Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.     

Modulation of Macrophage Activity by Proteolytic Enzymes. Differential Regulation of IL-6 and Reactive Oxygen Intermediates (ROIs) Synthesis as a Possible Homeostatic Mechanism in the Control of Inflammation

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-alpha production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.     

A simplified method for the preparation of EAC14 intermediate cells using human serum treated with methylprednisolone

The inhibitory effect of methylprednisolone 21-succinate ester (MPS) on the activities of complement components in human serum was studied by incubating human serum with various concentrations of MPS at 37 degrees C for 30 min and then measuring the residual activity of each component in human serum. The formation of EAC1 and EAC14 by C1 and C4 respectively, were only weakly inhibited by MPS at a final concentration of 10 mg/ml. In contrast, the same concentration of MPS completely inhibited the capacity of C2, C3, C5 and C6-9 to induce respective succeeding intermediates. On the basis of these findings a simplified method was devised for the preparation of EAC14 intermediates using human serum pretreated with MPS.     

2-Mercapto-ethanol enhancement of agglutination reaction--a possible in vitro serological correlate for assessment of functional immunity in simian malaria

Conventional indirect haemagglutination test was performed in rhesus monkey sera (collected from Plasmodium knowlesi infected animals) with and without prior treatment of sera with 2-mercapto-ethanol (2-ME). Surprisingly, many sera samples showed significant enhancement of final titre with 2-ME. The 2-ME enhancement effect was more pronounced in the sera of hyperimmune monkeys on further injection of antigen or parasites. It was also noticeable in the sera during primary drug-suppressed P. knowlesi infection and appeared to have a bearing on the immune status of the animals to rechallenge. The use of a soluble antigen prepared from P. knowlesi infected erythrocytes was found to be essential in IHA test to demonstrate the 2-ME enhancement effect. Antigen prepared from freed parasites (commonly used) failed to show a similar effect in IHA. The possible role of certain T-lymphocyte products - antigen binding, non-agglutinating, 2-ME sensitive molecules - in malarial immunology has been proposed.     

Prosopo-Thoracopagus Conjoined Twins and Other Cephalopagus-Thoracopagus Intermediates: Case Report and Review of the Literature

Prosopo-thoracopagus twins are united from the face down to the umbilicus, none with union in the brain but all with visceral anomalies intermediate between those of cephalopagus and thoracopagus. In a review of over 1200 cases of conjoined twins reported during the past 100 years, there were 14 that illustrate the continuum between cephalopagus and thoracopagus, including three that were united only from the cervical region to the umbilicus. Classic cephalopagus twins are joined from the top of the head to the umbilicus, sharing a single foregut as well as two relatively normal hearts, the “posterior” one often diminished. Typical thoracopagus, however, are conjoined only from the upper thorax to the umbilicus, each twin with a normal foregut but both sharing a single complex multiventricular heart. The intermediate cases shared either a single very abnormal heart or two hearts united by double aortic arches, and all except one had a single foregut. It is these cases intermediate between cephalopagus and thoracopagus which are the subject of this report. Received September 11, 1996; accepted December 16, 1996     

Capturing fleeting intermediates in a catalytic C–H amination reaction cycle

We have applied an ambient ionization technique, desorption electrospray ionization MS, to identify transient reactive species of an archetypal C–H amination reaction catalyzed by a dirhodium tetracarboxylate complex. Using this analytical method, we have detected previously proposed short-lived reaction intermediates, including two nitrenoid complexes that differ in oxidation state. Our findings suggest that an Rh-nitrene oxidant can react with hydrocarbon substrates through a hydrogen atom abstraction pathway and raise the intriguing possibility that two catalytic C–H amination pathways may be operative in a typical bulk solution reaction. As highlighted by these results, desorption electrospray ionization MS should have broad applicability for the mechanistic study of catalytic processes.     

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine.METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization.RESULTS: IFN-γ at 0.1 to 5 μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells.At 5 μg/L, IFN-γ also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γ showed no additive effect,sequential treatment first with lamivudine and then IFN-γ was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 μmol/L lamivudine for two days, followed by 1 μg/L IFN-γ for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 μmol/L lamivudine for two days, followed by 5 μg/L IFN-γ for six days showed a 72% reduction in HBV cccDNA pool.CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γ and lamivudine,especially in IFN-α non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.     

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.Many dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1–6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7–9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the short-tail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From low-resolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-arm, E-loop, A-domain, and P-domain, which together place the gp10 protein in the HK97 fold category (2, 13, 14). The T7 procapsid, capsid I, contains 110–140 molecules of scaffolding protein, gp9 (4, 15, 16). After scaffolding protein expulsion the spherical T7 capsid I expands to more angular intermediates, which are collectively called capsid II (2, 4, 14, 16–18).Two DNA-free capsid IIs are purified in quantity sufficient for structural studies by cryo-EM (16). Both are produced during the normal process of wild-type T7 DNA packaging in vivo. One has an unusually low density during buoyant density centrifugation in a metrizamide density gradient (1.086 g/mL; metrizamide low density, or MLD, capsid II) and the other has a density as expected for hydrated proteins (1.28 g/mL; metrizamide high density, or MHD, capsid II) (16). The low density of MLD capsid II is caused by impermeability to metrizamide (789 Da) (16). The MLD capsid II particles are produced before MHD capsid II particles based on kinetic studies (16).The DNA packaging of T7 phage starts at capsid I state where the DNA is packaged by the ATPases (gp18 and gp19) to pass through the portal (gp8) apparatus (19). By analyzing kinetics of in vivo-produced capsids, MLD capsid II was found to be the first postcapsid I capsid. MLD capsid II appears with the kinetics of an intermediate (16) but is obviously no longer in the DNA packaging pathway because it has detached from the DNA molecule that it was packaging. MLD capsid II is not produced when a nonpermissive host is infected with a T7 amber mutant defective in DNA packaging (summarized in ref. 16). Thus, MLD capsid II is an intermediate that has been altered during either cellular lysis or subsequent purification. MHD capsid II also has the appearance kinetics of an intermediate of packaging, but one that occurs later (16). Whereas MLD capsid II has the internal core stack including proteins gp8, gp14, gp15, and gp16 (16), MHD capsid II does not have the internal core stack proteins, which were presumably lost when packaged DNA exited the capsid (16).The existence of these various capsids provides an opportunity to obtain a high-resolution (3–4 Å) analysis of structural dynamics that occur in vivo. Here we report cryo-EM structures of the shells of the following bacteriophage T7 capsids: capsid I (4.6 Å), MLD capsid II (3.5 Å), MHD capsid II (6.6 Å), and phage (3.6 Å). The two capsid II shells are the first postprocapsid, in vivo-generated shells (for any packaging system) to be subjected to high-resolution structural analysis, to our knowledge. The results reveal (i) an HK97-fold shell protein with an intracapsomere, noncovalent topological linking and another intercapsomere, joint interaction, neither interaction having been found for other dsDNA tailed phages; (ii) details of the interaction of gp9 scaffolding protein with the inner surface of the capsid I shell; (iii) a novel refolding and externalization of the N terminus of major capsid protein, gp10; and (iv) a subtle, surprising contraction of the gp10 shell in transit from MLD capsid II to phage. Based on these observations, we propose a general procapsid assembly and maturation pathway for dsDNA viruses.