Prunus Armeniaca Hydroxynitrile Lyase (ParHNL) Catalyzed Asymmetric Synthesis of δ,ε-Unsaturated Cyanohydrins

Abstract

Hydroxynitrile lyases (HNL) are one of the key enzymes in cyanogenic plants, catalyzing the final step in the biodegradation pathway of cyanogenic glycosides releasing HCN and the corresponding carbonyl components. We have been able to find some new plant HNL from drupe available in the northern part of the ­Indian subcontinent. Asymmetric cyanohydrin synthesis from γ,δ-unsaturated aldehydes by applying those new HNL is reported in this communication.

Key Words

hydroxynitrile lyase - cyanohydrins - asymmetric synthesis

References and Notes

• 1a Kobler C. Bohrer A. Effenberger F. Tetrahedron  2004,  60:  10397 
• 1b Solís A. Luna H. Manjarrez N. Pérez HI. Tetrahedron  2004,  60:  10427 
• 1c Avi M. Fechter MH. Belaj F. Pöchlauer P. Griengl H. Tetrahedron  2004,  60:  10411 
• 1d Kobler C. Effenberger F. Tetrahedron: Asymmetry  2004,  15:  3731 
• 1e Han S. Chen P. Lin G. Huang H. Li Z. Tetrahedron: Asymmetry  2001,  12:  843 
• 1f Cruz Silva MM. Melo ML. Parolin M. Tessaro D. Riva S. Danieli B. Tetrahedron: Asymmetry  2004,  15:  21 
• 1g Li N. Zong M.-H. Peng H.-S. Wu H.-C. Liu C. J. Mol. Catal. B: Enzym.  2003,  22:  7 
• 1h Li N. Zong M.-H. Liu C. Peng H.-S. Wu H.-C. Biotechnol. Lett.  2003,  25:  219 
• 1i Fröhlich RFG. Zabelinskaja-Mackova AA. Fechter MH. Griengl H. Tetrahedron: Asymmetry  2003,  14:  355 
• 1j Hernández L. Luna H. Ruíz-Terán F. Vázquez A. J. Mol. Catal. B: Enzym.  2004,  30:  105 
• 1k Cyanide in Biology   Vennesland B. Conn EE. Knowels CJ. Westley J. Wissing F. Academic Press; New York: 1981. 
• 2 Kruse CG. Chiral Cyanohydrins - Their Manufacture and Utility as Chiral Building Blocks, In Chirality in Industry (The Commercial Manufacture and Applications of Optically Active Compounds)   Collins AN. Sheldrake GN. Crosby J. John Wiley and Sons; New York: 1992.  Chap. 14. p.279-299  
• 3a Garcia-Urdiales E. Alfonso I. Gotor V. Chem. Rev.  2005,  105:  313 
• 3b Gregory RJH. Chem. Rev.  1999,  99:  3649 
• 3c North M. Tetrahedron: Asymmetry  2003,  14:  147 
• 4a Smitskamp-Wilms E. Brussee J. Van der Gen A. van Scharrenburg GJM. Sloothaak JB. Recl. Trav. Chim. Pays-Bas  1991,  110:  209 
• 4b Gerstner E. Kiel UZ. Physiol. Chem.  1975,  356:  1853 
• 4c Xu L.-L. Singh BK. Conn EE. Arch. Biochim. Biophys.  1986,  250:  322 
• 4d Yemm RS. Poulton JE. Arch. Biochim. Biophys.  1986,  247:  440 
• 4e Albrecht J. Jansen I. Kula M.-R. Biotechnol. Appl. Biochem.  1993,  17:  191 
• 4f Wajant H. Förster S. Selmar D. Effenberger F. Pfizenmaier K. Plant Physiol.  1995,  109:  1231 
• 4g Wajant H. Mundry K.-W. Plant. Sci.  1993,  89:  127 
• 4h Bove C. Conn EE.
  J. Biol. Chem.  1961,  236:  207 
• 4i Kuroki GW. Conn EE. Proc. Natl. Acad. Sci. U.S.A.  1989,  86:  6978 
• 4j Wajant H. Förster S. Bottinger H. Effenberger F. Pfizenmaier K. Plant. Sci.  1995,  108:  1 
• 4k Hughes J. Lakey JH. Hughes MA. Arch. Biochim. Biophys.  1994,  311:  496 
• 5a Nanda S. Kato Y. Asano Y. Tetrahedron: Asymmetry  2006,  17:  735 
• 5b Forster S. Roos J. Effenberger F. Wajant H. Sprauer A. Angew. Chem., Int. Ed. Engl.  1996,  35:  437 
• 5c Trummler K. Roos J. Schwaneberg U. Effenberger F. Forster S. Pfizenmaier K. Wajant H. Plant Sci. (Shannon, Irel.)  1998,  139:  19 
• 5d Ognyanov VI. Datcheva VK. Kyler KS. J. Am. Chem. Soc.  1991,  113:  6992 
• 5e Griengl H. Klempier N. Pochlauer P. Schmidt M. Shi N. Zabelinskaja-Mackova AA. Tetrahedron  1998,  54:  14477 
• 5f Klempier N. Pichler U. Griengl H. Tetrahedron: Asymmetry  1995,  6:  845 
• 5g Warmerdam EGJC. van den Nieuwendijk AM CH. Kruse CG. Brussee J. van der Gen A. Recl. Trav. Chim. Pays-Bas  1996,  115:  20 
• 6 Marcus J. Johannes B. Van der G en A. Eur. J. Org. Chem.  1998,  2513 
• 7 Srikrishna A. Nagaraju S. Kondaiah P. Tetrahedron  1995,  51:  1809 
• 8 Mancuso AJ. Brownfain DS. Swern D. J. Org. Chem.  1979,  44:  4148 
• 9 Nanda S. Kato Y. Asano Y. Tetrahedron  2005,  61:  10908 
• 10 Avi M. Fechter MH. Gruber K. Belaj F. Pochlauer P. Griengl H. Tetrahedron  2004,  60:  10411 

11

Enzyme Extraction from Prunus armeniaca (Shakarpara Spricot) Ripened fruits were taken and the fleshy cover was removed to obtain the seeds. The upper layers of the seeds were cracked with a hammer to obtain the soft kernels. The kernels were homogenized at 4 ˚C, with aq K₃PO₄ buffer (10 mM, pH = 6.0), to give a milky suspension. The suspension was filtered through four layers of cheese cloth to remove the insoluble part. After that it was centrifuged (18800 g, 30 min), and removal of the residue gave a crude preparation of HNL. The crude preparation was fractionated with (NH₄)₂SO₄. Proteins precipitating with 30% saturation were collected by centrifugation (18800 g, 20 min), dissolved in the minimum volume of phosphate buffer and dialyzed against the same buffer with three changes. The dialyzed soln was then centrifuged and the supernatant was stored at 4 ˚C and assayed for HNL activity.
ParHNL Assay In a typical assay reaction 1.0 M of benzaldehyde soln (in DMSO, 40 µL) was dissolved in 400 mM citrate buffer (760 µL, pH = 4.0), followed by addition of enzyme soln (100 µL) and 1.0 M NaCN soln (100 µL, total reaction volume 1 mL), and the reaction mixture was incubated in a rotary shaker. After 5 min, the 100 µL of the reaction mixture was removed and extracted with 900 µL hexane-2-PrOH (9:1), the organic layer was analyzed with chiral HPLC for the formation of (R)-mandelonitrile. A blank reaction was also performed without enzyme, and the amount of mandelonitrile obtained was deducted from the biocatalyzed reaction product. One unit of the enzyme is defined as the amount of the enzyme that produces 1 mmol of (R)-mandelonitrile under the above reaction conditions in 1 min. The protein content in all the HNL was measured by the Bradford method using a Bio-Rad protein assay kit with BSA as the standard.
General Procedure for the Synthesis of δ,ε-Unsaturated Cyanohydrins by ParHNL
To a soln of γ,δ-unsaturated aldehyde 1-9 in DIPE, a soln of ParHNL (300 IU/mmol of aldehyde, DIPE/enzyme; 1:1 v/v) was added, and the resulting mixture was stirred vigorously until an emulsion was formed (the pH of the enzyme soln having been previously adjusted to 4.0 with 10% citric acid soln). Freshly prepared HCN in DIPE (2 equiv) was added to the mixture, and the temperature was kept at 10 ˚C. After completion of the reaction it was extracted thoroughly with Et₂O several times, and the organic layer was dried (Na₂SO₄). Evaporation of the solvent yielded the crude cyanohydrins, which were purified by chromatography.
Preparation of HCN in DIPE
Sodium cyanide (10 g) and citric acid (0.1 g) were dissolved in H₂O (100 mL). The soln was cooled in an ice-water bath and extracted with DIPE (50 mL), while acidifying with 33% HCl until pH 5.5. The H₂O layer, which contained a suspension of NaCl, was extracted twice with DIPE (25 mL). The combined DIPE layers were stored in a dark bottle. The above procedure must be performed in a well-ventilated fume hood and impermeable gloves must be worn.

12

Cyanohydrin from 2 ^¹H NMR (400 MHz, CDCl₃): δ = 5.8 (dd, J = 17.7, 10.6 Hz, 1 H), 5.1 (dd, J = 10.6, 1.4 Hz, 1 H), 5.00 (dd, J = 17.7, 1.4 Hz, 1 H), 4.48 (t, J = 7.6 Hz, 1 H), 1.88 (m, 2 H), 1.1 (s, 6 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 121.8, 119.4, 113.4, 59.8, 48.7, 36.5, 25.8, 24.6. [α]_D ^²7 +8.8 (c 1.0, CHCl₃).
Cyanohydrin from 3 ^¹H NMR (400 MHz, CDCl₃): δ = 5.7 (dd, J = 17.6, 10.6 Hz, 1 H), 5.22 (dd, J = 10.6, 1.4 Hz, 1 H), 5.00 (dd, J = 17.6, 1.4 Hz, 1 H), 4.49 (t, J = 7.6 Hz, 1 H), 1.99 (m, 2 H), 1.45 (m, 4 H), 0.86 (m, 6 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 145.1, 120.5, 114.7, 58.6, 41.9, 27.67, 27.0, 7.5. [α]_D ^²7 +4.4 (c 1.0, CHCl₃).
Cyanohydrin from 4 ^¹H NMR (400 MHz, CDCl₃,): δ = 7.50-7.10 (m, 10 H), 6.6 (dd, J = 17.6, 10.6 Hz, 1 H), 5.32 (dd, J = 10.6, 1.4 Hz, 1 H), 4.98 (dd, J = 17.6, 1.4 Hz, 1 H), 4.42 (t, J = 7.6 Hz, 1 H), 2.80 (m, 2 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 145.0, 144.7, 143.2, 128.6, 128.5, 128.2, 127.7, 127.6, 119.9, 114.5, 59.3, 52.64, 44.8. [α]_D ^²7 +10.5 (c 0.5, CHCl₃).
Cyanohydrin from 5 ^¹H NMR (400 MHz, CDCl₃): δ = 5.16 (t, J = 6.4 Hz, 1 H), 4.46 (t, J = 7.6 Hz, 1 H), 2.2 (m, 2 H), 1.85 (m, 2 H), 1.75 (s, 3 H), 1.7 (s, 3 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 134.6, 123.2, 119.4, 61.6, 36.7, 27.6, 23.4, 18.8. [α]_D ^²7 +2.1 (c 1.0, CHCl₃).
Cyanohydrin from 6 ^¹H NMR (400 MHz, CDCl₃): δ = 5.75 (dd, J = 17.4, 10.6 Hz, 1 H), 5.20-5.08 (m, 2 H), 4.45 (t, J = 6.8 Hz, 1 H), 2.01 (d, J = 6.8 Hz, 2 H), 1.80-1.50 (m, 8 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 143.7, 120.7, 113.9, 59.3, 47.8, 45.4, 37.0, 36.7, 23.0, 22.9. [α]_D ^²7 +6.4 (c 0.8, CHCl₃).
Cyanohydrin from 7 ^¹H NMR (400 MHz, CDCl₃): δ = 5.66 (dd, J = 17.4, 10.6 Hz, 1 H), 5.20 (d, J = 10.6 Hz, 1 H), 5.08 (d, J = 17.4 Hz, 1 H), 4.48 (t, J = 7.2 Hz, 1 H), 1.9 (d, J = 7.2 Hz, 2 H), 1.70-1.46 (m, 10 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 144.7, 120.8, 115.2, 58.5, 46.3, 39.2, 36.0, 35.5, 26.1, 21.9, 21.8. [α]_D ^²7 +4.8 (c 1.2, CHCl₃).
Cyanohydrin from 8 ^¹H NMR (400 MHz, CDCl₃): δ = 5.75 (dd, J = 17.4, 10.6 Hz, 1 H), 5.2 (dd, J = 10.6, 1.4 Hz, 1 H), 5.05 (dd, J = 17.6, 1.4 Hz, 1 H), 4.48 (m, 1 H), 1.9 (m, 2 H), 1.70-1.40 (m, 12 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 146.1, 120.7, 113.4, 58.8, 46.9, 42.3, 38.0, 37.2, 30.0, 22.3. [α]_D ^²7 +22.2 (c 0.6, CHCl₃).
Cyanohydrin from 9 ^¹H NMR (400 MHz, CDCl₃): δ = 5.75 (dd, J = 17.4, 10.6 Hz, 1 H), 5.15 (dd, J = 10.6, 1.4 Hz, 1 H), 5.00 (dd, J = 17.6, 1.4 Hz, 1 H), 4.48 (m, 1 H), 2.0 (m, 2 H), 1.60-1.20 (m, 9 H), 0.90 (d, J = 7.6 Hz, 3 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 147.2, 120.5, 112.9, 58.8, 40.4, 38.1, 34.6, 33.9, 31.9, 29.9, 29.8, 21.9. [α]_D ^²7 +11.5 (c 1.2, CHCl₃).