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A practical strategy to reconstitute the Fc functions of nanobody was developed by nanobody C-terminal dinitrophenylation. The Fc functions are successfully reinstated as proved by the potent ADCC and CDC in vitro and anti-tumor efficacies in vivo., Nanobodies are a class of camelid-derived single-domain antibodies that have many potential advantages over conventional antibodies and have been utilized to develop new therapeutic strategies for cancer and other diseases. However, nanobodies lack the Fc region of a conventional antibody, which possesses many functions, e.g., eliciting antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), essential for effective immunotherapy. The small molecular size of nanobodies also leads to poor pharmacokinetics, such as short in vivo half-life. To address these deficiencies, an endogenous antibody-based strategy to reconstitute the Fc functions for nanobodies was developed. As a proof-of-principle, an anti-human EGFR nanobody, 7D12, was selected to conduct C-terminal modification with the dinitrophenyl (DNP) hapten through Sortase A-mediated site-specific ligation. It was expected that the DNP motif would recruit endogenous human anti-DNP antibodies to indirectly reinstate the Fc functions. The resultant nanobody-DNP conjugates were shown to exhibit specific and high affinity binding to human EGFR expressed on target cancer cells. It was further proved that in the presence of anti-DNP antibody, these conjugates could mediate potent ADCC and CDC in vitro and exhibit significantly elongated half-life in vivo. Ultimately, it was proven in severe combined immunodeficiency (SCID) mice that treatment with the nanobody 7D12-DNP conjugate and anti-DNP mouse serum could inhibit xenograft tumor growth efficiently. In view of the abundance of anti-DNP and other endogenous antibodies in the human blood system, this could be a generally applicable approach employed to reconstitute the Fc functions for nanobodies and develop nanobody-based cancer immunotherapy and other therapies.

During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production capacity of microbial cells. To elucidate the acid stress-tolerance mechanisms of microbial cells, we performed genome mutagenesis combined with high-throughput technologies to screen acid stress-tolerant strains. Mutant strain Lactococcus lactis WH101 showed a 16,000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 and maintained higher ATP, NH4+, and intracellular pH (pHi) levels during acid stress. Additionally, comparative transcriptomics analysis revealed enhanced regulation of carbohydrate metabolism and sugar transport to provide additional energy, amino acid metabolism and transport to maintain pHi homeostasis and ATP generation, and fatty acid metabolism to enhance cellular acid tolerance. Moreover, overexpression of identified components resulted in 12.6- and 12.9-fold higher survival rates after acid shock for 3 h at pH 4.0 in L. lactis (ArcB) and L. lactis (MalQ) compared to the control strain, respectively. These findings provide valuable insight into the acid stress-response mechanisms of L. lactis and promote the further development of robust industrial strains.

The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81-90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.

Additional file 1: Fig. S1. Overall differentially expressed genes during acid stress. Fig. S2. Heatmaps of common differentially expressed genes in recombinant strains when compared to control strain. Table S1. Primers used in PCR amplifications. Table S2. The numbers of upregulated and downregulated genes through the eight groups.

Dextrans, as natural and biocompatible polysaccharides, are a promising candidate as PEG alternatives to improve the therapeutic profiles of biotherapeuticals. In this work, we chemically mutated the lysine residue in native Ex4 to aminooxyl containing lysine (K*) and three Ex4 analogs (Ex4-12K*, Ex4-27K*, Ex4-12K*-27K*) were synthesized and site-selectively modified with variable Mw of dextrans by facile oxime-ligation chemistry. The biological activities of six dextran-Ex4 conjugates were studied by in vitro and in vivo experiment. It was demonstrated that dextranylation at the 12-site of Ex4 determined the in vitro potency as GLP-1R agonist and the in vivo acute glucose-lowering activity. Furthermore, the in vivo long-acting antidiabetic experiment demonstrated that hypoglycemic effect was strongly dependent on the dextranylation site of Ex4 as well as the size of dextran. And Dextran-Ex4-12K*(20,000) exhibited the best long acting hypoglycemic activity in STZ/HFD-induced T2D mice and is a promising once daily reagent for T2D treatment.

In this study, 7-S-glycosides of kojic acid were designed and synthesized as mimics of its 7-O-glycosides to improve its water solubility and metabolic stability. To achieve this synthesis, a one-p...

Three 14-mer macrocyclic peptides 1–3 containing mono-, di- and tri-RGD structure motif were designed and synthesized by sortase A-mediated ligation in good yields. The results of in intro cell-based biological assays indicated that linear peptide 5 and macrocyclic peptide 1, containing di-RGD and mono-RGD motif respectively, showed remarkable potency and selectivity to αvβ3 integrin.

Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.

Background Sortase A (SrtA) is a transpeptidase found in Staphylococcus aureus, which is widely used in site-specific protein modification. However, SrtA was expressed in Escherichia coli (E. coli) in rather low level (ranging from several milligrams to 76.9 mg/L at most). The present study aims to optimize fermentation conditions for improving SrtA expression in E. coli. Results Under the optimized media (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, PBS buffer 180 mL/L) and condition (30 °C for 8 h) in a 7-L fermentor, the enzyme activity and the yield of SrtA reached 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively, which were higher by 5.8- and 4.5-folds compared with initial conditions, respectively. The yield of SrtA also represented threefold increase than the previously reported maximal level. In addition, the enzymatic characterizations of SrtA (optimal temperature, optimal pH, the influence of metal irons, and tolerance to water-soluble organic solvents) were determined. Conclusions Enhanced expression of SrtA was achieved by optimization of medium and condition. This result will have potential application for production levels of SrtA on an industry scale. Moreover, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application. Electronic supplementary material The online version of this article (doi:10.1186/s40643-017-0143-y) contains supplementary material, which is available to authorized users.

Three 14-mer macrocyclic peptides 1-3 containing mono-, di- and tri-RGD structure motif were designed and synthesized by sortase A-mediated ligation in good yields. The results of in intro cell-based biological assays indicated that linear peptide 5 and macrocyclic peptide 1, containing di-RGD and mono-RGD motif respectively, showed remarkable potency and selectivity to α

Additional file 1. Additional tables.

In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 °C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3-polystyrene beads.

In the current study, we demonstrate that the silencing of protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) and activating transcription factor 6 (ATF4) (using small interfering RNA expression constructs) inhibits the chondrocyte cell cycle and proliferation in vitro and ex vivo. The silencing of PERK alone using siRNA against PERK (siPERK) led to arrest in the G1 phase, it decreased the number of cells in the S phase, and delayed progressoin to the G2‑M phase. Co-transfection with siRNA against ATF (siATF4) led to a more profound inhibitory effect on cell cycle progression. Moreover, transfection with siPERK was associated with enhanced endoplasmic reticulum (ER) stress‑induced apoptosis during bone morphogenetic protein 2 (BMP2)‑induced chondrogenesis, and transfection with siATF4 exacerbated ER stress‑related cell death. Data from flow cytometry (FCM), immunohistochemistry and TUNEL assays supported these findings in vitro and ex vivo. As shown by our results, the combined effect of the silencing of ATF4 and PERK led to the activation of an ER stress‑specific caspase cascade in the cartilage tissue. On the whole, these findings reveal a new crucial combined effect of the silencing of PERK and ATF4 in modulating ER stress‑mediated apoptosis during chondrocyte differentiation and proliferation.

In this study, 7-S-glycosides of kojic acid were designed and synthesized as mimics of its 7-O-glycosides to improve its water solubility and metabolic stability. To achieve this synthesis, a one-pot approach involving S-glycosyl isothiouronium salts generated in situ as key intermediates was developed by using 7-chloro-kojic acid as the alkylation reagent. A series of water soluble 7-S-glycosides of kojic acid, incorporating monosaccharides and disaccharides, were prepared using this protocol. Thus, this work offers a mild, convenient, and efficient approach for the synthesis of 7-S-glycosides of kojic acid in medium to good yields.

Chitosan macro-particles prepared by the neutralization method were applied to Sortase A (SrtA) immobilization using glutaraldehyde as a crosslinking agent. The particles were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Response surface methodology (RSM) was employed to optimize the immobilization process. An average specific activity of 3142 U (mg protein)−1 was obtained under optimized immobilization conditions (chitosan concentration 3%, SrtA concentration 0.5 mg·mL−1, glutaraldehyde concentration 0.5%, crosslinking and immobilization at 20 °C, crosslinking for 3 h, and an immobilization time of 8 h). The transpeptidase activity of immobilized SrtA was proved by a peptide-to-peptide ligation with a conversion yield approximately at 80%, and the immobilized catalyst was successfully reused for five cycles without obvious activity loss. Moreover, the scale-up capability of using immobilized SrtA to catalyze a head-to-tail peptide cyclization was investigated in a batch reaction and the conversion yield was more than 95% when using 20 mg of peptide as a substrate.

Manganese(III)-mediated free-radical reaction of allenes with dimethyl malonate or ethyl cyanoacetate in acetic acid produces furan-2-(5//)-one derivatives (but-2-en-4-olides) or dimethyl 2-(2-oxoethylidene)malonates in moderate yields. The possible reaction mechanism is also discussed.

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world’s population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892−6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC(90) values below 0.01 μg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16−2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.

Our previous research has shown that the spliced isoform of XBP1 (XBP1s) is an important downstream mediator of BMP2 and is involved in BMP2-stimulated chondrocyte differentiation. Herein, we report that ATF6 and its cleaved N-terminal cytoplasmic domain (known as ATF6a) are expressed in growth plate chondrocytes. We find that these proteins are differentially induced during BMP2-triggered chondrocyte differentiation. This differential expression probably results from the activation of the ATF6 gene by Runx2 and its repression by the Sox6 transcription factor. Runx2 and Sox6 act through their respective binding elements on the ATF6 gene. When overexpressed, ATF6 and ATF6a intensify chondrogenesis; our studies demonstrate that under the stimulation of ATF6 and ATF6a, chondrocytes tend to be hypertrophied and mineralized, a process leading to bone formation. By contrast, lowering expression of ATF6a by use of its specific siRNA suppresses chondrocyte differentiation. Moreover, ATF6a interacts with Runx2 and augments the Runx2-mediated hypertrophication of chondrocytes. Importantly, overexpression and knockdown of ATF6a during the chondrocyte hypertrophy process also led to altered expressions of IHH and PTHrP (also known as PTHLH). Taken together, these findings indicate that ATF6a favorably controls chondrogenesis and bone formation (1) by acting as a co-factor of Runx2 and enhancing Runx2-incited hypertrophic chondrocyte differentiation, and (2) by affecting IHH and PTHrP signaling.

Hydrogenated silicon thin films (Si:H) have been deposited by using very high-frequency plasma-enhanced chemical vapor deposition (VHF PECVD). The structural, electrical and optical properties of the films were characterized. The transition process and the effect of pressure were studied. Results suggest that a narrow region, in which the transition from microcrystalline to amorphous growth takes place, exists in the regime of silane concentration (SC). This region is influenced by the working pressure (P). At lower pressure, the transition region is shifted to higher SC. Microcrystalline silicon (μ c-Si:H ) thin films deposited near transition region was applied as i-layer to the p-i-n solar cells. An efficiency of about 5.30% was obtained.

The hydrochalcogenation of allenyl sulfones with sodium organyl chalcogenolates can proceed smoothly to give β‐phenylchalcogenyl allyl sulfones in high yields with exclusive regioselectivity. For the asymmetric allenyl sulfone, the reaction has both high regio‐ and stereoselectivity with the predominace of E‐isomer. Some applications of β‐phenyltelluro and β‐phenylselanyl allyl sulfone in organic synthesis are also investigated.

Highly stable hydrogenated amorphous silicon (a- Si:H ) films were developed by very high frequency plasma enhanced chemical vapor deposition (VHF PECVD). Their electrical and structural properties were studied. The films were applied as i-layers for p-i-n solar cells. The stability of intrinsic films as well as solar cells was studied. Results suggest that a- Si:H films prepared at high hydrogen dilution ratio (R) and low plasma power (PW) have low hydrogen content (CH) and small microstructure factor (RH) and show high stability against light illumination. The device with i-layer prepared at PW=5 W and R=10 shows a high stability with degradation in fill factor and efficiency of 3.23% and 11.64%, respectively, over 1000 hours illumination. However, the device with i-layer prepared at higher plasma power (PW=25 W ) and lower hydrogen dilution ratio (R=5) was much less stable. The stability of the devices is directly related to the stability of the intrinsic materials.

Hydrogenated microcrystalline silicon films were prepared by very high-frequency plasma-enhanced chemical vapor deposition (VHF-PECVD) at 180 °C. The optical emission spectroscopy (OES) was used to monitor the plasma during the deposition process. When the pressure was enhanced from 50 to 80 Pa, the intensities of SiH*, H α * and H β * increase at first and then decrease for the pressures higher than 80 Pa. The transition from microcrystalline to amorphous phase occurs when increasing the pressure at silane concentration of 5%. It was found that the intensity ratio of I Hα* / I SiH* affects the growth of microcrystalline silicon. With the increase of I Hα* / I SiH* the crystallnity of films was improved. There are no direct relations between the SiH* intensity and the deposition rate. The deposition rate and dark conductivity are also discussed.

Intrinsic microcrystalline silicon films have been prepared with very high frequency plasma enhanced chemical vapor deposition (VHF-PECVD) from silane/hydrogen mixture at 180°C. The effect of silane concentration and discharge power on the growth of silicon films was investigated. Samples were investigated by Fourier transform infrared spectroscopy, Raman scattering and X-ray diffraction. The Raman spectrum shows that the morphological transition from microcrystalline to amorphous occurs under conditions of high silane concentration and low discharge power. X-ray diffraction spectra indicate a preferential growth direction of all microcrystalline silicon films in the (111) plane. In addition, a solar cell with an efficiency of 5.1% has been obtained with the intrinsic microcrystalline layer prepared at 10W.

We have examined the deposition of highly conductive boron-doped microcrystalline silicon (μc- Si:H ) films for application in solar cells. Depositions were conducted in a very high frequency plasma enhanced chemical vapor deposition (VHF PECVD) chamber. In the deposition processes, various substrate temperatures (TS) were applied. Highly conductive p-type microcrystalline silicon films were obtained at substrate temperature lower than 210°C. The factors that affect the conductivity of the films were investigated. Results suggest that the dark conductivity, which was determined by the Hall mobility and carrier concentration, is influenced by the structure. The properties of the films are strongly dependent on the substrate temperature. With TS increasing, the dark conductivity (σd) increases initially; reach the maximum values at certain TS and then decrease. Also, we applied the boron-doped μc- Si:H as p-layers to the solar cells. An efficiency of about 8.5% for a solar cell with μc- Si:H p-layer was obtained.

Intrinsic microcrystalline silicon films have been prepared using very high frequency plasma enhanced chemical vapor deposition (VHF-PECVD) at low temperature. The effect of silane concentration and power density on the deposition rate and crystallinity of silicon films has been investigated. The Raman spectra indicates a phase transition from microcrystalline to amorphous phase at silane concentrations of higher than 6%. A growth rate of microcrystalline films as high as 6.6 A/s is achieved at a silane concentration of 6% and the crystalline volume fraction Xc is 39%. We have also observed that the decrease of power density shifts the phase transition to low silane concentration.

Green fluorescent protein and a glycosylphosphatidylinositol (GPI) anchor containing the common core structure and a lipid chain were synthesized and then coupled together under the promotion of bacterial sortase A (SrtA), which was the first example for the synthesis of a full-size GPI-anchored protein by SrtA, demonstrating that this can be a generally useful method for GPI-anchored protein synthesis.

Analogs of the human CD52 and CD24 antigens carrying the common core structure of glycosylphosphatidylinositol (GPI) anchors and the intact polypeptide sequences of CD52 and CD24 were chemoenzymatically synthesized. CD52 and CD24 proteins were obtained by solid-phase peptide synthesis and then coupled to chemically synthesized GPI anchors under the influence of a bacterial enzyme, sortase A, to afford the target molecules in good yields.

A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biologically engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymatic reaction products and the resultant fluorescent liposomes. It was observed that increasing the concentration and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.

Sortases are a family of transpeptidases found in gram-positive bacteria responsible for covalent anchoring of cell surface proteins to bacterial cell walls. It has been discovered that sortase A (SrtA) of Staphylococcus aureus origin is rather promiscuous and can accept various molecules as substrates. As a result, SrtA has been widely used to ligate peptides and proteins with a variety of nucleophiles, and the ligation products are useful for research in chemical biology, proteomics, biomedicine, etc. This review summarizes the recent applications of SrtA with special emphasis on SrtA-catalyzed ligation of carbohydrates with peptides and proteins.

A chemoenzymatic method was developed for the synthesis of macrocyclic peptides and glycopeptides. Sortase A was found to mediate either head to tail cyclization or oligomerization and then head to tail cyclization of peptides and glycopeptides, depending on the peptide length, to produce 15-mer or higher cyclic peptides and glycopeptides.

Several peptides/small proteins and glycosylphosphatidylinositol (GPI) derivatives were synthesized and compared as substrates of sortase A (SrtA), a bacterial transpeptidase, for enzymatic coupling. It was observed that peptides containing the LPKTGGS and LPKTGGRS sequences as sorting signals at the peptide C-terminus were effectively coupled to GPI derivatives having one or two glycine residues attached to the phosphoethanolamine group at the nonreducing end. This reaction was employed to prepare several analogues of the human CD52 and CD24 antigens, which are naturally GPI-anchored glycopeptides/glycoproteins. It was further observed that the trisaccharide GPI analogues 5 and 6 were better SrtA substrates than monosaccharide GPI analogue 4, suggesting that steric hindrance of the GPI analogues does not affect their peptidation reaction mediated by SrtA. Therefore, this synthetic strategy may be useful for the preparation of more complex GPI-anchored peptides, glycopeptides, proteins, and glycoproteins.

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N(9)-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N(4)-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N(3) or cytosine O(2) atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory "cyclic" cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.

Background: Following the example of L-valine prodrugs of antiviral nucleoside analogues, L-valine ester of cyclopropavir (valcyclopropavir) was synthesized. Methods: The known tetrahydropyranylcyclopropavir was transformed to N-( tert-butoxycarbonyl)-L-valine ester, which was deprotected to valcyclopropavir. Results: Stability of valcyclopropavir towards hydrolysis at pH 7.0 roughly corresponded to that of valganciclovir. Valcyclopropavir inhibited replication of human cytomegalovirus (HCMV, Towne and AD169 strains) to approximately the same extent as the parent drug cyclopropavir. Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir was 95%. Conclusions: The prodrug valcyclopropavir offers some improved therapeutic parameters over the parent compound cyclopropavir.

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N(9)-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N(4)-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N(3) or cytosine O(2) atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory "cyclic" cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.

The hydrochalcogenation of allenyl sulfones with sodium organyl chalcogenolates can proceed smoothly to give β‐phenylchalcogenyl allyl sulfones in high yields with exclusive regioselectivity. For the asymmetric allenyl sulfone, the reaction has both high regio‐ and stereoselectivity with the predominace of E‐isomer. Some applications of β‐phenyltelluro and β‐phenylselanyl allyl sulfone in organic synthesis are also investigated.

(β-Organochalcogenyl)allyl phenyl sulfoxides 2 were prepared by treatment of allenyl sulfoxides 1 with sodium organyl chalcogenolates in good yields. The reaction was regioselective, giving exclusively one isomer in all cases. The applications of (β-phenyltelluro)allyl phenyl sulfoxide 2d and (β-organosulfanyl)allyl phenyl sulfoxide 2m in organic synthesis were also described.

Bisubstituted or 1,2,2-trisubstituted vinyl phosphine oxides were prepared stereoselectively by the carbocupration of acetylenic phosphine oxides followed by hydrolysis or by reacting with other electrophiles.

MUC1 glycopeptide was efficiently coupled to glycosylphosphatidylinositol (GPI) derivatives by sortase A (SrtA), verifying that SrtA can accept sterically hindered glycopeptide as substrate for ligation with GPIs. This work has established a practical method for the chemoenzymatic synthesis of GPI-linked glycopeptides.