Complete Stereochemistry and Preliminary Structure–Activity

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Complete Stereochemistry and Preliminary Structure−Activity Relationship of Rakicidin A, a Hypoxia-Selective Cytotoxin from Micromonospora sp. Naoya Oku,† Shouhei Matoba,† Yohko Momose Yamazaki,‡ Ryoko Shimasaki,† Satoshi Miyanaga,†,§ and Yasuhiro Igarashi*,† †

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan ‡ Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan S Supporting Information *

ABSTRACT: The complete stereochemistry of rakicidin A, a hypoxia-selective cytotoxin produced by Micromonospora sp., was unambiguously established by extensive chemical degradation and derivatization studies. During the PGME derivatization-based configurational analysis of 3-hydroxy2,4,16-trimethylheptadecanoic acid, an irregular Δδ distribution was observed, which necessitated further acylation of the 3-hydroxy group to resolve the inconsistency. A hydrogenated derivative of rakicidin A, its ring-opened product, and two congeners with different alkyl chain lengths were tested for hypoxia-selective cytotoxicity. The results indicated that both the conjugated diene unit and appropriate chain length are essential for the unique activity of rakicidin A.

L

pound was originally isolated from a culture broth of Micromonospora sp. as a moderate cytotoxin;7 later, rakicidin A was shown to have antiallergy activity in a mouse asthma model.8 Apart from rakicidin congeners,9,10 two groups of actinomycetesderived depsitripeptides, the vinylamycin-microtermolide A class11,12 and the BE-43547 complexes,13 contain the vDha residue (Figure 1). Although 15 years have passed since their discovery, the absolute stereochemistry of this class of

ocally advanced solid tumors usually contain hypoxic regions (oxygen tension ≤2.5 mmHg) due to unorganized vasculature and extremely high metabolic requirements.1 Classically, neoplastic cells in hypoxic loci resist radiotherapy, which utilizes oxygen as the sensitizer.2 However, evidence shows that these cells activate adaptive responses to withstand the adverse condition and undergo mutation caused by defective DNA repair and subsequent clonal evolution, resulting in the development of drug resistance, acquisition of aggressive phenotype, and poor prognostic outcome.3 Therefore, hypoxia has great potential as a target for the definitive therapy of cancer, and attempts to develop hypoxia-activated prodrugs or inhibitors of hypoxia-associated proteins have identified several promising drug candidates.4 Rakicidin A is a hypoxia-selective cytotoxin identified through a hypoxia/normoxia differential cytotoxicity screen of 20 000 microbial broths.5 Although the activity of most anticancer agents significantly decreases under hypoxic conditions, rakicidin A exhibited enhanced activity against many cancer cell lines, with the highest hypoxia/normoxia selectivity indices being achieved with PANC-1 human pancreatic carcinoma (14.9) and HCT-8 human ileocecal adenocarcinoma cells (17.5).5 Moreover, the hypoxia-selectivity was valid for myelogenous leukemic cells, thus adding to the list of potential applications.6 Rakicidin A is a depsitripeptide composed of three unusual amino acids, sarcosine, β-hydroxyasparagine (βOHAsn), and vinylogous dehydroalanine (vDha), and a β-hydroxy fatty acid, 3hydroxy-2,4,16-trimethylheptadecanoic acid (Htha). This com© XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Rakicidin A and related metabolites. Received: March 27, 2014

A

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compounds is available only for the two common amino acid residues in vinylamycin. This is probably because of the very poor solubility of this metabolite class in most common organic solvents. In fact, pure rakicidin A is practically insoluble in EtOAc, Et2O, and THF and poorly soluble in alcohols, aqueous MeCN, and chloroform. DMSO was used as the solvent for the NMR experiments of rakicidin A; however, once the solvent is evaporated, flaky residues are formed, which can be partially dissolved in the same solvent. The vDha residue may be responsible for this solubility problem because the replacement of other residues in the related metabolites did not remarkably improve the solubility. A series of preliminary mode of action studies revealed that the unique activity of rakicidin A is not the result of inhibition of hypoxia-adaptive responses mediated by hypoxia-inducible factor-1 (HIF-1), inhibition of the transcriptional activation of this pivotal protein, or the production of oxygen radicals.5 Thus, a novel mechanism is indicated. Although rakicidin A was found to be inactive in vivo against both the murine M109 lung carcinoma and P388 leukemia tumor models,7 the assignment of its absolute stereochemistry is still of great benefit in identifying new therapeutic targets and developing new anticancer drugs. Rakicidin A has five chiral centers, and they are present in the βOHAsn and Htha residues. Attempts to derivatize rakicidin A by common derivatization procedures, including saponification (aqueous LiOH/DMF), methanolysis (NaOMe/MeOH), or double-bond cleavage (RuCl3/NaIO4 or O3/NaBH4), to obtain the derivatives or fragments suitable for chiral analysis failed, leaving rakicidin A virtually unreacted. Because semitranslucent lumps, supposedly generated from the flakes of rakicidin A, remained persistently insoluble during the reactions, we attributed the chemical inertness of this peptide to its poor solubility in the reaction solvents. The only exception was the acid hydrolysis at elevated temperature, affording substantial amounts of products in the lipophilic extract. However, the expected product, Htha (7), was contaminated by impurities, preventing its reliable characterization by NMR. Obviously, a better solvent for rakicidin A was needed, and we found that pyridine followed by DMF satisfied this requirement. An ideal derivatization would be to modify the vDha residue while using these solvents. Two options were possible: hydrogenation and cleavage of the olefin moieties. The former would be preferable because it conserves the cyclic structure and should facilitate a structure−activity relationship (SAR) study, which may elucidate the role of vDha. The hydrogenation of rakicidin A using Pd/C-catalyst in pyridine/DMF14 afforded tetrahydrorakicidin A (2) as a single peak during HPLC purification (Scheme 1, 52%). As expected, this derivative was readily soluble in previously incompatible solvents and thus opened a way for further transformation. Methanolysis of 2 with NaOMe afforded carboxylic acid 3, which was methylated with TMS-diazomethane to give methyl ester 4. Derivatization of the two carbinol carbons with (S)- or (R)-αmethoxyphenylacetic acid (MPA)15 was not successful, presumably due to the steric hindrance exerted by the flanking methyl groups.16 Then, we applied the previously successful acid hydrolysis to 2. Extraction of the hydrolysate solution with Et2O separated βOHAsp (6) in the 6 N HCl layer and Htha (7) in the Et2O layer, the yield of the latter being almost quantitative (97%, Scheme 2).17 The assignment of NMR signals was possible without purifying the products. The relative stereochemistry of 6 was determined by silica gel TLC operating in the hydrophilic

Scheme 1. Preparation of a Solubility-Improved Derivative, Tetrahydrorakicidin A (2), and Attempt at Chemical Conversion to Establish the Absolute Configuration of Two Chiral Centers in 2

Scheme 2. Determination of the Relative Stereochemistry of βOHAsp (6) and Htha (7)a

a

Data reproduced from ref 20.

interaction liquid chromatography mode. A sample from the HCl layer was spotted on a TLC plate and developed with i-PrOH/ 25% aqueous NH3/2 mM CuSO4 (4:2:1). Upon visualization with ninhydrin, a yellow spot appeared at Rf 0.35, which was identical to that for a threo isomer (Rf 0.35 for L-threo; 0.25 for Lerythro). Thus, the original βOHAsn residue in rakicidin A should have a threo-configuration.18 To establish its absolute configuration, the HCl layer was dried and reacted with a chiral derivatizing reagent, 5-fluoro-2,4-dinitrophenyl-5-L-leucine amide (FDLA).19 A complete set of authentic standards were prepared by derivatizing L-erythro- and L-threo-βOHAsp with both the enantiomers of FDLA. The reversed-phase LC-MS analysis exhibited the coelution of the L-DLA-derivatized 6 with D-DLA-L-threo-βOHAsp. Because the latter was a chromatographic equivalent of L-DLA-D-threo-βOHAsp, the configuration of the βOHAsn residue in rakicidin A was concluded to be Dthreo. B

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To establish the relative stereochemistry of 7, the chemical shift data for this fatty acid were compared with those available for the four diastereomers of 3-hydroxy-2,4,6-trimethylheptanoic acid.20 However, none of the profiles matched that of 7 (Figure S6). The lack of diagnostic validity in this protocol is probably because of the difference in aliphatic chain lengths between the three contiguous chiral centers and the terminal isopropyl group. Then, we carefully examined the NMR data of the model compounds and found that the spin multiplicities of the 3oxymethine protons significantly discriminate the three configuration classes. In particular, a pair of small (∼3 Hz) and large (∼8 Hz) coupling constants indicates a 2,4-anti-dimethyl relationship, whereas two middle values (∼6 Hz) indicate a 2,4-syn-dimethyl relationship. The related proton H3 in 7 (δH 3.65 ppm) exhibited a combination of small (3.7 Hz) and large (7.9 Hz) values (Scheme 2), indicating a 2,4-anti configuration. An alternative decision between (2R*,3R*,4S*) and (2S*,3R*,4R*) was made based on a pair of homonuclear decoupling experiments to identify coupled partners associated with these values. Upon selective irradiation of H2 (δH 2.68 ppm), the H3 resonance was modulated into a doublet with a 3.7 Hz splitting, while irradiation of H4 (δH 1.66 ppm) resulted in a residual splitting of 7.9 Hz (Figure S22). This allowed the assignment of 3JH3,H4 = 3.7 Hz and 3JH2,H3 = 7.9 Hz, and hence the relative stereochemistry of 7 as (2R*,3R*,4S*). The absolute stereochemistry of Htha was established by chiral anisotropy analysis using PGME (phenylglycine methyl ester).21 Crude 7 was reacted with either (S)- or (R)-PGME, and the chemical shift differences of the products 8a and 8b (Δδ = δH(S) − δH(R)) were calculated to deduce the absolute configuration at the C2. However, the signs for Δδ values around the same chiral center were irregularly distributed (Scheme 3), preventing the

Figure 2. Different conformations proposed for 8b (upper) and 9b (lower). Note that the presence (8b) or absence (9b) of a hydrogen bond (dotted lines) on the acyl group defines the orientation of the PGME group along the C1−C2 axis (yellow bond). As a consequence, H3 and H4 (yellow atoms) in these derivatives are coplaced in the opposite compartments divided by the chiral anisotropy group.

the structure of microtermolide A12 tentatively assigned the configuration of its fatty acid unit as (2R*,3R*,4R*), based on the computation of the minimum energy conformation. However, the JH2,H3 and JH3,H4 values (10.0 and 1.0 Hz, respectively) are very similar to those reported for the Htha unit in rakicidin A (10.2 Hz, ∼0 Hz),7 and considering the global resemblance between the two metabolites, the proposed stereochemistry should be examined by chemical methods. The hypoxia-selective cytotoxicities of tetrahydrorakicidin A (2), its acyclic derivative 3, rakicidin B,7 and a new congener, rakicidin E (1: Supporting Information), were evaluated along with rakicidin A (Table 1). A significant decrease in the cytotoxicity against PANC-1 and HCT-8 cells under both the oxygenation conditions was observed for 2 and 3, indicating that the conjugated diene functionality in the vDHA residue is essential for cytotoxicity. Moreover, the hypoxia selectivity gradually decreased with the extension of the aliphatic chain. These functionalities should be conserved when exploiting the structure of rakicidin A for drug development and designing bioprobes to study its mode of action.

Scheme 3. Determination of the Absolute Stereochemistry of Htha (7)

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EXPERIMENTAL SECTION

General Experimental Procedures. Cosmosil 75C18-PREP (Nacalai Tesque Inc., 75 μm) was used for ODS flash chromatography. Semipreparative HPLC was performed on an Agilent Technologies 1260 Infinity LC-DAD system with an automated fraction collector using a Cosmosil Cholester column (Nacalai Tesque Inc., 1 × 25 cm) for purification of rakicidins A, B, and E (1) and a Shimadzu LC-10AT pump equipped with a Shimadzu SPD-10A UV−vis detector using a Cosmosil AR-II column (Nacalai Tesque Inc., 1 × 25 cm) for purification of 2, 8a/8b, and 9a/9b. NMR spectra were obtained on a Bruker AVANCE 500 spectrometer at 500 MHz for 1H. Residual solvent peaks at δH 3.31, 7.27, and 2.50 ppm and HSQC cross-peaks δH/δC 3.31/49.2, 7.27/77.0, and 2.50/39.5 ppm in CD3OD, CDCl3, and DMSO-d6, respectively, were used as chemical shift reference signals. TLC silica gel 60 F254 (Merck KGaA) was used for thin-layer chromatography. Analysis of βOHAsp derivatized with FDLA was performed on an Agilent 1100 LC-DAD system using a Cosmosil 5PEMS column (Nacalai Tesque Inc., 4.6 × 250 mm). HR-ESITOFMS were recorded on a Bruker micrOTOF focus mass spectrometer. Optical rotation and UV and CD spectra were recorded on a JASCO DIP-3000 polarimeter, a Hitachi U-3210 spectrophotometer, and a JASCO J720W spectropolarimeter, respectively. Extraction and Isolation. Fermentation of the rakicidins producer strain Micromonospora sp. TP-A0860 was described in our previous article.22 The 1-butanol extract (59.9 g) from the fermentation broth (17.6 L) was chromatographed on silica gel, eluting with a step gradient

assignment of its absolute configuration. This anomalous result was attributed to the conformational restriction caused by a hydrogen bond formed between the carboxylate oxygen and 3hydroxy group (Figure 2). To remove this constraint, 8a and 8b were acetylated to afford esters 9a and 9b. As expected, a systematic distribution of the signs for Δδ values was obtained, which supported a 2S-configuration. By correlating this assignment to the already established relative stereochemistry, the absolute configuration of 7 was concluded to be (2S,3S,4R). The stereochemistry established for rakicidin A is readily applicable to rakicidin B and rakicidin D because of the similarity in the NMR data and optical properties. A recent publication on C

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Table 1. Cytotoxicity (IC50: μM) of Rakicidins A, B, and E (1), Tetrahydrorakicidin A (2), and Carboxylic Acid (3) under Two Oxygenation Conditions and Indices of Hypoxia Selectivity cell

condition

rakicidin A

rakicidin B

1

2

3

Doxa

HCT-8

normoxia hypoxia SIb normoxia hypoxia SIb

3.9 0.30 12.8 2.3 0.37 6.3

2.1 0.21 10.0 3.1 0.71 4.3

9.4 1.8 5.2 17.9 4.7 3.8

27.7 6.5 4.3 19.3 75.4 0.26

33.9 63.4 0.54 27.4 >80

2.4 19.6 0.12 1.1 5.4 0.21

PANC-1

a

Doxorubicin. bIndex of hypoxia selectivity calculated from the equation IC50(normoxia)/IC50(hypoxia). TFA, 4.0 mL/min, 210 nm) to afford 3 (0.5 mg, 0.8 μmol): 1H NMR (500 MHz, CD3OD) δ 5.01 (d, J = 14.6 Hz, 1H, βOHAsn α-CH), 4.63 (d, J = 18.5 Hz, 1H, βOHAsn β-CH), 4.14 (s, 2H, Sar α-CH2), 3.92 (m, 1H, Apa H-4), 3.58 (m, 1H, Htha H-3), 3.04 (s, 3H, Sar N-CH3), 2.44 (m, 1H, Htha H-2), 1.55 (m, 1H, Htha H-4), 1.50 (m, 1H, Htha H-16), 1.30 (brs, 22H, Htha H-5−H-15), 1.17 (m, 2H, Apa, H-3), 1.15 (m, 3H, Apa H-5), 1.08 (m, 3H, Htha H-19), 0.87 (d, J = 7.0 Hz, 6H, Htha H-17, H-18), 0.86 (m, 3H, Htha H-20); 13C NMR (data assigned from HSQC, CD3OD) δ 76.6 (Htha C-3), 72.7 (βOHAsn β-CH), 56.3 (βOHAsn αCH), 51.5 (Sar α-CH2), 46.0 (Apa C-4), 45.4 (Htha C-2), 40.1 (Apa, C3), 36.8 (Sar N-CH3), 29.5 (Htha C-5 - C-15), 23.1 (Htha C-17, C18), 21.1 (Apa C-5), 15.7 (Htha C-19), 13.1 (Htha C-20). Apa CH2-2 was not observed. Methyl Ester Formation of 3. To a solution of 3 (0.3 mg, 0.48 μmol) in MeOH (150 μL) was added a 10% n-hexane solution of TMSdiazomethane (150 μL), and the reaction mixture was stirred for 30 min at room temperature. The solution was then quenched by AcOH (150 μL) and concentrated in vacuo to afford 4 (0.3 mg, 0.47 μmol): 1H NMR (500 MHz, CD3OD) δ 4.15 (s, 1H, Apa Sar α-CH2b), 4.08 (s, 1H, Sar αCH2a), 3.92 (m, 1H, Apa H-4), 3.72 (s, 3H, βOHAsn α-CO2CH3), 3.58 (m, 1H, Htha H-3), 3.09 (s, 3H, Sar N-CH3), 2.47 (m, 2H, Apa H-2), 2.44 (m, 1H, Htha H-2), 1.56 (m, 1H, Htha H-4), 1.31 (brs, 22H, Htha H-5−H-15), 1.17 (m, 3H, Apa H-5), 1.17 (m, 2H, Apa H-3), 1.08 (d, J = 7.1 Hz, 3H, Htha H-19), 0.87 (d, J = 6.7 Hz, 6H, Htha H-17, H-18), 0.86 (m, 3H, Htha H-20); 13C NMR (data assigned from HSQC, CD3OD) δ 75.5 (Htha C-3), 51.3 (βOHAsn α-CO2CH3), 49.2 (Sar α-CH2), 44.7 (Apa C-4), 44.3 (Htha C-2), 39.0 (Apa C-3), 36.0 (Sar N-CH3), 35.0 (Htha C-4), 29.6 (Apa C-2), 21.9 (Htha C-17, C-18), 19.9 (Apa C-5), 14.5 (Htha C-19), 11.9 (Htha C-20). Signals for α- and β-methines βOHAsn and Htha CH-16 were not observed. Acid Hydrolysis of 2. Compound 2 (3.4 mg, 5.57 μmol) was placed in a hydrolysis tube and dissolved in 6 N HCl (600 μL). The tube was degassed, sealed, and heated at 110 °C for 18 h. After leaving the hydrolysis tube until cool, the solution was extracted with Et2O. The aqueous layer was concentrated in vacuo to give a residue containing amino acid 6, while the combined organic extracts were concentrated in vacuo to afford crude fatty acid 7 (1.8 mg, 5.49 μmol) as a yellow oil: 1H NMR for 7 (500 MHz, CDCl3) δ 3.65 (dd, J = 2.8, 8.3 Hz, 1H, H-3), 2.68 (m, 1H, H-2), 1.63 (m, 1H, H-4), 1.53 (m, 1H, H-16), 1.27 (brs, 20H, H-5−H14), 1.22 (m, 3H, H-19), 1.16 (m, 2H, H-15), 0.90 (d, J = 7.1 Hz, 3H, H-20), 0.87 (d, J = 6.7 Hz, 6H, H-17, H-18); 13C NMR for 7 (data assigned from HSQC, CDCl3) δ 75.6 (C-3), 42.2 (C-2), 38.7 (C15), 34.7 (C-4), 29.7 (C-5−C-14), 27.6 (C-16), 22.3 (C-17, C-18), 13.8 (C-19), 12.7 (C-20). Derivatization of 8 with Modified Marfey’s Reagents. To a solution of 6 in 0.1 N NaHCO3 (100 μL) was added a solution of LFDLA (50 μg, 0.16 mol) in acetone (50 μL) at room temperature. The reaction mixture was heated at 60 °C for 40 min and then quenched with 0.2 N HCl (50 μL) to afford 6a. Derivatization of L-threo β-OHAsp Standards with Modified Marfey’s Reagents. To a solution of L-threo β-OHAsp (1.0 mg) in 0.1 N NaHCO3 (100 μL) was added a solution of L-FDLA (50 μg, 0.16 mol) in acetone (50 μL). The reaction mixture was stirred for 30 min at 50 °C and then quenched with 0.1 N HCl (50 μL). In an entirely analogous manner, D-DLA-L-threo β-OHAsp was prepared using D-FDLA. HPLC Analysis of FDLA Derivatives. The quenched reaction solutions were diluted with 50% aqueous MeCN containing 0.05% TFA

of CHCl3/MeOH from 1:0 to 0:1 (v/v). Fractions eluted with CHCl3/ MeOH = 1:1 and 0:1 were combined (total 7.028 g) and then separated on ODS by a stepwise elution with aqueous MeCN. A fraction eluted between 70% and 80% aqueous MeCN (628.1 mg) was purified by reversed-phase HPLC on a Cosmosil Cholester column using 70% aqueous MeCN as a solvent (4.0 mL/min, UV = 280 nm) to afford rakicidin A (10.8 mg, tR = 12.7 min), rakicidin B (7.3 mg, tR = 17.9 min), and a new congener, rakicidin E (1) (1.8 mg, tR = 25.2 min). The physicochemical properties of rakicidins A and B were essentially the same as those previously reported.7,10 Rakicidin E (1): white floc; [α]20D −4.3 (c 0.1, benzene); UV (EtOH) λmax nm (log ε) 260 (4.8), 219 (4.7); CD (EtOH) λ nm (Δε) 259 (+0.2), 219 (−0.5); HRESITOFMS 633.4223 [M − H]−, C34H58N4O7; calcd 633.4223; 1H and 13C NMR data, see Supporting Information. Evaluation of Hypoxia-Selective Cytotoxicity. Human colorectal adenocarcinoma cell line HCT-8 or human pancreatic adenocarcinoma cell line PANC-1 was seeded in duplicated 96-well microplates at a density of 2 × 104/100 μL medium per well and preincubated for 24 h to ensure complete adherence to the substratum. To these microcultures were added serially diluted sample solutions, and test cultures thus prepared were separately incubated in either a normoxic (5% CO2) or hypoxic condition (1% O2). Hypoxic conditions were achieved using a MIC-101 modular incubator chamber (BillupsRosenberg, Del Mar, CA, USA) equipped with an oxygen indicator. After 2 d of incubation, cytotoxicity at both the oxygenation conditions was compared by the crystal violet method under normoxic conditions.23 Hydrogenation of Rakicidin A. To a solution of rakicidin A (2.5 mg, 4.12 μmol) in a mixture of pyridine (150 μL) and DMF (200 μL) was added Pd/C (2.5 mg). The reaction mixture was stirred for 2 h under a H2 atmosphere at room temperature and then filtered through Celite to remove Pd/C. The filtrate and an ethanolic wash of the retentate were combined, and the resulting concentrate was purfied by HPLC (Cosmosil Cholester column, 60% aqueous MeCN for 5 min and 1%/min concentration ramp to 85%, 4.0 mL/min, 210 nm) to give tetrahydrorakicidin A (2) (1.3 mg, 2.14 μmol): 1H NMR (500 MHz, CD3OD) δ 5.25 (d, J = 1.9 Hz, 1H, βOHAsn α-CH), 4.97 (dd, J = 3.2, 8.7 Hz, 1H, Htha H-3), 4.77 (d, J = 1.9 Hz, 1H, βOHAsn β-CH), 4.40 (d, J = 18.6 Hz, 1H, Sar α-CH2b), 3.93 (m, 1H, Apa H-4), 3.88 (d, J = 18.6 Hz, 1H, Sar α-CH2a), 2.96 (s, 3H, Sar N-CH3), 2.84 (m, 1H, Htha H-2), 2.22 (m, 1H, Apa H-2b), 2.10 (m, 1H, Apa H-2a), 1.98 (m, 1H, Htha H4), 1.53 (m, 1H, Htha H-16), 1.30 (brs, 20H, Htha H-5−H-14), 1.18 (m, 2H, Apa H-3), 1.18 (m, 2H, Htha H-15), 1.16 (m, 3H, Apa H-5), 1.15 (m, 3H, Htha H-19), 0.91 (d, J = 6.7 Hz, 3H, Htha H-20), 0.88 (d, J = 6.6 Hz, 6H, Htha H-17, H-18); 13C NMR (data assigned from HSQC, CD3OD) δ 81.3 (Htha C-3), 71.0 (βOHAsn β-CH), 55.2 (βOHAsn αCH), 45.4 (Apa C-4), 43.0 (Htha C-2), 38.9 (Htha C-15), 35.4 (Htha C4), 35.1 (Sar N-CH3), 32.8 (Htha H-15), 29.5 (Htha C-5−C-14), 28.8 (Apa C-2), 28.0 (Htha C-16), 21.8 (Htha C-17, C-18), 19.7 (Apa H-5), 14.6 (Htha C-20), 14.5 (Htha C-19). Saponification of 2. Compound 2 (0.8 mg, 1.31 μmol) in a mixture of MeOH (220 μL) and 1 N LiOH (693 μL) was stirred overnight at room temperature. To quench and further acidify the reaction, 1 N HCl (1.5 mL) was added to the solution, and this was extracted with Et2O. The combined organic extracts were concentrated and then purified by HPLC (Cosmosil AR-II column, 60% aqueous MeCN containing 0.05% D

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(100 μL), and 5 μL aliquots were analyzed by HPLC (Cosmosil 5PEMS column, 9% aqueous MeCN containing 50 mM AcONH4, 1.0 mL/ min, 340 nm). The retention times for the D-FDLA and L-FDLA derivatives of L-threo β-OHAsp were 18.8 and 21.3 min, respectively, whereas that for 6a was 18.8 min. Preparation of Htha (R)-PGME Amide (8a). To a solution of 7 (0.4 mg, 1.22 μmol) in DMF (10 μL) was added (R)-PGME·HCl (0.6 mg, 2.99 μmol), PyBOP (1.2 mg), HOBt (0.5 mg), and TEA (200 μL) at room temperature. The reaction mixture was stirred for 1 h and then concentrated in vacuo. The dried residue was directly purified by HPLC (Cosmosil 5C18-AR-II column, 90% MeCN to 100% MeCN) to afford compound 8a as a colorless oil (0.4 mg, 0.89 μmol): 1H NMR (500 MHz, CD3OD) δ 7.37 (m, 6H, PGME Phe), 5.48 (s, 1H, PGME β-CH), 3.70 (s, 3H, PGME OCH3), 3.56 (dd, J = 4.1, 8.3 Hz, 1H, H-3), 2.61 (m, 1H, H-2), 1.56 (m, 1H, H-4), 1.52 (m, 1H, H-16), 1.28 (brs, 20H, H-5 H-14), 1.17 (m, 2H, H-15), 1.10 (m, 3H, H-19), 0.87 (d, J = 6.6 Hz, 6H, H-17, H-18), 0.84 (d, J = 6.9 Hz, 3H, H-20); 13C NMR (data assigned from HSQC, CD3OD) δ 129.0 (PGME Phe), 76.7 (C-3), 58.0 (PGME β-CH), 52.8 (PGME OCH3), 44.8 (C-2), 40.1 (C-15), 36.1 (C-4), 30.6 (C-5−C-14), 22.9 (C-17, C-18), 13.6 (C-20). C-16 and C-19 were not observed. Preparation of Htha (S)-PGME Amide (8b). Compound 7 (0.4 mg, 1.22 μmol) was derivatized with (S)-PGME·HCl (2.4 mg, 12.0 μmol) by essentially the same procedure using PyBOP (4.8 mg), HOBt (2.2 mg), and TEA (160 μL) to give 8b (0.3 mg, 0.67 μmol): 1H NMR (500 MHz, CD3OD) δ 7.36 (m, 6H, PGME Phe), 5.48 (s, 1H, PGME βCH), 3.69 (s, 3H, PGME OCH3), 3.54 (m, 1H, H-3), 2.62 (m, 1H, H-2), 1.56 (m, 1H, H-4), 1.52 (m, 1H, H-16), 1.28 (brs, 20H, H-5−H-14), 1.17 (m, 2H, H-15), 1.06 (m, 3H, H-19), 0.87 (m, 6H, H-17, H-18), 0.87 (m, 3H, H-20); 13C NMR (data assigned from HSQC, CD3OD) δ 130.0 (PGME Phe), 77.0 (C-3), 58.0 (PGME β-CH), 52.9 (PGME OCH3), 44.7 (C-2), 40.1 (C-15), 36.0 (C-4), 30.5 (C-5−C14), 27.8 (C-16), 22.7 (C-17, C-18), 14.1 (C-19), 13.4 (C-20). Acetylation of 8a. To a solution of compound 8a (0.4 mg, 0.89 μmol) in pyridine (400 μL) was added Ac2O (200 μL) at room temperature. The reaction mixture was stirred for 1 h, lyophilized, and then purified by HPLC with the same condition adopted for the purification of 8a to afford compound 9a (0.1 mg, 0.20 μmol) as a colorless oil: 1H NMR (500 MHz, CD3OD) δ 7.37 (m, 6H, PGME Phe), 5.46 (s, 1H, PGME β-CH), 5.08 (dd, J = 2.2, 10.3 Hz, 1H, H-3), 3.68 (s, 3H, PGME OCH3), 2.83 (m, 1H, H-2), 1.88 (s, 3H, OAc), 1.73 (m, 1H, H-4), 1.28 (m, 20H, H-5−H-14), 1.16 (m, 2H, H-15), 1.09 (d, J = 7.2 Hz, 3H, H-19), 0.89 (m, 3H, H-20), 0.87 (m, 6H, H-17, H-18); 13C NMR (data assigned from HSQC, CD3OD) δ 127.8 (PGME Phe), 76.7 (C-3), 56.4 (PGME β-CH), 51.4 (PGME OCH3), 38.6 (C-15), 29.3 (C5−C-14), 21.6 (C-17, C-18), 12.1 (C-20). C-2, C-3, C-16, and C-19 were not observed. Acetylation of 8b. Compound 9b (0.2 mg, 0.41 μmol) was prepared by essentially the same procedure described above except for the amounts of reagents used: 8b (0.3 mg, 0.67 μmol), pyridine (300 μL), and Ac2O (150 μL). 1H NMR (500 MHz, CD3OD) δ 7.36 (m, 6H, PGME Phe), 5.41 (s, 1H, PGME β-CH), 5.14 (dd, J = 2.4, 10.0 Hz, 1H, H-3), 3.67 (s, 3H, PGME OCH3), 2.84 (m, 1H, H-2), 1.88 (s, 3H, OAc), 1.74 (m, 1H, H-4), 1.51 (m, 1H, H-16), 1.28 (m, 20H, H-5−H-14), 1.17 (m, 2H, H-15), 1.09 (d, J = 7.2 Hz, 3H, H-19), 0.91 (m, 3H, H-20), 0.87 (m, 6H, H-17, H-18); 13C NMR (data assigned from HSQC, CD3OD) δ 127.7 (PGME Phe), 76.8 (C-3), 57.0 (PGME β-CH), 52.6 (PGME OCH3), 42.2 (C-2), 39.0 (C-15), 33.8 (C-4), 29.4 (C-5−C-14), 27.8 (C-16), 21.8 (C-17, C-18), 13.3 (C-19), 12.2 (C-20).

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Note

AUTHOR INFORMATION

Corresponding Author

*E-mail (Y. Igarashi): [email protected]. Present Address

§ Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo, Hokkaido 060-0812, Japan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a fund from Research Supporters Association for Toyama Prefectural University FY2011. REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

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H NMR chemical shift differences of 7 relative to literature values, results of hypoxia/normoxia differential cytotoxicity testings, comparison of 1H and 13C NMR data between 1 and rakicidin A, NMR spectra for 1−4, 7, 8a/b, and 9a/b. This material is available free of charge via the Internet at http://pubs. acs.org. E

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