Bioconjugate chetn. 1002, 3,540-548
540
Preparation and Characterization of Biologically Active 6'-0-(6-Aminocaproyl)-4'- 0-monophosphoryl Lipid A and Its Conjugated Derivative Kent R. Myers,'vt J. Terry Ulrich,? Nilofer Qureshi,*rs Kuni Takayama,tps Rong Wang,ll Ling Chen,ll W. Bart Emary,ll and Robert J. Cotter11 Ribi ImmunoChem Research, Inc., 553 Old Corvallis Road, Hamilton, Montana 59840, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 53705, Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, and Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Received August 7, 1992 N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-0-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli 55 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)z-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)z-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 pg/mL or less and only one-half as active at 100 pg/mL. In the induction of tumor necrosis factor release by RAW 264.7 murine macrophage cell line, the biotin-(Caph-MLA showed 7-9-fold lower activity than MLA at the concentration range of 0.1-1.0 pgImL. These results showed that Cap-MLA is a biologically active lipid A derivative that can be conjugated to other compounds through its free amino group to form new and active derivatives. It should thus be a useful reagent to study the biological properties of lipid A.
INTRODUCTION The lipopolysaccharide (LPS) of Gram-negative bacteria plays a key role in the pathophysiology of Gramnegative sepsis and septic shock (1). This potent molecule is able to stimulate a wide range of responses in humans at levels as low as 1ng/kg (2). I t is now well-established that essentially all of the biological activity of LPS resides in the lipid A portion of the molecule, although other structures present in LPS can potentiate these activities ( 3 , 4 ) . The medical importance of Gram-negative sepsis
* Author to whom correspondence should be addressed. Telephone: (406) 363-6214. Fax: (406) 363-6129. + Ribi ImmunoChem Research, Inc. * University of Wisconsin. 5 William S. Middleton Memorial Veterans Hospital. 11 Johns Hopkins University. 1 Abbreviationsused LPS, lipopolysaccharide;125I-ASD-LPS, 1251-iodinatedform of 2- [2-@-azidosalicylamido)ethyl]-3-dithio-
propionate-coupled LPS; SASD, sulfosuccinimidyl 2- [%@-midosalicy1amido)ethyll-3-dithiopropionate; MLA, monophosphoryl lipid A (hexaacyl); t-Boc, tert-butoxycarbonyl;Cap, 6-aminocaproyl; TLC, thin-layer chromatography; HPLC, highperformance liquid chromatography; LDMS, laser desorption mass spectrometry; PDMS, plasma desorption mass spectrometry;FAB-MS,fast atom bombardment mass spectrometry; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay.
and septic shock makes the mode of action of LPS and lipid A a topic of significant research interest. The exquisite sensitivity of certain cell types to LPS suggests that recognition occurs via one or more highaffinity receptors. Considerable effort has been directed at identifying the receptor structures that mediate LPS recognition by responsive cells (5). An important strategy for identifying potential LPS receptors involves the use of probes prepared by conjugation of the LPS molecule to appropriate reporter functionalities. One particularly useful derivative is 1251-ASD-LPS,which can be used to covalently label LPS binding structures with lZ5I(6).This derivative has made it possible to identify and initiate characterization of several candidate receptors (7, 8). Preparation of derivatives such as '25I-ASD-LPS requires the presence of free amino groups in LPS that can react with appropriately activated groups such as N-hydroxysuccinimide esters. Amino-containing substituents, such as phosphorylethanolamine and 4-deoxy-4-aminoarabinose, do occur in the core and/or the lipid A regions of LPS from many species of bacteria (9,101. However, these substituents are generally present in nonstoichiometric amounts and also may occur at more than one position in the LPS molecule (9,10). In addition, it is not known if the fatty acid distribution in the lipid A portion of LPS varies depending on the presence and location of amino-containing substituents; this possibility is suggested 0 1992 American Chemlcal Society
(Amlnocaproyl)monophosphoryl LipM A
fi'
(H0)2-P-0
Bloconlugete Chem., Vol. 3, No. 6, 1992
*:*
0
Rl
NH I R2
R3
NH
OH
I
R4
Figure 1. Structure of MLA from E. coli. R1 and Rz are 3-(acyloxy)myristoylgroups; Ra and Rd are 3-hydroxympistoyl groups. See Table I for further definition of R1 and RP.
by the observation that the polysaccharide and fatty acid contents of LPS are inversely correlated (11). These ambiguities complicate the interpretation of experiments performed with derivatives such as 1251-ASD-LPS. We now report on the selective introduction of a 6-aminocaproyl group to the 6'-O-position of 4'-O-monophosphoryl lipid A (MLA) obtained from the LPS of Escherichia coli (Figure 1). The product, 6'-0-(6-aminocaproy1)-MLA(Cap-MLA), contains a free amino group to which substituents such as SASD can be attached. As an example of the utility of this derivative, the conjugate of biotin and Cap-MLA was prepared. The resulting material, biotin-(Cap)~-MLA, retained the biological activity of unconjugated MLA with respect to activation of murine B-cells and macrophages, and exhibited high affinity for streptavidin. An additional result of this study was the observation of heterogeneity in the lipid A structure due to variability in the (acy1oxy)acylresidues at both the 2'-0- and 3'-0positions. This heterogeneity is evidently of biosynthetic origin. EXPERIMENTAL PROCEDURES
Reagents and Supplies. 6-Aminocaproic acid, 2[ [( tert-butoxycarbonyl)oxylimino] -2-phenylacetoni-
trile, dicyclohexylcarbodiimide,trifluoroacetic acid, and HCl(g) were obtained from Aldrich Chemical Co. BiotinCap N-hydroxysuccinimide ester and polymixin B were purchased from Sigma Chemical Co. All reagents were of the highest grades available, and were used without further purification. HPLC grade solvents were purchased from J. T. Baker, Inc. Chloroformand pyridineused in reactions were dried by storage over molecular sieves (4A). Chloroform treated in this way was free of residual alcohols or moisture asjudged by IR spectroscopy. Chromatographic media used in this study were Kieselgel 60 TLC plates, 250 pm (EM Science); Empore soft TLC plates, silica (Analytichem International, Inc.); Biosil HA silicic acid (Bio-Rad Laboratories-Chemical Division); Nova-Pak radial compression cartridge, CIS-bonded and capped 4 pm silica, 8 mm X 10 cm (Waters Associates, Inc.); and Accell Plus QMA ion-exchange packing (Waters Associates, Inc.). ELISA binding assays were performed on Immulon 2 microtiter plates (Dynatech Laboratories, Inc.). Culture reagents were RPMI-1640, antibiotics (Sigma Chemical Co.); fetal calf serum (Hyclone Laboratories, Inc.); I3H1thymidine (NEN Research Products); and antilipid A monoclonal antibodies DS77 and DS35 (Ribi ImmunoChem Research, Inc.). Female C3H/HeJ and C3H/HeSnJ mice 6-8 weeks of age were obtained from Jackson Laboratories. ICR mice were obtained from the mouse colony at Ribi ImmunoChem. Analytical Techniques. All TLC analyses were carried out using a solvent system consisting of chloroform/ methanol/water/ammonium hydroxide (50:31:6:2, v/v). Bands on the developed plates were visualized by spraying with 10% (w/v) phosphomolybdic acid in ethanol followed by charring or 0.2% (w/v) ninhydrin in n-butanollwaterl
541
acetic acid (95:4.5:0.5, v/v) followed by brief heating at 100 "C. TLC plates (visualized with phosphomolybdic acid) were analyzed by scanning densitometry with a Shimadzu CS9OOOU dual-wavelength flying spot scanner (Shimadzu Corp.), using a scanning wavelength of 520 nm. Phosphorus was determined by the method of Bartlett (121,and free amino groups were assayed by the procedure of Ghuysen and Strominger (131,using the modification of Jiao et al. (11). Amino sugar and amino acid analyses were carried out using Pico-Tag chemistry (Waters Associates, Inc.). Growth of Bacteria. E. coli J5 cells were grown in sparged culture at 37 "C in M9 media containing 16 g/L glucose. Cells were harvested at stationary phase with an Amicon hollow fiber system (Amicon Corp.), recovered as a pellet, and lyophilized. Isolation of LPS and Preparation of MLA. The rough chemotype LPS (Rc chemotype LPS) was extracted from the lyophilized cells of E. coli 55 by the method of Galanos et al. (141, as modified by Qureshi et al. (15).This crude RcLPS preparation (614 mg) was hydrolyzed in 0.1 N HC1 (3 mg/mL) at reflux for 20 min. The reaction mixture was cooled in an ice bath and then extracted with 2.5 volumes of chloroform/methanol (2:1, v/v) to yield crude MLA (290 mg). This material was applied to an anion-exchange column (Accell Plus QMA) and MLA was eluted using chloroform/methanol/60 mM ammonium acetate (2:3:1, v/v). The recovery of the purified MLA was 154.5 mg. A portion of this material (152 mg) was applied to a 3 cm X 30 cm Biosil HA silicic acid column and eluted with a linear gradient of 0-24% methanol/ water (955, v/v) in chloroform over 3.6 L a t a flow rate of 2 mL/min. Fractions (12 mL) were collected and analyzed for MLA content by TLC. Fractions 132-187 were pooled, evaporated to dryness, and weighed, yielding 78 mg of the desired pure MLA (hexaacyl). Preparation of t-Boc-Cap-MLA. t-Boc-Cap-MLA was prepared by reaction of MLA with 6-(t-Boc-amino)caproic anhydride. 6-Aminocaproic acid was first N-protected by reaction with 2 4 [(tert-butoxycarbonyl)oxy]iminol-2-phenylacetonitrile,using the procedure of Paleveda et al. (16). The anhydride was formed using dicyclohexylcarbodiimidein chloroform, and was recrystallized from diethyl ethedhexane (approximately 65:5, v/v). MLA (69.1mg, 40 pmol) and 6-(t-Boc-amino)caproic anhydride (53.9 mg, 121 pmol) were then combined in 3.4 mL of chloroform/pyridine (l:l,v/v) and stirred at 23 "C for 24 h. The reaction was quenched by addition of 3.4 mL of 0.1 M Na2C03(pH 10) followed by vigorous stirring for 30 min. The resulting mixture was extracted with 8.5 mL of chloroform/methanol (2:1, v/v). The phases were separated by centrifugation and the lower (organic) phase was washed with four 5-mL portions of 1.0 N HCl and once with 5 mL of water. The organic phase was then evaporated to dryness, yielding 90.7 mg of crude product mixture. A portion (84.5 mg) of this material was purified on Biosil HA silicic acid using the procedure described above, except that the linear gradient in this case was 0-18% methanol/water (95:5, v/v) in chloroform over 2.7 L at a flow rate of 2 mL/min. Fractions (10 mL) were collected and were pooled on the basis of their appearance on TLC. Three pools were obtained: I, fractions 70-72, 2.4 mg; 11,fractions 73-76, 12.8 mg; and 111,fractions 77170, 51.1 mg. Pool I11 represented the desired product, t-Boc-Cap-MLA. Deprotection of t-Boc-Cap-MLA. In general, t-BocCap-MLA was deprotected either by dissolving in trifluoroacetic acid at -20 "C and stirring for 20 min or by
542
Bioconlugete Chem., Vol. 3, No. 6, 1992
bubbling HCl(g) through a solution of the Boc-protected material in chloroform at -5 "C. The following is a representative example of the latter method: a solution of 14.0 mg of t-Boc-Cap-MLA (pool 111, 7.2 pmol) in 1.0 mL of anhydrous chloroform was stirred at -5 "C while being sparged with HCl(g). The reaction was followed by analytical TLC and was complete within 40 min. Two milliliters of 5 5% NaHCOsand5 mL of chloroform/methanol (2:1, v/v) were then added to the reaction solution, and the resulting mixture was vortexed and centrifuged. The organic phase was recovered, washed once with 2 mL of water, and evaporated to dryness. The yield was 12.5 mg of Cap-MLA (6.8 pmol, 94% yield), which appeared as a single ninhydrin-positive spot by analytical TLC. Preparation of Biotin-(Cap)z-MLA. Cap-MLA (9.5 mg, 5.2 pmol) and biotin-Cap N-hydroxysuccinimide ester (4.1 mg, 9.0 pmol) were stirred in 0.8 mL of chloroform/ pyridine (l:l, v/v) for 41 h at 23 "C. The reaction was then worked up by adding 5 mL of chloroform/methanol (2:1, v/v) and washing twice with 2 mL of 1.0 N HC1 and once with water. The organic phase was evaporated, yielding 9.9 mg of crude product. The major product was purified by preparative TLC using the solvent system described earlier, resulting in 6.2 mg of purified biotin(Cap)2-MLA. HPLC Fractionation of MLA Derivatives. HPLC was performed with two Waters 6000A solvent-delivery systems (Waters), a Waters 660 solvent programmer, a Waters U6K Universal liquid chromatograph injector, a variable-wavelength detector (Model LC-85B, PerkinElmer Corp.), and a radial compression module (Model RCM-100, Waters Associates, Inc.). Cap-MLA or t-Boc-Cap-MLA were first converted to the free acid form by passage through sulfonic acid resin (H+ form) followed immediately by methylation of the phosphate group with diazomethane. Samples were then analyzed by reverse-phase HPLC using a Nova-Pak cartridge (8 mm X 10 cm). A linear gradient of 20-80% 2-propanol in acetonitrile over 60 min at a flow rate of 2 mL/min was used. The wavelength of the detector was set at 210 nm. Typically, 2-3 mg of sample were injected per run, and an AUFS setting of 0.15 was used. LDMS. Laser desorption mass spectra were obtained on a CVC-2000 (Rochester, NY) time-of-flight mass spectrometer equipped with a Tachisto (Needham, MA) Model 215G pulsed carbon dioxide laser as previously described (17, 18). PDMS. Californium plasma desorption mass spectra were obtained on a BIO ION Nordic (Uppsala, Sweden) BIN-1OK time-of-flight mass spectrometer, with a 252Cf fission-fragmentionization source. Samples were dissolved in chloroform/methanol (l:l, v/v) saturated with the tripeptide glutathione (19)to a concentration of approximately 0.5 pg of sample/mL, and 10 pL of this sample was electrosprayed on the aluminum foil sample holder. Mass spectra were obtained by accumulating the ion signal to a preset value of 5 X 106 primary ion events. FAB-MS. Fast atom bombardment mass spectra were obtained on a AEI/Kratos (Manchester, England) mass spectrometer using 8 KeV xenon atoms (15, 20). The matrix consisted of dithiothreitol/dithioerythritol(3:1, v/v). Mass calibration was achieved by measurement of cesium iodide ions over the mass range from 800 to 2300. Proton NMR Spectroscopy. Spectra were recorded on a Bruker AM 500 spectrometer operating at 500 MHz. The spectrometer was equipped with an Aspect 3000 computer and digital pulse shifter. HPLC-purified dimethyl-t-Boc-Cap-MLA, peak III-C (8.0 mg; see below),
Myers et al.
was dissolved in 0.5 mL of CDCl&D30D (81,v/v). A two-dimensional proton correlation (COSY)spectrum was obtained under conditions described previously (21,22). ELISA Binding Assay. Immulon 2 microtiter plates were washed with 15% l-propanol and then coated for 2 h a t 37 "C with 100 pL/well of either MLA or biotin(Cap)Z-MLAat 10 pg/mL in 0.05 M sodium bicarbonate/ carbonate (pH 9.5) buffer. The plates were then washed three times with water, and 100 pL of antibody in ELISA buffer (0.01 M Tris, pH 7.2,25 % v/v FCS, 2 mg/mL EDTA, and 0.13'% v/v Tween 20) or ELISA buffer alone was added to each well. The plates were incubated for 1h and washed as above. Horse radish peroxidase-goat anti-mouse IgG (100 pL/well in ELISA buffer) was added to wells containing antibody. Horse radish peroxidase-streptavidin (100 pL/well in ELISA buffer) was added to wells to be analyzed for streptavidin binding. Horse radish peroxidase-streptavidin concentrations were tested in quadruplicate. Plates were incubated at 37 "C for 1h and then washed. o-Phenylenediamine reagent (0.4 mg/mL in 0.1 M citrate phosphate buffer, pH 5.0, containingO.Ol% v/v HzOZ;see ref 23) was added to each well, and plates were incubated for an additional 15 min. The reaction was stopped by addition of 50 pL of 1N H2S04, and the optical density was read at 490 nm. Stimulation of Lymphocyte Blastogenesis. Mitogenic activities of test materials were assessed according to standard procedures (24). Splenocytes were obtained from either C3H/HeJ, C3H/HeSnJ, or ICR mice, and were cultured in 96-well plates at a density of 2-3 X 105 cells/ well in RPMI-1640 media containing 10% fetal calf serum, 2 pg/mL Fungizone, 50 pg/mL gentamycin, 2 mM Lglutamine, and varying concentrations of test materials. Test materials were assayed in triplicate at each concentration. One group of three wells, to be used for estimation of background activity, received all media components but did not receive any added mitogens. After 36-48 h, 200 pL (1pCi) of r3H1thymidine was added to each well, and the cells were incubated for an additional 6-8 h. Cells were then harvested onto fiberglass filter pads and their i3H1thymidine uptake was determined by liquid scintillation counting. Stimulation indices were calculated as the ratio of 3H counts in cells exposed to mitogen versus media alone. For assays involving polymixin B inhibition, test materials were combined with appropriate concentrations of polymixin B in ELISA buffer and incubated at 37 "C for 30 min prior to addition to microtiter wells. Induction of TNF-a. The abilities of various materials to induce release of TNF-a were assessed in the RAW 264.7 cell line. Cells were cultured according to the procedures of Virca et al. (251, in 24-well plates at a cell density of 5 X lo6 cells/well. Experiments involved exposing cells to varying amounts of test materials in serum- and antibiotic-free RPMI- 1640 media containing 2 mM L-glutamate for 8 or 20 h. Cells exposed to media alone, without added test substances, were used to estimate background TNF-inducing activity. Conditioned supernatant fractions were analyzed in duplicate for the presence of TNF-a using an ELISA assay (obtained from Genzyme, Inc.). The TNF-a induced by each test substance was calculated as the difference between TNF-a elicited in the presence of the test material and TNF-a elicited by media alone. RESULTS
Preparation of CAP-MLA and Derivatives. The reaction of MLA (69.1 mg) with the t-Boc-protected anhydride of 6-aminocaproic acid proceeded readily at
Bloconlugate Chem., Voi. 3, No. 6, 1992 543
(Aminocaproyl)monophosphoryiLipid A
room temperature in the presence of pyridine to yield t-Boc-Cap-MLA. TLC of the product mixture followed by charring and scanning densitometry revealed one major product spot (Rf= 0.59,80 % 1, a smeared region containing one distinct spot (Rf = 0.65, 14% for the smear), and a minor spot (Rf= 0.69,4% 1. A small amount of material migrating with the same Rf as the starting material (Rf= 0.33,2 % ) was also evident. In other experiments, addition of further anhydride and incubation for longer times did not reduce the intensity of the lower spot, suggesting that it did not correspond to MLA starting material. Fractionation of the crude t-Boc-Cap-MLA on a silicic acid column yielded three major fractions: pool 111, the largest fraction with Rf = 0.59; pool I, a minor component with Rf = 0.65; and pool 11, a mixture of pools I and 111. Pools I and I11 were further analyzed. The t-Boc-Cap-MLA (pool 111) was deprotected by treatment with HCl(g) in chloroform at -5 "C. The yield of Cap-MLA was greater than 90%. On TLC, this product appeared as a single, ninhydrin-positive spot with an Rf slightly less than that of the starting MLA. It was determined to have a molar ratio of phosphorus to free amino groups of 1.00:1.13, whereas the starting MLA had aratio of 1.oO:O.Ol. Amino acid analysis of acid-hydrolyzed sample confirmed the presence of glucosamine and 6-aminocaproic acid in the Cap-MLA product (data not shown). The reaction of Cap-MLA with 1.7 equiv of biotin-Cap N-hydroxysuccinimide ester yielded a product with an Rf intermediate between that of the two starting materials. This product was identified to be biotin-(Cap)~-MLA.The reaction reached completion in 18 h at 23 "C. STRUCTURAL CHARACTERIZATION OF CAP-MLA AND DERIVATIVES
HPLC Purification of Dimethyl-t-Boc-Cap-MLA. Pools I and I11 from the silicic acid column fractionation were methylated with diazomethane and analyzed by reverse-phase HPLC. Methylated pool I gave a single major peak (LA) which was recovered and analyzed by FAB-MS (to be discussed later). The chromatographic profile of pool I11 is shown in Figure 2. It contained two prominent and two minor peaks (III-A through III-D). The relative abundance5 of these four components, based on integrated absorbance at 210 nm, are given in Table I. Mass Spectrometryof HPLC-PurifiedPools I and 111. LDMS of the major HPLC peak III-C (Figure 2) gave a molecular ion M + K+ at 1999 and M + Na+ at 1983 (Figure 3). The molecular weight of this product is 1960. A major fragmentation resulted from cleavage of the hydroxymyristoyl group (R3) at the 3'-O-position of the glucosamine disaccharide with proton transfer from the sugar (243 amu + H)to yield peaks at 1754 and 1738 (containing K+ and Na+, respectively). The loss of both R3 and t-Boc-Cap (243 + 230 amu) from 1999 gave a peak at 1524. The second major fragmentation pathway was cleavage of the 0-C1 and C& bonds of the reducing-end sugar to give the "distal ions" at 1426 (17,181.The mass of this ion suggests that the t-Boc-Cap group is attached to the distal glucosamine. If the group were attached to the reducing-end glucosamine, then the distal ion would have a mass of 1213, which is not observed. The proposed structure of t-Boc-Cap-MLA is shown in Figure 4. The origin of the peak at 1451 is not clear. However, we suggest that it is a two-bond ring cleavage carrying an additional ethylene group. The three peaks at 1426,1451, and 1524 all represent ions in which the only ester-linked fatty acid on the reducing end (R3) has already been lost.
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Figure 2. HPLC chromatogram of dimethyl-t-Boc-Cap-MLA, pool 111. A Cls-bonded silica cartridge was used. The mobile phase was a linear gradient of 20-80 % 2-propanol in acetonitrile at a flow rate of 2 mL/min over a period of 60min. The absorbance units a t full scale was set at 0.064 for 2.0 mg of dimethyl-t-BocCap-MLA. Baseline correction was made by the Perkin-Elmer LC-85B detector.
The only possible additional fragment ions would involve losses of the myristic and lauric acids attached to R1 and Rz, respectively. These are lost as the neutral acids (228 and 200 amu). All of the remaining peaks in the mass spectrum can be explained as losses of either lauric acid, myristic acid, or both. If one observes the loss of R3 from the peak at 1999,then one should also observe an additional loss of R1 (i.e., from the peak at 1754). This minor peak appeared at 1315. LDMS of the HPLC peak III-B gave a molecular ion M + K+ at 1971 and M + Na+ at 1955 (Figure 5). The molecular weight of this product is 1932. A major fragmentation resulted from cleavage of R3 to yield peaks at 1726 and 1710 (containing K+ and Na+, respectively). The loss of both R3 and t-Boc-Cap gave a peak at 1494. The peak at 1398is the distal portion plus OCH&H=O plus K+. The mass again suggests that the t-Boc-Cap is attached to the distal sugar and that the 28 amu difference between peaks III-B and III-C is located on the distal portion. The peak at 1382 is the distal portion plus OCH2CH=O plus Na+. The 1398ion further fragments to form the ions at 1196and 996, representing the successivelosses of two laurate groups. Similarly, the ion at 1494 showed the same two losses of lauric acid to yield fragments at 1296 and 1095; the ion at 1420 lost successive lauric acid units to form the ions at 1224 and 1022. These fragment ions would suggest that both RI and R2 are 3-(lauroyloxy)laurate, so that the 28 amu difference between this product and III-C resulted from the substitution of lauric acid for myristic acid in R1. It could be argued that R1 is 3-(myristoyloxy)myristate and Rz is 3-(caproyloxy)myristate. The ion at 1224 would then represent the loss of caproic acid from 1398,followed by the loss of myristic acid to give the peak at 996. However, if that were the case, one should also observe the loss of myristic acid from 1398 first at 1170, which is
Bioconlugete Chem., Vol. 3, No. 6, 1992
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Myers et at.
Table I. Structure of Dimethyl-t-Boc-Cap-MLA Species Isolated from Pool I11 by HPLC, As Determined by Mass SDectrometrv and Nuclear Mametic Resonancea position relat fatty acid distributionb HPLC peak Mr of t-Boc-Cap group abund Ri Rz R3 R4 111-A 1960 unk 6 c14oc14 ClZOCl4 111-B 1932 6‘ 38 c12oc14 c12oc14 OHCi4 OHCi4 111-c 1960 6’ 100 c14oc14 c12oc14 OHCi4 OHCir 111-D 1988 6’ 4 c14oc14 c14oc14 0HCi.i OHCir a HPLC chromatogram of pool I11 is shown in Figure 2. R1, Rz, RB,and Rd correspond to fatty acyl groups shown in Figure 4. Abbreviations used: OHC14, 3-hydroxymyristate; c12oc14, 3-(lauroyloxy)myristate;C14OC14, 3-(myristoyloxy)myristaate;unk, unknown. ~~
~~
100
Y
