Anaerobic biotransformation of hydrocarbons in the subsurface is an important research area given both the economic value of energy resources and fundamental science regarding the biochemistry of the requisite microorganisms ^[1]. In the last two decades, anaerobic degradation of petroleum hydrocarbons has been demonstrated ^{[1, 2, 3, 4]}. Various degradation mechanisms of petroleum hydrocarbons were proposed based on functional genes and metabolite evidences ^{[1, 4, 5]}. One of the most prevalent mechanisms is named fumarate addition pathway ^{[3, 4, 5]}. In this pathway, 2-(1-methylalkyl)succinates are produced in the initial activation, and recognized as biomarkers for identification of anaerobic hydrocarbon degradation ^[6]. The proposed metabolic pathway of fumarate addition is shown in Fig. 1. Further studies proposed skeletal rearrangement accompanying hydrogen atom migration from g position to β position,and a hydrogen elimination in β-oxidation stage (Fig. 1) ^[7]. So the β-C as marked with an asterisk is an important reaction centre. However,few direct observations on these further degradation products were reported,and few available substrates could be used. In a previous study,we have developed a synthetic route of 2-(1-methylalkyl)succinates,and made a contribution to the field by providing mass spectral data for the recognition of these compounds ^[8] and this paper is an extension of our previous work. Due to the wide application in biology ^{[9, 10, 11]},the use of isotope tracer could be a useful method for the demonstration of biodegradation pathway of alkanes.

Fig. 1

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Fig. 1. Proposed fumarate addition pathway of anaerobic degradation of alkanes. The carbon marked with an asterisk is a key position in the C-skeleton rearrangement and β-oxidation stage.

Thus a modified synthesis method was developed and a set of 2-[²H]-2-(1-methylalkyl)succinate homologs were prepared and characterized by gas chromatography-mass spectrometry and ¹H NMR. These deuterium compounds are new,and to the best of our knowledge,though Krapcho reaction was widely applied for decarboxylation of esters such as β-ketoesters,malonic esters, α-cyanoesters,or a-sulfonylesters ^{[12, 13]},the application of this reaction in the preparation of deuterated compounds is not common. The deuterated position is proved at the reaction centre. So,these deuterated compounds could be used as tracers not only for the identification of the degradation pathway,but also for investigation of the hydrogen exchange during the degradation process with an advantage of low isotopic effects,in biologic experiments than perdeuterated ones. 2. Experimental

The chemical reagents used in this work included bromoethane, 2-otcanol,diethyl malonate,Na,ethanol,dimethyl sulfoxide (DMSO),hexane,ethyl acetate,LiCl,NaOH,H₂SO₄,Na₂SO₄,HCl, HBr,and D₂O. All reagents purchased from commercial companies were of analytical grade.

GC-MS analyses were performed on an Agilent 6890 GC instrument equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) and mass detector (MSD 5975). The injection temperature was 280 °C. The oven temperature was held at 70 °C for 2 min,then increased at 10 °C per min to 260 °C and held for 30 min. The MS detector acquired data in the scan mode,from 30 to 1000 mass units. EI was operated at 70 eV, and the ion source temperature was held at 230 °C. 1HNMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz) at room temperature. Chemical shifts (in ppm) were referenced to tetramethylsilane in CDCl₃ as an internal standard.

The synthetic route of the target compounds is outlined in Scheme 1.

Scheme 1

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Scheme 1. Synthesis route of 2-[2H]-2-(1-methylalkyl)succinic acid.

Preparation of 2-bromooctane (2): 2-Otcanol (1) (0.035 mol, 4.55 g),HBr (0.0385 mol,6.63 g),and H₂SO₄ (0.049 mol,4.90 g) were mixed in a 100 mL flask at 0 °C. With vigorous stirring,the system was heated at 120 °C for 3 h. After cooled to room temperature,water was added to the mixture. Then the product was purified via steam distillation. The oil phase was separated and the water phase was extracted with hexane (10 mL × 3). Then the organic phase was combined together,washed with dilute NaHCO3 solution,dried over Na₂SO₄ and solvent was removed. The yield is 90.0%.

Preparation of triethyl 1,1,2-ethanetricarboxylate (5): A solution of sodium ethoxide was first prepared from Na (0.035 mol, 0.80 g) and absolute ethanol (10 mL) at 0 °C. Then,diethyl malonate (4) (0.035 mol,5.61 g) was added to the solution,and kept stirring for 30 min at 0 °C. Ethyl bromoacetate (3) (0.035 mol, 5.84 g) was then added drop wise to the mixture. The mixture was kept stirring for 6 h,and then heated at 80 °C for 2 h. The product was treated by filtration and solvent was removed. Dilute hydrochloric acid solution was added to the raw product. The oil phase was separated and the water phase was extracted with ethyl acetate (10 mL × 3). Then the organic phase was combined together,dried over Na₂SO₄ and solvent was removed. The yield is 90.0%.

Preparation of triethyl 3-methylalkane-1,2,2-tricarboxylate (6a,6b): For preparation of compound 6b,a solution of 0.035 mol/10 mL CH₃CH₂ONa/CH₃CH₂OH was prepared as described in step (b),Part B,Scheme 1. Compound 5 (0.035 mol, 8.62 g) and the sodium ethoxide solution were mixed at 0 °C. To the mixture was added 2-bromooctane (2b) (0.035 mol,6.76 g) which is the product of step (a),Part A,Scheme 1 and heated at 80 °C for 10 h (in the preparation of 2-[²H]-2-ethylsuccinic acid, bromoethane was purchased directly). When no more precipitate was produced,the mixture was filtered,and the solvent was removed. Dilute hydrochloric acid solution was added to the raw product. The oil phase was separated and the water phase was extracted with ethyl acetate (10 mL × 3). Then the organic phase was combined together,dried over Na₂SO₄ and solvent was removed.

Compound 6a could be synthesis via the same method when bromoethane (2a) (0.035 mol,3.81 g) was employed. The yield of 6a is 93.0% and 6b 86.0%.

Preparation of diethyl 2-[²H]-2-alkylsuccinate (7a,7b): For preparation of compound 7b,25 mL DMSO (DMSO was dried with CaH₂ and then distilled in reduced pressure,as described in Ref. ^[14]),triethyl 3-methylnonane-1,2,2-tricarboxylate (6b) (0.035 mol,12.55 g),LiCl (0.07 mol,2.96 g),and D₂O (0.035 mol, 0.70 g) were placed in a 100 mL flask and the mixture was heated at 150 °C for 8 h. The mixture was then cooled to room temperature. Water was added,and the ester was extracted with ethyl acetate (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated.

Compound 7a could be synthesis via the same method when compound 6a (0.035 mol,3.81 g) was employed. The yield of 7a is 80.1% and 7b 69.6%.

Preparation of 2-[²H]-2-alkylsuccinic acid (8a,8b): For preparation of compound 8b,in a 100 mL flask was placed diethyl 2-(1- methylheptyl)succinate (0.035 mol,10.05 g) produced in step d, NaOH (0.035 mol,1.40 g) and H₂O (20 mL). The mixture was refluxed for 4 h. When cooled to room temperature,the mixture was treated with 10 mL hexane three times to extract undissolved organic substances. Then the water phase was acidified with HCl, and extracted with ethyl acetate (10 mL × 3). The ethyl acetate solution was dried over Na₂SO₄ and concentrated.

Compound 8a could be synthesis via the same method when compound 7a (0.035 mol,7.08 g) was employed. The yield of 8a is 95.1% and 8b 88.9%. The 2-[²H]-2-(1-methylalkyl)succinic acids were purified via column chromatography (hexane:ethyl acetate = 2:3).

2-[²H]-2-Ethylsuccinic acid (8a): white solid with a total yield (3.2 g,63.8%). ¹H NMR (400 MHz,CDCl₃,TMS): δ 0.982 (t,3 H, J = 7.6 Hz),1.59-1.78 (m,2H),2.46-2.55 (m,1H),2.70-2.77 (m, 1H).

Diethyl 2-[²H]-2-(1-methylheptyl)succinate (7b): Yellow liquid. ¹H NMR (400 MHz,CDCl₃,TMS): δ 0.78-0.85 (m,6H),1.16- 1.25 (m,16H),1.66-1.86 (m,1H),2.20-2.32 (m,1H),2.59-2.71 (m, 1H),4.02-4.13 (m,4H). 3. Results and discussion

According to the method as described in Scheme 1,2-[²H]-2- ethylsuccinic acid (8a) and 2-[²H]-2-(1-methylheptyl)succinic acid (8b) have been synthesized. Traditionally,there are some methods to synthesize deuterium labelled fatty acids,such as H/D exchange, reduction,hydrolysis,chemical synthesis and biological synthesis ^[15]. In this study,the synthesis of the target compounds was achieved by four or five steps,including double alkylation of diethyl malonate,Krapcho reaction with D₂O,and hydrolysis as described in Scheme 1. Firstly,due to the particular structure (e.g. 8b) of the target compounds,2-alkanols were bromized for preparing the alkyl moiety in a yield of 92% (2-bromoalkanes could be used directly if available). Secondly,the succinate moiety was prepared by step b via alkylation of malonate in 90% yields,and then the two moieties combined together. Notably,the key step in this method is step δ decarboxylation via Krapcho reaction as the introduction of deuterium. One carboxyl group of substituted malonate diesters dissolved in dimethyl sulfoxide could be replaced by an H atom in the presence of LiCl and H₂O ^[16]. According to the reaction mechanism (see Supporting information) ^{[17, 18]},a deuterated method could be developed by a modification with the use of D₂O instead of H₂O in Krapcho reaction and this strategy was applied in this study to prepare diethyl 2-[²H]-2- ethylsuccinate (7a),and diethyl 2-[²H]-2-(1-methylheptyl)succinate (7b). Finally,2-[²H]-2-alkylsuccinic acids were achieved by treating 7a,7b with NaOH. The total yields of 8a and 8b are 63.7% and 43.1%,and isotopic purities were 97% and 95%,respectively.

After synthesis,the two products were characterized by mass spectrometry and ¹H NMR. The mass spectra of 8a and 8b diethyl esters are shown in Fig. 2a and c. In Fig. 2,the m/z of four characteristic fragments in deuterium-labelled 2-ethylsuccinate and 2-(1-methylheptyl)succinate have an additional mass-unit than non-labelled ones,resulting in m/z 175,130,158,116 and 129,175,200,242,respectively (the characteristics ions of diethyl 2-alkylsuccinate in EI-MS please see Ref. ^[8] and Supporting information). These results indicate that a hydrogen was substituted by a deuterium atom. Also,according to the ¹H NMR spectra of 8a and 7b,hydrogen of the methine group with a shift at d 2.9 was absent. Thus,based on these results,2-[²H]-2-(1- methylalkyl)succinates were produced via the method described in this work.

Fig. 2

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Fig. 2. Mass spectra characteristics of deuterated and undeuterated hydrocarbon-derived succinate ethyl esters. (a) Diethyl 2-[2H]-2-ethylsuccinate; (b) diethyl 2-ethylsuccinate; (c) diethyl 2-[2H]-2-(1-methylheptyl)succinate; (d) diethyl 2-(1-methylheptyl)succinate.

In conclusion,a synthesis method of specific deuteriumlabelled 2-(1-methylalkyl)succinic acids was developed in this work. Two compounds,2-[²H]-2-ethylsuccinic acid and 2-[²H]-2- (1-methylheptyl)succinic acid,were prepared. This method could provide tracers for illustrating the mechanism of anaerobic degradation of alkanes and the hydrogen exchange during the degradation.

This work was supported by the National Natural Science Foundation of China (No. 41373070,51174092) and the National Natural Science Foundation of China/Research Grants Council Joint Research Fund (No. 41161160560).

Supplementary data associated with this article can be found, in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.01. 010.