Urea-Appended Amino Acid To Vitalize Yeast Growth, Enhance

Aug 7, 2019 - The development of sustainable protocols for enhancing the production of ethanol is highly important for its utilization in automotive ...
0 downloads 0 Views 3MB Size
Download PDF
This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: ACS Omega XXXX, XXX, XXX−XXX

http://pubs.acs.org/journal/acsodf

Urea-Appended Amino Acid To Vitalize Yeast Growth, Enhance Fermentation, and Promote Ethanol Production Supriya Sasmal, Srayoshi Roy Chowdhury, Debasish Podder, and Debasish Haldar* Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur 741246, West Bengal, India

Downloaded via 46.161.63.223 on August 11, 2019 at 05:48:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: The development of sustainable protocols for enhancing the production of ethanol is highly important for its utilization in automotive transportation and energy sector. Up to 15% ethanol can blend with diesel to make e-diesel that can be used in fuel compression ignition engine. Urea-modified amino acids can be used as a very good vitalizer for yeast ( Saccharomyces cerevisiae, Baker’s yeast (ATCC 204508)) growth and thus promote ethanol production. A simple, one-step, room-temperature synthetic procedure has been developed for urea-appended α-amino acids from amino acid by treatment with KCNO. Single-crystal X-ray studies confirm the successful synthesis and molecular structures of the urea-appended α-amino acids. Out of 20 urea-appended amino acids, Arg-, Pro-, His-, and Gln-containing compounds promote yeast growth significantly after 12 h at pH 6.8 and 38 °C. These compounds are nontoxic. The urea-appended Arg shows 2-fold increase of yeast growth. However, urea-appended maminobenzoic acid and p-aminobenzoic acid inhibit yeast growth. NMR experiments confirmed the enhanced production of ethanol by glucose fermentation in the presence of 2.5 μmol urea-appended Arg. Not only glucose but also commercially available sugars and feedstock of the starch slurry drained out after boiling of rice exhibit significant enhancement of ethanol production under same conditions.

■

INTRODUCTION With rapidly growing urbanization and enhancement in living standards, a rapid growth in energy and fuel consumption has been observed in the last century.1 The demand of energy for transportation, heating, and industrial processing is increasing dramatically.2 But the reserve of fossil fuels is limited, and the use of fossil fuel is not environment-friendly. In this critical situation, conversion of biomass into fuel is highly important.3−7 Biomass has long been used widely to extract valuable products such as medicine, flavors, and fragrances.8 It is a renewable, cheap, and highly available natural resource, which is generally employed for the production of biodiesel and other commodity chemicals.9,10 Arguably, ethanol is one of the environment-friendly bioenergy sources with high efficiency.11 The annual production of ethanol is approximately 51 000 million liters worldwide,12 of which fuel encompasses 73% of ethanol obtained, industry uses 10%, and beverages consume 17%.12 However, over 98% of bioethanol is made from corn.13 As a result, the competition between fuel and food increases. Hence, the production of biodiesel from nonedible biomass or kitchen waste is highly important. Woodson and Jablonowskiy reported that “as an additive (ethanol), serves as a fuel volume extender, an oxygenate and an octane enhancer”.14 According to Sánchez and Cardona, most of the countries in the world use ethanol-mixed gasoline or directly ethanol as fuel.15 Generally, ethanol is a product of microbe-assisted fermentation. Microorganisms meet their © XXXX American Chemical Society

energy demand by converting carbon-containing compounds into carbon dioxide, lactic acid, and ethanol.16 Fermentation of carbohydrates into alcohol is one of the most effective and cheapest methods to produce ethanol with negligible environmental pollution. But due to the complex structure and very high stability, complex carbohydrates can hardly be degraded by most of the microorganisms under normal fermentation. Therefore, to enhance efficiency, pretreatment is necessary. Various pretreatment methods, including diluted acid pretreatment, liquid hot water pretreatment, steam explosion, wet oxidation, and biological treatment, have been reported.17−20 But pretreatments are costly and cause serious environmental concerns.21 In this context, we have adopted a sustainable method to vitalize yeast growth and promote carbohydrates/biomass fermentation. The objective was not to prepare isolated, pure chemicals like petrochemical industries or traditional organic synthesis. In general, we are interested in degradation and conversion of biomass/carbohydrates through fermentation to obtain ethanol. We are developing designer amino acids and analogues.22−24 Herein, we have designed and synthesized a series of urea-appended α-amino acids. Although the ureaappended m-aminobenzoic acid and p-aminobenzoic acid have Received: May 2, 2019 Accepted: July 25, 2019

A

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

been investigated in materials science,25 very little is known about the structure and function of the urea-appended αamino acids. The natural abundance and excellent biological importance of urea and α-amino acids motivated us to examine the conformational preferences and properties of these hybrid compounds (Figure 1).26 Urea is a popular fertilizer as a source

Figure 2. (a) Solid-state conformation of compound 1. (b) Intermolecular hydrogen-bonded hexagonal honeycomb-like structure of compound 1 along crystallographic a and b directions. Hydrogen bonds are shown as dotted lines.

Figure 1. (a) Chemical structure of urea-modified α-amino acid. (b) General synthesis scheme for 20 urea-modified amino acids used in this study.

directions (Figure 2b). The hydrogen-bonding parameters are listed in Supporting Information Table S1. Colorless monoclinic crystals of Arg-containing compound 2 were obtained from water solution by slow evaporation. There is one molecule of compound 2 along with one molecule of water in the asymmetric unit (Figure 3a). In higher-order

of nitrogen and is an important raw material for the chemical and cosmetic industry.27 Also, urea serves a crucial role in the metabolism of nitrogen-containing compounds. On the other hand, amino acids are the building blocks of proteins and enzymes. Due to their biological importance, amino acids are used in nutritional supplements and for the development of drugs, biodegradable polymers, and so on. So, the fusion of urea and amino acids will be fascinating (Figure 1). Singlecrystal X-ray diffraction analysis confirms the successful synthesis and molecular conformations of the urea-appended α-amino acids. Out of 20 urea-appended amino acids, Arg-, Pro-, His-, and Gln-containing compounds promote yeast growth significantly after 12 h at pH 6.8 and 38 °C. The ureaappended amino acids are nontoxic. The Arg-containing compound 2 shows twofold increase of yeast growth. However, urea-appended m-aminobenzoic acid and p-aminobenzoic acid inhibit yeast growth drastically. NMR experiments and kinetics studies confirmed the enhanced production of ethyl alcohol (EtOH) by glucose fermentation in the presence of 2.5 μmol compound 2 at 38 °C and pH 6.8. Not only glucose but also commercially available sugar and kitchen waste exhibit significant conversion to ethanol under the same condition.

Figure 3. (a) Solid-state conformation of Arg-containing ureamodified α-amino acid 2 with one molecule of water in the asymmetric unit. (b) Multiple intermolecular hydrogen-bonded cagelike structure of compound 2 along crystallographic b and c directions. Hydrogen bonds are shown as dotted lines.

■

RESULTS AND DISCUSSION Urea-appended amino acids were synthesized by the reaction between KCNO and the corresponding amino acid in water at 25 °C for 4 h, followed by acidification with hydrochloric acid to pH 2−3 (Figure 1b).28 The obtained white precipitate was collected by filtration. The synthesized compounds were characterized by 1H NMR, 13C NMR, and mass spectrometry techniques. The Leu- and Arg-containing urea-appended amino acids were analyzed by single-crystal X-ray diffraction analysis. From growth inhibition zone studies of Escherichia coli bacteria and the mammalian cell uptake experiments, the compounds are nontoxic (Supporting information Figures S83 and S84). Colorless orthorhombic crystals of Leu -containing compound 1 were obtained from dimethyl sulfoxide− tetrahydrofuran (DMSO-THF) solution by slow evaporation. There is one molecule of compound 1 in the asymmetric unit (Figure 2a). The packing diagram shows that each compound 1 molecule self-assembles with six molecules through N−H···O and O−H···O hydrogen bonds and thus forms a hexagonal honeycomb-like structure along crystallographic a and b

packing, compound 2 forms a cagelike structure with eight more molecules and waters through multiple intermolecular hydrogen-bonding interactions. Table S2 in the Supporting Information shows the hydrogen-bonding parameters of compound 2. So, compound 2 has many active sites to recognize and interact with other molecules. We have adopted a method to enhance carbohydrates/ biomass conversion using urea-appended amino acids. The objective was not to prepare isolated, pure chemicals that could be obtained from petrochemical industries or traditional synthesis. Rather we are interested in the degradation and conversion of biomass/carbohydrates through fermentation to obtain valuable functional end-products such as ethanol. Currently, ethanol is the most widely used biofuel in combination with diesel. Our approach will dramatically decrease the biomass processing time and cost, which is a major concern in this industry.29,30 For that purpose, we have used Lysogeny broth (LB) containing 2% dextrose as a medium. Although yeast nitrogen base (YNB) is a highly referenced growth medium for yeast cultivation, it contains B

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

for Arg-containing urea-appended amino acid 2. From Figure 4b, only urea, only Arg, or urea and Arg mixture has no effect on the growth of yeast. But Arg-containing urea-appended amino acid 2 increased the rate of yeast growth within 20 h. Similarly, Figure 4c shows the control yeast growth for Procontaining urea-appended amino acid, which increased the rate of yeast growth within 30 h. However, from Figure 4d, only urea, only Gln, or urea and Gln mixture have almost no effect on the growth of yeast. But Gln-containing urea-appended amino acid has increased the rate of yeast growth within 20 h (Figure 4d). Figure 4e shows that the His-containing ureaappended amino acid has increased the growth rate of yeast within 20 h. Figure 5a shows the pH optimization for Arg-containing urea-appended amino acid 2 after 12 h. LB medium (10 mL)

amino acids, nitrogen, vitamins, trace elements, and salts. But we want to measure the effect of urea-appended amino acid on yeast growth. So we have opted Lysogeny broth (LB) medium, which contains less amount of yeast growth nutrient and no amino acid. The medium (10 mL) was taken in a test tube and 2 mg of urea-appended amino acid was added. To this solution, 10 μL of yeast culture was added and incubated for 12 h at 35 °C. For control, we have used only urea and yeast following the same procedure and under the same condition. After 12 h, the absorption spectra were recorded. The absorbance at 600 nm was plotted (Figure 4a). From Figure

Figure 4. (a) Response of 20 urea-modified amino acids toward yeast growth. Only Arg-, His-, Gln-, and Pro-containing urea-modified amino acids are promoting the growth of yeast. The m-ABA- and pABA-containing urea-modified amino acids inhibited the growth of yeast. (b) Control yeast growth for Arg-containing urea-modified amino acid. (c) Control yeast growth for Pro-containing ureamodified amino acid. (d) Control yeast growth for Gln-containing urea-modified amino acid. (e) Control yeast growth for Hiscontaining urea-modified amino acid.

Figure 5. (a) pH optimization, (b) temperature optimization, (c) concentration optimization, (d) effect of urea-modified amino acids on yeast growth, (e) rate of yeast growth, and (f) yeast growth for the second cycle.

4a, we conclude that out of 20 urea-appended amino acids, Arg-, His-, Gln-, and Pro-containing urea-appended amino acids promote the growth of yeast, whereas urea-appended maminobenzoic acid (m-ABA) and p-aminobenzoic acid (pABA) inhibited the growth of yeast. Only urea has almost no effect on the growth of yeast. The Arg-containing ureaappended amino acid 2 exhibits maximum enhancement of yeast growth. For further clarification, we carried out the control experiments for Arg-, His-, Gln-, and Pro-containing urea-appended amino acids. LB medium (10 mL) containing 2% dextrose was taken in each test tube. Arg/His/Gln/Procontaining urea-appended amino acid, only urea, only amino acid, or urea and amino acid mixture (2 mg) was added. To that solution, 10 μL of yeast culture was added and incubated for 50 h at 40 °C. The absorbances at 600 nm were noted in a regular time interval. Figure 4b shows the control yeast growth

containing 2% dextrose was taken in test tubes, and 2 mg of Arg-containing urea-appended amino acid 2 and 10 μL of yeast culture were added in respective test tubes. The pH of the test varies from 3 to 11. The solutions were incubated for 12 h at 35 °C and studied by absorption spectroscopy. From Figure 5a, we conclude that the growth of yeast in the presence of Arg-containing urea-appended amino acid 2 is maximum at pH 6.8. We have also done temperature optimization for Argcontaining urea-appended amino acid 2. LB medium (10 mL) containing 2% dextrose was taken in each test tube, and 2 mg of Arg-containing urea-appended amino acid 2 and 10 μL of yeast culture were added at pH 6.8. The test tubes were incubated for 12 h at a different temperature ranging from 30 to 55 °C, and the absorbance was examined at 600 nm after 12 C

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

h. From Figure 5b, yeast growth in the presence of Argcontaining urea-appended amino acid 2 is maximum at 38 °C. Further, we have performed the concentration optimization for Arg/His/Gln/Pro-containing urea-appended amino acid after 12 h. The LB medium containing 2% dextrose was taken in a 96-well plate. Urea-appended Arg/His/Gln/Pro was taken at various concentrations (making concentration gradient: 0.1− 50 μmol), and 10 μL of yeast culture was added. The solution was incubated for 50 h at 38 °C and pH 6.8 and studied by absorption spectroscopy (Figure 5c). From Figure 5c, we conclude that the optimized concentration for Arg-containing urea-appended amino acids is 2.5 μmol, 16 μmol for Hiscontaining urea-appended amino acids, 9.5 μmol for Glncontaining urea- modified amino acids, and 25 μmol for Procontaining urea-appended amino acids. Then, we have studied the effect of Arg/His/Gln/Pro-containing urea-appended amino acid on yeast growth at pH 6.8 and temperature 38 °C. LB medium (10 mL) containing 2% dextrose was taken in test tubes and urea-appended Arg (2.5 μmol), His (16 μmol), Gln (9.5 μmol), and Pro (25 μmol) were added respectively. Yeast culture (10 μL) was added in each tube, the test tubes were incubated for 50 h at 38 °C and pH 6.8, and absorbance was examined at 600 nm in regular intervals. From Figure 5d, we conclude that for each case, yeast growth promoted for the respective urea-appended amino acid at respective optimized concentration. We also calculate the approximate growth rate as shown in Figure 5e. Moreover, we have studied the yeast growth for second cycle after reaching first time saturation at pH 6.8 and 38 °C in the presence of 2.5 μmol Arg-containing urea-appended amino acid 2. LB medium (10 mL) containing 2% dextrose was taken in the test tubes and 2.5 μmol Argcontaining urea-appended amino acid 2 and 10 μL of yeast culture were added. The test tubes were incubated at 38 °C and pH 6.8 for 50 h. The absorbance at 600 nm was recorded after 50 h. After that, the respective absorbance becomes saturated and we consider this absorbance as the absorbance data for 0 h for the second time addition of 2% dextrose (second cycle). Again the test tubes were incubated at 38 °C and pH 6.8 for 50 h and the absorbance at 600 nm was noted regularly. From Figure 5f, we conclude that the Arg-containing urea-appended amino acid 2 can facilitate yeast growth for the second cycle. Spot assay on Petri dishes (Figure 6) shows accelerated yeast growth by urea-appended amino acids after 24/48 h cell culture with several serial dilutions of cells.31

To study the kinetics of glucose fermentation by yeast and ethanol production, NMR experiments were performed. LB medium (50 mL) containing 2% dextrose was taken in a 100 mL sealed conical flask. Arg-containing urea-appended amino acid 2 (2.5 μmol) and 10 μL of yeast culture were added. The solution was incubated at 38 °C and pH 6.8. 1H NMR spectra were recorded in 5 h interval. From the 1H NMR stack plot (Figure 7a), we conclude that in the presence of Arg-

Figure 7. (a) 1H NMR kinetics study of glucose fermentation by yeast in the presence of Arg-containing urea-modified amino acid 2 and ethanol production. (b) Yield calculation for glucose fermentation and ethanol production. (c) Catalytic cycles.

containing urea-appended amino acid 2, the yeast fermented the dextrose (glucose) with consecutive generation of ethanol and the amount of ethanol increases with increasing time. The same procedure was followed for the control (without ureaappended amino acid). Figure 7b shows simultaneously the degradation (fermentation) of dextrose and the formation (generation) of ethanol. Figure 7b exhibits the higher rate of degradation of glucose by yeast in the presence of Argcontaining urea-appended amino acid 2 and the rate of formation of ethanol. We have also performed the catalytic cycle experiments. Interestingly, Figure 7c depicts that the degradation of glucose by yeast in the presence of Argcontaining urea-appended amino acid 2 works nicely up to three cycles. We have also examined the degradation of other carbohydrates. For that, 10 mL of LB medium containing 2% commercially available sugar was taken in the test tubes, and 10 μL of yeast culture was added. Arg/His/Gln/Procontaining urea-appended amino acids were taken respectively in test tubes and incubated at 38 °C and pH 6.8 for 12 h. The absorbance at 600 nm was recorded for different carbohydrates. We have also examined the EtOH formation for feedstock of the starch slurry drained out after boiling of rice through 1H

Figure 6. Spot assay (on Petri dishes) showing accelerate yeast growth. Range from 10 × 7 to 10 × 3/mL. D

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S14): 172.46, 162.77, 47.93, 38.17, 21.39, 19.93, 18.69. FTIR (KBr) (Supporting Information Figure S15): 3460, 3293, 2959, 1691, 1640, 1571, 1311, 1017, 719 cm−1. ESI-MS (Supporting Information Figure S16): m/z 175.7202 [M + H]+; Mcalcd: 175.10, 213.0808 [M + K]+; Mcalcd: 213.0612. H2N-U-Arg-OH (2). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S57): 4.67 (s, 1H, −CαH), 3.04 (m, 2H, −CH2), 1.64−1.49 (m, 4H, −2CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S58): 174.61, 163.23, 158.49, 60.6, 41.18, 29.51, 25.76. FTIR (KBr) (Supporting Information Figure S59): 3449, 1650, 1401, 850, 652, 611 cm−1. Mass spectra (Supporting Information Figure S60): m/z: 218.03 [M + H]+, 239.99 [M + Na]+, 255.95 [M + K]+; Mcalcd: 217.12. H2N-U-His-OH (3). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S45): 12.85 (s, 1H, −NH), 12.71 (s, 1H, −COOH), 8.83 (s, 1H, −ArH), 7.53 (s, 1H, −ArH), 6.76 (s, 1H, −NH), 5.54 (s, 2H, −NH2), 3.99 (m, 1H, −CαH), 2.88−2.76 (m, 2H, −CH2). 13C NMR (100 MHz, D2O, δ in ppm) (Supporting Information Figure S46): 180.37, 163.67, 135.86, 133.15, 118.45, 57.04, 29.85. FTIR (KBr) (Supporting Information Figure S47): 3413, 1656, 1583, 1396, 1011, 835, 699 cm−1. ESI-MS (Supporting Information Figure S48): m/z 198.0805 [M]+; Mcalcd: 198.08, 237.0141 [M + K]+; Mcalcd: 237.0411. H2N-U-Pro-OH (4). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S49): 3.97 (m, 1H, −CαH), 3.27−3.18 (m, 2H, −CH2), 2.21−1.84 (m, 4H, −2CH2). 13C NMR (100 MHz, D2O, δ in ppm) (Supporting Information Figure S50): 174.73, 156.49, 61.37, 46.26, 29.02, 23.82. FTIR (KBr) (Supporting Information Figure S51): 3445, 3196, 1714, 1646, 1418, 1231, 1116, 1075, 752, 617, 565 cm−1. ESIMS (Supporting Information Figure S52): m/z 159.9355 [M]+; Mcalcd: 157.0715. H2N-U-Gln-OH (5). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S65): 3.99−3.68 (m, 1H, −CαH), 2.21−1.8 (m, 4H, −2CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S66): 174.39, 173.3, 162.51, 58.55, 31.12, 25.53. FTIR (KBr) (Supporting Information Figure S67): 3225, 2945, 2726, 2622, 1655, 1400, 990, 834, 699 cm−1. ESI-MS (Supporting Information Figure S68): m/z 189.0146 [M + H]+; Mcalcd: 189.0711, 211.0052 [M + Na]+; Mcalcd: 211.06. H2N-U-Val-OH (6). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S1): 12.48 (s, 1H, −COOH), 6.16 (d, 1H, −NH J = 8.63), 5.6 (s, 2H, −NH2), 3.98 (s, 1H, −CαH), 1.97 (m, 1H, −CH, J = 5.36), 0.82 (t, 6H, −CH3, J = 8.39). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S2): 174.20, 158.54, 57.31, 30.3, 19.21, 17.66. FTIR (KBr) (Supporting Information Figure S3): 3456, 3342, 3290, 2961, 2469, 1938, 1678, 1626, 1564, 1398, 1304, 1169, 1023, 732, 607, 576, 503 cm−1. ESIMS (Supporting Information Figure S4): m/z 183.238 [M + H]+; Mcalcd: 183.07. H2N-U-Try-OH (7). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S5): 9.23 (s, 1H, −OH), 6.95 (d, 2H, −ArH J = 8.63), 6.63 (d, 2H, −ArH J = 7.64), 6.17 (d, 1H, −NH, J = 6.64) 5.54 (s, 2H, −NH2), 4.21 (t, 1H, −CαH, J = 4.32), 3.37 (s, 1H, −CH2), 2.8 (t, 1H, −CH2, J = 8.39). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S6): 175.35, 157.22, 156.13,

NMR (Figure 8). For that purpose, 10 mL of LB medium containing 2% starch slurry was taken in test tubes and 10 μL

Figure 8. Kinetics study of EtOH production by yeast in the presence of Arg-containing urea-appended amino acid 2 using 1H NMR experiments.

of yeast culture was added. Arg-containing urea-appended amino acid was taken respectively in test tubes and incubated at 38 °C and pH 6.8 for 12 h. NMR spectra were measured with respect to time. Figure 8 exhibits higher rate of formation of EtOH by yeast in the presence of Arg-containing ureaappended amino acid 2.

■

CONCLUSIONS In conclusion, we have designed and synthesized ureaappended α-amino acids. We have also discussed their effect on vitalization of yeast and enhancement of fermentation and ethanol production. In solid state, Leu-containing ureaappended amino acid 1 forms a hydrogen-bonded honeycomb-like structure. But Arg-containing urea-appended amino acid 2 forms water-mediated cagelike structure through multiple intermolecular hydrogen-bonding interactions. From the urea-appended amino acids library, Arg-, Pro-, His-, and Gln-containing compounds vitalize yeast significantly after 12 h at pH 6.8 and 38 °C. The compounds are nontoxic. The Argcontaining compound 2 exhibits twofold increase of yeast growth. The NMR studies exhibit the enhanced production of ethanol due to fermentation of glucose in the presence of 2.5 μmol compound 2 at 38 °C and pH 6.8. Moreover, commercially available sugar and kitchen waste like feedstock of starch slurry drained out after boiling of rice exhibit significant enhancement of ethanol production under same conditions. These developments pointed to a sustainable way for fuels and commodity chemical industries.

■

EXPERIMENTAL SECTION General. α-Amino acids and other reagents were procured from Sigma Chemicals. Synthesis. Amino acid (1 mmol) was dissolved in hot water and cooled to 5 °C, and KCNO (6 mmol) was added slowly. After complete addition of KCNO, the solution was stirred for 4 h at room temperature. The resultant mixture was cooled to 5 °C and acidified with HCl to pH = 1. The obtained white precipitate was filtered and dried under vacuum. The compounds were fully characterized by 1H NMR (400 MHz) spectroscopy, 13C NMR (100 MHz) spectroscopy, mass spectrometry, and Fourier-transform infrared (FTIR) spectroscopy analyses. Further, X-ray crystallography was performed to characterize compounds 1 and 2. H2N-U-Leu-OH (1). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S13): 6.19 (d, 1H, −NH, J = 6.8), 5.54 (s, 2H, −NH2), 4.05 (t, 1H, −CαH, J = 4.04), 1.44−1.4 (m, 2H, −CH2), 0.87 (m, 6H, −CH3). 13C E

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

NMR (100 MHz, D2O, δ in ppm) (Supporting Information Figure S42): 179.21, 161.95, 159.33, 59.29, 39.92, 28.96, 28.42, 20.39. FTIR (KBr) (Supporting Information Figure S43): 3440, 3200, 1754, 1641, 1402, 1215, 767, 632 cm−1. ESIMS (Supporting Information Figure S44): m/z 255.3092 [M + Na]+; Mcalcd: 255.1101. H2N-U-Asn-OH (14). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S53): 3.87 (t, 1H, −CαH, J = 3.07), 2.8−2.72 (m, 2H, −CH2). 13C NMR (100 MHz, D2O, δ in ppm) (Supporting Information Figure S54): 174.59, 173.38, 154.9, 51.51, 34.68. FTIR (KBr) (Supporting Information Figure S55): 3425, 1647, 1398, 992, 836, 701 cm−1. ESI-MS (Supporting Information Figure S56): m/z 175.9841 [M]+; Mcalcd: 175.0614. H2N-U-Asp-OH (15). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S61): 3.86 (t, 1H, −CαH, J = 6.72), 2.8−2.76 (m, 2H, −CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S62): 176.52, 175.11, 162.32, 56.56, 39.56. FTIR (KBr) (Supporting Information Figure S63): 3444, 3226, 1718, 1656, 1583, 1406, 1199, 637 cm−1. ESI-MS (Supporting Information Figure S64): m/z 176.2942 [M + H]+; Mcalcd: 176.04, 199.3502 [M + Na]+; Mcalcd: 199.0311. H2N-U-Ser-OH (16). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S69): 4−3.99 (m, 2H, −CH2), 3.26−3.08 (m, 1H, −CαH). 13C NMR (100 MHz, D2O, δ in ppm) (Supporting Information Figure S70): 171.52, 163.12, 59.26, 55.7. FTIR (KBr) (Supporting Information Figure S71): 3440, 1730, 1642, 1413, 612 cm−1. ESI-MS (Supporting Information Figure S72): m/z 149.1403 [M + H]+; Mcalcd: 149.0524. H2N-U-Aib-OH (17). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S73): 1.28 (s, 6H, −CH3). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S74): 176.8, 163.22, 58.79, 25.11. FTIR (KBr) (Supporting Information Figure S75): 3440, 3218, 1721, 1638, 1398, 1055, 764, 483 cm−1. ESI-MS (Supporting Information Figure S76): m/z 147.0455 [M]+; Mcalcd: 146.0711, 185.8586 [M + Na]+; Mcalcd: 185.0314. H2N-U-Ala-OH (18). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S77): 3.26 (q, 1H, −CαH, J = 7.32), 1.22 (d, 2H, −CH3, J = 7.93). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S78): 174.7, 161.23, 56.02, 16.35. FTIR (KBr) (Supporting Information Figure S79): 3446, 1626, 1398, 1055, 780, 483 cm−1. ESI-MS (Supporting Information Figure S80): m/z 136.4013 [M]+; Mcalcd: 136.0524, 303.3250 [2M + Na]+; Mcalcd: 303.0714. H2N-U-pABA-OH (19). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S21): 10.63 (br, 1H, −COOH), 7.87−7.29 (m, 4H, −ArH, J = 8.59), 6.27 (d, 1H, −NH, J = 6.23), 5.48 (s, 2H, −NH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S22): 167.17, 152.12, 145.04, 130.59, 130.44, 122.72, 118.06, 116.59. FTIR (KBr) (Supporting Information Figure S23): 3438, 1722, 1670, 1556, 1410, 1327, 1255, 1203, 859, 766 cm−1. ESIMS (Supporting Information Figure S24): m/z 182.0931 [M + H]+; Mcalcd: 181.0118. H2N-U-mABA-OH (20). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S33): 10.29 (br, 1H, −COOH), 8.99 (s, 1H, −ArH), 8.08 (d, 1H, −ArH, J = 8.56), 7.61−7.4 (m, 2H, −ArH), 7.32 (s, 1H, −NH), 5.71 (br, 2H, −NH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm)

130.72, 125.5, 114.91, 58.69, 39.09. FTIR (KBr) (Supporting Information Figure S7): 3443, 3297, 1738, 1696, 1634, 1519, 1436, 1384, 1249, 1114, 1092, 833, 771, 646, 573 cm−1. ESIMS (Supporting Information Figure S8): m/z 225.2024 [M + H]+; Mcalcd: 225.08, 247.0648 [M + Na]+; Mcalcd: 247.0711. H2N-U-Phe-OH (8). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S9): 7.19 (m, 5H, −ArH), 6.17 (d, 1H, −NH, J = 6.43), 5.63 (s, 2H, −NH2), 4.31 (t, 1H, −CαH, J = 4.13), 3.37 (s, 1H, −CH2), 2.92 (m, 2H, −CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S10): 174.08, 158.23, 137.78, 129.30, 128.11, 126.3, 53.97, 37.74. FTIR (KBr) (Supporting Information Figure S11): 3456, 3310, 1695, 1643, 1570, 1414, 1310, 1248, 1165, 1019, 832, 697, 583, 499 cm−1. ESI-MS (Supporting Information Figure S12): m/z 209.9738 [M + H]+; Mcalcd: 209.08, 248.9338 [M + Na]+; Mcalcd: 248.05. H2N-U-Ile-OH (9). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S17): 12.47 (br, 1H, −COOH), 6.16 (d, 1H, −NH, J = 9.16), 5.58 (s, 2H, −NH2), 4.02 (t, 1H, −CαH, J = 5.32), 1.69 (m, 1H, −CH), 1.35 (m, 1H, −CH), 1.12 (m, 1H, −CH), 0.84 (m, 6H, −CH3). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S18): 174.18, 158.42, 56.55, 39, 24.62, 15.72, 11.51. FTIR (KBr) (Supporting Information Figure S19): 3453, 308, 2976, 2945, 1697, 1634, 1567, 1395, 1302, 1167, 1011, 782, 730, 615, 501 cm−1. ESI-MS (Supporting Information Figure S20): m/z 176.2235 [M + H]+; Mcalcd: 176.1013, 370.1475 [2M + Na]+; Mcalcd: 370.19. H2N-U-Phgly-OH (10). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S25): 7.35−7.32 (m, 5H, −ArH), 6.81 (d, 1H, −NH, J = 8.4), 5.72 (s, 2H, −NH2), 5.15 (d, 1H, −CαH, J = 7.6). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S26): 172.90, 157.89, 138.56, 128.56, 127.77, 127.12, 56.69. FTIR (KBr) (Supporting Information Figure S27): 3457, 3405, 3239, 1710, 1638, 1544, 1450, 1388, 1295, 1253, 1180, 1074, 1024, 733, 691, 567, 504 cm−1. ESI-MS (Supporting Information Figure S28): m/z 195.1868 [M + H]+; Mcalcd: 195.0899, 217.1768 [M + Na]+; Mcalcd: 217.06. H2N-U-Met-OH (11). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S29): 6.08 (s, 1H, −NH), 5.58 (s, 2H, −NH2), 3.63 (m, 1H, −CαH), 2.45−2.25 (m, 2H, −CH2), 1.98 (s, 3H, −CH3), 1.85−1.68 (m, 2H, −CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S30): 173.86, 158.59, 54.46, 38.87, 29.89, 14.74. FTIR (KBr) (Supporting Information Figure S31): 3427, 2920, 1646, 1581, 1399, 1009, 840, 697 cm−1. ESI-MS (Supporting Information Figure S32): m/z 193.1455 [M + H]+; Mcalcd: 193.06. H2N-U-Trp-OH (12). 1H NMR (400 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S37): 10.84 (br, 1H, −COOH), 7.49−6.96 (5H, −ArH), 6.01 (d, 1H, −NH, J = 4.2), 5.59 (s, 2H, −NH2), 4.36 (t, 1H, −CαH, J = 6.12), 3.04 (m, 2H, −CH2). 13C NMR (100 MHz, DMSO-d6, δ in ppm) (Supporting Information Figure S38): 174.29, 163.28, 136.07, 127.6, 124.21, 123.71, 120.97, 118.33, 111.32, 109.58, 106.55, 60.25, 27.73. FTIR (KBr) (Supporting Information Figure S39): 3430, 3388, 3221, 1709, 1651, 1540, 1241, 740 cm−1. ESI-MS (Supporting Information Figure S40): m/z 247.527 [M + H]+; Mcalcd: 2471017. H2N-U-Lys-OH (13). 1H NMR (400 MHz, D2O, δ in ppm) (Supporting Information Figure S41): 4.25 (t, 1H, −CαH, J = 4.24), 3.05 (m, 2H, −CH2), 1.83−1.33 (m, 8H, −4CH2). 13C F

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

ORCID

(Supporting Information Figure S34): 167.49, 140.90, 131.21, 128.82, 123.86, 123.13, 122.01, 118.46. FTIR (KBr) (Supporting Information Figure S35): 3439, 3231, 3116, 3013, 2815, 2617, 2573, 1713, 1640, 1557, 1401, 1276, 1131, 759 cm−1. ESI-MS (Supporting Information Figure S36): m/z 181.0288 [M + H]+; Mcalcd: 181.05, 248.9338 [M + Na]+; Mcalcd: 248.05. NMR Experiments. All NMR spectroscopy experiments were done on a 400 MHz JEOL spectrometer. Compound concentrations were in the 1−10 mM range in DMSO-d6. FTIR Experiments. FTIR spectroscopy experiments in solid state were performed with a PerkinElmer Spectrum RX1 spectrophotometer using the KBr disk method. Absorption Spectroscopy. The absorption spectra of peptides were measured on a PerkinElmer UV/Vis spectrometer (Lambda 35) using quartz cell having 1 cm path length. Mass Spectrometry. Mass spectrometry was carried out on a Waters Corporation Q-Tof Micro YA263 high-resolution mass spectrometer by electrospray ionization (positive-mode). X-ray Crystallography. Intensity data for compounds 1 and 2 were recorded on a Bruker high-resolution X-ray diffractometer instrument. The structures were solved by a direct method and refined by least-squares calculations on F2 for all independent reflections (SHELXL-2014).32 Refinement of nonhydrogen atoms was performed using anisotropic thermal parameters. Crystal data of compound 1: C7H14N2O3, Mw = 170.20, P 21 21 21, a = 5.2432(9) Å, b = 10.3449(19) Å, c = 17.386(4) Å, α = 90°, β = 90°, γ = 90°, V = 943.0(3) Å3, Z = 4, dm = 1.227 Mg m−3, K = 198, R1 = 0.0676 and wR2 = 0.1915 for 1659 data with I > 2σ(I). Crystal data of compound 2: C7H15N5O3·H2O, Mw = 235.25, P1 21 1, a = 7.5481(2) Å, b = 8.9419(3) Å, c = 7.9881(2) Å, α = 90°, β = 94.066(2)°, γ = 90°, V = 537.8(3) Å3, Z = 2, dm = 1.453 Mg m−3, K = 100, R1 = 0.0314 and wR2 = 0.0800 for 1819 data with I > 2σ(I). The data were submitted at the Cambridge Crystallographic Data Centre with CCDC reference 1871263 and 1886141, respectively. Culture Preparations. We took 2.5 g of LB medium (casein enzymic hydrolysate, 10 g L−1; yeast extract, 2 g L−1; sodium chloride, 10 g L−1) and 2 g of dextrose in 100 mL of H2O and autoclaved it for 20 min in 15 lbs, 121 °C, 38 °C, 225 rpm, pH = 6.8. EtOH Measuring Methods. EtOH was measured through NMR experiment. Yeast growth was performed in a sealed NMR tube using an NMR reference and the production of EtOH was measured with time.

■

Debasish Haldar: 0000-0002-7983-4272 Author Contributions

S.S. synthesized and analyzed the compounds. S.R.C. and D.P. performed the experimental works. D.H. carried out the analysis and wrote the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS S.S. acknowledges IISER Kolkata for fellowship. D.P. and S.R.C. acknowledge CSIR, India, for research fellowship.

■

(1) Dudley, B. Global Primary Energy Consumption Grew Strongly in 2017, Led by Natural Gas and Renewables, with Coal’s Share of the Energy Mix Continuing to Decline; BP Statistical Review of World Energy, 2018, pp 1−53. (2) Caetano, N. S.; Mata, T. M.; Martins, A. A.; Felgueiras, M. C. New trends in energy production and utilization. Energy Procedia 2017, 107, 7−14. (3) Gallezot, P. Conversion of biomass to selected chemical products. Chem. Soc. Rev. 2012, 41, 1538−1558. (4) Huber, G. W.; Iborra, S.; Corma, A. Synthesis of Transportation Fuels from Biomass: Chemistry, Catalysts, and Engineering. Chem. Rev. 2006, 106, 4044−4098. (5) Corma, A.; Iborra, S.; Velty, A. Chemical Routes for the Transformation of Biomass into Chemicals. Chem. Rev. 2007, 107, 2411−2502. (6) Chheda, J. N.; Huber, G. W.; Dumesic, J. A. Liquid-phase catalytic processing of biomass-derived oxygenated hydrocarbons to fuels and chemicals. Angew. Chem., Int. Ed. 2007, 46, 7164−7183. (7) Climent, M. J.; Corma, A.; Iborra, S. Converting carbohydrates to bulk chemicals and fine chemicals over heterogeneous catalysts. Green Chem. 2011, 13, 520−540. (8) Anastas, P.; Eghbali, N. Green Chemistry: Principles and Practice. Chem. Soc. Rev. 2010, 39, 301−312. (9) Voss, B.; Andersen, S. I.; Taarning, E.; Christensen, C. H. C factors pinpoint resource utilization in chemical industrial processes. ChemSusChem 2009, 2, 1152−1162. (10) Danner, H.; Braun, R. Biotechnology for the production of commodity chemicals from biomass. Chem. Soc. Rev. 1999, 28, 395− 405. (11) Balat, M.; Balat, H.; Ö z, C. Progress in bioethanol processing. Prog. Energy Combust. Sci. 2008, 34, 551−573. (12) Arapoglou, D.; Varzakas, T.; Vlyssides, A.; Israilides, C. Ethanol production from potato peel waste (PPW). Waste Manag. 2010, 30, 1898−1902. (13) Zhao, Y.; Damgaardb, A.; Christensen, T. H. Bioethanol from corn stovera review and technical assessment of alternative biotechnologies. Prog. Energy Combust. Sci. 2018, 67, 275−291. (14) Woodson, M.; Jablonowski, C. J. An economic assessment of traditional and cellulosic ethanol technologies. Energy Sources, Part B 2008, 3, 372−383. (15) Sánchez, O. J.; Cardona, A. C. Trends in biotechnological production of fuel ethanol from different feedstock. Bioresour. Technol. 2008, 99, 5270−5295. (16) Lichtenthaler, F. W.; Peters, S. Carbohydrates as green raw materials for the chemical industry. C. R. Chim. 2004, 7, 65−90. (17) Sun, Y.; Lu, X.; Zhang, S.; et al. Intensification of Cellulose Hydrolysis Process by Supercritical Water. Obtaining of Added Value Products. Bioresour. Technol. 2011, 102, 2936−2942. (18) Bondesson, P.; Galbe, M.; Zacchi, G. Comparison of energy potentials from combined ethanol and methane production using steam-pretreated corn stover impregnated with acetic acid. Biomass Bioenergy 2014, 67, 413−424.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01260. Crystallographic data for UARG (CIF) Crystallographic data for UL_DMSO_THF_1 (CIF) 1 H NMR, 13C NMR, FTIR, and mass spectra; hydrogenbonding parameters; concentration optimization; kinetics study of EtOH production by yeast; and torsional angle parameter (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. G

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX

ACS Omega

Article

(19) Barakat, A.; Monlau, F.; Steyer, J. P.; Carrere, H. Effect of lignin-derived and furan compounds found in lignocellulosichydrolysates on biomethane production. Bioresour. Technol. 2012, 104, 90− 99. (20) Chen, L.; Chen, R.; Fu, S. FeCl3 Pretreatment of Three Lignocellulosic Biomass for Ethanol Production. ACS Sustainable Chem. Eng. 2015, 3, 1794−1800. (21) Chaturvedi, V.; Verma, P. An overview of key pretreatment processes employed for bioconversion of lignocellulosic biomass into biofuels and value added products. 3 Biotech 2013, 3, 415−431. (22) Debnath, M.; Sasmal, S.; Haldar, D. Fabrication of egg shell like nanovesicles from thiocoumarin based e-amino ester: a potential carrier. J. Mater. Chem. B 2017, 5, 5450−5457. (23) Paikar, A.; Debnath, M.; Podder, D.; Sasmal, S.; Haldar, D. Synthesis and structural investigation of 2-aminomethyl-3-(4methoxy-phenyl)-propionic acid containing a peptide analogue of the amyloidogenic AS(6−7) sequence: inhibition of fibril formation. Org. Biomol. Chem. 2017, 15, 4218−4225. (24) Maity, S.; Kumar, R.; Maity, S. K.; Jana, P.; Bera, S.; Haldar, D. Synthesis and study of 2-Acetyl amino-3-[4-(2-amino-5-sulfo-phenylazo)-phenyl]-propionic acid: A new class of inhibitor for HEWL amyloidogenesis. MedChemComm. 2013, 4, 530−536. (25) Hooper, A. E.; Kennedy, S. R.; Jones, C. D.; Steed, J. W. Gelation by supramolecular dimerization of mono(urea)s. Chem. Commun. 2016, 52, 198−201. (26) Hecht, S.; Huc, I. Foldamers: Structure, Properties, and Applications; Wiley-VCH, Weinheim, 2007. (27) Nickerson, D. M.; Angeles, V. V.; Auvil, T. J.; Sonia, S. S.; Mattson, A. E. Internal Lewis acid assisted ureas: tunable hydrogen bond donor catalysts. Chem. Commun. 2013, 49, 4289−4291. (28) Sharma, R.; Vohra, R. M. Microbial transformations: Production of D-amino acids using hydantoinase. Curr. Sci. 1999, 77, 127−136. (29) Gallezot, P. Alternative value chains for biomass conversion to chemicals. Top. Catal. 2010, 53, 1209−1213. (30) Gallezot, P. Direct routes from biomass to end-products. Catal. Today 2011, 167, 31−36. (31) Outten, C. E.; Falk, R. L.; Culotta, V. C. Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae. Biochem. J. 2005, 388, 93−101. (32) Sheldrick, G. M. SHELXL-2014/7, Program for the Refinement of Crystal Structures; University Göttingen: Göttingen, Germany, 2014.

H

DOI: 10.1021/acsomega.9b01260 ACS Omega XXXX, XXX, XXX−XXX