Syntheses and monolayer properties of vitamin B12 derivatives with seven alkyl chains

Colloids and Surfaces A: Physicochemical and Engineering Aspects 169 (2000) 47 – 58 www.elsevier.nl/locate/colsurfa

Syntheses and monolayer properties of vitamin B12 derivatives with seven alkyl chains Katsuhiko Ariga a,*, Keizo Tanaka a, Kiyofumi Katagiri a, Jun-ichi Kikuchi a, Eiji Ohshima b, Yoshio Hisaeda b a

Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), 8916 -5 Takayama, Ikoma, Nara 630 -0101, Japan b Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu Uni6ersity, 6 -10 -1 Hakozaki, Higashi-ku, Fukuoka 812 -8581, Japan

Abstract The heptapropyl and heptaoctyl esters of vitamin B12 derivatives with a Co(II) or Co(III) center have been synthesized and their monolayer properties have been investigated. The effect of the esters’ chain length on stability and orientation of the vitamin B12 in a lipid monolayer is discussed on the basis of their surface pressure molecular area (p-A) isotherms. Their isotherms significantly depend on the length of the side chains regardless of the oxidation state of the center cobalt. The isotherm of the heptaoctyl derivatives has a relatively steep shape with a molecular area of ca. 3 nm2 which is fairly close to the molecular area estimated by molecular modeling for face-on orientation. In contrast, the heptapropyl derivatives showed unstable characteristics with collapsing even at low pressures. The heptaoctyl derivatives can also be stably incorporated in the DPPC matrix monolayer, while the heptapropyl derivatives were squeezed out from the same matrix upon compression at high pressures. The effect of coordination of the center cobalt to the matrix lipid was also investigated. Mixing of the heptapropyl Co(II) derivatives with a lysine-functionalized lipid significantly improved the preservation of the vitamin B12 function in the monolayer due to coordination of the lysine amino group to the open axial position of Co(II). The obtained results in this study indicate that stable accommodation of the vitamin B12 functions in the lipid assembly can be achieved by the introduction of a long chain and/or coordination to the matrix lipid. The present study is the first example of a monolayer system of vitamin B12 mimics with a core structurally identical to the naturally occurring one. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Vitamin B12; Monolayer; p-A Isotherm; Coordination; Orientation

1. Introduction

* Corresponding author. Tel.: +81-743-72-6091; fax +81743-72-6099. E-mail address: [email protected] (K. Ariga)

For more than 50 years [1–3], vitamin B12 has been a research target attracting physicists, chemists, and biologists, because it uniquely possesses a naturally occurring carbonmetal (cobalt)

0927-7757/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 4 1 6 - 7

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bond. Two of its biologically active forms, coenzyme B12 (5%-deoxyadenosylcobalamin) and methylcobalamin catalyze isomerization reactions in enzymes such as dioldehydrase, methylmalonyl CoA mutase, and ribonucleotide reductase. Structural analyses [4 – 8], investigation of the catalytic mechanism [7,9 – 20], total syntheses [21], and application to modified electrodes [22– 27] have been performed. Various cobalt complexes have been synthesized as a model of vitamin B12 including cobaloxime (bis(dimethylglyoximato)cobalt), the cobalt complex of 1,19-dimethyl-AD-didehydrocorrin, and costa-type complexes [28 – 33]. However, they do not satisfy all the requirements with respect to redox behavior, steric hindrance, and microenvironment which are indispensable in order to completely mimic the vitamin B12 function. This fact suggests that preservation of the core structure seems to be a sure way to construct B12-based artificial enzymes. In the naturally occurring enzymes, the apoprotein provides a hydrophobic and organized environment as the reaction medium for vitamin B12. Immobilization of vitamin B12 mimics in the appropriate environment is also necessary to construct the artificial catalysis. Therefore, we prepared vitamin B12 derivatives which have a core structurally identical with vitamin B12 and seven ester chains at the outside, and immobilized them in synthetic bilayer membranes [34 – 43]. Stable preservation of the vitamin B12 derivatives in the lipid assembly is an important key to achieve successful catalysis. Incorporation of the vitamin B12 derivatives from the aqueous phase to lipid bilayers was investigated by gel-filtration chromatography and electronic spectroscopy [34,41]. Partition of the hexacoordinated Co(III) species of vitamin B12 derivatives to the bilayer apparently depends on the chain length of the side chains. The methyl ester derivative is not included in the bilayer vesicle, while the propyl and butyl esters are quantitatively incorporated into the same bilayer. For the partition of the Co(II) derivatives with an open axial site, coordination with a lipid component is a decisive factor. The bilayer of the alanine-containing

lipid did not show any ability to incorporate the methyl ester of the Co(II)-derivative. In contrast, the same derivative was quantitatively incorporated into the bilayer of the histidine-containing lipid. Apparently, coordination of imidazole with Co(II) promotes incorporation of the vitamin B12 derivative into the bilayers. The obtained information is crucial for stable immobilization of the vitamin B12 derivatives in the hydrophobic environment formed by the artificial bilayers. However, the partition experiment gives only information on the total amount of the incorporation. In this paper, we present the synthesis and monolayer properties of the propyl and octyl esters of vitamin B12 derivatives with a Co(II) or Co(III) center (Charts 1 and 2). Especially, the synthesis of the heptaoctyl derivatives have not been reported so far. The monolayer study presented in this paper can supply additional insight into the orientation of vitamin B12 derivatives incorporated in the lipid film. The effects of chain length and coordination on the relative stability and orientation of the vitamin B12 in the lipid monolayer are discussed on the basis of the surface pressure-molecular area (p-A) isotherms. The present study is the first example of a monolayer of vitamin B12 mimics with a core structurally identical to the naturally occurring one. Immobilization of the vitamin B12 derivatives in the lipid monolayer will provide a new methodology for constructing B12-functionalized electrodes, which have the potential to transform environmentally toxic chemicals through a chemical-free electrochemical process.

2. Experimental

2.1. Syntheses of 6itamin B12 deri6ati6es The syntheses of heptapropyl esters (1a and 2a) were described elsewhere [41,42]. The hydrophobic vitamin B12 derivatives were prepared by a method similar to that previously reported as shown in Chart 2 [41]. Dicyano heptamethylcobyrinate 5 was prepared from cyanocobalamin [44].

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2.1.1. Heptaoctyl dicyanocobyrinate (2b) Heptamethylcobyrinate 5 (1.0 g, 0.92 mmol) in dry 1-octanol (90 ml) was mixed with a solution of dry 1-octanol (10 ml) and concentrated sulfuric acid (6 ml). The solution was deoxygenated by bubbling nitrogen gas through it for 20 min and then heated to 40°C for 45 h in the dark under a nitrogen atmosphere. The reaction mixture was cooled and extracted with dichloromethane (200

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ml) and washed with water (200 ml× 3) and 4% sodium carbohydrate aqueous solution (200 ml× 3). The organic layer was shaken with aqueous potassium cyanide (2.0 g 100 ml − 1) and dried over sodium sulfate. After the layer was separated, the organic layer was concentrated in vacuo. The residue was then subjected to gelfiltration chromatography on Sephadex LH-20 with methanol as the eluent. The major fraction

Chart 1.

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Chart 2.

was collected and evaporated to dryness to afford a viscous dark purple: yield 1.56 g (96%). Found: C, 69.42; H, 9.57; N, 4.76%. C103H171CoN6O14 requires C, 69.64; H, 9.70; N, 4.73%; m/z (FAB), calculated for 1775.2185 (M+), found 1775.2 (M+), 1749.2 (M+-CN), 1723.2 (M+-2CN); nmax (neat)/cm − 1 2120 (CN str.) and 1730 (ester C0 str.); lmax (C6H6)/nm 279 (o/dm3 mol − 1 cm − 1 1.1× 104), 317 (1.0× 104), 373 (2.8×104), 425 (3.4× 103), 553 (8.5×103), 592 (1.1×104).

2.1.2. Heptaoctyl cyaneaquacobyrinate (6) Heptaoctyl Dicyanocobyrinate (2b) (500 mg, 0.28 mmol) was dissolved in 100 ml of dichrolomethane, and the resulting purple solution was treated with 100 ml of 30% aqueous perchloric acid. The orange dichloromethane

layer was separated from the acidic aqueous layer and washed with water. After being dried over sodium sulfate, the organic layer was evaporated to dryness at room temperature to obtain heptaoctyl cyanoaquacobyrinate (6) in a nearly quantitative yield. nmax (nujol)/cm − 1 2140 (CN str.), 1740 (ester CO str.), 620 (ClO− 4 str.), and; lmax (C6H6)/nm 324, 354, 405, 488.

2.1.3. Heptaoctyl cobyrinate (1b) The above monocyano complex 6 was dissolved in 100 ml of methanol. The solution was deoxygenated by bubbling nitrogen gas through it for 10 min, and sodium tetrahydroborate (100 mg, 2.7 mmol) was then added under a nitrogen atmosphere. At the instance that the color of the solution changed to dark green (cobalt(I) state), 3

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ml of 60% of aqueous perchloric acid was carefully added dropwise to decompose any excess sodium tetrahydrobrate and to convert it into the cobalt(II) state. The resulting cobalt complex was extracted with dichloromethane and washed with water. After being dried over sodium sulfate, the solvent was removed in vacuo to afford a viscous brown oil: yield 455 mg (89%); found: C, 65.06; H, 9.15; N, 3.28%. C101H171ClCoN4O18·2H2O requires C, 65.22; H, 9.48; N, 3.01%; lmax (C6H6)/ nm 316, 411, 473.

program were used without any modification. We used the following structure as an initial conformer for the optimization based on requirement that the alkyl chains in the vitamin B12 derivatives must be apart from the water phase. Conformation of the core structure was first optimized, and then seven alkyl chains in the all-trans conformation were attached to the core. In the initial conformer, the ester conformation was adjusted so that all the chains pointed out in the same direction, nearly vertically to the core.

2.2. Surface pressure-molecular area (p-A) isotherm measurement

3. Results and discussion

Water used for the subphase was distilled and deionized using an Autostill WS33 (Yamato Scientific, Japan) and Milli-Q Labo (Nihon Millipore, Japan), respectively, until the specific resistance of ca. 18 MV·cm was obtained. p-A Isotherms were measured with a computer-controlled FSD-300 film balance system (USI System, Fukuoka). Spectral grade benzene or a benzene/ ethanol mixture (80/20) was used as the spreading solvent. The starting trough area was 150 × 463 mm2, and ca. 100 ml of the mixed solution was spread. Compression was started about 10 min after spreading at a rate of 0.2 mm·s − 1 (or 30 mm2·s − l based on area). The subphase temperature was kept at 20 9 0.2°C. The surface pressures were measured by a Wilhelmy plate, which had been calibrated with the transition pressure of an octadecanoic acid monolayer.

2.3. Other measurements The electronic spectra of the benzene solutions of the vitamin B12 derivatives were measured by a UV-2400PC spectrometer (Shimadzu, Japan). Molecular conformations of the vitamin B12 derivatives were estimated by a Cerius2 calculation (version 3.8, Molecular Simulations Inc.) based on the UNIVERSAL force field (version 1.02) [45]. The structural error was minimized in bond, angle, torsion, inversion, van der Waals, and Coulomb terms, then, the conformational energy was optimized (minimum RMS gradient, 0.001). The UNIVERSAL parameters given in the

3.1. Single component monolayers Many studies have been made to evaluate the orientation of macrocyclic compounds on water with and without matrix lipids [46–55]. The molecular area in a p-A isotherm provides information on their molecular orientation on water and it also reveals their mixing ability with the other lipid components. We applied this methodology to the newly synthesized vitamin B12 derivatives. The present study is the first example of a monolayer system of vitamin B12 mimics with a core structurally identical to the naturally occurring one. The p-A isotherm of single component monolayers of the vitamin B12 derivatives are shown in Fig. 1. The isotherms strongly depend on the length of the alkyl chains regardless of the oxidation state of the center cobalt. Long-chain derivatives of 1b and 2b have relatively steep isotherms with a molecular area of ca. 3 nm2. Fig. 2 shows the packing motif of 2b on the basis of a simple molecular model where all the hydrophobic chains are assumed to be directed to the air phase. When the 2b molecules are hexagonally packed, the area of each 2b molecule is estimated to be 2.5 nm2. This estimated value is fairly close to the molecular area observed in the p-A isotherms of 1b and 2b at high pressures ( \ 15 mN m − 1). Therefore, 1b and 2b have a face-on orientation on water. The face-on orientation is favorable for these long-chain derivatives to avoid unfavorable contact of the hydrophobic chains with water. A

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similar molecular area was reported for the longchain phthalocyanine with a face-on orientation on water [46,47]. In contrast, the short-chain derivatives of 1a and 2a have a less steep isotherm. These molecular areas are apparently smaller than the area expected for the face-on orientation. The observed molecules might have an edge-on orientation and/or collapse upon compression. The repeated compression-expansion experiment provides information about the collapse behavior. Two cycles of repeated compression expansion at pressures below inflection pressure (apparent collapse pressure) were carried out (Figs. 3 and 4). Disagreement in the isotherms upon the repeated compression-expansion was clearly observed for the monolayers of 1a and 2a. Irreversible collapse during the first compression would lead to lack of reproducibility in the second scan. In contrast, the long-chain derivatives (1b and 2b) hardly show Fig. 2. Molecular models of 2b: (A), top view; (B), side view; (C), bottom view; (D), hexagonal packing of seven molecules of 2b, bottom view.

any hysteresis behavior as no area loss was detected upon the repeated compression expansion. This result confirms that the long-chain derivatives 1b and 2b form a stable monolayer with a face-on orientation on water.

3.2. Monolayers mixed with phospholipid

Fig. 1. p-A Isotherms of the vitamin B12 derivatives on pure water at 20°C: (A) (a) 1a and (b) 1b; (B) (a) 2a and (b) 2b.

Vitamin B12 derivatives were mixed with DPPC (Nichiyu Liposome Co, Japan) which was not expected to have a specific interaction with the vitamin B12. Therefore, investigation on the mixed monolayer of the vitamin B12 derivatives and DPPC would give general information on the miscibility of the derivatives to organized lipid assemblies. The isotherms of the mixed monolayers are shown in Figs. 5 and 6. Since the abscissa of these isotherms is based on the molecular area of DPPC, deviation from the isotherm of DPPC alone indicates the presence of the vitamin B12 derivatives in the DPPC monolayer. A significant difference between the short-chain derivatives (1a

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and 2a) and the long-chain derivatives (1b and 2b) is recognized in the molecular area at high surface pressures. The presence of the long-chain vitamin B12 derivatives clearly expands the DPPC monolayer even at high surface pressures. This result indicates that the long-chain derivatives are stably incorporated in the DPPC monolayer upon compression. Both the monolayers of 1b/DPPC and 2b/DPPC with a mixing ratio of 1/10 have inflection points at ca. 20 mN m − 1. This pressure is relatively close to the collapse pressure of the single-component monolayers of 1b and 2b. Therefore, some of the vitamin B12 molecules are phase separated at this mixing ratio and the phase of the vitamin B12 only is collapsed at this pressure [56]. In contrast, such an inflection point was hardly observed in the monolayer with mixing ratios of 1/50 and l/30. This result implies that the

Fig. 4. Hysteresis (compression-expansion) behaviors of p-A isotherms on pure water at 20°C: (A), 2a; (B), 2b. Each isotherm represents (a) first compression, (b) first expansion, (c) second compression, and (d) second expansion.

Fig. 3. Hysteresis (compression-expansion) behaviors of p-A isotherms on pure water at 20°C: (A), 1a; (B), 1b. Each isotherm represents (a) first compression, (b) first expansion, (c) second compression, and (d) second expansion.

long-chain derivatives can be molecularly accommodated in the DPPC matrix below the mixing ratio of 1/30. A similar tendency was reported for the mixed bilayer of the vitamin B12 derivatives and artificial lipids [34,41]. Vitamin B12 based catalysis was reported to be decreased when the mixing ratio was over 1/30. Results obtained for the mixed monolayers of the short-chain derivatives (1a and 2a) with DPPC are apparently different from those of the long-chain derivatives. Deviation in the molecular area of the mixed monolayer from that of the DPPC alone is very small at high surface pressures. This fact means that the short-chain derivatives are squeezed out from the DPPC matrix upon compression. The excluded vitamin B12 derivatives would come out of the monolayer plane of the DPPC. This instability probably originated due to the insufficient length of the side

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chains. Based on the assumption of a negligible interaction between the vitamin B12 derivatives and the matrix lipids, we can estimate the molecular area occupied by the vitamin B12 derivatives using the following equation: Aobsd = Amatrix +aAVB12

(1)

AVB12 =(Aobsd −Amatrix)/a

(2)

In these equations, Aobsd, Amatrix and AVB12 are the molecular areas of the mixed monolayer, matrix lipid, and vitamin B12 derivatives, respectively. Coefficient a denotes the mixing ratio of the vitamin B12 to the matrix lipid. The assumption of the negligible interaction may be wrong, because the collapse pressures of the matrix are apparently influenced by mixing the vitamin B12 derivatives. Here, we aim to make a rough estima-

Fig. 6. p-A Isotherms of mixed monolayers of the vitamin B12 derivatives and DPPC on pure water at 20°C: (A), 2a and DPPC; (B), 2b and DPPC. Abscissa is based on molecular area per DPPC molecule. Mixing ratios of the vitamin B12 derivatives to DPPC are (a) 0, (b) 1/50, (c) 1/30, and (d) 1/10.

Fig. 5. p-A Isotherms of mixed monolayers of the vitamin B12 derivatives and DPPC on pure water at 20°C: (A), 1a and DPPC; (B), 1b and DPPC. Abscissa is based on molecular area per DPPC molecule. Mixing ratios of the vitamin B12 derivatives to DPPC are (a) 0, (b) 1/50, (c) 1/30, and (d) 1/10.

tion of the orientation of the vitamin B12 derivatives in the matrix. The molecular areas of 1a and 1b in the DPPC matrix with a mixing ratio of 1/50 were calculated at 20 mN m − 1 (Table 1). The obtained molecular area of 1b is 2.70 nm2 which is close to that observed in the single-component monolayer of 1b (Fig. 1A) and that calculated with a molecular model (Fig. 2). Therefore, the long-chain derivative 1b is incorporated into the DPPC matrix with a face-on orientation. In contrast, the calculated molecular area of the shortchain derivative 1a is only 0.79 nm2 at 20 mN m − 1. This value is significantly smaller than the cross-section of the vitamin B12 core. It also suggests that 1a is easily squeezed out from the DPPC plane upon compression. The obtained results in this research indicate that the presence of long-side chains is advanta-

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geous in keeping the vitamin B12 function in the lipid membranes. Studies in aqueous vesicles have shown that the incorporation of the vitamin B12 function deep into the organized bilayer structure is required to obtain reactivity similar to naturally occurring systems. Therefore, the introduction of long chains into the vitamin B12 core must also be important to achieve effective reactivity of the vitamin B12 function in the bilayer system under various conditions. In the previous partition experiments, propyl and butyl esters showed 100% incorporation into the bilayer membrane. This fact suggested that the propyl chain is hydrophobic enough to be partitioned into the bilayer membrane. However, the present monolayer study demonstrates that the newly synthesized octyl esters of the vitamin B12 derivatives have an apparent superiority in incorporation into the DPPC monolayer. In the p-A isotherm measurement, the apparent molecular area is mainly affected by the vitamin B12 derivatives incorporated in the hydrophobic region. The derivatives just attached to the monolayer surface may not contribute to the molecular area. Therefore, isotherm measurements can provide information on the amount of the vitamin B12 derivatives deeply incorporated into the lipid assembly. The abovementioned monolayer study revealed that the octyl ester derivatives are more stably incorporated in the lipid membranes than the propyl derivatives, i.e. elongation of the ester chains from

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propyl to octyl is meaningful to obtain a stable hybrid of vitamin B12 and lipid membranes.

3.3. Monolayers mixed with lysine-functionalized lipid The vitamin B12 derivatives 1a and 1b have an axially-opened cobalt which can coordinate to basic components. On the other hand, 2a and 2b possess strong cyano ligands on both sides of Co(III) and thus they hardly interact with a base. A previous study of the bilayer system showed that coordination of an amine in the lipid was effective for preserving the vitamin B12 derivatives in the lipid matrix [34,41]. In addition, the role or meaning of the axial ligand has been extensively discussed in order to understand the catalytic mechanism of the B12-dependent enzymes [7,12,14,16,17]. A coordinate complex between the vitamin B12 derivatives and amine-possessing lipid in the monolayer would supply unique models to this field. Therefore, we investigated the miscible behavior between the lysine-functionalized lipid and the shortchain vitamin B12 derivatives (1a and 2a) which showed unstable behavior in the DPPC monolayer. Fig. 7A shows the electronic spectra of mixtures of 1a and 3 [57] in benzene. The spectrum of 1a alone has a peak at around 470 nm and the addition of equimolar 3 in the free base form changed the shape of the spectrum. The intensity at around 450–500 nm decreased and a new peak

Table 1 Calculated molecular areas (nm2) of vitamin B12 derivatives in mixed monolayers at 20°C at 20 mN m−1 Vitamin B12

Lipid

Mixing ratio (vitamin Bl2/lipid)

Molecular area for vitamin B12 nm2

1a 1a 1a 1b 1b 1b 1a 1a 1a 2a 2a 2a

DPPC DPPC DPPC DPPC DPPC DPPC 3 3 3 3 3 3

1/50 1/30 1/10 1/50 1/30 1/10 1/50 1/30 1/10 1/50 1/30 1/10

0.79 0.72 0.66 2.70 2.20 2.00 2.36 2.36 2.01 1.10 1.02 0.97

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show an increment in area upon the addition of 1a even at high surface pressure. The fluid nature of matrix 3 would be one of the key factors to accommodate 1a molecules. However, coordination between the amino group of 3 and Co(II) would have a large contribution in the stable incorporation of the short-chain vitamin B12 derivative. The effect of the amine-Co(II) interaction can be confirmed more clearly upon comparison of this result with the isotherms of the mixed monolayers of 2a and 3. The addition of 2a to the monolayer of 3 induced the area increase, but the increment apparently decreased as the surface pressure increased (Fig. 8B). The area occupied by the vitamin B12 derivatives was estimated by Eq. (2) (Table 1). The estimated molecular areas of 1a and 2a in the matrix 3 are 2.36 and 1.10 nm2, respectively, at a mixing ratio of 1/50 (1a or 2a/3)

Fig. 7. UV spectra of mixtures of vitamin B12 derivatives and 3 in benzene at 20°C: (A), 1a and 3; (B), 2a and 3. Concentration of the vitamin B12 derivatives were kept at 5.33 mM and mixing ratio of 3 to the vitamin B12 derivatives are (a) 0/1, (b) 1/1, (c) 1.5/1, and (d) 2/1 (mol/mol).

at around 550 nm was observed. The spectral shifts are quite similar to those observed when an amine was coordinated to heptamethyl cobyrinate perchlorate [43]. The amino group of the lysine residue in 3 is probably coordinated to Co(II) in 1a. Since the presence of an additional 3 molecule did not cause further changes in the spectrum, strong binding of 3 to 1a in equimolar stoichiometry was achieved. The same experiment was carried out for the combination of 2a and 3 (Fig. 7B). However, spectral changes upon the addition of 3 to 2a were hardly detected. The amino group of the Iysine residue in 3 cannot interact with Co(III) in 2a. The p-A isotherms of the mixed monolayers of 1a and 3 are shown in Fig. 8A where the molecular area is based on the matrix 3. All the isotherms have only an expanded phase and they

Fig. 8. p-A Isotherms of mixed monolayers of the vitamin B12 derivatives and 3 at pH 12 at 20°C: (A), 1a and 3; (B), 2a and 3. Abscissa is based on molecular area per 3 molecule. Mixing ratios of the vitamin B12 derivatives to 3 are (a) 0, (b) 1/50, (c) 1/30, and (d) 1/10.

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at 20 mN m − 1. The molecular area estimated for 1a indicates that the matrix 3 stably incorporated the vitamin B12 derivative 1a with an almost face-on orientation. In contrast, the estimated area of 2a is apparently smaller than its cross-section. The derivative with Co(III) (2a) tends to be squeezed out from the monolayer of 3 upon compression. The difference observed between the 1a/ 3 monolayer and 2a/3 monolayer confirms the importance of coordination of the lysine amine to Co(II) for preservation of vitamin B12 in lipid assemblies. 4. Conclusion and future perspective The present study is the first example of a monolayer system of vitamin B12 mimics with a core structurally identical to the naturally occurring one. The results presented here show the importance of long alkyl chains and axial coordination upon immobilization of the vitamin B12 derivatives in lipid assemblies. Evaluation of the molecular area confirms that the vitamin B12 derivatives can be stably immobilized in monolayers in a face-on orientation. As shown in this paper, the monolayer study provides a more quantitative interpretation of the lipid/B12 interaction. Here, we want to add the interesting possibility that the monolayer system will act as B12-mimic artificial enzymes. Upon compression of the monolayers, orientation, compressibility, and deformation of the vitamin B12 derivatives can be easily controlled. Various B12-dependent enzymatic mechanisms including the effect of corrin ring distortion and Co – N elongation have been proposed, and some recent studies have questioned them [11,12,14]. With the monolayer system presented here, such mechanical changes in the vitamin B12 moiety can be artificially regenerated. Therefore, it would be a good model in the study of the mechanism of B12 functions. Besides that, we can place the vitamin B12 function in a desirable environment in terms of orientation, polarity, and mobility. This fact would lead to a novel function of the vitamin B12 derivatives which have not been seen in naturally occurring systems.

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The monolayer study is indispensable research for immobilization of the vitamin B12 function on solid supports, leading to developing novel molecular devices. For example, an electrode system modified with vitamin B12 would be a key to solve the pollution problem as Rustling et al. proposed [23]. The vitamin B12 derivatives have a high potential to transform alkyl halides possibly including trihaloethane and mustard gas. Electrodedriven transformation is clean, because chemical redox reagents are not basically required. In order to make facile contact of the chemical substance, vitamin B12 and the electrode, immobilization of vitamin B12 on the electrode as an ultrathin film is indispensable. In this regard, we are now investigating stable immobilization of the vitamin B12 derivatives on an oxide electrode with the aid of silanol-functionalized monolayers.

Acknowledgements We would like to thank Kentaro Fukuda for structural optimization of the vitamin B12. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (A, No. 282) from the Ministry of Education, Science, Sports, and Culture, Japan.

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