Letter pubs.acs.org/OrgLett
Chemical Synthesis of Biosurfactant Succinoyl Trehalose Lipids Santanu Jana, Sumana Mondal, and Suvarn S. Kulkarni* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India S Supporting Information *
ABSTRACT: Herein, we report, for the first time, a strategy to differentiate O4/O4′ positions of 1,1′-α,α-trehalose via regioselective protection or site-selective functionalization and its application in the first chemical synthesis of succinoyl trehalose lipids. The biosurfactant glycolipids were obtained in 11−12 steps starting from trehalose in time span of 8−10 days and 11−12% overall yields.
B
iosurfactants have attracted increasing attention in recent years as alternative surfactants due to their high biodegradability, low toxicity, and their versatile biological functions.1 Glycolipids consisting of carbohydrates connected to long-chain aliphatic acids, as hydrophilic and hydrophobic groups, respectively, are the most promising biosurfactants for applications in the agriculture and food industry.2 To date, only a few glycolipids such as the mannosylerythritol lipid from Candida antarctica, sophorose lipids from Candida bombicola, and the rhamnolipids secreted by Pseudomonas aeruginosa have been chemically synthesized and studied well.3 Yet another group of glycolipid biosurfactants are formed by succinoyl trehalose lipids (STLs) mainly produced by rhodococci from n-alkanes (Figure 1). STLs have one or two succinic acids and two or three fatty acids on the 1,1′-α,α-trehalose core.4 The major component of STL-1 is characterized as 3,4-di-Opalmitoyl-2,2′-di-O-succinoyl-α,α-trehalose,5 and that of STL-2 is identified as 2,3,4-di-O-alkanoyl-O-succinoyl-α,α-trehalose,6
whereas STL-3 is shown to be 2,3,4,2′-mono-O-succinoyl-tri-Oalkanoyl-trehalose (Figure 1).7 The exact location of the acyl chains in STL-2 and STL-3 is not confirmed. However, based on the structure of STL-1, it is believed that the succinic acid is present at the O2 position. It has been shown that STL-1 and its sodium salt exhibit excellent surface activity at remarkably low concentrations which makes it a potential environmentally friendly anionic biosurfactant.5 Likewise, a mixture of STLs has been shown to exhibit potential emulsifying and dispersing activities.8a In addition to the improved biosurfactant properties, STLs show cell-differentiation induction toward human leukemia cells.8 Biophysical studies have shown that the trehalose lipid is able to incorporate into phosphatidylethanolamine bilayers and affect the structural and functional properties of the membranes.9 In 2015, the Stoineva group characterized a trehalose lipid tetraester (THL) biosurfactant produced by Nocardia farcinica strain BN26 as 3,4,2′-tri-O-alkanoyl-2-O-succinoyl-α,α-trehalose and also evaluated its antitumor activity.10 The major component of THL was shown to have one octanoic acid and two decanoic acid residues as the three fatty acids at the 3,4,2′-positions; however, their individual positions were not defined. THL displayed differential cytotoxic activity against human tumor cell lines, making it a potential antineoplastic drug candidate. In order to further evaluate these glycolipids and carry out detailed SAR studies, access to chemically pure, homogeneous, and structurally well characterized analogues is essential. There are a few reports on the production of STLs using bacterial cultures.11 Although these methods provide good quantities of glycolipids, separation of the various isomers by column chromatography is quite tedious; heterogeneity still exists in the lipid portion with respect to the chain length and their positions. Thus, bioproduction is not a viable option for obtaining pure glycolipids and especially their analogues for biological studies. To date, there are no reports on the chemical synthesis of succinoyl trehalose lipids, perhaps due to the lack of suitable methodology to functionalize the trehalose core. This is because
Figure 1. Structures of succinoyl trehalose lipids.
Received: February 23, 2017
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.7b00550 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 1. Synthesis of Succinoyl Trehalose Lipids-1 (STL-1)
juggling with six secondary hydroxyls of similar reactivity of trehalose is a difficult task.12 On the other hand, construction of unsymmetrically substituted 1,1′-α,α-linked trehalose is a challenge.13 Not surprisingly, synthesis of a trehalose glycolipid carrying a lipid chain at O4 has not been reported, although a number of reports exist regarding O2, O3, and O6 functionalization.12b As a part of a research program directed toward the synthesis of trehalose glycoconjugates,14 we report herein a strategy for the selective O4-functionalization of trehalose and its application in the first synthesis of STLs as well as THL. For the synthesis of STLs including THL, it was necessary to develop a strategy to differentiate the O4 and O4′ positions of trehalose. Selective functionalization of the O4/O4′ position of an unsymmetrically substituted trehalose is difficult. So, a protecting group that will allow selective masking of 4′-OH while leaving 4-OH free was required. Prior to this, the 6-OH has to be protected with such a protecting group that permits its removal under near-neutral conditions, so as to arrest the possible migration of acyl groups from the O4 to O6 position. Alternatively, a protecting/functional group pattern that would lead to a selective acylation of O4 over O4′ of the 4,4′-diol is highly desired. Further, the strategy should be general, short, and efficient to gain rapid access to various lipid chain analogues of STLs. Consequently, the synthetic route should involve direct, site-selective functionalization of the hydroxy groups and minimal use of protecting groups. With these considerations in mind, we began our exploration of the synthesis of complex STLs.
Our synthesis of STL-1 (Scheme 1) started with the known trehalose tricyclohexylidene acetal derivative 1,15 which can be conveniently obtained from trehalose in a single step in 69% yield.14c Regioselective 2-O-acylation of 1 with succinic acid monobenzyl ester (SAMBE)16 via a DCC-mediated coupling furnished the monoacylated compound 2 in good yield (75%), along with a corresponding diacylated compound (5%) and the recovered starting material 1 (15%). The remaining 3-OH in 2 was acylated with palmitic acid under similar conditions to obtain compound 3 in 97% yield. The O2′ and O3′ positions of 3 were selectively exposed employing a two-step procedure.14e First, all three cyclohexylidene acetals were removed using TFA-mediated hydrolysis, and then selective 4,6,4′,6′-dicyclohexylidenation of the crude hexa-ol was performed using 1,1-dimethoxy cyclohexane (DMC) (5 equiv) and cat. p-TsOH at rt to obtain the desired 2′,3′ diol 4 in overall 71% yield. Regioselective 2′-Oacylation of 4 with SAMBE via a DCC-mediated coupling furnished the triester 5 in good yield (71%), along with the corresponding tetra-acylated compound (8%) and the recovered starting material 4 (15%). Hydrolysis of the cyclohexylidene acetals in 5 using aq TFA furnished penta-ol 6 (75%), which upon selective silylation of primary hydroxyl groups using TBSCl and imidazole, followed by cyclic protection of the trans vicinal diol with the tetraisopropyldisiloxane-1,3-diyl (TIPDS)17 group, afforded compound 7 in 74% yield over two steps. The remaining 4-OH was acylated with palmitic acid to obtain compound 8 in 97% yield. Finally, removal of both silyl protecting groups in 8 using TBAF in AcOH (93%) followed by debenzylation of the B
DOI: 10.1021/acs.orglett.7b00550 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 2. Synthesis of Succinoyl Trehalose Lipids-2 (STL-2) Analogue
Scheme 3. Synthesis of Trehalose Lipid Tetraester (THL)
Regioselective 2-O-acylation of 9 with SAMBE via a DCCmediated coupling furnished the monoacylated compound 10 in 68% yield along with the corresponding diacylated compound (14%) and the recovered starting material 9 (10%). The remaining 3-OH was acylated with n-decanoic acid to obtain compound 11 (97%), which upon hydrolysis using aq TFA, afforded tetraol 12 in 80% yield. Next, the primary hydroxy groups in 12 were protected as TBS ethers using TBSCl and imidazole to obtain compound 13 in good yield (95%). With the requisite 4,4′diol 13 in hand, the stage was set for a regioselective O4 acylation. Earlier we have observed that acylation of a similarly function-
intermediate under catalytic hydrogenation conditions afforded STL-1 in 88% yield. All the synthetic intermediates and the target molecule were characterized using 1H, 13C and detailed 2D NMR analysis; our spectral data for STL-1 were in good agreement with the reported data as well (see Supporting Information). Scheme 2 delineates a novel strategy for the synthesis of a shorter chain analogue of trehalose triester STL-2 via a direct functionalization of O4/O4′. Dibenzylation of compound 1 followed by hydrolysis of tricyclohexylidene acetals using aq TFA and concomitant dicyclohexylidenation using 1,1-DMC (5 equiv) and p-TsOH afforded compound 9 in 75% yield over three steps. C
DOI: 10.1021/acs.orglett.7b00550 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters ORCID
alized 2,3-palmitoyl trehalose 6,6′-diol preferentially proceeds on the acylated sugar to furnish O6-acylated products in 60−70% yields.14e With the hope of obtaining similar selectivity we attempted a DCC mediated regioselective acylation of the 4,4′diol using n-caprilic acid. This reaction afforded the O4-acylated compound 14 as a major isomer in 50% yield along with a 27% yield of the corresponding O4′-acylated regioisomer, a trace amount of a diacyl compound, and the recovered starting material 13 (10%) which could be easily separated by silica gel column chromatography. Removal of the silyl protecting groups in 14 using aq TFA (85%) followed by debenzylation of the intermediate under catalytic hydrogenation conditions afforded the STL-2 analogue in 95% yield. For the synthesis of trehalose lipid tetraester (THL) biosurfactant produced by Nocardia farcinica strain BN26 isolated from soil, we employed the same strategy used for STL-1 synthesis (Scheme 3). We started our synthesis from previously prepared compound 2. The remaining 3-OH in 2 was acylated with n-decanoic acid to obtain compound 15 in 97% yield. Hydrolysis of tricyclohexylidene acetals using aq TFA and cyclohexylidene acetal protection using 1,1-DMC (5 equiv) and p-TsOH afforded dicyclohexylidene derivative 16 in 67% yield over two steps. Regioselective 2′-O-acylation of 16 with n-caprilic acid via a DCC-mediated coupling furnished the triacylated compound 17 in good yield (72%). Hydrolysis of cyclohexylidene acetals in 17 using aq TFA furnished 18 in 81% yield. Regioselective protection of primary hydroxyl groups with TBS using TBSCl and imidazole, followed by cyclic protection of transvicinal diol with the TIPDS group, gave compound 19 in 80% yield over two steps. The remaining 4-OH was acylated with ndecanoic acid to obtain compound 20 in 96% yield. Finally, removal of the silyl protecting groups in 20 using TBAF in AcOH (85%) followed by debenzylation of the intermediate under catalytic hydrogenation conditions afforded the desired trehalose lipid tetraester (THL) in 93% yield. In conclusion, we have established, for the first time, efficient conditions for differentiation of O4 and O4′ of trehalose via regioselective protection as well as through direct, site-selective functionalization. The regioselective protection strategy was applied to the first total synthesis of STL-1 and THL whereas a direct functionalization route was employed in the synthesis of the STL-2 analogue. In general, the schemes involve 11−12 steps each from cheaply available trehalose and the target glycolipids were obtained in excellent overall yields of 11−12%. Moreover, each sequence involves only three protecting groups and can be repeated in 8−10 days. The synthesized glycolipids which are available in pure form for the first time can now be tested for various applications. More importantly, the general and expedient route allows a rapid access to various lipid chain analogues of trehalose glycolipids.
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Suvarn S. Kulkarni: 0000-0003-2884-876X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Board of Research in Nuclear Sciences (Grant No. 2013/37C/51/BRNS) and SERB, DST (Grant No. EMR/2014/ 000235), and University Grants Commission (UGC-ISF Project F. No. 6-2/2016(IC)) for financial support. S.J. thanks CSIR− New Delhi for fellowships.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00550. 1 H and 13C NMR spectra for all compounds and 1H−1H COSY spectra for all new compounds (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
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DOI: 10.1021/acs.orglett.7b00550 Org. Lett. XXXX, XXX, XXX−XXX
