Synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally functionalized with hemagglutinin-binding Peptide.

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Synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally-functionalized with hemagglutinin-binding peptide Ken Hatano, Teruhiko Matsubara, Yosuke MUramatsu, Masakazu Ezure1, Tetsuo Koyama, Koji Matsuoka, Ryunosuke Kuriyama, Haruka Kori, and Toshinori Sato J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Sep 2014 Downloaded from http://pubs.acs.org on September 28, 2014

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Journal of Medicinal Chemistry

Revised Synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally-functionalized with hemagglutinin-binding peptide

Ken Hatano1,*, Teruhiko Matsubara2, Yosuke Muramatsu1, Masakazu Ezure1, Tetsuo Koyama1, Koji Matsuoka1, Ryunosuke Kuriyama2, Haruka Kori2, Toshinori Sato2,*

1

Division of Material Science, Graduate School of Science and Technology, Saitama

University, 255 Shimo-Ohkubo, Sakura-ku, Saitama 338-8570, Japan. 2

Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi,

Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.

* To whom correspondence should be addressed. *(K.H.) Phone/Fax: +81-48-858-3535. E-mail:

[email protected],

*(T.S.)

Phone:

+81-45-566-1795. E-mail: [email protected].

1

ACS Paragon Plus Environment

+81-45-566-1795.

Fax:


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Abstract A series of carbosilane dendrimers uniformly functionalized with hemagglutinin (HA)-binding

peptide

(sialic

acid-mimic

peptide;

Ala-Arg-Leu-Pro-Arg)

was

systematically synthesized and their anti-influenza virus activity was evaluated. The carbosilane-based peptide dendrimers, unlike sialylated dendrimers, cannot be digested by virus neuraminidases. The peptide dendrimers exhibited intriguing biological activities depending on the form of their core frame, with a dumbbell-type peptide dendrimer showing particularly strong inhibitory activities against two human influenza viruses, A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2). The IC50 values of the dumbbell-type peptide dendrimer for both strains were 0.60 µM, the highest activity among the HA-binding peptide derivatives. The results suggest that a dumbbell-shaped carbosilane dendrimer is the most suitable core scaffold for HA-binding peptide dendrimers.

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1. Introduction An average of 226,000 patients are currently hospitalized every year in the USA with pathogenic influenza virus infections.1 Seasonal influenza epidemics can be prevented by vaccination. Influenza vaccination decreases both the rate of influenza infection and the severity of the disease symptoms, but the effectiveness of vaccination is dependent on matching the influenza vaccine to the circulating virus.2 A typical seasonal influenza vaccine contains inactivated viruses composed of two type A subtypes and one type B strain that are thought most likely to circulate in the coming season. Subtypes of influenza A virus are classified based on the antigenicity of two major surface glycoproteins, hemagglutinin (HA)a and neuraminidase (NA).3 The treatment of influenza with antiviral drugs is becoming an increasingly promising therapy against pandemic and seasonal epidemics.4,5 Over the past few decades, several antiviral drugs against the flu have been developed that are designed to inhibit one or more steps of the viral life cycle. NA inhibitors such as zanamivir and oseltamivir have been widely used as therapeutic agents in the clinical treatment of influenza A and B virus during seasonal epidemics and the 2009 pandemic.

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The characteristic glycoproteins, HA and NA, play different roles on the surface of the virus.6 HA binds to sialyl oligosaccharide receptors on host cells and is involved in the initial stage of viral infection. The progeny viruses are released from the infected cells by the enzymatic cleavage by NA of sialic acid residues of sialyl oligosaccharides on the cell surface. The molecular design of NA inhibitors is based on the transition state analogue of sialic acid generated by the enzymatic hydrolysis of sialoside by NA.5,7,8 Although these drugs initially showed high potency for inhibiting the release of influenza virions from infected cells, oseltamivir-resistant viruses have emerged.9,10 The emergence of NA inhibitor-resistant influenza virus, as well as new mutations of influenza A virus, require the development of novel therapeutic agents. Recent advances in glycoscience have revealed that glycoconjugates generally located on the cell surface, such as glycoprotein and glycolipid, play a critical role in the process of cell adhesion with the proteins of pathogens.11-14 It is known that the early stage of cell adhesion involves the carbohydrate-mediated specific recognition of pathogens. Lee discovered that the clustering of carbohydrates enhances individual interactions between carbohydrates and proteins.15,16 A combination of specific recognition and the carbohydrate cluster effect has been applied to the molecular design of artificial inhibitors and neutralizing agents of pathogens, such as toxins, bacteria and

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viruses.17-19 To date, several forms of glycoclusters have been developed.20,21 We previously reported the synthesis of several glycoclusters in which carbosilane dendrimers were employed as carbohydrate scaffolds, and demonstrated the biological activities of some of these glycoclusters.22 HAs have a trimeric structure on the viral particle and contain three sialic acid-binding sites on each unit separated by approximately 45 Å.3 Assembling sialic acid residues to target the trimeric binding sites of HAs is a valuable approach for preventing infection by influenza A viruses. Significant effort has been made to prepare multivalent glycomaterials as HA blockers for inhibiting the adhesion of viruses to host cells. We previously developed a series of influenza virus HA blockers comprising carbosilane

dendrimers

peripherally-functionalized

[(Neu5Acα2–3Galβ1–4Glcβ1]

and

demonstrated

with

sialyl

lactoses

particularly

strong

inhibitory

activities against influenza viruses A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2).22 Sato et al. identified artificial HA-binding peptides (sialic acid-mimic peptides) that inhibit the interaction between sialylglycoconjugates and the HAs of H1 and H3 through multiple serial selections from phage-displayed random peptide libraries.23 Peptides modified with an N-stearoyl group to induce molecular self-assembly in aqueous solution showed high inhibitory activities against influenza virus infection; the

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activities were believed to be enhanced by the multivalency of the peptidic sections.24 The pentapeptide Ala-Arg-Leu-Pro-Arg (ARLPR) provided the highest inhibitory activity. Since peptides can be readily synthesized by solid-phase peptide synthesis, developing peptides as HA blockers has an obvious advantage over using sialyl lactose derivatives and motivated us to assemble the inhibitory peptide on carbosilane dendrimers. The advantages of using carbosilane dendrimers as the core framework are 1）facile synthesis, 2) potential for custom design, 3) charge neutrality, 4) chemical and biological stability, 5) highly flexible structure, and 5) biological inertness. Carbsilane dendrimers peripherally functionalized with aqueous peptides on line- and point-symmetrical dendrimers are predicted to be water-soluble, giving a spatially homogeneous distribution of the peptides on the dendrimer. The solubility and symmetric structure of these functionalized dendrimers may enhance their affinity (and inhibitory activity) for the target influenza virus. In this report, we present the synthesis of these dendrimers and their inhibitory activities as HA blockers against influenza A virus.

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2.

Results and Discussion

We

have

reported

the

synthesis

and

biological evaluation

of

carbosilane

glycodendrimers with several different frameworks.22 A bioactive carbohydrate moiety, sialyl lactose, is conjugated to the terminal end of these dendrimers. The resulting glycodendrimers showed binding activity towards influenza HAs,25 and inhibitory activities against influenza neunaminidase26 and virus infection.27 In addition, the shape of the framework in ether- and amide-elongated carbosilane glycodendrimers significantly influences binding to the active site on pathogens. In the present study, a series of carbosilane dendrimer frameworks was used as the core scaffold to prepare HA-binding peptide dendrimers. The HA-binding peptide, ARLPR, had been identified as a sialic acid-mimic peptide by affinity selection from random peptide libraries.23 The HA-binding peptide is thought to bind to a receptor (sugar)-binding site on HA, such as a sialic acid-containing oligosaccharide, so we expected that the peptide dendrimer would demonstrate potential as an HA blocker. In addition, since the peptide is not digested by viral neuraminidase, strong inhibitory activity of the peptide dendrimer against infection was anticipated. We chose three parent dendrimer frameworks, Fan(0)3, Ball(0)4, and Dumbbell(1)6, without elongation; three, four, and six peptide units, respectively, can link per framework

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(Figure 1). These frameworks are named by their shapes, generation number, and number of terminal ends.22 Since the three dendrimers have the same constituent atoms from the branching silicon to the C-terminus of the peptide, the distance between the peptide units is the same in all the dendrimers.

2.1. Synthesis of peptide dendrimers Three fundamental core structures of carbosilane dendrimers (fan, ball, and dumbbell shapes) were used as scaffolds for the synthesis of HA-binding peptide cluster compounds. The synthesis of related ω-amino carbosilane dendrimers (6–8) was described

in

our

previous

report

(Figure

2).28

Briefly,

hydrosilation

of

dichlorodimethylsilane with trichlorosilane using H2PtCl6·6H2O as a catalyst and the succeeding Grignard reaction with allylmagnesium bromide provided dumbbell-shaped framework 12.29 The resulting dendrimer 12 was treated with dicyclohexylborane, followed by hydrolysis with hydrogen peroxide in alkaline solution to afford a hexahydroxy derivative 13, which further underwent O-mesylation and treatment with sodium azide (Scheme S1). Staudinger reaction of the azide derivative 15 with triphenyl phosphine afforded the resulting dumbbell-shaped ω-amino carbosilane dendrimer 8. The N-terminal amino group of the carbosilane dendrimer condensed with the

8


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C-terminal

carboxylic

group

of

Fmoc-Ala-Arg(Pmc)-Leu-Pro-Arg(Pmc)-OH

5

Fmoc-protected via

peptide

HBTU/HOBt/DIEA-mediated

coupling reaction. The C-terminal carboxylic group reacted with the fan-shaped core 6 to give the Fmoc-protected fan-shaped cluster 9, with ball-shaped core 7 to yield the protected ball-shaped cluster 10, and with the dumbbell-shaped core 8 to afford the corresponding dumbbell-shaped conjugate 11 in yields of 24%, 36%, and 6%, respectively (Scheme 1). The products were purified by silica gel column chromatography followed by gel permeation chromatography (GPC, recycling preparative HPLC). The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) spectrum of the Fmoc-protected fan-shaped cluster 9 shows a peak at m/z 4326.5 due to the [M+H]+. The peaks for ball-shaped 10 and dumbbell-shaped 11 were observed at m/z 5657.4 and 8639.7, respectively. To remove the protecting groups from the Fmoc-protected peptide dendrimer, the Fmoc group at the N-terminus of the peptide and the Pmc group of the Arg side chain

were

removed

using

piperidine/DMF

and

cleavage

cocktail

(TFA/thioanisole/1,2-ethanedithiol/ethylmethylsulfide/thiophenol/water, 825:50:25:30:20:50), respectively. The de-protection of the Fmoc and Pmc groups was repeated until all the protected groups were removed, as confirmed by HPLC and MS

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analyses. The crude peptide dendrimer was further purified by gel filtration to remove low molecular weight decomposition products of Fmoc and Pmc. Peptide dendrimers (2–4) were obtained as a white powder following lyophilization from acetonitrile-water solutions. The expected structures of the peptide dendrimers were verified by MALDI-TOF MS.

2.2. Biological evaluation of the activities of carbosilane dendrimers derivatized with HA-binding peptide against human influenza virus To investigate the inhibitory activity of the carbosilane dendrimers derivatized with HA-binding peptide, the inhibition of Mardin-Darby canine kidney (MDCK) cell infection by influenza virus was determined using a plaque assay. Two seasonal influenza viruses, A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2), were used. Prior to assay, H1N1 or H3N2 virus (50–200 pfu) was mixed with peptide dendrimer for 30 min at room temperature (pre-incubation). The mixed solution was incubated for 30 min at 37 ºC with MDCK cells, excess virus and peptide dendrimer were removed by washing, and then the MDCK cells were cultured for 48 hours. The number of plaques formed was counted and the percentage of infected cells was plotted against peptide dendrimer concentration (Figure 3).

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The fan- and dumbbell-type peptide dendrimers (2 and 4) showed inhibitory activity towards both H1N1 and H3N2 strains. The IC50 values on a per-pentapeptide basis of fan-type peptide dendrimer 2 for H1N1 and H3N2 were 12.6 µM and 5.7 µM, respectively, whereas those of monomeric peptide 1b were >100 µM for both strains (Table 1). Furthermore, the activities of dumbbell-type dendrimer 4 were about 9.5–17.5-fold higher than that of fan-type 2 (0.72 µM for H1N1 and 0.60 µM for H3N2). The dumbbell-type dendrimer is composed of two fan-type peptide clusters linked through a dimethylsilane; therefore, these results indicated that clustering of the peptide unit was effective for enhancing the inhibitory activity of the peptide against viral infection. Furthermore, the dumbbell-type peptide dendrimer (IC50 values of about 0.60 µM) showed the highest activity among the HA-binding peptide derivatives.23 The carbosilane-based peptide dendrimers showed higher inhibitory activities than a previously reported N-stearoyl pentapeptide (C18-ARLPR-NH2; IC50 of 1.9 µM for H1N1)23 and a carbosilane-based sialyl lactose dendrimer (dumbbell-type: IC50 of 4 µM for H3N2).27 Molecular modeling was performed to elucidate the interaction between the peptide dendrimers and the receptor-binding site of HA. Three peptide dendrimers, Fan(0)3-peptide, Ball(4)-peptide, and Dumbbell(1)6-peptide, were built using modeling

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software and a simple minimization was performed (Figure 4A). Modeling suggested that the peptide backbones of Fan(0)3-peptide and Dumbbell(1)6-peptide could freely move independently around their tether-point. The dumbbell-type peptide dendrimer was superimposed on the HA structure (PDB entry: 1HGG.pdb) (H3N2), obtained from the protein data bank (PDB) (Figure 4B). When one of the peptide units of the dumbbell-type peptide dendrimer was placed in the receptor-binding site of HA, the dumbbell-type dendrimer blocked the binding site. In addition to the cluster effect, the molecular assembly of peptide dendrimers may contribute to the inhibitory activity. In our previous study, a stearoyl group was conjugated to peptides at their N-terminus, then the N-stearoyl peptides (C18-peptides) were evaluated using the plaque assay.23,24 The N-stearoylation of a peptide induced its self-assembly, thereby enhancing the inhibitory activity of the peptide through multivalency. In the present study, the self-assembly of the peptide dendrimers was detected using a fluorescent reagent, N-phenyl-1-naphthylamine, and then the critical micelle concentration (CMC) was determined. Figure S2 shows the results of the determination of the CMC of the peptide dendrimers. The CMC values of the fan- and dumbbell-type peptide dendrimers were determined to be 3.9 µM and 4.0 µM, respectively. The CMC value of the self-assembled dumbbell-type dendrimer (4 µM)

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was 40-fold larger than the original IC50 values based on dendrimer concentration (0.1 µM for H1N1). We concluded that the assembly of the dendrimer is not directly related to its inhibitory activity.

2.3. Importance of the carbosilane framework for inhibitory activity In our previous study, three carbosilane-based glycodendrimers (fan, ball, and dumbbell types) were shown to be effective in supporting multivalency for affinity and inhibitory activity.24 However, ball-type dendrimer 3 did not prevent infection (Figure 3 and Table 1), suggesting that the characteristic feature(s), e.g., binding selectivity, of the peptide slightly differs from that of sialyllactose. The differences between fan- and ball-type dendrimers are their framework and the number of peptide units. The molecular models shown in Figure 4 suggest that one of the most likely reasons for the inertness of the ball-type dendrimer is that steric hindrance by the peptide units inhibits interaction between the peptide dendrimer and the HA of the virus. The peptide units in the ball-type dendrimer are crowded, so the limited flexibility of the peptide units may prevent interaction with the receptor-binding site of HA.

2.4. Cytotoxicity of dendrimers

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To investigate the cytotoxicity of the peptide dendrimers, MDCK cells and erythrocytes were exposed to the dendrimers. The peptide dendrimers were incubated with MDCK cells for 2 days, then the tetrazolium dye (MTT) based assay was performed. The fanand dumbbell-type peptide dendrimers (2 and 4) induced no significant toxicity at concentrations over ten-fold higher than the IC50 of each virus, while the ball-type peptide dendrimer 3 induced no significant toxicity at the highest concentration tested (200 µM; Figure 5A). Hemolysis assays were also performed. The damaging effect of the dendrimers on erythrocytes was estimated from the release of hemoglobin, determined by absorbance at 540 nm. The peptide dendrimers were incubated with chicken erythrocytes for 24 hours with shaking; the fan- and ball-type peptide dendrimers (2 and 3) had no hemolytic effect at concentrations up to 30–40 µM (Figure 5B). On the other hand, the dumbbell-type peptide dendrimer (4) caused 46% and 85% hemolysis at 48 and 240 µM, respectively. Thus, although the peptide dendrimers induced slight toxicity at the highest concentration tested, the toxicity of the dendrimers is negligibly small at concentrations ten-fold higher than their respective IC50.

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3.

Conclusion

We have systematically synthesized a series of carbosilane dendrimers uniformly functionalized with HA-binding peptide. Biological evaluation of the dendrimers against two human influenza viruses showed that the dumbbell-type carbosilane-based dendrimer exhibited the strongest activity (IC50 of 0.72 and 0.60 µM for H1N1 and H3N2, respectively). The dumbbell-shaped carbosilane is thus a promising scaffold for HA-binding peptide dendrimers against influenza viral infection.

4. Experimental section 4.1. General methods 1

H and

13

C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker

DRX-400, Bruker AVANCE 500, or Bruker AVANCE 500T spectrometer at 400, 500, and 500 MHz for protons and at 100, 100, and 125 MHz for carbons, respectively. Proton chemical shifts are given in parts per million using tetramethylsilane (0 ppm) or residual solvent peaks as an internal standard. NMR signals were assigned by 1H, 13C, HH, and HC COSY measurements. Infrared (IR) spectra were recorded on a Shimadzu IR-Prestage 21. Recycling preparative high-performance liquid chromatography

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(HPLC) was performed with a LC-908 and LC-918W system (Japan Analytical Industry Co., Ltd.) connected to an RI-5 RI detector. Reversed phase HPLC was performed with a binary pump system connected to an UV detector operating at 220 nm (Gilson, Inc.). The purity (>95%) of the final compounds (2–5) was verified by HPLC.

4.1.1. Synthesis of Fmoc-protected peptide (5). 9-Fluorenylmethoxycarbonyl

(Fmoc)-protected

pentapeptide

Fmoc-Ala-Arg(Pmc)-Leu-Pro-Arg(Pmc)-OH (5) was prepared by solid-phase peptide synthesis using standard Fmoc chemistry.30 Fmoc amino acid derivatives and a coupling reagent, benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), were purchased from Novabiochem (Merck). Fmoc-Arg(Pmc)-OH, in which the side chain of Arg is protected by a 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc) group, was loaded onto 2-chlorotrityl chloride resin (0.76 mmol/g). The peptide was elongated manually in multiple batches on a 0.15 mmol scale, each using a PyBOP/N,N-diisopropylethylamine (DIEA) mixture for coupling and a 20% piperidine (PIP)/N,N-dimethylformamide (DMF) mixture for deprotection of the Fmoc group. To cleave the protected peptide from the resin, the resin (0.3 mmol) was treated with 5 mL of 1% trifluoroacetic acid (TFA)/dichloromethane (DCM) for 1 minute. The

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cleaved peptide was immediately neutralized with 1 mL of 10% pyridine/methanol. The cleavage was repeated ten times. The ten cleaved peptide solutions were combined (ca. 60 mL) and the solvent removed by evaporation. To remove the pyridine completely, the residue was dissolved in ethyl acetate, then the ethyl acetate phase was washed with water. After evaporation of the solvent, the crude peptide was obtained as a white solid, then further purified by reversed phase HPLC on a C4 column (250 x 20 mm) with a linear gradient of water containing 0.1% TFA and acetonitrile containing 0.1% TFA at a flow rate of 10 mL/min. The major fractions were lyophilized and the protected peptide 5 was obtained in 34% yield based on the first amino acid loading on the resin (0.28 g, purity >95% by reversed phase HPLC). The protected peptide 5 was characterized by electrospray ionization mass spectrometry (ESI-MS) (m/z: calcd exact mass C69H95N11O14S2 for [M+H]+ 1366.7, found 1366.7).

4.1.2. Fan(0)3-peptide-Fmoc (9) Fmoc-protected peptide Fmoc-Ala-Arg(Pmc)-Leu-Pro-Arg(Pmc)-OH (5) (177 mg, 0.130 mmol, 4.8 equiv.) was added to a solution of tris(3-aminopropyl)phenylsilane28 (6, Fan(0)3-NH2;

7.54

mg,

27.0

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

µmol,

1.0

hexafluorophosphate

17


equiv.) (HBTU,


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4.8 equiv.), N-hydroxybenzotriazole (HOBt, 4.8 equiv.), and DIEA (9.6 equiv.) in dimethyl sulfoxide (DMSO, 0.20 mL). The reaction mixture was stirred for 2 weeks at r.t. The solvents were evaporated and the residue was subjected to silica gel column chromatography followed by gel permeation chromatography (GPC) using a recycling preparative HPLC system. After lyophilization, Fan(0)3-peptide-Fmoc 9 (28 mg, 24%) was obtained as a yellow powder: 1H NMR (500 MHz, DMSO-d6): δ 7.93 (m, NH amide (Pro-Arg) (Ala-Arg), 6H); 7.87 (d, CH aromatic, J = 6.5 Hz, 6H); 7.80 (s, NH amide (Arg-NH), 3H );

7.71 (d, CH aromatic, J = 7.5 Hz, 6H); 7.61 (d, NH amide

Arg-Leu, J = 7.0 Hz, 3H); 7.53 (d, NH amide carbamate, J = 6.5 Hz, 3H); 7.41-7.31 (m, CH aromatic, 17H); 6.64 (s br, NH2 guanidine, 12H); 6.41 (s br, NH guanidine, 6H); 4.49 (m, Leu α-H, 3H); 4.32 (m, Pro α-H, 3H); 4.24 (d, Fmoc CH2, J = 7.0 Hz, 6H); 4.19 (t, Fmoc CH, J = 7.0 Hz, 3H); 4.12-4.03 (m, Ala α-H, Arg α-H, 9H); 3.60 (m, Pro CH2N, 6H); 3.00 (br, Arg CH2NH, SiCH2CH2CH2, 18H); 2.54 (m, Pmc CH2, 12H); 2.46 (s br, Pmc CH3Ar, 36H); 2.00 (s, Pmc CH3Ar, 18H); 1.84 (m, Pro CH2, 6H); 1.72 (m, Pmc CH2Ar, 12H); 1.59 (br, SiCH2CH2, Leu CH, 9H); 1.38 (m, Arg CH2CH2, Leu CH2, 30 H); 1.22 (s, Pmc CH3, 36H); 1.19 (d, Ala CH3, J = 7.0 Hz, 9H); 0.85-0.81 (m, Leu CH(CH3)2, 18H); 0.71 (m, SiCH2, 6H). MALDI TOFMS (m/z: calcd Mol. Wt. for C222H308N36O39S6Si [M+H]+ 4326.5, found 4326.5).

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4.1.3. Ball(0)4-peptide-Fmoc (10) The condensation reaction between Fmoc-protected peptide 5 (140 mg, 0.102 mmol, 6.4 equiv.) and tetrakis(3-aminopropyl)silane28 (7, Ball(0)4-NH2; 4.06 mg, 15.6 µmol, 1.0 equiv.) was carried out essentially as described for the preparation of 9 to give Ball(0)4-peptide-Fmoc 10 (32 mg, 36%) as a yellow powder: 1H NMR (500 MHz, DMSO-d6): δ 7.94 (m, NH amide (Ala-Arg) (Pro-Arg), 8H); 7.87 (m, CH aromatic, 8H); 7.80 (m, NH amide Arg-NH, 4H); 7.70 (m, CH aromatic, 8H); 7.61 (m, NH amide Arg-Leu, 4H); 7.53 (m, NH amide carbamate, 4H); 7.39 (m, CH aromatic, 8H); 7.30 (m, CH aromatic, 8H); 6.64(s br, NH2 guanidine, 16H); 6.39 (a br, NH guanidine, 8H); 4.50 (m, Leu α-H, 4H); 4.33 (m, Pro α-H, 4H); 4.23 (m, Fmoc CH2, 8H); 4.18 (m, Fmoc CH, 4H); 4.13 (m, Ala α-H, 4H); 4.03 (m, Arg α-H, 8H); 3.59 (m, Pro CH2N, 8H); 3.00 (br, Arg CH2NH, SiCH2CH2CH2, 24H); 2.55 (br, Pmc CH2, 16H); 2.45 (s br, Pmc CH3Ar, 48H); 1.99 (s, Pmc CH3Ar, 24H); 1.86 (br, Pro CH2, 8H); 1.71 (br, Pmc CH2Ar, 16H); 1.61 (br, SiCH2CH2, Leu CH, 12H); 1.38 (m, Arg CH2CH2, Leu CH2, 40 H); 1.21 (m, Pmc CH3, Ala CH3, 60H); 0.81 (m, Leu CH(CH3)2, 24H); 0.43 (m, SiCH2, 8H). MALDI TOFMS (m/z: calcd Mol. Wt. for C288H404N48O52S8Si [M+H]+ 5656.2, found 5657.4).

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Page 20 of 40

4.1.4. Dumbbell(1)6-peptide-Fmoc (11) The condensation reaction between Fmoc-protected peptide 5 (622 mg, 0.455 mmol, 9.6 equiv.) and bis[tris(3-aminopropyl)silylpropyl]dimethylsilane28 (8, Dumbbell(1)6-NH2; 26 mg, 47 µmol, 1.0 equiv.) was carried out essentially as described for the preparation of 9 to give Dumbbell(1)6-peptide-Fmoc 11 (25 mg, 6%) as a yellow powder: 1H NMR (500 MHz, DMSO-d6): δ 8.12-7.97 (m, 18 H, Arg, Arg, Leu-amide), 7.91-7.89 (m, 12 H, Fmoc), 7.75-7.72 (m, 12 H, Fmoc), 7.57-7.56 (m, 6 H, Ala-amide), 7.43-7.41 (m, 12 H, Fmoc), 7.36-7.34 (m, 12 H, Fmoc), 6.91-6.36 (br, 36 H, NH-C(NH)-NH), 4.53 (br, 6 H, CH-CH2CH(CH3)2), 4.37 (br, 6 H, N-CH-CO-NH-), 4.28 (br, 12 H, CH2-O-NH-), 4.24-4.22 (m, 6 H, -CH-CH2O-NH-), 4.18 (br, 18 H, NHCH(CH2)3-), 4.10 (m, 12 H, SiCH2CH2CH2NH-), 3.04 (m, 24 H, -CH2NHCNHNH-), 2.03 (m, 36 H, Pmc(-CH3)), 1.90 (br, 12 H, -NCH2CH2CH2-), 1.79-1.75 (m, 24 H, Pmc(aroma. -CH2-)), 1.65 (br, 30 H, -CH(CH3)2, -NHCH(CH2CH2CH2-)CO-), 1.43 (br, 48 H, SiCH2CH2CH2NH-, -CH2CH(CH3)2, -NHCH(CH2CH2CH2-)CO-), 1.31-1.15 (m, 90 H, Pmc((CH3)2), -NHCH(CH3)CO-),

0.852

(br,

36

H,

-CH2CH(CH3)2),

0.451

(br,

24

H,

SiCH2CH2CH2SiCH2, CH3SiCH2CH2), -0.07 (s, 6 H, SiCH3). MALDI TOFMS (m/z: calcd Mol. Wt. for C440H624N72O78S12Si3 [M+H]+ 8640.1, found 8639.7).

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4.1.5. Fan(0)3-peptide (2) To remove the Fmoc group, Fan(0)3-peptide-Fmoc 9 (10 mg, 2.3 µmol) was dissolved in 20% PIP/DMF (0.5 mL). After 1 hour, the solvents were removed completely by evaporation under vacuum. The residue was dissolved in cleavage cocktail (TFA/thioanisole/1,2-ethanedithiol/ethylmethylsulfide/thiophenol/water, 825:50:25:30:20:50) and the mixture was allowed to stand at room temperature for several hours until the cleavage was complete. After evaporation of the cocktail, the de-protected dendrimer was purified by gel filtration chromatography on a Sephadex LH-20 column using methanol as a solvent to give de-protected Fan(0)3-peptide 2 (3.4 mg, 70%) as a white powder after lyophilization from 50% acetonitrile: MALDI TOFMS (m/z: calcd exact mass for C93H170N36O15Si for [M+H]+ 2060.3, found 2060.7).

4.1.6. Ball(0)4-peptide (3) The de-protection of Ball(0)4-peptide-Fmoc 10 (4.5 mg, 0.80 µmol) was carried out essentially as described for the preparation of 2 to give de-protected Ball(0)4-peptide 3 (1.0 mg, 48%) as a white powder after lyophilization from 50% acetonitrile: MALDI TOFMS (m/z: calcd exact mass for C116H220N48O20Si [M+H]+ 2634.8, found 2634.1).

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4.1.7. Dumbbell(1)6-peptide (4) The de-protection of Dumbbell(1)6-peptide-Fmoc 11 (12 mg, 1.4 µmol) was carried out essentially

as

described

for

the

preparation

of

2

to

give

de-protected

Dumbbell(1)6-peptide 4 (1.8 mg, 32%) as a white powder after lyophilization from 50% acetonitrile: MALDI TOFMS (m/z: calcd Mol. Wt. for C182H348N72O30Si [M+H]+ 4110.4, found 4110.4).

4.2. Plaque assay To determine the inhibitory activity of each peptide dendrimer, the infection of MDCK cells by influenza virus was evaluated using a plaque assay.23 Briefly, the peptide dendrimer solution (4 mM) in 50% methanol was serially diluted with PBS (0.3 mL each in a 1.5-mL tube) and mixed with 0.3 mL of influenza A/Puerto Rico/8/34 (H1N1) or A/Aichi/2/68 (H3N2) virus solution containing 50–200 plaque forming units (pfu). After 30 min at room temperature, the mixture was incubated with a MDCK monolayer for 30 min at 37 °C under 5% CO2 (0.2 mL/well, n=3). After washing, the MDCK cells were incubated for 2 days and the number of plaques was counted using crystal violet. The percentage of inhibition was calculated using the equation 100(1–(N/N0)), where N and N0 are the number of plaques in the presence and in the absence of inhibitor,

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respectively. The IC50 value (50% inhibitory concentration) of the peptide dendrimer was obtained as described previously.23

4.3. Detection of self-assembly of the peptide dendrimer To confirm the formation of the dendrimer assembly, the CMC value of each peptide dendrimer was measured. The CMCs of the peptide dendrimers were determined using a fluorescent probe, N-phenyl-1-naphthylamine (NPN).24 Peptide dendrimer was dissolved in PBS containing 1 µM NPN using a vortex mixer and diluted to 10 nM – 5 µM. Fluorescence intensity was measured on a fluorescence spectrophotometer (FL-2500, Hitachi) using a 5 mm-cuvette at 25 ºC. In the presence of dendrimer assemblies, the fluorescence emission has a maximum at 450 nm upon excitation at 350 nm. Plotting the NPN fluorescence intensities against dendrimer concentrations allowed the CMC to be determined (Figure S2).

4.4. Molecular modeling Molecular modeling was performed using Discovery Studio 2.1 software (Accelrys, Inc.).

The

peptide

dendrimers

of

Fan(0)3-peptide,

Ball(4)-peptide,

and

Dumbbell(1)6-peptide were built using the DS Biopolymer module. Simple

23



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minimization was performed using the CHARMm force field; the structures obtained are drawn in Figure 4. The crystal structure of a target HA (H3N2) (PDB entry: 1HGG.pdb) was obtained as described previously.23 To compare the sizes of the molecules, Dumbbell(1)6-peptide and HA were superimposed, then one dendrimer peptide unit was manually placed in the receptor-binding site of HA.

4.5. Cytotoxicity assay The sensitivity of MDCK cells to peptide dendrimers was measured using a cell-counting kit (WST-1 kit) according to the manufacturer’s protocol (Dojindo, Japan). This methodology is a refinement of the tetrazolium dye (MTT) based colorimetric assay and monitors the toxic effects of compounds on cells as reflected by mitochondrial succinate-tetrazolium reductase activity. MDCK cells were seeded at 3x104 cells/well in a 96-well plate and grown overnight in minimum essential media (MEM) containing 10% fetal bovine serum. Cells were incubated with the dendrimers for 2 days at 37 ºC under 5% CO2, then 10 µL of reagent was added to each well and the plates were incubated for 2 hours. The absorbance of each well was measured using a microplate reader at 450/630 nm and percent viability was calculated by: (Adendr/Actrl) x 100, where Adendr and Actrl are the absorbance of a well containing the dendrimer and the

24


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MEM control, respectively. The hemolytic assay was performed as described previously.31 Chicken erythrocytes were separated from preserved blood in Alsever's solution (Kohjin Bio Co., Ltd., Japan) by centrifugation at 1000 x g and washed five times with isotonic PBS (pH 7.4). Erythrocytes were incubated with the dendrimers for 24 hours at 37 ºC with shaking at 180 rpm. After centrifugation at 5000 x g for 5 min, the absorbance of the supernatant was measured at 540 nm. The absorbance obtained in the presence of 1% Triton X-100 was taken as 100% hemolysis.

25



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Page 26 of 40

SUPPORTING INFORMATION AVAILABLE Synthetic scheme for Dumbbell(1)6-NH2 8 (Scheme S1), formation of the molecular assembly of peptide dendrimers (Figure S1), molecular modeling of the peptide dendrimers (Figure S2), NMR spectra (compounds 9–11), and MS spectra (compounds 2–4 and 9–11). This material is available free of charge via the Internet at http://pubs.acs.org.

The abbreviations used HA, hemagglutinin; NA, neuraminidase; Glc, glucose; Gal, galactose; Neu5Ac, N-acetylneuraminic acid; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;

HOBt,

N,N-diisopropylethylamine;

GPC,

high-performance

liquid

gel

chromatography;

N-hydroxybenzotriazole;

DIEA,

permeation

HPLC,

chromatography;

MALDI-TOF,

matrix-assisted

laser

desorption/ionization-time of flight; MS, mass spectrometry; MDCK, Mardin-Darby canine kidney; IC50, 50% inhibitory concentration; PDB, protein data bank; CMC, critical

micelle

concentration;

NMR,

nuclear

magnetic

resonance;

Fmoc,

9-fluorenylmethoxycarbonyl; ESI, electrospray ionization; PIP, piperidine; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; DCM, dichloromethane; DMSO, dimethyl sulfoxide.

26


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References (1)

Smith, N. M.; Bresee, J. S.; Shay, D. K.; Uyeki, T. M.; Cox, N. J.; Strikas, R. A. Prevention and Control of Influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm. Rep. 2006, 55, 1-42.

(2)

Hilleman, M. R. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 2002, 20 (25-26), 3068-3087.

(3)

Wiley, D. C.; Skehel, J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 1987, 56, 365-394.

(4)

Barik, S. New treatments for influenza. BMC medicine 2012, 10, 104.

(5)

Ward, P.; Small, I.; Smith, J.; Suter, P.; Dutkowski, R. Oseltamivir (Tamiflu) and its potential for use in the event of an influenza pandemic. J. Antimicrob. Chemother. 2005, 55 Suppl 1, i5-i21.

(6)

Webster, R. G.; Bean, W. J.; Gorman, O. T.; Chambers, T. M.; Kawaoka, Y. Evolution and ecology of influenza A viruses. Microbiol. Rev. 1992, 56, 152-179.

(7)

von Itzstein, M.; Wu, W. Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Van Phan, T.; Smythe, M. L.; White, H. F.; Oliver, S. W.; et al. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 1993, 363, 418-423.

(8)

von Itzstein, M. The war against influenza: discovery and development of sialidase inhibitors. Nature Rev. Drug Discover 2007, 6, 967-974.

(9)

Le, Q. M.; Kiso, M.; Someya, K.; Sakai, Y. T.; Nguyen, T. H.; Nguyen, K. H.; Pham, N. D.; Ngyen, H. H.; Yamada, S.; Muramoto, Y.; Horimoto, T.; Takada, A.;

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Goto, H.; Suzuki, T.; Suzuki, Y.; Kawaoka, Y. Avian flu: isolation of drug-resistant H5N1 virus. Nature 2005, 437, 1108. (10) de Jong, M. D.; Tran, T. T.; Truong, H. K.; Vo, M. H.; Smith, G. J.; Nguyen, V. C.; Bach, V. C.; Phan, T. Q.; Do, Q. H.; Guan, Y.; Peiris, J. S.; Tran, T. H.; Farrar, J. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N. Engl. J. Med. 2005, 353, 2667-2672. (11) Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 1993, 3, 97-130. (12) Angata, T.; Varki, A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem. Rev. 2002, 102, 439-469. (13) Karlsson, K. A. Animal glycosphingolipids as membrane attachment sites for bacteria. Annu. Rev. Biochem. 1989, 58, 309-350. (14) Esko, J. D.; Cold Spring Harbor Laboratory Press: 1999, p 429-440. (15) Lee, Y. C.; Townsend, R. R.; Hardy, M. R.; Lonngren, J.; Arnarp, J.; Haraldsson, M.; Lonn, H. Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J. Biol. Chem. 1983, 258, 199-202. (16) Lee, Y. C. Biochemistry of carbohydrate-protein interaction. FASEB J. 1992, 6, 3193-3200. (17) Lundquist, J. J.; Toone, E. J. The cluster glycoside effect. Chem. Rev., 2002, 102, 555-578. (18) Roy, R. A decade of glycodendrimer chemistry. Trends Glycosci. Glycotechnol., 2003, 15, 291-310.

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(19) Schengrund, C. –L. “Multivalent” saccharides: development of new approach for inhibiting the effects of glycosphingolipid-binding pathogens. Biochem. Pharmacol., 2003, 65, 699-707. (20) Chabre, Y. M.; Roy, R. Design and creativity in synthesis of multivalent neoglycoconjugate. Adv. Carbohydr. Chem. Biochem., 2010, 63, 165-393. (21) Eds by Renaudet, O.; Roy, R. Themed issue on “Multivalent scaffold in glycosciences”. Chem. Soc. Rev. 2013, 42(11), 4509-4841. (22) Hatano, K.; Matsuoka, K.; Terunuma, D. Carbosilane glycodendrimers. Chem. Soc. Rev. 2013, 42, 4574-4598. (23) Matsubara, T.; Onishi, A.; Saito, T.; Shimada, A.; Inoue, H.; Taki, T.; Nagata, K.; Okahata, Y.; Sato, T. Sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy. J. Med. Chem. 2010, 53, 4441-4449. (24) Matsubara, T.; Sumi, M.; Kubota, H.; Taki, T.; Okahata, Y.; Sato, T. Inhibition of influenza virus infections by sialylgalactose-binding peptides selected from a phage library. J. Med. Chem. 2009, 52, 4247-4256. (25) Oka, H.; Onaga, T.; Koyama, T.; Guo, C. T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.; Matsuoka, K. Sialyl alpha(2-->3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers. Bioorg. Med. Chem. Lett. 2008, 18, 4405-4408. (26) Sakamoto, J.; Koyama, T.; Miyamoto, D.; Yingsakmongkon, S.; Hidari, K. I.; Jampangern, W.; Suzuki, T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.; Matsuoka, K. Thiosialoside clusters using carbosilane dendrimer core scaffolds as a new class of influenza neuraminidase inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 717-721.

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(27) Oka, H.; Onaga, T.; Koyama, T.; Guo, C. T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.; Matsuoka, K. Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose moieties as inhibitors for human influenza viruses. Bioorg. Med. Chem. 2009, 17, 5465-5475. (28) Sakamoto, J.; Koyama, T.; Miyamoto, D.; Yingsakmongkon, S.; Hidari, K. I.; Jampangern, W.; Suzuki, T.; Suzuki, Y.; Esumi, Y.; Nakamura, T.; Hatano, K.; Terunuma, D.; Matsuoka, K. Systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties. Bioorg. Med. Chem. 2009, 17, 5451-5464. (29) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma, D.; Kuzuhara, H. Synthetic assembly of trisaccharide moieties of globotriaosyl ceramide using carbosilane dendrimers as cores. A new type of functional glyco-material. Tetrahedron Lett. 1999, 40, 7839-7842. (30) Fujitani, N.; Shimizu, H.; Matsubara, T.; Ohta, T.; Komata, Y.; Miura, N.; Sato, T.; Nishimura, S. Structural transition of a 15 amino acid residue peptide induced by GM1. Carbohydr. Res. 2007, 342, 1895-1903. (31) Rodi, P. M.; Bocco Gianello, M. D.; Corregido, M. C.; Gennaro, A. M. Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane. Biochim. Biophys. Acta 2014, 1838, 859-866.

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Figure legends Figure 1. A series of carbosilane dendrimers derivatized with the HA-binding pentapeptide. Ala-Arg-Leu-Pro-Arg (ARLPR) (1). The shapes of the dendrimers are fan (2), ball (3), and dumbbell (4).

Figure 2. A series of ω-amino carbosilane dendrimers. Fan(0)3-NH2 (6), Ball(0)4-NH2 (7), and Dumbbell(1)6-NH2 (8).

Scheme 1. Reagents and conditions: (i) HBTU/HOBt/DIEA; (ii) 20% PIP/DMF, then cleavage cocktail.

Figure 3. Inhibition of influenza virus infection of MDCK cells by peptide dendrimers Fan(0)3-peptide 2, Ball(0)4-peptide 3, and Dumbbell(1)6-peptide 4. The mixture of peptide dendrimer and H1N1 (A/Puerto Rico/8/34) or H3N2 (A/Aichi/2/68) strain was incubated with MDCK cells for 30 min. After washing, the MDCK cells were cultured for 2 days. The number of plaques was counted and the percentage inhibition was plotted against the concentrations of the peptide dendrimers. The concentrations of the

31



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peptide dendrimers are represented by [peptide], and were calculated based per peptide (ARLPR) unit. The data are average values ± the standard deviation (n = 3).

Figure 4. Modeling of peptide dendrimers and carbohydrate recognition by the dendrimer–HA complex. (A) Molecular modeling of the peptide dendrimers shown as space-filling representations. The carbosilane frameworks and silicone (Si) atoms are shown in navy-blue and light-blue, respectively. The dendrimer conformations were obtained by a simple minimization. (B) Illustration of carbohydrate recognition by the dendrimer–HA complex (superimposed image). One peptide unit of Dumbbell(1)6-peptide was placed manually in the receptor-binding site of H3 HA (PDB entry: 1HGG.pdb).

Figure 5. Cytotoxicity of peptide dendrimers. (A) Cytotoxic effect of dendrimers on MDCK cells. MDCK cells were incubated with the peptide dendrimers for 2 days, then cell viability was measured. (B) Hemolysis assay of dendrimers against erythrocytes. Chicken erythrocytes were incubated with the peptide dendrimers for 24 hours, then the percent hemolysis was

32


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estimated. Concentrations of the peptide dendrimers are represented by [peptide], and were calculated based per peptide (ARLPR) unit. The data are average values ± the standard deviation (n = 3).

33



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Page 34 of 40

Table 1. Inhibitory activities of peptide dendrimers against infection of MDCK cells by influenza virus. Code

Structures a

Infection b

Number of

IC50 (µM) c

peptide units H1N1

2 3 4 a b c

Fan(0)3-peptide Ball(0)4-peptide Dumbbell(1)6-peptide

3 4 6

12.6 >400 0.72

Peptide, Ala-Arg-Leu-Pro-Arg. Plaque assay. H1N1, A/Puerto Rico/8/34; H3N2, A/Aichi/2/68. IC50 values are calculated based per peptide (ARLPR) unit.

34


H3N2 5.7 >400 0.60

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Figure 1.

Figure 2.

35



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Scheme 1.

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Figure 3.

37



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Figure 4.

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Figure 5.

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TOC graphic

40


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Amphiphilic cationic carbosilane-PEG dendrimers: synthesis and applications in gene therapy.

Antiviral mechanism of polyanionic carbosilane dendrimers against HIV-1.

Inhibitory activity of selenium nanoparticles functionalized with oseltamivir on H1N1 influenza virus.

Synthesis of novel peptide dendrimers PDL-GB2 and PDL-G2.

Elucidation of the interaction mechanism with liposomes of gH625-peptide functionalized dendrimers.

Evaluation of the activity of new cationic carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga.

Synthesis and protein-tyrosine kinase inhibitory activities of flavonoid analogues.

Carbosilane dendrimers as gene delivery agents for the treatment of HIV infection.

In vivo anti-influenza virus activity of plant flavonoids possessing inhibitory activity for influenza virus sialidase.

Synthesis, Radiolabeling, and Biological Evaluation of Peptide LIKKPF Functionalized with HYNIC as Apoptosis Imaging Agent.

Design and synthesis of norendoxifen analogues with dual aromatase inhibitory and estrogen receptor modulatory activities.

Dendrimers with Oligospiroketal (OSK) Building Blocks: Synthesis and Properties.

Biological activities of 'noninfectious' influenza A virus particles.

Triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent HIV-1 sexual transmission.

An expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of highly functionalized hexahydro-1,6-naphthyridines.

Copper(II) complexes with 4-carbomethoxypyrrolidone functionalized PAMAM-dendrimers: an EPR study.

Synthesis, characterization, and antileishmanial activities of gold(I) complexes involving quinoline functionalized N-heterocyclic carbenes.

Poly(amidoamine) Dendrimers with Carbonic Anhydrase Inhibitory Activity and Antiglaucoma Action.

Synergistic activity profile of carbosilane dendrimer G2-STE16 in combination with other dendrimers and antiretrovirals as topical anti-HIV-1 microbicide.

Chemical modification of the sugar part of methyl acarviosin: synthesis and inhibitory activities of nine analogues.

Synthesis of 2-(N-arylcarboxamide)-3-substituted ethoxyindoles and their monoamine oxidase inhibitory and anticonvulsant activities.

Microwave-assisted synthesis, molecular docking, and cholinesterase inhibitory activities of new ethanediamide and 2-butenediamide analogues.

Synthesis of some novel hydrazone and 2-pyrazoline derivatives: monoamine oxidase inhibitory activities and docking studies.

A Tricholoma matsutake Peptide with Angiotensin Converting Enzyme Inhibitory and Antioxidative Activities and Antihypertensive Effects in Spontaneously Hypertensive Rats.