Synthesis of lesquerella α-hydroxy phosphonates

Industrial Crops and Products 53 (2014) 236–243

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Synthesis of lesquerella ␣-hydroxy phosphonates夽 John S.P. Cusimano a , Margaret M. Hart a , Diana M. Cermak a,∗ , Steven C. Cermak b,∗∗ , Amber L. Durham b,∗ ∗ ∗ a b

Knox College, Department of Chemistry, 2 E. South St., Galesburg, IL 61401, United States Bio-Oils Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, 1815 N. University St., Peoria, IL 61604, United States

a r t i c l e

i n f o

Article history: Received 29 August 2013 Received in revised form 18 December 2013 Accepted 20 December 2013 Keywords: Lesquerella oil Lesquerolic acid ␣-Hydroxy phosphonates

a b s t r a c t Hydroxy fatty acids (HFAs) have found a number of uses in today’s market, with uses ranging from industrial materials to pharmaceuticals. Castor oil, which is obtained from castor seeds, has served as a source of a versatile HFA; its principle component, ricinoleic acid, can be isolated from castor oil and has been modified extensively for a number of applications. Unfortunately, castor seeds also contain several undesirable compounds which pose severe health risks, including ricin, an unusually stable, deadly protein; ricinine, a poisonous alkaloid; and several allergens. Lesquerella oil, obtained from seeds of the Lesquerella fendleri species, has been identified as the most promising alternative source of HFAs. Lesquerolic acid, the primary HFA found in lesquerella oil, is also most homologous to ricinoleic acid; the only structural difference between the two is that lesquerolic acid possesses two additional methylene groups on the carboxyl end of the molecule. This structural and chemical similarity further suggests lesquerella oil and its derivatives may function as a competitive alternative to castor oil. Unfortunately, the desired HFA, lesquerolic acid, constitutes approximately 50% of lesquerella oil where castor oil contains approximately 90% of the desired HFA. This difference in desired HFA abundance in lesquerella oil required more extensive purification measures and careful identification of the correct component. Additionally, ␣-hydroxy phosphonates and their corresponding phosphonic acids are functional moieties that have been shown to display a wide variety of biological activities, as enzyme inhibitors, pesticides, antibiotics and anti-cancer therapeutics. We previously reported the synthesis of two families of ␣-hydroxy phosphonic acids based on ricinoleic acid: a family that retains the cis alkene found in ricinoleic acid and one where the alkene has undergone hydrogenation to produce a saturated ␣-hydroxy phosphonic acid. We now report the purification of the desired lesquerolic acid component of lesquerella oil and synthesis of lesquerolic acidderived ␣-hydroxy phosphonates, both the unsaturated and saturated families. As with the ricinoleic acid-based compounds, the lesquerolic acid-based compounds have been produced in high yields and high purity, and the synthesis of these compounds is reported in this manuscript. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hydroxy fatty acids (HFAs) represent a highly versatile family of compounds derived from triacylglycerols (TAGs), esters of glycerol and three fatty acids. TAGs serve as depots of metabolic fuel as fat droplets in animals’ adipose cells and in plants’ seeds; TAGs are thus the main constituents of vegetable oils (Nelson and

夽 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. ∗ Corresponding author. Tel.: +1 309 341 7434; fax: +1 309 341 7083. ∗∗ Corresponding author. Tel.: +1 309 681 6233; fax: +1 309 681 6524. ∗ ∗ ∗Corresponding author. Tel.: +1 309 681 6233; fax: +1 309 681 6524. E-mail addresses: [email protected] (D.M. Cermak), [email protected] (S.C. Cermak). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.12.031

Cox, 2008). Currently HFAs and their derivatives have been utilized in high-performance lubricants, cosmetics, waxes, nylons, plastics, drying agents, protective coatings, surfactants, and pharmaceuticals (Powell, 2009; Salywon et al., 2005; Glaser et al., 1992). Additionally, estolide derivatives of HFAs, oligomeric fatty acid esters, have demonstrated superior oxidative stability, pour points, and low temperature viscosity than their vegetable oil counterparts (Cermak et al., 2006; Isbell et al., 2006). These properties make HFAs particularly attractive as additives in low lubricity diesel fuels and ultralow sulfur diesel fuels (Prasad et al., 2012; Lapuerta et al., 2010; Moser et al., 2008). However, in spite of HFAs seemingly ubiquitous prevalence, the only HFA currently available in sufficient quantities for commercial purposes is ricinoleic acid [(9Z)-12-hydroxy-9-octadecenoic acid], derived from castor oil (1, Fig. 1) (Severino et al., 2012; Salywon et al., 2005). Castor oil comes from the the seeds of the Ricinus communis plant, more commonly known as the castor plant, and although the

J.S.P. Cusimano et al. / Industrial Crops and Products 53 (2014) 236–243

Fig. 1. Hydroxy fatty acids: ricinoleic acid [(9Z)-12-hydroxy-9-octadecenoic acid] (1), densipolic acid [(9Z,15Z)-12-hydroxyoctadeca-9,15-dienoic acid] (2), auricolic acid [(11Z,17Z)-14-hydroxyeicos-11,17-dienoic acid] (3), and lesquerolic acid [(11Z)-14-hydroxy-11-eicosenoic acid] (4).

annual shrub is native to Africa, it is capable of thriving in temperate climates worldwide. Accordingly, India now accounts for 70% of all castor exports, followed distantly by China, Brazil, and Thailand (Mutlu and Meier, 2010). Fortunately, castor seeds contain over 50% oil by weight, nearly 90% of which is the highly desired ricinoleic acid. This is attributed to the castor plant’s ability to incorporate ricinoleic acid at all three acyl positions of the glycerol backbone of the TAG molecules; the remaining ∼10% is a mixture of saturated, unsaturated and hydroxy fatty acids (Table 1) (Cermak et al., 2006; Glaser et al., 1992). Unfortunately, castor seeds also contain several undesirable compounds which pose severe health risks. These include ricin, an unusually stable, deadly protein that inhibits protein synthesis by depurinating a specific adenosine residue on the eukaryotic 60S ribosomal subunit; ricinine, a poisonous alkaloid; and several allergens, the most potent of which, CB-1A (castor bean allergen 1), exhibits an extraordinary ability to sensitize individuals exposed Table 1 Chemical composition of castor and Lesquerella fendleri oils, determined by GC and taken from Cermak and Evangelista (2011) and Salywon et al., 2005. Fatty acid Methyl esters

Castor oil (mass%)

Lesquerella oil (mass%)

16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 18:1 Hydroxy (1)a 18:2 Hydroxy (2)a 20:1 Hydroxy (4)a 20:2 Hydroxy (3)a

1.0 – – 3.7 4.4 – – – – 89.0

1.1 0.7 1.8 15.4 6.9 12.2 0.2 1.0 0.2 0.6 0.1 55.4 3.8

a

See Fig. 1.

1.1 –

237

to small amounts of the dust (Mutlu and Meier, 2010; Daussant et al., 1976). Because the hazards imposed upon workers by these compounds is caused by dust produced during the harvesting and refining process, castor has not been grown commerically in the United States since the early 1970s (Glaser et al., 1992). Accordingly, due to high demand and large array of applications for castor oil and the ricinoleic acid it contains, the United States now ranks among the top three importers of castor oil worldwide with a demand that exceeds 45,000 metric tons annually (Mutlu and Meier, 2010; Salywon et al., 2005). These drawbacks have motivated the United States to seek alternatives to the HFAs found in castor oil and in 1985 the United States began an active breeding program to explore alternatives including species within the Lesquerella genus (Salywon et al., 2005). Thus far, the Lesquerella fendleri species, hereafter referred to as lesquerella, has been identified as the most promising alternative source of HFAs (Dierig et al., 2011). The seeds of this North American winter annual possess roughly 30% oil by weight and contain roughly 55–64% HFAs (Cermak et al., 2006). Although lesquerella contains comparatively less desired oil than the castor plant, it is a highly cross-pollinated species which suggests breeding can lead to varieties with improved oil content, an increased amount of HFAs in the oil, higher yield, erect growth, and other traits desired by oilseed crops (Cermak et al., 2006; Glaser et al., 1992). Furthermore, previously mentioned breeding efforts stretching back several decades have already determined harvesting, water, fertilization, and herbicide requirements as well as planting methods and salt tolerance for the species (Glaser et al., 1992). These efforts have made it apparent that the arid climate of the American Southwest is a well suited environment for lesquerella and that traditional farm equipment can be used to harvest with no new investment. Additionally, lesquerella is not subject to attack by any notable diseases or pests, it requires fewer pesticides than either cotton or wheat, and does not possess ricin or any other potent allergens (Salywon et al., 2005). The primary HFAs found across the plants of the Lesquerella genus include densipolic acid (2), auricolic acid (3), and lesquerolic acid (4), all structural homologs to ricinoleic acid (1) (Fig. 1) (Salywon et al., 2005). The most promising HFA of the three is lesquerolic acid because it is the most abundant, comprising ∼55% of the total HFA content of the L. fendleri seeds (Cermak and Evangelista, 2011). However, the total percentage of lesquerolic acid (4) available in the crude oil is limited to 66% because plants of the Lesquerella genus are unable to incorporate lesquerolic acid at the sn-2, or middle position, of the TAG backbone. This is likely due to the fact that seeds of the lesquerella plant, unlike those of castor, lack or do not sufficiently express the PDAT 1–2 genes (Kim et al., 2011). These genes encode two phospholipid:diacylglycerol acyltransferase isozymes, enzymes that catalyze the final step in the acyl-CoA independent TAG biosynthetic pathway (Brown et al., 2012). Future breeding efforts or genetic modifications may be able to circumvent this current limitation. Lesquerolic acid is also most homologous to ricinoleic acid. In fact, the only structural difference between the two is that lesquerolic acid possesses two additional methylene groups on the carboxyl end of the molecule. This structural and chemical similarity further suggests lesquerella oil and its derivatives may function as an alternative to castor oil especially considering it can undergo the same chemical reactions as castor oil (Mutlu and Meier, 2010). A comparison of the fatty acid profiles of castor and lesquerella can be found in Table 1. ␣-Hydroxy phosphonates and their corresponding phosphonic acids are widely recognized as an important structural moiety. They have been shown to exhibit a wide variety of biological activity as enzyme inhibitors of renin, enzyme 5-enolpyruvylshikimate-3phosphate (EPSP) synthase, human immunodeficiency virus (HIV) protease, and farnesyl protein transferase (FPTase) (Wiemer, 1997; Cermak et al., 1999; Pompliano et al., 1992). They have also

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Fig. 2. Monounsaturated and saturated ␣-hydroxy phosphonates from lesquerolic acid.

been utilized as pesticides, antibiotics and anticancer and antiviral agents (Wu et al., 2010). ␣-Hydroxy phosphonates have also shown use as synthetic intermediates in the synthesis of other ␣substituted phosphonates and phosphonic acids, most commonly ␣-amino phosphonic acids, which have been shown to exhibit a wide variety of biological activity of their own (Kafarski and Lejczak, 1991). We have previously shown that ricinoleic acid, the principal component of castor oil, can be chemically modified to produce ␣hydroxy phosphonate and phosphonic acid analogs (Cermak et al., 2012). Noting the structural similarities between ricinoleic acid (1) and lesquerolic acid (4) and expected similarities in reactivity, it was anticipated that the same synthetic route used to produce the castor phosphonate derivatives could be utilized to produce monounsaturated and saturated lesquerella phosphonate derivatives (5a and 5b, respectively; Fig. 2). 2. Materials and methods 2.1. Materials Lesquerella oil was obtained by USDA-ARS (Peoria, IL) and was refined, bleached and deodorized (RBD) before use (Cermak and Evangelista, 2013). All other chemicals used were purchased from Aldrich Chemical Co. (Milwaukee, WI) and no further purification was necessary. Diethyl ether and tetrahydrofuran (THF) were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were dried by VAC solvent purifier (Vacuum Atmospheres Co.). Dichloromethane was obtained from AAPER Alcohol and Chemical (Shelbyville, KY) and triethylamine was obtained from Aldrich Chemical Co. (Milwaukee, WI); both solvents were freshly distilled from calcium hydride. Methanol was obtained from Aldrich Chemical Co. (Milwaukee, WI) as an anhydrous solvent. All reactions in non-hydroxylic solvents were conducted in oven-dried glassware under a positive pressure of nitrogen. Solvents for extraction and flash-column chromatography were reagent grade or better and were used without further purification. Flash-column chromatography was carried out on SiliCycle 40–63 ␮m (230–400 mesh) silica gel obtained from SiliCycle Inc. (Quebec City, Quebec, Canada). Thin layer chromatography (TLC) was performed on Aldrich silica gel 60F254 pre-coated TLC plates of 0.2 mm thickness. TLC plates were visualized using ultraviolet light and stained with phosphomolybdic acid (PMA) in ethanol. The acronyms used are defined as the following: DIBAL; diisobutylaluminum hydride; DMSO, dimethyl sulfoxide; TBDMS, tert-butyldimethylsilyl; TBAF, tetrabutylammonium fluoride; TMSBr, trimethylsilyl bromide. 2.2. Instrumentation 2.2.1. Nuclear Magnetic Resonance (NMR) 1 H, 13 C and 31 P NMR spectra were obtained on a Bruker ARX-500 (Karlsruhe, Germany) with a 5 mm dual proton/carbon probe (1 H at 500.11 MHz, 13 C at 125.77 MHz, 31 P at 202.44 MHz) using CDCl3 as solvent unless otherwise noted. Chemical shifts for 1 H NMR spectra were reported in ppm relative to Me4 Si (ı 0.00), chemical shifts for 13 C NMR spectra (1 H decoupled) were reported in ppm relative to

the center line of the triplet corresponding to CDCl3 (ı 77.00), and chemical shifts for 31 P NMR spectra (1 H decoupled) were reported in ppm relative to phosphoric acid (ı −0.12). Splitting patterns for 1 H NMR spectral absorptions are denoted as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets. Assignment of hydrogens and carbons are specified for ester 10 and hydroxy phosphonates 13a and 13b; other compounds are similar in assignment to these compounds and are thus not specified. 2.2.2. Gas chromatography Gas chromatography (GC). GC was performed with a HewlettPackard 5890 Series II gas chromatograph (Palo Alto, CA) equipped with a flame-ionization detector and an autosampler/injector. Analyses were conducted on a SP-2380 30 m × 0.25 mm i.d. column (Supelco, Bellefonte, PA). Saturated C8 –C30 FAMEs provided standards for calculating equivalent chain length (ECL) values, which were used to make fatty acid and by-product assignments. Parameters for SP-2380 analysis were: column flow 1.0 mL/min with helium head pressure of 15 psi; split ratio 50:1; programmed ramp to 120–135 ◦ C at 20 ◦ C/min, 135–265 ◦ C at 7 ◦ C/min, hold 5 min at 265 ◦ C; injector and detector temperatures set at 250 ◦ C. Retention times for eluted peaks of crude lesquerella oil transesterfication product; parentheses include compound description and ECL values as a %: methyl palmitate 7.52 min (16:0, 1.1%), methyl palmitoleate 8.10 min (16:1, 0.6%), methyl stearate 9.30 min (18:0, 1.9%), methyl oleate 9.85 min (18:1, 16.1%), methyl linoleate 10.61 min (18:2, 6.9%), methyl arachidate 11.26 min (20:0, 0.7%), methyl cis-11-eicosenoate 11.45 min (20:1, 0.8%), methyl linolenate 11.54 min (18:3, 10.7%), methyl ricinoleate 17.09 min (18:1 OH, 0.5%), methyl lesqueroleate 18.53 min (20:1 OH, 9, 56.8%) and methyl auricoleate 19.25 min (20:2 OH, 3.5%). Retention times for eluted peaks of purified methyl lesqueroleate with ECL values in parentheses were: methyl ricinoleate 17.10 min (0.9%), methyl lesqueroleate 18.53 min (9, 93.5%) methyl auricoleate 19.22 min (5.6%). 2.3. Methods 2.3.1. Procedure for esterification of lesquerella oil: preparation of ester 9 A mixture of lesquerella oil (242.09 g, 0.253 mol) and BF3 ·etherate (426 mL, 0.5 M in methanol, 0.213 mol) was heated to reflux for 10 h. The mixture was then cooled to rt and washed with pH 5 buffer (130 g NaH2 PO4 /1 L H2 O) until it reached pH 5. The reaction mixture was then dried (Na2 SO4 ), filtered, and the methanol was removed in vacuo. The remaining mixture was then distilled via Kugelrohr up to a temperature of 180 ◦ C (at 70 mTorr). The distillate was then purified by flash column chromatography (5% ethyl acetate/hexanes), yielding ester 9 as a clear, yellow oil (42.4 g, 89%): 1 H NMR (CDCl ) ı 5.30–5.58 (m, 2H), 3.65 (s, 3H), 3.60 (quintet, 3 J = 6.0 Hz, 1H), 2.29 (t, J = 7.5 Hz, 2H), 2.20 (t, J = 6.5 Hz, 2H), 2.03 (q, J = 6.9 Hz, 2H), 1.68 (br s, 1H), 1.56–1.64 (m, 2H), 1.39–1.49 (m, 3H), 1.21–1.37 (m, 19H), 0.87 (t, J = 6.9 Hz, 3H); 13 C NMR (CDCl3 ) ı 174.3, 133.3, 125.2, 71.5, 51.4, 36.8, 35.3, 34.1, 31.8, 29.6, 29.4, 29.4, 29.3, 29.2, 29.2, 29.1, 27.4, 25.7, 24.9, 22.6, 14.0; GC 18.53 min.

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2.3.2. Procedure for TBDMS protection of lesquerella ester 9: preparation of TBDMS protected ester 10 To a solution of lesquerella ester 9 (6.329 g, 18.58 mmol) in THF (130 mL) was added imidazole (3.199 g, 46.99 mmol) and TBDMSCl (3.036 g, 20.14 mmol) and the solution was heated to reflux for 48 h. The reaction mixture was cooled to rt and hexanes (160 mL) was added. The reaction mixture was washed with water (100 mL), dried over Na2 SO4 , filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (5% ethyl acetate/hexanes) to yield protected ester 10 (7.431 g, 88%) as a clear yellow oil: 1 H NMR (CDCl3 ) ı 5.35–5.45 (m, 2H, CH CH ),3.66 (s, 3H, OCH3 ), 3.62–3.71 (m, 1H, CH2 CH(OSi) CH2 ), 2.30 (t, J = 7.6 Hz, 2H, (C O) CH2 CH2 ), 2.18 (t, J = 5.9 Hz, 2H, CH CH2 CH(OSi) ), (q, J = 6.8 Hz, 2H, CH(OSi) CH2 CH2 ), 1.98–2.05 1.58–1.65 (m, 2H, CH CH2 CH2 ), 1.21–1.50 (m, 22H, (C O) CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH(OSi) CH2 CH2 CH2 CH2 CH2 CH3 ), 0.86–0.92 (m, 12H, Si C(CH3 )3 , CH2 CH3 ), 0.05 (s, 3H, SiCH3 ), 0.04 (s, 3H, SiCH3 ); 13 C NMR (CDCl3 ) ı 174.2 (C O), 131.4 ( CH CH ), 125.9 ( CH CH ), 72.4 ( CH(OSi) ), 51.3 ( OCH3 ), 36.9, 35.2, 34.1, 31.9, 29.7, 29.5, 29.4, 29.3, 29.2, 29.1, 27.4, 25.9 (3C, Si C(CH3 )3 ), 25.4, 24.9, 22.6, 18.1 ( Si C(CH3 )3 ), 14.1 ( CH2 CH3 ), −4.4 ( Si CH3 ), −4.6 ( Si CH3 ).

239

2.3.4. Hydrogenation of alcohol 11a: preparation of saturated alcohol 11b Protected alcohol 11a (2.503 g, 5.865 mmol) was dissolved in methanol (75 mL) and 10% Pd/C (0.246 g) was added in one portion. The flask was fit with a balloon containing excess H2 and the mixture was stirred vigorously at rt. After stirring for 41 h under H2 atmosphere, the solution was vacuum filtered over a pad of Celite, rinsed with ethyl acetate and concentrated in vacuo to give saturated alcohol 11b as a pale yellow semi-solid (2.309 g, 92%): 1 H NMR (CDCl3 ) ı 3.59–3.68 (m, 3H), 1.53–1.62 (m, 2H), 1.22–1.46 (m, 35H), 0.87–0.93 (m, 12H), 0.05 (s, 6H); 13 C NMR (CDCl3 ) ı 72.4, 63.0, 37.2, 32.8, 31.9, 29.9, 29.7, 29.6 (3C), 29.6 (2C), 29.6, 29.5, 29.4, 25.9 (3C), 25.7, 25.3, 25.3, 22.6, 18.1, 14.0, −4.4 (2C).

clear solution was stirred for 5 min, at which time a solution of protected alcohol 11a (1.863 g, 4.365 mmol) in CH2 Cl2 (15 mL) was added via cannula to provide a cloudy solution. After stirring at −80 ◦ C for 15 min, Et3 N (4.7 mL) was added and the reaction mixture was allowed to warm to 0 ◦ C, water (12 mL) was added, and the layers were separated. The aqueous layer was extracted with CH2 Cl2 (65 mL) and the organics were combined. The combined organics were washed with 0.5 M HCl (2 × 65 mL) and brine (65 mL), dried over MgSO4 , filtered, and concentrated in vacuo to give a clear, yellow oil. The resulting oil containing aldehyde 12a was dissolved in THF (50 mL) and dimethyl phosphite (0.90 mL, 9.8 mmol) was added. The mixture was cooled to −40 ◦ C, and a saturated solution of NaOMe [made by addition of sodium metal slivers to 12 mL anhydrous methanol at 0 ◦ C] was added via cannula. The reaction was stirred for 15 min at −40 ◦ C and sat. aq. NH4 Cl (40 mL) was added and allowed to warm to rt. The resulting solution was extracted with ethyl acetate (4 × 65 mL) and the combined organics were dried over MgSO4 , filtered, and concentrated in vacuo to give a crude oil. Purification by flash column chromatography (20% acetone/CH2 Cl2 ) gave ␣-hydroxy phosphonate 13a as a clear, yellow oil (1.866 g, 80% over two steps). ␣-Hydroxy phosphonate 13a, clear yellow oil: 80% over two steps, 1 H NMR (CDCl3 ) ı 5.28–5.43 (m, 2H, CH CH ), 4.36 (br s, 1H, (P O)CH(OH)CH2 ), 3.83–3.90 (m, 1H, (P O)CH(OH)CH2 ), 3.78 (d, J = 10.3 Hz, 3H, (P O)(OCH3 )2 ), 3.76 (d, J = 10.3 Hz, 3H, (P O)(OCH3 )2 ), 3.62 (quintet, J = 5.8 Hz, 1H, CH2 CH(OSi) CH2 ), 2.16 (t, J = 5.6 Hz, 2H, =CH-CH2 CH(OSi) CH2 CH2 ), CH(OSi) ), 1.98 (q, J = 6.9 Hz, 2H, 1.54–1.75 (m, 2H, CH(OH) CH2 CH2 ), 1.16–1.48 (m, 24H, CH(OH) CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH(OSi) CH2 CH2 CH2 CH2 CH2 CH3 ), 0.82–0.90 (m, 12H, Si C(CH3 )3 , CH2 CH3 ), 0.02 (s, 3H, SiCH3 ), 0.01 (s, 3H, SiCH3 ); 13 C NMR (CDCl3 ) ı 131.4 ( CH CH ), 125.8 ( CH CH ), 72.4 ( CH(OSi) ), 67.5 (d, JCP = 160.5 Hz, (P O)CH(OH) ), 53.2 (d, JCP = 7.1 Hz, (P O)(OCH3 )2 ), 53.1 (d, JCP = 7.1 Hz, (P O)(OCH3 )2 ), 36.8, 35.2, 31.8, 31.3, 29.7, 29.6, 29.5, 29.4, 29.4, 29.3, 29.2, 27.4, 25.9 (3C, Si C(CH3 )3 ), 25.6 (d, JCP = 13.5 Hz, (P O)CH(OH)CH2 ), 25.3, 22.6, 18.1 ( Si C(CH3 )3 ), 14.0 ( CH2 CH3 ), −4.4 ( Si CH3 ), −4.6( Si CH3 ); 31 P NMR (CDCl3 ) ı 27.7. ␣-Hydroxy phosphonate 13b, pale yellow semi-solid: 74% over two steps, 1 H NMR (CDCl3 ) ı 4.08 (br s, 1H, (P O)CH(OH)CH2 ), 3.85–3.92 (m, 1H, (P O)CH(OH)CH2 ), 3.80 (d, J = 10.3 Hz, 3H, (P O)(OCH3 )2 ), 3.78 (d, J = 10.3 Hz, 3H, (P O)(OCH3 )2 ), 3.60 (quintet, J = 5.8 Hz, 1H, CH2 CH(OSi) CH2 ), 1.54–1.78 (m, 2H, CH(OH) CH2 CH2 ), 1.16–1.44 (m, 32H, CH(OH) CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH(OSi) CH2 CH2 CH2 CH2 CH2 CH3 ), 0.84–0.92 (m, 12H, Si C(CH3 )3 , CH2 CH3 ), 0.02 (s, 6H,–Si(CH3 )2 ); 13 C NMR (CDCl3 ) ı 72.4 ( CH(OSi) ), 67.6 (d, JCP = 160.4 Hz, (P O)CH(OH) ), 53.2 (d, JCP = 7.2 Hz, (P O)(OCH3 )2 ), 53.1 (d, JCP = 7.2 Hz, (P O)(OCH3 )2 ), 37.1, 31.9, 31.4, 29.9, 29.7, 29.6, 29.6 (2C), 29.6 (2C), 29.5, 29.4, 29.3, 25.9 (3C, Si C(CH3 )3 ), 25.6 (d, JCP = 13.5 Hz, (P O)CH(OH)CH2 ), 25.3, 25.2, 22.6, 18.1 ( Si C(CH3 )3 ), 14.1 ( CH2 CH3 ), −4.44 (2C, Si CH3 ); 31 P NMR (CDCl3 ) ı 27.7.

2.3.5. Swern oxidation and subsequent phosphite addition: preparation of aldehydes 12a and 12b and ˛-hydroxy phosphonates 13a and 13b A solution of oxalyl chloride (3.3 mL, 2.0 M, 6.6 mmol) was dissolved in CH2 Cl2 (50 mL) and the mixture was cooled to −80 ◦ C. After 5 min at this temperature, DMSO (1.0 mL, 14 mmol) was added. After initial bubbling and turning cloudy, the resulting

2.3.6. Deprotection of TBDMS protecting group: preparation of phosphonates diols 5a and 5b ␣-Hydroxy phosphonate 13a (1.604 g, 2.999 mmol) was dissolved in 1% conc. HCl (12 M) in 95% ethanol (55 mL) and the cloudy mixture was stirred for 3 h. Sat. aq. NaHCO3 (230 mL) was added slowly to the clear solution and the mixture was extracted with ether (4 × 150 mL), dried over MgSO4 , filtered and concentrated

2.3.3. Procedure for DIBAL reduction of ester 10: preparation of alcohol 11a Protected ester 10 (2.016 g, 4.433 mmol) was dissolved in THF (86 mL) and the solution cooled to −78 ◦ C. DIBAL (28 mL, 1.0 M in THF, 28 mmol) was added dropwise maintaining the temperature below −78 ◦ C. The solution was stirred at −78 ◦ C for 2.5 h. The mixture was then quenched by dropwise addition of sat. aq. NH4 Cl (14 mL), maintaining the temperature below −78 ◦ C. The mixture was then warmed to rt and vacuum filtered, rinsing the resulting white solids with ethyl acetate (200 mL). The filtrate was washed with brine (60 mL), dried over Na2 SO4 , filtered and concentrated in vacuo. The residue was purified by flash column chromatography (gradient of 5% ethyl acetate/hexanes to 20% ethyl acetate/hexanes) to yield alcohol 11a (1.818 g, 96%) as a clear, yellow oil: 1 H NMR (CDCl3 ) ı 5.32–5.48 (m, 2H), 3.61–3.72 (m, 3H), 2.19 (t, J = 6.1 Hz, 2H), 1.98–2.08 (m, 2H), 1.53–1.61 (m, 2H), 1.22–1.52 (m, 25H), 0.87–0.92 (m, 12H), 0.04–0.07 (m, 6H); 13 C NMR (CDCl3 ) ı 131.4, 125.9, 72.4, 63.0, 36.9, 35.3, 32.8, 31.9, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 27.5, 25.9 (3C), 25.7, 25.4, 22.6, 18.1, 14.1, −4.4, −4.6.

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O CH3O

OTBDMS

7

5

6 1.1 eq DIBAL THF, -78 oC

O H

OTBDMS

OTBDMS +

7

7

5

HO

+

ester 6

7

8

5

1 : 1 : 2 ratio (7 : 8 : 6) Fig. 3. Reduction of methyl ricinoleate (6) with 1.1 equivalents of DIBAL at −78 ◦ C.

in vacuo. Purification by flash column chromatography (gradient of 30% acetone/CH2 Cl2 to 40% acetone/CH2 Cl2 ) gave phosphonate diol 5a as a clear, yellow oil (1.182 g, 94%) Phosphonate diol 5a, clear yellow oil: 94%, 1 H NMR (CDCl3 ) ı 5.46–5.55 (m, 1H), 5.33–5.41 (m, 1H), 4.14 (br s, 1H), 3.82–3.89 (m, 1H), 3.77 (d, J = 10.3 Hz, 3H), 3.75 (d, J = 10.3 Hz, 3H), 3.54–3.62 (m, 1H), 2.18 (t, J = 6.0 Hz, 2H), 2.01 (q, J = 6.9 Hz, 2H), 1.53–1.74 (m, 3H), 1.18–1.48 (m, 24H), 0.85 (t, J = 6.8 Hz, 3H); 13 C NMR (CDCl3 ) ı 133.1, 125.2, 71.5, 67.5 (d, JCP = 160.4 Hz), 53.2 (d, JCP = 7.4 Hz), 53.1 (d, JCP = 7.4 Hz), 36.8, 35.3, 31.8, 31.3, 29.6, 29.4, 29.4, 29.4, 29.3, 29.2 (2 C), 27.4, 25.7, 25.6 (d, JCP = 13.8 Hz), 22.6, 14.0; 31 P NMR (CDCl3 ) ı 27.7. Phosphonate diol 5b, pale yellow wax: 76%, 1 H NMR (CDCl3 ) ı 3.84–3.96 (m, 2H), 3.79 (d, J = 10.3 Hz, 3H), 3.78 (d, J = 10.3 Hz, 3H), 3.53–3.60 (m, 1H), 2.28 (br s, 1H), 1.89 (br s, 1H), 1.54–1.78 (m, 3H), 1.20–1.48 (m, 31H), 0.87 (t, J = 6.9 Hz, 3H); 13 C NMR (CDCl3 ) ı 71.9, 67.6 (d, JCP = 160.1 Hz), 53.2 (d, JCP = 7.0 Hz), 53.1 (d, JCP = 7.0 Hz), 37.4, 31.8, 31.3, 29.7, 29.6, 29.6, 29.5 (2 C), 29.5, 29.4, 29.4, 29.2, 25.7, 25.6 (d, JCP = 13.0 Hz), 25.6, 25.6, 22.6, 14.0; 31 P NMR (CDCl3 ) ı 27.7. 3. Results and discussion As mentioned previously, our goal was to produce monounsaturated and saturated lesquerella ␣-hydroxy phosphonate derivatives (5a and 5b, respectively; Fig. 2). Though there are several methods known for the synthesis of ␣-hydroxy phosphonates, the Pudovik reaction—the addition of a dialkyl phosphite to an aldehyde—is one of the most straightforward methods of introduction of the phosphonate functionality (Li and Hammerschmidt, 1993; Pudovik and Konovalova, 1979). Thus, it was necessary to modify lesquerella oil to provide the appropriate aldehyde for the Pudovik reaction. The most effective method for doing this would involve reducing an ester directly to the desired aldehyde with DIBAL at low temperatures. However, previous efforts by Cermak et al. (2012) demonstrated that when 1.1 equivalents of DIBAL was used to reduce the ricinoleic methyl ester 6 to its homologous unsaturated aldehyde 7, an undesirable mixture of multiple products and starting material was produced, as determined by

GC/MS (Fig. 3). To circumvent this issue, the ester underwent complete reduction to the corresponding alcohol (8) and followed by followed by Swern oxidation and subsequent Pudovik reaction, without purification of the intermediate aldehyde, the desired ␣hydroxy phosphonates of ricinoleic acid derived from castor oil were synthesized in acceptable yields, 80% and 74%, over two steps. One significant difference between castor oil and lesquerella oil is the distribution of fatty acids present in the oil, and the significant disparity in HFA availability in lesquerella oil versus castor oil. The desired HFA in lesquerella oil, lesquerolic acid, is only present in 55.4 mass %. Additionally, there are three other HFA’s present in lesquerella oil (see Fig. 1 and Table 1), with auricolic acid being present in the highest abundance (3.8 mass%). Purification of the desired HFA from lesquerella oil proved more difficult than purification of ricinoleic acid from castor oil. After transesterification of the triglyceride, the resulting methyl esters produced from castor oil can be distilled by Kugelrohr distillation and provide a quite pure fraction of methyl ricinoleate (>90% by GC). In contrast, lesquerella oil has a much wider fatty acid distribution. The crude transesterification product is a very dark brown oil. Kugelrohr distillation does help to purify the crude transesterification product in the effect that it is a light yellow oil, though the actual composition of the fatty acid distribution is surprisingly similar to that of the crude product (Table 2). Unlike with castor after transesterification and distillation, the distilled lesquerella product is not yet pure enough to use for the synthesis of lesquerolic acid derivatives. The Kugelrohr-distilled material was further purified by flash column chromatography followed by NMR analysis to determine which component was the desired methyl ester. GC was again used to determine the purity of the correct component, which was now present in >93% purity. The GC results from this purification process are compiled in Table 2. The first step in synthesizing the ␣-hydroxy phosphonates 5a/5b involves the generation of methyl lesqueroleate 9 from lesquerella oil (which had previously been RBD; Cermak and Evangelista, 2013) via Lewis acid catalyzed transesterfication as described by Cermak et al. (2006) (Fig. 4). Although ester 9 is the most plentiful ester found in the crude lesquerella oil, it is far from the only one. As mentioned above, purification involving Kugelrohr

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241

Table 2 Chemical composition of lesquerella oil after transesterification and purification. Fatty acid Methyl esters

Lesquerella oil post transesterification (mass%)

Lesquerella oil post Kugelrohr (mass%)

Lesquerella oil post column chromatography (mass%)

16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 18:1 Hydroxy (1)a 18:2 Hydroxy (2)a 20:1 Hydroxy (4)a 20:2 Hydroxy (3)a

1.1 0.6 1.9 16.1 6.9 10.7 0.7 0.8 – 0.5 – 56.8 3.5

1.1 0.6 2.0 16.4 7.1 11.2 0.8 0.8 – 0.6 – 55.3 3.7

– – – – – – – – – 0.9 – 93.5 5.6

a

See Fig. 1.

Fig. 4. Transesterification, protection and reduction of lesquerella oil.

distillation to remove colored impurities followed by flash silica gel chromatography provided pure methyl ester 9 in an 89% yield. This same method of transesterification was used with castor oil to produce methyl ricinoleate (Cermak et al., 2012). GC analysis of the castor transesterification reaction shows one peak with retention time of 17.1 min. In order to determine if alkene isomerization had occurred during this reaction, GC analysis of methyl ricineliadate, the trans alkene isomer of methyl ricinoleate, gave a retention time of 16.8 min. Because there was no sign of such a GC peak in the castor transesterification product, it can be assumed that alkene isomerization is not occurring during this reaction. Likewise, it would be assumed that such isomerization should also not be occurring during the transesterification of lesquerella oil and that the cis alkene is retained through the process. Finally, the hydroxy functionality then underwent protection as the TBDMS ether under standard conditions (Corey and Venkateswarlu, 1972) to provide protected ester 10 in an 88% yield. The reduction/oxidation/Pudovik sequence utilized with the castor-based sequence as described above was used with the lesquerella-based pathway (Cermak et al., 2012). The newly protected ester 10 was reduced to the corresponding alcohol 11a with excess of DIBAL at −78 ◦ C (Davis et al., 1993) followed by a careful quench with sat. aq. NH4 Cl to ensure that the TBDMS protecting group was not removed, resulting in alcohol 11a in a 96% yield. At this point in the synthesis, protected unsaturated alcohol 11a underwent hydrogenation under mild conditions to produce the homologous protected saturated alcohol 11b in a 92% yield (Fig. 5). Hydrogenation was performed at this point because working with the unsaturated lesquerella ester is substantially easier considering it is completely liquid at rt, as opposed to a waxy semi-solid. Both saturated and unsaturated protected alcohols 11a/b subsequently underwent Swern oxidation (Mancuso et al., 1978) to produce their corresponding aldehydes (12a/b) immediately followed by addition of the dialkyl phosphate under basic conditions (Li and Hammerschmidt, 1993) yielding phosphonates 13a/b in 80% and 74% yields, respectively, over two steps. After forming the protected ␣-hydroxy phosphonates 13a/b it was necessary to remove the TBDMS protecting group. This was accomplished by treatment of phosphonates 13a/b in 1% conc. HCl in 95% ethanol to produce phosphonate diols 5a/b in yields of 94% and 76%, respectively (Cunico and Bedell, 1980). When looking at the structure of lesquerolic acid, it is noticeable that it contains a chiral center at the C14 position. It is known that this fatty acid exists as one enantiomer having the R configuration at this position. When protected aldehydes 12a/b, which should still exist as one enantiomer, undergo phosphite addition to produce phosphonates 13a/b, this addition should be

242

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Fig. 5. Synthesis of diol phosphonates 5a and 5b.

non-stereospecific, resulting in a mixture of diastereomers. GC analysis was done at this point to see if evidence of diastereomers was present; GC results displayed one peak for phosphonate 13a (14.89 min) as well as one peak for subsequent deprotection of the alcohol contained in ␣-hydroxyphosphonates 5a and 5b (19.18 min for unsaturated diol 5a and 18.88 min for saturated diol 5b). The location of the chiral center located so far away from the reaction site (13 carbons away) would seem to have had little influence on either the pending nucleophilic addition of the phosphite to the aldehyde or the retention times of the two resulting diastereomers.

of lesquerella oil, two families of phosphorus derivatives have been prepared: those with the alkene functionality intact and a saturated version lacking this alkene. The pathway containing the alkene produced phosphonate diol 5a in 6 steps from lesquerella oil in a 57% overall yield; the pathway lacking the alkene added one step to the synthetic scheme (the hydrogenation step), thus producing phosphonates diol 5b in 7 steps from lesquerella oil in a 39% overall yield. Given the limited use of lesquerella oil as a source of hydroxy fatty acids, this chemistry has the potential to highlight their use as an interesting, synthetically-useful source of hydroxy fatty acids from a US-based crop that may serve as an alternative to castor oil.

4. Conclusions Acknowledgements Novel ␣-hydroxy phosphonates have been prepared from methyl lesqueroleate in respectable yields. After careful purification of methyl lesqueroleate from transesterification

The authors are grateful to Dr. Karl Vermillion (ARS-NCAUR) for his help with the 500 MHz NMR spectra. This research was

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