Anal. Chem. 1994,66, 782-792
Phospholipid Immobilization on Solid Surfaces Shaowei Ong, SongJun Cal, Candldo Bernai, Dongml Rhee, Xlaoxlng Qiu, and Charles Pldgeon' Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Purdue University, West Lafayette, Indiana 47907
Single chain ether phospholipids (PLs) containing w-carboxyl groups in the alkyl chain were immobilized on silica propylamine (SPA) to form IAM chromatography packing material. The PL ligands are analogs of phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA). All of these PLs contain polar functionalgroups in the lipid head group that require protection prior to PL immobilization and then deprotection after immobilization. The IAM surface was prepared in four steps: (i) the w-carboxyl group was activated with carbonyldiimidazole, (ii) the activated PL-imidazolide ligand was bonded to SPA, (iii) the surface was end capped with a long chain anhydride and then end-capped with a short chain anhydride, and (iv) protecting groups were removed to form the IAM surface. The extent of deblocking the protecting groups was typically 290%. This immobilization strategy generated a phospholipid surface that was stable when solvated with all organic solvents and aqueous buffers between pH 2 and 8. Both FT-IR spectroscopy and elemental analysis indicated that the bonding densities were 64-83 mg of PL/g of SPA, which corresponds to an area per molecule of 66-104 A2. These bonding densities for the immobilized PLS are very close to the area per molecule of mobile phospholipidscomprising liposome membranes. The similar areas per molecule of immobilized PLs and mobile phospholipid in liposomes indicate that the lipid environments are similar. Immobilized artificial membranes (IAMs) are solid-phase membrane mimetics whereby cell membrane phospholipid molecules are covalently bonded to silica particles at high molecular surface During the last few years, IAM chromatography particles have been prepared from double chain PC (phosphatidylcholine) IAM surfaces have been used to purify membrane predict the transport of solutes across human skin8 and other biological barriers? determine binding constants of biomolecules to US. Pat. 4, 931, 498, 1990. (2) Pidgeon, C. Method for Solid Membrane Mimetics. US.Pat. 4, 927, 879, (1) Pidgeon, C. Immobilized Artificial Membrane. 1990.
(3) Pidgeon, C.; Venkatarum, U. V. Anal. Biochem. 1989, 176, 36-47. (4) Otto, S.;Marcus, C.; Pidgeon, C.; Jcfcoate, C. Endocrinology 1991, 129,
970-982.
S.; Bhattacharyya, K. K.; Jcfcoate, C. R. Polycyclic Aromatic Hydrocarbon Metabolism in Rat Adrenal, Ovary, and Tatis Microsoma is Catalyzed by the Same Novel Cytochrome P450 (P45ORAP). Enzymology, in press. (6) Pidgeon, C.; Stevens, J.; Otto, S.; Jcfcoate, C.; Marcus, C. Anal. Biochem. 1991,194, 163-173. (7) Thurnhofer, H.; Schnabcl, J.; Betz, M.; Lipka, G., Pidgeon, C.; Hauser, H. Biochim. Biophys. Acta 1991, 1064, 275-286. (8) Alvarez, F. M.; Bottom, C. B.; Chickale, P.; Pidgeon, C. In Molecular Interactions in Biosepararions;Ngo,T., Ed.; Plenum Publishing Corp.: New York (in press). (9) Pidgcon, C.; Marcus, C.; Alvarez, F. M. In Applications of Enzyme Biofechnology; Kelly, J. W., Baldwin, T. O., Eds.; Plenum Prcss: New York, 1991; pp 201-220.
Chart 1. GewaI Struetwos d PL Ligand# Contalnlng Protoctlng Group8 In the Polar Hoed Oroup Rogkna 0 II 'RP PLCOOH
'iH2-O--co-Rp
CyOCH
I
-CHz-qH-?Hz 0 0
PG(IP)cooH
x
CHZ
I
CHzCH-COOC(CH3)3
I
PS(BOC)(~BU)COOH
NH-COOC(CH&
-CH,CH,-NHCOOC(CH,),
C d'bH
-CH,C Hz
PE(BOC)CCOH
PA(NPE)cooH
is the protectedfunctbnalgroup. The acronymfor each protecting group Is glven In parenthesis, Le., lsopropylldene(IP), n-butybxycarbonyl (Boc), tert-butyl (t-Bu), and @.nltrophenyl)ethyl(NE).
phospholipid membranes,l0J synthesize phospholipids,l2 predict pathophysiological effects of bile salts,13 and stabilize functional enzymes immobilized on the IAM s ~ r f a c e . 1 ~ 1 ~ Prior to the present work, only PC analogs have been immobilized. Although PC is the major phospholipid component found in virtually all cell membranes, PG, PS, PE, and PA are also important membrane constituents but they are present at much lower membrane surface densities compared to PC. In contrast to PC ligands, PE, PG, PS, and PA ligands contain functional groups (i.e., amines, carboxyls, hydroxyls, or phosphoryl groups) that must be protected during the PL immobilizationprocess. Protectionof thesegroups is necessary to eliminate the possibility of intermolecular bonding between PLs during the immobilization process and also to assure that the PLs bond to SPA with the polar head groups protruding away from the surface. Chart 1 shows the PL ligands and the blocking (or protecting) groups associated with each different lipid head group.
( 5 ) Otto,
782
Analytical Chemistty, Voi. 66, No. 6, March 15, 1994
(10) Chae, W. G.; Luo, C.; Rhce, D. M.; Lombardo, C. R., Low, P., Pidgeon, C. In Modern Phytochemical Methods; Fischer, N.H.; Isman, M. B.; Stafford, H. A., Eds.; Plenum Prcss: New York, 1991; Chapter 5 . (1 1) Markovich,R. J.;Qiu,X.;Invergo,B.;Nichols,D. E.,Alvarcz, F.A.;Pidgeon, C. Anal. Chem. 1991, 63, 1851-1860. (12) Pidgeon,C.;Markovich,R. J.;Liu,M.D.;Holzer,T. J.;Novak,R. M.;Keyer, K.A. J. Biol. Chem. 1993, 268,1173-1778. (13) Cohen, D. E.; Leonard, M. R.; Leonard, A. N.; Donovan; Carey, M. C. Gastroenferology 1993, 104, A889. (14) Chui, W. K.; Wainer, I. W. Anal. Biochrm. 1992, 201, 237-245. (15) Kolbah, T. A.; Felix, G.; Wainer, I. W. Chromatographia 1993,35,264-268. (16) Zhang, X-M.; Wainer, I. W . Tetrahedron Left. 1993, 34, 4731-4734. (17) Kolbah, T. A.; Wainer, 1. W. J . Chromatography 1993, 646, 289.
0003-2700/94/0360-07%2$04.60/0
0 1994 Amerlcan Chemkal Soclety
Silica surfacesare very stable at low pH, and the protecting groups shown in Chart 1 were chosen because they can be removed either under acidic conditions (PE, PS, and PG analogs) or by using nonnucleophilic bases &e., for the PA analog). In addition to designing acid labile protecting groups for the reactive functional groups in the polar head group, the molecular design criteria for the PL ligands included (i) an w-carboxyl group for immobilization, (ii) a single alkyl chain to assure high bonding density, and (iii) an ether linkage between the alkyl chain and the glycerol backbone to increase the ligand stability both during synthesis and after immobilization. Although these PL ligands are designed for high ligand density after bonding to SPA, they are also suitable for bonding to many other chromatographic surfaces including soft gels and synthetic polymers. Furthermore, we anticipate that these ligands will find many nonchromatographic applications.'*
EXPERIMENTAL SECTION Chemicals and Reagents. The following chemicals were purchased from Aldrich: ethanol-free CHCl3 (in a Sure-Seal bottle) used for CDI activation of the PLs, 1,l'-carbonyldiimidazole (CDI), dimethyl sulfoxide (DMSO, in a Sure-Seal bottle), decanoic anhydride, propionic anhydride, 1,8diazobicyclo[5.4.0]undec-7-ene (DBU), and IRC-50 (weak cation exchange resin). Ninhydrin and Phospray were purchased from Supelco. SPA (12 pm, 300 %L2) was obtained from Regis Chemical Co. Immediately prior to PL bonding, the SPA was degassed by preparing a CHC13 suspension and sonicating (- 15-30 s) followed by rotoevaporation ( 5 min). A bath sonicator (Branson 2200) was used for this procedure. IAM.PCC10/C3used for all solid-phase adsorption syntheses was purchased from Regis Chemical Co. Prior to use, the IAM.PCC10/C3powder was washed with 30 mL of acetone. The synthesis of the w-carboxyl PL ligands shown in Chart 1 was described in detail e1~ewhere.l~ The w-carboxyl PLs that were bonded to SPA include PG(IP)COOH, PS(Boc)(t-Bu)COOH, PE(Boc)COOH, and PA(NPE)COOH. FTIR spectroscopy was used to verify that the o-carboxyl group of each PL ligand was in the non ionized form prior to activation with CDI. If necessary, the carboxyl PLs can be passed through a weak cation exchange column to protonate the ionized carboxyl. Strong cation exchange resins cannot be used because they cause deprotection of the blocking groups (in particular IP). Elemental Analysis. Elemental analysis was performed on a Perkin-Elmer PE 240 in the Microanalytical Laboratory at the Purdue University Chemistry Department using approximately 10-15 mg of IAMs. Surface Area Measurements. A nitrogen adsorption/ desorption isotherm of SPA was obtained on Quantachrome Autosorb-1 with out-gassed samples at 298 K for 24 h. The total surfacearea was calculated to be 107 m2/g SPA according to the Brunauer-Emmett-Teller (BET) method.20 (18) Gabcr, B. P.; Peek,B. M.;Turner, D. C.; Brandow, S.L.; Leach-Scampvia, D. Formation and enzymatic modification of a silicon-bound lipid monolayer. AbsrracfsofPaprs,2OthNational Mcetingofthe AmericanChmicaI Society, Chicago, IL, Aug 22-27, 1993; American Chemical Society: Washington, DC, 1993; Poly 0203. (19) Qiu, X.;Ong, S.;Bernal, C.; Rhce, D.; Pidgeon, C. J. Org. Chcm. 1994.59, 537-543. (20) Brunauer, S.;Emmett, P. H.; Teller, E. J. Am. Chem.Soc. 1938,60,309-319.
Phospray and Ninhydrin Analysis of IAM Powder. IAM powder (- 1-2 mg) was placed in a test tube (13 mm X 100 mm), and then 2-3 drops of ninhydrin or Phospray wereadded. 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 pink" when unreacted surface amines on SPA are available to solvent penetratingthe IAM interfacial region (see footnoted in Table 2). IAM powder that remains white over 12 h is considered to be ninhydrin negative. All white IAM powders became bright blue after exposure to Phospray. FT-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 described in detail previously.l1P2l 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. FT-IR spectra of the IAM wafers were taken at a resolution of 4 cm-l using 256 scans. Except for the spectrum of the nonbonded PLs and SPA, all infrared spectra are difference spectra (Le., the SPA spectrum has been subtracted from the FT-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 infrared spectra of all soluble ligands and reagents (e.g., CDI) were obtained by depositing the compounds onto CaF2 plates. The FT-IR transmission spectra of the PL ligands and reagents were obtained from 32 scans at a resolution of 4 cm-1. To quantitate hydrocarbon content, the integrated intensity of the hydrocarbon (HC) stretch region was divided by the integrated intensity between 1945 and 1780 cm-l, which corresponds to the Si-0 combination band. The HC integration limits used for each IAM surfaces areas follows: 30152825 cm-l for eWAM.PGC10/C3 and e*erIAM.PEC10/C3;29952825 cm-l for etherIAM.PAC10/C3; 3020-2825 cm-l for ahaIAM.PSC10/C3. Integrated band intensitieswere performed in absorbance units and measured using Nicolet Omnic software. This method of quantitating the hydrocarbon content of IAMs is similar to that of our earlier work.21 However, it should be noted that elemental analysis measures carbon content, whereas FT-IR assay measures hydrocarbon content. For instance, the quaternary carbon of the tertbutyl group of ethCrIAM.PS(Boc)(t-Bu)c10~c3 can be detected by elemental analysis, but this is not reflected in the IR integrated HC band. Therefore, a correction was made according to the following equation: normalized IR HC area = r(HC are4Si-O area)
(1)
The correction factor y is defined as the total number of carbons of the PL divided by the number of aliphatic hydrocarbon carbons of the PL. For instance, y is 22/20 for etherIAM.PG(IP),23/20 for etherIAM.PE(Boc),28/23 for etherIAM.PS(Boc)(t-Bu),and 23/18 for e'hwIAM.PA(NPE). 'H NMR Spectroscopy. A lH 500-MHz NMR spectrometer (Varian VXR 500) was used for quantitating the CDI activation of the PL carboxyl ligands at 25 OC. The CDI (21) Markovich, R. J.; Stevens, J. M.;Pidgcon, C. AMI. Biochrm. 1989, 182, 237-244.
Amlyricel Chemlsby, Vol. 88, No. 8, March 15, 1994
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Scheme 1. Bonding Stratogy Used To Prepare IAMs from PLCOOH Ligands' 0 DMSO N A ~ - ~ - ~ or, IAM.PCC'o'C3 A N PLCOOH+
-
4
Step (ii)
0 yH,.O(CHZ)llC-N A N
Step (i)
4 H,N-OSi (SPA)
CH,OCH
1 I
9 I1
CHCl3 or DMSO
C H, -0PO-R,
CDI
I.
I 0PL-imidazolide
0
II
CH, -O(CH,)llC-NH-OSi
II
CH-OCH
.
. 3 1C H2 -0PO-Rp 7 I
I 2
0
Step (iii) Endcapping Step (iV) Deprotection
II CH2 -O(CH2)llC-NH-OSi
I
DCH307H P
C H, -0PO-Rd
PLCOOH ligands are shown in Chart 1. R, is the deblocked functionalgroup. CDI activation of each PLCOOH llgand (step i) was performed In solution either using DMSO or using IAM.PCC1o/aparticles to facilitate the reaction (see the solid-phase adsorption synthesis method in the text). Bondlng of the PL-imidazolides (step Ii) was performedfor 24-48 h. Step iil was end-capping with first decanoic anhydride and then propionic anhydride. Deblocking of the lipid protecting groups (step iv) was usually performed either in aqueous acidlc conditions or under mildly basic Conditions.
activation of the PL carboxyl group at 25 OC was monitored by the disappearance of CDI using the imidazole 'H resonances, as described in the Results and Discussion. The rate of reaction between the PL carboxyl ligand and CDI was monitored under the same conditions used for the solid-phase adsorption synthesis described below. Briefly, -20 mg of PLs dissolved in methanol were loaded on -50 mg of IAM.PCC10/C3powder. After removal of methanol, the powder was suspended in -0.5 mL of CDC13in a 5-mm NMR tube. lH NMR spectra were recorded as a function of time after the addition of 1.5 equiv of CDI to the NMR tube. General Bonding Strategy. The o-carboxyl group of each PL must be activated to facilitate the amidation reaction between the PL o-carboxyl and the surface amine of SPA (step i in Scheme 1). CDI activation of the PL o-carboxyl group was facile using the IAM solid-phase adsorption synthesis method. Following activation (step i in Scheme l), the PL-imidazolides were bonded to SPA (step ii in Scheme 1). The expensive synthetic PL carboxyl ligands were recovered and reused after each reaction. After end-capping the residual amines with decanoic (C10) and propionic (C3) anhydrides (step iii in Scheme 1),l1 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 of etherIAM.PGC10/C3, etherIAM,pSClO/C3,etherIAM.pEClO/C3,and etherIAM.pAClO/C3 are given below. Synthesis of etherIAM.PGC1o/a.IR analysis (not shown) demonstrated that during the purification of the PG(1P)COOH ligand, thecarboxyl group was quantitatively ionized." Thus the PG carboxyl ligand, Le., PG(1P)COO-, was converted into PG(1P)COOH by ion exchange chromatography using a weak cation exchange column. The mobile phase was 90% 704
Analytical Chemistty, Vol. 66,No. 6, March 15, 1994
CH3OH/water (v/v) and the protonated PG(1P)COOH ligand was obtained from pooling the fractions eluting from the column and drying by rotoevaporation and then high vacuum at 45 "C. The PG(1P)COOH free acid was bonded to SPA using CDI as the coupling reagent. PG(1P)COOH (4.76 g, 0.009 58 mol) and CDI (1.89 g, 0.01 1 67 mol) were dissolved in 110 mL of DMSO/CHC13 60/50 (v/v) and stirred under nitrogen at 25 "C for 10 h. After 10 h, CDI was totally consumed and approximately 60% of the PG(1P)COOH starting material was converted into the desired imidazolide. The reaction mixture was transferred under a nitrogen atmosphere, via cannula, to a 1000-mL round bottom (rb) flask containing 30 g of SPA suspended in 100 mL of dry CHCl3. The reaction was gently shaken on an orbit shaker for 36 h. The IAM CHC13 suspension was then filtered through a fine 300-mL sintered glass suction filter and washed with 300 mL each of CHCl3, methanol, and acetone. This IAM powder is denoted as etherIAM.PG(IP)and has not been end-capped with alkyl anhydrides. etherIAM.PG(IP)was end-capped with C10 and C3 anhydrides exactly as described previously.ll Typically, 1 g of anhydride is reacted with 10 g of IAM powder containing the immobilized phospholipid. The end-capped IAM material is denoted as ethcrIAM.PG(IP)C10/C3 and was washed with 300 mL of CHC13, methanol, and acetone. The IP group that protected the PG head groupglycerol moiety during the above bonding reactions was removed by adding 100 mL of 1 N HCI and 100 mL of THF.22 Deprotection was complete in 2 h at 25 "C. After the reaction was complete (based on FT-IR spectra; see Results and Discussion), the powder was washed (22) Green, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; John Wiley & Sons: New York, 1991.
with distilled water (- 300 mL) until the pH of the filtrate was neutral. Additional washes included 300 mL each of CH30H,THF, and acetone. The final IAM packing material was completely dried of residual solvents by high vacuum at 45 OC for 18-24 h. After concentration of the wash filtrates, thin layer chromatography (TLC) using Phospray for phospholipid detection demonstrated that no phospholipid leached during either the C10 and C3 end-capping reactions or the deprotection of the IP group. Synthesis of *rIAM.PSc10/c3. PS(Boc)(t-Bu)COOH was activated by CDI via solid-phase adsorption synthesis as follows. Dry IAM.PCC10/C3 powder (10 g) was placed in a fine 30-mL sintered glass suction filter and washed with acetone. A methanol solution (3 mL) of PS(Boc)(t-Bu)COOH (2.87 g, 0.00458 Mole) was slowly loaded into the center of the IAM.PCC10/C3 powder over 2 h. It is critical not to let the PL methanol solution drip through the powder or touch the sidesof the glass filtration funnel. If the PL methanol solution solvates the glass funnel and evaporates, the PL compounds adsorb to the sides of the glass funnel and are not available for activation by CDI. The IAM.PCC10/C3 powder, containing PS(Boc)(t-Bu)COOH adsorbed to the surface, was then transferred to a 500-mL rb flask and pumped in high vacuum at 45 OC overnight to remove residual CH3OH. This material was suspended in 100 mL of CHC13 containing CDI (1.25 g, 0.007 71 mol); the reaction was shaken at 25 OC for 5 h. From both FT-IR and NMR analysis, CDI was totally consumed after 5 h, and approximately 60% of the PS(Boc)(t-Bu)COOH starting material was converted into the desired PS-imidazolide product. PS-imidazolide was then coupled to 18 g of SPA, followed by C10 and C3 end-cappings using the same reaction conditions as described above for ctherIAM.PGC10/C3. After end-capping, the IAM phase is denoted as etherIAM .PS(Boc) (~ - B U ) ~ ~ O / ~ ~ . Both the Boc and 2-Bu protecting groups were simultaneously deblocked from etherIAM.PS(Boc)(t-Bu)c10~c3 using 4 mL of concentrated HC1 (37% wt) and 100 mL of ethyl acetate (at 25 OC, 2 h).22 The workup procedure after deblocking was similar to that used during the preparation of etherIAM.PGC10/C3.The final IAM powder containing immobilized PS is denoted as ethcrIAM.PSC10/C3. Synthesis of e*IAM.PEC10/C3. PE(Boc)COOH (4.0 g, 0.0076 mol) was (i) activated by CDI (1.47 g, 0.009 12 mol) using the solid-phase adsorption synthesis, (ii) coupled to 25 g of SPA, and (iii) end-capped with C10 and C3 anhydrides as described above for etherIAM.PSC10/C3. The Boc group of C~herIAM.PE(Boc)C10~C3 was deprotected using 8 mL of concentrated HC1(37% wt) and 92 mL of ethyl acetate (25 OC, 30 min). The workup procedure after deblocking was similar to t h a t used during the preparation of ctherIAM.pGC1O/C3, Synthesis of etherIAM.PAC10/C3. PA(NPE)COOH (3.55 g, 0.006 66 mol) was (i) activated with CDI (1.6 g, 0.010 mol) using the solid-phase adsorption synthesis, (ii) coupled to 30 g of SPA, and (iii) end-capped with C10 and C3 anhydrides as described above for the preparation of cthcrIAM.PSC10/C3. This IAM material, denoted as etherIAM.PA(NPE)C10/C3, was treated with a nonnucleophilic base (DBU) to remove the NPE group and generate a free
phosphoric acid on the IAM surface.23 Prior to the removal of NPE, 20 g of ethCrIAM.PA(NPE)C10/C3 was dried in a high vacuum at 45 OC for 24 h to completely remove residual water. To the dried CthCrIAM.PA(NPE)C10/C3 particles, 40 g of DBU and 40 mL of anhydrous CHCl3 were added. The reaction suspension was stirred at 25 "Cunder positive N2 pressure for 5 h. The ethcrIAM.PAC10/C3 powder was then washed with 500 mL each of acetonitrile, THF, CH3OH, and acetone. DBU hasvery high affinity for the cthcrIAM.PAC10/C3 surfaces and it is difficult to remove DBU after NPE deprotection. Most likely, the high affinity, noncovalent adsorption of DBU onto the etherIAM.PAC10/C3 surface is due to the electrostatic interactions between the DBU and the deblocked PA head group. However, DBU was removed from the ethcrIAM.PAC10/C3 powder by washing with 800 mL of 2 M NaCl solution followed by 500 mL of water and then 500 mL of acetone. The etherIAM.PAC10/C3 powder was dried by a high vacuum pump at 45 OC overnight.
RESULTS AND DISCUSSION Although activation of carboxyls to imidazolides with CDI is typically a facile reaction, the w-carboxyls of the single chain ether PLs shown in Chart 1 were difficult to convert into imidazolides using CDI. Two factors contributed to the poor reactivity of the PLs toward CDI: (i) after purification, the w-carboxyl was deprotonated (typically >50% carboxylate form),18 and (ii) the ether PLs are insoluble in many aprotic organic solvents. Problem i was solved by converting the carboxylate ion into the free acid using ion exchange chromarography on a weak cation exchange resin. However, we note that many of the PL ligands shown in Chart 1contain acid labile protecting groups that may be removed even under mildly acidic conditions of cation exchange resins. For instance, we found that the IP protecting group is unstable when PG(1P)COO- was chromatographed on a strong cation exchange resin that is in the protonated form. After ion exchange chromatography, FT-IR spectroscopy was used to confirm that the o-carboxyl was the protonated PLCOOH species by the IR absorption band centered at 1710 cm-l; the deprotonated PLCOO- species absorbs at 1550 cm-l. Conversion of the o-carboxylate into a free acid did not solve problem ii; the PLs in the free acid form are poorly soluble in CHC13, THF, and several other aprotic organic solvents. The PLs shown in Chart 1 are very soluble in alcohols and DMSO. Although DMSO can be used as a solvent for activation of PLs with CDI, a better alternative is to use the solid-phase adsorption synthesisdeveloped in our laboratory. l 2 The solid-phase adsorption synthesis involves dissolving the PLs in a minimal volume of CH3OH and dripping the CH3O H solution onto IAM particles; inexpensive and chemically neutral IAM particles like IAM.PCC10/C3 should be used. As much as 0.5 g of reactants (i.e,, PLs) can be loaded per gram of IAM.PCC10/C3 powder. Evaporation of the CH3OH causes the PL ligand to benoncovalently adsorbed to the IAM packing material, and when this is suspended in a CHCl3 solution of CDI, rapid formation of the PL-imidazolide occurs. After each solid-phase adsorption synthesis, the IAM.PCC'o/C3 powder can be reused by washing out the unreacted reactants
--
~~
~~~
~
(23) Uhlmann, E.; Pfleidcrer, W. Tetrahedron Left. 1980, 21, 1181-1184.
Analytlcal Chemlstty, Vol. 66,No. 6,March 15, 1994
785
(Le., PLs) or any other reagents adsorbedon the IAM.PCC10/C3 surfaces with organic solvents such as CH30H, CHCl3, and acetone. Because the IAM.PCC10/C3 powder is very stable in organic solvents,t1J4it can be reused many times for solid phase adsorption synthesis. Two advantages of using the solid-phase adsorption synthesis method for forming PL-imidazolides were immediately apparent. (1) Dispersing the PLs on IAM particles facilitatesdrying of the oily viscous PL ligands prior to reacting with CDI. (2) The unreacted carboxyl PLs are expensive synthetic intermediates that can be conveniently recovered from the solid-phasematerial by decanting thereaction solvent followed by washing with CHCl3 and methanol. Imidazolides and other impuritiesare soluble in the CHC13 filtrate, whereas unreacted PLs are recovered in the MeOH filtrate (>go% purity). These experimental advantages do not exist when the CDI activation is performed in DMSO. The syntheticPLs are expensiveand therefore it is desirable to efficiently bond them to SPA. An excess of CDI may be used to quantitatively activate the o-carboxyl groups, but unreacted CDI will couple to the amines of the SPA. The undesired side reaction of CDI and the surface amines comprising SPA is very rapid (C. Pidgeon, unpublished). Because surface amines rapidly reacted with CDI, we are very careful to assure that unreacted CDI is never present when the SPA is added to the PL-imidazolide solutions. TLC cannot be used to monitor unreacted CDI because CDI itself is hydrolyzed in the TLC solvent system (e.g., CHC13/ methanol/HzO 65/25/4). Thus FT-IR and NMR spectroscopy were used to evaluate the rate of CDI activation and the disappearance of CDI from the reaction mixture. Figure 1 shows that CDI has a strong IR band at 1770 cm-I, and the complete disappearance of this peak is direct evidence that no detectable CDI remains in the reaction mixture. The data in Figure 1 were routinely obtained for all CDI activation reactions with each different PL carboxyl ligand. All lipid bonding conditions were performed after it was confirmed by IR spectroscopy that no detectable CDI remained in the reaction mixture. IR spectroscopy is convenient for confirming the end of the CDI reaction, but it is very difficult to use IR spectroscopy to evaluate the kinetics of the CDI activation. The time course for the CDI activation was monitored by NMR spectroscopy. Typical NMR spectra are shown in Figure 2A for the CDI activation of PA(NPE)COOH. The formation of the PL imidazolide is apparent by the increase in peak d of Figure 2A. Thus, NMR was a convenient method to quantitate the kinetics of the CDI reaction, as discussed in the figure legend of Figure 2A. Figure 2B shows the time course from the activation of the four PL carboxyl ligands. Thedisappearance of CDI from the reaction mixture is log linear and the halflife (Tip) of CDI is 40 min for the activation of PA(NPE)COOH, 50 min for the activation of PE(Boc)COOH, 180 min for the activation of PS(Boc)(t-Bu)COOH, and 300 min for the activation of PG(1P)COOH (Figure 2B). After CDI was completely consumed, on the basis of FT-IR and NMR data, the PL-imidazolides were bonded to SPA. Typically, CDI activation of the PLCOOH ligands resulted in 90% (24) Qiu, X.; Pidgeon, C. J. Phys. Chem. 1993, 97, 12399-12407.
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AnaWcal Chembtry, Vol. 60, No. 0, March 15, 1994
9
?
A
A2000
1900
1800
1700
1600
Wavenumber (cm-' )
Flgure 1. Infrared spectra demonstrating that CDI Is completely consumed dwing the actlvatbn of PLCOOH to the PL-lmidazolide. Flgure 1A shows the strong 1770.0-cm-I peak of neat CDI, and Fbure l B shows that a new peak at 1740.0 cm-' exists which corresponds to the stretch'of PL-lmldazollde. Complete dlsappearanceof the CDI peak at 1770 cm-I was used to confirm that COI was completely consumed during the CDI acthratbn reaction.
conversion into the PL-imidazolide for the PE and PA carboxyl analogs, however only 60% formation of PL-imidazolide for the PG and PS analogs. For unknown reasons, the PG and PS carboxyl ligands formed carboxylate ions during the CDI reaction, and this is the reason for the lower yields. Unlike the CDI activation reaction, the PL ligand bonding to SPA cannot be monitored by NMR. However, PLimidazolide bonding to SPA can be convenientlyevaluated by FT-IR. Figure 3 demonstrates how FT-IR spectra were used to evaluate the IAM surface after each lipid bonding reaction. The nonbonded PG(1P)COOH ligand has a strong band at 1710 cm-1 corresponding to the protonated carboxyl group, and no COO- bands near 1550 cm-1 exist indicating that the carboxyl group is completed protonated. After bonding of PG(1P)-imidazolide to SPA, the spectrum of cthcrIAMPG(IP) shows the typical amide I (1640 cm-l) and amide I1 (1550 cm-l) bands, as well as a large increase in the hydrocarbon stretching bands found in the region between 3015 and 2825 cm-l (Figure 3). The amide I and I1 bands confirm the covalent linkage between the PG ligand and the SPA surface amines. As expected after bonding the PG ligand, the protonated carboxyl band centered at 1710 cm-l is not present (compare the free PG(1P)COOH ligand to the immobilized PG(1P) ligand in Figure 3).
B
A
CDI
PL-lmklazollde
2.2
51 1.2 1 74
h
*
b
1
1
0.8 -50
d
0
50
100 150 200 250 300
Time (min)
140 82
80
78
76
7 4 ppm
Flgurr 2. Time course for the reaction of carboxyl PL llgands with CDI. CDI ie present In 50% molar excess but Is completely consumed dwlng the reaction. Figure 2A shows typlcai NMR spectra obtained for the coupling between PA(NP€)COOH and CDI. CDI has 'H NMR resonancesat 8.17 ppm (peak a) and 7.50 ppm (peak c). The desked product, PA-lmldazolkle, has resonances at -8.14 ppm (peak b) end 7.45 ppm (peak d). Figure 28 shows the time dependent change In the CDI concentratlon as the reaction proceeds. The percentage of CDI left was calculated from the Integrated Intensity of the CDI peak c (7.50 ppm) dhrided by the sum of the Integrated intenstty of peak c and peak d (-7.45 ppm) that corresponds to CDI and the PL-imidazoilde, respectively.
The IR spectra obtained after the bonding reactions for preparing ethcrIAM.PSC10/C3 (Figure 4), cthcrIAM.PE10/C3 (Figure 5 ) , and cthcrIAM.PAClo/C3 (Figure 6) show not only the characteristic IR absorption bands for each immobilized lipid but also strong amide I (near 1640 cm-l) and amide I1 (near -1550 cm-l) bands which confirm that covalent bonding of the PLs to the SPA have occurred. Deprotection Reactions. This is the first report demonstrating that phospholipids with reactive functional groups in the polar head group region can be immobilized. The key concept is that efficient deblocking reactions are needed after the immobilization process to convert R, to Rd and to generate the desired immobilized lipid functional groups on the IAM surface. It is critical that the lipid R, protecting groups shown in Chart 1 remain intact during the initial PLCOOH immobilization. If the protecting groups are lost during the immobilization process (step ii in Scheme l), then the PLCOOH ligands may piggy back from the SPA surface and/or the alkyl end-capping reagents may piggy back from the deprotected PLs immobilized on the silica surface. The delicate surface chemistry of protecting the polar head groups until the final IAM structure is prepared was evaluated by IR spectroscopy. Table 1 shows the IR bands used to quantitate deprotection reactions. For instance, for the etherIAM.PGC10/C3 surface, the characteristic gem dimethyl bands (1380.0 cm-1, 1371.1 cm-l) of the IP blocking group were used to confirm that, during both PL bonding to SPA and end-capping with
-
alkyl anhydrides, the PG head group remained with an intact IP group. Thus, the intensity of the gem dimethyl bands did not change after end-capping (Figure 3), which confirmed the stability of the IP protecting group to the end-capping reaction conditions. However, after removal of the IP group to form the PG head group, the gem dimethyl groups were virtually completely removed (Figure 3). On the basis of the IR spectra in Figure 3, greater than 90%of the PG polar head groups were generated on the IAM surface after the IP d e p r o t e c t i o n r e a c t i o n . T h e s t a b i l i t y of t h e cthcrIAM.PG(IP)C10/C3 surface to acidic conditions should be noted. Deprotection of the IP group was performed under strongly acidic conditions (aqueous 1.0 N HCl in THF for 2 h), and no phospholipid leaching from the surface occurred during this reaction. The above analysis of the deprotection reaction for preparing the ctherIAM.PGC10/C3 surface was also performed for each other IAM surface. The relevant information concerning the IR bands used to follow the deprotection reactions is given in the figure legends (Figures 3-6). Unlike all other IAM surfaces, the lipid head groups of the CtherIAM.PA(NPE)C10/C3 surface were deblocked by a 0eliminationmechanism using DBU as a nonnucleophilicbasee23 DBU efficiently removes NPE from ctherIAM.PA(NPE)C1o/C surfaces but anhydrous conditions are critical for the deprotection reaction. Consequently the C*mIAM,PA(NPE)C10/C3 surfaces were dried in high vacuum at 45 OC for 24 h to completely remove residual water before deprotection with AnalyHcal Chemlstty, Vol. 00, No. 0, M r c h 15, 1994
707
After deprotection
-
Before deprotection
1420 1400 1380 1380 1340
Wavenumber (cm')
PO(I P)COOH
SPA
3500
3000
2500
2000
1500
Wavenumber (cm") Flguro 3. FT-IR spectra of SPA, W ligands, and 1AM.W. The spectra of *IAM.W(IP) and *IAM.Wc'o'cS are difference spectra (Le., the SPA spectrum has been subtracted from the FT-IR spectra of *IAM.WIP) and *IAM.WC1o/CS). The characteristicabsorptions of IP groups ) the doublet at 1380 and 1371.1 cm-l completely dlsappeared In the IR spectrum of *IAM.W~"J'~S, in whlch at 2985 cm-1 ( u ~and the small residual peak at 1371 cm-l Is the symmetric bending vibration from methyl groups of ImmoMWzed C10 OT C3 alkyl groups. The inserts at the rlght show the intensity of the gem dimethyl doublet at 1380 and 1371.1 cm-' before and after doprotection.
(am-)
After deprotection
1 Before deprotectlon
3500
3000
2500
2000
1500
Wavenumber (cm" )
Ft@wo4. FT-IR spectraof PS(eoCKf-BupOOH,*IAM.PS(eoCKt-Bu), and*IAM.PSC1OKJ. Thespectreof *IAM.PS(BOcKf&r)and*IAM.PSC'O/oS are difference spectra (la., the SPA spectra has b w n subtracted from the FT-IR spectrum of *IAM.PS(Boc)(t-Bu) and *IAM.PSclO/@).
The
characteristic absorptions of the t a u protecting groups at 2983.2 cm-l and the doublet at 1398.0 and 1373.2 om-', as wen as the strong ester band centwedat 1720 cm-' are absent in the IR spectrum of -IAM.PSC1o'Q, in which the rman reolducll peak at 1373.2 cm-' is the symmetric bending vibratlon from methyl group of immobilized C10 and C3 alkyl groups. The spectrum of *IAM.PSclo'cS also shows a new IR absorption centered 1750 cm-', which le the G-0 stretching band of the free carboxyl of the PS head group. The extent of doprotodon was monitored using the absorption of the t a u groups at 1380 and 1371.1 cm-? before and after deprotectlon.
-
DBU. Excess DBU was used to scavenge protons that may be generated from the CthCrIAM.PA(NPE)C'o/C3 surfaces duringdeprotection. Approximately90%oftheNPE blocking groups were removed from the ctherIAM.PA(NPE)C10/c3 surfaces, as shown by IR spectroscopy (Figure 6 ) . Reproducible deprotection of the blocking groups in the polar head group region of the immobilized PL ligands is 708 Analytical Chemistry, Vola66, No. 6, Wrch 15, 1994
critical for preparing IAMs with the same physico-chemical properties. As shown in Figures 3-6, and Table 3, deprotection of the blocking groups is usually greater than 90% efficient, except for ethcrIAM.PSC10/C3 which exhibited only 83% deprotection of the blocking groups. In addition to the cthcrIAM.PSC10/C3 surface, all of the other ethmIAMsurfaces also contain a small population of bonded PL ligands in silica
m,,,MA
3
After deprotectlon
Before &protection
-
0
II
-NH - C - 0 4(C H&
Boc group
1800
1754
1700
1860
1800
Wavenumber (cm”)
PE(Boc)COOH 3500
3000
2500
2000
1500
Wavenumber (cm“ )
Plguro 5. FT-IR spectra of PE(Boc)COOH, *IAM.PE(Boc), and *IAM.PEC1O/w. The spectra of *IAM.PE(Boc) and *IAM.pEO1olw are difference spectra(Le., the SPA spectrum has been subtractedfrom tho FT-IR spectra of *IAM.PE(Boc)and*IAM.pED1o~). The charactorbtic absorptions of Boc group at 2983.2 cm-l and the doublet at 1398.0 and 1373.2 cm-l as well as the strong Boc band centered at 1710 cm-1 are absent In the IR spectrum of *IAM.pEclo~w. As shown In the Inaorta at the right, the analyds of the integrated lntendty of the Boc band (1750-1675 cm-l) showed that before doprotection the I R Integrated intensity (ester area /Si-0 area) was 0.718, which decreased to 0 after doprotection, indkatlng 100% removal of the Boc group.
After deprotection
Before deprotectlon
II
3500
3000
lux)
2500
2000
1377
1353
1330
1500
Wavenumber (cm”)
Flguro 6. FT-IR spectra of PA(NPE)COOH, *IAM.PA(NPE), and *IAM.PAC1o/CS. The spectra of *IAM.PA(NPE) and *IAM.PAC1o/CS are dlffmnm spt3ctra (Le., the SPA spectrum has beensubtractedfromthe FT-IR spectraof *IAM.PA(NPE)and *IAM.PAClO’OS). The characte&tk absorptbns of the aromatlc NO1 at 1527.0 cm-1 (asymmetrical N-0 stretching) and 1349.0 cm-l (symmetrical N-0 etretchlng)almost totally dkeppewed after doprotection wlth DBU. As shown in the inserts at the right, analysb of the integrated intensities of the 1349.9 cm-1 band (1361-1331 cm-l) showed that before doprotectbn the IR Integrated IntensHy (NO2area/Sl-O area) was 0.474, whlch decreased to 0.042 after doprotection. Thb indkates that 89% of the NPE protecting group was removed.
cavities that are inaccessible to the solventsand reagents used for the deprotection reactions. Surface Density of Immobilized PL, C10, and C3 Lipids. We are attempting to emulate the lipid environment of fluid artificial membranes by immobilizing phospholipids on solid surfaces at high ligand densities. Both the biochemical and
physico-chemical properties of membranes depend on the surface densities of the immobilized lipids. The chemical characterization of the surface during each bonding reaction is given in Table 2, and the surface parameters calculated from these data for each PL ligand are given in Table 3. Figure 7 shows that FT-IRspectroscopyand elementalanalysis AnalytlcalChemistry, Voi. 66, No. 6, March 15, 1994
’189
Table 1. IR Frequencler Used To MonHor Doprotection Reactlonr
IAM surface e*erIAM.PG(IP)
functional group gem dimethyl'
frequency (cm-1)
6(C-H) 1380.0, 1371.1
u(C-H) 2985.0 6G-H) 1398.0,
e*e'IAM.PS(BoC)(t-Bu) t-BUb eWAM.PE (Boc)
1373.2
v(C-H) 2983.2 t-Bu esters & Bocb u(C=Ol 1750-1675 t-BV 6iC-H) 1398.0, 1373.2
u(C-H) 2983.2 e*erIAM.PA(NPE)
BocC
aromatic nitro groupd
u(C=O) 1710
vm(N=O)1527.0 u.(N=O) 1373.2
a See structure in Fi re 3. b See structures in Figure 4. See structure in Figure 5. g e structure in Figure 6.
Table 2. Chmlcal Analyrlr of IAM Surfacer durlng Bonding Reactlonr
bonding reactionsa
SPA and IAM surfaces
% Cb
HC area/ ninSi-0 areac hydrind 0.657 4.479 4.553 4.624 4.675 3.434
purple pink
step iv step ii
1.26 4.93 5.16 5.35 4.87 4.49
step iii
4.86
3.591
pink
4.96
4.660
white
4.24 5.3 5.31 5.42 4.63 4.52 4.53 4.76 4.01
3.393 4.800 5.021 5.154 4.078 3.186 3.092 3.184 3.010
purplee pink light pink White purplee
step ii step iii
step iv step ii step iii step iv step ii step iii
step iv
light pink
white white
pink
pink
pink white white
a See Scheme 1. * % C was determined b elemental analysis. IR inte ation limits for the Si-0 band of S%Aand the hydrocarbon banfof all IAM surfaces were given in the Experimental Section. d Qualitatively,the percentage of residual surface amines remaining on IAM surfaces corresponds to a ninhydrin test as follows: purple >40%; pink -10-20%; light pink >I2 h), all of the IAM surfaces become slightly pink, indicating that ninhydin can slowly diffuse into "crevices" on the IAM surface that contain unreacted amines. Undoubtedly, many of the surface amines are buried in these "crevices" (Chart 2) that are accessible to nitrogen gas used for the surface area measurements and C3 anhydrides used for the end capping reaction. From BET analysis, the total surface area of SPA is 107 m2/g, but because -40% of the amines are in the small crevices that sterically limit the acess of PLCOOH ligands, only about -60% of the total SPA surface (Le., 64 m2/g) is accessible for PL bonding. Thus the PL area per molecule given in Table 3 was calculated from the SPA surface area available for PL bonding which is 64 m2/g (Table 3). The area per molecule of the immobilized PLs ranged from 66 A2/molecule
Table 3. Molecular Propertler of I A M Surfaces.
IAM surface
mg of PL/ g of SPAb
PL:ClOC3C pmokpmokpmol
% reacted surface aminesd
phospholipid surface density" (A2/molecule)
etherIAM.pGClO/C3 etherIAM.pSClO/C3 etherIAM.pEClO/C3 etherWM.pAClO/C3
74.4 64.3 83.0 67.0
149:1853 102:2928 162:1:30 1261:64
62.8 45.4 55.2 54.6
72 104 66 84
deprotection % blocking groups removed
0 All surface properties were calculated from the data in Table 2 except footnote g, as described in this table. b The PL weight per gram of SPA was calculated from [ x % C J ( l + x ) ] = A% C, where x is the PL weight, A % C is the net carbon gain from elemental analysis, and % C, is the carbon content per PL molecule. The PL weight x is the value given in the table. The molar ratio of lipids bonded to 1 g of SPA. PL denotes either PG, PS, PE, or PA. For instance, 1491853 reflects 149 pmol of PG, 18 pmol of C10 alkyl chains, and 53 pmol of C3 alkyl chains per gram of SPA. The micromoles of each lipid were calculated from the weight of each lipid (PLs, C10, or C3) calculated according to the equation given in footnote b of this table. The initial SPA contained 350 pmol of pro ylamines per gram. Sequential bonding of PL, then C10, and then C3 lipids converted some of the amines into amides. The percent reactegsurface amines was calculated from the total micromoles of lipid bonded per gram of SPA divided by the total micromoles of propylamine per 1 g of SPA. e SPA contained 350 pmol of propylamine, but only 50430% of the surface amines are reactive to lipid bonding. We assume that the 40-5076 of the unreacted amines are in surface crevices that are accessible to small molecules like nitrogen (that was used to measure the surface area) but inaccessible to lar er molecules. On the basis of this assumption, the surface area available to bonding PLs can be estimated from 60/100 X 107 m2/ = 64 m!f/g. The PL surface density was calculated as (64 X 1020)/(ML X 6.023 X where MPLis the moles of PL bonded per gram of 8PA. If this assumption is not made, the surface densities are 110-173 12/molecule.f The weight of protective group removed from 1 g of SPA was calculated from elemental analysis data, and the percent of blocking groups removed was calculated from this weight. 8 Measured from FT-IR data as described in the legends to Figures 5 and 6.
t
A
Chart 2. IAM Surface Showing Crevices.
/
/
hr IAM.PE(Boc)o
2705
2255
-
1805
IAM.PS(Boc)(t-Bu)
Wavenuber (cm.')
PLs C10 C3
0
"
"
~
'
"
2
'
~
'
'
3
"
"
"
~
'
'
4
'
'
I
Large crevice
a Small crevicesare accessibleto only N2gas that was used for surface area measurements. Large crevicesare accessible to both N2gas and the C3 alkyl chain. The remainingsilica surface is accessible to the PL ligands, C10, and C3 alkyl groups.
/
5
Small crevices
6
% C by elemental analysis
Flgure 7. Correlation of immobilized HC content measured by FT-IR spectroscopyand % C measuredby elementalanalysis.The normalized I R HC area was calculated according to eq 1. The insert shows the bands used for the IR analysis.
(for etherIAM.PEc10/c3)to 104 A2/molecule (for etherIAM.PSC10/C3). This range of bonding densities is close to the phospholipid densities found in liposome membranes, and this corroborates our earlier 31PNMR studies demonstrating that IAMs emulate the lipid environment of fluid artificial membranes.24 etherIAM.PSC10/C3 exhibited the largest area per molecule of the immobilized PLs and this is most likely due to the bulky protecting groups in PS(Boc)(t-Bu)COOH. From Table 3, the rank order of the milligrams of PL bonded per gram of SPA is
PE > PG > PA > PS which also is the rank order of the size of the lipid head groups which include the blocking groups. The PL bonding density thus depends on the size of the phospholipid molecule; as the
size of the PL ligand increases, the bonding density decreases. It is interesting that the immobilization of single chain PL analogs is sterically limited by the size of the polar lipid head group, whereas the immobilizationof double chain PL analogs is sterically limited by the conformational free alkyl chain, as described above. Chart 2 summarizes the theoretical distribution of PLs, C10 chains, and C3 chains immobilized on the SPA surfaces. The surface area of SPA available for bonding is -60% of the total surface area because the N2 gas used for the surface area measurement can access small crevices that the larger C3, C10, and PL ligands cannot. The initial bonding of the PL ligands causes the formation of an immobilized lipid monolayer on the molecularly "flat" silica; the molecularly flat silica denotes the portion of the silica surface that is flat compared to the size of the phospholipid molecule. The single chain PLs are bonded sufficientlyclose that the C 10anhydride can not efficiently bond to the flat silica surfaces and the C10 alkyl chains cannot bond to either the small crevices or the large crevices. This is true for all the PLCOOH ligands, as shown by the low bonding densities of the C10 groups (Table 3, column 3). However, C3 end-capping always results in a large molar fraction of the surface amines covered because AnaMical Chemistrv. Vol. 66. No. 6. March 15- 1.9.94
191
the “large crevices” (inaccessible to the PL or C10 ligands) are accessible to the C3 anhydride used for end-capping. The low molar ratio of C10 bonding relative to C3 bonding is consistent with the general IAM structure given in Chart 2. CONCLUSIONS Immobilization methods were established for several single chain ether phospholipid ligands that contain amines, alcohols, and carboxyl groups in the polar head group region and an w-carboxyl group. Protecting groups for the functional groups in the polar head group region were utilized to protect the phospholipid during the immobilization process and to assure that immobilization was via the lipid alkyl chain. Covalent immobilization of the various phospholipids was by amidation of the w-carboxyl groups with the primary amines of silica propylamine. Activation of the w-carboxyl group of the PL ligands with carbonyldiimidazole was efficient using a novel solid phase adsorption synthetic method. The bonding density of the immobilized phospholipids is similar to the phospholipid density of fluid artificial membranes. The lipid blocking groups in the head group region were stable during both the immobilization process and the end capping reactions. The phospholipid ligands and bonding reactions described in this report are applicable to many different phospholipid ligands and many different surfaces. ABBREVIATIONS BET Boc t-Bu c3 c10 CDI DBU
Brunauer-Emmett-Teller N-tert-butoxycarbonyl tert-butyl propion yl decanoyl carbonyldiimidazole
DMSO
dimethyl sulfoxide
792
1,8-diazobicyclo[5.4.0]undec-7-ene
Analytical Chemistry, Vol. 66,No. 6,March 15, 1994
ET-IR Fourier transform infrared IAM(s) immobilized artificial membrane(s) IP isopropylidene CH,OH methanol HC hydrocarbon NMR nuclear magnetic resonance NPE @-nitropheny1)ethyl PA phosphatidic acid PA(NPE)- 0- [ 1-Q( 1 l-carboxyundecyl)-2-O-methyl-sn-glycero-3COOH phosphoryl]@-nitropheny1)ethanol PE phosphatid ylethanoamine PE(Boc)- 0-[1-0-(1l-carboxyundecyl)-2-O-methyl-sn-glycero-3COOH phosphoryl]-N-(tert-butoxycarbony1)ethanolamine PG phosphatidylglycerol PG(1P)- 0-[1-Q( 1 l-carboxyundecyl)-2-O-methyl-sn-glycero-3COOH phosphoryl]-2’,3’-isopropylidene-sn-glyceroI PL(s) phospholipid(s) PLCOO PL in the carboxylate form PLCOOH PL in the free acid form PS phosphatidylserine PS(Boc)- 0-[1-0-(1 l-carboxyundecyl)-2-0-methyl-sn-glycero-3(t-Bu)phosphoryl]-N-(tert-butoxycarbonyl)serinetert-butyl COOH ester rb round bottom SPA silica propylamine THF tetrahydrofuran TLC thin layer chromatography
ACKNOWLEDGMENT We are very grateful for the support from Eli Lilly and Co. This work was also supported by NSF (CTS 9214794), NIH (A13303I), and Regis Chemical Co. (2R446M3022-02). Received for revlew November 8, 1993. Accepted January 10, 1994.” *Abstract published in Adoance ACS Absrrocrs. February 15, 1994.
