Non-aqueousenzymology Jonathan S. Dordick U n i v e r s i t y o f I o w a , I o w a City, I o w a , U S A Compelling evidence has been obtained during the past year that enzymes retain their native active-site structure in organic solvents, and yet the properties of the solvent significantly affect enzyme kinetics. Fundamental advances in enzymatic catalysis in monophasic organic media are discussed and selected applications in the areas of asymmetric, polymer and chemoenzymatic syntheses are highlighted. Current Opinion in Biotechnology 1991, 2:401-407 Introduction
Enzymes occupy a unique position in synthetic chemistry. They are highly respected catalysts for their exquisite selectivities under mild reaction conditions (e.g. ambient temperatures and pressures, neutral solutions). Nevertheless, synthetic chemists have been reluctant to employ enzymes as reagents in organic synthesis, although there is certainly a need for high chemical selectivity in synthetic chemistry, especially in the synthesis of pharmaceuticals, chiral intermediates, specialty polymers and biochemicals. The main reason for this reluctance has been the strict adherence by both chemists and biochemists to the conventional notion that enzymes function only in aqueous solutions. Indeed, it is stated in virtually every biochemistry textbook that enzymes are aqueous-based and require water for catalysis. This is unfortunate because, although water is an ideal solvent for the predominantly polar species required for life (e.g. amino and nucleic acids, carbohydrates, cofactors and proteins), it is a poor solvent for nearly all applications in industrial chemistry. Most organic compounds of commercial interest are spa.tingly soluble or chemically unstable in water, and most synthetic reactions take advantage of thermodynamic equilibria that are unfavorable in aqueous media. Recently, it has become clear that enzymes can function in organic as well as aqueous media [1-3]. The astonishing realization that enzymes can retain their high degree of reaction specificity in a nearly anhydrous milieu has improved dramatically the prospect of employing enzymes in synthetic chemistry. Synthetic chemists are now more likely to include enzymes in their catalytic repertoire and for good reason. From a biotechnologica] standpoint, there are numerous potential advantages to be gained from employing enzymes in organic media (Table 1) [1-3]. These potential advantages have been the driving force behind the development of numerous strategies for carrying out enzymatic reactions in organic media including biphasic, aqueous-organic reaction me-
dia, reverse micelle-containing media and monophasic reaction media [4,5]. Only in the last example, however, is the enzyme in a predominantly non-aqueous environment. Because of this, all of the potential advantages listed in Table 1 have been realized. Table 1. The potential advantages of employing enzymes in
monophasic organic solvents [1,2,6.].
1. Increasedsolubility of non-polar substrates. 2. Shifting thermodynamic equilibria to favor synthesisover hydrolysis. "3. Suppressionof water-dependent side reactions. 4. Alteration in substrateand enantioselectivity. 5. Immobilizationis often unnecessarybecauseenzymesare insoluble in organic solvents. 6. Enzymescan be reco'veredby simple filtration. 7. Easeof product recovery from low boiling, high vapor pressure solvents. 8. Enhanced thermostability. 9. Eliminationof microbial contamination. 10. Potential for enzymesto be used directly within a new or existing chemical process.
This brief review highlights research carried out in the past 13 months (since December 1989) in the specific area of monophasic organic enzymology. Earlier references are included for completeness only. The reviewwill
Abbreviations
CCL--Candida cylindracea lipase; DMSO~dimethylsulfoxide; ESR---electron spin resonance; MAS-~magic angle spinning; NMR--nuclear magnetic resonance. (~) Current Biology Ltd ISSN 0958-1669
401
402
Biochemicalengineering focus mainly on the fundamentals of catalysis in organic solvents, including the effects of organic solvents on enzyme structure and function. Selected applications that have been developed in the areas of asymmetric catalysis, polymer synthesis, and chemoenzymatic synthesis (the last example taking advantage of the high selectivity of enzymatic catalysis coupled with the high reactivity and flexibility of chemical synthesis), will be summarized in a brief applications section. The reader is referred to several recently published reviews that deal with general monophasic organic enzymologT [1,6,] or with specific synthetic applications [7"*,8,9*].
Fundamentals of enzymes in monophasic organic media Prior to 1989, the field of non-aqueous enzymology was mainly phenomenological. A large number of novel enzymic reactions had been examined, yet very few studies were commenced to elucidate the effect of the organic solvent on enzyme structure and function. Nevertheless, the following vitally important information was gleaned from these earlier studies [1,2,6.]. First, water bound to the enzyme is vital for enzyme function and stripping this enzyme-bound water leads to inactivation of the enzyme. Second, Michaelis-Menten kinetics are observed in organic media. Third, for highly active enzymes, intraparticle diffusional limitations can be overcome by immobflizing the enzyme as a monolayer onto a solid support. Fourth, enzyme activity can be increased substantially by lyophilizing the enzyme from an aqueous solution at the optimal catalytic pH and, in some cases, by lyophilizing the enzyme in the presence of an active-site inhibitor. Presumably the former results in a solid enzyme preparation with the proper ionogenic state of the charged functional groups of the enzyme, and the latter produces a highly rigid active site that is 'molded' into the proper conformation for catalysis to take place. Fifth, enzymes are highly rigid in nearly anhydrous organic media. This reduces their ability to accommodate bulky substrates. Finally, enzymes are more thermostable in nearly anhydons media. For a full description of these phenomena, the reader is directed to [1,2,6.] and citations therein.
Solvent effects on enzyme structure As a result of three separate studies [10"*,11o*,12.*], it has become clear that organic solvents that sustain catalytic activity do not substantially alter the active-site structure of an enzyme. For example, solid-state magic angle spinning (MAS) 15N nuclear magnetic resonance (N/VlR) studies of ~-lytic protease (a serine protease) enriched with 15N at the active site histidine revealed similar tautomeric structures and hydrogen-bonding interac-
tions for the enzyme in acetone, octane and water [10oo]. These results indicate that the catalytic triad of the enzyme's active site is intact in the organic solvent and is similar to that in water. In dimethylsulfoxide (DMSO), a solvent that does not sustain enzymatic catalysis, the catalytic triad structure is perturbed. The kinetic isotope study performed by Klibanov et a t [11 °'] supports further the maintenance of the catalytic triad in serine proteases. Virtually no difference in the magnitude of the primary deuterium isotope effect is observed in different organic solvents, suggesting that the transition state structure of the enzymatic process is independent of the nature of the reaction medium. These findings show, for the first time, that enzyme function in organic solvents is a result of the native catalytic machinery of the enzyme. In agreement with 15N NMR results for wlytic protease, electron spin resonance (ESR) analysis of an unrelated enzyme, horse liver alcohol dehydrogenase, showed that the enzyme did not denature when suspended in nearly anhydrous organic solvents [12°°], but became substantially more rigid. Addition of water to the solvent did result in an increase in the active-site spin-label flexibility, although increased catalytic activity was not obtained. The increase in protein flexibility with higher solvent water content is consistent with observed decreases in the thermostability of enzymes in partially hydrated organic media. For example, ATPase is highly stable at 58"C in toluene containing 0.32% v/v water, with a halflife of 74 min. In aqueous buffer, the half-life of the enzyme drops to 2 min [13"]. The rigidity of enzymes in low-water environments is directly responsible for the activating effect of activesite ligands (inhibitors) during lyophilization. This phenomenon is known as ligand memory [14]. The ligand binds to the active site of an enzyme, makes a structural imprint in the active site, and is washed away from the enzyme prior to addition of substrate. The molecular imprint of the active-site ligand remains and provides a 'frozen' conformation that readily accepts the substrate structure leading to enhanced activity on the given substrate. In addition to enhancing the enzymatic activity on natural substrates, this molecular imprinting technique Can be used as a method to alter dramatically the substrate specificity of enzymatic catalysis. For exampie, Mosbach and coworkers [15"] found that precipitation of chymotrypsin with n-propanol in the presence of N-acetyl-D-tryptophan (chymotrypsin is normally highly selective to the L-isomer) enabled the enzyme to catalyze the esterification of N-acetyl-D-tryptophan ethyl ester in cyclohexane. Enzyme precipitation in the absence of the ligand or in the presence of the L-isomer did not result in esterification of the D-isomer. Molecular imprinting has also been carried out on inert proteins to convert them into abiotic receptors [16"]. Such imprinted proteins have relatively high affinities toward the organic compound used as a template and structurally similar compounds, in nearly anhydrous solvents. The phenomenon of molecular imprinting has been suggested as a simple approach to the development
Non-aqueous enzymology Dordick
of bioadsorbents or novel biocatalysts. The enhanced rigidity of proteins in organic solvents is responsible for the molecular imprinting phenomenon. Addition of water to the solvent, which causes an increase in the flexibility of the protein in the organic solvent, results in the relaxation of the imprinting phenomenon.
Solvent effects on enzyme function Substrate specificity and catalytic efficiency depend on the ability of the-enzyme to use the free energy of binding with the substrate [17]. This binding energy reflects the difference between binding energies of substrate-enzyme and substrate-solvent interactions [18]. Thus, kinetic parameters describing enzyme function, such as binding constant with substrate, Ks, Michaelis constant, Kin, and catalytic turnover, kcat or Vmax, depend strongly on the solvent. It might be expected that the replacement of water with an organic solvent would lead to profound changes in the observed kinetics of enzymatic catalysis. A profound effect of the organic solvent is illustrated by the dramatic loss in"catalytic efficiency (kcat/Km) observed in low-water environments
[1,6,1. One hypothesis regarding the decrease in catalytic activity in organic media, compared with water, is that partitioning of the substrate from the enzyme's active site to the hydrophobic organic solvent occurs, thereby lowering the binding affinityof substrate with enzyme, while increasing the affinity of substrate with solvent. Our group has tested such a hypothesis using linear free energy relationships with peroxidase catalysis in non-aqueous media [19°',20]. Phenolic substrates that differed only in the hydrophobicity of the alkyl substituent groups were reacted in a variety of water-miscible and immiscible organic solvents. Hydrophobic substrates were found to be less reactive in an organic solvent than in water. Furthermore, as both the substrate and solvent hydrophobicities increased, the reactivities of the phenols decreased. These findings are consistent with the partitioning behavior of hydrophobic phenols between the bulk reaction medium and the peroxidase's active site. This partitioning is likely to diminish as substrate and solvent hydrophobicities increase, thereby necessitating a larger concentration of a given phenol to saturate the enzyme. This results in a higher apparent substrate, Kin, in organic versus aqueous media and a lower catalytic efficiency. Substrate and solvent hydrophobicities also affect the regio- and enantio-selectivity of lipase catalysis. Specifically, the Pseudomonas fluorescens lipase-catalyzed transesterification of 1,4-dibutyryloxy-2-octylbenzene with n-butanol in a hydrophobic solvent such as toluene, results in the formation of the 4-butyryloxy-3-octylphenol, the sterically favored reaction [21.]. However, the re-
gioselectivity of lipase catalysis is reversed in acetonitrile wherein the sterically unfavored 4-butyryloxy-2-octylphenol is produced. The hydrophobicity of the solvent controls the regioselectivity of catalysis; the more hydrophobic the solvent, the more favored the formation of 4-butyryloxy-3-octylphenol. Furthermore, replacing the octyl group with a methyl moiety results in the formation of the 3-methylphenol product, even in acetonitrile. These results suggest that, in hydrophilic solvents, the octyi moiety can favorably partition into a hydrophobic cleft in the lipase's active site that positions the transacylation reaction to produce 4-butyrytoxy-2-octylphenol. In more hydrophobic media, the free energy of partitioning of the octyl group into the hydrophobic active site is less favorable and the sterically favored reaction pathway ensues. Enantioselectivity of lipase catalysis is also affected by the nature of the substrate and solvent. The lipase from Candida cylindracea (CCL) catalyzes the enantioselecfive esterification of 2-hydroxyacids with primary alcohols in nearly anhydrous solvents [22.]. Straight-chain 2-hydroxy- acids are esterified with nearly 100 % enantioselectivity in toluene to give the S-isomer. However, the more hydrophilic the solvent, and also the more hydrophobic, the lower the enantioselectivity of CCL catalysis. The loss in enantioselectivity in hydrophilic solvents is caused by a dramatic drop in the catalytic efficiency of the S-isomer, while that of the R-isomer remains unchanged. In solvents more hydrophobic than toluene (e.g. cyclohexane), the reverse is true and the catalytic efficiency of the R-isomer increases relative to that of the S-isomer. This shift may be a consequence of the cyclohexane being more hydrophobic than the enzyme's active site. In addition to solvent hydrophobicity, the polarity of the reaction medium has a major influence on the enantioselectivity [23"]. For example, subtilisin-catalyzed transesterification between seophenethanol and vinyl butyrate showed greatest enantioselectivity in less polar solvents. The correlation between enantioselectivity and dipole moment or dielectric constant was far greater than that observed between enantioselectivity and solvent hydrophobicity. These results suggest that reversal of enzyme enantioselectivity may be controlled by the nature of the organic solvent on enzyme kinetics rather than on enzyme structure.
Applications of enzymes in monophasic organic media The advantages of performing enzymatic catalysis in nonaqueous media (Table 1) have led to a variety of potential commercial applications. This section summarizes the major advances made during the past year in the areas of asymmetric, polymer and chemoenzymatic synthesis. Specifics concerning some of the more seminal individual syntheses can be found in the annotated references at the end of this review.
403
404
Biochemical engineering
CHzOH
CHzOH
OH
o
~ O
o
+ H2OH
OH
o
n . CF~CH20-CI[CHz-]4C-OCHzCF3
OH Fig. 1. Enzymatic polymerization of sucrose to poly(sucrose adipate) in anhydrous pyridine. An alkaline protease catalyzes the polycondensation of sucrose with bis(2,2,2-trifluoroethyladipate) to
~ Enzyme Pyridine 45"C O II
O II
Lf-C-iCH2F4C-O O-CHz O~
OO-~H CH2OOCHO2OH ] n
polymers in excess of 13000D. The regioselectivity of the enzyme recognizes sucrose as a diol rather than a molecule with eight free hydroxyl groups, and linkages form at the 1' and 6 positions of the sugar. (See [38,'] for details).
Asymmetric syntheses
Chemoenzymalic syntheses
Nowhere is the power of enzymatic catalysis more evident than in asymmetric synthesis, particularly in the reactions catalyzed by hydrolases such as lipases and proteases. For the purpose of this review, the term 'asymmetric' includes stereoselective as well as regioselective syntheses. From a preparative perspective, the most important classes of reactions catalyzed by enzymes in asymmetric organic synthesis include site-selective acylation of polyols (e.g. sugars [24.,25,26,27], nucleosides [28] and alkaloids [29,30°], peptide synthesis [8,31%32%33], optical resolutions [34,35"'], lactone synthesis [36"'] and interesterifications [37].
Although enzymes are highly selective catalysts, their productivities and scope of reactivities do not often match those of their chemical counterparts. The potential to use enzymes in non-aqueous media enables enzymatic syntheses to be performed in media compatible with chemical syntheses and a number of combined enzyme/chemical processes have been developed. Chemoenzymatic syntheses are characterized by the high reaction selectivity afforded by enzymatic catalysis and the fast reaction rates and wide substrate specificities furnished by traditional chemical catalysts. Some examples include optically active 1,2-diol synthesis (Fig. 2), enantioselective synthesis of 3-hydroxypropionic acids [41] and production of optically active polyacrylates [42"].
Polymer syntheses Polymer synthesis has traditionally been outside the realm of the enzymologist. Most polymers are produced easily and cheaply using conventional chemical approaches and for the most part this has not changed. The use of enzymes in speciality polymer synthesis, however, does have several potential advantages including high regioselectivity over polyfunctional monomers (Fig. 1) [38"'], high stereoselectivity for use in the synthesis of chiral polymers [39"] and, potentially, a fine degree of control over pol~mer molecular weight and polydispersity, particularly in phenolic resins [40,P1.].
Fulure prospects Enzymatic catalysis in non-aqueous media is already having an impact on the way in which biochemists, chemists and engineers view applied enzymology. Biochemists are no longer limited to dilute aqueous solutions for biocatalysis. Chemists may now take advantage of enzymic specificity to catalyze reactions formerly limited to expensive and tedious chemical processes. Engineers can employ chemoenzymatic processes with the specific advan-
(a) CCL
(R,S)-R1CH(OH)COOH + R'OH
(S).RICH(OH)COOR, + (R)-RICH(OH)COOH + H20
(b) LiAI(OcH3)3H (S)-R~CH(OH)COOR'
I
>
(S)-R~CH(OH)CH2OH + R'OH
Fig. 2. General scheme for the chemoenzymatic synthesis of optically active diols using lipase from Candida cylindracea (CCL). (a) The first step in the reaction is the lipase-catalyzed esterification of a racemic 2-hydroxyacid. (b) The resulting S-ester is then reduced to give the 1,2-diol product. The following diols (all S-isomers) can be synthesized in this way: 1,2propanediol (R1 -- CH3); 1,2-butanediol (R1 = CH3CH2); 3-methyl-l,2-butanediol' [R1 = (CH3)2CH]; 1,2-hexanediol [R1 = CH3(CH2)3]; 4-methyl-l,2-pentanediol [R1 -= (CH3)2 CHCH2]; 3-phenyl-l,2-propanediol (R1 ----PhCH2).See [22"] for further details.
Non-aqueousenzymologyDordick rages of both enzyme and chemical catalysts. New reactions and processes are being developed at an accelerated pace. From a fundamental level, several questions remain to be addressed before a comprehensive understanding of non-aqueous enzylnology can be reached. For example, what is the effect of water on the activesite structure and function in enzymatic catalysis? How do the physicochemical properties of the solvent govern enzyme structure and kinetics? Can the effects of an orgarlic solvent on the catalytic efficiency and specificity of an enzyme b e predicted? Research addressing these questions is presently underway in a number of laboratories around the world,
MAS solid-state tSN NMR w~as run on tSN-enriched ct-bxic protca.se in wuter, octane, acetone and DMSO. In the first three soh-ents, the MAS spectra were nearly identical, suggesting that enz)anes in organic solvents maintain the same active-site structure as in x~uter. Only in a denaturing sob,~ent such as DMSO did perturbation of the MAS spectra result. This is the first direct indication that enz)ane active sites under non-denaturing conditions are not significantly pertubed by organic media. 11. ,.
ADAMSKAtt, CttUNG S-H, KLmANOVAM: Kinetic Isotope Effect Investigation of Enz)Tne Mechanism in Organic Solvents. J Am Omm Soc 1990, 112:9418-9419. For the first dme, a kinetic isotope investigation of an enz)xne is performed in nearly anhydrous media_ Deuterium isotope effects o f subtilism Carlsberg and a bacterial lipase showed no effect of soh'ent polarity on the ratio of vH/v D or HKm/DKm. 12. .,
have been highlighted as:
GUL'ZNRM, SKERKERPS, KAVANAGtlP, CLARKDS: Activity and Flexibility o f Alcohol Dehydrugenase in Organic Soh-cnts. Biotechnol Bioeng 1991, 37: in press. ESR studies of active-site spin-labeled alcohol dehydrogenase in anhydrous and partially hydrated organic media have been used for the first time to elucidate the clynamics of the enz)anic active site. The enz)~ne's active site w~ts found to become more rigid in less polar solvents with lower ~xuter contents, as determined by a decrease in the flexibility of the spin h b d .
• •,
of interest of outstanding interest
13. •
1.
DORDICKJS: Enz)anatic Catalysis in Monophasic Organic SOlvents. Enz3~ne Microb Technol 1989, 11:194-211.
2.
KLm&'~OV AM: Enzymatic Catalysis in Anhydrous Organic Solvents. Trends Biod.~m Sci 1989, 14:141-144.
3.
HAI~'~GPJ: Biocatalysis in l~ulti-Phase Reaction Mixtures Containing Organic Liquids. Biotedmol Adv 1987, 5:47-84.
4.
LqJ_V MD: Two-Liquid Phase Biocatalytic Reactions. J Cbem Tecb Biotechnol 1982, 32:162-169.
5.
M.ARTLNEKK, LEV&SHOVAV, KLYACItKO N, KHMEI.NITSKI YL, BERET_.LN IV: MiceUar E ~ o l o g y . Eur J Biod)em 1986, 155:453-468.
References and recommended reading Papers of special interest, published vdthin the annual period o f review,
6. •
DORDICKJS: Principles and Applications of Nonaqueous Enzymology. In Applied Biocatal3~'is edited by Blanch HW, Clark DS [book]. New York: Marcel Dekker 1991, 1:1-51. This re, Sew, with 190 references, covers the field of monophasic nonaqueous enz~xnology in depth up until 1989. The state o f fundamental knowledge of these enz}~ne s3~tems is described and put into perspective ~Sth the potential commercial applications of this emerging technology.
7. KtmANOVAM: Asymmetric Transformations Catal)-zed by Ene. zymes in Organic Solvents. Acc Clam Res 1990, 23:114-120. Highlights the practice and potential of the use of enzymes as as)mmetric catab~ts in nearly arthydrous orgamc media (92 references). 8.
KISE H, HAYAKAWAA, NORITOMI H: Protease-Catalyzed Synthetic Reactions and Immobilization-Activation o f Enzymes in Hydrophilic Organic Solvents. J Biotechnol 1990, 14:239-254.
9. .
PdVA S, SECtmqX) F: Selective Enzymatic Acylations and Deacylations o f Carbohydrates and Related Compounds. 03imicaoggi 1990, 8:9-16. A description, x~ith 38 references, of the use of enz)rnes as highly selective catalystsfor the modification of carbohydrates and their derivatives. A detailed summary o f enzyme-catalyzed sugar acTLations in organic solvents is presented from a chemist's perspective. 10. •o
BURKEPA, SMITHSO, BACHOVCHINWW, KtIBA.',;OV_42,1:Demonstration of Structural Integrity o f an Enzyme in Organic Solvents by Solid-State NMK J Am Chem Soc 1989, 111:8290-8291.
GARZA-RAMo5G, D ~ O N A, DE GOMEZ-PUYOU MT, GOMF.ZPOYOUA: Enz)ane Catalysis in Organic Soh-ents with Low Water Content at High Temperatures. The Adenosinetriphosphatase of Submitochondrial Particles. Biochemistry 1990, 29:751-757. Addition of x~ter to toluene activates ATPase and leads to reduced thermostability. When 3.8% (v/v) w~ter is added, the enz)rne exhibits saturation kinetics tow"ard Mg-ATP, with a Km of 0.3 mM (virtually the same as in ~ t e r ) , but a Vrra_xapproximately 100-fold lower than in ~uter. 14.
RUSSELLAJ, KLm.~XOVAM: Inhibitor-lnduced Enzyme Activation in Organic Solvents. J Biol Cl)em 1988, 263:11624-11626.
15. ,
STMILM, MANSSON1~,|-O, MOSBACH K: The Synthesis of a DAmino Acid Ester in an Organic Media with Alpha-Chymotrypsin Modified by a Bio-Imprinting Procedure. Biotecl> nol Lett 1990, 12:161-166. Freeze-drying en~Tnes in the presence of an ace-e-site ligand (first demonstrated by RusseU and Klibanov [14]) is used to reverse the selec~'ity of ch)Tnotr~psin to accept D-tt)ptophan substrates in anhydrous organic media. 16. •
B~CO k DABUUS K, Klm&'~OV AM: Production o f Abiotic Receptors by Molecular Imprinting of Proteins. Proc Natl Acad Sci USA 1990, 87:274-277. When a protein is dissoh'ed in a concentrated aqueous solution of an organic compound, freeze-dried, and washed ~ t h an organic st)gent to remove the organic compound (much as is done with ligand-acti~ t i o n of enz)Tnes in organic solventS), a molecular imprint results on the protein. Prexiously inert proteins, such as bo~Sne serum albumin can be converted into selective binding materials (adsorbents) in dry organic solvents. Addition o f even a small amount of water severely compromises the imprinting effectiveness. 17.
JENCKS WP (ED): Catalysis in Chemistry and Enz)maology [book]. New York:. McGraw-Hill 1969.
18.
FE~HT AR (ED): Enzyme Structure and Mechanism [book]. New York: \VII Freeman 1985.
19. ,,
RVIJ K, DORDICKJS: Free Energy Relationships o f Substrate and Solvent Hydrophobicities with Enzymatic Catalysis in Organic Media. J Am C79em Soc 1989, 111:8026-8027. Substrate and soh'ent h)xlrophobicities govern the catabxic efficiency of horseradish peroxidase in non-aqueous media. The primary effect o f a h)xtrophobic solvent is to increase the apparent substrate, Km. Linear free energy relationships suggest that ac~'ities of peroxidase in various organic media and xMth different substrates can be predicted on the basis of substrate and sob,'ent h)xlrophobicities alone. 20.
RYu K, DORDICK JS: Kinetic Behavior and Substrate Specificity of Horseradish Peroxidase in Water-Miscible Organic Solvents. Resources Conserv Rec)v:l 1990, 3:177-185.
405
406
Biochemical engineering 21. ,,
RUBIOE, FER,'O2,'DEZ-MAYORAt~A, KtmAxov AM: Effect of the Solvent on Enzyme Regioselectivity. J Am Cbem Soc 1991, 113:695--696. The hydrophobicity of the organic sob:ent affects the regioselectivity of lipase-catalyzed t ~ t e r i f i c a t i o n . The alteration in selec~'ity is caused by a drop in the free energy of partitioning of a hydrophobic side group of the substrate (the oct)t moiety of 1,4-dibut)a31oxy-2-oct)tbertzene) in more hydrophobic media, thereby favoring an altema~-e regioselectMty to that occurring in h)xtrophilic media. 22. •
PARIDAS, DORDXCKJS: Substrate Structure and Solvent Hydrophobicity Control Lipase Catalysis and Enantioselectivity in Organic Media. J A m Cbem Soc 1991, 113:2253-2259. CCL catab'zes the enantioselective estefification of 2-h)xlroxy acids in nearly anhydrous media. Straight-chain acids are highly reactive in toluene and give nearly 100% enantioselec~ities for the S-isomer. Branched acids, or any straight-chain acids in solvents less hydrophobic than toluene exhibit a loss in catabxic efficiency and a drop in enantioselec~ty, as the formation of the S isomer decreases and that of the R isomer remains constant. Sok'ents more hydrophobic than toluene also afford reduced enantioselectivities, and yet increased catablJc el~ciendes, primarily as a result of an increase in the reactivities of both the S and R isomers. The data tend to support the notion that the active site of CCL is as hydrophobic as toluene. This paper also describes a two-step chemoenzymatic wnthesis of optically active 1,2-diols from 2-hydroxy acids. 23. **
FITZPATR/CKPA, Ktm&'~ov AM: ttow can the Solvent Affcct Enzyme Enantioselectivity? J Am Ctoem Soc 1991, 113:3166-3171. Enantioselectivity of subtilisin Cadsberg in the transesterification between a chiral alcohol and vinyl but)rate is greatly affected by the polarity of the solvent. Correlations between enantioselectivity and solvent polarity (dielectric and dipole moment) am much greater than between enantioselec~ity and sok-ent hydrophobicity; the lower the polarity, the greater the enantioselectivity. Water, and w-ater mimics such as formamide, relax enantioselectivity in non-polar solvents such as dioxane, and yet do not affect significantly the enantioselectivity (already low) in polar soh'ents such as acetonitrile. 24. •
CIUFFREDAP, COLOMBO D, RONCttETI'I F, TO.XL~. I.: Regioselective Acylation of 6-Deoxy-L- and D-hexosides Through Lipase-Catalyzed Transesterilication. Enhanced Reactivity of the 4-OH Function in the L Series. J Org O?em 1990, 55:4187--4190. A number of commercially m'aJlable lipases are shoran to ac31ate bhexose monosaccharides at the relatively unreactive 4-OH position when the 6-OH position is blocked. Interestingly, D-sugars are acylated primar@ at the 2-OH or 3-OH positions. Such regiospecificity shifts allow the rational design of sugar acylation procedures to be implemented.
Castanosperrnine, a potent inhibitor of the endoplasmic reticulum enz3rne cz-glucosidase I, is modified by a novel three-step enzymatic synthesis culminating in the production of the 7-O-but)ayl defi~a~-e. The enz3anatic synthesis initially invok-es subtilisin to catab'ze the ac51afion of the 4-OH in pyridine, foUowed by a bacterial lipase (from Clam mobacterium viscosum) to catabxe the ac3~ationof the 7-OH in tetrahydrofuran and, finally, subtilisin in aqueous solution to hydrob'ze the 4-ester leaving the buB'rate at the 7-OH position. 31. •
GAERTNERHI:, FERJANCICA, PUIGSERVERAJ: Papain-Catalyzed Peptide Synthesis and Oligomerization of Amino Acid Amides in Organic Solvents. Biocatal3~is 1990, 3:197-205. Papain, either immobilized to porous glass beads or modified vdth pob~ethylene glyeol), is used to catab'ze peptide bond formation between N-acyl-L-aminoacid esters and bphenylalanine amide in 1,1,1trichloroethane. A x~trietyof dipeptides and oligopeptides are produced, some in yields of 95 %. The largest molecule produced is Bz-Tyr-(Phe) 4NH2 with yields approaching 10%. The oligomerization takes place vdth aromatic substrates that are substantially less reactive than charged amino acid derix~ttives,the natural substrates for papain. 32. •
BLA.'qCORM, GUISANJM, HAU~G PJ: Agarose-Chymoto'psin as a Catalyst for Peptide and Amino Acid Ester Synthesis in Organic Media. Biotecbnol Lett 1989, 11:811--816. Ch)rnotrypsin immobilized to an agarose support by multi-point attachment catalyzes peptide wnthesis in eth)t acetate, 1,1,1-trichloroethane, or 3-pentanone, once the support is moistened. Yields of N-acetyI-TrpI.eu-NH2 of 20 % are obtained in ethyl acetate. The enzyme is found to be highly stable in the hydrated agarose beads.
33.
TAI D-F, FU S-L, CHUANG S-F, TS.,d H: Papain-Catalyzed Esterification in Polar Organic Solvents. Biotedmol Lett 1989, 11:173-176.
34.
LAUMENK, SEEMAYERR, SCHNEIDERMP: Enz)anic Preparation of EnantiomericaU¥ Impure Cyclohexanols: Ester Synthesis by Irreversible Acyl Transfer. J Cbem Soc Cbem Commun 1990, 49-51.
35. ••
WALLACE JS, REDAKB, \VII//AMSME, MORROWCJ: Resolution of a Chiral Ester by Lipase-Catalyzed Transesterification with Poly(Ethylene Glycol) in Organic Media. J Org Ooem 1990, 55:3544-3546. Porcine pancreatic lipase in v'arm diisopropyl ether is used to catalyze the enantioselective transesterification of the S isomer from racemic 2,2,2-trichloroethyl 3,4-epoxybutanoate with poly(eth)tene glycol) as a nucleophile. The unique aspect of this w~ork is that a pob'meric nudeophile is used as the acyl acceptor which is insoluble in the reaction mixture and thus simple to separate from the unreac~-e isomer. Such reactions that have a direct impact on downstream processing should be very useful in future applications of enzymatic catalysis in non-aqueous media.
25.
COLO.~mOD, RONCHETn F, Toxt', I= Enz)anic Acylation of Sugars. Rationale of the Regioselective Butyrylation of Secondary Hydroxy Groups of D- and L-Galacto and Mannopyranosides. Tetrahedron 1991, 47:103-110.
26.
ADELHORSTK, BJORKLL';GF, GODTFREDSENSE, KJ.RKO: Enzyme Catalysed Preparation of 6-O-Acylglucopyranosides. S3mtl~ s/s 1990, 112-115.
27.
NICOTRAF, RNA S, SECtn';DO F, ZUCC)n~tU L: w-Functionalyzed Esters by Enzymatic AcylatiorL S)~ztbetic Commun 1990, 20:679--685.
37.
28.
NOZAKIK, UEMURAA, YAMASHn'AJ-I, YASUMOTO /%I: Enzymatic Regioselective Acylation of the 3'-Hydroxyl Groups of 2'-Deoxy-5-fluorouridine (FUdR) and 2'-Deoxy-5-trifluoromethyleneuridine (CF3UdR). Tetrabedron Lett 1990, 31:7327-7328.
38. ••
29.
I/ATOLlM, NICOLOSIG, PIATrEI// /%1:Enzyme-Catalyzed Alcoholysis of Flavone Acetates in Organic Solvents. Tetrabedron Lett 1990, 31:7371-7374.
30. •
MARGOLL-NAI., DELLNCKDL: Enzymatic Synthesis of Biologically Active Compounds: Synthesis of Castanospermine Derivatives. Biotecbnol Prog 1990, 6:203-204.
36. •.
Gtrr~t~NAL, ZUOB1 K, BRA~,a)OT: IApase-Catalyzed Preparation of Optically Active Gamma-Butyrolactones in Organic Sob:ents. J Org Chem 1990, 55:3546-3552. Stereospecific lactone synthesis is carried out using porcine pancreatic lipase in hexane. Chiral lactones are produced from racemic y-hydroxyesters. Furthermore, the enz3rne exhibits prochiral selectivity and catabxes the enantioconvergent lactonization of s37nmetrical y-hydroxy diesters. MACFm.L&'~'EELA, ROBERTSSM, TURNERNJ: Enzyme-Catalysed Interesterification Procedure for the Preparation of Esters of a Chiral Secondary Alcohol in High Enantiomeric Purity. J Cbem Soc Ctoem Commun 1990, 569-571.
PATILDR, RE'I'H~ISCHDG, DORDICKJS: Enzymatic Synthesis of a Sucrose-Containing Linear Polyester in Nearly Anhydrous Organic Media. Biotechnol Bioeng 1991 37, in press. An alkaline protease catabxes the pob'condeusation of sucrose with bis(2,2,2-trifluoroeth~tadipate) in a 1:1 ratio to give a molecular weigl/t ofMw = 2100 and Mn = 1600 and with pobxners in excess of 13000D. The exquisite regioselec~ityof the enz)rne recognizes sucrose as a diol rather than a molecule with eight free h~roxTl groups, with linkages forming at the 1' and 6' positions of the sugar. The poIymer is highly w~ter-souble and is degraded by the alkaline protease to free sucrose in aqueous solutions.
N o n - a q u e o u s e n z y m o l o g y Dordick 39. ••
WALLACEJS, MORROWCJ: Biocatalytic Synthesis of Polymers. II. Preparation of [AA-BB]x Polyesters by Porcine Pancreatic Lipase Catalyzed Ttansesterification in Anhydrous Organic Solvents. J Pol27n Sci: Part A. PolMn Chem 1989, 27:3271-3284. For the first time, an enzymatic process has been used to produce high-molecular-weight chrial pobxosters with Isln up to 14 900. The reaction takes place in tetrahydrofuran with octanediol and bis(2,2,2trichloroeth)t) alkane- dioates as substrates. Other diols and sob:ents have also been used. This method has produced the highest molecular weight polymers by purely enz3rnatic means in organic media. 40.
41.
PIETIKAINENP, ADLERCREtrrz P: Influence of the Reaction Medium on the Product Distribution of Pcroxidase-Catalyzed Oxidation of p-CresoL Appl Microbiol Biotechno11990, 33:455-458. ATSUU~,US, NAK&',;O M, KOmE Y, TANAKA S, OKKUBO M, YONEZAWAT, FUNAB,~StllH, HASHIMOTOJ, MORISHL%IAtt: An Efficient Enantioselectb,,e Preparation of 2-Substituted-3-Hydroxypropionic Acids via Chemoenzymatic Reaction. Tetrabed)on Left 1990, 31:1601-1604.
GHOGARE A, KUMAR GS: Novel Route to Chiral Pol¥mers Involving Biocatalytic Transesterilication of O-Acrylo}t Oximes. J Cbem Soc ClJem Commun 1990, 134-135. Chiral polyacrylates are prepared using a two-step, chemoenzymatic approach. The first step is the lipase-catab-zed acTlation ofa chiral alcohol
~xith an O-act5to)t oxime to give a chrial acr}]ate, and this is followed by polymerization w~th benzo)t peroxide. PolD-nerswith M,~.sup to 77 000 are prepared.
Annotated patents • •.
of interest of outstanding interest
P1. •
POKORAAR, CYRUSWL: Biocatalytic Process for Preparing Phenolic Resins Using Peroxidase or Oxidase Enzyme. 1990, US Patent 4900671. Horseradish peroxidase is used to catabxe the pob~nerization of phenols in organic solvents without the need for formaldehyde. These polymers have properties similar to novolak and resole phenol formalde-
hydes.
42. o.
JS Dordick, Department of Chemical and Biochemical Engineering and Center for Biocatab~is and Bioprocessing, Universityof Iowa, Iowa City, l o w 52252, USA.
407
Enzymes occupy a unique position in synthetic chemistry. They are highly respected catalysts for their exquisite selectivities under mild reaction conditions (e.g. ambient temperatures and pressures, neutral solutions). Nevertheless, synthetic chemists have been reluctant to employ enzymes as reagents in organic synthesis, although there is certainly a need for high chemical selectivity in synthetic chemistry, especially in the synthesis of pharmaceuticals, chiral intermediates, specialty polymers and biochemicals. The main reason for this reluctance has been the strict adherence by both chemists and biochemists to the conventional notion that enzymes function only in aqueous solutions. Indeed, it is stated in virtually every biochemistry textbook that enzymes are aqueous-based and require water for catalysis. This is unfortunate because, although water is an ideal solvent for the predominantly polar species required for life (e.g. amino and nucleic acids, carbohydrates, cofactors and proteins), it is a poor solvent for nearly all applications in industrial chemistry. Most organic compounds of commercial interest are spa.tingly soluble or chemically unstable in water, and most synthetic reactions take advantage of thermodynamic equilibria that are unfavorable in aqueous media. Recently, it has become clear that enzymes can function in organic as well as aqueous media [1-3]. The astonishing realization that enzymes can retain their high degree of reaction specificity in a nearly anhydrous milieu has improved dramatically the prospect of employing enzymes in synthetic chemistry. Synthetic chemists are now more likely to include enzymes in their catalytic repertoire and for good reason. From a biotechnologica] standpoint, there are numerous potential advantages to be gained from employing enzymes in organic media (Table 1) [1-3]. These potential advantages have been the driving force behind the development of numerous strategies for carrying out enzymatic reactions in organic media including biphasic, aqueous-organic reaction me-
dia, reverse micelle-containing media and monophasic reaction media [4,5]. Only in the last example, however, is the enzyme in a predominantly non-aqueous environment. Because of this, all of the potential advantages listed in Table 1 have been realized. Table 1. The potential advantages of employing enzymes in
monophasic organic solvents [1,2,6.].
1. Increasedsolubility of non-polar substrates. 2. Shifting thermodynamic equilibria to favor synthesisover hydrolysis. "3. Suppressionof water-dependent side reactions. 4. Alteration in substrateand enantioselectivity. 5. Immobilizationis often unnecessarybecauseenzymesare insoluble in organic solvents. 6. Enzymescan be reco'veredby simple filtration. 7. Easeof product recovery from low boiling, high vapor pressure solvents. 8. Enhanced thermostability. 9. Eliminationof microbial contamination. 10. Potential for enzymesto be used directly within a new or existing chemical process.
This brief review highlights research carried out in the past 13 months (since December 1989) in the specific area of monophasic organic enzymology. Earlier references are included for completeness only. The reviewwill
Abbreviations
CCL--Candida cylindracea lipase; DMSO~dimethylsulfoxide; ESR---electron spin resonance; MAS-~magic angle spinning; NMR--nuclear magnetic resonance. (~) Current Biology Ltd ISSN 0958-1669
401
402
Biochemicalengineering focus mainly on the fundamentals of catalysis in organic solvents, including the effects of organic solvents on enzyme structure and function. Selected applications that have been developed in the areas of asymmetric catalysis, polymer synthesis, and chemoenzymatic synthesis (the last example taking advantage of the high selectivity of enzymatic catalysis coupled with the high reactivity and flexibility of chemical synthesis), will be summarized in a brief applications section. The reader is referred to several recently published reviews that deal with general monophasic organic enzymologT [1,6,] or with specific synthetic applications [7"*,8,9*].
Fundamentals of enzymes in monophasic organic media Prior to 1989, the field of non-aqueous enzymology was mainly phenomenological. A large number of novel enzymic reactions had been examined, yet very few studies were commenced to elucidate the effect of the organic solvent on enzyme structure and function. Nevertheless, the following vitally important information was gleaned from these earlier studies [1,2,6.]. First, water bound to the enzyme is vital for enzyme function and stripping this enzyme-bound water leads to inactivation of the enzyme. Second, Michaelis-Menten kinetics are observed in organic media. Third, for highly active enzymes, intraparticle diffusional limitations can be overcome by immobflizing the enzyme as a monolayer onto a solid support. Fourth, enzyme activity can be increased substantially by lyophilizing the enzyme from an aqueous solution at the optimal catalytic pH and, in some cases, by lyophilizing the enzyme in the presence of an active-site inhibitor. Presumably the former results in a solid enzyme preparation with the proper ionogenic state of the charged functional groups of the enzyme, and the latter produces a highly rigid active site that is 'molded' into the proper conformation for catalysis to take place. Fifth, enzymes are highly rigid in nearly anhydrous organic media. This reduces their ability to accommodate bulky substrates. Finally, enzymes are more thermostable in nearly anhydons media. For a full description of these phenomena, the reader is directed to [1,2,6.] and citations therein.
Solvent effects on enzyme structure As a result of three separate studies [10"*,11o*,12.*], it has become clear that organic solvents that sustain catalytic activity do not substantially alter the active-site structure of an enzyme. For example, solid-state magic angle spinning (MAS) 15N nuclear magnetic resonance (N/VlR) studies of ~-lytic protease (a serine protease) enriched with 15N at the active site histidine revealed similar tautomeric structures and hydrogen-bonding interac-
tions for the enzyme in acetone, octane and water [10oo]. These results indicate that the catalytic triad of the enzyme's active site is intact in the organic solvent and is similar to that in water. In dimethylsulfoxide (DMSO), a solvent that does not sustain enzymatic catalysis, the catalytic triad structure is perturbed. The kinetic isotope study performed by Klibanov et a t [11 °'] supports further the maintenance of the catalytic triad in serine proteases. Virtually no difference in the magnitude of the primary deuterium isotope effect is observed in different organic solvents, suggesting that the transition state structure of the enzymatic process is independent of the nature of the reaction medium. These findings show, for the first time, that enzyme function in organic solvents is a result of the native catalytic machinery of the enzyme. In agreement with 15N NMR results for wlytic protease, electron spin resonance (ESR) analysis of an unrelated enzyme, horse liver alcohol dehydrogenase, showed that the enzyme did not denature when suspended in nearly anhydrous organic solvents [12°°], but became substantially more rigid. Addition of water to the solvent did result in an increase in the active-site spin-label flexibility, although increased catalytic activity was not obtained. The increase in protein flexibility with higher solvent water content is consistent with observed decreases in the thermostability of enzymes in partially hydrated organic media. For example, ATPase is highly stable at 58"C in toluene containing 0.32% v/v water, with a halflife of 74 min. In aqueous buffer, the half-life of the enzyme drops to 2 min [13"]. The rigidity of enzymes in low-water environments is directly responsible for the activating effect of activesite ligands (inhibitors) during lyophilization. This phenomenon is known as ligand memory [14]. The ligand binds to the active site of an enzyme, makes a structural imprint in the active site, and is washed away from the enzyme prior to addition of substrate. The molecular imprint of the active-site ligand remains and provides a 'frozen' conformation that readily accepts the substrate structure leading to enhanced activity on the given substrate. In addition to enhancing the enzymatic activity on natural substrates, this molecular imprinting technique Can be used as a method to alter dramatically the substrate specificity of enzymatic catalysis. For exampie, Mosbach and coworkers [15"] found that precipitation of chymotrypsin with n-propanol in the presence of N-acetyl-D-tryptophan (chymotrypsin is normally highly selective to the L-isomer) enabled the enzyme to catalyze the esterification of N-acetyl-D-tryptophan ethyl ester in cyclohexane. Enzyme precipitation in the absence of the ligand or in the presence of the L-isomer did not result in esterification of the D-isomer. Molecular imprinting has also been carried out on inert proteins to convert them into abiotic receptors [16"]. Such imprinted proteins have relatively high affinities toward the organic compound used as a template and structurally similar compounds, in nearly anhydrous solvents. The phenomenon of molecular imprinting has been suggested as a simple approach to the development
Non-aqueous enzymology Dordick
of bioadsorbents or novel biocatalysts. The enhanced rigidity of proteins in organic solvents is responsible for the molecular imprinting phenomenon. Addition of water to the solvent, which causes an increase in the flexibility of the protein in the organic solvent, results in the relaxation of the imprinting phenomenon.
Solvent effects on enzyme function Substrate specificity and catalytic efficiency depend on the ability of the-enzyme to use the free energy of binding with the substrate [17]. This binding energy reflects the difference between binding energies of substrate-enzyme and substrate-solvent interactions [18]. Thus, kinetic parameters describing enzyme function, such as binding constant with substrate, Ks, Michaelis constant, Kin, and catalytic turnover, kcat or Vmax, depend strongly on the solvent. It might be expected that the replacement of water with an organic solvent would lead to profound changes in the observed kinetics of enzymatic catalysis. A profound effect of the organic solvent is illustrated by the dramatic loss in"catalytic efficiency (kcat/Km) observed in low-water environments
[1,6,1. One hypothesis regarding the decrease in catalytic activity in organic media, compared with water, is that partitioning of the substrate from the enzyme's active site to the hydrophobic organic solvent occurs, thereby lowering the binding affinityof substrate with enzyme, while increasing the affinity of substrate with solvent. Our group has tested such a hypothesis using linear free energy relationships with peroxidase catalysis in non-aqueous media [19°',20]. Phenolic substrates that differed only in the hydrophobicity of the alkyl substituent groups were reacted in a variety of water-miscible and immiscible organic solvents. Hydrophobic substrates were found to be less reactive in an organic solvent than in water. Furthermore, as both the substrate and solvent hydrophobicities increased, the reactivities of the phenols decreased. These findings are consistent with the partitioning behavior of hydrophobic phenols between the bulk reaction medium and the peroxidase's active site. This partitioning is likely to diminish as substrate and solvent hydrophobicities increase, thereby necessitating a larger concentration of a given phenol to saturate the enzyme. This results in a higher apparent substrate, Kin, in organic versus aqueous media and a lower catalytic efficiency. Substrate and solvent hydrophobicities also affect the regio- and enantio-selectivity of lipase catalysis. Specifically, the Pseudomonas fluorescens lipase-catalyzed transesterification of 1,4-dibutyryloxy-2-octylbenzene with n-butanol in a hydrophobic solvent such as toluene, results in the formation of the 4-butyryloxy-3-octylphenol, the sterically favored reaction [21.]. However, the re-
gioselectivity of lipase catalysis is reversed in acetonitrile wherein the sterically unfavored 4-butyryloxy-2-octylphenol is produced. The hydrophobicity of the solvent controls the regioselectivity of catalysis; the more hydrophobic the solvent, the more favored the formation of 4-butyryloxy-3-octylphenol. Furthermore, replacing the octyl group with a methyl moiety results in the formation of the 3-methylphenol product, even in acetonitrile. These results suggest that, in hydrophilic solvents, the octyi moiety can favorably partition into a hydrophobic cleft in the lipase's active site that positions the transacylation reaction to produce 4-butyrytoxy-2-octylphenol. In more hydrophobic media, the free energy of partitioning of the octyl group into the hydrophobic active site is less favorable and the sterically favored reaction pathway ensues. Enantioselectivity of lipase catalysis is also affected by the nature of the substrate and solvent. The lipase from Candida cylindracea (CCL) catalyzes the enantioselecfive esterification of 2-hydroxyacids with primary alcohols in nearly anhydrous solvents [22.]. Straight-chain 2-hydroxy- acids are esterified with nearly 100 % enantioselectivity in toluene to give the S-isomer. However, the more hydrophilic the solvent, and also the more hydrophobic, the lower the enantioselectivity of CCL catalysis. The loss in enantioselectivity in hydrophilic solvents is caused by a dramatic drop in the catalytic efficiency of the S-isomer, while that of the R-isomer remains unchanged. In solvents more hydrophobic than toluene (e.g. cyclohexane), the reverse is true and the catalytic efficiency of the R-isomer increases relative to that of the S-isomer. This shift may be a consequence of the cyclohexane being more hydrophobic than the enzyme's active site. In addition to solvent hydrophobicity, the polarity of the reaction medium has a major influence on the enantioselectivity [23"]. For example, subtilisin-catalyzed transesterification between seophenethanol and vinyl butyrate showed greatest enantioselectivity in less polar solvents. The correlation between enantioselectivity and dipole moment or dielectric constant was far greater than that observed between enantioselectivity and solvent hydrophobicity. These results suggest that reversal of enzyme enantioselectivity may be controlled by the nature of the organic solvent on enzyme kinetics rather than on enzyme structure.
Applications of enzymes in monophasic organic media The advantages of performing enzymatic catalysis in nonaqueous media (Table 1) have led to a variety of potential commercial applications. This section summarizes the major advances made during the past year in the areas of asymmetric, polymer and chemoenzymatic synthesis. Specifics concerning some of the more seminal individual syntheses can be found in the annotated references at the end of this review.
403
404
Biochemical engineering
CHzOH
CHzOH
OH
o
~ O
o
+ H2OH
OH
o
n . CF~CH20-CI[CHz-]4C-OCHzCF3
OH Fig. 1. Enzymatic polymerization of sucrose to poly(sucrose adipate) in anhydrous pyridine. An alkaline protease catalyzes the polycondensation of sucrose with bis(2,2,2-trifluoroethyladipate) to
~ Enzyme Pyridine 45"C O II
O II
Lf-C-iCH2F4C-O O-CHz O~
OO-~H CH2OOCHO2OH ] n
polymers in excess of 13000D. The regioselectivity of the enzyme recognizes sucrose as a diol rather than a molecule with eight free hydroxyl groups, and linkages form at the 1' and 6 positions of the sugar. (See [38,'] for details).
Asymmetric syntheses
Chemoenzymalic syntheses
Nowhere is the power of enzymatic catalysis more evident than in asymmetric synthesis, particularly in the reactions catalyzed by hydrolases such as lipases and proteases. For the purpose of this review, the term 'asymmetric' includes stereoselective as well as regioselective syntheses. From a preparative perspective, the most important classes of reactions catalyzed by enzymes in asymmetric organic synthesis include site-selective acylation of polyols (e.g. sugars [24.,25,26,27], nucleosides [28] and alkaloids [29,30°], peptide synthesis [8,31%32%33], optical resolutions [34,35"'], lactone synthesis [36"'] and interesterifications [37].
Although enzymes are highly selective catalysts, their productivities and scope of reactivities do not often match those of their chemical counterparts. The potential to use enzymes in non-aqueous media enables enzymatic syntheses to be performed in media compatible with chemical syntheses and a number of combined enzyme/chemical processes have been developed. Chemoenzymatic syntheses are characterized by the high reaction selectivity afforded by enzymatic catalysis and the fast reaction rates and wide substrate specificities furnished by traditional chemical catalysts. Some examples include optically active 1,2-diol synthesis (Fig. 2), enantioselective synthesis of 3-hydroxypropionic acids [41] and production of optically active polyacrylates [42"].
Polymer syntheses Polymer synthesis has traditionally been outside the realm of the enzymologist. Most polymers are produced easily and cheaply using conventional chemical approaches and for the most part this has not changed. The use of enzymes in speciality polymer synthesis, however, does have several potential advantages including high regioselectivity over polyfunctional monomers (Fig. 1) [38"'], high stereoselectivity for use in the synthesis of chiral polymers [39"] and, potentially, a fine degree of control over pol~mer molecular weight and polydispersity, particularly in phenolic resins [40,P1.].
Fulure prospects Enzymatic catalysis in non-aqueous media is already having an impact on the way in which biochemists, chemists and engineers view applied enzymology. Biochemists are no longer limited to dilute aqueous solutions for biocatalysis. Chemists may now take advantage of enzymic specificity to catalyze reactions formerly limited to expensive and tedious chemical processes. Engineers can employ chemoenzymatic processes with the specific advan-
(a) CCL
(R,S)-R1CH(OH)COOH + R'OH
(S).RICH(OH)COOR, + (R)-RICH(OH)COOH + H20
(b) LiAI(OcH3)3H (S)-R~CH(OH)COOR'
I
>
(S)-R~CH(OH)CH2OH + R'OH
Fig. 2. General scheme for the chemoenzymatic synthesis of optically active diols using lipase from Candida cylindracea (CCL). (a) The first step in the reaction is the lipase-catalyzed esterification of a racemic 2-hydroxyacid. (b) The resulting S-ester is then reduced to give the 1,2-diol product. The following diols (all S-isomers) can be synthesized in this way: 1,2propanediol (R1 -- CH3); 1,2-butanediol (R1 = CH3CH2); 3-methyl-l,2-butanediol' [R1 = (CH3)2CH]; 1,2-hexanediol [R1 = CH3(CH2)3]; 4-methyl-l,2-pentanediol [R1 -= (CH3)2 CHCH2]; 3-phenyl-l,2-propanediol (R1 ----PhCH2).See [22"] for further details.
Non-aqueousenzymologyDordick rages of both enzyme and chemical catalysts. New reactions and processes are being developed at an accelerated pace. From a fundamental level, several questions remain to be addressed before a comprehensive understanding of non-aqueous enzylnology can be reached. For example, what is the effect of water on the activesite structure and function in enzymatic catalysis? How do the physicochemical properties of the solvent govern enzyme structure and kinetics? Can the effects of an orgarlic solvent on the catalytic efficiency and specificity of an enzyme b e predicted? Research addressing these questions is presently underway in a number of laboratories around the world,
MAS solid-state tSN NMR w~as run on tSN-enriched ct-bxic protca.se in wuter, octane, acetone and DMSO. In the first three soh-ents, the MAS spectra were nearly identical, suggesting that enz)anes in organic solvents maintain the same active-site structure as in x~uter. Only in a denaturing sob,~ent such as DMSO did perturbation of the MAS spectra result. This is the first direct indication that enz)ane active sites under non-denaturing conditions are not significantly pertubed by organic media. 11. ,.
ADAMSKAtt, CttUNG S-H, KLmANOVAM: Kinetic Isotope Effect Investigation of Enz)Tne Mechanism in Organic Solvents. J Am Omm Soc 1990, 112:9418-9419. For the first dme, a kinetic isotope investigation of an enz)xne is performed in nearly anhydrous media_ Deuterium isotope effects o f subtilism Carlsberg and a bacterial lipase showed no effect of soh'ent polarity on the ratio of vH/v D or HKm/DKm. 12. .,
have been highlighted as:
GUL'ZNRM, SKERKERPS, KAVANAGtlP, CLARKDS: Activity and Flexibility o f Alcohol Dehydrugenase in Organic Soh-cnts. Biotechnol Bioeng 1991, 37: in press. ESR studies of active-site spin-labeled alcohol dehydrogenase in anhydrous and partially hydrated organic media have been used for the first time to elucidate the clynamics of the enz)anic active site. The enz)~ne's active site w~ts found to become more rigid in less polar solvents with lower ~xuter contents, as determined by a decrease in the flexibility of the spin h b d .
• •,
of interest of outstanding interest
13. •
1.
DORDICKJS: Enz)anatic Catalysis in Monophasic Organic SOlvents. Enz3~ne Microb Technol 1989, 11:194-211.
2.
KLm&'~OV AM: Enzymatic Catalysis in Anhydrous Organic Solvents. Trends Biod.~m Sci 1989, 14:141-144.
3.
HAI~'~GPJ: Biocatalysis in l~ulti-Phase Reaction Mixtures Containing Organic Liquids. Biotedmol Adv 1987, 5:47-84.
4.
LqJ_V MD: Two-Liquid Phase Biocatalytic Reactions. J Cbem Tecb Biotechnol 1982, 32:162-169.
5.
M.ARTLNEKK, LEV&SHOVAV, KLYACItKO N, KHMEI.NITSKI YL, BERET_.LN IV: MiceUar E ~ o l o g y . Eur J Biod)em 1986, 155:453-468.
References and recommended reading Papers of special interest, published vdthin the annual period o f review,
6. •
DORDICKJS: Principles and Applications of Nonaqueous Enzymology. In Applied Biocatal3~'is edited by Blanch HW, Clark DS [book]. New York: Marcel Dekker 1991, 1:1-51. This re, Sew, with 190 references, covers the field of monophasic nonaqueous enz~xnology in depth up until 1989. The state o f fundamental knowledge of these enz}~ne s3~tems is described and put into perspective ~Sth the potential commercial applications of this emerging technology.
7. KtmANOVAM: Asymmetric Transformations Catal)-zed by Ene. zymes in Organic Solvents. Acc Clam Res 1990, 23:114-120. Highlights the practice and potential of the use of enzymes as as)mmetric catab~ts in nearly arthydrous orgamc media (92 references). 8.
KISE H, HAYAKAWAA, NORITOMI H: Protease-Catalyzed Synthetic Reactions and Immobilization-Activation o f Enzymes in Hydrophilic Organic Solvents. J Biotechnol 1990, 14:239-254.
9. .
PdVA S, SECtmqX) F: Selective Enzymatic Acylations and Deacylations o f Carbohydrates and Related Compounds. 03imicaoggi 1990, 8:9-16. A description, x~ith 38 references, of the use of enz)rnes as highly selective catalystsfor the modification of carbohydrates and their derivatives. A detailed summary o f enzyme-catalyzed sugar acTLations in organic solvents is presented from a chemist's perspective. 10. •o
BURKEPA, SMITHSO, BACHOVCHINWW, KtIBA.',;OV_42,1:Demonstration of Structural Integrity o f an Enzyme in Organic Solvents by Solid-State NMK J Am Chem Soc 1989, 111:8290-8291.
GARZA-RAMo5G, D ~ O N A, DE GOMEZ-PUYOU MT, GOMF.ZPOYOUA: Enz)ane Catalysis in Organic Soh-ents with Low Water Content at High Temperatures. The Adenosinetriphosphatase of Submitochondrial Particles. Biochemistry 1990, 29:751-757. Addition of x~ter to toluene activates ATPase and leads to reduced thermostability. When 3.8% (v/v) w~ter is added, the enz)rne exhibits saturation kinetics tow"ard Mg-ATP, with a Km of 0.3 mM (virtually the same as in ~ t e r ) , but a Vrra_xapproximately 100-fold lower than in ~uter. 14.
RUSSELLAJ, KLm.~XOVAM: Inhibitor-lnduced Enzyme Activation in Organic Solvents. J Biol Cl)em 1988, 263:11624-11626.
15. ,
STMILM, MANSSON1~,|-O, MOSBACH K: The Synthesis of a DAmino Acid Ester in an Organic Media with Alpha-Chymotrypsin Modified by a Bio-Imprinting Procedure. Biotecl> nol Lett 1990, 12:161-166. Freeze-drying en~Tnes in the presence of an ace-e-site ligand (first demonstrated by RusseU and Klibanov [14]) is used to reverse the selec~'ity of ch)Tnotr~psin to accept D-tt)ptophan substrates in anhydrous organic media. 16. •
B~CO k DABUUS K, Klm&'~OV AM: Production o f Abiotic Receptors by Molecular Imprinting of Proteins. Proc Natl Acad Sci USA 1990, 87:274-277. When a protein is dissoh'ed in a concentrated aqueous solution of an organic compound, freeze-dried, and washed ~ t h an organic st)gent to remove the organic compound (much as is done with ligand-acti~ t i o n of enz)Tnes in organic solventS), a molecular imprint results on the protein. Prexiously inert proteins, such as bo~Sne serum albumin can be converted into selective binding materials (adsorbents) in dry organic solvents. Addition o f even a small amount of water severely compromises the imprinting effectiveness. 17.
JENCKS WP (ED): Catalysis in Chemistry and Enz)maology [book]. New York:. McGraw-Hill 1969.
18.
FE~HT AR (ED): Enzyme Structure and Mechanism [book]. New York: \VII Freeman 1985.
19. ,,
RVIJ K, DORDICKJS: Free Energy Relationships o f Substrate and Solvent Hydrophobicities with Enzymatic Catalysis in Organic Media. J Am C79em Soc 1989, 111:8026-8027. Substrate and soh'ent h)xlrophobicities govern the catabxic efficiency of horseradish peroxidase in non-aqueous media. The primary effect o f a h)xtrophobic solvent is to increase the apparent substrate, Km. Linear free energy relationships suggest that ac~'ities of peroxidase in various organic media and xMth different substrates can be predicted on the basis of substrate and sob,'ent h)xlrophobicities alone. 20.
RYu K, DORDICK JS: Kinetic Behavior and Substrate Specificity of Horseradish Peroxidase in Water-Miscible Organic Solvents. Resources Conserv Rec)v:l 1990, 3:177-185.
405
406
Biochemical engineering 21. ,,
RUBIOE, FER,'O2,'DEZ-MAYORAt~A, KtmAxov AM: Effect of the Solvent on Enzyme Regioselectivity. J Am Cbem Soc 1991, 113:695--696. The hydrophobicity of the organic sob:ent affects the regioselectivity of lipase-catalyzed t ~ t e r i f i c a t i o n . The alteration in selec~'ity is caused by a drop in the free energy of partitioning of a hydrophobic side group of the substrate (the oct)t moiety of 1,4-dibut)a31oxy-2-oct)tbertzene) in more hydrophobic media, thereby favoring an altema~-e regioselectMty to that occurring in h)xtrophilic media. 22. •
PARIDAS, DORDXCKJS: Substrate Structure and Solvent Hydrophobicity Control Lipase Catalysis and Enantioselectivity in Organic Media. J A m Cbem Soc 1991, 113:2253-2259. CCL catab'zes the enantioselective estefification of 2-h)xlroxy acids in nearly anhydrous media. Straight-chain acids are highly reactive in toluene and give nearly 100% enantioselec~ities for the S-isomer. Branched acids, or any straight-chain acids in solvents less hydrophobic than toluene exhibit a loss in catabxic efficiency and a drop in enantioselec~ty, as the formation of the S isomer decreases and that of the R isomer remains constant. Sok'ents more hydrophobic than toluene also afford reduced enantioselectivities, and yet increased catablJc el~ciendes, primarily as a result of an increase in the reactivities of both the S and R isomers. The data tend to support the notion that the active site of CCL is as hydrophobic as toluene. This paper also describes a two-step chemoenzymatic wnthesis of optically active 1,2-diols from 2-hydroxy acids. 23. **
FITZPATR/CKPA, Ktm&'~ov AM: ttow can the Solvent Affcct Enzyme Enantioselectivity? J Am Ctoem Soc 1991, 113:3166-3171. Enantioselectivity of subtilisin Cadsberg in the transesterification between a chiral alcohol and vinyl but)rate is greatly affected by the polarity of the solvent. Correlations between enantioselectivity and solvent polarity (dielectric and dipole moment) am much greater than between enantioselec~ity and sok-ent hydrophobicity; the lower the polarity, the greater the enantioselectivity. Water, and w-ater mimics such as formamide, relax enantioselectivity in non-polar solvents such as dioxane, and yet do not affect significantly the enantioselectivity (already low) in polar soh'ents such as acetonitrile. 24. •
CIUFFREDAP, COLOMBO D, RONCttETI'I F, TO.XL~. I.: Regioselective Acylation of 6-Deoxy-L- and D-hexosides Through Lipase-Catalyzed Transesterilication. Enhanced Reactivity of the 4-OH Function in the L Series. J Org O?em 1990, 55:4187--4190. A number of commercially m'aJlable lipases are shoran to ac31ate bhexose monosaccharides at the relatively unreactive 4-OH position when the 6-OH position is blocked. Interestingly, D-sugars are acylated primar@ at the 2-OH or 3-OH positions. Such regiospecificity shifts allow the rational design of sugar acylation procedures to be implemented.
Castanosperrnine, a potent inhibitor of the endoplasmic reticulum enz3rne cz-glucosidase I, is modified by a novel three-step enzymatic synthesis culminating in the production of the 7-O-but)ayl defi~a~-e. The enz3anatic synthesis initially invok-es subtilisin to catab'ze the ac51afion of the 4-OH in pyridine, foUowed by a bacterial lipase (from Clam mobacterium viscosum) to catabxe the ac3~ationof the 7-OH in tetrahydrofuran and, finally, subtilisin in aqueous solution to hydrob'ze the 4-ester leaving the buB'rate at the 7-OH position. 31. •
GAERTNERHI:, FERJANCICA, PUIGSERVERAJ: Papain-Catalyzed Peptide Synthesis and Oligomerization of Amino Acid Amides in Organic Solvents. Biocatal3~is 1990, 3:197-205. Papain, either immobilized to porous glass beads or modified vdth pob~ethylene glyeol), is used to catab'ze peptide bond formation between N-acyl-L-aminoacid esters and bphenylalanine amide in 1,1,1trichloroethane. A x~trietyof dipeptides and oligopeptides are produced, some in yields of 95 %. The largest molecule produced is Bz-Tyr-(Phe) 4NH2 with yields approaching 10%. The oligomerization takes place vdth aromatic substrates that are substantially less reactive than charged amino acid derix~ttives,the natural substrates for papain. 32. •
BLA.'qCORM, GUISANJM, HAU~G PJ: Agarose-Chymoto'psin as a Catalyst for Peptide and Amino Acid Ester Synthesis in Organic Media. Biotecbnol Lett 1989, 11:811--816. Ch)rnotrypsin immobilized to an agarose support by multi-point attachment catalyzes peptide wnthesis in eth)t acetate, 1,1,1-trichloroethane, or 3-pentanone, once the support is moistened. Yields of N-acetyI-TrpI.eu-NH2 of 20 % are obtained in ethyl acetate. The enzyme is found to be highly stable in the hydrated agarose beads.
33.
TAI D-F, FU S-L, CHUANG S-F, TS.,d H: Papain-Catalyzed Esterification in Polar Organic Solvents. Biotedmol Lett 1989, 11:173-176.
34.
LAUMENK, SEEMAYERR, SCHNEIDERMP: Enz)anic Preparation of EnantiomericaU¥ Impure Cyclohexanols: Ester Synthesis by Irreversible Acyl Transfer. J Cbem Soc Cbem Commun 1990, 49-51.
35. ••
WALLACE JS, REDAKB, \VII//AMSME, MORROWCJ: Resolution of a Chiral Ester by Lipase-Catalyzed Transesterification with Poly(Ethylene Glycol) in Organic Media. J Org Ooem 1990, 55:3544-3546. Porcine pancreatic lipase in v'arm diisopropyl ether is used to catalyze the enantioselective transesterification of the S isomer from racemic 2,2,2-trichloroethyl 3,4-epoxybutanoate with poly(eth)tene glycol) as a nucleophile. The unique aspect of this w~ork is that a pob'meric nudeophile is used as the acyl acceptor which is insoluble in the reaction mixture and thus simple to separate from the unreac~-e isomer. Such reactions that have a direct impact on downstream processing should be very useful in future applications of enzymatic catalysis in non-aqueous media.
25.
COLO.~mOD, RONCHETn F, Toxt', I= Enz)anic Acylation of Sugars. Rationale of the Regioselective Butyrylation of Secondary Hydroxy Groups of D- and L-Galacto and Mannopyranosides. Tetrahedron 1991, 47:103-110.
26.
ADELHORSTK, BJORKLL';GF, GODTFREDSENSE, KJ.RKO: Enzyme Catalysed Preparation of 6-O-Acylglucopyranosides. S3mtl~ s/s 1990, 112-115.
27.
NICOTRAF, RNA S, SECtn';DO F, ZUCC)n~tU L: w-Functionalyzed Esters by Enzymatic AcylatiorL S)~ztbetic Commun 1990, 20:679--685.
37.
28.
NOZAKIK, UEMURAA, YAMASHn'AJ-I, YASUMOTO /%I: Enzymatic Regioselective Acylation of the 3'-Hydroxyl Groups of 2'-Deoxy-5-fluorouridine (FUdR) and 2'-Deoxy-5-trifluoromethyleneuridine (CF3UdR). Tetrabedron Lett 1990, 31:7327-7328.
38. ••
29.
I/ATOLlM, NICOLOSIG, PIATrEI// /%1:Enzyme-Catalyzed Alcoholysis of Flavone Acetates in Organic Solvents. Tetrabedron Lett 1990, 31:7371-7374.
30. •
MARGOLL-NAI., DELLNCKDL: Enzymatic Synthesis of Biologically Active Compounds: Synthesis of Castanospermine Derivatives. Biotecbnol Prog 1990, 6:203-204.
36. •.
Gtrr~t~NAL, ZUOB1 K, BRA~,a)OT: IApase-Catalyzed Preparation of Optically Active Gamma-Butyrolactones in Organic Sob:ents. J Org Chem 1990, 55:3546-3552. Stereospecific lactone synthesis is carried out using porcine pancreatic lipase in hexane. Chiral lactones are produced from racemic y-hydroxyesters. Furthermore, the enz3rne exhibits prochiral selectivity and catabxes the enantioconvergent lactonization of s37nmetrical y-hydroxy diesters. MACFm.L&'~'EELA, ROBERTSSM, TURNERNJ: Enzyme-Catalysed Interesterification Procedure for the Preparation of Esters of a Chiral Secondary Alcohol in High Enantiomeric Purity. J Cbem Soc Ctoem Commun 1990, 569-571.
PATILDR, RE'I'H~ISCHDG, DORDICKJS: Enzymatic Synthesis of a Sucrose-Containing Linear Polyester in Nearly Anhydrous Organic Media. Biotechnol Bioeng 1991 37, in press. An alkaline protease catabxes the pob'condeusation of sucrose with bis(2,2,2-trifluoroeth~tadipate) in a 1:1 ratio to give a molecular weigl/t ofMw = 2100 and Mn = 1600 and with pobxners in excess of 13000D. The exquisite regioselec~ityof the enz)rne recognizes sucrose as a diol rather than a molecule with eight free h~roxTl groups, with linkages forming at the 1' and 6' positions of the sugar. The poIymer is highly w~ter-souble and is degraded by the alkaline protease to free sucrose in aqueous solutions.
N o n - a q u e o u s e n z y m o l o g y Dordick 39. ••
WALLACEJS, MORROWCJ: Biocatalytic Synthesis of Polymers. II. Preparation of [AA-BB]x Polyesters by Porcine Pancreatic Lipase Catalyzed Ttansesterification in Anhydrous Organic Solvents. J Pol27n Sci: Part A. PolMn Chem 1989, 27:3271-3284. For the first time, an enzymatic process has been used to produce high-molecular-weight chrial pobxosters with Isln up to 14 900. The reaction takes place in tetrahydrofuran with octanediol and bis(2,2,2trichloroeth)t) alkane- dioates as substrates. Other diols and sob:ents have also been used. This method has produced the highest molecular weight polymers by purely enz3rnatic means in organic media. 40.
41.
PIETIKAINENP, ADLERCREtrrz P: Influence of the Reaction Medium on the Product Distribution of Pcroxidase-Catalyzed Oxidation of p-CresoL Appl Microbiol Biotechno11990, 33:455-458. ATSUU~,US, NAK&',;O M, KOmE Y, TANAKA S, OKKUBO M, YONEZAWAT, FUNAB,~StllH, HASHIMOTOJ, MORISHL%IAtt: An Efficient Enantioselectb,,e Preparation of 2-Substituted-3-Hydroxypropionic Acids via Chemoenzymatic Reaction. Tetrabed)on Left 1990, 31:1601-1604.
GHOGARE A, KUMAR GS: Novel Route to Chiral Pol¥mers Involving Biocatalytic Transesterilication of O-Acrylo}t Oximes. J Cbem Soc ClJem Commun 1990, 134-135. Chiral polyacrylates are prepared using a two-step, chemoenzymatic approach. The first step is the lipase-catab-zed acTlation ofa chiral alcohol
~xith an O-act5to)t oxime to give a chrial acr}]ate, and this is followed by polymerization w~th benzo)t peroxide. PolD-nerswith M,~.sup to 77 000 are prepared.
Annotated patents • •.
of interest of outstanding interest
P1. •
POKORAAR, CYRUSWL: Biocatalytic Process for Preparing Phenolic Resins Using Peroxidase or Oxidase Enzyme. 1990, US Patent 4900671. Horseradish peroxidase is used to catabxe the pob~nerization of phenols in organic solvents without the need for formaldehyde. These polymers have properties similar to novolak and resole phenol formalde-
hydes.
42. o.
JS Dordick, Department of Chemical and Biochemical Engineering and Center for Biocatab~is and Bioprocessing, Universityof Iowa, Iowa City, l o w 52252, USA.
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