromocyclopropane 30; 2) a Pd-catalyzed coupling reaction of vinyl bromide 17 with a copper enolate to synthesize ketoester 16; 3) a RCM reaction to generate oxabicyclononenol 10 from diene 11; 4) a cyclopentenone fragment in substrate 8 was constructed through a Co-thiourea-catalyzed Pauson-Khand reaction (PKR); 5) a Dieckmann-type condensation to successfully form the A ring of schindilactone A (1). The chemistry developed for the total synthesis of schindilactone A (1) will shed light on the synthesis of other family members of schindilactone A.

Diastereoselective Total Synthesis of (+/-)-Schindilactone A, Part 1: Construction of the ABC and FGH Ring Systems and Initial Attempts to Construct the CDEF Ring System

First-generation synthetic strategies for the diastereoselective total synthesis of schindilactone A (1) are presented and methods for the synthesis of the ABC, FGH, and CDEF moieties are explored. We have established a method for the synthesis of the ABC moiety, which included both a Diels-Alder reaction and a ring-closing metathesis as the key steps. We have also developed a method for the synthesis of the FGH moiety, which involved the use of a Pauson-Khand reaction and a carbonylative annulation reaction as the key steps. Furthermore, we have achieved the construction of the central 7-8 bicyclic ring system by using a [3,3]-rearrangement as the key step. However, unfortunately, when this rearrangement reaction was applied to the construction of the more advanced CDEF moiety, the anticipated annulation reaction did not occur and the development of an alternative synthetic strategy would be required for the construction of this central core.

Class IV-G and IV-S lupus nephritis in Chinese patients: a large cohort study from a single center.

This study is to assess the difference between IV-G and IV-S in a large cohort of Chinese patients with lupus nephritis. The detailed data of patients with subclass IV-G and IV-S were retrospectively analyzed. Serum ANCA and anti-C1q antibodies were detected. A total of 172 cases were classified as class IV, including 152 cases with IV-G and 20 cases with IV-S. In IV-S, the level of haemoglobin was significantly higher (P = 0.024), proteinuria was milder (P = 0.003), serum creatinine was lower (P = 0.021), serum C3 was higher (P = 0.045) and the ratio of positive serum anti-cardiolipin antibodies was higher (P = 0.014). On pathological evaluation, the ratio of glomerular fibrinoid necrosis was significantly higher (P = 0.006) and the score of endocapillary hypercellualrity, interstitial inflammation and total activity indices was significantly lower (P < 0.001, P = 0.01, P = 0.006, respectively) in IV-S. The frequency of serum ANCA was significantly higher in IV-S than that in IV-G (20 vs 4.6%, P = 0.008). The frequencies of anti-C1q IgG1 and IgG3 subclass were significantly higher in IV-G (P = 0.006, P = 0.011, respectively). There are significant differences in clinical and pathological manifestations between IV-S and IV-G lupus nephritis which need further investigation.

Synthesis of Catechins via Thiourea/AuCl3-Catalyzed Cycloalkylation of Aryl Epoxides

A diversity-oriented approach for the synthesis of structurally diverse catechins was achieved in good yields via thiourea/AuCl(3)/AgOTf-catalyzed annulations of aryl epoxides under mild conditions. This new protocol provides a highly efficient entry to a library of catechins-derived natural products, notably anti-HIV agent 8-C-ascorbyl-(-)-epigallocatechin.

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO–ANCA‐positive vasculitis. The production of anti‐MPO antibodies in patients with PTU‐induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU‐induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti‐MPO antibodies (Pab1‐HRP, Pab2‐HRP) in a competitive inhibition enzyme‐linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1‐HRP and Pab2‐HRP were affinity purified from plasma exchanges of a patient with PTU‐induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44·7% ± 5·0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35·6% ± 6·0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68·4% ± 16·1% (P < 0·01) and 62·2% ± 17·2% (P < 0·01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1‐HRP and the inhibition rates were 81·4% ± 9·4%versus 86·6% ± 17·2% (P > 0·05). However, the average inhibition rate for Pab2‐HRP in the MPA group was significantly higher than that in the PTU group (76·3% ± 7·8%versus 58·9% ± 15·5%, P < 0·01). We conclude that anti‐MPO antibodies from patients with PTU‐induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti‐MPO antibodies from patients with PTU‐induced vasculitis might be more restricted.

The glycans deficiencies of macromolecular IgA1 is a contributory factor of variable pathological phenotypes of IgA nephropathy

Recent evidence has suggested that IgA1‐containing macromolecules and the glycosylation of IgA1 in sera from patients with IgAN might involve the pathogenesis of IgAN. However, whether the different histological phenotypes can be attributed or not to the aberrant glycosylation of macromolecular IgA1 has not yet been elucidated. The aim of the current study is to investigate the glycosylation of IgA1 molecules in serum IgA1‐containing macromolecules and their association with pathological phenotypes of IgAN. Sera was collected from 40 patients with IgAN and 20 donors. Twenty patients had mild mesangial proliferative IgAN, the remaining 20 had focal proliferative sclerosing IgAN. Polyethylene glycol 6000 was used to precipitate the macromolecules from sera of patients and controls. Biotinylated lectins were used in an enzyme‐linked immunosorbent assay (ELISA) to examine different glycans on IgA1 molecules. The α2,6 sialic acid was detected by elderberry bark lectin (SNA) and the exposure of terminal galactose (Gal) and N‐acetylgalactosamine (GalNAc) were detected by Arachis hypogaea (PNA) and Vilsa villosa lectin (VVL), respectively. The IgA1 glycans levels corrected by IgA1 concentrations were compared between patients and controls. Reduced terminal α2,6 sialic acid of IgA1 (79·89 ± 25·17 versus 62·12 ± 24·50, P = 0·034) was demonstrated only in precipitates from sera of patients with focal proliferative sclerosing IgAN, compared with those from controls. Reduced galactosylation of IgA1 molecules in precipitates was demonstrated in patients with both mild mesangial proliferative IgAN and focal proliferative sclerosing IgAN compared with normal controls (24·52 ± 18·71 versus 76·84 ± 32·59 P = 0·000 and 33·48 ± 25·36 versus 76·84 ± 32·59 P = 0·000). However, no significant difference was found in IgA1 glycosylation in the supernatant between patients and normal controls (P > 0·05). The glycosylation deficiency of IgA1 existed only in serum IgA1‐containing macromolecules of patients with IgAN, and was associated with the renal pathological phenotypes. This suggests that aberrant glycosylation of IgA1 in serum macromolecules might be a contributory factor in the pathogenesis of IgAN.