Rh-catalyzed hydrogenation of amino acids to bio-based amino alcohols

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Rh-catalyzed hydrogenation of amino acids to bio-based amino alcohols: tackling challenging substrates and application to protein hydrolysates Annelies Vandekerkhove, Laurens Claes, Free De Schouwer, Cédric Van Goethem, Ivo F. J. Vankelecom, Bert Lagrain, and Dirk De Vos ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01546 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Sustainable Chemistry & Engineering

Rh-catalyzed hydrogenation of amino acids to biobased amino alcohols: tackling challenging substrates and application to protein hydrolysates Annelies Vandekerkhove, Laurens Claes, Free De Schouwer, Cédric Van Goethem, Ivo F. J. Vankelecom, Bert Lagrain and Dirk E. De Vos* Affiliation: Department of Microbial and Molecular Systems, Centre for Surface Chemistry and Catalysis KU Leuven Celestijnenlaan 200F, post box 2461 3001 Heverlee, Belgium * Corresponding author: Prof. Dr. Dirk E. De Vos; E-mail: [email protected] KEYWORDS. Hydrogenation – amino acids – amino alcohols – biomass – rhodium

ABSTRACT. While protein-rich biomass waste is nowadays mainly used for animal feed, conversion of its amino acid constituents to nitrogenous chemicals is a potential higher value route. To that end, the hydrogenation of amino acids to amino alcohols was studied in this work. Using a bimetallic Rh-MoOx/SiO2 catalyst, glutamic acid was for the first time hydrogenated to the aminodiol in high yield. By minimizing partial reduction and consecutive hydrogenolysis,

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and by suppressing the competitive cyclization to pyroglutamic acid (and derivatives thereof), glutamidiol was obtained in 77% yield at 70 bar H2 and 80 °C. High yields (typically > 80%) and selectivities were also achieved for most other natural amino acids, except for the S-containing amino acids cysteine and methionine, which act as catalyst poisons. This limitation was overcome by applying a simple oxidation step with performic acid prior to the hydrogenation. The system was applied successfully to a mixture of amino acids obtained by hydrolysis of preoxidized bovine serum albumin. Amino alcohols were produced with high overall conversion (> 90%) and selectivity (88%) without the need for an intermediate, expensive and difficult separation step. The reaction proceeds with very high atom economies for both carbon and nitrogen, and generates only water as a by-product.

INTRODUCTION β-Amino alcohols are an interesting class of compounds with a broad range of applications, for instance as intermediates in the production of pharmaceuticals (e.g. nelfinavir, ethambutol),1,2 as chiral auxiliaries (e.g. pseudoephedrine)3 and as polymer precursors (e.g. lysinol).4 Classical synthetic approaches often start from fossil hydrocarbon resources and consist of multiple steps involving the use of homogeneous catalysts, toxic reagents and hazardous solvents.2,5–9 Alternatively, the one-step reduction of natural α-amino acids provides a bio-based route towards β-amino alcohols, with excellent atom economy for both carbon and nitrogen. Moreover, many amino acids are inexpensive and safe reagents, which are nowadays produced from renewable resources by large-scale fermentation processes1,10,11 or can be recovered from cheap agricultural protein-rich waste, such as animal slaughter waste, algae, sugar beet vinasses etc.12 This reaction may therefore be particularly attractive regarding the chemocatalytic upgrading of protein-rich


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biomass into nitrogenous chemicals, being highly complementary to recently developed strategies for selective defunctionalization of amino acids.13-28 The reduction of carboxylic acids is not straightforward. Transition-metal catalyzed hydrogenation, where nucleophilic hydride species are produced in situ by the dissociation of molecular hydrogen (H2), is the most attractive approach in terms of green chemistry because only water is generated as a co-product. Besides homogeneous catalysts also supported monometallic (e.g. Re/TiO2)29 and bimetallic (e.g. Pd-Re, Ru-Re, Pt-Mo etc.) hydrogenation catalysts have been reported.30 The latter are much more effective for this purpose, because the more oxophilic metal activates the carbonyl carbon for a hydride attack.31–34 However, α-amino acids and ester derivatives thereof are even more challenging substrates. The reduction can be performed either with metal hydride reagents (e.g. LiAlH4, NaBH4), or by homogeneous and heterogeneous transition metal-catalyzed hydrogenation. The first approach typically proceeds under mild conditions (< 80 °C and atmospheric pressure), but involves the use of hazardous reactants and solvents (e.g. THF).35 Moreover, the hydride reagents are used in excess, with concomitant salt waste generation.1,36–40 Amino acid esters have been hydrogenated using homogeneous Ru and heterogeneous Cu catalysts.41–43 Although amino alcohols were obtained in good to excellent yields, the methods proceed in organic solvents under 40-50 bar H2 and eventually require a protecting group on the amine moiety in the substrate. The first attempts towards the hydrogenation of free amino acids in aqueous media with heterogeneous Ru and Ni catalysts required very high pressures (200 bar at 100 °C) and long reaction times (up to 30 h); however, amino alcohols were only obtained in moderate yields.44 Bimetallic Ru-Re catalysts, either unsupported45 or immobilized on carbon46, operate under slightly milder conditions (150200 bar H2 at 80-120 °C) and amino alcohols have been produced in higher yields (31-80%);


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however, the product obtained from glutamic acid, the most abundant constituent in plant-based proteins, was not unambiguously characterized. Moreover, sintering is a major disadvantage under these conditions.47 The supported Ru/C catalyst is more resistant to sintering and facilitates the hydrogenation at substantially lower pressures. The product selectivity is strongly influenced by the reaction conditions: amino alcohols have been obtained in high yields (> 92%) at 70 bar H2 and 100 °C,48 whereas the hydrogenation-decarbonylation to primary amines occurs as a competitive side reaction at 40 bar H2 and 150 °C.22 However, in both cases highly acidic conditions are required to protonate the carboxylate group in amino acids (pKa ≈ 2-3)49 in order to increase the electrophilic character of the carbonyl group for a hydride attack. Nevertheless, the highest activity in amino acid hydrogenation has been observed for a bimetallic RhMoOx/SiO2 catalyst.50,51 In this material, metallic Rh and MoOx species are expected to be in close proximity and the adsorption of amino acids on the catalyst surface is facilitated by stabilizing the adsorbed substrate via hydrogen bonding between MoOx species and the carboxylic acid group. In this way, amino alcohols have been obtained in > 90% yield under very mild conditions (80 bar H2 at 50 °C); primary amines have been observed as minor side products as a result of the consecutive C-O hydrogenolysis of amino alcohols.50,51 Remarkably, these bimetallic Rh-based catalysts show also excellent performance in C-O hydrogenolysis reactions and have been applied successfully in the valorization of biomass-derived (poly)alcohols and ethers.52–58 Despite the development of several performant catalytic systems for amino acid hydrogenation, the substrate scope remains an important challenge. Up to now only a few simple amino acids (viz. glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine and lysine) or ester derivatives thereof have been modified successfully to the corresponding amino


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alcohols.3,4,42,45,46,48,50,51,59,60 The hydrogenation of other amino acids is more challenging due to the presence of multiple and potentially interfering functional groups in the side chain. For instance, the aromatic moieties in phenylalanine, tyrosine and tryptophan are preferentially reduced compared to the carboxylic acid61 and glutamic acid is converted readily to pyroglutamic acid under typical reaction conditions.59,62 Moreover, the hydrogenation of mixtures is currently limited to synthetic mixtures of three amino acids with an aliphatic side chain.60 In this work, we optimized a chemocatalytic system for the hydrogenation of many natural amino acids, even the challenging ones, under mild conditions. An oxidative pre-treatment of sulfur-containing amino acids has been developed to avoid catalyst poisoning. The successful application to both pure amino acids and protein hydrolysates demonstrates the potential of this system for the valorization of biomass waste. Indeed, a mixture of amino alcohols provides potential applications as precursors for epoxy resins. These polymers are obtained upon reaction with polyoxiranes under basic conditions and have applications in e.g. coatings, adhesives, electrical insulators.4,63 EXPERIMENTAL SECTION Catalyst synthesis A Rh-MoOx/SiO2 catalyst (4 wt% Rh, Mo/Rh molar ratio = 1:8) was synthesized by two impregnation steps according to a procedure reported by Tomishige and coworkers.50 First, an aqueous solution of RhCl3.3H2O (25 mM, 20 ml) was added to Aerosil 380 silica powder (1 g). This suspension was stirred at ambient temperature to evaporate water and afterwards the precatalyst was dried overnight in an oven at 60 °C. In the second step, the pre-catalyst was


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contacted with an aqueous solution of (NH4)6Mo7O24.4H2O (0.36 mM, 20 ml) and dried in a similar manner. Finally, the material was granulated (250-500 µm), calcined at 500 °C (2.5 °C min-1, 100 ml min-1 O2, 3 h) and reduced at 500 °C (2.5 °C min-1, 100 ml min-1 H2, 3 h) in a quartz U-tube. A Ru-Re black catalyst was prepared in situ by treating a suspension of Re2O7 (0.0074 g) and RuO2 (0.0077 g) in water (10 ml) at 80 bar H2 and 120 °C for 1 h.45 The synthesis was performed in a 50 ml high-pressure Parr batch reactor. The pre-reduced catalyst was used as such for the hydrogenation of amino acids (vide infra). Procedures for the synthesis of other supported Rh- and Pt-based catalysts studied in this work are provided in the supplementary information. Catalyst characterization The Rh-MoOx/SiO2 catalyst was characterized by (scanning) transmission electron microscopy ((S)TEM) and energy-dispersive X-ray spectroscopy (EDX). TEM specimens were prepared by depositing catalyst particles on a Lacey carbon-coated TEM grid (300 mesh Cu grid, Pacific Grid Tech, USA). TEM and STEM images as well as EDX elemental maps were collected on a JEOL ARM200F TEM instrument operated at 200 kV and equipped with a cold field emission gun and a probe aberration corrector. Hydrogenation of amino acids Reactions were performed in a 50 ml high-pressure Parr batch reactor equipped with a Teflon liner. In a typical reaction, the Teflon liner was charged with the amino acid(s) (0.11 M), the RhMoOx/SiO2 catalyst (4 wt% Rh, Mo/Rh = 1:8, Rh/amino acid(s) = 3.5 mol%) and water (15 ml). A highly acidic medium (pH 1.8) was obtained by the addition of phosphoric acid (0.3 M) to the aqueous suspension. Experiments with the Ru-Re black catalyst were performed by the addition of the amino acid (0.11 M) and phosphoric acid (0.3 M) to the aqueous solution containing the


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pre-reduced catalyst (vide supra, Ru/amino acid = 3.5 mol%). After insertion of the Teflon liner, the reactor was sealed, purged 3 times with N2, 3 times with H2 and finally pressurized with 65 bar H2. The reactor was heated to 80 °C while stirring mechanically (350 rpm). During the heating step, the hydrogen pressure increased to 70 bar. After the applied reaction time, the reactor was allowed to cool down in an ice bath. The gas was released and the solid catalyst was removed by centrifugation. Finally, the crude reaction mixture was analyzed by nuclear magnetic resonance (NMR) spectroscopy and high-pressure liquid chromatography (HPLC) to determine both the amino acid conversion and the product selectivity. Oxidation of sulfur-containing amino acids Amino acids with thiol and thioether moieties were oxidized prior to hydrogenation. First, performic acid was produced in situ by reacting hydrogen peroxide (29-32 wt% aqueous solution, 1 ml) and formic acid (9 ml) at ambient temperature for 1 h, and then this mixture was cooled to 0 °C.64 The substrate (cysteine, methionine or a protein) was added to the cold oxidant solution (molar ratio performic acid/sulfur = 3:1) and the reaction proceeded for 4 h in an ice bath. Finally, the excess of formic acid and water were evaporated by heating under vacuum and the solid residue was dried further overnight in a vacuum oven at 100 °C. Protein hydrolysis A standard procedure for protein hydrolysis was followed.65 To that end, a sample of bovine serum albumin (1 g; either as such, or pre-treated with performic acid) was dissolved in aqueous hydrochloric acid (6 M, 100 ml). The sample was incubated under nitrogen to prevent amino acid oxidation by flushing the headspace with N2 for 60 s and subsequently the sample was heated to 110 °C for 24 h. Afterwards, the hydrolysates were evaporated to dryness at 110 °C and the residue was suspended in water (5 ml, ± 1.5 M) and filtered to remove any remaining solid


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particles. Amino acid levels were determined by high-performance liquid chromatography (HPLC) after 2- to 10-fold dilution of the samples, in order to have an excess of the derivatisation agent (vide infra). Product analysis and identification Samples for NMR spectroscopy were prepared by mixing 300 µl of the crude reaction mixture with 300 µl of deuterium oxide. The spectra were recorded on a Bruker Ascend 400 MHz NMR spectrometer equipped with a BBO 5 mm atma probe and a sample case. The broad water signal at δ = 4.7 ppm was suppressed by applying a zgpr pulse program: p1 8 µs, ns 32, d1 5 s, aq 2.55 s, plw1 15 W, plw9 5.7·10-5 W, o1P on the resonance signal of water, determined and selected automatically. In addition, three types of 2D NMR experiments were performed to identify products derived from glutamic acid, tyrosine and arginine. 1H-1H NMR (COSY) according to the following pulse program: p0 9.75 µs, p1 9.75 µs, ns 32, d1 1.89 s, plw1 15 W; 1

H-13C NMR over 1 bond (HSQC) according to the pulse program: p1 9.75 µs, p2 19.50 µs, p3

7.50 µs, p4 15 µs, ns 24, d1 1.44 s, plw1 15 W, plw2 120 W, plw12 1.05 W; and 1H-13C NMR over 2 or 3 bonds (HMBC) according to the pulse program: p1 9.75 µs, p2 19.50 µs, p3 7.50 µs, ns 40, d1 1.39 s, plw1 15 W, plw2 120 W. ICP-OES analyses were used to determine the Rh and Mo content of the reaction mixture of leucine hydrogenation (6 h, 80 °C, 70 bar H2) using a Varian 720-ES equipped with a doublepass glass cyclonic spray chamber, a Sea Spray concentric glass nebulizer and a high solids torch. HPLC was used for both qualitative and quantitative analysis of protein hydrolysates and crude reaction mixtures after hydrogenation. Measurements were performed on an Agilent 1200 Series SL binary system equipped with a G1322A degasser, a G1312B binary pump, a G1367A


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automated sample injector, a G1316A thermostatted column compartment and a G1314A variable wavelength detector (VWD). Data were processed with Agilent Chemstation software version B.04.03. The method was adapted from an Agilent Application Note.66,67 An automated pre-column derivatization procedure was applied to convert the amine groups in amino acids, amino alcohols and amines into chromophoric moieties, in order to increase the sensitivity for UV detection. Primary amines were modified with o-phthalaldehyde (OPA) and 3mercaptopropionic acid, whereas secondary amines were treated with 9-fluorenylmethyl chloroformate (FMOC). These chromophores were detected at 338 nm and 262 nm, respectively. Pyroglutamic acid and pyroglutaminol, which contain a lactam moiety, were detected at 212 nm without any derivatisation. Separation of these compounds was achieved on an Agilent ZORBAX Eclipse Plus C18 reversed phase column (250 mm x 4.6 mm i.d., 5.0 µm particles), maintained at 40 °C. The aqueous mobile phase (A) consisted of a 40 mM solution of NaH2PO4.H2O in Milli-Q water at pH 7.8 and the organic mobile phase (B) consisted of a mixture of methanol, acetonitrile and Milli-Q water (45:45:10 v/v/v). Elution conditions: a flow rate of 1.5 mL min-1 and a mixture of 98% A and 2% B was used as eluent from 0 to 0.84 min. Then a linear gradient was applied from 2% to 20% B in 0.84 to 10 min, 20% to 50% B in 10 to 45 min, 50% to 100% B in 45 to 54 min, 100% B in 54 to 56.6 min, and 100% to 2% B in 56.6 to 57 min. The injection volume was 1.0 µl. After each analysis, the column was purged with the initial solvent mixture (98% A, 2% B) for 5 min.

RESULTS AND DISCUSSION Hydrogenation of glutamic acid: catalyst screening and optimization of reaction parameters


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The hydrogenation of glutamic acid (1a) was studied as a model reaction, thereby aiming at the selective production of glutamidiol (1d), a useful polymer building block e.g. for the production of epoxy resins. The product mixture was expected to be complex (Scheme 1). Glutamic acid contains two distinct carboxylic acid groups with different reactivity (pKa,α-COOH = 2.2 and pKa,γCOOH

= 4.3), which both can be reduced to an alcohol under highly acidic conditions; hence

besides glutamidiol also the monohydrogenated intermediates 4-amino-5-hydroxy-pentanoic acid (1b) and 5-hydroxy-norvaline (1c) may be present. Moreover, according to earlier observations, either alcohol group in glutamidiol is susceptible to C-O hydrogenolysis as well,50,51 thereby generating 2-amino-1-pentanol (1e) and 4-amino-1-pentanol (1f) and eventually also 2pentanamine (1g) as side products. The selectivity can also be affected by the intramolecular condensation of glutamic acid to pyroglutamic acid (2a), which occurs in parallel to the hydrogenation and proceeds easily upon heating the reaction mixture.23,68 Finally, pyroglutamic acid can be reduced to pyroglutaminol (2b) and further to prolinol (2c).59

Scheme 1. Possible reaction pathways in the hydrogenation of glutamic acid (1a): reduction to 4amino-5-hydroxy-pentanoic acid (1b), 5-hydroxy-norvaline (1c) and glutamidiol (1d); consecutive C-O hydrogenolysis of 1d to 2-amino-1-pentanol (1e), 4-amino-1-pentanol (1f) and


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2-pentanamine (1g). Pyroglutamic acid (2a) is obtained by thermal lactamisation of 1a and can be reduced to pyroglutaminol (2b) and further to prolinol (2c). Several Pt-, Ru- and Rh-based catalysts were evaluated on their performance in the model reaction under selected conditions (Table 1 and ESI, Table S1). The hydrogenation did not proceed in the absence of a catalyst; in this case however glutamic acid was converted selectively to pyroglutamic acid (Table S1, entry 1). Among the known catalysts for amino acid hydrogenation, only the bimetallic Rh-MoOx/SiO2 catalyst showed a reasonable activity, and the desired glutamidiol was obtained in 34% yield at near-complete conversion (Table 1, entry 3). In contrast, Ru-Re black and monometallic Ru-, Rh- and Pt-based catalysts were almost inactive: the reaction mixtures consisted mainly of pyroglutamic acid, and eventually also traces of the monohydrogenated products 1b and 1c were present (Table 1, entries 1, 2 and 4; Table S1, entries 9-12). The selectivity to 1b was always much higher than to 1c, as the carboxylic acid at the α-carbon is more reactive due to the close presence of a positively charged, electron withdrawing amine, rendering the carbonyl carbon more susceptible to a hydride attack. Substitution of Rh by Pt in the bimetallic catalyst was also not beneficial (Table S1, entries 2-3). The bimetallic Rh-MoOx catalyst was therefore selected for further optimization (Table S1, entries 4-8). However, the catalytic performance could not be improved upon variation of the metal loading or by using other support materials. In the latter case, the activity of the Rh-MoOx catalysts generally decreased with the surface area (SiO2 > zeolite beta >> TiO2, ZrO2) and hence with the dispersion of the active sites on the support. The best performance was achieved by immobilizing the Rh-MoOx species on a SiO2 support and by applying a Rh loading of 4 wt%, which is in good agreement with the observations of Tomishige and coworkers.50,51 Table 1. Catalytic hydrogenation of glutamic acid (1a): catalyst screening.[a]


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Entry Catalyst

X1a (%) S1b-1c (%) S1d-1f (%) S1g (%) S2a-2c (%)

1

Ru/C (5 wt% Ru)

61

9

99%

12

[b]

13[b]

14[b]

X16a > 99%


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Table 3. Rh-catalyzed hydrogenation of amino acids to amino alcohols (all reaction products are shown).[a] (continued)

Entry

Amino acid

Products

15[b]

X17a > 99%

S15b = 94%

S15c = 6%

X18a > 99%

S18b = 75%

S18c = 25%

S19b = 31%

S19c = 18%

S18b = 47%

S18c = 4%

16

17

[b]

X19a > 99%

[a]

Conditions: amino acid (0.11 M), Rh-MoOx/SiO2 (4 wt% Rh, Mo/Rh = 1:8, Rh/amino acid= 3.5 mol%), H3PO4 (0.3 M), water (15 ml), H2 (70 bar), 80 °C, 6 h. Conversion (X) and selectivity (S) were determined by 1H NMR spectroscopy and HPLC. [b] Reaction for 17 h.


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Catalyst recycling The stability of the Rh-MoOx/SiO2 catalyst under typical reaction conditions was demonstrated by recycling the material in the hydrogenation of isoleucine (7a) for three times (ESI, Figure S5). After each run the catalyst was recovered by centrifugation, washed with deionized water and dried at 60 °C. Elemental analysis of the reaction medium obtained after the first run revealed that Rh leaching was negligible (0.01%), whereas Mo leaching was more pronounced (8.2%). Nevertheless, recycling was successful since both the conversion of 7a and the selectivity to isoleucinol (7b) remained constant throughout the four runs. Influence of sulfur-containing compounds on amino acid hydrogenation Platinum group metal catalysts are known to be inhibited by irreversible coordination of sulfur-containing groups with lone pairs to the metal surface.69–71 Freifelder suggested that catalyst poisoning can be avoided by oxidation of thiols and thioethers to sulfonic acids and sulfones, respectively.71 This hypothesis was verified by applying the hydrogenation procedure on mixtures of threonine (10a) and various sulfur-containing additives. Threoninol (10b) was obtained in 89% yield in the presence of sulfolane and methanesulfonic acid (Table 4, entries 3,4); these results are nearly identical to the yield in the absence of any additive (entry 1). However, the reaction did not proceed at all in the presence of dimethyl sulfoxide, which still contains a lone pair (entry 2). These experiments clearly demonstrate that complete oxidation of the sulfur-containing moieties in cysteine and methionine is the key to prevent deactivation of the hydrogenation catalyst.


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Table 4. Rh-catalyzed hydrogenation of threonine (10a) in the presence of several sulfurcontaining additives.[a] Entry

Additive

X10a (%)

S10b (%)

1

-

> 99

90

2

Dimethyl sulfoxide

99

89

4

Methanesulfonic acid

> 99

89

[a] Conditions: 10a (0.11 M), Rh-MoOx/SiO2 (4 wt% Rh, Mo/Rh = 1:8, Rh/10a = 3.5 mol%), additive (5 mol%), H3PO4 (0.3 M), water (15 ml), H2 (70 bar), 80 °C, 6 h. Conversion (X) and selectivity (S) were determined by 1H NMR spectroscopy. Oxidation and subsequent hydrogenation of cysteine and methionine The oxidation of sulfur-containing compounds has already been studied extensively. Nevertheless, the method of choice for the oxidation of cysteine and methionine should be highly efficient, allow to modify thiols and thioethers simultaneously, proceed in aqueous conditions and preferably use an environmentally benign oxidant, such as hydrogen peroxide, or even oxygen. Catalytic oxidation of thioethers is often limited to the sulfoxide stage and this approach is therefore considered to be less useful for our purpose.72–76 Inspired by the analytical determination of sulfur-containing amino acids in proteins, oxidation with performic acid is expected to be more promising. This unstable oxidant is produced in situ from hydrogen peroxide and formic acid, and is able to transform cysteine and methionine selectively into cysteic acid and methionine sulfone, respectively. In this way even cystine can be converted to cysteic acid. The excess of formic acid can be easily removed afterwards via evaporation under vacuum. First, the hydrogenation of cysteic acid (24a) and methionine sulfone (25a), which were prepared by oxidation with performic acid for 4 h, was studied with Ru- and Rh-based catalysts.


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Again, Rh-MoOx/SiO2 outperformed Ru/C and after 16 h the amino alcohols derived from cysteic acid (24a) and methionine sulfone (25a) were obtained in about 90% yield at complete conversion (Table 5). These experiments demonstrate that the noble metal catalysts are not poisoned by the sulfonic acid or the sulfone moieties present in the substrates. Table 5. Catalytic hydrogenation of cysteic acid (24a) and methionine sulfone (25a).[a] Amino acid

Amino alcohol

Catalyst

Time (h)

X (%)

S (%)

6

59

88

16

> 99

87

6

13

96

16

78

94

6

57

90

16

> 99

90

6

18

96

16

48

95

Rh-MoOx/SiO2

Ru/C 24a

24b Rh-MoOx/SiO2

Ru/C 25a

25b

[a]

Conditions: pre-oxidized amino acid (0.11 M), catalyst (3.5 mol% Rh or Ru), H3PO4 (0.3 M), water (15 ml), H2 (70 bar), 80 °C. Conversion (X) and selectivity (S) were determined by 1H NMR spectroscopy. The duration of the oxidative pre-treatment has a large impact on the outcome of the hydrogenation (Figure 2). Indeed, by extending the oxidation time to 6 h, the conversions of cysteic acid and methionine sulfone in the hydrogenation already decreased to respectively 91% and 22%, whereas the catalytic activity was completely inhibited after 16 h of oxidation. The decrease in catalytic activity can probably be attributed to overoxidation at longer pre-treatment times, which has been suggested earlier for the performic acid-mediated oxidation of cysteine and methionine.64,77 Additional signals were observed in the 1H NMR spectra of the oxidized amino acids (Figures S6-S7), but the identity of the degradation products remains unclear. We


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suspect that the amino acid moiety is susceptible to oxidation, possibly concurring with decarboxylation as was also observed in previous work by our research group.14 Consequently, the oxidative pre-treatment step should be limited to 4 h in order to maximize amino acid conversions. 100 80

X (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 40 20 0 4

6

16

Pretreatment time (h)

Figure 2. Effect of the pre-treatment time during performic acid mediated oxidation of cysteine and methionine on the conversion (X) in the Rh-catalyzed hydrogenation of cysteic acid (blue) and methionine sulfone (orange). Conditions: pre-oxidized amino acid (0.11 M), Rh-MoOx/SiO2 (3.5 mol% Rh), H3PO4 (0.3 M), water (15 ml), H2 (70 bar), 80 °C, 6 h.

Hydrogenation of a protein hydrolysate Finally, the hydrogenation procedure was applied to mixtures of amino acids obtained by acidmediated protein hydrolysis, in order to demonstrate the potential for biomass valorization. Bovine serum albumin (BSA), which is present in cow’s blood plasma and has a well-known amino acid composition,78–83 was selected as a model protein for animal slaughter waste. The hydrolysis was preceded by an oxidative treatment with performic acid in order to eliminate potential catalyst poisons in proteins, in particular the sulfur-containing amino acid residues. The composition of the BSA hydrolysate and the product mixtures obtained after hydrogenation was


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determined by reversed phase HPLC (Figure 3, Table 6 and ESI, Tables S2-S4). The mixtures contained about 60 compounds, which were separated based on differences in polarity, with the more polar amino acids eluting first (4.3-34.4 min), followed by amino alcohols (11.4-50.8 min) and the least polar amines (17.3-52.4 min). The chromatograms obtained after several reaction times demonstrate that the amino acids were progressively converted into a mixture of amino alcohols and amines, hence that the catalyst was not poisoned (Figure S9). However, when the oxidative pre-treatment was omitted, the hydrogenation did not proceed at all (Figure S8).

Figure 3. Reversed phase HPLC analysis of amino acids after oxidation and hydrolysis of BSA (A), and subsequent hydrogenation for 48 h (B). The signals and elution ranges of amino acids (▲, red), amino alcohols (●, green) and amines (♦, blue) are highlighted in and below the chromatograms. Gradient elution was performed by combining an aqueous mobile phase (A; 40 mM NaH2PO4 in Milli-Q water at pH 7.8) and an organic mobile phase (B; methanol,


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acetonitrile and Milli-Q water (45:45:10 v/v/v)). Conditions: 1.5 mL min-1; 98% A and 2% B from 0 to 0.84 min, from 2% to 20% B in 0.84 to 10 min, 20% to 50% B in 10 to 45 min, 50% to 100% B in 45 to 54 min, 100% B in 54 to 56.6 min, and 100% to 2% B in 56.6 to 57 min. Several side products of reactions that coincide with the hydrogenation products were taken into account when calculating the amino acid conversions and the amino alcohol selectivities. Hydrogenation of some natural amino acids may generate other non-natural amino acids, which were observed as reaction intermediates: e.g. 3-amino-4-hydroxybutanoic acid and 4-amino-5hydroxy-pentanoic acid by reduction of the carboxylic acid at the α-carbon of aspartic acid and glutamic acid, but also α-amino-cyclohexanepropanoic acid and α-amino-4-hydroxycyclohexanepropanoic acid by hydrogenation of the aromatic moieties in phenylalanine and tyrosine. Similarly, C-O hydrogenolysis of aminodiols may produce other amino alcohols: e.g. 3amino-1-butanol and 2-amino-1-butanol from aspartidiol (15b), 4-amino-1-pentanol and 2amino-1-pentanol from glutamidiol (1d), 2-amino-1-propanol from serinol (9b), 3-amino-2butanol from threoninol (10b), and 4-(2-aminopropyl)-cyclohexanol from β-amino-4-hydroxycyclohexanepropanol (19b). Some amino alcohols may even originate from different amino acids: 2-amino-1-propanol can be generated by reduction of alanine (4a) or by C-O hydrogenolysis of serinol (9b), whereas prolinol (2c) can be obtained by reduction of either proline (8a) or pyroglutaminol (2b). Since both pathways cannot be distinguished in these mixtures, 2-amino-1-propanol was entirely assigned to alanine, and prolinol to proline. The mixture of amino acids was hydrogenated more slowly compared to single-compound experiments (Table 6). Trace amounts of residual formic acid after oxidation could slow down the hydrogenation as was observed in the discrepancy between the hydrogenation rates of cysteic acid and methionine sulfone obtained either commercially or after oxidation of cysteine and


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methionine (ESI Table S5). Also, amino acid hydrogenation can be affected by degradation products of tryptophan. For instance, a mixture of leucine, isoleucine, valine, glycine and tryptophan was hydrogenated more slowly compared to the same mixture in which tryptophan replaced by commercial cysteic acid (Table S6). Accordingly, the overall conversion was only 20% after 6 h (Table 6). Intrinsic differences in reactivity observed earlier were even more pronounced. For instance, the conversions of valine and isoleucine were poor after 6 h, 4% and < 1% respectively, due to steric hindrance in these substrates. Nevertheless, the selectivity to amino alcohols was excellent (> 99% in most cases) because C-O hydrogenolysis was not prominent yet. A strong increase in conversion was obtained by extending the reaction time to 48 h. As a drawback, the selectivity towards amino alcohols decreased as a result of C-O hydrogenolysis, especially for alaninol. Nevertheless, the overall conversion of the amino acid mixture amounts up to 90%, with an overall high selectivity of 88% to amino alcohols. Table 6. Rh-catalyzed hydrogenation of amino acids obtained by oxidation and hydrolysis of BSA.[a] Amino acid Mass fraction in BSA (wt%)

X (%)

SAmino alcohols (%)

SAmines (%)

6h

48 h

6h

48 h

6h

48 h

Ala

6.17

11

90

> 99

60

99

80

82

20

18

Asx[b]

10.65

99

77

99

94

92

6

8

Glx[d]

11.66

1

66

> 99

97

99

> 99

83

99

77

99

90

99

82

91

18

9

Metox[e]

0.41

14

> 99

> 99

76

99

91

99

> 99

> 99

99

99

99

> 99

Rh-catalyzed hydrogenation of amino acids to bio-based amino alcohols

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