Plan
Comptes Rendus
no. 3

Antimicrobial activity of calixarenes
Roger Lamartine1  ; Masuhiro Tsukada2, 3  ; Donna Wilson3  ; Akira Shirata2
1 UMR CNRS 5078 ‘SROMB’, université Claude-Bernard, Lyon-1, 43, bd du 11-Novembre-1918, 69622 Villeurbanne cedex, France
2 National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan
3 Department of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA
Comptes Rendus. Chimie, Volume 5 (2002) no. 3, pp. 163-169.

Résumés

The pursuit of antimicrobially active compounds against a variety of microorganisms is an area of intense and important research. In the present study, we examined the relative antimicrobial activity of calixarenes having different side chains, moieties and/or substitution groups against a diverse set of bacteria and fungi. Antimicrobial activity against the various species was evaluated by growth rate and inhibition rate comparisons to distinguish between the compounds for this desired property. Preliminary screening of 57 calixarenes was conducted to assay their potential as antimicrobially active compounds against Corynebacterium. Of these compounds, seven calixarenes numbered 25, 26, 27, 28, 30, 34 and 50 were found to exhibit suitable antimicrobial activity. These seven samples were then further tested to elucidate any antimicrobial activity they might have versus additional species. After examining the growth and inhibition values of these selected compounds, calixarenes sample numbers 25, 26, 27, 28 and 50 were shown to also display antimicrobial activity against Fusarium solani f. sp. mori 〚F.s.-26〛 with an inhibition range of approximately 60–70%. Additionally, sample numbers 25, 26, 27, and 28 exhibited excellent and selective antimicrobial activity against the fungal strains, Rosellinia necatrix 〚R-8〛, and Colletotrichum dematium 〚C.d. 8901〛.

La recherche de composés à activité antimicrobienne vis-à-vis de divers microorganismes demeure un défi important. Dans cette étude, nous déterminons et présentons l’activité antimicrobienne de calixarènes de chaînes latérales et/ou de groupements substitués différents vis-à-vis de différentes bactéries et champignons, par comparaison des vitesses de croissance et d’inhibition. Un criblage préliminaire des 57 calixarènes vis-à-vis de Corynebacterium a été effectué pour déterminer les composés présentant une activité antimicrobienne. Sept d’entre eux présentent une activité antimicrobienne satisfaisante. On a ensuite étudié l’activité antimicrobienne de ces derniers vis-à-vis d’autres espèces. La détermination des vitesses d’inhibition et de croissance montre que les calixarènes 25, 26, 27, 28 et 50 présentent une activité antimicrobienne vis-à-vis de fusarium solani f. sp. mori 〚F.s.-26〛 avec une vitesse d’inhibition de 60–70%. Par ailleurs, les calixarènes 25, 26, 27 et 28 possèdent une activité antimicrobienne très forte et sélective vis-à-vis de souches de champignons Rosellinia necatrix 〚R-8〛 et Colletotrichum dematium 〚C.d. 8901〛.

Métadonnées
Reçu le :
Accepté le :
Publié le :
DOI : 10.1016/S1631-0748(02)01354-1
Keywords: antimicrobial activity, macrocycles, calixarenes, pesticide
Mots clés : activité antimicrobienne, macrocycles, calixarènes, pesticides
Roger Lamartine 1 ; Masuhiro Tsukada 2, 3 ; Donna Wilson 3 ; Akira Shirata 2

1 UMR CNRS 5078 ‘SROMB’, université Claude-Bernard, Lyon-1, 43, bd du 11-Novembre-1918, 69622 Villeurbanne cedex, France
2 National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan
3 Department of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA 
@article{CRCHIM_2002__5_3_163_0,
     author = {Roger Lamartine and Masuhiro Tsukada and Donna Wilson and Akira Shirata},
     title = {Antimicrobial activity of calixarenes},
     journal = {Comptes Rendus. Chimie},
     pages = {163--169},
     publisher = {Elsevier},
     volume = {5},
     number = {3},
     year = {2002},
     doi = {10.1016/S1631-0748(02)01354-1},
     language = {en},
}
TY  - JOUR
AU  - Roger Lamartine
AU  - Masuhiro Tsukada
AU  - Donna Wilson
AU  - Akira Shirata
TI  - Antimicrobial activity of calixarenes
JO  - Comptes Rendus. Chimie
PY  - 2002
SP  - 163
EP  - 169
VL  - 5
IS  - 3
PB  - Elsevier
DO  - 10.1016/S1631-0748(02)01354-1
LA  - en
ID  - CRCHIM_2002__5_3_163_0
ER  - 
%0 Journal Article
%A Roger Lamartine
%A Masuhiro Tsukada
%A Donna Wilson
%A Akira Shirata
%T Antimicrobial activity of calixarenes
%J Comptes Rendus. Chimie
%D 2002
%P 163-169
%V 5
%N 3
%I Elsevier
%R 10.1016/S1631-0748(02)01354-1
%G en
%F CRCHIM_2002__5_3_163_0
Roger Lamartine; Masuhiro Tsukada; Donna Wilson; Akira Shirata. Antimicrobial activity of calixarenes. Comptes Rendus. Chimie, Volume 5 (2002) no. 3, pp. 163-169. doi : 10.1016/S1631-0748(02)01354-1. https://comptes-rendus.academie-sciences.fr/chimie/articles/10.1016/S1631-0748(02)01354-1/

Version originale du texte intégral

1 Introduction

Calixarenes are a very important class of compounds in supramolecular chemistry 〚1–5〛. As shown in Fig. 1, calixarenes are cavity-shaped cyclic molecules made up of phenol units linked via the ortho positions by methylene bridges. Calixarenes are synthetic macrocycles produced by condensation of p-substituted phenols with formaldehyde. They have an original molecular architecture and are considered to be important starting materials in the design of novel host-molecules for molecular recognition.

Fig. 1

Representation of the calixarenes and designation of the faces.

In the past ten years, parent calixarenes have been widely used as molecular platforms for the synthesis of selective molecular receptors for cations, anions and neutral molecules, in order to develop applications 〚6〛 in the fields of separation 〚7〛 purification 〚8〛, recovery of metals 〚9,10〛, pollution control 〚11〛 membranes for electronic devices 〚12,13〛, and phase transfer agents 〚14〛.

Although some calixarenes have been quoted for their antitumor and antiviral properties, very few publications appeared in the literature on their biological activities. Only recently, in August 2000, a communication 〚15〛 on the cellular toxicity of calixarenes stated that calixarene sulfonates, the calix〚6〛 and 〚8〛 derivatives display the same level of toxicity as glucose, while slight toxicity associated with the calix〚4〛arene sulfonate does exist.

In the present study 57 kinds of calixarenes were screened to find antimicrobially active compounds toward a variety of fungal and bacterial microorganisms. These novel compounds are not only important as new antimicrobial substances but are also attractive from a materials-based perspective in that calixarenes have metal binding properties.

2 Experimental

Materials

The calixarenes synthesized for these experiments are listed in Table 1, along with the different side chain moieties and/or substitution groups they contain.

Table 1

Synthesized calixarenes having different side chain moieties and/or substitution groups (Ph: phenyl; Ad: adamantyl; Py: pyridyl; bPy: bipyridyl).

CalixarenesxR1R2CalixarenesxR1R2
11C(CH3)3H301N=N–PhH
21C(CH3)3H311NO2H
Homooxaether bridge
32C(CH3)3H321CH2–CH=CH2H
43C(CH3)3H331C-ethyl bridges
Resorcin.
54C(CH3)3H341C-nonyl bridges
Resorcin.
65C(CH3)3H351C-benzylbridges
Resorcin.
76C(CH3)3H361CH(CH3)2C- CH 3 ∥O
87C(CH3)3H373N=N–PhH
91HH381CH(CH3)2CH3
102HH393CH(CH3)2CH3
113HH401CH(CH3)2CH2–CH3
125HH411CH(CH3)2CH2–CH=CH2
131CH(CH3)2H423CH(CH3)2C- Ph ∥O
141CH(CH3)2H431CH(CH3)2 CH 2 -C-O-C 2 H 5 ∥O
Homooxaether bridge
153CH(CH3)2H443CH(CH3)2 CH 2 -C-O-C 2 H 5 ∥O
165CH(CH3)2H455CH(CH3)2 CH 2 -C-O-C 2 H 5 ∥O
174CH2–CH3H461CH(CH3)2 CH 2 -C-O-C 2 H 5 ∥O
183CH2–CH2CH3H475CH(CH3)2 CH 2 -C- CH 3 ∥O
195CH2(–CH2)6–CH3H483CH(CH3)2 CH 2 -C- Ph ∥O
202C(CH3)2–CH2–H491CH(CH3)2 CH 2 -C- Ad ∥O
C(CH3)3
213C(CH3)2–CH2–H501CH(CH3)2 CH 2 -C-NC 2 H 5 2 ∥O
C(CH3)3
225C(CH3)2–CH2–H511C(CH3)3 CH 2 -C-O-C 2 H 5 ∥O
C(CH3)3
235CH2–(CH2)12–CH3H521CH(CH3)23 CH3, 1H
243CH2–(CH2)16–CH3H531N=N-Ph-NO2H
251SO3HH541C(CH3)32 Py + 2 bPy
263SO3HH551C(CH3)32 Py + 2bPy,Cu(I)
275SO3HH5612 NO2,2 C(CH3)3H
283N=N–Ph–SO3HH5713 C(CH3)3,1 H3 C- Ph , 1H∥O
291N=N–Ph–SO3HH

All even numbered parent calixarene samples 1, 4, 6, 8, 13, 15, 16 have been synthesized according to the Gutsche procedures 〚16–18〛. Odd numbered parent calixarene samples 3, 5, 7 were produced according to modified synthesis procedures 〚19,20〛. Dihomooxacalixarenes 2, 14 were synthesized in basic conditions by optimised synthetic procedures 〚21〛, while C-alkylcalix〚4〛resorcinarene 33, 34, 35 were obtained by acid-catalysed procedures 〚1〛. The upper rim tert-butyl groups were removed 〚22〛 by retro Friedel–Crafts reactions to form H-calixarenes 9–12.

Additionally, the alkylated upper rim calixarene samples 17–23, 29, 32 were obtained by Friedel–Crafts alkylation of H-calixarenes with alkyl chlorides in the presence of AlCl3 in CHCl3 solution 〚3〛. Sulfonato-calixarene samples 25–27 were produced by ipso-sulfonation 〚23〛, and phenylazocalixarene samples 28–30, 37, 53 were formed via the diazo coupling scheme 〚24,25〛. Functional groups were introduced into the lower rim of isopropylcalixarenes by means of Williamson-type OH-modifications. Literature procedures were used to introduce on the p-isopropylcalix〚4〛arene lower rim, ether, ester, acyl, ketone and amine groups for samples 36, 38, 40, 41, 43, 46, 49, 50. The exhaustive functionalisation of p-isopropylcalix〚6〛 and 〚8〛arene samples 39, 42, 44, 45, 47, 48, utilised literature procedures that were subsequently modified and optimised for these specific reactions 〚14〛.

3 Measurement

3.1 Antimicrobial activity towards bacteria

The antimicrobial activity of calixarenes was assessed against a variety of microorganisms, including several kinds of bacteria 〚26〛 and fungi, listed in Table 2. Phytoanthorogenic microorganisms were mainly used to evaluate antimicrobial activity of these compounds. Factors for these choices included ease of experiment, non-toxic quality and prevalent existence in nature.

Table 2

Microorganisms examined in the present study.

MicroorganismsStrain number of MAFF
Bacteria from Plant pathogens
Agrobacterium tumefaciens302307
Clavibacter michiganensis subsp. michganensis301368
Pseudomonas cichorii118079
Fungi from Plant pathogens
Colletotrichum dematium840066
Fusarium solani f. sp .mori840046
Rosellinia nocatrix840051

3.2 Antimicrobial activity against bacteria

Initial antimicrobial studies screened 57 types of calixarenes against the specific microbe, Corynebacterium, chosen for its high sensitivity to a wide variety of antimicrobial substances, such as Ag ions, and phytoalexin. After screening, the antimicrobial activity of the calixarenes was examined against many strains of bacteria and fungi.

From a Corynebacterium cell culture with a cell density of 100 cells/ml, 2 ml were taken and mixed with 25 ml agarose containing King B growth medium (Wako Pure Chemical Industries, Ltd.) at 55 °C, poured into a glass Petri dish and allowed to solidify at 25 °C. The calixarenes (approximately 0.5 mg) in powder form were placed onto the surface of the solid gel and incubated at 25 °C for two days. The antimicrobial activity was evaluated by measuring the radius of growth inhibition around the sample.

3.3 Antimicrobial activity against fungi

The antimicrobial activity of various calixarenes against several fungi, including Rosellinia sclerotiorum (R-8), Colletotrichum dematium (C.d.8901) and Fusarium solani f. sp. mori (F.s.26) was evaluated according to the following procedures. Solidified potato sucrose agar medium (A medium) was prepared by pouring liquid potato sucrose agar into a glass Petri dish at 25 °C. One of the fungi selected was inoculated at 25 °C on the surface of the fresh A medium. All fungal culture strains were thus inoculated on the A medium and allowed to grow at room temperature until a mycelia growth radius of approximately 3 cm had been reached (usually four days after inoculation). Another batch of solidified potato sucrose agar (B medium) was prepared according to the same methods as those described above. From the initial mycelia growths, a small portion from the mycelial periphery (size: 0.5 mm × 0.5 mm × 0.5 mm) was removed with a scalpel and transferred to a different place on the surface of the solidified B medium. Approximately 0.5 mg of calixarenes in powder form was then placed on these mycelia to assess antifungal activity. Growth rate (GR) and inhibition rate (IR) were both monitored for three days as a measure of the antifungal activity according to the equation below. The results presented here are the average of four mean values.

Growth Rate (GR)

GR =B/A×100 %
A: diameter size (mm) of control fungal colony without sample exposure.

B: diameter size of fungal colony with exposure to calixarene material.

Inhibition Rate (IR)

IR =1-B/A×100 %
A: diameter size (mm) of control fungal colony without sample exposure.

B: diameter size of fungal colony with exposure to calixarene material.

3.4 Antimicrobial activity

A preliminary evaluation of the antimicrobial activity of calixarenes against Corynebacterium was performed and gauged according to the following three stage criteria:

++ high antimicrobial activity; size of inhibition zone is approximately 3.5 mm;

+ relatively high antimicrobial activity; size of the inhibition zone is approximately 2.5 mm;

± weak antimicrobial activity; size of inhibition zone is less than 1.5 mm;

– no antimicrobial activity.

4 Results and discussion

For the preliminary screening studies of the 57 calixarenes, the antimicrobial activities of all samples were determined against Corynebacterium. Corynebacterium is useful because of its high sensitivity toward a wide variety of microorganisms of which the results are listed in Table 3.

Table 3

Preliminary screening data for antimicrobial activity of calixarenes against Corynebacterium.

Sample No.25262728303450
Activity++++±±±

From Table 3, it was found that the calixarenes exhibited a range of antimicrobial activities from complete growth inhibition to little or no inhibition. Sample numbers 25–28 all exhibit relatively high antimicrobial behaviour, and contain a common SO3H group in the basic form, which might play a role in this behaviour. The other calixarenes do not show any antimicrobial activity against Corynebacterium. It is of interest to note that calixarenes are generally able to selectively combine with metals; this feature could possibly lead to additional significant increases in the antimicrobial activity already displayed.

The extent of antimicrobial activity as a function of material concentration was also examined using the various samples in aqueous solution (Table 4). The 0.1% calixarene solution concentration corresponds to 1000 ppm.

Table 4

Antimicrobial activity of selected calixarenes against Corynebacterium as a function of material concentration.

Concentration (%)25262728
1+++++
0.1–––+
0.01––––
0.001––––

As it is clear from Table 4, calixarene sample numbers 25–27 have slight antimicrobial activity against Corynebacterium when the sample concentration is above 1%, while calixarene sample number 28 has a much stronger antimicrobial activity beyond concentrations of 0.1%. Therefore, calixarene 28 has the strongest antimicrobial potential toward Corynebacterium among the 57 calixarenes studied. From an application-based perspective, the desired concentration for an effective pesticide is considered to be less than 100 ppm. The experimental data indicate that these calixarenes do not display antimicrobial activity below 0.1%, 1000 ppm. Therefore, it seems that these compounds cannot be used as effective pesticides at the moment; however, we should bear in mind that most of the calixarenes listed in Table 1 are safe for the human body.

Growth rate and inhibition rate of fungi from plant

The comparison of the growth and inhibition rates is an important measure to distinguish the antimicrobial activity of the samples against specific fungi and bacteria. Figs. 2 and 3 demonstrate the growth and inhibition rates of various calixarenes against differing fungal strains, respectively.

Fig. 2

Growth rates of various calixarenes against differing fungal strains.

Fig. 3

Inhibition rates of various calixarenes against differing fungal strains.

Calixarene sample numbers 25–27 and 50 displayed a range of antimicrobial activity against Fusarium solani f. sp. mori (F.s.-26), having an inhibition rate of approximately 60–70%, while calixarene sample numbers 25–28 showed excellent and selective antimicrobial activity against fungal strains Rosellinia necatrix (R-8), and Colletotrichum dematium (C.d.8901).

5 Conclusion

Approximately 57 calixarenes were screened to determine compounds having antimicrobial activity against fungal and bacterial microorganisms. Sample numbers 25–28 all showed slight antimicrobial behaviour, and contain a common SO3H group in the basic form, which might play a role in this aspect. The compounds in Table 5 were found to be the prominent calixarenes exhibiting antimicrobial activity.

Table 5

Calixarenes exhibiting prominent character of antimicrobial activity.

Sample No.NameCharactern
25p-sulfonatocalix〚4〛areneOH, SO3H4
26p-sulfonatocalix〚6〛areneOH, SO3H6
27p-sulfonatocalix〚8〛areneOH, SO3H8
28p-phenylazocalix〚6〛areneOH, N=N–Ph–SO3H6
30p-phenylazocalix〚4〛areneOH, N=N–Ph4
34C-nonylresorcin〚4〛areneOH, OH4
50Aminocarbonyl p-iPr-calix 〚4〛areneCH2CON(C2H5)24

Here we report the slight antimicrobial behaviour of calixarene sample numbers 25–28, which all contain a common SO3H group in the basic form. Additionally interesting is the fact that the antimicrobially active samples 28 and 30 contain a common –N=N–phenyl group.

This essential preliminary information should be further investigated as to why the above calixarenes are antimicrobially active towards microorganisms, as well as to elucidate the activity mechanisms leading to these observations.

These experimental results indicate that several kinds of calixarenes possess antimicrobial activity towards Fusarium solani f. sp. mori (F.s-26). Because F.s.-26 is a strong pathogen and produces harmful plant injuries, these calixarenes are very important substances to consider when developing new functional pesticides. At present, there are no pesticides against F.s-26, indicating that calixarenes comprise an exciting family of compounds that could be a leading materials substance for new pesticide developments.

Normally pesticides that are effective for plants contain a large quantity of harmful halogens such as sulphur and chloride. The advantage presented by these compounds for pesticide production is that calixarenes do not contain halogens in such high amounts. Therefore, it is hypothesized that calixarenes operate under a different antimicrobial mechanism as compared with that of commercialised pesticides. Since calixarenes combine specifically with metal ions, we can produce new antimicrobially active materials based from calixarenes combined with metals, such as Ag. Ag-combined calixarene could offer a safe alternative pesticide, which would be non-toxic to the human body, and a more durable antimicrobial material, which could be used widely in several applications. With this information in place, it will be possible to develop new techniques and materials utilizing antimicrobial calixarenes in the industrial and medical fields.

Acknowledgements

This work was supported by the COE (Centre of Excellence), Special Coordination Funds for promoting Science and Technology, of the Science and Technology Agency, Japan.

Bibliographie

[〚1〛] C.D. Gutsche (J.F. Stoddart, ed.), Calixarenes, Monographs in Supramolecular Chemistry, The Royal Society of Chemistry, 1, 1989

[〚2〛] J. Vicens; V. Böhmer Calixarenes: a Versatile Class of Macrocyclic Compounds, Kluwer, Dordrecht, The Netherlands (J.E. Davies, ed.), 1990

[〚3〛] C.D. Gutsche (J.F. Stoddart, ed.), Calixarenes revisited, Monographs in Supramolecular Chemistry, The Royal Society of Chemistry, 6, 1998

[〚4〛] V. Böhmer Angew. Chem. Int. Ed. Engl., 34 (1995), p. 713

[〚5〛] A. Ike; S. Shinkai Chem. Rev., 97 (1997), p. 1713

[〚6〛] R. Perrin; R. Lamartine; M. Perrin Pure Appl. Chem., 7 (1993), p. 1549

[〚7〛] F. Vocanson; R. Lamartine; C. Duchamp; J.B. Regnouf de Vains Chromatographia, 41 (1995), p. 204

[〚8〛] J.B. Regnouf de Vains; R. Lamartine; B. Fenet Helv. Chim. Acta, 81 (1998), p. 661

[〚9〛] S. Pellet-Rostaing; J.B. Regnouf de Vains; R. Lamartine; P. Meallier; S. Guittonneau; B. Fenet Helv. Chim. Acta, 80 (1997), p. 1229

[〚10〛] P. Leverd; I. Dumazet; R. Lamartine; M. Nierlich Chem. Commun. (2000), p. 493

[〚11〛] A.W. Coleman; M. Munoz; P. Goreloff; R. Lamartine; F. Lequevre; J. Mugnier Condensed Matter News, 6 (1997), p. 3

[〚12〛] R. Mlika; I. Dumazet; H. Ben Ouada; N. Jaffrezic; R. Lamartine; M. Gamoudi; G. Guillaud Sensor. Actuat. B-Chem., 62 (2000), p. 8

[〚13〛] R. Mlika; M. Gamoudi; G. Guillaud; M. Charbonnier; M. Romand; J. Davenas; N. Jaffrezic; R. Lamartine; A. Touhami Mater. Sci. Eng., C 11 (2000), p. 129

[〚14〛] R. Lamartine; H. Meziani Supramol. Chem., 13 (2001), p. 143

[〚15〛] F. Perret; M. Mazzorana; P. Shahgaldian; A.W. Coleman XIth International Symposium on Supramolecular Chemistry, Fukuoka, Japan, 30 July 4 August 2000

[〚16〛] C.D. Gutsche; M. Iqbal Org. Synth., 68 (1990), p. 234

[〚17〛] C.D. Gutsche; B. Dhawan; M. Leonis; D. Stewart Org. Synth., 68 (1990), p. 238

[〚18〛] J.M. Munch; C.D. Gutsche Org. Synth., 68 (1990), p. 243

[〚19〛] I. Dumazet; N. Ehlinger; F. Vocanson; S. Lecocq; R. Lamartine; M. Perrin J. Incl. Phenom., 29 (1997), p. 175

[〚20〛] I. Dumazet; J.B. Regnouf de Vains; R. Lamartine Synth Commun., 27 (1997), p. 2547

[〚21〛] F. Vocanson; R. Lamartine Acros Org. Acta, 21 (1996), p. 6

[〚22〛] B. Yao; J. Bassus; R. Lamartine An. Quim., Int, 93 (1997), p. 165

[〚23〛] R. Lamartine, J.-B. Regnouf de Vains, US Patent, 08-671-170, 1996

[〚24〛] S. Bouoit-Montesinos; F. Vocanson; J. Bassus; R. Lamartine Synth. Commun., 30 (2000), p. 911

[〚25〛] S. Bouoit-Montesinos; J. Bassus; M. Perrin; R. Lamartine Tetrahedron Lett., 41 (2000), p. 2563

[〚26〛] T. Arai; G. Freddi; G.M. Colonna; E. Scotti; A. Boschi; R. Murakami; M. Tsukada J. Appl. Polymer Sci. (2000) (in printing)

Commentaires - Politique

Ces articles pourraient vous intéresser

Design, synthesis and anticoagulant activity of new flexible calix[8]arene sulfonic acids

Seifeddine Rekkab; Mesbah Lahouel; Taibi Ben Hadda; ...

C. R. Chim (2013)

Extraction and complexation of lanthanide ions by dihomooxacalix[4]arene and calix[4]arene tetraketone derivatives: An experimental and molecular dynamics investigation

Joel D. Fonseca; Paula M. Marcos; José R. Ascenso; ...

C. R. Chim (2019)

Synthesis and structural studies of a lanthanide complex of a calix[4]arene tris-amide

Mark I. Ogden; Brian W. Skelton; Allan H. White

C. R. Chim (2005)