Interactions of a Tetrazine Derivative with Biomembrane Constituents

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Interactions of a Tetrazine Derivative with Biomembrane Constituents: A Langmuir Monolayer Study Hiromichi Nakahara, Masayori Hagimori, Takahiro Mukai, and Osamu Shibata Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00997 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Interactions of a Tetrazine Derivative with Biomembrane Constituents: A Langmuir Monolayer Study Hiromichi Nakahara,1 Masayori Hagimori,2 Takahiro Mukai,2 and Osamu Shibata*,1 1

Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Nagasaki International University; 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan, 2

Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama Kitamachi, Higashinada-ku, Kobe 658-8558, Japan

*Corresponding author: *Osamu SHIBATA Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan Tel: +81-956-20-5686 Fax: +81-956-20-5686 E-mail: [email protected] Website: http://www.niu.ac.jp/~pharm1/lab/physchem/indexenglish.html Running Title: Interfactal properties of rTz-C18 and lipids Keywords:

Tetrazine; DPPC; Sphingomyelin; Cholesterol; Surface pressure; Surface potential 1 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 48

ABSTRACT Tetrazine (Tz) is expected to be used for bioimaging and analytical reagents. It is known to react very fast with trans-cyclooctene under water in organic chemistry. Here, to understand interactions between Tz and biomembrane constituents, we first investigated the interfacial behavior of a newly synthesized Tz derivative comprised of a C18-saturated hydrocarbon chain (rTz-C18) using a Langmuir monolayer spread at the air−water interface. Surface pressure (π)−molecular area (A) and surface potential (∆V)−A isotherms have been measured for monolayers of rTz-C18 and biomembrane constituents such as DPPC, DPPG, DPPE, PSM, and Cholesterol (Ch). The lateral interaction between rTz-C18 and the lipids was thermodynamically elucidated from the excess Gibbs free energy of mixing and two-dimensional phase diagram. The binary monolayers except for the Ch system indicated high miscibility or affinity. In particular, rTz-C18 was found to interact more strongly with DPPE, which is a major constituent of the inner surface of cell membranes. The phase behavior and morphology upon monolayer compression were investigated by using Brewster angle microscopy (BAM), fluorescence microscopy (FM), and atomic force microscopy (AFM). The BAM and FM images for the DPPC/, DPPG/, and PSM/rTz-C18 systems exhibited a coexistence state of two different liquid-condensed domains derived from mainly phospholipids

and

phospholipids−rTz-C18

monolayers.

From

these

morphological

observations, it is worthy to note that rTz-C18 is possible to interact with a limited amount of 2 ACS Paragon Plus Environment

Page 3 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the lipids except for DPPE.

INTRODUCTION Inverse electron-demand Diels−Alder (IEDDA) reactions of tetrazine (Tz) and trans-cyclooctene derivatives give stable adducts in high yield and N2 of the only byproduct in organic solvents, water as well as cell media.1

The reaction is possible to achieve the fast

reaction kinetics and the high selectivity without catalysis, and is thus well known as a method of rapid bioconjugation. Currently, this reaction has been applied to amine sensing2 and protein modification towards a tumor imaging.3-5

Molecular imaging techniques will

become important role in the clinic and in drug discovery and development.6, 7

However, it

has been largely unknown if the obtained product conjugated on the tumor cell via the IEDDA reaction and unreacting Tz can interact with the surrounding biomembrane constituents. The structure of cell membranes, which is well-established by Singer and Nicolson,8 is constructed by asymmetric two leaflets of biological membranes.9,

10

Among lipids,

phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Ch) are enriched on the outer surface of the bilayers. In particular, Ch is an essential component for maintenance of SM-rich microdomains or lipid rafts, which are thought to have important biological functions such as membrane signaling and protein trafficking.11, 12

In contrast, the inner surface of the

bilayers consists mainly of phosphatidylethanolamine (PE) and phosphatidylserine (PS). 3 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 48

Phosphatidylglycerol (PG) is a minor component in plasma membranes but exists specifically in pulmonary surfactants.13-15

It is noticed that pulmonary surfactants comprise PC (in

particular, dipalmitoyl-PC or DPPC) to a large extent. The Langmuir monolayer technique at the air−water interface is one of the simple and powerful methods to understand interactions among different molecules.16

The

monolayer, which is adopted as experimental paradigm of biomembranes, is an optimal model to examine the lateral interaction among them as a function of intermolecular distance. Furthermore, the physical behavior of biomembranes (or bilayers) is directly linked to that of monolayers at surface pressures (π) of 30−35 mN m−1.17, 18

Thus, many researchers have

employed the technique to elucidate the interfacial behavior of surfactants and the interactions (or mechanisms) between compounds such as natural lipids,19-21 synthesized lipids,22, 23 and proteins.24, 25

Much useful information on the phase variation of monolayers at the micro

and nano scales can be derived from microscopic observations with Brewster angle microscopy (BAM),26, 27 fluorescence microscopy (FM),28-30 and atomic force microscopy (AFM).31 In the present study, we have investigated a reduced Tz comprised of a single stearoyl (C18) chain (abbrev. rTz-C18) because of the rapid reaction kinetics (or the instability) and the aqueous solubility of Tz molecules. The objective in the present study is to clarify the lateral interaction between biomembrane constituents and rTZ-C18, which is 4 ACS Paragon Plus Environment

Page 5 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

assumed to be conjugated on tumor cells. The interfacial behavior of two-component monolayers of rTz-C18 and five different lipids such as DPPC, dipalmitoyl-PE (DPPE), -PG (DPPG), palmitoyl-SM (PSM), and Ch has been examined employing the Langmuir monolayer technique. The lipids except for Ch have the same hydrophobic chain, which is easy to understand the interaction between the Tz moiety and the headgroups of the lipids. The surface pressure (π)–molecular area (A) and surface potential (∆V)–A isotherms for the binary mixtures were measured on 0.02 M Tris buffer with 0.13 M NaCl (pH 7.4) at 298.2 K. The phase change and morphology of the monolayer upon compression were visualized with BAM, FM, and AFM.

EXPERIMENTAL Materials N-(6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)stearamide, abbreviated as rTz-C18 (see Fig. 1(2)), was newly synthesized as the procedure mentioned in the next section. The obtained rTz-C18 was purified by repeated recrystallizations from methanol and its identification was checked by 1H NMR,

13

C NMR (JNM-AL400, Jeol,

Tokyo, Japan), FT-IR (JASCO FT/IR-4200, Tokyo, Japan), and FAB-MS (SX102A, Jeol); m/z 559.4042

[M+Na+2H]+.

For

synthesis

of

rTz-C18,

5 ACS Paragon Plus Environment

stearic

acid,

DMF,

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Page 6 of 48

hydrochloride

(EDC

HCl),

and

1-hydroxybenzotriazole (HOBT) were purchased from nacalai tesque (Kyoto, Japan). N-Ethyldiisopropylamine was obtained from Wako Chemical (Osaka, Japan). These were of analytic grade and used as received. As the reagents for measurements of physico-chemical properties,

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC;

purity

>99%),

1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG; purity >99%), N-palmitoyl-D-erythro-sphingosylphosphorylcholine

(PSM;

purity

>99%),

and

fluorescent

the

probe,

1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphoc holine

(NBD-PC)

were

purchased

from

Avanti

Polar

Lipids

(Alabaster,

AL).

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE; purity >99%) was obtained from NOF Corporation (Tokyo, Japan). Cholesterol (Ch; purity >99%) was from Sigma (Sigma-Aldrich, Inc, St Louis, MO). These lipids were used without further purification. Chloroform (99.7%) and methanol (99.8%) were purchased from Kanto Chemical Co., Inc (Tokyo, Japan) and nacalai tesque, respectively. The chloroform/methanol (2/1, v/v) for mixtures were used as a spreading solvent. Tris(hydroxymethyl) aminomethane (Tris) and acetic acid (HAc) of guaranteed reagent grade for the preparation of the subphase were obtained from nacalai tesque. Sodium chloride (nacalai tesque) was roasted at 1023 K for 24 h to remove all surface-active organic impurities. The substrate solution was prepared using 6 ACS Paragon Plus Environment

Page 7 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

thrice-distilled water (surface tension = 72.0 mN m–1 at 298.2 K; electrical resistivity = 18 MΩ cm). The pH of the subphase (0.02 M Tris buffer and 0.13 M NaCl) was adjusted to 7.4 with an adequate amount of HAc.

Methods Synthesis of rTz-C18 (2) To a solution of 6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-amine (1) (50 mg, 0.20 mmol) in DMF was added stearic acid (129 mg, 0.45 mmol), EDC HCl (33 mg, 0.45 mmol),

HOBT (70 mg, 0.45 mmol), and N-ethyldiisopropylamine (59 mg, 0.45

mmol), and the reaction mixture was stirred for 24 h at room temperature. Water (100 mL) was added to the mixture and the precipitate formed was collected by filtration. Purification by column chromatography on silica gel (hexane/EtOAC 7/3) gave 2 (41 mg, 39% yield) as a white pellet, mp 55-56°C.

Surface pressure–area isotherms

The surface pressure (π) of monolayers was measured using a commercially available film balance system (KSV Minitrough, KSV Instruments Ltd., Finland).32

The

surface pressure sensor had a resolution of 0.004 mN m−1. The pressure-measuring system was equipped with filter paper (Whatman 541, periphery = 2.0 cm). The trough was made 7 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

from Teflon (area = 273cm2), and Teflon barriers were used in this study. The π–molecular area (A) isotherms were recorded on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 ± 0.1 K. Stock solutions of DPPC (1.0 mM), DPPE (1.0 mM), DPPG (0.5 mM), PSM (1.0 mM), Ch (1.0 mM), and rTz-C18 (1.0 mM) were prepared in chloroform/methanol (2/1, v/v). The spreading solvents were allowed to evaporate for 15 min prior to compression. The monolayer was compressed at a barrier speed of 10 mm min−1, or a compressing speed of ∼ 0.08 nm2 molecule−1 min−1.

Surface potential–area isotherms The surface potential (∆V) was recorded simultaneously with surface pressure, when the monolayer was compressed at the air−water interface. It was monitored with a Kelvin probe system (KSV SPOT1, KSV Instruments Ltd.) at 1–2 mm above the interface, while a counter electrode was dipped in the subphase.32

The Kelvin probe had a resolution of 1 mV.

Brewster angle microscopy (BAM) The monolayers were imaged directly at the air−water interface using a Brewster angle microscope (KSV Optrel BAM 300, KSV Instruments Ltd., Finland) coupled to the KSV Minitrough.33, 34 Using a 20 mW He–Ne laser, which emitting p-polarized light with a wavelength of 632.8 nm, and a 10× objective lens in combination allowed for a lateral 8 ACS Paragon Plus Environment

Page 8 of 48

Page 9 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

resolution of ~2 µm. The angle of the incident beam to the interface was fixed to the Brewster angle (53.1o) at 298.2 K. The reflected beam was recorded with a high-grade charge-coupled device (CCD) camera (EHDkamPro02, EHD Imaging GmbH, Germany).

Fluorescence microscopy (FM) The KSV Minitrough was mounted on the stage of an Olympus microscope BX51WI (Tokyo, Japan) that was equipped with a 100 W mercury lamp (USH-1030L), an objective lens (SLMPlan; 50×, working distance = 15 mm) and a 3CCD camera with a camera control unit (IKTU51CU, Toshiba, Japan).35

Prior to spreading, the stock solution of the samples

was doped with 1 mol% of the fluorescence probe(NBD-PC). Image processing and analysis were performed using Adobe Photoshop Elements ver. 7.0 (Adobe Systems Inc., CA). The total amount of ordered domains (dark-contrast regions or those not containing NBD-PC) was determined and expressed as a percentage per frame by dividing the respective frame into dark and bright regions.

Atomic force microscopy (AFM) Langmuir-Blodgett (LB) films were prepared using the KSV Minitrough. Freshly cleaved mica (Okenshoji Co., Tokyo, Japan) was used as a supporting solid substrate for film deposition, which was performed through the vertical dipping method. The transfer velocity 9 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

during the single layer deposition process, which was performed at selected surface pressures, was 5 mm min−1. During the transfer process, the hydrophilic part of the monolayer is in contact with the mica substrate while the hydrophobic part is exposed to air. LB films deposited at a rate of ~1 were used in the subsequent experiments. The images were obtained in the air at room temperature using an SPA 400 instrument (Seiko Instruments Co., Chiba, Japan) in the tapping mode.36, 37

RESULTS AND DISCUSSION π−A and ∆V−A isotherms The surface pressure (π)–molecular area (A) and surface potential (∆V)–A isotherms for the binary monolayers (DPPC/, DPPE/, DPPG/, PSM/, and Ch/rTz-C18) are shown in Fig. 2. rTz-C18 (curve 7) forms a typical liquid-condensed (LC) monolayer under the present condition. The π–A isotherm of rTz-C18 monolayers rises up at ~0.45 nm2 upon lateral compression and then reaches a collapse pressure (πc) of ~50 mN m−1, where the monolayer state converts to the three-dimensional (3D) bulk state at the air−water surface. The limiting molecular area of rTz-C18 monolayers is ~0.38 nm2, which is larger than the cross-sectional area, ~0.20 nm2, of one saturated aliphatic chain. Considering the area, ~0.30 nm2, of a six-membered ring, the orientation of rTz-moiety is likely to be turned toward the bulk at the 10 ACS Paragon Plus Environment

Page 10 of 48

Page 11 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

close-packed monolayer state. Under the monolayer compression, the ∆V value increases monotonously up to ~350 mV. The ∆V value of monolayers is interpreted as a combination of the vertical dipole moments of molecules in the subphase (layer 1), polar head group of monolayer-forming compounds (layer 2), and terminal group of their hydrophobic chain (layer 3).38

Independent dipole moments and effective local dielectric constants are

attributed to each of the three layers. Thus, the ∆V–A isotherms indicate changes in molecular orientation upon compression. The monotonous increment in ∆V indicates an improved orientation of C18 chains toward the vertical direction. The π–A isotherm of DPPC (curve 1 in Fig. 2A) exhibits a first-order transition from a liquid-expanded (LE) to an LC phase at πeq = ~11 mN m−1 (a dashed arrow). Similarly, DPPG (curve 1 in Fig. 2C) and PSM (curve 1 in Fig. 2D) monolayers undergo the transition at πeq = ~17 and ~21 mN m−1, respectively. DPPE (curve 1 in Fig. 2B) and Ch (curve 1 in Fig. 2E) form typical LC monolayers. As seen in the steepness of π–A isotherms, Ch monolayers are found to be more rigid than DPPE. The ∆V value of DPPE monolayers at the close-packed state is ~600 mV and this value is the largest among the phospholipid monolayers here. This is attributed to the difference in headgroup species as well as orientations of the hydrophobic chains, which are constricted by the spatial size of headgroups. Detailed analyses and discussions on monolayer properties of pure lipids were mentioned elsewhere.33, 34 Analyses of the isotherm for two-component systems provide fruitful information on 11 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mutual interactions from a thermodynamic aspect. The π–A isotherms for the binary lipids/rTz-C18 monolayer (except for the Ch system) regularly shift within those for pure components (curves 1 and 7). Apparently, all the systems indicate the πc variation with mole fraction of rTz-C18 (XrTz-C18). In addition, the incorporation of rTz-C18 induces a solidification of the lipid monolayer for the DPPC, DPPG, and PSM systems, which is characterized by a decrease in πeq with increasing XrTz-C18. These variations imply the miscibility between the two components in the monolayer state. On the other hand, the complicated behavior is observed in the Ch/rTz-C18 system (Fig. 2E). The π–A isotherms at XrTz-C18 = 0.1 and 0.3 overlap completely with that of single Ch monolayers (data not shown). The further addition of rTz-C18 slightly shifts the isotherm to larger molecular areas (see curve 2). The difference in A at 40 mN m−1 between the isotherms at XrTz-C18 = 0 (curves 1) and 0.5 (curve 2) is no more than 0.006 nm2, which is not a significant value in terms of the resolution of molecular areas. At XrTz-C18 = 0.7, the isotherm lies between those of the pure components. Judging from the appearance of the π–A isotherms, it is found that Ch dominates surface activity for the binary Ch/rTz-C18 monolayer with respect to A or its surface density.

Excess Gibbs free energy of mixing A lateral interaction between lipids and rTz-C18 monolayers can be analyzed exc thermodynamically by the excess Gibbs free energy of mixing ( ∆Gmix ), which is estimated


Page 12 of 48

Page 13 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

exc from the π−A isotherms in Fig. 2. The ∆Gmix value is calculated from the following equation

(Eq. 1),39 π

exc ∆Gmix = ∫ ( A12 − X 1 A1 − X 2 A2 )dπ 0

(1)

where Ai and Xi are the molecular area and mole fraction of component i, respectively, and A12 is the mean molecular area in the binary monolayer. When the intermolecular interactions between components in the mixed monolayer are the same as those in the respective one-component films (two-dimensional ideal solution) or when the film components are exc exc completely immiscible, the value of ∆Gmix is zero.40, 41. While the negative value of ∆Gmix

indicates that the interactions between components of the mixed film are more attractive or exc less repulsive as those in respective one-component monolayers. The ∆Gmix value at

selected surface pressures is plotted as a function of XrTz-C18 in Fig. S1 in the Supporting exc Information. The binary systems except for the Ch system show negative ∆Gmix values at

each surface pressure. The minimum values near −600 J mol−1 are observed at XrTz-C18 = 0.9 for the DPPC system (Fig. S1A) and at XrTz-C18 = 0.6 for the PSM system (Fig. S1D). The exc ∆Gmix value for the DPPE (Fig. S1B) and DPPG (Fig. S1C) systems decreases with an

increase in surface pressure and then reaches the minimum values of −1350 and −1150 J mol−1 at 45 mN m−1, respectively. On the contrary, the Ch/rTz-C18 system (Fig. S1E) exhibits exc the values between −200 and 200 J mol−1, which are not significant in terms of the ∆Gmix

change. This provides evidence of ideal mixing or immiscibility between Ch and rTz-C18 13 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

within a monolayer state. As for the phospholipids/rTz-C18 systems, it is found that the miscibility and affinity of rTz-C18 for the phospholipids become stronger in the following exc order: DPPE > DPPG> DPPC ≈ PSM. The ∆Gmix analysis has been performed even at

middle surface pressures equivalent to those of biological membranes (e.g., 30−35 mN m−1).17, 18

Thus, these results suggest that rTz-C18 interacts less strongly with the constituents of the

outer surface of lipid bilayers or biological membranes. In particular, there are few interactions of rTz-C18 with Ch, which is thought to serve as a regulator of fluidity in lipid rafts of plasma membranes.11, 12

Two-dimensional (2D) phase diagrams Shown in Fig. 3 are 2D phase diagrams for the present binary systems at 298.2 K. The diagram was constructed by plotting the πeq and πc values against XrTz-C18. The diagram allows us to understand the phase behavior and interaction mode between the two components in the whole surface pressure regions (particularly low and high surface pressures). Attention is paid to the πeq behavior (Fig. 3A, 3C, and 3D), the addition of rTz-C18 reduces πeq values derived from the respective phospholipids, which evidences the solidifying effect of rTz-C18 c on DPPC, DPPE, and PSM monolayers. The critical mole fraction X rTz− C18 , above which the

monolayer is in LC phases at finite surface pressures, is obtained by the extrapolation to πeq = c 0: X rTz− C18 = 0.20 for DPPC/rTz-C18, 0.25 for DPPG/rTz-C18, and 0.15 for PSM/rTz-C18.


Page 14 of 48

Page 15 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Moreover, a rate of πeq change (∂πeq/∂XrTz-C18) is respectively estimated to 55, 68, and 140 mN m−1. It is found from these values that the solidification property of rTz-C18 is exerted most effectively against PSM monolayers. The higher πeq value of pure PSM monolayers than DPPC is considered to be relevant to a hydroxy group in a PSM molecule because of their similar chemical structures. That is, these results imply that a hydrogen bonding between the hydroxy group and rTz moiety is newly generated upon lateral compression of the binary monolayer. This means that the hydrophobicity near the headgroup of PSM becomes higher to promote the monolayer solidification. On the other hand, the experimental πc values for all the systems except for the Ch system (Fig. 3E) also varied with XrTz-C18, supporting the two-component miscibility in the monolayer state. As mentioned above, the Ch/rTz-C18 systems have both possibilities of ideal mixing and immiscibility. This unclearness is also supported by the fact of slight πc changes against XrTz-C18 in the phase diagram (Fig. 3E). The coexistence phase boundary between the monolayer phase (2D) and the bulk phase (3D) of the molecules spread on the surface can be theoretically simulated using the Joos equation42, 43 and assuming a regular surface mixture:

{(

)

} (

)

{(

)

} (

1 = X 1 exp π c − π 1c A1 / kT exp ξ ⋅ X 2 + X 2 exp π c − π 2c A2 / kT exp ξ ⋅ X 1 2

2

)

(2)

where X1 and X2 represent the mole fractions of components 1 and 2, respectively, in the two-component monolayer; π 1c and π 2c are the collapse pressures of components 1 and 2, respectively; Α1 and Α2 are the molecular areas of components 1 and 2 at π 1c and π 2c , 15 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 48

respectively; ξ is the interaction parameter; and kT is the product of the Boltzmann constant and the temperature in Kelvin. A solid curve could be obtained at higher surface pressures by adjusting ξ in Eq. (2) so as to achieve the best fit for the experimentally determined πc values. Accordingly, the interaction energy (∆ε) is given as follows: ∆ε = ξRT/z

(3)

where z is the number of nearest neighbors per molecule (equal to 6 in this case) in a closely packed monolayer. The expression for the interaction energy can be rewritten as ∆ε = ε12 – (ε11 + ε22)/2,42

where ε12 denotes the potential interaction energy between components 1 and

2. Note here that the Ch/rTz-C18 monolayer is treated as a miscible system to elucidate the

ξ value between the two components and to compare it with those for the other systems. When the amount of rTz-C18 in the DPPC (Fig. 3A) and PSM (Fig. 3D) systems increases, the πc values change upward up to XrTz-C18 = 0.4−0.5 and then decrease to that of rTz-C18 monolayers so that the both systems have the values of ξ = −4.40 (∆ε = −1.82 kJ mol−1) and

ξ = −3.50 (∆ε = −1.45 kJ mol−1), respectively. The DPPG system shows the similar variation, however, the πc−XrTz-C18 plot can be divided into two regions at the boundary of XrTz-C18 = 0.5:

ξ = −1.96 (∆ε = −0.81 kJ mol−1) for 0 ≤ XrTz-C18 ≤ 0.5 and ξ = −1.20 (∆ε = −0.50 kJ mol−1) for 0.5 ≤ XrTz-C18 ≤ 1. As for the DPPE system (Fig. 3B), the monolayer collapse behavior against XrTz-C18 is complicated: ξ = −1.32 (∆ε = −0.55 kJ mol−1) for 0 ≤ XrTz-C18 ≤ 0.25, ξ = −2.53 (∆ε = −1.05 kJ mol−1) for 0.25 ≤ XrTz-C18 ≤ 0.5, and ξ = −3.13 (∆ε = −1.29 kJ mol−1) for 0.5 ≤ 16 ACS Paragon Plus Environment

Page 17 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

XrTz-C18 ≤ 1. It is noteworthy that the πc profile has a minimum value of ~36 mN m−1 at XrTz-C18 = 0.25. This value is smaller than those of pure DPPE and rTz-C18 monolayers, which means that a small amount of rTz-C18 makes DPPE monolayers easy to transfer from the monolayer to the 3D bulk state. In other words, this is interpreted as an attenuation of monolayer stability against lateral pressure. Considering the fact that the minimum value is almost the same pressure as the π value of biological membranes, the rTz moiety may induce the instability of biomembranes from the inside by the attractive interaction the rTz and PE groups. The Ch/rTz-C18 monolayer (Fig. 3E) has a positive ξ value of 0.66 (∆ε = 0.27 kJ mol−1) differently from the other systems. This indicates that the interaction between the same molecules (Ch−Ch or rTz-C18−rTz-C18) occurs more favorably than that of Ch−rTz-C18. All of the ∆ε values here were smaller than mean thermal energy (2RT = ~5.0 kJ mol−1 at 298.2 K), which suggests that the molecular interaction related with the binary miscibility at high surface pressures is based on non-bonding intermolecular forces not functional chemical bond such as a covalent bond.

Morphological observations Figure 4 and S2 show FM images for the DPPC/rTz-C18 monolayers containing different amounts of rTz-C18 in situ at the air−water interface. In the FM observation, the monolayer contains a small amount of a fluorescent probe (1 mol% NBD-PC). In common, 17 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the fluorescent probe is selectively dissolved in LE phases of monolayers because of its spatially bulky structure. Thus, bright and dark contrasts in the FM image respectively correspond to LE and LC phases. The FM images of DPPC monolayers below the πeq of ~11 mN m−1 are homogeneously bright like the image at 10 mN m−1 (Fig. S2). On further compression above the πeq, the coexistence of the LE and LC phases is observed (see the image at 15 mN m−1). The anticlockwise arms of the LC domain are characteristic of L-DPPC monolayers.44-47

At XrTz-C18 = 0.1, the LC domain shape does not change significantly in

shape or appearance, apart from the reduction in πeq. However, the further addition of rTz-C18 (XrTz-C18 = 0.3 and 0.5), to a great extent, causes a morphological variation of the LC domains: a shrink in size and an ununiform of domain shapes (Fig. 4). It is well-known that a domain formation is controlled by balance of a line tension at the boundary between disordered and ordered domains and a long-range dipole-dipole interaction between ordered domains.48-54 The apparent heterogeneity is resulted from the existence of two kinds of LC domains. One is the LC domain rich in DPPC (less in rTz-C18) and the other is the domain formed by the miscible monolayer of DPPC and rTz-C18, which is indicated by white arrows. This phenomenon is found not to result from the interaction with the probe because the same morphological behavior is observed in the in situ BAM images, which will be discussed in the latter section. Both of the LC domains grow in size with an increase in surface pressure. That is, the domain growth (e.g. size and shape) evidences the miscible interaction between them, 18 ACS Paragon Plus Environment

Page 18 of 48

Page 19 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

not their immiscibility. At XrTz-C18 = 0.5, the coexistence of the LC domains is also observed and the latter domain tends to form a network between the same LC domains. However, it can be said that the LC domain of the mixed monolayers predominates compared to the DPPC-rich LC domain. This implies the limited ratio that rTz-C18 is possible to interact with DPPC at the surface. In the DPPG/rTz-C18 system (Fig. 5 and S3), rTz-C18 interacts less strongly with DPPG below XrTz-C18 = 0.1. Similarly to the DPPC system, the LC domain (white arrows) of mixed DPPG/rTz-C18 monolayers is visualized in the image beyond XrTz-C18 = 0.3 (Fig. 5). However, as opposed to the DPPC system, the LC domain is hard to form the domain network due to the repulsive force derived from a positive charge of DPPG headgroups. On the other hand, the size of LC domains at XrTz-C18 = 0.1 in the PSM system (Fig. 6 and S4) becomes larger compared to that of pure PSM monolayers. In addition, the appearance of the domains transforms to a noncircular form. This means the enhancement of dipole-dipole repulsive interaction among the LC domains by the rTz-C18 incorporation. At XrTz-C18 = 0.3, the dipole-dipole interaction is improved further so that some LC domains adhere to each other (white arrows). The images at XrTz-C18 = 0.5 come to show the two different LC phases: the domain forming the network (PSM−rTz-C18) and that with the diameter of less than 10 µm (mainly, PSM). The results suggest the limited quantitative ratio of interaction of rTz-C18 with DPPC, DPPG, and PSM. 19 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7 shows BAM images for the above-mentioned three system. There indicate the three distinct phases of dark, white, and bright contrasts (white arrows) in each image. As opposed to the FM image, the dark contrast corresponds to LE phases of monolayers in the BAM image.26, 27

The domains with white contrasts in Fig. 7(a), (b), and (c) are almost the

same as those for single DPPC, DPPG, and PSM monolayers in terms of size and appearance, respectively.33, 35

Therefore, the bright domain with interference fringes is found to be the

LC domains of phospholipids−rTz-C18 monolayers, which are also seen in the corresponding FM images (Figs. 4−6). Comparison of FM photographs with BAM images evidences the followings: (1) the coexistence of two different LC domains is not induced by the interaction with the fluorescent probe and (2) the LC domains derived from phospholipids−rTz-C18 mixed monolayers are more rigid and higher density than those for single phospholipid monolayers. In the binary DPPE/rTz-C18 and Ch/rTz-C18 systems, the FM and BAM images exhibit no phase variations upon compression because of the same LC phase for DPPE, Ch, and rTz-C18 monolayers. Thus, to clarify the mutual interaction for the systems, we have performed AFM observations, which provide powerful information on micro- and nano-scale molecular distribution based on the height difference in the monolayer state. Shown in Fig. 8 are the AFM images for the DPPE/rTz-C18 system at 35 mN m−1. The image at XrTz-C18 = 0.5 indicates dark and bright contrasts. The dark domain reflects DPPE monolayers due to the fact 20 ACS Paragon Plus Environment

Page 20 of 48

Page 21 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

that the occupied area of the domain decreases when XrTz-C18 increases to 0.7 and that DPPE is lower in molecular size or height than rTz-C18. In addition, the increment in XrTz-C18 induces a dispersion of the DPPE domain. Considering the length of an ethylene group (~0.3 nm), the larger difference between the two monolayers (1.3 and 1.6 nm) means the vertical orientation of the rTz moiety on the mica substrate. These results reveal that rTz-C18 exerts the most highly dispersing effect on DPPE monolayers among the present systems, not the solidifying effect. Furthermore, rTz-C18 is possible to interplay with DPPE over the whole XrTz-C18 without the extra amount of the components. Here we turn our attention to the structural difference between DPPC and DPPE. The both lipids are zwitterion under the present experimental condition. The difference is only the end of their headgroups: a choline group (DPPC) and amino group (DPPE). In the present study, however, the additional effect of rTz-C18 on DPPC and DPPE monolayers is directly opposite. Expectedly, the rTz moiety is easier to approach the nitrogen atom of DPPE headgroups upon lateral compression. Therefore, a clue to exert the dispersing effect of the rTz group on biomembranes is an existence of interaction sites (here, amino group) in lipid structures. On the other hand, the Ch/rTz-C18 system at XrTz-C18 = 0.5 (Fig. 9) is highly dispersed, where the dark domain represents Ch monolayers because of the molecular size. However, as XrTz-C18 increases to 0.7, the extra amount of rTz-C18 (indicated by a straight-line arrow) is squeezed out of the mixed Ch−rTz-C18 monolayer. The exclusion does not mean the 21 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

bilayer formation of rTz-C18 because the mean height difference (1.6 nm) between the two components

is quite low in comparison to the bilayer thickness of roughly 5 nm.

Considering the difference between 0.9 and 1.6 nm, it is speculated that the rTz moiety lifts up from the surface to the air location due to the π−π interaction between the Ch steroid backbone and the rTz moiety. In this regard, it is suggested that the lateral interaction between Ch and rTz-C18 is quantitatively limited but they are almost ideally miscible with each other rather than the immiscibility.

CONCLUSION rTz-C18 forms a stable monolayer characterized of typical LC phases and takes on the miscibility with all of the lipids treated in this study in the monolayer state. The two-component miscibility of rTz-C18 with the lipids becomes higher in the following order: DPPE > DPPG > DPPC ≈ PSM > Ch. From the thermodynamical analyses, it is suggested that rTz-C18 interacts less strongly with the constituents (DPPC, PSM, and Ch) of the outer surface of biological membranes. The solidification of monolayer LC phases occurs for the DPPC, DPPG, and PSM systems. Furthermore, in these systems, two different LC phases of the phospholipid-rich domains and the domains consisting of the binary components coexist 22 ACS Paragon Plus Environment

Page 22 of 48

Page 23 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

at middle surface pressures. The LC domains (phospholipids−rTz-C18) are more rigid and higher density. Nevertheless, the coexistence of the two different LC phases suggest the quantitative limited ratio of the interaction between the two components. On the contrary, rTz-C18 interacts slightly with Ch, which is based on the π−π interaction between the Ch steroid backbone and the rTz moiety. In the DPPE system, a small-amount addition of rTz-C18 induces the instability of DPPE monolayers near the surface pressure (~35 mN m−1) of biological membranes. Whereas the rTz moiety exerts a highly dispersing effect on DPPE monolayers in the large XrTz-C18 region. This effect is likely to be exerted by the interaction of the rTz group with the amino group of DPPE headgroups. In summary, rTz derivative more strongly interacts DPPE monolayers compared with other components of biological membranes (DPPC, DPPG, PSM and Ch). The outstanding properties of this composite rTz will offer useful aspects on Langmuir monolayer recognition and interaction mechanism against biomembrane constituents.

ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research 26350534 and 16K08216 (H.N.) from the Japan Society for the Promotion of Science (JSPS). 23 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

. Plots of the excess Gibbs free energy of mixing with XTz-C18 and FM images for

the two-component systems (PDF)

REFERENCES (1) Blackman, M. L.; Royzen, M.; Fox, J. M., Tetrazine ligation: Fast bioconjugation based on inverse-electron-demand Diels-Alder Reactivity. J. Am. Chem. Soc. 2008, 130, 13518-13519. (2) Malinge, J.; Allain, C.; Galmiche, L.; Miomandre, F.; Audebert, P., Preparation, photophysical, electrochemical, and sensing properties of luminescent tetrazine-doped silica nanoparticles. Chem. Mater. 2011, 23, 4599-4605. (3) Rossin, R.; Renart Verkerk, P.; van den Bosch, S. M.; Vulders, R. C. M.; Verel, I.; Lub, J.; Robillard, M. S., In vivo chemistry for pretargeted tumor imaging in live mice. Angew. Chem. Int. Ed. 2010, 122, 3447-3450. (4) Roberts, D. A.; Pilgrim, B. S.; Cooper, J. D.; Ronson, T. K.; Zarra, S.; Nitschke, J. R., Post-assembly modification of tetrazine-edged FeII4L6 tetrahedra. J. Am. Chem. Soc. 2015, 137, 10068-10071. (5) Ni, Z.; Zhou, L.; Li, X.; Zhang, J.; Dong, S., Tetrazine-containing amino acid for peptide modification and live cell labeling. PLOS One 2015, 10, e0141918. 24 ACS Paragon Plus Environment

Page 24 of 48

Page 25 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(6) Hargreaves, R. J., The role of molecular imaging in drug discovery and development. Clin. Pharmacol. Ther. 2008, 83, 349-353. (7) Matthews, P. M.; Rabiner, E. A.; Passchier, J.; Gunn, R. N., Positron emission tomography molecular imaging for drug development. Br. J. Clin. Pharmacol. 2011, 73, 175-186. (8) Singer, S. J.; Nicolson, G. L., The fluid mosaic model of the structure of cell membranes. Science 1972, 175, 720-731. (9) Bretscher, M. S., Asymmetrical lipid bilayer structure for biological membranes. Nature 1972, 236, 11-12. (10) Daleke, D. L., Regulation of transbilayer plasma membrane phospholipid asymmetry. J. Lipid Res. 2003, 44, 233-242. (11) Simons, K.; Ikonen, E., Functional rafts in cell membranes. Nature 1997, 387, 569-572. (12) Simons, K.; Toomre, D., Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 2000, 1, 31-39. (13) Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F., The role of lipids in pulmonary surfactant. Biochim. Biophys. Acta 1998, 1408, 90-108. (14) Krüger, P.; Baatz, J. E.; Dluhy, R. A.; Lösche, M., Effect of hydrophobic surfactant protein SP-C on binary phospholipid monolayers. Molecular machinery at the air/water interface. Biophys. Chem. 2002, 99, 209-228. (15) Yu, S.-H.; Possmayer, F., Lipid compositional analysis of pulmonary surfactant monolayers and monolayer-associated reservoirs. J. Lipid Res. 2003, 44, 621-629. (16) Gaines, G. L., Jr. , Insoluble monolayers at liquid-gas interfaces. Interscience Publishers: New York, 1966; p 1-386. (17) Marsh, D., Lateral pressure in membranes. Biochim. Biophys. Acta - Reviews on Biomembranes 1996, 1286, 183-223. (18) Conrad, M. J.; Singer, S. J., Evidence for a large internal pressure in biological membranes. Proc. Natl. Acad. Sci. USA 1979, 76, 5202-5206. (19) Petrov, J. G.; Pfohl, T.; Möhwald, H., Ellipsometric Chain Length Dependence of Fatty Acid Langmuir Monolayers. A Heads-and-Tails Model. J. Phys. Chem. B 1999, 103, 3417-3424. (20) Shah, D. O.; Schulman, J. H., Influence of calcium, cholesterol, and unsaturation on lecithin monolayers. J. Lipid Res. 1967, 8, 215-226. (21) Chou, T. H.; Chang, C. H., Thermodynamic behavior and relaxation processes of mixed DPPC/cholesterol monolayers at the air/water interface. Colloids Surf. B 2000, 17, 71-79. (22) Hönig, D.; Overbeck, G. A.; Möbius, D., Morphology of pentadecanoic acid monolayers at the air/water interface studied by BAM. Adv. Mater. 1992, 4, 419-424. (23) Viseu, M. I.; Gonçalves da Silva, A. M.; Costa, S. M. B., Reorganization and desorption of catanionic monolayers. Kinetics of π−t and A−t relaxation. Langmuir 2001, 17, 1529-1537. (24) Leblanc, R. M., Molecular recognition at Langmuir monolayers. Curr. Opin. Chem. Biol. 25 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2006, 10, 529-536. (25) Orbulescu, J.; Micic, M.; Ensor, M.; Trajkovic, S.; Daunert, S.; Leblanc, R. M., Human cardiac Troponin I: A Langmuir monolayer study. Langmuir 2010, 26, 3268-3274. (26) Hénon, S.; Meunier, J., Microscope at the Brewster angle: Direct observation of first-order phase transitions in monolayers. Rev. Sci. Instrum. 1991, 62, 936-939. (27) Hönig, D.; Möbius, D., Direct visualization of monolayers at the air-water interface by Brewster angle microscopy. J. Phys. Chem. 1991, 95, 4590-4592. (28) von Tscharner, V.; McConnell, H. M., Physical properties of lipid monolayers on alkylated planar glass surfaces. Biophys. J. 1981, 36, 421-427. (29) Peters, R.; Beck, K., Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis. Proc. Natl. Acad. Sci. USA 1983, 80, 7183-7187. (30) von Tscharner, V.; McConnell, H. M., An alternative view of phospholipid phase behavior at the air-water interface. Microscope and film balance studies. Biophys. J. 1981, 36, 409-419. (31) Josefowicz, J. Y.; Maliszewskyj, N. C.; Idziak, S. H. J.; Heiney, P. A.; McCauley Jr., J. P.; Smith III, A. B., Structure of Langmuir-Blodgett films of disk-shaped molecules determined by atomic force microscopy Science 1993, 260, 323-326. (32) Nakahara, H.; Nakamura, S.; Okahashi, Y.; Kitaguchi, D.; Kawabata, N.; Sakamoto, S.; Shibata, O., Examination of fluorination effect on physical properties of saturated long-chain alcohols by DSC and Langmuir monolayer. Colloids Surf. B 2013, 102, 472-478. (33) Nakahara, H.; Krafft, M. P.; Shibata, A.; Shibata, O., Interaction of a partially fluorinated alcohol (F8H11OH) with biomembrane constituents in two-component monolayers. Soft Matter 2011, 7, 7325-7333. (34) Nakahara, H.; Lee, S.; Shibata, O., Pulmonary surfactant model systems catch the specific interaction of an amphiphilic peptide with anionic phospholipid. Biophys. J. 2009, 96, 1415-1429. (35) Nakahara, H.; Lee, S.; Krafft, M. P.; Shibata, O., Fluorocarbon-hybrid pulmonary surfactants for replacement therapy - a Langmuir monolayer study. Langmuir 2010, 26, 18256-18265. (36) Nakahara, H.; Lee, S.; Sugihara, G.; Chang, C.-H.; Shibata, O., Langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: Comparison of new preparations with Surfacten (Surfactant TA). Langmuir 2008, 24, 3370-3379. (37) Nakahara, H.; Lee, S.; Sugihara, G.; Shibata, O., Mode of interaction of hydrophobic amphiphilic α-helical peptide/dipalmitoylphosphatidylcholine with phosphatidylglycerol or palmitic acid at the air-water interface. Langmuir 2006, 22, 5792-5803. (38) Demchak, R. J.; Fort, T., Jr., Surface dipole moments of close-packed nonionized monolayers at the air-water interface. J. Colloid Interface Sci. 1974, 46, 191-202. 26 ACS Paragon Plus Environment

Page 26 of 48

Page 27 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(39) Goodrich, F. C. In Proceeding of 2nd International Congress on Surface Activity, J.H.Schulman ed., London, 1957; Butterworth & Co.: London, 1957; p 85. (40) Marsden, J.; Schulman, J. H., Trans. Faraday Soc. 1938, 34, 748-758. (41) Shah, D. O.; Schulman, J. H., J. Lipid Res. 1967, 8, 215-226. (42) Joos, P.; Demel, R. A., The interaction energies of cholesterol and lecithin in spread mixed monolayers at the air-water interface. Biochim. Biophys. Acta 1969, 183, 447-457. (43) Savva, M.; Acheampong, S., The interaction energies of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in spread mixed monolayers at the air-water interface. J. Phys. Chem. B 2009, 113, 9811-9820. (44) Weis, R. M.; McConnell, H. M., Two-dimensional chiral crystals of phospholipid. Nature 1984, 310, 47-49. (45) Cruz, A.; Vázquez, L.; Vélez, M.; Pérez-Gil, J., Influence of a fluorescent probe on the nanostructure of phospholipid membranes: dipalmitoylphosphatidylcholine interfacial monolayers. Langmuir 2005, 21, 5349-5355. (46) Leiske, D. L.; Meckes, B.; Miller, C. E.; Wu, C.; Walker, T. W.; Lin, B.; Meron, M.; Ketelson, H. A.; Toney, M. F.; Fuller, G. G., Insertion mechanism of a poly(ethylene oxide)-poly(butylene oxide) block copolymer into a DPPC monolayer. Langmuir 2011, 27, 11444-11450. (47) Scholtysek, P.; Li, Z.; Kressler, J.; Blume, A., Interactions of DPPC with semitelechelic poly(glycerol methacrylate)s with perfluoroalkyl end groups. Langmuir 2012, 28, 15651-15662. (48) McConnell, H. M., Structures and transitions in lipid monolayers at the air-water interface. Annu. Rev. Phys. Chem. 1991, 42, 171-195. (49) Keller, D. J.; McConnell, H. M.; Moy, V. T., Theory of superstructures in lipid monolayer phase transitions. J. Phys. Chem. 1986, 90, 2311-2315. (50) Keller, D. J.; Korb, J. P.; McConnell, H. M., Theory of shape transitions in two-dimenslonal phospholipid domains. J. Phys. Chem. 1987, 91, 6417-6422. (51) Moy, V. T.; Keller, D. J.; McConnell, H. M., Molecular order in finite two-dimensional crystals of lipid at the air-water interface. J. Phys. Chem. 1988, 92, 5233-5238. (52) McConnell, H. M., Harmonic shape transitions in lipid monolayer domains. J. Phys. Chem. 1990, 94, 4728-4731. (53) Benvegnu, D. J.; McConnell, H. M., Line tension between liquid domains in lipid monolayers. J. Phys. Chem. 1992, 96, 6820-6824. (54) Benvegnu, D. J.; McConnell, H. M., Surface dipole densities in lipid monolayers. J. Phys. Chem. 1993, 97, 6686-6691.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 48

FIGURE LEGENDS Synthesis

FIGURE 1

of

N-(6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)stearamide (2). FIGURE 2

The π–A and ∆V–A isotherms of the two-component (A) DPPC/rTz-C18, (B) DPPE/rTz-C18, (C) DPPG/rTz-C18, (D) PSM/rTz-C18, and (E) Ch/rTz-C18 monolayers on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K.

FIGURE 3

Two-dimensional phase diagrams based on the variation of the transition pressure (πeq: open circle) and collapse pressure (πc: solid circle) on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K as a function of XrTz-C18. In the πc region, the dashed lines were calculated according to Eq. (2) for ξ = 0. The solid lines were obtained by curve fitting of experimental πc values to Eq. (2). “M” indicates a mixed monolayer formed by each lipid and rTz-C18 species, whereas oblique-line areas show a bulk phase of the two components (“bulk phase” may be called “solid phase” not monolayer state) : (A) DPPC/rTz-C18, (B) DPPE/rTz-C18, (C) DPPG/rTz-C18, (D) PSM/rTz-C18, and (E) Ch/rTz-C18.

FIGURE 4

FM images of the DPPC/rTz-C18 monolayers for XrTz-C18 = 0.3, and 0.5 at 10 and 15 mN m−1 on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K. The monolayers contained 1 mol% Fluorescent probe (NBD-PC). The 28 ACS Paragon Plus Environment

Page 29 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

scale bar in the lower right represents 100 µm. FIGURE 5

FM images of the DPPG/rTz-C18 monolayers for XrTz-C18 = 0.3, and 0.5 at 15 and 20 mN m−1 on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K. The monolayers contained 1 mol% Fluorescent probe (NBD-PC). The scale bar in the lower right represents 100 µm.

FIGURE 6

FM images of the PSM/rTz-C18 monolayers for XrTz-C18 = 0.3, and 0.5 at 15 and 20 mN m−1 on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K. The monolayers contained 1 mol% Fluorescent probe (NBD-PC). The scale bar in the lower right represents 100 µm.

FIGURE 7

BAM images of the (a) DPPC/rTz-C18, (b) DPPG/rTz-C18, and (c) PSM/rTz-C18 monolayers for XrTz-C18 = 0.3 at typical surface pressures on 0.02 M Tris buffer solution with 0.13 M NaCl (pH 7.4) at 298.2 K. The scale bar in the lower right represents 100 µm.

FIGURE 8

Typical AFM topographic images of the binary DPPE/rTz-C18 monolayers for XrTz-C18 = 0.5 and 0.7 at 35 mN m−1. The cross-sectional profiles along the scanning line (white line) are given just below the respective AFM images. The height difference between the arrows is indicated in the cross-sectional profile.

FIGURE 9

Typical AFM topographic images of the binary Ch/rTz-C18 monolayers for XrTz-C18 = 0.5 and 0.7 at 35 mN m−1. The cross-sectional profiles along the 29 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

scanning line (white line) are given just below the respective AFM images. The height difference between the arrows is indicated in the cross-sectional profile.

Table of Contents Graphic


Page 30 of 48

Page 31 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.1 227x83mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 32 of 48

Page 33 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig.2A 192x281mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.2B 192x276mm (300 x 300 DPI)


Page 34 of 48

Page 35 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig.2C 193x278mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.2D 194x288mm (300 x 300 DPI)


Page 36 of 48

Page 37 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig.2E 192x277mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.3A 152x218mm (300 x 300 DPI)


Page 38 of 48

Page 39 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig.3B 152x213mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.3C 152x216mm (300 x 300 DPI)


Page 40 of 48

Page 41 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig.3D 152x215mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.3E 152x213mm (300 x 300 DPI)


Page 42 of 48

Page 43 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 4

10 mN m−1

XrTz-C18 = 0.3

XrTz-C18 = 0.5


15 mN m−1

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48

Figure 5

15 mN m−1

20 mN m−1

XrTz-C18 = 0.3

XrTz-C18 = 0.5


Page 45 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 6

15 mN m−1

20 mN m−1

XrTz-C18 = 0.3

XrTz-C18 = 0.5


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7

15 mN m−1 (a)

20 mN m−1 (b)

20 mN m−1 (c)


Page 46 of 48

Page 47 of 48

Figure 8

XrTz-C18 = 0.5

0

XrTz-C18 = 0.7

1.3 nm

[m]

3.0

0 [nm] 2.5

0 [nm] 2.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0


1.6 nm

[m]

3.0

Langmuir

Figure 9

XrTz-C18 = 0.5

0

XrTz-C18 = 0.7

0.9 nm

[m]

3.0

0 [nm] 2.5

0 [nm] 2.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 48

0


1.6 nm

[m]

3.0

Interactions of a Tetrazine Derivative with Biomembrane Constituents

Recommend Documents