the journal of vol. 267, no 11, of 15, 722&7239,1992 1992 ... · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No . 11, Issue of April 15, pp. 722&7239,1992 Printed in U.S.A.

Substrate Specificity of Saccharomyces cerevisiae Myristoyl-CoA:Protein N-Myristoyltransferase ANALYSIS OF FATTY ACID ANALOGS CONTAINING CARBONYL GROUPS, NITROGEN HETEROATOMS, AND NITROGEN HETEROCYCLES IN AN IN VITRO ENZYME ASSAY AND SUBSEQUENT IDENTIFICATION OF INHIBITORS OF HUMAN IMMUNODEFICIENCY VIRUS I REPLICATION*

(Received for publication, November 22,1991)

Balekudru Devadas$, Tianbao Lug, Akira Katohp, Nandini S . Kishore$, Arlene C. Wade$, Promod P. Mehta$, David A. RudnickT, Martin L. Bryant$, Steven P. Adam&, Qi Lij, George W. Gokelp, and Jeffrey I. Gordonlll From the SMonsanto Company, St. Louis, Missouri 63198, the §Department of Chemistry, Uniuersity of Miami, Coral Gables, Florida 33124, and the TDepartment of Molecular Biology and Pharmacology, Washington Uniuersity School of Medicine, St. Louis, Missouri 631 10

Covalent attachment of myristic acid (C14:O) to the amino-terminal glycine residue of a variety of eukar- yotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identi- fied as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl- CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 66 analogs of myristic acid (C14:O) to further charac- terize the acyl-CoA binding site of Saccharomyces ccrevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr65gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme’s binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C6-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-160°. The activities of NMT’s acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between

* This work was supported by Grants AI-27179 and AI-30188 from the National Institutes of Health and Monsanto Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(1 To whom correspondence should be addressed: Dept. of Molec- ular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 So. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243: Fax: 314-362-7058.

polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide- containing tetradecanoyl-CoA derivatives.

Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr6SgW for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 PM without associated cellular toxicity.

Myristoyl-CoA:protein N-myristoyltransferase (NMT,’ EC 2.3.1.97) catalyzes the cotranslational (Wilcox et al., 1987; Deichaite et al., 1988) transfer of myristate (tetradecanoate, C14:0), a rare cellular fatty acid (Orme et al., 1972; Awaya et al., 1975) to the amino-terminal glycine residues of proteins with a wide range of functions (reviewed in Towler et al., 1988a; James and Olsen, 1990; Gordon et al., 1991). These proteins include, for example, serine/threonine and tyrosine kinases (e.g. p60”*“, Cross et al., 1984; Kamps et al., 1985; Resh and Ling, 1990), protein phosphatases (e.g. calcineurin, Aitken et al., 1982; Cyert et al., 1991), yeast as well as mammalian guanine nucleotide binding a subunits of heterotri- meric G proteins (Blumer and Thorner, 1991; Stone et al., 1991; Mumby et al., 1990), several retroviral gag polyprotein precursors (e.g. the Pr55gag of human immunodeficiency virus I, Gottinger et al., 1989; Bryant and Ratner, 1990; Bryant et al., 1991), and the capsid proteins of a number of picornavi- ruses and papovaviruses (e.g. VP4 of poliovirus, Chow et al., 1987). Genetic and biochemical studies have indicated that the myristoyl moiety is essential for full expression of the biological properties of several of these proteins (e.g. Cross et al., 1984; Rein et al., 1986; Rhee and Hunter, 1987; Marc et al., 1989; Linder et al., 1991; Stone et al., 1991).

The best characterized NMT is from Saccharomyces cereuisiae. The monomeric 53-kDa enzyme has been purified to

The abbreviations used are: NMT, myristoyl-CoA:protein N- myristoyltransferase; HIV-I, human immunodeficiency virus I; HPLC, high-performance liquid chromatography; CPE, cytopathic effect; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; TCID50, dose of virus that infects 50% of cells in culture; TDsI, dose of analog that kills 50% of noninfected cells in culture; AZT, 3‘- azido-3’-deoxythymidine.

7224

Substrate Specificity of N-Myristoyltransferae 7225

homogeneity (Towler et al., 1987b). The single copy, haploid essential, S. cerevisiae NMTl gene has been meiotically mapped and isolated (Duronio et al., 1989, 1991b). NMTl specifies a 455-residue protein that has no obvious primary structural similarity to any entry in current databases. The Saccharomyces enzyme has been efficiently expressed in Esch- erichia coli, allowing isolation of large quantities of active NMT (Duronio et al., 1990a, 1990b; Rudnick et al., 1990). Detailed in vitro kinetic analyses (Rudnick et al., 1991) indicate that its mechanism of catalysis is ordered Bi Bi with myristoyl-CoA binding to NMT occurring prior to binding of peptide and CoA release taking place prior to release of its acylpeptide product as follows.

myr CoA 1 NMT - NMT-myristoyl-CoA

peptide catalysis - myristoylCoA-NMT-peptide “--+ 1

myristoylpeptide-NMT-CoA + 1

CoA NMT-myristoylpeptide > NMT

1 myristoylpeptide

Protein N-myristoylation appears to be an irreversible covalent modification (Olson, 1988; James and Olson, 1989), although exceptions may exist (da Silva and Klein, 1990). Interestingly, the enzyme is able to catalyze the reverse reaction in vitro as follows, Myristoylpeptide + CoA + NMT

+ NMT-myristoyl-CoA + peptide albeit a t a rate that is significantly slower than that of the forward reaction (Rudnick et al., 1991). This deacylation, together with the results of biophysical studies (Rudnick et al., 1990; 1991), provide additional evidence for a high affinity myristoyl-CoA-NMT intermediate.

The peptide and acyl-CoA substrate specificities of S. cerevisiae NMT have been explored using a discontinuous in vitro assay of enzyme activity. Analyses of over 100 synthetic peptides (Towler et al., 1987a, 1987b; 1988a, 1988b; Duronio et al., 1991a) indicate that NMT monitors the physicochemical properties of amino acids positioned over a broader area of the amino terminus than other S. cerevisiae amino-terminal modifying enzymes (e.g. Ne-acetyltransferase and methio- nylaminopeptidase, Tsunasawa et al., 1985; Lee et al., 1988, 1989a, 1989b, 1989c, 1990a, 1990b; Chang et al., 1990; Moer- schell et al., 1990).

Little is known about how this enzyme recognizes the coenzyme A moiety of its myristoyl-CoA substrate. Limited studies with 3’-dephospho-CoA suggest that the 3”phosphate is not necessary for peptide acylation (or deacylation of the myristoylpeptide) but may facilitate the formation of the acyl- CoA-NMT intermediate (Rudnick et al., 1991).

Considerably more is known about structure/activity relationships in the fatty acid chain of myristoyl-CoA. I n vitro kinetic studies of >90 fatty acid analogs with one or more oxygen or sulfur atoms, trans (E) or cis (Z) double bonds, triple bonds, or aromatic systems have helped define the basis for the enzyme’s acyl-CoA selectivity (Heuckeroth et al., 1988, 1990; Kishore et al., 1991; Gokel et al., 1992). These analyses indicate that hydrophobicity is less important than chain length. The acyl chain of myristoyl-CoA substrate appears to be present in a bent conformation with the principal bend (approximately 140-150”) occurring near C5-C6 (numbered from carboxyl). The binding pocket seems to have three reference points: (i) at the carboxyl terminus (S-CoA); (ii) at

TABLE I Sample structures and abbreviations of keto, ester, amide, and

acylamino acid analogs of myristic acid Functional Abbrevi-

group ations Example Code

0

-C- II

0 II

-0- c - 0 II

-NH- C -

0 II

- C -NH- ~~~

the omega methyl group, and (iii) the bend which, in turn, affects the distance measurement in both directions from the bend. The acyl-CoA binding pocket possesses a sensor that detects the terminal methyl group. The apparent conical shape of this sensor also permits it to monitor bulk (Kishore et al., 1991).

We have now synthesized 56 myristic acid analogs containing ketocarbonyl, ester, amide, acylamino acid, alkyl azido, and a variety of heterocycles including imidazole, triazole, and tetrazole. This broad family of compounds permitted us to explore the effects of polarity, basicity, and the related issues of branching and bulk on the physicochemical basis of the host-guest relationships. Biological studies of members of the panel of analogs revealed that 12-azidododecanoic acid was a potent inhibitor of HIV-I replication in both acutely and chronically infected T-lymphocytes at concentrations that were not associated with cellular toxicity.

EXPERIMENTAL PROCEDURES’ Compound Structures and Abbreviations-The single number fol-

lowing the abbreviation CO indicates the methylene group of myristic acid replaced by the carbonyl group. The two numbers that follow the abbreviations EST, AMD, and AAA indicate the two methylene groups which have been replaced. The numbers preceding IM, TRI, and TET indicate positional isomers. Sample compound structures and abbreviations are shown in Tables I, IV, and V.

Syntheses of Myristic Acid Analogs-Each of the ketocarbonyl compounds used in this study was prepared by one of four basic methods. These include the malonic ester acylation-decarboxylation sequence (CO-3); the acetoacetic ester synthesis ((20-4, CO-5, CO- 13); enamine acylation followed by hydroxide-induced ring cleavage (CO-6, CO-7); and coupling of a Grignard-derived cadmium salt to an acyl chloride (CO-9, CO-10, CO-11, CO-12). The ester analogs were prepared by mono-esterification of the appropriate diacid. Like- wise, the amide derivatives were obtained by formation of the mon- oamide of a diacid. Acylamino acid containing analogs were obtained by acylation of an w-amino acid derivative. w-Azido substituted alkanoic acids were prepared by azide displacement on the corresponding w-iodoalkanoic acid. The thia- and oxa-azido alkanoic acids were prepared in a similar fashion from the appropriate oxa- or thia-w- iodocarboxylic acid. The imidazole, triazole, and tetrazole derivatives were all synthesized by alkylation of the heterocycle using appropriate protection when necessary. Further details of the preparation, purification, and characterization of the analogs prepared for this study may he found in the miniprint.

Synthesis of Radiolabeled O~tapeptides-Gly-Ala-Arg-[~H]Ala-Ser- Val-Leu-Ser-NH2 (GAR[3H]ASVLS-NH2) representing residues 2-9 of the HIV-I Pr55g”K was synthesized and purified as described in

Portions of this paper (including part of “Experimental Proce- dures”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

7226 Substrate Specificity of N-Myristoyltramferase

Kishore et al. (1991). Its specific activity was 1.2 Ci/mmol. Radi- ochemical and chemical purity was >95%. Synthesis and purification of Gly-Ser-['H]Ala-Ala-Ser-Ala-Arg-Arg-NHz (GS['H]AASARR- NH2; specific activity, 1.1 Ci/mmol) is detailed in Heuckeroth et al. (1990).

In Vitro Pseudomonas Acyl-CoA Synthetase Assay-Conditions used to monitor the efficiency of conversion of analogs to their CoA thioesters are described in Kishore et al. (1991). The 100-pl reaction mixture contained fatty acid (final concentration, 160 p ~ ) , unlabeled CoA (1 mM), [3H]CoA (1 pCi, 1.2 Ci/mmol, see Kishore et al., 1991), dithiothreitol (3 mM), Triton X-100 (0.05%), Tris (5 mM, pH 7.4), MgClz (2.5 mM), EGTA (50 p ~ ) , and Pseudomonas aeruginosa acyl- CoA synthetase (0.3 units/ml; Sigma). Following a 25-min incubation at 30 "C, an equal volume of ethanokacetic acid (1:l) was added. The mixture was cooled on ice for 10 min and then fractionated by ClS reversed-phase HPLC using gradient conditions detailed in Kishore et al. (1991). The amount of 'H-labeled analog-CoA produced was quantitated using an in-line detector (Kishore et al., 1991) and compared to the amount of myristoyl-CoA produced in a parallel reaction. All assays were performed at least in duplicate.

In Vitro Discontinuous NMTAssay-A comprehensive description of this assay and the concentrations of each reactant is provided in earlier publications (Towler and Glaser, 1986; Duronio et al., 199Ob; Kishore et al., 1991). Briefly, an initial, single point assay was performed as follows: myristic acid or its analog (final concentration, 15 MM) was converted to its CoA thioester using 0.3 units/ml of Pseu- domonas acyl-CoA synthetase. Following a 25-min incubation at 30 "C, E. coli-derived, purified S. cerevisiue NMT (final concentration, 0.5 pg/ml) was added together with either one of the two tritiated peptides (final concentration, 25 p ~ ) . After an additional 10-min incubation at 30 "C, the enzymatically generated acylpeptide was purified using Cla reversed-phase HPLC and quantitated using an in- line scintillation counter. A reference control reaction was always run using myristic acid so that the amount of analog peptide produced could be expressed as a percentage of myristoylpeptide (Kishore et al., 1991). Assays were run in duplicate or triplicate for each experiment.

Selected analogs were characterized further by more detailed kinetic studies. The apparent K, and V,,, were first determined using saturating concentrations of analog. Apparent acyl-CoA K, and V, were then calculated using the ['Hloctapeptide at its K,. Myristic acid was used as a reference control in each experiment. All V, data were expressed as a percentage of the V, obtained with myristoyl- CoA. All assays were performed in duplicate for each experiment. All experiments were repeated at least twice.

Inhibition Assays-Several of the acylamino acid and amide containing myristic acid analogs were also tested as inhibitors of E. coli- derived S. cerevisiue NMT using the in vitro discontinuous assay. These compounds were tested either directly (1-100 pM; AMD 617, 516,415; AAA 617,516,415) or after conversion to the corresponding CoA derivatives (10 p ~ ; AAA 13/12, 10/9,9/8, AMD 6/7, 516, 4/51, in the presence of 0.23 p~ ['Hlmyristoyl-CoA and 180 p~ unlabeled GNAAAARR-NHz (Towler et al., 1987a).

Acute Virus Replication Assays-The effect of each analog on virus replication was assessed in two acute infectivity assays. The first assay was adapted from Pauwels et al. (1988). Assays were performed in 96-well tissue culture plates. CEM cells were grown in RPMI-1640 medium (GIBCO) supplemented with 10% fetal calf serum. Following treatment of the plate with polybrene (2 pg/ml), an 80-pl aliquot containing 1 X lo' cells was dispensed into each well of the plate. One hundred p1 of RPMI-1640 plus analog (dissolved in dimethyl sulfoxide) was then added and the cells were incubated at 37 "C for 1 h. (Note that the final concentration of dimethyl sulfoxide did not exceed 1.5%). A frozen stock of HIV-I was thawed and diluted in culture medium to a concentration of 5 X lo' TCIDW/ml (TCIDM equals the dose of virus that infects 50% of cells in tissue culture). Twenty pl of the virus sample (containing 1000 TCIDW of virus) were added to wells containing analog and to wells containing only medium plus dimethyl sulfoxide (infected control cells). This results in a multiplicity of infection of 0.1 (MOI = number of infectious units/ number of cells in culture). Several wells received culture medium without virus (uninfected control cells). Likewise, the intrinsic toxicity of the test compound was determined by adding medium without virus to several wells containing analog.

Following addition of virus, cells were incubated at 37 "C in a humidified atmosphere containing 95% air/5% COZ for 7 days. Ad- ditional aliquots of test compounds were added on days 2 and 5. On day 7, the cells in each well were resuspended and a 100-pl aliquot of

each suspension was removed for assay. Twenty pl of a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each 100 pl of suspended cells. The mixture was incubated for 4 h at 37 'C in 95% air/5% COz. During this incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is met- abolically reduced by living cells resulting in the production of an intracellular colored formazan product. One hundred pl of 10% sodium dodecyl sulfate in 0.01 N HCl was added to lyse the cells. Following an overnight incubation, the absorbance of each sample was determined at 590 nm.

The percentage reduction of the virus-induced cytopathic effect (CPE) by analog was determined using the following formula.

Absorbance of analog-treated infected sample - absorbance of virus control

Absorbance of cell control x 100

- absorbance of virus control

The dose that inhibits 50% of the cytopathic effect is referred to as IDso.

The second assay has been described in detail elsewhere (Bryant et al., 1989). It involves (i) infection of CD4+ H9 cells with HIV-I; (ii) addition of analog to serum-containing RPMI 1640 medium within 1 h after exposure of cells to the virus; (iii) replacement of the medium with fresh medium plus analog every 48 h; and (iv) measurement of virus production in days 8-10 medium by monitoring reverse transcriptase (RT) levels, and by determining p24 antigen concentrations (Bryant and Ratner, 1990). Data were referenced to three negative controls: cells treated with no additions, cells treated with ethanol alone (0.1%, the vehicle used to prepare stock solutions of the fatty acids for these experiments), or cells treated with a naturally occurring saturated fatty acid whose hydrophobicity is equivalent to that of the analog (Le. C100 or C120) but which is known not to be an active NMT substrate (Heuckeroth et al., 1988). Analog toxicity was assessed by (i) pulse labeling an aliquot of analog-treated and control cells on the last day of the experiment with ['Hlleucine or ['HI thymidine for 2-4 h and measuring incorporation into protein and DNA, respectively, and (ii) measuring viable cell number by trypan blue exclusion (Bryant et al., 1989).

Chronic Virus Replication Assay-Details of this assay are provided in Bryant et al. (1991). H9IIIB cells which chronically produce HIV- I were incubated with RPMI 1640 medium with or without analog (cell density, 2 X 106/ml in each well of a 48-well culture plate). After 2 days, the medium was removed and replaced with fresh medium. At the end of the fourth day of the experiment, medium was harvested, filtered (0.22 pm, Millipore) and assayed for reverse transcriptase and p24 virus antigen. Analog toxicity was assessed at the end of the treatment period by metabolic labeling with ['Hlleucine and ['HI thymidine (see above) and by measuring cell viability by trypan blue exclusion. Negative controls included treatment with medium alone plus or minus ethanol (0.1%) or C100. 13-Oxatetradecanoic acid was included as a positive control (Bryant et al., 1991).

RESULTS AND DISCUSSION

Our understanding of the interactions of purified S. cerevisiue NMT with myristoyl-CoA has resulted in part from in vitro studies of various structural analogs. As noted in the Introduction, these included compounds longer and shorter than myristate, compounds with double and triple bonds, and various aromatic residues. Certain of the aromatic subunits contained oxygen or sulfur. They were explored as were ether and thioether analogs (Heuckeroth et al., 1988,1990; Kishore et al., 1991). To extend these analyses, we decided to incor- porate oxygen in its carbonyl form (ketones, esters, amides). The amides also contained nitrogen which prompted a study of the effects of this atom in a variety of forms.

Myristic Acid Analogs with Ketocarbonyl for Methylene Sub- stitutions-A panel of 11 analogs was prepared in which each -CH2- group of C14:O (from C3-Cl3) was replaced by the carbonyl function (see "Experimental Procedures"). The ketocarbonyl analogs differ from previously reported oxatetradecanoic acid analogs (Heuckeroth et aL, 1988; 1990; Kishore et al., 1991) by the location and oxidation state of the oxygen atom. The carbonyl oxygen protrudes from the main chain at

Substrate Specificity of N-Myristoyltransferase 7227

a distance of -1.3 A in contrast to ether oxygen which is integral to the chain. The carbonyl group is a polar cylinder with electronegative oxygen at one terminus and electron- rich T bonds connecting it to the main hydrocarbon chain. This group also exhibits fixed directionality, i.e. the oxygen of the carbonyl is coplanar with the attached carbon and its adjacent carbons. The sulfoxide, 11-thiatetradecanoic acid S- oxide, is the only previously reported analog whose structure resembles that of these ketocarbonyl compounds. However, it is not a substrate for S. cerevisiae NMT in vitro. By contrast, 11-thiatetradecanoic acid and 11-oxatetradecanoic acid have activities which are, within experimental error, identical to that of myristate (Kishore et al., 1991). The different activities of these three compounds could reflect differences in their hydrophobicities, polarities, and/or bulk. Replacement of carbonyl functions at C3 through C12 of myristic acid provided a panel of “probes” of defined directionality, bulk, and polarity which allowed us to “sweep” the acyl-CoA binding site of NMT and thereby infer its geometric, steric, and polar properties.

The coupled, discontinuous in vitro NMT assay requires an initial conversion of the analog to its acyl-CoA derivative by the “nonspecific” Pseudomonas acyl-CoA synthetase (Shim- izu et al., 1980; Kishore et al., 1991). We, therefore, first measured the efficiency of this reaction using radiolabeled CoA and the bacterial enzyme. The extent of CoA acylation for 10 of the 11 compounds was similar to that of myristate: 88-139% (Fig. 1A). Only 3-oxotetradecanoic acid had a conversion efficiency less than 50%. Next, NMT-dependent transfer of unlabeled analog from CoA to a radiolabeled octapeptide substrate (GAR[3H]ASVLS-NH,) representing residues 2-9 of the gag polyprotein precursor encoded by HIV- I, was assessed using the single point assay described under “Experimental Procedures.” The results are presented in Fig. 2. Fig. 2A compares the amount of analogpeptide produced with the amount of myristoylpeptide generated in a parallel incubation that contained identical concentrations of reactants. The data indicate that the carbonyl group is accommodated by the binding pocket at all positions surveyed. However, activity was position-dependent. Location of the ketocarbonyl at position 6 (near the presumed bend) produced the most active analog (CO-6) while displacement of the C=O by one carbon in either direction diminished activity (e.g. CO- 5 = 175% of myristate; CO-6 = 250%; CO-7 = 150%). Activity generally diminished as the carbonyl was placed closer to the C14 position (CH3) of myristate. Similar results were obtained using a different radiolabeled octapeptide substrate (GS[3H] AASARR-NHJ, and selected members of this series (Fig.

An unexpected observation was made with 4-oxotetradecanoic acid. Two acylpeptide species were noted after this analog was introduced into the coupled in vitro NMT assay. This occurred with either octapeptide substrate. By contrast, all other oxotetradecanoic acids surveyed produced a single peak of radioactivity (see Fig. 2 4 ) . NMT was required for generation of the two peaks: addition of 4-oxotetradecanoic acid to Pseudomonas acyl-CoA synthetase yielded a single acyl-CoA derivative (see Fig. 3).3

).

Since CO-4 contains two carbonyl groups (a ketone and a thioester), the possibility of base-catalyzed, intramolecular, aldol-type reactions exists. It, therefore, seems possible that the two peaks observed for CO-4 actually reflect the analog and a self-condensation product or even two self-condensation products. Thus far, we have been unable to confirm this possibility, although numerous plausible condensation products have been considered. It is also possible that CO-5 could undergo similar self-condensation reactions but no evidence of such a process has been observed.

160

120

80

40

n ” 1312 11 10 9 8 7 6 5 4 3

FIG. 1. Conversion of myristic acid analogs to their CoA derivatives by Pseudomonae acyl-CoA synthetase. Details of the in vitro assay are provided under “Experimental Procedures.”

More detailed kinetic analyses (Table 11) revealed that the apparent peptide (GARASVLS-NH2) K, in the presence of saturating amounts of CO-6 was indistinguishable from that obtained with myristoyl-CoA, although the peptide V,,, was -3-fold greater with the analog. The apparent K , and V, for the analog-CoA were remarkably similar to myristoyl-CoA. Analyses of 5-oxo- and 7-oxotetradecanoic acid (CO-5 and CO-7) indicated that they had comparable kinetic properties: their K , for the enzyme was similar to myristoyl-CoA and their effects on peptide (GAR[3H]ASVLS-NH2) catalytic efficiency ( V,/K,) were essentially equivalent (see Table 11).

As was the case for 6-oxotetradecanoic acid (C0-6), 6- oxatetradecanoic acid (0-6) showed the greatest activity in the analogous panel of ether derivatives. While the activities of ketocarbonyl analogs diminished as the functional group approached either the methyl or carboxyl terminus, the effect of moving the ether (C-0-C) unit was different when the ether linkage approached the carboxyl group a diminution in activity was observed as in the carbonyl series but no pro- gressive decline was seen in the 0-6-0-13 analogs (compare A and C in Fig. 2).

Myristic Acid Analogs Containing Ester Functional Groups-The ester functional group was incorporated into the myristic acid backbone by esterifying a series ofdicarbox-

7228 Substrate Specificity of N-Myristoyltransferase

-C- ? I

Position of Substitution

FIG. 2. Screening assay to determine the substrate properties of myristic acid analogs with keto, ester, and ether functional groups. All assays were performed in duplicate or triplicate using [3H]octapeptides representing residues 2-9 of the HIV-I Pr55gag (GARASVLS-NH,, Ratner et al., 1985) or a derivative of residues 2- 9 of the catalytic subunit of mouse PK-A (GNAAAARR-NH, Towler and Glaser, 1986). The concentrations of reactants in the single point discontinuous in vitro NMT assay are described under “Experimental Procedures.” The amount of analogpeptide produced is expressed as a percentage of myristoylpeptide generated in a reference incubation conducted in parallel. The interassay variation in the amount of radiolabeled myristoyl-GARASVLS and myristoyl-GNAAAARR produced in these reference incubations was <30% (Kishore et al., 1991).

ylic acids. Thus, the orientation of the ester functional group (-OC-OR) is opposite to that of the myristic acid carboxyl (-CO-OH). The ester functional group is, in a sense, a com- posite of the carbonyl and ether. Like the carbonyl group, there is a polar oxygen protruding at a distance of -1.3 A from the main hydrocarbon chain. The cylindrical bulk of the carbonyl portion of the ester group is identical to that of an isolated carbonyl. The adjacent oxygen is less basic and less flexible than the corresponding ether oxygens in the oxatetradecanoic acid series because of resonance between it and the carbonyl group. Thus, several comparisons are possible: the ether with the ester and the ester with the carbonyl compounds. Such comparisons allow one to assess the effects of hydrophobicity on substrate activity, ester being the most hydrophilic of the three followed by carbonyl and ether. The effects of rigidity can also be assessed the ester functional group constrains the positions of five contiguous atoms, the carbonyl four, and the ether three. Finally, since the polarity of these three functional groups varies, an additional comparison is possible. The ether functional group is the least polar, but the relative polarity of ester versus carbonyl will depend upon the context: the ester carbonyl is less reactive toward nucleophiles than is an isolated carbonyl but is a stronger Lewis base donor at the oxygen end.

The efficiency of conversion of each of the 10 ester analogs to its CoA thioester is at least as good as that of myristate (with the exception of EST3/4, Fig. 1B). Fig. 2B compares the activities of the esters in the single point NMT assay using the HIV-I-derived octapeptide substrate. Interestingly, monoheptyl adipate (EST6/7) was the most active ester analog. Moving the ester functional group closer to the carboxyl end of the fatty acid produced a more dramatic reduction in activity than was observed with either the ether or the carbonyl group (which are the elements that comprise the ester). The activity profile of the position 6 to position 13 ester analogs resembled the ether analogs more closely than the

AcylCoA Acylpeptide

I

0 0 15 30 45 60 0 15 30

Retention Time (min)

FIG. 3. CIS reversed-phase HPLC of oxotetradecanoyl-CoAs and oxatetradecanoylpeptides. The left-hand column shows the HPLC elution profile of analog-CoA derivatives obtained after incubation of the unlabeled analog with [3H]CoA and Pseudomonas acyl- CoA synthetase. The upper panel also shows the gradient used to separate the reactants (buffer B = acetonitrile). The right-hand column demonstrates the elution profiles of ~ ~ ~ ~ o ~ - G A R [ ~ H ] A S V L S produced in the coupled in vitro NMT assay. Comparison of the left- and right-hand columns demonstrates that the two species obtained with 4-oxotetradecanoic acid were not generated by the acyl-CoA synthetase but rather were dependent upon addition of purified E. coli-derived S. cereuisiue NMT to the reaction mixture. Note also that the appearance of the two species was a phenomenon unique to this oxotetradecanoic acid analog: e.g. unique analogpeptide peaks are seen with 5-OXO-, 6-oxo-, and 7-oxotetradecanoic acid derivatives as well as all the other ketocarbonyl compounds analyzed (data not shown).

carbonyl derivatives (Fig. 2, A-C). More detailed kinetic studies of monononyl succinate

(EST4/5), monooctyl glutarate (EST5/6), and monoheptyl adipate (EST6/7) revealed that the affinities (apparent K,,,) of their CoA thioesters for NMT were similar to each other, to myristoyl CoA, to the corresponding carbonyls, and to the ether derivatives (Table 11). Only modest (-2-fold) reductions in acyl-CoA V,,, were noted with EST4/5 and 516 compared to 6/7. Similar effects on peptide binding were also observed with this series of ester analogs (Table 11).

Taken together, these results suggest that the enzyme can tolerate relatively nonpolar functional groups, even protruding ones, throughout most of the acyl chain. It should be noted that the position of the carbonyl group relative to the carboxyl will determine whether these two carbonyl groups are pointed in the same, or opposite, directions (assuming a fully extended conformation of the myristic acid chain). Thus,


TABLE I1 Kinetic studies of keto and ester anobgs of myristic acid

Kinetic studies were performed using GAR[3H]ASVLS-NH2. The apparent peptide K,,, was determined using an excess of fatty acyl- CoA (15-45 PM). The apparent acyl-CoA K,,, was subsequently ascer- tained using a peptide concentration equal to its experimentally determined K,. V,,, is expressed as a percentage of the value obtained with C14:O (peptide V,,, = 182 & 102 pmol/min/pg of purified E. coli- derived S. cereuisiae NMT; myristoyl-CoA V,,, = 120 + 54 pmol/min/ Ire of enzvme: n = 22 exDeriments. see Kishore et al.. 1991).

Compound

MY r CO-5 CO-6 CO-7 EST 4/5 EST 5/6 EST 6/7

Peptide

K, V, V, /Km %

9 + 2 100 11 25 + 6 204 f 24 8 1 8 + 6 3 0 4 2 14 17 37 + 5 203 f 57 6

15 + 3 4 3 + 13 3 37 + 15 108 + 62 3 14 + 2 115 + 13 8

Acyl-CoA

K , Vm %

6 + 2 100 2 161 4 162 3 243 4 66 5 59 4 141

Chain Length

FIG. 4. CIS reversed-phase HPLC of peptides containing ketocarbonyl or ester functional groups. The HPLC conditions used are described in Kishore et al. (1991). Peptide = GARASVLS- NHZ.

a carbonyl group at position 5 will be oriented with a dihedral angle of zero (synclinal) with respect to the Cl carbonyl and at C6 the carbonyl angle will be 180” (antiperiplanar) in the absence of other forces. There may, however, be severe con- straints placed upon the conformation by the residues which form the acyl-CoA binding site of S. cerevisioe NMT.

Our analog panel revealed one other positional effect (Fig. 4). Hydrophobicity, as judged by acylpeptide retention time on the CIS reversed-phase column, varied as a function of distance between the carboxyl and functional group. Thus, when the ester group in the analogGARASVLS was moved farther from the amide linkage, retention time decreased in a monotonic fashion. For example, EST3/4-peptide behaved on this chromatographic matrix as if it were a dodecanoylpeptide, while the ESTll/lZ-peptide resembled a decanoylpeptide. Comparable differences in hydrophobicities were apparent with the corresponding oxa- and thia-tetradecanoylpeptides (e.g. see Kishore et al., 1991 for an analysis of 3-oxatetra- decanoyl- through 13-oxatetradecanoylGARASVLS-NHJ. It is unclear at the present time why such dramatic differences in retention time would be caused by “simple” displacement of functional groups in an acylpeptide that has high overall polarity. The demonstration of such sensitivity may provide a powerful model system for (i) operationally defining the conformation of acyl chains in acylpeptides; (ii) exploring the contribution of acyl chains to the interactions of acylpeptides and acylproteins with model or naturally occurring mem- branes; and (iii) describing the environment “experienced” by the acyl chains within these membrane systems.

Analyses of the Peptide Functional Group in Two Isomeric Arrangements-The ability of the enzyme to accommodate the carbonyl group either in its “simple” form or as part of the ester functional group raised the question of how varied the carbonyl-containing functional group could be. In addition, we wondered whether or not the enzyme would be “confused” by the presence of an amide functional group. Although the enzyme possesses two functionally distinguish- able binding sites (Towler et al., 1987a, 1987b; Heuckeroth et al., 1988, 1990; Heuckeroth and Gordon, 1989; Rudnick et al., 1990,1991; Kishore et al., 1991), their structural relationships have not been well characterized. The introduction of a peptide functional group (i.e. amide) in the fatty acid chain, challenges the system to distinguish whether it is a component of a peptide or a myristic acid analog. Therefore, two series of analogs were synthesized (i) the amides (AMD) -NH-CO-

and (ii) the acylamino acids (AAA) in which the amide functional group is oriented -CO-NH-. The size and shape of these amide derivatives are expected to be similar to the esters while their polarity and rigidity (due to resonance) are greater than the esters, irrespective of the amide’s isomeric arrangement. All nine of the amide derivatives surveyed were substrates for the acyl-CoA synthetase (Fig. IC). However, as with EST3/ 4, AMD3/4 was barely accommodated. Unlike EST4/5 and EST5/6, the corresponding amide analogs (AMD4/5 and AMD5/6) had activities of <20% of myristate. Any position- ing of the amide group closer to the w terminus yielded analogs that were considerably more active substrates for the synthetase (activity, 55 & 12% of C14:O). Despite the relatively efficient conversion of AMD6/7-AMD12/13, none of their CoA derivatives yielded detectable levels of analogpeptide after incubation with NMT (limits of detection 25% of myristoylpeptide). AMD3/4-516 appeared to be poor substrates for both acyl-CoA synthetase and NMT (i.e. no analogpeptide production was detected in the single point assay).

Of the eight acylamino acid analogs tested, only three were converted to their CoA derivatives at measurable levels. The three analogs in which the functional group was most remote from carboxyl were the only ones thioacylated. Fig. 1D illus- trates the sharpness of the positional boundary between active and inactive substrates (compare the remarkable and dramatic difference in activities between AAA7/8 and AAA8/9).

The CoA thioesters of AAA8/9-AAA12/13 were further characterized in the discontinuous in vitro NMT assay. None yielded detectable levels of analogpeptide (data not shown).

Together, these results allowed us to draw two conclusions. First, irrespective of the orientation of the isomeric amide groups, the CoA derivatives are not substrates for S. cerevisiae NMT. Second, the amides were generally thioacylated whereas the acylamino acids generally were not. Given the ordered Bi Bi reaction mechanism of NMT, failure of these compounds to yield analogpeptide could reflect their inability to bind to the enzyme’s acyl-CoA binding site or their failure to be transferred to bound peptide. We examined these pos- sibilities as follows: the fatty acid or the enzymatically generated analog-CoA was tested for its ability to inhibit transfer of [3H]myristate from myristoyl-CoA to a well characterized (Towler et al., 1987a, 1987b) octapeptide substrate derived from residues 2-9 of the catalytic subunit of CAMP-dependent protein kinase (GNAAAARR-NHp). A 4-400-fold molar excess of AMD4/5, AMD5/6 or AMD6/7 (or AAA4/5, AAA5/6, or AAA6/7) over myristoyl-CoA, did not reduce the amount


of [3H]myristoylpeptide generated compared to control incubations containing no analog (n = 3 independent experiments with each assay done in duplicate). In addition, no inhibition of NMT activity was observed with a 40-fold molar excess (relative to myristoyl-CoA) of the CoA derivatives of AAA8/ 9-12/13 or AMD4/5-6/7 (n = 2 experiments). Thus, these compounds fail to bind to purified, E. coli-derived, S. cerevisiae NMT in vitro.

Myristic Acid Analogs Containing Terminal Azido Groups- The azide function is unique in organic chemistry. The three nitrogen atoms, N-=N=N'- are a less reactive linear array than such linear functional groups as -N=C=O or -N=C=S. The -NB functional group is a ?r electron-rich cylinder although it is attached at an angle to carbon. Azide is relatively nonpolar: the dipole moment for phenylazide (C6HB-NB) is -1.440 (Debye) compared to C,H,-C=N, where g = -4.050. The molecular volume of azide is also relatively small. The conformational preference (-AGO) for the equatorial position of cyclohexane is 1.70 kcal/mol for -CHs and 1.75 kcal/mole for -CH,CH3. Azide has the cylindrical symmetry of cyano (NsC-) or acetylene (HC=C-) which show conformational preferences of less than 0.5 kcal/mol.

Compared to a simple hydrocarbon (i.e. (CH2),CH3), azide is more reactive, more polar, and smaller, but the polarity and reactivity of the system are not dramatic. Azide occupies less volume than propyl but is similar in overall length. It is a poor H-bond acceptor although superior to a simple hydrocarbon.

Like the acetylene linkage that has previously been explored (Kishore et al., 1991), azide, when attached to carbon, is a linear array of four atoms. Unlike the acetylene group, the azide function must be at the end of the alkyl chain rather than within it. Azide was included at the w terminus of four N3-(CH),-COOH analogs (where n = 8, 10, 11, and 12). In contrast to the acylamino acid derivatives, each of these compounds was an excellent substrate for the Pseudomonas acyl-CoA synthetase (percentage of conversion to analog- CoA = 150-200% that of myristate, see Table 111). 12-Azido- dodecanoic acid (CIPN3) has the same overall length as pen- tadecanoic acid and was the most active substrate in the single point assay with either of the two octapeptides (Fig. 5). Detailed kinetic studies (Table 111) confirmed these observa- tions. The relative activities of the azide derivatives were similar to that observed with saturated fatty acids of equivalent chain length (i.e. C12:0, C14:0, C15:0, and C160, see Kishore et al. (1991)). These data indicate that the nonbulky, nonpolar, and nonhydrogen bonding properties of the azide function emulate the methyl terminus of a saturated fatty acid of comparable chain length.

Nitrogen Heterocycle Containing Myristic Acid Analogs- Previous studies of 10-(2-thienyl)decanoic acid and GSAA- SARR-NHz or GARASVLS-NH, indicated that it was more

TABLE 111 Kinetic studies of fatty acids containing terminal azido groups

See legend to Table I1 for experimental details. The abbreviations are provided in Table V.

Peptide Acyl-CoA AnalogCoA formation (% myris- Compound ____

K m V , V J K , K , V , toyl-CoA)

% 9AZ 31 117" 4 2 91

l l A Z 20 120 6 151

12AZ 66 159 2 3.5 98 193 4 145 173

13AZ 92 64 0.7 27 86 200 V , is expressed as a percentage of the V, observed with myristoyl-

CoA.

= GSAASARR

13AZ 12AZ 1 1AZ QAZ FIG. 5. Comparison of the activities of azido-fatty acid an-

alogs having different chain lengths. The numbers inparentheses refers to the retention time (in min) of the analogpeptide on the CIS

reversed-phase column relative to the retention time of the corresponding myristoylpeptide. The negative numbers refer to the fact that the analogpeptide was retained for a shorter period of time using the HPLC conditions described in Kishore et al. (1991). See Fig. 4 for the relative retention time of heptanoyl- through hexatetradeca- noylpeptides.

active than myristate, whereas 12-(2-thienyl)dodecanoic acid was considerably less active (Kishore et al., 1991). In contrast, 12-(2-furyl)dodecanoic acid showed almost identical activity to myristate (Table IV and Kishore et al., 1991). A final group of analogs was therefore synthesized in which the hydrocarbon chain was terminated by a nitrogen heterocycle. The three heterocycles, imidazole, triazole, and tetrazole, are all five-membered rings and contain 2, 3, or 4 nitrogen atoms, respectively. The heterocycles were alkylated at either nitrogen or carbon. The overall shape is similar for 0-, s-, and N- containing heterocycles.

We anticipated that since C-alkylation, rather than N- alkylation, of the nitrogen heterocycles results in a free NH residue, hydrophilicity and polarity would be greater than for the N-alkylated isomers. The results of the analog surveys described above and in earlier publications (Heuckeroth et al., 1988, 1990; Kishore et al., 1991) have suggested an inverse correlation between the overall polarity of the analog (exclu- sive of protein) and its activity in the in vitro NMT assay. This led us to predict that the N-alkylated isomers would be less active than the C-alkylated compounds. All of these compounds were substrates for the Pseudomonas acyl-CoA synthetase (activity varied between 20 and 60% of myristate, Table IV). Whenever a free NH was present, the activity of the analog CoA in the presence of NMT and either GSAA- SARR-NH2 or GARASVLS-NH2 was far lower than the N- alkylated isomer. For example, imidazole derivatives of un- decanoic and dodecanoic acid were more active when N- alkylated (compare analogs 2-IM-11 and 1-IM-11 with 2-1" 12 and 1-IM-12 in Table IV). The tetrazole derivatives of dodecanoic acid (compounds 5-TET-12 and 1-TET-12) provide a dramatic illustration of this difference: the activities of these two compounds differ by an order of magnitude in the presence of either GSAASARR-NH, or GARASVLS-NH, even though they have comparable reactivity in the acyl-CoA synthetase assay (31-49% that of myristate, see Table IV).

Conclusions from the Coupled in Vitro NMT Assay-These studies indicate that the acyl-CoA binding site of S. cerevisiae NMT has a remarkable spectrum of sensitivities. Analyses of the ketocarbonyl-, ester-, and amide-containing analogs indicate that the binding site is able to accommodate a dipolar protrusion from C4 through C13 of myristoyl-CoA analogs. This protrusion is tolerated without compromising activity on either side of the main chain and even where the chain is bent in the vicinity of C5-C6. In a qualitative sense, the


TABLE IV Comparison of the activities of fatty acid analogs with 2-thienyl, 2-fury1, and nitrogen heterocycle groups

Abbreviationb

TH-10

TH-12

FUR-12

2-IM-11

1-IM-11

2-IM-12

1-IM-12

2-MIM-12

1-TRI-12

1-TRI-12A

1-TRI-13

2-TRI-13

5-TET-12

1-TET-12

Analogpeptide Formed Analog-CoA (O/o Myristoylpeptide) Formation

GSAASARR GARASVLS (Yo Myristoyl-CoA)

160 120 6

30 45 178

90 85 126

c5 C 19

15 59

c5 23

19 23

c5 32

214 58

23 26 54

9 54

14 45

<5 c5 31

46 102 49

a Taken from Kishore et al., 1991. TH, thiophene; FUR, furan; IM, imidazole; TRI, triazole; TET, tetrazole. -, not determined.

activities of NMT's acyl-CoA substrates diminish with increasing polarity. This relationship was apparent in analogs containing nitrogen heterocycles in which C- and N-alkylated isomers could be compared. Comparison of the closely related ester- or amide-containing tetradecanoyl-CoAs further confirm this inverse relationship between polarity and activity.

The ketocarbonyl-, ester-, azido-, and nitrogen heterocycle- containing analogs were all substrates for the reportedly nonspecific Pseudomonas acyl-CoA synthetase. However, analyses of isomeric amide-containing tetradecanoic acid analogs revealed a striking sensitivity of the synthetase for the orientation of the carboxyl group in a fatty acid. This sensitivity extends from C3 to C8 but is lost thereafter. The fact that none of the amide (AMD, AAA) CoA derivatives can bind to NMT, in contrast to the corresponding EST-CoA derivatives,

suggests a fundamental difference in the way each enzyme senses its substrate.

Biological Activities of Myristic Acid Analogs-Recent metabolic labeling studies using tritiated oxatetradecanoic acids with oxygen substitutions at C6, C11, and C13 and several cultured mammalian cell lines indicated that these low molecular weight (Mr 230-250) compounds readily traverse the cell membrane and are substrates for both acyl-CoA synthetase and NMT (Heuckeroth and Gordon, 1989; Johnson et al., 1990). Analogs are selectively incorporated into distinct yet overlapping subsets of cellular N-myristoylproteins. This selective, analog-specific incorporation presumably arises because of the cooperative interactions that occur between NMT's acyl-CoA and peptide binding sites and the enzyme's ordered Bi Bi reaction mechanism (i.e. different analog CoAs


TABLE V Antiviral activity of myristic acid analogs

Compound abbreviation Structure

Ketocarbonyl analogs C013 C08 C06 C03

Ester-containing analogs EST12/13 ESTll i l2 EST10/11 EST9/10 EST8/9 EST718 EST617 EST516 EST415 EST314

Amide-containing analogs AMD12/13 AMD11/12 AMD10111 AMD9/10 AMD8/9 AMD7/8 AMD6/7 AMD5/6 AMD4/5 AMD3/4

Acylamino acids AAA12/13 AAA9/10 AAA8/9 AAA7/8 AAA6/7 AAA5/6 AAA4/5 AAA3/4

Azido-substituted analogs 13AZ 12AZ 11AZ

Triazolyl and tetrazolyl-substituted 1-TRI-12 1-TRI-12A 1-TRI-13 2-TRI-13 TET-12 TET-13 1-TET-12

Analogs with imidazolyl groups 2-MIN-12 2-IM-12 2-IM-9

12(N-l-Methyl-2-imidazolyl)-(CHZ)llCOOH 12(Imidazol-2-yl)-(CHz)ll-COOH 9-(Imidazol-2-~l)-(CH~)~ COOH

Dose of analog (pg/ml)

1 3 10 % reduction in cytopathic

effect’

6 14 40 0 5 30 6 8 34 0 5 7

17 27 0 0 0 0 0 0 0 9

31 34 39 T 0 0 0 0 0 0 0 0 0 0 0 2 0 3

20 30

5 0

2 0 0 3 2 0 1 0 0 2 0 0 2 3 3 4 0 0 0 0 0 0

d -

-

-

6 0 1 0 0 2 0 1 2 0 4 0 2 24 8 0 4 0 0 0 0

-

-

-

0 0 82 72 38 16 35

-

-

19 24 T 12 0 0

12

- 31 66 T

0 16 65

- - - -

- - -

15 31 0 3 5 0 0

-

-

45 15 32 18

>loo 6

>loo >loo >loo >loo >loo >loo

32 32

165

>loo >loo >loo >loo >loo

26 16 12

107 >loo

95 >loo

100 65 66 23

17 46 26

10 27 <1 22

168 179 34

62 >loo >500

See “Experimental Procedures” for details of this assay. *Dose of analog that produces a 50% reduction in the number of viable CME lymphocytes (cells were not

e T, toxic; defined as producing a >50% killing of uninfected cells at this concentration. infected with HIV-I when determining the TDm of analog).

-, not determined.

produce different effects on the peptide binding site). For (selective incorporation and selective perturbation of func- some cellular N-myristoylproteins, incorporation leads to an- tion) probably accounts for their lack of cellular toxicity. alog-specific and -dependent redistribution from membrane Some, but not all, oxatetradecanoic acid analogs can inhibit to cytosolic fractions (i.e. a given protein may undergo redis- replication of HIV-I in acutely and chronically infected CD4+ tribution with one, but not another, analog depending upon H9 cells (Bryant et al., 1989,1991). 13-Oxatetradecanoic acid the location of the heteroatom for -CH2- substitution, com- is the most potent member of this group of compounds. Its pare with Johnson et al. (1990)). This dual level of selectivity maximum inhibitory effect was observed at 20-40 p~ and was


not accompanied by cellular toxicity. Its mechanism of action involves, at least in part, incorporation into the Pr55gag and nef proteins of HIV-I (Bryant et al., 1991). The 13-oxatetra- decanoyl-containing gag polyprotein precursor undergoes redistribution from membrane to cytosolic fractions and a reduction in its proteolytic processing which is possibly due to an effect of the analog on dimerization of gag-pol and subsequent release of viral protease (Naria et al., 1989; Bryant et al., 1991).

These results suggested that cellular acyl-CoA synthetase and NMT activities could be exploited to deliver these analogs to target proteins and indicated that HIV-I replication was a sensitive and useful model system for assaying such events. We, therefore, screened members of the panel of 56 analogs described above in an acute viral replication assay to infer whether they may function as alternative substrates for these two enzymes and whether their incorporation into viral/ cellular proteins could interfere with viral assembly. Since Pr55gag and nef are both substrates for S. cereuisiae NMT (Jacobs et al., 1989; Bryant et al., 1991), we also tried to ascertain whether the activity of an analog in the coupled in uitro enzyme assay containing Pseudomonas acyl-CoA synthetase, S. cereuisiae NMT, and GARASVLS-NH2 could accurately forecast its antiviral activity.

The results of this antiviral screen are presented in Table V. Compounds were tested at concentrations of 1, 3, and 10 pg/ml (approximately 4-40 p ~ ) using acutely infected CEM cells and an assay adapted from Pauwles et al. (1988, see “Experimental Procedures”). Inhibition of viral replication was quantitated by observing the percentage of reduction of virus-induced cytopathic effect (CPE). Several of the ketocar- bony1 containing analogs that were good substrates for Pseu- domonas acyl-CoA synthetase and S. cereuisiae NMT (C013, C08, C06) produced 30-40% reductions in virus-induced cytopathicity at a concentration (40 JLM) that was 2-4-fold lower than the dose that killed 50% of noninfected cells (defined as the TD50). 3-Oxotetradecanoic acid (C03) which is the least active NMT substrate in the series (Fig. 2 A ) was the least active in the acute viral replication assay ( 4 0 % reduction in CPE. at 40 pM, a dose close to its TD50, see Table V). The most active ester analog in the coupled in uitro NMT assay, monoheptyl adipate (EST6/7, see Fig. 2B) had no antiviral activity, as was the case for most of the 10 analogs in this series. It is interesting to note that of the two EST compounds with modest, specific inhibitory effects (ESTlZ/ 13 and EST3/4, Table V), the latter was a very poor substrate for both the prokaryotic acyl-CoA synthetase and the yeast acyltransferase (see Figs. 1B and 2B). As expected from the in uitro assay results, analogs containing the peptide functional groups in either of the two isomeric arrangements were generally inactive ( 4 0 % inhibition of CPE at concentrations up to 40 p ~ ) . The only exception appeared to be AAA6/7 which produced a 24% reduction in CPE at a dose that was 10-fold below its TDso. The triazoles tested were quite toxic at low concentrations (the TD50 ranged from <4-100 p ~ ) . The analogs surveyed with tetrazolyl or imidazoyl groups were inactive or toxic, except for 12(N-l-methyl-2-imidazoyl) dodecanoic acid (cf. Table IV) which produced a modest 31% reduction in CPE at 40 JLM, a concentration which was 6-fold lower than its TD5o (Table V).

The most active compound of the 45 surveyed in the acute viral propagation assay was 12-azidododecanoic acid (12AZ): at 4 pM it produced an -80% reduction in cytopathic effect. The IDSO of 12AZ was <4 JLM while its TDso was 184 p ~ . These later values were comparable to those of 13-oxatetradecanoic acid (IDGo (4 p ~ , TDSo = 90 PM). The antiviral

2.0

3 1.0

1 10 50 100

D ” 10 50 100

” 10 50 100

Concentration (pM)

FIG. 6. Effect of myristic acid analogs containing terminal azido groups on replication of HIV-I in H9 cells. A, H9 cells were acutely infected with HIV-I (see Bryant et a!., 1989) and treated with myristic acid analogs, decanoic acid (ClO:O, hydrophobicity similar to that of 11-azidoundecanoic acid (11-AZ) and 12-azidododecanoic acid (12-AZ), see Fig. 4), or AZT at the indicated concentrations in serum-containing medium for 10 days. Medium was changed every 2 days. The antiviral effect of these compounds was determined by measuring reverse transcriptase activity (Poiesz et al., 1980) in cell culture supernatants prepared on the last day of the experiment (ie. containing any virus produced between days 8-10). AZT was used as a positive control. No additions or 0.1% ethanol (its concentration in media containing 100 p M analog) were employed as negative controls (earlier experiments indicated ethanol had no effect on virus production; cf. Bryant et al., 1989 and data not shown). The mean plus S.E. are shown; triplicate samples were assayed, all assays were performed in triplicate. B, the same media collected for reverse transcriptase assays were also used for measurement of the p24 viral antigen by enzyme-linked immunosorbent assay (Bryant et al., 1989). Note that the concentration of p24 antigen in cultures incubated with serum-containing media alone or with medium plus 0.1% ethanol was 2 pg/ml. C and D, the toxicity of 11-azidoundecanoic acid and 12- azidododecanoic acid was assessed by measuring viable cell number on the last day of the experiment (viability defined by trypan blue exclusion, control = cells incubated with medium alone, C ) and by incorporation of [3H]leucine into cellular protein after a 4-h pulse labeling (results expressed as a percentage of the incorporation observed in untreated cells, D).

activity of 12-azidododecanoic acid contrasted with the lack of activity of 13-azidotridecanoic acid and the modest reductions in CPE produced by 11-azidoundecanoic acid (16% at 4 p ~ , 35% at 10 PM, TDGo = 100 KM, see Table V). The great differences in biological activities of these analogs was not anticipated from their relative activities in the coupled in vitro NMT assay system (Fig. 5 ) .

To verify this finding, a second assay for acute viral infection was employed that used a different T-lymphocyte cell line (H9) and different parameters for measuring viral replication (reverse transcriptase activity and p24 antigen accu- mulation in culture supernatants). The results (Fig. 6) confirmed that 12-azidododecanoic acid inhibited viral replication in acutely infected cells more effectively than 11-


A Reverse Transcriptase 100

= 80 60

0 40 E 20

0

B P24 100

= 80 2

0 40 5 60

E 20 0

C 3H-Leucine Incorporation

D Viable Cell Count 100

= 80 2 5 60 0 40 s 20

n EtOH C1O:O 013 AZT 12AZ

FIG. 7. Effect of myristic acid analogs containing terminal azido groups or an oxygen for methylene group substitution on the propagation of HIV-I in chronically infected HOIIIB cells. The antiviral effects of 11-azidoundecanoic acid (11-AZ, 50 /IM) and 12-azidododecanoic acid (12-AZ, 50 p ~ ) were compared to 13-oxatetradecanoic acid (013, 50 p ~ ) , an analog previously shown to inhibit viral propagation in this assay system (Bryant et al., 1991), decanoic acid (ClO:O, 50 pM), AZT (5 pM), or ethanol (0.1%). Chron- ically infected cells were treated with the compounds over a 12-day period using a protocol described in an earlier publication [Bryant et al., 1991). Medium containing serum and the compound was replaced every other day. On day 12, medium was harvested and assayed for reverse transcriptase activity ( A ) and p24 antigen ( B ) . Cell viability was assessed by trypan blue exclusion ( C ) and by incorporation of [3H]leucine (D). Control = H9IIIB cells that had been incubated with serum-containing medium alone. Reverse transcriptase and p24 assays were performed on triplicate samples and the results expressed as the mean plus S.E. Measurements of cell viability were done on duplicate samples and the results were averaged.

azidoundecanoic acid (60% uers'sus <lo% at 10 p ~ ) and with a dose-response (from 1 to 100 p ~ ) that was comparable to that of 13-oxatetradecanoic acid (see Fig. 6, A and B ) . The antiviral effect of 12-azidodecanoic acid observed at the concentrations tested was not accompanied by significant cellular toxicity, as judged by the number of viable cells remaining after 10 days of treatment (no detectable differences from control, Fig. 6D) or by the incorporation of tritiated leucine into newly synthesized proteins (no difference at 10 pM, 20- 30% inhibition at 50-100 p ~ , Fig. 6C).

We do not have any labeled 12-azidododecanoic acid to perform metabolic labeling studies that might define its mechanism of action. However, we were able to confirm that this analog, like 13-oxatetradecanoic acid, can inhibit virus production by 60-70% (at 50 p ~ ) in chronically infected H9IIIB cells without appreciable associated cellular toxicity (less than 20% reductions in viable cell count or tritiated leucine incor-

poration, Fig. 7). This contrasts with the inability of AZT, an inhibitor of viral reverse transcriptase, to produce reductions in virus production in this model assay system (see Bryant et al., 1991).

12-Azidododecanoic acid thus represents the second myristic acid analog which has demonstrated efficacy in blocking viral production in acutely and chronically infected human lymphocyte cell lines (the other being 13-oxatetradecanoic acid). Its ability to serve as an excellent substrate for both Pseudomonas acyl-CoA synthetase and S. cereuisiae NMT is consistent with the notion that it enters lymphocytes, is converted to its CoA thioester, and serves as a substrate for human NMT. However, this proposed mechanism must be directly tested, especially in light of the fact that for many of the other types of analogs examined there was a poor correlation between activity in the coupled in vitro assay system and their antiviral effects. The lack of such correlation em- phasizes the need to compare and contrast the substrate specificities of human acyl-CoA synthetase and human NMT with those of the corresponding Pseudomonas and yeast enzymes. This information, together with an assessment of the mechanism of analog uptake into lymphocytes and the path- ways of their subsequent metabolic processing, should allow us to develop systems that will more accurately forecast the ability of different classes of myristic acid analogs to inhibit HIV-I replication.

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Substrate Specificity of N-Myristoyltransferase 7237 (6.70 g. 35%): bp: 122-126°C/0.12 torr: I R 3000 (broad). 1740. and 1710 m i l : 'H-NMR 0.93 13H. 1. J& Hz). 1.23-1.33 (8H. m). 1.51-1.69 16H. m). 2.30 (2H. 1. J=7 Hz]. 2.37 (2H. 1. J=7 Hz). 4.09 12H. 1. Jr6.5 Hz). and 10.7 IIH. brs): Anal. Calcd for C13H2,Oa: C. 63.91: H. 9.9096. Found: C. 63.78: H. 9.94%.

Monopropyl deemcdloate (ESTlO/ll). Dlprapyl decanedloate was prepared In 95% geld from decanedrolc acld 110.0 g. 0.050 moll In propanol 150 mL1 In the presence ofH2S0, (1 mL1: bp: 108-1 12 %/O.l2 tom: 'H-NMR: 0.92 16H. 1. J=7 Hz]. 1.2-1.8 116H. ml. 2.27 (4H. 1. J.6.5 Hz) and 4.02 (4H. t. J=6.5 HZ). A mlxture of decanedlolc acld 113.5 g. 0.067 mol). dlpropyl decanedloate I1 1.1 g. 0.039 moll. HCI 11.7 mL) In butyl ether 13.4 mL1 was treated as descnbed above. Rapanal was added (4.0 g. 0.067 mal and then 1.4 g. 0.022 mol alter 30 mlnl. The crude product was punned by short path IKugelrohr) dlstlllauon to afford EST10/11 (5.0 g. 51%): bp: 128-132 0c/0.07 tom: I R 3000 (broadl. 1745. and 1720 cm": IH-NMR 0.94 13H. 1. J.7 Hz). 1.23-1.44 18H. m). 1.53-1.65 (6H. ml. 2.29 (2H. 1. J=7 Hz). 2.34 (2H. 1. 5-7 Hz). 4.03 (2H. t.J=7 Hz). and 9.65 11H. brsl: Anal. Calcd for C I ~ H ~ ~ O , : C. 63.91: H. 9.9096. Found: C. 64.00: H. 9.92%.

Mon~ethylundeeancdlonte @ST11/12). Diethyl undecanedloate was prepared in 85gmyleld from undecanedlolc acid 11.5 g. 0.007 moll In ethanol (30 mL1 In the presence of HzSO, (0.5 mLI: bp: 136.140 "C/0.25 tom: 'H-NMR: 1.25 (6H. 1. J=7 Hz), 1.3-1.8 114H. ml. 2.20 14H. 1. J=7 Hz) and 4.02 (4H. t. J=7 Hz). A mlxture of undecanedlolc add (1.55 g. 7.2 mmol). dlethyl undecanedloate (1.14 g. 4.2 mmoll. HCI (0.2 mLI In butyl ether (0.5 mL1 was treated as d c r n b e d above. Ethanol was added 10.33 g. 7.2 mmol and then 0.1 1 g. 2.4 mmol after 30 mIn~.Thecrudeproductwaspun~edbyshortpath~Kugelrohr)distlllaaontoaff~rdE8T11/12 ~0.98g.48%1,mp:41-42°C.bp: l38-142~C/0.15tom:IR3000(broadl. 1725. 1 6 9 0 ~ m ~ ~ : ~ H - NMR:1.17-1.37110H.m1,1.28l3H,t,J=7Hz),1.62~2H,q,J=7.5Hz),1.6412H,q,J=7.5Hz), 2.28~2H,t.J=7.5Hz).2.3412H.t,J=7.5Hz],4.1312H.q.J=7Hzl.9.9611H.brsl.AnaLCalcd for CI3H2,O4: C. 63.91: H. 9.9Wh. Found: C. 63.81: H. 9.834.

Manomethyl dodeemcdbate lEST12/13): Dlmethyl dodecanedloate was prepared In 85% yleld from dodecanedloic add (5.0 g. 0,022 moll In methanol (60 mL] In lhhe presence ofqso, 10.5 mL1: bp: 136-140 "C/O.Z5 torr: IH-NMR 1.2-1.7 116H. m). 2.29 (4H. 1. ,1=7 H z ) and 3.62 (3H. SI. A mlxture of dodecanediolc add (3.99 g. 0.017 m01). dlmethyl dodecanedloate (5.16 g, 0.02 moll. HCI (0.8 mL1 In butyl ether (3.5 mL1 was treated as descnbed above. Methanol was added I1 mL and then 0.05 mL after 30 mlnl. The Crude product was purllled by short path (Kugelrohrl dlstlllatlon to afford FST12/13 (0.84 g. 9%1: mp: 49-51 'C (from hexane) (lit. ILyCan and Adams 1192911 mp: 51 'CI: I R 3000 lbraad). 1745. and 1715 cm": IH-NMR 1.1- 1.4~12H.rn1.1.5-l.7l4H1mm).2.3012H,t.J=6Hz-lzl.2.32~2H.t.J=6Hz~.3.6813H,s).and10.5 IIH. brsl.

2-Ov-DeeyUclrbrmoykcetIc acld [AMD3/4): Decylamlne (3.14 g. 20 mmol). monoethyl Amide Analogs of Myristic Acid (''AMD'']

malonate 12.64 g. 20 mmoll. and N-hydroxysucclnlmlde (2.41 g. 21 mmol) were dissolved m dry N.N-dimethylfomamide (DMF. 30 mL]. The solutlon was stirred and cooled to -10 OC dunng the addltlan of dlcyclahexylcarbadllmlde (4.33 g. 21 mmol) In DMF 110 mL). Afler 2h at -10 'C and another 24h a t room temperature. the solvent was removed under reduced pressure. The residue was dlssolved in EtOAc (200 mL). The arganlc phase was washed succe6s1vely wlth 5% HCI (3x50 mL). 5% NaHC03 l3x50mL). water 12x50 mL]. bnne (50 m ~ ) . and dried lNa2SOJ. The crude product was purtned by short path (Kugelrohr] dlSUllaUon to

give the coupllng product. ethyl 2-(N-decyl)carbamoylethanoate (4.1 g. 76%). mp: 41-43 Oc; 1H-NMR0.87I3H,t.J=6Hzl.1.1-1.6(I9H.m).3.I9(2H.q.J=6.5Hz).3.27(2H.s).4.16(2H. q. J=7 HZ). 7.13 (1H. bnl . To a solutlon of ethyl 2-(N-decyllcarbamoylethanoate (2.71 g. 10.0 mmoll In methanol (20 mL) was added 1M NaOH 121 mL. 2 1 mmol]. The reactlon mixture was S m e d for 2h at mom temperature. and then acldlned with 10% HCI (pH=II. After v ~ a c u o n with EtOAc I150 mL]. the organic phase was washed wlth water (2x50 mL). bnne ( 5 0 rn~], and dned (Na$3O,I. The crude product was punned hy short path [Kugelrahr) dlsullauon and subsequent recrystalllzauon from an EtOAc-hexane mixture to afford AMD3/4 [ I . 18 g, 5411: mp: 76-78OC: I R 3310. 3050 (broad). 1710. 1625 cm-l: 'H-NMR (400 MHz. 20% DMSO-de LnCDCl3I:0.88~3H.t.J=6Hzl.1.16-1.38(14H.m~.1.5412H.q.J~6.5~~.3.19(2~.q,J~6.5

Found: C. 63.65 H. 10.42: N. 6.04%. Hz]. 3.23 (2H. SI. 7.83 (1H. brsl. Anal. Calcd For C,aH25N03: C. 64.16: H. 10.36. N. 5.76%.

S ( n r - N o n g ~ e ~ o y l p m p ~ o l c add IAMDr/Sl: Nanylamlne 12.75 g. 19 mmoll. monomethyl Succinate 12.54 g. 19 mmol). and N-hydroxysucdnlmlde (2.33 g. 20 mmol) were dlssolved In dry DMF (45 mL1. m e solutlon was stirred and cooled to -10 'C during the addltlon of dlcyclohexylcarbodllde 14.16 g. 20 mmal) ~n DMF 115 mL1. After 2 h s t -10 OC and another

dissolved In EtOAc (200 mL1. The organlc phase was washed successively with 5% HCI (3x50 36h at room temperature. the solvent was removed under reduced pressure. The residue was

mLI. 5% NaHC03 13x50 mL1. water (2x50 mL1. brine 150 mL1. and dned (Na2S0,1. The -de product was p m n e d by short path (Kugelrohrl dlstlllauon to glve the coupllngproduct. methyl 3-1N-nonyl)carbamoyIproplonate (4.3 g. 87%). mp: 52-53 "C 'H-NMR 0.87 13H. 1. J.6 Hz).

5.78 IIH. brsl. To a soluuon of methyl 3-lN-nonyl)carbamoylpraplonate 13.0 g. I2 mrnal] In 1.1-1.6(14H.m1.2.5l 12H.t. J=7Hzl.2.57(2H. t. J=7HzI. 3.21 (2H. q. J=6Hz1.3.6613H. s).

methanol 125 mL1 was added 1M NaOH (23 mL. 23 mmol). Thhe reacuon mlxture was stlmed for 2h at room temperature. and then acldlned with 10% HCI lpH=l). After exlractlon with

EtOAc~150mLl,thearganlcphasewaswashedwlthwater~2x50mL).brtnel50mL).anddned

subsequent recryStalllzaUon from an EtOAc-hexane mmure to afford AMDQ/S (2.10 g. 74%): lNa+041. The crude product was punned by short path IKugelrohrl dlsUllatlon and

mp: 111-111.5 OC: IR: 3310. 3000 (broad). 1695. 1645 Cm~l: 'H-NMR (10% DMSO-de In CDCI3l:0.9013H,t.J=6Hz).1.20-1.39(12H,ml.1.4812H.q.J=6.5Hz).2.4612H,t.J~7Hz). 2.56 (2H. g. J=6.5 Hz). 3.15 (2H. q. J=6.5 Hz). 7.48 IIH. brs). Anal. Calcd For C,3HzsN03: C. 64.16: H. 10.36. N. 5.76%. Found: C. 64.09 H. 10.41. N. 5.72%.

pentanedloate 13.66 g. 15 mmoll. and N-hydroxysucclnimide (1.81 g. 16 mmol) weredlssolved 4-(N-~tyl lcub~oytbvtrnolc lcld WdDI/61: Octylamlne (1.94 g. 15 mmol). monoo~tyl

In dry DMF (30 mL). The solution was stirred and cooled to -10 'C during the addluon of dlcyclohexylcarbodIIrnlde (3.25 g. 16 mmol) In DMF (10 mLI. After 2h at -10 OC and another 24h at rWm temperature. the solvent was removed under reduced pressure. The resldue was dlssalved In EtOAc I150 mL). The organlc phase was washed successlvely wlth 5% HCI (3x50 mL1. 5% NaHC03 13x50 mL1. water 1 2 x 5 0 mLI. bane I50 mLI. and dned lN,+OI1. The crude product was pumllned by short path IKugelrohrl dlstlllatlan to glve the couplmg product. octyl 4-lN-octyyllcarbamoylbutan~~t~ 14.41 g. 83%): bp: 166-170 'C/0.25 tom; 'H-NMR 0.88 (6H. 1. J=6 Hz]. 1.1-1.7 l24H. ml. 1.96 (2H. 1. J=7 Hz]. 2.14 (2H. 1. J=7 H z ) , 2.37 12H. 1. J=7 Hz]. 3.18 12H. q. J=7 Hz). 4.04 12H. 1. J=6 Hz). 5.80 IIH. brsl. To a soluuon of octyl 4-IN- actyllcarbamoylbutanoate (3.55 g. 10 mmol) In methanol 125 mLI was added 1M NaOH (20 mL. 20 mmoll. The reactlon mlxture was stlrred for 2h at room temmrature. and then acldlned

wllh 10% HCI (pH=l). After eXtraCUon with EtOAc (150 mL1. the organlc phase was washed wlth water (2x50 mL). bnne 150 mL). and dned INa2S041. The crude product was punned by short path IKugelrohrI dlsullatlon and subsequent recrystalllzatlon from an EtOAc-hexane mlxturetoaffordMm)5/611.59g.651):bp:176-180YC/O.15torr:mp:61.5-63aC:IR:3345. 3000 (broad). 1705. and 1640cm I : 'H-NMR 0.89 13H. 1. J=6 Hz). 1.18-1.36 IlOH. ml. 1.46 (2H. q. J=7 Hz). 1.94 12H. q. 5.7 Hz). 2.29 (2H. t. J=7 Hz], 2.40 l2H. 1. J=7 Hzl. 3.20 IZH. q. J=7 Hz). 6.39 [IH. brsl. 9.20 (IH. brsl. Anal. Calcd for Cl3HZ5NO3: C. 64.16: H. 10.36. N. 5.76%. Found: C. 63.10 H. 10.40. N. 5.65%

S-(N-Hcptyl)clrbrmoyIpcntmolc add (AMD6/71: Heptylamlne 11.73 g. 15 mmoll. monoheptyl hexanedioate (3.66 g. 15 mmol). and N-hydroxysucclnlmlde (1.81 g. 16 mmoll were dlssolved In dry DMF I30 mLI. The soluuan was SUrred and cooled to -1OOC dunng the additlonofdlcyclohexylcarbodllmide(3.25g. 16mmol11nDMF110mL1.After2hat-10°Cand another 24h at room temperature. the solvent was removed under reduced pressure. Thhe resldue was dissolved in EtOAc 1150 mLI. The organlc phase was washed successively wlth 5% HCI (3x50 mL1. 5% NaHC03 (3x50 ml,L water 12x50 mL1. bnne 150 mL1. and dned lNa,SO,). me crude product was punned by short path IKugelrohrI dlstlllauan to glve the coupling product. heptyl5-(N-heptylJcarbamoylpentanOate14.32g. 84%1: bp: 145~l5O0C/O.IOtorr; 'H- NMR: 0.91 (6H. t. Jr6 H z ) , 1.1-1.5 (20H. m). 2.25 [ZH. I, J=7 Hz). 2.33 12H. t. J=7 Hzl. 3.25

heptyllcarbamoylpentanoate (2.10 g. 6 mmoll In methanol (15 mL1 was added IM NaOH 112 12H. q. J=7 Hz). 4.03 12H. 1. J.6 H z ] , 5.70 I1H. brsl. TO a SOlUUOn of heptyl 5-IN-

mL. 12 mmol). The reaction mlxture was stirred for 2h at room temperature. and then acldlfied wlth 10% HCI (pH=Il. After ertractlon with EtOAc 1100 mL). the organlc phase was washed wlth water 12x50 mL). hnne 150 mL). and dned lNa2S041. The crude product was punned by short path IKugelrahrl disullatlon and subsequent recrystallmtion from an EtOAc-hexane mlxture toaNordAMD6/7 (1.19g. 8281: bp: 194~198°C/0.20 tom: mp: 98-98.5OC: I R 3325, 3000 (broad). 1700. and 1650 cm": 'H~NMR 0.89 13H. 1. J=6 Hz). 1.19-1.38 18H. ml. 1.48

Hz]. 5.89 IIH. brsl. 8.89 IIH. brsl: Anal. Calcd for Cl3HZ5NO3: C. 64.16: H. 10.36. N. 5.76%. l2H.q.J=7Hz~.1.59~1.74~4H.ml.2.2212H.t,J=7Hz1.2.38~2H,t.J=6Hzl.3.23l2H.q.J=6

Found: C. 64.10; H. 10.32, N, 5.74%

heptanedioate (4.88 g. 20 mmoll. and N-hydroxysucclnlmlde 12.42 g. 21 mmol) were dlssolved 6-(N-Hcryl )eubnmoylh~~~ole add (AMD7/81: Hexylamine (2.02 g. 20 mmoll. monohexyl

in dry DMF (30 mL1. The soluhon was stlmed and coaled to ~ 1 0 'C dudng the addltlon of dlcyclohexylcarbodilmlde (4.33 g. 21 mmo1)In DMF 110 mL1. After 2h at -10 'C and another 24h at room temperature. the solvent was removed under reduced pressure. me resldue was

dlssolved In EtOAc I I50 mL1. The arganlc phase was washed successlvely with 5% HCI 13x50 mL1. 5% NaHC03 13x50 mLI. water (2x50 mL1. bnne (50 mL1. and dned (Na,SO,l. The crude product was punned by short path [Kugelrohrl dlstllladon to glve the coupllng product, hexyl 5-lN-hexyllcarbamoylhexanoale 14.65 g. 7104: hp: 158-162 'C: 'H-NMR 0.91 16H. t. J=6HzI.

J=6 H z ] , 5.60 IIH. brsl. To a solution of hexyl 6-[N~hexyl)ca~bam0ylhexan~~t~ 13.27 g. 10

1.1-1.8 122H. m). 2.18 12H. t. 5.7 Hz). 2.28 12H. t. J=7 Hz). 3.22 12H. q. J=6 Hz). 4.05 l2H. 1.

mmdl In methanol (20 mL1 was added IM NaOH I20 mL. 20 mmoll. The reactlo" mlxture was

stlrred for 2h at room temperature. and then acldlned wlth 10% HCI lpH=Il. After ULtracUon with EtOAc (100 mL). the organlc phase was washed with water 12x50 mL1. brtne I50 mL1. and drted lNa,SO,). The crude product was purified by short path IKugelrohr) dlsullauon and subsequent recrystallnauan from an EtOAc-hexane minure to afford AMD7/0 II .57 g. 6511:

NMR:0.89(3H.t.J=7Hzl.l.22-1.43(8H.m).1.48~2H.q.J=7Hzl.1.66l4H.q.J=7Hz~).2.21 bp: 193-197 oC/0.20 tom: mp. 51-52 'C : I R 3340. 3000 (braadl. 1710 and 1645 c m ~ l : 'H-

12H. t. J=7 Hz). 2.32 (2H. 1. Jr7 Hz). 3.25 12H. q. J=6 Hz). 6.03 [IH. brsl. and 10.0 IIH. brsl: Anal. Calcd for CI3HZ5NO3: C. 64.16: H. 10.36. N. 5.76% Found: C. 63.03: H. 10.38. N. 5.63%.

7-Ov-PcntyUeublmogIhept.nolc add (AMDSlSI: Pentylamine 11.74 g. 20 mmall. monopengl

In dry DMF (30 mL). The soIuUon was surred and cooled to - 10 OC during the addltlon of octanedloate (4.88 g. 20 mmoll. and N-hydroxysucclnlmlde 12.42 g. 21 mmol) were dlssolved

dicyclohexylcarbodlimide (4.33 g. 21 mmol) In DMF (IO rnL1. After 2h at -10 OC and another 24h at room temperature. t h e 501vent was removed under reduced r essure. The residue was dtssolved in EtOAC (150 mL). T h e organlc phase was washed successively wlth 5% HCI 13x50 mL). 5% NaHC03 13x50 mL). water 12x50 mLI. brine (50 mLI. and dned lNaZSO,I. The crude product was punned by short path IKugelrohr) disUllaUon to give the coupling product. pntyl 7-IN-pentyllcarbamoylheptanoate (5 11 g. 82W: bp: 160- 164 W0.20 torr: 'H-NMR: 0.94 16H. 1. J.6 Hz). 1.1b1.8 (20H. m). 2.19 (2H. 1. J=7 Hz). 2.30 12H. t. J=7 Hz). 3.22 (2H. q. J=6 lh l , 4.07 12H. 1. J=6 H z ) , 5.80 IIH. brs). To a solution ofpentyl 5-(N-pentyllcarbam~ylh~ptan~~t~ (3.13 g. IO mmol) In methanol (20 mL] was added 1M NaOH (20 mL, 20 mmol). The reactton mlxture was sumed far 2h at room temperature. and then acidifled with 10% HCI (pH=l). After exlracuon with EtOAc (LOO mLI. the organlc phase was washed wlth water (2x50 mL1. brlne 150 mLI. and dned (Na2S0,1. The crude product was purtned by short path IKugelrohrl dlstlllaUon and subsequent recry6tallbatJonfroman EtOAc-hexanemlxture toalfordAMD0/9 11.50 g. 62%): bp: 190-194 oC/O.ZO torr: mp: 73-74.5'C: I R 3320. 3000 broad). 1700. and

q.J=7Hzl.2.1612H.t.J=7.5Hzl.2.30~2H.t.J=7.5Hzl.3.1712H,q.J=7Hz).5.95I1H.brs~. 1650 cm.': 'H-NMR 0.88 13H. 1. J=6 Hz). 1.22-1.41 (8H. ml. I 47 12H. q. J=7 Hz). 1.58 (4H.

9.80 IIH. brs). Anal. Calcd forCI3Hz5NO3: C. 64.16: H. 10.36. A. 5.76%. Found: C. 64.14: H. 10.37. N. 5.81%

S-lN-Butyl)carbunoyloet.nole add lMIIDO/lOl: Butylamine 11.45 g. 20 mmol). methyl nonanedloate (4.0 g. 20 mmoll. and N-hydroxysucclnlmlde 12.4 g. 21 mmol) were dlssalved In dry DMF 130 mL1. The soluuon was surred and cooled to -10 O C dunng the addltlon of dlcyclohe~~carbodllmldeL4.3g.21mmol~lnDMFl10mL1.After2hat-10~Candanother24h at room temperature. the solvent was removed under reduced pressure. Thhe residue was dissolved In EtOAc I200 mL1. The organlc phase was washed successlvely wlth 5% HCI 13x50 mLl.5% NaHC03 (3x50 mL1. water (2x50 mL1. bnne (50 mL1. and dned INa2so,). The crude product was punned by short path (Kugelrohr) disUllaUon to glve the coupling product. butyl

1. J.6 Hz). 1.1-1.7 114H. m). 2.16 I2H. 1. J=7 HZ). 2.28 (2H. 1. Jr7 Hz). 3.25 (2H. t. J=6 Hz). 8-IN-butyilcarbamoyloctan~~t~ 12.80 g. 54%): bp: 144-148 DC/0.20 torr: 'H-NMR 0.92 [3H.

3.64 13H. SI. and 6.2 I1H. brsl. To a solutlon of butyl 8-IN-butyllcarbam0ylo~tnnoate 12.7 g.

was surred for 2h at room temperature. and then acldlned with 10% HCI (pH=l). After 10.5 mmoll In methanol I20 mL) was added IM NaOH (21 mL. 2 1 mmoll. The reactlon mlxture

extraction with EtOAc 1150 mL). the arganlc phase was washed wlth water (2x50 mL). brlne (50 mL1. and dned INa2S041. The crude product was punned by short path [Kugelrohr)

-/lo 12.10 g, 86%): bp: 180-186 'C/O.ZO tom: mp: 61-62 OC: IR: 3325. 3000 broad]. dlstlllatlon and subsequent recrystallbatlon from an EtOAc-hexane mlxture to afford

1710. and 1650 Cm~': IH-NMR: 0.93 l3H. 1. J=6 Hz]. 1.24-1.42 (8H. ml. 1.51 12H. q. J=7 Hz]. 1.56-1.69 14H. ml. 2.19 I2H. 1. J=7 Hz), 2.34 (2H. t. J.7 Hz). 3.25 (2H. q. J=6 Hz]. 5.84 (1H. brsl. and 9.29 11H. brs). Anal. Calcd for C13H25N03: C. 64.16: H. 10.36. N. 5.76%. Found: C. 64.19 H. 10.39. N. 5.73%.

Substrate Specificity of N-Myristoyltransferae

s-(N-Ropyl]cublmoy~onuroic add WdDlO/ll]: Prapylamlne (0.59 g. 10 mmol). propyl decanedloate (2.44 g. 10 mmoll, and N-hydraxysuccmlmlde 11.21 g. 1 1 mmol) were dissolved In dry DMF (20 mLl. The Solution was stirred and coaled to - 10 "C during the addluon of dlcyclohexylcarbodllmlde (2.16 g. 11 mmoll In DMF [IO mL1. After 2h at -10 'C and another 24h at room temperature. the solvent was removed under reduced pressure. me resldue was

mLI. 5% NaHC03 13x50 mLl. water (2x50 mLl. brine 150 mL1. and dried (Na,SO,I. me crude dlssolved In EtOAc (200 mL1. The organic phase was washed succcsslveIy wlth 5% HCI (3x50

product was purlfied by short path (Kugelrohrl dlstlllation to give the coupling praduet. propyl 9-[N-propyllcarbam~ylnanan~~t~ 11.41 g. 50%); bp: 148-152°C/0.20 tom: 'H-NMR 0.92 16H. t. J=6 Hz). 1.1-1.8 (16H. ml. 2.17 12H. t. J=7 Hz). 2.28 12H. t. J=7 Hz). 3.13 12H. t. J.6 Hz]. 4.00 (2H. t. J=6 Hz). and 5.80 (IH. brsl. To a solution of propyl 9-Wpm pyllcarbamoylnonanoate (1.14 g. 4.0 mmol) In methanol (20 mLI was added 1M NaOH (8 mL.

wlth 10% HCI (pH=ll. After extraetlon with EtOAc (100 mL1. the organle phase was washed 8.0 mmoll. The reaction mlxture was stirred for 2h at room temperature. and then acldlfied

Nth water 12x50 mL1. brlne (50 mL1. and dried INa,SO,I. The crude product was purified by Short path (Kugelrohrl dlstlllatlon and subsequent recrystallhatlon from an EtOAc-hexane mixturetaaflardAMDIO/Il(O.85g.87~~l;bp:188-1920C/0.20torr:mp:86-87nC:IR3320. 3000 (broadl. 1700. and 1650 Cm.': IH-NMR 0.90 (3H. t. J=6 Hz). 1.23-1.37 (8H. m). 1.44-

and9.10l1H.brsl .Anol .CalcdforC13H,5N03:C.64.16;H.10.36.N.5.76~~.Found:C.64.16 1.64 (6H. ml. 2.15 (2H. t. J=7 Hz). 2.33 12H. 1. J=7 H z l . 3.17 12H. q. J=6 Hz). 5.69 (IH. brsl.

H. 10.36. N. 5.76%

P(N-Mcthyl)clrbunoylundeepnolc ncld (MdD12/13): Methylamhe (3.2 mL. 30 mmoll. ethyl dodecanedlaate~5.16g.20mmoll.andN-hydroxysucclnlmlde~2.42g.2lmmollweredlssalved

dlcyclohexylcarbodllmlde 14.33 g. 21 mmoll In DMF (IO mL1. After 2h at -10 OC and another In dly DMF (30 mL). The solution was stirred and cooled to -10 OC during the addltlan of

24h at room temperature. the solvent was removed under reduced pressure. The resldue was dlssolved In EtOAc 1200 mLl. The organlc phase was washed successively with 5% HCI (3x50 mL1. 5% NaHC03 (3x50 mL1. water (2x50 mL1. hnne 150 mLI. and dried INazS041. The crude praduct was purified by short path IKugelrohrl dlstlllauon to glve the couplmg product. ethyl

119H,ml.2.1714H.t. J=7Hz1,2.78(3H.d. J=5HzI.4.12L2H.q. J=7Hz.and5.40I1H. brsl. 11-(N-methyllcarbamoylundecanoate(2.37g.44%l:mp: 65-66OC/0.20torr: IH-NMR 1.1-1.8

To a saluuon of ethyl 1 l-(N-methyllcarbamoylundecanaate (2.0 g. 7.4 mmoll In methanol 120 mL1 was added IM NaOH (15 mL. 15 mmol). The reaction mixture was surred for 2h at rwm temperature. and then acldlfied with 10% HCI IpH=ll. After extracum wlth EtOAc (150 mLl. the organlc phase was washed with water (2x50 mL1, brine (50 mLI. and dried (Na,SO,l. The crude product was purified by short path (Kugelrohrl dlstlllatlon and subsequent recrystalllzatlon from an EtOAc-hexane mixture to afford AMDl2/13 (1.42 g. 79%): mp: 9 7 ~ 98.5 OC: I R 3310. 2950 Ihroadl. 1690. and 1635 cm I : 'H-NMR 110% DMSO-d6 In CDCl,l:

Hz]. and 7.14 (1H. brsl. Anal. Calcd for C,3H25N03: C. 64.16; H. 10.36. N. 5.76%. Found: C. 1.19-1.38 (12H. m). 1.57 L4H. ml. 2.15 (211. t. 5=7 MI. 2.24 (2H. t. J=7 H z ) , 2.72 (3H. d. J=5

64.06 H. 10.41. N. 5.71%

Acylamino Acid Analogs of Myristic Acid P'AAA") 2-lundccnno~llunlno~cctle Add IAAA3/41: To a solution of aminoaceuc acld (3.42 g. 0.046 moll In water 115 mL) was added 5M NaOH 110 mL1. and the stirred solution was cooled In an le-water bath. Undecanoyl chloride 110.23 g. 0.050 moll and 2M NaOH (28 mL) were added alternatlvely In 5 mm at IO OC. After surnng for I h at rwm temperature. the reaction mixture was washed with ether 13x50 mL1. acidified (pH-21 with 10% HC1 and extracted wlth EtOAc

Na2S04. The crude product was recrystallbed from EtOAc-hexane to give AAA3/4 (5.3 g. I1 50 mL1. The organlc phase was washed with water 12x50 mL1. bdne (50 mL1 and dried over

48%). mp: 114-114.5°C: I R : 3310.3050 (broad]. 1690. 1635cm-I: 'H-NMR15%DMSO-d6h CDC131: 0.89 (3H. t. J=6 Hz). 1.19-1.23 (14H. ml. 1.62 (2H. q. J=7 H z l . 2.24 12H. t. J=7 Hz]. 3.9512H.d. J=6Hz1.6.90(1H. brsl .Anal .Calcdf~rCI3Hz5NO3:C.64.16:H. 10.36.N. 5.76%. Found: C. 64.26: H. 10.39. N. 5.811.

3-lDcc~n0yll~mln0p~oplonle Add LAAA4/5): To a solution of 3-amlnoproplonlc acld (4.45 g. 0.050 moll In water (15 mL1 was added 5M NaOH 110 mL1. and the stlrred soIuuon was

were added alternatively In 5 mln at IO 'C. After sumng for I h at room temperature. the cooled In an Ice-water bath. Decanoyl chloride 110.48 g. 0.055 moll and 2M NaOH 128 mL1

reaction mixture was washed with ether (3x50 mL1. acldlfied [pH=2) with 1 0 % HCI and extracted with EtOAc (150 mLI. The organlc phase was washed with water (2x50 mL). brine (50 mL1 and dried over Na,S04. The crude product was recrystallized from EtOAc to glve m 4 / 5 19.11 g. 75W: mp: 103-103.5°C: I R 3290.3000 (braadl. 1690. 1630 cm-': 'H-NMR (20% DMSO-d, In CDCI31: 0.88 13H. t. J.6.5 H z . 1.16-1.32 (12H. ml. 1.55 12H. q. 5.6.5 Hz]. 2.09 (2H. t. J=7 Hz). 2.47 (2H. t. J=6.5 &I, 3.40 12H. q, J=7 =I. 7.22 (IH. brsl. AWL Calcd forC13H25N03: C. 64.16: H. 10.36. N. 5.76%. Found: C. 64.04: H. 10.33. N. 5.75%.

4-(NonmoyI)Uncnobutyrk Acld (AAMIBI: To a solution of 4-amlnobuQ~lc acld (5.15 g. 0.050 moll in water (15 mL1 was added 5M NaOH (IO mL1. and the stlrred solution was cooled In an Ice-water bath. Nonanoyl chloride 19.70 g. 0.055 moll and 2M NaOH 128 mL1 were added alternatively In 5 mln at IO 'C. After stlrrlng for Ih a t room temperature, the reacuon mmure was washed wlth ether (3x50 mL1. acldlfied [pH=21 with 10% HCI and extracted with EtOAc

NazSOw The crude product was recrystallized from EtOAc-hexane to give A A M / B 19.80 g. 1150 mL). The organlc phase was washed with water 12x50 mL1, brine 150 mL) and dded over

81%). mp: 71-72 OC: I R 3290,3000 (broadl. 1685. 1625 Cm-': IH-NMR 0.89 (3H. t. J=6 Hz). 1.17-1.36llOH.ml.1.59~2H.q.J=6.5Hz~.1.8312H,q.J=6.5Hzl.2.2012H.t.J=6.5Hzl.2.38 (2H.t . J=6.5Hzl.3.29(2H. q. J=6.51121.6.47I1H.brsl.Anal.CalcdfarC13HzSN03:C,64.16 H. 10.36. N. 5.76%. Found: C. 64.19 H. 10.36. N. 5.73%.

g. 0.043 moll In water 115 mLl was added 5M NaOH 110 mL1. and the stlrred solution was 5-~Octmoyll~mlnopcntsnolc Add (AAAW71: To a solution of 5-aminopentanolc aCld 15.07

coaled In a n Ice-water bath. Octanoyl chloride (7.75 g. 0.048 moll and 2M NaOH (28 mL1 were added alternatively In 5 mln at IO OC. After stlmng for Ih at room temperature. the reaction

EtOAc (150 mL1. The organlc phase was washed withwater (2x50 mL1. brine (50 mL1 and dried mixture was washed with ether 13x50 mL1. acldlfied lpH.21 with 10% HCI and &acted with

over N*SO,. The crude product was recrystallkd from EtOAc-hexane to glve M 0 / 7 19.25 g. 88%). mp: 98-98.5 OC: I R 3310. 3050 broadl. 1685. 1630 cm.': 'H-NMR (400 MHz. 10%

q.J=6.5Hzl.2.1412H.t.J=6.5Hz~.2.2812H.t.J=6.5Hz~.3.20~2H,q.J=6.5Hz).7.13(1H. DMSO-d6InCDCl~l:0.88(3H.t .J=6Hzl , l .18-l .36(8H.ml.1.52(2H.q.J=6.5Hz). l .62(4H.

brsI.Ana1. CalcdforC,,H,,NO,: C. 64.16 H. 10.36. N. 5.76%.Faund: C.64.23:H. 10.37. N. 5.73%

6 - l H e p U n o y U ~ n o h e ~ ~ n o l c Acld [AAA7/8): To a solutlon of 6-mlnohexanolc acld 16.55 g. 0.050 mall In water (15 mL1 was added 5M NaOH 110 mL1. and the stirred solution was cooled In an Ice-water b a t h Heptanoyl chloride (8.17 g. 0.055 mol) and 2M NaOH (28 mLl were added alternatively In 5 mln at 10 'C. After stlmng for Ih at room temperature. the reaction

mixture was washed with ether (3x50 mL1. acldlfied lpH.21 with 10% HCI and extracted wlth EtOAc (150 mLI. The organlc phase was washed wlth water (2x50 mL1. brine 150 mL) and dded overNazSO4.Th~crudeproductwasrecrystallbedfromEtOAc-hexanetogiveAM7/8~10.17 g. 84961; mp: 58.5-59.5OC. IR: 3320.3000 lbroadl. 1690. 1630 c m ~ l : 'H NMR 0.90 13H. t. J=6 H z l . 1.24- 1.33 16H. ml. 1.37 (2H. q. Jz6.5 MI. 1.52 pH. q. JS.5 Hz). 1.65 14H. q. &6.5 Hz).

brsl. Anal. Calcd forCI3HzSNO3: C. 64.16 H. 10.36. N. 5.76%. Found: C. 64.08: H. 10.39. N. 2.19~2H,t.J=6.5Hz~.2.35~2H,t.J=6.5Hzl.3.2712H.q.J=6.5Hzl.5.96~1H.brsl.9.20~1H.

5.79%.

7-(Hex~oylllmmohcptlnole Add LAMLI/Ol: To a solutlon oi7-amlnoheptanolc acld (1.17 g. 0.008 mall In water (3 mLl was added 5M NaOH 12 mLl. and the stirred solution was cooled In an Ice-water bath. Hexanoyl chlande (1.19 g. 0,009 moll and 2M NaOH 15 mL1 were added

was washed wlth ether (3x20 mL1. acldlfied (pH=21 wlth 10% HCI and extracted with EtOAc alternatively In 5 mln at 10 'C. After sumng far Ih at room temperature. the reaction mlxture

(100 mL). The organlc phase was washed with water 12x20 mL). brine (20 mLI and dried over Na2S0,. The crude product was recrystallhd from EtOAc-hexane to give (1.61 g. 82%1: mp: 76~77 OC: I R 3280.3000 (braadl. 1685. 1620 cm": 'H-NMR 0.88 13H. 1. J=6 Hz).

12H. I. J=6.5 Hz). 3.24 (2H. q. J=6.5 Hz). 5.92 IlH. hrsl. 9.72 (1H. bra). AnaL Calcd for 1.24-1.38~8H,mJ.1.4912H,q.J=6.5Hz1.1.63~4H.q.J=6.5Hzl.2.17(2H.t.J=6.5Hzl.2.35

CI3Hz5NO3: C. 64.16: H. 10.36. N. 5.76%. Found: C. 64.21: H. 10.37, N. 5.79%.

&IheptnnoylllmtnDhcx.noie Aeld LAL¶O/lOl: To a ~olution of 8-amlnaoctanolc a d d (5.02 g. 0.031 moll In water (10 mL1 was added 5M NaOH (6.5 mL1. and the surred soluUon was cwled In an Ice-water bath. Pentanoyl chloride 14.17 g. 0.035 moll and 2M NaOH 118 mLl were added alternatively In 5 mi" at IO OC. After st imng for Ih at room temperature. the reaction mixture was washed with ether 13x30 mL1. acldlfled (pH.21 with 10% HCI and extracted with EtOAc 1150 mL1. T h e organic phase was washed with water (2x30 mLl. brine 130 mL1 and dried over Na2S04. The crude product was rccrystallwd from EtOAc-hexane to glvc AMB/IO (5.80 g. 76%); mp: 52-53 "C: IR 3320, 3050 (broadl. 1690. 1630 cm.': 'H-NMR: 0.92 13H. t. J.6.5 H z ] , 1.24-1.39 (8H. ml. 1.49 (2H. q. J.6.5 Hz]. 1.66 (4H. q. J.6.5 H z l , 2.20 12H. t. J.6.5 Hz). 2.34 12H. t. J=6.5 H z l . 3.24 (2H. q. J.6.5 Hz). 5.95 (1H. brs). 9.93 IIH. brsl. AnaL Calcd for CI3H2,NO3: C. 64.16; H. 10.36. N. 5.76% Found: C. 64.20 H. 10.40. N. 5.74%

11-lAcetyl)amlnoundcclnole Add (AM12/131: To a solution of 11-amlnoundecanalc acid

was coaled In an Ice-water bath. Acetyl chloride (32 g. 0.055 moll and 2M NaOH I28 mL1 were 110.05 g. 0.050 moll in water I15 mL1 was added 5M NaOH 140 mL1. and the stirred solution

added alternatively In 5 mi" at 10 OC. After stimng for Ih a t room temperature. the reaction mmure was washed with ether 13x50 mL1. acidifled lpH=2) with 10% HCI and extracted wlth EtOAc 1150 mL). T h e organlc phase was washed with water (2x50 mL1. brine (50 mLI and dried over NalSOl. The crude product was recrystallbed from EtOAc-hexane to glve AM12/13 11.0 g. 8 0 4 mp: 80.5-81.5 'C Illt. IFabnchnyl el (11. 1195811 mp: 83-84 OCI; I R 3305. 2950 (broadl. 1690.1635cm":'H~NMR:1.21-1.38(12H.m1.1.48(2H.q.J=6.5Ml.1.6412H.q.J=6.5Hzl. 1.99 13H. 51. 2.32 (2H. t. J=6.5&1. 324 I2H. t. J=6.5HzI. 5.84 IIH. brsl. 8.90(lH. brsl.

R-Azido Analogs ("AZ') PIodononanolc add. A mUcture of 9-bromononanolc acld (3.0 g. 0.013 moll. KI 13.0 g. 0.018 moll and 18-crown-6 (0.5 g. 1.9 mmol) In DMF was stirred at 60 OC for I2 h. The solvent was removed by dlstlllatlon I" u m u o and the resldue was partitioned between EtOAC I50 mLl and 2N HCI (25 mL). The organlc phase was washed (3x25 mL H,Ol. dried [Na2S0,1. concentrated

give 9-Iodononanok acid (2.5 g. 70%) as shiny. whlte flakes: mp: 54-55 OC. I R 2910. 2845. In DQCUO. flash chromatographed (10% EtOAc-hexanel. and then crystallized from hexane to

(1. 2H. 5.7.2 WZ). FAEIMS m/z = 291 IM++UI. 1690. 930cm". IH~NMR 1.33(m.8H). 1.64(m.2Hl. 1.82Im. 2HI. 2.36It.2H. J-7.2Hzl. 3.19

PMdOnO-oIc add. Az9. A mixture ai9-lodononanolc add (1.8 g. 0.0064 mol). sodlum azlde (1.2 g. 0.019 moll and 18-crown-6 10.5 g. 0.0019 moll In DMF I25 mL1 was surred at rwm temperature for 16 h. After removal of the solvent under vacuum. the resldue was partltloned between IN HCI (25 mLl and CH2Cll 125 mL1. The organlc phase was washed wlth water (3x25 mLl. dded (N*SOd and concentrated to give a pale yellow Uquld (3 9) whlch was punfied hy flash chromatography [slllca gel1 uslng 15% EtOAc In hexane to give A29 (0.98 g. 77%) as a ~ o l o r l e ~ s OIL. FT-IR 2940, 28M), 2100 lazldel, 1710 lacldl. 1280 and 1260 cm.': 'H NMR: 3.26 [t. 2H. J=7 H z ) : 2.36 k2H. 5.5.4 Hz): 1.62 (m. 4H1: 1.33 lm. 8Hl. FABMS m/z: 212 IM+HI and 206 IM+Ul. HRMS. Calcd m/z for C9H1,N3O2W: 206.1481%. Found 206.1485 (M++Ul.

Il-AIldo~d~cnnOIC Add. Az11. was prepared as described for A29 except that 11- lodoundecanolc acid [prepared as described by Patuson et a!. 1195611 was subsututed for an

asacolorlessoll.yleld64%.~-IR2910.28M).21001azlde1.17101acld1. 1280and1260cm~1: equivalent amount of l2-lodadodeeanalc add . The pmduct lB&a et aL 11988ll was abtaIned

CHz).FABMS.m/~:228(M+H):200and182.HRMSCalcdm/zforClIH2IN3O2U:234.1794. 'H NMR 3.28 It. 2H. J=6.8 H z , CH21; 2.35 k2H. 5.7.7 H z , CH,): 1.6 Im. 4Hl: 1.29 Im. 12H.

Found: 234.1821.

lodommethylsllane 10.5 mL1 in CCl, I3 mLl was stlrred at rwm temperature for 16 h. The 12- lodotrId~c .~0I~ Acld. A mixture of 12-hydroxymdecanolc acld (0.3 g. 0.0013 mol. and

soluuon was concentrated. cold water (10 mLl was added. and the mmure was extracted with

concentrated under reduced pressure. m e resulting resldue was punfled by flash chromatog- EtOAc (2x10 mL1. The organlc phase was washed wlth water (2x15 mL1. dried lNa2S0,1 and

raphy (sfllca gel) uslng 15% EtOAc In hexane to give the TMS ester (0.2 9) as a pale yellow solld IFABMS: m/z. 565 (M+2U-H). 559 lM+UlI whlch \MS suhJected to bask hydrolysis by stlmlng In 1N NaOH (3 mL) and THE (2 mL) at room temperature for 16 h. The solutlon was cwled. acldlfied with 2N HCI (1.5 mLl and extracted wlth EtOAc 12x10 mL1. The organlc phase was washed with water (2x10 mu. dned (N%SO,I. concentrated under reduced pressure and the resldue was punned by flash chromatography (sllaa gel1 uslng 10% EtOAc In hexane to W e

5-7.5 Hzk 1.85 Im. 2H): 1.64 lm. 2Hl. 1.27 (m. 16Hl: FABMS : m/r . 353 IM+PU-HI: 347 IM+Ul product 10.15 g. 34%) as a clear. colorless oll. 'H NMR 3.19 It. 2H. J=6.9 Hz: 2.35 It. 2H.

and 225. HRMS mlz. Calcd for C,3H2s102U. 275.2059. Found: 275.2004 (MIWI.

12-AzIdododecmole Add. Azla. A mlxture of 12-lododadecanolc acld 12.1 g. 0.0064 moll. sodlum azlde (1.2 g. 0.019 moll and 18-crown4 (0.5 g. 0.0019 moll tn DMF (25 mLI was SUrred at mom temperature for 16 h. After removal ofthe solvent under vacuum. the resldue was partltloned between IN HCI (25 mL1 and CqCl, 125 mL1. The organlc phase was washed with water (3x25 mL1. dned IN&$KI,l and concentrated to give a pale yellow llquld (3 9) whlch was pudfied by flash chromatography lsillca gel1 uslng 15% EtOAc In hexane to give 12- mdododecanolc acld [Alan and Sangkot (198611 (1.3 g. 84%1 as a colorless llquld F T I R 2930. 2860. 2100 (azide]. 1710. 1280 and 1260 cm": 'H NMR 3.19 (1. 2H. J.6.9 Hz. CH& 2.28 (1. 2H. J.7.5 Hz. CH,): 1.53 [m. 4H. 2xCHZ1; 1.21 (m. 14Hl. FABMS Im/zl: 254 (M+SU-HI: 248 IM+Ul and 226.

Substrate Spec.ificity of N-Myristoyltransferae 7239 lS-AIldotrldecMOlcAeld,~l3.Amlxtureof 13-lodomdecanolcacldII(ruhlngaandKellOgg (198611 (0.15 g. 0.44 mmol) sodium azide (0.09 g. 1.4 mmoll and 18-cram-6 (0.015 g. 0.057 mmol) In DMF (3 mL) was stirred at room temperature for 16 h. DMF was dbtilled under vacuum. cold 2N HCI (2 mL) was added and the mixture was extracted with EtOAc 110 mL1. The organlc phase was washed with water (2x10 mLI. dried lNa,SO,l and concentrated under

gel1 uslng 20% EtOAc In hexane to afford AZl3 (0.075 g. 67%) as a white solid. mp 38-40 'C. reduced pressure to glve a pale yellow solid whlch was purified by flash chromatography (slllca

'H NMR: 3.26 It. 2H. J=6.9 Hz): 2.35 It. 2H. J=7.5 H z ) : 1.62 (m. 4H1: 1.35 lm. 14Hl. FABMS Im/zl. 256 IM+H): 230 and 210. HRMS calcd for C,3H25N302U: 262.2107. found: 262.2164 (M+LI).

Heterocyclic Analogs

et al.. 1991. 1~(2-Thlenyl]-deo~noIc Acld. TH-10. This compound was prepared as described In KLshore

12-12-~enylI-dodeeanolc Add. M - 1 2 . Thls compound was prepared as described In Klshore et 01.. 1991.

12<2-FiryU-dodcemoleAcld. FUR-lZ.Thl~~0rnpund was prepared as described InKIshore etaL. 1991.

1 l-(2-lmld~tolyl)-undeepnole add. 2-18-1 1, Thls compound was prepared In 62%yleld: W- IR: 2920. 2850. 1640. 1560 and 900 cm.': W (MeOH): '216 lnm): 'H NMR (DMSO-%): 1.25 (br. s. 12HI: 1.49 [m. 2H): 1.60 [m. 2HI: 2.2 [t. 2H. A 7 . 2 Hz): 2.57 (1. 2H. 5.7.2 Hz): 6.85 (s.

2HI: FABMS: m/z=253 (M+H); HRMS: Calcd forCllH25Nz02 IMIH) 253.1916. found 253.1970.

g. 0.64 mmoll In DMF (5 mL) was added Imidazole (0.0969, 1.4 mmol), 18-crown-6 (59 mg. l l-( l-~ldlroIyl)-undec.nole add. 1-IM-11. To a soluuon of 11-ladoundecanalc acld (0.29

0.02 m o l ) . and NaH (69 mg. 80% suspenslon In 011). The mlxture was SUrred at room temperature (under A r t for 2.5 h. The solvent was removed in U(ICL(O. the resldue was dlssolved In H 2 0 I5 mL1. and the pH adjusted to 5.8 by treatment with IN HCI and IN NaOH while

yleld 100 ma, and crystallhed from MeCN to give I-IM-l I ITsuchlda et (11. (198911 as a whlte eaollng In an Ice bath. The preclpltate was filtered. washed with H20. dried lyellaw solld. crude

solid I79 mg. 43%). mp 119-120 'C. 'H-NMR (DMSO-$1: 1.2 lm. 12HI: 1.45 lm. 2H): 2.20 It. 2Hl: 3.95 (t. 2H): 6.85 lbrs. 1H): 7.15 (brs. 1H): 7.60 mrs. IHI. FABMS m/z 259 IMILII: 253 IM+H) and 160 H R M S Calcd for C,,,HZ4N2OZ UIMIUJ 259.1998: found: 259.1984,

l2-(2-Imldlrolyl)-dodcemole acld. 2-IM-12. To a solution of imldazale (1.2 g. 7.05 mmoll In dry THF (15 mL) cooled to -50 "C. was added dropwise a soIution of BuU 13.22 mL. 7.73 mmol). The resulting yellow s ~ l ~ t i o n was stlired at -40 OC for 30 mi". and added a sol~UOn

of 12-lodododecanolc acld (1.04 g. 3.2 mmol) In HMPA (4 mL). The reaction mlxture was then

h at -25 O C . the reaction temperature was a g l n ralsed to -5 'C and stirred at -5 'C for I .5 h. stirred at -40 OC for 15 min and the temperature was raised to -25 OC. After sumng for 1.5

The reaction mlxture was cooled to -40 "C and added cold 2N HCI 14.5 mL) and then paured onto a mlxture of crushed Ice. water ( I O mL) and EtOAc I25 mLI. After repeated extracUOnS with EtOAc. the pH of the aqueous phase was brought to 3.5 with IN NaOH. when a solld began to separate. It was filtered. washed thoroughly with water and acetonluile and finally erystalllzed from acetonltrile-water to afford 2-W-12 (0.44 g. 51%) as shlny whlte flakes. FT- IR: 2920.2850. 1640. 1570 and 900 c d l : W [MeOHI 210 nm: 'H NMR D M S O - 4 1 : 1.24 (br. 5, 14H1: 1.48 lm. 2H): 1.6 (m. 2H): 2.18 [t. 2H. J.7.2 H z ) : 2.57 It. 2 H . J=7.5 Hz): 6.84 Is. 2H): FABMS: rnlr=267 (M+H): H R M S Calcd for CISH2,N202 lM+H) 267.2073 found: 267.2175.

12-(l-~ld.rolyl)-dodecpnole Add. 1-IM-11. This compound was prepared as descrihed for

acetonlwlle-water. Yield. 52% mp 127 OC. F T I R : 3120. 2910. 2840. 1700. 1190. and 820 cm 1-"12 [above). The product. 1-W-11 ITanihlra et .I. 11978). was crystalllzed from

I. IH-NMR (DMSO-$): 1.23 lm. 14H): 1.5 Im. 2H): 1.68 Im. 2H): 3.93 It. 2H): 6.88 (brs. 1H1: 7.16 (brs. IH). FABMS: m/z = 273 IM+LO. 267 (M+Hl. HRMS: Calcd for C15H2hNz02LI(M+UI 273.2154: found 273.2147.

12-(2-N-Mcth~Umld~=~lyl~-dodceanole Add. 2-MIM-12. A suspenslon of 2-IM-12 10.09 g. 0.34 mmol). NaH (25 mg. 8(wo suspenslon In all) In DMF 12 mL). and HMPA (1.5 mLI contalnlng ICH3 (0. 106 g. 2.2 mmol) was stlrred at room temperature far 16 h. The reaction mlxture was dlluted with H 2 0 I10 mL). extracted with EtOAc 13x15 mLl. washed with brine

NaOH (1.5 mLl In MeOH (0.5 mL). After stlmng for 16 h at room temperature. the reaction (3x10 mL1. dried Na2S0,. and concentrated In vacuo. The resldue was hydrolyzed uslng IN

mlxture was acldlfied I1N HCI) to pH 5 and extracted with EtOAc (3x15 mLI. The comblned organlc extract was washed with water 13x10 mL). dried INa2SOJ. and concentrated Ln muo.

53%) as a pale. yellow powder. mp 84-85 'C. FTIR: 2910. 2840. 1700. 1500. 1460. 1180. 1100. and 740 cm.'. 'H-NMR (DMSO-&): 1.25 (m. 14H): 1.48 (m. 2H): 1.6 Im. ZH); 2.18 It. ZH. J=7.2 Hz): 2.58 It. 2H. J=7.2 H z ) : 3.53 (5. 3H): 6.72 lbrs. IH): 6.97 Ibrs. IH). FABMS m/r - 287 lM+W). 281 IM+H). and 263: HRMS: Calcd for C,6H28N202U 287.23 11: found 287.2346

Mld. 12M123. To a suspenslon of sodium hydride (0.13 g. of 80% suspension In 0111 Ln DMF 12-Il-~l.2.3-~oly~)l)I-aoaee.nolc Acld. 1-TRI-12. l2-lrc-2(l.2.3)mrrolyld~d~~Molc

I4 mLl coaled to 0 "C, was added dropwise a solution of 1.2.3-mazole (0.28 g. 0.004 mmoll In DMF I1 mLl. After 0.5 h. 18~crown-6 (0.025 g. 0.095 mmal) and 12-lodododecanalc acld 10.5 g. 0.0016 mal) were added and the mwure was stirred for 1 h at room temperature and 1.5 h a t 60 'C under nltrogen atmosphere. The reaction mlxture was concentrated under vacuum. the resldue was dlssolved In water ( lo mL). acldlfied with Cold I N HCI and the resulting

mLl. dried lNa$5041. concentrated under reduced pressure and the resldue was crystalllzed mlxturewasextractedwithEtOAc(3x15mL).Thearganlcphasewaswashedwithwater~2x10

from EtOAc to afford 12M124 10.12 g. 28%) as white crystals. mp: 79-80 OC: FT-IR: 2930. 2920. 2845. 1690 (acld). 1485. 1300 and 970 C m ': W IMeOHI kax: 222 nm: 'H NMR lCDCl3) :7 .59 l s .2H) .4 .44 ( t .2H.J=7 .2~) :2 .35 ( t .2H.J=7 .2Hz) .1 .95 [m.2H):1 .63Im.2Hl .

274.2105 lM++U). 1.26 lm. 14Hl. FABMS: m/z=274 IM+U). HRMS: Calcd for ClrHz5N3O2U: 274.2107. F o u n d

?he resulting substance M S purified by clystalllzation from EtOAc to 2"Ib¶-ll (0.050 g.

12-(1.2.4l~zolyIdodeeanole Add. l2M124. To a suspenslon of sodlum hydride 10.05 g. of 80% suspension In 011) In DMF cooled to 0 'C, was added dmpwise a solution of 1.2.4- m a d e 10.095 g. 1.38 mmoll In DMF 11.5 mL). The reaction mlxture was sllrred a1 0 OC for 30

mmoll In DMF (1 mL). was added. and stlrred at room temperature for 1 h and at 60 OC for mln. a solution of 12~lodododecanolc acid 10.2 g. 0.6 mmol) and 18-crown-6 (0.01 g. 0.038

1.5 h. DMF was distilled under reduced pressure. t h e resldue was dissolved In water (5 mL).

washed with water @x I O mLl. dried lNa2S041. and concentrated under reduced pressure. The aadlfied with I N HCI to pH 6 and extracted with EtOAc 12x15 mL1. The organlc phase was

residue was purified by clystalllzatlon from EtOAc hexane (1:11 to glve 12M124 (0.055 g. 34%) as a whlte powder. F T - I R : 2920. 2660. 1720. 1510. 1480. 1290 and 1190 Cm~l: W (MeOHl Lax: 214 nm: 'H NMR: 8.10 1s. IHI: 7.96 Is. IH): 4.17 It. 2H. J=7.2 Hz): 2.35 (1. 2H. J=7.5 Hz): 1.88 lm. 2H); 1.63 (m. 2Hl: 1 26 lm. 14HI. FABMS mlz.268 lM+HI.

Add. 2-TRI-13. A suspenslon of 1.2.3-triazole 10.12 g. 1.74 mmall and NaH (0.12 g. 80% 13-11-~1.2.3-~r0lyl)l-t.ldec.nOicAcld. l-TRI-13.nd 1~12<1.2.3"n~oly~1-tridce.nole

suspension In (111) In dry DMF (5 mLI was stirred at 10 'C under Ar 13-lodowldecanolc acld (0.4 g. 1.17 mmoll and I$-crown-6 10.02 91 were added after 0.5 h and the mlxture was heated at 70 'C for 3 h. The DMF was removed in vacuo. the resldue was dlssolved In H 2 0 I15 mL). acldlfied with I N HCI to pH 5.5. emacted wth EtOAc (3x10 mL). washed with QO (2xlOmL). dried IN+S04), concentrated under reduced pressure and clystallized from EtOAc to glve a mlxture of 1-TRI-13 and 2-TRI-13 10.17 g. 52%) whlch was separated by flash ehromatogra- phyusing5%methanollnCH2CI,tog~ve1-TRI-13Imp104-105oCIandZ-TRI-l3Imp81-82

OC). 1-TRI-13: F T - I R 3120.2920.2840. 1680. 1460. 1090.960. and 840 em-'. 'H-NMR 1.3 ~ m . 1 6 H 1 : 1 . 6 4 I m . 2 H l : 1 . 9 I m . 2 H 1 : 2 . 3 5 I t . 2 H . J = 7 . 5 H i ~ ; 4 . 3 9 l t . 2 H . J = 7 . 5 ~ ~ . 7 7 . 5 3 I s . 1H): 7.71 Is. 1H). FABMS: m/z = 294 IM+PLL-H). 288 lM+Ul. 202 and 1 6 0 HRMS Calcd for C,5H27N302Ll(M+L11 288.2263 found 288.2301.

2-TRI-13: IH-NMR: 1.35 Im. 16Hl: 1.64 Im. 2H): 1.95 Im. 2HI: 2.35 It. 2H. J = 7.2 Hz): 4.44 It. 2H. J = 7.2 Hz): 7.59 Is. 2HI: FABMS m / r E 294 IMIPU-HI. 288 lM+U1. 202. and 160. HRMS: Calcd for C15H27N302L11M+Ll) 288.2263: found: 286.2281

12-I5-11.2.3.4-Tetr~~~lyl~l-dodee.nolc Add. 5-TET-12. Melhyl 1Z-bmmododeconwte. To a cold IO 'C) solution ofdlcyclobexylcarbodllmlde 11.6

g. 7.85 mmoll In CH2Clz I15 mL1 and CH30H (1.2 mL. 30 mmoll. was added dropwise. a

g. 1.4 mmdl In CHlC12 (15 mL). m e reactants were stirred for I h a t 0 OC and then for 16 h solution of 12-bromododecanolc acld 12.0 g. 7.2 mmoll and 4-dlmethylamlnopyndlne 10.175

at roam temperature. The reaction mlxture was filtered. the n h t e was surred with 5% aceuc acld for 30 mln and filtered agln. The filtrate was washed with saturated NaHC03 (3x20 mL). H 2 0 (3x20 mL1: dried (Na2S0,1. and concentrated In v o w . The resldue was subJected to purincation by flash chromatography using 5% EtOAc In hexane to glve methyl 12- bramododecanoate IMaklta et al. 1198611 (1.9 g. 90%) as a colorless 011. IH-NMR 1.3 (m. 14H):

m/r = 299 IM+LI) and 293 lM+Hl. 1.4[m.2H1:1.85Im.2HI:2.3lt.2H.J=7.5~~:34(t.2H.J=6.9Hz~:3.67Is.3HJ.FABMS:

mmoll. potasslum cyanlde 10.14 g. 2.15 mmol). and 18-crown-6 10.03 g. 0.114 mmal) In dry Methyl 12-cyanalodeconwte. A mlxture of methyl 12-bromododecanoate 10.32 g. 1.1

DMF was heated at 70 'C for 16 h under Ar. After removal of DMF in v-, the resldue was

water 12x10 mLI. dried lNa,SO,). concentrated In -0. and punned by flash chromatography partihoned between H 2 0 I10 mLl and CH2C12 (25 mLI. The organlc phase was washed with

120% EtOAc/hexane) to give methyl 12-cyanododecanoate 10.21 g. 80%. Greene et aL (1969)l a5 a ~Olorless oil. FT-IR: 2920. 2860. 2240. 1740. 1440. 1170 cm~' . 'H-NMR 1.28 (m. IZH]: 1.45 lm. 2H1: 1.65 lm. 4H): 2.3 lm. 4H). FABMS: m/z = 246 [M+LO. 240 IM+H). and 208. HRMS: Calcd for C14H25N02LI[M+LIJ 246.2045: found 246.2088.

Methyl15-l1.23.4ltetrazolyl~od~~te.Amlxtureofmethyl 12-cyancdcdecanoate(0.24 g. I .5 mmoll and trimethylun azlde (0.62 g. 3.0 mmol) in dry toluene 15 mL) was heated at 100 OC for 24 h under Ar. Toluene was removed in "-and the resldue was dlssalved In THF 120 mL1. treated with 4N HCI In dloxane 10.5 mL) and surred for 30 mln. The solution was washed with water (2x10 mL1. dried IN~SO,). concentrated in mcuo. and purifled by nasb chromatography I5W EtOAc/hemd to glve the ester 10.30 g. 71%) as a whlte powder. mp 69-70'C. F T - 1 8 [KEIrl: 2920. 2840.2730.2620 and 1720 cm". IH-NMR: 1.4 Im. 14H): 1.62 lm. 2HI: 1.83 lm. 2HI: 2.35 It. ZH. J = 7.5 Hz): 3.03 (1. 2H. J = 7.5 Hz): 3.1 (s. 3H. OCH31. FABMS: m/z 289 IM+LI): H R M S Calcd for C,4H26N402U M+U) 289.2216. Found: 289.2257.

(1.2.3.4)tetrazoIyIIdodecanoate 10.25 g. 0.9 mmol. see above) In IN methanallc NaOH 16 mLl 12~15-11.2,3.4~TelrazolyV~-dodeca~l~ A M . 5-TET-12. A soluuon of methyl 15-

w a s stirred at room temperature for 3h. Solvent was removed In vacuo. the residue was dlssolved In H 2 0 110 mL). acldlned with IN HCI. extracted with EtOAc 13x10 mLl. washed with HzO (3x10 mL). dried [Na2S04). concentrated in vacmand clystalllzed from EtOAc to glve 5- TET.12 (0.12 g. 52%) as a whlte powder. mp 119~120 OC. FT-IR IKBr): 2915. 2850. 2840. 1690. 1570. 1460. 1250. 1100. and 910 cm". 'H-NMR (DMSO-&): 1.24 (m. 14Hl: 1.45 Im. 2H): 1.65 Im. 2HI: 2.18 It. 2H. J = 7.2 H z ) : 2.85 It, 2H. J = 7.5 Hz). FABMS m/r 275 [M+UI: HRMS: Calcd for C,3HZIN402U IMtLI): 275.2059. Found: 275.2047.

1 2 - ~ 1 - ( 1 , 2 . 3 . 4 - T e t r ~ 1 0 l y l ) ~ ~ d ~ d ~ c M 0 1 ~ Add. 1-TET-12. 12-(TetrProlyI]dodcelnOLe Add.

solution of tetrazale (0.095 g. 1.35 mmoll in DMF (1 mLl. After stlmng the reactants for 15 12TET. A suspenslon of NaH 10.045 g. 80% suspenslon In oil1 In DMF I1 mLl was added to a

min. a solution of 12-lodododecanolc acld (0.2 g. 0.6 mmol) was added. The resulting mlxture was stirred at room temperature for 1 h and at 55 'C for 1.5 h and then concentrated Ln ~(ICIIO. The resldue was treated with IN HCI I5 mL1 and extracted with EtOAc 12x10 mL1. The

and the residue was crystallized from EtOAc/Hexane to give the utle compound 10.095 g. 26%) organic layer was washed with water. dried IN+O,). concentrated under reduced pressure

as a 1:3 mlxture of N-3 and N-I Isomers. respectively. F l - I R 2920. 2850. 1720 laeldl and

Ut. 2H. J=7.2 Hz): 2.35 It. 2H. J=7.5 Hz): 1.95 Im. 2H): 1.63 Im. 2HI: 1.26 (m. 14H). FABMS: I180 c m ~ l : W IMeOHl Lmar 232 and 256 nm: 'H NMR 8.59 and 8.5 (2s. 1H1: 4.65 and 4.43

m/z. 269 [MIHI: 251 and 241. H R M S m/z=C13H,,N,0,U. calc: 275.2059. found 275.2004 IMILI).

the journal of vol. 267, no 11, of 15, 722&7239,1992 1992 ... · the journal of biological...

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