Anal. Chem. 1994,66, 2701-2709
Preparation of Mixed Ligand Immobilized Artificial Membranes for Predicting Drug Binding to Membranes Charles Pidgeon,' Shaowel Ong, Heesung Chol, and Hanlan Liu
Depatfment of Medicinal Chemistry, School of Pharmacy, Purdue Universiv, West Lafayet& Indiana 47907 Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are designated as either the primary phospholipid or the secondary phospholipid. The primary immobilized phaspholipid refersto the immobilized phospholipid that has the highestsurface density. For this work, the primary immobilized phospholipid was a single-chain ether phosphatidylcholine (PC) analog. Four mixed-ligand IAMs were prepared by use of immobilizedPC as the primary immobilized phospholipid. The secondary immobilizedphospholipid ligand was either phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or phosphatidicacid. All of these secondary phospholipids are bonded at approximately6-10 mol 5% relative to the molar amount of immobilized PC. Each secondary phospholipid containsfunctionalgroups in the polar head group regionthat require protectinggroups during the immobilization process. The four-step synthetic strategy to prepare mixedligand IAMs involves (i) immobilization of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followed by end capping with a short-chainanhydride, and (iv) deprotection of the polar head group protecting groups. The surface density of the mixed PLs bonded to the silica support was approximately 130 Hmol of PLs/g of SPA. Highperformance liquid chromatography using these mixed lipid IAMscan be exploitedto rapidly predict the membrane binding properties of drugs. Immobilized artificial membranes (IAMs) contain different types of phospholipid monolayers that are covalently bonded to silica particles.'" A variety of chemical and biological applications have beendes~ribed.~-~~ All of the IAMs prepared to date contain a single phospholipid analog immobilized on E-mail addrcss:
[email protected]. (1) Pidgcon, C. US. Patent 4,931,498, 1990, (2) Pidgeon, C. US. Patent 4,927,879, 1990, (3) Pidgeon, C.; Venkatarum, U. V. AMI. Biochem. 1989, 176, 36-47. (4) Qiu, X.; Ong, S.;Bcrnal, C.; Rhee, D.; Pidgeon, C. J. Org. Chem. 1994,59,
537-543.
( 5 ) Ong, S.;Cai, S.J.; Bernal, C.; Rhee, D.; Qiu, X.; Pidgeon, C. Anal. Chem.
1994.66, 782-792. (6) Rhee, D.; Markovich, R. J.; Chae, W. 0.; Qiu, X.; Pidgeon, C. Anal. Chim. Acta submitted. (7) Stevens, J. M.; Markovich, R. J.; Pidgcon, C. Biochromatography 1989, 4,
192-205. 970-982.
(8) Otto, S.; Marcus, C.; Pidgeon, C.; Jefcoate, C. Endocrinology 1991, 129,
(9) Pidgcon. C. Enzyme Microb. Techno/. 1990, 2, 149-150. (10) Pidgeon, C.; Stevens, J.; Otto, S.;Jcfcoate, C.; Marcus, C. Anal. Biochem. 1991, 194, 163-173. (1 1) Pidgcon, C.; Marcus, C.; Alvarcz, F. Immobilized Artificial Membranes Chromatography: Surface Chemistry and Applications; Plenum Press: New York, 1991; pp 201-220. (12) Kollbach, T.A.; Wainer, I. J. Chromatogr. 1993, 653, 122-129.
0003-2700l94I03662701~~4.5~lO 0 1994 American Chemical Society
a silica surface; these JAMS were prepared from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylserine (PS) ligands.l4 Biological membranes contain many phospholipids, but, typically PC is the major constituent. The minor membrane lipids of most cell membranes are PE, PG, PA, and PS,and although they are present in small amounts relative to PC, they frequently have a critical function in the membrane physiology of the cell. In order to design mixed lipid IAMs that can potentially emulate the lipid environment of cell membranes, we developed the bonding strategy to first immobilize a PC ligand at a high density and then immobilize PE, PG, PS, or PA analogs, at a low density. An important application of mixed-ligand IAMs is to predict drug transport across biological barriers. For diffusioncontrolled drug transport through membranes, the drug binding to the membrane can be the rate-limiting step, and consequently, membrane binding studies using fluid liposome membranes attempted to correlate the membrane drug transport proper tie^.^^-^^ Using immobilized membranes instead of fluid artificial membranes, we have recently demonstrated that IAM HPLC chromatography is a rapid, simple method to predict transport properties of drugs and in fact, for some classes of compounds, IAM columns can predict the bioavailability of the experimental compounds (Pidgeon et al., in preparation). The mixed-ligand IAMs developed in this work are suitable for these types of studies because most biological membranes are negatively charged surfaces. The preliminary data in this report demonstrate that mixed-ligand IAMs can predict the binding of drugs to fluid membranes. In addition to predicting drug transport and drug binding to membranes, mixed-ligand IAMs are expected to have applications for enzyme immobilization. Noncovalent im(13) Pidgeon,C.;Markovich,R. J.;Liu,M. D.;Holzcr,T. J.;Novak,R.M.;Kcyer,
K. A. J. Biol. Chem. 1993, 268, 7773-7778. (14) Qiu, X.; Pidgeon, C. J. Phys. Chem. 1993,97, 12399-12407. (15) Thumhofer, H.; Schnabel, J.; Betz, M.; Lipka, G.; Pidgeon, C.; Hauscr, H. Biochim. Biophys. Acta 1991, 1064,275-286. (16) Zhang, X.-M.; Waincr, I. W. Tetrahedron Lett. 1993, 34, 47314734. (1 7) Kolbah, T.A.; Felix, G.; Waincr, I. W. Chromatographla 1993,35,264268. (18) Kolbah, T.A,; Wainer, I. W. J. Chromatography 1993, 646, 289. (19) Chae. W. G.; Luo, C.; Rhee, D. M.; Lombardo, C. R.; Low, P.; Pidgcon, C. Immobilized Artificial Membrane Chromatograpy; Plenum Rcss: New York, 1991. (20) Chui, W. K.; Wainer. I. A w l . Biochem. 1992, 201, 237-245. (21) Cohcn, D. E.; Leonard, M. R.; Leonard, A. N.; Carey, M. C. Gastroenterology 1993, 104, A889. (22) Alvarcz, F. M.; Bottom, C. B.; Chickale. P.; Pidgwn, C. In Molecular Interactions in Bioseparations; Ngo, T.,Ed.;Plenum Press: New York, 1993; pp 151-167. (23) Bctagcri, G. V.; Rogers, J. A. Pharm. Res. 1989, 6, 39943. (24) Choi, Y. W.; Rogers, J. A. Pharm. Res. 1990, 7, 508-512. (25) Bctagcri, G. V.; Rogers, J. A. In?. J. Pharm. 1987,36, 165-173. (26) Rogers, J. A.; Choi, Y. W. Pharm. Res. 1993, 10, 913-917.
Analyrlcel Chemktry, Vol. 66, No. 17, September 1, 1994
2701
mobilization of enzymes on IAM HPLC columns has been successful at determination of the enantioselectivity of the immobilized enzyme.16-18*20 For these HPLC enzymereactors, noncovalent enzyme immobilization does not permit organic solvents or detergents to be included in the mobile phase because these solution conditions will cause leaching or elution of the enzyme. Covalent immobilization of the enzyme does not suffer from this limitation, and all organic solvents and detergents can be used. Thus, some IAM applications will require covalent immobilization of the enzyme. Recently, it has been established unequivocally that enzymes can act as catalysts in anhydrous organic solvents and that in this unnatural milieu enzymes exhibit a number of new prop e r t i e ~ . ~ ~Enzymatic -~l reactions in nonaqueous media are becoming increasingly important in synthetic organic chemi ~ t r y and , ~ ~ nondenaturing surfaces suitable for enzyme immobilization are essential. Mixed-ligand IAMs allow covalent immobilizationof the enzymesthrough the functional groups of the secondary phospholipids (such as the amino group on the PE head group). Most important, IAMs are gentle chromatography surfaces that do not denature the proteins during the protein adsorption step,1”18*20and this may make IAMs the preferred surface for the immobilization of labile enzymes. In addition, IAMs have excellent mechanical and chemical stability in both aqueous and organic solvents.5~6J4J3 EXPERIMENTAL SECTION Chemicals and Reagents. The following chemicals were purchased from Aldrich: ethanol-free CHCl3 (Sure-Seal bottle), used for activation of the PLs carboxyl group; 1,l’carbonyldiimidazole (CDI); decanoic anhydride (C10); propionic anhydride (C3); 1.8-diazobicyclo[5.4.O]undec-7-ene (DBU); and IRC-50 (weakcation exchangeresin). Phosphatebuffered saline (PBS) and a-adrenoceptor agonists including tetrahydrozoline, clonidine, naphazoline, xylometazoline, and oxymetazoline were purchased from Sigma. Ninhydrin and Phospray were purchased from Supelco. Spherical silica propylamine, (SPA) was 12 pm in diameter and contained 300-A pores. SPA was obtained from Regis Chemical Co. Immediately prior to PL bonding, the SPA was degassed by preparing a CHC13 suspension and sonicating for 15-30 s and then the suspension was rotoevaporated to dryness (- 5 min). A bath sonicator (Branson 2200) was used for the degassing procedure. IAM.PCC10/C3used for all solid-phase adsorption synthesis was purchased from Regis Chemical Co. Prior to use, the IAM.PCC10/C3powder was washed with acetone. The w-carboxyl PLs that were bonded to SPA include PCCWH,PGp, PSp, PEP, and PAP are shown in Chart 1. The subscript P in PGp, PSp, PEP, and PAP denotes that these
-
(27) Russell, A. J.; Trudel, L. J.; Skipper, P. L.; Groopman, J. D.; Tannenbaum, S. R.;Klibanov, A. M. 1989, 1 5 8 , 8 0 4 5 (28) Janda, K.D.; Ashley, J. A,; Jones, T.M.; Mclcod, D. A,; Schloeder, D. M.; Wcinhousc, M.J. J. Am. Chem. Soc. 1990,88864888. (29) Kirchner, G.; Scollar, M. P.; Klibanov, A. M. J . Am. Chem. Soc. 1985,107, 7072-7076. (30) Saini, S.; Hall, G. F.;Downs, M. E.; Turner, A. P. F.Anal. Chim. Acto 1991, 219, 1-15. (31) Zaks, A.; Klibanov, A. Science 1984, 224. 1249-1251. (32) Chen, C.; Sih, C. J. Angew. Chem., Int. Ed. Engl. 1989, 28, 695-707. (33) Markovich, R. J.; Stevens, J. M.; Pidgeon, C. Anal. Blochem. 1989, 182, 237-244.
2702
Ana!ytlcalChemisfry, Vol. 66, No. 17, September 1, 1994
Chart 1. General Structures ot the PL Ugandr Including the Prknary Phoqhoilpld Llgad (E-) and the Secondary Phorphdlpkl Llganda Contalnlng Protoctlng aroupr In the Pdar Head Group Region.
0
II CHrO-f‘-O-R
1 -
CHqOCH
“ I
0
First Ligand (PC) Prote cted Second Ligand PLI, PE,
R=Rp -CH,CH,-NHCOOC(CH -CH,CH-COOC(CH
PSP
I
3)3
R = -CHzCHzN(CH3)3+
DeprotectedSecond Ligand PL
R
PE
-CHZCH~-NH3’
3)3
PS
CH,
NH-COOC(CH 3)3 -CH,-CH.CH PG P
4
I
1
,
Rd
FH-C O O NH3’
PG
PA
-CH&H-CH I
2
I
OH OH
H
Plp denotes the entire phosphollpldcontalnlng a protecting group, & demtes only the protectedpolar headgroup, and&denotesthe deprotected polar head groups.
w-carboxyi PLs contain “protecting” groups in the polar head group region. The synthesis of these w-carboxyl PL ligands was described in detai1.6J4 FT-IR spectroscopy was used to verify that the w-carboxyl groups of each PL ligand were in the nonionized form prior to activation with CDI. If necessary, thecarboxyl PLs can be passed through a weak cation exchange column to protonate the ionized carboxyl prior to CDI activation. Elemental Analysis. Elemental analysis was performed on a Perkin-Elmer PE 240 in the Microanalytical Laboratory at Purdue University Chemistry Department using approximately 10-15 mg of IAMs. Phospray and Ninhydrin Analysis of IAM Powder. IAM powder (- 1-2 mg) was placed in a test tube (1 3 mm X 100 mm), and then two or three drops of ninhydrin or Phospray were added. The powder was allowed to air-dry at room temperature, and color changes were noted over 12 h. Ninhydrin causes the IAM chromatography packing material to become pink33when unreacted surface amines on SPA are available to solvent penetrating the IAM interfacial region (see footnote d in Table 2). IAM powder that remains white over 12 h is considered to be ninhydrin negative, and this indicates that there are no residual surface amines on the silica surface. All white IAM powders became bright blue after exposure to Phospray, indicating that a high surface density of immobilized phosphate exists on the IAM surface. IT-IR Spectroscopy. A Nicolet Magna 550 FT-IR spectrometer equipped with a Spectratech IR-Plan I microscope was used to analyze all IAM powders in the reflectance mode as d e ~ c r i b e d . ~Typically, , ~ ~ . ~ ~ -500 pg of IAM powder was pressed into a wafer using a hand press, and the infrared spectra were obtained after focusing the infrared beam on the surface of the gold mirror. IR spectra of the IAM wafers (34) Markovich,R. J.;Qiu,X.;Invergo, B.;Nichols,D.E.;Alvarez,F.M.;Pidgeon, C. Anal. Chem. 1991, 63, 1851-1860.
Schomo 1. Bondlng Wrategy Used To koprr Mbwd Lband IAMs from PL Ligan*
Step 1. Bonding of the first ligand 1) IAM.PC c10’c3
0
Solid phase adsorption
11
PCCooH d N - 2) CDI/CHCI - c -3
Si-o/\/\Nh
-
c
m
-
(SPA)’
p
CHC13
e M e r t ~ ~ . ~ ~
Step ii. Bonding of the secondary ligand 1) IAM.PC c10/c3
?I”
Solid phase adsorption
2) CDVCHCI
PLp
pLp-c
e m e r ~ ~ ~ . ~ ~ L e
-Nu’CHCl,
’ h e r ~ ~ ~ . ~ ~ / ~ ~
Step iii. ClWC3 end capping
Stepiv. Deprotection of the second ligand head group
e
m
e
r
pc10/c3 ~ ~
1 N HCVEtOAc
~
. ~ ~ or DBU
~ e~ ’ h e r ~ c10/c3 ~ ~ . ~ ~ / ~ ~
PL ligands are shown In Chart 1. CDI activation of each PL ligands to prepare the PL-lmidarolides (step I) and the solid-phase adsorption synthssis was performed uslng IAM.P@lom particles to facilitate the reaction. Bonding of the PL-lmidazoiides to silica propylamlnes was performed for 48 h (steps I and 11). Step ill was end capplng with first decanoic anhydride then propionic anhydride. Deblocking of the llpld protecting groups (step iv) was usually performed either In aqueous acidic conditions or under mildly basic conditlons as descrlbed in the text.
were taken at a resolution of 4 cm-l with 256 scans using a Happ-Gentzel apodization function. Except for the spectrum of the nonbonded PLs and the SPA particles, all infrared spectra are difference spectra (Le., the SPA spectrum has been subtracted from the IR spectrum of the IAM surface). The silica S i 4 combination band, centered at 1870 cm-l, was used as the reference band for each subtraction. The IR spectra of all soluble ligands and reagents (e.g., CDI) were obtained by evaporating microliter volumes of the solubilized ligandson CaF2 plates. IR spectra of the ligands and reagents were taken at a resolution of 4 cm-l with 32 scans using a Happ-Gentzel apodization function. To quantitate hydrocarbon content, the integrated intensity of the hydrocarbon (HC) stretch region (2995-2825 cm-l) was divided by the integrated intensity of the S i 4 combination band between 1945 and 1780 cm-l. The Si-0 combination band centered at 1870 cm-l was also used as the reference band for each spectral subtraction. General Bonding Strategy. The primary PL (Le., PCCOOH) was first bonded to SPA (step i in Scheme 1) at high density followed by the bonding of the secondary phospholipid, Le., either PEP, PSp, PGp, or PAp (step ii in Scheme 1). The bonding of both the primary and secondary PL ligands to SPA requires that the w-carboxylgroup of each PL be activated to facilitate the amidation reaction between the PL w-carboxyl group and the surface amine of SPA. CDI activation of the PL w-carboxyl group was facile using the IAM solid-phase adsorption synthesis method.s Following activation, the PLimidazolides were bonded to SPA. The surface was then end
capped with decanoic (C10) and propionic (C3) anhydrides (step iii in Scheme l), and finally, the protecting groups were removed either under aqueous acidic conditions or by using the nonnucleophilic organic base, DBU (step iv in Scheme 1). Specific details for the synthesis procedure of each step are given below. Bonding of the Primary Ligand Preparation of *IAM.PC. PCCWH(4.98 g, 10.6 mmol) was dissolved in 6 mL of CH3OH and adsorbed onto 7 g of IAM.PCC10/C3chromatographic particles. This loading step is critical for efficient reaction of the carboxyl groups and should be done as described.5 The I AM.PCC chromatography particles with adsorbed PCCWHwere placed in a 250-mL round-bottomed flask and dried in vacuo overnight to remove all traces of residual methanol. The dried IAM.PCC10/C3particles were suspended in 80 mL of CHCl3 and reacted with CDI (2.06 g, 12.7 mmol) at room temperature for 4 h to form the activated ligand which is PC-imidazolide. The bonding surface for preparing IAMs is silica propylamine (SPA). SPA (50 g) was suspended in 200 mL of CHCl, in a 1000-mL round-bottomed flask and sonicated for 5 min to degas the particles. The IAM.PCC10/C3suspension containing the PC-imidazolide ligand was filtered directly into the SPA suspension, followed by washing with 70 mL of additional dry CHCl3 through the IAM.PCC10/C3suspension into the SPA suspension. The SPA/PC-imidazolide mixture was shaken under nitrogen at room temperature for 45 h to bond PC to SPA. The product is denoted as CthCrIAM.PC and was recovered by filtering and washing with 150 mL each of Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
2703
the following solvents: CHCl3, CH3OH, THF, CH2C12, CH3OH, and acetone. TheetherIAM.PCpowder was dried in vacuo overnight to remove residual water and prepare the surface for bonding of the secondary phospholipid. Bonding of the Secondary Phospholipid Ligand (PLp): Preparation of e'kIAM.PC/PLp. The preparation of IAMs containing immobilized monolayers of PE, PS, PA, or PG was described previously described in detail.5 Although these IAMs are not mixed-ligand IAMs, the same bonding strategy and deprotectionstrategy was applied to prepare mixed-ligand IAMs. The o-carboxyl of the secondary PLs (Chart 1) was activated using the solid-phaseadsorptionmethod as described above for the CDI activation of the PCCWHligand. The secondary PL (200 mg) was dissolved in a minimum amount of CHsOH (0.35-0.7 mL) and adsorbed onto 450 mg of IAM.PCC10/C3 chromatography particles. The 4-h activation at room temperature utilized 3 mL of CHCl3 containing a 1-3-foldexcess of CDI. This CDI reaction was performed for PEP, PSp, PAP, and PGp to form the imidazolides of each secondaryPL ligand. These activated PL ligandswere bonded to ethCrIAM.PC. CtherIAM.PC chromatography particles (10 g, prepared in step i in Scheme 1) were suspended in 30 mL of CHCl3 in a 250-mL round-bottomedflask and sonicatedfor 1 min to degas the particles. The CDI-activated PL ligands were transferred to the CthCrIAM.PC chromatography particles as described above. Briefly, after solid-phaseadsorption synthesis, the PLimidazolideligands were directly filtered into the CtherIAM.PC suspension; the PL-imidazolide ligand transfer was completed by washing with 50 mL of additional CHCl3. The reaction mixture was mildly shakenunder nitrogen at room temperature for 48 h. The mixed-ligand IAM products were filtered and washed with 100mL each of CHCL, CH3OH, THF, CH2C12, CH3OH, and acetone. The final mixed-ligand IAMs having the general formula CtherIAM.PC/PL,were dried in vacuo overnight. The four mixed-ligandctherIAM.PC/PLpsurfaces are denoted as CthCrIAM.PC/PEp, ethCrIAM.PC/PGp, CthCrIAM.PC/PSp, and ctherIAM.PC/PAp. C10 and C3 End Capping: Preparation of **IAM.PC/ PLpc10/c3* Each of the etherIAM.PC/PLppacking materials (10 g) was suspended in a separate 250-mL round-bottomed flask using 60 mL of CHCl,; the suspensionswere sonicated for 1 min to degas the particles. Decanoic anhydride (2 mL) was added to eachsuspension,and the suspensions were shaken under nitrogenfor 24 h. The products were filtered and washed with 100 mL each of CHC13, CH30H, THF, CH2C12, CH3OH, and acetone and dried in vacuo overnight. The wash solutions were concentrated and checked by TLC for phospholipid leaching from the IAM surface during the endcapping reaction. None of the previously immobilized phospholipids leached from the surface during the end-capping reaction using Phospray positive spots for detection. The decanoic anhydride end-capped IAMs were subjected to an identical end-capping reaction using propionic anhydride to obtain the desired ClO/C3 end-capped products. Deprotection of the Immobilized Secondary PL Ligands. After end capping with long- and short-chain-length linear anhydrides, the final synthetic step is to remove the protecting groups tethered to the polar head group of the immobilized 2704
Analytical Chemistry, Vol. 66,No. 17, September 1, 1994
secondary phospholipids. The same deprotectionstrategy was used for immobilized PEP, PSp, and PGp. The chromatography particles were first suspended in 60 mL of 1 N HCl/ EtOAc by mildly shaking. After being sonicated for 1 min, the suspension was then shaken at room temperature for 2 h. The IAMs were filtered to remove the acidic reaction solvent, and then the particles were washed with 100 mL of EtOAc, 20 mL of CH30H, and finally with 500 mL of H20 to completely remove residual surface-associated acid. The IAMs were further washed with 100 mL each of CH3OH, THF, CH3OH, and acetone and dried in vacuo overnight. All of the wash solutions were combined with the acidic reaction solvent and concentrated to check for phospholipid. No phospholipid was detected by TLC in the washes/reaction solvent, which indicates that no phospholipids leached from the surface during the strongly acidic deprotection reaction conditions. For CthCrIAM.PC/PApC10/C3, a nonnucleophilic base, DBU, was used as the deprotecting reagent. The CthCrIAM.PC/ P A P ~ ' Oparticles /~~ were suspended in 30 mL of CHCl3 in a 100-mLround-bottomed flask and sonicated for 1 min. DBU (2 mL) was added, and the mixture was stirred under nitrogen at rt for 5 h. The product was filtered and washed with 500 mL each of acetonitrile, 2 N NaCl solution, and H 2 0 for the complete removal of excess DBU. The product was further washed with 100 mL each of CHsOH, THF, CH2C12, CH3OH, and acetone and dried in vacuo overnight. The wash solutions were concentrated and checked for phospholipid leaching on TLC. No Phospray positive spots were detected. HPLC Conditions. ethcrIAM.PCC10/C3 and *IAM.PC/ PLc10/c3chromatography particles suspended in acetone (10% w/v) were packed in 4.6 mm X 3 cm columns at Regis Chemical Co. All IAM HPLC columns were 4.6 mm X 3 cm and contained a void volume V ,of -410 pL. These columns were used to evaluate the ability of the mixed-ligand IAMs to predict the binding of drugs to artificial membranes using a-adrenoceptor agonists as model compounds. Choi and Rogers24have measured the binding of these experimental drugs to fluid artificial membranes that contain either neutral or negatively or positively charged membrane surfaces, and we have used Choi and Roger's data to validate the concept that IAMs can predict drug binding to fluid membranes. The injection volume was 15 pL of the a-adrenoceptor agonist solution (1 pg/pL) in 0.01 M phosphate-bufferedsaline (PBS) buffer (pH 7.4). Citric acid (0.1 pg/pL) in 0.01 M PBS buffer (pH 7.4) was used as an internal standard to measure the relative retention of the model compoundson the IAM chromatography surfaces. The flow rate was either 1 or 3 mL/min and detection was at 220 nm. The buffered aqueous mobile phase was 0.01 M PBS (pH 7.4). Chromatograms were obtained by using a Rainin HPLC pumping system equipped with a Knauer Model 87 detector and interfaced with a Macintosh computer. Rainin Dynamax software was used to record the chromatograms on the computer.
-
RESULTS AND DISCUSSION Although the phospholipid composition in natural biological membranes varies, typically the phospholipid content contains 10-30% negatively charged phospholipids and the remainder
-
-
503
0
85
90
95 100 105 110 115 120 pmol-PC/g-SPA
Figure 1. Dependence of the bonding density of the second ligand (PEp) on the bonding density of the first ligand (PC). The ligand density was Calculated on the basis of elemental analysis. The PL weight per x) = A% C, where gram of SPA was calculated from 4%C,)/(l x is the Plp weight, A% C is the net carbon gain from elemental analysis, and % C, is the carbon content per PL molecule. The relative amount of the second ligand bonded showed linear dependence on the amount of the first ligand bound to SPA (R > 0.99).
+
Table 1. Chemkai Analyrk d Mlxed-Nand IAM Surfaces during Bonding Reactions
bonding reactions‘ step i step ii step iii step iv step ii step iii step iv step ii step iii step iv step ii step iii step iv
IAMs
% C6
H - C areac Si - 0 area
dmIAM.PC *IAM.PC/PEp hIAM.PC/PEpC1O *=IAM.PC/PEpC10/C3 a”IAM.PC/PEC1O/C3 cWAM.PC/PGp dWIAM.PC/PGpC1o hIAM.PC/PGpC10/C3 *IAM.PC/PGCio/C3 cthmIAM.PC/PAp *IAM.PC/PApClO “WAM.PC/PApCIO/Cf &IAM.PC/PAC101C3 *IAM.PC/PSp *IAM.PC/PSpCio *IAM.PC/PSpClO/C3 *h~IAM,PC/PSCIO/C3
4.25 4.60 4.74 4.85 4.80 4.57 4.75 4.88 4.82 4.59 4.73 4.84 4.76 4.52 4.74 4.85 4.80
3.46 3.80 3.94 4.00 3.89 3.59 3.96 4.16 4.14 3.54 3.73 3.93 3.90 3.69 3.86 4.09 4.01
ninhydrind purple purple lavender white lavender purple lavender white white purple purple white white purple lavender white purple
mixed-ligand IAM surfaces containing -90% PC as the primary immobilizedlipid and 10%as secondary PL ligands, which include PG, PS, PA and PE analogs. The bonding chemistry of the first ligand (Le., PC) and the surface properties of ethcrIAM.PCC10/C3 have been described in detail elsewhere.6 It was found that a monolayer surface density (127 pmol of PC/g of SPA) of immobilized PC ligand could be achieved by using 1.2-1.5-fold excess of PC for CDI activation and subsequent bonding to SPAa6 However, synthesis of mixed-ligand IAMs requires bonding of the first ligand at a submonolayerdensity so that bonding space remains on the chromatographic surface for the bonding of the second ligand. Thus, the bonding density of the second ligand is expected to depend on the bonding density of the first ligand. To test this concept, the dependence of the bonding density of the secondary ligand (PLp) on the bonding density of the primary ligand ( P C c o o ~ was ) studied. PEP was used as the secondary ligand to evaluate how the surface density of the primary PC ligand effects the bonding density of the secondary PLp ligand. Figure 1 shows that the bonding density of PEP linearly decreases with increasing surface density of PC. Although Figure 1 was obtained for only PEP, we assumed that PSp, PGp, and PAp would have had similar results and we used this assumption in designing a synthetic route. Thus to obtain a 10% bonding density of the secondary PLp ligands, we estimate from Figure 1 that the primary bonding step should result in 115pmol of PC/g of SPA. Thus, we reacted 5 g of the CDI-activated PCcoo~ligand with 50 g of SPA for 48 h to produce a submonolayer surface density of 116.3 pmol of PC/g of SPA. This IAM surface, denoted as CthCrIAM.PC, was used as a matrix for bonding the secondary PLp ligands. During the bonding of the secondary PLp’s to CthCrIAM.PC, the CDI-activated PL,’s were used in a 1.2-1.5-fold excess of the secondary ligand. Although the surface density of the primary PC phospholipid is high, it did not inhibit the binding of the secondary PL,’s. As de~cribed,~ FT-IR spectroscopy and elemental analysis were used to quantitate the chemical compositions of each IAM surface after each bonding reaction; the results are given in Table 1. The surface parameters calculated from the data given in Table 1 are listed in Table 2. Table 2 shows that the surface density of the secondary ligand is 10% of the surface density of the primary ligand, except for the CthCrIAM.PC/ PSc*0/c3surface in which the secondary PS ligand is -6.5% of the primary ligand. From Table 2, the rank order of the
-
0 See Scheme 1. b % C data obtained from elemental analysis. FTIR peak area obtained from the following region for each band. C-H band, 2995-2825 cm-’; S i 4 band, 1945-1780 cm-*. Color changes were monitored 6 h after reacting with ninhydrin.
of endogenous membrane phospholipids are phosphatidylcholine analogs. Therefore, our initial goal was to synthesize
-
Table 2. Molecular Propertlet of Mixed-Ligand IAM Surfaces* mixed-ligand mg of IAM surfaces PL/g of SPAb PC:PL:ClO:C3C
dhQIAM.PC/PEC10/C3 dhcfIAM.PC/PGC10/C3 cthcrIAM.pC/pAC1O/C3 cthcrIAM,pC/pSCIO/C3
6.1 6.0 6.3 5.0
116.3:12.7:13.8:35.0 116.3:12.1: 16.3:42.6 116.3:11.8:13.8:35.0 116.3:8.0:21.1:35.0
% reacted surface aminesd
PL surface density’ (A*/moIecule)
50.8 53.5 50.5 51.5
82.0 82.5 83.0 85.0
*
0 All surface properties were calculated from the data in Table 1. PL denotes either PG, PS, PE, or PA. The PL weight per gram of SPA was calculated from the equation given the figure legend of Figure 1. The mole ratio of lipids bonded to 1 g of SPA. For instance, 116.3:12.7:13.8:35.0 reflects 116.3 pmol of PC, 12.7 @molof PE, 13.8 @molof C10 alk I chains, and 35.0 pmol of C3 alkyl chains per gram of SPA. The micromoles of each lipid were calculated from the weight of each lipid (PC, PL, &O, or C3) according to the equation given in footnote b of this table. d The initial SPA contained 350 pmol of propylamines per gram. Sequential bonding of PC, then PLp, then C10, and then C3 lipids converted some of the amines into amides. The percent reacted surface amines was calculated from the total micromoles of lipids bonded per gram of SPA divided by the total micromoles of pro ylamine of 1 g of SPA. * It was shown that the surface area of SPA available to bonding PLs was 64 m g.5 The phospholipid surface density was calcukted as (64 X 10zo)/(MpL X 6.023 X loz3), where MPLis the sum of the moles of PC and PL bon ed per gram of SPA.
a
Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
2705
r
Before deprotection
-NH-C-O-C(CH&
ekIAM.PC/PEC'w3
~
~
~
3150
'
I
2700
"
'
I
"
'
I
'
I
2250
1800 Wavenumber (cm" )
I
I
-
1350 1800 1750 1700 1650 1600 Wavenumber (em"
)
Figure 2. FT-IR differencespectra of *IAM.PC/PEp and *IAM.PClPEC10/C3. Differencespectra were obtained by subtracting the IR spectrum of *IAM.PC. The characteristic I R absorptions of the Boc group are the gem dimethyl doublet at 1398.0 and 1373 cm-l as well as the strong carbamate band centered at 1710 cm-l. The IR-active bands elicited by the Boc groups are clearly seen in the IR spectrum of *IAM.PC/PEp. As shown In the inset in the right, after the deprotectionof the BOCgroup (step iv In Scheme I), the Boc I R bands are absent from the I R spectrum of *IAM.PClPEC10'C3, which Indicates complete removal of the BOCgroups. Before deprotection
n
slheriAMpCpGClO/C3
-
After deprotection
I
3150
I
'
I
I
I
I
J
2700
I
2250
'
I
I
I
I
I
1800
Wavenumber (cm")
I
I
1350
3075 3000 2925 2850
Wavenumber (cm"
)
Figure 3. FT-IR differencespectra of *IAM.PC/POp and *IAM.PC/m101C3. Differencespectra were obtained by subtracting the I R spectrum of *IAM.PC from the spectrum of mixed-ligand IAM. The Characteristic absorption of the IP blocking group at 2992 cm-1 is clearly seen in the spectrum of *IAM.PC/POp after bondingof the secondary Pop ligand (step ii in Scheme 1). As shown In the inset in the rigM, this characteristic IR band of the IP group is absent In the I R spectrum of *IAM.PClP@10/C3 after the deprotection of the BOCgroup (step iv in Scheme 1) indicating the complete removal of the IP group.
surface density of the secondary PL,'s is
PE > PG > PA > PS which is also the rank order of the size of the protected polar lipid head groups. The same rank order of the surface density of bonded ligand was also observed in the preparation of singleligand IAMs containing PE, PG, PA, and PS analog^.^ This indicates that we have achieved sterically limited coupling of the secondary PLp's and the larger the secondary PLp the less efficiently it couples to the IAM surface that already contains a high surface density of PC. From Table 2, the total amount of immobilized mixed phospholipids forming the IAM surface is 130 pmol-PL/ g-SPA, and this corresponds to an average area/molecule of approximately 82-85 A2/molecule. This range of bonding densities is very close to that obtained for single-ligand IAMsS and is also close to the phospholipid densities found in liposome
-
2106
AnalytlcalChemisby, Vol. 66, No. 17, September 1, 1994
membranes. This indicates that both the mixed-ligand IAMs have approximately a monolayer density of immobilized phospholipids and may be good models for mixed lipid fluid membrane systems such as liposomes. Deprotection Reactions. As shown in Chart 1, the secondary PLp's, Le., PEP, PGp, PSp, and PAP, contain functional reactive groups (Le., amines, carboxyls, hydroxyls, or phosphoryl groups) that must be protected during the immobilization process. Protection of these groups is necessary to eliminate the possibility of intermolecular bonding between PLs during the immobilization process and also to assure that the phospholipids bond to SPA with the polar head groups protruding away from the surface. In Chart 1, R, denotes only the protected polar head group and &denotes the polar head group after deprotection. By using efficient deblocking reactions to convert R, to &,we have demonstrated that PLs with reactive functional groups in the polar head group region can be imm~bilized.~ Details of these deblocking reactions
I
l
1
*IAM.PCIPA~
~
1
2960
l
I
~
l
I
8
E
l
~
2220
2590
I
I
l
[
I
I
14'0
1850
Wavenumber (cm" )
I
Before deproteciion
~
I
1400 1380 1360 1340 1320 1300
Wavenumber (cm" ) Figure 4. FT-IR difference spectra of *IAM.PC/PAp and *IAM.PC/PAC10'C3. Dlfferencespectra were obtained by Subtracting the I R spectrum of *IAM.PC from the spectrum of mixed-ligand IAM. The characteristic absorptions of the N E group at 1528 and 1348 cm-I are clearly seen after bonding of the secondary PAp ligand (step li in Scheme 1). As shown In the Inset in the right, the In the spectrum of -IAM.PC/PAp characteristic I R band of the NPE group (- 1348 cm-I) is reduced to 10% of the original peak area in the I R spectrum of *IAM.PC/PAC10'C3 after the deprotectlon of the NPE group (step Iv in Scheme l), indicating the -90% removal of the NPE group.
-
Before deprotection . I 0
IAM.PC/PSp
t-Butyl
~
3150
I
I
I
2700
~
I
I
1
~
1
2250 I800 Wavenumber (cm" )
1
1
~
1350 1417
l
1
1400
1
1382
~
1365
Wavenumber (an" )
Figure 5. FT-IR dlfference spectra of *IAM.PC/PSp and * I A ~ V ~ . P C / P S ~ Dlfference ~~'~. spectra were obtained by subtractingthe IR spectrum of *IAM.PC from the spectrum of mlxed-ligand IAM. After bonding of the secondary PSp ligand (step ii In Scheme l),the characteristic I R absorptlon bands of the Boc and Bu blocking groups at 2982 cm-I, the doublet at 1398.0 and 1373 cm-I, and the strong Boc band centered at 1710 cm-l are clearly seen In the spectrum of *IAM.PC/PSp. As shown In the Inset in the right, these characteristic IR bands of the blocking groups are absent In the I R spectrum of *IAM.PC/PSC10'C3 after the deprotectlon of the BOCgroup (step lv in Scheme 1)indicatingthe complete removal of the BOCand Bu groups.
and the use of FT-IR to monitor these deblocking reactions on IAM surfaces were described.5 The IR signal intensities used to evaluate the R, to & deprotection reactions were weak because the secondary PLp ligands were immobilized at only -10% surface density compared to the immobilized PC ligand. Therefore, the spectrum of CthCrIAM.PC was subtracted from each mixedligand spectrum; the resultant IR difference spectra clearly showed the weak IR-active vibrations elicited by the immobilized PLp's. Figures 2-5 show the difference spectra before and after deprotection for each mixed-ligand IAM. The IR bands used to quantitate the deprotection reactions
are summarized in Table 3 and given in the legends of Figure 2-5. Briefly, IR spectroscopy of the mixed-ligand IAMs demonstrated that the R, protecting groups were virtually quantitatively converted into the deprotected & groups. Correlation of Drug Binding to Mixed-Ligand JAMS with Negatively Charged Liposome Membranes. Drug binding to liposome membranes has been used to predict the biological activity of a-adrenoceptor agonists.24 Although neutral liposome membranes prepared from pure dimyristoylpohosphatidylcholine (DMPC) can be used to predict the biological activities of the a-adrenoceptor agonists, it was found that negatively charged liposome membranes give better correlaAnalytical Chemistry, Vol. 66, No. 17, September 1, 1994
2707
Table 3. I R Frequencies Used To Monltor Deprotectlon Reactions
IAM surfaces
functional group(s)”
ethCrIAM.PGp eth=IAM.PSp
gem dimethyl r-Bu
“““IAM.PEp
1-Bu esters and Boc ~-Bu
C*erIAM.PAp
aromatic nitro group
BW
a
frequency (cm-l) v(C-H) 2992 6(C-H) 1398,1373 v(C-H) 2982 v(C=O) 1717 6(C-H) 1398,1372 v(C-H) 2982 v(C=O) 1710 v,(N=O) 1528 v,(N=O) 1348
The structures of these functional groups are given in Chart 1.
ti on^.^^-^^ This indicates that electrostatic interactions provided by negatively charged membrane lipids are important for optimum predictions of the binding and activity of a-adrenoceptors, For the first application of mixed-ligand IAMs, we demonstrate that negatively charged mixed-ligand IAMs emulate mixed lipid liposomal membrane systems; this is based on correlating the partition coefficient of drugs to negatively charged liposomal membranes with the capacity factors (k’values) of the drugs obtained on mixed-ligand IAM columns. The retention times ( t r ) of the a-adrenoceptor agonists on mixed-ligand IAMs were used to calculate the drug capacity factors (k’) with the following equation:
where t, is the retention time in minutes of the test compound
and to is the retention time of an unretained compound, Le., citric acid in this case. For chromatographic analysis, K is the equilibrium binding constant of a solute between the stationary phase and the mobile phase. The capacity factor k’ is related to K by
where Vm is the total volume of solvent within the HPLC column and V, is the volume of the interphase created by the immobilized phospholipids in the mixed-ligand IAMs. The phase ratio Vs/ Vmis a constant for a given IAM column, and therefore, k’ is directly proportional to K. The membrane binding partition coefficient for drugs to liposome membranes is defined as K, and Km reflects the affinity of the drug for the membrane.24 It should be noted that the K in eq 2 is equivalent to the Km’measured with liposome partitioning systems. However, K was measured on a system containing a monolayer of immobilized lipids,whereas Km’ was measured on a system containing a bilayer of fluid lipids. The Km’ values of the a-adrenoceptor agonists were greater for negatively charged liposomes compared to neutral liposomes because of attractive electrostatic interactions between the cationic imidazoline group in the a-adrenoceptor agonists and the anionic surface of the negatively charged liposomes (Table 4). The k’ values of the a-adrenoceptor agonists measured on mixed-ligand IAMs also gave the same result; i.e., k’ of each a-adrenoceptor agonist was greater on the negatively charged mixed-ligand columns than K on the
Table 4. Capaclty Factor (k’) Measured with IAM HPLC and Apparent Partltlon Coefflcknts of aAdrenoceptor Agonlrtr In Liposome/ Buffer Systems
Km‘
capacity factor (k’)
drug
structure
oxymetazoline
negatively charged liposomes neutral IAMs’ negatively charged IAMs neutral liposome PC PC/PE PC/PS PC/PA PC/PG DMPC (3.5:l) 25.86
29.55
31.80
35.00
32.21
87.10
870.96
316.23
xylometazoline
26.86
28.95
28.71
30.56
28.52
87.10
630.95
251.19
naphazoline
12.18
15.61
16.48
17.03
16.29
21.88
131.83
131.82
3.92
4.91
5.00
5.174
4.97
14.12
40.74
40.74
3.50
4.65
4.92
4.92
4.82
8.91
26.92
26.92
HO
CHI
H
clonidine
+=(I C
I
tetrahydrozoline
H
H
a The preparation of the neutral PC IAM surface, denoted as cthcrIAM.PCC10/C3, was described in ref 6. The K,‘values for liposomeswere obtained from Choi and Ro~ers.2~
2708
Ana&ticalChemlstty, Vol. 66, No. 17, September 1, 1994
3f .-a
30
I.I
*
ry" 20
20
CI
i
/
c)
. I
Ya
A IAM.FC/PC ~ 0 . 9 5 9
J 10 M
E n
0
200 400 600 800 Liposome Drug Partitlon CoeMcient (Km')
1
I
I
I
I
0
10
20
30
40
Liposome Drug Partition Coemcient (Km')
Figure 8. Correlatlon of IAM capacity factor k' with the drug blndlng equlllbrlum constants K,' obtained on liposome membranes. The data for the llposomes was obtained from Chol and Rogers.*' The composltlons of the neutral and negatively charged liposome membranes are given In Table 4. The composltlon of the positively charged liposome membranes was DMPC/STA 3:l mole ratlo).
neutral IAM column. The correlation of drug K values measured with neutral and negatively charged IAM surfaces and Km measured in neutral and negatively charged liposome membranes is shown in Figure 6A. As shown in Figure 6A, there is an excellent correlation for all membrane systems, which suggests specific drug-membrane binding interactions. This was confirmed by the control experiment shown in Figure 6B; a poor correlation exists when the liposome membrane has a positive surface charge and the IAM membrane has a negative or neutral surface charge. In other words, if the artificial membrane has a positive charge, then a negatively charged or neutral IAM surface can not predict drug binding. We note that the charge density of the fluid liposomal membranes in Choi and Roger's work is not identical to the charge density of the mixed-ligand IAMs prepared in this work. The fluid artificial membrane system contains a much higher charge density compared to our immobilized membranes. Consequently, when drug binding to a fluid membrane is directly dependent on the membrane surface charge, the immobilized membrane system gives a very good correlation, but the ability of IAMs to "predict" the actual membrane affinity will depend on how close the charge density of the immobilized membrane is relative to the charge density of the fluid membranes. These studies are in progress. CONCLUSIONS
We have developed a general method to immobilize two phospholipid ligands on solid surfaces to make mixed-ligand IAM surfaces. The mixed-ligand IAMs emulate the lipid environment of mixed-lipid artificial membranes. IAM HPLC is a rapid, simple method to measure the membrane partition coefficients of drugs and other solutes. ACKNOWLEDGMENT
We are very grateful for the support from Eli Lilly and Co. This work was also supported by NSF (Grant CTS 9214794), NIH (Grant AI33031), and Regis Chemical Co. (2R446M3022-02).
GLOSSARY
BW
N-tert-butoxycarbonyl tert-butyl propionyl decanoyl carbonyldiimidazole cholesterol CH 1,8-diazobicyclo[5.4.01undec-7-ene DBU DCP dicetyl phosphate Fourier transform infrared FT-IR high-performanceliquid chromatography HPLC IAM(s) immobilized artificial membrane@) IAM HPLC with a stationary phase composed of IAM packing HPLC materials IP isopropylidene HC hydrocarbon (pnitropheny1)ethyl NPE phosphatidic acid PA 0-[ 1 -0( 1 1 -carboxy)undecyl-2-Omethyl-sn-glycero-3PAP phosphoryl] @-nitropheny1)ethanol phosphate-bufferedsaline PBS PC phosphatidylcholine 1-0-( 1 1 -carboxyl)undecyl-2-O-methyl-sn-3-glycerophosPCCOOH phocholine PE phosphatidylethanoamine 0-[ 1-0-(1l-carboxy)undecyl-2-Omethyl-sn-glycero-3PEP phosphoryl]-N-(tert-butoxycarbony1)ethanolamine phosphatidylglycerol PG 0-[ 1 -0( 1 1-carboxy)undecyl-2-Omethyl-sn-glycero-3PGP phosphoryl]-2',3'-isopropylidene-sn-glycerol phospholipid(s) PLb) protected secondary immobilized phospholipid PLP PS phosphatidylserine 0-[ 1-0-(1l-carboxy)undecyl-2-Omethyl-sn-glycero-3PSP phosphoryl]-N-(rert-butoxycarbonyl)serine tert-butyl ester SPA silica propylamine stearylamine STA t-Bu c3 c10 CDI
Recelved for review April 4, 1994. Accepted May 17, 1994.' .Abstract published in Aduance ACS Abstracts, July 1, 1994.
Analytical Chemlstty, Vol. 66, No. 17, September 1, 1994
2709
